3-Amino-4-aminoximidofurazan derivatives: small molecules possessing antimicrobial and antibiofilm activity against Staphylococcus aureus and Pseudomonas aeruginosa.

AIM: The therapeutic treatment of microbial infections involving biofilm becomes quite challenging because of its increasing antibiotic resistance capacities. Towards this direction, in the present study we have evaluated the antibiofilm property of synthesized 3-amino-4-aminoximidofurazan compounds having polyamine skeleton. These derivatives were synthesized by incorporating furazan and biguanide moieties. METHODS AND RESULTS: Different 3-amino-4-aminoximidofurazan derivatives (PI1-4) were synthesized via protic acid catalysis and subsequently characterized by (1) H NMR and (13) C NMR spectra, recorded at 400 and 100 MHz respectively. We have tested the antimicrobial and antibiofilm activities of these synthetic derivatives (PI1-4) against both Staphylococcus aureus and Pseudomonas aeruginosa. The compounds so tested were also compared with standard antibiotics namely Tobramycin (Ps. aeruginosa) and Azithromycin (Staph. aureus) which were used as a positive control in all experimental sets. All these compounds (PI1-4) exhibited moderate to significant antimicrobial activities against both micro-organisms wherein compound PI3 showed maximum activity. Biofilm inhibition of both micro-organisms was then evaluated by crystal violet and safranin staining, estimation of biofilm total protein and microscopy methods using sub-MIC dose of these compounds. Results showed that all compounds executed anti biofilm activity against both Staph. aureus and Ps. aeruginosa wherein compound PI3 exhibited maximum activity. In relation with microbial biofilm inhibition, we have observed reduction in bacterial motility, proteolytic activity and secreted exo-polysaccharide (EPS) from both Staph. aureus and Ps. aeruginosa when they were grown in presence of these compounds. While addressing the issue of toxicity on host, we have observed that these molecules exhibited minimum level of R.B.C degradation. CONCLUSION: These findings establish the antibacterial and anti biofilm properties of 3-amino-4-aminoximidofurazan derivatives (PI1-4). SIGNIFICANCE AND IMPACT OF THE STUDY: Therefore, our current findings demonstrate that 3-amino-4-aminoximidofurazan derivatives (PI1-4) may hold promise to be effective biofilm and microbial inhibitors that may be clinically significant.

Stability studies of topical carbonic anhydrase inhibitor 6-hydroxyethoxy-2-benzothiazole sulfonamide.

A new carbonic anhydrase inhibitor, 6-hydroxyethoxy-2-benzothiazole sulfonamide (6-hydroxyethyoxyzolamide), was studied to determine its stability in aqueous solution from pH 2.9 to 9.2 at a constant ionic strength of 0.15 M. This newly synthesized derivative of ethoxyzolamide has demonstrated clinical efficacy for use as an ophthalmic drug to lower intraocular pressure. Drug solution in sealed ampules was placed in a constant temperature over either at two temperatures (75 and 85 +/- 0.2 degrees C) or four temperatures (75, 80, 85, and 90 +/- 0.2 degrees C). Samples were analyzed by known HPLC methods. The results indicated that 6-hydroxyethoxyzolamide is most stable at pH 4 to 5.5. The aqueous drug solutions at pH 7.0 and 8.0 were, nevertheless, sufficiently stable, based on extrapolation of kinetic data at high temperatures using the experimentally determined Arrhenius equation. The degradation compound was identified by spectral analysis to have a hydroxyl group substituting for the original -SO2NH2 group.

Biopharmaceutical explanation for the topical activity of 6-hydroxyethoxy-2-benzothiazolesulfonamide in the rabbit eye.

6-Hydroxyethoxy-2-benzothiazolesulfonamide (compound 1), 6-hydrogen-2-benzothiazolesulfonamide (compound 2) and ethoxzolamide (compound 3) are carbonic anhydrase inhibitors, of which only compound 1 is active in lowering intraocular pressure when administered to the rabbit eye. They were compared for potency by inhibiting carbonic anhydrase I (CAase I) using the pH stat procedure. No differences were detected. Binding affinity and number of binding sites were identical; only a single binding site was operative. 14C-labelled compound 1 was used to measure the presence of an ocular metabolite which ranged from 17 to 57% in cornea, iris/ciliary body and aqueous humor. By maintaining drug solutions on either the corneal or conjunctival/scleral surfaces of the eye of anesthetized rabbits for 120 minutes, steady state concentrations of compounds 1-3 were determined in cornea, aqueous humor and iris/ciliary body. It was concluded that compound 1 penetrated both routes equally well and also accumulated at the active site in considerably higher concentrations than compounds 2 and 3. Compounds 2 and 3 did not accumulate in the iris/ciliary body nor did compound 3 penetrate the conjunctival/scleral route very well, approximately 5-20 fold lower than from the corneal route.

Characterization of arylamine acetyltransferase in the rabbit eye.

The activity of arylamine acetyltransferase with p-aminobenzoic acid (PABA), sulfamethazine (SMZ), and aminozolamide as substrates was studied in rabbit tissue homogenates of the corneal epithelium, stroma-endothelium, iris-ciliary process, and liver. Rabbits were classified as rapid or slow acetylators with respect to their rate of hepatic acetylation of SMZ. The ocular disposition of aminozolamide in the two phenotypes was compared using a topical ocular infusion method that permitted a constant concentration to remain in contact with the intact cornea. The effect of hepatic-acetylator phenotype on the intraocular pressure (IOP) recovery rate and drug concentrations in tissues after single-dose administration of aminozolamide also was studied. In general, the rank order of arylamine acetyltransferase activity regardless of substrate was liver greater than iris-ciliary process greater than corneal epithelium greater than stroma-endothelium. The specific activity with aminozolamide as substrate was greater than that with SMZ in each tissue homogenate and greater than with PABA as substrate in all tissues except the stroma-endothelium of slow hepatic-acetylator rabbits. Very low enzyme activity ratios for ocular acetylation between rapid and slow hepatic-acetylating rabbits indicated that acetylation in the ocular tissues did not correspond with the acetylation phenotype. At various times during and after topical infusion to the anesthetized rabbit, assay determinations of drug and metabolite in ocular tissues indicated that there were no significant differences between phenotypes in the disposition of either drug or metabolite. These results correlate with the IOP measurements after topical infusion; they also showed no difference in the effect of aminozolamide between hepatic-acetylator phenotypes. These results indicate that the ocular disposition and the decrease in IOP from topical application of aminozolamide is independent of the hepatic-acetylation phenotype in the rabbit. There are significant amounts of acetyltransferase activity in the ocular tissues of the rabbit with these three substrates, indicating that acetylation may be occurring for other arylamine drugs used in the eye.

The study of ocular hypotensive effect of 6-hydroxyethoxy-2-benzothiazole sulfonamide: a topical carbonic anhydrase inhibitor.

A newly synthesized topical carbonic anhydrase inhibitor, 6-hydroxyethoxy-2-benzothiazole sulfonamide (6-HS), was administered systemically and topically to alpha-chymotrypsin-induced glaucoma rabbits to evaluate its ocular hypotensive effect. A significant IOP lowering effect was observed after topical application of 50 microL of 3% 6-HS gel, but a dose of 50 microL of 3% 6-HS suspension failed to reduce IOP. The maximal magnitude of reduced IOP after topical gel instillation was 24.4%, very close to the result obtained following intravenous injection of 6 mg/kg of 6-HS (23.3%). However, the blood levels of 6-HS after topical instillation with 3% 6-HS gel was much lower than that following 6 mg/kg of 6-HS injected intravenously (less than 5%). Since a lower dose of 6-HS (1 mg/Kg) administered intravenously did not cause a significant drop in IOP, it is reasonable to deduct that the ocular hypotensive effect of 6-HS applied topically can then be attributed to the inhibition of intraocular carbonic anhydrase activity. It was also noted that a larger dose of intravenous administration of 6-HS (20 mg/Kg) had a more profound IOP and blood pressure reducing effect with moderate metabolic acidosis.

Corneal and scleral penetration studies of 6-hydroxyethoxy-2-benzothiazole sulfonamide: a topical carbonic anhydrase inhibitor.

A newly synthesized topical carbonic anhydrase inhibitor, 6-hydroxyethoxy-2-benzothiazole sulfonamide (6-HS), was used as a model drug to determine its corneal and scleral permeabilities in rabbit eyes. The corneal permeability coefficient of 6-HS for short duration glaucoma and normal rabbit eye was not significantly different (its mean value was around 2.9 x 10(-6) cm/sec), while the corneal permeability coefficient for long duration glaucoma rabbit eye was 1.8 times greater than that for the normal eye. The sclera was found to have a higher permeability than the cornea in that after four hours perfusion the amount of drug which passed through the sclera was 11 times greater than that of the cornea. In addition, it was also noted that after topical instillation of 50 microL of 3% 6-HS gel the aqueous humor concentrations of 6-HS in short duration glaucoma eye and normal eye were not statistically different.

6-amino-2-benzothiazole-sulfonamide. The effect of a topical carbonic anhydrase inhibitor on aqueous humor formation in the normal human eye.

The carbonic anhydrase inhibitor, 6-amino-2-benzothiazolesulfonamide, formulated as a 3% suspension in a gel vehicle was instilled in one eye of 21 human subjects in a single dose study to determine its effect on aqueous dynamics. A small but statistically significant effect on aqueous humor flow was observed 2 to 7 hours after application. By 8 hours, the effect had disappeared, and intraocular pressure (IOP) measured 8 hours after application of a single dose was unchanged in these normal volunteers. The drug and its vehicle caused local side effects including irritation, hyperemia, and blurred vision. The authors wondered if multiple doses would produce a greater effect. Four subjects received up to four doses of the drug over 2 days and were restudied. Marked bulbar injection and follicular conjunctivitis, attributable to either the drug or the vehicle, developed in two of the subjects, both contact lens wearers. A milder form of bulbar injection and follicular conjunctivitis developed in a third subject, who received three doses of the drug and was not a contact lens wearer. These side effects precluded additional multiple-dose testing of this formulation of the drug, and no conclusions about the effect of the drug on aqueous flow can be drawn from this portion of the study.

Aminozolamide suspension: the role of the vehicle in a topical carbonic anhydrase inhibitor.

Aminozolamide (6-amino-2-benzothiazolesulfonamide) is a carbonic anhydrase inhibitor derived from ethoxzolamide that has been shown in gel formulation to lower IOP in rabbits, primates and humans. This study was designed to determine whether aminozolamide in suspension was also effective in lowering IOP. In separate single dose and multiple dose studies, patients with ocular hypertension were tested over 24 hours. No statistically significant drop in intraocular pressure was noted between the treated and control eye. In addition, conjunctival injection was noted in three of eleven subjects after multiple dosing. These studies suggest that retention of aminozolamide at the active site is inadequate when delivered in a suspension vehicle. In the past 10 years, efforts to develop an effective topical carbonic anhydrase inhibitor have been vigorously pursued. This has been aided by advancements in drug delivery and chemical synthesis involving molecular modification of existing carbonic anhydrase inhibitors. Various compounds have been developed that retain carbonic anhydrase inhibitory activity, penetrate the cornea and optimize the other important pharmacokinetic properties to lower intraocular pressure. One such compound, aminozolamide (6-amino-2-benzothiazolesulfonamide) is derived from ethoxzolamide. In suspension, it has been shown to lower IOP in rabbits and primates. It also has been shown to lower IOP in patients with ocular hypertension when delivered in a gel vehicle. The carbomer gel vehicle, a drug delivery system also used with pilocarpine (Pilopine HS), is used to prolong ocular contact and enhance penetration. The importance of the role of the vehicle in a topical carbonic anhydrase inhibitor has not been assessed.(ABSTRACT TRUNCATED AT 250 WORDS)

Ocular distribution studies of the topical carbonic anhydrase inhibitors L-643,799 and L-650,719 and related alkyl prodrugs.

The ocular distribution of the carbonic anhydrase inhibitors 6-hydroxybenzothiazide-2-sulphonamide (L-643,799) and 6-hydroxybenzothiophene-2-sulphonamide (L-650,719) has been investigated in albino rabbits after conjunctival administration of these compounds or related alkyl prodrugs. The ocular penetration of L-650,719 has been compared in ocular normotensive rabbits and in animals whose intraocular pressure has been experimentally elevated.

Ocular disposition of aminozolamide in the rabbit eye.

We have previously determined that 6-amino-2-benzothiazolesulfonamide (aminozolamide) significantly lowers IOP in rabbits and, more importantly, in ocular hypertensive human subjects. Results from in vitro experiments established that both the inhibitory activity of aminozolamide against carbonic anhydrase B as well as the penetration rate across excised rabbit corneas were equivalent to ethoxzolamide. Consequently, we have investigated the ocular disposition of aminozolamide to explain its activity when instilled topically to the eye. A constant concentration of 67.4 micrograms/ml of drug was applied for 90 min to the left eye of anesthetized rabbits. Drug and metabolite were measured in both aqueous humor and iris/ciliary body over time. The metabolite was collected and purified. Identification using mass spectroscopy, high pressure liquid chromatography (HPLC) and fluorescence scans indicated that the metabolite was 6-acetamido-2-benzothiazolesulfonamide. Relatively high levels of metabolite were identified in the cornea and iris/ciliary body but were much lower in aqueous humor. Tissue concentrations over time for the metabolite in iris/ciliary body were approximately 2-fold higher than levels of metabolite measured in aqueous humor. When compared to drug levels measured in either aqueous humor or iris/ciliary body, metabolite levels in these respective tissues were much higher. It is hypothesized that topical activity is a consequence of both metabolite retention in the iris/ciliary body as well as inhibition of 99+% of carbonic anhydrase. Both of these events must occur over a sufficient time period to effect a significant lowering of IOP.

Aminozolamide gel. A trial of a topical carbonic anhydrase inhibitor in ocular hypertension.

A topical carbonic anhydrase inhibitor, 6-amino-2-benzothiazolesulfonamide (aminozolamide), which is an analogue of ethoxzolamide, was studied in 18 patients with ocular hypertension. Significant lowering of intraocular pressure was achieved with a single 50-microL gel application compared with the untreated control eye. The onset of action occurred within two hours and lasted at least eight hours. No systemic side effects were elicited. Topical carbonic anhydrase inhibitors may be an important alternative in the medical management of glaucoma.

Topical carbonic anhydrase inhibitors. III: Optimization model for corneal penetration of ethoxzolamide analogues.

An analogue series representing modification to the benzene ring of ethoxzolamide has been evaluated for solubility, pKa, partitioning, and permeability across excised rabbit corneas. These physical parameters were correlated to Hammett sigma (para) and/or Hansch pi parameter values for each compound. From these correlations, a mathematical model was developed relating corneal permeability to molecular modifications of ethoxzolamide. A three-dimensional plot of maximum attainable penetration rate versus sigma (para) and pi yielded an optimal range of pi and sigma values from which an optimally penetrating analogue could be designed.

Ethoxzolamide analogue gel. A topical carbonic anhydrase inhibitor.

An analogue of ethoxzolamide, 6-hydroxyethoxzolamide, was synthesized to enhance corneal permeability yet retain carbonic anhydrase inhibitory activity for use in lowering intraocular pressure. In a 1% suspension, the analogue caused a small but statistically significant unilateral reduction of IOP when applied to one eye of normal rabbits. When formulated in a gel vehicle, 6-hydroxyethoxzolamide caused a more prolonged and larger reduction in IOP in normal and ocular hypertensive rabbits compared with its effect in suspension or with the parent compound.

Topical carbonic anhydrase inhibitors.

Ethoxzolamide and several derivatives (1-6) were synthesized and evaluated for carbonic anhydrase inhibition (CAI), solubility, pKa, distribution, and corneal permeability. The 6-hydroxy (5) and, particularly, the 6-chloro (6) analogues have the best combination of properties for penetrating the site of action and reducing intraocular pressure. Both 5 and 6 exhibited topical effectiveness in the normal rabbit, with 6 showing greater potency.