Investigating the bioactivation of simvastatin in vitro

Margarita Kate Ojeda Alimario

A thesis submitted in partial fulfilment of the requirements for the degree of

Honours in Pharmacology

The University of Auckland

November 2015

i

Abstract

Simvastatin (SMV) is a prodrug that is used for the management of hypercholesterolemia.

The active metabolite simvastatin β-hydroxy acid (SMVA) is essential for the therapeutic

action of this statin on the rate limiting enzyme for cholesterol synthesis HMG-CoA

reductase. However, the enzyme(s) involved in this bioactivation have not been fully

elucidated. This hydrolysis reaction has been speculated to be catalysed by ester hydrolase

enzymes such as carboxylesterase (CES) and paraoxonase (PON). The insufficient data

regarding the role and activity of the enzymes involved in this hydrolysis reaction could be

one of the factors which contribute to the variation observed in simvastatin disposition.

Therefore, this dissertation has focused on investigating the hydrolysis of simvastatin in vitro,

and investigating the possible role of ester hydrolases in this bioactivation.

Incubation experiments were used to determine whether simvastatin was hydrolysed to the

active metabolite in vitro. Initial experiments were undertaken to confirm the previously

reported extensive hydrolysis of SMV to SMVA in rat plasma. After a 30 minute incubation,

more than 80% loss of the prodrug and extensive formation of the β-hydroxy acid was

observed. Subsequent incubation experiments were conducted in human plasma, pooled

human liver microsomes and pooled human liver cytosol. Findings from these experiments

consistently demonstrated that there was no detectable hydrolytic loss of SMV compared to

non-enzymatic controls at the substrate concentration tested (48 µM). To confirm this data,

simvastatin was incubated with the purified ester hydrolase enzymes, CES1, CES2, PON1

and butyrylcholinesterase (BChE) as well as human serum albumin (HSA). Incubations with

purified ester hydrolase enzymes also demonstrated no appreciable hydrolysis of simvastatin

and no formation of simvastatin β-hydroxy acid, relative to non-enzymatic controls. This

suggests that the esterases under investigation were not able to hydrolyse simvastatin at the

substrate concentration tested. However, the relatively high substrate concentrations tested

may have contributed to autoinhibition of the reaction and future work should assess lower

and more clinically relevant simvastatin concentrations. The identity and extent of enzymatic

bioactivation of simvastatin in humans remains to be elucidated.

ii

Acknowledgements

I would like to express my special thanks to the following people for their contribution

towards this thesis:

First and foremost to my supervisor Associate Professor Nuala Helsby. Thank you for always

being available when I needed your help. Your invaluable guidance, knowledge and constant

support throughout this project has been both inspiring and admirable and it has been a great

pleasure to be supervised by you over the course of this degree.

I would also like to thank Associate Professor Malcolm Tingle, for providing access to his rat

plasma, and for his constant advice and guidance not only throughout this project but also in

my personal life. I am immensely grateful for your words of wisdom and listening ear

whenever I needed it.

A special thanks to Junpeng Yang for helping me with my HPLC assay and assisting me with

settling in to the laboratory environment, and to Dr Kathryn Burns for your patience and

guidance, always willing to answer all my questions.

To the Helsby group, the Tinglets, and the Paxton group, especially to Dannel, Liam and

Tharaka. I am truly blessed to have been surrounded with such an amazing, welcoming group

of people. Thank you for all the advice and constantly keeping me well fed and caffeinated

throughout this project. Your friendship, support and encouragement has made this year very

memorable and enjoyable. To Mike, your stories and food deliveries on my desk kept me

going and alive throughout the year. Thanks.

To my friends, thank you for taking me out when my project was getting the best of me. Your

words of encouragement has been amazing. To Jude, thank you for teaching me that life is

full of surprises and that I am capable of standing and succeeding on my own.

Finally to my family, especially Mum and Dad, your constant support throughout this journey

has been simply amazing. You taught me the importance of hard work and to not only strive

for greater things but also to keep my faith in everything I do. I love you.

iii

Table of Contents

ABSTRACT ........................................................................................................................................... I

ACKNOWLEDGEMENTS ................................................................................................................. II

TABLE OF CONTENTS ................................................................................................................... III

LIST OF FIGURES ............................................................................................................................ IV

ABBREVIATIONS ............................................................................................................................. VI

1 INTRODUCTION .......................................................................................................................... 1

1.1 MECHANISM OF SIMVASTATIN .................................................................................................... 1 1.2 BIOTRANSFORMATION AND TRANSPORT OF SIMVASTATIN ....................................................... 3 1.3 ADVERSE EFFECTS ASSOCIATED TO TREATMENT WITH SIMVASTATIN .................................... 8 1.4 FACTORS THAT MAY INFLUENCE SIMVASTATIN-RELATED TOXICITY ...................................... 9 1.5 ESTER HYDROLASES ................................................................................................................... 17 1.6 POSSIBLE DRUG-DRUG INTERACTIONS BETWEEN SIMVASTATIN AND CO-MEDICATIONS

WHICH ARE ESTERASE SUBSTRATES ......................................................................................... 24 1.7 GOAL OF THIS PROJECT ............................................................................................................. 26

2 MATERIALS AND METHODS ................................................................................................. 27

2.1 MATERIALS ................................................................................................................................. 27 2.2 METHODS .................................................................................................................................... 28 2.3 DATA ANALYSIS ......................................................................................................................... 35

3 RESULTS ...................................................................................................................................... 36

3.1 DETECTION OF SIMVASTATIN AND SIMVASTATIN ACID USING HPLC .................................... 36 3.2 SPONTANEOUS HYDROLYTIC CONVERSION OF SIMVASTATIN TO SIMVASTATIN β-HYDROXY

ACID ............................................................................................................................................. 41 3.3 CALIBRATION CURVE FOR SIMVASTATIN AND SIMVASTATIN ACID ........................................ 42 3.4 BIOACTIVATION OF SIMVASTATIN IN SUBCELLULAR FRACTIONS .......................................... 44 3.5 BIOACTIVATION OF SIMVASTATIN IN THE PRESENCE OF ESTER HYDROLASES ..................... 53 3.6 UNKNOWN PEAKS ....................................................................................................................... 64

4 DISCUSSION ................................................................................................................................ 68

4.1 THE LACK OF SIMVASTATIN BIOACTIVATION IN HUMAN LIVER AND PLASMA ...................... 68 4.2 THE LACK OF SIMVASTATIN BIOACTIVATION IN THE PRESENCE OF PURIFIED ESTER

HYDROLASE ENZYMES ............................................................................................................... 72 4.3 UNKNOWN PRODUCTS ................................................................................................................ 76 4.4 LIMITATIONS .............................................................................................................................. 77 4.5 FUTURE WORK ............................................................................................................................ 80 4.6 SUMMARY ................................................................................................................................... 80

REFERENCES .................................................................................................................................... 82

iv

List of Tables and Figures

Figure 1. The rate limiting reaction of cholesterol synthesis catalysed by HMG-CoA reductase and the chemical structures of simvastatin-lactone and simvastatin β-hydroxy acid, which inhibits the rate limiting enzyme HMG-CoA reductase. ............................... 2

Figure 2. Chemical structures of the oxidative metabolites of simvastatin (Elsby et al., 2012; Mauro, 1993; Prueksaritanont et al., 1997). ...................................................................... 5

Figure 3. A summary of the metabolic route and transport of simvastatin and simvastatin β-hydroxy acid based on the literature evidence and known location of enzymes transporters (Giacomini et al., 2010). ................................................................................ 7

Figure 4. The role of OATP1B1 in the hepatic uptake of simvastatin and simvastatin acid. Genetic variants of SLCO1B1 results in elevated circulating simvastatin β-hydroxy acid (SMVA) concentrations and are associated with an increased risk of myotoxicity. ....... 14

Figure 5. The chemical structure of the prodrug aspirin and its active metabolite salicylic acid and the enzymes reported to be involved in the bioactivation of aspirin. ....................... 25

Figure 6. Representative chromatogram of simvastatin standard. .......................................... 36

Figure 7. Chromatography of the reaction mixture after treatment with NaOH and HCl. ...... 37

Figure 8. Comparison of the normalised spectra of simvastatin (red) and simvastatin acid (blue dashed). .................................................................................................................. 38

Figure 9. Chromatography of the reaction mixture after treatment with NaOH. .................... 38

Figure 10. Chromatogram of simvastatin acid formed using the alternative method. ............ 39

Figure 11. Hydrolytic conversion of simvastatin (0.1 mg/mL) in 67 mM phosphate buffer (pH 7.4) for 300 minutes. ....................................................................................................... 41

Figure 12. Representative calibration curve of simvastatin (12.5 µg/mL) in acetonitrile. ...... 42

Figure 13. Representative calibration curve of simvastatin β-hydroxy acid (12.5 µg/mL) in 10% NaOH - acetonitrile. ................................................................................................ 43

Figure 14. Loss of simvastatin (A) and formation of simvastatin β-hydroxy acid (B) in the presence of rat plasma (26 mg/mL). ................................................................................ 45

Figure 15. Loss of simvastatin (A) and formation of simvastatin β-hydroxy acid in human plasma (26 mg/mL). ....................................................................................................... 46

Figure 16. The effect of calcium fortification (2 mM CaCl2) on simvastatin hydrolysis in denatured and non-denatured human plasma at a protein concentration of 9 mg/mL. ... 47

v

Figure 17. Chromatogram of the reaction mixture consisting of human liver microsomes (0.4 mg/mL) and the internal standard, ivermectin, in the absence of simvastatin. ............... 48

Figure 18. Loss of simvastatin (A) and formation of simvastatin β-hydroxy acid (B) in pooled human liver microsomes (2 mg/mL) in the absence and presence of calcium chloride. . 49

Figure 19. Loss of simvastatin (A and C) and formation of simvastatin β-hydroxy acid (B and D) in incubations of increasing protein concentrations of human liver microsomes. ..... 50

Figure 20. Loss of simvastatin (A) and formation of simvastatin β-hydroxy acid (B) in cytosol (5 mg/mL) in the absence and presence of calcium chloride. ............................. 52

Table 1. Simvastatin remaining (%) compared to non-enzymatic (denatured) controls in human liver and plasma. .................................................................................................. 53

Figure 21. Loss of simvastatin (A) and formation of simvastatin β-hydroxy acid (B) in the presence of CES1 and CES2. .......................................................................................... 55

Figure 22. Loss of simvastatin (A) and formation of simvastatin β-hydroxy acid (B) in the presence of PON1-QQ and PON1-RR. ........................................................................... 57

Figure 23. Representative chromatograms showing A) spontaneous hydrolysis of simvastatatin and B) the hydrolysis of simvastatin in the presence of paraoxonase 1 (RR). ................................................................................................................................ 58

Figure 24. Loss of simvastatin acid in the presence of PON1-QQ. ........................................ 59

Figure 25. Loss of simvastatin (A) and formation of simvastatin β-hydroxy acid (B) in the presence of increasing concentrations of BChE. ............................................................. 61

Figure 26. Loss of simvastatin (A) and formation of simvastatin β-hydroxy acid (B) in the presence of increasing concentrations of human serum albumin (HSA). ....................... 63

Figure 27. Representative chromatogram of the reaction mixture containing denatured pooled human liver microsomes and simvastatin. ....................................................................... 64

Figure 28. Comparison of the normalised UV spectra of the unknown product at retention time of 18.7 minutes (black dash) with simvastatin (red) after incubations with human liver microsomes. ............................................................................................................ 65

Figure 29. Formation of unknown product B in pooled human liver microsomes (2 mg/mL) in the absence and presence of calcium chloride. ................................................................ 65

Figure 30. Formation of the unknown product B in incubations of increasing concentrations of human liver microsomes with simvastatin. ................................................................. 66

vi

Abbreviations

ABC – ATP-binding cassette

AChE – Acetylcholinesterase

ACOT1 - Acyl-CoA thioesterase 1

ASA – Acetylsalicylic acid

AUC – Area-under-the-curve

BChE - Butyrylcholinesterase

BSA – Bovine Serum Albumin

CaCl2 – Calcium chloride

CEL - Carboxyl ester lipase

CES1 – Carboxylesterase 1

CES2 – Carboxylesterase 2

Cmax – Maximum plasma concentration of drug in plasma

CYP – Cytochrome P450

DMSO – Dimethyl sulfoxide

HCl – Hydrogen chloride

HDL – High density lipoprotein

HLM – Human liver microsomes

HMG-CoA – 3-hydroxy-3-methylglutaryl-coenzyme A

HPLC – High Performance Liquid Chromatography

HSA – Human Serum Albumin

IS – Internal standard

KH2PO4 – Potassium dihydrogen phosphate

Ki – Dissociation constant of the enzyme-inhibitor complex

Km – Michaelis constant

LDL – Low density lipoprotein

LIPA – Lysosomal acid cholesterol lipase/esterase

MRP2 – Multidrug resistance associated protein 2

Na2HPO4 – di-Sodium hydrogen phosphate

NaOH – Sodium hydroxide

OATP1B1 – Organic anion-transporting polypeptide 1B1

P-gp – Permeability glycoprotein

PA – Peak area

PAR – Peak area ratio

PON – Paraoxonase

Rt – Retention time

SIAE - Sialic acid acetylesterase

SLCO1B1 – Solute carrier organic anion transporter 1B1

SMV – Simvastatin

SMVA – Simvastatin β-hydroxy Acid

SNP – Single nucleotide polymorphism

UGT – UDP-glucuronosyl transferase enzyme

v/v – volume-to-volume ratio

1

1 Introduction

1.1 Mechanism of simvastatin

Simvastatin (SMV) is primarily used for the management of hypercholesterolemia; a disease

caused by elevated levels of cholesterol and consequently, an established risk factor for

multiple cardiovascular diseases including coronary heart disease (Kjeckshus et al., 1997,

Plosker & McTavish, 1995; Prueksaritanont et al., 2003; Slater & MacDonald, 1988; Stancu

& Sima, 2001).

Simvastatin is an inactive lactone prodrug which undergoes hydrolysis to form the active

metabolite simvastatin β-hydroxy acid (SMVA), which resembles the endogenous substrate

3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) (Figure 1) (Plosker & McTavish,

1995; Prueksaritanont et al., 2003; Prueksaritanont et al., 2001; Stancu & Sima, 2001; Todd

& Goa, 1990). Simvastatin is a competitive inhibitor (Ki = 0.12 nM) (Plosker & McTavish,

1995; Prueksaritanont et al., 2003; Stancu & Sima, 2001) of HMG-CoA reductase, the rate

limiting enzyme in cholesterol synthesis. Since the affinity of this enzyme for the endogenous

substrate (KM = 3 mM) is much lower than the affinity of simvastatin for this enzyme, SMV

acts as an efficient HMG-CoA inhibitor, decreasing the synthesis of mevalonic acid, thereby

resulting in the beneficial effect of decreasing hepatic cholesterol synthesis (Corsini et al.,

1995; Stancu & Sima, 2001). This depletion in cholesterol levels is therapeutic as it decreases

the progression of atherosclerosis and hence cardiovascular morbidity and mortality is also

reduced (Kjekshus et al., 1997; Pietro & Mantell, 1990; Slater & MacDonald, 1988; Todd &

Goa, 1990).

2

Figure 1. The rate limiting reaction of cholesterol synthesis catalysed by HMG-CoA reductase and the chemical structures of simvastatin-lactone and simvastatin β-hydroxy acid, which inhibits the rate limiting enzyme HMG-CoA reductase.

Simvastatin is orally administered, given at 5-80 mg daily depending on the severity of the

disease (Neuvonen et al., 2008; Pasanen, Neuvonen et al., 2006; Plosker & McTavish, 1995).

Simvastatin has an oral bioavailability of <5% (Neuvonen et al., 2008; Pasanen et al., 2006;

Plosker & McTavish, 1995) and undergoes extensive first pass metabolism which limits its

availability in the circulation, and is then excreted in faeces (60%) and urine (13%) (Bellosta

et al., 2004; Plosker & McTavish, 1995; Todd & Goa, 1990). Simvastatin has a half-life of 2-

3 hours and peak plasma concentrations (Cmax) of 6-9 ng/mL is achieved within 1-4 hours

after a 40 mg dose (Backman et al., 2000; Bellosta et al., 2004; Mauro, 1993; Pasanen et al.,

2006; Plosker & McTavish, 1995; Shitara & Sugiyama, 2006; Todd & Goa, 1990; Winsemius

et al., 2014).

3

As a prodrug, simvastatin is able to undergo pH dependent, non-enzymatic hydrolysis or

alternatively, be hydrolysed by ester hydrolase enzymes after blood sample collection. The

unstable nature of simvastatin allows for the detection of both lactone and acid form in

systemic circulation in humans (Backman et al., 2000; Winsemius et al., 2014) and even in

samples ex vivo.

1.2 Biotransformation and transport of simvastatin

1.2.1 Biotransformation

Simvastatin undergoes a hydrolysis reaction to form the active metabolite simvastatin β-

hydroxy acid (SMVA) (Billecke et al., 2000; Goswami et al., 2013; Plosker & McTavish,

1995; Prueksaritanont et al., 2001; Stancu & Sima, 2001; Todd & Goa, 1990) and the

presence of both lactone and acid forms have been detected in the systemic circulation in

humans (Ahmed et al., 2013; Backman et al., 2000; Kim et al., 2011; Najib et al., 2003; Vree

et al., 2001). SMVA has a half-life of approximately 1.9 hours, reaching Cmax of 2-3 ng/mL

within 4-6 hours after a 40 mg dose (Backman et al., 2000; Mauro, 1993; Plosker &

McTavish, 1995; Winsemius et al., 2014).

It is typically stated that simvastatin undergoes hydrolysis in the liver to the active β-hydroxy

metabolite (SMVA) (Pasha et al., 2006; Sirtori, 1990). Since this bioactivation is considered

to occur predominantly in the liver, this is assumed to target the therapeutic effects to the

organ largely responsible for the synthesis of cholesterol. In addition, it has been speculated

that “serum esterases” catalyse this bioactivation of simvastatin to simvastatin β-hydroxy

acid (Casey et al., 2013; Corsini et al., 1995; Elsby, Hilgendorf, & Fenner, 2012; Pasanen et

4

al., 2006; Wilke et al., 2012; Winsemius et al., 2014). Although the specific hydrolytic

esterase enzymes involved in this bioactivation have not been fully elucidated, an assessment

of the possible enzymes involved in this bioactivation is covered later in this chapter (Section

1.5).

Furthermore, simvastatin-lactone (the prodrug) is also susceptible to spontaneous hydrolysis

(Elsby et al., 2012; Tubic-Grozdanis et al., 2008) however, the extent of this hydrolysis

reaction after blood sample collection is not clear. Therefore, to overcome this spontaneous

conversion between the prodrug (SMV) and the active metabolite (SMVA), early

pharmacokinetic and drug metabolism studies have reported the ‘total’ simvastatin

concentrations after conversion of the β-hydroxy acid to the lactone form, by addition of acid

to samples (Vickers et al., 1990; Vickers et al., 1990). More recent studies report the

concentrations of both the prodrug and the active metabolite (Backman et al., 2000; Vree et

al., 2001; Winsemius et al., 2014). Nonetheless, many articles do not distinguish between

these two compounds when describing the pharmacological disposition of ‘simvastatin’.

Both the inactive lactone prodrug (SMV) and the active β-hydroxy metabolite (SMVA) can

undergo further oxidative reactions. These are predominantly (>80%) carried out by the

CYP3A enzyme subfamily, with CYP3A4 isozyme exhibiting greater affinity than CYP3A5

(Prueksaritanont et al., 2003; Prueksaritanont et al., 1997). These enzymes are present in the

liver and the intestinal wall and metabolism of the lactone prodrug produces a range of

metabolites (Figure 2) (Kim et al., 2011; Mauro, 1993; Prueksaritanont et al., 2003;

Prueksaritanont et al., 1997).

5

Figure 2. Chemical structures of the oxidative metabolites of simvastatin (Elsby et al., 2012; Mauro, 1993; Prueksaritanont et al., 1997).

Simvastatin β-hydroxy acid (SMVA) can also undergo further oxidative metabolism in

human liver microsomes, producing three metabolites (M1, M2, M3) however none of these

metabolites have been characterised (Prueksaritanont et al., 2003).

6

Recently, it has been suggested that the active metabolite simvastatin acid may also be

converted back to the lactone form subsequent to conjugation with glucuronic acid catalysed

by UGT, specifically the isoforms UGT1A1 and UGT1A3 (Pasanen et al., 2006;

Prueksaritanont et al., 2002). The formation of the acyl-glucuronide form of SMVA is

unstable thus resulting in spontaneous cyclization to release the lactone (SMV), although this

is believed to be a very minor pathway (Elsby et al., 2012; Prueksaritanont et al., 2002;

Tsamandouras et al., 2014). Interestingly, a related statin, atorvastatin acid has also been

shown to undergo a similar reaction pathway (Elsby et al., 2012; Riedmaier et al., 2011).

1.2.2 Drug uptake and efflux transporters

The clinical disposition of both simvastatin and simvastatin β-hydroxy acid is governed by

several drug uptake and efflux transporters. Among these transporters, the most extensively

studied is the organic anion transporting polypeptide 1B1 (OATP1B1) (Neuvonen et al.,

2008; Pasanen et al., 2006). OATP1B1 facilitates the uptake of simvastatin into hepatocytes

while efflux transporters namely the multidrug resistance-associated protein 2 (MRP2) clears

simvastatin from the portal circulation and into the bile (Neuvonen et al., 2008; Pasanen et

al., 2006).

It has been reported that simvastatin may be a substrate or an inhibitor of some of these

transporters, consequently influencing their mechanism. For instance the lactone prodrug

(SMV) preferentially inhibits MRP2, while the active β-hydroxy acid metabolite (SMVA)

exhibits greater inhibition of OATP1B1 relative to the lactone form (Chen et al., 2005). In

addition, both SMV and SMVA can inhibit another major transporter, the ATP-binding

cassette (ABC) transporter P-glycoprotein (P-gp), with the lactone form displaying greater

potency towards inhibition of P-gp (Badhan et al., 2009; Chen et al., 2005). Nonetheless

7

directional transport studies have demonstrated that there is an insignificant P-gp mediated

efflux of simvastatin and moderate P-gp transport of simvastatin acid, thus suggesting that

inhibition of the transporter does not indicate that the drug itself is a substrate for that

transporter (Hochman et al., 2004). Figure 3 below summarises the information presented in

this section regarding the disposition of simvastatin and simvastatin β-hydroxy acid which is

influenced by these transporters.

CH3

CH3

O

O

O

O

CH3CH3 CH3

OH

CH3

CH3

O

O

CH3

CH3CH3

OH

OOH

OH

Simvastatin(inactive lactone form)

Oxidative Metabolites

CYP3A4CYP3A5

'Esterases'

Simvastatin Acid(active metabolite)

Simvastatin(inactive lactone

form)

Simvastatin acid(active metabolite)

P.O.Simvastatin

'Est

eras

es'

BileGut wall Liver

OATP1B1

OATP1B1

MRP2

MRP2

Oxidative Metabolites

CYP3A4

UGT(<1%)

UDP-GA

UDPGA - SMVA conjugate

P-gpP-gp

Figure 3. A summary of the metabolic route and transport of simvastatin and simvastatin β-hydroxy acid based on the literature evidence and known location of enzymes transporters (Giacomini et al., 2010).

8

1.3 Adverse effects associated to treatment with simvastatin

Although statins are generally well tolerated, several adverse side effects related to

simvastatin drug treatment have been reported with the most common being abdominal pain

(7.3%), constipation (6.6%), nausea (5.4%) and headache (2.5%-7.4%) (Pietro & Mantell,

1990; Stancu & Sima, 2001). Reports of more serious adverse effects include muscle-related

toxicity, for instance rhabdomyolysis which only occurs in approximately 0.4% of

individuals receiving the highest given dosage of simvastatin (80 mg/day) (Pietro & Mantell,

1990; Stancu & Sima, 2001). Even though reports of rhabdomyolysis associated with statin

treatment is scarce, subclinical indicators of muscle damage observed are more prevalent

(Chapman & Carrie, 2005; Urso et al., 2005). For example, myalgia, defined as muscle ache

or weakness without changes in creatinine levels is the most common type of muscle related

toxicity reported due to statin treatment (Rosenson, 2004; Ucar et al., 2000), with incidence

rates of approximately 0.8%-8.4% (Chapman & Carrie, 2005; Ucar et al., 2000; Urso et al.,

2005).

Despite the frequent occurrence of this toxicity, the determinants of statin-induced myopathy

have not been extensively studied. This occurrence of muscle toxicity has been largely

attributed to elevated levels of the active metabolite simvastatin β-hydroxy acid and is

considered one of the many possible predisposing factors for this adverse effect of this statin

prodrug.

9

1.4 Factors that may influence simvastatin-related toxicity

1.4.1 Dose

The incidence of muscle toxicity observed related to simvastatin treatment appears to be

influenced by the dose. In a double-blinded randomised trial, 53 cases of myopathy (0.9%)

were reported in participants receiving 80 mg/day of simvastatin versus 2 reports (0.03%) in

those that received 20 mg/day of simvastatin (Meade et al., 2010). In addition, 7 participants

that were taking 80 mg/day of simvastatin were diagnosed with rhabdomyolysis, while none

of the participants taking 20 mg/day of simvastatin were diagnosed with rhabdomyolysis

(Meade et al., 2010).

Several other factors relating to changes in simvastatin disposition which may result in

elevated concentrations of the prodrug or the active metabolite have been associated with the

adverse effects observed in patients receiving simvastatin treatment. In particular, this statin-

induced toxicity has been attributed to drug-drug interactions and genetic polymorphisms in

enzymes and transporters involved in the clinical disposition of simvastatin and simvastatin

β-hydroxy acid (Becker et al., 2009; Becker et al., 2010; Todd & Goa, 1990).

1.4.2 Drug-drug interactions

Compounds which are known inhibitors of CYP enzymes, particularly CYP3A4, can

influence the oxidative metabolism of simvastatin (Todd & Goa, 1990). Drugs such as

erythromycin, verapamil, itraconazole and cyclosporin undergo extensive metabolism by and

inhibition of CYP3A4 enzymes and consequently may lead to drug-drug interactions with

simvastatin (Kantola et al., 1998; Mauro, 1993; Neuvonen et al., 1998; Pasanen et al., 2006;

10

Pasha et al., 2006). For instance, the presence of erythromycin (1.5 g) increased the Cmax of

simvastatin (40 mg) by 3.4 fold and simvastatin β-hydroxy acid by 5 fold (Kantola et al.,

1998). Verapamil (240 mg) also displays the same effect as erythromycin, increasing the Cmax

of simvastatin and simvastatin acid by 2.6 fold and 3.4 fold, respectively (Kantola et al.,

1998). Itraconazole (200 mg) administered with simvastatin (40 mg) resulted in at least a 10-

fold increase in Cmax of simvastatin and a 17-fold increase in Cmax of simvastatin β-hydroxy

acid (Neuvonen et al., 1998). It was suggested that these elevated levels of both the inactive

lactone form and the active SMVA metabolite were due to inhibition of CYP3A4 enzymes

during first pass metabolism leading to increased bioavailability of ‘total’ simvastatin.

Cyclosporin, another CYP3A4 inhibitor increases maximum plasma concentrations of

simvastatin (5-10 mg) by approximately 6-8 fold when both drugs are co-administered

together (Ichimaru et al., 2001; Lennernäs & Fager, 1997; Neuvonen et al., 2006; Pasanen et

al., 2006). However, cyclosporin can also act as a substrate of transporters involved in the

clinical disposition of simvastatin such as OATP1B1 and P-gp (Neuvonen et al., 2006), hence

the mechanism of this interaction is not clear.

Similarly, grapefruit juice, another inhibitor of CYP3A enzymes and some drug transporters

(Bailey, 2010; Lilja et al., 1998; Lilja et al., 2004; Wang et al., 2001), when given

concomitantly with simvastatin (60 mg) causes a 9-fold increase and a 7-fold increase in the

maximum serum concentrations of simvastatin and simvastatin β-hydroxy acid respectively

(Lilja et al., 1998; Lilja et al., 2004).

Thus, CYP3A4 inhibitors co-administered with simvastatin can influence the disposition of

both the lactone prodrug and the active β-hydroxy metabolite which may consequently lead

to interindividual variability in response to the both pharmacological and toxicological effects

of simvastatin (Arnadottir et al., 1993; Neuvonen et al., 2008). Furthermore, compounds that

11

cause drug-drug interactions at the transporter level, independent of CYP metabolism, may

also affect the influx and efflux of simvastatin and its metabolites to and from the site of

action, potentially altering patients’ response to simvastatin treatment (Lennernäs & Fager,

1997; Neuvonen et al., 1998; Neuvonen et al., 2006; Todd & Goa, 1990). For example,

changes in the plasma concentrations of simvastatin may be related to decreased biliary

clearance of the prodrug caused by drug-drug interactions which inhibit the transporter-

mediated efflux of simvastatin into the bile (Lennernäs & Fager, 1997; Pasanen et al., 2006;

Todd & Goa, 1990). Inhibition of CYP3A4 enzymes and transporters can influence the

biotransformation of simvastatin to simvastatin β-hydroxy acid and therefore cause

unexpected elevations in the serum concentrations of both lactone form and the active

metabolite. It is not known if this also plays a contributory role in the associated increased

risk in the development of skeletal muscle toxicity (Neuvonen et al., 1998).

1.4.3 Genetic Variation

Genetic variants in the oxidative enzymes and transporters which are important in simvastatin

disposition may also play a contributing factor in the interindividual differences observed

when patients receive simvastatin treatment. For instance, inherited genetic polymorphisms

of CYP3A enzymes which result in a decreased enzymatic activity can increase

concentrations of both simvastatin and simvastatin β-hydroxy acid, potentially resulting in

unwanted and unexpected toxicity (Becker et al., 2010). Conversely, polymorphic variants

that increase CYP3A activity may decrease concentrations of both the lactone form and the

active metabolite which may consequently lead to therapeutic failure (Becker et al., 2010).

Furthermore, genetic variants in transporters involved in the disposition of SMV may also

contribute to therapeutic failure or statin-induced muscle toxicity. Therefore, genetic

12

variations in both bioactivation enzymes and transporters may partially cause the

interindividual variability observed during simvastatin therapy, regarding both therapeutic

and adverse events.

1.4.3.1 Genetic variation in CYP enzymes

Analysis of CYP3A isozymes and their polymorphic variants have been conducted to

determine their possible role in influencing the toxicity associated with elevated simvastatin

plasma levels. Individuals with the variant CYP3A4*22 allele (rs35599367) for instance, has

been associated with increased bioavailability of simvastatin and thus have higher

concentrations of simvastatin and simvastatin β-hydroxy acid (Tsamandouras et al., 2014). In

contrast to this, individuals with the wildtype gene CYP3A5*1/*1 exhibited extensive

clearance of SMVA and had a lower mean AUC for simvastatin in comparison to

CYP3A5*3/*3 (rs776746) carriers; however neither the half-life nor the peak plasma

concentrations of simvastatin were affected (Kim et al., 2007). There were no associations

found between CYP3A4-392A>G (rs2740574) and CYP3A5*3 allele variants on the efficacy

and tolerability of simvastatin (Fiegenbaum et al., 2005). These findings indicate that

polymorphic variants of CYP3A4 and CYP3A5 genes may alter the disposition of simvastatin

thus contributing to the interindividual variability in the response to simvastatin therapy.

Albeit a minor pathway, the potential for simvastatin β-hydroxy acid (SMVA) to undergo

lactonization back the parent compound suggests that variation in the gene encoding for UDP

glucuronosyltransferases (UGTs) may possibly influence levels of simvastatin and SMVA

which could be another potential determinant of simvastatin-induced myopathy (Iwuchukwu

et al., 2014). Moreover, it has been recently reported that genetic variation in the UGT1A

13

gene may also be associated with alterations in the clinical efficacy of simvastatin

(Iwuchukwu et al., 2014).

1.4.3.2 Genetic variation in transporters

The absorption, elimination and exposure of tissues to drugs are largely influenced by drug

transport proteins. Simvastatin is a known substrate and possible inhibitor of various

transporters, requiring hepatic uptake in order to produce lipid-lowering effects (Badhan et

al., 2009; Chen et al., 2005; Hochman et al., 2004; Neuvonen et al., 2008; Pasanen et al.,

2006). Thus, polymorphisms of specific drug transport proteins have been strongly associated

with elevated plasma concentrations of simvastatin β-hydroxy acid as well as statin-induced

myopathy. More specifically, the single nucleotide polymorphism (SNP) rs4149056

(c.521T>C) which results in a Val174Ala substitution produces an OATP1B1 transporter

with a complete loss of function (Neuvonen et al., 2008; Postmus et al., 2014; SEARCH

Collaborative Group et al., 2008). Thus SLCO1B1 c.521C has been associated with an

increased risk of statin-induced myopathy, since it has a great impact on influencing SMVA

concentrations (Tsamandouras et al., 2014). For example, peak plasma concentrations

observed for SMVA in individuals who were TT homozygotes was 2.2 ± 0.9 ng/mL, in

comparison to 6.6 ± 1.1 ng/mL in individuals with the SNP variant rs4149056 (Pasanen et al.,

2006). This 200% increase in peak plasma concentrations (Cmax) of the active metabolite may

suggest that individuals with this specific allele variant are more predisposed to statin-

induced muscle toxicity since they have higher exposure to simvastatin β-hydroxy acid

(Pasanen et al., 2006). Interestingly, this particular polymorphism does not affect plasma

concentrations of the simvastatin lactone (Pasanen et al., 2006).

14

Figure 4. The role of OATP1B1 in the hepatic uptake of simvastatin and simvastatin acid. Genetic variants of SLCO1B1 results in elevated circulating simvastatin β-hydroxy acid (SMVA) concentrations and are associated with an increased risk of myotoxicity.

In addition to higher plasma concentrations of the β-hydroxy acid metabolite (SMVA), the

SNP variant SLCO1B1 c.521C (rs4149056) has been correlated with an increased risk of

reported occurrences of myopathy, particularly in patients given 80 mg/day of simvastatin

 

 

 

portal blood SMV SMVA

SMV SMVA HMGCoA

OATP1B1

hepatocyte

intestinalwall

SMVpo

bile

SMV-->SMVA SMV>>SMVA

[SMVA]increased

MRP2

SLCO1B1 Null variant

SLCO1B1 wildtype

increased risk ofmyotoxicity

portal blood SMV SMVA

SMV HMGCoA

OATP1B1 null

hepatocyte

intestinalwall

SMVpo

bile

SMV-->

SMVA

MRP2

SMVA

 

15

(Ieiri et al., 2009). Genome-wide association studies have reported that TT homozygotes had

a 0.6% cumulative risk of developing myopathy when treated with a daily dose of 80 mg of

simvastatin in comparison to the 13% cumulative risk reported in CC homozygotes (Ieiri et

al., 2009). In addition to this were findings that show individuals with the SLCO1B1 c.621C

variant have a 200% increase in simvastatin β-hydroxy acid plasma concentrations (Pasanen

et al., 2006). Therefore it has been surmised that OATP1B1 may have an important role in

the hepatic uptake of SMVA. Thus individuals with polymorphic variants that result in total

loss of transport function are predicted to have lower hepatic uptake of the prodrug leading to

elevated plasma concentrations of the active metabolite (SMVA) and therefore an increased

risk of developing myotoxicity (Figure 4) (Pasanen et al., 2006; Postmus et al., 2014;

SEARCH Collaborative Group et al., 2008).

Polymorphisms in other drug transport proteins may also contribute to statin-induced

myopathy. Variants in the ABCB1 gene (which encodes for P-gp) such as the ABCB1 1236T

(rs1128503, Gly412Gly) variant allele are associated with a considerable decrease in total

cholesterol and low density lipoproteins (LDLs) after treatment with simvastatin

(Fiegenbaum et al., 2005). These variant allele has also been associated with a higher risk of

adverse events occurring during simvastatin therapy (Fiegenbaum et al., 2005). In addition,

carriers of the ABCB1 c.1236T-c.2677T-c3435T (TTT) haplotype (rs1128503, Gly412;

rs2032582, Ser893Ala/Thr; rs1045642, Ile1145Ile) exhibited an increased AUC0-12h of

simvastatin β-hydroxy acid but this was not associated with any significant effect on either

the half-life or the peak plasma concentration of SMVA (Keskitalo et al., 2008). This

suggests that the role of P-gp in the disposition and transport of simvastatin β-hydroxy acid is

minor, irrespective of polymorphic variants in the gene (Hochman et al., 2004; Keskitalo et

al., 2008).

16

Genetic alterations in the ABCC2 gene encoding for MRP2 have also been attributed to

higher risk or occurrences of adverse effects, with H12 haplotypes more at risk than H2

haplotypes (Becker et al., 2013). Due to the lactone prodrug showing preferential inhibition

of MRP2, genetic variances in the expression or activity of this transporter can also

contribute to changes in plasma concentrations of simvastatin and thus simvastatin acid.

These numerous genetic studies indicate that polymorphisms in enzymes or transporters

largely responsible for metabolism and transport of simvastatin and simvastatin acid may

influence the pharmacokinetics of both the inactive lactone prodrug and the active metabolite.

Further investigations should be undertaken to ascertain if these genetic variances are also

great contributors to simvastatin-induced muscle toxicity.

1.4.4 Demographic factors

Aside from drug-drug interactions and genetic variances, other factors such as environmental

chemicals, diet, age and gender may influence the disposition and bioactivation of

simvastatin (SMV) to the β-hydroxy acid form (SMVA). For instance, it has been reported

that sexual dimorphism can influence the efficacy and safety of simvastatin, in which women

had higher reported incidences of myalgia (25.9 %) in comparison to men (9.0 %) while

abnormal liver function was higher in men (17.9 %) compared to women (7.6 %) (Smiderle

et al., 2014). Comorbidities such as hepatic dysfunction, renal insufficiency, hypothyroidism,

advanced age, diabetes mellitus and other serious infections are a few of other additional risk

factors which should be considered that may affect the pharmacological action of simvastatin

(Bellosta et al., 2004; Stancu & Sima, 2001).

17

1.5 Ester hydrolases

Ester hydrolases are enzymes which can hydrolyse compounds containing esters, amides and

thioester bonds (Fukami & Yokoi, 2012). A myriad of hydrolytic ‘esterase’ enzymes are

present in humans including acetylcholinesterase (AChE), acyl-CoA thioesterase 1 (ACOT1),

butyrylcholinesterase (BChE), carboxyl ester lipase (CEL), lysosomal acid cholesterol

esterase (LIPA), sialic acid acetylesterase (SIAE), human serum albumin (HSA),

carboxylesterase (CES1, 2, 3, 4A and 5A) and paraoxonase (PON1, 2, and 3) (Berry et al.,

2009; Darvesh et al., 2004; Fukami & Yokoi, 2012; Li et al., 2005). These ester hydrolases

have the capacity to hydrolyse numerous drug substrates depending on their structure. For

instance, esterases show selectivity towards different types of esters which can affect the

hydrolysis of several drug substrates. PON is known to catalyse the hydrolysis of cyclic-

esters (lactones), organic-esters and alkyl esters (carboxylesters) (Draganov & La Du, 2004;

Fukami & Yokoi, 2012; Tougou et al., 1998) whereas both CES (Imai, 2006; Takai et al.,

1997; Tang et al., 2006) and BChE (Masson et al., 1998) can catalyse the hydrolysis of alkyl

esters.

1.5.1 The role of esterases in the bioactivation of simvastatin

Since simvastatin is synthesised as a prodrug, the bioactivation of the lactone form to the β-

hydroxy acid active metabolite is important in order to produce pharmacological effects. This

bioactivation of SMV to SMVA is a hydrolysis reaction and much of the literature reviewed

for this dissertation states that this reaction is catalysed by ester hydrolases (Casey et al.,

2013; Corsini et al., 1995; Elsby et al., 2012; Prueksaritanont et al., 2001; Vree et al., 2001;

Wilke et al., 2012; Winsemius et al., 2014). However, the specific hydrolytic esterase

enzymes involved in this bioactivation have not been fully elucidated. Since there is known

18

variation in the expression and activity of many ‘esterase’ enzymes, this could potentially

play a role in the variable or elevated simvastatin β-hydroxy acid metabolite concentrations

observed in some individuals.

Preclinical models (rats and rabbits) have demonstrated that incubation of simvastatin in

plasma results in the in vitro hydrolysis of simvastatin, but when carried out in similar

conditions in dog and human plasma, no apparent hydrolytic loss of simvastatin was observed

(Vickers et al., 1990). However, prior to analysis of this reaction in dog and human plasma,

the addition of acid to these samples was carried out. Since this is known to convert

simvastatin β-hydroxy acid (SMVA) back to the parent compound SMV, the ability of human

or dog plasma to hydrolyze simvastatin to simvastatin β-hydroxy acid is not clear.

1.5.2 Carboxylesterase (CES)

Carboxylesterase enzymes are classified as serine esterases, which can hydrolyze numerous

compounds as well as alkyl esters (Fukami & Yokoi, 2012). In humans, CES enzymes can be

further categorized into 5 subfamilies: CES1, CES2, CES3, CES4A and CES5A (Holmes et

al., 2010). Human CES1 is predominantly expressed in the liver and lung, while CES2 is

highly abundant in the liver, small intestine and the kidneys (Imai, 2006). Both isozymes are

also expressed in human liver cytosol and it has been postulated that CES1 and CES2 are

localized in the lumen side of the endoplasmic reticulum (Fukami & Yokoi, 2012; Potter et

al., 1998; Tabata et al., 2004; Xu et al., 2002). These two CES isozymes are known to

catalyse the biotransformation reaction of several drugs and prodrugs. For instance, CES1

preferentially hydrolyses compounds with a small alcohol group and large acyl group. Drugs

such as clopidogrel, imidapril and methylphenidate are known substrates of CES1 (Sun et al.,

2004; Takai et al., 1997; Tang et al., 2006). Conversely CES2 substrates generally contain a

19

large alcohol group and a small acyl group, thus CES2 shows preference towards hydrolyzing

drugs such as irinotecan and prasugrel (Humerickhouse et al., 2000; E. T. Williams et al.,

2008).

Numerous studies have speculated that carboxylesterase enzymes are responsible for

catalyzing the bioactivation reaction of simvastatin (Casey et al., 2013; Kim et al., 2011;

Pasanen et al., 2006; Vree et al., 2001; Wilke et al., 2012). Nonetheless, the only direct study,

which has shown the role of CES in the bioactivation of simvastatin, has demonstrated that

purified carboxylesterase (CES1) was not capable of hydrolyzing simvastatin to simvastatin

β-hydroxy acid (Wang et al., 2015).

Be that as it may, there is some evidence that simvastatin may be an inhibitor of CES. Co-

incubation of clopidogrel with simvastatin in human liver S9 fractions led to a significant

increase in the formation of the, 2-oxo-clopidogrel, intermediate metabolite of clopidogrel

(Wang et al., 2015). In addition, simvastatin inhibited the CES1 hydrolysis of clopidogrel, 2-

oxo-clopidogrel and the active metabolite (Wang et al., 2015). Thus, although CES1 may not

be the primary enzyme involved in the bioactivation of simvastatin to simvastatin β-hydroxy

acid, drug-drug interactions caused by co-administration of a statin and a known substrate of

carboxylesterase could potentially cause unwanted or unexpected toxicity.

Several genetic polymorphisms have been identified in CES however evidence regarding the

contribution of these genetic variants on the disposition of drugs is limited. 16 SNPs in the

CES1 gene and 11 SNPs in CES2 have been determined (Marsh et al., 2004). In particular,

changes in the catalytic efficiency of carboxylesterase have been attributed to polymorphic

variants of the CES1 gene which result in p.Gly143Glu and p.Asp260fs mutations (Zhu et al.,

2008). These variant CES1 genes may consequently affect the pharmacokinetic profile of

20

drug substrates that are specifically hydrolyzed by CES1. This may also influence their

therapeutic effects, as several of these substrates are prodrugs, which require bioactivation.

1.5.3 Paraoxonase (PON)

The paraoxonase (PON) family is classified as a class A-esterase and is made up of three

enzymes (PON1, PON2 and PON3) (Billecke et al., 2000; Khersonsky & Tawfik, 2005;

Précourt et al., 2011). Paraoxonases are calcium dependent serum enzymes, known to

hydrolyze lactones, organophosphates and different types of esters (Billecke et al., 2000;

Khersonsky & Tawfik, 2005; Précourt et al., 2011). PON1 and PON3 are synthesized and

expressed in the liver and secreted into plasma while PON2 is predominantly expressed in

human tissues but not in plasma (Fukami & Yokoi, 2012; Ng et al., 2001). In the liver, PON

enzymes are located specifically in the endoplasmic reticulum (Fukami & Yokoi, 2012;

Gonzalvo et al., 1998). Among the three isoforms, PON1 in particular has been extensively

studied and has been shown to exhibit lactonase activity, able to metabolise drugs and

prodrugs with a lactone moiety (Billecke et al., 2000). In addition, PON1 can hydrolyze

organophosphates such as paraoxon as well as drugs containing a cyclic carbonate group such

as prulifloxacin (Tougou et al., 1998), pilocarpine (Hioki et al., 2011) and olmesartan

medoxomil (Ishizuka et al., 2012). PON3 is also capable of hydrolyzing drugs with a lactone

ring such as lovastatin and spironolactone. While PON2 has one known substrate, N-(3-

oxododecanoyl)-L-homoserine lactone, there are no known drug substrates that are

specifically hydrolysed by this isoform of paraoxonase (Teiber et al., 2008).

It has been reported that purified PON1 was able to hydrolyze simvastatin at a rate of 684.5 ±

34.5 pmol/min/mg (Billecke et al., 2000). Further assessment of the esterase activity of PON,

also revealed that simvastatin may be a substrate of PON3. Lactone forms of other statins

21

such as lovastatin and atorvastatin have been also reported as substrates for PON (Riedmaier

et al., 2011).

Thus, since PON is expressed in the intestinal mucosa, the liver and in blood plasma in

humans, it is not known whether the instability of simvastatin ex vivo is due to either the

chemical (hydrolytic) instability of the lactone or the catalytic action of esterases such as

PON in the blood plasma. While PON has the capacity to catalyse the hydrolysis reaction of

numerous drugs and compounds, literature surrounding drug-drug interactions on

paraoxonase-mediated bioactivation of simvastatin is scarce.

Over 160 polymorphic mutations have been discovered in the PON1 gene but the most

common genetic variants are L55M and Q192R (Costa et al., 2005; Fukami & Yokoi, 2012;

Précourt et al., 2011). Some of these genetic variants alter both activity and the protein

expression of PON. For instance, the alloform PON1Q192 displayed greater efficiency at

metabolizing oxidized HDL or LDL compared to PONR192 (Précourt et al., 2011).

Polymorphic -108C allele results in PON1 expression that is approximately two times greater

in comparison to the -108T allele (Aviram et al., 2000; Brophy et al., 2001). PON2 and

PON3 polymorphisms also exist however the effect of these polymorphisms on drug

metabolism have not been elucidated (Précourt et al., 2011). Currently, there is no literature

evidence which show that genetic variants in the PON gene can influence simvastatin-lactone

hydrolysis however it has been reported that PON1 and PON3 polymorphisms can affect the

hydrolysis of another statin, atorvastatin lactone (Riedmaier et al., 2011). Furthermore, no

association studies have assessed the effect of genetic variants of PON with the

pharmacokinetic disposition or the therapeutic outcome of simvastatin.

Although the physiological role of PON1 has not been fully explored, it has been suggested

that this particular ester hydrolase may have a cardio-protective role by preventing oxidation

22

of LDLs (Draganov et al., 2005; Précourt et al., 2011; Tougou et al., 1998). For instance,

differences in PON1 activity has been associated with the risk of developing cardiovascular

disease, atherosclerosis and other disease related to oxidative stress (Draganov et al., 2005;

Khersonsky & Tawfik, 2005; Précourt et al., 2011). Thus some individuals with

cardiovascular disease may have variants of this gene, which could potentially affect

simvastatin disposition.

It is also evident from several in vivo and in vitro studies that simvastatin has the potential to

influence the activity and expression of PON1 (Balogh et al., 2001; Mirdamadi et al., 2008;

Tomas et al., 2000). Interestingly, simvastatin therapy was shown to modulate the expression

of PON1 (assayed in vitro) (Deakin et al., 2003) and increased serum PON1 activity in vivo

after 3-4 months of treatment (Mirdamadi et al., 2008; Tomas et al., 2000). Hence, if the

bioactivation of simvastatin is potentially catalyzed by paraoxonase, the duration of

simvastatin treatment alone could contribute to the interindividual variability in plasma

pharmacokinetics of simvastatin. Moreover, modulation of PON activity caused by

simvastatin may cause changes in the protective role of PON on LDL oxidation, which may

also potentially affect the clinical efficacy of the drug in cardiovascular diseases.

1.5.4 Butyrylcholinesterase (BChE)

Butyrylcholinesterase (BChE) is one of the two primary human cholinesterases (Fukami &

Yokoi, 2012; Li et al., 2005). It is synthesized in the liver and secreted into plasma, but is

also present in other tissues such as lungs, brain and heart (Jbilo et al., 1994). The catalytic

properties of BChE are similar to acetylcholinesterase (AChE) (Chatonnet & Lockridge,

1989) and BChE has several known drug substrates such as bambutarol (Tunek et al., 1988),

irinotecan (Morton et al., 1999) and succinylcholine (Levano et al., 2005).

23

It has been demonstrated that simvastatin is a concentration-dependent inhibitor of BChE,

with a Ki value of 4.5 µM (Darvesh et al., 2004). However, there are no reports in the

literature indicating that BChE can hydrolyze simvastatin to simvastatin β-hydroxy acid.

Recently, it has been reported that dosing with simvastatin can modulate the expression and

activity of BChE in vivo, in normolipidemic rats (Macan et al., 2015). The activity of BChE

in these rats was increased in plasma by 29% and in the liver by 18% after a 21-day treatment

with simvastatin at a dose of 10 mg/kg/day (Macan et al., 2015). Intriguingly, in humans,

increased BChE activity has been correlated with abnormal lipid metabolism and BChE

activity is increased in patients diagnosed with hypercholesterolemia, hypertension, obesity

and diabetes (Rustemeijer et al., 2001). It has been proposed that this increase in enzyme

activity may be associated with increased serum levels of LDL-cholesterol and

triacylglycerols (Rustemeijer et al., 2001).

1.5.5 Albumin

Albumin is the most abundant protein in blood plasma and human serum albumin (HSA) is

known to have an extensive role in drug transport (Yang et al., 2007). However albumin may

also have a role in drug metabolism as it has been reported to have “esterase-like” activity

(Morgan & Truitt, 1965; Morikawa et al., 1979). In particular, HSA can hydrolyse alkyl

esters such as the prodrug aspirin (Morgan & Truitt, 1965; Morikawa et al., 1979).

The esterase activity of HSA towards aspirin is due to the binding of the acetyl group of

aspirin with the lysine residue of HSA (Bahar & Imai, 2013). Acylation of HSA then occur,

releasing the hydrolysed aspirin (Bahar & Imai, 2013). Acyl-glucuronides of other drugs such

as fenoprofen (Volland et al., 1991), etodolac (Smith et al., 1992) and gemfibrozil (Sallustio

et al., 1996) are known to undergo de-glucuronidation by human serum albumin. Currently,

24

there is no literature available which examines the ability of albumin to hydrolyse simvastatin

to simvastatin β-hydroxy acid. Nevertheless, changes in the expression or activity of these

ester hydrolases could potentially influence the bioactivation and efficacy of simvastatin.

However, very little is known about the bioactivation of simvastatin, the esterase enzymes

involved in this bioactivation step and the factors that which may influence this hydrolysis

reaction.

1.6 Possible drug-drug interactions between simvastatin and co-medications which are esterase substrates

Although simvastatin is hydrolysed to simvastatin β-hydroxy acid, the role of esterase

enzymes such as CES, PON and albumin in this reaction is not clear. However, a number of

common co-medications used in the treatment of cardiovascular disease (Stancu & Sima,

2001) are also substrates for these enzymes. Nonetheless it is not known whether taking a

combination of these drugs leads to a risk of drug-drug interactions.

For instance, statin combined with an anti-hypertensive drug (ACE inhibitor) and aspirin are

commonly used concomitantly in individuals which show a high risk of developing

cardiovascular diseases (Lonn & Yusuf, 2009; Stancu & Sima, 2001). In fact, the concept of

a polypill where a mixture of three or more of these type of drugs in a single pill has been

demonstrated to improve patient compliance (Selak et al., 2014; Soliman et al., 2011).

However, several of these co-medicated drugs such as ACE inhibitors and aspirin are ester

prodrugs which are also bioactivated by ester hydrolases (Bahar & Imai, 2013; Dhareshwar,

2007), which may result in drug-drug interactions.

Aspirin (acetylsalicylic acid; ASA) is an alkyl ester prodrug that is hydrolysed to the active

metabolite salicylic acid by several esterase enzymes (Figure 5) (Bahar & Imai, 2013).

25

Known as the most widely used drug globally, aspirin is one of the common co-medications

for the management of cardiovascular-related diseases (Bahar & Imai, 2013; de Cates et al.,

2014).

The bioactivation of aspirin to the active metabolite is catalysed by CES1 and CES2 in the

liver and intestine (Inoue et al., 1980; Takai et al., 1997; Tang et al., 2006; Williams et al.,

1989) and by PON (Santanam & Parthasarathy, 2007) and BChE (Masson et al., 1998) in

plasma. Albumin has also been shown to display catalytic activity towards the hydrolysis of

aspirin (Bahar & Imai, 2013; Fukami & Yokoi, 2012).

Figure 5. The chemical structure of the prodrug aspirin and its active metabolite salicylic acid and the enzymes reported to be involved in the bioactivation of aspirin.

Aspirin also has the potential to induce the expression of PON which can consequently lead

to elevations in PON activity and thus potentially cause further bioactivation of aspirin

(Jaichander et al., 2008). A similar effect of simvastatin on PON activity has also been

reported (Section 1.5.3).

26

Since aspirin is a substrate for several enzymes which have been speculated to catalyse the

bioactivation of simvastatin, a drug-drug interaction may feasibly occur due to possible

competition for these hydrolytic esterase enzymes. It is not known whether the efficacy and

safety of either of these drugs is compromised if such an interaction occurs when these drugs

are administered together.

1.7 Goal of this project

This first step in converting SMV to SMVA is not only essential for the therapeutic action of

this statin but may also be a contributing factor associated with muscle toxicity due to

elevated SMVA levels. Therefore, the goal of this project was to characterise the

bioactivation of simvastatin in human liver and plasma and to identify the esterase enzyme(s)

involved. The experiments undertaken to achieve this goal are summarised below.

In order to examine the bioactivation of the prodrug simvastatin to simvastatin β-hydroxy

acid, an HPLC assay was established based on a literature method (Carlucci et al., 1992). A

positive control (rat plasma) was used to demonstrate the ability of the assay to detect this

hydrolysis reaction. This HPLC assay was then used to detect the enzymatic loss of SMV

prodrug and formation of the active metabolite (SMVA) in human liver subcellular fractions

and human plasma. Finally, purified forms of several commercially available esterases were

incubated with simvastatin to attempt to identify the role of any of these enzymes in this

reaction.

The results of this project may provide evidence to support the common assumption that

simvastatin is a substrate for human liver CES enzymes, as currently the hydrolytic esterase

enzymes involved in this bioactivation has not been elucidated.

27

2 Materials and methods

2.1 Materials

Simvastatin (SMV) was purchased from Toronto Research Chemicals Inc. Ivermectin was

purchased from Sigma Aldrich, Australia. Acetonitrile analytical grade was obtained from

Scharlab, S.L., Spain. Sodium dihydrogen phosphate (NaH2PO4) and calcium chloride

(CaCl2) were purchased from Riedel-deHaën, Germany. Sodium hydroxide (NaOH) was

purchased from J.T. Baker Chemical Co., Phillipsburg, USA. Hydrogen chloride (HCl) was

purchased from Biolab Ltd., Australia. Chloroform was purchased from Ajax Chemicals,

Australia and methanol from VWR Chemicals, USA. Potassium dihydrogen orthophosphate

(KH2PO4) was purchased from BDH Laboratory Supplies., England and di-sodium hydrogen

orthophosphate (Na2HPO4) from UNILAB, Australia. Dimethyl sulfoxide (DMSO) HPLC

grade was obtained from Scharlau Chemie., Spain. Ammonium for was purchased from

Acros OrganicsTM., Pittsburgh, USA. Water, unless otherwise stated refers to ultra-pure water

obtained through a Milli-Q ultrapurification system (Merck Millipore | Billerica, MA, USA).

Pooled human liver microsomes (HLM) were purchased from BD Biosciences, USA (ethics

number AKL/98/040/AM). Pooled human cytosol HL5-39 (ethics number AKL/98/040/AM)

and rat plasma was kindly provided by Dr. M D Tingle, Department of Pharmacology (ethics

number AEC R824). Expired human plasma was purchased from NZ Blood service (ref

2013/32).

Purified human carboxylesterase 1 isoform b (CES1) and carboxylesterase 2 (CES2),

butyrylcholinesterase (BChE) from human serum and albumin (HSA) from human serum

were purchased from Sigma Aldrich, Co., USA. Purified paraoxonase 1 (PON1) standards

QQ and RR phenotypes were purchased from ZeptoMetrix Corporation (Buffalo, NY).

28

2.2 Methods

2.2.1 Preparation of stock solutions

2.2.1.1 Simvastatin Standard

A stock solution of simvastatin (1.0 mg/mL) was prepared by dissolving 1 mg of simvastatin

in 1 mL acetonitrile and stored at -80°C. Working solutions were then prepared by further

dilution (1:10, v/v) of simvastatin into acetonitrile to a final concentration of 0.1 mg/mL.

Aliquots of this working solution were used as an HPLC reference standard or were diluted

further into 67 mM phosphate buffer (pH 7.4) for use as a stock solution for incubations

(Section 2.2.4).

2.2.1.2 Internal Standard

Ivermectin was chosen as the internal standard due to having a log P value of 5.83. A stock

solution of ivermectin (1.0 mg/mL) was prepared by addition of 1 mg of ivermectin into 1

mL of acetonitrile. This was stored at -80°C. Working solutions were prepared by further

dilution (1:10 v/v) of ivermectin into acetonitrile to a final concentration of 0.1 mg/mL.

Addition of 50 µL of ivermectin (0.1 mg/mL) was used as the internal standard during

extraction of incubation samples.

29

2.2.1.3 Simvastatin (SMVA) reference standard

Based on a method from the literature, (Bhatia et al., 2011) chemical synthesis of simvastatin

acid was attempted by dissolving 25 mg of simvastatin in 25 mL of methanol followed by the

addition of 12.5 mL of NaOH (2 M). The solution was then placed in a water bath for 1 hour

at 45°C (Semco Temperature controlled water bath – Global Sciences). The solution was

cooled to room temperature and then neutralized with 12.5 mL of HCl (1 M). Simvastatin

acid was extracted from the solution by adding 10 mL of chloroform followed by separation

of the chloroform from the aqueous layer, repeated three times. The extracted layer was then

evaporated to dryness (Eppendorf Concentrator 5301, Eppendorf, Hamburg, Germany). Solid

crystals were resuspended in 25 mL of methanol and aliquots of the material was analysed by

HPLC.

Alternatively, a solution of simvastatin β-hydroxy acid was prepared as follows. 100 µL of

NaOH (2 M) was added to 100 µL of simvastatin dissolved in acetonitrile (1 mg/mL). This

solution was neutralized by addition of 130 µL of HCl (1 M) and diluted by addition of 670

µL of acetonitrile. Assuming 100% conversion of the starting material, the nominal

concentration of simvastatin β-hydroxy acid (SMVA) was 0.1 mg/mL. This solution was

used as a chromatography reference standard to determine the relative retention time of

SMVA.

30

2.2.2 Chromatographic system and conditions

The HPLC system was based on the method from the literature (Carlucci et al., 1992). The

chromatographic apparatus consisted of a Hewlett-Packard 110 diode array detector

(Hewlett-Packard, USA) and the separation was performed on a 150 mm x 4.60 mm, 5

micron Gemini C18 column (Phenomenex Inc., USA). The mobile phase comprised of a

linear gradient of 50 mM ammonium formate (pH 4.5) and acetonitrile, delivered at a flow

rate of 1.5 mL/min. The gradient conditions were: 55% acetonitrile for 0-13 minutes,

increasing to 90% acetonitrile by 16 minutes. This was maintained until 24 minutes and then

returned to initial conditions at 25 minutes. This included a minor modification of the original

method (replacement of 25 mM sodium dihydrogen phosphate (pH 4.5) as the aqueous

mobile phase). The mobile phase was prepared weekly and filtered with 0.2 µM pre-cut

membrane filters (Alltech Associates Inc., USA) before use. The column was maintained at

room temperature and the compounds were detected at a wavelength of 238 nm with a

bandwidth of 10 nm and a reference wavelength 550 nm.

2.2.3 Method to determine the limit of detection of the HPLC assay

A solution of simvastatin (12.5 µg/mL) was prepared by dilution of 75 µL of simvastatin (0.1

mg/mL) into 525 µL of acetonitrile. Aliquots (1-80 µL) of this solution were directly injected

onto the column and the peak area of the eluent recorded. A solution of simvastatin β-

hydroxy acid (12.5 µg/mL) was prepared and analysed in a similar manner.

31

2.2.4 Enzymes

As reported by the supplier, CES1 (5 mg/mL) has an activity of ≥500 units/mg protein while

CES2 (5 mg/mL) has an activity of ≥1000 units/mg protein, where one unit (U) will

hydrolyse 1 nmol of 4-nitrophenyl acetate per minute at pH 7.4 at 37°C.

Purified butyrylcholinesterase (BChE) was supplied at ≥50 units/mg protein (168

µmol/min/mg), where 1 unit of specific activity will hydrolyse 1 µmol of butyrylthiocholine

per minute at pH 8.0 at 37°C.

Human paraoxonase 1 (PON) was supplied as 40 kU/mL of (QQ) with a specific activity of

171-200 kU/L or PON (RR) with a specific activity of 54-66 kU/L. PON1 (QQ) has been

reported to have an arylesterase activity of 870 µmol/min/mg while PON1 (RR) has an

arylesterase activity of 731 µmol/min/mg.

Microsomes, cytosol, purified enzymes and plasma were stored at -80°C and were thawed

immediately prior to use.

Protein concentration was determined using the Direct DetectTM Spectrometer (EMD

Millipore Corporation, Billerica, MA, USA). For non-enzymatic conditions, proteins were

denatured by heating in a water bath (Semco Temperature controlled water bath – Global

Sciences) at 80°C for a minimum of 5 minutes.

Phosphate buffer (67 mM, pH 7.4) was prepared by dissolving 4.775 g of di-sodium

hydrogen phosphate (Na2HPO4) and 0.885 g of potassium dihydrogen phosphate (KH2PO4)

into 500 mL of water.

32

2.2.5 Analysis of the hydrolysis of simvastatin by plasma or subcellular fractions of liver

All incubations (n=4) were carried out in 67 mM phosphate buffer (pH 7.4), at a final

incubation volume of 0.5 mL. The enzyme source under investigation (e.g. microsomes,

cytosol or plasma) was added and pre-incubated for 5 minutes. The reaction was then

initiated by the addition of 100 µL of simvastatin (0.1 mg/mL in phosphate buffer) to give a

final concentration of 20 µg/mL (48 µM) in the incubation. The reaction mixture was

incubated at 37°C, for 30 minutes, with gentle agitation (300 rpm) in a thermomixer

(Eppendorf Thermomixer® Comfort, Eppendorf, Hamburg, Germany). After 30 minutes, ice-

cold acetonitrile (0.5 mL) was added to stop the reaction.

Internal standard, ivermectin (50 µL of 0.1 mg/mL) was then added to the samples. The

sample was then stored at -20°C for a minimum of 1 hour to precipitate the proteins followed

by centrifugation (IEC micromax microcentrifuge, Thermoelectron Corporation, Milford,

USA) at 20,000g for 10 minutes to ensure protein precipitation. The clear supernatant (970

µL) was then aspirated into a clean tube and evaporated to dryness under vacuum (Eppendorf

Concentrator 5301, Eppendorf, Hamburg, Germany). All samples were resuspended in a 100

µL solution of DMSO: ammonium formate (50 mM, pH 4.5): acetonitrile (20:35:45 v/v).

Aliquots of each sample were then analysed by HPLC.

Pooled human liver microsomes, pooled human liver cytosol and human plasma were

fortified with calcium by adding 10 µl of 2 mM CaCl2 in the solution.

To investigate the hydrolysis of simvastatin in human and rat plasma, 250 µL of rat plasma

(50 mg/mL) or human plasma (47 mg/mL) was suspended in 150 µL of 67 mM phosphate

buffer (pH 7.4) to yield a final concentration of 26 mg/mL and 23 mg/mL in solution,

respectively. To investigate the hydrolysis of simvastatin in human plasma fortified with 2

33

mM CaCl2, 100 µL of human plasma (47 mg/mL) was suspended in 290 µL of 67 mM

phosphate buffer to yield a final concentration of 9 mg/mL.

To investigate the hydrolysis of simvastatin in pooled human liver microsomes fortified with

2 mM CaCl2, 50 µL of pooled HLM (20 mg/mL) was suspended in 340 µL of 67 mM

phosphate buffer to yield a final concentration of 2 mg/mL.

To investigate the hydrolysis of simvastatin in pooled human liver cytosol fortified with 2

mM CaCl2, 149 µL of cytosol (17 mg/mL) was suspended in 241 µL of 67 mM phosphate

buffer to yield a final concentration of 5 mg/mL.

Similar incubations and extraction conditions were undertaken for the assessment of the

ability of BChE and human serum albumin (HSA) to hydrolyse simvastatin. To investigate

the hydrolysis of simvastatin in the presence of butyrylcholinesterase (BChE), BChE (5 U)

was dissolved in 1 mL of phosphate buffer (67 mM, pH 7.4) to have a working stock solution

of 5 U/mL of BChE. Increasing volumes of BChE (5 U/mL) was added into phosphate buffer

(67 mM, pH 7.4) to yield a final concentration of 0.02, 0.2 and 1 U/mL.

To investigate the hydrolysis of simvastatin in the presence of human serum albumin (HSA),

400 mg of HSA was dissolved in 2 mL of phosphate buffer (67 mM, pH 7.4) to have working

stock solution of 200 mg/mL HSA. Increasing volumes of HSA (200 mg/mL) was added into

phosphate buffer (67 mM, pH 7.4) to yield a final concentration of 5 mg/mL – 40 mg/mL of

HSA.

Non-enzymatic controls (n=4) were included to take into account spontaneous hydrolysis of

simvastatin in aqueous buffer. These were either heat denatured microsomes, cytosol or

plasma, or zero protein (as indicated in each experimental section).

34

2.2.6 Analysis of the hydrolysis of simvastatin in the presence of paraoxonase

Incubation conditions for the analysis of simvastatin hydrolysis in the presence of

paraoxonase (PON) differed from the conditions in Section 2.2.5 to reflect the method

reported in the literature (Billecke et al., 2000). Briefly, the incubation consisted of 30 µL of

the enzyme solution, 5 µL of simvastatin in acetonitrile (1 mg/mL), in a final volume of 300

µL of 67 mM phosphate buffer (pH 7.4) containing 1 mM CaCl2. The final substrate

concentration was 16.7 µg/mL (40 µM) and the final enzyme concentration was 4 U/mL. The

reaction mixture was then incubated at 37°C for 30 minutes with gentle agitation (300 rpm)

and the reaction was terminated by the addition of acetonitrile (300 µL). The samples were

then immediately centrifuged at 20,000 g for 10 minutes and aliquots (100 µL) were kept on

ice until HPLC analysis. Identical incubations were undertaken to assess the ability of PON

to biotransform simvastatin β-hydroxy acid (final substrate concentration of 16.7 µg/mL)

The ability of CES1 and CES2 to catalyse the hydrolysis of simvastatin was also undertaken

using this method. Based on the literature (Kim et al., 2014), 6 µL of purified

carboxylesterase solution (5 mg/mL) was diluted into a final volume of 300 µL of 67 mM

phosphate buffer (pH 7.4) to give a final concentration of 0.1 mg/mL of CES protein in the

incubation. The hydrolysis of simvastatin was investigated at two substrate concentrations: 5

µL of 0.1 mg/mL or 1 mg/mL simvastatin solution to give a final substrate concentration in

the incubation of 1.67 µg/mL and 16.7 µg/mL (4 µM and 40 µM) respectively.

35

2.3 Data Analysis

The peak area of simvastatin, simvastatin β-hydroxy acid and ivermectin (internal standard)

was detected at 238 nm and the HPLC chromatograms were analysed using Agilent

ChemStation software (Agilent Technologies, USA). Parameters of interest were the area-

under-the-curve (AUC) and the retention times of the fluorescence signal for each analyte

peak. Retention times of the analytes were determined following injection of authentic

standards. Fluorescence AUC data was obtained by manual integration of analyte peaks.

The data were presented as analyte/internal standard peak area ratio (PAR) relative to non-

enzymatic control, or as analyte peak area (PA) relative to non-enzymatic control.

Chromatograms were exported into GraphPad Prism 6.0 (GraphPad Software Inc., La Jolla,

CA) and graphs were drawn using the same software. Data are represented as mean ±

standard deviation (SD) of n=4 replicates. To determine statistical significance, between the

mean of the treatment conditions being tested and their relative controls, t-tests and one-way

repeated measures ANOVA testing was performed using GraphPad Prism. Statistical

significance was accepted when p<0.05

36

3 Results

3.1 Detection of simvastatin and simvastatin acid using HPLC

Simvastatin standard (0.1 mg/mL) was analysed using the HPLC based on the method in the

literature (Carlucci et al., 1992). A typical chromatogram of the simvastatin standard is

shown (Figure 6), demonstrating the peak resolution and retention time (Rt) of 11.4 minutes.

0 5 10 15 20

0

100

200

300

R e te n t io n tim e (m in )

Absorbance

(mA

U 2

38

nm

)

S M V

Figure 6. Representative chromatogram of simvastatin standard.

Since simvastatin β-hydroxy acid (SMVA) was not commercially available, chemical

synthesis of SMVA was attempted using the method described in the literature (Bhatia et al.,

2011). Briefly, a solution of simvastatin (1 mg/mL) in methanol was treated with sodium

hydroxide (NaOH, 2 M) for 1 hour at 45°C and was neutralised with hydrochloric acid (1 M),

extracted with chloroform and then evaporated to dryness. When the material was

resuspended in methanol, three peaks were observed on the HPLC, with the detection of

simvastatin β-hydroxy acid observed at a retention time of 4.80 minutes as well as

37

simvastatin (the starting material) and the formation of an additional unknown product A at

1.09 minutes (Figure 7).

0 5 1 0 1 5 2 0

0

2 0

4 0

6 0

8 0

R e te n tio n tim e (m in )

Ab

so

rba

nc

e(m

AU

23

8 n

m)

S M V

S M V A

A

Figure 7. Chromatography of the reaction mixture after treatment with NaOH and HCl

Simvastatin (1 mg/mL) was dissolved in methanol and then treated with NaOH (2 M). The solution was placed in a water bath for 1 hour at 45°C followed by the addition of HCl to neutralise the base. Extraction of simvastatin acid was carried out by the addition of chloroform and evaporation of the chloroform layer. This extraction method by chloroform was repeated three times. The reaction mixture was then injected onto the HPLC column and generated three peaks consistent with simvastatin at Rt = 11.42 minutes, simvastatin acid at Rt = 4.80 minutes and an unknown product at Rt = 1.09 minutes.

The detection of SMVA at retention time of 4.80 minutes was confirmed by comparing its

UV spectra with the parent compound SMV (Figure 8). Since simvastatin and simvastatin β-

hydroxy acid have identical UV spectra, it is possible to use simvastatin as a reference

calibration standard to quantify the relative formation of simvastatin acid.

38

2 0 0 2 5 0 3 0 0 3 5 0 4 0 00

5 0

1 0 0

W av e le n g th (n m )

Absorbance

(mA

U 2

38

nm

)

S im va s ta tin

S im v a s ta tin A c id

Figure 8. Comparison of the normalised spectra of simvastatin (red) and simvastatin acid (blue dashed).

0 5 1 0 1 5 2 0

-2

0

2

4

6

8

R e te n tio n tim e (m in )

Ab

so

rba

nc

e(m

AU

23

8 n

m)

S M V A

A

Figure 9. Chromatography of the reaction mixture after treatment with NaOH

Simvastatin (1 mg/mL) was dissolved in methanol and then treated with NaOH. The solution was placed in a water bath for 1 hour at 45°C. Extraction of simvastatin acid was carried out by addition of chloroform and evaporation of the chloroform layer. The reaction mixture was injected onto the HPLC column and generated two peaks consistent with simvastatin acid at Rt = 4.77 minutes and the unknown product A at Rt = 1.10 minutes.

39

It was assumed that was simvastatin observed in this mixture due to the excessive addition of

HCl. When HCl was not added to the reaction, this resulted in the absence of simvastatin and

only two peaks were seen at a retention time of 4.77 minutes and 1.10 minutes, consistent

with SMVA and the unknown compound A, respectively (Figure 9). In addition, a substantial

amount of the unknown product A was detected in the chromatogram, with comparable peak

areas to SMVA. Thus, due to this presence of the unknown compound A, as well as time

constraints, no further attempts to produce pure simvastatin acid were undertaken using this

method in the literature (Bhatia et al., 2011).

0 5 1 0 1 5 2 0

0

5 0

1 0 0

1 5 0

R e te n tio n tim e (m in )

Ab

so

rba

nc

e(m

AU

23

8 n

m) S M V A

Iv e rm e c tin

S M V A

T im e : 4 .5 9

A re a : 3 6 5 2 .3 0

IS

T im e : 1 9 .0 6 9

A re a : 1 3 1 5 .9 0

Figure 10. Chromatogram of simvastatin acid formed using the alternative method

Simvastatin (1 mg/mL) was treated with NaOH (2 M), incubated at room temperature for 20 minutes followed by the addition of HCL (1 M). The internal standard ivermectin (1 mg/mL) was added into the solution and 10 µL was injected onto the HPLC column. The reaction mixture generated two peaks consistent with simvastatin acid at Rt = 4.59 minutes and the internal standard ivermectin at Rt = 19.07 minutes.

40

However an alternative method (Section 2.2.1.3), was carried out which involved the addition

of NaOH (2 M) to simvastatin (1 mg/mL) in acetonitrile followed by the addition of HCl (1

M) to neutralise the base. The internal standard, ivermectin, was added to the solution and the

sample was analysed using HPLC. Two peaks were observed which were consistent with

simvastatin β-hydroxy acid at retention time 4.59 minutes and ivermectin at retention time

19.07 minutes (Figure 10). Using this method did not result in the formation of the unknown

product A.

Based on the method described in the literature, (Carlucci et al., 1992) termination of the

reaction and extraction of simvastatin from incubations was carried out by the addition of ice-

cold acetonitrile. However, the method sections of several literatures have stated that

acetonitrile was acidified during this extraction procedure (Riedmaier et al., 2011; Vickers et

al., 1990; Vickers et al., 1990). Thus, to investigate the effect of acidification during the

sample work up, simvastatin (20 µg/mL) in 67 mM phosphate buffer (pH 7.4) was first

incubated for 30 minutes to detect the spontaneous pH dependent hydrolytic formation of

SMVA followed either by the addition of ice-cold acidified (1% of 0.3 M HCl) or non-

acidified acetonitrile. Addition of acidified acetonitrile resulted in no detection of SMVA

after the 30 minute incubation of simvastatin. In contrast, SMVA was detectable (PA = 928.1

units) after extraction with acetonitrile. Therefore, to ensure detection of any simvastatin acid

formed in incubations, the extraction procedure for all further experiments was carried out by

the addition of (non-acidified) ice-cold acetonitrile.

41

3.2 Spontaneous hydrolytic conversion of simvastatin to simvastatin β-hydroxy acid

0 6 0 1 2 0 1 8 0 2 4 0 3 0 0

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

T im e (m in )

Pe

ak

are

a a

t 2

38

nm

SM V

SM VA

Figure 11. Hydrolytic conversion of simvastatin (0.1 mg/mL) in 67 mM phosphate buffer (pH 7.4) for 300 minutes.

A solution of simvastatin in aqueous buffer was prepared and left to equilibrate for 1 hour at

room temperature, and sequential samples were analysed by HPLC. After 1 hour, hydrolytic

conversion of simvastatin to simvastatin β-hydroxy acid was detectable at a ratio of 1:0.18.

Substantial conversion of simvastatin to the active metabolite was observed after 5 hours at

room temperature (Figure 11).

42

3.3 Calibration curve for simvastatin and simvastatin acid

Figure 12. Representative calibration curve of simvastatin (12.5 µg/mL) in acetonitrile.

Simvastatin (12.5 µg/mL) in acetonitrile was directly injected onto the column at decreasing volumes (from 80 µL to 1 µL). Graph (i) shows the amount of SMV on column at volume injections of 1 µL – 6 µL.

0.00 0.25 0.50 0.75 1.000

1000

2000

3000

Amount of SMV on column (µg)

Peak

are

a at

238

nm

0.000 0.025 0.050 0.075 0.100 0.125 0.1500

100

200

300

400

Amount of SMV on column (µg)

Peak

are

a at

238

nm

i

43

Figure 13. Representative calibration curve of simvastatin β-hydroxy acid (12.5 µg/mL) in 10% NaOH - acetonitrile.

Simvastatin β-hydroxy acid (12.5 µg/mL) in 10% NaOH - acetonitrile was directly injected onto the column at decreasing volumes (from 80 µL to 1 µL). Graph (i) shows the amount of SMVA on column at volume injections of 1 µL – 6 µL.

To determine the limit of detection of the established HPLC assay, decreasing volumes (from

80 µL to 1 µL) of simvastatin (12.5 µg/mL) in acetonitrile and simvastatin acid (12.5 µg/mL)

in 10% NaOH-acetonitrile were injected onto the HPLC column. Linear calibration curves (r2

= 1.00) were observed (Figure 12 and Figure 13). The lowest amount of simvastatin and

simvastatin β-hydroxy acid that was tested was 0.0625 µg on column.

0.00 0.25 0.50 0.75 1.000

1000

2000

3000

Amount of SMVA on column (µg)

Peak

are

a at

238

nm 0.000 0.025 0.050 0.075 0.100 0.125 0.150

0

100

200

300

400

Amount of SMVA on column (µg)

Peak

are

a at

238

nm

i

44

3.4 Bioactivation of simvastatin in subcellular fractions

3.4.1 Hydrolysis of simvastatin in rat plasma, human plasma and human red blood cells

Based on the evidence presented in the literature (Vickers et al., 1990) rat plasma is able to

hydrolyze simvastatin to simvastatin acid. Thus, to ensure that the extraction procedure and

the HPLC assay was able to detect loss of simvastatin and formation of simvastatin acid, the

hydrolysis of simvastatin in rat plasma and human plasma was investigated.

Incubations (n=4) of rat plasma (26 mg/mL) resulted in a significant (p<0.005) quantifiable

loss of simvastatin (0.208 ± 0.036 PAR) in comparison to non- enzymatic (denatured) control

(1.304 ± 0.372 PAR) (Figure 14A). The product had a retention time of 2.899 minutes, which

was confirmed as simvastatin acid by comparing it to the retention time and the UV spectra

obtained from the previous experiment. The 80% loss of the parent compound was accounted

for by the enzymatic formation of simvastatin acid (1.66 ± 0.201 PAR) above denatured

control (Figure 14B).

At the same concentration of human plasma (26 mg/mL) however, there was no significant

loss of simvastatin observed (1.321 ± 0.115 PAR) in comparison to its denatured control

(1.145 ± 0.123 PAR) (Figure 15A). Furthermore, Figure 15B shows that enzymatic formation

of the active metabolite was less (0.828 ± 0.130 PAR) than the non-enzymatic formation

(0.927 ± 0.115 PAR). The overall formation of simvastatin acid in human plasma at 26

mg/mL (Figure 15) was not significantly different (p>0.05) to the amount of simvastatin acid

produced non-enzymatically in the rat plasma at the same protein concentration (Figure 14).

45

C o n tro l R a t 0 .0

0 .5

1 .0

1 .5

2 .0S

imv

as

tati

n r

em

ain

ing

(SM

V /

IS

pe

ak

are

a r

atio

)

A**

C o n tro l R a t0 .0

0 .5

1 .0

1 .5

2 .0

Sim

va

sta

tin

ac

id f

orm

ati

on

(SM

VA

/ I

S p

ea

k a

rea

ra

tio)

B **

Figure 14. Loss of simvastatin (A) and formation of simvastatin β-hydroxy acid (B) in the presence of rat plasma (26 mg/mL).

Simvastatin (20 µg/mL) was incubated with rat plasma (26 mg/mL) at 37°C for 30 minutes (n = 4). For non-enzymatic controls, rat plasma was denatured by applying heat at 55°C for 30 minutes in a water bath. ** (p<0.005)

46

C o n tro l H u m a n0 .0

0 .5

1 .0

1 .5

2 .0S

imv

as

tati

n r

em

ain

ing

(SM

V /

IS

pe

ak

are

a r

atio

)

A

c o n tro l H u m a n0 .0

0 .5

1 .0

1 .5

2 .0

Sim

va

sta

tin

ac

id f

orm

ati

on

(SM

VA

/ I

S p

ea

k a

rea

ra

tio)

B

Figure 15. Loss of simvastatin (A) and formation of simvastatin β-hydroxy acid in human plasma (26 mg/mL)

Simvastatin (20 µg/mL) was incubated with human plasma (26 mg/mL) at 37°C for 30 minutes (n = 4). For non-enzymatic controls, human plasma was denatured by applying heat at 55°C for 30 minutes in a water bath.

47

Since one of the hydrolytic esterase enzymes that may catalyse the conversion of simvastatin

to simvastatin β-hydroxy acid is paraoxonase (PON) which is Ca2+ dependent, the effect of

CaCl2 fortification on the formation of SMVA was determined in human plasma. Incubations

of human plasma (9 mg/mL) fortified with 2 mM CaCl2 at 37°C for 30 minutes were

undertaken. As shown in Figure 16, the presence of Ca2+ did not increase the hydrolytic loss

of simvastatin prodrug, which suggests that human plasma esterases such as PON may not be

involved in the bioactivation of simvastatin. Preliminary assessment of esterase activity of

red blood cells (5 mg/mL) using the same incubation conditions indicated a similar amount of

simvastatin remaining (1.604 ± 0.066 PAR) (Data not shown).

C o n tr o l C o n tr o l + C a C l2

P la s m a P la s m a + C a C l2

0 .0

0 .5

1 .0

1 .5

2 .0

Sim

va

sta

tin

re

ma

inin

g(S

MV

/ I

S p

ea

k a

rea

ra

tio)

Figure 16. The effect of calcium fortification (2 mM CaCl2) on simvastatin hydrolysis in denatured and non-denatured human plasma at a protein concentration of 9 mg/mL.

Simvastatin (20 µg/mL) was incubated with human plasma (9 mg/mL) at 37°C for 30 minutes, in the absence and presence of 2 mM CaCl2 (n = 4). For non-enzymatic controls, human plasma was denatured by applying heat at 55°C for 30 minutes in a water bath.

48

3.4.2 Hydrolysis of simvastatin in pooled human liver microsomes

Evidence in literature has stated that several esterase enzymes are catalytically active in

human liver microsomes. To identify the possible catalytic activity of esterase enzymes on

simvastatin, formation of simvastatin acid was determined in pooled human liver

microsomes. Again, the effect of CaCl2 fortification was also investigated in human liver

microsomes as paraoxonase is known to be synthesized in the liver.

Incubations of human liver microsomes in the absence of the drug did not generate any

endogenous interfering peaks that eluted at retention times similar to simvastatin or

simvastatin acid and the internal standard, ivermectin (Figure 17).

0 5 1 0 1 5 2 0 2 5

0

2 0 0

4 0 0

1 0 0 0

2 0 0 0

R e te n t io n tim e (m in )

Absorbance

(mA

U 2

38

nm

) IS

Iv e rm e c t in

T im e : 1 9 .1 7 5

A re a : 4 0 1 8 .8

Figure 17. Chromatogram of the reaction mixture consisting of human liver microsomes (0.4 mg/mL) and the internal standard, ivermectin, in the absence of simvastatin.

Incubations of simvastatin (20 µg/mL) with human liver microsomes (2 mg/mL) did not

produce any significant loss of simvastatin under all conditions tested (Figure 18A) and there

was no increase in the formation of simvastatin acid in the presence of 2 mM CaCl2 (0.089 ±

0.0095 PAR) compared with microsomes alone (0.088 ± 0.028 PAR) (Figure 18B). In

49

addition, unlike in human plasma, the production of the active metabolite was greater under

enzymatic conditions (0.088 ± 0.028 PAR) than non-enzymatic conditions (0.060 ± 0.005

PAR), however this was not significant (p>0.05).

C o n tro l M ic ro s o m e s M ic ro s o m e s + C a C l2

0 .0

0 .4

0 .8

1 .2

Sim

va

sta

tin

re

ma

inin

g (

SM

V /

IS

pe

ak

are

a r

atio

)

C o n tro l M ic ro s o m e s M ic ro s o m e s + C a C l2

0 .0 0

0 .0 5

0 .1 0

0 .1 50 .4

0 .8

1 .2

Sim

va

sta

tin

ac

id f

orm

ati

on

(SM

VA

/ I

S p

ea

k a

rea

ra

tio)

A

B

Figure 18. Loss of simvastatin (A) and formation of simvastatin β-hydroxy acid (B) in pooled human liver microsomes (2 mg/mL) in the absence and presence of calcium chloride.

Simvastatin (20 µg/mL) was incubated with pooled human liver microsomes (2 mg/mL) at 37°C for 30 minutes, in the absence and presence of 2 mM CaCl2 (n = 4). For non-enzymatic controls, microsomes were denatured by heating at 80°C for 5 minutes.

50

At a protein concentration of 2 mg/mL, the formation of simvastatin acid was hardly

detectable. Thus, to investigate whether formation of the active metabolite is concentration

dependent, simvastatin (20 µg/mL) was incubated with increasing protein concentrations

(0.5, 1.0, 2.0 and 5.0 mg/mL) of pooled human liver microsomes (Figure 19).

0 .5 1 2 50 .0

0 .5

1 .0

1 .5

2 .0

2 .5

P ro te in C o n c e n tra t io n (m g /m l)

Sim

va

sta

tin

re

ma

inin

g(S

MV

/ I

S p

ea

k a

rea

ra

tio

)

E n zy m a tic

A

0 .5 1 2 50 .0

0 .5

1 .0

1 .5

2 .0

2 .5

P ro te in C o n c e n tra t io n (m g /m l)

Sim

va

sta

tin

ac

id f

orm

ati

on

(SM

VA

/ I

S p

ea

k a

rea

ra

tio

)

E n zy m a tic

B

0 .5 1 2 50 .0

0 .5

1 .0

1 .5

2 .0

2 .5

P ro te in C o n c e n tra t io n (m g /m l)

Sim

va

sta

tin

re

ma

inin

g(S

MV

/ I

S p

ea

k a

rea

ra

tio

)

N o n - E n zy m a tic

C

0 .5 1 2 50 .0

0 .5

1 .0

1 .5

2 .0

2 .5

P ro te in C o n c e n tra t io n (m g /m l)

Sim

va

sta

tin

ac

id f

orm

ati

on

(SM

VA

/ I

S p

ea

k a

rea

ra

tio

)

N o n - E n zy m a tic

D

Figure 19. Loss of simvastatin (A and C) and formation of simvastatin β-hydroxy acid (B and D) in incubations of increasing protein concentrations of human liver microsomes.

Simvastatin (20 µg/mL) was incubated with increasing concentrations (0.5, 1.0, 2.0 and 5.0 mg/mL) of human liver microsomes at 37°C for 30 minutes (n=4). The loss of simvastatin and formation of simvastatin acid is shown under enzymatic conditions (A and B) and under non-enzymatic conditions (C and D). Non-enzymatic conditions were achieved by heating pooled human liver microsomes at 80°C for 5 minutes.

Increasing concentrations of protein resulted in no observable loss of simvastatin levels in

both enzymatic and non-enzymatic conditions (Figure 19A and 19C). However, levels of

simvastatin appear to be slightly higher in functional human liver microsomes than in

51

denatured human liver microsomes. The hydrolysis of simvastatin to simvastatin acid under

enzymatic conditions (Figure 19B) seem to be protein concentration dependent with a

significant difference between the amount of simvastatin acid formed at 0.5 mg/mL and 5

mg/mL (p<0.001). Nonetheless, this apparent increase in formation of the hydroxy acid

seems to be very minor in comparison to that which was observed in rat plasma (Figure 14).

There was no concentration dependent increase observed in simvastatin acid formation under

non-enzymatic conditions (Figure 19D). However, the formation of simvastatin acid in

denatured human liver microsomes appears to be slightly higher in comparison to what is

observed in the presence of functional human liver microsomes.

3.4.3 Hydrolysis of simvastatin in pooled human liver cytosol

The lack of formation of simvastatin acid in microsomal proteins led to the investigation of

possible esterases located in cytosol which could potentially catalyse the conversion of the

prodrug to the active metabolite. Incubations of cytosol (5 mg/mL) indicated that formation

of simvastatin acid (0.104 ± 0.19 PAR) was elevated above the formation detected in

denatured cytosol (non-enzymatic hydrolysis) (0.073 ± 0.018 PAR) but again, this was not

significant (p>0.05). Although the presence of CaCl2 increased the formation of simvastatin

acid (Figure 20B) significantly (p<0.05), again this formation was considered very minor in

comparison to the amount of SMVA formed in rat plasma (Figure 14). The formation of

simvastatin acid in cytosol at 5 mg/mL was also similar to the amount of simvastatin acid

formed in microsomal protein at 5 mg/mL which was 0.21 ± 0.004 PAR (Figure 19B). Both

sources of esterase enzyme appear to be forming very minor amounts of simvastatin acid and

this formation is not dependent on the presence of Ca2+ which further suggests that PON may

not be involved in this hydrolysis reaction.

52

C o n tro l C y to s o l C y to s o l + C a C l2

0

1

2

3

4

5S

imv

as

tati

n r

em

ain

ing

(SM

V /

IS

pe

ak

are

a r

atio

)

A

C o n tro l C y to s o l C y to s o l + C a C l2

0 .0 0

0 .0 5

0 .1 0

0 .1 51

3

5

Sim

va

sta

tin

ac

id f

orm

ati

on

(SM

VA

/ I

S p

ea

k a

rea

ra

tio)

B

*

Figure 20. Loss of simvastatin (A) and formation of simvastatin β-hydroxy acid (B) in cytosol (5 mg/mL) in the absence and presence of calcium chloride.

Simvastatin (20 µg/mL) was incubated with cytosol (5 mg/mL) at 37°C for 30 minutes, in the absence and presence of 2 mM CaCl2 (n=4). For non-enzymatic controls, cytosol was denatured by heating at 80°C for 5 minutes. * (p<0.05)

53

In summary, the hydrolysis of simvastatin in the subcellular fractions investigated resulted in

the very minor loss of simvastatin and formation of simvastatin acid. Although enzymatic

hydrolysis was generally always greater than non-enzymatic hydrolysis, the overall formation

of simvastatin acid in the above experiments was very low - from 0.9 to 1.6-fold increase. In

addition, simvastatin loss is not significantly different when enzymatic conditions were

compared with non-enzymatic (denatured) controls (Table 1). Thus, this suggests that

simvastatin may not a substrate for human liver and plasma esterases at the substrate

concentration tested.

Table 1. Simvastatin remaining (%) compared to non-enzymatic (denatured) controls in human liver and plasma.

Human subcellular fractions Simvastatin remaining (%) compared to control

Plasma (26 mg/mL) 115.4 %

Plasma (9 mg/mL) 100.5 %

Cytosol (5 mg/mL) 129.9 %

Microsomes (2 mg/mL) 105.1 %

3.5 Bioactivation of simvastatin in the presence of ester hydrolases

The results thus far suggests that the conversion of simvastatin to simvastatin β-hydroxy acid

is not catalysed by esterases expressed in human liver nor plasma. To confirm these findings,

several purified ester hydrolase enzymes were incubated with simvastatin. To take into

account the spontaneous hydrolytic conversion of simvastatin to simvastatin β-hydroxy acid,

SMV was incubated alone in aqueous phosphate buffer (67 mM, pH 7.4) as a zero-enzyme

control.

54

3.5.1 Hydrolysis of simvastatin in the presence of carboxylesterase

The hydrolysis of SMV was initially tested in the presence of two isoforms of

carboxylesterase, CES1 and CES2. The major hydrolytic esterase enzyme speculated to be

involved in the bioactivation step of SMV is CES1, and CES2 is considered to be the major

intestinal esterase in humans. Based on the literature (Kim et al., 2014), a final concentration

of 0.1 mg/mL of CES1 and CES2 was chosen. The hydrolysis of simvastatin was not

observed in the presence of either isoforms of carboxylesterase (Figure 21). It is interesting to

note that although there was no significant formation of simvastatin in the presence of CES1,

analysis of these data sets show that there was a statistical significant formation (p< 0.05) of

the β-hydroxy acid metabolite in the presence of CES2 (Figure 21B). Nonetheless, this

formation of the acid metabolite above its control is minor compared to that which was

observed in rat plasma (Figure 14).

The catalytic activity of carboxylesterase was also tested at a 10-fold lower substrate

concentration of simvastatin (1.67 µg/mL) and again, it was observed that there was no

significant hydrolysis of simvastatin by CES1 and CES2 in comparison to control. In contrast

to the previous experiment, a lower substrate concentration resulted in no significant

difference between the formation of simvastatin acid via CES1 or CES2 (data not shown).

Findings from this experiment indicate that human carboxylesterase may have a little or no

role in the bioactivation of simvastatin.

55

C o n tro l C E S 1 C E S 20

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0S

imv

as

tati

n r

em

ain

ing

(Pe

ak

are

a)

A

c o n tro l C E S 1 C E S 20

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

Sim

va

sta

tin

ac

id f

orm

ati

on

(Pe

ak

are

a)

B

**

Figure 21. Loss of simvastatin (A) and formation of simvastatin β-hydroxy acid (B) in the presence of CES1 and CES2.

Simvastatin (16.7 µg/mL) was incubated with CES1 (0.1 mg/mL) and CES2 (0.1 mg/mL) at 37°C for 30 minutes (n=4). For non-enzymatic controls, simvastatin (16.7 µg/mL) was suspended in 67 mM phosphate buffer (pH 7.4) to take into account for spontaneous hydrolysis with zero protein. ** (p<0.05)

56

3.5.2 Hydrolysis of simvastatin in the presence of paraoxonase

It has been demonstrated that incubating simvastatin with purified paraoxonase 1 resulted in

the hydrolysis of the lactone form (Billecke et al., 2000). Thus, based on the method

established by these authors, simvastatin (1 mg/mL) in acetonitrile was incubated with two

genetic variants of PON1, phenotype QQ and RR, in 67 mM phosphate buffer with 1 mM

CaCl2, to assess and confirm the catalytic activity of paraoxonase.

Figure 22 shows that there were no significant changes in simvastatin levels between control

conditions (1739.2 ± 350.4 PA) and in the presence of either PON-QQ (1655.5 ± 214.4 PA)

and PON-RR (1775.6 ± 206.6 PA). Formation of simvastatin acid was slightly lower in the

control conditions, with a peak area of 81.4 ± 22.3, in comparison to PON-RR, which

resulted in a peak area of 76.3 ± 16.3. The phenotype QQ however had slightly greater

formation of simvastatin acid (113.1 ± 20.9 PA) in comparison to controls. Nonetheless

hydrolysis of simvastatin and formation of simvastatin β-hydroxy acid was not significantly

different (p>0.05) in the presence of either phenotypes of paraoxonase.

Interestingly, in the presence of PON-RR, an unknown peak X with retention time of 2.1

minutes was detected, with a peak area of 69.7 ± 2.3 (Figure 23).

57

c o n tro l P O N 1(Q Q )

P O N 1(R R )

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0S

imv

as

tati

n r

em

ain

ing

(pe

ak

are

a)

A

c o n tro l P O N 1(Q Q )

P O N 1(R R )

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

Sim

va

sta

tin

ac

id f

orm

ati

on

(pe

ak

are

a)

B

Figure 22. Loss of simvastatin (A) and formation of simvastatin β-hydroxy acid (B) in the presence of PON1-QQ and PON1-RR

Simvastatin (16.7 µg/mL) was incubated with PON-QQ (4 U/mL) and PON-RR (4 U/mL) in 67 mM phosphate buffer (pH 7.4) with 1 mM CaCl2 at 37°C for 30 minutes (n=4, except for PON (RR) where n=2). For non-enzymatic controls, simvastatin (16.7 µg/mL) was suspended in 67 mM phosphate buffer (pH 7.4) with 1 mM CaCl2 to take into account for spontaneous hydrolysis with zero protein.

58

0 5 1 0 1 5 2 0

0

5 0

1 0 0

R e te n t io n tim e (m in )

Absorbance

(mA

U 2

38

nm

)

S M V

S M V A

A

0 5 1 0 1 5 2 0

0

5 0

1 0 0

R e te n t io n tim e (m in )

Absorbance

(mA

U 2

38

nm

)

S M V

S M V AX

B

Figure 23. Representative chromatograms showing A) spontaneous hydrolysis of simvastatin and B) the hydrolysis of simvastatin in the presence of paraoxonase 1 (RR).

Simvastatin (16.7 mg/mL) was incubated with PON1-RR (4 U/mL) at 37°C for 30 minutes in the presence of 1 mM CaCl2 (n=2). Under zero-enzyme conditions, the sample generated two peaks consistent with simvastatin β-hydroxy acid at Rt = 3.9 minutes and simvastatin lactone at Rt = 11.4 minutes. In the presence of the PON-RR, three peaks were observed, with the unknown peak X detected at Rt = 2.1 minutes.

59

The lack of hydrolysis of simvastatin in the presence of PON1 as well as the detection of this

unknown peak X may suggest that simvastatin β-hydroxy acid is acting as the substrate for

enzymatic hydrolysis. Thus a follow-on experiment was carried out to investigate whether

incubations with the phenotype QQ using simvastatin acid as the substrate could also

generate the same unknown peak X.

C o n tr o l P O N (Q Q )

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

Sim

va

sta

tin

ac

id r

em

ain

ing

(Pe

ak

are

a)

Figure 24. Loss of simvastatin acid in the presence of PON1-QQ.

Simvastatin acid (16.7 mg/mL) was incubated with PON1-QQ (4 U/mL) in 67 mM phosphate buffer (pH 7.4) with 1 mM CaCl2 at 37°C for 30 minutes (n=4). For non-enzymatic controls, simvastatin acid (16.7 µg/mL) was suspended in 67 mM phosphate buffer (pH 7.4) with 1 mM CaCl2 to take into account for spontaneous hydrolysis with zero protein.

It is evident that when simvastatin acid (16.7 µg/mL) was used as the substrate (Figure 24),

PON-QQ was unable to hydrolyse it to the unknown compound X, as no observable loss of

SMVA was detected compared to control. In fact, with simvastatin β-hydroxy acid as the

60

substrate, the unknown compound X was not detected in these conditions. Interestingly, the

active metabolite also did not undergo relactonization back to the parent compound.

Given the above results, it can be inferred that simvastatin is most likely not a substrate for

human liver esterase enzymes such as carboxylesterase and paraoxonase.

3.5.3 Hydrolysis of simvastatin in the presence of butyrylcholinesterase

Several of the esterases previously mentioned are known to be synthesised and secreted from

the liver and is actively expressed in human plasma. Although there was a lack of hydrolysis

of simvastatin observed in human plasma in the absence and presence of Ca2+ from previous

experiments (Section 3.4.1), this suggests that PON may have a minor role in this

bioactivation. However, another esterase expressed in human plasma is butyrylcholinesterase

(BChE) and it has been shown that simvastatin is a concentration-dependent inhibitor of

BChE (Darvesh et al., 2004). Thus to investigate whether SMV may be a possible substrate

for BChE, the following experiment was undertaken.

Figure 25 shows that butyrylcholinesterase did not have a concentration-dependent effect on

the hydrolysis of simvastatin in comparison to the control conditions. Although it appears that

simvastatin remaining has decreased as the concentration of BChE increased from 0.02 U/mL

to 0.2 U/mL, simvastatin remaining at 1 U/mL of BChE (1.155 ± 0.102 PAR) was not

significantly different relative to its control (1.090 ± 0.057 PAR).

61

C o n tro l 0 .0 2 0 .20 .0

0 .5

1 .0

1 .5

B C h E e n z y m e u n its /m l

Sim

va

sta

tin

re

ma

inin

g(S

MV

/ I

S p

ea

k a

rea

ra

tio)

A

C o n tro l 10 .0

0 .5

1 .0

1 .5

B C h E e n z y m e u n its /m l

Sim

va

sta

tin

re

ma

inin

g(S

MV

/ I

S p

ea

k a

rea

ra

tio)

C

C o n tro l 0 .0 2 0 .20 .0

0 .5

1 .0

1 .5

B C h E e n z y m e u n its /m l

Sim

va

sta

tin

ac

id f

orm

ati

on

(SM

VA

/ I

S p

ea

k a

rea

ra

tio)

**** ****

B

C o n tro l 10 .0

0 .5

1 .0

1 .5

B C h E e n z y m e u n its /m l

Sim

va

sta

tin

ac

id f

orm

ati

on

(SM

VA

/ I

S p

ea

k a

rea

ra

tio)D

*

Figure 25. Loss of simvastatin (A and C) and formation of simvastatin β-hydroxy acid (B and D) in the presence of increasing concentrations of BChE.

Simvastatin (20 µg/mL) was incubated with increasing concentrations of butyrylcholinesterase (0.02, 0.2, 1 U/mL) at 37°C for 30 minutes (n=4). For non-enzymatic controls, simvastatin was suspended in aqueous buffer to take into account for spontaneous hydrolysis with zero protein. **** (p<0.0001), * (p<0.05)

3.5.4 Hydrolysis of simvastatin in the presence of human serum albumin

Due to the results thus far which have consistently shown a lack of hydrolysis of simvastatin

in human liver and plasma as well as several purified esterase enzymes, these findings give

an indication that the bioactivation of simvastatin is unlikely to occur in the liver nor plasma

in humans. However, since human serum albumin is the most abundant protein in human

plasma and has been reported to display esterase-like activity (Section 1.5.5), a final

experiment was carried out to determine whether HSA has a possible role in converting

simvastatin to simvastatin β-hydroxy acid.

62

Incubations of simvastatin (20 µg/mL) and increasing concentrations (5, 10, 20, 30 and 40

mg/mL) of human serum albumin at 37°C for 30 minutes were undertaken (Figure 26).

Figure 26B shows that the formation of simvastatin acid appears to have decreased in a

protein concentration dependent manner, with a peak area ratio (PAR) of 0.151 ± 0.034 at 5

mg/mL of HSA and 0.101 ± 0.019 at 40 mg/mL of HSA, however the differences between

the means of each concentration is not significant (p = 0.28).

The peak area ratios of simvastatin after incubations of increasing concentrations of human

serum albumin are generally higher or comparable to the levels observed under control

conditions (Figure 26A). This suggests that simvastatin was not hydrolysed by HSA thus no

loss of simvastatin is detected. However, the peak area ratios of simvastatin acid observed in

the presence of HSA were considerably much lower than the control conditions (Figure 26B)

(p<0.0001). This may suggest that the β-hydroxy acid is sequestered or bound to the protein

and not efficiently extracted therefore levels of simvastatin acid formation is much higher in

the absence of protein (zero-enzyme conditions).

63

c o n tro l 5 1 0 2 0 3 0 4 00 .0

0 .5

1 .0

1 .5

2 .0S

imv

as

tati

n r

em

ain

ing

(S

MV

/ I

S p

ea

k a

rea

ra

tio

)

A

H S A p r o t e in c o n c e n t r a t io n(m g /m l)

c o n tro l 5 1 0 2 0 3 0 4 00 .0

0 .5

1 .0

1 .5

2 .0

Sim

va

sta

tin

ac

id f

orm

ati

on

(SM

VA

/ I

S p

ea

k a

rea

ra

tio

)

B

H S A p r o t e in c o n c e n t r a t io n(m g /m l)

**** **** ******** ****

Figure 26. Loss of simvastatin (A) and formation of simvastatin β-hydroxy acid (B) in the presence of increasing concentrations of human serum albumin (HSA).

Simvastatin (20 µg/mL) was incubated with increasing protein concentrations (5.0, 10, 20, 30 and 40 mg/mL) of human serum albumin (HSA) at 37°C for 30 minutes (n=4). For controls, simvastatin was suspended in 67 mM phosphate buffer (pH 7.4) to take into account for spontaneous hydrolysis with zero protein. **** (p<0.0001)

64

3.6 Unknown peaks

Treatment of simvastatin in numerous subcellular fractions as well as with purified esterase

enzymes have generated some unknown compounds.

When determining the role of microsomal esterase enzymes on the hydrolysis of simvastatin,

formation of an unknown peak B with retention time of 18.7 minutes was detected, in both

enzymatic and non-enzymatic conditions (Figure 27).

0 5 1 0 1 5 2 0 2 5

0

2 0 0

4 0 0

1 0 0 0

2 0 0 0

R e te n tio n tim e (m in )

Ab

so

rba

nc

e(m

AU

23

8 n

m)

S M V A

S M V

B

IS

Figure 27. Representative chromatogram of the reaction mixture containing denatured pooled human liver microsomes and simvastatin

Simvastatin (20 µg/mL) was incubated with denatured human liver microsomes (0.5 mg/mL) at 37°C for 30 minutes (n=4). The reaction mixture generated four peaks at retention times of 4.62 minutes for simvastatin acid, 11.76 minutes for simvastatin, 18.67 minutes for the unknown product B and 19.21 minutes for the internal standard ivermectin.

Figure 28 shows the normalized UV spectra of the unknown peak B, in comparison to the UV

spectra of simvastatin. Simvastatin β-hydroxy acid formed using method I resulted in an

identical UV spectra as the parent compound (Figure 8). Contrastingly, this unknown product

B has a λmax = 234 nm while simvastatin has a λmax = 238 nm.

65

200 250 300 350 4000

50

100

W a v e le n g th (n m )

Absorbance

(mA

U 2

38

nm

)S im va s ta tin

U n kno w n

Figure 28. Comparison of the normalised UV spectra of the unknown product at retention time of 18.7 minutes (black dash) with simvastatin (red) after incubations with human liver microsomes.

N o n -e n z ym a tic M ic r o s o m e s M ic r o s o m e s + C a C l2

0 .0 0

0 .0 5

0 .1 0

0 .1 5

Un

kn

ow

n B

fo

rma

tio

n(B

/ I

S p

ea

k a

rea

ra

tio

)

Figure 29. Formation of unknown product B in pooled human liver microsomes (2 mg/mL) in the absence and presence of calcium chloride.

Simvastatin (20 µg/mL) was incubated with human liver microsomes (2 mg/mL) at 37°C for 30 minutes, in the absence and presence of 2 mM CaCl2 (n=4). For controls, human liver microsomes were denatured by applying heat for 5 minutes at 80°C.

66

0 .5 1 .0 2 .0 5 .00 .0

0 .5

1 .0

1 .5

P ro te in C o n c e n tra t io n (m g /m l)

Un

kn

ow

n/I

S P

AR

A E n zy m a tic

0 .5 1 .0 2 .0 5 .00 .0

0 .5

1 .0

1 .5

P ro te in C o n c e n tra t io n (m g /m l)

Un

kn

ow

n/I

S P

AR

B N o n - e n z ym a tic

Figure 30. Formation of the unknown product B in incubations of increasing concentrations of human liver microsomes with simvastatin.

Simvastatin (20 µg/mL) was incubated with increasing protein concentrations (0.5, 1.0, 2.0 and 5.0 mg/mL) of A) human liver microsomes and B) denatured human liver microsomes at 37°C for 30 minutes (n=4). For non-enzymatic conditions, human liver microsomes were denatured by applying heat at 80°C for 5 minutes.

67

Incubations of simvastatin with pooled human liver microsomes (2 mg/mL) resulted in the

slight formation of unknown product B (Rt = 18.7 minutes) above denatured (non-enzymatic)

control, with peak area ratio (PAR) of 0.058 ± 0.002. Fortification with CaCl2 did not

substantially increase this formation (Figure 29).

This unknown product B was again detected when simvastatin was incubated with increasing

concentrations of pooled human liver microsomes. Although this unknown peak was hardly

detectable at 0.5 mg/mL of microsomal protein (Figure 30A), it appears that formation of this

unknown product was concentration dependent. Furthermore, the same unknown compound

was also observed in non-enzymatic incubations with denatured human liver microsomes

(Figure 30B), again in a concentration-dependent manner. Generation of unknown product B

was significantly greater at 5 mg/mL compared to 0.5 mg/mL (p<0.05) in non-enzymatic

conditions. Furthermore, this formation was substantially greater in these non-enzymatic

conditions (PAR at 5 mg/mL = 0.992 ± 0.332) than in the presence of functional microsomes

(PAR at 5 mg/mL = 0.162) which suggests that perhaps the catalytic activity of the enzymes

in the denatured conditions were not completely abolished when heat was applied but

furthermore, their catalytic activity increased after heat was applied to the microsomes.

The production of the unknown product B was not detected in cytosol, plasma or red blood

cells, or when simvastatin was incubated with purified esterase enzymes CES, PON, BChE

and HSA.

68

4 Discussion

Previous reports in the literature have stated that ester hydrolases can bioactivate simvastatin,

however surprisingly, there is little direct evidence to support this statement. The aim of this

study were to establish an HPLC assay that was able to detect the loss of simvastatin and

formation of simvastatin β-hydroxy acid, to determine the bioactivation of simvastatin in

human liver and plasma and to assess the ability of purified ester hydrolase enzymes to

bioactivate this statin. The results, limitations and future prospects of this study will be

discussed below.

4.1 The lack of simvastatin bioactivation in human liver and plasma

In order to examine the bioactivation of simvastatin in vitro, confirmation was needed to

ascertain whether the HPLC established based on a literature method (Carlucci et al., 1992)

could detect simvastatin loss and in turn, the formation of simvastatin β-hydroxy acid. Thus

rat plasma was used as a positive control, as it is known to have the capacity to hydrolyse the

lactone prodrug to the active metabolite (Vickers et al., 1990). Indeed, it was evident from the

data generated that simvastatin was efficiently hydrolysed in rat plasma and this hydrolysis

resulted in the formation of the active metabolite. Not only do these results support the

existing literature that the hydrolysis of the lactone prodrug occurs in rat plasma, this also

provided substantial verification that this literature-based HPLC assay was able to detect both

the loss of the lactone prodrug and formation of active metabolite.

This bioactivation of simvastatin which was observed in rat plasma may be attributed to

carboxylesterases. The major isoform in rat plasma is Ces1 (Bahar et al., 2012; Berry et al.,

2009), thus this hydrolysis of SMV to SMVA is consistent with the data in the literature

which states simvastatin is a substrate for rat plasma Ces (Vickers et al., 1990). The human

69

isoform CES1, is an ester hydrolase that has been shown to bioactivate several prodrugs.

(Tang et al., 2006; Wang et al., 2015; Williams et al., 2008) However, hydrolysis of

simvastatin did not occur in human plasma at the same concentration which may be because

human plasma does not express CES (Li et al., 2005). Metabolic disposition studies of

simvastatin in several species have also established similar conclusions, presenting data

demonstrating the hydrolysis of simvastatin in rat plasma, and lack of hydrolysis of the

lactone prodrug in both dog and human plasma (Vickers et al., 1990; Vickers et al., 1990). It

is possible that the absence of CES expression in human plasma results in inefficient

hydrolysis of the lactone prodrug in this biological matrix.

Nonetheless, other ester hydrolases such as paraoxonase (PON), which are known to

hydrolyze lactones are active in human plasma (Billecke et al., 2000; Fukami & Yokoi, 2012;

Li et al., 2005). PON enzymes are calcium-dependent, and have a maximum catalytic activity

at a calcium concentration of 20 µM (Fukami & Yokoi, 2012; Kuo & La Du, 1998). In

plasma, the serum concentration of calcium is approximately 1 mM, (Kuo & La Du, 1998)

however during preparation of plasma from whole blood, depletion of calcium can occur.

Therefore, human plasma was fortified with 2 mM CaCl2 which did not result in any

increased hydrolytic loss of simvastatin compared with denatured controls. This is supported

by data from the literature (Hioki et al., 2010) which also observed that the addition of CaCl2

did not affect the the hydrolysis of SMV in human plasma. Red blood cells were also unable

to hydrolyse simvastatin which suggests that the bioactivation of SMV is unlikely to occur in

the systemic circulation in humans. This initial data also suggests that PON may not be the

primary enzyme which catalyses the hydrolysis of simvastatin.

Simvastatin is administered as an oral prodrug, and numerous reports have stated that the

hydrolysis of this lactone prodrug occurs in the liver (Pasha et al., 2006; Sirtori, 1990).

70

Although evidence is lacking, the justification for this hypothesis is that several esterase

enzymes, such as CES and PON, are expressed in the liver (Fukami & Yokoi, 2012; Imai,

2006; Jbilo et al., 1994; Ng et al., 2001). Although the hepatic endoplasmic reticulum has a

calcium concentration of approximately 0.1 to 1 µM (Kuo & La Du, 1998) which is lower

than in plasma, hepatic PON is still expected to be active.

Examination of bioactivation of simvastatin in human liver microsomes was subsequently

carried out. Once again, fortification of calcium was required in order to see if paraoxonase in

the liver has a role in the hydrolysis of the lactone prodrug. At a protein concentration of 2

mg/mL, human liver microsomes did not result in any noticeable loss of simvastatin and no

significant increase in the formation of simvastatin β-hydroxy acid compared with denatured

controls. More importantly, the addition of calcium (2 mM) to microsomes did not have any

significant effect in either loss of simvastatin or formation of simvastatin β-hydroxy acid.

Results from this experiment and the plasma data thus far suggests that paraoxonase is not the

major enzyme responsible for the bioactivation of simvastatin. This does not support the

findings from the literature (Hioki et al., 2015) which reported that human liver microsomes

were able to hydrolyse simvastatin efficiently and fortification of calcium increased this

hydrolysis.

As no detectable loss of prodrug or the formation of the active metabolite was observed, it

was considered that perhaps the protein concentration used in this particular experiment was

too low to distinguish any minor changes in simvastatin or simvastatin β-hydroxy acid levels.

Thus simvastatin was incubated with increasing protein concentrations of human liver

microsomes. However, there was still no appreciable loss of simvastatin suggesting that this

hydrolysis reaction is unlikely to be catalysed by human liver microsomes under these

incubation conditions. Furthermore, similar levels of simvastatin remaining were observed

71

under non-enzymatic (denatured enzyme) conditions, which was not expected, considering

denatured microsomes were assumed to have no enzymatic activity. This was also observed

in both enzymatic and non-enzymatic conditions in human plasma. It may be that heat-

denaturation does not completely abolish or eliminate the catalytic activity of the esterases

involved in this hydrolysis reaction. Data from this experiment revealed that the hydrolysis of

simvastatin in human liver microsomes was not concentration dependent and the same

observations could be made with regards to the formation of simvastatin β-hydroxy acid.

Additionally, the inability of pooled human liver microsomes to hydrolyse SMV also implies

that this bioactivation step is not catalysed by microsomal esterase enzymes.

Since the specific esteras(s) involved in the hydrolysis of simvastatin are not known, the

subcellular location of these enzymes may not be the endoplasmic reticulum/plasma

membrane (microsomal) fraction. Therefore, the subsequent experiment examined the

hydrolysis of simvastatin in pooled human liver cytosol, with and without fortification of

calcium. Similar to previous experiments, there was no detectable loss of simvastatin

observed in comparison to non-enzymatic (denatured) controls. Along with the results

obtained from incubations with human liver microsomes, it can be surmised from these

findings that the bioactivation of simvastatin does not occur in the liver and that perhaps

simvastatin is not a substrate for human liver esterase enzymes such as carboxylesterase or

paraoxonase.

72

4.2 The lack of simvastatin bioactivation in the presence of purified ester hydrolase enzymes

The lack of bioactivation of simvastatin in the pooled human liver microsomes and cytosol

suggests that human liver esterases are not responsible for catalysing the hydrolysis of the

parent compound simvastatin to the active metabolite simvastatin β-hydroxy acid. In

addition, the lack of bioactivation of simvastatin in human plasma even in the presence of

calcium further suggests that plasma paraoxonase may also have little or no role in this

bioactivation step. To confirm these results, simvastatin was incubated with purified CES and

PON, as well as other ester hydrolases that may potentially catalyse this hydrolysis reaction.

Purified CES1, which is the major enzyme speculated to be responsible for the hydrolysis of

SMV (Casey et al., 2013; J. Kim et al., 2011; Pasanen et al., 2006; Vree et al., 2001; Wilke et

al., 2012) did not result in any significant loss of simvastatin or any formation of SMVA

compared to control. These results support the limited data from the literature which

demonstrated that s9 fractions of CES1 overexpressed in human liver cells did not hydrolyse

SMV (Wang et al., 2015). Therefore, although numerous published articles in the literature

state that the bioactivation of the lactone prodrug is catalysed by carboxylesterases, it seems

that in humans, simvastatin is not a substrate for CES1.

Although bioactivation of simvastatin did not occur in the subcellular fractions of liver as

well as in human plasma, it is not known if this bioactivation occurs in the intestines.

Therefore, the major isoform of carboxylesterase, CES2, which is expressed in human

intestines (Imai, 2006) was also incubated with simvastatin. However, CES2 also did not

hydrolyse simvastatin relative to control in the incubation conditions used. Interestingly,

CES2 did lead to a significant formation of simvastatin β-hydroxy acid above the control.

Although this is statistically significant, this formation of the active metabolite was not

comparable to that which was observed when SMV was incubated in rat plasma thus offering

73

no biological significance. Furthermore, the conversion of SMV to SMVA clinically appears

to be approximately 20-40% of the prodrug concentration (Backman et al., 2000; Winsemius

et al., 2014), which is much greater than the formation seen following the incubation with

purified CES2. Therefore it also appears that CES2 is not the enzyme responsible for the

bioactivation of simvastatin.

The lack of bioactivation of simvastatin in human plasma, liver microsomes and cytosol, as

well as lack of activity of both isoforms of CES was unexpected, particularly as the

hydrolysis of another statin, lovastatin has been reported to occur in microsomes as well as

cytosol at a substrate concentration of 2.2 µg/mL (equivalent to 5.4 µM). This led to the

query that perhaps the substrate concentration being tested (48 µM) was saturating the

enzyme(s) therefore causing auto-inhibition of their catalytic activity. The experiments thus

far have investigated the hydrolysis of simvastatin at a substrate concentration of 16.7 - 20

µg/mL (approximately 40 – 48 µM). This prompted a subsequent experiment to investigate

the hydrolysis of simvastatin with CES1 and CES2 at a 10-fold lower simvastatin

concentration (1.67 µg/mL, equivalent to 4 µM). However, even at this lower substrate

concentration, there appeared to be no significant hydrolysis of SMV in the presence of either

isoforms and unlike the previous experiment, there was no significant formation of SMVA in

the presence of CES2.

It has been reported that CES enzymes display differences in substrate specificity in humans

and experimental animals (Fukami & Yokoi, 2012). Although this is not well characterized,

rat Ces enzymes are able to effectively hydrolyse the leukotriene receptor antagonist

pranlukast in the liver, while human CES enzymes cannot (Luan et al., 1997). Thus the

difference between the hydrolysis of simvastatin in human and rat plasma may be explained

by substrate specificity of carboxylesterase in various species.

74

Purified paraoxonase has been shown to have catalytic activity for the lactone forms of

several statins, including simvastatin (Billecke et al., 2000). Although the hydrolysis of

simvastatin was not increased when human plasma, microsomes and cytosol were fortified

with calcium, based on this report, two genetic variants of purified paraoxonase 1, PON1-QQ

and PON1-RR were incubated with simvastatin to rule out the possible role of PON1 in this

bioactivation step. Incubation conditions were also adapted to replicate the method used by

these authors (Billecke et al., 2000). However, consistent with the results from previous

experiments, there was no significant loss of simvastatin compared to control in the presence

of either variant of PON1. More importantly, this is in direct contrast to the data reported in

the literature (Billecke et al., 2000). This study prepared and purified PON1 from human

plasma and reported rates of 684.5 ± 34.5 and 568.3 ± 11.7 pmol/min/mg for type QQ and

RR, respectively. These rates appear to be very low specific activities for a purified enzyme.

However, another study (Hioki et al., 2010) which looked at recombinant human PON1,

PON2 and PON3, established that only PON3 exhibited simvastatin hydrolase activity, which

may explain the lack of simvastatin hydrolysis when incubated with PON1. Human PON3 is

thought to be expressed in human liver (Fukami & Yokoi, 2012; Ng et al., 2001) which does

not justify the ineffective hydrolysis observed in human liver microsomes in this dissertation.

An alternative explanation for this lack of hydrolysis however, could be the possible

autoinhibition (saturation) of the enzyme at high substrate concentration.

Butyrylcholinesterase is a major esterase that is present in human plasma (Li et al., 2005),

which is also produced by the liver. Simvastatin has been reported to exhibit concentration-

dependent inhibition of BChE, at a range of 30 µM – 160 µM substrate concentration

(Darvesh et al., 2004). Incubations of simvastatin with BChE generated no significant

75

difference in the loss of simvastatin compared to controls, which suggests that SMV is not a

substrate of BChE. In contrast, simvastatin acid formation appeared to be lower in the

presence of BChE, in comparison to non-enzymatic hydrolysis. This may suggest that SMVA

is tightly sequestered, possibly by covalent binding to BChE protein. Another posibility could

be that simvastatin β-hydroxy acid is a substrate and is further metabolised by BChE to

products which are not detectable by UV-HPLC.

Since human serum albumin is the most abundant protein in human plasma, with plasma

concentrations of approximately 0.6 mM in humans (Yang et al., 2007), the potential role of

human serum albumin in the bioactivation of SMV was also investigated. Studies have also

shown albumin has “esterase-like” activity that is heat stable. For example bovine serum

albumin (BSA) can catalyse the hydrolysis of esters even after pre-treatment at temperatures

of up to 160°C (Córdova et al., 2008). Since the preliminary data using microsomal fractions

of human liver indicated that the denatured microsomes behaved in a similar manner to the

non-heat treated material, the esterase activity of albumin in these preparations could play a

role in the hydrolysis observed in non-functional microsomes.

Incubations of simvastatin with increasing concentrations of HSA demonstrated no

significant concentration-dependent effect on the hydrolytic loss of simvastatin. However the

levels of simvastatin β-hydroxy acid was lower than observed in the zero protein controls.

Again, this suggests that this metabolite is sequestered or covalently bound to the protein in a

similar way as observed in the BChE experiments. Nonetheless, the minor changes in the

levels of simvastatin observed even at the highest concentration of HSA tested implies that

the hydrolysis of simvastatin is not efficiently catalysed by human serum albumin. This is in

contrast to the report that has shown serum albumin could hydrolyse simvastatin in both

human and rat (Hioki et al., 2010).

76

4.3 Unknown products

During some of the incubation experiments, the production of two unknown compounds were

observed. An unknown peak in pooled human liver microsomes (unknown product B) and the

one following incubation of SMV with PON1-RR (unknown product X).

The presence of the unknown product B was observed in both functional microsomes and in

heat-denatured controls. Since this unknown compound was observed even in heat-denatured

(non-enzymatic) conditions, this again suggests that the “esterase activity” may be heat

tolerant, similar to the heat stability reported for BSA (Córdova et al., 2008). Furthermore,

BSA has also exhibited an increased catalytic activity towards certain substrates at

temperatures between 70-150°C (Córdova et al., 2008). Therefore if human serum albumin

displays similar catalytic profile, applying heat to denature microsomes may have increased

the catalytic activity of HSA, resulting in much greater formation of the unknown compound

B under these “non-enzymatic” conditions. However, the formation of product B was not

detected when SMV was incubated with increasing concentrations of purified HSA, nor was

it seen in human liver cytosol or plasma. This unknown product was unlikely to be due to

NADPH-microsomal metabolism of SMV to one of its oxidative metabolites as this cofactor

was not included in the incubations. However, small contamination with the cofactor cannot

be discounted and further investigation of the addition of NADPH to examine whether the

productino of this unknown compound is enhanced in the liver is required.

Another unknown product, X, was observed when SMV was incubated with PON1-RR.

Since there was no indication of a significant loss of the parent compound, it was postulated

that perhaps the small amount of non-enzymatic SMVA in the incubation was acting as the

substrate that was further metabolized to this unknown compound. However, direct

incubations of SMVA with PON1-QQ did not generate this unknown product, which

suggests that perhaps the formation of this product is specific to one genetic variant. Future

77

experiments incubating SMVA with PON1-RR should be undertaken to confirm this. It is

interesting to note however, that there was no detection of the lactone prodrug which

indicates that when SMVA is placed in aqueous buffer, relactonization back to the parent

compound did not ccur, as suggested in the literature (Iwuchukwu et al., 2014; Tsamandouras

et al., 2014).

The presence of these unknown products gives an indication that the metabolic disposition of

SMV and SMVA may be complex, as both compounds could act as substrates for different

enzymes. Any variation in the reactions which lead to changes in the levels of either SMV or

SMVA, could be a potential factor not only the therapeutic actions of this statin (if the active

metabolite is metabolized further to compounds that are less active) but also the toxicity

associated with elevated levels of SMVA.

4.4 Limitations

Several limitations in this project need to be addressed. Firstly, the initial aim of this project

was to establish an HPLC assay which could detect both simvastatin and simvastatin β-

hydroxy acid. This HPLC assay was based on literature (Carlucci et al., 1992) and although it

was confirmed with rat plasma that this method could detect both loss of SMV and formation

of SMVA, it would be ideal if further assessment of this assay was carried out. However, due

to time constraints, only minor adjustments were performed (such as switching the mobile

phase from 25 mM sodium dihydrogen phosphate buffer (pH 4.5) to 50 mM ammonium

formate (pH 4.5) buffer). For instance, direct assessment of the inter- and intra- day

variability were not undertaken and this should be addressed in future work.

78

Since simvastatin β-hydroxy acid was not commercially available, attempts were made at

synthesising SMVA from SMV, again using a published method (Bhatia et al., 2011).

However, this method proved to be unsatisfactory as it resulted in a mixture of SMV, SMVA

and an unknown product A. Therefore, since SMVA was not available as a purified solid

form, quantification of the amount of SMVA formed was not possible. An alternative method

was used which involved dissolving SMV in 10% NaOH solution to produce an SMVA

solution. Nonetheless, although the chromatogram detected only a single peak at a retention

time consistent with SMVA, it can only be assumed that this method results in 100%

conversion from the lactone prodrug to the active metabolite. Therefore, this SMVA standard

was used only as a chromatographic reference standard to determine the relative retention

time of SMVA. Future work should quantify the molar loss of SMV and obtain simvastatin

acid so as to quantify the molar formation of the active metabolite.

Another limitation in this project was that only a single substrate concentration was tested. A

number of reports have indicated that microsomes are capable of hydrolysing statins to their

corresponding hydroxy acids (Riedmaier et al., 2011; Vickers et al., 1990) as well as plasma

(Hioki et al., 2010). These reports use 5-10 µg/ml (equivalent to 10–24 µM) substrate

concentrations. Although a lower substrate concentration was used in the incubations with

CES1 and CES2 (1.67 µg/mL, equivalent to 4 µM), which still did not result in any

significant hydrolysis compared to control, this could simply mean that simvastatin is a poor

substrate for human carboxylesterases. On the other hand, the lack of bioactivation of SMV

in human plasma, liver microsomes or cytosol or even PON1 which has been shown to have

the capacity to hydrolyse SMV, could instead be due to autoinhibition of the enzyme at such

high substrate concentrations rather than SMV not acting as a substrate. Thus it would be

interesting to see if the hydrolysis of the lactone prodrug is detectable in these incubation

experiments when a wide range of substrate concentrations are tested.

79

The inability of human plasma as well as several purified ester hydrolase enzymes to

effectively hydrolyse simvastatin compared to control was also quite unexpected. With

regards to human plasma, the hydrolysis of simvastatin was only tested in a single donor.

This individual could possibly have inefficient PON or BChE activities, so confirming this

lack of hydrolysis in other plasma donors is necessary to fully verify that SMV is not

hydrolysed in human plasma.

In addition, there are other isoforms of purified esterase enzymes which have not been

assessed. For instance there is evidence that PON3 displays catalytic activity for simvastatin

(Hioki et al., 2010). This isoform was not examined in this project, therefore it cannot be

completely ruled out that paraoxonase is not capable of bioactivating SMV. Furthermore,

although several of the purified esterase enzymes used in these experiments have been

recently shown in the research lab to have catalytic activity for the prototype substrates p-

nitrophenyl acetate and butyrylthiocholine (Zhang et al., 2015), the use of these positive

controls should have been directly integrated into the experiments in this project, to ensure

that these esterase enzymes still retained their catalytic activity during the incubation.

Finally, several reports from the literature (Riedmaier et al., 2011; Vickers et al., 1990;

Vickers et al., 1990) include the addition of acid during the extraction procedure prior to

HPLC analysis of the incubations. This is concerning as noted in the preliminary results for

this project, the addition of acidified acetonitrile resulted in conversion of any SMVA formed

in the incubation back to the parent compound. Thus, it is not clear how these literature

reports could detect formation of SMVA after addition of acid.

80

4.5 Future work

This dissertation has demonstrated that simvastatin was not hydrolysed in human liver

microsomes, cytosol and human plasma nor in the several purified ester hydrolase enzymes,

at the substrate concentration tested. Therefore further studies should be conducted to

examine this hydrolysis at lower substrate concentrations. Furthermore, although CES2 did

not have any catalytic activity for simvastatin hydrolysis, further examination of whether the

bioactivation of the lactone prodrug occurs in human intestinal microsomes or cytosol should

also be undertaken.

Another aspect which needs to explored further is the possibility that simvastatin β-hydroxy

acid is a substrate for these esterase enzymes. It would be interesting to see if further

metabolism of the active metabolite is observed if SMVA was used as a substrate in human

liver microsomes, cytosol and plasma, as well as BChE and HSA. In addition, further

characterization of the unknown peaks detected during incubations with microsomes and

PON1-RR using mass spectrometry could give an indication which product, SMV or SMVA,

is undergoing further metabolism to these unknown products.

4.6 Summary

To conclude, results obtained from this project indicate that at the substrate concentration

tested, simvastatin bioactivation does not occur in human liver microsomes, cytosol and

human plasma. Furthermore, carboxylesterase which is the major enzyme speculated to be

responsible for this bioactivation did not display catalytic activity for simvastatin. This

indicates that simvastatin was not a substrate for human CES1 and CES2, which does not

support the common assumption that human CES enzymes catalyse this hydrolysis reaction.

The lack of hydrolysis observed in vitro even after fortification with calcium also suggests

81

that paraoxonase has no role in this bioactivation step. This was confirmed when SMV was

incubated with purified PON1, which is again not consistent with the evidence that has been

shown in the literature. Other plasma ester hydrolases such as BChE and HSA also did not

hydrolyse the lactone prodrug at the substrate concentration tested but may sequester SMVA.

Therefore it is still not clear where this bioactivation step occurs and which esterase enzymes

are responsible for the hydrolysis of this lactone prodrug. The possible effect of auto-

inhibition by the metabolite (SMVA) on the hyrolysis of the prodrug should be investigated

in future experiments.

82

References

Ahmed, T. A., Hayslip, J., & Leggas, M. (2013). Pharmacokinetics of high-dose simvastatin in refractory and relapsed chronic lymphocytic leukemia patients. Cancer Chemotherapy and Pharmacology, 72(6), 1369-1374.

Arnadottir, M., Eriksson, L., Thysell, H., & Karkas, J. (1993). Plasma concentration profiles of simvastatin 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitory activity in kidney transplant recipients with and without ciclosporin. Nephron, 65(3), 410-413.

Aviram, M., Hardak, E., Vaya, J., Mahmood, S., Milo, S., Hoffman, A., . . . Rosenblat, M. (2000). Human serum paraoxonases (PON1) Q and R selectively decrease lipid peroxides in human coronary and carotid atherosclerotic lesions: PON1 esterase and peroxidase-like activities. Circulation, 101(21), 2510-2517.

Backman, J. T., Kyrklund, C., Kivistö, K. T., Wang, J., & Neuvonen, P. J. (2000). Plasma concentrations of active simvastatin acid are increased by gemfibrozil. Clinical Pharmacology & Therapeutics, 68(2), 122-129.

Badhan, R., Penny, J., Galetin, A., & Houston, J. B. (2009). Methodology for development of a physiological model incorporating CYP3A and P‐glycoprotein for the prediction of

intestinal drug absorption. Journal of Pharmaceutical Sciences, 98(6), 2180-2197.

Bahar, F. G., & Imai, T. (2013). Aspirin hydrolysis in human and experimental animal plasma and the effect of metal cations on hydrolase activities. Drug Metabolism and Disposition: The Biological Fate of Chemicals, 41(7), 1450-1456.

Bailey, D. G. (2010). Fruit juice inhibition of uptake transport: a new type of food–drug interaction. British Journal of Clinical Pharmacology, 70(5), 645-655.

Balogh, Z., Fülöp, P., Seres, I., Harangi, M., Katona, E., Kovacs, P., . . . Paragh, G. (2001). Effects of simvastatin on serum paraoxonase activity. Clinical Drug Investigation, 21(7), 505-510.

Becker, M. L., Visser, L. E., van Schaik, R. H., Hofman, A., Uitterlinden, A. G., & Stricker, B. H. (2009). Common genetic variation in the ABCB1 gene is associated with the cholesterol-lowering effect of simvastatin in males. Pharmacogenomics, 10(11), 1743-1751.

Becker, M. L., Visser, L. E., van Schaik, R. H., Hofman, A., Uitterlinden, A. G., Stricker, C., & Bruno, H. (2010). Influence of genetic variation in CYP3A4 and ABCB1 on dose decrease or switching during simvastatin and atorvastatin therapy. Pharmacoepidemiology and Drug Safety, 19(1), 75-81.

83

Becker, M., Elens, L., Visser, L., Hofman, A., Uitterlinden, A., van Schaik, R., & Stricker, B. (2013). Genetic variation in the ABCC2 gene is associated with dose decreases or switches to other cholesterol-lowering drugs during simvastatin and atorvastatin therapy. The Pharmacogenomics Journal, 13(3), 251-256.

Bellosta, S., Paoletti, R., & Corsini, A. (2004). Safety of statins: focus on clinical pharmacokinetics and drug interactions. Circulation, 109(23 Suppl 1), III50-7.

Berry, L. M., Wollenberg, L., & Zhao, Z. (2009). Esterase activities in the blood, liver and intestine of several preclinical species and humans. Drug Metabolism Letters, 3(2), 70-77.

Bhatia, M. S., Jadhav, S. D., Bhatia, N. M., Choudhari, P. B., & Ingale, K. B. (2011). Synthesis, characterization and quantification of simvastatin metabolites and impurities. Scientia Pharmaceutica, 79(3), 601-614.

Billecke, S., Draganov, D., Counsell, R., Stetson, P., Watson, C., Hsu, C., & La Du, B. N. (2000). Human serum paraoxonase (PON1) isozymes Q and R hydrolyze lactones and cyclic carbonate esters. Drug Metabolism and Disposition: The Biological Fate of Chemicals, 28(11), 1335-1342.

Brophy, V. H., Jampsa, R. L., Clendenning, J. B., McKinstry, L. A., Jarvik, G. P., & Furlong, C. E. (2001). Effects of 5′ regulatory-region polymorphisms on paraoxonase-gene (PON1) expression. The American Journal of Human Genetics, 68(6), 1428-1436.

Carlucci, G., Mazzeo, P., Biordi, L., & Bologna, M. (1992). Simultaneous determination of simvastatin and its hydroxy acid form in human plasma by high-performance liquid chromatography with UV detection. Journal of Pharmaceutical and Biomedical Analysis, 10(9), 693-697.

Casey Laizure, S., Herring, V., Hu, Z., Witbrodt, K., & Parker, R. B. (2013). The Role of Human Carboxylesterases in Drug Metabolism: Have We Overlooked Their Importance? Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy, 33(2), 210-222.

Chapman, M. J., & Carrie, A. (2005). Mechanisms of statin-induced myopathy: a role for the ubiquitin-proteasome pathway? Arteriosclerosis, Thrombosis, and Vascular Biology, 25(12), 2441-2444.

Chatonnet, A., & Lockridge, O. (1989). Comparison of butyrylcholinesterase and acetylcholinesterase. The Biochemical Journal, 260(3), 625-634.

84

Chen, C., Mireles, R. J., Campbell, S. D., Lin, J., Mills, J. B., Xu, J. J., & Smolarek, T. A. (2005). Differential interaction of 3-hydroxy-3-methylglutaryl-coa reductase inhibitors with ABCB1, ABCC2, and OATP1B1. Drug Metabolism and Disposition: The Biological Fate of Chemicals, 33(4), 537-546.

Córdova, J., Ryan, J. D., Boonyaratanakornkit, B. B., & Clark, D. S. (2008). Esterase activity of bovine serum albumin up to 160 C: a new benchmark for biocatalysis. Enzyme and Microbial Technology, 42(3), 278-283.

Corsini, A., Maggi, F. M., & Catapano, A. L. (1995). Pharmacology of competitive inhibitors of HMG-CoA reductase. Pharmacological Research, 31(1), 9-27.

Costa, L. G., Vitalone, A., Cole, T. B., & Furlong, C. E. (2005). Modulation of paraoxonase (PON1) activity. Biochemical Pharmacology, 69(4), 541-550.

Darvesh, S., Martin, E., Walsh, R., & Rockwood, K. (2004). Differential effects of lipid-lowering agents on human cholinesterases. Clinical Biochemistry, 37(1), 42-49.

de Cates, A. N., Farr, M. R., Wright, N., Jarvis, M. C., Rees, K., Ebrahim, S., & Huffman, M. D. (2014). Fixed‐dose combination therapy for the prevention of cardiovascular disease.

The Cochrane Library,

Deakin, S., Leviev, I., Guernier, S., & James, R. W. (2003). Simvastatin modulates expression of the PON1 gene and increases serum paraoxonase: a role for sterol regulatory element-binding protein-2. Arteriosclerosis, Thrombosis, and Vascular Biology, 23(11), 2083-2089.

Dhareshwar, S. S. (2007). Case study: enalapril: a prodrug of enalaprilat. Prodrugs (pp. 1221-1229) Springer.

Draganov, D., & La Du, B. N. (2004). Pharmacogenetics of paraoxonases: a brief review. Naunyn-Schmiedeberg's Archives of Pharmacology, 369(1), 78-88.

Draganov, D. I., Teiber, J. F., Speelman, A., Osawa, Y., Sunahara, R., & La Du, B. N. (2005). Human paraoxonases (PON1, PON2, and PON3) are lactonases with overlapping and distinct substrate specificities. Journal of Lipid Research, 46(6), 1239-1247.

Elsby, R., Hilgendorf, C., & Fenner, K. (2012). Understanding the critical disposition pathways of statins to assess drug–drug interaction risk during drug development: it's not just about OATP1B1. Clinical Pharmacology & Therapeutics, 92(5), 584-598.

85

Fiegenbaum, M., Silveira, F. R., Van der Sand, Cézar R, Van der Sand, Luiz Carlos, Ferreira, M. E., Pires, R. C., & Hutz, M. H. (2005). The role of common variants of ABCB1, CYP3A4, and CYP3A5 genes in lipid‐lowering efficacy and safety of simvastatin

treatment. Clinical Pharmacology & Therapeutics, 78(5), 551-558.

Fukami, T., & Yokoi, T. (2012). The emerging role of human esterases. Drug Metabolism and Pharmacokinetics, 27(5), 466-477.

Giacomini, K. M., Huang, S., Tweedie, D. J., Benet, L. Z., Brouwer, K. L., Chu, X., . . . Hillgren, K. M. (2010). Membrane transporters in drug development. Nature Reviews Drug Discovery, 9(3), 215-236.

Gonzalvo, M. C., Gil, F., Hernandez, A. F., Rodrigo, L., Villanueva, E., & Pla, A. (1998). Human liver paraoxonase (PON1): subcellular distribution and characterization. Journal of Biochemical and Molecular Toxicology, 12(1), 61-69.

Goswami, S., Wang, W., Arakawa, T., & Ohtake, S. (2013). Developments and challenges for mAb-based therapeutics. Antibodies, 2(3), 452-500.

Hochman, J. H., Pudvah, N., Qiu, J., Yamazaki, M., Tang, C., Lin, J. H., & Prueksaritanont, T. (2004). Interactions of human P-glycoprotein with simvastatin, simvastatin acid, and atorvastatin. Pharmaceutical Research, 21(9), 1686-1691.

Holmes, R. S., Wright, M. W., Laulederkind, S. J., Cox, L. A., Hosokawa, M., Imai, T., . . . Perkins, E. J. (2010). Recommended nomenclature for five mammalian carboxylesterase gene families: human, mouse, and rat genes and proteins. Mammalian Genome, 21(9-10), 427-441.

Humerickhouse, R., Lohrbach, K., Li, L., Bosron, W. F., & Dolan, M. E. (2000). Characterization of CPT-11 hydrolysis by human liver carboxylesterase isoforms hCE-1 and hCE-2. Cancer Research, 60(5), 1189-1192.

Ichimaru, N., Takahara, S., Kokado, Y., Wang, J., Hatori, M., Kameoka, H., . . . Okuyama, A. (2001). Changes in lipid metabolism and effect of simvastatin in renal transplant recipients induced by cyclosporine or tacrolimus. Atherosclerosis, 158(2), 417-423.

Ieiri, I., Higuchi, S., & Sugiyama, Y. (2009). Genetic polymorphisms of uptake (OATP1B1, 1B3) and efflux (MRP2, BCRP) transporters: implications for inter-individual differences in the pharmacokinetics and pharmacodynamics of statins and other clinically relevant drugs. Expert Opinion on Drug Metabolism & Toxicology, 5(7), 703-729

86

Imai, T. (2006). Human carboxylesterase isozymes: catalytic properties and rational drug design. Drug Metabolism and Pharmacokinetics, 21(3), 173-185.

Inoue, M., Morikawa, M., Tsuboi, M., Ito, Y., & Sugiura, M. (1980). Comparative study of human intestinal and hepatic esterases as related to enzymatic properties and hydrolizing activity for ester-type drugs. The Japanese Journal of Pharmacology, 30(4), 529-535.

Ishizuka, T., Fujimori, I., Nishida, A., Sakurai, H., Yoshigae, Y., Nakahara, K., . . . Izumi, T. (2012). Paraoxonase 1 as a major bioactivating hydrolase for olmesartan medoxomil in human blood circulation: molecular identification and contribution to plasma metabolism. Drug Metabolism and Disposition: The Biological Fate of Chemicals, 40(2), 374-380.

Iwuchukwu, O. F., Feng, Q., Wei, W., Jiang, L., Jiang, M., Xu, H., . . . Roden, D. M. (2014). Genetic variation in the UGT1A locus is associated with simvastatin efficacy in a clinical practice setting. Pharmacogenomics, 15(14), 1739-1747.

Jaichander, P., Selvarajan, K., Garelnabi, M., & Parthasarathy, S. (2008). Induction of paraoxonase 1 and apolipoprotein A-I gene expression by aspirin. Journal of Lipid Research, 49(10), 2142-2148.

Jbilo, O., Bartels, C. F., Chatonnet, A., Toutant, J., & Lockridge, O. (1994). Tissue distribution of human acetylcholinesterase and butyrylcholinesterase messenger RNA. Toxicon, 32(11), 1445-1457.

Kantola, T., Kivistö, K. T., & Neuvonen, P. J. (1998). Erythromycin and verapamil considerably increase serum simvastatin and simvastatin acid concentrations. Clinical Pharmacology & Therapeutics, 64(2), 177-182.

Keskitalo, J. E., Kurkinen, K. J., Neuvonen, P. J., & Niemi, M. (2008). ABCB1 haplotypes differentially affect the pharmacokinetics of the acid and lactone forms of simvastatin and atorvastatin. Clinical Pharmacology & Therapeutics, 84(4), 457-461.

Khersonsky, O., & Tawfik, D. S. (2005). Structure-reactivity studies of serum paraoxonase PON1 suggest that its native activity is lactonase. Biochemistry, 44(16), 6371-6382.

Kim, K., Park, P., Lee, O., Kang, D., & Park, J. (2007). Effect of Polymorphic CYP3A5 Genotype on the Single‐Dose Simvastatin Pharmacokinetics in Healthy Subjects. The

Journal of Clinical Pharmacology, 47(1), 87-93.

Kim, J., Ahn, B., Chae, H., Han, S., Doh, K., Choi, J., . . . Yim, D. (2011). A population pharmacokinetic-pharmacodynamic model for simvastatin that predicts low-density lipoprotein-cholesterol reduction in patients with primary hyperlipidaemia. Basic \& Clinical Pharmacology \& Toxicology, 109(3), 156-163.

87

Kjekshus, J., Pedersen, T. R., Olsson, A. G., Færgeman, O., & Pyörälä, K. (1997). The effects of simvastatin on the incidence of heart failure in patients with coronary heart disease. Journal of Cardiac Failure, 3(4), 249-254.

Kuo, C. L., & La Du, B. N. (1998). Calcium binding by human and rabbit serum paraoxonases. Structural stability and enzymatic activity. Drug Metabolism and Disposition: The Biological Fate of Chemicals, 26(7), 653-660.

Lennernäs, H., & Fager, G. (1997). Pharmacodynamics and pharmacokinetics of the HMG-CoA reductase inhibitors. Clinical Pharmacokinetics, 32(5), 403-425.

Levano, S., Ginz, H., Siegemund, M., Filipovic, M., Voronkov, E., Urwyler, A., & Girard, T. (2005). Genotyping the butyrylcholinesterase in patients with prolonged neuromuscular block after succinylcholine. Anesthesiology, 102(3), 531-535.

Li, B., Sedlacek, M., Manoharan, I., Boopathy, R., Duysen, E. G., Masson, P., & Lockridge, O. (2005). Butyrylcholinesterase, paraoxonase, and albumin esterase, but not carboxylesterase, are present in human plasma. Biochemical Pharmacology, 70(11), 1673-1684.

Lilja, J. J., Kivistö, K. T., & Neuvonen, P. J. (1998). Grapefruit juice—simvastatin interaction: Effect on serum concentrations of simvastatin, simvastatin acid, and HMG‐

CoA reductase inhibitors. Clinical Pharmacology & Therapeutics, 64(5), 477-483.

Lilja, J. J., Neuvonen, M., & Neuvonen, P. J. (2004). Effects of regular consumption of grapefruit juice on the pharmacokinetics of simvastatin. British Journal of Clinical Pharmacology, 58(1), 56-60.

Lonn, E., & Yusuf, S. (2009). Polypill: the evidence and the promise. Current Opinion in Lipidology, 20(6), 453-459.

Luan, L., Sugiyama, T., Takai, S., Usami, Y., Adachi, T., Katagiri, Y., & Hirano, K. (1997). Purification and characterization of pranlukast hydrolase from rat liver microsomes: the hydrolase is identical to carboxylesterase pI 6.2. Biological & Pharmaceutical Bulletin, 20(1), 71-75.

Macan, M., Vukšić, A., Žunec, S., Konjevoda, P., Lovrić, J., Kelava, M., . . . Bradamante, V. (2015). Effects of simvastatin on malondialdehyde level and esterase activity in plasma and tissue of normolipidemic rats. Pharmacological Reports,

Marsh, S., Xiao, M., Yu, J., Ahluwalia, R., Minton, M., Freimuth, R. R., . . . McLeod, H. L. (2004). Pharmacogenomic assessment of carboxylesterases 1 and 2. Genomics, 84(4), 661-668.

88

Masson, P., Froment, M., Fortier, P., Visicchio, J., Bartels, C. F., & Lockridge, O. (1998). Butyrylcholinesterase-catalysed hydrolysis of aspirin, a negatively charged ester, and aspirin-related neutral esters. Biochimica Et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology, 1387(1), 41-52.

Mauro, V. F. (1993). Clinical pharmacokinetics and practical applications of simvastatin. Clinical Pharmacokinetics, 24(3), 195-202.

Meade, T., Sleight, P., Collins, R., Armitage, J., Bowman, L., Parish, S., . . . Wincott, E. (2010). Intensive lowering of LDL cholesterol with 80 mg versus 20 mg simvastatin daily in 12,064 survivors of myocardial infarction: a double-blind randomised trial. Lancet, 376(9753), 1658-1669.

Mirdamadi, H. Z., Sztanek, F., Derdak, Z., Seres, I., Harangi, M., & Paragh, G. (2008). The human paraoxonase‐1 phenotype modifies the effect of statins on paraoxonase activity

and lipid parameters. British Journal of Clinical Pharmacology, 66(3), 366-374.

Morgan, A. M., & Truitt, E. B. (1965). Evaluation of acetylsalicylic acid esterase in aspirin metabolism. Interspecies comparison. Journal of Pharmaceutical Sciences, 54(11), 1640-1646.

Morikawa, M., Inoue, M., Tsuboi, M., & Sugiura, M. (1979). Studies on aspirin esterase of human serum. The Japanese Journal of Pharmacology, 29(4), 581-586.

Morton, C. L., Wadkins, R. M., Danks, M. K., & Potter, P. M. (1999). The anticancer prodrug CPT-11 is a potent inhibitor of acetylcholinesterase but is rapidly catalyzed to SN-38 by butyrylcholinesterase. Cancer Research, 59(7), 1458-1463.

Najib, N. M., Idkaidek, N., Adel, A., Admour, I., Astigarraga, R. E., Nucci, G. D., . . . Dham, R. (2003). Pharmacokinetics and bioequivalence evaluation of two simvastatin 40 mg tablets (Simvast & Zocor) in healthy human volunteers. Biopharmaceutics & Drug Disposition, 24(5), 183-189.

Neuvonen, P. J., Backman, J. T., & Niemi, M. (2008). Pharmacokinetic comparison of the potential over-the-counter statins simvastatin, lovastatin, fluvastatin and pravastatin. Clinical Pharmacokinetics, 47(7), 463-474.

Neuvonen, P. J., Kantola, T., & Kivistö, K. T. (1998). Simvastatin but not pravastatin is very susceptible to interaction with the CYP3A4 inhibitor itraconazole. Clinical Pharmacology & Therapeutics, 63(3), 332-341.

Neuvonen, P. J., Niemi, M., & Backman, J. T. (2006). Drug interactions with lipid‐lowering

drugs: Mechanisms and clinical relevance. Clinical Pharmacology & Therapeutics, 80(6), 565-581.

89

Ng, C. J., Wadleigh, D. J., Gangopadhyay, A., Hama, S., Grijalva, V. R., Navab, M., . . . Reddy, S. T. (2001). Paraoxonase-2 is a ubiquitously expressed protein with antioxidant properties and is capable of preventing cell-mediated oxidative modification of low density lipoprotein. The Journal of Biological Chemistry, 276(48), 44444-44449.

Pasanen, M. K., Neuvonen, M., Neuvonen, P. J., & Niemi, M. (2006). SLCO1B1 polymorphism markedly affects the pharmacokinetics of simvastatin acid. Pharmacogenetics and Genomics, 16(12), 873-879.

Pasha, M., Muzeeb, S., Basha, S. J. S., Shashikumar, D., Mullangi, R., & Srinivas, N. R. (2006). Analysis of five HMG‐CoA reductase inhibitors—atorvastatin, lovastatin,

pravastatin, rosuvastatin and simvastatin: pharmacological, pharmacokinetic and analytical overview and development of a new method for use in pharmaceutical formulations analysis and in vitro metabolism studies. Biomedical Chromatography, 20(3), 282-293.

Pietro, D. A., & Mantell, G. (1990). Simvastatin: A New HMG CoA Reductase Inhibitor. Cardiovascular Drug Reviews, 8(3), 220-228.

Plosker, G. L., & McTavish, D. (1995). Simvastatin. Drugs, 50(2), 334-363.

Postmus, I., Trompet, S., Deshmukh, H. A., Barnes, M. R., Li, X., Warren, H. R., . . . Donnelly, L. A. (2014). Pharmacogenetic meta-analysis of genome-wide association studies of LDL cholesterol response to statins. Nature Communications, 5

Potter, P. M., Wolverton, J. S., Morton, C. L., Wierdl, M., & Danks, M. K. (1998). Cellular localization domains of a rabbit and a human carboxylesterase: influence on irinotecan (CPT-11) metabolism by the rabbit enzyme. Cancer Research, 58(16), 3627-3632.

Précourt, L., Amre, D., Denis, M., Lavoie, J., Delvin, E., Seidman, E., & Levy, E. (2011). The three-gene paraoxonase family: physiologic roles, actions and regulation. Atherosclerosis, 214(1), 20-36.

Prueksaritanont, T., Ma, B., & Yu, N. (2003). The human hepatic metabolism of simvastatin hydroxy acid is mediated primarily by CYP3A, and not CYP2D6. British Journal of Clinical Pharmacology, 56(1), 120-124.

Prueksaritanont, T., Gorham, L. M., Ma, B., Liu, L., Yu, X., Zhao, J. J., . . . Vyas, K. P. (1997). In vitro metabolism of simvastatin in humans [SBT] identification of metabolizing enzymes and effect of the drug on hepatic P450s. Drug Metabolism and Disposition: The Biological Fate of Chemicals, 25(10), 1191-1199.

90

Prueksaritanont, T., Ma, B., Fang, X., Subramanian, R., Yu, J., & Lin, J. H. (2001). beta-Oxidation of simvastatin in mouse liver preparations. Drug Metabolism and Disposition: The Biological Fate of Chemicals, 29(10), 1251-1255.

Prueksaritanont, T., Subramanian, R., Fang, X., Ma, B., Qiu, Y., Lin, J. H., . . . Baillie, T. A. (2002). Glucuronidation of statins in animals and humans: a novel mechanism of statin lactonization. Drug Metabolism and Disposition: The Biological Fate of Chemicals, 30(5), 505-512.

Riedmaier, S., Klein, K., Winter, S., Hofmann, U., Schwab, M., & Zanger, U. M. (2011). Paraoxonase (PON1 and PON3) Polymorphisms: Impact on Liver Expression and Atorvastatin-Lactone Hydrolysis. Frontiers in Pharmacology, 2, 41.

Rosenson, R. S. (2004). Current overview of statin-induced myopathy. The American Journal of Medicine, 116(6), 408-416.

Rustemeijer, C., Schouten, J., Voerman, H., Beynen, A., Donker, A., & Heine, R. (2001). Is pseudocholinesterase activity related to markers of triacyglycerol synthesis in Type II diebetes mellitus? Clinical Science, 101(1), 29-35.

Sallustio, B. C., Fairchild, B. A., Shanahan, K., Evans, A. M., & Nation, R. L. (1996). Disposition of gemfibrozil and gemfibrozil acyl glucuronide in the rat isolated perfused liver. Drug Metabolism and Disposition: The Biological Fate of Chemicals, 24(9), 984-989.

Santanam, N., & Parthasarathy, S. (2007). Aspirin is a substrate for paraoxonase-like activity: implications in atherosclerosis. Atherosclerosis, 191(2), 272-275.

SEARCH Collaborative Group, Link, E., Parish, S., Armitage, J., Bowman, L., Heath, S., . . . Collins, R. (2008). SLCO1B1 variants and statin-induced myopathy--a genomewide study. The New England Journal of Medicine, 359(8), 789-799.

Selak, V., Elley, C. R., Bullen, C., Crengle, S., Wadham, A., Rafter, N., . . . Rodgers, A. (2014). Effect of fixed dose combination treatment on adherence and risk factor control among patients at high risk of cardiovascular disease: randomised controlled trial in primary care. BMJ (Clinical Research Ed.), 348, g3318.

Shitara, Y., & Sugiyama, Y. (2006). Pharmacokinetic and pharmacodynamic alterations of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors: drug–drug interactions and interindividual differences in transporter and metabolic enzyme functions. Pharmacology & Therapeutics, 112(1), 71-105.

Sirtori, C. R. (1990). Pharmacology and mechanism of action of the new HMG-CoA reductase inhibitors. Pharmacological Research, 22(5), 555-563.

91

Slater, E. E., & MacDonald, J. S. (1988). Mechanism of action and biological profile of HMG CoA reductase inhibitors. Drugs, 36(3), 72-82.

Smiderle, L., Lima, L. O., Hutz, M. H., Sand, Cézar Roberto Van der, Van der Sand, Luiz Carlos, Ferreira, M. E. W., . . . Fiegenbaum, M. (2014). Evaluation of Sexual Dimorphism in the Efficacy and Safety of Simvastatin/Atorvastatin Therapy in a Southern Brazilian Cohort. Arquivos Brasileiros De Cardiologia, 103(1), 33-40.

Smith, P. C., Song, W. Q., & Rodriguez, R. J. (1992). Covalent binding of etodolac acyl glucuronide to albumin in vitro. Drug Metabolism and Disposition: The Biological Fate of Chemicals, 20(6), 962-965.

Soliman, E. Z., Mendis, S., Dissanayake, W. P., Somasundaram, N. P., Gunaratne, P. S., Jayasingne, I. K., & Furberg, C. D. (2011). A Polypill for primary prevention of cardiovascular disease: a feasibility study of the World Health Organization. Trials, 12(3)

Stancu, C., & Sima, A. (2001). Statins: mechanism of action and effects. Journal of Cellular and Molecular Medicine, 5(4), 378-387.

Sun, Z., Murry, D. J., Sanghani, S. P., Davis, W. I., Kedishvili, N. Y., Zou, Q., . . . Bosron, W. F. (2004). Methylphenidate is stereoselectively hydrolyzed by human carboxylesterase CES1A1. The Journal of Pharmacology and Experimental Therapeutics, 310(2), 469-476.

Tabata, T., Katoh, M., Tokudome, S., Nakajima, M., & Yokoi, T. (2004). Identification of the cytosolic carboxylesterase catalyzing the 5'-deoxy-5-fluorocytidine formation from capecitabine in human liver. Drug Metabolism and Disposition: The Biological Fate of Chemicals, 32(10), 1103-1110.

Takai, S., Matsuda, A., Usami, Y., Adachi, T., Sugiyama, T., Katagiri, Y., . . . Hirano, K. (1997). Hydrolytic profile for ester- or amide-linkage by carboxylesterases pI 5.3 and 4.5 from human liver. Biological & Pharmaceutical Bulletin, 20(8), 869-873.

Tang, M., Mukundan, M., Yang, J., Charpentier, N., LeCluyse, E. L., Black, C., . . . Yan, B. (2006). Antiplatelet agents aspirin and clopidogrel are hydrolyzed by distinct carboxylesterases, and clopidogrel is transesterificated in the presence of ethyl alcohol. The Journal of Pharmacology and Experimental Therapeutics, 319(3), 1467-1476.

Teiber, J. F., Horke, S., Haines, D. C., Chowdhary, P. K., Xiao, J., Kramer, G. L., . . . Draganov, D. I. (2008). Dominant role of paraoxonases in inactivation of the Pseudomonas aeruginosa quorum-sensing signal N-(3-oxododecanoyl)-L-homoserine lactone. Infection and Immunity, 76(6), 2512-2519.

92

Todd, P. A., & Goa, K. L. (1990). Simvastatin. Drugs, 40(4), 583-607.

Tomas, M., Senti, M., Garcia-Faria, F., Vila, J., Torrents, A., Covas, M., & Marrugat, J. (2000). Effect of simvastatin therapy on paraoxonase activity and related lipoproteins in familial hypercholesterolemic patients. Arteriosclerosis, Thrombosis, and Vascular Biology, 20(9), 2113-2119.

Tougou, K., Nakamura, A., Watanabe, S., Okuyama, Y., & Morino, A. (1998). Paraoxonase has a major role in the hydrolysis of prulifloxacin (NM441) a prodrug of a new antibacterial agent. Drug Metabolism and Disposition: The Biological Fate of Chemicals, 26(4), 355-359.

Tsamandouras, N., Dickinson, G., Guo, Y., Hall, S., Rostami‐Hodjegan, A., Galetin, A., &

Aarons, L. (2014). Identification of the Effect of Multiple Polymorphisms on the Pharmacokinetics of Simvastatin and Simvastatin Acid Using a Population‐Modeling

Approach. Clinical Pharmacology & Therapeutics, 96(1), 90-100.

Tubic-Grozdanis, M., Hilfinger, J. M., Amidon, G. L., Kim, J. S., Kijek, P., Staubach, P., & Langguth, P. (2008). Pharmacokinetics of the CYP 3A substrate simvastatin following administration of delayed versus immediate release oral dosage forms. Pharmaceutical Research, 25(7), 1591-1600.

Tunek, A., Levin, E., & Svensson, L. (1988). Hydrolysis of 3 H-bambuterol, a carbamate prodrug of terbutaline, in blood from humans and laboratory animals in vitro. Biochemical Pharmacology, 37(20), 3867-3876.

Ucar, M., Mjörndal, T., & Dahlqvist, R. (2000). HMG-CoA reductase inhibitors and myotoxicity. Drug Safety, 22(6), 441-457.

Urso, M. L., Clarkson, P. M., Hittel, D., Hoffman, E. P., & Thompson, P. D. (2005). Changes in ubiquitin proteasome pathway gene expression in skeletal muscle with exercise and statins. Arteriosclerosis, Thrombosis, and Vascular Biology, 25(12), 2560-2566.

Vickers, S., Duncan, C. A., Chen, I. W., Rosegay, A., & Duggan, D. E. (1990). Metabolic disposition studies on simvastatin, a cholesterol-lowering prodrug. Drug Metabolism and Disposition: The Biological Fate of Chemicals, 18(2), 138-145.

Vickers, S., Duncan, C. A., Vyas, K. P., Kari, P. H., Arison, B., Prakash, S. R., . . . Duggan, D. E. (1990). In vitro and in vivo biotransformation of simvastatin, an inhibitor of HMG CoA reductase. Drug Metabolism and Disposition: The Biological Fate of Chemicals, 18(4), 476-483.

93

Volland, C., Sun, H., Dammeyer, J., & Benet, L. Z. (1991). Stereoselective degradation of the fenoprofen acyl glucuronide enantiomers and irreversible binding to plasma protein. Drug Metabolism and Disposition: The Biological Fate of Chemicals, 19(6), 1080-1086.

Vree, T. B., Dammers, E., Ulc, I., Horkovics-Kovats, S., Ryska, M., & Merkx, I. (2001). Variable plasma/liver and tissue esterase hydrolysis of simvastatin in healthy volunteers after a single oral dose. Clinical Drug Investigation, 21(9), 643-652.

Wang, E., Casciano, C. N., Clement, R. P., & Johnson, W. W. (2001). Inhibition of P-glycoprotein transport function by grapefruit juice psoralen. Pharmaceutical Research, 18(4), 432-438.

Wang, X., Zhu, H., & Markowitz, J. S. (2015). Carboxylesterase 1-Mediated Drug--Drug Interactions between Clopidogrel and Simvastatin. Biological & Pharmaceutical Bulletin, 38(2), 292-297.

Wilke, R., Ramsey, L., Johnson, S., Maxwell, W., McLeod, H., Voora, D., . . . Cooper‐

DeHoff, R. (2012). The clinical pharmacogenomics implementation consortium: CPIC guideline for SLCO1B1 and simvastatin‐induced myopathy. Clinical Pharmacology &

Therapeutics, 92(1), 112-117.

Williams, F. M., Mutch, E., Nicholson, E., Wynne, H., Wright, P., Lambert, D., & Rawlins, M. (1989). Human liver and plasma aspirin esterase. Journal of Pharmacy and Pharmacology, 41(6), 407-409.

Williams, E. T., Jones, K. O., Ponsler, G. D., Lowery, S. M., Perkins, E. J., Wrighton, S. A., . . . Farid, N. A. (2008). The biotransformation of prasugrel, a new thienopyridine prodrug, by the human carboxylesterases 1 and 2. Drug Metabolism and Disposition: The Biological Fate of Chemicals, 36(7), 1227-1232.

Winsemius, A., Ansquer, J., Olbrich, M., van Amsterdam, P., Aubonnet, P., Beckmann, K., . . . Lehnick, D. (2014). Pharmacokinetic interaction between simvastatin and fenofibrate with staggered and simultaneous dosing: Does it matter? The Journal of Clinical Pharmacology, 54(9), 1038-1047.

Xu, G., Zhang, W., Ma, M. K., & McLeod, H. L. (2002). Human carboxylesterase 2 is commonly expressed in tumor tissue and is correlated with activation of irinotecan. Clinical Cancer Research : An Official Journal of the American Association for Cancer Research, 8(8), 2605-2611.

Yang, F., Bian, C., Zhu, L., Zhao, G., Huang, Z., & Huang, M. (2007). Effect of human serum albumin on drug metabolism: structural evidence of esterase activity of human serum albumin. Journal of Structural Biology, 157(2), 348-355.

94

Zhang R. (2015). The human plasma esterase enzymes involved in bioactivation of the prodrug irinotecan (CPT-11) (Unpublished masters thesis). University of Auckland, New Zealand.

Zhu, H., Patrick, K. S., Yuan, H., Wang, J., Donovan, J. L., DeVane, C. L., . . . Sweet, D. H. (2008). Two CES1 gene mutations lead to dysfunctional carboxylesterase 1 activity in man: clinical significance and molecular basis. The American Journal of Human Genetics, 82(6), 1241-1248.