A phase I study of the oral combination of CI-994, a putative histone deacetylase inhibitor, and capecitabine.

BACKGROUND: This study was conducted to determine the toxicity profile, maximum tolerated dose (MTD) and pharmacokinetics of the putative histone deacetylase inhibitor CI-994 in combination with capecitabine. PATIENTS AND METHODS: Fifty-four patients were treated according to three different dosing schemes in which the capecitabine dose was fixed and the CI-994 dose was escalated. Capecitabine was administered in twice daily divided doses, and CI-994 was given as a single daily dose. In schedule A, 26 patients were treated with capecitabine 1650 mg/m2/day and CI-994 for 2 weeks of a 3-week cycle. In schedule B, six patients received capecitabine 1650 mg/m2/day for two 3-week cycles and CI-994 for 5 of 6 weeks. In schedule C, 22 patients were treated with capecitabine 2000 mg/m2/day and CI-994 for 2 of 3 weeks. RESULTS: At the MTD, the principal dose-limiting toxicity was thrombocytopenia. The pharmacokinetics of CI-994 were unaltered by capecitabine, and there was no correlation between body surface area and major pharmacokinetic parameters. Platelet count nadir was best predicted by the observed maximal concentration (C(max)) of CI-994. CONCLUSIONS: The recommended phase II dose is 6 mg/m2 (or 10 mg) of CI-994 in combination with capecitabine 2000 mg/m2/day for 2 weeks of a 3-week cycle.

Quinapril and its metabolite quinaprilat in human milk.

AIMS: To measure the milk to plasma ratio (M/P) of quinapril and its active metabolite quinaprilat in lactating mothers and to assess likely infant exposure. METHODS: A single dose of quinapril 20 mg was administered to six healthy mothers who had been breastfeeding their infants for at least 2 weeks. Blood was sampled for the measurement of quinapril and quinaprilat at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 16 and 24 h. Milk was collected for measurement of quinapril and quinaprilat concentrations over the periods -4-0, 0-4, 4-8, 8-12, 12-18, 18-24 h. The areas under the plasma and milk concentration-time curves were estimated and an M/P ratio derived for both quinapril and quinaprilat. RESULTS: The M/P ratio for quinapril was 0.12 (95% CI 0.09,0.14). No quinapril was detected in milk after 4 h. No quinaprilat was detected in any of the milk samples. The estimated 'dose' of quinapril that would be received by the infant was 1.6% (95% CI 1.0,2.2) of the maternal dose, adjusted for respective weights. CONCLUSIONS: Quinapril appears to be 'safe' during breastfeeding according to conventional criteria, although as always, the risk:benefit ratio should be considered when it is to be given to a nursing mother.

LC/MS/MS quantitation of an anti-cancer drug in human plasma using a solid-phase extraction workstation: application to population pharmacokinetics.

A liquid chromatographic/mass spectrometric (LC/MS/MS) method to quantitate an anti-cancer drug in human plasma was validated. The method has proven suitable for routine quantitation of the experimental anti-cancer compound at concentrations from 1 to 400 ng/ml. Retention times of the compound and internal standard (compounds I and II, respectively) were 1.8 and 2.1 min, respectively. No interfering endogenous peaks were observed throughout the validation process. Precision estimates for this approach were typically less than 5% relative standard deviation (RSD) across the calibration range. Other validation parameters studied included specificity, system reproducibility, limit of quantitation, accuracy, linear range, and stability of the compound and internal standard in plasma and injection solvent. This method was used to quantify drug for population pharmacokinetic studies.

Induced cytoskeletal changes in bovine pulmonary artery endothelial cells by resveratrol and the accompanying modified responses to arterial shear stress.

BACKGROUND: Atherosclerosis and coronary heart disease (CHD) are significant contributors to morbidity and mortality in developed countries. A noted exception is the low mortality of CHD in France, particularly the southwest region. This phenomenon, commonly referred to as the French paradox, may be associated with high consumption of red wine. We investigate whether the cardioprotective activity of red wine may involve the grape skin-derived polyphenol, resveratrol. We further test the possibility that resveratrol acts by modulating structural and functional changes in endothelial cells lining the blood vessel wall. RESULTS: Bovine pulmonary artery endothelial cells (BPAEC) were incubated with resveratrol, with and without concurrent exposure to simulated arterial shear stress. Resveratrol significantly affected proliferation and shape of BPAEC; growth was suppressed and cells became elongated, based on morphologic analysis of rhodamine-conjugated phalloidin stained F-actin by confocal microscopy. Using selective signaling inhibitors, we showed that the resveratrol-induced cellular phenotype was dependent on intracellular calcium and tyrosine kinase activities, and assembly of actin microfilaments and microtubules, but was unrelated to PKC activity. Exposure to simulated arterial flow revealed that, whereas controls cells easily detached from the culture support in a time-dependent manner, resulting in total cell loss after a 5 min challenge with simulated arterial flow conditions, a significant percentage of the treated cells remained attached to the cultured plastic coverslips under identical experimental conditions, suggesting that they adhered more strongly to the surface. Western blot analysis shows that whereas cells treated with 25 microM and 100 microM resveratrol had no change in total ERK1/2, treatment did result in an increase in phosphorylated ERK1/2, which probably involved stabilization of the active enzyme. An increase in nitric oxide synthase expression was detected as early as 6 h and persisted for up to 4 days of treatment. CONCLUSIONS: Results of our studies show that resveratrol interacts with endothelial cells in vitro to elicit morphological and structural changes; the observed changes support the interpretation that resveratrol acts as a cardioprotective agent.

Impact of population pharmacokinetic-pharmacodynamic analyses on the drug development process: experience at Parke-Davis.

BACKGROUND: Continued scepticism about the benefits of population pharmacokinetics and/or population pharmacodynamics, here referred to collectively as the population approach, hampers its widespread application in drug development. At the same time the sources of this scepticism have not been clearly defined. In an attempt to capture and clearly define these concerns and to help communicate the value of the population approach in drug development at Parke-Davis we conducted a survey of customers within the company. The results of this survey are presented here. METHODS: All drug development programmes conducted over the past 10 years that included a population approach in data analysis and interpretation were identified. A brief description of the population analysis was prepared together with a brief description of how the resulting information was used in each drug development programme. These synopses were forwarded to relevant members of each drug development team together with a survey designed to solicit opinions as to the relevance and impact of these analyses. RESULTS: The most frequent use of information derived from population-based analysis was in labelling. In all cases of drugs making to New Drug Application (NDA) submission the analyses resulted in information that was included in approved or proposed labelling. In almost half of the cases summarised here (5 of 12), population-based analysis was perceived to have resulted in information that influenced the direction of individual development programmes. In many of these cases the information was serendipitous. It is also noted that most of these analyses were not the result of clearly defined objectives and prospective analysis plans. CONCLUSIONS: Use of the population approach, even when applied retrospectively, may have value in complementing or supporting interpretation of other data collected during the course of a trial. Atypical systemic exposure is quickly and easily assessed for correlation with adverse events or exceptional efficacy in retrospective or ad hoc evaluation. Although we know of no direct evidence, it is possible that such use of population pharmacokinetic data has facilitated NDA review and approval by providing insight into the role of atypical systemic drug exposure in otherwise spurious events.

Pharmacodynamics and pharmacokinetic-pharmacodynamic relationships of atorvastatin, an HMG-CoA reductase inhibitor.

The objective of this study is to determine the relationships between plasma atorvastatin concentrations, LDL (low-density lipoprotein) cholesterol reduction, and atorvastatin dose; the earliest time at which lipid levels change when atorvastatin treatment is initiated or discontinued; and alterations in LDL particle composition. Twenty-four subjects with elevated LDL-cholesterol were treated with escalating daily doses of 5, 20, and 80 mg atorvastatin for 6 weeks each. Serial plasma lipid and lipoprotein analyses were performed during the initiation and discontinuation of atorvastatin therapy, as well as at steady state. LDL-apolipoprotein B and LDL-cholesterol were measured directly after ultracentrifugation, and LDL-cholesterol also was estimated by the method of Friedewald. Steady-state atorvastatin pharmacokinetic parameters were estimated on the last day of each dosing period. LDL-cholesterol (Friedewald) reductions of 34%, 43%, and 57% were produced by atorvastatin doses of 5, 20, and 80 mg, respectively. The mean dose-response relationship was log linear, and almost all individual dose-response curves paralleled the mean curve. LDL-apolipoprotein B reductions were slightly less than those of LDL-cholesterol. Atorvastatin area under the curve (AUC(0-24) values increased proportionally with dose, while values of Cmax (maximum concentration) increased more than proportionally, and Cmin (minimum concentration) increased less than proportionally. Following initiation of dosing, statistically significant decreases in total cholesterol, LDL-cholesterol (beta quant), and LDL-apolipoprotein B were observed within 24 hours and in LDL-C (Friedewald) within 72 hours. Following discontinuation of drug dosing, statistically significant increases were observed in total cholesterol and LDL-cholesterol (Friedewald) within 48 hours and in LDL-cholesterol (beta quant) and LDL-apolipoprotein B within 72 hours. At each dose, an individual's LDL-cholesterol response was not correlated with AUC(0-24). In conclusion, atorvastatin produces marked LDL-cholesterol reductions, the mean dose-response relationship is log linear, almost all individual dose-response curves parallel the mean dose-response curve, onset and cessation of action are rapid, the estimated and measured LDL-cholesterol are the same, LDL-cholesterol and LDL-Apo B reductions are similar, and plasma concentrations are not correlated with LDL-cholesterol reduction at a given dose.

Antisense oligonucleotides to c-fos and c-jun inhibit intimal thickening in a rat vein graft model.

BACKGROUND: C-fos and c-jun are 2 immediate early genes that have been implicated in the stimulation of vascular smooth muscle cell proliferation and migration. In previous experiments in our laboratory with a rat vein graft model a 2- to 3-fold increase of messenger RNA of c-fos and c-jun were noted 1 hour after vein graft perfusion. Because c-fos and c-jun are up-regulated after the perfusion of vein grafts, the purpose of this study was to delineate the temporal expression of c-fos and c-jun protein and to study the effect of antisense oligonucleotides (ASO) to c-fos and c-jun on intimal thickening observed in this model. METHODS: Sprague-Dawley rats underwent bilateral interposition femoral artery grafts with use of the superficial epigastric vein, which was harvested from 15 minutes up to 2 weeks and analyzed by Western blot for Fos and Jun protein. Additional rats underwent bypasses and at the time of the procedure 1 graft was treated with a pluronic gel containing an ASO to c-fos, c-jun, or sense and the contralateral side was treated with pluronic gel only. The vein grafts were harvested 2 weeks after the procedure and perfusion fixed. After longitudinal sectioning, the intimal and total wall thicknesses were measured in the perianastamotic and midgraft regions by a morphometric digitizing microscope and the statistics were analyzed by a paired Student's t test. RESULTS: Protein analysis by Western blot showed that c-fos levels rose quickly within 2 hours and leveled at 6 hours 40-fold above basal levels after vein graft perfusion. Similarly, c-jun levels rose 10-fold above basal levels after 15 minutes and peaked at 2 hours 120-fold above basal levels. The treatment of the vein grafts with these ASOs resulted in a reduction of about 30% in the thickness of the intimal layer and the total wall thickness in both the perianastomotic and the midgraft regions, which was statistically significant different from control veins. CONCLUSION: These results indicate a possible therapeutic role for ASO to immediate early genes in the treatment of vein graft intimal hyperplasia.

Erythromycin coadministration increases plasma atorvastatin concentrations.

The effect of erythromycin on the pharmacokinetics of atorvastatin, an inhibitor of HMG-CoA reductase, was investigated in 12 healthy volunteers. Each subject received a single 10 mg dose of atorvastatin on two separate occasions, separated by 2 weeks. Erythromycin (500 mg qid) was given from 7 days before through 4 days after the second atorvastatin dose. Atorvastatin concentrations were determined by an enzyme inhibition assay, which measured both atorvastatin and active metabolites. When erythromycin was coadministered with atorvastatin, mean Cmax and AUC(0-infinity) increased by 37.7% and 32.5%, respectively. Mean terminal half-life was similar following each atorvastatin dose. Possible mechanisms for this interaction include erythromycin inhibition of first-pass conversion of atorvastatin to inactive metabolites and erythromycin inhibition of P-glycoprotein-mediated intestinal or biliary secretion.

Atorvastatin does not produce a clinically significant effect on the pharmacokinetics of terfenadine.

The effect of atorvastatin, a CYP3A4 substrate, on the pharmacokinetics of terfenadine and its carboxylic acid metabolite, fexofenadine, were evaluated. Single 120-mg doses of terfenadine were given 2 weeks apart to healthy volunteers with 80-mg daily doses of atorvastatin administered from 7 days before through 2 days after the second terfenadine dose. Concentrations of terfenadine and fexofenadine were measured for 72 hours after each terfenadine dose. Administration of terfenadine alone or in combination with atorvastatin produced no alterations in the QTc interval. For terfenadine, atorvastatin coadministration produced an 8% decrease in maximum concentration (Cmax), a 35% increase in area under the concentration-time curve extrapolated to infinity (AUC0-infinity), and a 2% decrease in elimination half-life (t1/2). For fexofenadine, atorvastatin coadministration produced a 16% decrease in Cmax, a 2% decrease in AUC0-infinity and a 51 % increase in t1/2. None of these changes achieved statistical significance. Coadministration of atorvastatin with terfenadine does not result in a clinically significant drug interaction. Because 80 mg is the highest atorvastatin dose used clinically, drug interactions mediated by CYP3A4 inhibition are unlikely in clinical practice.

Regulation of G1/S transition and induction of apoptosis in HL-60 leukemia cells by fenretinide (4HPR).

We previously reported that all-trans retinoic acid (RA) and fenretinide (4HPR) suppress HL-60 leukemia cell growth and cause partial cell arrest in the G1-to-S phase. Moreover, 4HPR but not RA induces apoptosis in HL-60 cells. To investigate further the observed biological effects, cyclin D1 and cdk4 expression and the level of phosphorylation of the retinoblastoma protein Rb were assessed. Cyclin D1 and cdk4 expression and Rb phosphorylation were significantly reduced, by 40-75%, after 24 hr of treatment with RA or 4HPR; these decreases were either transient, e.g., only at 24 hr for cdk4, or sustained for 72 hr. In general, more pronounced decreases were seen in the 4HPR-treated cells. Evidence for 4HPR-induced apoptosis comes from (1) cleavage of the enzyme poly(ADP-ribose) polymerase (PARP) to an 89-kDa truncated product, (2) appearance of DNA ladders on agarose gel electrophoresis, and (3) higher incorporation in situ of digoxigenin nucleotides into the free 3'-ends of DNA. Overnight pretreatment with 0.5-5.0 microM of the CPP32 inhibitor DEVD, but not the ICE inhibitor YVAD, significantly reduced the specific processing of PARP, suggesting that CPP32 is involved in the mechanism of action of 4HPR. Analysis of 2 lipid-derived second messengers, ceramide and diacylglycerol (DAG), as a function of time of treatment with RA or 4HPR, showed ceramide but not DAG to be significantly albeit transiently increased 2-fold at 3 hr, by 4HPR. To test further whether ceramide may be involved in the signaling cascade that culminates in the induction of apoptosis in 4HPR-treated HL-60 cells, the effects of fumonisin B1, an inhibitor of ceramide synthase, were studied. Simultaneous treatment of cells with 4HPR and 25-100 microM fumonisin B1 resulted in a dose-dependent reduction in the elevation in ceramide, the extent of PARP cleavage, and induction of apoptosis. Pretreatment with DEVD or YVAD, on the other hand, had no effect on the 4HPR-induced increase in ceramide.

Neuropsychological correlates of negative, disorganized and psychotic symptoms in schizophrenia.

Recent studies suggest that three dimensions (negative, disorganized and psychotic) categorize schizophrenic symptoms. A developing literature indicates distinct cerebral correlates of each symptom cluster, but few investigations have determined their neuropsychological correlates. In the present study, the Schedules of Negative and Positive Symptoms measured symptom severity in 62 schizophrenics, and a subsequent principal components analysis revealed three symptom dimensions. Factor scores, age and parental socio-economic status were simultaneously entered into regression equations to explain variance across a broad neuropsychological test battery. Negative symptoms were associated with deficits involving intelligence, executive function, memory, sustained-attention and sensory-motor function, whereas disorganized symptoms correlated with decreased intelligence, attention-span and sensory-motor function. Psychotic symptoms were unrelated to deficits. These data are consistent with hypotheses that these three symptom dimensions have distinct neurobehavioral correlates.

Cross-reactivity of fosphenytoin in two human plasma phenytoin immunoassays.

The cross-reactivity of fosphenytoin, a phosphate ester prodrug of phenytoin, was investigated in the Abbott phenytoin TDx/TDxFLx fluorescence polarization immunoassay (TDx) and the Behring Diagnostics phenytoin Emit 2000 enzyme-multiplied immunoassay (Emit). The first part of our study investigating cross-reactivity utilized in vitro correlation of the two immunoassays with a validated and specific phenytoin HPLC method used to assay plasma samples prepared in several phenytoin and fosphenytoin concentration combinations. Fosphenytoin cross-reacted with both immunoassays, but to a greater extent with TDx. In the second part of the study, empirically-derived models that best explained the in vitro data were used to predict "immunoassay-derived" phenytoin concentrations in plasma samples collected from actual patients after intravenous (i.v.) or intramuscular (i.m.) fosphenytoin dosing. The greatest degree of phenytoin concentration overestimation occurred at times when fosphenytoin concentrations were highest: within 1 to 2 h after i.v. infusion or during the first 2 to 4 h after i.m. injection. It is recommended that phenytoin concentrations not be monitored using these or other potentially nonspecific immunoanalytical methods for at least 2 h after i.v. fosphenytoin infusion or 4 h after i.m. fosphenytoin injection.

In vitro metabolism of ceftiofur in bovine tissues.

The metabolism of ceftiofur in bovine kidney, liver, muscle and lung, and the effects of the presence of cystine and glutathione in the media were evaluated using S-9 and microsomal tissue fractions. Conversion of ceftiofur to desfuroylceftiofur (DFC) was catalyzed by an esterase which was most active in kidney, followed by liver. It was not very active in muscle and lung. After DFC was liberated, it rapidly bound primarily to tissue proteins (> 56%), and was also conjugated to cysteine and glutathione. Production of DFC-cysteine by disulfide exchange of DFC with cystine and production of DFC-glutathione by conjugation of DFC to glutathione occurred in buffer if glutathione and cystine were present in the medium. These conjugations were also observed in incubations with tissue fractions, indicating that they were not inhibited by the tissues endogenous molecules. In addition, the metabolism of DFC-glutathione to DFC-cysteine was observed when tissue proteins were present. The metabolism of DFC-glutathione to DFC-cysteine was faster in kidney than in liver. Metabolites devoid of an intact beta-lactam ring were not observed in these in vitro studies.

ANG II stimulates endothelial nitric oxide synthase expression in bovine pulmonary artery endothelium.

Although angiotensin II (ANG II) is a known pulmonary vasoconstrictor, the purpose of this study was to examine the effect of ANG II on pulmonary artery endothelial cell nitric oxide synthase (ecNOS) mRNA and protein expression. Cultured bovine pulmonary artery endothelial (BPAE; passages 5-8) cells were incubated for 0-12 h with 10(-6) M ANG II. Total RNA was extracted, and ecNOS expression was assessed by Northern blot analysis. In BPAE cells, ecNOS mRNA was significantly increased 2.4 +/- 0.3-fold (P < 0.05 vs. basal; n = 5) 6 h after the addition of ANG II over basal levels. In & similar time course, it was found that ecNOS protein concentrations are increased 247 +/- 62% (P < 0.05 vs. basal; n = 8) over basal levels 4 h after ANG II addition. There is a second protein peak 8 h after ANG II addition in which ecNOS was increased 333 +/- 145% over basal (P < 0.05, n = 3). These data suggest that ANG II stimulates ecNOS mRNA expression and are followed by increased levels of ecNOS protein in cultured BPAE cells, consistent with an observed increase in nitrite production. Both the increase in ecNOS protein and mRNA expression could be inhibited with the ANG II receptor antagonist saralasin. Additionally, actinomycin D, an inhibitor of transcription, prevented the rise in mRNA at 6 h while cycloheximide inhibited the initial protein peak. The effects of ANG II on ecNOS were specific for the pulmonary artery endothelium. Addition of ANG II did not increase ecNOS protein or mRNA expression in parallel studies in bovine coronary artery endothelium. The stimulation of ecNOS by ANG II may act to protect the lung and maintain low pulmonary artery pressures in the renin-angiotensin model of systemic hypertension.

Cognitive deficits distinguish patients with adolescent- and adult-onset schizophrenia.

Recent studies have shown that patients with schizophrenia who have an adolescent-symptom onset (before age 21) have a worse clinical course and greater frequency of cerebral abnormalities than those with an adult-onset (after age 25). However, little is known about the neuropsychological functioning of these groups. A comprehensive neuropsychological examination was administered to groups of patients with schizophrenia with either an adolescent- or adult symptom-onset and a healthy control group. The adolescent-onset group performed worse than the adult-onset and control groups, particularly on measures of memory and executive function. The adult-onset group also performed worse than the controls, but to a lesser extent than did the adolescent-onset group. Results are discussed with reference to hypotheses that adolescent-onset schizophrenia represents a distinct neurodevelopmental disease entity.

The volume of the entorhinal cortex in schizophrenia: a controlled MRI study.

1. The entorhinal cortex (EC), part of the limbic temporal lobe, is a critical link between the cerebral cortex and the hippocampus, and is considered one of the most important cortical "association" areas. Several postmortem abnormalities in the EC have been reported. 2. Here, the authors report the first in vivo study of the volume of the EC in schizophrenia using magnetic resonance imaging (MRI) scans. 3. The authors compared 57 schizophrenic patients and 35 healthy controls. No overall difference in the mean EC volume was found between controls and schizophrenic patients, but there was a strong trend (p = .078) for the schizophrenic females to have a large mean EC than control females and for the early onset schizophrenia group to have a smaller (EC (p = .07) than late onset schizophrenia subjects. 4. The implications of the findings are discussed.

Renal dysfunction does not alter the pharmacokinetics or LDL-cholesterol reduction of atorvastatin.

The objective of this study was to determine the effects of renal dysfunction on the steady-state pharmacokinetics and pharmacodynamics of atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. Nineteen subjects with calculated creatinine clearances ranging from 13 mL/min to 143 mL/min were administered 10 mg atorvastatin daily for 2 weeks. Pharmacokinetic parameters and lipid responses were analyzed by regression on calculated creatinine clearance. Correlations between steady-state atorvastatin pharmacokinetic or pharmacodynamic parameters and creatinine clearance were weak and, in general, did not achieve statistical significance. Although the elimination rate constant, lambda z (0.579), was significantly correlated with creatinine clearance, neither maximum plasma concentration (Cmax, -0.361) nor oral clearance (Cl/F, 0.306) were; thus, steady-state exposure is not altered. Renal impairment has no significant effect on pharmacodynamics and pharmacokinetics of atorvastatin.

Tubular transport mechanisms of quinapril and quinaprilat in the isolated perfused rat kidney: effect of organic anions and cations.

The clearance mechanisms of quinapril and quinaprilat were probed using an isolated perfused rat kidney model. Sixty-four experiments were performed with drug in the absence and presence of classic inhibitors of the organic acid (i.e., probenecid and p-aminohippurate) and organic base (i.e., tetraethylammonium and quinine) transport systems of the proximal tubule. Initial perfusate concentrations of quinapril and quinaprilat were approximately 2.36 microM (or 1000 ng/ml), and transport inhibitors were coperfused at 100-10,000 times the drugs' initial microM concentrations. Quinapril and quinaprilat concentrations were determined in perfusate, urine, and perfusate ultrafiltrate using a reversed-phase HPLC procedure with radiochemical detection, coupled to liquid scintillation spectrometry. Perfusate protein binding was determined using an ultrafiltration method at 37 degrees C. Overall, the clearance ratios of quinapril (total renal clearance divided by fu x GFR) and quinaprilat (urinary clearance divided by fu. GFR) were significantly reduced, and in a dose-dependent manner, by the coperfusion of organic acids but not organic bases. The data demonstrate that the organic anionic secretory system is the primary mechanism by which quinapril and quinaprilat are transported into and across renal proximal cells.

Biochemistry and cell biology of phospholipase D in human neutrophils.

Neutrophils play a major role host defense against invading microbes. Recent studies have emphasized the importance of the phospholipase D (PLD) in the signalling cascade leading to neutrophil activation. Phospholipase D catalyzes the hydrolysis of phospholipids to generate phosphatidic acid with secondarily generation of diradylglycerol; both of these products have been implicated as second messengers. Herein, we discuss the regulation and the biochemistry of the receptor-regulated PLD in human neutrophils. In vivo and in vitro studies suggest an activation mode in which initial receptor-linked activation of phospholipase C generates diacylglycerol and inositol trisphosphate. The resulting calcium flux along with the diacylglycerol activate a conventional isoform of protein kinase C (PKC), probably PKC beta 1. This PKC, in turn phosphorylates a plasma membrane component resulting in PLD activation and a second outpouring of diradylglycerol. The small GTP-binding proteins, RhoA and ARF, also participate in this process, and synergize with a 50 kDa cytosolic regulatory factor.

Disposition of quinapril and quinaprilat in the isolated perfused rat kidney.

An isolated perfused rat kidney model was used to probe the renal disposition of quinapril and quinaprilat after separate administration of each drug species. Control studies were performed with drug-free perfusate (n = 8) and perfusate containing quinapril (n = 9) or quinaprilat (n = 7) at initial drug concentrations of 1000 ng/ml (including corresponding tracer levels of tritiated drug). Physiologic parameters were within the normal range of values for this technique and were stable for the duration of each experiment. Quinapril and quinaprilat concentrations were determined in perfusate, urine, and perfusate ultrafiltrate using a specific and sensitive reversed-phase HPLC procedure with radiochemical detection, coupled to liquid scintillation spectrometry. Perfusate protein binding was determined using an ultrafiltration method at 37 degrees C. The total renal clearance of quinapril (CLr) was calculated as Dose/AUC(0-infinity), and is represented by the sum of its urinary and metabolic clearances. The urinary clearances (CLe) of quinapril and quinaprilat were calculated as urinary excretion rate divided by midpoint perfusate concentration for each respective species. Of the total renal clearance for quinapril (CLr = 4.49 ml/min), less than 0.1% was cleared as unchanged drug (CLe = 0.004 ml/min); over 99% of the drug was cleared as quinaprilat formed in the kidney. The clearance ratio of quinapril [CR = CLr/(fu.GFR)] was 41.0, a value representing extensive tubular secretion into the renal cells. Following quinaprilat administration, the clearance ratio of metabolite [CR = CLe/(fu.GFR)] was 3.85, indicating a net secretion process for renal elimination.