Extended-release formulations

Abstract

The present invention relates to a sustained release formulation.The present invention also relates to the use of the repellent formulation, its method of use, and compositions containing the formulation.The present invention further relates to a PMD glucovanillin conjugate compound of formula (II), a process for preparing the compound of formula (II), and further the use of the compound of formula (II) for the controlled release of PMD.The present invention also relates to a process for isolating racemic cis / trans-PMD from an extract containing PMD. TIFF2024542634000037.tif86138

Description

[Technical field]

[0001] The present invention relates to sustained release formulations, particularly insect repellent formulations having long-lasting effect. [Background technology]

[0002] Terrestrial and airborne arthropods are the most common vectors of disease transmission to humans, and rising global temperatures are expanding the number of areas affected by these diseases as they provide more suitable habitat for their respective vectors.

[0003] One way to combat these arthropods, and more specifically insect bites, is through the use of insect repellents. Currently, the active ingredients used in most commercial topical insect repellents are spatial repellents. Several approved synthetic topical insect repellents are commercially available, including DEET (N,N-diethyltoluamide), picaridin, and IR3535, but none are ideal. For example, DEET has a pungent odor, leaves a waxy feel, and can dissolve plastics and synthetic fibers. Furthermore, these synthetic molecules are generally perceived to be harmful to health, resulting in a rapidly expanding market for natural alternatives.

[0004] A wide range of natural molecules are known to provide insect repellency, but the majority are too volatile to provide protection for only a very short period of time. In fact, p-menthane-3,8-diol (PMD), which is active against a wide variety of insects, is the only natural insect repellent recognized by the World Health Organization that has a sufficiently high boiling point to allow slow evaporation over several hours.

[0005] Commercially available repellent products typically have a duration of effect between 4-6 hours after each application. However, when problematic insect species are replaced by other invasive species in a given environment, established commercial repellent products may no longer provide adequate protection. To use one non-limiting example, commercially available topical insect repellents are designed to combat mosquitoes that bite in the morning or evening, where a short duration of action is appropriate. The unprecedented increase in population of day-feeding Aedes mosquitoes (commonly known as Asian tiger mosquitoes), which are the source of infection for the Zika, Dengue and Chikungunya viruses, necessitates multiple applications of currently available commercial repellents and formulations. Due to busy lifestyles, regular repellent application is easily forgotten until a bite occurs, leaving users vulnerable to disease. To mitigate this problem, traditional repellent products with high concentrations of active ingredients may be applied, but increased exposure can cause multiple problems, such as toxicity and irritation.

[0006] Topical and spatial repellents are volatile molecules that, upon evaporation, provide a protective zone on or around the skin that prevents mosquito accretion and / or biting by complex mechanisms that are not fully understood. Each active ingredient has a Minimum Effective Evaporation Rate (MEER). Evaporation rates above this threshold result in non-productive decline, shortening the period during which the repellent is effective, after which concentrations are not sufficient to meet the MEER.

[0007] The development of an insect repellent product that allows for the controlled release of active ingredients would overcome many of the problems associated with current products by extending duration while at the same time reducing the concentration of active ingredient required, which would provide additional benefits such as reduced odor, reduced irritation, and avoidance of the greasy feel common with such products.

[0008] Furthermore, such methods are attractive because they may allow the use of other natural repellents that cannot currently be used due to their high volatility, which is important because a broad range of effective repellent molecules may reduce the incidence of resistance.

[0009] For the sustained release of insect repellents, two main technical solutions have been disclosed in the art: the encapsulation approach and the precursor approach.

[0010] In the encapsulation approach, the repellent product is applied to the entire surface or, in the case of repellent bracelets, to a body section, after which the active ingredient is slowly released from the target surface over time, for example by encapsulation within a microcapsule (e.g. EP-A-0348550 (Patent Document 1)), liposphere (EP-A-0502119 (Patent Document 2)), polymer (US4774082 (Patent Document 3)) or copolymer (US6180127 (Patent Document 4)) matrix, resulting in a sustained release of the insect repellent.

[0011] In the precursor approach, altering the physicochemical characteristics of the active ingredient by preparation of a prodrug or conjugation to a suitable carrier molecule offers an alternative method of controlled release, where the active ingredient is released by environmental mechanisms, extending the time that the MEER is maintained while avoiding the need to apply a large excess of the active ingredient.

[0012] The use of precursors such as prodrugs and conjugates is well known in the pharmaceutical field. With regard to insect repellent actives, the conjugation of volatile insect repellent molecules to compounds that reduce water solubility, such as fatty acids (US-A-2004 / 014811 (Patent Document 5)), preparation of water-soluble ester and ether prodrugs (EP-A-2439188 (Patent Document 6)), 5- and 6-membered cyclic acetals as insect repellent precursors (WO-A-99 / 00377 (Patent Document 7)), water-soluble conjugates to one or more sugar residues (JP-A-2000-096078 (Patent Document 8), JP-A-H01-213291 (Patent Document 9)), conjugates to polycarboxylic acids or polyols to improve water solubility (WO-A-2016 / 07152 (Patent Document 9)), and the like are well known in the pharmaceutical field. 1 (Patent Document 10)), conjugates to hyaluronic acid (WO-A-2016 / 071521 (Patent Document 10)), conjugates to crystalline carriers (WO-A-2010 / 144755 (Patent Document 11)), conjugates to crosslinked polymer gel compositions (US6846491 (Patent Document 12)), conjugates to vanillin (WO-A-2007082306 (Patent Document 13), WO-A-2017 / 081445 (Patent Document 14)), conjugates to ketones or aldehydes (GB-A-2581375 (Patent Document 15)), and 3'-ketoglycosides (WO-A-2021 / 160670 (Patent Document 16)).

[0013] European Patent Application EP-A-2862442 (Patent Document 17) discloses a composition comprising at least one monoterpenoid, at least one acid and at least one emulsifier.

[0014] Such prior art methods exhibit deficiencies with respect to effective long-term release application of insect repellents.

[0015] Despite the various proposals available in the state of the art, there is still a need for new formulations for use in the sustained release of active materials that exhibit a release rate of the active substance sufficient to be effective, in particular a repellent effect.

[0016] The selective isolation of cis-PMD and / or trans-PMD is a challenge for those skilled in the art, and several proposals have been disclosed in the art.

[0017] For example, CA-A-108341740 (Patent Document 18) discloses a method for preparing PMD, which is characterized in that citronellal is used as a raw material, graphene oxide is used as a catalyst, and water is used as a solvent, and PMD is dissolved in an organic solvent, which is ethyl acetate, n-hexane or a mixture thereof, at a temperature of 50 to 80°C, and then the temperature is lowered to room temperature to obtain crystals of cis-PMD, and trans-PMD is obtained from the mother liquor, thereby purifying the cis and trans forms of PMD.

[0018] US5959161 (Patent Document 19) discloses a method for producing para-menthane-3,8-diol, which comprises treating citronellal with an aqueous sulfuric acid solution having a concentration of 0.02 to 1.0% by weight, and then separating cis- and trans-PMD by column chromatography using a mixed solvent of ether and n-hexane.

[0019] Therefore, there is a need for an efficient process for isolating cis- and / or trans-PMD. [Prior art documents] [Patent documents]

[0020] [Patent Document 1] EP-A-0348550 [Patent Document 2] EP-A-0502119 [Patent Document 3] US4774082 [Patent Document 4] US6180127 [Patent Document 5] US-A-2004 / 014811 [Patent Document 6] EP-A-2439188 [Patent Document 7] WO-A-99 / 00377 [Patent Document 8] JP-A-2000-096078 [Patent Document 9] JP-A-H01-213291 [Patent Document 10] WO-A-2016 / 071521 [Patent Document 11] WO-A-2010 / 144755 [Patent Document 12] US6846491 [Patent Document 13] WO-A-2007082306 [Patent Document 14] WO-A-2017 / 081445 [Patent Document 15] GB-A-2581375 [Patent Document 16] WO-A-2021 / 160670 [Patent Document 17] EP-A-2862442 [Patent Document 18] CA-A-108341740 [Patent Document 19] US5959161 Summary of the Invention

[0021] Object of the invention The object of the present invention is a sustained release formulation.

[0022] Another aspect of the invention is the use of said formulation.

[0023] Another aspect of the invention is a sustained release formulation for use as a pharmaceutical.

[0024] Another aspect of the invention is a method of using the formulation.

[0025] Another aspect of the invention is a composition comprising the formulation.

[0026] Another aspect of the present invention is the use of the PMD glucovanillin acetal compound of formula (II) for the controlled release of PMD.

[0027] Another aspect of the invention is a process for isolating racemic cis / trans-PMD. [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 shows the eight stereoisomers of PMD. [Figure 2.1] Figures 2.1 and 2.2 show the chemical structures of additional insect repellent compounds. [Figure 2.2] See legend to Figure 2.1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description of the Invention The object of the present invention is to a) p-menthane-3,8-diol (PMD), b) glycosides, c) organic acids, and d) Divalent metal halide salts It is a sustained release formulation comprising:

[0030] The present authors have developed a formulation comprising a PMD, a glycoside, an organic acid and a divalent metal halide salt, which is capable of releasing the PMD on a surface such as human skin. Surprisingly, due to the more linear release profile, the formulation of the present invention is capable of releasing effective concentrations of the PMD, a volatile alcohol, on human skin over an extended period of time, for example to act as an insect repellent, which typically lasts for at least 24 hours, i.e., longer than observed with the delivery of free repellent alcohol.

[0031] The present authors have developed formulations comprising glycosides which, when combined with free PMD, an organic acid and a divalent metal halide salt, provide extended release compared to free PMD.

[0032] As shown in the examples, different glycosides, such as methyl-α-glucoside, hexyl glucoside, phenyl-β-glucoside, geranyl-β-glucoside, methyl-α-D-mannopyranoside, methyl-α-D-galactopyranoside, and PMD glucovanillin acetal, are suitable for the sustained release of PMD.

[0033] In this specification, as well as in the claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Ranges defined by the prepositional terms "between" or "to" are inclusive. The term "about" refers to a deviation of plus / minus 10%, preferably plus / minus 5%. Percentages are expressed as % by weight (wt %) unless otherwise specified.

[0034] According to IUPAC, the term "moiety" is used to denote a part of a molecule.

[0035] The term "repellent" as used herein refers to any substance or mixture of substances that is used to repel any insect.Repellent as defined herein has a preferred use for repelling flying insects (e.g., mosquitoes).However, it is expected that the insecticide formulations described can also be effective against mites, ticks (Lyme disease), and many other insects and their eggs and larvae.

[0036] PMD The alcohol 2-(2-hydroxypropan-2-yl)-5-methylcyclohexan-1-ol, also known as p-menthane-3,8-diol (PMD), has the following structure (I): Equivalent to TIFF2024542634000002.tif23128.

[0037] In the context of the present invention, the term "PMD" refers to p-menthane-3,8-diol and represents any stereoisomer, racemic mixture or diastereomeric mixture derived from the compound of formula (I).

[0038] PMD for use in the present invention may be derived from natural sources, synthetic, or a mixture thereof. PMD for use in the present invention may be a substantially pure form of the compound, i.e., greater than 95%, 97%, or 99% NMR or GC purity, a crude extract, or a crude extract that has been chemically modified, for example, by treatment under acidic conditions of a crude extract from a natural source.

[0039] The purity of PMD may be determined by GC using methods disclosed in the literature, for example, Barasa et al., Repellent Activities of Stereoisomers of p-Menthane-3,8-diols Against Anopheles gambiae (Diptera: Culicidae), J. Med. Entomol., 2002, 39(5), 736-741.

[0040] The purity of PMD may be determined by NMR using methods disclosed in the literature, for example, Borrego et al., Effect of the Stereoselectivity of para-Menthane-3,8-diol Isomers on Repulsion toward Aedes albopictus, J. Agric. Food Chem., 2021, 69(37), 11095-11109.

[0041] PMD is known to exist in two geometric isomers, namely cis and trans isomers. Overall, there are eight isomers of PMD, as disclosed in Figure 1. The present invention encompasses any single isomer and any combination of one or more isomers.

[0042] Racemic-cis-PMD is the enantiomer of: It consists of a mixture of TIFF2024542634000003.tif23128.

[0043] Racemic-trans-PMD is the enantiomer of: It consists of a mixture of TIFF2024542634000004.tif23128.

[0044] Different stereoisomers of PMD, either alone, as racemic mixtures or as diastereomeric mixtures, exhibit repellent effects as disclosed in Barasa, op. cit. According to Borrego, op. cit., different isomers exhibit differential repellent effects against Aedes albopictus mosquitoes. The insect repellent properties of PMD are also disclosed in Carroll et al., PMD a Registered Botanical Mosquito Repellent with DEET-Like Efficacy, J. Am. Mosquito Control Assoc., 2006, 22(3), 507-514) or Barnard et al., Laboratory Evaluation of Mosquito Repellents Against Aedes albopictus, Culex nigripalpus, and Ochlerotatus triseriatus (Diptera: Culicidae), J. Med. Entomol., 2004, 41(4), 726-730.

[0045] The preservative, antibacterial, fungicidal, bactericidal, antiviral, and hair growth activities of PMDs have also been disclosed, for example, in US-A-2004 / 0247708, US-A-2007 / 0178048, EP-A-0934741, WO-A-01 / 05226, Smither et al., Investigative study into whether an insect repellent has virucidal activity against SARS-CoV-2, Microbiol. Soc., 2021, 102(4) (DOI 10.1099 / jgv.0.001585).

[0046] Racemic PMD and its enantiomers are commercially available, for example, from Takasago, Sigma-Aldrich, Parchem, or BOC Sciences, or can be prepared according to the procedures disclosed in Barasa et al., supra, or Zimmerman et al., J. Am. Chem. Soc., 1953, 75, 2367-2370, or Yuasa et al., Org. Proc. Res. Devel., 2000, 4(3), 159-161. PMD may also be derived by acid modification of the oil of Corymbia citriodora, also known as hydrated and cyclized lemon eucalyptus (Eucalyptus citriodora) oil, which is commercially grown (e.g., in Brazil and other Latin American countries). Citriodiol® (Citrefine) is a product produced by simple chemical modification of the extract of the leaves of Corymbia citriodora under acidic conditions. It contains a mixture of racemic cis- and racemic trans-PMD at approximately 70% by weight. This commercial product also contains small amounts of linear and cyclic terpenoid components, such as citronellal, citronellol and isopulegol isomers. Hydrated and cyclized lemon eucalyptus oil is also commercially available from other suppliers, such as Chemian Technology SARL (on behalf of Chemian Technology Limited (UK)) and Fulltec GmbH.

[0047] In one embodiment, PMD is selected from any of the eight stereoisomers of PMD, racemic-cis-PMD, racemic-trans-PMD, and mixtures thereof.

[0048] In one embodiment, the PMD is a mixture of racemic-cis-PMD and racemic-trans-PMD.

[0049] In one embodiment, the PMD is selected from racemic-trans-PMD and racemic-cis-PMD to provide a low odor, odorless controlled release formulation.

[0050] In one embodiment, the PMD is racemic-trans-PMD.

[0051] In one embodiment, the PMD is racemic-cis-PMD, which is simpler to prepare than racemic-trans-PMD, is released more slowly from the formulations of the invention than pure cis / trans-PMD mixtures, and has a much lower odor than Citriodiol®.

[0052] In one embodiment, the PMD is selected from an enantiomer of formula (Ia), an enantiomer of formula (Ib), an enantiomer of formula (Ic), and an enantiomer of formula (Id).

[0053] In one embodiment, the PMD is a chemically modified crude natural extract of Corymbia citriodora.

[0054] Glycosides The formulation of the present invention comprises glycoside.The glycoside incorporated in the sustained release formulation of the present invention can effectively release PMD at a controlled rate when applied to a surface and when present in combination with organic acid and divalent metal halide salt.This PMD present in free form in sustained release formulation is an efficient insect repellent with long-lasting effect as shown in the examples, and also shows antiseptic activity, antibacterial activity, fungicidal activity, bactericidal activity, antiviral activity and hair growth activity as revealed above.

[0055] In the sense of the present invention, the term "glycoside" refers to a mixed acetal (ketal) derived from a cyclic form of a reduced sugar compound, in which the anomeric hydroxy group is replaced by a group -OR. The compound ROH is called the "aglycone" and the carbohydrate residue itself is called the "glycone".

[0056] When the sugar is glucose, the terms "glucoside" and "glucopyranoside" are used interchangeably.

[0057] In one embodiment, a glycoside is a reducing sugar attached to a non-carbohydrate via a glycosidic bond, where the glycosidic bond can be either alpha (α) or beta (β), and where the sugar is a monosaccharide, a sugar having a degree of polymerization comprised between 1.1 and 1.9, a disaccharide, or a trisaccharide.

[0058] In one embodiment, the reducing sugar is selected from a monosaccharide, a disaccharide, and a trisaccharide.

[0059] In one embodiment, the reducing sugar is a monosaccharide selected from L- and D-sugars.

[0060] In one embodiment, the reducing sugar is a monosaccharide selected from, for example, glucose, galactose, allose, altrose, mannose, gulose, idose, talose, fucose, xylose and 2-deoxyglucose, N-acetylglucosamine, and mixtures thereof; preferably, D-glucose, D-galactose, D-allose, D-altrose, D-mannose, D-gulose, D-idose, D-talose, and mixtures thereof; more preferably, D-glucose or D-galactose; and even more preferably, the sugar is D-glucose.

[0061] In one embodiment, the reducing sugar is a disaccharide selected from, for example, lactose, maltose, cellobiose, chitobiose, kojibiose, nigerose, isomaltose, sophorose, laminaribiose, gentiobiose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, mannobiose, melibiose, rutinose, rutinulose, xylobiose, and mixtures thereof; preferably lactose or maltose; more preferably lactose.

[0062] In one embodiment, the reducing sugar is a trisaccharide selected from, for example, nigerotriose, maltotriose, maltotriulose, and mixtures thereof; preferably, maltotriose.

[0063] In one embodiment, the primary hydroxy groups present on the sugar may be functionalized as ethers, amines, esters, amides, sulfates, phosphates, carbonates or carbamates.

[0064] In one embodiment, the non-carbohydrate (aglycone) moiety is an alcohol residue (-OR). The alcohol (ROH) may be selected from aliphatic or aromatic alcohols. Sugars with a degree of polymerization between 1.1 and 1.9, preferably between 1.2 and 1.6, have alcohol residues of C8 to C6. 18 When selected from the hydrocarbon chains, they are the sugar moieties of alkyl polyglycosides, which are industrial products obtained from renewable raw materials. Preferably, the sugar moiety is glucose.

[0065] In a preferred embodiment, the glycoside is selected from methyl-α-glucoside, hexyl glucoside, phenyl-β-glucoside, geranyl-β-glucoside, methyl-α-D-mannopyranoside, methyl-α-D-galactopyranoside, and PMD glucovanillin conjugates; preferably, selected from methyl-α-glucoside and PMD glucovanillin conjugates.

[0066] In one embodiment, the glycoside is methyl-α-glucoside.

[0067] In one embodiment, the glycoside is a PMD glucovanillin conjugate.

[0068] PMD Glucovanillin Conjugate Compound The PMD glucovanillin conjugate compound of formula (II) is The file is TIFF2024542634000005.tif74128.

[0069] In this description, the compound of formula (II) may be interchangeably named as: PMD glucovanillin conjugate, where the relative stereochemistry is not defined; cis-PMD glucovanillin conjugate, cis-PMD acetal or cis-acetal, where the compound is produced using cis-PMD; trans-PMD glucovanillin conjugate, trans-PMD acetal, or trans-acetal, where the compound is produced using trans-PMD.

[0070] In one embodiment, the PMD is selected from any of the eight stereoisomers of PMD, racemic-cis-PMD, racemic-trans-PMD, and mixtures thereof, and preferably, the PMD is a mixture of racemic-cis-PMD and racemic-trans-PMD, wherein the sugar moiety is D-glucose.

[0071] In one embodiment, the PMD is racemic-trans-PMD.

[0072] In a preferred embodiment of the PMD glucovanillin conjugate, the PMD is a mixture of racemic cis-PMD and racemic trans-PMD and the sugar moiety is glucose.

[0073] In a more preferred embodiment, the PMD glucovanillin conjugate compound of formula (II) is a conjugate in which the PMD is a mixture of racemic-cis-PMD and racemic-trans-PMD and the sugar moiety is glucose, preferably D-glucose.

[0074] Glucovanillin Glucovanillin is a compound obtained by functionalizing vanillin (4-hydroxy-3-methoxybenzaldehyde, CAS Nr. 121-33-5) with a sugar moiety. When the sugar moiety is D-glucose, the product is called glucovanillin (vanillin 4-O-β-D-glucoside, CAS Nr. 494-08-6).

[0075] Glucovanillin is the major natural product in many plants, including the vanillin pod, and is hydrolyzed by a combination of endogenous and exogenous enzymes during the production of natural vanillin.

[0076] However, natural vanillin, and therefore glucovanillin, is very expensive due to its limited supply. Therefore, vanillin is mainly produced synthetically and, in a stepwise manner, biosynthetically, as disclosed, for example, in Garcia-Bofill et al., Enzymatic synthesis of vanillin catalysed by a eugenol oxidase, Applied Catalysis A, 2019, 582, 117117, neither of which uses glucovanillin as a precursor.

[0077] Glucovanillin can be produced from vanillin using traditional chemical methods, as disclosed, for example, in Fischer et al., Synthesis of certain glucosides, Berichte, 1909, 42(2), 1465-76. Furthermore, chemical methods for O-glycosylation of aromatic alcohols (phenolic substrates) with various sugars are well known and are disclosed, for example, in Jacobsson et al., Aromatic O-glycosylation, Carbohydrate Research, 2006, 341, 1266-1281.

[0078] Glucovanillin is also commercially available, for example, from Carbosynth Ltd.

[0079] The glucovanillin conjugate compounds of formula (II) incorporated into the sustained release formulations of the present invention are effective insect repellents and therefore can effectively release PMD at a controlled rate.

[0080] Process for preparing PMD glucovanillin conjugate compound of formula (II) The process for preparing the PMD glucovanillin conjugate compound of formula (II) comprises the reaction of glucovanillin with PMD in the presence of a catalytic amount of an organic or inorganic acid, optionally in the presence of a solvent.

[0081] In a preferred embodiment, the acid catalyst is selected from sulfonic acid compounds such as citric acid and Dowex acidic resins.

[0082] In one embodiment, the formation of the acetal is carried out in the absence of any solvent, in another embodiment, the reaction is carried out in the presence of a solvent, preferably selected from acetonitrile and C1-C4 alcohols, more preferably selected from acetonitrile and ethanol, even more preferably the solvent is ethanol.

[0083] Typically, the reaction is carried out at a temperature between about 30° C. and about 60° C., preferably between about 40° C. and about 55° C., and more preferably at about 50° C. In solventless embodiments, initial heating is carried out to obtain a molten liquid from the starting product.

[0084] Considering that the sugar is not protected, it is surprising that by NMR no other acetals other than that of formula (II) are observed to be formed to any significant extent.

[0085] The cis / trans-PMD glucovanillin conjugate mixture can be adjusted to the desired ratio by appropriate selection of acid and solvent.

[0086] In one embodiment, using an excess of cis / trans-PMD mixture produces almost exclusively cis-PMD glucovanillin conjugates using citric acid as catalyst and acetonitrile as solvent, whereas using a strong acid (e.g., Dowex acidic resin) in the same solvent gives a 1:0.5 mixture of cis / trans-PMD glucovanillin conjugates. In contrast, using the same acid and ethanol as solvent gives 1:0.7 and 1:1 mixtures of cis / trans-PMD glucovanillin conjugates, respectively. Thus, the reaction can be easily tuned to give the desired isomeric product ratio by appropriate selection of reaction conditions such as acid and solvent.

[0087] In one embodiment, the PMD glucovanillin conjugate is obtained from Citriodiol® and glucovanillin using Dowex acidic resin, filtering the resin and then dissolving it in water.

[0088] In one embodiment, the PMD glucovanillin conjugate may also be produced in neat Citriodiol® or Citriodiol® in a solvent and used without isolation.

[0089] Use of the PMD glucovanillin conjugate compound of formula (II) Another aspect of the subject matter of the present invention is the use of a PMD glucovanillin conjugate compound of formula (II) for the controlled release of PMD from the sustained release formulation of the present invention.

[0090] The PMD glucovanillin conjugates contained in the sustained release formulations of the present invention may be applied in the treatment of a wide variety of indications, such as the repelling of mosquitoes, flies, ticks, mites, midges, biting midges, head lice, and their eggs and larvae.

[0091] organic acid The formulation of the present invention comprises organic acid.The organic acid incorporated in the sustained release formulation of the present invention can effectively release PMD when present in combination with glycoside and divalent metal halide salt when applied to a surface at a controlled rate.The free PMD contained in the formulation is an efficient insect repellent with long-lasting effect.

[0092] Organic acids suitable for inclusion in the formulation are sulfonic acids, monocarboxylic acids, dicarboxylic acids, tricarboxylic acids, hydroxycarboxylic acids, and amino acids.

[0093] In one embodiment, the organic acid is a sulfonic acid, such as p-toluenesulfonic acid, benzenesulfonic acid, or taurine.

[0094] In one embodiment, the organic acid is a carboxylic acid, such as oxalic acid, malonic acid, succinic acid, fumaric acid, or benzoic acid.

[0095] In one embodiment, the organic acid is a hydroxycarboxylic acid, such as lactic acid, tartaric acid, glycolic acid, malic acid, mandelic acid (2-hydroxy-2-phenylacetic acid), or citric acid.

[0096] In one embodiment, the organic acid is an amino acid, such as glycine, alanine, valine, leucine, isoleucine, aspartic acid, asparagine, glutamic acid, glutamine, serine, threonine, methionine, cysteine, lysine, arginine, histidine, proline, phenylalanine, tyrosine, or tryptophan.

[0097] In a preferred embodiment, the organic acid is selected from citric acid, succinic acid and their anhydrous forms or hydrates. In one embodiment, citric acid is used in anhydrous form or as a hydrate, preferably as a monohydrate. In one embodiment, succinic acid is used.

[0098] In one embodiment, the organic acid may be in equilibrium with the corresponding conjugate base, where the ratio between the species depends on the pH value of the formulation.

[0099] Divalent metal halide salts The formulations of the present invention include divalent metal halide salts. The divalent metal halide salts incorporated in the sustained release formulations of the present invention, when applied to a surface at a controlled rate, can effectively release PMDs when present in combination with glycosides and organic acids, and are efficient insect repellents.

[0100] Divalent metal halide salts suitable for inclusion in the formulations of the present invention are, for example, calcium chloride, magnesium chloride, magnesium bromide, zinc iodide, zinc chloride, zinc bromide, or cobalt chloride.

[0101] In one embodiment, the divalent metal halide salt is selected from zinc chloride, magnesium chloride and calcium chloride.

[0102] In one embodiment, the divalent metal halide salt is calcium chloride.

[0103] In one embodiment, the divalent metal halide salt is magnesium chloride.

[0104] In one embodiment, the divalent metal halide salt is zinc chloride.

[0105] Extended-release formulations Sustained release formulations include PMDs, glycosides, organic acids, and divalent metal halide salts.

[0106] In one embodiment, the sustained release formulation contains a) Between 0.01 and 2 mol, preferably between 0.02 and 1.5 mol, more preferably between 0.03 and 1.2 mol, more preferably between 0.05 and 1 mol, and even more preferably between 0.07 and 0.75 mol of glycoside. b) between 0.05 and 3 mol, preferably between 0.06 and 2.5 mol, more preferably between 0.08 and 2 mol, more preferably between 0.09 and 1.7 mol, more preferably between 0.1 and 1.5 mol and even more preferably between 0.12 and 1.24 mol of an organic acid, and c) between 0.01 and 5 mol, preferably between 0.02 and 2 mol, more preferably between 0.03 and 1 mol, and even more preferably between 0.06 and 0.58 mol of a divalent metal halide salt Includes.

[0107] In one embodiment, the sustained release formulation comprises PMD; a glycoside selected from methyl-α-glucoside, hexylglucoside, phenyl-β-glucoside, geranyl-β-glucoside, methyl-α-D-mannopyranoside, methyl-α-D-galactopyranoside, and PMD glucovanillin conjugate, preferably selected from methyl-α-glucoside and PMD glucovanillin conjugate; an organic acid selected from citric acid and succinic acid; and a divalent metal halide salt selected from calcium chloride, magnesium chloride, magnesium bromide, zinc iodide, zinc chloride, zinc bromide, and cobalt chloride.

[0108] In one embodiment, the sustained release formulation comprises PMD, a glycoside selected from PMD glucovanillin conjugate and methyl-α-glucoside, an organic acid selected from citric acid and succinic acid, and a divalent metal halide salt selected from calcium chloride, magnesium chloride, and zinc chloride.

[0109] In one embodiment, the sustained release formulation comprises PMD, a PMD glucovanillin conjugate, citric acid, and a divalent metal halide salt selected from calcium chloride, magnesium chloride, and zinc chloride.

[0110] In one embodiment, the sustained release formulation comprises PMD, methyl-α-glucoside, citric acid, and a divalent metal halide salt selected from calcium chloride, magnesium chloride, and zinc chloride.

[0111] In one embodiment, the sustained release formulation comprises PMD, a PMD glucovanillin conjugate, succinic acid, and a divalent metal halide salt selected from calcium chloride, magnesium chloride, and zinc chloride.

[0112] In one embodiment, the sustained release formulation comprises PMD, methyl-α-glucoside, succinic acid, and a divalent metal halide salt selected from calcium chloride, magnesium chloride, and zinc chloride.

[0113] In one embodiment, the content of PMD in the sustained release formulation is capable of delivering at least 0.1, preferably 0.2 mg of PMD per square centimeter of surface.

[0114] The delivery of PMDs is generally accomplished by a variety of cosmetic compositions and devices that are widely used and known to those skilled in the art. Non-limiting examples include solutions, creams, powders, pastes, sprays, lotions, films, or impregnated into or onto materials such as wipes, masks and sticks, medical devices or surfaces.

[0115] Another aspect of the present invention is the use of the sustained release formulation as an insect repellent.

[0116] Sustained release formulations for pharmaceutical use also form part of the invention, in particular for use as antiseptics, antibiotics, fungicides, bactericides or antivirals.

[0117] As shown in the Examples section, the sustained release formulation of the present invention surprisingly provides a slow and prolonged release of PMD. The effectiveness of the sustained release formulation is shown by evaluating the insect repellent effect of PMD. This formulation allows a significantly improved long-lasting insect repellent effect compared to commercial products such as Mosiguard (registered trademark) and DEET (see Examples 11 and 17). As shown in Examples 13 and 19, trans-PMD and cis-PMD provide controlled release repellents that have much lower odor compared to Citriodiol (registered trademark)-containing repellents, respectively.

[0118] Also forming part of the invention is a method of using the sustained release formulation, which comprises applying the formulation, either directly or in the form of a composition, onto a surface.

[0119] The formulations of the present invention may be applied to any surface, such as skin, hair, clothing, plants or soft surfaces such as crops, grain storage, or hard surfaces such as wood, synthetic materials, or ceramic materials. In one embodiment, the formulation is applied to the skin.

[0120] The preparation of formulations containing PMDs, glycosides, organic acids and divalent metal halides is a matter of routine for those skilled in the art. Information on the preparation of compositions in the form of solutions, creams, pastes, powders, sprays, lotions and films can be found, for example, in Remington The Science and Practice of Pharmacy, 20 th Ed., Lippincott Williams & Wilkins, 2000, Philadelphia, Part 5, Pharmaceutical Manufacturing, and other well-known handbooks.

[0121] Surprisingly, the sustained release formulation, when combined with a glycoside, an organic acid and a divalent metal halide salt, is capable of releasing the free PMD present therein.

[0122] As shown in the examples, sustained release is obtained with different glucopyranosides (Example 7), different glycosides (Example 16), different organic acids (Examples 9 and 14), and different divalent metal halides (Example 15). Both in vitro (Example 17) and in vivo (Example 11) studies show the advantages of sustained release formulations containing either Citriodiol® or the pure PMD of the present invention compared to commercially available compositions.

[0123] composition Compositions comprising sustained release formulations also form part of the subject matter of the present invention.

[0124] In one embodiment, the composition comprises a sustained release formulation and a cosmetically acceptable ingredient.

[0125] As used herein, "cosmetically acceptable" means that the product or compound it describes is suitable for use in contact with tissue (e.g., skin) without undue toxicity, incompatibility, instability, irritation, allergic response, etc. The term is not intended to limit the ingredient / product it describes to cosmetic uses only.

[0126] In one embodiment, the composition comprises: a) between 5% and 80% by weight of a sustained release formulation, and b) Between 20% and 95% by weight of cosmetically acceptable ingredients where the percentages of ingredients are adjusted to balance 100%.

[0127] The composition preferably comprises between 10% and 75% by weight of the sustained release formulation, more preferably between 15% and 70% by weight, more preferably between 25% and 65% by weight, and even more preferably between 30% and 60% by weight of the sustained release formulation.

[0128] The composition comprises a cosmetically acceptable ingredient, which in the context of the present invention is selected from a cosmetically acceptable medium, a cosmetic ingredient, and mixtures thereof.

[0129] A cosmetically acceptable medium (or carrier) is a medium in which a cosmetic ingredient is dissolved, emulsified, dispersed, or suspended. This medium is selected from water, water-miscible non-aqueous medium such as ethanol or isopropanol, and water-immiscible non-aqueous medium such as vegetable oils, fatty esters, medium-chain triglycerides, or alkanes. Preferably, the composition comprises water as a medium.

[0130] In one embodiment, the cosmetic ingredient is preferably selected from surfactants (emulsifiers), lipid compounds, emollients, density agents, thickeners, stabilizers, hydrotropes, preservatives, essences, colorants, silicone compounds, fats, waxes, lecithin, phospholipids, UV sun protection agents, film formers, and mixtures thereof.

[0131] The physical form of the composition for use according to the invention is not important and the composition may be made into a variety of product types, including, but not limited to, solid and liquid compositions such as solutions, lotions, creams, gels, sticks, sprays, ointments, cleansing liquid washes and solid bars, shampoos, pastes, powders, foams, mousses, milks, emulsions, dispersions, suspensions, or wipes.

[0132] Topical compositions useful in the present invention may be formulated as a solution. The solution may preferably include an aqueous solvent (e.g., about 0% to about 95% by weight or about 20% to about 85% by weight of a cosmetically acceptable aqueous solvent). More preferably, such compositions may include about 20% by weight of an aqueous solvent, but this may vary depending on the formulation. Such solvents may include ethanol, isopropanol, glycerol (glycerin), propylene glycol, polyethylene glycol, mixtures thereof, and the like, in combination with water. In one embodiment, topical compositions useful in the present invention may be formulated as a solution including an emollient. Such compositions preferably include about 2% to about 50% by weight of an emollient. As used herein, "emollient" refers to a material used to prevent or relieve dryness as well as to protect the skin. In one embodiment, the solution is an anhydrous mixture including a non-aqueous solvent, e.g., about 20% to about 85% by weight of a cosmetically acceptable non-aqueous solvent.

[0133] The lotion may be made from a solution. The lotion typically comprises from about 1% to about 20% by weight of an emollient and from about 50% to about 90% by weight of water.

[0134] Another type of product may be a cream, which typically comprises from about 5% to about 50% by weight of an emollient and from about 45% to about 85% by weight of water.

[0135] Another type of product may be an ointment. An ointment may be composed of a simple base of vegetable oil or semisolid hydrocarbon. An ointment may contain about 2% to about 100% by weight of an emollient, and about 0.1% to about 5% by weight of a thickening agent.

[0136] The topical compositions useful in the present invention may also be preferably formulated as emulsions. In one embodiment, the compositions contain water and lipophilic phases and are presented in the form of emulsions or dispersions, such as oil-in-water (O / W), water-in-oil (W / O), multiple emulsions (W / O / W), or PIT emulsions, or microemulsions. When the carrier is an emulsion, about 1% to about 10% by weight of the carrier should be composed of one or more emulsifiers. The emulsifiers may be nonionic, anionic, cationic, or amphoteric.

[0137] The topical compositions useful in the present invention may be formulated as gels. Suitable gelling agents for aqueous gels include, but are not limited to, natural gums, acrylic acid and acrylate polymers and copolymers, and cellulose derivatives (e.g., hydroxymethylcellulose and hydroxypropylcellulose). Suitable gelling agents for oils (such as vegetable oils, esters, etc.) include, but are not limited to, waxes, modified silicas, cellulose derivatives, polyamides, polyurethanes, and L-glutamic acid derivatives. Such gels typically contain between about 0.1% and 20% by weight of such gelling agents.

[0138] Compositions useful for use in the present invention are generally prepared by conventional methods, such as those known in the art of making topical compositions. Such methods may typically involve mixing the ingredients in one or more steps to a relatively homogeneous state, with or without heating, cooling, application of vacuum, etc.

[0139] Surfactants (emulsifiers) In one embodiment, the composition may include a surfactant (emulsifier) ​​to facilitate dissolution, emulsification, dispersion, or suspension of the cosmetic ingredients.

[0140] The surfactants can be anionic, nonionic, cationic and / or amphoteric.

[0141] The surfactant content is usually comprised between 1% and 30% by weight, preferably between 2% and 20% by weight, more preferably between 3% and 10% by weight, and even more preferably between 4% and 8% by weight.

[0142] Typical examples of anionic surfactants are, for example, soaps, sulfonated alkanes, sulfonated olefins, alkyl sulfates, fatty alcohol ether sulfates, glycerol ether sulfates, fatty acid ether sulfates, mono- and dialkyl sulfosuccinates, mono- and dialkyl sulfosuccinamates, ether carboxylic acids and their salts, fatty acid isethionates, fatty acid sarcosinates, fatty acid taurides, N-acylamino acids.

[0143] Typical examples of nonionic surfactants are, for example, polyalkoxylated fatty alcohols, polyalkoxylated fatty acids, polyalkoxylated fatty acid amides, polyalkoxylated fatty amines, alkoxylated triglycerides, mixed ethers, alkyl polyglycosides, N-alkyl sorbitan esters, fatty acid esters polyethoxylated sorbitan, and amine oxides.

[0144] Typical examples of cationic surfactants are, for example, quaternary ammonium compounds and the quaternized salts of esters of trialkanolamines with fatty acids, such as the esterquats.

[0145] Typical examples of amphoteric surfactants are, for example, alkylbetaines, alkylamidobetaines, aminopropionates, aminoglycinates, imidazolinium betaines and sulfobetaines.

[0146] The abovementioned surfactants are primarily known compounds, whose structure and preparation are known to those skilled in the art and are described, for example, in X. Domingo's book, A guide to the surfactants world, Proa, Barcelona, ​​1995.

[0147] Lipid components and softeners The compositions usually contain additional lipid compounds and emollients to optimize their organoleptic and dermatological properties.

[0148] The lipids and softening compounds are usually present in a total amount between 1% and 50% by weight, preferably between 5% and 25% by weight, and more preferably between 5% and 15% by weight.

[0149] Suitable lipid compounds are Guerbet alcohols, which are based on aliphatic alcohols having 6 to 18 carbon atoms, preferably 8 to 10 carbon atoms (such as the product Eutanol® G from BASF); linear C 6~22 Linear fatty acid esters with alcohol C 6~22 , C 6~22 C with linear alcohols 6~13 Branched carboxylic acid esters, for example, myristyl myristate, myristyl palmitate, myristyl stearate, behenyl stearate, behenyl isostearate, behenyl oleate, behenyl behenate, behenyl erucate, or erucyl myristate.

[0150] In addition, C 6~22 Linear fatty acid esters, in particular 2-ethylhexanol (e.g. the product Cetiol® 868 from BASF), and isopropyl myristate; linear or branched C 6~22 C with fatty alcohols 18~38Alkyl hydroxycarboxylic acid esters; linear and / or branched fatty acid esters with polyhydric alcohols and / or Guerbet alcohols; triglycerides of fatty acids with 6 to 10 carbon atoms (e.g. the product Mirytol® 318 from BASF); liquid mixtures of mono-, di- and triglycerides of fatty acids with 6 to 18 carbon atoms; esters of aliphatic alcohols and / or Guerbet alcohols with aromatic carboxylic acids, in particular benzoic acid, with dicarboxylic acids with 2 to 12 carbon atoms and linear or branched alcohols with 1 to 22 carbon atoms or polyols having 2 to 10 carbon atoms and 2 to 6 hydroxyl groups; vegetable oils; branched primary alcohols; substituted cyclohexanes; linear and branched C 6~22 Suitable are fatty alcohol carbonates, such as dicaprylyl carbonate (for example the BASF product Cetiol® CC); symmetrical or asymmetrical linear or branched dialkyl ethers having 6 to 22 carbon atoms per alkyl group, such as dicaprylyl ether (for example the BASF product Cetiol® OE); aliphatic or naphthenic hydrocarbons, such as squalane, squalene or dialkylcyclohexanes, as well as mixtures thereof.

[0151] Density Factors and Viscosifiers Density factors and thickeners are generally used in compositions to adjust viscosity and rheological behavior.

[0152] Among the density factors taken into account are, firstly, fatty alcohols with a chain length of 12 to 22 carbon atoms, preferably 16 to 18 carbon atoms, and also partial glycerides, fatty acids or hydroxylated fatty acids.

[0153] Suitable thickening agents are, for example, hydrophilic silicic anhydrides (such as the Aerosil® products from Evonik); polysaccharides, in particular xanthan gum, guar gum, agar, alginates and tyloses, carboxymethylcellulose and hydroxyethylcellulose, as well as polyethylene glycol mono- and high molecular weight fatty acid diesters; polyacrylates (for example Carbopol® and Pemulen® types from Lubrizol; Synthalene® from Sigma, Keltrol® types from CP Kelco; Sepigel® and Simulgel® types from Seppic; Salcare® types from Allied Colloids), polyacrylamides, polyvinyl alcohols, and polyvinylpyrrolidones.

[0154] Hydrotropes To improve the flowability of the composition, it is also possible to use hydrotropes, such as, for example, ethanol, isopropyl alcohol, or polyols. The polyols considered here preferably have 2 to 15 carbon atoms and at least two hydroxyl groups. The polyols may also contain other functional groups, in particular amino groups, or be modified with nitrogen. Typical examples are glycerol; alkylene glycols, such as ethylene glycol, diethylene glycol, propylene glycol, butylene glycol, hexylene glycol, and also polyethylene glycols with an average molecular weight of 100 to 1,000 daltons; technical oligoglycerol mixtures with a degree of self-condensation of 1.5 to 10, for example technical diglycerol mixtures with a diglycerol content of 40% to 50% by weight; methylol compounds, such as, in particular, trimethylolmethane, trimethylolpropane, trimethylolbutane, pentaerythritol and dipentaerythritol; alkyl glycosides with a chain of 1 to 8 carbon atoms in the alkyl part; C glycerides, such as sorbitol or mannitol. 5~12sugar alcohols; sugars having 5 to 12 carbon atoms, such as glucose or sucrose; amino sugars, such as glucamine; and dialcoholamines, such as diethanolamine or 2-amino-1,3-propanediol.

[0155] In one embodiment, glycerol, propylene glycol, and mixtures thereof are used as hydrotropes in the composition.

[0156] Preservatives Preservatives suitable for use in the compositions include, by way of example, phenoxyethanol, 3-(4-chlorophenoxy)-1,2-propanediol (chlorphenesin), pentanediol or sorbic acid, and the classes of additional substances set out in Annex V of Regulation (EC) No 1223 / 2009 of the European Parliament and of the Council.

[0157] A composition comprising the sustained release formulation of the present invention in combination with a film-forming polymer, e.g., polyvinyl acetate and polyvinylpyrrolidone, and a cosmetic ingredient such as an oil, e.g., isopropyl myristate, provides a much longer repellent effect compared to commercially available products (see Example 11).

[0158] In one embodiment, the composition comprises an additional insect repellent compound selected from essential oils, natural insect repellents, synthetic repellents, and mixtures thereof.

[0159] Further insect repellent compounds include, for example, DEET (N,N-diethyl-meta-toluamide), Icaridin (picaridin), DEPA (N,N-diethylphenylacetamide), IR3535, N-butyl-acetanilide, MGK Repellent 264, N-methylneodecanamide, AI3-35765, AI3-37220 (SS220), MGK Repellent 326, geraniol, thymol, eugenol, carvacrol, vanillin, limonene, nootkatone, spathulenol, dibutyl phthalate (DBP), dimethyl phthalate (DMP), dimethyl carbonate (DMC), dioctyl phthalate, benzyl benzoate, indalone, nepetalactone, methyl anthranilate, ethyl anthranilate, cyclohexanepropionic acid, nonanoic acid, 2-ethylhexanoic acid, undecylenic acid, lactic acid, diol, 2-butyl-2-ethyl-1,3-propanediol, Rutgers 612, pyrethroids, carbamates, and mixtures thereof. The chemical structures of some insect repellent compounds are shown in Figures 2.1 and 2.2. Preferably, the composition comprises a further insect repellent compound selected from geraniol, nootkatone, neem oil, DEET, icaridin, IR3535, and mixtures thereof.

[0160] Process for isolating cis / trans-PMD Another aspect of the invention is a process for isolating racemic cis / trans-PMD from an extract containing PMD.

[0161] The process is 1) adding a salt selected from calcium chloride, magnesium chloride, and mixtures thereof to a solution of an extract containing PMD; 2) isolating the solid obtained in step 1), and 3) isolating racemic cis / trans-PMD from the solid separated in step 2). Includes.

[0162] In a preferred embodiment, the process comprises: a) The extract is dissolved in a solvent, preferably C5-C 12dissolving in a mixture of a hydrocarbon and an aliphatic alcohol; preferably the aliphatic alcohol is selected from methanol, ethanol, n-propanol, i-propanol, n-butanol, i-butanol, sec-butanol, n-pentanol, i-pentanol, amyl alcohol, neopentyl alcohol, sec-amyl alcohol, 3-pentanol, methyl isopropyl carbinol, and 2-methyl-2 butanol; more preferably the solvent is a mixture of hexane and ethanol; b) adding a salt selected from calcium chloride, magnesium chloride, and mixtures thereof; c) stirring the mixture obtained in step b) at room temperature; d) separating the solid obtained after the stirring of step c); e) dissolving the solid obtained in step d) in a mixture of water and a water immiscible solvent; preferably, the water immiscible solvent is selected from hexane, cyclohexane, dichloromethane, 1,2-dichloroethane, chloroform, diethyl ether, t-butyl methyl ether, ethyl acetate, benzene, toluene, xylene, 1-butanol, 2-butanol, n-pentanol, i-pentanol, amyl alcohol, neopentyl alcohol, sec-amyl alcohol, 3-pentanol, methyl isopropyl carbinol, and 2-methyl-2 butanol; more preferably, the water immiscible solvent is hexane. f) separating the hexane phase from step e); and g) drying and removing the hexane by distillation Includes.

[0163] In one embodiment, the aliphatic alcohol in step a) is present in a catalytic amount, for example between 0.01 and 0.1 vol. %, preferably between 0.02 and 0.07 vol. %, more preferably about 0.05 vol. %, based on the total amount of solvent.

[0164] The process makes it possible to isolate racemic cis / trans-PMD from an extract containing PMD, with substantially quantitative recovery of the PMD contained in the extract.

[0165] In one embodiment, the extract containing PMD is the commercially available product Citriodiol®.

[0166] The following examples provide experiments for the preparation of compounds of the invention and performance testing of sustained release formulations. EXAMPLES

[0167] 1 H and 13 C NMR spectra were recorded on a Varian Mercury 400 ( 1 H is 400.1Mz, 13 C was obtained at 100.6 MHz. High-resolution mass spectra (HRMS) were obtained using an LC / MSD-TOF mass spectrometer (Agilent Technologies).

[0168] Reaction progress and product mixtures were monitored by thin layer chromatography (TLC) on commercially available silica gel 60 plates or by using the previously described instruments. 1 The chromatography was performed using column grade silica gel (0.040-0.063 mm mesh size).

[0169] Racemic cis / trans-PMD of approximately 95% purity was purchased from Boc Sciences and either used without further treatment or the cis and trans isomers (approximately 6:4 mixture) were separated by column chromatography on silica with ethyl acetate / hexane 1:3. The faster eluting cis isomer was separated from the standard (1S,2R,5R)-2-(1-hydroxy-1-methylethyl)-5-methylcyclohexanol purchased from Sigma-Aldrich (Merck). 1 H-NMR showed they were identical.

[0170] In the examples, the terms "cis / trans-PMD", "cis-PMD" and "trans-PMD" refer to "racemic cis / trans-PMD", "racemic cis-PMD" and "racemic trans-PMD", respectively, unless otherwise specified.

[0171] Citriodiol®, containing approximately 70% by weight of a mixture of racemic cis- and racemic trans-PMD with a racemic cis-PMD / racemic trans-PMD ratio of approximately 1.9:1, was a gift from Citrefine Ltd.

[0172] Glucovanillin was purchased from Carbosynth Ltd or prepared in-house using standard literature procedures.

[0173] All other chemicals were purchased from Sigma-Aldrich (Merck) or from various commercial sources through Cymit Quimica.

[0174] Hemotek membrane, a thin collagen membrane, was purchased from Hemotek Ltd. (UK).

[0175] Incubations were carried out at 32°C in a standard apparatus.

[0176] Example 1: Preparation of racemic cis-PMD glucovanillin conjugates TIFF2024542634000006.tif59128 A mixture of glucovanillin (1.03 g, 3.2 mmol), racemic cis-para-menthane-3,8-diol (2.04 g, 11.9 mmol) and citric acid monohydrate (0.24 g, 1.2 mmol) was suspended in acetonitrile (10 mL) and stirred at 50 °C for 21 h.

[0177] The resulting solution was concentrated by distillation under reduced pressure and purified by chromatography on silica using 1:1 EtOAc / hexanes to remove residual diol, followed by gradient elution with 1:10 MeOH / dichloromethane to give the title compound as an odorless white solid (1.42 g, 92%).

[0178] mass spectrometry, 1 H-NMR and 13 The compounds were characterized using C-NMR.

[0179] Example 2: Preparation of racemic trans-PMD glucovanillin conjugates TIFF2024542634000007.tif60128 A mixture of glucovanillin (0.5 g, 1.6 mmol), racemic trans-para-menthane-3,8-diol (1.0 g, 5.8 mmol) and citric acid monohydrate (0.1 g, 0.52 mmol) was suspended in acetonitrile (2 mL) and stirred at 50 °C for 22 h.

[0180] The resulting solution was cooled to room temperature and purified by chromatography on silica using 1:1 EtOAc / hexane to remove residual diol, followed by gradient elution with 1:10 MeOH / dichloromethane to give the title compound as an odorless white solid (0.73 g, 74%). The solid (50 mg) was found to be readily soluble in water (0.1 mL).

[0181] mass spectrometry, 1 H-NMR and 13 The compounds were characterized using C-NMR.

[0182] Example 3: General Procedure for the Preparation of Racemic cis / trans-PMD Glucovanillin Conjugates from Citriodiol® Citriodiol® is a commercial product that contains a mixture of about 70% by weight of racemic cis- and racemic trans-PMD with a racemic cis-PMD / racemic trans-PMD ratio of about 1.9:1.

[0183] A mixture of glucovanillin, Citriodiol® and Dowex® 50WX8, a sulfonic acid resin, was suspended in ethanol and stirred at 50° C. for 23 hours. The resulting solution was cooled to room temperature and filtered to obtain the PMD glucovanillin acetal crude reaction mixture as the filtrate. The residue was washed with ethanol (2×1 mL) and the filtrates were combined to give a pale yellow solution.

[0184] A certain amount of the solution was taken, and the solvent was removed by distillation under reduced pressure to obtain PMD glucovanillin acetal crude reaction concentrate. 13 In the C-NMR spectrum, integration of the C11 signal (sum of the signals at 95.6 ppm for the cis-PMD acetal and 94.8 ppm and 94.9 ppm for the trans-PMD acetal) according to the aforementioned numbering system revealed a cis / trans acetal ratio of 1:2.6. The material was then concentrated by distillation under reduced pressure and the residue was purified by chromatography on silica using 1:1 EtOAc / hexane to remove residual diol, followed by gradient elution with 1:10 MeOH / dichloromethane to give the title compound as a glassy solid.

[0185] The materials used in the following Examples 5, 6, 9 and 10 were prepared according to this general procedure but from different batches with different Citriodiol® / glucovanillin ratios, as shown in Table I.

[0186] [Table I] N / A: the material was not isolated, only the PMD / acetal ratio was determined.

[0187] Example 4: PMD reduction from paper discs containing racemic cis / trans-PMD isomer mixture and trans-PMD glucovanillin acetal with different amounts of citric acid and calcium chloride. 1 H-NMR measurements To measure the PMD reduction from paper discs containing isomeric mixtures of cis / trans-PMD and trans-PMD-glucovanillin acetal (hereafter referred to as trans-acetal) with different amounts of citric acid and calcium chloride, the following mixtures were prepared as shown in Table II.

[0188] [Table II]

[0189] The stock solutions used in the above mixtures contained the following components: Stock solution 1: 200 mg of cis / trans-PMD and 130 mg of pure trans-PMD glucovanillin acetal in 0.5 mL of MeOH-d4. Stock solution 2: 200 μL of stock solution 1 diluted with 200 μL of MeOH-d4. Stock solution 3: 400 mg citric acid in 1 mL MeOH-d4. · Stock solution 4: 0.7 g of anhydrous calcium chloride was dissolved in 1 mL of deuterium oxide.

[0190] The stock solutions were added to glass vials in the order and amounts shown in Table I above.

[0191] After thorough mixing, 50 μL of each mixture was dropped onto the entire surface of parallel 5 cm diameter paper towel disks, followed by incubation at 32°C.

[0192] After 2 and 18.5 hours, approximately one-quarter of each paper disc was cut and washed with DMSO-d6 (0.9 mL). 1 The mixture was analyzed by H-NMR. Another quarter of the paper treated with the mixture from experiment number 2 was treated similarly after 90.5 hours and analyzed. The cis-PMD / trans-acetal ratio was 1 H-NMR spectra were determined by comparison of the integrals of their methyl protons at 1.17 and 1.26 ppm, respectively.

[0193] In run 4.1, a small amount of cis / trans-acetal isomerization was observed, and therefore the cis-PMD and tran-PMD ratios were determined for the combination of acetal integral values ​​of 1.41 and 1.26 ppm. The results obtained at different time points are shown in Table III below.

[0194] [Table III] NR = Not implemented

[0195] The results of experiment 4.1 (cis / trans-PMD, trans-PMD glucovanillin acetal, citric acid and calcium chloride) clearly show that both cis-PMD and trans-PMD are reduced from the mixture of PMD isomers and trans-acetals (0.27 equivalents relative to total PMD) at a much slower rate in the presence of citric acid (1 equivalent relative to total PMD) and calcium chloride (1.7 equivalents relative to total PMD) than in their absence (experiments 4.2, 4.3 and 4.4).

[0196] In all subsequent examples using cis- and trans-PMD mixtures (e.g., Citriodiol®), 1 Only cis-PMD reduction was analyzed because it is easier to analyze due to the low overlap of H-NMR peaks, but in all cases the ratio of PMD isomers is quantitatively similar (i.e., both PMD isomers are retained to approximately the same extent as shown in this example).

[0197] Example 5: PMD reduction from paper disks or Hemotek membranes containing cis / trans-PMD isomeric mixtures and pure trans-PMD glucovanillin acetal (trans-acetal) or PMD glucovanillin acetal crude reaction concentrate in ethanol and water. 1 H-NMR measurements To measure PMD reduction from paper disks or Hemotek membranes containing cis- and trans-PMD and trans-PMD glucovanillin acetal in ethanol and water, with and without citric acid and calcium chloride, the following mixtures were prepared, as shown in Table IV.

[0198] [Table IV]

[0199] Run No. 5.1 is a repeat of Run No. 4.1 from Example 4, but note that ethanol was used instead of MeOH-d4.

[0200] The following stock solutions were prepared: Stock solution 1: 400 mg of cis / trans-PMD and 260 mg of trans-PMD glucovanillin acetal in absolute ethanol (1 mL). · Stock solution 2: 400 mg citric acid in 1 mL absolute ethanol. · Stock solution 3: 0.7 g anhydrous calcium chloride dissolved in 1 mL deuterium oxide. Stock solution 4: 200 mg of PMD glucovanillin acetal crude reaction concentrate from Example 3 dissolved in 200 μL of absolute ethanol.

[0201] The stock solutions were added to glass vials in the order and amounts shown in the table above, and after thorough mixing, clear, colorless solutions were obtained, with the exception of experiment number 5.3, which was a pale purple solution.

[0202] 50 μL of each mixture was applied dropwise to the entire surface of parallel 5 cm diameter paper towel disks or Hemotek membranes as indicated in the table above, followed by incubation at 32°C.

[0203] After 17.5 hours, approximately half of each disk was cut and washed with DMSO-d6 (0.9 mL). 1 In experiments 3 and 4, where the starting mixture was in equilibrium, the ratio of cis-PMD / trans-acetal was 1The 1H-NMR spectra were determined by comparison of the integrals of their methyl protons at 1.17 and 1.26 ppm, respectively. In runs 1 and 2, some cis / trans isomerization occurred, and therefore the cis-PMD / total acetal ratio was determined from a combination of the integrals of the signal at 1.17 ppm and the integrals of the signals at 1.41 and 1.26 ppm.

[0204] Stock solution 1 was measured as the ratio of time zero of experiments 1 and 2. 1 H-NMR showed a cis-PMD / acetal ratio of 3.07.

[0205] Stock solution 4 was measured as the ratio of time zero of reactions 3 and 4. 1 H-NMR showed a cis-PMD / acetal ratio of 1.17.

[0206] Table V shows the results obtained in this example.

[0207] [Table V]

[0208] The following can be observed: - The cis-PMD reduction from run no. 5.1 was comparable to run no. 4.1 of Example 4, indicating that the results were reproducible. - The cis-PMD reduction from paper (experiment no. 5.1) and Hemotek membrane (experiment no. 5.2) was very similar, indicating that the effect is independent of the type of surface. - The cis-PMD reduction from the Citriodiol® / trans-acetal mixture in the presence of citric acid and calcium chloride (run no. 5.3) was substantially lower than in their absence (run no. 5.4).

[0209] Example 6: Measurement of PMD reduction from PMD glucovanillin acetal crude reaction mixture using different amounts of citric acid and calcium chloride added on paper As shown in Table VI, the PMD reduction from paper disks containing cis-PMD and trans-PMD glucovanillin acetal was measured using different doses of citric acid and calcium chloride. 1 The following compositions were prepared for measurement by H-NMR:

[0210] Table VI 1 Time: 20.5 hours; 2 Time: 19 hours; 3 Duration: 21 hours

[0211] A composition was prepared by mixing 1 mL of Stock Solution 1, containing the PMD glucovanillin acetal crude reaction mixture prepared as shown in Example 3, with citric acid and calcium chloride in the amounts disclosed in Table VI.

[0212] 50 μL of each mixture was dropped onto the entire surface of parallel 5 cm diameter paper towel disks as shown in the table above, followed by incubation at 32°C.

[0213] At the indicated time points, approximately one-quarter of each disk was cut and washed with DMSO-d6 (0.9 mL). 1 The cis-PMD / trans-acetal ratio was 1 H-NMR spectra were determined by comparison of the integrals of their methyl protons at 1.17 and 1.26 ppm, respectively.

[0214] Stock solution 1 was measured as a ratio at time zero 1 H-NMR showed a cis-PMD / trans-acetal ratio of 10.18. The % cis-PMD remaining was calculated using the value at 20.5 hours, taking into account the value at time zero.

[0215] This experiment demonstrated that the combination of citric acid and calcium chloride provided a slow PMD reducing effect.

[0216] Example 7: Measurement of PMD reduction from paper discs containing a mixture of cis / trans-PMD isomers, citric acid, calcium chloride, and different glucopyranosides To measure the PMD reduction from paper discs containing a mixture of cis / trans-PMD isomers, citric acid, calcium chloride and different glucopyranosides, the mixtures shown in Table VII were prepared.

[0217] [Table VII]

[0218] The following stock solutions were prepared: Stock solution 1: 300 mg of cis / trans-PMD dissolved in 0.8 mL of MeOH-d4. Stock solution 2: 400 mg citric acid dissolved in 1 mL MeOH-d4. · Stock solution 3: 0.7 g anhydrous calcium chloride dissolved in 1 mL deuterium oxide.

[0219] The glucopyranoside (20 mg) shown in the table above was weighed into a parallel glass vial and the stock solution was added where and in the amount shown in Table VII.

[0220] After thorough mixing, 50 μL of each mixture was dropped onto the entire surface of parallel 5 cm diameter paper towel disks, followed by incubation at 32°C.

[0221] After 3 and 21 hours, approximately one-quarter of each paper disc was cut and washed with DMSO-d6 (0.9 mL). 1 The compound was analyzed by H-NMR.

[0222] The ratio of cis-PMD / glucopyranoside was 1 The following signals were determined from the H-NMR spectrum: - Experiment 7.1: It was observed that some of the trans-acetal was converted to cis-acetal by equilibration with cis / trans-PMD in solution prior to application to the paper. By comparison of the signals at 5.70 and 6.68 ppm, it was observed that the ratio of trans-acetal / cis-acetal did not change significantly once applied to the paper surface. Therefore, the ratio of cis-PMD / total acetal was calculated as: 1 From the 1 H-NMR spectrum, it was determined by comparison of the integrals of the cis-PMD signal at 4.15 ppm and the combined acetal signals at 5.70 and 5.68 ppm. -Run 7.2: Peaks at 4.15 / 5.08 ppm - Run 7.3: 4.15 ppm peak / harmonic of 1.17 ppm solvent peak. - Experiment 7.4: Peak at 4.15 / 4.84 ppm -Run 7.5: Peak at 4.15 / 4.90 ppm -Test 7.6: Same as Test 7.1

[0223] The difference in the ratios was then used to determine the degree of PMD reduction, and is shown in Table VIII.

[0224] [Table VIII]

[0225] It is evident that cis-PMD is reduced from paper at a significantly slower rate with different glucopyranosides in the presence of citric acid and calcium chloride than in the absence of glucoside, and it is observed that all glucosides tested have a positive effect on the retention of PMD reduction.

[0226] Example 8: Measurement of PMD reduction from a mixture of cis-PMD, methyl-α-glucopyranoside, citric acid, and calcium chloride compared to a mixture of cis-PMD and methyl-α-glucopyranoside added to paper at different loadings. To measure the PMD reduction from mixtures of cis-PMD and methyl-α-glucoside in both the presence and absence of a combination of citric acid and calcium chloride, stock solutions were prepared by mixing the amounts of materials shown in Table IX, followed by gentle heating with a heat gun.

[0227] [Table IX]

[0228] 50 μL of each stock solution was applied dropwise to the entire surface of parallel 5 cm diameter paper towel disks, followed by incubation at 32°C.

[0229] After 19 hours, approximately one-quarter of each disk was cut and washed with DMSO-d6 (0.9 mL). Two drops of deuterium oxide were added to each sample, followed by 1 The ratio of cis-PMD / methyl-α-glucopyranoside was 1 The H-NMR spectra were determined by comparison of the integrals of their protons at 4.15 / 4.90 ppm, respectively. The data obtained for stock solutions 1 and 2 are reported in Table X below, as Experiments Nos. 8.1 and 8.4, respectively.

[0230] Time zero data was obtained by analysis of the stock solutions as described above.

[0231] Subsequent experiments were performed in the same manner as shown above by dropping 0.25 and 0.5 mL of stock solutions 1 and 2 onto parallel papers. Data obtained at 22.5 and 45 hours are also reported in Table X below.

[0232] [Table X] NR = Not implemented ND = Not Determined

[0233] The data clearly show that when different concentrations of mixtures were applied, significantly more PMD remained on the paper in the presence of methyl-α-glucopyranoside with citric acid and calcium chloride compared to the presence of methyl-α-glucopyranoside alone.

[0234] Example 9: Effect of alternative acids on PMD reduction from paper. To determine the effect of alternative acids on PMD reduction from paper, 50 μL of anhydrous calcium chloride / water solution 0.7:1 (w / v) was added to 0.5 mL of PMD glucovanillin acetal crude reaction mixture prepared as shown in Example 3.

[0235] To a 0.1 mL aliquot of the above mixture, 10 mg amounts of p-toluenesulfonic acid and tartaric acid were added to obtain mixtures 1 and 2, respectively.

[0236] All of each mixture was applied to three parallel 5 cm diameter paper towel discs and then incubated at 32°C.

[0237] After 27 hours, approximately one-quarter of each disk was cut and washed with DMSO-d6 (0.9 mL). 1 The cis-PMD / cis-acetal ratio was calculated as follows: 1 H-NMR spectra were determined by comparison of the integrals of their methyl protons at 1.17 and 1.26 ppm, respectively.

[0238] The time zero ratio was determined by integration of the same peak of the PMD glucovanillin acetal crude reaction mixture.

[0239] The results are shown in Table XI.

[0240] [Table XI]

[0241] A low level of cis-PMD reduction was observed in the presence of p-toluenesulfonic acid and tartaric acid.

[0242] Thus, diacids and acids with widely different pKa's can be used to provide the same sustained release effect.

[0243] Example 10: Measurement of cis-PMD reduction from PMD glucovanillin acetal crude reaction mixtures applied to the skin in both the presence and absence of polymer To measure PMD reduction from cis-PMD applied to the skin both in the presence and absence of a polymer such as Kollicoat® SR 30 D, the following mixtures were prepared: Mixture 1: Citric acid monohydrate (212 mg) was dissolved in 3 mL of PMD glucovanillin acetal crude reaction mixture prepared as shown in Example 3 and 150 μL of 0.7:1 (w / v) anhydrous calcium chloride / water solution with gentle heating using a heat gun. Mixture 2: Kollicoat® SR 30 D (0.2 g) and isopropyl myristate (30 mg) were diluted with 1 mL of Mixture 1 to give a viscous, slightly opaque solution after gentle heating with a heat gun.

[0244] Each mixture (50 μL) was applied to a circular area of ​​5 cm diameter on the skin of the forearm. The area treated with mixture 2 was noticeably less sticky than the area treated with mixture 1.

[0245] After 4 hours, each area was tape stripped by successive application of 2 x 2 cm x 5 cm pieces of Sellotape® (Miarco Gamma Azul). The two tapes used to strip each area were then combined and washed with DMSO-d6 (1 mL), and the resulting solution was 1 The compound was analyzed by H-NMR.

[0246] The ratio of cis-PMD / cis-acetal is 1 H-NMR spectra by comparison of the integrals of their methyl protons at 1.17 and 1.41 ppm, respectively.

[0247] The results are shown in Table XII.

[0248] Table XII

[0249] The cis-PMD / cis-acetal ratios were similar, indicating that the addition of film-forming polymers and isopropyl myristate had a positive effect on the skin feel of the repellent mixtures without significantly affecting the PMD reduction rate.

[0250] Example 11: In vivo repellency – comparative experiment The following series of experiments were carried out on different days but under similar conditions. Thus, known amounts of the mixtures shown in the table below were applied to one middle-aged male volunteer (approximate skin area of ​​1200 cm2). 2 A dose of 100 mg of 10 ...

[0251] At the time periods after mixture application shown in the table below, volunteers stood in a location known to contain Aedes albopictus mosquitoes (Sant Cugat del Valles, Spain). The volunteers timed when each mosquito landed on each leg, brushed them off, then moved to a slightly different location to ensure the same mosquito did not land twice, and resumed counting and moving for 30 minutes. If multiple mosquitoes landed at the same time, all were counted.

[0252] Experiment A: In vivo repellent effect of a Citriodiol® / methyl-α-glucopyranoside / citric acid / calcium chloride mixture compared to the commercial Mosiguard® The following mixtures were tested: Mixture 1: Mosiguard® was purchased online from Amazon and was manufactured by Citrefine Inc. Mosiguard® contained 30% Citriodiol® according to the label. Mixture 2: Citriodiol® (6.0 g), methyl-α-glucopyranoside (2.2 g) and citric acid monohydrate (2.2 g) were diluted with ethanol (14 mL) and 0.7:1 (w / v) anhydrous calcium chloride / water solution (2.0 mL) under gentle heating using a heat gun. Kollicoat® SR 30 D (8 mL) and isopropyl myristate (1.2 mL) were added to give a viscous, slightly opaque solution. The mixture was an unstable emulsion that slowly separated into two phases after a few hours and therefore required thorough shaking before application.

[0253] Mixture 1 (1 mL) was applied to the left leg and Mixture 2 (2 mL) was applied to the right leg as described above, with deposition over time reported in Table XIII below.

[0254] [Table XIII]

[0255] No skin irritation was observed on any of the legs during the experimental period. Significant mosquito infestation was observed on the areas treated with mixture 1 after 9 hours, whereas mosquito infestation was observed on the areas treated with mixture 2 only after 15 hours.

[0256] Mixture 2 clearly provides mosquito repellency for at least 3 hours longer than Mixture 1 at the applied dose.

[0257] Study B: In vivo repellent efficacy of Mosiguard® at twice the prescribed dose The following mixtures were tested: Mixture 1: 2 mL of Mosiguard® (Citrefine) was applied to the right leg as described above, the left leg was left untreated.

[0258] Accretion over time is reported in Table XIV below.

[0259] [Table XIV]

[0260] No skin irritation was observed on either leg during the duration of the experiment. The dose of Mosiguard® used was doubled compared to experiment A, resulting in only a 9.5 hour duration.

[0261] Experiment C: In vivo repellent effect of a mixture of cis-PMD, methyl-α-glucopyranoside, citric acid and calcium chloride The following mixtures were tested: Mixture 1: cis-PMD (0.6 g), methyl-α-glucopyranoside (0.22 g) and citric acid monohydrate (0.22 g) were diluted with ethanol (1.4 mL) and 0.7:1 (w / v) anhydrous calcium chloride / water solution (0.2 mL) under gentle heating using a heat gun. Kollicoat® SR 30 D (0.8 mL) and isopropyl myristate (0.12 mL) were added to obtain a viscous, slightly opaque solution. The mixture was an unstable emulsion that slowly separated into two phases after several hours and required thorough shaking before application. 2 mL of Mixture 1 was applied to the left leg as described above, the right leg was left untreated.

[0262] Accretion over time is reported in Table XV below.

[0263] [Table XV]

[0264] At the end of the experiment, when no further mosquito activity was observed, a circular area of ​​hairless skin of the treated left leg, approximately 5 cm in diameter, was tape stripped and treated as in Example 10. 1 The samples were analyzed by H-NMR, with the exception that two drops of deuterium oxide were added to the NMR samples before analysis. Comparing the integrals of the cis-PMD multiplet at 4.14 ppm and the methyl-α-glucoside doublet at 4.52 ppm, approximately 25% of the cis-PMD remained on the skin.

[0265] No skin irritation was observed on either leg during the experimental period. This mixture produced a much lower odor on the skin than the Citriodiol® mixture used in Experiment A.

[0266] Mixture 1 provided mosquito repellency for at least 14 hours, with 25% active remaining suggesting that even longer duration may have been possible if mosquito activity had not been lost.

[0267] Experiment D: In vivo repellent effect of 20% DEET by volume in ethanol The following mixtures were tested: Mixture 1: 20 mL of N,N-diethyl-meta-toluamide (DEET) was diluted to 100 mL with ethanol. 1 mL of Mixture 1 was applied to the left leg as described above, the right leg was left untreated.

[0268] Accretion over time is reported in Table XVI below.

[0269] [Table XVI]

[0270] It is observed that 20% by volume of DEET provides a significantly shorter period of protection than the mixture used in Experiments A and C.

[0271] Example 12: Measurement of reduction of cis-PMD from a mixture of cis-PMD, methyl-α-glucopyranoside, citric acid and calcium chloride on skin The mixture was prepared as described in Example 11, Experiment C.

[0272] The mixture (50 μL) was applied to a circular area of ​​7×5 cm diameter on the forearm skin.

[0273] After various times, each area was tape stripped as described in Example 10, washed with DMSO-d6 (1 mL), two drops of deuterium oxide were added, and the resulting solution was 1 The compound was analyzed by H-NMR.

[0274] The ratio of cis-PMD / methyl glucopyranoside was determined by comparison of the integrals of the cis-PMD multiplet at 4.14 ppm and the methyl-α-glucoside doublet at 4.52 ppm, respectively, and the remaining cis-PMD was calculated.

[0275] Table XVII shows the linearity of the reduction in cis-PMD.

[0276] [Table XVII]

[0277] With the exception of the outlier results obtained after 20 hours, it was clearly demonstrated that the reduction of cis-PMD from the skin occurred in a linear fashion.

[0278] Example 13: Measurement of reduction of cis-PMD and trans-PMD from a mixture of cis- or trans-PMD, methyl-α-glucopyranoside, citric acid, and calcium chloride on skin Parallel mixtures were prepared as described in Example 11, Experiment C, except that Mixture 1 contained cis-PMD and Mixture 2 contained trans-PMD.

[0279] Each mixture (50 μL) was applied to a circular area of ​​2×5 cm diameter on the forearm skin.

[0280] After time zero and 6 hours, each area was tape stripped as described in Example 10, washed with DMSO-d6 (1 mL), 2 drops of deuterium oxide were added, and the resulting solution was 1 The compound was analyzed by H-NMR.

[0281] The ratio of cis-PMD / methyl glucoside was determined by comparison of the integrals of the cis-PMD multiplet at 4.14 ppm and the methyl-α-glucoside doublet at 4.52 ppm, respectively, and the remaining cis-PMD was calculated.

[0282] The trans -PMD / methyl glucoside ratio was determined by comparison of the integral of the trans -PMD methyl signal at cis 1.05 ppm (divided by 3) with that of the methyl-α-glucopyranoside doublet at 4.52 ppm.

[0283] Table XVIII shows the results.

[0284] [Table XVIII]

[0285] Although the integration in this example was not particularly reliable due to the short duration, it is noteworthy that Mixture 1 had a PMD odor on the skin, whereas Mixture 2 was odorless, opening the possibility of developing odorless repellents.

[0286] Example 14: Measurement of reduction of cis-PMD from a mixture of cis-PMD, methyl-α-glucopyranoside, carboxylic acid, and calcium chloride on skin Parallel mixtures were prepared as described in Example 11, Experiment C, except that Mixture 1 contained citric acid monohydrate and Mixture 2 contained succinic acid (both in equal weight equivalents).

[0287] Each mixture (50 μL) was applied to a circular area of ​​3×5 cm diameter on the forearm skin.

[0288] At various time points, each area was tape stripped as described in Example 10, washed with DMSO-d6 (1 mL), 2 drops of deuterium oxide were added, and the resulting solution was 1 The compound was analyzed by H-NMR.

[0289] The ratio of cis-PMD / methyl-α-glucopyranoside was determined by comparison of the integrals of the cis-PMD multiplet at 4.14 ppm and the methyl-α-glucopyranoside doublet at 4.52 ppm, respectively, and the remaining cis-PMD was calculated.

[0290] Table XIX shows the results.

[0291] [Table XIX]

[0292] Comparable rates of PMD reduction from mixtures containing citric and succinic acids are observed, even though the pKa of citric and succinic acids are significantly different.

[0293] Example 15: PMD reduction from parallel paper disks containing a racemic cis / trans-PMD isomeric mixture and trans-PMD glucovanillin acetal (trans-acetal) with citric acid and various metal halides. 1 H-NMR measurements Parallel mixtures were prepared as shown in Table XX.

[0294] [Table XX]

[0295] The following stock solutions were prepared: Stock solution 1: 0.11 g cis / trans-PMD, 75 mg trans-PMD glucovanillin acetal and 90 mg citric acid monohydrate dissolved in 0.6 mL EtOH and 110 μL water.

[0296] Stock solution 1 (60 μL) was added to glass vials containing different salts (5 mg) as shown in Table XX above.

[0297] After thorough mixing and gentle heating to dissolve all solids, the contents of each vial were applied dropwise to the entire surface of parallel 5 cm diameter paper towel disks, followed by incubation at 32°C.

[0298] After 2 and 20.5 hours, approximately one-quarter of each paper disk was cut and washed with DMSO-d6 (0.9 mL). 1 The compound was analyzed by H-NMR.

[0299] The ratio of cis-PMD / total acetals was 1 From the H-NMR spectrum, it was determined by comparing the integrals of the cis-PMD signal at 4.15 ppm with the combined acetal signals at 5.70 and 5.68 ppm. Note that, as in Example 7, some acetal isomerization may be observed, but this does not correlate with the retention of PMD.

[0300] The difference in ratios between the two time points is shown below in Table XXI.

[0301] [Table XXI]

[0302] All divalent metal halides tested reduced PMD at lower rates than the salt-free mixtures. Zinc and cobalt salts were more effective than calcium or magnesium salts. Chlorides were more effective than bromides or iodides.

[0303] Example 16: PMD reduction from parallel paper discs containing racemic cis / trans-PMD isomer mixtures, citric acid, calcium chloride, and alternative glycosides 1 H-NMR measurements Parallel mixtures were prepared as shown in Table XXII.

[0304] [Table XXII]

[0305] The following stock solutions were prepared: Stock solution 1: A mixture of 0.12 g cis / trans-PMD, 90 mg citric acid monohydrate, and 77 mg anhydrous calcium chloride was dissolved in ethanol (0.6 mL) and deionized water (110 μL) with gentle heating using a heat gun. Stock solution 2: A mixture of 0.12 g of cis / trans-PMD and 90 mg of citric acid monohydrate was dissolved in ethanol (0.6 mL) and deionized water (110 μL).

[0306] The glycosides shown in Table XXII (5 mg) were weighed into parallel glass vials and 60 μL of stock solution 1 or 2 was added.

[0307] The resulting mixtures were gently heated with a heat gun to produce colorless solutions, and each entire mixture was applied dropwise to the entire surface of parallel 5 cm diameter paper towel disks, followed by incubation at 32°C.

[0308] After 2.25 and 19 hours, approximately one-quarter of each paper disk was cut out, washed with DMSO-d6 (1 mL), and two drops of deuterium oxide were added. 1 The compound was analyzed by H-NMR.

[0309] The ratio of cis-PMD / glycoside was determined from: - Experiment 16.1: Peak at 4.65 / 4.02 ppm - Experiment 16.2: As described in Example 7, there was some initial acetal isomerization in the solution before addition to the paper, so the ratio of cis-PMD / total acetals was 1 From the 1 H-NMR spectrum, it was determined by comparison of the integrals of the cis-PMD signal at 4.15 ppm and the combined acetal signals at 5.70 and 5.68 ppm. -Run 16.3: Peak at 4.14 / 4.48 ppm -Test 16.4: Same as Test 16.2 -Run 16.5: Peak at 4.14 / 4.51 ppm -Run 16.6: Peak at 4.14 / 4.50 ppm

[0310] The difference in the ratios was then used to determine the degree of PMD reduction, as shown in Table XXIII.

[0311] [Table XXIII]

[0312] It is apparent that all glycosides, when present, result in retention of PMD compared to control experiment 16.4 (no calcium chloride added).

[0313] Therefore, a wide range of glycosides with different aglycone forms (see Example 7), sugar stereochemistry (type of sugar) and stereochemistry of the glycosidic bond can be used to maintain the PMD sustained release effect.

[0314] Example 17: In vitro repellent effect of a mixture of Citriodiol®, methyl-α-glucopyranoside, citric acid, and calcium chloride compared to the commercially available Mosiguard® Aedes albopictus mosquitoes were reared and maintained at a temperature of 26 ± 2 °C, relative humidity of at least 80% ± 10%, and a 12:12 h (light:dark) photoperiod. Adult mosquito populations were fed sugar solution (10%) but not blood prior to testing.

[0315] The following mixtures were prepared: Mixture 1: Mosiguard® was purchased online from Amazon, manufactured by Citrefine and contained 30 v / v Citriodiol® according to the label. Mixture 2: Citriodiol® (6.0 g), methyl-α-glucopyranoside (2.2 g) and citric acid monohydrate (2.2 g) were diluted with ethanol (14 mL) and 0.7:1 w / v anhydrous calcium chloride / water solution (2.0 mL) with gentle heating using a heat gun. Kollicoat® SR 30 D (8 mL) and isopropyl myristate (1.2 mL) were added to give a viscous, slightly opaque solution. The mixture was an unstable emulsion that slowly separated into two phases after a few hours and required thorough shaking before application.

[0316] 60 μL of Mixture 1 was applied to each of four parallel Hemotek membranes stretched over 3.7 cm diameter metal Hemotek reservoirs containing sheep blood. Four more membranes were similarly treated with 60 μL of Mixture 2. One additional membrane was left untreated.

[0317] After allowing residual solvent to dry for approximately 5 min, samples were attached to a Hemotek heater unit at 37°C and the membrane-covered reservoirs were warmed for 5 min, followed by exposure of the membranes to active, host-seeking, 5- to 7-day-old female mosquitoes that were selected and collected using an aspirator and organized into batches (15 females) in 12 × 20 cm plastic test containers, and the number of accretions recorded over a 20-min period.

[0318] The membrane-covered reservoirs were then removed from the Hemotek heating apparatus and stored in an incubator at 32°C, periodically reinstalled in the heated Hemotek heating apparatus at various intervals, and mosquito accretion was determined for a fresh batch of mosquitoes as described above.

[0319] The membrane treated with Mixture 1 exhibited significant repellency compared to the untreated membrane for the first 4 hours, but after 6 hours the repellent activity decreased significantly, whereas the membrane treated with Mixture 2 maintained consistent repellent activity throughout the 6 hour test period.

[0320] It can be concluded that the mixture containing Citriodiol®, methyl-α-glucopyranoside, citric acid, and calcium chloride retained its effectiveness for a longer period of time than the same amount of Mosiguard®, despite its lower Citriodiol® content.

[0321] Example 18: In vivo repellency test In this example, the repellent efficacy of a mixture of Citriodiol®, methyl-α-glucopyranoside, citric acid, and calcium chloride was compared with the commercially available Mosiguard® in an in vivo arm-cage study using Aedes albopictus mosquitoes.

[0322] We designed a test for repellents against mosquitoes using the ECHA (European Chemicals Agency) procedure (last updated March 2022). The repellent activity was evaluated at approximately 78 m 3The study was carried out under controlled climatic conditions, with a chamber volume of 1000 μg / kg. All studies were performed with non-blood-feeding females of Aedes albopictus mosquitoes aged 5–10 days. Mosquitoes were reared under laboratory conditions with a temperature of 25 ± 2 °C, relative humidity of 60 ± 5%, and a photoperiod of 12:12 h (L:D). Sugar water (5%) was provided before and during the study period to promote good body condition. The study was performed with four participants (two males and two females, aged 19–54 years). All signed an informed consent form that explained the aims and procedures of the study as well as their role during the study. Participants were asked to avoid the use of fragrances, nicotine, alcohol, and repellents for 12 h prior to the study. Before applying the repellent, the skin to be exposed was washed with unscented soap, rinsed with water, rinsed with 70% ethanol, and then dried with a towel. A regular AIC (arm-in-cage) test (WHO, 2009 and EPA, 2010) was performed. Eight 30 × 30 × 30 cm cages (two per participant) were prepared 24 h before the study with a density of approximately 38 mosquitoes.

[0323] The following mixtures were prepared: Mixture 1: Mosiguard® was purchased online from Amazon, manufactured by Citrefine and contained 30 v / v Citriodiol® according to the label. Mixture 2: Citriodiol® (3.0 g), methyl-α-glucopyranoside (0.19 g), succinic acid (2.2 g) and anhydrous magnesium chloride (0.07 g) were diluted with absolute ethanol (3.00 g), Kollicoat® SR 30 D (2.30 g) and 2 M aqueous sodium hydroxide (0.25 mL). The mixture was gently heated with a heat gun to obtain a clear viscous liquid.

[0324] Left forearm 600cm with Mixture 1 2The right forearm was treated with mixture 1 at an application rate of 1 ml per arm, and the right forearm was treated similarly with mixture 2. The amount of repellent applied was calculated based on the dimensions of each participant's arm (circumference and length) and taking into account the area to be exposed. Repellent activity was measured by exposing each participant's forearm to mosquitoes in cages for 3 min per hour for up to 8 hours or until a decrease in repellent activity was detected by counting the number of landings during each exposure period. The first exposure (T1) was 1 hour after application of the mixture to the skin.

[0325] Alighting occurred when a flying mosquito landed on the skin without probing or biting. Probing occurred when a mosquito penetrated the skin with its mouthparts without ingesting blood. If alighting occurred, participants were allowed to slowly wave their arm or blow lightly to prevent the mosquito from biting and sucking blood.

[0326] A complete protection time (CPT) was estimated for each mixture. The CPT is the first probing event and must be confirmed by a second probing event during the same exposure period or a subsequent 3-minute exposure period.

[0327] Accretion over time is reported in Table XXIV below.

[0328] [Table XXIV]

[0329] Mixture 2 showed a significantly longer mean CPT (4.75 hours) compared to Mixture 1 (3.25 hours). Statistical analysis showed that Mixture 2 showed approximately 50% longer duration than commercial Mosiguard® (Mixture 1) at the same Citriodiol application dose.

[0330] No skin irritation was observed from any of the mixtures.

[0331] A different batch of the same mixture 2 was prepared on a 200 g scale, 1.4 ml / 600 cm 2The test was conducted on 10 volunteers over a 12-hour period, with a skin exposure area of ​​100 cm. 2 and 80 mosquitoes per box. The times to complete protection were 9, 11, 12, 12, 12, 12, 12, 12, 12, and 9 hours, with a mean protection of 11 hours but potentially longer if the trial was run for a longer period.

[0332] Example 19: In vivo repellency test In this example, an in vivo arm-cage test was performed with Aedes albopictus to compare the repellency of a mixture of cis-PMD, methyl-α-glucopyranoside, citric acid, and calcium chloride with that of trans-PMD, methyl-α-glucopyranoside, citric acid, and calcium chloride.

[0333] Arm-caging mosquito tests were performed using essentially the same procedure as described in Example 18, except that 1.5 ml of each mixture was administered to each 600 cm of the forearm of four volunteers. 2 The area was applied.

[0334] The following mixtures were prepared: Mixture 1: A mixture of cis-PMD (3.0 g), methyl-α-glucopyranoside (0.64 g), succinic acid (0.64 g) and anhydrous magnesium chloride (0.24 g) was diluted with absolute ethanol (2.92 g), 2 M aqueous sodium hydroxide (0.42 g), deionized water (1.30 g) and Kollicoat® SR 30 D (0.3 g). The mixture was gently heated with a heat gun to give a colorless, opaque liquid. Mixture 2: A mixture of trans-PMD (3.0 g), methyl-α-glucopyranoside (0.64 g), succinic acid (0.64 g) and anhydrous magnesium chloride (0.24 g) was diluted with absolute ethanol (2.92 g), 2 M aqueous sodium hydroxide (0.42 g), deionized water (1.30 g) and Kollicoat® SR 30 D (0.3 g). The mixture was gently heated with a heat gun to give a colorless, opaque liquid.

[0335] Accretion over time is reported in Table XXV below.

[0336] [Table XXV]

[0337] Mixture 1 showed a longer mean CPT (4 hours) than Mixture 2 (1.75 hours). Both mixtures performed similarly in male volunteers, with Mixture 1 performing only slightly better than Mixture 2 in female volunteers.

[0338] No skin irritation was observed from any of the mixtures.

[0339] Example 20: In vivo repellency test In this example, an arm-cage in vivo test with Aedes albopictus was used to simulate long-term testing of mixtures containing low concentrations of cis-PMD, cis / trans-PMD, and Citriodiol® in absolute ethanol compared to dilute solutions of Mosiguard®, and comparison solutions of cis-PMD and tran-PMD.

[0340] The aim of this experiment was to mimic a longer-lasting repellency effect by comparing mixtures containing low concentrations of cis-PMD, Citriodiol® and cis / trans-PMD but without reduced concentrations of methyl-α-glucopyranoside, succinic acid, magnesium chloride or Kollicoat® SR 30 D with Mosiguard®, cis-PMD and trans-PMD at similar active ingredient concentrations diluted in ethanol.

[0341] Arm-caging mosquito tests were performed using essentially the same procedure as described in Example 18, except that 1.5 ml of each mixture was administered to each 600 cm of the forearm of four volunteers. 2 The area was applied.

[0342] The following mixtures were prepared: Mixture 1 (comparison): 4ml of Mosiguard®, purchased online from Amazon, manufactured by Citrefine and containing 30 v / v Citriodiol® according to the label, was diluted in absolute ethanol (6ml). Mixture 2: A mixture of cis-PMD (1.44 g), methyl-α-glucopyranoside (1.32 g), succinic acid (1.30 g) and anhydrous magnesium chloride (0.50 g) was diluted with absolute ethanol (9.5 ml), deionized water (5.5 ml) and Kollicoat® SR 30 D (1.2 ml). The mixture was gently heated with a heat gun to give a colorless, opaque liquid. Mixture 3: A mixture of commercially available cis / trans-PMD (1.44 g), methyl-α-glucopyranoside (1.32 g), succinic acid (1.30 g) and anhydrous magnesium chloride (0.48 g) was diluted with absolute ethanol (8.3 ml), deionized water (1.8 ml), Kollicoat® SR 30 D (4.8 ml) and glycerin (1.2 ml). The mixture was gently heated with a heat gun to obtain a viscous liquid. Mixture 4: A mixture of Citriodiol® (1.44 g), methyl-α-glucopyranoside (1.32 g), succinic acid (1.30 g) and anhydrous magnesium chloride (0.48 g) was diluted with absolute ethanol (8.3 ml), deionized water (1.8 ml), Kollicoat® SR 30 D (4.8 ml) and glycerin (1.2 ml). The mixture was gently heated with a heat gun to obtain a viscous liquid. Mixture 5 (comparison): cis-PMD (0.72 g) was dissolved in absolute ethanol (9.6 ml) to obtain a clear liquid. Mixture 6 (comparison): Commercially available cis / trans-PMD (0.72 g) was dissolved in absolute ethanol (9.6 ml) to obtain a clear liquid.

[0343] Accretion over time is reported in Table XXVI below.

[0344] [Table XXVI] TIFF2024542634000034.tif218130 (comparative mixtures in italics, inventive mixtures in bold)

[0345] Mixture 2 showed a significantly slower decline in repellent activity compared to Mixture 5, indicating that cis-PMD, when formulated with methyl-α-glucopyranoside, succinic acid and magnesium chloride according to the present invention, has a significantly more favorable effect on the in vivo repellent performance of cis-PMD compared to the same concentration of cis-PMD formulated in a traditional manner (in alcohol). Mixture 3 showed a significantly slower decline in repellent activity compared to Mixture 6, indicating that cis / trans-PMD, when formulated with methyl-α-glucopyranoside, succinic acid and magnesium chloride according to the present invention, has a significantly more favorable effect on the in vivo repellent performance of cis / trans-PMD compared to the same concentration of cis / trans-PMD formulated in a traditional manner (in alcohol). On average, Mixture 4 showed a significantly slower decline in repellent activity compared to Mixture 1, demonstrating that when Citriodiol® is formulated with methyl-α-glucopyranoside, succinic acid and magnesium chloride in accordance with the present invention, it has a significantly positive effect on the repellent performance of Citriodiol® in vivo compared to the same concentration of Citriodiol® formulated in a traditional manner (diluted Mosiguard®).

[0346] Example 21: In vivo repellency test In this example, an in vivo arm-cage study with Aedes albopictus was used to compare the repellent efficacy of a mixture of cis-PMD, methyl-α-glucopyranoside, citric acid, and calcium chloride with and without pH adjustment.

[0347] Arm-caging mosquito tests were performed using essentially the same procedure as described in Example 18, except that 1 ml of each mixture was administered to each 600 cm of the forearm of four volunteers. 2 The area was applied.

[0348] The following mixtures were prepared: Mixture 1: A mixture of cis-PMD (1.2 g), methyl-α-glucopyranoside (0.64 g), succinic acid (0.64 g) and anhydrous magnesium chloride (0.24 g) was diluted with absolute ethanol (3.24 g), 2 M aqueous sodium hydroxide (0.42 g), deionized water (2.50 g) and Kollicoat® SR 30 D (0.30 g). The mixture was gently heated with a heat gun to give a colorless, opaque liquid. Mixture 2: A mixture of cis-PMD (1.2 g), methyl-α-glucopyranoside (0.64 g), succinic acid (0.64 g) and anhydrous magnesium chloride (0.24 g) was diluted with absolute ethanol (3.56 g), deionized water (2.50 g) and Kollicoat® SR 30 D (0.30 g). The mixture was gently heated with a heat gun to give a colorless, opaque liquid.

[0349] Accretion over time is reported in Table XXVII below.

[0350] [Table XXVII]

[0351] Mixture 1 showed a slightly slower rate of decline in repellent activity over time than Mixture 2.

[0352] No skin irritation was observed from any of the mixtures.

[0353] Example 22: Extraction of pure racemic cis / trans-PMD from Citriodiol® followed by selective crystallization of racemic cis-PMD Citriodiol® (30.0 g) was dissolved in hexane (200 ml). Ethanol (0.10 ml) was added, followed by anhydrous calcium chloride (19.3 g, approximately 1.4 equivalents relative to the total PMD in Citriodiol®), and the mixture was vigorously stirred at room temperature for 4 hours. The mixture was filtered and the white solid residue was washed with hexane (20 ml). The residue was then dissolved in a mixture of water (100 ml) and hexane (100 ml), the aqueous phase was removed by separation, the organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure to give racemic cis / trans-PMD (20.8 g, approximately 99% recovery based on the total PMD in Citriodiol®). This product was 1 It was identical to standard cis / trans-PMD by H-NMR and contained essentially the same Cis / Trans ratio as the starting Citriodiol® (about 6:4 cis / trans-PMD). Essentially the same results can be obtained using anhydrous magnesium chloride instead of anhydrous calcium chloride.

[0354] Racemic cis / trans-PMD (100.0 g) was dissolved in ethyl acetate (15 ml) with heating to 70° C. Hexane (30 ml) was added dropwise and the solution was cooled to room temperature and then further cooled to 15° C. Stirring was continued for 3 hours and the mixture was filtered to give racemic cis-PMD (33.0 g, approximately 53% based on cis-PMD in the starting material) as a white solid. This material was 1 H-NMR showed it to be identical to standard cis-PMD.

[0355] Similar results were obtained by recrystallization using 10 times the amount of heptane relative to the starting material.

Claims

1. (a) p-menthane-3,8-diol (PMD), (b) glycosides, (c) organic acids, and (d) Divalent metal halide salts A sustained release formulation comprising:

2. 2. The sustained release formulation of claim 1, wherein the PMD is selected from the eight stereoisomers of PMD: racemic-cis-PMD, racemic-trans-PMD, and mixtures thereof.

3. A glycoside is a reducing sugar linked to a non-carbohydrate via a glycosidic bond, where the glycosidic bond is either alpha (α) or beta (β), and where the sugar is a monosaccharide, a sugar having a degree of polymerization comprised between 1.1 and 1.9, a disaccharide, or a trisaccharide.

3. The sustained-release formulation according to claim 1 or 2, characterized in that:

4. The reducing sugar is selected from glucose, galactose, allose, altrose, mannose, gulose, idose, talose, fucose, xylose, 2-deoxyglucose, N-acetylglucosamine, and mixtures thereof; preferably from D-glucose, D-galactose, D-allose, D-altrose, D-mannose, D-gulose, D-idose, D-talose, and mixtures thereof; more preferably from D-glucose or D-galactose; even more preferably, the sugar is D-glucose.

4. The sustained-release formulation according to claim 3,

5. 2. The sustained-release formulation according to claim 1, characterized in that the glycoside is selected from methyl-α-glucoside, hexylglucoside, phenyl-β-glucoside, geranyl-β-glucoside, methyl-α-D-mannopyranoside, methyl-α-D-galactopyranoside, and PMD glucovanillin conjugates; preferably selected from methyl-α-glucoside and PMD glucovanillin conjugates.

6. 2. The sustained-release formulation according to claim 1, wherein the organic acid is selected from sulfonic acids, monocarboxylic acids, dicarboxylic acids, tricarboxylic acids, hydroxycarboxylic acids, and amino acids.

7. 7. The sustained release formulation of claim 6, wherein the organic acid is selected from citric acid, succinic acid and their anhydrous forms or hydrates.

8. 2. The sustained-release formulation of claim 1, wherein the divalent metal halide salt is selected from calcium chloride, magnesium chloride, magnesium bromide, zinc iodide, zinc chloride, zinc bromide, and cobalt chloride; preferably selected from calcium chloride, magnesium chloride, and zinc chloride.

9. per mole of PMD (a) between 0.01 and 2 mol, preferably between 0.02 and 1.5 mol, more preferably between 0.03 and 1.2 mol, more preferably between 0.05 and 1 mol, and even more preferably between 0.07 and 0.75 mol of glycoside; (b) between 0.05 and 3 mol, preferably between 0.06 and 2.5 mol, more preferably between 0.08 and 2 mol, more preferably between 0.09 and 1.7 mol, more preferably between 0.1 and 1.5 mol, and even more preferably between 0.12 and 1.24 mol of an organic acid; and (c) between 0.01 and 5 mol, preferably between 0.02 and 2 mol, more preferably between 0.03 and 1 mol, and even more preferably between 0.06 and 0.58 mol of a divalent metal halide salt 2. The sustained release formulation according to claim 1, comprising:

10. 10. The sustained release formulation of claim 1 for use as an insect repellent.

11. 10. The sustained release formulation of claim 1 for use as a pharmaceutical; preferably for use as an antiseptic, antibiotic, fungicide, bactericide, or antiviral agent.

12. 10. The sustained release formulation of claim 1, for application of said sustained release formulation onto a surface.

13. A composition comprising the sustained release formulation of claim 1 and a cosmetically acceptable ingredient.

14. For the controlled release of PMD from the formulation of claim 1, a compound of formula (II): The use of PMD glucovanillin conjugate, a compound of the present invention.

15. (1) adding a salt selected from calcium chloride, magnesium chloride, and mixtures thereof to a solution of an extract containing PMD; (2) separating the solid obtained in step (1); and (3) isolating racemic cis / trans-PMD from the solid separated in step (2). A process for isolating racemic cis / trans-PMD from an extract containing PMD, comprising:

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