Attempts to target antitumor drugs toward opioid receptor-rich mouse tumor cells with enkephalin-ellipticinium conjugates.

Human colorectal and pulmonary carcinomas have been shown to contain high levels of opioid peptides and their corresponding membrane-bound receptors. Therefore possible targeted drugs, consisting of modified enkephalins linked to cytotoxic drugs, were designed. Such conjugates were expected to be specifically internalized within opioid receptor-bearing cells. As a model to this approach, we have synthesized enkephalin-ellipticinium conjugates in which the D-Ala2-D-Leu5-enkephalin (DADLE) was coupled to the 2-nitrogen of either ellipticine or 9-hydroxyellipticine, two drugs acting through different mechanisms of cytotoxicity. These conjugates, DADLE-ellipticinium (NME) and DA-DLE-9-hydroxyellipticinium (NMHE), respectively, were previously shown to retain in vitro both opioid receptors and DNA affinities close to those of the parent compounds. In this paper, we first show that each individual moiety in the complexes remains capable of recognizing its cellular targets. Thus, pretreatment of NG108-15 cells containing delta-opioid receptors by the DADLE-ellipticinium conjugates induced a loss of opioid receptor (down-regulation), while the smaller peptide conjugates, tyrosinyl-D-alanylglycine-ellipticinium, prepared as control, do not. On the other hand, peptide-NMHE conjugates were able to induce DNA topoisomerase II-associated DNA strand breaks suggesting that they have a mode of action similar to that of their parent molecule, NMHE. We then examined whether or not these molecules could exert a specific toxicity on opioid receptor-bearing cells. However, when tested on NG108-15 tumor cells and L-fibroblasts as control, the enkephalin-ellipticinium conjugates (DADLE-NME and DADLE-NMHE) proved to be similarly more cytotoxic on both cell lines than their ellipticinium (NME and NMHE) precursors. In order to understand this apparent lack of specificity we examined the cellular accumulation and distribution of DADLE-NME by fluorescence techniques. These experiments revealed that an important intracellular overconcentration caused by a nonspecific process is probably masking the specific targeted effect of the conjugates. Hence, the project of linking DADLE to highly cytotoxic molecules which cannot cross the plasma membrane without site-directed targeting is discussed.

Comparative binding properties of linear and cyclic delta-selective enkephalin analogues: [3H]-[D-Thr2, Leu5] enkephalyl-Thr6 and [3H]-[D-Pen2, D-Pen5] enkephalin.

The range of delta-selectivity of linear and cyclic analogues of enkephalin in rat brain was found to be: [D-Pen2, L-Pen5] enkephalin (DPLPE) greater than [D-Pen2, D-Pen5] enkephalin (DPDPE) greater than [D-Thr2, Leu5] enkephalyl-Thr6 (DTLET) greater than [D-Ser2, Leu5] enkephalyl-Thr6 (DSLET). Saturation experiments performed with [3H]DPDPE and [3H]DTLET in NG108-15 cells and rat brain showed similar binding capacities for both the ligands, but the delta-affinity of [3H]DTLET (KD approximately 1.2 nM) was much better than that of [3H]DPDPE (KD approximately 7.2 nM). The rather low delta-affinity of DPDPE induced high experimental errors cancelling the benefit of its better delta-selectivity. Binding experiments in rat or guinea-pig brains showed, in both cases, the better delta-selectivity of [3H]DTLET compared to [3H]DSLET. The former peptide remains at this time the most appropriate radioactive probe for binding studies of delta-receptor.

Antibodies specific for the neuronal form of the Src protein elicited by an antigenized antibody.

To elicit antibodies directed specifically against the neuron-specific form of the c-src gene product, pp60c-src(+), we used an antigenized antibody comprising a decamer containing the amino acid sequence specific to pp60c-src(+) inserted into the third hypervariable loop of the heavy (H)-chain variable (V)-region. This was used to raise anti-idiotype antibodies reacting with the peptide epitope in rabbits. The antisera reacted with pp60c-src(+), as judged by immune blotting, immunoprecipitation, immune complex kinase assay, and indirect immunofluorescence staining, but did not react with the fibroblast form of the c-src gene product, pp60c-src. Antigenized antibody is a useful approach for producing antibodies able to distinguish between isoforms of the same gene product and specific for the neuronal form of the Src protein.

Antitumor activity of the new vinca-alkaloid S 12363 alone or in combination with verapamil on a human multidrug resistant renal carcinoma xenograft.

We have established a model of human renal cell carcinoma, Kgg2, transplanted into athymic nude mice which expressed P-glycoprotein (P-gp) (detected by flow cytometry) and a high level of mRNA transcript of mdr1 gene (Northern blot analysis). We have evaluated the antitumor activity of a new highly potent vinca-alkaloid derivative, S 12363, in comparison with the activity of the reference compound vinblastine (VLB), when used alone or in combination with verapamil (VRP). The influence of the calcium influx blocker verapamil on the activity of the combination of S 12363 with adriamycin (ADR) was also determined. The results showed that S 12363 at a dose of 0.05 mg/kg/day, administered alone by intraperitoneal route daily on days 1 to 5, induced a tumoral regression of 50% during the first days after treatment. This effect was potentialized by simultaneous treatment with verapamil at 20 mg/kg/day for 5 days, leading to a long-term reduction of 70% of tumor growth. Vinblastine at a dose of 0.4 mg/kg/day administered alone or in combination with verapamil, using the same protocol, was less efficient. The association of S 12363 at 0.075 mg/kg/day (on days: 1-5, 11, 21 and 31), adriamycin at 2 mg/kg/day (on days: 11, 21 and 31) and verapamil at 20 mg/kg/day (on days: 0-5, 11, 21 and 31) induced an important reduction of tumor growth of 80% at the end of the experiment. In conclusion, the new vinca-alkaloid derivative S 12363 could present a therapeutic advantage over the reference compound vinblastine in the treatment of renal cell carcinoma.(ABSTRACT TRUNCATED AT 250 WORDS)

The human saphenous vein in pharmacology: effect of a new micronized flavonoidic fraction (Daflon 500 mg) on norepinephrine induced contraction.

Local acidosis (pH 6.4) depresses reactivity of vascular smooth muscle and especially the response of human isolated saphenous veins to exogenous norepinephrine. Experiments were performed to study, under acidosis conditions, the interaction between Daflon 500 mg, a micronized fraction of 90% diosmin and 10% hesperidin, and norepinephrine on human rings of veins. Varicose veins were obtained by conservative varicose veins surgery and normal veins from patients undergoing coronary artery bypass graft surgery. Isometric tension was recorded from venous rings in organ chambers filled with Krebs-Henseleit solution (pH 7.4; 37 degrees C). Metabolic acidosis (from pH 7.4 to 6.4) was obtained by lowering the HCO3- concentration of the Krebs-Henseleit solution. Cumulative concentration-response curves for norepinephrine (10(-7) to 10(-5)M) were obtained at pH 6.4 in the presence or in the absence of Daflon 500 mg (10(-5)M) added 20 min previously to the organ bath. Under acidotic conditions, Daflon 500 mg induced a shift to the left of the concentration-response curves for norepinephrine. This potentiation was significant in both normal and varicose veins and was increased in proportion with the pathological status of the venous rings. These results support the therapeutic benefits of Daflon 500 mg in chronic venous insufficiency.

Synthesis and binding properties to DNA and to opioid receptors of enkephalin-ellipticinium conjugates.

In order to specifically direct cytotoxic agents against tumor cells bearing delta opioid receptors, the DNA intercalating agents ellipticine and 9-OH-ellipticine were coupled by quaternarization of the pyridine nitrogen to an enkephalin modified pentapeptide through a short chemical linker. The ellipticine ring of these conjugates was shown to intercalate into DNA, with DNA affinity constants close to those of the non-conjugated ellipticines. Despite the addition of a polycyclic ring to the C-terminal amino acid, the D-Ala2-D-Leu5-enkephalin-ellipticine conjugates bind to the opioid receptor from rat brain and NG 108-15 cells with an affinity constant close to 10(8) M-1. Other derivatives were synthesized as a control using a tripeptide which does not bind to the opioid receptor.