CAROTENOID MICROEMULSIONS

Abstract: Use of compositions comprising an oil soluble carotenoid agent in the form of a microemulsion for the treatment of cancer and novel spontaneously forming carotenoid gel formulations.

Description: CAROTENOID MICROEMULSIONS The present invention relates to compositions for use in cancer prevention and treatment and novel compositions and processes for preparing said compositions. A number of therapeutic agents which have been shown to have useful activity in cancer prevention and treatment based on in vitro studies or models are difficult to formulate for therapeutic use. In particular, agents which are oil soluble, such as carotenoids are relatively difficult to administer orally and/or topically, compared to more easily administered aqueous formulations. Carotenoids are naturally-occurring pigments of the terpenoid group which can be found in plants, algae and bacteria. They include 'carotene' (a mixture of alpha and beta-carotenes but mostly beta-carotene), alpha-carotene, beta-carotene, gamma-carotene, lycopene, zeaxanthin, lutein (alpha-xanthophyll), bixin (e.g, from solvent extracted annatto), capsanthin (paprika), canthaxanthin, astaxanthin, actinioerythrol, violerythrin, beta-8'-apo-carotenal (apo-carotenal), beta-12'-apo-carotenal and esters of hydroxyl- or carboxyl-containing members thereof. Many of the carotenoids occur in nature as cis and trans-isomeric forms, while synthetic compounds are frequently racemic mixtures. There are approximated to be around 100 carotenoids in the human diet however few. e.g, beta-caroiene, lutein and canthaxanthin (CTX) can be detected at significant levels in the plasma. It has already been widely established that beta-carotene levels correlate with dietary vitamin A intake (Van Eenwyk, J., Davis, F.G & Bowen, P.E. (1991) "Dietary and serum carotenoids and cervical intraepithelial neoplasia" Int. J. Cancer 48: 34-38; de Vet, H.C., Knipschild, P.G., Grol, M.E., Schouten, H.J & Sturmans, F. (1991) "The role of beta-carotene and other dietary factors in the aetiology of cervical dysplasia: results of a case-controlled study" Int. J. Epidemiol. 20: 603-610) and several studies have correlated low intake of vitamin A with increased risk of cervical dysplasia (Liu, T., Wilson, N.P., Craig, C.B., Tamura, T., Soong, S., Sauberlich, H.E., Cole, P & Butterworth, CE. (1992) "Evaluation of three nutritional assessment methods in a group of women. Epidemiology 3: 496-502; Liu, T., Soong, S.J., Wilson, N.P., Craig, C.B., Cole, P., Macaluso, M & Butterworth, CE. (1993) "A case- controlled study of nutritional factors and cervical dysplasia" Cancer Epidemiol Biomarkers Prev 2: 525-530). SmithKline Beecham pic (WO 94/06310 and WO 97/10725) provide a process of formulating oil soluble ingredients including carotenoids in a stable aqueous composition which is highly bioavailable. Beta-carotene has been shown to inhibit oral carcinogenesis in several animal models e.g, (Schwartz, J.L., Shklar, E., Flynn, E & Trickier, D. (1990) "The administration of beta- carotene, regression of oral carcinoma in the hamster cheek pouch and the associated enhancement of the immune response" Adv. Exp. Med. Biol. 262: 77-93) and a number of clinical studies have shown that beta-carotene can produce regression of oral leukoplakia (Kaugars, G.E., Silverman, S., Lovas, J.G., Brandt, R.B., Riley, W.T., Dao, Q., Singh, V.N & Gallo, J. (1994). Oral Surg. Oral Med. Oral Pathol. 78: 462-468; Garewal, H. (1994) "Chemoprevention of oral cancer: beta-carotene and vitamin E in leukoplakia" Eur. J. Cancer Prev. 3: 101-107; Toma, S., Benso, S. , Albanese, E., Palumbo, R., Nicolo, G & Mangiante, P. (1992) "Treatment of oral leukoplakia with beta-carotene" Oncology 49: 77-81; Malaker, K., Anderson, B.J., Beecroft, W.A & Hodson, D.I. (1991) "Management of oral mucosal dysplasia with beta-carotene: a pilot cross-over study" Cancer Detect Prev 15: 335-340; Stich, H.F., Matthew, B., Sankaranarayanan, R & Nair, M.K. (1991) "Remission of pre- cancerous lesions in the oral cavity of tobacco chewers and maintenance of the protective effect of beta-carotene or vitamin A" Am. J. Clin. Nutr. 53: 298S-304S; Leo, M.A., Seitz, H.K., Maier, H & Lieber, C.S. (1995) "Carotenoid, retinoid and vitamin E status of the oropharangeal mucosa in the alcoholic" Alcohol-Alcohol 30: 163-170), although the precise relationship between beta-carotene consumption and cancer incidence is not clear cut. Smokers have been shown to have significantly lower levels of endogenous carotenoids in the oral mucosa which may contribute to a greater risk of developing neoplastic disease. Although many carotenoids are known to be potent antioxidants (Lawlor, S.M & O'Brien, N.M. (1995) "Modulation of oxidative stress by beta-carotene in chicken embryo fibroblasts" J. Nut. 73: 841-850; Everett, S.A., Subhas, K.C, Maddix, S & Willson, R.L. (1995) "Mechanisms of free-radical scavenging by the nutritional antioxidant beta-carotene" - 2 - Biochem. Soc. Trans. 23: 230S), until recently their putative role as inhibitors of neoplastic transformation was thought to be mediated via retinoids. However, it has been shown that both beta-carotene and CTX are able to modulate this effect via a distinct pathway (s). For example, beta-carotene appears to enhance immune system performance by increasing NK cell proliferation and T-cells expressing surface MHC-Ia, therefore allowing them to present viral antigen (Prabhala, R.H., Garewal, H.S., Hicks, M.J., Sampliner, R.E & Watson, R.R. (1991) "The effects of 13-cis-retinoic acid and beta-carotene on cellular immunity in humans" Cancer 67: 1556-1560). Carotenoids can inhibit chemically induced neoplastic transformation in vitro, probably via positive regulation of connexin gene expression, in particular connexin-43, which is involved in cell-cell communication (Bertram, J.S. (1993) "Cancer prevention by carotenoids: Mechanistic studies in cultured cells" Ann. New York Acad. Sci. 691 : 177-191; Zhang, L.X., Cooney, R.V & Bertram, J.S. (1992) "Carotenoids up-regulate connexin-43 gene expression independent of their provitamin A or antioxidant properties" Cancer Res. 52: 5707-5712; Bertram, J.S., Chow, C.K., Cross, C , Krinsky, N & Packer, L. (1993) "Inhibition of chemically induced neoplastic transformation by carotenoids: Mechanistic studies" Ann. New York Acad. Sci. 686: 161-176; Mehta, P.P., Bertram, J.S and Lowenstein, W.R (1986) "Growth inhibition of transformed cells correlates with their junctional communication with normal cells" Cell 44: 187-196; Bertram, J.S (1996) "Inhibition of neoplastic transformation by β-carotene and other carotenoids: correlation with enhanced expression of connexin-43, a gene associated with growth control" Proceedings of the 3rd International Conference on: " Antioxidant Vitamins and Beta-Carotene in Disease Prevention") and which may restore the cell's ability to control the mitotic cycle. Furthermore, gap-junction communication is known to be perturbed upon transformation with HPV-16 (Ennaji, M.M., Schwartz, J.L., Mealing, G., Belbaraka, L., Parker, C, Parentaux, M., Jouishomme, H. , Arella, M., Whitfield, J.F and Phipps, J (1995) "Alterations in cell-cell communication in human papillomavirus type 16-transformed rat myoblasts" Cell Mol. Biol. 41: 481-498; Oelze, I., Kartenbeck, J., Crusius, K and Alonso, A "Human papillomavirus type- 16 E5 protein affects cell-cell communication in an epithelial cell line" J. Virol. 69: 4489-4494). - 3 - Although it has been suggested that carotenes could be effective in inhibiting and/or reversing cellular neoplastic transformation in a variety of tissues, many of these e.g, oro-pharangeal carcinoma, are typically difficult to treat using oral formulations and rely upon high systemic bioavailability and there are to date no effective formulations available to successfully treat such indications. For example, human volunteers subjected to a carotene-rich diet for 3 months demonstrated elevated serum and GI tissue concentrations of beta-carotene and up- regulation of Cx43 expression in colon (Frommel, T.O., Litez, H & Mobarhan, S. (1994) "Expression of mRNA for the gap-junction protein connexin-43 in human colonic tissue is variable in response to beta-carotene supplementation" Nutr. Cancer 22: 257-265). However, the variability of the clinical response is symptomatic of the problem of poor bioavailability of the agent in solution and/or poor drug targetting. WO 95/27483 (Schlipalius et at) describes a carotenoid agent for inhibiting the conversion of epithelial cells to tumours after being subjected to UV radiation in vitro. The agent was prepared by heating the crystalline suspension of beta-carotene in soyabean oil and adding glycerol monooleate. This is dispersed in the glycerol-water phase by high shear mixing followed by homogenisation (8,000 to 10,000 psi) at 60-70°C followed by heat processing. Optionally 0.3% (w/v) antioxidant tocopherol are added. However, it would follow that carotenoid in the form of a dispersion such as this would be poorly bioavailable. WO 95/18605 (Marigen S.A) claims beta-carotene 'ultramicroemuls ions' for the treatment of some tumours, psoriasis and eczema. The concentrates claimed comprise 0.1 to 10% (w/v) carotene , 0 to 10% (w/v) active pharmaceutical substance, 1 to 40% (w/v) co-emulsifier solvent, 0.1 to 90 % (w/v) surfactant, 0 to 10% (w/v) vitamin and 0 to 10% (w/v) radical catcher, as well as carrier substances and/or diluents. All the examples provided contain between 5 and 90% of a powerful surfactant Invadin JFC 800% which is a drug compatible tertiary octylphenylpolyoxyethylene with 9 or 10 oxyethylene groups. The concentrates disclosed show some efficacy in in vitro proliferation tests on cell lines from pulmonary cancers and leukaemias. The concentrates are not spontaneously gel forming and there is no provision for their use in the area of preventative treatments or pre-cancerous states. The present invention provides compositions for use in the treatment and/or prevention of cancer in particular of the cervix, skin, oral cavity, oro-pharynx and/or gastrointestinal tract (including the upper and lower bowels). In one aspect, the present invention provides the use of a composition comprising an effective amount of an oil soluble carotenoid agent in the form of a microemulsion, obtainable by dispersing the agent in emulsifers and heating to 140 degrees C to obtain a transparent mixture and diluting with water, in the manufacture of a medicament for the treatment of cancer. Suitably the compositions are as provided in WO 94/06310 and WO 97/10725, the subject matter of which is incorporated herein by reference. In a further aspect, the present invention provides new highly bioavailable carotenoid gels, in particular beta-carotene gels, for treatment and/or prevention of HPV-mediated transformation in cervical epithelium. The compositions of the present invention are particularly advantageous in the treatment and/or prevention of low-grade cervical cancer and/or cervical dysplasia. Physicians and opinion leaders agree that dysplasia or atypias of the cervical epithelium, detected by cervical smear test, pose a significant clinical management problem. Current recommended practice is surveillance of the cervix for up to two years, during which time there is no available treatment until such time as the lesion progresses to CIN I , at which time surgical excision or ablation using laser therapy can be applied (Shafi, M.I & Luesley, D.M. (1995) "Management of low grade lesions: follow-up or treat?" Baillieres Clin. Obstet. Gynaecol. 9: 121-131). The difficulty is that overall, approximately 50% of lesions will regress, however it is not possible to predict which cases these are and therefore, there is a tendency to treat all lesions by surgery, an unsatisfactory practice in cost-benefit terms and despite the fact that spontaneous regression of cervical dysplasia is not uncommon (Mitchell, M.F., Hittleman, W.K., Lotan, R., Nishioka, K., Tortolero-Luna, G., Richards-Kortum, R., Wharton, J.T and Hong, W.H (1995) "Chemoprevention trials and surrogate end-point biomarks in the cervix" Cancer 76: 156S-1977S). Topical treatment of these types of lesions with 5-fIuoracil or trichloroacetic acid have to date, proven ineffective. Suitably the spontaneously forming gels of the invention are obtainable by mixing 1-2% of beta-carotene as a 30% dispersion in oil with 20-50% of an emulsifier or emusifier mix, heating to 140°C with stirring and adding water slowly with stirring as the mixture thickens. The invention extends to the spontaneously forming gels for use in therapy. The advantage of the present invention is that treatment and/or further prophylaxis can be conveniently commenced immediately upon diagnosis (using oral preparations, gels, pessaries etc). Particularly advantageous are topically applied beta-carotene and/or other carotenoid- containing gels which are specifically applied to the site of the lesion and permit highly effective tissue penetration and high local tissue bioavailability of the active ingredients, enabling clinical effects to be observed more readily than via supra-dietary ingestion of beta- carotene. These treatments could also be used as an adjunct to conventional surgical or laser therapies. The compositions of the present invention can be administered topically, either across mucosal surfaces or the stratum corneum; enterally (oral or rectal) or parenterally. The dosage of the active ingredient depends on the age and condition of the subject, lesion severity as well as the route of administration. Preferred formulation presentations may include: an intra-vaginal cap covered with gel, a gel for topical application on the skin, a cream, ointment, lotion, foam, pessary, suppository, capsule, lozenge, mouthwash, spray, aerosol, medicine, or beverage suitably prepared using methods known in the art. The following examples illustrate the invention. EXAMPLES Example 1: Microemulsion formulations prepared for in vitro studies in dermal epithelial cells. Formulation Code Formulation Type Final [beta-carotene] (w/v) and raw material source (see Example 4) 1437 Clear microemulsion 0.02% 2856 Clear microemulsion 0.02% 3290 Clear microemulsion 0.02% 5428 Opaque cloudy 0.02% dispersion 1793 Opaque cloudy 0.02% dispersion 9381 Opaque cloudy 0.02% dispersion 6420 Clear microemulsion Example 2: Beta-carotene gel composition % ( /v) beta-carotene (30% w/v, dispersion) in 1.65 corn oil (Roche) emulsifier (polysorbate 80) 25.0 emulsifier (sorbitan monooleate) 0.75 water 72.6 The beta-carotene solution is dispersed in the two emulsifiers and the mixture is heated to 140°C with stirring before dilution by slowly combining with hot or cold water with stirring as the mixture gradually thickens to a gel. Example 3: Beta-carotene gel composition %w/w beta-carotene (30% dispersion) 1.85 emulsifier (polysorbate 80) 36.5 water 61.65 The beta-carotene is dispersed in the emulsifier and the mixture is heated to 140°C with stirring. At this point the mixture should remain transparent. Hot (or cold) water is slowly combined with this mixture with stirring as the mixture gradually thickens to a gel. Example 4: Preparing samples for stability testing The carotene sources were as follows: All-trans beta-carotene (Roche CWD beadlets obtained from Hoffman-La Roche, Basel , Switzerland)(1437 & 5428); Cis and trans beta-carotene (Henkel CWS/F beadlets, obtained from Henkel, Little Island, Co Cork, Ireland)(2856 & 1793); Alpha-carotene, cis and trans beta-carotene (Quest Caroteen powder, obtained from Quest, Kilnagleary, Co Cork, Ireland)(320 & 9381) . These were formulated into dispersions (opaque, cloudy) or microemulsions as follows. Dispersions The samples were weighed directly into a 500ml volumetric flask and made up to near volume with warm water (deionised water). To aid dispersion, the flasks were sonicated for 5-10 minutes, then when cool they were made up to volume. These solutions were used directly for the preparation of all the cold water dispersible samples. Preparation of Microemulsions 1.665g of carotene source and 12.515g of the emulsifier mixture (the emulsifier mixture was 30.018g Tween 80 + 0.909g span 80 and was common to all microemulsions) was heated to 140°C and boiling water added in approximately 75mls. The microemulsion was cooled rapidly and made to lOOmls. 20mls of the concentrated microemulsion was diluted to volume in a 500ml volumetric flash with deionised water to give solution containing 20mg/ 100ml beta- carotene. 8 - Small samples were prepared in 35ml Wheaton vials and prior to crimping 35ul velcorin (preservative) was added and the headspace was flushed with nitrogen. After preparation the vials were stored in the dark at 4°C awaiting analysis by HPLC Example 5: Uptake and Expression Studies 5.1 : Materials and Methods A. Total cellular uptake of carotenoids. This was measured in human dermal fibroblast monolayer cultures and in organotypic keratinocyte (HaCAT's) cultures. The latter is a particularly useful system in which to work, since to gain access to the cells, carotenoids must be able to flux through a semi-permeable membrane and the layer of collagen on which cells are grown (see Figure 1). This means that measurement of cellular uptake by HPLC can provide an indication as to the tissue levels achievable following topical application in vivo. B. Connexin 43 (Cx43) Expression. This was investigated in: i. Normal Human Dermal Fibroblasts (n.b. cytokeratins are not expressed in these cells and hence were not studied). ii. Normal Human Keratinocytes (HaCaT cells, immortalised through spontaneous loss of p53 (Khan, M.A., Jenkins, G.R., Tolleson, W.H., Creek, K.E and Pirisi, L (1993) "Retinoic acid inhibition of human papillomavirus type 16-mediated transformation of human keratinocytes" Cancer Res. 53: 905-909). iii. HPV-Transformed Human Keratinocytes, immortalised by transfection of full-length HPV- 16 DNA (Creek, K.E., Jenkins, G.R., Khan, M.A., Batova, A., Hodam, J.R., Tolleson, W.H and Pirisi, L (1994) "Retinoic acid suppresses human papillomavirus type 16-mediated transformation of human keratinocytes and inhibits the expression of the HPV-16 oncogenes" In: M.M.Jacobs (ed.), Diet and Cancer: Markers, Prevention and Treatment, pp.19-35, New York, Plenum Press). C. Basal e.g, Kl or K10 and Suprabasal e.g, K5, Cytokeratin Expression. This was investigated in: i. Normal Human Keratinocytes (HaCaT cells). ii. HPV -Transformed Human Keratinocytes. All beta-carotene formulations were studied at 10 "5 , 10" 6 and 10" 7 M following appropriate dilution in serum-free medium and in all experiments, all-trans retinoic acid (10 -6 , 10 -7 , 10 -8 and 10" 9 M) was used as an internal positive control to demonstrate the responsiveness of the cell lines used. Cultures were treated with test formulations every two days for 10 days i.e, on days 0, 2, 4, 6 and 8 then harvested on day 10. For Cx43 and cytokeratin analysis, total protein lysates were prepared from cell suspensions and analysed by SDS-PAGE (D. Rickwood & B.D. Hames (eds, 1982), Gel Electrophoresis: a practical approach, IRL Press, Oxford) followed by Western blotting, according to standard procedures (Perbal, B (ed., 1988), A practical guide to molecular cloning, 2nd edn, John Wiley & Sons, New York). Filters were then probed using appropriate antisera against Cx43, Kl and K5. Autoradiographs were then scanned using a Hewlett Packard ScanJet lie and integrated density data for the immunostained bands calculated using the NIH, Image 1.41 software. 5.2: Results Statistical analyses were carried out using the Mann- Whitney U Test (Siegel, S (1956). Non- parametric Statistics for the Behavioural Sciences. Series in Psychology, McGraw-Hill Book Co., New York.). A. Bioavailability of Carotenoids in Normal Human Fibroblasts and Keratinocytes When the mean carotenoid uptake data are ranked it is clear that in both cell types, uptake is markedly greater from the microemulsion formulations compared to simple dispersions (Figure 2). In fibroblasts, uptake from the 'Quest' microemulsion (3290) was significantly greater than from the corresponding dispersion (9381)(p< 0.05) and in HaCaT cells, both the 'Henkel' and 'Roche' microemulsions (2856 & 1437) demonstrated superiority over the corresponding - 10 - dispersions (1793 & 5428) (p< 0.05) (Figure 3). Notably, there was no carotenoid uptake from the 'Henkel' dispersion (1793) in keratinocytes. These data reflect previous human oral dosing studies in which a 10 fold greater serum bioavailability of beta-carotene was achieved with microemulsion formulations compared with simple dispersions. B. Effect of Beta-Carotene Formulations on Connexin 43 (Cx43) Expression i. Normal Human Dermal Fibroblasts Several of the beta-carotene formulations tested induce a modest increase in Cx43 expression (Figure 4), most notably the 'Quest' microemulsion (3290), which exhibited a dose-responsive effect and also produced the highest intracellular carotenoid levels, although the high endogenous levels of Cx43 expression in untreated cells meant that none of the increases reached statistical significance. Interestingly, the 'Henkel' microemulsion (2856) was found to be superior to the corresponding 'Henkel' dispersion (1793), demonstrating a significant increase in Cx43 expression (p< 0.01) compared to the latter. Table 5.21 p Values for Cx43 ID Data in HaCaT's Formulation p Value (Mann Significance Whitney U Test base Level on Mean Values) Quest microemulsion (3290) 0.095 NS Quest dispersion (9381) 0.071 NS Roche microemulsion (1437) 0.036 p< 0.05 Roche dispersion (5428) 0.036 p< 0.05 Henkel microemulsion (2856) 0.071 NS Henkel dispersion ( 1793) 0.133 NS -ve control microemulsion (6420) 0.036 p< 0.05 Retinoic acid in acetone base 0.024 p< 0.05 In HaCaT cells, all formulations tested resulted in a numerical increase in Cx43 expression over and above untreated cells (Figure 5). The microemulsions generally appeared to be superior to the dispersions in increasing Cx43, although no statistically significant differences were detected. iii. HPV-Transformed Human Keratinocytes Although the sample size from this experiment was too small to apply statistical analysis, these data suggest that with the exception of the microemulsion control (6420) and the 'Roche' microemulsion (1437), there is a modest increase in Cx43 expression in cultures treated with beta-carotene formulations. (Figure 6) C. Effect of Beta-Carotene Formulations on Basal and Suprabasal Cytokeratin Expression i. Normal Human Keratinocytes It is now well-established that aberrant epidermal cytokeratin expression occurs in a variety of different situations. For example, tumourigenic clones of HaCaT cells transformed with the ras oncogene demonstrate increased expression of the suprabasal keratins 1 and 10 when grown in vitamin A-depleted medium, a trend that can be reversed by supplementation with all-trans retinoic acid. An identical effect is observed for the basal keratins 5 and 14 in cultures of vitamin A-depleted hamster, metaplastic epithelial cells (Lancillotti, F., Darwiche, N., Celli, G and De Luca, L.M (1992) "Retinoid status and the control of keratin expression and adhesion during the histogenesis of squamous metaplasia of tracheal epithelium" Cancer Res 52: 6144- 6152). Thus, compounds which mimic the negative effects of retinoic acid in this respect might restore control of cellular differentiation. All formulations tested resulted in substantial decreases in Kl expression (Figure 7). Several beta-carotene formulations, notably the Henkel dispersion, as well as the retinoic acid control demonstrated dose-responsive decreases in Kl expression, however, due to the small sample size of the test population (n=2) and the lack of data points for the 10" 5 M concentration of the Henkel dispersion, only the retinoic acid was found to be statistically significantly different to the corresponding acetone control (p<0.05). The K5 expression data was unequivocal. As expected, given that uptake was negligable, with the exception of the 'Henkel' and 'Roche' dispersions (1793 & 5428), all formulations dramatically reduced K5 expression (Figure 8), although no statistically significant differences were obtained when comparing any given microemulsion with the corresponding dispersion (see Table 5.22 for p values). Table 5.22 p Values for K5 Data in HaCaT's Formulation p Value (Mann Significance Level Whitney U Test base on Mean Values) Quest microemulsion (3290) vs 0.469 NS dispersion (9381) Roche microemulsion (1437) vs 0.066 NS dispersion Henkel microemulsion (2856) vs 0.129 NS dispersion ii. HPV-Transformed Keratinocytes Although quantitative, integrated density data were only obtained on Kl expression, these data unequivocally demonstrate that all beta-carotene formulations dramatically reduce Kl expression in these cells (Figure 9). Qualitative assessment of gels containing material from HPV-transformed keratinocytes also suggest a decrease in K5 expression in all beta-carotene treated cultures (data not shown). 5.3: Data Analysis, Conclusions and Discussion These data clearly demonstrate the superior bioavailability of carotenoids from microemulsion formulations over simple dispersions in two distinct dermal cell types. Numerically, most of the beta-carotene formulations were associated with modest increases in Cx43 expression over - 13 - and above the untreated fibroblasts. In keratinocytes, all formulations increased Cx43 expression compared to untreated cells. Numerically, it would also appear that in these cells the microemulsions are superior to the dispersions, however, a larger sample size is required to achieve statistical significance. All beta-carotene formulations appear to exhibit a significantly greater positive effect than retinoic acid, supporting literature reports that carotenoids are able to exert some biological effects independent of their retinoid metabolites. Experiments carried out in HPV-transformed cells suggest that with the exception of the 'Roche' microemulsion (1437), there is a modest increase in Cx43 expression in cultures treated with all other beta-carotene formulations. This represents indirect evidence that beta- carotene can exert a biological effect in cells infected with HPV. All of the carotene formulations studied appeared to reduce suprabasal and basal keratinocyte expression in non-HPV transformed keratinocytes. Qualitative assessment of gels containing material from HPV-transformed keratinocytes suggests a decrease in K5 expression in all beta- carotene treated cultures, a feature which would be consistent with the desired effect of 'normalising' turnover of the cervical epithelium and which would contribute to a positive clinical effect.

Claims: CLAIMS 1. Use of a composition comprising an effective amount of an oil soluble carotenoid agent in the form of a microemulsion, obtainable by dispersing the agent in emulsifers and heating to 140 degrees C to obtain a transparent mixture and diluting with water, in the manufacture of a medicament for the treatment of cancer. 2. Use according to claim 1 wherein the carotenoid agent is beta-carotene. 3. Use according to claim 1 wherein the composition is in the form of a beverage, gel, spray capsule, spray or pessary. 4. Use according to claim 1, 2 or 3 wherein the treatment is of cancer of the cancerous lesion present on the cervix, anus, recmm, mouth, oro-pharynx, skin, bowel, lung or gastrointestinal tract. 5. Use according claim 4 wherein the treatment is administered on pre-cancerous or dysplastic lesions. 6. Use according to any one of claims 1 to 5 wherein the composition is adapted for oral or topical administration. 7. A microemulsion which spontaneously forms a gel obtainable by mixing 1-2% of an oil soluble carotenoid agent as a 30% dispersion in oil, with 20-50% of an emulsifier or emulsifier mix and heating to 140°C with stirring and adding water slowly with stirring. 8. A microemulsion according to claim 7 wherein the carotenoid agent is beta-carotene. 9. Microemulsions of claim 7 or 8 for use in therapy.

Inventors: MEYERS NICHOLAS LESLIE, VAN DEN BRAAK MAARTEN, MEYERS, NICHOLAS, LESLIE, VAN DEN BRAAK, MAARTEN
Applicants: DEN BRAAK MAARTEN VAN, MEYERS NICHOLAS LESLIE, SMITHKLINE BEECHAM PLC, MEYERS, NICHOLAS, LESLIE, VAN DEN BRAAK, MAARTEN
Application year: 1998
Main classification: A61K9
IPC codes: A61K9, A61K31, A61K47, A61P35