Biomedical subjects
Publications and source records attributed to G N Lam.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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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.
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.
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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)
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A sensitive and specific capillary gas chromatographic assay is reported for the quantitation of oxycodone in human plasma. The technique involves a single extraction of oxycodone and internal standard (hydrocodone) from plasma by toluene containing 1% isopropanol. Separation is achieved on a methyl silicone (HP-1) fused-silica capillary column (25 m x 0.2 mm I.D., 0.33 microns film thickness) and detection is by nitrogen-phosphorus selective mode. The minimum quantifiable limit is 1.8 ng/ml using 2 ml of plasma. The method is applicable to characterize the plasma profile of oxycodone in humans after a single oral 5-mg oxycodone hydrochloride tablet.
The effects of the novel cognitive enhancer linopirdine [3,3-bis(4-pyridinylmethyl)-1-phenylindolin-2-one] on hepatic cytochromes P-450 (CYP) and linopirdine metabolism were determined in female mice fed 0, 10, 50, and 250 mg/kg/day of linopirdine in the diet for 4, 7, 14, and 28 days. Linopirdine induced CYP maximally by day 4 only at the highest dose, as demonstrated by significant (p < 0.05) increases in total spectral CYP and liver weight. SDS-PAGE revealed induced 52 kDa microsomal protein(s), identified as CYP2B and 3A by immunoblotting. Linopirdine also increased the rates of reactions selectively catalyzed by CYP2B and 3A (pentoxyresorufin O-dealkylation, benzphetamine N-demethylation, erythromycin N-demethylation, and testosterone 2 beta-, 6 beta-, 16 beta-hydroxylation), 1.7- to 3.0-fold vs. control, similar to increases produced by the prototypical CYP2B and 3A inducers phenobarbital and dexamethasone. No increase in microsomal CYP1A or 2E levels was demonstrated by immunoblotting or selective substrate assays. CYP induction increased the metabolism of linopirdine. The area under the plasma concentration-time curve of linopirdine after a 250 mg/kg/day dose decreased 11-fold from day 1-28, and microsomes from a parallel 250 mg/kg/day group metabolized linopirdine 1.9-fold faster than control (p < 0.05). This autoinduction was due primarily to the induced CYP3A, because antibodies recognizing CYP3A inhibited the microsomal metabolism of linopirdine by 85%, whereas antibodies to CYP2B were not inhibitory. In summary, the dietary consumption of 250 mg/kg/day of linopirdine by female mice coinduced CYP2B and 3A maximally by day 4, and resulted in an increased rate of metabolism of linopirdine, predominantly due to CYP3A.