Pre-clinical methods for detecting the hypersensitivity potential of pharmaceuticals: regulatory considerations.

Immune-based systemic hypersensitivities account for a significant number of adverse drug reactions. There appear to be no adequate nonclinical models to predict systemic hypersensitivity to small molecular weight drugs. Although there are very good methods for detecting drugs that can induce contact sensitization, these have not been successfully adapted for prediction of systemic hypersensitivity. Several factors have made the development of adequate models difficult. The term systemic hypersensitivity encompases many discrete immunopathologies. Each type of immunopathology presumably is the result of a specific cluster of immunologic and biochemical phenomena. Certainly other factors, such as genetic predisposition, metabolic idiosyncrasies, and concomitant diseases, further complicate the problem. Therefore, it may be difficult to find common mechanisms upon which to construct adequate models to predict specific types of systemic hypersensitivity reactions. There is some reason to hope, however, that adequate methods could be developed for at least identifying drugs that have the potential to produce signs indicative of a general hazard for immune-based reactions.

Assessment of immunosuppressant drug carcinogenicity: standard and alternative animal models.

Drugs intended for use in preventing allograft rejection in transplant patients are likely to be administered chronically; thus, it is normally expected that sponsors would conduct nonclinical studies to determine the carcinogenic potential of candidate compounds. For pharmaceuticals other than biologic agents, this would mean that rodent carcinogenicity bioassays would be performed under most circumstances. Immunosuppressant drugs have presented unique challenges with respect to the issue of carcinogenicity bioassays. The pharmacological activity of therapeutic immunosuppressants is thought to make them highly likely to act as promoters/cocarcinogens, even in the absence of genotoxic activity. Thus, it is assumed that this class of drug would represent a carcinogenic hazard in the absence of confirmatory standard rodent bioassay data. In addition, rodents typically have been sensitive to the pharmacological/toxicological effects of immunosuppressants. It has proven to be difficult, therefore, to conduct life-time bioassays at doses reasonably equivalent to those that would be used clinically. For this and other reasons, alternative models might be more appropriate for risk assessment with this class of drugs.

Inhibition of tumor necrosis factor alpha alters resistance to Mycobacterium avium complex infection in mice.

Increased production of tumor necrosis factor alpha (TNF-alpha) appears to play an important role in the progression of human immunodeficiency virus disease. One treatment strategy being explored is the use of TNF-alpha inhibitors. TNF-alpha also appears to be important in conferring resistance to infections, and the inhibition of this cytokine may exacerbate the emergence of opportunistic pathogens, such as Mycobacterium avium complex (MAC). The present study examines the possibility that inhibition of TNF-alpha will increase the progression of disease in mice infected with MAC. C57BL/6 beige (bg/bg) mice have been shown to be highly susceptible to infection with MAC and are routinely used for testing of antimycobacterial drugs. However, bg/bg mice are known to exhibit impaired phagocyte and natural killer cell function. Since these cell types are important sources of TNF-alpha, the susceptibility of the bg/bg strain to infection with MAC was compared with those of the heterozygous (bg/+) and wild-type (+/+) strains of C57BL/6 mice. The susceptibilities of the bg/bg and bg/+ strains of mice infected with MAC were found to be comparable. The +/+ strain was the least susceptible. Mycobacterial burden and serum TNF-alpha levels increased over time in all the strains of mice tested. The bg/+ strain of C57BL/6 mice was then chosen to measure the activity of TNF-alpha antagonists. Treatment with dexamethasone decreased serum TNF-alpha levels and increased mycobacterial burden. Treatment with anti-TNF-alpha antibody or pentoxifylline did not significantly alter serum TNF-alpha levels but increased mycobacterial burden. Treatment with thalidomide neither consistently altered mycobacterial burden in the spleens or livers of infected mice nor affected serum TNF-alpha levels.

Assessment of the skin sensitization potential of topical medicaments using the local lymph node assay: an interlaboratory evaluation.

The murine local lymph node assay (LLNA) is a method for the predictive identification of chemicals that have a potential to cause skin sensitization. Activity is measured as a function of lymph node cell (LNC) proliferative responses stimulated by topical application of test chemicals. Those chemicals that induce a threefold or greater increase in LNC proliferation compared with concurrent vehicle controls are classified as skin sensitizers. In the present investigations we have evaluated further the reliability and accuracy of the LLNA. In the context of an international interlaboratory trial the sensitization potentials of six materials with a history of use in topical medicaments have been evaluated: benzoyl peroxide, hydroquinone, penicillin G, streptomycin sulfate, ethylenediamine dihydrochloride, and methyl salicylate. Each chemical was analyzed in the LLNA by all five laboratories. Either the standard LLNA protocol or minor modifications of it were used. Benzoyl peroxide and hydroquinone, both human contact allergens, elicited strong LLNA responses in each laboratory. Penicillin G, another material shown previously to cause allergic contact dermatitis in humans, was also positive in all laboratories. Streptomycin sulfate induced equivocal responses, in that this material provoked a positive LLNA response in only one of the five laboratories, and then only at the highest concentration tested. Ethylenediamine dihydrochloride dissolved in a 3:1 mixture of acetone with water, or in 4:1 acetone:olive oil (one laboratory), was uniformly negative. However, limited further testing with the free base of ethylene diamine yielded a positive LLNA response when applied in acetone:olive oil (AOO). Finally, methyl salicylate, a nonsensitizing skin irritant, was negative at all test concentrations in each laboratory. Collectively these data serve to confirm that the local lymph node assay is sufficiently robust to yield equivalent results when performed independently in separate laboratories and indicate also that the LLNA is of value in assessing the skin sensitization potential of topical medicaments.

What are the prospects for regulation in immunotoxicology?

Evaluating the immunotoxic potential of investigational new drugs is a standard component of non-clinical safety assessment. Effects evaluated include the potential for drugs to induce hypersensitivity and/or autoimmune reactions or to produce unintended immunosuppression. The Center for Drug Evaluation and Research (CDER) is considering approaches for evaluating potential immunotoxicity. In particular, two methods are being examined for potential recommendation where indicated: immune cell phenotype determination and the murine local lymph node assay. Issues concerning immunotoxicology testing will be discussed.

Regulatory issues of importance to developing anti-HIV therapeutics.

In the United States, before clinical trials with new drugs can proceed, an Investigational New Drug (IND) application must be submitted to the Food and Drug Administration. Applications for drugs intended for the treatment of HIV-related diseases are reviewed by the Division of Anti-Viral Drug Products (DAVDP) within the Center for Drug Evaluation and Research. IND applications must contain adequate preclinical studies to support the safety of the proposed clinical trials. Essential for demonstrating safety are animal toxicology studies in which the drug has been administered in doses, by the route(s) of administration, and over a length of time equivalent to the corresponding parameters proposed for clinical trials. Reproductive toxicology/teratology studies should be conducted early in the development of the drug. Other nonclinical toxicology studies, such as carcinogenicity bioassays, are usually conducted concurrently with clinical trials. The DAVDP has developed a Pre-IND program to assist sponsors in preparing the best preclinical research to support the development of new drugs.

Halogenated anesthetics form liver adducts and antigens that cross-react with halothane-induced antibodies.

Two halogenated anesthetics, enflurane and isoflurane, have been associated with an allergic-type hepatic injury both alone and following previous exposure to halothane. Halothane hepatitis appears to involve an aberrant immune response. An antibody response to a protein-bound biotransformation product (trifluoroacetyl adduct) has been detected on halothane hepatitis patients. This study was performed to determine cross-reactivity between enflurane and isoflurane with the hypersensitivity induced by halothane. The subcellular and lobular production of hepatic neoantigens recognized by halothane-induced antibodies following enflurane and isoflurane, and the biochemical nature of these neoantigens was investigated in two animal models. Enflurane administration resulted in neoantigens detected in both the microsomal and cytosolic fraction of liver homogenates and in the centrilobular region of the liver. In the same liver, biochemical analysis detected fluorinated liver adducts that were up to 20-fold greater in guinea pigs than in rats. This supports and extends previous evidence for a mechanism by which enflurane and/or isoflurane could produce a hypersensitivity condition similar to that of halothane hepatitis either alone or subsequent to halothane administration. The guinea pig would appear to be a useful model for further investigations of the immunological response to these antigens.

Trifluoroacetylation potentiates the humoral immune response to halothane in the guinea pig.

Halothane hepatitis appears to result from an inappropriate immune response to the products of halothane metabolism. Attempts to produce an animal model for halothane hepatitis have been largely unsuccessful. Although guinea pigs produce neoantigens following treatment with halothane, the subsequent antibody response is weak, possibly accounting for the failure to produce halothane hepatitis in these animals. In order to increase the antibody response to halothane neoantigens, three methods for trifluoroacetylating proteins were used. Guinea pigs were either treated with S-ethylthiotrifluoroacetate, autologous lymphocytes trifluoroacetylated ex vivo, or immunized with trifluoroacetylated mycobacterial protein, followed by exposure to halothane, and examined for anti-halothane metabolite antibodies (anti-TFA antibodies). Animals treated with S-ethylthiotrifluoroacetate developed anti-TFA antibodies, and following exposure to halothane exhibited an enhanced antibody response. Treatment with trifluoroacetylated lymphocytes also resulted in an enhanced anti-TFA antibody response following halothane exposure. Immunization with trifluoroacetylated mycobacterial proteins resulted in very high anti-TFA antibody titers. However, subsequent exposure to halothane had no observable effect on specific antibody titers. Exposure to halothane, regardless of treatment, resulted in the production of anti-microsomal protein antibodies. Signs of halothane hepatitis were not observed, indicating that enhancement of the humoral immune response does not appear to be sufficient for production of halothane hepatitis.

Use of staphylococcal enterotoxin A-induced lymphoproliferation and interleukin 2 production as indicators of immunotoxicity.

Suppression of mitogen-induced splenocyte lymphoproliferation and interleukin 2 (IL-2) production can be used as indicators of immunotoxicity. Staphylococcal enterotoxin A (SEA) is both a potent mitogen and the most potent in vitro inducer of IL-2 production that has been described. An in vitro system was used to measure impairment of SEA-induced lymphoproliferation and IL-2 production using splenocytes from female C57BL/6 mice dosed with either cyclosporin A (30 mg/kg/day, 14 days), benzene (220, 440, or 880 mg/kg/day, 14 days), or vehicle. Splenocytes were stimulated with either concanavalin A (con A) or SEA. Benzene- and cyclosporin A-treated mice demonstrated significant decreases in splenocyte proliferation. IL-2 production was determined by incubating splenocyte culture supernatants with IL-2 dependent cytotoxic T-cells (CTLL-2), pulsing with 3H-thymidine, and determining amount of incorporated label. Cell proliferation and IL-2 production were inhibited by both benzene and cyclosporin A, effects more clearly demonstrated using SEA than con A. SEA was a superior mitogen compared to con A in the assays evaluated here.

Formation and identification of protein adducts to cytosolic proteins in guinea pig liver slices exposed to halothane.

The anesthetic halothane can be bioactivated to the reactive intermediate, trifluoroacetyl chloride, which can covalently bind to liver protein. The product of this reaction is trifluoroacetyl-N-epsilon-lysine which can act as a foreign epitope in altering both protein immunogenicity and antigenicity. An in vitro liver slice system was used to study the formation of protein adducts following exposure to halothane. Liver slices (30-35 mg wet weight, 250-300 microns thick) from adult male Hartley guinea pigs (600-800 g) were exposed to [14C]halothane (0.6-0.9 microCi, 1.0-1.7 mM) in 95% O2/5% CO2 for 1, 6 and 12 h. The slices were homogenized and subcellular fractions prepared. Proteins were resolved by electrophoresis and bound radioactivity was detected by scintillation counting and autoradiography. Greater than 80% of detectable radioactivity to whole liver cell protein was localized in the 20-30-kDa range and increased in a linear fashion over the 12-h incubation period. Covalent binding was localized to two proteins of 27 kDa and 26 kDa present in the cytosolic compartment. Purification followed by N-terminal amino acid sequence analysis of the 27-kDa protein has identified it to be homologous with glutathione-S-transferase b. This cytosolic protein appears to be the major target for trifluoroacetylation in liver slices exposed to halothane.

Screening for antibodies associated with halothane hepatitis.

The diagnosis of halothane hepatitis (HH) may be assisted by detection of antibodies reacting to trifluoroacetylated proteins (anti-TFA antibodies). An enzyme-linked immunosorbent assay (ELISA) utilizing trifluoroacetylated rabbit serum albumin (TFA-RSA) as antigen detected anti-TFA antibodies in 67% of sera from patients for whom a clinical diagnosis of HH was made. Anti-TFA antibodies were detected in 33% of sera when using an ELISA with liver microsomal protein from halothane-treated rabbits as antigen. Absorption of the sera with untreated rabbit liver microsomal protein before using the microsomal protein ELISA resulted in detection of anti-TFA antibodies in 42% of sera. Using the presumptive hapten N-epsilon-trifluoroacetyl-1-lysine to block antibody binding in an ELISA resulted in positive detection in 50% of sera: the results did not always agree with the other ELISA methods. The TFA-RSA ELISA was the most sensitive method and, combined with the TFA-lysine blocking ELISA, resulted in 92% of sera from HH patients testing positive for HH-associated antibodies.

Generation and detection of neoantigens in guinea pig liver slices incubated with halothane.

The volatile anesthetic halothane can be biotransformed by the hepatic cytochrome P-450 system to produce a reactive intermediate, trifluoroacetyl chloride, capable of covalently binding to liver proteins. The product of this reaction, the trifluoroacetyl lysinyl moiety, can act as an epitope to alter protein antigenicity. An in vitro system has been developed to produce halothane induced neoantigens and to study conditions for formation in the liver. Liver slices, capable of halothane biotransformation, provide a viable means for mechanistic studies. Liver slices (1 cm diameter, 300 microns thick) from male Hartley guinea pigs (600-800 g) were exposed to either 1.0 or 1.7 mM halothane (media concentration) in 95% O2/5% CO2 for 12 h. Covalent binding was determined using 14C-halothane. Neoantigens were detected by Western immunoblot analysis using rabbit anti-trifluoroacetylated albumin antiserum. Covalent binding was detected by 1 h of incubation and increased linearly through 12 h (20.7-48.5 nmole equiv/mg protein). Covalent binding preceded and correlated with the appearance of neoantigens. By 12 h of incubation, five neoantigens were seen with molecular weights ranging from 51 to 97 kD. These neoantigens have molecular weights similar to those seen in vivo. Liver slices exposed to deuterated halothane, which is oxidatively metabolized to a lower extent, did not develop neoantigens. This in vitro model system can be used to examine the mechanism for covalent binding and neoantigen production in the hepatocyte.

Induction of ethylenediamine hypersensitivity in the guinea pig and the development of ELISA and lymphocyte blastogenesis techniques for its characterization.

Ethylenediamine (EDA) is reported to be a poorly characterized iatrogenic and occupational contact sensitizer. To better characterize EDA hypersensitivity, a guinea pig model was employed in which the animals were exposed epicutaneously to simulate conditions of human exposure, and selected immune parameters were measured. Induction of hypersensitivity was by the Buehler occluded patch method (6 hr application/day, once a week for 3 consecutive weeks) to 10, 20, 30, or 40% EDA, using either an ethanol or acetone/corn oil vehicle. Fourteen days after the last induction, guinea pigs were challenged by patch application of 2% EDA (nonirritating). The incidence of responders for erythema in the 10% EDA (ethanol) treatment group was 83 and 50% at 24 and 48 hr, respectively. In the 10% EDA (acetone/corn oil) group the corresponding values were 50 and 17%. For 20, 30, and 40% EDA, in either vehicle, the incidence of erythema was 83 to 100%. Severity grades (scale = 0-3) for cutaneous reactions to increasing concentrations of EDA in ethanol ranged from 0.8 to 2.5; those for EDA in acetone/corn oil ranged from 0.6 to 2.8. Using an enzyme-linked immunosorbent assay developed to detect the predominant serum antibodies to EDA, it was shown that guinea pigs treated by patch application did not produce the main allergic antibody IgG specific for EDA. However, intradermal administration of an EDA-guinea pig serum albumin conjugate (EDA-GSA) to guinea pigs presensitized by patch application resulted in antibody production by 39 and 86% of the animals, at the initial and second dosing, respectively. An in vitro blastogenesis assay, using peripheral blood lymphocytes from EDA-sensitized guinea pigs, was developed to identify specific chemical allergens implicated in vivo sensitization. Maximum tritiated thymidine ([3H]TdR) incorporation by lymphocytes stimulated in vitro with EDA-GSA was observed on Day 7. Optimal antigen concentration for maximum lymphocyte proliferation ranged from 5 to 50 micrograms/ml, the major variation being attributable to interanimal differences. These results indicate that epicutaneous application of EDA in the guinea pig induces a Type IV delayed hypersensitivity; immunological memory to the hapten is maintained in cultured lymphocytes, suggesting the potential usefulness of the lymphocyte transformation test for in vitro diagnosis of chemically induced hypersensitivity in humans.