# Presentation: 880077

## Non-clinical Studies: Does One Size Fit All?  

- Martin D. Green, Ph.D.
- Associate Director P/T ODE 1 & 6

## Please Note the Following

- The views expressed in this presentation are the presenter’s and do not reflect the official policy of the FDA
- No official support or endorsement by the FDA is intended or should be inferred

## The Fundamental Problem

- Many of the familiar approaches and techniques for non-biological products are not directly applicable to large biological molecules
- Pharmacology and toxicity are often limited by receptors and physiological systems, e.g., function and specificity tightly related to the structure and form of protein molecules
- Concepts dictating testing paradigms which lead to interpretations and incorporate assumptions which are not necessarily valid for biological molecules

**Notes:**
Large molecular weight designated into domains with particular functions pk and pharmacology/toxicity.  Unlike drugs which do have pharmacophores by greater degree of unpredictability in terms of thermodynamics
Mutagenicity testing is important as it related to carcinogenicity testing because modification of DNA is the underlying event.

## The Fundamental Problem - Continued

- Additionally, certain aspects make standard testing for toxicity for biologicals unnecessary
- Not genotoxic by directly interfering with DNA – in vitro testing such as the Ames test not required
- Assessments for carcinogenicity using traditional methods not relevant
- Metabolism and the formation of active metabolites is not a concern – mass balance studies uninformative

## So Why Test At All?

- Biologics not given in a ‘natural’ way
- Not given in a manner similar to an endogenous substance as in an autocrine or paracrine exposure
- Given with systemic exposure like a hormone
- Reach a variety of targets and relative high levels
- Not in an appropriate physiological context (e.g., signal a proinflammatory response or growth signal)
- Regimen/exposure not physiologically appropriate
- Many biological products today are not natural – fusion protein, mixtures of isoforms, modified glycoforms, PEGylation

## The Fundamental Solution

- Specification based testing is more appropriate for synthetic, relatively small molecular weight drugs
- Performance based testing is more appropriate for cell based, relatively large molecular weight drugs
- Adaptive to constraints imposed by the pharmacological and toxicological properties

**Notes:**
Relevant animal model vs. 2 species, one being not a rodent

##  Basic Tools

- Main driver - Adequate models for the identification and extrapolation to the clinic
- Pharmacology
- Pharmacokinetics
- Toxicology
- General toxicity assessments
- Special toxicity assessments

**Notes:**
Main task issues – to identify target organs and toxicities; find a safe starting dose, dose escalation scheme and dosing regimen

##  Basic Tools

- Operating environment - Adequacy of supportive tools for scientific understanding and validity of information
- Assays
- Immunogenicity
- Pharmacokinetics
- GLP

**Notes:**
Environmental issues

##  Basic Tools

- Outcome measures - Interpretation of test results
- Toxicity, identification of organ systems at risk, e.g., testing for auto-antibodies, immune suppression, infection, secondary malignancy
- Pharmacokinetics, measurement of exposure, washout period
- Extrapolation of starting dose and dosing regimen

**Notes:**
Knowing the right questions; infection leading to TB or increase propensity to have the flu
Animals predictive of TPO MDGF
IL12

## Pharmacology

- Exaggerated pharmacology often an important aspect of safety
- Species restrictions in pharmacological actions due to receptor is the usual basis for selecting an animal model for toxicity testing
- Models of disease sometimes present unique situations to understand the expression of toxicity

## Exaggerated Pharmacology Leading to Toxicity

**Notes:**
CAMPATH-1H from Cambridge Pathology development started in 80’s(Alemtuzumab)

Chosen as an example that a high degree of specificity does not ensure safety particularly with a dosing regimen that does not consider pk and as an illustration of some factors influencing the pk of therapeutic proteins

Single high specificity does not preclude multiple plasma concentration relationships

Approved FOR THE TREATMENT OF PATIENTS WITH CLL WHO HAVE BEEN TREATED WITH ALKYLATING AGENTS AND WHO HAVE FAILED FLUDARABINE. CLL is a disease of malignant B cells rather than other leucocytes.
Also used for T-cell prolymphocytic leukaemia (T-PLL) and prevention of graft versus host disease (GVHD)

Clinical toxicity is characterized by first dose reaction due to cytokine release consisting of fever, rigors, rash and at time dyspnea and hypotension.  The most significant toxicity is profound, prolonged lymphopenia and subsequent increased risk of opportunistic infection.

Initial toxicity is controlled by initiating dosing a lower level of 3 mg daily until tolearated and increasing the dose to 10 mg daily.  When this is tolearated, a 30 mg dose 3 times weekly is instituted for 12 weeks.  

Operates through at least 3 different mechanisms

## CD52 Expression on Leukocytes

**Leukocytes**
**B- lymphocytes**
**T- lymphocytes**
**Monocytes**
**Macrophages**
**Thymocytes**
**Granulocytes (<5%)**

**Notes:**
For the approved indication of CLL only the CD52 receptors on B lymphocytes represent therapeutic target with the expression of CD52 at the other sites representing potential sites of toxicity

The approved dosing for CAMPATH includes a maintainence dosing phase of 30 mg/kg 3 times a week for a maximum of 12 total weeks inclusive of the dose escalation scheme.  (3 mg to 30 mg daily with 3 to 7 days)

5% detrermined in an environment of competition between the binding of CAMPATH and other WBC’s
So what’s happening to the bystander cells represented by granulocytes. 

## Expression on Other Non-Target Cell Types

**Male reproductive tract**
**Skin**
**Tested on a range of tissues, absent on**** **
**Erythrocytes**
**Platelets**
**Hematopoietic stem cells**

**Notes:**
Also not expressed on bone marrow prgenitor cells.

## Clinical Toxicity

**CAMPATH Related Mortality: 13 - 15% **
**Discontinuations for Treatment Related AEs: 21 - 25%**
**Drug related SAEs: 73- 88% of all SAEs**
**Opportunistic Infections: 28 - 42% study; 50% serious in nature**
**Hematological toxicity > 50%**
**Autoimmune toxicities**

**Notes:**
Not all biologic products are intrinsically safe (like Heceptin)
Pancytopenia / Aplasia:  (fatal)
Delayed recovery of neutrophils:
38% at two months of follow-up: 
25% at 4 months
Increased / new transfusion requirement during therapy ~ 68%
Prolonged CD4+ recovery: 27% < 200/ul at 4 months
Autoimmune – thrombocytopenia

## Pharmacokinetics

- Receptor mediated pharmacokinetics
- Mass effect pharmacokinetics – RES (liver, spleen), e.g. immunotoxins  conjugated to MoAb
- Dose saturation at lower levels
- Conversion from nonlinear to linear
- Molecular weight
- Organ systems – kidney
- Route of administration SC and IM distribution between lymphatics and blood stream

## Pharmacokinetics

- Nonclinical pk may be appropriate when receptors are not a factor – otherwise and relevant animal model
- Dose selection for initial clinical dose based on pk, pd and safety factor
- If large molecule like Ab scaling factor based on mg/kg with modification by uncertainty factors
- If smaller like cytokine mg/m2 to reflect first order elimination with uncertainty factors
- May be based on allometric model

## Toxicity - General

- Appropriate selection of a relevant animal species
- In cases in which other animal species are informative default to at least 2 species
- Antibody development may limit duration of dosing
- Additional tests should be incorporated in toxicity testing
- Core battery of safety pharmacology
- Aspect of immune function and cell markers

## Relative Sensitivity to IFN

- Tested by antiviral effect or rhIFN cytopathic effect reduction assay
- Human cell line 100%
- Rat <0.20%
- Guinea pig 30%
- Rabbit <0.12%
- Bovine 123%

## Relative Sensitivity

- Single dose, im
- Mouse 30,500 UX106 /kg
- Rat 30,100 UX106 /kg
- Rabbit 100 UX106 /kg
- Monkey 2.5 UX106 /kg

**Notes:**
Acute effects such as anemia and anorexia

## Importance of route of administration

- Toxicity studies should follow route of administration and duration as proposed for the clinical study
- Duration and regimen important, e.g. IL12
- Simply increasing exposure may not be a satisfactory answer to duration as peak and trough may influence receptor up and down regulation
- Conversion from IV to SC sometimes problematic

## Route of Administration

- Single dose toxicity for IFN
- Mouse IM 30,500 UX106 /kg
- Mouse SC 30 UX106 /kg
- Rat IM 30,100 UX106 /kg
- Rat SC 30 UX106 /kg
- Ferret IM 30 UX106 /kg
- Ferret SC 30 UX106 /kg
- Growth factors G-CSF

**Notes:**
Less sensitive species appear to have stronger effect perhaps pk mediated in addition to receptor sensitivity

## Toxicity - special

- Reproduction
- Commonly standard studies performed
- Immunogenicity sometimes abbreviates dosing
- Studies to confirm being member of a class may be performed for IFN’s

## Toxicity - Special

- Concerns related to neoplastic events
- Pulmozyme
- Secondary malignancy – OKT3 mitogenic activity
- New malignancy – immune alteration or suppression
- Current controversy – erythropoietin

## Toxicity – Special

- Immunogenicity
- Many biological products are foreign to standard test animals
- In a very limited number of cases chimps are requested
- Relative rates of immunogenicity may be considered in non-clinical studies (e.g., neoantigenicity)
- Autoantigenicity of pegylated form of thrombopoietin predicted by nonclinical studies in monkey
- PRCA and Eprex

**Notes:**
TPO auto-antibodies in 4% of healthy subjects PEG-rHuMGDF thrombocyopenia.  

## Toxicity Testing  

- Unresolved concerns testing for the unknown-unknowns
- Herceptin and cardiomyopathy
- G-CSF and splenic rupture
- TNF binders and TB
- IFN and suicidal ideation

## Conclusions

- Testing for pharmacology and toxicology of biological products is unique relative to other therapeutic drugs
- Targeting of toxicity testing to provide information rather than a collection of data is an important principle
- Increasing awareness of various types of toxicity such as the possibility of tumor promotion for use in chronic illnesses represents a challenge to both industry and regulators best suited to a scientific approach