Disposition Of Orally Administered Flavopiridol In The Intestine And Its Species And Gender Differences

Flavopiridol (FLAP,(-) cis-5,7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-hydroxy-1-methyl) piperidinyl]-4H-1-benzopyran-4-one), is a semisynthetic flavone structurally related to the natural alkaloid rohitukine from the Indian plant DysoxylumBinectariferum. The mechanism of action of FLAP involves the inhibition of phosphokinases, primarily cyclin-dependent kinases. FLAP inhibits proliferation and induces apoptosis in a variety of human cancer cells and cell lines, such as leukemia, esophageal and gastric cancer cells, among others; and has entered phase and phase Ⅱ clinical trials as a single agent and phase Ⅰ trials in combination with other chemotheraphy agentsAt present, different schedules of FLAP administration have been explored, including24h or72h continuous intravenous infusion (CIVI),1h intravenous bolus for3days (IVB) and a30min intravenous bolus followed by4h continuous intravenous infusion (IVB/CIVI).In allcurrent phase Ⅰ and phase Ⅱ clinical trials, intravenous infusion is the only medication route for FLAP. However, continuous and repetitive intravenous infusion leads to medication inconvenience and poor patient compliance. In addition, the FLAP dose is also limited because of hematologic and other toxicities such as diarrhea. On the other hand, oral administration is generally regarded as a safer, more convenient, and more acceptable strategy for therapy, especially for the treatment of digestive tract cancer, because the drug can directly affect the target tissues.Good oral bioavailability is the primary criterion in developing an efficient strategy for FLAP clinical treatment via oral administration. The low oral bioavailabilities (<10%) of flavonoids for extensive first-pass metabolism by phase II enzymes, including UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs), are a serious concern. Characterized as a flavonoid. FLAP also possesses a parent flavone structure, but with a heterocyclic ring substituent introduced at the8-position. This structural difference may affect the absorption and metabolism of FLAP and consequently alter its oral bioavailability. A number of studies have shown that FLAP is extensively metabolized in the rat liver into two monoglucuronides, Ml and M2, which are mainly excreted into the bile. whereas UGT1A9is the major UGT involved in the hepatic glucuronidation of FLAP in humans. However, research on the absorption and metabolism of FLAP in the intestine has not been conducted.In the present study, we determined dissolubility, stability and adsorbability of FLAP in various buffer solutions. We also determined absorption and disposition of FLAP in SD rats using in vivo dose (3and12mg·kg-1) oral and intravenous (3mg·kg-1) PK model, in situ four site rat intestinal perfusion model and in vitro a Caco-2cell culture model. Furthermore, we determined the species and gender differences in glucuronidation of FLAP using male and female dog, guinea pig, mouse, rat and human liver microsomes.1. The dissolubility, stability, adsorbability of FLAP.We investigate the FLAP’s stability and its solubility by using UPLC at wavelength X.=273nm. It was found that FLAP dissolve completely in HBSS buffer in the concentration range of0.625～40μM, all silica tube and EP tube used in the experiment didn’t show adsorbability to FLAP.2. Pharmacokinetics of FLAP in SD ratsWe detected the concentration of FLAP in plasma by LC/MS method at wavelength λ=273nm, the ionization method used was ESI source in the positive-ion mode and result verified by full scan mass spectrum. Results FLAP presented a good linear relation with the peak in the range of0.002～2.63μg·mL-1. Pharmacokinetic and bioavailability of FLAP were studied following an oral and intravenous adiminstrations in SD rats. All concentration-time curves correspond to the one-compartment model. Following a single intravenous injection of3mg·kg-1FLAP, the AUC0-∞, value was64.54±10.38mg·min·mL-1. the T1/2was173.73±31.97min. For the3and12mg·kg-1dosage oral administration, The Cmax values were127.52±45.54and329.34±37.68ng·mL-1. the Tmax values were18.00±6.71and11.00±5.48min, the AUC0-∞, values were48.98±11.88and200.36±60.51mg·min·mL-1.The oral bioavailability was75.89%.3. Absorptive characteristics of FLAP in Caco-2cell monolayer model.The concentration of FLAP across the Caco-2cell monolayer model was determined by UPLC method and the apparent permeability coefficient (P) of FLAP was calculated. PA-B increased from0.47×10-5cm·s-1to0.61×10-5cm·s-1as the donor side concentration increased from10μM to30μM, whereas PB-A significantly decreased from1.53×10-5cm·s-1to1.33×10-5cm·s-1(P<0.05), and the ratios of PB-A/PA-B were3.27and2.17. After the addition of cyclosporine A or verapamil, two typical P-gp inhibitors, the efflux ratios (PB-A/PA-B) decreased from3.27to1.12and1.35, respectively, the intracellular amounts of FLAP in the Caco-2cells also significantly increased.4. The absorption of FLAP in the intestine.The absorption of FLAP (40and10μM) were investigated by using the in situ intestinal perfusion in rats, the collected perfusate were detected by UPLC at wavelength273nm. The perfused FLAP was absorbed well, and its absorption at various regions of the intestine at both10and40μM showed no significant difference. The biliary metabolite excretion from the60min point to the150min point increased and was found significantly higher at40μM (12.47nmol to17.33nmol) than that at10μM (1.60nmol to2.84nmol) for each time point. However, only small amounts of the metabolite were found in the perfusate of all four intestinal segments at40μM (0.93nmol to1.23nmol).5. Species and gendercomparison on the glucuronidation of FLAP by liver microsomes in vitro. 10kinds of liver microsomal (male mouse, rat, guinea pig, dog, human and female mouse, rat, guinea pig, dog, human) incubation systems were used to investigate the UGT metabolism. The amounts of FLAP glucuronides in samples were determined by UPLC. FLAP was metabolized to FLAP glucuronide in each selected kind of microsomes. Species-dependent glucuronidation rates of FLAP displayed significant differences in the same gender (P<0.05). Significant difference was observed between the glucuronidation rates of dog and guinea pig (P<0.05), while not observed among those of human, rat and mouse.The present study aims to characterize the intestinal disposition of orally administered FLAP through pharmacokinetic studies in rats as well as absorption and metabolism studies using a Caco-2cell culture and four-site perfused rat intestinal models. Pharmacokinetic results show that FLAP has high bioavailability (>75%), long T1/2(>260min), and short peak time (<20min). In the Caco-2cell culture model, the bidirectional permeability of FLAP was0.47×10-5cm·s-1to1.53×10-5cm·s-1and the efflux ratios were3.27and2.17at10and30μM, respectively. Apical loading of two P-glycoprotein (Pgp) inhibitors, cyclosporine A and verapamil significantly increased the intracellular amount of FLAP and lowered its efflux ratio. In the four-site model,10and40μM FLAP perfusions were well absorbed at various regions of the intestine, and the biliary excretions of FLAP glucuronides were1.60to2.84nmol and12.47to17.33nmol, respectively. FLAP was metabolized to FLAP glucuronide in each selected kind of microsomes. Species-dependent glucuronidation rates of FLAP displayed significant differences in the same gender (P<0.05). Significant difference was observed between the glucuronidation rates of dog and guinea pig (P<0.05), while not observed among those of human, rat and mouse.Therefore, FLAP possesses high oral bioavailability and showed good absorption in the intestine, in which FLAP may be subjected to a Pgp efflux. Biliary excretion is the main elimination pathway for FLAP glucuronide and its enterohepatic cycling could be indicated. Glucuronidation is the dominant pathway in the metabolism of FLAP. There was no significant difference between the glucuronidation rate in human male liver microsomes and that in human female liver microsomes, while the species-dependence was markedly observed.