Biotransformation: Impact and Application of Metabolism in Drug Discovery.

Biotransformation has a huge impact on the efficacy and safety of drugs. Ultimately the effects of metabolism can be the lynchpin in the discovery and development cycle of a new drug. This article discusses the impact and application of biotransformation of drugs by mammalian systems, microorganisms, and recombinant enzymes, covering active and reactive metabolites, the impact of the gut microbiome on metabolism, and how insights gained from biotransformation studies can influence drug design from the combined perspectives of a CRO specializing in a range of biotransformation techniques and pharma biotransformation scientists. We include a commentary on how biology-driven approaches can complement medicinal chemistry strategies in drug optimization and the in vitro and surrogate systems available to explore and exploit biotransformation.

Generation, clearance, toxicity, and monitoring possibilities of unaccounted uremic toxins for improved dialysis prescriptions.

Current dialysis-dosing calculations provide an incomplete assessment of blood purification. They exclude clearances of protein-bound uremic toxins (PB-UTs), such as polyamines, p-cresol sulfate, and indoxyl sulfate, relying solely on the clearance of urea as a surrogate for all molecules accumulating in patients with end-stage renal disease (ESRD). PB-UTs clear differently in dialysis but also during normal renal function. The kidney clears PB toxins via the process of secretion, whereas it clears urea through filtration. Herein, we review the clearance, accumulation, and toxicity of various UTs. We also suggest possible methods for their monitoring toward the ultimate goal of a more comprehensive dialysis prescription. A more inclusive dialysis prescription would retain the kidney-filtration surrogate, urea, and consider at least one PB toxin as a surrogate for UTs cleared through cellular secretion. A more comprehensive assessment of UTs that includes both secretion and filtration is expected to result in a better understanding of ESRD toxicity and consequently, to reduce ESRD mortality.

Assessment of the mass balance recovery and metabolite profile of avibactam in humans and in vitro drug-drug interaction potential.

Avibactam, a novel non-ß-lactam ß-lactamase inhibitor with activity against Ambler class A, class C, and some class D enzymes is being evaluated in combination with various ß-lactam antibiotics to treat serious bacterial infections. The in vivo mass balance recovery and metabolite profile of [(14)C] avibactam (500 mg/1-h infusion) was assessed in six healthy male subjects, and a series of in vitro experiments evaluated the metabolism and drug-drug interaction potential of avibactam. In the mass balance study, measurement of plasma avibactam (using a validated liquid chromatography-tandem mass spectrometry method) and total radioactivity in plasma, whole blood, urine, and feces (using liquid scintillation counting) indicated that most of the avibactam was excreted unchanged in urine within 12 hours, with recovery complete (>97% of the administered dose) within 96 hours. Geometric mean avibactam renal clearance (158 ml/min) was greater than the product of unbound fraction of drug and glomerular filtration rate (109.5 ml/min), suggesting that active tubular secretion accounted for some renal elimination. There was no evidence of metabolism in plasma and urine, with unchanged avibactam the major component in both matrices. Avibactam demonstrated in vitro substrate potential for organic anion transporters 1 and 3 (OAT1 and OAT3) proteins expressed in human embryonic kidney 293 cells (Km > 1000 µM; >10-fold the Cmax of a therapeutic dose), which could account for the active tubular secretion observed in vivo. Avibactam uptake by OAT1 and OAT3 was inhibited by probenecid, a potent OAT1/OAT3 inhibitor. Avibactam did not interact with various other membrane transport proteins or cytochrome P450 enzymes in vitro, suggesting it has limited propensity for drug-drug interactions involving cytochrome P450 enzymes.

Alternate strategies to obtain mass balance without the use of radiolabeled compounds: application of quantitative fluorine (19F) nuclear magnetic resonance (NMR) spectroscopy in metabolism studies.

Nuclear magnetic resonance (NMR) spectroscopy is playing an increasingly important role in the quantitation of small and large molecules. Recently, we demonstrated that (1)H NMR could be used to quantitate drug metabolites isolated in submilligram quantities from biological sources. It was shown that these metabolites, once quantitated by NMR, were suitable to be used as reference standards in quantitative LC/MS-based assays, hence circumventing the need for radiolabeled material or synthetic standards to obtain plasma exposure estimates in humans and preclinical species. The quantitative capabilities of high-field NMR is further demonstrated in the current study by obtaining the mass balance of fluorinated compounds using (19)F-NMR. Two fluorinated compounds which were radio-labeled with carbon-14 on metabolically stable positions were dosed in rats and urine and feces collected. The mass balance of the compounds was obtained initially by counting the radioactivity present in each sample. Subsequently, the same sets of samples were analyzed by (19)F-NMR, and the concentrations determined by this method were compared with data obtained using radioactivity counting. It was shown that the two methods produced comparable values. To demonstrate the value of this analytical technique in drug discovery, a fluorinated compound was dosed intravenously in dogs and feces and urine collected. Initial profiling of samples showed that this compound was excreted mainly unchanged in feces, and hence, an estimate of mass balance was obtained using (19)F-NMR. The data obtained by this method was confirmed by additional quantitative studies using mass spectrometry. Hence cross-validations of the quantitative (19)F-NMR method by radioactivity counting and mass spectrometric analysis were demonstrated in this study. A strategy outlining the use of fluorinated compounds in conjunction with (19)F-NMR to understand their routes of excretion or mass balance in animals is proposed. These studies demonstrate that quantitative (19)F-NMR could be used as an alternate technique to obtain an estimate of the mass balance of fluorinated compounds, especially in early drug development where attrition of the compounds is high, and cost savings could be realized through the use of such a technique rather than employing radioactive compounds. The potential application of qNMR in conducting early human ADME studies with fluorinated compounds is also discussed.

Optimization of 5-vinylaryl-3-pyridinecarbonitriles as PKCtheta inhibitors.

Analog 8, a 3-pyridinecarbonitrile with an (E)-2-[6-[(4-methylpiperazin-1-yl)methyl]pyridin-2-yl]vinyl group at C-5, had an IC(50) value of 1.1 nM for the inhibition of PKCtheta and potently blocked the production of IL-2 in both stimulated murine T cells (IC(50)=34 nM) and human whole blood (IC(50)=500 nM).

ICI 182,780 penetrates brain and hypothalamic tissue and has functional effects in the brain after systemic dosing.

Previous reports suggest the antiestrogen ICI 182,780 (ICI) does not cross the blood-brain barrier (BBB). However, this hypothesis has never been directly tested. In the present study, we tested whether ICI crosses the BBB, penetrates into brain and hypothalamic tissues, and affects known neuroendocrine functions in ovariectomized rats. Using HPLC with mass spectrometry, ICI (1.0 mg/kg.d, 3 d) was detected in plasma and brain and hypothalamic tissues for up to 24 h with maximum concentrations of 43.1 ng/ml, and 31.6 and 38.8 ng/g, respectively. To evaluate antiestrogenic effects of ICI in the brain after systemic dosing, we tested its ability to block the effect of 17 alpha-ethinyl estradiol (EE) (0.3 mg/kg, 8 d) on tail-skin temperature abatement in the morphine-dependent model of hot flush and on body weight change. In the morphine-dependent model, EE abated 64% of the naloxone-induced tail-skin temperature increase. ICI pretreatment (1.0, 3.0 mg/kg.d) dose dependently inhibited this effect. ICI (3.0 mg/kg.d) alone showed estrogenic-like actions, abating 30% the naloxone-induced flush. In body weight studies, EE-treated rats weighed 58.5 g less than vehicle-treated rats after 8 d dosing. This effect was partially blocked by ICI (3.0 mg/kg.d) pretreatment. Similar to EE treatment, rats receiving 1.0 or 3.0 mg/kg.d ICI alone showed little weight gain compared with vehicle-treated controls. Thus, ICI crosses the BBB, penetrates into brain and hypothalamic tissues, and has both antiestrogenic and estrogenic-like actions on neuroendocrine-related functions.

P2Y1 receptor antagonists as novel antithrombotic agents.

The P2Y(1) and P2Y(12) purinergic receptors are responsible for mediating adenosine diphosphate (ADP) dependent platelet aggregation. Evidence from P2Y(1) knockout studies as well as from nucleotide-based small molecule P2Y(1) antagonists has suggested that the antagonism of this receptor may offer a novel and effective method for the treatment of thrombotic disorders. Herein, we report the identification and optimization of a series of non-nucleotide P2Y(1) antagonists that are potent and orally bioavailable.

A rational chemical intervention strategy to circumvent bioactivation liabilities associated with a nonpeptidyl thrombopoietin receptor agonist containing a 2-amino-4-arylthiazole motif.

The current study examined the bioactivation potential of a nonpeptidyl thrombopoietin receptor agonist, 1-(3-chloro-5-((4-(4-fluoro-3-(trifluoromethyl)phenyl)thiazol-2-yl)carbamoyl)pyridine-2-yl)piperidine-4-carboxylic acid (1), containing a 2-carboxamido-4-arylthiazole moiety in the core structure. Toxicological risks arising from P450-catalyzed C4-C5 thiazole ring opening in 1 via the epoxidation-->diol sequence were alleviated, since mass spectrometric analysis of human liver microsome and/or hepatocyte incubations of 1 did not reveal the formation of reactive acylthiourea and/or glyoxal metabolites, which are prototypic products derived from thiazole ring scission. However, 4-(4-fluoro-3-(trifluoromethyl)phenyl)thiazol-2-amine (2), the product of hydrolysis of 1 in human liver microsomes, hepatocytes, and plasma, underwent oxidative bioactivation in human liver microsomes, since trapping studies with glutathione led to the formation of two conjugates derived from the addition of the thiol nucleophile to 2 and a thiazole- S-oxide metabolite of 2. Mass spectral fragmentation and NMR analysis indicated that the site of attachment of the glutathionyl moiety in both conjugates was the C5 position in the thiazole ring. Based on the structures of the glutathione conjugates, two bioactivation pathways are proposed, one involving beta-elimination of an initially formed hydroxylamine metabolite and the other involving direct two-electron oxidation of the electron-rich 2-aminothiazole system to electrophilic intermediates. This mechanistic insight into the bioactivation process allowed the development of a rational chemical intervention strategy that involved blocking the C5 position with a fluorine atom or replacing the thiazole ring with a 1,2,4-thiadiazole group. These structural changes not only abrogated the bioactivation liability associated with 1 but also resulted in compounds that retained the attractive pharmacological and pharmacokinetic attributes of the prototype agent.

Determination of degradation pathways and kinetics of acyl glucuronides by NMR spectroscopy.

Acyl glucuronides have been implicated in the toxicity of many xenobiotics and marketed drugs. These toxicities are hypothesized to be a consequence of covalent binding of the reactive forms of the acyl glucuronide to proteins. Reactive intermediates of the acyl glucuronide arise from the migration of the aglycone leading to other positional and stereoisomers under physiological conditions. In order to screen for the potential liabilities of these metabolites during the early phase of pharmaceutical development, an NMR method based on the disappearance of the anomeric resonance of the O-1-acyl glucuronide was used to monitor the degradation kinetics of 11 structurally diverse acyl glucuronides, including those produced from the known nonsteroidal anti-inflammatory drugs (NSAIDs). The acyl glucuronides were either chemically synthesized or were isolated from biological matrices (bile, urine, and liver microsomal extracts). The half-lives attained utilizing this method were found to be comparable to those reported in the literature. NMR analysis also enabled the delineation of the two possible pathways of degradation: acyl migration and hydrolytic cleavage. The previously characterized 1H resonances of acyl migrated products are quite distinguishable from those that arise from hydrolysis. The NMR method described here could be used to rank order acyl glucuronide forming discovery compounds based on the potential reactivity of the conjugates and their routes of decomposition under physiological conditions. Furthermore, we have shown that in vitro systems such as liver microsomal preparations can be used to generate sufficient quantities of acyl glucuronides from early discovery compounds for NMR characterization. This is particularly important, as we often have limited supply of early discovery compounds to conduct in vivo studies to generate sufficient quantities of acyl glucuronides for further characterization.

Application of in-line liquid chromatography-accurate radioisotope counting-mass spectrometry (LC-ARC-MS) to evaluate metabolic profile of [3H]-mefenamic acid in rat plasma.

Profiling of rat plasma using a highly sensitive LC-ARC-MS technique showed that [(3)H] mefenamic acid was metabolized to several products, including a sulfate conjugate and a hydroxylated analogue as major metabolites. This technique of detecting low levels of radioactivity in plasma was superior to previously used methods, such as beta-RAM detectors.

Remote forest refugia for Fijian wildlife.

On Pacific islands non-native rats and mongooses threaten many native species. In Fiji we compared visitation rates of rats and mongooses at bait stations and measured biomass of leaf-litter invertebrates to assess the relative predation pressure from these species in forest areas at different distances from the forest edge. Forest areas over 5 km from the forest edge had significantly fewer baits encountered by rats or mongooses than did natural forest areas nearer agricultural and urban habitats. Remote forest areas may function as a last refuge for island species threatened by predation from non-native rats and mongooses. The biomass of leaf-litter invertebrates in remote forest areas was higher indicating a refuge effect for some taxa targeted by rats and mongooses. Protection of the few remaining large blocks of natural forests on Pacific islands may be the most cost-effective approach for conserving many island endemics threatened by rats and mongooses. Logging roads can compromise this refuge effect by acting as dispersal routes for rats into natural forests.

Inhibition of hepatobiliary transport as a predictive method for clinical hepatotoxicity of nefazodone.

Treatment with the antidepressant nefazodone has been associated with clinical idiosyncratic hepatotoxicty. Using membranes expressing human bile salt export pump (BSEP), human sandwich hepatocytes, and intact rats, we compared nefazodone and its marketed analogs, buspirone and trazodone. We found that nefazodone caused a strong inhibition of BSEP (IC(50) = 9 microM), inhibition of taurocholate efflux in human hepatocytes (IC(50) = 14 microM), and a transient increase in rat serum bile acids 1 h after oral drug administration. Buspirone or trazodone had no effect on biliary transport system. Nefazodone produced time- and concentration-dependent toxicity in human hepatocytes with IC(50) = 18 microM and 30 microM measured by inhibition of protein synthesis after 6 h and 24 h incubation, respectively. Toxicity was correlated with the amount of unmetabolized nefazodone. Partial recovery in toxicity by 24 h has been associated with metabolism of nefazodone to sulfate and glucuronide conjugates. The saturation of nefazodone metabolism resulted in sustained decrease in protein synthesis and cell death at 50 microM. The toxicity was not observed with buspirone or trazodone. Addition of 1-aminobenzotriazole (ABT), an inhibitor of CYP450, resulted in enhancement of nefazodone toxicity at 10 microM and was associated with accumulation of unmetabolized nefazodone. In human liver microsomes, ABT also prevented metabolism of nefazodone and formation of glutathione conjugates. We suggest that inhibition of bile acid transport by nefazodone is an indicator of potential hepatotoxicity. Our findings are consistent with the clinical experience and suggest that described methodology can be applied in the selection of nonhepatotoxic drug candidates.

Antiangiogenic and antitumor activity of a selective PDGFR tyrosine kinase inhibitor, CP-673,451.

CP-673,451 is a potent inhibitor of platelet-derived growth factor beta-receptor (PDGFR-beta) kinase- and PDGF-BB-stimulated autophosphorylation of PDGFR-beta in cells (IC(50) = 1 nmol/L) being more than 450-fold selective for PDGFR-beta versus other angiogenic receptors (e.g., vascular endothelial growth factor receptor 2, TIE-2, and fibroblast growth factor receptor 2). Multiple models have been used to evaluate in vivo activity of CP-673,451 and to understand the pharmacology of PDGFR-beta inhibition and the effect on tumor growth. These models include an ex vivo measure of PDGFR-beta phosphorylation in glioblastoma tumors, a sponge model to measure inhibition of angiogenesis, and multiple models of tumor growth inhibition. Inhibition of PDGFR-beta phosphorylation in tumors correlates with plasma and tumor levels of CP-673,451. A dose of 33 mg/kg was adequate to provide >50% inhibition of receptor for 4 hours corresponding to an EC(50) of 120 ng/mL in plasma at C(max). In a sponge angiogenesis model, CP-673,451 inhibited 70% of PDGF-BB-stimulated angiogenesis at a dose of 3 mg/kg (q.d. x 5, p.o., corresponding to 5.5 ng/mL at C(max)). The compound did not inhibit vascular endothelial growth factor- or basic fibroblast growth factor-induced angiogenesis at concentrations which inhibited tumor growth. The antitumor efficacy of CP-673,451 was evaluated in a number of human tumor xenografts grown s.c. in athymic mice, including H460 human lung carcinoma, Colo205 and LS174T human colon carcinomas, and U87MG human glioblastoma multiforme. Once-daily p.o. x 10 days dosing routinely inhibited tumor growth (ED(50) < or = 33 mg/kg). These data show that CP-673,451 is a pharmacologically selective PDGFR inhibitor, inhibits tumor PDGFR-beta phosphorylation, selectively inhibits PDGF-BB-stimulated angiogenesis in vivo, and causes significant tumor growth inhibition in multiple human xenograft models.