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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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the molecule being transported from the point of entry to the intended site of action,
the pharmacokinetic properties of the molecule (i.e., those related to the processes of
absorption, distribution, metabolism, and elimination) must be evaluated as these
factors are influenced by the route of administration. Additionally, economic factors
may also influence decisions regarding the ROA since if provided a choice between
similar drugs delivered either orally or subcutaneously, most patients would select an
oral formulation.
The various routes of administration may be categorized into three groups:
.
topical—applied directly to site of action
.
enteral—administered via the digestive tract
.
parenteral—administered via routes other than the digestive tract
Examples of various routes of administration are provided in Table 9.1.
When designing preclinical toxicology studies, for most cases the ROA will be the
same as the intended clinical route. Since the data generated from these preclinical
toxicology assays will be extrapolated to humans, using the same ROA eliminates an
unnecessary variable. Often, the ROAwill be determined by the therapeutic indication
of the compound. Treatments for localized skin disorders are usually applied
dermally, while a compound intended for use as an emergency treatment for acute
poisoning will likely be given intravenously. If similar drugs are already on the
market, then the ROA should be the same or less invasive than the current drug to be
a viable contender, as illustrated by the various oral and inhaled therapeutics that are
intended to replace injectable formulations.
9.2.1 Oral Route
The intended therapeutic route of administration for most compounds is oral, and this
route is mimicked in many preclinical toxicology studies. The drug to be tested may
either be weighed into inert, gelatin capsules or suspended or dissolved in a vehicle/
carrier for delivery to the animal model. The rate of dissolution of the compound is the
rate-limiting step for drug absorption and is determined by drug solubility (water), pH
TABLE 9.1 Examples of Various Routes of Administration
Topical Enteral Parenteral
Epicutaneous (local anesthesia) Orally (tablets or
suspensions)
Intravenous
Inhalation (asthma medications
and inhaled insulin)
Gastric feeding tube Intra-arterial
(vasodilators)
Intranasal route (nasal spray) Rectally (suppository
or enema)
Intramuscular (vaccines
and antibiotics)
Eye drops (antibiotics) Subcutaneous (insulin)
Ear drops (corticosteroids) Intraperitoneal
(peritoneal dialysis)
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of the environment, the chemical structure or form of the drug, and the drug
formulation (excipients, binders, enteric coating).
Following dissolution, the drug must pass through the semipermeable membrane
between the lumen of the gastrointestinal tract and the systemic circulation by passive
diffusion, active transport, or facilitated transport [1]. Passive diffusion follows
a concentration gradient and requires no energy, while active transport uses energy
to move compounds against the concentration gradient. Facilitated transport moves
molecules along the concentration gradient, across the cell membrane via special
transport proteins that are embedded within the cellular membrane, but at a much
faster rate than passive diffusion. Small molecules may be absorbed by either
mechanism. Molecules such as glucose, amino acids, and carbohydrates are transported by active or facilitated transpor t.
The pH of the gastrointestinal environment also affects the relative bioavailability
by altering the diffusion rates of compounds in the stomach and intestines. The rate of
diffusion is directly related to the ionization of the compound. The proportion of
a substance that is ionized at a given pH is determined using the Henderson–
Hasselbalch equation. In the stomach, drugs that are weak acids (such as aspirin)
will be present mainly in their nonionic form, and weak bases will be in their ionic
form. As nonionic compounds diffuse more readily through cell membranes, weak
acids will have a higher absorption in the highly acidic stomach. However, in the basic
environment of the intestines weak bases such as caffeine will diffuse more readily
since weak bases will be nonionic.
In addition to pH, the anatomical differences between the stomach and the small
and large intestines may influence the absorptive rates of compounds. The smooth
epithelium of the stomach and relatively fast gastric emptying rate (t
1/2
¼ 20–180 min
in the dog) [2] offers less opportunity for absorption when compared with the highly
invaginated small intestine, with a relatively long residence time of 4–6 h. For these
reasons, the small intestine is considered the most important absorptive site for drugs
administered orally.
Occasionally,animal studies are conducted to determine food effects (fasted versus
fed)on the absorption of the compound. Food maycause delayed orreduced absorption,
accelerated or increased absorption, or may have no effect on absorption. The food
effects on drug absorption must be determined before the compound is introduced
to humans. Basic animal model study designs provide for oral administration of a
compound to one group of fasted animals and one group of animals that had ingested
a knownamount of food. Samples are collected to determine absorption and the groups
are rotated the following week from fed to fasted and the process is repeated. Relative
bioavailabilityis determined and the food effectis evaluated.Fenofibrate is an example
of a compound due to its poor affinity for water and to its hydrophobic nature that is
much better absorbed after ingestion of food than in fasting conditions [3].
9.2.2 Intravenous Route
Some compounds that cannot be readily absorbed via the gastrointestinal tract or must
bypass the acidic conditions of the stomach (e.g., proteins) or delivered to the site of
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action immediately (i.e., emergency care drugs) are administered directly to the
circulatory system, usually intravenously. Whether delivered directly to a vein as a
bolus injection or infused over a period of time such as many of the chemotherapies,
study designs for compounds intended to be administered intravenously must be given
special considerations. For example, compounds may be administered to mice
intravenously, but their small size presents challenges that may confound the results
of the assay. Alternatively, the lack of easily accessible peripheral veins in guinea pigs
may limit their use on intravenous studies. For other species, either a temporary
peripheral catheter or a surgically implanted, indwelling venous access port may be
utilized to facilitate intravenous administration.
While bolus intravenous administration may be accomplished by skilled technicians, the use of catheters is recommended as it minimizes the chance of extravasation, accidental administration into the surrounding tissue. If the ROA is expected to
be intravenous infusion, surgically implanted venous access ports may be used in
preclinical studies to mimic the clinical route. The port is a central venous line
attached to a small reservoir that is covered with silicone rubber and is implanted
under the skin. Compounds are administered, usually with the aid of an infusion
pump, by connecting the pump to the port with an external catheter. Certain risks are
inherent with intravenous administration and include local or systemic infection and
phlebitis as well as irritation of a vein caused by the mechanical effects of the IV
catheter. Rodent studies require animals to be tethered, while ambulatory pumps are
used for delivery in larger mammals.
9.2.3 Dermal Route
The intended therapeutic indication for some compounds may require application
directly to the skin or dermally. In several cases, the intended site of action is the skin
such as Retin A compounds used to treat acne. Other examples include pain
medication, birth control, and other treatments delivered in a dermal patch in which
systemic absorption is the intended route. In either case, dermal application in the
preclinical toxicology studies can present challenges.
As with other routes of administration, the formulation of creams, gels, or
ointments may greatly influence the exposure levels to the animal model. Since low
viscosity preparations may be difficult to keep isolated at the dose site for the duration
of the dose period, a viscous preparation is preferred. Dermal application of these
types of preparations usually involves application with a syringe or tongue depressor
to the dorsal surface of the animal. Prior to the first application, the dose site should be
clipped free of hair to maximize skin contact and the site should represent approximately 10% of the total body surface of the animal. Although a variety of jackets and
collars are available to minimize the animal’s access to dose sites, formulations with
low viscosity may lead to inadvertent oral ingestion by the animal. Obviously, this
inadvertent ingestion would confound the toxicology and pharmacokinetic results
and could invalidate the entire study.
Another option for dermal delivery is the use of a transdermal patch with the patch
affixed to the dose site by means of an adhesive or hypoallergenic tape. This route of
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administration offers a more consistent and uniform delivery than applying a cream or
gel and the opportunities for inadvertent oral ingestion are reduced. If an adhesive is
used to secure the patch, care must be taken to determine if any observed dermal
irritation can be attributed to the test article or the adhesive. This irritation may be
minimized by rotating application sites on the animal.
9.3 FORMULATION ISSUES
The route of delivery will play a key role in determining the formulation. Compounds
intended to be delivered orally generally pose fewer problems than those administered
via a parenteral route since the latter needto besolutions. Additionally,thisformulation
will ultimately be used in human clinical trials, thus any vehicle or excipient in the
formulation must be selected as to not interfere with the toxicological interpretation.
To comply with GLP guidelines, the API (active pharmaceutica l ingredient)
should have an accompanying certificate of analysis (C of A). It is critical for the
compound to have a purity of close to 100%. However, any unknown impurities in
the API may confound the results of the assay and may require additional testing to
determine if toxicity is caused by the test article or the impurity. Additionally, the
storage conditions for the API and the control and test article formulations must be
controlled and documented.
A variety of vehicles are available for use, but it is advisable to use vehicles with
a known toxicity profile. For compounds to be administered orally, the test compound
may be dissolved (e.g., water, 0.9% saline) or suspended (e.g., 0.5% methylcellulose)
at the desired concentration levels with the target concentration levels based on the
actual purity of the compound or free base/free base acid content. If a suspension is
used, the mixture should be stirred continuously throughout the dosing process
to maintain homogeneity and prevent precipitation. Intravenous formulations are
commonly prepared with physiological compatible vehicles such as 0.9% saline or
lactated ringer’s solu tion. If the solution requires sterile filtration, solution samples
should be collected and analyzed before and after filtration to determine if the
compound adheres to the filter membrane.
Once the above issues have been addressed, the process for verifying the
formulation mus t be established. A requirement of all GLP toxicology studies is
verification of the concentration, stability, and homogeneity of the dose formulations
under their conditions of use. This task may be accomplished by pulling samples of
the dosing formulations, including the control material, immediately following
preparation and again at the end of the period of use for that particular batch. If
the formulation is a suspension, multiple samples would be required to demonstrate
homogeneity within the formulation container. These samples are then analyzed using
a validated analytical method according to GLP standards with the results included in
the study report. The results should demonstrate the following:
.
initial concentrations were within acceptable range of theoretical,
.
suspensions were relatively homogenous,
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.
all formulations were stable over the period of use,
.
the absence of test compound in the control material.
9.4 COMPOUND REQUIREMENTS
The amount of test article required to complete the IND supporting studies is often
either overlooked or underestimated. One must consider the size of the animal model,
the route of administration, and the proposed dose levels when calculating the amount
of required material cognizant that dose levels are based on toxicity and not efficacy.
Selection of animal models will be discussed in more detail in Section 9.5, but for
most IND filings, two models will be used, one rodent (mouse or rat) and one
nonrodent. Assuming an average body weight of 250 g for a rat, 10 animals/sex/group,
and dose levels of 10, 50, and 100 mg/kg of body weight, estimated requirements for
the rodent study given orally for 28 days would be near 23 g of the test article
(Table 9.2).
For the nonrodent species, common options include rabbits, dogs, monkeys, pigs,
and more frequently mini-pigs, with dogs and monkeys being the most commonly
selected nonrodent species. The daily compound requirement is calculated
for a standard oral dog study assuming 5 animals/sex/group, average weight of
8 kg/dog, and dose levels of 10, 50, and 100 mg/kg of body weight. Using these
assumptions, the daily compound requirements would be 12.8 g/day or 358.4 g for
a 28 day study (Table 9.3).
These estimates reflect only the theoretical amount delivered to the animals and do
not account for additional factors that will influence the actual amount. GLP studies
require a retention sample for all studies of longer than 4 weeks duration although an
amount is not specified (BASi generally retains 0.5 g). For studies that are delivered
via a solution or suspension, periodic samples of the formulated material will need to
be collected and analyzed using a GLP validated analytical method. The volume of
formulated material prepared must exceed the expected volume requirement to avoid
an interruption in the dosing regimen. The occurrence of unexpected spills can further
increase drug requirements. Many labs multiply the theoretical drug amount by
a factor of 1.5 to compensate for these unknowns. For the example cited above, the
total drug requirement to complete a 28 day oral toxicology studies in rats and dogs at
TABLE 9.2 Compound Requirement for a Standard Rat Study
Group ID Dose Level (mg/kg) Animals/Group Total Group Weight (kg) Total (mg/day)
Control 0 20 5 0
Low dose 10 20 5 50
Mid dose 50 20 5 250
High dose 100 20 5 500
800
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dose levels of 10, 50, and 100 mg/kg of body weight would be (23 þ 358.4)1.5 or
approximately 572 g of active compound which does not include a correction for
purity, water, salt weight, and so on.
9.5 ANIMAL MODELS
As wi th ROA, drug formulation and material requirements discussed in Sections 9.2,
9.3, and 9.4, respectively, selection of the animal models for the preclinical studies
requires some forethought. With some exceptions, such as large molecules, IND
requirements for the US Food and Drug Administration (FDA) require testing in two
species, one rodent and one nonrodent. Prior to selection of the animal models, the
ROA, physiological species variations and/or similarities, therapeutic indication, and
historical sensitivity to the class of compounds must be considered.
9.5.1 Mouse
The mouse is the most ubiquitous of the lab animals with a variety of inbred, outbred,
and genetically modified strains available along with volumes of historical data. First
utilized in the early 1900s as a laboratory model for genetic research, mice continue to
be used extensively for the study of genetics as well as research for cancer, toxicology,
metabolic disease, obesity,aging, and cardiovascular disease [4]. The recent mapping
of the mouse genome along with the ability to manipulate gene expression in mice
make the mouse unique to biomedical researchers and toxicologists. Their small size
reduces compound requirements and their short reproductive cycle adds to their value
as a research model. Mice are also relatively economical to acquire and maintain in the
laboratory compared with larger species and space requirements for housing are
minimal. One disadvantage of the mouse model is the minimal blood volume, which
can present difficulties in collecting multiple samples for toxicokinetics, hematology,
and clinical chemistry. Even with recent improvements in micromethodology,
approximately 0.5 mL that can be collected with a survival procedure is usually
insufficient to conduct more than a few of the endpoints. Dose administration can also
present challenges. Intravenous administration, often using a lateral tail vein which is
quite small, requires considerable practice. Oral gavage or com pound admixed into
the feed is the ROA for many 2 year oncogenecity studies, and with a lifespan of 2–3
years, the mouse, along with the rat, is the species of choice for these types of studies.
TABLE 9.3 Compound Requirement for a Standard Dog Study
Group ID Dose Level (mg/kg) Animals/Group Total Group Weight (kg) Total (mg/day)
Control 0 10 80 0
Low dose 10 10 80 800
Mid dose 50 10 80 4000
High dose 100 10 80 8000
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9.5.2 Rat
Rats, in addition to being used for oncogenecity studies, are also used for a variety of
studies to support biomedical research. As with the mouse, many inbred and outbred
strains have been developed since rats were first domesticated in the 1800s and an
extensive historical database is available [5]. As rats are generally larger than mice,
the test article requirements are slightly increased. The size of a rat does allow for
multiple blood sampling and easier access to the venous system. As a result , this animal
is the more prevalent rodent model used in support of an IND application. Among the
species variations of the rat to consider during the study design process are their
inability to regurgitate and their lack of a gall bladder. In addition, the rat’s coprophagic
behavior may have an impact on the test article exposure, particularly if the compound
is excreted in the feces. Lastly, in my experience, rats are also easier to handle and
manipulate. Compared with mice, incidents of rat bites are fairly uncommon.
9.5.3 Dog
Centuries of selective breeding have created hundreds of breeds of dog that differ in
size, shape, and behavior, but the beagle dog remains the most commonly used breed
for toxicology studies. The beagle dog offers several advantages for use in researc h:
1. their size and disposition allow for collection of multiple blood samples and
reduce the difficulty of dose administration;
2. an extensive historical database is available;
3. less expensive than nonhuman primates.
Similar to the rat and humans, dogs are obligate nasal breathers, which leads to
their use for inhalation studies, and aerosol deposition in the alveolar region of the dog
lung also closely correlates with humans (6). Among the disadvantages for the dog
model is their propensity for emesis following oral and often intravenous administration. Any emesis following an oral dose may confound study results.
9.5.4 Swine
The use of swine as the nonrodent model has increased significantly since the
development of the miniature swine in the 1950s [7]. The availability of several
breeds of mini-pigs (Gottingen, Hanford, Yucatan, Sinclair, and others) and public
pressure to avoid using dogs and monkeys has contributed to this trend. While most
often considered for use on dermal studies, the pig is a suitable surrogate for many
other types of toxicology studies, particularly drugs intended to treat cardiac disease.
In addition, since pigs and humans are both omnivores, the digestive system of the pig
is physiologically similar to that of humans and may be helpful in evaluating
compounds with an intended clinical route of oral administration. Economically,
mini-pigs are similar to dogs relative to the cost of acquiring and maintaining, but
depending on the breed, they will necessitate increased compound requirements.
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9.5.5 Nonhuman Primates
Nonhuman primates (NHP) are physiologically more similar to humans than any other
laboratory species and are used for a variety of general toxicology studies to support
registration of small molecules. In the last several years, breeding programs have
been established for the cynomolgus monkey, which has led to a readily available
supply of healthy animals. Rhesus monkeys and marmosets are also used occasionally.
The relatively small size of marmosets present challengesin multiple blood collections
and these animals tend to be less hardy than either the rhesus or cynomolgus. The
average weight of cynomolgus monkeys used for toxicology studies often ranges from
2 to 4 kg. Compared with other nonrodent models such as dog (8–10 kg) or mini-pig
(Gottingen—8–12 kg) compoundrequirements maybe reduced but animal cost may be
two to three times higher for NHPs. Difficultiesin restraint and handling, concerns over
zoonotic diseases including Herpes B, and sensitivity to the pressures of animal rights
activist all must be considered when opting to use an NHP model.
9.6 IND-SUPPORTING TOXICOLOGY STUDIES
A typical toxicology package to support filing an IND contains data from the
following stud ies:
.
Acute toxicity profile in rodent and one or more nonrodent species.
.
A dose ranging finding study in rodent and one or more nonrodent species.
.
A repeat dose toxicity study with recovery in one rodent and one nonrod ent
species.
.
Genotoxicity battery consisting of Ames test, chromosomal aberration, and
micronucleus assay.
9.6.1 Single-Dose Studies
Often the first mammalian toxicology studies will involve single-dose administrations
to mice or rats. While the specific names of the tests can vary based on the specific
design (i.e., dose range finding, dose escalation, etc.), these studies can generally be
categorized as acute studies as a single dose is typically involved. The objective of the
acute studies is to characterize the potential toxic effects of the compound as well as
establish dose levels for multiple dose studies. The study should be designed to
provide data on a range of dose levels delivered via the intended therapeutic ROA.
One example of an acute toxicology study is a dose escalation design. A dose
escalation study is designed to deliver a single dose to a group of animals (e.g.,
4–10 rats/group, 1–3 dogs/group) and closely observe the animals for several days
for changes in body weight and/or behavior. Following the dose administration, the
animals are returned to their cages and any unusual clinical signs of toxicity are
documented. It is imperative that all changes are noted accurately as this information
is useful in determining the possible site and mechanisms of action of the compound.
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Based on the results of this initial dose administration, subsequent dose levels will
be either decreased or increased with the goal of predicting dose levels in repeat
dose studies that will produce moderate toxicity in the high dose and no toxicity in the
low dose.
In some designs, blood sampling is added to the acute study to provide information
related to the kinetics of the compound. This information may be useful, especially if
correlated to clinical signs of toxicity. Unfortunately, the stress of blood collection to
the animal may confound results so the risk versus benefit should be considered.
Upon completion of single-dose studies, the results are evaluated to determine dose
levels for repeat-dose studies. Obviously dosages in the acute studies that caused
mortality or more than minimal toxicity following a single dose would not be suitable
for repeat-dose studies. The goal for selecting dose levels in repeat-dose studies is to
demonstrate a no-effect level at the lowest dose whereas establishing the potential
toxic effects of the compound at the highest dose level.
9.6.2 Repeat-Dose Studies
9.6.2.1 Objectives With the exception of drugs with an intended therapeutic use as
a single administration, most drugs require a series of repeated dose administrations
before the first dose to humans. The fundamental concept of repeat-dose studies is
administration of the drug by the anticipated ROA to multiple groups of experimental
animals, one dose level per group for a period of days to weeks. The actual duration of
the studies may be 7 days up to 90 days, but the clinical dosing regimen may not
exceed the duration of the preclinical studies. Avariety of endpoints may be evaluated
and the design of each repeat-dose study should be adapted on the basis of information
specific to each compound. Fundamental points that should be addressed in a study
protocol and specific parameters are tabulated in Table 9.4 and are described below.
9.6.2.2 Test System The animal models, acquired from approved commercial
vendors, should be laboratory strains of young healthy adult animals and the females
should be nonpregnant. The initial dose administration should ideally begin before the
animals are 9 weeks old (rats) or 8 months old (dogs), and physical examinations must
prove animals to be healthy for study inclusion. Animals found suitable for inclusion
to the study should be randomly assigned to the control or treated groups. The animal
weights should be stratified among groups with a target of 20% of the mean weight
of each sex.
9.6.2.3 Identification Appropriate animal identification methods such as permanent tattoos, ear tags, or subcutaneous transponders must be used to uniquely label
each animal individually, thereby minimizing mis-dosing of the animals and ensuring
the validity of the data. To reduce the effects of stress, animals should be acclimated to
their room and cage assignments several days before the first dose administration.
9.6.2.4 Study Groups At least 20 rodents (10 female and 10 male) and 6
nonrodents (3 female and 3 male) should be used at each dose level and consideration
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TABLE 9.4 Fundamental Repeat Dose Study Protocol and Specific Parameters
Rodent Nonrodent
Number of groups Control and low, mid, and high Control and low,
mid, and high
Number of main study animals 10/sex/group 3/sex/group
Number of recovery animals 5/sex/group 2/sex/group
Number of satellite animals 10/sex/group None
Body weight and feed
consumption
Yes Ye s
Daily clinical observations Yes Yes
Hematology
.
White blood cell count
.
Absolute differential
leukocyte count
.
Red blood cell count
.
Hemoglobin
.
Hematocrit
.
Mean cell volume
.
Mean cell hemoglobin
.
Mean cell hemoglobin
concentration
.
Platelet count
.
Reticulocyte count
Clinical chemistry
.
Sodium
.
Potassium
.
Chloride
.
Alkaline phosphatase
.
Alanine aminotransferase
.
Aspartate aminotransferase
.
Glucose
.
Blood urea nitrogen
.
Creatinine
.
Total cholesterol
.
Triglycerides
.
Total protein
.
Albumin
.
Globulin (calculated)
.
Albumin/globulin ratio (calculated)
.
Calcium
.
Inorganic phosphorus
.
Total bilirubin
Coagulation
.
Prothrombin time
.
Activated partial
thromboplastin time
ECGs None Pretest and study
termination
Macroscopic examination
of tissues
Yes Ye s
Standard organ weights Yes Yes
Histology 48–65 tissues/animal 48–65 tissues/animal
Pathology All animals All animals
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