WO2002039992A2

WO2002039992A2 - Adducts of quinolonic agents with natural polysaccharide polymers

Info

Publication number: WO2002039992A2
Application number: PCT/IB2001/002171
Authority: WO
Inventors: Piergiorgio Anzaghi; Rosanna Stefli
Original assignee: Pharma Biotech Limited
Priority date: 2000-11-17
Filing date: 2001-11-16
Publication date: 2002-05-23
Also published as: ITMI20002476A1; AU2002212631A1; WO2002039992A3; IT1319664B1

Abstract

Adducts of natural polyssacharide polymers with quinolonic antibacterial agents, in the form of aqueous solutions or solid powder form, possess an identical therapeutic efficacy if compared with the corresponding active ingredient alone, used at the same doses. Therefore they show lower toxicity with equal antimicrobial activity.

Description

ADDUCTS OF QUINOLONIC ANTIBACTERIAL AGENTS WITH NATURAL POLYSACCHARIDE POLYMERS.

FIELD OF THE INVENTION

The present invention concerns adducts of natural polysaccharide polymers with active ingredients belonging to the class of quinolonic drugs with improved therapeutic activity. STATE OF THE ART

Most of the problems that arise during treatment with chemotherapeutics derive from the fact that often the active principles do not possess optimum pharmacological and pharmacokinetic characteristics to carry out their antibacterial action. An approach used in the pharmaceutical field to overcome drawbacks due to poor bioavailability or limited action duration resides in binding the antibiotic molecule to natural or synthetic polymers which are inert and biocompatible. The formation of covalent or ionic bond complexes or conjugates sometimes proves advantageous. EP patent 0392487 in the name of Takeda describes a ionic bond complex between natural sulphonate polymers and an anthracycline which thus proves more stable at neutral pH. In Shionogi patent EP 0438747 a natural soluble polysaccharide is added to a glycopeptide antibiotic to be used as support of the finished product during freeze drying.

Sandoz in EP0428486 prolongs the half-life of the Polymyxin B by binding it to a natural or synthetic polymer with a covalent bond.

The polymer conjugated with the antibiotic can be active as such or, if the "linker" is biodegradable, it behaves as a prodrug that afterwards is bioactivated in vivo, releasing the antibiotic. However, this approach is expensive and complicated since it is necessary to chemically characterize the compound, in so far as it proves to be a new chemical product and therefore study once again its pharmacptoxicology, pharmacokinetics and clinical efficacy. SUMMARY OF THE INVENTION The Applicant has now surprisingly found some adducts of quinolonic antibacterial agents, characterized in that the percentage of said active ingredient is between 40 and 80% by weight calculated on the total weight of the adduct. The adducts according to the present invention are in powder form or in the form of an aqueous solution that preferably contains them in a concentration of between 0.1 and 1% by weight calculated on the total weight of the aqueous solution. The present invention further relates to the process for preparing the aforesaid adducts which, in particular, comprises the following steps: a) preparing the solution of the polysaccharide polymer in water in a percentage of ingredient of between 20 and 60% by weight on the total weight of the adduct and adding the active ingredient, thereby obtaining a concentration of the adduct being preferably comprised in the aforesaid intervals, b) filtering the aqueous solution thus obtained, in case the adduct in aqueous solution form is achieved, c) removing water thereby obtaining the adduct according to the present invention in powder form. The present invention further relates to pharmaceutical compositions containing the adducts according to the present invention in association with suitable excipients and/or diluents. DETAILED DESCRIPTION OF THE INVENTION

The present invention represents an optimum form for administering both orally and parenterally quinolonic antibacterial drugs to be used in the treatment of infections caused by Gram-negative and Gram-positive bacteria.

More specifically, the present invention concerns adducts between an active ingredient, belonging to the class of Quinolones, and a natural biocompatible polysaccharide, able to interact without forming covalent and ionic bonds. The adducts can be administered both orally and parenterally, depending on the physical-chemical characteristics and the pharmacodynamic and pharmacokinetic profile of the active ingredient.

The active ingredients used in the adducts according to the present invention are preferably nalidixic acid, pipemidic acid, cinoxacin, norfloxacin, cyprofloxacin, pefloxacin, enoxacin, ofloxacin and the levo form thereof levofloxacin. These are mainly used in the treatment of infections caused by Gram-negative and positive bacteria and their action mechanism consists in blocking DNA synthesis by inhibiting the DNA enzyme gyrase. More in detail, norfloxacin is effective in urinary, enteric and gonorrhea infections, at a dosage of 800-1200 mg/die through oral administration. Cyprofloxacin is suitable for urinary, respiratory, gastrointestinal and cutaneous infections and in osteomyelitis, since it is effective against Gram-negative organisms (E. Coli, Klebsiella, Salmonella typhi, Neisseria gonorrhoea et meningitidis, Haemophilus influentiae), Gram-positive (Staph. aureus et epidermidis, listeria) and Mycobacterium tubercolosis. It can be orally administered in the corresponding hydrochloride salt with daily doses of 500-1000 mg or, in more serious cases, intravenously in amount of 800-1200 mg/die in the corresponding lactate salt (A. Kucers et. N.Mck Bennett, The use of Antibiotics, 4^th ed., Heinemann Medical Books, 1989).

According to a particularly preferred solution of the present invention, the percentage of the quinolonic antibacterial agent calculated on the total weight of the adduct is 60%. Among the natural polysaccharide polymers dextrans are preferred, inulin and, for oral preparations, maltodextrins of pharmaceutical grade.

Dextrans are polymers formed by linear chains of molecules of α-D- glucose and may exhibit very different molecular weights, ranging from 1000 Dalton (dextran 1 ) to 110000 Dalton (dextran 110) and do not undergo enzymatic degradation. Hydrophilia and therefore the solubility in water decreases with the increase of molecular weight. The dextrans with molecular weights lower than 4000 are completely excreted with urine within 48 hours, while those having higher molecular weights remain in circulation for longer periods of time. Preferred in the present invention are 4-70 dextrans. The maltodextrins consist of a branched polymer of maltose and dextrins in which the D-glucose units are mainly linked by α bond (1-4), but also (1-6) in the branched portions. They show a molecular weights ranging from 900 to 9000 Dalton. Also inulin is a natural polysaccharide linear structure constituted by fructose molecules with a molecular weight of 5000, it does not undergo enzymatic degradation and is excreted with urine, so that it is used in the medical field as a diagnostic means of renal functionality. The selected natural polysaccharides are biocompatible and inert and therefore their quinolones adducts do not present problems with quinolones insofar as it concerns toxicity and also from an immunological stand point. The preparation of the adducts envisages the solution of the polymer in water, in a percentage that may vary between 20 and 60% in weight, and the subsequent addition of the active ingredient, allowing the hydrophilic interaction of carboxylic and aminic groups of the antibacterial agent with those of the polymer. Weak hydrogen type bonds are thus formed with lower interaction forces if compared to covalent or ionic type bonds (Remington's Pharmaceutical Science 18^th ed. p. 186).

The polymer and the drug are dissolved in distilled water (optionally with buffers and preservatives), filtering is performed to obtain a clear solution. Conversely for preparing the adduct in solid form, the solvent is removed from the adduct through a process of freeze-drying or nebulization (spray drying) thereby obtaining the adduct in solid form.

For the preparation of injectable forms, depyrogenated and sterile (w.f.p.) distilled water is used and the solution is filtered, with filters of from 0.1 to 0.2 μm porosity, then placed in depyrogenated and sterilized phialoids in a sterile environment, and preferably freeze-dried. The solution can be readily reconstituted by adding an aqueous solvent such as w.f.p. water or a physiological solution.

Distilled water is used for the preparation of oral forms and the solution is filtered on filters of 0.45 μm porosity. The solvent is removed preferably through nebulization, and a solid porous adduct is obtained suitable for the preparation of pharmaceutical forms for oral use, such as tablets, capsules and granules. The titre of the active ingredient in the adduct was detected by HPLC, using a Perkin-Elmer LC75 chromatograph with an LC Pump 414-T Karitron Analytical injector, Waters detector-integrator and a Lichrosorb column RP18. The in vitro microbiological activity of these adducts compared to that of the corresponding quinolones was determined both by the measurement of inhibition halos developed on plate as well as by the detection of the minimum concentration able to inhibit the bacterial growth in the sample tube. The in vivo activity was assessed in mice previously infected with a strain which is sensitive to the quinolones in question.

These adducts favourably alter the pharmacokinetic and pharmacological characteristics of the antibacterial agent.

In fact, an adduct with a polysaccharide of low molecular weight can increase the solubility of a poorly soluble antibacterial agent and accordingly, if orally administered the same adduct can increase its bioavailibility. In the event of parenteral administration an adduct with a high molecular weight polysaccharide can prolong the time the drug remains in circulation and therefore the same can prolong its effect. Moreover, the presence of reduced active ingredient doses lowers the toxicity with equal antibacterial activity.

The adducts in the form of aqueous solutions according to the present invention can be prepared with a process that in particular comprises the following steps: a') dissolving the active ingredient in distilled water, b') filtering the aqueous solution coming from the previous step, c') removing water, preferably by freeze-drying, from the product coming from in the previous step, d') reconstituting the aqueous solution by addition of water or other w.f.p. solvent, in which the polysaccharide polymer is dissolved, thereby obtaining a concentration of the adduct in said aqueous solution preferably comprised between 0.1 and 1% in weight.

The adducts of the present invention were obtained with simple, quick and economic processes if compared to the traditional synthetic approaches, which envisage the formation of a covalent or saline bond between the antibacterial agent and the polymer, therefore resulting less expensive.

Some examples of the separation of the adducts according to the present invention are reported hereinbelow for illustrative but not limitative purposes.

EXAMPLE 1 - PREPARATION OF THE SOLID ADDUCT 40% MALTODEXTRIN -

60% NORFLOXACIN 0.67 g of 500M maltodextrin are dissolved in distilled water under magnetic stirring. 1 g of norfloxacin is added to the solution while buffering with 1N HCI up to pH 4.8, and distilled water is added up to a final volume of 1 L. The solution, after being filtered on a filter with a porosity of 0.45 μm, is spray dried with a mini spray drier (Mini Buchi). The ejecting pressure is 800 mbar, the inlet temperature is 130°C and outlet temperature is 50°C and the suction is maintained at the maximum of capacity. 1.51 g of ivory coloured adduct are obtained in granular powder form, soluble in water and with a norfloxacin titre of 59.5% by weight. With the addition of suitable excipients the adduct is suitable to be formulated in capsules, sachets, and after being compressed in tablets.

EXAMPLE 2 - Preparation of the solid adduct: 50% dextran - 50% norfloxacin 1 g of dextran 5 is dissolved in 1 L of distilled water and 1 g of norfloxacin is added to the solution, bringing the pH to the value of 4.8 by the addition of 1N HCI. The solution is filtered with a filter of 0.45μm porosity and the solvent is removed by spraydrying as described in example 1. 1.87 g of whitish coloured adduct are obtained in the form of a light powder, with a norfloxacin titre of 49% in weight. The powder added with suitable excipients can be placed in sachets or formulated into capsules or directly compressed.

EXAMPLE 3 - Preparation of the solid adduct: 50% maltodextrin - 50% cyprofloxacin

2 g of 500M maltodextrin are solubilized in 1 L of distilled water under magnetic stirring. 2.5 g of cyprofloxacin lactate are added (corresponding to 2 g of active ingredient) while maintaining under stirring and adding 1N HCI up to total solubilization. The solution is filtered and spraydried following the same conditions of the previous example.

4.2 g of ivory coloured adduct are obtained as a light powder, with a cyprofloxacin titre of 49.7% by weight. The powder added with suitable excipients can be placed into sachets or formulated into capsules or directly compressed.

EXAMPLE 4 - PREPARATION OF THE ADDUCT: 40% INULIN - 60%

CYPROFLOXACIN

1.6 g of inulin are dissolved under magnetic stirring in 800 ml of w.f.p. water and added with 3 g of cyprofloxacin lactate (2.4 g cyprofloxacin) adding of 1N HCI up to complete solubilization. Additional w.f.p. water is added to adjust the total volume to 1 L. The solution is filtered with a sterilizing filter of 0.2 μm porosity and poured in a tray for bulk freeze-drying. The freezer-dryer is programmed to perform the freeze- drying cycle at the following temperatures: -35°C for prefreezing, -10°C during freeze-drying, +10°C for primary drying and +35°C for secondary drying, while maintaining the vacuum at 2.2 0^"2 mbar.

4.53 g of adduct are obtained in the form of a compact liophylized substance, soluble with addition of a physiological solution, and with a cyprofloxacin titre of 58.3% in weight. The bulk adduct is placed in containers under sterile environment conditions under a laminar flow hood. EXAMPLE 5 - Preparation of the solution of the adduct: 40% dextran - 60% cyprofloxacin

1.6 g of dextran 5 are dissolved in a volume of 800 ml of w.f.p. water under magnetic stirring 3.1 g of cyprofloxacin lactate equal to 2.43 g of cyprofloxacin are then added, while adjusting the pH with 1 N HCI until complete solution. The solution is brought to 1.5 L with w.f.p. water, filtered with a sterilizing filter with 0.2 μm porosity and placed in depyrogenated and sterilized vials, in a ratio of 37.5 ml/vial. The cyprofloxacin titre in the solution is 60.5%.

EXAMPLE 6 - PREPARATION OF THE SOLID ADDUCT: 40% DEXTRAN - 60% CYPROFLOXACIN The adduct solutions of example 5 are subjected to freeze-drying following the same cycle described in example 4. The freeze-dried substance obtained is compact with a cyprofloxacin titre of 60.4% by weight. It can be readily reconstituted by slow infusion with the addition of 30 ml of physiological solution. EXAMPLE 7 - Preparation of the solution of the adduct: 40% inulin - 60% cyprofloxacin

2 g of cyprofloxacin lactate, equal to 1.57 g of cyprofloxacin are dissolved in a volume of 500 ml of w.f.p. water under magnetic stirring, while adjusting the pH with 1N HCI until complete solution. The solution is brought to 520 ml with w.f.p. water, filtered with a sterilizing filter of 0.2 μm porosity and placed in depyrogenated and sterilized vials, in a ratio of 20 ml/vial. The vials are placed in a freeze-dryer programmed to perform the cycle described in example 4. The liophylized substance obtained is compact, with a cyprofloxacin titre of 60.3%. Dissolving the contents of each vial in 15 ml of a solvent based on a physiological solution and 40 mg of inulin the extemporaneous adduct solution is reconstituted. EXAMPLE 8 - Determination of the inhibition halos

As for the tests of in vitro antimicrobial activity of norfloxacin and cyprofloxacin, the inhibition halos were obtained with the diffusion method (FUI X ed., p. 135), using a Mueller-Hinton Medium Difco culture medium (with low thymine and thymidine content) conforming to the MG-P standards of the NCCLS in 9 cm diameter sterile Petri dishes and Tryptic soy broth (Difco). Small discs of filter paper were prepared for the sensitivity test, by soaking them in a quinolonic antibacterial agent at defined concentrations, and the corresponding adduct small discs were prepared, in which the total dose is the same, but the active ingredient content by weight is lower.

To produce the bacterial growth the following were used: 1 strain of Staphylococcus aureus ATCC 6538 P (strain A) and 2 field strains of Staphylococcus aureus (strain B and C).

Broth cultures were prepared placing a bacterial patina loop of a culture in agar of 12 h in a test tube containing 5 ml of Sterile Tryptic Soy Broth and then incubated at 37° C for 4 h. If necessary, the suspensions were diluted with sterile water to obtain a similar turbidity to the reference standard (0.5 Mc Fariand). 150 millilitres of Mueller- Hinton Medium were sterilized in autoclave at 121°C for 15,' a part was placed of in sterile Petri plates of 9 cm diameter and left to solidify, a part was cooled to = 45°C and inoculated with 1.5 ml of the bacterial suspensions previously prepared and poured on the surface of the solidified agar in a ratio of 10 ml per plate. The sown plates were kept open to dry under a laminar flow hood for 15' and, after such time, the discs were deposited on the surface of the agar with fine pointed tweezers, previously flame sterilized and cooled. 4 discs were placed in each plate: the adduct and the quinolonic antibacterial agent at the two concentrations that were to be compared. The plates thus prepared, 5 for each test, were incubated upside down at 37°C for 16 h and after this time the inhibition halos were calculated. The results obtained are contained in Table I Table

Antibiotic Adduct Dose Antibacterial agent Adduct halo

(μg) halo inhibition (mm) inhibition (mm) on on strain strain

A B C A B C

Norfloxacin Example 2 5 16.0 17.0 17.4 15.3 16.5 17.0

10 17.0 19.0 18.8 16.1 18.5 17.9

CyprofloxaExample 3 2 21.6 17.4 17.1 20.0 19.5 15.5 cin 6 21.9 18.0 18.0 20.5 20.0 16.0

EXAMPLE 9 - Determination of the in vitro minimum inhibiting concentration (MIC) The MIC values were calculated with the method of dilutions in liquid medium (F. Pasquinelli, Manual for laboratory technicians, vol. II, pp. 1286-89, 1968), using a Mueller-Hinton broth at pH 7.4, sterilized at 120°C for 20 minutes. In a series of test tubes containing a fixed medium volume of medium scalar concentrations by doubling the concentration of the active ingredient were added, from 0.1 μg/ml to 10 μg/ml. The same procedure was also followed for the adducts. A drop of 1 :10 dilution of the broth culture was added to all the test tubes and were incubated at 37°C for 18-24 hours. After this time, the test tube with the highest dilution in which turbidity was not observed was considered as the one containing the minimum inhibiting concentration. The results obtained are contained in Table II.

Table II

EXAMPLE 10 - In vivo activity

Solutions of the two antibacterial agents and the respective adducts ay various dilutions with purified water for oral use were administered to common albino mice each weighing 28 g, every 24 h for five days. They had previously been infected with Staphylococcus aureus (that did not show resistance to the antibacterial agents under consideration), and afterwards they had therefore developed an ulcerative dermatitis with localized abscesses.

After the treatment a bacteriological control was carried out by reisolation, taking some skin swabs of the animals and proceeding with the plate inoculum of the material taken. The results are contained in tables III and IV.

Table III

Table IV

The adducts of the present invention of quinolonic antibacterial agents and natural polysaccharide polymers show, in the in vitro and in vivo tests of antimicrobial activity in mice, a comparable activity or slightly higher than that of the active ingredient alone, although they contain it in lower amounts, thus reducing the cost of the therapy.

This translates into a reduction of adduct toxicity with equal efficacy.

In addition, these adducts are obtained with a quick, economic process which is suitable for the subsequent preparation of parenteral forms and, if combined with suitable excipients for the preparation, of oral forms for the treatment of infections due to Gram-positive and Gram-negative bacteria in animals and humans.

Claims

1. Adduct of a quinolonic type antibacterial agent with a polysaccharide polymer of natural origin, characterized in that the percentage of said active ingredient in the adduct is comprised between 40 and 80% by weight out calculated on the total weight of the adduct.

2. Adduct according to claim 1 wherein it is in the form of an aqueous solution.

3. Adduct according to claim 2 wherein it is contained in said aqueous solution in concentrations of between 0.1 and 1% by weight calculated on the total weight of said aqueous solution.

4. Adduct according to claim 3, wherein it is in powder form.

5. Adduct according to claim 4 wherein it is obtained by spraydrying or freeze- drying the aqueous solution according to claims 2 and 3.

6. Adduct according to any one of claims 1-5, wherein the content of quinolonic type antibacterial agent is equal to 60% by weight calculated on the total weight of the adduct.

7. Adduct according to any one of claims 1-6, wherein the quinolonic type antibacterial agent is selected from the group consisting of nalidixic acid, pipemidic acid, cinoxacin, norfloxacin, cyprofloxacin, pefloxacin, enoxacin, ofloxacin, levofloxacin.

8. Adduct according to any one of claims 1-7, wherein said polysaccharide polymers of natural type are selected from the group consisting of: dextrans, inulin, maltodextrins of pharmaceutical grade.

9. Adduct according to claim 8, wherein dextrans from 4 to 70 are used.

10. Process for preparing the adduct according to any one of claims 1-9, comprising the following steps: a) preparing a solution of the polysaccharide polymer in water in a percentage of between 20 and 60% by weight on the total weight of the adduct, and adding the active ingredient, b) filtering the aqueous solution thus obtained for preparing the adduct in aqueous solution form according to claim 2 or 3; c) removing water thereby obtaining the adduct in powder form according to claim 4.

11. The process according to claim 12 for preparing the adduct in the form of an aqueous solution according to claim 3, wherein in step (a) the concentration of the adduct in said aqueous solution is comprised between 0.1 and 1% in weight.

12. The process according to claim 10, wherein for the preparation of the adduct in the form of a sterile aqueous solution for injectable use in step (a) depyrogenated and w.f.p. sterile distilled water is used and in step (b) the solution is filtered with filters of a porosity from 0.1 to 0.2 μm, and step (c) is carried out by freeze-drying.

13. The process according to claim 10, wherein for the preparation of the adduct to be used for oral use in step (a) distilled water is used, in step (b) the solution is filtered through filters of 0.45 μm porosity, and in step (b) the solvent is removed by spraydrying.

14. Process for preparing the adduct in the form of an aqueous solution according to claim 2 or 3, comprising the following steps: a') dissolving the active ingredient in distilled water, b") filtering the aqueous solution coming from previous step, c') removing water, preferably by freeze-drying, from the product coming from previous step, d') reconstituting an aqueous solution by adding water or other w.f.p. solvent in which the polysaccharide polymer is dissolved.

15. The process according to claim 16, wherein the concentration of the adduct in the aqueous solution coming from step (d') is comprised between 0.1 and 1% in weight.

16. A pharmaceutical composition containing as the active ingredient at least one adduct according to any one of the claims 1-9, in association with suitable excipients and/or diluents.

17. The composition according to claim 15, for oral use in the form of tablets, capsules, granules.

18. The pharmaceutical composition according to claim 15 for injectable use in the form of aqueous solution that can be prepared at the moment of use by the addition of w.f.p. water to the adducts prepared with the process according to claim 11.