The initial method of analysis developed during the discovery phase of the molecule, with some modifications, may sometimes serve as a method of choice to begin with as the NCE enters the preclinical development stage. Since the complexity of development generally tends to increase as the lead candidate enters the toxicological and clinical phase of testing, it naturally calls for improved methods of analytical quantization, improvement in selectivity and specificity, and employment of sound and rugged validation tools to enable estimation of PK parameters that would also aid in the decision-making of the drug molecule's advancement in the clinic in addition to safety and tolerability data gathered at all phases of development. Additionally, it becomes necessary to quantify active metabolite(s) in both animals and humans.
ADME/PK screening is usually taken to mean in vitro systems for studying absorption and metabolism. However, in vivo studies still provide the definitive assessment of overall drug disposition, and progress has been made in overcoming some of the constraints associated with this approach. Previously, drug metabolism studies were performed at a late stage of drug development process and very often not until the phase of clinical studies. Therefore, inadequate metabolism and PK parameters were the major reason of failure for NCEs. Nowadays, introduction of in vitro approaches into drug metabolism enables the characterization of the metabolic properties of drug candidates at an earlier stage in the drug development process, at early preclinical studies performed during the drug discovery phase. Recently, the major reasons for high attrition rates have instead been identified to be lack of efficacy and safety, together accounting for approximately 60% of the failures. Cassette dosing is now an established method within the pharmaceutical industry as it provides a relatively quick way of ranking compounds according to their PK properties and requires the use of fewer animals.[53,54]
PBPK models are standard tools in pharmacology and are frequently used to support the prediction of clinical pharmacokinetics, drug-drug interactions, dose scaling for children, and formulation/adsorption development . Regulatory agencies such as the European Medicines Agency (EMA) and the Food and Drug Administration (FDA) encourage the use of PBPK models, and these models are often part of the submission of new medical entities [15, 16]. Also, World Health Organization (WHO) and the United States Environmental Protection Agency (US-EPA) underlines the necessity to establish common principles for their application in chemical hazard and risk assessment . Despite that the use of PBPK/TK models is encouraged in risk assessment by several recent reports and articles , their utilization is limited. This limited use is surprising given the potential for translating the information from standard laboratory species such as rats, rabbits, and mice to physiologically related small (wild) animals.
As a final step in the PBPK/TK model development, a local sensitivity analysis was performed. A sensitivity analysis is described as a crucial step in the best practices for modelling in risk assessment  and is recently used increasingly in numerous mechanism-based modelling efforts in PK/PD [58, 59]. A sensitivity analysis not only evaluates the robustness of the PBPK/TK models but in a PBPK/TK setting where several literature values are available for certain physiologically-based parameters, it reveals whether the choice of alternative values would significantly impact the observed behaviour. The results of the sensitivity analysis output parameters are shown in Fig 5 and S11 Fig for AUC, and Fig 5 and S12 Fig for Cmax. As determined by the change in AUC, the most sensitive parameter was the liver volume followed by the kidney and venous blood volumes. This result is expected since most of the compounds tested were partially or totally cleared through metabolism in the liver. Therefore, a relative change in the liver mass or volume would highly affect the amount of drug cleared and as such the AUC. Similarly, for the compounds cleared renally, either through GFR (inulin) or tubular secretion and GFR (acyclovir), the parameters highly affecting the AUC are kidney volume and filtration capacity (GFRspecific). The parameters such as gastric emptying time, or GIT-length and the effective surface area are sensitive only in scenarios involving oral administration (i.e., paracetamol PO, acyclovir PO). In contrast, gastric emptying time appears to be the most sensitive parameter regarding Cmax variation on compounds involving oral administration such as paracetamol and acyclovir. Furthermore, the muscle volume is the largest organ of the rabbit and the most important regarding distribution of drugs in the body hence it appears to be one of the most sensitive parameters. Overall, our analysis reveals sensitivities that are consistent with the biology and clearance pathways of each compound.
Preclinical development encompasses the activities that link drug discovery in the laboratory to initiation of human clinical trials. Preclinical studies can be designed to identify a lead candidate from several hits; develop the best procedure for new drug scale-up; select the best formulation; determine the route, frequency, and duration of exposure; and ultimately support the intended clinical trial design. The details of each preclinical development package can vary, but all have some common features. Rodent and nonrodent mammalian models are used to delineate the pharmacokinetic profile and general safety, as well as to identify toxicity patterns. One or more species may be used to determine the drug's mean residence time in the body, which depends on inherent absorption, distribution, metabolism, and excretion properties. For drugs intended to treat Alzheimer's disease or other brain-targeted diseases, the ability of a drug to cross the blood brain barrier may be a key issue. Toxicology and safety studies identify potential target organs for adverse effects and define the Therapeutic Index to set the initial starting doses in clinical trials. Pivotal preclinical safety studies generally require regulatory oversight as defined by US Food and Drug Administration (FDA) Good Laboratory Practices and international guidelines, including the International Conference on Harmonisation. Concurrent preclinical development activities include developing the Clinical Plan and preparing the new drug product, including the associated documentation to meet stringent FDA Good Manufacturing Practices regulatory guidelines. A wide range of commercial and government contract options are available for investigators seeking to advance their candidate(s). Government programs such as the Small Business Innovative Research and Small Business Technology Transfer grants and the National Institutes of Health Rapid Access to Interventional Development Pilot Program provide funding and services to assist applicants in preparing the preclinical programs and documentation for their drugs. Increasingly, private foundations are also funding preclinical work. Close interaction with the FDA, including a meeting to prepare for submission of an Investigational New Drug application, is critical to ensure that the preclinical development package properly supports the planned phase I clinical trial.
The drug development process is typically divided into three major steps: discovery, preclinical development, and clinical trial. The transition from discovery to preclinical development is a continuum, and results of preliminary pharmacology and toxicology testing often contribute to lead drug candidate selection. The boundary between preclinical development and clinical trial is sharply defined by the filing of an Investigational New Drug (IND; Table 1 lists preclinical development acronyms) application, which is required prior to initiation of the clinical trial. The activities supporting an IND application are the subject of this overview. The adage 'begin with the end in mind' is particularly appropriate for preclinical development, as the resulting IND must support the planned clinical trial design. For example, a clinical trial involving daily chronic administration requires repeat-dose toxicity studies in preclinical animal models.
Once a lead candidate is identified, a typical preclinical development program consists of six major efforts: manufacture of drug substance (DS)/active pharmaceutical ingredient (API); preformulation and formulation (dosage design); analytical and bioanalytical methods development and validation; metabolism and pharmacokinetics; toxicology, both safety and genetic toxicology and possibly safety pharmacology; and good manufacturing practice (GMP) manufacture and documentation of drug product for use in clinical trials (Figure 1) The IND application summarizes the results of the above activities for submission to the US Food and Drug Administration (FDA). Table 2 outlines the general organization of an IND application and lists many of the relevant Code of Federal Regulations (CFR) sections for each key component. These activities are seldom discrete and sequential; rather, they are interrelated and often concurrent, with results from each activity informing the other steps as the drug candidate progresses through characterization and optimization (Figure 2).
Preclinical drug development stages. Following identification of a drug target and candidate compounds, several early activities, such as pharmacology, in vivo efficacy, and experimental toxicology, can contribute to the selection of a lead candidate for preclinical development. These preclinical activities provide the basis for an Investigational New Drug (IND) application to the FDA for permission to initiate clinical testing in humans. ADME, absorption, distribution, metabol