„No matter how powerless we might think we are, we still should not, and must not remain indifferent

or silent when confronted by

injustice, corruption and lawlessness committed by the very men and women charged to protect and observe that same law.“
„When the rights and humanity of anyone is diminished then I am diminished, for am I not part of humanity? “
MICHAEL KAPOUSTIN JUNE 26, 2007
Answer to Bulgarian prosecutors and correctional officials when asked why he was defending the rights of fellow prisoners who
refused to help him
Excerpted from The Years in Solitary“

To the reader,

On the following pages are the U.S. and Bulgarian clinical results for what could have
been and still might be an exciting way to treat HIV/AID and Cancer patients in synergy
with the more conventional methods employed. The positive results here are only the tip
of an iceberg of discoveries that my company and I were pioneering in the early 1990’s.
We were recreating old knowledge for harnessing your body’s own immune system to
finish the work of the chemicals and other treatments. Why? Because in the end it’s
your body that has to do the final work of curing you, and keeping you healthy.

All the science we currently have now and had in the early 1990’s is there only to
reduce the disease or infection to a level where your immune system can take over. And
if it can’t, well we all know the consequences.

So what happened to this “miracle” of modern science and old world experience?

Well, I was arrested and imprisoned by the Bulgarian socialist [former communists]
government. They disliked me because I arrived in Bulgaria, recognized the science and
bought the technology for practically nothing. The now defunct Bulgarian communist
government and party had paid millions for development of this science but never saw
its potential. I did, so they hated me and made sure that I would not be allowed to bring
this technology to market.

With the secret assistance of Canada’s RCMP, the Bulgarian secret police had me
arrested, placed in solitary confinement, tortured for 3 years and then there was a circus
trial, a 23 year sentence and another 10 years in a maximum security prison.

I was finally released in July of 2008. And now, 1 year on, I am finally starting to share
all the bits and pieces of a complex story.

What follows is technical, and there is more in my archives. My prayers are to have the
time and resources to upload it all. The process just started.

The drawing to the left is mine, and it is my vision of myself in 1996 as I was dragged
down into the abyss of hell. I resurfaced stronger only thanks to God’s love and only
through His help.

Please read and write me if you wish, this document is not complete. I am still trying to
find the missing pages. I will.

Regards,
Michael Kapoustin
l

THYloIUS

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------------..-......-..:..~~====----- -
 Form Approved OM8 No 0910-0014
DEPARTMENT OF HEALTH AND HUMAN SERVICES Expiration Date: November 30, 1995
PUBLIC HEALTH SERVICE See OM8 Statement on Reverse.
FOOD AND DRUG ADMINISTRATION
NOTE. No drug may be shipped or clinical
INVESTIGATIONAL NEW DRUG APPLICATION (IND) investigation begun until an IND for that
(TITLE 21, CODE OF FEDERAL REGULATIONS (CFR) PART 312) investigation is in effect (21 CFR 312 40)

1. NAME OF SPONSOR 2. DATE OF SUBMISSION

L1FECHOICE INTERNATIONAL AD
3. ADRESS(Number. Street, City, State and Zip Code) 4. TELEPHONE NUMBER
(Include Area Code)
96-A Rakovsky Street
Sofia 1000, Bulgaria (*359-2) 883772
(*359-2) 801-236

5. NAME (S) OF DRUG (Include all available names. Trade. Generic. Chemical. Code) 6. IND NUMBER (if previously assigned)
Factor-R
7. INDICATION (S) (Covered by this submission) To prevent or to reverse AIDS - related immunological disorders

8. PHASE (S) OF CLINICAL INVESTIGATION TO BE CONDUCTED o PHASE1 o PHASE2 o PHASE3 o OTHER

L "'. LIST NUMBERS OF ALL INVESTIGATlONL NEW DRUG APPLICATIONS (21 CFR Parl312) . NEW DRUG OR ANTIBIOTIC APPLICATIONS
(21 CFR Parl314). DRUG MASTER FILES (21 CFR 314 420). AND PRODUCT LICENSE APPLICATIONS (21 CFR Parl601) REFERRED TO IN THIS
(Specify)

APPLICATION
-.
~

.
L 10. INO submission should be consecutively numbered. The initiallNO should be numbered
"Serial Number: 000. " The next submission (e.g., amendment, report, or correspondence) SERIAL NUMBER:

[ should be numbered "Serial Number: 001 . "Subsequent submissions should be numbered

consecutively in the order in which they are submitted. 0
- -0 -0
11 THIS SUBMISSION CONTAINS THE FOLLOWING (Check all that apply)
2'f INITIAL INVESTIGATIONAL NEW DRUG APPLICATION (IND) o RESPONSE TO CLINICAL HOLD

PROTOCOL AMENDMENT(~) . INFORMATION AMENDMENT(S). IND SAFETY REPORT(S).

o NEW PROTOCOL o CHEMISTRYIMICROBIOLOGY o INITIAL WRITTEN REPORT
o CHANGE IN PROTOCOL o PHARMACOLOGyrrOXICOLOGY o FOLLOW-UP TO A WRITTEN REPORT
o NEW INVESTIGATOR o CLINICAL
o RESPONSE TO FDA REQUEST FOR INFORMATION o ANNUAL REPORT o GENERAL CORRESPONDENCE
REQUEST FOR REINSTATEMENT OF IND THAT IS WITHDRAWN,
....J
INACTIVATED, TERMINATED OR DISCONTINUED
o OTHER
(Specify)
CHECK ONLY IF APPLICABLE

JUSTIFICATION STATEMENT MUST BE SUBMITTED WITH APPLICATION FOR ANY CHECKED BELOW. REFER TO THE CITED CFR SECTION FOR
FURTHER INFORMATION.

o TREATMENT IND 21 CFR 312.35(b) o TREATMENT PROTOCOL 21 CFR 312.35(a) o CHARGE REQUEST NOTIFICATION 21 CFR 312.7(d)
FOR FDA USE ONLY
CDRlDBIND/OGD RECEIPT STAMP DDR RECEIPT STAMP IND NUMBER ASSIGNED:

DIVISION ASSIGNMENT

FORM FDA 1571 (12192) PREVIOUS EDITION IS OBSOLETE PAGE 1 OF2

 I-

I
[ .

[
r
[ STUDY PROTOCOL
[
L
C
C
1--

l
 Fonn Approved OMB No 0910-0014
DEPARTMENT OF HEALTH AND HUMAN SERVICES Expiration Date: November 30, 1995
, PUBLIC HEALTH SERVICE See OMB Statement on Reverse.
FOOD AND DRUG ADMINISTRATION
STATEMENT OF INVESTIGATOR NOTE. No investigator may participate in an
investigation until he/she provides the sponsor
(TITLE 21, CODE OF FEDERAL REGULATIONS (CFR) PART 312) with a completed, signed Statement of
(See instructions on reverse side.) Investigator, Fonn FDA 1572 (21 CFR 312.53(c))
1. NAME ADDRESS OF INVESTIGATOR.

Prof. Bogdan Petrounov, MD, PhD, DSc

National Center of Infectious and Parasitic Diseases
26, Yanko Sakasov Str., Sofia 1504, Bulgaria
Phone (*359-2) 442 875, Fax (*359-2) 442 260
2. EDUCATION, TRAINING, AND EXPERIENCE THAT QUALIFIES THE INVESTIGATOR AS AN EXPERT IN THE CLINICAL INVESTIGATION OF THE
DRUG FOR THE USE UNDER INVESTIGATION. ONE OF THE FOLLOWING IS ATIACHED:
o CURRICULUM VITAE 0 OTHER STATEMENT OF QUALIFICATIONS

3. NAME AND ADDRESS OF ANY MEDICAL SCHOOL, HOSPITAL, OR OTHER RESEARCH FACILITY WHERE THE CLINICAL INVESTIGATION(S) WILL
BE CONDUCTED.

; Blackstock Family Health Center

[_ 4614 NIH 35
A4stin, TX. 78751

I 4. NAME AND ADDRESS OF ANY CLINICAL LABORATORY FACILITIES TO BE USED IN THE STUDY.

Clinical Pathology Laboratories

'Cross Park Drive, Austin, Texas 78754

l 5. NAME AND ADDRESS OF THE INSTITUTIONAL REVIEW BOARD (IRB) THAT IS RESPONSIBLE FOR REVIEW AND APPROVAL OF THE STUDY(IES).

r~
Western Institutional Review Board
PO. Box 10160
1006 W. 5th Ave.
Olympia, WA. 98502
[ 1-800-562-4789

I" , NAME OF THE SUBINVESTIGATORS (e.g., research fellows, residents, associates) WHO WILL BE ASSISTING THE INVESTIGATOR IN THE
CONDUCT OF THE INVESTIGATION(S).

David Wright, MD
,N6E: WlLL-It:\J.\5. c..C.~N

7. NAME AND CODE NUMBER, IF ANY, OF THE PROTOCOL(S) IN THE IND FOR THE STUDY(IES) TO BE CONDUCTED BY THE INVESTIGATOR

I
'-

FORM FDA 1572 (12192) PREVIOUS EDITION IS OBSOLETE. PAGE 1 OF2

 . , ..

8. ATIACH THE FOLLOWING CLINICAL PROTOCOL INFORMATION.

o FOR PHASE 1 INVESTIGATIONS, A GENERAL OUTLINE OF THE PLANNED INVESTIGATION INCLUDING THE ESTIMATED DURATION OF
THE STUDY AND THE MAXIMUM NUMBER OF SUBJECTS THAT WILL BE INVOLVED.

o FOR PHASE 2 OR 3 INVESTIGATIONS, AN OUTLINE OF THE STUDY PROTOCOL INCLUDING AN APPROXIMATION OF THE NUMBER OF
SUBJECTS TO BE TREATED WITH THE DRUG AND THE NUMBER TO BE EMPLOYED AS CONTROLS, IF ANY; THE CLINICAL USES TO BE
INVESTIGATED; CHARACTERISTICS OF SUBJECTS BY AGE, SEX, AND CONDITION; THE KIND OF CLINICAL OBSERVATIONS AND
LABORATORY TESTS TO BE CONDUCTED, THE ESTIMATED DURATION OF THE STUDY, AND COPIES OR A DESCRIPTION OF CASE
REPORT FORMS TO BE USED. ..

9 COMMITMENTS:

I agree to conduct the study(ies) in accordance with the relevant, current protocol(s) and will only make changes in a protocol after
notifying the sponsor, except when necessary to protect the safety, rights, or welfare of subjects.

I agree to personally conduct or supervise the described investigation(s).

I agree to inform any patients, or any persons used as controls, that the drugs are being used for investigational purposes and I wi" ensure
that the requirements relating to obtaining informed consent in 21 CFR Part 50 and institutional reView board (IRB) review and approval
in 21 CFR Part 56 are met.
I agree to report to the sponsor adverse experiences that occur in the course of the investigation(s) in accordance with 21 CFR 312.64.

I have read and understand the information in the investigators broshure, including the potential risks and side effects of the drug.

I agree to ensure that a" associates, colleagues, and employees assisting in the conduct of the study(ies) are informed about their
obligations in meeting the above commitments.
( ~:
I agree to maintain adequate and accurate records in accordance with 21 CFR 312.62 and to make those records available for inspection in
accordance with 21 CFR 312.68.

I will ensure that an IRB that complies with the requirements of 21 CFR Part 56 will be responsible for the initial and continuing review and
approval of the clinical investigation. I also agree to promptly report to the IRB all changes in the research activity and a" unanticipated
problems involving risks to human subjects of others. Additionally, I will not make any changes in the research without IRB approval,
except whe're necessary to eliminate apparent immediate hazards to human subjects.

I agree to comply with a" other requirements regarding the obligations of clinical investigators and all other pertinent requirements in 21
CFR Part 312.

[ INSTRUCTIONS FOR COMPLETING FORM FDA 1572

STATEMENT OF INVESTIGATOR:

L 1. Complete all sections. Attach a separate page if additional space is needed.

2. Attach curriculum vitae or other statement of qualifications as described in Section 2.

[ 3. Attach protocol outline as described in Section 8.

4. Sign and date below.

5. FORWARD THE COMPLETED FORM AND ATTACHMENTS TO THE SPONSOR. The sponsor will incorporate thi' .
information along with other technical data into an Investigational New Drug Application (IND).
INVESTIGATORS SHOULD NOT SEND THIS FORM DIRECTLY TO THE FOOD AND DRUG ADMINISTRATION.
[
10. SIGNATURE OF INVESTIGATOR 11. DATE

Public reporting burden for this collection of information is estimated to average 84 hours per res ponce, including
the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed
and completing reviewing the collection of information. Send comments regarding this burden estimate or any
other aspect of this col/ection of information, including suggestions for reducing this burden to:

Reports Clearance Officer, PHS and to: Office of Management and Budget

L Hubert H. Humphrey Building. Room 721-B

200 Independence Avenue. S.W
Washington, DC 20201
Paperwork Reduction Project (09100014)
Washington, DC 20503

Attn: PRA Pleace DO NOT RETURN this application to either of these addresses

L FORM FDA 1572 (12192) PAGE20F2

 I
r CURRICULUM VITAE
DAVID WRIGHT, M.D.

BIRTHPLACE Santa Monica, California

DATE OF BIRTH August 8, 1950

CURRENT ADDRESS 4614 N. I. H. 35 Austin, Texas 78751 (512) 458-9178

F MARITAL STATUS Married

EDUCATION Undergraduate : University of California at San Diego.

[ Degree : B.A., Biology, 1973

[ Medical: University of Texas Medical Branch, Galveston,

Texas, MD . 1980

[ RESIDENCY Farnily Practice Residency Program of Central Texas lv\edical,

Foundation/Brackenridge Hospital, Austin, Texas 1985-83

[ FElLOWSHIP Clinical Teaching Feliowship in Family Medicine, Fami!y PracticE'

Faculty Development Center, Waco, Texas 1985-86

! PRACTICE EXPERIENCE FamilyPractitioner/Ciinnical Director Mescalero Indian Health

Service Hospital, Mescalero, New Mexico

[. I\ssistant Director of Central Texas Medical Foundation's Family

Practice Residency Program, Austin, Texas 1983-85

Director of Central Texas Medica! Foundation's Familv, Practice

Residency Program, Austin, Texas 1987-Present

BOARD CERTIFICATION Family Practice- July 1983 Certified in Advance Cardiac Life
Support- 1983; Certified in Advance Trauma Life Support- 1984

SOCIETY MEMBERHIPS American Medical Association; Texas Medical Association;

Texas of Family Physician; Travis County Medical Society;
Fellow of the American Academy of Family Practice

CONSULTANT Physician Director

-Doctors House Call Service/Central East Austin

Community Organisation
-Austin Drug and Alcohol Abuse Program

Physician Consultant
-Austin Hospice Program

24
 r RESEARCH STUDIES "Evaluation of Magnitude and Duration Antipertensive Effect
of Tiamendinine in Patients with Essential Hypertension" 1987-88

"Study Comparing the Effectiveness of 20 mg Reglan Extentabs

and Placebo on Heartburn and Regurgitation Symptoms in
Patients with Gastroesophagel Reflux Disease" 1987-88

"Effects of Ethylene Oxide Exposure on Hospital Workers" 1986

"A Study to Compare the Efficacy of Carapho-Phenique Cold Sore

Gel Placebo and Blister Medicated Lip Ointment" 1991

"A Double-Blind Placebo-Controlled Study Comparing

Chloreseptic Spray vs. Placebo in Patients with Sore Throat Pain"
1990-91

"Investigator of a Double -Blind Placebo-Controlled Study

Comparing Ranitidine BID vs . Placebo in Patient with
Gastroesophgel Reflux Disease" 1990-91

AI DS-ASSOCIATED "Effects of Recombinant Erythropoietin on AIDS Patlenrs with

Anemia R With and Without AZr Use" 1990-91

[ )nvestigator of DOl and its Effect on AID Patientst/1989-92

[' "Ongoing Investigator of the Use of Alpha Interferon and its Effects
on H IV-Infected Patients With or without Kaposis Sarcoma" 1990

Principal Investigator

"An Open-Label Trial to Evaluate the Safety and Efficacy of

L Clarithromycin in the Treatment of Disseminated
MAC infection in AIDS Patients-M91-5 77" 1991

L "An open-Label Trial to Evaluate The Safety and Efficacy of

Clarithromycin in the Treatment of Disseminated
MAC infection in AIDS Patients-M91-577A for Patients who Have
[ Been on Clarithromycin Prior to study Enrolliment"

"A Treatment IND for 566C80 Therapy of Pneumocyslis Carinii

Pneumonia: Protocol 566-501" 1992

"Open label Oral 566C80 for the treatment of Patients with

Severe PCP who are intolerant and/on Unresponsive to Therapy
with Trimethopim/Sulpha methoxazole and Parenteral
Pentamidine" 1992
.....
"Single Patient Protocol for the Clinical Evaluation of
Intraconazole (R51, 211/CC) in the Compassionate Clearance
Treatment of System ic Mycoses" 1992

25
 Subinvestigator

i,Phase II Randomized Study to Evaluate the Safety and Efficacy of

Combination Therapy with AZT and intron A vs. AZT Alone in
Patients with Asymptomatic to Mildly Symptomatic HIV Infection"
1991

"Dideoxycytidine for Patients with AIDS or Advanced ARC who

r. Cannot be Maintained on Zidovudine (ZDV) for treatment of
Advanced HIV Diseases". 1992

~. "Open label Program of Dideoxycytidine (ddC) to be used in

Combination with Zidovudine (ZDV) for Tretment of AIDS-
Related Cryptococcal Meningitis". 1991
l "Double-Blind, Randomized, Comparative Study of Intraconazole
and Fluconnazole as Maintenance Therapy for Preventing Pelapse
L of AIDS-Related Cryptococcal Meningitis". 1992

r "Protocol TLC91 CG06: Multi-Center, Open Label Study of TLC G-

65 Single Agent Loading Dose with Subsequent Combination
Therapy in the Treatment of Disseminated MAl in AIDS Patients"
[' 1992

"A Community-Based Longitudal Study of HIV-Infected

[ Individuals (ODB)" 1992

"Open label Trial of Paromomycin for Cryptosporidiosis in I-IIV-

Positive Patients" 1992

"Rifabutin Therapy for the Prevention of Mycobactericurn Avium

Complex (MAC) Bacteremia in AIDS patients in
AIDS patients with CD4 counts < 200; A Double-Blind, Placebo-
Controlled Trial" 1990

"A study of Prophylactic Pyrimethamine Therapy for Prevention of

Toxoplasmosis Infection in individuals with Advanced HIV
infections" 1992

AIDS-RELATED CARE 1) Cared over 40 AIDS patients over the past 5 years;
ACTIVITIES
2) Cared for 12 patients receiving Gancyclovia and 3 patients
receiving Foscarnet over the past two years;

3) Care for over 150 H IV-positive patients at present;

4) Have an ongoing surveillance project to detect CMF Retinitis at

early stages.

26
 [
[
[ . CHEMISTRY, MANUFACTURING
AND CONTROL DATA
[
L
L
[-1
l ..!
 I
I GENERAL INVESTIGATIONAL PLAN

I PROTOCOL OF CLINICAL TRIALS WITH FACTOR-R

ON H/v-POSITIVE PEOPLE AND AIDS PA TlENTS

~ study will comprise a total of 100 persons, HIV-positive and AIDS patients divided in the
'ing groups:

) A - Patients with T-cells < 200 taki ng open label Factor-R;

b
~spira-
) 8 - Patients with T-cells < 200 - control;
Ie - Patients with T -cells 201-500 taking open label Factor-R;
r ;tab- I D - Patients with T-cells 201-500 - control;

of the tor-R in tablets of 10 mg is applied as follows: 3 tablets in the morning and 3 tablets in the
19 prior to meal in the course of 12 consecutive months.

19ainst lical observation.

3.inst
1,iicity nunological observation.
While
[ isor- nthly diary of self-observation of an HIV-positive or an AIDS patient.

t2spira-
r and

L
[ esof
/ other
[

28
 For Patients with T cell < 200
. Open label Factor-R
Patient would get Factor-R open label from the company and take standard dose.

Patient would be monitored monthly and also have:

(a) Karnofsky Scale q 2 months.

(b) Monthly CSC and SMAC.
(c) PPO q 6 months.
b (d) AFS blood cultures q 4 months or if they develop symptoms of MAC.

Patients may be on or off antiretroviral and will need to be on anti-PCP prophylaxis and may
[ be on other opportunistic prophylaxis, (fungal, MAC, viral).

[ Patients on open label will be compared to a control group matched for diseases state not
taking Factor-R and undergoing the state monitoring.

For patients with T cells 200-500

Open label Factor-R
Patient would get open label Factor-R for 12 months from the company in standard doses.

Patient would be monitored monthly and also have:

(1) Quality of life index questionnaire done quarterly.

c (2) T cells, CSC, SMAC done quarterly.

(3) Karnofsky Scale done q month.
(4) PPO q 6 months.

[ 1. Patient also needs to be on some antiretroviral treatment, (001, AZT, DOC orother mono or
combination therapy) .

2. Not on antibiotic prophylaxis for opportunistic infections.

Patient will be compared against a control group not on Factor-R on antiretrovirals and T cells
between 200-500.

32
 Control in the process of manufacturing of the
Factor-R's active substance

For the production of Factor-R 6 bacterial strains possessing the characteristic of the
species taxonomic symptoms are used. Each tablet contains 10 mg freeze-dr-ied lysate
and killed bacterial bodies of the following species: Streptococcus pneumonia,
Neisseria catarrhalis, Streptococcus pyogenes, Haemoplilus influenzae,
Staphylococcus aureus, Klebsiela pneumonia and other natural substances of the
purest and highest quality. A sample of each basic ingredient entering the
manufacturing facility is immediately analyzed for purity at Nationa! (enter of
Parasitic and Infection Diseases, Sofia, Bulgaria before it is approved for use in the
preparation .

Producing and keeping the "seed lot"

r
Ten typical colonies of a bacterial strain are cultivated on hard nutritive medium and
[. ·c ultured separately in test-tubes with the same nutritive medium: The bacterial
growth is washed with protective medium, poured out into ampules and freeze-

[ dried. After Iyophilisation a test is performed for viability, purity and taxonomic
characteristic. The freeze-dried strains are stored at temperatures from 20 to 80 (
and are used as "seed lots".

Controls during cultivation

[
A new ampule of the respective strain is opened for each cultivation . The content of
the ampule is cultured on hard nutritive medium. Uniform colonies with typical of
[ strain characteristics are used for subpopulation culture. The subcultures are tested
for purity by microscope analysis of the preparation coloured by Gram. The presence
of bacteria with characteristics not typical for the species is not allowed.

The density of the subpopulations is measured by spectrophotometer, after that each

subculture is then inoculated into the bioreactor. During bioreactor cultivation, the
development of the bacterial culture is followed by means of determinating density.

l- Samples are taken every hour. During the process of cultivation measurements of
technological parameters (temperature, pH and the level of the dissolved oxygen) are
automatically recorded. The cultivation is stopped at the beginning of the stationary
phase. At the end of the cultivation the purity of the bacterial suspension is checked
through microscopic analysis of preparations coloured by Gram and cultured on hard
nutritive medium. The presence of non typical bacteria and colonies with
characteristics not typical for the species is not permited . Bacterial density is also
determined.

35
 I
[ Control of the individual bacterial suspensions
Criteria for discarding
[ Three days after the addition of a killing agent, the suspensions are studied for sterility
by culturing on liquid nutritive medium. If necessary, depending on the bacterial
species, they are controlled for inactivation by culturing on specific hard nutritive
media. The presence of live bacterial cells is not allowed.

Stability of tablets

Factor-R's stability is investigated straight after the production of tablets and after 2
years of storage at temperature from 200 C to 25 0 C. The test are carried out with
Factr-R tablets of a series produced from active substances stored for 2 years at
temperature from 20 C to 80 C. For investigation of the stability the following criteria
are used:
- tablet's appearance
- tablet's solubility
- protein content, high effective liquid chromatography.

The methods here are the same as the ones lIsed for defying the quality of Factor-R
tablets.

r Table 1 displays the results of the investigation of the appearance, solubility and total
protein content of a Factol-R series straight after production in two years of storage.
[. No changes in the indicators tested at Faculty of Pharmacy, Department of Industrial
Pharmacy, Sofia, Bulgaria viewed after two years of storage at temperature in the
range of 200 C - 25 0 C.
[ Table 1
Stability data of Factor-R

[ Indicators Tablets after Tablets after

production 24 months

Appearance Enteric coated tablets Enteric coated tablets

Resistance in
gastric fluid 120 min. 120 min.

Total protein
by Lowry 4,20 mg/SO mg 4.21 mg/50 mg

Disintgration in
artificial enteric fluid to 30 min. to 30 min.

36
 I
r Figures 1 and 2 reveal results of high potency liquid chromatography of Factor-R
tablets straight after production and after two years of storage at temperatures of
e-
20 0 25°C. No changes in the retention times, number of components and their
concentrations where viewed. This is indicative of the good stability of the
preparation tested.

Factor-R tablets immediately after production

Absorbtion Units
35.00

I
I 28.75 1
(
[
22.50
22. 3~

r /1\
/ \

[
L
16.25 /\
' \121)
\f\
1_'
J
12.34

I: 10.00

0.00 22.50 45.00

Retention Time (minutes)

Figure 1

[
37

l:
 Factor-R tablets 24 months after production

Absorbtion Units
35.00

28.75 27.90

[ 22.50

[
r.
k
16.25

[ 1250

[
10.00

0.00 22.50 45.00

Retention Time (minutes)

Figure 2

38

._*- -.~.-.-~..... - - - -.. --.~- ---_.-_.-.-, ---..... ~-- ..... ----~-

 The results obtained show that Factor-R tablets possess good stability. No changes
related to the storage of the preparation after two years were observed. Estimation of
the specific biological activity - stimulation of the immunocompetent system using
Factor-R tablets stored for 22 months at room temperature was assessed.

The values of hemoagglutinating antibody against sheep erythrocytes in mice treated

perorally or subcutaneously with Factor-R tablets stored at room temperature for 22
months are presented in Table 2 (compare with Table 1 on page C - 8).

Table 2

Titre of hemoagglutination antibodies against sheep erythrocytes in mice treated

with Factor-R stored for 22 months at room temperature

Test groups of animals Antibody titre after administration

[ of 10 wh ite m ice each of the antigen sheep erythrocytes
treated with:

[ Primary antibody Secondary

response antibody
response

[ 7th day 14th day 7th day

L Factor-R perorally 7 days

before and 7 days after Ag 1 :256 1 :512 1 :2048

l Factor-R perorally 7 days

after administration of Ag 1 :128 1 :512 1 :2048

Factor-R s.c. 7 days

before and 7 days after Ag 1 :512 1 :2048 1 :4096
[
Factor-R s.c. 7 days
after administration of Ag 1 :256 1 :512 1 :2048

Control group - injected

with Ag only 1 :8 1 :16 1 :64

Ag - antigen - each mouse was injected i.v. with 0.2 ml of 50% suspension of sheep erythrocytes. On
the 28th day after the first administration the antigen was injected again in order to follow up the
secondary antibody response. Factor-R was administered in a daily dose of 2 mglO.5 ml physiological
solution perorally or s.c.

39
 As could be seen, no difference was observed between the capability for stimulation
of the immune system with freshly prepared Factor-R tablets and tablets stored for
22 months at room temperature and relative humidity 90% (table 1).

After storing Factor - R tablets for 22 months at room temperature the preservation of
specific biological activity was determined. Factor-R was applied orally to white mice
with staphylococcus infection in order to determine its protective effect.

These results can be seen in Table 3. No difference was found after the application of
freshly prepared or stored at room temperature for 22 months Factor-R tablets
(compare with Table 5 in part 4).

Table 3

Resu Its of the protective efficacy of Factor-R, stored for 22 months at room
temperature, administered perorally to white mice infected with staphylococcus.

Test groups of mice* Number of dead mice on:

treated with
1 2 3 4 5 6 7 8 9 10 11 12 day

L
Factor-R perorally 10
days before infection 2 2 2 2 1 1

Saline
perorally 10 days
before infection 8 1 1

* Each test group is of 10 white mice with body weight between 15 and 18 g each.

40
· .
PHARMACOLOGY ANDTOXICOLOGY
L DATA
L
[
[
 PHARMACOLOGY STUDY
I 1. Evidence of Bioavailability and Pharmacokinetics of
I the Active Component

1.1. Establishing the Immunostimulating Activity of Factor-R

1. 1. 1. Action on Antibody Formation

The immunostimulating activity of Factor-R was thoroughly examined. The following

tables represent several cases of studied immune response in mice resulting from
different schedules of administration of Factor-R. Some important conclusions about
the stimulating and prophylactic efficacy of Factor-R are set out based on the
described studies.
[
Table 1. represents the results of immune response to sheep erythrocytes in mice

r treated with Factor-R.

Table 1.
[ Titre of hemoagglutination antibodies against sheep erythrocytes in mice
treated with Factor-R.

Test groups of animals Antibody titre after administration

L of 8 wh ite m ice each
treated with:
of the antigen (sheep erythrocytes)

Primary antibody Secondary

l response antibody
response

7th day 14th day 7th day

Factor-R perorally 7 days

before and 7 days after Ag 1 :512 1:1024 1:2048

Factor-R perorally 7 days

after administration of Ag 1 :256 1:512 1 :2048

Factor-R S.c. 7 days

before and 7 days after Ag 1 :512 1 :2048 1 :4096

Factor-R S.c. 7 days

after administration of Ag 1:256 1:1024 1 :2048

Control group - injected

with Ag only 1 :32 1:128 1 :256

Ag - antigen - each mouse was injected i.v. with 0.2 ml of 50% suspension of sheep erythrocytes. On
the 28th day after the first administration the antigen was injected again in order to follow up the
secondary antibody response. Factor-R was administered in a daily dose of 2 mg/O.5 ml saline
perorally and s.c.

42
 I
f
The results of immune response to human serum albumin in mice is displayed In
Table 2.

Table 2.
Titre of antibodies against human serum albumin in mice treated with Factor-R

Test groups of animals Antibody titre after administration

of 8 wh ite m ice each of human serum albumin antigen
F treated with:
Primary antibody Secondary
response antibody
response

[ 7th day 14th day 7th day

[ Factor-R p.o. 7 days

before and 7 days after Ag 1 :64 1 :256 1:512

[ Factor-R p.o. 7 days

after administration of Ag 1:32 1 :128 1 :512
[.
Factor-R s.c. 7 days
before and 7 days after Ag 1:128 1:512 1 :1024

L Factor-R s.c. 7 days

r....:
J after administration of Ag 1 :64 1 :256 1:1024

Control group - injected

with Ag only 1 :8 1 :16 1:64

Ag - antigen - each mouse was injected i.v. with 0.2 ml human serum albumin. On the 28th day after
the first administration the antigen was injected again in order to follow up the secondary antibody's
response. Factor-R was administered in a daily dose of 2 mglO.S ml saline perorally and s.c. The
I, antibody titre was determined by passive hemoagglutination with sheep erythrocytes treated with
tannin.

The above results show that regardless of the schedule of application and method of
administration - perorally or subcutaneously - Factor-R evokes statistically significant
higher antibody titre against the antigen used than that of the control group. This
indicates that Factor-R has an obvious adjuvant action and stimulates the
immunocompetent cells which synthesize antibodies.

43
 1. 1.2. Determination of the spleen index in immunized mice

The stimulating activity of Factor-R was further substantiated by a higher spleen index
L
in mice fed with the polybacterial perorally or injected subcutaneously over the
r, ' course of 7 days compared with a control group (see Table 3).

Table 3
I. Spleen index of white mice immunized with human albumin and
treated with Factor-R
I
r
Test group of 8 white mice each Spleen index
treated with:

Factor-R perorally 7 days before and

[ 7 days after administration of Ag 1.660 mg

Factor-R orally 7 days after

[ administration of Ag 1.670 mg

Factor-R s.c. 7 days before and

7 days after administration of Ag 1.790 mg

Factor-R s.c. 7 days after

administration of Ag 1.400 mg

Control group injected with Ag only 1.030 mg

1 WK S.C. BEFORE AG

1 WK PERORALLY AFTER AG

1 WK PERORALLY BEFORE AG

1 WK S.C. AFTER AG

(AGONly)

Spleen of Index (mg)

Ag - antigen - each mouse was injected i.v. with 0.2 ml human serum albumin. Factor - R was
administered in a daily dose 2mg/O,Sml saline, perorally or s.c..The Spleen Index is the average mass
of the spleens of all mice in the group (the total spleen mass of the 8 mice is divided by 8). The Spleen
Index was determined 7 days after the second injection of Ag.

It is evident from these results that the intake of Factor-R leads to the activation and
involvement of the spleen in the immune defense of the organism.

44
 1. 1.3. Protective action on infections in white mice

In order to prove the stimulating action of Factor-R on the non-specific mechanisms

which determine the natural resistance of the body to infections a series of
experiments were carried out. Infection of white mice with a strain of Staphylococcus
aureus Sg 511, coagulase positive, which is pathogenic for mice, was used as a
model. Groups of 10 white mice each were injected intraperitoneally with Factor-R
in a dose of 5 mg/O.S ml saline solution 2 and 18 hours before being infected
intraperitoneally with a 25 million suspension/O.S ml of an 18 hour culture of
Staphylococcus aureus Sg 511.

Other groups of white mice (also 10 mice in a group) were fed in the course of 10
days with Factor-R also with a dose of 5 mglO.5 ml saline solution daily. On the 11th
day the mice were infected in the same way as the first group. All groups of mice
were followed up for 12 days after being infected. The dead mice were counted
every day. A Salmonella typhi Ty 2 glatt endotoxin solution according to Westphal
method (phenol-water) was used as a control. It was injected to groups of 10 white
l mice in a dose of 15 microgram/0.5 ml saline solution 2 and 18 hours respectively
before being infected with Staphylococcus aureus. The results of the experiments are

l presented in Tables 4. and 5.

[ Table 4.

Results of the protective efficacy of Factor-R administered subcutaneouslv to white

L Test group of
mice infected with Staphylococcus au reus. I

mice treated Number of dead mice on: (day)

( with:

2 3 4 5 6 7 8 9 10 11 12
Factor-R before
infection:
18 hrs 2 1 2 2
2 hrs 2 2 2 3
l Endotoxin before
infection:

L 18 hrs
2 hrs
1
2
2
2
2
1 2

Saline before
infection:
18 hrs 5 3
2 hrs 6 2

Each test group consisted of 10 white mice weighing between 15 and 18 grams each .

[
45
 Table 5

Results of the protective efficacy of Factor-R administered

perorally to white mice infected with staphylococcus

Test group of mice treated Number of dead mice on: (day)

with:

2 3 4 5 6 7 8 9 10 11 12

Factor-R 10 days
before infection:
2 2 2

Saline 10 days

[ before infection:
6 3

[ Each test group consisted of 10 white mice weighing between 15 and 18 grams each.

[ It is evident that when applied perorally and subcutaneously Factor-R has a clearly
expressed, favourable effect, on non- specific immune mechanisms. This is
evidenced by the longer survival of mice treated with Factor-R as compared to the
[ untreated control group. On the 4th day of the experiment when all control mice
had already died, 40 % of the mice treated with Factor-R were still alive when
injected 18 hours before the infection and 30% were alive when injected 2 hours
before the infection. The peroral test showed that all control mice died until the 3rd
day while 50 % of the mice treated with Factor-R were still alive. Since Factor-R
contains bacterial cellular walls and thus exerts a stronger endotoxic influence on the
organism these result are not surprising. These effects are evidenced by the
stimulation of various components of non-specific resistance to bacterial infections.

The protective activity of Factor-R administered intragastrically to mice weighing 22-

26 grams, each, against intraperitoneal infection with the K.pneumoniae "B" strain
which possesses a high virulence, was also investigated. The mice were immunized
during 9 days - each day with 10 mg Factor-R - and were infected on the 5th day
after the last dose. After the infection with 5 live bacterial cells, 48.5 % of the
immunized animals survived the infection. The K.pneumoniae "B" strain used is so
high in virulence that in the first case it destroyed the immune defense built as a
result of immunization with Factor-R.

Good results were later obtained after a double intragastrical immunization with 50
mg and 20 mg doses of Factor-R with an interval of 9 days between injections. 44 %
of the animals infected with 4 K.pneumoniae "B" cells were still alive during the 7
days (see Table 6).

46
 Table 6
Protective efficacy of Factor-R applied intragastrically on intraperitoneal infection
with K.pneumoniae "8"

I Group No. Number Infection dose Dead mice out of the Viability in %
of mice (live bacterial cells) total number

r 1. 7 5
on the 7th day

6 14
2. 6 10 5 17
3. 8 5 5 38
4. 7 10 6 14
5. 9 5 8 11
6. 9 5 5 44

[
No.1, 2, 5 are control groups of mice not treated with Factor-R.
No.3, 4 are groups of mice immunized intragastrically with 10 mg of Factor-R.
No.6 is a group of mice immunized intragastrically with 50 mg and 20 mg of Factor-R with time
interval of 9 days.

[
l

,.

1. 47
 Immunomorphologic studies of organs of mice treated
subcutaneously or perorally with Factor-R

Microscope and electron microscope studies were carried out on paraffin and
ultrathin sections of immunologically competent organs of 4 groups of mice treated
subcutaneously and perorally with Factor-R.

In the regional inguinal lymph nodes of mice treated subcutaneously with Factor-R a
manifested hyperplasia and highly dilated marginal and intermediary sinuses filled
with cell elements of the plasmocyte series were found microscopically (fig. 1).

r:
[
L
l.
I
l
Fig. 1. Inguinal lymph node of a mouse treated five times subcutaneously
with 1/10 human dose (5 mg) of Factor-R; on the 5th day after the last
treatment. The intermediary sinus was strongly extended and filled with cells
from the plasmatic line. Staining - H.E. ( Magnification - 140 X.) For a control
mouse - see fig. 16.

48
 The mesenteriallymph nodes were activated with highly expressed hyperplasia of the
lymph follicles. The marginal and intermediary sinuses were dilated and filled with
cells of the plasmocyte series (fig. 2)

r
[

I~
[ Fig. 2. Mesenterial lymph node o f a mouse t reated five times
subcutaneously with 1/10 human dose (5 mg) of Facto r-R; on the
5th day after the last treatm ent. The intermed iary sinus was
strongly extended and fill ed with cells from the plasmatic line.
Staining - H.E. (Magnification -140 X. )

In the spleen a hyperplasia of the white pulp and a. considerable number· of blast and
plasma cells in highly dilated marginal ~ inus es were found (fig. 3).

Fig. 3. Spleen of a mouse treated five times subcutaneously with 1/10

human dose (5 mg) of Factor-R; on the 5th day after the last treatment. The
marginal sinus was again strongly extended and filled with blast and plasma
cells. Staining - H.E. (Magnification - 140 X. )

49
I.
[ The small intestines showed preserved intestinal villi. Mononuclear infiltration of the
subepithelial tissue and mild edema of lamina propria mucosae with a considerable
number of plasmoblasts - precursors of the cells which synthesize specific
immunoglobulins - were present (fig.4).

[
[

o
r.
Fig. 4. Small intestine of a mouse treated five time!> subcutan€Ously with 1/10
human dose (5 mgl of Factor-R; on the 5th day after the last treatment. The brush
border is well preserved . ( Magnification - 30.000 x. )

Lymphoid tissue hyperplasia and an enlargement of the T-dependent zone were

established in the Pexer's patches. A considerable number of cells of the plasmocyte

c series were present (rig.5). .

Fig. 5. Peyer's patch of a mouse treated five times subcutaneously with 1/10 human
dose (5 mg) of Factor-R; on the 7th day after the last treatment. Numerous plasma
cells in the strong extended sinus. Staining - H.E. (Magnification - 640 X. )

50
r
I Immunomorphologic studies of organs of mice treated
subcutaneously or perorally with Fador-R

r Microscope and electron microscope studies were carried out on paraffin and
ultrathin sections of immunologically competent organs of 4 groups of mice treated
subcutaneously and perorally with Factor-R.

In the regional inguinal lymph nodes of mice treated subcutaneously with Factor-R a
manifested hyperplasia and highly dilated marginal and intermediary sinuses filled
with cell elements of the plasmocyte series were found microscopically (fig. 1).

r.

Fig. 1. Inguinal lymph node of a mouse treated five times subcutaneously

with 1/10 human dose (5 mg) of Factor-R; on the 5th day after the last
treatment. The intermediary sinus was strongly extended and filled with cells
from the plasmatic line. Staining - H.E. ( Magnification - 140 X.) For a control
mouse - see fig. 16.

48
 The mesenteriallymph nodes were activated with highly expressed hyperplasia of the
lymph fo llicles. The marginal and intermediary sinuses were dilated and filled with
cells of the plasmocyte series (fig. 2)

[
r
L Fig. 2. Mesenterial lymph node of a mouse treated five times
subcutaneously with 1/10 human dose (.5 mg) of Factor-R; on the
5th day after the last treatment. The intermediary sinus was
strongly extended and filled with cells from the plasmatic line.
[ Staining - H.E. (Magnification -140 X. )

In the spleen a hyperplasia of the white pulp and a considerable number of blast and
C plasma cells in highly dilated marginal sinuses were found (fig. 3).

l
I.
[

L Fig. 3. Spleen of a mouse treated five times subcutaneously with 1/10

human dose (5 mg) of Factor-R; on the 5th day after the last treatment. The
marginal sinus was again strongly extended and filled with blast and plasma
cells. Staining - H.E. ( Magnification - 140 X. )

49
I.
[ The small intestines showed preserved intestinal villi. Mononuclear infiltration of the
subepithelial tissue and mild edema of lamina propria mucosae with a considerable

c number of plasmoblasts - precursors of the cells which synthesize specific

immunoglobulins - were present (fig.4).

I
~

r.
r.
[ Fig. 4. Small intestine of a mouse treated five times subcutanE.ously with 1/10
human dose (5 mg) of Factor-R; on the 5th day after the last treatment. The brush
border is well preserved. ( Magnification - 30.000 X. )

Lymphoid tissue hyperplasia and an enlargement of the T-dependent zone were

established in the Pexer's patches. A considerable number of cells of the plasmocvte
series were present (rig.5). '

Fig. 5. Peyer's patch of a mouse treated five times subcutaneously with 1/10 human
dose (5 mg) of Factor-R; on the 7th day after the last treatment. Numerous plasma
cells in the strong extended sinus. Staining - H.E. (Magnification - 640 X. )

50
I
r Thickened alveolar septa and peribronchial mononuclear infiltrates were observed in
the lungs (fig.6).

Fig. 6. Lung of a mouse treated five times subcutaneously with 1/10 human dose (5
mg) of Factor R; on the 7th day after the last treatment. Peribronchial mononuclear
infiltration was observed . Stai ning - H.E. ( Magnification - 140 X. ) For a control rnOU 5C
[' - see fig. 17.

Well-formed lymphoid nodules with blast cells were also found. These findings showed that
L .' .
the lungs were actively involved in the immunogenesis as a secondary organ (fig.7) .

l~

Fig. 7. Lung of a mouse treated five times subcutaneously with 1/10 human dose (5
mg) of Factor-R; on the 7th day after the last treatment. Newly formed lymph nodes in
the parenchyma of the lung were observed. Staining - H.E. ( Magnification - 140 X. )
For a control mouse - see fig. 17.

51
 The electron microscope examinations showed that the plasma cells localized in the
dilated sinuses of the regional lymph nodes had a highly developed endoplasmic
reticulum and that they were in an active state (fig.7). The same types of plasma cells
were found in the mesenteriallymph nodes (fig.8) and in the spleen (fig.9).

[
r··· Fig. 8. Inguinal lymph node of a mouse treated five times subcutaneously vvith
1/10 human dose (5 mg) of Factor-R; on the 7th day after the treatment. Plasma cells
with a very well developed endoplasmic reticulum were observed . ( Magnification -
6.000 X. )

Fig.9. Mesenteriallymph node of a mouse five times treated subcutaneously with

1/10 human dose (5 mg) of Factor-R; on the 7th day after the last treatment.
Functionally active plasma cells with a very well developed endoplasmic reticulum
were observed. Magnification - 10.000 X.

52
 Mature plasma cells in an functionally active state with highly dilated cisterns filled
with secretory immunoglobulins were also found in the lamina propria mucosae of
the small intestines and in the Peyer's patches (fig. 10).

'-

Fig. 10. Spleen of a mouse five times treated subcutdneously with 1/10 human
L dose (5 mg) ; on the 7th day after the last treatment. Plasma cells with a very well
developed endoplasmic reticulum were obSf'rved. ( Magnification - 30.000 X. )

In the small i ntesti nes the vi II i were well preserved tfig. 11). The electron microscope
examinations proved that the so-called "brush border" was also fully preserved . No
shortening of the villi and no structural changes in the epithelial cells were found . A
great number of subepithelially located lymphocytes were observed .
[
[

Fig. 11 . Peyer's patch of a mouse five times treated subcutaneously with 1/10
human dose (5 mg ) of Factor-R; on the 7th day after the last treatment. Plasma
cells in a very good functional state and with a very well developed
endoplasmic reticulum were observed. ( Magnification - 40.000 X. )

53
 In the Peyer's patches a hyperplasia, an enlargement ofthe T- dependent zone and a
considerable number of cells of the plasmacyte series were found in the dilated
sinuses (fig. 12).

[
r Fig. 12. Small intestine of a mouse five times treated per os with 1/10 human dose (5
mg) of Factor-R; on the 7th day after the last treatment. A well-preserved brush
border was observed . (Magnification - 40.000 X.)

The mesenterial lymph nodes were also highly activated. The sinuses were dilated
and full of blast and plasma cells(fig. 13).

Fig. 13. Peyer's patch of a mouse five times treated subcutaneously with 1/10 human
dose (5 mg) of Factor-R; on the 7th day after the last treatment. Plasma cells with very
well developed endoplasmic reticulum were observed in a functionally active state.
( Magnification - 10.000 X. )

54
I
r In the inguinal lymph nodes a less expressed hyperplasia was found but with dilated
sinuses filled with cell elements of the plasmocyte series (fig. 14).

[
C Fig. 14. Mesenterial lymph node of a mouse treated five times per os with '!/10
human dose (5 mg) of Factor-R; on the 7th day after the last treatment. The
intermediary sinus was strongly extended and filled with the cells from the
plasmocyte line.5taining - H.E. Magnification .. 640X . . .

The same picture was seen in the spleen - hyperplasia of the white pulp, dilated
marginal sinuses filled with lymphocytes, blast cells and plasmocytes.

In the I.ungs of 5-times perorally trea.ted mice ~he alv~olar s~pta wer.e thickened with
a considerable number of lymphoid formations with peribronchial mononuclear
infiltrations (fig 15).

Fig. 15. Lung of a mouse treated five times per os with 1/10 human dose (5 mg) of
Factor-R; on the 7th day after the treatment. Newly formed lymph nodes localized
peribronchially were obseNed. Staining - H.E. Magnification -: 140 X. For a control
mouse - see fig. 17.

55
 The findings were similar to those seen in the mice subcutaneously treated with
Factor-R.

In the control mice the inguinal lymph nodes, the mesenterial lymph nodes, the
Peyer's patches and the lungs were calm. In the lymphoid organs single functionally
inactive plasma cells were found (fig. 16).

[
L·.
[
Fig. 16. Inguinal lymph node of an untreated mouse. Only separate plasmoblasts
[ were seen. Plasma cells with very well developed endoplasmic reticulum were not
seen . ( Magnification - 20.000 X. )

No thickened alveolar septa, peribronchial mononuclear infiltrations and lymphoid

nodules were observed in 'the lungs (fig. 17).

Fig. 17. lung of an untreated mouse. Peribronch ial mononuclear infiltrations and
newly formed lymph nodes we re not seen. Staining - H.E. ( Magnification - 140 X. )

56
 I
r The results indicate that Factor-R stimulates to a considerable degree the
immunologically engaged organs: lymph nodes, spleen and lung and preserves the
"brush border" of the small intestines. As well a lack of pathological changes in cell
elements of the other organs was observed.
,-
I

57
 Some investigations on the influence of Fador-R on the
pathenogenesis of pneumocystis carinii pneumonia
Prof. J. Cvetanov, Vet. med. PhD, DSc; Prof. B. Petrunov, MD, PhD, DSc;
Prof. P. Nenkov, MD, PhD, DSc
National Center of Infectious and Parasitic Diseases, Sofia

Pneumocystis carinii is a frequent cause of serious fatal interstitial pneumonia in

patients with congenital defects of the immune system or with acquired
immunodeficiency. In addition, the high frequency of pneumocystic pneumonia
among patients with acquired immunodeficiency syndrome (AIDS) has become an
object of public attention because of its high mortality rate.
The experiment was conducted on 20 rats, Sprague-Dawley of both sexes, with body
weight of 200-240 grams each, divided in two groups - control and experimental. The
control group was immunosuppressed wifh prednisolon (5 mg) twice a week
subcutaneously for eight weeks.The animals from the experimental group were fed 4
[ times per day in the course of 10 days with 25 mg of Factor-R (40 stimulations in all)
after that they were immunosuppressed with prednisolon (5 mg) weekly
subcutaneously for eight weeks. Both control and experimental groups were put on a
low protein diet and were given 1 mg tetracycline/ml drinking water. The animals
with pneumonia symptoms were- s.acri.fied using phenobarbital anesthesia. The lungs
were perfused through the right camera with 2.5 % glutaraldehyde containing 2 %
tannic acid.

Fig. 9 Rat lung trophozoites (T) with altered shape in close contact with one
another. Pneumocyte with opticaly empty mitohondria (M) was observed .
(Magnification - 8000 X )

Small pieces of rat lungs were immersed in fixative 2.5 % glutaraldehyde in 0.1 M
cacodylate buffer and postfixed in 2 % osmium tetroxide in 0.1 M cacodylate buffer
(pH 7.2) for one hour. The tissue blocks were stained on block with uranyl acetate in
0.1 M barbital buffer (pH 5.4) dehydrated in through graded ethanols and
embedded in durcopans. Sections were cut on a Reichert ultramicrothome stained
with lead citrate and examined with a JEM 100 C electron microscope.

58
 · ... ' .' ... --- .. -_.- . _- ..... _-.. .. .. ...- .. ." ...... .".- '.'~ ....- .; .. .... -.. ........... _.... -
- -

c Fig. 10 Lung from the rat A trophozoite (T) lines up along the alveolar wall on the
surface of a type I pneumocyte. ( Magnification- 16000 X )

[ Observation on ultrathin sectioned P. carinii rat lung revealed many trophozoites in

the alveolar space . There was great variety in size and shape. By altering their
shapes, the trophozoites were in very close contact with one another (Fig.9). In the
rat lungs the trophozoites lined up along the alveolar wall on the surface of type I
L pneumocytes (Fig.10) and type II pneumocytes (Fig.11). The type I and type II
pneumocytes which were in close contact with the trophozoites they showed
intracellular vacuoles and the majority of their mitochondria were optically empty
r. (Fig.10). A steady size increase of the lamelar bodies of type II pneumocytes and
degenerative changes of these cells were observed.

Fig.11 Rat lung Trophozoites (T) with different shapes in close contact with one
another and with a pneumocyte II type. ( Magnification - 20 000 X )

59
 • -~.- - - ' - . - - - - - - - - - , ..... ' •• '-!.... ...... - ••

c fig. 12 Rat lung Tubulomyelin structure (TS) included in its own vacuole. A
trophozoite
16000 X )
m
in dose contact with the vacuole wall was observed . ( Magnification -

In the alveolar space the trophozoites were in a very close contact with the
tubulornyelin structures considered to be pulmonary surfactanl. Moreover,
tubulomyelin structures were included in their own vacuoles and th e trophozoite
l. were tightly attached to the membrane of the vacuole (Fig.12).

l
(1
.- . ' 11.
'~~ 1' r-

fig. 13 Lung from a rat treated fi rst with factor-R perorally for 10 days with 2S mg
daily dose. Then subsequently treated for eight weeks sc. with 5 mg prednisolon.
The majority of the trophozoites m
were localized in the alveolar space.
( Magnification - 30 000 X )
In animals of the experimental group the symptoms of pneumonia appeared later.
After sectioning the lungs of the rats the pneumonial foci were not very large and the
lungs were not involved in a massive pneumonia. The majority of the trophozoites
were localized in the alveolar space (Fig.13).

60
I
r

[
Fig. 14 Lung from a rat treated first with Factor-R perorally for 10 days with 25 mg

r. daily dose. Then subsequently treated for eight weeks s. c. with 5 mg prednisolon .
The trophozoites (T) were not seen to be in close contact with tubulomyelinstructure
(TS) . ( Magnification - 12 000 X )

They were not localized intracellularly and were not seen to be in close contact with
tubulomyelin structures and lamelar bodies of pneumocystes of second type
(Fig. 14, 15).
L

Fig. 15 Lung from rat treated first with Factor-R 10 days perorally with 25 mg daily
dose. Then subsequently eight weeks s. c. with 5 mg prednisolon. The trophozoites
are localized in the alveolar space, but not intracelulary. ( Magnification - 50 000 X )

61
 '
I. :..;.:.. :'.;..~.'- .:-::.,; ;:";::;;;;':".;;::~:;!.:::;':;;~f:.!.;;~:-;;;:;"-"::~:~:.2~:%t~::~ •..:..:.:~~~::.::.::.-._ ~' •..

r These are, of course, preliminary investigations but we could suggest that treatment
with Factor-R prevents active development of Pneumocystis carinii in the lungs of
experimental rats.

The appearence of intracellular vacuoles in type I and type /I pneumocystes, the

increase of the lamelar bodies of type /I pneumocystes and degenerative changes of
these cells observed in ultrathin sectioned P. carinii rat lung in the animals from the
experimental group shows the efficacy of Factor-R on the Pneumocystis carinii
pneumonia.

[
[
c
c

[
[
L
[
62
I:
[
I
L
1.2. Changes in the pulmonary surfactant system in rats
stimulated with the polybacterial immunostimulant
Factor-R

Experiment:

Bronchiole-alveolar inflammatory processes owing to different respiratory infections

affect the pulmonary surfactant system and develop a respiratory distress syndrome.

[ Factor-R provokes humoral immune response in which the lymphoid tissue of the
intestines and bronchi are involved as well as the lung where lymphoid nodules are
formed, functioning as independent lymph follicles. It is known that the pulmonary
surfactant takes part in the protectio.n of the lung against the influence of different
non-bacterial and bacterial factors on it.
[
The level of pulmonary surfactant in-rats stimulated with Factor-R, concurrent with
other immunological changes occurring after stimulation, such as secretion, which
play a decisive role in the protection of the lung against different morbid external
influences, including bacterial, was studied.
[~

Materials and Methods:

[ The experiments were conducted on 20 male rats, Wistar breed, with body weight of
150-180 grams divided into two groups: a control group of 8 rats and an experimental
group of 12 rats .The animals were fed 4 times daily in the course of 5 days with 25
mg Factor-R - 20 stimulations in all. On the 5th day after the last stimulation the
animals were tracheotomized after a preliminary local anaesthesia with 1 % solution
of procaine. The trachea was cannulated with plastic cannula for realization of
broncho-alveolar lavage by standard method. A part of the lavage liquid was
separated for studying lung macrophages. The lungs were taken out for the purpose
of studying their phospholipid content. At the section material was taken for
histological and electron-m icroscopical study of the lungs, Peyer's patches,
mesenterial lymph nodes, spleen and bronchial lymph nodes. The materials were
processed by the usual histological and electron-microscopical methods and were
observed on an ordinary light microscope and on an electron microscope JAM 100 C
at different magnifications.

63
I . ----- '-'
r The phospholipid extraction was carried out by the method of Folch et al. (1947).
The phospholipid fractions were separated by thin layer chromatography and the
inorganic phosphorus was determined by the method of Kahovkova and Odavic
(1969). The protein was determined by the method of Lowry et al. (1951). The results
were processed statistically using the T-criterion of Student.

Results and Conclusions:

L At the peroral immunization the immunomorphological changes occurring in the

Peyer's patches, mesenterial lymph nodes, small intestines, bronchial lymph nodes
[ and the lung were examined.

(i) . Significant immunomorphological changes were observed in the

Peyer's patches. The lymphoid tissue localized in their T - and S-
dependent area was strongly hyperplasied . A significant number of
plasmatic cells with strongly developed endoplasmatic reticulum were
[ observed (Fig.18).

r.
L-
[

Fig.l B Peyer's patch of a rat treated with Factor-R; on the 5th day after the last dose .
Plasmatic cell with strongly developed endoplasmatic reticulum . (Magnification -
20,000 X)

There is morphological evidence that the Peyer's patches are not only
a source of cell predecessors of the plasmatic cells observed in the
lamina propria of the small intestines but that the Peyer's patches w ith
the plasmatic cells occurring therein directly take part in the secretion
of specific immunoglobulins. The activated lymphOid cells in the
Peyer's patches indicate a functional state conditioning th e
generalization of the immune response.

64
 I
r (i i). In the lamina propria mucosa of the small intestines a great number of
plasmatic cells with a strongly developed endoplasmatic reticulum
(Fig.19) was detected .

r
I

[
r
Fig.19 Small intestine of a·rat treated with Factor-R; on the 5th day after the last dose. In
[ the lamina propria mucosa plasmatic cells in a very aaive functional stat~ wit~.a strongly
developed endoplasmatic reticulum were observed . (Magnification- 12,OOO X) ,

L (ii i). The mesenterial lymph nodes were also strongly activated and in their
enlarged sinuses plasmatic cells in a very active functional state with a
strongly developed endoplasmatic reticulum (Fig. 20) we re observed.
[
r.

Fig. 20 Mesenterial lymph node of a rat treated w ith Factor-R; on the 5th day after
the last dose. Strongly activated lymphoid cells w ith numerous ribosomes in their
cytoplasm and a plasmatic cell with a strongly developed endoplasmatic reticulum.
(Magnification - 16,000 X)

65
I.
I,
(iv). Important immunomorphological changes were observed in the
I- parenchyma of the lung in which were detected formed lymphoid
nodules with plasmatic cells in an active functional state. This finding
/_. was similar in the bronchial lymph nodes where plasmatic cells with a
strongly developed endoplasmatic reticulum were observed

r
F

[
f

Fig. 21 Bronchial lymph node of a rat treated with Factor-R on the 5th day after the
last dose. A significant number of plasmatic cells with a strongly developed
endoplasmatic reticulum in an active functional state was observed. (Magnification -
[ 16,000 Xl

In rats treated with Factor-R the total phospholipids as well as the separated
phospholipid fractions were increased significantly. The phosphatidylchoiine and
phosphatidylglycerol possessing the most expressed surface active properties were
increased twice. The svingomyelin, phosphatidylserin , phosphatidylinositol and
phosphatidylethanolamin manifested statistically significant increases. An exception
in this respect was the diphosphatidylglycerol (cardiolipin) the increase of which was
l not statistically significant. The phospholipid content of the iung presented statistically
significant changes (Table 5).
[
[

66
 [
Table 5

Phospholipid content of alveolar surfactant and lung frpm rats, treated with Factor-R

Alveolar surfactat Lung

Phospholipids

L! Controls Experimental Controls Experimental

(mcglmg protein) % (mcglmg protein) % (mglg tissue) % (mglg tissue) %
b Lyso-
phospha-
thidyl- 1.1 ± 0.21 7 0.81 ± 0.2 4.4
choline

[ Sphingo-
myelin 20.16 ± 1.89 1.8 27.56 ± 2.15"· 2 1.45 ± 0.28 9.2 2.13 ± 0.3" 11.5
_._--_._- -
Phospha-
thidyl- 963.20 ± 21.8 86 1116.2 ± 23.6*** 81 6.71 ± 0.98 41.4 5.86 ± 1.78 31.7

L choline

Phospha-

l thidyl-
serin
11 .2 ± 0.97 20.67 ± 1.82"** 1.5 1.23 ± 0.25 7.8 3.33 ± 0.4** 18

r
Phospha-
thidyl- 5.6 ± 0.8 0.5 8.26 ± 1.21 ** 0.6 0.45 ± 0.09 2.8 0.61 ± 0.12 3.3
inositol

Phospha-
thidyl-
ethanol- 89.6 ± 5.9 8 150.2±7.1 10.9 3.4 ± 0.37 21.3 3.64 ± 0.43 19.7
amine

Phospha-
thidyl- 24.64 ± 1.97 2.2 48.35 ± 3.2 ...... 3.5 1.29 ± 0.2 7.6 1.48 ± 0.28 8
glycerol

Diphospha-
thidyl- 5.6 ± 0.92 0.5 6.9 ± 0.97 0.5 0.46 ± 0.09 0.9 0.63 ± 0.15 3.4
glycerol

*p < 0.05; **p < 0.01; ***p < 0.001

67
 The increase in the phospholipid fractions of the alveolar surfactant could be due to
stimulated synthesis, activated transport of the phospholipids effected by lipid
carrying proteins or to inhibited catabolism of the phospholipid molecules.

The described data speaks convincingly that peroral immunization with ·Factor-R
provokes a generalized humoral immune response. The hyperplasia of the bronchial
r increase lymph nodes, the observed immunomorphological changes in the lung and
I the increase of the phospholipid fractions in the alveolar surfactant system represents
a steady evidence of positive immunomodulating action of Factor-R in perorally
treated animals.

[
[
l

c
[
[
[
L
L:
I'

L. 68

I:
 I
~
1. Evidence of Efficacy of Factor-R.
r~ K. Kassabov, I. Stoichkov, D. Todorov
Medical Academy, Department of Experimental Cancer Therapy

I. 1.1lntrodudion

F In the following chapter some experiments which undoubtedfully reveal the efficacy
of Factor-R in its multiple applications are set out. There are some important notes
that you should consider before reading the information provided in this chapter.
t In the experiments that follow some other products have been used to show, through
I
a comparative study, the efficacy of Factor-R.
L
One of these products is the synthetic immunomodulator DTC
[ (diethylditiocarbamate) . Although this was not taken into account when experiments
were carried out, DTC has an interesting history as a proposed HIV therapy. The

[' study of this immunomodulator under the brand name Immuthiol, was claimed to
have shown some success in slowing the progression of HIV-related symptoms. In
October, 1990, Immuthiol was licensed by New Zealand as a treatment for HIV-
[ infected people in whom AZT therapy was ineffective. However, the FDA turned
down the treatments INO application (application for an "investigational new drug")
submitted by the patent-owing company, citing insufficient evidence of efficacy in
laboratories. In some of the experiments with Factor-R, treatment with DTC was used
as a comparative treatment, and in others a combined treatment with Factor-R and

L DTe was studied and compared to the respective single treatments.

Another pharmacological agent that has been used in the experiments summarized in
[ this chapter was Cyclophosphamide. This drug, being a typical immunosupressor,
was utilized to demonstrate the immunosuppressive nature of chemotherapy versus
the strong immunostimulative index of Factor-R. Moreover, the study of the
combined treatment with Factor-R and Cyclophosphamide allowed for a very strong
representation of the complete neutralizing effect of Factor-R with respect to the

L negative effect of immunosupressors. These experiments also provide an immune

model similar to that of human AIDS sufferers.

[ The main purpose of this study is to explore the possibility of the combined
therapeutical application with a polybacterial immunostimulant, Factor-R and
synthetic immunomodulator DTC, in experimental tumors and to examine their
L effects. For this we studied:

I' 1. The effect of Factor-R alone and in combination with DTC on immune
responce in normal and immunosuppresed with cyclophosphamide
mice (C57BU6), (BDF1).

69
 I -.. --- - --
(.
2. The influence of Factor-R and DTC on the growth and metastatic
acitivity of transplantanted tumors (Lewis lung carcinoma).

3. The immune mechanism, activated by Factor-R and DTC in tumor

bearing mice (C57BU6).

Materials and methods

[
Animals:
f- Normal and tumor-bearing mice (C57BL/6)

Tumors:
In this study we used Lewis lung carcinoma (LLC), lasting cellular lines
(YAC-1) and primary cultures from lung metastasis of LLC.

f. Agents:
- Factor-R;
- Cyclophosphamide (CY);
- DTC (diethyldithiocarbamate);

L - nutrium medium; chemicals; reactivs.

Treatment of animals:
L - Factor-R is disolved in saline and given peroraly in a daily dose of
50 mglkg for 10 consecutive days;
[ - DTC was applied s;c. at a Single dose of 25 rnglkg or i.p. (4 times
dosage of 10 mglkg);
- Cyclophosphamide at a single dose of 50, 100 and 150 mglkg.
I
b

Spontaneous lung metastases:

",
To produce spontaneous metastases LLC cells were injected into one
hind footpad of each mouse. At this place a progressive growing local
tumor has developed, providing spontaneously metastases in the lung.
After 11 or 12 days the primary tumor was removed by amputating the
tumor-bearing leg. Mice were autopsied after 22 or 25 days and their
lungs were fixed in Boun's fixative. The metastases were counted and
measured using a stereomicroscope.

Methods for the immune reactivity:

- DHR (delayed hypersensitivity reaction);
- cytolytic test in vitro;
- standard test for NK-cells cytolytic;
- rosette-formation with target cells.

70
 I
r~ In order to achieve more flexibility in the representation of the information, the
following abbreviations are used in the information provided in this chapter:
C Control group
FR Factor-R
CY Cyclophosphamide
I .
OTC - Oiethylditiocarbamate
T/C - Treated / Controls
Statistical Processing:
The statistical processing of information was carried out through the
methods of variation analysis. The reliability of data was analyzed according
to the t-criterion of Student.

1.2. Examination of the effect of Factor-R on mice

r 1.2.1. Effed of diethyldithiocarbamate (OTC) and Fador-R on the immune response of

normal and immunosuppressed mice (with cyclophosphamide (CY))
[ Fig. 1 shows the results of the effect of Factor-R and DTC on the primary
humoral immune response of normal mice BDF1. It was established that a 10-
l. day peroral treatment with Factor-R caused a significant increase (P < 0.01) in
the number of antibody forming cells (AFC) in the spleen. On the contrary, the
injection of OTC brought a decrease in the number of AFC. When the two
immunomodulators were applied in combination a statistically significant
increase (P < 0.001) in the number of AFC, even more expressed than the one
[ caused by Factor-R, was observed.

Effect of Factor-R (Ffl) and the DTC on the number of antibody

forming cell in the spleen

Antibody forming cells - Treated Mice/Controls (%)

r ..---··----··- --.-.-------. -------.. ----.----------..-:

250 -
I r-----.-------.. ---- - - - - ..---.-.--------
I
200 ~
-----._- - - - - - - - - - - - . - -

r
L •
150 ~

100 ~
I

o
C FR DTC FR-DTC

Fig.1 Effect of Factor-R (F-R) and DTC on Antibody Forming Cells in the spleen of normal mice
BDF1 .

71
I
r The evidence of the effect of Factor-R and OTC on OHR (delayed hypersensitivity
reaction) of normal mice BDF1 is displayed on fig.2/A/. The peroral treatment with Factor-R
led to a insignificant growth of the plantar edema. The fourfold administration of
OTC, however, significantly increased OHR. The combined treatment with the two
agents caused an even more expressed (P < 0.001) growth of the plantar edema.

r Plantar Edema
(A)
----
f- 1. 6 1
1.4 ~

1.2 -
1 ..

0.8 .

r 0.6 -1
0.4
1
I

r 0.2 -1
o
C FR DTC FR+DTC

[
A significant increase (P<0.001) of DHR was establis.hed in the injected with
cyclophosphamide (CY) (100 mglkg) animals as compared to the untreated controls
(fig.2/B!). The combination of the cytostatic with Factor-R led to a more expressed
growth of the plantar edema in comparison with the anirnals treated with CY only.
[ The involvement of OTC in the therapeutic scheme did not cause a significant change
of OHR in the animals treated with CY or with CY and Factor-R.

(8)
,._---------_._.. _-------_._-_.- ._-----_ .. _---,
I

0.5

o
C CY+FR CY+DTC CY+FR+DTC

Fig.2 Effect of Factor-R (FR) and OTe on OHR of normal (A) or injected with CY (B) mice BOF1.

72
 Fig. 3 shows the effect of Factor-R and DTC on the primary humoral immune
response of BDF1 mice immunosuppressed with CY. The injection of CY caused a
great reduction in the number of AFC in the spleen (36.3 ± 6.2) in comparison with
the untreated normal animals (451.6 ± 29.8), also BDF1 mice. The combined therapy
with CY and Factor-R led to a significant reinforcement of humoral response
suppressed by the cytostatic. A significant increase in the number of AFC was also
established in the group treated with CYand DTe. The combined treatment with the
three agents caused the most strongly expressed (P < 0,001) restoration of humoral
immune response suppressed by CY. In this case a significant increase in the number
f of AFC was observed as compared to the administration of CY with DTC (P < 0.01) or
with Factor-R only (P < 0.05).

r- Treated/Controls (%)
------------- -
100 -

90

80 -

l. 70 ""

60 - ,- -----_._-----_._----- - - . - - -.. - - - - - - - - - -
50 -

C 40 -

30 -
-- ------------- ------_.

[ 20 -

10 ..,
I

o
l Fig. 3
C CY CY+FR CY+DTC CY+FR+DTC
Effect of Factor-R (FR) and DTC on AFC in the spleen of immunosuppressed with
cyclophosphamide (CY) mice.
[ While analyzing the effect of DTC on The Natural killer cells function of hybrid mice
BDF1 we established that the splenocytes of the treated animals manifested an
intensifed rosette-forming and cytolitic activity in vitro towards target YAC-1 cells
(table 1).

Table 1
Effect of DTC on the spleen natural killer cells (NK cells) of immunosuppressed with
cyclophosphamide mice

NK cells-cytotoxicity (%)
Treatment spleno1Xtes bound with ._---_.. _ - - - - - -
Y C-1(%) 100:1 50 :1

No treatment 7.6+/-2 .1 32 .6 +/-2.4 22.9+/-1.9

DTC 21.1 + /-4.0** 39.7 +/-2 .0* 27 .6+/-1.3*

CY 5.0+/-1.7 16.1 +/-0.8*** 13.2+/-1.1**

CY+DTC 9.8+/-1.9+ 20.6 + /-2 .6** + 18.1 + /-2.4 +

*p<0.05, **p<O.Ol, ***p<O.OOl

significant differences to treated with CYanimals: + p < 0.05

73
I
I

I
r
[-:

f.
l.

L
C

6
 The injection of CY (100 mg/kg) caused a slight lowering of the binding ability of the
NK cells and a strong inhibition of their cytotoxicity. When a combined treatment
with the two agents was carried out we observed a significant increase both of the
cytotoxicity and the rosette-formation of the NK cells with target YAC-1 cells.

In Table 2, the spleen NK cells of treated with Factor-R mice manifested a slight

r intensification of the rosette-formation with target YAC-1 cells but their cytolytic
activity in vitro towards the same target cells marked with 51 Cr was significantly
reduced (table 2).
f- Table 2

[ Effect of Factor-R on the spleen NK cells of normal mice BDF1.

[ Treatment Splenocytes bound with N K cells-cytotoxicity (%)

---
YAC-1(%) 100:1 50:1
I
Saline 5.8+/-1 .1 39.1 +/-2.1 20.5 + /-1.3

L Factor-R 9.7+/-1.7* 27.6+/-1.7" 11.0 + /0.8"*

[ *p<O.05, "p<O.01, "*p<0.001

Fig.4 shows the results of the rosette-formation test of the spleen NK cells with

r. target YAC-1 cells.

Rosettes with YAC-l

[ ._-------
8-
~i --------

7 -"

6-

[ 5-

4-

l 3-

2~

L
1-

0
C FR CY CY+FR

Fig. 4 Effect of Factor-R on NK cell activity of injected with cyclophosphamide (CY) mice BDF1.

74
I. .o:::;.:i:i:.=,,:=:c~·-,._· ..

" We observed significantly (P < 0.001) reduced rosette-formation with the target cells
in injected with CY animals. The peroral treatment with Factor-R led to a complete
normalization of suppressed rosette-formation by CY.

Conclusions:

(i) . The research on the effect of Factor-R on spleen NK cells produced the
[ first evidence of influence of this preparation on the effector cell
population . The established increase in the rosette-formation of NK

t cells with target YAC-1 cells implies an increased number of surface

receptors, whereas suppressed cytolytic activity most probably marks a
hyperactivity of NK cells in the spleen. It may be explained with the
[ activation of cells with suppresive functions.

L (i i). While the effect of Factor-R on the immune response of CY

immunosuppressed mice was being examined it was established that

c the preparation twice increased the suppresed humoral response and

stimulated DHR (delayed hypersensitivity reaction).

[ 1.2.2. Effect of Factor-R and DTe on animals with implanted tumors.

The association between AIDS and cancer has been clear from the early days of the
epidemic when physicians in several cities began reporting numerous cases of
cancer. Scientists knew that suppression of the immune system could tigger
[ malignancies in transplant patients. They were not suprised therefore, that the
damage inflicted by HIV on the immune system could also cause cancer.

The limited success to date of conventional cancer therapy in the treatment of

d issem i nated malignancies has prom pted many atte m pts at deve lopi ng
immunotheraupetic regimes which attempt to activate host defense "in situ". As the
lung is frequently a site for the development of primary and/or metastatic tumors,
many efforts have been made to activate cells found in this organ which have potent
antitumor activity, such as natural killer (NK) cells or macrophages.

Clinical and experimental evidence suggesting that Factor-R may increase non-
specific defense mechanisms in the lung, the most frequently targeted organ for
haematogenous metastases, encouraged us to explore the possibility of the
immunostimulant inhibiting the formation and growth of pulmonary metastases.
Continuous peroral treatment with Factor-R may reinforce the immune mechanisms
of the lung against circulating tumor cells and may also prevent lung parenchyma
from localization of metastatic foci. White mice treated with Factor-R were used to
test these hypotheses.

75
 I .:.,
r·
Spontaneous metastasis model
I Lewis lung carcinoma (LLC) was implanted in the hind footpads of mice where a
local tumor was growing, metastasizing spontaneously in the lungs. The tumor-
I bearing legs of the mice were amputated 22 days after tumor implantation, .and their
lungs were fixed overnight in Bouin's fixative.

r Agents:
- Factor-R;

1- - Cyclophosphamide at a single dose of 50, 100 or 150 mglkg was injected i.p.

Animals - normal and tumor-bearing mice (C57BU6).

Results:

[ It was found that peroral treatment with Factor-R caused significant reduction of the
number of lung metastases in mice. The therapeutic efficacy of Factor-R was greater
when the treatment was initiated prior to or immediately after the implantation of
the tumor (fig 5).
Number of Lung metastases of LLC (%)
[. 100
*

90 -
80 -
r 70 -
60 -
50
40 -
30 -
20 -
10 -
0
C FR: 1-10 FR: 5-14 FR: 10-19

Fig. 5 Effect of Factor-R on the number of lung metastases of llC as a result of different schemes of
L • administration (1 st-1 Oth day after tumor implantation; 5th-14th day after tumor implantation; 10th-
19th day after tumor implantation)

Factor-R demonstrated an inhibitory effect not only on the number, but also on the
volume of metastases. In this case early prophylaxis with Factor-R immediately after
the implantation of the primary tumor was more effective than that applied on
preexisting pulmonary metastases. The efficacy of Factor-R depended on the degree
of dissemination of LLC- cells.

Peroral administration of Factor-R was significantly more effective in cases of

comparatively minor secondary tumors.

76
 r Twenty to seventy % of the animals treated with Factor-R were without visible
lung metastases on the 22-nd day after the tumor implantation. The greatest
r reduction of the number and volume of lung metastases was achieved in the
early phases of tumor dissemination. Administration of Factor-R after surgical
resection of the primary tumor also produced a significant effect in animals with
l spontaneous metastases.

r All these results demonstrated that Factor-R was effective against lung
metastases when administered perorally and that the maximum activity was

r- limited by the metastatic tumor burden. Therefore, the therapeutic potential

of Factor-R was examined in combination with chemotherapy to determine if
there was adjuvant activity for the treatment of well established metastatic
r lesions.

l Combined prophylaxis with cyclophosphamide and Factor-R

L
The effects of combined therapy with cyclophosphamide and Factor-R are
[ summarized in Table 3. The development of pulmonary metastases was inhibited
by each of these treatments alone. Cyclophosphamide demonstrated a strong
inhibitory effect in a dosedependent manner against lung metastases and the
dose of 150 mglkg was curative for 40% of the animals. Peroral immunotherapy
with Factor-R had comparatively minor therapeutic activity on the large tumor
l burden of preexisting pulmonary metastases. The combination of
cyclophosphamide at each dose tested with Factor-R had additional inhibitory
effects on lung metastases. The mice receiving a single dose of
cyclophosphamide of 50 mg/kg in combination with Factor-R showed a
significantly reduced number and volume of lung metastases as compared to
animals injected with cyclophosphamide only. This combined therapy
prolonged significantly the survival of the mice as compared to chemotherapy
r alone. In contrast, the addition of Factor-R to the treatment did not prolong the
survival of mice injected with 100 mglkg cyclophosphamide.

However, in this case a significant reduction of the number and volume of lung
metastases was also observed. Four out of ten mice receiving a single dose of
cyclophosphamide of 150 mglkg were freed from lung metastases, while no
metastatic nodules could be seen in six or seven of ten mice treated with
cyclophosphamide and Factor-R respectively. In short, these results
demonstrated that the potential therapeutic effect of cyclophosphamide on
preexisting pulmonary metastases was enhanced by subsequent peroral
treatment with Factor-R.

77
I
I
Table 3

Combination therapy of spontaneous lung metastases with cyclophosphamide (CY)

and Factor-R

CY Factor-R Lung metastases Incidence Survival· time

(mglkg) (Number of mice (days)
with pulmonary
[ Number Total volume
metastases /
number of mice
(cubic mm) examined)

t No 46.1 ± 13.6 67.6 ± 18 .2 10/10 28.2±3 .1

[ 50 No 19.3±7.1 23 .2±9.1 10/10 35 .8 ± 3.6

[ 100 No 9.5 ± 4.2 5.6 ± 2.0 10/10 43 .9 ± 5.6

r 150 No 4.0 ± 2.2 2.1±1 .0 6/10 ND

r
Yes 30.3 ± 8.8 38.5 ± 11.4 10/10 33.1 ± 4.2

50 Yes 9.4 ± 3.4*** 10.8 ± 2.8*** 10/10 43.0 ± 3.8

r 100 Yes 4.7 ± 1.2** 4.2 ± 2.1 9/10 47.7 ± 6.3

[ 150 Yes 1.8 ± 0.7** 1.9±O.6** 3/10 ND

[ *p < 0.05; **p < 0.01; ***p < 0.001

[
2. Immune mechanisms in the activity of Factor-R
[
The number of alveolar macrophages (AM) in the tumor-bearing mice was less than
the number of isolated AM in mice treated with Factor-R (both normal and tumor-
[ bering mice). In mice with i.m. implants of Lewis lung carcinoma (LLC) cell there was
a correlation between the increase of AM tumoricidal activity and the inhibition of

[ metastatic tumor growth.

Another interesting observation was the difference in the number of AM isolated

L from the control and those treated with Factor-R animals. In confirmation of this
observation two experiments were carried out - the effect of Factor-R on the
number of AM in normal (fig. 6) and implanted with LLC (fig. 7) mice C57BU6.

78
 Number of AM per Mouse 4(xl 0)
[ 1-
25

15 -;
I
I

[ o~

Factor-R Treatment

[ 2
5 Control group
Days of AM Harvest 10

[
Fig.6 Effect of peroral treatment with Factor-R on the number of alveolar macrofages (AM) in
normal C57BL/6 mice. Factor-R at 50 mglkg was administered perorally for 10 days, AM were
collected on days 1, 2, 5 and 10

f. Number of AM per Mouse 4(xl0)

[ 20 -
18 -

L 16 .
14 -
12 ·
10 -
8 -'
6-
4-

L 2-
0-

11 Factor-R Treatment

[ 12
15 Control group
20
Days of AM Harvest

Fig.7 Effect of peroral treatment with Factor-R on alveolar macrophages (AM) in LLC bearing
C57BL/6 mice. Samples of cells were inoculated i.m. on day o. Factor-R at 50 mglkg was administered
for 10 days, beginning on day 11. The AM were collected on days 11, 12, 15 and 20

79
L
[ Results:

I. (i) . In normal mice 24 hours after the first administration of Factor-R (on
the 2nd day) the number of isolated AM increased significantly. On the
fifth and tenth days this increase became even more pronounced. Ten
I days after the end of the treatment there was a great d~fference
between the numbers of AM harvested from the lungs of the treated

r (ii) .
and control animals.

In tumor-bearing mice peroral administration of Factor-R caused an

F increase in the number of AM which was insignificant at the beginning
of the treatment but on the 10th day was nearly as great as that in
normal animals.

In another experiment we followed the effect of Factor-R on induced by CY changes

[ in the normal composition of cells isolated by broncho-alveolar lavage (fig. 8)

Alveolar Cells per mouse 4(xl0)

60 -;

r. so ,
I

r- - - - - - - - - - - - - - - - - - - ._,

,,
[ 40 '-:
------------~

----.-------------~

r
30 ..,

20 ~
:

[
o
c FR CY CY+FR

• AM e Ly • PMN

[ --.----

[ Fig. 8 Effect of Factor-R on the composition of the broncho-alveolar cellular populations of mice
C57BL/6 immunosuppressed with cyclophosphamide (CY). AM, alveolar macrophages; LY,

L lymphocytes; PMN, polymorphonuclears.

L In the animals treated with Factor-R we observed a significant (760%) increase in the
total number of lavage isolated cells. The increase was significant (P < 0.001) for all
cellular populations (AM, lymphocytes and polymorphonuclears).

80
I:
[ The number of AM and polymorphonuclears was particularly increased in
comparison with normal controls. In the immunosuppressed with CY mice we
observed a strongly reduced number of AM whereas the number of lymphocytes
was not changed significantly. The two fold administration of Factor-R to the
injected with CY animals led to a restoration of the normal number of alveolar
I. cells.

[ In a separate experiment we examined the effect of Factor-R on the peritoneal

macrophages (PM) of normal and implanted with LLC mice BDF1 <table 4).

Table 4
r· Activation of peritoneal macrophages (PM) by Factor-R in normal and implanted with
LLC mice BDF1.

Therapy LLC Average number Cytotoxicity of PM

of PM per mouse (0/0 )
[ (100,000)

[ Saline No 1.2 ± 0.3 6.0 ± '1.1

Factor-R No 2.2 ± 0.5* 19.9 ± 2.4*

Saline Yes 0.6 ± 0.1 ** 2.2 ± 0.9*

Factor-R Yes 0.7 ± 0.1 18.6 ± 2.4* +

C *Significant difference to treated with saline normal mice

+ Significant difference to implanted with LLC mice
L It was established that the peroral therapy with Factor-R caused a significant increase
of cytotoxicity of PM. It was observed that the cytotoxicity of PM in the tUlllor-
bearing control animals was decreased in comparison with that of the normal
animals. However, the treatment with Factor-R led to a significant rise in the
tumoricidal properties of macrophages as compared to both tumor-bearing and
[ normal controls.

Conclusions:
u (i) . The activation of AM is the basic mechanism through which the
peroral i m m u nostim u lant Factor-R in h i bits the growth and
L dissemination of pulmonary metastases. The accumulation of
activated AM in the lungs and respiratory pathways shows the
defensive mechanisms of Factor-R against chronic respiratory
infections (fig. 6), which are the common life-threatening opportunistic
infection in AIDS sufferers.

81
I.
[ (i i). The peroral medication has antimetastatic activity in animals with
spontaneous pulmonary metastases. The greatest reduction of the
number and volume of lung metastases was achieved when the
treatment began in the early phases of tumor dissemination or after
surgical resection of the primary tumor (fig. 7). The antimetastatic
r activity of Factor-R increases host defence against tumors, .affecting
AIDS patients.

r (i i i). The combined therapy with Factor-R and cyclophosphamide led to

more effective inhibition of the metastatic growth in comparison with

E chemotherapy alone. This combined therapy could avoid the severe

side effects associated with high doses of cyclophosphamide . These
results suggested that the application of Factor-R could be successful in
the therapy of secondary pulmonary micrometastases. The success of
immunotherapy of tumor metastases depends on the capacity of

[ Factor-R to activate immune mechanisms in the target organ which

contains metastatic foci. The prooved effectivity of combined Factor-R
and cytostatic therapy may reflect in reduction of the dosage of
cytostatic agents in patients with AIDS, leading to the increase of their
toxic effects

[ (iv) . The cytolytic activity of AM recovered after peroral therapy with Factor-R for ten
days was higher than that of the control AM. The increase of cytolitic activity of
phagocytes may result in stimulation of host defence against tumors and certain
L intracellular microorganisms.

c (v). It was proven that there was a correlation between the capacity of Factor-R to
induce the accumulation of tumoricidal AM in the lower respiratory path ways and
lungs and the substances anti metastatic properties.

L In conclusion we can say that the activation of AM is one of the basic means of
inhibition of the growth of neoplasms.

[
[

82
I.
r
r TOXICITY STUDY

r 1. ACUTE TOXICITY

1. 1 Acute toxicity on albino mice

As experimental animals 60 albino mice of the strain ICR were used, weighting
18-20 g each. Twenty of them were fed per os through a probe with Factor-R - 50 mg
in capacity of 0,5 ml physiological solution 5 days. The control group of 10 mice were
fed in the same way with physiological solution only. The dose of 50 mg Factor-R
administered to mice is equivalent to about 5 000 daily human doses. All mice were
controlled regarding their general state, feeding and weight, and blood probes were
taken for determination of the main hematological indices (erythrocytes, leukocytes,
[ hemoglobin and differential counting).

The mice were sectioned under light ether narcosis on the 2nd day after the last
C·. feeding and a macroscopic view was made too. Material was collected for histologic
and electron-microscopic examination of the main parenchymal organs (lung, liver,
spleen and brain).
r.
No changes in general state, weight and behaviour, of the fed with Factor-R
compared with those from the control group were observed. The hematological
indices were in the limits of norm in both groups. The histological and electron-
microscopic study did not establish pathological changes in the examined organs.

r1 1.2 Acute toxicity on rabbits

As experimental animals 15 rabbits of the "Chinchilla" race were used, weighing

1.500-1.600 g each . Ten of them were fed through probe with Factor-R - 2 g, in
capacity of 2 ml physiological solution 5 days. This dose of Factor-R is equivalent to
about 2,000 daily human doses. Five rabbits were fed with 2 ml physiological
solution on the same scheme. All rabbits were controlled daily regarding their general
state, feeding and weight, and blood probes were taken for determination of their
main hematological indices (erythrocytes, leukocytes, hemoglobin and differential
counting).

The rabbits were sectioned after treatment with 300 mg hexobarbital sodium
intracutaneously on the second day after the last feeding. A macroscopic view was
made of the sectioned animals and material was collected for histological and
electronmicroscopic examination of their parenchymal organs (lung, liver, spleen and
brain).

83
 Compared with those of the control group, no changes in the general state, weight
and behaviour, of the animals fed with Factor-R were observed. The hematological
indices were in the limits of norm in both groups. No pathological changes were
observed in the organs examined through histological and electron-microscopic
studies.

r 2. CHRONIC TOXICITY

Chronic Toxicity in Rats

E 30 rats of the Wistar race, weighing 180 - 200 g each were used as experimental
animals. 20 of them were fed per os through probe with 0.5 g of Factor-R in capacity
of 1 ml physiological solution daily in the course of 4 months. One dose per rat is
equivalent to 5 000 daily human doses. The remaining 10 rats were fed with 1 ml
[ physiological solution on the same scheme.

The rats were controlled daily with regard to their general state, weight, feeding,
L morbidity and mortality. Their hematological indices were examined every month
during the 4-month period. The animals were sectioned under light ether narcosis 7

r. days after the chronical trials had been completed. A macroscopic view was made
and material for hematological and electron-microscopic examination of the main
parenchymal organs (lung, liver, spleen and brain) was collected.
[ As a result from the studies no changes in the general state (weight, feeding,
morbidity and mortality) of the animals fed with Factor-R was observed compared to
l those of the control group. The hematological indices were within the limits of norm
in both groups. No pathological changes were observed in the examined organs
through histological and electron-microscopic studies.
l

l
[

84
I
I
Name of drug: SUMMARY TABLE Processing number
FACTOR-R ref. to
III. A. ..I........
Generic name Page Number

Report date/Number:

r
Study period IV. 1987
SINGLE DOSE TOXICITY Referring to documentation
Volume: Page: to
Addendum No. :

F Species/Strain: White mice/JCR

Administration route: Number of animals: 60

per os

Treatment of controls: Dose <0.05 g>: max. non lethal: 5000 human doses
[ 10 white mice strain JCR
were treated per os with 0.5 ml
min. lethal:

saline and observed for 7 days Observation period: (Appl. day = Day 1)

r Study group (1) Contr. (2) (3) (4) (5) (6)

Dosage <0.01 g and 0.05 g > 0 1000 h. 5000 h

f. doses doses

Sex (m/t) m f m f m f m f m f m f
[ Animals per dosage 10 20 10 25 5

[ within 6 hours - - - - -
-
til
.c
ca
CD
7 - 24 hours
day 2-7
-
-
-
-
-
-
- -
- -
l C
I
day 8 - end of observ. - - - - -
Summary of salient findings: All mice were under daily control regarding their general
state, feeding and weight and blood testing was made for determination of the main hemato-
logical indices (erythrocytes, leukocytes, hemoglobin and differential counting). On the second
day after the last feeding the animals were sectioned under light ether narcosis and macro-
scopically viewed. Samples were taken from the main parenchymal organs (lung, liver, spleen and
brain) for histological and electron-microscopical testing . No changes were observed in the
general state (weight and behaviour) of the animals fed with Factor-R in comparison with
l hat of the control group. The hematological indices in both groups were within the normal
imits. No pathological changes in the examined organs were observed during the histological and
electron-microscopical studies.

Study conducted by the applicant: yes < > no < X >

If "no", indicate the name and address of the institute that conducted the study:
National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria
Study in compliance with GLP: yes < X> no

Page

85
I:

I.
Name of drug: SUMMARY TABLE Processing number

[ FACTOR-R ref. to
III. B .... .1.....
Generic name Page Number

I. REPEATED DOSE TOXICITY Report date/Number:

Study period IV. 1987

r 1. Subacute toxicity
(up to 3 months)
Referring to documentation
Volume: Page:
Addendum No.:
to

Species/Strain: Rabbits/Chinchilla

Number of animals: 15 Duration of treatment: 5 days

L Observation period after the end of dosing: 3 days
Administration route: per as
[ Treatment of controls: 5 rabbits
were treated per os with 2 ml
Age: at study
Body weight: 1500 - 1600 g initiation
saline for 5 days
Treatment days Der week: 5 consecutive davs
Study group (1) Contr. (2) (3) (4)

Dosage <2.0 g> 0 2000 human

L doses

Sex (m/t) m f m f m f m f
[ Number of test animals 5 - 10 5 ,
-

c Number of animals died

or sacrificed in extremis
- - - -

l Clinical observations: yes < X > no< > Clin. chemistry: yes < > no< >

1
Food consumption: yes < X > no < > Urinalysis: yes < > no< >
I
Water consumption: yes < X > no< > Organ weights: yes < X > no < > I

l Body weights: yes < X > no < >

Haematology: yes < X > no < >

Necropsy: yes < X > no < >

Histology: yes < X > no < >

L Additional examination: electron microscopy of the main parenchymal organs I

Additional information:

Histology performed according to EEC Note for Guidance: yes < > no< >
Study conducted by the applicant: yes < > no < X >
If "no", indicate the name and address of the institute that conducted the study:
National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria
Study in compliance with GLP: yes < X > no < > Page

86
 Name of drug: Processing number
FACTOR-R
referring to
III. b
Generic name SUPPLEMENTARY SHEET
REPEATED DOSE TOXICITY Report date/Number:
SUPPLEMENTAY SHEET
Study period (years): -
to III. b Referring to documentation
Volume: Page: to
Page from Addendum No. :

Important findiings Group 2 Group 3 Group 4

l M F M F M F

I[
I[
I[
I[
[

l
Il
Explanations: 0 = decrease 1= increase p= permanent t= transitory
ns = not significant * = p<O.05 ** = p<O.01 + = mild
+ + = moderate + + + = severe n = no. of animals Page
{ } = due to abnormal control values

l 87
I
I
Name of drug: Processing number
FACTOR-R
referring to
Generic name III
SUPPLEMENTARY SHEET
SUPPLEMENTARY SHEET Report date/Number:
TO SUMMARY TABLE .

r: III.
to register important
findings
Study period (years):
Referring to documentation
Volume: Page:
-

to
Page from Addendum No.:
~ The rats were under a daily control regarding their general state (weight, feeding,
morbidity and mortality). the hematological indices of both groups were investigated every
[ month during a 4-month period. Seven days after the chronic trial was completed the animals
were sectioned under a light ether narcosis. They were macroscopically viewed and

[ samples were taken for histological and electron-microscopical examination of the main
parenchymal organs (lung, liver, spleen and brain). No changes in the general state (weight

r
and behaviour) of the animals fed with Factor-R were observed in comparison with that of
the control group of rats. In both groups hematological indices were within the normal
limits. No pathological changes in the examined organs were observed during the histological
and electron-microscopical studies.

[
[
[.

[
L

L Page

89
r

I
~
I
[
f. RESULTS OF CLINICAL TRIALS
L
L
[
l

[
[
L
I
I. CLINICAL TRIALS

I A. Description of subject population

r The stimulating effect of Factor-R on the immune system was confirmed in extensive trials on
large contingents of subjects with various diseases.

r. B. Eligibility criteria

~ Trials were conducted in subjects with the following diseases:

- non-specific respiratory diseases,

[
- syphilis,

[ - lung cancer,

- HIV infection.
[
Although our investigations were conducted in subjects with different nosologic entities, the

[ common eligibility criterion was the presence of immunodeficiency in all of the remaining groups.

C. Study design
[ Subjects were treated with Factor-R perorally at the following doses: 25 mg, 40 g, 50 mg, 60 mg,
and 80 mg daily.
[ D. Objective parameters for both laboratory and clinical improvement

[ The clinical monitoring of subjects included: case history, physical examination and objective
condition, results of laboratory tests and some immunologic indices.

E. Toxicity

No adverse effects of toxicity induced by Factor-R were observed in any of the subjects.

F. Concomitant therapies allowed and utilized

Different groups of subjects received the following therapeutic agents in combination with Factor-
R, depending of the specificity of disease:

Antibiotics and chemotherapeutic drugs in subjects with syphilis and 8 AIDS subjects
out of 17;

Immunosuppressive therapy in subjects with lung cancer;

9 subjects with HIV-infection - antiretroviral drugs;

All subjects with nonspecific diseases and 9 HIV-infected subjects out of 17 received
no etiologic therapy to the date of clinical trials.
91
 G. Subject status at the conclusion of trial

The following effects were observed in subjects with chronic nonspecific pulmonary diseases
following Factor-R treatment:

beneficial effect on the clinical characteristics of the disease (severity, course and
outcome);

improvement of spirometric indices;

shortening of treatment duration;

[ favourable anti recurrent effect in long-term treated subjects;

immunologic indices showed an increase of non-specific defence mechanisms

[ (phagocytosis and stimulation of local and systemic humoral immune response).

c The conducted study shows that treatment with Factor-R has a stimulating effect on haemopoesis.
This reflects on the haemoglobin level and may lead to the improvement of the anaemic syndrome
in HIV infected persons.

[ The increase in the level of neutrophil granulocites and monocytes contribute to the activation of
the intracellular killing of phagocyted infectious agents. It also leads to the restoration of the
participation ofmonocytes and neutrophil granulocytes in the induction of humoral and cell-mediated
L immune responces. As a result of the stimulation of the immune system against secondary
opportunistic infections, the application of Factor-R will lead to significant reduction in the use of

[ antibiotics and chimiotherapeutics for the treatment of AIDS-subjects.

There is a demonstrabale increase in the level of total protein is especially beneficial in AIDS
[ subjects, due to their multiple reasons to develop deep hypoproteinemia.

These diverse effects of Factor-R have led to the improvement in the quality of life for HIV -infected
people and AIDS sufferers. The lack of stimulating effect on CD4 Iymphocites when accompanied
by the activation of the CDB Iymphocites, that produce IL-4 and IL-l0 could lead to an important
reduction in viral replication in host cells.
L Negative side effects were not observed in any of the subjects both during the appllication of F actor-
R as monotherapy and during its combined application with antiretroviral drugs. The foregoting
L logically leads to the suggestion that future wider research of the effect of Factor-R in combination
with antiretroviral agents could produce promising results. Although the research to date can be
regard as preliminary, we can suggest that the multiple effect of Factor-R on the protective
L mecanisms may optimize the treatment of HIV infected and AIDS sufferes.

92
 !._ ....

I. I. Examination of the effect of Factor-R on non-specific

respiratory diseases in humans.

r 1. Broad base clinical application of Factor-R for prophylaxis of broncho-

pulmonary infections in children

A basic problem of the medical practice in cases of chronic and recurrent broncho-
r pulmonary infections is prophylaxis. Modern research has entirely rejected the
preventive antibiotic treatment because of possible allergies or secondary activation
of pathogenic flora. Yet, HIV and AIDS sufferers receive such treatment as standart
course.

[ This necessitates the application of bacterial vaccines which through activation of the
immunocompetent system lead to the creation of short-term or long-term, local or

[ systemic specific or non-specific immunity. Factor-R is intended for strengthening of

the body's defensive mechanisms against acute and chronic diseases including
frequently occurring pulminary diseases in children.
( The similarity between this condition and the frequent occurence of pulmonary
disorders in HIV/AIDS sufferers makes the subject of this examination vital.
[
Clinical Experiment:

L 50 children at the age of 4 to 12 registered as frequently suffering from acute

broncho-pulmonary infections were observed for three months.

[ The immunostimulant was administered according to the following dosage scheme:

1 tablet of 25 mg daily in the morning before breakfast for 30 days, then followed a
[ 30-day period of rest, afterwards according to the above dosage scheme the first 10
days in 3 consecutive months. Side effects of the preparation were not observed.
,.
,
The children were divided into two groups:

[ (1) A group of 30 children treated with Factor-R

(2) Placebo group of 20 children

L For proper observation of the clinical effect of the application of Factor-R the
following indices were examined:

(1) The rate of secretory IgA in saliva

(2) The bactericidal activity of the serum

93
 I
[
Serum and saliva were examined before the course of treatment, on the tenth,
I thirtieth and ninetieth day after its beginning.

Results:

The results before the course of treatment and those from the tenth day were
compared with a placebo group of children whose serum and saliva were observed.

The results of the clinical effect of the administration of Factor-R are shown on table 1.

I Table 1
Diseases, antibiotic treatment and number of days of stay in hospital of the examined

[ children for a 3-month period of the previous year and the period of observation -
controlled study.

c For the previous

year
For the period of
observation

Studied patients Factor-R placebo Factor-R placebo

L Inflammatory disease
(total number) 95 65 37 61
[
Days of antibiotic
[ treatment (total number) 505 285 123 261

r
I ,
Number of days of
stay in hospital 138 127 33 137

[
n - number of observations
Studied patients: with Factor-R - 30, with placebo - 20

It is evident that there is a significant decrease in the number of diseases and a

reduction of the period of antibiotic treatment which is a marker of the seriousness of
L
the diseases.

In support of the results of the clinical effect are the results of the dynamic
examination of several immunologic parameters (see Table 2).

94
 I
[
Table 2
Dynamics of the level of IgA in the saliva of the children treated with F-R and those
from the control (placebo) group.

Before 10th day 30th day 60th day 90th day

treatment

Children treated 9.64 14.58 15 .36 14.2 13.95

with Factor-R

[ Control group 8.92 10.18 10.18 10.18 10.18

[
[ Conclusions:

r (i) . The application of Factor-R for prophylaxis of broncho- pulmonary

infections in frequently ailing children led to a significant decrease in
the number of diseases.

L (i i). The administration of the immunostimulant led to a considerable

increase of the level of IgA in the saliva of the children for the whole
period of observation.

r 1
(i i i) . We observed an increase in phagocytal and bactericydal activity
towards the bacterial strains contained in the vaccine.

1.2 Prophylaxis of broncho-pulmonary infections with Factor-R in adults

IgA has a great importance for the protection against bacterial infections of the
respiratory system. The immunological strength of a given immunostimulant can be
estimated by the increase of the IgA concentration in the serum and bronchial secretion.
In case of peroral administration of Factor-R a protection of the respiratory tract is
realized as a result of considerable increase of secretory IgA, e.g. Factor-R induces
resistance in patients suffering from chronic bronchitis.

Clinical Experiment:

34 patients with chronic bronchitis (bronchial form of chronic obstructive pulmonary

disease) at the age of 19 to 65 were treated with Factor-R. Factor-R was administered
during the period of clinical stabilization of the patients.

95
 I
I
During the first month the patients were given 1 tablet daily in the morning before
breakfast. Then followed a 30-day period of rest and in the end 1 tablet was
f
administered during the first 10 days of the third, forth, fifth months out of a total

r treatment period of six months. Every month the following indices were examined:
from the anamnesis - cough, expectoration: from a physical study of the lung -
evidence of bronchial obstruction, functional study of the breathing - vital capacity,

[ forced expiratory volume per second and Tiffno index; laboratory studies -
quantitative determination of IgA, IgG, IgM in the blood serum, of IgA, IgG, of the
secretory component SC and of albumin in the bronchial secretion using Mancini's
method.
I:
The changes in the characteristic of the passing of the chronic bronchitis were
[ determined by the frequency and duration of the fits as well as by the duration of the
temporary incapacity for work compared with the same period of the previous year.
With each observation the tolerability and the possible side effects of the preparation
[ were taken into consideration.

Results:

After the treatment with Factor-R the clinical picture changed. In the beginning all the
[ patients had a cough . At the end of the first month it stopped in 20 of the patients
(58 .8%) and at the end of the sixth month only 4 of them had a cough (1,1 %).
Similar changes were observed in the character of the expectoration . At the
beginning of the treatment course all patients had mucous expectoration (up to 20
cm 3 - 22 and above 20 cm 3 - 12 patients). At the end of the first month only 2
patients (0.6%) remained with more abundant expectoration (above 20 cm 3). The
expectoration was scarce in 20 (58 .8 %) and completely disappeared in 12 patients
(35.3%).
[
In the beginning while a physical study of the lungs was carried out 17 patients had

[ symptoms of bronchial obstruction . At the end of the first month obstruction was
established only in 4 patients (23 .5 %) and at the end of the treatment in none of
them.
[
Important changes in the basic functional indices of breathing were observed. The
[ vital capacity (VC) was increased from 2,500 up to 3,500 cm3 . Similar were the
changes in the forced expiratory volume (FEV1) and Tiffno index. They were a result
of an influence of the inflammatory component on the bronchial obstruction.
L The changes in serum immunoglobulins can be seen on Fig. 1. A certain increase in

L IgA and IgM was observed. The stimulating effect was slow and long. The decrease in
IgG probably reflected a tendency toward stabilization of the inflammatory process.

96
I
r 2.0 1
200
..
6--
I
-r
----¢:>--
__ ,,~
laoo; _ _

1'00 -
,
~

1200 -
150

.-----
t -- ~ .--- - -lgM

--0-- IgA I
1000 -

800 -

r
lOa
600 -

.0 400 -

200 -

--- -
r 0 ~1---------------------
30 60
D.,..
90 120
-----------
30 eo
D.,.. " 020

Fig. 1 Levels of the serum immunoglobulins (IgC IgA and IgM) during the 1st 2nd,
F I l

3rd and 4th months

[ .:.----.---~,

,
, ,
----.----
i

[
[
[
[
1st month 2 nd month 3rd month
__ .--.,
.

[ O lgA • IgG •
.-----.--~
Alb ~ SC i

[ Fig. 2 Levels of the immunoglobulins (lgA, Ige), of the albumin (Alb) and of the free
secretory component (5C) in the bronchial secretion during the 1st 2nd and 3rd
months.

The changes in the local immunity had a crucial importance for the passing of the
chronic bronchitis. Fig. 2 shows the changes which occurred in some of the basic
components of the bronchial secretion. The quantity of albumin and of IgG
decreased. This reflected a decrease of the degree of inflammation of the bronchial
mucouse membrane. The stimulation of local immunity is presented with an
increased level of IgA no matter that a certain decrease of the secretory component
could be observed. This is probably a result of the general stabilization of the local
inflammatory changes. A certain stimulation of cellular immunity could be observed
during the treatment period.

97
I
r Significant changes were established in the passing ofthe chronic bronchitis. Number
of fits as compared to those observed for the same period of the previous year
decreased from 3.1 to 1.3 and their duration - from 14.6 to 6.8 days. The temporary
incapacity for work for the two compared years decreased from 45.5 to 22.7 days.
Side effects as a result of the administration of the preparation were not observed.
[
Conclusions:

The clinical treatment that was carried out gave reason for the following conclusions:

(i). In the treatment of patients with chronic bronchitis with Factor-R it was
established that the main clinical indices changed for the better. The
bronchial obstruction was influenced significantly which was a result
[ of changes in its inflammatory component.

[ (ii). Changes in the levels of the serum immunoglobulins were observed

(stimulation of the humoral immunity). Some components of the
bronchial secretion which reflects the condition of the local immunity

C changed significantly. Mainly the production of IgA was being

stimulated. A certain stimulation of the cellular immunity could be
observed during the treatment period .

(iii) . As a result of the treatment the number and duration of the bronchial
fits decreased. The temporary incapacity for work dropped with 50%.
l (iv). The preparation has a very good tolerability.
[
[
[
[
L
L
L

98
 r-- II II LJ LJ r-l r-:J [-_-I II lTl ~ ~
~ [J

To Investigation: Project Participants, Diagnosis Period Test product. Criterion of evaluation Results Side
Page Investigator, number, and of Dosage and effects
Part Coordinating age, sex criteria for treatment administration
Organization admittance
1 2 3 4 5 6 7 8 9 10
K. Kisiova, The immunostimulating 161 patients (73 4 months Factor-R: 50 mg daily in Patients with data of immun- Factor-R Not
Zl. Zlatanov, activity of Factor-R was female and 88 the morning before deficiency suffering from provoked an observed
M. Peneva, compared in patients with male) aged 18 to 60 breakfast for 30 days, reccurent chronic non-specific antirecurrent
K. Yankov, afterwards according to pulmonary diseases were effect in 66.2%
recurrent non-specific selected. Before the treatment
An. Tzonev, diseases with data of the above dosage scheme and every following month an of the treated
\0 T. Mavrodiev. immune deficiency 10 days in 3 consecutive immunological study was made patients, in
\0 month including: phagocytic index, 82.25% the
Estimation of Some complement, serum IgG, IgA, period of
Immunostimulants IgM, circulating immune treatment was
Applied in Pulmonary complexes, intradermal skin tests shortened and
Practice. with kits of bacterial and mould in 30.6% it led
antigens for the determination of to reduction of
cell immunity state, of E-RFC-
Department of Internal total and active, the bronchial the treatment. It
Diseases, secretion obtained by was established
Clinic of Pneumology, fibrobronchoscopy was tetsted that some of the
Medical University, for alveolar macrophages, immunol;ogical
Varna, Bulgaria. protease inhibitors, lysozymes, indices,
immunoglobulin. The clinical especially the
effect was estimated taking into phagocytic
account the less frequency of the index and the
exudates, the shortening of
treatment periods and the rate ofIgA were
reduction of treatment in an influenced.
acute phase.
 ........---,
~ Il Il r-------'
J LJ rJ rJ rl 1I fTI :---l ~ 1I 1I

To Investiga tion: Project Participants, Diagnosis Period Test product. Criterion of evaluation Results Side
Page Investigator, number, and of Dosage and effects
Part Coordinating age, sex criteria for treatment administration
Organization admittance
1 2 3 4 5 6 7 8 9 10
K. Kissiova,
D. Petkova,
The clinical effectiveness of
Factor-R in patients with
93 patients (42
female and 51
Chronic
obstructive
I 4 months Factor-R : 50 mg
daily in the morning
The patients observed were
divided into 4 groups: with
I
In 81.9% of the Not
patients the period observed
before breakfast for chronic obstructive pulmonary of antibacterial
J. Radkov, recurrent chronic non- male) aged 47 !o pulmonary disease, chronic bronchitis with
A. Tzonev, specific pulmonary diseases 65, divided into 4 diseases, 30 days, afterwards recurrences, infectious bronchial treatment was
...... M. Peneva. was evaluated. groups pneumosclerosis, according to the asthma, morbus broncho- shortened and a
o infectious above dosage scheme ectaticus. All patients had data good antirecur-
o Factor-R in Clinical bronchial 10 days in 3 of suppression of the immune ren t effect was
asthma, morbus consecutive month. system determined by phagocytic obtained. Only in
Practice - Present and index, titer of complement, rate
Future Prospects. bronchoectaticus. of serum IgA, IgM, IgG, 30.1 % of the
circulating immune complexes, patients the basic
Department of Internal E-RFC and cell immunity using treatment with
Diseases, kits of bacterial and mould bronchodilating
Clinic of Pneumology, antigens injected intradermally. and inspectorat-
After the completion of ing agents was
Medical University, treatment these immunological
Varna, Bulgaria indices were observed monthly reduced. From
for a period of 6 months. The the immunolo-
clinical indices were also gical indices the
investigated: shortening of the increase of phago-
exudates, red uction of the cytic activity, rate
treatment, shortening of the of complement
treatment periods. The patients
treated with Factor-R were and serum IgA
compared with those undergoing titer was statist-
conventional therapy. ically reliable.
 .---,
. ----, 1I
LJ LJ r:J .. !l rTl ~ ~ II

To Investigation: Project Participants, Diagnosis Period Test product. Criterion of Results Side
Page Investigator, number, and of Dosage and evaluation effects
Part Coordinating age, sex criteria for treatment administration
Organization admittance
1 2 3 4 5 6 7 8 9 10
I. Iliev, Clinical immunological 37 ailing and 10 Non-specific 4 months Factor-R: 25 mg daily The treated children with Application of Not
V. Radanova, criteria for immuno- clinical healthy recurrent in the morning before immunodeficiency data were Factor-R leads to an observed
P. Georgieva, stimulating treatment with children aged 3 pulmonary breakfast for 30 days, selected among 153 increase in the
Factor-R of various to 7 tuberculosis afterwards according examined ones. They were number of T-
P. Petrova
infections of the respiratory accompanied by to the above dosage divided into 3 groups: 1st- lymphocytes, IgG,
...... 25 children with non-specific
o Clinical system in children had to be recurrent non- scheme 10 days in 3 IgA rate and of
...... found specific viral consecutive months. recurrent pulmonary phagocytic activity in
Immunological processes, 2nd - 12 children
Results of the and bacterial the treated children
with primary pulmonary from both groups. A
Application of infections. tuberculosis, 3rd - control general clinical
Factor-R to Children group of 10 healthy children.
estimation showed, in
With Various Before the treatment and on
the 14th and 30th day after statistical values of
Pulmonary Diseases
its completion the clinical significance
state was evaluated. The rate compared with those
Clinic of Pediatry,
of IgG, IgA, IgM in serum, before the treatment,
Medical Academy undoubtedly
Sofia, Bulgaria the number of T- and B-
limphocytes, the immune favourable effect on
complexes, the phagocytic the characteristics of
index and the state of cell the disease process
immunity by intradermal (heaviness, course
tests with kits of bacterial and and outcome) in
mould antigens were also more than 50% of the
determined. children.
 r-- r--o r-- ~ ~. r--l ~ IJ r-J r--
r-l I"j L"l II ITJ ~ ---""]

To Investigation: Project Participants, Diagnosis Period Test product. Criterion Results Side
Page Investigator, number, and of Dosage and of evaluation effects
Part Coordinating age, sex criteria for treatment administration
Organization admittance
1 2 3 4 5 6 7 8 9 10
K. Kissiova, An impartial assessment of 64 patients (30 Recurrent 4 months Factor-R: 50 mg daily, The patients were The favourable clinical Not
D. Kovachev, the effect of polybacterial female and 34 male) chronic nOn - in the morning before observed before effect of Factor-R was observed
specific treatment, the first and proved. It was found
B. Petrunov, immunostimulant Factor- aged in 18 to 65, breakfast for 30 days, u~ to the 3rd month that the titers of
P. Nenkov, R applied to patients with taking Factor-R pulmonary afterwards according to a ter the treatment 's specific antibodies of
D. Petkova, recurrent chronic non - perorally and 14 diseases. the above dosage completion, regardin IgG, IgA, IgM classes
o M. Pencheva. specific pulmonary patients, aged 25 to scheme 10 days for 3 the clinical indices an
paying attention to the
8 to bacterial compo-
nents significantly
tv diseases was made by 40, taking Factor-R consecutive months.
antirecurrent effect of increased at the end of
Optimisation determination of the titer through inhalations. factor-R as well as to the the 30day course and
of Factor-R of specific antibodies to its titers of complement decreases, through not
Therapeutical Schemes components in order to fIX and C3, pha!ocytic to the initial low values,
by Determination the right dosage scheme. index, rate 0 serum at the end of the 3-
of the Titer of Specific ISG, Ig.A , ISM and month supporting
circulatIng Immune course. The obtained
Antibodies to Its complexes, and the titer results led to the
Bacterial Components. of specific antibodies to creation of an im-
bacterial components of roved scheme of
Department of Factor-R were actor-R administra-
Internal Diseases, determined by ELISA. tion. It was concluded
Factor-R was applied that inhalatory treat-
Clinic of Allergy and through inhalations to a ment could be used
Clinical Immunology, grou~ of 14 patients and when an urgent effect
Medical University, on t e 7th, 14th, 21st is needed in patients
Varna, Bulgaria. and 28th day the above without bronchial
mentioned results were hyperreactivity.
fixed.
 [~ LJ L.l II :--J L-:l 1 1 ~ ~ ~

To Investiga tion: Project Participants, Diagnosis Period Test product. Criterion of Results Side
Page Investigator, number, and of Dosage and evaluation effects
Part Coordinating age, sex criteria for treatment administra tion
Organization admittance
1 2 3 4 5 6 7 8 9 10
Y. Petrovska, The clinical effectiveness 30 patients aged 29 Infectious 4 months Factor-R: 50 mg daily in In the beginning featients The results of the study Not
L. Tzvetkova, of Factor-R and changes to 57. bronchial the morning before with ~roved in ectious showed a considerable observed
bronc ial asthma were improvement of the
O. Petrova of some functional and asthma. breakfast for 30 days, examined on the 45th, clinical parameters even
imm uno logical indices afterwards according to 120th and 240th daf of as early as the 45th day
Clinical were studied in patients the above dosage the treatment. he from the beginning of
o Immunological with infectious bronchial scheme 10 days for 3 following clinical symp- treatment (in more than
w consecutive months. toms were under obser- 70% of the patients) but
Studies of Patients asthma.
vation: intensity and the most remarkable
With Infectious frequency of asthma results were observed on
Bronchial Asthma attacks, cough, rhinitis, the 240th (in more than
Treated With the expectoration, weakness, 80% ofthe patients). The
Polybacterial physical overtaxing, Ftre- s~irometrlc indices
Immunostimulant sence of rales. The un- s owed a considerable
ctional examination of improvement on the 240
Factor-R. the lung durin the same th day in more than 50%
Clinic of Pulmonary
3
periods inclu ed deter-
mination of YC, FEY,
of the treated patients.
The immunological
Diseases, FEY3, EMD, EMD50, studies showed a
Research Institute of EMD75, MEMD 25-75 decrease of bronchial
and the immunological mucose sensitivity to
Resort, Health and examination included inflammatory agents and
Physiotherapy, determination of serum an imlirovement of non-
Medical Academy, IgG, IgM, IgA in saliva - ~peci ic indices of the
Sofia, Bulgaria. IgA, IgG, and albumin, Immune system,
complement and C3, cell including the cell
immunity level. immunity.
 r-- ~ I.:'! I! ~ ["J r-l r--] Ll ...--, ---, ~ -------, --.

To Inves tiga tion: Project Participants, Diagnosis Period Test product. Criterion of Results Side
Page Investigator, number, and of Dosage and evaluation effects
Part Coordinating age, sex criteria for treatment administration
Organization admittance
1 2 3 4 5 6 7 8 9 10
P. Dobrev, The clinical effectiveness, 34 patients (17 Chronic 4 months Factor-R: 50 mg daily in Patients with chronic The main clinical Not
N. Nikolova the influence on the famale and 17 bronchitis the morning before bronchitis (bronchial indices changed to the observed
Sn. Stoyanova, functional indices of male) aged 19 to 65. (bronchial form breakfast for 30 days, form of chronic better and the bronchial
V. Maksimov, breathing and on some of chronic afterwards according the obstructive pulmonary obstruction was influ-
...... B. Petru nov, immunological parameters obstructive above dosage scheme 10 disease) were treated enced significantly.
o R. Radanova, of Factor-R in patients pulmonary days for 3 consecutive throughout a year and Some components of the
~ bronchial secretion
D. Mincheva, with chronic bronchitis disease). months. were examined
B. Popov. were studied. monthly: anamnestic- characterizing the state
ally - cough, expectora- of the local immunity
tion; physically - data also changed to a great
Prophylaxis of
extent. The production
Broncho-Pulmonary for bronchial obstruc-
of IgA was mainly
Infections with the tion; functionally -
stimulated. During the
Bulgarian investigation of breath-
treatment certain
Immunostimulant ing-VC, FEV, RIF; stimulation of the cell
Factor-R. immunolo-gical studies- immunity was observed.
quantitative deter- As a result of the
Research Institute of mination of serum IgG, treatment the number
Pulmonary Diseases, IgM, IgA , of IgA, IgG, and duration of exu-
Medical Academy, SC-component and of dates decreased. The
Sofia, Bulgaria. albumine in the temporalry incapacity
bronchial secretion for work dropped with
were made. 50%.
 ,
r~ rJ LJ l.J LI IJ r-l [J ----rI ~ ~ ~ ~

To Investigation: Project Participants, Diagnosis Period Test product. Criterion of Results I Side
Page Investigator, number, and of Dosage and evaluation effects
Part Coordinating age, sex criteria for treatment administration
Organization admittance
1 2 3 4 5 6 7 8 9 I 10
S. Garov. A study was made of the
clinical effectiveness of
24 patients (12
females and 12
Gingivitis
catarrhal is
4 months Factor-R: 50 mg daily,
in the morning before
The treated patients
were divided into 2 groups
I
Factor-R application Not
gave very good clinical Observed
Clinical Results Factor-R in the treatment of males) aged to 12 chronica. breakfast for 30 days, - 1st with Factor-R results. On the 30th day
of the Administration gingivitis catarrhalis to 24. afterwards according applied simultaneously from the beginning of the
chronica in patients with the above dosage with the undergoing local treatment 100% ofthose
of the New Bulgarian
established immuno- taking it came through
o Immunostimulant scheme 10 days for 3 and general treatment the illness. From those
VI Factor-R deficiency. consecutive months. and 2nd - without undergoing a convent-
in Complex Factor-R. Both groups ional treatment only 75%
Treatment consisted of 12 patients. got better. The favour-
of Gingivitis They were followed up able antirecurrent effect
Catarrhalis Chronica. monthly for a period of Factor-R was strongly
of 2 years from the expressed when the
Stomatological beginning of the results of both groups,
Faculty, treatment. The effect was which were 92% and
Medical University, reviewed under the fol- 25% two years after the
Plovdiv, Bulgaria. lowing criteria: clinical - beginning of the treat-
local state, antirecurrent ment compared. Data
about the stimulation of
effect and shortening of non-specific immuno-
the treatment period; logical indices - phago-
immunological - pha- cytic activity and rate of
gocytic index, rate of serum IgA, were
sera IgG, IgA, IgM. established.
 r-- ~ I::! II r-:=J r--' LJ ~ ~
~
,...----, ,..------.. --r--' ---. ----,

To Investigation: Project Participants, Diagnosis Period Test product. Criterion of evaluation Results Side
Page Investigator, number, and of Dosage and effects
Part Coordinating age, sex criteria for treatment administration
Organization admittance
1 2 3 4 5 6 7 8 9 10
M. Vassileva. The possibility of Factor-R 158 children at 10 Recurrent 4 months Factor-R : 25 mg for The study included children at 10 The clinical Not
application to children in months to 3 years non-specific children above 13 kg months to 3 years of age with observations oIRrvOO.
Immunoprophylaxis their early age as well as of age (83 boys and infections of and of 12.5 mg for recurrent infections of the and the physic-
and Immunotherapy its effectiveness in cases of 75 girls). the respiratory children under 13 kg, respiratory tract, recurrent al examinations
with Factor-R recurrent infections of the tract, otitis, daily, in the morning bronchitis with obstructive showed that in
o of Children up to respiratory tract was rhinitis. before breakfast for 30 syndrome, otitis and rhinitis. They 80% of the
0\ 3 years of Age. studied. days , afterwards were under observation for 6 children a very
according to above months after the treatment 's good clinical
Department dosage scheme 10 days complection. The effectiveness of effect was
of Pediatrics, in 3 consecutive Factor-R was evaluated by clinical achieved and
Medical University, months. indices. no side effects
Varna, Bulgaria. were observed.
It is proposed
that Factor-R
can be used in
immunotherapy
and immuno-
prophylaxis of
children in their
early age.

--
 I
I Immunoreactivity in cases of lung cancer,

r treated with cytostatics and Factor-R.

(preliminary report)

r K. Kassabov 1, D . Damianov2, A. Mihailova 3, I. Stoichkov 1

Introduction

Currently the leading oncological disease among .males in Bulgaria is lung cancer. The average
%
rate of contraction among Bulgarian males is 60 0, equal to 2600 new cases registered each
year. Nearly 80% of these cases constitute the non-small cell lung cancer clinical group. Only
20% to 27% of these cases are detected in clinical stages I or II. Consequently, more than 70%
of the patients with lung cancer cannot be subjected to radical operative programs. The treatment
of these patients, especially of those in stages III B and IV, is a challenge to clinical oncology. The

l ambition to improve the patients' quality of life in the long run stimulates the search for new
methods and preparations to optimise the complex treatment of non-small cell lung cancer.

r Some lung cancer patients demonstrate digressions and disturbances in cell mediated and humoral
immune response (9)*. Radiation therapy and/or combined chemotherapy also lead to changes
in immune status, mostly in a negative direction over different periods of time (1, 10). Consequently,
[ the complex treatment of lung cancer should include a simultaneous stimulation of the immune
system, aimed at restoration or reinforcement of its normal functions. This practice would improve

[ the final results of the treatment (2, 11).

The aim of this study is to determine some essential parameters which characterise the immune

[~ status of patients with locally advanced and/or metastasid non-small cell lung cancer after a
combined treatment with cytostatics and Factor-R immunotherapy.

[ This clinical study is based on previous experimental data on the inhibition of the growth of the
lung metastasis in patients with Lewis's carcinoma, caused by treatment with the Respivax
preparation (4, 5, 6, 8) .

Material and methods

Patients.
Seven patients, 6 men and 1 woman aged from 44 to 71, are included in the clinical study so far.
This number to increase to 20. In all cases the diagnosis has been morphologically confirmed -
flat-cell lung cancer. The apportionment of the cases in terms of clinical stage of the disease is as
follows:

Two patients in stage II (inoperable according to functional parameters or refusal of operative

treatment),4 patients in stage III, and 1 patient in stage IV. The combined chemotherapy applies
a quadruple combination designed by the Clinic of Medicinal Oncology, National Oncological
Center. The combination includes Vinblastin, Endoxan, Etoposide and Cisplatin (chart 1).

1 Tumor immunology and therapy division, Department of clinical immunology and laboratory studies, Medical Academy, Sofia
2 Clinic of Medicinal Onkotherapy, National Oncological Institute, Sofia .
3 Division ofTransplantation Immunology, Department of clinical immunology and laboratory research, Medical Academy, Sofia
* See section reference of this chapter

107
 · ....... , . . .... -.... .,. ..... ~-.. . ....... ~--~ ... ' , ...

On the Bth day of treatment a simultaneous peroral immunotherapy with Factor-R (during a

r period of 2B days, 40 gm per day in two applications) is introduced. After that a combined chemo
- immunotherapy is applied according to the same scheme. The above-mentioned scheme
provided for a blood examination of the patients one day before and after the combined
chemotherapy, as well as after the immunotherapy with Factor-R. In most of the cases the combi ned
chemotherapy lead to a salient leucopenia which prevented us from isolating the number of
r blood cells necessary to carry outall planned tests. For this reason we interrupted b~ood examination
immediately after the combined chemotherapy.

I Scheme of administration
1 course CT Factor-R (IT) 2 course CT Factor-R (IT) 3 course CT

Chart 1. Scheme of treatment and examination of patients suffering from non-small cell lung cancer undergoing
a combined type of chemical therapy (Cl) and a immunotherapy with Factor-R (IT)

l Isolation of peripheral mononuclear cells (PMC).

PMCs were isolated from heparinized venous blood according to the traditional method of gradient
centrofuge separation on Ficoll - Hypaque (Lymphocyte separation medium, ICN Flow). The
cells were resuspended in a nutritive environment RPMI1640, enhanced with 2rnM L -glutamin,
100 U penicillin/ml, 100 microg streptomycin /ml, and 10% heat inactivated fetal calf serum (ICN
Flow). The isolated cells were counted and used to determine the values of the following
immunological parameters:

[ Cytokines Secretion in vitro.

We tested the spontaneous and the inducted (with PHA and LPS) secretion of interleucine-2 (lL-
2) and the secretion of the tumor-necrocizing factor-alpha (TNF-alpha). The isolated PMC,
L concentrated to 2x10 6 /ml, were distributed among several 24-hole plates (Limbro, Flow
-Laboratories), 1 mllhole. The cells were cultivated for 24 hours with or without inductive agents in
a thermostat set on 37°C in a 5 % CO 2 environment. The resulting cell super-natants were frozen
[ and conserved at -20°C. The IL-2 and TNF-alpha levels were determined according to an
innunoenzymic method (ELISA) with standard kits (IntertestTM-2x IL-Elisa Kit, PredictaTM TFN-
alpha Elisa Kit, Genzyme).

E Activity of the natural killer cells (NK).

[ Determined according to the NK-sensitive erythroleucemic cell line K562. The target tumor cells
were counted, then concentrated to 105 cells/ml and spread on a 96-hole rounded-bottom plate
(Limro, Flow Laboratories), 100 microllhole. The effector cells were added to the target cells in E
: T proFortions of 50:1,25:1, 12:1, and 6:1. Enhanced RPMI1640 environment was added to the
L contro tumor cells at a rate of 100 microl. After a 20-hour treatment in a thermostat at 37°C and
5 % C02, the living tumor cells in the control (C) and test (T) holes were counted after the addition
of a 0.5 % solution of a tripan blau colouring agent (Flow Laboratories). The cytotoxicity was
L determined according to the following formula: % cytotoxicity = K - O/K x 100; K - number of the
living tumor cells in the control holes, T - number of the living tumor cells in the test cells.

L Surface leukocyte markers.

Determined with flowcytometer (FACStar, Becton Dickinson). We used a set of monoclonal

r antibodies (Monoclonal Antibodies Laboratory, National Institute of Contagious and Parasitic
Diseases), which allowed us to determine the general T lymphocytes (CD3 +), helper/inducer
(CD4 +) and suppressor/cytotoxic (COB +) T lymphocytes, NK-similar (COB + CDS7 +) and
activated (HLA-DR +) T lymphocytes, as well as the general (CDS7 +) and the typical (CDB-
CD57 +) NK cells.

108
 r Results and Discussion

r Activity of the NK cells.

r As indicated in Table 1, the examined patients show a decreased NK activity before the treatment
(as compared to healthy persons who volunteered for the tests). .

Table 1

Patient Time for E:T (%)

N! determination 50:1 25 :1 12 : 1 6 :1

1 at entering clinic 70.2 56.2 31.8 20.1

after 1 course of CT 28.4 16.9 21.2
after 1 course of IT 64.3 34.6 9.6
after 2 course of CT 13.7 6.6 14.3
after 2 course of IT 26.2 9.7 6.9

[ 2 at entering clinic
after 1 course of IT
88 .9
32.6
63 .5
18.6
36.5
4.7
7.9
2.3

3 at entering clinic 71 .6 50.2 31.4 18.5

after 1 course of IT 49.6 31 5.8 1.8
after 2 course of IT 48.4 54.6 59.3 28.1

4 at entering clinic 63 .2 32.4 20.1 9.4

after 1 course of CT 16.2 8.3 3
after 1 course of IT 21.7 16.4 9.9 1.8

[ 5
after 2 course of IT

at entering clinic
49.3

82.1
52

57.3
29.1

20.4
21.6

6.7
after 1 course of CT 30.1 38.4 14.9 7.2
after 1 course of IT 73.7 67.1 51.3 28.9

l 6 at entering clinic
after 1 course of IT
63.8
64
47.9
68.7
19.1
65.6
9
48.4

7 at entering clinic 45.8 30.2 19.7 8.3

[ X SD
after 1 course of IT

at entering clinic
after 1 course of cr
31.2

69.4 ± 12.9
30.1
31.2

48.2
27.7
± 11.7
± 9.1
35 .9

25.6
13.4
± 6.8
± 3.7
17.1

11.6 ± 5.3
6.6 ± 2.7
after 1 course of IT 45.5 ± 18.7 42.5 ± 21 .6 29.7 ± 22.1 15.7±16.3
after 2 course of IT 43.1 ± 8.2 44.3 ± 12.8 32.7 ± 20.4 18.9 ± 8.9

I-J Healty volunteers 74.5 ± 14.1 55 .3 ± 9.7 34.1 ± 8.6 20.1 ± 7.7
(n=6)

Table 1. Effects of the chemical therapy (eT) and immunotherapy with Factor-R (IT) on the NK activity of diseased suffering from
non-small cell lung cancer

Immediately after the cytostatic therapy most of the patients developed salient leucopenia,
which allowed us to determine the NK activity in only three of the cases. In all three
cases a decrease in the cytotoxicity of the NK cells in vitro was observed. After completion
of the 1st period of chemo-immunotherapy the NK activity was fully (4 cases) or partly (3
cases) restored. After completion of the 2nd period of treatment fully or partly restored
activity was observed in two of the cases. In lower concentrations of effector cells the
values came close to or even exceeded the initial values.

Four of the patients examined after immunotherapy (two of them after the 1st period of
treatment, and two - after the 2nd) showed a relatively higher NK cytotoxicity in lesser
effector cell concentrations. One of the possible explanations of this phenomenon is
stimulation of the monocytes in the peripheral blood. Some scholars affirm that monocytes
exert an inhibiting effect on the NK cells (1, 3) (especially higher concentrations of
monocytes). This statement might be verified by determining the activity of the two effector
populations together and separately.

109
 ' . .-..~ ....... ----- .... - . ---~.

Cytokines secretion in vitro.

[ Table 2 describes the spontaneous and the induced (with PHA and LPS respectively) secretion of
interleukine-2 (lL-2) and tumor-necrotizing factor-alpha (TNF-alpha).

f TABLE 2

PATIENT TIME FOR IL-2 [pglml THF-a [pglmll

r N! Determination Spontaneous Induced Spontaneous Induced

1 at entering clinic 527 .1 653 .2 380.5

after 1 course CT 607.7 968.9 509.4
after 1 course IT 709.7 >4000 166.7 306.3
after 2 course CT 13.7 6.6 14.3
after 2 course IT 116.2 294.6 160.8 328.8

2 at entering clinic 495 .8 333.7 > 1200 >1200

after 1 course CT 239.4 560.4 600.7
after 1 course IT 706 .9 1976.1 455.2 500.7

3 at entering clinic 578 505.7 103 .5 142.7

[ after 1 course CT
after 1 course IT
824.4
712.8
517.1
745 .1
116.7
246 .5
163.2
260 .1

4 at entering clinic 1314.6 2794.9 270.5 345.1

after 1 course CT 306 689.9 48.6 100.7

[ 5
after 1 course IT

at entering clinic
1167.3

862.7
1265

1804.7
97.4

347 .2
269.4

434.4
after 1 course CT 802 .1 1003.2 590.1 > 1200
after 1 course IT 463.5 612 537 .8 669.4

[ 6 at entering clinic 1221.1 1616.3 470.5 549.3

7 at entering clinic 675.7 1357.1 447 .9 567.7

[ X ± SO at entering clinic
after 1 course CT
after 1 course IT
811 ± 311
556 ± 244
752 ± 229
936 ± 592
795 ± 201
1720±1237
460 ± 322
365 ± 233
301 ± 169
540 ± 328
516±439
401 ± 160
Healthy volunteers 514 ± 242 854 ± 662 93 ± 77 203 ± 108

L
(N=6)

Table 2. Effect of chemical therapy (CT) and the immunotherapy with Factor-R (IT) on the secretion of interleukin-2 (IL-2) and
tumor-necrotising factor-alpha (TNF-a) from peripheral mononuclear cells

In most of cases there was an increased secretion of tested cytokines (compared to the healthy
volunteers). After the first period of chemotherapy the average values indicated decreased
secretion of the two cytokines (especially IL-2). After a one-month of peroral immunotherapy
with Factor-R the IL-2 secretion increased (especially the one induced with PHA). There was no
significant change in the TNF-alpha secretion.
Both in tested patients and healthy volunteers the levels of secretion showed a large range of
variation. The different patients responded in a rather individual way to the treatment which
accounts for the different models of secretion. For example, patients with an initially increased
level of secretion often reacted to the cytostatic therapy by an increase in the IL-2 and TNF-
alpha secretion. Patients with initially decreased level of secretion reacted Similarly after the
completion of the 1st period of chemotherapy. The overall analysis of the measured parameters
might help us give a satisfactory explanation of the observed phenomena.

Surface Leukocyte Markers

Table 3 describes the analysis of the percentage content of several group determinants (CD),
typical for the T- and NK- cell populations. The indicated surface markers were measured before
the beginning of the treatment and after the completion of the 1st and the 2nd period of chemo-
immunotherapy.

110
 . ~ -. _'. . . . ."... . . ,.... .. . . .a . ...

TABLE 3

r Patient
~
Time for
distribution
CD3+ CD4+ CD8+ CD8+
<D57+
HLA-
DR+
CD4/
CD8
General
<D57+
CD8-
<D57+

1 at entering 70.50 46 .00 24.80 4.60 3.20 1.90 15.00 10.40

after 1 course CIT 70.90 30.20 28.50 9.10 2.30 1.10 19.30 10.20
r after 2 ciurse CIT 68.40 20.70 34.50 9.70 1.60 0.60 . 22.50 12.80

2 at entering 69.50 47.80 22.40 4.00 2.90 2.10 15.40 11.40

after 1 course CIT 73.90 44 .50 24.90 6.80 2.20 1.80 16.20 9.40

r 3
after 2 ciurse CIT

at entering
66.00

68.50
38.10

47 .60
26.50

21.80
9.50

5.80
2.40

2.90
1.40

2.20
21 .60

15.20
12.10

9.40
after 1 course CIT 68.80 25.40 30.90 9.50 2.80 0.80 21.40 11 .90

~
after 2 ciurse CIT 63.50 36.20 18.40 9.60 3.10 2.00 20.90 11.30

4 at entering 66.70 45.30 21.50 3.80 2.70 2.10 13.00 9.20

after 1 course CIT 74.10 42 .50 22.90 6.40 2.00 1.90 15.80 9.40
after 2 ciurse CIT 57.40 34.50 20.80 9.80 2.60 1.70 21.60 11.80

[ 5 at entering
after 1 course CIT
67.40
58.80
45.80
37 .20
24.20
18.80
5.00
5.20
3.20
1.80
1.90
2.00
22.50
24.40
17.50
19.20

6 at entering 80.10 54.10 26.00 5.70 4.80 2.10 10.10 4.40

after 1 course CIT 87.50 62 .30 22.10 6.50 2.80 2.80 10.70 4.20
[ 7 at entering
after 1 course CIT
63.70
84.00
45.60
62.40
12.90
19.20
4.80
2.50
1.10
0.90
3.50
3.30
15.10
4.10
10.30
1.60

X ± SD at entering 69.50 47 .70 21.20 4.80 3.00 2.40 15.20 10.40

[ after 1 course CIT
± 6.3

74.00
±3.7

43 .50
± 5.0

24.90
± 0.8

6.60
± 1.3

2.10
± 6.0

2.00
± 5.3

16.00
±5.4

9.40
± 10.5 ± 13.4 ± 4.0 ± 2.6 ±0.7 ± 1.0 ± 7.5 ± 6.2

L NORM
after 2 course CIT

77.80
63 .80
± 4.1

43.10
32.40
± 6.9

25 .70
25.10
± 6.2

7.20
9.70
± 0.1

4.00
2.40
± 0.8

2.00
1.40
± 0.5

15.00
21.70
± 0.8

7.60
12.10
± 0.8

± 10.3 ± 8.2 ± 4.6 ±3.3 ± 3.5 ±0.7 ± 6.9 ± 4.7

[ Table 3. Effect of the combined chemical and immunotherapy (Cln with cytostatics and Factor-R on the percentage distribu-
tion of T-celi and NK-cell surface markers.

[ The presented data shows values within the range of normality with slightly decreased values of T
lymphocytes (C03 +) and NK-similar lymphocytes (C08 + COS7 +). After the 1st period of chemo-
immunotherapy the values of the two markers were slightly higher. The other markers remained
[ unchanged. In two of the cases (out of four examined after the 2nd period of therapy) there was
a slight decrease of the C03 and C04 positive cells, accompanied by a slight increase of the C08

[ population which led to an increase of the C04/C08 index. In the third case the value of the Thl
Ts relationship was greater in comparison to the previous test. In the fourth case the value remained
unchanged. All four patients demonstrated a tendency to increase the initial cell values describing
[ the NK marker COS7 - NK-similar lymphocytes (C08 + COS7 +) and typical NK cells (C08-
COS7 +).

[ Unfortunately the insufficient number of celis, isolated immediately after the end of chemotherapy,
did not allow for the determination of the direct effect of chemotherapy on the examined cell
populations. Consequently, it is very difficult to make a distinction between the effect of the
cytostatic treatment and the effect of the peroral immuno-treatment with Factor-R. A simultaneous
examination of the indicated leukocyte markers in a group of patients not treated with Factor-R
would clarify this question.
L This presentation is a preliminary report. The data included represent the initial results of stili
incompleted clinical research. The major goal of current ongoing research is to optimise the
L treatment of lung cancer patients through the incorporation of the Factor-R preparation into the
complex scheme of treatment. Despite the small number of patients tested so far, we believe that
the first results represent a good reason to continue the research and to expand the control group
to 20 patients.

111
 REFERENCES

1. Braun DP, J E Harris: Effect of chemotherapy on NK function in the periferal blood of

cancer patients. Cancer Immunology Immunotherapy, 21, 1986, 240-245.

r 2. Fishbein GE: Immunotherapy of Lung cancer. Seminars in oncology, 20, No4,1993,

351-358.
[
3. Gorelik E, R Herberman: Role of natural killer (NK) cells in the control of tumor growth
and metastatic spread. In: RB Herberman (ed) Cancer Immunology: Inovative approaches to
therapy. Martinus Nijhoff Publishers, Boston, 1986, pp 151-176.
'= 4. Kassabov K, J Stoychkov, B Petru nov: Inhibitory effect of the oral vaccine respivax on
[ pulmonary metastases of Lewis lung carcinoma in mice. Compt. Rend. Acad. Bul. Sci. 43, 1990,
XII, 117-119.

[ 5. Kassabov K, J Stoychkov: Enhancement of therapeutic effect of cyclophosphamide on

pulmonary metastases of Lewis lung carcinoma by the oral vaccine Respivax. Compo Rend. Acad.
[ Bul. Sci. 44,1991,1,93-95.

6. Kassabov K, J Stoychkov: Inhibition of spontaneous pulmonary metastases of Lewis

l. lung carcinoma by oral treatment with Respivax and Broncho-Vaxom: Cancer Immunology
Immunotherapy,33,1991,307-313.

[ 7. Mackensen A, C Galanos, R Engelhardt: Treatment of cancer patient with endotoxin

induces release of endogenous cytokines. Pathobiology, 59,1991,264-267.
8. Minev B, V Veleva, K Kassabov, J Stoychkov, H Neichev: Production of interleukin-1 by
[ Respivax stimulated peritoneal macrofhages. Compo Rend.Acad.BuI.Sci. 44,1991,IV,1 09-112.

9. Tsuyuguchi I, H Shiratsuchi, M Fukuoka: Increased circulating activated T cells in lung

[ cancer. Chest,89,1986,705-708.

[ 10. Un 5, Sm Lee, HT Kim et al: The effect of radiation therapy on immune function in
patients with squamous cell lung carcinoma. Chest, 105(1 ),1994,132-137.

[ 11. Wetanabe Y, T Iwa: Immunotherapy of lung cancer: A randomized clinical trial of OK-
432 immunotherapy and a review of the literature on nonspeCific immunotherapy. Int.J.
immunotherapy, 11(1 ),1986,51-67.

112
 I
f
Clinical and serological investigations of Factor-R
r action on different clinical forms of Syphilis
G. Spirov, O. Niagolova
(Preliminary report)

Nonspecific therapy of syphilis was a well known procedure prior to the discovery of Spirochaeta
pallida(1905) and the diagnostic serological reactions (1906). Historically different therapies were
used: malaria therapy, different pyrogenic substance (vaccine, 2 % sulfur emulsion, pyrogenic,
etc.), vitamins, biogenic stimulators (placenta, aloe, fibs), n.v. radiation, corticosteroids,
G balneotherapy etc.

[ For nearly a century, when discussing the problems of immunity in syphilis the fact that in the
evolution of the disease there are periods without any symptom (latent syphilis), could be explained
through relative immunity.
[ Even after treatment serological reactions for syphilis remain positive in a very high percentage of
treated patients - a disturbing fact for the doctors, the patients and their relatives . It is due to the
[ already accepted fact in syphilisology that alive, but non active spirochaetes in the lymphatic
nodes, produce of penicillinase, L-forms capable of reversion etc. According to Ljuben Popov
(1947) nonspecific treatment of syphilis is necessary because "without the participation of the
L organism in the treatment all the antisyphilitics means remain problematic".

The foregoing provided a motive to investigate the action of a modern immunostimulant in the
[ treatment of syphilis together with specific Penicillin therapy (Factor-R).

[ MATERIALS AND METHODS

Two groups of patients with contagious forms of syphilis were under observation, one of them
r was treated with crystalline Penicillin G (4x2 000 000 IU) per day - the other with crystalline
penicillin and Factor-R 50 mg daily. The duration of the treatment with Penicillin was 15 to 30
[ days according to the clinical form of the disease, and Factor-R with the first application on the
third day of Penicillin therapy for three month. Factor-R was appl ied by the following scheme:
30 days 50 mg in the morning before breakfast, 20 days rest, 10 days 50 mg, 20 days rest and again
[ 10 days rest.

The efficacy of applied treatment was valued with the help of the following criteria:
L a) clinical observation of persisting skin and mucose changes (in days);

L b) term of negativisation of the serological reaction for syphilis -microchlorholinic

test (VORL) and ReF "WASSERMANN" - type "KOLMER" (in days).

l c) immunological tracing

The observed patients are randomly chosen . The group treated only with Penicillin consist of 95
patients (38 female and 57 male with average age 22.6 years) divided in clinical forms : 30 syphilis
primaria seropositiva (4 female and 26 male), 35 syphilis secundaria recens (18 female and 17
male), 30 - syphilis secundaria recidiva (16 female and 14 male).

113
 The group treated with Penicillin G and Factor-R consist of 66 patients (25 female and 41 male)
average age 20.8 years divided by clinical forms in : syphilis primaria seropositiva 31 (4 female
and 27 male), syphilis secundaria recens 22 (12 female and 27 male), syphilis secundaria recidiva
- 13 (9 female and 4 male).
I
RESULTS

I. Clinical observation (persistence of the skin and mucose changes)

G Data for the duration of the persistence of the different skin and mucose changes (erosive or
ulcerous shancar, papulas, condylomata lata, changes on the palms and feet, angina syphilitica,
[ laryngitis catarrhalis, papulosa syphilitica, deflovium syphiliticum, alopecia syphilitica etc. were
calculated separately and after that for the different clinical forms were processed by the method
Wilkokson and White.
[ The results for the different forms are given in the tables 1,2, 3. They are approximately similar in
the two methods and the differences are statistically doubtful. These results show, that the clinical
[ observations conserning the persistence of skin and mucosa changes in contagious syphilis, treated
by the two methods, is not enough to give an impartial assessment for the effectiveness of the
treatment.

Table 1

STATISTICAL IMPORTANCE
[ OF DIFERENCE IN THE PERSISTENCE OF THE SKIN EFFLORESCENCE
r IN SYPHILIS PRIMARIA SEROPOSITIVA

Method of treatment:
1. Crystalline Penicillin "G" (4 x 2 mil E pro die)
2. Crystalline Penicillin "Gil (4 x 2 mil E pro die) +
Factor - R (50 mg pro die)

-
Method of Number of X (days)
L N~
Treatment patients X M1N - X MAX
9.10
1 Penicillin "G" 30
(6 - 13)

Penicillin "G" 9.16

2 31
+ FactQr - R (4 - 15)

Criteria P < 0.05

114
I

I Table 2

r STATISTICAL IMPORTANCE
OF DIFFERENCE IN THE PERSISTENCE OF THE SKIN EFFLORESCENCE
IN SYPHILIS SfCUNDARIA RfCfNS
r

Method of treatment:
1. Crystalline Penicillin "G" (4 x 2 mil E pro die)
2. Crystalline Penicillin "G" (4 x 2 mil E pro die) +
t Factor - R (50 mg pro die)

[ N!! Method of
Treatment
Number of
patients
X (days)

XM1N - xMAX

[ 1 Penicillin "G" 35
10.23
(6 - 16)

[ 2
Penicillin "G"
22
10.10
+ Factor - R (6 - 17)

[ Criteria P < 0.05

[ Table 3

[ STATISTICAL IMPORTANCE
OF DIFFERENCE IN THE PERSISTENCE OF THE SKIN EFflORESCENCE
IN SYPHILIS SfCUNDARIA RfClDlVA
[
Method of treatment:
[ 1. Crystalline Penicillin "G" (4 x 2 mil E pro die)
2. Crystalline Penicillin "G" (4 x 2 mil E pro die) +
[ Factor - R (50 mg pro die)

-
Method of Number of X (days)

E N2
Treatment patients XM1N - XMAX

12.23
L 1 Penicillin "G" 30
(7 - 18)

Penicillin "G" 11.48

L 2 + Factor - R 13
(6 - 18)

Criteria P < 0.05

115
 2. Term of negativisation (in days) of the serological reaction for syphilis.

We used as an objective criterion for the effectiveness of the application of the two methods for
f
treatment the period for negativisation of the following serological reactions: microchlorholnic
test (VORL) and ReF" WASSERMANN" (type "KOLMER") with cardiolipoidic agent.
I Sex, age and clinical forms are the same as in the clinical observations (the same patients). The

r results are given in table 4. The differences in the effectiveness of the two methods for treatment
are statistically doubtful only for VORL in syphilis primaria. ReF" Wassermann" even in this early
form becomes negative more quickly after the treatment with Penicillin G and Factor-R

t (P < 0.05). For the remaining later phases of contagious syphilis both reactions became negative
more quickly after the combined treatment (syphilis secundaria recens and syphilis secundaria
recidiva). These differences do not need statistical manipulation, but it gave the same results
(P < 0.5).

For instance after the treatment of Syphilis secundaria recens only with Penicillin G VORL becomes
[ negative for 112 days, and after the combined treatment with Factor-R for 84.90 Similar are the
differences for ReF" Wassermann" 101 days against 78.59 days.

[ The correlation in negativisation of the two reactions in syphilis secundaria recidiva are even
bigger: VORL - after 160.63 day against 109.30 and ReF "Wassermann" - 117.06 days against
109.30 days.
[
These differences are so great, that it is obvious, that the treatment with Penicillin G and Factor-R
must be preferred for the early forms of contagious syphilis.
L:
Table 4
l NEGATIVISATION OF THE SEROLOGIC REACTION FOR SYPHILIS

VDRL and ReF Wassermann (type Kolmer), in days in patients with, Syphilis primaria seropositiva, Syphilis
secundaria recens and Syphilis secundaria recidiva, treated with crystalline Penisillin "G" (8 mil E pro die)
and cryF -16stalline Penisillin "G" (8 mil E pro die) + Factor-R (50 mg pro die)

Penicillin "G" Penicillin "G" + Factor - R

Number of MlcrochroIin ReF Wassermann Number of Microchrolin ReF Wassermann
patients Microchrolinlc test type Kolmer with patients Mlcrochrolinlc test type Kolmer with
(VORL) cardlollpoidlc (VORL) cardlollpoidlc
CLINICAL FORMS antigen antigen

Total days Avr. days Total days Avr. days Total days Avr.days Total days A'T. days
per patient per patient per patient per patient

Syphilis primaria 30 2158

71.93
1770
59.00
31 1993
64.29
1530
4935
(2.4) (1.96) (2.14) (1.96)
seropositiva
Syphilis secundaria 35 3920
112.00
3535
101.00
ZZ 1886
112.00
1729
78.59
(3.73) (3.36) (2.83) (336)
recens
Syphilis secundaria 30 4819
160.63
3512
117.06
13 1421
10930
1104
84.92
(535) (3.90) (3.64) (2.83)
recidiva
104.17 92.81 (:iJ SO.03 (:iJ.10
TOTAL 95 9897 (3.47)
8817 (3.09) 5Z82
(3.47) 4363 (3.09)

116
 Investigations on later forms of syphil is: syphilis latens recent and syph His latens tarda show si milar
l results at the end, but the necessity of one year and longer tracing and the small number of
patients currently prevent interpretation.

f It was observed however that in two female patients with late latent syphilis (4 year period) and
several treatments with Penicillin G, cardiolipoid reactions were neutralized within 6 months

I after adding Factor-R to the treatment. We are continuing the immune tracing of those subjects.

In conclusion, although the authors do not make definite conclusions, it is strongly suggested
that Factor-R in combination with classical antisyphilitic treatment is more effective for the
treatment of contagious forms of syphilis and has in test subjects led to greater percentage of
recovered patients.
[
[
[
[
[
[
[
[
[

I
L

117
I
r TRIALS ON HIV/AIDS SUBJECTS

l The following information is the chronological subjective and objective evaluation of our clinical
experience in the therapeutic application of Factor-R in 133 seropositive subjects. All participants
in these trials were volunteers.
r Introduction

While there is a wealth of information on the effects of Factor-R, results, prior to the
r commencement of these studies, were derived primarily from numerous laboratory experiments
with animals. Even though immune deficiency was induced in these early studies animals have
typically failed to provide adequate research profiles to satisfy HIV investigation. The substantial
f clinical application of the substance in non-HIV/AIDS subjects, all with a demonstrated therapeutiC
benefit, also fails to provide the needed substantiation. Because of this history, the Investigators
encountered significant resistance from clinicians and researchers due to a lack of demonstrable
results in seropositive and AIDS subjects. The lack of these studies in HIV/AIDS sufferers provided
support for critics of the proposed therapeutic procedure. It also allowed purists to challenge the
theoretical hypothesis put forth by the investigators.
r In order to meet these challenges and to demonstrate both subjectively and objectively the

r therapeutic benefit of this form of immuno-modulation, the investigators accepted the applications
of two subjects in highly advanced stages of AIDS to participate in an early study. Since the
substance, Factor-R, has not at any time demonstrated any toxic effects on human subjects, the
investigators elected to proceed .

First application of Factor-R in HIV-positive subjects begun in 1993. The product was initially
given to two men:

Subject 1.

A 39-year-old, male, who was diagnosed HIV-positive on or about 1990 has suffered from fungal
infection (seborrhea dermatitis) and shingles during the past three years. In the preceeding year
he suffered from recurrent sinusitis, ocular herpes, weight loss, fatigue and diarrhea. In the two
months prior to commenCing the study he had salmonella sepsis. He has previously been treated
with AZT, antibiotics and antifungals. His very poor health condition reqUired an
exsangvinotransfusion some days before the commencement of Factor-R treatment. As a result
base statistics are skewed.

The subject took Factor-R (80 mglday) beginning October 15, 1993. From the laboratory data
presented in Fig. 1 - 10 it is evident that Factor-R stimulated leucopoiesis (fig. 1, 2). Lymphocytes
increased significantly and then stabilized (fig. 4). This resulted from a rise in CD8 cells (fig. 9).
Factor-R did not influence the number of CD4 lymphocytes and they stayed at very low levels
during observation (fig. 8). Although some laboratory data such as WBC (fig. 1), platelets
(fig. 6) and hemoglobin (fig. 5) remained below the normal, they did increase in number after two
months of treatment with Factor-R and to the end of the investigation the subjects general health
improved significantly. The subject had no further sinusitis or opportunistic infections, or chronic
diarrhea. The ocular herpes subsided . The subject demonstrated an unusually rapid recovery
from Salmonella sepsis.

Subject 2.

A 34-year-old, male, HIV-positive since 1987 had for almost two years persistent diarrhea and
weight loss. As well as Factor R (80 mglday) the Subject also took antiretroviral medication and
vitamins. From the laboratory data presented in Fig. 1,5 and 6 it is obvious that factor R stimulated
leucopoiesis, erythropoiesis and thrombocytopoiesis. It is very-important to note that after the
initial low values of WBC (fig. 1), neutrophils (fig. 2), thrombocytes (fig. 6) and hemoglobin (fig. 5)
these populations, after treatment, entered the normal range.

118
 I "','..

r Factor-R did not influence the total number of lymphocytes (fig. 4), including CD4's (fig. 8). At the
same time the number of CD8 lymphocytes (fig. 9) and monocytes (fig. 3)increased considerably
[ and constantly during the 6-month period of 06servation. A clearly expressed rise of protein level
was also observed (fig. 7). As a result the Subject reported an increased sense of well-being. Of
particular interest, and as demonstrated in Subject 1, an unexpected effect was that after almost

r two years of persistent diarrhea and the loss of 45 pounds, his weight stabilized at 150-155 lb. and
the diarrhea in fact stoped.

r woe
Range 4 8- 10 8 k/cumm 5p
5.5
E 5 ~Q
----
.'

/~-
[ 4.5

4 L/
[ 3.5
/
_? c/'/ _~1
3 -
[
)(
-
2.5
_2J
--
[ 2 "'----
- -
Starting Factor R 90th day 180th day
- Subject 1
- )(- Subject 2

r WBC in two subjects increased after treatment with F-R. The initial WBC values in both
Figure 1

were below referent ranges. In Subject 2 WBC reached normal range (5,5 k!cumm). The
l increase in Subject 1 was also considerable (from 2,0 to 3,1 k!cumm).

NEUTROPHILS
[ Range: 40-74 %11

[
60
55
50
ii----- .---- .-' - -
-.-.-.
54 Ji

[ 45
_12
40 .-
J(- - .- -

[ 35
30
//
1/
25
[ 20
/
15
\V
I Starting Factor-R 90th day 180th day
L
-.-
- )(- '
Subject 1
Subject 2
Figure 2

Neutrophils increased significantly in both subjects. In. Subject 1 cell populations rose
from 46 to 56 % and in Subject 2 the initial low count (16 %) reached the normal range
(41 %).

119
 MONOCYfES
[ Range: 3-11 %

r 11

10
10
----- --- --------------- -- -
II
10

I 9

t 7
6
r .§..
5
..1,_--- -
- "
4 - "
)t ~ -

l 3
Starting Factor-R 90th day 180th day
-II Subject 1
- )(
Subject 2
Figure 3
r

[ Observations varied slightly within the limits of the normal. In Subject 2, who demonstrated
the lowest count, experianced a consistent elevation, from 3,4 to 5 %, during a 6 month
period of treatment.

f
LYMPHOCYfES
L Range: 19-48 %

[ 36
~ ,-
~l:;

1- - - - -
32
..-' .-.- ....---------
[
/-

28 ii-,--./ --'
24

[ 20
16

[ 12
8
8 - - an 7

[ 4
Starting Factor R 90th day 180th day
Subject 1
Subject 2 Figure 4
L
Varieties of lymphocyte levels differed in the two subjects. In Subject 1 a significant increase
was evident after 90 days oftreatment and remained high (35 %) on 180th day_ The low
initial level of lymphocytes (8%) in Subject 2 was not substantially effected throughout
observation.

120
 - - '. -'-~'-"- - - - -'-~--

HEMOGLOBIN
Range: 14-18 g/d/
[ 16
/~
15 ----------- ~o
r /
/
14
1~V
r 13

12

11

[ 10

9
t!~ ________

Starting Factor R
-----._~L
90th day
_.---- --- --- 9.9

180th day
[ --- - -)(-
Subject 1
Subject 2 FigureS

[ A significant increase in the level of hemoglobin level was observed in Subject 2 - from
13,3 to 15,6 gldl on the 90th day. A slight Decrease occurred on the 180th day (14,6 gldl)
but within normal limits as compared to the initial value. In Subject 1 the hemoglobin did
[ not change Significantly although some increase was shown at the end of the investigation.
It should- be noted that Subject 1 had, only days before the initial draw, undergone
exsanguinotransfusion. All values in Subject 1 remained below normal ones.

l PlATELET COUNT
Range: 130-400 klcumm

[ 180
179
~./"
./'"
160
[ 140 04""
i(7
./
//~

120 0404 ..

~
-
qL
100
.~
...............-
[ 80 . ............. ~~~
..., ~
~---!j>
-
'

60 -
40
Starting Factor R 90th day 180th day
I .
Subject 1
Subject 2 Figure 6

Both Subjects entered the study with a platelet count under the normal. A significant

L increase of platelet count within the limit of the referent ranges (114 to 134 klcumm), was
evident after a 90-day course of treatment in Subject 2. This tendency remained stable
and Subject 2 demonstrated a platelet count of 179 klcumm platelets. In Subject 1 a
decrease from 97 to 59 and 66 klcumm on day 90th and 120th respectively was observed.
In Subject 1 the platelet count and hemoglobin level were most likely influenced by the
exsanguinotransfusion made immediatly before the begining of treatment. It is quite
probable that Subject 1 had an effectivly lower count prior to tranfusion and that the
values reflected on the 90th and 180th days may reflect an actual improvement.

121
 PROTEIN TOTAL
Range: 6-8 gldl
8)
r 8.2

8
/
7.8
/
/
7.6 /

~ 7.4
7,;_____.
/
7/ 7';;'
[ 7.2 -- ---- >-:::.
.- _. - ....
.-'

l _- .- --.. . -J..--.. -
7 -
[ 6.8
Starting Factor R 90th day 180th day

[ ---
- )(- -
Subject 1
Subject 2 Figure 7

Protein levels were found to be normal at all times during observation in both Subjects.
[ There was a demonstratable tendency for protein levels to increase (from 7 to 7,2 and 8,2
gldl) in both Subjects, most significantly in Subject 2.

[
[
T Helper/Inducer (CD4)

[ Range: 370-1482 percumm

,.,.,.
28
-l*
- -
ir---- ---~
24

20

16

12

8
.,. .,. ."
~
,...
~- - )(- -_.-

4
Starting Factor-R 90th day 180th day

----
- J (- .
Subject 1
Subject 2 Figure 8

Factor R treatment did not influence the number of CD4 cells in either subject. Their
values were very low and characteristic for advanced stage subjects.

122
I
f
T suppressor/cyto (CD8)

[ Range: 162-682 percumm

560
r 520 - - -
_~4

4~8 __ ~::----
[ 480

440
/ ---- ----.
~~1

400
V
//
360 ./

3~'
320 -
[ Starting Factor-R 90th day 180th day

• Subject 1
Figure 9
[ )( Subject 2

CD8 cell values were found to be at normal levels at all times during observation. A
considerable increase however was found in Subject 1. A slight decrease from 496 to 441
[ cells/cumm on day 180 after initial rise was found in Subject 2.

[ IN CONCLUSION Factor-R prOVided demonstrable efficacy in Subjects 1 and 2. Both demonstrated

a reduction in the number of and recovery period from existing and later infections (herpes,
sinusitis, fungal infection etc.) . Subject 1 recovered rapidly from salmonella sepsis. As well both
[ subjects were relieved of long term diarrhea. An obvious weight gain in both subjects was an
additionally encouraging sign of improved overall health. As a result of the stimulation of
hemopoiesis in both subjects WBC and hemoglobin increased, and in all subjects platelet count
[ increased by the 3rd draw. The increase of both neutrophils and monocytes (in Subject 2) is
indicative of a heightened capability for phagocytosis against extra- and intracellular parasites
and is a prerequisite forthe development of cell-mediated immunity against infections and tumors.
According to recent findings the elevation in CD8 is very important for conducting antiviral, cell-
mediated immunity against HIV. The subjective results in combination with improved laboratory
parameters provided positive early evidence that Factor-R treatment in advanced cases of AIDS
altered measurable parameters. It must be remembered that these subjects were extremely ill at
the time they commenced treatment. They had attempted and later abandoned all other accepted
treatment modalities. The overall improvement in quality of life in such advanced cases was in
and of itself an encouraging sign. This motivated investigators to consider further extended
studies in an attempt to reproduce and verify the reported results.

EXTENDED STUDY 133 SUBJECTS

Here we provide a profile of Subject populations. The distribution of all subjects is compared to
those for which Investigators currently have results and are used as an early sampling. Subjects in
the sampling are almost evenly divided according to applied therapy (anti-retrovirals, antibiotics,
etc.).

The sampling population is representative of the general population as far as sex, period of
seropositivity, age and secondary infections/malignancies.

123
 EVALUATION OF EFFECTS
[ OF FACTOR-R ON PATIENTS WITH HIV-INFECTION

(Preliminary report)
I Current results in the treatment of HIV-infected patients with antiretroviral drugs has, regretfully,
but finally, been recognized as limited in effect and not the sole solution to AIDS. While there is
some evidence that antiviral treatment may prolong the lives of some patients it has proven to be
[ too toxic for prolonged use. The profound depletion of a variety of defensive capabilities of the
organism, reflected in secondary immunodeficiency, imposes the need to apply preparations,
that restore HIV damaged defensive mechanisms and stimulate others unaffected by the virus.
[-:
The objective of our study was to examine the effect of a polybacterial immunomodulating
preparation, Factor-R, on some biological parameters in a statistically significant number of
[ HIV-infected patients.

[ MATERIALS AND METHODS

Our study included 133 subjects with HIV-infection. To the moment there are results after one
90-day course of treatment for 40 % of them. Patient age varies between 24 and 64 years. Duration
[ of seropositivity is between 1 and 11 years. 59,1 % of the participants in the study are undergoing
treatment with antiretroviral drugs, which is now combined with Factor-R, 8 of the patients had
never taken antiretrovirals.

r Subjects were divided in several groups according to the following criteria;

- Group 1, including patients with CD4 from 0 to 200 lymphocytes;

[ - Group 2, including patients with CD4 from 201 to 499 lymphocytes;
- Group 3, including patients with CD4 more then 500 lymphocytes.

[ The following parameters were initially studied in all subjects:

- CBC with manual different.ial;

l - platelet count;
- total protein;
- CD4 absolute number;
- CD8 absolute number.
[ At the time of this report investigators had complete data on only 17 subjects. Partial data for 44
subjects was obtained on the number of CD4 and CDB.

L
% Subjects Recieving Antiretroviral Therapy
(AZT, DOC, 001)

GeneraL population Sampling

L
L 50.00% --~ ---50.00%
64.66%

Treated with antiretrovirals Not treated with antiretroviral

124
 % Subjects Recieving Antibiotics And Other Therapies,

r Used for secondary opportunistic infections in patients with AIDS:

GeneraL popuLation Sampling

I 41.6
21.31
r 78.69%
58.33%
L

L
• With ethiological therapy Without ethiological therapy

Age
From 24 to 64 years old
Mean range 37,5 years old

GeneraL population Sampling

r
[ 40.98% 0.82%
16.67%
10.66%

[ 47.22% 36.11 %

•
C
From 24 to 34 years old
From 46 to 56 years old •
C
From 35 to 45 years old
57 and more years old

Sex of Subjects

GeneraL popuLation Sampling

91.80% 88.89%

Female
• Male

125
 Duration of Sero Positivity:
(as reported by subjects)
From 0.5 to 11 years
r Mean range: 4.4 years

r GeneraL population SampLing

51.64% 43.44% 33.33%

61.11%

I
•o From 0.5 to 5 years From 6 to 9 years

[ More than 10 years

The percentage of the more than 10 year survivors within the subject population and sampling
r is representative of the same group in the general seropositive population.

r AIDS related diseases

[ Secondary infections
(as reported by subjects)

[ GeneraL popuLation Sampling

72.22 %
[
[ 47.54%
27.78%

With secondary infections • Without secondary infections

[
L Malignancies

GeneraL population Sampling

82.50% 86.11 %
17.50% 13.89%

With • Without

126
 RESULTS AND DISCUSSIONS

5.4 ...-------,----1
5 .2 ~-------:----'" After a 90-day course of treatment with
Factor-R; in Group 1 the average values of
5·1- - - - - - - - - - ' --I- - - - - - - i white blood cell (WBC) count increased by
35 %. An increase in the absolute values of
WBC count was observed in 75 % of the
4.8-1- - - - - - - - t -1-- - - -
subjects in this group. In Group 2 the
average values decreased by 4.25 %; the
4.6-1- - - - - -~ .......;::--- -
increase in absolute values was observed
however in 55.9 % of the subjects in this
4.4 -f- - - -
group.

4.2 - - Before treatment the average values of WBC

in subjects of Group I were below the
4·1- - - --11.._- referent values (4.8 - 10.8 klcumm) . After
[ 3.8 L -_ _ _ _ _ _ _ _ _ __ _ _ __
Factor-R treatment WBC increased and
reached normal levels, Fig. 1.

r
Starting Factor R 90th day
___ Group 1 - subjects with CD4 from 0 to 200
...... Group 2 - subjects with CD4 from 201 to 499

[ Figure 1
WBC population after treatment with Factor-R.
Referent values 4.8-10.8 k/cumm 45 .6 ,----

[ 45.2 ~-------+--

[ In Group 1 the average percentage of 44.8 -1--- --

neutrophils increased by 1.69 % for the
group as a whole . An increase of
44.4 i-------'~----c-
neutrophil percentage was observed in
50% of the subjects in this group. In
Group 2 the average percentage of ~ +-------~-+--
neutrophils decreased by 3.24 % for the
group as a whole. But an increase in the
43.6 ·1- - - - · - - - - - - ;
percentage of neutrophils was observed
in 55 .6 % of the subjects in this group.
43.2 ·1- - - -
Average values remained normal in both
groups, Fig. 2. 42.8 L -_ _ _ _ _ _ _ _ _ _ _ _ _ __

Starting Factor-R 90th day

___ Group 1 - subjects with CD4 from 0 to 200

...... Group 2 - subjects with cn4 from 201 to 499

Figure 2

NEUTROPHIL population after treatment with Factor-R

Referent values: 40-74%

127
[ 10

9.6

/ 9.2 +------~---:---------

8.8 '

8.4 -

8 -

7.6 --
Monocytes decreased in all subjects after
7.2 Factor- R treatment. All values however
remained within the limits of normal ,
6.81...----- - - -- - - - - -- -- for both group. Fig. 3.
Starting Factor-R 90th day
_ Group 1 - subjects with CD4 from 0 to 200

[ Group 2 - subjects with CD4 from 201 to 499

Figure 3

[: MONOCYTES after treatment with Factor-R

Referent valus: 3-11 %
38

[ 36 - - -Jt-_____ --

[ 34 ------------.-------.-- .-- -------

[ 32 ---- ---------------..--. -- -.- __ ·._00 __

[ While lymphocytes levels did not change

30 ------. -- ......-- ..... --.--.. -...- - - - - - - - - - -

significantly in both groups, their average

28 · - - - . - - - - - - - -.. - - - - - - - - - -----
[ values did tend to increase - from 25,13
to 27,21 in subjects of Group 1 and from
36,20 to 36,97 in subjects of Group 2. The 26 - - -- -- --::;;;.",--.,

L observed values were all in the limits of

normal, Fig. 4. 241...-------------------
Starting Factor R 90th day

_ Group 1 - subjects with CD4 from 0 to 200

___ Group 2 - subjects with CD4 from 201 to 499

Figure 4
LYMPHOCYTES after treatment with Factor-R
Referent values: 19-48%

128
r 13.6 , . . - - - - - - - -- - - - - - - -

13.4-i-- - -Aoi;:--- - - - : - - - - - - - - - - - - ;

13 .2 -

13 ---------;
There were no considerable alterations in
the hemoglobin levels of the Subjects. All
12.8 ---------.-.--
remained below referent values. However
in Subjects of Group 1 it increased from
12,46 to 12,70. In Subjects of Group 2 it
12.6
decreased from 13,41 to 13,05. These
changes still remained below referent
L 12.4 ' - - - - - - - - --
Starting Factor R
- - - -- -- -
90th day
values, Fig. 5.

[ .....
___
Group 1 - subjects with CD4 from 0 to 200
Group 2 - subjects with CD4 from 201 to 499

[ FigureS
HEMOGLOBIN after treatment with Factor-R
Referent values: 14-18 g/dl

[ 200 ... -- . - - . - - - - - - - - - - - - - - - - -

After a 90-day course of treatment with 180 . - . - - - - - - - - - - - - ------.- . ___ .____ ---1
Factor-R, in Group 1 the average values of
platelet count decreased by 7.53 %. An
increase in the absolute values of platelet 170 ·· .. --. ---. -.--- .- .----.--- -. - . - . - - - - - - ,
count was observed in 25 % of the subjects in
this group. In Group 2 the average values
increased by 2.86 %.; the increase in absolute 160 · ----------,
values was observed in 44.4 % of the subjects
in this group.

The decrease in Group 1 was within normal

limits, Fig. 6.
140~----------------
Starting Factor-R 90th day

..... Group 1 - subjects with CD4 from 0 to 200

...... Group 2 - subjects with CD4 from 201 to 499

Figure 6
PLATELET COUNT after treatment with Factor-R
Referent values:' 130-400 k/cillnm

129
 I

r 8.6,----------------,

8.4 -

8 . 2 - 1 - - - - - - - - - ' - - - - 1 -- - - - - 1

8 -1 ---------~-I --~r----~

7.8-1- - - - ---!I
I
7.6 1-- - - -.".-- #-- - - - - 1i
I

7.4-1- - - - - _·_--_·1 !
Total protein increased in 50% of subjects
7.2-1- - - - - ---.1r-- - --I in Group 1 and in 88,9% of subjects in
Group 2. It was particularly pronounced
7+-- - -
in subjects of Group 2. The increase in

[ 6.8 ' - - - - - - - - - - - - - - - - - - "

average values was evident in all groups,
Fig. 7.
Starting Factor R 90th day

[ - Group 1 - subjects with CD4 from 0 to 200

..... Group 2 - subjects with CD4 from 201 to 499

[ Figure 7
PROTEIN TOTAL after treatment with Factor-R

[ Referent values: 6-8 gldl

360 r ---------------- -

[ i

3201----

[ 280 ------

[ Of particular interest were the resu Its 2401- - - - - - - - - - - ' - - - - - - - - - - - - - - - -

concerning the T-helper subpopulation of

lymphocytes-CD4. Despite the decrease of 200 - - - - - - - - - - i---------------~
[ average values from 89.37 to 66.00 for
,
patients of Group 1 and from 349.44 to 1601- - - - - - - - - - - -- - - - - - - - - - - -
1-- - 257.22 for those of Group 2 both average
[ values of CD4 did not reach normal ranges 120
,
-_._.-_ .. _._----_._------

after initial treatment with Factor-R. This

substantiates that while Factor-R stimulated 80 I----~~~......J..L-;;::------ -----
CD8 populations if had little or no effect in
directly stimulating CD4 production. Fig. 8. 40~------~-------~

Starting Factor R 90th day

_ Group 1 - subjects with CD4 from 0 to 200

...... Group 2 - subjects with CD4 from 201 to 499
FigureB
T helper/inducer (CD4) lymphocytes after treatment with Factor-R
Referent values: 370-1482 per cumm

130
 I
1 1100,------

[ 1000-l- - - - - - - - - : - j--~~-- I
i !
900-1- - - - - - i-------- -----~

There was an obvious tendency in the

800 -1- - - - - - - - - - - - - --- -------c level of T-suppressor lymphocyte
subpopulation - CD8 count to increase
in 10 subjects of both groups: in
700 -1- - - - - - - - - - - - -
4 subjects of Group 1 and in 6 subjects
, of Group 2. The decrease in the average
i
600 -1- - - - - - - - - - - - - - - - - - - ~ values of CD8 in patients of Group 1
from 517 to 438 was still in the referent
I
range. In Group 2 the average values of
500 -- - - ---=--=-""""""=
CD8 (820 to 1014) exceeded
[ 400~----------------------------~
considerably the top referent values,
Fig9.
Starting Factor R 90th day

[ _
-+-
Group 1 - subjects with CD4 from 0 to 200
Group 2 - subjects with CD4 from 201 to 499

[ Figure 9
T suppressorlcyto (COB) lymphocytes after treatment with Factor-R
Referent values: 162-682 per cumm

[
IN CONCLUSION the treatment of 17 subjects with Factor-R provided substantial evidence for
its stimulatory effect, particulary on T suppressor/cyto (COB) lymphocytes. The increase in the
I
I level of neutrophil granulocites would contribute to the activation of intracellular activity against
phagocyted infectious agents. This resulted in an increase of non specific resistance against
secondary opportunistic infections. The elevated number of CD8 lymphocytes in the group with
CD4 from 201 to 499 is important with respect to anti-viral and anti-tumor cell-mediated immunity.
This therapeutic effect could prove effective in the suppression of viral (HIV) replication and
should provide an improved defense against malignancies. The increased level of total protein is
especially favorable in AIDS patients due to their tendency to develop deep hypoproteinemia.
Besides, it could be assumed that the increase in total proteins is a result of accelerated formation
of specific antibodies against separate bacterial components of Factor-R.

The diverse effects of Factor-R lead to an improvement in the quality of life for the subjects
treated. The lack of direct stimulation of the CD41ymphocyte population supports the hypotheSiS
that viral replication is suppressed in host cells as a result of the accompanied activation of CD8
lymphocytes that produce IL-4 and IL-1 o. It is evident from the observations that some correlation
between subject CD4 count and the efficacy of Factor-R exist.

Factor-R's effect on WBC (Fig. 1) and Neutrophil populations (Fig. 2) is obviously more pronounced
in Subjects with CD4 < 200. This could provide hope that Factor-R treatment may be beneficial
even in more advanced cases than first antiCipated. Also the increase in blood protein (Fig. 7) is
very substantial in this subpopulation of subjects. However levels of CD8 (Fig. 9) on average in
this group are not substantially effected according to this criteria. However some did display
improvement individually (4 subjects), the CD4 200> subpopulation displayed the expected
increase and quite dramatically.

131
 A further interesting observation is that the subpopulation whose COB count was most effected
was already at the higher end of or infact over the normal referent values for COB. This
r subpopulation also provided the best subjective reports according to their overall health . This
group also consisted of all those having long term non-progressor status. This observation was
r critical in the Investigators later grouping based on COB population (see follo~ing section) and
has provided the Investigators an essential criteria for determining Subject response to this type of
therapeuticum.

Negative effects were not observed in any of the subjects either during the application of
Factor-R as monotherapy or its combined application with anti retrovi ral drugs. The foregoing
leads to the suggestion that future, better observation and wider research into the effects of
Factor-R in combination with antiretroviral agents could produce promising results.

I Although the research to date can be regarded as preliminary, we can suggest that the multiple
effect of Factor-R on protective mechanisms may optimize the treatment of HIV-infected and

L AIDS sufferers.

[
[
[
[
[

132
I
r
STATISTICALLY SIGNIFICANT EVIDENCE
I. OF CDB POPULATION STIMULATION
Recent research observations indicate a distinct relationship between long term survivability and
I. C08 populations in subjects who have survived over 5 years with little apprecia~le loss in quality
of life. The number of C04 and C08 cells in 44 of the subjects before and after treatment with
Factor-R are shown on Tables 1,2 and 3. As in the previously discussed 17 subjects, a considerable
I~ increase in the number of C08 cells was observed, Table 4. The increase of C08 lymphocytes
varied from 42,1 to 56,2 % of the subjects (average 50,0%) . The changes were similar in all

~
3 groups. The highest rise (in 56,2 %) was found in Group 2 which coincided with the highest
increase of C04 cells (in 31,3 %) of the same group. The more significant decrease of C08 was
observed in Group 1 - in 36,8 %. Increases during study period are highlighted.
[ Table I

[ CD4* CD8*
(per cumm) (percumm)
[ base line 90th day base line 90th day

[ Group l(C04: 0-199)

Subject 1 11 18 570 466

[ Subject 2 25 25 320 496
Subject 3 19 30 60 260
Subject 4 62 86 371 571
[ Subject 5 39 42 618 700
Subject 6 35 46 345 561
Subject 7 23 32 69 94
Subject 8 144 106 475 430
Subject 9 21 15 255 234
Subject 10 99 19 634 618
Subject 11 157 129 569 372
Subject 12 72 47 697 553
Subject 13 200 150 1300 200
Subject 14 184 66 790 963
Subject 15 101 63 888 1047
Subject 16 130 100 781 840
Subject 17 0 0 100 50
Subject 18 150 150 300 200
Subject 19 7 7 488 496

n = 19
78 60 507 482
X ±S ±313 ±270

There is no evident correlation between increase and decrease in C04 population and an increase
in C08 population.

133
 Table 2

[ CD4* CD8*
(per cumm) (per cumm)
base line 90th day base line 90th day
I
r Group2 (CD4 : 200-499)

Subject 1
Subject 2
264
357
273
510
1075
2307
1286
3386
~ Subject 3
Subject 4
286
340
309
578
1088
680
1139
1155
Subject 5 291 324 874 876
[ Subject 6
Subject 7
228
251
230
281
1119
767
1337
1161
Subject 8 220 250 400 800
Subject 9 325 100 250 250
[ Subject 10 281 245 791 552
Subject 11 471 324 2099 1651
Subject 12 325 250 900 500
[ Subject 13
Subject 14
400
283
100
228
875
634
750
783
Subject 15 378 346 654 918
[ Subject 16 375 200 425 800

[ n= 16
XiS 317 284 934 084
±555 ±706

[
Table 3

[ CD4* CD8*
(per cumm) (per cumm)
[ base line 90th day base line 90th day

Group 3 (CD4 > 500)

l Subject 1 518 562 1425 1569
592
t Subject
Subject
Subject
2
3
4
516
543
650
499
512
817
747
1015
1184
687
1002
Subject 5 615 609 1410 1265
L Subject
Subject
6
7
596
601
404
552
519
1442
571
1946
Subject 8 603 394 893 1339
L Subject 9 564 501 3243 3279

n=9
XiS 578 513 1279 1426
±761 ±765

134
I
r- Table 4. Changes in CD4 and CD8 cells of 44 HIV infected subjects after treatment with
Factor-R during a period of 90 days.
[
CD4* CD8*

r Group 1(CD4: 0-199)

incr.

5(26,3)
decr.

9(26,3)
stable

5(26,3)
incr.

8(42,1 )
decr.

7(36,8)
stable

4(21,0)

r n= 19
Group 2 (CD4: 200-499) 5(31,3)
n= 16
7(43,7) 4(25,0) 9(56,2) 4(25,0) 3(18,8)

t Group 3 (CD4 > 500) 1(11,1)

n=9
6(66,7) 2(22,2) 5(55,5) 2(22,2) 2(22,2)

TOTAL n=44 11(25,0) 22(44,0) 11 (22,0) 22(50,0) 13(29,5) 9(20,0)

[
* Number of subjects (percentage).
[ In general CD4 decreased in all groups. In 22 (44,0%) subjects CD4 decreased, in 25,0% CD4
increased or remained stable.

[ Influences of antiretroviral and ethiological therapy, as well the daily dose and duration of HIV
infection on the stimulatory effect of Factor-R were of particular interest in the study. The findings
are listed on Table 5. In reviewing subjects with antiretroviral and ethiological therapy we restricted
[ 36 subjects to dosages of 60-80 mg daily Factor-R. This was made in order to standardize the
conditions of the study.

[ Table 5. Influences of antiretroviral and ethiological therapy, the daily dosage and duration of
HIV infection on CD8 cells after treatment with Factor-R by group.

[ C08 cells*
increase decrease stable
[ On antiretrovirals 12(66,7) 2(11,1) 4(22,2)
n = 18
[ Without antiretrovirals
n= 18
8(44,4) 5(27,8) 5(27,8)

L On ethiological therapy
n =21
14(66,7) 6(28,6) 1(4,8)

Without ethiological therapy 10(66,7) 4(26,7) 1(6,7)

l n = 15

DOSE : 40 mg (n = 8) 2(25) 5(62,5) 1(12,5)

60 mg (n = 34) 22(64,7) 10(29,3) 2(5,9)
80 mg (n = 2) 1(50) 1(50)

Duration of HIV infection

0-5 years (n = 22) 14(63,6) 7(31,8) 1 (4,5)
6-11 years (n = 14) 9(64,3) 4(28,6) 1(7,1)

* Number of subjects (percentage).

135
 I
I. The combined therapy with Factor-R and antiretroviral preparations led to a considerable elevation
in COB cells - 66.7 % from 1B of the treated subjects. The rise of COB cells in subjects on
[ monotherapywith Factor-R was 47.40/0 in 1B of them.

r The percentages of elevation of COB in subjects on Factor-R and ethiological therapy (antibacterial,
antifungal, antiprotosoal and antiviral) coincided with those of subjects treated only with
Factor-R - 66.7 0/0. COB cell increase was highest in subjects treated with 60 mg of Factor-R per
day - 64.7 % of the subjects.

Bearing in mind the important role of COB cells on antiviral immunity these changes were examined
in subjects with different durations of seropositivity. Great similarities in the percentages of increase
(63.6 -64.3) and decrease (31.B-2B.6) of COB cells were observed in groups of subjects with different
durations of seropositivity. Eight subjects comprised the group of so called "Long Term Non-
[ Progressors" - subjects with seropositivity duration more than 5 years and C04 > 500 cells per
cumm. In this group we observed the highest increase of average values of COB cells with 20.70/0
(from 1136 to 1433 cells per cumm). A tendency to increase the average values of C08 cells was

l also found in the group with a duration of HIV infection less than 5 years and with different C04
cell counts (Table 6). Increase in COB of < 200 subjects seems to have some correlation with the
pretreatment levels of COB.
[
Table 6. Average values of COB cells in subjects with H IV infection less than 5 years
and treated with Factor-R, grouped according to C04 criterion.
[
Group CD4 cells CD8B CD81 Percent of Duration of HIV +
[ (per cumm) (per cumm) (per cumm) increase period (years)

[ 1 0-199 463 ± 264 504 ± 262 B,1 3,47

[ 2 200-499 1041 ± 468 106B ± 352 2,5 3,43

3 >500 1059 ± 337 10BB ± 450 2,5 6,44

F
'--

Among the 29 subjects who do not belong to the group of long-term non-progressors 6 subjects
were distinguished by their average COB values which exceeded 900 and surpassed minimum
baseline COB values of Long-Term Non-Progressors. The presence of such high COB values, in
subjects who in fact have HIV-positivity under 5 years, gives reason to consider them "Potential
Long-Term Non-Progressors", Table 7 and Figures 1, 2, 3,1.1,2.1,3.1.

136
I.

[
[
I

~
[
r
[
[
[
[
[
E
[
E

137
 II.
Ie Changes in average of COB population (per cumm)
Criterion: general population

Ie A
v
2090

1800
r-------------~------------~----------__,

t - - ---i
e
r 1600 t -- - - - i
a
9 1400
e
1200
IC v
a
I 1000
u
e 800
s
600
Potential long term non-progressors

I[
Long term non-progressors Other

• Before treatment
• After treatment

Figure 1
[ Changes in average of COB population (per cumm)
Criterion: treated with anli-retrovirals

I[ A
v
e
1800r-------------r-------------r------------ .

1600 1-------1
a
I[ g
e
1400 ~-----I

1200
v

Il a
I
u
1000

800
e
s
[ 600

400
Potential long term non-progressors
Long term non-progressors Other

[
• Before treatment
• After treatment
Figure 2

Changes in average of COB population (per cumm)

Criterion: treated without anti-retrovirals

A
2400 r-------------,-------------r-----------~
l v
e
r
a
2200 r----~~--1--------+-------~
2000
g
e 1BOO
1600
v
a 1400
I

l u 1200
e 1000
s
BOO

l
600
Potential long term non-progressors
long term non-progressors Other

• Before treatment
• After treatment

l We can observe from the above that treatment with Anti-Retrovirals seems to act as a catalyst
Figure 3

[ to the efficacy of Factor-R therapy in all subject subpopulations.

138
 I).

[[ IN CONCLUSION, although the data is of a preliminary character, results from the first 44 subjects
treated with Factor R provides demonstrable evidence of the stimulatory effect of this preparation.
1[ It is obvious from the results obtained todate that Factor-R stimulates leucopoiesis, erythropoiesis
and thrombocytopoiesis. Of great importance is that in a substantial number of subjects with
initiallowvaluesofWBC neutrophils, thrombocytes, a recovery to the normal range after treatment
IT can be achieved.

1[ Factor-R did not however influence the total number of lymphocytes. Yet at the same time the
number of COB lymphocytes increases considerably and constantly in 56.B % of the subjects
observed. According to recent findings an elevation in COB is very important in conducting antiviral
I~ cell-mediated immunity, including that against HIV. The data suggests that subjects with COB
from 200 to 499 are the most receptive for treatment with Factor-R according to this immunological
parameter, although a similar tendency was demonstrated in the other two groups.
I[
[ A decrease in C04 cells for many of the subjects was observed: an average of 44,0%. If the
investigators hold on to the hypothesis that the stimulation ofT-helper/inducer lymphocytes (CD4
cells) may lead to accelerated HIV replication, then the absence of such effect should be favorable.

I[ In the alternative there does seem to be some increase of C04 which correlates in some Subjects
to the increase in COB. If the COB are infact surrpressing viral replicational in infected CD4 cells
as is hypothesised than the associated increase may well prove a positive, rather than negative
I[ indicator of recovery.

I[ As well combined therapy with Factor-R and antiretroviral agents may provide an effective means
to achieve agressive stimulation of COB cell production. The results clearly demonstrate that, •
when Factor-R alone or Factor-R and antiretrovirals are applied jointly, COB population will increase
,[ accordingly. There is an observable link between the volume of COB increase and antiretroviral
therapy. We may suggest that future studies will demonstrate a possibility for dose reduction of
highly toxic antiretroviral preparations and achievement of increased therapeutic benefit. In this
[ way it may become possible to stimulate defense mechanisms and be simultaneously less harmful
therapy for inhibition of HIV replication.

[
Although the subjects in groups treated with 40 and BO mg respectively were not sufficient to
make comparison, it is evident that the rise of COB was dose dependent. The highest elevation of
I[ COB was connected with doses - 60 mg daily or more.

~ The substantial increase in CD8 cells of subjects with different duration of H IV positivity gives
reason to consider that treatment with Factor-R, by means of its immunomodulating action, on
the CD8 cell population, may lead to an extension and improvement in life as well as contribute
l to a transition of a number of subjects into Long-Term Non-Progressors. It is in this group that the
highest rise of C08 population was observed, followed by an B,1 % increase in subjects with CD4
cell from a to 200. This fact gives encouragement that the latter subjects may achieve a life style
l similar to that observed to date in Long-Term Non-Progressors.

l The observed increase of neutrophils and monocytes (in some subjects) is indicative of heightened
capability for phagocytosiS against extra- and intracellular parasites and is a prerequisite for the
[ development of cell-mediated immunity against infections and tumors.

140
 Increase in average of CDB count (%)
Criterion: general population

f 24

20
P
e
r 16
c
e 12
n

If g
e
t
a 8

:r 0
Before treatmentD After treatment

long term non,progressors

Potential long term non,progressors
Other
Figure 1.1.

Increase in average of CDB count (%)

Criterion ' treated with anti-retrovjrals

I 32

28
P 24
Ij e
20
c
e 16
n
t 12
a
g 8
e
4

0
Before treatmentO Mer treatment

Figure 2.1.

Increase in average of COB count (%)

Criterion ' not treated with antj-retroyjra/s
12 --------_.. - ----_._------

P 10
e
8
e
6 1 - -· - - -·
a
g 4
e
2 1---------7J~--l_- --------- . -.-

0r-----~------~
~~------------------------------------~
.. ====~------
Before treatmentO Mer treatment

-- Figure 3.1.

The same "catalytic" effect is evident in the percentage of increase in the various subpopulations.

139
 [!

[ [

[ [

II
[I

[I

II

iI

II

I I
1
\ II
I
r IN CONCLUSION, although the data is of a preliminary character, results from the first 44 subjects
treated with Factor R provides demonstrable evidence of the stimulatory effect of this preparation .
[ It is obvious from the results obtained todate that Factor-R stimulates leucopoiesis, erythropoiesis
and thrombocytopoiesis. Of great importance is that in a substantial number of subjects with
initial low values of WBC, neutrophils, thrombocytes, a recovery to the normal range after treatment
I can be achieved.

r Factor-R did not however influence the total number of lymphocytes. Yet at the same time the
number of COB lymphocytes increases considerably and constantly in 56.B % of the subjects
observed. According to recent findings an elevation in COB is very important in conducting antiviral
~ cell-mediated immunity, including that against HIV. The data suggests that subjects with CD8
from 200 to 499 are the most receptive for treatment with Factor-R according to this immunological

[ parameter, although a similar tendency was demonstrated in the other two groups.

[ A decrease in CD4 cells for many of the subjects was observed: an average of 44,00/0 . If the
investigators hold on to the hypothesis that the stimulation ofT-helper/inducer lymphocytes (CD4
cells) may lead to accelerated H IV repl ication, then the absence of such effect should be favorable.

E In the alternative there does seem to be some increase of CD4 which correlates in some Subjects
to the increase in COB. If the CD8 are infact surrpressing viral replicational in infected CD4 cells
as is hypothesised than the associated increase may well prove a positive, rather than negative
[ indicator of recovery.

[ As well combined therapy with Factor-R and antiretroviral agents may provide an effective means
to achieve agressive stimulation of COB cell production . The results clearly demonstrate that, •
when Factor-R alone or Factor-R and anti retrovi rals are applied jointly, CD8 population will increase
[ accordingly. There is an observable link between the volume of CD8 increase and anti retrovi ral
therapy. We may suggest that future studies will demonstrate a possibility for dose reduction of
highly toxic antiretroviral preparations and achievement of increased therapeutic benefit. In this
[ way it may become possible to stimulate defense mechanisms and be simultaneously less harmful
therapy for inhibition of HIV replication.

[
Although the subjects in groups treated with 40 and 80 mg respectively were not sufficient to
make comparison, it is evident that the rise of CD8 was dose dependent. The highest elevation of
CD8 was connected with doses - 60 mg daily or more.

The substantial increase in COB cells of subjects with different duration of HIV positivity gives
reason to consider that treatment with Factor-R, by means of its immunomodulating action, on
the CD8 cell population, may lead to an extension and improvement in life as well as contribute
l to a transition of a number of subjects into Long-Term Non-Progressors. It is in this group that the
highest rise of C08 population was observed, followed by an 8,10/0 increase in subjects with CD4
cell from a to 200. This fact gives encouragement that the latter subjects may achieve a life style
similar to that observed to date in Long-Term Non-Progressors.

The observed increase of neutrophils and monocytes (in some subjects) is indicative of heightened
capability for phagocytosis against extra- and intracellular parasites and is a prerequisite for the
development of cell-mediated immunity against infections and tumors.

140
I
r As a result of the improved hematological and immunological parameters Subjects report feeling
better. The existing clinical data shows a reduction in the number and the recovery time of existing
[ and later infections - bacterial, fungal and viral. Of particular interest is that in some subjects life
treatening conditions such as dramatic weight lost, bacterial sepsis, and chronic diarrhea with

r malabsorbtion are influenced very well. Most of the subjects reported a better quality of life.

Negative effects were not observed in any of the patients both during the application of Factor-R

r as monotherapy and during its combined application with antiretroviral drugs. The foregoing
leads to the suggestion that future wider research of the effect of Factor-R in combination with
antiretroviral agents could produce promising results.

E These results are currently unique. Even at the most recent AIDS conference (Yokohama, Japan)
no other product demonstrated such statistically significant effects on COB population in
[ seropositive subjects. The obvious connection between long-term survivability and excalated COB
count provides intriguing possibility concerning the quality of life for AIDS subjects. Substantially
prolonged and improved life could unto itself be an enormous boom to most seropositive patients
[ and AIDS suffers. More interestingly the increase in CD4 witnessed in some of the subjects seems
to be directly connected to the increase in COB. If these can be established as a healthy uninfected
new cell population then it may well be possible ~o stabilize the subjects overall immune profile.
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