M5076 Sarcoma Research Articles

Preparation, In Vivo Administration, Dose-Limiting Toxicities, and Antineoplastic Activity of Cytochalasin B

An effective and inexpensive protocol for producing cytochalasins A and B is being disclosed to propose a viable method by which to examine the in vivo antineoplastic activity of these congeners in preclinical tumor-bearing mammalian models. In addition, we determine the maximum tolerated doses of cytochalasin B using multiple routes and formulations, characterize the tissue distribution of intravenous bolus cytochalasin B, and assess the in vivo antineoplastic activity of cytochalasin B in comparison in doxorubicin in Balb/c mice challenged intradermally with M109 murine lung carcinoma. We also examine the effects of cytochalasin B against several other murine neoplastic cell lines (Lewis lung, LA4, B16F10, and M5076). Finally, we examine a potential mechanism of the antimetastatic activity of cytochalasin B by observing the effects of the agent on the secretion of N-acetylglucosaminidase (GlcNACase) by B16BL6 and B16F10 murine melanomas in vitro. The results of the study can be summarized as follows: 1) Cytochalasin B can be safely administered intravenously, intraperitoneally, and subcutaneously in murine models, with the maximum tolerated dose of all routes of administration being increased by liposome encapsulation. 2) Cytochalasin B can significantly inhibit the growth of tumors in mice challenged with M109, Lewis lung, LA4, B16F10, or M5076, producing long-term survival against lung carcinomas and adenocarcinomas (M109, Lewis lung, and LA4) and B16F10 melanoma, but not M5076 sarcoma. These effects were comparable to intraperitoneally administered doxorubicin. 4) Low concentrations of cytochalasin B inhibit the secretion of GlcNACase, indicating that cytochalasin B may inhibit metastatic progression by mechanisms not directly associated with its influence on cell adhesion and motility.

Crosstalk between Tumor Cells and Macrophages in Stroma Renders Tumor Cells as the Primary Source of MCP-1/CCL2 in Lewis Lung Carcinoma.

The chemokine MCP-1/CCL2 is produced by a variety of tumors and plays an important role in cancer progression. We and others previously demonstrated that the primary source of MCP-1 in several mouse tumors, including 4T1 breast cancer, M5076 sarcoma, and B16 melanoma, was stromal cells. In the present study, we identified that tumor cells were the primary source of MCP-1 in Lewis lung carcinoma (LLC), because MCP-1 mRNA was highly expressed in tumors grown in both wild type (WT) and MCP-1−/− mice with elevated serum MCP-1 levels. Since LLC cells isolated from tumors expressed low levels of MCP-1 in vitro, it appeared that the tumor–stromal cell interaction in a tumor microenvironment increased MCP-1 expression in LLC cells. In fact, co-culture of LLC cells with normal mouse peritoneal macrophages or normal lung cells containing macrophages increased MCP-1 expression by LLC cells. Macrophages from TNFα−/− mice failed to activate LLC cells and anti-TNFα neutralizing antibody abolished the effect of WT macrophages on LLC cells. When LLC cells were transplanted into TNFα−/− mice, the levels of MCP-1 mRNA in tumors and serum MCP-1 levels were markedly lower as compared to WT mice, and importantly, tumors grew more slowly. Taken together, our results indicate that TNFα released by tumor cell-activated macrophages is critical for increased MCP-1 production by tumors cells. Thus, disruption of tumor–stromal cell interaction may inhibit tumor progression by reducing the production of tumor-promoting proinflammatory mediators, such as MCP-1.

Abstract B198: The pattern of in vivo biological activity of Zalypsis® is different from that of trabectedin

Abstract Trabectedin is a tetrahydroisoquinoline molecule that was originally derived from the Caribbean marine tunicate Ectenaiscidia turbinata and is now produced by chemical synthesis. The compound is approved in the European Union and a number of other countries for use in patients with advanced soft tissue sarcomas. Zalypsis® (PM00104) is a novel synthetic tetrahydroisoquinoline structurally related to jorumycin. Both compounds have a similar A and B ring structures that bind DNA but differ in their C ring which protrudes out of DNA and is possibly relevant for the interaction with DNA binding proteins, including DNA repair proteins and transcription factors. In the present study, we characterized the in vivo antitumor activity compared to trabectedin. The antitumor activity of Zalypsis® was investigated in two murine models, M5076 reticulosarcoma and MNMCA1 fibrosarcoma, respectively metastasizing liver and lung. Zalypsis® (0.9 mg/kg) and trabectedin (0.15 mg/kg) were administered intravenously every week for three times (q7dx3). In both models Zalypsis® was very effective in inhibiting the primary tumors (T/C 18% and 20% respectively). In the case of MNMCA1, it was also effective against metastasis whereas it was inactive against liver metastasis of M5076 sarcoma. In the two experiments, trabectedin was very effective against metastasis although against M5076 sarcoma it was only marginally able to inhibit primary tumor growth (T/C 52%). In Cen3 tel sarcoma, of human origin, Zalypsis® was very effective with a T/C of 19% whereas trabectedin was essentially inactive (T/C 78%). In contrast Zalypsis® was less effective than trabectedin against HOC22 ovarian carcinoma. In summary it appears that Zalypsis® is biologically different from trabectedin when tested on in vivo tumor models, therefore its clinical development should not necessarily addressed to the same tumors that have shown sensitivity to trabectedin. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B198.

Interleukin-12 genetic administration suppressed metastatic liver tumor unsusceptible to CTL

A cytokine gene therapy approach was conducted against metastatic lesions of cytotoxic T lymphocyte (CTL)-unsusceptible tumor in mice. The EBV-based and conventional plasmid vectors that encode murine interleukin-12 (IL-12) gene (pGEG.mIL-12 and pG.mIL-12, respectively) were intravenously transfected into the mice that had received a subcutaneous inoculation of M5076 sarcoma cells. The pGEG.mIL-12 transfection drastically suppressed the subcutaneous as well as hepatic metastatic tumors, resulting in significant prolongation of survival period of the animals. Although single administration with pG.mIL-12 was not effective, repetitive transfection with the plasmid significantly prolonged the longevity of the mice-bearing the metastatic liver tumors. Multiple transfection with either pGEG.mIL-12 or pG.mIL-12 also suppressed peritoneal carcinomatosis in mice that had been injected with M5076 cells into the peritoneal cavity. It was suggested that a high level IL-12 production elicited by the intravenous delivery of the cytokine gene may be quite effective in inhibiting metastatic and CTL-unsusceptible neoplasms.

Synthesis and evaluation of novel pyrimido-acridone, -phenoxadine, and -carbazole as topoisomerase II inhibitors.

As part of a series of studies to discover new topoisomerase II inhibitors, novel pyrimidoacridones, pyrimidophenoxadines, and pyrimidocarbazoles were synthesized, and in vitro and in vivo antitumor activities and DNA-protein and/or DNA-topoisomerase II cross-linking activity as an indicator of topoisomerase II-DNA cleavable complex formation were evaluated. The pyrimidocarbazoles possessed high in vitro and in vivo potencies. Compound 26 (ER-37326), 8-acetyl-2-[2-(dimethylamino)ethyl]-1H-pyrimido[5,6,1-jk]carbazole-1,3(2H)-dione, showed in vitro growth inhibitory activity with respective IC(50) values of 0.049 microM and 0.35 microM against mouse leukemia P388 and human oral cancer KB. In vivo, this compound inhibited the tumor growth of mouse sarcoma M5076 implanted into mice with T/C values of 42% and 13% at 3.13 and 6.25 mg/kg/d respectively without significantly affecting the body weight. In addition, compound 26 (ER-37326) increased the formation of DNA-topoisomerase II cross-linking in P388 cells.

Contribution of specific transport systems to anthracycline transport in tumor and normal cells.

Anthracycline antibiotics are very effective neoplastic agents widely used clinically. However, because of their many adverse effects (e.g. cardiotoxicity, leukopenia and alopecia), their clinical use has been limited. In order to minimize their adverse effects in clinical cancer chemotherapy, anthracyclines must be selectively transported into tumor cells. If there are differences in transport characteristics between tumor and normal cells, it should be possible to establish a strategy for selectively delivering anthracyclines to tumor cells on the basis of the differences. In human cultured leukemia HL60 cells, as tumor cells, and human fresh mononuclear cells, as normal cells, doxorubicin, pirarubicin, daunorubicin and idarubicin were incorporated via a common carrier-mediated system, but the carriers were different in the two cell types. In HL60 cells, it was indicated that a nucleoside transport system contributed, at least in part, to the transport of doxorubicin and pirarubicin, but not daunorubicin and idarubicin, and its contribution to pirarubicin transport was found in other tumor cells, i.e. mouse ovarian sarcoma M5076 and Ehrlich ascites carcinoma cells. On the other hand, in mononuclear cells, there was no involvement of a nucleoside transport system for the four anthracyclines examined. Therefore, we thought that with the modification of an anthracycline molecule as a substrate for the nucleoside transport system, the anthracycline could be delivered selectively to tumor cells.

Preclinical antitumor activity of two novel taxanes.

Two taxane analogs, BMS-184476 and -188797, were evaluated for their in vitro cytotoxicity and in vivo antitumor activity, and compared with paclitaxel and occasionally docetaxel (Taxotere). Cytotoxicity was assessed in vitro using a panel of human tumor cell lines. Several different murine and human tumor models were used in vivo to evaluate the taxane analogs. Both compounds were found to have cytotoxic potency similar to paclitaxel and to partially overcome two different forms of paclitaxel resistance. BMS-184476 was found to be clearly superior to paclitaxel in three human xenograft tumor models: A2780 ovarian carcinoma; HCT/pk, a moderately paclitaxel-resistant colon carcinoma; and L2987 lung carcinoma. Additionally, in the clinically derived TAXOL-unresponsive ovarian carcinoma, HOC79, BMS-184476 performed slightly better than paclitaxel and Taxotere. BMS-184476 and paclitaxel were inactive in two murine model systems, M5076 sarcoma and the paclitaxel-resistant M109/txlr lung carcinoma. Against the parental M109 tumor, both BMS-184476 and paclitaxel performed comparably. BMS-184476 was never found to be inferior to paclitaxel. The other taxane analog, BMS-188797, displayed efficacy superior to paclitaxel in four in vivo tumor models: HOC79, HCT/ pk, M109, and L2987 carcinomas. Like paclitaxel and BMS-184476, BMS-188797 was inactive versus M5076 sarcoma. Two new taxane analogs were characterized as superior to paclitaxel or Taxotere in several in vivo tumor models. Both BMS-184476 and -188797 are currently in phase I or II clinical trials.

新規Ｘａｎｔｈｉｎｅ誘導体ＭＰＢＸによるＤｏｘｏｒｕｂｉｃｉｎ制癌効果増強作用

We previously reported that 1-methyl-3-propy1-7-butylxantlaine (MPBX), a novel xanthine derivative, enhanced the antitumor activity of doxorubicin (DOX) due to inhibition of the DOX efflux from tumor cells. In this study, we investigated the effects of MPBX not only on drug sensitive tumor but also on multidrug resistant or metastatic tumors with the aim of developing its clinical use. In Ehrlich carcinoma bearing mice, MPBX enhanced antitumor activity of DOX 1.8-fold and increased the DOX concentration in the tumor 1.6-fold. Moreover, on multidrug resistant P388 leukemia (P388/DOX), DOX + MPBX significantly reduced the tumor weight to 48% of control level, whereas DOX alone did not reduce the tumor weight. MPBX increased the DOX influx into P388/DOX cells and inhibited the DOX efflux in vitro, supporting the increase in the DOX concentration in the tumor induced by MPBX in vivo. Furthermore, MPBX enhanced the therapeutic efficacy of pirarubicin, an anthracycline antibiotic, against metastatic M5076 sarcoma. In conclusion, MPBX enhanced the antitumor activity of anthracycline agents on sensitive, multidrug resistant or metastatic tumors. Thus, we expected that MPBX would be used as a biochemical modulator in clinical cancer chemothrapy.

FK317: a novel substituted dihydrobenzoxazine with potent antitumor activity which does not induce vascular leak syndrome.

FK973, a substituted dihydrobenzoxazine, is an antitumor antibiotic which has shown high therapeutic efficacy in a phase I study, but its development has been abandoned because of the side effect of vascular leak syndrome (VLS) in the clinical study. This study was performed to investigate whether or not FK317, a new benzmethoxy derivative of FK973, retains the antitumor activity of FK973 without the side effect of VLS. VLS was evaluated by the volume of pleural effusion in rats. Cytotoxic activities were determined by a tetrazolium-based colorimetric assay (MTT assay) against murine (B16, P388) and human (HeLa S3, KB) tumor cell lines. Antitumor activities against murine ascitic leukemia (P388, L1210), murine solid tumors (reticulum cell sarcoma M5076, Colon 38 carcinoma) and human xenografts (mammary carcinoma MX-1, lung carcinoma LX-1) were examined. FK973 (1.8 mg/kg) given i.v. to rats induced pleural effusion, one of the elements of VLS, 36 days after the first dosing, but did not 28 days after dosing. This model reflects clinical VLS delayed-type effusion with high protein concentrations. In contrast, FK317 (1.0-3.2 mg/kg) did not induce pleural effusion at all. FK317 had stronger cytotoxic effects against in vitro cultured B16, P388, HeLa S3 and KB tumor cell lines, and in in vivo experiments, FK317 showed equivalent antitumor activity against P388, M5076 and MX-1, and more potent antitumor activity against L1210, Colon 38 and LX-1 compared with FK973. These results suggest that FK317 retains the antitumor activity of FK973 and does not induce VLS, and FK317 is a drug with high clinical potential for treating tumors in humans.

Combination chemotherapy involving orally administered etoposide and JM-216 in murine tumor models.

Orally administered VP-16 (etoposide) was evaluated in combination with an orally administered platinum analog, JM-216 [ammine/cyclohexylamine diacetatodichloride Pt(IV)], in mice bearing murine tumors, for therapeutic synergy. The treatment schedules used involved two courses of therapy, each course consisting of administration every day for 5 days beginning on either day 4 or day 5 posttumor implantation, and again on day 11 or day 12 postimplantation. The amounts of each drug tolerated in the combination treatment setting were much less than their individual maximum tolerated doses (MTDs). Thus, to be used safely, each drug's dose had to be greatly reduced from the amount tolerated when the drugs were given individually. Multiple experiments using a staged P388 leukemia model implanted intravenously yielded confirmatory data supporting the existence of a therapeutic synergy for the drug combination. Identical regimens applied in the staged M5076 sarcoma model implanted subcutaneously, however, were not considered to have yielded data indicative of therapeutic synergy. A clinical phase I study using this combination chemotherapy can be recommended on the basis of the results obtained in the leukemia model.

ChemInform Abstract: Synthesis and Antitumor Activity of Duocarmycin Derivatives: Modification of Segment A of Duocarmycin B2.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Tyrosine kinase inhibitors. 6. Structure-activity relationships among N- and 3-substituted 2,2'-diselenobis(1H-indoles) for inhibition of protein tyrosine kinases and comparative in vitro and in vivo studies against selected sulfur congeners.

A small series of 2,2'-diselenobis(1H-indoles) was synthesized as redox-modified congeners of our earlier reported 2,2'-dithiobis(1H-indole) series. Utilizing chemistry similar to that developed earlier for the disulfur series, compounds were made from 2-halogeno-3-indolecarboxylic acid precursors bearing various polar functionality at the C-3 position and small alkyl substituents at the N-1 position of the indole nucleus. Additional compounds were derived from (R)- or (S)-tryptophan via a novel application of diselenium dichloride as an electrophilic source of diselenium, and a much improved process to a 2,2'-dithiobis(1H-indole) congener was developed utilizing disulfur dichloride as a source of disulfur. Against isolated epidermal growth factor receptor (EGFr), platelet-derived growth factor receptor (PDGFr), and v-src tyrosine kinases, compounds in this series displayed broad inhibitory activity with IC50 = 0.9 to > 100 microM vs EGFr, 3.4 to > 50 microM vs PDGFr, and 0.4-6.7 microM vs v-src. In general, compounds derived from tryptophan displayed the greatest potency against EGFr and those from 2-halogeno-3-indolecarboxylic acids greater potency against PDGFr and v-src. Enzyme kinetics studies showed that both classes of compounds display primarily noncompetitive inhibition with respect to either ATP or peptide substrate. The sulfhydryl reducing agent dithiothreitol (DTT) caused a general decrease in inhibition of the EGFr and v-src tyrosine kinases by both the diselenium and disulfur series with the reversal of enzyme inhibition occurring less readily within the diselenium series. In whole cell studies, compounds of this class were growth inhibitory against Swiss 3T3 mouse fibroblasts with IC50 values from 0.5 to 19.5 microM, and the observed SAR was different from that of the 2,2'-dithiobis(1H-indoles). A comparative study in the same cell line on the effects of the 2,2'-diselenobis(1H-indole) derived from (R)-tryptophan vs its disulfur congener on growth factor mediated tyrosine phosphorylation showed that this compound significantly inhibited EGFr and PDGFr (in response to its ligand) autophosphorylation with complete suppression at 25 and 5 microM, respectively. Tyrosine phosphorylation of an 85 kDa protein typically phosphorylated in response to bFGF was also exquisitely sensitive to this compound, and it displayed inhibitory effects on DNA, RNA, and protein synthesis at submicromolar concentrations. The disulfur congener exhibited a qualitatively similar pattern; however, its potency was 10-fold less. This same diselenium/disulfur pair was evaluated in vivo against the B16 melanoma, colon carcinoma 26, and M5076 sarcoma murine tumors, and the A431 epidermoid, and C6 glioma human tumor xenografts. At maximum tolerated doses (1.8 and 5.0 mg/kg/injection, respectively), neither the diselenium nor disulfur congener was effective against the C6 glioma when administered intraperitoneally on a d1-9 schedule. Studies were also carried out against the A431 epidermoid xenograft to evaluate the same pair of compounds via continuous subcutaneous infusion from Alzet miniosmotic pumps. The maximum dose that could be administered daily was limited by compound solubility. Neither compound produced an antitumor effect in a 7-day continuous infusion study. In the 27-day study, the disulfur compound was inactive whereas the diselenium compound produced a 10.8-day growth delay without appreciable treatment related weight loss. The in vitro and in vivo findings offer a mechanistic rationale as to why the 2,2'-diselenobis(1H-indoles) are more potent inhibitors than their disulfur congeners.

Nucleosides and nucleotides. 155. synthesis, antitumor effects, and possible enzymatic activation mechanism of 5′-phosphatidyl-2′-deoxy-2′-methylenecytidine (DMDC)

2′-Deoxy-2′-methylenecytidine (DMDC, 1) and its 5-fluoro congener (5-F-DMDC, 2), potent antitumor nucleosides developed by us, were efficiently converted to their 5′-phosphatidyl derivatives bearing palmitoyl residues ( 3 and 4, respectively) as novel antitumor phospholipids by phospholipase D-catalyzed trans-phosphatidylation. These phospholipids 3 and 4, administered i.p., remarkably prolonged the life-span of mice which were i.p.-inoculated with M5076 sarcoma, and the effects were clearly superior to that of DMDC. Compound 3 was a good substrate for phospholipase A 2 from bovine pancreas as well as phospholipase D from Streptomyces, while it was slightly hydrolyzed by phospholipase C from Bacillus cereus.

Nucleosides and nucleotides. 150. Enzymatic synthesis of 5′-phosphatidyl derivatives of [formula omitted] (CNDAC) and their notable antitumor effects in mice 1

1-(2-C- Cyano-2-deoxy-β- d -arabino- pentofuranosyl)cytosine (CNDAC, 1) is a potent antitumor nucleoside developed by us. A series of 5′-phosphatidyl derivatives of CNDAC bearing various fatty acyl residues, namely dimyristoyl derivative 3a, dipalmitoyl derivative 3b, distearoyl 3c, dioleoyl derivative 3d, and dilinoleoyl derivative 3e, was synthesized by phospholipase D-catalyzed transphosphatidylation. All of these 5′-phosphatidyl-CNDACs ( 3a-e) administered ip almost perfectly inhibited the growth of sc-implanted M5076 sarcoma in mice, and the effects were clearly superior to that of CNDAC.

Lipid emulsions of palmitoylrhizoxin: effects of composition on lipolysis and biodistribution.

Four types of lipid emulsion for highly lipophilic antitumour agent RS-1541 (13-O-palmitoylrhizoxin) with mean particle diameters of 200-260 nm were prepared using soybean oil (SOY) or dioctanoyldecanoylglycerol (ODO) for the oil phase and lecithin (LEC) or polyoxyethylene-(60)-hydrogenated castor oil (HCO-60) for surfactants. The lipolysis rate of HCO-60-emulsified emulsions by lipoprotein lipase was much slower than that of LEC-emulsified emulsions. Particle sizes of emulsions incubated in plasma with the lipase for six hours were 75%, 79%, 101%, and 93% of initial values for SOY/LEC, ODO/LEC, SOY/HCO-60, and ODO/HCO-60 emulsions, respectively, showing an apparent size decrease for LEC-emulsified emulsions. In rats, uptake clearance values of SOY/LEC and ODO/LEC emulsions of RS-1541 in the reticuloendothelial system (RES) were 81.2 and 135.3 mL h(-1), respectively, and AUC values were 4.0 and 1.3 microg h mL(-1), respectively. In contrast, RES uptake clearances of HCO-60 emulsions of RS-1541 were considerably lower (4.2 mL h(-1) for SOY/HCO-60; 2.2 mL h(-1) for ODO/HCO-60), resulting in high AUC values (35.4 microg h mL(-1) for SOY/ HCO-60; 63.9 microg h mL(-1) for ODO/HCO-60). The concentrations of RS-1541 in tumour tissues after an intravenous administration of ODO/HCO-60 emulsions of RS-1541 to mice bearing solid tumour M5076 sarcoma were about ten times higher than those after the administration of SOY/LEC emulsions. These results indicate that HCO-60 emulsions, compared with conventional LEC emulsions, are more stable to lipoprotein lipase and show low uptakes by RES organs, long circulations in the plasma, and high distributions in tumours. Thus, these sterically stabilized emulsions could show potential as effective carriers for highly lipophilic antitumour agents to enhance the drug delivery in tumours.

Synthesis and antitumor activity of duocarmycin derivatives: modification of segment A of duocarmycin B2.

Several A-ring pyrrole derivatives of duocarmycin B2 were synthesized effectively from the 3-hydroxy compounds by utilizing an interesting acid-catalyzed rearrangement, their anticellular activity was preliminarily evaluated by assays of growth inhibition of HeLa S3 cells (in vitro) and antitumor activity against murine sarcoma 180 (in vivo). The 8-O-N,N-dialkylcarbamoyl derivatives of the A-ring pyrrole compound showed remarkably potent in vivo antitumor activity, superior to that of duocarmycin B2. these derivatives were subjected to further biological evaluation. They exhibited potent antitumor activity toward murine solid tumors including M5076 sarcoma, B-16 melanoma and Colon 26 adenocarcinoma. Their most noteworthy feature was their efficacy against various human xenografts including LC-6 (lung), St-4 (stomach), and Co-3 (colon).

Enhanced tumor delivery and antitumor activity of palmitoyl rhizoxin using stable lipid emulsions in mice.

A highly lipophilic antitumor agent, 13-O-palmitoyl-rhizoxin (RS-1541), was incorporated into lipid emulsions of various sizes consisting of triglyceride ODO and surfactant HCO-60. Pharmacokinetics, toxicities, and antitumor activities were evaluated after intravenous administration to mice bearing subcutaneously inoculated M5076 sarcoma cells. The levels of RS-1541 in the plasma and tissues including tumor, were determined by HPLC. The maximum tolerated dose (MTD) was estimated by toxic death and change in body weight. The decrease in tumor diameter was measured for antitumor activity. There existed large variations in pharmacokinetics of RS-1541, depending on the size of emulsion particles. Compared with a colloidal solution (reference solution), the small (110nm) and medium (230nm) size emulsions showed high concentrations of RS-1541 in the tumor, while the large emulsions (350nm-630nm) exhibited low concentrations. The MTD of RS-1541 was reduced, when incorporated in the emulsions larger than 220nm in size. At MTD, each size of emulsions (70nm-380nm) effectively retarded the tumor growth and increased survival time. The maximum effect was achieved for the 220 nm emulsions. When particle size is properly selected, these emulsions could be promising and effective as an injectable carrier for lipophilic antitumor agents in order to enhance the tumor delivery and efficacies while reducing toxicities.

Antitumour activity and retinotoxicity of ethyldeshydroxy-sparsomycin in mice.

The colony formation in agar of human tumour xenografts was used as a test system to study the cytostatic activity of ethyldeshydroxy-sparsomycin (EdSm) at the cellular level. EdSm was additionally studied in vivo in human tumour xenografts and murine tumour models. EdSm showed a clear dose-response effect in vitro. At continuous exposure with 0.01 micrograms/ml, 2 out of 11 of the tumours responded (a gastric and a small cell lung carcinoma). At 0.1 mu/ml EdSm, the tumour response was 5/11 tumours and at 1 microgram/ml the compound was active in all tumours. The maximal tolerable doses of EdSm in vivo have been determined in non-tumour bearing CDF1 mice. In the intraperitoneally (i.p.) given multiple dose schedules the respective LD10 doses indicated that the tolerable cumulative dose increases when lower doses are given more frequently. This also enhances the antitumour activity in L1210 leukaemia to 172% T/C. On the other hand, continuous infusion strongly diminished the tolerable dose as well as the antitumour activity. EdSm was also active against i.p. inoculated P388 leukaemia (150% T/C), B16 melanoma (156% T/C), and RC carcinoma (197% T/C), and the subcutaneously (s.c.) inoculated L1210 (139% T/C) and RC (138% T/C). Absence of tumour responses was found in the following s.c. implanted murine tumours: M5076 sarcoma, osteosarcomas C22LR and CP369, and the LL carcinoma, as well as in the human tumour xenografts: LXFG 529, a non-small cell lung carcinoma; GXF 251, a gastric carcinoma; and FMa, an ovary carcinoma. Possible long-range retinotoxic effects of EdSm were investigated in tumour-bearing mice, cured after surviving treatment with LD50 doses of EdSm, by assaying the protein biosynthetic capacity of the retinal by assaying the ocular rhodopsin and opsin levels as parameters. In none of these cases could a significant reduction in either opsin or rhodopsin levels be measured and no changes were seen histologically.

Plasma lipoproteins as targeting carriers to tumour tissues after administraion of a lipophilic agent to mice

We synthesized 14C-warfarin hexadecyl ether (14C-WHE) by addition of a palmityl moiety to the hydroxyl group at the 4-position of 14C-warfarin, a compound known to bind to serum albumin. 14C-WHE preferentially bound to the lipoproteins, low-density lipoprotein (LDL) and high-density lipoprotein (HDL), in mouse plasma both in vitro and in vivo. 14C-Warfarin mainly concentrated in the liver immediately after intravenous administration to mice bearing M5076 sarcoma, and was found at only low concentrations in other tissues including the tumour. 14C-WHE highly distributed to the tumour, adrenal, and spleen, as well as the liver. These tissues coincided with those in which human 125I-LDL was vigorously incorporated. The results indicate that chemical modification of an agent, giving it high lipophilicity, will enable it to bind to lipoproteins after intravenous administration. These modifications raise the possibility of lipoproteins as endogenous targeting carriers into tumour cells, which have high LDL-receptor activity.

Tumor selective effect of RS-1541 (palmitoyl-rhizoxin) in M5076 sarcoma and host tissues in vivo.

RS-1541 is a 13-O-palmitoyl derivative of rhizoxin, an inhibitor of tubulin polymerization. After intravenous administration of RS-1541 to mice bearing M5076 sarcoma, the maximal inhibitory effect of RS-1541 on DNA synthesis in the tumor was observed 24 h after administration, in agreement with the Cmax of rhizoxin produced from RS-1541, but not with the Cmax of RS-1541. The inhibitory effect after RS-1541 was much higher than that after rhizoxin itself. In the spleen, thymus and bone marrow, DNA synthesis was strongly inhibited by rhizoxin but not by RS-1541. After administration of RS-1541, no significant amounts of rhizoxin were detected in the tissues, except for the tumor. In acute toxicity tests, RS-1541 appeared to be less toxic than rhizoxin. These results indicate that RS-1541 possesses a high tumor-selective effect compared with rhizoxin, because of the selective production of rhizoxin in the tumor after administration of RS-1541.