The small organo-cobalt complex, CTC-96 (Doxovir™), is under development for prophylactic use in a topical vaginal microbicide. Such an anti-STD microbicide should be effective against a range of microbes, especially viruses, but harmless to the host, to her natural microbial flora and to her reproductive potential. The suitability of CTC-96 is supported by findings on its toxicology and anti-viral effects summarized here. CTC-96 is active against herpes simplex virus (HSV), human immunodeficiency virus (HIV), and human papillomavirus (HPV), which are the causative agents of genital herpes, AIDS, and anogenital warts, respectively, and there are preliminary indications of CTC-96 activity against other STD pathogens, too. Documentation of anti-HIV activity has been extended from laboratory strains to clinical isolates. We also report that CTC-96 is very weakly spermicidal in the Sanders-Cramer assay, and is not genotoxic either in the Ames mutagenesis test (with metabolic activation) or in the mouse clastogenesis (micronucleus) test. Toxicity is seen in mice injected (i.p.) with 80mg/kg body weight, but not with 40mg/kg. Obtaining this lower concentration in humans would require complete adsorption of 100 times the CTC-96 present in the most concentrated anticipated microbicide formulation. 0.1% CTC-96 has proven completely effective against infection by HSV in mouse and guinea pig models, and HIV or HPV in cultured cell or human tissue explant models, respectively. Experiments are now underway to better characterize CTC-96 potency against clinical HIV isolates, against infection by fusion with infected cells, and to evaluate CTC-96 for treatment of HPV infected human tissue explants in SCID mice. A preliminary human safety trial of CTC-96 has been conducted in eyes for indications of ocular herpes and inflammation. CTC-96 showed no ill effects when an eye drop containing 50μg/ml CTC-96 was given to healthy volunteers. This, together with animal and in vitro evidence, suggests that a CTC-96 based microbicide can be both innocuous and effective.

Activated bleomycin is the drug species seen to be kinetically competent to initiate DNA degradation in the reaction of bleomycin, Fe(II), and O(2) It also forms in reactions of bleomycin with Fe(III) and peroxide, or bleomycin with superoxide and either Fe(III) or Fe(II). Efforts to characterize this transient species proceeded by kinetic and spectroscopic strategies. Activated bleomycin now appears to be a drug-ferric-peroxide complex, but this may not be the proximate active drug species. Assuming that activated bleomycin peroxide cleavage yields a reactive product analogous to peroxidase compound I explains many characteristics of bleomycin-mediated DNA degradation reactions. DNA degradation is responsible for the cytotoxic and antitumor activities of this clinically useful antibiotic.

Purpose A new class of antiviral agent, cobalt chelates (the CTC series), was evaluated for treating epithelial herpetic keratitis, consequent stromal disease being the major infectious cause of blindness in industrial nations. Methods Effects of CTC complexes were monitored in cell cultures and in a rabbit eye model, either infected with herpes simplex virus type 1 (HSV-1) or uninfected. Several antiviral concentrations of CTC complexes nontoxic to Vero cells were administered to rabbit eyes with HSV-1-induced keratitis. Corneal surface virus titers were measured, and corneal lesions of epithelial keratitis were monitored by slit-lamp microscopy and scored. Recovery rates and incidence were compared in eyes treated with CTC complexes, placebo, or clinically formulated trifluorothymidine (Viroptic), using nonparametric statistics. Results All CTC complexes inhibited HSV-1 replication in vitro, CTC-96 being best. CTC-96, CTC-23, and CTC-67 eliminated (<1 plaqueforming unit[pfu]) corneal surface HSV-1 (otherwise >105 pfu) in order of descending potency, but CTC-82 was ineffective. CTC-96 (either 5 μg/ml six times daily or 10 μg/ml five times daily) accelerated herpetic dendritic keratitis recovery better than or the same as trifluorothymidine (10 mg/ml nine times daily). CTC complexes were nontoxic to Vero cells continuously exposed to <25 μg/ml; 50 μg/ml of CTC 96 nine times daily did not irritate uninfected rabbit eyes. Conclusion Topical CTC-96 applications were at least as effective as Viroptic in diminishing disease signs and corneal surface virus at concentrations less than one-thousandth that of Viroptic.

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The action of iron-bleomycin and O2 in cleaving DNA has been resolved into two kinetic events following the initial attack on DNA by the kinetically competent drug species, "activated bleomycin." At 4 degrees C, DNA strand scission, monitored both viscometrically and fluorimetrically (t1/2 = 2.5-5 min), precedes the release from DNA of nucleic base propenals, which is half complete in about 40 min. Therefore, a moderately stable intermediate consisting of cleaved DNA bearing a base propenal precursor is formed. The release of tritium from deoxyribose carbon-2 occurs at the time of DNA scission, which is consistent with the base propenal precursor retaining the deoxyribose-3'-phosphate bond. Specific mechanistic proposals are discussed.

Orthophosphate and phosphate derivatives including pyrophosphate, hexametaphosphate, ATP, ADP, and inositol hexaphosphate enhance the extent of DNA degradation by iron(II) bleomycin. These phosphate-containing compounds increase both the release of free nucleic base and that of base propenals which are DNA cleavage products, probably by enhancing the efficiency with which Fe(II) is recruited into the drug. Phosphate action occurs during drug activation prior to the attack on DNA. In addition, phosphates affect the stability of the activated drug complex, overcome the inhibition observed with high concentrations of DNA, and reduce the size of the DNA fragment necessary for reacting with the drug. Phosphate derivatives bind to iron(II) bleomycin and alter its optical spectrum. An analysis of titration data for pyrophosphate and inositol hexaphosphate indicates that each phosphate compound binds to more than one iron(II) bleomycin molecule. With ATP, ADP, and 2,3-diphosphoglycerate, only a single phosphate-containing compound binds to the ferrous drug complex. The affinity for ATP is sufficiently high as to suggest that the ternary complex formed in vitro may exist physiologically.

Activated bleomycin appears to have two more oxidizing equivalents than the Fe(III).bleomycin to which it spontaneously decays. Activated bleomycin reacts with NADH and thio-NADH, two-electron reductants, and with KI, a one-electron reductant, to yield Fe(III).bleomycin. The observed stoichiometries were 0.85 +/- 0.07 eq of thio-NADH oxidized or 1.5 +/- 0.25 eq of KI oxidized per mole of activated bleomycin. None of these reactions requires the presence of a redox mediator, as does the reduction of Fe(III).bleomycin by NADH or thio-NADH. The oxidations of both pyridine nucleotide coenzymes and of KI are inhibited by DNA, the usual bleomycin target.

Using Mössbauer spectroscopy, we have examined iron-bleomycin in various oxidation states and in complexes with dioxygen or carbon monoxide. Ferrous bleomycin is a high spin ferrous complex. Addition of O2 converts it into an EPR-silent oxygenated complex. Mössbauer studies in strong applied magnetic fields show that oxygenated bleomycin is diamagnetic. At 4.2 K, the quadrupole splitting delta EQ = -2.96 mm/s and the isomer shift delta = 0.16 mm/s suggest that its electronic structure is best described as low spin ferric iron bound to superoxide anion. A single electron reduction yields activated bleomycin, an EPR-active form that still retains oxygen and which is kinetically competent to initiate DNA cleavage. We have produced this complex by exposing ferrous bleomycin to O2 or by reacting ferric bleomycin with H2O2. The Mössbauer spectra give convincing evidence that the iron of activated bleomycin is low spin ferric. The decay of activated bleomycin yields low spin ferric bleomycin, a complex with Mössbauer parameters nearly identical with those reported for ferric cytochrome P-450. Although iron bleomycin does not have a polyaromatic structure like heme, many features of its electronic structure at the iron are very similar to those produced by the sulfur-coordinated heme iron of ferric cytochrome P-450, a protein that catalyzes a similar oxygen-dependent reaction.

The degradation of DNA by iron-bleomycin requires the formation of a complex with reduced oxygen species. Such complexes can result from reactions of Fe(III)·bleomycin with peroxides, or by reduction of O2 with Fe(II)·bleomycin. In the absence of both peroxides and reductants Fe(III)·bleomycin is normally inactive in cleaving DNA, but, like Co(III)·bleomycin [1], some activity is detected upon exposure to strong light. This activity of the ferric complex is now shown to result from the formation of Fe(II)·bleomycin. Anaerobic solutions of Fe(III)·bleomycin, when exposed to ultraviolet light, produce a stable complex which forms a characteristic chromophore (λmax = 500 nm) with the O2-analogue ethyl isocyanide. This reacts with O2 to cause DNA cleavage with the expected yield of ∼20%, with the regeneration of Fe(III)·bleomycin as indicated by optical and EPR spectroscopy.

The antitumor drug, bleomycin, interacts with either Fe(II) and O 2 or Fe(III) and H2O2 to form an activated complex which attacks DNA. Under aerobic conditions, both reactions yield similar quantities of free bases and products consisting of base plus deoxyribose carbon atoms 1 to 3. Under anaerobic conditions, activated bleomycin releases only free base. The yield of free base is the same under aerobic or anaerobic conditions, provided DNA is furnished in excess. When the DNA concentration is limiting, more base is released under anaerobic than under aerobic conditions. Drug self-destruction proceeds as quickly and completely in the presence or absence of O2.

A colorimetric assay of DNA breakage by bleomycin has been standardized and indicates that strand scission is stoichiometric with the formation of a single equivalent of an aldehyde compound consisting of base plus deoxyribose carbons 1' to 3'. Both strand scission and aldehyde formation require the presence of O2. An alternate DNA lesion inflicted by bleomycin, alkali labilization, is O2-dependent, as is the accompanying release of free bases.

We have studied the Cu(II), Co(II), and Fe(III) complexes of the antineoplastic drug bleomycin by using electron spin--echo envelope spectroscopy. For all three complexes, nitrogen coordination of the metal ions is demonstrated. For the Cu(II)-- and Co(II)--drug complexes, we have been able to identify imidazole as a metal ligand.