Pharmacokinetics and tissue distribution of the nonadecapeptide Moli1901 in rats and mice.

1. Administration of aerosolized, radiolabelled Moli1901 (duramycin, 2622U90), a 19 amino acid polycyclic peptide, to rats resulted in the deposition of high amounts of radiolabel in the respiratory tract, with deposited radiolabel persisting almost unchanged through 7 days after dosing. Little to no radiolabel was present in the bloodstream of these rats. 2. Rats absorbed little radiolabel after p.o. administration, with nearly all of the dose excreted in the faeces by 2 days after dosing. 3. At 7 days following an intravenous dose, rats excreted 54% of the radiolabel in faeces and 5.4% in the urine, with 44% remaining in the carcass, primarily in the liver (33%). 4. Following an intratracheal instillation dose to rats, radiolabel was eliminated from the pulmonary system with a half-life of 64 days. Excretion was almost exclusively via faeces, with an elimination half-life of 52 days. Plasma and blood concentrations in these animals were uniformly <1 ng eq. ml(-1) at all sampling times. 5. Results in mice given intravenous and oral doses were consistent with those observed in rats. 6. Prolonged retention of Moli1901 in pulmonary tissue supports its use in the treatment of respiratory diseases.

Comparison of celecoxib metabolism and excretion in mouse, rabbit, dog, cynomolgus monkey and rhesus monkey.

1. The metabolism and excretion of celecoxib, a specific cyclooxygenase 2 (COX-2) inhibitor, was investigated in mouse, rabbit, the EM (extensive) and PM (poor metabolizer) dog, and rhesus and cynomolgus monkey. 2. Some sex and species differences were evident in the disposition of celecoxib. After intravenous (i.v.) administration of [14C]celecoxib, the major route of excretion of radioactivity in all species studied was via the faeces: EM dog (80.0%), PM dog (83.4%), cynomolgus monkey (63.5%), rhesus monkey (83.1%). After oral administration, faeces were the primary route of excretion in rabbit (72.2%) and the male mouse (71.1%), with the remainder of the dose excreted in the urine. After oral administration of [14C]celecoxib to the female mouse, radioactivity was eliminated equally in urine (45.7%) and faeces (46.7%). 3. Biotransformation of celecoxib occurs primarily by oxidation of the aromatic methyl group to form a hydroxymethyl metabolite, which is further oxidized to the carboxylic acid analogue. 4. An additional phase I metabolite (phenyl ring hydroxylation) and a glucuronide conjugate of the carboxylic acid metabolite was produced by rabbit. 5. The major excretion product in urine and faeces of mouse, rabbit, dog and monkey was the carboxylic acid metabolite of celecoxib.

Validation of immunoassays for bioanalysis: a pharmaceutical industry perspective.

Immunoassays are bioanalytical methods in which quantitation of the analyte depends on the reaction of an antigen (analyte) and an antibody. Although applicable to the analysis of both low molecular weight xenobiotic and macromolecular drugs, these procedures currently find most consistent application in the pharmaceutical industry to the quantitation of protein molecules. Immunoassays are also frequently applied in such important areas as the quantitation of biomarker molecules which indicate disease progression or regression, and antibodies elicited in response to treatment with macromolecular therapeutic drug candidates. Currently available guidance documents dealing with the validation of bioanalytical methods address immunoassays in only a limited way. This review highlights some of the differences between immunoassays and chromatographic assays, and presents some recommendations for specific aspects of immunoassay validation. Immunoassay calibration curves are inherently nonlinear, and require nonlinear curve fitting algorithms for best description of experimental data. Demonstration of specificity of the immunoassay for the analyte of interest is critical because most immunoassays are not preceded by extraction of the analyte from the matrix of interest. Since the core of the assay is an antigen-antibody reaction, immunoassays may be less precise than chromatographic assays; thus, criteria for accuracy (mean bias) and precision, both in pre-study validation experiments and in the analysis of in-study quality control samples, should be more lenient than for chromatographic assays. Application of the SFSTP (Societe Francaise Sciences et Techniques Pharmaceutiques) confidence interval approach for evaluating the total error (including both accuracy and precision) of results from validation samples is recommended in considering the acceptance/rejection of an immunoassay procedure resulting from validation experiments. These recommendations for immunoassay validation are presented in the hope that their consideration may result in the production of consistently higher quality data from the application of these methods.

Development of a novel scintillation proximity competitive hybridization assay for the determination of phosphorothioate antisense oligonucleotide plasma concentrations in a toxicokinetic study.

A novel scintillation proximity competitive hybridization assay was developed for determining plasma concentrations of compound 4003W94, a 15-base phosphorothioate antisense deoxyribonucleotide that is currently under preclinical evaluation for the treatment of restenosis following coronary artery angioplasty. The principle of the assay involves the hybridization binding of antisense (4003W94) to a biotinylated sense oligonucleotide to form a double-stranded nucleic acid complex on the surface of scintillation proximity beads derivatized with streptavidin. As in a competitive radioimmunoassay, there is an inverse relationship between the amount of radioactivity in the final binding complex and the amount of 4003W94 present in the sample being analyzed. Because this is a homogenous assay, no physical separation of bound from free radioligand is necessary. Conventional cross-reactivity studies with either 3'- or 5'-deletion oligomers of 4003W94 indicated that cross-reactivity generally decreased with each base deletion. The assay was used to determine plasma concentrations of 4003W94 equivalents in rhesus monkeys during an exploratory 14-day toxicity study. This method conceivably could be adapted for use as an effective in vitro screening tool for the identification of potential antisense oligonucleotide drug candidates or as a diagnostic tool for the detection of pathological disorders.

Balance/excretion of 3H- and 14C-tyloxapol in the male rabbit after intratracheal administration.

1. Tyloxapol, trace-labelled (50-100 microCi/animal) with 3H or 14C, was administered intratracheally in a surfactant formulation (EXOSURF Neonatal) to the male rabbit in a total tyloxapol dose of 5 mg/kg. Urine, faeces, expired air, and blood were collected for up to 10 days following tyloxapol administration. 2. Over 5 days, 3H-tyloxapol-related radioactivity in the urine (13.4%) and faeces (27.4%) accounted for a major fraction of the labelled dose. However, urine also contained an additional 13% of the dose as tritiated water. Expired air accounted for only 4.2% of the dose. At the end of the study, an additional 35.6% of the radioactive dose was found in tissues and the carcass, mainly in the lung (27.4%) and to a lesser extent in the liver (2.8%) and kidney (0.4%). Levels of radioactivity in other tissues, including whole blood, were low. 3. Over a separate 10-day study, faecal (30.4%) and renal (9.7%) elimination of 14C-tyloxapol accounted for 40% of the radioactive dose, with expired air accounting for much less (2.7%). At the end of the study, additional radioactivity was recovered from the lung (43.9%) and to a lesser extent from the liver (3.8%) and kidney (0.3%). The half-life for the elimination of total radioactivity from the lung was estimated to be 10-12 days. 4. These data indicate that, following intratracheal administration, tyloxapol and metabolites were retained by the lung, released slowly into the systemic circulation, and eliminated through faecal and renal excretion.

A phase II study of vinblastine in combination with acrivastine in patients with advanced renal cell carcinoma.

Renal cell carcinoma exhibits chemoresistance attributable in part to the P-glycoprotein drug efflux mechanism. Acrivastine is a hydrophylic antihistamine that has been shown in vitro to reverse this form of resistance. After five patients were treated on a dose-finding study, seventeen patients with metastatic or unresectable renal cell carcinoma were entered into a phase II study of vinblastine in combination with acrivastine. Patients received oral acrivastine at doses of 400 mg every 4 hours for 6 days and a 96-hour continuous infusion of vinblastine at a dose of 1.6 mg/m2/24 h. Of 15 evaluable patients, no tumor responses were seen. The regimen was well-tolerated with the majority of toxicities being gastrointestinal and hematologic. Serum levels of acrivastine, its principal metabolite (270C81) and vinblastine were measured during the study. Based on in vitro data, the plasma levels of acrivastine were within a range adequate to block P-glycoprotein activity. High doses of acrivastine were well-tolerated clinically, however, the combination of acrivastine and vinblastine was not active against renal cell carcinoma.

Codeine toxicokinetics in rats during a two-year dosed feed study.

Codeine toxicokinetics in F344 rats of both sexes were determined during a 2-year chronic toxicology study using dosed feed as the exposure route with a 12-hr light/dark cycle starting at 7:00 a.m. Rats were allowed to access to dosed feed formulations ad libitum with codeine concentrations at 0, 400, 800, and 1600 ppm. Blood samples were collected from individual rat on days 7, 21, and 90 at 7:00 p.m., 11:00 p.m., 3:00 a.m., and 7:00 a.m. Additional samples were collected at 16 and 24 months between 6:00-8:00 a.m. Plasma concentrations of codeine and morphine were determined directly by radioimmunoassay. Concentrations of their conjugates were determined indirectly by measuring the total amount of free codeine and morphine released after samples were treated with beta-glucuronidase. Results indicated that plasma concentrations of both codeine and morphine steadily decreased from day 7 to 16 months and then rebounded at 24 months. Results also indicated that plasma concentrations of both codeine and morphine correlated well with the amounts of codeine added to the feed. Bioavailability of codeine using the dosed feed route increased with dose, varying from 10% to 25%, which was somewhat higher than the previously reported approximately 8% bioavailability using the gavage route. Concentrations of conjugated codeine were very low, whereas concentrations of conjugated morphine were very high. These results suggested that demethylation of codeine to morphine in rats is the main metabolic pathway and was maintained over the course of the study.

Metabolism of synthetic surfactants.

This article provided a brief overview of synthetic surfactants and DPPC metabolism, and summarized disposition data on the most widely used synthetic surfactant, CPHT (colfosceril palmitate, hexadecanol, and tyloxapol; Exosurf Neonatal, Burroughs Wellcome Co.). In separate experiments, young male rabbits were given intratracheal administrations of CPHT trace-labeled with either hexadecanol-3-[14C], choline-labeled [14C]DPPC, or [3H]tyloxapol. In mass balance and tissue distribution studies, radioactivity was quantitated in excreta and selected tissues over 5 days. Hexadecanol entered the pathways of intermediary lipid metabolism, via oxidation to palmitic acid, which was then utilized in the synthesis of phospholipids. After 5 days, most of the radiolabeled dose (56%) was distributed throughout the body, with renal (4%) and fecal (2%) excretion being minor routes of elimination compared with expired air (28%). The active component, DPPC, was still retained by the body (72%) after 5 days, having entered the pathways of lipid metabolism to become tissue associated. At this time, the lung and liver each contained approximately 10% of the labeled dose, whereas elimination in excreta (8%) was minimal compared with that in expired air (17%). Tyloxapol and metabolites were retained by the lung, released slowly (t1/2 = 5 to 6 days) into the systemic circulation, and eliminated through fecal (27%) and renal (26%) excretion.

Disposition of digoxin immune Fab in patients with kidney failure.

Digoxin and digoxin immune Fab, its antidote, are eliminated renally. However, the disposition of Fab in severe kidney disease is poorly described. Therefore, the disposition of Fab and its relationship to total and free digoxin were studied in five digoxin-toxic patients with end-stage renal disease (n = 4) or severe renal dysfunction (n = 1) with a mean (+/- SD) serum creatinine of 5.9 +/- 1.2 mg/dl (four patients were receiving long-term hemodialysis). Serum was drawn after a clinically neutralizing Fab dose (80 to 160 mg) every 12 to 24 hours for 204 to 327 hours. Fab concentrations were assessed by radioimmunoassay, whereas total digoxin concentrations were assessed with a modified radioimmunoassay or fluorescence polarization immunoassay. The concentration-time profile of Fab appeared to be similar to the concentration-time profile of total digoxin. The mean (+/- SD) half-lives of the alpha and beta disposition phases of Fab were 13 +/- 5 hours and 96 +/- 31 hours, respectively, which were similar to the alpha and beta parameter estimates of total digoxin (14 +/- 4 and 123 +/- 16 hours, respectively). Steady-state volume of distribution and systemic clearance of Fab were 0.29 +/- 0.11 L/kg and 0.057 +/- 0.022 ml/min/kg, respectively. Thus, in comparison to values reported in patients with normal renal function, the elimination of Fab and total digoxin are markedly delayed in patients with end-stage renal disease, which may necessitate prolonged clinical monitoring.

Radioimmunoassay for the chemical stable prostacyclin analog, 15AU81: a preliminary pharmacokinetics study in the dog.

15AU81 is a chemically stable tricyclic benzindene analog of prostacyclin, with possible application to the treatment of congestive heart failure. Effective pharmacological doses in a dog model are in the microgram to sub-microgram/kg range, necessitating an analytical method of high sensitivity for determination of the drug in plasma. This report describes the development and validation of a radioimmunoassay for 15AU81, and its application to a pharmacokinetic study in the beagle dog. An antiserum elicited by immunization with a 15AU81-bovine thyroglobulin conjugate was employed, along with 3H-15AU81, in the radioimmunoassay. Analogs of 15AU81, as well as a glucuronide metabolite produced by a dog liver subcellular fraction in vitro, were used to demonstrate the specificity of the radioimmunoassay. Specificity was confirmed by comparative analysis by radioimmunoassay and by a quantitative GC/MS procedure of plasma samples from dogs dosed with 15AU81. The limit of quantitation in dog plasma was 1.6 ng/ml; accuracy and precision were both acceptable. The assay was applied to a study of the pharmacokinetics of 15AU81 in the beagle dog after intravenous or intratracheal administration of a single 20-micrograms/kg dose. Following intravenous dosing, 15AU81 was eliminated rapidly from plasma (t1/2, 2.8 min), while after intratracheal administration, clearance appeared to be somewhat slower and bioavailability was appreciable (mean, 46%), suggesting that this route of administration may be worthy of further evaluation.

Disposition and metabolism of triprolidine in mice.

The disposition of the antihistamine, triprolidine, was studied in male and female CD-1 mice after a single oral 50 mg/kg dose of [14C]triprolidine HCl. Urine and feces collected over 72 hr postdosing were analyzed for total radiocarbon, and for parent drug and metabolites by radiochromatography. Structures of metabolites were determined by GC/MS, direct probe MS, FAB/MS, LC/MS, NMR, and IR techniques. More than 80% of the dose was recovered in the urine, with the remainder recovered in the feces. The carboxylic acid analog of triprolidine (219C69) was found to be the major metabolite in urine and feces, accounting for an average of 57.6% of the administered dose. Three minor metabolites were identified as a gamma-aminobutyric acid analog of triprolidine, a pyrrolidinone analog of 219C69, and a pyridine-ring hydroxylated derivative of triprolidine. Parent drug could only be detected in urine and accounted for 0.3% (females) to 1.1% (males) of the dose. The results of this study showed that triprolidine was absorbed well but extensively metabolized when administered orally to mice.

Disposition of triprolidine in the male beagle dog.

Three male beagle dogs were given 2.5 mg/kg doses of [14C]triprolidine HCl monohydrate (2.09 mg/kg of the free base) by intravenous and oral routes, in a nonrandomized cross-over experiment. After either route of administration, approximately 75% of the dose was excreted in the urine, and the remainder was excreted in the feces. Triprolidine was extensively metabolized, with less than 1% of the parent drug recovered in the excreta after either route of administration. Three metabolites were isolated from excreta and identified, including the major metabolite (metabolite 1, 219C69), in which the toluene ring methyl group was oxidized to a carboxylic acid, a metabolite (metabolite 2) in which the pyrrolidine ring was opened with oxidation of the terminal carbon to a carboxylic acid (a gamma-aminobutyric acid), and a metabolite (metabolite 3) that was a pyrrolidinone derivative of 219C69. Other metabolites in urine and feces were present in amounts too small for quantitation or identification. Route of administration had little effect on the metabolic pattern of triprolidine. Thus, after oral administration of triprolidine, a mean of 49.1% of the dose was excreted as 219C69, 12.0% as metabolite 2, 3.4% as metabolite 3, and 0.6% as triprolidine, while after intravenous administration, a mean of 50.8% of the dose was excreted as 219C69, 11.1% as metabolite 2, 4.2% as metabolite 3, and 0.8% as triprolidine. Plasma contained triprolidine, 219C69, and metabolite 2, as well as other apparent metabolites that were present at levels too low for quantitation. Mean pharmacokinetic parameters calculated for triprolidine after intravenous dosing were: CL = 24.4 ml/min/kg, Vdss = 5.8 liters/kg, and Vc = 1.6 liters/kg.(ABSTRACT TRUNCATED AT 250 WORDS)

Disposition of acrivastine in the male beagle dog.

Three male beagle dogs were given 10 mg/kg iv and oral doses of [14C]acrivastine, a novel nonsedating antihistaminic agent, in a nonrandomized crossover experiment. Urine and feces were collected for 72 hr after dosing. After iv dosing, a mean of 34% was recovered in the urine, and 63% was recovered in the feces. After po dosing, a mean of 29% of the radiocarbon was recovered in the urine, and 63% was recovered in the feces (dose adjusted for 14% lost in vomitus). Acrivastine and three major metabolites were detected in the excreta. The metabolites were identified as a side-chain-reduced analog of acrivastine (metabolite 3, 270C81), a gamma-aminobutyric acid analog of 270C81 (metabolite 2), and a benzoic acid analog of 270C81 (metabolite 1). After iv dosing, 34% of the dose was excreted as parent drug, 21% as metabolite 3, 15% as metabolite 2, and 6% as metabolite 1, while after po dosing, 35% of the dose was excreted as parent drug, 18% as metabolite 3, 11% as metabolite 2, and 7% as metabolite 1. Pharmacokinetic analysis of acrivastine plasma concentration-time curves after both routes of administration indicated a mean total body clearance of 17.3 ml/min/kg, a Vss of 0.93 liter/kg, a terminal half-life of 0.7 hr, and an oral bioavailability of 40%. The apparent plasma half-life of the metabolite, 270C81, was 1.5 hr. Analysis of AUC values indicated that greater amounts of 270C81 than acrivastine circulated in plasma after both iv and po dosing, and that first-pass metabolism of acrivastine to 270C81 occurred. The results indicated that acrivastine was extensively metabolized in the dog to 270C81 and suggested that 270C81 itself underwent further metabolism to metabolites 1 and 2.

Clinical and pharmacokinetic profiles of digoxin immune Fab in four patients with renal impairment.

Minimal pharmacokinetic data on digoxin immune Fab are currently available, especially in patients with impaired renal function. The serum concentration-time profiles of total digoxin, free digoxin, and digoxin immune Fab in four patients with moderate to severe renal impairment who received digoxin immune Fab are presented. The calculated elimination half-life of digoxin immune Fab was 25-73 hours. The calculated elimination half-life of total digoxin was 24-72 hours. Free digoxin concentrations rebounded to a peak of 1-2.9 ng/mL 44-97 hours after the administration of digoxin immune Fab. The areas under the curve for digoxin immune Fab were 213-1026 micrograms.h/mL, and total body clearances were 2.3-7.1 mL/min. The total digoxin concentrations peaked at 14-33 times the pre-Fab digoxin concentrations 5-30 hours after digoxin immune Fab administration. In comparing these data with data available from patients with normal renal function, the half-life of digoxin immune Fab and total digoxin was longer, the peak total digoxin concentration occurred later, the ratio of the peak total digoxin concentration to pre-Fab digoxin concentration was larger, and the rebound in free digoxin occurred later in patients with renal impairment. The Fab dose should not be reduced in patients with renal impairment; however, post-Fab monitoring should be extended to compensate for the prolonged half-life of Fab and later rebound of free digoxin.

Immunofluorometric assay for lamotrigine (Lamictal) in human plasma.

An immunofluorometric assay (IFA) has been developed for the potential antiepileptic agent, lamotrigine (Lamictal). The assay involves competition between lamotrigine free in solution and bound to a bovine thyroglobulin conjugate on the surface of microtiter strip wells for a limited amount of polyclonal lamotrigine antisera. The end-point of this reaction, which indicates the concentration of lamotrigine present in the solution under analysis, is detected by adding Eu(3+)-labelled anti-rabbit IgG, followed by an enhancement solution to produce a fluorescent product. Thus, the higher the concentration of lamotrigine in the sample, the less intense the fluorescence produced. The assay displays minor cross-reactivity (0.05%) by the major glucuronide metabolite (in humans) and moderate cross-reactivity (2.7%) by a minor N-oxide metabolite (in rats) of the parent drug. No interference from these sources in the analysis of plasma samples from clinical trials was demonstrated by comparative sample analysis by IFA and high-performance liquid chromatography. Intraassay accuracy and precision were excellent, greater than 90% and less than 5% coefficient of variation (CV), while interassay accuracy was greater than 95% and interassay precision (CV) was 8.8-17.0%. This assay is suitable for analysis of lamotrigine in plasma samples collected during clinical trials.

Pharmacokinetics of oral and transdermal triprolidine.

In this open, nonrandomized, three-way crossover study, six healthy male volunteers received single doses of triprolidine (TPL) hydrochloride syrup orally (2.5 mg) and wore transdermal TPL patches (5 mg and 10 mg doses) to compare the pharmacokinetic profiles and dose tolerance of the two formulations. A washout period of at least 1 week was scheduled between the three dosing periods. Blood samples were collected at defined times, and plasma concentrations were determined using a radioimmunoassay. Maximum plasma drug concentration (Cmax) decreased from 5.6 +/- 2.9 ng/mL (mean +/- SD) with oral dosing to 2.0 +/- 1.0 ng/mL and 4.2 +/- 2.0 ng/mL following 5 mg and 10 mg transdermal doses, respectively. Time to reach peak concentration (tmax) increased from 2.0 +/- 1.2 hours with oral dosing to 12.0 +/- 5.9 and 14.3 +/- 9.9 hours following 5 mg and 10 mg transdermal doses, respectively. The differences between AUC0-alpha values with the oral syrup and the 5 mg and 10 mg transdermal doses were not significant when normalized to 2.09 mg (TPL base). The bioavailabilities of the 5 mg and 10 mg transdermal doses relative to the oral 2.09 mg doses were 0.89 +/- 0.32 and 1.04 +/- 0.33, respectively. Mild erythema and pruritus were the most common adverse effects secondary to TPL transdermal application. Drowsiness observed following oral TPL, was not evident following either transdermal dose. The results of this study, therefore, indicate that TPL can be absorbed transdermally, providing consistent plasma concentrations.

Treatment of digoxin intoxication in a renal failure patient with digoxin-specific antibody fragments and plasmapheresis.

A patient with renal failure due to myeloma kidney and coincident digitalis intoxication due to prescribed daily digoxin administration was treated with digoxin-specific antibody fragments and plasmapheresis. Rapid response to therapy was noted, removal of digoxin-antidigoxin antibody complexes was confirmed, and prevention of delayed rebound toxicity was documented. We suggest that this is the therapy of choice in similar individuals.

Pharmacokinetics of acrivastine after oral and colonic administration.

Six healthy male volunteers participated in this randomized, crossover open-label pharmacokinetic study consisting of two dosing segments separated by a washout period of at least 5 days. During each dosing segment, each volunteer received 12 mg of acrivastine, an investigational histamine H1-receptor antagonist, in a syrup form either orally or by colonic administration in random order. After oral and colonic administration, respectively, the following mean +/- SD pharmacokinetic parameters were obtained: Cmax 179 +/- 11 and 13.8 +/- 5.2 ng/ml; tmax, 0.85 +/- 0.13 and 3.60 +/- 0.56 hr; AUC0-12 hr, 576 +/- 57 and 104 +/- 46 hr.ng/ml. Differences between the oral and colonic administration for all three parameters were statistically significant (P less than 0.001). The mean +/- SD relative bioavailability of acrivastine from colonic compared to oral dosing was 0.18 +/- 0.09. It may be concluded, therefore, that appreciable absorption of acrivastine from the colon does not take place. These results suggest that comparison of pharmacokinetic profiles of some drugs after oral and colonic administration may be a useful technique for predicting bioavailability from a sustained release oral formulation.