Preclinical Studies Required for Obtaining FDA IND Approval
Authors from Mesa Science Associates: Kaitlyn Wylie, Kenneth Dretchen
Authors from MedSurgPI: Gerald Klein, Roger Morgan, Lee Schacter, Freddy Byrd, Devsmita Das
Introduction
The FDA issuance of an Investigational New Drug (IND) application is dependent upon the successful completion of multiple preclinical studies including both pharmacology and toxicology evaluations. These studies consist of pharmacodynamic, pharmacokinetic, and toxicology studies that should be completed prior to submission and other additional required studies after initial FDA discussions.
Pharmacology
· Pharmacodynamics
Pharmacodynamic studies are investigations that describe and confirm the basic mechanism of action of a new drug candidate. These involve either or both in vivo or in vitro experiments. It is essential that the physiological response generated by the chemical entity can be measured with high precision and remains stable during the course of the experiment in in vivo studies. This includes heart rate, blood pressure, cardiac contractility, respiration rate, tidal volume, gastrointestinal motility, skeletal muscle contracture and urinary outflow. A dose/response or a dose/escalation study which measures the quantity of the drug received versus the initiated response should be undertaken. For example, an agonist will produce a standard S-shaped curve when comparing increasing doses to increasing responses including a threshold point, a rising phase, and a celling.
The other graphical evaluation that is useful in determining the duration of action is referred to as a time-action curve that compares the response obtained over time following the administration of a fixed dose of a drug. Increasing doses of the drug should generate parallel curves until the maximum response is obtained. Additional information that can be obtained are an estimate of the duration of the drug and inferences regarding any overt adverse events.
If at all possible, in vitro confirmation of the site and mechanism of action would be invaluable (Moctezuma-Ramirez et. al 2023).
· Pharmacokinetics (PK)
These experiments characterize the absorption, distribution, metabolism, and excretion (ADME) of the chemical entity. The administration of the drug in these experiments should mimic the intended route for human use. Since most drugs are either weak acids or weak bases, the PK is critically important to determine the degree of ionization which contributes to the drug’s ability to cross lipid membranes. Orally administrated drugs that are acidic would be preferentially absorbed in the stomach whereas those drugs that are basic would be preferentially absorbed in the small intestine. When considering the parenteral administration of drugs, it is imperative to use the correct length/caliber of needle to ensure the drug is in the intended compartment. Regarding a drug’s distribution, calculating the Volume of Distribution (VD) will indicate if the drug is confined to the plasma, the extracellular space, or the intracellular space (total body of water). Most drugs are metabolized in the liver by first order elimination kinetics. An estimate of metabolism can be obtained by calculating the Elimination Constant (Ke).
The gold standard of pharmacokinetic testing is the administration of a fixed dose of a drug and the measurements of the resultant plasma levels. The three critical parameters are the Maximum Concentration (Cmax), Time to Maximum Concentration (Tmax) and Area Under the Curve (AUC), which allows assessment of the amount of drug delivered to the bloodstream over time. The graphical representation of the plasma levels over time allows for the calculation of the half-life (T1/2). If a drug is administered by any route other than intravenously, the Bioavailability (F) should be calculated. Bioavailability assesses how much of an orally administered drug is absorbed into the blood. This is defined as the AUC obtained by the intended route of administration (typically oral), divided by AUC of the intravenously administered drug. This is also essential for topical drugs, to determine if significant amounts of the drug reach the systemic circulation and depends on whether the drug was intended for local use, or systemic effects.
Toxicology
These experiments are designed to evaluate higher doses of the drug in order to determine the incidence of adverse side effects. One of the most important of these tests is the No Observed Adverse Effects Level (NOAEL) which represents the highest dose prior to the appearance of an adverse effect. The safety of a drug can also be obtained by calculating the Therapeutic Index (TI). This is the ratio of the dose that is lethal to fifty percent of animals tested (LD50), divided by the dose that is effective in fifty percent of the animals (ED50). It should be noted that standard toxicology testing involves both histologic and pathologic examination of all organ systems.
It is recommended that in preparation for the initial meeting with the FDA a dose range finding study in two animal species should be conducted, one in a rodent model and the other in a larger animal species with the drug administered in the intended route of administration. Evaluations which will include clinical pathology, ocular examinations, EKG, toxicokinetic assessments, necropsy and histopathology. A toxicokinetic evaluation (similar to a pharmacokinetic study) should be conducted. Clinical pathology should include hematology, coagulation, clinical chemistry assessment and urinalysis in addition to histopathology. The duration of the study will be dependent on whether the drug is intended for a single or multiple dose administration. Another required test is the AMES study which employs a bacterial reverse mutation assay, to assess the potential of a chemical to cause DNA mutations. Another recommended test is the in vitro micronucleus assay in TK6 cells which is used to evaluate the genotoxicity of the chemical agent. The hERG study is also used to monitor dysfunction of potassium channels which could lead to prolongation of the QT segment on EKG, which can result in sudden death from arrhythmia.
Additional studies will be required following the initial meeting with FDA. These consist of toxicity screening in two species of animals, , conducted under Good Laboratory Practice (GLP) conditions using Good Manufacturing Practice (GMP) grade material of the drug substance. Conducting studies by GLP/GMP international standards requires a higher level of quality control than other studies. These studies will be of a longer duration than those previously described. In addition, studies will be required to assess the safety of the drug on the cardiovascular, respiratory and central nervous system.
Ensuring a Successful Study Outcome
· Protocol
One of the most important aspects is the generation of a comprehensive protocol prior to beginning the study. It is critical that the hypothesis is clear and that the experiments are sufficiently designed to either prove or disprove the hypothesis (Huang, W., et. 2020). No detail should be overlooked, including every component of a drug solution. All details such as the ambient temperature, humidity and lighting need to be recorded.
· Animal Model
The animal models selected for the study should be based upon the known comparability of the physiologic responses to the effects observed in humans. For example, studies evaluating the cardiovascular system are often done using pigs since the cardiac anatomy and physiology is comparable to humans (Moctezuma-Ramirez et. al 2023). Although rodents are predominantly used for initial screening of new drugs, there are large differences in the physiology of many organ systems between the two species.
· Animal Housing and Welfare
A high degree of non-reproducibility of experimental results between two different laboratories can often be attributed to differences in animal housing and the handling of the animals during acclimation prior to conducting the experiments. The quality of the animal care and use program and in life portion of the study contributes in large measure to the overall quality of an animal experiment (Everitt 2015). From the time of the animal’s arrival at the facility, actions must be put in place to properly prepare the animal for the study during this time, that animal’s baseline vitals and general condition can be assessed (Moctezuma-Ramirez et. al 2023).
· Statistical analysis
A critical aspect is the adequacy of the sample size (Lazic et. al 2018). The use of an inadequate number of animals might fail to detect a significant difference due to factors such as minimizing animal to animal variability (Bonapersona et al., 2019; Carneiro et al., 2018; Howells et al., 2014). It is often advisable to calculate power and sample size which is based upon known variability and probability. One way to increase external validity is to conduct identical studies at multiple sites, emulating an approach already applied in clinical trials (Dechartres et al., 2011; Friedman et al., 2015). Randomization and blinding should also be used in the allocation and administration, respectively, of the new treatment in order to reduce bias in the study and increase the validity of the results (Aban and George 2015). Most of these studies involve administering both drug and placebo to animals, so one may determine what the background incidence of findings are in untreated animals.
· GMP Drug Substance and GLP Requirements
It is acceptable to use non-GMP grade material under non-GLP conditions for the initial experiments that confirm the site and mechanism of action as well as the preliminary dose-response and ADME (absorption, distribution, metabolism, and excretion) experiments. However, all of the pharmacokinetic experiments that calculate the Cmax, Tmax and AUC should be done under GLP conditions. The requirements of a GLP study are much more rigorous than those of non-validated exploratory studies because in GLP studies, the researchers are held accountable for the reliability, reproducibility, and validity of their study techniques, quality control features, and results (Moctezuma-Ramirez et. al 2023). Finally, all of the experiments required for FDA approval of an IND required the use of a GMP drug substance with experiments conducted under GLP conditions. Companies should understand the importance and requirements for GLP/GMP studies. These studies are more expensive but can result in delay of acceptance of an IND for human study initiation of the package of preclinical studies if it does not meet regulatory requirements.
Note: This is an abridged version of the white paper. The full-length version is available on the Mesa Science Associates, Inc. website: https://www.mesascience.com/
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