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ABSTRACT

Drug kinetics explains the rate of change of drugs with time. Most drugs are chemically unstable and the result obtained from the study of chemical kinetics of such drug is used to predict the length of time within which the pure drug or formulation will remain therapeutically potent and effective at a specified temperature.

Efavirenz and sparfloxacin are classified in class II of the Biopharmaceutical Classification System, which means they have poor water solubility and are highly permeable.

This study was aimed at determining the influence of cosolvents and surfactants on the rate constant K, of efavirenz and sparfloxacin.

The kinetic study of the drugs was done by introducing 1ml of the drug solution (20µg/ml) into various percentage solutions of the cosolvent or surfactant as applicable.  The solutions were transferred into vials, and placed into a water bath at a temperature of 600C. At intervals of 30mins and up to 2hrs, samples were taken from the water bath and analyzed spectrophotometrically at a maximum wavelength of 304nm for sparfloxacin and 250nm for efavirenz. A plot of log percent drug concentration remaining of each drug versus time was carried out to obtain the rate constant K.

The log K of both drugs studied decreased considerably in the cosolvents and surfactants such as glycerol, propylene glycol, tween 80 and SLS employed in this study.

Altogether, the results suggests that glycerol, propylene glycol, polysorbate (tween) 80 and sodium lauryl sulphate (SLS) with 0.1M HCl and 0.1M NaOH  within the pharmaceutically acceptable non-toxic concentration can enhance the stability of sparfloxacin and efavirenz respectively when it is incorporated during formulation.

 

 

 

TABLE OF CONTENTS

Title page

Approval page

Dedication

Acknowledgement

Table of contents

List of figures

List of tables

Abstract

CHAPTER ONE

  • Introduction …………………………………………………………………………………………………………1

1.1. Stages of stability studies ………………………………………………………………………………………3

1.2. Relevance of stability studies …………………………………………………………………………………4

1.3. Methods of stability studies ……………………………………………………………………………………4

1.4. Factors influencing drug stability ……………………………………………………………………………8

1.5. Force degradation studies ………………………………………………………………………………………9

1.6. Objective of forced degradation studies……………………………………………………………………9

1.7. Selection of drug concentration……………………………………………………………………………..11

1.8. Degradation conditions………………………………………………………………………………………..11

1.9. Stability indicating method…………………………………………………………………………………..14

1.10. Drug Kinetics……………………………………………………………………………………………………14

1.11. Types of reaction order……………………………………………………………………………………….15

1.12. Surfactants ……………………………………………………………………………………………………….16

1.13. Classification of surfactants ……………………………………………………………………………….17

1.14. Pharmaceutical Application of surfactants…………………………………………………………….19

1.15. Cosolvents………………………………………………………………………………………………………..20

1.16. Biopharmaceutical classification system ………………………………………………………………21

1.17. Sparfloxacin……………………………………………………………………………………………………..23

1.18. Efavirenz………………………………………………………………………………………………………….26

1.19. Statement of purpose………………………………………………………………………………………….30

1.20. Aims and objective of study………………………………………………………………………………..30

 

CHAPTER TWO

2.0. Materials and methods ………………………………………………………………………………………..31

2.1. Materials…………………………………………………………………………………………………………….31

2.2. Apparatus…………………………………………………………………………………………………………..31

2.3. Instruments…………………………………………………………………………………………………………31

2.4. Preparation of calibration curves……………………………………………………………………………31

2.5. Preparation of 0.1M HCl and 0.1M NaOH solutions………………………………………………..32

2.6. Preparation of the different concentrations of the cosolvents…………………………………….32

2.7. Preparation of the different concentrations of the surfactants……………………………………32

2.8. Kinetic study………………………………………………………………………………………………………32

 

CHAPTER THREE

3.1. Calibration curve…………………………………………………………………………………………………34

3.2. Degradation kinetics…………………………………………………………………………………………….38

3.3. Determination of log K………………………………………………………………………………………..48

 

CHAPTER FOUR

4.1. Discussion………………………………………………………………………………………………………….54

4.2. Conclusion………………………………………………………………………………………………………….57

REFERENCES………………………………………………………………………………………………………..53

APPENDICES…………………………………………………………………………………………………………69

 

 

CHAPTER ONE

1.0 INTRODUCTION

Pharmaceutical product stability refers to the ability of such a product to retain its physical, microbiological, toxicological, chemical and preservative information specified on the container or closure system. (Kommanaboyina and Rhodes, 1999; Bajaj et al, 2012). It can also be defined as the time span within which a pharmaceutical product retains the same characteristics and properties it possessed at the time of packaging within specific limits throughout the period of its storage and usage. Stability test therefore assess how environmental factors affects the quality of a formulated product or drug substance, then the findings are used to predict the drug’s shelf life, determine appropriate storage requirements and recommend labelling instructions. Also for the regulatory approval of any formulation or drug, the results obtained during stability testing is a vital requirement. (Singh and Bakshi, 2000; Bajaj et al., 2012).

Conducting stability testing for pharmaceuticals involves complicated procedures which are expensive, time consuming and requires scientific expertise to ensure that quality, efficacy and safety is built into the drug formulation. The commercial and scientific success of a pharmaceutical product can be assured with the proper understanding of the process of drug development with the difficult and enormous procedures necessary to achieve a detailed development plan. Stability studies and pharmaceutical analysis are very crucial steps in the developmental stages of drugs as they are needed to determine and assure the quality, potency and purity of pharmaceutical ingredients as well as that of the products formulated. (Singh and Bakshi, 2000).

Stability testing can also be explained as a complicated process due to the fact that diverse number of factors influences the stability of pharmaceutical products. Stability of active ingredients, excipient and active ingredients interaction, the type of manufacturing process, type of dosage form, type of container or closure system used for packaging, light conditions such as heat and moisture encountered while shipping, handling and storage are some of the factors that influence stability. Also, degradation reactions such as reduction, oxidation, racemisation or hydrolysis which play significant roles in the stability of a pharmaceutical product also depend on other conditions such as pH, reactants concentration, catalyst, radiation etc., in addition to the nature of raw materials that was used and the period of time between the product manufacturing and final usage. Pharmaceutical products could also experience changes in clarity (for solutions), consistency, appearance, content uniformity, particle size and shape, moisture contents, pH and package integrity which can affect its stability. Those physical changes could be due to vibration, impact, abrasion and temperature changes such as freezing, thawing and shearing with chemical reactions like oxidation, solvolysis, racemisation, reduction etc which occur in pharmaceutical products that can lead to degradation product formation, loss of potency of active pharmaceutical ingredients (API), loss of excipient activity such as antimicrobial preservative action, antioxidants etc. (Carstensen et al., 2000). Microbiological changes such as microorganism’s growth in non sterile products and changes in the efficacy of the preservative can also affect pharmaceutical products stability. (Matthews, 1999; Bajaj et al., 2012).

It is very important to ensure that a pharmaceutical formulation remain stable throughout its shelf life in its packaging (Khalil, 2008; Naveed et al, 2016),  this is because changes in its physical, chemical, microbiological and therapeutic qualities in any constituent of the drug be it active or excipient will lead to its instability. (Yoshioka, 2000; Carstensen, 2000; Rockville, 2006; Naveed et al., 2016).

Therefore due to this fact, special considerations are given while designing and developing new dosage forms that both the active ingredients and excipient retain their properties and remain stable throughout the entire period of their storage. Such product should:

  • Should be composed of not less than 90% of its therapeutic quality.
  • Have at least 90% of the concentration stated.
  • Have preservatives added in effective concentration.
  • Not have visible change like discoloration, development of odours and

precipitation.

  • Not contain irritancy and toxicity.

The United States Pharmacopeia (USP), defined stability as “the ability of a product to retain its characteristics that it possessed during its manufacturing (physical, chemical, microbiological, therapeutic properties) within specified limits throughout its period of storage and use” (Attwood and Florence, 2008;  Naveed et al., 2016).

The ICH guidelines defined pharmaceutical stability testing as “ a systematic experiments conducted on pharmaceutical products to understand and provide evidence how the quality of a drug product varies under the influence of variety of environmental factors such as temperature, humidity, and light and to set re-test period for the drug or a shelf life for the drug product and recommend good storage condition” (Kim, 2009; Naveed et al., 2016)

1.1 Stages of stability studies

Stability studies are carried out at every stage of the drug’s life cycle from the early stages of product development to the late stage of follow up studies.

There are 6 different stages:

Stage1: Early stage- Stress and accelerated testing with drug substances.

Stage2: Stability on pre-formulation lotes/batches.

Stage3: Stress test done on scale up batches.

Stage4: Accelerated and long term testing for registration purposes.

Stage5: Enduring stability testing.

Stage6: Follow up studies.

  • Relevance of stability studies

The most important reason while stability testing is conducted is to ensure the welfare of the patient that is discomforted by the disease which the pharmaceutical product is designed for. Apart from the formation of toxic decomposition products from the degradation of the unstable product, loss of therapeutic efficacy up to a level of 85% of what was specified on the label can lead to therapy failure which could result in death, for example, the case of nitroglycerine tablets for cardiac arrest and angina. Consequently, the result from certain types of stability tests has become a legal requirement for the approval of a new product by regulatory agencies. Secondly, the protection of the manufacture’s prestige by ensuring that the product remains fit for use as it relates to all the relevant functional qualities throughout their stay in the market is very important. Stability studies done during the developing or marketing stage of products also help to provide a collection of data that may be useful in the selection of suitable formulations, excipient and packaging for a newly developed product, to predict its life span and conditions of storage, to justify the alleged shelf life for the registration report and to confirm that there has not been introduction of changes during the formulation or manufacturing process which can affect the product’s stability adversely. (Singh, 2000; Carstensen, 2000; Bajaj et al., 2012). Stability studies play an important role in the life span of a pharmaceutical product or formulation’s success. Apart from the physiochemical properties of a drug, stability studies also explain the efficacy and safety of the product throughout its storage. (Matthews, 1999; Singh, 2000; Reynolds et al, 2002; Alsante et al., 2003; Baertschi et al., 2011; Naveed et al., 2016).

1.3. Methods of stability testing

Testing the stability drug substances and products are performed routinely at different product development stages. During the early stages, accelerated stability testing (which are done at very high temperature/humidity) is used to find out the type of products it would degrade to after a long period of storage, conducting test under conditions that are less rigorous such as those proposed for shelf storage on long time basis, at temperatures that are slightly elevated are used to predict the shelf life and date of expiration of a product.

Stability testing of pharmaceuticals is done mainly to give satisfactory assurance that the products quality/fitness will remain at acceptable levels during the entire period they will be in the market for supply to patients and remains fit for consumption until the last unit of the product is used. (Kommanaboyina and Rhodes, 1999; Bajaj et al, 2012).

Based on the purpose for stability testing and the procedures followed, the following are the categories of the procedures for stability testing:

Real-Time stability testing

This type of stability testing is typically conducted over a longer interval of the examination phase so as to permit the degradation of a considerable product under suggested storage environment. The duration of the assessment depends on the stability of the product which should be sufficient enough to signify clearly that no significant degradation takes place and must allow one to differentiate degradation from inter-assay alteration. During the process of testing, information is collected at a suitable frequency such that a trend examination is able to tell the difference between instability from day-to-day uncertainty. The validity of data elucidation can be improved by including a single batch of reference material for which stability characteristics have already been established. Stability of the reference material also includes the stability of reagents as well as uniformity of the performance of the instrument to be used all through the period of the stability testing. Though, performance of the system, drift control and discontinuity as a result of changes in both reagents and instrumentation must be monitored. (Anderson and Scott, 1991; Bajaj et al., 2012).

 

Accelerated stability testing

Here, the product is subjected to stress at higher (warmer than ambient) temperatures and the quantity of heat necessary to cause product breakdown is determined. By so doing the product is subjected to a condition that hastens degradation. The resulting data is then proposed to forecast shelf life or used to contrast the relative stability of substitute formulations. This more often than not provides an early clue about the shelf life of the product and thus cutting short the development timetable. Aside temperature, other stress situations such as agitation, light, moisture, ph, gravity and package are applied during accelerated stability testing (Kommanaboyina and Rhodes, 1999; Bajaj et al, 2012). In accelerated stability testing the samples are made to undergo stress, cooled after stressing, and then analysed concurrently. Due to the short length of the analysis, the chance of instability in the measurement system is less when compared to the real-time stability testing. Furthermore, in accelerated stability testing, contrast is done between unstressed material and stressed product in the same assay and the stressed sample recovery is expressed as percent of unstressed sample recovery. For statistical reasons, the treatment in accelerated stability projections is recommended to be conducted at four different stress temperatures. However, for thermo labile and proteinaceous components, relatively precise stability projections are obtained when denaturing stress temperatures are avoided (Anderson and Scott, 1991; Bajaj et al., 2012) . Accelerated stability testing idea is based on the Arrhenius equation (1) and modified Arrhenius equation. (Connors  et al., 1973; Anderson and Scott, 1991; Bajaj et al., 2012).

………….. (1)

The equation describes the connection linking storage temperatures and degradation rate. Arrhenius equation can be used to project stability from the degradation rates observed at high temperatures for some degradation processes. Also the degradation rate at low temperatures may be projected from those observed at “stress” temperatures when the activation energy is known. (Connors and Amidon, 1973; Lachman, 1976; Bajaj et al., 2012).

Retained sample stability testing

Retained sample stability testing is a practice usually conducted for every product that is marketed especially when its stability data is requisite. In this type of testing, stability samples, for retained storage for at least a batch per year are selected. If more than 50 batches are marketed, it is recommended that stability samples from two batches should be taken. At the time of first introduction of the product in the market, the stability samples of every batch may be taken, which may be decreased to only 2% to 5% of marketed batches at a later stage. Here, the stability samples are tested at preset intervals i.e. If a product has shelf life of 5 years, it is conventional to test samples at 3, 6, 9, 12, 18, 24, 36, 48, and 60 months. This conventional process of obtaining stability figures on retained storage samples is known as constant interval method (Carstensen, 1993; Kommanaboyina and Rhodes, 1999; Bajaj et al., 2012). The modified way of stability testing is done by evaluation of market samples and it involves collecting samples that were previously in the market place and assessing their stability properties. This method of testing is intrinsically more practical as it challenges the product not just in the idealized retained sample storage conditions, but also in the real market place (Kommanaboyina and Rhodes, 1999; Bajaj et al., 2012).

Cyclic temperature stress testing

This type of stress testing is not an everyday testing process for marketed products. Here, repeated temperature stress tests are premeditated on understanding of the product so as to imitate probable conditions in market place storage. The duration of cycle commonly considered is usually 24 hours given that the earth has a diurnal rhythm of 24 hours, which the marketed pharmaceuticals are most probably to experience during storage. It is recommended that the minimum and maximum temperatures for the cyclic stress testing is to be selected on a product-by-product basis and taking into consideration factors like suggested storage temperatures for the product and particular physical and chemical degradation properties of the products. It is also suggested that the test should usually have 20 cycles (Kommanaboyina and Rhodes, 1999; Carstensen, 2000a; Bajaj et al., 2012).

1.4. Factors that influence drug stability

Moisture: Solid dosage forms that are water soluble will dissolve once it get in touch with any deposit of moisture, this leads to creating several physical and chemical changes in the dosage form that could lead to lose of its properties (Bakshi and Singh, 2002;  Naveed et al., 2016)

Excipients: The high water content of excipients such as starch and povidone can have an effect on the stability of products by escalating the water content of the formulation. Also, some chemical interactions that occur between the excipients and the drug can lead to decline in stability (Singh et al, 2013; Naveed et al., 2016)

Temperature: Temperature changes could sometimes have serious consequence on the stability of drugs. An increase in temperature often causes increase in hydrolysis rate of drugs. Arrhenius equation describes the effect of temperature on stability (Tollefson et al., 1998; Naveed et al., 2016)

pH: The effect of pH on the decomposition rate of drugs that undergo hydrolyzed in solution enormous. As a way of reducing this effect, drugs are formulated at the pH of their maximum stability using buffers (Marin and Barbas, 2004; Naveed et al., 2016).

Oxygen: Oxidation in some drugs is promoted by the presence of oxygen. Drugs with a higher rate of decomposition when exposed to oxygen are stabilized by replacing the oxygen in the storage container with nitrogen or carbon dioxide (Brümmer, 2011; Singh, 2012; Naveed et al., 2016).

Light: Some drugs that are photosensitive have their rate of decomposition increased when they are exposed to light. Their vulnerability can be tested by comparing its stability when exposed to light to that when stored in the dark. Drugs that are photosensitive should be stored in amber glass containers and should be kept in dark (Sanjay et al., 2012; Naveed et al., 2016).

 

1.5. Force degradation studies

Force degradation studies is a term used to describe the situation where a drug in bulk or product is subjected to accelerated stress conditions for two reasons: 1. When developing stability indicating methods specially when very little information is available about the degradation products and 2. To get information about the degradation pathways and degradations products that might affect it during storage (Lucas et al, 2004; Bouligand et al., 2005; Alsante et al., 2007; Naveed et al., 2016).

The studies also helps to facilitate pharmaceutical development, manufacturing, production and packaging since the drug product can be improved by the knowledge of its chemical behaviour (Naveed et al., 2014; Naveed et al., 2014; Naveed et al, 2014; Naveed et al., 2016).

 

1.6. Objective of forced degradation studies

Forced degradation studies are carried out to achieve the following:

  • To establish/interpret possible pathways for degradation of drug substances and products.
  • To differentiate the degradation products that is related to drug products from those that are generated from non-drug product in a formulation.
  • To identify degradation products that may be generated during real time stability and elucidate the structure of the degradation products.
  • To develop a method to quantify degradation impurities and separate excipient peaks (placebo) form impurities.
  • To develop/validate a stability indicating method and establish the nature of a developed method.
  • To determine the intrinsic stability of a drug substance in formulation.
  • To generate more stable formulations.
  • To reveal the degradation mechanisms such as hydrolysis, oxidation, thermolysis or photolysis of the drug substance and drug product.
  • To understand the chemical properties of drug molecules.
  • To produce a degradation profile similar to that of what would be observed in a formal stability study under ICH conditions.
  • To solve stability-related problems. (Reynolds et al, 2002; Brummer, 2011; Blessy et al, 2014; Sharma and Murugesan, 2017).

The chemical stability of pharmaceutical molecules is a very important matter because it affects the safety and efficacy of the drug product. The FDA and ICH guidance states the requirement of stability testing data to understand how the quality of a drug substance and drug product changes with time under the influence of various environmental factors.

For the selection of proper formulation and packaging as well as providing appropriate storage situation and shelf life, an understanding of the stability of the molecule is important, it is also necessary for regulatory certification. Forced degradation as a term explains the degradation of drug products substances at more severe circumstances than that experienced in accelerated conditions which thus generates degradation products that can be studied to decide the stability of the molecule. The ICH guideline states that the intention of stress testing is to make out the possible degradation products which further helps in the establishment of the inherent stability of the molecule and degradation pathways, and to authenticate the stability indicating procedures used (Blessy et al., 2014). But these rules are universal in carrying out forced degradation but they do not give facts about the realistic approach towards stress testing. Even though forced degradation studies are a regulatory prerequisite and scientific obligation during drug development, it is not considered as a must for formal stability program. (Blessy et al., 2014)

1.7. Selection of drug concentration

It has not been specified in regulatory guidance the concentration of drug to be used for degradation study but it is suggested that the studies should be began at a concentration of 1mg/mL (Singh and Bakshi, 2000; Blessy et al., 2014). By using the drug concentration of 1 mg/mL, it is more often than not likely to obtain even negligible decomposition products in the range of detection. It is recommended that some degradation studies should also be done at a concentration which the drug is expected to be present in the final formulations (Bakshi and Singh, 2002; Blessy et al., 2014). The reason for proposing this is the examples of aminopenicillins and aminocephalosporins where a range of polymeric products have been found to be formed in commercial preparations containing drug in high concentrations (Larsen and Bundgaard, 1978; Blessy et al., 2014).

 

1.8. Degradation conditions

Hydrolytic conditions

Hydrolysis is one of the commonest chemical degradation reactions that take place over a broad range of pH. It is a chemical procedure that involves breakdown of a chemical compound by reaction with water. Hydrolytic study under acidic and basic state involves catalysis of ionizable functional groups present in the molecule. Acid or base stress testing involves forced degradation of a drug substance by exposure to acidic or basic conditions which generates primary degradants in desirable range. The choice of the type and concentrations of acid or base depends on the stability of the drug substance. Hydrochloric acid or sulfuric acids (0.1–1M) for acid hydrolysis and sodium hydroxide or potassium hydroxide (0.1–1M) for base hydrolysis are suggested as suitable reagents for hydrolysis (Singh and Bakshi, 2000; Alsante et al., 2007; Blessy et al., 2014) . When conducting stress test for compounds that have poor water solubility, they can be dissolved with cosolvents in HCl and NaOH. The structure of the drug substance forms the basis for the choice of cosolvent. Stress testing test is normally initiated at room temperature and if no degradation takes place, high temperature (50–700C) is applied. Stress testing should not go beyond 7 days. To avoid additional decomposition, the degraded sample is neutralized by the use of a appropriate acid, base or buffer.

Oxidation conditions

In forced degradation studies, oxidation of drug substances are done using oxidizing agents such as metal ions, oxygen, and radical initiators (e.g., azobisisobutyronitrile, AIBN) but hydrogen peroxide is the most extensively used. Selection of an oxidizing agent, its concentration and conditions depends on the drug substance. It is reported that subjecting the solutions to 0.1–3% hydrogen per- oxide at neutral pH and room temperature for seven days or up to a maximum 20% degradation could potentially produce pertinent degradation products (Alsante et al., 2007). The oxidative degradation of drug substance involves an electron transfer mechanism to form reactive anions and cations. Amines, sulfides and phenols are susceptible to electron transfer oxidation to give N-oxides, hydroxylamine, sulfones and sulfoxide (Gupta et al., 2011). The functional group with labile hydrogen like benzylic carbon, allylic carbon, and tertiary carbon or α-positions with respect to hetro atom is susceptible to oxidation to form hydro peroxides, hydroxide or ketone (Boccardi, 2005; Alsante et al, 2003; Blessy et al., 2014).

Photolytic conditions

The photo stability testing of drug substances is carried out to show that an exposure to light does not result in intolerable change. This study is performed to create main degradation products of a drug substance by exposure to UV or fluorescent environment. Some suggested conditions for photo stability testing are described in ICH guidelines. Samples of drug substance and solid/liquid drug product should be exposed to at least 1.2 million lx h and 200Wh/m2 light. The usually accepted wavelength of light is in the range of 300–800 nm to cause the photolytic degradation (Allwood and Plane 1986; Baertschi and Thatcher, 2006). The limit of light exposure suggested is 6 million lx h (Alsante et al, 2003). Light stress circumstances can induce photo oxidation by free radical mechanism. Functional groups like carbonyls, nitro aromatic, N-oxide, alkenes, aryl chlorides, weak C–H and O–H bonds, sulfides and polyenes are likely to introduce drug photosensitivity (Ahuja and Scypinski, 2001; Blessy et al., 2014).

Thermal conditions

Thermal degradation (caused by exposure to heat either dry or wet) have to be conducted at more strenous conditions than those recommended in ICH Q1A accelerated testing conditions. Samples of solid-state drug substances and products should be exposed to dry and wet heat, while liquid drug products should be exposed to dry heat. Studies may be conducted at higher temperatures for a shorter period (Alsante et al, 2007). Arrhenius equation below explains the effect of temperature on thermal degradation of a substance.

K=Ae-Ea/RT

Where k = specific reaction rate, A = frequency factor, Ea = activation energy, R = gas constant (1.987 cal/deg mole) and T = absolute temperature (Alsante et al, 2003; Qiu and Norwood, 2007; Trabelsi et al, 2005). Thermal degradation study is conducted out at 40–800C (Blessy et al., 2014).

1.9. Stability indicating method

A stability indicating method (SIM) is a method of analysis that is used for quantifying the decline in the amount of active pharmaceutical ingredient (API) in drug product as result of degradation. An FDA guidance document defines stability-indicating method as a validated quantitative analytical process that can be used to investigate the stability of the drug substances and products with changes in time. It accurately measures the changes in the concentration of active ingredients without interfering with other products of degradation, impurities and excipient. Stress testing is carried out to show the specificity of the developed method to measure the changes in concentration of drug substance when little knowledge is available about possible degradation product. The development of an appropriate stability indicating method provides a backdrop for the pre-formulation studies, stability studies and the development of suitable storage requirements. Bakshi and Singh (Bakshi and Singh, 2002) discussed some significant issues about developing stability indicating methods. Dolan (Dolan, 2002) made comments and suggestions on stability indicating assays. Smela (Smela, 2005) discussed from a regulatory point of view about stability indicating analytical methods. The reverse phase high performance liquid chromatography (RP-HPLC) is a most extensively used analytical tool for separation and quantifying the impurities and it is most often coupled with a UV detector (Trabelsi et al., 2005). The following are the steps involved for development of SIM on HPLC which meets the regulatory requirements. (Blessy et al., 2014).

 

 

1.10. Drug Kinetics

Drug kinetics explains the rate of change of drugs with time. Most drugs are chemically unstable and the result obtained from the study of chemical kinetics of such drug is used to predict the length of time within which the pure drug or formulation will remain therapeutically potent and effective at a specified temperature.

1.11. Orders of reactions

(a)Zero order reaction

This is the type of reaction where the concentration of reactants does not affect the rate of the reaction, meaning there is no change in the concentration or the amount of change is negligible. For example; fading of dyes.

 (b) First order reaction                                  

This is the commonest pharmaceutical reactions. It includes reactions such as absorption of drug and degradation of drug. The rate of change of the reaction is proportional to drug concentration i.e. drug concentration is not constant.

Special Case

Apparent zero order of reaction

For drugs in aqueous suspensions, more drug dissolve to maintain drug concentration as the dissolved drug decomposes, that means that the drug concentration is kept constant and once all the undissolved drug is dissolved, the rate of the reaction becomes first order.

Another special case: Pseudo 1st order:

This occurs where two components, one of which is changing appreciably from its initial concentration and the other is present in excess that it is considered constant or nearly constant. Note: In first order reactions, neither K or nor t1/2 is dependent on concentration.

(c) 2nd Order reaction

This occurs when two components in a reaction are reacting with each other or one component reacting with itself in a reaction. (https://www.scribd.com/doc/76640703/Drug-stability-and-kinetics)

 

1.12. Surfactants

Surfactants are referred to as surface-active agents, they are known to adsorb at 2 interfaces: the oil and water interfaces. As a result of the properties of both ends of the molecule, their methods of adsorption are well defined. The hydrophobic end is oil loving while the hydrophilic end is water loving. Surfactants are said to form oriented, stabilizing films. (Manisha et al., 2009).

Mode of Action

Surfactants may act in three diverse methods:

  1. Roll-up mechanism
  2. Emulsification of oil
  3. Solubilisation

(a) Roll-up mechanism: in the roll-up mechanism, the surfactant lowers the interfacial tension between the oil in solution and the fabric in solution, through this means the stain is lifted off the fabric by it.

(b)Emulsification: Here the interfacial tension of the oil’s solution is lowered by the surfactant making it easy for the oil to be emulsified.

(c)Solubilisation: Here substances dissolve spontaneously to form clear and stable solution as a result of the interaction between them and the micelles of a surfactant in a solvent (water).

Surfactants can also be known as foam formers and wetting agents. They are used for preparing emulsion as well as to remove stains and dirt from fabrics. When they are dissolved, they lower the surface tension of the medium by decreasing the interfacial tension between two interfaces or media such as air and water, water and stain and stain and fabric. Surfactants also play an important role in entrapping oil phase. In the cleaning of dirt and grease from dirty dishes, clothes and other surfaces, surfactants lowers the surface tension of the water and makes it easier to lift oil from it thereby helping to remove the oily dirt or grease suspended in the water thus forming emulsion. The hydrophilic or water-loving head remains in the water while it pulls the oil towards the water. (Manisha et al., 2009)

Surfactants play an important role in different drug deliveries. When formulating compounds that are sparingly water-soluble, surfactants and cosolvents are employed at pharmaceutically acceptable levels to enhance solubility.

Surfactants are amphipathic organic compounds that contain both hydrophilic groups (“heads”) and hydrophobic groups (“tails”), so they are soluble in both water and organic solvents. Surfactants are composed of both polar and non-polar regions. Their molecules are formed by two parts with different affinities for the solvents. One of them has affinity for oil (non-polar solvents) and the other for water (polar solvents). A little quantity of surfactant molecules rests upon the water-air interface and decreases the water surface tension value (the force per unit area needed to make available surface). Upon mixing of surfactant, water and oil, the surfactant rests at the water-oil interface. Depending on the stability of these systems they are called emulsions or micro emulsions (thermodynamically stable). Although, the properties of emulsions and micro emulsions are different, they both obey the same principle. They try to form enough interface for preventing the polar non-polar solvent contact. (Manisha et al., 2009).

In pharmaceutical sciences, surfactants are used as wetting agents, emulsifiers, solubilizers amongst others. Most surfactants are mainly derived from petroleum although some may be gotten from natural fats or sugars. (Manisha et al., 2009).

1.13 Classification of surfactants:

Surfactant can be classified according to the charged groups present in their head. A surfactant that does not have any charge group over its head is called a non-ionic surfactant. If it carries a negative charge, it is called an anionic surfactant while it is a cationic surfactant if it carries a positive charge.  If the head of the surfactant carries two oppositely charged groups, it is termed a zwitterion. (Manisha et al., 2009)

(a)Anionic surfactants

In solution, the head of this class of surfactant carry a negative charge. This class of surfactants are the most extensively used surfactant for preparing shampoos due to their outstanding cleaning quality and high hair conditioning property. They are especially effectual in oily cleaning and oil/clay suspension. The reaction between the positively charged water hardness ions (calcium and magnesium) in wash water can result to partial deactivation. As the calcium and magnesium molecules in the water increases, the more the anionic surfactant system suffers from deactivation. To avoid this, the anionic surfactants require help from other ingredients such as builders (Ca/Mg sequestrants) and also more detergent dosed in the hard water. Anionic surfactants that are most frequently used are alkyl sulphates, alkyl ethoxylate sulphates and soaps. They mostly include carboxylate, sulfate and sulfonate ions. (Remington, 1995; Manisha et al., 2009). The straight chain of surfactants is either a saturated or unsaturated C12 to C18 aliphatic group. The presence of double bonds determine their water solubility potential. (Zagrafti, 1995; Manisha et al., 2009).

(b) Cationic Surfactants: in this case the head of this type of surfactant carry a positive charge in solution. They are quaternary ammonium compounds which are chiefly used for their antiseptic and preservative property for the reason that they have high-quality bactericidal properties. They are used on the skin for cleaning wounds or burns. Predominantly used cationic surfactants are cetrimide which has tetradecyl trimethyl ammonium bromide with least amount of dodecyl and hexadecyl compounds. (Carter, 2008) Others are benzalkonium chloride, cetylpyridinium chloride etc. (Manisha et al., 2009).

(c) Non-Ionic Surfactants: Non-ionic surfactant carries no electrical charge on their head; this makes them opposed to water hardness deactivation. They are less irritating when compared to other surfactants. Their hydrophilic part contains the polyoxyethylene, polyoxypropylene or polyol derivatives, while their hydrophobic part consists of saturated or unsaturated fatty acids or fatty alcohols. They are brilliant grease/oil removers and emulsifiers. Non-ionic surfactants contribute to making the surfactant system less hardness responsive. The non ionic surfactant can be classified as polyol esters, polyoxyethylene esters, and poloxamers. The polyol esters comprise glycol and glycerol esters and sorbitan derivatives. Polyoxyethylene esters consist of polyethylene glycol (PEG 40, PEG -50, PEG- 55).  The most frequently used non-ionic surfactants are ethers of fatty alcohols. (Manisha et al., 2009)

(d) Amphoteric/Zwitterionic Surfactants: These class surfactants are very mild; this makes them mainly appropriate for use in individual care preparations over sensitive skins. They can be cationic (positively charged), anionic (negatively charged) or non-ionic (no charge) in solution, depending on the pH of the water. These surfactants can contain two charged groups with different signs. The positive charge is almost all the time ammonium but the source of the negative charge may vary (carboxylate, sulphate, sulphonate). These surfactants have very good dermatological properties. They are usually used in shampoos and other cosmetic products as well as in hand dishwashing liquids owing to their high foaming properties. (Manisha et al., 2009).

1.14. Pharmaceutical uses of surfactants

  • As percutaneous absorption enhancers

The use of certain adjuvant known as enhancers can increase the transport of molecules through the skin. Transdermal absorption is enhanced by ionic surfactants through the disordering of the lipid layer of the stratum corneum and the denaturing of keratin. Enhancers can boost drug penetration by causing the stratum corneum to swell up and maybe leak out some of the structural components, thereby decreasing the diffusional resistance and increasing the permeability of the skin. (Choi et al, 2009; Manisha et al., 2009).

  • As agents of flocculation

To retard the sedimentation of floccules, a suspending agent is frequently added. Such agents include carboxy methyl cellulose, tragacanth, veegum, carbopol 934, or bentonite. They are either employed alone or in combination. This may lead to incompatibilities, depending on the initial particle charge and the charge carried by the flocculating agent and the suspending agent. Positively charged particles are flocculated by addition of an anionic electrolyte such as monobasic potassium phosphate. (Li et al., 2009).

  • In mouth washes

Mouthwashes are aqueous solutions mostly found in concentrated form containing one or more active ingredients or excipient. They are used by swirling the liquid in the mouth. Mouthwashes can be used for two purposes: therapeutic and cosmetic. Therapeutic mouth rinses or washes are formulated to reduce gingivitis, plaque, dental caries, and stomatitis while cosmetic mouthwashes are formulated to decrease bad breath by the use of antimicrobial and/or flavouring agents in the formulation. Surfactants are used because they help to solubilise flavours and remove debris by their foaming action. (Reshad et al., 2009; Manisha et al., 2009).

  • In respiratory distress therapy, surfactants find use
  • As suppository bases
  • For influencing drug absorption
  • They also aid in transdermal penetration of drugs. (Manisha et al., 2009).

 

1.15. Cosolvents

Cosolvents are water-miscible organic solvents that are used in the formulation of liquid drugs to increase the solubility of substances with poor water solubility and to increase the chemical stability of a drug. (Rubino, 2013). Cosolvency (mixing a permissible non-toxic organic solvent with water) is the most common and feasible technique to enhance the aqueous solubility of drugs. The common cosolvents utilized are glycerol, ethanol, polyethylene glycols (mainly 200, 300 and 400), polyethylene glycol ether, propylene glycol and tetrahydrofurfuryl alcohol.

1.16. Biopharmaceutical classification system

Biopharmaceutical Classification System is a scientific schematic proposed by Amidon and his co-workers in 1995, which classifies drugs based on their solubility and intestinal permeability parameters into four classes (Amidon et al, 1995, Arrunategui et al., 2015). It is a scientific scheme that divides drugs according to their solubility and permeability and has been used by various guides as a criterion for bio waiver. (Arrunategui et al., 2015).

The four possible categories for a drug according to the BCS are in Table 1.

Table 1. The Biopharmaceutical Classification System scientific framework

Class Solubility Permeability
I High High
II Low High
III High Low
IV Low Low

(Arrunategui et al., 2015).

Insufficient bioavailability is often caused by poor solubility and low dissolution rate of drugs that have poor water solubility in aqueous gastrointestinal fluids. Drugs in class II with low solubility and high permeability according to BCS classification may have their bioavailability enhanced by increasing the solubility and dissolution rate of the drug in gastrointestinal fluids. As for drugs classified in class II, their rate limiting step is the drug release from the dosage form and solubility in the gastric fluid and not their absorption, so their bioavailability can be increased by increasing the solubility. (Sharma et al., 2009; Yellela, 2010; Kumar et al., 2011; Savjani et al., 2012).

Compounds with low solubility has negative effects such as: poor absorption and bioavailability, burden shifted to patient in form of frequent high-dose administration, insufficient solubility for IV dosing, development challenges leading to increase in the development cost and time.  (Edward and Li, 2008; Savjani  et al., 2012).

 

 

1.17. SPARFLOXACIN

Sparfloxacin, chemically known as 5-amino-1-cyclopropyl-7-(cis-3, 5-dimethyl-1- piperazinyl)-6,8-difluoro-1, 4-dihydro-4-oxo-3-quinolinecarboxylic acid is a difluoroquinolone and an antibacterial agent belonging to the third generation quinolones. It is clinically used in the treatment of streptococci infections. It is insoluble in water. (Mbah and Ozuo, 2011).

Pharmacological properties

About 37-45% of Sparfloxacin is bound to proteins in the blood. (Shimada et al., 1993; Psaty, 2008).

Apart from the central nervous system, the rate of penetration of Sparfloxacin into most tissues is high.

After a single oral dose of 400 mg sparfloxacin, the mean peak concentration in cantharides-induced inflammatory fluid is 1.3 lA-g per ml after a mean duration of 5 h post-dose. Therefore, overall sparfloxacin penetration into inflammatory fluid is 117% and the mean elimination half-life from this fluid is 19.7 h. (Montay, 1996).

Sparfloxacin penetrates the skin well having skin plasma ratios of 1.00 at 4 h (time of peak plasma concentration) and 1.39 at 5 h. Following single oral doses of 100 or 200 mg, concentrations in skin of 0.56 and 0.82-1.31 lA-g per g, respectively, can be expected. (Nogita and Ishibashi, 1991). Sparfloxacin excellently penetrates into human polymorphonuclear white blood cells in vitro. (García et al., 1992).

Sparfloxacin achieves high concentrations in sinus and respiratory tissues. Following an oral loading dose of 400 mg followed by 200 mg daily, mean concentrations of sparfloxacin (2.5 to 5 h after dosing) in bronchial mucosa, epithelial lining fluid and alveolar macrophages are 4.4 µg/g, 15.0 µg/ml and 53.7 µg/g, respectively. The mean sparfloxacin concentration in maxillary sinus mucosa, 2-5 h after a single 400 mg dose, is 5.8 µg/g. (Shimada et al. 1993;

The summary of most of the researches published in Japanese concerning the distribution of sparfloxacin in the tissues has been reported. (Wise and Honeybourne, 1996)

High concentrations are achieved in sputum, pleural fluid, skin, lung, prostate, gynaecological tissues, breast milk and otolaryngological tissues. Salivary concentrations are 66-70% of plasma levels, while CSF penetration appears to be sort of limited with CSF plasma concentration ratios of only 0.25-0.35.

Sparfloxacin achieves concentrations in bile and gallbladder of 7.1- to 83-fold the concurrent serum levels.

In rabbits, sparfloxacin achieves very good penetration into the ocular vitreous (54%), cornea (76%) and lens (36%). (Cochereau et al., 1993).

Medical uses

Sparfloxacin is used for the treatment of treat community-acquired lower respiratory tract infections such as acute sinusitis, exacerbations of chronic bronchitis  which is caused by susceptible bacteria and community-acquired pneumonia. (Rubinstein, 1996; Goa et al., 1997; Stein and Havlichek, 1997; Zhanel et al., 2002).

Mechanism of action

A review was conducted with 2081 patients of adult age participating in a Phase III clinical trial of sparfloxacin in lower respiratory tract and community-acquired infections, sparfloxacin (200- or 400 mg loading dose then 100 or 200 mg daily; i.e. 200/100 mg and 400/200 mg) had a similar incidence of adverse events as the comparator agents (Rubinstein, 1996). The overall rates of drug-related adverse reactions for sparfloxacin 400/200 mg versus comparators and 200/100 mg versus the comparator (amoxicillin/clavulanic acid) were 13.7 versus 17.7%, and 9.5 versus 13.2%, respectively. However, many of these reported reactions were very minor; discontinuation of the antibacterial agent because of drug-related adverse reactions occurred in 1.6 versus 1.6%, and 1) versus 1.1%, respectively. Some adverse reactions that affect the nervous system was reported in 5.7% of the sparfloxacin group, the most common being insomnia and other disorders in sleep.

2.0% of the recipients of sparfloxacin recorded photo toxicity, and the average delay in onset being 6.3: t 4.5 days (range 1–14 days) after commencing sparfloxacin. The phototoxicity incidence that accompanies sparfloxacin seems to be lower than what was reported for enoxacin, fleroxacin, nalidixic acid and pefloxacin but higher than what was observed with ofloxacin and ciprofloxacin.

Most importantly, features of the haemolytic-uremic syndrome such as that associated with temafloxacin have not been reported. (Rubinstein, 1996).

 

 

Structure of sparfloxacin

 

 

 

1.18. EFAVIRENZ

Efavirenz is a non nucleoside reverse transcriptase inhibitor which is used commonly in the therapeutic procedures for the treatment of patients with HIV (Barbaro et al., 2005). It is a crystalline lipophilic solid with aqueous solubility of 9.0μg/ml. (Bindu et al., 2011).

Chemically, efavirenz is known as (S)-6-chloro-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one. It has an empirical formula of C14H9ClF3NOwith a molecular mass of 315.68 g/mol. Efavirenz is a crystalline powder that could be white or slightly pink. It has a very low solubility in water.

Efavirenz gained approval from FDA on September 21, 1998, then it became the 14th approved drug for retroviral infections.

The generic tablet produced by Mylan was approved by FDA On February 17, 2016.

Medical uses

The United States Department of Health and Human Services Panel on Antiretroviral Guidelines encourages that efavirenz should be administered in combination with tenofovir/emtricitabine (Truvada) as it is among the preferred NNRTI-based regimens for adults, adolescents and children especially for HIV patients that has not previously received treatment.

Efavirenz is also combined with other antiretroviral as part of a spread out post-exposure prophylaxis plan to decrease the risk of HIV infection in vulnerable people who are exposed to risks like needle stick injuries, certain types of unprotected sex, etc. (Kuhar et al., 2013).

Pregnancy and breastfeeding

During pregnancy, efavirenz is safe for use in the first trimester. (Ford et al., 2014). Breastfed babies may experience exposure to efavirenz as it passes into the mother’s breast milk. (Waitt et al., 2015).

 

Contraindications

People who have experienced allergic reactions on previous intake of efavirenz should avoid taking further doses. Intolerance reactions include Steven-Johnson syndrome, toxic skin eruptions, and erythema multiforme. (FDA, 2016).

Adverse effects

A usual adverse effect of efavirenz is neuropsychiatric effects with symptoms like disturbed sleep (including nightmares, insomnia, disrupted sleep, and daytime fatigue), dizziness, headaches, vertigo, blurred vision, anxiety, and cognitive impairment (including fatigue, confusion, and memory and concentration problems), and depression, including suicidal thinking. (Treisman and Soudry, 2016; Apostolova et al., 2015). Some other patients experience euphoria, which leads to abuse and diversion of the drug. (Treisman and Soudry, 2016).

Rash and nausea may occur. (FDA, 2016).

The intake of efavirenz has given wrong positive results in some urine test for marijuana. (Rossi et al., 2006; Röder et al., 2007)

People with or at risk of Torsades de Pointes should avoid the use of efavirenz as it may lengthen the QT interval. (Abdelhady et al., 2016).

It is also advisable to avoid its usage in adult and paediatric populations with a history of seizures as it could cause convulsions. (FDA, 2016).

Drug interactions

Enzymes like CYP2B6 and CYP3A4 that are known to belong to the cytochrome P450 system and they breakdown efavirenz in the liver. However, efavirenz can reduce the metabolism of other drugs due to the fact that they need the same enzymes and efavirenze is a substrate of the enzymes. (FDA, 2016). Also, those enzymes are also induced by efavirenz thereby enhancing the activity of the enzyme and it can also enhance the metabolism of other drugs that are broken down by CYP2B6 and CYP3A4. (FDA, 2016). Therefore, patients taking efavirenz alongside other drugs that are metabolised by the same enzymes may have to increase or decrease their dose of those drugs.

Protease inhibitors, used for the treatment of HIV/AIDS are one of the groups of drugs affected by efavirenz. The blood levels of most of the protease inhibitors such as indinavir, atazanavir and aprenavir are lowerd by efavirenz. (FDA, 2016). At such reduced levels protease inhibitors may become ineffective for persons taking them together, which imply that the virus causing HIV/AIDS will continue replicating and could even start showing resistance to protease inhibitor. Antifungal drugs that are used to treat fungal infections such as urinary tract infections are also affected by efavirenz. Just like the effect of efavirenz on protease inhibitors, the blood levels of antifungal drugs like ketoconazole, voriconazole, posaconazole and itraconazole is lowered by efavirenz. (FDA, 2016). Due to the lowered blood levels, persons taking both drugs may find antifungal drugs ineffective which may lead to resistance of the fungi causing the infection to the antifungal.

Mechanism of action

Anti-HIV effects

Efavirenz belongs to the group of antiretrovirals called non nucleoside reverse transcriptase inhibitors (NNRTI). Nucleoside and non-nucleoside RTIs inhibit the same target, which is the reverse transcriptase enzyme and it is a vital enzyme that transcribes viral RNA to DNA. In contrast with nucleoside RTIs that bind at the active site of the enzyme, NNRTIs acts when they bind to a distinct site different from the active site called the NNRTI pocket.

Efavirenz is ineffective for the treatment of HIV-2, this is because the pocket of the reverse transcriptase for HIV-2 has a different structure, and this confers inherent resistance of HIV-2 to the NNRTI class of antiretrovirals. (Ren et al., 2002).

Since most of the NNRTI”s bind inside the same pocket, strains of virus that show resistance to efavirenz also do same to other NNRTI such as delavirdine and nevirapine. K103N is the commonest mutation that is observed after treatment with efavirenz and it is also observed when other NNRTI are used. (FDA, 2016). The binding target of nucleoside RTI and that of efavirenz are different, so it is unlikely to experience cross resistance, this also true about protease inhibitor and efavirenz.

Neuropsychiatric effects

The mechanism of the neuropsychiatric adverse effect of efavirenz was not understood as at 2016. (Treisman and Soudry, 2016; Apostolova et al., 2015).

Efavirenz seems to have neurotoxicity maybe due to interference with the function of mitochondria, this could possibly also be due to the inhibition of creatine kinase, and it may also be due to the disruption of the membranes of the mitochondria or by interference with nitric oxide signalling. Cannabinoid receptors may mediate some of the neuropsychiatric adverse effect or through activity at the 5-HT2A receptor, but many receptors of the CNS experience interaction with efavirenz, although it is not clear. Neuropsychiatric adverse effects are dependent on dose. (Treisman and Soudry, 2016).

 

Structure of efavirenz

 


1.19. STATEMENT OF PURPOSE

Efavirenz and sparfloxacin are classified in class II of the Biopharmaceutics Classification System, which means they are poorly water-soluble and highly permeable. Their low solubility in aqueous medium limits the absorption and bio distribution of the drug from the gastrointestinal tract.

The use of surfactants to improve the solubility of sparingly-soluble or water-insoluble drugs has been reported (Alkhamis et al., 2003; Zhao et al., 1999; Li and Zhao 2003). Various studies have reported the influence of surfactants on dissolution of pharmaceutical active ingredients (Crison et al., 1997; Chowdary and Manjula, 2000; Jinno et al., 2000; Balakrishnan et al., 2004; Park and Choi 2006). Mbah and Ozuo stated that surfactants are employed in dissolution studies because natural surfactants in the body aid in the dissolution and subsequent absorption of drugs with limited aqueous. (Mbah and Ozuo, 2011). However no study has been done to investigate the influence of cosolvents and surfactants on the log K of both drugs.

1.20. AIMS AND OBJECTIVE OF STUDY

This study is aimed at determining the influence of cosolvents and surfactants on the rate constant K, of efavirenz and sparfloxacin.

To achieve this aim the following objectives were set:

  • The effect of cosolvents and surfactants on their degradation kinetics was investigated.
  • The influence of cosolvents and surfactants on the log K of both drugs was also determined.

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