1.

0 Introduction
1.1 Drug Discovery and Development

A drug discovery program initiates because there is a disease or clinical condition without
suitable medical products available and it is this unmet clinical need which is the
underlying driving motivation for the project. The initial research, often occurring in
academia, generates data to develop a hypothesis that the inhibition or activation of a
protein or pathway will result in a therapeutic effect in a disease state [1]. The drug
development process is typically divided into three major steps: discovery, preclinical
development, and clinical trial. The transition from discovery to preclinical development
is a continuum, and results of preliminary pharmacology and toxicology testing often
contribute to lead drug candidate selection [2].
Discovery and development of a new drug is a long, labour-demanding process. Recent
studies revealed that the average time to discover, develop and approve a new drug in the
United States has steadily increased from 8.1 years in the 1960s to 14.2 years by the
1990s. Typically, the whole process is fragmented into Discovery, Development and
Registration phases.

The Discovery phase, routinely three to four years, involves identification of new
therapeutic targets, lead finding and prioritization, lead optimization and nomination
of new chemical entities (NCEs).
The Development phase, drug candidates are subject to preclinical testing in animals
(also known as Phase I) for about two years. In addition, the preliminary clinical
trials and the subsequent full clinical trials (known as Phase II and III) will take a
much longer time (~8.6 years).
The Registration phase averaged around 1.8 years in the late 1990s. Each
pharmaceutical firm has their own collection of drug candidates which are being
allocated to different phases of the discovery and development process, often referred
to as the pipeline.

Discovery scientists primarily focus on hunting for drug candidates, whereas in

Development one is trying to promote NCEs as novel and safer commercial medicines
[3]. Pharmacokinetics (PK) and toxicology traditionally had a minor role during
discovery. Organizations implemented rigorous testing during candidate selection to
ensure that compounds with poor properties did not advance. Implementation of this
strategy revealed another need: series structure-activity relationship (SAR) was improved
during early stages but the candidates were later rejected for inadequate PK or safety.
These failures consumed resources, time and enthusiasm that could be expended on other
series. Studies of drug databases showed that successful drugs tend to have drug-like
properties. Drug-likeness, when viewed at the in vivo level, is thought of in terms of PK
and safety. These complex in vivo properties result from an interaction of
physicochemical and structural properties, such as solubility, permeability and stability,
which are studied in vitro. These properties are, in turn, dictated by fundamental
molecular properties, such as molecular weight, hydrogen bonding and polarity which are

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studied in silico. As a result of the importance of properties, a new strategy emerged:
testing the drug-like properties of compounds during early discovery using high
throughput property methods in silico, in vitro and in vivo (often termed pharmaceutical
profiling) [4].

Key: IND: Investigational New Drug Application, NDA: New Drug Application, BLA: biologics Licence Application.
Figure 1.1 (a) Representation of drug discovery and development

1.2 Key pharmacokinetic parameters

1.2.1 Bioavailability

Bioavailability (F) is the extent to which an active moiety is absorbed from a

pharmaceutical dosage form and becomes available in the systemic circulation. Oral
bioavailability is one of the key considerations for discovery and development of NCEs.
Bioavailability is usually determined by calculating the respective plasma drug exposure
assessed as the total area under the drug plasma concentration versus time curve profile
termed as 'area under the curve (AUC)' after oral and intravenous (IV) administration as:

Absolute Bioavailability =

1.2.2 Clearance

Clearance describes the process of drug elimination from the body or from a single organ
without identifying the individual processes involved. Clearance may be defined as the
volume of fluid cleared the drug from the body per unit of time. The units for clearance
are milliliters per minute (mL/min) or liters per hour (L/h) [5]. Assuming liver and /or
kidney as major organs involved in drug elimination, prediction of hepatic and renal
clearance are considered most important in predicting PK characteristics of new chemical
entities.

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1.2.3 Apparent Volume of distribution

The concentration of drug in the plasma or tissues depends on the amount of drug
systemically absorbed and the volume in which the drug is distributed. The apparent
volume of distribution, in general relates the plasma drug concentration to the amount of
drug present in the body. Vapp is used to represent the apparent volume of distribution [5].

Where, Cp is the plasma drug concentration, C t is the tissue drug concentration, DB is the
amount of drug in the body, Vp is the plasma fluid volume, Vt is the tissue volume.

1.2.4 Elimination Half-Life

The relationship between half-life (t1/2), volume of distribution (Vd) and systemic
clearance (Cl) can be shown in the following equation:

Thus, the half life of any drug is a function of its blood and tissue binding as well as its
total clearance and is a derived parameter from Cl and Vd. For drugs with high clearance,
the half-life is restively independent of changes in intrinsic clearance, whereas for drugs
with low clearance, an increase in intrinsic clearance results in decreased half-life.

1.3 Preclinical pharmacokinetics

In drug discovery, initial PK studies are most commonly conducted in rodents and/or the
other species for the assessment of in-vivo efficacy. Subsequently, experiments in a large
animal species such as dog or monkey are performed to better describe the disposition of
a compound (in multiple species), support toxicology studies, and generate data useful in
predicting human PK parameters [6].
The drug development process involves several steps, from target identification,
screening, lead generation and optimization, preclinical and clinical studies to final
registration [7]. The average time to discover, develop and approve a new drug in the
United States has steadily increased from 8.1 years in the 1960s to 14.2 years by the
1990s. The actual time may be even longer as the above calculation considered the
starting point from chemical synthesis, thereby not including the time for target
identification and lead finding/selection process, which in general takes a minimum of
two more years [8]. The costs of discovering and developing a drug were of the order of
US $804 million [9]; current estimates are closer to about US $900 million. Despite the
huge investment on research and development the average success rate for drug
candidates entering development Phase I is only around 11%, which means that most of
the compounds that are being worked on will end up in the waste bin, with no return on
the expenditure for the investment [8]. The failure of drug candidates can be attributed to
numerous reasons such as:

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 The selection of improper targets in early phases that lack proof of concept in man.
High attrition rate during development phases due to poor PKs.
Poor toxicological and safety-related pharmacological properties.
Elongated discovery and development time course.

Kennedy analyzed the causes by which 198 NCEs failed in clinical development [10] and
found that the most prominent cause of the failures was associated with poor PK and
ADME properties as shown in the figure 1.3 (a). Although lack of efficacy was still one
of the main reasons for terminations, the unsatisfactory PK/ADME, toxicology and
adverse effects accounted for up to two-thirds of the total failures [8].

Figure 1.3 (a) Kennedy analysis of the reasons for failure of 198 drug candidates in
clinical development

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 Figure 1.3 (b) Bar representations of reasons for drug attrition (1991-2000)
In 1991, adverse PK and bioavailability results were the most significant cause of
attrition and accounted for ~40% of all attrition as shown in the figure 1.3 (b). During the
past years, pharmaceutical companies have invested and introduced a number of new
approaches dedicated to improving the rate of success of development of new drugs.
Careful pre-clinical PK assessment was one among the approaches to improve success in
drug development. As a result by 2000, poor PK as a cause of attrition in drug
development had dramatically reduced and contributed less than 10% [11].
The sole purpose of preclinical ADME work is to ensure that compounds do not fail in
the clinic due to ADME reasons [12]. Preclinical PK helps in sorting the compounds for
further clinical studies. Considering the above statistics, the one can realize the
importance of proper PK in the success of a drug. Hence, to ensure the success of a drug
development, the drug candidates must possess good bioavailability and appropriate t 1/2,
this attribute to a better understanding of the PK data and factors that affect the PKs will
guide the rational drug design. The drug should possess the desirable t 1/2 of plasma is one
of the important determinant factors of a dosage regimen. The short t1/2 of drugs attributes
to frequent dosing, poor patient compliance. The metabolic stability and t 1/2 of the drug
can be improved for drugs which are highly cleared and short t 1/2 by chemical
modification approach of drug in drug design. The drugs having a long apparent half-life
in tissues than the plasma half-life is responsible for the potential accumulation in the
body and leads to the toxicity of specific drugs. Therefore, an understanding of PK data
of a drug and their metabolite formation is crucial for anticipating therapeutic efficacy
and also useful in explaining the toxicity of particular drugs.
The ultimate goal of PK is to give patients maximal benefits of the drug by close and
accurate measurement of the drug and/or its metabolites in different biological matrices,
thus offering optimal drug management and patient care. The use of PK for better patient
care includes:

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 Individualization of patient dose and dosing regimen.
Assessment of the bioavailability and bioequivalence of the drug by the proposed
routes.
Aid in determining the mechanism of drug-drug interaction and their avoidance.
Prediction of PK in man, from results obtained in animals.
Identification of optimum methods to accelerate drug elimination from the body in the
case of toxicity and/or over dosage. Identification of active metabolites of drugs and
quantification of their role in producing the overall response following drug
administration.

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2.0LITERATURE REVIEW
2.1 Importance of Pharmacokinetic Profiling in Drug Discovery
The low success rate of drug discovery programs in the present scenario can be attributed
to various factors such as; complexities of current diseases, high entry bar for new drugs
because of enhanced standards of care and stringent regulatory guidelines. What used to
be acceptable earlier is no more acceptable. The prime objective of a regulatory authority
is to improve public life and therefore the regulators try to evaluate the important aspects
of safety and efficacy and measure the risks and benefits of a new drug [6]. ADME work
generally begins with the identification of a hit and continues throughout the lead
optimization, selection and development stages. To a large extent the outcome of a drug
development program is predetermined at the point of candidate selection. In the early
pre-clinical phase, it is not essential to obtain a complete pharmacokinetic (PK) profile of
a compound but rather a rank order would suffice. At this juncture, the pace at which the
weak compounds are identified and eliminated governs the speed of a drug discovery
program. The candidates that exhibit undesirable PK profile are of little or no interest and
their elimination enables the discovery scientists to concentrate on fewer potential lead
compounds. Full PK screening of selected compounds can be finally performed on
relatively large number of animals (n=10) by administering a single dose of a compound,
to reconfirm the results of in-vivo high throughput PK and generate statistically accurate
PK parameters. This would also facilitate the identification of in vivo metabolites, which
are quite difficult to identify in a jumbled up chromatogram of a cassette comprising of
several compounds. The candidates that successfully clear the in vivo screen can be
subjected to various in vitro assays which have been designed to determine the rate and
extent of metabolism across various species and predict the likelihood drug-drug
interactions (DDIs) in human. The use of large number of animals for repeat PK in order
to reconfirm the results can be questioned from an ethical and cost perspective which is
of growing importance to the current pharmaceutical industry. The solution to this
problem probably lies with the acceptability of preclinical human micro-dosing (HMD)
studies early in the preclinical stage [6].

2.2 Pharmaceutical Industry as a Key Asset to Scientific and Medical

Progress
Thanks to advances in science and technology, the researchbased pharmaceutical
industry is entering an exciting new era in medicines development. Some major steps in
biopharmaceutical research, complemented by many smaller steps, have allowed for
reductions in mortality, for instance from HIV/AIDSrelated causes and a number of
cancers. High blood pressure and cardiovascular disease can be controlled with
antihypertensive and cholesterollowering medicines [7].

2.3 Importance of Pharmaceutical Research and Development

In 2014, the pharmaceutical industry invested nearly 30,900 million in research and
development (R&D) in Europe. A decade of strong US market dominance led to a shift of
economic and research activity towards the US from 19952005. Additionally, Europe is

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now facing increasing competition from emerging economies: rapid growth in the market
and research environments in countries such as Brazil and China is contributing to the
move of economic and research activities to nonEuropean markets. The geographical
balance of the pharmaceutical market and ultimately the R&D base is likely to shift
gradually towards emerging economies.

Figure 2.3 Allocation of R&D Investment by function (%).

2.4 Indian Pharmaceutical Industry Analysis

The Indian pharmaceuticals market increased at a compound annual growth rate (CAGR)
of 17.46% during 2005-16 with the market increasing from US$ 6 billion in 2005 to US$
36.7 billion in 2016 and is expected to expand at a CAGR of 15.92% to US$ 55 billion by
2020. By 2020, India is likely to be among the top three pharmaceutical markets by
incremental growth and sixth largest market globally in absolute size. Indias cost of
production is significantly lower than that of the US and almost half of that of Europe. It
gives a competitive edge to India over others [8].

(US$)

Figure 2.4 Revenue of Indian pharmaceutical sector.

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2.5 Bioanalytical method development
Bioanalytical methods are employed for the quantitative determination of drugs and their
metabolites in biological matrices, in all stages of the drug development process [11].
These methods such as gas chromatography (GC), high-pressure liquid chromatography
(HPLC), combined GC and LC mass spectrometric (MS) procedures, such as LC-MS,
LC-MS-MS, GC-MS, and GC-MS-MS, ligand binding assays (LBAs), immunological
and microbiological assays are performed for the quantitative determination of drugs
and/or metabolites, and therapeutic proteins in biological matrices, such as blood, serum,
plasma, urine, tissue, and skin [12]. Bioanalytical method consists of two main
components:

2.5.1 Sample preparation

Sample preparation is a technique used to clean up a sample before analysis and/or to

concentrate a sample to improve its detection. When samples are biological fluids such as
plasma, serum or urine, this technique is described as bioanalytical sample preparation
[13]. The determination of drug concentrations in biological fluids yields the data used to
understand the time course of drug action, or PK, in animals and man and is an essential
component of the drug discovery and development process [14].

2.5.2 Detection of the compound

The detector of choice is a mass spectrometer. Currently, the principle technique used in
quantitative bioanalysis is LC-MS/MS using either electrospray ionization (ESI) or
atmospheric pressure chemical ionization (APCI) techniques [14]. Before a bioanalytical
method can be implemented for routine use, it is widely recognized that it must first be
validated to demonstrate that it is suitable for its intended purpose. A Good Laboratory
Practices (GLP) validated bioanalytical method is needed to support all development
studies (e.g., toxicology studies and human clinical trials).

2.6 Method validation

The concept of validation has expanded through the years to embrace a wide range of
activities from analytical methods used for the quality control of drug substances and
drug products to computerized systems for clinical trials, labelling or process control,
Validation is founded on, but not prescribed by regulatory requirements and is best
viewed as an important and integral part of current good manufacturing practices (cGMP)
[15]. Method validation is the process of establishing documented evidence which
provides high degree of assurance that product (equipment) will meet the requirements
for the intended analytical applications [16].

2.6.1 Importance of validation

Assurance of quality
Time-bound
Process optimization

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 Reduction of quality cost
Reduction in rejections
Increased output
More rapid and reliable start-up of new equipment
Government regulation (Compliance with validation requirements is necessary for
obtaining approval to manufacture and to introduce new products) [17, 18].

2.6.2 Full validation

Full validation is important when developing and implementing a bioanalytical method

for the first time analysis of novel compound. A full validation of the revised assay is
important if metabolites are added to an existing assay for quantification [12].

2.6.3 Partial validation

Partial validations are conducted to validate modifications of previously fully validated

bioanalytical methods. Partial validations can range from an intra- or inter-assay
precision and accuracy experiment to nearly a full validation. Partial validations are
performed to assess the validity of a change in method but not a change to the type of
method used, (e.g. LC-MS to ligand binding) where a full validation would be required
[19].

2.6.4 Cross-validation

Cross-validation is a comparison of validation parameters when two or more

bioanalytical methods are used to generate data within the same study or across different
studies. An example of cross-validation would be a situation where an original validated
bioanalytical method serves as the reference and the revised bioanalytical method is the
comparator. The comparisons should be done both ways [12].

2.7 Validation Parameter

As per USFDA, the following validation parameters should be evaluated for quantitative
procedures: accuracy (bias, precision) selectivity, calibration model, stability, and limit of
quantification. Additional parameters which might have to be evaluated include limit of
detection, recovery, reproducibility and ruggedness (robustness).

2.7.1 Linearity

Linearity assesses the ability of the method to obtain test results that are directly
proportional to the concentration of the analyte in the sample. The linear range of the
method must be determined regardless of the phase of drug development. The linearity
solutions are prepared by performing serial dilutions of a single stock solution;
alternatively, each linearity solution may be separately weighed. The resulting active
response for each linearity solution is plotted against the corresponding theoretical
concentration. The linearity plot should be visually evaluated for any indications of a
nonlinear relationship between concentration and response. A statistical analysis of the

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regression line should also be performed, evaluating the resulting correlation coefficient,
Y intercept, slope of the regression line, and residual sum of squares. A plot of the
residual values versus theoretical concentrations may also be beneficial for evaluating the
relationship between concentration and response [20].

2.7.2 Selectivity

Selectivity is the ability of the bioanalytical method to measure unequivocally and to

differentiate the analyte(s) in the presence of components, which may be expected to be
present. Typically, these might include metabolites, impurities, degradants, matrix
components, etc [21].

2.7.3 Accuracy

The accuracy of an analytical procedure expresses the closeness of agreement between

the value which is accepted either as a conventional true value or an accepted reference
value and the value found [22]. Accuracy should be measured using a minimum of five
determinations per concentration. A minimum of three concentrations in the range of
expected concentrations is recommended. The mean value should be within 15% of the
actual value except at lower limit of quantification (LLOQ), where it should not deviate
by more than 20%. The deviation of the mean from the true value serves as the measure
of accuracy [23].

2.7.4 Precision

The precision of a bioanalytical method is a measure of the random error and is defined
as the closeness of agreement between a series of measurement obtained from multiple
sampling of the same homogeneous sample under the prescribed conditions [24].
Precision should be measured using a minimum of three concentrations in the range of
expected concentrations is recommended. Precision is expressed as the percentage of
coefficient of variance (%CV) or relative standard deviation (R.S.D.) of the replicate
measurements.

The precision at every concentration should not exceed 15% of the CV except for the
LLOQ, where it should not exceed 20% of the CV. Precision or reproducibility, which
measures precision with time, and may involve different analysis, equipment, reagents,
and laboratories [25].

2.7.4.1 Within-run precision

For the validation of the within-run precision, there should be a minimum of six samples
per concentration level at LLOQ, low, medium and high quality control (QC) samples in

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a single run. The within-run CV value should not exceed 15% for the QC samples, except
for the LLOQ which should not exceed 20%.

2.7.4.2 Between-run precision

For the validation of the between-run precision, LLOQ, low, medium and high QC
samples from at least six analyzed on at least three different days should be evaluated.
The between-run CV value should not exceed 15% for the QC samples, except for the
LLOQ, which should not exceed 20%.

2.7.5 Robustness

The robustness of an analytical procedure is a measure of its capacity to remain

unaffected by small, but deliberate variations in method parameters and provides an
indication of its reliability during normal usage [22].

2.7.6 Recovery

Recovery of the analyte and internal standard (IS) must be evaluated to determine loss
during sample preparation and matrix ionization. Recovery can be defined as the detector
response from a sample of a given concentration of analyte added to the biological matrix
prior to sample preparation compared to the detector response from a sample of the same
concentration of analyte added to the biological matrix after sample preparation [26].
Recovery experiments should be carried out by comparing the analytical results for
extracted samples at three concentrations (low, medium and high) with unextracted
standards that represent 100% recovery.

Recovery indicates the degree of extraction: in general recovery is impacted by the

interaction of the analyte with endogenous and/or exogenous components may not be
completely extracted. An extraction method need not result in 100% recovery of the
analyte; however, the degree of recovery must be consistent, precise and reproducible
[27].

2.7.7 Calibration curve

Calibration curve is the relationship between instrument response and known

concentrations of the analyte. Standard or calibration curve should be described
preferably by a simple monotonic (i.e. strictly increasing or decreasing) response function
that gives reliable measurements [24]. It should be prepared in the same biological matrix
as the intended study samples; however, they may be prepared in a surrogate matrix if the
study matrix is rare [26]. A calibration curve should consist of six to eight non-zero
samples covering the entire range, including LLOQ, and blank sample (matrix sample
processed without IS) and zero samples (matrix processed with IS).
The following conditions should be fulfilled in the calibration curve, 20% of deviation is
allowed to the LLOQ from the nominal concentration and 15% deviation for standards
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other than LLOQ from the nominal concentration. Among six non-zero standards at least
acceptance criterion for the standard curve is that at least 75% of non-zero standards
should meet the above criteria, including the LLOQ. Acceptability of the linearity data is
often depending on the correlation coefficient and y-intercept of the linear regression line
of the calibration curve. The best fit of linearity between response and analyte
concentration is considered when the R2 value is >0.999 or 1.

2.7.7.1 Lower limit of quantification

The lowest standard on the calibration curve should be accepted as the lower limit of
quantification (LLOQ) if the following conditions are met:
The analyte response at the LLOQ should be at least 5 times the response compared to
blank response.
Analyte peak (response) should be identifiable, discrete, and reproducible with a
precision of 20% and accuracy of 80-120% [12].
2.7.7.2 Upper limit of quantification

The upper limit of quantification (ULOQ) is the maximum analyte concentration of a

sample that can be quantified, with acceptable precision and accuracy. The ULOQ is
identical with the concentration of the highest calibration standards [24]. The back-
calculated concentration should have precision that does not exceed 15% of the CV and
accuracy within 15% of the minimal concentration.

2.7.7.3 Quality control samples

There are three concentrations of quality control sample (QCs) in duplicate should be
incorporated into each run as follows:
Low QC: one within three times the LLOQ.
Middle QC: one in the midrange.
High QC: one approaching the high end of the range of the expected study
concentrations.
At least 67% (e.g. at least four out of six) of the QCs concentration results should be
within 15% of their nominal concentrations. A confidence interval approach yielding
comparable accuracy and precision in the run is an appropriate alternative.
QC sample should be at least 5% of the number of unknown samples or six total QCs
whichever is greater. It is recommended that calibration standards and QCs be prepared
from stock solution. Samples involving multiple analytes should not be rejected based on
the data from one analyte failing the acceptance criteria. The data from rejected runs need
not be documented, but the fact that a run was rejected and the reason for failure should
be recorded.

2.7.8 Stability

The chemical and physical stability of an analyte in given matrix under specific
conditions for given time intervals. The aim of a stability test is to detect any degradation
of the analyte of interest during the entire period of sample collection, processing,
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storing, preparing and analysis. The condition under which the stability is determined is
largely dependent on the nature of the analyte, the biological matrix, and the anticipated
time period of storage [24].
There are several kinds of stability that should be examined during the validation.
Suggested experiments to determine stability are stated below.

2.7.8.1 Short-term stability

The stability of the analyte in biological matrix at ambient temperature should be

evaluated. Three aliquots of low and high concentrations should be kept for at least 24
hours and then analyzed [28].

2.7.8.2 Long-term stability

The stability of the analyte in the matrix should equal or exceed the time period between
the date of first sample collections and date of last sample analysis [27].

2.7.8.3 Freeze-thaw stability

During freeze-thaw stability evaluations, the freezing and thawing of stability samples
should mimic the intended sample handling conditions to be used during sample analysis.
Stability should be assessed for a minimum of three freeze-thaw cycles [27].

2.7.8.4 Bench-top stability

Bench top stability experiments should be designed and conducted to over the laboratory
handling conditions that are expected for study samples. Replicate (e.g., triplicate) QC
samples in matrix at a minimum of 2 concentrations are analyzed after keeping them at
ambient temperature for 4 to 24 hours to cover at least the duration of time it takes to
extract the samples. The observed sample concentrations are compared with their
nominal values. This experiment can be combined with that for the extracted
samples/autosampler tray stability above to demonstrate overall process stability if
desired.

2.7.8.5 Stock solution stability

The stability of stock solutions of drug should be evaluated. When the solutions exist in a
different state or in a different buffer composition from the certified reference standard,
the stability data on this stock solution should be generated to justify the duration of stock
solution storage stability [24].

2.7.9 Dilution integrity

Precision and accuracy should not effect from the dilution of samples. If applicable,
dilution integrity should be demonstrated by spiking the matrix with an analyte
concentration above the LLOQ and diluting this sample with blank matrix (at least five
determinations per dilution factor). Accuracy and precision should be within 15%.
Evaluation of dilution integrity may be covered by partial validation. Use of another
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matrix may be acceptable, as long as it has been demonstrated that it does not affect
precision and accuracy.

2.7.10 Limit of detection

The limit of detection (LOD) of an individual analytical procedure is the lowest amount
of analyte in a sample which can be detected but not necessarily quantified as an exact
value. Several approaches for determining the detection limit are possible, depending on
whether the procedure is a non-instrumental or instrumental [15]. A signal-to-noise ratio
between 3 or 2:1 is generally considered acceptable for estimating the detection limit.

2.7.11 Limit of quantification

The quantification limit of an individual analytical procedure is the lowest amount of

analyte in a sample which can be quantitatively determined with suitable precision and
accuracy. The quantification limit is a parameter of quantitative assays for low levels of
compounds in sample matrices and is used particularly for the determination of impurities
and/or degradation products [15]. A typical signal-to-noise ratio is 10:1 is considered
acceptable for quantification limit.

2.8 Bioanalysis
Bioanalysis is a term generally used to describe the quantitative measurement of a
compound (drug) or their metabolite in biological fluids, primarily blood, plasma, serum,
urine or tissue extracts [10].

2.9 In vitro pharmacokinetics

2.9.1 Serum stability

Among various bio-matrices used for the compounds quantification, blood is the most
preferred matrix. Even in blood samples, serum and plasma are most commonly
employed samples because of their easiness sampling, processing. Stability of the
compound in the bio-matrix used is the prime requisite for its quantification, instability of
compound in matrix misleads the results and gives altered PK. This stability study in the
bio-matrix is done at ambient temperature and processed in the same way as that of the
real samples. The known concentration of the analyte is added to bio-matrix and the
sampling is done at regular time points to find the difference in the amount of analyte.

2.9.2 Gastrointestinal fluid stability

Among various drug administration routes oral route is most preferable for patient
satisfaction and compliance hence, to achieve good bioavailability of compounds
administered orally gastrointestinal stability is a prequisite. In the early drug discovery
process the determination of gastrointestinal (GI) stability is an important and common
step for screening of new chemical entities (NCEs). This stability assessment will provide
valuable information about suitability of the compound for oral administration.

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Gastrointestinal fluid stability consists of stability study in the presence of simulated
gastric fluid and simulated intestinal fluid individually.

2.9.3 Metabolic stability

Metabolism of a drug can decrease its half-life and often determines whether it will be
effective and safe. For this reason, candidate drug compounds are often screened early in
the discovery process for metabolic stability. In vitro metabolic stability studies are
generally performed by liver microsomes, liver slices and hepatocytes of human and
preclinical animal models. In vitro metabolic stability in presence of microsomes
provides useful information about the inter species differences in metabolism and aids in
identifying the right animal model for toxicity studies. The metabolites obtained in vitro
should be in good agreement with those obtained in vivo, or else the in vivo data should
be given precedence [4].

2.10 In vivo pharmacokinetics

PK is more accurately described by the acronym ITE. In this case, I stands for input
into the body, since some routes of administration do not have absorption components
(e.g., intravenous administration), T for transfer of drug within the body, and E for
elimination from the body (which includes metabolism, since metabolism is a major route
of drug elimination) [9]. It also characterizes the rate and extent of drug absorption, the
concentrations and time course of drug in the body, and the rate and mechanisms of
elimination. This information allows the identification of features limiting the desired
route of administration.

2.10.1 Pilot pharmacokinetic study

Pilot PK study carried out in a small number of test animals to get a better choice of
ADME of the novel compound as a guide to design the definitive PK study. This study is
performed in identical to the definitive PK study (same species, dose, formulation, bio-
samples, sample processing, quantification) but in a small number of animals.

2.10.2 Definitive pharmacokinetic

In this PK study, novel compound is formulated into a suitable formulation and then
administered through the route. Apart from the conventional full PK studies, newer
techniques are used in preclinical PK studies which have time, animals, cost and also are
fast, rapid and results are equally predictive and reliable.

2.11 Leishmaniasis
Leishmaniasis is a neglected tropical and subtropical disease caused by trypanosomatids
from the genus Leishmania, which are transmitted to humans by the bite of infected
female phlebotomine sand flies [29]. As per WHO, an estimated 1.3 million new cases of
visceral and cutaneous leishmaniasis are reported annually and 20,00030,000 deaths
occur worldwide each year [30]. India witnessed the occurrence of 50% of the worlds

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leishmaniasis cases and 70% of those are in Bihar [31]. Poverty-allied malnutrition, poor
housing and sanitary conditions, weak immune system and lack of resources are the
major risk factors associated with the leishmaniasis [30]. Leishmania parasite lives a
digenetic life cycle. It exists in promastigote and amastigote form in insect and human
vector, respectively. The phlebotomine sand flies are exclusively responsible for
transmission as well as proliferation of promastigotes. After biting by phlebotomine sand
flies, the parasite multiplies within mammalian macrophages, dendritic cells and/or
neutrophils as intracellular amastigotes [32].
The disease can be characterized by skin ulcers (cutaneous leishmaniasis), mucous
degeneration, especially from the mouth and internal nose (mucocutaneous
leishmaniasis), and visceral organ damage (visceral leishmaniasis), which is lethal if
untreated. The different forms of leishmaniasis manifestation depend mainly on the
species of parasite but are also related to the host immune system [33].
Cutaneous leishmaniasis (CL) is a most common form of leishmaniasis and causes ulcers
on exposed parts of the body, leaving life-long scars and serious disability. About 95% of
CL cases occur in the America, Mediterranean basin, and the Middle East and Central
Asia. CL in the Old World is caused by Leishmania tropica, L. major, and L. aethiopica.
New World leishmaniasis is caused by L. Mexicana, L. amazonensis, and L. Braziliensis
[34]. CL is endemic to the Thar Desert of Rajasthan, Bikaner, India. In a study describes
clinico-epidemiologcial data of all cases of CL in this region during 20012011. A total of
1,379 patients with 2,730 lesions were reported during the study period [35].
Mucocutaneous leishmaniasis (ML) is a known risk from Leishmania species of the
Viannia subgenus, typically found in the Americas (L. (V) braziliensis, Leishmania
(Viannia) amazonensis, L. (V) panamensis, and Leishmania (V) guyanensis). Clinical
progression to mucosal disease is dependent on a combination of host cell-mediated
immunity and parasite virulence [36].
Visceral leishmaniasis (VL), also known as Kala azar, is a protozoan systemic infection
which is always fatal if left untreated [37]. The number of cases of VL is calculated to be
as high as 0.20.4 million people per year, with more than 90% of these occurring in
India, Bangladesh, Sudan, Ethiopia, and Brazil, with a mortality estimated at 10%20%,
especially in poor areas [38].

17
2.11.1 Life cycle of leishmaniasis

The life cycle of parasite Leishmania represented in following figure 2.11 (a).

Figure 2.11 (a) Diagrammatic representation of life cycle of parasite Leishmania.

The stages of the above picture are as follows:

1. When biting their hosts, infected sandflies regurgitate Leishmania promastigotes into
the skin.
2. Which invade or are phagocytosed by local or recruited host cells, mainly
macrophages.
3. Within the phagolysosomes of resident macrophages, promastigotes become
amastigotes.
4. Amastigotes replicate and may then infect additional macrophages, either locally or
in distant tissues after dissemination.
5. When blood-feeding on an infected host.
6. Naive sandflies become infected with amastigotes.
7. Which transform back into promastigotes in the sandflys gut (depending on
Leishmania spp, different regions of the gut will be parasitized).
8. The parasites then migrate to the sandflys proboscis.

Thus, complete the Leishmania life cycle [39, 40].

2.11.2 Symptoms

In CL lesions may begin as small red papules (510 mm initially). Depending on the
species of Leishmania, over 13 months, they can progress into erythematous nodules,
indurated plaques, scaly plaques {Fig. 2.11 (b) (A)}, or ulcers with raised, rolled dusky
borders {Fig. 2.11 (b) (B)} [41-43]. Lesions may be dry and crusted or accompanied by
exudates [44]. Satellite lesions and local lymphadenopathy are sometimes present.

18
Lesions may leave depigmented retracted scars [44]. Acutely, lesions of CL often are
mistaken for furuncles and methicillin-resistant Staphylococcus aureus infections [36].

(A) (B)
Figure 2.11 (b) CL lesions. (A) Scaly raised plaques caused by infection with
Leishmania major. (B) Crusted ulcer with rolled indurated borders caused by infection
with Leishmania mexicana.

Ninety percent of patients have a scar from a prior episode of CL [45]. Early ML can
commence with erythema and ulceration of the nares. The mid-to-late disease can present
with erythema and edema of the nares, nasal septum perforation, palatal ulceration,
gingival edema and periodontitis [46].

(A) (B)
Figure 2.11 (c) ML lesions. (A) Crusted papules and plaques around the nose and lips
caused by infection with Leishmania (Viannia) braziliensis. (B) Deeper, wet appearing
ulcers on the legs of the same patient.

VL affects the internal organs especially the spleen, liver, and bone marrow. Latent cases
may remain undiagnosed from years to decades until the individual becomes
immunocompromised (especially with HIV) then the individual will typically develop
fever, weight loss, hepatosplenomegaly, and pancytopenia [47].

2.11.3 Diagnosis

Tests that may be done to diagnose the condition include:

Biopsy of the spleen and culture

Bone marrow biopsy and culture
Direct agglutination assay

19
 Indirect immunofluorescent antibody test
Leishmania-specific PCR test
Liver biopsy and culture
Montenegro skin test (not approved in the USA)
Skin biopsy and culture
Other tests that may be done include:
Complete blood count
Serologic testing
Serum immunoglobulin levels
Serum protein
2.11.4. Treatment

There are number of therapies for various forms of leishmaniasis and the preferences for
first line and second line treatment vary on the type of disease and are often guided by
regional practice. The following is a general description of the drugs used in clinical
practice:

2.11.4.1 Amphotericin B

Amphotericin B (AmB) is a polyene derived from Streptomyces nodosus. This compound

was discovered in the mid-1950s and remains a first-line agent for the treatment of
invasive aspergillosis and other life-threatening invasive fungal infections [48, 49]. AmB
has approximately 100% cure rate at a dose of 0.75-1mg/kg daily or alternate days [50].
AmB binds with ergosterol, a component of parasite cell membranes, forming pores that
cause rapid leakage of monovalent ions (K+, Na+, H+ and Cl) and subsequent cell
death. There are four formulations of AmB for clinical use: deoxycholate AmB,
liposomal AmB, amphotericin colloidal suspension and AmB lipid complex.
Side effects: Most of the patient's experience infusion reactions e.g., fever, chills, and
thrombophlebitis and occasionally serious toxicitye.g. hypokalemia, nephrotoxicity,
myocarditis, and even death [50].

2.11.4.2 Pentamidine

Pentamidine is an aromatic diamidine. This drug is used in antimonial resistance cases. It

was originally used in the treatment of VL via an intramuscular route, but because of
increasing resistance and toxicity, its use was forfeited. It also has potential as a useful
drug for maintenance treatment in immunocompromised hosts [51]. Its primary
mechanism of action is unknown and yet to be explored, it is thought that the drug is
accumulated in the parasite, with effects including binding to kinetoplast DNA. It also
has been reported that the drug enters promastigotes through arginine and polyamine
transporters and is accumulated in mitochondria, acting to inhibit mitochondrial
topoisomerase II [52]. This drug can be administered parenterally, by intramuscular or
intravenous route. It has a half-life is 5 to 15 minutes and 54 minutes, respectively. Drug
distribution shows their concentration to be considerable higher in the liver, kidneys,
adrenal glands and spleen, while only a small amount is found in lungs [53].

20
Side effects: Treatment with pentamidine may cause myalgia, pain at the injection site,
nausea, headache, and, less commonly, results in a metallic taste, a burning sensation,
numbness and hypotension, irreversible insulin-dependent diabetes mellitus, and death.
Increasing unresponsiveness in India, emergence of drug resistance, especially in HIV co-
infections, and toxicity (reversible hypoglycemia and nephrotoxicity) are some other
limitations to its usefulness [54, 55].

2.11.4.3 Miltefosine

Miltefosine, 2-(hexadecoxy-oxido-phosphoryl)oxyethyltrimethyl-azanium, is the first

reported oral drug for both visceral and cutaneous leishmaniasis [56]. Its use as an
antileishmanial agent was initiated in the mid-1980s. It is the most recent antileishmanial
drug to enter the market [52]. Mechanism of action of this compound can be extrapolated
from its effect on mammalian cells, where it causes modulation of cell surface receptors,
inositol metabolism, phospholipase activation, protein kinase C and other mitogenic
pathways, eventually culminating in apoptosis [57].
Side effects: Gastrointestinal disturbances, renal toxicity, hepatotoxicity and
teratogenicity. it is contraindicated in pregnancy and women of childbearing age group,
not observing contraception [52, 58].

2.11.4.4 Paromomycin

Paromomycin, an aminoglycoside antibiotic, is a relatively new broad-spectrum

antibiotic drug that has been used for treatment of leishmaniasis. It can be used for
treatment of both types of leishmaniasis (VL as well as CL), although paromomycin is
more effective for CL. Limited availability restricts its use in endemic regions [59, 60].
The mechanism of action of paromomycin in Leishmania requires further elucidation and
it inhibits protozoan protein synthesis. It binds to the 30S ribosomal subunit, interfering
with initiation of protein synthesis by fixing the 30S-50S ribosomal complex at the start
codon of mRNA, leading to accumulation of abnormal initiation complex [59]. It is
available as an intramuscular injection for parenteral administration to treat systemic
infections (i.e. VL), and as an ointment formulation to treat local skin infections (i.e. CL)
[61, 62]. The drug is poorly absorbed into systemic circulation after oral administration
but rapidly absorbed from intramuscular sites of injection. Peak concentration in plasma
occurs in 30-90 min and its apparent volume of distribution is 25 % of body weight. The
half-life varies between 2 and 3 hours in patients with normal renal function. Their
clearance is almost entirely by glomerular filtration [63].
Side effects: Ototoxicity, liver dysfunctioning, skin rashes, local pruritus and burns [53,
64].

2.11.4.5 Azoles

The imidazoles and triazoles are well known oral antifungal agents that are well tolerated.
They also have antileishmanial activity against certain species as they inhibit 14-
demethylase, a key enzyme in the sterol biosynthesis pathway, thereby interfering with
Leishmanial cell membrane biosynthesis [58].

21
2.11.4.6 Combination therapy

Due to unresponsiveness to most of the monotherapeutic regimens, the combination

therapy has found new scope in the treatment of leishmaniasis. The combination of
antileishmanial drugs could reduce the potential toxic side effects and prevent drug
resistance. Some studies have shown that some drugs increase their antileishmanial effect
in conjunction [65].

Paromomycin has been used extensively in Sudan in combination with sodium

stibogluconate for the treatment of VL in a period of 17 days [66]. Combined
chemotherapy against VL in Kenya was evaluated using oral allopurinol (21 mg/Kg,
three times a day for 30 days) with endovenous pentostam (20mg/Kg once a day). The
therapy was efficient, but relapses were found in the first month after treatment [67].

2.11.5 Remarks of treatment of leishmaniasis in categories of patients

Leishmaniasis is generally associated with severe immunodeficiency (AIDS; renal, liver

and heart transplantations; haemopoietic malignancies). More rarely it can be related to
an immunotolerance status such as pregnancy. In these patients, differences in the
response to treatment of leishmaniasis have demonstrated the complexity of managing
infections between different individuals [68]. Infection with Leishmania during
pregnancy is rare and deserves special attention since little information is available
regarding the occurrence of visceral leishmaniasis during gestational period and the real
possibility of vertical transmission of this disease. Currently, AmB is strongly
recommended as the first choice drug due to their activity and it's fewer maternal-fetal
adverse effects [69].

2.12 CDRI compound

For the treatment of leishmaniasis, CDRI developed novel and potent anti-leishmanial
agent. This present study focuses on in vitro and in vivo studies of a compound in order to
aid its development as potential candidate drug.

22
3.0 OBJECTIVE OF THE WORK
To develop reverse phase high performance liquid chromatography (RP-HPLC)
method of the compound.
To develop and validate liquid chromatography-tandem mass spectrometry (LC-
MS/MS) method for the quantification of the compound in rat serum
Investigation of drug like properties in regards to in vitro studies (such as: serum
stability simulated gastric fluid stability, simulated intestinal fluid stability and
metabolic stability) using LC-MS/MS.
Investigation of per oral and intravenous pharmacokinetics of the compound
Estimation of pharmacokinetic parameters of the compound

23
4.0 BASIS OF WORK
The discovery and development of drugs for parasites diseases has been neglected due to
the economic reasons. Only 1% of new drugs introduced in the market between 1975 and
1996 were for the treatment of tropical diseases that together contributed to 5% of the
global diseases burden. The leishmaniasis is a public health problem in many countries of
the world. It is endemic in 98 countries and is closely associated with poverty. More than
a million new cases are reported per year and 350 million people are at risk of contracting
the infection. For the most severe form of leishmaniasis, visceral leishmaniasis (VL),
300 000 new cases are estimated to occur annually resulting in 40 000 deaths [70].
The large spectrum of clinical manifestations reflects leishmaniasis complexity, mainly
associated with the number of Leishmania species that cause disease, as well as many
sandfly and mammalian species indicated as vectors and reservoirs, respectively. The
existing therapy is far from satisfactory owing to the emergence of resistances, toxicity
and its limited efficacy due to disease exacerbation, mainly associated with compromised
immune capability.
The drug pipelines for leishmaniasis are very thin: very few compounds are in
development and drug discovery efforts are limited, one compound for VL (nifurtimox)
making the need for enriching the pipeline with novel chemical entities of critical
importance.

24
5.0 MATERIALS AND MEDIA

5.1 Chemicals and reagents

The CDRI compound is obtained from Pharmaceutics Division, Central Drug Research
Institute, Lucknow. Phenacetin (internal standard, IS) and LCMS grade acetonitrile
(ACN) and methanol (MeOH) were purchased from Sigma-Aldrich (St. Louis, USA).
The ultrapure water of resistivity 18.2 M cm at 25C was from Milli-Q PLUS PF
(Billerica, USA) water purification system. Male Sprague Dawley rats weighing 250 25
g and male golden hamsters (Mesocricetus auratus) weighing 110-120 g are obtained
from laboratory animal division of the institute housed in plastic cages and were given
rodent food and water ad libitum. Drug free rat serum was separated after centrifuging
the collected blood at 3000xg for 10 min at 4C. The collected serum from all rats was
pooled and stored at -20C till use.

5.2 Instruments

Table 5.2 (a) Instruments used for sample preparation

S. No. Instrument Model/ specification Company

1 Micropipette 10 L, 100 L,200 Brandtech scientific

l,1000l Inc. USA
2 Electronic Weighing ML204/AO1,Switzerland (Mettle Toledo)
Balance,max.220g
5 Vacuum dryer SVC 2009 Savant instruments
Inc. USA
6 Ultrasonic bath XUBA3 Grant Instruments,
Cambridgeshire,
UK
7 Multi-Tube vortexer BV1010 Benchmark
scientific Inc. New
jerky, USA
10 Centrifuge Sigma 3-16 KL IVD Sigma
Laborzentrifugen,
Germany

5.3 Plastic ware and glassware

Table 5.3 (a) List of plastic ware and glassware

S. No. Material Company
1 Microcentrifuge tubes Axygen
2 10 mL volumetric flask Borosil
3 Graduated tubes Vencil

25
 4 Test tube Borosil
5 Bottles Borosil
6 Gloves Cole-Parmer

5.4 Preparation of various media and buffers

5.4.1 Sodium hydroxide (NaOH 0.2 N) solution

Sodium hydroxide (0.4 g) was dissolved in 50 mL Milli-Q water.

5.4.2 Simulated gastric fluid (pH 1.2)

Sodium chloride (0.2g) was dissolved in 50 ml Milli-Q water, to this 0.7 ml conc. HCl
was added and the final volume was made upto100ml with Milli-Q water. The pH of this
was adjusted to about 1.2 using 0.1N HCl.

5.4.3 Simulated intestinal fluid (pH 6.8)

Potassium di-hydrogen phosphate (0.68g) was dissolved in 75 mL of Milli-Q water; to

this solution, 7.7 mL of 0.2N NaOH was added and the pH was adjusted to 6.8 using
0.2N NaOH. Finally, the volume was made up to 100mL by water.

5.4.4 Tris buffer (0.1M, pH7.4)

Tris base (12.1g) was weighed and dissolved in 1 litre of Milli Q water followed by
filtration and adjustment of pH to 7.4using HCl.

5.4.5 Microsomal reaction mixture

Microsomal reaction mixture was prepared by addition of 449 L of tris buffer, 20 L of

MgCl2 (500mM) and 4 L of rat liver microsomes (0.5 mg/mL) in to a test tube and
gently mixed with pipette. The mixture should not be subjected to heavy vortexing, since
the microsomes may get de-activated due to froth formation.

26
6.0 EXPERIMENTAL
6.1 Chromatographic condition

Different conditions used in the development of HPLC-UV method as follows:

Table 6.1 (a) Conditions used in the development of HPLC-UV method

Instrument Shimadzu, Japan
Column Discovery HS C-18 (5m,1004.6 mm id) preceded with
a guard column packed with same material (5m, 204.0
mm, id)
Detector UV-Vis
Virtual Analyser Class VP (6.12 SP5)
Flow Isocratic
Injection Volume 100 L
Detection Wavelength 230 nm

6.2 Preparation of primary stock solution

The primary stock solution (1 mg/ml) was prepared by dissolving the compound in
acetonitrile (ACN) and di-methylsulfoxide (DMSO). The working stock solution (100
g/mL and 10 g/mL) were prepared by diluting of 1 mg/mL in ACN.

6.2.1 Optimization of mobile phase for HPLC method

The mobile phase was optimized by trial of different composition of solvents in different
proportions. Concentration of compound in every mobile phase was 2 g/mL. It was
prepared by taking 100 L of 100 g/mL solution in each mobile phase.

Table 6.2 (a) Different mobile phase tested for the compound
Mobile Phase RT Peak
Height
ACN: Methanol: Ammonium Acetate buffer (30:30:40); %v/v 1.875 24563
ACN: Methanol: Ammonium Acetate buffer (40:40:20) ); %v/v 2.375 533676
ACN: Methanol: Ammonium Acetate buffer (10:50:40) ); %v/v 2.375 5416462
ACN: Ammonium Acetate buffer (60:40) ); %v/v 0.950 151306
Methanol: Ammonium Acetate buffer (60:40) ); %v/v 1.983 5713
Methanol: Milli Q water (60:40) ); %v/v 2.134 234125
0.1% formic acid in Methanol: Milli Q water (60:40) ); %v/v 2.212 154557

Since the HPLC-UV method was not suitable as the retention time was very low a more
sensitive LC-MS/MS method has been developed for the compound.

27
6.3 LC-MS/MS Bioanalytical method development for compound

LC-MS/MS evolved as a gold standard technology to support the pharmacokinetic (PK)

and drug metabolism studies with significant improvement in the assay sensitivity and
specificity in addition to reducing the assay time per sample. To study the oral and
intravenous PK of compound, a LC-MS/MS method was developed and validated.

6.3.1 Instrumentation

MS/MS analysis was performed using API 4000 QTrap mass spectrometer (Applied
Biosystems, Toronto, Ontario, Canada) which includes a hybrid triple quadrupole/LIT
(linear ion trap) and a turbo VTM Ion Source. The LC system consists of a Shimadzu
UFLC pump (LC-20AD) with online degasser (DGU-20A3), an autosampler (SIL-HTc)
with a temperature- controlled Peltier-tray and a column oven (CTO-10AS).
Chromatographic separation of the compound and IS (Phenacetin) were achieved on a
supelco Discovery HS C-18 column (5 m, 100 x 4.6 mm id) preceded with a guard
column (5 m, 50 x 4.0 mm, id) packed with the same material under isocratic condition
at a flow rate of 0.6 mL/min. The mobile phase [0.1% formic acid in Milli-Q water:
MeOH (10:90, %v/v)] was degassed by ultrasonication for 15 min before use. LC-
MS/MS system was equilibrated for approximately 20 min before commencement of
analysis. Total run time was 5 min. The mass spectral analysis was performed using
multiple reaction monitoring mode [m/z 419.3 267.2; compound and m/z
180.1138.1; IS] in positive electrospray ionization (ESI) mode at 4500 V spray voltage.
Data acquisition and quantification were performed using AnalystTM (version 1.4.2
software; Applied Biosystems, Toronto, Ontario, Canada). A specific, sensitive and
reliable method was developed and validated as per US FDA guidelines and successfully
applied for the PK study of compound.

6.3.2 Preparation of analytical standards

The Analytical standards (AS) in the range of 1-200 ng/mL of compound was prepared
by appropriately diluting the 10 g/mL solution with the mobile phase, 0.1% formic acid
in Milli-Q water: MeOH (10:90, %v/v). The procedure of preparing the AS is shown in
the table 5.5.2 (a).

Table 6.3 (a) Analytical standard preparation of compound for LC-MS/MS

Code Concentration Volume of stock Volume of mobile
(ng/mL) solution to be diluted phase to be added
AS 8 200 6L ( WS: 294L
10g/mL)
AS 7 100 100L (AS 8) 100L
AS 6 50 100L (AS 7) 100L
AS 5 25 100L (AS 6) 100L
AS 4 10 100L (AS 5) 150L
AS 3 5 150L (AS 4 ) 150L

28
 AS 2 2.5 200L (AS 3) 200L
AS 1 1 200L (AS 2) 300L

6.3.3 Optimization of the extraction process and solvent(s)

Extraction means the extracting out of a specific active agent from a mixture with
extracting media. Most often, liquid-liquid extraction is performed owing to its
advantages like easiness, clean sample and cost effectiveness when compared to solid
phase extraction and protein precipitation.

6.3.3.1 Liquid-liquid extraction

The principle involved in liquid-liquid extraction (LLE) method is partitioning of the

sample between two immiscible liquids or phases which differ in polarity, one of the
phases is aqueous and the other is an immiscible organic solvent. A pre-determined
concentration of bio-samples is achieved by spiking known volume of working stock into
measured volume of serum. These bio-samples of known concentration are extracted by
various solvents such as
Hexane: Ethyl Acetate (60:40, %v/v)
n-Hexane
n-Hexane containing 3% Isopropyl alcohol (IPA)
Ether: Ethyl Acetate (60:40, %v/v)
tert-Butyl methyl ether (TBME)
Ethyl Acetate
Different extraction solvents were tried. Bio-samples (50 L) of known concentration
(200 ng/mL) were extracted with 1 mL of each of the above mentioned solvents
individually and vortexed for 5 min, followed by centrifugation at 3000 rpm for 10 min
and the supernatant after snap freezing over liquid nitrogen were collected into graduated
tubes (Vencils) which were subjected to drying. The aqueous layer was extracted again
using the similar procedure and supernatants were transferred in same vencils and then
dried as was done during first extraction. After completion of double extraction, the dried
residues in the vencils were reconstituted with 100 L of mobile phase and vortexed.
Then clear samples were transferred to inserts and analyzed for the concentration of the
analyte.

Table 6.3 (b) Optimization of the extraction solvent

Extraction Solvent % Recovery
Hexane: Ethyl Acetate (60:40) 36
n-Hexane 65
n-Hexane containing 3% IPA 51
Ether: Ethyl Acetate 60:40 26
tert- Butyl methyl ether (TBME) 34

29
 Ethyl Acetate 31

n-Hexane was selected as the extraction solvent for the compound as it gives maximum
and reproducible recovery with minimal interferences.

6.3.2 Preparation of calibration standards and quality control samples

Calibration curve (CS) and quality control (QC) samples were prepared by spiking
Normal hamster serum (NHS) with the stock solution of compound and IS. The CS in the
range of 1-200 ng/mL was prepared by serial dilution method. The preparation of
calibration standards was simplified and shown in table 6.3 (c).

Table 6.3 (c) Procedure for the preparation of calibration standards

Code Concentration Volume of solution Volume of NHS to
(ng/mL) to be added be added
CS 8 200 6L ( WS: 294 L
10g/mL)
CS 7 100 100L (CS 8) 100 L
CS 6 50 100L (CS 7) 100 L
CS 5 25 100L (CS 6) 100 L
CS 4 10 100L (CS 5) 150 L
CS 3 5 150L (CS 4 ) 150 L
CS 2 2.5 200L (CS 3) 200 L
LLOQ 1 200L (CS 2) 300 L
HQC 180 9L ( WS: 500 L
10g/mL)
MQC 90 150 L (HQC) 150 L
LQC 1.75 125 L CS2+ 125
-
L LLOQ

6.4 In vitro pharmacokinetic study

6.4.1 Serum stability

1. Normal rat serum (500 L) was taken into a test tube and incubated at 37C for 15
min.
2. To this 2 L of drug stock solution (100 g/mL) was added, vortex mixed for about 1
min and kept for incubation.
3. Aliquots of 50 L were collected at regular time intervals (0, 2, 15, 30, 60 min) in
separate microcentrifuge tubes.
4. The collected samples were processed (extracted as per the method developed earlier)
and analyzed for compounds concentration.
5. Compound concentration obtained at 0 min. is considered as 100% of the compound
remained, amount of the compound remained at various time points was calculated
with respect to it.

30
6. A curve is plotted by taking percent (%) compound remained on Y-axis and time on X-
axis. The protocol is simplified in table 6.4 (a).

6.4.2 Gastrointestinal fluid stability

1. SGF/SIF was prepared as per the procedure is given in 5.4.2 and 5.4.3.
2. Media (SGF/SIF) was taken into test tubes and incubated in shaking incubator at
37C.
3. Required volume of the compounds working stock solution was added to this pre-
incubated media and vortexed properly.
4. Aliquots of 50L were collected at regular time intervals up to 60 and 120 min. for
SGF and SIF respectively.
5. The collected aliquots were diluted up to 150 L with use of mobile phase and
transferred to vials.
6. Concentration of the compound was analyzed in the collected samples over different
time points.
7. Concentration of compound at 0 min. is considered as 100% compound remained and
the concentration obtained by rest of the samples is calculated in accordance with it.
8. Percent compound (%) compound remained versus time is plotted to see the
degradation if any.
9. The protocol is simplified in table 6.4 (a).

Table 6.4 (a) protocol for SGF, SIF and Serum stability study
SGF USP without SIF USP without
Media (pH) Serum
enzymes (1.2) enzymes (6.8)
Volume taken
800 800 500
(L)
Conc.(ng/mL) 100 100 100
Duration of
60 120 60
study (minutes)
Sample volume
50 50 50
(L)
Sample Extracted and
Diluted up to 150 L with mobile phase
processing analyzed

The stability studies were investigated by determining the disappearance of parent

compound from the incubation mixture. The results of the experiments were expressed in
the percent of parent compound remaining in the incubation mixture which was
calculated by

31
6.4.3 Metabolic study

9.1 500 L of reaction mixture (prepared by procedure given in 5.4.5) was taken in a test
tube.
9.2 To this 25 L of NADPH solution (2 mM, prepared previously) was added and
mixed without vortexing.
9.3 For negative control 100 L of Tris buffer was added instead of NADPH.
9.4 The above mixture was incubated at 37C for 15 min.
9.5 To this mixture required volume of compounds primary stock (100 g/mL) was
added and mixed properly.
9.6 Aliquots of 50 L were withdrawn at regular time intervals (0, 2, 5, 10, 15, 30, 45,
60min).
9.7 These aliquots were taken in microcentrifuge tubes contained with equivalent
amount (150 L) of ice cooled ACN to terminate the reaction.
9.8 These microcentrifuge tubes were vortex mixed and subjected to centrifugation at
10,000 rpm for 10 min.
9.9 Supernatant of 80 L was collected in the test tubes and analyzed.
9.10Percent compound remained at 0 min. was considered as 100, based on this %
compound remained at various time points was calculated
9.11Natural log of % compound remained at each time point was calculated.
9.12Then a curve was plotted with natural log of % compound remained versus time.
Protocol for this study is summarized into table 6.4 (b).

Hepatic elimination rate constant (k) was calculated directly from the slope of ln (percent
drug remaining) and time profile. In-vitro half-life (t1/2), intrinsic clearance (Clint) and
hepatic clearance (Clint hep) were calculated from the following equations:

Where K is the rate constant, V= incubation volume (mL), P= Protein concentration

(mg).

32
 Table 6.4 (b) Protocol for metabolic stability studies
Factors Net condition
Compound 1 M
Rat liver microsomes 0.5 mg/mL
MgCl2 (500mM) 20 mM
NADPH (20mM) 25 L
Tris buffer pH 7.4 q.s. upto 500 L
Duration 60 min.
Sample volume 50 L
Reaction termination By 150 L CAN
Positive control Testosterone
Negative control Compound without NADPH

6.5 In vivo pharmacokinetic study

6.5.1 Per oral pharmacokinetic

Per oral PK of the compound was carried out in young and healthy male golden Syrian
hamsters weighing 110-120 g, the animals were housed in plastic cages under standard
laboratory condition with a regular 12 h day-night cycle. Standard pelleted laboratory
chow (Goldmohar Laboratory Animal Feed, Lipton India Ltd, Chandigarh, India) and
water were allowed ad libitum. The hamsters were acclimatized to this environment for at
least two days before conducting the experiments.

6.5.1.1 Preparation of formulations

Following per oral dosing was given as suspension prepared by triturating the drug along
with gum acacia (1 %w/v). Drug (37.5 mg) was triturated with 30 mg gum acacia by
dropwise addition of water in a mortar and pestle and final volume was made upto 3mL
with Milli-Q water.

6.5.1.2 Sampling procedure

Following per oral administration, blood samples were withdrawn at 0.5, 1, 1.5, 2, 4, 6,
10, 24 h post dose. Blood (~100 L) was collected in microcentrifuge tubes by retro-
orbital plexus. The blood was allowed to clot, centrifuged at 3000 rpm for 10 min at 4C
and the serum was separated and stored at -20C until analysis.

33
6.5.1.3 Study design

In vivo per oral PK studies of compound were performed in golden Syrian hamsters
(weighing 110-120 g, n=5, each). The protocol followed was framed beforehand and is
shown in tabulated form below for simplification. The method of sampling and the type
of bio-matrix to be used were optimized and the design of study was done accordingly.
All experiments, euthanasia and disposal of carcasses were carried out as per the
guideline of the local ethics committee for animal experimentation.

Table 6.5 (a) Protocol for the in vivo (per oral) study of compound in golden Syrian
hamsters
Parameter Per oral
Dose (mg/kg) 50
Biosample Serum
Sampling Retro-orbital plexus
Sample analysis LC-MS/MS

6.5.1.4 Preparation of samples

To the bio-matrix i.e. serum (50 L; blank spiked or test), 1 mL of n-Hexane was added,
vortex-mixed for 5 min and centrifuged at 3000 rpm for 10 min. The supernatant was
withdrawn after snap freezing the sample and transferred to the vencils which are
subjected to dry. The extraction of the sample was done once more using the same
procedure. The residue after drying was reconstituted with 100 L mobile phase and
vortexed. The clear samples were transferred to the inserts and analyzed for the
concentration of analyte.

6.5.1.5 Data analysis

Data acquisition and quantification were performed using Analyst TM (version 104.2
software; Applied Biosystems, Toronto, Ontario, Canada). The concentration-time data of
compound was subjected to two compartmental analysis using Phoenix WinNonlin
(version 6.3; Certara Inc, Missouri, USA) to calculate different PK parameter like
clearance (CL), volume of distribution (Vd), elimination half-life (t1/2), and area under
curve (AUC).

6.5.2 Intravenous pharmacokinetics

Intravenous (IV) PK of the compound was carried out in young and healthy male
Sprague Dawley rats weighing 300 25 g. The formulation was given at a dose of 10
mg/kg. The animals were housed in plastic cages under standard laboratory condition
with a regular 12 h day-night cycle. Standard pelleted laboratory chow (Goldmohar
Laboratory Animal Feed, Lipton India Ltd, Chandigarh, India) and water were allowed
ad libitum. The rats were acclimatized to this environment for at least two days before
conducting the experiments.

34
6.5.2.1 Sampling procedure

Following IV administration, blood samples were withdrawn at 5 min, 15 min, 30 min, 1,

2, 4, 6, 10, 24 h post dose. Blood (~100 L) was collected in microcentrifuge tubes by
retro-orbital plexus. The blood was allowed to clot, centrifuged at 3000 rpm for 10 min at
4C and the serum was separated and stored at -20C until analysis.

6.5.2.2 Study design

In vivo IV PK studies of compound were performed in male Sprague Dawley rats

(weighing 300 25 g, n=5, each). The protocol followed was framed beforehand and is
shown in tabulated form below for simplification. The method of sampling and the type
of bio-matrix to be used were optimized and the design of study was done accordingly.
All experiments, euthanasia and disposal of carcasses were carried out as per the
guideline of the local ethics committee for animal experimentation.

Table 6.5 (b) Protocol for the in vivo IV study of compound in Sprague Dawley rats
Parameter Intravenous
Dose (mg/kg) 10
Bio sample Serum
Sampling Retro-orbital plexus
Sample analysis LC-MS/MS

6.5.2.3 Preparation of samples and data analysis

To the bio-matrix i.e. serum (50 L; blank spiked or test), 1 mL of n-Hexane was added,
vortex-mixed for 5 min and centrifuged at 3000 rpm for 10 min. The supernatant was
withdrawn after snap freezing the sample and transferred to the vencils which are
subjected to dry. The extraction of the sample was done once more using the same
procedure. The residue after drying was reconstituted with 100 L mobile phase and
vortexed. The clear samples were transferred to the inserts and analyzed for the
concentration of analyte.

35
7.0 RESULTS AND DISCUSSION
7.1 Optimization of LC-MS/MS conditions
Initially, MS and MS/MS spectra were recorded after direct infusion of compound
solution in MeOH into the electrospray source via syringe pump (Havard Scientific,
Holliston, MA, USA) at a rate of 10 L/min. The parent ion spectra of the analytes are
shown in Fig 7.1 (a). For the robust and reliable quantification of drugs in complex
matrices, it is recommended to monitor more than one ion transition, but the analytes
resulted in only one ion transition with sufficient sensitivity. Among the various
modification tried, LC condition were optimized to 0.1% formic acid with 90% methanol
in 10% Milli-Q water at a flow rate of 0.6 mL/min with the use of a Discovery HS C-18
(Superlco, Bellefonte, USA) column (5 m, 50 x 406 mm id). In the positive ion mode,
the protonated species [M + H]+ at m/z 419.3 and 180.1 were observed for compound and
IS, respectively, under the above LC conditions. The curtain, GS1 and GS2, were set at
10, 45, 45 psi respectively. While the ion spray voltage (ISV) and the source temperature
(TEM) were 5000 V and 550C, respectively. The declustering potential (DP), entrance
potential (EP), collision energy (CE) and collision cell exit potential (CXP) were
optimized and shown in Table 7.1 (a). The instrument was interfaced to a workstation
running AnalystTM (version 1.6) software. MS/MS spectra of Q1 and product are shown
in Fig 7.1 (a, b).
Table 7.1 (a) Optimized LC-MS/MS parameters for compound and IS
Analyte Q1 Q3 Declustering Entrance Collision Collisio
Precursor Product potential Potential energy n cell
ion Ion (DP) (EP) (CE) exit
potentia
l (CXP)
Compound 419.3 267.2 74 11 18.2 5.3
Phenacetin 180.1 138.1 60 10 25 10
(IS)

Parent ion

Figure 7.1 (a) Parent ion spectra of compound

36
 Product ion Parent ion

Figure 7.1 (b) Product ion spectra of compound

7.2 Validation of compound

7.2.1 Specificity and matrix effect

The technique of LC-MS/MS itself offers a high degree of selectivity and specificity due
to its ability to operate in multiple reactions monitoring (MRM), which greatly reduces
the effect of matrix interferences. Selectivity of the method was established by the
analysis of blank samples of drug-free hamster serum from six individual hamsters and
from pooled samples. Representative chromatograms of blank serum, serum spiked with
IS and serum Spiked with compound, are shown in the Figures 7.2 (a, b, c).
XIC of +MRM (3 pairs): 419.300/127.000 Da from Sample 5 (CS 0) of CS.wiff (Turbo Spray) Max. 1330.0 cps.

0.04
1300
1250
1200
1150
1100
1050
1000
950
1.88 4.45
900 3.37
3.06
850 4.24
4.19
800 1.93 3.52 3.81
In te n s ity , c p s

750 4.13
700 2.17 2.57 2.95
2.37 4.92
650 3.45
2.31
600
3.30
550 1.81
3.70 4.04
500
1.30 2.10 2.71 4.52
450 0.66
0.43 1.43 1.68 4.88
2.02 4.71
400 1.09
0.22
350
1.49
300
0.81
250 0.92
200 0.36 1.21
150
100
50
0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
Time, min

Figure 7.2 (a) Chromatogram of blank hamster serum

37
 Figure 7.2 (b) Chromatogram of serum spiked with IS

Figure 7.2 (c) Chromatogram of serum spiked with compound at LLOQ

7.2.2 Linearity and reproducibility

The linearity of the standard curve of the compound was established over the
concentration range 1-200 ng/mL, R2 of the curve was found to be 0.999 as shown in
Figure 7.2 (c).

38
 Figure 7.2 (d) Calibration curve of compound in normal hamster serum showing
linearity of the developed and validated method

7.2.3 Lower limit of quantification

The lower limit of quantification (LLOQ) was found to be 1 ng/mL as the response at this
concentration was 10 times higher than that of the blank serum (S/N ratio > 10).

7.3.4 Recovery, accuracy and precision

The recovery, accuracy and precision were determined at LLOQ, LOQ, MQC, HQC of
compound, and the detailed information is shown in table 7.2 (a). Recoveries of the
analytes from the serum were always >90%. The percentage bias and %RSD varies from
8.64 to 0.39% and 9.76 to 4.68 %, respectively. The LC-MS/MS method for the
determination of compound was reliable and reproducible as percentage bias and %RSD
for all estimated concentrations of compound were within acceptance criteria for assay
validations.
Table 7.2 (a) The recovery, accuracy and precision of the assay method
Absolute recovery Accuracy (%bias) Precision (%RSD)
Concentration
Mean CV(%) Intra-day Inter-day Intra-day Inter-day
1 ng/mL 102.3 8.1 0.8 0.6 4.8 8.9
1.75 ng/mL 102.0 8.0 1.1 2.8 5.9 8.8
90 ng/mL 111.2 10.8 8.6 7.9 9.7 6.6
180 ng/mL 98.9 7.6 3.1 0.3 8.7 4.6

39
7.3 In vitro pharmacokinetic study

7.3.1 Serum stability

Figure 7.3 (a) Curve showing serum stability of compound in normal rat serum

Serum stability assessment of compound was performed in doublets at a concentration of

100 ng/mL. The study was performed 370.5C in a bench-top Lab-Line shaker (julabo
SW23, Germany) for 1 h. The results depicted that the compound was stable in serum under the
studied conditions, as shown in figure 7.3 (a) and table 7.3 (a).

40
 Reaction concentration- 100 ng/mL
Time % Compound remained SD
(MIN)
0 100 0
5 93.8 4.3
15 90.3 2.5
30 83.2 5.0
45 85.6 1.6
60 83.0 1.8
Table 7.3 (a) Serum stability data of compound

7.3.2 Gastrointestinal fluid stability

Since oral route is most preferred route of drug delivery it is important to assess the
compound stability in simulated gastrointestinal fluids. Stability of compound was
carried out in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) without
enzymes at 370.5C in a bench-top Lab-Line shaker (julabo SW23, Germany) for 1 h
and 2 h respectively which operated at 90 rpm stability study of compound was
performed in doublets at a concentration of 100 ng/ml.
Table 7.3 (b) Stability study data of compound in SGF
Reaction concentration- 100 ng/ml
Time (min) % Compound remained SD
0 100 0
5 106.5 4.6
10 104.0 1.7
15 98.4 4.0
30 101.3 2.0
45 102.2 0.6
60 95.2 4.9

Table 7.3 (c) Stability study data of compound in SIF

Reaction concentration- 100 ng/ml
Time (min) % Compound remained SD
0 100 0
5 94.8 3.6
10 96.7 1.3
15 88.7 5.6
30 95.0 4.4
45 88.7 4.4
60 83.8 3.4
90 100.2 11.5
120 97.6 1.8

41
 Figure 7.3 (b) Curve showing stability of compound in SGF and SIF
The results show that the compound was stable in simulated gastrointestinal fluids under
studied conditions as shown in the tables 7.3 (b) and 7.3 (c).
7.3.4 Metabolic study

In vitro metabolic stability of compound was conducted at a concentration of 1 mM in the

presence of rat liver microsomes using a bench-top Lab-Line shaker (Julabo SW23,
Germany) for 1 h at 370.5oC. Testosterone was used as in-house positive control and
compound without NADPH was used as negative control in same incubation condition.
The metabolic profile of the positive control was within the acceptable in-house limits
suggesting that the enzymes are active, as shown figure 7.3 (c).
There was no significant change in the % negative control remained suggesting the
absence compounds degradation in the system and the compound was not found
unchanged at the end of 1 min was due to microsomal metabolism indicating 99% of
compound was metabolized. Natural log of % compound remained at each point Vs.
respective time was plotted from this elimination rate constant was calculated and the
parameters estimated were as in vitro half-life, intrinsic clearance (Clint) intrinsic hepatic
clearance (Clint hep) 0.46 ml/min, 828 mL/min/kg respectively as shown in the table 7.3
(d).
Table 7.3 (d) In vitro metabolic stability profile of compound in rat liver microsomes.
Time Percent % SD K (min-1) Clint Clint hep t1/2
(minutes) compound (mL/mi (mL/min/kg (min)
remained n*mg of of body
protein) weight)
0 100 0 0.46 0.001 0.46 828 0.003 0.11
2 57.7 1.8 0.001 0.01
5 12.5 1.9
10 0.5 0.1
15 0.1 0.027

42
 Each value represents the average of three experiments; values are shown as mean
SEM; Abbreviations: k = in vitro hepatic elimination rate constant, t1/2 = in vitro hepatic
microsomal half-life, Clint = intrinsic Clearance and Clint,h= intrinsic hepatic Clearance0.

Figure 7.3 (c) Time-dependent metabolic depletion of compound in rat liver microsomes.
Bar represents SEM (n=2).

** The variations in the stability studies represent both stability parameter and inherent
intra/ inter batch variations.
7.4 In vivo Pharmacokinetic study
7.4.1 Per oral pharmacokinetic study
In vivo per oral pharmacokinetic (PK) study was conducted in golden Syrian hamsters but
the concentration in serum for compound was below LLOQ concentration and therefore it
cant be quantified with accuracy. So that to determine the serum concentration of
compound alternative route such intravenous route of administration has to be performed.
7.4.2 Inraveneous pharmacokinetic study
Intraveneous (IV) PK study of compound at the dose of 10 mg/kg was performed in male
Sprague Dawley rats and the results revealed that the animals tolerated the treatment as
no peculiarities in their behavior were observed. The serum concentration-time profile
was subjected to compartmental analysis using Phoenix WinNonlin (version 6.3; Certara
Inc, Missouri, USA). The PK models were compared according to maximal correlation
between observed and predicted concentration, and minimal sum of squared residuals,
Akaikes Information Criterion (AIC) and Schwarz Bayesian Criterion (SBC) [71]. The
concentration-time data was best described by a two-compartmental open model and the
calculated PK parameters are shown in Table 7.4. The volume of distribution (34.8
2.3L/kg) is larger than the total blood volume of rat (0.054 L/kg; [2]) and systemic
clearance (5.4 0.3 L/h/Kg) is also higher than the total hepatic blood flow in rats (2.9
L/h/kg; [72]) indicating extravascular distribution along with the extrahepatic elimination
of compound.

43
 Figure 7.4 Concentration- time profile of compound after IV dose (10 mg/kg) in male
Sprague Dawley rats (n=3). Bar represents SEM.

Table 7.4 Pharmacokinetic parameters of compound in male Sprague Dawley rats

Parameters Values
Cmax (ng/mL) 761.2 45.7
AUC(ng h/mL) 1878.4113.4
Vss (L/kg) 34.82.3
Clearance (L/h/kg) 5.40.3
t1/2 (h) 7.31.2

Each value represents the average of five rats dosed intravenous (10 mg/kg); values are
mean SEM
Abbreviations: Cmax= Peak serum concentration, AUC= area under the serum
concentration-time curve, Vss = volume of distribution at steady-state andt1/2= terminal
elimination half-life.

44
8.0 Conclusion
The subject area of Bioanalysis covers a very broad range of assays for the quantitative
measurement of drugs and their metabolites in various biological systems like whole
blood, plasma, serum, urine, feces and tissues. These methods of measuring drugs and
their metabolites have become a vital part in the process of drug discovery and
development. Hence sensitive and selective analytical methods for the quantitative
evaluation of drugs and metabolites have become very important in both pre-clinical and
clinical drugs.
LC-MS/MS methods was developed and validated for the quantitative determination of
compound in samples. Validation has been performed for the method and was found to be
sensitive, accurate and precise. This method was employed for the quantification of
compound in in-vitro stability in pooled normal rat serum and also in simulated
gastrointestinal fluids.
Oral bioavailability of the compound was estimated following suspension and solution
administration through per oral and intraveneous route to golden Syrian hamsters and
Sprague-Dawley rats respectively. Analyte was below LOD in serum through oral
administration of compound in hamsters. Metabolic stability study in presence of rat liver
microsomes demonstrated that unchanged compound was not found after 30 min with
half-life of 0.11 min. The serum concentration-time profiles of the compound were
analyzed by two-compartmental open model approach. Volume of distribution (Vss
34.82.3) is greater than the total blood volume and systemic clearance is also higher
than the total hepatic blood flow in rats indicating extravascular distribution along with
the extrahepatic elimination of compound.

45
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