ABSTRACT
Various c-mesenchymal-to-epithelial transition (c-MET) inhibitors are effective in the treatment of non-small cell lung cancer; however, the inevitable drug resistance remains a challenge, limiting their clinical efficacy. Therefore, novel strategies targeting c-MET are urgently required. Herein, through rational structure optimization, we obtained novel exceptionally potent and orally active c-MET proteolysis targeting chimeras (PROTACs) namely D10 and D15 based on thalidomide and tepotinib. D10 and D15 inhibited cell growth with low nanomolar IC50 values and achieved picomolar DC50 values and >99% of maximum degradation (Dmax) in EBC-1 and Hs746T cells. Mechanistically, D10 and D15 dramatically induced cell apoptosis, G1 cell cycle arrest and inhibited cell migration and invasion. Notably, intraperitoneal administration of D10 and D15 significantly inhibited tumor growth in the EBC-1 xenograft model and oral administration of D15 induced approximately complete tumor suppression in the Hs746T xenograft model with well-tolerated dose-schedules. Furthermore, D10 and D15 exerted significant anti-tumor effect in cells with c-METY1230H and c-METD1228N mutations, which are resistant to tepotinib in clinic. These findings demonstrated that D10 and D15 could serve as candidates for the treatment of tumors with MET alterations.
ABSTRACT
Physiologically based pharmacokinetic (PBPK) modeling and simulation can be used to predict the pharmacokinetic behavior of drugs in humans using preclinical data. It can also explore the effects of various physiologic parameters such as age, ethnicity, or disease status on human pharmacokinetics, as well as guide dose and dose regiment selection and aid drug-drug interaction risk assessment. PBPK modeling has developed rapidly in the last decade within both the field of academia and the pharmaceutical industry, and has become an integral tool in drug discovery and development. In this mini-review, the concept and methodology of PBPK modeling are briefly introduced. Several case studies were discussed on how PBPK modeling and simulation can be utilized through various stages of drug discovery and development. These case studies are from our own work and the literature for better understanding of the absorption, distribution, metabolism and excretion (ADME) of a drug candidate, and the applications to increase efficiency, reduce the need for animal studies, and perhaps to replace clinical trials. The regulatory acceptance and industrial practices around PBPK modeling and simulation is also discussed.
ABSTRACT
In the present study, the specifically knockdown models of P-gp or MRP2 were constructed by using a series of chemically synthesized small interfering RNA (siRNA) in vitro. The expression of P-gp and MRP2 was measured by real-time PCR and Western blot, and the function was evaluated by applying P-gp and MRP2 substrate, rhodamine and methotrexate. The results showed that MRP2 siRNA-3 or P-gp siRNA-2 significantly decreased the mRNA expression of MRP2 or P-gp, the inhibition ratio was 68% or 84%; MRP2 siRNA-3 or P-gp siRNA-2 at a dose of 80 nmol x L(-1) significantly reduced the protein expression of MRP2 or P-gp at 48 h after treatment, the inhibition ratio was 62% or 70%. Meanwhile, other transporters were not influenced by siRNA. When pretreatment with MRP2 siRNA-3 or P-gp siRNA-2, the efflux of methotrexate or rhodamine decreased significantly and the intra-cellular concentration increased. The results suggested that chemically synthesized siRNA could significantly inhibit the expression and function of MRP2 and P-gp, and the model of RNAi in vitro could be used to evaluate the role of efflux transporters in transportation of drugs.
ABSTRACT
Inflammatory conditions are associated with most diseases. Phenoconversion of drug-metabolizing enzymes(DME) leads to altered drug metabolism and disposition. It has profound impact on the pharmacotherapy of widely used clinically relevant medications in terms of safety and efficacy. More and more evidence has proved that elevated levels of proinflammatory cytokines may downregulate the expression and the activity of many Phase Ⅰ and Phase Ⅱ DME, which are involved in complex regulation mechanisms of drug disposition. The aim of this review is to present the recent findings in this area. Clinical practice based on personalized medicine according to DME phenotype with improved safety and efficiency can yield robust efficacy outcomes of drug treatment and has promising future prospects.
ABSTRACT
Objective To develop a steady-state brain distribution model in rats and to assess the blood brain barrier(BBB) penetration of antipyrine, atenolol and a group of ZZB candidate compounds. Methods Antipyrine, atenolol and ZZB compounds were administered to rats by an initial iv bolus dose (loading dose) followed by iv infusion at a constant rate for 30-40 min to reach steady-state plasma kinetics. The blood and brain tissue samples were then collected. The steady-state concentrations of the samples were measured by LC-MS/MS. The steady-state ratio of brain to plasma concentration (Kp) was calculated. The drugs and candidate compounds were also tested with Caco-2 cell model and the apparent bidirectional transport permeability coefficient (Papp) was obtained. Results Antipyrine and atenolol were known as drugs with high and low BBB penetration properties respectively. The mean brain concentrations of antipyrine and atenolol at steady-state were(2561 ± 125) and(20.1±0.8)ng/g with the Kp values of 0.93 ± 0.04 and 0.015 ± 0.002, respectively. The Kp value of antipyrine was about 60 folds higher than that of atenolol. Despite the similar structures of ZZB compounds, the Kp values were varied in the range from 0.044 to 6.41. The Kp values were not correlated with Papp values yielded from Caco-2 cell model. Conclusion The established rat steady-state brain distribution model is simple, reliable and could significantly reduce the animal use. It is a practical in vivo model for assessment of BBB penetration of drugs.
ABSTRACT
OBJECTIVE To investigate and compare the enzyme kinetic characters of psoralen (PRN)and isopsoralen(IPRN)in rat and human liver microsomes. METHODS PRN and IPRN in liver microsomes incubates were determined using LC-MS/MS. The enzyme kinetic and metabolic stability of PRN and IPRN were investigated by employing the optimized rat and human liver microsomes incubations. The Vmax and Km values were calculated using the nonlinear regression method. RESULTS The quanti?tative method showed good linearity within the range of 0.1-50.0 μmol · L-1 and was suitable for the assay in biological samples. The in vitro elimination was linear with the substrate concentrations lower than 1 μmol,the protein concentration within 0.5 g · L-1,and the incubation time within 40 min. The t1/2 values of PRN and IPRN in rat and human liver microsomes were 74.5,95.0,74.5 and 173.3 min, respectively. The Vmax values of PRN in rat and human liver microsomes were(1.140±0.080)μmol·min-1·g-1 protein,(0.620±0.060)μmol·min-1·g-1 protein,while Km values of PRN in rat and human liver microsomes were (12.9 ± 0.3)μmol · L- 1,(7.4 ± 1.3)μmol · L- 1,respectively. The Vmax values of IPRN in rat and human liver microsomes were(0.251±0.012)and(0.103±0.014)μmol·min-1·g-1 protein,while Km values of IPRN in rat and human liver microsomes were (3.0 ± 0.4)μmol · L-1,(3.4 ± 0.7)μmol · L-1,respectively. CONCLUSION The enzyme kinetic characters and metabolic stability of PRN and IPRN show species and chemical structures related differences. Interestingly,the metabolic eliminations of PRN and IPRN are similar in rats. However,the metabolic elimination of IPRN in humans involved in CYP enzymes may be much slower than that of PRN.
ABSTRACT
OBJECTIVE To investigate the effect of ketoconazole on the pharmacokinetic (PK) behaviors of midazolam and its metabolite through intranasal and intragastric(ig) routes in rats. METHODS Twenty-four rats were evenly divided into 4 groups. Two groups of rats were administrated singly with midazolam (1 mg?kg-1) through intranasal or ig route. The other two groups were concomitant with CYP3A inhibitor,ketoconazole(30 mg?kg-1),midazolam(1 mg?kg-1)through the same two routes. Blood samples were collected from different time points. Plasma concentration of midazolam and 1′-hydroxymidazolam was determined. Major pharmacokinetic parameters were calculated and statistical tests were performed by using t test. RESULTS Tmax was about 2 and 25 min for rats administered singly with midazolam via intranasal or ig routes,respectively and AUC was 296 and 179 μg?L-1?h, respectively. When concomitant with ketoconazole,AUC increased to 2.1 and 3.3 folds the original value for intranasal and ig routes,respectively. However,the Tmax value of midazolam via intranasally didn′t change after being coadministrated with ketoconazole,but Tmax increased to 1.14 h via ig. CONCLUSION Compared with administration via ig,intranasal route administrated midazolam displays significant advantages of faster absorption and higher exposure,which are vital for the first aid. Concomitant with CYP3A inhibitor and midazolam via intranasal route,the absorption speed is not affected,but with the metabolism blocked,the systemic exposure is greatly elevated. While via ig,both absorption speed and metabolism are inhibited. The dose should be cut down or the dosing interval increased in clinic practice in this concomitant situation.
ABSTRACT
Objective To establish and optimize the rat jugular vein catheterization model in our lab, and perform a cross-over study using this model to compare the pharmacokinetic characters of a newly developed midazolam formulation to the existing preparation. Methods Six SD rats (half male and half female) received the right jugular vein catheterization when the rats were sufficiently anesthetized. One week after the operation, all the rats were used to conduct a cross-over double period pharmacokinetic study. Totally1.33 mg/kg midazolam solutions from automatic needle and clinic available injection were adminisered to the jugular vein catheterization rats via im route. The washout period was 5 days. Exact volume of blood samples at designed time points were taken through the catheter. After preparation, the concentrations of midazolam in rat plasma were determined by using established LC-MS/MS method. The corresponding pharmacokinetic parameters were calculated by WinNolin software. Results The rat jugular vein catheterization model was successfully built. Blood was easily sampled and rats were well tolerated, meeting the requirement of repeated blooding. This model solved the bottleneck of cross-over study performed in rats. The pharmacokinetic behavior of newly developed midazolam formulation had no difference with that of clinic injections. The relative bioavailability was around 99%. Conclusion Rat jugular vein catheterization model is proved to be that of a propagating technique to do the cross-over study and to evaluate the pharmacokinetic characters of novel formulations.
ABSTRACT
Aim To investigate the inhibitory and in-duction effects of the active components of SIWU de-coction on cytochrome P450 enzymes ( CYP ) and as-sess the CYP based drug interaction. Methods Ac-tive components fructose, ferulic acid, paeoniflorin, li-gustrazine and their combinations were incubated sepa-rately with human liver microsomes ( HLM) and probe substrates. Metabolites of the CYP probe substrates were determined by LC-MS/MS to assess the inhibitory activities on human CYP1 A2 , 2 B6 , 2 C9 , 2 C19 , 2 D6 and 3 A4 . Sandwich-cultured rat hepatocytes model was used to evaluate the CYP1 A2 and CYP3 A1/2 induc-tion. Results The inhibitory rates on CYP1A2, 2B6, 2 C9 and 2 C9 by the test groups at 100 μmol · L-1 were all < 62%, while the activities of CYP3 A4 and 2D6 were not affected. The CYP1A2 activities in the test groups of peoniflorin and its combinations ( 50μmol·L-1 ) were significantly enhanced, with the in-creasing fold more than 40% of positive control group. No significant induction on rat CYP3 A1/2 was ob-served for four principles and their combinations. Con-clusions The active components of SIWU decoction do not show significant inhibitory effects on six CYP isoforms. Peoniflorin could induce the CYP1A2 activity in rat hepatocytes. The induction activity is enhanced by the concomitant use of peoniflorin with ferulic acid and/or ligustrazine.
ABSTRACT
The metabolic characteristics of ligustrazin (TMPz) in liver microsomes were investigated in the present study. The reaction phenotyping of TMPz metabolism was also identified by in vitro assessment using recombinant human cytochrome P450 enzymes (CYP) and UDP glucuronosyltransferases (UGT). TMPz was incubated at 37 degrees C with human (HLM) and rat liver microsomes (RLM) in the presence of different co-factors. The metabolic stability and enzyme kinetics of TMPz were studied by determining its remaining concentrations with a LC-MS/MS method. TMPz was only metabolically eliminated in the microsomes with NADPH or NADPH+UDPGA. In the HLM and RLM with NADPH+UDPGA, t1/2, K(m) and V(max) of TMPz were 94.24 +/- 4.53 and 105.07 +/- 9.44 min, 22.74 +/- 1.89 and 33.09 +/- 2.74 micromol x L(-1), 253.50 +/- 10.06 and 190.40 +/- 8.35 nmol x min(-1) x mg(-1) (protein), respectively. TMPz showed a slightly higher metabolic rate in HLM than that in RLM. Its primary oxidative metabolites, 2-hydroxymethyl-3, 5, 6-trimethylpyrazine (HTMP), could undergo glucuronide conjugation. The CYP reaction phenotyping of TMPz metabolism was identified using a panel of recombinant CYP isoforms (rCYP) and specific CYP inhibitors in HLM. CYP1A2, 2C9 and 3A4 were found to be the major CYP isoforms involved in TMPz metabolism. Their individual contributions were assessed b) using the method of the total normalized rate to be 19.32%, 27.79% and 52.90%, respectively. It was observed that these CYP isoforms mediated the formation of HTMP in rCYP incubation. The UGT reaction phenotyping of HTMP glucuronidation was also investigated preliminarily by using a panel of 6 UGT isoforms (rUGT). UGT1A1, 1A4 and 1A6 were the predominant isoforms mediated the HTMP glucuronidation. The results above indicate that the metabolism of TMPz involves multiple enzymes mediated phase I and phase II reactions.
ABSTRACT
3-Hydroxymethyl-4-methyl-DCK (, HMDCK) was discovered previously as a potent HIV non-nucleoside reverse transcriptase inhibitor (NNRTIs) (EC: 0.004 μM, TI: 6225) with a novel mechanism of action. It exerts anti-HIV activity by inhibiting the production of HIV-1 double-stranded viral DNA from a single-stranded DNA intermediate, rather than blocking the generation of single-stranded DNA from a RNA template, which is the mechanism of action of current HIV-1 RT inhibitors. However, the insufficient metabolic stability oflimits its further clinical development. In the current study, a series of ester prodrugs ofwas designed and synthesized to explore the new drug candidates as NNRTIs. The l-alanine ester prodrugexhibited desirable pharmacokinetic propertiesandand showed improved oral bioavailability of 26% in rat, and would be a potential clinical candidate as a new anti-AIDS drug.
ABSTRACT
The inter-species differences of thienorphine metabolism were investigated in human, Beagle dog and rat liver microsomes, by comparing enzyme kinetics of the parent drug and the formation of its major metabolites. The incubation systems of thienorphine with liver microsomes of the three species were optimized in terms of thienorphine concentration, microsomal protein content and incubation time. The concentrations of thienorphine and its metabolites in incubates were measured by a LC-MS/MS method. The biotransformation of thienorphine by human liver microsomes was the lowest among the three species. The Km, Vmax, CLint and T1/2 of thienorphine obtained from human liver microsomes were (4.00 ? 0.59) ?mol?L-1, (0.21 ? 0.06) ?mol?L-1?min-1, (117 ? 3.19) mL?min-1?kg-1 and (223 ? 6.10) min, respectively. The corresponding kinetic parameters for dog and rat liver microsomes were (3.57 ? 0.69) and (3.28 ? 0.50) ?mol?L-1, (0.18 ? 0.04) and (0.14 ? 0.04) ?mol?L-1?min-1, (213 ? 1.06) and (527 ? 7.79) mL?min-1?kg-1, (244 ? 1.21) and (70.7 ? 1.05) min, respectively. A total of six phase I metabolites were observed in liver microsomes, including one N-dealkylated metabolite, three oxidative metabolites and two N-dealkylated oxidation metabolites. All these six metabolites were detected in the liver microsomes of the three species. However, the relative amounts of the metabolites generated were different in three species. The results indicated that the major phase I metabolic pathway of thienorphine was similar in the liver microsomes from all three species. However, the inter-species differencesobserved were relative amounts of the metabolites as well as the metabolic characteristics of thienorphine in liver microsomal incubates.
ABSTRACT
OBJECTIVE To explore the absorption mechanism of thiophenorphine, and its effect on P-glycoprotein (P-gp) expression by using Caco-2 cell monolayer model. METHODSThe LC-MS-MS method was applied to determine thiophenorphine concentration in millicell system. The bi-directional permeability studies were performed to investigate the potential involvement of efflux carriers in the intestinal absorption. P-gp inhibition was studied by flow cytometry using calcein-AM as P-gp substrate.The expression of P-gp was evaluated using Western blotting. RESULTSThiophenorphine transport in Caco-2 cells was in time-dependent manner. Its average apparent permeability coefficient (P_(app)) was 2.338×10~(-6) cm·s~(-1). P_(app) was increased 2.8 folds by P-gp inhibitor ciclosporin A, and 2.3 folds by mulitdrug resistance-associated protein2 (MRP2) inhibitor MK571. The accumulation of calcein-AM and the expression of P-gp in Caco-2 cell line wasn't changed noticeably by thiophenorphine. CONCLUSION Thiophenorphine is a common substrate of P-gp and MRP2 and it shows normal transport in millicell system. The expression of P-gp doesn't induce by thiophenorphine.
ABSTRACT
The biotransformation, CYP reaction phenotyping, the impact of CYP inhibitors and enzyme kinetics of 3-cyanomethyl-4-methyl-DCK (CMDCK), a new anti-HIV preclinical candidate belonging to DCK analogs, were investigated in human intestinal microsomes and recombinant cytochrome P450 (CYP) enzymes. CMDCK (4 micromol L(-1)) was incubated with a panel of rCYP enzymes (CYP1A2, 2C9, 2C19, 2D6 and 3A4) in vitro. The remaining parent drug in incubates was quantitatively analyzed by a LC-MS method. CYP3A4 was identified as the principal CYP isoenzyme responsible for its metabolism in intestinal microsomes. The major metabolic pathway of CMDCK was oxidation and a number of oxidative metabolites were screened with LC-MS. The Km, Vmax, CLint and T1/2 of CMDCK obtained from human intestinal microsome were 45.6 micromol L(-1), 0.33 micromol L(-1) min(-1), 12.1 mL min(-1) kg(-1) and 25.7 min, respectively. Intestinal clearance of CMDCK was estimated from in vitro data to be 3.3 mL min(-1) kg(-1), and was almost equal to the intestinal blood flow rate (4.6 mL min(-1) kg(-1)). The selective CYP3A4 inhibitors, ketoconazole, troleandomycin and ritonavir demonstrated significant inhibitory effects on CMDCK intestinal metabolism, which suggested that co-administration of CMDCK with potent CYP3A inhibitors, such as ritonavir, might decrease its intestinal metabolic clearance and subsequently improve its bioavailability in body.
ABSTRACT
Rotundine (1 micromol L(-1)) was incubated with a panel of rCYP enzymes (1A2, 2C9, 2C19, 2D6 and 3A4) in vitro. The remained parent drug in incubates was quantitatively analyzed by an Agilent LC-MS. CYP2C19, 3A4 and 2D6 were identified to be the isoenzymes involved in the metabolism of rotundine. The individual contributions of CYP2C19, 3A4 and 2D6 to the rotundine metabolism were assessed using the method of total normalized rate to be 31.46%, 60.37% and 8.17%, respectively. The metabolites of rotundine in incubates were screened with ESI-MS at selected ion mode, and were further identified using MS2 spectra and precise molecular mass obtained from an Agilent LC/Q-TOF-MSMS, as well as MS(n) spectra of LC-iTrap-MS(n). The predominant metabolic pathway of rotundine in rCYP incubates was O-demethylation. A total 5 metabolites were identified including 4 isomerides of mono demethylated rotundine and one di-demethylated metabolite. The results also showed that CYP2C19, 2D6 and 3A4 mediated O-demethylation of methoxyl groups at different positions of rotundine. Furthermore, the ESI-MS cleavage patterns of rotundine and its metabolites were explored by using LC/Q-TOF-MSMS and LC/iTrap-MS(n) techniques.
ABSTRACT
The inter-species differences of thienorphine metabolism were investigated in human, Beagle dog and rat liver microsomes, by comparing enzyme kinetics of the parent drug and the formation of its major metabolites. The incubation systems of thienorphine with liver microsomes of the three species were optimized in terms of thienorphine concentration, microsomal protein content and incubation time. The concentrations of thienorphine and its metabolites in incubates were measured by a LC-MS/MS method. The biotransformation of thienorphine by human liver microsomes was the lowest among the three species. The K(m), V(max), CL(int) and T1/2 of thienorphine obtained from human liver microsomes were (4.00 +/- 0.59) micromol x L(-1), (0.21 +/- 0.06) micromol x L(-1) x min(-1), (117 +/- 3.19) mL x min(-1) x kg(-1) and (223 +/- 6.10) min, respectively. The corresponding kinetic parameters for dog and rat liver microsomes were (3.57 +/- 0.69) and (3.28 +/- 0.50) micromol x L(-1), (0.18 +/- 0.04) and (0.14 +/- 0.04) micromol x L(-1) x min(-1), (213 +/- 1.06) and (527 +/- 7.79) mL x min(-1) x kg(-1), (244 +/- 1.21) and (70.7 +/- 1.05) min, respectively. A total of six phase I metabolites were observed in liver microsomes, including one N-dealkylated metabolite, three oxidative metabolites and two N-dealkylated oxidation metabolites. All these six metabolites were detected in the liver microsomes of the three species. However, the relative amounts of the metabolites generated were different in three species. The results indicated that the major phase I metabolic pathway of thienorphine was similar in the liver microsomes from all three species. However, the inter-species differences observed were relative amounts of the metabolites as well as the metabolic characteristics of thienorphine in liver microsomal incubates.