ABSTRACT
Rat CYP2D1 has been considered as an ortholog of human CYP2D6 To assess the role of CYP2D1 in physiologic processes and drug metabolism, a CYP2D1-null rat model was generated with a CRISPR/Cas9 method. Seven base pairs were deleted from exon 4 of CYP2D1 of Sprague-Dawley wild-type (WT) rats. The CYP2D1-null rats were viable and showed no abnormalities in general appearance and behavior. The metabolism of venlafaxine (VLF) was further studied in CYP2D1-null rats. The V max and intrinsic clearance of the liver microsomes in vitro from CYP2D1-null rats were decreased (by â¼46% and â¼57% in males and â¼47% and â¼58% in females, respectively), while the Michaelis constant was increased (by â¼24% in males and â¼25% in females) compared with WT rats. In the pharmacokinetic studies, compared with WT rats, VLF in CYP2D1-null rats had significantly lower apparent total clearance and apparent volume of distribution (decreased by â¼36% and â¼48% in males and â¼23% and â¼25% in females, respectively), significantly increased area under the curve (AUC) from the time of administration to the last time point, AUC from the start of administration to the theoretical extrapolation, and C max (increased by â¼64%, â¼59%, and â¼26% in males and â¼43%, â¼35%, and â¼15% in females, respectively). In addition, O-desmethyl venlafaxine formation was reduced as well in CYP2D1-null rats compared with that in WT rats. Rat depression models were developed with CYP2D1-null and WT rats by feeding them separately and exposing them to chronic mild stimulation. VLF showed better efficacy in the WT depression rats compared with that in the CYP2D1-null rats. In conclusion, a CYP2D1-null rat model was successfully generated, and CYP2D1 was found to play a certain role in the metabolism and efficacy of venlafaxine. SIGNIFICANCE STATEMENT: A novel CYP2D1-null rat model was generated using CRISPR/Cas9 technology, and it was found to be a valuable tool in the study of the in vivo function of human CYP2D6. Moreover, our data suggest that the reduced O-desmethyl venlafaxine formation was associated with a lower VLF efficacy in rats.
Subject(s)
Antidepressive Agents, Second-Generation/pharmacokinetics , Cytochrome P450 Family 2/genetics , Gene Knockout Techniques/methods , Liver/metabolism , Microsomes, Liver/metabolism , Venlafaxine Hydrochloride/pharmacokinetics , Animals , Antidepressive Agents, Second-Generation/therapeutic use , Body Weight/drug effects , Depression/drug therapy , Disease Models, Animal , Female , In Vitro Techniques , Liver/enzymology , Male , Metabolic Clearance Rate , Microsomes, Liver/enzymology , Motor Activity/drug effects , Rats , Rats, Sprague-Dawley , Venlafaxine Hydrochloride/therapeutic useABSTRACT
This study was carried out to investigate the effect of oral administration of Dendrobium huoshanense on the expressions and activities of hepatic microsomal cytochrome P450s in mice, and to provide a reference for the evaluation of drug-drug interactions between D. huoshanense and clinical drugs. The C57BL/6 mice were randomly divided into blank control group, D. huoshanense low dose group (crude drug 1.25 g·kg⻹), D. huoshanense high dose group (crude drug 7.5 g·kg⻹), and phenobarbital positive control group (0.08 g·kg⻹). Each group was intragastrically administered with drugs for 2 weeks. The mice were sacrificed and their liver microsomes were prepared. The expressions of major subtypes of P450 enzyme were determined by Western blot and the probe drugs were used to detect the enzyme activities of P450 subtypes with protein expression changes. Western blot analysis showed that the protein expressions of CYP1A1, CYP1A2 and CYP2B in liver tissues were up-regulated in D. huoshanense-treated group. In vitro enzyme activity tests showed that there were no significant difference in metabolism of 7-ethoxyresorufin (a probe drug for CYP1A1) and bupropion (a probe drug for CYP2B) between D. huoshanense group and control group. The metabolism of phenacetin (a probe drug for CYP1A2) showed a statistical difference in rate Vmax, and it was significantly increased by approximately 20% in D. huoshanense group as compared with the blank control group, and the clearance CLint in treated group was also increased by about 32%. Therefore, oral administration of D. huoshanense had no effects on the activities of most hepatic P450 enzymes in mice, with no drug-drug interaction related to the P450 enzyme system in most clinical drugs theoretically. However, oral administration of D. huoshanense may accelerate the metabolism of CYP1A2-catalyzed drugs, which needs to be considered in clinical practice.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Dendrobium/chemistry , Drugs, Chinese Herbal/pharmacology , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Animals , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP1A2/metabolism , Mice , Mice, Inbred C57BL , Random AllocationABSTRACT
CYP2C11 is involved in the metabolism of many drugs in rats. To assess the roles of CYP2C11 in physiology and drug metabolism, a CYP2C11-null rat model was generated using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9method. A 2-base pair insertion was added to exon 6 of CYP2C11 in Sprague-Dawley rats. CYP2C11 was not detected by western blotting in liver microsomes of CYP2C11-null rats. No off-target effects were found at 11 predicted sites of the knockout model. The CYP2C11-null rats were viable and had no obvious abnormalities, with the exception of reduced fertility. Puberty in CYP2C11-null rats appeared to be delayed by â¼20 days, and the average litter size fell by 43%. Tolbutamide was used as a probe in this drug metabolism study. In the liver microsomes of CYP2C11-null rats, the Vmax and intrinsicclearance values decreased by 22% and 47%, respectively, compared with those of wild-type rats. The Km values increased by 47% compared with that of wild types. However, our pharmacokinetics study showed no major differences in any parameters between the two strains, in both males and females. In conclusion, a CYP2C11-null rat model was successfully generated and is a valuable tool to study the in vivo function of CYP2C11.
Subject(s)
Aryl Hydrocarbon Hydroxylases/genetics , CRISPR-Cas Systems/genetics , Cytochrome P450 Family 2/genetics , Steroid 16-alpha-Hydroxylase/genetics , Animals , CRISPR-Cas Systems/drug effects , Female , Inactivation, Metabolic/physiology , Male , Microsomes, Liver/drug effects , Models, Animal , Rats , Rats, Sprague-Dawley , Tolbutamide/pharmacologyABSTRACT
This study aims to investigate the protective effect of Dendrobium huoshanense, D.officinale(Huoshan), D.officinale(Yunnan), D.moniliforme and D. henanense on CCl4-induced hepatic damage in mice. C57BL/6 mice were randomly divided into control group, model group, high-dose(7.5 gâ¢kg⻹) and low-dose (1.25 gâ¢kg⻹) groups of the five Dendrobium. Each group was intragastrically administered with drugs for 2 weeks. The control group was intraperitoneally injected with Olive oil solution, while the other groups were intraperitoneally given 0.5%CCl4combined with Olive oil solution 2 h later after the last administration. Subsequently, ALT and AST activities in serum, SOD activities and MDA contents in liver tissues were determined in all groups 16 h later after administration. The liver index was calculated, and hepatic histopathological examination was performed. The mRNA expressions of IL-1ß, IL-6 and TNF-α were analyzed by Real-time PCR. Compared with the CCl4 model group, the activities of ALT and AST in serum decreased significantly in the five different Dendrobium groups. Meanwhile, in liver tissues, the levels of MDA reduced obviously, while the SOD activities markedly increased. Furthermore, liver tissue damage induced by CCl4 was ameliorated according to the histopathological examination. IL-1ß, IL-6 and TNF-α mRNA expressions in D.huoshanense-treated liver tissues were significantly decreased. In conclusion, the five different Dendrobium groups showed hepatoprotective effects on CCl4-induced acute liver injury in mice. However, there were differences among Dendrobium of different types and origins. The protect effect of D.huoshanense is the most obvious, and the order of the protective effect of the other Dendrobium from high to low is D.officinale(Yunnan), D. officinale(Huoshan), D.henanense and D.moniliforme. The differences between the different types of Dendrobium might be related to their chemical components.