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PubMed1991

Temperature dependence of melphalan efflux kinetics in Chinese hamster ovary cells.

The temperature dependence of the kinetics of efflux of melphalan from Chinese hamster ovary (CHO) cells was studied from 4 degrees to 47 degrees. Time courses for melphalan efflux showed an initial rapid phase of efflux followed by a plateau. The data for melphalan concentration (c) versus efflux time (t) were described by the equation c(t) = A + B exp(-kt), where A is the final steady-state melphalan concentration, B is the total change in melphalan concentration from time zero until steady-state conditions are reached, and k is the rate constant for the efflux process. The plateau level obtained was not dependent on temperature and corresponded to 22 +/- 3.2% of the drug remaining in the cells after efflux. The time for melphalan efflux to reach the plateau level was dependent on temperature. This was reflected by an increase in the rate constants for melphalan efflux with increasing temperature from 30 degrees to 47 degrees. The rate constant for melphalan efflux at 37 degrees was 0.045 +/- 0.002 min-1. Efflux of melphalan occurred much more slowly at lower temperatures such as 4 degrees and 20 degrees. An Arrhenius plot for melphalan efflux showed a linear and decreasing trend at temperatures between 30 degrees and 47 degrees with an activation energy of 1.046 x 10(3) J/mol.

Animals↗
PubMed1998

Isolated lung perfusion with melphalan and tumor necrosis factor for metastatic pulmonary adenocarcinoma.

BACKGROUND: Isolated left lung perfusion with melphalan and human tumor necrosis factor-alpha for pulmonary metastatic adenocarcinoma in the WAG/Rij rat was studied. METHODS: Survival was determined for melphalan, human tumor necrosis-alpha. Lung, pulmonary effluent, and serum melphalan were analyzed by chromatography after isolated lung perfusion or intravenous injection. On day 0, rats were injected with 2.0 x 10(6) CC531S cells intravenously. On day 7, rats underwent sham thoracotomy, received melphalan intravenously, or underwent isolated left lung perfusion with saline, melphalan, tumor necrosis factor, and a combination of the latter two. On day 14, tumor nodules were counted. RESULTS: For the doses of 400 microg tumor necrosis factor, 1,000 microg tumor necrosis factor, or both melphalan and tumor necrosis factor (2 mg + 200 microg), survival rates after contralateral pneumonectomy were 33%, 17%, and 80%, respectively. Survival in all other groups was 100%. Left lung melphalan level was significantly higher after isolated lung perfusion compared to intravenous administration. Significantly fewer left lung nodules were found for 0.5 mg isolated lung perfusion with melphalan (28+/-17) compared to isolated administration (200+/-0) (p = 0.001), and for 1.0 mg intravenous lung perfusion with melphalan (16+/-10) compared to controls (171+/-65) (p = 0.00047). Tumor necrosis factor showed no significant effect. CONCLUSIONS: Isolated lung perfusion with melphalan is an effective treatment for pulmonary metastases from adenocarcinoma in the rat.

Adenocarcinoma↗
PubMed2002

Reduced liver uptake of arterially infused melphalan during retrograde rat liver perfusion with unaffected liver tumor uptake.

Isolated hepatic perfusion (IHP) with melphalan is used for patients with nonresectable metastases confined to the liver. To improve the efficacy of IHP and to reduce the toxicity to the liver, reversion (retrograde perfusion) of the bloodstream through the liver in a rat model was studied. For liver tumor induction male WAG/Rij rats were inoculated with CC531 cells, a colorectal tumor cell line. After 11 to 12 days the tumor-bearing rat livers were perfused by single-pass perfusion through either the portal (orthograde) or caval vein (retrograde) for different time periods. During perfusion melphalan (160 Schultze) was infused in the hepatic artery. Melphalan concentrations were measured by high-performance liquid chromatography. A rapid extraction of melphalan by the liver occurred in the first 5 min, reaching steady state after 10 to 20 min for both perfusion directions. The melphalan concentration of the outflow perfusate was significantly higher in the retrograde perfusion compared with the orthograde perfusion. The melphalan content of the tumor tissue was unaffected by perfusion direction at any time point. To the contrary, the melphalan uptake in liver tissue was strongly influenced: the melphalan content after 40-min retrograde perfusion was 12% of that after orthograde perfusion. The average tumor/liver concentration ratio was 6 for orthograde perfusion and 30 for retrograde perfusion. In conclusion, retrograde IHP with continuous melphalan infusion in the hepatic artery provides a high tumor uptake of melphalan with potentially reduced liver toxicity compared with orthograde IHP.

Animals↗
PubMed2005

Synergistic antitumor response of interleukin 2 with melphalan in isolated limb perfusion in soft tissue sarcoma-bearing rats.

The cytokine interleukin 2 (IL-2) is a mediator of immune cell activation with some antitumor activity, mainly in renal cell cancer and melanoma. We have previously shown that tumor necrosis factor (TNF)-alpha has strong synergistic antitumor activity in combination with chemotherapeutics in the isolated limb perfusion (ILP) setting based on a TNF-mediated enhanced tumor-selective uptake of the chemotherapeutic drug followed by a selective destruction of the tumor vasculature. IL-2 can cause vascular leakage and edema and for this reason we examined the antitumor activity of a combined treatment with IL-2 and melphalan in our well-established ILP in soft tissue sarcoma-bearing rats (BN175). ILP with either IL-2 or melphalan alone has no antitumor effect, but the combination of IL-2 and melphalan resulted in a strong synergistic tumor response, without any local or systemic toxicity. IL-2 enhanced significantly melphalan uptake in tumor tissue. No signs of significant vascular damage were detected to account for this observation, although the tumor sections of the IL-2- and IL-2 plus melphalan-treated animals revealed scattered extravasation of erythrocytes compared with the untreated animals. Clear differences were seen in the localization of ED-1 cells, with an even distribution in the sham, IL-2 and melphalan treatments, whereas in the IL-2 plus melphalan-treated tumors clustered ED-1 cells were found. Additionally, increased levels of TNF mRNA were found in tumors treated with IL-2 and IL-2 plus melphalan. These observations indicate a potentially important role for macrophages in the IL-2-based perfusion. The results in our study indicate that the novel combination of IL-2 and melphalan in ILP has synergistic antitumor activity and may be an alternative for ILP with TNF and melphalan.

Animals↗
PubMed1988

The systemic administration of intravenous melphalan.

Melphalan (L-phenylalanine mustard) is a bifunctional alkylating agent that is commonly administered orally to treat a wide variety of malignancies, including cancers of the breast and ovary, as well as multiple myeloma. Although commercially available in Europe and Canada, intravenous (IV) melphalan remains investigational in the United States. The role of IV melphalan in cancer chemotherapy is not well defined, despite its manageable toxicity and higher and more predictable blood levels following IV administration compared with oral administration. In addition, unlike oral melphalan, an extensive phase I evaluation of IV melphalan has not been undertaken. At lower doses (eg, 30 to 70 mg/m2), both as a single agent and in combination, the activity of IV melphalan has been evaluated in only a limited number of diseases. However, striking activity has been observed in previously untreated patients with rhabdomyosarcoma, a disease not generally considered responsive to alkylating agents. When administered at high doses (greater than 140 mg/m2) requiring bone marrow reinfusion, melphalan effects a high response rate (but no improvement in survival) in a variety of nonhematologic tumor types, including resistant tumors such as melanoma and colon carcinoma. In contrast, in poor-prognosis patients with non-Hodgkin's lymphoma, Hodgkin's disease, multiple myeloma, or neuroblastoma, high-dose melphalan-containing regimens have yielded both high response rates and improved survival, despite considerable toxicity. Additional clinical trials will be necessary to define the spectrum of activity of lower doses of IV melphalan and to define subgroups of patients most likely to benefit from high-dose melphalan.

Administration, Oral↗
PubMed1995

A phase I trial of amifostine (WR-2721) and melphalan in children with refractory cancer.

Melphalan has a steep dose-response curve, but the use of high doses results in unacceptable myelosuppression. Strategies to circumvent this dose-limiting myelosuppression would allow for the administration of higher, more effective doses of melphalan. Amifostine (WR-2721) has been shown in preclinical studies to protect the bone marrow from the myelotoxicity of melphalan, and in clinical trials, to protect from the myelotoxicity of other alkylating agents. A Phase I trial of the combination of amifostine and melphalan was performed in children with refractory cancers to: (a) define the acute toxicities of amifostine and its maximum tolerated dose (MTD); and (b) to determine whether the dose of melphalan could be safely escalated when administered in combination with amifostine. Amifostine was administered i.v. as a 15-min infusion 30 min before melphalan. The starting dose of amifostine was 750 mg/m2, with planned dose escalations in 30% increments. Melphalan was administered as a 5-min infusion using the previously defined MTD in heavily pretreated patients, 35 mg/m2, as the starting dose. The dose of melphalan was escalated by 30% increments. Nineteen patients, ranging in age from 3 to 24 years (median, 15 years), were entered on trial. The dose of amifostine was escalated to 2700 mg/m2, which is approximately 3-fold higher than the adult recommended dose, without reaching a MTD. Fifteen patients experienced nondose-limiting (< 25%), transient decreases in blood pressure after the amifostine infusion. Other nondose-limiting toxicities of amifostine included mild nausea and vomiting, flushing, anxiety, diarrhea, and urinary retention. Six patients, three each at the 2100 and 2700 mg/m2 amifostine dose levels were treated with an escalated dose of melphalan (45 mg/m2). All of these patients experienced grade 4 neutropenia (< 500/mm3), and five of six patients had grade 4 thrombocytopenia. The duration of this dose-limiting myelosuppression exceeded 7 days in four of six patients. Although no dose-limiting (grade 3 or 4) toxicity was attributed to amifostine, significant anxiety and reversible urinary retention occurred at the two highest amifostine dose levels. A dose of 1650 mg/m2 for pediatric Phase II trials is recommended. High doses of amifostine, however, do not appear to allow for escalation of melphalan beyond its single agent MTD of 35 mg/m2.

Adolescent↗
PubMed1988

Degradation of melphalan in vitro: rationale for the use of continuous exposure in chemosensitivity assays.

The hydrolysis of melphalan in cell culture medium at 37 degrees C has been studied. Degradation of melphalan proceeded via monohydroxy-melphalan (MOH) to dihydroxymelphalan [M(OH)2] with a half-life of 66 min for melphalan and 58 min for MOH. The half-life for melphalan was similar to the terminal half-life of the drug in vivo. The effect of the two metabolites, MOH and M(OH)2, on the chemosensitivity of K562 leukaemia cells during continuous exposure to melphalan was also examined. M(OH)2 had no potentiating effect on melphalan cytotoxicity at concentrations up to 100 micrograms/ml. MOH also had little effect on cell kill at concentrations higher than those commonly achieved during in vitro chemosensitivity assays. The LD50 for 1 h exposure to melphalan was twice that for continuous exposure: this also suggests no interference by MOH and M(OH)2. These data suggest that continuous exposure of melphalan in in vitro chemosensitivity assays is probably preferable to the arbitrary 1 h drug exposure time commonly employed.

Culture Media↗
PubMed1991

Enhanced expansion of the thymic CD8+ cell subset as a potential mechanism for the generation of enhanced antitumor cytotoxicity by thymocytes from low-dose melphalan-treated MOPC-315 tumor bearers.

We have previously shown that thymocytes from low-dose melphalan (L-phenylalanine mustard)-treated MOPC-315-tumor-bearing mice (melphalan TuB) are able to generate an enhanced level of anti-MOPC-315 cytotoxicity, as compared to thymocytes from untreated MOPC-315-tumor-bearing mice or thymocytes from untreated or low-dose melphalan-treated normal mice, upon in vitro stimulation with MOPC-315 tumor cells in the presence of a low concentration of recombinant interleukin-2 (rIL-2). Here we show that the generation of enhanced anti-MOPC-315 cytotoxicity by melphalan TuB thymocytes depends on the ability of the thymocytes to proliferate. In addition, the ability of melphalan TuB thymocytes to generate an enhanced level of anti-MOPC-315 cytotoxicity correlated with their ability to proliferate more readily than thymocytes from untreated tumor-bearing mice and thymocytes from untreated or melphalan-treated normal mice in response to stimulation with MOPC-315 tumor cells plus a low concentration of rIL-2. Moreover, although fresh melphalan TuB thymocytes do not contain a higher percentage of phenotypically mature cells (i.e., CD4-/CD8+ or CD4+/CD8-) than do thymocytes from normal mice or untreated tumor-bearing mice, after a 5-day culture with both MOPC-315 tumor cells and a low concentration of rIL-2, cultures of thymocytes from melphalan TuB contained a much higher percentage of CD4-/CD8+ (but not CD4+/CD8-) cells than did cultures of thymocytes from the other two sources. Since CD4-/CD8+ cells were previously shown to be responsible for the exertion of antitumor cytotoxicity by thymocytes stimulated with MOPC-315 in vitro, our results indicate that the enhanced antitumor cytotoxicity exerted by melphalan TuB thymocytes following in vitro stimulation with MOPC-315 tumor cells in the presence of a low concentration of rIL-2 is due, at least in part, to an expansion of the pool of CD4-/CD8+ effector cells.

Animals↗
PubMed1989

Enhancement of the anti-tumor effect of melphalan in experimental mice by some vaso-active agents.

A comparison is made between the vaso-active agents hydralazine, nifedipine, and verapamil for their ability to increase the anti-tumor effectiveness of melphalan. Treatment of mice with hydralazine (5 mg/kg) 15 mins after melphalan increases by a factor of approximately 2.5 melphalan-induced delay in growth of either the RIF-1 or KHT tumors. Similar enhancements are obtained when measurement is made of the surviving fraction of tumor cells in vitro following treatment in vivo with hydralazine and melphalan. Further, tumor cell kill is also increased when nifedipine is administered with melphalan compared with the effect of melphalan alone. These enhanced effects are observed if the vaso-active agents are given before or after melphalan. Hydralazine (5 mg/kg) induces close to 100% radiobiological hypoxia in the RIF-1 and KHT tumors. In contrast, Nifedipine has no effect on tumor hypoxic fraction at a dose of 10 mg/kg although the anti-tumor effectiveness of melphalan is substantially increased. However, a higher dose of 50 mg/kg nifedipine causes a large increase in tumor hypoxic fraction, an effect that persists for several hours. Verapamil causes no change in the fraction of hypoxic cells in the KHT tumor and increases, only slightly, the anti-tumor effect of melphalan.

Animals↗
PubMed2005

Metabolism of melphalan by rat liver microsomal glutathione S-transferase.

One of the major problems in the treatment of human cancer is the phenomenon of drug resistance. Increased glutathione (gamma-glutamylcysteinylglycine, GSH) conjugation (inactivation) due to elevated level of cytosolic glutathione S-transferase (GST) is believed to be an important mechanism in tumor cell resistance. However, the potential involvement of microsomal GST in the establishment of acquired drug resistance (ADR) remains uncertain. In our experiments, a combination of liquid chromatography/electrospray ionization/mass spectrometry (LC/ESI/MS) was employed for structural characterization of the resulting conjugates between GSH and melphalan, one of the alkylating agents. The spontaneous reaction of 1mM melphalan with 5mM GSH at 37 degrees C in aqueous phosphate buffer for 1h gave primarily the monoglutathionyl and diglutathionyl melphalan derivatives, with small amounts of mono- and dihydroxy melphalan derivatives. We demonstrated that rat liver microsomal GST presented a strong catalytic effect on the reaction as determined by the increase of monoglutathionyl and diglutathionyl melphalan derivatives and the decrease of melphalan. We showed that microsomal GST was activated by melphalan in a concentration- and time-dependent manner. Microsomal GST which was stimulated approximately 1.5-fold with melphalan had a stronger catalytic effect. Thus microsomal GST may play a potential role in the metabolism of melphalan in biological membranes, and in the development of ADR.

Animals↗
PubMed1993

Characteristics of the glutathione/glutathione-S-transferase detoxification system in melphalan resistant human prostate cancer cells.

Glutathione (GSH) and glutathione-S-transferases (GST) have been implicated in resistance of tumor cells to certain alkylating agents, including melphalan. Glutathione levels and GST activities were determined in melphalan-resistant sublines of the human prostate carcinoma cell lines DU 145, PC-3 and LNCaP produced by serial treatment with melphalan at progressively increasing concentrations. The resistant sublines M4.5DU145, M5DU145, M6DU145, M6PC-3 and M6LNCaP were 27-, 7-, 3-, 6- and 2-fold more resistant to melphalan than the parental lines. The melphalan-resistant DU 145 and PC-3 lines showed cross-resistance to cisplatin and tetraplatin, but retained sensitivity to vinblastine, colchicine and etoposide. Interestingly, both sublines were also resistant to methotrexate and adriamycin. The melphalan-resistant LNCaP line showed slight resistance to cisplatin and adriamycin, but remained sensitive to tetraplatin and methotrexate. This line also retained sensitivity to vinblastine while developing resistance to colchicine. Intracellular GSH levels were increased 2.8 fold for M5DU145, 1.7 fold for M6PC-3 and 2.1 fold for M6LNCaP compared to the parental lines, whereas GST activity using chlorodinitro-benzene as a substrate was comparable for all lines. When cumene hydroperoxide was used as a substrate, an increase in GST activity was noted only in the M6PC-3 line as compared with the parent line. Western blot analysis showed no change in GST isozyme profile between parent and resistant DU 145 lines; however a mu class isoenzyme was detected in the resistant, but not in the parent PC-3 line, using a Yb1 antibody. M5DU145 cells maintained in the absence of melphalan for seven months maintained their resistance to melphalan. Depletion of GSH, with buthionine sulfoximine, to control levels reversed melphalan resistance to control levels.

Antineoplastic Agents↗
PubMed2001

Microdialysis and response during regional chemotherapy by isolated limb infusion of melphalan for limb malignancies.

This study sought to use a microdialysis technique to relate clinical and biochemical responses to the time course of melphalan concentrations in the subcutaneous interstitial space and in tumour tissue (melanoma, malignant fibrous histiocytoma, Merkel cell tumour and osteosarcoma) in patients undergoing regional chemotherapy by Isolated Limb Infusion (ILI). 19 patients undergoing ILI for treatment of various limb malignancies were monitored for intra-operative melphalan concentrations in plasma and, using microdialysis, in subcutaneous and tumour tissues. Peak and mean concentrations of melphalan were significantly higher in plasma than in subcutaneous or tumour microdialysate. There was no significant difference between drug peak and mean concentrations in interstitial and tumour tissue, indicating that there was no preferential uptake of melphalan into the tumours. The time course of melphalan in the microdialysate could be described by a pharmacokinetic model which assumed melphalan distributed from the plasma into the interstitial space. The model also accounted for the vascular dispersion of melphalan in the limb. Tumour response in the whole group to treatment was partial response: 53.8% (n = 7); complete response: 33.3% (n = 5); no response: 6.7% (n = 1). There was a significant association between tumour response and melphalan concentrations measured over time in subcutaneous microdialysate (P< 0.01). No significant relationship existed between the severity of toxic reactions in the limb or peak plasma creatine phosphokinase levels and peak melphalan microdialysate or plasma concentrations. It is concluded that microdialysis is a technique well suited for measuring concentrations of cytotoxic drug during ILI. The possibility of predicting actual concentrations of cytotoxic drug in the limb during ILI using our model opens an opportunity for improved drug dose calculation. The combination of predicting tissue concentrations and monitoring in microdialysate of subcutaneous tissue could help optimise ILI with regard to post-operative limb morbidity and tumour response.

Adult↗
PubMed1995

Effect of melphalan and hyperthermia on cell cycle progression and cyclin B1 expression in human melanoma cells.

We have investigated the effect of mild hyperthermia (42 degrees C) on the cytotoxic activity of a 1 h melphalan exposure in human melanoma cell lines. Hyperthermia did not affect cell growth of any culture, but it increased, to a different extent, melphalan cytotoxicity in all cell lines, with a reduction in the IC50 of 1.7 to 2.6-fold. Flow cytometric analysis showed that in normal temperature conditions melphalan caused S phase cell accumulation, which was evident only at 24 h in JR8, M14 and 2/21 cell lines and was still persistent at 72 h in 2/60 cells. Moreover, in all cell lines, the delay in S phase was paralleled, or followed, by an accumulation of cells in G2+M, which was transient in JR8 and M14 cells and persisted until 72 h in 2/21 and 2/60 melanoma clones. Hyperthermia caused a stabilization and prolongation of melphalan induced G2+M accumulation in JR8 and M14 cells. Conversely, in 2/21 and 2/60 clones, cell cycle perturbations induced by the drug were similar under normothermic or hyperthermic conditions. Specifically, in JR8, for which the maximum enhancement by hyperthermia on melphalan cytotoxicity was observed, cell accumulation in G2+M was still present 120 h after treatment. The accumulation was accompanied by an inhibition in the G2-M transition, as demonstrated by the significant reduction in the mitotic index of cells exposed to combined treatment compared to controls. Moreover, a bivariate distribution of cells stained for DNA and cyclin B1 showed that, following melphalan and hyperthermia treatment, the fraction of cyclin B1-expressing cells paralleled the fraction of G2+M phase cells, thus indicating that the inability of cells to enter mitosis was not ascribable to a reduction of cyclin B1 expression. On the whole, our results indicate that hyperthermia can stabilize the G2 accumulation induced by melphalan in human melanoma cells. Such a stabilization could contribute to the enhancement of melphalan cytotoxicity by heat, even though a strict correlation was not observed between the magnitude and persistence of the cell cycle perturbations and the extent of melphalan activity.

Cell Cycle↗
PubMed1992

The effect of an amino acid-lowering diet on the rate of melphalan entry into brain and xenotransplanted glioma.

Melphalan (L-phenylalanine mustard, L-PAM, alkeran; molecular weight, 305,000) is transported across tumor cell membranes and the blood-brain barrier by the large neutral amino acid (LNAA) transport system. Normally, plasma LNAA levels are high enough and the affinity low enough that this system does not transport much melphalan into the brain. However, plasma amino acids can be reduced by fasting and protein-free diet. We used this method to reduce competition and to increase melphalan transport into brain tumors. In nude mice fasted for 12 h and then fed a protein-free diet for 2 and 6 h, mean plasma LNAA levels were 46% and 42% of control values. Nude mice with xenotransplanted D-54MG human gliomas were used to study tissue distribution and uptake kinetics of [3H]melphalan in a control group and a diet group (after a 12-h fast and 2 h of a 0% protein diet). The K1 (blood-to-tissue transfer constant) of melphalan, determined by graphical analysis and by nonlinear fitting to a 2-compartment model, was higher in the diet group in all tumor regions except the necrotic center of subcutaneous tumors; the increase was significant in the tumor periphery of brain and s.c. tumors. The ratio of K1s (diet to control) varied from 1.2 to 1.3 in brain tumors, 1.9 to 2.1 in subcutaneous tumors, and 1.8 to 3.1 in tumor-free brain. The apparent [3H]melphalan distribution space was significantly higher in the tumor periphery of both brain and subcutaneous tumors of the 15- and 30-min diet group. We also measured blood-brain barrier transport of [alpha-14C]aminoisobutyric acid and blood flow (with [131I]iodoantipyrine): the K1 of [alpha-14C]aminoisobutyric acid was 28.1 +/- 6.6 (SE) in brain tumors and 24.3 +/- 8.9 microliters/g/min in subcutaneous tumors. Blood flow was 58.2 --> 3.9 in brain tumors and 5.2 +/- 0.4 ml/100 g/min in subcutaneous tumors. Fasting, when combined with a protein-free diet, reduces plasma amino acid levels and thereby reduces competition between melphalan and LNAAs. This may increase the amount of melphalan that can enter a brain tumor without increasing the administered drug dose and suggests a therapeutic manipulation that can be used to increase the delivery of melphalan.

Amino Acid Transport Systems↗
PubMed1988

Melphalan transport, glutathione levels, and glutathione-S-transferase activity in human medulloblastoma.

Melphalan transport, glutathione levels, and glutathione-S-transferase activity were measured in two continuous human medulloblastoma cell lines and transplantable xenografts in athymic nude mice, TE-671 and Daoy. In vitro mean glutathione levels were 10.06 nmol/10(6) cells in TE-671 and 2.96 nmol/10(6) cells in Daoy. In vitro mean glutathione-S-transferase values were 91.52 nmol/min/mg protein in TE-671 and 50.31 nmol/min/mg protein in Daoy. Transport studies revealed kinetic parameters of Km = 108.3 microM, Vmax = 363.1 pmol/10(6) cells/min in TE-671 and Km = 111.7 microM, Vmax = 180.6 pmol/10(6) cells/min in Daoy. Melphalan transport was inhibited by both DL-alpha-2-aminobicyclo[2.2.1]heptane-2- carboxylic acid and sodium ion depletion in TE-671 and Daoy cells in vitro, indicating that both systems of amino acid transport are functional in these medulloblastoma lines. In vivo s.c. xenograft glutathione values were lower (7.79 nmol/mg protein) in TE-671 than in Daoy (13.68 nmol/mg protein). The mean plasma concentration in mice given a 10% lethal dose (71.3 mg/m2) of melphalan i.p. was 50.3 microM at 10 min, with the half-life of 29.9 min. At this dose, s.c. xenograft levels were 2- to 3-fold higher in TE-671 than in Daoy tumors for the 3-h period measured. These studies demonstrate transport parameters confirming facilitated transport of melphalan in human medulloblastoma, a mean murine plasma melphalan concentration (following treatment with melphalan) above the in vitro drug dose at which there is a 90% reduction in the number of colonies in comparison to controls for TE-671 and Daoy for 2 h, and glutathione and glutathione-S-transferase levels in the same range previously reported in other melphalan-sensitive and melphalan-resistant human tumors. Future work with spontaneous and acquired melphalan-resistant human medulloblastoma cell lines and xenografts will define the role of these mechanisms in mediating drug resistance.

Animals↗
PubMed1981

Mechanism of melphalan resistance developed in vitro in human melanoma cells.

Melphalan resistance developed previously in a human melanoma cell line (MM253) could not be further increased. Cross-resistance was found to nitrogen mustard but not to ultraviolet light radiation. A clone of MM253 had the same drug sensitivity and heterogeneous chromosome complements as did the parent culture. The melphalan-resistant cells (MM253-12M) had 2.6-fold the D0, 1.5-fold the size, 1.3-fold the RNA content, 1.4-fold the protein content, and 2.6-fold the DNA content of the sensitive parent line. There was no evidence for activation or detoxification of melphalan by intact melanoma cells or by mouse liver microsomes competent for the activation of other drugs. Melphalan transport was similar in both cell lines, reaching a steady-state level 3 times the concentration in the medium after 2.5 min. Both lines covalently bound the same total amount of [3H]melphalan per cell, but in MM253-12M a 50% decrease in binding to DNA was almost sufficient to account for the increase in resistance. The level of melphalan-induced DNA interstrand cross-links, which were heat labile but not alkali labile, reached a maximum during the 4-hr treatment period and then declined slowly. The degree of cross-linking in MM253-12M was 50% less than that in MM253. Unlike ultraviolet light, methyl methanesulfonate, and nitrogen mustard, melphalan at equitoxic doses did not damage the DNA sufficiently to immediately inhibit DNA synthesis. Although both lines were proficient for repair of ultraviolet light and methyl methane sulfonate damage, melphalan did not induce significant levels of DNA repair synthesis and had little effect on the rate of DNA chain elongation. In MM253 cells, strand breaks were detected only at high melphalan doses; MM253-12M formed breaks more readily. This evidence suggests that the cross-linking events and that developed resistance arises from decreased susceptibility to DNA to this damage.

Biological Transport↗
PubMed1984

Potentiation of melphalan cytotoxicity in human ovarian cancer cell lines by glutathione depletion.

The effectiveness of alkylating agents in the treatment of ovarian cancer is limited by the frequent development of drug resistance. In order to examine the mechanisms of resistance and potential ways in which this resistance could be overcome, we have developed a human ovarian cancer cell line, 1847ME, resistant to the bifunctional amino acid nitrogen mustard, melphalan. A 4-fold higher concentration of melphalan was required to produce an equivalent reduction in tumor colony formation in 1847ME cells as compared to the parent melphalan-sensitive line A1847. The magnitude of resistance in 1847ME was similar to that observed in the cell lines NIH:OVCAR-2, NIH:OVCAR-3, and NIH:OVCAR-4 which were derived from ovarian cancer patients clinically resistant to alkylating agents. There was no detectable difference in melphalan uptake between A1847 and 1847ME. The cellular content of the inactive dihydroxy melphalan metabolite, however, was two times greater in 1847ME compared to A1847. Levels of the principal intracellular thiol, glutathione, were found to be 2-fold greater in 1847ME than in A1847, and to be similarly elevated in the OVCAR lines. Depletion of glutathione by incubation of the cells in cystine-free medium or in the presence of the specific inhibitor of glutathione synthesis, DL-buthionine-S,R-sulfoximine, was accompanied by a marked increase in melphalan cytotoxicity. Doses of DL-buthionine-S,R-sulfoximine which were only minimally cytotoxic were associated with complete reversal of the induced resistance to melphalan in 1847ME. Synergism between melphalan and DL-buthionine-S,R-sulfoximine was also demonstrated in the OVCAR cell lines derived from previously treated ovarian cancer patients. The reversal of induced resistance to melphalan by modulation of glutathione levels is of potential clinical relevance. In addition, these cell lines provide a useful model system in which to study further the mechanisms of alkylating agent resistance in human tumors.

Buthionine Sulfoximine↗
PubMed1994

Intrathecal melphalan therapy of human neoplastic meningitis in athymic nude rats.

We report the activity and toxicity of intrathecal melphalan in the treatment of human neoplastic meningitis in the subarachnoid space of athymic nude rats. Animals received injections via chronic indwelling subarachnoid catheters with 5 x 10(5) or 5 x 10(6) TE-671 human rhabdomyosarcoma cells or 5 x 10(6) D-54 MG human glioma cells and were treated with melphalan on days 8, 5, or 5, respectively. Melphalan toxicity in nontumor-bearing rats was assessed at single doses of a 2.0, 3.0, 4.0, or 5.0 mM solution, with clinical and histological evidence of neurotoxicity observed at the 4.0 and 5.0 mM levels. Multiple-dose toxicity studies using a dosing schedule of twice a week for two weeks with a 0.25, 0.5, 0.75, 1.0, 1.5, or 2 mM solution revealed dose-dependent clinical and histological evidence for toxicity at all dosages. Treatment of TE-671 with a single dose of 2.0 mM intrathecal melphalan produced an increase in median survival of 442% compared with saline controls (P < 0.003). Comparison of a single dose of 1.0 or 2.0 mM melphalan with a multiple dose regimen at 0.25 or 0.5 mM melphalan in the treatment of TE-671 revealed increases in median survival of 50% for 1.0 mM, 57% for 2.0 mM, 79% for 0.5 mM, and 111% for 0.25 mM concentrations. Comparison of a single dose of 1 mM melphalan with multiple doses of 0.25 mM melphalan in the treatment of D-54 MG revealed an increase in median survival of 475+% for each of the regimens. Intrathecal melphalan may be an important new addition in the treatment of neoplastic meningitis and is currently being evaluated clinically in a Phase 1 trial.

Animals↗