| First Author | Moiseeva EV | Year | 1997 |
| Journal | Mouse Genome | Volume | 95 |
| Issue | 4 | Pages | 895-97 |
| Mgi Jnum | J:45475 | Mgi Id | MGI:1195496 |
| Citation | Moiseeva EV, et al. (1997) Testing of Liposomal formulations of DL-Melphalan and Rubomycin Lipid derivatives on new Breast Cancer Mouse Model. Mouse Genome 95(4):895-97 |
| abstractText | Full text of Mouse Genome contribution: TESTING OF LIPOSOMAL FORMULATIONS OF DL-MELPHALAN AND RUBOMYCIN LIPID DERIVATIVES ON NEW BREAST CANCER MOUSE MODEL. Moiseeva, E.V., Vodovozova, E.L., Mikhalyov, I.I., Molotkovsky, J.G. Shemyakin-Ovchinnikov. Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117871 Moscow GSP-7, Miklukho-Maklaja str., Russia1 INTRODUCTION The BLRB-Rb (8.17)lIem mouse strain (hereafter called BLRB) with a high incidence of spontaneous mammary adenocarcinomas, bred in our Institute, was used for the testing of some chemical and natural substances with assumed anticancer activity [1, 2, 3]. Our particular approach to the testing procedure consists of drug anticancer activity analysis, performed progressively in three stages. Investigations of any drug influence are carried out (i) on mice with grafted tumours, taken from syngeneic female spontaneous adenocarcinoma (transplanted model) (ii) on female mice with tumours (spontaneous model) and (iii) on pretumour female mice (prophylactic model). Recently we proposed and tested a new effective way for cancer treatment based on saccharide- vectored delivery of cytotoxic liposomes to malignant cells [4]. Generally drug targeting is performed by loading a macromolecular carrier with a drug, and increasing the efficacy of its delivery to a target tumour with the help of a specific ligand (vector). Liposomes have obvious advantages as carriers for drug delivery (see eg [5]). They are accessible, biodegradable and allow the delivery of lipid derivatives of cytotoxic drugs (or drugs lipophilic originally) contained in the liposomal bilayer. This feature appears to be of great importance since it allows at least two obstacles in the route of quantitative liposomal drug delivery to be overcome: undesirable leakage of a water soluble drug encapsulated in the liposomal interior when circulating in blood, and the losses connected with the stage of liposome-cell fusion and transmembrane crossing. Such considerations prompted synthesis of a number of lipid derivatives of cytotoxic agents which have appeared to be more effective than parent drugs in many cases [6]. It is thought that, after penetration of the derivative into the cell interior, its active cytotoxic part is released by cellular enzymes (esterases, phospholipases etc.) Among specific ligands that can be used to direct vehicles to the target, tumour-specific antibodies are the most often used (see eg [7]). Another way of targeting exists: a large number of mammalian cells express sugar-binding proteins (lectins) on the cell surface [8]. The lectin allows reception of specific glycoconjugates that mediate endocytosis and different cellular interactions, believed to be involved in the formation of metastases [9, 10]. The over expression of lectins can be used for targeting of the cytotoxic drug carriers equipped with carbohydrate ligands [9]. Here we apply the liposomes loaded in the lipid phase with the lipid derivatives of DL-melphalan or rubomycin and equipped with a saccharide vector for the treatment of mice with breast cancer, and represent the data obtained on the transplanted step of our new drug testing approach (see above). MATERIALS AND METHODS Reagents and Chemicals The lipophilic derivative of DL-melphalan (octadecylmelphalan, ODM) was synthesized from 1-octadecanol and DL-melphalan, as the corresponding dioleoylglycerol derivative [11]. Synthesis of the rubomycin (daunorubicin) derivative, N-(l,2-dioleoylglycero-3-oxalyl)rubomycin (DOR), will be described elsewhere. Saccharide vector as a conjugate of N-acetyl-Beta-glucosamine with phosphatidylethanolamine derivatized short polyacrylamide, [12] was a generous gift of Dr N V Bovin. Liposomes consisting of the mixture of egg yolk phosphatidylcholine, phosphatidylglycerol, cholesterol, and DL-alpha-tocopherol (112:9:45:1, by mol) with 10 mol % ODM or 5 mol % DOR and saccharide vector (0.4 mol % of BetaGlcNAc residue to matrix lipid) were prepared by sonication with a titanium probe. Final concentrations of drug derivatives in the liposomal preparations were measured by UV absorption ([ODM] = 0.44 mM, [DOR] = 0.22 mM), the completeness of the derivatized drug inclusion in liposomes was controlled by gel filtration. Average size of liposomes (120-150 nm) was controlled by laser light scattering. The liposome suspension was stored in phosphate buffered saline, pH 7.0 (PBS), at 4 degrees C and used within 24 h. 1-This work was supported by grants from the International Science Foundation of George Soros (No. MGS300) and the Russian Foundation for Basic Research (No. 9704-46421). Mice and Tumour Cells Inoculation Three independent experiments (in summer, winter and spring) were carried out. Each experimental group contained 10 BLRB males at the age of 6-8 months. Mice were inoculated s.c. near the right flank with tumour cell (t.c.) suspensions, performed from syngeneic spontaneous female breast carcinoma according to the conventional procedure (about 10(7) cells per mouse in 0.5 m PBS). Tumour growth was observed in 100% BLRB males with grafted t.c. Tumourigenesis Parameters The animals were examined daily to observe mammary cancer appearance and survival of mice and once a week for the score of tumour growth rate. Emerging tumours were measured with callipers by one person with no knowledge of the treatment. A three-dimensional size measurement for each tumour was performed to calculate median tumour diameter using the average of these mutually perpendicular parameters. The significance of our data was determined by the parametric Student t-test and the nonparametric Wilcoxon U-criterion. The data on relative tumour growth and relative percentage survival dynamics were calculated according to the formula: (parameter in experiment/parameter in control - 1) 100%. The mean relative values of three independent experiments are represented (Table 2). The comparison of dynamic parameter curves for different experimental groups was made by Wilcoxon U-criterion. Treatments Beginning on the third day following t.c. inoculation, males of the 1st experimental group were given two i.p. injections of liposomes (1 ml/mouse) consisting only of matrix lipids (on the third and sixth days). Animals of the 2nd experimental group were treated with DL-melphalan or rubomycin solutions in PBS in the same manner. Mice of the 3rd experimental group were treated with the liposomal ODM, or DOR. Animals of the 4th experimental group were treated with the same drug derivatives included in vectored liposomes. In the same way control mice were given injections of PBS. RESULTS AND DISCUSSION The results of three independent experiments with ODM are summarized in Table 1. Table 1: Average survival time and maximal median mammary tumour diameter of BLRB males under different treatment protocols (experiments with ODM)a. Group: 1; Experiment 1: maximal diameter mm: 24.7 +/- 1.3; survival time days: 99.8 +/- 7.7; Experiment 2: maximal diameter mm: 24.9 +/- 0.5; survival time days: 66.1 +/- 3.5; Experiment 3: maximal diameter mm: 16.4 +/- 1.3; survival time days: 53.0 +/- 4.5. Group: 2; Experiment 1: maximal diameter mm: 24.1 +/- 1.8; survival time days: 98.8 +/- 14.3; Experiment 2: maximal diameter mm: 24.6 +/- 1.0; survival time days: 68.3 +/- 4.2; Experiment 3: maximal diameter mm: 14.1 +/- 2.1; survival time days: 50.9 +/- 4.8. Group: 3; Experiment 1: maximal diameter mm: 22.3 +/- 0.9; survival time days: 99.6 +/- 4.3**; Experiment 2: maximal diameter mm: 23.9 +/- 0.9; survival time days: 75.1 +/- 4.3+; Experiment 3: maximal diameter mm: 14.9 +/- 1.4; survival time days: 67.4 +/- 5.0+. Group: 4; Experiment 1: maximal diameter mm: 22.2 +/- 2.2; survival time days: 127.3 +/- 18.1**; Experiment 2: maximal diameter mm: 24.2 +/- 1.4; survival time days: 75.5 +/- 4.1+. Group: Control; Experiment 1: maximal diameter mm: 23.2 +/- 1.4; survival time days: 82.7 +/- 3.0; Experiment 2: maximal diameter mm: 22.5 +/- 0.6; survival time days: 66.3 +/- 3.9; Experiment 3: maximal diameter mm: 16.4 +/- 1.0; survival time days: 51.9 +/- 4.1. aMean +/- S.E. *P<0.05, **P<0.01 by t-test; +P<0.05 by U-criterion Two of the tumourigenesis parameters are reported. Liposomes themselves (1st group) did not result in any effect on tumour growth rate and life-span of experimental mice. DL-Melphalan (according to the treatment protocol used for the 2nd group) did not have a statistically significant effect on tumour growth and survival course, as compared to controls. The average maximal tumour diameter did not differ from one group to another within each experiment. But different times were required for tumours to achieve this final size. BLRB males treated twice with liposomal ODM (the 3rd group) demonstrated a statistically significant extension of the average life-span, related to the control one: by 21%, 14% and 30% in 1st, 2nd and 3rd experiments. Mammary tumours in these mice appeared and then grew more slowly. So the animals survived better under all observation periods, as related to control ones (data not shown). Only males under this treatment protocol (the 3rd group) demonstrated the pronounced decrease in relative tumour growth and relative survival under all observation periods represented (see Table 2). These dynamic parameter curves differed from those obtained for groups treated with original DL-melphalan, the differences being statistically significant. Table 2: The dynamics of relative mammary tumour growth and relative survival of BLRB males under different treatment protocols with ODM Week group: 1; Relative tumour growth, %: 1: +19; 2: +11; 3: +7; 4: -2; 5: -15; 6: -15; 7: -7; 8: -7; Relative survival, %: 9: +4; 10: +12; 11: +10; 12: -. Week group: 2; Relative tumour growth, %: 1: -32; 2: -47; 3: -35; 4: -25; 5: -21; 6: -12; 7: -5; 8: -6; Relative survival, %: 9: -2; 10: +12; 11: +9; 12: -1. Week group: 3++; Relative tumour growth, %: 1: -66; 2: -59; 3: -52; 4: -52; 5: -48; 6: -43; 7: -34; 8: -33; Relative survival, %: 9: +43; 10: +66; 11: +47; 12: +77. Week group: 4; Relative tumour growth, %: 1: -62; 2: -32; 3: -40; 4: -25; 5: -27; 6: -33; 7: -30; 8: -23; Relative survival, %: 9: +17; 10: +8; 11: +52; 12: +137. ++ Difference from the 2nd group by U-criterion (P<0.01) Analogous studies with liposomal formulations of lipid-derived rubomycin showed statistically significant improvement of the survival course (but without tumour growth inhibition) as compared to that obtained for control mice (data not shown). Next, the experimental mice, treated with the DL-melphalan lipid derivative in the vectored liposomal formulation (the 4th group) demonstrated an average life-span extending from 53% to 14%, related to that of control mice for 1st and 2nd experiments respectively (see Table 1). These experimental groups demonstrated relative tumour growth decreasing and survival extending under all observation periods. During time intervals represented here, these parameters did not differ statistically significantly from those for mice treated with unvectored liposomal drug (the 3rd group, Table 2). However it revealed a very interesting distinction. Some individual mice (one per group) treated with the drug in the vectored liposomal formulation showed slight tumour growth followed by decrease, and complete disappearance of tumour for 6 months and more. Only after this long period did mammary carcinomas appear again and grow according to the conventional process. We suppose that the prolonged suppressive effect of the drug formulation with the vectored liposomes on experimental mammary tumourigenesis is related not only to the influence on the growth of malignant cells, but also to the tumour infiltrating lymphocyte interaction. REFERENCES 1. Kramnik, I.B., Moiseeva, E.V. and Slatinova, O.V. (1989) MNL, 85: 10. 2. Festing, M.F.W. (1993) International Index of Laboratory Animals, 6th ed., p. 22. 3. 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