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The Preclinical Minefield: A Guide to the Possible Pitfalls

 

From the moment a new compound is championed for drug development, the possibility of failure is continually present.  This article attempts to point out the likelihood of this occurrence and possible remedies.

Investigational New Drug Submission (IND)

The typical preclinical pathway to an IND submission involves the following studies covering Safety Pharmacology, Genotoxicity and Toxicology:

Safety Pharmacology

                Rat Behavioral Study

                Rat Respiratory Study

                Non-rodent Cardiovascular Study (non-rodent encompasses canines or non-human primates)

                In vitro Human ether-a-gogo related gene Assay (hERG)

Genotoxicity

                In vitro Reverse Bacterial Mutation Assay (Ames Test)

                In vitro Chromosome Aberration Assay

                In vivo   Rodent Micronucleus Assay

Toxicology

                Rat Range Finder via intended therapeutic route (p.o., i.v. s.c. etc.)

                Non-rodent Range Finder via intended therapeutic route

                14/28 Day Rat Toxicity Study

                14/28 Day Non – rodent Toxicity Study

 

Thus, there are at least eleven (11) opportunities for an adverse result to hinder the progress towards an IND submission.

As the candidate drug would have undergone extensive pharmacological evaluation prior to nomination, the likelihood of significant issues arising from the rat behavioral and respiratory studies are minimal unless the compound exhibits unexpected profound neurological problems.  Occasionally these types of issues do arise but are commonly evaluated in relation to the pharmacological profile of the compound and rarely affect further development of the compound.

The latter two studies in the Safety Pharmacology package however, can pose more serious problems.  The Non-rodent cardiovascular study involves using telemetered animals and can pick up subtle changes in cardiovascular parameters that could raise issues for the compound while the hERG assay is a sensitive predictor of QT prolongation and possible Torsades de Pointes that can lead to sudden cardiac death.  Thus, any adverse finding in these two studies need careful evaluation before proceeding further with the development of the compound.

However, if these hurdles are successfully cleared one moves on to the Genotoxicity package.  A positive finding in the Bacterial Reverse Mutation Assay is probably the biggest compound killer in Pharmaceutical development as it provides an easy GO/NOGO decision point in the life of the drug.  In the case of big Pharma where the drug is one of several, the choice is easy – a positive finding kills the compound and proceed with a compound that is negative in the assay.  For smaller companies for whom the drug is their “only child”, the decision is far harder and, given that the test can throw out false positives, alternative defining tests such as the Comet Assay can be utilized to support the false positive argument.

The Chromosome Aberration Assay is known to show false positives either alone or with metabolic activation and is not as common a showstopper.  However, in all of these genotoxicity cases the positives can act like an albatross around the development of the compound as, in the eyes of the regulators, they never go away.  In this case it is always good to try and add to the package with additional studies such as the Comet Assay which, if negative, will provide further evidence that the original assay had provided a false positive.

Overall, in genotoxicity, the take-home message is that three negatives ensures progression, two of three really needs to become three of four by the addition of a further assay to ensure progression.

Having gone so far, the next move is to define the toxicity of the compound thereby enabling the therapeutic index to be established so that dosing in the clinic (probably in Human Volunteers) can begin.

Range finders in two species (rat and non-rodent) are performed to set not only the dose levels for the definitive toxicology studies, but also to establish the limits (Maximum Tolerated Dose – MTD) at which the animals can be dosed.  Occasionally, surprising adverse findings are seen at unexpectedly low levels that would disqualify the compound because toxicity was seen at levels that were considered to be the efficacious clinical dose.  Again, given the extensive nature of pharmacological studies performed prior to nomination, this is unlikely in a study of this duration (typically 7 days).

The IND-enabling toxicity studies can be either of 14 or 28 days duration depending on the intended dosing envisaged in the clinic as regulators view the toxicology in comparison to clinic dosing on a like-for-like basis i.e. 14 day toxicology study enables 14 days in the clinic, etc.  The doses are set based on the prior range-finders and consist of undosed controls, low, intermediate and high dose levels.  The high dose level is set such that some evidence of toxicity is apparent; the low dose is set at around the anticipated therapeutic dose and the intermediate dose is normally the geometric mean between the two doses.

Ideally, the intermediate dose level will be considered the No Observed Effect Level (NOEL) and should provide a suitable therapeutic margin to enable safe dosing in the clinic.  Setting the NOEL lower than that however, would require much thought on the part of the clinicians as to whether lowering the intended human dose would still provide efficacy, or whether the development should be abandoned.

New Drug Application (NDA)

Assuming that all went well with both the IND application and the early clinical trials, the compound now has to embark on the long journey towards final submission driven by both the needs of the clinicians and the regulators.  Besides the longer term studies required in the toxicology area, reproductive studies are also needed along with carcinogenicity studies in two species and are outlined below:

Toxicology

                3 Month Rat Toxicology

                3 Month Non-rodent Toxicology

                6 Month Rat Toxicology

                9 Month Non-Rodent Toxicology

Reproductive

                Rat Fertility (Male and Female)

                Rat Embryotoxicity

                Rabbit Embryotoxicity

                Rat Peri- and Post-natal Toxicity

Carcinogenicity

                2 Year Rat Study

                2 Year Mouse Study

                OR

                6 Month Transgenic Mouse Study

The longer-term studies are designed to detect issues related to continuous exposure.  Thus, what might appear to be a slightly elevated liver enzyme in the short term studies could manifest itself as liver disease over a prolonged period.  However, as biomarkers are becoming more and more sensitive, the “unexpected” findings in these longer studies are becoming less prevalent.  Once completed, these become the definitive studies and clinical studies can now be dosed as long as necessary to establish primary endpoints, efficacy and safety.

Any compound that could be dosed to women of child-bearing potential (WOCBP) has to be evaluated for effects on reproductive parameters such as fertility and teratogenicity.  Adverse effects here could have implications for the product if it is intended to be dosed to WOCBP.  However, if the product is intended for an older population then a so-called “Black Box Warning” may be applied such as seen in the major Pfizer product, Lipitor which carries the following statement:

“Do not take LIPITOR if you are pregnant or think you may be pregnant, or are planning to become pregnant. LIPITOR may harm your unborn baby. If you get pregnant, stop taking LIPITOR and call your doctor right away.”

The final part of the NDA jigsaw puzzle relates to the potential for tumorigenicity.  For many years regulators insisted on whole lifespan studies in both rat and mouse.  These studies involved large numbers of animals exposed to the drug for approximately 2 years with histopathological review adding at least another six months to this timespan.  More recently, the use of a transgenic mouse model dosed for six months has been accepted as a replacement for the 2-year mouse study allowing a much faster decision point regarding carcinogenicity.  Adverse effects at this late stage in development can prove catastrophic, but again the margin of safety needs to be considered and a risk/benefit analysis performed before terminating the program.

The take-home message here is encapsulated in a quote by Paracelsus (1493-1541):

“Poison is in everything, and no thing is without poison. The dosage makes it either a poison or a remedy.”

Thus, it is no surprise that adverse effects may be seen in this journey towards an NDA.  However, they need not be showstoppers.  By evaluating mechanisms of action, risk/benefit analysis, therapeutic margins, clinical requirements and population targeting, the path to an NDA may still be achieved.