Noninvasive in vivo 13C-NMR spectroscopy in the rat to study the pharmacokinetics of 13C-labeled xenobiotics.

Noninvasive NMR methodology has been developed to enable monitoring of 13C-labeled xenobiotics in the rat in vivo. 2,2-Dichloro-1-(2-chlorophenyl)-1-(4-chlorophenyl)-[3-13C]-propane can be detected in the liver of intact rats by in vivo 13C surface coil NMR spectroscopy after ip administration of the compound. The experiments were performed at 1.9 and 9.4 Tesla. The intrahepatic changes of the signal intensity of the labeled compound were followed as a function of time. In the days following administration, the concentration decreased and dropped to values below the detection limit after 12 days. The study demonstrates the feasibility of studies on pharmacokinetics of 13C-labeled compounds in the rat using noninvasive, in vivo surface coil NMR spectroscopy in animals. The sensitivity allows the detection of a single dose of the drug of 200 mg/kg, but can be improved.     

K1K2Pu, a recombinant t-PA/u-PA chimera with increased thrombolytic potency, consisting of amino acids 1 to 3 and 87 to 274 of human tissue-type plasminogen activator (t-PA) and amino acids 138 to 411 of human single chain urokinase-type plasminogen activator (scu-PA). Purification in centigram quantities and conditioning for use in man.

K1K2Pu, a recombinant t-PA/u-PA chimera with increased thrombolytic potency in animal models of venous and arterial thrombosis, which consists of amino acids 1 to 3 and 87 to 274 of human tissue-type plasminogen activator (t-PA) and amino acids 138 to 411 of human single chain urokinase-type plasminogen activator (scu-PA), was produced and conditioned for use in patients. Chinese hamster ovary cells were transfected with an expression plasmid containing the K1K2Pu cDNA, high producer cell lines were selected and scaled up in 800 cm2 roller bottles, and 350 ml conditioned cell culture medium was harvested 3 to 7 times at 2 to 5 day intervals. Batches of 21 +/- 4 liter (mean +/- SD, n = 28) containing 1.8 +/- 0.6 mg/l of K1K2Pu related antigen were purified by chromatography on Copper chelate-Sepharose and immunoadsorption on an insolubilized murine monoclonal antibody (MA-1C8). Yields were 8.6 +/- 3.4 mg K1K2Pu per batch with a specific activity of 83,000 +/- 44,000 IU/mg. The final material, obtained at a concentration of approximately 0.7 mg/ml, was dialyzed against 0.3 M NaCl, 0.02 M Tris-HCl buffer, pH 7.5, containing 0.01% Tween 80 and 10 KIU/ml aprotinin. It was homogeneous on SDS-PAGE, contained 6.5 +/- 6.9 percent two chain material and the contamination with murine monoclonal antibody was less than 0.1 percent. After filtration of pools of 3 to 5 selected batches on 0.22 microns Millipore filters the material was sterile and virus free by routine screening; it was obtained at a concentration of approximately 0.5 mg/ml with a specific activity of 110,000 +/- 16,000 IU/mg (mean +/- SD, n = 3) and an endotoxin content of 0.5 to 7 units/mg. Bolus injection at a dose of 1 mg/kg in mice did not produce weight loss within 8 days. Thus, this material appears to be suitable for the investigation on a pilot scale of the pharmacokinetic and thrombolytic properties of K1K2Pu in patients with thromboembolic disease.     

Promoting clinically effective practice: general practitioners' awareness of sources of research evidence

BACKGROUND: Practitioners are being encouraged to base their clinical practice on research evidence. In order to do this, they must be aware of and use the sources of evidence. METHODS: A questionnaire survey was undertaken to establish GPs' awareness of research evidence in their clinical practice and, in fundholding practices, its influence on purchasing plans. Questionnaires were sent to 360 lead fundholders in North Thames Region and 440 of a random sample of the remaining general practitioners in the region for comparison. RESULTS: Questionnaires were returned by 62% of lead fundholders and 63% of GPs in the random sample. There was limited use of the electronic sources of clinical effectiveness. There was greater reported awareness of published sources of research evidence and fundholding GPs were significantly more likely to have referred to publications summarizing research evidence. CONCLUSIONS: GPs seem to make more use of published clinical effectiveness sources than the electronic databases. Consequently, they need educational and technical support if they are to make full use of the available sources of research evidence available in other media.      

Pythium insidiosum sp. nov., the etiologic agent of pythiosis.

Pythium insidiosum sp. nov., the etiologic agent of pythiosis, a cosmopolitan disease of horses, cattle, and dogs, is described and illustrated.