MANGIAFICO SERGIO (IT)
| CN107517963A | 2017-12-29 | |||
| GB2517107A | 2015-02-11 | |||
| JPS51100892A | 1976-09-06 |
| CLAIMS 1 . A process for preparing solid iodophor complexes comprising iodine and cyclodextrins , comprising the step of mixing molecular iodine , and possibly an iodide source , with cyclodextrin . 2 . The process according to the preceding claim, wherein said iodine and said iodide source are separately added to said cyclodextrin . 3 . A process according to claim 1 or 2 , wherein said cyclodextrin is a substituted cyclodextrin or an unsubstituted cyclodextrin or an unsubstituted a-cyclodextrin, β-cyclodextrin or γ- cyclodextrin . 4 . A process according to any one of the preceding claims , wherein said cyclodextrin is selected from the group comprising : a-cyclodextrins , β-cyclodextrins and γ-cyclodextrins , hydroxypropyl -α- cyclodextrin, methyl- β-cyclodextrins, sul f obutylether- β-cyclodextrin, hydroxypropyl - γ-cyclodextrins , me thyl-y- cyclodextrins , sul f obutylether-y-cyclodextr ins , methyl- carboxyl-thioether-y- cyclodextrin . 5 . A process according to any one of the preceding claims , wherein said iodide source is represented by an iodine salt or hydroiodic acid (HI ) possibly generated in si tu by an iodine salt and a reducing agent selected from the group comprising : carbonic acid, formic acid, oxalic acid, or an ammonium salt thereof or urea . 6. A process according to any one of the preceding claims, wherein said mixing is carried out inside a rotational or vibrational ball mill or in a mortar with pestle. 7. A process according to any one of the preceding claims 1 to 6, wherein said mixing is carried out in the absence of solvent. 8. A process according to any one of the preceding claims 1 to 6, wherein said mixing is carried out in the presence of a solvent, where said solvent is selected from the group comprising: water, alcoholic aqueous mixture, ethanol, isopropanol, tetrahydrofuran, ethyl ether, ethyl acetate, hexane. 9. A process according to the preceding claim, wherein said mixing is carried out in the presence of an amount of solvent of about 0.1-1.2 ml/g solid mixture. 10. A process according to any one of the preceding claims, which is carried out by heating the mixture to a temperature of about 40-120°C in a closed container. 11. A process according to any one of the preceding claims, wherein the molar ratios of cyclodextrin to molecular iodine and iodide are from about 1:0.05 to about 1:2 moles/moles for cyclodextrin and molecular iodine, and preferably in a ratio between about 1:0.25 and about 1:1.2. 12. A process according to any one of the preceding claims, wherein the molar ratios of molecular iodine to iodide ion are about 1:0 and about 1:4 moles/moles, and preferably in a ratio between about 1:0.25 and about 1:2. 13. A process according to any one of the preceding claims comprising the further grinding and/or microni zing step . 14 . Solid iodophor complexes of formula : CD/Ix , where CD is a cyclodextrin selected from the group comprising substituted cyclodextrins and unsubstituted cyclodextrins or unsubstituted a-cyclodextrins , β-cyclodextrins and y- cyclodex trins , x=2 and/or 3 , where when x=2 then CD is not a-cyclodextrin, p- cyclodextrin, hydroxypropyl -α- cyclodextrin, methyl- β-cyclodextr in obtained according to the process of any one of the preceding claims . 15 . Solid iodophor complexes of formula : CD/Ix , where CD is a cyclodextrin selected from the group comprising substituted cyclodextrins and unsubstituted cyclodextrins or unsubstituted a-cyclodextrins , β-cyclodextrins and y- cyclodextrins , x=2 or 3 , where when x=2 then CD is not : a-cyclodextrin, p- cyclodextrin, hydroxypropyl -a- cyclodextrin, methyl- p-cyclodextr in . 16. Solid iodophor complexes according to the preceding claim, wherein said cyclodextrin is selected from the group comprising : a-cyclodextrins , β-cyclodextrins and y-cyclodextrins , hydroxypropyl -α- cyclodextrin, methyl- p-cyclodextr in, sul f obutylether- p-cyclodextr in, hydroxypropyl -y-cyclodextrins , me thyl-y- cyclodextrins , sulf obutylether-y-cyclodextr ins , methyl- carboxyl-thioether-y- cyclodextrin . 17. Formulations comprising one or more of the solid iodophor complexes according to claim 14 at concentrations such as to obtain available iodine solutions between 0.01-2%. 18. Solid iodophor complexes according to claim 14 or 15 or 16 or formulations according to claim 17 for medical use. 19. Solid iodophor complexes according to claim 14 or 15 or 16 or formulations according to claim 17 for antiviral, antimicrobial, and antifungal medical use. 20. Use of the solid iodophor complexes according to claim 14 or 15 or 16 in agriculture, phytosanitary field, aquaculture, and animal husbandry. |
DESCRIPTION
Iodine is an element used in the chemical industry and especially in the pharmaceutical industry for the biocidal properties thereof (antibacterial, antiviral and antifungal) . Iodine has several disadvantages, as it is insoluble in water, its crystals sublimate at room temperature, the vapors are oxidizing and chemically aggressive. Therefore, it is difficult to store, handle and dissolve in water, a necessary requirement to obtain aqueous formulations of iodine-based pharmaceuticals. The poor solubility of iodine can be overcome by dissolving it in ethyl alcohol, but alcoholic iodine solutions have drawbacks especially for tolerability, irritability and toxicity, especially when the mucous membranes of various organs are treated. These problems can be reduced by adding iodide ion to iodine solutions to obtain the triiodide species (I 3 -) , which is instead water soluble. However, in this case as well, the irritant effects are not completely eliminated. Despite these drawbacks, Lugol's aqueous solution containing 5% iodine and 10% potassium iodide and the alcoholic iodine solution, known as iodine tincture, have been described for more than a century in several pharmacopoeias and are still in use today. In the last century several iodine-based preparations have been made with the aim of reducing the side effects while maintaining biocidal efficacy. One of the best results was achieved with polyvinylpyrrolidone-iodide (PVP-Iodine) , which is the most important commercial product today. One of the advantages of this product is that it is a powdered solid with an iodine content of about 10%, stable over time. There are several patents and scientific works in the literature which discuss the applications and properties of inclusion complexes between cyclodextrins (CDs) and Iodine (I2) in aqueous solutions. In fact, cyclodextrins are capable of solubilizing hydrophobic molecules in water in particular steric and energetic circumstances and among these also iodine. J. Chem. Educ. vol. 71, no. 8, 1 August 1994 pp 708-714 describes how to solubilize iodine in water with the use of β-cyclodextrin (βCD) . The iodine solution in water obtained by formation of the βCD/I2 complex is used today as a mouthwash for gargling, and is commercially available in Japan.
Another work (Drug Development and Industrial Pharmacy Vol. 28, No. 10, pp. 1303-1309, (2002) , reports the use of hydroxypropyl-a- cyclodextrin (HPαCD) as a better alternative to a-cyclodextrin (αCD) for the formation of the complex with Iodine which is 100 times more soluble with respect to αCD and also with respect to p- cyclodextrin (pCD) ; furthermore, it would have a greater antiseptic activity than 1 PVP-Iodine which is the most used Iodine-based product in the world. In the same work, reference is made to the greater stability of the HPαCD/Iodine complex; in fact, it is reported that the loss of iodine from an aqueous solution where hydroxypropyl-a-cyclodextrin is present is lower with respect to aqueous solutions of Iodine where a-Cyclodextrin or β-cyclodextrin is used and is also lower with respect to aqueous solutions where PVP or KI is present as a complexant . JP
No S51100892A describes the preparation of the solid complex βCD/ I2 , useful as an anti fungal antiseptic and as a preservative for food, by means of precipitation exploiting the poor solubility that these Cyclodextrin/ Iodine complexes have in water already at room temperature .
Chinese patent application CN 107517963A reports the preparation of the complex HPαCD/ I 2 by means of precipitation from an aqueous solution, in the presence of a Tween 80 surfactant . This cyclodextrin is more soluble than the respective native αCD, whereby the temperature of 0 ° C is necessary for it to precipitate as a complex with iodine . This procedure includes the presence of Potassium Iodide , in aqueous solution, not exceeding 30% of the molecular iodine . The preparation procedure for precipitation from saturated solutions as done for HPαCD becomes impossible , even at refrigerated temperature , for all substituted β-cyclodextrins which are very soluble such as hydroxypropyl- β-cyclodextrins , so similarly for all y-cyclodextrins and all those cyclodextrins substituted with groups which confer great solubility in water . As mentioned above , it is impossible to obtain solid complexes in powder form, CD/ I2 , for cyclodextrins such as hydroxypropyl- β- cyclodextrins (HPβCD) , methyl- β-cyclodextrins (RMβCD) and sul fobutylether- β-cyclodextrins ( SBEβCD) . It is even more di f ficult for hydroxypropyl-y-cyclodextrins (HPγCD) , methyl-y- cyclodextrins , sul fobutylether-y-cyclodextrins , and methyl- carboxyl-thioether-y-cyclodextrin ( sugammadex ) ; in fact , the existence and preparation of these cyclodextrins as solid complexes are not reported except for methyl-β-cyclodextrin (RMβCD) . The preparation of the solid complex, RMβCD/I 2 , by treating RMβCD with an excess of Iodine in a rotary evaporator i s reported in Tze-Loon ' s work; thereby, the formation of the complex is obtained by virtue of the inclusion of gaseous iodine , obtained by heating at high temperature , inside the cavity of the methyl-β- cyclodextrin . This preparation leads to the formation of the complex RMβCD/I 2 , with a procedure of only academic interest and not easily practicable from an industrial point of view, as in the case of precipitation (RMβCD would be too soluble for the precipitation method) .
In conclusion, the preparation of solid CD/I 2 complexes reported in the scienti fic and patent literature is substantially limited to those few complexes which can best form saturated solutions and thus precipitate .
Summary
The inventors of the present patent application have surprisingly developed a new process for preparing solid iodophors , comprising cyclodextrins .
Brief description of the drawings
Figure 1 shows the thermogram related to HPβCD used in the preparation of iodophors and figure 2 the related FTIR plot .
Figure 3 shows the thermogram related to HPβCD/I 3 K of Example 3 and figure 4 the related FTIR plot . Figure 5 shows the thermogram related to HPβCD/I 3 K of Example 4 and figure 6 the related FTIR plot.
Figure 7 shows the thermogram related to RMβCD used in the preparation of iodophors and figure 8 the related FTIR plot.
Figure 9 shows the thermogram related to RMβCD/I 3 H of Example 6 and figure 10 the related FTIR plot.
Figure 11 shows the thermogram related to αCD used in the preparation of iodophors and figure 12 the related FTIR plot.
Figure 13 shows the thermogram related to αCD/I 3 H of Example 10 and figure 14 the related FTIR plot.
Object of the invention
In a first object, a process for preparing solid complexes comprising iodine referred to as "iodophors" is described.
In a second object, solid complexes comprising iodine (referred to as "iodophors") obtained with the process of the invention and solid complexes comprising iodine (referred to as "iodophors") as such are described.
In a third object, formulations comprising the complexes of the invention are described.
In a fourth object, the complexes and formulations comprising the complexes of the invention are described for medical and veterinary use.
In particular, the complexes and formulations comprising the complexes of the invention are described for antiviral, antimicrobial and antifungal medical use. In a further object, the use of the solid complexes of the invention and formulations comprising them as antiviral, antimicrobial and antifungal agents is described.
In a still further object, the use of the solid complexes of the invention and formulations comprising them in agriculture, the phytosanitary field, aquaculture and animal husbandry is described .
Detailed description of the invention
Def ini tions
For the purposes of the present patent application, the following terms are defined:
Available iodine = iodine titratable with thiosulphate
Total iodine = iodide + titratable iodine
Free iodine = uncomplexed iodine which can be determined in a dialysis test or in an electrochemical model or iodine which can be extracted with heptane or cyclohexane from an aqueous solution of defined concentration .
In the continuation of the present patent application, the compounds described will be referred to with the term "iodophors".
In a first object, it is described a process for preparing the complexes of the invention.
In particular, such a process comprises the step of mixing molecular iodine with an iodide source and adding cyclodextrin (method A) .
Likewise, such a process can comprise the step of mixing only molecular iodine with cyclodextrin (method A' ) . Alternatively, the method can comprise a first step of mixing cyclodextrin with an iodide source to obtain a CD/ I- complex and a second step of adding molecular iodine (method Bl ) .
In particular, the second step can include heating the mixture of CD/ I- and molecular iodine at a temperature of about 40- 120 ° C for a few hours in a sealed heating container until the solid iodophor complex CD/ I 3 - is obtained .
Likewise , the method can comprise a first step of mixing cyclodextrin with molecular iodine to obtain a CD/ I2 complex and a second step of adding an iodide ion source (method B2 ) .
Therefore , in methods Bl and B2 described above , there is a first step in which one among molecular iodine and the iodide source is added to cyclodextrin and the other ( among molecular iodine and the iodide source ) is then added; therefore , in such methods , molecular iodine and the iodide source are added separately to cyclodextrin .
For the purposes of the present invention, in the described method, the cyclodextrin is preferably chosen from the group comprising : substituted cyclodextrins and unsubstituted cyclodextrins or unsubstituted a-cyclodextrins , β-cyclodextrins and y-cyclodextrins .
Examples of cyclodextrins are therefore : a-cyclodextrins , p- cyclodextrins and y-cyclodextrins , hydroxypropyl-a-cyclodextrin (HPαCD) , methyl- β-cyclodextrin (RM- p-CD) , sul fobutylether- p- cyclodextrin ( SBEpCD) , hydroxypropyl-y-cyclodextrins (HPγCD) , me thyl-γ- cyclodextrins , sul f obutylether-y-cyclodextr ins , me thy 1- carboxyl-thioether-y-cyclodextrin ( sugammadex ) .
As regards the source of iodide ions , this can be : an iodine salt , for example : iodide of an alkali or alkaline earth metal ; for example , it can be potassium iodide , or hydroiodic acid (HI ) .
According to an aspect of the present invention, the hydroiodic acid can be formed in si tu from an iodine salt and a reducing agent .
In particular, such a reducing agent can be an acid selected from the group comprising : carbonic acid, formic acid, oxalic acid, or an ammonium salt thereof ; alternatively, urea can be employed .
According to an aspect of the present invention, the mixing of the components can be carried out in a dry way .
In particular, such a mixing can be obtained inside a rotational or vibrational ball mill or in a mortar with pestle ; alternatively, equipment capable of shredding, crushing, grinding or microni zing can be employed .
In a particular aspect of the present invention, the dry process can be promoted with LAG ( Liquid Assisted Grinding) technology, soaking the solid mixture with an amount of a solvent of about 0 . 1- 1 . 2 ml/g of solid mixture .
The amount of solvent indicated above is minimal and is notsuf f icient to obtain a solution . In particular, such a solvent is selected from the group comprising: water, aqueous alcoholic mixture, ethanol, isopropanol, tetrahydrofuran, ethyl ether, ethyl acetate, hexane. The process can be carried out by heating the mixture at a temperature of about 40-120°C for a few hours in a sealed heating container until the solid iodophor complex CD/I 3 - is obtained.
In accordance with another aspect of the present invention, the mixing of the components can be carried out in a solvent.
In particular, such a solvent is selected from the group comprising: water, aqueous alcoholic mixture, ethanol, isopropanol, tetrahydrofuran, ethyl ether, ethyl acetate, hexane. For the purposes of the present invention, the molar ratios of cyclodextrin, molecular iodine and iodide ion in the mixture placed under mechanical stirring are preferably from about 1:0.05 to about 1:2 mol/mol for cyclodextrin and molecular iodine and preferably in a ratio between about 1:0.25 and about 1:1.2.
The molar ratios of molecular iodine to iodide ion (introduced for example as an iodide salt or as hydroiodic acid) can range from about 1:0 to about 1:4 mol/mol and are preferably in a ratio between about 1:0.25 and about 1:2.
If the iodide ion is produced in situ by reaction of iodine and organic acid the molar ratios can be as follows:
- between cyclodextrin and iodine are preferably from about 1:0.1 to about 1:4, and
- between iodine and organic acid, or the ammonium salts or amides thereof, from about 1:0.2 to about 1:0.8. The process of the invention leads to the preparation of solid iodophors .
According to an aspect of the present invention, such iodophors can be subj ected to a further grinding and/or microni zing step until the desired particle si ze is obtained .
In accordance with a second obj ect of the invention, solid complexes ( iodophors ) are described, obtained with the described process having formula :
CD/Ix , where
CD= cyclodextrin and x=2 or 3 .
When x=2 then reference is made to molecular iodine complexes (I 2 ) .
When x=3 then reference is made to triiodide ion complexes ( I 3 - ) .
According to a first aspect , the solid iodophor complexes of the invention have formula CD/ Ix, where x=2 ; therefore , such complexes have formula CD/ I2 .
In a second aspect of the present invention, the solid iodophor complexes of the invention have formula CD/ Ix, where x=3 ; therefore , such complexes have formula CD/ I 3 - .
In an aspect of the invention, the sol id iodophor complexes described above are obtained by the process of the invention .
For the purposes of the present patent application, solid iodophor complexes are presented with a granulometry which depends on the conditions applied during the process and, in particular, in the grinding step . Solid iodophor complexes described by this patent application comprise :
In an aspect of the invention, the solid iodophor complexes reported in the table above are obtained by the process of the invention :
In accordance with a particular aspect , complexes ( or " iodophors" ) are described comprising cyclodextrin, as such .
In particular, such complexes have the formula :
CD/Ix , where
CD= cyclodextrin and x=2 or 3 .
When x=2 then reference is made to molecular iodine complexes ( I2 ) . When x=3 then reference is made to triiodide ion complexes ( I 3 - ) . In a preferred aspect, CD is a cyclodextrin selected from the group comprising: substituted cyclodextrins and unsubstituted cyclodextrins or unsubstituted a-cyclodextrins, β-cyclodextrins and y-cyclodextrins .
Examples of cyclodextrins are therefore: a-cyclodextrins, p- cyclodextrins and y-cyclodextrins, hydroxypropyl-a-cyclodextrin (HPαCD) , methyl-β-cyclodextrin (RM-p-CD) , sulfobutylether-p- cyclodextrin (SBEpCD) , hydroxypropyl-y-cyclodextrins (HPγCD) , methyl -y-cyclodextrins , sulf obutylether-y-cyclodextr ins , me thy 1- carboxyl-thioether-y-cyclodextrin (sugammadex) .
For the purposes of the present patent application, in compounds having the above formula, when x=2 then CD is not: a-cyclodextrin (a-CD) , β-cyclodextrin (p-CD) , hydroxypropyl-a-cyclodextrin (HPαCD) , methyl-β-cyclodextrin (RM-p-CD) .
In accordance with a third object, there are described formulations comprising the solid iodophor complexes of the invention .
In particular, such formulations comprise the solid iodophor complexes of the invention CD/I 3 and CD/I 2 .
More in particular, the formulations of the invention comprise solid iodophor complexes at concentrations such as to obtain solutions of available iodine (titratable with thiosulfate) between 0.01 -2% (corresponding to an iodophor concentration which can range from about 0.1-30%) .
In particular, there can be prepared formulations in the form of solution, gel, hydrogel, emulsion, ointments, tablets or powders . For the purposes of the present invention, for example the emulsions contain an oil phase in water, a surfactant and/or an emulsifier .
Oils which can be employed to this end comprise: petrolatum, mineral oils, liquid waxes, long chain esters, lanolin, liquid paraffin, essential oils, vegetable oils, hydrogenated vegetable oils and long- and medium-chain triglycerides, natural or synthetic mono-, di-, tri-glycerides.
The formulations can contain organic and inorganic pH buffers which can confer a pH between 2.0 and 7.0 and preferably between 3.0 and 5.5.
In a preferred aspect, the buffer is citrate buffer or phosphate buffer .
According to the type of formulation, there can be included one or more other components selected among: osmotic agents, surfactants, emulsifiers, oils, viscosifying or gelling polymers, preservatives, cyclodextrins, complexing agents, inorganic salts and mixtures of these excipients.
Examples of solutions comprising iodophor complexes according to the present invention are reported in the following table:
Table 5.
*RβΒCD, HPβCD, βCD, SBEβCD, HPαCD, αCD, HPγCD
Examples of ointments and creams comprising iodophor complexes according to the present invention are reported in the following table :
Table 6 .
*RMβCD, HPβCD, βCD, SBEβCD, HPαCD, αCD, HPγCD
Examples of emulsions comprising iodophor complexes according to the present invention are reported in the following table :
Table 7 .
*RMβCD, HPβCD, βCD, SBEβCD, HPαCD, αCD, HPγCD
In accordance with a fourth obj ect , the complexes and formulations comprising the complexes of the invention are described for medical or veterinary use .
According to a particular aspect , the complexes and formulations comprising the complexes of the invention are described for biocidal medical or veterinary use .
More in particular, such a biocidal medical or veterinary use is an antiviral , antimicrobial and anti fungal use . For the purposes of the present invention, the term "biocidal" refers to activity against a broad spectrum of viruses, bacteria, fungi, spores, mycobacteria, parasites, prions and other microbes. With reference to viruses infecting animals, especially mammals or humans, these can include, by way of explanation: papovaviruses , Louis encephalitis viruses, retroviruses, rhabdoviruses , coronaviruses, rhinoviruses, orthomyxoviruses, caliciviruses , filoviruses, astroviruses, and potentially all viruses are susceptible to the compositions of this invention. Viruses such as that of influenza, especially H5N1 influenza, Herpes Simplex Virus, (HSV1 and HSV2 ) , Coxsackie virus, Humam immunodeficiency virus (I and II) , Andes virus, Dengue virus, Papilloma, Epstein- Barr virus, Variola and other pox viruses, West Nile virus are relevant targets of the compositions in accordance with this invention .
With reference to bacteria, these include: gram negative, gram positive and include antibiotic resistant comprising: MRSA, S. aureus; S. epidermidis ; S. saphrophyticus , and other bacteria responsible for infections in humans and animals.
With reference to fungi, these include: Aspergillus, Coccidioides , Histoplasma capsulatum and Candida.
The solid iodophor complexes of the present invention are further useful in the elimination of spores such as sporangiospores and zygospores from fungi, ascospores from ascomycetes, basidiospores from basidiomycetes , aeciospores, teliospores and uredeiospores from fungi; also included are meiospores, microspores, megaspores, mitospores , zoospores , aplanospores , autospores , ballistospores and statismospores .
In a further obj ect , there is described the use of the solid iodophor complexes of the invention and formulations comprising them as antiviral , antimicrobial and anti fungal agents , as described above .
Furthermore , there is described the use of the solid iodophor complexes of the invention for the removal of molds from surfaces that they can deteriorate .
With reference to mold, these include : Zygomicota and Ascomicota, Acrenomium, Alternaria, Aspergillus , Cladosporium, Fusarium, Mucor, Pennicillinum, Rhizopus , Trichoderma and Stachibotrys .
For the purposes of the present invention, the treated surfaces can be natural or arti ficial .
In a still further obj ect , there is disclosed the use of the solid complexes of the invention and formulations comprising them in agriculture , the phytosanitary field, aquaculture and animal husbandry .
In particular, in agriculture , the solid iodophor complexes of the invention find use as bioforti fying agents .
In particular, in the phytosanitary field, the solid iodophor complexes of the invention find use as antivirals .
In fact , the solid iodophor complexes of the invention can be employed against viruses in plants .
In particular, these can be plants used in agriculture also for food use . The target viruses can be chosen from the group comprising: Partitiviruses , Potyviruses, Bromoviruses, Comoviruses,
Geminiviruses , Rhabdoviruses , Reoviruses, Satellite Viruses, Tombosviruses , Sequiviruses , and other viruses which infect plants .
In particular, in aquaculture, the solid iodophor complexes of the invention find use as a preservative.
In particular, in animal husbandry, the solid iodophor complexes of the invention find use as a source of iodine.
Preparation examples
Preparation examples for HPγCD/I 3 K, HPγCD/I 3 H, HPβCD/I 3 K, HPβCD/I 3 H, HPβCD/I 2 , RMβCD/I 3 H, RMβCD/I 3 K, βCD/I 3 K, βCD/I 3 H, αCD/I 3 H are shown below .
Example 1. Preparation of HPγCD/I 3 K
Hydroxypropyl-γ-cyclodextrin, (154 g; 0.1 mol) and potassium iodide (12.5 g; 0.075 mol) are mixed dry with molecular Iodine (19.0 g; 0.075 mol) and then introduced into a rotating ball mill. Depending on the set power, the mill is left under mechanical action until the desired granulometry is obtained, at the end of the grinding the product is forced through a 0.038 mm sieve before being formulated. The percentage of available iodine (titratable with thiosulfate) was found to be 10.1%.
Example 1.1 Preparation of HPγCD/I 3 K
Hydroxypropyl-γ-cyclodextrin, (154 g; 0.1 mol) and potassium iodide (12.5 g; 0.075 mol) are mixed dry and then introduced into a rotating ball mill. Depending on the set power, the mill is left under mechanical action until a homogeneous mixture is obtained, then in a second stage the mixture is added with molecular iodine (19.0 g; 0.075 mol) . The mixture containing iodine is sealed in a screw-capped glass bottle and then heated at 60°C for 6 h. The product thus obtained after cooling is forced through a 0.038 mm sieve before being formulated. The percentage of available iodine (titratable with thiosulfate) was found to be 9.9%.
Example 2. Preparation of HPγCD/I 3 H
Hydroiodic acid, 20.2 g of 57% aqueous solution (equivalent to 11.5 g; 0.09 mol of hydroiodic acid) are introduced into 10 ml of ethanol in which 22.8 g (0.09 mol) of iodine have been previously suspended. The ethanol solution thus composed is poured onto 154 g (0.1 mol) of hydroxypropyl-y-cyclodextrin, then homogenized with a pestle in a mortar until it dries. The almost dry powdered product is introduced into a rotating mill with a 250 ml teflon container. The rotor acceleration of 1g and the rotation speed of the container is set at 180 rpm. 20 mm zirconia balls were used as grinding bodies. The mechanical process was continued for 30 minutes. The percentage of iodine available in the iodophor produced was found to be 11.8%. The powder obtained is forced through a 0.022 mm sieve before being formulated.
Example 3. Preparation of HPβCD/I 3 K
12.7 g of iodine (0.05 mol) and 8.3 g of potassium iodide (0.05 mol) are dissolved in 50 ml of ethanol and then the ethanol solution poured into 100 g of hydroxypropyl-β-cyclodextrin (0.067 mol) placed in a porcelain mortar. The mixture is pressed and homogenized with a pestle until a homogeneous paste is obtained. Ethanol is evaporated from the product at room temperature for 2 h and then shredded with a pestle until a powder is obtained. The percentage of iodine available in the iodophor produced was found to be 10.1%.
Example 3.1 Preparation of HPβCD/I 3 K
8.3 g of potassium iodide (0.05 mol) are dissolved in 40 ml of ethanol and then the ethanol solution poured into 100 g of hydroxypropyl-β-cyclodextrin (0.067 mol) placed in a porcelain mortar. The mixture is pressed and homogenized with a pestle until a homogeneous paste is obtained. Ethanol is evaporated from the product at 50°C for 2 h and then shredded with a pestle until a powder is obtained, to which 12.7 g of previously shredded iodine (0.05 mol) is added. The mixture containing iodine is placed in a closed glass vial with screw cap and heated for 8h at 60°C. The percentage of iodine available in the iodophor thus obtained was found to be 10.3%.
Example 3.2 Preparation of HPβCD/I 3 K
12.7 g of iodine (0.05 mol) are mixed with 100 g of hydroxypropyl - β-cyclodextrin (0.067 mol) placed in a closed glass container with cap and then heated at 70°C for 6h. The cyclodextrin iodine complex thus obtained is added with 8.3 g of potassium iodide (0.05 mol) and then 35 ml of ethyl alcohol are added. The ethanol mixture thus obtained is pressed and homogenized with a pestle until a homogeneous paste is obtained. Ethanol is evaporated from the product at room temperature for 2 h and then shredded with a pestle until a powder is obtained. The percentage of iodine available in the iodophor produced was found to be 10.3%.
Example 4. Preparation of HPβCD/I 3 H
15.2 g of iodine (0.06 mol) and 13.5 g of a 57% aqueous hydroiodic acid solution (equivalent to 7.7 g; 0.06 mol of hydroiodic acid) are dissolved in 30 ml of ethanol and then the ethanol solution poured into 100 g of hydroxypropyl-β-cyclodextrin (0.067 mol) placed in a porcelain mortar. The paste is pressed and homogenized by hand with a pestle until a powder is obtained. The product is left to dry again in air for 2 h or under vacuum in a rotary evaporator, then it is reduced to powder with a pestle. The percentage of iodine available in the obtained iodophor product was found to be 12.0%. The product is formulated as the product above .
Example 4.1 Preparation of HPβCD/I 3 H Hydroxypropyl-β-cyclodextrin (100 g; 0.067 mol) is suspended in 20 ml of water. Formic acid (1.5 g; 0.033 mol) is added to this solution and then the solution is brought to pH 7.5 with an ammonia solution. Iodine (25.4 g; 0.1 mol) is introduced into the suspension. The solution is left under mechanical stirring for about 2 hours until the foam produced by the development of CO2 is finished, after which the solution is heated to 65°C and allowed to evaporate until a pasty solid is obtained, which is mechanically treated with a pestle until a dry powder is obtained. The percentage of iodine available in the iodophor obtained was found to be 12.1%.
Example 4.2 Preparation of HPβCD/I 3 H
Ammonium formate (2.08 g; 0.033 mol) is dissolved in a mortar using 15 ml of water, the aqueous solution is brought to pH 7.6 with a concentrated ammonia solution and then iodine (25.4 g; 0.1 mol) and after 100 minutes hydroxypropyl-β-cyclodextrin (100 g; 0.067 mol) is added to the solution. The suspension is mixed continuously with a pestle for about 1 h, allowed to dry until a solid product is obtained, which can be easily reduced to a powder with a pestle. The percentage of available iodine (titratable with thiosulphate) in the iodophor thus obtained was found to be 12.3%.
Example 5. Preparation of HPβCD/I 2 Hydroxypropyl-β-cyclodextrin, (150 g; 0.1 mol) is mixed dry with molecular Iodine (20.3 g; 0.08 mol) and then introduced into a rotating ball mill. Depending on the set power, the mixture is left under the mechanical action of the mill until the desired particle size is obtained at the end of the grinding. The percentage of available iodine (titratable with thiosulphate) in the iodophor product obtained was found to be 11.3%.
Example 5.1 Preparation of HPβCD/I 2
The hydroxypropyl-β-cyclodextrin, (150 g; 0.1 mol) powder is mixed dry with molecular Iodine (25.4 g; 0.1 mol) previously pulverized with pestle in a mortar and then introduced into a tightly closed glass container that is heated at 60°C for 10 h, then the product obtained is left open until reaching room temperature. The percentage of available iodine (titratable with thiosulphate) in the iodophor product thus obtained was found to be 12.9%.
Example 6. Preparation of RMβCD/I 3 H
Hydroiodic acid, 20.2 g of 57% aqueous solution, (equivalent to 11.5 g; 0.09 mol of hydroiodic acid) are introduced into 50 ml of ethanol in which 22.8 g (0.09 mol) of iodine were previously dissolved. The ethanol solution thus composed is poured into 160 g (0.12 mol) of methyl-β-cyclodextrin, the paste is homogenized (if necessary adding a few ml of ethanol or water up to the right consistency) with a pestle in a mortar until it dries, the almost dry powder product is introduced into a rotating mill in a 400 ml teflon container. The rotor acceleration of 1g and the rotation speed of the container is set at 200 rpm. 20 mm zirconia balls were used as grinding bodies. The mechanical process was continued for 40 minutes. The percentage of iodine available in the iodophor obtained was found to be 11.9%. The powder obtained is forced through a 0.038 mm sieve before being formulated.
Example 6.1 Preparation of RMβCD/I 3 H 30.48 g (0.12 mol) of iodine and 160 g (0.12 mol) of methyl-p- cyclodextrin are mixed and gently homogenized with a pestle in a mortar, the mixture is transferred to a closed glass container with screw cap and then heated at 60°C for 8h. After allowing the Cyclodextrin Iodine complex thus obtained to cool in air, 26.9 g of a 57% aqueous solution of hydroiodic acid (equivalent to 15.3 g; 0.12 mol of hydroiodic acid) are added, then the water evaporated by rotary evaporation at room temperature and pressure of 100 mmHg. The percentage of iodine available in the iodophor thereby obtained was found to be 13.2%. The powder obtained is forced through a 0.038 mm sieve before being formulated.
Example 7. Preparation of RMβCD/I 3 K Methyl-β-cyclodextrin, (131 g; 0.1 mol) and potassium iodide (12.5 g; 0.075 mol) are mixed dry with molecular Iodine (19.0 g; 0.075 mol) and then introduced into a rotating ball mill. Depending on the set power, the mill is left under mechanical action until the desired granulometry is obtained, at the end of the grinding the product is forced through a 0.038 mm sieve before being formulated. The percentage of available iodine (titratable with thiosulfate) was found to be 10.4%.
Example 7.1 Preparation of RMβCD/I 3 K Methyl-β-cyclodextrin, (131 g; 0.1 mol) and potassium iodide (12.5 g; 0.075 mol) are mixed in 25 ml of an ethanol solution containing molecular Iodine (19.0 g; 0.075 mol) and then introduced into a rotating ball mill. Depending on the set power, the mill is left under mechanical action until the desired particle size is obtained. The percentage of available iodine (titratable with thiosulfate) was found to be 11.7%.
Example 8. Preparation of βCD/I 3 H
The β-cyclodextrin (113.5 g; 0.1 mol) is suspended in 35 ml of water. Ammonium formate (2.40 g; 0.037 mol) is added to this suspension and then the suspension is brought to pH 7.5 with an ammonia solution. Iodine (28.4 g; 0.112 mol) is introduced into the suspension. The pasty suspension is left under mechanical stirring for about 2 hours until the foam produced by the development of CO2 is finished, after this the solution is heated to 70°C by grinding the solid with a pestle from time to time until dryness and obtaining a powder. The percentage of iodine available in the iodophor obtained was found to be 13.4%.
Example 9. Preparation of βCD/I 3 K
25.4 g of iodine (0.1 mol) and 19.9 g of potassium iodide (0.12 mol) are dissolved in 45 ml of ethanol and then the ethanol solution poured onto 113 g of β-cyclodextrin (0.1 mol) previously placed in a porcelain mortar. The paste is pressed and homogenized by hand with a pestle until a powder is obtained. The product is allowed to air dry for 3 h. The percentage of iodine available was found to be 15.6%. The product is formulated as the product above.
Example 10. Preparation of αCD/I 3 H
Hydroiodic acid, 16.7 g of 57% aqueous solution (equivalent to 9.5 g; 0.075 mol of hydroiodic acid) are introduced into 40 ml of ethanol in which 19.0 g (0.075 mol) of iodine were previously dissolved. The ethanol solution thus composed is poured onto a- cyclodextrin (97.3 g; 0.1 mol) . The paste is homogenized (if necessary adding a few ml of ethanol or water to obtain the right consistency of the paste) with a pestle in a mortar until a dry powder product is obtained. The percentage of iodine available in the iodophor thus obtained was found to be 14.9%.
Each preparation example above can be applied starting from any unsubstituted or substituted a, p, and y-cyclodextrin .
Structural confirmation In accordance with the analyses ( thermogravimetric, infrared, and UV) , the iodophors obtained in the above examples are new inclusion structures .
Methods
Thermogravimetric analysis was conducted using a TGA Q500 ( TA Instruments , a division Waters ) using a platinum crucible , under nitrogen atmosphere ( flow 60 mL/min) in a temperature range from 50 ° C to 500 ° C with a heating rate of 5 ° C per minute . 2 to 8 mg of sample were weighed for the analyses . The percentage loss in weight and the corresponding derivative was recorded as a function of temperature .
IR analyses were conducted with a Perkin Elmer Spectrum 100 spectrometer ( Perkin Elmer, Waltham, MA, USA) reporting Fourier trans form spectra ( FTIR) . The analysis was conducted directly on the samples without preliminary treatments , us ing a sampling system for universal ATR acquiring the spectrum from 4000 to 600 cm -1 , with a resolution of 4 . 0 cm -1 .
The UV analysis in water of the new iodophors , not reported, shows for all inclusion complexes absorption spectra where the absorption band at 352 nm typical of the triiodide ion and the band between 450 and 550 nm typical of Iodine are present . The determined amount of iodine available in the various iodophors , by means of UV spectroscopy and/or thiosul fate titration, is always that expected from the stoichiometry used in the preparation . Similarly, aliquots of subsequent extractions with cyclohexane , from an aqueous phase containing any iodophor of the present patent application, collected in a single container until the discoloration of the aqueous phase provides an organic phase containing all the molecular iodine I2 which is expected from the stoichiometry of the preparation, this evaluation is performed by UV spectroscopy at the wavelength of 525 nm (maximum absorption of the molecular iodine in cyclohexane ) .
The figures show the thermograms and FTIR plots of some of the compounds exempli fied in the examples .
Table 1 below shows the data in the thermogravimetric analyses .
Table 1
Table 2 below shows the data of the evaluations using FTIR .
Table 2
Solid iodophor stability
The powder iodophor products obtained were placed in low density polyethylene containers at a temperature of 25°C and a relative humidity (RH) of 60%. The products were highly stable, as expected from the thermogravimetric studies. The iodine content after 24 months is 100% of the initial titer for all CD/I 3 K and CD/I 3 H products and greater than 90% for the CD/I 2 complexes with the exception of yCD/I 2 which reaches 82% of the initial iodine titer. The following table shows the controls at the production date (TO) and after 3 (T3) , 6 (T6) 12 (T12) and 24 (T24) months. Table 3 below shows the results of the solid iodophor stability study .
Table 3
Iodophor stability in aqueous solution
The iodophors were formulated in 0.025% aqueous solution of available iodine (about 0.25% by weight of iodophor) . The formulations were placed in 50 ml glass vials with a polypropylene screw cap and Teflon gasket and stored at a temperature of 25°C and a relative humidity (RH) of 60%. 1 ml is taken from the different formulations at the time of analysis.
The Table below shows the stability data of some iodophors in 0.25% aqueous solution.
Table 4.
Biocidal Activity
Determination of antiviral activity (Time killing assay)
Each virus suspension to be tested was replicated in the MRC-5 cell line , so as to produce a high titer of each virus . After virus replication, the cultures were frozen/ thawed almost 3 times to disrupt the cells and the cell debris was separated by centri fugation ( 400g for 15 minutes ) . For each viral strain, the TCID50/mL ( Tissue Culture Infection Dose 50 ) was determined by titration on a plate reader and was used to inoculate three di f ferent test solutions : the iodophor to be tested at 0 . 025% of available iodine , the blank for the negative control and the positive control consisting of the blank containing an antiviral drug at the concentration of the relative IC50 for the virus in question. Each viral strain was used to inoculate the three test solutions by adding 100 gL of each viral strain to tubes containing 900 gL of the iodophor solution to be tested, in 900 gL of the blank and in 900 gL of positive control (antiviral formulated in the blank) . The incubation times were: 5s, 10s, 30s, 1, 3, 6, 60 min, 3, 6, 12 hours. After the established contact times, the treated suspensions containing the viral strain were neutralized with a sterile solution of 0.5% (w/w) sodium thiosulfate by 1:10 (v/v) dilution. The logarithmic titer in
TCID50/mL was calculated using the Spearman-Karber method, as described in paragraph C.3.2 of UNI EN 14476 : 2013+A2 : 2019 (E) . The iodophors of this invention induce, at the concentration of 0.025 of available iodine, an inhibitory effect of: >3-LoglO after 5 seconds of exposure; > 4-LoglO after 1 minute of exposure and >5- LoglO after 6 hours of exposure.
Determination of antimicrobial activity (Time killing assay) The bacterial suspension for each reference strain was prepared from a 16 hour growth culture and then diluted to ~ 1.5 x 10 8 CFU/ml, estimated by UV spectrophotometer using as reference the standard turbidity of 0.5 McFarland units. Three types of samples were inoculated for each bacterial strain: the iodophor to be tested at 0.025% of available iodine, the blank for the negative control and the positive control, consisting of the blank containing an antibiotic at a concentration corresponding to the respective MIC of each microorganism evaluated. An intermediate suspension of about 7.5 x 10 6 CFU/ml was prepared for each strain, in order to obtain a final concentration of 5x10 5 CFU/ml applied in the test. Each bacterial suspension was used to inoculate the three samples, adding 100 pL of each bacterial suspension to the three tubes containing 1.9 mL of the iodophor to be tested, 1.9 mL of blank formulation, 1.9 mL of positive control, respectively. The final concentration of each bacterial suspension was ~5xl0 5 CFU/mL. The dilution effect of the inoculum did not affect the concentration of the tested solutions, as it did not exceed 5% of the total final volume of the test. Each of the three samples containing the inoculated bacterial suspension was incubated for the following times: 10, 20, 40 s, 1, 2, 4, 8, 60 min, 6 and 12h. After the set times, the suspension in the tube was diluted 1:10 (v/v) by withdrawing 100 pL and bringing to a final volume of 1000 pL of sterile 0.5% sodium thiosulfate solution. From this suspension, 100 pL were inoculated onto agar plate/with culture medium then the plates were incubated at 37°C for 24-48 hours. The control of the inoculum applied for the time killing assay was carried out as follows. From the last suspension of each bacterial strain used for the colony count calculation, two further 1:10 (v/v) dilutions were prepared in sterile saline and placed on agar/culture medium, so as to verify the inoculum used for the test. Dilutions were made so as to have a colony count between 20- 300 CFU/plate as an interval, but not less than 6. The plates were incubated at 37 °C for 24 hours prior to the colony count. The iodophors of this invention induce an inhibitory effect > 3-LoglO between 10s and 2 minutes exposure, but most bacterial strains are inhibited within 40s.
Determination of antifungal activity (Time killing assay)
The fungal suspension for each reference strain was prepared from a 5-7 day growth culture and then diluted to ~ 1.5 x 10 6 CFU/ml, estimated by UV spectrophotometer using as reference the standard turbidity of 0.5 McFarland units. Three types of samples were inoculated for each fungal strain: the iodophor to be tested at 0.025% of available iodine, the blank for the negative control and the positive control, consisting of the blank containing an antifungal at a concentration corresponding to the respective MIC of each microorganism evaluated. Each fungal suspension was used to inoculate the three samples, adding 100 pL of each bacterial suspension to the three tubes containing 1.9 mL of the iodophor to be tested, 1.9 mL of blank formulation, 1.9 mL of positive control, respectively. The final concentration of each fungal suspension was approximately 2x10 4 ÷ 1x10 5 CFU/mL. The dilution effect of the inoculum did not affect the concentration of the tested solutions, as it did not exceed 5% of the total final volume of the test. Each of the three samples containing the inoculated fungal suspension was incubated for the following times: 10, 20, 40 s, 1, 2, 4, 8, 60 min, 3, 6 and 12h. After the set times, the suspension in the tube was diluted 1:10 (v/v) by withdrawing 100 pL and bringing to a final volume of 1000 pL of sterile 0.5% sodium thiosulfate solution. From the latter by 1:10 (v/v) dilution, a suspension was prepared in sterile water from which 50 pL was taken for each fungal strain and plated on agar medium, the plates were incubated at 35°C for 24-72 hours. The control of the inoculum applied for the time killing assay was carried out concurrently with the test, so as to have a colony count ≤ 150 colonies, according to ISO 21527-2:2008: selecting and counting plates containing less than 150 colonies. The plates were incubated at 35°C for 24-72 hours. The non-linearity of the counts, by dilution, often occurs, i.e., 10-fold dilutions of the samples often do not translate into 10-fold reductions in the number of colonies recovered. This was attributed to the fragmentation of the mycelia and disruption of the spore clumps during dilution, in addition to competitive inhibition especially when large numbers of colonies are present on the plates. The iodophors of this invention showed efficacy in reducing fungal growth, in most of the above tested strains the reduction is 99.999% after 10 seconds of exposure.
From the above description, the advantages offered by the present invention will be immediately apparent to those skilled in the art .
In particular, it should be noted that the solid iodophor complexes described offer the possibility of having stable powder compositions available, containing high concentrations of iodine from which stable aqueous solutions can be obtained; this is highly advantageous with respect to iodophors formulated directly in water. A solid iodophor is preferable with respect to that in solution because it is easier to store , can be formulated in water or in other solvents in the desired concentrations , can be included in solid and semi-solid matrices or used directly in powder .
The present invention further provides a process for preparing solid complexes between cyclodextrin and molecular iodine CD/ I 2 which has general validity and is not constrained by the precipitation of these products in water .
With the method of the present invention it is also possible to produce complexes already known, but also and surprisingly, a new class of stable powdered solid iodophors consisting of any cyclodextrin and the triiodide ion ( CD/ I 3 - ) .
Since the biocidal activity depends on the free iodine obtained by dissociation of the complex with cyclodextrin, in the case of the iodophors of this invention the free iodine is provided by the dissociation equilibrium of the complexes CD/ I 3 K or CD/ I 3 H or CD/I 2 and the free iodine levels increase linearly with the iodophor concentration, this represents an advantage over the technology employing povidone iodide . In fact , to provide stable solutions such a technology requires being formulated at high concentrations , typically 10% , concentrations at which the polyvinylpyrrolidone polymer aggregates and traps iodine to provide a solution with only 2 -4 ppm of free iodine in a composition containing 15000 ppm of total iodine not used for biocidal activity . In the case of the present invention, the cyclodextrin-based iodophor complexes instead do not have aggregations. Therefore, for the same concentration, the iodophor complexes of the invention can provide stable formulations with a significantly greater amount of free iodine.