Nonclinical Development and Testing

Having moved beyond target and product discovery, the next steps in the development process involve obtaining a more complete picture of the activities and properties of the lead compound (Fig. 4.24). Most commonly, this involves a more thorough investigation of the compound in in vitro assays as well as more extensive evaluations in animals. Clearly, it is the goal of these "preclinical" studies to provide the information necessary to initiate clinical trials. As such, they are heavily driven by the clinical indication and seek to define what happens when the drug enters the body:

• Where does it go in general and any special tissue sites?

• What happens to it, especially the elimination steps in the liver, kidney, or elsewhere?

• Does it interact with compounds in the body or ones likely to be used in patients with this disease?

• What does it do in target sites and all other tissues?

r Activity and efficacy studies: c. In vitro testing:

> Affinity, potency, minimum active concentration, physical characteristics, stability, mechanism of action c. In vivo testing:

> Potency, dose-response, drug effects

> Models are chosen to best reflect the therapeutic indication r Pharmacokinetics:

c Effect the body has on the drug:

5> Clearance, distribution, degradation r Pharmacodynamics:

c Effect the drug has on the body:

> Impact on disease or disease markers, PK requirements

Non-clinical development is also referred to as "preclinical" development, since it takes place prior to initiating clinical trials.

Fig. 4.24. Non-clinical Development & Testing

Answers to these questions help guide toxicity studies and how the drug will be initially used in the clinic.

Clearly, the basic goal for the in vitro and animal tests is to help predict the actions of a lead compound in humans (Fig. 4.25). It should be emphasized that good activity in the test tube or even in animals is no guarantee of success in humans. This result is due to various aspects of in vitro and in vivo assays, neither of which may accurately reflect the disease process in humans. This slide presents four reasons for in vitro tests and three representative reasons for animal tests for the lack of their predictive ability in humans.

Although pharmacokinetics and pharmacodynamics are also monitored extensively in human clinical trials, they have a special significance for preclinical development. This follows from the fact that it is the early work in animals that is used to develop the toxicology studies, which in turn are used to select the dose and dose regimens that will be initially used in humans. The animal work also will help design the type of pharmacokinetic trials needed to be done in humans, especially any special studies related to unexpected movement of a product in tissues, or special tissue effects, or special route of administration issues. Animal studies are usually quite predictive of human trials for pharmacokinetics, but surprises can occur as well. For example, protein binding can differ between species impacting pharmacokinetic parameters.

And what effects does the drug have on the body, the phar-macodynamic effects, and how do they correlate with pharma-cokinetics? Again, animal research is intended to help predict human activities, and four key questions are listed on Figures 4.26 and 4.27. Ideally, there is a positive biological effect on the disease (though toxic effects must also be monitored), and it is r In vitro tests may not accuratelyr eflect disease process: C NO access or penetration issues C NO clearance C NO metabolism c NO toxicity r Animal models also may not accurately reflect disease process:

C Animal physiology and metabolism differ from that of humans

C Most models are acute, where as many human diseases are chronic o Clearance, distribution and metabolism differs between species

Generally, data from multiple in vitro and in vivo models are desirable, but are no guarantee of success in humans

Fig. 4.25. Activity & Efficacy Studies: Some Issues r Seeks to understand and predict product levels as function of dose and route of administration:

o Intravenous administration results in high initial concentrations in blood and then decreasing concentrations:

> Appropriate for products requiring high peak concentrations, rapid onset and/or short exposure o Subcutaneous administration results in lower & delayed peak concentrations in blood:

> Product concentrations often remain elevated for longer periods of time

> Appropriate for products where extended coverage is desirable or high plasma concentrations may cause safety concerns o Oral administration provides low peak concentrations and sustained product levels: S> Appropriate for drugs with good oral availability

> Inappropriate for most proteins, due to reduced stability at low pH and poor oral absorption r Important goal: Match route of administration to indication, drug, and patient population

Fig. 4.26. Pharmacokinetics (PK), Preclinical Work the preclinical work that must identify the desired drug concentrations and dosing frequencies necessary to produce that effect. Similarly, it is helpful if, in addition to having readily measurable effects on the disease, other markers of therapeutic efficacy can also be identified. Such "surrogate" markers of clinical efficacy can be extremely important in monitoring the effects of a new drug in humans, sometimes even early indicators of beneficial or untoward effects before full actions of the drug and the resulting change in disease pathogenesis occurs (remember the discussion of PSA with Fig. 7 on pg. 91).

As an example, let's consider the pharmacokinetics and pharmacodynamics of a new antibody therapeutic, Raptiva®, which was approved in 2004 for the treatment of moderate to severe psoriasis (Fig. 4.28). Raptiva® (here identified as hu1124) is a humanized monoclonal IgG1 antibody that binds to the CD-11a component of human LFA-1 (lymphocyte function-associated antigen 1), a surface structure on lymphocytes that participates in T-cell trafficking and activation. As a consequence of this binding, CD-11a is downregulated and its function inhibited.

Shown in the graph is the effect of a single 8 mg/kg intravenous injection in chimpanzees [32]. Immediately after the IV injection (time 0), the concentration of Raptiva in the blood increased to over 100 |g/mL and then decreased over the next 2 months. At the same time, the expression of lymphocyte CD11a immediately decreased and stayed suppressed for the same 2-month period. Note that when the level of circulating Raptiva® fell below 3 |g/mL, the clearance of Raptiva® was accelerated and CD11a levels began to return to normal. Based on these data, mathematical models were developed (solid and dashed lines) that described the dose-dependent effects of hu1124 on antibody clearance and CD11a expression in chimps, and these models were then used to predict probable hu1124/CD11a profiles in humans. Such data also helps select initial dosing schemes for human trials.

Of all the parameters studied prior to initiating human clinical trials, product safety remains one of the most important (Fig. 4.29). This focus is because the primary decision made by the company, the FDA, and the clinicians regarding the initiation of human trials is whether the product poses a safety risk. As a consequence, toxicity studies should be incorporated not only into the final product evaluation but also into the initial product evaluation and selection process. For compounds r Seeks to clarify and predict relationship between product concentration and biological effect:

c What blood concentration is required to achieve benefit? o How long is the effect be maintained?

o How frequently must I dose to maintain this concentration/effect? o How are peak and trough levels effected by route of administration?

r Helps identify surrogate markers of disease or therapeutic efficacy

Fig. 4.27. Pharmacodynamics (PD), Preclinical Work

Fig. 4.28. PK & PD Example (Reprinted with permission from Springer. Heidelberg, Germany. From Graph - Population pharmacokinetics and pharmacodynamics of the anti-CD11a antibody hu1124 in human subjects with psoriasis. Bauer RJ et al. J. Pharmacokin Biopharm 1999;27(4):397)

Nonclinical Development

Fig. 4.28. PK & PD Example (Reprinted with permission from Springer. Heidelberg, Germany. From Graph - Population pharmacokinetics and pharmacodynamics of the anti-CD11a antibody hu1124 in human subjects with psoriasis. Bauer RJ et al. J. Pharmacokin Biopharm 1999;27(4):397)

k Important in vivo tool in selection of lead candidates: o In vitro tools are being developed, but are not yet reliable k Can be monitored initially in (or prior to) efficacy studies

-« Single dose and multi-dose, depending upon indication

-« Dosing at multiples of expected human exposure and determination of maximum tolerated dose (MTD)

- Helps to define therapeutic index (range between effective and toxic doses of the drug)

Fig. 4.29. Toxicity Assessments, Preclinical Work such as the small-molecule drugs, initial screening in animals can help reduce the number of candidates that require further characterization. Animal toxicology work often can be predictive of many, but not all, the major side effects to be seen in humans. To help ensure this predictive capacity, two species, one non-rodent, are used often in the animal studies. Drug doses are given singly at multiples of the human dose, acutely (daily over a few days), subacutely (daily for a few weeks), and possibly chronically (daily for months), if the drug will be used in that fashion in humans. The formulation of the product in the animal study needs to be as similar as possible to the human forms, because often drug delivery and absorption depends on the formulation. For the biologics, where it is less typical to screen large numbers of potential product candidates, initial toxicity studies can help guide the doses used in animal efficacy studies and can identify areas of potential concern. Animal studies for toxicology pose complications for most protein biologics because they are foreign to the animal and will cause an immune reaction. In all cases, compounds with high maximum tolerated doses (MTDs) and wide therapeutic indexes (TI) are more easily moved along the development path. However, in some indications (for example, certain life-threatening diseases such as cancer) a less favorable safety profile may still be acceptable.

Toxicology work comprises the majority of studies and costs in the preclinical phase of research, as shown in figure 4.30 [33]. Most of these 10 different tox studies (Fig. 4.30) are prescribed in regulatory guidelines for IND submissions; note the varied lengths of treatment with study product and the species. Three special toxicology studies are required as well for mutagenicity, carcinogenicity (a very expensive research requirement and possibly time consuming before human trails are permitted), and reproductive performance on mother and fetus. Pharmacogenomic studies are a new, either toxicology or efficacy, parameter to document if genetics plays a significant role in adverse events or patient responsiveness to the product. Guidelines are currently voluntary until the value and role of pharmacogenomics is established for diseases and therapy. This long list of studies is not a surprise given the need to find significant toxicity as early as possible and kill poorly performing molecules, thereby avoiding the expensive late termination of a product in clinical trial or after marketing, as well as the mission of the regulatory bodies to protect the public. The costs are $2.5 million to $6 million for this work, as noted in


Cost Range (Euros)

Acute Toxicity (rodents)

3,900 -4,600

Subacute toxicity (4 weeks in rats-dogs)


Subacute toxicity (4 weeks in monkeys)


Subacute toxicity (13 weeks in rats-dogs)


Subacute toxicity (13 weeks in monkeys)


Chronic toxicity (26 weeks in rats & 39 weeks in dogs)


Chronic toxicity (39 weeks in monkeys)

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