GB2581375A - Insect repellent compositions and methods of use - Google Patents

Abstract

A composition of p-Menthane-3,8-diol (PMD, OLE, oil of lemon eucalyptus, citriodiol, citrepel, p-methane-3,8-diol), vanillin as an aldehyde fixative, sodium bisulphite (bisulfite) as an antioxidant and isopropanol as a delivery vehicle. The composition is applied in a dose less than 1.67 g/cm2. It may repel Scottish Highland midge (Meanbh-chuileag), Culex mosquito, Aedes Aegypti mosquitos, ticks and biting fly. This helps prevent transmission of diseases such as Dengue fever, Yellow fever, Zika virus, West Nile virus and Lyme disease. The composition is applied as a spray, lotion, gel or roll-on. It may also be applied to an article of clothing or material, such as a mosquito net or a dermal wipe. The PMD is present in at least 20% weight of the composition and provides protection for at least eight hours. The antioxidant prevents discolouration during storage for 14 days at 54°C. Vanillin is present between 3 to 15% and sodium bisulphite is present between 0.5% to 1.5% and isopropanol is present at 40%. The composition can also be defined by the weight percentage of PMD being 30% or more and the PMD:fixative ratio by weight being in the range of 3:1 to 10:1, where there is no alcohol.

Description

Intellectual Property Office Application No. GII1902071.8 RTM Date:21 November 2019 The following terms are registered trade marks and should be read as such wherever they occur in this document: Citriodiol Citrefine Intellectual Property Office is an operating name of the Patent Office www.gov.uk /ipo Insect Repellent Compositions and Methods of Use The present invention is concerned with insect repellent compositions based on p-methane-3,8-diol (PMD) and with methods of using these to repel insects over an extended period of time. The compositions of the invention are botanical insect repellents, as compared to synthetic chemical compositions such as those based on DEET, and can provide a prolongation of complete protection times to an 8 hour minimum thereby enabling a once daily dosage regime.

Certain botanical insect repellent compositions based on PMD have been disclosed in our earlier patent application W02017/081445, herein incorporated by reference, and to which reference can be made for further details. The present invention extends this earlier work, disclosing new botanical insect repellent compositions, and in particular new methods of use based upon further experimental findings.

W02017/081445 gives a detailed description of arm in cage tests which are used to test insect repellency. The World Health Organisation (WHO) insect repellent arm in cage tests are the standard measure used for assessing insect repellency. These tests describe using an insect repellant composition dose of 1.67 mg/cm2, that is, a dose of 1.67mg of product is applied for each square centimetre of skin surface, when comparing complete protection times (CPT). This dose is equivalent to 1 gm of product being applied to 600 square cm of skin -typically the area between wrist and elbow of the forearm skin of test subjects. Indeed, manufacturers of insect repellent compositions frequently describe a 'length of protection' in hours based on arm in cage tests performed at a dose application of 1.67 mg/cm2. This dosing quantity has thus come to be accepted as a standard measure within the industry.

The present inventor has appreciated that the above standard measure is subject to many environmental variables, and that, in practice, actual dosing levels of insect repellent compositions applied by users can very often be much lower than 1.67 mg/cm2-for example, at around 1 mg/cm2. This results in the problem that the claimed "protection time" of a particular commercial product is often over-stated on the label, and that users do not in fact achieve the protection times claimed.

The present inventor has further appreciated that it would be highly desirable to be able to provide an insect repellent which achieves an excellent level of protection -for example, a complete protection time of at least 8 hours, at lower dosing levels. It would also be highly desirable to be able to achieve this using a botanical insect repellent, due to the well documented problems, restrictions and ever increasing consumer cynicism seen with synthetic repellents such as DEET.

According to one aspect of the present invention, there provided a method of providing protection against insects, the method comprising administering to the skin of a user an insect repellent composition comprising p-methane-3,8-diol, a fixative, and a delivery vehicle, wherein the composition is applied at a dose of less than 1.67mg/cm2.

The invention provides protection against, or repels, insects especially biting insects such as flies from the family Culicidae or Ceratopogonidae, especially mosquitoes and midges. This includes the mosquito vector for malaria which is the Anopheles species, and the current vector for Dengue, Yellow fever and Zika which is Aedes Aegypti, and the Culex mosquito vector which is the cause of West Nile virus in the United States. It also provides protection against ticks which is the vector for the bacterial infection Lyme disease.

In another aspect, the present invention also provides an insect repellent composition comprising p-methane-3,8-diol, a fixative, and a delivery vehicle for use in providing protection against insects, or for repelling insects, wherein the composition is applied at a dose of less than 1.67mg/cm2 to the skin of a user.

In another aspect, the present invention also provides an insect repellent composition comprising p-methane-3,8-diol in an amount of 30% or more by weight of the composition, a fixative wherein the ratio of PMD: fixative (by weight) is in the range of 3:1 to 10:1, and a delivery vehicle, wherein the composition does not comprise any alcohol.

In a further aspect, the invention provides a method of preventing a disease state selected from the group consisting of: disease states caused by a biting fly, disease states caused by Aedes Aegypti, ticks, Dengue fever, Yellow fever and the Zika virus; or of repelling a biting fly including ticks, which method comprises administering to the skin of a user a composition according to the present invention as described herein.

FIGURES

The following Figures illustrate the present invention.

Figure 1: Dose response curve for DEET, from Buescher et al. Figure 2: CPT Performance of 30% PMD / 5% vanillin (both by weight of total composition) at different application doses, against Aedes Aegypti.

Figure 3: Non-linear regression analysis of CPT and TFB Performance of 30% PMD / 5% vanillin (both by weight of total composition) and 20% DEET (by weight of total composition) at different application doses, against Aedes Aegypti.

Figure 4: Non-linear regression analysis of TFB Performance of 30% PMD / 5% vanillin and 45% PMD / 7.5% vanillin (all by weight of total composition) and 20% and 50% DEFT (all weight of total composition) at different application doses, against Aedes Aegypti.

Fig 5: Decay curve for 30% PMD / 5% vanillin composition based on ED95 results -refer to Experiment 2.

Fig 6: ED95 and decay curves for 30% PMD / 5% vanillin composition and 20% DEFT composition based on results from Experiment 2.

Fig. 7: Decay curves for 30% PMD / 5% vanillin composition and 20% DEET composition at WHO recommended dose of 1.67 mg/cm2 showing Area under the Curve (AUC) comparison.

The present inventor has found that excellent levels of protection using compositions based on the natural compound PMD together with a fixative compound such as vanillin can, in fact, be achieved even at low dosing levels.

Preferably, in the present method, the composition is applied at a dose of less than 1.4mg/cm2, or at a dose of less than 1.2mg/cm2. In a preferred aspect, the composition is applied at a dose in the range 0.6mg/cm2 to 1.4mg/cm2, or is applied at a dose in the range 0.8mg/cm2 to 1.2 mg/cm2. A dose of around 1mg/cm2 is particularly preferred. The dosing refers to the amount of the composition (as a whole) that is applied to each square centimetre of the skin or other surface.

The desired dose will vary principally upon the concentration of active substance i.e. pmethane-3,8-diol (PMD) in the composition, as will be clear from Figures 3 and 4 which illustrate the dose response curves for compositions comprising different concentrations of PMD (30% and 45% respectively).

The p-methane-3,8-diol may be present in an amount of at least 20% by weight of said composition, or at least 25% by weight of said composition, or at least 30% by weight of said composition, or at least 40% by weight of said composition. Higher concentrations have been found to allow for lower product doses whilst still providing protection times of 8 hours or more.

In a preferred aspect, the composition comprises p-methane-3,8-diol as the sole insect repellent.

In the methods and compositions provided by the present invention it is highly preferred that the insect repellent composition provides a complete protection time of at least 8 hours. A complete protection time of at least 10 hours may also be achieved.

The composition for use in the present invention may further comprises an antioxidant, optionally wherein the antioxidant prevents discoloration of said fixative when said composition is stored at 54°C for 14 days. The antioxidant may, for example, comprises a bisulphite salt, such as sodium bisulphite.

Where present, the antioxidant may be present in an amount of about 0.05% to about 1.5% by weight % of the composition.

The composition for use in the invention comprises a fixative, and this has been found to aid in the protection performance provided by PMD, as described previously in our application W02017/081445 to which reference can be made for further details. In W02017/081445, we describe a key result that for example the compound vanillin in medium concentrations (<15%) can act as a highly effective fixative and prolong the repellent action of PMD way beyond 6 hours to achieve 12 hours or more repellency.

The fixative suitably comprises a compound which can reversibly form an acetal with PMD, such as a compound comprising an aldehyde or keto-functional group, although other compounds are not excluded. For example, the fixative may comprise a compound of the general formula RCHO or RCO, wherein R is alkyl, aryl, or heteroaryl. Aliphatic aldehydes with a carbon chain length of 14 or less are preferred. Aromatic aldehydes may also be used, including aromatic compounds where the aldehyde group is not directly attached to the aromatic ring.

The fixative is preferably present in an amount of about 3% to 15% by weight of said composition, optionally from 5% to 15% by weight of said composition.

We have found another advantageous way of defining the amount of fixative is by reference to the amount of PMD. Thus, in a preferred aspect, the ratio of PMD: fixative (by weight) is in the range of 4:1 to 8:1, alternatively in the range of 5:1 to 7:1, and most preferably the ratio of PMD: fixative (by weight) is about 6:1.

In a preferred aspect, the fixative comprises vanillin or a derivative thereof such as ethyl vanillin.

Suitably, in a composition for use in the present invention the fixative may comprise vanillin and the antioxidant, where present, prevents discoloration of said vanillin when said composition is stored at 54°C for 14 days.

Preferably, in the method of the invention, the dose is administered once or bi-daily. One daily application only can often be achieved because of the high protection levels (in terms of hours of protection) which can be provided using the presently described compositions.

The methods and compositions of the invention are suitable for the prevention of a disease state selected from the group consisting of: disease states caused by Aedes Aegypti, Dengue fever, Yellow fever and the Zika virus. They are also suitable for the prevention of a disease state caused by the Culex mosquito, or the West Nile virus, and also for repelling the Scottish Highland midge (Meanbh-chuileag). They are also suitable for providing protection against ticks which is the vector for the bacterial infection Lyme disease In the methods and compositions of the invention, preferably the delivery vehicle (which can be any suitable vehicle) comprises a pharmaceutically acceptable solvent, such as an aqueous solvent, or an alcoholic solvent or a hydroalcoholic solvent. Suitably, the delivery vehicle comprises a pharmaceutically acceptable alcohol, such as isopropyl alcohol.

In the methods and compositions of the invention, the insect repellent composition preferably comprises p-methane-3,8-diol, vanillin, sodium bisulfite, water and isopropyl alcohol.

The solvent, which is suitably isopropyl alcohol, may be present in an amount of about 40% or more by weight of said composition, depending upon the concentration of other ingredients, such as the active material.

In one aspect of the invention, especially where an aqueous or alcoholic or hydro-alcoholic solvent system is used, the insect repellent composition is in the form of a spray (such as a pump spray), an aerosol, lotion, gel or roll-on.

Preferably, the insect repellent composition comprises vanillin in an amount of about 5% or about 10% by weight of said composition.

Preferably, the insect repellent composition comprises a bisulphite, such as sodium bisulfite, in an amount of about 0.1% by weight of said composition.

Preferably, the insect repellent composition comprises an alcohol, such isopropyl alcohol, in an amount of about 40% by weight of said composition.

In a further aspect, the invention also provides a method of providing protection against insects, the method comprising impregnating an article of manufacture, such as a mosquito net or a dermal wipe, with an insect repellent composition comprising p-methane-3,8-diol, a fixative, and a delivery vehicle, wherein the composition is applied to the said article at a dose of less than 1.67mg/cm2.

In this method, the feature(s) of the dose application or the insect repellent composition or the disease state treated or prevented, may be according to any one or more of the features as defined above.

The invention also provides a method of preventing a disease state selected from the group consisting of: disease states caused by a biting fly, disease states caused by Aedes Aegypti, ticks, Dengue fever, Yellow fever and the Zika virus; or for repelling a biting fly including ticks, which method comprises administering to the skin of a user a composition as defined by any one or more of the features above, and as defined in the appended claims, at a dose of less than 1.67mg/cm2.

In such a method, the composition may be applied at a dose of less than 1.4mg/cm2, or at a dose of less than 1.2mg/cm2, or at a dose in the range 0.6mg/cm2 to 1.4mg/cm2, or at a dose in the range 0.8mg/cm2 to 1.2 mg/cm2.

The method is suitable for the prevention of a disease state selected from the group consisting of: disease states caused by the Culex mosquito, and West Nile virus, as well as, for example, for repelling the Scottish Highland midge (Meanbhchuileag). It also provides protection against ticks which is the vector for the bacterial infection Lyme disease We have also found that in practice at higher concentrations of active in a PMD-fixative system of the type described herein (and also as described in our earlier W02017/081445, to which reference can be made for further details), that there can be formulation difficulties when using an alcoholic or hydro-alcoholic solvent system, of the type typically employed for pump-sprays and aerosols. Such problems are generally not seen with DEET. Thus, we have found that levels of PMD at around 40% or more can lead to stability and/or dissolution problems. Having appreciated this problem, this has led to further investigation by the inventor, particularly at active levels of 30% PMD or more, or 40% PMD or more, or 50% PMD or more. The range of 40-60% PMD (by weight of the total formulation) is of particular interest.

Preliminary results also indicate that in a PMD-fixative system (for example, one comprising PMD and vanillin) the ratio of active material (PMD) to the fixative appears to be of importance.

Thus, in a further broad aspect of the invention, there is provided an insect repellent composition comprising p-methane-3,8-diol in an amount of 30% or more by weight of the formulation, a fixative wherein the ratio of PMD: fixative (by weight) is in the range of 3:1 to 10:1, and a delivery vehicle, wherein the formulation does not comprise any alcohol.

Preliminary results indicate that such a composition may suitably be formulated as a gel, or other similar formulations such as a cream, lotion, or wipe. A preferred formulation is the above insect repellent composition in the form of a gel.

Preferably, the composition also does not comprise any water.

Suitably, the composition may comprise p-methane-3,8-diol in an amount of 40% or more by weight of the composition, or may comprise p-methane-3,8-diol in an amount of 50% or more by weight of the composition, or may comprise p-methane-3,8-diol in an amount of 60% or more by weight of the composition.

In such a composition, the ratio of PMD: fixative (by weight) is preferably in the range of 4:1 to 8:1, or the ratio of PMD: fixative (by weight) is in the range of 5:1 to 7:1, or most preferably the ratio of PMD: fixative (by weight) is about 6:1.

The fixative may comprise vanillin, or a vanillin derivative such as ethyl vanillin.

The composition preferably comprises p-methane-3,8-diol as the sole insect repellent.

Suitably, the insect repellent composition provides a complete protection time of at least 8 hours, or a complete protection time of at least 10 hours.

As with the above-described compositions the fixative may comprise a compound which can reversibly form an acetal with PMD, such as a compound comprising an aldehyde or ketofunctional group. For example, the fixative may comprise a compound of the general formula RCHO or RCO, wherein R is alkyl, aryl, or heteroaryl. Aliphatic aldehydes with a carbon chain length of 14 or less are preferred. Aromatic aldehydes may also be used, including aromatic compounds where the aldehyde group is not directly attached to the aromatic ring.

The fixative may for example be present in an amount of about 3% to 15% by weight of said composition, optionally from 5% to 15% by weight of said composition.

The above composition is especially suited to be in the form of a gel, or a cream, or a lotion or is a composition suitable for application to a wipe such as a dermal wipe.

In a preferred aspect, the composition, which is preferably a gel, includes a delivery vehicle which comprises a thickening agent or a thixotropic agent, or both. Any suitable agent may be used, but preliminary testing shows that a delivery vehicle comprising fumed silica may be particularly suitable.

The invention provides a preferred composition which is an insect repellent composition comprising p-methane-3,8-diol in an amount of 40% or more by weight of the composition, vanillin wherein the ratio of PMD: vanillin (by weight) is in the range of 5:1 to 7:1, and a delivery vehicle comprising fumed silica, wherein the composition does not comprise any alcohol or water, and wherein the composition is in the form of a gel, a cream, a lotion or is a formulation suitable for application to a wipe such as a dermal wipe.

The above described composition may further comprises an antioxidant, optionally wherein the antioxidant prevents discoloration of said fixative when said composition is stored at 54°C for 14 days.

The antioxidant may for example comprise a bisulphite salt, such as sodium bisulphite. Such an antioxidant may for example be present in an amount of about 0.05% to about 1.5% by weight % of the composition.

In a preferred aspect, in the above composition, the fixative comprises vanillin and the antioxidant, where present, prevents discoloration of said vanillin when said composition is stored at 54°C for 14 days.

As with the other compositions disclosed herein, the above composition may be for once or bidaily administration.

The above composition may be used for the prevention of a disease state selected from the group consisting of: disease states caused by the Culex mosquito, and West Nile virus, as well as for repelling the Scottish Highland midge (Meanbh-chtilleacy). It may also be used to provide protection against ticks which is the vector for the bacterial infection Lyme disease.

The invention also provides, in a further aspect, a method of preventing a disease state selected from the group consisting of: disease states caused by a biting fly, disease states caused by Aedes Aegypti, ticks, Dengue fever, Yellow fever and the Zika virus; or of repelling a biting fly including ticks, which method comprises administering to the skin of a user a nonalcoholic composition as described above. The method may also encompass the prevention of a disease state selected from the group consisting of: disease states caused by the Cu/ex mosquito, or West Nile virus, or ticks; or for repelling the Scottish Highland midge (Meanbhchuileaa).

The present invention will now be further illustrated by the following results.

Experimental Protocol Experiment 1 Comparative arm-in-cage studies were carried out to compare a PMD formulation containing 30% by weight PMD, with a 20% by weight DEET formulation, to assess protection against Aedes Aegypti. The type of investigation test selected was the laboratory arm or hand in cage test and the mosquito species selected was Aedes Aegypti. The tests were undertaken to WHO standards in line with regulatory guidelines WHO protocol (HTM/NTD/VVHOPES/2009.4 and OPPTS 810.3700). The protocols used are summarised below.

Complete protection times are calculated as the number of minutes (or hours) elapsed between the time of repellent application and the first mosquito landing or probing. Complete Protection Times are reported herein as a median value of protection time given by each individual. Complete Protection Times are abbreviated herein as CPTs. The test employed to determine the same consisted of inserting a repellent treated arm into a cage measuring 35 cm on each side, containing laboratory bred 100 numbers of non-blood fed Aedes aegypti mosquitoes that are 5-7 days old, and measuring the elapsed time to first landing or probing (which refers to an insect landing and penetrating the skin with its mouthparts, without ingesting blood).

Time to first confirmed bite (herein abbreviated to TFB) as described above was also recorded.

The experiments were undertaken at the highly experienced, international centre of excellence, Ross Lifesciences, Pune, Maharashtra, India.

TEST SUBSTANCE CHARACTERIZATION

1) Active ingredient: PMD in formulation is 30% w/v (43% citriodiol containing 72% PMD). This PMD composition (Neo-Innova Healthcare (NIH) Neo-part® repellant, alternatively referred to herein as PMD-XL) comprised 30% by weight PMD (from "Citriodiol", approved in the UK and USA, and supplied by the company Citrefine International Limited) and 5% vanillin in isopropyl alcohol. 0.1 % (by weight of composition) sodium bisulphite was also present. IPA and citriodiol were mixed by simple stirring, followed by the addition of the remaining ingredients.

2) DEET formulation prepared In-house. 20% DEET by weight. Inert ingredients: Isopropyl alcohol.

REFERENCE SUBSTANCE CHARACTERIZATION Name: 20% w/v DEET formulation

CONTROL

One arm of every test subject will remain untreated, and this arm of test subject is the negative control.

TEST ROOM CONDITIONS

Temperature, 80±10°F (27± 5°C).

*50±20% Relative Humidity. ;*Artificial lighting.

SAMPLE STORAGE CONDITIONS

Samples (test formulations) will be stored at room temperature and ambient humidity before the test.

ARM IN CAGE METHOD

Complete protection time provided by each product was be evaluated with arm-in-cage studies modified after the WHO protocol (2009). There were 6 participants, with an equal number of each gender. The subjects will be aged between 18 and 60 years and will be selected based on formally signed Ross Lifescience informed consent prior to participation, which provides for the right of candidates to refuse to participate and the right of participants to withdraw. Participants will be instructed to avoid alcohol, caffeine and fragrance products for 12 hours before and during the test. For both treated and untreated (control) subjects, the forearms will be washed with unscented soap, rinsed with water and then washed with solution of 70% alcohol and 30% w/v water and dried with a clean towel.

Following scientific standards, treatments of 30% w/v PMD product to 600 square cm of the area between wrist and elbow of the forearm skin of test subjects will be applied at a dosing rate starting with the quantity calculated after range finding studies. An incremental dose of 0.03 grams will be applied on the test arm (after every 30 minutes) and monitored for the repellency until an ED90 value is obtained. The test substance will be sprayed/applied directly from the sample container onto the skin. After the application, the sample will be returned to the balance and the amount applied determined. After application on skin the test substance will be spread around the forearm to help ensure complete coverage using two fingers with a gloved hand of a member of the study staff (trained entomologist). Upon application of the repellent, subjects will be instructed not to rub, touch or wet the treated arm. Subjects can do light work or will be seated in a hall between exposures.

Cages with mosquitoes will be placed in a 30 cubic meter glass chamber for testing. Test exposures will consist of participants inserting their untreated control arm or treated arm into cages measuring 40 cm on each side, containing 50 laboratory bred non-blood fed adult female Aedes aegypti mosquitoes approximately 5-7 days post-eclosion. Before any application of test materials, the forearms of the subjects will be exposed to the mosquitoes in the test cage to gauge suitable attractiveness. A landing rate of 5 mosquitoes landing in 30 seconds on the untreated arm will be regarded as the minimum for the test subject to qualify for further participation.

Readiness of the mosquitoes to bite (biting pressure) will be checked by inserting the untreated arm for up to 30 seconds, prior to inserting the treated arm into the other test cage. Separate cages will be used for exposing untreated positive control versus negative control as well as the test arm of the treated subjects. Five or more landings in 30 seconds on the untreated arm will be again considered the minimum to initiate the test with the treated arm. Exposures of treated arms will last for 30 seconds and will be monitored for landing or probing activity. This procedure will be repeated every 30 minutes until the first landing or probing is observed. Complete Protection Time (CPT) will be calculated as the number of minutes (or hours) elapsed between the time of repellent application and the first mosquito probing. Landing without probing, which begins earlier than probes, will be also recorded in all the experiments.

TEST SYSTEM

Adult female mosquitos (Aedes aegypti) approximately 5-7 days post-eclosion. Source: Ross Lifescience Private Limited, Pune, India.

CONTROL OF BIAS

A population of only healthy vigorous insects will be selected for this study.

TEST REQUIREMENT

Mosquitos: Tests will be conducted with Aedes aegypti species of laboratory bred adult female mosquitos.

Rearing techniques: Mosquito Larvae reared under optimal conditions at temperature of 27±5°C, relative humidity 50±20%, and photoperiod 16:8 hours (light: dark).

Feeding of adults: Adult females fed with 10% sucrose and no blood meal before the test and starved for minimum of 12 hours before the test, will be used for this study.

Use of insect: Test insects will be used for only for 1 test and will be destroyed post testing by using electric bat and finally disposing it by incineration.

Density of Insects used in test: In each test cage a maximum of 50 mosquitos per cage will be ensured.

Test cage and testing conditions: Cages utilized will be 40 cm X 40 cm X 40 cm (LBH) consisting of one sleeved opening for the subjects arm.

DEFI NITIONS

Bite: Refers to an insect penetrating skin with its mouthparts and ingesting blood, with resulting abdomen swelling and color change.

First Confirmed bite: Refers to a bite followed by an additional bite during one exposure period or, when one bite occurs in such an exposure period and another bite occurs in the next exposure period. Once a first confirmed bite occurs on a subject, the test subject does not participate in any additional exposure periods on that day of testing.

Land: Refers to an insect that lands, but does not probe or bite.

Light or probe: Refers to an insect landing and penetrating the skin with its mouthparts, without ingesting blood.

Protection time (PT): It is the time when the first confirmed land occurred, and may not necessarily involve biting in case of PT Complete Protection time (CPT): Refers to the time from application of the repellent to the time until the first confirmed bite (FCB) occurred.

Repellency: Refers to a lack of insects probing or biting human skin where repellent has been applied.

LIBes: Landing with Intent to Bite 7. CANDIDATE SCREENING: RECRUITMENT, SCREENING, CONSENT, PRIVACY Inclusion Criteria: All subjects Age: 18-60 years Sex: Male/female Race: Any race Education: Some college or post-high school formal technical training. Regularly exposed in work or daily life to wild mosquitos.

Test subjects will be selected based on signing of informed consent form prior to any participation in the study. Subjects will be briefed about the experiment prior to them signing the consent form.

Test Subjects (Treated): The number of test subjects per formulation/test will be as follows: Test will comprise of 6 treated test subjects. Each subject will have one forearm treated with 30% w/v PM D formulation and the other arm will be the negative control. The same subjects will once again have one arm treated with 20% w/v DEET formulation and the other arm will be the negative control, on separate test days per subject. Equal numbers of adult male and female treated test subjects will be utilized.

Test Subjects (Untreated): The number of test subject per formulation/test will be as follows: One untreated control to monitor mosquito activity in the test cages throughout the test.

Test Subjects (Treated and Untreated): They will all be instructed to avoid alcohol, caffeine, and fragrance products (e.g perfume, cologne, hair spray, lotion, etc.) for 12 hours before, and during the test.

Preparation for All Test Subjects (Treated and Untreated): Dry control forearms will be washed, rinsed and dried exactly like treated forearms. To establish their attractiveness, forearms of the subjects will be exposed to the mosquitos in the test cage before the test could begin. The minimal landing rate of the mosquitos on the untreated arm will be considered as 5 LIBes ('Landing with Intent to Bite') per 30 seconds. A mean study LIBes rate of 5 LIBes per untreated (negative control) lower arm per 30 seconds is required for the test subject to qualify as a participant.

Preparation of Treated Test Subjects: Area from wrist to elbow will be used for applying test item in treated subjects. The test area will be washed with unscented soap, rinsed with water, then with a solution consisting of 70% ethanol and 30% w/v water, and dried with a towel.

Treatment area calculation: The surface area (in cm2) will be calculated for each test subject's forearm as per methodology given in WHO/HTM/NTD/WHOPES/2009.4 9. STUDY DESIGN Experiment 1: Repellence Dose response testing-Establishment of Dose Response curve for 30% PMD Neo-lnnova repellent versus 20% DEET (formulation prepared in-house) Objective The specific aim of these test is to estimate dose-response and effective doses (EDs) of the 30% PMD Neo-Innova repellent and a 20% DEET formulation corresponding to 50% (ED50) and 90% (ED90) protection from mosquito landing and/or probing compared to the number of mosquito landings on an un-treated control arm.

Testing of repellents on human subjects is the method of choice as it utilizes the repellent end-user in the testing process and yields results that are relevant to the actual conditions of use.

General Methodology 1. Tests are carried out on adult human volunteers who may be selected from among candidates exhibiting mild or no sensitivity to mosquito bites.

2. Equal numbers of male and female test volunteers are preferred. (10 nos) 3. In preparation for the laboratory studies, the test area of the volunteer's skin would be cleaned with unscented soap and rinsed with water, then rinsed with a solution of 70% ethanol or isopropyl alcohol in water and dried with a towel.

4. Standardized mosquito rearing and laboratory testing conditions are ensured to obtain reliability and reproducibility of data.

5. Mosquitoes will be reared, maintained and tested (in a separate space or room) at 27 ± 2 C temperature, E30 ± 10% relative humidity, and a 12:12 (light: dark) photoperiod.

6. Observations of repellency would be made using female mosquitoes starved for the preceding 12 hours 7. Mosquito repellency tests would be conducted with anthropophilic Aedes aegypti 8. Mosquitoes will be contained during testing using a cage (1 meter per side) with a solid bottom and top, screen or netting on the back, a clear acrylic sheet (for viewing) on the right and left sides, and a fabric sleeve for access on the front.

9. Female mosquitoes would be collected from a stock population cage in which both sexes have been maintained to allow mating to occur. They would be host-seeking, of uniform age, preferably 5-7 days post-emergence. Active host-seeking females be selected to ensure a good response from the test mosquitoes using an aspirator or an appropriate airflow apparatus.

10. Repellent product will be tested to identify an effective dose range. Dosages giving responses between 10% and 100% are used for this analysis, preferably 2-3 dosages that give <50% repellent response and 2-3 dosages that give >50% response.

11. Each volunteer uses incremental doses on the test forearm ranging from 0.05 g/600 sq.cm to 1 g/600 sq cm. A dose that achieves 100% repellence is used to determine the decay profile and half-life of the product.

12. Methodology: Before insertion of the arm into the cage containing 100 female mosquitoes, the hands are protected by gloves made of material through which the mosquitoes cannot bite. The first step is to insert the control forearm into the cage and to count the number of mosquitoes that land on and/or commence to probe the skin during a 30-second period. During testing, the volunteer should avoid movement of the arm. For the test to proceed, the biting rate must be a10 landings and/or probing in the 30-second period. The control arm of the volunteer would then be removed from the cage before mosquitoes had the chance to bite. The other arm (test arm) treated with the lowest dose of repellent per 600 sq.cm would then be introduced into the test cage. The treated arm is placed in the cage for a 30-second period and observed for mosquito landings and/or probing. After counting the number of mosquitoes, the arm would be retrieved from the cage, and the next incremental dose which would be applied on top of the last one to achieve the incremental dose as given in the testing plan (table below) would be applied on the test arm using a micropipette to dispense the formulation on the volunteer's arm. This procedure would be repeated until the lowest dose which gives 100% repellency is achieved. After this the control arm would be reintroduced and the number of mosquitoes landing/probing would be counted again to account for any change in activity. Successive tests should be carried out one after the other without delay (preferably in one day). At any time, in the test or control arm, if the landing and/or probing rate is too high to accurately count the number of mosquitoes landing and/or commencing to probe the skin or biting commences, the mean landing and/or probing rate for the test would be calculated from a series of three readings, each five seconds long, and the sum multiplied by two to estimate the landings and/or probings that would occur in a 30-second period.

Experiment 2 A further experiment was carried out to determine the ED95 value of the above prolonged action Neo-Innova Healthcare (NIH) mosquito repellent formulation containing 30% w/v PMD (and 5% vanillin) and compare the results with a 20% w/v DEET formulation prepared in-house.

This test was carried out with 10 volunteers under the same test conditions as Experiment 1 above. Incremental doses of repellent were applied until first landing, and the ED95 was calculated by Probit analysis. Final dose was at first landing left on arm, with reintroduction to cage every hour and the landings counted. We compared landings on a control with no repellent, and calculated repellence and decay with time.

Decay Curve for PMD and 20% DEET formulations: Leave the final 100% repellence dose on the test arm and check for its repellence after 30 minutes by counting landing/probing for 30 secs. Place un-treated arm in cage and record exact number of bites and landings for 30 secs and then withdraw. Place test arm in cage and record exact number of bites and landings for 30 seconds on test arm after another 30 minutes, and compare that reading with the control arm. This procedure will repeat after every 30 minutes intervals and the number of mosquitoes landing and/or commencing to probe the skin would be recorded in the data collection sheet for up to 7 hours.

The trained volunteer will record the number of landings and/or probing.

Protection (p) is expressed as a proportion of the number of mosquito landings and/or probings on the treated arm (T) in relation to the number of landings and/or probing on the control arm (C) of the same individual: p = 1 -(T / C) = (C -T) / C where C is the average of the landings/probings on untreated arms.

Results Fig 1 shows results obtained for DEET by Buescher in 1983, using different dose applications in mg/cm2. In the present study, similar results for DEET (20%) were obtained and these were analysed as described below for PMD. Regression analysis was performed (as for PMD) and the DEFT results are shown in Fig 3. These results were also extrapolated for a higher concentration of DEFT (50% by weight of the composition, in a standard ethanolic solution) and these results are shown in Fig 4.

Fig 2 shows the results obtained for a 30% PMD / 5% vanillin PMD-XL formulation as provided by the present inventor. It can be seen from the results in Figure 2 that the PMD-XL formulation provides excellent protection (i.e. repellency of around 8 hours or above) even at doses around or below 1mg/ cm2. It can also be appreciated that in the application dose range of 1 mg/ cm2 and above towards the WHO recommended level of 1.67 mg/ cm2, that the PMD-XL formulation provides increasing levels of protection well above 8 hours CPT, with doses of around 1.2 mg/ cm2 providing at least 10 hours protection. The level of protection shows the characteristics of an S-shaped curve, which eventually saturates, in contrast to DEFT which shows different (logarithmic) kinetics (Figs 1, 3 and 4).Previous results (also carried out at Ross Lifesciences, India) have shown that the complete protection time (CPT) obtained for a proprietary 20% DEET product applied at an application dose of 1.67 mg/ cm2 (i.e. the WHO recommended level) is around 5.7 hours. By contrast, the present results show that 30% PMD-XL provides a CPT of around 10.25 hours at an application dose of 1.67 mg/ cm2. That is, the CPT provided by PMD-XL is nearly twice that provided by DEET at the recommended application dose of 1.67 mg/ cm2. The current comparative tests, which employed 20% DEET made up in a standard ethanolic solution, gave a slightly higher measure of CPT for DEET, but still showed very significant differences compared to the PMD-XL solution.

The results obtained have been modelled using non-linear regression analysis and this is shown in Figure 3 for the PMD-XL (30%) and DEET (20%) compositions. The equations found to best fit the results are shown to the right of the Figure. It can be seen that PMD-XL shows exponential behavior (an S-shaped curve), providing a steep active range over which the repellency protection provided rises quickly to high levels, even at low doses (i.e. well below 1.67 mg/cm2. This contrasts with the logarithmic behavior of DEET which shows a decreasing rate of rise in protection as the dose is increased. Both CPT and TFB (time to first bite) measures show about 8 hours protection at a dosing level of around 1mg/cm2. Levels of PMDXL at doses of 1.4 mg/m2 or below or at 1.2 mg/m2 or below show significantly higher performance -i.e. well beyond 8 hours protection. By contrast 20% DEET shows a CPT of only 6.7 hours (401 minutes) at the WHO dosing level of 1.67mg/m2.

These results have been extrapolated to show the predicted level of protection at higher concentrations of both PMD (45%, with 7.5% of the fixative, vanillin) and DEET (50%) (all by weight of the total formulation), and these results are shown in Fig 4. Regression analysis has been performed in the same way as for Fig 3. Again, the superior performance of PMD-XL particularly at lower dosing levels is apparent. Thus, around 8 hours protection is seen for PMD (45%) at a dosing level of between 0.6 and 0.7 mg/m2. By levels of around 1mg/m2, the performance is close to saturation, providing protection of well over 11 hours. It can also be seen that for all doses from a dose of around 0.65 mg/m2 upwards, the PMD (45%) formulation is superior to even a DEET composition comprising 50% DEET (by weight).

The results from Experiment 2 are shown in Figures 5, 6 and 7 where the standard pharmaceutical measures of ED95 (effective dose to achieve desired result in 95% of population), -11/2 (half-life) and Area under the Curve (AUC) are shown for the same 30% PMD / 5% vanillin composition and 20% DEET composition tested in Experiment 1.

The ED96 for the DEET and PMD-XL formulations were calculated as 0.13mg/cm2 and 0.36 mg/cm2 respectively. As shown in Fig 6, the half-life for the PMD-XL composition, in terms of repellency, was found to be significantly higher than for DEET (3.94 hours versus 2.85 hours).

Fig. 7 illustrates further evidence for the superiority of the PMD-XL formulation over DEET at the higher WHO recommended dose of 1.67 mg.cm2. It can be seen that the area under the curve (AUC) for PMD-XL is about 1.5 times that for DEET (9.35 versus 6.39), which indicates a more prolonged repellency effect for the PMD-XL composition.

Claims (64)

1. Claims 1. A method of providing protection against insects, the method comprising administering to the skin of a user an insect repellent composition comprising p-methane-3,8-diol, a fixative, and a delivery vehicle, wherein the composition is applied at a dose of less than 1.67mg/cm2.
2. 2. A method according to claim 1 wherein the composition is applied at a dose of less than 1.4mg/cm2.
3. 3. A method according to claim 1 or 2, wherein the composition is applied at a dose of less than 1.2mg/cm2.
4. 4. A method according to claim 1, 2 or 3 wherein the composition is applied at a dose in the range 0.6mg/cm2 to 1.4mg/cm2.
5. 5. A method according to any preceding claim wherein the composition is applied at a dose in the range 0.8mg/cm2 to 1.2 mg/cm2.
6. 6. A method according to any preceding claim wherein the p-methane-3,8-diol is present in an amount of at least 20% by weight of said composition.
7. 7. A method according to any preceding claim wherein the p-methane-3,8-diol is present in an amount of at least 25% by weight of said composition.
8. 8. A method according to any preceding claim wherein the p-methane-3,8-diol is present in an amount of at least 30% by weight of said composition.
9. 9. A method according to any preceding claim wherein the p-methane-3,8-diol is present in an amount of at least 40% by weight of said composition.
10. 10. A method according to any preceding claim wherein the composition comprises p-methane-3,8-diol as the sole insect repellent.
11. 11. A method according to any preceding claim wherein the insect repellent composition provides a complete protection time of at least 8 hours.
12. 12. A method according to any preceding claim wherein the composition provides a complete protection time of at least 10 hours.
13. 13. A method according to any preceding claim, wherein the composition further comprises an antioxidant, optionally wherein the antioxidant prevents discoloration of said fixative when said composition is stored at 54°C for 14 days.
14. 14. A method according to claim 13 wherein the antioxidant comprises a bisulphite salt, such as sodium bisulphite.
15. 15. A method according to claim 13 or 14, wherein the antioxidant is present in an amount of about 0.5% to about 1.5% by weight % of the composition.
16. 16. A method according to any preceding claim, wherein the fixative comprises a compound which can reversibly form an acetal with PMD, such as a compound comprising an aldehyde or keto-functional group.
17. 17. A method according to claim 16 wherein the fixative comprises a compound of the general formula RCHO or RCOR, wherein R is alkyl, aryl, or heteroaryl; or an aliphatic aldehyde with a carbon chain length of 14; or aromatic aldehyde, including aromatic compounds where the aldehyde group is not directly attached to the aromatic ring.
18. 18. A method according to any preceding claim wherein the fixative is present in an amount of about 3% to 15% by weight of said composition, optionally from 5% to 15% by weight of said composition.
19. 19. A method according to claim 18 wherein the fixative comprises vanillin, or a vanillin derivative such as ethyl vanillin.
20. 20. A method according to any preceding claim wherein the fixative comprises vanillin and the antioxidant, where present, prevents discoloration of said vanillin when said composition is stored at 54°C for 14 days.
21. 21. A method according to any preceding claim, wherein the dose is administered once or bi-daily.
22. 22. A method according to any preceding claim, for the prevention of a disease state selected from the group consisting of: Lyme disease, disease states caused by Aedes Aegypti, ticks, Dengue fever, Yellow fever, and the Zika virus.
23. 23. A method according to any preceding claim, for the prevention of a disease state caused by the Culex mosquito, or the West Nile virus.
24. 24. A method according to any preceding claim, for repelling the Scottish Highland midge (Meanbh-chuileag).
25. 25. A method according to any preceding claim, wherein the delivery vehicle comprises a pharmaceutically acceptable aqueous solvent.
26. 26. A method according to claim 25, wherein the delivery vehicle comprises a pharmaceutically acceptable alcohol, such as isopropyl alcohol.
27. 27. A method according to any preceding claim, wherein the insect repellent composition comprises p-methane-3,8-diol, vanillin, sodium bisulfite, water and isopropyl alcohol.
28. 28. A method according to claim 25, 26, or 27, wherein the solvent, which is optionally isopropyl alcohol, is present in an amount of about 40% by weight of said composition.
29. 29. A method according to any preceding claim, wherein the insect repellent composition is in the form of a spray, lotion, gel or roll-on.
30. 30. A method of providing protection against insects, the method comprising impregnating an article of manufacture, such as a mosquito net or a dermal wipe, with an insect repellent composition comprising p-methane-3,8-diol, a fixative, and a delivery vehicle, wherein the composition is applied to the said article at a dose of less than 1.67mg/cm2.
31. 31. A method according to claim 30, wherein the dose application or the insect repellent composition or the disease state is as defined in any one of claims 2 to 29.
32. 32. A method of preventing a disease state selected from the group consisting of: Lyme disease, disease states caused by a biting fly, disease states caused by Aedes Aegypti, ticks, Dengue fever, Yellow fever and the Zika virus; or for repelling a biting fly, which method comprises administering to the skin of a user a composition as defined in any of claims 1 to 29 at a dose of less than 1.67mg/cm2.
33. 33. A method according to claim 32, wherein the composition is applied at a dose of less than 1.4mg/cm2, or at a dose of less than 1.2mg/cm2, or at a dose in the range 0.6mg/cm2 to 1.4mg/cm2, or at a dose in the range 0.8mg/cm2 to 1.2 mg/cm2.
34. 34. A method according to claim 32 or 33, for the prevention of a disease state selected from the group consisting of: disease states caused by the Culex mosquito, and West Nile virus.
35. 35. A method according to claim 32 or 33, for repelling the Scottish Highland midge (Meanbhchuileag).
36. 36. An insect repellent composition comprising p-methane-3,8-diol in an amount of 30% or more by weight of the composition, a fixative wherein the ratio of PMD: fixative (by weight) is in the range of 3:1 to 10:1, and a delivery vehicle, wherein the composition does not comprise any alcohol.
37. 37. A composition according to claim 36, which does not comprise any water.
38. 38. A composition according to claim 36 or 37, comprising p-methane-3,8-diol in an amount of 40% or more by weight of the composition.
39. 39. A composition according to claim 36, 37 or 38, comprising p-methane-3,8-diol in an amount of 50% or more by weight of the composition.
40. 40. A composition according to any one of claims 36 to 39, comprising p-methane-3,8-diol in an amount of 60% or more by weight of the composition.
41. 41. A composition according to any one of claims 36 to 40, wherein the ratio of PMD: fixative (by weight) is in the range of 4:1 to 8:1.
42. 42. A composition according to any one of claims 36 to 41, wherein the ratio of PMD: fixative (by weight) is in the range of 5:1 to 7:1.
43. 43. A composition according to any one of claims 36 to 42, wherein the ratio of PMD: fixative (by weight) is about 6:1.
44. 44. A composition according to any one of claims 36 to 43, wherein the fixative comprises vanillin.
45. 45. A composition according to any one of claims 36 to 44, wherein the composition comprises p-methane-3,8-diol as the sole insect repellent.
46. 46. A composition according to any one of claims 36 to 45 wherein the insect repellent composition provides a complete protection time of at least 8 hours.
47. 47. A composition according to any one of claims 36 to 46, wherein the composition provides a complete protection time of at least 10 hours.
48. 48. A composition according to any one of claims 36 to 47, wherein the fixative comprises a compound which can reversibly form an acetal with PMD, such as a compound comprising an aldehyde or keto-functional group.
49. 49. A composition according to any one of claims 36 to 48, wherein the fixative comprises a compound of the general formula RCHO, or RCOR wherein R is alkyl, aryl, or heteroaryl; or is an aliphatic aldehydes with a carbon chain length of 14; or an aromatic aldehydes including aromatic compounds where the aldehyde group is not directly attached to the aromatic ring.
50. 50. A composition according to any one of claims 36 to 49, wherein the fixative is present in an amount of about 3% to 15% by weight of said composition, optionally from 5% to 15% by weight of said composition.
51. 51. A composition according to any one of claims 36 to 50, wherein the fixative comprises vanillin, or a vanillin derivative such as ethyl vanillin.
52. 52. A composition according to any one of claims 36 to 51, wherein the formulation is in the form of a gel, a cream, a lotion or is a composition suitable for application to a wipe such as a dermal wipe.
53. 53. A composition according to any one of claims 36 to 52, wherein the delivery vehicle comprises a thickening agent or a thixotropic agent, or both.
54. 54. A composition according to claim 53, wherein the delivery vehicle comprises fumed silica.
55. 55. An insect repellent composition comprising p-methane-3,8-diol in an amount of 40% or more by weight of the composition, vanillin wherein the ratio of PMD: vanillin (by weight) is in the range of 5:1 to 7:1, and a delivery vehicle comprising fumed silica, wherein the composition does not comprise any alcohol or water, and wherein the composition is in the form of a gel, a cream, a lotion or is a formulation suitable for application to a wipe such as a dermal wipe.56. A composition according to any one of claims 36 to 53, wherein the composition further comprises an antioxidant, optionally wherein the antioxidant prevents discoloration of said fixative when said composition is stored at 54°C for 14 days.55. A composition according to any one of claims 36 to 54, wherein the antioxidant comprises a bisulphite salt, such as sodium bisulphite.
56. 56. A composition according to any one of claims 36 to 55, wherein the antioxidant is present in an amount of about 0.05% to about 1.5% by weight % of the composition.
57. 57. A composition according to any one of claims 36 to 56, wherein the fixative comprises vanillin and the antioxidant, where present, prevents discoloration of said vanillin when said composition is stored at 54°C for 14 days.
58. 58. A composition according to any one of claims 36 to 57, for once or bi-daily administration.
59. 59. A composition according to any one of claims 36 to 58, for the prevention of a disease state selected from the group consisting of: Lyme disease, disease states caused by a biting fly, disease states caused by Aedes Aegypti, ticks, Dengue fever, Yellow fever and the Zika virus; or for repelling a biting fly.
60. 60. A composition according to any one of claims 36 to 58, for the prevention of a disease state selected from the group consisting of: disease states caused by the Culex mosquito, and West Nile virus.
61. 61. A composition according to any one of claims 36 to 58, for repelling the Scottish Highland midge (Meanhh-chuileag).
62. 62. A method of preventing a disease state selected from the group consisting of disease states caused by a biting fly, disease states caused by Aedes Aegypti, ticks, Dengue fever, Yellow fever and the Zika virus; or of repelling a biting fly, which method comprises administering to the skin of a user a composition according to any one of claims 36 to 58.
63. 63. A method according to claim 62, for the prevention of a disease state selected from the group consisting of: disease states caused by the Culex mosquito, and West Nile virus.
64. 64. A method according to claim 63, for repelling the Scottish Highland midge (Meanbh-Chuileag).