INTRODUCTIONDiabetes Mellitus is a group of metabolic disorder of carbohydrate metabolism in

which glucose is underutilized, producing Hyperglycemia. It is accompanied by the presence of glucose in urine. It may result from either diminish production of Insulin from β-cells of Islets of Langerhans of Pancreas or resistance to its effect but this is not always the case. D.M. manifesting itself by hyperglycemia, glucosuria, High protein breakdown & Ketosis. Long term sequence of D.M. causes many complications like Retinopathy, Neuropathy, Angiopathy, Nephropathy and Atherosclerosis. It is most common disease in developed and developing country. For e.g. Diabetes affects approximately 17 million people (about 8% of the population) in the United Status. Diabetes is the third leading cause of death in the United Status after heart disease & cancer. That’s why cure and treatment of diabetes is very important. Anti-diabetic drugs play an important role in the treatment of diabetes. Besides it, anti-diabetic drugs also have side effects (like hypoglycemia). Therefore, discovery and development of safer & effective anti-diabetic drug is a challenge for researchers. Discovery and development of new drug is followed by Clinical trials. Clinical Research is a systematic study for new drugs/test drug in human subjects to generate to data for verifying the clinical, pharmacological or adverse effect with the objective of determining safety & efficacy of the new drug. Clinisys Clinical Research Limited conducts bioavailability studies through its world- class facilities and Bioanalytical laboratory. Clinsys Clinical Research Limited conducts bioavailability studies through its world-class facilities and Bioanalytical laboratory. It offers Bioavailability, Bioequivalence, Pharmacokinetic studies as per ICH-GCP and USFDA guidelines. Clinsys is a full-service clinical research organization (CRO) that provides pharmaceutical, biotechnology and medical device companies with a broad range of clinical research services in support of Phase I-IV drug and device development, including project management, clinical monitoring, scientific and medical support, investigator and patient recruitment, site management, biostatistics, data management, drug safety, quality assurance, regulatory affairs and medical writing. The present study is a part of above setup and is oriented toward bioanalysis i.e. analysis of a drug in human plasma.

Once the promising molecule is identified in the lab, it is subjected to preclinical studies or animal studies, where different aspects of drug including its efficacy & toxicity are studied. Data obtained after the preclinical studies are submitted as an IND (Investigational New Drug) to the regulatory authorities for the permission to conduct human studies/clinical trials. Clinical trials are carried out into 4 phases in the human subjects.

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1. Phase I trial : - This is the first time the new drug is administered to a small number, around 20-80 healthy informed volunteers under the close supervision of a doctor. This is mainly targeted at identifying the safety, tolerability and the general mechanism of action of the drug in the humans.

2. Phase II trial : - In second phase, the medicine is administered to a group of approximately 50-300 informed patients to determine its effects and also to check for any unacceptable side effect.

3. Phase III trial : - The third phase deals with the trial on more than 1000 patients. This phase is usually multicentric and focuses on the effects on drugs in different ethnic groups, comparison with the standard drugs on the market and also study of the effect of the drug on different variants of the diseases. Then, an NDA (New Drug Application) is filed to the regulatory authority containing the study data regarding permission to market the drug or for a commercial license.

4. Phase IV trial : - Fourth phase is usually conducted after the after the launch of the drug on the market. However, clinical research does not stop here; it continues throughout the lifetime of the drug to include post marketing surveillance where a periodic ‘Progress report’ is submitted to the regulatory authorities once every 2 year after the drug is released into the market and also into pharmacovigilance where safety of marketed drugs, biologics or medical devices are monitored.

In planning a clinical trial, the investigator first identifies the drug to be tested i.e. test drug and compare two medications or drug, in which one is test drug and another is reference or generic drug. To design the clinical trials/studies, one or more pilot experiments are conducted to gain insights for design of the clinical trial to follow. The field of pharmaceutical bioanalysis concerns with the analysis of drugs in human body fluids.1 Bioanalytical methods is used for the quantitation of drugs and their metabolites in biological matrices. In today’s drug development environment, highly sensitive and selective methods are required to quantify drugs in matrices such as blood, plasma, serum or urine. Chromatographic methods [high-performance liquid chromatography (HPLC) or gas chromatography (GC)] are widely used for the bioanalysis of small molecules, with liquid chromatography coupled to triple quadrupole mass spectrometry (LC/MS/MS) being the single most commonly used technology. The process to develop/design a method for BA/BE studies is called Method development After developing a method with desired attributes, the method is validated to establish accurate, precise and reproducible data during study-sample analysis.2 Bioanalytical method validation in the pharmaceutical industry is influenced by regulations from the US Food and Drug Administration (FDA), the UK Medicines Control Agency (MCA) and similar bodies from Canada, Japan and other countries.3 Method validation is a process that demonstrates that the method will successfully meet or exceed the minimum standards recommended in the Food and Drug Administration (FDA) Guidance 1 for accuracy,

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precision, selectivity, sensitivity, reproducibility and stability. While obtaining these parameters, other parameters are also determined during validation e.g., extraction efficiency, calibration range, response function (linear or nonlinear and dilution integrity). The validation is performed using a control matrix spiked with the compounds to be quantified.

After the validation of developed of a developed method, perform the BA/BE studies i.e. measure in vivo bioavailability or demonstrating bioequivalence. (Bioavailability means the rate and extent to which the active ingredient or active moiety is absorbed from a drug product and becomes available at the site of action. While Bioequivalence means the absence of a significant difference in the rate and extent to which the active ingredient or active moiety in pharmaceutical equivalents or pharmaceutical alternatives i.e. test drug & reference drug becomes available at the site of drug action when administered at the same molar dose under similar conditions in an appropriately designed study). The present study involves solid phase extraction method for the first time for Drug Analyte along with LC-MS/MS determination. Cmax, Tmax & AUC are analyzed by the data generated in BA/BE study, by which determine the safety & efficacy of the drug.

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Drug discovery & lead compound

Biological activity testing

Chemical synthesis & scale up

testing

Formulation development

Safety testing in animal

IND filling

IND application

IND approval Clinical hold

Phase I trial

Phase II trial

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FDA Approval

Market

Phase IV clinical trial post approval marketing

FDA Disapproval

Phase III Clinical trial

NDA Filling