Sterilization of heparinized Cuprophan hemodialysis membranes

The effects of sterilization of dry heparinized Cuprophan hemodialysis membranes by means of ethylene oxide (EtO) exposure, gamma irradiation, or steam on the anticoagulant activity and chemical characteristics of immobilized heparin and the permeability of the membrane were investigated. Sterilization did not result in a release of heparin or heparin fragments from heparinized Cuprophan. Sterilization of heparinized Cuprophan by means of EtO exposure and gamma irradiation induced a slight, insignificant decrease of the anticoagulant activity. In contrast, steam-sterilized heparinized Cuprophan showed a higher anticoagulant activity than unsterilized heparinized Cuprophan, which was most likely caused by cleavage of some of the covalent bonds between heparin and Cupropha. The effects of sterilization on the permeability of unmodified Cuprophan and heparinized Cuprophan were compared. The permeability of unmodified Cuprophan for vitamin B12 (Vit B12) and sulfobromophthalein (SBP) was reduced by 20–35% after EtO exposure and gamma irradiation and was reduced by 90–95% after steam sterilization. The water permeability of unmodified Cuprophan remained the same after EtO exposure and gamma irradiation but also dramatically reduced after steam sterilization. These reductions were ascribed to the collapse of pores of the membrane. The permeability of heparinized Cuprophan was not affected by EtO exposure and gamma irradiation but dramatically reduced after steam sterilization, although to a lesser extent than in the case of unmodified Cuprophan. Apparently, the presence of immobilized heparin (partially) prevented the collapse of pores during sterilization. Gamma irradiation was recommended as the preferred method of sterilization for heparinized Cuprophan.\u

Design of a new type of coating for the controlled release of heparin

Thrombus formation at the surface of blood contacting devices can be prevented by local release of heparin. Preferably, the release rate should be constant for prolonged periods of time. The minimum heparin release rate to achieve thromboresistance will be different for various applications and should therefore be adjustable. In this study a new type of heparin release system is presented which may be applied as a coating for blood contacting devices. The system is based on the covalent immobilization of heparin onto porous structures via hydrolysable bonds. This approach was evaluated by the immobilization of heparin onto a porous cellulosic substrate via ester bonds. Cuprophan was used as a model substrate and N,N¿-carbonyldiimidazole as a coupling agent. Heparinized Cuprophan incubated in phosphate buffered saline showed a release of heparin due to the hydrolysis of the ester bonds between heparin and Cuprophan. The release rate could be easily adjusted by varying the amount of coupling agent used during immobilization. Cuprophan with a rather stable heparin coating (release rate: 6.1 mU/cm2·h) and Cuprophan which shows a substantial release of heparin (release rate up to 23.0 mU/cm2·h) could be prepared. Except when the release was relatively high, release rates were constant for at least 1 week. Storage of the release system at ambient conditions up to 6 months or sterilization by means of steam, ethylene oxide exposure, or gamma irradiation did not affect the release properties. It was concluded that this concept for a heparin release system is highly promising to prepare thromboresistant surfaces for various blood contacting devices

The antifibrotic potential of a sustained release formulation of a PDGFβ-receptor targeted rho kinase inhibitor

Rho kinase activity in hepatic stellate cells (HSCs) is associated with activation, transformation and contraction of these cells, leading to extracellular matrix production and portal hypertension in liver cirrhosis. Inhibition of rho kinase activity can reduce these activities, but may also lead to side effects, for instance systemic hypotension. This can be circumvented by liver-specific delivery of a rho kinase inhibitor to effector cells. Therefore, we targeted the rho kinase inhibitor Y27632 to the key pathogenic cells in liver fibrosis, i.e. myofibroblasts including activated HSCs that highly express the PDGF beta-receptor, using the drug carrier pPB-MSA. This carrier consists of mouse serum albumin (MSA) covalently coupled to several PDGF beta-recognizing moieties (pPB). We aimed to create a prolonged release system of such a targeted construct, by encapsulating pPB-MSA-Y27632 in biodegradable polymeric microspheres, thereby reducing short-lasting peak concentrations and the need for frequent administrations. Firstly, we confirmed the vasodilating potency of PDGF beta-receptor targeted Y27632 in vitro in a contraction assay using HSCs seeded on a collagen gel. We subsequently demonstrated the in vivo antifibrotic efficacy of pPB-MSA-Y27632-loaded microspheres in the Mdr2 - / - mouse model of progressive biliary liver fibrosis. A single subcutaneous microsphere administration followed by organ harvest one week later clearly attenuated liver fibrosis progression and significantly suppressed the expression of fibrosis related genes, such as several collagens, profibrotic cytokines and matrix metalloproteinases. In conclusion, we demonstrate that polymeric microspheres are suitable as drug delivery system for the sustained systemic delivery of targeted protein constructs with antifibrotic potential, such as pPB-MSA-Y27632. This formulation appears suitable for the sustained treatment of liver fibrosis and possibly other chronic diseases

Inulin glasses for the stabilization of therapeutic proteins

Sugar glasses are widely used to stabilize proteins during drying and subsequent storage. To act successfully as a protectant. the sugars should have a high glass transition temperature (Tg). a poor hygroscopicity, a low crystallization rate, and contain no reducing groups. When freeze drying is envisaged as method of drying, a relatively high Tg of the freeze concentrated fraction (Tg') is preferrable. in this study, whether inulins meet these requirements was investigated. Inulins of various degrees of polymerisation (DP) were evaluated. Trehalose glass was used as a positive control. It was found that the Tg and the Tg' of inulins with a number/weight average DP (DPn/DPw) higher than 5.5/6.0 were higher than those of trehalose glass. Furthermore, inulin glasses showed a similar hygroscopicity to that of trehalose glass but crystallized less rapidly. Less than 6% of the sugar units of inulins with a DPn/DPw higher than 5.5/6.0 contained reducing groups. Trehalose contained no reducing groups. Freeze drying of an alkaline phosphatase solution without protectant induced an almost complete loss of the activity of the protein. In contrast, when inulins with a DPn/DPw higher than 5.5/6.0 or trehalose were used as stabilizer, the activity was fully maintained, also after subsequent storage for 4 weeks at 20 degreesC and 0, 45, or 60% RH, respectively. The stabilizing capacities of inulin with a lower DP and glucose were substantially less pronounced. After storage at 60 degreesC for 6 days, the activity of freeze dried samples containing inulins with a DPn/DPw higher than 5.5/6.0 was still about 50% whereas the activity of samples containing inulin with a lower DP, glucose, or trehalose was completely lost. It is: concluded that inulins with a DPn/DPw higher than 5.5/6.0 meet the physicochemical characteristics to successfully act as protectants for proteins. The stabilizing potential of these inulins was clearly shown using alkaline phosphatase as a model protein. (C) 2001 Elsevier Science B.V. All rights reserved

Incorporation of lipophilic drugs in sugar glasses by lyophilization using a mixture of water and tertiary butyl alcohol as solvent

In this study, anew and robust method was evaluated to prepare physically stable solid dispersions. Trehalose, sucrose, and two inulins having different chain lengths were used as carrier. Diazepam, nifedipine, Delta(9)-tetrahydrocannabinol, and cyclosporine A were used as model drugs. The sugar was dissolved in water and the drug in tertiary butyl alcohol (TBA). The two solutions were mixed in a 4/6 TBA/water volume ratio and subsequently freeze dried. Diazepam could be incorporated at drug loads up to 63% w/w. DSC measurements showed that, except in some sucrose dispersions, 97-100% of the diazepam was amorphous. In sucrose dispersions with high drug loads, about 10% of the diazepam had crystallised. After 60 days of exposure at 20degreesC and 45% relative humidity (RH), diazepam remained fully amorphous in inulin dispersions, whereas in trehalose and sucrose crystallization of diazepam occurred. The excellent physical stability of inulin containing solid dispersions can be attributed to the high glass transition temperature (T-g) of inulin. For the other drugs similar results were obtained. The residual amount of the low toxic TBA was only 0.1-0.5% w/w after freeze drying and exposure to 45% RH and 20degreesC. Therefore, residual TBA will not cause any toxicity problems. This study provides a versatile technique, to produce solid dispersions. Inulin glasses are preferred because they provide an excellent physical stability of the incorporated amorphous lipophilic drugs. (C) 2004 Wiley-Liss, Inc

Anomalous dissolution behaviour of tablets prepared from sugar glass-based solid dispersions

In this study, anomalous dissolution behaviour of tablets consisting of sugar glass dispersions was investigated. The poorly aqueous soluble diazepam was used as a lipophilic model drug. The release of diazepam and sugar carrier was determined to study the mechanisms governing dissolution behaviour. The effect of carrier dissolution rate and drug load was tested with four different sugars, in the order of decreasing dissolution rates: sucrose, trehalose and two oligo-fructoses; inulinDP11 and inulinDP23 having a number average degree of polymerization (DP) of 11 and 23, respectively. Diazepam was incorporated in these sugar glasses in the amorphous state by means of freeze drying using water and tertiary butyl alcohol (TBA) as solvents. None of the tablets disintegrated during dissolution. Dissolution of 80% of the lipophilic drug within 20 min was found when diazepam and sugar dissolution profiles coincided. The sugar carrier and diazepam dissolved at the same rate, which was constant in time and fast. This condition was met for relatively slow dissolving carriers like the inulins or for low drug loads. For relatively fast dissolving carriers like sucrose or trehalose with high drug loads, release profiles of diazepam and sugar did not coincide: diazepam dissolved much more slowly than the sugars. In case of non-coinciding release profiles, diazepam release was split into three phases. During the first phase non-steady-state dissolution was observed: diazepam release accelerated and a drug rich layer consisting of crystalline diazepam was gradually formed. This first phase determined the further release of diazepam. During the second phase a steady-state release rate was reached: zero-order release was observed for both drug and carrier. During this phase, the remaining (non-crystallised) solid dispersion is dissolved without the further occurrence of crystallisation. The third phase, starting when all carrier is dissolved, involved the very slow dissolution of crystallised diazepam, which was present either as the skeleton of a tablet resulting in a zero-order release profile or as separate particles dispersed in the dissolution medium resulting in a first-order release. To understand the anomalous dissolution behaviour, a model is proposed. It describes the phenomena during dissolution of amorphous solid dispersion tablets and explains that fast dissolution is observed for low drug loads or slow dissolving carriers like inulin. (C) 2004 Elsevier B.V. All rights reserved

Fabrication and characterization of an asymmetric polyurethane membrane for use as a wound dressing

To prevent wound dehydration and bacterial penetration, a wound dressing should be occlusive, but on the other hand it should also be permeable for wound exudate to prevent bullae formation. To meet these requirements a new type of polyurethane wound dressing which consists of a microporous top layer (pore size < 0.7 m) supported by a sublayer with a highly porous sponge-like structure containing micropores (pore size <10 m) as well as macropores (pore size: 50-100 m) was designed. The pores of both layers are interconnected and form a continuous structure in the membrane. Membranes according to this design were prepared either by means of a two-step or by means of a one-step casting process. Both fabrication methods are based on phase inversion techniques. Asymmetric polyurethane Biomer® membranes prepared by the two-step casting process were tested in vivo as full thickness skin substitutes using guinea pigs. Neither wound dehydration nor infections were observed while the drainage capacity of the wound dressing was effective in preventing bullae formation. Furthermore the wound dressing remained firmly adhered to the wound surface during the whole process of wound healing. In contrast to all other commercial wound dressings currently available the polyurethane wound dressing applied on excised clean wounds did not need to be replaced during healing but could be left on the wound until full regeneration of the skin had taken place after which it was spontaneously repelled