Lothar Biesert

Manufacturing of a Prothrombin Complex Concentrate Aiming at Low Thrombogenicity

The paper describes the production of a prothrombin complex concentrate (PCC) with high virus safety and a well-balanced content of vitamin K-dependent clotting factors and inhibitors. Solid-phase extraction is followed in a second step by optimized anion exchange chromatography using a radial column. A step for virus removal by nanofiltration is introduced in addition to the solvent/detergent step. By speeding up the chromatographic step, the period of time required for production is reduced considerably. The activities of the four vitamin K-dependent clotting factors II, VII, IX and X are in ratios of about 1:1:1:1. Protein C, Protein S, and Protein Z are also present in therapeutically effective concentrations. The product shows no thrombogenicity, in either in vivo nor in vitro models. Clinical investigations show that the PCC is a safe and efficient preparation for the substitutive treatment of FIX or FVII in patients suffering from the respective deficiencies. All bleeding episodes have been efficiently controlled with relatively low doses of the concentrate. The surgical procedures have been conducted without any problems in severely FIX and FVIII deficient patients.

Markers for HIV-disease progression in untreated patients and patients receiving AZT : evaluation of viral activity, AZT resistance, serum cholesterol, β2-microglobulin, CD4 + cell counts, and HIV antigen

SummaryIn order to find parameters which allow the assessment of the clinical state of HIV patients with or without antiviral therapy, viral cultures on lymphocytes and monocytes/macrophages, CD4-cell counts, HIV antigen, β2-microglobulin and serum cholesterol were evaluated for their predictive value. As had been shown previously for lymphocytes, the efficiency of viral isolation on macrophages also depends on the disease stage (CDC) of the patients and thus has a high predictive value. A multivariant discriminant analysis showed that the combination of β2-microglobulin, viral antigen, CD4+ cell count and HDL cholesterol predicted the outcome of viral cultures with 80% accuracy. While viral antigen, CD4+ cell counts and β2-microglobulin had been known, HDL cholesterol deserves further evaluation as prognostic parameter. The analysis of HIV derived from patients with AZT showed a 20–200-foldin vitro drug resistance after seven to 24 months of therapy. DNA sequence determination of such strains isolated from AZT patients over time showed only two of the amino acid exchanges described in the literature for resistant strains and an additional Val60-Ile transition after 32 months of therapy.ZusammenfassungUm Parameter zu finden, die es erlauben, den klinischen Status HIV-Infizierter exakt zu bestimmen, wurden die Virusanzucht auf Lymphozyten und Makrophagen, CD4+-Zellzahlen, Cholesterin-Werte, β2-Mikroglobulin-Spiegel und virales Antigen hinsichtlich ihres prädiktiven Wertes verglichen. Wie bereits für die Virusanzucht auf Lymphozyten beschrieben, war auch die HIV-Kultur auf Makrophagen vom Gesundheitszustand der Patienten (bestimmt nach CDC) abhängig und hat somit hohen prädiktiven Wert. Eine multivariante Diskriminanzanalyse zeigte, daß das Ergebnis der Viruskultur unabhängig vom Zellsystem durch eine Kombination von β2-Mikroglobulin, Virusantigen, CD4+-Zellzahlen und HDL-Cholesterin-Werten zu 80% voraussagbar ist. HDL-Cholesterin verdient daher, als neuer prognostischer Parameter weiter erforscht zu werden. Die Analyse von HIV-Isolaten aus AZT-behandelten Patienten zeigte eine 20–200- fachein vitro Resistenz gegenüber der Substanz nach sieben bis 24 Monaten der Therapie. Sequenzbestimmungen bei einem der 100fach resistenten Isolate ergaben nur zwei der in der Literatur beschriebenen Aminosäure-Austrausche und eine neue Mutation (Val60 zu Ile) in einem Virus nach 32 Monaten der Therapie.

Prion safety of transfusion plasma and plasma-derivatives typically used for prophylactic treatment

Reports about transfusion-related transmissions of variant Creutzfeldt-Jakob disease have urged the need for more information regarding the risk for prion contaminated units in the blood supply and the safety of transfusion plasma and biopharmaceuticals derived from this precious raw material. According to a possible epidemiological model, the risk in many European countries is the same or lower than that of human immunodeficiency virus. Comprehensive investigations have shown that the prion safety margin of both single-donor and pooled solvent/detergent treated transfusion plasma is high. Furthermore, prophylactic treatment using plasma-derivatives poses a very low risk in terms of prion disease despite extensive lifetime exposure.

Viral safety of a new highly purified factor VIII (OCTATE)

The inactivation of both transfusion‐relevant and model viruses by modified pasteurisation (10 hours at 63°C in solution) has been evaluated following the established guidelines of the EU CPMP Ad Hoc Working Party on Biotechnology/Pharmacy. This heat treatment was introduced into the manufacturing process of OCTAVI, a very high purity factor VIII concentrate stabilised only by von Willebrand factor, in the presence of a proprietary mixture of low molecular weight stabilisers. Both enveloped (human immunodeficiency virus, Sindbis virus, herpes simplex virus, pseudorabies virus) and nonenveloped viruses (poliovirus, Coxsackievirus, hepatitis A virus) were inactivated by this heating step by more than 4.7 log10. The combination of the solvent/detergent step used in the manufacture of OCTAVI with this modified pasteurisation leads to a double virus‐inactivated factor VIII concentrate (OCTATE) with a viral safety distinctly superior to monoinactivated products.

Anti-human immunodeficiency virus (HIV) drug HOE/BAY 946 increases membrane hydrophobicity of human lymphocytes and specifically suppresses HIV-protein synthesis

The polysulfated polyxylan HOE/BAY 946, which has been tested in two pilot studies in ARC/AIDS patients and in asymptomatic HIV carriers in Germany, was believed to act by inhibiting virus attachment to the cell. However, the drug was also found to reduce the amount of HIV particles released from infected peripheral blood mononuclear cells (PBMC) in vitro. Furthermore, preincubation of PBMC with the drug led to a partial inhibition of a following HIV infection, suggesting that the drug also affects virus entry. Electron Paramagnetic Resonance (EPR) measurements on uninfected human lymphocytes using 5-proxyl-nonane as spin label demonstrated smaller hyperfine coupling constant (aN) values in the presence of HOE/BAY 946 or dextran sulfate 5000. Accordingly, h-1P/h-1H ratios were decreased, indicating increased plasma membrane hydrophobicity and a membrane-stabilizing effect of the drugs. Culture of the chronically HIV-infected monocytic cell line U937/HIV-2D194 in the presence of HOE/BAY 946 specifically and drastically reduced the release of virions and the intracellular synthesis of viral proteins as determined by radioimmunoprecipitation and reverse transcriptase assays. In conclusion, although the EPR studies showed a physico-chemical effect on membrane polarity, HOE/BAY 946 and dextran sulfate clearly affect processes beyond the cell membrane. Thus, in contrast to previous reports suggesting that polysulfated sugars affect HIV only by inhibiting virus binding to uninfected cells, they clearly inhibit HIV in infected cells as well and appear to have a pleiotropic mode of action. Such drugs may be less likely to result in viral resistance after prolonged application than substances acting only on one step in the life cycle of the virus.