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,
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
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
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), 133 102-4.
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 P450 1 9 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
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
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 148.10 100 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 50 40 30 20 10 0 m/z 147 OH H 147.10 149.10 140.15 141.16 150.14 152.18 157.18 159.10 161.14 140 142 144 146 148 150 152 154 156 158 160 m/z H D k H k D 2-2 H-Quinoxaline O H M+H - 148 amu k H /k D = m/z148 / m/z147 M+H = 147 amu M+H - 148 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 4.7 5.1 5.0 2-2 H-Phthalazine 4.9 5.0 5.1
Inter-molecular KDIE on competitive first order elimination rate constants Substrate H k / D k with cytosolic AO from human rat guinea pig Quinoline 5.5 6.1 6.0 Carbazeran 4.8 5.0 6.0 Zoniporide 5.8 3.6 4.8
Inter-molecular KDIE on steady state kinetic constants v/s 250 200 inolone l/min/ml 2-qu pmol 150 100 50 0 Vmax 5:1 0 200 400 600 800 1000 1200 Quinoline [um] Quinoline 2-2 H-Quinoline K m (mm) 212 193 V max (pmol/min) 246 47 V max / K m 1.2 0.2 KDIE on Cl int = 6.0
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
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 1.2 07 0.7 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 0.2 0 10 20 Systemic half-life, AUC and C max may reflect the KDIE on the Cl int -0.3 CL int /Q
Pfizer drugs examined in-vitro and in-vivo Aldehyde Oxidase component to metabolism Monoamine Oxidase component to metabolism
Intrinsic clearance KDIE for Carbazeran Substrate Human Rat Guinea Pig Cytosol Hepatocytes Cytosol Hepatocytes Cytosol S-9 Carbazeran 4.8 1.5 5.0 4.6 6.0 5.0 Metabolite profile for carbazeran 100 Intensity 80 60 40 13.12 Glucuronide m/z 537 15.23 Carbazeran m/z 361 19.54 Hydroxy Carbazeran m/z 377 Human Hepatocytes Human S9 + cofactors 20 0 100 80 Intensity 60 40 12.27 13.71 15.76 17.38 18.80 21.30 22.51 24.08 25.06 19.53 15.23 Guinea Pig S9 + cofactors 20 0 100 Inten nsity 80 60 40 20 0 13.11 13.54 15.97 16.83 20.11 23.13 23.98 15.13 Rat Hepatocytes Rat S9 + cofactors 19.57 12.96 14.40 16.39 17.34 24.82 12 14 16 18 20 22 24 Time (min)
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)
KDIE on PK parameters for Carbazeran (Guinea pigs) KDIE (D/H) AUC T1/2 Mean 46 4.6 08 0.8 Std. Dev 0.7 0.1 (Guinea pigs) KDIE (D/H) AUC T1/2 Cmax Mean 21.9 0.5 22.5 Std. Dev 2.6 0.1 7.7 IV-dosed Orally-dosed (Rats) KDIE (D/H) AUC T1/2 Mean 20 2.0 11 1.1 Std. Dev 0.5 0.2 (Rats) KDIE (D/H) AUC T1/2 Cmax Mean 2.3 1.27 1.5 Std. Dev. 0.2 0.1 0.0 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
Intrinsic clearance KDIE for Zoniporide Substrate Human Rat Guinea Pig Cytosol Hepatocytes Cytosol Hepatocytes Cytosol S-9 supplemented Zoniporide 5.8 1.9 3.6 2.7 4.8 1.5 RT: 6.0-21.0 Intensity uau Metabolite profile for Zoniporide in rat hepatocytes 500000000 400000000 300000000 200000000 100000000 0 140000 120000 100000 80000 60000 40000 20000 0 13.2 M1 M10 14.44 M2 M3 15.6 16.0 16.3 M6 16.2 M5 M7 M8 M9 14.3 9.3 13.1 M4 14.6 15.9 17.1 18.8 L: 5.07E8 TIC F: + c ESI Full ms [50.00-800.00] MS data02 17.1 19.5 17.6 20.2 18.7 L: 1.57E5 nm=220.0-400.0 PDA data02 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Time (min) zoniporide
(Guinea pig) KDIE (D/H) AUC T1/2 Mean 11 1.1 12 1.2 Std. Dev 0.5 0.3 (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 09 0.9 14 1.4 Std. Dev 0.1 0.3 (Rat) KDIE (D/H) AUC T1/2 Cmax Mean 1.13 1.2 0.9 Mean 0.6 1.12 0.7 Std. Dev 0.1 0.2 0.1 Std. Dev 0.2 0.0 0.1 As predicted from in-vitro hepatoctyes experiments, essentially no effect is observed on the pharmacokinetics of Zoniporide in guinea pig and rat
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 64 1250 7.55 1410 8.5 9.6 OH 83.7 423 35.8 633 2.3 3.5 CF3 4.5 755 0.71 1190 6.3 9.9 F 111.5 1060 21.1 2050 5.3 10.2 Cl 26.6 320 3.45 380 7.7 9.2 Br 17.1 226 2.06 260 8.3 9.6 Me 18.6 150 2.05 152 9.1 9.2 O2 200 4820 38.3 8270 5.2 9.0 Taken from: andigama and Edmondson, Biochemistry (2000), 39(49), 15258-15265
Proposed Mechanism of MAO-catalyzed deaminations
Properties and clinical results for CP409092 P lasma Concent tration (ng/ml) of (1) (D 2 )H 2 C H CP-409092 O 1000 100 10 1 O 10 mg 30mg 100mg 300mg 1000mg 0.1 0 10 20 30 40 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 1000 800 600 400 200 0 10 100 1000 Dose (mg)
Low contribution of MAO to CP-409092 clearance in the rat Intrinsic clearance KDIE for CP409092 System H k/ D k Microsomes - ADPH 2.5 Microsomes + ADPH 1.1 Hepatocytes 1.1 Radiolabel Disposition of CP-409092 in Rat Metabolites Urine (U) Feces (F) U+F M1A 0.06 D 0.06 M1 0.74 6.47 7.21 M1B 0.04 0.62 0.66 M2 0.88 3.5 4.39 M2A 0.08 D 0.08 M4 (MAOpathway) 0.62 3.24 3.86 M5 0.02 0.22 0.24 (unchanged drug) 1.01 72.4 73.41 Total 3.45 66.5 89.9 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
IV and oral pharmacokinetic isotope effect for CP-409092in the rat 1000 Mean 100 IV CP-409092 PF-05136259 100 Oral CP-409092 PF-05136259 Conc ( n g /m L ) 10 centration (ng/ml) Plasma Conc 10 1 0 2 4 6 8 10 12 1 0 1 2 3 4 5 6 7 Time (hrs) Time (h) KDIE (IV) Rat# AUC T1/2 Mean 1.2 1.2 Std. Dev. 0.1 0.1
KDIE in human in vitro systems for CP409092 System H k/ D k Human Microsomes - ADPH 4.3 Microsomes + ADPH 3.8 Hepatocytes 5.7 rmao-a A 38 3.8 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
Sandwich cultured hepatocyte billiary excretion model (1) in BD109 Rosuvastatin in BD109 30 140 25 120 pmol/mg protein 20 15 10 +Ca -Ca pmol/mg protein 100 80 60 40 Ca+ Ca- 5 20 0 0 5 10 15 Time(min) i 0 0 5 10 15 Time Uptake, app Cl b BEI (%) (pmol/min/ (ul/min/mg mg) protein) Rosuvastatin 1 7.7 2.7 34 CP-409092 1.5 0.77 30
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
Rat IV and oral PK profiles for PF-X Conc (ng/ml) 10000 PF-04455242 IV PF-05094041 1000 100 10 1 0.1 0 4 8 12 16 20 24 Time (hrs) Plasma Con nc. of PF-4455242 (ng/ml L) Mean 1000 Oral PF-04455242 PF-04094041 100 10 1 0 4 8 12 Time (hrs) Essentially no KDIE on IV or oral PK parameters for PF-X
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