This project is aimed to address a current real world problem in the field of pharmaceutics. It involves the assessment of the stability of Nalidixic acid when combined with a commercially used excipient known as Polyvinylpyrrolidone (PVP). It will look to investigate the presence or absence of excipient-drug interactions at various conditions over an eight week time period. A comprehensive analysis of the literature will be carried out to provide a strong basis of a concise and well planned laboratory investigation.
Both excipient and drug will be exposed to varying conditions, in order to establish whether PVP is a suitable excipient for further drug development. These studies have been found to be crucial in assessing long term drug stabilities and successful excipient screening.
Nalidixic Acid is white or sometimes pale yellow crystalline powder known to be practically insoluble in water. It has a molecular mass of 232.2 and a melting point at approximately 230°c. (British Pharmacopoeia, 2003) They come under the class of quinolones antibiotics and their general indication is for urinary tract infections. Branded under the US brand name of 'Neggram', it is primarily found effective against gram negative bacteria. (Rx List, 2007)
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Figure 1: Structure of Nalidixic AcidPovidone is the stable structure of Polyvinylpyrrolidone (PYP). Povidone is followed by a K-value which characterises their viscosity in solution. As it is a polymer it follows the molecular formula (C6H9NO)n and its corresponding molecular weight (111.1)n. Its general characteristics are that of a hygroscopic nature and freely soluble in water. (British Pharmacopoeia, 2003)
Polyvinylpyrrolidone (Povidone) is a commercially used excipient commonly used in pharmaceutical dosage forms. 'It is now one of the most versatile and widely used pharmaceutical auxiliaries' It possesses a number of desirable properties ideal for formulations such as excellent water solubility, good film forming abilities and good adhesive properties to name a few. This makes it a popular excipient with pharmaceutical companies mainly due to its many functions and numerous applications in the pharmaceutical industry. PVP is available in a number of molecular weights which is normally selected according to its intended purpose. (Buhler, V, 2005).
Although all molecular weights of povidone portray good stability at room temperature, previous research carried out by (P. Mura et al, 1995) shows PVP-30 has proven to be suitable in investigating for drug-excipient screening using DSC. The use of PVP-30 in their studies has proven to lead to a reliable set of results. The experiment carried out with similar stability studies therefore it would be advisable use previously successful excipients. For the study, PVP with K-value characteristics would be ideal as at high viscosities such as PVP-90, it has been known to be unsuitable for stability testing research. (Buhler, V, 2005).
Stability tests are of increasing importance as they give an indication upon the rate of chemical degradation. As formulation degradation is dependent on several factors; conditions ideally need to be accelerated in order to attain desired information about stability more quickly rather than using real time studies. Real time stability studies although are known to offer a more concise method of determining product stability, they have the disadvantage of being extremely time consuming particularly for relatively stable formulations (Mehta, A.C. 1995). Therefore, accelerated conditions are proven to be of more benefit and are shown to a common approach to modern day stability testing (Fitzpatrick, S et al. 2002). Any accelerated stability should be backed up with real time data to ensure assumptions are valid (Aulton, 1998).
Previous research has showed stability testing should be undertaken at conditions close to the drug storage conditions. Also, "product licence purposes, the actual long-term stability data under ambient conditions are required by the regulatory authorities." (Mehta, A.C, 1995) Normal drug storage temperatures for Nalidixic Acid are room temperature. Therefore, the study will be carried out at temperatures +/- 10°c of room temperature. ICH guidelines also recommend 'stress testing is likely to be carried out on a single batch of the drug substance. It should include the effect of temperatures (in 10Â°C increments (e.g., 50Â°C, 60Â°C, etc.)' This further enforces the decision to carry out the study at 10°c intervals (ICH. 2003)
High-performance liquid chromatography (HPLC) has been found most suitable for stability indicating assays due to its ability to separate and measure the drug from its corresponding degradation products and excipients. Furthermore, HPLC is more rapid to carry out compared to other methods such as gas chromatography. Unlike GC, HPLC has the advantage where it can be used for polar, non-volatile and thermally labile compounds whereas with GC if a sample were to be non-volatile or thermally labile, it is required to be derivatised as an attempt to increase its volatility and stability. In turn this can be far more time consuming. (Mehta, A.C, 1995)
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HPLC allows much flexibility in the choice of stationary and mobile phases allowing several ways for a compound to be chromotographed. Separation can be altered by changes to the pH, polarity or ionic strength of the mobile phase. Retention and selectivity can also be varied so that decomposition products formed can be eluted before the drug, allowing the determination of minor components with the benefit of maximum sensitivity. Moreover, HPLC is well-suited to automation, which gives the opportunity to a large number of samples to be processed with ease. (Mehta, A.C, 1995)
Previous research has indicated reverse-phase HPLC as a stability indicating analytical assay is a common sight. Reviewing their work it has become apparent that HPLC has in turn provided a reliable good set of results. Therefore it would be favourable to incorporate reverse phase HPLC in our study. ( L.A. Shervington et al. (2005) and (Kopelent-Frank, H et al. (1996)
Research has shown although HPLC is an excellent method of analysis. Since HPLC, further technological advances have lead to the discovery of supercritical fluid chromatography (SFC) and capillary electrophoresis (CE). They possess all the positive qualities required for drug stability studies such as:
Mild conditions of analysis
No preliminary volatilization of sample required,
Good sensitivity and reproducibility (Mehta, A.C, 1995)
Aims and Objectives
Determine whether Povidone is a suitable excipient for Nalidixic Acid
Investigate whether there is evidence of drug-excipient interactions in the presence of Nalidixic Acid and PVP
Determine whether the presence of PVP affects Nalidixic Acids degradation products and rate
Establish if HPLC is a suitable method of analysis for evidence of drug-excipient interactions
Intended methods of Investigation:
For this study, Nalidixic Acid will be combined with the excipient Polyvinylpyrrolidone and the trials will be undertaken in a solution phase. The only variable to be modified is temperature. The temperature will be investigated at three different temperatures over eight weeks at set time intervals. As mentioned earlier, reading around stability studies has indicated the following temperatures (25°C, 35°C and 45°C) would be adequate in providing a reliable set of results. Also, regulatory bodies request stability studies to be carried out at temperatures around drug storage temperatures. Prior to the initiation of the experiment, we have been told the drug and excipient are relatively stable at room temperature therefore although accelerated studies are being carried out preliminary heating experiments are not needed to be undertaken to get an indication of the temperatures to be used. Both excipient and drug will not be subject to preparation or subsequent conditioning within the commencement of the study.
Reverse-phase High Performance Liquid Chromatography assay has been chosen for the main of method of analysis of the data, where 6ml vials per run will be used for the HPLC columns. The equipment must be validated against The International Conference on Harmonization (ICH) of Technical Requirements for the Registration of Pharmaceuticals for Human Use to ensure the equipment is performing according to its specification (ICH Guideline Q1A(R2) 2003). The primary objective here is to look for evidence of possible drug-excipient interactions further investigating their compatibility and stability. There is a large amount of possibility that thermal analysis using Differential Scanning Calorimetry will be our secondary method of analysis to further consolidate our results but this will be purely dependant laboratory time constraints. The calibration data obtained will be shared with another individual undertaking a similar experiment but the tests and samples will be run independently. The exchange of experimental data is purely to provide further evidence for our research leading to a more reliable set of results for analysis.
The research data acquired through our study will be subject to constant analysis to ensure through and comprehensive analysis particularly at every time point. A key tool in the methodology for analysis will be through the use of the Arrhenius equation. For each corresponding temperature, a calibration curve will be constructed where we will be looking to calculate an rate constant. The manipulation of kinetic calculations will be used to calculate the rate of degradation of any for each of the samples. The data for the drug stability on its own without PVP will be provided and used as a reference to the results obtained from our experiment.
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As a secondary measure of data analysis, the research data will be subject to computer statistical analysis at each time point through a software known as SPSS. This statistical package will be consulted to perform statistical tests such as correlations and means, descriptive/biovariate statistics and linear modelling and regression. In turn, this allows the simple identification of trends and acknowledges any significance of the data. Furthermore, SPSS has the advantage of providing verification in the validity and reliability of the data collected. The objective of the data analysis is to attain a time point where degradation becomes insignificant. Furthermore, a secondary objective would be to investigate whether the presence of PVP affects Nalidixic Acids' stand alone stability.
In order to carry out a successful project, project management is a fundamental factor to aid a successful research study. Adhering to specific time constraints forms the basis for effective project delivery. Below this is represented through a project schedule known as a Gantt Chart illustrating a breakdown of the components of the study. Furthermore, a research diary will be used to track the progress of findings.
The Department Of Health (2003). British Pharmacopoeia. London: The stationary office. 1296.
Used as a reference to find properties of Nalidixic Acid and Povidone
Rx List. (2007). Negram. Available http://www.rxlist.com/neggram-drug.htm . Last [Accessed 06 Decemeber 2009]
Specifically consulted for information on Nalidixic Acid
Figure 1: Mayer, G. (2009). Antibiotics - protein synthesis, nucleic acid synthesis and metabolism. Available: http://pathmicro.med.sc.edu/mayer/antibiot.htm. [ Last accessed 8 December 2009]
Provided adequate background on Nalidixic Acid. The image of the structure of Nalidixic Acid was retrieved from this website.
Buhler, V. (2005). Soluble Povidone. In: Buhler, V Polyvinylpyrrolidone Excipients for Pharmaceuticals. Germany: Springer. p5-124
Excellent found very useful in acquiring background information on Povidone regarding its properties, structure and characteristics
Mura, P et al. (1995). Utilization of differential scanning calorimetry as a screening technique to determine the compatibility of ketoprofen with excipients. International Journal of Pharmaceutics. 119 (1), p71-79
Useful journal in researching how previous compatibility testing has been carried out and its success.
Mehta,A.C. (1995). Analytical Issues in the Chemical Stability Testing of Drugs in Solution. Analytical Proceedings Including Analytical Communications. 32 (1), P67-70.
Extremely useful journal on analytical methods had been used continually throughout study. Invaluable journal, most of the information on stability and HPLC was retrieved from this source
Fitzpatrick,s et al. (2002). Effect of moisture on Polyvinylpyrrolidone in accelerated stability testing. International Journal of Pharmaceutics. 246 (1), p143-151
Good information on the application of accelerated stability testing and helped determine what grade of PVP was the ideal one to use
Aulton, M.E (1998). Pharmaceutics: The Science of Dosage Form Design. Churchill Livingstone, Edinburgh, p. 124-128.
Very reputatable book provided a good basis and knowledge on the important considerations in formulation development
ICH. (2003). Q1A(R2): Revision of the Stability Guideline: Stability Testing of New Drug Substances and Products. Available: http://www.ich.org/LOB/media/MEDIA419.pdf [Last accessed 07 December 2009]
Consulted to verify the conditions of validation and the requirements for accelerated/stressed stability studies.
L.A. Shervington et al. (2005). The simultaneous separation and determination of five quinolone antibiotics using isocratic reversed-phase HPLC: Application to stability studies on an ofloxacin tablet formulation. Journal of Pharmaceutical and Biomedical Analysis. 39 (1), p769-775
Good source of information for the application of reverse-phase HPLC to stability testing.
Kopelent-Frank, H et al. (1996). Stability-indicating high-performance liquid chromatographic assay for the simultaneous determination of dixyrazine and chlorprothixene in intravenous admixtures. Journal of Chromatography A. 729 (1-2), p201-206
Basic source consulted to establish details of previous Hplc analysis
Raymond C et al (2006). Handbook of Pharmaceutical Excipients. 5th ed. London: Pharmaceutical Press. p611-615.
Proved to be a very good book on important information on the properties of Povidone, how it is used commercially and its corresponding ratio.