|Title of Invention||
A METHOD OF PRODUCING A COSMETIC SYSTEM/FORMULATION
|Abstract||The present invention relates to development of system/formulation to protect skin from ultraviolet radiation (UVR) exposed damage, anti-ageing as well as controlling skin colour by differentially regulating genes responsible for such behaviour changes on exposure to UV. The invention also relates to identification and synthesis of novel genes for use in such system/formulation to control/monitor the above said skin conditions.|
|Full Text||FORM -2
THE PATENTS ACT, 1970 (39 of 1970)
(See Section 10)
GENES REGULATED BY UV
HINDUSTAN LEVER LIMITED, a company incorporated under the Indian Companies Act, 1913 and having its registered office at Hindustan Lever House, 165/166, Backbay Reclamation, Mumbai -400 020, Maharashtra, India
The present invention relates to development of system/formulation to protect skin from ultraviolet radiation (UVR) exposed damage, anti-ageing as well as controlling skin colour by differentially regulating genes responsible for such behaviour changes on exposure to UV. The invention also relates to identification and synthesis of novel genes for use in such system/formulation to control/monitor the above said skin conditions.
Background and Prior art:
The pigmentary system of the skin is based on two cell types, melanocytes and keratinocytes, interacting as a functional unit, the epidermal melanin unit (Jimbow et al, 1993). Ultraviolet (UVR) radiation is the main external stimulus for skin pigmentation. It increases proliferation and melanisation of melanocytes (Rosen ef a/, 1987). To stimulate melanogenesis, UVR can act directly on cultured malanocytes (Friedman and Gilchrest, 1987; Aberdam et af, 1993; Eller et al, 1996) or indirectly through the release of keratinocyte-dehved factors (Kupper et al, 1987; DeLeo et af, 1988; Kupper et af, 1989; Bos and Kapsenberg, 1993; Kondo ef al, 1993 and Gilchrest ef al, 1996) Protection against the damaging effects of UVR is provided predominantly by melanin, which is produced in the epidermis by the melanocytes and transferred to adjacent keratinocytes.
A wide variety of factors that influence melanocyte function at various levels, control visible pigmentation in the skin, hair and eyes. It has been known that following exposure to UVR, there is an increase in the number of active melanocytes in the basal epithelium, individual melanocytes are stimulated to produce more melanin and there is an increased rate of transfer of melanosomes to keratinocytes (Friedman and Gilchrest, 1987; Abdel-Malek ef al, 1994; Yohn ef al, 1992 and Barker ef al, 1995)
The physiological responses to UVR have been documented as erythema, heat, oedema, pain and puritis, followed by tanning and epidermal thickening.
However, there appear to be variations in responses to UV depending on the skin colour. Caucasian skin, which contains less melanin, tends to burn easily on UV exposure, whereas black or brown skin tends to tan first.
Effects of UV on keratinocytes is known to occur at 3 different levels i.e., membrane, cytosol and nucleus, all of which eventually play a role in melanogenesis. UVR can easily penetrate to any part of a cell and therefore it is necessary to investigate its effects at every stage to get a complete picture of UV mediated cellular effects. At the membrane level, it could affect phospholipid degradation and subsequent metabolism of fatty acids to active mediators. At the cytosolic level, there could be changes in the melanosomal size and shape, melanin degradation activation of cytosolic enzymes like cyclooxygenase. At the nuclear level, it could lead to increased deoxyribose nucleic acid (DNA) transcription, leading to up-regulation of cytokines and protein synthesis (Soriani et at, 1999; Avalos-Diaz et al, 1999; Djavaheri-Mergny et al, 1996; Grether-Beck etal, 1996)
A large number of investigations on melanogenesis have employed murine melanoma cell lines. Such cells respond to a variety of stimuli by secreting various cytokines with inflammatory, immunologic and proliferative properties (Kreider ef al 1973, Pawalek, 1976, Lotan and Lotan, 1981, Wick 1981, Schimizu et al 1999, Tsatmali et al, 2000). Studies have also been done to determine the signal transduction mechanism, as to whether melanocytes respond directly or via secondary signals from the surrounding UV-irradiated keratinocytes.
However, the mechanisms involved in the above processes are not clear and there has been no clear understanding of responses of melanocytes and keratinocytes to UVR, that impact skin pigmentation and skin conditions.
It is thus the basic objective of the present invention to provide system/formulations to protect skin from conditions such as UV exposed
damage, anti-ageing as well as controlling skin colour based on identification of cytokines and genes responsible for such skin conditions in response to UV exposure.
Another object of the present invention is directed to selectively identify and provide synthesis of novel genes/antisense vectors which would serve in altering cell functions to thereby assist in protecting skin from UV exposed damage, anti-ageing as well as controlling skin colour and skin conditions.
Yet another object of the present invention is directed to provide novel system of delivery of active through cells for cosmetologic application especially in protecting skin from UV exposure damage, anti-ageing as well as other skin/cosmetic benefit application.
Further object of the present invention is directed to provide for diagnostic kits involving identification of genes with specified nucleotide sequences and its related special characteristics and means to determine the skin/cosmetic conditions based on comparative study and also determining loads for regulation of such conditions by selective actives.
Yet further object of the present invention is directed to provide for system for diagnosis/treatment of diseases based on identification of genes/clones involved in such diseases not known to the art.
Description of the Invention:
Thus according to one aspect of the present invention there is provided a
system/formulation to protect skin from conditions such as UV exposed damages, anti-ageing as well as skin colour comprising actives such as nucleotide and translated amino acid sequences in particular enzyme Argininosuccinate synthetase (ASS).
According to another aspect of the present invention there is provided means for identifying, providing and synthesising nucleotide and translated amino acid sequences such as enzyme Argininosuccinate synthase (ASS) which can be used as actives for conditions such as sun-screening, anti-ageing agents to protect skin against UV exposed damages, anti-ageing and as well as regulation in skin colour.
According to another aspect the present invention is directed to a novel system of delivery of actives through cells comprising:
Synthesising nucleotides and translated amino acid sequences; and
delivery of the relevant DMA such as an antisense vector through cells using
liposomes to thereby regulate skin/cosmetic conditions.
According to yet further aspect of the present invention there is provided a diagnostic kit involving/providing nucleotide and/or translated amino acid sequences and its characteristics/representation conditions and means for comparing the sample nucleotide sequence to determine whether the same matches to thereby diagnose the condition of the sample tested and further determine the regulation of active through cells for protecting and/or improving skin conditions.
In particular, the present invention is directed to achieve the above objects and aspects by way of identification of the following genes/clones involved in melanogenesis and their related characteristics on UV radiation/niacinamide treatment as detailed.
Cell line: B16
Gene: Argininosuccinate synthetase (ASS)
This gene was induced upon exposure of cells to Ultraviolet radiation (UVR) when analyzed two hours after exposure. It was obtained through RAPD analysis using the random primer KD1 Nucleotide sequence: 1 to 185
AAGAGCCCGTGAGCATGAACGTGTAGGGTGATTATGAGCCAATTGATGCCA CCGGGTTCATCAATTCCCJAGGCTGAAGGAATAGGAATATCATCGTCTGCA GAGCAAGGTCACTGCCAAATAGACCCCGTACAATGAGGAGCTGGAGCCTC CTCAATTTGCAGATCCCC CAAGTACGGG CTCTT
Cell line: B16
Gene: Argininosuccinate synthetase (ASS)
This gene was induced upon exposure of cells to Ultraviolet radiation (UVR)
when analyzed I5min after exposure. It was obtained through RAPD analysis
using the random primer KD1
Nucleotide sequence - 1 to 193
It is possible by way of the present invention to provide systems/formulations directed to regulate the gene ASS involved in the nitric oxide (NO) pathway and serve as a rate limiting enzyme in the production of NO. Nitric oxide has been established to stimulate tyrosinase activity of melanocytes, the net result being more production of melanin and thereby, more color/pigmentation. Clones pBU4 and pBU14 represent the ASS gene, which was induced by exposure to ultraviolet radiation (UVR). The findings of the present invention are further directed to provide for diagnostic kits for identification of skin conditions based on differential regulation of ASS and also method of treatment of skin conditions
based on such enzyme (ASS) characteristics responsible for altering melanogenesis and controlling skin colour.
The above discussed various aspects of the present invention are explained in relation to the following non-limiting illustrative studies carried out.
B16 murine melanoma cells and HaCaT human keratinocytes were cultured in Dulbecco's minimal eagle media (DMEM) supplemented with 10 U/ml Penicillin G, 0.1 mg/ml streptomycin sulphate, 25 mM HEPES buffer and 10% heat inactivated foetal calf serum at 37°C in a water saturated, 5% CO2 in air atmosphere. Cells of passage numbers 5-20 were used in all assays.
B16 or HaCaT cells were washed with PBS and were exposed to UV for 60 seconds at a distance of 26 cm from the UV bulb. A Wotan UV lamp (peak emission 367 nm) was used. Control cells were not exposed to UV. The dose of UV exposure is UVA = 0.17 J/min and sun bum = 0.18 med/min (UVB = 28-35 mJ). The experiment was done as duplicates and one batch of cells were stained with ammoniacal silver nitrate and from the other batch, RNA was extracted.
Ribonucleic acid (RNA) Extraction
After UV exposure, the cells were incubated in the same old media for different time points i.e., 15 min, 1 hr, 2hr, 4 hr and overnight. Cells, which were not exposed to UVR, were included as controls. Total cellular RNA was then extracted form these cells using TRI reagent.
The cDNA synthesis was carried out using oligo (dT)i8 primer, which can anneal with poly A tail of mRNA, using cDNA synthesis kit from Clonetech, as per the protocol recommended by the supplier.
Conditions used: 72°C - 5 min (for primer annealing)
37°C - 10 min (during this time MMLV RT, dNTPs,
42°C - 60 min (for DNA synthesis)
4°C - storage
Random amplification of polymorphic DNA - Polvmerase chain reaction (RAPD -
Random amplified polymorphic DNA PCR was carried out using a single RAPD
primer (KD1) having the sequence, 5'
was set as follows:
Mastermix was prepared depending on the number of reactions. For 10 reactions, it was as follows:
10X PCR buffer
For 10 reactions :25µl
50mM Magnesium : 12.5 µl
Random primer : 10 ul
DNA : 2 µl
: 187.5 µl : 240 µl
5U/ul Taq polymerase Sterile H2O Total
For one reaction (2.5 ul/reaction, final
(1.25 µl/reaction, final
(1 µ I/reaction, final cone.
(0.3 µl/reaction, final
(0.2 ul/reaction, final
cone. -1 U)
: 1 ul/reaction.
(In serial dilutions)
PCR cycling conditions: 94°C-5min. 36°C-30sec. 72°C - 30 sec.
94°C - 30 sec.}
36°C - 30 sec.} 35 cycles
72°C-1 min. } 4°C - storage
DMA separation, elution and re-amplification
The amplified DMA fragments were analysed by separating the fragments on a 2.5% agarose gel, as well as by Polyacrylamide gel electrophoresis followed (PAGE) by silver nitrate staining as described by Blum et al, 1987 and Caetano-Anolles, 1991. The RAPD pattern on the agarose and acrylamide gels were documented and the bands which showed variations in intensity based upon their differential regulation upon UVR, were cut out from the gel using a sharp scalpel. The DMA from the gel piece was electro-eluted using the protocol described by Sambrook et al, 1989. In the case of acrylamide gel, the gel piece carrying the DMA was placed in a centrifuge tube and the DNA was eluted for atleast 1 hr. at 65°C into 50ul of tris-EDTA buffer (TE). The eluted DNA bands were rechecked on gel and amplified again by PCR to get more DNA. The DNA from the gel piece was then extracted using phenol - chloroform, and precipitated with ethanol and used for cloning.
The DNA fragments were cloned into a commercial vector pGEM-T, vector from Promega (Cat # A3610). This vector has a 3' terminal overhang (3' - T) at both ends and cloning PCR products with the pGEM-T vector system exploits the template-independent addition of a single deoxyadenosine residue to the 3' end
of the PCR fragment via the terminal transferase activity of certain thermostable enzymes including Taq polymerase (Starts and Mezei, CRC Press). In the case of Taq polymerase, this nucleotide is almost exclusively an adenosine due to the strong preference of this polymerase for dATP. The vector contains the origin of replication of the filamentous phage f1 and can be used to produce ssDNA. The plasmid also contains T7 and SPG RNA polymerase promoters flanking a multiple cloning region within the ct-peptide coding region for the enzyme p-galactosidase. Insertional inactivation of the a-peptide allows recombinant clones to be identified directly by colour screening on indicator plates.
The RAPD fragments were ligated to the vector using T4 DMA ligase from Gibco BRL, Cat # 15224-025) and DH5 alpha competent cells were transformed with the ligated mix and screened for recombinants using ampicillin as a marker on colour indicator plates containing isopropyl-p-thiogalactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl-p-Dgalactopyranoside (X-gal). The presence of the foreign DMA in the vector was confirmed by releasing the fragment by doing a double restriction digestion with two different enzymes Sac I and Sac II, flanking the insert in the multiple cloning site of the vector. The clones were named as pBU4 and pBU14 and they contained the DNA fragment in opposite orientations as revealed by restriction digestion using the above enzymes. Figure 1 shows a diagrammatic representation of pGEM-T vector and clones pBU4 and pBU14.
DNA Sequencing and comparison with nucleotide database The transformants were streaked on to plates and the plates bearing the colonies/clones were sent for DNA sequencing. The DNA sequences were then compared to those in the nucleotide database by doing a NCBI, Blast search to look for homology with reported sequences. On comparison with the database, the identity of the DNA fragment was established to be Argininosuccinate synthetase (ASS). The two clones were found to contain the partial ASS gene in opposite orientations and clone pBU14 was found to have ASS in the anti-sense orientation.
Construction of RNA anti-sense to argininosuccinate synthetase
The clone pBU14 was used for constructing RNA anti-sense to ASS. The DNA fragment was eluted from pBU14 using the restriction enzymes Apa I and Not I. The same restriction sites were also present in the mammalian expression vector pcDNAS. This eluted DNA fragment was ligated to Apa I - Not I linearised pcDNAS to form the anti-sense construct pcASS". Figure 2 shows a diagrammatic representation of pcASS".
Reverse transcriptase PCR (RT-PCR)
In order to be sure that the RAPD fragments picked up earlier were indeed differentially regulated, primers were synthesised for the clones and a semi-quantitative RT-PCR was done on cells subjected to UVR using two specific, forward and reverse primers for the respective clones. Slot blots were also done for the purpose of confirmation of the clones using RNA from UVR exposed cells.
In addition to this, RT-PCR was also done for cells ± UVR, using specific primers 1or enzymes such as Argininosuccinate Lyase (ASL) and inducibte Nitric Oxide Synthase (iNOS), to see if there was differential expression of genes. The following primers were chosen.
Beta actin control
F 5' - GTGGGCCGCTCTAGGCACCAA - 3'
R 5' - CCAAAGTAGACCTGCCCGGACTC - 3'
F 5' - GCTATAGGGGACCAGGGAAC
R 5J - TCGCAGAGCTCGTATCACAG - 3'
F 5' - AGAAGCTCCAGGAGGAGGAG - 3'
R 5' - CGGAGAGTTTTGAGCAGGTC - 3' Inducible Nitric Oxide Svnthase F 5' - TCCCAAGTACGAGTGGTTCC - 3' R 5' - GGCCTCAGCTTCTCATTCTG - 3'
Results and Discussion
The RAPD PCR method on cDNA from UVR exposed B16 cells mouse melanoma cells and HaCaT human keratinocytes was followed to identify the genes that were differentially regulated.
In cells regulating skin colour, UVR was found to induce genes, which were manifested by differences in the RAPD banding pattern.
Interestingly, the clones pBU4, pBU14 showed homology to the gene argininosuccinate synthetase (ASS), an enzyme which makes available more arginine for the formation of nitric oxide required for UV induced melanogenesis. Moreover, the two clones, pBU4 and pBU14 showing homology were from two different batches of cells indicating that the results were reproducible. This observation was further confirmed by RT-PCR, where ASS levels were found to be higher in UVR exposed cells. It is thus identified for the first time by way of present invention the contributions of UVR with increased ASS levels at the mRNA level.
Studies were carried out to analyse the levels not only ASS, but also other enzymes that are involved in the nitric oxide cycle, such as iNOS and ASL, by RT-PCR. The levels of these two enzymes were higher in UVR exposed cells when compared to unexposed controls. This substantiates the finding on increase in NO production upon UVR. NO is derived from the amino acid L-arginine in a five-electron oxidation reaction catalysed by NO synthase requiring reduced pyridine nucleotides, reduced biopteridines and calmodulin. The by-
product citrulline is recycled back to L-arginine by ASS and it is established by way of this invention the latter, to be upregulated by UVR.
It is thus possible based on the above findings of RAPD and RT-PCR to provide system/formulation/actives to regulate cells upon simulation by UVR.
In accordance with the further aspect, the role of NO in melanogenesis is ascertained by curtailing the by-product citrulline from being recycled to arginine, which is a prudent approach for controlling melanogenesis. It is proposed that controlling the rate-limiting enzyme ASS would help in controlling melanogenesis / pigmentation. Therefore, it is now possible by way of the present invention to block the ASS gene in pigmentation cells using antisense vectors to regulate melanogenesis. Blocking the effects of NO at the keratinocyte level will also provide for beneficial effects such as reduction of inflammation and melanogenesis signalled through nitric oxide.
Our RT-PCR experiments showed a decrease in the levels of ASL and iNOS, which are also key enzymes in the production of nitric oxide. It is thus possible by way of the invention to control UVR regulated cytokines and genes like for example, ASL and iNOS, that are involved in the autocrine-paracrine regulation leading to stimulation of pigmentation.
Thus, the present invention provides for identification of a clone matched with deposited sequences for which a function could be assigned. Importantly, by way of the above study one gene representing the enzyme Argininosuccinate synthetase (ASS), was found to be induced by UVR. ASS is a rate-limiting enzyme in the conversion of citrulline to arginine in the formation of nitric oxide, which induces melanogenesis. Therefore, an antisense vector (pcASS") was constructed for this gene in the expression vector pcDNAS. When pcASS" transfected B16 cells were exposed to UVR, the transfected cells were found to be morphologically different from control and UVR exposed cells. They had long;
thin and branched dendrites with central to overall distribution of melanosomes unlike UVR exposed cells, which had short stubby dendrites with peripheral distribution of melanosomes. Therefore, blocking the function of ASS in UVR exposed cells resulted in the formation of long dendrites, which is also a condition seen when cells are treated with niacinamide which is a very successful skin lightening agent. ASS' transfected cells were also lighter in colour as compared to their respective control cells, which did not contain the ASS" insert. This indicated that blocking ASS in the cell resulted in beneficial skin lightening effects.
The present invention findings and applications are directed to the fact that UVR regulates cytokines and genes that are involved in the autocrine-paracrine regulation leading to induced pigmentation. Further it would now be possible by way of this invention to provide kits for diagnosis of levels of regulation of the key factors that influence pigmentation, provide for new actives and its method of delivery for achieving skin lightening. The invention also provides for system for blocking a particular function of the cell using antisense vectors for altering cell functions to modify/regulate skin/cosmetic conditions.
Dated this 14th day of December 2001
|Indian Patent Application Number||1179/MUM/2001|
|PG Journal Number||21/2010|
|Date of Filing||14-Dec-2001|
|Name of Patentee||HINDUSTAN LEVER LIMITED|
|Applicant Address||HINDUSTAN LEVER HOUSE, 165/166, BACKBAY RECLAMATION, MUMBAI 400 020, MAHARASHTRA, INDIA|
|PCT International Classification Number||C12Q1/68|
|PCT International Application Number||PCT/US01/28214|
|PCT International Filing date||2001-09-07|