Mol Cancer Ther. 2003;2:1331-1339
© 2003 American Association for Cancer Research
Antitumor activity of the novel melphalan containing tripeptide J3 (L-prolyl-L-melphalanyl-p-L-fluorophenylalanine ethyl ester): Comparison with its m-L-sarcolysin analogue P2
Joachim Gullbo1,
Charlotta Wallinder1,
Marcus Tullberg2,
Henrik Lövborg1,
Hans Ehrsson3,
Rolf Lewensohn4,
Peter Nygren5,
Kristina Luthman2 and
Rolf Larsson1
1 Department of Medical Sciences, Division of Clinical Pharmacology, Uppsala University, Uppsala, Sweden; 2 Department of Chemistry, Medicinal Chemistry, Göteborg University, Göteborg, Sweden; 3 Karolinska Pharmacy and 4 Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden; 5 Department of Oncology, Radiology and Clinical Immunology, Uppsala University Hospital, Uppsala, Sweden
Requests for Reprints: Joachim Gullbo, Department of Medical Sciences, Division of Clinical Pharmacology, Uppsala University, SE-751 85, Uppsala, Sweden. Phone: 46-18-6115250; Fax: 46-18-518237. E-mail: Joachim.Gullbo{at}medsci.uu.se
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Abstract
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Peptichemio (PTC), a mixture of six oligopeptides all containing m-L-sarcolysin, has previously shown impressive results in clinical trials. The tripeptide P2 (L-prolyl-m-L-sarcolysyl-p-L-fluorophenylalanine ethyl ester) has been suggested as the main contributor to PTC activity. In contrast to its analogue melphalan, m-L-sarcolysin never reached clinical use. To allow a direct comparison, the corresponding melphalan containing tripeptide J3 (L-prolyl-L-melphalanyl-p-L-fluorophenylalanine ethyl ester) was synthesized and its activity was compared with that of P2; the activities of melphalan and m-L-sarcolysin were studied in parallel. Cytotoxic activity in human tumor cell lines and some fresh human tumor specimens were analyzed as well as effects on cellular metabolism, macromolecular synthesis, and preliminary evaluation of the cell death characteristics. The results show that melphalan and m-L-sarcolysin display similar activity in these systems and that the tripeptides were more active than their parent monomers. Surprisingly however, the melphalan containing tripeptide J3 demonstrated a significantly more rapid and stronger activity than the m-L-sarcolysin analogue P2. Finally, the in vivo toxicity and activity of melphalan and J3 were investigated in mice bearing human leukemia cells in s.c. fibers. The in vitro results seem translatable into the in vivo situation, demonstrating better antileukemic effect of J3 but similar side effects as melphalan.
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Introduction
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During the 1960s, an Italian company, Istituto Sieroterapico Milanese (S. Belfanti, Milan, Italy), under the supervision of Professor De Barbieri, synthesized a few hundred small peptides based on m-L-sarcolysin (1). A mixture of six of the most interesting compounds was marketed as a peptide cocktail under the name Peptichemio (PTC). De Barbieri stated that "the main idea was that of synthesizing molecules simultaneously endowed with a remarkable cytotoxic activity, due to an alkylating group present in same, and a selective carrier function towards special cells or structures, possibly neoplastic" (1). During the following two decades, PTC was used and tested successfully in clinical trials for several human malignancies. For example, PTC treatment yielded 25% partial responses while melphalan was ineffective in a clinical trial with advanced breast cancer patients previously treated with combination chemotherapy (2). In another phase 2 study, PTC treatment yielded eight complete and five partial responses in 15 patients with multiple myeloma, of whom seven were previously treated with alkylating agents, including melphalan (3). Later studies confirmed this lack of cross-resistance with classical alkylators in patients with breast cancer (4) and plasma cell neoplasms (5) as well as ovarian cancer (6).
More recently, two other m-L-sarcolysin containing tripeptides have been developed and studied thoroughly. The preclinical efficacy of Ambamustine (p-L-fluorophenylalanine-m-L-sarcolysyl-L-methionine ethyl ester, also named PTT-119) in mouse models (transplantable L1210 leukemia and virally induced leukemia) was clearly impressive (7). Clinically, the drug appeared useful in some tumors (e.g., non-Hodgkin's lymphoma; 8) but with no or only modest activity in others (e.g., pretreated small-cell lung cancer; 9). MF-13 (L-prolyl-m-L-sarcolysyl-L-norvaline ethyl ester) has been shown to selectively trigger apoptosis in malignant cells (10), and the activity shown in preliminary xenograft models in mice appeared promising (11).
Previous studies on the individual peptides of PTC has focused our attention on the tripeptide P2 (L-prolyl-m-L-sarcolysyl-p-L-fluorophenylalanine ethyl ester; 12–14), which appeared to be the most potent of the PTC peptides. In addition, P2 also showed interesting properties like high activity in slow proliferating cells and low dependence on reduced glutathione (GSH)-mediated drug resistance, thereby suggesting a potential clinical advantage in comparison with standard alkylating agents. Recently, we showed that the novel dipeptide J1 (L-melphalanyl-p-L-fluorophenylalanine ethyl ester), designed as an intermediate between melphalan and P2, was several-fold more active than P2 (15). The results prompted further investigation of J1 activity as well as the synthesis of the complete melphalan-based analogue of P2. The present study reports the synthesis of this novel compound, hereafter named J3 (L-prolyl-L-melphalanyl-p-L-fluorophenylalanine ethyl ester), as well as a presentation of its in vitro activity in comparison with P2. The activities of the corresponding amino acid derivatives, melphalan and m-L-sarcolysin, were investigated in parallel. Cytotoxic activity was assayed in primary cultures of human tumor cells as well as a panel of established cell lines expressing defined types of drug resistance using the fluorometric microculture cytotoxicity assay (FMCA), which has proven to be a valuable tool in both experimental and clinical purposes (16–19). Other aspects of the in vitro response to the test compounds were also assayed, including effects on macromolecular syntheses using radiolabeled substrates, extracellular acidification, mitochondrial potential, and nuclear fragmentation. Finally, the in vivo activity and toxicity of J3 in comparison with melphalan were investigated in mice. Antitumor activity was assayed in the hollow-fiber model with s.c. implantation of human tumor cells, considered to be a robust and resistant model with low activity of several standard cytotoxic drugs (20).
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Materials and Methods
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Human Tumor Cell Lines
Complete concentration-effect curves were obtained for all compounds in a panel of 10 human tumor cell lines, the composition of which has been discussed in detail previously (16). Table 1 summarizes some characteristics of this panel, designed to represent cells of different origin and common resistance mechanisms. Correlation analysis of log IC50 values (or "Delta"; defined as the deviation of the log IC50 value of one cell line from the mean log IC50 of the whole panel) has proven valuable in the early mechanistic classification of different cytotoxic agents (16, 17). Cells were maintained as described previously, and for experimental purposes, cells were harvested in log phase. RPMI 1640 (Sigma Chemical Co., St. Louis, MO) supplemented with 10% heat-inactivated FCS (Sigma), 2 mM glutamine, 100 µg/ml streptomycin, and 100 units/ml penicillin was used unless otherwise specifically indicated.
Primary Cultures of Human Tumor Cells
In addition to the complete dose-response analysis in the cell lines, primary cultures of human tumor cells from 21 patients were used to determine the cytotoxic activity of the test compounds at two to three different concentrations. The diagnoses were acute myelocytic leukemia (n = 3), acute lymphatic leukemia (n = 1), chronic lymphatic leukemia (CLL; n = 3), non-Hodgkin's lymphoma (n = 1), ovarian carcinoma (n = 3), colorectal cancer (n = 2), adenocarcinoma of the lung (n = 2), hepatocellular cancer (n = 1), breast cancer (n = 1), neuroblastoma (n = 1), renal fibroadenoma (n = 1), abdominal adenocarcinoma (n = 1), and solid child tumor (n = 1). The tumor samples were obtained by bone marrow/peripheral blood sampling (19), routine surgery, or diagnostic biopsy (18), and this sampling was approved by the local ethics committee at the Uppsala University Hospital. Cell viability was determined by the trypan blue exclusion test, and the proportion of tumor cells in the preparation was judged by visual inspection of May-Grünwald-Giemsa-stained cytospin preparations by a cytopathologist. RPMI 1640 (Sigma) supplemented with 10% heat-inactivated FCS (Sigma), 2 mM glutamine, 100 µg/ml streptomycin, and 100 units/ml penicillin was used for the experiments. Cells were in general cryopreserved in a culture medium containing 10% DMSO (Sigma) and 90% heat-inactivated FCS (Sigma), and this does not affect drug sensitivity (21).
Test Compounds
Melphalan was purchased from Sigma, m-L-sarcolysin was a kind gift from Karolinska Pharmacy (Sweden), and P2 (hydrochloride salt, 632.06 g/mol) was a kind gift from Istituto Sieroterapico Milanese. The synthesis of J3 (hydrochloride salt, 632.06 g/mol) is presented below (see also Fig. 1). Molecular structures of the test compounds are given in Fig. 2. The compounds were dissolved in warm absolute ethanol (concentrations from 10 to 4.0 mM) and further diluted with sterile water. The maximum ethanol concentrations (never exceeding 1% v/v) did not produce any effects in the assays used (data not shown). Time in aqueous solution was always kept minimal to limit the influence of mustard hydrolysis. For comparison, cytotoxic activity was also measured for the ethyl ester of p-fluorophenylalanine.
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Figure 1. Outline of the synthesis of J3. Reagents: (A) EDC/NMM, DCM; (B) HCl(g)/ethanol; (C) tBoc-Pro-OH, EDC/NMM, DCM.
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The Fluorometric Microculture Cytotoxicity Assay
The FMCA has been presented in detail previously (16, 18, 19). In summary, this 72-h nonclonogenic viability assay measures response to cytotoxic drugs in 96-well microtiter plates (Nunc, Roskilde, Denmark), with hydrolysis of fluorescein diacetate by cells with intact plasma membrane as the end point. Fluorescein diacetate (Sigma) was dissolved in DMSO to 10 mg/ml and kept frozen as a stock solution in the dark. Dose-response curves in the cell lines were obtained by testing each drug at six different concentrations in duplicates. All cell line experiments were performed thrice. Due to limited number of cells, patient tumor samples were only tested at two (melphalan and m-L-sarcolysin; 4.0 and 10 µM) or three (J3 and P2; 0.8, 4, and 10 µM) different concentrations (in duplicates).
The cytotoxic IC50 value for drugs in the cell lines was determined from log concentration effect (Survival Index %) curves in GraphPad Prism (GraphPad Software Inc., San Diego, CA) using nonlinear regression analysis. Comparison of activity were made with Student's t test (GraphPad Prism).
Measurement of DNA and Protein Synthesis
The effects on DNA and protein synthesis were assayed in U-937 cells using 14C-labeled thymidine and leucine in a Cytostar-T plate (with scintillants molded into the transparent polystyrene bottom, available in the "In situ mRNA Cytostar-T assay" kit; Amersham International, Buckinghamshire, United Kingdom; 22, 23). 14C-thymidine (Amersham CFA.532, 56 mCi/mmol, 50 µCi/ml) and 14C-leucine (Amersham CFB.183, 56 mCi/mmol, 50 µCi/ml) were handled according to the manufacturer's instructions and stored in refrigerated containers until use. The method and use of U-937 cells has been described in detail previously (24). Briefly, the cells were suspended (50 x 103 cells in 180 µl) in fresh medium containing 111-nCi/ml thymidine (for DNA experiments) or 222-nCi/ml leucine (for protein experiments) and seeded into the plate; blank wells received isotope-containing medium only. Drugs and PBS in test and control wells were added in duplicates (20 µl/well, yielding concentrations of 5.0 or 10 µM). Radioactivity was measured with a liquid scintillation counter (Wallac OY, Turku, Finland) at 24, 48, and 72 h. Between measurements, the plates were stored in an incubator at 37°C. During measurement, the plates were covered with a plate sealer to inhibit microbiological contamination. The experiment was performed thrice, and data are presented as substrate incorporation in percent of control cells (means ± SEM).
Measurement of Extracellular Acidification
The Cytosensor Microphysiometer (Molecular Devices, Inc., Sunnyvale, CA) method was used for the measurement of extracellular acidification originating from excreted acidic byproducts of cellular respiration, such as lactic acid and carbon dioxide, as described previously (25, 26). A complete low buffering RPMI 1640 (National Veterinary Institute, Uppsala, Sweden) was used for the experiments. U-937 cells were suspended in agarose matrix (25% agarose and 75% cell suspension) and placed in flow chambers. The flow chambers were perfused with complete low buffered cell medium, and every 90 s, the flow was stopped for 30 s and the extracellular acidification rate was measured with a light-sensitive potentiometric sensor (27). To establish a baseline for the experiment, cells were exposed to medium only during the first hour of the assay. Cells in two of the channels, one per workstation, in each experiment were kept unexposed and served as controls. Freshly prepared drug solution was added directly to the room-tempered medium container at the start of drug exposure. The concentration indicated (i.e., 5.0 µM) refers to this initial concentration and does not take mustard or peptide hydrolysis in the medium into consideration. Each experiment was run for 24 h with continuous drug exposure. The acidification rate was calculated by the Cytosoft program as -µV/s and was later normalized to a percent value, percent of control baseline (25). The experiment was performed thrice with similar results.
Mode of Cell Death
For the preliminary evaluation of cell death characteristics in this study, MitoTracker Red CMXRos (Molecular Probes, Eugene, OR) was used as an indicator of mitochondrial membrane potential, and Hoechst 33342 staining of nuclei was used for morphological evaluation of fragmentation and condensation.
U-937 cells were plated in flat-bottom 96-well plates and exposed to drugs (5 µM). After 6 or 8 h of incubation, cells were stained with MitoTracker Red CMXRos (at a final concentration of 1 µM) for 30 min, washed twice with PBS, and fixed in 3.7% formaldehyde with 10 µM Hoechst 33342. Image of the cells were acquired using an automated image capture and analysis equipment (ArrayScan II; Cellomics, Inc., Pittsburgh, PA) with suitable filter sets. Quantification of the mitochondrial membrane potential was based on automated image analysis. Cells were identified based on Hoechst 33342 staining and the intensity of each single cell (2000 cells/treatment) was measured. Mitochondrial data are presented as the mean cellular intensities in the red channel of these 2000 cells.
Activity and Toxicity in Vivo: Hollow-Fiber Model
The animal experiments were performed at Visionar Biomedical AB (Uppsala) and conducted according to national legislation and ethical guidelines. Male NMRI albino mice were purchased from Scanbur B&K Universal (Sweden) and kept under standard laboratory conditions. The hollow-fiber procedure was performed as described previously (20), and primary human CLL cells were used as the tumor model. Pieces (20 mm) of polyvinylidene fluoride hollow fibers (500-kDa molecular weight cutoff, 1 mm diameter; Spectrum, Laguna Hills, CA) were filled with cell suspension and the ends were sealed. After 48-h incubation in vitro, the fibers were implanted s.c. through a small skin incision on the back of the anesthetized animals. On the following day, animals were randomized into groups receiving J3 (n = 6; 25 µmol/kg), melphalan (n = 6; 25 µmol/kg), or vehicle only (n = 6) into the tail vein. This dosing is close to maximum tolerated dosage for both J3 and melphalan in this mouse strain (previous experiment; data not shown). Five days later, the animals were sacrificed and blood was withdrawn from the orbital plexus. The fibers were explanted and cell density was determined by staining with 3-(4,5-dimethylthiaxol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma); results are reported as net growth during the experiment. Toxicity was investigated by daily monitoring of the animals, by weighing before and after the experiment, and finally by analysis of blood cells. Data (presented as means ± SEM) were compared with unpaired two-tailed t tests of test versus control groups in GraphPad Prism.
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Synthesis of J3
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The synthesis of J3 is outlined in Fig. 1. The detailed synthetic procedure of compounds 1 to J1 has been presented elsewhere (28).
General
All solvents were of analysis or synthesis grade. Nuclear magnetic resonance (NMR) spectra were obtained on a Varian 400 NMR spectrometer and recorded at 400 MHz (1H NMR) or 100 MHz (13C NMR). The reactions were monitored by thin-layer chromatography on silica-plated aluminum sheets (Silica gel 60 F254, E. Merck, KgaA, Damstadt, Germany), detecting spots by UV light and/or 2% ninhydrin in ethanol followed by heating. Column chromatography was performed on wet-packed silica (Silica gel 60, 0.040–0.063 mm; E. Merck) using flash chromatography. Melting points were measured in a Büchi melting point B-540 apparatus and are uncorrected. Optical rotations were measured at room temperature with a Perkin-Elmer (Europe BV, The Netherlands) 341 LC polarimeter. Elemental analysis was performed at Mikrokemi AB (Uppsala, Sweden).
N-tert-Butoxycarbonyl-L-Prolyl-L-Melphalanyl-p-L-Fluorophenylalanine Ethyl Ester (4)
N-tert-Butoxycarbonyl-L-proline (245 mg, 0.38 mmol), compound J1 (94 mg, 0.38 mmol), and N-methylmorpholine (NMM; 0.15 ml, 1.31 mmol) were dissolved in dry CH2Cl2 (14 ml). The reaction was stirred for 30 min at 0°C whereupon 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC; 75 mg, 0.39 mmol) was added. The solution was stirred for 30 min at 0°C and then at room temperature overnight. The reaction mixture was diluted to 20 ml with CH2Cl2 and extracted with 10% aqueous citric acid (15 ml) and with saturated aqueous NaHCO3 (15 ml). The organic layer was dried (MgSO4) and concentrated under reduced pressure. The crude product was purified by flash chromatography using CH2Cl2/CH3OH/hexane (6:1:5) as eluent. Pure 4 was isolated as a white powder (220 mg; 82%).
Mp 139–144°C. [
]D 1° (c 1, CHCl3). 1H NMR (CDCl3) 
7.04–6.88 (m, 6H, Ph-H, Mel and Phe), 6.60–6.50 (m, 2H, Ph-H, Mel), 4.74–4.66 (m, 1H, -CH), 4.55 (app s, 1H, -CH), 4.17 (br s, 1H, -CH), 4.12–4.04 (m, 2H, CH2CH3), 3.69–3.61 (m, 4H, N-CH2), 3.59–3.52 (m, 4H, CH2-Cl), 3.39–3.21 (m, 2H, -CH2, Pro), 3.07–2.83 (m, 4H, CH2-Ph), 2.15–1.55 (m, 4H, ß- and 
-CH2, Pro), 1.42 (s, 9H, CH3-Boc), 1.17 (t, 3H, CH2CH3). 13C NMR (CDCl3) 172.05, 170.86, 170.62 (C = O, amide and ester), 161.91 (d, JC,F = 244.5 Hz, C-4), 155.02 (C = O, Boc), 145.03 (C-4'), 131.93 (C-1), 130.87 (2 C:s; d, J3C,F = 7.7 Hz, C-2), 130.58 (2 C:s; C-3'), 125.38 (C-1'), 115.28 (2 C:s; d, J2C,F = 21.5 Hz, C-3), 112.10 (2 C:s; C-2'), 80.52 (C-Boc), 61.46 (CH2CH3), 60.39 (-CH, Pro), 54.00, 53.61 (-CH, Mel and Phe), 53.46 (2 C:s; N-CH2), 47.14 (-CH2, Pro), 40.52 (2 C:s; CH2-Cl), 37.14 (2 C:s; CH2-Ph), 31.60 (ß-CH2, Pro), 28.35 (3 C:s; CH3-Boc), 22.68 (-CH2, Pro), 14.16 (CH2CH3).
J3 Hydrochloride
Compound 4 (200 mg, 0.273 mmol) was dissolved in HCl saturated ethanol (15 ml) and stirred for 4 h at room temperature. The crude product was concentrated under reduced pressure and recrystallized in ethanol to give pure J3 hydrochloride as white crystals (174 mg; 96%).
Mp 160–163°C. []D 1.4° (c 1.4, CH3OH). 1H NMR (CD3OD) 7.25–7.18 (m, 2H, Ph-H, Phe), 7.09 (d, J = 8.79 Hz, 2H, Ph-H, Mel), 7.02–6.97 (m, 2H, Ph-H, Phe), 6.64 (d, J = 8.79 Hz, 2H, Ph-H, Mel), 4.63–4.55 (m, 2H, -CH, Mel and Phe), 4.50–4.42 (m, 1H, -CH, Pro), 4.18–4.08 (m, 3H -CH, Pro and CH2CH3), 3.70 (t, J = 6.59 Hz, 4H, N-CH2), 3.63 (t, J = 6.59 Hz, 4H, CH2-Cl), 3.38–3.21 (m, 2H, -CH2, Pro), 3.15–2.76 (m, 4H, CH2-Ph), 2.39–2.30 (m, 1H, ß-CH2, Pro), 2.04–1.82 (m, 3H, -CH2, and ß-CH2, Pro), 1.19 (t, 3H, CH2CH3). 13C NMR (CD3OD) 173.28 (C = O, Pro), 172.50, 169.46 (2 C:s; C = O, amide and ester), 163.4 (d, JC,F = 243.4 Hz, C-4), 146.72 (C-4'), 134.18 (C-1), 132.32 (2 C:s; C-2) 131.56 (2 C:s; C-3'), 126.58 (C-1'), 116.18 (2 C:s; d, J2C,F = 22.2 Hz, C-3), 113.38 (2 C:s; C-3'), 62.61 (-CH, Pro), 60.95 (CH2CH3), 56.75 (-CH, Phe), 55.42 (-CH, Mel), 54.53 (2 C:s; N-CH2), 47.58 (-CH2, Pro), 41.81 (2 C:s; CH2-Cl), 38.14 (CH2-Ph), 37.82 (CH2-Ph), 31.24 (ß-CH2, Pro), 25.08 (-CH2, Pro), 14.64 (CH2CH3).
Anal. calc. for C29H38Cl3FN4O4: C, 55.1; H, 6.1; N, 8.9; Found C, 54.7; H, 6.1; N, 8.8.
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Results
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The effect of melphalan (4.0 and 10 µM) or m-L-sarcolysin in the primary human tumor cells is shown in Fig. 3, A and C. At 10 µM, melphalan was active in four and m-L-sarcolysin in three of the hematological samples, while both drugs only showed activity (defining activity as survival index < 50%; data not shown) in one solid tumor sample (the solid child tumor; suspected diagnosis at biopsy was Wilm's tumor). In contrast, J3 was active in all hematological tumors already at the lowest tested concentration and produced a clear concentration-dependent reduction of tumor cell viability in the solid tumor samples (active in 8 of 13 samples at 10 µM). The advantageous effects of J3 were statistically significant compared with P2 (Fig. 3, B and D).
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Figure 3. Activity in patient human tumor samples (mean ± SEM): melphalan and m-L-sarcolysin in eight hematological samples (A), J3 and P2 in eight hematological samples (B), melphalan and m-L-sarcolysin in 13 solid tumor samples (C), and J3 and P2 in 13 solid tumor samples (D). Statistical analysis performed with paired t tests (ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001).
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This pattern of potency was also apparent in the cell lines, where the average activity of melphalan (mean IC50 = 12.4 µM) was comparable with that of m-L-sarcolysin (mean IC50 = 16.5 µM). As might be expected from the "Delta" graphs in Fig. 4, the activity patterns of the two drugs were highly correlated (Pearson's correlation = 0.98). The two tripeptides displayed a difference in potency, J3 (mean IC50 = 0.52 µM) being significantly (P < 0.01) more active than P2 (mean IC50 = 3.1 µM) but with a high degree of correlation (0.98), indicating an identical mechanism of action. Quite surprisingly, the correlation analysis between the tripeptides and the single amino acid derivatives yielded only moderate correlation factors (0.79–0.84). The COOH-terminal amino acid residue, p-fluorophenylalanine ethyl ester, showed no toxicity (IC50 > 200 µM) in any of the cell lines (data not shown).
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Figure 4. Mean (Delta) graphs of the compounds. Delta, deviation of the log IC50 value of one cell line from the panel mean log IC50 value. Hence, bars deviating to the right indicate cell lines more sensitive than the panel mean and vice versa. The sum of all bars equals zero (for further reference on Delta mean graphs, see Boyd, M. R. and Paull, K. D. Drug Dev. Res. (1995) 34: 91–109.
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The cytotoxic activity in U-937 GTB cells also complies with this pattern (i.e., melphalan and m-L-sarcolysin show equal potency while J3 was more potent than P2; Fig. 5, A and B). Microphysiometric analysis of cellular metabolism (or extracellular acidification) in this cell line showed a sharp decrease of metabolic activity in response to J3 after 
5 h exposure (Fig. 5D). Melphalan and m-L-sarcolysin produced a gradual (but modest) decrease in metabolic activity over the 24-h period, while exposure to P2 resulted in a more pronounced gradual decrease (Fig. 5, C and D). Incorporation of 14C-labeled thymidine and leucine was inhibited in a dose-dependent manner. Melphalan and m-L-sarcolysin showed a similar degree of inhibition being 40% of control at 24 h for both DNA and protein synthesis (Fig. 5E). In contrast, J3 at equimolar concentrations showed an inhibition of DNA and protein synthesis of 80% and 75% of control, respectively. P2, on the other hand, showed a degree of inhibition similar to melphalan and m-L-sarcolysin (Fig. 5, E and F). Six or 8 h of exposure to 5 µM of the drugs (as in the experiments discussed above) resulted in fragmented nuclei and decreased mitochondrial potential for J3, apparently unaffected cells for melphalan and m-L-sarcolysin and intermediate results for P2 (Fig. 5, G and H).
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Figure 5. Activity in U-937 cells. A and B, dose-response curves (mean ± SEM). C and D, metabolic effects measured in the Cytosensor (one representative experiment). E and F, effects on macromolecular synthesis (mean ± SEM). G and H, cell death characteristics (one representative experiment; mitochondrial potential and nuclear fragmentation); insets, Hoechst-stained nuclei strongly fragmented after J3 exposure but unaffected by melphalan and m-L-sarcolysin.
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When studied in vivo, melphalan and J3 yielded similar reduction of peripheral blood cell count and weight during the study (Fig. 6A). The effect was most pronounced for leukocytes with a 85% reduction (from 11 to 1.5 x 106 cells/ml) at day 5 and minor changes in hemoglobin (data not shown), platelets, and weight gain. Only one animal (in the melphalan group) lost weight during the study. Considering antileukemic activity however, J3 produced a significant inhibitory effect compared with vehicle-treated cells (mean net growth of 54% vs. 161%; P < 0.05) while this was not seen in the melphalan-treated animals (109%; Fig. 6B).
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Figure 6. In vivo toxicity (A) and antileukemic activity (B) in mice. Statistical analysis was performed with unpaired t test versus vehicle-treated animals (i.e., control animals). ns, not significant; *, P < 0.05; ***, P < 0.001. The differences between melphalan and J3 did not reach statistical significance at this dose level (P = 0.055 for antileukemic activity). Absolute values for the toxicity parameters in vehicle-treated animals were as follows: leukocyte count, 10.9 billion cells/l; platelets, 1104 billion cells/l; weight gain, 2.0 g during 6 days.
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Discussion
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In the present study, the tripeptide derivatives J3 and P2 showed higher potency than their respective alkylating amino acid derivative. This is not surprising given previous reports on increased tumor cell uptake of various alkylating oligopeptides (29, 30). The exact mechanism for this phenomenon is not clear but may involve either passive diffusion or increased influx into the tumor cell by membrane-bound oligopeptide transporters. Such transport proteins have previously been suggested to be overexpressed in various tumor cell types (31). Alternatively, the increased cytotoxic potency of the tripeptides may reflect an increased activity of esterases and/or proteases in tumor cells leading to "ion trapping" of the alkylating moiety within the cell. Indeed, several reports have demonstrated an increased expression of such hydrolytic enzymes in different tumor tissues (e.g., breast and colon cancer; 32, 33). Moreover, recent results on the activity of an analogous dipeptide suggest that the difference in potency between this compound and melphalan could be correlated to esterase and protease activity in a cell line panel1. However, the mechanistic details for the increased antitumor activity of the tripeptides remain to be fully elucidated.
The activity of melphalan and m-L-sarcolysin appeared similar in all assays. This is consistent with a previous in vitro study (14). In addition, the acute toxicity in vivo also appears to be similar between the two compounds with LD10 values following i.p. administration to rats (34).
Interestingly, the correlation analysis of activities in the cell lines yielded high correlation factors between J3 and P2 on one hand and between melphalan and m-L-sarcolysin on the other but only moderate correlation between the two groups. Because high correlations between activity patterns in this cell line panel is indicative of mechanistic similarity (16), this might suggest an additional level of biological activity for the peptide derivatives. Interestingly, indications of antimetabolic properties of the PTC peptides has been observed previously. For example, following PTC exposure, the decrease of protein synthesis was more pronounced in tumoral systems than in normal ones; furthermore, m-L-sarcolysin alone could not produce this effect (1). The relative degree of DNA and protein synthesis inhibition observed in this study may support this notion. In addition, the more rapid onset of metabolic inhibition by the tripeptides measured by the Cytosensor adds to this potential explanation. Decreased acidification rate measured by the Cytosensor correlates well with reduced viability as judged by microscopic evaluation of May-Grünwald-Giemsa-stained cell preparations (26). However, similar to the case for melphalan, cell death in response to the tripeptides appears to occur by apoptosis.
Considering the structural similarities between J3 and P2, the pronounced difference in activity is surprising, especially when facing equipotency between melphalan and m-L-sarcolysin in these in vitro assays. Although the difference in potency was shown in all cell systems, a few other observations are worth mentioning. For example, the cytotoxic dose-response curves were in general steeper for P2 than for J3 (data not shown). This might be explained by a better transport of J3 into the cells (more potent) but a less efficient alkylation (reactivity) while inside. One could speculate that the para-substitution versus the meta-substitution pattern influences factors like the peptide susceptibility to hydrolytic enzymes or the reactivity of the aromatic bis(2-chloroethyl)amino moiety (in either tripeptide or liberated form). Interestingly, the same relationship, although less pronounced, was observed between melphalan and m-L-sarcolysin. Hypothetically, the advantageous transport of melphalan (via amino acid transporters?) is neutralized through its lower reactivity compared with m-L-sarcolysin, and the two compounds are thus equipotent in these in vitro systems. In addition, the variation of IC50 values between cell lines was less for P2 than for the other compounds (as seen by low absolute values in Fig. 4), the difference between the most sensitive (CEM/VM1) and the most resistant (8226/LR5) cell lines being 17-fold for P2 compared with 200-fold for J3. This pattern of low variation of activity in the cell lines also gives P2 an apparent low dependence on resistance mechanisms expressed in the panel. For example, the resistance factor for GSH-mediated drug resistance (comparing 8226/LR5 with RPMI 8226/S) is only 1.3 for P2 while 2.3 for J3 (and 2.7 and 3.2 for melphalan and m-L-sarcolysin, respectively). Alternatively, the low variation of P2 activity may reflect a higher degree of unspecific general toxicity potentially leading to reduced tumor selectivity. However, further explanations based on the present results become speculative and the exact cause of these differences must be clarified in future studies. In any case, the results indicate that J3 appears to be an interesting novel candidate as an antitumor agent.
Finally, the activity and toxicity in mice indicate that the promising in vitro data may, at least to some extent, be translatable into the in vivo situation. The hollow-fiber model with s.c. implantation of human tumor cells is considered to be a robust and resistant model with low activity of several standard cytotoxic drugs, which allows for evaluation of drug activity as well as toxicity in the same animal (20). As expected, both melphalan and J3 treatment result in reduced weight gain and hematopoietic depression measured 5 days after an i.v. injection. The amplitude of the registered side effects were similar for the two drugs while only J3 produced a significant antileukemic effect against the primary human CLL cells.
In conclusion, while the alkylating amino acids melphalan and m-L-sarcolysin expressed similar cytotoxic activities, the novel melphalan containing tripeptide J3 appeared more potent than its m-L-sarcolysin containing analogue P2. The results were consistent in all assays measuring cytotoxic activity as well as cellular metabolism and macromolecular synthesis. The results of the present study prompt further evaluation of J3, including more detailed mechanistic studies (e.g., DNA damage assays) as well as more traditional in vivo studies with xenograft tumor models.
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Footnotes
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Unpublished data. 
Received 7/21/03;
revised 9/15/03;
accepted 9/16/03.
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References
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Abstract
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