WO2005051365A1

WO2005051365A1 - Composition and device for facilitating venipuncture

Info

Publication number: WO2005051365A1
Application number: PCT/EP2004/013425
Authority: WO
Inventors: Carlo Ghisalberti; Mauro Marangoni
Original assignee: Medis S.R.L.
Priority date: 2003-11-27
Filing date: 2004-11-26
Publication date: 2005-06-09
Also published as: ITMI20032322A1

Abstract

A device for external use designed to improve the location of subcutaneous veins so as to facilitate the practice of venipuncture, which said device is based on substances able to highlight the venous system without damaging the skin application area.

Description

COMPOSITION AND DEVICE FOR FACILITATING VENIPUNCTURE

The invention relates to a device for external use designed to improve visualisation of the veins and facilitate venipuncture in infusion and blood sampling operations, which said device contains one or more substances that highlight the venous system and have no side effects, particularly with a significant survival of the epidermal nerve fibers (ENFs) in the area of application. The invention also relates to a method for highlighting and visualising the subcutaneous venoarterial structure, thus facilitating the performance of intravenous injection (i.v.), infusion and blood sampling in subjects in whom the injection site cannot easily be located using substances with a low toxicological impact. Difficulty in locating the vein at the time of venipuncture is common, and is due to the characteristics of the circulatory and muscle structure and the skin structure of each individual. A venous system that is hard to locate from the outside is found in subjects who have poor muscle tone and/or are overweight and/or have a thick skin, which may be subject to natural aging processes or aging processes accelerated by environmental factors (typically "skin photoaging"). The combination of these factors can produce a subcutaneous vein lattice that is particularly difficult to visualise, even using a tourniquet, which increases the local vein pressure. Under these conditions the operator is in difficulty and can cause problems to the patient subjected to venipuncture during blood sampling, injection of drugs, parenteral nutrition or the administration of various substances (e.g. contrast media). Low visibility can lead to clumsy attempts to find a vein, with the result that the operation has to be repeated several times, with the risk of dispersing the liquid (in the case of i.v. injection) or damaging tissues, nerve connections, blood vessels and capillaries. This problem is obviously felt most by patients who undergo repeated venipuncture sessions, such as cancer patients who undergo intensive treatments with oncological drugs and drips, and have numerous blood samples taken to monitor the progress (or regression) of the disease. Another example is patients suffering from haemoglobin disorders such as major thalassaemia, who undergo blood sampling to determine blood compatibility and periodic infusions of blood to compensate for the defective erythropoiesis. In practice, numerous categories of chronic and acute patients require numerous venipuncture operations, and locating veins can present a problem. Numerous patients with a low frequency of venipuncture can also present this problem, which mainly affects the operators, but also the patients. The practice of intravenous autoinfusion, performed by patients themselves, can be particularly critical when this difficulty arises. In our experience this need is largely unmet, as no satisfactory solution has been found to date which is practical and easy to use for operators and patients alike. Devices based on the method of "transilluminating" the skin with red and infrared light, already marketed as Venlite™ by Traslite (Sugar Land, TX, USA) or under study, for example in the US Air Force laboratories, involve expensive, complex apparatus, which is generally usable for the exploratory stage of surgery, phlebotomy, sclerotherapy, etc.. A device for venipuncture, Agaven™, by Regard s.a.s. (Bagnocavallo,

RA, Italy), seems to meet this need, at least to some extent. This device, disclosed in patents IT 1234578 and IT 1249406, substantially consists of a hydroalcoholic solution containing a fluid extract of capsicum. In addition to the capsicum extract, the device contains a combination of other natural extracts which potentially improve the required performance. The additional benefits claimed include an anaesthetising action and a disinfectant action by ethyl alcohol. However, we have found that in current practice, a number of patients present low tolerance to the action of capsaicin, the active constituent of capsicum. These patients experience a prolonged stinging sensation. Moreover, as extensively documented (Simone, D.A, et al., J. Neuros., 18(21): 8947-8959, 1998), inhibition of peripheral nociceptive activity can last for a long time, up to 6 months after application, so repeated use can lead to gradual desensitisation of the area. Moreover, in addition to chronic nociceptive desensitisation, capsaicin cause apoptosis (programmed cell death) in the cells of the nervous system, which lead to irreversible desensitisation (Sugimoto T. et al., Brain Research, 807: 147-154, 1998) by the destruction of the epidermal nerve fibers (ENFs) (Nolano et al., Pain 81 : 135- 145, 1998). We therefore felt the need to develop a device for external use designed to improve the visibility of the veins in order to facilitate venipuncture in i.v. infusion operations or blood sampling, which said device contains substances designed to highlight the venous system without the destruction of epidermal nerve fibers (ENFs) in the treated area. The invention therefore relates to a device and a method for highlighting and visualising the subcutaneous venoarterial structure with the use of innocuous substances so as to facilitate the injection practice of venipuncture. The device according to the invention consists of the following components: a) one or more substances with a selective vasodilation and/or vasoactive and/or local muscle-relaxant and/or astringent and/or local muscle-relaxant effect, b) a fluid vehicle able to dissolve/disperse substances (a), and possibly other dermatologically acceptable ingredients; c) an apparatus able to apply and cause the local action of an effective quantity of (a)+(b) in the area destined for venipuncture; wherein substances (a) are characterised by a low risk of toxicity, irritation and chronic desensitisation behaviour in the application area and, more particularly, by non-neurotoxic effect as expressed as epidermal nerve fibers (ENFs) survival value Δ^ENFs < 85% after 2 week of repeated applications. Solution or dispersion (a)+(b) may take various liquid and fluid forms, including hydroalcoholic solutions, gels, emulsions, creams, ointments, etc.. These substances can be contained in a dispensing container. In this case the device is called a "two-component" system, because (c) consists of a dispensing container which is separate from the application/rubbing medium. The dispensing container may be of various types, such as a spray container, bottle with dropper, bottle of flexible material from which the contents exit under pressure, jar, tube, or a any other container which allows application to the skin and/or to the application/rubbing medium of a quantity of (a)+(b) generally ranging between 0.3 and 3 ml. The application/rubbing medium could be enclosed in the pack or available at the user's premises. For example, it could be a tissue, a sponge made of non-toxic elastomer material, a non-woven material (such as cotton wool) or another medium which allows the application of (a)+(b) for rubbing on the area chosen for venipuncture, such as a swab on a stick (cotton wool buds or the like), or other similar means which ensure close contact between the skin and (a)+(b). Alternatively the device could be a "one-component system", wherein the application/rubbing medium is embedded with solution/dispersion (a)+(b). Said means could be a woven cloth, a sponge made of non-toxic elastomer material, a non-woven material (such as cotton wool) or other medium containing a quantity of (a)+(b) generally ranging between 0.3 and

3 ml which is immediately usable for rubbing onto the required area. In this context the medium will preferably be contained in monodose sachets or packaged in other sealed forms, such as (but not limited to)

"roll-on" sticks or other forms of ready-for-use packaging of solution/dispersion (a)+(b) which also prevents external contamination and loss of (a)+(b) due to evaporation. Fluid vehicle (b) could be constituted by physiological solvents such as DMSO, ethanol and hydroalcoholic mixtures able to dissolve substances (a). The quantity of fluid vehicle (b) ranges between 50% and 99.9%, and preferably between 80% and 95%, in weight. The use of physiological solvents with an anhidrotic action such as ethyl and isopropyl alcohol, which cause further skin transparency with a concomitant low sensitising and irritant effect, is particularly preferred. Fluid vehicle (b) can also be designed as a heterogeneous system, such as emulsions, suspensions, microemulsions, etc., and can benefit in this respect from the presence of cosmetic and dermatological ingredients that promote dispersion, with possible benefits in terms of applicability, increased penetration and increased efficacy. Substances (a) were identified by choosing from among drugs and plant extracts with a selective vasodilating, astringent, vasokinetic and local muscle-relaxant action. It was therefore necessary to identify active constituents which could perform these functions but entailed a low risk of chronic irritation/desensitisation and significant preservation of the epidermal nerve fibers (ENFs) in the application area. The function of vasokinetic agents (al) is to increase blood flow in the subcutaneous veins, which further increases their visibility. The presence of non-irritant vasodilators causes tumescence and an increase in the diameter of the vein, thus making it more visible. The astringent substances help to increase the transparency of the skin, providing a greater contrast with the underlying circulatory network. The function of the muscle-relaxant is to cause relaxation, enhancing vein dilation due to contraction of the musculocutaneous system. Substances (a) which are useful and devoid of irritant/desensitising and neurotoxic effects, were identified as the following categories: catechins, bioflavonoids, coumarins, monoterpenes, plant alkaloids, ginkgolides/ginsenosides, adrenergic alkaloids, xanthophylls, non-irritant vanilloids, polyunsaturated fatty acids, alphahydroxy acids and potassium salts. In particular, each of these classes of substances possesses a selective vasodilating, astringent, vasokinetic and local muscle-relaxant action to different extents and in different proportions, as illustrated by example 1 below. Examples for each of the categories cited include catechins such as tannic acid (of the ellagic or catechic type), gallic acid, oligomeric proanthocyanosides, catechin, epicatechin and the corresponding gallocatechins and gallate esters; bioflavonoids such as hesperidin, naringenin, rutin, troxerutin, diosgenin, myricetin, apigenin, quercetin, campherol and baicalein; coumarins such as khellin, visnadine, esculoside and esculetin; monoterpenes such as menthol, geraniine, beta- and alpha-pinene, camphene, beta-myrcene, limonene, cineol, camphor, linalol, bornyl acetate, terpinenol, and isoborneol monoterpenes; plant alkaloids such as vincamine, vincine, vincaminine, eburnamonine, vincinine, bulbocapnine, berbamine, berberastine and berberine, ginkgolides such as ginkgetin, and ginkgolides A, B and C; panax-ginsenosides such as ginsenosides RBI, RC, RE, RG and RG1; adrenergic alkaloids such as atropine, dopamine, raubasine, papaverine and histamine; xanthophylls such as pentoxyphylline, theobromine, theophylline, caffeine and pentifylline; polyunsaturated fatty acids such as ximeninic acid, gamma-linolenic acid, alpha-linoleic acid, gamma-homo-linolenic acid, docosahexaenoic acid, eicosapentaenoic acid and eicosahexaenoic acid; and alphahydroxy acids such as glycolic acid, d,l-lactic acid, malic acid, citric acid and tartaric acid. The above list comprises substances belonging to the class of metabolites of natural origin which present a low risk of irritant and toxic skin effects. In this context we should mention vanilloid receptor agonists (subsequently called "non-irritant vanilloids"), whose properties differ from those of capsaicin because they present no risk of irritation or irreversible desensitisation. Examples of non-irritant vanilloids include zingerone, olvanil and synthetic analogues, together with the natural vanilloid receptor agonists described by Sterner O and Szallasi A in Trends, Pharm. Sci., 2001, 20:459-

461, or the semi-synthetic vanilloids described by Appendino et al., J Med.

Chem., 2002 15:45(17):3739-45. Further examples of substances (a) are vasodilators with a rubescent effect such as nicotinic acid, its esters and alcohols, acetylcholine, resorcinol, pyroglutamic acid, reserpine, adenine, adenosine, arginine, cinnamic acid and aldehyde, cymarin, forscolin, esculoside, yohimbin and gamma-oryzanol. Synthetic vasoactive substances can be used according to this invention, provided that the dose is not such as to generate toxicity risks in topical use. Synthetic vasoactive substances (a) include: short- and long-acting alpha-blockers such as phenoxybenzamine, dibenamine, doxazosin, terazosin, phentolamine, tolazoline, prazosin, trimazosin, alfuzosin, tamsulosin and indoramin; ergot alkaloids such as ergotamine and ergotamine analogues such as acetergamine, brazergoline, bromerguride, cianergoline, delorgotrile, disulergine, ergonovine maleate, ergotamine tartrate, etisulergine, lergotrile, lysergide, mesulergine, metergoline, metergotamine, nicergoline, pergolide, propisergide, proterguride and terguride; vasodilators such as nimodepine, pinacidil, cyclandelate, dipyridamol and isoxsuprine; chlorpromazine; haloperidol; trazodone; prostaglandins such as prostaglandins El and E2, vasoactive amines such as histamine and serotonin, nitrates such as isosorbide dinitrate, erythritol tetranitrate, amyl nitrate, sodium nitroprusside, molsidomine, linsidomine hydrochloride and S-nitroso-N-acetyl-d,l- penicillamine, S-nitroso-N-cysteine and S-nitroso-N-glutathione and diazenium diolates, vasokinetics such as nonyl valeramide, cyclandelate, cinnarizine, tolazoline and cepharanthine; peripheral vasodilators such as buflomedil, butalamine, cetiedil, cyclonicate, cinnarizine, cyclandelate, diisopropylamine dichloroacetate, eledoisin, fenoxedil, phentolamine, flunarizine, ifenprodil, moxisylate, nafronyl, nicergoline, brovincamine, nicofuranose, nylidrin, piribedil, suloctidil, tolazoline and verapamil; nicotine derivatives such as methyl and benzyl nicotinate, nicotinic alcohol, beta- butoxyethyl nicotinate, ichthammol, tocopherol nicotinate, inositol hexanicotinate, etc.. The devices of the invention may advantageously include an homogeneous solution, usually hydroalcoholic, to be embedded into tissues or small sponges. Typical components and the corresponding quantitative ranges of said hydroalcoholic solutions are reported below: Menthol 0.5 - 2.5 g Eugenol 0.25 - 1 g Zingerone 0 - 2 g Visnadine 0 - 0.5 g L-Arginine 0 - 1 g Nicotinyl alcohol tartrate 0 - 0.25 g Caffeine 0 - 0.2 g Gallic acid 0 - 2 g Glycerol 2 - 4 g d,l-lactic acid 80% 2.5 - 7.5 g 65° ethanol, volume q.s. for 100 ml The hydroalcoholic solution can be suitably applied by an imbedded non-woven tissues, cotton wool ball, sponge or other device (c) suitable for local application to highlight the venous network under the skin. Fluid vehicle (b) can also include other dermatologically /cosmetically acceptable ingredients. These ingredients include oils and lipids, which can be combined with water by means of emulsifiers ("surfactants") to give O/W or W/O emulsions, depending on the hydrophilic/lipophilic balance (HLB) of the emulsifying system. The surfactants can be incorporated in quantities of between 0.5% and 30%, and preferably between 1% and 15%, in weight. Cationic, non-ionic, anionic and amphoteric surfactants and combinations thereof can be usefully employed. Non-ionic surfactants include alkoxylates of fatty acids and alcohols or of sorbitan, polyoxypropylene and polyoxyethylene, and alkyl polyglycosides; anionic surfactants include soaps of fatty acids, sodium lauryl sulphate or lauryl ether sulphate, alkyl benzene sulphonates, mono and/or dialkyl phosphates and the like; amphoteric surfactants include dialkylamine oxides, various types of betaine, phospholipids and natural ceramides. A thickener can be present in the quantity of 0.1 % to 10% of a composition with an aqueous base, and preferably 0.5% to 5%. Examples of thickeners are cross-linked polyacrylates (Carbopol ), and gums like xanthan, carrageenan, gelatin, karaya and pectin. The device in aqueous form with little or no alcohol must be protected against growth of micro-organisms with preservatives, for example with alkyl esters of p-hydroxybenzoic acid, hydantoins, parabens, imidazolidinyl urea, sodium dehydroacetate, benzyl alcohol, and a range of quaternary ammonium compounds, in the quantity up to 1% in weight or more. Anhydrous devices, can other contain other- than- water solvents, e.g. silicones, such as those with a viscosity ranging between 10 and 10⁷ centistokes at 25°C, and mixtures thereof, in the quantity of 5% to 95% in weight. Another category of functional ingredients which can be included in the device according to the invention is emollients. Under some circumstances emollients perform a dual function, acting both as vehicles (facilitating the dispersion of the ingredient according to the invention) and as skin softeners. Emollients can be incorporated in the device according to the invention in the quantity of 0.5% to 50% in weight. Emollients can be classified as fatty alcohols, esters and acids, polyols and hydrocarbons. Examples of fatty diesters are dibutyl adipate, diethyl sebacate, diisopropyl dimerate, propylene glycol myristyl ether acetate, diisopropyl adipate and dioctyl succinate. Examples of branched fatty esters are 2-ethyl-hexyl myristate, isopropyl stearate and isostearyl palmitate. Examples of fatty triesters are triisopropyl trilinoleate, trilauryl citrate, tributyrin and saturated and unsaturated vegetable oils. Examples of esters of fatty alcohols are lauryl palmitate, myristyl lactate, oleyl erucate, oleyl stearyl coco-caprylate/caprate, and cetyl octanoate. Examples of linear fatty alcohols are alcohols C10-C20 such as cetyl, myristyl, palmityl and stearyl alcohols. Examples of linear and branched polyols are polyhydroxyalkylated copolymers, such as propyl/butylene glycol, sorbitol glycerin, or pure polyols such as glycerine, polypropylene glycol and polyethylene glycol. Examples of hydrocarbons are linear hydrocarbons C12-C30, petrolatums, paraffin waxes and squalene. Other ingredients, including colorants and pigments, opacifiers, perfumes, etc., can also be incorporated in quantities of between 0.1% and 20% in weight. In addition to the substances indicated, the device according to the invention may contain substances allowed for cosmetic use, such as those included in the Annex to EU Directive 93/35/EEC, as amended. A wide range of devices according to the present invention can be made by methods known to the skilled practitioners. The following examples illustrate the invention. EXAMPLES Example 1 - Selection of active substances (a) by level and type of activity As substances (a) which are candidates for use in the invention possess one or more of the required activities, a simple use and visual/subjective evaluation test was conducted to establish the degree and type of action of said substances. For this purpose, one active constituent was taken as reference for each category. In particular zingerone was used for non-capsaicin vanilloids, tannic acid (tannin) from oak was used for catechins, neohesperidin was used for bioflavonoids, Amni visnaga extract was used for coumarins, menthol was used for monoterpenes, hydroalcoholic fluid extract of Vinca minor was used for plant alkaloids, dried extract of Ginkgo biloba was used for ginkgolides/ginsenosides, dopamine was used for adrenergic alkaloids, pentoxyphylline was used for xanthophylls, polyunsaturated fatty acids obtained from saponified grape pip oil were used for polyunsaturated fatty acids, lactic acid (racemate) was used for alphahydroxy acids, and potassium acetate was used for potassium salts. A 2% solution in 65° ethyl alcohol in volume is generally produced for each substance. The results are illustrated in Table I. TABLE I

Type of selective cutaneous vasokinetic muscle-relaxant substance vasodilation astringency action activity

Non-irritant vanilloids ** ***

Catechins *** **

Bioflavonoids ** ** ***

Coumarins ** ** *** **

Monoterpenes ** **

Plant alkaloids *** *** **

Ginkgolides/ginsenosides *** ** **

Adrenergic alkaloids ** ** **

Xanthophylls ** **

Polyunsaturated fatty acids **

Alpha-hydroxy acids *** ** Potassium salts *** **

Key: * = little or none * * = moderate * ** - high Example 2 - Two-component system with spray lotion and application with gauze A hydroalcoholic solution is prepared by dissolving the following ingredients: Gallic acid 2 g Ginkgo biloba dried extract 24% total ginsenosides 1 g 98%o hesperidin 0.5 g Nicotinyl tartrate 0.2 g Glycerin 3 g 65° ethanol volume q.s. for 100 ml The solution is then placed in a 100 ml PE bottle fitted with a dispenser with manual pressure valve, which sprays approx. 1 ml of solution in the venipuncture area. This is rubbed onto the skin with cotton gauze, with the effect of displaying the required vein. Example 3 - Two-component system with W/O emulsion and application with cotton wool An emulsion with a high inner phase is prepared by hot emulsifying the aqueous and oily phases of the following ingredients: Ginkgo biloba extract 12 g Adrenaline 0.2 g Fatty acids from grape pips 10 g Glycolic acid 5 g Glycerin 1 g 01eyl-(2)-POE 5 g Hydrogenated coconut oil 5 g Bentone 38 0.5 g CaS0₄ 7₂0 0.3 g Deionised water q.s. for 100 ml The emulsion is then placed in a 100 ml ceramic jar, and approx. 2 ml of cream is applied manually to the venipuncture area. The cream is then rubbed onto the skin with a sterile cotton wool ball, and has the effect of displaying the required vein. Example 4 - Two-component system with emulsion in tube and application with sponge An O/W emulsion is prepared by hot emulsifying of aqueous and oily phases of the following ingredients: Menthol 1.5 g Dopamine 0.2 g Periwinkle, fluid extract 2 g Cyclomethicone 2.0 g Cetearyl alcohol + PEG-40 hydrogenated castor oil + Na cetearyl sulphate 4.5 g Octyl stearate 3.0 g Castor oil 4.0 g Glycerin 3.0 g Carbopol 0.3 g Hydroxypropyl methylcellulose 0.3 g Sodium edetate 1.5 g Preservatives q.s. Deionised water q.s. for 100 ml The emulsion is then placed in a 100 ml flexible PE tube with nozzle, and approx. 2 ml of cream is squeezed onto the venipuncture area. The cream is rubbed onto the skin with a sponge, with the effect of displaying the required vein. Example 5 - One-component system with tissue embedded with alcoholic lotion A hydroalcoholic solution is prepared by dissolving the following ingredients: Tannic acid from Quercus robus 0.5 g Menthol i g Zingerone i g Periwinkle, fluid extract 2 g d,l-lactic acid, 80% in water 5 g Glycerin 2.5 g 65° ethanol volume q.s. for 100 ml 2 ml of the solution is placed in sachets containing a non-woven cellulose tissue. The sachets are then heat-sealed. At the time of application the sachets are opened and the tissue is rubbed on the skin, with the effect of displaying the required vein. Example 6 - One-component system with tissue embedded with alcoholic lotion A hydroalcoholic solution is prepared by dissolving the following ingredients: Gallic acid 1 g Terpene fraction from lemon 1 g Buflomedil 0.5 g Visnadine (Visnadex™, Indena, I) 1 g Adenosine 0.5 g Esculoside 0.5 g Glycerin 2 g 65° ethanol, volume q.s. for 100 ml 2 ml of the solution is placed in sachets containing a non-woven cellulose tissue. The sachets are then heat-sealed. At the time of application the sachets are opened and the tissue is rubbed on the skin, with the effect of displaying the required vein. Comparative example 1 - One-component system with tissue embedded with an alcoholic solution of capsaicin Agaven™ tissues (Regard s.a.s., Bagnocavallo, RA, Italy), packaged in heat-sealed sachets, contain the following ingredients: Extract of Formes officinalis 12 g Extract of Avena sativa 12 g Extract of Capsicum frutescens 10 g Extract of Cratageus oxyacantha 10 g Glycerin 1 g 70° ethanol volume q.s. for 100 ml At the time of application the sachets are opened and the tissue is rubbed on the skin, with the effect of displaying the required vein. Some of the patients treated experience severe stinging, which persists for a few hours after application. Example 7 - Test of cell survival on fibroblast culture to evaluate skin irritation potential and biocompatibility (MTT assay) The key reagent is 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide or MTT, a substance which gives a yellow colour in aqueous solution. The mitochondrial dehydrogenase of the viable cells splits the tetrazole ring, leading to the formation of purple-violet formazan crystals which are insoluble in water. The crystals are dissolved in acidified isopropanol, and the resulting violet solution is measured spectrophoto metrically. The test is conducted with the solution described in example 5. Under the test dilution conditions, the irritant power demonstrates the skin compatibility of the device according to the invention. Example 8 - Occlusive application test for clinical evaluation of irritant, allergenic and sensitising effects (patch test) An increase or reduction in viable cells leads to a concomitant change in the quantity of formazan formed, which can be considered as an indicator of the degree of cytotoxicity caused by exposure to irritant substances. This test enables the tolerability of a cosmetic product to be evaluated by identifying and classifying its potential irritant power, and at a preliminary level, identifying its potential sensitising power. The device will be applied to volunteers using "Finn Chambers", 7 mm diameter aluminium discs containing absorbent paper discs soaked in a known quantity of test sample. The skin area destined for the test (the skin of the back) is cleansed with a 70% alcohol solution, and the test product is then applied. Skin reactions will be evaluated 15 minutes, 1 and 24 hours after removal of the Finn Chambers. The test conducted using the device described in example 5 indicates a level of irritant and sensitising effects compatible with the use of the device. Example 9 - Assay on neurotoxicity safety by the epidermal nerve fibers (ENFs) survival The neurotoxicity safety of the device of the present invention and/or or the single component thereof in comparison with capsaicin-containing device are assessable by the method of Nolano et al. (Pain 81 : 135-145, 1998). Ten-healthy symptom-free volunteers enrolled after neurological examination of the peripheral nervous system and chack of the motor (ulnar, median, peroneal and tibial) and sensory (median and sural) nerve conduction normality are instruicted to apply the device of Example 7 (B) or the capsaicin-containing device (Agaven™, by Regard s.a.s., Bagnocavallo, RA, Italy) (C) four times daily for 2 weeks onto ea two separate 15 cm² area on the inner arm or volar forearm with vigorously applications. Skin blisters can be made by applying a suction capsule (W.R. Medical Electronics, Stillwater, MN) with double sided tape to cleansed, shaved skin. The surface of the capsule resting on skin contained a 3-mm diameter opening. The capsule can be secured to skin with an elastic bandage, the area warmed with a heating pad and the capsule evacuated to a negative pressure of 300 mm Hg. After a full blister had formed the capsule can be removed immediately to prevent overstretching. The blister roof can be excised with a microscissors, flattened between two microscope slides in a drop of cold Zamboni's fixative for approximately 10 min, placed in cold Zamboni's solution overnight, then cryoprotected with 20% sucrose in 0.1 M phosphate buffered saline (PBS) until processed. The whole blister roof can be frozen, then double immunostained to visualize ENFs using antibodies to the pan-neuronal marker protein gene product 9.5 (PGP 9.5, rabbit polyclonal) and to Langerhans cells using antibody to CD- la (mouse monoclonal) then reacted with secondary antibodies conjugated with cyanine 2, 3 or 5 fluorescent probes (Jackson Immunoresearch, West Grove, PA). Skin biopsies obtained with a 3-mm punch following local anesthesia lidocaine (1%) anesthesia can be fixed overnight in Zamboni's solution and then cryoprotected with 20% sucrose in 0.1 M PBS. Frozen sections, 100 mm thick, can processed for immunofluorescent localization of the antigens listed in Table 1 with cyanine 2, 3, and 5. Neural antigens included PGP 9.5, calcitonin gene related peptide (CGRP) and substance P (SP). Type IV collagen immunoreactivity (ir) can be used to identify basement membrane. Images were collected with a confocal microscope (BioRad, Boston,MA). Each image was comprised of a Z-series acquired in 2 mm increments throughout the thickness of the section. The number of ENFs in blisters can be counted using a x 20 objective with a Nikon Microphot-SA fluorescent microscope (Lake Success, NY). Five fields from each blister, each 0.40 x 0.30 mm, displayed on a Sony video monitor (San Jose, CA) and the ENFs were counted with the aid of a superimposed grid. Areas that contained tissue folds or defects from hair follicles should be avoided. ENFs in biopsies can be quantified as described (Kennedy, W.R., Wendelschafer-Crabb, G. and Johnson, T., Neurology, 47 (1996) 1042-1048.). Briefly, digitized confocal images of immunostained nerves in the epidermis can be traced using Neurolucida software (Micro-BrightField, Colchester, VT). Counts of nerve fibers in epidermis can be derived from the number of fibers crossing the dermal-epidermal basement membrane. ENFs can be expressed as fibers/mm length of section. The thickness and continuity of nerve bundles in the subepidermal plexus can be graded subjectively in four steps from normal to severely abnormal. Biopsy samples of skin obtained: (A) prior to treatment, and (B) with a capsaicin-free device of example 6 and, (C) with capsaicin-containing device of comparative example 1, can be examined. Results are summarized in Table II. TABLE II (A) (B) _ΔENFs_{(B) (c) A}ENFs_(c)

ENFs/mm²-blister 392 358 91.3% 111 28.3%

ENFs/mm biopsy 39 35 89.7% 22 56.4% (A) skin biopsy prior to treatment; (B) skin biopsy after 2 weeks of application of capsaicin-containing device; Δ^BNFs(B) percent survival of epidermal nerve fibers (ENFs) of (B) compared to control (A); (C) skin biopsy after 2 weeks of application of capsaicin-free device; Δ^ENFs(C) percent survival of epidermal nerve fibers (ENFs) of (C) compared to control (A). Samples (B) contained dermal PGP 9.5-ir bundles of unmyelinated nerves that ascended and branched to form an extensive subepidermal neural plexus (SNP) in the papillary dermis, before single nerve fibers crossed the dermal- epidermal junction to ascend in the epidermis which closely resembled those of samples (A). Substance P (SP) and calcitonin gene related peptide (CGRP) containing fibers were present in the nerve trunks and the SNP but seldom entered the epidermis. Examination of cutaneous innervation by immunolocalization of the PGP 9.5 of samples (C) revealed a striking loss of nerve fibers after 2 weeks. The most pronounced reduction of nerve fibers occurred in the epidermis with lesser involvement found deep to the dermal-epidermal junction. The epidermal nerve loss was clearly seen in blister preparations, and in biopsy sections. The latter included the SNP and dermal nerves. Under the test conditions, the demonstrates that the device according to the invention produce a neurotoxicity effect misurated on the epidermal nerve fibers (ENFs) survival which is consistent with the non-toxic behaviour required. More particularly the inventive device is devoid of significant neurotoxicity behaviour.

Claims

1. A device for external use designed to facilitate the practice of venipuncture by highlighting the subcutaneous venous system in subjects in whom it is difficult to locate the veins, said device comprising: a) one or more substances with a selective vasodilation and/or vasoactive and/or astringent and/or local muscle-relaxant effect, b) a fluid vehicle able to dissolve/disperse substances (a), and possibly other dermatologically acceptable ingredients; c) an apparatus for the application to the site of venipuncture of an effective quantity of (a)+(b); wherein substances (a) are characterised by a low risk of toxicity, irritation and chronic desensitisation behaviour in the application area as determined by non-neurotoxic effect expressed as epidermal nerve fibers (ENFs) survival value Δ^ENFs < 85% after 2 week of repeated applications.

2. Device as claimed in claim 1, wherein (a)+(b) are in the form of a hydroalcoholic solution, gel, emulsion, cream or ointment.

3. Device as claimed in claims 1 and 2, wherein (c) is a two-component system constituted by a dispensing container and by a separate application/rubbing system.

4. Device as claimed in claim 3, wherein the dispensing container is a spray container, bottle with dropper, jar, tube, or other container which allows application to the skin and/or to the application/rubbing medium of a quantity of (a)+(b) ranging between 0.3 and 3 ml.

5. Device as claimed in claims 1 and 2, wherein (c) is a one-component system wherein the application/rubbing medium is embedded with solution/dispersion (a)+(b).

6. Device as claimed in claim 4 wherein said medium is a tissue, a sponge made of non-toxic elastomer material, a non- woven material or other medium soaked in a quantity of (a)+(b) ranging between 0.3 and 3 ml.

7. Device as claimed in claim 5, wherein said medium is packaged in monodose sachets, "roll-on" sticks or other sealed ready-to-use forms.

8. Device as claimed in claim 1 where (a) is chosen from the group comprising catechins selected from tannic acid (of the ellagic or catechic type), gallic acid, oligomeric proanthocyanosides, catechin, epicatechin and the corresponding gallocatechins and gallate esters; bioflavonoids selected from hesperidin, naringenin, rutin, troxerutin, diosgenin, myricetin, apigenin, quercetin, campherol and baicalein; coumarins selected from khellin, visnadine, esculoside and esculetin; monoterpenes selected from menthol, geraniine, beta- and alpha-pinene, camphene, beta-myrcene, limonene, cineol, camphor, linalol, bornyl acetate, terpinenol and isoborneol monoterpenes; plant alkaloids selected from vincamine, vincine, vincaminine, eburnamonine, vincinine, bulbocapnine, berbamine, berberastine and berberine, ginkgolides such as ginkgetin, and ginkgolides A, B and C; panax-ginsenosides selected from ginsenosides RBI, RC, RE, RG and RG1; adrenergic alkaloids selected from atropine, dopamine, raubasine, papaverine and histamine; xanthophylls selected from pentoxyphylline, theobromine, theophylline, caffeine and pentifylline; polyunsaturated fatty acids selected from ximeninic acid, gamma- linolenic, alpha-linoleic, gamma-homo-linolenic, docosahexaenoic, eicosapentaenoic and eicosahexaenoic acids; alphahydroxy acids selected glycolic acid, d,l-lactic acid, malic acid, citric acid and tartaric acid; potassium salts, and mixtures thereof.

9. Device as claimed in claim 1, wherein (a) is chosen from the group comprising alpha-blockers selected from phenoxybenzamine, dibenamine, doxazosin, terazosin, phentolamine, tolazoline, prazosin, trimazosin, alfuzosin, tamsulosin and indoramin; ergot alkaloids selected from ergotamine and ergotamine-analogues; vasodilators selected from nimodepine, pinacidil, cyclandelate, dipyridamol and isoxs prine; chlorpromazine; haloperidol; trazodone; prostaglandins selected from prostaglandins El and E2, vasoactive amines selected from histamine and serotonin, nitrates selected from isosorbide dinitrate, erythritol tetranitrate, amyl nitrate, sodium nitroprusside, molsidomine, linsidomine hydrochloride and S-nitroso-N-acetyl-d,l- penicillamine, S-nitroso-N-cysteine and S-nitroso-N-glutathione; diazenium diolates, vasokinetics selected from nonyl valeramide, cyclandelate, cinnarizine, tolazoline and cepharanthine; peripheral vasodilators selected from buflomedil, butalamine, cetiedil, cyclonicate, cinnarizine, cyclandelate, diisopropylamine dichloroacetate, eledoisin, fenoxedil, phentolamine, flunarizine, ifenprodil, moxisylate, nafronyl, nicergoline, brovincamine, nicofuranose, nylidrin, piribedil, suloctidil, tolazoline and verapamil; and nicotine derivatives selected from methyl nicotinate, nicotinic alcohol, beta- butoxyethyl nicotinate, ichthammol, tocopherol nicotinate and inositol hexanicotinate.

10. Device as claimed in claim 1, wherein (a) is a non- irritant vanilloid selected from zingerone, olvanil and natural, synthetic and semi-synthetic vanilloid agonists and mixtures thereof, with the proviso it does not include capsaicine.

11. Device as claimed in any one of the claims from 1 to 8 comprising an hydroalcoholic solution having the following composition: Menthol 0.5 - 2.5 g Eugenol 0.25 - 1 g Zingerone 0 - 2 g Visnadine 0 - 0.5 g L-Arginine 0 - 1 g Nicotinyl alcohol tartrate 0 - 0.25 g Caffeine 0 - 0.2 g Gallic acid 0 - 2 g Glycerol 2 - 4 g d,l-lactic acid 80% 2.5 - 7.5 g 65° ethanol, volume q.s. for 100 ml

12. Device as claimed in claim 1, also containing dermatologically and pharmacologically acceptable active constituents, ingredients and excipients.

13. Method for improving the visibility of the veins in order to facilitate venipuncture in i.v. infusion or blood sampling operations in a subject, comprising the application of a device of claims 1-10 to the skin of said subject.