UPLC-MS/MS Analysis of Dextromethorphan-O-demethylation Kinetics in Rat Brain Microsomes

Formation of dextrorphan (DXT) from dextromethorphan (DXM) has been widely used to assess cytochrome P450 2D (CYP2D) activity. Additionally, the kinetics of CYP2D activity have been well characterized in the liver microsomes. However, studies in brain microsomes are limited due to the lower microsomal content and abundance of CYP2D in the brain relative to the liver. In the present study, we developed a micro-scale enzymatic incubation method, coupled with a sensitive UPLC-MS/MS assay for the quantitation of the rate of DXT formation from DXM in brain microsomes. Rat brain microsomes were incubated with different concentrations of DXM for various times. The reaction was stopped, and the proteins were precipitated by the addition of acetonitrile, containing internal standard (d3-DXT). After centrifugation, supernatant (2 μL) was injected onto a UPLC, C18 column with gradient elution. Analytes were quantitated using triple-quadrupole MS/MS with electrospray ionization in positive ion mode. The assay, which was validated for accuracy and precision in the linear range of 0.25 nM to 100 nM DXT, has a lower limit of quantitation of 0.125 fmol on the column. Using our optimized incubation and quantitation methods, we were able to reduce the incubation volume (25 μL), microsomal protein amount (5 μg), and incubation time (20 min), compared with reported methods. The method was successfully applied to estimation of the Michaelis-Menten (MM) kinetic parameters of dextromethorphan-O-demethylase activity in the rat brain microsomes (mean ± SD, n = 4), which showed a maximum velocity of 2.24 ± 0.42 pmol/min/mg and a MM constant of 282 ± 62 μM. It is concluded that by requiring far less biological material and time, our method represents a significant improvement over the existing techniques for investigation of CYP2D activity in rat brain microsomes

UPLC-MS/MS Analysis of the Michaelis-Menten Kinetics of CYP3A-mediated Midazolam 1′- and 4-hydroxylation in Rat Brain Microsomes

Midazolam (MDZ) is a short-acting benzodiazepine with rapid onset of action, which is metabolized by CYP3A isoenzymes to two hydroxylated metabolites, 1′-hydroxymidazolam and 4-hydroxymidazolam. The drug is also commonly used as a marker of CYP3A activity in the liver microsomes. However, the kinetics of CYP3A-mediated hydroxylation of MDZ in the brain, which contains much lower CYP content than the liver, have not been reported. In this study, UPLC-MS/MS and metabolic incubation methods were developed and validated for simultaneous measurement of low concentrations of both hydroxylated metabolites of MDZ in brain microsomes. Different concentrations of MDZ (1–500 µM) were incubated with rat brain microsomes (6.25 µg) and NADPH over a period of 10 min. After precipitation of the microsomal proteins with acetonitrile, which contained individual isotope-labeled internal standards for each metabolite, the analytes were separated on a C18 UPLC column and detected by a tandem mass spectrometer. Accurate quantitation of MDZ metabolism in the brain microsomes presented several challenges unique to this tissue, which were resolved. The optimized method showed validation results in accordance with the FDA acceptance criteria, with a linearity ranging from 1 to 100 nM and a lower limit of quantitation of 0.4 pg on the column for each of the two metabolites. The method was successfully used to determine the Michaelis-Menten (MM) kinetics of MDZ 1′- and 4-hydroxylase activities in rat brain microsomes (n = 5) for the first time. The 4-hydroxylated metabolite had 2.4 fold higher maximum velocity (p \u3c 0.01) and 1.9 fold higher (p \u3c 0.05) MM constant values than the 1′-hydroxylated metabolite. However, intrinsic clearance values of the two metabolites were similar. The optimized analytical and metabolic incubation methods reported here may be used to study the effects of various pathophysiological and pharmacological factors on the CYP3A-mediated metabolism of MDZ in the brain

A Comparison of Calcium Aggregation and Ultracentrifugation Methods for the Preparation of Rat Brain Microsomes for Drug Metabolism Studies

Preparation of brain microsomes by the calcium chloride aggregation method has been suggested as an alternative to the ultracentrifugation method. However, the effects of the calcium chloride concentration on the quality of the microsomal fractions are not known. Brain microsomes were prepared from the adult rat brains using the high-speed ultracentrifugation and low-speed calcium chloride (10–100 mM) aggregation methods (n = 5–6 per group). The microsomal protein yield (spectrometry), the cytochrome P450 reductase (CPR) activity (spectrometry), and the monooxygenase activities (UPLC-MS/MS) of CYP2D and CYP2E1 were determined in the obtained fractions. Increasing the concentrations of calcium chloride progressively increased the protein yield of the low-speed microsomal fractions. However, the increased yield was associated with a significant decrease in the activities of CPR, CYP2D, and CYP2E1. Additionally, the CYP2D and CYP2E1 activities were significantly correlated with the CPR activities of the fractions. In conclusion, when an ultracentrifuge is available, preparation of brain microsomes by the ultracentrifugation method might be preferable. However, the calcium aggregation method at a calcium chloride concentration of 10 mM is an acceptable alternative to the ultracentrifuge method

Kinetics of Dextromethorphan-O-Demethylase Activity and Distribution of CYP2D in Four Commonly-Used Subcellular Fractions of Rat Brain

The purpose of this study was to compare the enzymatic kinetics and distribution of cytochrome P450 2D (CYP2D) among different rat brain subcellular fractions. Rat brains were used to prepare total membrane, crude mitochondrial, purified mitochondrial, and microsomal fractions, in addition to total homogenate. Michaelis–Menten kinetics of the brain CYP2D activity was estimated based on the conversion of dextromethorphan (DXM) to dextrorphan using UPLC-MS/MS. Protein levels of CYP2D and subcellular markers were determined by Western blot. Microsomal CYP2D exhibited high affinity and low capacity, compared with the mitochondrial CYP2D that had a much lower (∼50-fold) affinity but a higher (∼six-fold) capacity. The apparent CYP2D affinity and capacity of the crude mitochondria were in between those of the microsomes and purified mitochondria. Additionally, the CYP2D activity in the whole homogenate was much higher than that in the total membranes at higher DXM concentrations. A CYP2D immune-reactive band in the brain mitochondria appeared at a lower MW but had a much higher intensity than that in the microsomes. Mitochondrial brain CYP2D has a much higher capacity than its microsomal counterpart. Additionally, brain homogenate is more representative of the overall CYP2D activity than the widely-used total membrane fraction

Effects of Chronic Cirrhosis Induced by Intraperitoneal Thioacetamide Injection on the Protein Content and Michaelis–Menten Kinetics of Cytochrome P450 Enzymes in the Rat Liver Microsomes

Chronic intraperitoneal injection of thioacetamide (TAA) in rats has been used as an animal model of human cirrhosis to study the effects of the disease on drug metabolism. However, TAA inhibits P450 enzymes directly and independently of cirrhosis. We investigated the effects of chronic cirrhosis in rats, induced by 10 weeks of intraperitoneal TAA, on the P450 enzymes after a 10-day washout period to eliminate TAA. Liver histology and serum biomarkers of hepatic function confirmed cirrhosis in all animals. Microsomal total P450 content, P450 reductase activity and ethoxycoumarin O-deethylase activity, a general marker of P450 activity, were significantly reduced by 30%–50% in cirrhotic animals. Additionally, the protein content and Michaelis–Menten kinetics of the activities of CYP2D, CYP2E1 and CYP3A were investigated. Whereas cirrhosis reduced the microsomal protein contents of CYP2D and CYP3A by 70% and 30%, respectively, the protein contents of CYP2E1 were not affected. However, the activities of all the tested isoenzymes were substantially lower in the cirrhotic livers. It is concluded that the TAA model of cirrhosis that incorporates a 10-day washout period after intraperitoneal injection of the chemical to rats produces isoenzyme-selective reductions in the P450 proteins or activities, which are independent of the direct inhibitory effects of TAA

\u3cem\u3eIn Vitro\u3c/em\u3e and \u3cem\u3eEx-Vivo\u3c/em\u3e Evaluation of Topical Formulations Designed to Minimize Transdermal Absorption of Vitamin K1

Topical application of Vitamin K1 has been demonstrated to effectively treat papulopustular skin rash, a serious and frequently encountered side effect of Epidermal Growth Factor Inhibitors (EGFRIs). Systemic absorption of vitamin K1 from skin and the resultant consequence of antagonizing EGFRIs anticancer effects jeopardizes the clinical acceptability of this rather effective treatment. The purpose of the present study was to rationally formulate and evaluate the release rate and transdermal absorption of a wide range of Vitamin K1 dermal preparations with a variety of physiochemical properties. A library of 33 formulations with were compounded and tested for Vitamin K1 permeation using hydrophobic membranes and porcine skin mounted in a Fran diffusion cells. Our results demonstrate the lowest diffusion for water-in-oil emulsions, which also demonstrated a negligible transdermal absorption. The statistical analysis showed a significant correlation between in vitro and ex vivo results. While viscosity did not have a significant impact on the diffusion or absorption of vitamin K1, an increase in the lipid content was correlated with an increase in transmembrane diffusion (not with transdermal absorption). Overall, formulation design significantly impacts the release rate and transdermal absorption of vitamin K1, and confirms the possibility of minimal systemic distribution of this vitamin for this specific purpose

Perinatal growth restriction decreases diuretic action of furosemide in adult rats

Perinatal growth restriction programs higher risk for chronic disease during adulthood via morphological and physiological changes in organ systems. Perinatal growth restriction is highly correlated with a decreased nephron number, altered renal function and subsequent hypertension. We hypothesize that such renal maladaptations result in altered pharmacologic patterns for life. Maternal protein restriction during gestation and lactation was used to induce perinatal growth restriction in the current study. The diuretic response of furosemide (2mg/kg single i.p dose) in perinatally growth restricted rats during adulthood was investigated. Diuresis, natriuresis and renal excretion of furosemide were significantly reduced relative to controls, indicative of decreased efficacy. While a modest 12% decrease in diuresis was observed in males, females experienced 26% reduction. It is important to note that the baseline urine output and natriuresis was similar between treatment groups. The in vitro renal and hepatic metabolism of furosemide, the in vivo urinary excretion of the metabolite, and the expression of renal drug transporters was unaltered. Creatinine clearance was significantly reduced by 15% and 19% in perinatally growth restricted male and female rats, respectively. Further evidence of renal insufficiency was suggested by decreased uric acid clearance. Renal protein expression of sodium-potassium-chloride cotransporter, a pharmacodynamic target, was unaltered. In summary, perinatal growth restriction could permanently imprint pharmacokinetic processes affecting drug response.Keywords: perinatal growth restriction, furosemide, pharmacokinetics, fetal programming, renal insufficiency, in utero growth restrictio

GLP-1-mediated delivery of tesaglitazar improves obesity and glucose metabolism in male mice

Dual agonists activating the peroxisome proliferator-activated receptors alpha and gamma (PPARɑ/ɣ) have beneficial effects on glucose and lipid metabolism in patients with type 2 diabetes, but their development was discontinued due to potential adverse effects. Here we report the design and preclinical evaluation of a molecule that covalently links the PPARɑ/ɣ dual-agonist tesaglitazar to a GLP-1 receptor agonist (GLP-1RA) to allow for GLP-1R-dependent cellular delivery of tesaglitazar. GLP-1RA/tesaglitazar does not differ from the pharmacokinetically matched GLP-1RA in GLP-1R signalling, but shows GLP-1R-dependent PPARɣ-retinoic acid receptor heterodimerization and enhanced improvements of body weight, food intake and glucose metabolism relative to the GLP-1RA or tesaglitazar alone in obese male mice. The conjugate fails to affect body weight and glucose metabolism in GLP-1R knockout mice and shows preserved effects in obese mice at subthreshold doses for the GLP-1RA and tesaglitazar. Liquid chromatography–mass spectrometry-based proteomics identified PPAR regulated proteins in the hypothalamus that are acutely upregulated by GLP-1RA/tesaglitazar. Our data show that GLP-1RA/tesaglitazar improves glucose control with superior efficacy to the GLP-1RA or tesaglitazar alone and suggest that this conjugate might hold therapeutic value to acutely treat hyperglycaemia and insulin resistance

Functional Activity of Renal Cytochrome P-450 1A1 Enzyme in Rats with Low Birth-Weight

To determine the effect of maternal low protein diet (LPD) administered during pregnancy on the status of renal cytochrome-P450 (CYP) enzymes in the offspring, pregnant rats were fed either a purified control diet (C76) or a low protein diet (L76) throughout pregnancy and lactation and offspring were weaned onto lab chow on postnatal day 28. The control diet contained 18% protein while the LPD contained 8% protein. One male and one female offspring were sacrificed and kidneys were collected and frozen from 28-day, 65-day, and 150-day old rats. Renal microsomes were prepared via differential centrifugation technique. For the current study, we examined CYP1A1 which is an important drug metabolizing enzyme involved in the generation of carcinogenic metabolites. Its main role is the oxidation of aromatic amines, polarizing such compounds so they may be readily metabolized and excreted from the body. CYP1A2 activity was measured via its ability to oxidize the substrate ethoxy-resorufin into the fluorescent product resorufin; activity was measured at 530/590 nm upon incubation of renal microsomes for 30 min. Preliminary analysis of day 28, 65, and 150 male and female rats suggests no alterations in CYP1A2 activity normalized to microsomal protein amount. However, upon normalization to total kidney mass, we found significant changes. In summary, renal CYP1A1 activity is permanently altered by early life nutritional insult. Honors: Thesis with Distinction Awar

Functional Activity of Renal Cytochrome P-450 1A1 Enzyme in Rats with Low Birth-Weight

To determine the effect of maternal low protein diet (LPD) administered during pregnancy on the status of renal cytochrome-P450 (CYP) enzymes in the offspring, pregnant rats were fed either a purified control diet (C76) or a low protein diet (L76) throughout pregnancy and lactation and offspring were weaned onto lab chow on postnatal day 28. The control diet contained 18% protein while the LPD contained 8% protein. One male and one female offspring were sacrificed and kidneys were collected and frozen from 28-day, 65-day, and 150-day old rats. Renal microsomes were prepared via differential centrifugation technique. For the current study, we examined CYP1A1 which is an important drug metabolizing enzyme involved in the generation of carcinogenic metabolites. Its main role is the oxidation of aromatic amines, polarizing such compounds so they may be readily metabolized and excreted from the body. CYP1A2 activity was measured via its ability to oxidize the substrate ethoxy-resorufin into the fluorescent product resorufin; activity was measured at 530/590 nm upon incubation of renal microsomes for 30 min. Preliminary analysis of day 28, 65, and 150 male and female rats suggests no alterations in CYP1A2 activity normalized to microsomal protein amount. However, upon normalization to total kidney mass, we found significant changes. In summary, renal CYP1A1 activity is permanently altered by early life nutritional insult. Honors: Thesis with Distinction Awar