Lc Ms Ms In Pharmacokinetics And Metabolism Biology Essay

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Bioanalysis deals with quantitative estimation of active drug ingredients as well as, bio-transformed products in various body fluids. Several analytical techniques have been employed for this purpose. LC-MS/MS has become the technique of choice for pharmaceutical industry for quantitative bio analysis of drugs and active metabolites in bio fluids such as plasma, serum to support clinical development and bio equivalence of new chemical entity and generics drug respectively. LC-MS/MS has been preferred over HPLC, GC due to selectivity and specificity which in turn results in shorter run time of the assays. Shorter run times in turn, help in high throughput analysis of samples which is the need of hour to support the faster development of molecules through clinical phases. The introduction of automated sample preparation techniques of solid phase extraction, liquid-liquid extraction and protein precipitation have brought about high-throughput approach to bio-analysis.

Selective and sensitive analytical methods for the quantitative evaluation of drugs and their metabolites (analytes) are critical for the successful conduct of preclinical and/or biopharmaceutics and clinical pharmacology studies. Bioanalytical method validation includes all the procedures that demonstrate usefulness of a particular method used for quantitative measurement of analytes in a given biological matrix, such as blood, plasma, serum, or urine, in a reliable and reproducible manner for the intended use. The fundamental parameters for this validation include (1) accuracy, (2) precision, (3) selectivity, (4) sensitivity, (5) reproducibility, and (6) stability.

BIO-ANALYTICAL METHOD DEVLOPMENT, VALIDATION AND ITS APPLICATIONS

LC-MS based screening approaches are extensively used to evaluate the potential of drug-drug interactions/beneficial either in vitro or in vivo. It is very important to understand potential for adverse or beneficial drug interactions in order to adjust the dosages clinically. Hence rugged and robust analytical methods will help in monitoring the levels in circulatory ingredients. An attempt has been made here to develop and validate bioanalytical methods for various classes of drugs that have potential to interact with each other in clinical scenario. Validation as per international regulatory guidelines involved documenting, through the use of specific laboratory investigations, that the performance characteristics of the method are suitable and reliable for the intended analytical applications. The acceptability of analytical data corresponds directly to the criteria used to validate the method using USFDA guidelines for bioanalytical method validation. Apart from these studies an attempt also being made to develop the simultaneous method for multi probe substrate estimation which can be used for in-vitro metabolism studies.

In vitro metabolism studies are performed to obtain the early indication of inhibition or induction of metabolism of compounds by cytochrome P450 (CYP isozymes). The latter can be assessed by measuring the influence of the drug on cytochrome P450 enzyme system, which is responsible for metabolism of most of the drugs. With this understanding, it has become essential to make educated predictions for new drugs in regards to the other drugs with which they are likely, or not likely, to interact.

Knowledge of drug interactions with the liver microsomal mixed-function oxidase system is becoming increasingly important in the safety assessment of new compounds developed for clinical use. It is of particular importance during multidrug treatment, because the pharmacodynamics of co administered drugs may be dramatically altered by the influence of one drug on the metabolism of another. CYP isoform selective substrates have been identified and are being commonly used for probing the role of specific CYP isoforms in drug metabolism.

Chapter 1: Simultaneous determination of celecoxib, erlotinib and its metabolite desmethyl erlotinib (OSI-420) in rat plasma by liquid chromatography/tandem mass spectrometry with positive/negative ion-switching electrospray ionization

The epidermal growth factor receptor (EGFR) is recognized as an important molecular target in cancer therapy. Erlotinib (ERT) is an orally active and potent inhibitor of the EGFR tyrosine kinase (TKI) used in lung cancer and several other cancers. Celecoxib (CCB) is a selective cyclooxygenase-2 (COX-2) inhibitor used in inflammatory disorders. However COX-2 is an inducible enzyme that is over expressed in pancreatic cancer. Through the conversion of arachidonic acid to prostaglandin, the COX-2 enzyme modulates angiogenesis and metastasis. Current ongoing clinical trial utilizes erlotinib and celecoxib in non small cell lung cancer/ head and neck cancer. A fast, sensitive and specific LC-MS/MS method for the simultaneous determination of ERT and its active metabolite OSI-420 and CCB in rat plasma was developed, validated and applied successfully for preclinical pharmacokinetic study. After administration of ERT and CCB, blood samples were periodically collected from male wistar rats. The pharmacokinetics parameters of ERT, OSI-420 and CCB were calculated.

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Fig. 1. Structures of (a) Erlotinib, (b) Desmethly Erlotinib(OSI-420) and (c) Celecoxib

Summary

The calibration curve was linear over the concentration range of 1.6-1144.4 ng/mL for CCB and 1.8-1289.2 ng/mL for ERT and 1.5- 1117.2 ng/mL for component OSI-420. Intra and inter-day accuracy analyzed at low (3.8 ng/mL), medium (400 ng/mL) and high (900 ng/mL) quality control samples were >85%. Extraction recovery assessed at low and high quality control samples was >73% using convenient and Simple protein precipitation method. Stability assessment including three freeze-thaw cycle, bench top, auto-sampler stability and long term storage stability.The method was good enough to detect low concentration of 1.5 ng/mL for all these analytes in 50 µL plasma and further can be improved by increasing the plasma volume. Intra- and inter-day accuracy and precision of the validated method were within the acceptable limits of <15% and 85t o 115% respectively at low and <10% at other concentrations. No significant matrix effect found (ion enhancement as well as suppression). Incurred sample reanalysis (ISR) was performed to reconfirm the initial values and to demonstrate that the assay was reproducible. The method was successfully applied to generate stability profile as well as PK evaluation of simultaneous administration of Erlotinib and Celecoxib in rat following oral administration.

Chapter 2 Liquid chromatography-tandem mass spectrometry for the simultaneous quantitation of glipizide, cilostazol and its active metabolite 3, 4-dehydro-cilostazol in rat plasma: application for a pharmacokinetic study

Therapeutic drug management of antidiabetic agents (ADA) and antiplatelet drugs is an aid in the clinical management of diabetic patients with peripheral arterial diseases (PAD). Monitoring these drugs is desirable for adjusting doses, to provide safe, effective, avoiding side effects and assessing patient compliance. Among ADAs, Glipizide, a second generation sulphonyl urea derivate, stimulates the release of insulin from beta cells of the pancreatic islet tissues. It increases pancreatic insulin secretion by competitively binding to sulphonyl urea receptors (SUR), thereby inhibiting KATP channels and thus reducing blood glucose levels in diabetic patients. The insulinotropic response to a meal occurs within 30 minutes after an oral dose of glipizide (GLZ) in diabetic patients, but elevated insulin levels do not persist beyond the time of the meal challenge. Extra pancreatic effects may play a part in the mechanism of action of sulphonylureas. It is metabolized predominantly by CYP2C9 to its hydroxy metabolites and defective CYP2C9*3 allele making the exposure of glipizide drastically lower than other subjects.

Cilostazol is the most often used drug for anti-platelet therapy in diabetics along with other ADAs. Cilostazol (CLZ), a selective PDE-III enzyme inhibitor, showed antithrombotic, vasodilator, antimitogenic and cardiotonic properties used for the treatment of intermittent claudication in PAD. CYP3A4 and CYP2C19 are the major enzymes involved in metabolism of CLZ that were found during the in vitro metabolism studies using human liver microsomes. The major active in vivo metabolite found in rat and human after administration of CLZ was 3, 4-dehydro cilostazol (DCLZ) and the minor was 4'-trans-hydroxy-cilostazol along with the other inactive metabolites.

The possibilities of drug-drug interactions are more common in diabetics since they are commonly prescribed with anti-platelet agents along with ADAs. The prescriptions of using GLZ and CLZ in combination are expected in treatment of diabetics and there is no such information of drug-drug interaction effect available on these two drugs. During literature survey, we found several publications quoting the methods for the determination of either individual or simultaneous estimation of multiple analytes in biological fluids by reverse phase chromatography.

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Fig. 2. Structures of (a) Glipizide (b) Cilostazol and (c) 3, 4-dehydro cilostazol

Summary

The calibration curve was linear over the concentration range of 25-2500 ng/mL (average r2 > 0.993, n=4). The method was good enough to detect low concentration of 25 ng/mL for all these analytes in 50 µL plasma and further can be improved by increasing the plasma volume. Analytes recovery from spiked control samples were >76%, using convenient and fast LLE method. Intra- and inter-day accuracy and precision of the validated method were within the acceptable limits of <15% and 85t o 115% respectively at low and <10% at other concentrations. Stability assessment includes three freeze-thaw cycle, bench top, auto-sampler stability and long term storage stability. No significant matrix effect found (ion enhancement as well as suppression). Incurred sample reanalysis (ISR) was performed to reconfirm the initial values and to demonstrate that the assay was reproducible. The method was successfully applied to generate stability profile as well as PK evaluation of simultaneous administration of Glipizide, Cilostazol and DCLZ in rat following oral administration.

Chapter 3 Simultaneous determination of methotrexate, dasatinib and its active metabolite N- dsehydroxyethyl dasatinib in rat plasma by LC-MS/MS: Method validation and application to pharmacokinetic study

Dasatinib (DSN) is a multiple kinase inhibitor that potently inhibits the Bcr-Abl, Src family and platelet-derived growth factor receptor kinases. Dasatinib ( Sprycel®)novel protein tyrosine kinase inhibitor targeting SFK and Abl family kinases, was recently approved for use in the treatment of adults with chronic myeloid leukemia (CML) and resistance or intolerance to prior therapy. In addition to potently inhibiting the constitutively active Bcr-Abl kinase, dasatinib inhibits Lck at low picomolar concentrations.

Methotrexate (MTX) is an antimetabolite and antifolate drug. It is used in treatment of cancer, autoimmune diseases, ectopic pregnancy, and for the induction of medical abortions. It acts by inhibiting the metabolism of folic acid. Folic acid is needed for the de novo synthesis of the nucleoside thymidine, required for DNA synthesis. Also, folate is needed for purine base synthesis, so all purine synthesis will be inhibited. MTX therefore, inhibits the synthesis of DNA, RNA, thymidylates, and proteins.

Philadelphia chromosome/BCR-ABL-positive acute lymphoblastic leukemia (ALL) is the largest genetically defined subtype in adult ALL. Several strategies have been tested to optimize the combination of DSN and chemotherapy. The combination of dasatinib with a variety of cytotoxic chemotherapy regimens both in younger and elderly patients with de novo or minimally pretreated Philadelphia positive ALL was explored in recent clinical trials. DSN and MTX combination have advanced to phase II and phase III clinical program particularly in Hyper-CVAD and Dasatinib in Patients with Philadelphia Chromosome Positive and/or BCR-ABL Positive ALL. However, these methods showed limited extraction procedure, and required a relatively long analysis time to attain sufficient chromatographic separation. We developed a reverse phase HPLC method for simultaneous estimation of MTX, DSN and M-4 on C18 column using tandem mass spectroscopy detection and validated before using in our preclinical experiments. The current study describes a fast, sensitive, specific and simple liquid-liquid extraction method using LC-MS/MS for the simultaneous determination of MTX and DSN along with its major active metabolite M-4 in rat plasma suitable for pharmacokinetic and drug-drug interaction studies.

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Fig. 3. Structures of (a) Methotrexate, (b) Dastanib and (c) Deshydoxyethyl dasatinib

Summary

The calibration curve was linear over the concentration range of 1-1000 ng/mL (average r2³0.99, n=4). Intra and inter-day accuracy analyzed at low (3 ng/mL), medium (400 ng/mL) and high (800 ng/mL) quality control samples were >85%. Extraction recovery assessed at low and high quality control samples was > between79- 96% across the analytes and Fast LLE method. Stability assessment includes three freeze-thaw cycle, bench top, auto-sampler stability and long term storage stability. The method was good enough to detect low concentration of 1 ng/mL for all these analytes in 50 µL plasma and further can be improved by increasing the plasma volume. Intra- and inter-day accuracy and precision of the validated method were within the acceptable limits of <15% and 85 to 115% respectively at low and <10% at other concentrations. No significant matrix effect found ( ion enhancement as well as suppression). Incurred sample reanalysis (ISR) was performed to reconfirm the initial values and to demonstrate that the assay was reproducible. The method was successfully applied to generate stability profile as well as PK evaluation of simultaneous administration of dasatinib and methotrexate in rat following oral administration.

Chapter 4. Simultaneous quantitation of IC87114, roflumilast and its active metabolite roflumilast n-oxide in plasma by LC-MS/MS: application for a pharmacokinetic study

Chronic obstructive pulmonary disease (COPD) is a serious and mounting global public health problem. Although its pathogenesis is incompletely understood, chronic inflammation plays an important part and so new therapies with a novel anti-inflammatory mechanism of action may be of benefit in the treatment of COPD. Roflumilast (RFM), an oral, selective phosphodiesterase 4 inhibitor, has been shown to reduce exacerbations and improve pulmonary function in patients with COPD. This study examined the efficacy, safety and tolerability of roflumilast in Asian patients with COPD. RFM and its primary metabolite roflumilast N-oxide (RFN) are targeted phosphodiesterase-4 (PDE4) inhibitors with similar in vivo potency. Roflumilast is being developed for the treatment of inflammatory airway diseases such as chronic obstructive pulmonary disease and asthma.

IC87114 was described as a p110δ selective inhibitor that reduces fmlp-induced PIP3 synthesis and chemotaxis in neutrophils. PI3K pathway has been implicated in many diseases including cancer, thrombosis, rheumatoid arthritis and asthma, and that PI3Ks are the focus of intensive drug discovery efforts, with first-generation drugs now in early clinical trials. trafficking and recruitment of eosinophils during allergic airway inflammation is mediated by the PI3K family of signaling molecules. PI3K delta inhibitor IC87114 also blocked TNF1 alpha-stimulated elastase exocytosis from neutrophils in a mouse model of inflammation. Thus PI3K delta and PDE4 plays an essential role in certain signalling pathways of neutrophil activation and appears to be an attractive target for the development of an anti-inflammatory therapeutic action and it can produce synergism in the activity. It will benefit in the reduction of dose in the roflumilast. We developed a reverse phase HPLC method for simultaneous estimation of IC87114, RFM and RFN on C18 column using tandem mass spectroscopy detection and validated before using in our preclinical experiments. The current study describes a fast, sensitive, specific and simple liquid-liquid extraction method using LC-MS/MS for the simultaneous determination of IC87114 and RFM along with its major active metabolite RFN in rat plasma suitable for pharmacokinetic and drug-drug interaction studies.

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Fig. 4. Structures of (a) IC87114, (b) Roflumilast and (c) Roflumilast N-oxide

Summary

The calibration curve was linear over the concentration range of 6.41 ng/mL to 2980.7 ng/mL , 0.101 ng/mL to 60.3 ng/mL and 0.111 ng/mL to 62.7 ng/mL respectively IC87114, RFM and RFN (average r2³0.99, n=4). Intra and inter-day accuracy analyzed at 6.4,15.9, 1242.0,2483.9 ng/mL for IC87114, 0.101, 0.322, 25.1, 50.2 ng/mL for RFM and 0.111, 0.334, 26.1, 52.2 ng/mL for RFN as lloqc, low , medium and high, respectively as quality control samples were >85%. Extraction recovery assessed at low and high quality control samples was between79- 94% across the analytes using the fast LLE method. Stability assessment includes three freeze-thaw cycle, bench top, auto-sampler stability and long term storage stability. The method was good enough to detect low concentration 6.4 ng/mL for IC87114 and 0.1 ng/mL for RFM and RFN in 50 µL plasma and further can be improved by increasing the plasma volume. Intra- and inter-day accuracy and precision of the validated method were within the acceptable limits of <15% and 85to 115% respectively at low and <10% at other concentrations. No significant matrix effect found ( ion enhancement as well as suppression). Incurred sample reanalysis (ISR) was performed to reconfirm the initial values and to demonstrate that the assay was reproducible. The method was successfully applied to generate stability profile as well as PK evaluation of simultaneous administration of dasatinib and methotrexate in rat following oral administration.

Chapter 5. Simultaneous determination of metabolites from multiple cytochrome p450 probe substrates by gradient liquid chromatography with LC-MS/MS detection

The "drug metabolizing enzymes" (DMEs) are a diverse group of proteins that are responsible for metabolizing a vast array of xenobiotic compounds including drugs, environmental pollutants, and endogenous compounds such as steroids and prostaglandins. From an enzymological point-of-view, they are most noted for their broad substrate specificity; some members of the Cytochrome P450 (P450 or CYP450) and flavin monooxygenase (FMO) families are known to metabolize more than 50 structurally diverse compounds.

The human CYP2C subfamily contains four highly homologous genes, CYP2C8, 2C9, 2C18, and 2C19, located in an approximate 500-kbp cluster on chromosome 10q24. The CYP2C subfamily accounts for about 18% of the total adult liver cytochrome P450 content, the major form being CYP2C9 followed by CYP2C19 and CYP2C8. Low levels of CYP2C mRNA and protein have also been detected in the small intestine and other extrahepatic tissues. Transcriptional regulation of both CYP2C9 and 2C19 is known to involve the constitutive androstane receptor (CAR) and pregnane X receptor (PXR) as well as the glucocorticoid receptor (GR). Clinically relevant genetic variability within the CYP2C locus has also been well documented.

Evaluation of enzyme activity is traditionally performed by using a single CYP isoform at a time, which is labor-intensive, time consuming and cost effective. Attempt has been made for the development for simultaneous determination of multiple probe substrates for CYP isoform in a single LC-MS/MS run.

Summary

For CYP 2C9, Tolbutamide was used as probe substrate and we monitored the Hydroxy tolbutamide formation. For CYP 2C19, Mephytoin was used as probe substrate and we monitored the formation of metabolite hydroxy mephytoin. Following parameters were validated for the CYP2C9 & CYP2C19 inhibition, determination of Protein concentration required, determination of incubation time required, determination of substrate concentration required and calculation positive standards. Assay was successfully applied for screening the test compounds for CYP2C9 and CYP219 inhibition, simultaneously. The validity of the method were established using the positive standards viz. Fluconazole for CYP2C9 and Ticlopidine for CYP 2C19 and the same method can be applied for determining the inhibition of these isozymes by unknown test compounds.

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