The discovery of [1-(4-dimethylamino-benzyl)-piperidin-4-yl]-[4-(3,3-dimethylbutyl)-phen yl]-(3-methyl-but-2-enyl)-amine, an N-type Ca+2 channel blocker with oral activity for analgesia.

Our drug discovery efforts for N-type calcium channel blockers in the 4-piperidinylaniline series led to the discovery of an orally active analgesic agent 26.1-[4-Dimethylamino-benzyl)-piperidin-4-yl]-[4-(3,3-dimethyl-but yl)-phenyl]-(3-methyl-but-2-enyl)amine (26) showed high affinity to functionally block N-type calcium channels (IC50=0.7 microM in the IMR32 assay) and exhibited high efficacy in the anti-writhing analgesia test with mice (ED50=12 mg/kg by po and 4 mg/kg by iv). In this report, the rationale for the design, synthesis, biological evaluation, and pharmacokinetics of this series of blockers is described.

Syntheses and biological activities of chiral piperidines-tachykinin NK3 antagonists.

AIM: To develop nonpeptide tachykinin NK3 antagonists. METHODS: Five tachykinin NK3 antagonists were synthesized. Receptor binding assay and oral absorption study were made. RESULTS: The 4,4-disubstituted piperidine compounds (1b, 1c, and 1d) showed stronger activities (IC50 = 5.9, 6.2, and 11 nmol.L-1, respectively) than the monosubstituted ring compound 1e (IC50 = 17 nmol.L-1). 4-Phenyl (1b) and 4-phenylsulfonylmethyl (1c) compounds were more active than the 4-fluorobenzyl compound (1d). All antagonists were found to be orally absorbable, the T1/2 of 1b (6.4 h) was more than three-fold longer than that of 1a (1.9 h). CONCLUSION: Compound 1b had the best binding activity (IC50 = 5.9 nmol.L-1) and the best AUC (2081 micrograms.h.L-1).

Sample pooling to enhance throughput of brain penetration study.

The advent of combinatorial synthesis and high throughput screening in pharmaceutical research has inevitably given rise to a large number of interesting prelead compounds that requires rapid analytical throughput for kinetic characterization. The traditional approach of one-compound-at-a-time bioanalysis has not been able to meet the demand for high productivity of pharmacokinetic screening. This report demonstrates the application of sample pooling in expediting the pharmacokinetic screening, including assessment of brain penetration, of six NK1 receptor antagonists in rats: CAM 6108 (C1), CAM 6122 (C2), CAM 6178 (C3), CAM 5825 (C4), CAM 6182 (C5), and CAM 6121 (C6). The approach was adopted to avoid complications associated with cocktail dosing where multiple compounds are administered to one animal. The present investigation features individualized dosing (one compound per animal), followed by sample pooling of brain and plasma and bioanalysis via a conventional LC-fluorescence method. Rats were dosed intravenously with each of the six NK1 receptor antagonists and blood and brain samples were harvested at suitable post-dose time intervals. Plasma or brain homogenate samples from the same time points were pooled into two groups (C1-C3 and C4-C6) for assay. Drug compounds in plasma or brain were extracted by protein precipitation and quantitated using a validated gradient HPLC/fluorescence method, which was made feasible for both groups of compounds with a modification in gradient scheme. Plasma assay precision and accuracy for C1-C6 were < or =4.7% and within +/-9.8%, respectively. Brain homogenate assay accuracy for C1-C6 was within +/-7.0%. Brain penetration of these compounds was evaluated as the AUC of brain and plasma and their respective brain/plasma AUC ratio. The sample pooling approach helped to quickly identify C1 as the NK1 receptor antagonist with the greatest extent of brain penetration, followed by C2, C6, C4, C5, and C3 in that order. By employing sample pooling approach, pharmacokinetic parameters and brain penetration of all six compounds were obtained in a fraction of the time required by conventional single compound dosing and analysis.

Development and application of sensitive HPLC assays for NK3 antagonists in rat plasma.

CAM 5500 and CAM 5187 are new nonpeptide tachykinin NK3 receptor antagonists with different lipophilicity and solubility. We have developed and validated two separate, simple HPLC methods for quantitation of these two compounds in plasma to support oral pharmacokinetic/bioavailability studies in rats. The two compounds in plasma were extracted on cyano SPE cartridges with different washing schemes to optimize extraction efficiency and chromatographic specificity. The analytes and internal standard in the resulting extracts were chromatographed on a C18 HPLC column, using mobile phases containing different phosphate buffer strengths and acetonitrile concentrations. Both compounds were detected using UV, Peak area ratios were proportional over the concentration range of 50-3000 ng ml-1 for CAM 5500, and 100-1500 ng ml-1 for CAM 5187. Stability profiles of both drugs and internal standard in rat plasma at 37 degrees C and in injection solvent at ambient temperature were good. Assay precision, based on quality controls, was < 5.6% and 13.4% (%RSD) for CAM 5500 and CAM 5187, respectively. Similarly, assay accuracy for both compounds was within +/- 7.1% and +/- 6.0% (%RE), respectively. The HPLC methods were successfully applied to assay samples from two oral bioavailability studies. Oral bioavailability studies were conducted for each compound in rats receiving a PO dose of 20 mg kg-1 or an i.v. dose of 5 mg kg-1. Despite their difference in lipophilicity and solubility, the absolute oral bioavailability of CAM 5500 (5.3 +/- 4.8%) is similar to that of CAM 5187 (8.8% +/-3.2%).

Sample pooling to expedite bioanalysis and pharmacokinetic research.

In the progression from drug discovery to development, not only pharmacokinetic (PK) characterization needed for lead compound selection often becomes a rate-limiting step, but also high volume of routine sample analysis ensued from numerous required biodisposition studies for the lead compounds and their back-ups often place a burdensome hurdle to the throughput of IND and NDA development phases. Higher throughput of PK screening via cocktail dosing has been reported to accelerate PK screening in the discovery phase. However, concerns on drug-drug interactions and other limitations associated with the cocktail M-in-One dosing (multiple compounds per dose per animal) has prompted the present investigation of sample pooling alongside One-in-One dosing strategy (one compound per dose per animal) as an alternative to the cocktail dosing approach. Using traditional HPLC for bioanalysis as an example, the present study illustrate the concept and usefulness of sample pooling that could facilitate the throughput of PK screening and characterization in both discovery and development phases. Six proprietary dopamine D4 receptor antagonist preleads representing three different chemical classes, used as model compounds (C1-C6), were administered orally to rats. One rat received one compound and three rats were used for each compound. Six unknown plasma samples from six different rats at each time point were pooled. The pooled plasma samples were extracted by a one-step liquid-liquid extraction and concentrations of the six preleads were quantitated simultaneously. By sample pooling, a substantial amount of PK information was obtained at the same time for the six preleads, which requires much less workload than when bioanalysis is dealt with one compound at a time. For the first time in one aspect of innovative bioanalysis, the present investigation has demonstrated that sample pooling following One-in-One dosing can be utilized to enhance the throughput rate in PK screening in discovery phase. The sample pooling approach is likely to be useful in enhancing the throughput of PK characterization in development phase. With the advent of LC-MS and its becoming user-friendly, where separation of drug compounds is no longer an issue, the uniqueness of sample pooling may also pose a new way of thinking in regard to the old ways of handling bioanalysis for traditional PK research.

Perturbation of epidermal growth factor clearance after radioiodination and its implications.

The clearance of human epidermal growth factor (hEGF1-53) has been thought to be mediated mainly by a high-capacity receptor system, yet relatively low in vivo clearance rates (<10 mL/min/kg) and long terminal elimination half-lives (>120 min) have been observed in rats receiving the peptide that was iodinated by the oxidative chloramine-T (CT) method. We investigated if a mild, less oxidative iodination by the lactoperoxidase (Enzymobeads, EB) method, which is known to yield an iodinated peptide with receptor-binding equivalence, could produce a labeled peptide that behaves pharmacokinetically similar to the native material. For comparison, a parallel study was also conducted with EB-125I-hEGF1-48, which in its native form has a much reduced receptor binding activity due to the loss of the C-terminal pentapeptide. Plasma radioactivity concentrations were determined by trichloroacetic acid (TCA) precipitation and immunoprecipitation. Rats cleared unlabeled hEGF1-53 and hEGF1-48 markedly faster (CL(tot) > 120 mL/min/kg) than their radiolabeled counterparts. Approximately 96% of the hEGF1-53 dose was cleared during the initial phase (0-4 min), as opposed to only 5-14% for the iodinated peptide. Similar change was also observed for EB-125I-hEGF1-48 and CT-125I-hEGF1-53. The pharmacokinetic behavior of EB-125I-hEGF1-53 was, in fact, comparable to that of CT-125I-hEGF1-53. These observations indicate that receptor-binding equivalence does not have direct relationship with in vivo EGF clearance. Both iodination methods (oxidative CT and less oxidative EB) might have perturbed one or more steps in the cascade of ligand-receptor internalization and intracellular procession, which in turn modified the disposition of the peptides. In addition, the two independent precipitation techniques for the same peptide generated different kinetic outcomes. The overall experimental results suggest that it is unacceptable to use an iodinated form to characterize the disposition of peptides/proteins like EGF with a specific receptor system mediating its clearance.

The tachykinin NK1 receptor antagonist PD 154075 blocks cisplatin-induced delayed emesis in the ferret.

The activity of a selective tachykinin NK1 receptor antagonist, PD 154075 ([(2-benzofuran)-CH2OCO]-(R)-alpha-MeTrp-(S)-NHCH(CH3) Ph), was examined in radioligand binding studies, in a [Sar9,Met(O2)11]substance P-induced foot-tapping model in the gerbil, and in cisplatin-induced acute and delayed emesis in the ferret. In radioligand binding studies, PD 154075 showed nanomolar affinity for the human, guinea-pig, gerbil, dog and ferret NK1 receptors with an approximate 300 times lower affinity for the rodent NK1 receptor. Using NK2,NK3 receptors and a range of other receptor ligands, PD 154075 was shown to exhibit a high degree of selectivity and specificity for the human type NK1 receptor. Following subcutaneous administration PD 154075 dose dependently (1-100 mg/kg) antagonised the centrally mediated [Sar9,Met(O2)11] substance P-induced foot tapping in the gerbil with a minimum effective dose (MED) of 10 mg/kg. The ability of PD 154075 to readily penetrate into the brain following oral administration was confirmed by its extraction and high performance liquid chromatography assay from the rat brain. PD 154075 was shown to achieve a relatively fast and sustained brain concentration (brain/plasma ratios ranged from 0.27 to 0.41 during the time period of 0.25-12 h). Further pharmacokinetic studies revealed that the absolute oral bioavailability of PD 154075 in the rat was (mean +/- S.D.) 49 +/- 15%. PD 154075 (1-30 mg/kg, i.p.) dose dependently antagonised the acute vomiting and retching in the ferret measured for 4 h following administration of cisplatin (10 mg/kg, i.p.) with a MED of 3 mg/kg. The administration of a lower dose of cisplatin (5 mg/kg, i.p.) in the ferret induces both an acute (day 1) and delayed (days 2 and 3) phase of emesis. The i.p. administration of PD 154075, 10 mg/kg three times a day for 3 days, almost completely blocked both the acute and delayed emetic responses. In the same study, the 5-HT3 receptor antagonist ondansetron (1 mg/kg, i.p., t.i.d.) was also very effective against the acute emetic response observed during the first 4 h following cisplatin, but it was only weakly active against the delayed response. In conclusion, PD 154075 is a selective and specific high affinity NK1 receptor antagonist with good oral bioavailability which is effective against both acute and delayed emesis induced by cisplatin in the ferret.

Development of HPLC plasma assays for CAM 4515 and CAM 4750, two new nonpeptide tachykinin antagonists, and application to bioavailability studies.

CAM 4515 and CAM 4750 are new nonpeptide tachykinin NK1 receptor antagonists with different lipophilicities. Two separate, simple, and sensitive HPLC methods for the quantitation of these two compounds in plasma and the evaluation of their oral bioavailability in rats were developed and validated. Extraction of CAM 4515 from plasma involved protein precipitation with acetonitrile, while that for CAM 4750 involved a one-step liquid-liquid extraction with methylene chloride. The analytes in extracts were chromatographed on a C18 column using two different separation buffers, 47% 0.02 M sodium citrate (pH 3.5)-53% acetonitrile for CAM 4515 and 59% 0.02 M potassium phosphate dibasic (pH 7.0)-41% acetonitrile for CAM 4750, and both compounds were detected by fluorescence (excitation 278 nm; emission 342 nm). Stability profiles of both drugs at -20 degrees C or room temperature in plasma and in reconstituted buffers were good. The limit of quantitation for both drugs was 5 ng ml-1 with good linearity from 5 to 1000 ng ml-1 using 100-200 microliters of plasma. Excellent precision (relative standard deviation < 8.3%) and accuracy (relative error +/- 9.2%) were observed for both CAM 4515 and CAM 4750. Oral bioavailability studies were conducted for each compound in rats receiving a p.o. dose of 20 mg kg-1 and an i.v. dose of 5 mg kg-1. The absolute oral bioavailability of CAM 4750 (80%) was estimated to be 40-fold greater than that of CAM 4515 (2%). The experimental results suggest that incorporation of a pyridine group into the structural backbone may greatly improve bioavailability.

Development and validation of two solid-phase enzyme immunoassays (ELISA) for quantitation of human epidermal growth factors (hEGFs).

PURPOSE: The purpose of the present investigation was to develop and validate two separate enzyme-linked immunosorbent assays (ELISA) for quantitation of exogenous human epidermal growth factor (hEGF1-53) and its truncated fragment (hEGF1-48) in rat plasma. METHODS: The present assay systems were based on the sandwiching of the antigen between a monoclonal mouse anti-hEGF1-53 antibody, pre-coated on a 96-well polystyrene plate, and a polyclonal rabbit anti-hEGF1-48 antibody, which is then detected with a peroxidase-labeled goat anti-rabbit antibody. RESULTS: The calibration curves for hEGF1-48 and hEGF1-53 in plasma were validated over a concentration range of 7.8-250 and 62.5-1000 pg/ml, respectively. Determined from replicate assays of hEGF1-48 quality control samples, the intra-assay precision and accuracy were < or = 8.8% RSD and within +/- 9.8%; and the inter-assay precision and accuracy were < or = 14.8% RSD and within +/- 9.7% RE, respectively. Determined from replicate assays of hEGF1-53 quality control samples, the intra-assay precision and accuracy were < or = 10.0% RSD and within +/- 8.5%; and the inter-assay precision and accuracy were < or = 10.0% RSD and within +/- 5.7% RE, respectively. The limit of quantitation of the hEGF1-48 and hEGF1-53 assay using 200 microliters plasma per well is 7.8 and 62.5 pg/ml, respectively. These two ELISA methods are specific to hEGFs and do not cross-react with mouse EGF or other growth factors (TGF alpha, TGF beta, PDGF, and FGF) or lymphokines (IL1 beta and TNF alpha). These validated methods have been routinely applied to assay of plasma samples from various pharmacokinetic studies in rats receiving intravenous hEGFs. Both assay methods were also adapted to assay endogenous hEGFs in biological fluids of different animal species. CONCLUSIONS: Two sensitive ELISA methods have been validated for quantitation of hEGF1-53 and hEGF1-48 in rat plasma. Their utility has been demonstrated in the application of assaying immunoreactive concentration of exogenous and endogenous epidermal growth factors.

Absorption of Cam-2445, and NK1 neurokinin receptor antagonist: in vivo, in situ, and in vitro evaluations.

Cam-2445 is a selective, high-affinity NK1 receptor antagonist that is a potentially useful treatment for arthritis, asthma, migraine, anxiety, psychosis, and emesis. Cam-2445 exhibits low aqueous solubility and high lipophilicity and has a molecular weight of 470. Cam-2445 has poor oral bioavailability and the purpose of this research was to examine the potential barriers to the oral bioavailability of Cam-2445. Cam-2445 was relatively stable at 37 degrees C in 0.1 N HCl, 5 microM alpha-chymotrypsin, rat intestinal perfusate, and in rat jejunal brush border membrane suspension. High permeability was observed from CACO-2 cells and from rat single-pass intestinal perfusions. Cam-2445 was administered as a solution to rats by intravenous (i.v.), oral (p.o.), intraduodenal (i.d.), and intraportal (i.p.v.) routes. The total oral bioavailability was poor at 1.4%. Absorption appeared to be rapid after i.d. dosing; bioavailability was 26%, and 54% of the dose was absorbed intact into the portal system. After i.p.v. dosing, 48% of the dose was available to the systemic circulation. The elimination t1/2 after i.d. dosing (2.91 h) was comparable to that i.v. dosing (2.93 h), whereas it was significantly longer after p.o. dosing (12.4 h). The p.o. dose apparently precipitated in the gastrointestinal (GI) tract, resulting in low oral bioavailability. These results indicated that neither stability in the GI tract nor membrane transport were major obstacles to the absorption of Cam-2445. While hepatic extraction of 52% was significant, the low aqueous solubility of Cam-2445, as well as the differences noted between p.o. and i.d. studies, strongly support GI dissolution and/or precipitation as the limiting factor for the oral bioavailability of the compound.

Disposition kinetics of human epidermal growth factor (hEGF1-53) and its truncated fragment (hEGF1-48) in rats.

Clearance of human epidermal growth factor (hEGF1-53) has been proposed to be mediated by a receptor pathway involving a typical cascade of ligand-receptor endocytosis and lysosomal degradation. Deletion of the C-terminal pentapeptide from hEGF1-53, which yields hEGF1-48, is known to be associated with a marked reduction in receptor binding. We defined the intravenous (iv)-bolus (acute exposure) and the iv-infusion (prolonged exposure) pharmacokinetics of hEGF1-53 and hEGF1-48 in rats to investigate the impact of the deletion of C-terminal pentapeptide on the EGF clearance using a validated, sensitive ELISA method for quantitation of the peptides in plasma. Both peptides at the low iv bolus dose of 10 micrograms/kg were cleared from plasma with unusually high clearances (CLtot: 128 +/- 31 ml/min/kg for hEGF1-53 and 168 +/- 47 ml/min/kg for hEGF1-48), which are virtually complete within 4-min postdose, and the difference in the overall pharmacokinetics is of minor significance. A 10-fold increase in bolus dose to 100 micrograms/kg decreased clearances 3- to 6-fold, indicating a nonlinear kinetics for both peptides; however, hEGF1-48 was cleared (52 +/- 11 ml/min/kg) 2.5-fold faster than hEGF1-53. A similar nonlinear kinetics was also noticed for both peptides when they were administered by a 2-hr iv infusion at 30 and 300 micrograms/kg doses. hEGF1-48 at the low and high infusion doses was cleared at 126 +/- 16 and 33.7 +/- 14.5 ml/min/kg, respectively, which are 4-fold greater than the corresponding clearance rates of hEGF1-53. These observations suggest that a) deletion of C-terminal pentapeptide is associated with a faster clearance of the growth factor and b) the receptor clearance pathway may be more sensitive to saturation with hEGF1-53 than with hEGF1-48 at low microgram dose levels. hEGF1-53 at the low infusion dose of 30 micrograms/kg was cleared (32.1 +/- 6.2 ml/min/kg) 4-fold slower in comparison with the low bolus dose of 10 micrograms/kg, indicating a remarkable injection mode-dependent disposition kinetics for hEGF1-53, which does not exist for hEGF1-48. The overall results suggest that deletion of C-terminal pentapeptide leads to faster clearance of the growth factor, and the degree of the impact of deletion of C-terminal pentapeptide on the global pharmacokinetics is also dependent on the length of exposure of the receptor to the ligand. The negative relationship between receptor binding and plasma clearance for the two peptides remains to be elucidated at the molecular and receptor levels.

Impact of receptor downregulation on clearance of two human EGFs with different receptor binding activity.

Human epidermal growth factor [hEGF(1-53)] has been thought to be cleared mainly via an EGF receptor (EGFR) endocytosis pathway. Pretreatment of rats with hEGF(1-53) has been shown previously to cause a dramatic reduction in clearance of the peptide contributable to EGFR downregulation. The impact of receptor downregulation has raised concerns for rational design of dosage regimen for this potential wound-healing therapeutic peptide. However, following a similar protocol, we could not reproduce the dramatic reduction in clearance reported previously mediated by an i.v. bolus acute dose. As EGFR downregulation may be sensitive to the length of exposure and to the activation of the receptor tyrosine kinase activity, two other pretreatment protocols were also evaluated: a 4-h i.v. infusion (prolonged exposure) of the peptide and an i.v. bolus of a potent synthetic kinase inhibitor pretreatment were evaluated for effects on clearance. However, neither pretreatment affected the peptide's clearance profile. Further, no effects on clearance and other kinetic parameters were observed for any pretreatment paradigms with a truncated analogue hEGF (1-48), whose EGF receptor binding activity is much weaker but plasma clearance is much higher than hEGF (1-53). In addition, a study in a second rat strain showed no difference in clearance profile of hEGF-(1-53) following pretreatment. Results of the present investigation suggest that receptor binding does not have a direct relationship with plasma clearance, and that the EGF clearance mechanisms is highly refractory with EGF receptors possibly recovering rapidly from downregulation through the recycling process.

Considerations in the development of a sensitive HPLC assay for human epidermal growth factors in human plasma.

A sensitive assay was developed for human epidermal growth factors (hEGF) 1-48 (dosed), hEGF 1-53 (endogenous), without interference from potential metabolites hEGFs 1-47 or 1-46. Spiked human plasma samples were injected directly, utilizing on-line immunoaffinity HPLC (anti-hEGF) clean-up. No change in capacity was noted after 81 cycles. After release from the immunoaffinity column, the fragments were further resolved by strong cation-exchange (SCX) via a column switching valve. Method development also required interfacing immunoaffinity, ion-exchange, and detection components. Immunoassays on collected fractions yielded a detection limit of 1 microgram ml-1, although a detection limit of 75 pg ml-1 appears feasible.

Quantification of a dual angiotensin I-converting enzyme-neutral endopeptidase inhibitor and the active thiol metabolite in dog plasma by high-performance liquid chromatography with ultraviolet absorbance detection.

MDL 100,240 ([4S-[4 alpha,7 alpha(R*), 12b beta]]-7-[[2- (acetylthio)-1-oxo-3-phenylpropyl]amino]-1,2,3,4,6,7,8,12b-octahyd ro-6-oxo- pyrido[2,1-a][2]benzazepine-4-carboxylic acid, I) is the thioacetyl prodrug of the active thiol, MDL 100,173 (II), a dual inhibitor of angiotensin-I converting enzyme (ACE) and neutral endopeptidase (NEP). A drug which simultaneously inhibits both ACE and NEP may provide a unique therapy for hypertension and congestive heart failure. Methods based on high-performance liquid chromatography with UV absorbance detection at 200 nm were developed to support preclinical pharmacokinetic investigations. One method is used to measure unchanged I and free II, while the second method is used to quantify the total level of the thiol II after the plasma is incubated with the disulfide reducing agent, dithiothreitol. By either method, the analytes are quantified over the range of 25-1000 ng/ml with good accuracy and precision. The overall extraction efficiencies of unchanged I and free II in dog plasma were 79% and 86%, respectively, while the extraction efficiency of total II averaged 75%. Described in this report are the results obtained in validating the assay methods for measuring the compounds in plasma. Pharmacokinetic data are presented which were obtained by applying these methods to plasma collected from dogs dosed with I.

Disposition and cardioselectivity of MDL 74,405, a vitamin E-like free radical scavenger, in rats and dogs after intravenous infusion.

The disposition kinetics of MDL 74,405, a potent free radical scavenger for cardiac reperfusion and a vitamin E analog, was investigated in rats (1.2, 6.0, and 12 mg/kg) and dogs (1 and 10 mg/kg) after an intravenous infusion. Because the heart is the target site of drug action, a tissue distribution study was also conducted in rats (1.2 mg/kg) to explore the affinity of the drug to rat heart. In both animal species, plasma drug concentrations declined rapidly in the early distribution phase and exhibited a multiexponential pattern of elimination. Of the total excretion (95-96% of the dose) in 120 hr in rats, 45-51% was excreted in urine and 45-50% in feces. Of the total excretion (86-89% of the dose) in 120 hr in dogs, 41-43% of the dose was excreted in urine and 41-43% in feces. The dose was excreted mainly unchanged (62-78% in rat and 80-86% of the dose in dog urine) with several potential minor metabolites, indicating that renal and biliary excretions are the two major, equally important, routes of drug elimination in both species. Rats cleared the drug from the body markedly faster than dogs: the mean residence time in rats. (1.9-3.2 hr) was 15-20 times shorter, the terminal elimination half-life in rats (3.6-7.0 hr) was 10 times shorter; and the total clearance in rats (148-216 ml/min/kg) was 6-9 times greater. The steady-state volume of distribution was very large for both species. (19-37 liters/kg for rats and 59-64 liters/kg for dogs), which is consistent with the extensive tissue uptake of the drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of bioavailability and systemically available fractions of drugs undergoing reversible metabolism: application to 4-amino-5-chloro-2-[2-(methylsulfinyl)ethoxy]-N-[2- (diethylamino)ethyl]benzamide and its sulfide and sulfone metabolites in rats.

Methods are discussed which permit the calculation of the bioavailability (F) and fraction of an oral dose entering the central circulation (f) of a drug and its interconversion metabolite. The interrelationships between the F and f and between the F and systemically available fractions afforded by reversible metabolism are also derived and described. The application of these principles is illustrated by the pharmacokinetic analysis of 4-amino-5-chloro-2-[2-(methylsulfinyl)ethoxy]-N-[2- (diethylamino)ethyl]benzamide (ML-1035, 1) and its sulfide (2) and sulfone (3) metabolites in rats. Like intravenous ML-1035, ML-1035 administered orally underwent metabolic interconversion with 2 but not with 3 in this species. Both ML-1035 and 2 were absorbed rapidly and are pharmacologically active. On average, 8.3 and 13% of an oral dose (152.4 mumol/kg) of ML-1035 were bioavailable as ML-1035 and its sulfide metabolite, respectively, while 23 and 65% of a molar equivalent dose of the sulfide metabolite were bioavailable as either compound, respectively. Thus, the sulfide metabolite is better absorbed than ML-1035 in rats. Following oral administration of either ML-1035 or 2, the systemically available fractions of both compounds were weakly to moderately influenced by the reversible metabolism process in rats. Moreover, the bioavailability of the sulfone metabolite was very poor (2.5-4%) following separate oral administration of ML-1035, 2, and 3.

Pharmacokinetics and metabolic interconversion of intravenous 4-amino-5-chloro-2-[(methylsulfinyl)ethoxy]-N-[2-(diethylamino)ethyl] benzamide and its sulfide and sulfone metabolites in rats.

The pharmacokinetics of a new 5-hydroxytryptamine (5HT3) receptor antagonist, 4-amino-5-chloro-2-[(methylsulfinyl)ethoxy]-N-[2-(diethylamino)ethyl] benzamide (ML-1035, 1), and its sulfone and sulfide metabolites were examined in 12 rats. Each of these compounds (25.4 mumol/kg) was administered to rats intravenously. Their plasma concentrations were measured by high-performance liquid chromatography. These plasma data revealed that 1, a sulfoxide, underwent interconversion with its sulfide metabolite. However, no interconversion was observed between 1 and its sulfone metabolite. Examination of mean times and additional properties of the 1/sulfide metabolite system revealed that total exposure times of 1 and the sulfide metabolite were moderately and weakly, respectively, influenced by the metabolic interconversion process. However, the tissue distribution process strongly influenced the total exposure times of both compounds. The disposition of the sulfone metabolite of 1 was also strongly influenced by the tissue distribution process. In addition, < 3% of the intravenous dose of 1 or the sulfide was available to the general circulation as the sulfone metabolite.

Simultaneous determination of free tissue-type and free urokinase-type plasminogen activators in biological fluids by a solid-phase immunoassay.

Tissue-type and urokinase-type plasminogen activators (t-PA and u-PA) coexist in numerous biological fluids, where their fibrinolytic activities are determined by the concentration of the free, uncomplexed species. A simple, sensitive method has been developed for the simultaneous determination of free t-PA and u-PA concentrations in biological fluids using a solid-phase immunoassay. Microtiter plates were coated with polyclonal goat antibodies and incubated with PA standards or unknown samples. The absorbed PAs were then assayed by incubation with a mixture of plasminogen, poly-L-lysine, and the chromogenic substrate H-D-norleucylhexahydrotyrosyllysine-p-nitroanilide. Free t-PA and free u-PA were detectable in human plasma and urine, and in conditioned media from different endothelial cell cultures. The method is sensitive, with lower limits of quantitation being 0.76 mU/ml (1.25 pg/ml) for free t-PA and 0.16 mU/ml (2.0 pg/ml) for free u-PA. There was no cross-reaction between the two PA species and the recovery in plasma was greater than 95% for both. The intra- and interassay coefficients of variation for t-PA and u-PA were 3.5-12.2 and 3.2-11.3%, and 2.4-11.8 and 1.6-10.4%, respectively. The presence of PA-inhibitor complexes and PA inhibitors in biological fluids did not interfere with the assay. Application of the assay has demonstrated that free u-PA levels are several fold higher than free t-PA levels in human plasma and in conditioned media of human vascular endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Disposition kinetics of ML-1035 sulfoxide enantiomers and the prochiral sulfide in rats.

ML-1035, 4-amino-5-chloro-2-[2-(methylsulfinyl)ethoxy]-N-[2- (diethyl-amino)ethyl]benzamide, is a sulfoxide compound and a racemic gastroprokinetic agent with a chiral center at the sulfur atom. We have investigated the disposition kinetics of (R)-ML-1035 sulfoxide (R) and (S)-ML-1035 sulfoxide (S) after the single enantiomers and the racemic mixture were administered to rats in separate experiments. There was no noticeable chiral inversion after either enantiomer dose. Both enantiomers were rapidly absorbed. After dosing with enantiomers or with the racemate, the resulting plasma concentration-time curve of R was closely parallel to that of S in both intravenous and oral experiments, suggesting that the two enantiomers have approximately the same disposition kinetics. After intravenous enantiomer doses, only S underwent conversion to sulfide, suggesting that sulfidation in the liver is enantioselective. However, the enantioselective sulfidation after intravenous dosing did not introduce a difference in the global plasma disposition profiles between R and S, since the reduction reaction is a minor metabolic process. Other metabolic reactions such as sulfonation and mono-N-desethylations were not enantioselective. After oral administration, conversion to sulfide was observed for both enationers, implicating the existence of a nonhepatic pathway in sulfidation. Administration of a prochiral sulfide dose was associated with an enantioselective sulfoxidation, in which the R/S concentration ratios increased as a function of time. In addition, enatiomeric interaction causing changes in pharmacokinetic parameters was observed after the oral racemate dose, while the interaction is negligible after an intravenous racemate dose, indicating a route dependency in enantiomeric interaction.

Validation of a column-switching high-performance liquid chromatographic (HPLC) method for determination of ML-1035 and its five metabolites in plasma.

A fully automated column-switching HPLC procedure has been developed and validated for quantitation of ML-1035 and its five metabolites in plasma employing direct injection. Plasma samples were injected onto a CN extraction column (4 x 4.6 mm, 5 microns) for micellar cleanup with 0.5% sodium dodecyl sulfate (SDS) in 50 mM phosphate. The proteinaceous components were solubilized and flushed out. The extracted compounds were then eluted by forward flush onto a C8 analytical column (150 x 4.6 mm, 5 microns) for further analysis using fluorescence detection (excitation, 308 nm; emission, 350 nm). After the subsequent washing and reequilibration with a sequence of three solvent mixtures, the extraction column was ready for the next injection. The limit of quantitation for all compounds of interest was about 10 to 15 ng/ml using 100 microliters of plasma. Excellent precision, accuracy, and linearity were obtained for all compounds over a range of 10 to 1500 ng/ml. The practicality of the HPLC method was also validated with plasma samples from dogs receiving ML-1035. Longevity for both extraction and analytical columns is excellent. Micellar cleanup coupled with the column-switching technique is a promising HPLC procedure when using direct injection of biological fluids.