Sults): Role of Genetic Polymorphisms

A Thesis

Entitled

A Systemic Investigation of the Sulfation of Opioid Drugs by the Human Cytosolic

Sulfotransferases (SULTs): Role of Genetic Polymorphisms

Andriy Chepak

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Master of Science Degree in Pharmaceutical Sciences (Pharmacology/Toxicology)

______Dr. Ming-Cheh Liu, Committee Chair

______Dr. Frederick E. Williams, Committee Member

______Dr. Caren L. Steinmiller, Committee Member

______Dr. Amanda C. Bryant-Friedrich, Dean College of Graduate Studies

The University of Toledo

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of

A Systemic Investigation of the Sulfation of Opioid Drugs by the Human Cytosolic Sulfotransferases (SULTs)

Andriy Chepak

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Pharmaceutical Sciences (Pharmacology/Toxicology)

The University of Toledo May 2020

Opioid drugs are of great importance in the management of acute and chronic pain conditions. They are recognized as being essential in the management of severe malignant and non-malignant pain (Mercadante, 2015). Pharmacokinetic differences among these drugs contribute to patients having differential responses, including bioavailability, metabolism, and elimination from the body. It is becoming more evident that genetics play a vital role in affecting the metabolism of opioid drugs. Three major enzyme systems, CYP450, UDP-glucuronosyltransferase (UGTs) and SULTs have been shown to be involved in their metabolism. Polymorphisms of these enzyme systems can result in an individual having distinct phenotypes: poor metabolizers which express two nonfunctional alleles, intermediate metabolizers at least one reduced functional allele, extensive metabolizers at least one functional allele, and ultra-rapid metabolizers express multiple copies of the functional allele. These genetic differences are partially explained by single nucleotide polymorphisms, (-SNPs), which encode for molecular entities involved in the pharmacodynamic and pharmacokinetic processes. Understanding how and to what degree the allozymes of different enzyme systems such as the CYPs, UGTs

iii and SULTs affect drug metabolism can add new insights into gene therapy-based approaches and greatly improve the treatment of chronic pain (Peiro, 2016.)

My thesis research was focused on the role of human SULTs in the metabolism of opioid drugs. Previous studies have demonstrated that of the eleven known SULTs,

SULT1A3 was found to be involved in opioid drug metabolism (Bairam, 2018). By carrying out in vitro assays, we first determined the sulfating activity of SULT1A3 toward opioid drugs and subsequently the effects of SNPs of human SULT1A3/SULT1A4 genes on the enzymatic characteristics of SULT1A3 allozymes in mediating hydromorphone and pentazocine, two commonly used opioid drugs. The results obtained provided valuable information relevant to the differential metabolism of hydromorphone and pentazocine in individuals having different SULT1A3/SULT1A4 genotypes.

iv Acknowledgements

Foremost, I would like to thank God and my family for their support and encouragement throughout my program at the University of Toledo, College of Pharmacy and Pharmaceutical Sciences. Very importantly, I would like to thank my research advisor Dr. Ming-Cheh Liu for being a great advisor and for his patience, enthusiasm, and great knowledge.

I am thankful to my thesis committee members: Dr. Frederick E. Williams and

Dr. Caren L. Steinmiller for their guidance and insightful comments. I am also very grateful to my academic advisor, Dr. Youssef Sari, for his friendly and positive attitude throughout my stay at U.T.

Last but not least, I would like to thank all my lab mates at Dr. Liu’s research lab for their support, friendly attitudes and help throughout this entire process.

v Table of Contents

Abstract...... iii

Acknowledgements...... v

Table of Contents...... vi

List of Tables ...... viii

List of Figures...... ix

List of Abbreviations...... x

List of Symbols...... xii

1. Introduction...... 1

1.1 Overview of Opioid Drugs...... 1

1.2 Opioids Metabolism...... 2

1.2.1 Metabolism of Opioid Drugs...... 3

1.2.2 Pharmacokinetics...... 5

1.3 Major Classes of Enzyme Involved in the Metabolism of Opioid Drugs...... 8

1.3.1 Role of the SULTs in Opioid Metabolism...... 11

1.4 Role of Pharmacogenetics in Opioid Metabolism...... 14

1.4.1 Pharmacogenetics...... 14

1.4.2 SULTs Allozymes...... 14

1.5 Objectives and Goals...... 17

vi 2. Materials and Methods...... 18

2.1 Materials...... 18

2.2 Methods...... 18

2.2.1 2.2.1 Analysis of the sulfating activity of the human SULTs toward

opioid drugs...... 18

2.2.2 Analysis of the kinetic activity of the human SULTs toward

opioid drugs...... 20

2.2.3 Analysis of the opioid drug-sulfating activity of the

human SULT1A3 allozymes...... 21

2.2.4 Statistical Analysis...... 21

3. Results and Discussion...... 22

3.1 Results...... 22

3.1.1 Differential sulfating activity of the human SULTs toward

hydromorphone, oxymorphone, and pentazocine...... 22

3.1.2 Kinetics Analysis of the Sulfation of Hydromorphone by

SULT1A3...... 24

3.1.3 Differential sulfating activities of human SULTs allozymes toward

hydromorphone...... 27

3.1.4 Differential sulfating activities of human SULTs allozymes toward

pentazocine...... 32

Discussion...... 36

References...... 42

vii List of Tables

1.1 Major Opioid Metabolites ...... 5

1.2 Recommendation of opioid use in patients with liver impairment ...... 7

1.3 Metabolic Pathway/Enzyme Involvement ...... 10

1.4 SULT1A3 Allozymes ...... 16

3.1 Kinetic Parameters of the wild-type SULT1A3 with hydromorphone as

substrate ...... 25

3.2 Differential sulfating activities of the human SULTs allozymes toward

hydromorphone ...... 29

3.3 Similar, higher, and lower specific activities of SULT1A3 allozymes with

hydromorphone as substrate ...... 30

3.4 Differential sulfating activities of the human SULT1A3 allozymes toward

pentazocine ...... 33

3.5 Similar, higher, and lower specific activities of SULT1A3 allozymes with

pentazocine as substrate ...... 34

viii List of Figures

1-1 Sulfation Reaction of Opioid Drugs ...... 12

1-2 Chemical structures of three opioid drugs (Hydromorphone, Pentazocine,

and Oxymorphone) ...... 13

1-3 Sulfation Reaction...... 13

3-1 X-ray film of Sulfated Drugs ...... 23

3-2 Hydromorphone Michaelis-Menten kinetics ...... 26

3-3 Hydromorphone Lineweaver-Burk double-reciprocal plot ...... 26

3-4 Differential sulfating activities of the human SULTs allozymes toward

hydromorphone ...... 28

3-5 Differential sulfating activities of the human SULTs allozymes toward

pentazocine...... 32

3-6 Allozyme sulfation difference towards pentazocine, highest vs lowest

sulfating allozymes...... 39

ix List of Abbreviations

ADP...... Adenosine-5’-Diphosphate APS ...... Adenosine-5’-Phosphosulfate ATP ...... Adenosine-5’-Triphosphate

CYP ...... Cytochrome P-450

DMSO ...... Dimethyl Sulfoxide DTT ...... Dithiothreitol

HEPES ...... N-2-Hydroxylpiperazine-N2-Ethanesulfonic

PAPS ...... 3‘-phosphoadenosine-5‘-phosphosulfate PCR ...... Polymerase Chain Reaction PPi ...... Pyrophosphate

S.D ...... Standard Deviation SDS ...... Sodium Dodecyl Sulfate SDS–PAGE ...... Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis SNPs...... Single Nucleotide Polymorphisms SULTs ...... Human Cytosolic Sulfotransferases Enzymes SULT1A1 ...... Human Cytosolic Sulfotransferases Family 1A Member 1 SULT1A2 ...... Human Cytosolic Sulfotransferases Family 1A Member 2 SULT1A3 ...... Human Cytosolic Sulfotransferases Family 1A Member 3 SULT1B1 ...... Human Cytosolic Sulfotransferases Family 1B Member 1 SULT1C2 ...... Human Cytosolic Sulfotransferases Family 1C Member 2 SULT1C3 ...... Human Cytosolic Sulfotransferases Family 1C Member 3 SULT1C4 ...... Human Cytosolic Sulfotransferases Family 1C Member 4 SULT1E1 ...... Human Cytosolic Sulfotransferases Family 1E Member 1 SULT2A1 ...... Human Cytosolic Sulfotransferases Family 2A Member 1 SULT2B1a ...... Human Cytosolic Sulfotransferases Family 2B Member 1a SULT2B1b ...... Human Cytosolic Sulfotransferases Family 2B Member 1b SULT4A1 ...... Human Cytosolic Sulfotransferases Family 4A Member 1 SULT6B1 ...... Human Cytosolic Sulfotransferases Family 6B Member

x TLC ...... Cellulose Thin-Layer Chromatography

UGTs...... Uridine 5'-diphospho-glucuronosyltransferase

xi List of Symbols

L ...... Liter ml ...... Milliliter µl ...... Microliter g...... Gram mg ...... Milligram µg ...... Microgram ng...... Nanogram mM ...... Millimolar µM ...... Micromolar

SO3-...... Sulfonate Group SO42-...... Inorganic Sulfate nmol ...... Nanomole mmol ...... Millimole pmol...... Picomole min ...... Minute

°C...... Celsius

Ci ...... Curie

Vmax ...... Maximum Velocity Km ...... Michaelis Constant Vmax/Km ...... Catalytic Efficiency

xii Chapter 1

Introduction

1.1 Overview of Opioid Drugs

Opioids are of great importance for the management of acute and chronic pain conditions. They have been recognized as essential drugs for severe malignant and non- malignant pain (Mercadante, 2015). They are very important in managing cancer pain and are included in the step II and III analgesics ladder. As designated by the World

Health Organization (WHO), step I uses non-opioid analgesics such as acetaminophen or

NSAIDs, step II uses “weak” opioids (hydrocodone, codeine, or tramadol), and step III uses “strong” opioids (morphine, hydromorphone, oxycodone, fentanyl, or methadone).

Pentazocine. Published clinical studies and extensive experience have shown that pentazocine, the first of the practical agonist/antagonist analgesics, is a potent analgesic with extensive application in clinical medicine (Goldstein, 1985). Pentazocine is a mixed agonist/antagonist that can bind to both μ and κ opioid receptors. It can be combined with naloxone via IV administration to mitigate the abuse of drug. It can provide analgesia as effective as the opioids including morphine and meperidine without producing anxiolytic effects in patients. Pentazocine is metabolized in the liver, mainly by oxidation of the terminal methyl groups of the dimethyl alkyl side chain to form alcoholic and carboxylic

1 acid metabolites; followed by conjugation to glucuronides in the liver and sulfation which terminates the effects of pentazocine.

Hydromorphone. Hydromorphone is an analog of morphine. Hydromorphone is approximately five-fold more potent than morphine at the μ opioid receptor, compared to dihydromorphine and norhydromoprhone which have same potency as morphine (Coller

2008). It is metabolized in the liver by glucuronidation utilizing the UGT2B7 enzymes as well as sulfation. Hydromorphone and its metabolites may accumulate in patients with renal failure, resembling the problems found with morphine and its metabolite elimination in similar circumstances (the mean ratio of hydromorphone-3 glucuronide

(H3G) to hydromorphone may increase to 100:1 in renal failure). In fact, these substances are active and may contribute to producing opioid toxic effects (Mercadante, 2015).

Oxymorphone. Oxymorphone, a 3-O-demethylation metabolite of oxycodone, is a potent opioid that displays three to five times higher μ opioid receptor affinity than that of morphine. Oxymorphone has been studied for treating postsurgical pain in an oral, immediate-release formulation and appears to be effective (Smith, 2009). Oxymorphone has a short half-life but has a prolonged duration of action due to slow dissociation from its cognate receptor. The main metabolic pathway is glucuronidation. As mentioned, it has a high affinity for μ-opioid receptors, being approximately 10 times more potent than morphine in the parenteral form. Like heroin, it has a high addiction potential (Waldman,

2009).

1.2 Opioid Metabolism

Metabolism refers to the process of biotransformation by which drugs are broken down so that they can be eliminated from the body. The majority of opioids undergo

2 extensive first-pass metabolism in the liver before entering the systemic circulation.

When the metabolism of these drugs is altered, they may be removed from body too rapidly, thus not reaching the therapeutic target, or they may stay in the body too long thereby producing toxic effects. First-pass metabolism reduces the bioavailability of the opioid. Opioids are typically lipophilic, which allows them to cross cell membranes and reach target tissues. Drug metabolism is ultimately intended to make a drug hydrophilic in order to facilitate its excretion in the urine. Opioid metabolism takes place primarily in the liver, which produces enzymes for this purpose. These enzyme systems promote two forms of metabolism: phase 1 metabolism (modification reactions) and phase 2 metabolism (conjugation reactions) (Mercadante, 2015).

1.2.1 Metabolism of Opioid Drugs

Certain opioids produce multiple active metabolites after administration (Table

1.1) (Smith, 2009). Altered metabolism due to medical conditions, genetic factors, or drug to drug interactions may disrupt the balance of metabolites, which may in turn alter the efficacy and/or the adverse effects of the drug. One of the primary metabolites of hydromorphone is hydromorphone-3-glucuronide which has a neuroexcitatory potential.

On the other hand, fentanyl, oxymorphone, and methadone do not produce metabolites that could potentially complicate treatment. Oxymorphone produces an active metabolite

6-hydroxy-morphone; however, it makes up less than 1% of the administered dose excreted in urine and is metabolized through the same pathway as parent compound, making an imbalance among metabolites unlikely. Pentazocine is extensively metabolized. Less than 13% of dose appeared in the urine as unchanged pentazocine and between 12% and 30% was excreted as glucuronide conjugate (Berkowitz, 1969).

3 Opioids that produce active metabolites that are structurally identical to other opioid medications can complicate efforts to monitor patients to prevent abuse and diversion

(Smith, 2009).

Oxymorphone primarily undergoes phase II metabolism via conjugation with glucuronic acid to form oxymorphone-3-glucuronide but is also metabolized to 6- hydroxy-oxymorphone, an active metabolite. The opioid also goes through sulfation as it

- has two hydroxyl groups which are able to undergo conjugation with a sulfate (SO3 ) group. Approximately 99% of the total administered dose is excreted in the urine in its conjugated form, while less than 1% is metabolized and undergoes renal elimination as 6-

OH-oxymorphone; the active metabolite thus does not reach levels that contribute meaningfully to drug effect. Therefore no dose adjustment is necessary for anticipated changes in cytochrome P450 activity as oxymorphone has no known pharmacokinetic drug-drug interactions (Hemmings, 2019).

4 Table 1.1: Major Opioid Metabolites (Smith, 2009). Opioid Inactive Active Active metabolites metabolites metabolites that are not identical to pharmaceutical pharmaceutical opioids opioids Morphine Normorphine Hydromorphone Morphone-3-G glucuronide Morphone-6-G glucuronide Hydromorphone Minor metabolites None Hydromorphone-3- glucuronide Hydrocodone Norhydrocodone Hydromorphone None Codeine Norcodeine Hydrocodone None Morphine

Oxycodone None Oxymorphone Noroxycodone Oxymorphone Oxymorphone-3- None 6-Hydroxy- glucuronide oxymorphone Fentanyl Norfentanyl None None Pentazocine Pentazocine- None None glucuronide Highlighted are the three drugs used in this study. All three drugs are active on their own (not pro-drugs), do not produce active metabolites, are eliminated mostly by glucuronidation and sulfation as demonstrated in this study.

1.2.2 Opioids Pharmacokinetics

Opioids present considerable physicochemical and pharmacokinetic differences, which largely explain the differential effects under different clinical conditions.

Pharmacokinetics contributes to the variability in response to opioid drugs, affecting the bioavailability of a drug, the production of metabolites with lingering clinical activity, and their elimination from the body. Understanding the metabolism of opioids is essential for clinicians who use this class of drugs, particularly in older and complicated patients.

This population often receives multiple medications and may have impaired renal and hepatic function (Table 1.2). Age, genetics, simultaneous chronic diseases or conditions,

5 along with concomitant medications may substantially influence opioid drug metabolism

(Mercadante, 2015). Since genetic factors play a significant role in opioid drug activation and metabolism it is crucial that we explore the area of pharmacogenetics and its relevance to opioid based pain management.

Clinicians understand that individual patients have varied responses to specific opioid analgesics and that patients may require trials of several opioids before finding an agent that provides effective analgesia with acceptable tolerability. Reasons for this variability include factors that are not yet clearly understood, such as allelic variants that dictate the complement of opioid receptors and subtle differences in the receptor-binding profiles of opioids (Smith, 2009). Alterations to opioid metabolism can influence the drugs response in terms of efficacy and tolerability. Having a strong understanding of the enzymes involved in the drug transformation process can greatly aid physicians and other medical professionals in treating their patients who suffer from acute and chronic pain.

This knowledge can help decrease the risk of drug interactions by knowing which enzyme systems may metabolize the opioid and how significant genetic factors are in drug metabolism.

6 Table 1.2: Recommendation of opioid use in patients with liver impairment (Mercadante 2015). Opioid Use Dosing

Morphine Needs careful use Use dosing intervals

Hydromorphone Needs careful use Use dosing intervals

Oxycodone Best to avoid

Codeine Best to avoid

Methadone Not recommended

Fentanyl Usually safe

Tramadol Best to avoid

Hydromorphone (Table 1.2), for example, has a direct effect on brain stem respiratory centers, depressing the respiratory drive and suppressing cough. This drug has a United States Black Box Warning indicating that: “Addiction, abuse, and misuse are all potential risks affecting hydromorphone users.” Individuals who chronically use this drug require regular monitoring and a clear plan for the duration of treatment (Abi-Aad and

Derian, 2019). Pregnant women should avoid prolonged treatment to avoid neonatal withdrawal syndrome. Oxymorphone has a high abuse potential with potential for respiratory depression. Pentazocine produces as much respiratory depression as morphine and also increases heart rate and blood pressure by increasing the release of norepinephrine (Craig and Stitzel, 2004). Knowing the genetic differences between individuals who are susceptible to the potentially serious adverse effects of these and

7 other opioid drugs could save lives and at the same time improve pain management in patients.

1.3 Major Classes of Enzymes Involved in the Metabolism of Opioid Drugs

Opioids undergo phase 1 metabolism by the CYPs and phase 2 metabolism by conjugation, or both (Table 1.3) Phase 1 metabolism typically subjects the drug to oxidation or hydrolysis. It involves the cytochrome P450 (CYP) enzymes, which facilitate reactions that include N-, O-, and S-dealkylation; aromatic, aliphatic, or N- hydroxylation; N-oxidation; sulfoxidation; deamination; and dehalogenation. Phase 2 metabolism conjugates the drug to hydrophilic groups/moieties, such as glucuronic acid, sulfate, glycine, or glutathione. The most important phase 2 reaction is glucuronidation, catalyzed by the enzyme uridine diphosphate glucuronosyltransferase (UGT).

Glucuronidation produces molecules that are highly hydrophilic and therefore can be easily excreted.

The extent to which these drugs are metabolized by phase 1 and phase 2 enzymes differs among opioid drugs. Genetic factors pertaining to the CYP enzyme CYP2D6 can have dramatic effects on the metabolism of opioid drugs such as hydrocodone, codeine, and dihydrocodeine. Individuals who possess allelic variants of the CYP2D6 gene found to be associated with either reduced clearance or rapid metabolism should be carefully monitored when using these drugs or be given substitute opioid drugs that are not completely dependent on CYP2D6, such as oxycodone (Smith, 2009). Hydromorphone is not metabolized by CYP450 to any great extent. Thus, genetic polymorphisms of

CYP450 isoenzymes have a limited effect on metabolism or clearance of this agent.

8 Morphine, oxymorphone, and hydromorphone are each metabolized by phase 2 glucuronidation catalyzed by the enzyme uridinediphosphateglucuronosyltransferase

(UGT) (Smith, 2009). In the liver, hydromorphone is transformed by UGT1A3 and

UGT2B7, like morphine, to glucuronides, at 3 and 6 positions (after hydroxylation), to form H3G and H6G respectively, which are polar substances that can be easily eliminated by the kidneys (Smith, 2011. The enzymes responsible for glucuronidation reactions may be subject to a variety of factors that in turn alter the metabolism of opioid drugs.

Substrates and inducers have been identified, although the significance of these interactions has not been well investigated. Although a genetic variability of UGT2B7 has been reported, the functional significance of these genetic variants has not been shown to alter significantly the levels of production of its metabolites or the clinical effects. Potentially, the inhibition of UGT2B7 could decrease morphine transformation to

M6G, a potent µ-agonist. However, the extent of this interaction has never been made clear. Opioids metabolized through phase II reactions have a lower potential for causing drug/drug interactions than those metabolized by CYP450 pathways (Mercadante, 2015).

It is noted that previous studies displayed the presence of a number of hydromorphone metabolites, including dihydromorphine, dihydroisomorphine, and norhydromorphone, in human urine. As these hydromorphone metabolites all contain hydroxyl group(s), it is possible that they may also be sulfated by human SULT enzyme(s) (Kurogi, 2014). Since opioids are possibly metabolized by SULTs via phase

II, it is important to verify the sulfation and then examine factors such as genetics that can influence SULT1A3 sulfation.

9 Table 1.3: Metabolic Pathway/Enzyme Involvement (Smith, 2009).

Highlighted are the three drugs studied that demonstrated to undergo sulfation by the SULT1A3 enzyme system (Kurogi 2014, Bairam 2018). All three undergo glucuronidation by the UGT2B7 and sulfation by SULT1A3, while pentazocine also undergoes oxidation by CYPs in phase 1 according to previous studies (Coller, 2008).

10 1.3.1 Role of the SULTs in Opioid Metabolism

In humans, eleven SULT forms have been distinguished: SULT1A1 (phenol- sulfating phenol sulfotransferase, thermostable phenol sulfotransferase 1, P-PST, ST1A3,

HAST1), SULT1A2 (thermostable phenol sulfotransferase 2, ST1A2, HAST4),

SULT1A3 (catecholamine-sulfating phenol sulfotransferase, thermolabile phenol sulfotransferase, M-PST, ST1A5, HAST3), SULT1B1 (thyroid hormone sulfotransferase,

ST1B2), SULT1C1 (ST1C2, SULT1C sulfotransferase), SULT1C2 (ST1C3, SULT1C sulfotransferase 2), SULT1E1 (estrogen sulfotransferase, EST, ST1E4), SULT2A1

(hydroxysteroid or dehydroepiandrosterone sulfotransferase, HST, ST2A3), SULT2B1a,

SULT2B1b, and SULT4A1 (SULT X3, brain SULT-like protein, ST5A1) (Glatt, 2000).

Human SULTs are distributed among various human tissues, e.g., kidney, stomach, thyroid gland, spleen, placenta, colon, etc. “SULT1A3 expression (on both mRNA and protein levels) is high in the gut, detectable in many other extrahepatic tissues, and negligible in liver. SULT1A1 and 1A3 protein and enzyme activities are present also in the platelets, which therefore have been used for phenotyping human subjects for these forms. SULT1A1 levels are very high in liver; whereas it is present at lower levels in many other tissues.”(Glatt, 2000). Such varying distribution is a very interesting aspect of the SULT enzymes. Their varying distribution in the tissues throughout the body can be an interesting area for future research. For example, high levels of SULT2A1 proteins are present in adrenal gland and liver; while low levels were detected in small intestine, and little were detected in other tissues (Glatt, 2000).

Previous studies have demonstrated the sulfation of opioid drugs, buprenorphine, pentazocine, and naloxone using human hepatocyte and tissue cytosols, thereby

11 identifying the responsible SULT enzymes (Kurogi 2014, Bairam 2018). It is therefore possible that SULT-mediated sulfation may play an important role in the metabolism of not only morphine, hydromorphone, levorphanol, nalorphine, but also other opioid drugs including oxymorphone, butorphanol, nalbuphine, nalorphine, and naltrexone. These initial studies can provide further research opportunities into the extent of the sulfation of numerous opioid drugs. Sulfation as catalyzed by the SULTs is an important process that serves for the biotransformation and homeostasis of endogenous steroid/thyroid hormones, catecholamines, and bile acids, as well as for the detoxification of xenobiotics including drug compounds. Sulfate conjugation generally inactivates the biologically active compounds and concomitantly increases their water solubility, thus facilitating their removal from the body (Figure 1-1).

Figure 1-1. Sulfation Reaction of Opioid Drugs (Kurogi, et al. 2014).

All three drugs examined in my thesis research carry hydroxyl groups attached to their aryl hydrocarbon rings along with amino groups (Figure 1-2). Sulfation reaction works by attaching a sulfonate group from the active sulfate, 3′-phosphoadenosine 5′- phosphosulfate (PAPS), to the substrate compound (Figure 1-3). This may result in the inactivation of the substrate compounds or increasing their water-solubility, which in turn facilitates their removal from the body. Since SULTs are involved in phase II conjugation which more often results in inactivation, these drugs would most likely lose their activity.

Of the thirteen know human SULTs, SULT1A3 was shown to be a major enzyme

12 responsible for the sulfation of these analgesic drugs (Bairam, Rasool et al. 2018).

Because of the widespread use of these drugs along with potential serious adverse effects, it is important to understand how these drugs are metabolized and what enzyme systems are responsible. SULTs are involved in phase II conjugation and substrates with carboxyl

(-COOH), hydroxyl (-OH), amino (NH2), and sulfhydryl (-SH) group(s) are more likely to undergo sulfation. The addition of the charged sulfate group results in increased molecular weight, lower activity, and increased elimination. The degree of sulfation is due to the difference in the chemical structures of the opioid drugs along with different molecular and allozyme properties of the SULTs.

Figure 1-2: Chemical structures of three opioid drugs. All three drugs contain Hydroxyl (- OH) and amino (-NH2) groups which are able to undergo conjugation with a sulfate - (SO3 ) making the drug charged, more polar and thus more readily excreted.

Figure 1-3: Sulfation Reaction. SO3- group from the donor compound PAPS to the hydroxyl (-OH) or amino (- NH2) group of the substrate compound. (Image from Bairam, Rasool et al. (2018)

13 1.4 Role of Pharmacogenetics in Opioid Metabolism

1.4.1 Pharmacogenetics

Painkillers are associated with wide inter-individual variability in the analgesic response. This is partially explained by the presence of single nucleotide polymorphisms in genes that encode molecular entities, i.e., enzymes (CYPs, UGTs, SULTs, etc.) involved in both pharmacodynamics and pharmacokinetics. A major goal in the field of pharmacogenomics is to achieve individualized therapy by boosting the efficacy of drugs and also minimizing potential toxicity. Genes affecting pharmacodynamics are based on the variations in drug target receptors and downstream signal transduction (i.e. μ-opioid receptor, OPRM1; enzyme catecholamine methyltransferase, COMT, etc.). Genes affecting pharmacokinetics (PK) apply to drug metabolism/elimination (i.e. cytochrome

P450 family of enzymes, enzymes responsible for glucuronidation, drug transporter proteins, COX enzymes, etc.) which alters the relationship between drug dose and steady state serum drug concentrations. Genetic factors are thought to be responsible for approximately 12%–60% of response variance in opioid based treatment (Peiro, 2016).

1.4.2 SULTs Allozymes

All cytosolic sulfotransferases studied are members of a single superfamily, now termed SULT, classified according to the similarities in the nucleotide sequences of their genes/cDNAs. A classification into families (indicated by a number after the superfamily name, e.g. SULT1), subfamilies (capital letter after family name, e.g. SULT1A), individual genes (number after subfamily name, e.g. SULT1A3), and different enzyme proteins due to splice variants (lower-case letter after gene name, e.g. SULT1A3a), alleles or allelic protein variants (asterisk and code apostrophed to gene or protein name,

14 e.g. SULT1A3*1) is in preparation, but not yet finalized. Nevertheless, it is already widely used, at least for human SULTs (Glatt 2000).

In humans, thirteen SULTs that fall into four distinct gene families have been identified and characterized. In a recent published study, SULT2A1 and SULT1A3 displayed the sulfating activities toward buprenorphine and pentazocine, respectively, while several SULTs, particularly SULT1A1, showed strong sulfating activity toward naloxone (Kurogi, 2014). In my thesis research, I studied the sulfation of three opioid drugs, pentazocine, hydromorphone, and oxymorphone by the wild type and 10 allozymes belonging to the SULT1A3 family, which were previously expressed and purified. This was accomplished by performing metabolic labeling which “allows to monitor the generation of radioactive [35S]sulfated products within the whole cell and enzymatic assay using radioactive sulfonate donor, PAP[35S], to detect the sulfating activity” (Kurogi, 2014). I had also analyzed the kinetics of the sulfation of the drug hydromorphone.

15 Table 1.4: SULT1A3 Allozymes (Bairam, 2018).

Of the 16 missense cSNPs found for SULT1A3, 13 distinct amino acid alterations were found. The table shows the designated names and their SNP ID number. Certain amino acids variations are located on parts close to the surface of the molecule which are thought to be involved in substrate selectivity.

16 1.5 Objectives and Goals

My thesis research aimed to identify the human SULT(s) that are responsible for the sulfation of opioid drugs hydromorphone, oxymorphone, and pentazocine. After determining that these drugs could be sulfated by SULT1A3 enzyme, I proceeded to determine the kinetic parameters of hydromorphone. Lastly, I investigated the effects of genetic polymorphisms of SULT1A3 allozymes on the sulfating activity of hydromorphone and pentazocine.

17 Chapter Two

Materials and Methods

2.1 Materials

Hydromorphone and Oxymorphone were purchased from Cayman Chemical

Company. Pentazocine was from Cerilliant. N-2-hydroxylpiperazine-N-2-ethanesulfonic acid (HEPES), dithiothreitol (DTT), dimethyl sulfoxide (DMSO) were from Merck

(EMD Millipore Corporation). POLYGRAM CEL 300 (TLC) plates from Macherey-

Nagel. Ecolume scintillation cocktail was purchased from MP Biomedicals (Solon, OH).

Butyl Alcohol and Isopropyl Alcohol from EMD Millipore Corporation. Formic acid purchased from Aqua Solutions. Premium Clear Blue X-Ray Film from Bioland and Fuji

Medical X-Ray Film from FUJIFILM. Recombinant human SULTs were cloned, expressed, and purified as described previously (Suiko, et al., 2000; Pai, et al., 2002;

Sakakibara, et al., 1998; Kurogi, et al., 2012; Bairam, et al., 2018).

2.2 Methods

2.2.1 Analysis of the sulfating activity of the human SULTs toward opioid drugs.

The complete series of the 13 human SULTs were previously prepared in Dr.

Liu’s Laboratory according to established procedures (Bairam, 2018). The sulfating

18 activity of SULT1A3 allozymes toward hydromorphone, oxymorphone, and pentazocine were analyzed using an established enzymatic assay procedure (Kurogi, 2014). The established enzyme assay procedure included the following steps. 16 µL of the Master

Mix was added to each of the test tubes, 2µl of DMSO to the control tubes, and 2µL of the appropriate substrate molarity to respective test tubes; followed by the addition 2µL of the substrate was then added into a 16µL mixture, called the Master Mix. This master mix consists of 8µL of water, 4µL of 50mM HEPES at pH7.4, µL14 µM of PAPS [35S]

(15 Ci/mmol), and 2µL of 1 mM DTT. After all of these components were added to each test tube, WT enzyme and appropriate allozymes were added to each test tube, however it was crucial that the enzyme did not touch the mixture or otherwise the reaction would initiate. Therefore, the enzyme was placed above the mixture and not mixed until it is placed into the hot water bath for 5 minutes at a temperature of 37 degrees Celsius, which initiates the reaction. After each test tube spends five minutes in the water bath, the test tube was then placed into a dry bath incubator at 100 degrees Celsius for 3 minutes which stops the reaction. The precipitates formed during heating are cleared by centrifugation at 13, 000 rpm for 3 min. The supernatant was then subjected to the analysis of [35S] sulfated product, by placing 2 dots for each test tube containing1µL on the POLYGRAM

CEL 300 TLC, and then putting the TLC’s into a 2:1:1:2 mixture containing Butyl

Alcohol (EMD), Isopropyl Alcohol (EMD), Formic acid (Aqua Solutions), and H2O, in appropriate order.

Afterwards, the TLC was saturated with the mixture until the mixture reached nearly the top of the TLC plate. It was then dried for 30 minutes and auto- radiographed by using an X-ray film. The film was then exposed for 2 days. The radioactive spot

19 corresponding to the sulfated product was located by using the X-ray film, cut out and eluted into a test tube containing 500uL of water and shaken for 30 min using an orbital shaker. 2ml of Ecolume scintillation liquid was added to each vial, mixed thoroughly and counted using a liquid scintillation counter machine. The results obtained were then calculated and expressed in nanomoles of sulfated product formed per min/mg of purified enzyme.

2.2.2 Analysis of the kinetic activity of the human SULTs toward opioid drugs.

To study the kinetic parameters of human SULT1A3 in mediating the sulfation of hydromorphone the following concentrations were used: 50 µM, 100 µM, 250 µM, 500

µM, 750 µM, 1000 µM. The reaction conditions were the same as those previously described in the sulfotransferase assay. Data obtained from kinetic experiments was analyzed based on Michaelis-Menten kinetics and Lineweaver-Burk double reciprocal plots were used to calculate kinetic constants of wild type and SULT1A3 allozymes in mediating the sulfation of tested substrate compounds. GraphPad Prism 7 software was used in data analysis. Data from scintillation studies was translated into graphs on Excel to show the specific activities of the allozymes with respect to the two drugs tested.

20 2.2.3 Analysis of the opioid drug -sulfating activity of the human SULT1A3 allozymes

Sets of SULT allozymes (SULT1A3-wt, T7P, S8P, R9C, P10L, V15M, V18F,

P101L, P101H, R144C, K234N, K235T, S209T) were prepared based on a previously established procedure as mentioned in section 2.1 (Bairam, 2018). Hydromorphone allozyme activity was tested using 50 µM, 500 µM, and 1000 µM. Pentazocine allozyme activity was tested using 500 µM, 1000 µM, and 2500 µM. Standard assay procedure was used as described in section 2.2.1. The data obtained from the liquid scintillation counter machine determined the amount of product formed per min/mg of purified enzyme. Once the specific activity was determined, the kinetic parameters of these allozymes was also able to be verified.

2.2.4 Statistical analysis

Data obtained from the kinetic experiments was analyzed based on the

Michaelis-Menten equation with nonlinear-regression and the Lineweaver-Burk double reciprocal plot to calculate the kinetic constants. GraphPad Prism 7 software was used for data analysis.

21 Chapter 3

Results and Discussion

3.1 Results

3.1.1 Differential sulfating activity of the human SULTs toward hydromorphone, oxymorphone, and pentazocine.

In a pilot experiment, wild-type SULT1A3 (SULT1A3-WT) was tested for sulfating activity toward hydromorphone, oxymorphone, and pentazocine at 1mM and

10mM substrate concentrations. As shown in Figure 3-1, SULT1A3-WT displayed activities toward all three opioid drugs tested as substrates. Among the three dugs, hydromorphone appeared to be the best substrate, followed by oxymorphone and pentazocine. Figure 3-1 shows the autoradiograph taken from the cellulose TLC plate used for the analysis of the sulfation reaction mixture. Hydromorphone had the darkest spot on the TLC, meaning it had the most sulfated metabolite present as compared to the other two drugs. The dark spots on the bottom of the TLC plates identify the origin where the mixture containing the controls and substrates was placed. The dark spots identified by the origin show unused PAPS that did not sulfate the substrates thus did not travel to the sulfated metabolite areas identified on the top of the TLC plates.

22 Figure 3-1: Analysis of the sulfating activities of the human SULTs toward hydromorphone, oxymorphone, and pentazocine. The figure shows the autoradiographs taken from the TLC plates used for the analysis of the reaction mixtures of SULT1A3- mediated sulfation of hydromorphone, oxymorphone, and pentazocine, respectively. Upper panel shows SULT1A3 mediated sulfation of hydromorphone and oxymorphone at 1 mM and 10 mM substrate concentrations, and lower panel shows SULT1A3-mediated sulfation of pentazocine at 10 mM substrate concentration. The solvent system used for the TLC separation was a 2:1:1:2 mixture of butyl alcohol, isopropyl alcohol, formic acid, and H2O.

23 3.1.2 Kinetic Analysis of the Sulfation of Hydromorphone by SULT1A3.

As hydromorphone produced the strongest visual evidence of sulfation, it was selected to investigate its kinetic parameters. Different concentrations of the drug (50

µM, 100 µM, 250 µM, 500 µM, 750 µM, 1000 µM) were tested in an enzymatic assay to determine the right substrate concentration to be used for the sulfating activity of human

SULT1A3 allozymes. Data obtained was then processed using GraphPadPrism 5 software program to generate the best fitting curves for the Michaelis-Menten equation with non-linear regression to calculate values of Km and Vmax for SULT1A3 in mediating the sulfation of the opioid. This data was fitted to hyperbolic kinetic curves (Michaelis-

Menten kinetics), and was also confirmed by the Lineweaver-Burk double-reciprocal plots. Results were compiled in Table 3.1, and the curves displayed in Figure3-2 and, 3-3. where the Lineweaver-Burk double-reciprocal plot has 1/[Hydromorphone] plotted against 1/v of the SULT1A3. Seven different concentrations show that 50 µM had the lowest but visible sulfating activity and 1000 µM had the highest sulfating activity, with

500 µM at the middle point of sulfating activity. Therefore, to test the sulfating activities of human SULTs allozymes with hydromorphone, three different concentrations (50µM,

500µM, 1000µM) of the substrate were used for the last step of the experiment. Higher concentrations were chosen for Pentazocine (500µM, 1000µM, and 2500µM) as it had a lower but still visible sulfating activity as seen in Figure 3.1.

24 Table 3.1: Kinetic Parameters of the wild-type SULT1A3 with hydromorphone as substrate. SULT Vmax Km (µM) Vmax / Km (nmol/min/mg)

SULT1A3-WT 29.83 (±1.452) 499.9 (±19.6) 0.061(±0.01)

The Km value of 499.9 was comparable to that 487.6 (±54.94) reported in previous studies (Kurogi, 2014). The Vmax was lower which could be due to different factors such as age of the enzyme used, enzyme concentration, lower drug molarity, etc.

SULT1A3 showed the lowest Km value of 499.9 µM compared to other reported values for different SULTs such as SULT1A1, SULT1A2, and SULT1C4 which had Km values of 670.9 (±1.0), 1146 (±59), and 826.7 (±23.3) respectively (Kurogi, 2014). In addition,

SULT1A3 dispalyed the highest catalytic efficiency (Vmax /Km) toward hydromorphone according to the study mentioned. This data confirms that SULT1A3 is involved in the sulfation of hydromorphone and is a solid candidiate for further studies. Beacause of its low Km value and high catalytics efficiency, SULT1A3 and hydromoprhone were good candidates for the allozyme analysis study.

25 Figure 3-2: The figure shows saturation curve analysis of sulfation of hydromorphone by human SULT1A3. The fitting curve was generated based on the Michaelis-Menten equation with non-linear regression using GraphPad Prism7 software. The velocity of the reaction is indicated as nmol/min/mg of the enzyme. Data shown represents the calculated mean ± S.D derived from the experiment.

Figure 3-3: Lineweaver-Burk double-reciprocal plot of human SULT1A3 using hydromorphone as substrate. The concentrations of the substrate were 50, 100, 250, 500, 750, and 1000 µM. The velocity of the reaction (v) is expressed as nmol/min/mg of enzyme. Each data point shown represents mean ± S.D of the mean derived from three measurements.

26 According to a previous study (Kurogi, 2014) the reported Km (µM) value for

Pentazocine was 1222 (±40) and 1782 (±76) for Oxymorphone. The experimental Km value for hydromorphone was around 499 µM. These values are consistent with the sulfation evidence produced on the X-ray films. Hydromorphone had the strongest presence of sulfated metabolites and displayed a high catalytic efficiency, while the other two drugs had a weaker presence of sulfated metabolites and also had a lower reported catalytic efficiency. This data is very useful in determining which SULTs are primarily responsible for the sulfation of a particular opioid and could have the strongest impact on the way a patient is able to metabolize a particular opioid drug.

3.1.3 Differential sulfating activities of human SULTs allozymes toward hydromorphone.

Purified SULT1A3 allozymes together with the wild type-enzyme were analyzed for their sulfating activity toward hydromorphone and pentazocine. In an initial study, three different concentrations (one well below reported Km, one close to Km, and one well above Km) of each of the two substrates (hydromorphone, pentazocine) were tested in the enzymatic assays. The activity data shown in Figures 3-4,3-5 is described below. It should be pointed out that considering the many steps involved in the sulfotransferase assay, and the subsequent TLC separation and scintillation counting, the data obtained should not be viewed as strictly quantitative, but rather semi-quantitative.

27 Hydromorphone 2 1.8 1.6 1.4 1.2 1 0.8 0.6 (nmol/min/mg) Specific Specific Activity 0.4 0.2 0

SULT1A3 allozymes

Figure 3-4: Specific activities of the sulfation of hydromorphone by human SULT1A3 allozymes. Concentrations of hydromorphone used in the enzymatic assays were 50 μM (blue), 500 μM (red) and 1000 μM (green). Specific activity refers to nmol hydromorphone sulfated/min/mg of purified allozyme. Data shown represent mean ± standard deviation derived from three determinations. WT refers to wild-type SULT1A3.

At low, mid, and high substrate concentrations (50 μM, 500 μM and 1000 μM respectively), similar patterns of hydromorphone sulfating activities were found for the

SULT1A3 allozymes analyzed. The allozymes increased in specific activity with increasing concentrations. Most of the allozymes had either similar or higher specific activity than the wild-type. SULT1A3 allozymes which appeared to have similar activity to that of the wild type include: V15M, R144C, R9C, P10L and S8P. The allozymes that displayed significantly higher activity vs the w.t. include: P101L and S290T (≈90-145% higher). The allozymes that displayed significantly lower activity as compared to the w.t. include: K234N and N235T (≈93-98% lower).

28 Table 3.2: Differential sulfating activities of the human SULTs allozymes toward Hydromorphone.

SUTL1A3 50 µM 500 µM 1000 µM 50 SD 500 SD 1000 SD

WT 0.323 0.582 0.765 WT 0.025 0.063 0.114

T7P 0.583 0.746 1.026 T7P 0.021 0.09 0.136

S8P 0.553 0.813 0.955 S8P 0.012 0.085 0.143

R9C 0.301 0.755 1.2 R9C 0.08 0.029 0.034

P10L 0.555 0.649 0.902 P10L 0.025 0.038 0.124

V15M 0.314 0.489 0.617 V15M 0.079 0.01 0.052

V18F 0.42 0.908 1.095 V18F 0.092 0.015 0.153

P101L 1.066 1.392 1.875 P101L 0.142 0.087 0.024

P101H 0.473 0.91 1.182 P101H 0.045 0.107 0.08

R144C 0.308 0.525 0.672 R144C 0.05 0.039 0.028

K234N 0.007 0.015 0.028 K234N 0.003 0.012 0.003

N235T 0.006 0.038 0.049 N235T 0.006 0.006 0.01 S290T 0.416 1.111 1.378 S209T 0.032 0.117 0.126

The concentrations of hydromorphone that were used in the enzymatic assay were 50, 500, and 1000 µM. Results show different specific activities toward hydromorphone for each concentration used. Specific activity refers to nmol hydromorphone sulfated/min/mg of purified allozyme.

29 Table 3.3: Similar, higher, and lower specific activities of SULT1A3 allozymes. Similar Activity: Specific Activity equal to or <30% difference vs Wild Type Higher Activity: Specific Activity > +30% difference vs Wild Type Lower Activity: Specific Activity > - 30% difference vs Wild Type 50 µM Percent 500 µM Percent 1000 µM Percent WT 0.323 100 0.582 100 0.765 100 T7P 0.583 180.4954 0.746 128.1787 1.026 134.1176 S8P 0.553 171.2074 0.813 139.6907 0.955 124.8366 R9C 0.301 93.18885 0.755 129.7251 1.2 156.8627 P10L 0.555 171.8266 0.649 111.512 0.902 117.9085 V15M 0.314 97.21362 0.489 84.02062 0.617 80.65359 V18F 0.42 130.031 0.908 156.0137 1.095 143.1373 P101L 1.066 330.031 1.392 239.1753 1.875 245.098 P101H 0.473 146.4396 0.91 156.3574 1.182 154.5098 R144C 0.308 95.35604 0.525 90.20619 0.672 87.84314 K234N 0.007 2.167183 0.015 2.57732 0.028 3.660131 N235T 0.006 1.857585 0.038 6.52921 0.049 6.405229 S209T 0.416 128.7926 1.111 190.8935 1.378 180.1307

At 50µM concentration Similar Activity Higher Activity Lower Activity V15M (-3) P101H (+46) N235T (-98) R144C (-5) S8P (+71) K234N (-98)

R9C (-7) P10L (+72)

S290T (+29) T7P (+80) V18F (+30) P101L (+230)

30 Table 3.3: Similar, higher, and lower specific activities of SULT1A3 allozymes (cont.). At 500µM concentration Similar Activity Higher Activity Lower Activity V15M (-16) S8P (+40) K234N (-97) R144C (-10) P101H (+56) N235T (-93)

P10L (+12) V18F (+56)

T7P (+28) S290T (+91) R9C (+30) P101L (+139)

At 1000µM concentration Similar Activity Higher Activity Lower Activity V15M (-19) T7P (+34) K234N (-96) R144C (-12) V18F (+43) N235T (-94) P10L (+17) P101H (+54) S8P (+25) R9C (+57) S290T (+91) P101L (+145)

The allozymes can vary in activity based on the concentration of the drug used.

For example, the T7P allozyme displayed a significantly higher activity at 50 µM concentration vs wild type enzyme (80% higher). However, once the concentration of hydromorphone was increased to 500 µM, the T7P allozyme had a decrease in activity compared to the wild type (28% higher). At an even higher concentration of 1000 µM the enzyme maintained a metabolism rate similar to that of the wild type (34% higher). On the other hand, allozymes such as S290T displayed significantly higher amounts of sulfated product vs the wild type with increasing concentrations, especially once the concentration was increased from 50µM to 500µM (90% higher). V18F allozyme displayed similar pattern to S290T, showing a significant jump in increase in specific

31 activity vs wild type once the concentration was increased from 50µM to 500µM (56% higher). K234N and N235T allozymes consistently displayed a much lower activity compared to wild type, by nearly a 100% lower specific activity.

3.1.4 Differential sulfating activities of human SULTs allozymes toward pentazocine.

0.45 Pentazocine

0.4

0.35

0.3

0.25

0.2 nmol/min/mg Specific Specific Activity 0.15

0.1

0.05

SULT1A3 allozymes Figure 3-5: Specific activities of the sulfation of pentazocine by human SULT1A3 allozymes. Concentrations of hydromorphone used in the enzymatic assays were 500 μM (blue), 1000 μM (red) and 2500 μM (green). Specific activity refers to nmol pentazocine sulfated/min/mg of purified allozyme. Data shown represent mean ± standard deviation derived from three determinations. WT refers to wild-type SULT1A3.

At low, mid, and high substrate concentrations, similar patterns of pentazocine- sulfating activities were found for the SULT1A3 allozymes analyzed. Among them,

SULT1A3-P101H was the only allozyme that had a similar but still significantly lower specific activity than the wild-type enzyme (30% lower). The rest of the SULT1A3 allozymes all displayed lower specific activities compared with the wild-type enzyme,

32 with SULT1A3-R9C, K234N, N235T, V15M, and S290T exhibiting the lowest specific activity (≈ 100-90% lower). S8P, P10L, P10L, and R144C displayed lower specific activity compared to the wild type (≈ 55-70% lower), however not as significant

SULT1A3-R9C, K234N and N235T.

Table 3.4: Differential sulfating activities of the human SULTs allozymes toward Pentazocine. 500 SD 1000 SD 2500 SD SUTL1A3 500 µM 1000µM 2500µM WT 0.006 0.042 0.021 WT 0.225 0.354 0.392 T7P 0.008 0.03 0.024 T7P 0.049 0.082 0.098 S8P 0.004 0.018 0.02 S8P 0.138 0.158 0.202 R9C 0.003 0.002 0.001 R9C 0.018 0.016 0.017 P10L 0.007 0.017 0.014 P10L 0.082 0.138 0.187 V15M 0.002 0.007 0.004 V15M 0.006 0.023 0.027 V18F 0.016 0.008 0.004 V18F 0.023 0.088 0.086 P101L 0.017 0.028 0.031 P101L 0.061 0.132 0.108 P101H 0.039 0.035 0.006 P101H 0.148 0.235 0.279 R144C 0.006 0.007 0.006 R144C 0.076 0.16 0.193 K234N 0.001 0.001 0 K234N 0.024 0.021 0.019 N235T 0.001 0.001 0.001 N235T 0.02 0.017 0.016 S209T 0.005 0.016 0.008 S290T 0.029 0.05 0.024 The concentrations of pentazocine that were used in the enzymatic assay were 500, 1000, and 2500 µM. Results show different sulfating activities toward pentazocine for the different concentrations. Highlighted is the SULT1A3 wild type and the P101H allozyme that had the closest metabolic activity level as compared to the other allozymes which produced a lower amount of pentazocine metabolite.

33 Table 3.5: Similar, higher, and lower specific activities of SULT1A3 allozymes. Similar Activity: Specific Activity equal to or < 40% difference vs Wild Type Lower Activity: Specific Activity > - 40%difference vs Wild Type

500 µM Percent 1000 µM Percent 2500 µM Percent WT 0.225 100 0.354 100 0.392 100 T7P 0.049 21.77778 0.082 23.16384 0.098 25 S8P 0.138 61.33333 0.158 44.63277 0.202 51.53061 R9C 0.018 8 0.016 4.519774 0.017 4.336735 P10L 0.082 36.44444 0.138 38.98305 0.187 47.70408 V15M 0.006 2.666667 0.023 6.497175 0.027 6.887755 V18F 0.023 10.22222 0.088 24.85876 0.086 21.93878 P101L 0.061 27.11111 0.132 37.28814 0.108 27.55102 P101H 0.148 65.77778 0.235 66.38418 0.279 71.17347 R144C 0.076 33.77778 0.16 45.19774 0.193 49.23469 K234N 0.024 10.66667 0.021 5.932203 0.019 4.846939 N235T 0.02 8.888889 0.017 4.80226 0.016 4.081633 S290T 0.029 12.88889 0.05 14.12429 0.024 6.122449

At 500 µM Similar Activity Lower Activity S8P (-39) V15M (-97) P101H (-34) R9C (-92) N235T (-91) V18F (-90) K234N (-89) S290T (-87) T7P (-78) P101L (-73) R144C (-66) P10L (-64)

34 Table 3.5: Similar, higher, and lower specific activities of SULT1A3 allozymes (cont.) At 1000 µM concentration Similar Activity Lower Activity P101H (-34) R9C (-95) N235T (-95) K234N (-94) V15M (-93) S290T (-86) T7P (-77) V18F (-75) P101L (-63) P10L (-61) S8P (-56) R144C (-55)

At 2500 µM concentration Similar Activity Lower Activity P101H (-29) R9C (-96) N235T (-96) K234N (-95) S290T (-94) V15M (-93) V18F (-78) T7P (-75) P101L (-72) P10L (-52) R144C (-51) S8P (-48)

As with the drug hydromorphone, certain allozymes displayed varying activity based on the concentration of pentazocine. For example, at lowest drug concentration

(500 µM) S8P displayed a specific activity level similar to that of the SULT1A3-wild type (39% lower). However, at 1000 and 2500 µM substrate concentrations, S8P had specific activity that was 56 and 48% lower vs w.t. While allozymes such as T7P, S8P,

P10L, P101H, and R144C displayed higher specific activity with increasing

35 concentrations other allozymes such as R9C, V15M, V18F, P101L, K234N, N235T, and

S290 reached their Vmax at either 1000 µM or 2500 µM indicating that they reach full saturation at these concentrations.

Discussion

The significance of single nucleotide polymorphisms (SNPs) is becoming increasing important in the field of medicine, particularly in pharmacogenomics. It has the potential to revolutionize the practice of medicine by individualizing treatment through the use of novel diagnostic tools. This new science has the potential to reduce the trial-and-error approach to the choice of treatment and thereby limit the exposure of patients to drugs that are not effective or are toxic for them. Single Nucleotide

Polymorphisms (SNPs) holds the key in defining the risk of an individual’s susceptibility to various illnesses and response to drugs. There is an ongoing process of identifying the common, biologically relevant SNPs, in particular those that are associated with the risk of disease (Alwi, 2005). The majority of SNPs are functionally silent meaning they occur in the non-coding or non-regulatory regions of the genome. Some SNPs can however lead to altered protein structure or expression. Such SNPs play a vital role in human diversity regarding health and disease. The changes in protein structure can have a significant impact on enzymes such as SULTs.

Deduced amino acid sequences of different SULTs provided useful information for not only the classification of the SULTs, but also the delineation of conserved sequences, particularly the so-called “signature sequences” that are involved in crucial step of the binding of PAPS, a co-substrate and sulfonate donor involved in the SULT- mediated sulfation reaction (Suiko, 2017). Three of the SULT1A3 allozymes examined,

36 SULT1A3-P101L, SULT1A3-P101H and SULT1A3-R144C, involve amino acid substitutions close to the catalytic residue (His108) and/or substrate binding residues

(residues Asp86 and Glu146) of the enzyme (Bairam, 2018). The amino acid substitutions at different locations can have various effects on the structure of the allozymes and be involved in substrate-binding and reshaping of the substrate pocket.

SULT1A3-V15M and SULT1A3-V18F involve the substitution of a valine residue with an S-methyl thioether side chain-containing or aromatic amino acid residue. This results in a decreased sulfating activity towards four analgesics from a previous study (APAP, morphine, tapentadol, and O-DMT). Both Methionine and Phenylalanine contain larger side chains than Val, which could result in more restricted conformations resulting in decreased sulfation. Thus, decreased sulfating activities of SULT1A3 with particular drugs could also be due to amino acid substitutions in the N-terminal region. Two of the allozymes tested, SULT1A3-K234 N and SULT1A3-N235T have amino acid substitutions which are close to the PAPS-binding site. These two allozymes exhibited lower sulfating activities with both hydromorphone and pentazocine, as well as three opioids (morphine, tapentadol, and O-DMT) analyzed in a previous study. The very low sulfating activity of SULT1A3-N235T might be due to the difficulty in accommodating the bulky side chain of the Threonine residue into the α-helical segment of the SULT1A3 molecule (Bairam, 2018).

Understanding SULT1A3 allozymes could prove very beneficial in opioid treatment with drugs such as hydromorphone and pentazocine, particularly in patients who have SNPs that code for an allozyme that significantly alters the SULT activity.

These two drugs are active on their own, unlike pro-drugs such as codeine, oxycodone,

37 hydrocodone, or tramadol which are dependent on CYP activation (Ruano, 2018). Since

SULTs are involved in phase II conjugation vs phase I, the main role of SULTs is in the elimination aspect of various substrates as opposed to phase I which can convert a pro- drug to an active metabolite. The metabolic activity levels of the wild type SULT1A3 enzyme and its corresponding allozymes is classified into four metabolic genotypes.

These include ultra-rapid metabolizer (UM), extensive metabolizer (EM), intermediate metabolizer (IM), and poor metabolizer (PM) phenotype (Blake 2013). Patients who have the genotype for poor SULT1A3 metabolism for these two drugs may accumulate toxic levels of the drug in their system and experience potential risk for addiction and serious adverse effects such as a depressed respiratory drive. On the other hand, patients who have the genotype for ultra-rapid metabolism (UM) might eliminate the opioid drugs at a very high rate and not allow the drugs to reach their therapeutic threshold.

The SULT1A3 allozymes had various specific activity levels compared to the wild type. Hydromorphone appeared to be a better substrate than pentazocine for

SULT1A3 allozymes. The allozyme specific activity levels were fairly evenly distributed between similar and higher activity levels. Compared to the wild-type, allozymes such as

R9C (CGC ⇒ TGC), V15M (GTG ⇒ ATG), R144C (CGT ⇒ TGT), P10L (CCG ⇒

CTG), and S8P (TCC ⇒ CCC) displayed similar activity levels, while enzymes such as

P101L (CCC ⇒ CTC) and S290 (AGC ⇒ ACC) displayed higher activity levels (≈90-

145% higher). On the other hand, the K234N (AAG ⇒AAT) and N235T (AAC ⇒ ACC) allozymes displayed a significantly lower activity level by nearly a 100% less.

It appeared that certain allozymes such as P101L and S290 displayed a significantly higher specific activity, especially at higher substrate concentrations

38 (500µM and 1000µM). Individuals with these alleles would likely display either ultra- rapid or extensive metabolizer phenotype. These individuals would be able to eliminate the drug fairly rapidly which could become a potential issue. If a drug is eliminated very rapidly, the patient might not be able to achieve adequate pain relief since the drug would quickly lose its efficacy.

Hydromorphone 2

1.5

0.5

0 W.T. P101L P101H S290T K234N N235T Wild Type/Allozymes Specific Specific (nmol/min/mg) Activity 50uM 500uM 1000uM

Figure 3-6: Differences in the capacity of sulfation of hydromorphone by SULT1A3 allozymes. Graph shows significant difference in specific activity between high specific activity allozymes P101L, P101H, and S290T vs low specific activity allozymes K234N and N235T.

The hydromorphone-sulfating activities of SULT1A3 allozymes P101L, P101H, and S290 were found to be significantly higher than that of the wild type. This means these allozymes may generate more sulfated drug product that would be more readily excreted via urine. Individuals that carry these alleles would metabolize/excrete hydromorphone at a faster rate, causing more drug to be excreted. On the other hand, individuals expressing of the K234N or N235T allozymes would fall under the poor metabolizer phenotype (PM). Such individuals would have hydromorphone in their system longer than individuals with an UM genotype and would have to be carefully

39 monitored to avoid the adverse effects/toxicities associated with the opioid drug hydromorphone. Individuals with genotypes for allozymes with similar sulfating activity to that of the wild type would display similar results to that of the wild-type enzyme.

While hydromorphone and pentazocine belong to the opioid category, it is clear that they differ dramatically in their metabolism via sulfation. SULT1A3 allozymes in general displayed hydromorphone-sulfating activity either similar to or higher than that of the wild-type enzyme. However, with pentazocine the only allozyme that showed similar, but somewhat lower sulfating activity was the P101H allozyme, whereas the rest of the allozymes all displayed significantly lower pentazocine-sulfating activity.

Individuals with genotypes coding for T7P, R9C, V15M, K234N, N235T, S290T) might be more susceptible to the adverse effects of pentazocine compared with individuals with wild type allele who would be able to metabolize and excrete pentazocine at a normal rate. Individuals with the genotype for above-mentioned allozymes would fall under a category of poor metabolizer (PM). Individuals who have the genotype for P101H allozymes would be able to metabolize and excrete pentazocine better than individuals who expresses allozymes such as K234N or N235T. Individuals who carry the P101H allele might fall under intermediate metabolizer (IM) category relative to the wild type.

Since pentazocine can produce respiratory depression just as the other opioids, it is important to know the SULT1A3 genotype of the patient (Goldstein, 1985).

In conclusion, genetic variations involving SNPs leading to l SULT1A3 allozymes may cause has a significant effect on the extent of hydromorphone and pentazocine metabolism. While enzyme systems such as the cytochrome P450 monooxygenase system (CYPs) involved in Phase 1 oxidation and UDP-

40 glucuronosyltransferases involved in Phase II glucuronidation are often thought to be the major players involved in opioid drug activation and metabolism, SULT allozymes should also be considered as playing a key role in phase II metabolism. Genetic polymorphisms among the SULTs as demonstrated by the data presented in this study may have a significant impact on the metabolism of opioids and possibly other groups of drugs. This knowledge can add to the field of pharmacogenomics whose goal is to develop rational methods to optimize drug therapy with respect to the patient's genotype, as well as to ensure maximum drug efficacy with minimal adverse effects in each individual (Zhang and Nebert, 2017). It is anticipated that a more extensive knowledge of the effect of genetic polymorphisms of SULT1A3 and other SULTs on opioids metabolism should greatly help improve personalized medicine and patient care.

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