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The aim of this study was to develop an in-vitro topical treatment for Acanthamoeba keratitis (AK) effective against cysts and trophozoites. Qualitative assays were performed with voriconazole, chlorhexidine, propamidine, cellulase, tobramycin, ciprofloxacin and paromomycin as monotherapy and various combinations. Riboflavin with ultraviolet-A (R + UV-A) as monotherapy or combined with voriconazole and moxifloxacin was also tested. Quantitative assays to assess cyst viability after treatment were performed for the chemicals that showed the highest activity in the qualitative assays. Paromomycin and propamidine did not show antiamoebic activity. Regardless of the total dose, no amoebicidal effect was observed for R + UV-A. Tobramycin, ciprofloxacin, voriconazole, chlorhexidine and cellulase were selected for quantitative assays because they appeared to cause greater damage to the structure of amoebae. Chlorhexidine and ciprofloxacin were the most active against Acanthamoeba spp. as monotherapy. Among the combinations evaluated, ciprofloxacin-voriconazole-chlorhexidine showed the greatest amoebicidal activity, with severe damage of the cellular membrane and an important decrease in cell concentration. In summary, ciprofloxacin as monotherapy and in combination with voriconazole and chlorhexidine has been classified as promising treatment. Additional in-vivo studies in animal models and clinical trials in patients with AK should be considered to confirm the efficacy of ciprofloxacin.

Propamidine is an aromatic diamidine compound. In the current study, baseline sensitivity of Sclerotinia sclerotiorum to propamidine was determined using 78 strains collected from the oilseed rape fields without a previous history of propamidine usage. The median effective concentration (EC50) values for propamidine inhibiting mycelial growth ranged from 0.406 to 3.647μg/mL, with a mean of 1.616±0.217μg/mL. There was no correlation between sensitivity to propamidine and sensitivity to dimethachlon or carbendazim. After treated with propamidine, mycelia were thinner with irregular distortion and more branches; cell wall became thicker with uneven distribution of cytoplasm than untreated control. In addition, sclerotia production, cell membrane permeability and oxalic acid content significantly decreased. On detached oilseed rape leaves, propamidine exhibited better control efficacy than carbendazim at the same concentration whether the leaves were inoculated with carbendazim-sensitive or resistant strains. All the results showed that propamidine exhibited strong antifungal activity and potential application in controlling S. sclerotiorum. Importantly, these data will provide more information on understanding the mode of action of propamidine against S. sclerotiorum and should be valuable for development of new antifungal drugs.

Purpose To characterize the risk factors, clinical course, treatment outcome and the association between in vivo resistance and in vitro sensitivity for subjects with persistently culture-positive Acanthamoeba keratitis. Design Retrospective noncomparative case series. Participants Eleven subjects with repeatedly positive cultures for Acanthamoeba treated between January 1990 and December 2000, were reviewed. Only subjects with 2 or more positive cultures, availability of the clinical data, and availability of the last Acanthamoeba isolate were included in this study. Methods The medical records were analyzed, and the last isolate from each case was tested in vitro for the antiamoebic drugs used clinically: polyhexamethylene biguanide (PHMB), chlorhexidine, propamidine and hexamidine. Main outcome measures Risk factors, the clinical outcome and in vitro cysticidal drug sensitivity assay. Results Eleven subjects (11/180, 6.1%) had 2 or more positive cultures of whom 8 eyes of 8 subjects (8/180, 4.45%) were included in this study. Seven of eight (87%) subjects were diagnosed over 1 month from onset (late diagnosis). The most common presenting findings were diffuse stromal infiltrate (5/8, 62.5%), ring infiltrate (5/8, 62.5%), and corneal ulceration (3/8, 37.5%). The clinical course of the disease in all subjects consisted of recurrent episodes of corneal and scleral inflammation, with a mean duration of 13.4 ± 9 months. All subjects received PHMB, and 5/8 (62.5%) chlorhexidine too; hexamidine was used in combination in 6/8 (75%), and propamidine in 1/8 (12.5%). All subjects had topical steroids, and 5/8 (62.5%) systemic immunosuppression. The disease resolved with corneal scarring in 3/8 (37.5%) subjects, corneal (or impending) perforation treated with therapeutic keratoplasty in 4/8 (50%), and enucleation in 1/8 (12.5%). Final visual acuity was 0.43 ± 0.37. In vitro most isolates were resistant to propamidine, hexamidine was cysticidal in high concentrations, and PHMB and chlorhexidine had excellent sensitivity profiles. Conclusions In our large series of Acanthamoeba keratitis with a positive microbiologic diagnosis at presentation, nearly 5% developed recurrent episodes of corneal and scleral inflammation with viable Acanthamoeba in the cornea despite prolonged treatment with biguanides and/or diamidines. There was no correlation between in vitro drug sensitivities and the in vivo response for biguanides.

Purpose To characterize patients with Acanthamoeba keratitis and to evaluate the safety and efficacy of propamidine isethionate 0.1% ophthalmic solution (Brolene) when administered concomitantly with neomycin-polymyxin B-gramicidin ophthalmic solution (Neotricin) in the treatment of Acanthamoeba keratitis. Design Prospective, noncomparative case series. Methods The authors report the clinical characteristics and outcomes of patients who entered this multicentered, open-label, clinical trial. Eighty-three patients with Acanthamoeba keratitis representing 87 infected eyes entered the trial. Results Sixty (69%) of the 87 eyes enrolled had data analyzed for treatment efficacy and safety. Of these 60 eyes, 50 (83%) experienced treatment success. Thirty (60%) patients successfully treated adhered to treatment protocol guidelines. Patients who broke protocol had disease exacerbation during the maintenance therapy phase. The only eyes lost/enucleated were 7 of 17 in which penetrating keratoplasty was performed before eradication of the infectious agent. Conclusion Propamidine isethionate and neomycin are an effective treatment for Acanthamoeba keratitis. Penetrating keratoplasty should be performed only for visual rehabilitation and not to "debulk" an active infection. The authors advocate treating patients with topical medications, mainly Brolene, until all organisms are eradicated. There should be no signs of infection for at least 3 months in the patients not receiving antiamebic medications before penetrating keratoplasty is performed.

Macrophages in the liver (Kupffer cells) can be depleted by a single intravenous injection with liposome encapsulated clodronate, an anionic bisphosphonate or by liposome encapsulated propamidine, a cationic diamidine. In the present study, it was investigated whether the efficacy of the liposome mediated depletion of Kupffer cells could be enhanced by modification of the phospholipid bilayers. The efficacy of liposome mediated depletion was evaluated by comparison of the percentages of ED2-positive Kupffer cells in the rat liver, depleted by the drugs encapsulated in the liposomes at different concentrations. It is demonstrated that anionic control liposomes, containing no drug at all and composed of phosphatidylcholine (PC), phosphatidylserine (PS) and cholesterol (C) in a molar ratio of 3:3:1, reduced the percentage of ED2-positive Kupffer cells in the rat liver to about 20% of their normal numbers. At certain drug concentrations, anionic liposomes are shown to be efficacious carriers for intraphagocytic delivery of clodronate, but not for propamidine. Optimal efficacy of the latter drug was achieved by encapsulation into neutral liposomes. Omission of cholesterol or mannosylation of the phospholipid bilayers did not improve the efficacy when compared to neutral liposomes. At high concentrations of encapsulated drugs, all liposome formulations induced a nearly complete depletion of Kupffer cells (>95%).

The hydration of the d(CGCAAATTTGCG)2 duplex and its complex with a propamidine reporter ligand has been examined in aqueous solution by two-dimensional NMR at two spectrometer frequencies and three temperatures. Quantitative analysis of ROESY and NOESY cross-peaks showed effective correlation times of approximately 0.5 ns at 283 K for DNA-water interactions in the major groove. In some cases the sign of the NOE inverts on changing either the temperature or spectrometer frequency. Larger effective correlation times of approximately 1 ns were observed for water interactions with A5(H2) and A6(H2) atoms located in the minor groove. Interproton NOEs and changes in chemical shifts showed that propamidine binds in the minor groove 5'-AATTT region of the host duplex, but does not displace waters adjacent to either A5(H2) or A6(H2). In the complex, the effective correlation times of these waters increase more than two-fold, possibly as a result of stabilisation due to H-bonded interaction with the amidine groups of the ligand. Hydration of the bound molecule was also found, suggesting that water may contribute to the DNA binding process for bis(amidine) drugs. Structure refinement by a NOE-restrained dynamic annealing procedure revealed that ligand binding is non-centrosymmetric with respect to the duplex, in accordance with the energetically favoured 5'-ATT (= 5'-AAT) sites predicted by analytical molecular modelling. In particular, the bound propamidine spans 3-4 base pairs in the A6-T7-T8 tract and makes close H-bonded contacts with A(N3/O4) acceptors positioned close to the minor groove floor. The refined NMR structure for the DNA-propamidine complex is compared with that determined recently using X-ray crystallographic methods.

A molecular modelling strategy has been developed to identify potential binding sites for bis(amidine) ligands in the minor groove of duplex DNA. Calculations of interaction energy for propamidine and pentamidine with d(CGCGAAT TCGCG)2 show that this duplex contains two symmetrically equivalent binding sites of identical affinity, each displaced by 0.3-0.4 bp from the centre of the AT segment. The ligands occupy groove sites spanning approximately 4 and 4-5 bp, respectively with asymmetric binding to the 5'-AATT sequence. The DNA-bis(amidine) interactions have been examined by high-resolution 1H-NMR. The patterns of induced changes in DNA proton chemical shift and the DNA-ligand NOEs confirm that both agents bind in the AT minor groove in a non-centrosymmetric fashion. Detailed structures were determined for each complex using a NOE-restrained simulated annealing procedure, showing that the B-type DNA conformation is not significantly altered upon complexation with either ligand. The free DNA duplex has previously been shown to be extensively hydrated in the minor groove [Kubinec, M.G. and Wemmer, D.E. (1992) J. Am, Chem. Soc. 114, 8739-8740 Liepinsh, E. Otting, G. and Wüthrich, K. (1992) Nucleic Acids Res. 20. 6549-6553]. We detect hydration water close to the A(H2) protons in the presence of propamidine, which may stabilise certain waters against exchange. This conclusion supports recent crystallographic analyses, suggesting that such ligands may use water molecules to bridge between amidinium protons and host DNA bases Details of the ligand interactions with AT-tract DNA duplexes can now be compared for the subsequences 5'-AAT, 5'-AATT and 5'-AAATTT.

The effects of Brolene Tm on adherence of Candida albicans blastospores to buccal epithelial cells and on hyphal development were investigated in vitro. Brolene TM significantly reduced the number of adherent blastospores/ epithelial cell, increased the number of ‘clear’ epithelial cells, decreased percentage germination of blastospores and subsequent hyphal development.

Chromatographic separations of the components of polymyxin B sulphate are reported and it is shown that the separations are such that two of the three major components detected are specific for the intact drug in the presence of chemical decomposition products. The specificity of the separations with respect to other antibacterials, propamidine, dibromopropamidine and trimethoprim is also reported. Validation of the stability indicating assay based on this separation is described. Rate constants for the decomposition of polymyxin at various temperatures between 32 and 52°C and in solutions of different pH values from 2.0 to 10.3 are determined and the t 90 value at pH 7 and 4°C is estimated.

We developed an intensive treatment regimen of topical neomycin, propamidine, and polyhexamethylene biguanide that was tapered to a maintenance level over a 14- to 28-day period as toxicity developed. Since July 1991, we used this treatment on six eyes of five patients in whom Acanthamoeba keratitis was diagnosed clinically. All patients had positive cultures for microorganisms from their corneas or contact lens cases or had pathognomonic findings of pseudodendritic subepithelial infiltrates and radial keratoneuritis. After therapy, all patients improved within two to four weeks, with regression or resolution of neuritis and infiltrates, healing of epithelial defects, and lessening of pain. By three to four months, visual acuity had returned to 20/20 in all eyes. We believe the addition of polyhexamethylene biguanide to our treatment regimen in Acanthamoeba keratitis dramatically aided and hastened the clinical improvement in five consecutive patients and may, with early diagnosis, increase the number of medical cures.

An arsenical resistant cloned line of Trypanosoma brucei brucei was derived from a parent sensitive clone by repeated selection in vivo with the pentavalent melaminophenyl arsenical, sodium melarsen. The melarsen-resistant line was tested in vivo in mice against a range of trypanocidal compounds and found to be cross-resistant to the trivalent arsenicals, melarsen oxide, melarsoprol and trimelarsen (33, 67 and 122-fold, respectively). A similar pattern of cross-resistance was found in vitro using a spectrophotometric lysis assay (greater than 200-fold resistance to melarsen oxide and greater than 20-fold resistance to both trimelarsen and melarsoprol). Both lines were equally sensitive to lysis by the lipophilic analogue phenylarsine oxide in vitro, suggesting that the melamine moiety is involved in the resistance mechanism. Although trypanothione has been reported to be the primary target for trivalent arsenical drugs [1], levels of trypanothione and glutathione were not significantly different between the resistant and sensitive lines. Statistically significant differences were found in the levels of trypanothione reductase (50% lower in the resistant clone) and dihydrolipoamide dehydrogenase (38% higher in the resistant clone). However, the Km for trypanothione disulphide, the Ki for the competitive inhibitor Mel T (the melarsen oxide adduct with trypanothione) and the pseudo-first order inactivation rates with melarsen oxide were the same for trypanothione reductase purified from both clones. The melarsen-resistant line also showed varying degrees of cross-resistance to the diamidines: stilbamidine (38-fold), berenil (31.5-fold), propamidine (5.7-fold) and pentamidine (1.5-fold). Cross-resistance correlates with the maximum interatomic distance between the amidine groups of these drugs and suggests that the diamidines and melaminophenyl arsenicals are recognised by the same transport system.

Polyhexamethylene biguanide (PHMB) is a polymeric biguanide disinfectant that has not previously been used in the treatment of infection. Six patients with confirmed Acanthamoeba keratitis were treated with PHMB 0.02%. All patients had uncontrolled keratitis refractory to therapy with multiple conventional antiamebic agents. The rationale for use and the dose of PHMB was determined by in vitro sensitivity testing of the Acanthamoeba corneal isolates to the drugs available for use. Trophozoite forms were sensitive to most agents. Only PHMB was cysticidal at low concentrations in all cases. Sensitivity to the other drugs, including propamidine, showed wide variation. In 5 of 6 cases, complete resolution of inflammation followed the introduction of PHMB. Toxicity to the ocular surface was not evident with PHMB, unlike propamidine or neomycin. The reasons for the treatment failure in one case, despite cyst sensitivity to both PHMB and propamidine, are not clear. PHMB is a promising new treatment for this infection.

The inhibitory ability of aromatic diamidines has been studied on porcine kidney diamine oxidase. The reversibility of drug-protein interactions has been tested by means of exhaustive dialysis experiments, showing in all cases a reversible binding pattern. Ki values obtained by means of Lineweaver-Burk plots were: stilbamidine 12 microM, 2-OH-stilbamide 8.5 microM, phenamidine 4 microM, propamidine 8 microM, dibromopropamidine 4.9 microM and amicarbalide 12 microM.

1. 1. The amount of valine/glutamic acid and aspartic acid/glutamic acid transaminases in resting cells of Escherichia coli, 15, M2, is not affected by growing the cultures in 10−3M isoniazid, 10−4M carbutamide, 5·10−5M pentamidine or 10−5M propamidine. 2. 2. The amount of β-galactosidase formed in resting cells of E. coli, 15, M2, is not affected when cultures are grown in 10−3M isoniazid, 10−4M carbutamide or 10−5M pentamidine. The amount of this enzyme formed in the logarithmic phase of growth is lower in carbutamide and pentamidine grown cells than in control cultures. 3. 3. The β-galactosidase activity of cell-free extracts of E. coli, 15, M2, is 75% and 99% inhibited by 3.3·10−3M propamidine and pentamidine respectively. The inhibition is competitive. Isoniazid and carbutamide at the same concentration are without effect on the activity of β-galactosidase.

1. 1. The effect of a series of aromatic amidines on arginase activity was investigated. 2. 2. The relative effectiveness of the compounds with respect to their inhibitory activity on arginase was found to be: p -guanylbenzylidene-3-methoxy-4-hydroxyphenylacetone 4,4′-diguanylchalcone isethionate γ-keto-α-guanyl-α-( p -benzamidino)-γ-( o -hydroxyphenyl)propane 4-guanyl-2′-hydroxychalcone isethionate bisamidinomethyldibenzyl bisguanidodiphenylmethane stilbamidine propamidine.

With the demonstration of their trypanocidal action by Lourie and Yorke in 1939 (1), a group of aromatic diamidines, notably stilbamidine (4,4'-stilbenedi-carboxamidine), propamidine (p, p'-trimethylenedibenzamidine) and pentamidine (pp'-pentamethylenedibenzamidine) has found a wide use in the treatment of trypanosomiasis. Subsequent studies revealed that these compounds possess as well activity against other protozoans as babesias and leishmanias, bacteria and fungi. Their development was recently reviewed by Schoenbach and Greenspan (2).

A particulate electron-transport system, consisting primarily of mitochondria and kinetoplasts, was prepared from Crithidia fasciculata by differential centrifugation. The cells were broken by grinding with alumina. The mitochondrial pellet sedimented at 12,000 g after centrifugation at 1,000 g and was used to study possible modes of action of trypanocides. Suramin inhibited succinic dehydrogenase activity 50% at 0.4 m M . Suramin and Antrycide were the most effective drugs against mitochondrial respiration, inhibiting at 1.0 m M . Respiration of the intact cells was most affected by 2-hydroxystilbamidine (81% at 1.0 m M ). Inhibition by other diamidines (e.g., pentamidine, propamidine, and stilbamidine isethionate) was reflected in the oxygen uptake of the intact cells. The energy-yielding reactions of trypanosomes may be the main target of certain trypanocides.

Demonstration of the fungistatic activity and therapeutic efficacy of stilbami-dine and 2-hydroxystilbamidine against Blastomyces dermatitidis in vitro and in vivo prompted an appraisal of the in vitro activity of other diamidine compounds. The objectives of this study were (1) to develop a reproducible in vitro technic for testing the drug sensitivity of fungi; (2) to investigate the mycelial and yeast phase sensitivities of the organisms simultaneously, under conditions as similar as possible; (3) to compare the fungistatic activity of the available diamidine compounds; and, (4) to compare the activity of all drugs employed against a number of strains of B. dermatitidis to determine the extent of strain variation in sensitivity.

The synthesis of propamidinef (4-4' diamidino-diphenoxypropane) was reported in 1942 by Ewins and his co-workers in the investigation of trypanocidal drugs (1). In 1943, the therapeutic activity of propamidine as a topical antiseptic against Cram positive cocci in chronic wound sepsis was demonstrated by Thrower and Valentine (2) and by other clinical investigators (3, 4, 5). In 1945, Elson reported the in vitro sensitivity of Btastomyces dermetitidis to propamidine (6). B. dermafitidis was completely inhibited by 3.75 micrograms of propamidine per cc. of yeast extract agar. Because of the marked in vitro sensitivity of B. dermatitidis and the absence of toxic symptoms in cases of wound sepsis treated with topical application of propamidine, therapeutic trial in eases of cutaneous blastomycosis seemed warranted. It should be emphasized that, in spite of the lack of toxic symptoms reported, when propamidine is used in concentration of 0.1% as a local antiseptic, this drug and related aromatic diamidines produce general toxic effects when given systemically. After the intravenous injection of 2 mgm. of propamidine per kg. of body weight in the treatment of African sleeping sickness, Lourie (7) noted the production of immediate and severe collapse which was alarming but transitory. Late toxic effects have also have been reported with the use of the aromatic diamidines. Delayed kidney and liver damage and dissociated anesthesia of the trigeminal nerve have been particularly serious. An excellent review of these compounds has been published recently by Sehoenbaeh and Greenspaa (5).