WO2018131058A1 - Pharmaceutical composition for the treatment of bee pathologies - Google Patents

Pharmaceutical composition for the treatment of bee pathologies Download PDF

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Publication number
WO2018131058A1
WO2018131058A1 PCT/IT2018/050004 IT2018050004W WO2018131058A1 WO 2018131058 A1 WO2018131058 A1 WO 2018131058A1 IT 2018050004 W IT2018050004 W IT 2018050004W WO 2018131058 A1 WO2018131058 A1 WO 2018131058A1
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WIPO (PCT)
Prior art keywords
acaricide
pharmaceutical composition
mixture
hydrocolloids
oxalic acid
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PCT/IT2018/050004
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French (fr)
Inventor
Emanuele Nizzetto
Original Assignee
Chemicals Laif S.P.A.
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Publication date
Application filed by Chemicals Laif S.P.A. filed Critical Chemicals Laif S.P.A.
Priority to EP18703861.7A priority Critical patent/EP3568013A1/en
Publication of WO2018131058A1 publication Critical patent/WO2018131058A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/36—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids

Definitions

  • Embodiments described here concern an acaricide pharmaceutical composition for the treatment of bee pathologies, in particular of the domestic bee Apis mellifera.
  • embodiments described here concern an acaricide pharmaceutical composition for the treatment of pathologies connected to the Varroa mite (Mesostigmata, varroa destructor).
  • embodiments described here concern the use of the acaricide pharmaceutical composition in a beehive, or in artificial means associated with the reproduction of bees, such as cells, observation hives, or in any other environment suitable for the reproduction of bees.
  • the acaricide pharmaceutical composition for the treatment of bee pathologies in particular of the domestic bee Apis mellifera, according to the embodiments described here, can be provided in liquid form, ready for use, or in the form of a support in strips.
  • Varroa mite (Mesostigmata, varroa destructor) is one of the worst enemies of honey bee (Apis mellifera) colonies. It was identified for the first time in Italy in the early eighties of the last century. In the following years, this parasite spread very rapidly throughout the peninsula, leading to the virtual extinction of all wild colonies of honey bees. Even today this parasite inflicts heavy damage on beekeeping worldwide, except for that carried out in Africa and Australia. The lack of knowledge of potential enemies of this parasite, and the failure of the efforts to eradicate it, oblige bees to coexist forcibly with this mite. This situation obliges beekeepers to carry out therapeutic chemical treatments, which are generally planned and carried out during the beekeeping season in the framework of pest control and which usually involve the use of acaricides.
  • acaricides that are currently registered can be used for therapeutic treatments during the active season of bee colonies but are not suitable for use during winter when the bees hibernate. This despite the fact that winter is the most suitable period for therapy because the characteristics of the parasite's biological cycle can achieve the highest levels of acaricide efficacy, so as to drastically reduce the population of mites and start again with the following season with the lowest level of infestation.
  • One of the few active principles that makes this possible is oxalic acid, which can be administered to the colonies by dripping its sugary solution between the frames occupied by the bees, or by sublimation of its crystals in powder form.
  • Oxalic acid is the most effective and widely used acaricide against the Varroa mite.
  • Applicant's known product Api-Bioxal 886 mg/g soluble powder for bees is the only oxalic acid-based product currently authorized in Italy for combatting bee varroosis.
  • Another great advantage of oxalic acid, the active principle contained in Api-Bioxal, is that it is a natural component of honey, where it can be found in variable concentrations with the botanical origin of the nectar. The treatments carried out correctly with Api-Bioxal in solution do not increase the concentration of oxalic acid in honey beyond natural limits.
  • beekeepers must prepare a sugar solution (syrup) with a desired concentration of oxalic acid (which can be for example 2.8%, 3.5% or 4.2% as in the case of Api-Bioxal) and 60% sugar (sucrose solution weight/volume) and then use this solution within 24 hours of its preparation.
  • the application is carried out by dripping this solution with a 5 ml syringe between the frames occupied by the bees;
  • sublimation by sublimation: beekeepers must buy a special sublimation apparatus, called a sublimator, a car battery or a generator in order to supply electric current to power the sublimator.
  • the dosage for each hive is, for example, 2.3 grams of Api-Bioxal 886 mg/g soluble powder, or 2 grams for pure oxalic acid raw material as such.
  • one purpose of the present invention is to provide an acaricide pharmaceutical composition for treating bee pathologies, in particular of the domestic bee Apis mellifera, whose formulation uses oxalic acid, or its salt or ester, or citric acid, or its salt or ester, which is effective against the specific pathology, which is stable over time and whose formulation is such that it does not generate HMF.
  • Another purpose is to provide an acaricide pharmaceutical composition for the treatment of bee pathologies that is ready for use in the correct proportions, without the operator or beekeeper needing to manually dose the oxalic acid.
  • Yet another purpose is to provide an acaricide pharmaceutical composition for the treatment of bee pathologies that is safe for the health of the operator or beekeeper.
  • Another purpose is to provide an acaricide pharmaceutical composition for the treatment of bee pathologies which is ready to be used in liquid form, or which can be provided in a ready-to-use form as it is formulated on a support in strips.
  • the Applicant has devised, tested and embodied the present invention to overcome the shortcomings of the state of the art and to obtain these and other purposes and advantages.
  • an acaricide pharmaceutical composition for the treatment of bee pathologies is provided, in particular of the domestic bee Apis mellifera.
  • the acaricide pharmaceutical composition comprises oxalic acid, or a salt or ester thereof, in the presence of a polyalcohol or a mixture of polyalcohols, or alternatively the acaricide pharmaceutical composition comprises oxalic acid, or a salt or ester thereof, in the presence of citric acid or a salt or ester thereof, and one or more hydrocolloids or a mixture of hydrocolloids.
  • the acaricide pharmaceutical composition according to embodiments described here can be in liquid form, ready to use, or in ready-to-use form formulated on a support in strips.
  • the present description also includes the ranges that derive from uniting or overlapping two or more ranges described, unless otherwise indicated.
  • the present description also includes the ranges that can derive from the combination of two or more values taken at different points, unless otherwise indicated.
  • Embodiments described here concern an acaricide pharmaceutical composition for treating bee pathologies, in particular of the domestic bee Apis mellifera.
  • the acaricide pharmaceutical composition comprises oxalic acid or a salt or ester thereof, in the presence of a polyalcohol or a mixture of polyalcohols, or alternatively, the acaricide pharmaceutical composition comprises oxalic acid or a salt or ester thereof, in the presence of citric acid or a salt or ester thereof, and one or more or a mixture of hydrocolloids.
  • polyalcohol or “polyalcohols” means diols, polyols not of sugars and alcohols of sugars.
  • an alcohol of sugars can be selected as polyalcohol, in particular selected from glycerol, mannitol or sorbitol.
  • the hydrocolloid can be selected from xanthan gum, starch, modified starch, pectin, locust bean, guar gum, gelatin, agar-agar, alginate, carrageenan, cellulose or Arabic gum or mixtures of two or more of them.
  • gelatin or pectin or a mixture of both can be used.
  • embodiments described here concern an acaricide pharmaceutical composition for treating bee pathologies connected to the Varroa mite (Mesostigmata, varroa destructor).
  • polyalcohol can be understood as a single polyalcohol, or a mixture of two or more polyalcohols.
  • hydrocolloid can be understood to be either a single hydrocolloid or a mixture of two or more hydrocolloids.
  • the polyalcohol present in the acaricide pharmaceutical composition according to the present description is highly hygroscopic, such as sucrose used as an excipient in the state of the art, but does not have the problems of HMF formation, thanks to the fact that the polyalcohol is stable in the presence of an acidic pH.
  • the hydrocolloids usable in embodiments described here are also stable in the presence of an acidic pH.
  • the oxalic acid is dihydrate.
  • oxalic acid in a dihydrate form since it is generally the product available on the market, usually in the form of odorless, colorless, water-soluble crystals, and in polar solvents.
  • the oxalic acid dihydrate is present in the above composition from 25 to 80 grams, in particular from 45 to 75 grams, more particularly from 50 to 70 grams, even more particularly from 55 to 65 grams for every 100 grams of polyalcohol or mixture of polyalcohols, and/or from 5 to 25 grams, in particular from 5 to 20 grams, more particularly from 5 to 15 grams for every 100 grams of hydrocolloid or mixture of hydrocolloids, according to the various embodiments.
  • some examples can be 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 grams of oxalic acid dihydrate for every 100 grams of polyalcohol.
  • hydrocolloid or mixture of hydrocolloids some examples can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 grams of oxalic acid dihydrate for every 100 grams of hydrocolloid.
  • the weight/weight ratio between oxalic acid dihydrate and polyalcohol, in the acaricide pharmaceutical composition according to the embodiments described here can range from 0.25 to 0.80, in particular from 0.45 to 0.75, more particularly from 0.50 to 0.70, even more particularly from 0.55 to 0.65 for 1 gram of polyalcohol and/or in the case of hydrocolloid or mixture of hydrocolloids
  • the weight/weight ratio of oxalic acid dihydrate and hydrocolloid or mixture of hydrocolloids can range from 0.05 to 0.25, in particular from 0.05 to 0.2, more particularly from 0.05 to 0.15, for every 1 gram of hydrocolloid or mixture of hydrocolloids, according to the various embodiments.
  • the weight/weight ratio between citric acid and hydrocolloid or mixture of hydrocolloids ranges from 0.005 to 0.15, in particular from 0.01 to 0.1. Some examples can be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 grams of citric acid for every 100 grams of hydrocolloid or mixture of hydrocolloids.
  • citric acid it is to be understood as a weak tricarboxylic acid in a colorless solid substance.
  • the acaricide pharmaceutical composition can be in liquid form, in particular in liquid solution, ready for use, that is, it already has the correct dosage and the correct proportion of the various components of which it consists, without the need for further intervention by the operator or beekeeper.
  • the pharmaceutical composition can be in the form of a support in strips, ready for use, that is, it already has the correct dosage and the correct proportion of the various components of which it consists, without the need for further intervention by the operator or beekeeper.
  • the acaricide pharmaceutical composition comprises water.
  • the water is distilled water.
  • the oxalic acid dihydrate is used in the quantities indicated above in relation to 100 grams of polyalcohol and enough water is added to bring the composition to a desired final volume of 1000 ml.
  • oxalic acid dihydrate for every 100 grams of polyalcohol or mixture of poly alcohols and/or from 5 to 20 grams, more particularly from 5 to 15 grams of oxalic acid dihydrate for every 100 grams of hydrocolloid or mixture of hydrocolloids.
  • the acaricide pharmaceutical composition according to the present description consists exclusively of oxalic acid or a salt or ester thereof, polyalcohol or a mixture of polyalcohols and water, and/or citric acid or a salt or ester thereof, hydrocolloid or a mixture of hydrocolloids and water.
  • oxalic acid or a salt or ester thereof and the polyalcohol or a mixture of polyalcohols in the proportions described above, enough water is present to reach a predefined and desired volume of the composition, for example 1000 ml of said composition, in the case of a liquid formulation, or 100 grams, in the case of a formulation on a support in strips.
  • acaricide pharmaceutical composition according to the present description there is only one poly alcohol and/or only one hydrocolloid or mixture of hydrocolloids.
  • the polyalcohol can be glycerol.
  • the oxalic acid dihydrate is present in the composition from 25 to 80 grams, in particular from 45 to 75 grams, more particularly from 50 to 70 grams, even more particularly from 55 to 65 grams for each 100 grams of glycerol.
  • Some examples can be 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 grams of oxalic acid dihydrate for every 100 grams of glycerol.
  • the weight/weight ratio between oxalic acid dihydrate and glycerol, in the acaricide pharmaceutical composition according to the embodiments described here can range from 0.25 to 0.80, in particular from 0.45 to 0.75, more particularly from 0.50 to 0.70, even more particularly from 0.55 to 0.65.
  • glycerol can be the only polyalcohol of the acaricide pharmaceutical composition.
  • the hydrocolloids can be gelatin and/or pectin or a mixture thereof.
  • 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16 grams of oxalic acid dihydrate can be provided for every 100 grams of gelatin or pectin or a mixture thereof.
  • gelatin and/or pectin can be the only hydrocolloids of the acaricide pharmaceutical composition.
  • the acaricide pharmaceutical composition according to the present description is free of added preservatives.
  • inventions also concern a method for the production of an acaricide pharmaceutical composition for the treatment of bee pathologies, said method providing to make a solution of oxalic acid or a salt or ester thereof, in the presence of a polyalcohol or a mixture of polyalcohols.
  • the method provides to use water as a medium to dissolve the oxalic acid or its salt or ester, in the presence of the polyalcohol or mixture of polyalcohols and/or citric acid or a salt or ester thereof, hydrocolloid or a mixture of hydrocolloids.
  • the quantities and the reciprocal proportions between the oxalic acid or its salt or ester, and polyalcohol or mixture of polyalcohols and/or citric acid or a salt or ester thereof, hydrocolloid or a mixture of hydrocolloids in the production method are essentially as described above, water being used to close the balance, to reach the final volume of, for example, 1000 ml as described above in the case of liquid formulation, or the final weight, for example, of 100 grams, in the case of a formulation on a support in strips as described above.
  • an esterification reaction takes place that involves the oxalic acid and the polyalcohol, it is provided to use, at the start, a slightly higher quantity than that which is desired to be the final quantity in the pharmaceutical composition, since part of the oxalic acid is consumed during the controlled esterification reaction itself.
  • the final pharmaceutical composition has to have a quantity of oxalic acid of 62 mg/ml of solution, it is provided to use about 65 mg of oxalic acid at the start.
  • inventions also concern a method to control the Varroa mite in a beehive, or in artificial means associated with the reproduction of bees, in particular of the domestic bee Apis mellifera.
  • the method comprises the application, distribution or delivery, for example by dripping, in said hive or in said artificial means, of an acaricide pharmaceutical composition according to the present description.
  • the Applicant has developed and tested by way of example the acaricide pharmaceutical composition according to possible embodiments, so that it can be formulated in liquid form, in particular in a liquid solution for beehives, or in the form of strips, ready for use in the correct and safe proportions of the various components, so that it contains the same concentration, or similar or near concentrations, of the active principle oxalic acid which is obtained by reconstituting in a solution the formulation of the product known as Api-Bioxal soluble powder for bees.
  • the Applicant has developed a possible formulation of the acaricide pharmaceutical composition according to the present description containing 62 mg/ml in a liquid solution for beehives which contains the same quantity of active principle as the known soluble product Api-Bioxal 886 mg/g powder for bees.
  • acaricide pharmaceutical composition according to the present description can be used, for example by dripping, in the same and/or similar concentration, in the strip form, as that of the reconstituted powder, we believe that the acaricide pharmaceutical composition according to the present description and the known formulation have the same effectiveness in combatting bee varroosis.
  • the acaricide pharmaceutical compositions according to the possible embodiments described here do not have dextrose or sucrose among their excipients and co-formulants and this makes the product stable in an acidic environment, since there is no possibility of HMF forming, which is the typical product of degradation obtained from two sugars in an acidic environment.
  • the Applicant has carefully selected the one or more polyalcohols, in particular glycerol, or mannitol or sorbitol, or also mixtures thereof, and/or the one or more hydrocolloids, in particular for example gelatin and pectin, or gelatin and/or pectin and/or sorbitol, or also mixtures thereof, used as excipients present in the acaricide pharmaceutical composition according to the present description, so as to:
  • VMP veterinary medicinal product
  • the Applicant found a validity of at least 24 months for the acaricide pharmaceutical composition according to the present description in all climatic areas.
  • a package intended as the primary packaging, containing the acaricide pharmaceutical composition for treating bee pathologies.
  • the package is made of polyethylene, in particular high density polyethylene.
  • primary packaging materials it is possible to use both polyethylene bottles and polyethylene cans, with polyethylene safety screw caps and warranty seal.
  • the choice of containers as primary packaging consisting of polyethylene bottles with polyethylene caps and polyethylene cans with polyethylene screw cap and warranty seal, ensures that the product is protected against light and guarantees the necessary stability.
  • the Applicant has conducted stability studies on the primary packaging. The inactivity of the primary packaging materials on the product has been tested with stability studies conducted on the formulations to be tested, with the purpose of obtaining the final formulation. The different packaging materials did not make any alterations to the formulations tested.
  • the Applicant has conducted studies to evaluate whether to include preservatives in the formulation of the acaricide pharmaceutical composition according to the present description, and concluded that there is no such need.
  • the degree of microbial presence in the acaricide pharmaceutical composition according to the present description was measured, following the method described in European Pharmacopoeia (Ph. Eur.). The following microorganisms were used:
  • Varroa mites were treated topically with oxalic acid solutions at different concentrations containing or not containing 10% glycerol. Mortality was measured at 24 and 48 hours after treatment.
  • the slope of the dose-effect curves obtained was higher at the post-treatment measurement for solutions containing glycerol.
  • the latter showed a lower dose-efficacy mean (ED50) compared to the opposite solutions (0.282 vs 0.357 micrograms/mite), indicating a higher short-term toxicity for the parasite (the Varroa mite dies more quickly).
  • the ED50 was very similar in both groups (respectively: 0.261 vs 0.262), the ED90 was found to be 4 times higher for the solutions not containing glycerol (respectively: 0.46 vs 1.85).
  • the calculated fiducial limits were normally narrower in the solutions with added glycerol, confirming not only a higher toxicity towards the mites but also a better repeatability compared to the non-added controls.
  • mannitol and sorbitol could be two polyalcohols useful to replace sucrose as excipients in the acaricide pharmaceutical composition according to the embodiments described here.
  • polyalcohols preferably including glycerol, or mannitol or sorbitol, can be an effective substitute for sucrose in oxalic acid formulations for the control and therapeutic treatment of the Varroa mite.
  • One possible embodiment was found to be an acaricide pharmaceutical composition of 62 mg/ml of oxalic acid dihydrate in the form of a liquid solution of glycerol ready for use and free of preservatives.

Abstract

Acaricide pharmaceutical composition for the treatment of bee pathologies, which comprises oxalic acid or a salt or ester thereof, in the presence of a polyalcohol or a mixture of polyalcohols.

Description

PHARMACEUTICAL COMPOSITION FOR THE TREATMENT
OF BEE PATHOLOGIES
FIELD OF THE INVENTION
Embodiments described here concern an acaricide pharmaceutical composition for the treatment of bee pathologies, in particular of the domestic bee Apis mellifera. In particular, but not restrictively, embodiments described here concern an acaricide pharmaceutical composition for the treatment of pathologies connected to the Varroa mite (Mesostigmata, varroa destructor). In particular, embodiments described here concern the use of the acaricide pharmaceutical composition in a beehive, or in artificial means associated with the reproduction of bees, such as cells, observation hives, or in any other environment suitable for the reproduction of bees. More particularly, the acaricide pharmaceutical composition for the treatment of bee pathologies, in particular of the domestic bee Apis mellifera, according to the embodiments described here, can be provided in liquid form, ready for use, or in the form of a support in strips.
BACKGROUND OF THE INVENTION
It is known that the Varroa mite (Mesostigmata, varroa destructor) is one of the worst enemies of honey bee (Apis mellifera) colonies. It was identified for the first time in Italy in the early eighties of the last century. In the following years, this parasite spread very rapidly throughout the peninsula, leading to the virtual extinction of all wild colonies of honey bees. Even today this parasite inflicts heavy damage on beekeeping worldwide, except for that carried out in Africa and Australia. The lack of knowledge of potential enemies of this parasite, and the failure of the efforts to eradicate it, oblige bees to coexist forcibly with this mite. This situation obliges beekeepers to carry out therapeutic chemical treatments, which are generally planned and carried out during the beekeeping season in the framework of pest control and which usually involve the use of acaricides.
Most of the acaricides that are currently registered can be used for therapeutic treatments during the active season of bee colonies but are not suitable for use during winter when the bees hibernate. This despite the fact that winter is the most suitable period for therapy because the characteristics of the parasite's biological cycle can achieve the highest levels of acaricide efficacy, so as to drastically reduce the population of mites and start again with the following season with the lowest level of infestation. One of the few active principles that makes this possible is oxalic acid, which can be administered to the colonies by dripping its sugary solution between the frames occupied by the bees, or by sublimation of its crystals in powder form.
Oxalic acid is the most effective and widely used acaricide against the Varroa mite. Applicant's known product Api-Bioxal 886 mg/g soluble powder for bees is the only oxalic acid-based product currently authorized in Italy for combatting bee varroosis. Another great advantage of oxalic acid, the active principle contained in Api-Bioxal, is that it is a natural component of honey, where it can be found in variable concentrations with the botanical origin of the nectar. The treatments carried out correctly with Api-Bioxal in solution do not increase the concentration of oxalic acid in honey beyond natural limits.
To date, there are only two known methods for the therapeutic application of oxalic acid in bee colonies against Varroa mites:
1) by dripping: beekeepers must prepare a sugar solution (syrup) with a desired concentration of oxalic acid (which can be for example 2.8%, 3.5% or 4.2% as in the case of Api-Bioxal) and 60% sugar (sucrose solution weight/volume) and then use this solution within 24 hours of its preparation. The application is carried out by dripping this solution with a 5 ml syringe between the frames occupied by the bees;
2) by sublimation: beekeepers must buy a special sublimation apparatus, called a sublimator, a car battery or a generator in order to supply electric current to power the sublimator. The dosage for each hive is, for example, 2.3 grams of Api-Bioxal 886 mg/g soluble powder, or 2 grams for pure oxalic acid raw material as such.
As a result of the above, beekeepers have a practical problem: the inconvenience of applying oxalic acid, both by dripping and by sublimation. Consequently, this situation means that the need to have a ready-to-use product available is extremely urgent in the sector.
In the first case of the sugar solution, that is, application by dripping, beekeepers must be careful to obtain the right proportion of sugar and oxalic acid, because, if it is wrong, there can be reduced acaricide efficacy or an increase in toxicity for the bees: therefore, in both cases there would be a negative impact on the probability of survival of the bee colonies.
Furthermore, there is no possibility of preserving the sugar solution with added oxalic acid because the acidic pH determines the oxidation of the sucrose, generating HMF (5-hydroxymethyl-2-furfural) which is extremely toxic to bees (30 ppm cause the death of up to 15% of adult bees and larvae in 20 days, 150 ppm cause the death of up to 58% of adult bees and larvae in 20 days).
In the second case, application by sublimation, there is an important workload for beekeepers with a high risk for their health because during the entire preparation and application process, and up to 15 days after application, beekeepers must wear protective gloves, goggles and a protective mask (with filters for organic acids) in order to avoid oxalic acid crystals which, if they reach the deep parts of the lungs (primary and secondary alveoli) can cause serious damage, in particular at the pulmonary and renal level.
The Milani document, "Activity of Oxalic Acid and Citric Acids on the Varroa mite Destructor in Laboratory Assays", 1 January 2001, describes a study to evaluate the activity of oxalic acid and citric acid on the Varroa mite in a laboratory test. This document, however, does not take into consideration the problems connected to the generation of toxic HMF for bees.
The document Maggi Matias et al. "A new formulation of oxalic acid for Varroa destructor control applied in Apis mellifera colonies in the presence of brood", Apidologie 2016, describes a formulation of oxalic acid to control the Varroa mite called Aluen CAP. Document EP-A-3.095.328 describes a similar or identical composition. These documents also do not mention the problem of HMF toxicity.
Document WO-A-2013/030854 describes a food supplement for bees used for the prophylaxis and treatment of Colony Collapse Disorder.
There is therefore a need to perfect an acaricide pharmaceutical composition for the treatment of bee pathologies that can overcome at least one of the disadvantages of the state of the art.
In particular, one purpose of the present invention is to provide an acaricide pharmaceutical composition for treating bee pathologies, in particular of the domestic bee Apis mellifera, whose formulation uses oxalic acid, or its salt or ester, or citric acid, or its salt or ester, which is effective against the specific pathology, which is stable over time and whose formulation is such that it does not generate HMF.
Another purpose is to provide an acaricide pharmaceutical composition for the treatment of bee pathologies that is ready for use in the correct proportions, without the operator or beekeeper needing to manually dose the oxalic acid.
Yet another purpose is to provide an acaricide pharmaceutical composition for the treatment of bee pathologies that is safe for the health of the operator or beekeeper.
Another purpose is to provide an acaricide pharmaceutical composition for the treatment of bee pathologies which is ready to be used in liquid form, or which can be provided in a ready-to-use form as it is formulated on a support in strips. The Applicant has devised, tested and embodied the present invention to overcome the shortcomings of the state of the art and to obtain these and other purposes and advantages.
Other limitations and disadvantages of conventional solutions and technologies will be clear to a person of skill after reading the remaining part of the present description with reference to the drawings and the description of the embodiments that follow, although it is clear that the description of the state of the art connected to the present description must not be considered an admission that what is described here is already known from the state of the prior art
SUMMARY OF THE INVENTION
The present invention is set forth and characterized in the independent claims, while the dependent claims describe other characteristics of the invention or variants to the main inventive idea.
In accordance with some embodiments, an acaricide pharmaceutical composition for the treatment of bee pathologies is provided, in particular of the domestic bee Apis mellifera. According to one embodiment, the acaricide pharmaceutical composition comprises oxalic acid, or a salt or ester thereof, in the presence of a polyalcohol or a mixture of polyalcohols, or alternatively the acaricide pharmaceutical composition comprises oxalic acid, or a salt or ester thereof, in the presence of citric acid or a salt or ester thereof, and one or more hydrocolloids or a mixture of hydrocolloids. Advantageously, the acaricide pharmaceutical composition according to embodiments described here can be in liquid form, ready to use, or in ready-to-use form formulated on a support in strips.
These and other aspects, characteristics and advantages of the present disclosure will be better understood with reference to the following description, drawings and attached claims. The drawings, which are integrated and form part of the present description, show some embodiments of the present invention, and together with the description, are intended to describe the principles of the disclosure.
The various aspects and characteristics described in the present description can be applied individually where possible. These individual aspects, for example aspects and characteristics described in the specification or in the attached dependent claims, can be the object of divisional applications.
It is understood that any aspect or characteristic that is discovered, during the patenting process, to be already known, shall not be claimed and shall be the object of a disclaimer.
DETAILED DESCRIPTION OF SOME EMBODIMENTS
We will now refer in detail to the various embodiments of the present invention, of which one or more examples are shown in the attached drawings. Each example is supplied by way of illustration of the invention and shall not be understood as a limitation thereof. For example, the characteristics shown or described insomuch as they are part of one embodiment can be adopted on, or in association with, other embodiments to produce another embodiment. It is understood that the present invention shall include all such modifications and variants.
Before describing these embodiments, we must also clarify that the present description is not limited in its application to details of the construction and disposition of the components as described in the following description using the attached drawings. The present description can provide other embodiments and can be obtained or executed in various other ways. We must also clarify that the phraseology and terminology used here is for the purposes of description only, and cannot be considered as limitative.
Unless otherwise defined, all the technical and scientific terms used here and hereafter have the same meaning as commonly understood by a person with ordinary experience in the field of the art to which the present invention belongs. Even if methods and materials similar or equivalent to those described here can be used in practice and in the trials of the present invention, the methods and materials are described hereafter as an example. In the event of conflict, the present application shall prevail, including its definitions. The materials, methods and examples have a purely illustrative purpose and shall not be understood restrictively.
All the measurements are made, unless otherwise indicated, at 25°C and at atmospheric pressure. All the temperatures, unless otherwise indicated, are expressed in degrees Celsius.
All percentages and ratios indicated are weight/volume ratios and refer to the volume of the total composition (w/v), unless otherwise indicated.
All the ranges reported here are understood to include the extremes, including those that show a range "between" two values, unless otherwise indicated.
The present description also includes the ranges that derive from uniting or overlapping two or more ranges described, unless otherwise indicated.
The present description also includes the ranges that can derive from the combination of two or more values taken at different points, unless otherwise indicated.
Embodiments described here concern an acaricide pharmaceutical composition for treating bee pathologies, in particular of the domestic bee Apis mellifera. According to one embodiment, the acaricide pharmaceutical composition comprises oxalic acid or a salt or ester thereof, in the presence of a polyalcohol or a mixture of polyalcohols, or alternatively, the acaricide pharmaceutical composition comprises oxalic acid or a salt or ester thereof, in the presence of citric acid or a salt or ester thereof, and one or more or a mixture of hydrocolloids.
According to some embodiments, the term "polyalcohol" or "polyalcohols" means diols, polyols not of sugars and alcohols of sugars.
According to a possible embodiment, which can be combined with all the embodiments described here, an alcohol of sugars can be selected as polyalcohol, in particular selected from glycerol, mannitol or sorbitol.
According to some embodiments, which can be combined with all the embodiments described here, the hydrocolloid can be selected from xanthan gum, starch, modified starch, pectin, locust bean, guar gum, gelatin, agar-agar, alginate, carrageenan, cellulose or Arabic gum or mixtures of two or more of them. For example, gelatin or pectin or a mixture of both can be used.
In particular, embodiments described here concern an acaricide pharmaceutical composition for treating bee pathologies connected to the Varroa mite (Mesostigmata, varroa destructor).
Hereafter, where not explicitly specified otherwise, the term "polyalcohol" can be understood as a single polyalcohol, or a mixture of two or more polyalcohols.
Moreover, hereafter, unless explicitly specified otherwise, the term "hydrocolloid" can be understood to be either a single hydrocolloid or a mixture of two or more hydrocolloids.
Advantageously, the polyalcohol present in the acaricide pharmaceutical composition according to the present description is highly hygroscopic, such as sucrose used as an excipient in the state of the art, but does not have the problems of HMF formation, thanks to the fact that the polyalcohol is stable in the presence of an acidic pH. Advantageously, the hydrocolloids usable in embodiments described here are also stable in the presence of an acidic pH.
According to a possible embodiment, which can be combined with all the embodiments described here, the oxalic acid is dihydrate. Here reference is made to oxalic acid in a dihydrate form since it is generally the product available on the market, usually in the form of odorless, colorless, water-soluble crystals, and in polar solvents.
In particular, where hereafter in the description reference is made to oxalic acid dihydrate, it should be noted that the weight-to-weight conversion factor between oxalic acid dihydrate and oxalic acid is 0.71.
In accordance with other embodiments, which can be combined with all the embodiments described here, the oxalic acid dihydrate is present in the above composition from 25 to 80 grams, in particular from 45 to 75 grams, more particularly from 50 to 70 grams, even more particularly from 55 to 65 grams for every 100 grams of polyalcohol or mixture of polyalcohols, and/or from 5 to 25 grams, in particular from 5 to 20 grams, more particularly from 5 to 15 grams for every 100 grams of hydrocolloid or mixture of hydrocolloids, according to the various embodiments.
In the case of polyalcohol or a mixture of polyalcohols, some examples can be 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 grams of oxalic acid dihydrate for every 100 grams of polyalcohol.
In the case of hydrocolloid or mixture of hydrocolloids, some examples can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 grams of oxalic acid dihydrate for every 100 grams of hydrocolloid.
In other words, the weight/weight ratio between oxalic acid dihydrate and polyalcohol, in the acaricide pharmaceutical composition according to the embodiments described here, can range from 0.25 to 0.80, in particular from 0.45 to 0.75, more particularly from 0.50 to 0.70, even more particularly from 0.55 to 0.65 for 1 gram of polyalcohol and/or in the case of hydrocolloid or mixture of hydrocolloids, the weight/weight ratio of oxalic acid dihydrate and hydrocolloid or mixture of hydrocolloids can range from 0.05 to 0.25, in particular from 0.05 to 0.2, more particularly from 0.05 to 0.15, for every 1 gram of hydrocolloid or mixture of hydrocolloids, according to the various embodiments.
In possible embodiments, the weight/weight ratio between citric acid and hydrocolloid or mixture of hydrocolloids ranges from 0.005 to 0.15, in particular from 0.01 to 0.1. Some examples can be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 grams of citric acid for every 100 grams of hydrocolloid or mixture of hydrocolloids.
It goes without saying that, according to the conversion ratio of 0.71 identified above, such quantities and values of weight ratios between oxalic acid dihydrate and polyalcohol and/or hydrocolloid can be traced back to corresponding quantities and values of weight ratios between oxalic acid and polyalcohol and/or hydrocolloid or mixture of hydrocolloids.
According to a possible embodiment, which can be combined with all the embodiments described here, where citric acid is used, it is to be understood as a weak tricarboxylic acid in a colorless solid substance.
In accordance with a possible embodiment, which can be combined with all the embodiments described here, the acaricide pharmaceutical composition can be in liquid form, in particular in liquid solution, ready for use, that is, it already has the correct dosage and the correct proportion of the various components of which it consists, without the need for further intervention by the operator or beekeeper. In other embodiments, the pharmaceutical composition can be in the form of a support in strips, ready for use, that is, it already has the correct dosage and the correct proportion of the various components of which it consists, without the need for further intervention by the operator or beekeeper.
According to a possible embodiment, which can be combined with all the embodiments described here, the acaricide pharmaceutical composition comprises water. In a possible implementation, the water is distilled water. For example, in possible implementations, to prepare 1000 ml of solution of the pharmaceutical composition according to the present description, the oxalic acid dihydrate is used in the quantities indicated above in relation to 100 grams of polyalcohol and enough water is added to bring the composition to a desired final volume of 1000 ml. Therefore, in 1000 ml of solution of the pharmaceutical composition, there can be present from 25 to 80 grams, in particular from 45 to 75 grams, more particularly from 50 to 70 grams, even more particularly from 55 to 65 grams of oxalic acid dihydrate for every 100 grams of polyalcohol or mixture of poly alcohols and/or from 5 to 20 grams, more particularly from 5 to 15 grams of oxalic acid dihydrate for every 100 grams of hydrocolloid or mixture of hydrocolloids.
According to a possible embodiment, the acaricide pharmaceutical composition according to the present description consists exclusively of oxalic acid or a salt or ester thereof, polyalcohol or a mixture of polyalcohols and water, and/or citric acid or a salt or ester thereof, hydrocolloid or a mixture of hydrocolloids and water. For example, considering the oxalic acid or a salt or ester thereof and the polyalcohol or a mixture of polyalcohols, in the proportions described above, enough water is present to reach a predefined and desired volume of the composition, for example 1000 ml of said composition, in the case of a liquid formulation, or 100 grams, in the case of a formulation on a support in strips.
According to a possible embodiment, which can be combined with all the embodiments described here, in the acaricide pharmaceutical composition according to the present description there is only one poly alcohol and/or only one hydrocolloid or mixture of hydrocolloids.
According to a specific possible embodiment, the polyalcohol can be glycerol. In some embodiments, the oxalic acid dihydrate is present in the composition from 25 to 80 grams, in particular from 45 to 75 grams, more particularly from 50 to 70 grams, even more particularly from 55 to 65 grams for each 100 grams of glycerol. Some examples can be 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 grams of oxalic acid dihydrate for every 100 grams of glycerol. In other words, the weight/weight ratio between oxalic acid dihydrate and glycerol, in the acaricide pharmaceutical composition according to the embodiments described here, can range from 0.25 to 0.80, in particular from 0.45 to 0.75, more particularly from 0.50 to 0.70, even more particularly from 0.55 to 0.65.
In particular, in a possible variant embodiment, glycerol can be the only polyalcohol of the acaricide pharmaceutical composition.
According to a specific possible embodiment, the hydrocolloids can be gelatin and/or pectin or a mixture thereof.
According to some examples, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16 grams of oxalic acid dihydrate can be provided for every 100 grams of gelatin or pectin or a mixture thereof.
Other examples can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 grams of citric acid for every 100 grams of gelatin or pectin or a mixture thereof. In particular, in a possible variant embodiment, gelatin and/or pectin can be the only hydrocolloids of the acaricide pharmaceutical composition.
In particular, a specific example of possible formulation of the acaricide pharmaceutical composition according to the present description provides:
- 62 grams of oxalic acid dihydrate, corresponding to 44.02 grams of anhydrous oxalic acid given the conversion factor 0.71 as above,
- 100 grams of glycerol FU/ Ph. Eur;
- distilled water as required to 1000 ml.
In particular, another specific example of possible formulation of the acaricide pharmaceutical composition according to the present description provides:
- from 2 to 10 grams of oxalic acid dihydrate, corresponding to 1.42 to 7.1 grams of anhydrous oxalic acid given the conversion factor 0.71 as above; - 2 to 10 grams of citric acid;
- 2 grams of pectin;
- 6 grams of gelatin;
- 35 grams of sorbitol;
- distilled water as required to 100 grams.
In accordance with possible embodiments, which can be combined with all the embodiments described here, the acaricide pharmaceutical composition according to the present description is free of added preservatives.
Other embodiments also concern a method for the production of an acaricide pharmaceutical composition for the treatment of bee pathologies, said method providing to make a solution of oxalic acid or a salt or ester thereof, in the presence of a polyalcohol or a mixture of polyalcohols. In possible embodiments, the method provides to use water as a medium to dissolve the oxalic acid or its salt or ester, in the presence of the polyalcohol or mixture of polyalcohols and/or citric acid or a salt or ester thereof, hydrocolloid or a mixture of hydrocolloids. The quantities and the reciprocal proportions between the oxalic acid or its salt or ester, and polyalcohol or mixture of polyalcohols and/or citric acid or a salt or ester thereof, hydrocolloid or a mixture of hydrocolloids in the production method are essentially as described above, water being used to close the balance, to reach the final volume of, for example, 1000 ml as described above in the case of liquid formulation, or the final weight, for example, of 100 grams, in the case of a formulation on a support in strips as described above. It should be noted that in some embodiments of the method, since an esterification reaction takes place that involves the oxalic acid and the polyalcohol, it is provided to use, at the start, a slightly higher quantity than that which is desired to be the final quantity in the pharmaceutical composition, since part of the oxalic acid is consumed during the controlled esterification reaction itself. For example, but not restrictively, in the case where the final pharmaceutical composition has to have a quantity of oxalic acid of 62 mg/ml of solution, it is provided to use about 65 mg of oxalic acid at the start.
Other embodiments also concern a method to control the Varroa mite in a beehive, or in artificial means associated with the reproduction of bees, in particular of the domestic bee Apis mellifera. The method comprises the application, distribution or delivery, for example by dripping, in said hive or in said artificial means, of an acaricide pharmaceutical composition according to the present description.
The Applicant has developed and tested by way of example the acaricide pharmaceutical composition according to possible embodiments, so that it can be formulated in liquid form, in particular in a liquid solution for beehives, or in the form of strips, ready for use in the correct and safe proportions of the various components, so that it contains the same concentration, or similar or near concentrations, of the active principle oxalic acid which is obtained by reconstituting in a solution the formulation of the product known as Api-Bioxal soluble powder for bees. For example, the Applicant has developed a possible formulation of the acaricide pharmaceutical composition according to the present description containing 62 mg/ml in a liquid solution for beehives which contains the same quantity of active principle as the known soluble product Api-Bioxal 886 mg/g powder for bees.
Moreover, since the acaricide pharmaceutical composition according to the present description can be used, for example by dripping, in the same and/or similar concentration, in the strip form, as that of the reconstituted powder, we believe that the acaricide pharmaceutical composition according to the present description and the known formulation have the same effectiveness in combatting bee varroosis.
Moreover, unlike the reconstituted powder of the known product Api-Bioxal, the acaricide pharmaceutical compositions according to the possible embodiments described here do not have dextrose or sucrose among their excipients and co-formulants and this makes the product stable in an acidic environment, since there is no possibility of HMF forming, which is the typical product of degradation obtained from two sugars in an acidic environment.
This allows the formulation of a ready-to-use liquid solution and/or support in strips based on oxalic acid, which can be stored for the entire period of validity proposed for this formulation, which can be for example 24 months or even more, for example 36 months.
The Applicant has carefully selected the one or more polyalcohols, in particular glycerol, or mannitol or sorbitol, or also mixtures thereof, and/or the one or more hydrocolloids, in particular for example gelatin and pectin, or gelatin and/or pectin and/or sorbitol, or also mixtures thereof, used as excipients present in the acaricide pharmaceutical composition according to the present description, so as to:
- obtain a stable medicinal product;
- enable the authorization to market a medicinal product that is ready for use, easier to use than the reconstituted powder and safer for the operator;
- allow a prompt and complete solubilization of the active principle in the final formulation of the veterinary medicinal product (VMP).
STABILITY ANALYSIS
A study was carried out on the development of the acaricide pharmaceutical composition according to the present description, taking as a reference model the knowledge deriving from the precursor VMP, that is, the known product Api- Bioxal soluble powder for bees, for example 886 mg/g.
The samples deriving from the development of the acaricide pharmaceutical composition according to the present description, in this case 62 mg/ml in liquid solution, were subjected to accelerated stability studies in order to verify the stability of the formulations and their compatibility with the primary packaging. In practice, accelerated aging tests were carried out over a period of 2 months, at a temperature of + 50°C with measurements taken at 4 different times (day 0, day 7, day 35 and day 75). At these times the following parameters were measured:
- physical-chemical characteristics (appearance and pH);
- titer of oxalic acid;
- titer of related substances.
From the results obtained, no significant changes in the pH value were recorded; the oxalic acid titer remains almost constant from day T7 onwards. At TO the value was 99.7%, while from T7 the value was about 95%. Regarding the related substances, at the same time the presence of an additional peak in the running of the chromatogram was recorded, close to the oxalic acid identification peak but completely separate, remaining around the 5% value of the peak of oxalic acid (about 0.2% of the formulation), up to the last analysis point; whereas in absolute terms the peak represents about 0.3% on the formulation.
Furthermore, stability studies have been carried out on the finished product corresponding to the acaricide pharmaceutical composition according to the present description, to evaluate the degree of chemical degradation or to test the physical and quality characteristics of the primary packaging, using extreme storage conditions.
In this case, for an accelerated stability study, high temperatures were evaluated: 40°C ± 2°C/RH 75% ± 5%. It was found that a temperature of + 40°C for 6 months does not change the physical-chemical characteristics of the acaricide pharmaceutical composition according to the present description. The high level of humidity does not cause any effects on the product.
For a long-term stability study, the following conditions of temperature and humidity were analyzed: 25°C ± 2°C / H 60% ± 5%. The finished product was stored at a controlled temperature for 24 months (at 25 °C ± 2°C / RH 60% ± 5%).
It should be noted that during each study, all parameters tested remained within the range of compliance limits defined for normal storage conditions. The product proved to be chemically stable.
From the results obtained, the Applicant found a validity of at least 24 months for the acaricide pharmaceutical composition according to the present description in all climatic areas.
PRIMARY PACKAGING
In accordance with other embodiments, which can be combined with all the embodiments described here, a package is provided, intended as the primary packaging, containing the acaricide pharmaceutical composition for treating bee pathologies. In possible embodiments, the package is made of polyethylene, in particular high density polyethylene. For example, as primary packaging materials it is possible to use both polyethylene bottles and polyethylene cans, with polyethylene safety screw caps and warranty seal.
The choice of containers as primary packaging consisting of polyethylene bottles with polyethylene caps and polyethylene cans with polyethylene screw cap and warranty seal, ensures that the product is protected against light and guarantees the necessary stability. The Applicant has conducted stability studies on the primary packaging. The inactivity of the primary packaging materials on the product has been tested with stability studies conducted on the formulations to be tested, with the purpose of obtaining the final formulation. The different packaging materials did not make any alterations to the formulations tested.
Moreover, the results of the stability studies, carried out on 3 batches of the final product in their final package, showed no alterations. The following parameters were studied: appearance, pH of the solution, titer of the active principle, related substances, titer of the antioxidants and microbiological controls. The evaluation after 18 months through HPLC analysis of the samples stored at 40°C/RH 75% and 25°C/RH 60% did not show other peaks different from those recorded at TO ("zero" or initial time). Therefore, the material described can be considered suitable for packaging this product.
MICROBIAL ANALYSIS
The Applicant has conducted studies to evaluate whether to include preservatives in the formulation of the acaricide pharmaceutical composition according to the present description, and concluded that there is no such need. In particular, the degree of microbial presence in the acaricide pharmaceutical composition according to the present description was measured, following the method described in European Pharmacopoeia (Ph. Eur.). The following microorganisms were used:
- Pseudomonas aeruginosa, ATCC 9027;
- Staphylococcus aureus, ATCC 6538;
- Aspergillus brasiliensis, ATCC 16404;
- Escherichia coli, ATCC 8739;
- Candida albicans, ATCC 10231.
Five packages of the acaricide pharmaceutical composition according to the present description were subjected to preservability tests according to the Ph. Eur method. The results obtained show a decrease in the microbial count: already at the control point TO the growth rate of the microorganisms Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa resulting from contamination of the placebo was complete, while with the presence of the product it was drastically reduced, settling at <1 cfu/ml on day 14 and day 28. As for the fungicidal activity, 14 days after contamination with Candida Albicans and Aspergillus niger it showed a drastic reduction, reaching <1 cfu/ml on day 14 and day 28. These results definitely justify that it is not necessary to add preservatives to the formulation.
CLINICAL FIELD TESTS
A first pre-clinical study conducted on an experimental basis showed that the addition of glycerol, in place of sucrose, increases the power of the action and the effectiveness of oxalic acid in the treatment of bee varroosis.
In this study, Varroa mites were treated topically with oxalic acid solutions at different concentrations containing or not containing 10% glycerol. Mortality was measured at 24 and 48 hours after treatment.
The slope of the dose-effect curves obtained was higher at the post-treatment measurement for solutions containing glycerol. At 24 hours, the latter showed a lower dose-efficacy mean (ED50) compared to the opposite solutions (0.282 vs 0.357 micrograms/mite), indicating a higher short-term toxicity for the parasite (the Varroa mite dies more quickly). On the contrary, at 48 hours the ED50 was very similar in both groups (respectively: 0.261 vs 0.262), the ED90 was found to be 4 times higher for the solutions not containing glycerol (respectively: 0.46 vs 1.85). Moreover, the calculated fiducial limits were normally narrower in the solutions with added glycerol, confirming not only a higher toxicity towards the mites but also a better repeatability compared to the non-added controls.
The advantage of replacing sucrose with glycerol as an excipient in a solution with oxalic acid has been further demonstrated in two other field trials carried out experimentally.
In a first of these two works, a group of bee colonies without operculated brood were treated with the known product Api-Bioxal 886 mg/g soluble powder for bees which uses sucrose as excipient and in which the oxalic acid dihydrate is therefore dissolved in a sugar solution, in this case 60% sucrose (group G). Furthermore, a group of bee colonies without operculated brood were treated with the acaricide pharmaceutical composition according to the present description in which the excipient, instead of sucrose, is glycerol 62 mg/ml (in this case 10% glycerol), in liquid solution for beehives, that is, in the ready-to- use liquid formulation (group S). In both cases in comparison with untreated controls. Subsequently, the acaricide efficacy against Varroa mites and tolerability on bees were measured.
In group G, the acaricide efficacy was higher than in the controls but not with a statistically significant difference compared with that recorded in group S (respectively: 92.9%, 5.3%, 94.5%). Bee mortality registered after treatment was similar without statistically significant differences (respectively: 153.4, 109.8, 146.5 dead bees in 16 days). In the same way, the size of the colonies after the winter, hence with the resumption of the beekeeping season, showed no statistically significant differences between the 3 groups (respectively: 5800, 6875, 5950 bees and 3676, 3400, 3910 brood cells).
In the second of these two works, through 6 different clinical field trials (3 summer and 3 winter), the acaricide efficacy obtained with the known product Api-Bioxal in powder dissolved in a sugar solution as indicated above (group AO - S) was compared with the acaricide pharmaceutical composition according to the present description in which, that is, oxalic acid dihydrate is dissolved in solution with glycerol (group AO - G). The tests were conducted on 82 hives (48 during the summer trials and 34 during the winter trials). During the trials the values of temperature and humidity inside the hives were recorded. The trials showed that there is no statistically significant difference between the efficacy obtained with the two different solutions of Api-Bioxal under different environmental conditions (season, temperatures, humidity), which shows that glycerol can be a valid substitute for sucrose, however, with the advantages connected to the fact that the acaricide pharmaceutical composition according to embodiments is ready for use in liquid form, in particular in liquid solution, and therefore avoids the risk of erroneous dosages and proportions of sugar and oxalic acid, it does not develop HMF and is not dangerous for the health and safety of operators, in particular beekeepers.
The Applicant found that also mannitol and sorbitol could be two polyalcohols useful to replace sucrose as excipients in the acaricide pharmaceutical composition according to the embodiments described here.
In light of the above, the experimental data and field observations, it is clear that polyalcohols, preferably including glycerol, or mannitol or sorbitol, can be an effective substitute for sucrose in oxalic acid formulations for the control and therapeutic treatment of the Varroa mite.
To the best of current knowledge, the use of polyalcohols, preferably including glycerol, or mannitol or sorbitol, as an excipient for the acaricide pharmaceutical composition in liquid solution for beehives has not caused problems from the point of view of acaricide efficacy and tolerability for bees.
One possible embodiment was found to be an acaricide pharmaceutical composition of 62 mg/ml of oxalic acid dihydrate in the form of a liquid solution of glycerol ready for use and free of preservatives.
It is clear that modifications and/or additions of parts can be made to the acaricide pharmaceutical composition for the treatment of bee pathologies as described heretofore, without departing from the field and scope of the present invention.
It is also clear that, although the present invention has been described with reference to some specific examples, a person of skill in the art shall certainly be able to achieve many other equivalent forms of acaricide pharmaceutical composition for the treatment of bee pathologies, having the characteristics as set forth in the claims and hence all coming within the field of protection defined thereby.

Claims

1. Acaricide pharmaceutical composition for the treatment of bee pathologies, said acaricide pharmaceutical composition comprising oxalic acid or a salt or ester thereof, in the presence of a polyalcohol or a mixture of polyalcohols chosen from diols, polyols not of sugars and alcohols of sugars, and/or in the presence of citric acid or a salt or ester thereof, one or more hydrocolloids or a mixture of hydrocolloids, in which:
- in the case of polyalcohol or mixture of polyalcohols, the weight/weight ratio between oxalic acid dihydrate and polyalcohol or mixture of polyalcohols goes from 0.25 to 0.80, in particular from 0.45 to 0.75, more particularly from 0.50 to 0.70, even more particularly from 0.55 to 0.65,
- in the case of hydrocolloids or a mixture of hydrocolloids, the weight/weight ratio of oxalic acid dihydrate and hydrocolloid and/or mixture of hydrocolloids goes from 0.05 to 0.25, in particular from 0.05 to 0.2, more particularly from 0.05 to 0.15.
2. Acaricide pharmaceutical composition as in claim 1, in which the weight/weight ratio between citric acid and hydrocolloid or mixture of hydrocolloids goes from 0.005 to 0.15, in particular from 0.01 to 0.1.
3. Acaricide pharmaceutical composition as in claim 1, in which the polyalcohol can be chosen from sugar alcohols, in particular glycerol, mannitol or sorbitol or mixtures thereof.
4. Acaricide pharmaceutical composition as in claim 1, in which the polyalcohol is glycerol.
5. Acaricide pharmaceutical composition as in any claim from 1 to 4, in which the hydrocolloid can be selected from xanthan gum, starch, modified starch, pectin, locust bean, guar gum, gelatin, agar-agar, alginate, carrageenan, cellulose or Arabic gum or mixtures of two or more of them.
6. Acaricide pharmaceutical composition as in any claim from 1 to 4, in which the hydrocolloid is gelatin or pectin or a mixture thereof.
7. Acaricide pharmaceutical composition as in any claim from 1 to 6, for the treatment of bee pathologies connected to the Varroa mite (Mesostigmata, varroa destructor).
8. Acaricide pharmaceutical composition as in any of the claims from 1 to 7, in which the oxalic acid is dihydrate.
9. Acaricide pharmaceutical composition as in any claim from 1 to 8, in which, for 1000 ml of said composition, the oxalic acid dihydrate is present in said composition from 5 to 80 grams, in particular from 25 to 80 grams, in particular from 45 to 75 grams, more particularly from 50 to 70 grams, even more particularly from 55 to 65 grams, for every 100 grams of polyalcohol or mixture of polyalcohols, while in the case of hydrocolloid or mixture of hydrocolloids from 5 to 20 grams, in particular from 5 to 15 grams of oxalic acid dehydrate, are provided for every 100 grams of hydrocolloid or mixture of hydrocolloids.
10. Acaricide pharmaceutical composition as in any of the claims from 1 to 9, in which said acaricide pharmaceutical composition is in liquid form, in particular in liquid solution, or in the form of a support in strips.
11. Acaricide pharmaceutical composition as in claim 10, in which said acaricide pharmaceutical composition comprises water.
12. Acaricide pharmaceutical composition as in claims 9 and 11, in which there is enough water present to reach 1000 ml of said composition in the case of a liquid formulation, and 100 grams in the case of a formulation on a support in strips.
13. Acaricide pharmaceutical composition as in any of the claims from 1 to 12, in which said acaricide pharmaceutical composition is without added preservatives.
14. Acaricide pharmaceutical composition as in claim 11 or 12, or claim 13 when it depends on claims 11 or 12, in which said acaricide pharmaceutical composition consists exclusively of oxalic acid or a salt or ester thereof, of a polyalcohol or a mixture of polyalcohols and water and/or citric acid or a salt or ester thereof, a hydrocolloid or a mixture of hydrocolloids and water.
15. Acaricide pharmaceutical composition as in any of the claims from 1 to 14, in which said acaricide pharmaceutical composition comprises a single polyalcohol and/or a single hydrocolloid, and/or a mixture of hydrocolloids.
16. Package or container comprising an acaricide pharmaceutical composition as in any of the claims from 1 to 15.
17. Method to make an acaricide pharmaceutical composition for the treatment of bee pathologies, said method providing to make a solution of oxalic acid or a salt or ester thereof, in the presence of a polyalcohol or a mixture of polyalcohols chosen from diols, polyols not of sugars and alcohols of sugars, and/or in the presence of citric acid or a salt or ester thereof, one or more hydrocolloids or a mixture of hydrocolloids, in which:
- in the case of polyalcohol or mixture of polyalcohols, the weight/weight ratio between oxalic acid dihydrate and polyalcohol or mixture of polyalcohols goes from 0.25 to 0.80, in particular from 0.45 to 0.75, more particularly from 0.50 to 0.70, even more particularly from 0.55 to 0.65,
- in the case of hydrocolloids and/or a mixture of hydrocolloids, the weight/weight ratio of oxalic acid dihydrate and hydrocolloid and/or mixture of hydrocolloids goes from 0.05 to 0.25, in particular from 0.05 to 0.2, more particularly from 0.05 to 0.15.
18. Method to control the Varroa mite in a hive, or in artificial means associated with the reproduction of bees, said method comprising the application, distribution or delivery in said hive or in said artificial means, of an acaricide pharmaceutical composition as in any of the claims from 1 to 15.
PCT/IT2018/050004 2017-01-16 2018-01-09 Pharmaceutical composition for the treatment of bee pathologies WO2018131058A1 (en)

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