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
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
The objective of this study was to investigate the stability of edaravone in dog plasma by using an added antioxidant stabilizer, with an ultimate goal of developing and validating a sensitive, reliable and robust LC-MS-MS method for determination of edaravone in plasma samples. Edaravone was unstable in plasma, but it presented a good stability performance in the plasma with sodium metabisulfite (SMB), an effective antioxidant. The blood sample was collected in the heparinized eppendorf tube containing SMB and plasma sample was deproteinized using acetonitrile containing 20 ng/mL of phenacetin (Internal standard). The chromatographic separation was performed on a Zorbax Extend-C18 analytical column (2.1 mm × 150 mm I.D., particle size 3.5 µm, Agilent Technologies, USA). The mobile phase consisted of 0.1% formic acid in water (v/v) and methanol, and gradient elution was used. The analyte detection was performed on a triple quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization by multiple reaction ion monitoring mode of the transitions at m/z [M + H]+ 175.1 â 77.1 for edaravone, and m/z [M + H]+ 180.2 â 110.0 for phenacetin. The linearity of this method was within the concentration range of 10-1000 ng/mL for edaravone in dog plasma. The lower limit of quantification was 10 ng/mL. The relative standard deviations of intra- and inter-precision were <10%. This method was successfully employed in the pharmacokinetics evaluation of edaravone in beagle dogs after intravenous administration.
Subject(s)
Antioxidants/analysis , Antioxidants/pharmacokinetics , Antipyrine/analogs & derivatives , Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Animals , Antioxidants/chemistry , Antipyrine/blood , Antipyrine/chemistry , Antipyrine/pharmacokinetics , Dogs , Edaravone , Female , Linear Models , Male , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Methylmercury (MeHg) is an environmental neurotoxicant that is especially harmful during brain development. Previously, we found greater sensitivity to MeHg-induced oxidative stress and greater loss of mitochondrial membrane potential in synaptosomes from early postnatal rats than in synaptosomes from older rat pups and adults. Here, we determine whether MeHg exposure also leads to greater changes in dopamine (DA) levels and dopamine transporter (DAT) function in synaptosomes from early postnatal rats. We report that MeHg exposure leads to DAT inhibition, and increases the levels of released DA compared to control; further, the effects are much greater in synaptosomes prepared from postnatal day (PND) 7 rats than in synaptosomes from PND 14 or PND 21 animals. In addition to the effects of MeHg in young rats, we observed age-dependent differences in dopaminergic function in unexposed synaptosomes: synaptosomal DA levels increased with age, whereas medium (released) DA levels were high at PND 7 and were lower in PND 14 and PND 21 synaptosomes. DAT activity increased slightly from PND 7 to PND 14 and then increased more strongly to PND 21, suggesting that higher DA release, in addition to the lower DAT activity seen in PND 7 animals, was responsible for the age differences in levels of released DA. These results demonstrate that MeHg affects the dopaminergic system during early development; it thus may contribute to the neurobehavioral effects seen in MeHg-exposed children.