In-silico physiological based pharmacokinetic modeling of prodrugs

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Content Copyright [2021] Sanjana Sanjay Parikh
In-silico Physiological Based Pharmacokinetic Modeling of Prodrugs

by

Sanjana Sanjay Parikh

A Thesis Presented to the
FACULTY OF THE USC SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
PHARMACEUTICAL SCIENCES

August 2021

ii

Acknowledgements

I am so grateful to my advisor and supervisor Dr. Rebecca Miranda Romero for her constant
support and guidance for the completion of my thesis. With her knowledge, expertise, and
constant learning, she has guided me through this research project.
I am extremely thankful to The University of Southern California, School of Pharmacy for
giving me this wonderful opportunity to work under such renowned faculties.
I would also like to thank all the faculty members that have guided me directly or indirectly
throughout my journey.
Last, by not the least, I would like to acknowledge my parents and family for constantly
supporting me emotionally even from afar.
Sincere gratitude to all my friends who always stayed by my side.

iii

Table of Contents
Acknowledgements ......................................................................................................................... ii
List of Tables .................................................................................................................................. v
List of Figures ................................................................................................................................ vi
Abstract ........................................................................................................................................ viii
CHAPTER 1 : INTRODUCTION TO PRODRUGS ..................................................................... 1
WHAT ARE PRODRUGS? ................................................................................................................... 1
CLASSIFICATION OF PRODRUGS ........................................................................................................ 3
DRAWBACKS OF THE PHARMACEUTICAL ACTIVE FORM .................................................................. 4
Aspect of prodrug formation ........................................................................................................... 5
Enzymes for prodrug conversion .................................................................................................... 6
PROCESS OF ACYLATED PRODRUG CONVERSION............................................................................... 7
CHAPTER 2 : ROLE OF METABOLIZING ENZYMES............................................................. 9
INTRODUCTION ................................................................................................................................ 9
ESTERASES ..................................................................................................................................... 10
CARBOXYLESTERASE ..................................................................................................................... 11
The Structure of Carboxylesterase (CES) ..................................................................................... 11
Classification of CES .................................................................................................................... 12
Function of Carboxylesterase (CES) ............................................................................................ 13
CHAPTER 3 : BASICS OF PHARMACOKINETICS ................................................................ 15
INTRODUCTION .............................................................................................................................. 15
MODELS: COMPARTMENT AND PBPK ............................................................................................ 15
ADME ........................................................................................................................................... 18
Bioavailability ............................................................................................................................... 18
Absorption..................................................................................................................................... 19
Metabolism ................................................................................................................................... 23
Elimination .................................................................................................................................... 25
PHARMACOKINETIC PARAMETERS .................................................................................................. 26
Clearance....................................................................................................................................... 26
Volume of distribution .................................................................................................................. 27
Half-life ......................................................................................................................................... 28
PHARMACOKINETIC OUTCOME ...................................................................................................... 29
CHAPTER 4 : PHYSIOLOGICAL BASED PHARMACOKINETIC MODELING- NOVEL
AND THRIVING AREA OF PHARMACOKINETICS. ............................................................. 31
GASTROPLUS™- A TOOL FOR PBPK MODELING ........................................................................... 33
EVALUATION OF DRUG MOLECULES ............................................................................................... 34
BUILDING OF A PBPK MODEL USING A PRODRUG .......................................................................... 35
CHAPTER 5 : OSELTAMIVIR CARBOXYLATE AND ITS PRODRUG: OSELTAMIVIR
PHOSPHATE ............................................................................................................................... 38
iv

GENERAL INFORMATION ................................................................................................................ 38
A BRIEF OVERVIEW ON OSELTAMIVIR ADME ............................................................................... 40
CHAPTER 6 : OSELTAMIVIR MODEL .................................................................................... 42
BACKGROUND ................................................................................................................................ 42
EXPERIMENTAL DATA .................................................................................................................... 43
PBPK MODELING OF OSELTAMIVIR PRODRUG ............................................................................... 45
Physiological parameters .............................................................................................................. 50
Pharmacokinetic parameters ......................................................................................................... 50
RESULTS ........................................................................................................................................ 53
One compartment model ............................................................................................................... 54
PBPK Model ................................................................................................................................. 55
CHAPTER 7 : CURCUMIN ......................................................................................................... 57
STEPS USED FOR THE SIMULATION: ................................................................................................ 59
PREDICTED GASTROPLUS™ DATA. ................................................................................................ 60
IN VITRO OBSERVED DATA: LOGP AND SOLUBILITY ....................................................................... 61
SIMULATION PARAMETERS ............................................................................................................. 62
RESULTS ........................................................................................................................................ 67
CHAPTER 8 : BIOEQUIVALENCE ........................................................................................... 69
CONCLUSION ............................................................................................................................. 73
REFERENCES ............................................................................................................................ 74

v

List of Tables
Table1.1: Different routes of drug administration. Each row describes the following: absorption
mechanism, advantages, onset and duration of action, and examples of the 3 different types of dosage
forms. .......................................................................................................................................................... 20

Table 6.1: Parameters of OP and OC considered for the one compartment and the PBPK model. Observed
data are experimental data from literature while the predicted values are default GastroPlus™ and
ADMET Predictor® data. ........................................................................................................................... 51
Table 6.2 Physicochemical and Pharmacokinetic data for the OP-OC simulation. .................................... 52
Table 6.3: Below are the factors responsible for active parent metabolite (OC) formation from
Oseltamivir Phosphate. ............................................................................................................................... 52

Table 7.1: Representation of some of the constant physiological, pharmacokinetic, and physicochemical
parameters of curcumin, CDD and CDG, either predicted or observed data of prodrug release model. .... 61
Table 7.2: Simulation data obtained from the physicochemical and pharmacokinetic profile of curcumin.
For the simulation, both observed and predicted values were considered .................................................. 63
Table 7.3: Simulation data obtained from the physicochemical and pharmacokinetic profile of curcumin
released from CDD. For the simulation, both observed and predicted values were considered ................. 64
Table 7.4: Simulation data obtained from the physicochemical and pharmacokinetic profile of curcumin
released form CDG. For the simulation, both observed and predicted values were considered ................. 65
Table 7.5: Represents the enzymes used for the simulation of curcumin, CDD and CDG plasma
concentration-time curves………………………………………………………………………………....66

Table 8.1:Bioequivalence study to determine statistical significance......................................................... 71

vi

List of Figures
Figure 1.1: Cycle of prodrug circulation, conversion to the parent active metabolite and lastly elimination
from the body. ............................................................................................................................................... 7

Figure 3.1: Representation of a one compartment model, where Ka is the rate of absorption into the
compartment and Ke is the rate of elimination from the compartment (adapted from Ref.21). ................. 16
Figure 3.2: Representation of a two-compartmental model, where K 12 represents the rate of drug
movement from central to the peripheral compartment, K 21 represents the rate of drug movement from the
peripheral to the central compartment and K e is the rate of elimination (adapted from Ref.21). ................ 16
Figure 3.3 Representation of a PBPK Model (adapted from Ref.21). ........................................................ 17
Figure 3.4: Plasma Concentration-time curve representing the maximum concentration, time at which
Cmax is achieved, AUC of the respective drug. ......................................................................................... 29

Figure 4.1: The large arrows display the circulation of the prodrug till its conversion to the active
metabolite. The thin smaller arrows show the circulation of the active form of the drug to the tissues.
arrow indicates the connection between stomach, spleen and pancreatic blood vessels.(adapted from Ref.
33). .............................................................................................................................................................. 37

Figure 5.1: A visual representation of oseltamivir phosphate (ethyl ester group) structure and its
metabolism by esterase enzyme to oseltamivir carboxylate (carboxylic acid group) (adapted from Ref.
40). .............................................................................................................................................................. 40

Figure 6.1: Experimental curve representing the plasma concentration-time curve of OP and OC on
administering 150 mg OP orally. The data in the curve is obtained from a normal human volunteer of
70kg weight (adapted from Ref.40). ........................................................................................................... 43
vii

Figure 6.2: The seven tabs are the representation of simulation in GastroPlus™ of the plasma
concentration-time curve of oseltamivir phosphate OP on oral administration. ......................................... 49
Figure 6.3: One compartment oseltamivir phosphate Concentration(ng/ml) vs Time(hr.) curve of the
experimental and simulated data. ................................................................................................................ 54
Figure 6.4: One compartment oseltamivir carboxylate Concentration(ng/ml) vs Time(hr.) curve of the
experimental and simulated data. ................................................................................................................ 54
Figure 6.5: PBPK model of oseltamivir phosphate Concentration(ng/ml) vs Time(hr.) curve of the
experimental and simulated data. ................................................................................................................ 55
Figure 6.6: PBPK model of oseltamivir carboxylate Concentration(ng/ml) vs Time(hr.) curve of the
experimental and simulated data. ................................................................................................................ 55
Figure 6.7: Representation of the process of OP absorption, circulation, metabolism to OC, and then OC
circulation to the site of action and ultimately elimination from the body (adapted from Ref.33,34). ....... 56

Figure 7.1: The structures are as follows: a. Curcumin, b. Curcumin diethyl disuccinate (CDD), ............ 58
Figure 7.2: Model of CDD conversion to curcumin, metabolism by various enzymes, distribution and
circulation in the body, and elimination and excretion (adapted from Ref. 51,52). The enzymes
responsible for the prodrug conversion and drug metabolism for curcumin, CDD and CDG is mentioned
in the appendix below. ................................................................................................................................ 59
Figure 7.3: Representation of the plasma concentration-time profile of simulated curcumin vs
experimental curcumin in Wistar rats. The concentration is measured as ng/ml and time as hours. .......... 67
Figure 7.4: Representation of the plasma concentration-time profile of simulated curcumin release from
CDD vs experimental curcumin release from CDD in Wistar rats. The concentration is measured as ng/ml
and time as hours. ....................................................................................................................................... 68
Figure 7.5: Representation of the plasma concentration-time profile of simulated curcumin release from
CDG vs experimental curcumin release from CDD in Wistar rats. The concentration is measured as ng/ml
and time as hours. ....................................................................................................................................... 68
viii

Abstract
The technology known as Physiological Based Pharmacokinetic (PBPK) modeling is an in-silico
tool used by researchers and scientists to derive the pharmacokinetic (PK) concentration-time
curve based on a drug molecule’s physicochemical properties, as well as on the species
physiological characteristics. In vitro to in vivo extrapolation (IVIVE) approaches along with in-
silico predictions on drug molecules help to parameterize the PBPK model. PBPK modeling
allows researchers to study the effect of changes in species physiology or a drug's physical
chemical properties in-silico and prior to dosing patient populations. This makes PBPK modeling
a cost-effective and time-saving predictive tool for bioavailability and pharmacokinetic studies.
Prodrugs are designed to overcome biopharmaceutical and pharmacokinetic drawbacks like poor
solubility or permeability which causes reduced oral bioavailability. Developing a PBPK model
for prodrugs is challenging because prodrugs are initially pharmacologically inactive, which on
systemic absorption and metabolism convert to the pharmacologically active molecule which is
difficult to predict with PBPK modeling tools. The prodrug PBPK model is based on both the
initial structural properties of an inactive prodrug form and on the active parent metabolite. In our
experimental model, we have considered two prodrugs: Oseltamivir and Curcumin prodrug
analogs. An In-silico PBPK model of the two prodrugs using their physicochemical and
physiological properties and comparing the results to experimental pharmacokinetic
characteristics of both the active and inactive parent molecule is the focus of our current research.
1
Chapter 1 : Introduction to Prodrugs
What are Prodrugs?
A prodrug, as the name suggests, is a form of drug delivery that enhances the drug
pharmacokinetic properties of an inactive moiety that is attached to the drug. A prodrug is said to
be an inactive form of the active metabolite, obtained naturally or formed through synthetic or
semisynthetic processes. The discovery of prodrugs was an accidental invention by scientists.
Prodrug formation is through a rational drug design process. The scientific idea of a prodrug was
developed to improve drug discovery and development. The oral form of a drug can often
experience issues with solubility, absorption and metabolism that lead to poor bioavailability.
Alternative formulations to overcome poor oral bioavailability related to low solubility, poor
absorption, or high first pass metabolism include parenteral such as intravenous infusion, IV
bolus, and intramuscular injections. These forms of drug administration can cause some
psychological drawbacks caused by fear of needles and pain at the injection site, increased cost,
as well as problems like risk of infection. The prodrug approach can overcome some of these
limitations in drug development. The role of a prodrug is to enhance the pharmacokinetic,
physicochemical, and pharmacological effect of a drug. As a prodrug is in an inactive form it
requires an external force for its conversion to the active form. Enzymatic or chemical activity
like pH, temperature, esterase, and CYPs enzymes in the body are some of the factors
responsible for conversion of prodrugs
1
.
The prodrug is formed by the process of a chemical modification, usually by attachment of a pro-
moiety to the parent drug. Examples include the addition of an ester functional group to a drug
containing a carboxylic acid forming an ethyl ester form. The inactive prodrug molecule formed
has no pharmacologic effect or activity of its own but acts as a non-pharmacological molecule.
2

Some prodrugs can have their own therapeutic activity, but the main substance responsible for the
pharmacological effect is the active molecule. The functional groups such as carboxylic acids,
amines, phosphates, carbonyls, and hydroxyls can be converted to the prodrug molecule on
attachment of these moieties. This process can optimize the pharmacokinetic properties of the
initial or parent drug molecules
2,3
.
The prodrug should be converted into the active product sufficiently and in an adequate time frame
to exert a therapeutic effect. An active molecule mainly responsible for the pharmacological effect
might face some pharmaceutical issues like solubility, lipophilicity, or stability, which can lead to
reduced bioavailability, and tissue distribution
4
. Metabolic degradation due to enzymatic action as
well as pharmacodynamic issues of toxicity can lead to formation of metabolites which also must
be considered prodrugs. Prodrugs like dabigatran etexilate, omeprazole, oseltamivir phosphate,
ramipril, and valganciclovir are designed for better absorption, distribution, clearance, solubility,
stability, and bioavailability, showing that the prodrug approach is an effective tool that reduces
the need to search for a whole new active moiety
5
.

3

Classification of prodrugs
Prodrugs are one of the pharmaceutical tools used in drug discovery to overcome some of the
biopharmaceutical disadvantages of the active drug molecule. Prodrugs can be classified in many
ways based on:
1. Type functional moiety
2. Therapeutic activity
3. Chemical changes
4. Site of conversion.
Upon oral administration, the prodrug distributes throughout the body and is exposed to various
tissue organs and metabolic sites. The prodrugs can be metabolized by metabolic enzymes present
at various sites in the body. Different prodrugs get converted by specific enzymes present in
different parts of the body. The type of functional group and moiety determines which enzymes
are responsible for its conversion, for example, phosphate prodrugs get metabolized by
phosphatases. The carrier-linked functional groups are esters, amides, oximes, carbamates, and
carbonates. Some prodrugs were developed to overcome the drawback of low solubility, too high
lipophilicity, or to enhance the drugs therapeutic efficacy and reduce toxicity. Prodrugs can be of
any therapeutic class, designed to treat or prevent various diseases such as hypertension, hormonal
malfunctions, or infections. The fourth classification is the newest and is based on the prodrug
conversion site i.e., either intracellular or extracellular. This classification is again divided into
Type IA, IB, and Type IA, IB, and IC. Type I is intracellular and mainly occurs within cellular
target cells and/or metabolic organs like the liver and intestines, while Type II is extracellular and
4

is converted in the gastrointestinal fluids and/or in the systemic circulation where therapeutic
targeting agents are activated (ADEPT, GDEPT).The active parent metabolite either undergoes a
second drug metabolism forming a daughter metabolite or exerts its therapeutic effect on the target
cells
2,6
.
Drawbacks of the Pharmaceutical Active Form\
According to the Biopharmaceutical classification system, or BCS classification, class I drugs have
high solubility and high permeability and are generally approved with fewer investigations on their
properties. Class II, III, and IV have biopharmaceutical limitations in which the active molecules
might benefit from being administered in a prodrug form. Prodrugs act at the molecular level and
are designed to overcome the limitations of an active molecule, but not the manufacturing
limitations of an oral dosage formulation, such as chipping, cracking, or bitterness. The
pharmaceutical limitations involve:
1. Low solubility causing problems in dissolution and affecting the bioavailability of drugs in the
plasma, ultimately showing less therapeutic efficacy.
2. Reduced lipophilicity of the pharmacologically active drug molecule, which increases the
difficulty of diffusion through cellular membranes of organs.
3. Increased Toxicity, which can escalate due to accumulation of the drug in the body
5,7

5

Aspect of prodrug formation
All 3 issues can be solved by designing a prodrug form of the active drug molecule. The result of
converting the polar functional groups like -OH -SH -NH -COOH containing drug molecules to
carbamates, carbonates, amides, esters, ethers, and phosphate forms overcomes all the
pharmaceutical and pharmacokinetic issues. This results in a better oral bioavailable drug form.
The promoiety attached, in a carrier mediated prodrug form of the parent molecule, enhances the
prodrug formation. The attached promoiety should be clinically safe, non-toxic, biocompatible,
and bio-convertible in nature. Sometimes, the increment in bioavailability of a drug is not as
expected in its prodrug form. In such cases, a pro-prodrug concept is introduced where the enzyme
activity along with chemical transformation, forms the pharmacological active moiety.
Important physicochemical parameters which regulate high or low solubility and lipophilicity are
based on the drug’s nature and include the partition coefficient and pKa. Converting a polar moiety
to a more non-polar carbon form can increase the logP of that molecule, transforming it to a
lipophilic molecule. Such a change can led to greater bioavailability because the drug can more
easily diffuse through the lipid membrane. An example is Tamiflu (Oseltamivir phosphate), an
ethyl ester prodrug of Oseltamivir carboxylate with increased lipophilicity (LogP) and better
bioavailability in the body
5
.
Solubility is one another important factor to consider while developing a drug molecule or dosage
form for a therapeutic effect. Solubility affects the dissolution of the molecule in the GI tract.
Esterification can improve the drugs aqueous solubility and metabolic stability, rendering it more
absorbable and more effective to the body. Conversion of prodrugs at the specific tissue of interest
leads to better pharmacological activity and reduced toxicity in other sites/organs
5
.
6

Enzymes for prodrug conversion
As we know, larger drug molecules with many carbons tend to be nonpolar in nature and difficult
to dissolve while smaller molecules with less carbons, tend to be more polar in nature and have
more difficulty penetrating through the lipophilic tissue membrane. So, to overcome such
drawbacks, prodrugs of interest are formed with a careful balance of good solubility and
dissolution, as well as the ability to penetrate membranes. It is helpful if the prodrug can be
converted in the liver by the first pass metabolism, which converts the prodrug into an active
therapeutic molecule, allowing biologic activity in the plasma. Most of the bioconversion to an
active metabolite occurs due to hydrolytic action of enzymes present in the liver, the systemic
circulation or at the local sites of action.
In pharmaceutical markets, about 10% of the active drug molecules are available in prodrug form.
The enzymatic action on prodrugs is to convert it to its original form. These enzymes are mainly
the esterase class of enzymes. Esterases are hydrolase enzymes responsible for breakage of ester
bonds between functional moieties. The most prominent metabolic sites of conversion are the liver,
intestines, plasma, and kidneys. The types of esterases present in the body that are responsible for
bioconversions are phosphatases, carboxylesterases, arylesterase, butylestrases, and
cholinesterase. Another class of enzymes are the cytochrome P-450 enzymes responsible for drug
bioconversions. For example, codeine, and the more potent morphine, gets demethylated by
CYP2D6
8
to morphine. CYPs and esterases are present abundantly in varied species. This
abundance helps to link the bioconversion to its therapeutic active form through the first-pass
metabolism. Lipase type esterase enzymes cleave the bond formed by carboxyl, hydroxyl, and
amine functional groups, and exert better lipophilicity, while the phosphate linkage in prodrugs
produce more water-soluble molecules which increase the bioavailability and show no inter
7

species variability
2
. The promoieties bind to the enzyme in a non-covalent fashion and biodegrade
in the body safely. The esterase conversion to an active form differs among the species contributing
to inter-variability as the number of enzymes present among species can play a rate limiting step
and alter the therapeutic activity of the drug. Prodrug transport across the membrane can also differ
and must be considered when attaching a chemical moiety, as a non-ionic molecule can passively
diffuse while an ionic molecule requires active or facilitated transport systems like transporters
9
.
Even with some limitations in conversion by enzymes, many of the pharmacokinetic,
pharmaceutical, and pharmacological limitations of drugs can be solved through prodrugs.
Prodrugs are developed to solve many of these limitations without affecting the efficacy and safety
of the drug substance. The prodrug approach is better than developing and inventing a whole new
drug molecule for improved therapeutic efficacy, which can cost much more than developing a
prodrug
10
.
Process of acylated prodrug conversion.

Figure 1.1: Cycle of prodrug circulation, conversion to the parent active metabolite and lastly elimination from the body.
8

On administration of an oral prodrug, as it passes through the GI tract, the oral formulation gets
disintegrated and dissolved. On dissolution, the drug molecule passes through the GI organs like
the stomach and small intestine due to its lipophilic nature. The lipophilic molecule can easily
diffuse through the membrane while the hydrophilic or ionic molecule takes time to diffuse and
may be actively transported. Through the portal circulation, the prodrug gets into the liver where
it experiences the first pass metabolism as the liver is the major site of drug conversion to the
active metabolite. The converted or hydrolyzed active metabolite gets absorbed into the plasma
through which it circulates throughout the body, to various tissues or organs. Some drug molecules
experience the enterohepatic circulation where the active molecule gets reabsorbed into the
intestinal region through bile via the gallbladder. It may be reabsorbed in the intestines or
eliminated as fecal waste. On circulation through the body, drugs of smaller molecular weight can
get absorbed into the urinary tract and thus get cleared out from the body through the kidneys. The
whole process of prodrug ADME is explained in figure 1
1,11
.
9

Chapter 2 : Role of Metabolizing enzymes
Introduction
Metabolism is the process of drug inactivation or prodrug activation. Drug metabolizing
enzymes play a crucial role in drug metabolism and body detoxification. Enzymes have protein-
like structures and on binding to its active site, the drug undergoes conversion. All the drugs and
xenobiotics present/administered in the body must pass through the metabolizing enzymes either
in the GI tract or in the liver, which are the major sites of metabolism. There are 2 phases of
metabolism. Phase I is regulated by either CYPs or esterases while the latter phase-II is regulated
by UGTs. Phase-I metabolism mainly causes molecular weight and solubility conversion like
oxidation, and reduction, while Phase-II conjugation, such as glucuronidation, and sulfation,
confers increased water solubility
9
.
Enzymes are biological proteins that catalyze and convert the substrate drug. Enzymes that
metabolize drugs are ubiquitously present in various species and are also responsible for
converting prodrugs into their active form. Prodrugs have various functional groups masked to
enhance their biopharmaceutical and pharmacokinetic properties. Masking the active moiety can
also protect it from other degradation reactions in the systemic circulation. The enzyme's action
on a specific substrate determines the drug molecules Vmax and Km i.e., maximum velocity of
drug metabolism and the concentration of drug at half of maximum concentration,
respectively
1,3,11
.

10

Esterases
The esterase enzyme is a type of hydrolase enzyme responsible for drug hydrolysis. Hydrolysis
is a process that splits a polar functional group like an ester into an alcohol and an acid. The
various types of esterases enzymes are located throughout the body. Esterases have a role in drug
metabolism, rendering the prodrug active. Esterase enzymes convert the prodrug to its active
metabolite leading to its elimination from the body. Esterases are responsible for cleavage of
esters, amides, peptides, and halides like functional groups. This distribution throughout the body
determines the esterase activity. Esterases present throughout the body are named according to
their substrate activity: acetylcholinesterase (hydrolysis of acetylcholine to choline),
carboxylesterase (hydrolysis of ethyl ester, amide, carbamates, thioesters. Environmental toxins),
aryl esterase (antioxidant activity on lipoproteins). The esterases are localized in various parts of
the body like the neuromuscular junctions in muscles and nerves, skin, liver, gastrointestinal
tract, plasma, and erythrocytes
12,13
.
In early studies, Esterases having drug metabolizing activity were divided into 3 categories based
on their substrate specificity and inhibitory substances. The categories or types were designated as
A, B and C esterases. Esterases A hydrolyze aromatic(organophosphate) esters while Esterases B
(carboxylesterases and cholinesterase) are inhibited by organophosphates, carbamates, and
organosulfur compounds. The third-class C can neither inhibit nor hydrolyze organophosphate
drugs. This classification is now not used by researchers as each individual esterase enzyme
activity is known
13,14
.
Esterase activity differs among people, leading to inter-individuality. Expression of
carboxylesterase subtypes in cytosolic and microsomal regions of tissues differs in individuals,
11

causing subsequent increase or decrease in therapeutic effect of the drug molecule. It is said that
esterases are responsible for autogenous and exogenous substance metabolism
13
.
Researchers have developed prodrugs (inactive forms) by considering the activity of esterase
enzymes in the body using their ability to cleave the drug into its active metabolite, rendering its
therapeutic effect. Thus, instead of the esterase enzyme causing inactivation of the active drug
molecule, it now converts the inactive form of the drug molecule into its active metabolic form.
Thus, with wide substrate specificity and abundant presence of esterases, the literature states that
over 20% of active drugs and 50% of prodrugs are hydrolyzed through esterase activity
15
.
Carboxylesterase
The Structure of Carboxylesterase (CES)
The carboxylesterase class of esterase enzymes are mainly responsible for drug metabolism
predominantly present in the GI tract and liver. The carboxylesterase enzyme has a range of
substrate specificity based on their amino acid sequence. The carboxylesterase enzymes can
hydrolyze functional moieties like esters, amides, thioesters, and carbamates. CESs, sometimes
called aliesterases, have a structure like an alpha/beta hydrolase and are from the serine family of
esterases and consist of a serine molecule. The catalytic structure of these enzymes is made up
of Ser (S), His (H) and Asp (D) as a catalytic triad. In two-steps, these enzymes can hydrolyze
the ester containing molecules into acid and alcohol
13
.
The steps of the reaction are:
Step 1. Release of an alcohol metabolite and an acylated enzyme due to formation of covalent
bond between the acid moiety of the drug molecule and the Ser residue at the catalytic site. The
first step is said to be a nucleophilic attack of catalytic serine –OH on the carbonyl carbon of ester
12

bond. The step is stabilized through hydrogen bonding to His (H) which in turn is stabilized by a
carboxylic group of acidic members of the catalytic triad.
Step 2: The His (H) residue of the catalytic triad shows affinity toward water molecules and it also
helps the enzyme to return to its active state with the release of an acid moiety
15
.
The whole process is thus responsible for specificity of carboxylesterase enzymes. This catalyzes
the hydrolysis reaction due to carboxylesterase enzymes forming a water-soluble compound
leading to renal elimination
16
.
Classification of CES
Carboxylesterase activity differs among species, developing inter-species variation.
Carboxylesterases are abundant in tissues like the liver, the GI tract or gut, and the kidneys. CEs
are mainly present in the tissues acting as a barrier against xenobiotics toxins. The five subtypes
of carboxylesterase are CES1, CES2, CES3, CES4 and CES5 classified according to their amino
acid sequence. CES1 and CES2 are the classes responsible for prodrug activation. Localization of
CES1 and CES2 is mainly on the endoplasmic reticulum of the cell membrane. The presence of
this enzyme causes the prodrug activation or drug metabolism in organs like the liver and the
intestines. CES1 enzymes are predominantly present in the liver microsomes and cytosols with
lesser amounts in the small intestine. While the situation is vice-a-versa for CES2, with higher
concentrations in the small intestine than in the liver. In mammals the classification of
carboxylesterases named ces1, ces2, ces3, ces4 and ces5 are based on its sequence of amino acids.
Humans’ predominant carboxylesterases enzymes are signified as: hCE1 and hCE2
15
. The amount
of esterases in rats differs from humans with a higher concentration in rats
16
. Over the years it has
been determined that humans lack the CES activity in the plasma in comparison to smaller
13

mammals like rats and mice. The protein activity is lower in humans, so when using rats for
experimental procedures, the researcher needs to consider how CES rates of hydrolysis might
differ between species. This variation may cause changes in the rate of hydrolysis, with faster rates
in rats than in humans. Thus, this may lead to not-so-good predictions of metabolism due to
esterases, specifically carboxylesterases. This may cause experimental error when doing in vivo
experiments with rats.
Now, the question arises as to how can we differentiate whether the drug gets metabolized by
hCE1 or hCE2 in humans? The answer to this question is its location in the body and the structure
of the ester group. An ester, in general, is made up of an acyl group and an alcohol, and a drug
with a bulky acyl group and small alcohol group is metabolized mainly by hCE1, and drugs with
small acyl groups and large alcohol groups are hydrolyzed by hCE2. Localization of hCE1 is
largely in the liver and very little in the small intestine, while hCE2 presence is mainly in the small
intestine with modest quantities in the small intestine. Drugs experience first-pass metabolism
when passed through the liver by hCE1. If the drug has an affinity for hCE2 then after absorption
into the enterocytes in the small intestine, the drug is metabolized. Both isoenzymes are membrane
bound within the liver and are present in the cytosolic and microsomal regions
11,16
.
Function of Carboxylesterase (CES)
The Carboxylesterases function is to protect the tissue organs against xenobiotic toxins, as CES
hydrolyzes the ester bond in substances available both naturally and synthetically. On prodrug
administration, the molecule faces metabolism due to carboxylesterases converting it into its
therapeutic active molecule. This addition of an ester linkage helps protect the active moiety and
enhances its biopharmaceutical properties. The ester functional group makes the drug lipophilic
14

thus enhancing its cell permeation
11
. One such example is oseltamivir, a prodrug of Oseltamivir
carboxylate. Oseltamivir carboxylate is a drug with low lipophilicity and thus poor oral
bioavailability. Oseltamivir has an ester linkage which on administration gets hydrolyzed to a
carboxylic acid forming oseltamivir carboxylate. This conversion to an inactive prodrug of
oseltamivir carboxylates is to increase its hydrophobicity and improve permeability in the GI tract.
Oseltamivir is the ethyl ester prodrug of oseltamivir carboxylate, hydrolyzed by hCE1 enzyme in
the liver
16
.
The carboxylesterase enzymes hydrolyze mainly the ester moiety thus formation of the ester
prodrug is a strategy to enhance the biopharmaceutical and pharmacokinetic properties of the
potent drug. The pharmaceutical companies are developing new drugs through synthetic
processes utilizing esters as their main functional group. The use of ester functional groups, in
general, leads to improved water solubility and better permeability with increased oral
bioavailability of the parent metabolite. Due to its abundance in the liver and intestine, it
concentrates the prodrugs bioconversion and activation
16
.
The function of esterases, apart from metabolizing the drug molecule, is to cause the metabolic
clearance of various toxins from the body. Esterase inhibitors are required to improve the
pharmacokinetics of some active drugs. A way to inhibit the esterase activity is to co-administer
an esterase inhibitor drug that protects the active moiety against the esterase activity. Inhibitors
can be used to enhance the biopharmaceutical properties of drugs that are metabolized by
esterases into inactive forms. Carboxylesterases are inhibited by organophosphorus compounds
like bisnitro-phenyl phosphate

as CES1 class of enzymes has the role of metabolizing the
cholesteryl esters and thus increase the risk of disease conditions like obesity, atherosclerosis,
and diabetes
13
.
15

Chapter 3 : Basics of Pharmacokinetics
Introduction
Pharmacokinetics is the term used to define what the drug does to the body. Pharmacokinetics is
a fundamental factor considered during drug development. PK parameters are monitored to
determine what, when and how the drug is prescribed to the patient for a better therapeutic effect
and less interaction between the medications. Pharmacokinetic factors are dependent on inter-
individual variability among patients
18,19
.
Pharmacokinetic considerations should not be limited to healthy volunteers but also the study
should be done on patients that have kidney and/or liver failure to determine the proper dosage
regime, route of administration, etc. All these parameters are primarily based upon the drugs
absorption, distribution, metabolism, and excretion from the body. Drug-drug interaction, tissue
distribution, protein binding, and clinical efficacy all play a role in considering the perfect dosage
form, drug molecule, route of administration, dosing time interval, and treatment duration
20
.
Models: Compartment and PBPK
The one compartmental model represents the body as one homogeneous compartment.
According to the one compartmental model, the drug is distributed equally to all the parts of the
body which is not always true as the amount of the drug at the time interval(t) in plasma is not
equal to that in the tissue. A linear relationship is observed between the concentration in the body
and time interval
21
.

16

Figure 3.1: Representation of a one compartment model, where Ka is the rate of absorption into the compartment and Ke is the
rate of elimination from the compartment (adapted from Ref.21).
Two compartmental models contain a central compartment having more perfused tissues like liver,
kidney, heart, and lungs, and a peripheral compartment having other tissues like fats and muscle.
The drug administered enters the central compartment to either distribute to the peripheral
compartment or to be eliminated out of the body. Initially, the drug equilibration is not observed
between the central and peripheral compartment leading to rapid decline in the drug plasma
concentration in the central compartment and distribution to the peripheral compartment. At a
certain time(t) the concentration leaving the central compartment will be equal to the amount
entering. Thus, directing the drug to eliminate from the central compartment
21
.

Figure 3.2: Representation of a two-compartmental model, where K12 represents the rate of drug movement from central to the
peripheral compartment, K21 represents the rate of drug movement from the peripheral to the central compartment and Ke is the
rate of elimination (adapted from Ref.21).

Physiologically Based Pharmacokinetic Modeling (PBPK) is a theoretical compartmental
technique in which each organ of the body is represented as compartments. The blood being
unidirectional flows through all the organs or tissues of the body as it gets distributed to different
chambers of the body. PBPK models consider the blood flow and volume of blood flow to each
tissue or organ of the body.
17

Figure 3.3 Representation of a PBPK Model (adapted from Ref.21).
Physiologically Based Pharmacokinetic Modeling (PBPK) is a mathematical tool used to
understand the pharmacokinetics of the drugs and prodrugs and thus simulate the plasma
concentration-time curve. PBPK is an approach used nowadays in the drug development process.
PBPK models consider all the tissues of the body and represents it as compartments, representing
the connection between blood plasma and tissue concentration of the drug of interest. Thus, PBPK
modeling is a structured model considering predicted, in vitro and preclinical in vivo data to
simulate the plasma concentration-time curve.
20
.
Drugs have a therapeutic window and when the plasma level is above the therapeutic window, it
leads to drug toxicity and when the plasma level is below the therapeutic window, it has no
pharmacological effect. The drugs that have narrow therapeutic windows may cause toxicity at a
slight change in drug concentration. The therapeutic index in humans, which is related to the
therapeutic window, may differ due to factors like age, weight, disease, sex, and gender
18
.

18

ADME
Bioavailability
Bioavailability is determined based upon the drug concentration rate of appearance in the plasma
upon absorption and distribution. The factors of pharmacokinetics should be considered when
determining the bioavailability of the drug. Either administered orally or intravenously, the drug’s
effect on the body is a result of the amount of bioavailable drug in the body. Dosage form, dose
administered, and form of administration all contribute to the bioavailability of the drug.
Intravenous administration of drugs is considered to have 100% bioavailability, as all the drug is
available for therapeutic activity, without first pass metabolism. On the other hand, orally
administered drugs undergo first-pass metabolism, which reduces the amount of bioavailable drug
in the body
18,22
.
As the study of absorption, distribution, metabolism, and excretion is said to regulate the dosage
regimen in the body, all these factors should be studied to develop a PBPK model.
In absorption, the diffusion of drugs through the membrane is the major factor to be considered
when researching a drug molecule (chemical entity) and the route of administration.
The drug must pass through various membranes like the blood-brain barrier or hepatocellular
membranes to enter the site of action. The various modes of diffusion are listed below:
I. Passive diffusion: the transport of a drug through the phospholipid membrane, which is
dependent upon the concentration gradient until equilibrium. More lipophilic drugs pass easier
through the membrane.
19

II. Facilitated diffusion: It involves the use of carrier macromolecules to pass through the
membrane and is not energy dependent.
III. Active transport: the drug molecule passes through the membrane, dependent on energy for
its transport.
Various other transporters which are present at the membrane help in the drug transport. The
crossing of drugs through the membrane with transporters present on the membrane depends upon
the ionization of drug molecules and requires energy for transporters such as OATP, P-gp, and
MCRp.
Other mechanisms for absorption across membranes include endocytosis and pinocytosis, which
are also responsible for transport using lipid vesicles in the cell cytoplasm
18
.
Absorption
The administration of the drug and the amount reaching the systemic circulation is dependent on
how well the drug is absorbed. The factors mainly responsible for absorption are solubility and
permeability which control the bioavailability of drugs. The absorption is dependent upon
lipidation of the drug, molecular size, pKa of a drug, and degree of ionization
22,23,24
.

20

Table1.1: Different routes of drug administration. Each row describes the following: absorption mechanism, advantages, onset
and duration of action, and examples of the 3 different types of dosage forms
25
.
Oral route Injection route Transdermal route
Administration of the drug orally
passes through GIT and enters the
liver for hepatic metabolism (first-
pass metabolism)
Injection directly into the plasma
eliminates the first-pass metabolism
in the liver and GI tract.
Transdermal administration of lipophilic
drugs passively absorbed through the
skin
Oral administration is convenient and
easy to use.
Greater bioavailability is observed
through this route of administration
rather than any other route (nearly
100%)
Can be delivered to the bloodstream
without injection
It is the most studied dosage form as
it is easy to manufacture, deliver, and
patient compliant and can be designed
to be released immediately as well as
sustain delivery
The vascularity impacts the onset of
action at the site of action as
subcutaneous has slow onset,
intramuscular is intermediate and
I.V is immediate.
Longer duration of action, therapeutic
effect can be immediate for potent
drugs, with the ability to sustain release
Oral dosage forms include syrup,
tablets, capsules.
Intravenous infusion, bolus,
intramuscular, subcutaneous.
It involves the use of patches, creams
and ointments.
After absorption, the drug distributes significantly into the systemic circulation and then enters the
tissues to exert its therapeutic effect. For this to happen, the drug needs to cross the tissue or organ
membrane. The drug, once administered, is diluted in the fluid volume converting the drug
concentration in the systemic circulation. Intravenous administration enables the drug to directly
enter the plasma without any metabolic barrier which causes a higher drug level in the plasma in
comparison to an oral dosage form.
21

Distribution to the tissues depends on the rate of blood flow to the tissues or organs, tissue binding,
regional pH, permeability of tissue membrane, and protein binding. The drug in the plasma flows
to the tissues and this diffusion is equilibrated once the concentration in blood is equivalent to that
in the tissues.
Perfusion is the term coined for the passage of blood to the tissue. Perfusion is measured as the
volume of blood per unit time per unit tissue mass. The perfusion of membranes is one factor that
determines the distribution level:
1. Highly perfused tissues: Kidney, liver, and lungs.
2. Low perfused tissues: Muscles, fats (adipose tissues), and the blood-brain barrier.
Highly perfused tissues allow more of the drug to cross the tissue membrane from the systemic
circulation. Lipophilic drugs are distributed faster as they are more permeable through the lipid
membrane of the organs. Low perfused tissues do not allow easy diffusion into the tissues/organs
leaving the drug concentration in unequilibrated form between plasma and tissues, leading to
distribution being a rate-limiting step.
Thus, the amount of drug dosing is regulated based on drug distribution to the tissues or site of
action to exert its therapeutic effect.
Plasma proteins, like albumin, glycoproteins, and lipoproteins, which can bind to drugs, are other
important factors to be considered when determining distribution. If the drug binds covalently to
the plasma proteins, then there would be no therapeutic efficacy of the drug on the body, however,
if the drug binds to the protein for a certain amount of time noncovalently, then its therapeutic
action could be delayed or extended, due to its slower distribution and less elimination form the
22

body. The irreversible binding of the drug to the protein causes the unequilibrated state between
plasma and tissue levels.
Conversely, if there is drug-drug interaction between drugs for binding to the plasma proteins then
this may affect the drug clearance and efficacy. If one drug has more binding affinity than the
other, this may lead to the less therapeutic effect of the former drug and a higher concentration of
the latter in the plasma
26
. The drug concentration is measured in plasma, serum, urine, saliva, and
cerebrospinal fluid. The drug concentration is determined by a combination of bound and free
drugs. The drug can be bound by proteins like albumin, glycoprotein or lipoproteins present in the
plasma. The bound form of the drug is said to be in equilibrium to the free(unbound) form. On
release of the free drug in the plasma, it can have a pharmacological effect.
It is rightly said that the drug concentration in plasma is not equivalent to the drug concentration
in the tissues at plasma steady state. The reason behind this is:
1. Differences in permeability and perfusion and the presence of uptake and efflux transporters at
membrane surfaces,
2. Drug binding to the proteins in plasma and,
3. Rapid clearance of drugs from the site of action because of activity of metabolic enzymes.
Thus, plasma proteins may help to extend the half-life of the drug in the body, affecting both
bioavailability and distribution. The ratio of plasma to tissue concentration may vary based on
drug nature and binding. Pharmacokinetic monitoring includes proper dosage regimen of drugs as
well regulation of serum drug levels
18
.
23

Metabolism
Metabolism, in general, is a process that renders a drug ineffective. A process of drug conversion
from parent molecule to an inactive metabolite. Inactive metabolites, in general, do not have any
therapeutic effect on the body. The structures responsible for metabolic activity include enzymes
that are proteins with metabolic functions in the body. Different types of enzymes are present
throughout the GIT, in the liver, kidney, intestine. The metabolite form of a drug, in general, has
the inactive characteristics of the parent molecule and is easier to eliminate from the body.
Lipophilic drug molecules can penetrate through the phospholipid membrane easier and into the
systemic circulation but can cause problem with clearance giving rise to toxicity. Therefore,
enzymatic activity renders the lipophilic drug more hydrophilic which helps to eliminate it out of
the body rather than reabsorption into membranes.
Looking at the process of metabolism, scientists decided to take advantage, so the concept of the
prodrug was introduced. A prodrug is an inactive form of the molecule which upon metabolism
gets converted to an active molecule rendering its pharmacological effect.
Some of the enzymes responsible for the biotransformation of drugs or prodrugs are CYPs,
esterases, phosphatases, and proteases through the process of hydrolysis, oxidation, condensation,
isomerization, and hydration.
Drug metabolism has been described as a 2-phase process:
1. Phase I - Inactivation of a drug molecule due to hydrolysis, oxidation, reduction reactions.
24

2. Phase II- Attachment of an inactive drug molecule to a hydrophilic molecule for better
elimination of the drug. Examples of hydrophilic molecules are sulfates, acetylates, glucuronic
acid
9
.
The rate of metabolism determines the drug conversion due to enzymatic activity. In first order
kinetics, the metabolic rate of conversion and substrate concentration are proportional. While if
the substrate concentration is increased, but the rate of reaction remains constant due to the limited
concentration of enzymes present, the increase in substrate concentration would not have any
effect on the rate of reaction
27
. This non-linear kinetics is also termed Michaelis-Menten kinetics.
The kinetics initially relates the rate of reaction with the concentration of substrate but with time
the enzyme can saturate, and the reaction becomes independent of the substrate concentration. The
maximum rate of reaction with 100% saturation of the enzymes is Vmax. The substrate
concentration at a 50% reaction rate is called the Km
18,28
.
The equation is:
𝑉 =
𝑉 𝑚𝑎 𝑥 𝐾𝑚 + 𝑆
Where, Km is equal to the substrate concentration at half of the maximum reaction rate; Vmax is
equal to the maximum rate of reaction; and S is the substrate concentration
28.

25

Elimination
Elimination is the process of clearance of a hydrophilic non-active metabolite out of the body to
avoid any toxicity due to accumulation.
The drug molecule has 2 main modes of drug clearance- one is renal, and the other is bile. Except
for this, the other routes of excretion are saliva, sweat, tears or breast milk.
The excretion from the kidney is through the process of glomerular filtration, reabsorption, and
secretion. The preferred metabolites for urinary clearance are water soluble and small in molecular
size.
The drug gets metabolized in the liver after which it gets eliminated. Larger and lipophilic
molecules are highly prone to metabolism and elimination through the liver because lipophilic
drugs tend to be difficult to eliminate from the body as they tend to be absorbed through
membranes. The concept of the prodrug was to convert perfusion limited drugs into more lipophilic
drugs (prodrugs) for better absorption and distribution to the tissues/organs, but the main drawback
in this discovery is its limitation for excretion if the prodrug cannot be efficiently metabolized in
the liver. Therefore, prodrugs for immediate release are designed to be metabolized and converted
into a more excretable form for elimination.
One other physiological factor which plays a role in determining the pharmacokinetic properties
of the drug is transporters. Hydrophilic and large ionic molecules are transported through
biological membranes using transporters. Transporters play a role in drug absorption and
clearance. There are different types of transporters known as efflux and influx or uptake
transporters. Efflux transporters are responsible for the function of uptake of the drugs from blood
to the epithelial tissues. While influx transporters move the drug from tissues to blood.
26

Transporters maintain the concentration of drugs in tissues as well as in the blood. The transporters
present on the membrane of excretory organs facilitate the clearing of drugs out of the body. With
efflux transporters present on kidney or liver organ membranes there can be a possibility of
reabsorption. The reabsorbed drug enters the portal circulation, circulating throughout the body.
Since they can reabsorb, the half-life of the drug increases and thus the drug remains in the body
longer
29
.
Pharmacokinetic parameters
Clearance
Clearance is measured as the amount of drug excreted out from the body in any form: bile, renal,
sweat, tears. Clearance is the volume of the fluid cleared of the drug from the body per unit time
due to various metabolic reactions. The units of clearance are ml/min and L/h.
Clearance (CL) is the pharmacokinetic term for elimination from the body. CL is measured as the
rate of elimination divided by the plasma concentration.
CL= ke/Cp
Where, ke= elimination rate constant(µg/min)
18
Cp= Plasma concentration (µg/ml)
The elimination rate constant is a time-dependent first-order process. The rate of elimination is
measured as the amount of drug administered orally or IV eliminated per unit time from the body.
ke= dE/dt
CL = dE/dt ÷ Cp
27

= ke*Cp*Vd ÷ Cp
= ke*Vd
30
Clearance is the constant term dependent on Vd and ke. As the plasma concentration of the drug
in the body decreases, the rate of elimination is reduced and attains zero once all the drug is cleared
out from the body. All the clearance processes sum up to the total clearance of the body.
CLtotal = CLrenal + CLurine + CLbody
22
Kinetics determines the rate of reactions, divided into zero, first, and second-order kinetics.
In first-order kinetics, the chemical reaction is dependent on only one reactant. If the elimination
rate constant is first-order kinetics, the rate of elimination will be dependent on only the
concentration of the drug in the plasma and the clearance rate will be constant. In general, most
drugs have first-order elimination rate constants. In zero-order kinetics, as it is independent of
reactant concentration, it does not make any difference in the rate of chemical reaction if the
amount or concentration of reactants is reduced or increased in the reaction
18,28
.
Volume of distribution
The volume of distribution is measured as the amount of the administered drug dissolved in the
body fluid converting to the total drug concentration in plasma. The unit for Vd is L/kg. As the
volume of fluid in the body is dependent on the body weight, the unit contains per kg. Different
tissues have different volumes of distribution of drugs as the concentration in one organ is not like
that in another organ or tissues. The volume of distribution is not based on the volume of the body
fluids, but it is the distribution of the drug within the entire body
22
.
28

The drug distribution is determined by the value of Vd; if the Vd is large it means that the drug is
highly distributed in the tissues of the body, while if the Vd value is small, it means that the drug
tends to be in the plasma with very little bound to the tissues. Drugs are distributed differently in
tissues with some drugs binding more to fats or adipose tissue, while others to other tissues.
Acidic and lipophilic drugs are highly protein-bound molecules, limiting the volume of distribution
of these drugs, while basic, lipophilic drugs are less protein bound, so they can permeate through
the tissues, increasing the apparent volume of distribution
18,26
.
Half-life
The half-life is the time it takes the drug to reach half of its initial concentration in the body. The
symbol used for half-life is t1/2 and the unit, in general, is hours. The concentration of drugs in
the body, or the amount to be reduced to half, is dependent on how much is cleared and
distributed. Therefore, half-life is the pharmacokinetic parameter dependent on clearance and
volume of distribution.
Both the clearance and volume of distribution are related to the half-life by the following equation:
T1/2 = 0.693 * Vd/CL.
where in the equation,
Vd= volume of distribution.
CL= Clearance
18
.
29

Half-life is increased if more of the drug is distributed (higher Vd) and less if the drug is cleared
per unit time, while on the other hand, faster clearance of the drug reduces the value for the half-
life and the time is lessened to reach half of the concentration of the drug.
In practical life, the actual half-life of a drug varies among individuals as it depends on several
patient and drug-specific variables
22
.
Pharmacokinetic Outcome

Figure 3.4: Plasma Concentration-time curve representing the maximum concentration, time at which Cmax is achieved, AUC of
the respective drug.

The concentration-time curve represents the amount of drug present in the plasma. The
concentration of the drug increases as per unit increase in time. On attaining the highest
concentration, a peak is obtained, and equilibrium is obtained at that time as the amount present in
the body becomes equivalent to the amount excreted from the body. Slowly with time then the
concentration reduces as the amount excreted will be more than that present in the plasma
31
. Some
of the common parameters measured are:
30

Cmax: Maximum observed concentration of the drug in the plasma is termed as Cmax. The unit
for concentration is ng/ml or mg/ml.
AUC: Area under the plasma concentration time curve with time from initial dose of
administration to the final time with drug concentration.
Tmax: Time at which the highest drug concentration is observed on the plasma concentration time
curve. Tmax is the term independent of the initial dose of administration
19,32
.

31

Chapter 4 : Physiological Based Pharmacokinetic modeling- Novel
and Thriving area of Pharmacokinetics.
Different PK models are used to measure the plasma concentration-time curve of drugs
corresponding to their absorption, rate of metabolism, and involvement of transporter proteins.
Physiological Based Pharmacokinetic Modeling is an in-silico method that uses mathematical
equations and parameters related to the physiology of the body to derive the plasma concentration-
time curve. The compartmental model divides the body as compartments with the following 2
compartments in the classic model: 1. central compartment and 2. peripheral compartments
20
.
The organs or tissues of the body are interpreted as compartments. with the blood flow rate
constants determined throughout the body for each organ. The organ compartments in the body
have different tissue volumes, partitioning, permeability, and blood flow according to the specific
species which vary due to age, weight, height, and sex
20,33
.
A plasma concentration-time curve (ng/ml vs hour) is obtained using these compartmental models.
It simulates the pharmacokinetic profiles of drugs with respect to individuals based on preclinical
ADME properties of drugs like the rate of absorption, distribution after administration through
different routes, metabolism by enzymes, and the site of elimination can be predicted. The
physicochemical parameters of the drugs considered for the simulation are molecular weight, logP,
pKa, solubility, permeability, while some of the pharmacokinetic properties to be considered are
Volume of distribution, Clearance, half-life, and blood/plasma ratio. Developing a PBPK model
with physiological parameters in mind for different species, different age groups, ethnicity, weight,
disease state, and so forth, will provide a path for further in-silico simulations
34
. The type of species
(rat, mouse, guinea pig, human) will influence the organ size, volume of blood and blood flow
32

rate, while the impact of fasting or the fed state can influence absorption and drug interactions with
food, and gastric emptying time. The PK parameters derived from one in vivo study can be
extrapolated to another study with demographic changes to estimate the correct dosing among
individuals. In PK modeling, various population-based PK models are developed to estimate the
plasma concentration-time profiles of individuals in the population who are separated by age,
weight, and ethnicity. The factor of variability among individuals decides the amount of dose
administration or dose selection for the individual
35
.
In PBPK modeling, each organ is distributed as a compartment, and the role of each compartment
differs based on the physiology of the human body. So, for example, the physiological data of the
renal diseased patient can be incorporated in the model with the dose of healthy volunteers and the
plasma concentration-time curve can be simulated for the former individual with kidney disease.
Therefore, the right dosage regimen for the diseased patient can be calculated using experimental
data from a healthy individual. Because of such advantages from PBPK modeling, it is now being
widely used by scientists in academia and in pharmaceutical companies. The major use of PK
models is to eliminate the unnecessary use of animals during experimental studies and to move
forward with only the best potential molecules for drug development. The use of in-silico PK
models in new drug applications to regulatory agencies such as the FDA is growing as well
20
.
A PBPK model is developed based on the blood flow, permeability, and metabolism in various
organ compartments and how the drug is distributed among these organs. The use of PBPK
modeling has been around for a long time but because of a lack of reliable information and
computer resources, it was not used by pharmaceutical industries. As time passed, scientists started
using in-silico modeling software more and more, to shorten the time for drug development and
reduce the resources needed for clinical trials
20
.
33

Ga s tr o P l u s ™- A tool for PBPK modeling
GastroPlus™ by Simulations plus is a mathematical PBPK modeling software used to measure the
pharmacokinetic plasma concentration-time profile of an orally or intravenously administered
drug. GastroPlus™ can mimic the human anatomy and physiological characteristics, with
compartments corresponding to humans’ organs. Along with humans, there are other species
considered in GastroPlus™ such as rats, mice, monkeys, guinea pigs, cats, and rabbits. One, two,
and multiple compartments can be developed using the software. An oral drug model accounts for
the following characteristics: release profile, pH for drug dissolution and solubility, permeability,
time to pass through the gastrointestinal tract, absorption to the intestinal wall, and first-pass
metabolism. The simulation and predictions are based on advanced compartmental absorption and
transit models (ACAT model). GastroPlus™ contains various modules like the ADMET
Predictor® module which helps to assess in-silico physicochemical properties of drugs through
the drug’s structure and extrapolates the properties into GastroPlus™ to simulate the PK curve.
There are 4 modes of simulation in GastroPlus™, single simulation, batch simulation, population
simulator, and parameter sensitivity analysis (PSA). The single simulation compares the predicted
individual drug data against the clinical individual data, while batch simulation can help to perform
a single simulation with each specific variable of the database, resulting in batch files of the drug.
The population simulator model considers several individuals as a population, with a drug
administered at a fixed dose (mg/kg or mg/m2). The PBPKPLUS™ module of GastroPlus™ helps
to define an individual or population using categories like species, population type, age, and health
conditions. The Parameter Sensitivity Analysis simulation compares various properties, such as
physicochemical, physiological, and various pharmacokinetic parameters based on an upper and
lower limit for a particular property and runs a range of values to determine how a change might
34

affect a specific parameter
36
. Parameter sensitivity analysis (PSA) is a good built-in tool in PBPK
modeling software like GastroPlus™, PK-Sim®, and other modeling software
34
.
Evaluation of drug molecules
The PK models use in vitro and in-silico data for determining the plasma concentration-time curve
in place of using traditional experimental methods. GastroPlus™ PBPK modeling software can
generate the physicochemical properties of drugs based on a text representation of the drug, known
as a string structure, with the help of ADMET Predictor®. Using these PK models, we can compare
the data sets of concentration-time profiles obtained through in vitro experiments with those
derived from simulated PK models like GastroPlus™. The predicted parameters and in vitro
experimental factors can be analyzed in PBPK modeling software using the parameter sensitivity
analysis
20
.
Physicochemical properties like logP, pKa, molecular weight, solubility, and physiological
characteristics like transporters, enzymes, transit time, and pharmacokinetics parameters like
clearance, the volume of distribution, and blood/plasma ratio, all have some specific effect on the
plasma concentration-time profile of the drug. Each parameter should be considered individually
to check its effect on the drug concentration. All drugs have certain characteristics which play a
major role in determining the drug concentration in the body
34
.
35

Building of a PBPK model using a prodrug
The prodrug is used in the field of drug development mainly to overcome the pharmacokinetic
and pharmaceutical drawbacks of the parent active moiety. Prodrugs are structural modifications
of the active drug molecule that might have poor solubility, lipophilicity, and/or poor
bioavailability. The prodrug strategy is a growing field in the drug development process.
Prodrugs are the masks of the parent active molecules, developed for their improvement in poor
solubility and permeability. Prodrugs along with their pharmacokinetic alterations also help
increase pharmacodynamic aspects which lead to improved therapeutic efficacy
37
.
Developing a prodrug PBPK model is a challenging and an interesting area of interest, where the
properties of not only the improved molecule are considered but also the active molecule PK
properties. Hence, simulating a pharmacokinetic time curve, keeping in mind both drug
characteristics, is an interesting area of research.
In vitro studies are thought to have poor predictive properties as they do not mimic the actual
human conditions but so do in vivo studies on rats, guinea pigs, or other animals, as they might
show higher activity levels of metabolism, clearance, and/or protein binding, in comparison to
humans
33
. For example, permeability studies performed on Caco-2 cells under in vitro conditions
for a specific lead molecule might help to determine a permeability value for that molecule, but
the results might not be accurate due to environmental factors such as the quality/stability of Caco-
2 cells. But Caco-2 cells can be a good tool to approximate the permeability of lead molecules.
Preclinical animal studies can also be done to determine metabolism; however, animals might have
higher, lower, or different concentrations of metabolizing enzymes in comparison to humans. This
difference could lead to faster or slower metabolism of a drug of interest in animals, when
36

compared to humans. The values of various properties can also be obtained or derived from a
literature review on experimental in vitro and/or in vivo studies on prodrugs and their active
molecule, which helps in building a PBPK model. Our aim of prodrug conversion is not just for
better solubility or permeability but also to minimize the first-pass metabolism of the parent moiety
which can lead to reduced efficacy.
While developing a PBPK model, many parameters must be considered because of their specific
effect on absorption, distribution, metabolism, and excretion of the drug from the body.
Consideration of all the parameters like ionization, permeability, and metabolic conversion
collectively in the PBPK model is a must for a desired pharmacokinetic outcome. Physiological
Based Pharmacokinetic Modeling along with in vivo in vitro correlation (IVIVE) is widely used
and integrated into the simulation method that is used to predict the pharmacokinetics of the drug
in the body. The process of integration collectively uses all the physicochemical properties derived
through in vivo and in vitro studies along with in-silico simulation with IVIVE approaches that
characterize the drug-related pharmacokinetic properties such as clearance, the volume of
distribution, protein binding, and half-life
38
. For the prodrug approach, prodrug-specific
physicochemical properties were derived to parametrize the PBPK model, thus simulating the PK
curve for the active moiety after oral administration. Thus, to be explained in a simpler way we
can just input the physicochemical data of a prodrugs derived from a literature review or from in-
silico predictions along with some pharmacokinetic data input to generate a PK curve of the active
moiety
37
.

37

The following diagram describes the process of prodrug conversion and circulation throughout the
body:

Figure 4.1: The large arrows display the circulation of the prodrug till its conversion to the active metabolite. The thin smaller
arrows show the circulation of the active form of the drug to the tissues. arrow indicates the connection between
stomach, spleen and pancreatic blood vessels. arrow indicates the circulation of the prodrug form till its conversion.
arrow indicates circulation of active parent metabolite after its conversion (adapted from Ref. 33).
As described in the figure of the prodrug upon oral administration, the inactive moiety of the drug
molecule undergoes metabolism either in the upper gastrointestinal tract and/or in the liver.
Prodrugs are designed to mask the active moiety and are developed to improve a drugs
bioavailability. The whole process of prodrug administration and conversion to the active parent
metabolite was developed in our PBPK model. After metabolism there is the step of elimination
or clearance from the body. In the process of excretion 2 organs can be considered, the liver and
kidney. In the liver, the metabolized parent drug can be excreted from the liver into the bile to the
feces, or enter the systemic circulation, circulate to body tissues, or get excreted through the
kidneys into the urine.

38

Chapter 5 : Oseltamivir carboxylate and its prodrug: Oseltamivir
Phosphate
General information
Oseltamivir carboxylate (OC) is the drug given to prevent or treat the deadly respiratory virus that
causes influenza. Influenza is a respiratory infection that causes an increased rate of mortality
among people around the world and was labeled as a pandemic in 2009. Oseltamivir carboxylate
is the active drug moiety having therapeutic efficacy to treat Influenza- a respiratory tract
infection
39
. Oseltamivir carboxylate (OC) is a molecule with a -COOH functional group attached
to its surface, that has low lipophilicity and reduced permeability properties. OC, due to its
unfavorable physical-chemical properties has poor oral bioavailability, therefore it is administered
in a prodrug form. The prodrug form masks the active drug molecule to enhance its oral
bioavailability. An oral prodrug, rather than a parenteral formulation, is both easy to administer
and cost-effective
40
.
Oseltamivir (OP) is the ethyl ester prodrug form of oseltamivir carboxylate (OC). Its carboxylic
acid functional moiety is converted to an ester, enhancing its lipophilicity. A prodrug is the inactive
form of the pharmacological active molecule, responsible for better therapeutic efficacy.
Oseltamivir is the prodrug form that renders the drug active due to the action of esterases present
in the liver and intestine
41
.
Oseltamivir phosphate, due to its good lipophilicity, gets absorbed into the tissues and has a higher
permeability than OC. Esterase substrate specificity for oseltamivir is due to its ester group, which
is converted to a carboxylic acid forming oseltamivir carboxylate. The CES1(hCE1) class of
esterase enzymes, most abundantly present in the liver, metabolize oseltamivir causing its hepatic
39

metabolism and excretion through urine and bile. Oseltamivir carboxylate is a small hydrophilic
molecule excreted mainly through the kidney
42
.
The Influenza virus has 2 subset forms: Influenza A and B. Oseltamivir is a drug capable of treating
both subtypes. The mechanism of action of oseltamivir is to act on the viral neuraminidase
enzymes. Neuraminidase enzymes are responsible for replicating the virions, increasing the
infection in the body and its transmission. Oseltamivir carboxylate binds to the active site of the
neuraminidase enzyme present on the surface of the virus responsible for the release of progeny
virions from the host cells and inhibits its action
42,43
.
Oseltamivir carboxylate was examined to check its action against other bacteria or viruses, but its
action is selective for mainly influenza virus, both for its treatment and prevention. Oseltamivir is
administered orally and forms a primary active metabolite, oseltamivir carboxylate, mainly due to
hepatic esterase enzymes. The prescribed dose of oseltamivir (Tamiflu) is 150mg daily or 75mg
twice a day for the initial treatment time which then can be reduced after 5-6 days of treatment
42
.
Associated severe effects of Influenza are pneumonia, bronchitis, and otitis media which are
prevented by taking Oseltamivir.

40

CH
3
CH
3
O
NH O
CH
3
NH
2
O
O
CH
3

CH
3
CH
3
O
O
OH
NH
2
NH C H
3
O

Figure 5.1: A visual representation of oseltamivir phosphate (ethyl ester group) structure and its metabolism by esterase enzyme
to oseltamivir carboxylate (carboxylic acid group) (adapted from Ref. 40). Oseltamivir phosphate is the phosphate salt form of
the prodrug but is not considered for simulation as GastroPlus™ cannot predict the salt form.
Oseltamivir capsules are considered to have good prophylactic activity against the influenza virus.
Oseltamivir does vary in its efficacy, when given to prevent influenza once daily for 42 days. The
pharmacokinetics of Oseltamivir is linear and dose proportional. A dose of up to 500 mg is well
tolerated in patients according to certain studies
43
.
A brief overview on Oseltamivir ADME
The oseltamivir carboxylate is converted to prodrug-oseltamivir to enhance the bioavailability of
the drug to 80%. The main goal of the prodrug formation for oseltamivir carboxylate is to mask
the low lipophilicity of the parent drug for a better absorption. Oseltamivir prescribed orally
converts to oseltamivir carboxylate in no more than 30 minutes while in 3-4 hours its full
conversion to OC is observed
43
.
Experimentally, oseltamivir is given orally at a dose of 75 mg or 150 mg and is absorbed through
the GIT where a minimum of 75% of the prodrug is hydrolyzed, some in the small intestines and
most mainly in the liver, to oseltamivir carboxylate by esterase enzymes. Due to its fast metabolism
to oseltamivir carboxylate, a dose higher than required was initially recommended to attain a better
therapeutic effect with severely ill patients. However, a higher dose increased the risk of drug
Esterase (CES1)
Oseltamivir Phosphate (OP)
Oseltamivir Carboxylate (OC)
41

induced adverse effects among some populations
44
. The majority of OC clearance is through urine
while <20% is through the bile and the feces. On the other hand, clearance in the OP form has
been found to be <5% in the feces
45,46
.
Oseltamivir, when tested for intra or inter- variability, showed less differences among healthy
patients in terms of its plasma concentration. While in patients with renal or liver disorder it may
lead to a change in plasma concentration of oseltamivir carboxylate in the body. In the geriatric
population differences in concentration of oseltamivir have been observed, as the rate of clearance
is reduced in elderly patients in comparison to young patients. Oseltamivir and oseltamivir
carboxylate do not show any interaction with human CYPs and UGTs while in the human body
43
.

42

Chapter 6 : Oseltamivir model
Background
Oseltamivir carboxylate is the active metabolite discovered to treat the Influenza virus
(respiratory infection). Oseltamivir is given to treat the viral infection during the
prophylaxis(preventive) state of the disease. The dose at which oseltamivir is administered is 75
mg twice daily for 5-6 consecutive days or 150my once daily. The prodrug form of oseltamivir
carboxylate; oseltamivir phosphate (OP) comes in a salt form and is administered orally to
enhance the therapeutic efficacy of the parent drug, increasing its bioavailability through better
absorption of the prodrug into the systemic circulation. The ethyl ester prodrug enhances the
lipophilicity of the active therapeutic parent form, thus causing rapid absorption into tissues
which then leads to its hydrolysis to OC
43
. Therefore, upon oral administration, OP undergoes
conversion to its active form by metabolic (hydrolytic) action of mainly hepatic esterases i.e.,
carboxylesterases I.
The Physiological Based Pharmacokinetic Modeling module found in GastroPlus™ was used for
the in-silico simulation of the plasma concentration-time curve of Oseltamivir carboxylate and its
prodrug Oseltamivir phosphate. The research aim was to develop a model for oseltamivir
carboxylate and its prodrug in GastroPlus™ and to compare the results with experimental data, to
validate the model by comparing the bioequivalence of the 2 methods. GastroPlus™ along with
ADMET Predictor® can predict the physicochemical and ADME properties of the two molecules
using the text known as smile string structures as input. Our goal was to build a PBPK model based
on a one-compartmental model of the respective drug molecules. The extrapolation from a one
compartment to a PBPK model was done because a PBPK model considers the role of all the
43

tissues or organs as different compartments and is a better representation than a single, one
compartment model.
Experimental data
Comparing the simulated data to the observed data helps to determine the bioequivalence of the
PBPK model. Concentration and time data were extracted from the data graph using the software
Engauge Digitizer
47
. The graph representing the experimental plasma concentration-time curve
has the concentration values in ng/ml units while the time interval is measured in hours.

Figure 6.1: Experimental curve representing the plasma concentration-time curve of OP and OC on administering 150 mg OP
orally. The data in the curve is obtained from a normal human volunteer of 70kg weight (adapted from Ref.40).
On extraction of the experimental data points from the graphical representation, we can save the
data in GastroPlus™ in the .opd file. The pharmacokinetic parameters of interest including Cmax,
Tmax, and AUC0-t, which were simulated using the prodrugs physicochemical properties and some
of the active drug’s ADME properties. After, the simulation of the plasma concentration-time
curve and the pharmacokinetic parameters were generated, the bioequivalence was calculated to
0.1
1
10
100
1000
0 5 10 15
OP-OC Concentration in plasma (ng/ml)
Time (hours)
Plasma Concentration-Time Curve
Experimental OP
Experimnetal OC
44

check whether the simulated prodrug and active drug were within the bioequivalence range of 80-
125%.
A one compartment model was structured first to determine the physicochemical, ADME, and
physiological parameters which could be extrapolated to construct a PBPK model, considering all
the tissues of the body. Extracting the in vitro and in vivo data through clinical experiments found
in the literature and developing a model in GastroPlus™ along with ADMET Predictor® predicted
data was the goal of the modeling. Our aim was to model the prodrug form of the drug in
GastroPlus™ and to simulate the prodrugs and their active forms PK concentration-time curve.
The purpose was to prepare a model which showed the increased bioavailability of the parent
compound when administered in the form of a prodrug rather than in its active form. The PBPK
modeling tools were inefficient in predicting the prodrug’s metabolic conversions and thus it was
a challenge to model the prodrugs and to simulate the active drug’s concentration-time profile.
Based on the in vitro experiments on oral administration of the prodrug, oseltamivir phosphate
reaches a maximum plasma concentration of 80ng/ml and that of oseltamivir carboxylate (initially
administered in the oral prodrug form) is 440ng/ml. Based on these results, we concluded that
OC, when administered as a prodrug, was absorbed better and was metabolized less than OP. The
maximum plasma concentration (Cmax) of OC was reached faster due to the immediate conversion
of oseltamivir-to-oseltamivir carboxylate on oral administration by intestinal and hepatic esterases.
The OP dose used for the model was 150mg in an immediate release or IR-capsule. Therefore,
when OC was given in the prodrug form it gave a better therapeutic efficacy against the
neuraminidase enzyme due to its higher concentration in the plasma
40
.

45

PBPK modeling of Oseltamivir prodrug
PBPK modeling was developed keeping in mind both the perfusion-rate-limited kinetics and
permeability-rate-limited kinetics. Perfusion-rate-limited kinetics can occur for a drug with lower
solubility, while permeability-rate-limited kinetics can occur for drugs that are hydrophilic in
nature, due to their limited entry through the lipid membrane. Both these modes of kinetics can
help determine the pharmacokinetics of the drugs. The novel approach used with GastroPlus™
was to start with the prodrug structure, already developed clinically to overcome perfusion or
permeability limited drawbacks
31
. Oseltamivir carboxylate is a lower lipophilic drug molecule
with poor permeability, but its prodrug is a lipophilic molecule. Thus, the main aim of our
research was to develop a human and rat prodrug model using GastroPlus™ as the modeling
tool. Our approach was to develop a model in GastroPlus™ which mimics the clinical in vivo
pharmacokinetic concept of prodrug absorption, distribution, conversion due to metabolic
enzymes, distribution of parent metabolite, and lastly elimination of both prodrug and parent
metabolite from the body of physiologic species like rat, mouse, cat, humans, guinea pig, dog,
rabbit.
Below is a flow chart diagram of various tabs of GastroPlus™ representing the living species'
physicochemical, physiological, and ADME properties. On importing the smile string structure
(saved as text files (.txt)) of the drug, the following tabs open which leads to simulating the plasma
concentration-time curve and pharmacokinetic properties of the following drug.

46

1. On import of the smile string, the following tab pops-up.

2. Compound tab signifying the physicochemical properties based on the chemical structure of drug/prodrug.

47

3. Physiology tab indicating the transit time and species name.

4. Pharmacokinetic tab (ADME properties).

48

5. Enzyme table representing the enzymatic conversion of prodrug OP by esterase (CES1).

6. Simulation tab representing the resultant PK parameters of the prodrug OP after its conversion to its active metabolite OC.

49

7. Graph of Plasma concentration(ng/ml) vs Time(hr). Note that the data points shown as squares are the experimental data and
the solid line is the predicted PK curve.

Figure 6.2: The seven tabs are the representation of simulation in GastroPlus™ of the plasma concentration-time curve of
oseltamivir phosphate OP on oral administration.
The panels in the figures are a short summary of the various parameters considered while preparing
a model in GastroPlus™. As per the figures, we can say that the PBPK model in GastroPlus™
takes into consideration all the required parameters of the human body to simulate an accurate
plasma-concentration time curve. Thus, it mimics the human body anatomically and
physiologically.
A PBPK model was developed in GastroPlus™ to assess the prodrug and the pharmacokinetics of
its active metabolite. The model focused on the role of: (1) LogP and permeability (Peff) of the
prodrug (OP); (2) Clearance of the prodrug and the drug from the liver and kidney; (3) Volume of
distribution of the prodrug and that of the active metabolite after conversion in tissues; and (4)
Enzymatic conversion (fitting Vmax and Km). All these steps sum up the building of the PBPK
model of oseltamivir.

50

Physiological parameters
An American human volunteer of 30 years and 70kg weight with no comorbidities was modeled
in GastroPlus™. On import of the oseltamivir phosphate structure, the default dosage form and
dose are changed from IR-Tablet (default dosage form in GastroPlus™) to an IR-capsule and a
100mg dose changed to a 150mg dose. The physiological parameters are parameterized based on
tissue volume and blood flow. Transit time of the stomach is thus set to be 0.15hours when the
dosage form is a capsule, as pharmaceutical products such as capsules are designed in a way that
do not stay too long in the gastric environment of the stomach.
Pharmacokinetic parameters
The prodrug was mainly developed to enhance the bioavailability and thus the bioavailability
factor(F) is positively optimized in the PBPK modeling of the prodrugs. The prodrug, when
converted into the parent drug, had better pharmacokinetic parameters (Table 2). The oral
bioavailability of OC might be slightly different from that of the experimental data because of the
cumulative effect of the PBPK model of the organs and tissues. The oral bioavailability in the
PBPK model was denoted as F%. The OC bioavailability (F%) was 77.68 based on oral OP
administration, while the experimental OC bioavailability was 75. A normal human volunteer of
30 years was considered for the model with an experimental clearance (CL) value of 18.8L/hr
39
.
The CL of OP and OC was mainly through the renal clearance process, with < than 20% through
feces. The blood to plasma ratio, and the fraction unbound are some of the pharmacokinetic
parameters predicted by GastroPlus™ with ADMET Predictor®. For OP and OC, transport
through the membrane was assumed to be a passive process as no transporters are involved in the
model because of the lipophilic nature of oseltamivir phosphate and the lack of experimental data
on transporters for OP. Oseltamivir phosphate is an ethyl ester prodrug that is metabolized by the
51

carboxylesterase family of enzymes. Oseltamivir phosphate is metabolized mainly in the liver,
and CES1 plays a foremost role in its conversion to its carboxylate parent metabolite. The
Michaels-Menten equation for the metabolism process played a role in the prodrug conversion in
the PBPK modeling. The Vmax and Km were the two respective parameters considered in the
enzyme table of GastroPlus™
28
.
Table 6.1: Parameters of OP and OC considered for the one compartment and the PBPK model. Observed data are experimental
data from literature while the predicted values are default GastroPlus™ and ADMET predictor® data.
Parameters Oseltamivir Phosphate Oseltamivir carboxylate
Observed value Predicted value Observed value Predicted value
Dose(mg) 150
40
100 150
40
100
Dosage form Capsule
40
Tablet Capsule
40
Tablet
LogP 0.4
48
1.14 -2.1
48
-1.85
pKa 7.7
48
7.37 3.8(acid),7.8(base)
48
3.38(acid),8.39(base)
Solubility
(mg/ml)
30
41
15.53 588
49
15.79
Permeability
(10
4
cm/s)
12 ± 22 · 10
−144
1.01
_
0.37
Stomach
Transit
Time(hours)
- 0.15 - 0.15
Clearance
(L/hr.)
20
40
14.71 18.8
40
8.93
Volume of
distribution
(L/kg)
_ 0.94 0.328-0.371
45
0.44
Half-life
(hours)
1-3
43
- 5-10
45
-

52

Table 6.2 Physicochemical and Pharmacokinetic data for the OP-OC simulation.

a- Predicted values from ADMET Predictor® used in Gastroplus™ simulation.
b- - Adjusted values for the simulation to obtain PK curve of OC in plasma considering the physicochemical properties of OP
(prodrug).
c- Vd calculated depended on the experimental Clearance value entered in the PBPK model.
d- Adjusted after determining the Vd with GastroPlus™ for the experimental Oseltamivir Carboxylate PK curve.

Table 6.3: Below are the factors responsible for active parent metabolite (OC) formation from Oseltamivir Phosphate.
Metabolic
Parameters
One compartment

PBPK model
OP OC with OP
parameters
OP OC with OP
parameters
Liver enzyme
Vmax (CES1)
0.318 0.001 0.000047 0.000001
Liver enzyme
Km (CES1)
2 2 2 2
Intestinal
%FPE
20 20 20 20

Parameters Oseltamivir Phosphate Oseltamivir Carboxylate
One
compartment
model
PBPK model One
compartment
model
PBPK model
LogP 1.14
a
1.14
a
1.14
b
1.14
b

Permeability(10
4
cm/s) 1.01
a
1.01
a
1.01
b
1.01
b

Stomach Transit time
(hours)
0.15 0.15 0.15 0.15
Clearance (L/hr.) 20 20 18.8 18.8
Volume of
distribution(L/kg)
0.94
a
1.12
c
2.4
d
1.9
c
Half-life (hrs.) 2.28 2.69 6.19 4.97
53

Results
Based on the added, physicochemical, physiological, and pharmacokinetic data of the drug and its
prodrug (Oseltamivir carboxylate and Oseltamivir phosphate, respectively), a predicted Cmax,
Tmax, AUC0-t, AUC0-inf, %F, and other parameters, were calculated. The calculated data was
then compared against the observed, experimental data. The process of prodrug pharmacokinetics
was thus explained through the modeling tool. The main step in the developmental process of any
prodrug is the absorption of the prodrug and then its conversion to the active parent metabolite.
The pharmacokinetics of the prodrug was designed in GastroPlus™ in a way that on absorption,
which is indicated as %Fa in GastroPlus™, it gets metabolized, some in the intestine denoted as
%FPE, and most in the liver by the esterase enzyme (CES1), which converts it the OC form. Once
OC is in the plasma, the clearance and the volume of distribution are similar to oseltamivir
carboxylate, as now the prodrug has been converted to its active form.
The graph representing the plasma concentration-time profile of oseltamivir phosphate and
oseltamivir carboxylate against the experimental data is shown below. One compartment and the
PBPK model both were taken into consideration for the concentration-time curves.
54

One compartment model

Figure 6.3: One compartment oseltamivir phosphate Concentration(ng/ml) vs Time(hr.) curve of the experimental and simulated
data.

Figure 6.4: One compartment oseltamivir carboxylate Concentration(ng/ml) vs Time(hr.) curve of the experimental and simulated
data.
0.1
1
10
100
0 5 10 15 20 25 30
Concentration(ng/ml)
Time(hr.)
One- Compartment
Plasma Concentration-Time Curve
Experimental OP Simulated OP
0.1
1
10
100
1000
0 5 10 15 20 25 30
Concentration(ng/ml)
Time(hr.)
One Compartment
Plasma Concentration-Time Curve
Experimental OC Simulated OC
55

PBPK Model

Figure 6.5: PBPK model of oseltamivir phosphate Concentration(ng/ml) vs Time(hr.) curve of the experimental and simulated
data.

Figure 6.6: PBPK model of oseltamivir carboxylate Concentration(ng/ml) vs Time(hr.) curve of the experimental and simulated
data.
The process of the prodrug to drug conversion and circulation is such that on administration,
oseltamivir phosphate gets absorbed and some of it gets metabolized in the intestine due to the
presence of trivial amounts of carboxylesterase enzyme. Most of the oseltamivir phosphate (ethyl
ester prodrug) escapes from the GUT and enters the liver through the portal vein where it
0.1
1
10
100
1000
0 5 10 15 20 25 30
Concentration(ng/ml)
Time(hr.)
Plasma Concentration-Time Curve
Experimental OC OC-PBPK
0.1
1
10
100
0 5 10 15 20 25 30
Concentration(ng/ml)
Time(hr.)
Plasma Concentration-Time Curve
Experimental OP OP-PBPK
56

experiences first-pass metabolism in the liver and where most of the prodrug (OP) gets converted.
Now, with most of the prodrug converted, the carboxylate metabolite has the potential to be
excreted in the bile from the gallbladder and could potentially be reabsorbed (note that absorption
is low for the metabolite) or eliminated in the feces. While OP remnants that were not converted
in the intestines or liver, can either be eliminated through the feces or by the kidneys and be
excreted through the urine. The whole operation of prodrug and drug pharmacokinetics can be
explained through the figure below.

Figure 6.7: Representation of the process of OP absorption, circulation, metabolism to OC, and then OC circulation to the site of
action and ultimately elimination from the body (adapted from Ref.33,34).
57

Chapter 7 : CURCUMIN
Curcumin is an anti-inflammatory, anti-oxidative therapeutic molecule used to treat disease
conditions like arthritis, and hyperlipidemia. It is a molecule present in turmeric which is a type of
spice used as an antiseptic in Indian culture. Curcumin is a good therapeutic drug molecule that
can treat diseases but has a drawback of poor bioavailability because of its very low solubility and
stability in the GI tract. Curcumin obtained from curcuma longa is a molecule affected by first
pass metabolism, affecting the bioavailability. It is a polyphenol drug molecule that has cellular
level drug activity to treat health conditions like cancer or cardiovascular diseases
50
. Due to its
poor oral bioavailability, curcumin prodrug derivatives were designed to enhance the oral
bioavailability and thus the therapeutic efficacy
51
.
A.
O
CH
3
O H
O
O
CH
3
OH
O
H

B.
O
CH
3
O
O
O
O
C H
3
O
CH
3
O
O
O
O
CH
3
O
H
O

58

C.
O
CH
3
O
O
O H
O
O
CH
3
O
O
O
H
O
OH
O

Figure 7.1: The structures are as follows: a. Curcumin, b. Curcumin diethyl disuccinate (CDD),
c. Curcumin diglutaric acid (CDG).
The curcumin derivatives studied were curcumin diethyl disuccinate and curcumin diglutaric acid.
Both prodrugs are designed to overcome poor bioavailability. Experimentally, the bioavailability
of curcumin is <1%, so it is not recommended or prescribed for more than a certain dose. The
reason for the low bioavailability of curcumin is because of its low water solubility. The absorption
profiles of the drug molecules were controlled through the physicochemical and metabolic
properties predicted from the ADMET Predictor® module in GastroPlus™.
The Physiological Based Pharmacokinetic Modeling in GastroPlus™ was used to simulate the
pharmacokinetic factors like Cmax, Tmax, AUC of the drug molecule which were validated with
the experimental drug properties of curcumin. Curcumin and its prodrug PBPK models were
designed in GastroPlus™ and were based on the rat physiology and experimental PK data from
rats. Both curcumin drug forms were compared to the experimental data and were checked for
bioequivalence with the simulated data. Curcumin and its 2 prodrugs, curcumin diethyl
disuccinate and curcumin diglutaric acid, were orally administered as a suspension to rats weighing
0.5kgs. The metabolic pathway of prodrug conversion was through hydrolysis by esterase
enzymes, to form curcumin and then glucuronidation leading to its clearance.

59

Steps used for the simulation:

Figure 7.2: Model of CDD conversion to curcumin, metabolism by various enzymes, distribution and circulation in the body, and
elimination and excretion (adapted from Ref. 51,52). The enzymes responsible for the prodrug conversion and drug metabolism
for curcumin, CDD and CDG is mentioned in the appendix below.

60

Predicted G a stro Plus™ data.
GastroPlus™ requires as input, a drug structure, which can be a simple text file known as a smile
strings (in the enol form) to represent the drug’s structure. The rat body weight, the molecular
weight, rat body mass index, organ weight, tissue volume and the blood flow to the tissues, were
automatically calculated. The simulations of curcumin and the prodrugs were based on an oral
suspension formulation, given at a 20mg dose. Physicochemical properties such as LogP,
solubility, permeability, particle size, pKa were predicted by the ADMET Predictor® module in
GastroPlus™. Physiological parameters like the fasted state in rats were predetermined in the
“Physiology tab” of GastroPlus™ All these parameters determined the pharmacokinetic values
like clearance, Vss and half-life of the drug in the Wistar rat with a weight of 0.5kgs. The
pharmacokinetic parameters in the “Pharmacokinetic tab” of GastroPlus™ were obtained through
in vitro studies and experimental data to optimize the pharmacokinetic data. The PBPK module,
which considers all the tissues or organs for the PK profile of curcumin, were selected from the
compartmental option of the “Pharmacokinetic tab”.

61

Table 7.1: Representation of some of the constant physiological, pharmacokinetic, and physicochemical parameters of curcumin,
CDD and CDG, either predicted or observed data of prodrug release model.
Parameter Curcumin Curcumin diethyl
disuccinate
Curcumin
diglutaric acid
Molecular weight 368.39 624.65 596.59
Initial dose (mg) 20 20 20
Dose volume (mL) 5 5 5
Dosage form Suspension Suspension Suspension
Effective permeability (cm/s) 6.56 x 10
4
1.58 x 10
4
1.09 x 10
4
Plasma fraction unbound (%) 5.12 5.08 5.11
Blood/plasma concentration
ratio
0.88 0.88 0.88

In vitro observed data: LogP and Solubility
Curcumin is a polyphenol medicinal drug derivative used to treat or prevent diseases. Curcumin,
present in turmeric, has a biopharmaceutical drawback of poor water solubility and stability,
therefore a succinate prodrug form i.e., curcumin diethyl disuccinate was developed with the aim
to enhance the bioavailability of curcumin. The low bioavailability of curcumin contributes to its
clinical insignificance and its low efficacy. The curcumin prodrug is such that it has better alkaline
stability in solution than curcumin but still low oral bioavailability. The reason for the low
bioavailability of curcumin from curcumin diethyl disuccinate (CDD) is its low aqueous
solubility
51
. To overcome this drawback another prodrug form, curcumin diglutaric acid (CDG)
was designed with a better solubility profile, thus it has the potential to optimize the
62

pharmacokinetic profile of curcumin upon release from CDG
53
. The LogP defines the lipophilicity
of the drug molecule. Lipophilicity of the drug molecule contributes to its permeability and its
volume of distribution (Vss) to the tissues.
Simulation parameters
A simulation of a single individual oral dose in rats was run to calculate the Cmax, Tmax, AUC0t,
and F% of the drug molecules based on data. A visual representation is presented in the form of
graphs of the plasma concentration-time curve (ng/ml vs hr.)
The predicted data from ADMET Predictor® and in vitro experimental data were combined to
simulate a PK plasma concentration-time curve for curcumin. The experimental PK data points
helped to validate the simulated PK curve of curcumin release in plasma. Validation of the model
is required as it makes sure the necessary parameters are playing a role in the pharmacokinetics of
curcumin. The curcumin prodrugs, curcumin diethyl disuccinate and curcumin diglutaric acid,
have different pharmacokinetic profile before their conversion to curcumin. The curcumin release
simulation is based on the 20 mg suspension form in Wistar rats. For the CDD and CDG PBPK
model, an approach of importing the smile string structure of curcumin was used, but the
physicochemical properties values like solubility, LogP, and permeability were from the respective
prodrugs. Curcumin experiences metabolism by lumped enzymes (UGTs and other CYPs) in small
amounts present in the liver and intestine, causing its conversion to glucuronide or sulfate
conjugates. The curcumin stepwise reduction to dihydrocurcumin-tetrahydrocurcumin-
hexahydrocurcumin-octahydrocurcumin-glucuronidederivative and glucuronidation leads to
glucuronide
52
. The metabolite formed in the liver can be reabsorbed back through the intestines by
enterohepatic circulation or eliminated by fecal excretion. Rats do not have gall bladders and thus
63

there can be a direct excretion with bile, then absorption back through the intestines, which causes
a second curve observed in the plasma concentration-time curve of curcumin
51
.
Table 7.2: Simulation data obtained from the physicochemical and pharmacokinetic profile of curcumin. For the simulation, both
observed and predicted values were considered:
Run Adjustment Solubility
(mg/ml)
Log P Clearance
(L/hr.)
Vd
(L)
Cmax
(ng/ml)
Tmax
(h)
AUC0-t
(ng-h/ml)
1
Default settings
0.048
a
3.52

0

0.357

1311.6 1.95 7330.3
2
Experimental
solubility
0.00043
51
3.52

0

0.357

9.94 3.6 176.29
3
Experimental
LogP
0.00043

2.23
55
0

0.098

8.66 2.83 53.04
4
Fitting to t1/2 of
1.98hrs.
0.00043

2.23

0.034

0.098 0.16 0.933 0.54

Experimental
data
0.00043 2.23 0.17
51
0.75
51
0.50
51
a-Solubility at pH 5.33.
Curcumin diethyl disuccinate and curcumin diglutaric acid prodrug forms can be hydrolyzed to
curcumin through the metabolic action of carboxylesterase-I enzyme present mainly in the liver
with low concentrations present in the intestines
51
. CDD is protected from GUT metabolic
enzymes like CYP2C9, CYP1A2, however, there is still some loss as a certain percent gets
converted in the intestine or excreted and was represented as %FPE (First Pass Extraction) in the
simulation. The solubility in both the prodrug forms were higher than the original curcumin drug.
But still, the bioavailability of curcumin released from CDD is no better than curcumin. The default
pharmacokinetic parameters were solely based on the predicted GastroPlus™ values while in
addition to in vitro PK data a PBPK model for curcumin was developed. The half-life (t1/2) was
64

fitted for all 3 simulations to the half-life of curcumin, or 1.98hrs
50
.. The bioavailability of
curcumin released from CDD was still <1% as per the simulation.
Table 7.3: Simulation data obtained from the physicochemical and pharmacokinetic profile of curcumin released from CDD. For
the simulation, both observed and predicted values were considered:
Run Adjustment Solubility
(mg/ml)
Log P Clearance
(L/hr.)
Vd
(L)
Cmax
(ng/ml)
Tmax (h) AUC0-t
(ng-h/ml)
1
Default settings
0.0033
a
4.37 0 0 696.75 8 3622.3
2
Experimental
solubility
0.000059
51

4.37 0 0 12.53 8 65.06
3
Experimental
LogP
0.000059 2.55
51
0 0.143 5.87 8 39.37
4
Fitting to t1/2
0.000059 2.55 0.034 0.098 28.57 2.75 159.47
5 Prodrug release
model
b

0.000059 2.55 0.034 0.098 0.12 0.75 0.35

Experimental
data
0.000059 2.55
0.11
51
0.75
51
0.23
51

a-Solubility at pH 6.17.
b-The Prodrug release model is curcumin released from CDD.
Curcumin diglutaric acid was another prodrug which was developed because CDD was still
inefficient in increasing the bioavailability of curcumin. The CDG pharmacokinetic parameters
were developed and based on the PK model used for CDD. There was no experimental PK data
or experimental evidence for CDG, therefore, CDD was used as a model. The simulated
65

bioavailability of curcumin released from CDG was still low. The simulated %F of curcumin
released from CDG was 0.024. All simulated rat PK parameters were compared among the 3
curcumin forms, with CDG showing the highest bioavailability. Curcumin diglutaric acid follows
the same metabolic pathway as that of CDD, where the main metabolic site is the liver with some
percent of CDG being converted in the intestine, which accounted for the %FPE in the simulation.
The simulated PK data obtained from the PBPK model of GastroPlus™ considering the rat
physiology is stated in the below table:
Table 7.4: Simulation data obtained from the physicochemical and pharmacokinetic profile of curcumin released form CDG. For
the simulation, both observed and predicted values were considered:
Run Adjustment Solubility (mg/ml) Log P Cmax
(ng/ml)
Tmax (h) AUC0-t
(ng-h/ml)
1
Default settings
0.0848
a
3.11 2.8*10
5
8 2.13*10
6
2
Experimental
solubility
0.00748
53
3.11 2.79*10
5
8 2.07*10
6
3
Experimental LogP
0.00748 1.79
49
2.7*10
5
8 2.03*10
6
4

Fitting to t1/2
0.00748 1.79 2.06*10
5
0.93 7.0*10
5
5 Prodrug release
model
b

0.00748 1.79 11.239 0.75 29.563

Experimental
CDD data
0.000059
51
2.55
51
0.11
51
0.75
51
0.23
51

Experimental
Curcumin data
0.00043
51
2.23
55
0.17
51
0.75
51
0.50
51

a-Solubility at pH 4.05.
b-The Prodrug release model is curcumin released from CDG.

66

Table 7.5: Represents the enzymes used for the simulation of curcumin, CDD and CDG plasma concentration-time curves.
Metabolic enzymes are listed with their respective Vmax and Km values in the above table. In the brackets are the site of
metabolism .
a-The enzymatic values in liver, GUT and PBPK sites are the general predicted values from ADMET Predictor®.
b- LumpedMP enzyme is manually added as to represent the remaining enzymes involved in curcumin metabolism.
c- CES1 is the esterase enzyme for the prodrug conversion.
d- %FPE for CDD and CDG is for loss in intestine either through hydrolysis or excretion.

Metabolic Enzymes Curcumin PBPK
model
Curcumin diethyl disuccinate
PBPK model
Curcumin diglutaric acid PBPK
model

Vmax
(mg/s)

Km
(mg/L)

Vmax
(mg/s)

Km
(mg/L)

Vmax
(mg/s)

Km
(mg/L)
CYP1a2(PBPK)
a
0.00453 16.66 0.00453 16.66 0.00453 16.66
CYP1a2(LIVER)
a
0.00162 16.66 0.00162 16.66 0.00162 16.66
CYP2C9(GUT)
a
0.00478 0.78 - - - -
CYP2C9(LIVER)
a
0.00478 0.78 0.00478 0.78 0.00478 0.78
CYP2C9(PBPK)
a
0.00141 0.78 0.00141 0.78 0.00141 0.78
LumpedMP
(PBPK)
b
0.000002 0.78 0.00041 0.78 0.00041 0.78
CES1(PBPK)
c
- - 0.001 0.78 0.001 0.78
Intestinal %FPE
d
17 17
67

Results
The graph representing the curcumin plasma concentration-time profile was obtained through
GastroPlus™. Using the PK curve, we compared the experimental data to that of the simulated
curcumin data. The curcumin concentration in the plasma after oral administration of curcumin
was compared to the in vivo experimental rat data and is shown in figure (7.3). The second
graphical representation (figure 7.4) is of curcumin release from the CDD prodrug form, compared
to the observed experimental data. The final figure (7.5) is of CDG released curcumin, designed
by using the prodrug model from CDD. The results represent the Cmax, Tmax, AUC0-t, %F and
other parameters of curcumin in the plasma. Based on the simulated data, it appears that CDG
might be a better prodrug than CDD but did not better than curcumin. Bioequivalence calculations
were then conducted to compare and validate the models and to compare them to the experimental
data.

Figure 7.3: Representation of the plasma concentration-time profile of simulated curcumin vs experimental curcumin in Wistar
rats. The concentration is measured as ng/ml and time as hours.
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
0 2 4 6 8 10
Concentration(ng/ml)
Time(hr.)
Plasma Concentration-Time Curve
Exp Curcumin
Simulated
Curcumin
68

Figure 7.4: Representation of the plasma concentration-time profile of simulated curcumin release from CDD vs experimental
curcumin release from CDD in Wistar rats. The concentration is measured as ng/ml and time as hours.

Figure 7.5: Representation of the plasma concentration-time profile of simulated curcumin release from CDG vs experimental
curcumin release from CDD in Wistar rats. The concentration is measured as ng/ml and time as hours.

0.0001
0.001
0.01
0.1
1
0 2 4 6 8 10
Concentration(ng/ml)
Time(hr)
Plasma Concentration-Time Curve
Experimental
Curcumin release
from CDD
Simulated
Curcumin released
from CDD
0.01
0.1
1
10
100
0 2 4 6 8 10
Concentration(ng/ml)
Time(hr.)
CDG Plasma Concentration-Time Curve
Experimental Curcumin
released from CDD
Simulated Curcumin
released from CDG
69

Chapter 8 : Bioequivalence
Bioequivalence is considered when comparing drug substances that are not statistically different
from each other. Bioequivalence between two drugs is measured using the same dosage form,
strength, dose, quality, purity, and manufacturing labelling. Bioequivalence is a term used when
the reference drug and the test drug are within the decided range of equivalence of 80%-125% In
other words, bioequivalence is the ratio of the reference drug and test drug, calculated through
pharmacokinetic parameters like Cmax, Tmax, AUC0-t, AUC0-inf
56,58
.
In our in-silico experiments, we compared the experimental drug parameters to the simulated
GastroPlus™ data and used bioequivalence to validate our results. Through bioequivalence
calculations we calibrated the PBPK model in GastroPlus™. The comparable bioequivalence
range is 80-125% for our study. The concept of bioequivalence was introduced to approve generic
drug products, and to avoid issues related to safety, efficacy, and the quality of generics.
Bioequivalent drugs are intended to show the same pharmacological effect and bioavailability by
having similar rates and extent of absorption when administered. Generic drugs marketed today
need to obtain permission from the FDA by conducting bioequivalence test developed by
regulatory bodies before they can be marketed at lower prices. The era of marketing generic drugs
was started when the cost of pharmaceutical medications escalated during a period late in the
1960s. In the 1970s and 80s a panel for bioequivalence was formed to discuss the regulations for
assessing the bioequivalence and bioavailability of drugs. Various statistical methods were
considered by the FDA like confidence interval, BE hypotheses, and Bayesian approaches to study
the bioequivalence of drugs
59
. In 1984, the FDA decided to approve generic drugs through various
applications submitted to regulatory agencies, with titles like abbreviated new drug application or
ANDA and ANADA, along with data that shows bioequivalence to the reference listed drug. These
70

applications should be completed before marketing the new reformulated drugs. Brand drugs are
given a monopoly to market the reference drugs for a certain period (~20 years) before the generic
drugs or similar products can enter the market
59,60
.
The GastroPlus™ PBPK model is an in-silico method that is currently used in place of animals for
discovering thousands of drug molecules and for the development process. A PBPK model can
extrapolate in vitro data of a drug substance and simulate a plasma concentration-time profile. One
way to validate the data is to measure the bioequivalence between the observed experimental data
and the GastroPlus™ simulated data. Bioequivalence is used to check if two drug molecules,
reference, and test drugs, give similar pharmacokinetic outcomes, which can be determined by
comparing pharmacokinetic endpoints like AUC0-t, Cmax, and Tmax
58,61
. All these parameters
thus help to compare the therapeutic and potential drug toxicity of the test drug to that of the
reference drug
61
.
In our model, the prodrugs were prepared based on the combined, predicted physicochemical
properties and extrapolated from in vitro and in vivo observed data. Our interest was to determine
how prodrugs behave in an in-silico environment and compare the results to experimental
conditions. We wanted to be able to predict the prodrug PK behavior in silico. GastroPlus™ is a
tool to measure the plasma concentration-time profile using predicted data points, but is unable,
currently, to predict a prodrug pharmacokinetic profile. Therefore, the model imports the drug
structure but uses the prodrug parameters of LogP, solubility, and permeability from the prodrug
and attempts to measure the active metabolite concentration profile in the plasma, using the
imported drug structure. Measuring bioequivalence of our model was a way to calibrate the
prodrug model as well as compare our model to the observed experimental data. The
bioequivalence range is set to be 80-125% by regulatory agencies and within that range our product
71

would not be considered significantly different. Before I begin a discussion of how the ratio of
the pharmacokinetic parameters were calculated (AUC0-t, Cmax, Tmax), I would like to specify
that the BE range is set to be 80-125% and not 80-120% because the pharmacokinetic parameters
we were considering for the BE study were in natural log-transformed form (arbitrary form) and
so for a normally distributed form we need to consider the range of BE to be 80-125%. For a
statistical calculation, the normally distributed form of data points is required as it gives better and
more reliable results
56,57
.
Table 8.1:Bioequivalence study to determine statistical significance
57
.
Drug Cmax Tmax AUC0-t
OP (one compartment) 80/80.14
= 99%
2/1.84
= 108%
291.18/232.4
= 125%
OC (one compartment) 440/459.6
= 95%
4/3.92
= 102%
6070.1/5802.3
= 104%
OP (PBPK) 80/80.35
= 98%
2/1.76
= 113%
291.18/241.42
= 120.6%
OC (PBPK) 440/505.75
= 86%
4/3.28
= 121%
6070.1/4652.9
= 130%
Curcumin (PBPK) 0.17/0.16
=100%
0.75/0.93
=80.3%
0.61/0.5
=112%
CDD(PBPK) 0.11/0.12 =89% 0.75/0.75 =100% 0.46/0.39
= 130.6%
From the table, OP = Oseltamivir phosphate, OC = Oseltamivir carboxylate, CDD = Curcumin Diethyl Disuccinate.
Table8.1, above shows the calculation of the percent bioequivalence of GastroPlus™ PK
parameters in correspondence to the experimental observed data. As the range of BE is set to be
80-125% by the federal agency, most of the PK parameters in table 8.1 are within the range. Only
the AUC0-t of OC(PBPK) and CDD (PBPK) are slightly above the given range
56
. Thus, we can
state that our model of Oseltamivir and curcumin prodrugs are bioequivalent to the experimental
72

procedure and can be used to simulate the plasma concentration-time profile of prodrugs.
Curcumin diglutaric acid (CDG) is not checked for the bioequivalence because there is not
experimental/test data for CDG releasing curcumin form.

73

Conclusion
In simple terms, physiological-based pharmacokinetic modeling is a tool that may be used to
reduce or eliminate clinical trials and wasteful experiments on humans or other species for the
development of drugs marketed in different formulations, different doses, different populations, or
for the development of generic forms. However, in-silico Physiological Based Pharmacokinetic
Modeling is a tool that has its drawbacks, such as not being able to predict the metabolic conversion
of a prodrug to an active parent metabolite. Thus, to overcome the drawback, we developed a
prodrug model in a way that attempts to predict the conversion. Replacement by modeling tools is
a good source of procuring pharmacokinetic knowledge based upon mathematical equations for
the drugs. For our prodrug model, initially, we chose the prodrug Oseltamivir phosphate, because
it can be absorbed by passive diffusion which avoids the use of transporters (both influx and efflux)
in our model. The reason for not considering a prodrug or drug absorbed by transporters in our
model was to not complicate the model and to minimize the number of pharmacokinetic
parameters in obtaining the parent drug concentration in plasma. The second prodrug model which
is of curcumin is based on the same ideology and approach. Thus, the pharmacokinetics of the
drug in the plasma is obtained through esterase (CES1) metabolism in the liver and passive
diffusion through the membrane. Based on the bioequivalence study of the prodrugs using
GastroPlus™ PK data and experimental data, we can conclude that the GastroPlus™ PK modeling
tool is a good resource to obtain pharmacokinetics properties of drugs. Thus, the predicted data
from ADMET predictor® and the in vitro and in vivo preclinical data from experiments combined,
facilitated the development of the prodrug PK model.
74

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Asset Metadata

Creator Parikh, Sanjana Sanjay (author)

Core Title In-silico physiological based pharmacokinetic modeling of prodrugs

Contributor Electronically uploaded by the author (provenance)

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Degree Conferral Date 2021-08

Publication Date 07/27/2021

Defense Date 07/23/2021

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag absorption,bioavailability,CES1,curcumin,curcumin diethyl disuccinate,curcumin diglutaric acid,distribution,esterase enzyme,excretion,metabolism,oai:digitallibrary.usc.edu:usctheses,OAI-PMH Harvest,oseltamivir carboxylate,oseltamivir phosphate,pharmacokinetics,physiological based pharmacokinetic modeling (PBPK),prodrug

Format application/pdf (imt)

Language English

Advisor Romero, Rebecca (committee chair), Haworth, Ian (committee member), Zaro, Jennica (committee member)

Creator Email parikhs@usc.edu,sanjana.parikh64@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC15623317

Unique identifier UC15623317

Legacy Identifier etd-ParikhSanj-9896

Document Type Thesis

Format application/pdf (imt)

Rights Parikh, Sanjana Sanjay

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given.

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Repository Email uscdl@usc.edu

Abstract (if available)

Abstract The technology known as Physiological Based Pharmacokinetic (PBPK) modeling is an in-silico tool used by researchers and scientists to derive the pharmacokinetic (PK) concentration-time curve based on a drug molecule’s physicochemical properties, as well as on the species physiological characteristics. In vitro to in vivo extrapolation (IVIVE) approaches along with in-silico predictions on drug molecules help to parameterize the PBPK model. PBPK modeling allows researchers to study the effect of changes in species physiology or a drug's physical-chemical properties in-silico and prior to dosing patient populations. This makes PBPK modeling a cost-effective and time-saving predictive tool for bioavailability and pharmacokinetic studies. Prodrugs are designed to overcome biopharmaceutical and pharmacokinetic drawbacks like poor solubility or permeability which causes reduced oral bioavailability. Developing a PBPK model for prodrugs is challenging because prodrugs are initially pharmacologically inactive, which on systemic absorption and metabolism convert to the pharmacologically active molecule which is difficult to predict with PBPK modeling tools. The prodrug PBPK model is based on both the initial structural properties of an inactive prodrug form and on the active parent metabolite. In our experimental model, we have considered two prodrugs: Oseltamivir and Curcumin prodrug analogs. An In-silico PBPK model of the two prodrugs using their physicochemical and physiological properties and comparing the results to experimental pharmacokinetic characteristics of both the active and inactive parent molecule is the focus of our current research.