Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0048—Eye, e.g. artificial tears
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents

Definitions

• the present invention relates to new pharmaceutical compositions for the specific treatment of ocular infectious diseases, particularly those affecting the ocular surface and its adnexa, and the internal districts of the eye.
• ocular infectious diseases particularly those affecting the ocular surface and its adnexa, and the internal districts of the eye.
• Ophthalmic infections are generally treated with topical compositions (e.g. eye drops or ophthalmic ointments) based on antimicrobial drugs that, applied directly to the eye, enable effective concentrations of the active ingredient to be reached in the affected tissues with a low systemic absorption and a consequently scarce incidence of unwanted systemic side-effects.
• topical compositions e.g. eye drops or ophthalmic ointments
• antimicrobial drugs e.g. eye drops or ophthalmic ointments
• broad-spectrum antibiotics are often used with, a view to preventing the potentially vision-impairing consequences of ocular infections that are not promptly and adequately treated.
• the choice of active ingredient depends on the patient's clinical picture and on the epidemiological data described in the references in relation to the bacterial strains typically involved-in ocular diseases.
• Gram-positive germs are also responsible in approximately 92-97% of cases of endophthalmitis (Recchia FM et al., Arch Ophthalmol, 2005, 123 (3): 341-346).
• endophthalmitis Recchia FM et al., Arch Ophthalmol, 2005, 123 (3): 341-346.
• gram-negative germs particular importance is attributed to H. influenzae, P. aeruginosa (Chalita MR et al., Am J Ophthalmol, 2004, 137 (1), 43- 51).
• the various structural changes made to the first-generation quinolones have given rise to several generations of quinolones (now in their fourth), in which each subsequent generation has been synthesised by adding substitutions in key positions on the molecule's base nucleus with a view-to extending its spectrum of action, improving its efficacy and/or pharmacokinetics, and preventing bacteria from becoming resistant to its effects.
• the "fluoroquinolones” are characterised by fluoridation at position 7 of the quinolone ring and they have a more intense and broader- spectrum antibacterial activity than the previous generation of quinolones.
• Fluoroquinolones have an excellent bioavailability when administered orally and parenterally, and their kinetics are excellent, with an effective endocellular penetration and a good tissue distribution.
• Gemifloxacin is a latest-generation fluoroquinolone used in the treatment of respiratory infections, such as acquired pneumonia and acute exacerbations of-chronic bronchitis. This antibiotic proves active against Streptococcus- pneumoniae and against the gram-negative respiratory pathogens (Haemophilus influenzae, Moraxella catarrhalis) andatypical pathogens such as Chlamydia pneumoniae, Legionella pneumophila and
• Mycoplasma pneumoniae Like other latest-generation fluoroquinolones (e.g. moxifloxacin, gatifloxacin), gemifloxacin has a dual mechanism of action (inhibiting the topoisomerases Il and IV, i.e. "gyrase” and “polyisomerase”, respectively), which makes it more difficult for the micro-organism to develop a resistance because two genetic mutations have to take place simultaneously for them to succeed in contrasting the effect of the antibiotic.
• gyrase i.e. "gyrase” and "polyisomerase”
• the fluoroquinolones are broad-spectrum antibiotics that have recently been applied in the ophthalmologic field too.
• the topical ophthalmic use of fluoroquinolones is generally associated, however, with ocular tissue toxicity, the severity of which is directly proportional to the concentration used in clinical practice (generally 0.3%-0.5%). This toxicity takes the form of corneal opacification, late corneal scarring and reepithelization, retinal toxicity, and so on.
• the fluoroquinolones are active ingredients that are fairly soluble in aqueous media, which acquire a moderately acid pH (4-5): although these acidity levels can be restored to the physiological levels of the lacrimal fluid, they are responsible for such products being scarcely tolerated at ocular level.
• compositions are characterised in that they contain gemifloxacin plus a polymer component chosen from among the following: polyethylene glycol, polypropylene glycol, polyvinylpyrrolidone, a block copolymer consisting of polyethylene glycol and polypropylene glycol, mixtures of polyethylene glycol with polyethoxylated fatty acids (or PEG/polyethoxylated fatty acid mixtures), cellulose or-carbomers.
• the compositions may also contain buffer, isotonizing and preserving agents.
• the ophthalmic preparations thus obtained show a remarkable selectivity of action on a specific compartment of the eye, depending on the polymer used, which gives rise to a particular distribution (penetration and accumulation) of the active ingredient on the superficial ocular tissues or in the internal districts of the eye.
• These compositions consequently enable a highly-effective, focused antibiotic-treatment with an ample margin of safety, suitable for preventing or treating bacterial ocular infections.
• Figure 1a Gemifloxacin accumulation in cornea after- treatment with different gemifloxacin compositions in different dosage regimens.
• Figure 1b Gemifloxacin .accumulation in aqueous humour after treatment with different gemifloxacin compositions in different dosage regimens.
• Figure 1c Standard spectrofiuorimetric curve for gemifloxacin
• Figure 2 Reduction of bacterial load after treatment with different gemifloxacin compositions in different dosage regimens in a model of keratitis.
• the object of the invention is a number of pharmaceutical compositions for ophthalmic use comprising gemifloxacin in a suitable solvent plus a polymer component chosen from among the following: polyethylene glycol, polypropylene glycol, polyvinylpyrrolidone, a block copolymer of polyethylene glycol and polypropylene glycol, mixtures of polyethylene glycol with polyethoxylated fatty acids, cellulose and carbomers, and possibly also buffer, isotonizing and preserving agents.
• a polymer component chosen from among the following: polyethylene glycol, polypropylene glycol, polyvinylpyrrolidone, a block copolymer of polyethylene glycol and polypropylene glycol, mixtures of polyethylene glycol with polyethoxylated fatty acids, cellulose and carbomers, and possibly also buffer, isotonizing and preserving agents.
• Gemifloxacin is used in its base form or, preferably, in the form of a pharmaceutically acceptable derivative, e.g. mesylate. Its concentration in the formulation depends on the type and severity of the condition to treat, and on the site of the infection: as a guideline, its concentration may be between 0.01% and 0.5% w/v, and preferably between 0.05% and 0.3%, or better still between 0.1% and 0.2%, e.g. 0.15%.
• the increase detected in the penetration and accumulation of the active ingredient on a level with the superficial or internal eye tissues is determined distinctively by the polymers used in the compositions forming-the object of ihe-invention, and the consequently greater efficacy of the preparation in these districts enables significantly lower doses of gemifloxacin to be administered than those generally used for fluoroquinolones in ophthalmic clinical practice.
• the quantity of gemifloxacin per single administration comes between 5 and 250 ⁇ g, and preferably between 25 and 150 ⁇ g, or better still between 50 and 100 ⁇ g.
• the invention thus includes ophthalmic compositions as defined above, characterised in that they are suitable for administering said doses of active ingredient.
• the preparations can be-given to patients in single-dose or multiple-dose packaging.
• the suitable polymers can be- used in their various commercially-available molecular weights and in different concentrations, thus enabling compositions of differing viscosity be obtained, suitably-chosen according to known criteria in relation to the preferred. mode of administration.
• polyethylene glycol (PEG) can be added to the mixture in its various molecular weights (e.g. from 300 to 10,000).
• a preferred example is Macrogol® 400, which has a molecular weight coming within the range 380-420; polypropylene glycol (PPG) can be used in molecular weights coming between 600 and 4,000; polyvinylpyrrolidone (Kollidon®, or povidone or PVP, not cross-linked) can be used in its various molecular weight, and particularly between 2,000 and 2,000,000.
• PPG polypropylene glycol
• PVP polyvinylpyrrolidone
• Examples of commercially-available PVP include Kollidon 12 PF, Kollidon 17 PT, Kollidon 25, Kollidon 30 and Kollidon 90 F (with mean molecular weights, Mv, coming between 1 ,000,000 and 1 ,500,000), the last of these being particularly preferred for the purposes of the present invention.
• PVP is used in the compositions in a range of concentrations coming between 1 and 10% w/v, and preferably between 2 and 5%, e.g. 3.5%.
• Block copolymers of polyethylene glycol and polypropylene glycol are commercially available in various molecular weights (e.g. Poloxamer®, Pluronic®, Lutrol®). Lutrol F-127 is particularly preferred, with a molecular weight coming between 9,840 and 14,600. These copolymers are added in quantities preferably coming between 2 and 20% w/v, or better still between 5 and 15%, e.g. 10%. The PEG/polyethoxylated fatty acid mixture is added in quantities preferably coming between 2 and 20% w/v, or better still between 5 and 15%, e.g. 12%.
• Poloxamer® Pluronic®
• Lutrol F-127 is particularly preferred, with a molecular weight coming between 9,840 and 14,600.
• These copolymers are added in quantities preferably coming between 2 and 20% w/v, or better still between 5 and 15%, e.g. 10%.
• the PEG/polyethoxylated fatty acid mixture is added in quantities
• Said mixture is composed of PEG and polyethoxylated fatty acids in w/w ratios preferably coming between 1 :2 and 1:20, or better still between 1 :3 and 1 :7, e.g. 1 :5.
• a preferred example of polyethoxylated fatty acid is Cremophor® RH40 (polyethoxylated castor oil or polyoxyethylene glycerol trihydroxystearate).
• the PEG contained in the mixture can be in various molecular weights, e.g. from 300 to 10,000.
• a preferred example is Macrogol® 400, which has a molecular weight coming within the range 380-420.
• cellulose such as hydroxypropyl cellulose (HPC, " Kiucei®), in a range of molecular weights coming between 80,000 and 1 ,150,000, or hydroxypropyl methyl cellulose (HPMC, Methocel®), in molecular weights coming between 10,000 and 1 ,500,000, or hydroxyethyl cellulose (HEC, Natrosol®) in an apparent viscosity range (measured at 20°C in a 2% solution) coming between 25 and 6.500 mPa x s ⁇ or carboxymethyl cellulose (CMC, Akucell®) in molecular weights coming between 90,000 and 700,000.
• HPMC hydroxypropyl cellulose
• HPMC hydroxypropyl methyl cellulose
• HEC hydroxyethyl cellulose
• CMC carboxymethyl cellulose
• Akucell® carboxymethyl cellulose
• the buffer agent can be chosen from among those used in the ophthalmic sector, e.g. phosphate, phosphate-citrate, Tris, NaOH, histidine, tricine, lysine, glycine, serine, possibly adjusted to the right pH with an acid component; the buffer is added in a sufficient concentration to bring/maintain the pH between 5 and 8, the range compatible with the ocular tissues.
• the isotonizing agent can be chosen from among the known options, e.g. sodium chloride or citric acid, glycerol, sorbitol, mannitol, ethylene glycol, propylene glycol, dextrose; this is added in a range of concentrations coming, for instance, between 0 and 1% w/v, enough to ensure that the formulation is isotonic with the lacrimal fluid (270-310 mOsm/kg).
• the above-mentioned buffer and isotonizing agents are useful and preferred, but they are not indispensable for the purposes of the present invention.
• the preparations forming the object of the invention that are formulated in multiple doses may -also contain antimicrobial preserving agents, such as: parabens, quaternary ammonium salts, poiyhexarnethylene biguanide (PHMB) and any others among those used in compositions for ophthalmic uses.
• the solvent used in the compositions is preferably water or an aqueous solution of one or more components suitable for topical ophthalmic uses.
• compositions in association with the gemifloxacin.
• anti-inflammatory agents may be chosen from among the corticosteroids, nonsteroidal anti- inflammatory drugs (NSAID), selective COX-2 inhibitors and molecules that, though they do not come within these categories, they exert a mechanism of action that is:
• anti-inflammatory in that they interfere with the inflammatory mediators, such as histaminer eicosanoids (prostanoids, leukotrienes), PAF (platelet activating factor), bradykinin, nitric oxide, neuropeptides (neuroquinine A, substance P, calcitonin gene-related peptide [-CGRP]) and cytokines (interleukins, chemokines, interferons, growth factors [GF], tumour necrosis factors [TNF], macrophage (M- CSF) or granulocyte (G-CSF) colony stimulating factors;
• histaminer eicosanoids prostanoids, leukotrienes
• PAF platelet activating factor
• bradykinin nitric oxide
• neuropeptides neuropeptides
• neuropeptides neuropeptides
• cytokines interleukins, chemokines, interferons, growth factors [GF], tumour necrosis factors [TNF], macrophage (
• immunosuppressive i.e. methotrexate, mesalazine and its derivatives (sulfasalazine, balsalazine, ...), cyclosporine, azathioprine, leflunomide, tacrolimus and its analogues (pimecrolimus, sirolimus, gusperimus, everolimus), mycophenolate mofetil, thalidomide, lenalidomide).
• antimicrobial agents that can be included in the preparations forming the object of the invention, the following are listed here as an example: beta- lactamines (penicillin, cephalosporin), macrolides, and natural polypeptides and their fragments with an antibacterial action.
• beta- lactamines penicillin, cephalosporin
• macrolides macrolides
• natural polypeptides and their fragments with an antibacterial action include, for instance, lysozyme, lactoferrin, melanocortin, thrombocidin, and other antimicrobial peptides that can be divided into three main groups according to their formulation and secondary structure: 1) peptides containing cysteines, i.e.
• peptides containing several disulfide bridges that adopt an antiparallel b-sheet structure (defensin, tachyplesin); (b) peptides with a loop structure containing only one disulfide bridge (bactenecin, brevinin, esculentin); 2) peptides containing a high percentage of specific amino acids (PR-39, apidaecin and the proline- and arginine-rich bovine peptides Bac5 and Bac7); 3) iinear-peptides that take on an ⁇ - helical organization in a hydrofobic environment and a random structure when in solution; this group is the most numerous and most thoroughly studied, to which the majority of the "classic" antimicrobial peptides belong, of which there are numerous examples (e.g. cecropin, melittin, magainin, dermaseptin, temporin, bombinin).
• the procedure for preparing the compositions described herein entails mixing gemifloxacin and the chosen polymer, component in a suitable solvent.
• This procedure constitutes a further aspect of the invention.
• the following preparation methods are described: 1) two separate solutions are prepared under stirring at a temperature generally coming between-2°C and 50 0 C: the firstsolution contains the polymer component and a ⁇ pa ⁇ t of any buffer an ⁇ isotonizing agents or other excipients; the second contains the gemifloxacin, any other active ingredient and the remainder of the buffer and isotonizing agent(s). The two solutions are then combined together under stirring to obtain the final formulation.
• the aforesaid compositions are preferably obtained as a solution for ophthalmic use (eye drops, eye baths, etc), but the invention also includes the preparation and use of alternative ophthalmic preparations, e.g. semisolid or solid systems such as eye gels, medicated patches, contact lenses containing the gemifloxacin carried by the aforesaid polymers on their surface or within their mass.
• alternative ophthalmic preparations e.g. semisolid or solid systems such as eye gels, medicated patches, contact lenses containing the gemifloxacin carried by the aforesaid polymers on their surface or within their mass.
• These alternative forms can be achieved with the compositions described herein by suitably adjusting the molecular weight/concentration of the polymer component (and consequently the viscosity of the resulting preparation) and, if necessary, by adding further components.
• the methods for obtaining these alternative compositions are already known to a person skilled in the art.
• compositions described herein are highly-effective antibacterial agents thanks to their capacity to penetrate and accumulate in specific ocular compartments, particularly on a level with the surface tissues or the internal districts of the eye, depending on which. polymer is used. They consequently afford an antibacterial ophthalmic -treatment that is strongly focused and effective, and particularly useful in conditions (diseases and/or surgical procedures) that require the treatment of specific ocular regions.
• a further object of the invention is therefore the use ⁇ of the above-described compositions in the preparation of medication for the treatment and prevention of ocular infections, particularly those affecting the surface and the internal districts of the eye.
• the invention also includes the use of the above-described compositions in the treatment and prevention of ocular infections, particularly affecting the ocular surface and the internal districts of the eye, comprising the administration of a therapeutically effective quantity of the above-described compositions in a patient requiring treatment. Said use also comprises curative treatment, prophylaxis and post-operative antibiotic coverage.
• the compositions forming the object of the invention can also be used in the case of ophthalmic surgery (e.g. refractive surgery, cataract removal, or vitrectomy), with a view to preventing the onset of keratitis or endophthalmitis, which are rare but severe complications associated with ophthalmic surgery.
• compositions for these treatments may, merely as an example, be those containing a block copolymer of polyethylene glycol and polypropylene glycol.
• infective uveitis a condition affecting the internal districts of the eye
• stromal keratitis infections due to deep trauma, corneal ulcers, and treatment after invasive surgery (e.g.- corneal transplants)
• the preferred compositions for these treatments may, merely as an example, be those containing a PEG/polyethoxylated fatty acid mixture.
• the compositions of the invention are also particularly effective in the treatment of keratitis (an infections traditionally difficult to cure without using intensive antibiotic treatments based on front-line drugs, such as cephalosporins or aminoglycosides, in "fortified" compositions, i.e.
• the purpose of the gemifloxacin treatment is to combat the infectious element character-ising-the condition.
• the antibacterial spectrum characterising the preparations forming the object of the invention is the same as the one already known for gemifloxacin.
• Streptococcus pneumoniae gram-negative respiratory pathogens (Haemophilus influenzae, Moraxella catarrhalis), atypical pathogens such as Chlamydia pneumoniae, Legionella pneumophila, and Mycoplasma pneumoniae, and other species including Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Serratia marcescens, etc.
• the marked activity of gemifloxacin achieved by the compositions involved in the invention means that it is also extremely useful to use the latter because the active ingredient formulated with each of the polymers chosen according to the invention still achieves concentrations in the various ocular districts that are always far higher than the minimum inhibitory concentrations (MIC) needed to control the principaLpalhogenic strains affecting the eye (Table 1).
• MIC minimum inhibitory concentrations
• the great efficacy observed for the preparations forming the object of the invention also enables the use of limited doses of active ingredient, with the consequent advantages of a greater ocular tolerability, fewer risks of overdosage and systemic absorption, and lower product costs.
• this greater efficacy- observed for the preparations forming the object of the invention enables a smaller quantity of the drug to be administered in each dose (concentration) ⁇ ap.d/or-a lower frequency of drug, administrations over time, by comparison with-Oonventional treatments (in aqueous solutions).
• the presence of the polymer also enables the ample use of buffer solutions with a physiological or near physiological pH, without running the risk of the active ingredient precipitating.
• the sodium chloride is weighed and dissolved in purified water.
• the resulting solution is divided into two aliquots.
• Kollidon 90 is added to one aliquot and-stirred- for at least 2 hours at ambient temperature until it has dissolved.
• Gemifloxacin is added to the other aliquot of solution under stirring (away from the light).
• the two solutions are then combined under stirring to obtain a monophasic solution (pale yellow in colour, pH 5.4-5.8; osmolarity 270-310 mOsm/kg).
• the resulting formulation is sterilised by-filtering through a polyvinyl difluoridine (PVDF) 0.22 ⁇ m filter.
• PVDF polyvinyl difluoridine
• the citric acid, Na 2 HPO 4 12H 2 O and NaCI are weighed and dissolved in purified water.
• the resulting solution is divided into two aliquots.
• Lutrol is added to one aliquot and stirred for at least 12 hours at a temperature of 2-8°C until it has dissolved.
• Gemifloxacin is added to "the other aliquot and stirred (away from the light) until it has dissolved.
• the two solutions are then combined under stirring to obtain a monophasic solution (pale yellow in colour, pH 5.4-5.8; osmolarity 270- 310 mOsm/kg).
• the formulation obtained is sterilised by filtering through a PVDF (poiivinyldifluoridine) 0.22 ⁇ m filter.
• the citric acid and Na 2 HPO 4 12H 2 O are weighed and dissolved in purified water.
• the Cremophor, PEG and gemifloxacin are first combined together, under stirring, and mixed for at least 30 minutes at ambient temperature (away from the light).
• the resulting semisolid mixture is added to the previously-prepared solution, stirring and heating to 37-4O 0 C, to obtain a monophasic solution (pale yellow in colour, pH 5.4-5.6; osmolarity 270-310 mOsm/kg).
• the formulation obtained is sterilised by filtering through a PVDF (poiivinyldifluoridine) 0.22 ⁇ m filter.
• Example 4 In-vivo efficacy test and tests on accumulation in cornea and aqueous humour
• the animals were stabled in conditions of humidity (40-60% RH), temperature (15-21 0 C) and natural lighting (12 hours of daylight and 12 hours of darkness each day) and they were fed a standard diet in pellet form (Mucedoia - Italia) with access to water ad libitum.
• the animals were weighed and examined by the vet, then a 7-day period of acclimatisation ensued before starting the study, during which time their state of health was monitored by the vet.
• Keratitis was induced using St. aureus 7786 ocular isolate.
• the strain was cultivated on Tryptic Soy Agar (TSA) + 5% sheep blood the night before the lesion was induced. On the first day of the experiment, several colonies were resuspended in PBS to obtain a density corresponding to the 0.5 McFarland standard (10 8 colony forming units/ml). This suspension was then diluted in PBS to obtain a final concentration of 10 5 CFU/ml, ascertained by means of a plate count. The minimum inhibiting concentration (MlC) of gemifloxacin for this ocular isolate (i.e. 0.016 ⁇ g/ml) was determined using the E-test method (see "In vitro antibacterial efficacy tests").
• the animals were anesthetized with intramuscular ketamine (37.5 mg/kg) and xylazine (10 mg/kg) and topical novesin (2 drops in each eye) before they were intrastromaily injected with the bacterial suspension prepared as explained above.
• 50 ⁇ l of bacterial suspension were injected intrastromaily in both corneas of all rabbits using an insulin syringe with a 30 gauge needle.
• the animals were then divided into six treatment groups as follow-Sr •Group 1 (untreated)
• aqueous humour was harvested using a hypodermic syringe. Samples were placed in previously-labelled and weighed tubes. After collecting the samples of aqueous humour, the palpebral conjunctiva was resected, the eyeball was removed and the cornea was harvested. The cornea was then washed with isotonic saline solution and placed in labelled tubes. All the test tubes were then stored at -20 0 C until the time of the analysis.
• the corneas were defrosted and weighed, then homogenised in 2 ml of sterile physiological solution (PS) with 0.1% of peptone at 4 0 C for 30". Of the 2 ml of homogenate, 1 ml was collected, diluted with 9 ml of PS and filtered (having already validated the 1:10 dilution ratio needed ' to inactivate the antibiotics). The filter was then placed on TSA agar plates with 5% of sheep blood. To ensure that the colonies could be counted accurately, the other 1 ml of homogenate was serially diluted in PS and peptone and each dilution was seeded in duplicate on TSA agar plates. After- overnight incubation of the plates at 37°C, the total bacterial load was measured by counting the CFU on the filter and on the plate. The bacterial loads were recorded as CFU/gr of tissue.
• PS sterile physiological solution
• SDS sodium dodecyl sulfate
• a standard curve for gemifloxacin fluorescence in the same medium had already been ascertained in a range of concentrations coming between 0 and 1000 ng/ml: the spectrofluorimetric readings were plotted on a graph against the corresponding concentrations in order to obtain the standard curve.
• One hundred ⁇ l of each sample of aqueous humour or corneal homogenate (coming from the 2 ml used for the bacterial count) were then added to 2 ml of micellar buffer; the fluorescence value obtained was interpolated with the corresponding standard curve and calculated as nanograms per ml or nanograms per g of tissue.
• the experimental data were plotted on a graph for statistical analysis (one-way ANOVA and Dunnet Post Test, Fig. 2).
• the 0.3% gemifloxacin compositions containing Kollidon, Lutrol or Cremophor-PEG enabled a reduction of more than two log units in the bacterial load after 7 administrations (at intervals of. one hour), which is comparable with the results achieved after 14 doses at 30-minute intervals of the 0.3% gemifloxacin -solution with no added polymers.
• FIG. 1a Spectrofluorimetric determination of gemifloxacin accumulation in cornea and aqueous humour
• the results relating to the accumulation of gemifloxacin in cornea and aqueous humour are given in figures 1a and 1b, while Fig. 1c shows the standard curve.
• the data in figure 1a identify a corneal accumulation of the active ingredient that ranges from significant to very high for all the compositions produced according to the invention.
• the compositions containing Lutrol or Cremophor revealed the greatest capacity for gemifloxacin accumulation in the cornea: the levels of active ingredient achieved in the cornea with these compositions were much the same as those reached after administering the aqueous solution of gemifloxacin at the same concentration with no added polymers, but using the double dosage regimen.
• Fig.1 b show an accumulation of gemifloxacin in aqueous humour that ranged from significant to very high for all the compositions prepared according to the invention!
• the compositions containing Cremophor/PEG showed the greatest capacity to accumuiate ' the-active ingredient in this district: in fact, the levels of accumulation were substantially the same as those obtained with the gemifloxacin at the same concentration in the solution containing no polymers, but using twice the dosage.
• the active ingredient remained stable in solution and there was no evidence of any precipitation.
• the method of E-test involves using a thin strip of inert, nonporous plastic loaded with a rising concentration gradient of the various antibiotics used, on which there is a graduated scale for reading the MIC in ⁇ g/ml.
• the strip for the E-test is placed on an agar surface seeded with a bacterial strain, the antibiotic gradient is transferred from the plastic strip onto the matrix on the agar surface.
• a stable, continuous and exponential gradient of concentrations of antibiotic thus forms underneath the strip.
• the MIC is measured directly on the graduated scale on the strip in terms of ⁇ g/ml and it coincides with the reading where the edge of the oval intersects the strip.
• the test was carried out using the E-test for gemifloxacin, moxifloxacin, ciprofloxacin, levofloxacin and ofloxacin using a concentration gradient coming between 0.002 and 32 ⁇ g/ml.
• ocular_bacterial isolates Eighty-five ocular_bacterial isolates were used, including: 27 St. aureus, 29 Sf. epidermidis, 16 coagulase-negative Staphylococci, 8 Ps. aeruginosa and 5 Serraiia marcescens. The following ATCC bacteriaLstrains. were also included: Staphylococcus aureus 25923, Staphylococcus aureus 29213, Staphylococcus aureus 43300, S. epidermidis 35984, Pseudomonas aeruginosa 27853 and Pseudomonas aeruginosa 9027.
• a sterile swab was steeped in the previously-prepared suspension of the inoculation. Excess fluid was removed by pressing the swab against the inside wall of the test tube. After seeding the agar plate with the swab, the E-test strip was placed on the surface of the agar. Different plates were chosen according to the strain being tested, e.g. Mueller Hinton agar plates were used for the Gram- negative strains and the methicillin-sensitive Staphylococci, while Mueller Hinton agar plates with 2% NaCI added were used for the methicillin-resistant Staphylococci.
• Table 1 shows the MIC values recorded for each antibiotic and for each group of bacterial isolates, in terms of MIC50 and MIC90 .

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