Pharmacokinetics and metabolism of a cysteinyl leukotriene-1 receptor antagonist from the heterocyclic chromanol series in rats: in vitro-in vivo correlation, gender-related differences, isoform identification, and comparison with metabolism in human hepatic tissue.

CP-199,331 is a potent antagonist of the cysteinyl leukotriene-1 (LT(1)) receptor, targeted for the treatment of asthma. The pharmacokinetic/metabolism properties of CP-199,331 were studied in rats and compared with those in human liver microsomes/hepatocytes. In vitro biotransformation of CP-199,331 in rat and human hepatocytes was similar, consisting primarily of CP-199,331 O-demethylation. Marked sex-related differences in plasma clearance (CL(p)) of CP-199,331 were observed in rats: 51 and 1.2 ml/min/kg in males and females, respectively. This difference in CL(p) was attributed to gender differences in metabolizing capacity because V(max) and K(m) values for CP-199,331 metabolism were 30-fold higher and 8-fold lower, respectively, in male rat liver microsomes compared with female microsomes. Scale-up of the in vitro microsomal data predicted hepatic clearance (CL(h)) of 64 and 2.5 ml/min/kg in male and female rats, respectively. These values were in close agreement with the in vivo CL(p), suggesting that CP-199,331 CL(p) in male and female rats was entirely due to hepatic metabolism. Studies with rat recombinant cytochromes P450 and anti-rat cytochrome P450 (CYP) antibodies revealed the involvement of male rat-specific CYP2C11 in the metabolism of CP-199,331. In contrast, CP-199,331 metabolism in human liver microsomes was principally mediated by CYP3A4. The projected human clearance in liver microsomes and hepatocytes varied 6-fold from low to moderate, depending on CYP3A4 activity. Considering that O-demethylation is the major route of elimination in humans, the in vivo clearance of CP-199,331 may exhibit moderate variability, depending on CYP3A4 abundance in the human population.

A high-capacity LC/MS system for the bioanalysis of samples generated from plate-based metabolic screening.

HPLC/MS is a linear technique characterized by serial injection and analysis of individual samples. Parallel-format high-throughput screens for druglike properties present a significant analytical challenge. Analysis speed and system ruggedness are key requirements for bioanalysis of thousands of samples per day. The tasks involved in LC/MS analysis are readily divided into three areas, sample preparation/liquid handling, LC/MS method building/sample analysis, and data processing. Several automation and multitasking strategies were developed and implemented to minimize plating and liquid handling errors, reduce dead times within the analysis cycle, and allow for comprehensive review of data. Delivering multiple samples to multiple injectors allows the autosampler time to complete its wash cycles and aspirate the next set of samples while the previous set is being analyzed. A dual-column chromatography system provides column cycling and peak stacking and allows rapid throughput using conventional LC equipment. Collecting all data for a compound into a single file greatly reduces the number of data files collected, increases the speed of data collection, allows rugged and complete review of all data, and provides facile data management. The described systems have analyzed over 40 000 samples per month for two years and have the capacity for over 2000 samples per instrument per day.

Pharmacokinetics and pharmacodynamics of the leukotriene B4 receptor antagonist CP-105,696 in man following single oral administration.

AIMS: CP-105,696, (+)-1-(3S,4R)-[3-(4-phenylbenzyl)-4-hydroxy-chroman-7-yl] cyclopropane carboxylic acid is a potent, novel LTB4 receptor antagonist advanced to clinical trials to determine its efficacy in inflammatory diseases. The pharmacokinetics and pharmacodynamics of CP-105,696 were investigated in healthy male volunteers following oral administration of single doses of 5 to 640 mg. METHODS: Forty-eight subjects participated in a randomized, double-blind, parallel group study. Plasma and urine concentrations of CP-105,696 were determined at intervals after drug administration. As an indication of LTB4 receptor antagonism following oral administration of CP-105,696, the inhibiton of LTB4-induced upregulation of the neutrophil cell surface complement receptor (CR3), CD11b/CD18, was monitored at 4 h following drug administration using an ex vivo whole blood flow cytometry assay. RESULTS: Cmax and AUC(0, infinity) increased in a dose-related manner. Respective mean Cmax values were 0.54 to 30.41 microg ml(-1) following doses of 5 to 640 mg. Respective mean AUC(0, infinity) values were 1337 to 16819 microg ml(-1) h for the 40 to 640 mg dose groups. Plasma concentrations declined in a monoexponential manner, with terminal elimination half-lives ranging from 289 to 495 h. Group mean terminal elimination half-lives were dose-independent. Urinary excretion of unchanged drug accounted for < 1% of the administered dose. A linear relationship was observed between CP-105,696 plasma concentrations and inhibition of LTB4-mediated CD11b upregulation on human neutrophils in whole blood. CP-105,696 plasma concentrations of 5-6 microg ml(-1) were necessary to elicit a two-fold shift to the right of the LTB4 concentration response curve for CD11b upregulation. CONCLUSIONS: These studies demonstrate pharmacologically significant LTB4-receptor antagonism following a single dose of CP-105,696 and pharmacokinetics consistent with once-daily dosing.

N-carbamoyl analogs of Zafirlukast: potent receptor antagonists of leukotriene D4.

Exploration of the indole nitrogen region of Zafirlukast (1) has uncovered a potent series of cysteinyl leukotriene D4 (LTD4) antagonists. These studies showed that a variety of functionality could be incorporated in this region of the molecule without sacrificing potency. Efforts to exploit this site in order to improve oral efficacy are discussed.

The prediction of human pharmacokinetic parameters from preclinical and in vitro metabolism data.

We describe a comprehensive retrospective analysis in which the abilities of several methods by which human pharmacokinetic parameters are predicted from preclinical pharmacokinetic data and/or in vitro metabolism data were assessed. The prediction methods examined included both methods from the scientific literature as well as some described in this report for the first time. Four methods were examined for their ability to predict human volume of distribution. Three were highly predictive, yielding, on average, predictions that were within 60% to 90% of actual values. Twelve methods were assessed for their utility in predicting clearance. The most successful allometric scaling method yielded clearance predictions that were, on average, within 80% of actual values. The best methods in which in vitro metabolism data from human liver microsomes were scaled to in vivo clearance values yielded predicted clearance values that were, on average, within 70% to 80% of actual values. Human t1/2 was predicted by combining predictions of human volume of distribution and clearance. The best t1/2 prediction methods successfully assigned compounds to appropriate dosing regimen categories (e.g., once daily, twice daily and so forth) 70% to 80% of the time. In addition, correlations between human t1/2 and t1/2 values from preclinical species were also generally successful (72-87%) when used to predict human dosing regimens. In summary, this retrospective analysis has identified several approaches by which human pharmacokinetic data can be predicted from preclinical data. Such approaches should find utility in the drug discovery and development processes in the identification and selection of compounds that will possess appropriate pharmacokinetic characteristics in humans for progression to clinical trials.

Granulomatous hepatitis and necrotizing splenitis due to Bartonella henselae in a patient with cancer: case report and review of hepatosplenic manifestations of bartonella infection.

Bacillary angiomatosis and the related disorders of bacillary peliosis hepatis and bacillary splenitis are manifestations of infection with Bartonella henselae and Bartonella quintana in immunocompromised persons. B. henselae infection, but not B. quintana infection, is linked to contact with cats and is presumed to cause visceral cat-scratch disease. We reports a case of visceral infection by B. henselae in an adult patient with cancer who was receiving chemotherapy and had had no contact with a cat or dog. The patient--whose illness was eventually diagnosed on the basis of findings of histologic, polymerase chain reaction, and serological studies--was treated with doxycycline and rifampin, and the infection resolved. In addition, 41 cases of documented or suspected bartonella infection of the liver, spleen, or both in immunocompetent or immunocompromised hosts are reviewed.

Multiple-dose pharmacokinetics and safety of trovafloxacin in healthy volunteers.

The multiple-dose pharmacokinetics and safety of trovafloxacin (CP-99,219), a new fluoroquinolone antibacterial agent, were evaluated in healthy male volunteers. Trovafloxacin was administered orally at 100 or 300 mg as a single dose followed by a 3 day washout period, and then was dosed once-daily for 14 consecutive days. Multiple serum and urine samples were collected on days 1 and 17 and were analysed for trovafloxacin concentrations by HPLC-UV. Following single doses, the mean Cmax values (mean +/- S.D.) were 1.0 +/- 0.3 and 2.9 +/- 0.4 mg/L for the 100 and 300 mg, respectively; those after 14-day consecutive daily dosing (day 17) were 1.1 +/- 0.2 and 3.3 +/- 0.5 mg/L, respectively. Trovafloxacin was rapidly absorbed and reached Cmax approximately 1 h after dosing. The mean values of T1/2 associated with the 100 and 300 mg doses were 9.2 +/- 1.2 on day 1 and 10.5 +/- 0.7 h on day 17; those after the 300 mg doses were 10.5 +/- 1.4 and 12.2 +/- 1.9 h, respectively. The cumulative urinary recovery of unchanged drug averaged 5.3% of the administered dose. Trovafloxacin renal clearance was 0.43 +/- 0.09 L/h. The free fraction of the drug in plasma was 23.8 +/- 6.1%. The renal clearance, half-life and unbound fraction did not change over the course of 2 weeks of multiple dosing. Steady-state serum concentrations were attained by the third daily dose, with approximately 1.3-fold accumulation. Both doses of trovafloxacin were well tolerated, and no significant changes in any laboratory safety parameters were detected. This study shows that the pharmacokinetics of trovafloxacin are linear and stationary and that steady-state serum concentrations above the MICs for most susceptible pathogens attained.

Pharmacokinetics of trovafloxacin (CP-99,219), a new quinolone, in rats, dogs, and monkeys.

The pharmacokinetics of trovafloxacin [CP-99,219; 7-(3-azabicyclo[3.1.0]hexyl)-naphthyridone] were studied in rats, dogs, and monkeys following oral and intravenous administration. After intravenous dosing, the systemic clearances of trovafloxacin in rats, dogs, and monkeys were 12.5, 11.1, and 7.2 ml/min/kg of body weight, respectively, and the respective volumes of distribution were 0.9, 1.7, and 4.3 liters/kg, with corresponding elimination half-lives of 0.7, 1.8, and 7.0 h. After the administration of oral doses of 50, 20, and 20 mg/kg to rats, dogs, and monkeys serum trovafloxacin concentrations reached a maximum at 0.6, 2.3, and 2.3 h, respectively, with respective maximum concentrations of trovafloxacin in serum of 11.5, 3.5, and 5.2 micrograms/ml; the corresponding elimination half-lives were 2.2, 2.5, and 7.5 h. The oral bioavailability of trovafloxacin was 68, 58, and 85% in rats, dogs, and monkeys, respectively. The binding of trovafloxacin to serum proteins was concentration independent, averaging 92, 75, and 66% for rats, dogs, and monkeys, respectively. Trovafloxacin penetrated well into tissues in dogs. The urinary recoveries of unchanged drug were less than 5% in dogs and monkeys, with or without incubation with alkali or Glusulase (beta-glucuronidase and sulfatase). In rats, 99.8% of the orally administered radioactivity was recovered in feces, while 20.6, 3.4, and 67.1% of the radioactive dose in bile duct-cannulated rats were recovered in feces, urine, and bile, respectively. These results suggest that the elimination of trovafloxacin from rats, and possibly from dogs and monkeys, is primarily through biliary excretion.

Determination of trovafloxacin, a new quinolone antibiotic, in biological samples by reversed-phase high-performance liquid chromatography.

A simple, accurate and precise high-performance liquid chromatographic method was developed and validated for the determination of trovafloxacin, a new quinolone antibiotic, in serum and urine. Following solid-phase extraction, chromatographic separation was accomplished using a C18 column with a mobile phase consisting of 0.04 M H3PO4-acetonitrile-tetrabutylammonium hydroxide-0.005 M dibutyl amine phosphate (D-4) reagent (83:16.85:0.05:0.1, v/v), pH 3. Trovafloxacin and the internal standard (a methyl derivative of trovafloxacin) were detected by ultraviolet absorbance at 275 nm. The lower limit of quantification for trovafloxacin was 0.1 microgram/ml and the calibration curves were linear over a concentration range of 0.1 to 20.0 micrograms/ml (r2 = 0.9997). The average recoveries were greater than 70% for both trovafloxacin and internal standard. The intra-day and inter-day coefficients of variation were generally less than 5% in urine and serum over the concentration range of 0.1 to 20.0 micrograms/ml. Human serum samples could be stored for up to 12 months at -20 degrees C and urine samples could be stored up to 18 months at -80 degrees C.

Discovery of the hemifumarate and (alpha-L-alanyloxy)methyl ether as prodrugs of an antirheumatic oxindole: prodrugs for the enolic OH group.

Ether, ester, and carbonate derivatives of the antirheumatic oxindole 1 were prepared and screened as potential prodrugs of 1. This effort led to the discovery of the (alpha-L-alanyloxy)-methyl ether and hemifumarate derivatives of 1 which deliver the drug efficiently into the circulation of test animals, are stable in the solid state, and possess good stability in solution at low pH as required to ensure gastric stability. Success in achieving acceptable bioavailabilities of 1 across species (rats, dogs, and monkeys) followed the inclusion of ionizable functionality within the promoiety to compensate for masking the polar enolic OH group of the free drug. However, the introduction of ionizable functionality was often associated with decreased stability, as demonstrated by the hemisuccinate, hemiadipate, hemisuberate, and alpha-amino ester derivatives of 1 which could not be isolated. A clear exception was the hemifumarate derivative of 1 which was not only isolable but actually more stable at neutral pH than the nonionizable ester analogues. The solution and solid state stability of the hemifumarate, together with its activity as a prodrug of 1, suggests that hemifumarate be considered as an alternative to hemisuccinate as a prodrug derivative for alcohols, particularly in situations where solution state stability is an issue.

Pharmacokinetics and safety of trovafloxacin (CP-99,219), a new quinolone antibiotic, following administration of single oral doses to healthy male volunteers.

Trovafloxacin (CP-99,219) is a new fluoroquinolone antibacterial agent with a broad spectrum of activity against Gram-positive and Gram-negative bacteria. The pharmacokinetics and safety of trovafloxacin were characterised in healthy male volunteers after administration of single oral doses of 30, 100, 300, 600 and 1000 mg. trovafloxacin was rapidly absorbed and serum concentrations reached a maximum approximately 1 h after dosing. The corresponding mean Cmax values (mean +/- SD) were 0.3 +/- 0.0, 1.5 +/- 0.5, 4.4 +/- 1.1, 6.6 +/- 1.4 and 10.1 +/- 0.5 mg/L. Terminal-phase half-life was independent of dose, with an overall mean of 9.9 +/- 2.5 h. Generally, Cmax and AUC0-infinity increased linearly with dose. Less than 10% of the administered dose was recovered unchanged in urine. Over the dosing range, trovafloxacin renal clearance was fairly constant, averaging 0.67 +/- 0.36 L/h. Trovafloxacin binding to serum proteins was moderate (70%). Trovafloxacin was well tolerated at doses of 300 mg or below. There were no significant changes in the clinical chemistry or haematology parameters evaluated over the entire dosing range.

Triacylglycerol lipase activity in the rabbit renal medulla.

Although the renal medulla is rich in triacylglycerols, the lipolysis of these intracellular triacylglycerols by a renomedullary triacylglycerol lipase has not been directly demonstrated. The present study demonstrates triacylglycerol lipase activity localized in the particulate subcellular fractions of rabbit renal medullae. Renomedullary triacylglycerol lipase activity, as determined by the hydrolysis of [14C]triolein to [14C]oleic acid, was observed to have a pH optimum of 5.8. Addition of cAMP/ATP/magnesium acetate resulted in an 80% activation of crude homogenate triacylglycerol lipase activity; addition of exogenous cAMP-dependent protein kinase resulted in a further activation of lipolysis. 3 mM CaCl2 had no effect on basal triacylglycerol lipase activity. 1 M NaCl did not inhibit lipolysis, suggesting that the lipase activity measured was not due to lipoprotein lipase. Endogenous renomedullary triacylglycerols were hydrolysed by a lipase in the 100,000 X g pellet of renomedullary homogenates, resulting in the release of free fatty acids including arachidonic and adrenic acids. Dispersed renomedullary cells were prepared to monitor hormone-sensitive triacylglycerol lipase activity in intact cells. Addition of 10 microM forskolin and 10 microM epinephrine resulted in 8-fold and 50-fold increases in triacylglycerol lipase activity, respectively, as defined by release of free glycerol from the cells. These studies demonstrate that a cAMP-dependent hormone-sensitive triacylglycerol lipase is present in the renal medulla, and is responsible for the hydrolysis of renomedullary triacylglycerols.

Metabolic fate of radiolabeled prostaglandin D2 in a normal human male volunteer.

50 microCi of [3H]prostaglandin D2 tracer (100 Ci/mmol) was infused intravenously into a normal human male volunteer. 75% of the infused radioactivity was excreted into the urine within 5 h. This urine was added to urine obtained from two mastocytosis patients with marked overproduction of prostaglandin D2. Radiolabeled prostaglandin D2 urinary metabolites were chromatographically isolated and purified and subsequently identified by gas chromatography-mass spectrometry. 25 metabolites were identified. 23 of these compounds comprising 37% of the recovered radioactivity had prostaglandin F-ring structures, and only two metabolites comprising 2.7% of the recovered radioactivity retained the prostaglandin D-ring structure. The single most abundant metabolite identified was 9,11-dihydroxy-15-oxo-2,3,18,19-tetranorprost-5-ene-1,20-dioic acid which was isolated in a tricyclic form as a result of formation of a lower side chain hemiketal followed by lactonization of the terminal carboxyl and the hemiketal hydroxyl. Different isomeric forms of several prostaglandin F-ring metabolites were identified. An isomer of prostaglandin F2 alpha was also excreted intact into the urine as a metabolite of prostaglandin D2. 15 PGF-ring compounds were treated with n-butylboronic acid and 13 failed to form a boronate derivative, suggesting that the orientation of the hydroxyl group at C-11 in these 13 metabolites is beta. This study documents that prostaglandin D2 is metabolized to prostaglandin F-ring metabolites in vivo in humans. These results also bring into question the accuracy of quantifying prostaglandin F2 alpha metabolites as a specific index of endogenous prostaglandin F2 alpha biosynthesis, as well as quantifying urinary prostaglandin F2 alpha as an accurate index of renal production of prostaglandin F2 alpha.

Transformation of prostaglandin D2 to 9 alpha, 11 beta-(15S)-trihydroxyprosta-(5Z,13E)-dien-1-oic acid (9 alpha, 11 beta-prostaglandin F2): a unique biologically active prostaglandin produced enzymatically in vivo in humans.

In vitro studies examining the metabolic transformation of prostaglandin D2 (PGD2) by human liver were conducted. PGD2 was found to be converted by a NADPH-dependent enzyme in the 100,000 X g supernatant of human liver exclusively to a single more polar compound that had a mass spectrum essentially the same as that of prostaglandin F2 alpha (PGF2 alpha). However, this compound could be chromatographically separated from PGF2 alpha and failed to form a butylboronate derivative. The structure of this compound was established as 9 alpha, 11 beta-(15S)-trihydroxyprosta-(5Z, 13E)-dien-1-oic acid (9 alpha, 11 beta-PGF2) by comparison of its chromatographic and mass spectral characteristics with authentic 9 alpha, 11 beta-PGF2. This compound was found to be biologically active by demonstrating increases in blood pressure in rats in a dose-related fashion following intravenous administration. By using a mass spectrometric assay, levels of this compound in plasma and urine from a normal volunteer were 6 pg/ml and 982 ng/24 hr, respectively. In a patient with systemic mastocytosis associated with overproduction of PGD2, urinary excretion of 9 alpha, 11 beta-PGF 2 was 6634 ng/24 hr and a circulating plasma level as high as 490 ng/ml was found during a severe episode of systemic mast cell activation. 9 alpha, 11 beta-PGF2 is structurally a unique prostaglandin, is enzymatically formed, is produced in vivo in humans, and is biologically active.

Bacteremia due to nontypable Haemophilus influenzae--three cases in a community hospital.

Nontypable Haemophilus influenzae is a normal inhabitant of the upper respiratory tract and a common pathogen in diseases limited to mucosal surfaces. Nontypable H. influenzae has only occasionally been reported to cause invasive disease locally or systemically. In a period of two years, three patients of 17 with positive blood cultures for H. influenzae were found to have nontypable strains, two of which were resistant to ampicillin. The presumed sites of entry were an oral mucosal lesion, sinus mucosa, and female genital tract. All three patients responded rapidly to antibiotics to which their isolates were susceptible.

Pathogenesis and prevention of recurrent infection after Haemophilus influenzae bacteremia.

Recurrence of disease after Haemophilus influenzae bacteremia is relatively uncommon and may often be preventable. Three previously unreported and 11 reported occurrences in ten patients were evaluated in regard to pathogenesis. Recrudescence can be prevented by adequate culturing prior to therapy, proper treatment based on complete sensitivity testing and pharmacologic principles, and careful evaluation of clinical and microbiologic response. Relapse may be prevented in some instances by administering prophylactic rifampin to patients and close contacts who may be carriers of an infecting strain. Reinfections may be prevented through public health measures and the development of effective vaccines.