[Progress on pharmacokinetic study of antibody-drug conjugates].

Antibody-drug conjugate (ADC) is a new class of therapeutics composed of a monoclonal antibody and small cytotoxin moieties conjugated through a chemical linker. ADC molecules bind to the target antigens expressed on the tumor cell surfaces guided by the monoclonal antibody component. The binding ADC molecules can be internalized and subsequently the toxin moieties can be released within the tumor cells via chemical and/or enzymatic reactions to kill the target cells. The conjugation combines the merits of both components, i.e., the high target specificity of the monoclonal antibody and the highly potent cell killing activity of the cytotoxin moieties. However, such complexities make the pharmacokinetic and metabolic studies of ADCs highly challenging. The major challenges should include characterization of absorption, distribution, metabolism and excretion, investigation of underlying mechanisms, assessment of pharmacokinetic- pharmacodynamic relationship, and analytical method development of ADC drugs. This review will discuss common pharmacokinetic issues and considerations, as well as tools and strategies that can be utilized to characterize the pharmacokinetic and metabolic properties of ADCs.

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.

An in-vitro cocktail assay for assessing compound-mediated inhibition of six major cytochrome P450 enzymes.

An efficient screening assay was developed and validated for simultaneous assessment of compound-mediated inhibition of six major human cytochrome P450 (CYP) enzymes. This method employed a cocktail of six probe substrates (i.e., phenacetin, amodiaquine, diclofenac, S-mephenytoin, dextromethorphan and midazolam for CYP1A2, 2C8, 2C9, 2C19, 2D6 and 3A4, respectively) as well as individual prototypical inhibitors of the six CYP enzymes in human liver microsomes under optimized incubation conditions. The corresponding marker metabolites (i.e., acetaminophen, N-desethylamodiaquine, 4-OH-diclofenac, 4-OH-S-mephenytoin, dextrorphan and 1-OH-midazolam) in the incubates were quantified using LC-MS/MS methods either by an internal standard (IS) calibration curve or a simplified analyte-to-IS peak area ratio approach. The results showed that the IC50 values determined by the cocktail approach were in good agreement with those obtained by the individual substrate approach as well as those reported in the literature. Besides, no remarkable difference was observed between the two quantification approaches. In conclusion, this new cocktail assay can be used for reliable screening of compound-mediated CYP inhibition.

Pharmacokinetics, distribution, and metabolism of [14C]sunitinib in rats, monkeys, and humans.

Sunitinib is an oral multitargeted tyrosine kinase inhibitor approved for the treatment of advanced renal cell carcinoma, imatinib-refractory gastrointestinal stromal tumor, and advanced pancreatic neuroendocrine tumors. The current studies were conducted to characterize the pharmacokinetics, distribution, and metabolism of sunitinib after intravenous and/or oral administrations of [(14)C]sunitinib in rats (5 mg/kg i.v., 15 mg/kg p.o.), monkeys (6 mg/kg p.o.), and humans (50 mg p.o.). After oral administration, plasma concentration of sunitinib and total radioactivity peaked from 3 to 8 h. Plasma terminal elimination half-lives of sunitinib were 8 h in rats, 17 h in monkeys, and 51 h in humans. The majority of radioactivity was excreted to the feces with a smaller fraction of radioactivity excreted to urine in all three species. The bioavailability in female rats was close to 100%, suggesting complete absorption of sunitinib. Whole-body autoradioluminography suggested radioactivity was distributed throughout rat tissues, with the majority of radioactivity cleared within 72 h. Radioactivity was eliminated more slowly from pigmented tissues. Sunitinib was extensively metabolized in all species. Many metabolites were detected both in urine and fecal extracts. The main metabolic pathways were N-de-ethylation and hydroxylation of indolylidene/dimethylpyrrole. N-Oxidation/hydroxylation/desaturation/deamination of N,N'-diethylamine and oxidative defluorination were the minor metabolic pathways. Des-ethyl metabolite M1 was the major circulating metabolite in all three species.

Assessment of three human in vitro systems in the generation of major human excretory and circulating metabolites.

An early understanding of key metabolites of drugs is crucial in drug discovery and development. As a result, several in vitro models typically derived from liver are frequently used to study drug metabolism. It is presumed that these in vitro systems provide an accurate view of the potential in vivo metabolites and metabolic pathways. However, no formal analysis has been conducted to validate their use. The goal of the present study was to conduct a comprehensive analysis to assess if the three commonly used in vitro systems, pooled human liver microsomes, liver S-9 fraction, and hepatocytes, adequately predict in vivo metabolic profiles for drugs. The second objective was to compare the overall capabilities of these three systems to generate in vivo metabolic profiles. Twenty-seven compounds in the Pfizer database and 21 additional commercially available compounds of diverse structure and routes of metabolism for which the human ADME data was available were analyzed in this study to assess the performance of the in vitro systems. The results suggested that all three systems reliably predicted human excretory and circulating metabolite profiles. Furthermore, the success in predicting primary metabolites and metabolic pathways was high (>70%), but the predictability of secondary metabolites was less reliable in the three systems. Thus, the analysis provides sufficient confidence in using in vitro systems to reliably produce primary in vivo human metabolites and supports their application in early discovery to identify metabolic spots for optimization of metabolic liabilities anticipated in humans in vivo. However, the in vitro systems cannot solely mitigate the risk of disproportionate circulating metabolites in humans and may need to be supplemented with metabolic profiling of plasma samples from first-in-human studies or early human radiolabeled studies.

Identification of primary and sequential bioactivation pathways of carbamazepine in human liver microsomes using liquid chromatography/tandem mass spectrometry.

Carbamazepine (CBZ)-induced idiosyncratic toxicities are commonly believed to be related to the formation of reactive metabolites. CBZ is metabolized primarily into carbamazepine-10,11-epoxide (CBZE), 2-hydroxycarbamazepine (2-OHCBZ) and 3-hydroxycarbamazepine (3-OHCBZ), in human liver microsomes (HLM). Over the past two decades, the 2,3-arene oxidation has been commonly assumed to be the major bioactivation pathway of CBZ. Recently, CBZE has been also confirmed to be chemically reactive. In order to identify other possible primary and sequential CBZ bioactivation pathways, individual HLM incubations of CBZ, CBZE, 2-OHCBZ and 3-OHCBZ were conducted in the presence of glutathione (GSH). In the CBZ incubation, a variety of GSH adducts were formed via individual or combined pathways of 10,11-epoxidation, arene oxidation and iminoquinone formation. In the CBZE incubation, the only detected GSH adducts were CBZE-SG1 and CBZE-SG2, which represented the two most abundant conjugates observed in the CBZ incubation. In the incubation of either 2-OHCBZ or 3-OHCBZ, a number of sequential GSH adducts were observed. However, none of the 2-OHCBZ-derived GSH adducts were detected in the CBZ incubation. Meanwhile, several GSH adducts were only observed in the CBZ incubation. In conclusion, CBZ can be bioactivated in HLM via 10,11-epoxidation, 2,3-arene oxidation, and several other pathways. In addition, the sequential bioactivation of 3-OHCBZ appeared to play a more important role than that of either CBZE or 2-OHCBZ in the overall bioactivation of CBZ in HLM. The identification of several new bioactivation pathways of CBZ in HLM demonstrates that possible CBZ bioactivation can be more complex than previously thought.

Evaluation of capravirine as a CYP3A probe substrate: in vitro and in vivo metabolism of capravirine in rats and dogs.

Metabolism of [(14)C]capravirine was studied via both in vitro and in vivo means in rats and dogs. Mass balance was achieved in rats and dogs, with mean total recovery of radioactivity >86% for each species. Capravirine was well absorbed in rats but only moderately so in dogs. The very low levels of recovered unchanged capravirine and the large number of metabolites observed in rats and dogs indicate that capravirine was eliminated predominantly by metabolism in both species. Capravirine underwent extensive metabolism via oxygenation reactions (predominant pathways in both species), depicolylation and carboxylation in rats, and decarbamation in dogs. The major circulating metabolites of capravirine were two depicolylated products in rats and three decarbamated products in dogs. However, none of the five metabolites was observed in humans, indicating significant species differences in terms of identities and relative abundances of circulating capravirine metabolites. Because the majority of in vivo oxygenated metabolites of capravirine were observed in liver microsomal incubations, the in vitro models provided good insight into the in vivo oxygenation pathways. In conclusion, the diversity (i.e., hydroxylation, sulfoxidation, sulfone formation, and N-oxidation), multiplicity (i.e., mono-, di-, tri-, and tetraoxygenations), and high enzymatic specificity (>90% contribution by CYP3A4 in humans, CYP3A1/2 in rats, and CYP3A12 in dogs) of the capravirine oxygenation reactions observed in humans, rats, and dogs in vivo and in vitro suggest that capravirine can be a useful CYP3A substrate for probing catalytic mechanisms and kinetics of CYP3A enzymes in humans and animal species.

Ocular disposition, pharmacokinetics, efficacy and safety of nanoparticle-formulated ophthalmic drugs.

Ophthalmic drugs are delivered to ocular tissues predominantly via relatively simple formulations, such as topically dosed water-soluble drug solutions and water-insoluble drug suspensions in ointments. An ideal topical drug delivery system should possess certain desirable properties, such as good corneal and conjunctival penetration, prolonged precorneal residence time, easy instillation, non-irritative and comfortable to minimize lachrymation and reflex blinking, and appropriate rheological properties. In general, ocular efficacy is closely related to ocular drug bioavailability, which may be enhanced by increasing corneal drug penetration and prolonging precorneal drug residence time. To improve ocular bioavailability of topically dosed ophthalmic drugs, a variety of ocular drug delivery systems, such as hydrogels, microparticles, nanoparticles, microemulsions, liposomes and collagen shields, have been designed and investigated. These newer systems may, to some extent, control drug release and maintain therapeutic levels in ocular tissues over a prolonged period of time. This review focuses on the in vitro, ex vivo and in vivo studies of ophthalmic drugs formulated in nanoparticles published over the past two decades. The progress and development issues relating to ocular disposition, pharmacokinetics, efficacy and safety of the nanoparticle-formulated ophthalmic drugs are specifically addressed. Information and discussions summarized in this review are helpful for pharmaceutical scientists to develop better ophthalmic therapeutics.

A unique example of drug metabolism: tetra- and penta-oxygenation reactions of capravirine in rats, dogs and humans.

Six tetra- and two penta-oxygenated capravirine metabolites observed in rats, dogs and humans represent the maximum numbers of isomers that can be predicted since oxygenations are restricted at the pyridinyl nitrogen (N-oxidation), sulfur (sulfoxidation), and isopropyl group (hydroxylation), exemplifying a unique case that is very unusual for sequential drug metabolism.

Identification of enzymes responsible for primary and sequential oxygenation reactions of capravirine in human liver microsomes.

Capravirine, a new non-nucleoside reverse transcriptase inhibitor, undergoes extensive oxygenation reactions, including N-oxidation, sulfoxidation, sulfonation, and hydroxylation in humans. Numerous primary (mono-oxygenated) and sequential (di-, tri-, and tetraoxygenated) metabolites of capravirine are formed via the individual or combined oxygenation pathways. In this study, cytochrome P450 enzymes responsible for the primary and sequential oxygenation reactions of capravirine in human liver microsomes were identified at the specific pathway level. The total oxygenation of capravirine is mediated predominantly (>90%) by CYP3A4 and marginally (<10%) by CYP2C8, 2C9, and 2C19 in humans. Specifically, each of the two major mono-oxygenated metabolites C23 (sulfoxide) and C26 (N-oxide), is mediated predominantly (>90%) by CYP3A4 and slightly (<10%) by CYP2C8, the minor tertiary hydroxylated metabolite C19 by CYP3A4, 2C8, and 2C19, and the minor primary hydroxylated metabolite C20 by CYP3A4, 2C8, and 2C9. However, all sequential oxygenation reactions are mediated exclusively by CYP3A4. Due to their relatively insignificant contributions of C19 and C20 to total capravirine metabolism, no attempt was made to determine relative contributions of cytochrome P450 enzymes to the formation of the two minor metabolites.

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.

A simple sequential incubation method for deconvoluting the complicated sequential metabolism of capravirine in humans.

Capravirine, a non-nucleoside reverse transcriptase inhibitor for the treatment of human immunodeficiency virus type 1, undergoes extensive oxygenations to numerous sequential metabolites in humans. Because several possible oxygenation pathways may be involved in the formation and/or sequential metabolism of a single metabolite, it is very difficult or even impossible to determine the definitive pathways and their relative contributions to the overall metabolism of capravirine using conventional approaches. For this reason, a human liver microsome-based "sequential incubation" method has been developed to deconvolute the complicated sequential metabolism of capravirine. In brief, the method includes three fundamental steps: 1) 30-min primary incubation of [(14)C]capravirine, 2) isolation of (14)C metabolites from the primary incubate, and 3) 30-min sequential incubation of each isolated (14)C metabolite supplemented with an ongoing (30 min) microsomal incubation with nonlabeled capravirine. Based on the extent of both the disappearance of the isolated precursor (14)C metabolites and the formation of sequential (14)C metabolites, definitive oxygenation pathways of capravirine were assigned. In addition, the percentage contribution of a precursor metabolite to the formation of each of its sequential metabolites (called sequential contribution) and the percentage contribution of a sequential metabolite formed from each of its precursor metabolites (called precursor contribution) were determined. An advantage of this system is that the sequential metabolism of each isolated (14)C metabolite can be monitored selectively by radioactivity in the presence of all relevant metabolic components (i.e., nonlabeled parent and its other metabolites). This methodology should be applicable to mechanistic studies of other compounds involving complicated sequential metabolic reactions when radiolabeled materials are available.

Metabolism and excretion of capravirine, a new non-nucleoside reverse transcriptase inhibitor, alone and in combination with ritonavir in healthy volunteers.

Metabolism and disposition of capravirine, a new non-nucleoside reverse transcriptase inhibitor, were studied in healthy male volunteers who were randomly divided into two groups (A and B) with five subjects in each group. Group A received a single oral dose of [(14)C]capravirine (1400 mg) and group B received multiple oral doses of ritonavir (100 mg), followed by a single oral dose of [(14)C]capravirine (1400 mg). Mean total recoveries of radioactivity for groups A and B were 86.3% and 79.0%, respectively, with a mean cumulative recovery in urine comparable with that in feces for both groups. Excretion of unchanged capravirine was negligible in urine and low in feces for both groups. The results suggest that capravirine was well absorbed, with metabolism as the principal mechanism of clearance. Capravirine underwent extensive metabolism to a variety of metabolites via oxygenations (mono-, di-, tri-, and tetra-) representing the predominant pathway, glucuronidation, and sulfation in humans. No useful plasma profiles of group A were obtained due to extremely low levels of plasma radioactivity. Analysis of group B plasma indicated that unchanged capravirine was the major radiochemical component, with three monooxygenated products and a glucuronide of capravirine as the major circulating metabolites. Nineteen metabolites were identified using liquid chromatography-multistage ion-trap mass spectrometry methodologies. In summary, coadministration of low-dose ritonavir (a potent CYP3A4 inhibitor) drastically decreased the levels of sequential oxygenated metabolites and markedly increased the levels of the parent drug and primary oxygenated metabolites overall in plasma, urine, and feces.

Development and validation of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of ribavirin in human plasma and serum.

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the analysis of antiviral agent ribavirin in human plasma and serum. The samples (0.1 ml) were extracted from the matrix using a simple protein precipitation procedure. The supernatants were evaporated to dryness, reconstituted and injected onto the LC/MS/MS system. The chromatography separation was achieved on a silica column operated with an aqueous-organic mobile phase. The use of a silica column not only provided adequate retention for the extremely polar compound of ribavirin, but also enhanced electrospray ionization sensitivity with the use of high percentage organic solvent in the mobile phase. The method has been validated over the concentration range of 10-10000 ng/ml ribavirin in human plasma and serum. Bamethan was used as the internal standard. The protein precipitation extraction has been automated based on 96-well format with the use of robotic liquid handlers to improve the overall throughput of the analysis.