Determination of the antihypertensive agent 1-(2-aminoethyl)-3-(2,6-Dichlorophenyl)thiourea in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and specific normal-phase (adsorption) high-performance liquid chromatographic (HPLC) assay was developed for the determination of 1-(2-aminoethyl)-3-(2,6-dichlorophenyl)thiourea [I] in plasma and urine. The assay involves the extraction of the compound into methylene chloride from plasma or urine buffered to pH 10, and the HPLC analysis of the residue dissolved in methylene chloride—methanol—heptane (85:10:5). A 10-μm silica gel column was used with methylene chloride—methanol—heptane—ammonium hydroxide (85:10:5:0.1) as the eluting solvent. The effluent was monitored at 254 nm and quantitation was based on the peak height vs. concentration technique. The assay has a recovery of 64.5 ± 4.5% (S.D.) from plasma and 96.0 ± 6.3% (S.D.) from urine in the concentration range of 0.1–2 μg per ml and 2–40 μg per 0.1 ml of plasma and urine, respectively, with a limit of detection of 0.05–0.1 μg [I] per ml of plasma using a 1-ml specimen and 0.1 μg per ml urine using a 0.1-ml specimen, respectively. The assay was applied to the determination of plasma levels and urinary excretion of the compound [I] in dog following the oral administration of 28.8 mg of [I] · maleate per kg body weight.The HPLC assay was also used to determine the stability of [I] and for the measurement of a potential degradation product, clonidine [II] [2-(2,6-dichlorophenylamino)-2-imidazoline] in pooled human plasma stored at ?17°C, and pooled human urine stored at ?17°C and ?90°C, respectively.     

Determination of the antiallergenic agent, N-[4-(1H-imidazol-1-YL)butyl]-2-(1-methylethyl)-11-oxo-11H-pyrido[2,1,-b]quinazoline-8-carboxamide, in plasma by reversed-phase high-performance liquid chromatographic analysis using fluorometric detection

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of the antiallergenic compound N-[4-(1H-imidazol-1-yl)butyl]-2-(1-methylethyl)-11-oxo-11H-pyrido[2,1-b] quinazoline-8-carboxamide (I), and its major metabolite, 2-(1-methylethyl)-11-oxo-11H-pyrido[2,1-b] quinazoline-8-carboxylic acid (I-A), in plasma. The assay involves precipitation of the plasma proteins with aceto-nitrile—methanol (9:1), followed by the analysis of an aliquot of the protein-free filtrate by reversed-phase ion-pair HPLC with fluorescence detection for quantitation. The analogous compound, N-[6-(1H-imidazol-1-yl)hexyl]-2-(1-methylethyl)-11-oxo-11H-pyrido[2,1-b]-quinazoline-8-carbonxamide (II), is used as the internal standard. The overall recovery of compounds I and I-A from plasma is 107.0 ± 8.6% and 107.0 ± 10.0%, respectively. The sensitivity limits of quantitation are 20 ng of I, and 10 ng of I-A per ml of plasma using a 0.5-ml aliquot. The assay was used to monitor the plasma concentrations of I and of I-A in a dog following a 5 mg/kg intravenous infusion of I · 2HCl, a 10mg/kg oral dose of I · 2HCl and of metabolite I-A.     

Determination of cyclizine and norcyclizine in plasma and urine using gas—liquid chromatography with nitrogen selective detection

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of 8-chloro-6-(2-chlorophenyl)-4H-imidazo-[1,5-a]-[1,4]-benzodiazepine-3-carboxamide [I] and its 4-hydroxy metabolite, 8-chloro-6-(2-chlorophenyl)-4-hydroxy-4H-imidazo-[1,5-a][1,4]-benzodiazepine-3-carboxamide [II] in whole blood, plasma or urine. The assay for both compounds involves extraction into diethyl ether—methylene chloride (70:30) from blood, plasma, or urine buffered to pH 9.0. The overall recoveries of [I] and [II] are 92.0 ± 5.4% (S.D.) and 90.3 ± 4.9% (S.D.), respectively. The sensitivity limit of detection is 50 ng/ml of blood, plasma, or urine using a UV detector at 254 nm. The HPLC assay was used to monitor the blood concentration—time fall-off profiles, and urinary excretion profiles in the dog following single 1 mg/kg intravenous and 5 mg/kg oral doses, and following multiple oral doses of 100 mg/kg/day of compound [I].     

Metabolism of the carbocyclic analogue of (E)-5-(2-iodovinyl)-2'-deoxyuridine in herpes simplex virus-infected cells. Incorporation of C-IVDU into DNA

The carbocyclic analogues of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU), in which the sugar moiety is replaced by a cyclopentane ring and which have been designated as C-BVDU and C-IVDU, respectively, are, like their parent compounds BVDU and IVDU, potent and selective inhibitors of herpes simplex virus type 1 (HSV-1) and, to a lesser extent, herpes simplex virus type 2 (HSV-2) replication. We have now synthesized the radiolabeled C-IVDU analogue, C-[125I]IVDU, and determined its metabolism by HSV-infected and mock-infected Vero cells. C-[125I]IVDU was effectively phosphorylated by HSV-1-infected cells and, to a lesser extent, HSV-2-infected cells. C-[125I]IVDU was not phosphorylated to an appreciable extent by either mock-infected cells or cells that had been infected with a thymidine kinase-deficient mutant of HSV-1. Furthermore, C-[125I]IVDU was incorporated into both viral and cellular DNA of HSV-1-infected Vero cells. This finding represents the first demonstration of the incorporation of a cyclopentylpyrimidine into DNA.     

Synthesis, radiolabelling and biological characterization of (D)-7-iodo-N-(1-phosphonoethyl)-5-aminomethylquinoxaline-2,3-dione, a glycine-binding site antagonist of NMDA receptors

(D)-7-Iodo-N-(1-phosphonoethyl)-5-aminomethylquinoxaline-2,3 -dione (I-PAMQX), is a potent, in vivo active antagonist acting at the glycine binding site of the NMDA receptor complex. Radioiodinated [131I]I-PAMQX was prepared with good yields and high specific activity from its 7-bromo analogue. Biodistribution studies of [131I]I-PAMQX in mice showed a relatively slow clearance from the blood. The uptake of radioactivity was highest in the kidneys, moderate in the heart, lung, liver and bones, and low in the brain.     

Synthesis of long-chain amide analogs of the cannabinoid CB1 receptor antagonist N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716) with unique binding selectivities and pharmacological activities

An extended series of alkyl carboxamide analogs of N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl- 1H-pyrazole-3-carboxamide (SR141716; 5) was synthesized. Each compound was tested for its ability to displace the prototypical cannabinoid ligands ([3H]CP-55,940, [3H]2; [3H]SR141716, [3H]5; and [3H]WIN55212-2, [3H]3), and selected compounds were further characterized by determining their ability to affect guanosine 5'-triphosphate (GTP)-gamma-[35S] binding and their effects in the mouse vas deferens assay. This systematic evaluation has resulted in the discovery of novel compounds with unique binding properties at the central cannabinoid receptor (CB1) and distinctive pharmacological activities in CB1 receptor tissue preparations. Specifically, compounds with nanomolar affinity which are able to fully displace [3H]5 and [3H]2, but unable to displace [3H]3 at similar concentrations, have been synthesized. This selectivity in ligand displacement is unprecedented, in that previously, compounds in every structural class of cannabinoid ligands had always been shown to displace each of these radioligands in a competitive fashion. Furthermore, the selectivity of these compounds appears to impart unique pharmacological properties when tested in a mouse vas deferens assay for CB1 receptor antagonism.     

Validation of liquid chromatography assay for the quantitation of (Z)-3-[2,4-dimethyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]propionic acid (SU006668) in human plasma and its application to a phase I clinical trial

The validation of an analytical method to quantify the antiangiogenic, (Z)-3-[2,4-dimethyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]propionic acid (SU006668) for pharmacokinetic determination in a phase I clinical trial, is described. HPLC, with a gradient mobile phase and UV detection at 440 nm, was used. SU006668 was extracted from plasma by precipitation of proteins with acetonitrile. The assay was linear from 25 to 2000 ng/ml (r(2)=0.997); sensitive (limit of quantification 25 ng/ml), accurate (RE 2.6-11.9%) and reproducible (inter-batch precision C.V. 3.2%). Pharmacokinetic data for six patients are presented. They show linear pharmacokinetics with a low volume of distribution and induction at doses of 50, 100 and 200 mg/m(2).     

N-succinimidyl 5-(trialkylstannyl)-3-pyridinecarboxylates: a new class of reagents for protein radioiodination

N-Succinimidyl 5-(trialkylstannyl)-3-pyridinecarboxylates (alkyl = Me, Bu) have been prepared and used as a precursor to label N-succinimidyl 5-[131I]iodo-3-pyridinecarboxylate (SIPC). SIPC was obtained in greater than 80% yield from either the methyl or butyl precursor with N-chlorosuccinimide and heating at 60-65 degrees C. Significantly lower yields were observed with tert-butyl hydroperoxide. After a 30-min incubation with [131I]SIPC at pH 8.5, goat IgG, an intact monoclonal antibody (MAb), and a MAb F(ab')2 fragment were labeled in 60-65% yield. Specific binding of the MAb and MAb fragment after SIPC labeling was identical with that observed with N-succinimidyl 3-iodobenzoate and higher than that reported previously for these MAbs after labeling by using the Iodogen method. When 5-[131I]iodonicotinic acid was injected into normal mice, thyroid uptake was less than 0.2% of the injected dose, reflecting the inertness of this compound to deiodination. Paired-label biodistribution studies indicate that for both the MAb and the F(ab')2 labeled by using SIPC, accumulation of activity in the thyroid and other tissues is comparable to that observed when these proteins were labeled by using N-succinimidyl 3-iodobenzoate. The results of this study suggest that SIPC may be a reagent for labeling MAbs with halogen nuclides.     

5-[3-(E)-(4-azido-2,3,5,6-tetrafluorobenzamido)propenyl-1]-2'-deoxy- uridine-5'-triphosphate substitutes for thymidine-5'-triphosphate in the polymerase chain reaction.

The DNA targets may be labeled and simultaneously amplified in the polymerase chain reaction (PCR) using a pair of respective primers after elongation with nucleoside-5'-triphosphates carrying photoreactive groups. The amplified DNA may be subsequently photoactivated by irradiation above 300 nm, resulting in photo-cross-linking of the strands. For this goal 5-[3-(E)-(4-azido-2,3,5,6-tetrafluorobenzamido)propenyl-1]-, 5-{N-[N'-(4-azido-2,3,5, 6-tetrafluorobenzoyl)-3-aminopropionyl]aminomethyl}-, and 5-{N-[N'-(2-nitro-5-azidobenzoyl)-3-aminopropionyl]aminomethyl}-2'-de oxyuridine-5'-triphosphate (VII, VIa, and VIb) derivatives have been synthesized. It was found that VII is capable of efficiently elongating DNA primers with both Klenow fragment DNA polymerase I and Thermus aquaticus DNA polymerase. Thereto, it turned out to provide quantitative incorporation in DNA as revealed by the formation of the full-length amplificate by PCR in the presence of this photoreactive analogue without any dilution with natural dTTP. On the contrary, it was found, that incorporation of VIa and VIb do not permit further DNA replication.     

A microplate binding assay for the somatostatin type-2 receptor (SSTR2)

The clinical importance of somatostatin type-2 receptors (SSTR2) and the study of novel analogues of somatostatin such as OctreoScan or [Tyr3]-octreotide containing DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) as metal chelator led us to develop a methodology to monitor the expression of SSTR2 on tumours of pancreatic origin (e.g. rat AR4-2J cancer cells). Usual binding assay protocols using the commercial [125I][Tyr1]-somatostatin radioligand failed, even in the presence of a cocktail of protease inhibitors with a broad spectrum of activity, possibly due to the high susceptibility of this tracer to proteases expressed in pancreatic cells. We prepared our own radioligand [125I][Tyr2]-octreotide which was shown to be much more resistant to degradation after incubation with AR4-2J plasma membranes. As expected, the increased stability of [125I][Tyr3]-octreotide was associated with good binding to SSTR2. Addition of appropriate protease inhibitors further increased the specific binding of [125I][Tyr3]-octreotide to AR4-2J plasma membranes without affecting the stability of the tracer, suggesting that the protease inhibitors also protect the integrity of SSTR2. Optimal conditions (time, temperature, medium) were developed for a binding assay in 96-well plates using AR4-2J plasma membranes in order to make the assay suitable for high-throughput analysis. This protocol was the basis for studying the in vivo regulation of SSTR2 expression in AR4-2J cells implanted into scid mice after exposure to different compounds.     

Activation of 2-5A-dependent RNase by analogs of 2-5A (5'-O-triphosphoryladenylyl(2'----5')adenylyl(2'----5')adenosine ) using 2',5'-tetraadenylate (core)-cellulose

A variety of 2-5A (px(A2'p)nA; x = 2 or 3, n greater than or equal to 2) analogs were assayed for their abilities to activate murine 2-5A-dependent RNase (subsequently "the nuclease") using a recently developed method. This technique consists of immobilizing and partially purifying the nuclease using core-cellulose [A2'p)3A-cellulose) and then monitoring the breakdown of poly(U)-3'-[32P]Cp into acid-soluble fragments. Several 5'-adenosinecapped analogs of 2-5A (containing a tetra-, tri-, or diphosphate) were analyzed, and it was found that reducing the number of phosphoryl groups between the 5' to 5'-diadenosine linkages resulted in a progressive loss of activity. Because A5' pppp(A2'p)3A was a potent activator of the nuclease yet stable during the assay these results suggested that a free 5'-phosphoryl group may not be required for the activation of the nuclease. A number of 8-bromoadenosine-substituted analogs of 2-5A were also studied. Curiously, the brominations decreased the activities of the 5'-di- and triphosphorylated molecules while substantially increasing the activities of the 5'-monophosphorylated species. The results indicated that a tri- or diphosphate moiety on the 5'-end of 2-5A or the presence of ATP is not absolutely required for the nuclease to be active. Furthermore, the ATP analog, beta, gamma-methylene ATP, did not inhibit the activity of the nuclease. Finally, a 3',5'-phosphodiester linkage isomer of 2-5A and a 3'-deoxy (cordycepin) analog of 2-5A were tested, and both were found to be completely without activity.     

Effects of ciprofibrate and 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA) on the distribution of carnitine and CoA and their acyl-esters and on enzyme activities in rats. Relation between hepatic carnitine concentration and carnitine acetyltransferase activity.

The effects of feeding the peroxisome proliferators ciprofibrate (a hypolipidaemic analogue of clofibrate) or POCA (2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate) (an inhibitor of CPT I) to rats for 5 days on the distribution of carnitine and acylcarnitine esters between liver, plasma and muscle and on hepatic CoA concentrations (free and acylated) and activities of carnitine acetyltransferase and acyl-CoA hydrolases were determined. Ciprofibrate and POCA increased hepatic [total CoA] by 2 and 2.5 times respectively, and [total carnitine] by 4.4 and 1.9 times respectively, but decreased plasma [carnitine] by 36-46%. POCA had no effect on either urinary excretion of acylcarnitine esters or [acylcarnitine] in skeletal muscle. By contrast, ciprofibrate decreased [acylcarnitine] and [total carnitine] in muscle. In liver, ciprofibrate increased the [carnitine]/[CoA] ratio and caused a larger increase in [acylcarnitine] (7-fold) than in [carnitine] (4-fold), thereby increasing the [short-chain acylcarnitine]/[carnitine] ratio. POCA did not affect the [carnitine]/[CoA] and the [short-chain acylcarnitine]/[carnitine] ratios, but it decreased the [long-chain acylcarnitine]/[carnitine] ratio. Ciprofibrate and POCA increased the activities of acyl-CoA hydrolases, and carnitine acetyltransferase activity was increased 28-fold and 6-fold by ciprofibrate and POCA respectively. In cultures of hepatocytes, ciprofibrate caused similar changes in enzyme activity to those observed in vivo, although [carnitine] decreased with time. The results suggest that: (1) the reactions catalysed by the short-chain carnitine acyltransferases, but not by the carnitine palmitoyltransferases, are near equilibrium in liver both before and after modification of metabolism by administration of ciprofibrate or POCA; (2) the increase in hepatic [carnitine] after ciprofibrate or POCA feeding can be explained by redistribution of carnitine between tissues; (3) the activity of carnitine acetyltransferase and [total carnitine] in liver are closely related.     

(E)-5-(2-bromovinyl)-2'-deoxyuridine-5'-triphosphate as a DNA polymerase substrate.

Time course of incorporation and the effect of 5'-triphosphate of the selective antiherpetic agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (bv5dUTP) on the incorporation of dTTP and dATP into template-primers of different structure were studied in E. coli DNA polymerase I Klenow fragment enzyme-catalyzed reactions. bv5dUTP could substitute for dTTP depending on the structure of template-primer. E.g. into calf thymus DNA incorporation of bv5dUMP was around 80% of that of dTMP at 30 minutes of incubation. The analog has also inhibited dTMP incorporation, net DNA synthesis, however, was hardly affected. The substrate properties of the analog were studied with [2-14C]-labelled bv5dUTP.     

In-vitro cytotoxicity and cell cycle analysis of two novel bis-1,2, 4-triazole derivatives: 1,4-bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4-(p-tolyl)-1,2,4-triazol-3-yl]-butane (MNP-14) and 1,4-bis[5-(carbethoxy-methyl)-thio-4-(p-ethoxy phenyl)-1,2,4-triazol-3-yl]-butane (MNP-16)

In the present study, we have tested the cytotoxic and DNA damage activity of two novel bis-1,2,4 triazole derivatives, namely 1,4-bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4-(p-tolyl)-1,2,4-triazol-3-yl]-butane (MNP-14) and 1,4-bis[5-(carbethoxy-methyl)-thio-4-(p-ethoxy phenyl) -1,2,4-triazol-3-yl]-butane (MNP-16). The effect of these molecules on cellular apoptosis was also determined. The in-vitro cytotoxicity was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as well as Trypan blue dye exclusion methods against human acute lymphoblastic leukemia (MOLT4) and lung cancer cells (A549). Our results showed that MNP-16 induced significant cytotoxicity (IC(50) of 3-5 μM) compared with MNP-14. The cytotoxicity induced by MNP-16 was time and concentration dependent. The cell cycle analysis by flow cytometry (fluorescence-activated cell sorting [FACS]) revealed that though there was a significant increase in the apoptotic population (sub-G(1) phase) with an increased concentration of MNP-14 and 16, there was no cell cycle arrest. Further, the comet assay results indicated considerable DNA strand breaks upon exposure to these compounds, thereby suggesting the possible mechanism of cytotoxicity induced by MNP-16. Hence, we have identified a novel molecule (MNP-16) which could be of great clinical relevance in cancer therapeutics.     

In vitro and in vivo binding of (E)- and (Z)-N-(iodoallyl)spiperone to dopamine D2 and serotonin 5-HT2 neuroreceptors

Apparent affinities (Ki) of (E)- and (Z)-N-(iodoallyl)spiperone [E)- and (Z)-NIASP) for dopamine D2 and serotonin 5-HT2 receptors were determined in competition binding assays. (Z)-NIASP (Ki 0.35 nM, D2; Ki 1.75 nM, 5-HT2) proved slightly more potent and selective for D2 sites in vitro than (E)-NIASP (Ki 0.72 nM, D2; Ki 1.14 nM, 5-HT2). In vivo, radioiodinated (E)- and (Z)-[125I]-NIASP showed regional distributions in mouse brain which are consonant with prolonged binding to dopamine D2 receptors accompanied by a minor serotonergic component of shorter duration. Stereoselective, dose-dependent blockade of (E)-[125I]-NIASP uptake was found for drugs binding to dopamine D2 sites, while drugs selective for serotonin 5-HT2, alpha 1-adrenergic and dopamine D1 receptors did not inhibit radioligand binding 2 hr postinjection. Specific binding in striatal tissue was essentially irreversible over the time course of the study, and (E)-[125I]-NIASP gave a striatal to cerebellar tissue radioactivity concentration of 16.9 to 1 at 6 hr postinjection. Thus, (E)-[125I]-NIASP binds with high selectivity and specificity to dopamine D2 sites in vivo.     

In-Vitro Cytotoxicity and Cell Cycle Analysis of Two Novel bis-1,2, 4-triazole derivatives: 1,4-bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4-(p-tolyl)-1,2,4-triazol-3-yl]-butane (MNP-14) and 1,4-bis[5-(carbethoxy-methyl)-thio-4-(p-ethoxy phenyl)-1,2,4-triazol-3-yl]-butane (MNP-16)

In the present study, we have tested the cytotoxic and DNA damage activity of two novel bis-1,2,4 triazole derivatives, namely 1,4-bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4-(p-tolyl)-1,2,4-triazol-3-yl]-butane (MNP-14) and 1,4-bis[5-(carbethoxy-methyl)-thio-4-(p-ethoxy phenyl) -1,2,4-triazol-3-yl]-butane (MNP-16). The effect of these molecules on cellular apoptosis was also determined. The in-vitro cytotoxicity was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as well as Trypan blue dye exclusion methods against human acute lymphoblastic leukemia (MOLT4) and lung cancer cells (A549). Our results showed that MNP-16 induced significant cytotoxicity (IC₅₀ of 3–5 μM) compared with MNP-14. The cytotoxicity induced by MNP-16 was time and concentration dependent. The cell cycle analysis by flow cytometry (fluorescence-activated cell sorting [FACS]) revealed that though there was a significant increase in the apoptotic population (sub-G₁ phase) with an increased concentration of MNP-14 and 16, there was no cell cycle arrest. Further, the comet assay results indicated considerable DNA strand breaks upon exposure to these compounds, thereby suggesting the possible mechanism of cytotoxicity induced by MNP-16. Hence, we have identified a novel molecule (MNP-16) which could be of great clinical relevance in cancer therapeutics.     

Vasopressin(1-8) (des-glycinamide9-[Arg8]vasopressin) is an endogenous peptide present in rat plasma

Using a radioimmunoassay for [Arg8]vasopressin(1-8) (des-glycinamide9-[Arg8]vasopressin; DGAVP) endogenous immunoreactive DGAVP (IR-DGAVP) was detected in extracts of plasma prepared from trunk blood of male Wistar rats. The IR-DGAVP was further characterized by reversed-phase high pressure liquid chromatography (HPLC). One of the two immunoreactive peaks obtained by HPLC coeluted with synthetic DGAVP and did not cross-react in a radioimmunoassay specific for [Arg8]vasopressin(1-9) (AVP). The other showed the chromatographical and radioimmunological characteristics of AVP. Analysis by HPLC of plasma prepared from fresh blood spiked with 3H-AVP indicated that under the experimental conditions employed no DGAVP was formed during extraction. The results indicate that DGAVP is present in rat plasma, possibly as an endogenous metabolite of AVP.     

5 alpha-Cholest-8(14)-en-3 beta-ol-15-one. In vivo conversion to cholesterol upon oral administration to a nonhuman primate

The metabolism of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one (I), a potent inhibitor of cholesterol synthesis with marked hypocholesteremic activity, has been studied in a nonhuman primate. A mixture of [2,4-3H]-I and [4-14C]-cholesterol was administered to a male baboon in the form of a feedball. Blood was samples at 4, 8, 12, 16, and 24 hr. Detailed analyses of the plasma lipids indicated very rapid absorption of I (relative to cholesterol) and metabolism to cholesterol, cholesteryl esters, and esters of I. The labeled cholesterol was characterized by chromatographic techniques and by purification by way of its dibromide derivative. The levels of 3H in plasma associated with I, esters of I, cholesterol, and cholesteryl esters each showed a different time course. By 24 hr after the administration of [2,4-3H]-I, most of the 3H in plasma was associated with cholesterol and cholesteryl esters. The levels of total 3H and 14C in plasma at various times after the administration of the mixture of [2,4-3H]-I and [4-14C]-cholesterol differed markedly with 3H showing a maximum value at 4 hr and 14C showing a maximum value at 24 hr.     

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine photolabels a substrate-binding site of rat hepatic cytochrome P-450 form PB-4

Hepatic microsomes isolated from untreated male rats or from rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (3-MC) were labeled with the hydrophobic, photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). [125I]TID incorporation into 3-MC- and PB-induced liver microsomal protein was enhanced 5- and 8-fold, respectively, relative to the incorporation of [125I]TID into uninduced liver microsomes. The major hepatic microsomal cytochrome P-450 forms inducible by PB and 3-MC, respectively designated P-450s PB-4 and BNF-B, were shown to be the principal polypeptides labeled by [125I]TID in the correspondingly induced microsomes. Trypsin cleavage of [125I]TID-labeled microsomal P-450 PB-4 yielded several radiolabeled fragments, with a single labeled peptide of Mr approximately 4000 resistant to extensive proteolytic digestion. The following experiments suggested that TID binds to the substrate-binding site of P-450 PB-4. [125I]TID incorporation into microsomal P-450 PB-4 was inhibited in a dose-dependent manner by the P-450 PB-4 substrate benzphetamine. In the absence of photoactivation, TID inhibited competitively about 80% of the cytochrome P-450-dependent 7-ethoxycoumarin O-deethylation catalyzed by PB-induced microsomes with a Ki of 10 microM; TID was a markedly less effective inhibitor of the corresponding activity catalyzed by microsomes isolated from uninduced or beta-naphthoflavone-induced livers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiophen-2-yl]cyclopent-3-enecarboxylic acid (CP-191,166), an angiotensin II antagonist, in dog and rat plasma by high-performance liquid chromatography

A simple and precise high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of a novel angiotensin II antagonist, 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiopen-2-yl)cyclopent-3-enecarboxylic acid (CP-191,166, I), in dog and rat plasma. The internal standard (II, a saturated derivative of I) and analyte were extracted by liquid-liquid extraction using methyl tert.-butyl ether. Samples were analyzed by reversed-phase HPLC using a Zorbax C₈ narrow-bore column with ultraviolet detection at 289 nm. The quantitation limit of I was 10 ng/ml and the calibration curve was linear over the range of 0.01–10.0 μg/ml (r²>0.99). In dog and rat plasma, intra- and inter-assay precision ranged from 0.00 to 3.36% and 0.00 to 4.95%, respectively. The average recoveries were similar (73%) for both I and II and the upper limit of quantification of I can be as high as 500 μg/ml. The method described has been successfully applied to the quantification of I in about 2000 dog and rat plasma samples over a nine-month period.