Phase 1 trial of the antiangiogenic peptide ATN-161 (Ac-PHSCN-NH(2)), a beta integrin antagonist, in patients with solid tumours.

To evaluate the toxicity, pharmacological and biological properties of ATN-161, a five -amino-acid peptide derived from the synergy region of fibronectin, adult patients with advanced solid tumours were enrolled in eight sequential dose cohorts (0.1-16 mg kg(-1)), receiving ATN-161 administered as a 10-min infusion thrice weekly. Pharmacokinetic sampling of blood and urine over 7 h was performed on Day 1. Twenty-six patients received from 1 to 14 4-week cycles of treatment. The total number of cycles administered to all patients was 86, without dose-limiting toxicities. At dose levels above 0.5 mg kg(-1), mean total clearance and volume of distribution showed dose-independent pharmacokinetics (PKs). At 8.0 and 16.0 mg kg(-1), clearance of ATN-161 was reduced, suggesting saturable PKs. Dose escalation was halted at 16 mg kg(-1) when drug exposure (area under the curve) exceeded that associated with efficacy in animal models. There were no objective responses. Six patients received more than four cycles of treatment (>112 days). Three patients received 10 or more cycles (> or =280 days). ATN-161 was well tolerated at all dose levels. Approximately, 1/3 of the patients in the study manifested prolonged stable disease. These findings suggest that ATN-161 should be investigated further as an antiangiogenic and antimetastatic cancer agent alone or with chemotherapy.

Safety, tolerability and pharmacokinetics of subcutaneous A6, an 8-amino acid peptide with anti-angiogenic properties, in healthy men.

AIMS: To assess the safety, tolerability and pharmacokinetics of subcutaneous A6, an 8-amino acid peptide with anti-angiogenic properties, in healthy men. METHODS: Double-blind, placebo-controlled, parallel-group, dose-rising, phase I study of single and repeated doses. In the single dose phase, successive groups of 5 subjects received A6 15, 35, 75, 150, 300 mg, or placebo, as subcutaneous injections in the upper thigh. In the repeat dose phase, 2 groups of 6 subjects received repeat doses of A6 35 mg and 75 mg, or placebo, and 1 group of 5 subjects received 150 mg, or placebo, 12-hourly for 6 days (11 doses in total). In each group, 4 subjects received active treatment, the remainder placebo. Pharmacokinetics of A6 were assessed up to 24 h after single doses, for 12 h after the first of the repeated doses, and up to 24 h after the last of the repeated doses. MATERIALS: A6 for subcutaneous injection in phosphate buffer, pH 5.6-6.0. Phosphate-buffered saline was used as placebo. RESULTS: All dose regimens of A6 were safe and well-tolerated, both systemically and locally. Time to peak plasma concentration was similar (0.5-2.1 h) in all dosage groups. Cmax and AUC(0-inf) were linearly proportional to dose. Mean Cmax ranged from 454-10,333 ng/ml and mean AUC(0-inf) from 1,690-43,371 ng x h/ml after the 15 and 300 mg single doses, respectively. Terminal t(1/2) was 1.4-1.8 h, and there was no evidence of unexpected drug accumulation. Urinary excretion of unchanged A6 was 94.6% (SD 20.7) after the 300 mg single dose (0-24 h collection), and 78.4% (SD 13.0) after the 150 mg repeated dose (0-12 h collection). A6 did not trigger production of anti-A6 IgG antibodies within 14 days of the first dose. CONCLUSION: Single doses of A6 up to 300 mg, and repeated doses up to 150 mg, were well-tolerated and safe in healthy young men. A6 was rapidly absorbed; it was eliminated, mainly unchanged, in urine. Plasma concentrations were dose-proportional. A6 did not trigger an early immunogenic response.

A high-performance liquid chromatography-tandem mass spectrometric method for the determination of pharmacokinetics of ganaxolone in rat, monkey, dog and human plasma.

A method for determining concentration levels of ganaxolone in rat, monkey, dog and human plasma was validated in the range of 5-1500 ng/ml using a 200-microl plasma sample volume. This validation report describes the linearity, specificity. sensitivity, reproducibility, accuracy, recovery and stability of the analytical method. The inter-day C.V. ranged from 0.5 to 9.2%, intra-day C.V. from 0.7 to 8.8% and intra-day accuracy (mean absolute percentage difference) ranged from 0.0 to 14.0% for rat, monkey, dog and human plasma. The method was used for the routine analysis of ganaxolone in rat, monkey, dog and human plasma and summary of the pharmacokinetic data are presented.

Development of a high-performance liquid chromatographic-tandem mass spectrometric method for the determination of pharmacokinetics of Co 102862 in mouse, rat, monkey and dog plasma.

A method for determining concentration levels of Co 102862 in mouse, rat, monkey and dog plasma was validated in the range of 5 to 2000 ng/ml using 200 microl plasma sample volume. This validation report describes the linearity, specificity, sensitivity, reproducibility, accuracy, recovery and stability of the analytical method. The inter-day RSD ranged from 3.5 to 10.1%, intra-day RSD from 0.6 to 5.7% and intra-day accuracy (mean absolute percent difference) ranged from 2.2 to 14.9% for rat, monkey and dog plasma. A mini-validation (5-2000 ng/ml) of Co 102862 was performed in mouse plasma using the same methods. Additionally, the assay range at the low end was successfully extended to 0.5 ng/ml for monkey plasma. The method was used for the routine analysis of Co 102862 in mouse, rat, monkey and dog plasma and summary of the pharmacokinetic data are presented.

In vivo metabolism and mass balance of 4-[4-fluorophenoxy]benzaldehyde semicarbazone in rats.

The pharmacokinetics, mass balance, tissue distribution, and metabolism of Co 102862 was investigated in rats after a single oral dose. [(14)C]Co 102862 showed multiexponential pharmacokinetics in rat plasma with an extensive distribution phase. After p.o. administration (approximately 10 mg/kg), the half-lives were long for total radioactivity compared with unchanged Co 102862. Profiles of rat urine and bile suggest that Co 102862 is extensively metabolized in vivo. [(14)C]Co 102862 was extensively distributed into all tissues, with the fatty tissues and secretory glands tissues containing the highest radioactivity. Elimination of radioactivity from the tissues had an estimated half-life of 14 days. A total of 91% of the administered radioactivity was recovered in both intact and bile-duct cannulated rats over 120 and 48 h, respectively, with the majority ( approximately 74%) of the radioactivity being excreted in the urine. Approximately 10% of the total radioactivity remained in the tissues on day 5 and decreased with time to approximately 3% on day 28. Bile-duct cannulated experiments show the enterohepatic circulation is an important route of elimination and reabsorption. Six metabolites were identified in the urine and bile of which the carboxylic acid was the major metabolite. The carboxylic acid was the only metabolite found in plasma and was probably responsible for the radioactivity in the tissues.

Preparation of pyrrolidine and isoxazolidine benzamidines as potent inhibitors of coagulation factor Xa.

The serine protease factor Xa is a critical enzyme in the blood coagulation cascade. Recently, the inhibition of factor Xa has begun to emerge as an attractive strategy for the discovery of novel antithrombotic agents. Here we describe pyrrolidine and isoxazolidine benzamidines as novel and potent inhibitors of factor Xa.

Nonsymmetric P2/P2' cyclic urea HIV protease inhibitors. Structure-activity relationship, bioavailability, and resistance profile of monoindazole-substituted P2 analogues.

Using the structural information gathered from the X-ray structures of various cyclic urea/HIVPR complexes, we designed and synthesized many nonsymmetrical P2/P2'-substituted cyclic urea analogues. Our efforts concentrated on using an indazole as one of the P2 substituents since this group imparted enzyme (Ki) potency as well as translation into excellent antiviral (IC90) potency. The second P2 substituent was used to adjust the physical and chemical properties in order to maximize oral bioavailability. Using this approach several very potent (IC90 11 nM) and orally bioavailable (F% 93-100%) compounds were discovered (21, 22). However, the resistance profiles of these compounds were inadequate, especially against the double (I84V/V82F) and ritonavir-selected mutant viruses. Further modification of the second P2 substituent in order to increase H-bonding interactions with the backbone atoms of residues Asp 29, Asp 30, and Gly 48 led to analogues with much better resistance profiles. However, these larger analogues were incompatible with the apparent molecular weight requirements for good oral bioavailability of the cyclic urea class of HIVPR inhibitors (MW < 610).

Preparation of meta-amidino-N,N-disubstituted anilines as potent inhibitors of coagulation factor Xa.

The serine protease factor Xa is a critical enzyme in the blood coagulation cascade. Recently, the inhibition of factor Xa has begun to emerge as an attractive strategy for the discovery of novel antithrombotic agents. Here we describe a series of meta-amidino-N,N-disubstituted anilines as structurally simple and very potent inhibitors of factor Xa.

Biological matrix-dependent pharmacokinetic and pharmacodynamic parameters of a novel platelet glycoprotein IIB/IIIA receptor antagonist, XU063, in beagle dogs.

The pharmacokinetic-pharmacodynamic (PK/PD) relationship of a novel platelet glycoprotein IIb/IIIa receptor antagonist, XU063, was evaluated as a function of biological matrix in beagle dogs. The disposition of 14C-radioactivity in various blood or plasma matrices and kinetics of inhibition of adenosine diphosphate (ADP) induced platelet aggregation were determined in beagle dogs following an intravenous infusion of 14C-XU063 at 2 micrograms/kg for 45 min. The 14C-radioactivity was maximum in platelet poor plasma (PPP) harvested from blood collected in EDTA and lowest in PPP harvested from blood collected in citrated vacutainers over the entire concentration versus time profile during and post infusion. The 14C-radioactivity values in blood and platelet rich plasma (PRP) were comparable and were between EDTA PPP and citrated PPP values. The resultant estimates of the PK and PD parameters of 14C-XU063 varied widely depending on the type of matrix used. The systemic clearance values for 14C-XU063 were 1 and 10 mL/min/kg for EDTA and citrated PPP, respectively. The values for the volume of distribution at steady-state were 0.2 and 1.3 L/kg, for EDTA and citrated PPP, respectively. The terminal elimination half-life appeared independent of the matrix with a median value of 2 h. The estimated ex vivo IC50 values of XU063 ranged from 0.4 ng/mL (citrated PPP, platelet free drug) to 7 ng/mL (EDTA PPP, total drug). These results demonstrated the dependence of PK and PD parameters of antiplatelet agent XU063 on the type of biological matrix used to determine concentrations of XU063. The pros and cons of various blood sample collection methods for the evaluation of PK/PD relationship of potential antiplatelet agents are presented.

Improved P1/P1' substituents for cyclic urea based HIV-1 protease inhibitors: synthesis, structure-activity relationship, and X-ray crystal structure analysis.

We present several novel P1/P1' substituents that can replace the characteristic benzyl P1/P1' moiety of the cyclic urea based HIV protease inhibitor series. These substituents typically provide 5-10-fold improvements in binding affinity compared to the unsubstituted benzyl analogs. The best substituent was the 3,4-(ethylenedioxy)benzyl group. Proper balancing of the molecule's lipophilicity facilitated the transfer of this improved binding affinity into a superior cellular antiviral activity profile. Several analogs were evaluated further for protein binding and resistance liabilities. Compound 18 (IC90 = 8.7 nM) was chosen for oral bioavailability studies based on its log P and solubility profile. A 10 mg/kg dose in dogs provided modest bioavailability with Cmax = 0.22 microg/mL. X-ray crystallographic analysis of two analogs revealed several interesting features responsible for the 3,4-(ethylenedioxy)benzyl-substituted analog's potency: (1) Comparing the two complexes revealed two distinct binding modes for each P1/P1' substituent; (2) The ethylenedioxy moieties are within 3.6 A of Pro 81 providing additional van der Waals contacts missing from the parent structure; (3) The enzyme's Arg 8 side chain moves away from the P1 substituent to accommodate the increased steric volume while maintaining a favorable hydrogen bond distance between the para oxygen substituent and the guanidine NH.

Pharmacokinetics and metabolism of EXP921, a novel cognitive enhancer, in rats.

EXP921, 5,5-bis(4-pyridinylmethyl)-5H-cyclopenta[2,1-b:3,4-b']-dipyridine, was a potential drug candidate for the improvement of cognitive performance in patients with Alzheimer's-type dementia. It has been shown to improve cognitive performance in rodent and primate models of learning and memory. To characterize the disposition of EXP921, the pharmacokinetics and metabolism of this compound were studied in rats after oral and intravenous administrations. EXP921 exhibited good bioavailability, 43% at 3 mg/kg and 61% at 10 mg/kg and was rapidly eliminated with a terminal half-life ranging from 1.28 to 2.29 hr after oral doses. Absorption from oral doses was rapid, as peak plasma levels were reached within 1 hr. A major metabolite was identified in plasma as the pyridinyl mono-N-oxide of EXP921. This metabolite (EXP696) was rapidly formed, and significant levels were detected in rat plasma after oral or intravenous administration. Its terminal half-life was slightly longer than that of EXP921. EXP696 was found to be reduced back to EXP921, demonstrating that the N-oxidation at the pyridyl ring is reversible. The interconversion favored the oxidation of EXP921 to EXP696. Two additional metabolites were identified in rat plasma at doses higher than or equal to 30 mg/kg. They result from despicolylation, followed by hydroxylation in the cyclopentane ring.

Nonpeptide angiotensin II receptor antagonist: pharmacokinetics and pharmacodynamics in rats of EXP3174, an active metabolite of losartan.

The pharmacokinetics and pharmacodynamics of EXP3174 (2-n-butyl-4-chloro-1-[(2'-(1H-tetrazole-5-yl)biphenyl-4- yl-)methyl]imidazole-5-carboxylic acid), an angiotensin II receptor antagonist, were studied in conscious rats. Elimination half-life, systemic clearance, and apparent volume of distribution of EXP3174 at a dose of 10 mg/kg i.v. were 2.9 h, 1.8 ml/min/kg, and 0.25 l/kg, respectively. Inhibition of the angiotensin II pressor response correlated with the log of the steady state plasma EXP3174 concentration in a sigmoidal fashion with an IC50 of about 200 ng/ml. When corrected for plasma protein binding, the IC50 (free) for EXP3174 was 0.4 ng/ml (0.9 nmol/l). This study indicates a predictable plasma concentration-effect relationship of EXP3174 in rats which would be helpful in designing more rational dosing schemes for pharmacodynamic studies.

Determination of brequinar in rat plasma by direct deproteinization and reversed-phase high-performance liquid chromatography with ultraviolet absorbance detection.

A direct deproteinization method for the determination of brequinar in rat plasma by high-performance liquid chromatography (HPLC) has been developed. This assay avoids the use of dichloromethane, a known carcinogen, in an existing extraction method. Acetonitrile was used to denature plasma proteins and the supernatant was injected onto the HPLC column. Chromatographic separation of brequinar was conducted on a Biophase octyl column using a mixture of acetonitrile and 0.1 M phosphoric acid (50:50, v/v) as the mobile phase and detection of brequinar was by UV absorbance at 254 nm. The method has been validated in rat plasma over the concentration range of 0.05-50.00 micrograms/ml which is adequate for the determination of pharmacokinetics of brequinar in animals.

Dose and species dependent pharmacokinetics of a novel sigma receptor antagonist, DuP 734.

The pharmacokinetics of a novel sigma receptor antagonist, DuP 734, was evaluated in mice, rats, beagle dogs and cynomolgus monkeys at various intravenous and oral doses utilizing a specific reversed-phase HPLC assay. Following intravenous dosing, the disposition of DuP 734 in all species was characterized by high total body systemic plasma clearance (46 to 87 ml/min/kg) and large steady-state volume of distribution (3.6 to 6.8 l/kg). The terminal elimination half-life ranged from 50 to 83 min. The gastrointestinal absorption from an aqueous solution was very rapid in mice and rats with peak DuP 734 plasma concentrations attained within 5 and 20 min following administration, respectively. The peak plasma concentrations in dogs and monkeys were attained within 45 and 130 min, respectively. The absolute bioavailability in mice ranged from 29 to 46% at doses of 3.1 to 30.1 mg/kg. The bioavailability increased from 4 to 10% and from 14 to 72% when doses were increased from 12.5 to 50 mg/kg and 1 to 3 mg/kg of DuP 734 in rats and dogs, respectively. The bioavailability in monkeys was 30.5% at 9.3 mg/kg DuP 734 dose. The dose dependent pharmacokinetics of DuP 734 was observed within narrow dose ranges in all animal species investigated.

Determination of a novel cognitive enhancer, X9121, and its mono N-oxide metabolite, XG696, in dog plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection.

A selective and sensitive high-performance liquid chromatographic assay for a novel cognitive enhancer, X9121 (I), and its mono N-oxide metabolite, XG696 (II), in dog plasma has been developed. Compounds I, II and internal standard (I.S.) were first extracted from dog plasma using a solid-phase Bond Elut Certify I 10-ml LRC reservoir extraction cartridge. Chromatographic separation of I, II and I.S. was conducted on a reversed-phase Zorbax Stable Bond cyano column. Ammonium acetate buffer (0.05 M, pH 6)-acetonitrile-triethylamine (75:25:0.1, v/v) was used as the mobile phase. Detection of all three compounds was by UV light absorbance at 313 nm. Using 0.5 ml of dog plasma for extraction, the minimum quantifiable limit was 10 ng/ml and the assay was linear from 10 to 5400 ng/ml. The coefficients of variation for intra-day precision ranged from 2.2 to 8.5% for I and from 2.5 to 9.8% for II. The coefficients of variation for the inter-day precision for these two compounds ranged from 2.6 to 9.0% and from 3.6 to 16.2%, respectively. The absolute percent differences for the accuracy results were within 11.0% of the spiked concentrations. Compounds I and II were stable in frozen plasma at -20 degrees C for at least 67 days.

The pharmacokinetics and pharmacodynamics of the angiotensin II receptor antagonist losartan potassium (DuP 753/MK 954) in the dog.

The pharmacokinetics and plasma concentration-effect relationship for the nonpeptide angiotensin II (Ang II) receptor antagonist losartan potassium (losartan) have been determined with conscious and anesthetized dogs. The p.o. bioavailability of single doses of 5 to 20 mg/kg was low, 23 to 33%, and independent of the dose. Absorption was rapid, with peak plasma levels observed within 1 hr, and the Cmax and area under the concentration vs. time curve to infinity were proportional to the dose, P < .05. The elimination half-life, 108 to 153 min, was longer than that observed after a single i.v. dose, 41 min, and may reflect both continuous absorption and enterohepatic recirculation because the major route of excretion was via the bile. Single i.v. doses were eliminated rapidly, with a systemic plasma clearance of 22.2 ml/min/kg. When corrected for the blood:plasma distribution ratio, 0.66 to 0.72, the systemic clearance approximates hepatic blood flow, suggesting that clearance is primarily via hepatic metabolism and biliary excretion. Losartan was not distributed extensively to tissues; apparent volume of distribution at steady-state of 0.30 liters/kg and was highly but not extensively bound to plasma proteins; 2.7 to 2.9% unbound (free). The plasma concentration vs. blockade of exogenous Ang II-induced vasopressor response was also determined after a single 3-mg/kg i.v. dose of losartan with a sigmoidal Emax model. Blockade of the pressor response was rapid, 89% at 5 min, and declined to 11% at 240 min postdose. The relationship between concentration and effect was highly significant (r = 0.922, P < .01), with an IC50 (total) of 96 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of a novel sigma receptor antagonist, DuP 734, in rat plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection.

A selective high-performance liquid chromatographic (HPLC) assay for a sigma receptor antagonist, DuP 734 (I), in rat plasma has been developed. Compound I and internal standard, XC031 (I.S.), were first extracted from plasma into an ethyl acetate-toluene mixture (3:7, v/v) and then back-extracted into freshly prepared phosphoric acid (0.03 M). Separation of I and I.S. with no interference from endogenous substances was achieved on a reversed-phase octyl column and detection was by UV at 229 nm. The mobile phase consisted of acetonitrile-glacial acetic acid-triethylamine-0.05 M ammonium acetate (670:4:2:2000, v/v). Using 0.5 ml of rat plasma for extraction, the limit of quantitation was 43 ng/ml and the assay was linear from 43 to 8536 ng/ml. The intra- and inter-day coefficients of variation ranged from 0.7 to 3.0%, and from 1.4 to 14.5%, respectively, over the entire concentration range. The accuracy was within 16.1% of the spiked concentrations. I was stable in frozen plasma at -20 degrees C for at least 68 days.