Interference of mouse mammary tumor virus replication by PTT.119 [p-fluoro-L-phe-m-bis-(2-chloroethyl)amino-L-phe-met ethoxy HCl]--an antitumor agent

PTT.119 [p-fluoro-L-Phe-m-bis-(2-chloroethyl)-amino-L-Phe-Met ethoxy HCl] is a new bifunctional alkylating compound that possesses both cytolytic and antiviral activities. Continuous treatment of mouse mammary tumor cells with 15 microM PTT.119 reduced production of the B-type retrovirus murine mammary tumor virus (MuMTV). MuMTV levels in PTT.119-treated cultures were reduced by 31% in the first 24 hours; an additional 24 hours of treatment resulted in a further reduction of 70%. These reductions were significantly greater than could be accounted for by the effects of PTT.119 tumor cell metabolism and viability. Under identical treatment conditions, equimolar concentrations of L-phenylalanine mustard reduced MuMTV production by only 3%. PTT.119 inhibition of MuMTV replication was also apparent when mammary tumor cells were exposed for periods as short as 0.5-4 hours; persistent decreases in virus production were detected even 7 days following tripeptide treatment. Analyses of the polypeptide composition of MuMTV by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that virions from these PTT.119-treated mammary tumor cultures contained significant reductions in the relative level of the nonglycosylated 24,000-dalton (p24) viral polypeptide associated with the nucleoprotein core. Decreases in p24 were observed in MuMTV produced in the presence of the tripeptide and 1-7 days after removal of PTT.119. Examination of the steady-state levels and rates of intracellular MuMTV protein synthesis suggested that PTT.119 interferes with late steps in MuMTV processing and maturation.     

Evaluation of 3-(p-fluorophenyl)-l-alanyl-3-[m-bis(2-chloroethyl)aminophenyl]-l-alanyl-l-methionine ethyl ester HCl (PTT.119) against xenografts of human rhabdomyosarcoma

Summary PTT.119 [p-F-phe-m-bis(2-chloroethyl)amino-l-phe-met ethoxy HCl], a synthetic tripeptide mustard, was evaluated for therapeutic efficacy against a spectrum of childhood rhabdomyosarcomas (RMS) maintained as xenografts in immune-deprived mice. These xenografts were established from previously untreated tumors, and sublines were selected in mice for resistance to l-phenylalanine mustard (L-PAM). PTT.119 caused regression of four of six RMS lines established from untreated tumors, and demonstrated activity similar to that of L-PAM in this model. Against tumors Rh18/L-PAM and Rh28/L-PAM, selected in situ for L-PAM resistance, PTT.119 had no significant activity. Rh28/L-PAM was cross-resistant also to oxazophosphorine mustards (ifosfamide, cyclophosphamide), and both tumors were cross-resistant to adriamycin and vincristine. PTT.119 caused hematologic toxicity similar to thet of L-PAM, characterized by a marked decrease in white blood cells and thrombocytopenia.Abbreviations PTT.119 3-(p-fluorophenyl)-L-alanyl-3-[m-bis-(2-chloroethyl)-aminophenyl]-L-alanyl-L-methionine ethyl ester HCl - L-PAM L-phenylalanine mustard - VCR vincristine - ADR adriamycin - CTX cyclophosphamide - Ifos ifosfamide Supported by PHS grant CA23099 from the National Cancer Institute and by American Lebanese Syrian Associated Charities Offprint requests to: Peter J. Houghton, Laboratories for Developmental Therapeutics, Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Box 381, Memphis, TN 38101, USA     

Cytokinetic changes in two cases of plasma cell leukemia treated with a multipeptide derivative of m-[di(2-chloroethyl)amino]-l-phenylalanine (Peptichemio)

In two patients with plasma cell leukemia (PCL) the cytokinetics of blood and bone marrow plasmocytomatous cells was examined shortly after Peptichemio (PTC 40 mg/alternate days for 6–7 doses) therapy by combined DNA cytofluorometry and in vitro tritiated thymidine (³H-TdR) cytoautoradiography. In both cases a rapid fall in circulating plasma cells was induced by the treatment and in one of them the percentage of bone marrow plasma cells was also diminished. After treatment the values of labeling index (LI) and growth fraction (GF) were increased both in blood and bone marrow plasma cells and proliferating bone marrow plasma cells were mostly in early S phase. Their mean grain count (MGC) did not change. Increased population of U-like cells. i.e. damaged cells, was found in the bone marrow. By examining the possible explanations of the findings, the authors relate them to a cellular recruitment following tumor mass reduction. DNA content only was also determined in the blood during the treatment and an increased percentage of 2n–4n plasma cells was found. The significance of this last finding is not known at the present.     

Antitumor activity of homo-aza-steroidal esters of [p-[bis(2-chloroethyl)amino]phenyl]acetic acid and [p-[bis(2-chloroethyl)amino]phenyl]butyric acid

Summary Three new modified steroidal alkylating agents, 3-hydroxy-13-amino-13,17-seco-5-androstan-17-oic-13,17-lactam-p-bis(2-chloroethyl)aminophenylacetate, 3-hydroxy-13-amino-13,17-seco-5-androstan-17-oic-13,17-lactam-p-bis-(2-chloroethyl)aminophenylbutyrate, and 17-hydroxy-3-aza-A-homo-4-androsten-4-one-p-N,N-bis(2-chloroethyl)-aminophenylacetate are active in treatment of L1210 and P388 leukemias. A stereoisomer of the first compound, 3-hydroxy-13-amino-13,17-seco-5-androstan-17-oic-13, 17-lactam-p-bis(2-chloroethyl)aminophenylacetate, was tested in L1210 leukemia. This stereoisomer, in which the alkylating agent is linked to the modified steroid in the axial position, is active only at much higher doses in L1210 leukemia. The results of testing these compounds and previous results from similar compounds allow certain conclusions to be drawn regarding structure-activity relationships. The presence of the lactam moiety is the major structural feature that confers activity in the murine leukemias. The steric arrangement of the alkylating moiety at position 3 and the hydrogen atom at position 5 influence toxicity and antileukemic activity.     

Toxicological study on a new nitrosourea derivative, 1-(2-chloroethyl)-3-isobutyl-3-(beta-maltosyl)-1-nitrosourea

TA-077, 1-(2-chloroethyl)-3-isobutyl-3-(beta-maltosyl)-1-nitrosourea, is a new water-soluble nitrosourea derivative which is disubstituted at the N-3 position of the structure, and is activated by a unique mechanism whereby organic isocyanates can never be produced. In order to clarify the biological functions of the organic isocyanates which common nitrosourea derivatives generate, TA-077 was tested in BDF1 mice for lethality, weight loss and hematological toxicity. TA-077 showed qualitatively and quantitatively similar toxicity to other nitrosourea derivatives which generate organic isocyanates, such as 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosour ea (ACNU), methyl 6-[3-(2-chloroethyl)-3-nitrosoureido ]-6-deoxy-alpha-D-glucopyranoside (MCNU), and 1-(2-chloroethyl)-3-(beta-D-glucopyranosyl)-1-nitrosourea (GANU), indicating that the organic isocyanates do not play an important role in the biological activity of the nitrosourea derivatives. Furthermore, the toxicity of TA-077 did not increase significantly (except for weight loss) when the drug was administered daily for five days, and TA-077 given according to this schedule showed far more effective antitumor activity than when given as a single treatment. These results suggested that TA-077 is an analog suitable for consecutive administration in cancer treatment.     

Interactions of 1-3 bis-(2-chloroethyl)-nitrosourea (NSC 409962) (BCNU) with clone DS19 of murine erythroleukemia cells (MELC)

Murine erythroleukemia cells (MELC) were incubated with BCNU in concentrations from 1 to 160 mu molar. The cytotoxicity was linear with concentration for 10 mu mol ar(GID15) to 40 mu molar (GID95). Incubation of MELC with BCNU induced striking morphological changes in the cell which include multinucleation and cell enlargement which appear to be linear with BCNU concentration in the range from 10 to 40 mu molar. Polykaryon formation was induced in 85% of MELC when exposed to 40 mu molar BCNU and was accompanied by marked increases in chromatin condensation and cellular fragility. The mean generation time for MELC(MGT) was increased from 14 h in control cultures to greater than 48 h in 40 mu molar BCNU. Hemoglobin synthesis in the presence of Me2SO was inhibited by 50% by exposure to 10 mu molar BCNU and to zero by exposure to 40 mu mo lar BCNU. These effects can be observed at 96 h after as little as 3 h of exposure to BCNU. The effects reported above are not reversible after washing cultures and reseeding them in fresh medium lacking BCNU. This is in contrast to cisplatin, another divalent alkylating agent in which the half-life of the crosslinks appears to be much shorter and in which washing and resuspension in fresh unsupplemented medium permits recovery of ability to replicate and to synthesize hemoglobin in the presence of Me2SO. The data supports the idea that the presence of single stranded breaks in DNA are required for induction of hemoglobin synthesis.     

Antitumor activity of 2'-deoxy-2'-methylidenecytidine, a new 2'-deoxycytidine derivative

The antitumor activity of 2'-deoxy-2'-methylidenecytidine (DMDC), an inhibitor of DNA synthesis, was examined and compared with that of 1-beta-D-arabinofuranosylcytosine (ara-C) against various murine tumors and human tumor xenografts. Against P388 murine leukemia, repeated treatments of DMDC were more effective than its single administration. Interestingly, DMDC was effective against colon 26 murine carcinoma, M5076 murine reticulum cell sarcoma, LX-1 human lung cancer xenograft, and SK-Mel-28 human melanoma xenograft, which are less sensitive or refractory to ara-C, while DMDC was not more potent against murine leukemias P388 and L1210 than ara-C. The in vitro cytotoxic effects of DMDC and ara-C against L1210 leukemia cells were prevented dose dependently by deoxycytidine, suggesting that DMDC, like ara-C, may require phosphorylation by deoxycytidine kinase for antitumor activity. DMDC was effective against human and murine experimental tumor models, especially nonleukemic tumors refractory to ara-C, suggesting that DMDC will be a promising agent for the treatment of cancer.     

Radiotherapy of metastatic bone tumor with a synthetic calcitonin derivative (Elcatonin) with irradiation

Twenty cases of metastatic bone tumors were treated with Elcatonin together with irradiation, achieving pain relief in 47.3% of cases, improved bone scintigrams in 16.7% of cases and radiographic improvement of invaded bone in 7.7% of cases. It has reported that calcitonin is effective for pain relief in 50% of cases but for the radiographic improvement of bone lesions in only 0-10%. The life quality of patients with bone metastases is controversial since treatment techniques for advanced oncological patients has progressed remarkably. The pain relief obtained with calcitonin contributes significantly to improving the quality of life for such patients when administered in combination with conventional treatment modes.     

CI-973, a new platinum derivative with potential antileukemic activity.

We examined the effects of CI-973 (supplied by Parke-Davis) on several human leukemia cell lines and a Chinese hamster ovary (CHO) line and their drug-resistant counterparts. The cell lines used were HL-60, HL-60/mAMSA, HL-60/DOX, KBM3, KBM3/mAMSA 6, KBM3/mAMSA 6(85), CHO, and CHO/AC-7. DOX, mAMSA, and AC-7 indicate resistance to doxorubicin, amsacrine, or 1-beta-D-arabinofuranosylcytosine, respectively. Cells were incubated with CI-973, and the effect was evaluated by two methods: growth inhibition assay and inhibition of colony formation. All cell lines examined were inhibited by CI-973; two of three amsacrine-resistant lines and the one cytarabine-resistant line demonstrated collateral sensitivity. At equivalent dosages, a 4-day exposure provided much greater cell kill than a 1-h exposure. Clonogenic assay showed exponential killing over 3 log units. Maximum CI-973 levels required to kill 50% of cells were 10-fold lower than the peak plasma levels achieved in a phase I solid tumor study. A continuous infusion phase I study in acute leukemia has been initiated.     

Antitumor activity of BOF-A2, a new 5-fluorouracil derivative

A compound containing both CNDP (3-cyano-2,6-dihydroxypyridine), an inhibitor of 5-fluorouracil (5-FU) degradation, and EM-FU (1-ethoxymethyl-5-fluorouracil), a masked form of 5-FU, was synthesized and named BOF-A2 (3-[3-(6-benzoyloxy-3-cyano-2-pyridyloxycarbonyl)benzoyl]-1-ethoxy methyl-5- fluorouracil). The antitumor activity of BOF-A2 was investigated in sarcoma-180-bearing mice and Yoshida sarcoma-bearing rats. The ED50 (the dose for 50% inhibition) values of BOF-A2 were 25 mg/kg against sarcoma-180 and 15 mg/kg against Yoshida sarcoma. In vitro studies showed that BOF-A2 was rapidly degraded to EM-FU and CNDP in homogenates of the liver and small intestine of mice and rats, and in sera of mice, rats and human, and the conversion of EM-FU to 5-FU occurred only in the microsomal fraction of rat liver in the presence of NADPH. After oral administration of BOF-A2 at 15 mg/kg to Yoshida sarcoma-bearing rats, BOF-A2 was hydrolyzed to EM-FU, CNDP and 5-FU, and their maximum concentrations in the blood were 2000 ng/ml, 300 ng/ml and 40 ng/ml, respectively. Moreover when BOF-A2 was given at the same dose to tumor-bearing mice and rats, the 5-FU levels in the tumor tissue increased much more than those in the blood and persisted for more than 8 h, whereas those in the blood decreased more rapidly. This accumulation and maintenance of a high level of 5-FU in the tumor tissue are concluded to be related to the high antitumor activity of BOF-A2.     

6-[Bis(2-chloroethyl)amino]-6-deoxygalactopyranose hydrochloride (C6-galactose mustard), a new alkylating agent with reduced bone marrow toxicity

We have previously reported that chloroethyl nitrosourea and nitrogen mustard bone marrow toxicity can be selectively reduced by placement of the cytotoxic group on specific positions of a glucose molecule. We have now synthesized and evaluated a new drug in which the mustard cytotoxic group is attached to the carbon-6 position of galactose (C6-GLM). C6-GLM, administered i.p. as a single 10% lethal dose of 15.5 mg/kg, produced a 121% increase in life span (ILS) in mice bearing the ascitic P388 leukemia, compared to a 60% ILS with a 10% lethal dose of nitrogen mustard (P less than 0.01). A single p.o. dose of C6-GLM, 16 mg/kg, produced an ILS of 58%. Against i.p.-implanted B-16 melanoma, i.p. C6-GLM produced a 56% ILS compared to 30% with an equitoxic dose of nitrogen mustard (P less than 0.01). The activity of the two drugs for Ehrlich ascites was comparable, with 60% survivors with the galactose mustard. A single 10% lethal dose of C6-GLM reduced the white blood cells to 74% of control; circulating granulocytes remained at 91% of initial values. With nitrogen mustard, the nadir white blood cell count was 57% of control with an absolute granulocyte count of 70% of initial values (P less than 0.01). The toxicity of melphalan was considerably greater, with a lower and more protracted while blood cell nadir and an absolute neutrophil count nadir of 49% of control. These findings paralleled the relative decrements in bone marrow DNA synthesis produced by the three drugs. Measurement of human bone marrow granulocyte-macrophage colony-forming units, following in vitro exposure to graded concentrations of the three mustards, confirmed the bone marrow sparing properties of C6-GLM. At the highest concentration, 1 X 10(-2) mM, the latter drug produced only a 33% reduction in colonies compared to a 75% reduction with nitrogen mustard and a virtual elimination of activity of colony-forming units with melphalan. The demonstration of antitumor activity, at least equivalent to nitrogen mustard, without the necessity of significant bone marrow toxicity supports the development of C6-GLM for clinical trials in humans.     

Alpha-bromoacryloyl derivative of distamycin A (PNU 151807): a new non-covalent minor groove DNA binder with antineoplastic activity.

PNU 151807 is a new synthetic alpha-bromoacryloyl derivative of distamycin A. In the present study we investigated the DNA interaction and the mechanism of action of this compound in parallel with the distamycin alkylating derivative, tallimustine. PNU 151807 possesses a good cytotoxic activity in in vitro growing cancer cells, even superior to that found for tallimustine. By footprinting experiments we found that PNU 151807 and tallimustine interact non-covalently with the same AT-rich DNA regions. However, differently from tallimustine, PNU 151807 failed to produce any DNA alkylation as assessed by Taq stop assay and N3 or N7-adenine alkylation assay in different DNA sequences. PNU 151807, like tallimustine, is able to induce an activation of p53, and consequently of p21 and BAX in a human ovarian cancer cell line (A2780) expressing wild-type p53. However, disruption of p53 function by HPV16-E6 does not significantly modify the cytotoxic activity of the compound. Flow cytometric analysis of cells treated with equitoxic concentrations of PNU 151807 and tallimustine showed a similar induction of accumulation of cells in the G2 phase of the cell cycle but with a different time course. When tested against recombinant proteins, only the compound PNU 151807 (and not tallimustine or distamycin A) is able to abolish the in vitro kinase activity of CDK2-cyclin A, CDK2-cyclin E and cdc2-cyclin B complexes. The results obtained showed that PNU 151807 seems to have a mechanism of action completely different from that of its parent compound tallimustine, possibly involving the inhibition of cyclin-dependent kinases activity, and clearly indicate PNU 151807 as a new non-covalent minor groove binder with cytotoxic activity against cancer cells.     

1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylamino)carbonylhydrazine (101M): a novel sulfonylhydrazine prodrug with broad-spectrum antineoplastic activity

Our laboratory has synthesized and evaluated the anticancer activity of a number of sulfonylhydrazine DNA modifying agents. As a class, these compounds possess broad spectrum antitumor activity, demonstrating significant activity against a variety of experimental murine tumors, including the P388 and L1210 leukemias, B16 melanoma, M109 lung carcinoma, and M5076 reticulum cell sarcoma, as well as against the human LX-1 lung carcinoma xenograft. The current report describes the activity of a more recently synthesized member of this class, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylamino)carbonylhydrazine (101M). 101M was active in mice against the i.p. implanted L1210 leukemia over a wide range of doses and produced long-term survivors when administered as a single i.p. bolus of 10, 20, 40, 60, or 80 mg/kg, demonstrating a wider margin of safety than the nitrosourea, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Curative therapy was achieved with doses of 101M that did not produce depression of the bone marrow. 101M was also highly effective against the L1210 leukemia when administered by the oral route. The ability of 101M to penetrate the blood-brain barrier and eradicate leukemia cells in the brain was remarkable (>6 log kill). This agent was also curative against L1210 variants resistant to cyclophosphamide, BCNU, or melphalan. Mice implanted with the murine C26 colon carcinoma were also cured by two injections of 10 or 20 mg/kg of 101M. Administration of 101M by two different well-tolerated regimens caused complete regression of established human glioblastoma U251 xenografts in 100% of treated mice, and significant responses were also obtained with 101M against advanced murine M109 lung carcinomas in mice. The broad spectrum of anticancer activity of the sulfonylhydrazine prodrug 101M coupled with the wide range of therapeutic safety exhibited by this agent, makes 101M particularly attractive for further development and clinical evaluation.     

Clinical study of a synthetic calcitonin derivative (elcatonin) in patients with metastatic bone tumors

Forty-three cases of metastatic bone tumor were treated with Elcatonin. The agent was injected intramuscularly to each patient at a dose of 40 units twice daily. Twelve patients (28%) experienced pain relief within 4 days after treatment and after 4 weeks, twenty-eight patients (65%) had palliation of pain. In patients with hypercalcemia (4 cases), a decrease of serum Ca was observed one week after administration of Elcatonin. Improved bone scintigram was observed in 37.5% of cases, and radiographic improvement in 20% of cases. These data indicate that Elcatonin is effective for achieving pain relief and in improving the state of invaded bones when administered in combination with conventional treatment modalities for patients with metastatic bone tumors.     

In vitro and in vivo activity of 1-aryl-3-(2-chloroethyl) urea derivatives as new antineoplastic agents

A few 1-aryl 3-(2-chloroethyl)ureas (CEU) were synthesized and screened in vitro for their cytotoxicity. Some of these derivatives were assayed for their mutagenicity, their in vivo toxicity and their antineoplastic activity. Methyl 4-(p-(3-(2-chloroethyl) ureido) phenyl) butyrate, 4-methyl and 4-tertbutyl (3-(2-chloroethyl) ureido) phenyl) butyrate, 4-methyl and 4-tert-butyl (3-(2-chloroethyl) ureido) benzene had an ID50 of 28, 20 and 4 microM respectively when tested on LoVo cells, while chlorambucil (CBL) and CCNU had an ID50 of 21 and 45 microM. These 3 chloroethyl urea derivatives were not toxic when injected i.p. at doses up to 220 mg/kg, whereas chlorambucil was already toxic at 18.5 mg/kg. The survival time of BDF1 mice bearing L1210 leukemia tumors was significantly enhanced by intraperitoneal injections of CBL and CEU. The most cytotoxic derivative (tert-butyl derivative) gave the best antineoplastic activity with a median survival time 1.77 times that of the control at 10 mg/kg/day and was not toxic, whereas CBL at this concentration enhanced survival time by a factor of 1.6 and presented important side effects. The 4-tert-butyl (3-(2-chloroethyl) ureido) benzene and the methyl 4-(p-(3-(2-chloroethyl) ureido) phenyl) butyrate showed no mutagenicity when assayed on TA-97, TA-98, TA-100 and TA-102, four strains of S. thyphimurium, while CBL had a weak effect on TA-102 and CCNU was highly mutagenic on TA-100 and TA-102.     

Disposition of the prodrug 4-(bis (2-chloroethyl) amino) benzoyl-L-glutamic acid and its active parent drug in mice

A novel therapy for improving selectivity in cancer chemotherapy aims to modify distribution of a cytotoxic drug by generating it selectively at tumour sites. In this approach an antibody-enzyme conjugate is allowed to localise at the tumour sites before injecting a prodrug which is converted to an active drug specifically by the targeted enzyme in the conjugate. We present here pharmacokinetic studies on the prodrug 4-(bis (2-chloroethyl) amino) benzoyl-L-glutamic acid and its activated derivative, benzoic acid mustard. The glutamic acid is cleaved from the prodrug to form the active drug by carboxypeptidase G2 (CPG2), an enzyme from Pseudomonas sp., which is not found in mammalian cells. The prodrug and its parent active drug were rapidly distributed in plasma and tissues after administration of prodrug or active drug (41 mumol kg-1 intraperitoneally) to mice bearing human choriocarcinoma xenografts. Prodrug and active drug both followed a two-compartment kinetic model. Prodrug was eliminated more rapidly (t1/2 alpha = 0.12 h, t1/2 beta = 0.70 h) than active drug (t1/2 alpha = 0.37 h, t1/2 beta = 1.61 h). Conversion of the prodrug to the activated parent drug was detected within 5 min of administration to mice which had previously received a F(ab')2-anti-human chorionic gonadotrophin antibody (W14A) conjugated to the enzyme, CPG2 (1,000 U kg-1). Tumour was the only tissue that activated all the prodrug reaching the site. It contained the highest concentration of targeted enzyme conjugate capable of catalysing the reaction of prodrug to drug. Plasma and other tissues were also capable of activating the prodrug but active drug production was limited by the amount of enzyme present. The active drug measured in plasma and tissues other than tumour was attributable to residual antibody-enzyme conjugate at non-tumour sites. Low levels of conjugate in tissues and plasma militate against the advantage of tumour localised enzyme therefore necessitating removal of non-localised enzyme.