Search

Introduction. Telcagepant (MK-0974) is a novel, orally active and selective CGRP receptor antagonist being investigated for acute treatment of migraine. Early clinical data suggested greater than dose proportional increases in exposure following oral administration. The aim of the present studies was to definitively characterize the oral and IV dose proportionality of telcagepant. Methods. Healthy adult subjects were enrolled in two separate open-label randomized dose proportionality studies: 1) single oral dose crossover from 50 to 600 mg (N = 19); 2) single IV dose parallel group from 5 to 250 mg (N = 10 per dose). Blood samples were collected at time points from 0 to 48 hours postdose. Results. Telcagepant was rapidly absorbed with a Tmax of approximately 1 to 2 hours after oral administration. The terminal half-life was approximately 8 to 9 hours after IV dosing and approximately 4 to 7 hours after oral dosing. Oral administration of telcagepant resulted in greater than dose proportional increases in exposure, while IV administration resulted in approximately dose proportional increases in exposure. Conclusions. Telcagepant was generally well tolerated. Oral telcagepant exhibits non-linear pharmacokinetics.

A method coupling hydrophilic interaction chromatography (HILIC) with tandem mass spectrometry (MS/MS) has been developed for the simultaneous determination of three polar non-structurally related compounds--a carbapenem antibiotic, imipenem (IMP), a renal dehydropeptidase inhibitor, cilastatin (CIL), and an investigational beta-lactamase inhibitor, MK-4698 (BLI), in rat plasma, monkey plasma and mouse blood. The analytes were extracted through protein precipitation, chromatographed on a Waters Atlantis HILIC column, and detected on a Sciex API4000 mass spectrometer using a Turbo-Ion Spray ion source in positive ionization mode following multiple-reaction monitoring (MRM). The assay dynamic range was 0.1-100 microg/mL for IMP, CIL and BLI, respectively, using a total of 20-25 microL biologic samples, and the total HPLC/MS/MS run time was 4 min/injection. The assay was found to be sensitive, selective and reproducible. The challenges, namely, sample stability, blood sample processing, matrix effect in monkey study samples, and dilution re-assays for the limited mouse blood samples, are resolved and discussed. This technique allowed rapid analysis of polar compounds in biologic matrixes with satisfactory chromatographic retention and increased throughput.

To eliminate the diastereomer interference on Telcagepant (MK-0974) determination during clinical study support, on-line high turbulent-flow liquid chromatography (HTLC) methods, HTLC-A and HTLC-B that covered dynamic range of 0.5-500 nM and 5-5000 nM, respectively, were developed. To meet the requirement of rapid assay transfer among multiple laboratories and analysts, a solid-phase extraction (SPE) assay was derived from the existing HTLC-B assay under the same dynamic range. The on-line HTLC assays were achieved through direct injection of plasma samples, extraction of analyte with a Cohesive C18 column (50 mm x 0.5 mm, 50 microm), followed by HPLC separation on a FluoPhase RP column (100 mm x 2.1 mm, 5 microm) and MS/MS detection. The off-line SPE assay used Waters Oasis HLB microElution plate to extract the analytes from plasma matrix before injecting on a FluoPhase RP column (150 mm x 2.1 mm, 5 microm) for LC-MS/MS analysis. Under both on-line and off-line assay conditions, the diastereomer 1c was chromatographically separated from MK-0974. Cross-validation with the pooled samples demonstrated that both on-line and off-line assays provided comparable data with a difference of2.6%. The assays were proved to be specific, accurate and reliable, and have been used to support multiple clinical studies. The pros and cons of on-line and off-line assays with regard to man power involved in sample preparation, total analysis time, carryover, cost efficiency, and the requirement for assay transfer are discussed.

Telcagepant (MK-0974) is a novel oral calcitonin gene-related peptide (CGRP) receptor antagonist and is currently under clinical development. Results from phases II and III clinical trials have suggested that telcagepant is effective for migraine treatment. A reliable and high throughput protein precipitation (PPT) method for determination of telcagepant in human plasma using liquid chromatography coupled with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry has been developed. Clinical samples, internal standard (IS) and acetonitrile are transferred into 96-well plates using a robotic liquid handling system. An aliquot of 10 microL supernatant is directly injected into the LC-MS/MS system where separation is performed on a FluoPhase RP (150 x 2.1mm, 5 microm) column with an isocratic mobile phase (60% acetonitrile with 0.1% formic acid and 40% water with 0.1% formic acid) at 0.2 mL/min. The interfering 3S-diastereomer of telcagepant, which is observed in clinical samples, is chromatographically resolved from telcagepant. The PPT procedure significantly reduces the time required for sample processing and the assay is sufficiently sensitive for detection using both API 4000 and API 3000 mass spectrometers. The linear calibration range is 5-5000 nM using 200 microL of plasma. Assay intraday validation was conducted using six calibration curves derived from six lots of human control plasma. Calibration standard accuracy did not deviate by more than 3% and 6% of nominal values, and precision did not exceed 4% coefficient of variation (CV) and 10% CV, respectively on the API 4000 and API 3000. Several clinical phases IIb and III studies have been successfully supported with this assay.

Studies using in-tip solid phase microextraction (in-tip SPME) in a 96-well plate format are conducted to investigate the feasibility of SPME automation. The sample preparation process, including extraction and desorption, was fully automated and coupled with currently commercially available automated liquid handling systems. Several process parameters including extraction time and speed, and desorption time were investigated. An LC-MS/MS method has been developed and validated to determine the levels of a drug compound (MK-0533) in human plasma that demonstrates the suitability of this new approach. The developed method has a lower limit of quantitation (LLOQ) of 5 ng/mL when 0.25 mL of human plasma is processed and is validated in the concentration range of 5-2, 000 ng/mL. The successful application of the assay in clinical sample analysis indicates that in-tip SPME can be easily automated and has great potential to be used for high throughput quantitative determination of drugs in pharmaceutical industry.

Calcitonin gene-related peptide (CGRP) has been implicated in the pathogenesis of migraine. Herein we describe optimization of CGRP receptor antagonists based on an earlier lead structure containing a (3R)-amino-(6S)-phenylcaprolactam core. Replacement of the phenylimidazolinone with an azabenzimidazolone gave stable derivatives with lowered serum shifts. Extensive SAR studies of the C-6 aryl moiety revealed the potency-enhancing effect of the 2,3-difluorophenyl group, and trifluoroethylation of the N-1 amide position resulted in improved oral bioavailabilities, ultimately leading to clinical candidate 38 (MK-0974).

Pyrimidino-thiazolyl carbonitriles were prepared that are potent VEGFR-2 (KDR) kinase inhibitors. The modification of lead structures resulted in 3m which exhibited the best overall profile in KDR inhibitory activity, iv/po pharmacokinetics, and reduced hERG affinity.

In this study, we have demonstrated that the critical hydrogen bonding motif of the established 3-aminopyrazinone thrombin inhibitors can be effectively mimicked by a 2-aminopyridine N-oxide. As this peptidomimetic core is more resistant toward oxidative metabolism, it also overcomes the metabolic liability associated with the pyrazinones. An optimization study of the P(1) benzylamide delivered the potent thrombin inhibitor 21 (K(i) = 3.2 nM, 2xaPTT = 360 nM), which exhibited good plasma levels and half-life after oral dosing in the dog (C(max) = 2.6 microM, t(1/2) = 4.5 h).

A series of N-(1,3-thiazol-2-yl)pyridin-2-amine KDR kinase inhibitors have been developed that possess optimal properties. Compounds have been discovered that exhibit excellent in vivo potency. The particular challenges of overcoming hERG binding activity and QTc increases in vivo in addition to achieving good pharmacokinetics have been acomplished by discovering a unique class of amine substituents. These compounds have a favorable kinase selectivity profile that can be accentuated with appropriate substitution.

Modification of lead compound 1 by reducing lipophilicity in the P3 group produced a series of low molecular weight thrombin inhibitors with excellent potency in functional assays, metabolic stability, and oral bioavailability. These modifications led to the identification of two optimized compounds, 14 and 16.

In this manuscript we demonstrate that a modification principally directed toward the improvement of the aqueous solubility (i.e., introduction a P3 pyridine N-oxide) of the previous lead compound afforded a new series of potent orally bioavailable P1 N-benzylamide thrombin inhibitors. An expedited investigation of the P1 SAR with respect to oral bioavailability, plasma half-life, and human liver microsome stability revealed 5 as the best candidate for advanced evaluation.

Recent efforts in the field of thrombin inhibitor research have focused on the identification of compounds with good oral bioavailability and pharmacokinetics. In this manuscript we describe a metabolism-based approach to the optimization of the 3-(2-phenethylamino)-6-methylpyrazinone acetamide template (e.g., 1) which resulted in the modification of each of the three principal components (i.e., P1, P2, P3) comprising this series. As a result of these studies, several potent thrombin inhibitors (e.g., 20, 24, 25) were identified which exhibit high levels of oral bioavailability and long plasma half-lives.

A sensitive and specific assay has been developed and validated for the separation of a chiral sulfoxide drug candidate with simultaneous determination of the corresponding sulfide and sulfone in plasma by normal-phase LC–MS–MS. Separation was achieved on a Chiralpak AD (100×2.1 mm) column with a 2-propanol–hexane (80:20) mobile phase within 7 min. Aqueous mobile phase (2-propanol–10 m M ammonium acetate, 75:25) was added post-column prior to introduction into the heated nebulizer interface of a Sciex API 3plus mass spectrometer, to avoid the explosion hazard of hexane-containing mobile phases in the presence of a corona discharge. The linear range of this assay was 5–2500 ng/ml. The accuracy and precision of the chiral sulfoxides, the sulfide and the sulfone were within ±15% across the linear range. The limit of quantitation for each component was 5 ng/ml based on the extraction of 0.25 ml plasma. The recovery for each component was between 82 and 120%. This assay was sufficiently sensitive and specific to support pre-clinical development studies in rats, dogs and monkeys.

The Aria LX4 staggered parallel high performance liquid chromatography (HPLC) system is evaluated for application to good laboratory practice (GLP) level quantitative analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS). This system consists of four fully independent binary HPLC pumps, a modified autosampler, and a series of switching and selector valves all controlled by a single computer program. The system improves sample throughput without sacrificing chromatographic separation or data quality. Validation results for four different compounds, each analyzed on a separate channel of the Aria system, show precision and accuracy equivalent to that required of a single-channel system. The results show that sample throughput can be increased nearly four-fold without requiring significant changes in current analytical procedures. The flexibility and ease of use of the Aria system suggest that it should be possible to quickly implement it in any analytical LC/MS/MS environment.

We show results from experiments designed to determine the relative importance of gas phase processes and solution phase processes into ionization suppression observed in biological sample extracts. The data indicate that gas phase reactions leading to the loss of net charge on the analyte is not likely to be the most important process involved in ionization suppression. The results point to changes in the droplet solution properties caused by the presence of nonvolatile solutes as the main cause of ionization suppression in electrospray ionization of biological extracts.

A series of tricyclic CGRP receptor antagonists was optimized in order to improve oral bioavailability. Attenuation of polar surface area and incorporation of a weakly basic indoline nitrogen led to compound 5, a potent antagonist with good oral bioavailability in three species.

Several high performance liquid chromatographic methods utilizing a variety of fluorescence derivatization schemes have been summarized both for the detection and routine quantitation of peptide drugs in biological fluids. The derivatization reactions have been carried out either in pre- or post-column modes with various commercially available reagents and targeted primarily at α- and ϵ-amino groups, guanidino group of arginine and sulfhydryl groups present in the peptide chain. These analytical methods are very robust and comparable to immuno assays in sensitivity and have been used for quantification of peptide analytes in various pharmacokinetic and toxicokinetic studies. This discussion also summarizes the derivatization schemes that have not been fully explored to date but may find utility in future for development of highly sensitive analytical methods for peptide analytes.

A high-performance liquid chromatographic (HPLC) method was developed for the determination of SK&F 105058 (I), the N-ethyl tricarbamate ester pro-drug of fenoldopam, in dog plasma. Fenoldopam is a selective agonist of peripheral dopaminergic (DA-1) receptors and has been shown to improve blood flow and lower blood pressure. The method involves isolation of I and the internal standard (I.S.) from plasma by solid-phase extraction prior to chromatographic separation on an octyl silica column. Following chromatographic separation, the carbamate esters of I and I.S. were simultaneously hydrolyzed and the liberated alkylamines were derivatized, by mixing the column effluent with an alkaline solution of o -phthaldialdehyde and a thiol, to generate a highly fluorescent isoindole product which was subsequently detected with a fluorometer. Optimization of chromatographic and post-column reaction conditions resulted in an on-column detection limit of 0.4 ng. The recoveries for I and I.S. from plasma were 80.0 ± 5.0% and 62.0 ± 4.0%, respectively. The limit of quantification for I using 0.2 ml of plasma was 5 ng/ml. Linear response was observed for concentrations of I ranging from 5 to 2000 ng/ml plasma. The method was suitably specific and sensitive for pharmacokinetic and metabolic studies of I in dogs. The methodology developed should be generally applicable to the determination of carbamate-type pro-drugs in biological media.

A sensitive and selective high-performance liquid chromatographic method was developed for the determination of SB 107647 (I), a novel synthetic hematoregulatory peptide, in plasma samples of dog and rat. The method involves isolation of I and the internal standard (SB 203285, IS) from plasma by a solid-phase anion-exchange extraction column prior to reversed-phase ion-pair chromatographic separation on an octyl silica column. Following separation, a selective post-column reaction of the epsilon-amino groups of the lysine moieties of the peptide with o-phthaldialdehyde and a thiol under basic conditions was used to generate a highly fluorescent isoindole product, which was subsequently detected on-line with a fluorometer. Optimization of chromatographic conditions resulted in an on-column detection limit of 1 ng. The recovery of I from dog plasma at 20 and 4000 ng/ml was 50.0 +/- 5.94 and 56.6 +/- 1.45% (Mean +/- S.D.), respectively. The limit of quantification for I, for 0.25-ml plasma samples, was 20 ng/ml. Linear response was observed for concentrations of I ranging from 20 to 4000 ng/ml of plasma. The assay was sufficiently sensitive, accurate and precise to support toxicokinetic studies in animal species.

A sensitive and selective high-performance liquid chromatographic method was developed for the determination of SK&F 106203 3-(2-carboxyethylthio)-3-[2-(8-phenyloctyl)phenyl]propanoic acid, a potent peptidoleukotriene end organ receptor antagonist, in human plasma. The method involves isolation of SK&F 106203 and the internal standard (SK&F 104736) from plasma samples by liquid-liquid extraction prior to derivatization with 9-anthryldiazomethane. The derivatized samples were first subjected to a solid-phase extraction procedure prior to injection onto a short silica column, which is part of a chromatographic system equipped with an automated column-switching device. Column switching was used to heart-cut the chromatographic zone containing the peaks of interest from this first column and transfer it to an analytical silica column for further chromatographic separation. The peaks were quantified with an in-line fluorometer by measuring the fluorescence emission intensity at 415 nm after excitation at 365 nm. An on-column detection limit of 0.625 ng was achieved for SK&F 106203 by optimizing the derivatization and chromatography conditions. The limit of quantification for SK&F 106203, using 250 μl of plasma, was 20 ng/ml. Linear response in SK&F 106203/internal standard peak-height ratios was observed for SK&F 106203 concentrations ranging from 10 to 5000 ng/ml of plasma. Precision and accuracy were within 5% across the calibration range. The assay was sufficiently sensitive, accurate, and precise to support pharmacokinetic studies in humans.

Although the S3 pocket of the thrombin active site is lined with lipophilic amino acid residues, the accommodation of polarity within the lipophilic P3 moiety of small molecule inhibitors is possible provided that the polar functionality is capable of pointing away from the binding pocket outwards toward solvent while simultaneously allowing the lipophilic portion of the P3 ligand to interact with the S3 amino acid residues. Manipulation of this motif provided the means to effect optimization of functional potency, in vivo antithrombotic efficacy and oral bioavailability in a series of 3-aminopyrazinone thrombin inhibitors which contained non-charged groups at the P1 position.

A novel high-performance liquid chromatographic method was developed for the determination of monohydroxy compounds using the combined approach of pre-column derivatization and post-column reaction with fluorescence detection. Monohydroxy-containing drugs were modified by pre-column derivatization with propyl isocyanate (in pyridine, 50°C, 1 h) to form the corresponding n -propyl carbamate esters. Excess of reagent, reagent impurities and solvent were then removed by evaporation. Following chromatographic separation on a reversed-phase octadecylsilica column, the n -propyl carbamate ester derivative was subjected to post-column reaction and detection using alkaline hydrolysis to generate free propylamine, which was subsequently derivatized in-line using o -phthaladehyde and 3-mercaptopropionic acid to form a fluorescent isoindole. The fluorescence emission of the isoindole product was measured at 455 nm following excitation at 340 nm. As propyl isocyanate is highly volatile and physico-chemically different to many drug molecules, problems associated with reagent impurities and reaction by-prodycts were substantially minimized. The method was highly sensitive, allowing detection of sub-nanogram amount of monohydroxy-containing drugs, hydroxy steroids and hydroxamic acids. The utility of this derivatization scheme was demonstrated by the highly sensitive measurement in human plasma at oxiracetam (4-hydroxy-2-oxo-1-pyrrolidineacetamide), an investigational drug inteded for use in dementia and other memory disorders.

The underlying mechanism for low oral bioavailability of MK-0974, a potent calcitonin-gene related peptide (CGRP)-receptor antagonist, in monkeys and for species-dependent non-linear pharmacokinetics in monkeys and rats were investigated. In monkeys, MK-0974 displayed moderate clearance (14-20 ml min(-1) kg(-1)), while oral bioavailability was 6%. The pharmacokinetics of MK-0974 remained linear across 0.5-10 mg kg(-1) intravenous dose in monkeys, but the oral area under the plasma concentration-time curve (AUC) increase (5-30 mg kg(-1)) was 15-fold over dose-proportional. Based on a comparison of AUC following hepatic portal vein administration and cephalic vein infusion, MK-0974 exhibited a low-to-moderate hepatic extraction ratio (36%) in monkeys. Following oral dose of [14C]MK-0974 to monkeys, the hepatic portal AUC ratio of MK-0974 versus total radioactivity was 0.32, and the total radioactivity recovered in bile and urine was 45-83%. MK-0974 undergoes significant oxidative metabolism (cytochrome P450 (CYP) 3A) in monkey intestinal microsomes. In contrast, oral AUC of MK-0974 in rats was near dose-proportional (15-100 mg kg(-1)). Following oral administration of [14C]MK-0974 to rats, the hepatic portal AUC ratio of MK-0974 to total radioactivity (0.67) was higher than in monkeys. Additionally, the metabolic rate of MK-0974 was slower in rat than in monkey intestinal microsomes. Collectively, intestinal first-pass metabolism played a significant role in the low oral bioavailability in monkeys and contributed to the species-dependent non-linear oral pharmacokinetics in rats and monkeys of MK-0974.

In our effort to find potent, orally bioavailable CGRP receptor antagonists for the treatment of migraine, a novel series based on a pyridinone template was investigated. After optimizing the privileged structure and the placement of the attached phenyl ring, systematic SAR was carried out on both the N-alkyl and C-5 aryl substituents. Several analogs with good potency and pharmacokinetic profiles were identified.

A rapid analogue approach to identification of spirohydantoin-based CGRP antagonists provided novel, low molecular weight leads. Modification of these leads afforded a series of nanomolar benzimidazolinone-based CGRP receptor antagonists. The oral bioavailability of these antagonists was inversely correlated with polar surface area, suggesting that membrane permeability was a key limitation to absorption. Optimization provided compound 12, a potent CGRP receptor antagonist (Ki = 21 nM) with good oral bioavailability in three species.

Optimization of a previously reported thrombin inhibitor, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-trans-4-aminocyclohexylmethylamide (1), by replacing the aminocyclohexyl P1 group provided a new lead structure, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-2-aminomethyl-5-chlorobenzylamide (2), with improved potency (K(i) = 0.49 nM for human thrombin, 2x APTT = 0.37 microM in human plasma) and pharmacokinetic properties (F = 39%, iv T(1/2) = 13 h in dogs). An effective strategy for reducing plasma protein binding of 2 and improving efficacy in an in vivo thrombosis model in rats was to replace the lipophilic fluorenyl group in P3 with an azafluorenyl group. Systematic investigation of all possible azafluorenyl P3 isomers and azafluorenyl-N-oxide analogues of 2 led to the identification of an optimal compound, 3-aza-9-hydroxyfluoren-9(R)-ylcarbonyl-l-prolyl-2-aminomethyl-5-chlorobenzylamide (19b), with high potency (K(i) = 0.40 nM, 2x APTT = 0.18 microM), excellent pharmacokinetic properties (F = 55%, T(1/2) = 14 h in dogs), and complete efficacy in the in vivo thrombosis model in rats (inhibition of FeCl(3)-induced vessel occlusions in six of six rats receiving an intravenous infusion of 10 microg/kg/min of 19b). The stereochemistry of the azafluorenyl group in 19b was determined by X-ray crystallographic analysis of its N-oxide derivative (23b) bound in the active site of human thrombin.

In an effort to discover potent, clinically useful thrombin inhibitors, a rapid analogue synthetic approach was used to explore the P(1) region. Various benzylamines were coupled to a pyridine/pyrazinone P(2)-P(3) template. One compound with an o-thiadiazole benzylic substitution was found to have a thrombin K(i) of 0.84 nM. A study of ortho-substituted five-membered-ring heterocycles was undertaken and subsequently demonstrated that the o-triazole and tetrazole rings were optimal. Combination of these potent P(1) aryl heterocycles with a variety of P(2)-P(3) groups produced a compound with an extraordinary thrombin inhibitory activity of 1.4 pM. It is hoped that this potency enhancement in P(1) will allow for more diversification in the P(2)-P(3) region to ultimately address additional pharmacological concerns.

Chemotherapy of prostate cancer with antimitotic agents such as vinblastine and doxorubicin is only marginally effective, due to dose-limiting systemic toxicity. Herein we report the development of peptidyl conjugate 5 of the cytotoxic agent vinblastine (1), along with the results of its in vitro and in vivo evaluation as a pro-drug targeted at prostate cancer cells. Prostate-derived tumors are known to produce significant amounts of prostate specific antigen (PSA), a serine protease with chymotrypsin-like properties. Earlier work in these laboratories established that an appropriately engineered peptidyl pro-drug will release active cytotoxic agent strictly within the microenvironment of the tumor tissue (Garsky, V. M., et al. J. Med.Chem. 2001, 44, 4216-4224). Conjugate 5, which features an octapeptide segment attached by an ester linkage at the 4-position of vinblastine (1), undergoes rapid cleavage by PSA (T(1/2) = 12 min) between the Gln and Ser residues. In nude mouse xenograft studies, 5 reduced circulating PSA levels by 99% and tumor weight by 85% at a dose just below its MTD. By contrast, the putative end-point metabolite, the cytotoxic agent des-acetyl vinblastine (1b), was ineffective in reducing PSA levels and tumor burden at its maximum tolerated doses. Additional data from metabolism studies on 5 support the supervention of a novel in vivo processing mechanism, the spontaneous release of 1b from a dipeptidyl intermediate driven by favorable diketopiperazine formation.

An alternative on-line automated sample enrichment technique useful for the direct determination of various drugs and their metabolites in plasma is described for rapid development of highly sensitive and selective liquid chromatographic methods using mass spectrometric detection. The method involves direct injection of plasma onto an internal surface reversed-phase (ISRP) guard column, washing the proteins from the column to waste with aqueous acetonitrile, and backflushing the analytes onto a reversed-phase octyl silica column using switching valves. The analytes were detected using a tandem mass spectrometer operated in selected reaction monitoring (SRM) mode using atmospheric pressure chemical ionization (APCI). Use of two ISRP guard columns in parallel configuration allowed alternate injections of plasma samples on these columns for sample enrichment and shortened the column equilibration and LCMSMS analysis times, thereby increasing the sample throughput. The total run time, including both sample enrichment and chromatography, was about 6 min. Using this technique, an analytical method was developed for the quantitation of granisetron and its active 7-hydroxy metabolite in dog plasma. Granisetron is a selective 5-HT3 receptor antagonist used in the prevention and treatment of cytostatic induced nausea and vomiting. Recovery of the analytes was quantitative and the method displayed excellent linearity over the concentration ranges tested. Results from a three day validation study for both compounds demonstrated excellent precision (1.3–8.7%) and accuracy (93–105%) across the calibration range of 0.1 to 50 ng/ml using an 80 μl plasma sample. The automated method described here was simple, reliable and economical. This on-line approach using ISRP columns and column switching with LCMSMS is applicable for the quantification of other pharmaceuticals in pharmacokinetics studies in animals and humans which require high sensitivity.

An highly sensitive and selective high-performance liquid chromatographic method was developed for the determination of epristeride [17 beta-(N-tert.-butyl carboxamido)-androst-3,5-diene-3-carboxylic acid, SK&F 105657], a potent inhibitor of the prostatic steroid 5 alpha-reductase enzyme, in human plasma samples. Epristeride is currently in development for the treatment of benign prostatic hyperplasia. The analytical method involves isolation of epristeride and the internal standard [17 beta-(N,N-diisopropyl carboxamido) estra-1,3,5 (10)-triene-3-carboxylic acid, SK&F 105419] from plasma by solid-phase extraction prior to chromatographic separation on an aminopropyl silica column, using hexane-methylene chloride-2-propanol-acetic acid as the mobile phase, with subsequent ultraviolet absorption detection. The absolute recovery of epristeride from plasma was 90.2 +/- 2.96. The limit of quantification for epristeride was 2.5 ng/ml. Linear response was observed for concentrations of epristeride ranging from 1 to 500 ng/ml plasma. The assay was sufficiently sensitive, accurate and precise to support pharmacokinetic studies in human subjects.

Peptides containing a free alpha- or epsilon-amino group react with fluorescamine under mild alkaline conditions to generate a highly fluorescent but unstable reaction product and, consequently, practical high-performance liquid chromatographic (HPLC) approaches to analysis have typically involved the use of postcolumn derivatization. An automated precolumn approach is reported in which peptides are reacted with fluorescamine just prior to HPLC analysis by a commercially available autoinjector with derivatization capabilities. The autoinjector added base and fluorescamine reagent solutions to a sample vial containing peptide analytes, and the derivatization reaction was allowed to proceed for 5 min at room temperature prior to injection into the HPLC system. The derivatized peptides were analyzed by reversed-phase HPLC with fluorescence detection (excitation at 390 nm; emission 470-nm cut-off filter) on an octylsilica column. Optimization of the precolumn reaction conditions and the use of narrower HPLC columns (2 mm I.D.) resulted in a typical on-column detection limit of 30-50 fmol of peptide, which was substantially lower than that in previously reported post-column methods. This approach was applied to the HPLC of several naturally occurring and synthetic peptides containing alpha- and epsilon-amino groups. In combination with solid-phase extraction, prior to automated precolumn fluorescence derivatization and chromatographic analysis, the methodology was used for the determination of a synthetic growth hormone-releasing peptide in plasma samples.

Given the prominent role of CYP3A in the metabolism of drugs, it is important to identify whether new chemical entities will affect this enzyme system and produce clinically relevant drug interactions. This study evaluated concomitant administration of intravenous [14C N-methyl] erythromycin (3 microCi) (erythromycin breath test; EBT) and 2 mg oral midazolam as probes of systemic and of systemic plus presystemic CYP3A activity, respectively. Twelve males received the probes in a two-period crossover fashion: one period included the probes on two occasions, 5 days apart; in the second period, 200 mg ketoconazole was given orally 2 hours prior to the probes. The within-subject CV for EBT (%14CO2/h) and midazolam AUC0-last was 4.9% and 16.9%, respectively. Ketoconazole reduced %14CO2/h by 43% and increased midazolam AUC0-last by approximately fivefold. In a nonrandomized third period (N = 5), ketoconazole was given simultaneously with midazolam (no EBT); midazolam AUC0-last was similar whether ketoconazole was given 2 hours prior to or simultaneously with the midazolam. The low midazolam dose was generally well tolerated; mild sedation was occasionally seen. Concurrent administration of the EBT and oral midazolam is a sensitive and reproducible tool to screen new chemical entities for potentially important CYP3A interactions.