Timothy W. Harper

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.

Reaction Phenotyping: Current Industry Efforts to Identify Enzymes Responsible for Metabolizing Drug Candidates

Reaction phenotyping studies to identify specific enzymes involved in the metabolism of drug candidates are increasingly important in drug discovery efforts. Experimental approaches used for CYP reaction phenotyping include incubations with cDNA expressed CYP enzyme systems and incubations containing specific CYP enzyme inhibitors. Since both types of experiments present specific advantages as well as known drawbacks, these studies are generally viewed as complementary approaches. Although glucuronidation pathways are also known to present potential drug–drug interaction issues as well as challenges related to their polymorphic expression, reaction phenotyping approaches for glucuronidation are generally limited to cDNA expressed systems due to lack of availability of specific UGT inhibitors. This article presents a limited review of current approaches to reaction phenotyping studies used within the pharmaceutical industry.

In Silico Prediction of Biliary Excretion of Drugs in Rats Based on Physicochemical Properties

Evaluating biliary excretion, a major elimination pathway for many compounds, is important in drug discovery. The bile duct-cannulated (BDC) rat model is commonly used to determine the percentage of dose excreted as intact parent into bile. However, a study using BDC rats is time-consuming and cost-ineffective. The present report describes a computational model that has been established to predict biliary excretion of intact parent in rats as a percentage of dose. The model was based on biliary excretion data of 50 Bristol-Myers Squibb Co. compounds with diverse chemical structures. The compounds were given intravenously at <10 mg/kg to BDC rats, and bile was collected for at least 8 h after dosing. Recoveries of intact parents in bile were determined by liquid chromatography with tandem mass spectrometry. Biliary excretion was found to have a fairly good correlation with polar surface area (r = 0.76) and with free energy of aqueous solvation (ΔGsolv aq) (r = −0.67). In addition, biliary excretion was also highly corrected with the presence of a carboxylic acid moiety in the test compounds (r = 0.87). An equation to calculate biliary excretion in rats was then established based on physiochemical properties via a multiple linear regression. This model successfully predicted rat biliary excretion for 50 BMS compounds (r = 0.94) and for 25 previously reported compounds (r = 0.86) whose structures are markedly different from those of the 50 BMS compounds. Additional calculations were conducted to verify the reliability of this computation model.

Pyrazole-based sulfonamide and sulfamides as potent inhibitors of mammalian 15-lipoxygenase.

A series of inhibitors of mammalian 15-lipoxygenase (15-LO) based on a 3,4,5-tri-substituted pyrazole scaffold is described. Replacement of a sulfonamide functionality in the lead series with a sulfamide group resulted in improved physicochemical properties generating analogs with enhanced inhibition in cell-based and whole blood assays.

Beta 3 agonists. Part 1: evolution from inception to BMS-194449.

Screening of the BMS collection identified 4-hydroxy-3-methylsulfonanilidoethanolamines as full beta 3 agonists. Substitution of the ethanolamine nitrogen with a benzyl group bearing a para hydrogen bond acceptor promoted beta(3) selectivity. SAR elucidation established that highly selective beta(3) agonists were generated upon substitution of C(alpha) with either benzyl to form (R)-1,2-diarylethylamines or with aryl to generate 1,1-diarylmethylamines. This latter subset yielded a clinical candidate, BMS-194449 (35).(1)

BMS-196085: A potent and selective full agonist of the human β3 adrenergic receptor

A series of 4-hydroxy-3-methylsulfonanilido-1,2-diarylethylamines were prepared and evaluated for their human beta(3) adrenergic receptor agonist activity. SAR studies led to the identification of BMS-196085 (25), a potent beta(3) full agonist (K(i)=21 nM, 95% activation) with partial agonist (45%) activity at the beta(1) receptor. Based on its desirable in vitro and in vivo properties, BMS-196085 was chosen for clinical evaluation.

Optimization of 1,2,4-Triazolopyridines as Inhibitors of Human 11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD-1).

Small alkyl groups and spirocyclic-aromatic rings directly attached to the left side and right side of the 1,2,4-triazolopyridines (TZP), respectively, were found to be potent and selective inhibitors of human 11β-hydroxysteroid dehydrogenase-type 1 (11β-HSD-1) enzyme. 3-(1-(4-Chlorophenyl)cyclopropyl)-8-cyclopropyl-[1,2,4]triazolo[4,3-a]pyridine (9f) was identified as a potent inhibitor of the 11β-HSD-1 enzyme with reduced Pregnane-X receptor (PXR) transactivation activity. The binding orientation of this TZP series was revealed by X-ray crystallography structure studies.

Discovery of 3-hydroxy-4-cyano-isoquinolines as novel, potent, and selective inhibitors of human 11β-hydroxydehydrogenase 1 (11β-HSD1).

Derived from the HTS hit 1, a series of hydroxyisoquinolines was discovered as potent and selective 11β-HSD1 inhibitors with good cross species activity. Optimization of substituents at the 1 and 4 positions of the isoquinoline group in addition to the core modifications, with a special focus on enhancing metabolic stability and aqueous solubility, resulted in the identification of several compounds as potent advanced leads.

Synthesis and structure–activity relationship of 2-adamantylmethyl tetrazoles as potent and selective inhibitors of human 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1)

A series of 2-adamantylmethyl tetrazoles bearing a quaternary carbon at the 2-position of the adamantane ring (i.e. structure A) have been designed and synthesized as novel, potent, and selective inhibitors of human 11β-HSD1 enzyme. Based on the SAR and the docking experiment, we report for the first time a tetrazole moiety serving as the active pharmacophore for inhibitory activity of 11β-HSD1 enzyme. Optimization of two regions of A, R(1) and R(2) respectively, was explored with a focus on improving the inhibitory activity (IC50) and the microsomal stability in both human and mouse species. These efforts led to the identification of 26, an orally bioavailable inhibitor of human 11β-HSD1 with a favorable development profile.

Design, synthesis, and SAR studies of novel polycyclic acids as potent and selective inhibitors of human 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1).

Starting from high throughput screening hit 2-adamantyl acetic acid 3, a series of polycyclic acids have been designed and synthesized as novel, potent, and selective inhibitors of human 11β-HSD-1. Structure-activity relationships of two different regions of the chemotype (polycyclic ring and substituents on quaternary carbon) are discussed.