1.
Gold Standard Physiological
Measurements and Novel Drug
Delivery Methods – Session 2
Sponsored by:
Dr. Robert Doyle
Professor of Chemistry & Biology,
Syracuse University

2.
InsideScientific is an online educational environment
designed for life science researchers. Our goal is to aid in
the sharing and distribution of scientific information
regarding innovative technologies, protocols, research
tools and laboratory services.

3.
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

4.
Obesity
CDC Behavioral Risk Factor Surveillance System, 2012, http://www.cdc.gov/obesity/data/adult.html

5.
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 PYY3-36
2
1 Ekblad et al. Peptides 2002, 23 (2), 251–261.
2 Batterham et al. Nature 2002, 418 (6898), 650−654.

6.
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.

7.
Vitamin B12 (B12/Cobalamin)
1 Nexø et al. Nat. Rev. Gastroentero. 2012, 9 (6), 345-354.
2 Russell-Jones et al. Bioconjugate Chem. 1995, 6 (1), 34-42.
3 Russell-Jones et al. Bioconjugate Chem. 1999, 10 (6), 1131-1136.
onsible
ceptor-
ntial for
only the
cfactor
issyn-
omach,
sidethe
highly
transcobalamin synthesis.Theaffectedchilddisplaysfew
symptomsat birth,but within monthsaseveredeficiency
developsand,if left untreated,it leadstolifelongimpair-
mentsdueto neurological damage.23–27
Several different
kindsof mutationsleading to alack of transcobalamin
havebeen identified, includingdeletionsand mutations
resultingin erroneousRNA editing.23–27
Haptocorrin isheavilyglycosylated and isexpressed in
many,but not all,mammals.28
In humans,haptocorrin is
b
Cytosol
Mitochondrion
FolatePurines, pyrimidines TH-
Folate5-methyl TH-
Methionine synthase Methylcobalamin
Homocysteine
Methionine
Adenosylcobalamin
Methylmalonyl-CoA
Succinyl-CoA
Methylmalonyl-CoA
mutase
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 conversion of methylmalonyl-CoA to succinyl-CoA by mitochondrial
inked to folate metabolism because the methyl group transferred to
thyl tetrahydrofolate to tetrahydrofolate. Tetrahydrofolate is essential

8.
B12 Dietary Uptake Pathway
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.
B12$
HC$
B12$
HC$
B12$
B12$
B12$ B12$
B12$
B12$
IF$
IF$
CB$
AM$
B12$
IF$
CB$
AM$
to$ileum$
to$stomach$
Kd$≈$0.01$pM$
$
to$duodenum$
$pH$>$5$
!
Dietary$source$of$
B12$is$broken$in$
mouth$releasing$
B12;$bound$by$HC$
ileal$enterocyte$B12$
TCII$
MRP1$
B12$
TCII$
?$
B12$
TCII$
CD320$
MG$
Kd$≈$1.0$pM$
Kd$≈$0.005$pM$
pH$<$3$
B12$
Average$daily$
uptake$of$B12$is$
about$1O5$μg3$
Dietary source
of B12 is broken
down in the
mouth, releasing
B12; bound by HC

9.
Hypothesis
Conjugation of B12 to PYY3-36 will have
positive pharmacodynamic and
pharmacokinetic effects in vivo upon
subcutaneous (sc) administration

10.
Specific Aims
1. Synthesize and characterize B12-PYY3-36
conjugates via a series of B12-alkyne precursors
2. Test B12-PYY3-36 conjugatesfor 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

11.
Synthesis of B12-Alkyne Precursors
Doyle et al. Synlett. 2012, 23 (16), 2363-2366.
Yield (%) MW (g/mol)
84 1406
79 1420
75 1434 EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimideHOBt: hydroxybenzotriazole

12.
Structure and Modification of PYY3-36
PDB: 2DF0
N term. β-Turn α helix C term.
I K P E A P G E D A S P E E L N R Y Y A S L R H Y L N L V T R Q R Y
Pederson et al. J. Pept. Sci. 2009, 15 (11), 753-759.

13.
Synthesis of B12-PYY3-36 Conjugates (1-3)
n
1
2
3
Yield (%) MW (g/mol)
93 5481
95 5495
90 5509
TBTA: tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine

14.
Representative Purification (1)
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.
tR = 23.1 min
5456.008
MALDI-ToF MS: 1:1 sample:matrix ratio, CHCA matrix, 10 mg/mL, 50:50 H2O:MeCN with 0.1% TFA.
Expected m/z:
5481 (parent)
5455 (-CN)
0
200
400
600
800
1000
1200
Intens.[a.u.]
2000 4000 6000 8000 10000
m/z

15.
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

16.
1 Jacoby et al. ChemMedChem 2006, 1 (8), 760-782.
2 Herzog et al. PNAS 1992, 89 (13), 5794-5798.
GPCR Signal Transduction
Plasma Membrane
Gq-coupled Gs-coupled Gi-coupled
αq
*αq
αs *αs αi βββ
γ γγ
PIP2 IP3 + DAG
+
+ PLCβ
+
Adenylate
Cyclase
ATP
IP3
Ca++
Ca++
+
PKC
β γ
--
cAMP
+
PKA
*αi
Transcrip on
factors
Promoters
CRE, SRE
Gene expressionDNABP
nucleus
ER
biological response

17.
CICR Signaling and Detection
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.
O O
N
O
N O
N
O O
OO
O
O
O
O
COO-
Ca2+
O
N
O
O
O
O
O
O
N
O
O
O
O
O
O
O
O
O
N
O
O
O
O
O O
O
O
CytosolSES
Fura-2AM Fura-2 bound to Ca2+

18.
Y2 Receptor-Stimulated CICR
1 vs. 2 vs. 3
Beck-Sickinger et al. J. Pept. Sci. 2000, 6 (3), 97-122.
PYY3-36
1
2
3
Compound EC50 (nM)
PYY3-36 16
1 72
2 27
3 127

19.
Y1 Receptor-Stimulated CICR
PYY1-36
PYY3-36
2
Beck-Sickinger et al. J. Pept. Sci. 2000, 6 (3), 97-122.
Compound EC50 (nM)
PYY1-36 10
PYY3-36 620
2 2200

20.
Y1 vs. Y2 Receptor
Nygaard et al. Biochemistry 2006, 45 (27), 8350-8357.
PYY1-36
PYY3-36
PYY1-36
PYY3-36

21.
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.

22.
Y2-Receptor Stimulated CICR
PYY3-36 & 2 vs. PYYC36 & 4
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.
PYY3-36
2
PYYC36
4
Compound EC50 (nM)
PYY3-36 16
2 27
PYYC36 762
4 1809

23.
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

24.
Dose Escalation Study with 2
Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749.

25.
Thermal/Solution Stability of 2
*All samples ran at 300 nM
Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749.

26.
Implanting Microinfusion Pumps

27.
Dosing Schedule
Baseline
PYY3-36
Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749.
Baseline
2
Baseline
PYY3-36

28.
Food Intake Trends
4
2
PYY3-36
4
2
PYY3-36
* P < 0.05
Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749.

29.
Food Intake Trends
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
4 2 PYY3-36
4 2 PYY3-36
10 day treatment
• 2 (n = 6)
• PYY3-36 (n = 4)
• 4 (n = 4)
5 day treatment
• 2 (n = 9)
• PYY3-36 (n = 8)
• 4 (n = 5)
* P < 0.05
** P < 0.01
*** P < 0.001
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.

30.
* P < 0.05
** P < 0.01
Body Weight Gain
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.
4 2 PYY3-36

31.
Pulses of Drugs and Time of Action
Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749.
PYY3-36 B12-PYY3-36PYY3-36 B12-PYY3-36
PYY3-36 B12-PYY3-36 PYY3-36 B12-PYY3-36PYY3-36 2 PYY3-36 2
PYY3-36 2 PYY3-36 2
* P < 0.05
2
PYY3-36
4

32.
In Vivo Uptake Studies
Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749.
** *
**
10 nmol/kg 6
10 nmol/kg PYY3-36
10 nmol/kg 2 (n = 4)
10 nmol/kg PYY3-36 (n = 3)
Drug AUC0-∞ (pg/h/ml) Cmax (pg/mL) t1/2 (h) VD/F (L/kg) CL/F (mL/min/kg)
PYY3-36 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
Tmax = 1 h
* P < 0.05
** P < 0.01

33.
PYY3-36: Mechanisms of Action
BRAIN GUT BLOOD
Vagal nerve carries sensory
information from the Y2
receptors in the gut to
solitary tract nucleus (NTS)2
Circumventricular
organs3
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.
CENTRAL PERIPHERAL
PYY3-36 crosses BBB and
activates Y2 receptors in
the arcuate nucleus (ARC)1

34.
C-Fos Immunohistochemistry
PYY3-36 2 4 Saline
Y2 Receptor Activation
!
!Y2 Receptor Activation
Vagus Nerve
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.
* P < 0.05
2 (n = 9)
PYY3-36 (n = 8)
4 (n = 5)

35.
Design of NOTA-2
Doyle R.P. et al. unpublished data.

36.
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.

37.
Zucker Rats: FI Trends
Average
FoodIntake(g/day)
Baseline 4d Treatment
0
10
20
30
40
B12-PYY3-36
PYY3-36
*
*
2
PYY3-36
* P < 0.05
2 (n = 3)
PYY3-36 (n = 5)
Doyle R.P. et al. unpublished data.

38.
Zucker Rats: BW Trends
BodyWeight(g)
Day 0 Day 10 Day 20 Day 30
750
800
850
900
950
1000
B12-PYY3-36
PYY3-36
Baseline Treatment Compensation
6
PYY3-36
2
PYY3-36
* P < 0.05
DBodyWeight(g)
4 day 10 day
-30
-20
-10
0
B12-PYY3-36
PYY3-36
*
*
*p<0.05 compared to pretreatment
2
PYY3-36
Average
FoodIntake(g/
Baseline 4d Treatment
0
10
20
30
B12-PYY3-36
PYY3-36
*
*
2
PYY3-36
Doyle R.P. et al. unpublished data.

39.
Conclusions and Summary
** *
**
10 nmol/kg 6
10 nmol/kg PYY3-36
10 nmol/kg 2
10 nmol/kg PYY3-36
4 2 PYY3-36
2
PYY3-36
4
Average
FoodIntake(g/day)
Baseline 4d Treatment
0
10
20
30
40
B12-PYY3-36
PYY3-36
*
*
DBodyWeight(g)
4 day 10 day
-30
-20
-10
0
B12-PYY3-36
PYY3-36
*
*
*p<0.05 compared to pretreatment

40.
Future Work: SUPER PYY!
GLP1-R agonism
Y2-R biased agonism
Doyle R.P. et al. unpublished data; Patent Filed Sept. 2015

41.
Thank you to our event sponsor
Innovative drug infusion technology for laboratory animals.

42.
Dr. Robert Doyle
rpdoyle@syr.edu
Thank You!
For additional information on iPrecio
infusion pumps and Innovative drug
infusion technologies for laboratory
animals please visit:
http://www.iprecio.com/