Assessment of Genetic Deterministic Processes of Drug Metabolism

The Ministry of Science and Higher Education of the Russian Federation 
Kazan National Research Technological University 
S. Garmonov, L. Nugbienyo
ASSESSMENT OF GENETIC 
DETERMINISTIC PROCESSES 
OF DRUG METABOLISM 
Monograph 
Kazan 
KNRTU Press 
2022 
1 

UDC 615:577.1 
Published by the decision of the Editorial Review Board  
of the Kazan National Research Technological University 
Reviewers: 
PhD., Professor G. Budnikov 
PhD., Professor S. Egorova 
Garmonov S. 
Assessment of Genetic Deterministic Processes of Drug Metabolism : monograph / S. Garmonov, L. Nugbienyo; The Ministry of Science and Higher 
Education of the Russian Federation, Kazan National Research Technological University. – Kazan : KNRTU Press, 2022. – 120 p. 
ISBN 978-5-7882-3223-2 
This monograph contains review of the current state and prospects of 
development of analytical methods for the study of genetic deterministic processes of drug metabolism in the human body. It also discusses the role of 
analytical methods in determining the activity of enzyme systems that control 
the processes of biotransformation of xenobiotics and endogenous compounds in the body. The monograph also demonstrates the application of 
chemical and physico-chemical methods of analysis for direct and indirect 
determination of metabolic and exogenous markers of biotransformation. 
It is intended to serve as a teaching and learning material for postgraduate and PhD students of Russian universities, studying Pharmacy. 
Prepared at the Department of Analytical Chemistry, Certification and 
Quality Management. 
UDC 615:577.1 
ISBN 978-5-7882-3223-2 
© S. Garmonov, L. Nugbienyo, 2022 
© Kazan National Research Technological 
University, 2022 
2 

CONTENTS 
List of abbreviations 
...................................................................................... 4 
FOREWORD ................................................................................................ 5 
INTRODUCTION 
......................................................................................... 7 
Chapter 1. PHARMACOKINETICS AND METABOLISM 
OF DRUGS ................................................................................................. 12 
Chapter 2. OXIDATIVE METABOLISM AND METHODS OF 
DETERMINATION OF MICROSOMAL OXIDASE ACTIVITY ........... 24 
Chapter 3. PHASE II REACTIONS OF BIOTRANSFORMATION. 
ACETYLATION 
......................................................................................... 35 
Chapter 4. METABOLISM OF MESALAZINE AND ITS 
PRODRUGS ............................................................................................... 48 
Chapter 5. METABOLISM OF SULFONAMIDES 
................................... 55 
Chapter 6. PHARMACOKINETICS AND METABOLISM OF 
PROCAINAMIDE ...................................................................................... 67 
Chapter 7. QUANTITATIVE DETERMINATION OF DRUGS 
IN BIOLOGICAL FLUIDS ........................................................................ 75 
Chapter 8. RELATIONSHIP OF ACETYLATION RATE 
WITH VARIOUS DISEASES .................................................................... 82 
CONCLUSION ........................................................................................... 89 
References ................................................................................................... 90 
3 

List of abbreviations 
ААТ – arylamine acetyltransferase 
ASA – aminosalicylic acid 
ASDM – acetylated sulfadimezine 
CIA – cyclic injection analysis 
CNS – central nervous system 
DES – deep eutectic solvent 
DMSO – dimethyl sulfoxide 
DNA – deoxyribonucleic acid 
HPLC – high performance liquid chromatography 
INH – isonicotinic acid hydrazide 
LE – liquid extraction 
LOD – limit of detection 
MO – microsomal oxidase 
MOS – monooxygenase system 
NAPA – N-acetylprocainamide 
NAT – N-acetyltransferase 
NSAID – non-steroidal anti-inflamatory drugs 
PA – procainamide 
PCR – polymerase chain reaction 
RNA – ribonucleic acid 
SDM – sulfadimezine 
SDS – sodium dodecyl sulfate 
SLE – systemic lupus erythematosus 
XO – xanthine oxidase 
4 

FOREWORD 
The number of medicines being used currently is in hundreds of thousands. 
In connection with such a significant expansion and dynamism of drug therapy, 
the problem of safe and effective use of medicines is increasingly attracting global attention.  
The safety of medicines is determined by their quality on the pharmaceutical market. An equally important aspect of their safety, as well as effectiveness is the metabolic 
processes they undergo in the body. Besides the recovery of the normal course and rhythm 
of body functions, a number of qualitative and quantitative changes occur with medicinal 
substances. Herewith, the biological activity of drugs may decrease or completely be lost, 
and in some cases, on the contrary, increase, leading to undesirable toxic effects. In this 
regard, keen interest of chemists, physicians and pharmacists in the problem of drug 
safety is due to the fact that, this area, which occupies a multidisciplinary position, covering a number of scientific disciplines, enables new scientific foundations of the most 
rational, effective and safe applications of medicinal substances to the human body. 
Currently, the concept of personalization of the use of drugs extends to those that 
were once considered universal for treating specific diseases. It is becoming clear, that 
patients with the same disease react differently to the same drug, depending on the pharmacokinetic characteristics, as well as the metabolism of the drug or its metabolites, 
which mostly,  are under the biochemical control of a person’s genetic predisposition. 
The genes encode proteins, some of which are enzymes and receptors, that are involved 
in the processes of drug metabolism. Indeed, significant variations are possible in the effectiveness and safety of a drug used to treat a particular disease in different patients. 
Personalized medicine is a set of approaches for prevention, diagnosis and treatment of diseases, based on evaluation of the individual characteristics of the patient. 
Among them are pharmacogenetic characteristics of drug metabolism, which lead to 
differences in pharmacokinetic parameters; and thus, require optimization of drug dosing, depending on the geno- and phenotypes of a particular patient. The implementation 
of this approach plays an important role in ensuring effectiveness and safety of drugs, 
as well as dealing with individual drug tolerance. The technologies and approaches 
applied in personalized medicine include: 
•
biopharmaceutical analysis;
•
establishment of genetic polymorphism of drug-metabolizing enzymes by
molecular genetic methods, as well as with the help of cytogenetic and molecular-cytogenetic technologies;
5 

•
therapeutic drug monitoring based on foundations of pharmacogenomics,
pharmacogenetics and pharmacoproteomics.
Methods of biopharmaceutical analysis, aimed at detecting genetic deterministic 
processes of biotransformation, are able to show biochemical phenotype. Indeed, 
the importance of biopharmaceutical analytical methods is steadily rising in the diagnosis of hereditary disorders, assessment of risk of toxicity and carcinogenesis. The reason 
for this is the need to supplement molecular genetic methods, in most cases, with methods of biopharmaceutical analysis, since the former describe genotype, while the latter 
determine phenotype. In turn, the characteristics of the course of disease and the toxic 
effects of drugs are ultimately consequences of the phenotype. It should be noted that 
gene action is a complex process; so the fact of not detecting a mutation by a molecular 
genetic method is not always a complete guarantee of a normal phenotype.  
This monograph discusses methods of evaluation of the activity of enzymes in 
human metabolic systems, with the aim of determining genetic characteristics at 
the level of detection of products of gene expression, using methods of biopharmaceutical analysis, detection of specific components of biochemical processes (endogenous 
and medicinal compounds, as well as their metabolites), arising as a result of genetic 
determination. The monograph also discusses the application of medicinal substances 
as biopharmaceutical markers for establishing biochemical phenotypes of metabolism. 
Indeed, in order to establish the foundation for the application of personalized 
medicine within the framework of traditional medicine, it is necessary to develop new 
technologies, including biopharmaceutical analysis, carry out educational activities 
among health workers and sensitize the public about its capabilities. An integral part of 
drug therapy in the future is a deep understanding of the principles and genetic basis of 
the functionalities of drugs in the body. 
This book was composed by a team of authors, working with problems inspired 
by the needs of personalized medicine. The area of their professional interests is in 
the field of pharmaceutical and analytical chemistry, medical diagnostics and drug development. The team of authors include: Prof. S. Yu. Garmonov, PhD. Chemical Sciences (Kazan National Research Technological University, Russia) and L. Nugbienyo, 
PhD. Chemical Sciences (Accra Technical University, Ghana).  
6 

 
INTRODUCTION 
Biopharmaceutical analysis plays the role of ensuring the safety and 
effectiveness of drug administration, which is an important part of personalized medicine. The use of methods of biopharmaceutical analysis enables 
the establishment of individual characteristics of metabolism and pharmacokinetics of drugs for specific patients, as well as the optimization of drug 
dose regimen for various diseases. 
Biopharmaceutical analysis can be said to be a type of pharmaceutical 
analysis, involving the extraction, preconcentration, and determination of 
drug substances and their metabolites in biological materials, including biological fluids (urine, saliva, blood, plasma or serum, cerebrospinal fluid), and 
tissues of internal organs, as well as the study of properties of human genetic 
deterministic processes of drug metabolism (see Table) [1].  
 
Enzymatic Systems of Drug Metabolism 
Enzymatic System 
Drug Substances 
Metabolic Process 
Acetylation 
N-acetyltransferase 
Amines, amides, indoles, 
hydrazines, hydrazides 
Catalase 
Peroxides 
Disintegration of 
peroxides 
Glucuronide formation 
Glucuronosyltransferase 
Antibiotics, barbiturates, 
opiates, sulfanilamides 
Arylesterase 
Esters 
Hydrolysis of esters 
Liver monooxygenase system Various drugs 
Microsomal oxidation 
 
Biopharmaceutical analysis is a tool for biopharmaceutical studies of 
drugs, as well as for the development of individualized treatment for patients. 
In fact, the development of methods for drug analysis in biological samples 
plays an important role in evaluating bioequivalence and, in general, ensuring the safety, effectiveness and personalization of drug administration. 
The major objects of biopharmaceutical analysis are biological fluids, 
having very complex composition. These are multicomponent mixtures, containing a very large number of inorganic and, especially, organic compounds, 
having various chemical properties: trace elements, carbohydrates, polypeptides, lipids, nucleic acids, etc. Their concentration ranges from a few nanograms to 10 mg/mL. Thus, several hundreds of organic compounds have been 
7 

 
identified in urine; blood plasma contains more than 200 different proteins. 
Indeed, a biological object is a very dynamic system. Its condition and chemical composition depend on individual characteristics of a person, as well as 
the influence of environmental factors (food composition, physical and psychological stress, etc.). This complicates the execution of biopharmaceutical 
analysis to determine low concentrations of drugs against the background of 
such a large number of organic substances of complex chemical structure. 
The process of biotransformation of drugs in biological fluids results in 
the formation of metabolites, the number of which can be in the range of 
several dozens. The extraction of these substances from complex mixtures, 
their preconcentration and the establishment of their chemical composition 
remain a difficult task. 
Biopharmaceutical analysis enables the attainment of information on 
drug activity in the body, depending on physicochemical properties, such as 
degree of dispersion of drug components (including excipients) and the manufacturing technologies used in drug production processes. Quantitative assessment and the knowledge of processes and mechanisms of biotransformation enable the personalization of drug dosages, assessment of risk of possible undesirable side effects, as well as  the administration of the right combination of drugs. The study of mechanisms of biotransformation of drugs in 
the human body is mainly approached through the establishment of the structure of metabolites, as well as the calculation and evaluation of pharmacokinetic parameters. 
In personalized medicine, biopharmaceutical analysis is widely applied 
in diagnosis of genetic deterministic characteristics of patients, using test 
markers, followed by prescription of individual therapy, corresponding to 
the molecular profile of patients [2]. In this regard, personalized medicine 
has prospects of improving quality of medical care and, in some cases, reducing health care costs in general [3]. Knowledge of the molecular basis of 
diseases enables the identification of biotargets, responsible for biotransformation of compounds, and thus, improve the prescription of drugs, which 
fundamentally changes pharmaceutical practice [4]. Currently, the development of individualized approaches to drug therapy represents an important 
part of personalized medicine, which is being applied increasingly in many 
areas of clinical practice, as genes associated with certain diseases are found. 
In some areas of medicine, prognosis of risks for patients is systemically and 
directly applied during the treatment process, while in other areas, more 
8 

 
research is required for the clarification of the molecular basis of diseases for 
individual treatment [5]. 
The effectiveness of medical treatment for most human diseases is confirmed by evidence-based medicine. Therapeutic strategy, including medication, is also determined by  principles and clinical standards, developed on 
the basis of evidence-based medicine for the treatment of diseases. Personalized approaches to therapy provide partial solutions to the problem of undesirable adverse drug reactions. Indeed, the safety of drug therapy depends on 
the individual characteristics of a person; thus, the use of drug therapy requires 
a personalized approach to each individual patient. This means personalized 
medicine can improve the safety of drug therapy, as well as reduce the cost of 
correcting adverse reactions. In addition to the development of molecular genetic approaches in personalized medicine, there are studies of biomarkers in 
biological fluids (usually, specific proteins), which enables prognosis of the 
development of certain diseases. Moreover, such areas as the study of gene 
activity based on the study of mRNA and processes of drug metabolism have 
emerged as modern tools of personalized medicine [6]. Genetic characteristics 
of metabolic processes, age, sex, presence of ailment and other environmental 
factors significantly affect the pharmacodynamics and pharmacokinetics, as 
well as the therapeutic effect of drugs. In this regard, the study of these factors 
is a prerequisite for the personalization of therapy [7]. 
Through metabolism, changes to pharmacological activity of  drugs occur as follows: 
• a pharmacologically active substance is converted into an inactive 
form;  
• a pharmacologically active substance is converted into another active substance, i. e. an active metabolite is formed; 
• pharmacologically inactive substances, so-called prodrugs, are converted into active forms.  
Drug metabolism reactions are divided into two phases — I and II. 
Phase I reactions are non-synthetic reactions. By this, hydrophilic compounds are formed from drugs through the addition or removal of active functional groups. Phase I reactions are mainly oxidation reactions, of which  hydroxylation reaction is most common. The catalysts for these reactions are 
oxidases, the substrate specificity of which is very low. Consequently, oxidases are involved in oxidation of drugs of various chemical structures. Reduction and hydrolysis reactions are less common. Phase II metabolic reactions involve the conjugation of drugs or their metabolites with endogenous 
9 

 
substances to form water-soluble polar conjugates, which are easily excreted 
by the kidneys or with bile. The most common phase II reactions include 
acetylation, sulfation, glucuronidation, methylation, and aqueous conjugation. Xenobiotics, as a result of phase II reactions, may lose biological activity or form  active metabolites [8]. Drug metabolism can occur by phase I or 
II reactions, by reactions of both phases simultaneously (one part of the drug 
in the first, the other in the second),  or by reactions of both phases sequentially [9]. 
Acetylation reaction, catalyzed by N-acetyltransferase of hepatocytes, 
is one of the important pathways of biotransformation of drugs. The rate of 
the process of acetylation is genetically determined, and  polymorphism of 
the genes of the enzyme, N-acetyltransferase, results in metabolic polymorphism with bimodal distribution (slow and rapid acetylation phenotypes). 
As a phenotypic marker, determination of acetylation phenotypes serves as 
a tool for evaluation of a person’s predisposition to disease, as well as for 
prognosis of side effects of some drugs [7]. 
Problem solving in personalized medicine requires the use of technologies and approaches of biopharmaceutical analysis [10] and molecular genetic 
methods, that are applied in pharmacogenetics [11], pharmacogenomics [12] 
and pharmacoproteomics [13]. Biopharmaceutical analysis methods enable 
the detection of genetic deterministic processes of biotransformation, and indicate biochemical phenotypes [14]. Human biological fluids, as the main object of biopharmaceutical analysis, are complex multicomponent matrices [15–17], which complicates the implementation of biopharmaceutical 
analysis in practice. The expression of genes, that are responsible for the synthesis of metabolic enzymes affects pharmacokinetics and pharmacodynamics 
of drug substances [18–20]. Furthermore, drug metabolism processes can 
cause side effects for patients with high enzyme activity in the functionalization stage and low enzyme activity in the conjugation stage [21]. 
Molecular genetic methods enable the determination of variations in 
the primary structure of deoxyribonucleic acid (DNA) fragments by identifying their nucleobase sequences. In this regard, molecular genetic analyses 
include nucleic acid (DNA or RNA) sample preparation, restriction of nucleic acid into fragments and their subsequent identification by various physicochemical methods [22]. The determination of metabolic phenotype varies 
from the level of gene expression to the final metabolites of a drug. Accordingly, the use of methods of biopharmaceutical analyses to complement molecular genetic methods is essential, as the latter determines genotype while 
10