Toxicokinetics and Safety Ratios
Pharmacokinetics is essentially the study of “how a substance gets into the body and what happens to it in the body.” Similarly, toxicokinetics deals with what the body does with a drug or other substance when given a relatively high dose relative to the therapeutic dose. Toxicokinetic studies are generally carried out at much higher doses than those used in pharmacokinetic studies, and this dose information is critical for predicting the safety of substances. The primary objective of toxicokinetics is to describe the systemic exposure achieved in animals and its relationship to dose level and the time course of the toxicity study. The ratio of drug exposure in animals at the no observed adverse effect level (NOAEL) and in humans at the expected therapeutic dose is one of the precautionary principles to determine the risk benefit profile of pharmaceuticals.
Purpose and Rationale
Pharmacokinetics is essentially the study of “how a substance gets into the body and what happens to it in the body.” Similarly, toxicokinetics deals with what the body does with a drug or other substance when given a relatively high dose relative to the therapeutic dose. Toxicokinetic studies are generally carried out at much higher doses than those used in pharmacokinetic studies, and this dose information is critical for predicting the safety of substances (Welling 1995).
To relate the exposure achieved in toxicity studies to toxicological findings and contribute to the assessment of the relevance of these findings to clinical safety
To support the choice of species and treatment regimen in nonclinical toxicity studies
To provide information which, in conjunction with the toxicity findings, contributes to the design of subsequent nonclinical toxicity studies
These data may be used in the interpretation of toxicology findings and their relevance to clinical safety issues (ICH Guidance on Toxicokinetics 1994).
The ratio of drug exposure in animals at the no observed adverse effect level (NOAEL) and in humans at the expected therapeutic dose is one of the precautionary principles to determine the risk benefit profile of pharmaceuticals. For this ratio the expressions “safety ratio” and “safety margin” were also used. It is usually based on area under plasma concentration-time curve (AUC) – which is a measure of the exposure – between animal and human (animal/human AUC ratio). However, depending on the mode of action or whichever is smaller, the ratio can also be based on the maximum concentration in plasma (animal/human Cmax ratio).
Number of Animals and Time Points
In the ICH Guidance on Toxicokinetics (1994), it is stated that “the number of animals to be used should be the minimum consistent with generating adequate toxicokinetic data” and that “the area under the matrix level concentration-time curve and/or the measurement of matrix concentrations at the expected peak-concentration time Cmax, or at some other selected time C(time), are the most commonly used parameters.” In large animals (like dogs), the number of animals is usually fixed by the number of animals that are necessary for safety evaluation. The withdrawal of a sufficient number of blood samples (six to nine) per animal is not a problem. However, in small animals like rodents, it is recommended not to collect more than 10% of the blood volume during the AUC sampling interval (BVA/FRAME/RSPCA/UFAW Working Group of Refinement 1993; Cayen 1995). A good practice guide to the administration of substances and removal of blood, including routes and volumes, is described in the similar paper (Diehl et al. 2001). The authors limit the total daily volumes of multiple sampling to 7.5% of the circulatory blood volume at a recovery period of 1 week, 10–15% at a recovery period of 2 weeks, and 20% at a recovery period of 3 weeks. The optimum number of time points is always a compromise between blood volume restrictions and reliable assessment of toxicokinetic parameters (AUC and Cmax) (Diehl et al. 2001).
A more recent report of the European Partnership for Alternative Approaches to Animal Testing urges the regulatory acceptance of alternatives to animal testing: networking and communication (including cross-sector collaboration, international cooperation, and harmonization), involvement of regulatory agencies from the initial stages of test method development, and certainty on prerequisites for test method acceptance including the establishment of specific criteria for regulatory acceptance. Data sharing and intellectual property issues affect many aspects of test method development, validation, and regulatory acceptance. In principle, all activities should address replacement, reduction, and refinement methods (albeit animal testing is generally prohibited in the cosmetics sector) (Ramirez et al. 2015).
The ICH “Focus on microsampling” Implementation Working Group also stresses out the important contribution to 3R benefits (replacement, reduction, and refinement) by reducing or eliminating the need for toxicokinetic animals. Nevertheless, the today’s common analytical method sensitivity (such as that of liquid chromatography/mass spectrometry) has been improved, allowing microsampling techniques (very low volume sampling) to be widely used in toxicokinetic assessment (ICH Focus on microsampling 2017). For three different compounds, Pai et al. (1996) compared the AUCs from intensive (full) (10–15 time points with 4 to 5 rats per time point) sampling schemes with sparse sampling schemes (5 time points with 2 rats per time point). Using Monte Carlo simulation, Pai et al. (1996) could show that the deviation of AUC estimation of the sparse sampling scheme from the full sampling scheme was not larger than 10%. Thus it is seen that a sparse sampling scheme with five to seven time points with two to three animals per time point is well suited for the reliable determination of systemic exposure in small animal toxicity studies.
Main Group or Satellite Animals?
Whenever possible, toxicokinetic measurements are performed on all the animals in the toxicity study. This is the most representative approach, and it allows the individual pharmacokinetic data to be directly correlated with the toxicological findings. The second choice is toxicokinetic measurement in representative subgroups or satellite groups. Satellite groups are animals that are treated and housed under conditions identical to those of the main study animals. The use of satellite animals is indicated, for example, in small animals, where the collection of a relatively large volume of blood may influence the toxicological findings.
Integration of pharmacokinetics into toxicity testing implies early development of analytical methods for which the choice of analytes and matrices should be continually reviewed as information is gathered on metabolism and species differences. The analytical methods to be used in toxicokinetic studies should be specific for the entity to be measured and of adequate accuracy and precision. The limit of quantification should be adequate for the measurement of the range of concentrations anticipated to occur in the generation of the toxicokinetic data (ICH Guidance on Toxicokinetics 1994). The today’s common analytical methods, such as ultra(high)-performance liquid chromatography (U(H)PLC) and liquid chromatography-mass spectrometry (LC-MS), have excellent performance of sensitivity and specificity, and the limit of quantification can be low to very low (Lee and Kerns, 1999).
Pharmacokinetic profile of the compound (exposure)
Dose dependency of AUC and Cmax
Changes of exposure during the course of the toxicity study
Pharmacokinetic Profile of the Compound (Exposure)
For toxicokinetic purposes it is usually sufficient to describe the systemic burden in plasma or serum of the test species with the test compound and/or its metabolites. The AUC and/or the measurement of matrix concentrations at the expected peak-concentration time, Cmax, or at some other selected time (e.g., C(24h) as trough value), C(time), is the most commonly used parameter. According to the supplementary notes in the ICH Guidance on Toxicokinetics (1994), for a profile (e.g., four to eight) matrix, samples during a dosing interval should be taken to make an estimate of Cmax and/or C(time) and the AUC.
Dose Dependency of AUC and Cmax
Changes of Exposure During the Course of the Toxicity Study
According to the ICH Guidance on Toxicokinetics (1994), the description of the relationship of exposure to the time course of the toxicity study belongs to the primary objectives of toxicokinetics. This objective may be achieved by deriving pharmacokinetic parameters from measurements made at appropriate time points during the course of the individual studies. In short-term studies (1 month or shorter), day 1 and a day at the end of the toxicity study may be appropriate profiling days. In long-term studies, day 1, a day after one third of the study duration, and a day at the end of the toxicity study may be appropriate sampling days. Increasing exposure may occur during the course of a study for those compounds that have a particularly long plasma half-life. Conversely, unexpectedly low exposure may occur during a study as a result of auto-induction of metabolizing enzymes. However, other facts can also play a role in changes of exposure during the course of the study. Very often, rats and mice are used at an age at which they are not sexually mature, and during the study, sexual maturation takes place in the first 2 months, with its known impact on the rate and extent of metabolism. The harm of elimination pathways (e.g., nephro- or hepatoxicity) by the test compound can be another reason for changes in exposure. A more trivial reason such as aging or change of the administered batch with impact on bioavailability should also be considered.
According to the ICH Guidance on Toxicokinetics (1994), it is normal to estimate exposure in animals of both sexes unless some justification can be made for not doing so. For evaluation both sexes should be evaluated separately. The assessment of exposure data of the two sexes can be performed by calculating the ratio of AUC, Cmax, and elimination half-life in males and females. However, additional factors such as size of the investigated groups (with respect to random variation) and sexual maturity have to be considered. As a rule of thumb, it can be stated that in rodents sex difference is quite common when CYP metabolism is involved as a major elimination pathway, whereas in non-rodents distinct sex differences are rather rare.
Comparison between animal and human exposure is generally based on AUC, but sometimes it may be more appropriate to use Cmax. The synonyms “safety ratio” “safety margin” and “margin of safety” are frequently used for animal/human exposure ratio.
Most Sensitive Species
The animal/human ratios are always estimated in a conservative way, which means that the lowest exposure data (most sensitive animal species and sex) in animals and the human exposure data at the maximum recommended human dose (MRHD) are taken for calculating the ratio.
The unbound drug in plasma is thought to be the most relevant indirect measure of tissue concentrations of unbound drug. The rules on how to deal with the protein-binding issue are clearly defined (Note 8 of the ICH Topic S1C(R2) 2008).While in vivo determinations of unbound drug might be the best approach, in vitro determinations of protein binding using parent and/or metabolites as appropriate (over the range of concentrations achieved in vivo in rodents and humans) might be used in the estimation of unbound AUC. When protein binding is low in both humans and rodents, or when protein binding is high and the unbound fraction of drug is greater in rodents than in humans, the comparison of total plasma concentration of drug is appropriate. When protein binding is high and the unbound fraction is greater in humans than in rodents, the ratio of the unbound concentrations should be used.
The AUC value used for the ratio calculation is generally AUC0–24 under steady-state condition for animals as well as for humans. Even if the drug is administered more than once daily to either species (e.g., the frequency of administration in laboratory animals may be increased compared to the proposed schedule for the human clinical studies in order to compensate for faster clearance rates or low solubility of the active ingredient), the exposure per day should be calculated and compared. For the rare cases in which the dosing interval is longer than 24 h, an appropriate calculation has to be performed and mentioned along with the value.
Duration of Treatment
Usually, data of several toxicity studies in the same species, but with different dosing duration, are available. The ratio estimation should be done in the most conservative way, which means that the lowest exposure data in animals under steady-state conditions should be used whenever the exposure is determined. It is not recommended that exposure be determined at the end of the life span, and exposure monitoring is not considered essential beyond 6 months (ICH Guidance on Toxicokinetics 1994). Careful attention should be paid to the interpretation of toxicokinetic results in animals if there are already some toxicological findings. For example, very often it is not clear if the high exposure observed in these animals is the reason for their bad state or rather the consequence of it. Therefore, the direct link between observed exposure in already impaired animals and toxicological finding should be avoided.
When the administered compound acts as a “prodrug” and the delivered metabolite is acknowledged to be the primary active entity
When the compound is metabolized to one or more pharmacologically or toxicologically active metabolites, which could make a significant contribution to tissue/organ responses
When the administered compound is very extensively metabolized and the measurement of plasma or tissue concentrations of a major metabolite is the only practical means of estimating exposure following administration of the compound in toxicity studies
In the issued FDA Guidance for Industry Safety Testing of Drug Metabolites (2008), it was additionally emphasized that it is crucial to gather toxicokinetic data from disproportionate metabolites in toxicity studies with direct dosing of the metabolite.
In the FDA Guidance for Industry S6 Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals (1997), it is repeatedly emphasized that systemic exposure should be monitored during the toxicity studies. Thus, there is no difference compared to low-molecular-weight compounds. However, where a product has a lower affinity to, or potency in, the cells of the selected species than in human cells, testing of higher doses may be important. The multiples of the human dose that are needed to determine adequate safety margins may vary with each class of biotechnology-derived pharmaceutical and its clinical indication or indications. In addition, the effects of antibody formation on pharmacokinetic/pharmacodynamic (PK/PD) parameters, incidence and/or severity of adverse effects, complement activation, or the emergence of new toxic effects should be considered when interpreting the data.
Critical Assessment of the Method
Systemic Exposure as Surrogate for Exposure in All Other Tissues
The concept of safety margins based on exposure data is based on the assumption that plasma concentrations of a compound are the surrogate for exposure in all other tissues, including the target organ of toxicity. This approach is justified in the majority of cases. However, in some cases, the systemic exposure in plasma may go in the opposite direction to the specific exposure in the target organ. For example, strong first-pass hepatic extraction may increase the exposure in the target organ liver and concomitantly trigger the toxicity but decrease the systemic exposure in plasma. Another example for systemic exposure going in the opposite direction to target organ exposure was given by Lacy et al. (1998). Probenecid, a competitive inhibitor of organic anion transport in the proximal tubular epithelial cells, was evaluated for its effect on the chronic toxicity and pharmacokinetics of cidofovir in monkeys. Nephrotoxicity was present only in monkeys receiving cidofovir without probenecid. The co-administration of probenecid resulted in an inhibition of the active tubular secretion of cidofovir into the kidneys and concomitantly in a shift from local exposure in the kidney toward higher systemic exposure to cidofovir (as measured by AUC in plasma). The decrease of specific exposure in the kidneys is most likely the reason for the protection against nephrotoxicity.
How to Deal with Small Safety Factors
Can reversibility of effects be demonstrated in repeated dose toxicity studies that include a drug-free period, which may provide reassurance that the findings will not be irreversible?
If good mechanistic data for toxic effects are available, it may help in the assessment of relevance to human safety.
A smaller safety factor might also be used when toxicities produced by the therapeutic are easily monitored by relevant and valid biomarkers, are predictable, and exhibit a moderate-to-shallow dose–response relationship with toxicities that are consistent across the tested species (both qualitatively and with respect to appropriately scaled dose and exposure).
A predicted safety margin close to 1 or even less in a clinical dose escalation study does not necessarily force a stop to the trial but requires a slower dose progression.
References and Further Reading
- EMEA CHMP SWP Reflection Paper on PPARs (peroxisome proliferator activated receptors); Doc Ref: EMEA/341972/2006 (2006)Google Scholar
- FDA Guidance for Industry S6 Preclinical Safety Evaluation of Biotechnology- Derived Pharmaceuticals (1997)Google Scholar
- FDA Guidance for Industry Safety Testing of Drug Metabolites (2008)Google Scholar
- ICH Focus on Microsampling (2017) Note for guidance on toxicokinetics: the assessment of systemic exposure – focus on microsampling (CHMP/ICH/320985/2016); adopted by CPMP December 2017Google Scholar
- ICH Guidance on Toxicokinetics (1994) ICH topic S3 A; toxicokinetics: a guidance for assessing systemic exposure in toxicology studies (CPMP/ICH/384/95); approval by CPMP November 1994Google Scholar
- ICH Topic S1C(R2) (2008) Dose selection for carcinogenicity of pharmaceuticals (EMEA/CPMP/ICH/383/1995); approval by CPMP April 2008Google Scholar
- Ramirez T, Beken S, Chlebus M, Ellis G, Griesinger C, de Jonghe S, Manou I, Mehling A, Reisinger K, Rossi LH, van Benthem J, van der Laan JW, Weissenhorn R, Sauer UG (2015) Knowledge sharing to facilitate regulatory decision-making in regard to alternatives to animal testing: report of an EPAA workshop. Regul Toxicol Pharmacol 73:210–226CrossRefGoogle Scholar