WO2002005644A1

WO2002005644A1 - Compositions against blood sucking or biting insects comprising insulin and/or insulin like substances

Info

Publication number: WO2002005644A1
Application number: PCT/SE2001/001649
Authority: WO
Inventors: Lars Wahlberg; Bengt Furberg; Jan MÅRDH
Original assignee: Lars Wahlberg; Bengt Furberg; Maardh Jan
Priority date: 2000-07-18
Filing date: 2001-07-18
Publication date: 2002-01-24
Also published as: AU2001271223A1; SE0002716D0; SE0002716L

Abstract

The invention relates to the use of insulin or insulin like substances and degradation products thereof for the preparation of a composition against blood sucking or biting insects. The invention also relates to a composition against blood sucking or biting insects comprising insulin and/or insulin like substances and/or degradation products in an amount sufficient for repelling or preventing the insects from sticking or biting. The invention may be used against mosquitoes, flies, ticks, fleas, lice and ants.

Description

Compositions against blood sucking or biting insects comprising insulin and/or insulin like substances.

The present invention relates to the use of insulin or insulin like substances and degradation products thereof for the preparation of a composition against blood sucking or biting insects and to compositions comprising such substances

Despite significant advances in the techniques used for its control during recent decades, the mosquito continues to pose serious public health problems. In addition to the persistent irritation they cause humans and animals simply by virtue of their bloodsucking behaviour and the itching this causes, mosquitoes are also the principal vector of a variety of serious diseases, including malaria, yellow fever, dengue, and encephalitis.

Forty percent of the world's population lives in regions where malaria is endemic. According to WHO, malaria affects 500 million people annually and kills approximately 2.7 million humans each year solely as a result of malaria transmitted by mosquitoes. The world- wide incidence of the disease has increased, and resistance to available drugs for prevention and treatment is growing rapidly.

Although substantial need has existed for products which control or eradicate these insects, prior art attempts have failed to provide effective formulations which are capable of fully eradicating or controlling insects, while also being non-toxic to humans and animals.

In an attempt to meet the consumer demand for products of this nature, various pesticides, insecticides and insect repellent formulations have been developed. However, these prior art formulations typically incorporate active ingredients, which are highly toxic to humans and to many animals. Consequently, the usable concentration of these toxic chemicals must be reduced, typically to the point of rendering the resulting formulation ineffective in providing the desired killing or repelling effect. Another problem that exists with prior art pesticides is the ability of the insects to become resistant or immune to the pesticide. Due to the highly adaptive nature of most insects and the manner in which pesticides typically function, many prior art insecticides are effective initially, but lose their effectiveness over time due to insect developed immunity or resistance.

One most well known organic pesticide which has been developed and which had been particularly effective is dichlorodiphenyltrichloroethane (DDT). Although this compound was found to have extraordinary insecticidal properties and had been widely used as one of the most effective pesticides, its use has now been banned in the United States and in other countries due to its more recently discovered toxicity to humans and animals.

Following the discovery of the efficacy of DDT, numerous other organochlorides, such as cyclodienes were developed as practical pesticides, with further discoveries revealing organophosphate compounds as highly effective pesticides. However, as use of these chemical compounds grew, the deleterious effects of these compounds were also discovered and their use was restricted.

While other attempts have been made to eliminate the toxicity of pesticides or develop non-toxic pesticides, these attempts have failed to provide a completely effective, non- toxic pesticide. In particular, most of these prior art pesticides are non-biodegradable, causing the pesticide to continuously build up in the soil and, subsequently, in the food chain. This long-term build up has been found to be particularly hazardous to the long- term health of the population. As a result, use of many such pesticides has been either banned or severely restricted by recent legislation.

Although the need for an effective, non-toxic, biodegradable pesticide has existed for decades, an effective pesticide has not been realised. As an alternative, non-chemical methods have been used. However, little success has been attained with non-chemical methods. As a result, in addition to these non-chemical methods, pesticides continue to be used in limited concentrations with reduced efficacy.

Another groups of pesticides that have been employed in the prior art comprise pyrethroids or pyrethrins. Compounds, coming within this classification, have been widely used in a wide variety of insecticides and insect repellent products. However, although originally effective, the pyrethroids are examples of pesticides, which are now increasingly ineffective, due to acquired resistance by the insects. Presently, many insects have developed resistance or immunity to the insecticidal effect of the pyrethroids and, as a result, these compounds are increasingly unusable for effectively controlling undesirable insects and pests.

Another repellent agent is N,N-diethyl-m-toluamid, the active substance in the marketed products Off, US 622 and jungle oil. However it smells badly and irritates skin and eyes.

Also volatile oils have been used such as oils from roses, geranium and lavender to minimise the area that needs to be covered by the repellent agent.

It has now turned out that insulin or insulin like products or the degradation products thereof prevent biting, sticking and blood-sucking insects from attacking the human or animal body.

The invention therefore relates to the use of insulin or insulin like substances and degradation products thereof for the preparation of a composition against any sticking behaviour such as biting, sticking or blood sucking insects. By "insulin like substances" is meant any substance with different chemical structure or slightly different chemical structure and degradation products or parts thereof that have the effect of insulin and that has an effect against the above mentioned insects. Also functional analogues of insulin may be used. Moreover, any substance that is involved in or influences the mechanism of action of insulin such as any intermediate, by-product, secondary product, spin-off product or end product of the metabolic pathway of insulin and that inhibit the insects mentioned above may be used according to the invention.

The use of insulin like substances as a repellent agents for certain insects has the advantage that the substances are not toxic to humans or animals and do not irritate the skin. Moreover, the substances do not produce any toxic degradation products.

Insects that can be treated according to the invention may belong to any group of insects that bites such as flies, e.g. horse fly and gad fly; ants; or any group of insects that comes into contact with the blood system and sticks or bites the host skin and/ or sucks blood such as ticks, fleas and lice.

Insulin or any insulin like substances that prevent insects from sticking or biting may be used according to the invention. The practitioner may easily test if a substance has such an effect by performing the test described in the following examples 1-6 or according to a mosquito Repellent Testing Protocol established for products in lotion, pump, aerosol, liquid, and cream formulations (see Gerberg et at. 1994. Manual for Mosquito Rearing and Experimental Techniques. AMCA Bulletin No. 5). Briefly this is made as follows.

GENERAL CONSIDERATIONS Number of test subjects:

• Three

• Test subjects should avoid alcohol, caffeine, and fragrance products for 12 hours before testing

Test area size and preparation:

• Test area: one forearm, from the wrist to the elbow

• (a) wash forearm with unscented soap

(b) rinse with water

(c) dry

• Calculate and record surface area of forearm to be treated for each subject

• Protect hand from mosquito bites with vinyl, latex, or plastic glove • Test subjects are to avoid exertion during test (to minimize perspiration), as well as abrasion, rubbing, touching, or wetting of treated area

Amount of repellent applied:

• 1 ml of repellent material per 650 cm² of test area.

MOSQUITOES

Species tested:

• Aedes aegypti (yellow fever mosquito)

• Anopheles quadrimaculatus (North American vector of malaria)

• Culex quinquefasciatus (southern house mosquito)

Stage, age, sex

• Female mosquitoes, 5 to 8 days old, 200 per test cage

• Reared under optimal conditions for each species (see Gerberg et al. 1994. Manual for Mosquito Rearing and Experimental Techniques. AMCA Bulletin No. 5)

TEST CAGES AND TESTING ENVIRONMENT:

• Test cages are approximately 14 x 14 x 14 inches with clear acrylic panels (for observation) on right and left sides; a sheet aluminum bottom, screen (16 mesh) on back and top; cotton stockinette access sleeve on front

• Maintain room temp, and RH at 22° to 27° C and 50% to 80%, respectively

Controls

• Record the number of landing/probing mosquitoes on exposed skin of untreated forearm when inserted into test cage for 5-10 seconds

Exposure period

• 30 minutes after treatment, insert forearm into cage for 3 minutes

• Repeat exposure at 60 minute intervals until first land/probe recorded up to a maximum of 8 hours

Mature insulin consists of two polypeptide chains, A and B, joined in a specific manner. However, the initial protein product of the insulin gene in .beta.-cells is not insulin, but preproinsulin. This precursor differs from mature insulin in two ways. Firstly, it has a so-called N-terminal "signal" or "pre" sequence which directs the polypeptide to the rough endoplasmic reticulum, where it is proteolytically processed.

The product, proinsulin, still contains an additional connecting peptide between the A and B Chains, known as the C-peptide, which permits correct folding of the whole molecule. Proinsulin is then transported to the Golgi apparatus, where enzymatic removal of the C-peptide begins. The processing is completed in the so-called secretory granules, which bud off from the Golgi, travel to, and fuse with, the plasma membrane, thus releasing the mature hormone. Any type of insulin precursors such as preproinsulin or proinsulin may be used according to the invention. Such insulin precursors are described e.g.in USP 5,202,415.

Insulin purified from other non-human species may also be used such as domestic animals, e.g. oxen, pigs, dog, rabbit and sheep se e.g. USP 4,677,192 and USP 5,245,008. The insulins from certain domestic animals are very similar in structure to human insulin, and may be converted into human insulin by semisynthetic procedures as described by Morihara et al., Nature 280 (1979), 412-413 and Marcussen (U.S. Pat. No. 4,343,898).

Such insulin may exist in an equilibrium between monomeric, dimeric, tetrameric, hexameric and even polymeric insulin. The invention also relates to any polymeric forms of insulin and iOnsulin like substances.

The invention also relates to analogues of the insulin molecule obtained by insertions, deletions, additions and substitution of one or more amino acids in the insulin molecule as described in WO 86/05497, European Patent Application No. 214,826, USP 5,716,927, USP 4,992,418 and USP 4,992,417, USP 5,164,366.

According to the invention insulin produced synthetically or by recombinant technology may be used e.g. as described in USP 5.015.575.

The invention also relates to derivatives of insulin e.g. as described in USP 5,55,506,202 such as salts described in USP 5,430,016 and substitution derivatives described in USP 3,950,517.

Degradation products of insulin or insulin like substances may also be used according to the invention such as short peptides with insulin activity such as bombyxins "Bombyxin, an insulin-related peptide of insects, reduces the major storage carbohydrates in the silk-worm Bombyx mod" Comp Biochem Physiol B Biochem Mol Biol 1997 Oct; 118, or the peptides described in USP 5.461,035, USP 5.008.241 and USP 4,946,828.

Insulin products may produced as described above or bought on the marked such as Actrapid, Humulin, Humalog, Humulin, Mixtard, Insulatard Velosulin from Novo nordisk, Pharma AB Murmansgatan 126, Box 50587, 20215 Malmδ, Humalog, Humulin, Humulin NP, Humutard, Humulin Mix from Eli Lilly Sweden AB, Warfvings vag 25, Box 30037, 10425 Stockholm, Isuhuman Basal and -Comb, -Infusat and -Rapid, from Hoechst Marion Rousell AB, Bryggvagen 16-18, 117 68 Stockholm.

Other insulin like substances such as chloromethyl ketones may be used, such as phenylalanine chloromethyl ketone and leucine chloromethyl ketone.

The invention also relates to insulin like hormones such as insulin like neuro hormone and insulin-like growth factor-I (IGF-I) and IGF-II.

Insulin-like growth factors (IGFS) have been identified in various animal species as polypeptides that act to stimulate growth of cells in a variety of tissues (see Baxter et al., Comp. Biochem. Physiol. 91B:229-235 (1988); and Daughaday et al., Endocrine Rev. 10:68-91 (1989) for reviews), particularly during development (see D'Ercole, J. Devel. Physiol. 9:481-495 (1987) for review). The IGFs, each of which has a molecular weight of about 7,500 daltons, are chemically related to human proinsulin: i.e. they possess A and B domains that (1) are highly homologous to the corresponding domains of proinsulin, and (2) are connected by a smaller and unrelated C domain. A carboxyl-terminal extension, the D domain, is also present in IGFs but is not found in proinsulin. All publications mentioned herein are hereby incorporated by reference.

The insulin or insulin like substance according to the invention may be administrated in any form. Preferably the insulin is administrated topically. The active substances are dissolved or suspended in a cosmetically or pharmaceutically acceptable diluting agent such as water, saline, or in an organic solution such as an alcohol, acetonitrile, ethyl acetate, hexane and the like. The alcohol may be selected from primary, secondary or tertiary, non-aromatic, unsubstituted, saturated or unsaturated, branched or unbranched alcohols, and have an overall chain length of 2 to 16 carbons. In a particular embodiment, the alcohol is nonaromatic, unsubstituted, unbranched and has 2-5 carbon atoms, and in a more particular embodiment, the alcohol ethanol.

The composition may also contain cosmetically or pharmaceutically acceptable carriers or excipients.

The active substance may also be mixed into a cosmetically or pharmaceutically acceptable body, face or sun lotion such as Delial, solution ointment or paste.

The composition may also comprise other cosmetically or pharmaceutically acceptable insect repellent substances that may be allowed today or in the future.

Volatile oils may be added to the composition in order to spread the active ingredient around the body. They may be selected from the group consisting of: almond bitter oil, anise oil, basil oil, bay oil, caraway oil, cardamon oil, cedar oil celery oil, camomile oil, cinnamon oil, citronella oil, clove oil, coriander oil, cumin oil, dill oil, eucalyptus oil, fennel oil, ginger oil, grapefruit oil, lemon oil, line oil, mint oil, parsley oil, peppermint oil, pepper oil, rose oil, spearmint oil, menthol, sweet orange oil, thyme oil, turmeric oil, and oil of wintergreen.

The man of art may establish the amount of active substance to be dissolved or mixed with the diluting agent or carrier by performing tests according to the ones described in examples 1-6. When insulin or an insulin like compound is dissolved in water 0,001-100 IE of insulin or an insulin like compound per ml of the total composition may be used, preferably 0,01-1 and especially 0,1 -HE per ml.

The invention will no be described but not limited to the following examples.

Example 1. Preparation of a water solution of insulin.

Humalog, Lilly ( Eli Lilly Sweden AB, Warfvings vag 25. Stockholm), injection solution 100 E/ml (IE/ml) was fully emptied into 200 ml water. The solution was sprayed or applied all over the body of a test person, who then spent 30 minutes on mire, where there were thousands of mosquitoes.

The mosquitoes did settle on the body but never stuck during the test period.

Example 2. Preparation of a water solution of insulin and degradation products of insulin.

Humalog, Lilly ( Eli Lilly Sweden AB, Warfvings vag 25. Stockholm), injection solution 100 E/ml (IE/ml) was fully emptied into 200 ml water. The solution was left to stand for 24 hours of which 8 hours were in sunshine in order to degrade the insulin by the sunshine and the heat. Then it was sprayed or applied all over the body of a test person, who then spent 30 minutes on a mire, where there were thousands of mosquitoes.

The mosquitoes did settle on the body but never stuck during the test period.

Example 3. Preparation of a ethanol solution of insulin.

Humalog, Lilly ( Eli Lilly Sweden AB, Warfvings vag 25. Stockholm), injection solution 100 E/ml (IE/ml) was fully emptied into 200 ml ethanol 40% (vol/vol). The solution was sprayed or applied all over the body of a test person, who then spent 30 minutes on mire, where there were thousands of mosquitoes.

The mosquitoes did settle on the body but never stuck during the test period.

Example 4. Preparation of an ethanol solution of insulin and degradation products of insulin.

Humalog, Lilly ( Eli Lilly Sweden AB, Warfvings vag 25. Stockholm), injection solution 100 E/ml (IE/ml) was fully emptied into 200 ml ethanol. 40% (vol/vol The solution was left to stand for 24 hours of which 8 hours were in sun shine in order to degrade the insulin by the sun shine and the heat. Then it was sprayed or applied all over the body of a test person, who then spent 30 minutes on mire, where there were thousands of mosquitoes.

The mosquitoes did settle on the body but never stuck during the test period.

Example 5. Preparation of a sun lotion of insulin.

Humalog, Lilly ( Eli Lilly Sweden AB, Warfvings vag 25. Stockholm), injection solution 100 E/ml (IE/ml) was fully emptied into a teaspoon (15ml) of Delial sun lotion. The lotion was applied all over the body of a test person, who then spent 30 minutes on mire, where there were thousands of mosquitoes.

The mosquitoes did settle on the body but never stuck during the test period.

Example 6. Preparation of sun lotion of insulin and degradation products of insulin.

Humalog, Lilly ( Eli Lilly Sweden AB, Warfvings vag 25. Stockholm), injection solution 100 E/ml (IE/ml) was fully emptied into 200 ml water. The lotion was left to stand for 24 hours of which 8 hours were in sunshine in order to degrade the insulin by the sunshine and the heat. Then it was applied all over the body of a test person, who then spent 30 minutes on mire, where there were thousands of mosquitoes.

The mosquitoes did settle on the body but never stuck during the test period.

Claims

1. Use of insulin or insulin like substances and degradation products thereof for the preparation of a composition against blood sucking or biting insects.

2. Use according to claim 1, where the blood sucking or biting insects are selected from mosquitoes, flies, ticks, fleas, lice and ants.

3. Use according to anyone of claims 1 and 2, where the insulin like substance is a hormone, such as a neuro hormone; an insulin analogue, an insulin derivative

4. A composition against blood sucking or biting insects, characterised in that it comprises insulin and/or insulin like substances and/or degradation products in an amount sufficient for repelling or preventing the insects from sticking or biting.

5. A composition according to claim 4, characterised in that it comprises one or more other insect repellent agents.

6. A composition according to claim 4 or 5, characterised in that it comprises a diluent and or carrier and or an adjuvant

7. A composition according to anyone of claims 4-6, characterised in that it comprises 0,001-100 IE of insulin or insulin like compound per ml of the total composition, preferably 0,01-1 and especially 0,1- HE per ml.

8. A composition according to anyone of claims 4 to 7, characterised in that the diluent is water, saline or an organic solution such as an alcohol.