EP0296205A1 - Procede et composition pour la pousse des cheveux dans des calvities de type commun - Google Patents
Procede et composition pour la pousse des cheveux dans des calvities de type communInfo
- Publication number
- EP0296205A1 EP0296205A1 EP88900664A EP88900664A EP0296205A1 EP 0296205 A1 EP0296205 A1 EP 0296205A1 EP 88900664 A EP88900664 A EP 88900664A EP 88900664 A EP88900664 A EP 88900664A EP 0296205 A1 EP0296205 A1 EP 0296205A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- composition
- lipids
- isolated
- omental
- growth
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
- A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q7/00—Preparations for affecting hair growth
Definitions
- This invention relates to a method and composition for stimulating new hair growth in mammals.
- a composition including isolated lipids from mammalian omenta is used to prevent hair loss and stimulate new hair growth in mammals exhibiting common pattern baldness.
- compositions and claims for promoting hair growth are applied in cream form and contains principally marrow, such as beef marrow, with additions of bergamot oil, alcohol, oil and sulfur as described in U.S. Patent No. 4,520,012 issued on May 28, 1985 to Alfonsi.
- the patent teaches that the composition enhances blood circulation in the scalp dermis to nourish the hair, possibly due to the effects of the bergamot oil. It is noted, however, that bergamot oil is known to be a skin irritant and Alfonsi also warns that more than 5% bergamot oil in the composition can actually cause hair loss. (Alfonsi, col. 2, lines 6-12).
- a pharmaceutical composition claimed to promote hair growth was patented in U.S. Patent No. 4,139,619 issued on February 13, 1979 to Chidsey.
- This pharmaceutical composition is known under the generic name Minoxidil and is marketed by Upjohn Company of Kalamazoo, Michigan.
- the preparation is claimed to increase the rate of terminal hair growth (the broad diameter and colored hair that is readily seen) and to convert vellus hair (fine, colorless hair) to terminal hair.
- Minoxidil is a potent antihypertensive substance which has been documented in studies to cause hair growth in virtually all patients that were orally treated with the drug for more than one year. de Villez, R. L., Arch. Dermatol. 121: 197-202 (1985). However, within four months of discontinuing the Minoxidil treatment all of the newly grown hairs had fallen out. Id. at 197.
- Patterns of hair loss are classified in the now universally accepted system described in Hamilton, James B. Annals of N.Y. Acad. of Sci. 53: 708-628 (1951).
- Types I-III represent non balding scalps
- types IV-V represent slight balding
- types VI-VIII represent horse shoe outlined baldness.
- Types VII and VIII representing the most advanced stages of baldness were not included in the minoxidil study. Of the 64 study participants, only 3 showed an excellent hair growth result. De Villez, supra at page 199. This study indicates that those subjects having advanced baldness, i.e., the greatest degree of baldness and the longest duration of baldness, were the least response to the treatment.
- inventive composition minus sodium hyaluronate greatly diminishes the effectiveness of the composition, indicating that the hair growth activity may be the result of a synergistic effect of the omental extract and sodium hyaluronate. It is noted that the foregoing analysis represents only one theory of the activity of the inventive product and is not meant to be limiting to the invention.
- new hair growth refers to new hair grown in balding areas genetically programmed for hair follicles, as opposed to merely lengthening already grown hair.
- the term also refers to terminal hair, or the hairs of the scalp having the larger diameter and being pigmented, as opposed to vellus hair, or the fine colorless hairs.
- Topical application of a composition including omental lipid material and sodium hyaluronate promotes new terminal hair growth, while at the same time preventing further hair loss in the progression of androgenetic alopecia.
- Additional ingredients include vitamins A, D and E, silicone, emulsifiers, anti-oxidants, skin fillers, hygroscopic agents and preservatives.
- the composition may be applied to the skin in the form of lotions, ointments, gels, sticks, or creams, although lotion form is preferable.
- the composition applied to balding or bald areas of mammalian skin has been observed to stop further hair loss and to promote new hair growth of terminal hairs to enhance hair distribution. In areas not genetically coded for terminal hair growth such as facial or arm skin areas it is observed that application of the composition did not initiate hair growth.
- Figure 1 is a diagrammatic flow chart of the fractionation method of porcine omentum hexane extracts.
- Figure 2 is a photograph of the vertex of the head of a test subject exhibiting Hamilton type VII-VIII male pattern baldness after application of the inventive composition designated as Lotion 2-36A, for six weeks according to the protocol of Example I infra.
- Figure 3 is a photograph comparing the same area of the same test subject as shown in Figure 1 after application of the inventive composition Lotion 2-36A according to the Example I protocol infra, for an additional two and a half months.
- Figure 4 is a second comparison photograph of the same test subject's baldness area as shown in Figures 2 and 3 after 5 months of applying the inventive composition Lotion 2-36A.
- the principal ingredient of the inventive composition is isolated omental lipid, preferably extracted from mammals.
- the omental material may be combined with conventional cosmetic forms for applying the material to the area to be treated, such as ointments, lotions, paste, gels, aerosols, sticks and the like which are known in the art.
- the preferred embodiment is a lotion form which combines the omental material with emulsifiers, antioxidants, hygroscopic agents, chelating agents, skin fillers., silicon and preservatives.
- the novel composition When applied to areas genetically coded for hair growth, the novel composition promotes new hair growth and prevents further hair loss in the progression of alopecia.
- Omental material may be extracted as described in Goldsmith, et al.,.J. Amer. Med. Assoc., 252: 2034-36 (1984).
- a variety of mammalian sources may be used to supply the material, such as porcine, bovine, ovine, or feline omentum.
- omental materials are also used in angiogenesis for myocardial conditions such as myocardial infarcts, angina, vascular or coronary implants and angioplasty as described in copending U.S. Patent Application, Serial No. 811,375, filed on December 20, 1985 entitled “Method for Treatment of Angina and Myocardial Infarctions with Omental Lipids” by Catsimpoolas, et al., herein incorporated by reference.
- the preferred method for preparation of omentum for use in the hair growth composition is a brittle grinding technique (also know as a cryogenic technique) combined with direct hexane extraction of the thus obtained omentum powder.
- Porcine omentum is preferred for use in the composition, however, bovine, feline, or any other type of mammalian omentum may be used.
- the porcine omentum is cut or broken into pieces. Freezing the omentum is accomplished by a method described in copending U.S. Patent Application, Serial No. 811,507, filed on December 20, 1985 by Kamarei, et al. entitled "Brittle Grinding and Extraction of Animal and Plant Derived Material”; herein incorporated by reference.
- the omental material is frozen to lower its temperature to an empirically critical "Brittleness Temperature" which converts the unbreakable, viscous, and sticky material into an extremely brittle and fragile substance.
- Freezing can be accomplished via various methods, including:
- the brittle pieces of tissues may be transferred to any size reduction equipment and homogenized (ground) for the desired length of time, i.e., a few minutes at 22,000 RPM.
- ground i.e., a few minutes at 22,000 RPM.
- roll mills with both attrition and impact grinding could be used. It is important that the tissue be kept at or below its brittle temperature throughout the grinding process. Grinding below the brittleness temperature is required in order to produce the necessary small particle size. For this purpose one could occasionally add, if needed, quantities of LN 2 to the size-reduction equipment, provided that there is a vent to allow exhaustion of the nitrogen vapor generated during the process.
- tissue powder an extremely fine, and free-flowing (non-sticky) cryoground tissue
- tissue powder an extremely fine, and free-flowing (non-sticky) cryoground tissue
- the resultant powder obviously may contain some granular lumps as well as fine indiscrete particles.
- density of omentum powder is 0.44 ( ⁇ 5%) g/ml which is almost half of the density of lipids extracted from omentum.
- tissue powder should be sieved at or below its brittleness temperature.
- stacked stainless steel standard sieves having standard designations as shown in Table 1.
- some chemical changes e.g., oxidation of unsaturated lipids, especially because of tremendous surface area generated by cryogrinding; insolubilization or destabilization of proteins; and degradation of pigments and vitamins and other biomolecules
- Reduction of freezer temperature will cause decline of the rates of the above reactions.
- tissue powder be stored at -40°C, under vacuum or inert gas, and in the dark, (to prevent any possible photo-catalytic reactions).
- Preliminary evaluation shows that various tissue powders stored under the above conditions for up to 2 months, did not show any physical changes (texture, color, odor, etc.) in the product.
- tissue powder To use the uniform cryoground product, one should desirably "thaw" the tissue powder. Since thawing of non-fluid tissues is inherently slower than freezing, when comparable temperature differentials are employed (due to different thermal properties of ice vs. water). Hence, tissue powders may be subject to damage by chemical or physical (and less microbial or enzymatic) means. In light of these considerations, one skilled in the art will recognize that the thawing process must be carefully considered.
- porcine omentum Two samples of porcine omentum were prepared by the preferred brittle grinding technique followed by hexane extraction as discussed in section (Id) supra. Also see McCluer, R., et al, Lipids, (May 1987) reporting feline omentum extract characterization which may be modified for porcine omentum lipids.
- the two porcine hexane extracts (known as HxFrl and HxFr III) were fractionated by a method diagrammed in Figure 1 and described infra.
- Neutral lipids obtained from the evaporation of the hexane phase were next fractionated on a silica gel column, such as a Unisil column, to remove a large quantity of triglycerides eluted with benzene from minor neutral lipids eluted with a benzene/ethyl/acetate/acetic acid mixture.
- Fatty acids of the triglycerides were analyzed by gas chromatography (GC) and mass spectrometry (MS) based on methyl esters obtained by alkaline methanolysis.
- Polar lipids obtained from the ethanol phase were partitioned according to the Folch method following Folch et al., J. Biol. Chem. 226: 497-509 (1957) and described in copending U.S. patent application, serial no. 811,505 filed on December 20, 1985 by Catsimpoolas et al and entitled "Lipids from Omentum and Methods for Cosmetic Use", herein incorporated by reference (i.e., the fraction was dissolved in chloroform/methanol (2:1, 20 volumes, v/w) and 0.2 volumes of water were added. Phases were thoroughly mixed, and allowed to separate.)
- Chloroform, methanol and water were used to obtain upper and lower phase components.
- the lipids in the upper phase were absorbed onto a C-18 reverse phase cartridge; eluted with methanol, acidic lipids (i.e., gangliosides) and neutral lipids (i.e., complex glycolipids); and separated on a DEAE - sephadex column.
- the Folch lower phase lipids were run on a Unisil chromatography column and the following three fractions were obtained: (1) minor neutral lipids eluted with chloroform;
- the triglyceride fraction eluted by benzene constituted about 97% of the first hexane extract (HXFrl).
- the remaining ten (10) percent consisted of 14:0 and fatty acids that were present at less than 1% of the total.
- the minor neutral lipids were further fractionated by preparative thin layer chromatography (TLC) into 13 components.
- the major components of the minor neutral lipids were free fatty acids, cholesterol and diglycerides in the appropriate ratio of 1:1:0.5, as estimated by the TLC charring.
- the remaining components were present in trace amounts identified as various diglyceride molecular species determined by direct probe mass spectrometry and fatty acid analysis. Most of the monoglycerides distributed into the ethanol phase were seen in the chloroform fraction but were eluted from the Unisil column. Analysis of Ethanol Phase Lipids
- a chloroform fraction of approximately 0.44% by weight of the total HXFrl fraction was obtained and analyzed by TLC. This fraction was found to contain the same neutral lipids found in the hexane layer discussed supra.
- Cholesterol and free fatty acids make up about 51% of this fraction; monoglycerides represent 12%; diglycerides represent 4%; and triglycerides represent 26%.
- Neutral glycolipids in the acetone fraction were determined by TLC analysis to represent less than .001% of the HxFrl extract.
- glycolipids in this fraction were only 0.05% of the total fraction weight. Several non-polar components were present in this fraction which represent the majority of the fraction weight.
- the phospholipid content of the HxFrl was approximately
- the neutral fraction of the HxFrl extract sample is less than 0.001% of the total HxFrl, and contains globosides and three other polyglycosyl ceramides.
- the glycolipids of this neutral fraction represent about 16% by weight. The majority of this percentage is contributed to phospholipid, which is partitioned into the upper phase with the gangliosides and polyglycolyl ceramics.
- Gangliosides represent less than 0.001% of the total HxFrl extract in this fraction as analyzed by TLC. This fraction contains 4 major gangliosides. Approximately 33% of the ganglioside was GM3 , 33% GDla 25% GMl and 8% GD3.
- the second hexane extract sample (HxFrlll) composition was found to be almost" identical to the composition of the first sample, except a significant amount of volitiles, probably moisture, was found in the initial preparation.
- the amount of 18:2 fatty acid in the triglyceride fraction (5%) was decreased and the quantity of the polar lipids (i.e., phospholipids and glycolipids) was almost doubled as determined from fraction weights and TLC analysis.
- the ganglioside content of HxFrlll was estimated to be 0.001%.
- the solvent extract was subjected to rotary evaporation (under vacuum, 37 °C) until dryness, i.e., neither any solvent condensation occurs, nor any solvent odor is present.
- the whitish CMFr weighing 70+2% of the omentum was then collected.
- the total hexane phase is then subjected to rotary evaporation (under vacuum, 37°c) until a translucent hexane extract of chloroform/methanol fraction is obtained (HxCMFr).
- HxCMFr translucent hexane extract of chloroform/methanol fraction
- a supercritical fluid such as SC-CO 2 is preferably used to extract omentum.
- An SCF has increased solvation power at temperatures above the critical pressure (Pc) and critical temperature (Tc).
- Polar materials such as gangliosides remain in the residue while the extract contains the more non-polar or lipid materials such as triglycerides. Temperatures used for example are 38-39 °C and extractor pressure is about 3500 psig. Thus these conditions can avoid extraction using toxic materials, or inefficient extraction or use of expensive and time-consuming extractions and materials.
- a process development unit is used.
- the PDU consists of an extractor and three separators, which are housed in an oven at a predetermined temperature.
- a supercritical solvent in this case, CO 2
- CO 2 is pumped into the extractor and then flows, sequentially, through separators, and the "knock out" vessel.
- Pressure is maintained by back pressure regulators. Gas, e.g., CO 2 , at approximately normal atmospheric pressure, exits the vent, when the extraction is completed.
- the vessels were purged with low pressure CO 2 , and were then brought to the temperatures and pressures indicated in Tables 2-3. CO 2 was then pumped at a rate of about 0.3 lb/min through the system until a weight of about 200 times the sample weight was pumped. Pressure was bled, and samples in each of the vessels and extractor were removed, weighed, and analyzed.
- the residues obtained from the extractor were found to be insoluble in CO 2 .
- Polar portions of the material, such as gangliosides were expected to remain in the fraction, while extract fractions were expected to be rich in neutral, non-polar components. This has been observed to be the case, as the insoluble residue is found to contain polar materials, such as gangliosides, while non-polar materials, such as triglycerides, are found in the extracts .
- Lipids are displaced from homogenized cell membranes, or other complexes involving proteins, by amphipathic detergent molecules which render the proteins "soluble” in aqueous media. The released lipid material is recovered by flotation after centrifugation.
- the omentum was weighed, spread out on to a plastic surface and cut into approximately 4 cm pieces using surgical scissors.
- the omental pieces (which range in weight from 30 to 66 gm per cat) were placed in a Waring Blender containing 300 ml of phosphate buffered saline (PBS) precooled to 4°C.
- PBS phosphate buffered saline
- homogenization was performed for 5 minutes at 20,500 rpm.
- the resulting homogenate was centrifuged in 250 ml plastic bottles at 1600 g in a refrigerated centrifuge at 4°C for 20 minutes. After centrifugation, three fractions were visible in the bottles, i.e. a pellet, a turbid homogenate and a floating cream colored cake.
- the cake was removed after decantation of the homogenate and weighed.
- the blender as well as all the solutions were pre-cooled at 4°C for one hour before use.
- the cake was homogenized in a Waring blender for 2 min with chloroform/ methanol (2:1, v/v) solvent at room temperature at a ratio of 1 gm cake to 10 ml of solvent.
- the particulate matter was removed by centrifugation at 2000 g in a clinical centrifuge at room temperature for 10 minutes. The clear supernatant was then subjected to rotary evaporation at 37 °C under vacuum to remove the chloroform- methanol mixture.
- total lipid extract also known as the lipid extract and/or the chloroform-methanol extract or CME, and the CMFr or chloroform-methanol extracted fraction of the omentum.
- This hexane fraction is also known as the the neutral lipid fraction from omentum or the HxCMFr (hexane phase material from the 95% Ethanol Hexane partition of the CMFr).
- HxCMFr hexane phase material from the 95% Ethanol Hexane partition of the CMFr.
- Other hydrocarbons e.g., pentane, cyclohexane, cyclopentane, benzene, etc.
- Methanol, n-propanol, and acetoni-trile can be used instead of ethanol for the above separation.
- Other methods can also be used as seen and obvious to those skilled in the art in Kates, M.
- a N 2 atmosphere for example can be used to prevent oxidation during extraction.
- the neutral lipid fraction was obtained by applying 200 mg of a total omentum lipid extract (CMFr) dissolved in chloroform to a 10 gm silica gel column and eluting with 25 column volumes of chloroform. This fraction was analyzed for fatty acid content by gas chromatography-mass spectrometry (GC-MS). In order to liberate the fatty acids and form their methyl esters an aliquot (2.5 mg) of the neutral lipid fraction was dissolved in 0.67 ml of chloroform and 0.33 ml of 0.5 N methanolic sodium hydroxide was added. The sample was heated at 60°C for one hour, cooled and mixed with 0.2 ml of water to form two phases. The upper phase was removed and discarded.
- CMFr total omentum lipid extract
- the lower phase was evaporated to dryness with a stream of nitrogen.
- the fatty acid methyl esters (FAME) were redissolved in n-hexane for analysis by GC-MS.
- a Finnigan 4500 GC-MS was used to analyse the FAME.
- Gas chromatography was performed on a 30 M x 0.25 mM methyl silicone bonded phase fused silica capillary column (DB-1, J & W Scientific, Inc.) with helium as the carrier gas at 12 psi.
- the column oven temperature was programmed from 205°C to 300°C at 2°C/min with the exit end placed directly into the ion source of the mass spectrometer.
- Mass spectrometry was performed in the electron impact mode (70 eV) with positive ion detection. Spectra were collected and stored at 0.5 sec intervals.
- composition formulation Various omental material samples which may also be used in the composition formulation may be prepared as follows:
- the hair growth composition may be formulated in any conventional topical form such as lotions, creams, ointments, aerosols, sticks, gels, and other forms known in the art.
- a lotion is formulated having a percentage by weight of mammalian omental material ranging from about 5% to 20% of the topical preparation, preferably from about 15-20%.
- Carbomer 940 (2% soln.) Carbopol 940 (2% soln. ) B.F. Goodrich 5.0 Water Purified Water USP 48.4
- Carbomer 940 (2% soln. ) Carbopol 940 (2% soln..) B.F. Goodrich 0.07 Water Purified Water USP 40.16
- ACTIMOIST R owned by Active Organics of Van Nuys, California.
- the preferred sodium hyaluronate used in the manufacturing of the hair growth composition has the following chemical analysis:
- sodium hyaluronate content not less than 95%
- nucleic acid not more than 0.2;
- Vitamin A and Vitamin D are Vitamin A and Vitamin D
- Vitamins A & D preferably all-trans vitamin A palmitate and vitamin D 3 , respectively, are used in a ratio of 1 million units/gm to 200,000 units/gm to equal about 0.10% of the total solution by weight.
- a carbomer 940 solution which is 2% by weight of the composition formula is prepared. To prepare the solution the following ingredients are needed.
- Porcine omentum extract prepared by brittle grinding and direct hexane extraction was used in the preparation of this lotion.
- the ingredients of the oil phase are added as follows: Omentum, Sorbitan Stearate, Proplyparaben, Polysorbate 60, Glyceryl Stearate, Tocopheryl Acetate (Vitamin E), Vitamin A, Vitamin D, and Dow Corning 200 Fluid.
- the mixture is heated to preferably 70 °C while mixing to achieve uniformity of solution. DO NOT OVERHEAT.
- the oil phase is then added into the main kettle while mixing. Temperatures should be adjusted to 75°C. Triethanolamine, 99% and Phenoxyethanol are added into the combined mixture and the hot water from the jacketed kettle is drained to begin cooling the batch.
- the batch is continually mixed and cooled and at approximately 45 °C, sodium hyaluronate is added.
- the user wash his or her hair once a day, with a mild acting shampoo, such as a baby shampoo. Once the hair is dry, the lotion should be applied to bald and balding areas of the scalp and gently rubbed for approximately ten seconds.
- a mild acting shampoo such as a baby shampoo.
- lotion 2-36A (lotion I) is applied topically once or twice a day: in the morning after showering and in the evening before bedtime.
- This group consisted of men ranging in age from 23-59 years old having Hamilton types of IV to VIII.
- 17 reported new hair growth.
- 15 reported retardation or stoppage after use of the lotion.
- FIG. 2 The growth of new terminal hair on this test subject is illustrated in comparison Figures 2-4.
- Figure 2 At the time Figure 2 was taken, the subject had been using the Lotion 2-36-A for about six weeks. Distinctive new terminal hairs can be seen in the vertex area below the area containing transplanted hairs.
- Figure 3 includes two photographic views of the test subject's vertex after 2 1 ⁇ 2months of applying the Activa TM lotion to the balding area and shows new hair growth.
- Figure 4 is a photograph taken of the subject's vertex after about five months of treatment illustrating additional new hair, growth.
- Figures 2-4 one sees an increase of new hair growth in the bald area of the subject's vertex after only five months of topically applying the Lotion 2-36A to his balding vertex.
- the overall results of the new hair growth study indicate that approximately 80% of the subjects participating in these studies reported that hair loss had stopped after treatment with the inventive composition for only three weeks. Approximately 32% of the subjects reported new hair growth following treatment for
- Example I manufacturing method supra minus polysorbate 60 Fifty-eight (58) healthy male subjects ranging in age from 24-72 and having Hamilton types ranging from II -
- inventive composition may be made by combining sodium hyaluronate with any type of lipid or fatty material known in the art.
- lipids may be isolated from mammalian tissue, as well as plant tissue, according to techniques known in the art.
- EXAMPLE III A preferred cream formulation known as Activa TM Cream is as follows :
- Carbomer 940 (2% soln.) Carbopol 940 (2% soln.) B.F. Goodrich 12.50
- Trisodium EDTA Trisodium EDTA Dow 0.10
- Another preferred cream formulation is as follows for the inventive composition:
- inventive composition is preferably used in lotion form as cream formulations have been found to have a tacky or greasy feeling when applied to scalp areas.
- inventive composition is preferably used in lotion form as cream formulations have been found to have a tacky or greasy feeling when applied to scalp areas.
- variations of the proportions of formula will be obvious to those skilled in the art.
- the Ames mutagenicity test (Ames et al., (1975) Mutation Research 31: 347-364) performed on the porcine omentum extract (POCMFr) by Product Safety Laboratories of 725 Cranbury Rd, East Brunswick, New Jersey 08816.
- the mutagenic potential of the test material was measured by its ability to induce reverse "(Back") mutations in specially constricted strains of the bacterium Salmonella Typhimuruim.
- Mutagens which require activation by enzymes found in mammalian tissues are detected in the bacterial assay by the addition of mammalian microsomes (S-9 fractions).
- N-Methyl-N-nitro-N-nitrosoguanidine MNNG
- 9-amino- acridine 9AA
- 2-nitroflourene 2NF
- the Salmonella/mammalian microsome assay of Ames utilizes specially constructed strains of the bacterium, Salmonella typhimurium, to detect mutagenic activity of chemicals.
- the rationale for the test lies in the established correlation between mutagenic activity and carcinogenic potential, and, similarly, with birth defects and heritable disease.
- Gene (point) mutations are usually recognized as change in a phenotypic characteristic.
- Ames assay the dependence of the Salmonella tester strains on an exogenous source of histidine (auxotrophy) is altered by reverse mutation to histidine independence (prototrophy).
- auxotrophy an exogenous source of histidine
- prototrophy histidine independence
- the reversion occurs by either base-pair substitution or frameshift mutation, depending upon the strain.
- the various strains carry additional genetic markers which enhance their sensitivity to mutagens.
- tester strains are exposed to a series of doses of the test article at concentrations up to the highest practicable does within the limitations of toxicity or solubility. Mutagenic activity is manifested by an increase in the numbers of mutant colonies which grow in the absence of histidine, compared to the spontaneous (background) number of mutants. Mutagens which require activation by enzymes found in mammalian tissues, are detected in the bacterial assay by the addition of mammalian microsomes (S-9 fractions). Ames Test Materials: Bacterial Indicator Strains
- Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were originally obtained from Dr. Bruce N. Ames, Berkeley, California. Strains were propagated in nutrient broth containing 0.5% NaCl, checked for appropriate genetic markers, and stored frozen (Revco freezer) after addition of DMSO (dimethyl sulfoxide) to approximately 8% final concentration.
- DMSO dimethyl sulfoxide
- Top agar contained 0.6% Difco agar, 0.5% Nacl, 0.5 mM biotin, and 0.5 mM L-histidine-HCl.
- Minimal-glucose agar medium contained 1.5% Bacto-Difco agar in Vogel-Bonner Medium E (Vogel and Bonner, 1956) with 2% glucose.
- N-methyl-N-nitro-N-nitrosoguanidine MNNG
- 9-aminoacridine 9-AA
- 2-nitrofluorene 2-NF
- 2-aminoanthracene (2-AA) was used with all strains in the assay with metabolic activation as shown in Appendix 2.
- S-9 was thawed on the day of assay, diluted first in Tris-KCl buffer to a protein concentration of 10 mg/ml, then added to S-9 buffer to give a final concentration of 100 microliter S-9/ml.
- the complete S-9 mix was passed through a 0.45 um disposable Nalgene filter unit for sterilization and stored at approximately 4°C until use on the same day.
- the cultures used in this assay were started from the frozen permanents in 21 ml of nutrient broth with 0.5% NaCl and incubated 12-16 hours on a shaker water bath at 37°C.
- test article The effect of the test article on the survival of the bacterial strains was determined prior to the Ames bioassay. This was accomplished by adding 0.1 ml of different levels of the test article and solvent to tubes containing 2.0 ml top agar (at 45°C) and 0.1 ml of the tester strain. After mixing, the tube contents were poured onto the surface of tryptone agar plates. The plates were incubated at 37°C for 40-48 hours and the background lawn of bacteria in test article plates was compared to the bacterial lawn in the solvent plates. Toxicity on the tryptone agar plates was detectable by a thinning or disappearance of this background lawn of bacteria.
- Each tube was prepared individually, immediately vortexed, and the contents poured over the surface of a minimal-glucose agar plate. Each plate was rotated to evenly distribute the top agar before it hardened. All plates were incubated for approximately 48 hours at 37 °C. After incubation, the plates were observed for revertant colonies and the colonies were counted and recorded.
- test article Five doses of test article were tested in triplicate with each strain. Positive control articles for each strain and spontaneous revertant controls consisting of the indicator strains and solvent were run concurrently. The solvent and the highest dose level of the test material were checked for sterility by adding 0.1 ml of each to.2 ml of top agar without the test organisms and pouring the entire contents over minimal-glucose agar plates.
- the positive control article for each strain and spontaneous revertant controls consisting of the indicator strains and S-9 mix were run concurrently.
- the S-9 mix was checked for sterility by adding 0.5 ml to 2 ml of top agar without the test organisms and pouring the entire contents over minimal-glucose agar plates. All plates were incubated approximately 48 hours at 37°C.
- test material should exhibit a dose response effect
- test sample should produce a positive dose response over three concentrations with the lowest increase in revertants/plate greater than or equal to 3x the solvent control value or the S-9 fraction control value, as applicable.
- test sample should produce a positive dose response over three concentrations with at least one dose producing an increase in revertants/plate greater than or equal to 3.5 x the solvent control value or the S-9 fraction control value, as applicable.
- MMNG N-methyl-N-nitro-N-nitrosoguanidine
- 9-AA 9- aminoacridine
- 2-NF 2-nitrofluorene
- DMSO dimethyl- sulfoxide
- mice were each uniquely identified and individually housed in stainless steel wire bottomed cages in an environmentally controlled room with a 12 hour light/dark cycle. Feed and water were provided ad-libitum after dosing.
- FHSLA 16 CFR 1500.3.
- the rats were fasted for 18 hours and then individually and singly dosed by gavage with 5.0 g/kg body weight of test material.
- the rats were individually caged and observed for mortality or other signs of gross toxicity for 14 days. Feed and water were provided ad-libitum.
- the oral LD 50 of the Porcine HxFr test material is greater than 5.0 g/kg.
- test material was administered orally at a dose of 5g/kg to a group of 15 male and 15 females rats as described supra in Example XI.
- the MMTS of the PO HxCMFr was 1.00.
- the material is considered to be practically non-irritating (PN) .
- the MMTS of the BO HxCMFr was 0.00.
- the test material is considered to be non-irritating.
- test is considered positive if four or more of the animals in the test group exhibited a positive reaction. If only one an-imal exhibits a positive reaction, the test shall be regarded as negative. If two or three animals exhibit a positive reaction, the test is repeated using a different group of six animals.
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Dermatology (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Immunology (AREA)
- Virology (AREA)
- Birds (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Cosmetics (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Procédé et composition favorisant la pousse des cheveux et empêchant la chute des cheveux chez les mammifères présentant un type commun de calvitie. En particulier, la composition contient une substance lipide omentale isolée incorporée dans des compositions telles que des lotions, des crèmes, des pommades, des gels, ou sous la forme d'atomiseurs ou de ''sticks'' (bâtonnets).
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US94801786A | 1986-12-31 | 1986-12-31 | |
US948017 | 1986-12-31 | ||
US4690987A | 1987-05-05 | 1987-05-05 | |
US46909 | 1987-05-05 |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0296205A1 true EP0296205A1 (fr) | 1988-12-28 |
EP0296205A4 EP0296205A4 (fr) | 1989-03-14 |
Family
ID=26724431
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP19880900664 Withdrawn EP0296205A4 (fr) | 1986-12-31 | 1987-12-23 | Procede et composition pour la pousse des cheveux dans des calvities de type commun. |
Country Status (11)
Country | Link |
---|---|
EP (1) | EP0296205A4 (fr) |
JP (1) | JPH01502591A (fr) |
KR (1) | KR890700029A (fr) |
AU (1) | AU1100488A (fr) |
DK (1) | DK482588A (fr) |
FI (1) | FI883989A (fr) |
HU (1) | HUT48121A (fr) |
IE (1) | IE873549L (fr) |
IL (1) | IL84864A0 (fr) |
NO (1) | NO883848D0 (fr) |
WO (1) | WO1988004931A1 (fr) |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4814351A (en) † | 1987-06-26 | 1989-03-21 | Redken Laboratories, Inc. | Scalp treatment |
IE891173L (en) * | 1988-04-18 | 1989-10-18 | Univ Utrecht | Composition for promoting hair growth in androgenetic¹alopecia and method thereof |
GB2228736A (en) * | 1989-02-10 | 1990-09-05 | Sterivet Lab Ltd | Cosmetic formulation |
JP2005047876A (ja) * | 2003-07-31 | 2005-02-24 | P & P F:Kk | 液状組成物 |
KR100780577B1 (ko) * | 2005-12-23 | 2007-11-29 | 주식회사 바이웰 | 탈모 예방 및 양모 촉진용 천연 복합 조성물 |
EP2688576A4 (fr) | 2011-03-25 | 2014-12-03 | Cooperlabs Ltd | Extraits d'épiploon extraits à la chaleur, compositions, procédés de préparation et leurs utilisations |
MX2015015676A (es) | 2013-05-16 | 2016-03-04 | Procter & Gamble | Composiciones de espesamiento para el cabello y metodos de uso. |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1986001111A1 (fr) * | 1984-08-20 | 1986-02-27 | Trustees Of Boston University | Facteur d'angiogenese et procede d'obtention d'angiogenese |
WO1987001939A1 (fr) * | 1985-10-01 | 1987-04-09 | Angio-Medical Corporation | Compositions contenant des molecules de lipides ayant une activite angiogenique amelioree |
EP0237644A2 (fr) * | 1985-12-20 | 1987-09-23 | Trustees Of Boston University | Lipides de l'omentum et leurs applications cosmétiques |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4215178A (en) * | 1978-04-28 | 1980-07-29 | Chloeta F. Martin | Non-stick rubber liner |
SU812291A1 (ru) * | 1979-05-28 | 1981-03-15 | Институт Микробиологии Ан Узбекскойсср | Способ получени липидов |
FR2492659A1 (fr) * | 1980-10-27 | 1982-04-30 | Wiel Gaston | Composition destinee a etre utilise |
DE3480171D1 (en) * | 1983-06-20 | 1989-11-23 | Kao Corp | Hair growth promoting composition |
EP0158090B1 (fr) * | 1984-03-07 | 1990-08-29 | Roshdy Dr. Ismail | Médicament pour le traitement et la protection de la peau |
JPS60186949U (ja) * | 1984-05-21 | 1985-12-11 | 新日本製鐵株式会社 | 連続鋳造用鋳鍋装架台車 |
FR2569347B1 (fr) * | 1984-08-22 | 1987-10-23 | Mollet Marc | Composition lipidique favorisant la pousse des phaneres et produits cosmetiques en renfermant |
US4695452A (en) * | 1984-10-10 | 1987-09-22 | Gannis Peter M | Cosmetic stick |
IT1177276B (it) * | 1984-11-20 | 1987-08-26 | Farmaka Srl | Composizioni cosmetiche dermotrope |
JPH0721113A (ja) * | 1993-06-30 | 1995-01-24 | Sega Enterp Ltd | マルチプロセッサシステム |
-
1987
- 1987-12-17 IL IL84864A patent/IL84864A0/xx unknown
- 1987-12-23 KR KR1019880701059A patent/KR890700029A/ko not_active Application Discontinuation
- 1987-12-23 JP JP63501210A patent/JPH01502591A/ja active Pending
- 1987-12-23 EP EP19880900664 patent/EP0296205A4/fr not_active Withdrawn
- 1987-12-23 AU AU11004/88A patent/AU1100488A/en not_active Abandoned
- 1987-12-23 WO PCT/US1987/003429 patent/WO1988004931A1/fr not_active Application Discontinuation
- 1987-12-23 HU HU881285A patent/HUT48121A/hu unknown
- 1987-12-30 IE IE873549A patent/IE873549L/xx unknown
-
1988
- 1988-08-29 NO NO883848A patent/NO883848D0/no unknown
- 1988-08-30 FI FI883989A patent/FI883989A/fi not_active IP Right Cessation
- 1988-08-30 DK DK482588A patent/DK482588A/da unknown
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1986001111A1 (fr) * | 1984-08-20 | 1986-02-27 | Trustees Of Boston University | Facteur d'angiogenese et procede d'obtention d'angiogenese |
WO1987001939A1 (fr) * | 1985-10-01 | 1987-04-09 | Angio-Medical Corporation | Compositions contenant des molecules de lipides ayant une activite angiogenique amelioree |
EP0237644A2 (fr) * | 1985-12-20 | 1987-09-23 | Trustees Of Boston University | Lipides de l'omentum et leurs applications cosmétiques |
Non-Patent Citations (1)
Title |
---|
See also references of WO8804931A1 * |
Also Published As
Publication number | Publication date |
---|---|
NO883848L (no) | 1988-08-29 |
FI883989A0 (fi) | 1988-08-30 |
DK482588D0 (da) | 1988-08-30 |
AU1100488A (en) | 1988-07-27 |
WO1988004931A1 (fr) | 1988-07-14 |
IE873549L (en) | 1988-06-30 |
HUT48121A (en) | 1989-05-29 |
KR890700029A (ko) | 1989-03-02 |
NO883848D0 (no) | 1988-08-29 |
IL84864A0 (en) | 1988-06-30 |
EP0296205A4 (fr) | 1989-03-14 |
JPH01502591A (ja) | 1989-09-07 |
DK482588A (da) | 1988-08-30 |
FI883989A (fi) | 1988-08-30 |
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