Elucidating the structure and cytochrome P450-mediated mechanism for novel metabolites of GDC-0575 in rats.

1. GDC-0575 is an ATP-competitive small-molecule inhibitor of ChK1 that is being developed by Genentech for the treatment of various human malignancies.2. In a radiolabeled mass balance study of GDC-0575 in rats, two novel metabolites, named M12 (-71 Da,) and M17 (+288 Da), were detected as abundant circulating metabolites.3. Subsequent mass spectrometry and nuclear magnetic resonance analysis showed that M12 was a cyclized metabolite of GDC-0575, whereas M17 was its heterodimer to the parent. We further determined that M12 was mainly generated by cytochrome P450 (Cyp) 2d2.4. We proposed the potential mechanism was initiated by the oxidation on the pyrrole ring and subsequent cyclisation of the free primary amine onto C-3 of the pyrrole ring. This was followed by expulsion of cyclopropylcarboxamide and a loss of water to form intermediate I, which can be further oxidised to form M12, or dimerise with another molecule of GDC-0575 as nucleophile to form M17.5. To verify this hypothesis, we attempted to trap the intermediate I with glutathione (GSH) trapping assay and the GSH conjugate on the pyrrole ring was identified. This suggests the oxidation on the pyrrole led to reactive metabolite formation and supported this proposed mechanism.

Absorption, metabolism and excretion of pictilisib, a potent pan-class I phosphatidylinositol-3-Kinase (PI3K) inhibitor, in rats, dogs, and humans.

The absorption, metabolism and excretion of pictilisib, a selective small molecule inhibitor of class 1 A phosphoinositide 3-kinase (PI3K), was characterized following a single oral administration of [14C]pictilisib in rats, dogs and humans at the target doses of 30 mg/kg, 5 mg/kg and 60 mg, respectively.Pictilisib was rapidly absorbed with Tmax less than 2 h across species. In systemic circulation, pictilisib represented the predominant total radioactivity greater than 86.6% in all species.Total pictilisib and related radioactivity was recovered from urine and faeces in rats, dogs, and human at 98%, 80% and 95%, respectively, with less than 2% excreted in urine and the rest excreted into faeces.In rat and dog, more than 40% of drug-related radioactivity was excreted into the bile suggesting biliary excretion was the major route of excretion. Unchanged pictilisib was a minor component in rat and dog bile. The major metabolite in bile was O-glucuronide of oxidation on indazole moiety (M20, 21% of the dose) in rats and an oxidative piperazinyl ring-opened metabolite M7 (10.8% of the dose) in dogs.Oxidative glutathione (GSH) conjugates (M18, M19) were novel metabolites detected in rat bile, suggesting the potential generation of reactive intermediates from pictilisib. The structure of M18 was further confirmed by NMR to be a N-hydroxylated and GSH conjugated metabolite on the moiety of the indazole ring.

Characterizing the in vitro species differences in N-glucuronidation of a potent pan-PIM inhibitor GNE-924 containing a 3,5-substituted 6-azaindazole.

1. Glucuronidation of amines has been shown to exhibit large species differences, where the activity is typically more pronounced in human than in many preclinical species such as rat, mouse, dog and monkey. The purpose of this work was to characterize the in vitro glucuronidation of GNE-924, a potent pan-PIM inhibitor, to form M1 using liver microsomes (LM) and intestinal microsomes (IM). 2. M1 formation kinetics varied highly across species and between liver and intestinal microsomes. In LM incubations, rat exhibited the highest rate of M1 formation (CLint,app) at 140 ± 10 µL/min/mg protein, which was approximately 30-fold higher than human. In IM incubations, mouse exhibited the highest CLint,app at 484 ± 40 µL/min/mg protein, which was >1000-fold higher than human. In addition, CLint,app in LM was markedly higher than IM in human and monkey. In contrast, CLint,app in IM was markedly higher than LM in dog and mouse. 3. Reaction phenotyping indicated that UGT1A1, UGT1A3, UGT1A9, UGT2B4 and the intestine-specific UGT1A10 contributed to the formation of M1. 4. This is one of the first reports showing that N-glucuronidation activity is significantly greater in multiple preclinical species than in humans, and suggests that extensive intestinal N-glucuronidation may limit the oral exposure of GNE-924.

Solid-State Characterization of Novel Propylene Glycol Ester Solvates Isolated from Lipid Formulations.

The purpose of this study was to identify and characterize precipitates obtained from a liquid formulation of GNE068.HCl, a Genentech developmental compound, and lipophilic excipients, such as propylene glycol monocaprylate, and monolaurate. Precipitates were characterized using powder X-ray diffractometry (PXRD), differential scanning calorimetry, thermogravimetry, microscopy, nuclear magnetic resonance spectroscopy (NMR; solution and solid-state) and water sorption analysis. PXRD and NMR revealed the precipitates to be crystalline solvates of propylene glycol esters. The solvates (capryolate and lauroglycolate) were isomorphic and stable up to 70 °C, beyond which melting of the lattice occurred with subsequent dissolution of the active ingredient in the melt (microscopy and variable temperature PXRD). They were found to be mechanically stable (no change in PXRD pattern upon compression) and were nonhygroscopic up to ∼70% RH (25 °C). Our results highlight the outcome of inadvertent drug-excipient interactions in two separate lipid solution formulations with good solid-state properties and, thus, potential for further development.

Elucidating the mechanism of cytochrome P450-mediated pyrimidine ring conversion to pyrazole metabolites with the BACE1 inhibitor GNE-892 in rats.

We investigated an uncommon biotransformation of pyrimidine during the metabolism of GNE-892 ((R)-2-amino-1,3',3'-trimethyl-7'-(pyrimidin-5-yl)-3',4'-dihydro-2'H-spiro[imidazole-4,1'-naphthalen]-5(1H)-one), a ß-secretase 1 inhibitor. Three novel metabolites, formed by conversion of pyrimidine to pyrazole, were observed in the (14)C-radiolabeled mass balance study in rats. Their structures were characterized by high-resolution mass spectrometry and nuclear magnetic resonance. Although these metabolites accounted for <5% of the administered dose, their unique nature prompted us to conduct further investigations. The pyrazole-containing metabolites were formed in vitro with rat hepatocytes and liver microsomes, which supported that they were formed during hepatic metabolism. Further, their generation was inhibited by 1-aminobenzotriazole, indicating involvement of cytochrome P450s. Studies with rat recombinant enzymes identified that CYP2D2 generated the N-hydroxypyrazole metabolite from GNE-892. This biotransformation proceeded through multiple steps from the likely precursor, pyrimidine N-oxide. On the basis of these data, we propose a mechanism in which the pyrimidine is activated via N-oxidation, followed by a second oxidative process that opens the pyrimidine ring to form a formamide intermediate. After hydrolysis of the formamide, a carbon is lost as formic acid, together with ring closure to form the pyrazole ring. This article highlights a mechanistic approach for determining the biotransformation of the pyrimidine to a pyrazole for GNE-892.

Pharmacokinetics, metabolism, and excretion of [14C]axitinib, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, in humans.

The disposition of a single oral dose of 5 mg (100 µCi) of [(14)C]axitinib was investigated in fasted healthy human subjects (N = 8). Axitinib was rapidly absorbed, with a median plasma Tmax of 2.2 hours and a geometric mean Cmax and half-life of 29.2 ng/ml and 10.6 hours, respectively. The plasma total radioactivity-time profile was similar to that of axitinib but the AUC was greater, suggesting the presence of metabolites. The major metabolites in human plasma (0-12 hours), identified as axitinib N-glucuronide (M7) and axitinib sulfoxide (M12), were pharmacologically inactive, and with axitinib comprised 50.4%, 16.2%, and 22.5% of the radioactivity, respectively. In excreta, the majority of radioactivity was recovered in most subjects by 48 hours postdose. The median radioactivity excreted in urine, feces, and total recovery was 22.7%, 37.0%, and 59.7%, respectively. The recovery from feces was variable across subjects (range, 2.5%-60.2%). The metabolites identified in urine were M5 (carboxylic acid), M12 (sulfoxide), M7 (N-glucuronide), M9 (sulfoxide/N-oxide), and M8a (methylhydroxy glucuronide), accounting for 5.7%, 3.5%, 2.6%, 1.7%, and 1.3% of the dose, respectively. The drug-related products identified in feces were unchanged axitinib, M14/15 (mono-oxidation/sulfone), M12a (epoxide), and an unidentified metabolite, comprising 12%, 5.7%, 5.1%, and 5.0% of the dose, respectively. The proposed mechanism to form M5 involved a carbon-carbon bond cleavage via M12a, followed by rearrangement to a ketone intermediate and subsequent Baeyer-Villiger rearrangement, possibly through a peroxide intermediate. In summary, the study characterized axitinib metabolites in circulation and primary elimination pathways of the drug, which were mainly oxidative in nature.

Chemoselective sp2-sp3 cross-couplings: iron-catalyzed alkyl transfer to dihaloaromatics.

The chemoselective functionalization of a range of dihaloaromatics with methyl, cyclopropyl, and higher alkyl Grignard reagents via iron-catalyzed cross-coupling is described. The site selectivity of C-X (X = halogen) activation is determined by factors such as the position of the halogen on the ring, the solvent, and the nucleophile. A one-pot protocol for the chemoselective synthesis of mixed dialkyl heterocycles is achieved solely employing iron catalysis.

Degradation of a pharmaceutical in HPLC grade methanol containing trace level formaldehyde.

An anomalous peak was observed in the HPLC/UV analysis of a developmental drug product. High resolution LC/MS revealed that the mass of this degradant was 12 Da greater than the drug substance, corresponding to a net gain of a single carbon atom. The degradant was reproduced by incubating the drug substance with formaldehyde, followed by isolation using normal phase chromatography and structure elucidation by NMR. It was determined to be an analytical artifact caused by the nucleophilic reaction of the drug substance with trace levels of formaldehyde in the methanol diluent. Typical formaldehyde levels in various grades of methanol were determined, leading to the adoption of spectrophotometric purity solvent to mitigate the recurrence of this artifact. This work demonstrates that even ppm levels of impurities in solvents can cause significant degradation of drug product and the HPLC grade solvents are not always suitable for HPLC analysis in drug product development.

An isolable acyclic hemiacetal of ansamitocin P-3.

During impurity analysis of maytansinol (2), produced from the reduction of ansamitocin P-3 (AP-3, 1), a surprisingly stable acyclic hemiacetal (4) was isolated. A combination of 1D and 2D NMR experiments, along with liquid chromatography-mass spectrometry data was used to confirm the structure. Comparison of NMR data to the previously reported bridged acetal (3), a by-product of AP-3 reduction, supports reassignment of the latter to the former. Additionally, ROESY data, in conjunction with minimum energy calculations, support intramolecular hydrogen bonding that is involved in stabilizing the hemiacetal. This report adds another example to the very short list of isolable acyclic hemiacetals.

Strategies for the analysis of highly reactive pinacolboronate esters.

Pinacolboronate esters (or boronic acid, pinacol esters) are widely used in the Suzuki coupling reaction to connect organic building blocks for the total synthesis of complex molecules. The 2-aminopyrimidine-5-pinacolboronate ester was used as a starting material in the synthesis of a development compound, necessitating a chromatographic purity method to assess its quality. This aryl pinacolboronate ester posed unique analytical challenges due to its facile hydrolysis to the corresponding boronic acid, which is nonvolatile and poorly soluble in organic solvents. This made GC and normal-phase HPLC analysis unsuitable. In reversed-phase mode, typical sample preparation and analysis conditions promoted rapid sample degradation to the boronic acid. To overcome these challenges, unconventional approaches were necessary in order to stabilize 2-aminopyrimidine-5-pinacolboronate ester, adequately solubilize its boronic acid, and produce acceptable separation and retention. The final method employed non-aqueous and aprotic diluent, and a reversed-phase separation using highly basic mobile phases (pH 12.4) with an ion pairing reagent. These strategies were successfully applied to several other reactive pinacolboronate esters for purity analysis, demonstrating broad applicability to this unique class of compounds.

Novel mechanism for dehalogenation and glutathione conjugation of dihalogenated anilines in human liver microsomes: evidence for ipso glutathione addition.

The objective of the present study was to investigate the influence of halogen position on the formation of reactive metabolites from dihalogenated anilines. Herein we report on a proposed mechanism for dehalogenation and glutathione (GSH) conjugation of a series of ortho-, meta-, and para-dihalogenated anilines observed in human liver microsomes. Of particular interest were conjugates formed in which one of the halogens on the aniline was replaced by GSH. We present evidence that a (4-iminocyclohexa-2,5-dienylidene)halogenium reactive intermediate (QX) was formed after oxidation, followed by ipso addition of GSH at the imine moiety. The ipso GSH thiol attacks at the ortho-carbon and eventually leads to a loss of a halogen and GSH replacement. The initial step of GSH addition at the ipso position is also supported by density functional theory, which suggests that the ipso carbon of the chloro, bromo, and iodo (but not fluoro) containing 2-fluoro-4-haloanilines is the most positive carbon and that these molecules have the favorable highest occupied molecular orbital of the aniline and the lowest unoccupied orbital from GSH. The para-substituted halogen (chloro, bromo, or iodo but not fluoro) played a pivotal role in the formation of the QX, which required a delocalization of the positive charge on the para-halogen after oxidation. This mechanism was supported by structure-metabolism relationship analysis of a series of dihalogenated and monohalogenated aniline analogues.

Significant species difference in amide hydrolysis of GDC-0834, a novel potent and selective Bruton's tyrosine kinase inhibitor.

(R)-N-(3-(6-(4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenylamino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide (GDC-0834) is a potent and selective inhibitor of Bruton's tyrosine kinase (BTK), investigated as a potential treatment for rheumatoid arthritis. In vitro metabolite identification studies in hepatocytes revealed predominant formation of an inactive metabolite (M1) via amide hydrolysis in human. The formation of M1 appeared to be NADPH-independent in human liver microsomes. M1 was found in only minor to moderate quantities in plasma from preclinical species dosed with GDC-0834. Human clearance predictions using various methodologies resulted in estimates ranging from low to high. In addition, GDC-0834 exhibited low clearance in PXB chimeric mice with humanized liver. Uncertainty in human pharmacokinetic prediction and high interest in a BTK inhibitor for clinical evaluation prompted an investigational new drug strategy, in which GDC-0834 was rapidly advanced to a single-dose human clinical trial. GDC-0834 plasma concentrations in humans were below the limit of quantitation (<1 ng/ml) in most samples from the cohorts dosed orally at 35 and 105 mg. In contrast, substantial plasma concentrations of M1 were observed. In human plasma and urine, only M1 and its sequential metabolites were identified. The formation kinetics of M1 was evaluated in rat, dog, monkey, and human liver microsomes in the absence of NADPH. The maximum rate of M1 formation (V(max)) was substantially higher in human compared with that in other species. In contrast, the Michaelis-Menten constant (K(m)) was comparable among species. Intrinsic clearance (V(max)/K(m)) of GDC-0834 from M1 formation in human was 23- to 169-fold higher than observed in rat, dog, and monkey.

Absorption, distribution, metabolism, and excretion of [¹4C]GDC-0449 (vismodegib), an orally active hedgehog pathway inhibitor, in rats and dogs: a unique metabolic pathway via pyridine ring opening.

2-Chloro-N-(4-chloro-3-(pyridin-2-yl)-phenyl)-4-(methylsulfonyl)-benzamide (GDC-0449, vismodegib) is a potent and selective first-in-class small-molecule inhibitor of the Hedgehog signaling pathway and is currently in clinical development. In this study, we investigated the metabolic fate and disposition of GDC-0449 in rats and dogs after a single oral administration of [¹4C]GDC-0449. An average of 92.4 and 80.4% of the total administered radioactivity was recovered from urine and feces in rats and dogs, respectively. In both species, feces were the major route of excretion, representing 90.0 and 77.4% of the total dose in rats and dogs, respectively. At least 42.1 and 30.8% of the dose was absorbed in rats and dogs, respectively, based on the total excretion of radioactivity in bile and urine. GDC-0449 underwent extensive metabolism in rats and dogs with the major metabolic pathways being oxidation of the 4-chloro-3-(pyridin-2-yl)-phenyl moiety followed by phase II glucuronidation or sulfation. Three other metabolites resulting from an uncommon pyridine ring opening were found, mainly in feces, representing 1.7 to 17.7% of the dose in total in rats and dogs. In plasma, the total radioactivity was absorbed quickly in both rats and dogs, and unchanged GDC-0449 was the predominant circulating radioactive species in both species (>95% of total circulating radioactivity). Quantitative whole-body autoradiography in rats showed that the radioactivity was well distributed in the body, except for the central nervous system, and the majority of radioactivity was eliminated from most tissues by 144 h.

Formation of a quinoneimine intermediate of 4-fluoro-N-methylaniline by FMO1: carbon oxidation plus defluorination.

Here, we report on the mechanism by which flavin-containing monooxygenase 1 (FMO1) mediates the formation of a reactive intermediate of 4-fluoro-N-methylaniline. FMO1 catalyzed a carbon oxidation reaction coupled with defluorination that led to the formation of 4-N-methylaminophenol, which was a reaction first reported by Boersma et al. (Boersma et al. (1993) Drug Metab. Dispos. 21 , 218 - 230). We propose that a labile 1-fluoro-4-(methylimino)cyclohexa-2,5-dienol intermediate was formed leading to an electrophilic quinoneimine intermediate. The identification of N-acetylcysteine adducts by LC-MS/MS and NMR further supports the formation of a quinoneimine intermediate. Incubations containing stable labeled oxygen (H(2)(18)O or (18)O(2)) and ab initio calculations were performed to support the proposed reaction mechanism.

Bioactivation of flutamide metabolites by human liver microsomes.

Flutamide, a widely used nonsteroidal antiandrogen drug for the treatment of prostate cancer, has been associated with rare incidences of hepatotoxicity in patients. It is believed that bioactivation of flutamide and subsequent covalent binding to cellular proteins is responsible for its toxicity. A novel N-S glutathione adduct has been identified in a previous bioactivation study of flutamide (Kang et al., 2007). Due to the extensive first pass metabolism, flutamide metabolites such as 2-hydroxyflutamide and 4-nitro-3-(trifluoromethyl)phenylamine (Flu-1) have achieved plasma concentrations higher than the parent in prostate cancer patients. In vitro studies in human liver microsomes were conducted to probe the cytochrome P450 (P450)-mediated bioactivation of flutamide metabolites and identify the possible reactive species using reduced glutathione (GSH) as a trapping agent. Several GSH adducts (G1, Flu-1-G1, Flu-1-G2, Flu-6-Gs) derived from the metabolites of flutamide were identified and characterized. A comprehensive bioactivation mechanism was proposed to account for the formation of the observed GSH adducts. Of interest were the formation of a reactive intermediate by the desaturation of the isopropyl group of M5 and the unusual bioactivation of Flu-1. Studies using recombinant P450s suggested that the major P450 isozymes involved in the bioactivation of flutamide and its metabolites were CYP1A2, CYP3A4, and CYP2C19. These findings suggested that, in addition to the direct bioactivation of flutamide, the metabolites of flutamide could also be bioactivated and contribute to flutamide-induced hepatotoxicity.

Identification of a novel glutathione conjugate of flutamide in incubations with human liver microsomes.

Flutamide, a nonsteroidal antiandrogen drug widely used in the treatment of prostate cancer, has been associated with rare incidences of hepatotoxicity in patients. It is believed that bioactivation of flutamide and subsequent covalent binding to cellular proteins is responsible for its toxicity. Current in vitro studies were undertaken to probe the cytochrome P450 (P450)-mediated bioactivation of flutamide and identify the possible reactive species using reduced glutathione (GSH) as a trapping agent. NADPH- and GSH-supplemented human liver microsomal incubations of flutamide gave rise to a novel GSH conjugate where GSH moiety was conjugated to the flutamide molecule via the amide nitrogen, resulting in a sulfenamide. The structure of the conjugate was characterized by liquid chromatography-tandem mass spectrometry and NMR experiments. The conjugate formation was primarily catalyzed by heterologously expressed CYP2C19, CYP1A2, and, to a lesser extent, CYP3A4 and CYP3A5. The mechanism for the formation of this conjugate is unknown; however, a tentative bioactivation mechanism involving a P450-catalyzed abstraction of hydrogen atom from the amide nitrogen of flutamide and the subsequent trapping of the nitrogen-centered radical by GSH or oxidized glutathione (GSSG) was proposed. Interestingly, the same adduct was formed when flutamide was incubated with human liver microsomes in the presence of GSSG and NADPH. This finding suggests that P450-mediated oxidation of flutamide via a nitrogen-centered free radical could be one of the several bioactivation pathways of flutamide. Even though the relationship of the GSH conjugate to flutamide-induced toxicity is unknown, the results have revealed the formation of a novel, hitherto unknown, GSH adduct of flutamide.

In vitro metabolic activation of thiabendazole via 5-hydroxythiabendazole: identification of a glutathione conjugate of 5-hydroxythiabendazole.

Thiabendazole (TBZ) is a broad-spectrum antihelmintic used for treatment of parasitic infections in animals and humans and as an agricultural fungicide for postharvest treatment of fruits and vegetables. It is teratogenic and nephrotoxic in mice, and cases of hepatotoxicity have been observed in humans. Recent reports have demonstrated a correlation between 5-hydroxythiabendazole (5-OHTBZ) formation, a major metabolite of TBZ, and covalent binding of [(14)C]TBZ to hepatocytes, suggesting another pathway of activation of TBZ. Current in vitro studies were undertaken to probe the bioactivation of TBZ via 5-OHTBZ by cytochrome P450 (P450) and peroxidases and identify the reactive species by trapping with reduced glutathione (GSH). Microsomal incubation of TBZ or 5-OHTBZ supplemented with NADPH and GSH afforded a GSH adduct of 5-OHTBZ and was consistent with a bioactivation pathway that involved a P450-catalyzed two-electron oxidation of 5-OHTBZ to a quinone imine. The same adduct was detected in GSH-fortified incubations of 5-OHTBZ with peroxidases. The identity of the GSH conjugate suggested that the same reactive intermediate was formed by both these enzyme systems. Characterization of the conjugate by mass spectrometry and NMR revealed the addition of GSH at the 4-position of 5-OHTBZ. In addition, the formation of a dimer of 5-OHTBZ was discernible in peroxidase-mediated incubations. These results were consistent with a one-electron oxidation of 5-OHTBZ to a radical species that could undergo disproportionation or an additional one-electron oxidation to form a quinone imine. Overall, these studies suggest that 5-OHTBZ can also play a role in TBZ-induced toxicity via its bioactivation by P450 and peroxidases.

Human in vitro glutathionyl and protein adducts of carbamazepine-10,11-epoxide, a stable and pharmacologically active metabolite of carbamazepine.

The clinical use of carbamazepine (CBZ), an anticonvulsant, is associated with a variety of idiosyncratic adverse reactions that are likely related to the formation of chemically reactive metabolites. CBZ-10,11-epoxide (CBZE), a pharmacologically active metabolite of CBZ, is so stable in vitro and in vivo that the potential for the epoxide to covalently interact with macromolecules has not been fully explored. In this study, two glutathione (GSH) adducts were observed when CBZE was incubated with GSH in the absence of biological matrices and cofactors (e.g., liver microsomes and NADPH). The chemical reactivity of CBZE was further confirmed by the in vitro finding that [14C]CBZE formed covalent protein adducts in human plasma as well as in human liver microsomes (HLMs) without NADPH. The two GSH adducts formed in the chemical reaction of CBZE were identical to the two major GSH adducts observed in the HLM incubation of CBZ, indicating that the 10,11-epoxidation represents a bioactivation pathway of CBZ. The two GSH adducts were isolated and identified as two diastereomers of 10-hydroxy-11-glutathionyl-CBZ by NMR. In addition, the covalent binding of [14C]CBZE was significantly increased in the HLM incubation upon addition of NADPH, indicating that CBZE can be further bioactivated by HLMs. To our knowledge, this is the first time the metabolite CBZE has been confirmed for its ability to form covalent protein adducts and the identity of the two CBZE-glutathionyl adducts has been confirmed by NMR. These represent important findings in the bioactivation mechanism of CBZ.