David R. Worthen

An in vivo evaluation of the antiseizure activity and acute neurotoxicity of agmatine.

Agmatine, an endogenous cationic amine, exerts a wide range of biological effects, including modulation of glutamate-activated N-methyl-D-aspartate (NMDA) receptor function in the central nervous system (CNS). Since glutamate and the NMDA receptor have been implicated in the initiation and spread of seizure activity, the capacity of agmatine to inhibit seizure spread was evaluated in vivo. Orally administered agmatine (30 mg/kg) protected against maximal electroshock seizure (MES)-induced seizure spread in rats as rapidly as 15 min and for as long as 6 h after administration. Inhibition of MES-induced seizure spread was also observed when agmatine was administered intraperitoneally. Agmatines antiseizure activity did not appear to be dose-dependent. An in vivo neurotoxicity screen indicated that agmatine was devoid of any acute neurological toxicity at the doses tested. These preliminary data suggest that agmatine has promising anticonvulsant activity.

Development of a peptide-containing chewing gum as a sustained release antiplaque antimicrobial delivery system.

The objective of this study was to characterize the stability of KSL-W, an antimicrobial decapeptide shown to inhibit the growth of oral bacterial strains associated with caries development and plaque formation, and its potential as an antiplaque agent in a chewing gum formulation. KSL-W formulations with or without the commercial antibacterial agent cetylpyridinium chloride (CPC) were prepared. The release of KSL-W from the gums was assessed in vitro using a chewing gum apparatus and in vivo by a chew-out method. A reverse-phase high-performance liquid chromatography method was developed for assaying KSL-W. Raw material stability and temperature and pH effects on the stability of KSL-W solutions and interactions of KSL-W with tooth-like material, hydroxyapatite discs, were investigated.KSL-W was most stable in acidic aqueous solutions and underwent rapid hydrolysis in base. It was stable to enzymatic degradation in human saliva for 1 hour but was degraded by pancreatic serine proteases. KSL-W readily adsorbed to hydroxyapatite, suggesting that it will also adsorb to the teeth when delivered to the oral cavity. The inclusion of CPC caused a large increase in the rate and extent of KSL-W released from the gums. The gum formulations displayed promising in vitro/ in vivo release profiles, wherein as much as 90% of the KSL-W was released in a sustained manner within 30 minutes in vivo. These results suggest that KSL-W possesses the stability, adsorption, and release characteristics necessary for local delivery to the oral cavity in a chewing gum formulation, there-by serving as a novel antiplaque agent.

The antiproliferative and immunotoxic effects of L-canavanine and L-canaline

L-Canavanine and its arginase-catalyzed metabolite, L-canaline, are two novel anticancer agents in development. Since the immunotoxic evaluation of agents in development is a critical component of the drug development process, the antiproliferative effects of L-canavanine and L-canaline were evaluated in vitro. Both L-canavanine and L-canaline were cytotoxic to peripheral blood mononucleocytes (PBMCs) in culture. Additionally, the mononucleocytes were concurrently exposed to either L-canavanine or L-canaline and each one of a series of compounds that may act as metabolic inhibitors of the action of L-canavanine and L-canaline (L-arginine, L-ornithine, D-arginine, L-lysine, L-homoarginine, putrescine, L-ω-nitro arginine methyl ester and L-citrulline). The capacity of these compounds to overcome the cytotoxic effects of L-canavanine or L-canaline was assessed in order to provide insight into the biochemical mechanisms that may underlie the toxicity of these two novel anticancer agents. The results of these studies suggest that the mechanism of L-canavanine toxicity is mediated through L-arginine-utilizing mechanisms and that the L-canavanine metabolite, L-canaline, is toxic to human PBMCs by disrupting polyamine biosynthesis. The elucidation of the biochemical mechanisms associated with the effects of L-canavanine and L-canaline on lymphoproliferation may be useful for maximizing the therapeutic effectiveness and minimizing the toxicity of these novel anticancer agents.

A novel technique for visualizing the intracellular localization and distribution of transported polyamines in cultured pulmonary artery smooth muscle cells

The use of a combination of monofluorescein adducts of spermidine (FL-SPD) and spermine (FL-SPM) with confocal laser scanning microscopy (CLSM) provides a useful means for monitoring the fate and time-dependent changes in the distribution of transported polyamines within living cells. Polyamine-fluorescein adducts were synthesized from fluorescein isothiocyanate and the appropriate polyamine. Monofluorescein polyamine adducts (ratio 1:1) were isolated using thin layer chromatography, and the structure and molecular weight of the monofluorescein polyamine adducts were confirmed using NMR and mass spectroscopy, respectively. The covalent linkage of the fluorescent adduct moiety to SPD and SPM did not influence their rate of uptake by bovine pulmonary artery smooth muscle cells (PASMC). Similar to 14C-SPD and 14C-SPM, the rate of uptake of 14C-FL-SPD and 14C-FL-SPM in PASMC was temperature-dependent. Treatment for 24 h with difluoromethylornithine (DFMO), a selective blocker of the enzyme ornithine decarboxylase and an inducer of the polyamine transport system, significantly increased the cellular uptake of 14C-FL-SPD and 14C-FL-SPM compared to that of control cells. When compared to control cells, treatment of PASMC with the pyrrolizidine alkaloid monocrotaline for 24 h also significantly increased the cellular uptake of 14C-FL-SPD and 14C-FL-SPM. On the other hand, 24 h treatment of PASMC with a polymer of SPM, a selective blocker of the polyamine transport system, or with free spermine, markedly reduced the cellular accumulation of 14C-FL-SPD and 14C-FL-SPM. After a 20-min treatment of PASMC with FL-SPD or FL-SPM, CLSM revealed that adduct fluorescence was localized in the cytoplasm of living cells. Treatment with DFMO increased the cytoplasmic accumulation of both FL-SPD and FL-SPM. In addition, the fluorescence observed in the cytoplasm of chinese hamster ovary cells (CHO) was significantly higher than that detected in the cytoplasm of their polyamine transport deficient variants (CHOMGBG). The results of this study provide the first evidence of the utility of a novel method for visualizing the uptake, distribution, and cellular localization of transported polyamines in viable cultured mammalian cells.

Formulating a sulfonated antiviral dendrimer in a vaginal microbicidal gel having dual mechanisms of action.

SPL7013 is the sodium salt of a sulfonated dendrimer that has potent antiviral properties. VivaGel®, a topical gel containing 3% (wt/wt) SPL7013, is in development as a vaginal microbicide. BufferGel® is a Carbopol®-based acidic buffering gel that enhances the natural protective action of the vagina to produce a broad-spectrum microbicidal environment. The positive attributes of both gels were combined into a combination vaginal microbicidal gel having dual mechanisms of action. A 3% (wt/wt) SPL7013 combination gel, pH 3.7, was developed and fully characterized and was shown to have more than twofold greater acidic buffering capacity than BufferGel. Ultracentrifugation experiments demonstrated that SPL7013 was not sequestered or entropically trapped in the viscous gel, thereby confirming, along with viral challenge studies, that SPL7013 has sufficient mobility in the viscous gel to exert antiviral properties.

l-Canavanine modulates cellular growth, chemosensitivity and P-glycoprotein substrate accumulation in cultured human tumor cell lines

L-Canavanine (L-CAV) is a naturally occurring L-arginine analog that induces the formation of non-functional proteins in a variety of organisms. Previous studies have shown that L-CAV is cytotoxic for several human tumor cell lines. In this study, we have evaluated the cytotoxicity of L-CAV for both parental and multi-drug resistant (MDR) human tumor cells. We have also determined the effect of L-CAV exposure on cellular expression and activity of the MDR P-glycoprotein (P-gp) membrane efflux pump, and the effect of L-CAV on cellular accumulation of P-gp substrates. The effect of pre-treatment with non-cytotoxic doses of L-CAV on cellular sensitivity to ten standard antineoplastic agents was also evaluated, in order to assess the chemosensitization potential of L-CAV. 3-(4,5-Dimethylthiazol-)2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity assays revealed that the MDR variants of human uterine sarcoma and leukemic cells were equally sensitive to L-CAV as compared with their respective parental controls. Although the presence of free L-CAV in the uptake media did not influence cellular accumulation of P-gp substrates, cells cultured for 72 h in 250 microM L-CAV accumulated from 16 to 23% less P-gp substrate than untreated controls. Although L-CAV-cultured sarcoma cells accumulated 17% less doxorubicin (DOX) than untreated controls, they were three times more sensitive to its cytotoxic effects. L-CAV-treated cells were also significantly more sensitive to cisplatin, 5-fluorouracil, mitoxantrone and bleomycin than were untreated controls. Indirect immunofluorescence revealed that 72-h exposure to as much as 1000 microM L-CAV did not alter cellular expression of P-gp. These studies suggest that L-CAV may be equally cytotoxic for both parental and MDR tumor cells, and that L-CAV neither induces the expression of, nor is a substrate for, P-gp. The observation that L-CAV pre-treatment reduces cellular accumulation of DOX, yet sensitizes tumor cells to DOX and other DNA-targeting antineoplastic drugs, suggests a role for L-CAV as a chemosensitizer for the chemotherapy of cancer.

Endogenous indoles as novel polyamine site ligands at the N-methyl-D-aspartate receptor complex

High-throughput ligand displacement screens of a series of endogenous indoles revealed that tryptamine, serotonin and 5-methoxytryptamine readily displace [3H]spermidine and [3H]MK-801 from their respective binding sites in rat brain homogenate. These data, coupled with their potent inhibition of spermidine-potentiated [3H]MK-801 binding, suggest that certain endogenous indoles may act as ligands to one or more polyamine binding sites in the brain, including those on the N-methyl-D-aspartate receptor complex.

Methods for the Recovery and Purification of Polyene Antifungals

Despite the development of newer antifungal drugs, the polyene antifungals continue to be the most potent broad-spectrum fungicides available for clinical use. The incidence and severity of fungal infections are on the rise, underscoring the need for new and more effective antifungal drugs. Thus, the search for new polyene antifungals is ongoing. The limited solubility, polymorphic character, and inherent chemical instability of these compounds make their economical recovery and purification from mass culture challenging problems in biotechnology. This article provides a comprehensive review of the methods that have been developed for the recovery and purification of amphotericin B and nystatin, the two most important polyenes currently in clinical use.

Aminoanthraquinones as novel ligands at the polyamine binding site on the N-methyl-D-aspartate receptor complex.

As part of a drug discovery program using high-throughput radioligand-binding assays, aminoanthraquinones were identified as potential modulators of N-methyl-D-aspartate (NMDA) receptor function. Aminoanthraquinones may represent a novel class of polyamine binding site ligands with a unique pharmacophore and may facilitate the rational design of novel NMDA-receptor modulators.

A Unique Interaction between Polyamine and Multidrug Resistance (P-glycoprotein) Transporters in Cultured Chinese Hamster Ovary Cells Transfected with Mouse mdr-1 Gene

We have shown that a functional link exists between the polyamine transporter and the multi-drug resistance (MDR) efflux transporter (P-glycoprotein, P-gp) in MDR-positive cancer cells. To further explore the nature of this interaction, we have examined the effect of reduced polyamine transport activity on cellular expression and activity of P-gp acquired by either selection or transfection. Chinese hamster ovary (CHO) cells and their polyamine transport-deficient mutants (CHOMGBG) were transfected with mouse mdr-1b gene. The activity of P-gp in these cells was quantified by measuring cellular accumulation of radiolabeled taxol and etoposide in the presence and absence of the P-gp modulator SDZ PSC-833 (valspodar; a semisynthetic undecapeptide derived from cyclosporin D). The mdr-1b-transfected CHO cells accumulated 2- to 3-fold less taxol and etoposide than the controls, an accumulation defect reversed by the potent MDR modulator PSC-833. Despite expression of P-gp on the surface of mdr-1b-transfected CHOMGBG cells, this classic MDR phenotype was not observed. Similarly, CHO cells, but not CHOMGBG cells, showed MDR activity after selection with doxorubicin as determined by reduced accumulation of radiolabeled taxol. Treatment with 50 microM of reduced polymer of spermine and glutaraldehyde, a selective blocker of the polyamine transport system, reduced MDR activity in mdr-1-transfected CHO cells and restored cellular accumulation of etoposide and taxol to control levels, effects not observed in mdr-1-transfected CHOMGBG cells. Notably, mdr-1-transfected CHO cells were 4- to 16-fold more resistant to the cytotoxic effects of the P-gp substrates doxorubicin, taxol, and etoposide than were the mdr-1-transfected CHOMGBG cells. CHO cells transfected with the mdr-1 gene exhibited a 23% reduction in cellular uptake of [14C]spermidine compared with untransfected controls; spermidine accumulation in CHOMGBG cells was no different than that in untransfected controls. These data suggest that the existence of a functioning polyamine transport system may be a requirement for MDR transporter activity, while the expression of functioning P-gp appears to reduce polyamine transporter activity.