Eight novel mutations in MLC1 from 18 Iranian patients with megalencephalic leukoencephalopathy with subcortical cysts.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) (MIM #604004) is a rare autosomal recessive neurological disorder characterized by macrocephaly, motor and cognitive decline, ataxia, spasticity and occasional seizures. Magnetic resonance imaging (MRI) shows diffusely abnormal and swollen white matter of the cerebral hemispheres and subcortical cysts in the anterior temporal and frontoparietal region. Mutations in MLC1(22q13.33) and GLIALCAM have been identified in patients with MLC. Mutations in MLC1 account for approximately 75% of the cases. MLC was suspected in eighteen Iranian patients from sixteen families based on positive clinical findings including macrocephaly beginning in the first year, neurocognitive deterioration, seizure or loss of consciousness after minor head trauma. All except two were born to consanguineous parents. Brain MRI images were compatible with MLC and confirmed the diagnosis. Sequencing of entire coding region of MLC1 was performed for seventeen patients and mutations in MLC1 were detected in all of them. Eight novel mutations and seven previously reported mutations were identified. This report shows that MLC is relatively common in Iranian population, as expected for rare diseases with high inbreeding, with a surprisingly high frequency of novel mutations.

Persistent bisphosphonate use and the risk of osteoporotic fractures in clinical practice: a database analysis study.

INTRODUCTION: International guidelines on the treatment and prevention of osteoporosis recommend the use of bisphosphonates to prevent fractures in this population. However, low persistent use of bisphosphonates could considerably limit the prevention of fractures in clinical practice. OBJECTIVE: This study aimed to investigate the association between persistent use of bisphosphonates and the risk of osteoporotic fractures in clinical practice. METHODS: Data were obtained from the PHARMO Record Linkage System, which includes, among other databases, drug-dispensing records from community pharmacies linked to hospital discharge records of more than two million subjects in defined areas in the Netherlands. Persistence with bisphosphonate therapy was assessed during a period of 3 years. A nested matched case control study (cases:controls = 1:10) was performed to study the association between persistent bisphosphonate use and hospitalisation for osteoporotic fractures and analysed by conditional logistic regression analysis. The analyses were adjusted for patient characteristics such as previous hospitalisations for fractures, co-morbidity and co-medication. RESULTS: 14,760 new female users of bisphosphonates were identified of which 541 women had a hospitalisation for osteoporotic fracture after start of bisphosphonate treatment (1-3 years follow-up). One-year persistence rates increased from 33% with alendronate daily to 48% with alendronate weekly, an increase of 15%. Similar results were obtained with risedronate daily and weekly. One year persistent use of bisphosphonates resulted in a statistical significant 26% lower fracture rate (OR 0.74; 95%CI 0.57-0.95) whereas 2 year persistent use resulted in a 32% lower rate (OR 0.68; 95%CI 0.47-0.96). CONCLUSIONS: Persistent use of bisphosphonates decreases the risk of osteoporotic fractures in clinical practice. Approximately 6% of fractures among users of bisphosphonates could be prevented if persistence was improved by 20%. However, current persistence with bisphosphonate therapy is suboptimal and strategies that further increase persistence are likely to further prevent the number of fractures.

Utilisation pattern of fentanyl transdermal system in The Netherlands.

PURPOSE: To describe the utilisation pattern of TTS fentanyl in daily practice. METHODS: A retrospective cohort study was performed with data from the Dutch PHARMO system, including medication and hospital admission records of 850,000 inhabitants of 25 Dutch cities. New starters of TTS fentanyl with at least two consecutive prescriptions in the period of 1 January 1996 through 31 December 2001 were included in the study cohort. Patients were distinguished in having non-cancer or cancer pain. RESULTS: About 61% of the patients suffered from non-cancer pain and 60% used other opioids before start of TTS fentanyl. The majority of the patients used other pain medication during the first treatment episode. Most patients (74%) started treatment with the lowest dose of TTS fentanyl (25 microg/hour), patients with cancer pain more often started with higher doses. About half of the patients changed type of patch during the first treatment episode, 80% of these patients had an increase in dose of TTS fentanyl. Fifty percent of all patients had a first treatment episode of less than 2 months and more than one third did not renew their prescription within two months. The median number of days of use per patch was 2.2 days for all patients. CONCLUSIONS: The use of TTS fentanyl is limited to a short period of time for a substantial percentage of patients starting treatment. The median duration of use per TTS fentanyl patch i.e. 2.2 days, was lower than the 3 days application period recommended in the summary of product characteristics.

Persistence with inhaled corticosteroid therapy in daily practice.

OBJECTIVE: To quantify persistence with inhaled corticosteroids (ICS) among new users in daily practice and identify determinants of persistence. METHODS: A retrospective cohort study was performed with data from the Dutch PHARMO system. This system consists of medication and hospital admission records of 325,000 inhabitants of 12 Dutch cities. In patients who were already using other drugs with a labeled indication of obstructive lung diseases (ATC: R03), individuals with a first dispensing of ICS between January 1, 1994 and December 31, 2000 were identified. Persistence with ICS was defined as the number of days on ICS treatment in the first year of use. Determinants of persistence were identified one year before start of the first dispensing of ICS. RESULTS: Approximately 50% of the patients used inhaled corticosteroids (ICS) for less than 200 days, while 18% continued treatment for one year. One-year persistence rates increased to 40% in patients with a history of multiple respiratory disease related drugs. Persistence rates also increased with lower initial doses, if the initial prescription was instituted by a medical specialist, if a patient was previously hospitalized for obstructive lung diseases, and with increasing age. CONCLUSION: The persistence rate of ICS is poor. Preventing early treatment discontinuation may be important to ensure maximal benefit from ICS treatment.

[Rising costs of drugs in hospitals in the period 1996-2000 and over the next few years]. / Stijegende kosten van geneesmiddelen in ziekenhuizen in 1996-2000 en in de komende jaren.

OBJECTIVE: To obtain an impression of drug expenditure in hospitals and in particular the costs due to novel more expensive drugs both in the past and in the future. DESIGN: Descriptive. METHOD: Data on intramurally supplied drugs were collected from 6 of the 95 general hospitals, 5 of the 14 top clinical hospitals, 4 of the 8 university hospitals and I of the 13 categorical hospitals for the period 1 January 1996-31 December 2000. The data were extrapolated to the entire of the Netherlands per hospital category and per year on the basis of the adherence figures. The drug costs were calculated on the basis of cost prices. For the most important new potential drugs it was ascertained whether they would actually become available and if they would substantially contribute to the drug expenditure. RESULTS: In 2000, the total drug expenditure within all Dutch hospitals was estimated to be [symbol: see text] 402 million. A substantial part of the costs could be attributed to the use of anticancer drugs (19%) and antibiotics (14%). In 2000, about 12% of the drug expenditure could be attributed to the use of novel, expensive drugs. The total costs of drugs increased on average by 8% per year during the period 1996-2000. The contribution of novel, expensive drugs nearly doubled during this period, and anticancer drugs were the most significant factor in this. A large proportion of the potentially new drugs were innovative anticancer drugs and drugs used to treat immune diseases. These compounds are likely to be expensive. Based on these findings it is expected that over the next few years, drug expenditure within hospitals to grow by at least 20% per year.

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.

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.

Maintenance of the synthesis of large proteoglycans in anatomically intact murine articular cartilage by steroids and insulin-like growth factor I.

OBJECTIVES: The exact regulation of the synthesis of cartilage specific molecules, such as collagen type II and aggrecan, by articular chondrocytes is unknown, but growth factors and hormones probably play an important part. The effects of glucocorticosteroids (prednisolone and triamcinolone), in combination with insulin-like growth factor I (IGF-I), on the synthesis and hydrodynamic volume of proteoglycans from murine patellar cartilage were investigated. METHODS: The in vitro effect of IGF-I and steroids on proteoglycan synthesis in murine patellar cartilage was evaluated by [35S]sulphate incorporation in combination with dissociative gel chromatography using a Sephacryl S-1000 column. The impact of in vivo prednisolone (0-5 mg/kg) on proteoglycan synthesis in murine patellar cartilage was analysed by [35S]sulphate incorporation immediately after dissection from the knee joint. RESULTS: Prednisolone stimulated proteoglycan synthesis in murine patellar cartilage from normal knees and in cartilage from knees injected with papain in vitro in the absence and presence of IGF-I. Moreover, oral administration of prednisolone for seven days to C57Bl10 mice resulted in enhanced proteoglycan synthesis in patellar cartilage. The incubation of patellar cartilage for 48 hours without serum or growth factors led to the synthesis of proteoglycans with a smaller hydrodynamic volume than those synthesised immediately after dissection of the patellae. This could either be circumvented by the addition of IGF-I or by the addition of glucocorticosteroids (prednisolone or triamcinolone) to the culture medium. CONCLUSIONS: These results show that in a dose range of 0.0003-0.3 mmol/l, glucocorticosteroids, like IGF-I, stimulate proteoglycan synthesis and maintain the synthesis of hydrodynamically large proteoglycans by chondrocytes from murine articular cartilage. This indicates that glucocorticosteroids might play a part in the preservation of matrix integrity in articular cartilage.

Stereospecific in vitro embryotoxicity of l-homocysteine in pre- and post-implantation rodent embryos.

Recently a derangement of homocysteine metabolism has been suggested as a possible risk factor for neural tube defects and recurrent spontaneous abortion. To investigate a possible role of homocysteine in the aetiology of neural tube defects we tested the in vitro embryotoxicity of l-homocysteine by culturing day 10 post coitum post-implantation rat embryos in whole embryo culture (WEC) for 24 hr and day 2 post coitum pre-implantation mouse embryos for 48 hr. With an area under curve (AUC) of 6.3 mm/hr, l-homocysteine significantly reduced the percentage of mouse embryos that developed into blastocysts. In rat WEC, an AUC for l-homocysteine of 3.6 mm/hr reduced the mitotic index of the neural epithelium of the rhombencephalon and the cell density of the mesenchyme adjacent to it, while at an AUC of 7.2 mm/hr l-homocysteine reduced the total morphological score and the number of malformations was increased. Malformations most often seen were transparent rhombencephalon, no or delayed formation of forelimb buds, dysmorphogenesis of the somites, and blister formation dorso-laterally of the place of forelimb bud formation. The embryotoxicity of l-homocysteine was stereospecific since d-homocysteine caused no embryotoxic effects. Also the oxidation product l-homocystine (AUC, 72 mm/hr) and the metabolite l-methionine (AUC, 144 mm/hr) were not embryotoxic. Both stereoisomers of homocysteinethiolactone were embryotoxic at an AUC of 72 mm/hr. The results are discussed in relation to the metabolism of homocysteine and methionine and their possible role in the neurulation process.