No abstract

The blood-brain barrier restricts the passage of substances into the brain. Neuropeptides, such as enkephalins, cannot be delivered into the brain when given systemically because of this barrier. Therefore, there is a need to develop efficient transport systems to deliver these drugs to the brain. Recently, we have demonstrated that conjugation of doxorubicin or penicillin to peptide vectors significantly enhances their brain uptake. In this study, we have conjugated the enkephalin analog dalargin with two different peptide vectors, SynB1 and SynB3, to improve its brain delivery and its pharmacological effect. We show by in situ brain perfusion that vectorization markedly enhances the brain uptake of dalargin. We also show using the hot-plate model that this enhancement in brain uptake results in a significant improvement in the observed antinociceptive effect of dalargin. These results support the usefulness of peptide-mediated strategies for improving the availability and efficacy of central nervous system drugs.Brain delivery is one of the major challenges for the neuropharmaceutical industry since increasing number of hydrophilic therapeutic agents, such as anticancer drugs, antibiotics, and antiviral drugs are unable to cross the blood-brain barrier (BBB). The BBB represents a complex endothelial interface in vertebrates that separates the blood compartment from the extracellular fluid compartment of the brain parenchyma. The capillaries in the brain parenchyma possess a high electrical resistance due to tight junctions between the endothelial cells and also lack pores. Thus, the brain capillary endothelium behaves like a continuous lipid bilayer, and diffusion through this BBB layer is largely dependent on the lipid solubility of the drug. Because peptides are hydrophilic, biologically unstable, and large molecules, it is difficult for them to penetrate the BBB. Even though their brain uptake is not so high, some peptides and proteins are delivered into the brain by carrier-mediated transport, receptor-mediated transport, or adsorptive-mediated transport mechanisms. One of the problems associated with the inability of many peptides and proteins to accumulate in the brain in therapeutically meaningful amounts is the efflux transport systems. For example, it has been shown that the selective ␦-opioid receptors against [D-Pen 2 ,D-Pen 5 ]-enkephalin (DPDPE) has a poor BBB permeability that is explained in part by P-glycoprotein (P-gp)-mediated efflux, and DPDPE is also a substrate of the rat organic anion transporting polypeptide 2 (OATP2) and human OATP-A (Kakyo et al., 1999;Gao et al., 2000).To overcome the limited access of drugs to the brain, various strategies have been applied to direct central nervous system (CNS) drugs into the brain . Most of these methods are invasive, such as surgical implantation of an intraventricular catheter followed by drug infusion into the ventricular compartment, transient opening of the tight junctions by the intracarotid infusion of a hypertonic solution (Chamberl...

Ellipticine derivatives have potential as anticancer drugs. Their clinical use has been limited, however, by poor solubility and host toxicity. As N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-anticancer conjugates are showing promise in early clinical trials, a series of novel HPMA copolymer conjugates have been prepared containing the 6-(3-aminopropyl)-ellipticine derivative (APE, NSC176328). Drug was linked to the polymer via GFLG or GG peptide side chains. To optimize biological behavior, HPMA copolymer-GFLG-APE conjugates with different drug loading (total APE: 2.3-7% w/w; free APE: <0.1% w/w) were synthesized. Conjugation of APE to HPMA copolymers considerably increased its aqueous solubility (>10-fold). HPMA copolymer-GG-APE did not liberate drug in the presence of isolated lysosomal enzymes (tritosomes), but HPMA copolymer-GFLG-APE released APE to a maximum of 60% after 5 h. The rate of drug release was influenced by drug loading; lower loading led to greater release. Whereas free APE (35 microg/mL) caused significant hemolysis (50% after 1 h), HPMA copolymer-APE conjugates were not hemolytic up to 300 microg/mL (APE-equiv). As would be expected from its cellular pharmacokinetics, HPMA copolymer-GFLG-APE was >75 times less cytotoxic than free drug (IC(50) approximately 0.4 microg/mL) against B16F10 melanoma in vitro. However, in vivo when tested in mice bearing s.c. B16F10 melanoma, HPMA copolymer-GFLG-APE (1-10 mg/kg single dose, APE-equiv) given i.p. was somewhat more active (highest T/C value of 143%) than free APE (1 mg/kg) (T/C =127%). HPMA copolymer-APE conjugates warrant further evaluation as potential anticancer agents.

Norfloxacin (Nflx), an antibiotic which is active against some intracellular bacteria, was coupled to a polymeric carrier, namely poly(L-lysine citramide) via a lysine or an ethylcarbamate spacer to obtain a macromolecular prodrug. The carrier, which derived from the two metabolites citric acid and L-lysine, is known to be biocompatible and slowly degradable under slight acidic conditions. Conjugates were characterised by UV, 1H and 13C NMR and SEC. The presence of Norfloxacin and the lysine type spacer caused chain aggregation, due to a probable physical cure. The release of Norfloxacin from these prodrugs and from a prodrug where Norfloxacin is bound to the carrier backbone without spacer arm was investigated comparatively in vitro. Conjugation via a carbamate-type linkage appeared as a method to achieve the release of Norfloxacin from a PLCA-type conjugate at neutral.

Oligosaccharides are implicated in the development of the immune response notably in complement activation. Anti-tumoural immunotherapy by monoclonal antibodies (mAbs) offers some advantages to chemotherapy including cell targeting but some of them are inefficient to generate cytotoxicity dependent complement (CDC) known to be important in the antibody's efficacy. The aim of this study is to give a CDC activity of mAb by linkage of a complement activating oligosaccharide to this antibody via a hetero-bifunctional linker allowing control of the conjugation reaction. We worked on non Hodgkin Burkitt's lymphoma as cancer source, Fab fragments of rituximab devoid of complement activity as mAb and the trisaccharide Galα(1→3)Galβ(1→4)GlcNAc as immunogenic glycan. The bioconjugate Fab-Gal was characterized by biochemical methods and we demonstrated that the α-Gal epitope was recognized by seric immunoglobulins. After checking the recognition capacity of the FabGal conjugate for the CD20 epitope, in vitro assays were performed to evaluate the activation of the complement cascade by the Fab-Gal conjugate. The effect of this bioconjugate was confirmed by the evaluation of the proliferation response of Burkitt's cell line. The relative facility realization of this strategy represents new approaches to increase activities of mAbs.