Preclinical antitumor activity of the azonafide series of anthracene-based DNA intercalators.

The azonafides are a series of anthracene-based DNA intercalators which inhibit tumor cell growth in vitro at low nanomolar concentrations and are not affected by the multidrug resistance phenomenon (MDR). Prior studies have described antitumor efficacy in murine tumor models including L-1210 and P-388 leukemias, and B-16 melanoma. The current results extend these cell line observations to human tumors tested in the NCI panel of 56 cell lines, in freshly isolated tumors tested in colony-forming assays in soft agar and in several animal models. In the NCI panel, the overall mean 50% cell kill (LC50) for the unsubstituted azonafide, AMP-1, was 10(-5.53) M, with some selectivity noted in melanomas (10(-6.22) M). The mean LC50 for the 6-ethoxy substituted analog, AMP-53, was 10(-5.53) M, with some selectivity found in non-small cell lung cancer (10(-5.91)) and renal cell carcinoma (10(-5.84)). In freshly isolated human tumors tested in soft agar, there was marked activity (mean IC50 in microg/ml) for AMP-53 in four cell types: breast cancer (0.09), lung cancer (0.06), renal cell carcinomas (0.06) and multiple myeloma (0.03). These effects were superior to doxorubicin and to several other azonafides, including AMP-1, AMP-104 and the 6-hydroxyethoxy derivative, AMP-115. Compound AMP-1 was shown to be superior to amonafide in the mammary 16C breast cancer model in B6CF31 mice, but it had little activity in Colon-38 nor in M5076 ovarian sarcomas in vivo. Nine azonafides were evaluated in the Lewis lung cancer model in C57/bl mice, but only AMP-53 demonstrated significant efficacy with a treated/control x 100% (T/C) value of 30%. Because AMP-53 demonstrated the greatest breadth of activity, it was then evaluated in several human tumor cell lines growing in mice with severe combined immunodeficiency disease (SCID). Only three tumors were sensitive (T/C<42%), including HL-60 leukemia (T/C=39%), MCF-7 breast cancer (T/C=39%) and A549 non-small cell lung cancer (T/C=37%). Overall, these results demonstrate that the 6-ethoxy substituted azonafide, AMP-53, has consistent (in vitro and in vivo) experimental antitumor activity in human breast and lung cancer, and could be considered for clinical testing in patients with MDR tumors.

Dermal toxicity of topical (-)epigallocatechin-3-gallate in BALB/c and SKH1 mice.

(-)Epigallocatechin-3-gallate (EGCG), the major polyphenolic component of green tea, inhibits experimental chemical and physical carcinogenesis, yet little toxicological data has been reported. Therefore, we performed studies on the dermal toxicity of EGCG applied in an ointment formulation in mice. Female BALB/c mice were dehaired with a topical depilatory and administered 75 microl EGCG in hydrophilic Ointment U.S.P. at three concentrations (10, 3, and 1%, all w/w) daily for 30 days. At the 10% concentration, gross toxicity was manifested by the formation of erythema and papular lesions by day 5. A 7% reduction in weight was observed by day 15. No toxicity was observed at the two lower concentrations or in the vehicle control group. Also, no toxicity was observed when mice were dehaired by shaving. This study was repeated in female SKH1 mice, an outbred hairless strain that does not require depilation. No toxicity was observed in the SKH1 mice, indicating that daily topical EGCG appears non-toxic in normal skin. However, use of topical depilatories may potentiate dermal toxicity of EGCG.

The state-of-the-art in chemoprevention of skin cancer.

The incidence of skin cancer (both melanoma and non-melanoma) continues to grow at an alarming rate. Our chemoprevention strategies include the development of novel agents evaluated by (1) preclinical mechanistic studies in models of ultraviolet (UV) radiation-induced skin carcinogenesis; (2) clinical studies of immunohistochemical surrogate endpoint biomarkers in high-risk patients; and (3) randomised, placebo-controlled phase I, II and III clinical chemoprevention trials. Recent clinical results validate this development model. Molecular targets of chemopreventive strategies for melanoma and non-melanoma skin cancers include the ras and activator protein-1 (AP-1) signal transduction pathways. A transgenic murine melanoma model has been developed for evaluating potential agents in vivo. Agents at various stages of study include the green tea catechin epigallocatechin gallate (EGCG), the limonene derivative perillyl alcohol, the ornithine decarboxylase inhibitor alpha-difluoromethylornithine (DFMO), selenium, retinoids and salicylates. New chemopreventive agents that can be used to complement sunscreens may result in decreased incidence, morbidity and mortality of skin cancer.

Pharmacokinetics of the green tea derivative, EGCG, by the topical route of administration in mouse and human skin.

PURPOSE: (-)-Epigallocatechin gallate (EGCG), the main physiologically active polyphenol of green tea, is associated with antitumor and antimutagenic activities. The goal of this study was to determine the stability and pharmacokinetic parameters of pure EGCG administered topically to human and mouse skin. METHODS: EGCG was investigated by measuring drug levels of a 10% ointment formulation stored under different conditions over a period of 6 months. To determine pharmacokinetic parameters of EGCG following topical application. EGCG was applied as 10% EGCG in hydrophilic ointment USP to full-thickness mouse or human skin in vitro. The transdermal and intradermal. Penetration of EGCG was measured by reverse phase HPLC assays at different time-points. RESULTS: The stability of EGCG in hydrophilic ointment USP was dependent on time, temperature and the degree of oxidation. For example, 10% EGCG was lost after 2 days at 37 degrees C, but the same formulation supplemented with 0.1% butylated hydroxytoluene (BHT) had significantly longer stability with > or =90% EGCG remaining after 130 days at 37 degrees C. Topical application of EGCG in hydrophilic ointment USP to human or mouse skin resulted in substantial intradermal uptake of up to 1-20% of the applied dose. However, transdermal penetration was observed only in mouse skin. CONCLUSION: The present study showed that topical application of EGCG in hydrophilic ointment USP achieved high concentrations in skin but negligible systemic availability. The drug was susceptible to oxidation, but if supplemented with BHT, the hydrophilic ointment formulation could potentially be used in clinical trials of skin cancer prevention.

Liposomal amikacin: improved treatment of Mycobacterium avium complex infection in the beige mouse model.

Disseminated Mycobacterium avium complex (MAC) infection has reached epidemic proportions and is a major cause of morbidity and mortality in AIDS patients. We have developed a liposomal preparation of amikacin, VS107, which incorporates the drug in 54-65 nm diameter unilameller phospholipid vesicles and is stable at 4 degrees C for more than 4 months. VS107 exhibits superior microbiological and pharmacological activity over the free amikacin and improves the survival of mice in the established model for MAC infection. The serum half-life of VS107 in mice was 9.1 h and a peak serum level of 730 mg/L was obtained after administering three doses of 160 mg/kg. For the therapeutic study, beige mice infected with 10(7) cfu M. avium complex strain 101 were randomised to be treated with placebo liposomes, buffer, free amikacin or VS107 The drugs were administered via the caudal vein thrice weekly for 1, 3, 5 or 7 weeks beginning 5 days after infection. After 51 days of treatment with VS107, the number of viable M. avium in the liver and spleen was a 100 fold lower than was achieved with conventional amikacin (P < 0.01), and more than six decimal logarithms lower than was found untreated controls (P < 0.001). VS107 was well tolerated and might be a suitable candidate for treating human MAC infections.

Antidote studies of vinorelbine-induced skin ulceration in the mouse.

The new cantharanthine-modified vinca alkaloid vinorelbine (Navelbine) was administered intradermally (ID) to dehaired BALB/c mice. Dose-dependent skin lesions were produced over the range 0.01-0.5 mg/mouse, with complete healing after 9-35 days. Local (ID) injections of hydrocortisone and saline were ineffective at blocking vinorelbine-induced skin ulceration. Topical skin heating to 43 degrees C or cooling to 10 degrees C were also ineffective. In contrast, hyaluronidase, 15 Units ID, following vinorelbine significantly reduced skin lesions. These results show that vinorelbine is a vesicant and that inadvertent extravasations may be managed with subcutaneous injection of the spreading factor enzyme, hyaluronidase.

Chemoprotectants for cancer chemotherapy.

Maximal dosing of cytotoxic chemotherapy drugs is often limited by the development of severe nonmyelosuppressive toxicities. Numerous studies have demonstrated that sulfur-containing nucleophiles can antagonize the dose-limiting effects of alkylating agents on the genitourinary tract. Examples include the use of sodium thiosulfate to prevent cisplatin-induced renal tubular necrosis and the use of sulfhydryl-containing compounds like N-acetylcysteine and 2-mercaptoethanesulfonate (mesna) to block oxazophosphorine-induced bladder toxicity. Mesna does not block the antitumor action of oxazophosphorines due to its rapid formation of the inactive dimer dimesna in the bloodstream. The active monomer is selectively reduced from dimesna in renal tubule cells, thereby limiting the inactivation of toxins like acrolein to the genitourinary tract. Recent clinical trials suggest that oral mesna has adequate bioavailability (roughly 50% by urinary thiol measurements) to prevent urotoxicity in high-dose ifosfamide regimens. In addition, mesna is stable in aqueous oral formulations. This may facilitate more convenient oral mesna dosing in protocols using high-dose cyclophosphamide or ifosfamide. Whereas agents like mesna and sodium thiosulfate complex directly with activated (electrophilic) alkylator species, chemoprotectants for the anthracyclines appear to complex with metal cofactors like iron, which are required for the production of cardiotoxicity. Several ethylenediaminetetraacetic-like agents have been evaluated, and a water-soluble piperazinyl derivative, ICRF-187, is currently undergoing clinical evaluation in patients receiving large cumulative doxorubicin doses. An initial clinical trial suggests that ICRF-187 can prevent doxorubicin-induced cardiomyopathy. As with mesna, ICRF-187 does not block the myelosuppressive or the antitumor effects of doxorubicin. Overall, these studies show that site-selective chemoprotection is now feasible for at least two major classes of anticancer agents.

Mitomycin C toxicity and pharmacokinetics in mice given sulfur nucleophiles.

The sulfur nucleophiles, sodium thiosulfate (Na2S2O3) and N-acetylcysteine (NAC) given in maximally tolerated doses did not reduce the hematologic toxicity of high dose mitomycin C (MMC) in normal mice. In addition, neither sulfur nucleophile significantly altered the antileukemic activity of MMC. Pharmacokinetic studies of MMC in normal mice, demonstrated rapid plasma elimination (T1/2 beta = 0.53 hrs) and substantial drug distribution to the bone marrow which was enhanced by NAC. These results demonstrate a lack of MMC antidotal activity for Na2S2O3 and NAC.

Lack of experimental vesicant activity for the anticancer agents cisplatin, melphalan, and mitoxantrone.

Cisplatin and L-PAM are DNA-crosslinking anticancer agents which have not been systematically studied for vesicant potential. Mitoxantrone is a new active anthracene-based, DNA intercalator which is undergoing widespread clinical testing for antitumor efficacy in man. These three agents were tested for vesicant activity in dehaired BALB/c mice given ID injections equivalent to human clinical doses. Neither cisplatin (up to 150 mg/m2) nor L-PAM (up to 71 mg/m2) produced any skin necrosis in the mice. The L-PAM solvent (acid/alcohol in propylene glycol) was ulcerogenic if injected undiluted. Mitoxantrone (up to 14 mg/m2) was not ulcerogenic in the mice, although the skin site retained a blue drug discoloration for several weeks. It is concluded that in clinically relevant doses, cisplatin, L-PAM, and mitoxantrone are not vesicants.

Cold protection and heat enhancement of doxorubicin skin toxicity in the mouse.

A series of experiments were performed in a BALB/c mouse model to evaluate the efficacy of topical heating and cooling on doxorubicin (DOX) ulceration of the skin. Unanesthetized mice were administered a dose of 0.05 mg or 0.5 mg of DOX intradermally, followed by topical heating (43 degrees C-44 degrees C) or cooling (8 degrees C-10 degrees C) of the skin area for up to 1 hour. DOX disposition from skin and plasma was studied by high-pressure liquid chromatography in both cooled and uncooled groups of animals. Human tumor clonogenic cells were exposed to DOX for 1 hour at different temperatures to determine the direct effect of heat and cold on DOX-induced lethality in vitro. Skin temperature of 17 degrees C +/- 2.3 degrees C was achieved with cooling and skin temperature of 38.5 degrees C +/- 1.2 degree C was achieved with heating, compared to control intradermal skin temperature of 32 degrees C +/- 0.5 degree C. Local heating caused duration-dependent DOX lethality: 20% after 20 minutes, 40% after 45 minutes, and 80% after 1 hour. There were no deaths in the control groups. A 20% lethality rate was constant in the cooled groups. Skin lesions were approximately fourfold larger in the heated groups receiving 0.5 mg of DOX (P less than 0.05). In contrast, the application of cold significantly reduced intradermal DOX skin toxicity following the lower DOX dose of 0.05 mg (P less than 0.05). There was no consistent benefit for cooling beyond a 45-minute duration, which achieved maximal protection against ulceration. Pharmacokinetic studies of DOX disposition in skin and blood failed to show a significant difference for total tissue concentrations or plasma levels between cooled and uncooled animals. However, clonogenic human tumor cells (HEC-1A endometrial cells) did demonstrate significantly reduced DOX effects when exposed to the drug at reduced temperatures. These results confirm the biologic efficacy of local cooling and clearly contra-indicate the use of local heating to treat inadvertent DOX extravasations in the clinic. Instead, cold should be applied immediately and maintained for 45 minutes or longer to reduce or prevent serious DOX skin ulcerations in patients.

Bleomycin compatibility with selected intravenous medications.

Three separate in vitro admixture experiments were accomplished with bleomycin and other common parenteral medications. Bleomycin was assayed by a radioimmunoassay procedure. Bleomycin was found to be compatible with a majority of drugs including the aminoglycoside antibiotics, the vinca alkaloid antineoplastics, the antimetabolite fluorouracil, several phosphate salt corticosteroids, diphenhydramine and the anticoagulant heparin sulfate. Incompatible admixtures include the acidic cephalosporin and penicillin-derived antibiotics, the antineoplastics, methotrexate, and mitomycin C, the sodium succinate salt of hydrocortisone as well as ascorbic acid (vitamin C).

The limited role of corticosteroids in ameliorating experimental doxorubicin skin toxicity in the mouse.

Local skin necrosis is a serious complication of doxorubicin (Adriamycin) infiltration in man. Intradermal (ID) doxorubicin infection was used in the mouse to create skin lesions, after which a number of local corticosteroid interventions were studied. The most effective local antidote was low-dose (2.5 mg) hydrocortisone (HC), but only against the low-dose doxorubicin challenge (0.05 mg). Other andidotes with lesser effectiveness included dexamethasone-sodium bicarbonate and an intermediate dose of HC (6.25 mg). Larger doses of ID HC did not prevent local doxorubicin toxicity and were inherently toxic to the skin. Systemic corticosteroids were similarly ineffective. The superiority of the low ID HC dose, the ineffectiveness of additions (sodium bicarbonate, topical HC cream), and the resistance of the high-dose doxorubicin ID challenge (0.5 mg) suggests a limited role for corticosteroids in the management of experimental doxorubicin skin toxicity.

The current status of laetrile.

Amygdalin at various concentrations and with numerous impurities is the most common cyanogenic glycoside found in laetrile samples. Its chemical properties were first described in 1837, and pharmacologic studies have shown that ultimately it is broken down to HCN, benzaldehyde, and glucose by enzymes found in gut bacteria but not intracellularly in humans. Fatal and nonfatal toxicities to orally ingested cyanogenic glycosides have been reported worldwide. We review here the signs and symptoms of acute cyanide toxicity and its treatment. Substantial in-vitro and in-vivo testing in animal tumor systems has shown that amygdalin is entirely devoid of significant anticancer activity. Control animals often have lived longer than those treated with various doses and schedules of amygdalin. Acceptable clinical studies in humans are lacking, but such ventures would appear to be contraindicated from animal studies and observed human toxicities. We also discuss current legal-judicial aspects of laetrile therapy for cancer.