Development of An Educational Tool

to Simulate ADME Processes In Vitro

By:

Ashadi Ashadi

A dissertation submitted to the School of Applied Science In partial fulfillment of the requirements for the degree of

Master of Science In

Pharmaceutical and Analytical Science

Supervisor:

Dr. Jordi Morral-Cardoner

Dr. Karl Hemming

School of Applied Science University of Huddersfield

August 2013

 

ii  

Table of Contents  

ABSTRACT .......................................................................................................................................... vi 

ACKNOWLEDGEMENT .................................................................................................................... vii 

Chapter 1. Introduction ........................................................................................................................ 1 

1.1 Background ................................................................................................................................. 1 

1.2 Aim and Objective .................................................................................................................... 3 

Chapter 2. Literature Review .............................................................................................................. 4 

2.1 Aspects in Pharmacokinetics ................................................................................................... 4 

2.1.1 Compartment Models in Pharmacokinetics .................................................................... 4 

2.1.2 Linear and Non Linear Pharmacokinetics ...................................................................... 5 

2.2 Test Drugs .................................................................................................................................. 6 

2.2.1 Aspirin® ............................................................................................................................... 6 

2.2.2 Zidovudine ........................................................................................................................... 7 

2.3. Materials and Reagents ........................................................................................................... 9 

2.3.1 Esterase, immobilised on Eupergit® C ........................................................................... 9 

2.3.2 Tris-HCL buffer ................................................................................................................... 9 

2.3.3 Silica gel............................................................................................................................... 9 

2.3.4 Alumina .............................................................................................................................. 10 

2.3.5 Amberlite® resin ............................................................................................................... 10 

2.3.6 Iron (III) chloride ............................................................................................................... 10 

2.3.7 Triphenylphosphine (PPh3) ............................................................................................. 10 

2.3.8 Dithiothreitol (DTT) ........................................................................................................... 10 

2.3.9 Ninhydrin ............................................................................................................................ 11 

2.3.10 Glutathione ...................................................................................................................... 11 

Chapter 3. Experimental Methodology............................................................................................ 12 

3.1 Instrument and apparatus ...................................................................................................... 12 

3.1.1 UV-Visible spectrophotometry ........................................................................................ 12 

3.1.2 Infra Red (IR) Spectroscopy ........................................................................................... 13 

3.1.3 ADME Simulator ............................................................................................................... 14 

3.2 Experiment Apparatus and Materials ................................................................................... 16 

3.3 Experimental procedure ......................................................................................................... 16 

3.3.1 Pharmacokinetic Simulation of Aspirin® ....................................................................... 16 

 

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3.3.2 Hydrolysis of Aspirin® ..................................................................................................... 17 

3.3.3 Reduction of AZT ............................................................................................................. 20 

Chapter 4. Result and Discussion ................................................................................................... 24 

4.1 Pharmacokinetic Simulation of Aspirin® .............................................................................. 24 

4.2 Hydrolysis of Aspirin® ............................................................................................................. 26 

4.2.1 Hydrolysis of Aspirin® with immobilised esterase in medium of DI water ............... 26 

4.2.2 Hydrolysis of Aspirin® with immobilised esterase in medium of Tris-HCL buffer .. 26 

4.2.3 Hydrolysis of Aspirin® with silica gel ............................................................................. 28 

4.2.4 Hydrolysis of Aspirin® with Alumina .............................................................................. 28 

4.2.5 Hydrolysis of Aspirin® with Amberlite® resin ............................................................... 28 

4.3 Reduction of AZT ..................................................................................................................... 28 

4.3.1 Reduction via Staudinger Reaction ............................................................................... 28 

4.3.2 Reduction with Thiols (Dithiothreitol and Glutathione) ............................................... 31 

Chapter 5. Conclusion ....................................................................................................................... 35 

References .......................................................................................................................................... 36 

 

 

iv  

List of Figures

 

Figure 2.1 The one-compartment model (Rosenbaum, 2011) ............................................... 5

Figure 2.2 The two-compartment model (Rosenbaum, 2011) ............................................... 5

Figure 2.3 The three-compartment model (Rosenbaum, 2011) ............................................. 6

Figure 2.4 Aspirin molecular structure (BP 2012) .................................................................. 7

Figure 2.5 Hydrolysis of Aspirin to Salicylic acid .................................................................... 7

Figure 2.6 Zidovudine molecular structure (BP 2012) ........................................................... 8

Figure 2.7 Reduction of Zidovudine (3'-azidothymidine) to 3'-aminothymidine ...................... 9

 

Figure 3. 1 Shimadzu Portable Ultraviolet-Visible Spectrophotometer (University of Huddersfield) ....................................................................................................................................... 13 

Figure 3. 2 Thermo Infra Red Spectrophotometer (University of Huddersfield) ....................... 14 Figure 3. 3 (A) Schematic processes in ADME simulator and (B) the simulator appearance (University of Huddersfield) .............................................................................................................. 15 Figure 3. 4 The ADME simulator parts. (C1a) the drug container, (C1b) 45 cm porous tube, (C2) separating column, (C3 and C4a) peristaltic pump, (C4b) 500 ml measuring flask, (C4c and C5b) waterbath contains 7 L water, (C5a) water circulator and (C6) measuring flask contains the 'urine'. ............................................................................................................................ 15 Figure 3. 5 Standard calibration curve of Acetylsalicylic Acid obtained from absorbance versus concentration by UV-Vis spectroscopy. ............................................................................. 17 Figure 3. 6 Standard's calibration curve of salicylic acid (+ iron (III) chloride) obtained from absorbance versus concentration by UV-Vis spectroscopy. ....................................................... 20 

Figure 4.1 Cumulative amount of Aspirin® excreted into measuring flask (simulating urine in

human body) over the time. .................................................................................................. 25

Figure 4.2 UV-Vis spectrum of (A) Aspirin (ASA) standard , (B) UV-Vis spectrum of Salicylic

acid standard with FeCL3, (C) UV-Vis spectrum of Aspirin with FeCL3 and (D) UV-Vis

spectrum of Aspirin (after hydrolysed with immobilised esterase) with FeCL3. .................... 27

Figure 4.3 IR spectrum of (A) AZT , (B) AZT reaction with PPH3 at 37 °C and (C) AZT

reaction with PPH3 at 80 °C. ................................................................................................ 30

Figure 4.4 IR spectrum of (A) AZT, (B) AZT reaction with DTT at 37 °C and (C) Aniline (from

Orgchem, 2013) .................................................................................................................... 32

Figure 4.5 UV-Vis spectrum of (A) AZT with 1% Ninhydrin , (B) DTT with 1% Ninhydrin and

(C) AZT (reacted with DTT) with 1% Ninhydrin. .................................................................... 33

 

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List of Tables

Table 4.1 Absorbance of Aspirin® sample solutions from measuring flask (simulating urine

in human body) over the time measured by UV-Vis spectroscopy and Aspirin®

concentrations obtained by calculation using standard regression line. ............................... 25

Table 4.2 Number of spots and retention factor of AZT, PPH3 and AZT reaction with PPH3 at

37 °C ..................................................................................................................................... 29

Table 4.3 Number of spots and retention factor of AZT, PPH3 and AZT reaction with PPH3

at 80 °C ................................................................................................................................. 30

Table 4.4 Number of spots and retention factor of AZT, DTT and AZT reduction with DTT at

37 °C ..................................................................................................................................... 31

Table 4. 5 Number of spots and retention factor of AZT, Glutathione and AZT with

Glutathione reaction at 37 °C ................................................................................................ 34

 

vi  

Development of An Educational Tool

to Simulate ADME Processes In Vitro

by Ashadi Ashadi (u1274952)

Supervisor: Dr. Jordi Morral-Cardoner, Dr. Karl Hemming

ABSTRACT

Understanding of pharmacokinetics (sometimes refer to what the body does to the

drug) is crucial for pharmacy and medical students. Doses, regimens and drug formulations

are determined by using data obtained from pharmacokinetic studies. In context of education

for students, the majority of university give pharmacokinetics practice based on animal

experimentation.

We describe herein development of a new educational tool to simulate absorption,

distribution, metabolism and elimination (ADME) in vitro for undergraduate student. In the

present work, the ADME simulator performance has been examined by using Aspirin® as

model drug.

In order to simulate metabolism process, preliminary experiment has been done by

using Aspirin® and Zidovudine as drug models. Hydrolysis of Aspirin® by using Immobilised

esterase on Eupergit® in medium of Tris-HCL buffer has been performed and shows a good

result of the reaction. We have also performed reduction of Zidovudine (azido thymidine or

AZT) by using Dithiothreitol as the reducing agent. A simple UV-Vis spectroscopy method is

used to distinguish and to calculate the concentration of the drug and the metabolite, such

as between Aspirin® and salicylic acid and AZT and AMT (aminothymidine).

 

 

 

 

 

vii  

ACKNOWLEDGEMENT  

 

I would never been able to finish my dissertation without assistance of others. Firstly, I would like to express my deepest gratitude to my supervisor, Dr. Jordi Morral-Cardoner and Dr. Karl Hemming, for their ideas, excellent guidance, patience, and supports to me in this research. Secondly, I would like to thank Hayley Markham and other university staff for their excellent works, helps, advices and good atmosphere in the laboratory.

I would also like to thank my beloved wife, our parents, my kids, all family and friends for infinite supports, encouragements and prayers to me in study and works.

Lastly, I would like to thank my sponsor the Government of Indonesia, especially NA-DFC, and all co-workers for the financial aid, support and opportunity to me in performing study in University of Huddersfield. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Chapter 1. Introduction

1.1 Background

Derived from the Greek words, pharmakon for drug and kinetikos for moving, the term pharmacokinetics was originally been introduced by F.H. Dost in 1953. Pharmacokinetics examines the motion of a drug over time through the body or concerns on how drugs go into the body, spread throughout the body, and depart the body (Harvey and Champe, 2009). To put simply, it includes the study of drug absorption, distribution, metabolism and excretion (ADME) (Rosenbaum, 2011).

Firstly, absorption of drug from the location of administration (absorption) allows entry of the drug into blood stream. Absorption is complete for intravenous and intra-arterial dispatch while partial absorption and thus lower bioavailability is resulted from drug delivery by other routes as in the oral route. Based on their chemical characteristics, drugs may be absorbed from the gastro intestinal (GI) tract by either passive diffusion, active transport (involving specific carrier proteins) or endocytosis and exocytosis. There are also another types of drug administration that do not involve absorption in GI tract such as inhalation, rectal and transdermal administration (Harvey and Champe, 2009).

Secondly, the drug may reversibly distribute in between bloodstream, extracellular, intracellular fluids and/or the cells of the tissues (distribution). Primary factors of the drug distribution are blood flow, capillary permeability, the extent of drug binding to plasma and tissue proteins, and the relative hydrophobicity of the drug (Harvey and Champe, 2009).

Thirdly, the drug may be metabolised mainly in the liver but may also metabolised in kidney or in other tissues. The purpose of metabolism process in the body is bioinactivation, detoxification and/or elimination by converting lipophilic drugs into products which is more in polarity and thus readily to excrete. There are two general types of metabolism reaction, called phase I and phase II. Phase I reactions involve cytochrome P450 system and others enzymatic reactions (oxidation, reduction or hydrolysis), resulting more polar molecules by inserting or uncovering a polar functional group such as -OH and -NH2. Phase II reaction involve conjugation of a reactive group on the metabolite from phase I metabolism. It is a subsequent conjugation reaction with an endogenous substrate, such as glucuronic acid, resulting polar, usually more water-soluble and pharmacologically inactive compounds. The example of conjugation reactions are glucuronidation acetylation and methylation (Harvey and Champe, 2009; Rang and Dale's, 2007).

Lastly, the drug and/or its metabolites are eliminated from the body via kidney, bile , intestine, lung or milk in nursing mother. The most important elimination of a drug from the body is via kidney into the urine. Glomerular filtration, active tubular secretion and passive diffusion across tubular epithelium are three fundamental processes for renal drug excretion. Biotransformation and/or excretion into the urine are the most often drug elimination pathway (Rang and Dale's, 2007).

 

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Understanding of pharmacokinetics (sometimes refer to what the body does to the drug) is crucial for pharmacy and medical students. Students must be aware that pharmacological, and/or toxicological, effects in therapy are principally correlated to the plasma concentrations of drugs. A sufficient doses and an appropriate administration of the drug is required so that therapeutic yet nontoxic levels of drug in target tissues can be achieved (Rosenbaum, 2011). In the field of drug development, such as in preclinical toxicity testing and in studies of efficacy in order to find minimum effective concentration (MEC) and maximum tolerated concentration (MTC), doses and formulations of drugs are obtained using data from pharmacokinetics studies. Understanding the general principal of pharmacokinetics is also essential in clinical field. For example, clinicians are often dealing with a severely ill patient with the need of special dosing regimen, such in situations where rapid therapeutic concentrations in plasma are required and in situations where liver and renal disease that can inhibit the body's clearance of the drug are present (Rang and Dale's, 2007).

In order to train pharmacokinetic for undergraduate students, practical by using laboratory animals is usually required. This type of practical has some limitations such as the need for animals handling, the number and the type of animal used is also limited. The main animal used is a laboratory rat of the species Rattus norvegicus (Sprague-Dawley). Generally, there is one laboratory rat prepared to every two students resulting up to twenty rats can be sacrificed in a single laboratory practical (e.g. involving forty students) (Ward and Reilly, 1981).

The outcomes animal experimentation is the severity and the death of the animal. The experiment can be rather cruel. For example to study drug absorption with in situ rat gut technique, a solution of drug is passed through a tube connected to the start of the duodenum and the end of the ileum while the rat is kept alive under general anesthesia (e.g Doluisio et al, 1969). In relation to drug metabolism, most of in vitro drug metabolism studies include liver or liver tissues from mice (e.g. Mitchell et al., 1973), rats, species of duck, rainbow trout and flounder (e.g. Murk et al., 1994). In addition, to study drug metabolism in undergraduate laboratory practical, sacrificing of previously starved overnight Rattus norvegicus (Wistar) is needed (e.g. Ward and Reilly, 1981). These types of experiment are not only an ethical issue, but can also interfere with the student’s ability to concentrate on the practical. In addition, the experiment with isolated animal organ shows only a particular step rather than the whole processes of ADME.

The purpose of this project is to find a realistic and accessible in vitro method to simulate the whole processes of drug absorption, metabolism and excretion, by using simple porous tube to simulate absorption, derivatised solid phase resins (and silica) to simulate the metabolism and conveniently placed tubes and manifolds to simulate drug excretion. A peristaltic pump and a fish tank filter will simulate the heart pumping and blood flow..

Despite there are well established alternative in vitro methods to study drug absorption (e.g. Waterbeemd et al., 2001) and an alternative methods to study drug metabolism based on computer programs (e.g. Jolivette and Ekins, 2007), these experiments are not very realistic and far too advanced for undergraduate practical (Johansson et al., 2007; Linnet, 2004; and Guengerich, 1996). At the present time, from the author’s knowledge, there are no studies that combines drug absorption, metabolism and

 

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excretion that similar to this project, and also cheaply and easily enough to be used in undergraduate practical without having to harm any animals.

The University has Infra Red (IR) and UltraViolet-Visible (UV-Vis) spectroscopy apparatus that can be used in undergraduate practical. IR spectroscopy will allow the identification of a particular expected metabolite, and the quantification of the proportion of each metabolite using UV-Vis spectroscopy will be very likely. By studying the structure of the different metabolites with straightforward spectroscopic methods (UV-Vis or IR), the student will have a clear insight into the whole process of drug metabolism. The student also should be able to quantify some of the pharmacokinetic characteristics of the drug (e.g. half-life, volume of distribution, clearance, etc) by quantifying the amount of metabolite (or unaltered drug) excreted and the remaining concentration of drug.

Once the model is fully functional, the student will be able to perform the simulation in laboratory practical session with a reasonable amount of time. This project will be the first simplified model combining drug absorption, metabolism and excretion studies that could give the student a more realistic idea of the absorption, distribution, metabolism, and excretion (ADME) processes, together with the opportunity to practice similar mathematical equations to the ones used in in vivo studies.

1.2 Aim and Objective  

As mentioned above, the aim of the project is to develop a new system as educational tool for undergraduate student that can simulate ADME process in vitro. Aspirin® and Zidovudine (AZT) is selected as drug models in this project because of the importance of these drugs for pain and anti-HIV therapy respectively and with the chemical properties of these drugs for hydrolysis and reduction reactions (Moffat et al., 2013). The objective is to find methods to simulate Absorption, Distribution, Metabolism and Excretion (ADME) processes. The absorption process is simulated by using a porous tube as similitude of the GI tract. The distribution process is simulated by using a waterbath and water circulator as similitude of the body compartment. The metabolism process is simulated by using separating funnel containing a resin as similitude of the liver. The excretion process is simulated by using a tubing system with flow regulator as similitude of the kidney. The systemic circulation is simulated by using a tubing system connected to a peristaltic pump as similitude of the heart. We want to simulate the hydrolysis of Aspirin® using esterase linked to solid phase or alternatively using chemical substances such as silica, Amberlite® resin and alumina. We also wanted to simulate the reduction of Zidovudine using thiols (Dithiothreitol or Glutathione) or the reduction via Staudinger reaction.  

 

 

 

4  

Chapter 2. Literature Review  

2.1 Aspects in Pharmacokinetics  

The objective of pharmacokinetics is study on how the fate of drugs in the body, ranging from the process of absorption, distribution, metabolism, and excretion (ADME). These processes will been described quantitatively or can be calculated through formulas obtained from mathematical equations. Pharmacokinetics applies mathematical and biochemical techniques in relationship with a physiological and pharmacological context (Gibaldi and Levy, 1976). A suitable mathematical model is used to analyse and convert such data to some meaningful parameter values (Wagner, 1975).   

2.1.1 Compartment Models in Pharmacokinetics  

Pharmacokinetics model is a simplification of complex body systems into a simple mathematical equation that describes the fate of drugs in the body in a simple way to understand. The easiest way is using a compartment model such as one-compartment, two-compartment and three-compartment model. A compartment is an imaginary unit that used to indicate a group of tissues with similar paces of drug distribution and it is a homogeneous unit where the drug concentration is uniform throughout at all times (Wagner, 1975). The one-compartment model consists only of a central compartment and distribution to those tissues that the drug can access occurs rapidly and appears to be an instantaneous process (Figure 2.1). Paracetamol is an example of drug that follows one-compartment model (Anderson et al., 1998). The two-compartment model consists central and peripheral compartments where in this model the drug distribution occurs very rapidly in the central compartment tissues but a noticeable distribution of a significant amount of the drug to other tissues occurs at slower rate (Figure 2.2). Digoxin is an example of drug that follows two-compartment model (Bauer, 2013). The three-compartment model has three groups of tissues, which are central, peripheral and the deep tissues. It is an addition of the two-compartment model, where a considerable amount of the drug distributes, at an extremely slow rate, to definite very poorly perfused tissues (such as fat and bone) (Figure 2.3), Propofol is an example of drug that follows three-compartment model (Rxlist, 2013). The appropriate model can be used to summarise a drug's properties and to estimate the model parameters (e.g. clearance, volume of distribution, etc.) (Rosenbaum, 2011). In order to find pharmacokinetic profile of a drug, a plot of the drug's plasma concentration against time is needed. Since most of drug's concentration in plasma changing exponentially, the linear relationship of the concentration versus time is obtained by converting the concentration value to the natural logarithms (Ln) or base 10 logarithms (Log). The pharmacokinetics parameters can be calculated base on this linear relationship.

 

 

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6  

biotransformation or elimination) is the most example of nonlinear pharmacokinetic observed clinically. Phenytoin is an example of drug that shows this pharmacokinetics (Rosenbaum, 2011).

 Figure 2. 3 The three-compartment model (Rosenbaum, 2011)

(A) The two-compartment model consists of the central, peripheral and deep tissue compartment. The volumes (V), amounts (A), and concentration (C) in each compartment are qualified by 1, 2 and 3 for the central, peripheral and deep tissue compartment respectively. Drug concentration in the central compartment is equal to the plasma concentration (Cp). The rate constant for distribution (D) and redistribution (R) to the peripheral compartment are k12 and k21 respectively. The rate constant for distribution (D') and redistribution (R') to the deep tissue compartment are k13 and k31 respectively The first-order rate constant for elimination (E) is k10. (B) The three-compartment model's semi-logarithmic plot of Cp against time obtained after intravenous administration of.

2.2 Test Drugs  

In this experiment we want to mimic the similar metabolism process of Aspirin® and Zidovudine in human body. In the liver, Aspirin® undergoes hydrolysis to salicylic acid (Rowland et al.,1972) while Zidovudine is reduced to the 3'-amino-3'- deoxythymidine (AMT) (Ghodke et al, 2012). We wanted to simulate the metabolism process of these drugs that can be used in the developed simulator tool.

2.2.1 Aspirin®  

Aspirin® is one of the most consumed drugs in the world, approximately 100 billion tablets are taken worldwide per year (Aspirin-Foundation, 2013). Aspirin®, or acetylsalicylic acid (ASA), has pharmacologic effect as an analgesic to reduce slight aches and acute pains, as an antipyretic to decrease fever, and as an anti-inflammatory medication in rheumatic condition. Aspirin® also has anti platelet activity for the prevention and treatment of thromboembolic diseases (Rainsford, 2004).

Aspirin® is white or almost white, crystalline powder or colourless crystals with molecular formula and molecular weight of C9H8O4 and 180.2 respectively. It is slightly soluble in water, freely soluble in ethanol, soluble in chloroform and ether. It slowly hydrolyses in contact with moisture to form acetic and salicylic acids but is stable in dry air (British Pharmacopoeia, 2012; Moffat et al. 2004).

 

 

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8  

AZT is a white to yellowish or brownish odourless crystalline powder with molecular formula and molecular weight of C10H13N5O4 and 267.2 respectively. It is sparingly soluble in water but freely soluble in alcohol. (British Pharmacopoeia, 2012; Moffat et al., 2004).

The anti-HIV drug Zidovudine (ZDV), also known as 3'-azidothymidine (AZT), has three ways of clearance. The first metabolic pathway is intracellular phosphorylation through three cellular kinases, thymidine kinase, thymidylate kinase, and diphosphate kinase resulting the active triphosphate metabolite (ZDP-TP) (Peter and Gambertoglio, 1998). The second way is inactivation of ZDV by glucuronidation resulting 5'-glucuronyl zidovudine (GZDV) (Good et al, 1990). The last pathway involves reduction of the azido functional group via a P450-type reductive reaction forming a toxic metabolite 3'-amino-3'- deoxythymidine (AMT) (Pan-Zhou et al, 1998). The last metabolism reaction forming AMT is the one that we want to simulate in this project.

 

Figure 2. 6 Zidovudine molecular structure (BP 2012)

There are many methods to reduce an azide but only few are susceptible to use in undergraduate practical (mainly because of time consuming). However, we can simulate the reduction of azido fuctional group of AZT via Staudinger reaction or alternatively by using Thiols compound. Staudinger reaction shows a selective reductive reaction of an azide functional group to an amine. The azide group reacts with PPh3 to form an phosphazide, which sequentially loses nitrogen gas to form an iminophosphorane. The iminophosphorane is then hydrolysed by water to produce an amine and triphenylphosphine oxide (Fox and Edgar, 2012). The ability of dithiothreitol (DTT) and Glutathione to reduce 3'-azidothymidine to 3'-aminothymidine, both in physiological and non-physiological conditions, has been examined. DTT shows reduction of AZT in a phosphate buffer solution at room temperature (Handlon and Oppenheimer, 1988).

 

9  

 Figure 2. 7 Reduction of Zidovudine (3'-azidothymidine) to 3'-aminothymidine (stucture from BP 2012)

2.3. Materials and Reagents  

2.3.1 Esterase, immobilised on Eupergit® C  

Esterase on Eupergit® C is esterase from hog liver and is immobilised on copolymer of methacrylamide, allyl-glycidylether and methylene-bis-acrylamide. It is in form of moist pearls (dried material ~35%, pearl diameter ~150 μm) and is used as reagent for the preparation of chiral building blocks. It contains ~200 U/g moist material where 1 U corresponds to the amount of enzyme which hydrolyzes 1 μmol ethyl valerate (Fluka No. 30784) per minute at pH 8.0 and 25 °C (Sigma-Aldrich a, 2013).

2.3.2 Tris-HCL buffer  

Tris (hydroxymethyl) aminomethane (MF C4H11NO3, MW 121.14) is a colourless white crystalline substance and has pH 10.5 to 12. It is used as buffers in many of biological systems such as pH control in vitro and in vivo and as an alkalizing agent in blood acidosis treatment. The Tris-HCl buffer is been made by dissolving in water nearly to volume; adjusted with gradual addition of hydrochloric acid to the desired pH, and then diluted to the appropriate final volume (Sigma-Aldrich b, 2013).

2.3.3 Silica gel  

Silica gel (MF SiO2, MW 60.08) is a transparent, tasteless crystals or amorphous powder and is insoluble in water, alcohol and in other organics solvent. It is used as dessicant, suspending and viscosity-increasing agent in pharmaceutical and is also used in chromatography technique as stationary phase due to its polarity (O'Neil et al,, 2013; Sigma-Aldrich c, 2013). The silica used in this experiment is silica gel for chromatography, it has property with pore size 60 A, 70-230 mesh, 63-200 µm.

 

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2.3.4 Alumina  

Alumina (MF Al2O3, MW 101.96) is a white crystal powder and is insoluble in water but very hygroscopic. It is used as dessicant, chromatographic matrix and as catalyst for organic reactions (O'Neil et al., 2013). In this experiment, Alumina is activated by soaking it in water overnight and then is dried. 2.3.5 Amberlite® resin  

Amberlite is a pale yellow translucent spherical bead. It has trimethyl ammonium as functional group on matrix of styrene divinylbenzene copolymer. Amberlite® IRA402 Cl resin is a type 1 strongly basic anion exchange resin and is used in water treatment applications since it can remove both strong and weak acids (Dow chemical,2013). It is also used as chelating resin in analytical chemistry (Tewari and Singh, 2002)

2.3.6 Iron (III) chloride  

Iron (III) chloride hexahydrate (MF Cl3Fe.6H2O, MW 270.29) is a brownish-yellow or orange monoclinic crystals and is readily soluble in water, alcohol, acetone and ether. It is used as dye, as catalyst in organic reactions, as reagent in clinical practise (O'Neil et al., 2013) and in analytical chemistry (Sarhan and Bolm, 2009)

2.3.7 Triphenylphosphine (PPh3)  

Triphenylphosphine (MF C18H15P, MW 162.20) is a hexagonal, dark leaflet or plates odourless monoclinic platelets or prisms from ether and is freely soluble in ether; soluble in benzene, chloroform, glacial acetic acid; less soluble in alcohol; practically insoluble in water. It is used in organic synthesis as polymerization initiator (O'Neil et al., 2013). It is also used as reducing agent in Wittig reaction (Shimojuh et al., 2011) and Mitsunobo reaction (Chen and Luh, 2008).

2.3.8 Dithiothreitol (DTT)

Dithiothreitol (MF C4H10O2S2, MW 154.25) is a slightly hygroscopic needles from ether and is freely soluble in water, ethanol, acetone, ethyl acetate, chloroform and ether. It has reduction potential of -0.33 volts at pH 7 and is used in organic synthesis as reagent for protection of SH groups (O'Neil et al., 2013). It is used extensively in biochemistry as a reducing agent (Santos et al., 2005)

 

11  

2.3.9 Ninhydrin  

Ninhydrin (MF C9H6O4, MW 178.14) is a pale yellow prism from water or alcohol and is freely soluble in water. It is used as reagent for detection of free amino and carbonyl groups in proteins and peptides, yielding a blue colour under the proper conditions (O'Neil et al., 2013).

2.3.10 Glutathione  

Glutathione (MF C10H17N3O6S, MW 307.32) is a major low molecular weight thiol compound of the living plant or animal cell. The crystals form from 50% ethanol is freely soluble in water, dilute alcohol liquid ammonia and dimethylformamide (O'Neil et al., 2013). It contains a sulfur-hydrogen bond and acts as endogenous antioxidant (reducing reactive oxygen species formed during cellular metabolism and the respiratory burst) (Sigma-Aldrich d, 2013). Glutathione involves in phase II metabolism. It can conjugate drug metabolites via its sulfhydryl group, such as in the detoxification of paracetamol (Rang and Dale's, 2007)  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

12  

Chapter 3. Experimental Methodology   

3.1 Instrument and apparatus  

Ultraviolet-Visible (UV-Vis) and Infra Red (IR) spectroscopy are approved methods in official monograph such as Pharmacopoeia. UV-Vis is used both for identification and for quantitative determination of pharmaceutical products (Hansen et al., 2012), since most of the products have chromophores that can absorb electromagnetic radiation in the UV-Vis wavelength region (200-400 nm). IR is primarily used for identification of pharmaceutical products, and it is used to determine functional groups and bonds of the products that can absorb electromagnetic radiation in the IR wavelength region (2500-25000 nm) (Hansen et al., 2012). UV-Vis and IR spectroscopy are rapid and easy to perform with good accuracy and precision, and the costs are also relatively low. These methods are suitable to be used in practical laboratory for undergraduate students. 3.1.1 UV-Visible spectrophotometry UV-visible spectroscopy is possibly the most common quantitative analysis techniques used in chemical, environmental, forensic and clinical laboratories all over the world (Skoog et al., 2004). Atoms, molecules, or other chemical species can absorb electromagnetic radiation differently. Each species has characteristic energy states that correlated with changes in the energy states of the interacting chemical species. Molecules having such functional groups and have ability to absorb ultraviolet-visible radiation are called chromophores (Robinson, 1987). Spectroscopy is used to measure and to interpret this phenomenon and uses it to identify the interacting species and to obtain quantitative information. The measurement of the transmittance (T) or the absorbance (A) of solutions contained in transparent cells is a fundamental of molecular absorption spectroscopy. Generally, the concentration of analyte is linearly related to absorbance as describe by Beer's law :

A = -log T = log P0/P = abc

where A= absorbance, T = Transmittance, P0 = Incident radiant power, P = transmitted radiant power, a = Molar absorptivity, b = path length of sample, and c = concentration of absorbing analyte (Robinson, 1987). A calibration curve of absorbance versus the concentrations from several standards with known concentration is commonly used in order to find the concentration of unknown samples. The concentration of calibration standards for a spectrophotometer method should approximate as nearly as possible to the concentration of the actual samples and should cover a realistic range of analyte concentrations (Skoog et al., 2004).

 

13  

 Figure 3. 1 Shimadzu Portable Ultraviolet-Visible Spectrophotometer (University of Huddersfield)

UV-Vis spectroscopy can be used to determine Aspirin® and Zidovudine since both of drugs are absorb electromagnetic radiation in UV wavelength region (Moffat et al., 2013). With colorimetry technique, salicylic acid (the metabolite) can be reacted with Iron(III) chloride to form a colour complex that has ability to absorb the radiation in visible region wavelength (400-800 nm), while Aspirin® does not give the same reaction (Ogawa and Tobe, 1966). This technique can also be applied to Zidovudine, where the metabolite (3'-amino-3'- deoxythymidine or AMT) can be reacted with Ninhydrin (MacFadyen, 1950) and form a colour complex that can measured in the visible region, Zidovudine does not give the same reaction. 3.1.2 Infra Red (IR) Spectroscopy Modern IR spectroscopy is a reliable method for the qualitative and quantitative determination of all organic and inorganic molecular since these molecular species absorb radiation in the IR region, despite the exception of homonuclear molecules (N2, O2, Cl2, etc.). The applications of IR spectrophotometry is divide into three main categories based on the IR spectral regions which are the near IR region (extends from 4000 to 14000 cm -1 or 0.75 to 2.5 µm), mid-IR region (from 670 to 4000 cm -1 or 2.5 to 14.9 µm) and the far IR region (from 15 to 1000 µm). The mid-IR is the most important region and is most important tools for determining the structure of organic and biochemical species (Skoog et al., 2004). Infrared radiation release energy that can excite electronic vibrational and rotational transitions but it is not enough to excite electronic transitions. The number of atoms and the number of bonds in molecule affects the number of ways a molecule can vibrate or rotate. A molecule can have numbers of vibrational modes and most differing from each other in energy but not all of these vibrations produce infrared peaks (Skoog et al., 2004).

 

14  

 Figure 3. 2 Thermo Infra Red Spectrophotometer (University of Huddersfield)

IR spectroscopy can be used to detect the presence of particular functional group on a molecule. Zidovudine has an azide functional group which transforms to an amine after reduction. The disappearance of the azide and the presence of the amine as the result of the reduction can be clearly seen from IR spectrum obtained, azide functional group has absorption at 2098 cm-1 while amine functional group has absorption at 3300-3500 cm-1. The IR method is used as confirmation whether the reduction of AZT to AMT is successfully done or not with the proposed procedures.

3.1.3 ADME Simulator Absorption, Distribution, Metabolism and Excretion (ADME) simulator was developed as a tool to teach pharmacokinetics to pharmacy undergraduates. The simulator consists of a 7 L water bath with water circulator pump to simulate blood in the body, a peristaltic pump to simulate heart, a 45 cm porous tube to simulate GI tract, a 50 ml of separating column (contains solid phase resin) to simulate liver, 500 ml measuring flask to simulate bladder. All the parts are connected with a tubing system to simulate blood stream (Figure 3.3 B). The simulation is performed using water as a medium with the aid of a pump and a filter tank for the circulation to simulate the heart pumping and drug flow of the body. The test drug is placed in a funnel and is then carried to the porous tube by the water flows. The tube is made of porous hose that have some incisions on it and thus small particles of the drug are expected to get filtered by the tube and some of the drug are also expected to dissolve while in the tube. The drug then enters separating column containing solid phase resin and is expected to react with the resin before entering water bath. From the water bath, the drug solution (unaltered and/or metabolite resulting from reaction) is separated into two parts where one part is disposed to the 'urine' container (a measuring flask) and the other circulates again to the drug container (the funnel) and then the processes are repeated (Figure 3.3 A and Figure 3.4).

 

 

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16  

3.2 Experiment Apparatus and Materials Apparatus

ADME Simulator Ultra Violet-Visible Spectrophotometer Infra Red Spectrophotometer Digital Hot Plate with Thermometer probe

Materials

Aspirin® tablet Zidovudine capsule Acetylsalicylic acid Salicylic acid Iron (III) chloride Amberlite® IRA402 Cl resin Alumina activated Silica Gel, pore size 60 A, 70-230 mesh, 63-200 µm Tris (hydroxymethyl) aminomethane Triphenylphosphine Dithiothreitol Ninhydrin Glutathione

3.3 Experimental procedure 3.3.1 Pharmacokinetic Simulation of Aspirin® In order to check the simulator, a simulation was performed without metabolism process. Metabolism process is going to simulate by using solid phase chemistry technique and it will be studied in the sequel. Aspirin® tablet (contains 300 mg of Aspirin®) was powdered and was placed into sample container. Simulation process was started once the pump switched on. The pump was running on scale 0.7 in which produced 60 ml/min of water circulation. The simulation performed at 37 °C to simulate temperature of the body and used 7 L of DI water to simulate the blood volume. Discharge rate of water into measuring flask to simulate renal excretion was approximately 10 ml/min. 3 ml of sample solution is collected from measuring flask (simulate drug in urine) every 5 minutes up to 50 minutes. Then samples are analysed by UV-Vis spectroscopy method at 289.5 nm to determine the concentrations of Aspirin® (Acetylsalicylic acid). Acetylsalicylic Acid (ASA) Standard Solution Preparation. Stock standard solution of ASA (100 μg/mL) was prepared by dissolving 25 mg ASA with ethanol : DI water (1 :1) as the solvent in 250 ml volumetric flask and was shaken vigorously. This solution was gradually diluted to get required concentrations range. The solutions were prepared ranging from 1 to 80 μg/ml. The absorbance of these solutions was measured at wavelength 289.5 which is the ʎmax of ASA. The relationship between

 

17  

absorbance versus concentration of ASA standard was described in regression line equation of y=0.0040 Conc. - 0.0124 with correlation coefficient (r) =0.9922 (Figure 3.4)

 

Figure 3. 5 Standard calibration curve of Acetylsalicylic Acid obtained from absorbance versus concentration by UV-Vis spectroscopy.

Calibration curve of ASA standard was used to calculate the concentration of Aspirin® in the samples, with a strong positive linear relationship (r = 0.9922) within the range of the standard's concentration (1-80 µg/ml). The changes of Aspirin® concentration in 'urine' was determined to see the elimination of it by excretion. The elimination was solely influenced by the flow of water discharged and thus followed zero-order kinetics (running at constant rate).

3.3.2 Hydrolysis of Aspirin®

Hydrolysis to salicylic is the main route of elimination of Aspirin®. Aspirin® experiences both chemical and enzymatic hydrolysis to salicylic (Rainsford, 2004). Enzymatic hydrolysis by esterase plays the main role in the hydrolysis process (Inoue et al., 1980). We was conducted simulation of Aspirin® hydrolysis by using immobilised esterase on solid phase in different medium (DI water and Tris-HCl buffer) Hydrolysis reaction of Aspirin® by catalytic action involving silica surface has been found (Demianenko, 2011). Aspirin® also experiences catalytic hydrolysis by acid and alkaline aqueous (Edwards, 1952). We was also conducted an alternative simulation of Aspirin® hydrolysis by catalytic action using silica gel, alumina and Amberlite® resin

y = 0.0040x + 0.0124r = 0.9922

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 10 20 30 40 50 60 70 80 90

Ab

sorb

ance

Concentration (µg/ml)

Calibration Curve of Acetylsalicylic Acid Standard

 

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3.3.2.1 Hydrolysis of Aspirin® with immobilised esterase in medium of DI water Procedure A powder of Aspirin® (26 mg, 0.144 mmol) was dissolved in 8 ml of DI water pH 8.0 (adjusted with addition of 0.1 M NaOH). As much as 2 (two) of the same solutions were made and placed into 50 ml bottle flask with stopper (Solution A, B). Each solution was differently treated as follows; there was no further treatment for solution A while 10 mg of Immobilise esterase on Eupergit® was added into solution B. The solutions (A, B) then incubated in water bath at 37 °C for 1 hour. As much as 2.0 ml of each solution was collected after 1 hour and was placed into 10 ml volumetric flask. 0.5 ml of 0.02 M FeCl3 solution was added into solution and sufficient water was added to produce 10 ml of solution. Colour changes were observed indicating that the reaction takes place. 3.3.2.2 Hydrolysis of Aspirin® with immobilised esterase in medium of Tris-HCL buffer The purified esterase from human intestinal mucosa was found to hydrolyse ester-type drugs, such as Aspirin®, clofibrate, indanyl carbenicillin and procaine (Inoue et al, 1979). This in vitro experiment was carried out in Tris-HCL buffer as medium and was using incubation procedure at 37 °C. Procedure 0.1 M Tris-HCL buffer ph 8.0 was prepared by dissolving 0.6057 g of tris(hydroxymethyl)aminomethane dissolve in 45 ml DI water, adjust pH with 1 M HCL to pH 8.0. Add water to volume 50 ml.

A powder of Aspirin® (26 mg, 0.144 mmol) was dissolved in 8 ml of 0.1 M Tris-HCl buffer pH 8.0. As much as 3 (three) of the same solutions were made and placed into 50 ml bottle flask with stopper (Solution A, B, and C). Each solution was differently treated as follows; there was no further treatment for solution A, 10 mg of Immobilise esterase on Eupergit® was added into solution B and 30 mg of Immobilise esterase on Eupergit® was added into solution C. The solutions (A, B, and C) then incubated in water bath at 37 °C for 1 hour. As much as 2.0 ml of each solution was collected after 1 hour and was placed into 10 ml volumetric flask. 0.5 ml of 0.02 M FeCl3 solution was added into solution and sufficient water was added to produce 10 ml of solution. Analysis with UV-Vis Spectroscopy was carry out to find amount of salicylic resulted from the enzymatic hydrolysis process using absorbance at 530.5 nm as lambda maximum of Fe-Salicylic complex formed.

3.3.2.3 Hydrolysis of Aspirin® with silica gel Hydrolysis reactions of Aspirin® involving silica surface has been studied. The hydrolysis occurs due to the interaction between silica surface and acetylsalicylic acid molecule that produces catalytic effect (Demianenko, 2011).

 

19  

Procedure A powder containing Aspirin® (100 mg, 0.555 mmol) was dissolved in 5 ml of ethanol 50%. As much as 2 (two) of the same solutions were made and placed into 50 ml bottle flask with stopper (Solution A and B). Each solution was differently treated as follows; there was no further treatment for solution A while 200 mg of silica gel was added into solution B. The solutions (A, B) then incubated in water bath at 37 °C for 1 hour. As much as 2.0 ml of each solution was collected after 1 hour and was placed into 10 ml volumetric flask. 0.5 ml of 0.02 M FeCl3 solution was added into solution and sufficient ethanol 50% was added to produce 10 ml of solution. Colour changes were observed as indication that the reaction takes place.

3.3.2.4 Hydrolysis of Aspirin® with Alumina Procedure A powder containing Aspirin® (100 mg, 0.555 mmol) was dissolved in 5 ml of ethanol 50%. As much as 2 (two) of the same solutions were made and placed into 50 ml bottle flask with stopper (Solution A and B). Each solution was differently treated as follows; there was no further treatment for solution A while 1000 mg of alumina (activated previously with water and allow to stand overnight) was added into solution B. The solutions (A, B) then incubated in water bath at 37 °C for 1 hour. As much as 2.0 ml of each solution was collected after 1 hour and was placed into 10 ml volumetric flask. 0.5 ml of 0.02 M FeCl3 solution was added into solution and sufficient ethanol 50% was added to produce 10 ml of solution. Colour changes were observed as indication that the reaction takes place.

3.3.2.5 Hydrolysis of Aspirin® with Amberlite® resin Procedure A powder containing Aspirin® (100 mg, 0.555 mmol) was dissolved in 5 ml of ethanol 50%. As much as 2 (two) of the same solutions were made and placed into 50 ml bottle flask with stopper (Solution A and B). Each solution was differently treated as follows; there was no further treatment for solution A while 850 mg of Amberlite® resin (activated by washing sequentially with 1 N NaOH solution) was added into solution B. The solutions (A, B) then incubated in water bath at 37 °C for 1 hour. As much as 2.0 ml of each solution was collected after 1 hour and then was placed into 10 ml volumetric flask. 0.5 ml of 0.02 M FeCl3 solution was added into solution and sufficient ethanol 50% was added to produce 10 ml of solution. Colour changes were observed as indication that the reaction takes place.

Salicylic acid reacted with Iron (III) chloride standard solution preparation. Hydrolysis of Aspirin® results salicylic acid and acetic acid. Salicylic acid formed from the reaction can be detected with adding iron (III) chloride to produce colour complex (Fe-Salicylic) and afterwards the concentration of salicylic acid can be determinate using UV-Vis spectroscopy method.

 

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Stock standard solution of salicylic acid (1000 μg/mL) was prepared by dissolving 25 mg ASA with ethanol : DI water (1 :1) as the solvent in 25 ml volumetric flask and was shaken vigorously. This stock solution was pipetted ranging from 10 to 1000 μl and placed into 10 ml volumetric flask. 1 ml of 0.02 M Iron (III) chloride was added into volumetric flask, then solution mix diluted with DI water to the volume. The final concentration of salicylic acid in the solutions was ranging from 1 to 100 μg/ml. The absorbance of these solutions was measured at wavelength 531.5 nm, which is the ʎmax of iron-salicylic complex. The relationship between absorbance versus concentration of salicylic standard (+ iron (III) chloride) was described in regression line equation of y=0.0120 Conc. - 0.0035 with correlation coefficient (r) =0.9998 (Figure 3.5).

 

Figure 3. 6 Standard's calibration curve of salicylic acid (+ iron (III) chloride) obtained from absorbance versus concentration by UV-Vis spectroscopy.

Calibration curve of salicylic acid standard (+iron (III) chloride) was used to calculate the concentration of salicylic acid (after reaction with the iron) in the samples, with a strong positive linear relationship (r = 0.9998) within the range of the standard's concentration (1-100 µg/ml).

3.3.3 Reduction of AZT

The anti-HIV drug zidovudine (AZT) experiences reduction on the azide functional group via a P450-type reductive reaction to form a toxic metabolite 3'-amino-3'- deoxythymidine (AMT) in its metabolism process (Pan-Zhou et al, 1998). In order to simulate similar reaction, we conducted experiments by using triphenylphosphine (Staudinger reaction) and by using thiol compounds (dithiothreitol and gluthatione) as the reducing agent. Staudinger reaction has showed a selective reductive reaction of an azide functional group to an amine in biomedical application (Fox and Edgar, 2012). The thiols has also showed reduction of AZT in a phosphate buffer solution at room temperature (Handlon and Oppenheimer, 1988).

y = 0.0120x + 0.0035r = 0.9998

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 20 40 60 80 100 120

Ab

sorb

ance

Concentratin (µg/ml)

Calibration Curve of Salicylic acid (+iron (III) chloride)

 

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3.3.3.1 Reduction via Staudinger Reaction

Staudinger reaction shows a selective reductive reaction of an azide functional group to an amine. The azide group reacts with PPh3 to form an phosphazide, which sequentially loses nitrogen gas to form an iminophosphorane. The iminophosphorane is then hydrolysed by water to produce an amine and triphenylphosphine oxide (Fox and Edgar, 2012) 3.3.3.1.1 Reduction at 37 °C Procedure A powder containing AZT (100 mg, 0.37 mmol) was dissolved in 10 ml of THF. The solution was filtered using cotton wool and placed into 100 ml round bottom flask. Then into solution PPh3 (0.1 g, 0.38 mmol) was added and after that the solution allowed to stand overnight with stirring. Thereafter, 1 ml of water was added and the solution was stirred at 37 °C. After two hours, aliquot was collected and analysed by Thin Layer Chromatography (TLC) method using AZT in THF and PPh3 in THF as sample comparisons and Acetic Acid : Petroleum ether 40/60 (2 : 1) as the mobile phase. The remaining reacted solution evaporated with rotary evaporator. Then the reacted solution evaporated with rotary evaporator. Hereafter, an oily liquid resulted from evaporation process was dissolved in 25 ml of Dichlormethane and 2 g of Magnesium Sulphate was added into solution. Then the solution stirred for 5 minutes and filtered to get clear solution. The solution evaporated again to obtain an oily liquid which then analysed by IR spectroscopy from 4000 to 400 cm-1 wavenumbers. 3.3.3.1.2 Reduction with reflux at 80 °C Procedure A powder containing AZT (100 mg, 0.37 mmol) was dissolved in 10 ml of THF. Solution was then filtered using cotton wool and placed into 100 ml round bottom flask. Then into solution PPh3 (0.1 g, 0.38 mmol) was added and after that the solution allowed to stand overnight with stirring. Thereafter 1 ml of water was added and the solution was refluxed at 80 °C. After two hours aliquot was collected and analysed by TLC method using AZT in THF and PPh3 in THF as sample comparisons and Acetic Acid : Petroleum ether 40/60 (2 : 1) as the mobile phase. Then the reacted solution evaporated with rotary evaporator. Hereafter, an oily liquid resulted from evaporation process was dissolved in 25 ml Dichlormethane and 2 g of Magnesium Sulphate was added into solution. Then the solution stirred for 5 minutes and filtered to get clear solution. The solution evaporated again to obtain an oily liquid which then analysed by IR spectroscopy from 4000 to 400 cm-1 wavenumbers.

 

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3.3.3.2 Reduction with Thiols (Dithiothreitol and Glutathione)

The ability of dithiothreitol (DTT) and Glutathione to reduce 3'-azidothymidine to 3'-aminothymidine, both in physiological and non-physiological conditions, has been examined. DTT and Glutathione has showed reduction of AZT in a phosphate buffer solution at room temperature (Handlon and Oppenheimer, 1988).

3.3.3.2.1 Reduction with DTT Procedure AZT powder from capsule dosage form (contains 100 mg, 0.37 mmol) was dissolved in 10 ml of DI water and filtered using cotton wool to produce clear solution. 5 ml of the solution (contains 0.050 g, 0.187 mmol) was taken and placed into 50 ml bottle flask with stopper. Dithiothreitol (0.144 g, 0.933 mmol) was added into solution and stirrer at 37 °C. After one hour, aliquot was analysed by TLC method using AZT in water and DTT in water as comparisons and Ethyl acetate : Acetic acid (2 : 1) as the mobile phase. For next step, 2 ml of reacted solution was collected, added with 2 ml of 0.1% Ninhydrin solution and then heat to 80 °C for 10 minutes. Solution was allowed to cool in room temperature and was transferred to 10 ml of volumetric flask and then diluted with water to the mark. Colour changes of solutions were observed and then the solution was analysed with UV-Vis spectrophotometer at lambda maximum of 566.0 nm. 3.3.3.2.2 Reduction with DTT, Carbon active and Amberlite® resin Procedure AZT powder from capsule dosage form (contains 100 mg, 0.37 mmol) was inserted into separating column containing 0.144 g DTT mixed with 25 g Amberlite® resin at the top and active charcoal at the bottom. 1 L of water in beaker glass was circulated through the column up to 1 hour. 2 ml of reacted solution was collected, added with 2 ml of 0.1% Ninhydrin solution and then heat to 80 °C for 10 minutes. Solution was allowed to cool in room temperature, transferred to 10 ml of volumetric flask and then diluted with water to the mark. Colour changes of solutions were observed and then the solution was analysed with UV-Vis spectrophotometer. 3.3.3.2.3 Reduction with Glutathione Procedure AZT powder from capsule dosage form (contains 100 mg, 0.37 mmol) was dissolved in 10 ml of DI water and filtered using cotton wool to produce clear solution. 5 ml of the solution (contains 0.050 g, 0.187 mmol) was taken and placed into 50 ml bottle flask with stopper. Glutathione (0.144 g, 0.933 mmol) was added into solution and stirrer at 37 °C. After one hour, aliquot was analysed with TLC method using AZT in water and Glutathione in water as comparisons and Ethyl acetate : Acetic acid (2 : 1) as the mobile phase. For next

 

23  

step, 2 ml of reacted solution was collected, added with 2 ml of 0.1% Ninhydrin solution and then heat to 80 °C for 10 minutes. For next step, 2 ml of remaining reacted solution was collected, added with 2 ml of 0.1% Ninhydrin solution and then heat to 80 °C for 10 minutes. Solution was allowed to cool in room temperature, was transferred to 10 ml of volumetric flask and then diluted with water to the mark. Colour changes of solutions were observed and then the solution analysed with UV-Vis spectrophotometer.  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Chapter 4. Result and Discussion  

4.1 Pharmacokinetic Simulation of Aspirin® A simplified tool to simulate ADME process that cheaply and easily enough to be used in undergraduate practical was studied. Before simulating metabolism process, it is important to check the simulator's performance and to find concentrations of tested drug using simple UV-Vis spectroscopy.

Generally, when a drug is administered orally, it will enter through the mouth, experience absorption in the stomach and/or intestines (GI tract), metabolism in the liver, distribution throughout the body in the blood, and then excreted through the urine, respectively. The drug in the blood, will continue to circulate in the general circulation by the heart pumping and experience the process of elimination (metabolism and excretion) repeatedly until disappears from the blood.

The system used in simulator has several parts as similitude of the human body. It consists of a drug container (funnel) as similitude of the mouth, a porous tube as similitude of the GI tract, a separating funnel (will be filled with resin) as similitude of the liver, a water bath contains DI water (with the aid of a fish tank filter that stirs the waters) as similitude of the body compartment with the blood, and an urine container (measuring flask) as similitude of the bladder. It is connected with a circulated tubing system and a peristaltic pump as similitude of the general circulation and the heart.

The fact that the system as such simulates a one-compartment model because it only has one reservoir (the water bath) that is well stirred, and there is no separate reservoir for distribution and retention of the drug such as in two-compartment model. And since there is no metabolism in this simulation, the elimination was exclusively determined by the excretion through urine and it follows zero-order kinetics (at constant rate). In the future, studies to simulate a two and three compartment models will be realised.

The simulation was performed as described in procedure 3.3.1, the samples from measuring flask (simulates urine) were collected and the accumulated volume of water was recorded. Absorbance of the samples were observed using UV-Vis spectrophotometer instrument. The absorbance obtained was used to calculate the concentrations of Aspirin® in the samples using regression line equation of Aspirin (ASA) standard solutions (y=1.509 Conc. - 0.018).

As the result, there is an increasing in the concentration of Aspirin® excreted in the 'urine' (Table 4.1). The “quantity of aspirin eliminated” increases but the concentration of aspirin in urine remains more or less constant. It can be clearly seen from the accumulated amount of Aspirin® (Figure 4.1) that the elimination was taken place at constant rate (follows zero-order kinetics). The deviations are due to the fact that we started collecting 'urine' when part of the drug has not been completely absorbed (sample was not from the bath).

From the results of this experiment, the only pharmacokinetic parameter could be

calculated is the rate of elimination (k) and the half-life (t1/2). Since the process followed zero-order kinetics, the k value is the slope of the regression equation, which is 0.259

 

25  

mg/min. The half-life is equals to the original amount of drug (300mg) divided by two times of elimination rate, resulting value of 579.2 minutes or 9.6 hours.

Table 4. 1 Absorbance of Aspirin® sample solutions from measuring flask (simulating urine in human body) over the time measured by UV-Vis spectroscopy and Aspirin® concentrations obtained by calculation using standard regression line.

'Urine' Data

Time (min)

Abs Concentration

(mg/l) Cumulative Volume

Excreted (ml) Cumulative Amount

Excreted (mg) 2.5 0.0517 9.8 25 0.2

5 0.0664 13.5 50 0.7

10 0.0939 20.4 100 2.0

15 0.1082 24.0 145 3.5

20 0.1135 25.3 185 4.7

25 0.1180 26.4 225 5.9

30 0.1232 27.7 265 7.3

35 0.1235 27.8 300 8.3

40 0.1282 29.0 335 9.7

45 0.1344 30.5 365 11.1

50 0.1405 32.0 395 12.6

 

Figure 4. 1 Cumulative amount of Aspirin® excreted into measuring flask (simulating urine in human body) over the time.

We could not have more than zero order because there was no metabolism and we could not have more than one compartment because there is no place in the simulator apart

y = 0.259x ‐ 0.518R² = 0.999

0

2

4

6

8

10

12

14

0 10 20 30 40 50 60

Cu

mu

lati

ve a

mo

un

t ex

cret

ed

(mg

)

Time (minute)

Aspirin concentrations versus time from 'urine sample'

 

26  

from the bath for the drug to be retained. More works have to be done for the model to be completed. However, we saw that the simulator have potential to become really valuable educational tool.

We envision that when a suitable method to simulate metabolism of Aspirin® in the simulator has been found, we will be able to simulate the pharmacokinetics of it. In addition, the salicylic acid formed as the result of the metabolism (hydrolysis reaction) can be detected by using UV-Vis spectroscopy. Aspirin® and salicylic acid can be distinguished by reacting them with iron (III) chloride. Aspirin® does not react with iron (III) chloride, while salicylic acid will reacted and gives purple colour of complex iron-salicylic (Ogawa and Tobe, 1966). This method will be very useful in practical because the metabolism process can be clearly seen by the students. Furthermore, iron-salicylic complex also has absorption at different wavelengths with Aspirin® (Aspirin® at 289.5 nm whereas Iron-salicylic complex at 531.5 nm) that can be detected and quantified by using UV-Vis spectroscopy.

4.2 Hydrolysis of Aspirin®

4.2.1 Hydrolysis of Aspirin® with immobilised esterase in medium of DI water Considering that the aim of the project is to make an ADME simulation as an educational tool for ADME undergraduate student, we tried to use simple materials and instrumentation and reactions that can finished in less than two hours.. The primary choice for use as a medium is DI water for the reason that water is a universal solvent, neutral, non-toxic and easily available to be used in the laboratory practical. In this experiment (Procedure 3.3.2.1) we tried to use water as the medium of Aspirin® hydrolysis reaction. After incubation at 37 °C for one hour, iron (III) chloride was added into the solution. There was no change in the colour of the sample solution observed which proves that salicylic acid was not formed as the result of the hydrolysis reaction. We conclude that the water cannot be used as medium for hydrolysis of Aspirin® by immobilized esterase on Eupergit®.

4.2.2 Hydrolysis of Aspirin® with immobilised esterase in medium of Tris-HCL buffer Based on unsuccessful experiments with the water, we tried to use another media namely Tris-HCl buffer for the hydrolysis. Tris ,or tris(hydroxymethyl)aminomethane, is an organic compound widely used in the biology/biochemistry practises as a component of buffer solution to simulate the physiological pH (7-9). Tris-HCl buffer was used as enzyme medium in the study of enzymatic hydrolysis of aspirin by human esterase (Inoue et al, 1979). This experiment can be considered successful in the study for the hydrolysis of Aspirin®. According to procedure 3.3.2.2 described previously, a solution of 0.02 M iron (III) chloride was added into each sample after the incubation process. At each sample tested, the purple colour was formed. This colour was a proof that the hydrolysis reaction of Aspirin® occurs and salicylic acid resulted from the reaction formed coloured complex (iron-salicylic) with the addition of iron (III) chloride. Coloured complex formed were analysed by UV-visible spectroscopy method for the determination of salicylic acid produced (Figure 4.2).

 

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Using 531.5 nm as lambda maximum of the complex, the sample solution A (without immobilized esterase on Eupergit®) produced absorbance of 0.0317. Calculation by using calibration curve of salicylic acid standard (reacted with iron (III) chloride), gave 0.094 mg of salicylic acid concentration. Solution B (using 10 mg immobilized esterase) produced absorbance of 0.0983 and Solution C (using 30 mg immobilized esterase) produced absorbance of 0.2211. Using the standard calibration, concentration of salicylic acid obtained from solution B and C were 0316 mg and 0725 mg respectively. We used 10 mg of Immobilised esterase based on information from the producer (Sigma-Aldrich) that the product contains ~242 Unit/gram moist material, where 1 unit corresponds to the amount of enzyme which hydrolyses 1 μmol ethyl valerate per minute at pH 8.0 and 25°C. Assuming that Aspirin® is hydrolysed at same speed, 10 mg enzyme will hydrolyse 2.42 μmol/min or 145.2 μmol/h (equals to 26.2 mg of Aspirin®) In summary, using Tris-HCl buffer as a medium can also lead to hydrolysis reaction of Aspirin® with 0.47% yield. However, the use of 10 mg and 20 mg of immobilized esterase on Eupergit® result greater changes of Aspirin® with 1.59% and 3.64% yield respectively. The amount of enzyme used is very small, roughly we need at least 600 mg of enzyme to get over 95% yield from 26 mg of Aspirin®. Considering the amount of Aspirin® used in simulation (300 mg), in the future we should use a larger amount of enzyme to simulate the hydrolysis. It can be concluded from these experiment results that the immobilised esterase can be considered as the material for the metabolism simulation of Aspirin® in the developed simulator. However, there are differences in the conditions in which this experiment performed with that on the simulator. Contact between Aspirin® and immobilised esterase is continues over time in the experiment above, while in the simulator the reaction will be performed in circulation. With the volume of separating column (immobilised esterase container in the simulator) of 50 ml and the medium flow rate of 60 ml/min, the contact between the immobilised esterase and Aspirin® will lasted for less than a minute (although part of it will contact the resin later, like in the body). Further study is need to be conducted to see the effectiveness of hydrolysis reaction of Aspirin® with immobilised esterase in circulatory system as intended will be done in the simulator.

 Figure 4. 2 UV-Vis spectrum of (A) Aspirin (ASA) standard , (B) UV-Vis spectrum of Salicylic acid standard

with FeCL3, (C) UV-Vis spectrum of Aspirin with FeCL3 and (D) UV-Vis spectrum of Aspirin (after hydrolysed with immobilised esterase) with FeCL3.

 

28  

4.2.3 Hydrolysis of Aspirin® with silica gel In this experiment, we tried to find an alternative ways of hydrolysis of Aspirin® without the use of esterase. Silica has proved in causing hydrolysis of Aspirin®, so it is advised not to use it as adjuvant in the manufacturing process of drugs containing Aspirin® (Demianenko, 2011). The experiment was carried out using the procedure 3.3.2.3 and as the result there was no purple colour formed. This suggests that silica may not cause hydrolysis of Aspirin® within the timeframe set of one hour. Hydrolysis of Aspirin® may occur in a state without water with silica but this process takes place very slowly. This is in good agreement with the results of studies by Demianenko (2011) that Aspirin® hydrolysis by silica takes place at a slower rate than Aspirin® hydrolysis by water.

4.2.4 Hydrolysis of Aspirin® with Alumina Another experiment to find an alternative ways of hydrolysis of was conducted with the use of alumina. Aluminium oxide is an amphoteric substance, which means can react with both acids and bases to producing a salt (Chemguide, 2013). Since Aspirin® is a weak acid, we tried to see if the hydrolysis of Aspirin® may occur with the use of alumina. As the result of the experiment (procedure 3.3.2.4), there was no purple colour formed in sample solution after the addition of iron (III) chloride. This suggests that there was not hydrolysis reaction of Aspirin® with the use of alumina within the timeframe set of one hour.

4.2.5 Hydrolysis of Aspirin® with Amberlite® resin Amberlite® IRA402 Cl is an anion exchange and is a strong basic. It has trimethyl ammonium functional group and used in demineralization of water (Dow Chemical Company, 2013). As the result of the experiment (procedure 3.3.2.5), there was no purple colour formed in sample solution after the addition of iron (III) chloride. This suggests that there was not hydrolysis reaction of Aspirin® with the use of Amberlite® within the timeframe set of one hour.

4.3 Reduction of AZT

In this project, we also wanted to try to simulate the metabolism of zidovudine (AZT). In particular, reduction of the azide functional group on AZT to the primary amine functional group on 3'-amino-3'-deoxythymidine (AMT) metabolite.

4.3.1 Reduction via Staudinger Reaction

The first experiment we did was the reduction of azide functional group via Staudinger reaction procedure. Staudinger reaction to convert azide to amine is widely used

 

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in the organic synthesis process (Murphy et al., 2008), in peptide synthesis (Nilsson, 2001) and oligonucleotide mimics synthesis (Worthington et al., 2007). 4.3.1.1 Reduction at 37 °C This experiment was conducted using triphenyl phosphine as a reductor agent and THF as the solvent (Procedure 3.3.3.1.1). Reaction process carried out for two hours at 37 ° C and an amine formation was expected to occur in the presence of water. TLC method was used as an initial detection that reduction reaction of AZT by PPh3 occurs. As the result from observation of TLC plat under UV light 254 nm, there was a change in the number of spot of AZT which only one spot of the original sample AZT turned into four spots after the reaction (Table 4.2). This was proving the occurrence of reactions leading to molecular changes of AZT.

Table 4. 2 Number of spots and retention factor of AZT, PPH3 and AZT reaction with PPH3 at 37 °C

Sample Number of Spots Rf AZT 1 0.61 PPH3 1 0.95

AZT+PPH3 4

0.04 0.37 0.80 0.95

We conducted further tests using Infra Red (IR) spectroscopy to detect the changes in azide functional groups to an amine on AZT as a the results of the reduction. From the IR spectra of sample obtained, there was no absorption of azide functional group (2109 cm-1) as in the AZT sample and also no absorption of amine functional group (3300-3500 cm-1) (Figure 4.3). It proves that there was no amine formation in this experiment. This may be due to the reaction of amine formation occurs very slowly, as the experiment that performed by Fox and Edgar (2012) which lasted over the night. An overnight reaction will not serve the purpose of the simulator that so the procedure is excluded.

4.3.1.2 Reduction at 80 °C Experiment was conducted using the same procedure as above but using a higher temperature of 80 °C. We tried to see if amine-forming reaction can take place rapidly at higher temperature (Procedure 3.3.3.1.2). The observation using a TLC method showed a change in the spot of AZT which originally one spot changed to four spots (Table 4.3). Further test using IR spectroscopy was showed that there was no absorption of azide functional group (2109 cm-1) and also no absorption of amine functional group (3300-3500 cm-1) (Skoog et al., 1998). There was no amine formation resulting from this experiment, thus it can be concluded that using higher temperature (80 °C) was also not effective to increase the process of amine formation (Figure 4.3). Furthermore, using 80 °C reaction will not serve the purpose of the simulator (not possible in practical) that so the procedure is excluded.

 

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Table 4. 3 Number of spots and retention factor of AZT, PPH3 and AZT reaction with PPH3 at 80 °C

Sample Spot Rf AZT 1 0.58 PPH3 1 0.92

AZT+PPH3 4

0.04 0.29 0.79 0.94

 Figure 4. 3 IR spectrum of (A) AZT , (B) AZT reaction with PPH3 at 37 °C and (C) AZT reaction with PPH3 at 80 °C.

 

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4.3.2 Reduction with Thiols (Dithiothreitol and Glutathione) 4.3.2.1 Reduction with Dithiothreitol (DTT)  

This experiment was conducted at 37 °C by using DTT as a reducing agent and DI water as medium (Procedure 3.3.3.2.1). After one hour of reaction process, sample was analysed by TLC method. As the result from observation of TLC plat under UV light 254 nm, there was a change in the number of spot of AZT which only one spot of the original sample AZT turned into five spots after the reaction (Table 4.4). This was proving the occurrence of reactions leading to molecular changes of AZT.

Table 4. 4 Number of spots and retention factor of AZT, DTT and AZT reduction with DTT at 37 °C

Sample Spot Rf AZT 1 0.70 DTT 1 0.75

AZT+ DTT 5

0.01 0.40 0.56 0.76 0.83

We conducted further tests using IR spectroscopy to detect the changes in azide functional groups to an amine on AZT as the results of the reduction. From the IR spectra of sample obtained, there was no absorption of azide functional group (2098 cm-1) as in the AZT sample and there was a presence of absorption in 3295 and 3419 cm-1 which is within range of amine functional group (3300-3500 cm-1) (Figure 4.4). It can be concluded from the result that reduction with DTT can be used as method for reduction of azide to amine functional group on AZT.

Since AZT and AMT metabolite has absorption at the same wavelength in ultra violet region, we need to find a method to distinguish between these compounds with UV-Vis spectroscopy. In this experiment, colorimetry technique was used since the AMT has amine functional group that can be reacted with ninhydrin to form a coloured compound called Ruhemann’s purple (MacFadyen, 1950). As the results, there was a purple colour formed in solution of AMT (AZT with DTT reduction) while solution AZT does not give the same colour. A further analysis with UV-Vis spectrophotometer scanning within range of 200-600 nm was performed to determine the spectrum differences between AZT-Ninhydrin and AMT-Ninhydrin solution. The scanning results had showed differences in the spectrum form and the absorbance of the peak. AZT-Ninhydrin solution gave the peak absorption at 296.5 nm while AMT-Ninhydrin solution gave that at 565.0 nm (Figure 4.5).

 

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Figure 4. 4 IR spectrum of (A) AZT, (B) AZT reaction with DTT at 37 °C and (C) Aniline (from Orgchem, 2013)

Although this experiment has shown successful reduction of AZT to AMT, the amount of AMT produced could not be calculated due to the absence of standard AMT. We need to purchase or prepare the standard in the future. This metabolism procedure may be possible to be used in practical. The student can clearly seen that the metabolism takes place by observing the change in the concentration of AZT during the course of time and detection (also calculation) of the AMT (metabolite formed) using UV-Vis spectroscopy as describe above.

 

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 Figure 4. 5 UV-Vis spectrum of (A) AZT with 1% Ninhydrin , (B) DTT with 1% Ninhydrin and (C) AZT (reacted with DTT) with 1% Ninhydrin.

4.3.2.2 Reduction with DTT, Active charcoal and Amberlite® resin Experiment was conducted in order to see the reduction of AZT to AMT with system used in ADME simulator where the reaction is carried out in the separating column and lasts by circulation (Procedure 3.3.3.2.2). In this experiment, the separating column was filled with cotton wool and active charcoal at the bottom. The charcoal was added to reduce pungent odour of DTT. DTT was mixed with the Amberlite® resin and then the mixture was transferred into the column. Amberlite® resin has granules form and fills the separating column properly. The mixing was expected to help spreading DTT along the column. By spreading the DTT, we can be sure that the AZT will reacted with it while going down along the column After reaction for one hour, sample was collected and ninhydrin solution was added into the sample. As result, there was no purple colour occurs so that as conclusion there was no amine resulted from the reaction process. This may occur due to the reaction between DTT and Amberlite since Amberlite is a strong alkaline substance (Dow Chemical, 2013) which can possibly causing the loss of the ability of DTT to reduce AZT. Another possibility is that DTT and the amine may be absorbed by the charcoal, since it is used in medicine to absorb poisons (including drugs) (Medicinescomplete, 2013)

 

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4.3.2.3 Reduction with Glutathione

The aim of this experiment is to see whether the reduction of AZT to AMT can also performed by using glutathione as reducing agent. Glutathione is also a thiol group (such as DTT) and has been also reported to reduce AZT to AMT (Handlon and Oppenheimer, 1988). Glutathione also provides reducing capacity for several reactions in the body, such as the formation of deoxyribonucleotides by ribonucleotide reductase and the reduction of dehydroascorbate to ascorbate (Pastore et al., 2003). We performed experiment with use of Glutathione by consideration that it has less smell than Dithiothreitol to be used in practical. Sample was collected after one hour of the reaction and was analyzed using TLC technique (procedure 3.3.3.2.3). The observation of TLC plat under UV light 254 nm showed that there was no changing of AZT spot (Table 4.5). From this result, it can be concluded that glutathione does not reacted with AZT in the experiment and a further study is needed to explain this result.

Table 4. 5 Number of spots and retention factor of AZT, Glutathione and AZT with Glutathione reaction at 37 °C

Sample Spot Rf AZT 1 0.73 Glutathione 1 0.01

AZT+ Glutathione 2 0.01 0.71

 

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Chapter 5. Conclusion  

In the present work, the simulator was successfully demonstrated the process of absorption and excretion by using Aspirin® as model drug (simulation with Zidovudine was not conducted). However, this simulation was only showed a one compartment pharmacokinetic model since there was no part of the simulator for the drug to be retained and zero-order elimination process since there was no metabolism process (no resin were used). Hydrolysis of Aspirin® by using Immobilised esterase on Eupergit® in medium of Tris-HCL buffer showed a good result of the reaction, and reduction of Zidovudine (azido thymidine or AZT) was also successfully done by using Dithiothreitol as the reducing agent. A simple UV-Vis spectroscopy method can be used to distinguish and to calculate the concentration of drug and the metabolite, such as between Aspirin® and salicylic acid and AZT and AMT (aminothymidine). In future, further studies are need to be conducted to apply this metabolism procedure in simulator by means solid phase reaction method that previously planned. A two-compartment model simulator is also possible to be studied in the future

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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