Purpose: The goals of this study were (1) to compare the injury at the basement membrane zone (BMZ) of rabbit corneal organ cultures exposed to half mustard (2 chloroethyl ethyl sulfide, CEES) and nitrogen mustard with that of in vivo rabbit eyes exposed to sulfur mustard (SM); (2) to test the efficacy of 4 tetracycline derivatives in attenuating vesicant-induced BMZ disruption in the 24-h period postexposure; and (3) to use the most effective tetracycline derivative to compare the improvement of injury when the drug is delivered as drops or hydrogels to eyes exposed in vivo to SM. Methods: Histological analysis of hematoxylin and eosin-stained sections was performed; the ultrastructure of the corneal BMZ was evaluated by transmission electron microscopy; matrix metalloproteinase-9 was assessed by immunofluorescence; doxycycline as drops or a hydrogel was applied daily for 28 days to eyes exposed in vivo to SM. Corneal edema was assessed by pachymetry and the extent of neovascularization was graded by length of longest vessel in each quadrant. Results: Injury to the BMZ was highly similar with all vesicants, but varied in degree of severity. The effectiveness of the 4 drugs in retaining BMZ integrity did not correlate with their ability to attenuate matrix metalloproteinase-9 expression at the epithelial-stromal border. Doxycycline was most effective on organ cultures; therefore, it was applied as drops or a hydrogel to rabbit corneas exposed in vivo to SM. Eyes were examined at 1, 3, 7, and 28 days after exposure. At 7 and 28 days after SM exposure, eyes treated with doxycycline were greatly improved over those that received no therapy. Corneal thickness decreased somewhat faster using doxycycline drops, whereas the hydrogel formulation decreased the incidence of neovascularization. Conclusions: Corneal cultures exposed to 2-chloroethyl ethyl sulfide and nitrogen mustard were effective models to simulate in vivo SM exposures. Doxycycline as drops and hydrogels ameliorated vesicant injury. With in vivo exposed animals, the drops reduced edema faster than the hydrogels, but use of the hydrogels significantly reduced neovascularization. The data provide proof of principle that a hydrogel formulation of doxycycline as a daily therapy for ocular vesicant injury should be further investigated.

The design of specific DNA cleavers is a growing field of research for several reasons: (i) DNA is the biological target of a large number of antitumor agents which are able to cleave double-stranded nucleic acids, bleomycin,* 1 neocarzinostatin,2 and enediyne molecules,3 (ii) transition metal complexes linked to a DNA recognizing molecule (intercalating agents,4 peptides,5 or oligonucleotides6) are also able to cleave selectively single-or double-stranded nucleic acids, and (iii) some of these hybrid molecules, "DNA cleaver-vector", might have a future as antiviral or antitumoral agents.7 Cationic manganese-porphyrin complexes are among the efficient DNA cleavers because of their affinity for nucleic acids and their capacity to oxidize the sugar carbon-hydrogen bonds of deoxyribose units.8 Mechanistic studies on DNA breaks mediated by cationic manganese-porphyrins strongly suggest that high-valent manganese-oxo species might be responsible for DNA cleavage instead of diffusible oxygen species like hydroxyl radicals.8•9 Such metal-oxo species were also proposed for "activated bleomycin", an antitumoral antibiotic able to cleave DNA.la'cl°DNA cleavage selectivity can be modulated by linking the tris(methylpyridiniumyl)porphyrinatomanganese motif to different vectors: an intercalating agent like 9-methoxyellipticine11 or an oligonucleotide.12 This DNA cleaver exhibits also an anti-HIV activity alone or linked to 9-methoxyellipticine.13 For these different reasons, we decided to undergo the preparation of a series of water-soluble, cationic porphyrin ligands based on the tris(methylpyridiniumyl)porphyrin motif (Table I). All these unsymmetric porphyrins contain a * Author to whom correspondence should be addressed.(1) (a) Hecht, S.

volume of the loaded VPI-5 was only about one third of that of an unloaded sample (unloaded: 0.184 ~m -~, loaded 0.065 cm3).Besides the proof of the inclusion of C,, in the micropores, this study also included preliminary investigations on the dynamics of the adsorbed C60. For this purpose solid-state I3C N M R spectra both with and without rotation about the magic angle (magic angle spinning, MAS) were recorded for aluminophosphate samples that have no detectable bulk C,, on the outer surface according to XRD. These spectra showed that the C,, molecules rotate freely within the channels of the aluminophosphates, because the line-broadening expected for restricted rotationf51 was not unambiguously observed. Nevertheless, the lines were broadened in those spectra recorded without MAS, from which a partial restriction of the free isotropic rotation can be deduced. A further, though only slight, broadening of the lines from 600 to 700 Hz was obtained by cooling the sample from 298 to 230 K. This might point to a transition from the "rotator" phase to the "rachet" phase (the low-temperature phase according to ref. (51) in the aluminophosphate lattice at relatively high temperatures.['] It should, however, be mentioned that coadsorbed water can influence the dynamics significantly.At present we are studying in more detail the electronic and optical properties of the molcular sieves doped with C,, .In particular, we are attempting to separate C,,, C,,, and higher fullerenes in the gas phase by these methods. This would be an alternative to the usual separation by liquid chromatography['. and is also applicable on larger scales. E.xperinrenta1 ProcedureSample preparation: AIPO,-5 and AIPO,-8 were activated by simple heating to 200 C in air to remove adsorbed water. Under analogous treatment VPI-5 was transformed irreversibly into AIPO,-X and had therefore to be dehydrated under high vacuum Torr) [4]. The aluminophosphate samples were placed in one arm of an inverted glass U-tube. and the other was charged with C60. The arm containing aluminophosphate was heated separately. and the sample chamber was evacuated prior to calcination through the opening in the center of the tube. At lo-' Torr the aluminophosphates were first activated at 280 "C for 12 hand then at 480 "C for 1 h. Thereafter under maintenance of the vacuum the glass tube was fused, and the glass ampule containing the activated aluminophosphate and C,, was heated for 10 h at 450 C. The tube was opened, and the sample washed in toluene. filtered off'. and again washed for three periods of 15 min in toluene (200 mL portions per 1 g loaded aluminophosphate) in a ultrasonic bath. This procedure removed the last residues of C,, from the crystal surface (determined by XRD). The IR spectra were recorded on crushed crystal aggregates with an Analytical Plan IR microscope (SpectraTec) coupled to a Nicolet 5SXB FTIR spectrometer. The N L sorption measurements were performed at 77 K with a volumetric sorption apparatus (Omnisorp 100). To remove adsorbed water and toluen...

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