Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Fatemeh Akhlaghi

Abstract

Cardiovascular disorders are the leading cause of death in the United States. Members of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) class of lipid-lowering drugs are used worldwide for the prevention and treatment of cardiovascular disorders. Cardiovascular disorders are the primary cause of morbidity and mortality in patients with diabetes mellitus and obesity as well as renal and liver transplantation. The rising global burden of these chronic disorders has resulted in long-term use of statins to prevent and treat cardiovascular disorders in diverse patient populations. Several statins are commercially available; however, atorvastatin calcium (Lipitor®, Pfizer Pharmaceuticals, NY) is the world’s top selling medication of all time; whereas, rosuvastatin calcium (Crestor®, AstraZeneca, DE) is the most efficacious member of statin family.

Although, statins are well-tolerated, approximately 7% of patients on statin therapy experience myotoxicity, which is ranging from a mild condition called myalgia to a rare but potentially fatal rhabdomyolysis requiring hospitalization. A meta analysis study reported by the United States FDA indicated three times higher incidence of rhabdomyolysis in patients with diabetes mellitus. Previously published in vitro and clinical studies identified the role of lactone metabolites in myopathy. Our group found significantly elevated plasma concentrations of atorvastatin lactone metabolites in the stable kidney transplant recipients with diabetes mellitus. Our study indicated that reduced clearance of lactone could be attributed to decreased activity of cytochrome P450 (CYP) 3A4, which is the main drug metabolizing enzyme. Prior studies assessed the effect of genetic polymorphism in drug metabolizing enzymes and transporters on pharmacokinetics and toxicological properties of parent drug and lactone metabolite in healthy Finish and Korean populations.

Currently, limited information is available on the effect of concurrent diseases and genetic polymorphisms of drug metabolizing enzymes and transporters on pharmacokinetics of acid and lactone forms of atorvastatin. Altered pharmacokinetics of atorvastatin acid or lactone in concomitant diseases possibly influences the clinical outcome, resulting in unfavorable benefit/risk ratio. In this study, we have assessed the impact of inherent demographic characteristics in conjunction with coexisting diseases and genetic polymorphisms using a population pharmacokinetic analysis utilizing a nonlinear mixed effect model to identify potential covariates that explain the variability in pharmacokinetic properties of acid and lactone forms of atorvastatin.

A physiologically-based pharmacokinetic modeling approach was used for the prediction of pharmacokinetics of orally administered atorvastatin acid and rosuvastatin acid along with their major metabolites from in vitro data allowing mechanistic characterization of the observed concentration-time profile. A mechanistic modeling is needed to provide insights into the interplay of various phenomena involved in oral absorption and metabolism of drugs. Additionally, simulations through virtual patients using physiologically-based pharmacokinetic modeling will allow to design a population pharmacokinetic study and to determine significant covariates.

Manuscript I of this dissertation provides detailed information of pharmacokinetic and pharmacological properties of atorvastatin and rosuvastatin acid. Moreover, it describes the effect of various factors such as age, gender, race, food, liver and kidney diseases, time of drug administration, and genetic polymorphism of drug metabolizing enzymes and transporters on the pharmacokinetic properties of both statins. Atorvastatin acid is significantly metabolized by CYP3A4, and it is more likely to cause clinically important drug-drug interaction. Previously reported drug-drug interactions of atorvastatin are discussed in this manuscript. This manuscript will be submitted for publication to “Clinical Pharmacokinetics” as a review article.

Due to lack of simple and sensitive methods for simultaneous quantification of atorvastatin and its five metabolites in human plasma, a liquid chromatography tandem-mass spectrometry (LC-MS/MS) bioanalytical method was developed.

Manuscript II explains in detail about the development and validation of a sensitive, selective and simple LC-MS/MS assay for simultaneous quantitative determination of parent drug and its five metabolites (published in Anal Bioanal Chem. 2011 Apr;400(2):423-33).

Esterase activity is a key reason of instability of ester-containing drugs in biological matrices. The effect of several anticoagulants on lactone to acid interconversion was investigated by comparing different types of plasma (sodium heparin, K2EDTA and sodium fluoride/potassium oxalate) and serum. No, statistically significant difference was found between serum and plasma with various anticoagulants; however, sodium fluoride (esterase inhibitor) plasma was preferred to ensure stability of lactone upon long-term storage of clinical study samples. Comprehensive stability studies were conducted prior to the method validation to establish stability conditions for unstable lactone analytes during the extraction steps as well as storage duration of clinical samples. The method was validated according to recent FDA guidelines. The post-column infusion test was performed to assess matrix effect. Ortho- and para-hydroxy analytes have similar precursor ion-product ion transitions. Additionally, the acid form could possibly undergo in-source fragmentation and, following the loss of water, the resulting product would interfere with their respective lactone forms. For these combined reasons, chromatographic conditions that would assure baseline chromatographic separation of the respective analytes were determined. This goal was achieved using a narrow-bore Zorbax-SB phenyl column. Because this column provided excellent peak focusing a high S/N was obtained that enabled us to use a simple protein precipitation extraction procedure without performing pre-concentration steps for sample clean up. The extraction method contained a simple protein precipitation step requiring only 50 μL of plasma and achieved a lower limit of quantification of 50 pg/mL for all six analytes.

To the best of our knowledge, no published bioanalytical method has described a fully validated LC-MS/MS assay for the quantification of rosuvastatin lactone metabolite in human plasma. A sensitive and simple LC-MS/MS assay was developed for the simultaneous quantification of parent drug and its two metabolites in human plasma.

Manuscript III describes the development and validation of an LC-MS/MS method for the simultaneous quantification of rosuvastatin acid and its two metabolites; N-desmethyl rosuvastatin and rosuvastatin lactone (published in Anal Bioanal Chem. 2012 Jan; 402(3):1217-27).

Like atorvastatin lactone, rosuvastatin lactone is also very unstable. Stability of all the three analytes was tested in various conditions such as non-buffered human plasma and buffered human plasma (human plasma diluted 1:1 with 0.1 M, pH 4.0 sodium acetate buffer). For the proposed assay, plasma was diluted 1:1 with 0.1 M, pH 4.0 sodium acetate buffer due to prevent the loss of lactone form (25%) that occurred in non-buffered plasma after 1 month storage at -80 °C. Furthermore, to ensure stability of rosuvastatin lactone metabolite during extraction of samples, 0.1% v/v glacial acetic acid in methanol was used as precipitating agent to minimize the interconversion of lactone to acid and vice versa. With the use of narrow-bore Zorbax-SB Phenyl column, lower limit of quantification of 0.1 ng/mL for acid and lactone forms of rosuvastatin, and 0.5 ng/mL for N-desmethyl rosuvastatin acid were achieved using 50 μL of buffered human plasma.

The impact of concurrent chronic disorders and polymorphisms in genes coding for drug metabolizing enzymes and transporters involved in the parent drug and the major metabolite elimination were investigated through a population pharmacokinetic modeling approach using NONMEM software (Manuscript IV). The plasma concentrations of parent drug and metabolite of one hundred and thirty two, male (n=77) or female (n=55) non-transplant (diabetic, n=46; non-diabetic, n=53) or the stable kidney transplant (diabetic, n=22; non-diabetic, n=11) recipients receiving a single oral dose or multiple oral doses of Lipitor (atorvastatin calcium) were included in the study. The study samples were analyzed using an LC-MS/MS method described in Manuscript II.

A complex parent-metabolite combined population pharmacokinetic model was developed by analyzing a total of 639 concentrations including both acid (n=322) and lactone (n=317) forms of atorvastatin through a nonlinear mixed-effects modeling approach to identify and interpret the genetic, demographic, physiological and pathological factors that significantly affect the pharmacokinetic properties of atorvastatin acid and its major metabolite. Concentration-time profiles of atorvastatin acid and lactone metabolite were adequately described respectively, using a two-compartment model with first-order oral absorption and a one-compartment model with linear elimination, with some degree of interconversion between the two forms. Covariate model building was conducted to investigate and determine sources of variability (covariates) that elucidate differences in pharmacokinetic parameters between patients, through univariate analysis followed by stepwise forward addition and backward elimination. Covariate analysis identified the kidney transplantation status and lactate dehydrogenase (liver enzyme) as significant covariates affecting the apparent clearance of atorvastatin lactone metabolite. Renal transplant recipients had 50% lower metabolite clearance than non-transplant patients. However, polymorphisms in genes coding for enzymes and transporters as well as biomarkers of diabetes such as HbA1c and serum glucose levels did not significantly affect the pharmacokinetics of either parent drug or metabolite. The final model was validated using a visual predictive check method and nonparametric bootstrap analysis, which guaranteed robustness of the present population pharmacokinetic model. The finding of the study indicated the need of careful monitoring while prescribing atorvastatin treatment to the kidney transplant population. This manuscript will be submitted for publication to “Clinical Pharmacology and Therapeutics” as a research article.

Manuscript V (to be submitted to “Molecular Pharmaceutics”) presents a whole-body physiologically-based pharmacokinetic (PBPK) model for atorvastatin and rosuvastatin acid with their respective metabolites allowing a mechanistic characterization of observed plasma concentration-time profiles of parent drug and its metabolites. Plasma samples obtained from the stable kidney transplant recipients with diabetes mellitus were analyzed using LC-MS/MS methods described in Manuscripts II and III.

The GastroPlus (Simulations Plus, Inc., USA) advanced compartmental absorption and transit (ACAT) model, generic PBPK module and population estimates for age-related physiology feature were used to predict systemic exposure of both statins following an oral administration in stable kidney transplant recipients with diabetes mellitus. The required number of input parameters was obtained experimentally, in silico and from the literature. Atorvastatin acid undergoes extensive gut and hepatic metabolism mainly by CYP3A4. In vitro Km and Vmax values of metabolic clearance of atorvastatin acid previously determined in our laboratory using diabetic human liver microsomal fractions were implemented in the model. The model used a built-in utility for conversion of in vitro Km and Vmax values to in vivo values. To predict observed large volume of distribution of both statins, the Berezhkovskiy algorithm was utilized to determine tissue distribution of perfusion-limited tissues. The observed mean plasma concentration-time curves for both statins and their metabolites were adequately described by the proposed PBPK model. A parameter sensitivity analysis identified that systemic exposure of both statins is significantly affected by changes in intestinal transit time. Part of the validation process included virtual trial simulations, which allowed incorporation of inter-subject variability. The virtual trial simulation results showed that the observed mean plasma concentration-time curves of both statins lay between 90% confidence interval of simulated concentrations of ten virtual patients. The present whole-body PBPK model demonstrated that in vitro metabolic clearance data generated from a specific disease tissue were superior for adequate prediction of systemic exposure of an extensively metabolized drug that might have modified due to altered activity of drug metabolizing enzyme in a specific disease state.

In summary, the work presented in this dissertation evaluate the effect of preexisting disease states including diabetes mellitus and renal transplant as well as genetic polymorphisms in drug metabolizing enzymes and transporters that predominantly contribute to overall disposition of atorvastatin acid and its lactone metabolite. The study indicated that the clearance of lactone, a myotoxic metabolite of atorvastatin acid, is significantly decreased by 50% in the stable kidney transplant recipients. However, the unbalanced sample size in this study restricts to delineate the individual influence of renal transplant and diabetes mellitus on clearance of parent drug and its metabolite. In addition, for the first time the disposition of atorvastatin acid and rosuvastatin acid as well as their respective metabolites was characterized using a mechanistic modeling approach, to predict observed plasma concentration-time profiles in the kidney transplant recipients with diabetes mellitus. The PBPK model that integrated in vitro kinetic parameters for metabolic clearance of atorvastatin acid measured in human liver microsomal fractions of diabetic livers were superior for the prediction of observed plasma concentration-time curve of atorvastatin acid.

Overall, this study demonstrated a significant reduction of clearance of atorvastatin lactone metabolite in patients with the kidney transplant and thus they might be at higher risk of developing myotoxicity. This finding indicated the need of careful monitoring of atorvastatin acid therapy in the kidney transplant recipients who are on multiple medications and also have lifetime co-morbidities. Moreover, mechanistic modeling suggested that the systemic exposure of both statins is very sensitive to change in intestinal transit time. It would be beneficial to study the activity of intestinal drug metabolizing enzymes and transporters in diabetes mellitus and its impact on pharmacokinetics of statins.

Share

COinS