Effect of tranexamic acid incorporated in fibrin sealant clots on the cell behavior of neuronal and nonneuronal cells.

Fibrin sealants are commonly used for hemostasis following surgery on various types of tissues. Aprotinin, an effective fibrinolysis inhibitor, is one of the components in some fibrin sealant products currently available. Tranexamic acid (tAMCHA) is another fibrinolysis inhibitor and is used as an alternative to aprotinin. Recent studies on fibrin sealant products containing tAMCHA indicate that it may be responsible for various adverse reactions when used in neurological applications. To determine a possible mechanism for such adverse reactions, we examined the effect of tAMCHA on the behavior of neuronal and nonneuronal cells using in vitro assays. The data indicate that different concentrations of tAMCHA incorporated in fibrin clots had no effect on the initial cell adhesion of either proliferative cells (glial cells and fibroblasts) or nonproliferative cells (neuronal cells) to the fibrin clots. Moreover, a high concentration of tAMCHA (300-450 mM) incorporated in the fibrin clots increased glial and fibroblast proliferation on fibrin clots. However, because tAMCHA is known to leach out of the fibrin clots, we have also examined the effect of solubilized tAMCHA in a growth medium on cells seeded on matrix-coated surfaces. A high concentration (300-450 mM) of tAMCHA detached all cell types from matrix-coated dishes. Our model suggests that tAMCHA in fibrin clots has no adverse effect on cells bound to the fibrin clots; however, tAMCHA leaching out from the fibrin clots reduces adhesion of adjacent cells bound to their natural extracellular matrix. Thus, a high concentration of tAMCHA should not be used as a fibrinolysis inhibitor in fibrin sealant products, especially in neurosurgery.

In vitro stability of recombinant human factor VIII (Recombinate).

Factor VIII (FVIII) is currently administered in diverse settings and by a range of methods, and it is important that the stability of specific FVIII preparations be documented for these varying uses. This study of Recombinate recombinant human FVIII (rhFVIII) evaluated: (i) thermostability; (ii) photostability; (iii) stability during simulated continuous infusion; and (iv) stability after dilution. This evaluation was conducted over a range of initial rhFVIII potencies and under differing conditions of temperature, light exposure, dilution and heparin usage. FVIII biological activity was measured by one-stage and chromogenic substrate assays. Microbiological assessment was also performed. Lyophilized rhFVIII was found to be highly thermostable, as evidenced by an energy of activation (Ea) of 16.2 kcal mol-1 and recovery of 99.3% of initial activity after incubation for 6 months at 40 degrees C and 93.8% at 60 degrees C for 2 months. No significant loss of activity could be detected after accelerated simulated natural daylight exposure of lyophilized rhFVIII, although partial activity loss was observed after similar exposure of reconstituted rhFVIII. Shielding in foil wrap effectively prevented such photodegradation of reconstituted rhFVIII. Based upon these results, exposure of lyophilized rhFVIII to sunlight is unlikely to affect stability adversely. Activity of reconstituted rhFVIII (22-106 IU mL-1) remained stable during simulated continuous infusion for 96 h at ambient (20-25 degrees C) and elevated (28-32 degrees C) temperature, and in the presence or absence of 1 U mL-1 heparin. After dilution of reconstituted rhFVIII, an immediate 14-42% loss of expected rhFVIII activity was observed depending upon diluent composition. Accordingly, potential partial loss of rhFVIII activity should be taken into account when dilution is being considered. rhFVIII remained sterile at least 96 h during simulated continuous infusion. rhFVIII is a robust preparation exhibiting biological stability under a wide array of clinically relevant conditions.

Stability assessment of lyophilized intravenous immunoglobulin after reconstitution in glass containers and poly(vinyl chloride) bags.

Human intravenous immunoglobulin (IGIV) has been in use for the past 20 years. This biological product is commonly provided in liquid or lyophilized dosage form. When the lyophilized product is rehydrated, it is usually administered within 2-3 h from time of complete dissolution. While this practice is advisable whenever possible, occasionally the patient or care-giver may need to delay the infusion. Hence, a study of the stability of lyophilized IGIV after reconstitution with water for injection was conducted. The reconstituted product was stored either in its original glass container or pooled into poly(vinyl chloride) (PVC) bags. The effect of extended storage on the active ingredient (IgG), excipients (glucose, albumin) and extractables [sodium from glass vials, and di-(2-ethyl-hexyl) phthalate and cyclohexanone from PVC bags] was evaluated. The stability of the active ingredient was evaluated by physico-chemical tests (molecularsize distribution, pH, appearance, total protein), monitoring titres of a specific antibody (hepatitis B surface antigen) and an antibody functional test (bacterial opsonization). To evaluate the risk of microbial contamination during reconstitution and pooling procedures, sterility, pyrogen and animal-safety tests were included in the protocol. The potential of IgG polymerizing in solution during storage and subsequent complement activation was evaluated by assaying for non-specific binding of complement (anti-complement activity). Results show that aseptically reconstituted IGIV is stable and remains sterile up to 48 h at 5 degrees C. The reconstituted product was also found to be stable at room temperature (25 degrees C) up to 12 h.

Acid hydrolysis of monoclonal antibodies.

For analysis of monoclonal antibodies using polyacrylamide gel electrophoresis, two hydrolytic fragments derived from the heavy chain of mouse IgG1 were produced during incubation of the antibodies in Laemmli reducing sample buffer at 100 degrees C for 5 min. The cleavage sites were identified by amino terminal sequencing. Results indicate that the final pH of the mixture is critical for the production of the fragments which are generated when the pH is approximately 6.0. At pH 8.0, no fragments are detected. The relevance of this finding to those working with monoclonal antibodies is discussed.

Long-term use of IgA-depleted intravenous immunoglobulin in immunodeficient subjects with anti-IgA antibodies.

The use of intravenous immunoglobulin is standard practice for antibody replacement in the humoral immunodeficiency diseases. Most infusions proceed uneventfully, but a proportion of infusions (5-8%) produces some degree of an infusion reaction. While the cause of most of these infusion reactions is unknown, an established, but rare cause of reactions is IgA antibodies in the serum of the patient, which apparently forms an immune complex with the traces of IgA in the infused immunoglobulin. This article describes our studies of five immunodeficient patients who had high-titered anti-IgA antibodies and a history of severe infusion reactions to intravenous immunoglobulin products not depleted of IgA (IgA content, 270-720 micrograms/ml). Over a 6-year period we gave these patients IgA-depleted intravenous immunoglobulin for a total of 170 infusions. These infusions were generally well tolerated; however, mild to moderate infusion reactions did occur in 9 of the 170 infusions (5.3%). These reactions were not related to the IgA content of the immunoglobulin solutions used--ascertained to vary between 0.4 and 2.9 micrograms/ml of IgA. Levels of plasma C3a and C4a increased after immunoglobulin infusions but the appearance of these components was not accompanied by any infusion reaction. We conclude that the long-term infusions of IgA-depleted intravenous immunoglobulin, within the range of IgA concentrations investigated, into patients with even very high-titered antibodies to IgA, is a safe practice.

The pharmacokinetics of total IgG, IgG subclasses, and type specific antibodies in immunodeficient patients.

The advent of immunoglobulin concentrates suitable for intravenous administration has greatly improved the clinical management of patients with a primary immunodeficiency syndrome. However, proper treatment requires understanding of the pharmacokinetics of the infused IgG and its components. We review here the work that has been conducted in this area. In particular, two studies have shown that these concentrates have adequate catabolic properties with regards to total IgG, IgG subclasses, and specific antibodies. We conclude that careful evaluation of the pharmacokinetics of a given IgG preparation is necessary in order to determine an appropriate treatment regimen.

The half-lives of IgG subclasses and specific antibodies in patients with primary immunodeficiency who are receiving intravenously administered immunoglobulin.

With the increased use of immunoglobulin for intravenous use (IGIV) as replacement therapy for patients with primary immunodeficiencies, a natural concern is whether such preparations demonstrate a normal turnover rate with regard to total IgG, individual IgG subclasses, and specific antibody titers. We have conducted such a pharmacokinetic study on a cohort of eight patients with an IGIV preparation, Gammagard. For total IgG, the half-life found was 25.8 days; for IgG1 it was 29.7 days; for IgG2 it was 26.9 days; and for IgG3 it was 15.7 days. The results are similar to those reported for endogeneous IgG. Half-lives for antibodies to S. minnesota (Re 595 mutant), cytomegalovirus, and S. pneumoniae were of the same order of magnitude as that for total IgG. We conclude that this IGIV preparation is catabolized in patients with primary immunodeficiency at a rate similar to that of native IgG in normal individuals.