Interferons toxicity testing

Each interferon preparation was ultracentrifuged at 20,000 revolutions per minute for one hour to remove tissue debris and inactivated virus. The supernatant was dialyzed against distilled water (1 400) for 24 hours at4°C. The material was then freeze-dried. The dried product was reconstituted in one-tenth of the original volume in distilled water and dispensed into ampoules. Reconstituted solutions were assayed for interferon activity, examined for toxicity, and tested for sterility. 

This approach appears somewhat irrational and without much scientific merit, since many of these new molecules are minimally toxic or nontoxic by this sort of acute evaluation. As in the case of interferons or monoclonal antibodies, the toxic effects observed in humans might not be predicted from safety assessments in rodents. An appropriate test species should be selected. Is the rat or mouse the appropriate species to evaluate a species-specific rDNA protein such as human growth hormone or interferons, or would nonhuman primates be more suitable Does the nonhuman primate really offer any advantages There is some consensus that the nonhuman primate may be a more appropriate species for testing some rDNA human proteins. 

The synthetic polymeric components as well as their combinations with proteins such as human serum albumin (HSA), bovine serum albumin (BSA), human serum albumin/a-interferon mixtures (HSA-IFNa) and myoglobin (MYO) did not give any negative response to in vitro and in vivo biocompatibility tests, such as platelet aggregation, complement activation, acute toxicity, and acute thromboembolic potential. 

Due to the toxicity of the co-(cyclopolymer) 8, the capability of interferon induction of a great number of polymers has been tested. Thus, interferon is formed by the presence of natural double helix RNA, which occurs in some viruses (Reo-viruses), but also by the synthetically produced complex (40) from poly(inosinic acid) and poly(cytidylic acid) (1 1). An improved and extended interferon induction of such poly(nucleic acid)s can be achieved by complexing with synthetic polycations, as for example diethylaminodextrane (41) or with a polycation (42) of composition 9. 

Evaluating the immune response to the vaccine at the same time as evaluating toxicity is recommended in a 2003 WHO guideline (Table 31.1). This makes good sense because any toxicity observed with vaccines is frequently related to the immune response, and correlation of these two endpoints provides a fuller explanation of the test results. Assays to measure the immune response include those which measure antigen-specific antibody responses (e.g., ELISA) and cell-mediated responses (e.g., y-interferon—ELISPOT). Developing these... 

In early trials, aldesleukin was given with lymphokine-activated killer (LAK) cells or tumor-infiltrating lymphocytes. However, later data showed that the addition of LAK cells does not improve the therapeutic response in renal cell carcinoma and can produce more pulmonary toxicity and hypotension (1). Compared with aldesleukin or interferon alfa alone, the combination of aldesleukin plus interferon alfa produces a significantly longer event-free survival without effect on the overall survival, but induces substantial toxicity with severe and resistant hypotension (3). The optimal safe and effective dose and schedule of administration of aldesleukin is not yet well defined, and a variety of regimens have been tested, with doses of 600 000 units/kg by intermittent bolus intravenous infusion or 18 106 units/m by continuous subcutaneous or intravenous infusion. 

Although inhibition of stem-cell proliferation is the most likely mechanism of hematological toxicity, increased platelet hepatic uptake has been suggested to account for thrombocytopenia (227). Raised serum thrombopoietin concentrations were found in patients with interferon alfa-induced thrombocytopenia (228). However, there is evidence that serum thrombopoietin concentrations in patients who have had thrombocytopenia during interferon alfa treatment for chronic viral hepatitis C either do not increase (in patients with compensated cirrhosis) or increase only moderately and less than expected (in non-cirrhotic patients) (229). The authors proposed that interferon alfa impairs liver production of thrombopoietin, raising the possibility of testing thrombopoietin administration in patients with severe thrombocytopenia before or during treatment with interferon alfa (230). 

German and other European physicians also latched on to combination therapy of Proleukin with Interferon-alpha as helpful to outpatient or home care.35 A series of reports in German medical journals suggested that subcutaneous infusion and combination therapy would result in fewer side effects, less strain on patients, and more successful treatment of cancer.36 The approach drew sufficient interest from physicians that the company sponsored tests to document its efficacy and eventually presented data to the BGA supporting this method of application. Cetus used a database of 425 patients to compare 225 patients who received Proleukin as an intravenous drip infusion with 200 patients who injected themselves with Proleukin combined with Interferon-alpha. The two approaches did not result in statistically different outcomes in terms of cancer remission, but were strikingly different in their toxicity. Intravenous application had a remission rate of 15 percent, while 30 percent of the patients suffered from severe toxicity. In contrast, the subcutaneous application had a 20 percent remission rate with only 5 percent of patients experiencing severe toxicity.37... 

The interferon inducer and immune modulator, poly(ino-sinic)-poly(cytidylic) acid, complexed with poly(lysine) and carboxymethyl cellulose, poly(ICLC) was administered i.m., plus AZT, orally to patients with advanced AIDS. The purpose of this part of the study was to evaluate the safety of the treatment in such a series of patients, to test for toxic effects, and to see if there were any alterations in some of the subsets of lymphocytes. There did not appear to be any significant clinical or laboratory indications of toxicity. The disease was not worsened by the treatment. There were transient boosts in a number of immune parameters, such as NK cell activity, T4 and T8 cells, and the expression of DR antigens on both B and T lymphocytes. These data will be used in the next phase of the study to determine a rational dose and time schedule to maximize effectiveness. 

The toxicity of TSST-1 and HI 35 A was tested in 20 g BALB/c mice (n=15 per dose) injected intraperitoneally (ip) with either protein diluted in PBS. Four hours later, mice were injected ip with 75 pg of E. coli 055 B5 LPS (Difco Laboratories, MI) and lethality was observed over 72 h. The serum cytokine levels of tumor necrosis factor a and P (TNF), as well as interferon gamma (IFNy), were measured over 14 h (2 h intervals) in mice injected with either 15 pg TSST-1, 15 pg TSST-1 plus 75 pgLPS, 15 pgH135Aplus LPS, or 75 pg LPS only. Serum levels of TNF were determined by an ELISA (Genzyme, M A) with triplicate samples the standard deviation (SD). Data for IFNy were collected from an L929 cell lysis assay with duplicate samples 10%. 

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