Low molecular weight proteins as carriers for renal drug targeting. Preparation of drug-protein conjugates and drug-spacer derivatives and their catabolism in renal cortex homogenates and lysosomal lysates.

Low molecular weight proteins (LMWPs) are known to be reabsorbed and catabolized primarily by the proximal tubular cells of the kidneys. As such, LMWPs might serve as drug carriers that release drugs site-specifically in the kidney. We tested this concept in vitro by coupling different drugs to the LMWP lysozyme both directly (amide bond) and via different spacers: oligopeptides (amide bond), (poly-)alpha-hydroxy acids (ester bond), and a pH sensitive cis-aconityl spacer (amide bond). The capability of the kidney to release the parent drug from such drug-spacer derivatives and drug-LMWP conjugates by enzymatic or chemical hydrolysis of the bond was tested by incubation experiments in renal cortex homogenates and lysosomal lysates. Directly coupled conjugates of terminal carboxyl group containing drugs and lysozyme were catabolized to single amino acids, but did not result in release of the parent drug. The amide bond between the drug and the final amino acid (lysine) appeared to be stable in the incubation milieu. Different oligopeptide spacers coupled to the drugs showed similar results: the oligopeptide itself was cleaved but the amide bond between the drug and different single amino acids remained untouched. Only amide bonds of derivatives of carboxylic drugs with peptide structures themselves were cleaved. Some of the directly coupled conjugates of terminal amino drugs and oligopeptides showed clear release of the parent drug whereas others were stable. Terminal amino drugs were rapidly released from an acid-sensitive cis-aconityl spacer. Terminal carboxyl group containing drugs were enzymatically released from their glycolic and lactic ester spacers at different rates. These kinds of drugs were also released as parent drug from LMWP conjugates with ester spacers like L-lactic acid. Increasing spacer length by intercalating a tetra(L-lactic acid) molecular between the drug and the protein further increased the extent and rate of drug release, indicating increased accessibility of the bond to the enzymes. Terminal amino group containing drugs were rapidly generated as parent drug from LMWP conjugates using an acid-sensitive spacer. In addition the conjugates were found to be adequately stable in plasma, considering their rapid clearance from the bloodstream. It is concluded that LMWPs may indeed be of use as carriers for specific renal delivery of drugs, since renal cortex homogenates and lysosomal lysates are able to catabolize the protein and generate the parent drug from drug-LMWP conjugates bearing suitable spacers. The option of enzymatic release is limited by the narrow specificity of the lysosomal enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis and identification of twelve A-ring reduced 6 alpha- and 6 beta-hydroxylated compounds derived from 11-deoxycortisol, corticosterone and 11-dehydrocorticosterone.

The synthesis and identification of 12 A-ring reduced 6 alpha-(and 6 beta-)hydroxylated compounds derived from 11-deoxycortisol (S), corticosterone (B) and 11-dehydrocorticosterone (A) are reported here. These steroids were prepared in two steps from the corresponding 6 6 alpha-(and 6 beta-)hydroxy-4-pregnene-3-ones. Selective reduction of the 4,5 double bond yielded 12 6 alpha-(and 6 beta)hydroxy-5 alpha-(and 5 beta)pregnane-3,20-diones. Enzymatic reduction of these compounds with NADH and 3 alpha-hydroxysteroid dehydrogenase yielded the corresponding tetrahydro steroids. The steroids were characterized by high performance liquid chromatography (HPLC), gas chromatography mass spectrometry (GC and GC/MS) and in part by 1H-NMR. 6 beta OH-THS and 6 beta OH-5 alpha THS were identified by 1H-NMR. The structures of the two precursors, i.e. 6 beta OH-5 beta DHS and 6 beta OH-5 alpha DHS were confirmed by 1H-NMR using two-dimensional spectra. 6 alpha OH-THS was identified by comparing its HPLC, GC and MS data with those of the steroid obtained by enzymatic oxidation of the standard reference steroid 6 alpha OH-20 beta HHS to the corresponding 20-ketosteroid. The other steroids, e.g. 6 alpha OH-THB and 6 alpha OH-5 alpha THB were identified by using the proved sequence of elution of each of the epimer pairs on the normal phase HPLC column (5 alpha < 5 beta), and by the reversed order of elution of the same epimer pair as the methoxime-trimethylsilyl ethers on the GC column (5 alpha > 5 beta) and by the mass spectra, with the exception of 6 beta OH-THA.

Isolation of two cytotoxic diterpenes from the fern Pteris multifida.

From aerial parts of the fern Pteris multifida Poir. (Polypodiaceae) two diterpenes, entkaurane-2 beta, 16 alpha-diol and ent-kaur-16-ene-2 beta, 15 alpha-diol, were isolated by repeated column chromatography using silica gel and silica gel impregnated with silver nitrate. The structures were confirmed by spectroscopic methods. Both compounds showed a moderate cytotoxicity to Ehrlich ascites tumour cells.

Electron-dense granules in Desulfovibrio gigas do not consist of inorganic triphosphate but of a glucose pentakis(diphosphate).

Under certain growth conditions the sulfate-reducing bacterium Desulfovibrio gigas forms electron-dense granules in the cells which had been claimed to consist of a magnesium triphosphate). We observed granules after cultivation in media with a low Fe2+ or NH4+ concentration and reinvestigated the nature of the electron-dense bodies. Energy-dispersive X-ray analysis of the granules in the cells showed that they contain large amounts of P, Mg, and K. Gel electrophoresis and chromatographic analyses of isolated granules which had been dissolved in 20 mM EDTA, however, revealed discrepancies with commercially available polyphosphates. 31P-NMR spectra also lacked the peaks in the -22-ppm region which are characteristic for inner phosphates of polyphosphates confirming that the phosphocompound as isolated from the electron-dense bodies of D. gigas did not consist of polyphosphates. Using multinuclear NMR spectroscopy we showed that the electron-dense bodies of D. gigas contained a novel metabolite which was identified as alpha-glucose 1,2,3,4,6-pentakis(diphosphate).

31phospho-NMR demonstration of phosphocysteine as a catalytic intermediate on the Escherichia coli phosphotransferase system EIIMtl.

The mannitol-specific phosphotransferase system transport protein, Enzyme IIMtl, contains two catalytically important phosphorylated amino acid residues, both present on the cytoplasmic part of the enzyme. Recently, this portion has been subcloned, purified, and shown to be an enzymatically active domain. The N-terminal half has also been subcloned and shown to be the mannitol-binding domain. When combined the two domains catalyze mannitol phosphorylation at the expense of phospho-HPr (van Weeghel, R. P., Meyer, G. H., Pas, H. H., Keck, W. H., and Robillard, G. T., Biochemistry in press). The phospho-NMR spectrum of the purified phosphorylated cytoplasmic domain, taken at pH 8.0, shows two signals, one at -6.9 ppm compared with inorganic phosphate resulting from phosphohistidine and one at +11.9 ppm originating from phosphocysteine. Addition of mannitol plus membranes containing the N-terminal mannitol-binding domain results in the formation of mannitol 1-phosphate and the disappearance of the two signals at -6.9 and +11.9 ppm.