Atiprimod (AnorMED).

Atiprimod is a macrophage-targeting oral cytokine inhibitor which is being developed by AnorMED as a potential treatment for rheumatoid arthritis and other autoimmune diseases. Phase I trials have been successfully completed and phase II multicenter trials have been approved by the FDA [303260]. The compound was discovered in a joint research and development program with SmithKline and French (now SmithKline Beecham, SB) but following acceptance of the phase II protocol by the FDA, SB decided not to proceed with further development of atiprimod as a result of an internal restructuring program [350042]. All rights to atiprimod and other azaspiranes developed in this program have reverted to AnorMED, which is seeking corporate partners for further development of the compound [303260,337657]. The compound was originally disclosed in European patent, EP-00310321, entitled 'Preparation of N-aminoalkyl-2-azaspiro[4.5]decanes and analogs as immunosuppresants', while a cost-effective, efficacious pilot plant synthesis has also been described [298722].

Development of a sensitive activity assay for high-volume evaluation of human renin inhibitory peptides in rat serum: results with U-71,038.

A sensitive activity assay for high volume evaluation of human renin inhibitory peptides (RIPs) in rat sera (range 2-80 ng/ml) was developed based on the low affinity of RIPs to rat renin and their high affinity to human renin. The utility of this activity assay was tested by measuring concentrations of a human RIP, U-71,038 (BOC-Pro-Phe-N-MeHis-Leu psi [CHOHCH2]Val-Ile-Amp), in rat sera, determined by the activity assay, by a sensitive radioimmunoassay (RIA), and by tracking tritiated drug. Rats were given radiolabeled drug as an intravenous bolus, and blood samples were collected at various times after dosing. The serum level of U-71,038 equivalents was determined by the three techniques. Whole blood was also counted for total radioactivity to evaluate the potential for U-71,038 incorporation into red blood cells. Results from the three serum assays indicate good agreement between the calculated U-71,038 equivalents for the 30 min and 1 hr collection times. The 2 and 4 hr collection times show excellent agreement for the activity assay and RIA; [3H]-U-71,038 determinations gave substantially higher values. Serum levels for U-71,038 determined 30 min after dosing averaged less than 300 ng equivalents/ml suggesting that less than 1% of the administered dose was in the systemic circulation at that time. Thus, U-71,038 was rapidly cleared. At the 4 hr collection time, the level of U-71,038 equivalents, as determined by activity assay and RIA, was ten times the in vitro IC50 for the renin inhibitory activity of U-71,038.(ABSTRACT TRUNCATED AT 250 WORDS)

A primate bioassay for the determination of renin inhibitory peptides in serum.

The study of renin inhibitory peptides (RIPs) in rodents and primates requires the establishment of a simple, high volume method for determining the concentration of RIPs in serum after intravenous or oral dosing. The human renin inhibition assay useful for rodents is not directly applicable to primates due to inherent production of angiotensin I from the primate serum angiotensinogen and added recombinant human renin. Therefore, a novel approach to analyze the serum concentrations of RIPs in primates is described based on in vitro studies with monkey serum. The procedure involves the inactivation of monkey angiotensinogen and monkey renin by thermal denaturation prior to analysis. Application of this assay was demonstrated by analyzing serum samples from an in vivo study in monkeys using ditekiren (U-71,038), a renin inhibitory peptide, and by validation of the assay and results using a tritium-based radioimmunoassay (RIA) for ditekiren. The minimum detectable limit of ditekiren for both the RIA and the bioassay for primates was 10ng/ml serum. The reported bioassay should be of value for monitoring serum levels of thermostable RIPs from pharmacokinetic, bioavailability, and pharmacodynamic studies in primates as well as in humans.

Multiple dose trial of the thromboxane synthase inhibitor furegrelate in normal subjects.

Furegrelate sodium, a pyridinyl derivative thromboxane synthase inhibitor, was evaluated for its effects on thromboxane synthesis in normal volunteers after multiple dose administration. Twenty-four subjects were randomized to 200, 400, 800 or 1600 mg furegrelate or placebo treatment BID for 4 1/2 days. Furegrelate (800 or 1600 mg) significantly inhibited thromboxane synthesis throughout the dosing interval as assessed by thromboxane B2 generation from platelet-rich plasma challenged with arachidonic acid or from serum. Platelet aggregation was inhibited, but the effect was variable and a clear dose response relationship was not apparent. Bleeding times were also variable but tended to increase at the higher doses. There was no clinically significant change in any coagulation parameters or in any safety laboratory evaluations. Peak serum concentrations occurred approximately 1 h after dosing; t1/2ke was approximately 2 h. There was no significant change in furegrelate's effects or pharmacokinetics over time (ie. Day 1 vs Day 5).

Determination of pioglitazone in dog serum using solid-phase extraction and high-performance liquid chromatography with ultraviolet (229 nm) detection.

An analytical method is described for the determination of the free base of pioglitazone hydrochloride (U72, 107A, AD-4833) in dog serum. The method used solid-phase extraction of pioglitazone from serum followed by high-performance liquid chromatographic analysis on an octadecylsilane column with an eluent of acetonitrile-water (41:59, v/v) containing 1.2 ml/l acetic acid (pH 6.0 +/- 0.05). The column effluent was monitored at 229 nm. The analytical procedure has a linear range of 25 ng/ml to 20 micrograms/ml, a minimum quantifiable level of 25 ng/ml, absolute recovery of greater than 90% (n = 15), and precision of less than or equal to 8.8% (n = 45). The method was used in a preliminary dose proportionality study in the dog.

Thromboxane synthase activity and platelet function after furegrelate administration in man.

Furegrelate sodium (U-63,557A), a pyridine-derivative thromboxane synthase inhibitor, was administered orally in single doses of 200 to 1600 mg to normal male subjects. Furegrelate produced a dose-related inhibition of thromboxane synthesis for 8-12 hours when measured either ex vivo from platelet-rich plasma (PRP) or in vivo from urine. In general, the extent of thromboxane synthesis inhibition was greater in PRP than in urine. Furegrelate significantly inhibited platelet aggregation, but the effect was variable and measurements of thromboxane synthase did not predict the impact on platelet aggregation. Bleeding times and coagulation parameters were not altered significantly. Furegrelate was well absorbed orally with Tmax = 1 hr and t1/2 = 3.5 to 5 hrs. There was no marked metabolism; elimination was primarily by renal excretion of parent compound. Thus, furegrelate is an effective inhibitor of thromboxane synthase in man with a relatively long biologic and circulating half-life.

Pharmacokinetics of furegrelate after oral administration to normal humans.

Furegrelate sodium is a thromboxane synthetase inhibitor with potential for the treatment of various diseases including hypertension, thrombosis, and renal disorders. The absorption and disposition of the parent drug in normal male volunteers have been studied after single- and multiple-dose oral administration. The results from the single-dose study indicate that furegrelate is rapidly absorbed, with a Tmax of 1.0-1.7 hr, has an apparent terminal disposition rate constant of 0.12-0.17 hr-1, and is eliminated primarily by the kidney, with 62-78% of the dose excreted as parent drug. After multiple-dose oral administration for 4.5 days using a b.i.d. dosing regimen, no apparent change in the absorption, disposition, and elimination kinetics is detected and only a slight potential for drug accumulation is observed.

High-performance liquid chromatographic method for the determination of eclanamine and its N-desmethyl and N,N-didesmethyl metabolites in urine.

A high-performance liquid chromatographic method has been defined for the determination of eclanamine (free base of eclanamine maleate) and two of its metabolites, N-desmethyleclanamine and N,N-didesmethyleclanamine in urine. The method employs 10-ml urine samples, has a linear range from 5 to 500 ng/ml for the three compounds, and has a detection limit of 0.5 ng/ml for each compound. Sample preparation uses a cyanopropylsilane extraction column with washes of water, acetonitrile-water (30:70, v/v), and acetonitrile, and elution with 2% trifluoroacetic acid in acetonitrile. The eluate is evaporated to dryness, the residue dissolved in 1.0 ml acetonitrile-water (10:90, v/v) and 100 microliter are injected onto a Supelcosil LC-CN column. Eclanamine and its metabolites are eluted with an acetonitrile-water (35:65, v/v) eluent containing 0.01 M triethylamine and adjusted to pH 7.0 with phosphoric acid. The method has been validated by preparing and analyzing a series of fortified urines (range 2-500 ng/ml for each compound) on four separate days. Good linearity, precision, reproducibility, and specificity were obtained. Certification of the analytical method was accomplished by analyzing urine specimens collected from one volunteer administered a single oral dose of 45 mg eclanamine maleate. The data suggest that the metabolites of eclanamine have long elimination half-lives with levels still quantifiable in the 72-96 h collection interval.

Tolerance and disposition of cefpimizole in normal human volunteers after intramuscular administration.

Cefpimizole sodium (AC-1370, U-63196E) was administered intramuscularly in doses from 100 mg (0.5 ml) to 2,000 mg (two 3.5-ml doses) to healthy human volunteers in three double-blind placebo and positive-controlled (cefotaxime, cephalothin) single-dose studies and in two multiple-dose studies. Mild transient pain was observed at the injection site, but no erythema, petechia, necrosis, or atrophy was noted. Creatinine phosphokinase values were increased during the study in the cefpimizole- and placebo-treated groups but began to return to normal toward the end of the study period (day 5). They were not paralleled by a similar magnitude of elevation in serum glutamic oxalacetic transaminase and lactate dehydrogenase or by pain and tenderness. There were no clinically meaningful or statistically significant changes (P greater than 0.5) or trends in vital signs and no other patterns of drug-related clinical abnormalities noted in any of the laboratory measurements evaluated (hematology, chemistry, urinalysis). No serious side effects occurred during or after the study. Cefpimizole was well tolerated locally and systemically by all the subjects at all administered dosage levels. Cefpimizole concentrations in serum (microbiological assay) remained above 1 microgram/ml at 12 h after drug administration for all dose levels. The median peak concentrations in plasma for the 500- and 1,000-mg twice-daily dosages of cefpimizole were, respectively, 21.6 and 45.5 micrograms/ml on day 1, 16.2 and 43.7 micrograms/ml on day 3, and 20.1 and 41.4 micrograms/ml on day 6 of the study. The apparent terminal disposition half-life throughout the study was about 2.0 h. The median amounts of cefpimizole excreted in the urine for the first 12 h of each day evaluated were 370 and 1,071 mg on day 1, 416 and 972 mg on day 3, and 370 and 975 mg on day 6 for the 500- and 1,000-mg twice-daily dosages, respectively. Dose proportionally of cefpimizole was obtained for the 500- and 1,000-mg and the 2,000-mg groups. The absorption, distribution, and elimination of cefpimizole after multiple-dose intramuscular administration were uniform, were linear in relation to dose, and did not result in drug accumulation.

Pharmacokinetics and dose proportionality of cefpimizole in normal humans after intramuscular administration.

The pharmacokinetics of cefpimizole (free acid equivalents of cefpimizole sodium), a broad-spectrum cephalosporin antibiotic, were evaluated after intramuscular administration of single doses (dose range, 100 to 1,000 mg) and multiple doses (dose range, 500 to 2,000 mg) given b.i.d. for 6 or 11 days. The kinetics after intramuscular administration correspond to a one-compartment model with first-order input. The apparent volume of distribution of the absorbed dose averaged 18.6 +/- 3.4 (standard deviation) liters for 58 individuals; the absorption-phase and elimination-phase rate constants averaged 2.53 +/- 1.16 h-1 (half-life, 0.27 h) and 0.338 +/- 0.041 h-1 (half-life, 2.05 h), respectively; and the mean residence time was 3.43 +/- 0.43 h. The total body clearance of the absorbed dose after single-dose intramuscular administration was 102 +/- 13 ml/min. The primary route of elimination was renal with 73 to 83% of the administered dose excreted in the urine as unchanged drug. Renal clearance averaged 81 +/- 13 ml/min. Dose proportionality was obtained from area under the plasma curve, concentration maximum in plasma, and cumulative urinary excretion levels. Multiple-dose evaluation of intramuscular administration of cefpimizole indicated no apparent change in the absorption or elimination phases after b.i.d. dosing for 6 or 11 days. The kinetic parameters determined from multiple-dose plasma and urine levels were in close agreement with the same parameters calculated from single-dose results. No apparent accumulation of cefpimizole occurred, and nondetectable levels of drug were observed in the 24-h plasma and 24- to 48-h urine specimen after administration of the last dose. The kinetics of cefpimizole after intramuscular administration were similar to the kinetics obtained after intravenous infusion.

Liquid chromatographic-ultraviolet methods for furegrelate in serum and urine: preliminary pharmacokinetic evaluation in the dog.

Analytical methods have been developed for the quantitative determination of furegrelate (1), a thromboxane synthetase inhibitor, in dog serum and urine specimens. The methods use ion-pairing reversed-phase high-performance liquid chromatography (HPLC) with UV detection at 268 nm and have the necessary specificity, linearity, and sensitivity to quantitate 100 ng/mL of the drug in serum and 20 micrograms/mL in urine. Preliminary pharmacokinetic and bioavailability evaluations in the dog indicate that 1 was rapidly distributed and had a terminal half-life of 132 min after an intravenous dose. The primary route of elimination was renal with approximately 70% of the parent drug excreted in urine. Fasted dogs dosed orally with the drug in solution or capsule had similar absorption and elimination kinetics and agreed favorably with the intravenous results. The bioavailability of 1 dosed orally was 77-80% based on serum area under curve and greater than 90% based on urinary excretion of the parent compound. The serum kinetics, but not the urinary elimination, of 1 appeared to be altered in fed dogs given drug in capsule.

Pharmacokinetics of cefpimizole in normal humans after single- and multiple-dose intravenous infusions.

The pharmacokinetics of cefpimizole (free acid equivalents of cefpimizole sodium), a broad-spectrum cephalosporin antibiotic, were determined after single- and multiple-dose 20-min intravenous infusions of 1, 2, and 4 g. The kinetics of single-dose administration of cefpimizole correspond to a two-compartment model with an average apparent volume of distribution of 20.0 +/- 3.5 liters, a distribution rate constant of 2.24 +/- 1.00 h-1, and a terminal rate constant of 0.358 +/- 0.036 h-1 (half-life, 1.9 h). The total body clearance was 118.6 +/- 20.2 ml/min. The primary route of elimination for cefpimizole was the renal route, with approximately 80% of the administered dose excreted as the parent compound. The elimination rate constant, as calculated from urinary excretion data, was 0.339 +/- 0.043 h-1, which is in close agreement with the terminal rate constant for plasma. Renal clearance of cefpimizole was 96.2 +/- 17.3 ml/min. Dose proportionality over the three dose levels was obtained from area under the plasma curve and cumulative urinary excretion data. The results of the multiple-dose study indicated that no apparent change in the distribution or elimination kinetics of cefpimizole occurred after the administration of 1-, 2-, and 4-g doses for 7 days, three times a day. The kinetics from the multiple-dose study were in close agreement with those from the single-dose study. No accumulation of cefpimizole occurred, and nondetectable levels was observed 24 h after administration of the last dose. Peaks that could be attributed to metabolites of cefpimizole were not observed during high-pressure liquid chromatographic analysis of either plasma or urine specimens.

High-performance liquid chromatographic methods for the determination of cefpimizole in plasma and urine.

Quantitative analytical methods have been defined for the determination of cefpimizole, a new broad-spectrum cephalosporin antibiotic, in plasma and urine specimens. The methods employ ion-pair reversed-phase high-performance liquid chromatography with both ethylene-diaminetetraacetic acid (EDTA) and tetrabutylammonium hydroxide as pairing agents for separation and ultraviolet detection at 254 nm. Sample preparation for plasma aliquots consisted of acetonitrile protein precipitation followed by phase separation; the aqueous phase was filtered and assayed. For urine, sample preparation consisted of diluting an aliquot with chromatographic eluent, filtering, and assaying. The methods had a linear range of 17-0.3 micrograms/ml for plasma and 800-15 micrograms/ml for urine and had sufficient precision and accuracy to provide quantitative data. Stability studies in plasma and urine indicated that cefpimizole degraded rapidly at room temperature. Addition of EDTA to the physiological fluid substantially increased the stability at room temperature, and little or no degradation was observed in plasma or urine stored at -30 degrees C for over 100 days. Utility of the methods was demonstrated by assaying plasma and urine specimens obtained from a human volunteer receiving three dose levels. Estimates of various pharmacokinetic parameters are presented.

Quantitative determination of N-(trans-2-dimethylaminocyclopentyl)-N-(3',4'-dichlorophenyl) propanamide and its N-demethyl metabolite in dog serum by gas chromatography.

A gas chromatographic-electron capture (GC-EC) method has been developed for the determination of N-(trans-2-dimethylaminocyclopentyl)-N-(3',4'-dichlorophenyl)pr opanamide, a potential antidepressant drug, and its N-demethyl metabolite in serum. The GC-EC system employed a 3% OV-17 on 100/120 mesh Supelcoport, 2-m X 2-mm i.d. glass column and an isothermal temperature of 195 degrees C. The parent drug and metabolite were extracted from alkalinized serum (pH approximately 13) with toluene, back-extracted into an acidic solution (pH approximately 1), and finally, after adjusting to pH 13, extracted again with toluene. The extensive sample cleanup was necessary to remove serum components which interfered with the analysis. The analytical method was shown to give quantitative recovery of the drug and metabolite, to be linear over a 100-fold concentration range, and to have the necessary precision and sensitivity to detect and quantify as little as 1 ng/mL of the drug or its metabolite. The method has been employed to determine the serum level of drug and metabolite in dogs receiving a single oral dose and to determine the possible correlation between the administered dose and serum levels.

High-performance liquid chromatographic microdetermination of indoprofen in human milk.

A previously reported high-performance liquid chromatographic (HPLC) method for indoprofen determination in physiological fluids was modified and extended to provide quantitative data on drug concentrations in human milk samples at a low nanogram per milliliter level. The reversed-phase HPLC technique was modified to give a better separation of the drug and milk components. To achieve the necessary cleanup for low level determination, the milk samples required protein precipitation, liquid-liquid drug extraction, and concentration. Excellent indoprofen recovery was obtained with this technique; the average recovery from 20 milk samples spiked with various nanogram drug levels was 95%. The analytical technique showed excellent reproducibility; the calibration solutions over 15 days had a relative standard deviation of 3.2%. Results for indoprofen levels in milk and plasma samples from seven subjects who received either a single or multiple oral drug dose are presented.

Determination of indoprofen in physiological fluids by reversed-phase liquid chromatography.

A rapid, sensitive, reversed-phase high-performance liquid chromatographic procedure was developed for the quantitative analysis of indoprofen in plasma and urine. Minimal sample preparation is required for the analysis of unconjugated urinary or plasma drug levels. The method provided quantitative results for indoprofen levels of 0.5-50 microgram/ml of plasma and 0.5-200 microgram/ml of urine and had a lower detection limit of 1 ng. Total urinary indoprofen levels required enzymatic hydrolysis of the conjugated drug prior to analysis. Results are presented for the plasma and urinary excretion levels of indoprofen for a patient receiving a single oral dose.