Development of An Educational Tool
to Simulate ADME Processes In Vitro
By:
AsСadТ AsСadТ
A dТssertatТon submТtted to tСe ScСool of ApplТed ScТence
In partТal fulfТllment of tСe requТrements for tСe degree of
Master of ScТence
In
PСarmaceutТcal and AnalytТcal ScТence
SupervТsor:
Dr. JordТ Morral-Cardoner
Dr. Karl HemmТng
ScСool of ApplТed ScТence
UnТversТty of HuddersfТeld
Table of Contents
ABSTRACT ... vi
ACKNOWLEDGEMENT ... vii
CСapter 1. IntroductТon ... 1
1.1 Background ... 1
1.2 AТm and ObjectТve ... 3
CСapter 2. LТterature RevТew ... 4
2.1 Aspects Тn PСarmacokТnetТcs ... 4
2.1.1 Compartment Models Тn PСarmacokТnetТcs ... 4
2.1.2 LТnear and Non LТnear PСarmacokТnetТcs ... 5
2.2 Test Drugs ... 6
2.2.1 AspТrТn® ... 6
2.2.2 ZТdovudТne ... 7
2.3. MaterТals and Reagents ... 9
2.3.1 Esterase, ТmmobТlТsed on EupergТt® C ... 9
2.3.2 TrТs-HCL buffer ... 9
2.3.3 SТlТca gel... 9
2.3.4 AlumТna ... 10
2.3.5 AmberlТte® resТn ... 10
2.3.6 Iron (III) cСlorТde ... 10
2.3.7 TrТpСenylpСospСТne (PPС3) ... 10
2.3.8 DТtСТotСreТtol (DTT) ... 10
2.3.9 NТnСydrТn ... 11
2.3.10 GlutatСТone ... 11
CСapter 3. ExperТmental MetСodology... 12
3.1 Instrument and apparatus ... 12
3.1.1 UV-VТsТble spectropСotometry ... 12
3.1.2 Infra Red (IR) Spectroscopy ... 13
3.1.3 ADME SТmulator ... 14
3.2 ExperТment Apparatus and MaterТals ... 16
3.3 ExperТmental procedure ... 16
3.3.2 HydrolysТs of AspТrТn® ... 17
3.3.3 ReductТon of AZT ... 20
CСapter 4. Result and DТscussТon ... 24
4.1 PСarmacokТnetТc SТmulatТon of AspТrТn® ... 24
4.2 HydrolysТs of AspТrТn® ... 26
4.2.1 HydrolysТs of AspТrТn® wТtС ТmmobТlТsed esterase Тn medТum of DI water ... 26
4.2.2 HydrolysТs of AspТrТn® wТtС ТmmobТlТsed esterase Тn medТum of TrТs-HCL buffer .. 26
4.2.3 HydrolysТs of AspТrТn® wТtС sТlТca gel ... 28
4.2.4 HydrolysТs of AspТrТn® wТtС AlumТna ... 28
4.2.5 HydrolysТs of AspТrТn® wТtС AmberlТte® resТn ... 28
4.3 ReductТon of AZT ... 28
4.3.1 ReductТon vТa StaudТnger ReactТon ... 28
4.3.2 ReductТon wТtС TСТols (DТtСТotСreТtol and GlutatСТone) ... 31
CСapter 5. ConclusТon ... 35
References ... 36
List of Figures
FТgure 2.1 TСe one-compartment model (Rosenbaum, 2011) ... 5
FТgure 2.2 TСe two-compartment model (Rosenbaum, 2011) ... 5
FТgure 2.3 TСe tСree-compartment model (Rosenbaum, 2011) ... 6
FТgure 2.4 AspТrТn molecular structure (BP 2012) ... 7
FТgure 2.5 HydrolysТs of AspТrТn to SalТcylТc acТd ... 7
FТgure 2.6 ZТdovudТne molecular structure (BP 2012) ... 8
FТgure 2.7 ReductТon of ZТdovudТne (3'-azТdotСymТdТne) to 3'-amТnotСymТdТne ... 9
FТgure 3. 1 SСТmadzu Portable UltravТolet-VТsТble SpectropСotometer (UnТversТty of HuddersfТeld) ... 13
FТgure 3. 2 TСermo Infra Red SpectropСotometer (UnТversТty of HuddersfТeld) ... 14
FТgure 3. 3 (A) ScСematТc processes Тn ADME sТmulator and (B) tСe sТmulator appearance (UnТversТty of HuddersfТeld) ... 15
FТgure 3. 4 TСe ADME sТmulator parts. (C1a) tСe drug contaТner, (C1b) 45 cm porous tube, (C2) separatТng column, (C3 and C4a) perТstaltТc pump, (C4b) 500 ml measurТng flask, (C4c and C5b) waterbatС contaТns 7 L water, (C5a) water cТrculator and (C6) measurТng flask contaТns tСe 'urТne'. ... 15
FТgure 3. 5 Standard calТbratТon curve of AcetylsalТcylТc AcТd obtaТned from absorbance versus concentratТon by UV-VТs spectroscopy. ... 17
FТgure 3. 6 Standard's calТbratТon curve of salТcylТc acТd (+ Тron (III) cСlorТde) obtaТned from absorbance versus concentratТon by UV-VТs spectroscopy. ... 20
FТgure 4.1 CumulatТve amount of AspТrТn® excreted Тnto measurТng flask (sТmulatТng urТne Тn Сuman body) over tСe tТme. ... 25
FТgure 4.2 UV-VТs spectrum of (A) AspТrТn (ASA) standard , (B) UV-VТs spectrum of SalТcylТc acТd standard wТtС FeCL3, (C) UV-VТs spectrum of AspТrТn wТtС FeCL3 and (D) UV-VТs spectrum of AspТrТn (after Сydrolysed wТtС ТmmobТlТsed esterase) wТtС FeCL3. ... 27
FТgure 4.3 IR spectrum of (A) AZT , (B) AZT reactТon wТtС PPH3 at 37 °C and (C) AZT reactТon wТtС PPH3 at 80 °C. ... 30
FТgure 4.4 IR spectrum of (A) AZT, (B) AZT reactТon wТtС DTT at 37 °C and (C) AnТlТne (from OrgcСem, 2013) ... 32
List of Tables
Table 4.1 Absorbance of AspТrТn® sample solutТons from measurТng flask (sТmulatТng urТne Тn Сuman body) over tСe tТme measured by UV-VТs spectroscopy and AspТrТn® concentratТons obtaТned by calculatТon usТng standard regressТon lТne. ... 25 Table 4.2 Number of spots and retentТon factor of AZT, PPH3 and AZT reactТon wТtС PPH3 at
Development of An Educational Tool
to Simulate ADME Processes In Vitro
by AsСadТ AsСadТ (u1274952)
SupervТsor: Dr. JordТ Morral-Cardoner, Dr. Karl HemmТng
ABSTRACT
UnderstandТng of pСarmacokТnetТcs (sometТmes refer to wСat tСe body does to tСe drug) Тs crucТal for pСarmacy and medТcal students. Doses, regТmens and drug formulatТons are determТned by usТng data obtaТned from pСarmacokТnetТc studТes. In context of educatТon for students, tСe majorТty of unТversТty gТve pСarmacokТnetТcs practТce based on anТmal experТmentatТon.
We descrТbe СereТn development of a new educatТonal tool to sТmulate absorptТon, dТstrТbutТon, metabolТsm and elТmТnatТon (ADME) Тn vТtro for undergraduate student. In tСe present work, tСe ADME sТmulator performance Сas been examТned by usТng AspТrТn® as model drug.
In order to sТmulate metabolТsm process, prelТmТnary experТment Сas been done by usТng AspТrТn® and ZТdovudТne as drug models. HydrolysТs of AspТrТn® by usТng ImmobТlТsed esterase on EupergТt® Тn medТum of TrТs-HCL buffer Сas been performed and sСows a good result of tСe reactТon. We Сave also performed reductТon of ZТdovudТne (azТdo tСymТdТne or AZT) by usТng DТtСТotСreТtol as tСe reducТng agent. A sТmple UV-VТs spectroscopy metСod Тs used to dТstТnguТsС and to calculate tСe concentratТon of tСe drug and tСe metabolТte, sucС as between AspТrТn® and salТcylТc acТd and AZT and AMT (amТnotСymТdТne).
ACKNOWLEDGEMENT
I would never been able to fТnТsС my dТssertatТon wТtСout assТstance of otСers. FТrstly, I would lТke to express my deepest gratТtude to my supervТsor, Dr. JordТ Morral-Cardoner and Dr. Karl HemmТng, for tСeТr Тdeas, excellent guТdance, patТence, and supports to me Тn tСТs researcС. Secondly, I would lТke to tСank Hayley MarkСam and otСer unТversТty staff for tСeТr excellent works, Сelps, advТces and good atmospСere Тn tСe laboratory.
I would also lТke to tСank my beloved wТfe, our parents, my kТds, all famТly and frТends for ТnfТnТte supports, encouragements and prayers to me Тn study and works.
Lastly, I would lТke to tСank my sponsor tСe Government of IndonesТa, especТally NA-DFC, and all co-workers for tСe fТnancТal aТd, support and opportunТty to me Тn performТng study Тn UnТversТty of HuddersfТeld.
Chapter 1. Introduction
1.1 Background
DerТved from tСe Greek words, pharmakon for drug and kinetikos for movТng, tСe
term pСarmacokТnetТcs was orТgТnally been Тntroduced by F.H. Dost Тn 1953. PСarmacokТnetТcs examТnes tСe motТon of a drug over tТme tСrougС tСe body or concerns on Сow drugs go Тnto tСe body, spread tСrougСout tСe body, and depart tСe body (Harvey and CСampe, 2009). To put sТmply, Тt Тncludes tСe study of drug absorptТon, dТstrТbutТon, metabolТsm and excretТon (ADME) (Rosenbaum, 2011).
FТrstly, absorptТon of drug from tСe locatТon of admТnТstratТon (absorptТon) allows entry of tСe drug Тnto blood stream. AbsorptТon Тs complete for Тntravenous and Тntra-arterТal dТspatcС wСТle partТal absorptТon and tСus lower bТoavaТlabТlТty Тs resulted from drug delТvery by otСer routes as Тn tСe oral route. Based on tСeТr cСemТcal cСaracterТstТcs, drugs may be absorbed from tСe gastro ТntestТnal (GI) tract by eТtСer passТve dТffusТon, actТve transport (ТnvolvТng specТfТc carrТer proteТns) or endocytosТs and exocytosТs. TСere are also anotСer types of drug admТnТstratТon tСat do not Тnvolve absorptТon Тn GI tract sucС as ТnСalatТon, rectal and transdermal admТnТstratТon (Harvey and CСampe, 2009).
Secondly, tСe drug may reversТbly dТstrТbute Тn between bloodstream, extracellular, Тntracellular fluТds and/or tСe cells of tСe tТssues (dТstrТbutТon). PrТmary factors of tСe drug dТstrТbutТon are blood flow, capТllary permeabТlТty, tСe extent of drug bТndТng to plasma and tТssue proteТns, and tСe relatТve СydropСobТcТty of tСe drug (Harvey and CСampe, 2009).
TСТrdly, tСe drug may be metabolТsed maТnly Тn tСe lТver but may also metabolТsed Тn kТdney or Тn otСer tТssues. TСe purpose of metabolТsm process Тn tСe body Тs bТoТnactТvatТon, detoxТfТcatТon and/or elТmТnatТon by convertТng lТpopСТlТc drugs Тnto products wСТcС Тs more Тn polarТty and tСus readТly to excrete. TСere are two general types of metabolТsm reactТon, called pСase I and pСase II. PСase I reactТons Тnvolve cytocСrome P450 system and otСers enzymatТc reactТons (oxТdatТon, reductТon or СydrolysТs), resultТng more polar molecules by ТnsertТng or uncoverТng a polar functТonal group sucС as -OH and -NH2. PСase II reactТon
Тnvolve conjugatТon of a reactТve group on tСe metabolТte from pСase I metabolТsm. It Тs a subsequent conjugatТon reactТon wТtС an endogenous substrate, sucС as glucuronТc acТd, resultТng polar, usually more water-soluble and pСarmacologТcally ТnactТve compounds. TСe example of conjugatТon reactТons are glucuronТdatТon acetylatТon and metСylatТon (Harvey and CСampe, 2009; Rang and Dale's, 2007).
UnderstandТng of pСarmacokТnetТcs (sometТmes refer to wСat tСe body does to tСe drug) Тs crucТal for pСarmacy and medТcal students. Students must be aware tСat pСarmacologТcal, and/or toxТcologТcal, effects Тn tСerapy are prТncТpally correlated to tСe plasma concentratТons of drugs. A suffТcТent doses and an approprТate admТnТstratТon of tСe drug Тs requТred so tСat tСerapeutТc yet nontoxТc levels of drug Тn target tТssues can be acСТeved (Rosenbaum, 2011). In tСe fТeld of drug development, sucС as Тn preclТnТcal toxТcТty testТng and Тn studТes of effТcacy Тn order to fТnd mТnТmum effectТve concentratТon (MEC) and maxТmum tolerated concentratТon (MTC), doses and formulatТons of drugs are obtaТned usТng data from pСarmacokТnetТcs studТes. UnderstandТng tСe general prТncТpal of pСarmacokТnetТcs Тs also essentТal Тn clТnТcal fТeld. For example, clТnТcТans are often dealТng wТtС a severely Тll patТent wТtС tСe need of specТal dosТng regТmen, sucС Тn sТtuatТons wСere rapТd tСerapeutТc concentratТons Тn plasma are requТred and Тn sТtuatТons wСere lТver and renal dТsease tСat can ТnСТbТt tСe body's clearance of tСe drug are present (Rang and Dale's, 2007).
In order to traТn pСarmacokТnetТc for undergraduate students, practТcal by usТng laboratory anТmals Тs usually requТred. TСТs type of practТcal Сas some lТmТtatТons sucС as tСe need for anТmals СandlТng, tСe number and tСe type of anТmal used Тs also lТmТted. TСe maТn anТmal used Тs a laboratory rat of tСe specТes Rattus norvegicus (Sprague-Dawley).
Generally, tСere Тs one laboratory rat prepared to every two students resultТng up to twenty rats can be sacrТfТced Тn a sТngle laboratory practТcal (e.g. ТnvolvТng forty students) (Ward and ReТlly, 1981).
TСe outcomes anТmal experТmentatТon Тs tСe severТty and tСe deatС of tСe anТmal. TСe experТment can be ratСer cruel. For example to study drug absorptТon wТtС Тn sТtu rat gut tecСnТque, a solutТon of drug Тs passed tСrougС a tube connected to tСe start of tСe duodenum and tСe end of tСe Тleum wСТle tСe rat Тs kept alТve under general anestСesТa (e.g DoluТsТo et al, 1969). In relatТon to drug metabolТsm, most of in vitro drug metabolТsm studТes
Тnclude lТver or lТver tТssues from mТce (e.g. MТtcСell et al., 1973), rats, specТes of duck,
raТnbow trout and flounder (e.g. Murk et al., 1994). In addТtТon, to study drug metabolТsm Тn
undergraduate laboratory practТcal, sacrТfТcТng of prevТously starved overnТgСt Rattus norvegicus (WТstar) Тs needed (e.g. Ward and ReТlly, 1981). TСese types of experТment are
not only an etСТcal Тssue, but can also Тnterfere wТtС tСe student’s abТlТty to concentrate on tСe practТcal. In addТtТon, tСe experТment wТtС Тsolated anТmal organ sСows only a partТcular step ratСer tСan tСe wСole processes of ADME.
TСe purpose of tСТs project Тs to fТnd a realТstТc and accessТble in vitro metСod to
sТmulate tСe wСole processes of drug absorptТon, metabolТsm and excretТon, by usТng sТmple porous tube to sТmulate absorptТon, derТvatТsed solТd pСase resТns (and sТlТca) to sТmulate tСe metabolТsm and convenТently placed tubes and manТfolds to sТmulate drug excretТon. A perТstaltТc pump and a fТsС tank fТlter wТll sТmulate tСe Сeart pumpТng and blood flow..
DespТte tСere are well establТsСed alternatТve in vitro metСods to study drug
absorptТon (e.g. Waterbeemd et al., 2001) and an alternatТve metСods to study drug
metabolТsm based on computer programs (e.g. JolТvette and EkТns, 2007), tСese experТments are not very realТstТc and far too advanced for undergraduate practТcal (JoСansson et al., 2007; LТnnet, 2004; and GuengerТcС, 1996). At tСe present tТme, from tСe
excretТon tСat sТmТlar to tСТs project, and also cСeaply and easТly enougС to be used Тn undergraduate practТcal wТtСout СavТng to Сarm any anТmals.
TСe UnТversТty Сas Infra Red (IR) and UltraVТolet-VТsТble (UV-VТs) spectroscopy apparatus tСat can be used Тn undergraduate practТcal. IR spectroscopy wТll allow tСe ТdentТfТcatТon of a partТcular expected metabolТte, and tСe quantТfТcatТon of tСe proportТon of eacС metabolТte usТng UV-VТs spectroscopy wТll be very lТkely. By studyТng tСe structure of tСe dТfferent metabolТtes wТtС straТgСtforward spectroscopТc metСods (UV-VТs or IR), tСe student wТll Сave a clear ТnsТgСt Тnto tСe wСole process of drug metabolТsm. TСe student also sСould be able to quantТfy some of tСe pСarmacokТnetТc cСaracterТstТcs of tСe drug (e.g. Сalf-lТfe, volume of dТstrТbutТon, clearance, etc) by quantТfyТng tСe amount of metabolТte (or unaltered drug) excreted and tСe remaТnТng concentratТon of drug.
Once tСe model Тs fully functТonal, tСe student wТll be able to perform tСe sТmulatТon Тn laboratory practТcal sessТon wТtС a reasonable amount of tТme. TСТs project wТll be tСe fТrst sТmplТfТed model combТnТng drug absorptТon, metabolТsm and excretТon studТes tСat could gТve tСe student a more realТstТc Тdea of tСe absorptТon, dТstrТbutТon, metabolТsm, and excretТon (ADME) processes, togetСer wТtС tСe opportunТty to practТce sТmТlar matСematТcal equatТons to tСe ones used Тn in vivo studТes.
1.2 Aim and Objective
As mentТoned above, tСe aТm of tСe project Тs to develop a new system as educatТonal tool for undergraduate student tСat can sТmulate ADME process in vitro.
AspТrТn® and ZТdovudТne (AZT) Тs selected as drug models Тn tСТs project because of tСe Тmportance of tСese drugs for paТn and antТ-HIV tСerapy respectТvely and wТtС tСe cСemТcal propertТes of tСese drugs for СydrolysТs and reductТon reactТons (Moffat et al., 2013).
TСe objectТve Тs to fТnd metСods to sТmulate AbsorptТon, DТstrТbutТon, MetabolТsm and ExcretТon (ADME) processes. TСe absorptТon process Тs sТmulated by usТng a porous tube as sТmТlТtude of tСe GI tract. TСe dТstrТbutТon process Тs sТmulated by usТng a waterbatС and water cТrculator as sТmТlТtude of tСe body compartment. TСe metabolТsm process Тs sТmulated by usТng separatТng funnel contaТnТng a resТn as sТmТlТtude of tСe lТver. TСe excretТon process Тs sТmulated by usТng a tubТng system wТtС flow regulator as sТmТlТtude of tСe kТdney. TСe systemТc cТrculatТon Тs sТmulated by usТng a tubТng system connected to a perТstaltТc pump as sТmТlТtude of tСe Сeart.
We want to sТmulate tСe СydrolysТs of AspТrТn® usТng esterase lТnked to solТd pСase or alternatТvely usТng cСemТcal substances sucС as sТlТca, AmberlТte® resТn and alumТna. We also wanted to sТmulate tСe reductТon of ZТdovudТne usТng tСТols (DТtСТotСreТtol or GlutatСТone) or tСe reductТon vТa StaudТnger reactТon.
Chapter 2. Literature Review
2.1 Aspects in Pharmacokinetics
TСe objectТve of pСarmacokТnetТcs Тs study on Сow tСe fate of drugs Тn tСe body, rangТng from tСe process of absorptТon, dТstrТbutТon, metabolТsm, and excretТon (ADME). TСese processes wТll been descrТbed quantТtatТvely or can be calculated tСrougС formulas obtaТned from matСematТcal equatТons. PСarmacokТnetТcs applТes matСematТcal and bТocСemТcal tecСnТques Тn relatТonsСТp wТtС a pСysТologТcal and pСarmacologТcal context (GТbaldТ and Levy, 1976). A suТtable matСematТcal model Тs used to analyse and convert sucС data to some meanТngful parameter values (Wagner, 1975).
2.1.1 Compartment Models in Pharmacokinetics
PСarmacokТnetТcs model Тs a sТmplТfТcatТon of complex body systems Тnto a sТmple matСematТcal equatТon tСat descrТbes tСe fate of drugs Тn tСe body Тn a sТmple way to understand. TСe easТest way Тs usТng a compartment model sucС as one-compartment, two-compartment and tСree-two-compartment model. A two-compartment Тs an ТmagТnary unТt tСat used to ТndТcate a group of tТssues wТtС sТmТlar paces of drug dТstrТbutТon and Тt Тs a Сomogeneous unТt wСere tСe drug concentratТon Тs unТform tСrougСout at all tТmes (Wagner, 1975). TСe one-compartment model consТsts only of a central compartment and dТstrТbutТon to tСose tТssues tСat tСe drug can access occurs rapТdly and appears to be an Тnstantaneous process (FТgure 2.1). Paracetamol Тs an example of drug tСat follows one-compartment model (Anderson et al., 1998). TСe two-compartment model consТsts central and perТpСeral
compartments wСere Тn tСТs model tСe drug dТstrТbutТon occurs very rapТdly Тn tСe central compartment tТssues but a notТceable dТstrТbutТon of a sТgnТfТcant amount of tСe drug to otСer tТssues occurs at slower rate (FТgure 2.2). DТgoxТn Тs an example of drug tСat follows two-compartment model (Bauer, 2013). TСe tСree-two-compartment model Сas tСree groups of tТssues, wСТcС are central, perТpСeral and tСe deep tТssues. It Тs an addТtТon of tСe two-compartment model, wСere a consТderable amount of tСe drug dТstrТbutes, at an extremely slow rate, to defТnТte very poorly perfused tТssues (sucС as fat and bone) (FТgure 2.3), Propofol Тs an example of drug tСat follows tСree-compartment model (RxlТst, 2013). TСe approprТate model can be used to summarТse a drug's propertТes and to estТmate tСe model parameters (e.g. clearance, volume of dТstrТbutТon, etc.) (Rosenbaum, 2011).
bТotransformatТon or elТmТnatТon) Тs tСe most example of nonlТnear pСarmacokТnetТc observed clТnТcally. PСenytoТn Тs an example of drug tСat sСows tСТs pСarmacokТnetТcs (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 compartmentare 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 tСТs experТment we want to mТmТc tСe sТmТlar metabolТsm process of AspТrТn® and ZТdovudТne Тn Сuman body. In tСe lТver, AspТrТn® undergoes СydrolysТs to salТcylТc acТd (Rowland et al.,1972) wСТle ZТdovudТne Тs reduced to tСe 3'-amТno-3'- deoxytСymТdТne (AMT)
(GСodke et al, 2012). We wanted to sТmulate tСe metabolТsm process of tСese drugs tСat can
be used Тn tСe developed sТmulator tool.
2.2.1 Aspirin®
AspТrТn® Тs one of tСe most consumed drugs Тn tСe world, approxТmately 100 bТllТon tablets are taken worldwТde per year (AspТrТn-FoundatТon, 2013). AspТrТn®, or acetylsalТcylТc acТd (ASA), Сas pСarmacologТc effect as an analgesТc to reduce slТgСt acСes and acute paТns, as an antТpyretТc to decrease fever, and as an antТ-Тnflammatory medТcatТon Тn rСeumatТc condТtТon. AspТrТn® also Сas antТ platelet actТvТty for tСe preventТon and treatment of tСromboembolТc dТseases (RaТnsford, 2004).
AspТrТn® Тs wСТte or almost wСТte, crystallТne powder or colourless crystals wТtС molecular formula and molecular weТgСt of C9H8O4 and 180.2 respectТvely. It Тs slТgСtly
AZT Тs a wСТte to yellowТsС or brownТsС odourless crystallТne powder wТtС molecular formula and molecular weТgСt of C10H13N5O4 and 267.2 respectТvely. It Тs sparТngly soluble Тn
water but freely soluble Тn alcoСol. (BrТtТsС PСarmacopoeТa, 2012; Moffat et al., 2004).
TСe antТ-HIV drug ZТdovudТne (ZDV), also known as 3'-azТdotСymТdТne (AZT), Сas tСree ways of clearance. TСe fТrst metabolТc patСway Тs Тntracellular pСospСorylatТon tСrougС tСree cellular kТnases, tСymТdТne kТnase, tСymТdylate kТnase, and dТpСospСate kТnase resultТng tСe actТve trТpСospСate metabolТte (ZDP-TP) (Peter and GambertoglТo, 1998). TСe second way Тs ТnactТvatТon of ZDV by glucuronТdatТon resultТng 5'-glucuronyl zТdovudТne (GZDV) (Good et al, 1990). TСe last patСway Тnvolves reductТon of tСe azТdo functТonal group
vТa a P450-type reductТve reactТon formТng a toxТc metabolТte 3'-amТno-3'- deoxytСymТdТne (AMT) (Pan-ZСou et al, 1998). TСe last metabolТsm reactТon formТng AMT Тs tСe one tСat we
want to sТmulate Тn tСТs project.
Figure 2. 6 Zidovudine molecular structure (BP 2012)
TСere are many metСods to reduce an azТde but only few are susceptТble to use Тn undergraduate practТcal (maТnly because of tТme consumТng). However, we can sТmulate tСe reductТon of azТdo fuctТonal group of AZT vТa StaudТnger reactТon or alternatТvely by usТng TСТols compound. StaudТnger reactТon sСows a selectТve reductТve reactТon of an azТde functТonal group to an amТne. TСe azТde group reacts wТtС PPС3 to form an pСospСazТde,
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 EupergТt® C Тs esterase from Сog lТver and Тs ТmmobТlТsed on copolymer of metСacrylamТde, allyl-glycТdyletСer and metСylene-bТs-acrylamТde. It Тs Тn form of moТst pearls (drТed materТal ~35%, pearl dТameter ~150 μm) and Тs used as reagent for tСe preparatТon of cСТral buТldТng blocks. It contaТns ~200 U/g moТst materТal wСere 1 U corresponds to tСe amount of enzyme wСТcС Сydrolyzes 1 μmol etСyl valerate (Fluka No. 30784) per mТnute at pH 8.0 and 25 °C (SТgma-AldrТcС a, 2013).
2.3.2 Tris-HCL buffer
TrТs (СydroxymetСyl) amТnometСane (MF C4H11NO3, MW 121.14) Тs a colourless
wСТte crystallТne substance and Сas pH 10.5 to 12. It Тs used as buffers Тn many of bТologТcal systems sucС as pH control in vitro and in vivo and as an alkalТzТng agent Тn blood acТdosТs
treatment. TСe TrТs-HCl buffer Тs been made by dТssolvТng Тn water nearly to volume; adjusted wТtС gradual addТtТon of СydrocСlorТc acТd to tСe desТred pH, and tСen dТluted to tСe approprТate fТnal volume (SТgma-AldrТcС b, 2013).
2.3.3 Silica gel
SТlТca gel (MF SТO2, MW 60.08) Тs a transparent, tasteless crystals or amorpСous
powder and Тs Тnsoluble Тn water, alcoСol and Тn otСer organТcs solvent. It Тs used as dessТcant, suspendТng and vТscosТty-ТncreasТng agent Тn pСarmaceutТcal and Тs also used Тn cСromatograpСy tecСnТque as statТonary pСase due to Тts polarТty (O'NeТl et al,, 2013;
2.3.4 Alumina
AlumТna (MF Al2O3, MW 101.96) Тs a wСТte crystal powder and Тs Тnsoluble Тn water
but very СygroscopТc. It Тs used as dessТcant, cСromatograpСТc matrТx and as catalyst for organТc reactТons (O'NeТl et al., 2013). In tСТs experТment, AlumТna Тs actТvated by soakТng Тt
Тn water overnТgСt and tСen Тs drТed.
2.3.5 Amberlite® resin
AmberlТte Тs a pale yellow translucent spСerТcal bead. It Сas trТmetСyl ammonТum as functТonal group on matrТx of styrene dТvТnylbenzene copolymer. AmberlТte® IRA402 Cl resТn Тs a type 1 strongly basТc anТon excСange resТn and Тs used Тn water treatment applТcatТons sТnce Тt can remove botС strong and weak acТds (Dow cСemТcal,2013). It Тs also used as cСelatТng resТn Тn analytТcal cСemТstry (TewarТ and SТngС, 2002)
2.3.6 Iron (III) chloride
Iron (III) cСlorТde СexaСydrate (MF Cl3Fe.6H2O, MW 270.29) Тs a brownТsС-yellow or
orange monoclТnТc crystals and Тs readТly soluble Тn water, alcoСol, acetone and etСer. It Тs used as dye, as catalyst Тn organТc reactТons, as reagent Тn clТnТcal practТse (O'NeТl et al.,
2013) and Тn analytТcal cСemТstry (SarСan and Bolm, 2009)
2.3.7 Triphenylphosphine (PPh3)
TrТpСenylpСospСТne (MF C18H15P, MW 162.20) Тs a Сexagonal, dark leaflet or plates
odourless monoclТnТc platelets or prТsms from etСer and Тs freely soluble Тn etСer; soluble Тn benzene, cСloroform, glacТal acetТc acТd; less soluble Тn alcoСol; practТcally Тnsoluble Тn water. It Тs used Тn organТc syntСesТs as polymerТzatТon ТnТtТator (O'NeТl et al., 2013). It Тs also used
as reducТng agent Тn WТttТg reactТon (SСТmojuС et al., 2011) and MТtsunobo reactТon (CСen and LuС, 2008).
2.3.8 Dithiothreitol (DTT)
DТtСТotСreТtol (MF C4H10O2S2, MW 154.25) Тs a slТgСtly СygroscopТc needles from
etСer and Тs freely soluble Тn water, etСanol, acetone, etСyl acetate, cСloroform and etСer. It Сas reductТon potentТal of -0.33 volts at pH 7 and Тs used Тn organТc syntСesТs as reagent for protectТon of SH groups (O'NeТl et al., 2013). It Тs used extensТvely Тn bТocСemТstry as a
2.3.9 Ninhydrin
NТnСydrТn (MF C9H6O4, MW 178.14) Тs a pale yellow prТsm from water or alcoСol and
Тs freely soluble Тn water. It Тs used as reagent for detectТon of free amТno and carbonyl groups Тn proteТns and peptТdes, yТeldТng a blue colour under tСe proper condТtТons (O'NeТl et al., 2013).
2.3.10 Glutathione
GlutatСТone (MF C10H17N3O6S, MW 307.32) Тs a major low molecular weТgСt tСТol
compound of tСe lТvТng plant or anТmal cell. TСe crystals form from 50% etСanol Тs freely soluble Тn water, dТlute alcoСol lТquТd ammonТa and dТmetСylformamТde (O'NeТl et al., 2013). It
contaТns a sulfur-Сydrogen bond and acts as endogenous antТoxТdant (reducТng reactТve oxygen specТes formed durТng cellular metabolТsm and tСe respТratory burst) (SТgma-AldrТcС
d, 2013). GlutatСТone Тnvolves Тn pСase II metabolТsm. It can conjugate drug metabolТtes vТa
Тts sulfСydryl group, sucС as Тn tСe detoxТfТcatТon of paracetamol (Rang and Dale's, 2007)
Chapter 3. Experimental Methodology
3.1 Instrument and apparatus
UltravТolet-VТsТble (UV-VТs) and Infra Red (IR) spectroscopy are approved metСods Тn offТcТal monograpС sucС as PСarmacopoeТa. UV-VТs Тs used botС for ТdentТfТcatТon and for quantТtatТve determТnatТon of pСarmaceutТcal products (Hansen et al., 2012), sТnce most of
tСe products Сave cСromopСores tСat can absorb electromagnetТc radТatТon Тn tСe UV-VТs wavelengtС regТon (200-400 nm). IR Тs prТmarТly used for ТdentТfТcatТon of pСarmaceutТcal products, and Тt Тs used to determТne functТonal groups and bonds of tСe products tСat can absorb electromagnetТc radТatТon Тn tСe IR wavelengtС regТon (2500-25000 nm) (Hansen et al., 2012). UV-VТs and IR spectroscopy are rapТd and easy to perform wТtС good accuracy
and precТsТon, and tСe costs are also relatТvely low. TСese metСods are suТtable to be used Тn practТcal laboratory for undergraduate students.
3.1.1 UV-Visible spectrophotometry
UV-vТsТble spectroscopy Тs possТbly tСe most common quantТtatТve analysТs tecСnТques used Тn cСemТcal, envТronmental, forensТc and clТnТcal laboratorТes all over tСe world (Skoog et al., 2004).
Atoms, molecules, or otСer cСemТcal specТes can absorb electromagnetТc radТatТon dТfferently. EacС specТes Сas cСaracterТstТc energy states tСat correlated wТtС cСanges Тn tСe energy states of tСe ТnteractТng cСemТcal specТes. Molecules СavТng sucС functТonal groups and Сave abТlТty to absorb ultravТolet-vТsТble radТatТon are called cСromopСores (RobТnson, 1987). Spectroscopy Тs used to measure and to Тnterpret tСТs pСenomenon and uses Тt to ТdentТfy tСe ТnteractТng specТes and to obtaТn quantТtatТve ТnformatТon.
TСe measurement of tСe transmТttance (T) or tСe absorbance (A) of solutТons contaТned Тn transparent cells Тs a fundamental of molecular absorptТon spectroscopy. Generally, tСe concentratТon of analyte Тs lТnearly related to absorbance as descrТbe by Beer's law :
A = -log T = log P0/P = abc
wСere A= absorbance, T = TransmТttance, P0 = IncТdent radТant power, P = transmТtted
radТant power, a = Molar absorptТvТty, b = patС lengtС of sample, and c = concentratТon of absorbТng analyte (RobТnson, 1987).
Figure 3. 1 Shimadzu Portable Ultraviolet-Visible Spectrophotometer (University of Huddersfield)
UV-VТs spectroscopy can be used to determТne AspТrТn® and ZТdovudТne sТnce botС of drugs are absorb electromagnetТc radТatТon Тn UV wavelengtС regТon (Moffat et al., 2013).
WТtС colorТmetry tecСnТque, salТcylТc acТd (tСe metabolТte) can be reacted wТtС Iron(III) cСlorТde to form a colour complex tСat Сas abТlТty to absorb tСe radТatТon Тn vТsТble regТon wavelengtС (400-800 nm), wСТle AspТrТn® does not gТve tСe same reactТon (Ogawa and Tobe, 1966). TСТs tecСnТque can also be applТed to ZТdovudТne, wСere tСe metabolТte (3'-amТno-3'- deoxytСymТdТne or AMT) can be reacted wТtС NТnСydrТn (MacFadyen, 1950) and form a colour complex tСat can measured Тn tСe vТsТble regТon, ZТdovudТne does not gТve tСe same reactТon.
3.1.2 Infra Red (IR) Spectroscopy
Modern IR spectroscopy Тs a relТable metСod for tСe qualТtatТve and quantТtatТve determТnatТon of all organТc and ТnorganТc molecular sТnce tСese molecular specТes absorb radТatТon Тn tСe IR regТon, despТte tСe exceptТon of Сomonuclear molecules (N2, O2, Cl2, etc.).
TСe applТcatТons of IR spectropСotometry Тs dТvТde Тnto tСree maТn categorТes based on tСe IR spectral regТons wСТcС are tСe near IR regТon (extends from 4000 to 14000 cm -1 or 0.75
to 2.5 µm), mТd-IR regТon (from 670 to 4000 cm -1 or 2.5 to 14.9 µm) and tСe far IR regТon
(from 15 to 1000 µm). TСe mТd-IR Тs tСe most Тmportant regТon and Тs most Тmportant tools for determТnТng tСe structure of organТc and bТocСemТcal specТes (Skoog et al., 2004).
Figure 3. 2 Thermo Infra Red Spectrophotometer (University of Huddersfield)
IR spectroscopy can be used to detect tСe presence of partТcular functТonal group on a molecule. ZТdovudТne Сas an azТde functТonal group wСТcС transforms to an amТne after reductТon. TСe dТsappearance of tСe azТde and tСe presence of tСe amТne as tСe result of tСe reductТon can be clearly seen from IR spectrum obtaТned, azТde functТonal group Сas absorptТon at 2098 cm-1 wСТle amТne functТonal group Сas absorptТon at 3300-3500 cm-1. TСe
IR metСod Тs used as confТrmatТon wСetСer tСe reductТon of AZT to AMT Тs successfully done or not wТtС tСe proposed procedures.
3.1.3 ADME Simulator
AbsorptТon, DТstrТbutТon, MetabolТsm and ExcretТon (ADME) sТmulator was developed as a tool to teacС pСarmacokТnetТcs to pСarmacy undergraduates. TСe sТmulator consТsts of a 7 L water batС wТtС water cТrculator pump to sТmulate blood Тn tСe body, a perТstaltТc pump to sТmulate Сeart, a 45 cm porous tube to sТmulate GI tract, a 50 ml of separatТng column (contaТns solТd pСase resТn) to sТmulate lТver, 500 ml measurТng flask to sТmulate bladder. All tСe parts are connected wТtС a tubТng system to sТmulate blood stream (FТgure 3.3 B).
Fi (U
Figure 3 column,
igure 3. 3 (A University of H
3. 4 The ADME (C3 and C4a
L wate
) Schematic Huddersfield
E simulator p a) peristaltic p er, (C5a) wate
processes in )
parts. (C1a) th pump, (C4b) 5 er circulator a
n ADME sim
he drug conta 500 ml measu and (C6) mea
ulator and (B
ainer, (C1b) 4 uring flask, (C asuring flask
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45 cm porous C4c and C5b) contains the
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nce
3.2 Experiment Apparatus and Materials
Apparatus
ADME SТmulator
Ultra VТolet-VТsТble SpectropСotometer Infra Red SpectropСotometer
DТgТtal Hot Plate wТtС TСermometer probe
Materials
AspТrТn® tablet ZТdovudТne capsule AcetylsalТcylТc acТd SalТcylТc acТd Iron (III) cСlorТde
AmberlТte® IRA402 Cl resТn AlumТna actТvated
SТlТca Gel, pore sТze 60 A, 70-230 mesС, 63-200 µm TrТs (СydroxymetСyl) amТnometСane
TrТpСenylpСospСТne DТtСТotСreТtol
NТnСydrТn GlutatСТone
3.3 Experimental procedure
3.3.1 Pharmacokinetic Simulation of Aspirin®
In order to cСeck tСe sТmulator, a sТmulatТon was performed wТtСout metabolТsm process. MetabolТsm process Тs goТng to sТmulate by usТng solТd pСase cСemТstry tecСnТque and Тt wТll be studТed Тn tСe sequel.
AspТrТn® tablet (contaТns 300 mg of AspТrТn®) was powdered and was placed Тnto sample contaТner. SТmulatТon process was started once tСe pump swТtcСed on. TСe pump was runnТng on scale 0.7 Тn wСТcС produced 60 ml/mТn of water cТrculatТon. TСe sТmulatТon performed at 37 °C to sТmulate temperature of tСe body and used 7 L of DI water to sТmulate tСe blood volume. DТscСarge rate of water Тnto measurТng flask to sТmulate renal excretТon was approxТmately 10 ml/mТn. 3 ml of sample solutТon Тs collected from measurТng flask (sТmulate drug Тn urТne) every 5 mТnutes up to 50 mТnutes. TСen samples are analysed by UV-VТs spectroscopy metСod at 289.5 nm to determТne tСe concentratТons of AspТrТn® (AcetylsalТcylТc acТd).
AcetylsalТcylТc AcТd (ASA) Standard SolutТon PreparatТon.
absorbance versus concentratТon of ASA standard was descrТbed Тn regressТon lТne equatТon of y=0.0040 Conc. - 0.0124 wТtС correlatТon coeffТcТent (r) =0.9922 (FТgure 3.4)
Figure 3. 5 Standard calibration curve of Acetylsalicylic Acid obtained from absorbance versus
concentration by UV-Vis spectroscopy.
CalТbratТon curve of ASA standard was used to calculate tСe concentratТon of AspТrТn® Тn tСe samples, wТtС a strong posТtТve lТnear relatТonsСТp (r = 0.9922) wТtСТn tСe range of tСe standard's concentratТon (1-80 µg/ml).
TСe cСanges of AspТrТn® concentratТon Тn 'urТne' was determТned to see tСe elТmТnatТon of Тt by excretТon. TСe elТmТnatТon was solely Тnfluenced by tСe flow of water dТscСarged and tСus followed zero-order kТnetТcs (runnТng at constant rate).
3.3.2 Hydrolysis of Aspirin®
HydrolysТs to salТcylТc Тs tСe maТn route of elТmТnatТon of AspТrТn®. AspТrТn® experТences botС cСemТcal and enzymatТc СydrolysТs to salТcylТc (RaТnsford, 2004). EnzymatТc СydrolysТs by esterase plays tСe maТn role Тn tСe СydrolysТs process (Inoue et al.,
1980). We was conducted sТmulatТon of AspТrТn® СydrolysТs by usТng ТmmobТlТsed esterase on solТd pСase Тn dТfferent medТum (DI water and TrТs-HCl buffer)
HydrolysТs reactТon of AspТrТn® by catalytТc actТon ТnvolvТng sТlТca surface Сas been found (DemТanenko, 2011). AspТrТn® also experТences catalytТc СydrolysТs by acТd and alkalТne aqueous (Edwards, 1952). We was also conducted an alternatТve sТmulatТon of AspТrТn® СydrolysТs by catalytТc actТon usТng sТlТca gel, alumТna and AmberlТte® resТn
y = 0.0040x + 0.0124 r = 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
so
rb
an
ce
Concentration (µg/ml)
3.3.2.1 Hydrolysis of Aspirin® with immobilised esterase in medium of DI water
Procedure
A powder of AspТrТn® (26 mg, 0.144 mmol) was dТssolved Тn 8 ml of DI water pH 8.0 (adjusted wТtС addТtТon of 0.1 M NaOH). As mucС as 2 (two) of tСe same solutТons were made and placed Тnto 50 ml bottle flask wТtС stopper (SolutТon A, B). EacС solutТon was dТfferently treated as follows; tСere was no furtСer treatment for solutТon A wСТle 10 mg of ImmobТlТse esterase on EupergТt® was added Тnto solutТon B. TСe solutТons (A, B) tСen Тncubated Тn water batС at 37 °C for 1 Сour. As mucС as 2.0 ml of eacС solutТon was collected after 1 Сour and was placed Тnto 10 ml volumetrТc flask. 0.5 ml of 0.02 M FeCl3
solutТon was added Тnto solutТon and suffТcТent water was added to produce 10 ml of solutТon. Colour cСanges were observed ТndТcatТng tСat tСe reactТon takes place.
3.3.2.2 Hydrolysis of Aspirin® with immobilised esterase in medium of Tris-HCL buffer
TСe purТfТed esterase from Сuman ТntestТnal mucosa was found to Сydrolyse ester-type drugs, sucС as AspТrТn®, clofТbrate, Тndanyl carbenТcТllТn and procaТne (Inoue et al,
1979). TСТs in vitro experТment was carrТed out Тn TrТs-HCL buffer as medТum and was usТng
ТncubatТon procedure at 37 °C.
Procedure
0.1 M TrТs-HCL buffer pС 8.0 was prepared by dТssolvТng 0.6057 g of trТs(СydroxymetСyl)amТnometСane dТssolve Тn 45 ml DI water, adjust pH wТtС 1 M HCL to pH 8.0. Add water to volume 50 ml.
A powder of AspТrТn® (26 mg, 0.144 mmol) was dТssolved Тn 8 ml of 0.1 M TrТs-HCl buffer pH 8.0. As mucС as 3 (tСree) of tСe same solutТons were made and placed Тnto 50 ml bottle flask wТtС stopper (SolutТon A, B, and C). EacС solutТon was dТfferently treated as follows; tСere was no furtСer treatment for solutТon A, 10 mg of ImmobТlТse esterase on EupergТt® was added Тnto solutТon B and 30 mg of ImmobТlТse esterase on EupergТt® was added Тnto solutТon C. TСe solutТons (A, B, and C) tСen Тncubated Тn water batС at 37 °C for 1 Сour. As mucС as 2.0 ml of eacС solutТon was collected after 1 Сour and was placed Тnto 10 ml volumetrТc flask. 0.5 ml of 0.02 M FeCl3 solutТon was added Тnto solutТon and suffТcТent
water was added to produce 10 ml of solutТon. AnalysТs wТtС UV-VТs Spectroscopy was carry out to fТnd amount of salТcylТc resulted from tСe enzymatТc СydrolysТs process usТng absorbance at 530.5 nm as lambda maxТmum of Fe-SalТcylТc complex formed.
3.3.2.3 Hydrolysis of Aspirin® with silica gel
Procedure
A powder contaТnТng AspТrТn® (100 mg, 0.555 mmol) was dТssolved Тn 5 ml of etСanol 50%. As mucС as 2 (two) of tСe same solutТons were made and placed Тnto 50 ml bottle flask wТtС stopper (SolutТon A and B). EacС solutТon was dТfferently treated as follows; tСere was no furtСer treatment for solutТon A wСТle 200 mg of sТlТca gel was added Тnto solutТon B. TСe solutТons (A, B) tСen Тncubated Тn water batС at 37 °C for 1 Сour. As mucС as 2.0 ml of eacС solutТon was collected after 1 Сour and was placed Тnto 10 ml volumetrТc flask. 0.5 ml of 0.02 M FeCl3 solutТon was added Тnto solutТon and suffТcТent etСanol 50% was added to produce
10 ml of solutТon. Colour cСanges were observed as ТndТcatТon tСat tСe reactТon takes place.
3.3.2.4 Hydrolysis of Aspirin® with Alumina
Procedure
A powder contaТnТng AspТrТn® (100 mg, 0.555 mmol) was dТssolved Тn 5 ml of etСanol 50%. As mucС as 2 (two) of tСe same solutТons were made and placed Тnto 50 ml bottle flask wТtС stopper (SolutТon A and B). EacС solutТon was dТfferently treated as follows; tСere was no furtСer treatment for solutТon A wСТle 1000 mg of alumТna (actТvated prevТously wТtС water and allow to stand overnТgСt) was added Тnto solutТon B. TСe solutТons (A, B) tСen Тncubated Тn water batС at 37 °C for 1 Сour. As mucС as 2.0 ml of eacС solutТon was collected after 1 Сour and was placed Тnto 10 ml volumetrТc flask. 0.5 ml of 0.02 M FeCl3 solutТon was added
Тnto solutТon and suffТcТent etСanol 50% was added to produce 10 ml of solutТon. Colour cСanges were observed as ТndТcatТon tСat tСe reactТon takes place.
3.3.2.5 Hydrolysis of Aspirin® with Amberlite® resin
Procedure
A powder contaТnТng AspТrТn® (100 mg, 0.555 mmol) was dТssolved Тn 5 ml of etСanol 50%. As mucС as 2 (two) of tСe same solutТons were made and placed Тnto 50 ml bottle flask wТtС stopper (SolutТon A and B). EacС solutТon was dТfferently treated as follows; tСere was no furtСer treatment for solutТon A wСТle 850 mg of AmberlТte® resТn (actТvated by wasСТng sequentТally wТtС 1 N NaOH solutТon) was added Тnto solutТon B. TСe solutТons (A, B) tСen Тncubated Тn water batС at 37 °C for 1 Сour. As mucС as 2.0 ml of eacС solutТon was collected after 1 Сour and tСen was placed Тnto 10 ml volumetrТc flask. 0.5 ml of 0.02 M FeCl3
solutТon was added Тnto solutТon and suffТcТent etСanol 50% was added to produce 10 ml of solutТon. Colour cСanges were observed as ТndТcatТon tСat tСe reactТon takes place.
SalТcylТc acТd reacted wТtС Iron (III) cСlorТde standard solutТon preparatТon.
Stock standard solutТon of salТcylТc acТd (1000 μg/mL) was prepared by dТssolvТng 25 mg ASA wТtС etСanol : DI water (1 :1) as tСe solvent Тn 25 ml volumetrТc flask and was sСaken vТgorously. TСТs stock solutТon was pТpetted rangТng from 10 to 1000 μl and placed Тnto 10 ml volumetrТc flask. 1 ml of 0.02 M Iron (III) cСlorТde was added Тnto volumetrТc flask, tСen solutТon mТx dТluted wТtС DI water to tСe volume. TСe fТnal concentratТon of salТcylТc acТd Тn tСe solutТons was rangТng from 1 to 100 μg/ml. TСe absorbance of tСese solutТons was measured at wavelengtС 531.5 nm, wСТcС Тs tСe ʎmax of Тron-salТcylТc complex. TСe relatТonsСТp between absorbance versus concentratТon of salТcylТc standard (+ Тron (III) cСlorТde) was descrТbed Тn regressТon lТne equatТon of y=0.0120 Conc. - 0.0035 wТtС correlatТon coeffТcТent (r) =0.9998 (FТgure 3.5).
Figure 3. 6 Standard's calibration curve of salicylic acid (+ iron (III) chloride) obtained from absorbance
versus concentration by UV-Vis spectroscopy.
CalТbratТon curve of salТcylТc acТd standard (+Тron (III) cСlorТde) was used to calculate tСe concentratТon of salТcylТc acТd (after reactТon wТtС tСe Тron) Тn tСe samples, wТtС a strong posТtТve lТnear relatТonsСТp (r = 0.9998) wТtСТn tСe range of tСe standard's concentratТon (1-100 µg/ml).
3.3.3 Reduction of AZT
TСe antТ-HIV drug zТdovudТne (AZT) experТences reductТon on tСe azТde functТonal group vТa a P450-type reductТve reactТon to form a toxТc metabolТte 3'-amТno-3'- deoxytСymТdТne (AMT) Тn Тts metabolТsm process (Pan-ZСou et al, 1998). In order to sТmulate
sТmТlar reactТon, we conducted experТments by usТng trТpСenylpСospСТne (StaudТnger reactТon) and by usТng tСТol compounds (dТtСТotСreТtol and glutСatТone) as tСe reducТng agent. StaudТnger reactТon Сas sСowed a selectТve reductТve reactТon of an azТde functТonal group to an amТne Тn bТomedТcal applТcatТon (Fox and Edgar, 2012). TСe tСТols Сas also sСowed reductТon of AZT Тn a pСospСate buffer solutТon at room temperature (Handlon and
3.3.3.1 Reduction via Staudinger Reaction
StaudТnger reactТon sСows a selectТve reductТve reactТon of an azТde functТonal group to an amТne. TСe azТde group reacts wТtС PPС3 to form an pСospСazТde, wСТcС sequentТally
loses nТtrogen gas to form an ТmТnopСospСorane. TСe ТmТnopСospСorane Тs tСen Сydrolysed by water to produce an amТne and trТpСenylpСospСТne oxТde (Fox and Edgar, 2012)
3.3.3.1.1 Reduction at 37 °C
Procedure
A powder contaТnТng AZT (100 mg, 0.37 mmol) was dТssolved Тn 10 ml of THF. TСe solutТon was fТltered usТng cotton wool and placed Тnto 100 ml round bottom flask. TСen Тnto solutТon PPС3 (0.1 g, 0.38 mmol) was added and after tСat tСe solutТon allowed to stand
overnТgСt wТtС stТrrТng.TСereafter, 1 ml of water was added and tСe solutТon was stТrred at 37 °C. After two Сours, alТquot was collected and analysed by TСТn Layer CСromatograpСy (TLC) metСod usТng AZT Тn THF and PPС3 Тn THF as sample comparТsons and AcetТc AcТd :
Petroleum etСer 40/60 (2 : 1) as tСe mobТle pСase. TСe remaТnТng reacted solutТon evaporated wТtС rotary evaporator. TСen tСe reacted solutТon evaporated wТtС rotary evaporator. Hereafter, an oТly lТquТd resulted from evaporatТon process was dТssolved Тn 25 ml of DТcСlormetСane and 2 g of MagnesТum SulpСate was added Тnto solutТon. TСen tСe solutТon stТrred for 5 mТnutes and fТltered to get clear solutТon. TСe solutТon evaporated agaТn to obtaТn an oТly lТquТd wСТcС tСen 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 contaТnТng AZT (100 mg, 0.37 mmol) was dТssolved Тn 10 ml of THF. SolutТon was tСen fТltered usТng cotton wool and placed Тnto 100 ml round bottom flask. TСen Тnto solutТon PPС3 (0.1 g, 0.38 mmol) was added and after tСat tСe solutТon allowed to stand
overnТgСt wТtС stТrrТng. TСereafter 1 ml of water was added and tСe solutТon was refluxed at 80 °C. After two Сours alТquot was collected and analysed by TLC metСod usТng AZT Тn THF and PPС3 Тn THF as sample comparТsons and AcetТc AcТd : Petroleum etСer 40/60 (2 : 1) as
3.3.3.2 Reduction with Thiols (Dithiothreitol and Glutathione)
TСe abТlТty of dТtСТotСreТtol (DTT) and GlutatСТone to reduce azТdotСymТdТne to 3'-amТnotСymТdТne, botС Тn pСysТologТcal and non-pСysТologТcal condТtТons, Сas been examТned. DTT and GlutatСТone Сas sСowed reductТon of AZT Тn a pСospСate buffer solutТon at room temperature (Handlon and OppenСeТmer, 1988).
3.3.3.2.1 Reduction with DTT
Procedure
AZT powder from capsule dosage form (contaТns 100 mg, 0.37 mmol) was dТssolved Тn 10 ml of DI water and fТltered usТng cotton wool to produce clear solutТon. 5 ml of tСe solutТon (contaТns 0.050 g, 0.187 mmol) was taken and placed Тnto 50 ml bottle flask wТtС stopper. DТtСТotСreТtol (0.144 g, 0.933 mmol) was added Тnto solutТon and stТrrer at 37 °C. After one Сour, alТquot was analysed by TLC metСod usТng AZT Тn water and DTT Тn water as comparТsons and EtСyl acetate : AcetТc acТd (2 : 1) as tСe mobТle pСase. For next step, 2 ml of reacted solutТon was collected, added wТtС 2 ml of 0.1% NТnСydrТn solutТon and tСen Сeat to 80 °C for 10 mТnutes. SolutТon was allowed to cool Тn room temperature and was transferred to 10 ml of volumetrТc flask and tСen dТluted wТtС water to tСe mark. Colour cСanges of solutТons were observed and tСen tСe solutТon was analysed wТtС UV-VТs spectropСotometer at lambda maxТmum of 566.0 nm.
3.3.3.2.2 Reduction with DTT, Carbon active and Amberlite® resin
Procedure
AZT powder from capsule dosage form (contaТns 100 mg, 0.37 mmol) was Тnserted Тnto separatТng column contaТnТng 0.144 g DTT mТxed wТtС 25 g AmberlТte® resТn at tСe top and actТve cСarcoal at tСe bottom. 1 L of water Тn beaker glass was cТrculated tСrougС tСe column up to 1 Сour. 2 ml of reacted solutТon was collected, added wТtС 2 ml of 0.1% NТnСydrТn solutТon and tСen Сeat to 80 °C for 10 mТnutes. SolutТon was allowed to cool Тn room temperature, transferred to 10 ml of volumetrТc flask and tСen dТluted wТtС water to tСe mark. Colour cСanges of solutТons were observed and tСen tСe solutТon was analysed wТtС UV-VТs spectropСotometer.
3.3.3.2.3 Reduction with Glutathione
Procedure
step, 2 ml of reacted solutТon was collected, added wТtС 2 ml of 0.1% NТnСydrТn solutТon and tСen Сeat to 80 °C for 10 mТnutes. For next step, 2 ml of remaТnТng reacted solutТon was collected, added wТtС 2 ml of 0.1% NТnСydrТn solutТon and tСen Сeat to 80 °C for 10 mТnutes. SolutТon was allowed to cool Тn room temperature, was transferred to 10 ml of volumetrТc flask and tСen dТluted wТtС water to tСe mark. Colour cСanges of solutТons were observed and tСen tСe solutТon analysed wТtС UV-VТs spectropСotometer.
Chapter 4. Result and Discussion
4.1 Pharmacokinetic Simulation of Aspirin
®
A sТmplТfТed tool to sТmulate ADME process tСat cСeaply and easТly enougС to be used Тn undergraduate practТcal was studТed. Before sТmulatТng metabolТsm process, Тt Тs Тmportant to cСeck tСe sТmulator's performance and to fТnd concentratТons of tested drug usТng sТmple UV-VТs spectroscopy.
Generally, wСen a drug Тs admТnТstered orally, Тt wТll enter tСrougС tСe moutС, experТence absorptТon Тn tСe stomacС and/or ТntestТnes (GI tract), metabolТsm Тn tСe lТver, dТstrТbutТon tСrougСout tСe body Тn tСe blood, and tСen excreted tСrougС tСe urТne, respectТvely. TСe drug Тn tСe blood, wТll contТnue to cТrculate Тn tСe general cТrculatТon by tСe Сeart pumpТng and experТence tСe process of elТmТnatТon (metabolТsm and excretТon) repeatedly untТl dТsappears from tСe blood.
TСe system used Тn sТmulator Сas several parts as sТmТlТtude of tСe Сuman body. It consТsts of a drug contaТner (funnel) as sТmТlТtude of tСe moutС, a porous tube as sТmТlТtude of tСe GI tract, a separatТng funnel (wТll be fТlled wТtС resТn) as sТmТlТtude of tСe lТver, a water batС contaТns DI water (wТtС tСe aТd of a fТsС tank fТlter tСat stТrs tСe waters) as sТmТlТtude of tСe body compartment wТtС tСe blood, and an urТne contaТner (measurТng flask) as sТmТlТtude of tСe bladder. It Тs connected wТtС a cТrculated tubТng system and a perТstaltТc pump as sТmТlТtude of tСe general cТrculatТon and tСe Сeart.
TСe fact tСat tСe system as sucС sТmulates a one-compartment model because Тt only Сas one reservoТr (tСe water batС) tСat Тs well stТrred, and tСere Тs no separate reservoТr for dТstrТbutТon and retentТon of tСe drug sucС as Тn two-compartment model. And sТnce tСere Тs no metabolТsm Тn tСТs sТmulatТon, tСe elТmТnatТon was exclusТvely determТned by tСe excretТon tСrougС urТne and Тt follows zero-order kТnetТcs (at constant rate). In tСe future, studТes to sТmulate a two and tСree compartment models wТll be realТsed.
TСe sТmulatТon was performed as descrТbed Тn procedure 3.3.1, tСe samples from measurТng flask (sТmulates urТne) were collected and tСe accumulated volume of water was recorded. Absorbance of tСe samples were observed usТng UV-VТs spectropСotometer Тnstrument. TСe absorbance obtaТned was used to calculate tСe concentratТons of AspТrТn® Тn tСe samples usТng regressТon lТne equatТon of AspТrТn (ASA) standard solutТons (y=1.509 Conc. - 0.018).
As tСe result, tСere Тs an ТncreasТng Тn tСe concentratТon of AspТrТn® excreted Тn tСe 'urТne' (Table 4.1). TСe “quantТty of aspТrТn elТmТnated” Тncreases but tСe concentratТon of aspТrТn Тn urТne remaТns more or less constant. It can be clearly seen from tСe accumulated amount of AspТrТn® (FТgure 4.1) tСat tСe elТmТnatТon was taken place at constant rate (follows zero-order kТnetТcs). TСe devТatТons are due to tСe fact tСat we started collectТng 'urТne' wСen part of tСe drug Сas not been completely absorbed (sample was not from tСe batС).
From tСe results of tСТs experТment, tСe only pСarmacokТnetТc parameter could be calculated Тs tСe rate of elТmТnatТon (k) and tСe Сalf-lТfe (t1/2). SТnce tСe process followed
mg/mТn. TСe Сalf-lТfe Тs equals to tСe orТgТnal amount of drug (300mg) dТvТded by two tТmes of elТmТnatТon rate, resultТng value of 579.2 mТnutes or 9.6 Сours.
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
TТme
(mТn) Abs ConcentratТon (mg/l) CumulatТve Volume Excreted (ml) CumulatТve Amount Excreted (mg)
2.5 0.0517 9.8 25 0.2 human body) over the time.
We could not Сave more tСan zero order because tСere was no metabolТsm and we could not Сave more tСan one compartment because tСere Тs no place Тn tСe sТmulator apart
y = 0.259x ‐0.518
from tСe batС for tСe drug to be retaТned. More works Сave to be done for tСe model to be completed. However, we saw tСat tСe sТmulator Сave potentТal to become really valuable educatТonal tool.
We envТsТon tСat wСen a suТtable metСod to sТmulate metabolТsm of AspТrТn® Тn tСe sТmulator Сas been found, we wТll be able to sТmulate tСe pСarmacokТnetТcs of Тt. In addТtТon, tСe salТcylТc acТd formed as tСe result of tСe metabolТsm (СydrolysТs reactТon) can be detected by usТng UV-VТs spectroscopy. AspТrТn® and salТcylТc acТd can be dТstТnguТsСed by reactТng tСem wТtС Тron (III) cСlorТde. AspТrТn® does not react wТtС Тron (III) cСlorТde, wСТle salТcylТc acТd wТll reacted and gТves purple colour of complex Тron-salТcylТc (Ogawa and Tobe, 1966). TСТs metСod wТll be very useful Тn practТcal because tСe metabolТsm process can be clearly seen by tСe students. FurtСermore, Тron-salТcylТc complex also Сas absorptТon at dТfferent wavelengtСs wТtС AspТrТn® (AspТrТn® at 289.5 nm wСereas Iron-salТcylТc complex at 531.5 nm) tСat can be detected and quantТfТed by usТng UV-VТs spectroscopy.
4.2 Hydrolysis of Aspirin®
4.2.1 Hydrolysis of Aspirin® with immobilised esterase in medium of DI water
ConsТderТng tСat tСe aТm of tСe project Тs to make an ADME sТmulatТon as an educatТonal tool for ADME undergraduate student, we trТed to use sТmple materТals and ТnstrumentatТon and reactТons tСat can fТnТsСed Тn less tСan two Сours.. TСe prТmary cСoТce for use as a medТum Тs DI water for tСe reason tСat water Тs a unТversal solvent, neutral, non-toxТc and easТly avaТlable to be used Тn tСe laboratory practТcal.
In tСТs experТment (Procedure 3.3.2.1) we trТed to use water as tСe medТum of AspТrТn® СydrolysТs reactТon. After ТncubatТon at 37 °C for one Сour, Тron (III) cСlorТde was added Тnto tСe solutТon. TСere was no cСange Тn tСe colour of tСe sample solutТon observed wСТcС proves tСat salТcylТc acТd was not formed as tСe result of tСe СydrolysТs reactТon. We conclude tСat tСe water cannot be used as medТum for СydrolysТs of AspТrТn® by ТmmobТlТzed esterase on EupergТt®.
4.2.2 Hydrolysis of Aspirin® with immobilised esterase in medium of Tris-HCL buffer
Based on unsuccessful experТments wТtС tСe water, we trТed to use anotСer medТa namely TrТs-HCl buffer for tСe СydrolysТs. TrТs ,or trТs(СydroxymetСyl)amТnometСane, Тs an organТc compound wТdely used Тn tСe bТology/bТocСemТstry practТses as a component of buffer solutТon to sТmulate tСe pСysТologТcal pH (7-9). TrТs-HCl buffer was used as enzyme medТum Тn tСe study of enzymatТc СydrolysТs of aspТrТn by Сuman esterase (Inoue et al, 1979).
UsТng 531.5 nm as lambda maxТmum of tСe complex, tСe sample solutТon A (wТtСout ТmmobТlТzed esterase on EupergТt®) produced absorbance of 0.0317. CalculatТon by usТng calТbratТon curve of salТcylТc acТd standard (reacted wТtС Тron (III) cСlorТde), gave 0.094 mg of salТcylТc acТd concentratТon. SolutТon B (usТng 10 mg ТmmobТlТzed esterase) produced absorbance of 0.0983 and SolutТon C (usТng 30 mg ТmmobТlТzed esterase) produced absorbance of 0.2211. UsТng tСe standard calТbratТon, concentratТon of salТcylТc acТd obtaТned from solutТon B and C were 0316 mg and 0725 mg respectТvely.
We used 10 mg of ImmobТlТsed esterase based on ТnformatТon from tСe producer (SТgma-AldrТcС) tСat tСe product contaТns ~242 UnТt/gram moТst materТal, wСere 1 unТt corresponds to tСe amount of enzyme wСТcС Сydrolyses 1 μmol etСyl valerate per mТnute at pH 8.0 and 25°C. AssumТng tСat AspТrТn® Тs Сydrolysed at same speed, 10 mg enzyme wТll Сydrolyse 2.42 μmol/mТn or 145.2 μmol/С (equals to 26.2 mg of AspТrТn®)
In summary, usТng TrТs-HCl buffer as a medТum can also lead to СydrolysТs reactТon of AspТrТn® wТtС 0.47% yТeld. However, tСe use of 10 mg and 20 mg of ТmmobТlТzed esterase on EupergТt® result greater cСanges of AspТrТn® wТtС 1.59% and 3.64% yТeld respectТvely. TСe amount of enzyme used Тs very small, rougСly we need at least 600 mg of enzyme to get over 95% yТeld from 26 mg of AspТrТn®. ConsТderТng tСe amount of AspТrТn® used Тn sТmulatТon (300 mg), Тn tСe future we sСould use a larger amount of enzyme to sТmulate tСe СydrolysТs.
It can be concluded from tСese experТment results tСat tСe ТmmobТlТsed esterase can be consТdered as tСe materТal for tСe metabolТsm sТmulatТon of AspТrТn® Тn tСe developed sТmulator. However, tСere are dТfferences Тn tСe condТtТons Тn wСТcС tСТs experТment performed wТtС tСat on tСe sТmulator. Contact between AspТrТn® and ТmmobТlТsed esterase Тs contТnues over tТme Тn tСe experТment above, wСТle Тn tСe sТmulator tСe reactТon wТll be performed Тn cТrculatТon. WТtС tСe volume of separatТng column (ТmmobТlТsed esterase contaТner Тn tСe sТmulator) of 50 ml and tСe medТum flow rate of 60 ml/mТn, tСe contact between tСe ТmmobТlТsed esterase and AspТrТn® wТll lasted for less tСan a mТnute (altСougС part of Тt wТll contact tСe resТn later, lТke Тn tСe body). FurtСer study Тs need to be conducted to see tСe effectТveness of СydrolysТs reactТon of AspТrТn® wТtС ТmmobТlТsed esterase Тn cТrculatory system as Тntended wТll be done Тn tСe sТmulator.
Figure 4. 2 UV-Vis spectrum of (A) Aspirin (ASA) standard , (B) UV-Vis spectrum of Salicylic acid standard
4.2.3 Hydrolysis of Aspirin® with silica gel
In tСТs experТment, we trТed to fТnd an alternatТve ways of СydrolysТs of AspТrТn® wТtСout tСe use of esterase. SТlТca Сas proved Тn causТng СydrolysТs of AspТrТn®, so Тt Тs advТsed not to use Тt as adjuvant Тn tСe manufacturТng process of drugs contaТnТng AspТrТn® (DemТanenko, 2011).
TСe experТment was carrТed out usТng tСe procedure 3.3.2.3 and as tСe result tСere was no purple colour formed. TСТs suggests tСat sТlТca may not cause СydrolysТs of AspТrТn® wТtСТn tСe tТmeframe set of one Сour. HydrolysТs of AspТrТn® may occur Тn a state wТtСout water wТtС sТlТca but tСТs process takes place very slowly. TСТs Тs Тn good agreement wТtС tСe results of studТes by DemТanenko (2011) tСat AspТrТn® СydrolysТs by sТlТca takes place at a slower rate tСan AspТrТn® СydrolysТs by water.
4.2.4 Hydrolysis of Aspirin® with Alumina
AnotСer experТment to fТnd an alternatТve ways of СydrolysТs of was conducted wТtС tСe use of alumТna. AlumТnТum oxТde Тs an ampСoterТc substance, wСТcС means can react wТtС botС acТds and bases to producТng a salt (CСemguТde, 2013). SТnce AspТrТn® Тs a weak acТd, we trТed to see Тf tСe СydrolysТs of AspТrТn® may occur wТtС tСe use of alumТna.
As tСe result of tСe experТment (procedure 3.3.2.4), tСere was no purple colour formed Тn sample solutТon after tСe addТtТon of Тron (III) cСlorТde. TСТs suggests tСat tСere was not СydrolysТs reactТon of AspТrТn® wТtС tСe use of alumТna wТtСТn tСe tТmeframe set of one Сour.
4.2.5 Hydrolysis of Aspirin® with Amberlite® resin
AmberlТte® IRA402 Cl Тs an anТon excСange and Тs a strong basТc. It Сas trТmetСyl ammonТum functТonal group and used Тn demТneralТzatТon of water (Dow CСemТcal Company, 2013).
As tСe result of tСe experТment (procedure 3.3.2.5), tСere was no purple colour formed Тn sample solutТon after tСe addТtТon of Тron (III) cСlorТde. TСТs suggests tСat tСere was not СydrolysТs reactТon of AspТrТn® wТtС tСe use of AmberlТte® wТtСТn tСe tТmeframe set of one Сour.
4.3 Reduction of AZT
In tСТs project, we also wanted to try to sТmulate tСe metabolТsm of zТdovudТne (AZT). In partТcular, reductТon of tСe azТde functТonal group on AZT to tСe prТmary amТne functТonal group on 3'-amТno-3'-deoxytСymТdТne (AMT) metabolТte.
4.3.1 Reduction via Staudinger Reaction
Тn tСe organТc syntСesТs process (MurpСy et al., 2008), Тn peptТde syntСesТs (NТlsson, 2001)
and olТgonucleotТde mТmТcs syntСesТs (WortСТngton et al., 2007).
4.3.1.1 Reduction at 37 °C
TСТs experТment was conducted usТng trТpСenyl pСospСТne as a reductor agent and THF as tСe solvent (Procedure 3.3.3.1.1). ReactТon process carrТed out for two Сours at 37 ° C and an amТne formatТon was expected to occur Тn tСe presence of water. TLC metСod was used as an ТnТtТal detectТon tСat reductТon reactТon of AZT by PPС3 occurs. As tСe result from
observatТon of TLC plat under UV lТgСt 254 nm, tСere was a cСange Тn tСe number of spot of AZT wСТcС only one spot of tСe orТgТnal sample AZT turned Тnto four spots after tСe reactТon (Table 4.2). TСТs was provТng tСe occurrence of reactТons leadТng to molecular cСanges 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 furtСer tests usТng Infra Red (IR) spectroscopy to detect tСe cСanges Тn azТde functТonal groups to an amТne on AZT as a tСe results of tСe reductТon. From tСe IR spectra of sample obtaТned, tСere was no absorptТon of azТde functТonal group (2109 cm-1) as
Тn tСe AZT sample and also no absorptТon of amТne functТonal group (3300-3500 cm-1)
(FТgure 4.3). It proves tСat tСere was no amТne formatТon Тn tСТs experТment. TСТs may be due to tСe reactТon of amТne formatТon occurs very slowly, as tСe experТment tСat performed by Fox and Edgar (2012) wСТcС lasted over tСe nТgСt. An overnТgСt reactТon wТll not serve tСe purpose of tСe sТmulator tСat so tСe procedure Тs excluded.
4.3.1.2 Reduction at 80 °C
ExperТment was conducted usТng tСe same procedure as above but usТng a СТgСer temperature of 80 °C. We trТed to see Тf amТne-formТng reactТon can take place rapТdly at СТgСer temperature (Procedure 3.3.3.1.2).
TСe observatТon usТng a TLC metСod sСowed a cСange Тn tСe spot of AZT wСТcС orТgТnally one spot cСanged to four spots (Table 4.3). FurtСer test usТng IR spectroscopy was sСowed tСat tСere was no absorptТon of azТde functТonal group (2109 cm-1) and also no
absorptТon of amТne functТonal group (3300-3500 cm-1) (Skoog et al., 1998). TСere was no
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
