Nanocristais de besifloxacino para o tratamento de conjuntivite bacteriana: preparação, caracterização físico-química e toxicidade / Besifloxacin nanocrystals for treating bacterial conjunctivitis: preparation, physicochemical characterization, and toxicity

A conjuntivite bacteriana tem significante impacto na Saúde Pública. Essa infecção representa mais de um terço das doenças oculares relatadas em âmbito global. É uma doença altamente contagiosa causada por variedade de bactérias aeróbias e anaeróbias. Diferentes antibióticos empregados no tratamento dessa doença têm apresentado elevada incidência de resistência bacteriana. Dentre os antibióticos de última geração, destaca-se o besifloxacino, antibiótico de quarta geração da classe das fluoroquinolonas, indicado exclusivamente para uso oftálmico tópico. Entretanto, esse fármaco possui baixa solubilidade em água, diminuindo sua biodisponibilidade. Tendo em vista superar esse desafio, foi proposta abordagem nanotecnológica para o desenvolvimento de nanocristais desse fármaco. A preparação de nanocristais de besifloxacino empregando moagem via úmida em escala reduzida foi promissora empregando tensoativo Povacoat®. O Diâmetro hidrodinâmico médio (DHM) da partícula foi de aproximadamente 550 nm, com índice de polidispersão (IP) menor que 0,2. Esse resultado permitiu aumentar a solubilidade de saturação em aproximadamente duas vezes em relação a matéria-prima, possibilitando aumentar a velocidade de dissolução desse fármaco e melhorar sua biodisponibilidade e segurança. Além disso, foi validado o método para quantificação do besifloxacino por CLAE, apresentando especificidade, linearidade no intervalo de 20 a 80µg/mL (r= 0,9996), precisão por repetibilidade (DPR= 1,20%, 0,84% e 0,39%), precisão intermediária (DPR= 0,94%) e exatidão 99,03%. Estudo de estabilidade acelerado (90 dias) na condição 40°C±2°C/75%UR±5%UR e estudo de estabilidade de acompanhamento (150 dias) na condição: 25°C ± 2°C / 60% UR ± 5% UR evidenciaram a estabilidade do teor no período avaliado. Ainda, a nanossuspensão de besifloxacino 0,6% m/m (nanocristais) na dose máxima (500 mg/kg) e o estabilizante Povacoat® (750 mg/kg) não apresentaram toxicidade em larvas de G. mellonella. A concentração inibitória mínima (CIM) para a formulação inovadora foi de 0,0960 µg/mL e 1,60 µg/mL frente a Staphylococcus aureus e Pseudomonas aeruginosa, respectivamente, confirmando eficácia in vitro

Bacterial conjunctivitis greatly impacts the population's health, presenting more than a third of eye diseases reported worldwide. It is an infection caused by various aerobic and anaerobic bacteria and is highly contagious. Therefore, it presents a high incidence of bacterial resistance to the antibiotics commonly used for treatment. Among the most recent antibiotics, besifloxacin is a fourth-generation fluoroquinolone antibiotic indicated exclusively for topical ophthalmic use. Due to its importance in treating bacterial conjunctivitis and its low solubility in the water, a nanotechnological approach was proposed to develop besifloxacin nanocrystals. The preparation of besifloxacin nanocrystals using small-scale wet milling was promising using Povacoat® surfactant. The particle's average hydrodynamic diameter (DHM) was approximately 550 nm, with a polydispersity index (IP) of less than 0.2. This result increased the saturation solubility approximately two times concerning the raw material, making it possible to increase the dissolution rate of this drug and improve its bioavailability and safety. In addition, the method for quantification of besifloxacin by HPLC was validated, presenting specificity, linearity in the range of 20 to 80µg/mL (r= 0.9996), precision by repeatability (DPR= 1.20%, 0.84% and 0.39%), intermediate precision (DPR= 0.94%) and accuracy 99.03%. Accelerated stability study (90 days) at 40°C±2°C/75%RH±5%RH condition and follow-up stability study (150 days) at 25°C ± 2°C / 60% RH ± condition 5% RH showed the stability of content in the evaluated period. Furthermore, the 0.6% besifloxacin nanosuspension (nanocrystals) at the maximum dose (500 mg/kg) and the Povacoat® stabilizer (750 mg/kg) did not show toxicity in G. mellonella larvae. The minimum inhibitory concentration (MIC) to innovative formulation was 0.0960 µg/mL and e 1.60 µg/mL against Staphylococcus aureus and Pseudomonas aeruginosa, respectively, confirming in vitro efficacy

Levofloxacin Hemihydrate In Situ Gelling Ophthalmic Solution: Formulation Optimization and In Vitro and In Vivo Evaluation.

Bacterial conjunctivitis is a leading cause of ocular infections requiring short-term therapeutic treatment with frequent administration of drugs on daily basis. Topical dosage forms available in the market for the treatment of bacterial conjunctivitis such as simple drug solutions and suspensions are rapidly eliminated from the precorneal space upon instillation due to tear turn over and nasolacrimal drainage, limiting intraocular bioavailability of drug to less than 10% of the administered dose. To overcome issues related to conventional drop, an effort was made to design and evaluate prolong release ophthalmic solution of levofloxacin hemihydrate (LFH) using ion-sensitive in situ gelling polymer. Gellan gum was used as the in situ gelling agent. Formulations were screened based on in vitro gelation time, in vitro drug release, and stability towards sol to gel conversion upon storage. The prototype formulations exhibiting quick in vitro gelling time (< 15 s), prolonged in vitro drug release (18-24 h), and stability for at least 6 months at 25°C/40% relative humidity (RH) and 40°C/25% RH were evaluated for pharmacokinetic studies using healthy New Zealand white rabbits. Tested formulations were found to be well-tolerated and showed significant increase in AUC0-24 (22,660.39 h ng/mL) and mean residence time (MRT 12 h) as compared with commercially available solution Levotop PF® (Ajanta Pharma Ltd., India)(AUC0-24 6414.63 h ng/mL and MRT 4 h). Thus, solution formulations containing in situ gelling polymer may serve as improved drug delivery system providing superior therapeutic efficacy and better patient compliance for the treatment of bacterial conjunctivitis.

The antibacterial activity of levofloxacin eye drops against staphylococci using an in vitro pharmacokinetic model in the bulbar conjunctiva.

The incidence of fluoroquinolone-resistant staphylococcal isolates from the conjunctival sac is increasing. We compared pharmacological effects of levofloxacin (LVFX) against Staphylococcus epidermidis using an in vitro pharmacokinetic (PK) model simulating the concentration in the bulbar conjunctiva after applying eye drops of 0.5% and 1.5% LVFX. We used S. epidermidis conjunctival sac isolates [minimum inhibitory concentrations (MICs) of LVFX, 0.125 µg/mL]. LVFX-resistant strains were obtained from parental strains after culture with LVFX. The in vitro PK model simulated the concentration in the bulbar conjunctiva following three topical applications of 0.5% or 1.5% LVFX ophthalmic solution (0, 4, and 8 h) to rabbit eyes. Parental and LVFX-resistant strains were exposed to LVFX in the in vitro PK model, and changes in viable bacterial counts were evaluated for 12 h. The MICs of LVFX for the resistant isolates were 2-32 times higher than the parental strain, and those with MICs ≥2 ug/mL had mutations in the quinolone resistance-determining region. The PK model simulation predicts that 1.5% LVFX exerts bactericidal and bacteriostatic effects against strains with MICs of 0.125-2 and 4 µg/mL, respectively, whereas 0.5% LVFX would only be effective against strains with MICs of 0.125-1 µg/mL. The PK model predicts that the 1.5% LVFX ophthalmic solution exhibits a stronger bactericidal effect against resistant staphylococci in the bulbar conjunctiva than the 0.5% LVFX ophthalmic solution.

Preparation and in vitro/in vivo evaluation of antimicrobial ocular in situ gels containing a disappearing preservative for topical treatment of bacterial conjunctivitis.

The study aimed to formulate and evaluate levofloxacin hemihydrate ocular in situ gels along with freshly prepared disappearing preservative reported to be safer to human eyes. Formulae were prepared using thermosensitive (PF127 and PF68) or ion-activated (Gelrite) polymers. They were evaluated for gelation temperature (GT), capacity, content uniformity, pH, rheological behavior, in vitro drug release with kinetic analysis. Best formulae were exposed to storage effect to select the optimum formula that was subjected to different sterilization methods and in vivo evaluation. The prepared disappearing preservative (sodium perborate monohydrate) proved to be active oxidative preservative and compatible with our formulae. F9 (24% PF127, 15% PF 68, 0.5% levofloxacin hemihydrate, and 0.0025% sodium perborate monohydrate) showed prolonged drug release (12 h), acceptable GT, viscosity, and pH. It remained stable over 3 months at two temperatures and was best sterilized by filtration. It showed longer residence time (12 h) in rabbits' eye fluids compared with the Levoxin® eye drops (4 h). This successful attempt of using thermo-gelling system along with a disappearing type of preservatives would allow the use of these systems to achieve sustained release of antimicrobial drugs with minimum risk of eye damage improving patient compliance and treatment efficacy.

Homotrimeric macrophage migration inhibitory factor (MIF) drives inflammatory responses in the corneal epithelium by promoting caveolin-rich platform assembly in response to infection.

Acute inflammation that arises during Pseudomonas aeruginosa-induced ocular infection can trigger tissue damage resulting in long term impairment of visual function, suggesting that the appropriate treatment strategy should include the use of anti-inflammatory agents in addition to antibiotics. We recently identified a potential target for modulation during ocular infection, macrophage migration inhibitory factor (MIF). MIF deficiency protected mice from inflammatory-mediated corneal damage resulting from acute bacterial keratitis. To gain a better understanding of the molecular mechanisms of MIF activity, we analyzed the oligomeric states and functional properties of MIF during infection. We found that in human primary corneal cells infected with P. aeruginosa, MIF is primarily in a homotrimeric state. Homotrimeric MIF levels correlated with the severity of infection in the corneas of infected mice, suggesting that the MIF homotrimers were the functionally active form of MIF. During infection, human primary corneal cells released more IL-8 when treated with recombinant, locked MIF trimers than when treated with lower MIF oligomers. MIF promoted P. aeruginosa-induced IL-8 responses via the formation of caveolin-1-rich "signaling hubs" in the corneal cells that led to elevated MAPK p42/p44 activation and sustained inflammatory signaling. These findings suggest that inhibiting homotrimerization of MIF or the functional activities of MIF homotrimers could have therapeutic benefits during ocular inflammation.

Investigation of tear film change after recovery from acute conjunctivitis.

PURPOSE: To study the changes of tear film after recovery from acute conjunctivitis. METHODS: This study involved 73 eyes of 56 consecutive patients who complained of dry eye after recovery from acute conjunctivitis at the Zhongshan Ophthalmic Center. Excluded were other factors that could affect the stability of the tear film. Tear film breakup time (BUT), Schirmer 1 test (S1T), tear meniscus height (TMH), and fluorescein staining (FL) were performed on both recovered and healthy eyes. The scores were measured at 3, 7, 14, 21, and 30 days after recovery. RESULTS: Compared with the results of the healthy eyes, most scores of BUT, S1T, TMH, and FL were all abnormal until day 30 after recovery from acute conjunctivitis. BUT decreased at 3, 7, 14, and 21 days (P < 0.05). S1T decreased at 3, 7, and 21 days (P < 0.05). TMH values became less than normal at 3, 7, and 14 days (P < 0.05). FL increased significantly at 7, 14, and 21 days (P < 0.05). At 30 days after recovery, all of the test scores returned to normal (P > 0.05). CONCLUSIONS: During acute conjunctivitis, inflammation and topical therapeutic agents can alter the tear film secretion, resulting in dry eye for nearly 1 month in recovered eyes. To minimize the effect of topical agents to the tear film, individualized treatment instead of frequent instillation of topical agents is recommended for acute conjunctivitis.

Enhanced release of IL-6 and IL-8 into tears in various anterior segment eye diseases.

OBJECTIVE: To determine the levels of interleukin 6 (IL-6) and interleukin 8 (IL-8/CXCL-8) in tears collected from the eyes of normal individuals and of patients with different irritative eye diseases, in order to acquire information on the immunological changes occurring during the early postoperative period following various forms of eye surgery, including penetrating keratoplasty (PKP). METHODS: IL-6 and IL-8 levels were measured with the aid of human ultrasensitive ELISA kits in the non-stimulated tears of patients in the early postoperative period following PKP or cataract operation, and of patients with acute bacterial conjunctivitis or with a corneal foreign body. The IL-6 and IL-8 concentrations, the total amounts released in a given time and the rates of their release were calculated. RESULTS: A significant increase in IL-6 release was observed in all patient groups compared with the normal controls (p < or = 0.003). The IL-8 release levels were significantly higher in the tears of all patient groups (p < or = 0.03), except for the cataract operation group, where the IL-8 release was not significantly higher (p = 0.053) than in the control samples. No significant differences in IL-6 or IL-8 release were observed when the various patient groups were compared with each other. CONCLUSION: The release of IL-6 and IL-8 into the tears is enhanced in various anterior segment eye diseases, and this may be used as an indicator of various inflammatory reactions in the early postoperative period.

Quantitative determination of histamine in tears during conjunctivitis by a novel HPLC method.

The histamine content of tears of healthy sex- and age-matched subjects and patients affected by allergic or nonallergic inflammatory ocular diseases was determined through a new competitive reverse-phase high-performance liquid chromatography (HPLC) method. Tear samples from 50 healthy subjects, 30 patients affected by seasonal allergic conjunctivitis, 12 patients with bacterial conjunctivitis associated with Haemophilus influenzae and 8 patients with bacterial conjunctivitis associated with Streptococcus pneumoniae were analyzed for histamine concentration by O-phthaldialdehyde precolumn derivatization-based HPLC. In physiological conditions, the tear histamine content was low (2.26 ng/ml) and did not vary in relation to age and sex. Histamine levels were significantly higher in all the patients studied, to a greater extent in those affected by allergic (23.61 ng/ml) or Haemophilus influenzae-associated (21.53 ng/ml) conjunctivitis.

[Cytokine status of patients with chlamydial conjunctivitis]. / Tsitokinovyi status bol'nykh khamidiinym kon''iunktivitom.

The study of cytokine status in 37 patients with chlamydial conjunctivitis revealed its noticeable alteration: increment of production of serum IL-16 and IL-14 and IL-1 beta, IL-6 and TNF-alpha in the lacrymal fluid. The investigation of the cytokine status in patients with chlamydial conjunctivitis can be a valuable addition in the complex estimation of immunity to perform immunomodulating therapy.

Bacterial conjunctivitis in Muc1 null mice.

PURPOSE: In contrast to wild-type mice, genetically engineered Mucin1 (Muc1) null animals display a marked propensity for development of blepharitis and conjunctivitis. Molecular approaches confirmed the presence of Muc1 mRNA and protein in the conjunctival tissue of wild-type mice and identified the bacterial species in Muc1 null symptomatic mice. METHODS: Muc1 null animals housed in a conventional facility were examined for visually apparent inflammation of the eye and surrounding tissue. Blood taken from overtly affected animals was assayed for antibodies to common murine viral agents. Swabs of infected eyes and whole eye preparations were used to detect and speciate bacterial pathogens. Frozen sections of whole eye, lid margin, and Harderian gland were immunostained with antibodies to Muc1 and cytokeratin 14, both epithelial cell markers. Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) were performed on RNA isolated from conjunctiva and Harderian gland of wild-type mice to compare relative levels of transcript. RESULTS: Student's unpaired t-test performed on the eye inflammation frequency of Muc1 null mice confirmed a statistical significance (P < 0.01) when compared to wild-type background animals housed in the same room. Analysis of blood samples from affected Muc1 null animals detected no common murine viral pathogens. Bacterial analysis of conjunctival swabs and whole eye preparations demonstrated the presence of coagulase-negative Staphylococcus, Streptococcus type alpha, and Corynebacterium group G2. Muc1 antibody staining of wild-type sections revealed the presence of Muc1 on conjunctival goblet and non-goblet cells and on the epithelium of the Harderian gland. Serial sections stained with cytokeratin 14 antibody confirmed the epithelial nature of cells expressing the Muc1 protein. RNA from conjunctiva and Harderian gland subjected to RT-PCR and northern blot analysis showed an abundance of Muc1 transcript in these tissues. CONCLUSIONS: Muc1 mRNA and protein are present in murine conjunctival and Harderian gland epithelia. Animals lacking Muc1 mRNA and protein are predisposed to developing eye inflammation when compared to wild-type animals with an intact Muc1 gene. Muc1 appears to play a critical protective role at the ocular surface, presumably by acting as a barrier to infection by certain bacterial strains.

Collagen content and types in trachomatous conjunctivitis.

PURPOSE: To study alterations in conjunctival collagen in the conjunctiva of patients with active trachoma. METHODS: We studied conjunctival biopsy specimens obtained from nine subjects with active trachoma and from four control subjects. We used immunohistochemical techniques and a panel of monoclonal and polyclonal antibodies directed against types I, III, IV and V collagen. RESULTS: In normal conjunctiva, the staining for types I and III collagen was localised to the substantia propria. Type IV collagen was located in the epithelial and capillary endothelial basement membranes. The staining for type V collagen was absent. In trachoma biopsy specimens, staining for types I and III collagen showed collagen fibrils among epithelial cells, patchy increase in staining intensity in the upper stroma, and thicker and irregularly arranged collagen fibrils in the substantia propria. Staining for type IV collagen showed irregularly thickened epithelial basement membrane. Staining for type V collagen showed patchy staining in the upper substantia propria; it was also noted in the cytoplasm of fibroblasts, in the walls of blood vessels in the substantia propria, and in the walls of accessory lacrimal glands. CONCLUSIONS: Our data indicate new type V collagen formation, and increased types I, III and IV collagen content, in the conjunctiva from patients with active trachoma.

Tear stix tests for leucocyte-esterase, nitrite, haemoglobin, and albumin in normals and in a clinical series.

Tears are absorbed by a tuft of cotton and subjected to stix test for leucocyte-esterase (L), nitrite (N), haemoglobin (H), and albumin (A). Testing of 84 cases of infectious conjunctivitis and 282 normals revealed nosographic sensitivity to L in 89% and a specificity of 98%. By including N (only 26% positive with infectious conjunctivitis) and H the sensitivity rose to 98% while the specificity fell to 95%. A was generally raised in cases of infectious conjunctivitis. An additional number of 607 stix tests were carried out on a clinical series. The reaction was controlled before, during, and after cataract extraction. Conjunctivitis patients were observed for possible infection, the result of antibiotic treatment was studied, and contact lens wearers were controlled for infection. Predominantly stix-positive reaction was noticed in keratitis, allergic conjunctivitis, and ocular prosthesis socket. Predominantly negative reaction was seen in chronic simple conjunctivitis, sicca, scleritis, and iritis, the latter despite pronounced ciliary hyperaemia. Contralateral reflexly induced L and H were rendered probable. H-positive reaction predominated immediately after removal of suture. The tear stix test is easy to carry out, reasonably precise, and valuable in the clinical work.

Levels of erythromycin in tear fluid and serum in infants with conjunctivitis.

38 newborns with purulent conjunctivitis were treated with oral erythromycin ethylsuccinate 25 mg/kg every 12 h for 14 days. 3-4 days after initiation of therapy, erythromycin levels in serum and tear fluid were measured 1 and 12 h after the administration of erythromycin. The level of erythromycin in tear fluid was significantly higher than that in serum 1 and 12 h after administration of the antibiotic. On both occasions the concentrations of erythromycin in tear fluid and in serum exceeded the minimum inhibitory concentration (MIC) in vitro for Chlamydia trachomatis.