A randomized, intraindividual, non-inferiority, Phase III study comparing daylight photodynamic therapy with BF-200 ALA gel and MAL cream for the treatment of actinic keratosis.

BACKGROUND: The most effective treatment modality for actinic keratosis (AK) is photodynamic therapy (PDT). Major obstacles of PDT are the need of a special illumination device and pain accompanying the illumination. These issues may be overcome by replacing an artificial high-power light source with natural daylight for more extended illumination at lower light doses. OBJECTIVE: To determine whether BF-200 ALA (a nanoemulsion gel containing 7.8% 5-aminolaevulinic acid) is non-inferior to MAL (a cream containing 16% methyl-aminolaevulinate) in the treatment of mild-to-moderate AK with daylight PDT (dPDT). Non-inferiority of the primary efficacy variable (total lesion clearance rate per patient's side 12 weeks after PDT) is established if the mean response for BF-200 ALA is no worse than for MAL, within a statistical margin of Δ = -12.5%. METHODS: The study was performed as an intraindividual comparison with 52 patients in seven centres in Germany and Spain. Each patient received one dPDT. Results include clinical endpoints as well as 1-year follow-up results. RESULTS: Twelve weeks after a single dPDT, 79.8% of the AK lesions treated with BF-200 ALA gel and 76.5% of the lesions treated with MAL cream were completely cleared. The median of differences was 0.0 with a one-sided 97.5% CI of 0.0, establishing non-inferiority (P < 0.0001). Results for secondary efficacy parameters were in line with the primary outcome. Recurrence rates 1 year after the treatment were 19.9% for lesions treated with BF-200 ALA and 31.6% for lesions treated with MAL. Adverse reactions including pain were mostly mild and transient and identical to those previously described for dPDT. CONCLUSION: Daylight PDT of AK with BF-200 ALA is well-tolerated and non-inferior to MAL/dPDT. The study demonstrates a trend towards higher efficacies after 3 months and significantly lower recurrence rates after 1 year follow-up.

[Testing of medicinal products produced from pooled plasma]. / Prüfung von aus gepooltem Plasma hergestellten Produkten.

Medicinal products produced from human plasma fall under the administrative batch release procedure of the competent authority. In Germany, this has been carried out since 1995 by the Paul Ehrlich Institute (PEI), the responsible state control agency for blood products. Medicinal products released for the European and national market are tested for quality, efficacy and safety. Experimental testing of the final product and the starting materials, the plasma pools, as well as control of the production documentation guarantee a constantly high product safety. In the 28,000 batches tested since the beginning of the state controlled batch release testing of these blood products at the PEI, there has been no transmission of infectious viruses (HIV, HBV and HCV) to any patient. The batch release has made a contribution to the improvement of product quality. This procedure is still an important tool to ensure safety of blood products. The PEI is integrated in the batch release network of the European Directorate for the Quality of Medicines & Health Care (EDQM) in Strasbourg. Regulations and guidelines for official control authority batch release (OCABR) ensure harmonized procedures for mutual recognition of batch release on the European level. The EU certificates and German national certificates are requested and accepted in over 70 countries worldwide. Experimental testing in the EU and the requisite certificates have developed into a seal of quality for the world market.

Thromboembolic events associated with immunoglobulin treatment.

OBJECTIVE: Due to an increasing number of reported thromboembolic events (TEE) after the administration of one intravenous immunoglobulin (IVIG) and one subcutaneous immunoglobulin (SCIG), pharmacovigilance and laboratory data were collected to analyse the root cause and assess the reporting frequency of TEEs for various IG products. METHODS: Paul-Ehrlich-Institut retrospectively analysed 228 reports of TEEs associated with six different IG products and estimated annual TEE-reporting rates based on worldwide sale figures over a period of 6 years (2006-2011). In addition, non-activated partial thromboplastin time (NAPTT) testing was performed to capture pro-coagulant potential of six IG products (four IVIG and two SCIG). RESULTS: For three IVIGs, the drug-related TEE-reporting rates remained stable from 2006 to 2011 (0-0·83 cases per 1000 kg IVIG distributed). In contrast, the TEE rate of one IVIG increased significantly from 0·33 cases in 2006 to nearly nine cases in 2010 (P < 0·001). The NAPTT testing of IG products with a low TEE rate revealed a NAPTT time >200 s and a NAPTT ratio >0·8, whereas TEE-associated batches of IG products with an increased TEE rate had a NAPTT ratio <0·8. After modifications of manufacturing processes, a normalization of NAPTT results and a decrease in TEE rates could be demonstrated.

Factor VIII Bypassing Activity (FEIBA) assays: standardization and development of the 1st NIBSC Working Standard for FEIBA--results from a collaborative study.

Factor-Eight-Inhibitor-Bypassing-Activity (FEIBA) is a bypassing-agent used to control spontaneous bleeding or cover surgical interventions in Haemophiliacs who develop neutralizing antibodies against FVIII/FIX. The market lot-release of FEIBA is dependent on specific clot-based assays, carried out by both the manufacturer and regulatory authorities, relative to manufacturer's in-house standards, which are produced on a small-scale and are replaced frequently. We sought to standardize the FEIBA assay by developing a FEIBA primary standard which would be internationally available in sufficiently large quantities, with a predicted lifetime of many years. A collaborative study involving the manufacturer and three regulatory authorities, was carried out in which a candidate material, sample B (06/172), was calibrated by assays relative to the manufacturer's in-house FEIBA standards (C and D). All laboratories used their routine validated methods (16 APTT-assays, 8 ACTIN-FS-assays and 27 DAPTTIN-assays). Intra-laboratory geometric coefficients of variation (GCVs) for candidate B ranged from 3% to 29% (GCVs <9% from majority of labs). Assessment of inter-laboratory variability gave overall GCV values of 6.9% and 4.4% relative to standards C and D, respectively, for all methods. There was good agreement in potency estimation between laboratories using each of the three methods, with the overall potencies by the three methods differing by less than 10% of the overall mean, giving an overall combined potency of 28.0 units per ampoule. All participants agreed that candidate B (06/172) be established as the 1st NIBSC Working Standard for FEIBA with an assigned potency of 28.0 units per ampoule, based on combined results for both methods, relative to either standard C or D.

Identification of kallikrein and FXIa as impurities in therapeutic immunoglobulins: implications for the safety and control of intravenous blood products.

BACKGROUND AND OBJECTIVES: The occurrence of thromboembolic events (TEEs) with intravenous immunoglobulin lots (IVIGs) raised the question of the causative agent for these adverse events. We investigated the predominant plasma proteases in 19 IVIG lots from five manufacturers including three lots associated with adverse events. MATERIAL AND METHODS: The inhibitor profile of the amidolytic activity in IVIG lots was investigated with substrates S-2302 and S-2288. In immunocapture assays, prekallikrein and FXI antigen and respective active proteases were quantified. Non-activated partial thromboplastin time (NAPTT) and a modified FXIa PTT served as global and FXIa-specific clotting assays, respectively. RESULTS: Kallikrein was identified as one major contaminant activity in IVIGs. A second activity was seen in some IVIGs with substrate S-2288, but not with S-2302. Inhibition studies excluded FXIIa, thrombin or plasmin as contaminant activity. FXI antigen was seen in all 19 IVIG lots, and FXIa activity was found as second major impurity in some IVIGs, including all lots involved in TEEs. FXIa highly correlated with a short clotting time in NAPTT. CONCLUSIONS: Kallikrein and FXIa are the major contaminants in IVIGs. FXIa was highly procoagulant, with highest level in TEE-associated IVIGs. Since the NAPTT unambiguously identified FXIa procoagulant activity in IVIGs, its implementation as a release test would improve the safety of IVIGs.

Calibration of human coagulation factor VII concentrate Ph. Eur. BRP batch 2.

The potency assay of human coagulation factor VII concentrate preparations as described in the European Pharmacopoeia (Ph. Eur.) requires a reference preparation calibrated in International Units (IU). The current Ph. Eur. Biological Reference Preparation (BRP) batch 1 was established in 2005 during an international collaborative study. It has an assigned potency of 8.2 IU/vial for the chromogenic assay method. Stocks of this BRP are dwindling and a replacement batch needs to be established. A candidate material was produced by a manufacturer from a plasma-derived concentrate preparation, with the same formulation and approximately the same potency, in the interest of continuity. The candidate material fulfilled the requirements of a BRP with regard to precision and homogeneity of fill, residual water content and stability. The potency of the candidate BRP (cBRP) was determined using chromogenic assays as required by the Ph. Eur. and in-house clotting assays in an attempt to assign a potency for both methods, as is the case for the current batch. The statistical model used for most laboratories was the maximum likelihood of the parallel line model using a logarithmic transformation of the responses. In the chromogenic assay, a potency of 9.9 IU/vial (+/- 1.8 %) was obtained for the cBRP with a very good consistency between laboratories. The results from the clotting assay, however, were less homogenous and yielded consistently higher results (13 IU/vial +/- 12 %), probably due to a higher activated factor VII (FVIIa) content than in the current BRP (3 % as compared to 0.3 %). Due to the large difference between the values obtained with the 2 different methods, it was not possible to reconcile the outcomes with each other. On the other hand, the uncertainty observed with the clotting assay method was quite large and seemed questionable for a reference preparation. Therefore the use of BRP batch 2 as a reference for the clotting assay method is not recommended. Nevertheless, the results of the study showed that the candidate BRP (cBRP) is suitable as a reference standard for the chromogenic assay according to the Ph. Eur. general chapter 2.7.10 Assay of human coagulation factor VII. It was adopted by the Ph. Eur. Commission in December 2009 as an official Ph. Eur. BRP for human coagulation factor VII concentrate with an assigned potency of 9.9 IU/vial.

Are quality differences responsible for different adverse reactions reported for SD-plasma from USA and Europe?

Thromboembolic adverse reactions reported after transfusion of SD-plasma in the United States (US) prompted us to perform a comparative study with SD-plasma from the US and the European (EU) market. In SD-plasma from US, residual tri-N-butyl phosphate was found, and citrate concentrations were lower than in EU-plasma. Except for substantial losses of FV, FVIII and antiplasmin found for all SD-plasmas, clotting factor activities were mainly retained. However, for SD-plasma from US, markedly elevated concentrations of lipoprotein (a) [Lp(a)], fibrin monomer and a particularly high degree of complement activation (C3a des-Arg) were observed. Furthermore, pronounced differences were found for protein S. Although SD-plasma pools from US contained nearly normal concentrations of free and bound protein S antigen, protein S activities were almost completely absent. In contrast to this, SD-plasma from EU showed a moderate loss of both protein S activity and free antigen. Antitrypsin inhibitor activities were much more diminished in SD-plasma from US than from EU. In view of a possible thrombogenicity of SD-plasma from US, the loss of protein S and elevated Lp(a) concentrations could be of significance. The very high levels of C3a des-Arg in US plasma could possibly have an additional effect, through priming platelet activation after transfusion.

Establishment of the human coagulation factor VII concentrate European Pharmacopoeia biological reference preparation batch 1.

For the potency assay of human coagulation factor VII concentrate preparations according to the European Pharmacopoeia (Ph. Eur.) a reference preparation calibrated in International Units (IU) is needed. Currently, the 1st International Standard (97/592, potency: 6.3 IU/ampoule) but no Ph. Eur. reference preparation is available. A collaborative study was run to calibrate a candidate Ph. Eur. Biological Reference Preparation (BRP) for human coagulation factor VII concentrate against the 1st International Standard; the BRP is intended to be used as working standard. A candidate BRP batch 1 was produced from a plasma-derived human factor VII concentrate preparation available on the European market. It fulfilled the requirements of a BRP with regard to precision and homogeneity of fill, residual water content and stability. In addition, the content of activated factor VII was low. Sixteen laboratories from 9 countries participated in the collaborative study. The potency of the candidate BRP was determined using the participants' chromogenic assay based on the Ph. Eur. and their in-house clotting assay, if available. The statistical model used for analysis of the results from most laboratories was the maximum likelihood of the parallel line model following a logarithmic transformation of the responses. In the chromogenic assay, a potency estimate of 8.2 IU/vial (+/-3.7%) was obtained for the candidate BRP. Results from the clotting assay were lower and less homogenous (6.7 IU/vial+/-11.6%). The results from the collaborative study showed that the candidate BRP is suitable as a reference standard for the chromogenic assay according to the Ph. Eur. It was adopted by the Ph. Eur. Commission in March 2006 as official Ph. Eur. BRP for this purpose.

Factor VIII test in reference preparations: compensation for different dilutions.

An approach is described that aims to prevent the problems associated with factor VIII (FVIII) assays. This approach takes a number of factors into consideration: 2 different assay types are currently being performed (clotting and chromogenic), different standards coexist (for plasma and for concentrates) and not only are reference standards being diluted for test purposes, but likewise plasmas are diluted by adding reference standards which are significantly less concentrated than commercial concentrates. Moreover, artificially FVIII-depleted plasmas are diluted as compared to normal or haemophilic plasma with respect to non-FVIII proteins as well. Taken together, these observations have led us to re-examine FVIII assays and to establish an algorithm for FVIII measurements with plasma as the natural reference.

Activation of proPHBSP, the zymogen of a plasma hyaluronan binding serine protease, by an intermolecular autocatalytic mechanism.

The hyaluronic acid binding serine protease (PHBSP), an enzyme with the ability to activate the coagulation factor FVII and the plasminogen activator precursors and to inactivate factor VIII and factor V, could be isolated from human plasma in the presence of 6M urea as a single-chain zymogen, whereas under native conditions only its activated two-chain form was obtained. The total yield of proenzyme (proPHBSP) was 5-6 mg/l, corresponding to a concentration of at least 80-100nM in plasma. Upon removal of urea, even in the absence of charged surfaces a rapid development of amidolytic activity was observed that correlated with the appearance of the two-chain enzyme. The highest activation rate was observed at pH 6. ProPHBSP processing was concentration-dependent following a second order kinetic and was accelerated by catalytic amounts of active PHBSP, indicating an intermolecular autocatalytic activation. Charged macromolecules like poly-L-lysine, heparin, and dextran sulfate strongly accelerated the autoactivation, suggesting that in vivo proPHBSP activation might be a surface-bound process. The intrinsic activity of the proenzyme was determined to be 0.25-0.3%, most likely due to traces of PHBSP. The presence of physiological concentrations of known plasma inhibitors of PHBSP, like alpha2 antiplasmin and C1 esterase inhibitor, but not antithrombin III/heparin, slowed down zymogen processing. Our in vitro data suggest that the autoactivation of proPHBSP during plasma fractionation is induced by the removal of inhibitors of PHBSP and is accelerated by charged surfaces of the chromatographic resins.

Detection of a novel plasma serine protease during purification of vitamin K-dependent coagulation factors.

A novel serine protease (PHBSP) was purified from human plasma by two chromatographic steps with a final yield of 1.6 mg/l plasma. The protease consists of two disulfide-bridged chains of about 50 and 30 kDa with the light chain containing the active site of the enzyme. NH2-terminal sequence analysis revealed identity to the deduced amino acid sequence of HGFA-like mRNA. The activity of PHBSP is strongly dependent on Ca2+ ions and is efficiently inhibited by alpha2-antiplasmin and aprotinin. Possible functions of PHBSP in the hemostatic system are discussed.