[Nanofiltration in production of Beriplex P/N: increasing the capacity of virus elimination while maintaining product quality]. / Nanofiltration bei der Herstellung von Beriplex P/N: Erhöhung der Kapazität zur Viruseliminierung unter Beibehaltung der Produktqualität.

For the manufacture of the PCC Beriplex P/N, nanofiltration was introduced into the production process of Beriplex HS providing an additional means to heat treatment for the clearance/inactivation of viruses. By nanofiltration, large enveloped viruses (HSV-1, HIV-1) were completely eliminated by a factor of more than 7 log10. While medium-sized enveloped viruses (HBV, BVDV) were cleared by a factor of approximately 4 log10, small non-enveloped viruses (poliovirus) were not removed. The product profile remained, no thrombogenic activities were detected.

Investigation of activated prothrombin complex concentrate as potential hirudin antidote in animal models.

The specific thrombin inhibitor r-hirudin (HBW 023) has been demonstrated to be effective in preventing thrombosis in preclinical models. Up to now, no bleeding complications have been observed using therapeutically effective doses in animals studies. However, in case of inadvertent overdosing the occurrence of undesired impairment of coagulation cannot be excluded. As a potential antidote an activated prothrombin complex concentrate (APC) was tested on its ability to normalize blood coagulation. APC given s bolus injections 5 min and 3.0 mg/kg neutralized the r-hirudin-induced prolongation and 3.0 mg/kg neutralized the r-hirudin-induced prolongation of whole blood coagulation time in rabbits completely within 5 min without any clot formation in the blood vessels or capillaries of the heart, kidneys, or lungs. Furthermore, bleeding time prolongation induced by bolus application of 3.0 and 30.0 mg/kg r-hirudin was significantly inhibited by APC within 5 min. These results suggest that administration of APC may be an effective way to reverse the effects of r-hirudin in the coagulation system in case of inadvertent overdosing of r-hirudin.

Functionally inactive S. aureus alpha-toxin containing a single amino acid substitution: potential usefulness as a vaccine.

Substitution of His-35 with arginine in staphylococcal alpha-toxin leads to loss of its membrane perturbating properties in vitro. In this study, we report that the inactive mutant toxin is also devoid of toxic properties when injected intravenously into rabbits. Whereas a single application of native toxin at a dose of 20 micrograms/kg proved uniformly lethal within 3 hours (n = 3), all animals receiving 20, 100 or 200 micrograms/kg mutant toxin (n = 3 for each group) remained in unaltered, healthy states over a 15 day period of observation. Furthermore, all animals receiving inactive toxin developed antibody titers on day 10-15. No dose dependency was noted in the tested range. Thus, the site-directed alpha-toxin mutant (His-35:Arg) is an excellent vaccine candidate.

Activity of coagulation and fibrinolysis parameters in animals.

Using diagnostics for the determination of clotting factors and fibrinolytic parameters in human plasma, samples from rat, guinea pig, rabbit, cat, dog, sheep, cattle, horse, pig, and monkey were analysed. The human system was employed even for standard curves and controls. Results obtained in this way are relative values in relation to pooled fresh human plasma of healthy donors which is defined to contain 100% of the norm or 1 unit of each factor per 1 ml. Under these conditions, marked differences between the human clotting system and those of different animal species appear. Thus, rabbits have an about 50 times higher activity of factor V than humans and guinea pigs only 6% of the factor VII activity of human plasma. Plasma levels of fibrinogen, alpha 2-antiplasmin and antithrombin III in plasma samples of the investigated animal species are in the range of human plasma. Differences in the plasma level of single factors as compared to human plasma are reflected by clotting times of the screening tests. For the determination of plasminogen, streptokinase was used as activator of the fibrinolytic system. Hence, the results obtained by this method merely reflect the activatability of the animal plasminogen in comparison to the human system, however, do not allow statements concerning the real plasminogen content of the plasma sample from the animal species. For pharmacological investigations a proper selection of the animal species is important to prevent wrong conclusions.

In vivo effects of intravascularly applied Escherichia coli hemolysin: dissociation between induction of granulocytopenia and lethality in monkeys.

The effects of intravascular application of endotoxin-depleted Escherichia coli hemolysin (HlyA) was studied in rabbits and monkeys. In rabbits, bolus application of HlyA calculated to effect final blood levels of approximately 2-3 HU/ml (200-300 ng/ml) caused an acute fall of polymorphonuclear blood leukocytes to less than 20% of starting levels within 5 min. Additionally, platelet counts dropped to approximately 30% of starting levels, whereas lymphocyte counts varied considerably and seldom fell to less than 50%. Nine out ten animals that received 2-4 HU/ml toxin died within 90 min post application. These animals presented with signs of acute respiratory failure and post mortem inspection of the internal organs revealed hemorrhagic pulmonary edema. Other internal organs appeared unaffected. Application of less than 1 HU/ml HlyA was never fatal (n = 9), and only transient leukopenia was noted. Monkeys presented with a remarkable and different response. Two animals were repeatedly given HlyA at high doses ranging from 3 to 10 HU/ml. Both animals developed selective granulocytopenia, but following a short, transient drop in blood pressure they showed no severe clinical signs of cardiovascular or pulmonary malfunction. Histological examinations revealed accumulation of polymorphonuclear granulocytes in both animals in liver, lung and spleen. Very high leukocyte elastase levels were measured in one animal over a period of 1.5 h. The present results demonstrate a remarkable tolerance of monkeys towards the leukocidal effects of E. coli hemolysin. Lethality in rabbits must be due to additional effects of the toxin, possibly on cells in the pulmonary vasculature. Neither pulmonary sequestration of granulocytes nor massive release of elastase from these cells is in itself sufficient to provoke pulmonary dysfunction in monkeys.

[In vitro and in vivo efficacy of a toxin-neutralizing human staphylococcal immunoglobulin]. / In vitro- und In vivo-Wirksamkeit eines Toxin neutralisierenden humanen Staphylokokken-Immunglobulins.

The pathogenic relevance of alpha-hemolysin of Staph. aureus in human infections is up to the present in discussion. Numerous therapeutic human trials to modify the outcome of a Staph. aureus infection by giving passively hyperimmune sera did not clarify the situation. First the work of Bhakdi22 has provided us with a rational approach for a systemic use of specific immunoglobulin preparations. A highly purified Staph. aureus alpha-hemolysin was carefully detoxified and injected into volunteers. From these sera were collected by plasmapheresis and a 5S immunoglobulin was prepared. In preclinical trials the antitoxin efficacy was evaluated. We examined in vitro cultures of human thrombocytes and monocytes, and of porcine pulmonary arterial endothelial cells. As in vivo models we used the intoxication of mice and monkeys and an intradermal test in rabbits. In all these models we were able to demonstrate the high antitoxic efficacy of a human anti-alpha-hemolysin-5S-immunoglobulin preparation.

Toxicological testing of monoclonal antibodies.

Toxicity studies with monoclonal antibodies for in vivo diagnostic or therapeutic administration must be designed case by case to take into account the intended use in humans and the type of structure (nude or coupled to immunotoxins or radionuclides). The design of experiments is influenced by the origin of the clone (murine or human with different problems of antigenicity) and the type of culture (ascites or fermentation with various kinds of possible contaminations). Therefore, routine animal tests must be supplemented by analytical procedures and assays of pharmacological quality control (pyrogenicity, abnormal toxicity). The antigenicity of these antibodies for experimental animals imposes restrictions concerning duration of experiments and extrapolation of the results to humans. Selection of appropriate species is difficult, they should have identical target cells if possible. Sometimes the choice is limited to non-human primates. Toxicological experiments should be based on pharmacokinetic studies. Formal tests on mutagenicity, teratogenicity and cancerogenicity are, in most cases, irrelevant. Antibody formation may make it impossible to carry out longer-term studies.

Human hyperimmune globulin protects against the cytotoxic action of staphylococcal alpha-toxin in vitro and in vivo.

Alpha-toxin, the major cytolysin of Staphylococcus aureus, preferentially attacks human platelets and cultured monocytes, thereby promoting coagulation and the release of interleukin-1 and tumor necrosis factor. Titers of naturally occurring antibodies in human blood are not high enough to substantially inhibit these pathological reactions. In the present study, F(ab')2 fragment preparations from hyperimmune globulin obtained from immunized volunteers were tested for their capacity to inhibit the cytotoxic action of alpha-toxin in vitro and in vivo. These antibody preparations exhibited neutralizing anti-alpha-toxin titers of 80 to 120 IU/ml, whereas titers in commercial immunoglobulin preparations were 1 to 4 IU/ml. In vitro, the presence of 2 to 4 mg of hyperimmune globulin per ml protected human platelets against the action of 1 to 2 micrograms of alpha-toxin per ml. Similarly, these antibodies fully protected human monocytes against the ATP-depleting and cytokine-liberating effects of 0.1 to 1 microgram of alpha-toxin per ml. Intravenous application of 0.5 mg (85 to 120 micrograms/kg of body weight) of alpha-toxin in cynomolgus monkeys elicited acute pathophysiological reactions which were heralded by a selective drop in blood platelet counts. Toxin doses of 1 to 2 mg (170 to 425 micrograms/kg) had a rapid lethal effect, the animals presenting with signs of cardiovascular collapse and pulmonary edema. Prior intravenous application of 4 ml of hyperimmune globulins per kg inhibited the systemic toxic and lethal effects of 1 mg (200 micrograms/kg) of alpha-toxin. In contrast, normal human immunoglobulins exhibited no substantial protective efficacy in vitro and only marginal effects in vivo. It is concluded that high-titered anti-alpha-toxin antibodies effectively protect against the cytotoxic actions of alpha-toxin.

Identification of immunotoxic effects of chemicals and assessment of their relevance to man.

Immunotoxicity is defined as the adverse effects of foreign substances (xenobiotics) on the immune system. Two types of effects are possible: immunosuppression (which may result in an increased susceptibility to infection or to the development of tumours) and immunopotentiation (which may manifest as an allergy or as autoimmunity). There is, as yet, little evidence that well controlled occupational exposure to industrial chemicals has led to clinically significant immunosuppression. In contrast, a number of industrial chemicals have been shown to cause immunopotentiation in exposed populations, producing occupational asthma and contact dermatitis and possibly autoimmunity. In experimental models, immunosuppression (usually assessed by in vivo or in vitro immune function tests) has been induced by a wide range of chemicals but there are a few reports of the immunosuppression leading directly to an increased susceptibility to infection or to the development of tumours. Predictive experimental models are available for type IV allergic reactions, but the identification of chemicals that have a potential to cause other types of allergy or autoimmune reactions requires further research and the development and validation of new animal models. It is considered that routine subacute and chronic toxicity studies should include a full gross and histopathological assessment of the lymphoid organs to more accurately detect the potential of a chemical to cause immunotoxicity. Should such studies indicate that a substance has affected the immune system directly, an assessment of overall immune competence and function tests may be necessary using dose levels below those which cause frank toxicity. However, precise interpretation of immune function tests in terms of their relevance to human health requires an improved understanding of the extent of the functional reserve of the immune system. A strategy for assessing immunotoxicity in exposed human populations demonstrates a need for reliable clinical assessment, accurate medical record-keeping, an environmental and biological monitoring for levels of contaminating chemicals and the judicious use of well-validated immune function tests.

Quality criteria for i.v. immunoglobulins: importance of tests and product properties.

Quality criteria for i.v. immunoglobulins (i.v. Igs) are critically discussed laying emphasis on spontaneous anticomplementary activity (aca) and size-dependent composition. Considering the percentage of fractions obtained by gel filtration (Ultrogel AcA 34), however, the monomeric IgG containing fraction contributed the major part of aca under the experimental conditions chosen. Subclass IgG3 seems to contribute considerably to aca. No correlation was found between aca and the percentage of fractions containing components larger in size than IgG dimers in various commercially available Igs. Moreover, the subclass distribution in different batches of individual products showed considerable variations. The amount of IgG4 is correlated with that of IgA in chemically unmodified products relatively poor in IgA (approx. less than or equal to 10 mg/5 g Ig), indicating that attempts to reduce IgA consequently result in removal of IgG4.

Quality criteria for i.v.-immunoglobulins: importance of tests and product properties.

Quality criteria for i.v.-immunoglobulins (i.v.- Igs) are critically discussed laying emphasis on spontaneous anticomplementary activity (aca) and size-dependent composition. Considering the percentage of fractions obtained by gel filtration (Ultrogel AcA 34), however, the monomeric IgG containing fraction contributed the major part of aca under the experimental conditions chosen. Subclass IgG3 seems to contribute considerably to aca. No correlation was found between aca and the percentage of fractions containing components larger in size than IgG dimers in various commercially available Igs. Moreover, the subclass distribution in different batches of individual products showed considerable variations. The amount of IgG4 is correlated with that of IgA in chemically unmodified products relatively poor in IgA (approx. less than or or equal to 10 mg/5 g Ig), indicating that attempts to reduce IgA consequently result in removal of IgG4.

Assay of possible formation of antigenic components in heat-treated plasma protein preparations.

To eliminate the risk of hepatitis infections, human plasma protein preparations can be heated in solution to 60 degrees C for 10 h thus inactivating several viruses. Preclinical safety experiments were performed in order to exclude the possibility of the formation of antigenic components, not present in normal human plasma, through this pasteurization step. Rabbits were immunized with either the heated or the unheated product. Sera were investigated in the Ouchterlony test against the homologous antigen to demonstrate precipitating antibodies. Antisera of rabbits immunized with the heated product were adsorbed with the preparation without the heating step. After centrifugation, the incubates were retested in the Ouchterlony assay. In passive cutaneous anaphylaxis assays guinea-pigs received the adsorbed antisera i.v., and both preparations were injected intracutaneously. The diameters of skin reactions were measured and did not show differences between those treated with the heated and non-heated product. No neoantigens could be detected with the following heated plasma protein fractions: prothrombin, coagulation factors VIII, IX, X, XIII, antithrombin III, fibrinogen, and Cl-inactivator. These results did not apply to heat-denaturated rabbit serum in a validation experiment.

Influence of antithrombin III on carbon tetrachloride intoxication of rabbits.

Intraperitoneal application of carbon tetrachloride into rabbits caused high mortality due to liver damage with increase of serum transaminases, coagulation disorders, and decrease of antithrombin III (AT III) blood levels. Groups of animals were treated i.v. either with heparin (200 NIH-U/kg), AT III from human plasma (25 or 50 U/kg), heparin plus AT III (200 NIH-U +25 U/kg), or isotonic saline as control substance 6 hours after intoxication and 2 times daily during the following 5 days (10 applications). The therapy with saline or heparin was ineffective. AT III 25 U/kg and in combination with heparin showed marginal effects, 50 U/kg AT III resulted in increased survival rate and time, improvement of pathological changes of coagulation parameters and of blood urea nitrogen and serum creatinine levels. These experimental results confirmed clinical effects of AT III in acute hepatic intoxications with coagulopathy in humans.

[Effect of antithrombin III on experimental hepatotoxin poisoning in dogs]. / Wirkung von Antithrombin III auf experimentelle Intoxikationen mit Hepatotoxinen bei Hunden.

Carbon tetrachloride (CCl4) produces hepatic necrosis and Galactosamine (GALN) causes acute hepatocellular injury in dogs. 8 Beagle dogs were treated orally twice with 0.4 ml/kg CCl4 and 12 Beagle dogs with 200 mg/kg GALN i.v. After intoxication, groups of dogs were given antithrombin III (AT III) (Kybernin) from human plasma (25-100 U/kg i.v., days 1-3). Serum enzymes (GOT, GPT) were elevated, alkaline phosphatase values and serum bilirubin were increased in all animals. Dogs developed severe coagulation disorders reflecting intravascular coagulation and depressed levels of factors produced by the liver, such as AT III or fibrinogen. First toxic symptoms were seen after 48 h. Untreated dogs died between 48 and 72 h after GALN. AT III reduced the toxic effects on the coagulation system dose-dependently (minimal effective dose 3 X 50 U/kg). Decrease of fibrinogen and of platelet count were less pronounced, coagulation tests (Quick, TEG) less altered than in untreated dogs. Death rate was reduced. In CCl4 intoxicated animals also serum enzyme levels normalized after AT III. In GALN treated animals serum glucose levels were decreased in control dogs. These experimental results confirmed clinical effects of AT III in acute hepatic intoxications of humans.

[Studies of a fibrin adhesive on punch wounds in rats]. / Untersuchungen mit einem Fibrin-Kleber an Rattenhaut-Stanzwunden.

The efficacy of fibrin adhesive in routine quality control was examined in experimentally induced skin wounds of rats with fixation of punched-out skin pieces. In this rat model the influence of factor XIII, different fibrinogen concentrations, the stability of the dissolved lyophilized components, variation of adhesion time, and of CIG (cold insoluble globulin) was investigated. The adhesive strength was improved by factor XIII (optimum 60 U/ml). Concentrations of fibrinogen lower than 40 mg/ml resulted in insufficient adhesion effects. The stability of the dissolved components could be demonstrated up to 24 h after reconstitution. The adhesive strength improved with increasing time, it should be at least 15 min. No negative influence on tear-off weights were seen after 24 h caused by fibrinolysis. In a modification of the experimental design without fixation of the punched out skin pieces the adhesive was applied once or repeated times on the open wounds. No alteration or support of the normal wound healing was observed.

A new inactivated tissue culture rabies vaccine for use in man. Evaluation of PCEC-vaccine by laboratory tests.

A new inactivated rabies vaccine (purified chick embryo cell vaccine) has been developed using the Flury LEP-C 25 strain of rabies virus propagated in primary chick embryo cell cultures. The antigen was purified and concentrated by continuous density gradient centrifugation and inactivated by betapropiolactone. This vaccine was tested for innocuity, tolerability and protective capacity in a series of laboratory tests and compared with human diploid cell strain (HDC)-vaccines of similar antigenicity. The results indicated that this new vaccine was excellently tolerated and that its protective activity met the high standard of HDC-vaccine, conditions which were imposed on this vaccine before entering clinical trials in man.

Toxicity studies with human fibroblast interferon.

Acute toxicity studies of interferon derived from human fibroblasts (FHIF) were performed in mice and rats with doses between 6 X 10(4) and 480 X 10(4) IU/kg i.v. and an observation period of 14 days. No severe toxic effects, such as loss of weight or other pathological clinical signs were observed. In local innocuity studies (i.v., i.a., paravenous) in rabbits FHIF proved to have no adverse effects. 180,000 IU/kg were pyrogen free in rabbits, in the limulus test the content of lipopolysaccharides (LPS) was 208 pg/ml as compared to the FDA standard EC-2. Studies were performed to assess the pharmacological safety of FHIF in 10 monkeys (Macaca fascicularis). The i.v. injection of 60,000 IU/kg had no pathological effects on circulation, respiration, number of blood cells, or body temperature. In six monkeys a short decrease of arterial blood pressure was noted a few minutes after injection of 120,000 IU/kg i.v. Both doses caused an increase of thrombelastographic time (TEG). The proposed single human dose of approximately 60,000 IU/kg has been proved safe in the acute toxicity studies and the pharmacological safety assessment in mice, rats, rabbits, and monkeys. The LPS content is low.