The international standard for human interleukin-2. Calibration by international collaborative study.

Three lyophilized preparations of interleukin-2 coded 86/500, 86/564 and 86/504 have been evaluated in an international collaborative study for their suitability as an international standard. All of the preparations performed well in the different bioassay systems included in the study, and showed excellent stability on accelerated temperature degradation. Material similar to that in preparation 86/504 has served well as an interim reference reagent for interleukin-2 for 3 years. Therefore with the agreement of the study participants and the authorization of the Expert Committee on Biological Standardization of the World Health Organization, the preparation coded 86/504 was established in 1987 as the 1st international standard for interleukin-2, with a defined potency of 100 IU/ampoule.

Preparation of T cell growth factor free from interferon and factors stimulating hemopoietic cells and mast cells.

A simple two-step method involving ammonium sulfate precipitation followed by hydrophobic chromatography is described for the separation of T cell growth factor (TCGF) from a number of other factors contained in medium conditioned by concanavalin A-stimulated spleen cells. Thus, granulocyte-macrophage colony-stimulating factor, P cell-stimulating activity, pluripotential stem cell-supporting activity and interferon activity were not detected in TCGF partially purified by these steps. T cell-replacing factor co-purified with TCGF. Macrophage activity factor (MAF) co-purified with TCGF, but the ratio of MAF to TCGF activities was reduced more than 20-fold relative to that in crude conditioned medium. All of the factors were present in the 50-80% saturated ammonium sulfate fraction, however, levels of concanavalin A were reduced by 98% in this step. TCGF, separated in this way from these other regulatory factors will be useful in experiments analyzing the actions of TCGF on mixed populations of cells.

Interleukin-2 liposomes for primary immune deficiency using the aerosol route.

This is the first report of aerosol interleukin 2 (IL-2) liposome administration to individuals with immune deficiency. Parenteral IL-2 therapy has shown beneficial effects in some patients with cancer, common variable immunodeficiency (CVID), and human immunodeficiency virus (HIV) but is problematic because of side effects including fever and malaise as well as local swelling (delayed type hypersensitivity like reaction) after each subcutaneous IL-2 injection. Provision of an IL-2:human albumin liposome formulation via the aerosol route had few side effects in a recent clinical trial in cancer patients. Details of good manufacturing practice (GMP) synthesis and analysis of IL-2 liposomes (N= 6 lots) made without albumin carrier protein and placebo liposomes (three lots) are presented. After centrifugation, IL-2 was closely associated with the liposome pellet (99%). Mean diameter of liposomes was 1.1 microm. Patient acceptance, safety, toxicity, and immune effects of IL-2 liposomes were studied in individuals with primary immune deficiency (N = 15) and subsequently, a larger cohort of patients with hepatitis C. Experience in the immune deficient patients is the subject of this report. Placebo liposomes (12 weeks) and IL-2 liposomes (12 weeks) were provided using a nebulizer. Aerosol placebo liposomes and IL-2 liposomes were well tolerated. No changes in chest X-ray or pulmonary function were seen. Since biologic activity of aerosol IL-2 liposomes has been seen in viral disease (hepatitis C), additional studies of aerosol IL-2 liposomes in individuals with hepatitis C and HIV are planned.

The 2nd International Standard for Interleukin-2 (IL-2). Report of a collaborative study.

Two candidate preparations of human sequence recombinant Interleukin-2 (IL-2) were formulated and lyophilized at NIBSC prior to evaluation in a collaborative study for their suitability to serve as a replacement international standard. The preparations were tested by eight laboratories using in vitro bioassays and immunoassays. The candidate preparation 86/500 was judged suitable to serve as a replacement international standard based on the data obtained for activity and stability. On the basis of the results reported here, the preparation coded 86/500 was established by the WHO Expert Committee on Biological Standardisation (ECBS) in 2012 as the WHO 2nd IS for human IL-2 with an assigned value for IL-2 activity of 210IU/ampoule. Calibration of the 2nd IS is primarily based on the bioassay in use in various laboratories and relies exclusively on the estimates calculated relative to the WHO 1st IS for continuity of the IU.

Interaction of interleukin 2 with cells: quantitative analysis of effects.

The response of T lymphocytes to interleukin 2 (IL 2) is accurately described by a four-parameter logistic function. Both data generated by a theoretical model of IL 2-driven proliferation and experimental data conformed to this function for all doses of IL 2. Assays measuring either the rate of DNA synthesis or cellular metabolism were well described. The variance of response was not constant but increased in a predictable way. Weighting was therefore included in deriving a nonlinear curve-fitting program. The effects on response of cell density, time, and the T lymphocyte line used were examined. Assays gave reproducible estimates of potency when test preparations were compared with a standard preparation, but not otherwise. A model for IL 2 proliferation was derived on the basis of the two-state model of the cell cycle, with cells leaving a quiescent state randomly and then traversing the other stages of the cell cycle in a determinate way.

Effects of cytokines on antiviral pharmacokinetics: an alternative approach to assessment of drug interactions using bioequivalence guidelines.

The effects of cytokines on the pharmacokinetics of nucleoside analogs were evaluated in two separate studies using zidovudine in combination with interleukin-2 and didanosine in combination with alpha interferon. In each study, drug interactions were evaluated by using both a standard method (Student's t test) and bioequivalence testing. Serial blood samples were collected from human immunodeficiency virus-infected patients prior to and during cytokine therapy for determination of nucleoside analog concentrations. Concentrations were fit separately to a two-compartment model by using the iterative two-stage approach to population analysis. No alterations in area under the curve or oral clearance were observed for either drug during combination therapy. In general, there was good agreement between statistical methods for determining if antiviral pharmacokinetic parameters were altered by concomitant cytokine therapy. However, large individual changes in the maximum concentration of zidovudine in serum were detected by bioequivalence testing but no difference was found by Student's t test. For didanosine, significant but clinically irrelevant decreases determined by standard hypothesis testing were seen for both the volume of the central compartment (1.91 to 1.86 liters) and the absorption rate constant (0.79 to 0.73 h-1) in the presence of alpha interferon. No interaction was noted for these parameters by using bioequivalence guidelines. Bioequivalence testing may provide an alternative approach to assessment of drug interactions. Interleukin-2 and alpha interferon do not alter the pharmacokinetics of zidovudine and didanosine, respectively.