Studies On Preclinical Pharmacokinetics Of IMM018 And Hydromorphone

Potent drug-drug interactions can result in serious side effects, as a result, preclinical （in vitro） and some clinical （in vivo） interaction studies are now important components of the drug candidate selection process. A rapid liquid chromatographytandem mass spectrometry （LC/MS/MS） method has been validated and employed routinely evaluate the potential for a new drug to modify cytochrome P450 （P450） activities by determining the effect of the drug on in vitro probe reactions that represent activity of specific P450 enzymes.IMM018, 1-[5-（4-Chloro-phenyl）-3-oxo-pent-4-enyl]-pipefidine-4-carboxylic acid ethyl ester, is a drug candidate which has been shown to have analgesic and antiinflammatory actions in rats. In present paper, pharmacokinetics of IMM018 in rats and dogs were investigated. An analytical method based on liquid chromatography coupled to tandem mass spectrometry detection was developed and validated for simultaneous quantification of IMM018 and its five metabolites in rat plasma, using diphenhydramine as the internal standard. The metabolism, excretion and tissue distribution of IMM018 in rats were studied to support the pharmacology and toxicology study of IMM018.Hydromorphone is a semi-synthetic opioid agonist. A sensitive, rapid and specific liquid chromatographic-tandem mass spectrometric （LC/MS/MS） method for the determination of hydromorphone in beagle dog plasma was developed. The method is proved to be suitable for hydromorphone control-release preparation pharmacokinetics and bioavailability in beagle dog.1. Determination of six CYPs probe metabolitesIn vitro drug interaction data can be used in guiding clinical interaction studies, or, the design of new candidates. To make such a claim, it must be assured that the in vitro data obtained is confident. To meet this need, a rapid liquid chromatography-tandem mass spectrometry （LC/MS/MS） method has been validated and employed for routine screening of new chemical entities for inhibition of six major human cytoohrome P450 （CYP） isoforms using cDNA-expressed CYPs. Probe substrates were used near the Michaelis-Menten constant （K_m） concentration values for CYP1A2 （phenacetin）, CYP2C9 （tolbutamide）, CYP2C19 （S-mephenytoin）, CYP2D6 （dextrornethorphan）, CYP2E1 （chlorzoxazone）, and CYP3A4 （midazolam and dextromethorphan）. The major metabolites of CYP-specific probe substrates were quantified. The LC/MS/MS method was found to be accurate and precise within the linear range of 1.0-2000 ng·mL^-1 for each analyte in enzyme incubation mixture. The lower limit of quantification （LLOQ） was 1.0 ng·mL^-1. The limit of detection （LOD） for the tested analytes was 0.48 ng·mL^-1 or better based on signal-to-noise ratio＞3. The inhibition potential of the six CYP isoforms has been evaluated using their known selective inhibitors. The 50% inhibitory concentrations (IC₅₀ values) measured by this method demonstrated high precision and are consistent with the literature values.2. Studies on effect of IMM018 on hepatic CYPsIMM018, a new anti-inflammatory agent, was being developed for the treatment of patients with rheumatoid arthritis. Drug-drug interaction studies using recombinant human cytochrome P450s were conducted to assess CYP1A2, CYP2C9, CYP2D6 and CYP3A4 inhibition potential; Induction potential of CYP1A, CYP2C, CYP2D and CYP3A by IMM018 was examined with liver microsomes from control rats or rats treated with IMM018 at 50 and 100 mg·kg^-1·d^-1 for 3 consecutive days. The assays that were validated are: phenacetin O-deethylase （CYP1A2）, tolbutamide methylhydroxylase （CYP2C9）, dextromethorphan O-demethylase （CYP2D6）, midazolam l’-hydroxylase （CYP3A4） and dextromethorphan N-demethylase （CYP3A4）. High-pressure liquid chromatography-tandem mass spectrometry was applied as the analytical method. IMM018 was a mixed-type inhibitor of CYP1A2 (K_i=13.1μmol·L^-1) and it also appeared to be noncompetitive inhibitor of CYP2C9 (K_i=20.2μmol·L^-1) and CYP2D6 (K_i=10.0μmol·L^-1). Furthermore, IMM018 showed time-dependent inactivation for CYP3A4. In the induction study, there were no significant difference in the enzymatic activities between the control and IMM018 treated rats except CYP3A （p＞0.05）. IMM018 is likely to inhibit the metabolism of comedications moderately, if their primary routes of elimination are via cytochrome. The induction of CYP3A activity by IMM018 was dose-dependent in Wistar rats.3. Studies on pharmacokinetics of IMM018 in rats and dogsA sensitive LC/MS/MS method was developed for the determination of IMM018 and its five major metabolites in the plasma using diphenhydramine as internal standard. The analytes and internal standard were extracted with redistilled ethyl acetate and chromatographed isocratically on a Gemini C₁₈ analytical column with a mobile phase composed of methanol: water: formic acid in the ratio of 50: 50: 0.25 （v/v/v）. All analytes were detected in the selected reaction monitoring mode using an electrospray ionization source. IMM018 and its five metabolites could be simultaneously determined within 6 min. Linear calibration curves were obtained in the concentration ranges of 3.0～4000 ng·mL^-1 for IMM018, M3, M4 and M5, 0.4～4000 ng·mL^-1 for M1, and 4.0～40000 ng·mL^-1 for M2. The intra- and inter-day precision （RSD）, calculated from quality control （QC） samples, was between 2.0%～14.7% for each analyte. The accuracy was between -4.1%～4.3%. The method was utilized to support preclinical pharmacokinetic and toxicokinetic studies of IMM018 in rats and dogs.The pharmacokinetics of IMM018 was investigated in rats and dogs by the LC/MS/MS method. After oral administration of IMM018 to rats (25 mg·kg^-1), the t_1/2 values for metabolites M1, M2 and M3 were 4.49 h, 2.67 h and 2.79 h, respectively. After intravenous injections of IMM018 to rats (25 mg·kg^-1), the t_1/2 values for metabolites M1, M2 and M3 were 5.69, 4.38 and 3.43 h, respectively.4. Studies on excretion and tissue distribution of IMM018 in ratsAn LC/MS/MS method for the simultaneous determination of IMM018 and its metabolites in rat urine, feces and bile was developed and validated. The excretion of IMM018 and its metabolites was studied by this method. According to the volume of urine and bile and the weight of feces, the total accumulated excretion of IMM018 and its metabolites were calculated.After oral administration of IMM018 at dosage of 50 mg·kg^-1 to rats, IMM018 was not found in urine sample, accumulated excretion of M2 in urine sample was 75.90%; M3 was 3.24%; M1 was 0.57%; M4 was 0.0097%; M5 was 0.012%. The accumulation excretion of IMM018 in feces was 0.050%; M2 was 2.17%; M3 was 0.17%; M1 was 0.00404%; M4 was 0.035%; M5 was 0.00091%. The accumulation excretion of IMM018 in bile was 0.17%; M2 was 1.97%; M3 was 0.15%; M1 was 3.69%; M4 was 0.01%. These results show that IMM018 was mainly excreted in urine in the form of metabolite M2.The rats were divided into 3 groups. After the oral administration of 50 mg·kg^-1 IMM018, the rats were sacrificed at 1.0, 4.0 and 12 h separately. The tissue samples were collected and used to prepare homogenates. A sensitive, specific and accurate method for simultaneous quantifying IMM018 and its metabolites in rat tissues and to study the distribution of IMM018 in rat tissues was developed. After oral administration of IMM018, the drug was distributed extensively in rats in vivo. IMM018 and its major metabolites （M1～M5） were found in gastric contents and intestinal content. The concentrations of M1～M5 in lung, uterus, heart, bladder, pancreas, and liver were higher than that of the other tissues. The concentration of IMM018 and its metabolites were low in brain, which show that the blood-brain barrier was hard to be passed through for IMM018 and its metabolites.5. Studies on pharmacokinetics of hydromorphone control-release preparationAn LC/MS/MS method for determination of hydromorphone （HYD） in Beagle dog plasma was established. After incubation withβ-glucuronidase for 16 h, an aliquot of 0.1 mL plasma was treated by liquid-liquid extraction. The analytes of interest were separated on a Zorbax SB C₈ column with the mobile phase consisting of methanol: water: formic acid （65: 35: 1）. Atmospheric pressure chemical ionization source of MS was applied and operated in positive ion mode. The linear calibration curve was obtained in the concentration range of 0.80～200.0 ng·mL^-1. The lower limit of quantification was 0.80 ng·mL^-1. The inter-day and intra-day precision （RSD） was below 6.0%, and the accuracy （RE） was between-0.2%～1.0% calculated from QC samples. The method was used to determine the pharmacokinetic parameters of HYD after a single oral administration of 4 mg HYD sustained release tablets to Beagle dogs. The method was proved to be special, sensitive, and suitable for the pharmacokinetic study of HYD sustained release formulation.