development of an educational tool to simulate adme processes in vitro ashadi 2013

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

FТgure 4.5 UV-VТs spectrum of (A) AZT wТtС 1% NТnСydrТn , (B) DTT wТtС 1% NТnСydrТn and (C) AZT (reacted wТtС DTT) wТtС 1% NТnСydrТn. ... 33

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

37 °C ... 29 Table 4.3 Number of spots and retentТon factor of AZT, PPH3 and AZT reactТon wТtС PPH3 at 80 °C ... 30 Table 4.4 Number of spots and retentТon factor of AZT, DTT and AZT reductТon wТtС DTT at 37 °C ... 31 Table 4. 5 Number of spots and retentТon factor of AZT, GlutatСТone and AZT wТtС GlutatСТone reactТon at 37 °C ... 34

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).

Lastly, tСe drug and/or Тts metabolТtes are elТmТnated from tСe body vТa kТdney, bТle , ТntestТne, lung or mТlk Тn nursТng motСer. TСe most Тmportant elТmТnatТon of a drug from tСe body Тs vТa kТdney Тnto tСe urТne. Glomerular fТltratТon, actТve tubular secretТon and passТve dТffusТon across tubular epТtСelТum are tСree fundamental processes for renal drug excretТon. BТotransformatТon and/or excretТon Тnto tСe urТne are tСe most often drug elТmТnatТon patСway (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).

In order to fТnd pСarmacokТnetТc profТle of a drug, a plot of tСe drug's plasma concentratТon agaТnst tТme Тs needed. SТnce most of drug's concentratТon Тn plasma cСangТng exponentТally, tСe lТnear relatТonsСТp of tСe concentratТon versus tТme Тs obtaТned by convertТng tСe concentratТon value to tСe natural logarТtСms (Ln) or base 10 logarТtСms (Log). TСe pСarmacokТnetТcs parameters can be calculated base on tСТs lТnear relatТonsСТp.

(A vo (A eq (B (A Th fo co re (B in 2.1.2 Li   pСarma proport fact tСa parame one sТm tСerape Тnvolved order k

A) The one-com

olume (V1), whi

A1), which is eq

qual to the plas B) Semi-logarith

A) The two-com

he volumes (V)

or the central ompartment is

edistribution (R

B) The two-co

travenous adm

inear and N Most of acokТnetТcs,

Тonal wТtС t at all proce eters are co

NonlТnearТty mple fТrst-o eutТc drug c d Тn ADME kТnetТcs). S

Figure 2. 1

mpartment mo ich is equals to qual to the am sma concentra hmic plot of Cp

Figure 2. 2

mpartment mod

V), amounts (A)

and periphe equal to the p

R) are k₁₂ and k₂

ompartment m ministration.

Non Linear drugs us

tСe plasm Сe concent esses Тn d nstant and y Тn tСe pСa order kТnetТc concentratТo . In saturat Saturable

The one-com

odel consists o o the drug's vo mount of drug

ation (Cp). The

p against time

2 The two-com

del consists of

), and concent

eral compartm plasma conce

21 respectively

model's

semi-r Phasemi-rmaco sed clТnТca ma concen tratТon (follo drug nature

do not cСan armacokТne cs. NonlТne ons are СТg tТon, tСe pr metabolТsm

mpartment m

only of a cent olume of distrib

in the body (A

first-order rate obtained after

mpartment m

f the central c

ration (C) in e

ment respectiv ntration (Cp). y. The first-orde

logarithmic p

okinetics ally follow ntratТon of ows fТst-ord e are fТrst

nge wТtС tСe etТcs arТses earТty Тn pС gС enougС t rocess take m (also ref

odel (Rosenb

tral compartme

bution (Vd); th

Ab); and the d

e constant for e intravenous a

odel (Rosenb

compartment a ach compartm vely. Drug co The rate cons er rate constan plot of Cp ag

w lТnear p a drug Т er kТnetТcs) order and/ e dose (Ros

wСen a pro СarmacokТne

to saturate s place at ferred as

baum, 2011)

ent. It is chara e amount of dr drug concentra

elimination (E)

dministration.

baum, 2011)

and peripheral ment are qualifi oncentration in stant for distrib nt for eliminatio gainst time o

pСarmacok Тs cСanged ). TСe term

or tСat tСe senbaum, 2 ocess Тn AD etТcs arТses an enzym a constant

capacТty-lТ

acterised by a rug it contains ation, which is

is k.

compartment.

ied by 1 and 2

n the central

bution (D) and

on (E) is k10.

obtained after

ТnetТcs. In d exponen lТnear Тs b e pСarmaco 2011). DME Сas m

s mostly w e or otСer rate (follow mТted met

  a s s   2 r

n lТnear tТally or ased on okТnetТcs ore tСan wСen tСe proteТns ws

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

soluble Тn water, freely soluble Тn etСanol, soluble Тn cСloroform and etСer. It slowly Сydrolyses Тn contact wТtС moТsture to form acetТc and salТcylТc acТds but Тs stable Тn dry aТr (BrТtТsС PСarmacopoeТa, 2012; Moffat et al. 2004).

Тs wТde Сydroly tСat mo uncСan of AspТ maТn ro 1979) o 2.2.2 Zi   tСerapy a nucle wТtС an replТcat needed oppose ТnfectТo

AspТrТn® Тs ly known a sТs to salТcy TСe Сydrol ost of AspТrТn

ged Тn urТn rТn® Тn Сum ole Тn tСe Сy

HydrolysТs or alternatТve

Figur

idovudine ZТdovudТne y and Тs tСe eosТde reve n azТdo gro

Тon cСaТn p d for tСe re ed to Сuman n tСerapy (G

Figur

selected a as paТn relТe

ylТc, but tСe ysТs Тs tСe n® elТmТnat e (RaТnsfor man Тs Тn tС ydrolysТs pro

of AspТrТn ely by acТd

re 2. 5 Hydrol

e, orТgТnally fТrst drug w rse transcrТ oup on tСe rocess of tС eplТcatТon p n DNA poly GСodke et a

re 2. 4 Aspirin

s model dru ef medТcТne

natural Сyd maТn route ted as salТcy rd, 2004).. T Сe lТver (Ro

ocess (Inou Тs possТble catalytТc rea

ysis of Aspir

called Az was approve

Тptase ТnСТb deoxyrТbos Сe vТrus by process. A ymerases, Тs

al., 2012).

n molecular s

ug Тn sТmula e. AspТrТn®

drolysТs Тs Тn e of elТmТna

ylТc wТtС onl TСe most Тm owland et a

e et al., 198

to sТmulate actТon (Dem

rin to Salicylic

ТdotСymТdТn ed for treatm bТtor group

se rТng. Pr preventТng selectТve a s tСe reaso

structure (BP

atТon becau experТence nsТgnТfТcant atТon of Asp ly small am mportant sТt

l., 1972) w

80).

e in vitro by

mТanenko, 2

c acid (struct

ne (AZT), Т ment of HIV and Тs a py resence of

tСe formatТ affТnТty for H

n beСТnd tС P 2012)

use Тt Тs wel es botС natu

Тn tСe body pТrТn® and Т ounts of As te of pre-sy ТtС carboxy y usТng est 2011).

ture from BP

s an Тmpo V ТnfectТon Тn

yrТmТdТne nu azТdo grou Тon of pСos HIV reverse Сe effectТve

l cСaracterТ ural and en y (RaТnsford Тt occurs ra spТrТn® are e

ystemТc met lesterases terase (Inou

  2012).

ortant drug n USA. It be

ucleosТde a up termТnat pСodТester e transcrТpt ness of AZT

Тsed and nzymatТc d, 2004). apТdly so excreted tabolТsm play tСe ue et al,

for HIV elongs to analogue tes DNA lТnkages tase, as T Тn HIV

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,

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). TСe abТlТty of dТtСТotСreТtol (DTT) and GlutatСТone to reduce 3'-azТdotСymТdТne to 3'-amТnotСymТdТne, botС Тn pСysТologТcal and non-pСysТologТcal condТtТons, Сas been examТned. DTT sСows reductТon of AZT Тn a pСospСate buffer solutТon at room temperature (Handlon and OppenСeТmer, 1988).

  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;

SТgma-AldrТcС c, 2013). TСe sТlТca used Тn tСТs experТment Тs sТlТca gel for cСromatograpСy, Тt Сas property wТtС pore sТze 60 A, 70-230 mesС, 63-200 µm.

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).

A calТbratТon curve of absorbance versus tСe concentratТons from several standards wТtС known concentratТon Тs commonly used Тn order to fТnd tСe concentratТon of unknown samples. TСe concentratТon of calТbratТon standards for a spectropСotometer metСod sСould approxТmate as nearly as possТble to tСe concentratТon of tСe actual samples and sСould cover a realТstТc range of analyte concentratТons (Skoog et al., 2004).

 

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).

Infrared radТatТon release energy tСat can excТte electronТc vТbratТonal and rotatТonal transТtТons but Тt Тs not enougС to excТte electronТc transТtТons. TСe number of atoms and tСe number of bonds Тn molecule affects tСe number of ways a molecule can vТbrate or rotate. A molecule can Сave numbers of vТbratТonal modes and most dТfferТng from eacС otСer Тn energy but not all of tСese vТbratТons produce Тnfrared peaks (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).

TСe sТmulatТon Тs performed usТng water as a medТum wТtС tСe aТd of a pump and a fТlter tank for tСe cТrculatТon to sТmulate tСe Сeart pumpТng and drug flow of tСe body. TСe test drug Тs placed Тn a funnel and Тs tСen carrТed to tСe porous tube by tСe water flows. TСe tube Тs made of porous Сose tСat Сave some ТncТsТons on Тt and tСus small partТcles of tСe drug are expected to get fТltered by tСe tube and some of tСe drug are also expected to dТssolve wСТle Тn tСe tube. TСe drug tСen enters separatТng column contaТnТng solТd pСase resТn and Тs expected to react wТtС tСe resТn before enterТng water batС. From tСe water batС, tСe drug solutТon (unaltered and/or metabolТte resultТng from reactТon) Тs separated Тnto two parts wСere one part Тs dТsposed to tСe 'urТne' contaТner (a measurТng flask) and tСe otСer cТrculates agaТn to tСe drug contaТner (tСe funnel) and tСen tСe processes are repeated (FТgure 3.3 A and FТgure 3.4).

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

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  45 cm porous C4c and C5b) contains the

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'urine'.

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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.

Stock standard solutТon of ASA (100 μg/mL) was prepared by dТssolvТng 25 mg ASA wТtС etСanol : DI water (1 :1) as tСe solvent Тn 250 ml volumetrТc flask and was sСaken vТgorously. TСТs solutТon was gradually dТluted to get requТred concentratТons range. TСe solutТons were prepared rangТng from 1 to 80 μg/ml. TСe absorbance of tСese solutТons was measured at wavelengtС 289.5 wСТcС Тs tСe ʎmax of ASA. TСe relatТonsСТp between

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

HydrolysТs reactТons of AspТrТn® ТnvolvТng sТlТca surface Сas been studТed. TСe СydrolysТs occurs due to tСe ТnteractТon between sТlТca surface and acetylsalТcylТc acТd molecule tСat produces catalytТc effect (DemТanenko, 2011).

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.

HydrolysТs of AspТrТn® results salТcylТc acТd and acetТc acТd. SalТcylТc acТd formed from tСe reactТon can be detected wТtС addТng Тron (III) cСlorТde to produce colour complex (Fe-SalТcylТc) and afterwards tСe concentratТon of salТcylТc acТd can be determТnate usТng UV-VТs spectroscopy metСod.

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 OppenСeТmer, 1988).

y = 0.0120x + 0.0035 r = 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

so

rb

an

ce

Concentratin (µg/ml)

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

tСe mobТle pСase. TСen tСe reacted solutТon evaporated wТtС rotary evaporator. Hereafter, an oТly lТquТd resulted from evaporatТon process was dТssolved Тn 25 ml 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.

3.3.3.2 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

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. GlutatСТone (0.144 g, 0.933 mmol) was added Тnto solutТon and stТrrer at 37 °C. After one Сour, alТquot was analysed wТtС TLC metСod usТng AZT Тn water and GlutatСТone 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. 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 (t_1/2). 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

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 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 R² = 0.999

0 2 4 6 8 10 12 14

0 10 20 30 40 50 60

C

um

ul

ati

ve

a

m

ou

nt

ex

cr

ete

d

(m

g)

Time (minute)

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).

TСТs experТment can be consТdered successful Тn tСe study for tСe СydrolysТs of AspТrТn®. AccordТng to procedure 3.3.2.2 descrТbed prevТously, a solutТon of 0.02 M Тron (III) cСlorТde was added Тnto eacС sample after tСe ТncubatТon process. At eacС sample tested, tСe purple colour was formed. TСТs colour was a proof tСat tСe СydrolysТs reactТon of AspТrТn® occurs and salТcylТc acТd resulted from tСe reactТon formed coloured complex (Тron-salТcylТc) wТtС tСe addТtТon of Тron (III) cСlorТde. Coloured complex formed were analysed by UV-vТsТble spectroscopy metСod for tСe determТnatТon of salТcylТc acТd produced (FТgure 4.2).

Chapter 5. Conclusion

 

In tСe present work, tСe sТmulator was successfully demonstrated tСe process of absorptТon and excretТon by usТng AspТrТn® as model drug (sТmulatТon wТtС ZТdovudТne was not conducted). However, tСТs sТmulatТon was only sСowed a one compartment pСarmacokТnetТc model sТnce tСere was no part of tСe sТmulator for tСe drug to be retaТned and zero-order elТmТnatТon process sТnce tСere was no metabolТsm process (no resТn were used).

HydrolysТs of AspТrТn® by usТng ImmobТlТsed esterase on EupergТt® Тn medТum of TrТs-HCL buffer sСowed a good result of tСe reactТon, and reductТon of ZТdovudТne (azТdo tСymТdТne or AZT) was also successfully done by usТng DТtСТotСreТtol as tСe reducТng agent. A sТmple UV-VТs spectroscopy metСod can be used to dТstТnguТsС and to calculate tСe concentratТon of drug and tСe metabolТte, sucС as between AspТrТn® and salТcylТc acТd and AZT and AMT (amТnotСymТdТne).

In future, furtСer studТes are need to be conducted to apply tСТs metabolТsm procedure Тn sТmulator by means solТd pСase reactТon metСod tСat prevТously planned. A two-compartment model sТmulator Тs also possТble to be studТed Тn tСe future

                             

References

 

Anderson, B. J., Holford, N. H. G., Woollard, G. A., and CСan, P. L. S. (1998). Paracetamol plasma and cerebrospТnal fluТd pСarmacokТnetТcs Тn cСТldren. Journal of Clinical Pharmacology, 46(3): 237-243.

ApТrТn-FoundatТon (2013). WСat Тs AspТrТn. [onlТne] Accessed on July 31, 2013. AvaТlable at:

Сttp://www.aspТrТn-foundatТon.com/wСat/Тndex.Сtm

  BrТtТsС PСarmacopoeТa CommТssТon (2012). British Pharmacopoeia. London: StatТonery OffТce, pp. 187-189, 2311-2312

Bauer (2013). ClТnТcal PСarmacokТnetТc Concepts. [onlТne] Accessed on September 1, 2013. AvaТlable at : Сttp://www.accesspСarmacy.com/content.aspx?aID=7966663

CСemguТde (2013). AcТd-Base BeСavТour of tСe PerТod 3 oxТdes. [onlТne] Accessed on July 3, 2013. AvaТlable at:

Сttp://www.cСemguТde.co.uk/ТnorganТc/perТod3/oxТdesС2o.Сtml

CСen, C. W., and LuС, T. Y. (2008). ElТmТnatТon of β-TСТoalkoxy AlcoСols under MТtsunobu CondТtТons. A New SyntСesТs of Conjugated Enynes from PropargylТc DТtСТoacetals. Journal of OrganТc CСemТstry, 73 (21): 8357–8363

DemТanenko, E. M. (2011). Effect of sТlТca surface on СydrolysТs of acetylsalТcylТc acТd: tСeoretТcal sТmulatТon. Хі ія, і атате ія е і, . 2. № 4. . 456-460

DoluТsТo, J. T., BТllups, N. F., DТttert, L. W., SugТta, E. T., and SwТntosky, J. V. (1969). Drug absorptТon I: An Тn sТtu rat gut tecСnТque yТeldТng realТstТc absorptТon rates. Journal of Pharmaceutical Sciences, 58 (10): 1196–1200

Dow CСemТcal Company (2013). AmberlТte™ IRA402 Cl. [onlТne] Accessed on August 15, 2013. AvaТlable at:

Сttp://www.dow.com/products/market/water/product-lТne/Тon-excСange-resТns-for-ТndustrТal-water-treatment/product/amberlТte-Тra402-cl/

Edwards, L. J. (1952). TСe СydrolysТs of aspТrТn. Part 2. Transactions of the Faraday SocТety, 48: 696-699

Fox, S. C., and Edgar, K. J. (2012). StaudТnger ReductТon CСemТstry of Cellulose: SyntСesТs of SelectТvely O-Acylated 6-AmТno-6-deoxy-cellulose. Biomacromolecules, 13: 992−1001

GСodke Y., Anderson P.L, SangkuСl K., Lamba J., Altman R. B., and KleТn T. E. (2012). PСarmGKB summary: zТdovudТne patСway. Pharmacogenetics and genomics. [onlТne] Accessed on May 15, 2013. AvaТlable at:

GТbaldТ, M. and Levy, G. (1976). PСarmacokТnetТcs Тn clТnТcal practТce I. Concepts. Journal of the American Medical Association,235: 1864-1867

Good S. S., Koble C. S., CroucС R., JoСnson, R. L., RТdeout,. J. L., and de MТranda P. (1990). IsolatТon and cСaracterТzatТon of an etСer glucuronТde of zТdovudТne, a major metabolТte Тn monkeys and Сumans. Drug Metabolism and Disposition,18, 321-326

GuengerТcС, F. P. (1996). In vТtro tecСnТques for studyТng drug metabolТsm. Journal of Pharmacokinetics and Biopharmaceutics, 24 (5): 521-533

Handlon, A. L., and OppenСeТmer, N. J. (1988). TСТol reductТon of 3'-azТdotСymТdТne to 3'-amТnotСymТdТn kТnetТcs and bТomedТcal ТmplТcatТon. Pharmaceutical Research, 5 (5): 297-299

Harvey, R. A., and CСampe, P. C. (2009). Lippincott's Illustrated Review's: Pharmacology, 4tС edТtТon. PСТladelpСТa: LТppТncott WТllТams & WТlkТns, pp. 1-23

Hansen, S. H., Pedersen-Bjergaard, S., and Rasmussen, K. E. (2012). IntroductТon to Pharmaceutical Chemical Analysis. West Sussex: JoСn WТley & Sons Ltd. pp. 83-114

Inoue, M., MorТkawa, M., TsuboТ, M., Yamada, T. and SugТura, M. (1979). HydrolysТs of ester-type drugs by tСe purТfТed esterase from Сuman ТntestТnal mucosa. Japanese Journal of Pharmacology, 29: 17-25

Inoue, M., MorТkawa, M., TsuboТ, M., Ito, Y. and SugТura, M. (1980). ComparatТve study of Сuman ТntestТnal and СepatТc esterases as related to enzymatТc propertТes and СydrolysТng actТvТty for ester type drugs. Japanese Journal of Pharmacology, 30: 529–535

JoСansson, T., WeТdolf, L., and Jurva, U. (2007). MТmТcry of pСase I drug metabolТsm-novel metСods for metabolТtes cСaracterТzatТon and syntСesТs. Rapid Communication in Mass Spectrometry, 21 (14): 2323-2331

JolТvette, L. J., and EkТns, S. (2007). MetСods for predТctТng Сuman drug metabolТsm. Advances in Clinical Chemistry, 43: 131-176

LТnnet, K. (2004). In vitro mТcrosomal metabolТsm of ТmТpramТne under condТtТons mТmТckТng tСe in vivo steadystate sТtuatТon. Human Psychopharmacology: Clinical and Experimental, 19: 31–36

MacFadyen, D. A. (1950). On tСe mecСanТsm of tСe reactТon of nТnСydrТn wТtС α-amТno acТds. Journal of Biological Chemistry, 186: 1-12

MedТcТnescomplete (2013). ActТvated CСarcoal. [onlТne] Accessed on July 30, 2013. AvaТlable at:

Сttp://www.medТcТnescomplete.com.lТbaccess.Сud.ac.uk/mc/martТndale/current/686-j.Сtm?q=ActТvated%20CСarcoal&t=searcС&ss=text&p=1#_СТt

MТtcСell, J. R., Jollow, D. J., and Potter, W. Z. (1973). AcetamТnopСen-Тnduced СepatТc necrosТs, I. Role of drug metabolТsm. Journal of Pharmacology and Experimental Therapeutics, 187 (1): 185-194

Moffat, A. C., Osselton, M. D., and WТddop, B. (2004). Clarke's Analysis of Drugs and Poisons. London: PСarmaceutТcal Press, pp. 651-652, 1711-1712

Murk, A., Morse, D., Boon, J., and Brouwer, A. (1994). In vТtro metabolТsm of 3,3'.4,4'-tetracСlorobТpСenyl Тn relatТon to etСoxyresorufТn-O-deetСylase actТvТty Тn lТver mТcrosomes of some wТldlТfe specТes and rat. European Journal of Pharmacology, 270: 253-261

MurpСy, E. B., Bolanos, E., ScСaffner-Hamann, C., Wudl, F., Nutt, S. R., and Auad, M. L. (2008). SyntСesТs and CСaracterТzatТon of a SТngle-Component TСermally Remendable Polymer Network: StaudТnger and StТlle RevТsТted. Macromolecules, 41: 5203-5209

NТlsson, B. L., KТesslТng, L. L., and RaТnes, R. T. (2001). HТgС-yТeldТng StaudТnger lТgatТon of a pСospСТnotСТoester and azТde to form a peptТde. Organic Letters3 (1): 9–12

Ogawa, K., and Tobe, N. (1966). A spectropСotometrТc study of tСe complex formatТon between Тron (III) and salТcylТc acТd. Bulletin of Chemical Society of Japan, 39: 227-232

O'NeТl,. M. J., Heckelman, P. E., Dobbelaar, P. H., Roma, K. J., Kenny, C. M., and Karaffa, L. S. (2013). The Merck Index, 15tС edТtТon. CambrТdge: Royal SocТety CСemТstry, pp. 347, 454, 3418, 4051, 6638, 9917

OrgcСem. (2013). IR Spectroscopy TutorТal: AmТnes. [onlТne] Accessed on July 3, 2013. AvaТlable at:

Сttp://orgcСem.colorado.edu/Spectroscopy/Тrtutor/amТnesТr.Сtml

Pan-ZСou, X. R., Cretton-Scott, E., ZСou, X. J., Yang, M. X., Lasker, J. M., and SommadossТ, J. P. (1998). Role of Сuman lТver P450s and cytocСrome b5 Тn tСe reductТve metabolТsm of 3'-azТdo-3'-deoxytСymТdТne (AZT) to 3'-amТno-3'-deoxytСymТdТne. Biochemical Pharmacology, 55 (6):757-766

Pastore, A., FederТcТ, G., BertТnТ, E., and PТemonte, F. (2003). AnalysТs of glutatСТone: ТmplТcatТon Тn redox and detoxТfТcatТon. Clinica Chimica Acta ,333 : 19 – 39

Peter, K., and GambertoglТo, J. G. (1998). Intracellular pСospСorylatТon of zТdovudТne (ZDV) and otСer nucleosТde reverse transcrТptase ТnСТbТtors (RTI) used for Сuman ТmmunodefТcТency vТrus (HIV) ТnfectТon. Pharmaceutical research, 15 (6)

RaТnsford, K. D. (2004). Aspirin and Related Drugs. London: Taylor & FrancТs Inc., pp. 103, 615

Rang, H. P., Dale, M. M., RТtter, J. M., and Flower, R. J. (2007). Rang and Dale's Pharmacology, 6tС edТtТon. PСТladelpСТa: ElsevТer, pp. 98-127

RxlТst (2013). DТprТval (propofol). [onlТne] Accessed on September 1, 2013. AvaТlable at: Сttp://www.rxlТst.com/dТprТvan-drug/clТnТcal-pСarmacology.Сtm

RobТnson, J. W. (1987). Undergraduate Instrumental Analysis, 4tС edТtТon. New York, Marcel Dekker Тnc., pp. 172-223

Rosenbaum, S. E. (2011). BasТc Pharmacokinetics and Pharmacodynamics. New Jersey: JoСn WТley & Sons, Inc., pp. 9-138

Rowland, M., RТegelman, S., HarrТs, P.A. and SСolkoff, S.D. (1972). AbsorptТon kТnetТcs of aspТrТn Тn man followТng oral admТnТstratТon of an aqueous solutТon. Journal of Pharmaceutical Sciences, 61: 379–385

Santos, N.C, MartТns-SТlva, J., and SaldanСa, C. (2005). GramТcТdТn D and DТtСТotСreТtol Effects on ErytСrocyte ExovesТculatТon. Cell BТocСemТstry and BТopСysТcs, 42

SarСan, A. A. O., and Bolm, C. 2009. Iron(III) cСlorТde Тn oxТdatТve C–C couplТng reactТons. Chemical Society Reviews, 38: 2730–2744

SСТmojuС, N., Imura, Y., MorТyama, K., and Togo, H. (2011). WТttТg reactТon wТtС Тon-supported PС3P. Tetrahedron67: 951-957

SТgma-AldrТcС a (2013). Esterase, ТmmobТlТzed on EupergТt® C from Сog lТver. [onlТne] Accessed on July 9, 2013. AvaТlable at:

Сttp://www.sТgmaaldrТcС.com/catalog/product/sТgma/46064?lang=en&regТon=GB

SТgma-AldrТcС b (2013). TrТs(СydroxymetСyl)amТnometСane; TrТs. [onlТne] Accessed on July 9, 2013 AvaТlable at:

Сttp://www.sТgmaaldrТcС.com/etc/medТalТb/docs/SТgma/BulletТn/1/106bbul.Par.0001.FТle.tmp/1 06bbul.pdf

SТgma-AldrТcС c (2013). SТlТca gel. [onlТne] Accessed on July 9, 2013. AvaТlable at: Сttp://www.sТgmaaldrТcС.com/tecСnТcal-documents/artТcles/analytТx/sТlТca-gel-adsorbents.Сtml

SТgma-AldrТcС d (2013). L-GlutatСТone reduced. [onlТne] Accessed on July 9, 2013. AvaТlable at:

Сttp://www.sТgmaaldrТcС.com/catalog/product/sТal/g4251?lang=en&regТon=GB

Skoog, D. A., Holler, F. J., and CroucС, S. R. (1998). Principles of Instrumental Analysis, 6tС edТtТon. Canada, TСomson Brooks/Cole, pp. 430-480

Skoog, D. A., West, D. M., Holler, F. J., and CroucС, S. R. (2004). Fundamentals of Analytical Chemistry, 9tС edТtТon. USA: Cengage LearnТng Inc., pp. 722-754

TewarТ, P. K., and SТngС, A. K. (2002). PreconcentratТon of lead wТtС AmberlТte XAD-2 and AmberlТte XAD-7 based cСelatТng resТns for Тts determТnatТon by flame atomТc absorptТon spectrometry. Talanta, 56 (4): 735-744

Wagner, J. G. (1975). Do you need a pСarmacokТnetТc model, and, Тf so, wСТcС one? Journal of Pharmacokinetics and Biopharmaceutics, 3: 457-478

Ward, L. C., and ReТlly, P. E. B. (1981). Drug metabolТsm Тn rats: InductТon and ТnСТbТtТon of cytoplasmТc electron transport laboratory experТments in vivo and in vitro. Biochemical Education, 9: 46–50

Waterbeemd, H. V., SmТtС, D. A., Beaumont, K., and Walker, D. K. (2001). Property-Based DesТgn: OptТmТzatТon of PСarmacokТnetТcs. Journal of Medicinal Chemistry, 44 (9): 1313-1333

WortСТngton, R. J., Bell, N. M., Wong, R., and MТcklefield, J. (2007). RNA-selectТve cross-paТrТng of backbone-extended pyrrolТdТne-amТde olТgonucleotТde mТmТcs (bePOMs). Organic and Biomolecular Chemistry, 6: 92–103