The antifibrotic potential of a sustained release formulation of a PDGFß-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 PDGFß-receptor, using the drug carrier pPB-MSA. This carrier consists of mouse serum albumin (MSA) covalently coupled to several PDGFßR-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 PDGFß-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.

Pharmacokinetics of a sustained release formulation of PDGFß-receptor directed carrier proteins to target the fibrotic liver.

Liver fibrogenesis is associated with excessive production of extracellular matrix by myofibroblasts that often leads to cirrhosis and consequently liver dysfunction and death. Novel protein-based antifibrotic drugs show high specificity and efficacy, but their use in the treatment of fibrosis causes a high burden for patients, since repetitive and long-term parenteral administration is required as most proteins and peptides are rapidly cleared from the circulation. Therefore, we developed biodegradable polymeric microspheres for the sustained release of proteinaceous drugs. We encapsulated the drug carrier pPB-HSA, which specifically binds to the PDGFßR that is highly upregulated on activated myofibroblasts, into microspheres composed of hydrophilic multi-block copolymers composed of poly(l-lactide) and poly ethylene glycol/poly(ϵ-caprolactone), allowing diffusion-controlled release. Firstly, we estimated in mice with acute fibrogenesis induced by a single CCl4 injection the half-life of I125-labeled pPB-HSA at 40 min and confirmed the preferential accumulation in fibrotic tissue. Subsequently, we determined in the Mdr2 −/− mouse model of advanced biliary liver fibrosis how the subcutaneously injected microspheres released pPB-HSA into both plasma and fibrotic liver at 24 h after injection, which was maintained for six days. Although the microspheres still contained protein at day seven, pPB-HSA plasma and liver concentrations were decreased. This reduction was associated with an antibody response against the human albumin-based carrier protein, which was prevented by using a mouse albumin-based equivalent (pPB-MSA). In conclusion, this study shows that our polymeric microspheres are suitable as sustained release formulation for targeted protein constructs such as pPB-HSA. These formulations could be applied for the long-term treatment of chronic diseases such as liver fibrosis.

Polymeric microspheres for the sustained release of a protein-based drug carrier targeting the PDGFß-receptor in the fibrotic kidney.

Injectable sustained release drug delivery systems are an attractive alternative for the intravenous delivery of therapeutic proteins. In particular, for chronic diseases such as fibrosis, this approach could improve therapy by reducing the administration frequency while avoiding large variations in plasma levels. In fibrotic tissues the platelet-derived growth factor receptor beta (PDGFßR) is highly upregulated, which provides a target for site-specific delivery of drugs. Our aim was to develop an injectable sustained release formulation for the subcutaneous delivery of the PDGFßR-targeted drug carrier protein pPB-HSA, which is composed of multiple PDGFßR-recognizing moieties (pPB) attached to human serum albumin (HSA). We used blends of biodegradable multi-block copolymers with different swelling degree to optimize the release rate using the model protein HSA from microspheres produced via a water-in-oil-in-water double emulsion evaporation process. The optimized formulation containing pPB-HSA, showed complete release in vitro within 14days. After subcutaneous administration to mice suffering from renal fibrosis pPB-HSA was released from the microspheres and distributed into plasma for at least 7days after administration. Furthermore, we demonstrated an enhanced accumulation of pPB-HSA in the fibrotic kidney. Altogether, we show that subcutaneously administered polymeric microspheres present a suitable sustained release drug delivery system for the controlled systemic delivery for proteins such as pPB-HSA.

Robust neurite extension following exogenous electrical stimulation within single walled carbon nanotube-composite hydrogels.

UNLABELLED: The use of exogenous electrical stimulation to promote nerve regeneration has achieved only limited success. Conditions impeding optimized outgrowth may arise from inadequate stimulus presentation due to differences in injury geometry or signal attenuation. Implantation of an electrically-conductive biomaterial may mitigate this attenuation and provide a more reproducible signal. In this study, a conductive nanofiller (single-walled carbon nanotubes [SWCNT]) was selected as one possible material to manipulate the bulk electrical properties of a collagen type I-10% Matrigel™ composite hydrogel. Neurite outgrowth within hydrogels (SWCNT or nanofiller-free controls) was characterized to determine if: (1) nanofillers influence neurite extension and (2) electrical stimulation of the nanofiller composite hydrogel enhances neurite outgrowth. Increased SWCNT loading (10-100-µg/mL) resulted in greater bulk conductivity (up to 1.7-fold) with no significant changes to elastic modulus. Neurite outgrowth increased 3.3-fold in 20-µg/mL SWCNT loaded biomaterials relative to the nanofiller-free control. Electrical stimulation promoted greater outgrowth (2.9-fold) within SWCNT-free control. The concurrent presentation of electrical stimulation and SWCNT-loaded biomaterials resulted in a 7.0-fold increase in outgrowth relative to the unstimulated, nanofiller-free controls. Local glia residing within the DRG likely contribute, in part, to the observed increases in outgrowth; but it is unknown which specific nanofiller properties influence neurite extension. Characterization of neuronal behavior in model systems, such as those described here, will aid the rational development of biomaterials as well as the appropriate delivery of electrical stimuli to support nerve repair. STATEMENT OF SIGNIFICANCE: Novel biomedical devices delivering electrical stimulation are being developed to mitigate symptoms of Parkinson's, treat drug-resistant depression, control movement or enhance verve regeneration. Carbon nanotubes and other novel materials are being explored for novel nano-neuro devices based on their unique properties. Neuronal growth on carbon nanotubes has been studied in 2D since the early 2000s demonstrating increased outgrowth, synapse formation and network activity. In this work, single-walled carbon nanotubes were selected as one possible electrically-conductive material, dispersed within a 3D hydrogel containing primary neurons; extending previous 2D work to 3D to evaluate outgrowth within nanomaterial composites with electrical stimulation. This is the first study to our knowledge that stimulates neurons in 3D composite nanomaterial-laden hydrogels. Examination of electrically conductive biomaterials may serve to promote regrowth following injury or in long term stimulation.

Microsphere size influences the foreign body reaction.

Biodegradable poly-(DL-lactide-co-glycolide) (PLGA) microspheres (MSP) are attractive candidate vehicles for site-specific or systemic sustained release of therapeutic compounds. This release may be altered by the host's foreign body reaction (FBR), which is dependent on the characteristics of the implant, e.g. chemistry, shape or size. In this study, we focused on the characterisation of the influence of MSP size on the FBR. To this end we injected monodisperse MSP of defined size (small 5.8 µm, coefficient of variance (CV) 14 % and large 29.8 µm, CV 4 %) and polydisperse MSP (average diameter 34.1 µm, CV 51 %) under the skin of rats. MSP implants were retrieved at day 7, 14 and 28 after transplantation. The FBR was studied in terms of macrophage infiltration, implant encapsulation, vascularisation and extracellular matrix deposition. Although PLGA MSP of all different sizes demonstrated excellent in vitro and in vivo biocompatibility, significant differences were found in the characteristics of the FBR. Small MSP were phagocytosed, while large MSP were not. Large MSP occasionally elicited giant cell formation, which was not observed after implantation of small MSP. Cellular and macrophage influx and collagen deposition were increased in small MSP implants compared to large MSP. We conclude that the MSP size influences the FBR and thus might influence clinical outcome when using MSP as a drug delivery device. We propose that a rational choice of MSP size can aid in optimising the therapeutic efficacy of microsphere-based therapies in vivo.

[Modified polyurethane foam as a local hemostatic agent after dental extractions]. / Gemodificeerd polyurethaanschuim als lokaal hemostaticumn na gebitsextracties.

In this split mouth experiment, the feasibility ofpolyurethane foam as a local hemostatic agent after dental extractions was studied. Ten healthy patients underwent 2 extractions ofa dental element in 1 treatment session. The 10 patients were subsequently randomly divided in a gelatin group and a collagen group. In the gelatin group, a polyurethane foam (PU) was applied in 1 extraction socket, while in the other socket a commercially available gelatin foam was applied. In the collagen group, a PU was applied in 1 socket, and a collagen wadding in the other. All hemostats were removed after 2 minutes, after which the degree of coagulation was measured using a thrombin/antithrombin test and a fibrinogen test. This study suggests that polyurethane foam has hemostatic capacity. Large scale clinical research is needed to confirm this finding, and should indicate whether this hemostatic capacity is clinically relevant.

In vitro degradation of a biodegradable polyurethane foam, based on 1,4-butanediisocyanate: a three-year study at physiological and elevated temperature.

Biodegradable polyesterurethanes (PUs) may be used as scaffold materials for tissue regeneration applications, because of their excellent mechanical properties. In this study, the degradation of highly porous PU foams was evaluated in vitro. The PU had amorphous soft segments of DL-lactide/epsilon-caprolactone and uniform hard segments, synthesized from 1,4-butanediisocyanate and butanediol. The foams were degraded for 3 years in a Sörensen buffer solution (pH 7.4) at 37 and 60 degrees C. Dimensions of the foams, intrinsic viscosity, mass loss, thermal properties, and composition of the remaining material were evaluated. Copolyester (CP) foams of DL-lactide/epsilon-caprolactone served as controls. The PU foams kept their dimensions for 20 weeks at 37 degrees C, whereas CP foams collapsed after 3 weeks. PU mass loss reached a maximum of 80% at both 37 and 60 degrees C. CP mass loss reached 99.9% at 60 degrees , and 92% at 37 degrees C after 3 years. The degradation processes at 37 and 60 degrees C are initially the same, but eventually degradation products with different thermal properties are being formed. (1)H NMR studies showed that the hard urethane segments of the PU do not degrade in vitro at pH 7.4. It was concluded that the PU material has favorable characteristics for a scaffold material. Compared to long-term in vivo results of the same PU these in vitro results are not representative for the in vivo situation and therefore total resorption has to be investigated in long-term in vivo studies.

In vivo resorption of a biodegradable polyurethane foam, based on 1,4-butanediisocyanate: a three-year subcutaneous implantation study.

Degradable polyurethanes (PUs), based on aliphatic diisocyanates, can be very useful in tissue regeneration applications. Their long-term in vivo degradation has not been extensively investigated. In this study a biodegradable PU with copolyester soft segments of DL-lactide/epsilon-caprolactone and hard segments synthesized from 1,4-butanediisocyanate was evaluated with regard to tissue response during degradation and, ultimately, the resorption of the material. Highly porous PU foam discs were subcutaneously implanted in rats and rabbits for intervals up to 3 years. A copolymer foam of DL-lactide and epsilon-caprolactone served as a control. The foams, the surrounding tissues and the draining lymph nodes were evaluated with light and electron microscopy. In the first stages of degradation the number of macrophages and giant cells increased. As the resorption stage set in their numbers gradually decreased. Electron microscopy showed macrophages containing pieces of PU. The size of the intracellular PU particles diminished and cells containing these remnants gradually disappeared after periods from 1 to 3 years. After 3 years an occasional, isolated macrophage with biomaterial remnants could be traced in both PU and copolymer explants. Single macrophages with biomaterial remnants were observed in the lymph nodes between 39 weeks and 1.5 years following implantation. It is concluded that the PU foam is biocompatible during degradation. After 3 years PU samples had been resorbed almost completely. These results indicate that the PU foam can be safely used as a biodegradable implant.

An animal model for oroantral communications: a pilot study with Göttingen minipigs.

A pilot study was performed to investigate whether the Göttingen minipig is a suitable animal model for creating and closing oroantral communications (OACs) and to test whether these defects can be closed with a biodegradable polyurethane (PU) foam. In three adult minipigs, an OAC was created on both sides of the maxilla. The left side was closed by a standard surgical buccal flap procedure, the right side by applying a PU foam. The pigs were killed after two weeks, one month and three months, respectively. Postmortem and histological examination showed that an OAC was created in only one of six cases. In the remaining cases, the infraorbital canal was perforated instead of the floor of the maxillary sinus. It was concluded that the Göttingen minipig is not a suitable animal model for OAC investigations. As a result, the closure of OACs with a biodegradable PU could not be evaluated.

Short-term in vitro and in vivo biocompatibility of a biodegradable polyurethane foam based on 1,4-butanediisocyanate.

In this study short-term in vitro and in vivo biocompatibility apects of a biodegradable polyurethane (PU) foam were evaluated. The PU consists of hard urethane segments and amorphous soft segments based on a copolyester of dl-lactide and epsilon-caprolactone. The urethane segments are of uniform length and synthesized with 1,4-butanediisocyanate. The foam has good mechanical properties and will be used for tissue regeneration applications. Degradation tests were carried out in a buffer solution for twelve weeks. Cytotoxicity was determined using extract and direct contact test methods with incubation periods varying form 24 to 72 h. The foam was implanted subcutaneously for one, four and twelve weeks and the tissue response to the material was histologically evaluated. In vitro, the mass loss was 3.4% after twelve weeks. In the cytotoxicity tests the PU caused no abnormal growth behaviour, nor morphological changes or inhibition in metabolic activity. The in vivo studies showed no toxic tissue response to the PU. Connective tissue ingrowth, accompanied by vascular ingrowth was complete at twelve weeks. In vivo degradation had started within four to twelve weeks. In conclusion, the PU shows a good in vitro and in vivo biocompatibility in these short-term experiments.

Thiolated recombinant human tumor necrosis factor-alpha protects against Plasmodium berghei K173-induced experimental cerebral malaria in mice.

The introduction of reactive thiol groups in recombinant human tumor necrosis factor (TNF) alpha (rhTNF-alpha) by the reagent succinimidyl-S-acetylthioacetate resulted in the formation of a chemically stabilized rhTNF-alpha trimer (rhTNFalpha-AT; as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis). rhTNFalpha-AT showed a substantially enhanced protective efficacy against the development of experimental murine cerebral malaria (ECM) after intravenous injection compared to the protective efficacy of nonmodified rhTNF-alpha. Administration of thiolated rhTNF-alpha with protected thiol groups (rhTNFalpha-ATA; no stabilized trimers in vitro) exhibited the same protective efficacy against ECM, while in vitro bioactivity was reduced. Parasitemia was significantly suppressed in rhTNF-treated mice that were protected against ECM but not in treated mice that developed ECM. Protection against ECM was not related to increased concentrations in plasma of soluble TNF receptor 1 and 2 directly after injection or at the moment of development of ECM in nontreated mice. The results indicate that thiolation of rhTNF-alpha leads to the formation of stable trimers with increased potential in vivo.

Treatment with recombinant human tumour necrosis factor-alpha reduces parasitaemia and prevents Plasmodium berghei K173-induced experimental cerebral malaria in mice.

The present study shows that treatment with recombinant human tumour necrosis factor-alpha (rhTNF-alpha) can suppress parasitaemia and prevents development of experimental cerebral malaria (ECM) in Plasmodium berghei K173-infected mice. Mice received rhTNF-alpha treatment either by subcutaneous injection of free or liposome-encapsulated rhTNF-alpha or sustained intraperitoneal administration of rhTNF-alpha given via mini-osmotic pumps. Low-dose treatment with a subcutaneous bolus injection of rhTNF-alpha protected against ECM when treatment was started on day 5 or 6 after infection. The same protective efficacy was obtained either by subcutaneous injection of liposome-encapsulated rhTNF-alpha or by sustained release from osmotic pumps, but in the latter case a 10-fold lower daily dose of rhTNF-alpha was sufficient. Treatment with rhTNF-alpha substantially suppressed parasitaemia in ECM-protected mice, but not in mice developing ECM. Thus, the rhTNF-alpha mediated suppression of parasitaemia is directly or indirectly involved in protection against ECM. Sustained delivery of rhTNF-alpha through osmotic pumps, but not by subcutaneous injection of liposome-encapsulated rhTNF-alpha, resulted in increased concentrations of soluble mouse TNF receptor R75 (sTNFR75) in plasma at day 9 after infection when non-treated mice die of ECM. Thus, protection against ECM is not directly correlated with the sTNFR75 concentrations at day 9 after infection.

Absorption and biodistribution of 111indium-labelled desferrioxamine (111In-DFO) after subcutaneous injection of 111In-DFO liposomes.

In the present study the kinetics of the release of desferrioxamine (DFO) from liposomes (fluid and rigid liposome type) at the subcutaneous (s.c.) injection site was studied. DFO was labelled with 111indium (111In-DFO) and the fate of s.c. administered liposomal 111In-DFO was monitored with a gamma camera. Free 111In-DFO rapidly disappeared from the s.c. injection site [90% of the injected dose (%ID) within 2 h]. After s.c. injection of the fluid liposome type, 20 %ID was released within 4 h and 50 %ID within 24 h. Approximately 25 %ID remained at the injection site 6 days after injection. With the rigid liposome type, no initial release (within 4 h) was observed. The rate of DFO release was similar to the fluid liposome type. Free drug was rapidly cleared from the bloodstream (within 2 h), while low, but detectable blood levels of 111In-DFO were maintained for 6 days after s.c. injection of liposomal drug. This resulted in higher peak levels of 111In-DFO in liver and kidney (4-6 %ID/g) compared with free drug (2-4 %ID/g), which were reached later in time. The present data point to sustained release of DFO from s.c. administered DFO-liposomes as the mechanism behind their enhanced therapeutic effect in murine malaria.

Treatment with liposome-bound recombinant human tumor necrosis factor-alpha suppresses parasitemia and protects against Plasmodium berghei k173-induced experimental cerebral malaria in mice.

Our study describes liposomes (conventional or sterically stabilized) as carrier systems for recombinant human tumor necrosis factor-alpha (rhTNF-alpha) to increase its protective efficacy against Plasmodium berghei-induced experimental cerebral malaria (ECM) in mice. rhTNF-alpha was either covalently coupled to the outer surface of preformed liposomes or encapsulated into the liposomes. For coupling to the liposomes, reactive thiol groups were introduced in rhTNF-alpha by reaction with N-succinimidyl S-acetylthioacetate. Intravenous injection of liposome-bound rhTNF-alpha substantially enhanced protection against ECM as compared with injection of free rhTNF-alpha. A similar protective efficacy against ECM was obtained by treatment with rhTNF-alpha coupled to either conventional or sterically stabilized liposomes. Encapsulation of rhTNF-alpha into liposomes did not improve the protective efficacy of rhTNF-alpha against P. berghei-induced ECM. Parasitemia was suppressed by treatment with either free or liposome-bound rhTNF-alpha in mice protected against ECM, but not in rhTNF-alpha-treated mice developing ECM. These data suggest that the effect of rhTNF-alpha on parasitemia plays a role in establishing protection against ECM. Our studies indicate that liposome-bound rhTNF-alpha exhibits an enhanced protective efficacy against ECM compared with free rhTNF-alpha. It is hypothesized that thiolation of rhTNF-alpha and coupling to the liposomal bilayer stabilizes the bioactive trimeric configuration of rhTNF-alpha.

Plasmodium vinckei: optimization of desferrioxamine B delivery in the treatment of murine malaria.

Optimization of desferrioxamine B (DFO) delivery for the treatment of malaria was studied in Plasmodium vinckei infected mice. DFO was administered by three different treatment regimens: (1) multiple subcutaneous injections of free DFO, (2) intraperitoneal infusion of free DFO, or (3) multiple subcutaneous injections of liposomal DFO. In a first series of experiments, DFO treatment was started prior to infection. Multiple subcutaneous injections of free DFO before and during infection suppressed parasitemia, whereas injections only prior to infection did not. Suppression of parasitemia and long-term survival (>1 month after infection) of mice were obtained by intraperitoneal infusion starting 1 day before infection (14 days, 130 mg DFO/kg/day) or by subcutaneous injections of liposomal DFO prior to infection (days -1 and 0, 400 or 800 mg DFO/kg/day). The efficacy of the antimalarial activity of liposomal DFO was influenced by the drug-to-lipid ratio but was hardly affected by bilayer rigidity. In a second series of experiments, DFO treatment was started at days 6 and 7 after infection. Parasitemia was reduced by all three treatment regimens; however, long-term survival was obtained only by treatment with liposomal DFO (days 7 and 8, 400 mg/kg/day). The present results indicate that continuous exposure of the parasite to low doses of DFO suffice to clear parasitemia, whereas high doses of free DFO administered intermittently do not. A right balance between dose of DFO, time of exposure to DFO, and parasitemia suppresses parasitemia even in the treatment of late-stage malaria. It was shown that liposomes are suitable carrier systems for DFO in experimental malaria therapy when given prior to infection and, moreover, in the treatment of advanced stages of malaria.

The influence of the route of administration and liposome composition on the potential of liposomes to protect tissue against local toxicity of two antitumor drugs.

The present paper reports on the influence of the route of administration and liposome stability on the protective effect of liposome encapsulation of two model antitumor agents, mitoxantrone and doxorubicin. The results demonstrate that liposome encapsulation can protect surrounding tissue from the cytotoxic effects of the drugs after subcutaneous (s.c.) and intramuscular (i.m.) administration. The route of administration is an important factor influencing tissue damage. Liposomal mitoxantrone caused much less tissue irritation after im injection than after s.c. injection. Liposome stability is also an important factor. Liposomes composed of 'fluid-state' phospholipids only delayed the damaging effects of doxorubicin when injected sc. Liposomes with a more rigid nature were much more effective in preventing local tissue damage over a longer period of time when administered sc. Results suggest that slow release of liposome-associated drugs may eventually cause severe local tissue damage. The incorporation of the hydrophilic lipid derivative distearoylphosphatidylethanolamine-poly(ethyleneglycol) (PEG-PE) had no apparent effect on the protective effect of liposomes after sc administration.

Lymphatic uptake and biodistribution of liposomes after subcutaneous injection. II. Influence of liposomal size, lipid compostion and lipid dose.

The present paper reports on the results of a systematic study on liposome variables potentially affecting lymphatic disposition and biodistribution of liposomes after sc injection. Liposomal size was found to be the most important factor influencing lymphatic uptake and lymph node localization of sc administered liposomes. Lymphatic uptake from the s.c. injection site of small liposomes (about 0.04 microm) was relatively high (76% of the injected dose (%ID)) as compared to large, non-sized liposomes, which remained almost completely at the site of injection. Small liposomes were less efficiently retained by regional lymph nodes than larger liposomes. Liposomal lipid composition did not influence lymphatic uptake with one exception: Lymphatic uptake was decreased in case of neutral liposomes composed of (DPPC). Lymph node localization was substantially enhanced by inclusion of phosphatidylserine (PS) into the liposomal bilayers. Saturation of lymphatic uptake and lymph node localization did not occur over a large liposomal lipid dose range, illustrating the efficient performance of lymph nodes in capturing s.c. administered particles.

Effect of septicaemia on the plasma levels of biopterin and nitric oxide metabolites in rats and rabbits.

Live Escherichia coli decreased mean arterial blood pressure in rabbits from 67 to 20 mmHg. E. coli did not affect blood pressure in rats but did significantly increase heart rate by 29%. To related the cardiovascular effects with putative relevant biochemical pathways, the plasma levels of nitrate + nitrite (NOx) and biopterin, representing the main metabolites of nitric oxide and tetrahydrobiopterin, respectively, were determined in conscious rats and rabbits after treatment with live E. coli. In rats, E. coli induced a rapid 43% increase in the plasma level of biopterin preceding the 7- to 26-fold increase in NOx level. In rabbits, no increase in the NOx level was observed despite a 3- to 5-fold increase in the biopterin level at 6-10 hr posttreatment. It is concluded that the synthesis of tetrahydrobiopterin precedes nitric oxide synthesis after induction of septicaemia in the rat. After the induction of septicaemia, rabbits show a clear hypotensive response and an increase in biopterin level but no concomitant increase in NOx. Biopterin apparently represents a more appropriate biochemical marker of septic shock than does NOx.

Oxidative stress in malaria; implications for prevention and therapy.

Malaria affects world-wide more than 200 million people, of which 1-2 million die every year. New drugs and treatment strategies are needed to face the rapidly increasing problems of drug resistance. During a malaria infection, both host and parasite are under oxidative stress. Increased production levels of reactive oxygen species (ROS, e.g superoxide anion and the hydroxyl radical) are produced by activated neutrophils in the host and during degradation of haemoglobin in the parasite. The effects of ROS in malaria can be both beneficial and pathological, depending on the amount and place of production. Enhanced ROS production after the administration of pro-oxidants, which is directed against the intra-erythrocytic parasite, inhibits the infection both in vitro and in vivo. However, ROS are also involved in pathological changes in host tissue like damage of the vascular endothelial lining during a malaria infection (cerebral malaria). Pro-oxidants support the host defense against the parasite when working in or near the infected cell but potentially cause vascular damage when working on or near the vascular lining. Examples of pro-oxidants are found among xenobiotics and food components. Important new drugs belonging to the class of pro-oxidants are artemisinin and its derivatives. Anti-oxidants potentially counteract these agents. Treatment with anti-oxidants or chelators of metals to prevent their catalytic function in the generation of ROS may prevent vascular pathology. In addition, the iron chelator desferrioxamine, exhibits an antiparasitic activity, because iron is also essential for the proliferation of the parasite. Cytokines play an important role in ROS-related pathology of malaria, though their mechanism of action is not completely elucidated. This field might bring up new treatment concepts and drugs. Drugs which prevent host pathology, such as the cerebral complications might be life saving.