UCN-01 dose-dependent inhibition of normal hyperproliferative cells in mice.

UCN-01 is a hydroxylated derivative of staurosporine and a potent protein kinase C (PKC) inhibitor. Interest in the potential usefulness of this compound as an anticancer drug stems mainly from its unique anti-signaling, growth-arresting properties on tumor cells. This include activation of CDC2 kinase (CDK1) which interacts with either cyclin A or cyclin B1 at the G1 or G2/M border, suggeting that this event is one of the major consequences of the drug action on eukaryotic cells. Nonetheless, the antiproliferative activity of UCN-01 on normal rapidly dividing cells (intestinal epithelial and bone marrow cells) is not well documented. Thus, the main objective of this study was to investigate the in vivo antiproliferative activity of UCN-01 on these normal hyperproliferative cells and evaluate whether cellular response to UCN-01 could be modulated in the presence of DNA damage. Mice were injected i.m. with a single dose of UCN-01 (2.5 mg/kg-20 mg/kg) followed 3 and 24 h later by in vivo BrdU labeling for 1 h. At autopsy, bone marrow cells were collected and fixed for dual parameter BrdU/DNA flow cytometry. Different regions of the gut were also fixed for immunoperoxidase BrdU assays. Newly replicated cells were mainly located in the lower compartments of the crypt columns and were scored for BrdU stained nuclei using an image analysis system. A comparison between groups showed that 5 mg/kg UCN-01 induced inhibition in BrdU incorporation at 3 and 24 h, as compared to the other groups injected with various doses of UCN-01. Flow cytometric analysis of bone marrow cells stained with fluorescein tagged anti-BrdU (FITC) along with propidium iodide (PI) also showed inhibition in BrdU incorporation of S phase fraction cells in mice treated with 5 mg/kg UCN-01. These bone marrow cells were arrested primarily in the G1 phase of the cell cycle. The colony-forming unit (CFU) assay of the bone marrow cells was then used to determine the level of drug interaction of UCN-01 and, topotecan, a topoisomerase I inhibitor, at a fixed dose ratio. An antagonistic drug interaction (CI > 1) was observed as determined by the median-effect analysis. However, an additive interaction (CI = 1) was obtained with the use of camptothecin or 10,11-methylenedioxycamptothecin and UCN-01. The results of the in vitro drug interaction with UCN-01 may predict protection from topotecan-induced bone marrow toxicity.

Tissue distribution and plasma pharmacokinetics of UCN-01 at steady-state and following bolus administration in rats: influence of human alpha1-acid glycoprotein binding.

The primary focus of this study was to investigate the role of human alpha1-acid glycoprotein (hAGP) on the pharmacokinetics and tissue distribution of the antitumor drug UCN-01 (7-hydroxystaurosporine) in rats, following bolus administration and at steady-state blood concentration. To evaluate plasma pharmacokinetics, the rats received UCN-01 alone, UCN-01 + hAGP (87:1 ratio), or UCN-01 + hAGP (26:1 ratio) i.v. Additional rats were studied after i.m. administration of UCN-01 and i.v. administration of human AGP (87:1 ratio). For tissue distribution, rats received UCN-01 alone, UCN-01 + hAGP (87:1 ratio). One hour after drug administration, blood samples as well as various tissues and organs were collected. Plasma concentrations of UCN-01 as well as tissue accumulation were measured by HPLC using a fluorescence detector. Following i.v. bolus administration, the UCN-01 concentration-time profile declined bi-exponentially. The distribution half-life was 0.2 hours, while the elimination half-life was 6.65 hours. The volume of distribution of the central compartment (Vc) was 1000 ml/kg and the volume of distribution during the elimination phase (Vdb) was 2551 ml/kg. The total body clearance (TBC) was 4.4 ml/min/kg. Co-administration of hAGP with UCN-01 at 1:87 ratio did not affect the elimination half-life of UCN-01 during our sampling period, however the distribution half-life was delayed by approximately 2.7-fold. Furthermore, the Vc, Vd(beta) and TBC were significantly reduced to 395 ml/kg, 735 ml/kg and 1.34 ml/min/kg, respectively. UCN-01 Vd's and TBC were reduced further by increasing human hAGP:UCN-01 ratio to 26:1. Also, hAGP administration did not significantly affect the pharmacokinetic profile of UCN-01 after i.m. administration, which was similar to that measured after i.v. administration. One hour after i.v. bolus administration, UCN-01 was distributed extensively to all tissues with a tissue/plasma ratio ranging from 10-times in the brain to more than 1000-times in the lungs. The presence of hAGP drastically reduced tissue accumulation of UCN-01, although the tissue to plasma ratio remained > 1.0 except for the brain. At steady-state blood concentration following the infusion of UCN-01 over 180 minutes, the ratio of the drug concentration to the concomitant plasma concentration remained > 1.0, even in the presence of hAGP. The data showed that the binding of UCN-01 to hAGP drastically altered its pharmacokinetics and tissue distribution, even at the plasma steady-state concentration.

UCN-01 and camptothecin induce DNA double-strand breaks in p53 mutant tumor cells, but not in normal or p53 negative epithelial cells.

Previous research has shown synergistic growth inhibition between UCN-01 and camptothecin (CPT) in tumor cells with mutant p53 versus tumor cells with wild-type p53. To determine the possible role of p53 in this drug combination, we tested the hypothesis that the synergistic growth inhibition is due to the absence of p53, and can result from the induction of DNA double-strand breaks (DSBs). Experiments were performed with the use of normal human mammary epithelial cells (HMEC); HMEC transfected with HPV16 E6 protein which inactivates p53 (HE6), or p53-mutant MDA-MB-231 tumor cells. CPT, UCN-01, or a 1:1 combination of both, in either HMEC or HE6 cells did not induce DSBs. In contrast, simultaneous treatment of MDA-MB-231 cells with both UCN-01 and CPT induced significant levels of DSBs while treatment with either drug alone did not. While UCN-01 was surprisingly potent against HMEC, the growth inhibition was only additive between UCN-01 and CPT against these cells. HE6 cells were much less sensitive than HMEC to UCN-01 and slightly less sensitive to the combined treatment with UCN-01 and CPT. The drug combination was synergistic against HE6 cells, due to their lower sensitivity to UCN-01. Unlike what was observed previously in MDA-MB-231 cells, UCN-01 did not abrogate CPT-induced inhibition of DNA synthesis in either HMEC or HE6 cells. These data indicate that synergistic growth inhibition by UCN-01 and CPT against p53 mutant MDA-MB-231 tumor cells may be due to induction of DSBs however the loss of p53 function alone does not sensitize normal cells to the combination of both drugs.

Enhancement of camptothecin-induced cytotoxicity with UCN-01 in breast cancer cells: abrogation of S/G(2) arrest.

PURPOSE: To determine the ability of UCN-01 to abrogate the cell cycle arrest induced by camptothecin (CPT) in tumor cells that lack p53 function, and therefore enhance the cytotoxicity of CPT in these cells in relation to normal cells with wild-type p53. METHODS: The responses of MDA-MB-231 and GI 101A breast cancer cells were compared to those of normal bovine endothelial cells. Cytotoxicity was assessed by the MTT assay, and the resulting data were modeled using median-effect analysis. Inhibition of DNA synthesis was determined by loss of [(3)H]thymidine incorporation, and cell cycle status was determined by flow cytometric analysis of propidium-iodide-stained nuclei. RESULTS: UCN-01, a specific inhibitor of protein kinase C (PKC) presently in clinical trials, abrogated CPT-induced activation of S and G(2) checkpoints in human MDA-MB-231 and GI 101A breast carcinoma cells, both of which are mutants for the p53 gene. This abrogation occurred with the use of sublethal doses (100 nM) of UCN-01 and correlated with the enhancement of CPT-induced cytotoxicity. Median-effect analysis showed that synergistic cytotoxic interactions existed between CPT and UCN-01 against these tumor cells. In normal cells, however, abrogation of the S phase arrest caused accumulation in G(0)/G(1) phase, perhaps by the presence of wild-type p53 activity, with no change in CPT-induced cytotoxicity. CONCLUSION: We have shown previously that the cytotoxicity of CPT is correlated with cell cycle response in normal and tumor cells. Low doses of CPT arrest cells in the G(2)/M phase and inhibit DNA synthesis, but higher doses cause arrest of cells in S phase. Thus modulation of events at the S and G(2) checkpoints may provide an opportunity to enhance CPT-induced cytotoxicity in tumor cells. The results of this study indicate that UCN-01 enhances the progression of tumor cells through S phase thus greatly increasing CPT-induced cytotoxicity. Normal cells, however, are able to arrest in G(0)/G(1) and thus avoid the increased toxicity induced by CPT. Our findings suggest potential usefulness of combining UCN-01 in topoisomerase I inhibitor-based drug therapy for the treatment of breast cancer with a dysfunctional p53 gene.

Antiangiogenic potential of camptothecin and topotecan.

PURPOSE: To determine the inhibitory nature of sublethal doses of camptothecin (CPT) and topotecan (TPT) treatments on normal human endothelial cells in vitro, as well as the in vivo antiangiogenic activity as compared to another antiangiogenic compound, TNP-470 and to a nonspecific cytotoxic agent, cisplatin. METHODS: Growth inhibition was determined by the crystal violet assay to measure relative cell numbers. (3)H-thymidine uptake was used to determine the inhibitory effect of CPT and TPT on DNA synthesis in vitro. Cell viability was determined using trypan blue exclusion assays. Cell cycle response to CPT was determined by flow cytometric analysis of propidium iodide-stained nuclei. In vivo inhibition of angiogenesis was determined by the disc angiogenesis system (DAS), where surgical sponge discs were placed subcutaneously in the rat dorsum and the ability of systemic treatment with liposomal CPT (LCPT), TPT, TNP-470 or cisplatin to inhibit vascular growth into the discs was evaluated. Quantitation of vascular growth was determined using toluidine blue staining of sectioned discs followed by digital image analysis. RESULTS: Treatment with 50 nM CPT or TPT inhibited human umbilical venular endothelial cell (HUVEC) growth as shown by crystal violet staining, but was not cytotoxic to the cells. This was evidenced by the fact that cell numbers did not increase or decrease with treatment, but remained static while cells were viable for over 96 h posttreatment. (3)H-thymidine uptake in HUVEC was inhibited as early as 5 min, reached a maximum inhibition at 24 h and lasted over 96 h posttreatment. Cell cycle analysis of CPT-treated HUVEC showed arrest in S-phase at 12 h with a concurrent decrease in population of cells in G(1). Accumulation of cells at the G(2)/M-phase was discernible at 24 h along with the S-phase inhibition. Treatment of rats with 1 mg/kg LCPT or TPT every other day for 14 days resulted in approximately 30% inhibition of vascular growth into the discs. This inhibition was similar to the inhibition seen with TNP-470, an established and potent angiogenic inhibitor. In contrast, cisplatin was not as effective in inhibiting vascular growth into the discs. CONCLUSIONS: In this work we showed that CPT and TPT inhibit human endothelial cell growth in vitro in a non-cytotoxic manner and that this inhibition lasts more than 96 h after drug removal. We also showed that LCPT and TPT, unlike a nonspecific cytotoxic agent, cisplatin, are as effective as TNP-470 in inhibiting angiogenic growth in the in vivo disc angiogenesis model. From this observation we propose that in addition to their proven tumoricidal activities, camptothecins may have an indirect in vivo antitumor effect mediated through the inhibition of angiogenesis.

Sensitivity to camptothecin of human breast carcinoma and normal endothelial cells.

PURPOSE: To assess parameters that might determine resistance to the topoisomerase I inhibitor, camptothecin (CPT), the sensitivities of three established human breast cancer cell lines (ER-) and of normal bovine endothelial cells to CPT in the free form and incorporated into liposomes (LCPT), were contrasted with topoisomerase I (topo I) content and activity, and with cell cycle response to CPT treatment. METHODS: Drug sensitivities were determined using the tetrazolium dye assay and by 3H-thymidine incorporation. Topo I levels were determined by Western blot analysis, and catalytic activity was determined with a plasmid relaxation assay, using nuclear protein from each cell line. CPT stabilization of cleavable complexes in nuclear extracts was determined using a labeled oligonucleotide with a specific topo I cleavage site. Cell cycle response to CPT was determined by flow cytometric analysis of propidium iodide-stained nuclei. RESULTS: CPT was extremely potent against MDA-MB-157 cells with an IC50 value of 7 nM compared with IC50 values of 150 nM for GI 101A and 250 nM for MDA-MB-231 cells. In contrast, CPT inhibited the incorporation of 3H-thymidine at very low doses in GI 101A and MDA-MB-231 cells with IC50 values of 9 nM and 5 nM, respectively; while MDA-MB-157 cells did not stop incorporating 3H-thymidine until very high doses (500 nM) of CPT were used. When incorporated into multilamellar liposomes (LCPT), CPT retained its potency, with IC50 values similar to that of the free drug. No correlation was found between CPT-induced cytotoxicity and any of the topo I parameters determined. Cell cycle analysis, however, showed an accumulation of cells in G2/M phase after 24 h treatment with low doses (5 nM) of CPT in only GI 101A and MDA-MB-231 cells with no arrest in normal endothelial or MDA-MB-157 cells. At higher doses (50 nM), however, a dramatic accumulation of cells in the S phase was observed in MDA-MB-157, MDA-MB-231 and GI 101A cells. In contrast, a G2/M phase block was seen with the normal bovine endothelial cells using the higher doses of CPT. CONCLUSIONS: The results suggest that cell cycle regulation plays an important role in determining the effect of CPT on malignant and normal cells. The possible mechanisms explaining the sensitivities of the two cellular compartments to the action of CPT are discussed.

Camptothecin exhibits selective cytotoxicity towards human breast carcinoma as compared to normal bovine endothelial cells in vitro.

We have reported earlier that camptothecin (CPT) incorporated into multilamelar liposomes of appropriate lipid composition displayed effective anti-tumor activity with minimal host toxicity in a nude mouse model xenographed with the human breast carcinoma Clouser nut 1. To investigate this observation further, we have determined the differential effects of CPT on the Clouser tumor cells as well as normal vascular (BVEC) endothelial cells in culture. We report here that Clouser cells are approximately 200-fold more sensitive to CPT (IC50 = 4.0 nM) than the normal endothelial cells (IC50 approximately 1 microM) as assayed by MTT; however, CPT demonstrates a potent anti-proliferative activity on both cell lines at low drug concentrations as measured by [3H]thymidine uptake. At higher concentrations (> 25.0 nM), however, the Clouser cells maintained a higher percentage of cells capable of incorporating [3H]thymidine. No significant differences in the levels of topoisomerase 1 protein and in vitro enzymatic activity were seen; although, the Clouser cells showed a 2-fold greater incidence of cleavable complex formation by CPT in vivo. Based on the data presented here, we propose that the selective cytotoxic activity of CPT towards tumor cells may be a function of the tumor cells' reduced ability to prevent cleavable complex formation. We also propose that the antitumor effect of CPT may be enhanced in vivo by its anti-proliferative effect on vascular endothelial cells which are normally solicited to promote tumor growth.

Polymerase chain reaction analysis of cisplatin-induced mitochondrial DNA damage in human ovarian carcinoma cells.

The purpose of this study was to determine whether the observed synergistic interaction between cisplatin and valinomycin (VM) in human ovarian carcinoma is the result of mitochondrial DNA (mtDNA) damage. A polymerase chain reaction (PCR)-based method was used to quantitate the lesion frequencies produced by cisplatin, VM and/or drug combination in a 1.1 kbp segment of mtDNA and a 0.536 kbp segment of the nuclear-located beta-globin gene in human ovarian CaOV-3 carcinoma cells. Our data indicates that the nuclear DNA (nDNA) received more cisplatin-induced damage at doses of 25 microM or less than did mtDNA. At higher cisplatin doses (50 microM or more), however, the damage was relatively equal in both segments. VM alone produced little or no damage on mtDNA, yet a significant amount of damage was detected within nDNA. However, when 1 microM VM was used in combination with low doses of cisplatin (0-40 microM), extensive mtDNA damage was detected as compared with the absence of detectable damage on nDNA. In mtDNA, the lesion frequency was 5.45 lesions/10 kb/10 microM cisplatin in the presence of 1 microM VM, whereas no detectable lesions were induced by cisplatin alone. This drug combination produced no detectable damage on DNA, indicating that cisplatin-induced mtDNA damage could be the basis for the observed synergistic interaction with VM. These results also correlate well with our recent in vivo study with the nude mice model of human ovarian cancer treated with a cisplatin/liposomal VM drug combination. Furthermore, this report shows evidence for the role of mitochondria and mtDNA as alternative targets for drug action in cancer therapy.

Antitumor effect of liposome-incorporated camptothecin in human malignant xenografts.

The nuclear enzyme topoisomerase I (topo I) has been recently recognized as the target for the anticancer drug camptothecin (CPT; NSC 94600) and its derivatives. This drug has been reported to display effective antitumor effects on a variety of human tumor models xenografted in nude mice. However clinical studies of sodium CPT have revealed that the open-ring form of the drug is a poor inhibitor of topo I and much less potent antitumor agent than CPT lactone. However, the insolubility of CPT lactone makes it difficult to devise a suitable formulation for further clinical testing. In view of these observations, we report here the successful incorporation of CPT into a liposome-based drug delivery system (LCPT) composed of DPPC:Sph:CHOL:PI (2.4:6.6:1.0:0.05 M ratio) that can be used as a suitable formulation for clinical testing of the drug. Higher incorporation efficiency was observed when the total phospholipids:drug ratio = 40 and the cholesterol content = 1%. Image analysis of the CPT-containing liposomes with freeze-fracture electron microscopy has indicated that CPT significantly increased the interlamellar space of the vesicles as a result of its intercalation between lipid bilayers. This has occurred with no major disruptive effects on the bilayer structure. The in vitro drug release study in human serum was characterized by an initial rapid loss-of 50% of contents during 4 h, followed by a slow leakage of the remaining 50% of the total drug over a 20 h period. When tested for its antitumor activity on nude mice xenografted with human malignant melanoma and breast carcinoma, LCPT displayed effective antitumor activity with minimal host toxicity. For example, single i.m. injection of LCPT at 10 mg/kg has produced complete tumor regression to nude mice xenografted with CLO breast carcinoma. Likewise, similar results were obtained with the nude mice xenografted with human malignant BRO cells melanoma. These results appear to suggest that i.m. administration of liposome-incorporated CPT has considerable potential for the treatment of human neoplastic diseases, especially lymph node metastases.

Combination chemotherapy of human ovarian xenografts with intraperitoneal liposome-incorporated valinomycin and cis-diamminedichloroplatinum(II).

Intraperitoneal administration of liposomal valinomycin (MLV-VM) with cis-diamminedichloroplatinum(II) (cDDP) had significant antitumor activity against murine P388 leukemia and inhibited the growth of OVCAR-3 tumors in a nude mouse model of human ovarian cancer. This tumor is a teratoma originating in the ovary with pathogenesis and metastatic properties similar to those of human ovarian cancer. Drug was given to the mice once every 5 days for 4 doses beginning 1 day after i.p. implantation of 10(7) or 5 x 10(7) OVCAR-3 tumor cells. For P388 leukemia, drug was given i.p. once or on days 1 and 5 after tumor inoculation. Despite the use of low doses of MLV-VM, the antitumor activity of the combination [increase in life span (%T/C), 289%-294%] represents a 4-log cell kill over the additive effect of the two drugs, indicating a synergistic interaction between MLV-VM and cDDP. Likewise, low doses of the drug combination produced a synergistic interaction on human ovarian OVCAR-3 tumors, and tumor-free, long-term survivors were obtained. Combined therapy of liposome-incorporated valinomycin and cisplatin was well tolerated and produced no overlapping nephrotoxicity, although a decrease in liver enzyme markers (alkaline phosphatase and/or alkaline aminotransferase) with MLV-VM was observed. These results appear to suggest that MLV-VM with cDDP may have considerable potential for the treatment of ovarian cancer disseminated within the peritoneal cavity, although the frequency and sequence of drug administration may need to be improved.

In vitro interaction of liposomal valinomycin and platinum analogs: cytotoxic and cytokinetic effects.

Cisplatin is the most active agent in the chemotherapy of ovarian cancer and this activity can be enhanced by liposomal valinomycin (MLV-VM) in vitro. To test whether MLV-VM is capable of augmenting the cytotoxic and cytokinetic effects of other platinum analogs, drug combinations of MLV-VM and platinum drugs were tested against two human ovarian cancer cell lines (OVCAR-3 and CaOV-3) and on Chinese hamster ovary (CHO) cells in vitro. MLV-VM enhanced the sensitivity to cisplatin, ormaplatin and carboplatin on human ovarian carcinoma cells that show various degrees of drug sensitivity. This interaction was shown to be truly synergistic by median-effect analysis up to 90% cell kill. The combination index at 50% cell kill (Cl50) was also used to quantitate the extent of drug synergy. In the OVCAR-3 cell line, for example, the Cl50s were 0.62, 0.85 and 0.8 for cisplatin, ormaplatin and carboplatin, respectively. DNA histograms obtained by flow cytometry showed that CHO cells treated with cisplatin alone accumulated in the S-G2 segment, with a partial G2 block. The addition of 2 microM VM with cisplatin, significantly enhanced the accumulation of cells at the G2/M phase. Our results further demonstrate that in vitro treatment with VM, cisplatin and/or combination is associated with an increase in protein kinase C (PKC) activity. These findings suggest that accumulation of cells at G2/M phases and modulation of PKC activity could be among the basis for the cytotoxic synergism observed between cisplatin and VM.

Cell membranes as targets for anti-cancer drug action.

A variety of studies strongly hint that cell membranes can be important targets for new and existing anti-tumor drugs. Traditionally, the search for new anti-cancer agents has focused on compounds designed to act on the biosynthesis, stability or function of DNA; it may be timely, however, to broaden our search horizon and consider targets outside of the nucleus. Thus, the cell membrane as well as membraneous organelles may well play a key role in the future of anti-tumor drug development.

Synergistic cytotoxic actions of cisplatin and liposomal valinomycin on human ovarian carcinoma cells.

We have previously shown that the toxicity of valinomycin (VM), a membrane-active agent with antineoplastic activity, can be dramatically reduced with no loss of the antitumor efficacy of the drug by incorporating it into liposomes. In the present study, we investigated the interaction between cis-diamminedichloroplatinum(II) (CDDP) and VM in terms of in vitro cytotoxicity to human ovarian tumor cells. Using the MTT assay and analyzing the data using the median-effect principle, we showed that synergistic cytotoxic interactions exist between CDDP and VM in their liposomal form. The degree of cytotoxic synergism was influenced by the duration of drug exposure and the dose ratio. The cellular accumulation of platinum by ovarian cells at 37 degrees C was slightly higher after exposure to VM as compared with controls; however, it is not clear that this accounts for the cytotoxic synergism. These results suggest that the combination of liposomal VM and CDDP may have merit as a form of localized drug delivery for the treatment of ovarian cancer disseminated within the peritoneal space.

Modulation of doxorubicin resistance by valinomycin (NSC 122023) and liposomal valinomycin in Chinese hamster ovary cells.

Recently, we have reported that the toxicity of the membrane-active agent valinomycin (VM) can be reduced with maintenance and/or enhancement of its antitumor activity by incorporation in liposomes (S. S. Daoud and Juliano, Cancer Res., 46:5518-5525, 1986). Since the underlying defect(s) in multidrug resistance reside mainly in the cell membrane, it seems reasonable to attempt to overcome multidrug resistance with membrane-active drugs. Here, we report on the in vitro restoration of Adriamycin (ADR) sensitivity in a resistant Chinese hamster ovary cell line (CHRC5) by treatment with nontoxic doses of valinomycin or of liposomal valinomycin. During a 1-h drug exposure, the sensitivity of CHRC5 to ADR was enhanced 21- to 28-fold when 20 or 40 nM VM was present, doses which are not toxic to CHRC5 cells. At the same time, modest synergistic toxicity could be seen in the parent drug-sensitive cell line (AUX B1). At 100 nM VM, the sensitivity of CHRC5 to ADR was restored to almost that of the sensitive AUX B1 cells. The effects of liposomal VM on ADR sensitivity were similar to the effects produced by free VM. At nontoxic doses and with continuous exposure of the drug, valinomycin was highly active in restoring ADR sensitivity in CHRC5 cells. In cells treated for 72 h, valinomycin enhanced the sensitivity to ADR 208- to 250-fold in CHRC5 and 3- to 5-fold in AUX B1 cells. Measurements of ADR uptake and efflux indicate that, unlike other multidrug resistance modifiers, valinomycin exerts its actions in modulating ADR resistance by mechanism(s) other than increasing intracellular accumulation of Adriamycin. The possible mechanisms of the restoration of ADR sensitivity by valinomycin are discussed.

In vitro effect of liposome-incorporated valinomycin on growth and macromolecular synthesis of normal and ras-transformed 3T3 cells.

Valinomycin is a depsipeptide antibiotic that selectively translocates potassium ion across biologic membranes. This drug has been reported to display antitumor effects, but its use has been limited by its extreme toxicity. However, its incorporation into lipid vesicles (liposomes) has resulted in a reduction in toxicity and in the enhancement of the drug's therapeutic index. As a preliminary investigation of the mechanistic basis for this enhancement, the in vitro response of normal 3T3 and ras-transformed cells to free (VM) and liposomal valinomycin (VM-MLV) was examined. The incorporation of [3H]-leucine and [methyl-3H]-thymidine was used to assess macromolecular synthesis, and the MTT vital dye assay was used to measure cell survival and growth. Pretreatment of exponentially growing NIH/3T3 cells with 20 nM VM for 1 h decreased [3H]-leucine and [methyl-3H]-thymidine incorporation by 90% and 80%, respectively. However, Ha-ras 3T3 cells showed resistance to VM treatment with inhibitory doses in the range of 200 nM. At equimolar VM concentrations, VM-MLV was found to be less inhibitory than VM for protein and DNA synthesis. Specifically, marked protective activity was apparent with normal 3T3 cells. In this report we also demonstrate that VM selectively killed normal cells compared with ras-transformed cells grown in vitro. However, VM-MLV displayed a modest cytotoxic selectivity (3- to 4-fold) to ras-transformed cells. Our data suggests that first, there is good correlation between growth inhibition and inhibition of DNA and protein synthesis by VM, and second, VM-MLV exhibits a modest, selective toxicity to the ras-transformed 3T3 cell line as compared with nontransformed 3T3 cells, whereas free VM has the opposite selectivity.

Synthesis and pharmacological effect of substituted phenyl-4-(2-chloroethyl)tetrahydro-1,4-oxazine hydrochlorides.

Substituted phenyl-4-(2-chloroethyl)tetrahydro-1,4-oxazine hydrochlorides were prepared by treating the corresponding amino alcohols with thionyl chloride. Since these compounds are considered to be monofunctional 2-haloalkylamine type agents, they were tested for possible alpha-adrenergic blocking activity using the rat anococcygeus muscle. The pharmacological action of this series of compounds is discussed.

Renal handling of adriamycin.

The excretion of the anticancer drug, adriamycin (ADR) was investigated in dogs. Its plasma half-life was found to be 8.4 hrs. 1. ADR was excreted in the bile against concentration gradient. The biliary clearance ratio between ADR and creatinine was ranged from 30-100. 2. Intra-arterial injection studies showed that ADR was both reabsorbed and secreted by the renal tubules. Stop-flow studies indicated that the secretion of ADR was localized at the proximal tubular section. 3. The tubular secretion of ADR is probably by an organic base secretory mechanism. Acidification of the urine would augment the urinary excretion of ADR and alkalinization reduces it. Probenecid has little effect on ADR excretion.

Reduced toxicity and enhanced antitumor effects in mice of the ionophoric drug valinomycin when incorporated in liposomes.

Valinomycin (NSC 122023) is a cyclic depsipeptide antibiotic with potassium selective ionophoric activity. This drug has been reported to display antitumor effects but its utilization has been limited by its extreme toxicity. Here we report that the incorporation of valinomycin into multilamellar liposomes composed of dimyristoyl phosphatidyl choline:cholesterol:phosphatidyl serine (10:4:1 M ratio) results in a profound reduction in toxicity with maintainence of antitumor efficacy. Thus the median lethal dose (LD50) for i.p. administered valinomycin (VM) in C57BL/6 X DBA/2 mice is 1.7 mg/kg whereas the LD50 for liposome incorporated valinomycin (MVL-VM) is in excess of 50 mg/kg. In like manner, the LD50 for i.v. administered VM is 0.18 mg/kg where the LD50 for MLV-VM preparations passed through a 0.6-micron filter is greater than 10 mg/kg. The antitumor efficacies of i.p. administered VM or MLV-VM against i.p. P388 mouse leukemia were similar in multiple dose formats using doses below the maximal tolerated dose for VM. However, since MLV-VM was substantially less toxic than VM, the liposomal drug also produced significant (170% median survival time of treated mice/median survival time of untreated control) antitumor effects when administered as a single dose at levels above the maximal tolerated dose for free VM; single doses of free VM at the maximal tolerated dose were ineffective in this context. In experiments with i.v. inoculated P388 leukemia, MLV-VM but not free VM, displayed antitumor activity (144% median survival time of treated mice/median survival time of untreated control) when administered i.v. at equitoxic doses. Thus the use of a lipid vesicle drug carrier system permits a reduction in the toxicity of valinomycin with maintainence or enhancement of antitumor activity against i.p. or i.v. P388 leukemia.