ABSTRACT
Compound LY335979 is a P-glycoprotein inhibitor currently entering phase I clinical trials for potential reversal of multidrug resistance to cancer chemotherapy. In early exploratory studies, LY335979 was found to be rapidly transformed in incubations with liver microsomes from rats, dogs, monkeys, and humans. Although the parent compound was completely metabolized, no prominent metabolite peaks were observed. One peak did appear early in the time course, but it did not increase over time. In another preliminary experiment, rats were treated iv with [3H]LY335979 (prepared for pharmacology studies), and urine and bile fractions were collected. Analysis of the urine by reverse-phase HPLC with UV and radioactivity detection revealed that almost all of the material eluted with the solvent front. More than half the radioactivity in bile was accounted for by two peaks eluting earlier than the parent compound (the rest eluted at the solvent front). With both bile and the incubations with microsomes, initial attempts to isolate metabolites were not successful. There was also evidence in both systems of products derived from cleavage of LY335979 (by both further metabolism and degradation). LC/NMR was thus used to analyze materials directly in their respective matrices. An N-oxide metabolite (LY389551) formed by oxidation of the quinoline nitrogen was identified in the microsomal incubations; in bile, three glucuronide metabolites were identified, all of which were conjugates of products formed by oxidation of the quinoline ring of LY335979. There have been few reports in the literature of LC/NMR analysis of bile, which is a more complex matrix than either urine or microsomal suspensions. However, the HPLC techniques developed in this work for the HPLC/UV and LC/MS analyses of LY335979 metabolites in the microsomal matrix and in bile proved readily adaptable for LC/NMR. Using a 500-MHz instrument, basic 1H NMR spectra could be obtained in 2-3 hr with approximately 100 ng of material in the LC/NMR microprobe. With approximately 1.5 microg of material injected onto the column, 1H-1H correlation spectroscopy spectra could be acquired overnight. Along with LC/MS data, the LC/NMR technique facilitated direct identification of a number of metabolites of LY335979 at a point at which their identification by traditional methods would not have been pursued.
Subject(s)
Bile/metabolism , Dibenzocycloheptenes/chemistry , Dibenzocycloheptenes/metabolism , Drug Resistance, Multiple , Microsomes, Liver/metabolism , Quinolines/chemistry , Quinolines/metabolism , Animals , Bile/chemistry , Catheterization , Chromatography, Liquid , Humans , Magnetic Resonance Spectroscopy , Male , Mass Spectrometry , Microsomes, Liver/chemistry , Rats , Rats, Inbred F344 , Time FactorsABSTRACT
The metabolism and disposition of LY231514 was studied in mice and dogs. LY231514 is a novel pyrrotopyrimidine-based multi-target antifolate (MTA) showing broad in vivo antitumor activity in mouse models and is currently in phase II human clinical trials. Doses (iv) of the compound showed high plasma levels, resulting in AUC values of 30-33 micrograms-hr/ml for mice and dogs after 20 and 7.5 mg/kg doses, respectively. The compound was eliminated rapidly. Half-life values for mice and dogs were about 7 and 2 hr, respectively. In vitro plasma binding measured 56% in mice, 46% in dogs, and 81% in humans. Fecal elimination was the major excretion pathway in mice after single iv doses of [14C]LY231514. Urine constituted the major route of excretion in dogs. Parent LY231514 accounted for the majority of urinary radiocarbon in mice (90%) and dogs (68%). Minor metabolites were found in urine, but the amounts were too small to isolate or identify. Based on an earlier observation that LY231514 photodegraded to produce reaction products having similar retention times as these minor urinary isolates, a photo-oxidation system was developed which in fact produced these metabolites. Subsequently, these photolytically-produced materials were used as standards to identify two novel in vivo metabolites formed by oxidation of the pyrrolo-pyrimidine ring system of LY231514. The oxidative transformations are similar to those observed for tryptophan and other indoles in that the pyrrole ring is oxidized to give an amide; further oxidation cleaves this ring, one ring carbon is lost, and a ketone is formed.
Subject(s)
Antineoplastic Agents/pharmacokinetics , Enzyme Inhibitors/pharmacokinetics , Folic Acid Antagonists/pharmacokinetics , Glutamates/pharmacokinetics , Guanine/analogs & derivatives , Animals , Area Under Curve , Dogs , Female , Guanine/pharmacokinetics , Half-Life , Male , Metabolic Clearance Rate , Mice , Pemetrexed , Thymidylate Synthase/antagonists & inhibitorsABSTRACT
The disposition and metabolism of LY295501 was studied in mice, rats, and monkeys. This novel diaryl sulfonylurea oncolytic agent is structurally related to sulofenur and shows excellent activity in a broad range of mouse antitumor models. The compound is well absorbed, giving plasma concentrations greater than 200 micrograms/ml after oral doses of 30-100 mg/kg, where it appears to be completely bound (> 99.9%) to plasma proteins. The high degree of protein binding may be a factor in its relatively long half-life, which ranges from about 8 hr in rats and 15 hr in mice to 50 hr in monkeys. While more material was excreted in feces than in urine from mice and rats given single oral doses of [14C]LY295501, urine was the major route of elimination in monkeys. Three major metabolites-all formed via oxidation of the saturated part of the benzodihydrofuran moiety-were characterized in the urine of mice, rats, and monkeys. It is interesting that two of these metabolites are derived from opening of this saturated ring, an unusual metabolic process which represents a significant part of the metabolism of LY295501. As with sulofenur, metabolites of 3,4-dichloroaniline formed after metabolic cleavage of the sulfonylurea linkage were also found in urine. Unlike sulofenur, these do not seem to have major toxicological significance, but their formation does explain the minor methemoglobinemia observed in toxicology studies of LY295501. Even though only trace amounts of LY295501 were found in urine, LY295501 is the predominant drug-related material in plasma, along with small amounts of other, relatively nonpolar, metabolites.
Subject(s)
Antineoplastic Agents/pharmacokinetics , Benzofurans/pharmacokinetics , Phenylurea Compounds/pharmacokinetics , Animals , Area Under Curve , Biological Availability , Female , Half-Life , Macaca mulatta , Male , Metabolic Clearance Rate , Mice , Mice, Inbred C3H , Rats , Rats, Inbred F344ABSTRACT
LY207702 (1) is a difluorinated purine nucleoside that exhibits impressive antitumor activity in preclinical models. This agent, however, also possesses cardiotoxicity which limits the potential clinical utility of this novel drug candidate. We therefore developed linker chemistry whereby regioselective N6-tritylation of LY207702 (1) allowed this drug to be coupled to epsilon-lysine amino groups of mAb's reactive with human tumor-associated antigens. The resulting immunoconjugates 3 possessed conjugation ratios ranging from 5 to 7 mol of LY207702/mol of mAb, minimal aggregate content (5-10%), and good immunoreactivity. The electronic nature of substituents on the aromatic rings of the trityl group dictated the degree of acid lability of the trityl linker. Increased electronic stabilization of the transient trityl carbocation led to increase in the release rate of free drug, i.e., m-DMT 10a = p-DMT 10b > p-MMT 10d > p-T 10f. Consequently, the more acid labile DMT conjugates 3a and 3b proved to be the most potent cytotoxic agents, and the most stable p-T conjugate 3f exhibited the least antitumor activity when evaluated in vitro and in vivo. p-MeT-linked conjugate 3e, the most stable construct that retained excellent in vivo antitumor activity, was selected for more extensive evaluation. No detectable free drug or metabolite was observed in mouse plasma at a single intravenous dose of p-MeT conjugate 3e, which was consistent with its predicted stability under physiological conditions. This construct did, however, exhibit significant antigen-mediated antitumor activity in vivo. No cardiotoxicity was detected in mice dosed with conjugate 3e (6 mg/kg free drug content per day for 21 days) equivalent to approximately 8 times the total dose required for complete regression of well-established (approximately 1 g) HC1 human colon tumor xenografts in nude mice. Cardiotoxicity was induced in 20% of free drug 1 treated group at the equivalent dose. Cardiomyopathy was, however, observed when the dose of conjugate 3e was increased to 8 mg/kg per day for 21 days. These data suggest that antitumor activity of LY207702 (1) was maintained and its cardiotoxic potential reduced when this agent was administered to human tumor xenograft bearing nude mice as COL1-N6-p-MeT-207702 conjugate 3e.
Subject(s)
Antineoplastic Agents/chemistry , Deoxyadenosines/chemistry , Animals , Antibodies, Monoclonal , Antigens, Neoplasm/chemistry , Antineoplastic Agents/adverse effects , Deoxyadenosines/adverse effects , Heart/drug effects , Humans , Immunoconjugates/chemistry , Mice , Mice, NudeABSTRACT
LY288513 [(4S,5R)-trans-N-(4-bromophenyl)-3-oxo-4,5-diphenyl-1- pyrazolidinecarboxamide] has been developed as a cholecystokinin-B antagonist for anxiolytic indications. In the drug disposition studies reported herein, we found LY288513 was metabolized rapidly in mice and rats to form a glucuronide adduct. This metabolite was isolated from rodent urine and further characterized to determine whether a ring-nitrogen or the oxygen atom in the pyrazolidinone nucleus of LY288513 was bonded to the glucuronide. Using 13C NMR plus two-dimensional rotating frame Overhauser enhancement spectroscopy analysis and IR analysis of model compounds, we determined the metabolite was an O-glucuronide. The major elimination pathway for [14C]LY288513 was fecal, with > 75% of total radiocarbon excreted within 24 hr. Plasma levels for parent and metabolite in the three species varied substantially. The plasma half-life was significantly longer in dogs (approximately 12 hr) than mice (approximately 24 hr) or rats (2.6-3.6 hr). Consistent with this, in dogs relatively little O-glucuronide was observed in plasma, but in rats and mice the AUCs for glucuronide were greater than the parent AUC. A dose-ranging study in rats revealed oral absorption of LY288513 is nonsaturated, even after 800 mg/kg oral doses. A plot of AUC vs. dose revealed a consistent overproportional increase in AUC for both parent and glucuronide metabolite with increasing dose.
Subject(s)
Cholecystokinin/antagonists & inhibitors , Hormone Antagonists/pharmacokinetics , Pyrazoles/pharmacokinetics , Animals , Biotransformation , Chromatography, High Pressure Liquid , Dogs , Female , Glucuronates/blood , Hormone Antagonists/blood , Magnetic Resonance Spectroscopy , Male , Mice , Pyrazoles/blood , Rats , Rats, Inbred F344 , Species Specificity , Spectrophotometry, Infrared , Spectrophotometry, UltravioletABSTRACT
The absorption and pharmacokinetics of sulofenur [N-(indan-5-sulfonyl)-N'-(4-chlorophenyl)urea, LY186641] and its major metabolites were examined in mice, rats, monkeys, and dogs. The compound is a diarylsulfonylurea currently being evaluated as an oncolytic agent in phase I and II trials. In all species, sulofenur was well absorbed after an oral dose, but over a prolonged period, and sulofenur exhibited a fairly long half-life of elimination from plasma. These values ranged from 6 h in rats up to 30, 110, and 200 h in mice, monkeys, and dogs, respectively, at doses (240-1000 mg/m2) within the range of those used in clinical trials. Experiments describing the high degree of binding of sulofenur to plasma proteins (consistently > 99%) help to explain these relatively long half-lives. There is, however, a large difference between these plasma half-lives in the species studied. Sulofenur was previously found to be extensively metabolized to products that are excreted primarily into the urine. In this study, its major metabolites, which are found mainly in the urine, were also minor components of the drug-related material (< 10% of the sulofenur concentrations) in the plasma of rats treated with sulofenur. The absorption, binding characteristics, and elimination of these major metabolites after their administration to rats were also compared with sulofenur.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Antineoplastic Agents/pharmacokinetics , Sulfonylurea Compounds/pharmacokinetics , Animals , Blood Proteins/metabolism , Dogs , Female , Half-Life , Macaca mulatta , Mice , Mice, Inbred C3H , Protein Binding , Rats , Rats, Inbred F344 , Species SpecificityABSTRACT
The metabolic formation of p-chloroaniline from the oncolytic agent sulofenur [N-(5-indanesulfonyl)-N'-(4-chlorophenyl)urea, LY186641,] and from similar diaryl-substituted sulfonylureas, and its possible relevance to the compound's toxicity, was studied. In previous studies it was found that significant amounts of metabolites such as 2-amino-5-chlorophenyl sulfate (II), which is also a metabolite of p-chloroaniline, are formed from sulofenur in mice, rats, monkeys, and humans. The metabolism of N-(4-tolyl)-N'-(2-hydroxy-4-chlorophenyl)-urea (V) was studied, and V was not found to be an intermediate in the metabolic formation of II from the sulfonylurea N-(4-tolyl)-N'-(4-chlorophenyl)urea (LY181984, III). The amounts of this p-chloroaniline metabolite (II) formed in C3H mice from a series of diarylsulfonylureas were found to correlate with the compound's propensities to form methemoglobin, one notable toxicity of p-chloroaniline. This metabolism was also found to correlate with the structure of the arylsulfonyl moiety of the sulfonylurea. Other evidence supports the hypothesis that p-chloroaniline is directly formed by metabolism of sulfofenur and similar diarylsulfonylureas as well. Metabolic formation of p-chloroaniline thus appears to be a plausible explanation for the methemoglobinemia and anemia found to be dose-limiting toxicities of sulofenur in Phase I trials.
Subject(s)
Aniline Compounds/metabolism , Antineoplastic Agents/toxicity , Sulfonylurea Compounds/toxicity , Animals , Antineoplastic Agents/metabolism , Female , Magnetic Resonance Spectroscopy , Mass Spectrometry , Methemoglobin/analysis , Mice , Mice, Inbred C3H , Structure-Activity Relationship , Sulfonylurea Compounds/chemistry , Sulfonylurea Compounds/metabolismABSTRACT
The rabbit cornea accumulates platelet activating factor (PAF) three hrs after alkali burn. PAF was isolated by HPLC and assayed by platelet aggregation. This bioactivity was blocked by the PAF receptor antagonists BN 52021 and alprazolam. Added PAF increases the chemiluminescence response of the cornea in vitro and BN 52021 inhibits this effect. In vivo experiments show that the synthesis of 5-HETE and 12-HETE is inhibited by the PAF antagonist BN 52021. It is concluded that a metabolic interrelationship may exist between the PAF cycle and the lipoxygenation of arachidonic acid, and that drugs that affect these lipid mediators may modulate the inflammatory response of the anterior segment of the eye.