Deuteration of Drugs for Pharmacokintic Enhancement: Considerations Essential for Success

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1 Deuteration of Drugs for Pharmacokintic Enhancement: Considerations Essential for Success Alvin D.. Vaz Alfin D. Vaz Pfizer Global Research and Development Groton, CT, USA Collaborators: Raman Sharma, Tim Strelevitz, Aarti Sawant, Alan Clark, Elaine Tseng, Hongying Gao, Klass Schildnegt, Patrick Verhoest, Vinod Parikh,

2 Uses for Deuterated Drugs As an internal standard in quantitative analysis Three or more non-exchangeable deuterium atoms incorporated to mass-differentiate the internal standard and analyte Alter pharmacokinetic or toxic properties Deuterium substitution for hydrogen does not appreciably change physicochemical properties such as polar surface area, molecular volume, hydrogen bonding, is naturally abundant to 0.09%, 09% and is OT radioactive. One or more deuterium atoms substituted t at specific sites can slow metabolic clearance and result in: Increases in C max, drug exposure (AUC), and systemic half- life (T 1/2 ) Decreased metabolically generated toxic metabolites

3 Origin of the Kinetic Deuterium Isotope Effect (KDIE) Difference in mass between deuterium and hydrogen results in a zero-point energy difference between C-H and C-D bonds, consequently an increase in energy required to break a C-D bond If the transition state involves a symmetrical breaking of a C-H bond, substitution of hydrogen by deuterium slows down the reaction rate by a factor of 5 9 KDIE = k H /k D ~ 5 _ 9 Enzyme intrinsic isotope effect = D k 1 / H k 1 The intrinsic isotope effect reflects the enzyme s commitment to catalysis In pharmacokinetics the intrinsic clearance isotope effect reflects the In pharmacokinetics the intrinsic clearance isotope effect reflects the kinetic isotope effect on the first order rate constant for disappearance of substrate or V max /K m

4 Press releases by Concert Pharmaceuticals 43% increase in preclinical (monkey) half life (4.5 to 6.3 hrs) for deuterated Linezolid Oct 27th 2008 Phase 1 start for deuterated Paroxetine Sept. 25th, 2008; announced protection against CYP2D6 inactivation Sept. 29th, 2009 Phase 1b multiple dose escalation study for deuterated Atazanavir ov 9th, 2009 Expectation: QD dosing without Ritonavir co-administration Effect of deuterium substitution on sympathomimetic amines on adrenergic responses. Belleau, B.; Burba, J.; Pindell, M.; Reiffenstein, J. Science (1961),

5 Systemic Clearance Mechanism and KDIEs on Pharmacokinetics CL systemic = CL H + CL extrahepatic CL urine + CL other Clearance enzymes where KDIEs apply Aldehyde Oxidase 4 6 Monoamine Oxidases 2 9 Cytochromes P Alcohol/aldehyde CL metabolic + CL bile Clearance enzymes where KDIEs do not apply Flavin monooxygenases Glucuronyl transferases Sulfotransferases Glutathione-S-transferases dehydrogenases 6 8 -acetyl trasferases For pharmacokinetic enhancement: Metabolic clearance must determine systemic clearance The enzyme(s) involved must have KDIEs on their intrinisic clearance

6 Why KDIEs with aldehyde oxidase? Aldehyde Oxidase (AO) is a cytosolic molybdopterin class oxidase, hydroxylates nitrogen heterocycles to the nitrogen Experience with human PK failure due to high clearance by AO Interspecies variability that results in failure of allometric scaling Lack of direct in vitro to in vivo correlation in clearance scaling possibly due to wide tissue distribution Proposed reaction mechanism involves a rate-limiting hydride/proton abstraction Potential to alter PK by use of KDIE

7 KDIEs to establish interspecies commonality in the AO reaction mechanism Intra-molecular isotope effect (intrinsic) H H Spectra of hydroxylated y metabolite of pthalazine after 30 minute incubation in guinea k H pig/ human cytosol D H k D OH D 90 H 1-2 H-Phthalazine 80 5:1 ratio D/H H O 70 H m/z 148 D H 60 O Intensity m/z 147 OH H m/z H D k H k D 2-2 H-Quinoxaline O H M+H amu k H /k D = m/z148 / m/z147 M+H = 147 amu M+H amu D k H /k D = m/z148 / m/z147 H O M+H = 147 amu Substrate H k / D k with cytosolic AO from Guinea pig Rat human 2-2 H-Quinoxaline H-Phthalazine

8 Inter-molecular KDIE on competitive first order elimination rate constants Substrate H k / D k with cytosolic AO from human rat guinea pig Quinoline Carbazeran Zoniporide

9 Inter-molecular KDIE on steady state kinetic constants v/s inolone l/min/ml 2-qu pmol Vmax 5: Quinoline [um] Quinoline 2-2 H-Quinoline K m (mm) V max (pmol/min) V max / K m KDIE on Cl int = 6.0

10 Conclusions from in-vitro KDIEs for AO- catalyzed reactions Across species the rate-limiting step in AO-catalyzed reactions is proton/hydride abstraction The KDIE for AO is fully expressed on the intrinsic clearance (V max /K m ) If systemic clearance of a drug is metabolically driven by AO, pharmacokinetics could be altered

11 Theoretical relationship between clearance (CL) and intrinsic clearance (CL int) ) for a KDIE of 7.0 CL = Q x CL int If CL int >> Q; CL ~ = Q Q + CL int Blood flow limit (Q) proto- deutero- Systemic half-life not expected to change for an IV or orally dosed drug. AUC and C max may reflect the KDIE on the extra-hepatic contributions to overall clearance CL If CL int << Q; CL = ~ Cl int Systemic half-life, AUC and C max may reflect the KDIE on the Cl int -0.3 CL int /Q

12 Pfizer drugs examined in-vitro and in-vivo Aldehyde Oxidase component to metabolism Monoamine Oxidase component to metabolism

13 Intrinsic clearance KDIE for Carbazeran Substrate Human Rat Guinea Pig Cytosol Hepatocytes Cytosol Hepatocytes Cytosol S-9 Carbazeran Metabolite profile for carbazeran 100 Intensity Glucuronide m/z Carbazeran m/z Hydroxy Carbazeran m/z 377 Human Hepatocytes Human S9 + cofactors Intensity Guinea Pig S9 + cofactors Inten nsity Rat Hepatocytes Rat S9 + cofactors Time (min)

14 Prediction of Carbazeran pharmacokinetic outcome from in-vitro assays In human: o PK enhancement In guinea pig and rat: o effect on systemic half-life (blood flow limited clearance ) Possible increases in Cmax and AUC due to KDIE on intrinsic i i clearance (extrahepatic AO contribution)

15 KDIE on PK parameters for Carbazeran (Guinea pigs) KDIE (D/H) AUC T1/2 Mean Std. Dev (Guinea pigs) KDIE (D/H) AUC T1/2 Cmax Mean Std. Dev IV-dosed Orally-dosed (Rats) KDIE (D/H) AUC T1/2 Mean Std. Dev (Rats) KDIE (D/H) AUC T1/2 Cmax Mean Std. Dev Despite a common metabolic pathway, the guinea pig and rat differ in the outcome of KDIEs on the pharmacokinetic parameters, suggesting a species difference in their systemic clearance mechanisms

16 Intrinsic clearance KDIE for Zoniporide Substrate Human Rat Guinea Pig Cytosol Hepatocytes Cytosol Hepatocytes Cytosol S-9 supplemented Zoniporide RT: Intensity uau Metabolite profile for Zoniporide in rat hepatocytes M1 M M2 M M M5 M7 M8 M M L: 5.07E8 TIC F: + c ESI Full ms [ ] MS data L: 1.57E5 nm= PDA data Time (min) zoniporide

17 (Guinea pig) KDIE (D/H) AUC T1/2 Mean Std. Dev (Guinea pig) KDIE (D/H) AUC T1/2 Cmax KDIE on PK parameters for Zoniporide IV-dosed Orally-dosed (Rat) KDIE (D/H) AUC T1/2 Mean Std. Dev (Rat) KDIE (D/H) AUC T1/2 Cmax Mean Mean Std. Dev Std. Dev As predicted from in-vitro hepatoctyes experiments, essentially no effect is observed on the pharmacokinetics of Zoniporide in guinea pig and rat

18 KDIEs on the steady-state kinetic parameters for oxidation of parasubstituted phenethylamines by Monoamine Oxidase -A proteo deutero isotope effects kcat Km kcat Km D kcat D (kcat/km) substituent (min-1) ( M) (min-1) ( M) D CL int H OH CF F Cl Br Me O Taken from: andigama and Edmondson, Biochemistry (2000), 39(49),

19 Proposed Mechanism of MAO-catalyzed deaminations

20 Properties and clinical results for CP P lasma Concent tration (ng/ml) of (1) (D 2 )H 2 C H CP O O 10 mg 30mg 100mg 300mg 1000mg Time (hr) Known attributes: MAO-A substrate Un-desirable clinical PK characterized by Clp/F> 200mL/min/kg; Plasma elimination t 1/2 : ~ 8.5 h Large variability in PK on-linear PK ast(ng*hr/m ml) (ng*h/ml L) AUC0-tl AUC Dose (mg)

21 Low contribution of MAO to CP clearance in the rat Intrinsic clearance KDIE for CP System H k/ D k Microsomes - ADPH 2.5 Microsomes + ADPH 1.1 Hepatocytes 1.1 Radiolabel Disposition of CP in Rat Metabolites Urine (U) Feces (F) U+F M1A 0.06 D 0.06 M M1B M M2A 0.08 D 0.08 M4 (MAOpathway) M (unchanged drug) Total Conclusion from in-vitro KDIE o effect on PK Solubility: > 5 mg/ml; Caco 2 : Papp AB : 1.2x 10-6 ; Papp: BA/AB: 7

22 IV and oral pharmacokinetic isotope effect for CP in the rat 1000 Mean 100 IV CP PF Oral CP PF Conc ( n g /m L ) 10 centration (ng/ml) Plasma Conc Time (hrs) Time (h) KDIE (IV) Rat# AUC T1/2 Mean Std. Dev

23 KDIE in human in vitro systems for CP System H k/ D k Human Microsomes - ADPH 4.3 Microsomes + ADPH 3.8 Hepatocytes 5.7 rmao-a A Large KDIE in human in vitro systems suggest possible PK enhacement The presence of other clearance routes (biliary/absorption) may not favor overall enhancement of pharmacokinetic parameters

24 Sandwich cultured hepatocyte billiary excretion model (1) in BD109 Rosuvastatin in BD pmol/mg protein Ca -Ca pmol/mg protein Ca+ Ca Time(min) i Time Uptake, app Cl b BEI (%) (pmol/min/ (ul/min/mg mg) protein) Rosuvastatin CP

25 Studies with PF-X KDIE in rat and human in vitro systems In Development 5X higher clearance at FIH than predicted MAO contributes tes to metabolism Known metabolic pathways System H k/ D k rmao-a 2.8 RLM + ADPH 1.09 Rat hepatocytes 1.06 Human hepatocytes 1.4 O X H R (D 2 )H 2 Ph H R (D 2 )H 2 CYP Ph O X O MAO-A OH O HO Ph O X Conclusion: o KDIE on the intrinsic clearance in rat hepatocytes and a small KDIE in human hepatocytes suggests deuteration will not enhance pharmacokinetics in either rat or humans

26 Rat IV and oral PK profiles for PF-X Conc (ng/ml) PF IV PF Time (hrs) Plasma Con nc. of PF (ng/ml L) Mean 1000 Oral PF PF Time (hrs) Essentially no KDIE on IV or oral PK parameters for PF-X

27 Considerations for deuteration as a PK enhancement strategy The identity of enzymes involved in the metabolic clearance Knowledge of their reaction mechanisms The extent of their contribution to the overall metabolic clearance Magnitude of the intrinsic i i clearance isotope effect tin hepatocytes (or equivalent) for multiple species Knowledge of other non-metabolic clearance mechanisms and the extent of their contribution to systemic clearance