title: 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.
abstract: 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)
authors.affiliation: Department of Pharmacology and Pharmacotherapy, University Center for Pharmacy, Groningen, The Netherlands.
authors.forename: E J
authors.initials: EJ
authors.lastname: Franssen
authors.forename: J
authors.initials: J
authors.lastname: Koiter
authors.forename: C A
authors.initials: CA
authors.lastname: Kuipers
authors.forename: A P
authors.initials: AP
authors.lastname: Bruins
authors.forename: F
authors.initials: F
authors.lastname: Moolenaar
authors.forename: D
authors.initials: D
authors.lastname: de Zeeuw
authors.forename: W H
authors.initials: WH
authors.lastname: Kruizinga
authors.forename: R M
authors.initials: RM
authors.lastname: Kellogg
authors.forename: D K
authors.initials: DK
authors.lastname: Meijer
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004337:Drug Carriers / Q000737:chemistry*; D010450:Endopeptidases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007672:Kidney Cortex / Q000378:metabolism*; D007773:Lactates / Q000737:chemistry; D019344:Lactic Acid; D007930:Leucine / Q000737:chemistry; D008247:Lysosomes / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D009288:Naproxen / Q000008:administration & dosage / Q000493:pharmacokinetics; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D013388:Succinimides
chemical_list: D000596:Amino Acids; D004337:Drug Carriers; D007773:Lactates; D009842:Oligopeptides; D011506:Proteins; D013388:Succinimides; D012710:Serum Albumin, Bovine; D019344:Lactic Acid; D009288:Naproxen; D009113:Muramidase; D010450:Endopeptidases; D007930:Leucine; C001426:N-hydroxysuccinimide