Comparison of the cytotoxic activity of melphalan with L-prolyl-m-L-sarcolysyl-L-p-fluorophenylalanine in human tumour cell lines and primary cultures of tumour cells from patients.

m-L-sarcolysin (m-L-SL) is an isomer of melphalan (Mel) with the di(2-chloroethyl) amino group being substituted in the meta position of phenylalanine. By covalent conjugation of amino acids to m-L-SL, a peptide complex consisting of six m-L-SL-based oligopeptides known as peptichemio (PTC) was developed previously. In the present study, the cytotoxic activity pattern of the different oligopeptides of PTC was investigated in ten human tumour cell lines representing different mechanisms of cytotoxic drug resistance using the fluorometric microculture cytotoxicity assay (FMCA). In the cell line panel, L-prolyl-m-L-sarcolysyl-L-p-fluorophenylalanine (P2) was the most active oligopeptide, showing slightly lower mean IC50 values (2.6 vs 3.9 and 4.1 microg ml(-1)) than Mel and m-L-SL. The other five oligopeptides were less active than Mel. All active oligopeptides showed mechanistic similarity to Mel as judged by the correlation analysis of the cell line panel log IC50 values (R > or = 0.90), although P2 appeared to be less sensitive to GSH-mediated drug resistance. The relative activity of Mel and P2 was found to be related to degree of proliferation, P2 being more active towards low-proliferating cell lines. P2 and Mel were then further characterized in 49 fresh human tumour samples. In these samples P2 was considerably more active than Mel and showed a higher relative solid tumour activity (2.7 to 4.5-fold). However, the correlation of log IC50s between P2 and Mel in patient cells was high (R = 0.79), indicating a similar mechanism of action in this tumour model too. Cross-resistance with other standard drugs was lower for P2 than Mel. The results show that P2 is the most potent component of PTC and demonstrates a favourable activity profile compared with Mel. These data suggest that further investigation of P2 as a potential anti-tumour agent is warranted.

Increased toxicity and DNA cross-linking by peptide bound m-L-sarcolysin (Peptichemio) as compared to melphalan and m-L-sarcolysin in human melanoma cell lines.

Alteration of the melphalan molecule by shifting the di(2-choroethyl)aminogroup from the para- to the meta-position of the phenylalanine residue results in m-L-sarcolysin. By covalent conjugation of different amino acids at the amino- and carboxylgroups of this molecule, a mixture of six peptides known as Peptichemio has been synthesized. In a previous investigation we found that Peptichemio was less toxic to human lymphoblasts than m-L-sarcolysin. In contrast, in the present investigation we found that Peptichemio has higher cytotoxic effect than m-L-sarcolysin on two human melanoma cell lines. The higher cytotoxicity was paralleled by a higher induction of DNA cross-links by Peptichemio as compared to m-L-sarcolysin. A comparative analysis of the six peptides on Peptichemio showed differences in cytotoxic effects on a melanoma cell line. One of the six peptides displayed a considerably higher cytotoxicity than peptichemio itself.

Efficacy of peptide bound m-L-sarcolysin (peptichemio) on melphalan resistant human myeloma cells in vitro.

Peptichemio (PTC) is a mixture of six synthetic oligopeptides, each containing the alkylating agent m-di(2-chloroethyl)amino-phenylalanine. Freshly obtained myeloma cell infiltrated human bone marrow specimens were in parallel exposed to melphalan and PTC. The cytotoxic effect of the drugs on the myeloma cells of each specimen was measured by the differential staining culture method (DISC). PTC displayed higher cytotoxicity to the myeloma cells as compared to melphalan in all 12 cases analysed. The increase of the cytotoxic effect of PTC compared to melphalan varied between different cases. In melphalan resistant cases the cytotoxic effect of PTC as compared to melphalan was clearly significant (P = 0.001).

Differential DNA cross-linking and cytotoxicity in PHA-stimulated human lymphocytes exposed to melphalan, m-L-sarcolysin and peptichemio.

DNA interstrand, as well as DNA protein cross-linking, was more efficient in phytohaemagglutinin-stimulated human lymphocytes exposed to m-L-sarcolysin as compared to melphalan at equivalent concentrations of the drug. The cellular uptake of m-L-sarcolysin was more efficient as compared to melphalan. With peptichemio, a mixture of 6 peptides containing m-L-sarcolysin, an intermediate level of DNA cross-linking was found. The differences in DNA cross-linking between the 3 drugs were parallel to differences in cytotoxicity. Peptichemio has been ascribed anti-metabolic properties in addition to the alkylating properties conferred by its m-L-sarcolysin content. In the phytohaemagglutinin-stimulated lymphocytes, however, the effect of peptichemio seems linked to its capacity for DNA cross-linking.

Inhibition of T and B lymphoblastic response by mithramycin, dacarbazine, prospidium chloride and peptichemio.

Four different cytostatic compounds (prospidium chloride, peptichemio, dacarbazine and mithramycin) have been assayed for their effect on lymphoblastic transformation of spleen cells from mice. The drugs did not affect cell viability at concentrations lower than 0.1-0.2 micrograms/ml (peptichemio and mithramycin) or than 10-20 micrometers/ml (prospidium chloride and dacarbazine). Except for peptichemio, which did not show any marked effect, these cytostatics acted more actively on B cells than on T cells at concentrations of drug not affecting lymphocyte viability. Mithramycin was the most active inhibitor of the mitogen-induced DNA synthesis in the cell. Concentrations of this drug to inhibit the blastogenic response to 50% (IC50) were lower than 0.1 microgram/ml. Inhibition of mitogenesis was less pronounced in the case of dacarbazine (IC50 = 50 and 10 micrograms/ml for T and B cells, respectively), prospidium chloride (IC50 greater than 50 and 50 micrograms/ml for T and B cells) and peptichemio (IC50 = 0.25 and 0.5 microgram/ml for T and B cells, respectively.

The combined action of X-irradiation, drug and hyperthermia on the DNA in mammalian cells in vitro.

The effects of the alkylating cytostatic drug PTC on the production of radiation-induced single-strand breaks in DNA, and on the repair in L cells incubated at 37 degrees or 42 degrees C were studied. The same amount of SSB is obtained when the cells are treated simultaneously with PTC and hyperthermia as when irradiated with 2 500 rad, but different rate in the rejoining was observed. There is no potentiation of SSB formation by pretreatment of cells with 10 microgram PTC/ml for 2 hours at 42 degrees C before irradiation, but in these cells less effective repair occurs. The effect of PTC on the rejoining of SSB induced with 2 500 rad depends on the drug's concentration and post-incubation time.

Effect of peptichemio and of peptichemio plus hydrocortisone on growth and alkaline phosphatase activity in HeLa cells in vitro.

The actions of Hydrocortisone, Peptichemio and of Peptichmio + Hydrocortisone mixture in HeLa cells have been experimented. The results show an increase of cell number in the controls and a lower, but not statistically different growth, in the Hydrocortisone-treated cultures. In the cultures treated with peptichemio the growth is hardly evident, in the ones treated with the Hydrocortisone + Peptichemio mixture, the cell number is decreasing with time. ALP activity in cells treated with drugs is much higher than in the controls ones. The results either on cellular growth and on ALP activity, are compared to the Ara C ones. No significant difference in media ALP activity between drug-treated cultures and controls is evidentiated. The results show in the Peptichemio + Hydrocortisone mixture, that the action of Hydrocortisone is overcome by the Peptichemio one. By the cytochemical technique the enzyme localization is particularly evident in the cytoplasmic inclusions, which are very numerous in the cells treated with Peptichemio.

Phase I trial with peptichemio.

Thirty-two patients with tumor progression, even after conventional cytostatic drug treatment, were treated with peptichemio, with increasing doses for groups of 4 patients. The maximum tolerated dose (with minimum hematological toxicity and without any other evident toxicity) with repeated administrations, was 1.2 mg/kg twice weekly. The recommended doses for phase II trials are, as shown by the detailed analysis of the results, 0.9 mg/kg, twice weekly and administered alone, and 1.3 mg/kg, once weekly combined with other cytostatic drugs, in 500 ml of infusion fluid, with 25 mg of heparin and 25 mg of hydrocortisone to minimize the freqent risk of local phlebosclerosis.

Effects of hyperthermia and drug in cultured mammalian cells.

Hyperthermia (42 degrees C) and peptichemio (PTC) applied simultaneously result in an increased killing effect and in a significant induction of single strand breaks (SSB) of DNA molecule. The enhanced killing effect was observed in all circumstances, whenever hyperthermia was applied before, during or after PTC treatment, but the most effective cell killing was obtained when hyperthermia and PTC were applied simultaneously. The results show that PTC concentrations used in these experiments do not induce SSB in DNA molecule during incubation for 2 hours at 37 degrees C. When the cells were exposed to PTC and hyperthermia the induction of SSB was observed. The number of SSB depends on the exposure time and PTC concentration. Cells with a greater number of SSB lost the capacity for repair during post-incubation at 42 degrees C.

Clinical trial of peptichemio in solid tumors of childhood.

Peptichemio (PTC) was utilized in various advanced and disseminated tumors in children. With the schedule that was used, toxicity was mild. Immediate side effects included anaphylactoid reactions which required close supervision of the patient during infusion of the drug. PTC proved efficacious in advanced neuroblastoma. In patients with bone metastases, PTC has a prompt analgesic effect which seems to occur independently of any antitumor action.

Effect of peptichemio upon localization of injected radiolabelled lymphocytes in the lymphatic organs of antigen-stimulated mice.

Increased localization (trapping) of lymphocytes occurs in lymphoid organs following antigenic challenge. The effect of peptichemio (PTC) upon lymphocyte trapping in lymph nodes and spleen was investigated: the results demonstrate that the drug diminishes trapping in lymphatic organs. The depression of lymphocyte trapping may provide at least one mechanism whereby PTC achieves it immunosuppressive effects.

Cytotoxic effects of peptichemio on synchronized EUE cells.

The effect of Peptichemio (PTC) on cellular growth and on macromolecular syntheses was analyzed through the cell cycle of EUE cells. The cell response to the various treatments was measured by determining plating efficiency, growth rates and incorporation of labelled precursors of DNA, RNA and protein syntheses. Three maximum inhibition points were found on cell survival, one corresponding to the early G1, another to the middle S and a last one to the late G2. Parallel experiments of incorporation of labelled precursors of DNA, RNA and proteins revealed an effect only on DNA during the early and middle S phase.