FORM - 2 THE PATENTS ACT, 1970 (39 of 1970) & The Patents Rules, 2003 COMPLETE SPECIFICATION (See Section 10 and Rule 13) APOCRINE CELL LINE HINDUSTAN UNILEVER LIMITED, a company Incorporated under the Indian Companies Act, 1913 and having its registered office at 165/166, Backbay Reclamation, Mumbai -400 020, Maharashtra, India The following specification particularly describes the invention and the manner in which it is to be performed WO 2008/116713 PCT/EP2008/052173 1 Apocrine Cell Line The present invention relates to an apocrine cell line and in particular to an apocrine cell line exhibiting long-term proliferation. In addition to eccrine glands, apocrine glands secrete aqueous fluid containing various lipidic and aminoacid solutes which, when exposed on human skin to skin bacterial populations, are transformed to malodorous compounds, including steroids and short chain fatty acids. In other words, apocrine glands contribute to human sweating. Considerable research continues to be carried out to find means to counteract the generation of malodorous compounds on skin, but this is hampered by the absence of an apocrine cell line that closely mimics the functions of an apocrine gland in situ, i.e. in human skin. The development of the latter would be very valuable because it would offer one or more of the benefits mentioned herein. It would enable a greater number of in vitro, as opposed to in vivo, studies to be carried out. This would (i) assist in identifying the transport mechanisms within the apocrine gland that lead to sweating, (ii) facilitate the screening and identification of substances for reducing the formation of malodorous compounds, and (iii) help determine their effective concentration, i.e. the concentration required to act as a deodorant when applied topically to the body. The existence of a suitable cell line would also provide the opportunity to identify novel glandular functions other then sweating per se. WO 2008/116713 PCT/EP2008/052173 2 In common with attempts to sustain other secretory cells in long-term culture, primary cell lines obtained from apocrine cultures have not been passaged more than a few times before losing morphological, phenotypic and/or functional respects characteristic of an apocrine gland. A paper by R Wicher et al in Andrologia 35, pp342-350 entitled "Establishing of two in vitro models of epithelial cells from the apocrine secreting rat coagulating gland" relates to apocrine cells from rats, and does not disclose a human-derived apocrine cell line exhibiting long term proliferation. Furthermore, the cell markers used therein to demonstrate functionality are not necessarily relevant to the human apocrine model. Moreover, the authors seemingly did not seek to passage their cells after freezing and thawing, and thereby did not demonstrate their long term proliferation capability. A paper by Z. Maras et al published in In Vitro Cell. Dev. Biol. Animal Vol 35, Nov-Dec (1999), pp 606-611 entitled "Cultivation of Epithelia from the Secretory Coil of the Ovine Apocrine Gland; Evidence of Secretory Cell Function and Ductal Morphogenesis in Vitro" relates to apocrine cells from sheep, and similarly does not disclose a human-derived apocrine cell line exhibiting long term proliferation. A paper by Dieter C. Gruenert et al published in In Vitro Cell. Dev. Biol. Vol 26, April (1990), pp 411-416 entitled "Long Term Culture of Normal and Cystic Fibrosois WO 2008/116713 PCTYEP2008/052173 3 Epithelial Cells grown under Serum-free Conditions" relates to eccrine cells instead of apocrine cells and therefore does not disclose a human-derived apocrine cell line exhibiting long term proliferation. Object of the present invention It is an object of the present invention to provide an apocrine cell line that mimics the sweating function of an apocrine gland, which can be maintained in culture through subcultures, and exhibits proliferation in the long term. Brief summary of the present invention According to the present invention there is provided a cultured apocrine cell line exhibiting long term proliferation. Proliferation in the present context indicates that the cells retain the capability to divide when cultured in a suitable culture medium, but does not imply that the cell line is immortalised. Long term, in the context of the present invention, indicates that cells retain their capability to proliferate after at least 30 sub-cultures (passages). In practice, when such long term proliferation has been achieved by a cell Line, it can commonly exhibit proliferation through at least 100 or 1000 passages. Proliferation through such a large number of proliferations is indicative of indefinite proliferation, at least for practical purposes for cultured apocrine cell lines according to the present invention. WO 2008/116713 PCT/EP2008/052173 4 In particular, there is provided a human apocrine cell line ASG5 which has been deposited with the European Collection of Cell Cultures,(ECACC), Porton Down, Salisbury, SP4 OJG, England, under the depository number 07021301 and which exhibits at least long term proliferation. Detailed Description of preferred embodiments of the invention and morphology This text describes the generation of a long term proliferative apocrine cell line, and subsequently, its characterisation and comparison with primary cultured apocrine cells via electrophysiological, molecular, and biochemical techniques. Particular interest is paid to steroid synthesis and the analysis of odour precursor compounds with express reference to the apocrine cell line ASG5, that has demonstrated at least long term proliferation. Isolation and primary culture of apocrine glands An apocrine cell line that exhibited long term proliferation capability was obtained by the following method. Whole apocrine glands were isolated by shearing the axillary skin of women aged between 30 and 70, having previously obtained permission from the relevant ethical committee and informed consent of the subjects. Some apocrine sweat glands were fragmented by the shearing process, and cells could be cultured from those fragments. Microscopic observation and Neutral Red uptake experiments showed that the apocrine gland isolated by shearing consisted purely of coil separated from duct. On average WO 2008/116713 PCT/EP2008/052173 5 between 10 and 100 apocrine coils were isolated from samples of axillary skin of about 50mm x 5mm. The apocrine coils were then incubated using a modified method, proposed by Lee et al in J. Cell Sci. 83:103-118, 1986, in a Williams E medium (herein for short WEM) -containing collagenase type II (at 2 mg ml"1) for 30 minutes at 37°c in 5% C02/air of 95% relative humidity. The coils were then washed in enzyme-free WEM supplemented with 10 ng ml"1 epidermal growth factor (EGF), 10 pg ml-1 insulin, 10 ng ml"1 hydrocortisone, 10 pg ml"1 holotransferrin, 1 mM L-glutamine, 100 U ml"1 penicillin and 100 pg ml-1 streptomycin. Approximately 15 apocrine glands were plated out per 25cm2 flasks in 1 ml of Mammary Epithelial Growth Medium (MEGM), supplemented with 10 ng ml-1 EGF, 10 pg ml"1 insulin, 0.5 pg ml""1 hydrocortisone, 30 pg ml-1 bovine pituitary extract, 50 pg ml""1 gentamicin and 50 ng ml"1 amphotericin. After 24hr incubation in a 95% air / 5% CO2 humidified incubator at 37°C, a further 3ml MEGM was added to each flask and thereafter the medium was changed every 3 days. Generation and maintenance of a long term proliferating apocrine cell line (A5G5) - indicative of an indefinitely proliferating cell line Primary sweat gland isolation and culture As indicated before, whole intact human apocrine sweat glands were isolated by shearing. Histological examination of apocrine glands revealed that shearing completely WO 2008/116713 PCT/EP2008/052173 6 removed the short absorptive duct leaving only the secretory coil portion intact. Isolated glands were plated out in supplemented MEGM. Approximately 50% of glands plated out generated outgrowths, routinely observed after 2-3 days. Outgrowths continued to proliferate for up to 20 days and displayed typical ^cobblestone' epithelial cell morphology (as shown in Figure 1, which show phase contrast micrographs of (A) cells growing from an apocrine coil nine days after explantation, and (B) proliferating apocrine cells). At early stages of culture, before outgrowths were observed, a small number of cells with an elongated fibroblastoid morphology were often observed around explanted glands. However, these cells did not proliferate in MEGM. A proliferating apocrine cell line was derived from primary cultures of apocrine secretory coils. After 7 days culture in MEGM, the potent phorbol ester, TPA (phorbol-12-myristate-13 acetate; ex Calbiochem, Nottingham, UK) was included in the medium at a concentration of 250 nM. It is an important feature of the process of obtaining a cell-line exhibiting long term proliferation capability that the culture medium contains an effective concentration of an ester of phorbol, phorbol being alternatively called 1, la, lb, 4, 4a, 7a, 7b, 8, 9, 9a-decahydro-4a,7b,9,9a-tetrahydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-5H-cyclopropa[3,4]benz[1,2-e]azulen-5-one. Commonly, the ester comprises at least aliphatic substituent particularly suitably at the 12 and/or 13 positions, such as a long chain alkyl (C7 to C24) and optionally also a short chain alkyl (C2 to Ce)• WO 2008/116713 PCT/EP2008/052173 7 Accordingly, the method herein enables the preparation of an apocrine cell line exhibiting indefinite proliferation. TPA induced premature multilayering 3-4 days after addition. However, the upper differentiated layers were removed by repeated washing to leave a proliferative monolayer of cells still attached to the flask. Cells of the proliferative layer were passaged into 96 well plates to generate clonal cell lines. Cells were maintained, passaged and continued to grow in MEGM containing TPA for approximately 2 months. After this period cells were cultured in MEGM alone and monitored for continued proliferation. Initially, many subclones continued to grow, but by the end of the next 10 passages, the majority had begun to senesce. One culture, in particular, ASG5, continued to grow and has now been maintained in culture, proliferating over more than 40 passages with no signs of a reduction in growth rate. This is indicative of exhibiting a proliferation capability indefinitely. This culture continues to proliferate in the absence of TPA, can survive cryopreservation and is considered to be stable. By comparison, primary apocrine sweat gland cultures grow for a maximum of 1 month and 4 passages and do not survive cryopreservation. The cell culture passaged herein exhibiting long term proliferation is morphologically similar to primary apocrine cultures, for example peak agonist responses. Such a cell culture is accordingly capable of being used to demonstrate whether and/or the extent to which a substance exhibits deodorising properties in vitro, i.e. is capable of acting as a personal deodorant. WO 2008/116713 PCI7EP2008/052173 8 Preparation of ASG5 apocrine cells for electrophysiology After 13-18 days, confluent ASG5 cultures were passaged onto Transwell collagen-coated permeable supports (0.33 cm^ area, Costar, cat. no. 3495, High Wycombe, Bucks, UK). This involved incubation in 2% EDTA for 20 minutes, followed by trypsin (0.5%)-EDTA (0.2%) for approximately five minutes. 3 xlO^ cells were transferred onto each Transwell supplemented with WEM. The Transwell supports were suspended in 24-well plates in 0.6 ml of the same medium. For primary cultures, only first passage cells were used in experiments. The medium bathing cultures on Transwells was changed every two days. Electrophysiology Transepithelial resistance (TER) across epithelia on Transwells was monitored using 'chopstick' electrodes (WPI, Alresford, Hants, UK) connected to an ohmmeter. The TER was estimated by subtracting the measurement for a moistened filter blank. At peak TER, the Transwell supports were mounted in an Ussing Chamber. Epithelia were bathed in a modified Krebs buffer, oxygenated and maintained at 37 °C, pH 7.4, with 95% 02/5% C02. The buffer consisted of (mM): NaCl, 117; KCl, 4.7; CaCl2, 1.5; MgS04, 1.2; NaHC03, 25; and glucose, 11.1. Voltage and current electrodes (calomel and silver/silver chloride respectively, ABB Kent Taylor, Stonehouse, Glos, UK), connected to a voltage/current clamp were extended into the bath via bridges containing agar dissolved in 3 M potassium chloride. WO 2008/116713 PCT/EP2008/052173 9 Voltage electrodes were balanced in the absence of a Transwell and fluid resistance between the voltage electrodes was compensated for by the injection of an arbitrary DC current.. After mounting a Transwell the epithelium was short-circuited, the current allowed to stabilise, typically for 15 minutes. Given the rarity of apocrine material, drugs were typically added in succession as disclosed by Brayden et al (1988) J. Physiol, 405: 657-675, Pedersen et al (1992) Exp. Physiol, 77; 863-871, and Shen et al, (1994) Am. J. Physiol, 266: L493-501 and changes in short circuit current (Isc) recorded. The duration of experiments was typically 15-60 min, TER was estimated using Ohm's Law, routinely by clanping the voltage at ± 10 mV and measuring the current required to achieve this. Unless otherwise stated all drugs listed in the legend were added to the basolateral side, except for amiloride that was added to the apical side. Amiloride is an inhibitor of the epithelial Na+ channel (ENaC) and the Na+/H+ exchanger. Modifications of Krebs Buffer Chloride substitution experiments were carried out in Krebs buffer in which sodium gluconate was substituted for NaCl, potassium gluconate for KC1 and CaS04 for CaCl2. Glucose-free experiments were carried out with ll.lmM mannitol as a replacement to maintain osmolality. Experiments in which barium was used as an inhibitor of potassium channels were carried out in the absence of sulphate ions, such that MgCl2 replaced MgS04. WO 2008/116713 PCT/EP2008/052173 10 Basal transepithelial properties Proliferating apocrine cultures were passaged onto permeable supports and transferred to an Ussing chamber at peak TER. The mean TER for such proliferating cultures was 395±40Hcm2 (P<0.001). Resting short circuit current (Isc) was 4.5+0.8 pA cm~2 (p<0.001) for proliferating apocrine cultures. Resting transepithelial potential difference (TEPD) was 1.0 mV for proliferating apocrine cultures, apically negative. Effects of ion transport inhibitors on resting Isc summarised in Table 1 below. Resting Isc in proliferating cultures was sensitive to 10 u,M amiloride confirming that sodium reabsorption is a significant component of ion transport in cultured sweat gland epithelia. Apically applied amiloride (10 JJM) reduced resting Isc by 4.511.0 uA cm" 2 in proliferating apocrine cultures (n=20,P<0.001). Dose response curves reveal that the IC^Q for amiloride is 2-4 \M and that 10 pM amiloride was a supramaximal dose. The effects of furosemide, an inhibitor of NKCC1, the epithelial Na+-K4-Cl" cotransporter (and hence transepithelial chloride transport) was also examined in apocrine cultures. Basolateral addition of 1 itiM furosemide reduced resting lsc in transformed cultures by 0.8+0.30.2 uA (n=20, P<0.001) . WO 2008/116713 PCT/EP2008/052173 11 Table 1. The effects of amiloride and furosemide on the short circuit current in cultured apocrine (primary and proliferating) and eccrine sweat gland cells. Primary Apocrine Proliferating Apocrine Primary Eccrine Resting Isa (]iK cm-2) 8.4+0.9 (n=57) 4.5±0.8 (n=40) 8.6±1.0 (n=22) Al$c ((J.A cm"2) Amiloride (10 HM) -6.0±0.9** (n=49) -4.211.0 (n=20) -7.1+0.7 (n=22) Furosemide (1 mW -0.5+0.2 (n=13) -0.8+0.3 (n=20) nd The role of sodium reabsorption in agonist responses Primary apocrine and proliferating apocrine cultures were pre-treated with 10 [iM amiloride to determine the role of sodium reabsorption in agonist responses. Appended Figure 2 shows Peak increases in Isc in primary apocrine, eccrine and proliferating apocrine cultures in the presence of amiloride. The agonists were added to the basolateral side of cultures on Transwells. Each bar in the Figure represents a minimum of n=l0. Peak increases in short circuit current in the presence of apical amiloride (10 uM) are shown. The agonists were added basolaterally: Carbachol (CCh; 20 pM), Isoprenaline (Iso; 10 pM) , L-bradykinin (LBK; 170 nM), Histamine (His; 200 pM) and Adenosine triphosphate (ATP; 100 pM). Significance was tested using unpaired Student's t test and the Wilcoxon tests. WO 2008/116713 PCT/EP2008/052173 12 Figure 2 demonstrates that amiloride pre-treatment significantly reduced responses to CCh, His and ATP in a long term proliferating apocrine cell line, ASG5, suggesting that sodium reabsorption is a component of these responses. Figure 3 shows peak increases in Isc in primary apocrine, eccrine and proliferating apocrine cultures. Agonists were added to the basolateral side of cultures on Transwells. Peak increases in short circuit current are shown. The agonists were added basolaterally: CCh (20 pM) , Iso (10 pM) , LBK (170 nM) , His (200 pM) and ATP (100 pM) . Figures 4A and 4B are representative Isc records (traces) on the effects of ion channel inhibitors on the response to carbachol in ASG5 cells. Figure 4A shows an apical pre-treatment with amiloride (10 pM) and the effects of addition of 1 mM furosemide (Fru) and 50 |j,M barium chloride (Ba; a maxi-K channel blocker), on the CCh (20 pM) response. Figure 4B shows an apical pre-treatment with 1 mM furosemide and the effects of amiloride and barium chloride on CCh response. The representative trace of proliferating apocrine cultures in Figure 4A also demonstrated that compared to the observed values for agonist stimulation in Figure 3, carbachol stimulation is reduced after amiloride pre-treatment . WO 2008/116713 PCT/EP2008/052173 13 In four cases, whole apocrine secretory coils were explanted directly onto permeable supports to determine if the primary culture and passage of apocrine cultures affected their electrophysiological properties. In the cultures examined, basal and agonist stimulated electrophysiological properties were not significantly different to primary cultures, whether in the presence or absence of amiloride (data not shown). The role of transepithelial chloride transport in transient agonist responses To further characterise the ionic basis of agonist induced responses in primary and proliferating apocrine cultures, the role of transepithelial chloride transport was examined. Under chloride-free conditions, resting IEC for primary and proliferating apocrine cultures was 6.0+0.8 |jA cm"2 and 3.1±0.6 |J.A cm~2 respectively. Resting TEPD was -0.9 mV for primary cultures and -1.8 mV for proliferating cultures. Addition of 10 |iM amiloride reduced basal Isc by 3.6 \iA cm-2 jn primary cultures and by 2.5 |oA cm-2 in proliferating cultures. Responses to carbachol, histamine and ATP were significantly reduced in both primary and proliferating apocrine cultures when compared to identical experiments in the presence of chloride (Table 2) suggesting that chloride transport does play a significant role in mediating agonist responses. Furthermore, Figure 4B shows a representative trace from proliferating cultures demonstrating that pre-treatment with furosemide reduces the magnitude of response to carbachol compared to the values in Figure 3. Addition WO 2008/116713 PCT/EP2008/052173 14 of furosemide after agonist stimulation induced a rapid reduction in responses in both primary and transformed apocrine cultures, demonstrating the role of NKCC1 in regulating chloride uptake. (Table 3). Table 2. Peak agonist responses of apocrine cultures in chloride-free buffer. Aj Primary apocrine cultures B: proliferating apocrine culture (ASG5). Agonist Chloride-free n Control n Isc responses pA cm~2 A Carbachol (20 pM) 0.7 ± 0.6 * 5 3.9 ± 0.7 34 Histamine (200 pM) 1.7 + 0.5 ** 5 6.4 ± 1.3 14 ATP (100 pM) 2.9 + 0.9 * 9 7.4 ± 1.8 10 B Carbachol (20 pM) 3.0 + 0.8 * 6 4.0 ± 0.7 20 Histamine (200 pM) 3.3 ± 0.8 ** 6 4.8 ± 1.3 20 ATP (100 pM) 4.0 + 0.5* 6 6.3 + 1.8 20 WO 2008/116713 PCT/EP2008/052173 15 Table 3. Furosemide inhibition of agonist responses in primary and long term proliferating (AGS5) apocrine cultures. Agonist Inhibition by furosemide (90 yM) A Isc , ]iA cm~2 Primary Proliferating Carbachol (> 20 uM) -2.0 ± 0.5 (n=4) -1.1±0.4 (n=10) Isoprenaline (10 uM) 0.8 ± 0.2