Gold Standard Physiological

Measurements and Novel Drug

Delivery Methods – Session 2
Dr. Robert Doyle
Professor of Chemistry & Biology,
Syracuse University

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 Synthetic, Structural, and Mechanistic
Investigations of Vitamin B12 Conjugates of
the Anorectic Peptide PYY3-36

Professor Robert P. Doyle

Syracuse University & SUNY, Upstate Medical University
November 12th 2015
 Obesity

CDC Behavioral Risk Factor Surveillance System, 2012, http://www.cdc.gov/obesity/data/adult.html

 PYY and Appetite Regulation

• PYY is a 36 aa intestinal hormone that belongs to

the pancreatic polypeptide family1
• Synthesized and released by specialized
enteroendocrine cells (L cells)1
• PYY has two main receptors, Y1 (orectic effect)
and Y2 (anorectic effect)2
• The active anorectic form of PYY is a truncated
form known as PYY3-362

1 Ekblad et al. Peptides 2002, 23 (2), 251–261.

2 Batterham et al. Nature 2002, 418 (6898), 650−654.
 PYY3-36 in Obesity Research
• Peripheral administration of PYY3-36 into rodents1
and primates,2 including humans,3 has resulted in
an observed reduction in food intake
• Infusion of PYY3-36 into obese individuals (BMI ≥ 30)4
results in a reduced caloric intake comparable to
individuals of lower BMI3
• Oral delivery of PYY3-36 via vitamin B12 has been
established by the Doyle group in clinically relevant
levels (> 180 pg/mL) in rodents5
1 Batterham et al. Nature 2002, 418 (6898), 650−654.
2 Moran et al. Am. J. Physiol.: Regul. Integr. Comp. Physiol. 2005, 288 (2), R384−R388.
3 Batterham et al. N. Engl. J. Med. 2003, 349 (10), 941–948.
4 http://www.nhlbi.nih.gov/health/health-topics/topics/obe/diagnosis.html
5 Doyle et al. J. Med Chem. 2011, 54 (24), 8707-8711.
 onsible transcobalamin synthesis. The affected child displays few
ceptor- symptoms at birth, but within months a severe deficiency
ntial for develops and, if left untreated, it leads to lifelong impair-
only the
c factor
Vitamin B12 (B12/Cobalamin)
ments due to neurological damage.23–27 Several different
kinds of mutations leading to a lack of transcobalamin
is syn- have been identified, including deletions and mutations
omach, resulting in erroneous RNA editing.23–27
side the Haptocorrin is heavily glycosylated and is expressed in
highly many, but not all, mammals.28 In humans, haptocorrin is

b
Cytosol
5-methyl TH- Folate Homocysteine

Methionine synthase Methylcobalamin

Purines, pyrimidines TH- Folate Methionine

Mitochondrion

Methylmalonyl-CoA

Methylmalonyl-CoA
Adenosylcobalamin
mutase

Succinyl-CoA

B12 structure. The core of B12 consists of a corrin ring that encircles a
n atoms from the corrin ring, as well as to a nitrogen atom from a
ositioned below the plane of the corrin ring and a variable group (R)
able group can be occupied by several ligands, including a hydroxyl,
matically active cofactor carries either a methyl or a 5' -deoxadenosyl
efers to all variants of the vitamin, unless otherwise stated.
two distinct enzymatic processes: the conversion of homocysteine to
he 1conversion
Nexø et al.ofNat. Rev. Gastroentero.
methylmalonyl-CoA 2012, 9 (6),
to succinyl-CoA by345-354.
mitochondrial
2 Russell-Jones
inked et al. Bioconjugate
to folate metabolism because theChem.
methyl1995,
group6transferred
(1), 34-42.to
3 Russell-Jones
thyl et al. Bioconjugate Chem. 1999, 10 (6),
tetrahydrofolate to tetrahydrofolate. Tetrahydrofolate 1131-1136.
is essential
 B12 Dietary Uptake Pathway
$
Dietary source
Dietary$ source$of$ to$stomach$ to$duodenum$
of12B$is$
B is broken
12 broken$in$ B12$ B12$ $>$5$ B12$
pH$<$3$ pH$
down in the
mouth$releasing$
HC$ HC$ ! IF$
mouth,
B releasing
12;$bound$by$HC$
B12; bound by HC Kd$≈$0.01$pM$ Kd$≈$1.0$pM$
CB$ CB$
to$ileum$

B 12$
I F$
AM$ AM$

Average$daily$
B12$ uptake$of$B12$is$
B12$ B12$ ileal$enterocyte$
about$1O5$μg3$
TCII$ IF$
?$
MRP1$

CD320$
B12$ B12$
B12$ B12$ B12$
TCII$ TCII$
Kd$≈$0.005$pM$ MG$
B12$

1 Nexø et al. Nat. Rev. Gastroentero. 2012, 9 (6), 345-354.

2 Alpers et al. Pharm. Biotechnol. 1999, 12, 493-520.
3 Banerjee et al. J. Biol. Chem. 2013, 288 (19), 13186-13193.
4 Doyle et al. Exp. Opin. Drug. Deliv. 2010, 8 (1), 127-140.
 Hypothesis

Conjugation of B12 to PYY3-36 will have

positive pharmacodynamic and
pharmacokinetic effects in vivo upon
subcutaneous (sc) administration
 Specific Aims
1. Synthesize and characterize B12-PYY3-36
conjugates via a series of B12-alkyne precursors
2. Test B12-PYY3-36 conjugates for binding,
selectivity, and agonism of the Y2 (anorectic)
and Y1 (orectic) receptors in vitro
3. Perform sc in vivo feeding studies with B12-PYY3-
36 conjugates
 Synthesis of B12-Alkyne Precursors

Yield (%) MW (g/mol)

84 1406

79 1420

HOBt: hydroxybenzotriazole
75 1434 EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

Doyle et al. Synlett. 2012, 23 (16), 2363-2366.

 Structure and Modification of PYY3-36

N term. β-Turn α helix C term.

IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY

Pederson et al. J. Pept. Sci. 2009, 15 (11), 753-759. PDB: 2DF0

 Synthesis of B12-PYY3-36 Conjugates (1-3)

n Yield (%) MW (g/mol)

1 93 5481

2 95 5495

3 90 5509

TBTA: tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine
 Representative Purification (1)

Intens . [a .u.]
5456.008

1200

1000
Expected m/z:
5481 (parent)
800 5455 (-CN) tR = 23.1 min

600

400

200

0
2000 4000 6000 8000 10000
m /z

RP-HPLC: C18 analytical column, flow rate 1 mL/min, 25 °C, UV detection at 280 nm.
A: 0.1% TFA in H2O, B: MeCN, Method: 10% B to 35% B over 25 minutes.
MALDI-ToF MS: 1:1 sample:matrix ratio, CHCA matrix, 10 mg/mL, 50:50 H2O:MeCN with 0.1% TFA.
 Aim 2: Binding, selectivity, and
agonism of the Y2 (anorectic) and
Y1 (orectic) receptors in vitro

Goals
1. Construct and optimize calcium-induced calcium
release (CICR) assay via Y2 and Y1 receptors to test
activity of conjugates 1-3 vs. PYY3-36 and PYY1-36
2. Confirm Y2 receptor agonism with synthesis and in
vitro characterization of a “null” conjugate
 GPCR Signal Transduction
Gq-coupled Gs-coupled Gi-coupled

Plasma Membrane
Adenylate
αq β PLCβ αs β *αs *αi αi β
+ Cyclase
γ *αq γ + - γ
-
+ β γ
PIP2 IP3 + DAG cAMP
ATP
+
+ PKA
Ca++ PKC
DNABP Gene expression

Transcrip on
factors
ER
IP3 Promoters
Ca++ CRE, SRE
nucleus
biological response

1 Jacoby et al. ChemMedChem 2006, 1 (8), 760-782.

2 Herzog et al. PNAS 1992, 89 (13), 5794-5798.
 CICR Signaling and Detection
SES Cytosol
O O O
O O O O
N Ca2+
O O N O O N
O O
O O
O O O
O O O
O O O
N O O
O
O N O
O N O
O O O
COO-
O

Fura-2AM Fura-2 bound to Ca2+

λex: 340 and 380 nm

λem: 510 nm
1 https://www.lifetechnologies.com/order/catalog/product/F1201
2 Herzog et al. PNAS 1992, 89 (13), 5794-5798.
 Y2 Receptor-Stimulated CICR
1 vs. 2 vs. 3

Compound EC 50 (nM)
PYY3-36 16
PYY3-36 1 72
1 2 27
2 3 127
3

Beck-Sickinger et al. J. Pept. Sci. 2000, 6 (3), 97-122.

 Y1 Receptor-Stimulated CICR

Compound EC 50 (nM)
PYY1-36 PYY1-36 10
PYY3-36 PYY3-36 620
2 2 2200

Beck-Sickinger et al. J. Pept. Sci. 2000, 6 (3), 97-122.

 Y1 vs. Y2 Receptor
PYY1-36 PYY1-36

PYY3-36 PYY3-36

Nygaard et al. Biochemistry 2006, 45 (27), 8350-8357.

 Synthesis of Null Conjugate B12-PYYC36 (4)

SPDP: 3-(2-pyridylthio)propionic acid N-hydroxysuccinimide ester

Doyle et al. ChemMedChem 2014, 9 (6), 1244-1251.

 Y2-Receptor Stimulated CICR
PYY3-36 & 2 vs. PYYC36 & 4

Compound EC 50 (nM)
PYY3-36 PYY3-36 16
2 2 27
PYYC36 PYYC36 762
4 4 1809

1 Beck-Sickinger et al. J. Pept. Sci. 2000, 6 (3), 97-122.

2 Pederson et al. J. Pept. Sci. 2009, 15 (11), 753-759.
3 Beck-Sickinger et al. Eur. J. Biochem. 1994, 225 (3), 947-958.
 Aim 3: In vivo feeding studies (sc)
with PYY3-36, 2, and 4 in rats*
Goals
1. Optimize dosing in lean (Sprague Dawley) male rats
2. Acclimate rats to experimental schedule
3. Pharmacodynamic (PD) analysis
4. Pharmacokinetic (PK) analysis
5. Elucidate mechanism of action
6. Repeat sc studies in obese (Zucker) male rats

*All animal studies performed in collaboration with Dr. Christian Roth and
Clinton Elfers at Seattle’s Children’s Research Institute in Seattle, WA
 Dose Escalation Study with 2

Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749.

 Thermal/Solution Stability of 2

*All samples ran at 300 nM

Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749.

Implanting Microinfusion Pumps
 Dosing Schedule

Baseline
2

Baseline
Baseline
PYY3-36
PYY3-36

Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749.

 Food Intake Trends

4
2
4
PYY3-36
2
PYY3-36

* P < 0.05

Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749.

 Food Intake Trends

4 2 PYY3-36
4 2 PYY3-36

5 day treatment 10 day treatment

* P < 0.05
• 2 (n = 9) • 2 (n = 6)
** P < 0.01
• PYY3-36 (n = 8) • PYY3-36 (n = 4)
*** P < 0.001
• 4 (n = 5) • 4 (n = 4)

23.7% reduction in food intake due to treatment with 2 and a 13.2% reduction in food
intake due to treatment with PYY3-36
1 Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749.
2 Reidelberger et al. Am. J. Physiol.: Regul. Integr. Comp. Physiol. 2006, 290 (2), R298-305.
3 Pittner et al. Int. J. Obes. Relat. Metab. Disord. 2004, 28 (8), 963-971.
 Body Weight Gain

4 2 PYY3-36

* P < 0.05
** P < 0.01

1 Henry et al. Endocrinology 2015, 156 (5), DOI: en.2014-1825.

2 Reidelberger et al. Am. J. Physiol.: Regul. Integr. Comp. Physiol. 2006, 290 (2), R298-305.
3 Pittner et al. Int. J. Obes. Relat. Metab. Disord. 2004, 28 (8), 963-971.
 Pulses of Drugs and Time of Action

PYY
PYY3-36 B12-PYY
2 3-36 PYY
PYY3-36 B12-PYY
2 3-36
3-36 3-36

2
PYY3-36
4

PYY 3-363-36 B12-PYY

PYY 2 3-36 PYY 3-36 3-36 B12-PYY
PYY 2 3-36

* P < 0.05

Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749.

 In Vivo Uptake Studies
Tmax = 1 h
**
* * *

1010nmol/kg
nmol/kg 6 = 4)
2 (n
1010nmol/kg
nmol/kgPYYPYY (n = 3)
3-363-36

* P < 0.05
** P < 0.01

Drug AUC0-∞ (pg/h/ml) C max (pg/mL) t 1/2 (h) V D /F (L/kg) C L /F (mL/min/kg)

PYY 3-36 3843 ± 1125 1680 ± 243 0.82 ± 0.16 12.8 ± 1.5 188.6 ± 65.6
2 7130 ± 2050 2520 ± 257 1.34 ± 0.28 15.0 ± 1.5 133 ± 32

Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749.

 PYY3-36: Mechanisms of Action

CENTRAL PERIPHERAL

BRAIN GUT BLOOD

PYY3-36 crosses BBB and Vagal nerve carries sensory Circumventricular

activates Y2 receptors in information from the Y2 organs3
the arcuate nucleus (ARC)1 receptors in the gut to
solitary tract nucleus (NTS)2

1 Nonaka et al. J Pharmacol. Exp. Ther. 2003, 306 (3), 948-953.

2 Abbott et al. Brain Res. 2005, 1044 (1), 127-131.
3 Koda et al. Endocrinology, 2005, 146 (5), 2369-2375.
 C-Fos Immunohistochemistry

Y2 Receptor Activation

Vagus Nerve

!
Y2
! Receptor Activation

PYY3-36 2 4 Saline

* P < 0.05
2 (n = 9)
PYY3-36 (n = 8)
4 (n = 5)
1 Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749.
2 Blevins et al. Peptides 2008, 29 (1), 112-119.
3 Schwartz et al. Nature 2000, 404 (6778), 661-671.
 Design of NOTA-2

Doyle R.P. et al. unpublished data.

 64Cu-NOTA-2 PET Scan

Administered Dose recovered in brain for 2 vs. PYY3-36. (2-tailed p=0.08). 15 μCi
injected dose 64Cu-labeled conjugate by iv.
3 h PET scan of Sprague Dawley rats (n = 3)

Doyle R.P. et al. unpublished data.

 Zucker Rats: FI Trends
40

Food Intake (g/day) 30

*
Average

*
20

10
B
212-PYY3-36
PYY3-36
PYY 3-36
0
Baseline 4d Treatment
* P < 0.05
2 (n = 3)
PYY3-36 (n = 5)

Doyle R.P. et al. unpublished data.

 30

Food Intake (g/

*

Average
*
Zucker Rats: BW Trends
20

D Body Weight (g)

0
4 day 10 day
10
B
212-PYY
B212-PYY
3-363-36
-10 PYY PYY3-36
PYYPYY
3-36 3-36
3-36
0
Baseline 4d*Treatment
-20
* P < 0.05
*
-30 *p<0.05 compared to pretreatment
1000
Body Weight (g)

62B12-PYY3-36
950 PYY
PYY3-36
PYY 3-36

900

850

800
Baseline Treatment Compensation
750
Day 0 Day 10 Day 20 Day 30

Doyle R.P. et al. unpublished data.

 Conclusions and Summary
40 2

Food Intake (g/day)

PYY3-36
4
30
*

Average
*
20

10
4 2 PYY3-36
B12 -PYY3-36
PYY3-36
0
Baseline 4d Treatment
**
* * *

D Body Weight (g)

0
4 day 10 day
10nmol/kg
10 nmol/kg2 6
10nmol/kg
10 nmol/kg PYY
PYY 3-363-36
B12-PYY3-36
-10 PYY3-36

*
-20

*
-30 *p<0.05 compared to pretreatment
 Future Work: SUPER PYY!

GLP1-R agonism

Y2-R biased agonism

Doyle R.P. et al. unpublished data; Patent Filed Sept. 2015

Thank you to our event sponsor

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For additional information on iPrecio
infusion pumps and Innovative drug
infusion technologies for laboratory
animals please visit: Dr. Robert Doyle
http://www.iprecio.com/ rpdoyle@syr.edu