Comparative efficacy of a recombinant feline interferon omega in refractory cases of calicivirus-positive cats with caudal stomatitis: a randomised, multi-centre, controlled, double-blind study in 39 cats.

Chronic caudal stomatitis with alveolar/buccal mucositis in calicivirus-positive cats is the most severe presentation of feline chronic gingivostomatitis. Refractory cases are helped by antibiotic and anti-inflammatory treatments often including glucocorticoids. In order to evaluate the comparative efficacy of oromucosal administration of recombinant feline interferon omega (rFeIFN-ω) versus oral administration of glucocorticoids, a randomised, multi-centre, controlled, double-blind study was performed in 39 cats. The progression of behavioural, clinical and lesional scores was assessed over 90 days. Daily oromucosal treatment with 0.1 MU of rFeIFN-ω was associated with a significant improvement of clinical lesions (caudal stomatitis and alveolar/buccal mucositis) and a decrease of pain scores from D0 to D90. Although no such statistical improvement was noticed in the prednisolone group, there was, however, no significant difference between the two groups for most of the parameters, except pain at D60 and D90.

Biological standardization of human interferon beta: establishment of a replacement world health organization international biological standard for human glycosylated interferon beta.

Human interferon beta (IFN-beta) has been developed as a major biotherapeutic agent for the treatment of multiple sclerosis. Since World Health Organization (WHO) international standards (IS) for IFN-beta were established several years prior to the development of clinical grade IFN-beta products, a number of scientific issues with regard to the biological standardisation of natural and recombinant IFN-beta products have emerged. In order to address these issues, an international collaborative study to evaluate WHO IS and candidate international standards (CIS) of IFN-beta was instigated by the National Institute for Biological Standards and Control (NIBSC) in 2000 and was carried out in the succeeding year. Sixteen expert laboratories from 8 countries worldwide participated in the study. They performed titrations on 8 different IFN-beta preparations, including IS and new CIS, in a variety of mainly antiviral- but also including some antiproliferative- and reporter gene-assays, and contributed raw data from these assays to NIBSC for statistical analysis and calculation of potencies. While both intra- and inter-laboratory variation of potency estimates was evident, overall validity of the study as a whole was clearly shown by comparison of two pairs of internal coded duplicates, which gave the expected relative potency of 1 and the lowest inter-laboratory variability of potency estimates in all assay types. The CIS containing Chinese hamster ovary (CHO) cell- or human fibroblast-derived, glycosylated, IFN-beta gave similar low inter-laboratory variation in potency estimates one to another as the coded duplicates, which was significantly less than to the 2nd WHO IS of IFN-beta, human fibroblast-derived, Gb23-902-531. One of these CIS, designated 00/572, containing CHO cell-derived IFN-beta and formulated with both bovine casein and human serum albumin, could be assigned a potency, consistent for all assay types, of 40,000 international units (IU) per ampoule relative to the IU of the 2nd IS of IFN-beta, Gb23-902-531. Other CIS containing glycosylated IFN-beta, either CHO cell- or human-fibroblast-derived, could also be assigned potency values that were continuous with the IU of Gb23-902-531 and 00/572. However, greater inter-laboratory variations in estimates were evident from comparisons of Gb23-902-531 or 00/572 with either the 1st IS for E. coli-derived, non-glycosylated, IFN-beta with serine substitution at position 17 (IFN-beta Ser 17 mutein), Gxb02-901-535, or with a CIS (00/574) containing IFN-beta Ser 17 mutein. Indeed, variations in potency estimates for preparations containing IFN-beta Ser 17 mutein were sufficiently large to indicate that assays could distinguish preparations of IFN-beta Ser 17 mutein from preparations of glycosylated IFN-beta. Thus, neither the 2nd IS of IFN-beta, Gb23-902-531, containing fibroblast-derived IFN-beta, nor CIS, 00/572, containing CHO cell-derived IFN-beta, was appropriate for standardisation of preparations of IFN-beta Ser 17 mutein. Conversely, neither the IS of IFN-beta Ser 17 mutein, Gxb02-901-535, or a CIS of IFN-beta Ser 17 mutein, 00/574, was appropriate for the standardisation of preparations of glycosylated IFN-beta. CIS 00/572, containing CHO cell-derived, glycosylated IFN-beta, was clearly shown to be suitable to serve as a primary standard for glycosylated forms of IFN-beta, especially clinical grade IFN-beta-1a products. It was further shown to exhibit high thermal and long-term stability. Since the CHO cell-derived IFN-beta used for preparation of 00/572 was of a greater purity than the IFN-beta used for the 2nd IS of IFN-beta, Gb23-902-531, it was recommended by the WHO Informal Consultation on the Standardisation of Cytokines, Growth Factors and Other Endocrinological Substances, which met in October 2003, that 00/572 should replace Gb23-902-531 as the IS for glycosylated IFN-beta. This recommendation was accepted by the WHO Expert Committee on Biological Standardization (ECBS) at its annual meeting in November 2003 and 00/572 was established as the 3rd IS for human glycosylated IFN-beta with an assigned potency of 40,000 IU. As this study identified no advantage to replacing the existing 1st IS for IFN-beta Ser 17 mutein, Gxb02-901-535, WHO ECBS accepted that this should continue to serve as the IS for this material.

Establishment of new and replacement World Health Organization International Biological Standards for human interferon alpha and omega.

The complexity of the human interferon-alpha (IFN-alpha) family, with its multiple molecular forms and various biological activities, raises a number of scientific issues with regard to the biological standardisation of natural and recombinant IFN-alpha products. To address such issues and to achieve an appropriate biological standardisation of human interferon-alpha (IFN-alpha) preparations, the National Institute for Biological Standards and Control (NIBSC) of the United Kingdom (UK), in association with the Centre for Biologics Evaluation and Research (CBER) of the United States of America (USA), organised an international collaborative study, which was subsequently divided into two parts. Ninety-three participating laboratories from 29 countries worldwide participated in the first part of the study. They performed titrations on up to 15 different IFN-alpha preparations and one IFN-omega (omega) preparation in a variety of assays, including those based upon antiviral, antiproliferative, and other biological activities of IFN, and contributed raw data from these assays to NIBSC for analysis and calculation of relative activities. Analysis of data from this part of the study showed a greater than expected assay-dependent disparity between the relative activities of different IFN-alpha preparations. This disparity was found when only antiviral assays were considered and even when there were only small molecular dissimilarities between two otherwise closely related IFN-alpha preparations. The lack of assay independence and relative activity equivalence has indicated that a single biological potency standard for all IFN-alpha subtypes and mixtures would be inappropriate. Hence, individual, homologous standards, each with a separate unitage, were required for biological standardisation and potency determinations of individual IFN-alpha subtypes. At this stage, potency assignments to the IFN-alpha and -omega preparations included in the study were made as far as possible on the basis of comparison of antiviral activity with that of the 1st International Reference Preparation (IRP) for IFN, human leukocyte, 69/19. However, it was recognised that other standards had been used in assays to estimate potencies of widely available, current, therapeutic IFN-alpha products. Thus, to ensure the continuity of unitages already in use for IFN-alpha products, the second part of the study, which involved 12 members of the International Federation of Pharmaceutical Manufacturers Association (IFPMA), was carried out using for calibration of antiviral assays those IFN-alpha preparations that most closely matched manufacturers' products or that had been previously used for assay calibration by a manufacturer for a particular product. On the basis of data analysis from the second part of the study, potency assignments to the IFN-alpha preparations, as made in the first part of the study, were either left unchanged or changed to potency assignments that ensured as far as possible continuity with existing unitages. From among the IFN preparations evaluated, the following were recommended as the most suitable to continue or replace existing WHO international standards (IS) and have subsequently been formally established as WHO IS at the 51st meeting (October 1999) of the WHO ECBS: 83/514, 1st WHO IS for human IFN-alpha1 8000 international units (IU); 95/650, 2nd WHO IS for human IFN-alpha2a, 63,000 IU; 95/566, 2nd WHO IS for human IFN-alpha2b, 70,000 IU; 95/580, 1st WHO IS for human IFN-alpha2c, 40,000 IU; 95/572, 1st WHO IS for human IFN-alpha1/8, 27,000 IU; 94/786, 1st WHO IS for human IFN-alphaCon1, 100,000 IU; 94/784, 2nd WHO IS for human IFN-alpha (leukocyte), 11,000 IU; 95/574, 1st WHO IS for human IFN-alpha (leukocyte n3), 60,000 IU; 95/568, 2nd WHO IS for human IFN-alpha (lymphoblastoid n1), 38,000 IU; 94/754, 1st WHO IS for human IFN-omega, 20,000 IU. These WHO IS are available upon request to NIBSC.

Interferon standardization and designations.

A large number of different human and nonhuman interferon (IFN) preparations are now available for either research purposes or commercial use. Consistency of results can be achieved only through rigorous application of biologic standards and individual species designation. International standards for the potency determinations of these preparations have been produced in accordance with World Health Organization (WHO) guidelines and are available for calibrating assays. Until recently, potency has been assessed purely as a measure of antiviral activity expressed in international units. Other biologic properties are now also being considered, including antiproliferation and immunomodulation. Indirect methods of measuring IFN, such as radioimmunoassay or enzyme immunoassay, if fully validated, may also provide useful estimates of function. Reference antisera are useful for characterizing IFN preparations and for monitoring neutralization assays for detecting anti-IFN antibodies but should not have a function in assay calibration. Factors to be considered when referring to specific designations for pure IFN species include distinctions for the species of origin, any mutant or hybrid forms, the method of production, and the presence of additional glycosylation.

[Recombinant probiotics: problems and prospects of their use for medicine and veterinary practice]. / Recombinantnye probiotiki: problemy i perspektivy ispolzovaniia v meditsine i veterenarii.

Approaches to designing a new probiotic class based on recombinant strains of bacteria that produce the predetermined therapeutic proteins are dealt with. The prospects of the approach are shown via studies of the biological properties of Bacillus subtilis 2335 strain transformed by the plasmid encoding the synthesis of human interferon alpha-2. The recombinant strain was demonstrated to preserve the high antagonistic activity of the parent culture (the bases of the probiotic biosporine) and to acquire marked antiviral properties due to interferon synthesis. The antiviral activity of the designed strain was shown by in vitro and in vivo experiments on experimental viral infections. By using this strain, the authors designed the new probiotic subaline, a promising biological agent for medicine and veterinary practice. Subalin has a number of advantages: it combines antibacterial and antiviral properties, is easy to use and prepare.

Effective natural interferon-alpha therapy in recombinant interferon-alpha-resistant patients with hairy cell leukemia.

To explore the relationship between anti-interferon-alpha (anti-IFN-alpha) antibodies and loss of clinical responsiveness to IFN-alpha treatment, we examined sera from 59 patients with hairy cell leukemia who responded to therapy with recombinant IFN-alpha-2a (rIFN-alpha-2a). During the first 2 years of therapy, 10 patients developed rIFN-alpha-2a-neutralizing and 15 rIFN-alpha-2a-binding antibodies. Nine of the 59 initially responding patients became resistant to rIFN-alpha-2a and suffered a relapse of the disease at 7 to 24 months of treatment. All nine relapsing patients tested positive for both neutralizing and binding antibodies with titers above 400 INU/mL, while none of the antibody-negative patients relapsed. Six patients with detectable binding antibody titers below 400 INU/mL continued to respond to treatment. By measuring the IFN kinetics and the levels of the IFN-induced Mx-homologous protein in mononuclear cells after a single injection each of rIFN-alpha-2a and nIFN-alpha the IFN antibodies of eight of the nine resistant rIFN-alpha patients were found to be highly specific for rIFN-alpha-2a. Therefore, these eight patients were switched to natural IFN-alpha (nIFN-alpha) therapy at doses of 3 million IU, three times a week. All eight patients responded to treatment with nIFN-alpha, achieving durable objective responses similar to those obtained previously with rIFN-alpha-2a. These data clearly demonstrate that rIFN-alpha antibody-positive patients can effectively be treated with nIFN-alpha.

[The sensitizing properties of the ballast admixtures in human leukocyte interferon]. / Izuchenie sensibiliziruiushchikh svoistv ballastnykh primesei chelovecheskogo leikotsitarnogo interferona.

Due to the features of its production technology, human leukocyte interferon (HLI) for intranasal administration is likely to contain ovalbumin (OA). Commercial batches of HLI were tested for the presence of OA responsible for the sensitizing effect of HLI given intranasally. The content of OA in commercial batches of HLI was shown to vary widely and to exceed by far the concentrations having the sensitizing effect. As a result of these studies, the upper limit of OA content was established and the technology of HLI production was modified. These measures permitted production of commercial batches of HLI with OA content not exceeding 0.05 micrograms/ml.

Highly sensitive enzyme immunoassay of human interferon-beta 1.

A highly sensitive sandwich enzyme immunoassay for human interferon-beta 1 (HuIFN-beta 1) was developed. HuIFN-beta 1-containing samples and horseradish peroxidase (HRP)-labeled mouse anti-HuIFN-beta 1 monoclonal antibody (Fab') were incubated overnight at 2-10 degrees C in the wells of a 96-well microtiter plate, onto which affinity-purified rabbit anti-HuIFN-beta 1 polyclonal antibody was coated. The EIA was able to detect 0.5 IU/ml of HuIFN-beta 1, thus showing higher sensitivity than bioassay. The values obtained by the EIA closely paralleled those obtained by bioassay in the concentration which bioassay can detect. In order to detect the concentration below 0.5 IU/ml of HuIFN-beta 1, the avidin/biotin-amplified EIA was also developed. The use of biotinylated mouse anti-HuIFN-beta 1 monoclonal antibody (F(ab')2) and HRP-avidin in the EIA made it possible to detect 0.1 IU/ml of HuIFN-beta 1. These EIAs were applied for the studies such as process control of HuIFN-beta 1 production, pharmacokinetics of HuIFN-beta 1, and determination of serum level of HuIFN-beta 1 in healthy subjects.

Development of a new type of drug by using cell technology; preparation of human interferon-beta from human diploid fibroblast cultures.

Several strains of human fibroblast were identified as good producers of human interferon-beta (HuIFN-beta), among DIP-2 cell was one of the best. We have developed an improved microcarrier culture system for both the mass culture of such cells and the large scale HuIFN-beta production. A routine pilot plant, and successively a large plant, have been accomplished for preparation, purification and preclinical or clinical trials of HuIFN-beta. The purified and lyophilized HuIFN-beta was assayed for its safety for clinical use under the regulation of the National Institute of Health of Japan. Using this HuIFN- preparation, the clinical trials on various viral diseases and malignant tumors were started from the middle of 1979. More recently, the Ministry of Health and Welfare approved this HuIFN-beta as a new drug against melanoma, glioblastoma and chronic active B type hepatitis.

Development of interferon-specific monoclonal antibody for in vitro interferon assays.

In this study, monoclonal antibodies (MABs) to human interferon alpha (HuIFN alpha) were evaluated for their suitability for the quantification of different HuIFN alpha preparations by conventional immunoassay methods. The results obtained using four different MABs were compared and suggest that whilst immunoassays can be highly sensitive and reproducible to perform, calibration of antigen content in relation to units of biological activity remains a problem, particularly because of the molecular and biological heterogeneity of HuIFN alpha preparations. Nevertheless, the judicious selection of MABs specific for an individual HuIFN alpha subtype has indicated that immunoassays may be accurately calibrated in units of biological activity for potency estimations of that HuIFN alpha subtype.

Evaluation of absorption with Limulus amebocyte lysate to remove contaminating endotoxin from interferon and lymphokine preparations.

Alpha and beta human interferon (IFN) preparations and lymphokines (supernatants of PHA-stimulated blood lymphocytes) were deliberately contaminated with endotoxin (20 ng/ml) and subsequently rendered endotoxin-free by absorption with Limulus amebocyte lysate (LAL). Absorption with LAL did not appreciably affect the antiviral activity of IFN and lymphokines in 8 experiments and caused a 30-50% reduction in two. The capacity of these agents to stimulate natural killer cell activity and monocyte cytotoxicity was not consistently modified by absorption on LAL. When the chemotactic activity of lymphokine for monocytes was measured, the maximal number of monocytes induced to migrate and the maximal active lymphokine concentration were not affected by absorption with LAL. LAL-treated lymphokines, however, showed a prozone phenomenon, presumably related to the release of chemotaxis inhibitor(s) from the LAL gel.

Laboratory evaluation of commercial interferon preparations.

The antiviral, antiproliferative and natural killer-cell (NKC) stimulatory activities of four commercial therapeutic interferon preparations were assayed in our laboratory. The antiviral and antiproliferative activities of each preparation were relatively similar, but an unexpectedly high NKC stimulatory activity was found in one of them. In-house determination of antiviral activity and evaluation of the antiproliferative and NKC stimulation potential of interferon preparations are essential before rational clinical trials of this agent are carried out.