Altersolanol A: a selective cytotoxic anthraquinone from a Phomopsis sp.

UNLABELLED: The cytotoxic compound Altersolanol A, an anthraquinone derivative was isolated from PM0409092 a fungus of Nyctanthes arbor-tristis (family Oleaceae). It was identified as a Phomopsis sp. by DNA amplification and sequencing of the ITS region. The chemical structure of Altersolanol A was elucidated from its physicochemical properties, 2D NMR spectroscopy and other spectroscopic data. The compound has in vitro cytotoxic activity against 34 human cancer cell lines with mean IC50 (IC70) values of 0.005 µg ml(-1) (0.024 µg ml(-1)) respectively. Altersolanol A, a kinase inhibitor, induces cell death by apoptosis through the cleavage by Caspase-3 and -9 and by decreased anti-apoptotic protein expression. There are several previous reports of the anticancer activity of Altersolanol A, but we report here an extensive study using 36 cell lines which gives wider spectrum of results. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms the cytotoxic potential of Altersolanol A isolated from the endophyte Phomopsis sp. (PM0409092) of the plant Nyctanthes arbor-tristis. The compound exhibits in vitro cytotoxicity against 34 human cancer cell lines with mean IC50 (IC70) value of 0.005 µg ml(-1) (0.024 µg ml(-1)). This is an in-depth report of Altersolanol A against a panel of 34 human cancer cell lines and extends observations from previous studies indicating that Altersolanol A can be used for the development of chemotherapeutics. Altersolanol A, a kinase inhibitor, induces cell death by apoptosis through the cleavage of Caspase-3 and -9 and by decreased anti-apoptotic protein expression.

Preclinical pharmacology of the pyrrolobenzodiazepine (PBD) monomer DRH-417 (NSC 709119).

The pyrrolobenzodiazepine monomer DRH-417 is a member of the anthramycin group of anti-tumor antibiotics that bind covalently to the N2 of guanine within the minor groove of DNA. DRH-417 emerged from the EORTC-Drug Discovery Committee and NCI 60 cell line in vitro screening programs as a potent antiproliferative agent with differential sensitivity towards certain cancer types such as melanoma, breast and renal cell carcinoma (mean IC(50) = 3 nM). DRH-417 was therefore tested for in vivo activity. The maximum tolerated dose (MTD) was established as 0.5 mg/kg given i.p. Marked anti-tumor activity was seen in two human renal cell cancers, one breast cancer and a murine colon tumor model (p<0.01). A selective HPLC (LC/MS) analytical method was developed and plasma pharmacokinetics determined. At a dose of 0.5 mg kg(-1), the plasma AUC was 540 nM h (197.1 ng h ml(-1)) and the peak plasma concentration (171 nM [62.4 ng ml(-1)]) occurred at 30 min., reaching doses levels well above those needed for in vitro antiproliferative activity. Genomic profiling of in vivo sensitive tumors revealed that the latter have an activated insulin-like growth factor signaling pathway.

Chemotherapeutic activity of new 2-chloroethylnitrosoureas in rat L5222 leukemia: comparison of bifunctional and water-soluble derivatives with 1,3-bis(2-chloroethyl)-1-nitrosourea.

The chemotherapeutic activity of eight nitrosourea derivatives was compared with that of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in transplantable rat leukemia L5222 cells. Bifunctional 1,1'-polymethylenebis-3-(2-chloroethyl)-3-nitrosoureas effected cure rates between 30 and 75% in single equitoxic doses in the therapy of advanced ip implanted L5222 [staging of L5222 leukemia development (hr before median day of death in controls): early = greater than 120; advanced = 120--61; late = 60--25; and preterminal = 24--0]. Of three water-soluble monofunctional alkylating 1-(omega-hydroxyalkyl)-3-nitrosoureas, 1-(2-hydroxyethyl)-3-(2-chloroethyl)-3-nitrosourea yielded more cures (90%) than did BCNU (cure rate, 70%) and was also superior to the other substances. Against preterminal ip implanted and late intracerebrally implanted L5222, the hydroxyethyl compound was significantly superior to BCNU.

Effects of cytostatic drugs and 40.5 degrees C hyperthermia on human bone marrow progenitors (CFU-C) and human clonogenic tumor cells implanted into mice.

Human spontaneous tumors after surgical resection were implanted into NMRI nude mice. Clonogenic cells from these tumors (4 malignant melanomas, 2 squamous cell carcinomas of the lung, and 1 small cell carcinoma of the lung) were cultured in a methylcellulose monolayer assay. Dose-response curves with 7 cytostatic drugs (doxorubicin, bleomycin, dactinomycin, cisplatin, vincristine, vinblastine, and melphalan) were assessed at 37 degrees C and after a 2-hour pulse at 40.5 degrees C. In addition, bone marrow cells (CFU-C) were plated in the same assay. Dose-response curves were assessed with the same drugs under the same conditions. Hyperthermic treatment without drugs did not alter the colony formation of tumor and bone marrow cells. Bone marrow samples from different donors showed homogeneous response patterns; the tumor probes revealed sensitivity patterns typical for each tumor. In 13 of 48 tumor-drug combinations a thermal enhancement of the drug effects was observed, whereas there was no significant difference between normothermic and hyperthermic cultures in the bone marrow cultures. A comparison of colony and bone marrow dose-response curves suggested that a hyperthermic enhancement can occur in single cases. However, this phenomenon seems to be due to individual properties of the tumor.

Chromosomal localization of the carcinoembryonic antigen gene family and differential expression in various tumors.

Carcinoembryonic antigen (CEA) is a glycoprotein which is important as a tumor marker for a number of human cancers. It is a member of a gene family comprising about 10 closely related genes. In order to characterize mRNAs transcribed from individual genes we have identified by DNA and RNA hybridization experiments, gene-specific sequences from the 3' noncoding regions of CEA, and of nonspecific cross-reacting antigen (NCA) mRNAs, which have been recently cloned. With these probes, CEA mRNAs with lengths of 3.5 and 3.0 kilobases and an NCA mRNA species of 2.5 kilobases were identified in various human tumors. A 2.2-kilobase mRNA species, however, could only be detected in leukocytes of patients with chronic myeloid leukemia by hybridization with a probe from the immunoglobulin-like repeat domain of CEA. This region is known to be very similar among the various members of the CEA gene family, and indeed the probe hybridizes with all four mRNA species. In situ hybridization with a cross-hybridizing probe from the NCA gene localized the members of the CEA gene family to the short and to the long arm of chromosome 19. In addition, a CEA cDNA probe was found to hybridize to the long arm of chromosome 19 only.

Phase II preclinical drug screening in human tumor xenografts: a first European multicenter collaborative study.

In a European joint project carried out in 6 laboratories a disease-oriented program was set up consisting of a panel of 7 tumor types, each represented by 4 to 8 different human tumor lines, for secondary screening of promising anticancer drugs. Human tumor lines were selected on the basis of differences in histology, growth rate, and sensitivity to conventional cytostatic agents. Xenografts were grown s.c. in nude mice, and treatment was started when tumors reached a mean diameter of 6 mm in groups of mice where at least 6 tumors were evaluable. Drugs were given at the maximum tolerated dose. For evaluation of drug efficacy, median tumor growth curves were drawn, and specific growth delay and treated/control x 100% were calculated. Doxorubicin (8 mg/kg i.v. days 1 and 8) was effective (treated/control < 50%, and specific growth delay > 1.0) in 0 of 2 breast cancers, 1 of 3 colorectal cancers, 2 of 5 head and neck cancers, 3 of 6 non-small cell lung cancers, 4 of 6 small cell lung cancers, 0 of 3 melanomas, and 3 of 6 ovarian cancer lines. Amsacrine (8 mg/kg i.v. days 1 and 8) was not effective, while datelliptium (35 mg/kg i.p. days 1 and 8) was active against 2 of 6 small cell lung cancer lines. Brequinar sodium (50 mg/kg i.p. days 1-5) showed efficacy in 4 of 5 head and neck cancers, 5 of 8 non-small cell lung cancers, and 4 of 5 small cell lung cancer lines. The project has been shown to be a feasible approach. Clinical activity for doxorubicin and inactivity for amsacrine against solid tumor types was confirmed in the human tumor xenograft panel. Additional anticancer drugs will be studied in the European joint project to further define the reliability of this novel, promising screening approach.

Predicting tumor responses to mitomycin C on the basis of DT-diaphorase activity or drug metabolism by tumor homogenates: implications for enzyme-directed bioreductive drug development.

Mitomycin C (MMC) is a clinically used anticancer drug that is reduced to cytotoxic metabolites by cellular reductases via a process known as bioreductive drug activation. The identification of key enzymes responsible for drug activation has been investigated extensively with the ultimate aim of tailoring drug administration to patients whose tumors possess the biochemical machinery required for drug activation. In the case of MMC, considerable interest has been centered upon the enzyme DT-diaphorase (DTD) although conflicting reports of good and poor correlations between enzyme activity and response in vitro and in vivo have been published. The principle aim of this study was to provide a definitive answer to the question of whether tumor response to MMC could be predicted on the basis of DTD activity in a large panel of human tumor xenografts. DTD levels were measured in 45 human tumor xenografts that had been characterized previously in terms of their sensitivity to MMC in vitro and in vivo (the in vivo response profile to MMC was taken from work published previously). A poor correlation between DTD activity and antitumor activity in vitro as well as in vivo was obtained. This study also assessed the predictive value of an alternative approach based upon the ability of tumor homogenates to metabolize MMC. This approach is based on the premise that the overall rate of MMC metabolism may provide a better indicator of response than single enzyme measurements. MMC metabolism was evaluated in tumor homogenates (clarified by centrifugation at 1000 x g for 1 min) by measuring the disappearance of the parent compound by HPLC. In responsive [T/C <10% (T/C defined as the relative size of treated and control tumors)] and resistant (T/C >50%) tumors, the mean half life of MMC was 75+/-48.3 and 280+/-129.6 min, respectively. The difference between the two groups was statistically significant (P < 0.005). In conclusion, these results unequivocally demonstrate that response to MMC in vivo cannot be predicted on the basis of DTD activity. Measurement of MMC metabolism by tumor homogenates on the other hand may provide a better indicator of tumor response, and further studies are required to determine whether this approach has real clinical potential in terms of individualizing patient chemotherapy.

Effects of human bone marrow stroma on the growth of human tumor cells.

As some tumors metastasize frequently to marrow we modified the clonogenic assay for human tumor cell growth by culturing tumor cells in the presence of human bone marrow stromal cells. In a bilayer soft agar assay, human tumor cells which had been passaged in nude mice were plated in the agar overlayer on an underlayer containing a suspension of trypsinized human bone marrow stromal cells. These marrow stromal cells stimulated the growth of tumor cells in a dose-dependent fashion, with a growth peak at a stromal cell density of 5-10 x 10(5)/ml. The maximal stimulation of tumour cell growth was 13-fold. We evaluated clonal growth of six separate tumors of five different histological types (small and large cell bronchogenic carcinoma; mammary carcinoma; malignant melanoma; pleural mesothelioma) and demonstrated that in 9 of 11 experiments tumor cell colonies formed in the absence of stromal cells, but colony growth was markedly stimulated by stromal cells in every case. Stromal stimulation persisted after irradiation of the stromal cells with 10 Gy. Growth of five fresh human tumor samples was similarly stimulated by the presence of human bone marrow stromal cells. Tumor cell colonies were characterized morphologically by Pappenheim stain and immunologically for surface antigens by peroxidase-antiperoxidase immunostaining utilizing monoclonal antibodies (carcinoembryonic antigen 26/3/13 and 26/5/1, EMA, HEA125, Sam 2 and Sam 10) which detected epithelial cell antigens. Colonies consisted of cytologically malignant cells which expressed epithelial cell antigens. Thus, the tumor cell origin of colonies from mammary carcinoma and bronchogenic small cell, large cell, and adenocarcinoma was proven. This tumor stem cell assay permits further analyses of human tumor cell biology and may be useful for testing drug sensitivity.

Anticancer activity of a lectin-rich mistletoe extract injected intratumorally into human pancreatic cancer xenografts.

BACKGROUND: In single case observations, tumour remissions after intratumoral injections of mistletoe extracts have been described. MATERIALS AND METHODS: We investigated the antitumour activity of intratumorally (i.t.)-injected lectin-rich mistletoe extract at different dosages and i.t.-injected mistletoe lectin I in comparison to intravenous (i.v.) Gemcitabine and i.t. treatment with placebo in a human pancreatic cancer xenograft. RESULTS: In a preliminary dose-response experiment, the most marked tumour inhibition was induced when mistletoe extract was given at 8 mg/kg body weight (BW) and mistletoe lectin I at 5.3 microg/kg BW. In a second experiment, bi-weekly i.t. injections of mistletoe extract over 8 weeks resulted in a very high antitumour activity with an optimal T/C value (=median relative tumour volume of the test group vs. the control) of 0.4% combined with 3/8 partial and 3/8 complete remissions. Gemcitabine was less active with 2/8 partial and 1/8 complete remissions and an optimal TIC of 4.6%. CONCLUSION: I.t.-injected lectin-rich mistletoe extract should be further evaluated in patients with inoperable locally advanced pancreatic cancer.

Flavopiridol (L86-8275): selective antitumor activity in vitro and activity in vivo for prostate carcinoma cells.

We have selected a panel of human tumor xenografts for in vitro and in vivo studies that allows an indication of selectivity of action of novel chemotherapeutic agents. We report here the antitumor activity of the flavone flavopiridol (previously designated L86-8275), which has been selected for further studies based in part on its behavior in the anticancer drug screening system of the United States National Cancer Institute. Eighteen human tumor and five cell line-derived xenografts established by serial passage in nude mice in our laboratory were used as tumor models for in vitro investigations using a modified double-layer soft agar assay. In vivo investigations were completed in nude mice bearing advanced-stage s.c. growing prostate cancer xenografts. Antitumor activity in vitro (test/control

BBR 3464: a novel triplatinum complex, exhibiting a preclinical profile of antitumor efficacy different from cisplatin.

Multinuclear platinum complexes represent a new class of anticancer agents, distinct in terms of DNA binding features and the profile of antitumor activity from their mononuclear counterparts, in particular cisplatin. Among complexes of this class, BBR 3464, a trinuclear platinum compound has been selected for preclinical development. In the present study, we describe the preclinical evaluation of BBR 3464 in a series of human tumor cell lines and tumor xenografts, with special emphasis on tumor types known to be resistant to cisplatin. In a panel of seven human tumor cell lines naturally resistant to cisplatin (three ovarian and four melanomas), BBR 3464 was extremely potent with IC50 values at least 20-fold lower than cisplatin. Against eight human tumor xenografts including four tumors refractory to cisplatin, BBR 3464 was confirmed to be very active with a tumor weight inhibition >80% in seven of them. The efficacy of BBR 3464 against cisplatin-resistant tumors was consistent with the ability of the drug to completely overcome resistance in three cell systems characterized by acquired resistance to cisplatin. Moreover, BBR 3464 caused a more prolonged effect than cisplatin, which was reflected by higher specific growth delay values. This prolonged effect is likely to be related to a more persistent perturbation of the cell cycle induced by BBR 3464 than by cisplatin, as shown in one ovarian tumor cell line. Finally, the profile of sensitivity to BBR 3464 within the 60-cell-lines screening panel of the National Cancer Institute, NIH (Bethesda, MD) differed from those of established drugs, thus supporting the hypothesis of a distinct mechanism of cytotoxic activity of BBR 3464. The novel trinuclear platinum complex, in light of its innovative antitumor activity profile, has the potential to become a useful clinical agent for the treatment of unresponsive tumors.

Phase I trial of methotrexate-albumin in a weekly intravenous bolus regimen in cancer patients. Phase I Study Group of the Association for Medical Oncology of the German Cancer Society.

Methotrexate-albumin conjugate (MTX-HSA) is a novel human albumin-based prodrug conjugate of methotrexate (MTX). A low MTX loading rate provided optimal tumor targeting and therapeutic efficacy during preclinical testing. The objectives of this first Phase I study of MTX-HSA were to determine dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) in a weekly regimen. Seventeen cancer patients who were no longer amenable to standard treatment were enrolled and were evaluable for DLT. Up to eight injections were performed in weekly intervals. Dose escalation was as follows: 20, 40, 50, and then 60 mg/m2 MTX-HSA (based on the amount of MTX bound to albumin). Additional MTX-HSA courses were feasible in case of tumor response. DLT (mainly stomatitis, Common Toxicity Criteria grade 3) occurred, beginning at the 50 mg/m2 dose level after repeated administrations; in one case, thrombocytopenia was dose-limiting. Two events of DLT occurred at the 60 mg/m2 dose level within the first two administrations. Mild anemia, transaminitis, and one case of skin toxicity were found. No significant leukopenia, nausea, renal toxicity, or other toxicities were observed. MTX-HSA was well tolerated. Drug accumulation occurred on the weekly schedule. The half-life of the drug was estimated to be up to 3 weeks. Tumor responses were seen in three patients: (a) a partial response was seen in one patient with renal cell carcinoma (response duration, 30 months, ongoing); (b) a minor response was seen in one patient with pleural mesothelioma (response duration, 31 months, ongoing); and (c) a minor response was seen in one patient with renal cell carcinoma (response duration, 14 months until progression). Poststudy treatment was administered at 2-4-week intervals. No signs of toxicity or drug accumulation were seen. Altered pharmacological properties of MTX-HSA such as plasma half-life, tumor targeting, or intracellular metabolism might have contributed to these responses. The MTD for weekly administration was 4 x 50 mg/m2 MTX-HSA during short-term treatment. A regimen with MTX-HSA injections of 50 mg/m2 every 2 weeks was recommended for a further clinical Phase I study.

Clonogenic assay with established human tumour xenografts: correlation of in vitro to in vivo activity as a basis for anticancer drug discovery.

Pluripotent cells can be grown in clonogenic assays. The tumour stem-cell fraction, which accounts for <0.4% of the total cells, and which is considered the most relevant cell type in the development of metastases and recurrences, is able to divide and to form colonies in a semisolid matrix (agar or methylcellulose). Major applications of the tumour clonogenic assay (TCA) are chemosensitivity testing of tumours and xenografts, and for assessments within drug discovery programmes. Of critical relevance for the usefulness of the TCA is whether it can predict sensitivity or resistance towards clinically used agents. When we compared the response of human tumours established as xenografts in nude mice in the TCA in vitro to that of the clinical response, 62% of the comparisons for drug sensitivity, and 92% of the comparisons for drug resistance were correct. The same percentage of true/false observations was found when tumours were tested after serial passage in nude mice in the TCA in vitro and their response compared to in vivo activity in corresponding xenografts (60% and 90%, respectively). The highest correct predictive values were, however, found when the clinical response of tumours was compared to their explants established in the nude mouse and treated in vivo. Of 80 comparisons performed, we observed a correct prediction for tumour resistance in 97% and for tumour sensitivity in 90%. In our opinion, the TCA with established human tumour xenografts has an important role in current drug discovery strategies. We therefore included the TCA as secondary assay in our approach to anticancer drug discovery and found that a number of novel agents were active; these are now in advanced preclinical development or clinical trials. Thus, the tumour clonogenic assay has proven predictive value in the chemosensitivity testing of standard and experimental anticancer drugs.

Cycloplatam: a novel platinum compound exhibiting a different spectrum of anti-tumour activity to cisplatin.

Cycloplatam is a novel platinum compound which has shown anti-tumour activity in murine tumour models. In this study, cycloplatam was found to have anti-tumour activity in vitro and in vivo in human tumour models. In 15 cell lines (mainly ovarian), cycloplatam showed similar cytotoxicity as cisplatin, using the sulphorhodamine B assay. Determination of the resistance factor (IC50 of cisplatin-resistant divided by IC50 of parental cell line) clearly showed lower values for cycloplatam than for cisplatin. In the parental ovarian cell line CH1 and the cisplatin-resistant CH1 cisR model, we observed no cross-resistance of cycloplatam and cisplatin. The in vitro anti-tumour activity was confirmed in human tumour xenografts using the clonogenic assay. Mean IC70 values of cycloplatam were 0.54 microgram/ml (1.25 microM) and of cisplatin 0.42 microgram/ml (1.4 microM), respectively. In the murine subcutaneously implanted ADJ/PC6 plasmacytoma in vivo cycloplatam showed less activity than cisplatin, with a 2-fold smaller therapeutic index than cisplatin. In ovarian cancer xenografts cycloplatam was less active than cisplatin. However, anti-tumour activity of cycloplatam in lung cancer xenografts was quite different from cisplatin. In LXFS 538, a model moderately sensitive to cisplatin, a partial remission was observed, but in LXFL 529, a cisplatin-sensitive model, cycloplatam was inactive, cycloplatam thus demonstrating a different spectrum of anti-tumour activity. Based on these results, further preclinical investigations with other tumours, such as cisplatin-sensitive and -resistant gastric cancer models, are warranted with cycloplatam.

Superior in vivo experimental antitumour activity of vinflunine, relative to vinorelbine, in a panel of human tumour xenografts.

The antitumour activity of vinflunine, 20',20'-dichloro-3',4'-dihydrovinorelbine, a fluorinated Vinca alkaloid obtained by reaction in superacid media, was evaluated in comparison with vinorelbine against a series of subcutaneously-implanted human tumour xenografts. The tumours studied were established from bladder (BXF1299), pancreas (PAXF546), kidney (RXF944LX), colon (DLD-1, HT-29, TC37), central nervous system (SF-295), small cell lung (NCI-H69) and prostate (PC-3). Vinflunine or vinorelbine was administered as four weekly intraperitoneal treatments, within dose ranges of 5-80 or 0.63-10 mg/kg/injection, respectively. The overall antitumour activity of vinflunine was superior to that of vinorelbine. Vinflunine showed high activity against RXF944LX and NCI-H69 xenografts and moderate activity against PAXF546, PC-3 and TC37 tumours, achieving an overall response of 64%. This contrasts with a 27% response with vinorelbine, which proved only moderately active against RXF944LX and TC37 xenografts. These results confirm and extend our previous report of the broad spectrum of in vivo antitumour activity of vinflunine and reinforce its potential as a valuable addition to current chemotherapeutic agents.

The antitumour activity of the prodrug N-L-leucyl-doxorubicin and its parent compound doxorubicin in human tumour xenografts.

The antitumour activity of the investigational agent N-L-leucyl-doxorubicin (Leu-DOX) was compared with that of doxorubicin (DOX) in human tumour xenografts growing subcutaneously in athymic nude mice. Leu-DOX was developed as a prodrug of DOX, and may be converted into the clinically active parent compound by hydrolytic enzymes present in or on tumour cells. It has been suggested that a better therapeutic index with a reduced cardiac toxicity and higher efficacy might be obtained. Both compounds were administered intravenously weekly for 2 weeks, each at maximum tolerated doses of 8 mg/kg and 28 mg/kg for DOX and Leu-DOX, respectively. The panel of xenografts represented three different tumour types. Leu-DOX showed antitumour activity, defined as tumour growth inhibition > 50% and specific growth delay > 1.0, in 10 of the 16 tumours, including two of five breast, five of seven small cell and three of four non-small cell lung carcinomas. In comparison, DOX was active in one breast, four small cell lung and two lung adenocarcinoma xenografts. In all the DOX sensitive lung tumours, Leu-DOX showed higher efficacy than the parent compound. Based on the results of the present study, and since phase I clinical trials with Leu-DOX have already been performed, phase II clinical evaluation of Leu-DOX in patients with breast and lung cancer is recommended.

The antitumour activity of alkylating agents is not correlated with the levels of glutathione, glutathione transferase and O6-alkylguanine-DNA-alkyltransferase of human tumour xenografts. EORTC SPG and PAMM Groups.

Twenty-three human xenografts, including five colon, five gastric, nine lung (three small cell lung cancer) and four breast carcinomas, were investigated for their sensitivity to nitrosoureas, dacarbazine (DTIC), cyclophosphamide (CTX) and cisplatin (DDP). In 12 cases, at least one of the drugs produced complete or partial remission, in 2, a minor regression was observed and in the other 9, treatment was ineffective. The level of sensitivity to each drug, using a score from 1 to 5, was correlated to three biochemical parameters reported to be involved in resistance to alkylating agents: glutathione (GSH), glutathione transferase (GST) and O6-alkylguanine-DNA-alkyltransferase (AGT). A wide variability was found in these parameters in the xenografts investigated. No correlation was found between any of the three parameters and sensitivity to the drugs used or between sensitivity to one drug and to any of the other drugs tested. These results illustrate the complexity of the question of resistance to alkylating agents and indicate that, at least in xenografts, the biochemical parameters examined are not predictive of response to alkylating agents.

Correlation of drug response in patients and in the clonogenic assay with solid human tumour xenografts.

The potential of evaluating the preclinical response of solid tumours was studied in human tumour xenografts in the clonogenic assay. Tumour specimens surgically removed from cancer patients were implanted subcutaneously into thymus-aplastic nude mice. Chemosensitivity of the mouse-grown tumours was tested with a modification of the double-layer soft-agar clonogenic assay. Tumour cells were tested against thirteen established cytostatic drugs at two dosages by continuous exposure. 62 retrospective in vivo/in vitro correlations were done. The clonogenic assay predicted correctly for clinical response in 16/27 (59%) and for resistance in 32/35 (91%). These correlation rates were similar to reported data for fresh solid human tumour specimens. The results support the clinical relevance of the nude mouse/clonogenic assay model.

Comparative antitumour activity of vinblastine-isoleucinate and related vinca alkaloids in human tumour xenografts.

The antitumour activity of the investigational agent vinblastine-isoleucinate (V-LEU) was compared with vintriptol, another investigational agent of the same series of vinblastine-23-oyl amino acid derivatives, and vinblastine, their clinically active parent compound, in a panel of nine human tumour xenografts growing subcutaneously in nude mice. Compounds were administered intravenously at equitoxic doses twice weekly. As assessed by optimal tumour growth inhibition and tumour growth delay, vinblastine, V-LEU and vintriptol exhibited antitumour activity in 8/9, 7/9 and 4/7 human tumour xenografts, respectively. When growth curves and numbers of complete remissions were compared, V-LEU was the most active agent in two malignant melanoma lines (THXO and LOX p28) and two small cell lung carcinoma lines tested (LXFS 538 and WX 322), whereas vinblastine was more active against the two colorectal carcinomas (CXF 243 and CXF 280). Notably, the non small cell lung carcinoma (NSCLC) line AHXOL was resistant to the three agents. The results of this study suggest that V-LEU was as active as vinblastine in most tumour lines, exhibiting superior antitumour activity in malignant melanoma, SCLC and breast cancer lines. The decision to bring this compound into clinical trial shall await further confirmation of these preclinical results and the evaluation of its toxicity profile in relation to other vinca alkaloids.

Cytotoxicity of TGF alpha-PE40 and correlation to expression of epidermal growth factor receptor.

TGF alpha-PE40 is a chimeric protein composed of transforming growth factor alpha (TGF alpha) linked to a modified Pseudomonas exotoxin (PE40). We tested the in vitro cytotoxicity of TGF alpha-PE40 on 23 different solid human tumour xenografts established in nude mice and human bone marrow cells from healthy donors, utilising a modified clonogenic assay. In order to distinguish non-specific toxicity from the targeted effects of TGF alpha-PE40, epidermal growth factor receptor (EGFR) expression of the tumours studied was assessed by Northern blot, slot blot and immunohistochemistry. TGF alpha-PE40 demonstrated differential cytotoxicity on human tumour xenografts in the clonogenic assay. No toxicity on human bone marrow cells was observed. In vitro activity of TGF alpha-PE40 showed a significant correlation with the expression of EGF receptors as determined by immunohistochemistry and slot blot. Further studies will be performed in order to determine the in vivo activity of this compound in tumour-bearing nude mice.