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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5571_Библиотеки_им_академика_М_И_Перельмана
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should be given to an additional satellite group of animals for at least 14 days check
Webster for all such entries posttreatment to observe for reversibility or delayed
toxicity [8, 9]. Gen erally, at least three test groups and a control group should be used
with dosages calculated from the range-finding studies. Except for treatment with the
test article, animals in the control group should be handled the same as the animals
given the test article. Preferably, all animals should receive the same volume with
dosages adjusted by varying the concentration of the dose formulation. If a study
design incorporates a vehicle to administer the test substance and different dose
volumes are used, the control group should receive the vehicle in the highest dose
volume.
9.6.2.5 Justification of Doses All existing toxicity and kinetic data available for
the test compound or related compounds should be considered when selecting dose
levels. The highest dose level should be selected with the intent of eliciting toxic
effects but not mortality or severe toxicity. Once the high dose is selected,
a descending sequence of dose levels should be chosen. Two- to fourfold intervals
are frequently used for setting the descending dose levels, but the lowest dose level
should demonstrate a no-observed-adverse effect (NOAEL).
9.6.2.6 Dosing The cont rol or test article is administered to each animal daily for
a period of up to 28 days. In some cases, the dosing regimen may be altered to mimic
the intended clinical use such as chemotherapy drugs, which may be given as an
infusion once or twice a week for 4 weeks. Regardless of the ROA, changes in dosages
should be achieved by adjusting the concentration as opposed to altering dose
volumes between groups.
9.6.2.7 Body Weight and Food Consumption Body weight and food consumption data are collected periodically to assess the health of the animal and to calculate
dosages (body weight). Baseline body weight values are collected during the pretest
period and then daily or weekly during the dose administration phase. Food
consumption is quantitated by weighing the feeder either daily or weekly and
subtracting the ending weight of the feeder with the initial weight and dividing by
the number of days the feed was offered. Occasionally, for species such as rabbits,
monkeys, and swine, only a limited amount of food is offered to control weight gain
and prevent food wastage.
9.6.2.8 Clinical Observations Careful observation of animals following dose
administration should always be included in a study design. The observer must be
trained to recognize normal animal behavior, and it is also helpful to note individual
behaviors a few days before dose administration has commenced. A standardized
menu of common clinical observations is useful in maintaining consistency and
accuracy in the recorded clinical signs of toxicity. These observations should
“include, but not be limited to, changes in skin, fur, eyes, mucous membranes,
occurrence of secretions and excretions and autonomic activity (e.g. lacrimation,
piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and
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response to handling as well as the presence of clonic or tonic movements,
stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g.
self mutilation, walking backwards) should also be recorded.” [10] no changes in
quoted text
9.6.2.9 Clinical Pathology Many options are available for evaluating clinical
pathology. Common tests include hematology, coagulation, clinical chemistry, and
urinalysis testing. For larger species, baseline samples are collected at pretest and
periodically during the dosing and recovery period. In consideration of blood volume
restrictions, sampling for rodent studies is often restricted to a terminal sample
collected at necropsy. The results from the clinical pathology coupled with clinical
observations recorded during the dosing period and gross and microscopic observation of the tissues at the end of the study can be useful in determining organs or organ
systems affected by administration of the test compound.
9.6.2.10 Electrocardiograph Electrocardiograph (ECG) data is usually restricted
to larger mammals although recent advances in technology have enabled researchers
to collect data from rodents. ECG data may be collected from either conscious
animals or animals that have been anesthetized. One or two ECGs are collected before
the first dose administration to screen for preexisting conditions and establish baseline
information, once following the first dose to capture acute changes, and at periodic
intervals during the study. If pharmacokinetic information is available, design the
study to collect ECGs on Day 1 at approximate C
max
to maximize the opportunity to
observe potential cardiotoxicity.
9.6.2.11 Postmortem Procedures and Evaluations At the end of the dosing or
recovery period, animals must be humanely euthanized and subjected to a gross
necropsy. The AVMA has published guidelines on acceptable methods of euthanasia
and review of this document is recommended [11]. The actual method of euthanasia
may cause gross and microscopic changes in tissues (i.e., sodium pentobarbital—
enlarged spleens in dogs), so euthanasia of the control and treated animals should be
identical. Immediately following euthanasia, appropriate blood and urine samples
should be collected and then all organs examined grossly. During this process, organs
or tissues as specified in the study protocol will be harvested and either fixed in
formalin or weighed and then fixed. A complete description of any abnormal tissue or
lesion is recorded and the finding is collected to ensure that the tissue will be examined
microscopically. Target organs may often be identified during the gross necropsy
process.
9.6.2.12 Organ Weight Immediately following tissue collection, organ weight
data is collected and recorded. The weight of the organ or tissue is recorded following
complete exsanguination and trimming of extraneous fat and tissue. Care must be
taken to be consistent in the trimming of tissues to increase the validity of the results.
The data are often reported as actual weights, relative to brain weight, or relative to
body weight with the latter requiring a fasted body weight. In the absence of control
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data for comparison, organ weight data for animals found dead or sacrificed moribund
are not normally collected.
9.6.2.13 Microscopic Pathology Once tissues have been preserved in 10% neutral
buffered formalin or another fixative, representative samples (3–5 mm) are taken from
each tissue and processed. Tissue processing involves taking tissue from a waterbased state, dehydrating it and infiltrating it with paraffin. Following processing, the
tissues are embedded in paraffin blocks or othe r embedding media and thin slices of
tissue are transferred to microscope slides. Avariety of specialized stains are available
today,but the most common stain is hematoxylin and eosin (H&E) stain. This staining
method involves application of the basic dye hematoxylin, which colors basophilic
structures with blue–purple hue, and alcohol-based acidic eosin Y, which colors
eosinophilic structures bright pink. The slides are then evaluated, preferably by
a board certified veterinary pathologist, to determine what changes, if any, can be
attributed to the administration of the test compound.
9.7 STUDY RESULT INTERPRETATION
The challenge of evaluating and interpreting data generated by a toxicology study is
an important process to recognize. Data collected from treated animals are compared, either individually or by group, to correlating data collected from control
animals to determ ine physiological or pathologica l alterations that can be attributed
to administration of test compound. Group mean values may be analyzed statistically, but it is equally important to evaluate any “outliers” that may artificially skew
the group mean. Each study design will be adapted to the specific compound to be
tested based on discovery data, therapeutic area, method or mode of action, and
results of studies conducted on similar class compounds and the endpoints from
these designs may be extensive. A few of the fundamental endpoints are discussed
below.
9.7.1 Clinical Observations
Careful observation of animals periodically during the study may reveal valuable
information as to the toxicity of the compound and the site of action. Technicians
are carefully trained to observe and record behavioral data following administration
of the test compound, preferably at or near C
max
and periodically throughout the
study. Evaluation of these observations may indicate whether the compound affects
systems such as digestive (diarrhea, emesis, dark stool) , nervous (ataxia, convulsions, paralysis), renal (urine output and coloration), anogenital (estrus, rectal
prolapse), oral/nasal (nasal discharge, discolored mucous membranes), and respiratory (dyspnea, cyanosis). In addition to recording the presence of the observation,
it is equally important to document the time (relative to dose administration) the
observation was noted, the severity of the observation, and when and if the
observation resolved.
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9.7.2 Body Weight/Feed Consumption
Examination of body weight and feed consumption data may often provide the first
indication of toxicity in repeat dose studies. Young healthy animals should exhibit
daily weight gain until they reach the adult stage. Thus, body weight loss may indicate
subtle toxicity that is otherwise not apparent. While decreased feed consumption
usually accompanies a decrease in body weight, some compounds may cause weight
reduction while normal feed consumption is maintained, either by increasing the
metabolism or increasing the rate of peristalsis. Reduced body weight coupled with
reduced food consumption could indicate inappetance due to either a neurological
effect or gastrointestinal irritation or bloc kage. Review of the clinical observations, in
particular incidences of loose stool/diarrhea, emesis, or no stool should be utilized to
differentiate this occurrence. Alternatively, a reduced/increased body weight coupled
with unchanged food consumption may indicate a metabolic change.
9.7.3 Clinical Pathology
Hematology test usually references a complete blood count (CBC) (with reticulocyte
count, platelet count, mean cellular volume (VCM), and differential leukocyte count).
This test includes the quantification of red blood cells (RBCs) and hemoglobin and
calculation of the percentage of blood volume that is occupied by RBCs (hematocrit).
Potential toxic effects include decreases in red blood cells, hemoglobin, and/or
hematocrit, which may be indicative of anemia, caused by hemorrhage, red cell
destruction, or decreased red cell production (bone marrow suppression). Anemia is
the most common hematologic change noted in toxicology studies [12]. Results from
the clinical observations and gross necropsy can usually identify the source of blood
loss, while evaluation of the reticulocytes or red cell distribution width (RDW) may be
used to distinguish cell destruction from cell production. Reticulocyte production
should increase as a normal adaptive response in anemic condition. Sometimes the
intended therapeutic use of a compound may cause anemia, such as the immunosuppressant drug Azathioprine [13] and other chemotherapy drugs. In rare cases,
increases in red blood cells, hemoglobin, and/or hematocrit are noted, but these
increases are usually related to hemoconcentration caused by dehydration.
The CBC also includes quantification and differentiation of leucocytes (neutrophils, lymphocytes, monocytes, eosinophils, and basophils). Changes in the number
of leukocytes or white blood cells may indicate an immune response to infection or
disease, effects of physiological stress, or may be a sign of immunotoxicity.
Examination of the changes in the numbers of each type of cell provides additional
information as to the cause or source of the effect. When interpreting the differential
results, the absolute counts as opposed to relative or percent counts are evaluated since
the latter have no inherent value in assessing the condition of an animal [14].
9.7.4 Clinical Chemistry
The analysis of serum may detect toxicity of the hepatic-biliary function, renal
function, carbohydrate, protein, and lipid metabolism, and balance of electrolytes.
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Elevations of alanine aminotransferase and aspartate aminotransferase may indicate
hepatic injury while increased alkaline phosphatase may be a sign of cholestasis or
biliary hyperplasia. Although uncommon, bilirubin increases suggest hepatic injury,
cholestasis, or both. Serum urea nitrogen and creatinine levels are used to evaluate
renal function but are not sensitive to subtle changes. Serum proteins (albumin and
globulin), glucose, and serum lipids (total cholesterol and triglycerides) are monitored to detect toxic effects on metabolism. Changes in serum levels of the electrolytes
(sodium, potassium, and chloride) may be attributed to dehydration from emesis or
diarrhea, decreased food intake, or renal failure. The clinical chemistry data are often
compared statistically (control versus treated groups) to determine if any changes
reach significance. For individual or groups of animals that do exhibit a significant
change, it is equally important to compare with pretest or historical data to verify the
biological significance of the finding.
9.7.5 Electrocardiograms
The ECG data collected from animals in a toxicology study are ideally analyzed by
a board certified veterinary cardiologist with the results appended as a contributing
scientific report. Cardiovascular (hemodynamic) function is part of the safety
pharmacology tests and limited to detecting cardiotoxicity following single administrations. These tests are not designed to capture cardiac changes caused by repeated
administrations. Baseline readings collected once or twice pretest indicate the
absence of preexisting abnormalities and are compared to a reading collected
following one dose and readings collected following multiple doses such as on Day
13 for a 2 week study. Parameters for evaluation may include blood pressure
(diastolic, mean, and systolic), heart rate, P duration, PR interval, QRS interval, R
amplitude, and QT interval. If it is determined that cardiac changes are present and
appear to be related to administration of the test article, ECG data should be collected
from recovery animals to determine the reversibility of the noted changes.
9.7.6 Organ Weights
Careful examination of organ weight data may often illustrate the site of action of
a drug as well as identify target organs for microscopic evaluation. After careful
trimming and removal of residual blood, organ weight data are collected. As a means
to normalize the data, the organ weights are also expressed relative to fasted body
weight and brain weight. One common finding noted in many studies is the incidence
of increased liver weights due to the relatively high doses that are used for toxicology
studies. This increase is not necessarily indicative of hepatic injury, but can typically
be attributed to increased activity of the drug-metabolizing enzymes present in the
liver [15]. The method of euthanasia may also affect organ weights. For example,
splenic weights from dogs euthanized with pentobarbital are increased. It is best
practice to evaluate all of the three options (actual organ weights, organ weight
relative to body weight, and organ weights relative to body) at the same time to
differentiate if any noted changes are due to actual changes in the weight of the organ
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or possibly related to changes in weight gain. The data from microscopic examination
of the tissues may be used to help assess the biological relevance of any differences
between organ weights that appear to be affected by drug administration.
9.7.7 Pathology
Similar to the evaluation of electrocardiograms, pathology slides are ideally examined by an expert in the field such as a board-certified veterinary pathologist.
Experience is invaluable in the evaluation of patholog y slides. The decision between
reporting a lesion as drug induced as opposed to a tissue collection or processing
artifact is not trivial and may be the determining factor for advancing into clinical
trials versus shelving the compound. The pathology report should be clear in stating
what lesions were present and whether these lesions were drug related, dose
dependent, and reversible. If results are questionable, it is advisable to consider
using a peer review pathologist to resolve any issues. The peer review process utilizes
an independent pathologist to review the slides and original report to verify or
question the findings. Upon review, the peer review pathologist consults with the
original pathologist to discuss any findings that may be questionable. In my
experience, the peer review process adds value to a study when pathology findings
are spurious and not supported by clinical pathology or organ weights.
9.8 GENETIC TOXICOLOGY STUDIES
In addition to the general toxicology studies, most small molecule IND applications
will also require inclusion of the results from a series of genetic toxicology tests. A
brief descrip tion of the guidelines promulgated by the ICH “Standard Battery of
Genotoxicity Testing of Pharmaceuticals” and its role in an IND submission are
presented. For a more detailed discussion, see Chapter 7. Three standard tests are
required
1. gene mutation test in bacteria;
2. in vitro test with evaluation of chromosomal damage in mammalian cells or in
vitro mouse lymphoma assay;
3. in vivo test to screen for chromosomal damage using rodent hematopoietic
cells.
9.8.1 Gene Mutation
The objective of the gene mutation or Ames assay is to evaluate the genotoxicity
potential of a drug by measuring its ability to induce reverse mutations at selected loci
in several bacterial strains in the presence and absence of a rat-liver-derived
metabolizing system (S9 mix). Several strains of Salmonella typhimurium and/or
Escherichia coli are genetically modified to require amino acids for growth. These
strains are then exposed to the drug along with appropriate positive and negative
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controls and evaluated for colonies that restore the functional capability to synthesize
the required amino acid [16]. Drugs that promote reverse mutations may be classified
as mutagens.
9.8.2 Chromosomal Aberration
Screening for chromosomal aberrations may be conducted either in vivo or in vitro.
The mouse lymphoma-TK assay is the standard in vivo test to examine mutations at
the thymidine kinase locus caused by base-pair changes, frameshift, and small
deletions [17]. Mutant cells, deficient in thymidine kinase due to the forward mutation
in the TK locus, are resistant to the cytotoxic effect of pyrimidine analogues such as
5-trifluorothymidine (TFT). The mutagenicity of the test article is indicated by the
increase in the number of mutants after treatment with test article.
The in vitro chromosome aberration test uses cultured mammalian cells to identify
test articles that cause structural chromosome aberrations. Two types of structural
aberrations may be observed: chromosome or chromatid [18]. The majority of
chemical mutagens are chromatid-induced aberrations. Some chromosome-type
aberrations also occur. While there is evidence that chromosome mutations causing
alterations in oncogenes and tumor-suppressor genes are involved in cancer induction
in humans and experimental animals, the incidence of false positives continues to fuel
debate on the validity of this assay.
9.8.3 In Vivo Mouse Micronucleus
The micronucleus test is used to screen for potential genotoxic compounds. There are
two versions of this test: in vivo and in vitro. The in vivo test is commonly used in
support of an IND. Groups of mice are exposed to control material or test article with
bone marrow smears collected from the mice at 24 or 48 h following administration.
One smear from each animal is examined for the presence of micronuclei in
polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes is assessed by examination. The values from the treated animals are compared
with control values. Detection of damage induced by the test substance to the
chromosomes or the mitotic apparatus of erythroblasts is reported [19]. This assay
is useful in predicting genotoxic carcinogens, that is, carcinogens that act by causing
genetic damage.
9.9 CONCLUSION
The toxicology studies to support filing of an IND are an integral part of the drug
development process for a new chemical entity. It is critical that all key aspects of the
plan be considered beforehand to prevent loss of time and resources and to avoid
mistakes that may prevent a drug from moving forward into clinical trials. This
chapter reviewed key aspects to consider based on my years of experience in the field
of drug development. While timing is always an important factor in filing an IND, the
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time and effort to prepare a well-thought and comprehensive IND plan will, in most
cases, save time and resources. Failure to consider these aspec ts may result in costly
(money and time) mistakes, which sometimes requires studies to be repeated.
REFERENCES
1. Nogrady, T. and Weaver, D. F., Medicinal Chemistry, A Biochemical Approach, Oxford
University Press, New York, 1988, pp. 431–432.
2. Dunlop, R. H. and Malbert, C. H., Veterinary Pathophysiology, Wiley, New York, 2004,
pp. 113–114.
3. Brunton, L., Blumenthal, D., Buxton, I., and Parker, K., Goodman and Gilman’s Manual of
Pharmacology and Therapeutics, McGraw-Hill, New York, 2008, p. 619.
4. Suckow, M. A., Danneman, P., and Brayton, C., The Laboratory Mouse, CRC Press, Boca
Raton, FL, 2001.
5. Sharp, P. E., LaRegina, M., and LaRegina, M. C., The Laboratory Rat, CRC Press, Boca
Raton, FL, 1998.
6. Hall, R. L., Principles and Methods of Toxicology, Hays, A. W. (ed.), CRC Press, Boca
Raton, FL, 2007, p. 1321.
7. Bollen, P., Hansen, A., and Rasmussen, H., The Laboratory Swine, CRC Press, Boca Raton,
FL, 2000.
8. OECD Guideline for the Testing of Chemicals 407—Repeated Dose 28-day Oral Toxicity
Study in Rodents, Adopted by the Council on 27th July 1995.
9. OECD Guideline for the Testing of Chemicals 409—Repeated Dose 90-day Oral Toxicity
Study in Non-Rodents, Adopted by the Council on 21st September 1998.
10. Code of Federal Regulations. Title 9: Animals and animal products. US Government
Printing Office. Revised 1 January 1998.
11. AVMA Guidelines on Euthanasia June 2007.
12. Jacobson-Kram, D., and Keller, K. A., Toxicology Testing Handbook: Principles, Appli-
cations, and Data Interpretation, Marcel Dekker, New York, 2001, pp. 62–68.
13. Maddison, J. E., Page, S., and Church, D., Small Animal Clinical Pharmacology, Elsevier
Health Sciences, Amsterdam, The Netherlands, 2002, p. 234.
14. Hall, R. L.(Author) and Hayes, A. W.(Editor), Principles and Methods of Toxicology, CRC
Press, Boca Raton, FL, 2001, pp. 1019, 1023.
15. Amacher, D. E., Schomaker S. J., and Burkhardt, J. E. The relationship among microsomal
enzyme induction, liver weight and histological change in rat toxicology studies. Food
Chem. Toxicol., 1998, 36(9–10), pp. 831–839.
16. OECD Guideline for the Testing of Chemicals 471—Bacterial Reverse Mutation Test,
Adopted by the Council on 21st July 1997.
17. OECD Guideline for the Testing of Chemicals 476—In Vitro Mammalian Cell Gene
Mutation Test, Adopted by the Council on 21st July 1997.
18. OECD Guideline for the Testing of Chemicals 473—In Vitro Mammalian Chromosome
Aberration Test, Adopted by the Council on 21st July 1997.
19. OECD Guideline for the Testing of Chemicals 474—Mammalian Erythrocyte Micronucleus Test, Adopted by the Council on 21st July 1997.
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10
PRECLINICAL CANDIDATE
NOMINATION AND DEVELOPMENT
NILS BERGENHEM
10.1 INTRODUCTION
The generic discovery phase starts with identifying a target, screening for compounds
that affect the target, identifying hits, understanding the structure–activity relationship to be able to modify the hits into potent lead molecules, and finally optimizing
those lead molecules to be as drug like and efficacious as possible (Figure 10.1). The
activities in this phase are performed under non-GLP (Good Laboratory Practice)
conditions, and involve a lot of problem solving and novel thinking. This is the phase
when the drug is invented.
With the selection of the preclinical candidate, the more constrained development
phase is initiated. In development, all the boxes are checked to insure that the drug is
likely to be safe when dosed in humans. The activities in this phase are performed
under GLP conditions to ensure the quality of the results meets the requirements
of the investigational new drug (IND) application. Moving a candidate into development will initiate a cascade of activities that are quite costly. Also, for preclinical
development to proceed as rapidly as possibl e, activities occur in parallel, leading
the process to be difficult to discontinue if a problem is identified. Therefore, the
selection of a preclinical development candidate is a critical step in drug discovery
and development.
ADMET for Medicinal Chemists: A Practical Guide, Edited by Katya Tsaioun and Steven A. Kates
Copyright Ó 2011 John Wiley & Sons, Inc.
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10.2 INVESTIGATIONAL NEW DRUG APPLICATION
AND CLINICAL DEVELOPMENT
While this chapter describes preclinical candidate selection and preclinical development, the outcome is the nomination of a clinical candidate, and a basic understanding of the clinical development phases is required to establish the goals for
preclinical development.
The goal of preclinical development is to generate the data and documentation
of the drug candidate required to complete an IND application. To administer a drug
candidate to a human, an (IND) application has to be approved by the FDA that
provides guidance for the content and format of the application [1]. Interestingly, the
IND application is a request for an exemption from the Federal statute that prohibits
an unapproved drug from being transported across state lines, which is typically
required to ship and subsequently provide clinical investigators drug product. An
IND is the documentation the Sponsor of the clinical trial submits to the FDA for this
exemption.
The IND application includes data and information in three broad areas:
.
Chemistry, Manufacturing, and Control (CMC) Information;
.
Animal Pharmacology and Toxicology Studies; and
.
Clinical Protocols and Investigator Information
Figure 10.1 The efficacy of a drug candidate is assessed during discovery and Phase 2 and 3
clinical trials. The safety of a drug candidate is assessed in preclinical development and
Phase 1 clinical trial. The cost increases dramatically during initiation of preclinical clinical
development.
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