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.

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.

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.

Time and cost analysis of repacking medications in unit-of-use containers.

OBJECTIVES: To evaluate the effects of repacking drugs in unit-of-use containers in community pharmacies. The purpose of this study was to examine whether unit-of-use repacking reduces routine mechanical "counting and pouring" to allow more time for pharmaceutical care. DESIGN: Simulation pilot study to evaluate the differences between the existing and proposed systems. Based on the literature, four variables--optimum pack size, time savings, packaging costs, and shelving requirements--were selected for evaluation. Historical prescription data from a chain were used in determining the sample drugs and their optimum pack sizes. Workflow analysis and time study were used to estimate the time savings. Manufacturer bottles, repack bottles, and shelves were measured to determine the impact of using unit-of-use containers on storage requirements. SETTING: Three community pharmacies in a major drugstore chain in Cincinnati, Ohio. RESULTS: The 25 fastest-moving solid oral dosage forms, representing 21.6% of all drugs dispensed by the chain, were selected as the sample drugs for the study. The workflow analysis and time study revealed that 0.79 minutes could be saved per prescription if repacked drugs were used. There was an increased cost of approximately $0.05 for every repack bottle used in place of a prescription vial. It was calculated that repacking in unit-of-use containers would increase storage requirements in the pharmacy by 2.5 times if current inventory levels were maintained. CONCLUSION: Repacking drugs in unit-of-use containers is potentially an inexpensive method to increase usable time in the pharmacy that does not require an increase in personnel or the purchase of additional technology at the store level.

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.

The in vitro anti-tumor activity of some crude and purified components of blackseed, Nigella sativa L.

A crude gum, a fixed oil and two purified components of Nigella sativa seed, thymoquinone (TQ) and dithymoquinone (DIM), were assayed in vitro for their cytotoxicity for several parental and multi-drug resistant (MDR) human tumor cell lines. Although as much as 1% w/v of the gum or oil was devoid of cytotoxicity, both TQ and DIM were cytotoxic for all of the tested cell lines (IC50's 78 to 393 microM). Both the parental cell lines and their corresponding MDR variants, over 10-fold more resistant to the standard antineoplastic agents doxorubicin (DOX) and etoposide (ETP), as compared to their respective parental controls, were equally sensitive to TQ and DIM. The inclusion of the competitive MDR modulator quinine in the assay reversed MDR Dx-5 cell resistance to DOX and ETP by 6- to 16-fold, but had no effect on the cytotoxicity of TQ or DIM. Quinine also increased MDR Dx-5 cell accumulation of the P-glycoprotein substrate 3H-taxol in a dose-dependent manner. However, neither TQ nor DIM significantly altered cellular accumulation of 3H-taxol. The inclusion of 0.5% v/v of the radical scavenger DMSO in the assay reduced the cytotoxicity of DOX by as much as 39%, but did not affect that of TQ or DIM. These studies suggest that TQ and DIM, which are cytotoxic for several types of human tumor cells, may not be MDR substrates, and that radical generation may not be critical to their cytotoxic activity.

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.

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.

Exploring a fourth dimension: spirituality as a resource for the couple therapist.

This article explores ways in which the therapist's own spirituality can serve as a resource in couple therapy. Spirituality is defined as subjective engagement with a fourth, transcendent dimension of human experience. This engagement enhances human life and evokes corresponding behavior. Spiritually based therapy may be influenced by three assumptions: that God or a Divine Being exists, that human-kind yearns innately for connection with this Being, and that this Being is interested in humans and acts upon and within their relationships to promote beneficial change. In therapy these assumptions affect how the therapist listens and responds throughout sessions. The authors incorporate a case example illustrating the application of this fourth dimension in couple therapy.

Structure-activity studies of L-canaline-mediated inhibition of porcine alanine aminotransferase.

L-Canaline [L-2-amino-4-(aminooxy)butanoic acid] (L-CAN) and a family of eleven structurally related analogs were synthesized and evaluated for their inhibitory effect on PLP-dependent alanine aminotransferase (AlaAT) (EC 2.6.1.2) obtained from porcine heart. These congeners were selected to determine the stereochemical, aliphatic chain length, and aminooxy substitutional effects on L-CAN-mediated inhibition of AlaAT activity. L-CAN was the most effective inhibitor of the tested compounds; 10(-7) M L-CAN elicited a 55% reduction in AlaAT activity after a 5 min exposure. This deleterious effect results from the ability of L-CAN to react avidly with PLP moiety of the enzyme to form a stable, L-CAN-PLP oxime. In contrast, the methyl and ethyl esters of L-CAN reduced AlaAT activity by only 8% and 6%, respectively. While all of the L-enantiomeric forms of the tested compound were more potent AlaAT inhibitors than their corresponding D-stereoisomers, the D-enantiomers, particularly D-canaline, were active. Chain shortening or lengthening dramatically curtailed L-CAN-mediated loss in AlaAT activity, but the replacement of the alpha-amino group with a hydrogen was of little consequence in this regard. AlaAT was treated with L-CAN in the presence of free PLP to assess PLP capacity to protect AlaAT against 10(7) M L-CAN-dependent inactivation. L-CAN retained approximately two-thirds of its inhibitory ability in the presence of equimolar PLP, but AlaAT inhibition was reduced 90% by a 10-fold excess of PLP over L-CAN.