Pre-clinical immunotoxicity studies of nanotechnology-formulated drugs: Challenges, considerations and strategy

Abstract

Assorted challenges in physicochemical characterization, sterilization, depyrogenation, and in the assessment of pharmacology, safety, and efficacy profiles accompany pre-clinical development of nanotechnology-formulated drugs. Some of these challenges are not unique to nanotechnology and are common in the development of other pharmaceutical products. However, nanoparticle-formulated drugs are biochemically sophisticated, which causes their translation into the clinic to be particularly complex. An understanding of both the immune compatibility of nanoformulations and their effects on hematological parameters is now recognized as an important step in the (pre)clinical development of nanomedicines. An evaluation of nanoparticle immunotoxicity is usually performed as a part of a traditional toxicological assessment; however, it often requires additional in vitro and in vivo specialized immuno- and hematotoxicity tests. Herein, I review literature examples and share the experience with the NCI Nanotechnology Characterization Laboratory assay cascade used in the early (discovery-level) phase of pre-clinical development to summarize common challenges in the immunotoxicological assessment of nanomaterials, highlight considerations and discuss solutions to overcome problems that commonly slow or halt the translation of nanoparticle-formulated drugs toward clinical trials. Special attention will be paid to the grand-challenge related to detection, quantification and removal of endotoxin from nanoformulations, and practical considerations related to this challenge.

Keywords: Anaphylaxis; Coagulopathy; Complement activation; Cytokines; Endotoxin; Hemolysis; Immunotoxicity; Nanoparticles; Phagocytosis; Pre-clinical; Protein binding; Thrombosis.

Fig. 1. Challenges in pre-clinical development of nanotechnology-formulated drugs

Pre-clinical studies encompass assorted challenges in four key areas: chemistry, efficacy, pharmacology and toxicology, and hematology and immunology. Critical attributes in particle characterization are summarized in this fishbone diagram according to the relevant area of pre-clinical research.

Figure 2. Challenges in analyzing nanomaterials for potential endotoxin contamination by LAL assay

Nanoparticle interference with Limulus amoebocyte lysate assays (LALs) can be detected by inhibition/enhancement controls (IECs), which are prepared by spiking a known amount of control standard endotoxin (CSE) into a quality control (water) or a test nanoparticle. The test results for each of the IECs are then compared to the theoretical value. The non-interfering sample is the one that demonstrates spike recovery between 50% and 200%, according to U.S. Pharmacopoeia Bacterial Endotoxins Test (USP BET) 85 [60]. Spike recovery below 50% is considered inhibition and means that the endotoxin in the test sample is underestimated; recovery above 200% is considered enhancement and signifies that the amount of endotoxin in the test sample may be overestimated or that the test sample is highly contaminated. Most interferences may be overcome by dilutions as long as the tested dilutions are within the maximum valid dilution (MVD) limit [60]. ^¶ Proteins and surfactants may either inhibit or enhance endotoxin detection depending on the concentration. Removing or diluting surfactants helps overcome the interference; protein interferences can be eliminated by either heating the sample at 75 °C for 15 min or digestion with endotoxin-free protease (e.g., BioDTech ESP); ^¶¶Reconstitution of the lysate in glucan-blocking buffer (e.g., Glucashield) or using recombinant factor C assay helps overcome this interference; ^¶¶¶ Proteases (e.g., trypsin) can be inactivated by heating the sample at 75 °C for 15 min; *If proteinaceous in nature, use either heat or endotoxin-free protease treatment to eliminate this interference; **The presence of Ca²⁺ may inhibit LAL, and in this case, adding low concentrations of EDTA may help overcoming the interference; ***Source specific, consider dilution, removal, or inhibition of the interfering substance.

Figure 3. Nanoparticle interference with LAL is not detectable by the IEC

Endotoxin may get trapped by lipid and/or hollow nanoparticles during synthesis. Only free endotoxin is detectable by the LAL; trapped endotoxin is masked from the recognition. Case studies show examples of 2 PEG-liposomes in which particle destruction by heat resulted in higher endotoxin recovery. In the case of liposome 1, the higher endotoxin level recovered from the particle is still within the endotoxin limit (EL), shown as a red line. In case of the liposome 2, the recovered endotoxin is above the EL. QC: quality control prepared by spiking a known amount of endotoxin into LAL-grade water; API: active pharmaceutical ingredient; PEG: polyethylene glycol.

Figure 4. Trend in pro-inflammatory cytokine induction by nanotechnology carriers

Of the nanomaterials tested by the NCL during its 10 years of operation, approximately 10% induced cytokines; many of them induced IL-8, and the majority of the IL-8 inducers did so exclusively (i.e., without inducing TNFα and IL-1β). These materials were typically liposomes, micelles, and nanoemulsions. The example of such a micelle which can induce IL-8 without triggering TNFα and IL-1β production is nanosized excipient Cremophor-EL commonly used to formulate hydrophobic drugs.

Figure 5. Combining a cytokine-inducing nanotechnology carrier with an pro-inflammatory API results in a pro-inflammatory formulation

Some nanoparticles are pro-inflammatory and induce cytokines. Using such carriers to formulate an API known to induce interferons (e.g., therapeutic nucleic acids) may cause the final formulation to induce both cytokines and interferons. Such a combination may be beneficial when immunostimulation is desirable (e.g., vaccines), but other carriers should be considered when the immunostimulation is unwanted.

Figure 6. Tiered approach for assessing nanoparticle compatibility with the immune system in vitro during early-phase pre-clinical development

This framework was developed based on the traditional approach used in discovery-level toxicology for xenobiotics and novel low-molecular-weight pharmaceuticals, and common toxicities for nanoparticle-formulated drug failure in pre-clinical development. The first tier is intended for the identification of microbial and endotoxin contamination, which, when present, may confound results of toxicity and efficacy studies. The second tier is focused on identifying toxicities commonly responsible for nanoparticle failure in pre-clinical studies. This tier focuses on identifying acute toxicities. The third tier is focused on myelo- and lymphotoxicities affecting the immune cell function. This tier aims at identifying potential concerns for the long-term toxicities. The fourth tier is intended for the identification and understanding of the mechanisms of toxicities. It may involve both in vitro and in vivo studies, and is used to inform lead candidate selection and to support the design of pre-clinical GLP studies required for regulatory filings of investigational drugs. The inclusion of individual components of the final formulation along with precursors is helpful in identifying the source of toxicity when the final formulation is found to be toxic. “(If feasible)” refers to the analysis of formulations, which, in these assays, is not trivial because of the particle physical properties and the limitations of currently available methodologies.