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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5849_Библиотеки_им_академика_М_И_Перельмана
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flows, etc. The body is considered to be a series of compartments (usually associa ted
with each of the organs; see also Figure 5.24) and relating CL and V
d
of a drug in the
rat to the physiology of the rat allows one to apply the same physiological rational
to humans to predict the properties of the drug in human.
A series of differential equations is written to account for the movement of the drug
from the arterial to the venous side taking into account the role of the clearing organs.
Fitting these differential equations to the data collected from each tissue is accomplished (Figure 5.25) and using various organ- and tissue-specific scaling factors the
corresponding profiles for the drug in human tissues can be simulated.
Many commercially available software packages assist with scaling using the
PBPK approach such as Gastroplus
. In this software, distribution characteristics of
a drug into various tissues have been extrapolated from the basic physicochemical
properties of the drug, thereby requiring minimum experimentation. Using this
approach, De Buck et al. [28] compared the predictions of 26 clinically tested drugs
with projections from Gastroplus
. They reported a 65–74% succe ss in predicting
various PK and PD parameters within twofold of the actual data. Although the
concepts used in PBPK modeling are scientifically the most relevant, the only way
an accurat e model can be constructed is if each tissue in the animal body is taken and
Figure 5.24 The physiological representation of the body for PBPK modeling [26].
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the drug content measured as a function of time. Since this is very tedious in a
discovery setting, it has found minimum favor among PK practitioners for the purpose
of scaling.
5.5.3 In Vitro–In Vivo Correlations
In discovery, there are many examples as well as published papers that have used
in vitro results as a guide to better predict and project in vivo data. A majority of these
methods have been developed to improve the ability to scale the CL of the compound.
Scaling the CL usin g the in vitro intrinsic CL values estimated using the well-stirred,
parallel tube, and dispersion models predicted that the scaling factors (CL
intrinsic in vivo
/
CL
intrinsic in vitro
) in humans were very varied. Using the in vitro human CL intrinsic
values along with the human scaling factors, the predictions were poor; incorporating
the animals scaling factors improved the predictions [29].
Other investigators have used sandwich-cultured human hepatocytes to predict
human biliary clearance [30] and in vitro expressed UDP-glucuronosyltransferase to
predict the Phase II glucuronidation [31] of compounds. Judicious decisions should be
made by project teams in deploying such techniques and methods to benefit the
various stages of discovery and development continuum.
5.6 PK PRACTICES
The following section is dedicated to describing practical considerations that should
be taken into account when designing and executing PK studies and when analyzing
and interpreting PK data. Because of the limited size of this chapter, only a brief
outline of the key issues will be given below. More relevant information can be found
in the references provided.
Figure 5.25 PBPK modeling of cocaine in the rat [27].
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5.6.1 PK Studies for Different Stages of Discovery Projects
Animal PK testing is widely used in pharmaceutical discovery. Although different
companies may utilize somewhat different approaches and processes in discovering
new drugs, the key stages of the discovery projects are fairly similar. A typical
discovery project involves four major stage gates: target identification, lead selection,
lead optimization, and preclinical development (Figure 5.26). An example of a similar
process can be found in the literature [32]. A similar discovery assay by stage (DABS)
approach is described by the group of the scientists from Millenium Pharmaceuticals [33] who broke the discovery process into four stages: (1) high-throughput
screening (HTS), hit-to-lead (HTL), lead optimization (LO), and development
candidate (DC).
5.6.1.1 Target Identification The target identification stage focuses primarily on
the biology of a selected target. Animal testing, primarily in rodents, is utilized for
preliminary evaluation of PK properties of screening hits and/or benchmark compounds described in the literature. At this stage, animal PK data can be used to select
an appropriate dosage and route of administration that would deliver sufficient
exposures in the animal efficacy models to build confidence in the rationale of the
biology of the target.
5.6.1.2 Lead Selection The primary purposes of the lead selection stage of the
discovery project are to (1) identify the spectrum of ADME properties of several
chemical templates (series) and (2) identify the drivers of efficacy by analyzing the
relationships between the extent and the duration of the in vivo exposures, efficacy,
and target modulation.
In this case, certain tissue distribution studies (e.g., brain penetration) may provide
valuable information. Some initial screening PK is used at this stage to quickly assess
Lead
Optimization
Preclinical
Development
Lead
Selection
Target
Identication
Characterize initial
hits and benchmark
compounds
Ensure sucient
exposures in animal
ecacy studies
Early exploration of
routes of
administration and
dose for ecacy
assessment
Identify ADME issues
with lead chemical series
Dierent routes of
administration for
ecacy models
Correlate extent and
duration of exposures
with ecacy and target
modulation (ecacy
drivers)
Target tissue distribution
to ensure exposure at site
of action
Routes of elimination
Design and test new
compounds to x
identied ADME
problems
Support of ecacy and
biomarker studies
Formulation evaluation
Characterize a few lead
compounds
Routes of elimination for
in vitro-in vivo
correlation
Support ecacy and
biomarker studies
Dose escalation studies
to support initial
toxicology assessment
Multi-species PK to
project human PK
In vivo DDI assessment
Enabling formulation
selection based on PK
In vivo biotransformation
assessment
Figure 5.26 The stages of the discovery process and primary purposes of animal PK studies.
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in vitro–in vivo correlation (IVIVC) and identify the reason for their absence. Some
initial in vivo mechanistic studies aimed at evaluation of the primary routes of
elimination may be conducted in bile-duct cannulated (BDC) rats or even dogs. The
lead selection stage of the project is probably the most critical for the success of the
entire program because it narrows down the chemical space in which the program will
operate and limits the opportunity to modulate and correct any critical pharmaceutical
properties (ADME, potency and efficacy, etc.).
5.6.1.3 Lead Optimization Selection of the lead series is a very significant
milestone for the discovery project, which now moves to the lead optimization stage.
At this stage, the primary efforts of animal tes ting are to fix any identified ADME
problems and to identify the best compounds for further advancement. Screening PK
studies aimed at driving the design of new compounds with better properties are
conducted and PK data cont inues to be used to support efficacy and biomarker studies
as well as find reasonable formulations for early safety assessments. A small number
of the most promising compounds are identified prior to moving to the next stage.
5.6.1.4 Preclinical Development In the preclinical development stage, research is
carried out to prepare for the first-in-human (FIH) dosing of the selected front-running
drug candidate. Animal studies may be conducted in a “definitive” rather than a
“screening” format because they are used to predict human PK and project an FIH
dose. A more detailed evaluation of PK/PD relationships is done in different animal
species using biomarkers and efficacy readouts. Usually a fair number of animal
studies are done to develop a formulation that will be used in investigational
toxicology (IVT) evaluations in rodent (usually rats) and nonrodent (usually dogs
but occasionally monkeys) species. As part of a risk assessment, some mechanistic
drug–drug interaction (DDI), in vivo biotransformation and metabolite identification
studies may be conducted in different animal species and compared to human in vitro
DDI studies. Such comparisons help properly select toxicology species.
Once the preclinical package is ready for filing in an investigational new drug
(IND) application, preclinical development may be considered complete. Nonetheless, a significant number of animal studies are usually performed to support further
development of the drug candidate. The goal of these studies may be to support a
broader safety evaluation of the drug candidate (e.g., reproductive toxicology,
carcinogenicity, etc.) or address some specific issues of clinical development (e.g.,
biotransformation).
5.6.2 Key Parameters of PK Studies
There are several key parameters that have to be taken into account when planning for
an animal PK study:
.
compound
.
formulation
.
animal species and strain
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.
dosing route and frequency
.
sample collection
.
bioanalysis
.
data analysis and reporting.
Those parameters are discussed in the subsequent sections.
5.6.2.1 Compounds There are several critical properties of the compound (sometimes referred to as “test compound” or “test article” or “test material”) that need to be
discussed and agreed upon between medicinal chemists and the ADME scientists
when planning and performing an animal PK study:
.
metabolic stability in expected biological matrices
.
properties related to formulations
.
purity
.
supply
.
physical form.
Compound Metabolic Stability in Expected Biological Matrices Compound stability in the biological matrices planned to be collected and analyzed is a property
that may either prevent conducting the study or correctly interpreting the PK data.
The metabolic stability of a newly synthesized compound can be measured in vitro in
metabolizing organs subcellular fractions (such as microsomes or S9 fractions) or
whole cells (such as hepatocytes). This stability may be estimated based upon
information obtained for closely related structural analogs or on general knowledge
of the metabolism of similar classes of compounds. Ester prodrugs and peptide drug
candidates are two well-known examples of compounds whose metabolic stability in
biological media needs to be evaluated prior to animal dosing. Although challenging,
PK studies of some metabolically unstable compounds can be conducted if some
special sample collection techniques are used. For example, exposure to ester
prodrugs can be assessed in animal blood or plasma samples if plasma esterases
are inhibited immediately upon sample collection by using esterase inhibitors such as
paraoxon [34], phenylmethylsulfonyl fluoride (PMSF), sodium fluoride or others [35],
or by mixing the blood or plasma samples with water-miscible quenching agents
(acetonitrile, methanol, acids, etc.). These agents coagulate proteins and destroy
enzymatic activity. However, there is a potential risk of re-esterification of the
prodrugs if alcohols are used as the blood- or plasma-quenching agents. While
technically feasible, such techniques need to be considered in the context of drug
development program, and a team should consider whether a molecule with such
metabolic liabilities is an appropriate clinical candidate.
Metabolic instability of test compounds in biological media may lead to an
overestimated total clearance of the compound, which may be higher than the total
hepatic blood flow in the animal species. When a new series of chemical compounds is
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chosen for evaluation in the lead selection stage of the project, metabolic instability
may become a deselecting criterion for the entire series or individual molecules.
In vitro metabolic stability in animal plasma is typically evaluated as the first
parameter for new compounds. The assay is fairly straightforward and may be
performed by spik ing a stock solution of the compound into animal plasma to
produce an incubation mixture maintained at 37
o
C and monitoring the disappearance
of the parent molecule or appearance of some known or expected metabolites usually
using liquid chromatography mass spectrometry (LC/MS) quantitation.
The use of at least one positive control (a compound with a known and accurately
measurable metabolic half-life) is highly recommended for these experiments since
enzymatic activity of the plasma may be reduced upon its storage and handling.
Also, some organic solvents (e.g., DMSO) used for preparation of the stock
solution may inhibit plasma enzymes responsible for biotransformation of the
compounds. As a rule of thumb, the maximum concentration of DMSO in the
incubation mixture should not exceed 0.1%. Higher percentages of organic solvents
can potentially be used for low-solubility compounds if a positive control with a
similar mechanism of metabolism is used as a reference.
Another potential source of data variability or error is evaporation from the
incubation mixture. This can happen if the mixture is sampled multiple times to
determine metabolic stability of the compounds at multiple time points. A better assay
format may utilize preparing a “bulk” amount of the incubation mixture sufficient for
generating multiple individual incubation samples in the tubes with caps or on the
plate with tight lids. These individual samples can be quenched at different time points
to establish a metabolic stability time course.
If the compound needs to be quantified in the animals organs and tissues, it may be
necessary to generate some data for metabolic stability in those tissues, homogenates,
or subcellular fractions.
Usually the PK matrix metabolic stability experiments are performed in a “quick
and dirty” format with a relative rather then absolute LC/MS quantitation. The analyte
peak area is used for quantification and ranking of compounds (see Section 5.6.2.7).
Compound Properties Related to Formulations The solubility and the chemical
and physical stability of the test compounds are critical for preparing a suitable
formulation for in vivo administration.
SOLUBILITY Sufficient solubility of the compound is especially important for intra-
venous formulations that require true solutions with no microparticles. Another
limitation for IV formulation is that the dose volumes are more restricted and typically
should not exceed 5 mL/kg. If the maximum solubility of a compound in a particular
IV formulation is 1 mg/mL, the maximum IV dose that can be achieved in the animal
study is 5 mg/kg.
CHEMICAL STABILITY Chemical stability of the compound may be experimentally
evaluated prior to preparing an appropriate formulation. Typically, acid- or basecatalyzed degradation of the compound may lead to its limited stability in solution or
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solid form. The stability of the compound in solution typically can be assessed at
pH 1–2 (representing gastric pH of human and some animal species) and pH 6–9
(representing intestinal pH of different sections of the intestine from duodenum to
colon). The duration of the stability experiments depends on the expected time
between the formulation preparation and animal dosing, which may range from
several minutes to 12–24 h.
If the compound is prepared as a salt of a strong acid (e.g., HCl or TFA) for PK, it is
recommended to assess its stability at least at room temperature with a typical ambient
humidity. It is strongly advised to conduct this stability if the salt form is hygroscopic.
Another source of compound instability is photodegradation due to light exposure.
In this case, the compound can be maintained in amber glass vials and handled in the
labs equipped with yellow lamps.
Oxidation by atmospheric oxygen may make handling the compound challenging
and reduce the probability for commercialization.
HYGROSCOPICITY Hygroscopicity of the compound may lead to higher instability in
solid form as well as to the absorption of atmospheric moisture that may change the
correction factor for the content of the parent compound content in the material used
for animal studies.
CRYSTALLINITY A crystalline form of the compound does not impact true solution
formulations but may have a profound effect on suspension formulations used for oral
dosing.
Usually, in the early stages of a project, a new compound is purified using liquid
chromatography followed by lyophilization of the collected fractions. Frequently this
process leads to an amorphous form of the compound with a lower melting point and
higher solubility. Higher solubility of the amorphous compound may facilitate its
dissolution in oral dosing suspensions which, in turn, may lead to a higher fraction
absorbed and higher oral bioavailability of the amorphous compound compared to
its crystalline form. If the compound may exist in different crystalline forms
(polymorphs), different polymorphs may exhibit different PK properties such as
ritonavir [36, 37].
ELECTROSTATICS If the compound is amorphous (frequently fluffy), it may be
electrostatic and require special handling to avoid loss of material when transferring
to a vial. When possible, transfer of the material should be minimized or avoided.
A potential good practice may be to determine the weight of the empty vials used for
lyophilization so the weight of the solid material can be accurately determined and
used for formulation work without material transfer. If transfer of solid material is still
required, certain devices reducing static electricity can be used to reduce the risk of
losing compound.
Compound Purity There are several aspects of compound purity that may impact PK
studies. The test compound must be pure enough for safe administration in animals.
Even if the impurities do not cause animal death, animal health and physiology can be
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impacted, which, in turn, may affect ADME properties of the compound. Both the
absolute content of the desired compound in the material prepared for animal dosing
as well as the quantity and spectrum of potential biological/toxicological properties
of undesired impurities must be well understood.
Typically, the total purity of the compound intended for animal administration in
preclinical PK is expected to be not less than 95%. Lower content of the test
compound may be scientifically justified if the impurities are represented by known
and fairly safe components (water, certain salts). Conversely, greater than 95% purity
of the test compound may not be sufficient for animal PK if the impurities are highly
toxic components of chemical synthesis or purification of the compounds (reagents,
catalysts, side products).
The total content of the test compound (purity) needs to be taken into account in
calculating the total amount of the material required for animal dosing. Total content
of the test compound may change due to sample storage and handling. For example, if
the acid in the salt form of the compound is not strongly associated with the parent
molecule, there is a possibility for the acid to be fully or partially released from the
test material leading to increased formal purity of the material. If the compound is
hygroscopic, it may absorb atmospheric moisture and reduce the percentage of the test
compound in the material leading to a reduced formal purity of the test material. It is
critical for medicinal chemists to clearly communicate to their ADME colleagues
the best practices in compound storage and handling.
Residual solvent in the amorphous samples of the compound needs to be calculated
to determine the purity of the compound. Proton or
13
C-NMR may provide qualitative
and in some cases quantitative inform ation about the residual solvent content.
Amount of Compound Required for Rat PK Studies The amount of compound
required for a particular PK study can be calculated using the following equation:
CR ¼
DL BW GS NA
P
Overage ð5:51Þ
where CR is the amount of the compound required for the study (mg), DL is a dose
level (mg/kg), BW is the animal body weight (kg), GS is an animal group size (the
number of animals on study), NA is the number of administrations (in case of a
multidose experiments), P is the purity of the test material.
Typical animal weights can be found in Appendix 5.A.1. However,it may be a good
practice to consult with ADME sci entists on the actual weight of the animals for a
particular study especially if less typical animal strains or younger or older animals
are used.
Additional material may be needed to be reserved for the formulation work if the
compound is new and a suitable formulation is not yet developed.
Some material needs to be reserved for preparation of the calibration standards
and quality control samples for bioanalysis of the PK samples. Usually this amount is
relatively low compared to the test material required for animal dosing. It is a good
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practice to reserve a small quantity (at least several milligrams) of the most pure
material as an analytical standard for bioanalysis.
The number of administrations depends on the durati on of the study and the
frequency of daily administration. Early PK studies typically utilize a single oncedaily (QD) administration of the compound. However, some efficacy or toxicology
studies may require multiday and more than once-daily administration.
Overage of the compound is needed due to the inevitable loss of material during
sample transfer, formulation preparation, and administration of the compound
especially in the case of oral gavage of suspensions. Studies with smaller animals
may require higher overage due to higher loss of material in the formulation vessels,
syringes, etc. Studies with different animals may require different overages typically
ranging from 1.1 to 1.5 with an average of about 1.2.
Example: The total amount of a 95% pure material for a single dose 1 mg/kg
PK study with two rats on study can be estimated based on a 0.3-kg rat weight as
CR ðmgÞ¼
1mg=kg 0:3kg=rat 2 rats 1 administration
0:95
1:2 ð5:52Þ
In this example, the minimum amount not including the material for the formulation development and bioanalysis is 0.75 mg.
Physical Form of Compounds The compound for animal dosing can be delivered to
ADME scientists in several forms such as solids, solutions, or oils.
SOLIDS Solid materials are perhaps most frequently used as a source of animal
dosing. Advantages of solid materials include ready transferability from one container to another (assuming they are not electrostatic) and the stability may potentially
be higher than a solution form. It is common in the pharmaceutical industry to
maintain the samples of small molecule drug candidates as a solid material in dry and
oxygen-free conditions. The disadvantage of solid material for animal dosing is that
more material is usually lost due to transfer between containers.
Careful consideration is required when several different batches of the compound
are combined in order to create a single batch for animal dosing. The advantage of
combining several batches is that the final batch has uniform purity and crystallinity if
the batches are dissolved together and recrystallized. When supplying compound to
the ADME scientist for animal dosing, avoid delivering several batches in separate
vials. This forces either the scientist preparing the dose or the analyst to determine
the percentage of parent compound out of a variety of salt forms and/or purities and
can lead to mathematical or dosing errors.
SOLUTIONS Dissolving a newly synthesized compound in certain organic solvents
(typically DMSO) to prepare a concentrated stock solution for initial testing is a
common practice in the pharmaceutical industry.The advantage of using a solubilized
form of the compound is that it is easy to transfer the solution from container to
container (e.g., from a master 96-well plate to secondary plates) with minimal loss.
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This process can be highly automated to increase efficiency and human error. The
disadvantage of using a solution of the compound is that different compounds may
require individualized selection of the best solvents for solubilization, which is
difficult to achieve in the modern industrialized pharmaceutical setting. Incomplete
solubility of the compounds in standard solvents (e.g., DMSO or acetonitrile) may
lead to erroneous initial concentrations of the stock solutions. Even if the compound
is initially fully soluble in the stock solution, it may absorb atmospheric moisture or
fall out of solution.
Incomplete solubility of the compound in the solution or its falling out of solution
upon storage may cause inconsistency of the PK data. A potential solution to
incomplete solubility of the compound may be to spin down the stock solution and
use the supernatant as a source for formulation work and as the stock solution for
preparing calibration curves in bioanalysis.
A compound may not be completely chemically stable in a solvent. Although
frequently used as a solvent, DMSO may chemically react with certain compounds
(e.g., thiol- or isothiocyanate-containing molecules).
Another potential disadvantage of using solutions as a source of animal testing is
that the formulation of the compound will contain the solvent that may not be
acceptable or preferred as a component of the formulation vehicle.
OILS Some compounds may exist in an oil form due to a low melting point (lower
than the ambient temperature) or due to the presence of some residual solvents used
for the synthesis or purification. Transferring and weighing oily compounds may be a
challenging task for ADME scientists especially if the oils are very viscous. Another
problem is that the oily compounds may contain a significant and/or not well-defined
amount of the residual solvents. Thus, it is difficult to calculate the purity of the
compound and the actual concentration in the dosing solution.
5.6.2.2 Formulations A very brief overview of formulation development for
animal dosing, some examples of the excipients and formulation recipes, and practical
suggestions for formulating discovery compounds are presented. The area of drug
formulations is large and more information can be found in the following publications [38–46]. Additionally, medicinal chemists should consult with their pharmaceutical science colleagues on the best approaches in formulation development.
Developing a suitable formulation of a compound for animal dosing is one of the
major limitations for early PK studies because the current information about the
solubility and chemical and physical stability of the compound may be very limited.
Evaluationof potential formulations may require a fairly substantial amount of the test
material and experimentation. Thus medicinal chemists should collect as much in vitro
ADME, solubility, and chemical and metabolic stabilityfor the compound series. Even
incomplete information or “educated guesses” may be valuable for initial solubilization of the compound that can then be diluted in some standard formulation vehicle.
Additional techniques can be used to solubilize the compound. Prolonged and
vigorous vortexing may be helpful if the dissolution kinetics is slow. Sonication
and heating may help dissolve the compound, although there is a risk of thermal
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