Experimental antitumor activity of BMY-28090, a new antitumor antibiotic

Summary BMY-28090 is a novel actinomycete fermentation derived antitumor agent. The cytotoxic effect of BMY-28090 was evaluated in two murine and eight human tumor cell lines in vitro. Following 72-hour exposures, BMY-28090 was cytotoxic for all of these cell lines with IC50 values of < 0.02 to 3.25 g/ml. BMY-28090 was evaluated for in vivo antitumor activity in a variety of experimental murine tumor and human tumor xenograft models. Initial testing against the murine tumor models was performed using BMY-28090 as the water insoluble free base whereas subsequent antitumor tests were performed using water soluble lactate or succinate salts. BMY-28090 administered ip demonstrated good, reproducible antitumor activity against ip implanted P388 leukemia, L1210 leukemia, B16 melanoma and M5076 sarcoma. The water soluble preparations of BMY-28090 were active iv against sc implanted B16 melanoma and M5076 sarcoma as well as subrenal capsule (src) M5076 sarcoma; activity against src implanted B16 was marginal. BMY-28090 lactate was also evaluated for activity against src implanted MX-1 human mammary tumor xenografts in nude mice and the HCT116 human colon tumor xenografts in immune-suppressed BDF mice. At maximum tolerated doses administered ip, BMY-28090 was active against the MX-1 xenograft in two of three tests, causing > 90% inhibition of tumor growth. BMY-28090 administered iv at maximally tolerated doses had marginal activity against the HCT116 xenografts, producing 61% and 68% inhibition of tumor growth in two tests. The results of these studies demonstrated that BMY-28090 has a broad spectrum of in vitro cytotoxicity against both murine and human tumor cell lines. Moreover, BMY-28090 had in vivo antitumor activity against murine leukemias, murine solid tumors and human tumor xenografts, including tumors located distal to the site of drug administration.A preliminary report of some of this research was presented at the Annual Meeting of the American Association for Cancer Research, Los Angeles, CA, May 1986 (1090).

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Experimental antitumor activity of new azathioprine analogues

Two newly synthesized azathioprine (AZA) analogues, 6-(1,2-dimethyl-4-nitro-5-imidazolyl)thiopurine (Met-AZA) and 6-(2-methyl-5-nitro-4-imidazolyl)thiopurine (IZO-AZA), were investigated against KB human tumor cells. In 5 transplantable murine tumor models, including sc Sa180, sc Ca755, ip LL and ip leukemias; L1210 and P388 both drugs were found to be antitumor active in all the experiments carried out regardless of dosing regimen or the route of administration. Similar good activity was shown in the KB, ip Sa180, and Ca755 systems and partly against LL as compared to AZA. However, Met-AZA against ip P388 demonstrated therapeutic advantage following qd 1-9 daily dosing, 0.33 log10 tumor cell kill; therapeutic index (TI = ILSmax/ILS 25) = 2, and ILS = 69% in comparison to AZA and IZO-AZA, TI = 1.3, 1.2, and ILS = 31%, 40%, respectively. Met-AZA is comparable to AZA, while seeming to display greater antileukemic activity than AZA.     

Antitumor activity of spergualin, a novel antitumor antibiotic

Spergualin (SGL), a novel antitumor antibiotic, exhibited strong antitumor activity against transplantable leukemias in mice: L1210, L1210(IMC), P388, P815, C1498, EL-4 and RL male 1. It also exhibited antitumor activity against M5076 fibrosarcoma, AH66 and AH66F rat hepatomas, but not against Meth-A fibrosarcoma, B16 melanoma, Lewis lung carcinoma (LL) and C26 colon adenocarcinoma. The antitumor activity of SGL was administration-schedule dependent. The strongest activity against L1210 was obtained by ip continuous infusion for 7 days or daily ip administration for 9 days. Single ip injection of SGL at 100 mg/kg to mice caused convulsion and death within 15 minutes after injection. Such acute toxicity was not observed by continuous infusion. SGL showed its strongest activity at subtoxic dose against sensitive tumors except for L1210(IMC). Mice implanted ip with L1210(IMC) were cured by treatment with SGL at 3.13 mg/kg/day for 9 days, but died from the tumor at 50 mg/kg/day X 9. The cured mice rejected a second inoculation of up to 10(6) tumor cells. The tumor cells isolated from mice after treatment with the high dose showed resistance to SGL in vivo. Mice implanted sc with L1210(IMC) were also cured by 9 daily ip administrations of SGL at 12.5 mg/kg/day, but solid tumor was observed at the implantation site until 3 days after the final injection of SGL in some cured mice. These results suggest that the therapeutic effect of SGL has a relatively high specificity for leukemias and that the immunological effect is involved in the antitumor activity.     

Dynemicins, new antibiotics with the 1,5-diyn-3-ene and anthraquinone subunit. II. Antitumor activity of dynemicin A and its triacetyl derivative.

Dynemicin A showed extremely potent in vitro cytotoxicity against a variety of murine and human tumor cells. In the experimental animal tumor models implanted ip with P388, L1210 leukemias and B16 melanoma cells, dynemicin A administered ip significantly prolonged life-span of tumor-bearing mice with the wide range of activity. This antibiotic administered iv was also active against iv implanted P388 and L1210 leukemias. In the macromolecule biosynthesis of B16 melanoma cells, dynemicin A inhibited DNA synthesis specifically. The triacetyl derivative exhibited similar in vitro and in vivo antitumor activities to those of the parent antibiotic.     

In vivo antitumor activity of sparsomycin and its analogues in eight murine tumor models

Summary Sparsomycin (Sm) is a known inhibitor of ribosomal protein synthesis with an attractive anticancer potential. Recently, several analogues of Sm which are more active than the parent drug were selected for further study on the basis of in vitro investigations. Six analogues as well as the parent drug were tested for their antitumor activity in eight in vivo murine tumor models: P388 and L1210 leukemias, RC renal cell carcinoma, B16 melanoma, C38 colon carcinoma, LL Lewis lung carcinoma, C22LR osteosarcoma and M5076 sarcoma. Sm itself appeared to have only borderline activity on L1210 leukemia. The analogues that were most active in vitro showed also the highest in vivo activity. The most sensitive tumors were RC, L1210 and P388. Minimal activity was found on B16 and no activity on C22LR, M5076, C38 and LL. The most active compounds are deshydroxy-Sm, ethyl-deshydroxy-Sm and n-pentyl-Sm. There was a considerable loss of activity when L1210 leukemia was implanted sc while the drugs were administered iv. Only one drug, ethyl-deshydroxy-Sm appeared to be active in this assay. No single most effective compound could be found in this study. The overall activity of Sm and its analogues is moderate. The three analogues which show high activity in three ascitic tumors will be further investigated using human tumor xenograft models.Part of this work was presented in the 5th EORTC/NCI symposium on New Drugs in Cancer Therapy, Amsterdam, 22–24 October 1986.     

Antitumor properties of 2(1H)-pyrimidinone riboside (zebularine) and its fluorinated analogues

2(1H)-Pyrimidinone riboside (zebularine, 1b) and its 5-fluoro (6b) and 2'-ara-fluoro (7b) analogues have been synthesized and evaluated in vivo as antitumor agents. Zebularine provides increase in life span (ILS) values of ca. 70% against intraperitoneal (ip) murine B16 melanoma and 50% against P388 leukemia. This compound is active when administered either ip or orally against ip or subcutaneously implanted L1210 leukemia, producing ILS values of about 100% at an optimum dose of 400 mg/kg. 1b is also active (60% ILS) against ara-C-resistant L1210. The analogous unsubstituted purine riboside nebularine (2) has modest activity against P388 leukemia (60% ILS). While 2'-ara-fluorozebularine (7b) is only marginally active (40% ILS) at high doses against L1210 leukemia, 5-fluoro analogue 6b is more active than zebularine and is ca. 100 times more potent. Although the activity of 6b is about the same as that of 1b against P388 leukemia, greater potency also is realized in this model. Zebularine is a strong inhibitor of cytidine deaminase, but in contrast to tetrahydrouridine, 1b is acid-stable. In an attempt to use this property to advantage in oral administration, 1b and ara-C have been orally coadministered to mice with ip L1210 leukemia. When zebularine is given in divided doses, up to a 2-fold increase in activity is realized, relative to treatment with the same dose of ara-C alone.     

In vivo antitumor activity of 6-benzyl-1,3-benzodioxole derivatives against the P388, L1210, B16, and M5076 murine models

A series of 6-benzyl-1,3-benzodioxoles have been synthesized and evaluated against the in vivo ip P388 murine lymphocytic leukemia. Selected activities against this system were tested against the additional in vivo systems L1210, B16, M5076, and MX1. The most active of the 6-benzyl-1,3-benzodioxoles tested were as effective as podophyllotoxin as experimental antitumor agents in vivo, but larger doses were required. Three of the P388-active series members were active against the in vitro astrocytoma assay, which detects compounds that interfere with or bind to tubulin.     

A new antitumor antibiotic, FR-900482. III. Antitumor activity in transplantable experimental tumors

FR-900482 (4-formyl-6,9-dihydroxy-14-oxa-1,11-diazatetracyclo[7.4.1.02,7, O10,12]tetradeca-2,4,6-triene-8-ylmethyl carbamate), a new antibiotic with antitumor activity was isolated from fermentation broth of Streptomyces sandaensis. Its antitumor activities were studied and compared with that of mitomycin C (MMC) in animals. FR-900482 in doses of 0.32 approximately 10 mg/kg (ip) prolonged the life of mice bearing ascitic P388, L1210, B16, MM46, Ehrlich or EL4 tumors and rats bearing ascitic AH130 or AMC60 tumors. FR-900482 in doses of 5.6 approximately 18 mg/kg (iv) inhibited human LX-1, MX-1, SC-6 and LC-6 tumors xenografted sc in nude mice. FR-900482 was more effective than or equally effective to MMC in all the tumors used. FR-900482 was ineffective against cyclophosphamide-resistant P388, but was effective against MMC- or vincristine-resistant P388. The results suggest that FR-900482 may have clinical potential.     

Potential CNS antitumor agents VI: aziridinylbenzoquinones III.

Thirty-one aziridinylbenzoquinones were compared against five murine tumor models in vivo. Two intracerebral (ependymoblastoma and L-1210 leukemia) and three intraperitoneal (P-388 and L-1210 leukemia and B16 melanoma) systems were utilized. Excellent activity was observed for many compounds. Multiple long-term survivors were produced in the ependymoblastoma, P-388, and intraperitoneal L-1210 systems. Diethyl 2,5-bis(1-aziridinyl)-3,6-dioxo-1,4-cyclohexadiene-1,4-dicarbamate demonstrated superior activity in all five test systems. This compound also was reproducibly active against two colon tumors, a mammary tumor, and the intracerebrally implanted P-388 leukemia model.     

Synthesis and antitumor activity of halogen-substituted 4-(3,3-dimethyl-1-triazeno)quinolines

Halogenated 4-(3,3-dimethyl-1-triazeno)quinolines were synthesized as potential antitumor agents on the basis of the biochemical pharmacological properties of existing triazenes, their structural-activity relationships, and the high melanin binding of chloroquine and iodoquine in vivo and in vitro. They were synthesized by diazotization of appropriate halogen-substituted 4-aminoquinolines in fluoboric acid at -5 degrees C followed by coupling with dimethylamine. Among these new compounds, 8-chloro-4-(3,3-dimethyl-1-triazeno)quinoline produces significant antitumor activity against both P-388 and L1210 murine leukemias. Although only marginally active or inactive against P-388, the other chloro, bromo, or iodo analogues show activity against L1210 comparable to that of dacarbazine (DIC). However, none of these compounds is active against B-16 melanoma. Compared with DIC these new agents demonstrate a higher in vitro affinity for melanin; however, this affinity is apparently not correlated with their antitumor activity.     

Potential central nervous system antitumor agents. Hydantoin derivatives.

Hydantoin derivatives of varying lipophilic character were prepared as nitrogen mustard carriers for CNS antitumor evaluation. Activity was studied in the murine ependymoblastoma brain tumor system. Multiple cures were observed for three of the four analogs examined. The compounds were also active in the intraperitoneal leukemia L1210 and P388 systems as well as in B16 melanoma and Lewis lung carcinoma.     

Preclinical pharmacokinetics and antitumor activity of imexon

Summary Imexon is an aziridine compound originally studied for immune-enhancing effects on lymphocytes. The drug was well-tolerated in humans and was shown to be active in a variety of animal tumor models. Recently, imexon has demonstrated antitumor activity in human multiple myeloma cell linesin vitro. The pharmacokinetics of the compound using a normal phase HPLC assay were studied in normal mice and in dogs with mast cell tumors. Doses of 100 mg/kg given intraperitoneally produced peak plasma levels over 100 (ml in mice and the drug was rapidly eliminated with half lives of 8 minutes ( phase) and 29 minutes ( phase). Only 20% of an oral imexon dose was absorbed in the mouse. In dogs, the and phase half lives ranged from 18–26 minutes and 91–110 minutes, respectively. Peak levels over 100 g/ml were obtained following intravenous doses of 12.5 mg/kg and 25 mg/kg. Imexon was active in mice bearing either P-388 or L-1210 leukemia, but not in mice with B-16 melanoma. These results suggest that cytotoxic drug concentrations can be obtainedin vivo and that imexon is active in lymphoproliferative tumors.     

WF-3405, a novel antitumor antibiotic. Taxonomy, isolation, structure elucidation and biological properties

A new antitumor antibiotic, WF-3405 was isolated from the culture of Amauroascus aureus F-3405. The structure has been determined as 1,5-dioxiranyl-1,2,3,4,5-pentanepentanol on the basis of spectroscopic and chemical evidence. WF-3405 exhibits strong inhibitory activity against various murine tumors including leukemia P388, leukemia L1210 and Lewis lung carcinoma.     

Antitumor spectrum of deoxyspergualin and its lack of cross-resistance to other antitumor agents

Antitumor activities of 15-deoxyspergualin (NKT-01), an analogue of spergualin (SGL), were examined in cultured tumor cells, transplantable murine tumors, and human tumor xenografts in nude mice. NKT-01 exhibited strong antitumor activity specifically against leukemias both in vitro and in vivo. Moreover, it also showed activity against AH66F hepatoma, M5076 fibrosarcoma and MH134 hepatoma. However, antitumor activity of NKT-01 against other non-leukemic tumors was marginal. Effective dose range of NKT-01 in sensitive tumors was so wide that the largest chemotherapeutic indexes were produced by NKT-01 in P388 and L1210 leukemias among 15 antitumor agents examined. The efficacy of NKT-01 against doxorubicin- and cytosine arabinoside-resistant P388 leukemias was comparable to that against parental sensitive P388 leukemia. NKT-01 also retained activity against other p388 leukemia sublines resistant to cisplatin, 5-fluorouracil or nimustine, although the effect was slightly decreased. In addition, in the in vitro and in vivo experiments using NKT-01-resistant P388 and SGL-resistant L1210(IMC) leukemias, no cross-resistance was observed. Moreover, collateral sensitivity was observed especially to alkylating agents in animal study.     

Activity of flavone acetic acid (NSC-347512) against solid tumors of mice

Flavone acetic acid (FAA) is a new antitumor agent that has recently entered Phase I clinical trials. In preclinical studies, we have found that FAA was broadly active against a variety of transplantable solid tumors of mice (colon #51, #07, #10, #26; pancreatic ductal adenocarcinomas #02 and #03; mammary adenocarcinoma #16/C/Adr; M5076 reticulum cell sarcoma and Glasgow's osteosarcoma). FAA was curative for colon adenocarcinoma # 10 and pancreatic ductal adenocarcinoma # 03. Thus, for the first time an agent has been identified with very broad, perhaps nearly universal solid tumor activity. FAA was also found to be orally active and stable in solution at 37 °C for 48 h. FAA was selectively cytotoxic in vitro for solid tumors over leukemias L1210 and P388 (in a soft-agar colony formation assay), thus correlating cellular selectivity in vitro with in vivo antitumor activity. The finding that FAA was active in vitro, established that the agent did not need metabolism (activation) outside the tumor cell. The main drawback of FAA was an unusual threshold behavior in which only a narrow range of doses were active and splitting the dose markedly decreased activity.     

Cytotoxic effects of glutathione synthesis inhibition by L-buthionine-(SR)-sulfoximine on human and murine tumor cells

The glutathione (GSH) synthesis inhibitor, buthionine sulfoximine (BSO) was tested for cytotoxicity and thiol depletion in murine and human tumor cells in vitro, and for its antitumor activity and toxicity in vivo. The cell lines used in these studies included murine L-1210 leukemia, human RPMI 8226 myeloma, MCF-7 breast cancer and WiDr colon carcinoma. Soft agar colony forming assays showed that BSO was most effective at reducing tumor colony formation when exposed continuously to cells in vitro. Drug concentrations which inhibited colony formation to 50% of control levels ranged from 2.0–6.2 mM (for 1 hour exposures), 2–100 mM for 24 hour exposures and 0.4–1.40 M (for continuous BSO exposures). Human myeloma cells proved most sensitive to BSO. In vitro cytotoxicity correlated with depletion of intracellular nonprotein sulfhydryls to 10% of control values in both L-1210 and 8226 cells. This was routinely achieved with prolonged exposures to mM BSO concentrations for > 24 hours. Normal mice tolerated high BSO doses (up to 5.0 g/kg) without evidence of acute toxicity. BSO was not active against L-1210 leukemia-bearing DBA/2 mice. When tested in vivo against MOPC-315 plasmacytoma-bearing BALB/c mice, BSO was not active at doses up to 4.0 g/kg. In contrast, the bifunctional alkylating agent melphalan (L-PAM) was active against MOPC-315 and this activity was enhanced by a 24 hour pretreatment of mice with 50 mg/kg of L-BSO. This BSO dose was shown to significantly reduce sulfhydryl levels in the liver (50% of control) and kidney (20% of control) but not in the bone marrow (100% of control). The enhancement by BSO was most significant only for the lower doses of L-PAM tested. These results suggest that BSO may not have direct antitumor activity, but that it can enhance cytotoxicity from a classic alkylating agent in vivo. Due to its low toxicity, BSO should be tested in combination with either anticancer agents which are dependent on (GSH) for detoxification and potential drug resistance.     

Isolation and characterization of terpentecin, a new antitumor antibiotic

A new antitumor antibiotic, terpentecin was isolated from the culture broth of strain MF730-N6. Strain MF730-N6, isolated from soil, was found to belong to the genus Kitasatosporia. The antibiotic was extracted with chloroform, purified by column chromatography using silica gel and Diaion HP-20 successively, and finally purified by high performance reverse-phase thin layer chromatography. The molecular formula of terpentecin was determined to be C20H28O6 (molecular weight, 364). The antibiotic inhibited the growth of Gram-positive and Gram-negative bacteria, and prolonged the survival period of mice bearing leukemia L-1210, P388 and Ehrlich ascites carcinoma.     

Preclinical antitumor activity and pharmacokinetics of methyl-2-benzimidazolecarbamate (FB642)

Methyl-2-benzimidazolecarbamate(carbendazim, FB642) is an anticancer agentthat induces apoptosis of cancer cells. Invitro, FB642 demonstrated potent antitumoractivity against both the murine B16melanoma (IC₅₀ = 8.5 m) andhuman HT-29 colon carcinoma(IC₅₀ = 9.5 m) cell lines. FB642was also highly active against both murinetumor models and human tumor xenografts atvarying doses and schedules. In the murineB16 melanoma model, T/C values > 200 wereobserved. In the human tumor xenograft,FB642 produced tumor growth inhibition ofgreater than 58% in five of the sevenxenograft models evaluated. Partial andcomplete tumor shrinkage was noted withFB642 against the MCF-7 breast tumor model.Pharmacokinetic studies in ratsdemonstrated that oral absorption of FB642was variable and may be saturated at the2000 mg/kg dose level since higher dosesfailed to produce a further increase in thearea under the time concentration curve. Toxicity of FB642 in vivo appeared to bedose-dependent. Lower doses in the range of2000–3000 mg/kg were better tolerated,while still preserving antitumor activity. Evaluation of FB642 in phase I clinicaltrials of adult patients with advancedmalignancies is currently ongoing.     

Preclinical antitumor activity of XK469 (NSC 656889)

XK469 (NSC 656889) is a water-soluble member of the novel quinoxaline family of antitumor agents. In vitro, XK469 demonstrated selective cytotoxicity for several murine solid tumors including colorectal and mammary adenocarcinoma cell lines, when compared to both leukemia and normal epithelial cells. In vivo, XK469 was active against 7/7 murine tumors tested, including pancreatic ductal carcinomas #02 and #03, colon adenocarcinomas #38 and #51/A, mammary adenocarcinoma #16/C and the Adriamycin resistant mammary adenocarcinomas #16/C/ADR and #17/ADR. XK469 was efficacious both intravenously and orally. Regardless of dosing schedule, conventional mice tolerated higher total doses than SCID or nu/nu mice did. Despite these reduced doses, XK469 was active against xenografts of 4/6 human tumor lines including mammary adenocarcinoma MX-1, the small cell lung cancer DMS 273, the prostate model LNCaP and the CNS tumor SF295. The lower doses in the xenograft studies were below curative levels. The dose-limiting toxicity appeared to be myelosuppression with rapid host recovery (5–8 days), and in vitro assays of XK469 toxicity to murine bone marrow neutrophil progenitors CFU-GM (colony forming unit-granulocyte/macrophage) demonstrated concentration-dependent toxicity from 0.5–30 g/mL. The difference in drug tolerance between BDF₁ and SCID mice was detected in vitro as a 3-fold difference in the IC₉₀ for CFU-GM, despite similar IC₅₀ values. Comparative in vitro hematotoxicology studies revealed that human bone marrow CFU-GM tolerated XK469 as well as their SCID counterparts (IC₉₀ values 5.7 vs. 7.4 g/mL). Based on comparison with previously tested anti-cancer agents, these data suggest that humans will be able to tolerate XK469 doses that are efficacious against human tumor xenografts.     

Morpholinylanthracyclines: cytotoxicity and antitumor activity of differently modified derivatives

Summary The relationship between different chemical modifications on morpholinylanthracyclines and their ability to overcome multidrug resistance (MDR) has been evaluated testing all compounds in vitro on LoVo and LoVo/DX human colon adenocarcinoma cells and in vivo on disseminated P388 and P388/DX murine leukemias.Results obtained led us to the following conclusions: 1) the insertion of the morpholinyl or the methoxymorpholinyl group on position 3 of the sugar moiety confers the ability to overcome MDR in vitro and in vivo; conversely, 4 morpholinyl compounds are effective on MDR cells only in vitro and result inactive in vivo on DX-resistant leukemia; 2) all chemical modifications performed on 3 morpholinyl or methoxymorpholinyl derivatives, that is substitutions on the aglycone or on position 2 of the morpholino ring, do not interfere with the activity of the compounds: all derivatives present in fact the same efficacy on sensitive and resistant models.It is concluded that position 3 in the sugar moiety plays a crucial role in the ability of morpholinylanthracyclines to overcome MDR.