The subject matter of the present invention is a non-aqueous pharmaceutical composition in the form of a suspension for oral administration comprising a b-lactam antibiotic and a b-lactamase inhibitor, a process of preparing and use thereof in treating bacterial diseases.

In the prior art, there are oral administration suspensions for children wherein active substances amoxicillin and clavulanic acid are in the powder form. Water should be added in sufficient quantity to the powder-containing packaging to ensure that the product concentration is suitable. Correct product concentration determines the administration of the right dose to the patient. The suspension is prepared ex tempore. The dry powder is stable for 2 years. The prepared suspension is stable for 7 days. The ready-to-use suspension (a drug after adding water) should be stored in the refrigerator (at the temperature from 2°C to 8°C), not longer than 7 days. If treatment for more than 7 days is necessary, the patient must buy another drug packaging. Often patients have difficulty preparing the suspension at the correct concentration because they struggle with the amount of water added. Often, when the patients are young children, the dose is not taken as a whole because the children choke, vomit, which results in the lack of curative effects and an increase in antibiotic resistance.

Often, veterinarians prescribe a human drug for animals, but the suspension contains (fruity) flavour enhancers which are unacceptable for animals.

In animals, the administration of antibiotic tablets twice a day for 7 to 21 days is basically infeasible. Animal owners resort to the administration of drugs in food. The tablet dissolves, it is spit out by the animal, very often it is not eaten or the animal takes it in a bite of food and then spits it out which the owner may not notice. The dose of the administered drug is then incorrect. The drug is ineffective and generates an increase in antibiotic resistance.

Administration of an antibiotic in the form of a solution for intramuscular injection to animals twice a day for 7 to 21 days is also not acceptable for animals because the intramuscular injection is very painful. Moreover, visiting the veterinarian twice a day is a nuisance for the owner.

WO 2008039472 A2 discloses a non-aqueous pharmaceutical composition comprising: amoxicillin (trihydrate), potassium clavulanate, silicon dioxide, colloidal silicon dioxide, medium-chain fatty acid triglycerides (Myritol 318PH), sorbitan monostearate, and BHT. In another variant of the invention, there is also a kit consisting of two containers, wherein, for example, the first container comprises amoxicillin and clavulanic acid, and the second container comprises medium-chain fatty acid triglycerides. The containers of the kit are mixed before the first use. The resulting preparation was shown to be stable for 21 days.

US 5122377 A discloses an oral delivery system for veterinary drugs that are unstable in an aqueous environment. It has been disclosed that an oral, non-aqueous gel can be prepared by combining vegetable or animal oil with silicon dioxide. For example, the composition comprises amoxicillin, peanut oil, cod liver oil and silicon dioxide. In another variant of the invention, the composition comprises one or more triglycerides (preferably capric and caprylic acid triglycerides and/or capric, caprylic and stearic acid triglycerides). For example, the composition for oral administration is in the form of a paste and comprises amoxicillin, Miglyol (capric and caprylic acid triglycerides), cod liver oil, silicon dioxide, a surfactant, and a flavour enhancer. For example, the composition for oral administration is in the form of a paste and comprises amoxicillin, Miglyol (capric and caprylic acid triglycerides), Softisan (capric, caprylic and stearic acid triglycerides), cod liver oil, silicon dioxide, a surfactant, sodium saccharin, and a flavour enhancer and a tBHQ-antioxidant. The composition does not comprise clavulanic acid or another b-lactamase inhibitor. The compositions can be administered with a dosage syringe.

The present application relates to a non-aqueous pharmaceutical composition in the form of a suspension for oral administration comprising both a b-lactam antibiotic and a b- lactamase inhibitor which, as has been shown during stability studies, is stable for 6 months at the temperature of 25 °C and does not lose its antibacterial properties even though it does not comprise preservatives. This allows the drug to be used for a longer time, especially in those patients in whom the administration of tablets or injections is difficult, e.g. children, the elderly and animals. The compositions of the present invention can be packaged in dosage syringes.

To prepare the compositions of the invention, olive oil or C8 and CIO fatty acid triglycerides are used, e.g. Labrafac Lipophile LW 1349 which is a specific liquid fat - easily absorbed, easily metabolised, it is a good, biologically inert source of energy. The form of the drug is a suspension in a lipophilic gel wherein a network structure is formed on the basis of colloidal silica (colloidal silicon dioxide, trade name Aerosil 200) that allows gelling of the liquid phase of the formulation (olive oil or Labrafac Lipophile LW 1349). The composition comprises flavour enhancers suited to the preferences of animals or humans. Alternatively, a mixture of C8 and CIO fatty acid triglycerides and C8-C18 fatty acid mono-, di- and triglycerides, e.g., Labrafac Lipophile WL1349 and Gelucire 43/01, is used to prepare the composition of the invention.

The composition of the invention is intended to treat bacterial diseases in humans and animals. Preferably, the preparation for humans comprises 875 mg of amoxicillin and 125 mg of clavulanic acid per sachet or 400 mg of amoxicillin and 57 mg of clavulanic acid in 5 mL. Preferably, the preparation for animals comprises 20 mg of amoxicillin and 5 mg of clavulanic acid in 1 mL or 100 mg of amoxicillin and 50 mg of clavulanic acid in 1 mL or 200 mg of amoxicillin and 50 mg of clavulanic acid in 1 mL.

The subject matter of the invention is a non-aqueous pharmaceutical composition in the form of a suspension for oral administration comprising a b-lactam antibiotic, a b-lactamase inhibitor, a filler, an antioxidant and a flavour enhancer, characterised in that the filler is a mixture of C8 and CIO fatty acid triglycerides and C8-C18 fatty acid mono-, di- and triglycerides.

Preferably, the b-lactam antibiotic is selected from the group consisting of amoxicillin, benzylpenicillin, phenoxymethylpenicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, ampicillin, pivampicillin, ticarcillin, carbenicillin, azlocillin, mezlocillin, piperacillin, apalcillin, mecillinam, pivmecillinam, cefadroxil, cefaloridine, cefazolin, cefradine, cefaglycine, cefalexin, cefalotin, cefapirin, cefacetrile, cefatril, cefuroxime, cefamandole, cefaclor, cefatrizine, cefonicid, ceforemide, cefotiam, cefprozil, cefotaxime, ceftriaxone, cefoperazone, ceftizoxime, cefsulodine, cefmenoxime, cefpiramide, ceftazidime, cefpirome, cefepime, ceftan, cefmetazole, cefoxitin, cefotetan, imipenem, aztreonam and mixtures thereof and pharmaceutically acceptable salts and hydrates thereof, whereas the b-lactamase inhibitor is selected from the group consisting of clavulanic acid, sulbactam, tazobactam, avibactam, vaborbactam, relebactam and pharmaceutically acceptable salts thereof.

Preferably, the pharmaceutical composition comprises the b-lactam antibiotic and the b-lactamase inhibitor in the amount of 2-25% by weight, and the mixture of C8 and CIO fatty acid triglycerides and C8-C18 fatty acid mono-, di- and triglycerides in the amount of 74-97% by weight.

Preferably, the ratio of the b-lactam antibiotic to the b-lactamase inhibitor is from 2: 1 to 14:1 w/w.

More preferably, the ratio of the b-lactam antibiotic to the b-lactamase inhibitor is 4:1 w/w.

Preferably, the ratio of the C8 and CIO fatty acid triglycerides to the C8-C18 fatty acid mono-, di- and triglycerides in the mixture is 0.75:0.25 w/w.

Preferably, the b-lactam antibiotic is amoxicillin and the b-lactamase inhibitor is clavulanic acid.

Preferably, the antioxidant is alpha-tocopherol.

Preferably, the pharmaceutical composition is stable for 6 months at the temperature of

25°C.

Preferably, the pharmaceutical composition is in a dosage syringe.

Preferably, the pharmaceutical composition is intended for mammals and reptiles.

Preferably, the mammal is a human.

Preferably, the mammal is a cat, a dog, primates (e.g., lemurs, capuchins, gibbons, chimpanzees, gorillas), diprotodonts (e.g., sugar glider, koala, kangaroo), even-toed ungulates (e.g., goats, sheep, camels, pigs, cows, alpacas, llamas), odd-toed ungulates (e.g., horses, ponies, tapirs, zebras), rodents (e.g., mice, rats, chinchillas, hamsters, guinea pig, gerbils), erinaceomorpha (e.g., hedgehogs), lagomorpha (e.g., rabbits), predators (e.g., tigers, pumas, panthers).

Another subject matter of the invention is a process of preparing the pharmaceutical composition of the invention, characterised in that it comprises the steps of: a) a b-lactam antibiotic and a b-lactamase inhibitor are mixed with a flavor enhancer; b) C8-C18 fatty acid mono-, di- and triglycerides are heated to melt and then slightly cooled; c) the molten mass obtained in step b) is mixed with C8 and CIO fatty acid triglycerides and an antioxidant is added; d) products obtained in steps a) and c) are mixed to obtain a homogeneous suspension.

Preferably, the heating temperature in step b) does not exceed 60°C. Preferably, the mixing in step d) is carried out at the speed of 700-800 rpm.

Another subject matter of the present invention is the pharmaceutical composition of the invention for use in treating bacterial diseases.

Another subject matter of the present invention is a non-aqueous pharmaceutical composition in the form of a suspension for oral administration comprising a b-lactam antibiotic, a b-lactamase inhibitor, a filler, a gelling substance, an antioxidant and a flavour enhancer, characterised in that the filler is selected from the group consisting of C8 and CIO fatty acid triglycerides and olive oil, and the gelling substance is colloidal silica.

Preferably, the b-lactam antibiotic is selected from the group consisting of amoxicillin, benzylpenicillin, phenoxymethylpenicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, ampicillin, pivampicillin, ticarcillin, carbenicillin, azlocillin, mezlocillin, piperacillin, apalcillin, mecillinam, pivmecillinam, cefadroxil, cefaloridine, cefazolin, cefradine, cefaglycine, cefalexin, cefalotin, cefapirin, cefacetrile, cefatril, cefuroxime, cefamandole, cefaclor, cefatrizine, cefonicid, ceforemide, cefotiam, cefprozil, cefotaxime, ceftriaxone, cefoperazone, ceftizoxime, cefsulodine, cefmenoxime, cefpiramide, ceftazidime, cefpirome, cefepime, ceftan, cefmetazole, cefoxitin, cefotetan, imipenem, aztreonam and mixtures thereof and pharmaceutically acceptable salts and hydrates thereof, whereas the b-lactamase inhibitor is selected from the group consisting of clavulanic acid, sulbactam, tazobactam, avibactam, vaborbactam, relebactam and pharmaceutically acceptable salts thereof.

Preferably, the pharmaceutical composition comprises the b-lactam antibiotic and the b-lactamase inhibitor in the amount of 2-25% by weight, the C8 and CIO fatty acid triglycerides or olive oil in the amount of 70-89% by weight and colloidal silica in the amount of 3-12% by weight.

Preferably, the ratio of the b-lactam antibiotic to the b-lactamase inhibitor is from 2: 1 to 14:1 w/w.

More preferably, the ratio of the b-lactam antibiotic to the b-lactamase inhibitor is 4:1 w/w.

Preferably, the b-lactam antibiotic is amoxicillin and the b-lactamase inhibitor is clavulanic acid.

Preferably, the antioxidant is alpha-tocopherol.

Preferably, the pharmaceutical composition is stable for 6 months at the temperature of

25°C.

Preferably, the pharmaceutical composition is in a dosage syringe.

Preferably, the pharmaceutical composition is intended for mammals and reptiles. Preferably, the mammal is a human.

Preferably, the mammal is a cat, a dog, primates (e.g., lemurs, capuchins, gibbons, chimpanzees, gorillas), diprotodonts (e.g., sugar glider, koala, kangaroo), even-toed ungulates (e.g., goats, sheep, camels, pigs, cows, alpacas, llamas), odd-toed ungulates (e.g., horses, ponies, tapirs, zebras), rodents (e.g., mice, rats, chinchillas, hamsters, guinea pig, gerbils), erinaceomorpha (e.g., hedgehogs), lagomorpha (e.g., rabbits), predators (e.g., tigers, pumas, panthers).

Another subject matter of the present invention is a process of preparing the pharmaceutical composition of the invention, characterised in that it comprises the steps of: a) a b-lactam antibiotic and a b-lactamase inhibitor are mixed with a flavor enhancer; b) a filler is heated and an antioxidant is added; c) products obtained in steps a) and b) are mixed to obtain a homogeneous suspension; d) a gelling substance is added portionwise to the product obtained in step c) and mixed to cool.

Preferably, the heating temperature in step b) is 35-39°C.

Preferably, the mixing in steps c) and d) is carried out at a speed of 500-800 rpm.

Another subject matter of the present invention is the pharmaceutical composition of the invention for use in treating bacterial diseases.

Brief description of figures

Fig. 1 shows changes in the amoxicillin and clavulanic acid content in the LH-25 preparation during storage.

Fig. 2 shows a comparison of the E. coli ATCC 25922 strain growth inhibition zones caused by the LH-25 formulation in ex tempore studies and after 1, 3 and 6 months of drug storage under accelerated ageing conditions.

Fig. 3 shows a comparison of the S. aureus ATCC 6538 strain growth inhibition zones caused by the LH-25 formulation in ex tempore studies and after 1, 3 and 6 months of drug storage under accelerated ageing conditions.

Fig. 4 shows changes in the amoxicillin and clavulanic acid content in the Ol-L-25 preparation during storage.

Fig. 5 shows changes in the amoxicillin and clavulanic acid content in the 01-0-25 preparation during storage.

Fig. 6 shows changes in the amoxicillin and clavulanic acid content in the LH-125 preparation during storage.

Fig. 7 shows changes in the amoxicillin and clavulanic acid content in the Ol-L-125 preparation during storage.

Fig. 8 shows changes in the amoxicillin and clavulanic acid content in the 01-0-125 preparation during storage.

Fig. 9 shows a comparison of the E. coli ATCC 25922 strain growth inhibition zones caused by the 01-0-125 formulation obtained in ex tempore studies and after 1, 3 and 6 months of drug storage under accelerated ageing conditions.

Fig. 10 shows a comparison of the S. aureus ATCC 6538 strain growth inhibition zones caused by the 01-0-125 formulation in ex tempore studies and after 1, 3 and 6 months of drug storage under accelerated ageing conditions. Fig. 11 shows changes in the amoxicillin and clavulanic acid content in the LH-250 preparation during storage.

Fig. 12 shows changes in the amoxicillin and clavulanic acid content in the Ol-L-250 preparation during storage.

Fig. 13 shows changes in the amoxicillin and clavulanic acid content in the 01-0-250 preparation during storage.

Fig. 14 shows a comparison of the E. coli ATCC 25922 strain growth inhibition zones caused by the 01-0-250 formulation obtained in ex tempore studies and after 1, 3 and 6 months of drug storage under accelerated ageing conditions.

Fig. 15 shows a comparison of the S. aureus ATCC 6538 strain growth inhibition zones caused by the 01-0-250 formulation in ex tempore studies and after 1, 3 and 6 months of drug storage under accelerated ageing conditions.

Fig. 16 shows dosage syringes with the formulations of the invention.

Examples Example 1

Ol-L-250 formulation

Preparing preparations with amoxicillin and clavulanic acid for dogs and cats at the dose of 250 mg/mL, a Labrafac Lipophile WL 1349-based lipophilic gel type Formula for 100 mL of the preparation:

Mixture of amoxicillin with clavulanic acid in the ratio of 4: 1 w/w 25.00 g

Filler (Labrafac Lipophile WL 1349) 75.14 g

Gelling substance (Aerosil 200) 6.38 g

Antioxidant (a-tocopherol) 0.20 g

Corrigens (flavour enhancer) 0.05 g

Process of preparing:

Mix amoxicillin and clavulanic acid with the corrigens. Heat the filler to the temperature of 35- 39°C, add the antioxidant. Mix amoxicillin with clavulanic acid and the corrigens with the filler to obtain a homogeneous suspension by mixing at the speed of 500-800 rpm. Without changing the mixing speed, add Aerosil 200 portionwise. Stir to cool. Then, transfer suitable portions to the unit packaging and secure with an outer foil. Store in a refrigerator.

Example 2

01-0-250 formulation

Preparing preparations with amoxicillin and clavulanic acid for dogs and cats at the dose of 250 mg/mL, an olive oil-based lipophilic gel type

Formula for 100 mL of the preparation: Mixture of amoxicillin with clavulanic acid in the ratio of 4: 1 w/w 25.00 g

Filler (olive oil) 74.70 g

Gelling substance (Aerosil 200) 6.00 g Antioxidant (a-tocopherol) 0.20 g Corrigens (flavour enhancer) 0.05 g

Process of preparing:

Mix amoxicillin and clavulanic acid with the corrigens. Heat the filler to the temperature of 35- 39°C, add the antioxidant. Mix amoxicillin with clavulanic acid and the corrigens with the filler to obtain a homogeneous suspension by mixing at the speed of 500-800 rpm. Without changing the mixing speed, add Aerosil 200 portionwise. Stir to cool. Then, transfer suitable portions to the unit packaging and secure with an outer foil. Store in a refrigerator.

Example 3

LH-250 formulation

Preparing preparations with amoxicillin and clavulanic acid for dogs and cats at the dose of 250 mg/mL, a triglyceride mixture type

Formula for 100 mL of the preparation:

Mixture of amoxicillin with clavulanic acid in the ratio of 4: 1 w/w 25.00 g

Filler (mixture of triglycerides*) 77.00 g

Antioxidant (a-tocopherol) 0.077 g

Corrigens (flavour enhancer) 0.05 g

* mixture of triglycerides comprises Labrafac Lipophile WL1349 and Gelucire 43/01 pellets in a quantity ratio of 0.75:0.25 w/w

Process of preparing:

Mix amoxicillin and clavulanic acid with the corrigens. Heat Gelucire 43/01 pellets to melt, not exceeding the temperature of 60°C. After slightly cooling Gelucire 43/01 pellets, mix the molten mass with Labrafac Lipophile WL 1349. Add the antioxidant to the mixture to obtain the base of the preparation. Mix the amoxicillin and clavulanic acid and the corrigens with the base (mixing speed 700-800 rpm) to obtain a homogeneous suspension. Then, transfer suitable portions to the unit packaging and secure with an outer foil. Store in a refrigerator.

Example 4

Ol-L-125 formulation

Preparing preparations with amoxicillin and clavulanic acid for dogs and cats at the dose of 125 mg/mL, a Labrafac Lipophile WL 1349-based lipophilic gel type

Formula for 100 mL of the preparation:

Mixture of amoxicillin with clavulanic acid in the ratio of 4: 1 w/w 12.5 g Filler (Labrafac Lipophile WL 1349) 82.24 g Gelling substance (Aerosil 200) 8.00 g Antioxidant (a-tocopherol) 0.20 g

Corrigens (flavour enhancer) 0.05 g

Process of preparing:

Mix amoxicillin and clavulanic acid with the corrigens. Heat the filler to the temperature of 35- 39°C, add the antioxidant. Mix amoxicillin with clavulanic acid and the corrigens with the filler to obtain a homogeneous suspension by mixing at the speed of 500-800 rpm. Without changing the mixing speed, add Aerosil 200 portionwise. Stir to cool. Then, transfer suitable portions to the unit packaging and secure with an outer foil. Store in a refrigerator.

Example 5

01-0-125 formulation

Preparing preparations with amoxicillin and clavulanic acid for dogs and cats at the dose of 125 mg/mL, an olive oil-based lipophilic gel type Formula for 100 mL of the preparation:

Mixture of amoxicillin with clavulanic acid in the ratio of 4: 1 w/w 12.5 g Filler (olive oil) 78.05 g

Gelling substance (Aerosil 200) 7.00 g

Antioxidant (a-tocopherol) 0.20 g

Corrigens (flavour enhancer) 0.05 g

Process of preparing:

Mix amoxicillin and clavulanic acid with the corrigens. Heat the filler to the temperature of 35- 39°C, add the antioxidant. Mix amoxicillin with clavulanic acid and the corrigens with the filler to obtain a homogeneous suspension by mixing at the speed of 500-800 rpm. Without changing the mixing speed, add Aerosil 200 portionwise. Stir to cool. Then, transfer suitable portions to the unit packaging and secure with an outer foil. Store in a refrigerator.

Example 6

LH-125 formulation

Preparing preparations with amoxicillin and clavulanic acid for dogs and cats at the dose of 125 mg/mL, a triglyceride mixture type

Formula for 100 mL of the preparation:

Mixture of amoxicillin with clavulanic acid in the ratio of 4: 1 w/w 12.5 g Filler (mixture of triglycerides*) 83.30 g

Antioxidant (a-tocopherol) 0.083 g Corrigens (flavour enhancer) 0.05 g

*mixture of triglycerides comprises Labrafac Lipophile WL1349 and Gelucire 43/01 pellets in the quantity ratio of 0.75:0.25 w/w

Process of preparing:

Mix amoxicillin and clavulanic acid with the corrigens. Heat Gelucire 43/01 pellets to melt, not exceeding the temperature of 60°C. After slightly cooling Gelucire 43/01 pellets, mix the molten mass with Labrafac Lipophile WL 1349. Add the antioxidant to the mixture to obtain the base of the preparation. Mix the amoxicillin and clavulanic acid and the corrigens with the base (mixing speed 700-800 rpm) to obtain a homogeneous suspension. Then, transfer suitable portions to the unit packaging and secure with an outer foil. Store in a refrigerator.

Example 7

Ol-L-25 formulation

Preparing preparations with amoxicillin and clavulanic acid for dogs and cats at a dose of 25 mg/mL, a Labrafac Lipophile WL 1349-based lipophilic gel type Formula for 100 mL of the preparation:

Mixture of amoxicillin with clavulanic acid in the ratio of 4: 1 w/w 2.50 g Filler (Labrafac Lipophile WL 1349) 78.7 g

Gelling substance (Aerosil 200) 10.00 g

Antioxidant (a-tocopherol) 0.20 g

Corrigens (flavour enhancer) 0.05 g

Process of preparing:

Mix amoxicillin and clavulanic acid with the corrigens. Heat the filler to the temperature of 35- 39°C, add the antioxidant. Mix amoxicillin with clavulanic acid and the corrigens with the filler to obtain a homogeneous suspension by mixing at the speed of 500-800 rpm. Without changing the mixing speed, add Aerosil 200 portionwise. Stir to cool. Then, transfer suitable portions to the unit packaging and secure with an outer foil. Store in a refrigerator.

Example 8

01-0-25 formulation

Preparing preparations with amoxicillin and clavulanic acid for dogs and cats at a dose of 25 mg/mL, an olive oil-based lipophilic gel type Formula for 100 mL of the preparation:

Mixture of amoxicillin with clavulanic acid in the ratio of 4: 1 w/w 2.50 g Filler (olive oil) 83.70 g

Gelling substance (Aerosil 200) 8.60 g

Antioxidant (a-tocopherol) 0.20 g Corrigens (flavour enhancer) 0.05 g

Process of preparing:

Mix amoxicillin and clavulanic acid with the corrigens. Heat the filler to the temperature of 35- 39°C, add the antioxidant. Mix amoxicillin with clavulanic acid and the corrigens with the filler to obtain a homogeneous suspension by mixing at the speed of 500-800 rpm. Without changing the mixing speed, add Aerosil 200 portionwise. Stir to cool. Then, transfer suitable portions to the unit packaging and secure with an outer foil. Store in a refrigerator.

Example 9

LH-25 formulation

Preparing preparations with amoxicillin and clavulanic acid for dogs and cats at a dose of 25 mg/mL, a triglyceride mixture type Formula for 100 mL of the preparation:

Mixture of amoxicillin with clavulanic acid in the ratio of 4: 1 w/w 2.50 g Filler (mixture of triglycerides*) 83.30 g

Antioxidant (a-tocopherol) 0.09 g

Corrigens (flavour enhancer) 0.05 g

*mixture of triglycerides comprises Labrafac Lipophile WL1349 and Gelucire 43/01 pellets in the quantity ratio of 0.75:0.25 w/w

Process of preparing:

Mix amoxicillin and clavulanic acid with the corrigens. Heat Gelucire 43/01 pellets to melt, not exceeding the temperature of 60°C. After slightly cooling Gelucire 43/01 pellets, mix the molten mass with Labrafac Lipophile WL 1349. Add the antioxidant to the mixture to obtain the base of the preparation. Mix the amoxicillin and clavulanic acid and the corrigens with the base (mixing speed 700-800 rpm) to obtain a homogeneous suspension. Then, transfer suitable portions to the unit packaging and secure with an outer foil. Store in a refrigerator.

Example 10

Qualitative studies of the drug form with amoxicillin and clavulanic acid

The task, constituting stage VI of the project, involved the durability and stability studies of amoxicillin and clavulanic acid in the formulations of the invention in a 6-month time, by the accelerated ageing method. The aim of stage VI of the study is to select a target pharmaceutical form on the basis of the conducted study and on the basis of the earlier study results.

Preparation of drug forms for durability studies

Drug forms, characterised by good rheology and texture parameters in the previous stages of the study, were prepared in the amounts necessary to conduct durability and stability studies at four time points of a six-month storage period: - ex tempore (immediately after the preparation and cross-linking of the drug form, i.e. no sooner than 24 hours since the preparation of the drug),

- 1 month after the preparation,

- 3 months after the preparation,

- after 6 months of storage.

9 drug forms were prepared: LH-25, Ol-L-25, 01-0-25, LH-125, Ol-L-125, 01-0-125, LH- 250, Ol-L-250, 01-0-250.

Storage of drug forms under accelerated ageing study conditions

Nine prepared formulations were packed in dispensing tubes (dosage syringes). Then, formulation samples were placed in a thermal test chamber (a climatic chamber type KBK- 100W, “Warned” Medical Equipment Plant, Warsaw). Standard humidity and temperature conditions required for durability and stability studies of drugs intended for storage in a refrigerator (accelerated ageing method) were used. Drug forms were stored at the temperature of 25°C at the humidity of 60%.

Structural viscosity study after the preparation of the formulation (ex tempore), after 1, 3 and 6 months of storage

Structural viscosity studies of the formulation were performed immediately after the preparations were produced (ex tempore) and after 1, 3 and 6 months of drug storage under accelerated ageing conditions. The study was performed using a digital cone-plate rheometer (Brookfield CAP 2000+ rheometer, Brookfield). Rheological studies of the formulations were performed on 0.5 cm ³ samples at the temperature of 25 ± 0.5°C. 6 replicates of the measurements were performed for each formulation. Viscosity curves were done in the shear rate range from 79.99 to 920 s ¹ .

Study of the change in the drug substance content immediately after preparation (ex tempore) and after 1, 3 and 6 months of storage

Evaluation studies of the amoxicillin and clavulanic acid content immediately after the preparations were produced (ex tempore) and after 1, 3 and 6 months of drug storage under accelerated ageing conditions were performed. Samples for the studies were prepared as follows:

- Drug forms for cats (kittens) and dogs (puppies) weighing 1 kg, for which the medicinal dose of 1 cm ³ comprises 20 mg of amoxicillin and 5 mg of clavulanic acid: 1 g of the drug form was weighed, dissolved in 20 mL of chloroform and shaken with 50 mL of distilled water for 10 min. After this time, the upper layer was collected with a syringe and filtered through a 0.22 pm pore size filter.

- Drug forms for cats and dogs weighing 10 kg, for which the medicinal dose of 1 cm ³ comprises 100 mg of amoxicillin and 25 mg of clavulanic acid: 1 g of the drug form was weighed, dissolved in 20 mL of chloroform and shaken with 250 ml of distilled water for 10 min. After this time, the upper layer was collected with a syringe and filtered through a 0.22 pm pore size filter. - Drug forms for dogs weighing 20 kg, for which the medicinal dose of 1 cm ³ comprises 200 mg of amoxicillin and 50 mg of clavulanic acid: 1 g of the drug form was weighed, dissolved in 20 mL of chloroform and shaken with 500 mL of distilled water for 10 min. After this time, the upper layer was collected with a syringe and filtered through a 0.22 pm pore size filter.

The evaluation of the amoxicillin and clavulanic acid content in the samples of the prepared formulations was conducted by the spectrophotometric method after the release of the active substances to the acceptor medium (Nicolet Evolution Spectrophotometer, Spectro Lab). Sample absorbance was tested at the wavelength of 272 nm. Based on the previously prepared calibration curve, the concentration of amoxicillin and clavulanic acid in the samples was determined and the dose coefficient in the sample was established as a percentage of the declared value (Q). Then the percentage of the dose change over time (% change) was calculated.

Antibacterial efficacy study after the preparation of the formulations (ex tempore) and after 1, 3 and 6 months of storage

Antibacterial efficacy studies were performed on the prepared formulations ex tempore (immediately after the preparation thereof) and after 1, 3 and 6 months of drug storage under accelerated ageing conditions. 2 standard bacterial strains were used in the studies. One was gram-negative - Escherichia coli ATCC 25922 and the other was gram-positive - Staphylococcus aureus ATCC 6538. Both strains came from the American Type Culture Collection (ATCC) and were in the spectrum of antibacterial activity of amoxicillin combined with clavulanic acid - the active substance in the analysed drug formulations. They may also be potential pathogens for farm and pet animals.

Discs soaked with the tested formulations were prepared as described below. The tested formulations were liquefied at the temperature of 35°C. Then, 20 pL of the formulation was transferred to the surface of a sterile paper disc (Oxoid) and placed in the refrigerator for a period of 30 minutes for the formulation mass to settle. A thin layer of the tested formulation was thus formed on the surface of each disc.

The antibacterial efficacy study was performed by a disc diffusion method which is commonly used to evaluate the antibacterial activity of the preparations comprising antimicrobial substances. The suspension of standard strain cells with the optical density of 0.5 according to the McFarland scale was inoculated on the surface of the Mueller-Hinton (BD) agar medium, on which discs soaked with the tested drug formulations were then placed. The inoculated plates were pre-incubated at room temperature for 30 minutes and then incubated under aerobic conditions at the temperature of 35°C for 16-18 hours. The evaluation of the antibacterial action of the formulation was assessed on the basis of the resulting growth inhibition zones used in the studies of standard bacterial strains. For each of the four time points in the six-month storage period, the study was performed in triplicate. When describing the obtained study results, mean values were used.

The following results were obtained and the following conclusions were drawn from the segment of qualitative studies of the drug form: Drug forms for cats (kittens) and dogs (puppies) weighing 1 kg, for which the medicinal dose of 1 cm ³ comprises 20 mg of amoxicillin and 5 mg of clavulanic acid

LH-25 formulation

The LH-25 formulation is not only characterised by the lowest viscosity, which facilitates dosing and application, but also by the highest physicochemical stability. The mean viscosity of the LH-25 formulation measured for the shear rate range from 440 to 920 s ¹ is 273.81 mPa s, while under the same study conditions the value of 1608.45 mPa s was obtained for the Ol-L-25 formulation and the value of 1180.42 mPa s was obtained for the 01-0-25 formulation. Characteristic variations in viscosity over the 6-month storage period are slight for the LH-25 formulation. After 6 months of storage, mean viscosity of this formulation determined for the shear rate range from 440 to 920 s ¹ is 373.93 mPa s and essentially does not differ from the result obtained in the ex tempore study which is 273.81 mPa s - Table 1.

Table 1. Comparison of the mean viscosity values of the LH-25 drug form after different storage times obtained in the shear rate range from 440 to 920 s ¹ .

The study of the amoxicillin and clavulanic acid content in the LH-25 formulation during the 6-month storage period shows a slight increase in the mean active substance content, wherein the change percentage does not exceed the low value of 3.8% after 6 months of storage under accelerated ageing conditions. With the standard deviation values of up to 13%, this constitutes the basis for drawing a conclusion on the stability of amoxicillin and clavulanic acid in the formulation and thus on its durability. The above is also confirmed by the lack of observed changes in the nature of the UV spectrum and the maximum absorbance of 272 nm unchanged during storage - Table 2, Fig. 1.

Table 2. Mean values of amoxicillin and clavulanic acid concentrations in the LH-25 preparation during storage.

The sizes of the growth inhibition zones of the standard strains - S. aureus ATTC 6538 and E. coli ATCC 25922 caused by the LH-25 formulation after 1, 3 and 6 months of drug form storage under accelerated ageing conditions do not differ from the sizes of the inhibition zones caused by the formulation in the ex tempore studies. The obtained study results clearly indicate that the LH-25 formulation subjected to the accelerated ageing test does not lose its antibacterial properties - Fig. 2 and Fig. 3.

Ol-L-25 formulation

At all four measuring points of the study (ex tempore, after 1, 3, 6 months of storage), the Ol-L-25 formulation is characterised by the highest viscosity. In the ex tempore study, the mean viscosity of this formulation obtained for the shear rate range from 440 to 920 s ¹ is 1608.48 mPa s, while the value of 1180.42 mPa s was obtained for 01-0-25 under the same study conditions and the value of 273.81 mPa s was obtained for LH-25. After a 6-month storage period at the same shear rates for the Ol-L-25 formulation, a mean viscosity value of 1982 mPa s was obtained, while for 01-0-25, it is 1405.28 mPa s, and for LH-25, it is 273.93 mPa s. It should be emphasized that after 3 months of storage, a sharp increase in the viscosity of the Ol-L-25 formulation was noted. At low shear rates from the 79.99-320 s ¹ range, the viscosity values are outside the measuring capabilities of the rheometer used. Also, after 6 months of storage, the Ol-L-25 drug form maintains very high viscosity parameters at low shear rates. In the shear rate range from 79.99 to 200 s ¹ , they are outside the measuring range of the apparatus and can only be measured at the shear rate of 320 s ¹ . Since the Ol-L-25 formulation is characterised by very high viscosity at the lowest shear rates, this translates into quite large initial forces needed to squeeze the formulation out of the tube. This phenomenon is a function of a small amount of active substances in the formulation (20 mg + 5 mg), which affects the liquefaction of the preparation, as well as the high content of excipients, especially Aerosil 200, which contributes to an increase in structural viscosity. Possible mutual influence of Labrafac Lipophile WL 1349 and Aerosil forming a lipophilic gel on the continuous cross-linking of the formulation over time should also be considered. Mean viscosity measured for the shear rate range from 440 to 920 s ¹ in the ex tempore study is 1608.45 mPa s, after 1 month of storage - 1836.01 mPa s, after 3 months - 2081.08 mPa s, and after 6 months - 1982.78 mPa s. - Table 3.

Table 3. Comparison of the mean viscosity values of the Ol-L-25 drug form after different storage times obtained in the shear rate range from 440 to 920 s ¹ .

Mean percent changes in the amoxicillin and clavulanic acid content measured at four time points of the storage period indicate amoxicillin and clavulanic acid durability in this formulation. The percentage of change in the drug substance content, as in the case of the Theologically stable LH-25 formulation, does not exceed the low value of 3,8 % after 6 months of storage in the thermal test chamber. Also in the case of the Ol-L-25 formulation, no changes were observed in the nature of the UV spectrum and maximum absorbance (272 nm) during the accelerated ageing test - Table 4, Fig. 4.

Table 4. Mean values of amoxicillin and clavulanic acid concentrations in the Ol-L-25 preparation during storage.

The sizes of the growth inhibition zones of the standard strains - S. aureus ATTC 6538 and E. coli ATCC 25922 caused by the Ol-L-25 formulation after 1, 3 and 6 months of drug form storage under accelerated ageing conditions do not differ from the sizes of the inhibition zones caused by the formulation in the ex tempore studies. The obtained study results clearly indicate that the Ol-L-25 formulation subjected to the accelerated ageing test does not lose its antibacterial properties.

01-0-25 formulation

The 01-0-25 drug form exhibits lower viscosity parameters than the Ol-L-25 formulation. The 01-0-25 formulation is easily squeezed out of the dosage syringe, it is convenient in application. In ex tempore studies, the mean viscosity of the 01-0-25 formulation obtained for the shear rate range from 440 to 920 s ¹ is 1180.42 mPa s, while for Ol-L-25 the viscosity value of 1608.45 mPa s was obtained under the same conditions. Compared to LH- 25, higher viscosity was noted in turn for 01-0-25. In ex tempore studies, the mean viscosity of the 01-0-25 formulation obtained for the shear rate range from 440 to 920 s ¹ is 273.81 mPa s, and the viscosity of 01-0-25 is 1180.42 mPa s. Despite such a significant difference in viscosity values, the 01-0-25 formulation, like LH-25, can be classified, in terms of rheological parameters, into the group of pharmaceutical preparations with beneficial usage properties. In turn, an increase in the viscosity of this formulation during storage in relation to the initial value (ex tempore), which is visible especially in the studies after 3 months, is not favourable. In the ex tempore viscosity studies in the shear rate range from 440 to 920 s ¹ , the viscosity of 01-0- 25 is 1180.42 mPa s. After 1 month of storage, viscosity under the same conditions is 1073.66 mPa s., after 3 months of storage, the viscosity value of 1686.91 mPa s. was obtained, while after 6 months - 1405.29 mPa s. The above does not in any way translate into the usage properties of the preparation: throughout the entire time of the durability and stability study (6 months), the 01-0-25 formulation is easily squeezed out of the dispenser - Table 5.

Table 5. Comparison of the mean viscosity values of the 01-0-25 drug form after different storage times obtained in the shear rate range from 440 to 920 s ¹ .

The mean amoxicillin and clavulanic acid content in the 01-0-25 preparation during storage changes in relation to the mean initial value by about 5%, with deviations of 9.9. Changes in the mean amoxicillin and clavulanic acid content in relation to the initial value are higher in the case of the 01-0-25 formulation than for the LH-25 formulation which has the best rheological properties in the group of drug forms with the lowest dose of drug substances. Nevertheless, the result of the study of amoxicillin and clavulanic acid content during storage of the 01-0-25 drug form is fully satisfactory and is at an acceptable level of durability and stability of amoxicillin and clavulanic acid in this pharmaceutical preparation - Table 6, Fig. 5.

Table 6. Mean values of amoxicillin and clavulanic acid concentrations in the 01-0-25 preparation during storage.

The sizes of the growth inhibition zones of the standard strains - S. aureus ATTC 6538 and E. coli ATCC 25922 caused by the 01-0-25 formulation after 1, 3 and 6 months of drug form storage under accelerated ageing conditions do not differ from the sizes of the inhibition zones caused by the formulation in the ex tempore studies. The obtained study results clearly indicate that the 01-0-25 formulation subjected to the accelerated ageing test does not lose its antibacterial properties. Discussion of the study results of the drug forms for cats and dogs weighing 10 kg, for which the medicinal dose of 1 cm ³ comprises 100 mg of amoxicillin and 25 mg of clavulanic acid

LH-125 formulation

The LH-125 pharmaceutical preparation is characterised by low viscosity values at both high and low shear rates compared to other preparations for which the medicinal dose of 1 cm ³ comprises 100 mg of amoxicillin and 25 mg of clavulanic acid. In the ex tempore studies, the mean viscosity of LH-125 obtained for the shear rate range from 440 to 920 s ¹ is 509.45 mPa s, while under the same study conditions the value of 1605.91 mPa s was obtained for Ol-L-125 and 1494.32 mPa s for 01-0-125. During the storage of the formulation, a decrease in its mean viscosity value was observed, not affecting the physicochemical stability of the preparation and the application properties thereof. Compared to the mean formulation viscosity of 509.45 mPa· s in ex tempore study conditions, a viscosity value of 415.68 mPa s was obtained after a 6-month storage period - Table 7.

Table 7. Comparison of the mean viscosity values of the LH-125 drug form after different storage times obtained in the shear rate range from 440 to 920 s ¹ .

The amoxicillin and clavulanic acid content changes in relation to the initial values by about 5.9% after a 6-month storage period. The change in the mean value of the amoxicillin and clavulanic acid content exhibits a linear nature increasing over time. The result of the study of the amoxicillin and clavulanic acid content during the LH-125 drug form storage is at an acceptable level of durability and stability of amoxicillin and clavulanic acid in this pharmaceutical preparation - Table 8, Fig. 6.

Table 8. Mean values of amoxicillin and clavulanic acid concentrations in the LH-125 preparation during storage.

The sizes of the growth inhibition zones of the standard strains - S. aureus ATTC 6538 and E. coli ATCC 25922 caused by the LH-125 formulation after 1, 3 and 6 months of drug form storage under accelerated ageing conditions do not differ from the sizes of the inhibition zones caused by the formulation in the ex tempore studies. The obtained study results clearly indicate that the LH-125 formulation subjected to the accelerated ageing test does not lose its antibacterial properties.

Ol-L-125 formulation

The Ol-L-125 formulation has the highest viscosity in the group of the preparations for which the medicinal dose of 1 cm ³ comprises 100 mg of amoxicillin and 25 mg of clavulanic acid. Under ex tempore study conditions, the mean 01-0-125 viscosity obtained for the shear rate range from 440 to 920 s ¹ is 1605.91 mPa· s. Under the same study conditions, the viscosity of the LH-125 formulation is 509.45 mPa s, and the viscosity of the 01-0-125 formulation is 1494.32 mPa· s. The viscosity of the Ol-L-125 formulation is high and reaches values similar to the analogous formulation with a lower content of drug substances (Ol-L-25). While under ex tempore study conditions, for the Ol-L-125 formulation for the shear rate range from 440 to 920 s ¹ , the mean viscosity value obtained was 1605.91 mPa s (Table 9), this value is 1608.48 mPa s for the Ol-L-25 formulation under the same study conditions. The Ol-L-25 formulation has higher viscosity values than OL-L-125 at the lowest shear rates used during the experiment. In the study, after 6 months of storage, the mean value of the Ol-L-25 viscosity at the shear rate of 79.99 s ¹ and 200 s ¹ is very high, impossible to test with the rheometer used because it lies outside its measuring range. For the Ol-L-125 formulation, the viscosity determined after 6 months of storage at a shear rate of 79.99 s ¹ is also high, but measurable and it is 23628.33 mPa s.

At the next shear rate of 200 s ¹ , the viscosity of Ol-L-125 is 7531.17 mPa s. During storage, viscosity value variations do not exceed 10%, which indicates the rheological stability of the formulation. In ex tempore studies, for the shear rate range from 440 to 920 s ¹ , the mean viscosity value of 1605.92 mPa s was obtained for the Ol-L-125 formulation. Under the same study conditions after 1 month of storage, the viscosity of this formulation is 1487.65 mPa s. After 3 months, the value of 1624.58 mPa s was obtained, whereas after 6 months - 1443.83 mPa s - Table 9.

Table 9. Comparison of the mean viscosity values of the Ol-L-125 drug form after different storage times obtained in the shear rate range from 440 to 920 s ¹ .

The content of amoxicillin and clavulanic acid is subject to slight variations, but their stability in the formulation is high. Changes in the content of amoxicillin and clavulanic acid during storage do not exceed 3,7 % in relation to the initial content. This confirms high durability and stability of amoxicillin and clavulanic acid during storage - Table 10, Fig. 7.

Table 10. Mean values of amoxicillin and clavulanic acid concentrations in the Ol-L-125 preparation during storage.

The sizes of the growth inhibition zones of the standard strains - S. aureus ATTC 6538 and E. coli ATCC 25922 caused by the Ol-L-25 formulation after 1, 3 and 6 months of drug form storage under accelerated ageing conditions do not differ from the sizes of the inhibition zones caused by the formulation in the ex tempore studies. The obtained study results clearly indicate that the Ol-L-25 formulation subjected to the accelerated ageing test does not lose its antibacterial properties.

01-0-125 formulation

The viscosity of the 01-0-125 preparation under ex tempore study conditions is lower than the viscosity of Ol-L-125. For the 01-0-125 formulation, for the shear rate range from 440 to 920 s ¹ , the mean viscosity value of 1494.32 mPa s was obtained. Under the same study conditions, for the Ol-L-125 formulation, the value of 1605 mPa s was obtained. Throughout the entire 6-month storage period of pharmaceutical preparations, the determined viscosity values of the 01-0-125 formulation at four time points of the study are as follows: 1494.32 mPa s, 1461.64 mPa s, 1739.52 mPa s and 1395.93 mPa s. For the Ol-L-125 formulation, the following values were obtained under the same conditions: 1605.91 mPa s, 1487.65 mPa s, 1624.58 mPa s and 1443.83 mPa s. Although the viscosity differences of both formulations are at a similar level, 01-0-125 is characterised by favourable rheological parameters: it is well squeezed out of the dosage syringe and well applied. The reason for this fact is related to the behaviour of preparation samples at low shear rates. At the shear rate of 79.99 s ¹ , which is the first shear rate used in the experiment, the viscosity of Ol-L-125 under ex tempore conditions is 26788.33 mPa s, after 1 month of storage - 29480 mPa s, after three months - outside the measuring capabilities of the apparatus and after 6 months - 23628.33 mPa s. For the 01-0-125 formulation, much lower values were obtained at the shear rate of 79.99 s ¹ : in the ex tempore studies, the value of 10009.83 mPa s was obtained, after 1 month of storage - 11941.67 mPa s, after 3 months - 11165 mPa s and after 6 months - 11622.86 mPa s. These differences in viscosity indicate a variation in the flow limit, understood as the shear stress value at which the preparation ceases to be a solid and begins to flow. The 01-0-125 formulation is characterised by much lower values of the flow limit which allows the formulation to deliquesce and makes application easy. As previously demonstrated, changes in the viscosity of the 01-0-125 formulation during storage are not a tendency by nature, but are only subject to fluctuations not affecting the application properties - Table 11.

Table 11. Comparison of the mean viscosity values of the 01-0-125 drug form after different storage times obtained in the shear rate range from 440 to 920 s ¹ .

Changes in the content of amoxicillin and clavulanic acid during storage of the 01-0- 125 formulation do not exceed 2,7 % in relation to the initial content. This indicates high durability and stability of amoxicillin and clavulanic acid during storage - Table 12, Fig. 8.

Table 12. Mean values of amoxicillin and clavulanic acid concentrations in the 01-0-125 preparation during storage.

The sizes of the growth inhibition zones of the standard strains - S. aureus ATTC 6538 and A. coli ATCC 25922 caused by the 01-0-125 formulation after 1, 3 and 6 months of drug form storage under accelerated ageing conditions do not differ from the sizes of the inhibition zones caused by the formulation in the ex tempore studies. The obtained study results clearly indicate that the 01-0-125 formulation subjected to the accelerated ageing test does not lose its antibacterial properties - Fig. 9 and Fig. 10.

Discussion of the study results of the drug forms for dogs weighing 20 kg, for which the medicinal dose of 1 cm ³ comprises 200 mg of amoxicillin and 50 mg of clavulanic acid

LH-250 formulation Like with the previous doses of drug substances, the LH-250 preparation has the lowest viscosity in the group of formulations with the highest dose of amoxicillin and clavulanic acid at all shear rates. The mean LH-250 viscosity for the shear rate range from 440 to 920 s ¹ is 635.11 mPa s under ex tempore study conditions. Under the same study conditions, the 01 -L- 250 formulation has the viscosity of 1885.67 mPa s, and the 01-0-250 formulation - the viscosity of 1282.07 mPa s. Changes in viscosity over time are visible and exceed 10% of the value during the 6-month storage period. When mean viscosity after producing and cross- linking of the preparation is 635.11 mPa s, it drops to the value of 530.05 mPa s after 1 month, after 3 months it is 570.59 mPa s, and after 6 months - 541.14 mPa s. Proven measured rheological instability has no influence on application difficulties. The preparation is easily squeezed out and dispensed - Table 13.

Table 13. Comparison of the mean viscosity values of the LH-250 drug form after different storage times obtained in the shear rate range from 440 to 920 s ¹ .

Changes in the mean content of amoxicillin and clavulanic acid during storage are slight, they do not exceed 4%. In the case of the LH-250 preparation, high values of standard deviation (even 12.39) should be emphasized, which may indicate the inhomogeneity of the preparation. The above can be eliminated under semi -technical and production conditions during the optimization of the mixing process - Table 14, Fig. 11.

Table 14. Mean values of amoxicillin and clavulanic acid concentrations in the LH-250 preparation during storage.

The sizes of the growth inhibition zones of the standard strains - S. aureus ATTC 6538 and E. coli ATCC 25922 caused by the LH-250 formulation after 1, 3 and 6 months of drug form storage under accelerated ageing conditions do not differ from the sizes of the inhibition zones caused by the formulation in the ex tempore studies. The obtained study results clearly indicate that the LH-250 formulation subjected to the accelerated ageing test does not lose its antibacterial properties.

Ol-L-250 formulation

Like with previous doses of amoxicillin and clavulanic acid, the preparation has the highest viscosity. For the shear rate range from 440 to 920 s ¹ , the mean viscosity value of 1885.67 mPa s was obtained in the ex tempore study for the Ol-L-250 formulation. Under the same study conditions, the LH-250 viscosity is 635.11 mPa s, and the Ol-L-250 viscosity - 1282.07 mPa s. At the measurement points of the storage time, large variations in the value of structural viscosity were noted, indicating the rheological instability of the Ol-L-250 drug form. In the study of the preparation after its production and cross-linking, mean viscosity in the established shear rate range was estimated at 1885.67 mPa s. After 1 month, the viscosity value was 1313.01 mPa s, after 3 months - 1283.42 mPa s, and after 6 months - 1682.38 mPa s - Table 15.

Table 15. Comparison of the mean viscosity values of the Ol-L-250 drug form after different storage times obtained in the shear rate range from 440 to 920 s ¹ .

As in the case of the LH-250 formulation, the change percentage of the amoxicillin and clavulanic acid content in the Ol-L-250 preparation is small and in this case does not exceed 3.71%, which indicates durability and stability in the range of the mean amoxicillin and clavulanic acid content. The standard deviation obtained when measuring the content of amoxicillin and clavulanic acid in Ol-L-250 is much lower than in the case of LH-250, which allows to predict better homogeneity of the Ol-L-250 formulation - Table 16, Fig. 12.

Table 16. Mean values of amoxicillin and clavulanic acid concentrations in the Ol-L-250 preparation during storage.

The sizes of the growth inhibition zones of the standard strains - S. aureus ATTC 6538 and E. coli ATCC 25922 caused by the Ol-L-250 formulation after 1, 3 and 6 months of drug form storage under accelerated ageing conditions do not differ from the sizes of the inhibition zones caused by the formulation in the ex tempore studies. The obtained study results clearly indicate that the Ol-L-250 formulation subjected to the accelerated ageing test does not lose its antibacterial properties.

01-0-250 formulation

The viscosity of 01-0-250 adopts mean values in the group of formulations with the highest content of amoxicillin and clavulanic acid. In the ex tempore study, the mean viscosity of the 01-0-250 formulation in the shear rate range from 440 to 920 s ¹ is 1282.07 mPa s. Under the same study conditions, the viscosity of 635.11 mPa s was obtained for the LH-250 formulation, and for the Ol-L-250 formulation - the viscosity of 1885.67 mPa s. The 01-0-250 drug form is characterised by high rheological stability. Viscosity changes at the time points of the storage process do not exceed 10%. When the mean viscosity of 01-0-250 in the shear rate range from 440 to 920 s ¹ under ex tempore conditions is 1282.07 mPa s, after 1 month of storage the value of 1149.83 mPa s was obtained, after 3 months - 1328.13 mPa s, and after 6 months - 1139.33 mPa s. The 01-0-250 preparation is convenient in application, rheological parameters facilitate its dosing from the dosage syringe - Table 17.

Table 17. Comparison of the mean viscosity values of the 01-0-250 drug form after different storage times obtained in the shear rate range from 440 to 920 s ¹ .

The percentage of change in the concentration of amoxicillin and clavulanic acid during storage of the 01-0-250 formulation is negligible and does not exceed 2.4%, which indicates high stability of amoxicillin and clavulanic acid in the drug form over time - Table 18, Fig. 13.

Table 18. Mean values of amoxicillin and clavulanic acid concentrations in the 01-0-250 preparation during storage.

The sizes of the growth inhibition zones of the standard strains - S. aureus ATTC 6538 and E. coli ATCC 25922 caused by the 01-0-250 formulation after 1, 3 and 6 months of drug form storage under accelerated ageing conditions do not differ from the sizes of the inhibition zones caused by the formulation in the ex tempore studies. The obtained study results clearly indicate that the 01-0-25 formulation subjected to the accelerated ageing test does not lose its antibacterial properties - Fig. 14 and Fig. 15.