Antitumor activity of SCH 66336, an orally bioavailable tricyclic inhibitor of farnesyl protein transferase, in human tumor xenograft models and wap-ras transgenic mice.

We have been developing a series of nonpeptidic, small molecule farnesyl protein transferase inhibitors that share a common tricyclic nucleus and compete with peptide/protein substrates for binding to farnesyl protein transferase. Here, we report on pharmacological and in vivo studies with SCH 66336, a lead compound in this structural class. SCH 66336 potently inhibits Ha-Ras processing in whole cells and blocks the transformed growth properties of fibroblasts and human tumor cell lines expressing activated Ki-Ras proteins. The anchorage-independent growth of many human tumor lines that lack an activated ras oncogene is also blocked by treatment with SCH 66336. In mouse, rat, and monkey systems, SCH 66336 has excellent oral bioavailability and pharmacokinetic properties. In the nude mouse, SCH 66336 demonstrated potent oral activity in a wide array of human tumor xenograft models including tumors of colon, lung, pancreas, prostate, and urinary bladder origin. Enhanced in vivo efficacy was observed when SCH 66336 was combined with various cytotoxic agents (cyclophosphamide, 5-fluorouracil, and vincristine). In a Ha-Ras transgenic mouse model, prophylactic treatment with SCH 66336 delayed tumor onset, reduced the average number of tumors/mouse, and reduced the average tumor weight/animal. In a therapeutic mode in which gavage treatment was initiated after the transgenic mice had developed palpable tumors, significant tumor regression was induced by SCH 66336 in a dose-dependent fashion. This was associated with increased apoptosis and decreased DNA synthesis in tumors of animals treated with SCH 66336. Enhanced efficacy was also observed in this model when SCH 66336 was combined with cyclophosphamide. SCH 66336 is presently being evaluated in Phase I clinical trials.

Potent, selective, and orally bioavailable tricyclic pyridyl acetamide N-oxide inhibitors of farnesyl protein transferase with enhanced in vivo antitumor activity.

We previously reported compound 1 as a potent farnesyl protein transferase (FPT) inhibitor that exhibited reasonable pharmacokinetic stability and showed moderate in vivo activity against a variety of tumor cell lines. The analogous C-11 single compound, pyridylacetamide 2, was found to be more potent than 1 in FPT inhibition. Further studies showed that modification of the ethano bridge of the tricyclic ring system by conversion into a double bond with concomitant introduction of a single bond at C-11 piperidine resulted in compound 3 that had superior FPT activity and pharmacokinetic stability. Compound 4, a 5-bromo-substituted analogue of 3, showed improved FPT activity, had good cellular activity, and demonstrated a remarkably improved pharmacokinetic profile with AUC of 84.9 and t1/2 of 82 min. Compound4 inhibited the growth of solid tumor in DLD-1 model by 70% at 50 mpk and 52% at 10 mpk.

Inhibitors of farnesyl protein transferase. 4-Amido, 4-carbamoyl, and 4-carboxamido derivatives of 1-(8-chloro-6,11-dihydro-5H-benzo[5,6]- cyclohepta[1,2-b]pyridin-11-yl)piperazine and 1-(3-bromo-8-chloro-6,11- dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl)piperazine.

The synthesis of a variety of novel 4-amido, 4-carbamoyl and 4-carboxamido derivatives of 1-(8-chloro-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl) piperazine to explore the SAR of this series of FPT inhibitors is described. This resulted in the synthesis of the 4- and 3-pyridylacetyl analogues 45a and 50a, respectively, both of which were orally active but were found to be rapidly metabolized in vivo. Identification of the principal metabolites led to the synthesis of a variety of new compounds that would be less readily metabolized, the most interesting of which were the 3- and 4-pyridylacetyl N-oxides 80a and 83a. Novel replacements for the pyridylacetyl moiety were also sought, and this resulted in the discovery of the 4-N-methyl and 4-N-carboxamidopiperidinylacetyl derivatives 135a and 160a, respectively. All of these derivatives exhibited greatly improved pharmacokinetics. The synthesis of the corresponding 3-bromo analogues resulted in the discovery of the 4-pyridylacetyl N-oxides 83b (+/-) and 85b [11S(-)] and the 4-carboxamidopiperidinylacetamido derivative 160b (+/-), all of which exhibited potent FPT inhibition in vitro. All three showed excellent oral bioavailability in vivo in nude mice and cynomolgus monkeys and exhibited excellent antitumor efficacy against a series of tumor cell lines when dosed orally in nude mice.

Structure-activity relationship of 3-substituted N-(pyridinylacetyl)-4- (8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene )- piperidine inhibitors of farnesyl-protein transferase: design and synthesis of in vivo active antitumor compounds.

Novel tricyclic Ras farnesyl-protein transferase (FPT) inhibitors are described. A comprehensive structure-activity relationship (SAR) study of compounds arising from substitution at the 3-position of the tricyclic pyridine ring system has been explored. In the case of halogens, the chloro, bromo, and iodo analogues 19, 22, and 28 were found to be equipotent. However, the fluoro analogue 17 was an order of magnitude less active. Whereas a small alkyl substituent such as a methyl group resulted in a very potent FPT inhibitor (SCH 56580), introduction of bulky substituents such as tert-butyl, compound 33, or a phenyl group, compound 29, resulted in inactive FPT inhibitors. Polar groups at the 3-position such as amino 5, alkylamino 6, and hydroxyl 12 were less active. Whereas compound SCH 44342 did not show appreciable in vivo antitumor activity, the 3-bromo-substituted pyridyl N-oxide amide analogue 38 was a potent FPT inhibitor that reduced tumor growth by 81% when administered q.i.d. at 50 mpk and 52% at 10 mpk. These compounds are nonpeptidic and do not contain sulfhydryl groups. They selectively inhibit FPT and not geranylgeranyl-protein transferase-1 (GGPT-1). They also inhibit H-Ras processing in COS monkey kidney cells and soft agar growth of Ras-transformed cells.

Influence of food on the oral bioavailability of loratadine and pseudoephedrine from extended-release tablets in healthy volunteers.

The effect of a high-fat breakfast on the bioavailability of the components of an extended-release tablet containing 10 mg loratadine in the immediate-release coating and 240 mg pseudoephedrine sulfate in the extended-release core was studied in 24 healthy male volunteers in a single-dose, two-way crossover study. The drug was administered after a 10-hour overnight fast or within 5 minutes of consuming a standardized high-fat breakfast. Serial blood samples were collected over a 48-hour period, and plasma was analyzed for loratadine and its active metabolite descarboethoxyloratadine (DCL), and pseudoephedrine. For pseudoephedrine, maximum concentration (Cmax) and area under the concentration-time curve extrapolated to infinity (AUCzero-infinity) were similar after both treatments, indicating no relevant food effect on the bioavailability of pseudoephedrine. Also, the absorption profiles of pseudoephedrine (from Wagner-Nelson analysis) were similar for the fed and fasted treatments, indicating no apparent differences in absorption. Plasma concentration-time profiles and values for Cmax and AUCzero-infinity of DCL were similar for the two treatments, indicating no relevant food effect on the pharmacokinetics of DCL. In contrast, for loratadine, administration with food resulted in a significantly increased mean Cmax (53%) and AUC from time zero to the final quantifiable sample (AUCif) (76%). However, the resultant Cmax and AUC of loratadine under fed conditions were well below those previously obtained at steady-state after multiple-dose administration of loratadine (40 mg/day) that were shown to be safe and well-tolerated in several clinical studies. The effect of food on the bioavailability and pharmacokinetic profiles of the components of a combination loratadine/pseudoephedrine extended-release tablet is not likely to be clinically significant.

Single-dose pharmacokinetics of isepamicin in young and geriatric volunteers.

Isepamicin is a new aminoglycoside antibiotic with activity against both gram-negative and gram-positive bacteria. The pharmacokinetics of isepamicin were evaluated after a 0.5-hour intravenous infusion of alpha 15-mg/kg dose to groups of young adults and geriatric volunteers. Isepamicin was safe and well tolerated. No adverse events related to the infusion were reported. As age increased, there were increases in the elimination phase half-life (t1/2 beta) and the area under the plasma concentration-time curve extrapolated to infinity (AUC0-infinity), and decreases in systemic (Cl) and renal clearance (Clr). The changes seen in Cl with age were a result of changes in renal function estimated by creatinine clearance (Clcr). There were no apparent correlations between age and maximum plasma concentration (Cmax), half-life of the tau-phase (t1/2 tau), volume of distribution at steady-state (Vdss), or the amount of isepamicin excreted in urine within 24 hours after dose administration (Ae24 hrs). When comparing the elderly (61-80 years old) with the younger (21-60 years) volunteers, the (AUC0-infinity), and t1/2 beta values were higher in the elderly and the Cl and Clr values were lower, but Cmax, t1/2 tau and Vdss were similar in the two age groups. The contribution of the tau-phase to the overall AUC was minimal and similar for the two age groups. Also, there were no gender effects on the pharmacokinetics of isepamicin in both the young and elderly volunteers. These results demonstrate that changes in the pharmacokinetics of isepamicin in the elderly are attributable to changes in renal function, whereas age, per se, is not a significant factor.

Effects of SCH 59228, an orally bioavailable farnesyl protein transferase inhibitor, on the growth of oncogene-transformed fibroblasts and a human colon carcinoma xenograft in nude mice.

The products of the Ha-, Ki-, and N-ras proto-oncogenes comprise a family of 21 kDa guanine nucleotide-binding proteins which play a crucial role in growth factor signal transduction and in the control of cellular proliferation and differentiation. Activating mutations in the ras oncogenes occur in a wide variety of human tumors. Ras proteins undergo a series of posttranslational processing events. The first modification is addition of the 15-carbon isoprene, farnesyl, to a Cys residue near the carboxy-terminus of Ras. Prenylation allows the Ras oncoprotein to localize to the plasma membrane where it can initiate downstream signalling events leading to cellular transformation. Inhibitors of the enzyme which catalyzes this step, farnesyl protein transferase (FPT), are a potential class of novel anticancer drugs which interfere with Ras function. SCH 59228 is a tricyclic FPT inhibitor which inhibits the farnesylation of purified Ha-Ras with an IC50 of 95 nM and blocks the processing of Ha-Ras in Cos cells with an IC50 of 0.6 microM. SCH 59228 has favorable pharmacokinetic properties upon oral dosing in nude mice. The in vivo efficacy of SCH 59228 was evaluated using a panel of tumor models grown in nude mice. These included several rodent fibroblast lines expressing mutationally-activated (val12) forms of the Ha-Ras oncogene. In some cases, these proteins contain their native C-terminal sequence (CVLS) which directs farnesylation. In one model, the C-terminal sequence was altered to CVLL, making the expressed protein a substrate for a distinct prenyl transferase, geranylgeranyl protein transferase-1. When dosed orally at 10 and 50 mg/kg (four times a day, 7 days a week) SCH 59228 significantly inhibited tumor growth of cells expressing farnesylated Ha-Ras in a dose-dependent manner; over 90% growth inhibition was observed at the 50 mg/kg dose. Tumor growth of cells expressing the geranylgeranylated form of Ha-Ras was less potently inhibited. Growth of tumors derived from a rodent fibroblast line expressing activated Ki-Ras containing its native C-terminal sequence (CVIM), which preferentially directs farnesylation, was also inhibited by SCH 59228. Inhibition in the Ki-Ras model was less than that observed in the Ha-Ras model. In contrast, tumors derived from cells transformed with the mos oncogene were not significantly inhibited even at the highest dose level. SCH 59228 also significantly and dose-dependently inhibited the growth of human colon adenocarcinoma DLD-1 xenografts (which express activated Ki-ras). These results indicate that SCH 59228 possesses in vivo antitumor activity upon oral dosing in tumor models expressing activated ras oncogenes. This is the first report of oral antitumor activity with an FPT inhibitor. These results are discussed in light of recent observations on alternative prenylation of some Ras isoforms.

Studies on the metabolism of the neurotoxic tri-o-cresyl phosphate. Synthesis and identification by infrared, proton nuclear magnetic resonance and mass spectrometry of five of its metabolites.

Five metabolites of the industrial neurotoxic chemical tri-o-cresyl phosphate (TOCP) were synthesized and their structures were verified by infrared, IR; proton nuclear magnetic resonance, 1H-NMR; and mass spectrometry. The 2 acids, o-cresyl dihydrogen phosphate and di-o-cresyl hydrogen phosphate were prepared in 2 steps. Step 1, POCl3 was reacted with o-cresol, using 1:1 and 1:2 molar ratios, in the presence of anhydrous AlCl3 as a catalyst, to form the 2 intermediates o-cresyl phosphorodichloridate and di-o-cresyl phosphorochloridate, respectively. Step 2, the chloridate intermediates were hydrolyzed under the appropriate condition to the corresponding acids. These acids were further derivatized to the corresponding methyl ester and the products were analyzed by the spectroscopic techniques. Saligenin cyclic-o-tolyl phosphate [2-(o-cresyl)-4H-1:3:2-benzodioxaphosphoran-2-one] was synthesized by reacting the potassium salt of o-hydroxybenzyl alcohol with o-cresyl phosphorodichloridate. Hydroxymethyl TOCP [di-o-cresyl o-hydroxymethylphenyl phosphate] and dihydroxymethyl TOCP [o-cresyl di-o-hydroxymethylphenyl phosphate] were synthesized by reacting di-o-cresyl phosphorochloridate with the potassium salt of o-hydroxybenzyl alcohol. The products were separated and purified by repeated preparative thin-layer chromatography (TLC) using 3 different solvent systems. The purity of the 5 metabolites, which was determined by high performance liquid chromatography (HPLC), ranged from 92% to 99%.

Studies on the metabolism of the neurotoxic tri-o-cresyl phosphate. Distribution, excretion, and metabolism in male cats after a single, dermal application.

The metabolism of a single, dermal dose of 50 mg/kg of [14C]tri-o-cresyl phosphate (TOCP) was studied in male cats. TOCP was applied to an unprotected, preclipped area on the back of the neck. Three animals were sacrificed on each of 0.5, 1, 2, 5, and 10 days following application. Radioactivity disappeared biexponentially from the dosing site with a faster initial rate; 73% of the dose disappeared in the first 12 h followed by a slower phase with a half-life of 2.03 days. No radioactivity was detected in the expired air. TOCP was absorbed from the skin and subsequently distributed throughout the body. Generally, the highest concentrations of radioactivity were associated with bile, gall bladder, urinary bladder, kidneys, and liver; the lowest were found in the neural tissues, muscle, and spleen. Within the 10-day experimental period, approximately 28% and 20% of the applied dose were recovered in the urine and feces, respectively. TOCP and its metabolites in the urine, feces, bile, and plasma were analyzed by high performance liquid chromatography and liquid scintillation counting. TOCP was the predominant compound in the feces (26.3% of total fecal radioactivity); it was detected in a smaller percentage in the urine (2.3% of total urinary radioactivity). The major metabolite in the urine was o-cresol followed by di-o-cresyl hydrogen phosphate and o-cresyl dihydrogen phosphate; in the feces di-o-cresyl hydrogen phosphate was the predominant metabolite followed by o-cresyl dihydrogen phosphate. Trace amounts of saligenin cyclic-o-tolyl phosphate, hydroxymethyl, and di(hydroxymethyl) TOCP were also detected in the urine and feces. Other metabolites identified in the urine and feces were the stepwise oxidation products of the methyl group of o-cresol. Unlike the feces, the bile contained mostly metabolites with only trace amounts of TOCP detected at 12 h and 24 h following application. o-Cresyl dihydrogen phosphate and di-o-cresyl hydrogen phosphate were the prevalent metabolites in the bile and plasma. While di(hydroxymethyl) TOCP was present in trace amounts in plasma, an appreciable amount of saligenin cyclic-o-tolyl phosphate, believed to be the active neurotoxic metabolite, was detected. This study shows that skin is an important port of entry for TOCP. Since TOCP represents organophosphorous chemicals capable of producing delayed neurotoxicity in test animals and in humans, it is essential that industrial hygiene control prevents skin contamination of workers handling these chemicals.

Absorption, distribution, and elimination of a single oral dose of [14C]tri-o-cresyl phosphate in hens.

The absorption, distribution, elimination, and metabolism of a single oral dose of 50 mg (4.6 microCi)/kg of uniformly phenyl-labeled [14C]tri-o-cresyl phosphate (TOCP) was investigated in adult chickens. Three treated hens were killed at each time interval: 0.5, 1, 2, and 5 days. TOCP was absorbed from the gastrointestinal tract and subsequently distributed throughout the body. Generally, the highest concentrations of radioactivity were associated with gastrointestinal tract parts, bile, kidneys, liver, and lungs. Most of the radioactivity (47%) was excreted in the combined fecal-urinary excreta during the first 12 h. Very small fractions of the dose were deposited in egg albumen and egg yolk, 0.12% and 0.24%, respectively during the 5-day study. After 5 days, 99% of the dose was eliminated in excreta. TOCP and its metabolites in bile and the combined fecal-urinary excreta were analyzed by high-performance liquid chromatography and liquid scintillation spectrometry. TOCP and nine of its metabolites were identified. In the bile a TOCP active metabolite, saligenin cyclic-o-cresyl phosphate, was the predominant compound found compared to the parent compound in the excreta. These results suggest that in the hen TOCP is excreted slower than the rat and also undergoes metabolic activation. The absorption, elimination, and metabolic profile of TOCP in the hen may contribute to its sensitivity to delayed neurotoxicity.

Absorption, distribution, excretion and metabolism of a single oral dose of [14C]tri-o-cresyl phosphate (TOCP) in the male rat.

A single oral dose of 50 mg/kg of [14C]TOCP was administered in corn oil to male rats. Three animals were sacrificed at each of 2, 6 and 12 h and 1, 2 and 5 days following dosing, and tissues and excreta were analyzed for 14C. Within 5 days, 63 and 36% of the dose were recovered in the urine and feces, respectively. Initially, the highest concentrations of radioactivity were observed in the gastrointestinal tract, its contents, the urinary bladder, liver and kidneys. Appreciable concentrations of 14C were detected in plasma, red blood cells, lungs and adipose tissues, while neural tissues, muscle, spleen and testes contained lower concentrations of radioactivity. Among neural tissues, the sciatic nerve contained the highest concentrations of 14C at all time points studied. The concentration of TOCP in plasma was at maximum by 6 h then declined biexponentially with terminal half-life of 46 h. The predominant metabolites in plasma were o-cresyl dihydrogen phosphate, di-o-cresyl hydrogen phosphate and o-hydroxybenzoic acid (salicylic acid). Small concentrations of the neurotoxic metabolite of saligenin cyclic-o-tolyl phosphate, were detected in plasma at all but the last time point analyzed. Most of the radioactivity extracted from the livers of rats sacrificed at 2 and 4 h were metabolites. No TOCP was detected in the urine or feces collected within 3 days after dosing. The major metabolite in the urine and feces was o-cresyl dihydrogen phosphate followed by di-o-cresyl hydrogen phosphate, salicylic acid, o-hydroxybenzyl alcohol and o-cresol. This study supports the hypothesis that the insensitivity of the rat to TOCP-induced delayed neurotoxicity may be attributed, in part, to the disposition and metabolism of this chemical.

Pharmacokinetic screening for the selection of new drug discovery candidates is greatly enhanced through the use of liquid chromatography-atmospheric pressure ionization tandem mass spectrometry.

Selection of a new drug discovery candidate from a series of compounds requires a means of performing rapid analytical method development and sensitive quantitation of each drug in serum, plasma or other biological matrices. Information on serum/plasma concentration, bioavailability and half-life can often aid the discovery process by selecting those candidates with the desired pharmacokinetic parameters. In one series of farnesyl protein transferase (FPT) inhibitors, gas chromatography with nitrogen-phosphorus detection (NPD) was initially used to analyze samples from pharmacokinetic studies in mice and monkeys. Typical turnaround times using this technique approached 2-4 weeks for method development, quantitation of study samples and calculation of pharmacokinetic parameters. Once LC-atmospheric pressure ionization (API) MS-MS analysis was implemented in these same studies, they could be completed in less than one week. The advantages of using LC-API-MS-MS to aid in the drug candidate selection process is demonstrated for one compound (SCH 44342) in this series of FPT inhibitors.

Permeability of lipophilic compounds in drug discovery using in-vitro human absorption model, Caco-2.

Highly lipophilic compounds are often encountered in the early stages of drug discovery. The apparent permeability (Papp) of these compounds in Caco-2 cell could be underestimated because of considerable retention by the Caco-2 monolayer and non-specific binding to transwell surface. We have utilized a general approach for the determination of permeability of these compounds, which includes the addition of 1-5% DMSO in the apical (AP) and 4% bovine serum albumin (BSA) in the basolateral (BA) side. Two highly lipophilic and highly protein bound Schering compounds, SCH-A and SCH-B, exhibited poor recovery and low Papp in the conventional Caco-2 system that included 1% DMSO in the AP and BA sides. In contrast, both compounds were well absorbed in cynomolgus monkeys. Inclusion of BSA (up to 4%) in the BA side provided necessary absorptive driving force similar to in vivo sink conditions improving both recovery and Papp of these compounds as well as progesterone, a model highly lipophilic and highly protein bound compound. Whereas, the recovery and Papp of mannitol (high recovery, low permeability) and propranolol (high recovery, high permeability) remained unaffected. The presence of 4% BSA increased Papp of SCH-A, SCH-B, and progesterone by five-, four-, and three-fold, respectively. We also compared this approach with a second, based on the disappearance of the compound from the AP side, which resulted in a reasonable estimate of the permeability (23.3x10(-6) cm/s) for SCH-A. The results demonstrated that the reliable estimates of permeability of highly lipophilic compounds that are subjected to considerable retention by the cell monolayer and exhibit non-specific binding are obtained by the addition of BSA to the BA side.

High-performance liquid chromatographic analysis and stability of anti-tumor agent temozolomide in human plasma.

Temozolomide (SCH 52365; TEMODAL) is an antineoplastic agent with activity against a broad spectrum of murine tumors. This compound is currently marketed in the European Union for the treatment of patients with glioblastoma multiforme and anaplastic astrocytoma, which are serious and aggressive types of brain cancers. It has been postulated that temozolomide exerts its in vivo activity via the decomposition product MTIC, which is believed to alkylate nucleophiles, and in the process is converted to AIC. A high-performance liquid chromatographic (HPLC) method was developed and validated for the analysis of temozolomide in human plasma. The determination of temozolomide involved extraction with ethyl acetate followed by separation on a reversed phase C-18 column and quantification by UV absorbance at 316 nm. The calibration curve was linear over a concentration range of 0.1-20 microg/ml. The limit of quantitation was 0.1 microg/ml, where the coefficient of variation (CV) was 0% and the bias was 10.0%. The method was precise with a coefficient of variation ranging from 2.5 to 6.9% and accurate with a bias ranging from 5.0 to 10.0%. Temozolomide was unstable at 37 degrees C in human plasma with a degradation t1/2 of 15 min; however, it was stable at 4 degrees C for at least 30 min. Temozolomide was stable in acidified human plasma (pH < 4) for at least 24 h at 25 degrees C, and for at least 30 days at -20 degrees C. Moreover, temozolomide was stable in acidified human plasma after being subjected to three freeze thaw cycles. The assay was shown to be specific, accurate, precise, and reliable for use in pharmacokinetic studies.

Liquid chromatographic analysis, stability and protein binding studies of the anti-HIV agent benzoic acid, 2-chloro-5[[(1-methylethoxy)thioxomethyl]amino]-,1-methylethyl ester.

A method was developed for the assay of benzoic acid, 2-chloro-5-[[(1-methylethoxyl)thioxomethyl]amino]-,1-meth yle thyl ester (NSC 629243) in hamster, mouse, human and, to a limited extent, dog plasma. Protein in 0.5 ml of plasma was precipitated with four volumes of methanol and the supernatant was analysed for NSC 629243 by LC. Liquid chromatography was carried out on a reversed-phase Nova-Pak C18 column, with a mobile phase of 60% acetonitrile in water at 1 ml min-1, and the compound was quantified with a UV detector set at 283 nm. Two standard curves of NSC 629243 were needed to cover a concentration range of 0.05-100 micrograms ml-1. All standard curves had correlation coefficients > 0.999. In practice, the minimum quantifiable concentration was approximately 0.05 microgram ml-1 in 0.5 ml of plasma. NSC 629243 appeared to have good stability at 37 degrees C in hamster, human and dog plasma at concentrations of 1 and 50 micrograms ml-1 (at least 80% remained in plasma after a 4 h incubation). Breakdown occurred in mouse plasma after 1 h at 37 degrees C, with extensive breakdown occurring after 24 h. NSC 629243 was extensively bound to plasma proteins of Syrian hamsters and humans. The extent of binding ranged from a minimum of 88.6% to a maximum of 99.9% over a concentration range of ca 1-100 micrograms ml-1.

Liquid chromatographic analysis in mouse, dog and human plasma; stability, absorption, metabolism and pharmacokinetics of the anti-HIV agent 2-chloro-5-(2-methyl-5,6-dihydro-1,4-oxathiin-3-yl carboxamido) isopropylbenzoate (NSC 615985, UC84).

NSC 615985 (UC 84) has demonstrated anti-HIV activity in the NCI-AIDS antiviral screen and was under consideration as an anti-AIDS drug. The compound was subsequently shown to be a non-nucleoside reverse transcriptase inhibitor (NNRTI). An HPLC method was developed for the analysis of NSC 615985 in mouse, dog and human plasma; and was used to study its stability in plasma and blood as well as its absorption and metabolism in mice. The method involved precipitation of plasma protein with three volumes of methanol followed by HPLC analysis of the supernatant. The HPLC analysis was carried out on a reversed-phase Nova-Pak C18 column with a mobile phase of KH2PO4 (0.01 M; pH 4.8)-acetonitrile (52:48, v/v) at a flow rate of 1 ml min-1 and quantification with a UV detector set at 259 nm. The lower limit of quantitation was 0.05 microgram ml-1 in 1 ml of dog or human plasma or 0.1 microgram ml-1 in 0.5 ml of mouse plasma. NSC 615985 was more stable in dog and human plasma than in mouse plasma, and was less stable in blood than in plasma of the three species investigated. Following bolus intravenous (i.v.) administration at 10 mg kg-1 to male CDF1 mice, NSC 615985 elimination followed biexponential kinetics with half-lives of 1 and 7 min, and was extensively metabolized. NSC 615985 was very poorly absorbed following oral (PO) administration as a suspension in water or in 20% lipid emulsion (Liposyn II). Following bolus subcutaneous (s.c.) administration of [14C]NSC 615985 at 10 mg kg-1, relatively low concentrations of the parent compound were observed in three of 36 mice. One metabolite was tentatively identified in plasma of both the i.v.- and s.c.-treated animals as the sulfoxide of the parent compound. No parent compound was detected in the urine of NSC 615985 dosed mice. At least seven metabolites were present in urine; one metabolite (constituting 8-14% of urinary radioactivity) was tentatively identified as the carboxylic acid resulting from the hydrolysis of the isopropyl group from the parent compound. In summary, NSC 615985 was poorly absorbed following oral administration and extensively metabolized and eliminated following i.v. or s.c. administration. This unfavorable pharmacokinetic profile of NSC 615985 as well as its pattern of activity against NNRTI-resistant strains of HIV-1 precluded its progression to clinical trial; however, other members of the general chemical class are currently being evaluated by the NCI.

Analysis of dimethyl hydrogen phosphite and its stability under simulated physiological conditions.

In a study by the National Toxicology Program, dimethyl hydrogen phosphite (DMHP) was shown to cause dose-related neoplastic lesions in the lungs and forestomachs of Fischer 344 rats. This investigation was carried out to study the stability of this carcinogen under conditions similar to those that the chemical may encounter in the physiological environment. To carry out these studies, capillary gas chromatography was utilized to analyze DMHP. The method was linear over a range of 10 to at least 1000 ng. High-performance liquid chromatography (HPLC) also was used to analyze DMHP and its degradation products. In aqueous solutions, DMHP was stable for a period of time before degradation began. Once the process began, degradation continued until 10-20% of the original concentration was reached and further decomposition was minimal. At 10% concentration in 0.1M phosphate buffer, pH 7.4, DMHP was stable for 3.6 h at 37 degrees C. This stability period increased at lower temperature, at lower DMHP concentration, and in slightly more alkaline buffer (pH 8). After the stability period, DMHP disappeared from the solution and this disappearance followed a first order kinetics with a rate dependent upon temperature, concentration of DHMP, and the pH of the solution. The half-time of disappearance of DMHP under the conditions mentioned above was 2.4 h, which increased at lower temperature, at lower DMHP concentration, and in slightly more alkaline buffer (pH 8). The decomposition products of DMHP were identified by HPLC and proton nuclear magnetic resonance (1H-NMR) spectroscopy as methanol, monomethyl hydrogen phosphite, and orthophosphorous acid.