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

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


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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.


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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


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


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


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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. 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. 2 Moran


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onsible ceptorntial for only the c factor is synomach, side the highly 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 impairments 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 Vitamin B12 (B12/Cobalamin) b Cytosol 5-methyl TH- Folate Homocysteine Methionine synthase Purines, pyrimidines TH- Folate Methylcobalamin Methionine Mitochondrion Methylmalonyl-CoA Methylmalonyl-CoA mutase Adenosylcobalamin 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 1 Nexø et al. Nat. Rev. Gastroentero. 2012, 9 (6), 345-354. conversion of methylmalonyl-CoA to succinyl-CoA by mitochondrial 2 Russell-Jones et al. Bioconjugate Chem. 1995, 6 (1), 34-42. inked to folate metabolism because the methyl group transferred to 3 tetrahydrofolate al. Bioconjugate Chem. 1999, 10 (6),essential thylRussell-Jones et to tetrahydrofolate. T etrahydrofolate is 1131-1136.


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B12 Dietary Uptake Pathway Dietary source of$ source$ Dietary$ of $12broken$ B12$ in$ B12Bis$is broken down in the releasing$ mouth$ HC$ mouth, by$ bound$ HC$ B12;$ releasing pM$ ≈$ B12; bound by HC Kd$ 0.01$ stomach$ to$ $ duodenum$ to$ $ >$ pH$ 5$ B12$ <$ pH$ 3$ CB$ ≈$ pM$ Kd$ 1.0$ CB$ AM$ B12$ TCII$ IF$ ?$ CD320$ B12$ B12$ TCII$ pM$ ≈$ Kd$ 0.005$ daily$ Average$ of$ uptake$ B12$is$ 1O μg about$ 5$ 3$ enterocyte$ ileal$ B12$ MRP1$ B12$ ileum$ to$ B 12$ I F$ B12$ IF$ ! HC$ AM$ B12$ B12$ B12$ TCII$ MG$ B12$ 1 Nexø et al. Nat. Rev. Gastroentero. 2012, 9 (6), 345-354. 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. 2


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


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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-PYY336 conjugates


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


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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


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


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Intens . [a .u.] Representative Purification (1) 5456.008 1200 1000 800 Expected m/z: 5481 (parent) 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.


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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


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


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CICR Signaling and Detection SES Cytosol O O Ca2+ N O O N O O N O O O O O O O O N O O O O N O O O O O O O O O O O O O O O O N O COO- O Fura-2AM Fura-2 bound to Ca2+ λex: 340 and 380 nm λem: 510 nm 1 2 https://www.lifetechnologies.com/order/catalog/product/F1201 Herzog et al. PNAS 1992, 89 (13), 5794-5798.


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Y2 Receptor-Stimulated CICR 1 vs. 2 vs. 3 PYY3-36 1 2 3 Beck-Sickinger et al. J. Pept. Sci. 2000, 6 (3), 97-122. Compound EC 50 (nM) PYY3-36 16 1 72 2 27 3 127


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Y1 Receptor-Stimulated CICR PYY1-36 PYY3-36 2 Beck-Sickinger et al. J. Pept. Sci. 2000, 6 (3), 97-122. Compound EC 50 (nM) PYY1-36 10 PYY3-36 620 2 2200


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


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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.


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Y2-Receptor Stimulated CICR PYY3-36 & 2 vs. PYYC36 & 4 PYY3-36 2 PYYC36 4 1 Beck-Sickinger et al. J. Pept. Sci. 2000, 6 (3), 97-122. Pederson et al. J. Pept. Sci. 2009, 15 (11), 753-759. 3 Beck-Sickinger et al. Eur. J. Biochem. 1994, 225 (3), 947-958. 2 Compound EC 50 (nM) PYY3-36 16 2 27 PYYC36 762 4 1809


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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


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


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


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Implanting Microinfusion Pumps


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


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


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


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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. 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. 2


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Pulses of Drugs and Time of Action PYY3-36 PYY3-36 B12-PYY3-36 2 PYY3-36 PYY3-36 B12-PYY3-36 2 2 PYY3-36 4 PYY3-363-36 B12-PYY3-36 PYY 2 * P < 0.05 Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749. PYY3-36 3-36 B12-PYY3-36 PYY 2


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In Vivo Uptake Studies Tmax = 1 h * * ** * 10 nmol/kg 6 10 nmol/kg 2 (n = 4) 10 nmol/kg PYY3-363-36= 3) 10 nmol/kg PYY (n * P < 0.05 ** P < 0.01 Drug AUC0-∞ (pg/h/ml) C max (pg/mL) PYY 3-36 2 3843 ± 1125 7130 ± 2050 t 1/2 (h) V D /F (L/kg) C L /F (mL/min/kg) 1680 ± 243 0.82 ± 0.16 12.8 ± 1.5 2520 ± 257 1.34 ± 0.28 15.0 ± 1.5 Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749. 188.6 ± 65.6 133 ± 32


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PYY3-36: Mechanisms of Action CENTRAL PERIPHERAL BRAIN PYY3-36 crosses BBB and activates Y2 receptors in the arcuate nucleus (ARC)1 1 GUT Vagal nerve carries sensory information from the Y2 receptors in the gut to solitary tract nucleus (NTS)2 Nonaka et al. J Pharmacol. Exp. Ther. 2003, 306 (3), 948-953. Abbott et al. Brain Res. 2005, 1044 (1), 127-131. 3 Koda et al. Endocrinology, 2005, 146 (5), 2369-2375. 2 BLOOD Circumventricular organs3


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C-Fos Immunohistochemistry Y2 Receptor Activation Vagus Nerve ! Y2 Receptor Activation ! PYY3-36 1 Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749. Blevins et al. Peptides 2008, 29 (1), 112-119. 3 Schwartz et al. Nature 2000, 404 (6778), 661-671. 2 2 * P < 0.05 2 (n = 9) PYY3-36 (n = 8) 4 (n = 5) 4 Saline


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


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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.


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


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Body Weight (g) * * Zucker Rats: BW Trends 20 D Body Weight (g) Average Food Intake (g/ 30 10 0 0 4 day 10 day B12-PYY3-363-36 2 2 B12-PYY PYY PYYPYY3-36 PYY3-36 3-36 3-36 -10 Baseline 4d*Treatment 1000 -20 * P < 0.05 -30 950 * *p<0.05 compared to pretreatment 6B12-PYY3-36 2 PYY PYY3-36 PYY 3-36 900 850 800 Baseline 750 Day 0 Doyle R.P. et al. unpublished data. Treatment Day 10 Compensation Day 20 Day 30


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4 PYY3-36 * 10 nmol/kg 10 nmol/kg 2 6 10 nmol/kg PYY3-36 10 nmol/kg PYY3-36 2 PYY3-36 4 40 30 * * 20 10 0 ** D Body Weight (g) * * 2 Average Food Intake (g/day) Conclusions and Summary 0 B12 -PYY3-36 PYY3-36 Baseline 4d Treatment 4 day 10 day B12-PYY3-36 PYY3-36 -10 * -20 -30 * *p<0.05 compared to pretreatment


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


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


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


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