Crohns disease

Crohn's disease is a type of inflammatory bowel disease (IBD) that may affect any
part of the gastrointestinal (GI) tract, from mouth to anus.[1] It was named after
gastroenterologist Burrill Bernard Crohn, in 1932, along with colleagues Leon
Ginzburg and Gordon Oppenheimer at Mount Sinai Hospital in New York, described
a series of patients with inflammation of the terminal ileum of the small intestine, the
area most commonly affected by the illness.[2]
While symptoms vary from patient to patient and some may be more common than
others, the tell-tale symptoms of Crohns disease include symptoms related to
inflammation of the GI tract and general symptoms that may also be associated with
IBD.[1] Symptoms often include: abdominal cramps and pain, persistent diarrhea
(which may be bloody if inflammation is severe), urgent need to move bowels,
sensation of incomplete evacuation and constipation (which can lead to bowel
obstruction). General symptoms include fever, weight loss, loss of appetite, fatigue,
night sweats and loss of normal menstrual cycle.[3]
Crohn's disease is caused by a combination of environmental, immune and bacterial
factors in genetically susceptible individuals.[4],[5],[6] It results in a chronic
inflammatory disorder, in which the body's immune system attacks the gastrointestinal
tract possibly directed at microbial antigens.[7] While Crohn's is an immune related
disease, it does not appear to be an autoimmune disease (in that the immune system is
not being triggered by the body itself). [8] The exact underlying immune problem is not
clear;

however,

it

may

be

an

immunodeficiency

state. [9]

About half of the overall risk is related to genetics with more than 70 genes found to
be involved.[3] Tobacco smokers are two times more likely to develop Crohn's disease
than nonsmokers.[10] It also often begins after gastroenteritis. Diagnosis is based on a
number of findings including: biopsy and appearance of the bowel wall, medical
imaging and description of the disease. Other conditions that can present similarly
include irritable bowel syndrome and Behcet's disease.[3]

There are no medications or surgical procedures that can cure Crohn's disease.
Treatment options help with symptoms, maintain remission, and prevent relapse.
In those newly diagnosed a corticosteroid may be used for a brief period of time to
quickly improve the disease with another medication such as either methotrexate or a
thiopurine used to prevent recurrence. An important part of treatment is the stopping
of smoking among those who do. One in five people with the disease are admitted to
hospital each year, and half of those with the disease will require surgery for the
disease at some point over a ten year period. While surgery should be used as little as
possible, it is necessary to address some abscesses, certain bowel obstructions, and
cancers. Checking for bowel cancer via colonoscopy is recommended every few
years, starting eight years after the disease has begun.[3]
Crohn's disease affects about 3.2 per 1,000 people in Europe and North America, [11]
but it is less common in Asia and Africa.[12] Crohns has historically been more
common in developed rather than undeveloped countries, in urban rather than rural
areas, and in northern rather than southern climates. [13] However, rates have been
increasing, particularly in the developing world since the 1970s. [14] Inflammatory
bowel disease resulted in 35,000 deaths in 2010[15] and those with Crohn's disease
have a slightly reduced life expectancy.[3] The disease tends to start in the teens and
twenties, although it can occur at any age.[1] Males and females are equally likely to
be affected.[1] Crohns tends to run in families, so if a person or a close relative have
the disease, the family members have a significantly increased chance of developing
Crohns. Studies have shown that 5-20% of affected individuals have a first degree
relative (parents, child, or sibling) with one of the diseases. The risk is greater with
Crohns disease than ulcerative colitis. The risk is also substantially higher when both
parents have IBD.

Crohns Disease Medication Options

Management of Crohn's disease involves first treating the acute symptoms of the
disease, then maintaining remission. Since Crohn's disease is an immune system
disease, it cannot be cured by medication or surgery. Treatment for Crohn's disease
usually involves drug therapy or, in certain cases, surgery. There is currently no cure
for the disease, and there is no one treatment that works for everyone. Doctors use one
of two approaches to treatment either step-up (starts with milder drugs first), or
top-down (gives people stronger drugs earlier in the treatment process).
The goal of medical treatment is to reduce the inflammation that triggers the signs and
symptoms. It is also to improve long-term prognosis by limiting complications. In the
best cases, this may lead not only to symptom relief but also to long-term remission.
Successful medical treatment accomplishes two important goals: it allows the
intestinal tissue to heal and it also relieves the symptoms of fever, diarrhea, and
abdominal pain. Surgery may be required for complications such as obstructions or
abscesses, or if the disease does not respond to drugs within a reasonable time.
However, surgery cannot cure Crohn's disease by any means - it involves removing
the diseased part of the intestine and rejoining the healthy ends, as Crohn's disease
tends to recur after surgery. Once the symptoms are brought under control (inducing
remission), medical therapy is used to decrease the frequency of disease flares
(maintaining remission or maintenance).[16]

There are several groups of drugs to treat Crohns disease today. They are:
Anti-inflammatory drugs
Anti-inflammatory drugs are often the first step in the treatment of inflammatory
bowel disease. They include Oral 5-aminosalicylates and corticosteroids.

Oral 5-Aminosalicylates (5-ASA): these drugs may be helpful if Crohn's disease

affects the colon, but they aren't helpful treating disease in the small intestine.
They are medications that contain 5-aminosalicylate acid (5-ASA), which helps
control inflammation. Health care providers use aminosalicylates to treat people
newly

diagnosed

with

Crohn's

disease

who

have

mild

symptoms.

Aminosalicylates include Sulfasalazine (Azulfidine) which contains sulfa,

Mesalamine (Asacol, Delzicol, Pentasa, Lialda, Apriso), Olsalazine and
Balsalazide.
These drugs are not specially approved by the Food and Drug Administration
(FDA) for use in Crohns. Especially sulfasalazine, have a number of side effects,
including nausea, diarrhea, vomiting, heartburn and headache. However, they can
work at the level of the lining of the GI tract to decrease inflammation. They are
thought to be effective in treating mild-to-moderate episodes of Crohns disease [17]
and useful as a maintenance treatment in preventing relapses of the disease.
They work best in the colon and are not particularly effective if the disease is
limited to the small intestine. These drugs have been widely used in the past but
now are generally considered of limited benefit.

Corticosteroids: they are a class of anti-inflammatory drug that are used primarily
for treatment of moderate to severe flares of Crohn's disease. Also known as
steroids, they are used more sparingly due to the availability of effective
treatments with fewer side-effects.[18]. Corticosteroids help reduce the activity of
the immune system and decrease inflammation. Health care providers prescribe
corticosteroids for people with moderate to severe symptoms. Corticosteroids
include Prednisone (the most commonly prescribed oral steroid), Budesonide,
Hydrocortisone and Methylprednisone. They can help reduce inflammation
anywhere in the body, but they have numerous side effects, including a puffy face,

excessive facial hair, night sweats, insomnia and hyperactivity. More-serious side
effects include high blood pressure, diabetes, osteoporosis, bone fractures,
cataracts, glaucoma and increased chance of infection.
Also, corticosteroids don't work for everyone with Crohn's disease. Doctors
generally use them only if you don't respond to other treatments. Budesonide
(Entocort EC), a newer type of corticosteroid, works faster than do traditional
steroids and appears to produce fewer side effects. However, it is only effective
for Crohn's disease that's in certain parts of the bowel. It is also useful when used
in combination with antibiotics for active Crohns disease.[19] Steroid enemas can
also be used for disease of the lower colon and rectum, in order to treat symptoms.
Hydrocortisone and budesonide liquid and foam enemas are being marketed for
these reasons.
Corticosteroids aren't for long-term use, but they can be used for short-term (three
to four months) symptom improvement, to induce remission and should not be
used as a maintenance medication. If a patient cannot come off steroids without
suffering a relapse of his/her symptoms, the doctor may need to add some other
medications to help manage the disease. Corticosteroids may also be used with an
immune system suppressor the corticosteroids can induce remission, while the
immune system suppressors can help maintain it.

Immune system suppressors

This class of medications modulates or suppresses the bodys immune system
response so it cannot cause ongoing inflammation. Immunomodulators generally are
used in people for whom aminosalicylates and corticosteroids havent been effective
or have been only partially effective. They may be useful in reducing or eliminating
the need for corticosteroids. They also may be effective in maintaining remission in
people who havent responded to other medications given for this purpose.
Immunomodulators may take several months to begin working.

For some people, a combination of these drugs works better than one drug alone.
Immunosuppressant drugs include:

Azathioprine (Imuran) and mercaptopurine (Purinethol): they are the most widely
used immunosuppressants for maintenance therapy of Crohons disease. Taking
them requires that the patient follow up closely with the doctor and have blood
checked regularly to look for side effects, such as a lowered resistance to
infection. Short term, they also can be associated with inflammation of the liver or
pancreas and bone marrow suppression. Long term, although rarely, they are
associated with certain infections and cancers including lymphoma and skin
cancer. They may also cause nausea and vomiting. The doctor will use a blood test
to determine whether you can take these medications.

Methotrexate (Rheumatrex): a folate anti-metabolite drug which is also used for

chemotherapy, and it is useful in maintenance of remission for those no longer
takinh corticosteroids.[20] This drug, which is used to treat cancer, psoriasis and
rheumatoid arthritis, is sometimes used for people with Crohn's disease who don't
respond well to other medications. Short-term side effects include nausea, fatigue
and diarrhea, and rarely, it can cause potentially life-threatening pneumonia.
Long-term use can lead to bone marrow suppression, scarring of the liver and
sometimes to cancer. There is need to be followed closely for side effects.

Cyclosporine (Gengraf, Neoral, Sandimmune) and Tacrolimus (Astagraf XL,

Hecoria): these potent drugs, often used to help heal Crohn's-related fistulas, are
normally reserved for people who haven't responded well to other medications.
Cyclosporine has the potential for serious side effects, such as kidney and liver
damage, seizures, and fatal infections. These medications aren't for long-term use.

Ustekinumab (Stelara): this drug is used to treat psoriasis. Studies have shown it's
useful in treating Crohn's disease as well and may be used when other medical
treatments fail.

Antibiotics
Antibiotics may be used when infections, such as abscesses, occur in Crohns disease.
They can also be helpful with fistulas around the anal canal and vagina. Antibiotics
used to treat bacterial infection in the GI tract that may play a role in activating the
intestinal immune system, leading to inflammation. They may be used in addition to
other medications or when infection is a concern, such as with perianal Crohn's
disease. However, there's no strong evidence that antibiotics are effective for Crohn's
disease. Frequently prescribed antibiotics include:

Metronidazole (Flagyl): at one time, it was the most commonly used antibiotic for
Crohn's disease. However, it can cause serious side effects, including numbness
and tingling in your hands and feet and, occasionally, muscle pain or weakness. If
these effects occur, the patient must stop the medication and call the doctor.

Ciprofloxacin (Cipro): this drug, which improves symptoms in some people with
Crohn's disease, is now generally preferred to metronidazole. A rare side effect is
tendon rupture, which is an increased risk if you're also taking corticosteroids.

Metronidazole and ciprofloxacin are antibiotics which are used to treat Crohn's that
have colonic or perianal involvement, although this use has not been approved by the
Food and Drug Administration.[21] They are also used for treatment of complications,
including abscesses and other infections accompanying Crohn's disease.[18]

Biologic Therapies
These medications represent the latest class of therapy used for people with Crohn's
disease who have not responded well to conventional therapy. These medications are
antibodies grown in the laboratory that stop certain proteins in the body from causing
inflammation.

Infliximab (Remicade), Adalimumab (Humira) and Certolizumab pegol (Cimzia):

These drugs, called TNF inhibitors or biologics, work by neutralizing an
immune system protein known as tumor necrosis factor (TNF). They are used for
adults and children with moderate to severe Crohn's disease to reduce signs and
symptoms. They also may induce remission. In order to compare their benefits,
researchers continue to study upon these drugs. TNF inhibitors may be used soon
after diagnosis, particularly if the doctor suspects that the person is likely to have
more severe Crohn's disease or a fistula. Sometimes they are used after other
drugs have failed. They also may be combined with an immunosuppressant in
some people, but this practice is somewhat controversial.
People with certain conditions can't take TNF inhibitors. Tuberculosis and other
serious infections have been associated with the use of immune-suppressing
drugs. The patient must talk to the doctor about the potential risks and have a skin
test for tuberculosis, a chest X-ray and a test for hepatitis B before starting these
medications. They are also associated with certain cancers, including lymphoma
and skin cancers.

Natalizumab (Tysabri) and Vedolizumab (Entyvio): these drugs work by stopping

certain immune cell molecules -integrins - from binding to other cells in the
intestinal lining.
Natalizumab is approved for people with moderate to severe Crohn's disease with
evidence of inflammation who aren't responding well to any other medications. [22]
A total of 3 large randomized controlled trials have demonstrated that natalizumab
is effective in increasing rates of remission [23] and maintaining symptom-free
status[24] in patients with Crohn's disease. Natalizumab has also been linked to
PML (though only when used in combination with interferon beta-1a). [25] The

label also recommends monitoring of liver enzymes due to concerns over possible
damage or failure. Because the drug is associated with a rare but serious risk of
progressive multifocal leukoencephalopathy (brain infection leading to death or
severe disability), the patient must be enrolled in a special restricted distribution
program to use it.
Vedolizumab recently was approved for Crohn's disease. It works like natalizumab
but appears not to carry a risk of brain disease.

Strucrure of Pharmaceutical drugs

Figure 1 - 5-Aminosalicylate (Mesalamine), Sulfasalazine, Balsalazide and Olsalazine

Figure 2 - Prednisone

Figure 3 Budesonide

Figure 4 Hydrocortisone

Figure 5 Methylprednisone

10

Figure 6 Azathioprine

Figure 7 Mercaptopurine

Figure 8 - Methotrexate

11

Figure 9 Cyclosporine

Figure 10 Tacrolimus

12

Figure 11 Ustekinumab

Figure 12 Metronidazole

Figure 13 Ciprofloxacin

13

The Role of HPLC in Drug Analysis

The most characteristic feature of the development in the methodology of
pharmaceutical and biomedical analysis during the past 25 years is that HPLC became
undoubtedly the most important analytical method for identification and
quantification of drugs, either in their active pharmaceutical ingredient or in their
formulations during the process of their discovery, development and manufacturing.
[26]-[32]

Figure 14 - Steps in a drug development and manucacturing in the pharmaceutical industry

The various stages of the pharmaceutical streamline of drug discovery, development

and manufacturing, are summarized in Figure 14. Drug development starts with the
discovery of a molecule with a therapeutic value. [33],[34] This can be done by high
throughput screening during which separations by fast or ultra-fast HPLC are
performed.[35] At the discovery stage there can be also characterizing synthetic or
natural products.[36]-[39] Drug metabolism and pharmacokinetics (DMPK) is the step
where the candidate compounds for drug are tested for their metabolism and
pharmacokinetics. The studies involve use of LC-MS or LC-MS/MS.[40]-[43]

14

The next stage is the development stage, where HPLC is used to characterize products
of the chemical synthesis, by analyzing the active pharmaceutical ingredients (API),
their impurities and/or degradation products generated by accelerated aging.[44]
The development of formulation requires also studies of the dissolution properties of
solid dosage forms as well as assays of the pharmaceutical formulations. [45]
Method for the verification of system's cleanliness during the manufacturing process
are developed and used at this stage.[46] All the HPLC methods that have been
finalized at the developmental stage are validated and transferred to the
manufacturing laboratories for a quality control analysis.[47]-[53]

15

HPLC
Assay of Corticosteroids
A mixture of six corticosteroids namely: deoxycortisone, hydrocortisone acetate,
cortisone, prednisone, hydrocortisone and prednisolone can be assayed by HPLC
method. Following are presented the chromatographic parameters for the assay:
Sample size: 10 L
Column: size-250 x 4.6 mm ID
Adsorbent: Lichrosorb DIOL: 10m
Mobile-phase: Gradient elution of A (n-Hexane) and B (Isopropanol)
Detector: UV-254 nm
The six well-elaborated and distinct peaks under the above HPLC parameters, are
clearly shown in Figure 15.

Figure 15 - HPLC Chromatogram of Corticosteroids

16

Budesonide
The methods available for determination of Budesonide, as per literature, include Uv
spectroscopy

[54]

UV detection

[58]

, Stability indicating HPLC

. One UV spectroscopy

[59]

[55]

, LC/MS method

[56],[57]

and TLC with

and HPLC [60] methods were also reported

for simultaneous determination of Budesonide in its combine dosage form.

One LC/MS/MS method was reported as well for determination of Budesonide in
human sputam sample.[61].Simultaneous HPLC analysis was also reported for
determination of Budesonide along with triamcinolone acetonide in microdialysate
and rat plasma.

[62]

Stability indicating LC

[63]

and visible spectroscopy.[64]

RP-HPLC is found to be the most effective and widely used method where sensitivity
as well as accuracy is concerned. Considering the wide importance of the novel
steroids in the treatment of respiratory disorders, we developed simple, sensitive,
accurate as well as precise RP-HPLC method for quantification of pure steroids and
application of the developed method for determination of their metered dose
commercial formulations were carried out. After development of the method, it was
successfully validated as per validation parameters of ICH guidelines.[65]
Budesonide, was soluble in methanol; therefore methanol was selected as common
solvent for this steroid. Attempts were made to optimize a common mobile phase for
the novel steroid which would give fast retention as well as detection at lower
concentration. Various mobile phases were tried and retention time as well as peak
area and peak symmetry was observed. Best results were obtained using Methanol:
ACN in the ratio of 60: 40 v/v as mobile phase using C-18 column. The retention time
(RT) of Budesonide was found to be 3.41610.1305. The selected mobile phase gives
best result in terms of faster retention, least tailing of the peaks as well as accurate
results for each kind of chromatographic measurements throughout the development
of the entire method. The chromatogram of Budesonide was represented in Figure 16.

17

Figure 16 - HPLC Chromatogram of Budesonide

18


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