Aberra Fura

Role of pharmacologically active metabolites in drug discovery and development

Pharmacologically active metabolites can contribute significantly to the overall therapeutic and adverse effects of drugs. Therefore, to fully understand the mechanism of action of drugs, it is important to recognize the role of active metabolites. Active metabolites can also be developed as drugs in their own right. Using illustrative examples, this paper discusses a variety of biotransformation reactions that produce active metabolites and their structure-activity relationships. The paper also describes the role and significance of active metabolites in drug discovery and development, various experimental observations that can be used as indicators of their presence, and methods that can be used to assess their biological activities and contribution to the overall therapeutic and adverse effects of drugs.

Shift in pH of biological fluids during storage and processing: effect on bioanalysis.

The pH of ex vivo plasma, bile and urine was monitored at different times and temperatures of storage, and following different sample processing methods such as ultrafiltration, centrifugation, precipitation and evaporation. The results showed that the pH of ex vivo plasma, bile and urine increased upon storage, and following sample processing and could lead to significant degradation of pH-labile compounds. Several compounds were used to illustrate the impact of pH shifts on drug stability and interpretation of results obtained from in vivo studies. For example, after 1 h of incubation (37 degrees C) in rat plasma (pH 8.3), about 60%, of I was lost. However, in phosphate buffer, losses were about 12% at pH 7.4 and 40% at pH 8.0. Plasma pH also increased during ultrafiltration, centrifugation and extraction. After methanol precipitation of plasma proteins, and evaporation of the supernatant and redissolution of the residue, the resulting solution had a pH of 9.5. Under these conditions, II was degraded by 60% but was stable when phosphate buffer was used to maintain the pH at 7.4. The shift in plasma pH can yield misleading results from in vivo studies if the pH is not controlled. For example, the major circulating metabolite of II was also formed in plasma ex-vivo. This ex vivo degradation was prevented when blood samples were collected into tubes containing 0.1 volume of phosphate buffer (0.3 M, pH 5). The pH of ex vivo plasma can best be stabilized at physiological conditions using 10% CO2 atmosphere in a CO2 incubator. Changes in pH of ex vivo urine and bile samples can have similar adverse effect on pH-labile samples. Thus, processing of plasma samples under a 10% CO2 atmosphere is a method of choice for stability or protein binding studies in plasma, whereas citrate or phosphate buffers are suitable for stabilizing pH in bile and urine and for plasma samples requiring extensive preparations or long term storage.

Pharmacokinetics of the Dipeptidyl Peptidase 4 Inhibitor Saxagliptin in Rats, Dogs, and Monkeys and Clinical Projections

Saxagliptin is a potent, selective, reversible dipeptidyl peptidase 4 (DPP4) inhibitor specifically designed for extended inhibition of the DPP4 enzyme and is currently under development for the treatment of type-2 diabetes. The pharmacokinetics of saxagliptin were evaluated in rats, dogs, and monkeys and used to predict its human pharmacokinetics. Saxagliptin was rapidly absorbed and had good bioavailability (50–75%) in the species tested. The plasma clearance of saxagliptin was higher in rats (115 ml/min/kg) than in dogs (9.3 ml/min/kg) and monkeys (14.5 ml/min/kg) and was predicted to be low to moderate in humans. The plasma elimination half-life was between 2.1 and 4.4 h in rats, dogs, and monkeys, and both metabolism and renal excretion contributed to the overall elimination. The primary metabolic clearance pathway involved the formation of a significant circulating, pharmacologically active hydroxylated metabolite, M2. The volume of distribution values observed in rats, dogs, and monkeys (1.3–5.2 l/kg) and predicted for humans (2.7 l/kg) were greater than those for total body water, indicating extravascular distribution. The in vitro serum protein binding was low (≤30%) in rats, dogs, monkeys, and humans. After intra-arterial administration of saxagliptin to Sprague-Dawley and Zucker diabetic fatty rats, higher levels of saxagliptin and M2 were observed in the intestine (a proposed major site of drug action) relative to that in plasma. Saxagliptin has prolonged pharmacodynamic properties relative to its plasma pharmacokinetic profile, presumably due to additional contributions from M2, distribution of saxagliptin and M2 to the intestinal tissue, and prolonged dissociation of both saxagliptin and M2 from DPP4.

Hepatitis C virus NS5A replication complex inhibitors: the discovery of daclatasvir.

The biphenyl derivatives 2 and 3 are prototypes of a novel class of NS5A replication complex inhibitors that demonstrate high inhibitory potency toward a panel of clinically relevant HCV strains encompassing genotypes 1-6. However, these compounds exhibit poor systemic exposure in rat pharmacokinetic studies after oral dosing. The structure-activity relationship investigations that improved the exposure properties of the parent bis-phenylimidazole chemotype, culminating in the identification of the highly potent NS5A replication complex inhibitor daclatasvir (33) are described. An element critical to success was the realization that the arylglycine cap of 2 could be replaced with an alkylglycine derivative and still maintain the high inhibitory potency of the series if accompanied with a stereoinversion, a finding that enabled a rapid optimization of exposure properties. Compound 33 had EC50 values of 50 and 9 pM toward genotype-1a and -1b replicons, respectively, and oral bioavailabilities of 38-108% in preclinical species. Compound 33 provided clinical proof-of-concept for the NS5A replication complex inhibitor class, and regulatory approval to market it with the NS3/4A protease inhibitor asunaprevir for the treatment of HCV genotype-1b infection has recently been sought in Japan.

Synthesis and SAR of azolopyrimidines as potent and selective dipeptidyl peptidase-4 (DPP4) inhibitors for type 2 diabetes.

Several pyrazolo-, triazolo-, and imidazolopyrimidines were synthesized and evaluated as inhibitors of DPP4. Of these three classes of compounds, the imidazolopyrimidines displayed the greatest potency and demonstrated excellent selectivity over the other dipeptidyl peptidases. SAR evaluation for these scaffolds was described as they may represent potential treatments for type 2 diabetes.

Discovery of 6-(Aminomethyl)-5-(2,4-dichlorophenyl)-7-methylimidazo[1,2-a]pyrimidine-2-carboxamides as Potent, Selective Dipeptidyl Peptidase-4 (DPP4) Inhibitors.

Continued structure-activity relationship (SAR) exploration within our previously disclosed azolopyrimidine containing dipeptidyl peptidase-4 (DPP4) inhibitors led us to focus on an imidazolopyrimidine series in particular. Further study revealed that by replacing the aryl substitution on the imidazole ring with a more polar carboxylic ester or amide, these compounds displayed not only increased DPP4 binding activity but also significantly reduced human ether-a-go-go related gene (hERG) and sodium channel inhibitory activities. Additional incremental adjustment of polarity led to permeable molecules which exhibited favorable pharmacokinetic (PK) profiles in preclinical animal species. The active site binding mode of these compounds was determined by X-ray crystallography as exemplified by amide 24c. A subsequent lead molecule from this series, (+)-6-(aminomethyl)-5-(2,4-dichlorophenyl)-N-(1-ethyl-1H-pyrazol-5-yl)-7-methylimidazo[1,2-a]pyrimidine-2-carboxamide (24s), emerged as a potent, selective DPP4 inhibitor that displayed excellent PK profiles and in vivo efficacy in ob/ob mice.

Identification and optimization of a novel series of [2.2.1]-oxabicyclo imide-based androgen receptor antagonists

A novel series of [2.2.1]-oxabicyclo imide-based compounds were identified as potent antagonists of the androgen receptor. Molecular modeling and iterative drug design were applied to optimize this series. The lead compound [3aS-(3aalpha,4beta,5beta,7beta,7aalpha)]-4-(octahydro-5-hydroxy-4,7-dimethyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl)-2-iodobenzonitrile was shown to have potent in vivo efficacy after oral dosing in the CWR22 human prostate tumor xenograph model.

N-Aryl-oxazolidin-2-imine Muscle Selective Androgen Receptor Modulators Enhance Potency through Pharmacophore Reorientation

A novel selective androgen receptor modulator (SARM) scaffold was discovered as a byproduct obtained during synthesis of our earlier series of imidazolidin-2-ones. The resulting oxazolidin-2-imines are among the most potent SARMs known, with many analogues exhibiting sub-nM in vitro potency in binding and functional assays. Despite the potential for hydrolytic instability at gut pH, compounds of the present class showed good oral bioavailability and were highly active in a standard rodent pharmacological model.

Prediction of human oral pharmacokinetics using nonclinical data: examples involving four proprietary compounds.

The oral pharmacokinetics (concentration‐time profile) of four proprietary compounds in humans were predicted using the Cvss‐MRT method. The first step was to demonstrate superposition of intravenous (i.v.) pharmacokinetic profiles of preclinical species following mathematical transformation of their respective concentration‐time curves using the corresponding Cvss (where Cvss=dose/Vss; Vss is the volume of distribution at steady state) and mean residence time (MRT) values. The resultant profiles were then back‐transformed to estimate human i.v. plasma concentration‐time profiles using human Cvss and MRT values. Human Cvss and MRT values were estimated from projected human Vss and CL values. Projection of CL was based on scaled (in vitro) metabolic clearance, simple allometry with and without various correction factors and the unbound fraction corrected intercept method. Vss values were estimated by allometric scaling with and without correction for interspecies differences in plasma protein binding. The predicted human i.v. profiles, in combination with the estimated mean absorption rate constants and bioavailability, were then used to simulate the oral pharmacokinetics in human using one‐ or multi‐compartment kinetic models. Overall, with this approach, key oral pharmacokinetic parameters such as AUC, Cmax, Cmin and oral plasma T½ were projected to be within two‐fold of the actual values in humans. Copyright

Optimization of Activity, Selectivity, and Liability Profiles in 5-Oxopyrrolopyridine DPP4 Inhibitors Leading to Clinical Candidate (Sa)-2-(3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-5-oxo-5H-pyrrolo[3,4-b]pyridin-6(7H)-yl)-N,N-dimethylacetamide (BMS-767778)

Optimization of a 5-oxopyrrolopyridine series based upon structure-activity relationships (SARs) developed from our previous efforts on a number of related bicyclic series yielded compound 2s (BMS-767778) with an overall activity, selectivity, efficacy, PK, and developability profile suitable for progression into the clinic. SAR in the series and characterization of 2s are described.