Gold Standard Physiological Measurements and Novel Drug Delivery Methods – Session 2

Sponsored by:

Dr. Robert DoyleProfessor 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 PYY3-36

2

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.html5 Doyle et al. J. Med Chem. 2011, 54 (24), 8707-8711.

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.

2 | ADVANCE ONLINE PUBLICATION www.nature.com/ nrgastro

and haptocorrin (also known as the R-protein or trans-

cobalamin I). These proteins share the same overall

structural scaffold and each carries a single B12

molecule

(Figure 2).12,13

Intrinsic factor has a crucial function in transporting

B12

to the intrinsic factor–B12

receptor, cubam, which is

expressed on enterocytes in the ileum and is responsible

for the absorption of the vitamin by means of receptor-

mediated endocytosis. Intrinsic factor is also essential for

the actual uptake process as cubam recognizes only the

intrinsic factor–B12

complex and neither intrinsic factor

nor free B12

alone.14,15 In humans, intrinsic factor is syn-

thesized and secreted by parietal cells of the stomach,

and only small amounts have been detected outside the

gastrointestinal tract.16 This carrier protein is highly

Key points

■ A coherent vitamin B12

(B12

) transport pathway from food to the body’s cells has

now been delineated; the pathway includes an ABC transporter for cellular B12

efflux and a receptor for uptake of B12

-bound transcobalamin

■ More than 15 gene products are involved in B12

transport and/ or processing;

several new genes encoding intracellular proteins (including a potential

lysosomal transporter of B12

) have been identified

■ Gastrointestinal uptake of B12

is via cubam, the complex of cubilin and

amnionless

■ Novel genetic causes of B12

deficiency disease have been clarified; many of the

new proteins have been identified by positional cloning of the genes harbouring

the disease-causing mutations

■ New diagnostic assays for B12

deficiency are being developed; plasma level

of holo-transcobalamin is a promising biomarker in combination with existing

markers

glycosylated, which, as well as its specific amino acid

sequence, is thought to protect it from digestion by

intestinal enzymes.17

Structurally, intrinsic factor features a two-domain

architecture where B12

binds at the interface between

the two domains.12 A similar mode of binding of B12

at the interface of two domains is reiterated not only

for transcobalamin and haptocorrin,13 but also in the

B12

-dependent enzymes methionine synthase18 and

methylmalonyl-CoA mutase.19

Owing to the critical role of intrinsic factor in B12

absorption, deficiency of this protein (caused by auto-

immune attack of the parietal cells or rare inborn errors

of synthesis) leads to severe B12

avitaminosis and classic

pernicious anaemia.20,21 Intrinsic factor was discovered

by Castle as the ‘intrinsic factor’ lacking in patients suf-

fering from pernicious anaemia despite normal supply

of the ‘extrinsic factor’ (that is B12

).22

Circulating transcobalamin has an essential role in

transporting B12

absorbed in the ileum to cells of the

body. The importance of transcobalamin is obvious

in the small number of children with inborn errors of

transcobalamin synthesis. The affected child displays few

symptoms at birth, but within months a severe deficiency

develops and, if left untreated, it leads to lifelong impair-

ments due to neurological damage.23–27 Several different

kinds of mutations leading to a lack of transcobalamin

have been identified, including deletions and mutations

resulting in erroneous RNA editing.23–27

Haptocorrin is heavily glycosylated and is expressed in

many, but not all, mammals.28 In humans, haptocorrin is

bCytosol

Mitochondrion

Folate

H3C

N

N

N

N

CH3

H3C

H3C

NH2

O

NH2

O

CH3

CH3

NH2O

Co

CH3

OHN

H2N

H3C

O

O

H2N

O

H2N

O

H3C

P

–O O

O

CH2OH

O

HO

N

N

CH3

CH3

R

Purines, pyrimidines TH-

Folate5-methyl TH-

Methionine synthase Methylcobalamin

Homocysteine

Methionine

Adenosylcobalamin

Methylmalonyl-CoA

Succinyl-CoA

Methylmalonyl-CoAmutase

a

Figure 1 | B12

structure and coenzyme function. a | B12

structure. The core of B12

consists of a corrin ring that encircles a

central cobalt ion. The latter is linked to four nitrogen atoms from the corrin ring, as well as to a nitrogen atom from a

5,6-dimethylbenzimidazole ribonucleotide moiety positioned below the plane of the corrin ring and a variable group (R)

positioned above the plane of the ring.5–8,10 The variable group can be occupied by several ligands, including a hydroxyl,

cyano, methyl, or 5’-deoxyadenosyl group. The enzymatically active cofactor carries either a methyl or a 5' -deoxadenosyl

group at this position. In this Review, the term B12

refers to all variants of the vitamin, unless otherwise stated.

b | Coenzyme function. B12

serves as a coenzyme in two distinct enzymatic processes: the conversion of homocysteine to

methionine by cytosolic methionine synthase and the conversion of methylmalonyl-CoA to succinyl-CoA by mitochondrial

methylmalonyl-CoA mutase. The former reaction is linked to folate metabolism because the methyl group transferred to

homocysteine is provided by the conversion of 5-methyl tetrahydrofolate to tetrahydrofolate. Tetrahydrofolate is essential

for the production of purines and pyrimidines. Prolonged B12

deficiency results in accumulation of 5-methyl tetrahydrofolate

with impaired DNA synthesis as a result. This scenario is known as the methyl-folate-trap. Abbreviation: TH, tetrahydro-.

REVIEWS

© 2012 Macmillan Publishers Limited. All rights reserved

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$

v! v!MG$

Kd$≈$1.0$pM$

Kd$≈$0.005$pM$

pH$<$3$

B12$

Average$daily$uptake$of$B12$is$about$1O5$μg3$

DietarysourceofB12isbrokendowninthe

mouth,releasingB12;boundbyHC

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 precursors2. 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

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

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.

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

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

Inte

ns

. [a

.u.]

2000 4000 6000 8000 10000m /z

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

1 Jacoby et al. ChemMedChem 2006, 1 (8), 760-782.2 Herzog et al. PNAS 1992, 89 (13), 5794-5798.

GPCR Signal Transduction

PlasmaMembrane

Gq-coupled Gs-coupled Gi-coupled

αq

*αq

αs *αs αi βββγ γγ

PIP2 IP3+DAG

+

+ PLCβ

+

AdenylateCyclase

ATP

IP3Ca++

Ca+++

PKC

βγ--

cAMP

+PKA

*αi

Transcrip onfactors

PromotersCRE,SRE

GeneexpressionDNABP

nucleus

ER

biologicalresponse

CICR Signaling and Detection

λex: 340 and 380 nmλem: 510 nm

1 https://www.lifetechnologies.com/order/catalog/product/F12012 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

OO

OO

O

O

O

O

O

N

O

OO

O

O O

OO

CytosolSES

Fura-2AM Fura-2boundtoCa2+

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

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

Y1 vs. Y2 Receptor

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

PYY1-36

PYY3-36

PYY1-36

PYY3-36

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

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

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

PYY3-36

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

Baseline

2

Baseline

PYY3-36

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.

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.

* 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

Pulses of Drugs and Time of Action

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

PYY3-36 B12-PYY3-36 PYY3-36 B12-PYY3-36

PYY3-36 B12-PYY3-36 PYY3-36 B12-PYY3-36 PYY3-36 2 PYY3-36 2

PYY3-36 2 PYY3-36 2

* P < 0.05

2 PYY3-36

4

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

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

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 < 0.052 (n = 9)

PYY3-36 (n = 8)4 (n = 5)

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 μCiinjected 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

Av

era

ge

Fo

od

In

take (

g/d

ay)

Baseline 4d Treatment0

10

20

30

40

B12-PYY3-36

PYY3-36

**

2 PYY3-36

* P < 0.052 (n = 3)

PYY3-36 (n = 5)

Doyle R.P. et al. unpublished data.

Zucker Rats: BW Trends

Bo

dy

We

igh

t (

g)

Day 0 Day 10 Day 20 Day 30750

800

850

900

950

1000B12-PYY3-36

PYY3-36

Baseline Treatment Compensation

6

PYY3-36

2

PYY3-36

* P < 0.05D

Bo

dy W

eig

ht

(g)

4 day 10 day

-30

-20

-10

0

B12-PYY3-36

PYY3-36

*

**p<0.05 compared to pretreatment

2

PYY3-36

Av

era

ge

Fo

od

In

take (

g/d

ay)

Baseline 4d Treatment0

10

20

30

40

B12-PYY3-36

PYY3-36

**

2 PYY3-36

Doyle R.P. et al. unpublished data.

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

Av

era

ge

Fo

od

In

take

(g

/da

y)

Baseline 4d Treatment0

10

20

30

40

B12-PYY3-36

PYY3-36

**

D B

od

y W

eig

ht

(g)

4 day 10 day

-30

-20

-10

0

B12-PYY3-36

PYY3-36

*

**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

Innovative drug infusion technology for laboratory animals.

Dr. Robert Doylerpdoyle@syr.edu

Thank You!For additional information on iPrecioinfusion pumps and Innovative drug infusion technologies for laboratory animals please visit:

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