NCL Objectives

1. Establish and Standardize an Analytical Cascade for Nanomaterial Characterization
   
   
2. Facilitate the Clinical Development and Regulatory Review of Nanomaterials for Cancer Clinical Trials
   
   
3. Identify and Characterize Critical Parameters Related to Nanomaterials' Absorption, Distribution, Metabolism, Excretion, and Toxicity Profiles of Nanomaterials Using Animal Models
   
   
4. Examine the Biological and Functional Characteristics of Multicomponent/Combinatorial Aspects of Nanoscaled Therapeutic, Molecular and Clinical Diagnostics, and Detection Platforms
   
   
5. Engage and Facilitate Academic and Industrial-Based Knowledge Sharing of Nanomaterial Performance Data and Behavior Resulting from Pre-Clinical Testing (i.e. Physical Characterization, In Vitro Testing, and In Vivo Pharmaco- and Toxicokinetics)
   
   
6. Interface with Other Nanotechnology Efforts

1. Establish and Standardize an Analytical Cascade for
Nanomaterial Characterization

Nanomaterials characterized by the NCL are intended for in vivo diagnostic and therapeutic purposes. To this end, the NCL will develop and perform a standardized analytical cascade that tests the pre-clinical toxicology, pharmacology, and efficacy of nanoparticles and devices. Nanomaterials received from academia, government, and industry will be subjected to this
assay cascade that characterizes nanoparticles' physical attributes, their in vitro biological properties, and their in vivo compatibility. The time required to characterize a nanoparticle from receipt through the in vivo phase is anticipated to be 1 year ultimately enabling a sponsor's filing of an Investigational New Drug (IND) or Investigational Device Exemption (IDE) application with the FDA. This sequence is shown in Figure 1.

  Physical Characterization
  In Vitro Characterization
  In Vivo Characterization

Physical Characterization

Current research on therapeutic and diagnostic applications for nanomaterial is helping to identify critical parameters for the material's compatibility with biological systems. Extant literature implicates physical attributes such as size, hydrophilicity, and surface chemistry as key factors contributing to nanomaterial's in vivo fate. The first phase of the analytical cascade will therefore focus on the characterizing of the material's physical properties. The goal of this phase is to determine the particle's size, size distribution, molecular weight, density, surface area, porosity, hydrophilicity, surface charge density, purity, sterility, surface chemistry, and stability. The batch-to-batch reproducibility of material as provided by the sponsor/vendor will also be addressed during this stage. NCL will rely heavily on the expertise and resources of the NIST for the physical characterization phase.

In Vitro Characterization

Prior to filing an Investigational New Drug (IND) or Investigational Device Exemption (IDE) application with the FDA, a new product must be adequately studied. For these products, toxicity or biocompatibility must first be characterized in animals and efficacy may be standardized in animal discovery models. The cost- and labor-intensiveness of these in vivo studies impel drug and device discovery efforts to utilize in vitro methodologies wherever technology permits. Refined in vitro protocols related to drug and device discovery allow researchers to make a first-order assessment of a material's in vivo pharmacokinetics, biocompatibility, and toxicity.

Nanoparticles' binding, pharmacology, and uptake properties, for example, will be monitored by common cell and molecular biology methods, such as ELISA and fluorescence microscopy. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) will also be used as tools to observe the particle's interaction with cellular-level components. Electron microscopy, chromatography and electrophoresis protocols allow the NCL to characterize the nanomaterial's blood contact properties, such as opsonization and macrophage phagocytosis as well as pinocytosis and uptake by nonphagocytic cells.

Also included in the in vitro characterization is a thorough examination of the nanoparticle's therapeutic and/or diagnostic functionality. For example, particles with imaging modalities will be examined for their signal intensity (i.e., signal-to-noise ratio); nanotechnology strategies that incorporate therapeutic or preventative agents will be characterized for their drug-release kinetics and ability to cross biological barriers. A nonexhaustive list of equipment used for the in vitro phase is shown in Table 1.

In vitro models can also serve as a gross approximation of a nanomaterial's absorption, distribution, metabolism, excretion and toxicity (ADME/Tox) properties. For example, an initial assessment of acute toxicity can be conducted using hepatic microsomes, primary bone marrow cultures (GM-CFU), or mitochondrial toxicity assays. Other cellular assays to monitor apoptosis and cytotoxicity are now commonplace. As an example of pharmacokinetic characterization, release curves from nanoparticles with drug delivery strategies will be obtained and then assessed against other standardized release models, such as insulin.

In Vitro Diagnostics (clinical work-up)

For products which are intended to be primary diagnostics either for use in conjunction with a therapeutic or for stand-alone use, the new test should be analytically and clinically well established and should be studied in the intended use population in a manner that allows the product to be used for clinical diagnostic use. Of particular importance is establishing how well the new nanotechnology-based diagnostic performs at discriminating between true versus false positive and negative results. Table 2 lists properties that are relevant to diagnostic nanodevices.

In Vivo Characterization

The primary goal of the in vivo characterization is to elucidate the nanomaterials' safety, efficacy, and toxicokinetic properties in animal models. As is the case with any new chemical entity (NCE), these properties and other ADME data must be obtained prior to transitioning the nanoparticles to clinical applications. This phase will leverage the plethora of knowledge and protocols used to characterize drugs and devices in vivo.

Animal studies conducted under the in vivo phase for the study of nanoparticles will be in support of the FDA's Guidance For Industry, Single Dose Acute Toxicity Testing For Pharmaceuticals (http://www.fda.gov/cder/guidance/pt1.pdf). The nanoparticle will be administered to animals to identify (1) doses causing no adverse effect and (2) doses causing life-threatening toxicity. The information obtained from these tests will provide preliminary identification of target organs of acute toxicity, and may aid in the selection of starting doses for Phase I human trials. Preliminary data on the nanoparticle ADME profile will also be obtained in this phase. in vivo studies will characterize the nanoparticle absorption, pharmacokinetics, serum half-life, protein binding, tissue distribution/ accumulation, enzyme induction or inhibition, metabolism characteristics and metabolites, and excretion pattern.

Studies conducted in the in vivo phase for diagnostic nanodevices should support the following applicable guidances:

• Multiplex Tests for Heritable DNA Markers, Mutations and Expression Patterns
• Guidance For Submission of Tumor Marker Premarket Notifications
• Statistical Guidance on Reporting Results from Studies Evaluating Diagnostic Tests

Given the multifunctional potential of nanoparticles, the in vivo characterization phase will also include an assessment of the strategy's targeting and/or imaging capabilities. Targeting will be assessed, for example, by comparing a nanoparticle distribution profile with a non-targeting nanoparticle from the same class. For those particles used with imaging modalities, the signal enhancement will be monitored using the appropriate magnetic resonance, ultrasound, optical, positron emission tomography imaging instrumentation. The NCL will actively collaborate with NCI's Cancer Imaging Program (CIP) to facilitate and harmonize the NCL studies with imaging strategies to be used in clinical trials.

NCI's Developmental Therapeutics Program (DTP) is an example of an in vivo program already in place at NCI-Frederick that can augment the NCL programs. DTP accepts candidate drugs from intramural and extramural investigators and then subjects these compounds to an extensive series of animal studies. These studies include determining the drug's maximum tolerated dose (MTD), its biological effective dose (BED), its toxicity to cardio-, hematopoetic, neurological, and nephritic tissues, and its efficacy in hollow-fiber protocols and xenograph implant models. The NCL will attempt to leverage DTP's protocols when resources permit, but may also outsource the in vivo animal studies when demand and schedule warrant.

Another NCI program that conducts pre-clinical studies using animal models is the Development of Clinical Imaging Drugs and Enhancers (DCIDE) program administered by the Cancer Imaging Program. DCIDE is a competitive program to expedite and facilitate the development of promising investigational imaging enhancers (contrast agents) or molecular probes from the laboratory to IND status. The DCIDE program provides pre-clinical pharmacokinetics, dosimetry, imaging feasibility, and provides assistance with regulatory affairs for IND filing. Through its ongoing collaboration with CIP, the NCL will leverage DCIDE's expertise, personnel, and other resources whenever the opportunity permits.

In addition to capitalizing on these existing animal protocols, early efforts at the NCL will also focus on standardizing the analytical and histopathological methods that are relevant, and perhaps unique, to nanoparticles. For example, several pre-clinical studies suggest a key role for macrophages in clearing nanoparticles from the blood. Similarly, a growing number of reports implicate the kidneys - rather than the liver - as the primary tissue responsible for excreting nanoparticles. Special attention will therefore be applied to standardizing assays associated with these components of the reticuloendothelial system (RES).