International standard drug development

Hundreds of national and international guidelines have been published, with the aim to standardize drug development on the basis of indication, duration and schedule of treatment of patients. However, it is the challenge and responsibility of the scientist to define finally the clinical profile of a drag candidate and to further profile it for a promising development up to approval. 

Too often, the analyst omits a very important step in method development. A standard should be selected which will assure that the desired separation is reproducible on a day-to-day, month-to-month basis. The internal standard (quantitative) discussed above is not satisfactory for this purpose because it is not one of the compounds that is a "potential" compound to be resolved. Consider, if you will, a situation wherein a method has been developed which will resolve a drug from a given metabolite. This metabolite is a product indicative of some toxic reaction within the body. Let us assume that the drug produces this reaction and, hence, the metabolite occurs only rarely in some people. Obviously, the internal standard (quantitative) must be resolved from the drug and the metabolite. However, because the toxic reaction occurs rarely, in most analyses, the metabolite will not be present. Could the analyst assume that because the retention volumes of the internal standard (quantitative), and the drug Standard, are about the same as they were the last time the analysis was done, that the metabolite would still be resolved, if present The answer is apparent The analyst must include a metabolite "standard" to reaffirm selectivity and sensitivity. 

In general, it is easier (and faster) to develop an HPLC-ESI-MS/MS method for multiple analytes with the matrix effects identified and under control as compared to IA (see below). Furthermore, selectivity and interference from matrix components and/or metabolites is less of a problem with an LC-MS/MS method compared to IA. With an appropriate internal standard (IS), LC-MS/MS methods are more precise than IA. Moreover, the LC-MS/MS calibration range is broader and can accommodate disproportionate concentration ranges of the drug compound and its metabolites. In contrast, most IA are geared to quantify only one analyte at a time because the method development of multiplex IA is complicated and often cannot be optimized for all analytes of interest. 

Foods and Veterinary Medicine The FDA s Center for Food Safety and Applied Nutrition (CFSAN) and Center for Veterinary Medicine (CVM) actively participate in the development of international standards by the Codex Alimentarius Commission (Codex). Codex is an international organization formed in 1962 to facilitate world trade in foods and to promote consumer protection. It is a subsidiary of two United Nations components, the Food and Agriculture Organization (FAO) and the WHO. Codex standards cover food commodity standards (similar to FDA standards of identity), food additives, food contaminants, and residues of veterinary drugs in food. FDA officials chair two Codex committees, the Food Hygiene Committee and the Residues of Veterinary Drugs in Foods Committee, and participate in many others. 

An isocratic HPLC method for screening plasma samples for sixteen different non-steroidal anti-inflammatory drugs (including etodolac) has been developed [29]. The extraction efficiency from plasma was 98%. Plasma samples (100-500 pL) were spiked with internal standard (benzoyl-4-phenyl)-2-butyric acid and 1 M HC1 and were extracted with diethyl ether. The organic phase was separated, evaporated, the dry residue reconstituted in mobile phase (acetonitrile-0.3% acetic acid-tetrahydrofuran, in a 36 63.1 0,9 v/v ratio), and injected on a reverse-phase ODS 300 x 3.9 mm i.d. column heated to 40°C. A flow rate of 1 mL/min was used, and UV detection at 254 nm was used for quantitation. The retention time of etodolac was 30.0 minutes. The assay was found to be linear over the range of 0.2 to 100 pg/mL, with a limit of detection of 0.1 pg/mL. The coefficients of variation for precision and reproducibility were 2.9% and 6.0%, respectively. Less than 1% variability for intra-day, and less than 5% for inter-day, in retention times was obtained. The effect of various factors, such as, different organic solvents for extraction, pH of mobile phase, proportion of acetonitrile and THF in mobile phase, column temperature, and different detection wavelengths on the extraction and separation of analytes was studied. 

A reverse-phase microbore HPLC method with photodiode-array detection and UV spectral library was developed for toxicological screening of various drugs in plasma including etodolac and its methyl ester [30]. Sample preparation involved addition of prazepam (internal standard) to 500 pL of plasma followed by addition of 30 pL of 1M sodium hydroxide and 5 mL of dichloromethane. After shaking the sample for 1 minute and centrifuging, the upper aqueous layer was discarded. The organic phase was evaporated and reconstituted with 50 pL methanol and 20 pL water ... 

When an appropriate internal standard could not be found, it is sometimes possible to develop a method without an internal standard [19], especially for usage in early drug discovery stage where less strict criteria could be used. Nevertheless, efforts should be made to minimize the variations in sample extraction, LC separation, and... 

LC-MS/MS assays typically rely on the use of an internal standard that mimics the performance of the analyte to improve the precision, reproducibility and reliability of the assay. An ideal internal standard candidate is a stable-isotope labeled ( stable labeled ) form of the drug. Because synthesizing stable labeled chemicals can be expensive and time-consuming, it is very common to use a chemically similar structural analog of the analyte(s) as the internal standard, especially during the early phases of drug development. 

An hplc assay was developed suitable for the analysis of enantiomers of ketoprofen (KT), a 2-arylpropionic acid nonsteroidal antiinflammatory drug (NSAID), in plasma and urine (59). Following the addition of racemic fenprofen as internal standard (IS), plasma containing the KT enantiomers and IS was extracted by liquid—liquid extraction at an acidic pH. After evaporation of the organic layer, the drug and IS were reconstituted in the mobile phase and injected onto the hplc column. The enantiomers were separated at ambient temperature on a commercially available 250 x 4.6 mm amylose carbamate-packed chiral column (chiral AD) with hexane—isopropyl alcohol—trifluoroacetic acid (80 19.9 0.1) as the mobile phase pumped at 1.0 mL/min. The enantiomers of KT were quantified by uv detection with the wavelength set at 254 nm. The assay allows direct quantitation of KT enantiomers in clinical studies in human plasma and urine after administration of therapeutic doses. 

The history of drug development, especially its preclinical aspects, has been one of irregular advances, often based on ad hoc means intended to detect recent clinical problems and adverse effects and commonly based on national expertise and practices. The result was a patchwork of overlapping and even conflicting but commonly mutually exclusive data requirements in different countries. Additional barriers to facilitating clinical development have been the various multiple national and local standards and guidance that often resulted in duplication, inefficiency, and delays. By common consent this internationally disharmonized state of drug development slowed and inhibited the development of new treatments for rare and common diseases and led to much waste of scarce and precious resources. 

Nakamura et al. [42] developed a simple and rapid semi-micro colunm HPLC method with UV detection for the simultaneous determination of lornoxicam and other oxicams in human blood samples. The drugs including isoxicam as an internal standard were extracted from buffered plasma samples (pH 3) with dichloromethane and the resulting extracts were subjected to HPLC analysis. The separation was performed with a Ci8 reversed-phase semi-micro column (25 cm x 1.5 mm, 5 pm) at 35 °C. The mobile phase used was a mixture of acetonitrile-0.1 M acetate buffer (pH 5)-methanol, and the detection wavelength was set at 365 nm. The drugs were separated within 30 min without interference by the blood components. The detection limits of lornoxicam were 6.4 ng/ml in serum and 9.3 ng/ml in plasma at a signal-to-noise ratio of 3. The method was applied to the determination of lornoxicam in the sera of the patients. 

Where it is possible to predict likely metabolites and a full interpretation of the parent drug spectrum has been made, the mass spectrometer can be used as a specific detection system in selected ion monitoring (SIM) mode (see Selected ion monitoring, p. 25). The early impetus provided by the development of mass fragmentography in the identification of chlorpromazine metabolites [64] has led to many further applications [65,66,68]. The extension of SIM to quantitative drug measurement with the incorporation of internal standards e.g. [88] has again opened up new possibilities in pharmacokinetic studies in clinical pharmacology, a topic discussed separately below. 

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