Biomedical subjects

R C Gupta

Publications and source records attributed to R C Gupta.

At least 37 records · Page 2Linked to original sources

PubMed2003

An HPLC-MS method for simultaneous estimation of alpha,beta-arteether and its metabolite dihydroartemisinin, in rat plasma for application to pharmacokinetic study.

This manuscript reports, the development and validation of a sensitive and selective assay method for simultaneous determination of alpha,beta-arteether and its metabolite dihydroartemisinin (DHA) in rat plasma by liquid chromatography-mass spectrometry. Chromatographic separations were achieved by gradient elution of the analytes with an initial composition of methanol-potassium acetate buffer (pH 4; 73:27, v/v) to 100% methanol in 3 min and maintained for 5 min on a Spheri-10, RP(18) (100 x 4.6 mm i.d.) column following an RP(18) (30 x 4.6 mm i.d.) guard column. The total effluent from the column was split so that one-tenth was injected into the electrospray LC/MS interface. ESI-MS analysis was performed using a Micromass Quattro II Triple Quadrupole Mass Spectrometer equipped with an electrospray source. The MS analysis was carried out at cone voltage of 22 V with a scan range of 200-500 Da. The analytes were quantified from the [M+ K](+) ion chromatograms of alpha,beta-arteether at m/z 352, DHA at m/z 323, artemisinin at m/z 321 and propyl ether analogue of arteether at m/z 365. Liquid-liquid extractions with a combination of n-hexane and hexane-ethyl acetate (8:2) were used to isolate alpha,beta-arteether and DHA from rat plasma. The method was validated and gave good accuracy and precision for the studied domain. Linearity in serum was observed over the range 4.375-70 ng/mL for alpha-arteether and 10-160 ng/mL for beta-arteether and DHA. Percentage bias (accuracy) and within- and between-assay precision were well within the acceptable range. This method was applied to study the pharmacokinetics following oral administration of alpha,beta-arteether (30 mg/kg) in rats.

Administration, Oral↗

PubMed2003

A sensitive and selective liquid chromatographic/electrospray ionization tandem mass spectrometric assay for the simultaneous quantification of alpha-,beta-arteether and its metabolite dihydroartemisinin in plasma, useful for pharmacokinetic studies.

A sensitive, selective, specific and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric assay method was developed and validated for the simultaneous quantitation of alpha-,beta-arteether (alpha-,beta-AE) and its metabolite alpha-dihydroartemisinin (DHA) in monkey plasma using the propyl ether analogue of beta-arteether (PE) as an internal standard. The method involves a simple two-step liquid-liquid extraction with hexane. The analytes were chromatographed on a C(18) reversed-phase chromatographic column by isocratic elution with methanol-ammonium acetate buffer (pH 4) (92 : 8, v/v) and analysed by mass spectrometry in the multiple reaction monitoring mode. The chromatographic run time was 7 min and the weighted (1/x(2)) calibration curves were linear over the range 0.78-200 ng ml(-1). The method was validated in terms of accuracy, precision, absolute recovery, freeze-thaw stability, bench-top stability and re-injection reproducibility. The limit of detection and lower limit of quantification in monkey plasma were 0.39 and 0.78 ng ml(-1) respectively for all the analytes. The intra- and inter-batch precision and accuracy were found to be well within acceptable limits (<15%). All three analytes were stable even after three freeze-thaw cycles (deviation < 15%). The average absolute recoveries of alpha-,beta-AE, DHA and PE, used as an internal standard, from spiked plasma samples were 85.85 +/- 6.56, 70.10 +/- 7.06, 54.37 +/- 3.39 and 93.90 +/- 6.9%, respectively. The assay method described here could be applied to study the pharmacokinetics of alpha-,beta-AE and DHA in rhesus monkeys.

Antimalarials↗

PubMed2003

Simultaneous quantitative analysis of three drugs by high-performance liquid chromatography/electrospray ionization mass spectrometry and its application to cassette in vitro metabolic stability studies.

A sensitive and selective liquid chromatography/electrospray mass spectrometry (LC/ESI-MS) method has been developed for the simultaneous quantitative determination of three new chemical entities (NCEs), of the class of aryloxy-substituted aryl piperazines, in rat liver S9 fraction. S9 fraction samples (0.1 mL) were simply extracted with 2% isopropanol in diethyl ether and the extracts analyzed by HPLC with the detection of the analytes in the selective ion recording (SIR) mode. The determination of the analytes was accurate and reproducible, with a limit of quantification of 50 ng/mL for all the analytes in rat liver S9 fraction. The standard calibration curve for the analytes was linear over the concentration range 50-4000 ng/mL. Analysis accuracy and precision over the concentration range were lower than +/-15%. This method offered significant increase in the analytical throughput, which is illustrated by the 'N-in-One' study of metabolic stability of the compounds in rat liver S9 fractions. The quantitative results from the 'N-in-One' procedure correlated well with those obtained from conventional discrete analyses. In addition, the samples were reanalyzed to allow for detection of the metabolites formed during the same incubation. The metabolites were first characterized by nominal mass measurement of the corresponding protonated molecules. Subsequent tandem mass spectrometry allowed confirmation of the detected metabolites.

Animals↗

PubMed2002

LC determination of the anti-ulcer agent CDRI-85/92 in rat serum.

CDRI-85/92 is a new potent anti-ulcer compound developed by Central Drug Research Institute, Lucknow (India). This compound is in advanced stage of preclinical trials. A High-performance liquid chromatographic (HPLC) method was developed and validated for the analysis of CDRI-85/92 using rat serum. The HPLC analysis, applicable to 0.5-ml volumes of serum, involved protein precipitation of serum samples with acetonitrile (1:3 v/v) followed by centrifugation and separation on a C-18 column and the use of UV detector at the wavelength 250 nm. The method was sensitive with a lowest limit of quantitation (LLOQ) of 1.25 ng ml(-1) in rat serum and the recovery was more than 96%. The linearity was satisfactory as indicated by correlation of >0.99, in addition to the visual examination of the calibration curves. The precision and accuracy were acceptable as indicated by relative standard deviation (R.S.D.) ranging from 4.15 to 8.21%, bias values ranging from 2.96 to 11.18%. In-process stability evaluation showed the stability of the compound in processed samples lasted up to 168 h. The method was applied for analysing CDRI-85/92 in rat serum after administration of single oral or iv bolus dose of 20 mg kg(-1). The robustness/ruggedness of the HPLC procedure was tested using different HPLC instrumentation and column of different make. The assay was found to be sensitive (limit of quantification was 1.25 ng ml(-1)), specific (retention time for CDRI-85/92 is 7.5 min), accurate (% bias is <12%), precise (% R.S.D. is <10%), robust (no significant change in peak profile in two HPLC Instruments) and reliable for use in pharmacokinetic or toxicokinetic studies.

Animals↗

PubMed2002

Evaluation of interaction potential of certain concurrently administered drugs with pharmacological and pharmacokinetic profile of centchroman in rats.

Centchroman (Ormeloxifene) is a nonsteroidal, selective estrogen receptor modulator, oral contraceptive and anticancer agent, and is intended for long-term use by women. In view of its vast clinical application and the interaction of steroidal oral contraceptives with certain commonly used therapeutic agents, evaluation of interaction of certain concomitantly administered therapeutic agents (ibuprofen, rifampicin, diazepam, salbutamol, nifedipine, paracetamol, haloperidol, and tetracycline), in terms of both the postcoital contraceptive efficacy and pharmacokinetic profile, with centchroman was undertaken in female Sprague-Dawley rats. Among the representatives from each commonly used therapeutic category, interaction (pharmacokinetic) was observed with ibuprofen (60 mg/kg, twice daily), haloperidol (0.7 mg/kg, twice daily), and tetracycline (140 mg/kg, twice daily) coadministration on Days 1 through 5 postcoitum. Of these three therapeutic agents, only tetracycline interfered with the contraceptive efficacy of centchroman. It reduced the bioavailability of centchroman and its active metabolite by increasing their excretion through bile and feces. Increased metabolite excretion on tetracycline coadministration indicates the enterohepatic recirculation of the metabolite, not the parent drug. However, the effect of tetracycline was negated by the inclusion of lactic acid bacillus spores in the regimen.

Animals↗

PubMed2002

Metabolism and excretion of centbutindole (neuroleptic) in rats after oral administration.

The metabolism and excretion of centbutindole was studied in Sprague-Dawley rats after oral administration. The percentage of dose excreted was monitored over 4 days. Efforts were also directed towards evaluating the stability of centbutindole in various in-vitro biomatrices. Centbutindole was found to be metabolised in rats and only a negligible amount of parent drug was excreted. The percentage of the dose excreted in bile and faeces was 0.2% and 0.6%, respectively. No drug was found in urine after oral administration, but after an intravenous dose only 0.0012% of the dose was eliminated through urine. Two metabolites, dealkylated metabolite and hydroxy metabolite, were identified by ion spray LC/MS/MS, using a combination of parent ion and product ion scanning techniques. The major routes of metabolism of centbutindole include reduction of the carbonyl functional group in the butyrophenone side chain and N-dealkylation of the butyrophenone side chain attached to the pyrazinopyridoindole ring nitrogen at position 2. The hydroxy metabolite was excreted negligibly in bile, although it was present in the form of glucuronide conjugates more in comparison to its free form.

Administration, Oral↗

PubMed2002

Human exposure to fipronil from dogs treated with frontline.

This investigation determined fipronil residues on gloves worn while petting dogs after Frontline application. Frontline contains 9.8% fipronil, which controls fleas and ticks on dogs for at least 30 d. Frontline (1.34 ml) was applied topically on adult household dogs and gloves worn for 5 min during pettingwere collected 24 hr and 1, 2, 3, 4 and 5 w post-Frontline application for fipronil residue determinations using GC/MS. The highest concentration of fipronil (589.3 +/- 205.7ppm) was detected 24 h after Frontline application and was undetectable in the gloves collected at 5w. Repeated exposure to such contamination can pose human health risks.

Animals↗

PubMed2001

Effect of chemopreventive agents on DNA adduction induced by the potent mammary carcinogen dibenzo[a,l]pyrene in the human breast cells MCF-7.

Over 1500 structurally diverse chemicals have been identified which have potential cancer chemopreventive properties. The efficacy and mechanisms of this growing list of chemoprotective agents may be studied using short-term bioassays that employ relevant end-points of the carcinogenic process. In this study, we have examined the effects of eight potential chemopreventive agents, N-acetylcysteine (NAC), benzylisocyanate (BIC), chlorophyllin, curcumin, 1,2-dithiole-3-thione (D3T), ellagic acid, genistein, and oltipraz, on DNA adduction of the potent mammary carcinogen dibenzo[a,l]pyrene (DBP) using the human breast cell line MCF-7. Bioactivation of DBP by MCF-7 cells resulted in the formation of one predominant (55%) dA-derived and several other dA- or dG-derived DNA adducts. Three test agents, oltipraz, D3T, and chlorophyllin substantially (>65%) inhibited DBP-DNA adduction at the highest dose tested (30 microM). These agents also significantly inhibited DBP adduct levels at a lower dose of 15 microM, while oltipraz was effective even at the lowest dose of 5 microM. Two other agents, genistein and ellagic acid were moderate (45%) DBP-DNA adduct inhibitors at the highest dose tested, while NAC, curcumin, and BIC were ineffective. These studies indicate that the MCF-7 cell line is an applicable model to study the efficacy of cancer chemopreventive agents in a human setting. Moreover, this model may also provide information regarding the effect of the test agents on carcinogen bioactivation and detoxification enzymes.

Anticarcinogenic Agents↗

PubMed2001

Alterations in cytochrome c oxidase activity and energy metabolites in response to kainic acid-induced status epilepticus.

The effects of kainic acid (KA)-induced limbic seizures have been investigated on cytochrome c oxidase (COx) activity, COx subunit IV mRNA abundance, ATP and phosphocreatine (PCr) levels in amygdala, hippocampus and frontal cortex of rat brain. Rats were killed either 1 h, three days or seven days after the onset of status epilepticus (SE) by CO2 and decapitation for the assay of COx activity and by head-focused microwave for the determination of ATP and PCr. Within 1 h COx activity and COx subunit IV mRNA increased in all brain areas tested between 120% and 130% of control activity, followed by a significant reduction from control, in amygdala and hippocampus on day three and seven, respectively. In amygdala, ATP and PCr levels were reduced to 44% and 49% of control 1 h after seizures. No significant recovery was seen on day three or seven. Pretreatment of rats with the spin trapping agent N-tert-butyl-alpha-phenylnitrone (PBN, 200 mg kg(-1), i.p.) 30 min before KA administration had no effect on SE, but protected COx activity and attenuated changes in energy metabolites. Pretreatment for three days with the endogenous antioxidant vitamin E (Vit-E, 100 mg/kg, i.p.) had an even greater protective effect than PBN. Both pretreatment regimens attenuated KA-induced neurodegenerative changes, as assessed by histology and prevention of the decrease of COx subunit IV mRNA and COx activity in hippocampus and amygdala, otherwise seen following KA-treatment alone. These findings suggest a close relationship between SE-induced neuronal injury and deficits in energy metabolism due to mitochondrial dysfunction.

Adenosine Triphosphate↗

PubMed2001

The synaptic activity of HsDmc1, a human recombination protein specific to meiosis.

Human Dmc1 protein, a meiosis-specific homolog of Escherichia coli RecA protein, has previously been shown to promote DNA homologous pairing and strand-exchange reactions that are qualitatively similar to those of RecA protein and Rad51. Human and yeast Rad51 proteins each form a nucleoprotein filament that is very similar to the filament formed by RecA protein. However, recent studies failed to find a similar filament made by Dmc1 but showed instead that this protein forms octameric rings and stacks of rings. These observations stimulated further efforts to elucidate the mechanism by which Dmc1 promotes the recognition of homology. Dmc1, purified to a state in which nuclease and helicase activities were undetectable, promoted homologous pairing and strand exchange as measured by fluorescence resonance energy transfer (FRET). Observations on the intermediates and products, which can be distinguished by FRET assays, provided direct evidence of a three-stranded synaptic intermediate. The effects of helix stability and mismatched base pairs on the recognition of homology revealed further that human Dmc1, like human Rad51, requires the preferential breathing of A small middle dotT base pairs for recognition of homology. We conclude that Dmc1, like human Rad51 and E. coli RecA protein, promotes homologous pairing and strand exchange by a "synaptic pathway" involving a three-stranded nucleoprotein intermediate, rather than by a "helicase pathway" involving the separation and reannealing of DNA strands.

Adenosine Triphosphatases↗

PubMed2001

Triplex-induced recombination in human cell-free extracts. Dependence on XPA and HsRad51.

Triple helix-forming oligonucleotides (TFOs) can bind to polypurine/polypyrimidine regions in DNA in a sequence-specific manner. Triple helix formation has been shown to stimulate recombination in mammalian cells in both episomal and chromosomal targets containing direct repeat sequences. Bifunctional oligonucleotides consisting of a recombination donor domain tethered to a TFO domain were found to mediate site-specific recombination in an intracellular SV40 vector target. To elucidate the mechanism of triplex-induced recombination, we have examined the ability of intermolecular triplexes to provoke recombination within plasmid substrates in human cell-free extracts. An assay for reversion of a point mutation in the supFG1 gene in the plasmid pSupFG1/G144C was established in which recombination in the extracts was detected upon transformation into indicator bacteria. A bifunctional oligonucleotide containing a 30-nucleotide TFO domain linked to a 40-nucleotide donor domain was found to mediate gene correction in vitro at a frequency of 46 x 10(-)5, at least 20-fold above background and over 4-fold greater than the donor segment alone. Physical linkage of the TFO to the donor was unnecessary, as co-mixture of separate TFO and donor segments also yielded elevated gene correction frequencies. When the recombination and repair proteins HsRad51 and XPA were depleted from the extracts using specific antibodies, the triplex-induced recombination was diminished, but was either partially or completely restored upon supplementation with the purified HsRad51 or XPA proteins, respectively. These results establish that triplex-induced, intermolecular recombination between plasmid targets and short fragments of homologous DNA can be detected in human cell extracts and that this process is dependent on both XPA and HsRad51.

Cell-Free System↗

PubMed2001

Identification and characterization of a novel benzo[a]pyrene-derived DNA adduct.

The aim of this study was to generate and identify a novel benzo[a]pyrene (BP)-derived DNA adduct found both in vitro and in vivo. To date, the majority of studies have focused on N(2)-[10 beta(7 beta,8a,9a-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene)yl]-deoxyguanosine (anti-BPDE-dG), the major adduct generated following bioactivation of BP. However, a second adduct is also formed following bioactivation of BP which has been speculated to result from further metabolism of 9-OH-BP. In order to identify this second reaction pathway, the ultimate DNA binding species, and the DNA base involved, we have synthesized and characterized a dG-derived DNA adduct arising from further bioactivation of 9-OH-BP in the presence of rat liver microsomes. Analysis of the adducted nucleotides was conducted using both the (32)P-postlabeling assay and capillary electrophoresis-mass spectrometry (CE-MS).

Animals↗

PubMed2001

1,2-dithiole-3-thione and its structural analogue oltipraz are potent inhibitors of dibenz.

Dithiolethiones are currently one of the most promising classes of cancer chemopreventive agents that exhibit antitumorigenic properties at numerous organ sites against several classes of carcinogens. In the current study, we examined the effects of 2 dithiolethiones, 1,2-dithiole-3-thione (D3T) and its structural analogue oltipraz, on DNA adduction induced by the potent mammary carcinogen dibenzo-[a,l]pyrene (DBP) in vivo. Female Sprague-Dawley rats were provided dietary D3T and oltipraz (500 ppm each) for I week followed by a single intragastric dose of DBP (8 micromol/kg body weight) and killed 5 days later. D3T inhibited DBP-DNA adduction from 78% to 82% in all tissues examined, while oltipraz was equally effective in the lung and liver but less effective in the mammary glands, inhibiting DBP-DNA adduction by nearly 60%. These data coupled with their broad anti-tumor specificity support the use of D3T and oltipraz as cancer-preventive agents in clinical trials.

Animals↗

PubMed2001

Nitric oxide modulates high-energy phosphates in brain regions of rats intoxicated with diisopropylphosphorofluoridate or carbofuran: prevention by N-tert-butyl-alpha-phenylnitrone or vitamin E.

Acute effects of seizure-inducing doses of the organophosphate compound diisopropylphosphorofluoridate (DFP, 1.25 mg/kg s.c.) or the carbamate insecticide carbofuran (CF, 1.25 mg/kg s.c.) on nitric oxide (NO) were studied in the brain of rats. Brain regions (pyriform cortex, amygdala, and hippocampus) were assayed for citrulline as the determinant of NO and for high-energy phosphates (ATP and phosphocreatine) as well as their major metabolites (ADP, AMP, and creatine). Rats, anesthetized with sodium pentobarbital (50 mg/kg i.p.), were killed using a head-focused microwave (power, 10 kW; duration, 1.7 s). Analyses of brain regions of controls revealed significantly higher levels of citrulline in the amygdala (289.8+/-7.0 nmol/g), followed by the hippocampus (253.8+/-5.5 nmol/g), and cortex (121.7+/-4.3 nmol/g). Levels of energy metabolites were significantly higher in cortex than in amygdala or hippocampus. Within 5 min of CF injection, the citrulline levels were markedly elevated in all three brain regions examined, while with DFP treatment, only the cortex levels were elevated at this time. With either acetylcholinesterase (AChE) inhibitor, the maximum increase in citrulline levels was noted 30 min post-injection (> 6- to 7-fold in the cortex, and > 3- to 4-fold in the amygdala or hippocampus). Within 1 h following DFP or CF injection, marked declines in ATP (36-60%) and phosphocreatine (28-53%) were seen. Total adenine nucleotides and total creatine compounds were reduced (36 58% and 28-48%, respectively). The inverse relationship between the increase in NO and the decease in high-energy phosphates, could partly be due to NO-induced impaired mitochondrial respiration leading to depletion of energy metabolites. Pretreatment of rats with an antioxidant, the spin trapping agent N-tert-butyl-alpha-phenylnitrone (PBN, 200 mg/kg i.p.), prevented DFP- or CF-induced seizures, while the antioxidant vitamin E (100 mg/kg i.p. per day for 3 days) had no anticonvulsant effect. Both antioxidants, however, significantly prevented the increase of citrulline and the depletion of high-energy phosphates. It is concluded that seizures induced by DFP and CF produce oxidative stress due to a marked increase in NO, causing mitochondrial dysfunction, and thereby depleting neuronal energy metabolites. PBN pretreatment provides protection against AChE inhibitor-induced oxidative stress mainly by preventing seizures. Additional antioxidant actions of PBN may contribute to its protective effects. Vitamin E has direct antioxidant effects by preventing excessive NO production.

Adenosine Triphosphate↗

PubMed2001

Optimization of contraceptive dosage regimen of Centchroman.

Centchroman (Ormeloxifene), a non-steroidal oral contraceptive, is used at a dose of 30 mg once a week. To prevent failures in the beginning of the therapy, it is recommended that a dose of 30 mg twice a week for 12 weeks be administered to build up adequate blood levels. The present study was undertaken to simplify the dosing schedule without sacrificing the purpose of twice a week dosing regimen, using modeling and measurement approaches. The drug was given to 60 female volunteers who were divided into seven groups: group I, 30 mg weekly; group II, 30 mg twice a week; group III, 30 mg twice a week for 12 weeks followed by 30 mg weekly; group IV, 30 mg twice a week for 6 weeks followed by 30 mg weekly; group V, 60 mg weekly; and groups VI and VII, single 60 mg loading dose followed by 30 mg weekly doses. The blood samples were collected and analyzed by HPLC. In group I, mean trough concentrations of centchroman and its active metabolite, 7-desmethyl centchroman, were comparable to the steady-state trough concentrations in groups III, IV, VI, and VII. The metabolite to parent drug ratio remained constant in all the groups. The pharmacokinetic parameters in group VII were comparable to those reported after a single 30 mg dose. Dosage regimen VI was more convenient and provided better pregnancy protection (Pearl index 1.18; unpublished report) than regimen III, which is currently on the market and, thus, could be effectively used for contraception.

Centchroman↗

PubMed2001

Depletion of energy metabolites following acetylcholinesterase inhibitor-induced status epilepticus: protection by antioxidants.

Status epilepticus (SE)-induced neuronal injury may involve excitotoxicity, energy impairment and increased generation of reactive oxygen species (ROS). Potential treatment therefore should consider agents that protect mitochondrial function and ROS scavengers. In the present study, we examined whether the spin trapping agent N-tertbutyl-alpha-phenylnitrone (PBN) and the antioxidant vitamin E (DL-alpha-tocopherol) protect levels of high-energy phosphates during SE. In rats, SE was induced by either of two inhibitors of acetylcholinesterase (AChE), the organophosphate diisopropylphosphorofluoridate (DFP, 1.25 mg/kg, sc)- or the carbamate carbofuran (1.25 mg/kg, sc). Rats were sacrificed 1 h or 3 days after onset of seizures by head-focused microwave (power, 10 kW; duration 1.7 s) and levels of the energy-rich phosphates adenosine triphosphate (ATP) and phosphocreatine (PCr) and their metabolites adenosine diphosphate (ADP) and adenosine monophosphate (AMP), and creatine (Cr), respectively, were determined in the cortex, amygdala and hippocampus. Within 1 h of seizure activity, marked declines were seen in ATP (34-60%) and PCr (25-52%). Total adenine nucleotides (TAN = ATP + ADP + AMP) and total creatine compounds (TCC = PCr + Cr) were also reduced (TAN 38-60% and TCC 25-47%). No changes in ATP/AMP ratio were seen. Three days after the onset of seizures, recovery of ATP and PCr was significant in the amygdala and hippocampus, but not in the cortex. Pretreatment of rats with PBN (200 mg/kg, ip, in a single dose), 30 min before DFP or carbofuran administration, prevented induced seizures and partially prevented depletion of high-energy phosphates. Pretreatment with the natural antioxidant vitamin E (100 mg/kg, ip/day for 3 days), partially prevented loss of high energy phosphates without affecting seizures. In controls, citrulline, a product of nitric oxide synthesis, was found to be highest in the amygdala, followed by hippocampus, and lowest in the cortex. DFP- or carbofuran-induced seizures caused elevation of citrulline levels seven- to eight-fold in the cortex and three- to four-fold in the amygdala and hippocampus. These results suggest a close relationship between SE, excitotoxicity and energy metabolism. The involvement of oxidative stress is supported by the findings that DFP and carbofuran trigger an excessive nitric oxide (NO) production in the seizure relevant regions of the brain.

Adenosine Triphosphate↗

PubMed2001

LC fluorescence method for multiple synthetic compounds to rapidly create in vivo pharmacokinetic database utilizing 'N-in-One' dosing.

This manuscript reports development and validation of an assay method in rat serum for the simultaneous estimation of C1, C2, and C3, in-house CDRI molecules, of the class of aryloxy-substituted aryl-piperazinyl derivatives. The assay was applied to determine pharmacokinetic data after simultaneous intravenous administration of these three compounds. A high-performance liquid chromatography assay method using isocratic elution and fluorescence (excitation, 250 nm; emission 350 nm) was developed for simultaneous estimation of all the three compounds in rat serum. Linearity was observed between 12.5 and 400 ng/ml for all the three compounds in serum. Recoveries were highly consistent over the concentration ranges for all the analytes. Variations in the intra- and inter-batch accuracy and precision were within the acceptable limits of +/-20% at the limit of quantitation, whereas at higher concentrations it was +/-15%. A mixture of the three compounds was administered intravenously to rats. Blood samples were collected over a period of 6 h and analyzed to determine serum levels and pharmacokinetics of each compound. The pharmacokinetics of the aforementioned three compounds was also determined after individual administration. The results obtained in the N-in-One dosing correlated well with discrete dosing of compounds. Based on the results obtained, C2 emerges to be the compound with appropriate pharmacokinetic parameters. Thus, the N-in-One method is a useful method for increasing the throughput to obtain the pharmacokinetic information.

Animals↗

PubMed2001

LC determination of the anti-ischemic and anti-hypertensive agent CDRI-93/478 in rat serum.

CDRI-93/478 is a potent anti-ischemic and anti-hypertensive agent. This compound is in advanced stage of pre-clinical trials. A high-performance liquid chromatographic (HPLC) method was developed for the analysis of CDRI-93/478 in rat serum, a species used for safety evaluation. The HPLC analysis, applicable to 1 ml volumes of serum, involved double extraction of serum samples with diethyl ether at alkaline pH followed by separation on a spheri-5 cyano column and the use of fluorescence detector at excitation wavelength 250 nm and emission wavelength 372 nm. The method was sensitive with a limit of quantitation of 10 ng ml(-1) in rat serum and the recovery was more than 84%. The linearity was satisfactory as indicated by correlation of >0.99, in addition to the visual examination of the calibration curves. The precision and accuracy were acceptable as indicated by relative standard deviation (R.S.D.) ranging from 1.73 to 9.51%, bias values ranging from -7.31 to 8.68%. Moreover, CDRI-93/478 was stable in rat serum after being subjected to three freeze-thaw cycles. In-process stability evaluation showed the stability of the compound in processed samples lasted up to 168 h. The assay was found to be sensitive, specific, accurate, precise, and reliable for use in pharmacokinetic or toxicokinetic studies.

Animals↗