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PubMed1997

Tissue and perfusate pharmacokinetics of melphalan in isolated perfused rat hindlimb.

Melphalan is commonly used as a cytotoxic agent in isolated limb perfusion for locally recurrent malignant melanoma. The time course of melphalan concentrations in perfusate and tissues during a 60-min melphalan perfusion and 30-min drug-free washout in the single-pass perfused rat hindlimb was examined using a physiologically based pharmacokinetic model. The rat hindlimbs were perfused with Krebs-Heinseleit buffer containing 4.7% bovine serum albumin (BSA) or 2.8% dextran 40 at a constant rate of 3.8 ml/min. The concentration of melphalan in perfusate and tissues was determined by high-performance liquid chromatography. The tissue concentrations of melphalan were significantly higher with the perfusate containing dextran than BSA during the 60-min perfusion. During the washout period, the melphalan concentration in the perfusates decreased rapidly in first few minutes, followed by a slower monoexponential decline. The estimated half life (t1/2) for melphalan removal from skin and fat was 59 +/- 2 min for both BSA and dextran perfusates. However, the estimated t1/2 for melphalan removal from muscle was 79 and 96 min for BSA and dextran washout perfusates, respectively. The predicted concentration-time profiles obtained for melphalan with BSA and dextran perfusates appear to correspond closely to the observed data. This study showed that the uptake of melphalan into perfused tissues is impaired by the use of perfusates in which melphalan is highly bound. Melphalan washout from muscle, but not skin and fat, was facilitated by the use of perfusates in which melphalan is highly protein bound.

Adipose Tissue↗
PubMed1997

Lack of antitumour activity of human recombinant tumour necrosis factor-alpha, alone or in combination with melphalan in a nude mouse human melanoma xenograft system.

The most promising developments in the field of isolated limb perfusion have centred around the use of the recombinant cytokine tumour necrosis factor-alpha (rTNF-alpha) in combination with melphalan. While the results of clinical trials are impressive, the exact antitumour mechanisms of rTNF-alpha and its role in combination with melphalan remain unclear. Our aim was to study the antitumour activity of human rTNF-alpha with or without the combination of melphalan in a nude mouse human melanoma xenograft system. In a first attempt to define the maximal tolerated single dose of rTNF-alpha in this setting, 15 animals were exposed to increasing doses of rTNF-alpha (60-2500 microg/kg intraperitoneally). All but one animal survived and tumour growth was not influenced by these single dose applications of rTNF-alpha even at the very high doses. Anti-tumour activity of repeated application of melphalan (three times 9 mg/kg in group 2 and three times 6 mg/kg in group 3), of rTNF-alpha alone (nine doses of 50 microg/kg in group 4), and of rTNF-alpha in combination with melphalan (nine doses of 50 microg/kg rTNF-alpha and three times 6 mg/kg melphalan in group 5) was further compared with non-treated animals (group 1). Tumour growth was significantly inhibited in all animals treated with melphalan (group 2, 3 and 5), but was not decreased in animals treated with rTNF-alpha alone (group 4). Mean final tumour volumes and mean tumour weight were not different in group 2 (789 +/- 836 mm3, 0.38 +/- 0.20 g), group 3 (1173 +/- 591 mm3, 0.55 +/- 0.29 g) and group 5 (230 +/- 632 mm3, 0.37 +/- 0.29 g), but significant lower than group 1 (3156 +/- 1512 mm3, 2.35 +/- 0.90 g) and group 4 (3228 +/- 1990 mm3, 2.00 +/- 1.16 g). There were no significant differences between high and low dose melphalan treatment and between melphalan treatment in combination with rTNF-alpha. Histological examination did not show differences between treated and non-treated animals besides slightly inhibited mitotic activities of tumour cells in melphalan-treated animals. While tumour growth of human xenotransplanted melanoma in nude mice could be inhibited by melphalan, we failed to demonstrate any antitumour effect of rTNF-alpha. The combination of melphalan and rTNF-alpha did not enhance the antiproliferative effect of melphalan alone. Human xenotransplanted tumours on nude mice might not be the ideal experimental setting for studies of potential direct antineoplastic activity of rTNF-alpha, and these results support the concept that TNF-alpha exerts its antitumour activity indirectly, possibly by impairing the tumour vasculature and by activating the immune system.

Animals↗
PubMed1997

Isolated limb reperfusion with tumor necrosis factor and melphalan in patients with extremity melanoma after failure of isolated limb perfusion with chemotherapeutics.

BACKGROUND: This retrospective study evaluated the benefit of using tumor necrosis factor (TNF) and melphalan administered via an isolated limb perfusion (ILP) in a series of patients with metastatic melanoma who failed initial ILP with chemotherapeutics. METHODS: Seventeen patients with extremity melanoma who underwent prior ILP with conventional chemotherapeutics (10 with melphalan; 4 with platinum; 2 with platinum, dacarbazine, thiotepa, actinomycin D, and nitrogen mustard; and 1 with thiotepa, actinomycin D, and nitrogen mustard) and had local recurrences were treated with a 90-minute isolated hyperthermic limb reperfusion with melphalan (10 mg/L limb volume) plus TNF (2-6 mg). Five prior ILPs were adjuvant and 12 were therapeutic. RESULTS: Reperfusion was associated with an overall 94% response rate and a 65% complete response (CR) rate. Of the patients who failed an initial ILP with melphalan alone the overall response rate was 90% after the reperfusion with TNF and melphalan. In patients who failed an initial ILP with agents other than melphalan the CR rate was 100% after ILP with TNF and melphalan. TNF/melphalan isolated limb reperfusion was found to be more effective in terms of CR after initial ILP regimens that did not utilize melphalan (100% CR after nonmelphalan ILP vs. 50% CR after melphalan ILP [P = 0.04]). Regional toxicity was comprised of mild skin blistering and peeling in 47% of patients. One patient developed Grade 3 (based on National Cancer Institute Common Toxicity Criteria) skin necrosis, and one developed Grade 5 muscle and nerve toxicity, requiring an amputation. CONCLUSIONS: Isolated limb reperfusion with TNF and melphalan can be performed safely with response rates similar to those of other trials of single perfusions. Repeat ILP using TNF and melphalan in patients with melanoma who have failed prior ILP with chemotherapeutics is justified. The utility of TNF (vs. melphalan alone) will be defined in ongoing Phase III trials.

Adult↗
PubMed1985

Antagonism of the cytocidal activity and uptake of melphalan by tamoxifen in human breast cancer cells in vitro.

The effect of the antiestrogen tamoxifen on the cytocidal activity and uptake of melphalan in human breast cancer cells was investigated. A clonogenic assay was used to obtain dose-survival curves of estrogen receptor-positive MCF-7 cells and of estrogen receptor-negative Evsa T cells following treatment with melphalan and/or tamoxifen. Isobolograms derived from these dose-survival curves were concave downward, suggesting that the drug interaction was antagonistic. The effect of tamoxifen on melphalan uptake by breast cancer cells was evaluated at steady-state conditions. Thin-layer chromatography revealed that the intracellular level of free intact melphalan (mean +/- S.E.) in control cells was 6.47 +/- 1.21 fmoles/cell and that in cells treated with tamoxifen was 3.60 +/- 0.35 fmoles/cell; this 44% reduction in cellular melphalan was statistically significant (P = 0.006). Thus, the antagonistic cytocidal effect of melphalan and tamoxifen against breast cancer cells appeared to be due to inhibition of melphalan uptake at the steady state by the antiestrogen. Further investigation revealed that tamoxifen inhibited unidirectional melphalan influx in human breast cancer cells both by the sodium-independent system L and by the sodium-dependent system ASC. Tamoxifen also appeared to stimulate melphalan efflux from human breast cancer cells. The first-order rate constant K for melphalan efflux from control cells was 0.085 +/- 0.008 and that from cells treated with tamoxifen was 0.129 +/- 0.005; the difference was highly significant (P less than 0.001). Therefore, the antagonistic effect of tamoxifen on the uptake and cytocidal activity of melphalan in breast cancer cells appeared to be due to inhibition of melphalan influx and stimulation of drug efflux.

Breast Neoplasms↗
PubMed1999

Melphalan resistance and photoaffinity labelling of P-glycoprotein in multidrug-resistant Chinese hamster ovary cells: reversal of resistance by cyclosporin A and hyperthermia.

The multidrug resistance phenotype is often associated with overexpression of P-glycoprotein, an energy-dependent efflux pump responsible for decreased intracellular accumulation of chemotherapeutic agents. The role of P-glycoprotein in the mechanism of cross-resistance to melphalan in multidrug-resistant Chinese hamster ovary cells (CH(R)C5) was investigated by photoaffinity labelling of P-glycoprotein using [3H]azidopine. We investigated whether the chemosensitiser cyclosporin A and hyperthermia, either used alone or combined, could reverse melphalan resistance and alter transport processes for [14C]melphalan in CH(R)C5 cells. Melphalan inhibited azidopine photolabelling of P-glycoprotein, implicating drug efflux mediated by P-glycoprotein in the mechanism of melphalan resistance in CH(R)C5 cells. Azidopine photolabelling also was inhibited by the chemosensitiser cyclosporin A, which binds to P-glycoprotein. Cyclosporin A alone reversed melphalan resistance in CH(R)C5 cells, but had no effect in drug-sensitive AuxB1 cells. Hyperthermia (40-45 degrees) alone increased melphalan cytotoxicity in both cell lines. When hyperthermia was combined with cyclosporin A, a large increase in melphalan cytotoxicity occurred, but only in CH(R)C5 cells. This effect increased with temperature and exposure time. Sensitisation to melphalan cytotoxicity by heat and cyclosporin A in CH(R)C5 cells appeared to be explained by altered drug transport processes. Lower accumulation of melphalan occurred in CH(R)C5 cells than in drug-sensitive cells. At 37 degrees, cyclosporin A increased drug accumulation in CH(R)C5 cells, but not in AuxB1 cells, by slowing drug efflux from cells. Heat alone increased both melphalan uptake and drug efflux for both cell lines. Our findings suggest that the combination of cyclosporin A and hyperthermia could be very useful in overcoming melphalan resistance by increasing intracellular drug accumulation in multidrug-resistant cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1986

Effects of verapamil and alcohol on blood flow, melphalan uptake and cytotoxicity, in murine fibrosarcomas and human melanoma xenografts.

Verapamil had previously been shown to increase cellular melphalan uptake and cytotoxicity in fibrosarcomas, and increased the area under the blood concentration versus time curve (AUC) for melphalan in CBA mice. Verapamil (10 mg kg-1 i.p.) had no effect on the fractional distribution of cardiac output (FDCO), measured with 86Rb-rubidium chloride, to subcutaneous fibrosarcomas. 14C-Melphalan uptake by FS13 fibrosarcomas was increased 60 min after verapamil (10 mg kg-1 i.p.), but not after lower doses which did not affect the AUC. Flunarizine (5 mg kg-1 i.p.) also had no effect on FDCO to FS13 fibrosarcomas, and tended to increase 14C-melphalan content of blood and the fibrosarcomas and to promote growth delay by melphalan. Alcohol increased FDCO to FS13 fibrosarcomas, maximally at a 1:20 dilution in saline, but had no effect on 14C-melphalan uptake or growth delay. Thus, melphalan cytotoxicity correlated with tumour melphalan uptake, and both followed changes in the AUC for melphalan but not changes in FDCO. In these murine fibrosarcomas melphalan uptake and cytotoxicity were not limited by blood flow. In subcutaneous human melanoma HX46 xenografts, verapamil had no effect on the FDCO, nor on 14C-melphalan uptake, and did not affect blood 14C-melphalan levels, suggesting absence of effects on the AUC and on cellular uptake. Alcohol did not increase the FDCO to HX46 xenografts, providing evidence for a different vascular supply.

Animals↗
PubMed1987

Effects of various oxygenation conditions on the enhancement by Fluosol-DA of melphalan antitumor activity.

The cytotoxicity of melphalan toward exponentially growing FSaIIC fibrosarcoma cells under hypoxia, normal aeration, hyperoxygenation, and stationary phase normally oxygenated cells was examined. Through 4 logs of cell kill by melphalan, there was no difference in survival of FSaIIC cells under any of the four conditions. In the fifth and sixth logs of cell kill, melphalan was most cytotoxic toward normally aerated cells. DNA alkaline elution was performed in FSaIIC cells treated for 1 h with melphalan under the various atmospheres. Both upon immediate elution and after a 6-h delay period the greatest number of DNA cross-links were formed in the normally oxygenated cells. Tumor growth delay studies of the FSaIIC fibrosarcoma treated with melphalan were performed under four levels of oxygenation. From air breathing to 100% oxygen at 3 atm, the tumor growth delay produced by melphalan increased from about 3 days to about 9 days. With the addition of Fluosol-DA, there was an increase in tumor growth delay by melphalan from about 6.5 days with air breathing to about 13 days with 100% oxygen at 3 atm (1 h). When FSaIIC fibrosarcoma tumors were treated with melphalan, and tumor cell survival was measured by colony formation in culture, increasing doses of melphalan produced increasing levels of tumor cell kill in a relatively log linear manner. The addition of Fluosol-DA to treatment with melphalan produced approximately 1 log greater tumor cell kill than melphalan and air breathing under the various oxygenation conditions. There was approximately a 4-fold increase in toxicity to bone marrow granulocyte-macrophage colony-forming units under both extended carbogen breathing conditions (6 h) and hyperbaric oxygenation conditions (100% oxygen, 1 h, 3 atm). The response of the spleen to these various treatment regimens appeared to be immediate and shortlived. Necrotic cells were seen on day 1 posttreatment, with a substantial reduction by day 4 posttreatment. Mitotic figures were essentially absent from the liver on day 1 posttreatment, but by day 4 were significantly increased in treatment groups receiving Fluosol-DA, with the largest number seen in the melphalan/Fluosol-DA with carbogen-breathing group. In conclusion, Fluosol-DA and 1 h of carbogen breathing significantly increases the antitumor activity of melphalan without a concomitant increase in normal tissue toxicity. Although increasing the oxygenation level increased the response of the tumor, normal tissue toxicity was also increased.

Animals↗
PubMed2006

Optimizing the factors which modify thermal enhancement of melphalan in a spontaneous murine tumor.

BACKGROUND: Hyperthermia enhances the cytotoxicity of some chemotherapeutic agents. Both clinical and laboratory studies suggest melphalan may be an important drug when hyperthermia is added to chemotherapy treatments. Factors that may modify the thermal enhancement of melphalan were studied to optimize its clinical use with hyperthermia. METHODS: The tumor studied was an early-generation isotransplant of a spontaneous C3Hf/Sed mouse fibrosarcoma, Fsa-II. All studies were performed under supervision of the Animal Care and Use Committee. Hyperthermia was administered by immersing the tumor-bearing foot into a constant temperature water bath. Four factors were studied: duration of hyperthermia, sequencing of hyperthermia and melphalan, intensity of hyperthermia, and tumor size. To study duration of hyperthermia tumors were treated at 41.5 degrees C for 30 or 90 min immediately after intraperitoneal administration of melphalan. For sequencing of hyperthermia and melphalan, animals received hyperthermia treatment of tumors for 30 min at 41.5 degrees C immediately after drug administration, both immediately and 3 h after administration of drug or only at 3 h after administration of drug. Intensity of hyperthermia was studied using heat treatment of tumors for 30 min at 41.5 or 43.5 degrees C immediately following drug administration. Effect of tumor size was studied by delaying experiments until three times the tumor volume (113 mm3) was observed. Treatment of tumors was for 30 min at 41.5 degrees C immediately following drug administration. Tumor response was studied by the mean tumor growth time. RESULTS: Hyperthermia in the absence of melphalan had a small but significant effect on tumor growth time at 43.5 degrees C but not at 41.5 degrees C. Hyperthermia at 41.5 degrees C immediately after melphalan administration doubled mean tumor growth time at 30 min and caused a threefold increase at 90 min (P=0.0002) when compared to tumors treated with melphalan alone at room temperature. Application of hyperthermia for one-half hour immediately following drug administration was the most effective in delaying tumor growth. No significant difference in mean tumor growth time was observed with an increase in temperature from 41.5 to 43.5 degrees C. For large tumors heat alone and melphalan alone caused a moderate increase in tumor growth delay. These effects in large tumors were greatly increased by a combination of chemotherapy and hyperthermia. CONCLUSIONS: From our data it would appear that the administration of intraperitoneal melphalan immediately prior to 90 min of heat at 41.5 degrees C may optimize anti-neoplastic activity. These data may be useful in formulating clinical protocols in which melphalan and heat are combined.

Animals↗
PubMed1996

Isolated lung perfusion with melphalan for the treatment of metastatic pulmonary sarcoma.

OBJECTIVE: Isolated lung perfusion allows the delivery of high-dose chemotherapy to the perfused lung and is an efficacious modality in the treatment of pulmonary metastases in the rat. Melphalan activity in this model was investigated. METHODS: TOXICITY STUDY: Maximum tolerated dose of melphalan delivered by means of isolated lung perfusion was determined by survival after contralateral pneumonectomy. PHARMACOKINETICS STUDY: Nineteen rats were treated with melphalan administered either by isolated lung perfusion (2 mg) or intravenously (2 mg or 1 mg). Lung, pulmonary effluent, and serum melphalan were analyzed by high-pressure liquid chromatography. EFFICACY STUDY: On day 0, 41 rats received an intravenous injection of 5 x 10(6) methylcholanthrene induced sarcoma cells. On day 7, rats either received intravenous melphalan (2 mg [n = 10]; 1 mg [n = 8]) or underwent left isolated lung perfusion with 2 mg of melphalan (n = 12). Isolated lung perfusion with buffered hetastarch in sodium chloride (Hespan, n = 11) was used as control. On day 14, pulmonary nodules were counted. RESULTS TOXICITY: Maximum tolerated dose of melphalan delivered buy means of isolated lung perfusion was 2 mg. PHARMACOKINETICS: Left lung melphalan level was significantly higher in the isolated lung perfusion group (62.2 +/- 34.3 microg/gm lung) than in the intravenous treatment groups (6.9 +/- 1.9 microg/gm lung and 3.3 +/- 0.9 microg/gm lung, respectively) (p = 0.0002). EFFICACY: Significantly fewer left lung nodules were found in animals receiving melphalan by means of isolated lung perfusion (7 +/- 10) than in the groups receiving intravenous melphalan (60 +/- 21) or buffered hetastarch by isolated lung perfusion (84 +/- 52) (p = 0.01 and p = 0.0001, respectively). CONCLUSION: Isolated lung perfusion with melphalan is safe and effective in the treatment of pulmonary sarcoma metastases in the rat.

Animals↗
PubMed2001

In vitro evidence for homologous recombinational repair in resistance to melphalan.

BACKGROUND: The generation of DNA interstrand cross-links is thought to be important in the cytotoxicity of nitrogen mustard alkylating agents, such as melphalan, which have antitumor activity. Cell lines with mutations in recombinational repair pathways are hypersensitive to nitrogen mustards. Thus, resistance to melphalan may require accelerated DNA repair by either recombinational repair mechanisms involving Rad51-related proteins (including x-ray repair cross-complementing proteins Xrcc2, Xrcc3, and Rad52) or by nonhomologous endjoining involving DNA-dependent protein kinase (DNA-PK) and Ku proteins. We investigated the role of DNA repair in melphalan resistance in epithelial tumor cell lines. METHODS: Melphalan cytotoxicity was determined in 14 epithelial tumor cell lines by use of the sulforhodamine assay. Homologous recombinational repair involving Rad51-related proteins was investigated by determining the levels of Rad51, Rad52, and Xrcc3 proteins and the density of nuclear melphalan-induced Rad51 foci, which represent sites of homologous recombinational repair. Nonhomologous endjoining was investigated by determining the levels of Ku70 and Ku86 proteins and DNA-PK activity. Linear regression analysis was used to analyze correlations between the various protein levels, DNA-PK activity, or Rad51 foci formation and melphalan cytotoxicity. All statistical tests were two-sided. RESULTS: Melphalan resistance was correlated with Xrcc3 levels (r =.587; P =.027) and the density of melphalan-induced Rad51 foci (r =.848; P =.008). We found no correlation between melphalan resistance and Rad51, Rad52, or Ku protein levels or DNA-PK activity. CONCLUSION: Correlations of melphalan resistance in epithelial tumor cell lines with Xrcc3 protein levels and melphalan-induced Rad51 foci density suggest that homologous recombinational repair is involved in resistance to this nitrogen mustard.

Antigens, Nuclear↗
PubMed2002

Repair of DNA interstrand crosslinks as a mechanism of clinical resistance to melphalan in multiple myeloma.

Melphalan is widely used as a preparative agent in patients with multiple myeloma (MM) undergoing autologous stem cell transplantation (SCT). Although disease relapse is the major cause of death after a melphalan-conditioned autograft, the mechanism remains unclear. Melphalan produces a number of DNA adducts with the DNA interstrand crosslink (ICL) considered to be the critical cytotoxic lesion. By using a modification of the single-cell gel electrophoresis (Comet) assay, we have measured formation and repair of DNA ICL in plasma cells from melphalan- naive and melphalan-treated patients (ie, those who have relapsed after a melphalan-conditioned autologous SCT or oral melphalan therapy). Similar levels of dose-dependent DNA interstand crosslinking were observed in cells from both melphalan-naive and -treated patients. However, marked differences in ICL repair were observed: cells from naive patients showed no repair, whereas those from treated patients exhibited between 42% and 100% repair at 40 hours. In vitro sensitivity to melphalan in plasma cells was found to correlate with ICL repair. These findings suggest that ICL repair may be an important mechanism by which melphalan resistance emerges after autologous SCT or oral therapy. This mechanism may have implications for MM patients undergoing melphalan therapy.

Adult↗
PubMed1992

Melphalan penetration of the blood-brain barrier via the neutral amino acid transporter in tumor-bearing brain.

Melphalan, a nitrogen mustard derivative of the neutral amino acid L-phenylalanine, was transported across the rat blood-brain barrier by the large (L-system) neutral amino acid transporter in tumor-bearing brain, but no evidence for blood-brain barrier transport by the alanine-serine-cysteine system carrier was obtained in the present study. The ability of melphalan to inhibit phenylalanine uptake was compared in rats implanted with two experimental CNS tumors: the C-6 glioma (a model of primary brain tumors) and Walker carcinoma (a model of metastatic brain tumors). The melphalan concentration which caused 50% inhibition of blood-brain barrier (BBB) phenylalanine uptake (Ki) was 0.49 +/- 0.18 mM in the Walker tumor, compared with 0.46 +/- 0.19 mM in the contralateral control brain. In the ipsilateral hemisphere (Ki = 0.59 +/- 0.25 mM) and contralateral hemisphere (Ki = 0.45 +/- 0.19 mM), drug entry was also via the neutral amino acid transporter. In C-6 gliomas (Ki = 0.77 +/- 0.20 mM) and contralateral control brain (Ki = 0.84 +/- 0.29 mM), melphalan also inhibited BBB phenylalanine transport. A major finding was that, at melphalan concentrations greater than 1.0 mM, BBB permeability of radiolabeled indium (chelated to EDTA) increased in proportion to melphalan concentration. In the contralateral hemisphere of rats implanted with C-6 gliomas, brain extractions of indium-EDTA measured 3 to 4% in the absence of drug, 5 to 6% at 2.5 mM melphalan, and 9 to 10% at 5 mM melphalan. A similar phenomenon was observed in the nontumoral brain regions of rats implanted with Walker carcinoma cells. In normal (nonimplanted) rats, melphalan's inhibition (Ki = 0.29 mM) of phenylalanine and tryptophan (Ki = 0.20 mM) uptake was confirmed, and brain extraction of sucrose (a nonspecific marker which does not penetrate the intact BBB) was observed to increase in proportion to melphalan concentration. We conclude that melphalan not only enters the brain via the neutral amino acid transporter, but at higher concentrations (greater than 1 mM) may open the blood-brain barrier in a nonspecific manner.

Amino Acid Transport Systems↗
PubMed1995

The effect of hyperthermia and verapamil on melphalan cytotoxicity and transport in multidrug-resistant Chinese hamster ovary cells.

The effect of both hyperthermia and verapamil on cytotoxicity and transport of melphalan was studied in a pleiotropic drug-resistant Chinese hamster ovary cell line (CHRC5) and in the drug-sensitive parent line (AuxB1). The CHRC5 cell line was selected for resistance to colchicine but is also cross-resistant to other drugs including melphalan. Verapamil (10 microM) increased melphalan cytotoxicity in drug-resistant cells but not in drug-sensitive cells. Hyperthermia (40 to 45 degrees C) increased melphalan cytotoxicity in both cell lines. In drug-resistant but not drug-sensitive cells, melphalan cytotoxicity was increased further when verapamil was combined with hyperthermia (40 to 45 degrees C). The increased cytotoxicity caused by verapamil in drug-resistant cells was accompanied by alterations in membrane permeability to melphalan. The cellular uptake of melphalan after 15 min increased in the presence of verapamil (7 to 30 microM) at 37 and 42 degrees C. When verapamil (10 microM) was present, the rate of efflux of melphalan from CHRC5 cells decreased by almost 40% at 37 degrees C. The rate of efflux was increased at 42 degrees C relative to 37 degrees C, but with verapamil the rate decreased to that obtained at 37 degrees C in CHRC5 cells. In drug-sensitive cells, verapamil (< or = 50 microM) did not affect either uptake or efflux of melphalan. These findings suggest that verapamil could be beneficial by increasing the effectiveness of melphalan in the elimination of multidrug-resistant cells. The combination of hyperthermia and verapamil could be especially advantageous by increasing melphalan cytotoxicity in a localized target region.

Animals↗
PubMed1997

Synergism of tumor necrosis factor-alpha and melphalan in systemic and regional administration: animal study.

Tumor necrosis factor (TNF) is a highly cytotoxic cytokine. However, due to its severe side effects, the only clinical situation allowing its administration in humans is isolated limb perfusion (ILP). Early studies have shown that TNF alone is of limited efficacy even at high doses via ILP, and that a chemotherapeutic agent needs to be added. The most commonly used drug in this setting is melphalan which is considered to be synergistic with TNF. However, since melphalan has not been commonly used in sarcoma, we believed that confirmation of its synergistic effect with TNF in an experimental sarcoma model could prove valuable for future drug choice. B16F10 melanoma and CT26 colon carcinoma cells were injected subcutaneously (s.c.) into mice, while GF fibrosarcoma cells were injected s.c. into the hindleg of Wistar rats. The animals were then divided into four treatment groups: TNF alone, melphalan alone, TNF and melphalan, and 0.9% NaCl controls. Mice were treated with intraperitoneal injections and rats by ILP. TNF dosage was 20 microgram for mice and 200 microgram for rats. Melphalan was given at 5-10 mg/kg for both mice and rats. Results showed synergism of TNF and melphalan in both modes of therapy. In the systemic administration groups (mice carrying B16F10 and CT26 tumors), tumors increased in size in all but the combined TNF-melphalan group. In the regional delivery groups (rats carrying GF sarcoma cells treated via ILP), there was a 16% decrease in tumor volume in rats treated with TNF alone, a 29% decrease in rats treated with melphalan, and a 75% decrease in the combined TNF-melphalan group. In conclusion, TNF and melphalan proved to be highly synergistic in both systemic and regional delivery. This fact makes melphalan an adequate choice for TNF perfusion in advanced limb malignancies.

Animals↗
PubMed1985

Effects of amino acids on the transport and cytotoxicity of melphalan by human bone marrow cells and human tumor cells.

In human tumor cells freshly obtained from patients with breast cancer, ovarian cancer, or adenocarcinoma of unknown etiology and in normal human bone marrow cells, the cell-to-medium ratio (intracellular/extracellular concentration) in vitro of 5.42 microM melphalan rose rapidly to levels of 6-17 after 35 min at 37 degrees C in Dulbecco's phosphate-buffered saline containing bovine serum albumin and glucose. Only patient C (breast cancer) had received chemotherapy. In all cells studied, L amino acids (1 mM) such as leucine, glutamine, tyrosine, and methionine reduced the cell-to-medium ratio of melphalan at 3 and 35 min. There was a good correlation between the reduction of melphalan transport at 35 min in the heterogeneous nucleated bone marrow cell population by amino acids and their effect on melphalan cytotoxicity in the CFU-C system. Aminoisobutyric acid (A1B), a specific substrate of the A system of amino acid transport, at a concentration between 1 and 50 mM had no significant effect on melphalan uptake at 3 min in any of the human cells studied except those of patient C. At 35 min A1B (10 or 50 mM) significantly reduced the intracellular melphalan concentration in normal bone marrow cells and tumor cells from patients B and C. At 2 mM, 2-aminobicyclo-(2, 2,1)-heptane-2-carboxylic acid (BCH), a specific substrate of the L system of amino acid transport, reduced the cell-to-medium ratio to 70% of control at 3 and 35 min in human bone marrow cells. In tumor cells from patients A, B, D, and F, 2 mM BCH had no significant effect on melphalan uptake at 3 min; it slightly decreased uptake in tumor cells from patient C. At 35 min, 2 mM BCH significantly reduced melphalan transport in tumor cells from patients C and F only. The lack of a BCH-suppressible component to melphalan uptake into human tumor cells freshly obtained from previously untreated patients contrasts with the presence of this component in murine L1210 leukemia cells, murine P388 leukemia cells, and human tumor cell lines. This suggests that minor differences in melphalan transport may exist amongst species and also between human tumor cells which are freshly obtained and cell lines maintained in culture.

Adenocarcinoma↗
PubMed1987

Melphalan concentration dependent plasma protein binding in healthy humans and rats.

The binding of melphalan to plasma proteins from four healthy humans and from rats was measured by centrifugal ultrafiltration. Melphalan concentrations were determined by HPLC and by measuring 14C-melphalan activity. In whole blood, melphalan was distributed preferentially in plasma. However, a constant fraction, 37%, which was independent of the total melphalan concentration in whole blood, was present within the red blood cells. The binding of melphalan to plasma proteins from humans was less than that from rats. In both, however, the fraction bound was constant throughout the concentration range (0.1 to 9.0 microM) that is achieved during standard-dose melphalan therapy. Albumin was the primary binding protein. At concentrations equal to or in excess of 33 microM, which have been achieved during high-dose melphalan therapy, free plasma melphalan concentrations were no longer linearly related to total drug concentrations, and the plasma protein binding of melphalan in the human became concentration dependent. This occurred at concentrations of 70 microM in the rat. Scatchard analysis of the data indicated the presence of 2 groups of binding sites. Class I sites had 0.03 and 0.4 binding sites per albumin molecule in humans and rats, with respective association constants of 4.43 X 10(4) M-1 and 1.92 X 10(4) M-1. Class II sites had 5.18 and 2.60 binding sites per molecule, with respective association constants of 3.82 X 10(2) M-1 and 2.01 X 10(2) M-1.

Animals↗
PubMed2004

Hyperthermia modifies pharmacokinetics and tissue distribution of intraperitoneal melphalan in a rat model.

BACKGROUND AND OBJECTIVES: Peritoneal surface malignancy is a common manifestation of failure of treatment for abdominal cancers. Best results of treatment have been achieved with complete cytoreduction followed by heated intraoperative chemotherapy. Melphalan is a chemotherapeutic agent that shows increased pharmacological activity with heat. But the combination of intraperitoneal administration and heat have never been tested for this drug. The purpose of this study was to evaluate the effect of hyperthermia on the pharmacokinetics and tissue distribution of intraperitoneal melphalan in a rodent model. METHODS: Melphalan was given by the intraperitoneal route to 20 Sprague-Dawley rats at a dose of 12 mg/kg over 90 min. Rats were randomized into two groups according to the temperature of the peritoneal perfusate: group NT received normothermic (33.5 degrees C) melphalan; group HT received hyperthermic (42 degrees C) melphalan. During the course of intraperitoneal chemotherapy, peritoneal fluid and blood were sampled at 5, 15, 30, 60 and 90 min. At the end of procedure, the rats were killed and tissues samples (heart, liver, ileum, jejunum, colon, omentum, and abdominal wall) were collected. Concentrations of melphalan were determined in peritoneal fluid, plasma, and tissues by high-performance liquid chromatography. RESULTS: The area under the curve (AUC) of peritoneal fluid melphalan was significantly lower in the HT group than in the NT group ( P=0.001), whereas no significant difference in plasma AUC was found. AUC ratios (AUC peritoneal fluid/AUC plasma) were 12.1 for the NT group and 12.3 for the HT group. The mean time to reach the plasma peak was shorter in the HT group than in the NT group ( P=0.004). The HT group exhibited increased melphalan concentrations in all intraabdominal tissues. These differences were significant for the ileum ( P=0.03) and jejunum ( P=0.04). CONCLUSION: Hyperthermia affected the pharmacokinetics of intraperitoneal melphalan by decreasing the AUC of peritoneal fluid melphalan without increasing the plasma AUC. It increased intraabdominal tissue concentrations.

Animals↗
PubMed1996

Hyaluronidase pretreatment produces selective melphalan enrichment in malignant melanoma implanted in nude mice.

Preclinical and clinical observations suggest that the administration of hyaluronidase (Hyase) shortly before that of chemotherapy increases the access and, thus, the effectiveness of anticancer drugs in tumors. To examine this hypotheses as well as the selectivity of such a therapeutic approach potentially beneficial in isolated limb perfusion, the Hyase-induced distribution of melphalan was measured in tumor-bearing nude mice with respect to the mode of drug administration using RP-18 ion-pair high-performance liquid chromatography (HPLC) with fluorimetric detection. Melphalan alone (50 micromol/kg) or a combination of melphalan (50 micro mol/kg) and Hyase (100,000 IU/kg) was injected either i.p. or s.c. in the vicinity of the tumors. The s.c. melphalan injection caused a 4-fold rise in melphalan concentration (59 microM) in the tumors as compared with i.p. application (15 microM). Only minor effects were observed with respect to the route of melphalan application on its distribution in other tissues (ca. 13 microM in plasma, 15 microM in muscle, 30 microM in the liver, 26 microM in the kidney, and 21 microM in the testicle). Irrespective of the route of Hyase coadministration, the enzyme increased the concentration of i.p. injected melphalan in all tissues to ca. 20 microM in the tumor, 15 microM in plasma, 27 microM in muscle, 40 microM in the liver, 29 microM in the kidney, and 28 microM in the testicle. In contrast, s.c. injected melphalan was selectively accumulated by the tumors after both s.c. and i.p. Hyase administration (462 and 388 microM, respectively). Melphalan enrichment in the tumors was higher (16- to 32-fold higher than in the other tissues) after i.p. administration of Hyase since, in contrast to s.c. injection of the enzyme, its i.p. administration caused a decrease in the concentration of the cytostatic in all other tissues as compared with the s.c. administration of melphalan alone.

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