Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
  • A61Q19/04—Preparations for care of the skin for chemically tanning the skin
• • 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
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
  • A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
• • 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
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
  • A61K8/494—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with more than one nitrogen as the only hetero atom
  • A61K8/4953—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with more than one nitrogen as the only hetero atom containing pyrimidine ring derivatives, e.g. minoxidil
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
  • A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/74—Biological properties of particular ingredients
  • A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
  • A61K2800/782—Enzyme inhibitors; Enzyme antagonists

Definitions

• The-, present invention generally relates to compositions for topical application on a subject's skin, and specifically, to pigmentation enhancers, and to methods for assaying the effects of compositions on skin.
• composition of matter for stimulating melanogenesis in human epidermal cells comprising an effective amount of a melanin-increasing agent wherein the agent comprises a phosphodiesterase inhibitor capable of increasing levels of melanin in a human melanocyte; and an effective amount of pharmaceutically acceptable topical carrier capable of delivering the phosphodiesterase inhibitor to the melanocyte under in vivo conditions.
• the agent may also comprise methylxanthine.
• a method for blocking ultraviolet radiation comprising providing a pigment enhancer prior to exposure to the ultraviolet radiation, the pigment enhancer comprising: an effective amount of an agent capable of increasing the amount of melanin in a human melanocyte; and an effective amount of a pharmaceutically acceptable topical carrier capable of delivering the agent to a melanocyte.
• the present invention preferably comprises a composition of matter comprising an effective amount of a phosphodiesterase-inhibitor.
• the phosphodiesterase-inhibitor may be capable of increasing levels of melanin in a human melanocyte.
• the composition may also comprise an effective amount of a pharmaceutically acceptable topical carrier.
• the carrier is capable of delivering the phosphodiesterase- inhibitor to the melanocyte under in vivo conditions.
• a composition of matter of the present invention may also comprise an effective amount of a methylxanthine which is capable of increasing levels of melanin in a human melanocyte.
• the composition also comprises an effective amount of a pharmaceutically acceptable topical carrier capable of delivering the methylxanthine to the melanocyte under in vivo conditions.
• the present invention also preferably comprises a method for at least partially shielding the skin from ultraviolet radiation in a subject.
• the subject's skin is treated with a pigment enhancer comprising an agent capable of increasing the amount of melanin in a human melanocyte, and a pharmaceutically acceptable topical carrier as previously described.
• Another method of the present invention is a method of promoting tanning of a subject's skin.
• the skin is treated with a pigment enhancer as previously described.
• the present invention is also directed to a human organ culture comprising a viable human foreskin tissue specimen having an epidermis and a dermis, a nutrient medium, and a means for supporting the foreskin in the medium.
• the foreskin is supported such that the dermis contacts the medium, and the epidermis is in substantially non-contacting relationship with the medium.
• the human organ culture may be used to assay the effects of agents on skin.
• a baseline level of a biological factor in the foreskin is observed.
• the foreskin is contacted with the agent, and a post-contact observation made of the biological factor in the foreskin.
• Figure 1 is a plan view of the organ culture system of the present invention.
• Figure 2 is a side cross-section view of the organ culture system shown in Figure 1 taken along line 2-2 shown in Figure 1. Detailed Description of the Preferred Embodiments
• the present invention in one version is a composition of matter for stimulating melanogenesis in human epidermal cells, the composition comprises (1) an effective amount of a melanin-increasing agent wherein the agent comprises a phosphodiesterase inhibitor capable of increasing levels of melanin in a human melanocyte; and (2) an effective amount of pharmaceutically acceptable topical carrier capable of delivering the phosphodiesterase inhibitor to the melanocyte under in vivo conditions.
• This composition may further comprise an effective amount of tyrosine.
• the phosphodiesterase inhibitor of the composition may be from about O.lmM to about lOOmM.
• the phosphodiesterase inhibitor of the melanin-increasing agent may further comprise at least one member of the group comprising theophylline, isobutyl methylxanthine, aminophylline, andpharmaceuticallyacceptable salts of theophylline, isobutyl methylxanthine and aminophylline which are capable of increasing melanin in the human melanocyte. More preferably, the composition may comprise an effective amount of (1) a methylxanthine capable of increasing levels of melanin in a human melanocyte; and (2) an effective amount of pharmaceutically acceptable topical carrier capable of delivering the methylxanthine to the melanocyte under in vivo conditions.
• the methylxanthine may comprise at least one member of the group comprising theophylline, isobutyl methylxanthine, and pharmaceutically acceptable salts of theophylline or isobutyl methylxanthine which are capable of increasing melanin in the human melanocyte.
• the composition comprising a methylxanthine may further comprise an effective amount of tyrosine.
• the composition may further comprise a composition theophylline, and may further comprise an effective amount of tyrosine.
• the methylxanthine is isobutyl methylxanthine, and the composition further comprises an effective amount of tyrosine.
• the composition may further comprise an effective amount of dibutyryl cAMP, in addition to the methylxanthine.
• the methylxanthine may be in a concentration of from about 0.02 percent to about 2 percent by weight.
• compositions of matter described herein which contain methylxanthine or other phosphodiesterase inhibitors may further comprise an effective amount of tyrosine for serving as a tyrosinase substrate; and an effective amount of a pharmaceutically acceptable topical carrier capable of delivering the phosphodiesterase inhibitor and tyrosine to the melanocyte under in vivo conditions.
• the present invention also discloses a method for at least partially shielding skin from ultraviolet radiation in a subject.
• the method comprises treating the skin with a pigment enhancer prior to exposure to the ultraviolet radiation, in which the pigment enhancer comprises (1) an effective amount of an agent capable of increasing the amount of melanin in a human melanocyte; and (2) an effective amount of a pharmaceutically acceptable topical carrier capable of delivering the agent to a melanocyte under in vivo conditions.
• the agent may be a phosphodiesterase inhibitor, or more specifically a methylxanthine or more specifically yet at least one member of the group comprising theophylline, isobutyl methylxanthine, and analogs, derivatives, isomers and pharmaceutically acceptable salts of theophylline or isobutyl methylxanthine capable of increasing melanin in the human melanocyte and may further comprise an effective amount of tyrosine.
• the invention also discloses a method of promoting the tanning of a subject's skin, comprising the step of treating the skin with a pigment enhancer comprising (1) an effective amount of an agent capable of increasing the amount of melanin in a human melanocyte; and (2) an effective amount of a pharmaceutically acceptable topical carrier capable of delivering the agent to a human melanocyte under in vivo conditions, wherein the agent is as described herein and which may further comprise an amount of tyrosine.
• the invention further comprises a human organ culture for testing the effects of an agent on a subject's skin.
• the culture comprises (1) a viable human foreskin sample having an exposed epidermis and an opposing exposed dermis, (2) a nutrient medium, and (3) means for supporting the foreskin in the medium such that the dermis contacts the medium, while the epidermis is in substantially non-contacting relationship to the medium.
• the medium may have a liquid phase and the means for supporting may comprise a buoyant float.
• the invention further comprises a method for assaying the effects of an agent on skin.
• the method has the steps of (1) providing a human skin organ sample comprising: a human foreskin tissue specimen having an exposed epidermis and an opposing exposed dermis, (2) observing a biological factor in the foreskin at a baseline level, (3) contacting the foreskin with the agent under study, while maintaining the foreskin's viability, (4) observing the biological factor in the treated foreskin, and (5) comparing the baseline and the post-contact observations of the biological factor.
• the viability of the foreskin may be maintained by contacting the dermis with a nutrient medium.
• the nutrient medium may have a liquid phase and the dermis may be contacted with the nutrient medium by supporting the foreskin in the medium on a buoyant float such that the dermis is contacted by the nutrient medium, while the epidermis is in substantially non- contacting relationship to the medium.
• Skin color in humans arises from a complex series of cellular processes which are carried out within a unique population of cells called elanocytes. Melanocytes are located in the lower part of the epidermis, and their function is to synthesize a brown pigment, melanin, which protects the body from the damaging effects of ultraviolet radiation.
• melanocytes When skin is exposed to ultraviolet radiation, such as that contained in sunlight, melanocytes increase their synthesis of melanin. Melanin is deposited in melanosomes, which are vesicles found within the cell. The melanosomes are extruded from the cell and carried to the surface of the skin by keratinocytes, which internalize the melanin containing melanosomes. The end result is that the visible layers of the skin exhibit a brown color typically known as a "tan".
• the darkness of the color observed in the skin is proportionate to the amount of melanin synthesized by melanocytes and transferred to the keratinocytes.
• skin can be at least partially shielded from ultraviolet radiation, and tanning of the skin can be achieved without exposure of the skin to potentially damaging ultraviolet radiation.
• the present invention is directed to compositions and methods which increase melanogenesis in subjects.
• Subjects as used herein mean mammals, and, more preferably, humans.
• the present invention comprises a composition of matter comprising an effective amount of an agent capable of increasing the amount of melanin in a melanocyte (hereafter "melanin-increasing agent") and, more preferably, in a human melanocyte.
• the melanin- increasing agent may function in any of the various ways that are believed to increase the amount of melanin: by increasing cAMP or derivatives of cAMP which function as cAMP in the melanocyte; by inhibiting phosphodiesterase; or by increasing tyrosinase activity.
• the melanin-increasing agent should be capable, alone or with the aid of a carrier as described hereafter, of crossing the melanocyte membrane, and more preferably, a human melanocyte membrane, in order to function as will be described.
• the melanin-increasing agent of the present invention comprises at least one phosphodiesterase- inhibitor.
• Phosphodiesterase-inhibitors are believed to increase the amount of cAMP in biological systems.
• cAMP is believed to increase the activity of tyrosinase, which is the rate limiting enzyme which transforms the substrate tyrosine into melanin.
• the melanin-increasing agent of the present invention comprises at least one methylxanthine, and preferably a methylxanthine capable of functioning as a phosphodiesterase inhibitor.
• the agent comprises theophylline, isobutyl methylxanthine, or a derivative thereof such as aminophylline.
• melanin-increasing agents of the present invention may be combined in order to enhance pigmentation of the individual.
• the melanin-increasing agent may also comprise any pharmaceutically acceptable salt, analog, derivative or iso er of any phosphodiesterase inhibitor or any methylxanthine capable of functioning as described herein.
• composition of matter of the present invention may additionally comprise an effective amount of a pharmaceutically acceptable topical carrier capable of delivering the agent to the melanocyte under in vivo conditions.
• the carrier may comprise any solution, suspension, emulsion or any other form which is capable of delivering the agent to the melanocyte under in vivo conditions.
• Capable of delivery means that the carrier should permit the agent to cross the stratum corneum and successive cell layers found epidermal to the melanocyte, and/or aids the agent in crossing the melanocyte membrane. Furthermore, the carrier should not substantially interact with the agent so that the agent may perform its function as described herein.
• the carrier will depend on the identity of the melanin-increasing agent used in the composition of the present invention. However, in many instances, the carrier will represent from about 95 percent to about 99 percent of the composition.
• the carrier may comprise 20% water, 50% isopropanol and 30% glycerol, combined with theophylline to produce a composition containing 0.2 percent by weight theophylline.
• the carrier may comprise 50% propylene glycol, 20% water and 30% isopropanol to produce a composition containing 0.2 percent by weight theophylline.
• the melanin-increasing agent is present in the composition of the present invention in any effective amount.
• An "effective amount" of the melanin-increasing agent is an amount which increases melanogenesis in the treated area of the subject. This amount may vary with, among other things, the identity of melanin-increasing agent and carrier, the subject's skin color and condition, and the degree of tanning and/or photoprotection sought.
• the melanin- increasing agent is present in the composition from about 0.01 mM to about 100 mM.
• 18 mg of theophylline may be combined with 10 ml of a pharmaceutically acceptable topical carrier to cover about a 50 square inch area of skin.
• 180 mg of theophylline may be combined with 10 ml of a pharmaceutically acceptable topical carrier to form a composition capable of covering about a 50 square inch area of skin.
• 2.2 mg of isobutyl methylxanthine may be combined with 10 ml of a pharmaceutically acceptable carrier to cover about a 50 square inch area of skin.
• the enzyme tyrosinase converts the substrate tyrosine into melanin.
• the composition of the present invention is believed to increase tyrosinase activity, the present invention preferably further comprises an effective amount of tyrosine so that the tyrosinase has as much substrate as possible to synthesize melanin.
• An "effective amount" of tyrosine means any amount which can be utilized by tyrosinase over the amount of tyrosine already present in the melanocyte.
• a preferable amount of tyrosine is about 0.01 mM to about 1 mM of the composition of the present invention.
• 1.8 mg of tyrosine may be combined with 18 mg of theophylline and 10 ml of a pharmaceutically acceptable topical carrier.
• composition of matter of the present invention is preferably applied directly to the skin of the individual seeking tanning of the skin and/or photoprotection from ultraviolet radiation.
• the treated area can be the entire skin surface of the subject or only those areas normally exposed to ultraviolet radiation. Since the composition of the present invention does not dye the skin, but rather permits the body chemistry to produce the tan, the tanning will not be streaked in the areas of application. Application of the composition may be repeated periodically for greater protection and/or tanning effect.
• composition of matter is preferably in a lotion or solution form which may be manually rubbed on the skin.
• Other means of application are acceptable such as aerosol sprays or the use of an applicator bottle.
• the present invention comprises cells in situ in an organ and maintains the viability of the organ in order to emulate the human skin in vivo. It is the development of a human organ culture system of the present invention which has permitted the adequate evaluation and development of the compositions and methods of treatment of the present invention.
• the human organ culture of the present invention employs a viable foreskin. "Viable" means there has been no substantial morphological change in the foreskin after surgical removal. Viability may be determined by changes in tissue ultrastructure determined through histochemical staining and/or dopa reaction staining, techniques which permit monitoring of any changes in the tissue ultrastructure.
• the foreskins may be obtained by circumcising male neonates by standard surgical procedures. After surgical removal, the foreskin is preferably prepared for the organ culture by injection intradermally with the medium described hereafter. This swells the mucous membrane and allows for the removal of the membrane thereby allowing adequate nutrient flow to the foreskin through the dermis.
• the foreskin comprises an epidermis which is normally exposed to the environment and a dermis opposing the epidermis. After surgical removal and preparation for the organ culture, the dermis, which is normally supplied nutrients by the body, is exposed to the environment. In order to maintain the viability of the foreskin, a nutrient medium supplies nutrients to the foreskin through the dermis as described hereafter.
• the nutrient medium is any composition which maintains the viability of the foreskin.
• the nutrient medium has a liquid phase such as a solution, suspension or emulsion.
• a portion of the medium may be obtained commercially, such as Iscove's modified Dulbecco's medium (IMDM) , Ham's nutrient mixture F-10 medium, Minimum essential media (MEM), RPMI media 1630 or 1640, Dulbecco's Modified Eagle Media (D-MEM) or Media 199 all of which are manufactured by Gibco Laboratories of Grand Island, New York as well as other companies, the specification sheets of which are hereby incorporated by reference.
• IMDM Iscove's modified Dulbecco's medium
• MEM Minimum essential media
• RPMI media 1630 or 1640 RPMI media 1630 or 1640
• D-MEM Dulbecco's Modified Eagle Media
• the medium comprises about 10% to about 30% horse serum and about 2% to about 10% fetal bovine serum; the serums may be purchased from Hyclone Lab Inc., of Logan, Utah. If necessary, an alkalizer such as sodium bicarbonate may be added until the medium achieves a preferred pH, preferably about a physiological pH. Antibiotics such as penicillin and/or streptomycin may also be added for microbial control.
• the foreskin is immediately placed on an absorbent support saturated with the nutrient medium.
• the foreskin is disposed in the medium within about 3-4 hours after surgical removal.
• the position of the foreskin in the medium should be that the dermis contacts the medium and the epidermis is not substantially contacted by the medium.
• the present invention comprises a foreskin 12, having an epidermis 14 and a dermis 15.
• the foreskin 12 is placed in the medium 16 so that the medium 16 contacts the dermis 15 but does not substantially contact the epidermis 14.
• the epidermis 14 is in contact with the environment as under in vivo conditions.
• the medium 16 nourishes the foreskin 12, and maintains its viability while the organ culture is under study. About 7 days or less are generally needed to complete such a study.
• the medium 16 comprises a liquid phase and is disposed in a container such as a petri dish 26. The amount of medium needed will depend upon, among other things, the container and the support means used. Generally about 5 ml to about 10 ml is a sufficient amount of medium for one foreskin in a petri dish having a diameter of 1.5 inches with the foreskin supported on a float as described hereafter.
• the foreskin 12 may be supported by any means which will permit positioning of the foreskin in the medium 16 as previously described.
• the foreskin 12 is disposed on a buoyant and permeable float 18 having a top 20 and a bottom 22; the dermis 15 contacts the top 20 of the float 18.
• the float 18 floats on top of the medium 16.
• the float 18 preferably comprises a sterile plastic screen such as a Swinnex 25 cm filter support screen manufactured by Millipore. More preferably, a permeable pad 24 is interposed between the dermis 15 and the top 20 of the float 18. The pad 24 functions to hold the foreskin 12 on the float 18.
• a pad 24 used in accordance with the present invention is a AP20 025 00 filter manufactured by Millipore. Because both the float 18 and pad 24 are permeable to the culture medium 16, the medium maintains contact with the dermis 15 while the foreskin is positioned on the float 18, as required to maintain the foreskins viability. The float 18 is placed on top of the medium 16 and may freely move about the top of the medium 16.
• the organ culture system is incubated during the length of observation of the foreskin.
• the medium is changed daily, since nutrients may be depleted over time, and the incubation causes degradation of medium components.
• the foreskin is surgically removed, prepared and disposed in the organ culture system as described herein.
• the foreskin should be observed to determine the amount and/or the condition of the biological factor under study to obtain a baseline measurement.
• the biological factor is again observed for a post-treatment measurement to be compared to the baseline measurement. For example, if the amount of tyrosinase in the foreskin is under study, the amount of tyrosinase is determined as a baseline measurement prior to application of the agent to the foreskin.
• the foreskin may be observed for DNA synthesis, protein synthesis, ATP synthesis, or any combination of these cell functions. If the permeability of an agent on skin is to be determined, the absence of the agent in the skin is the baseline measurement.
• biological factors which may be studied are tyrosinase, melanin, DNA, RNA, proteins, carbohydrates, or any other factor that can be measured or observed for differences between the treated and the untreated foreskins.
• the agent should be added to the medium 16, so that it comes into contact with the foreskin 12 through the medium 16.
• the agent can be placed directly on the epidermis 14.
• the treatment time will depend on the results sought, the identity of the agent under study, the time over which the foreskin can remain viable, and other variables.
• the foreskin may be observed and/or tested in any manner which will determine the differences in the foreskin from the observation and/or tests on the untreated foreskin.
• the activity of tyrosinase may be measured as described herein and correlated to an increase in melanogenesis; the rate of DNA synthesis can be measured by 3 H-thymidine uptake and compared to controls; or the increase or decrease in the synthesis of proteins and/or RNA can be measured by determining the rate of incorporation of [ 3 H] leucine (protein) or [ 3 H] uridine (RNA) into acid- precipitating material.
• the organ culture system of the present invention offers an opportunity to study the effects of many agents on human skin, and many biochemical processes of human skin. Factors other than pigmentation processes, and agents other than pigmentation enhancers may be evaluated for effects on the skin. For example, cosmetic agents or suspected toxic environmental compounds may be tested for their effects on skin morphology, DNA synthesis, abnormal chromosome appearance, skin cell viability, and protein and RNA synthesis. Also, the permeability of agents through the epidermis and the dermis may be studied in order to evaluate agents for use in transdermal delivery systems for systemic delivery of the agent.
• Example 1 illustrates the practice of the method and composition of the present invention.
• the organ culture medium was prepared from IMDM with glutamine supplemented with 20% horse serum, 5% fetal bovine serum, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and 3 mg/ml sodium bicarbonate.
• the serums were obtained from Hyclone Lab. Inc. of Logan Utah.
• Culture units were prepared by placing sterilized filters (AP20 025 00, Millipore) over sterilized support screens (25 cm Swinnex filter support screens, Millipore) in the wells of 6-well tissue culture plates (Falcon 3046) with medium added to the wells such that the skin support screens floated and the filter absorbed the medium from beneath.
• the tissue samples were placed, epidermis up, on top of the saturated filters and incubated at 37°C in a 5% C0 2 humidified atmosphere. The medium was changed everyday. Harvested cultures were frozen at -75°C.
• Tyrosinase activity in human skin organ cultures was determined by measuring the tyrosine hydroxylase activity of the enzyme. The assay measures the production of 3 H 2 0 during the conversion of [ 3 H]tyrosine to L-DOPA. Weighed skin preparations were incubated in 0.3 ml of a reaction mixture containing 0.01 mM of L-tyrosine, 5-6 uCi/ml of [ 3 H]tyrosine and O.lmM L-DOPA in 0.1 M of pH 6.8 phosphate buffer for 4 hours at 37°C.
• both black and white human foreskins were placed in culture and at 24 hours intervals skin sections were removed for histological examination by H&E staining and by dopa staining.
• the human foreskin retained its normal morphology throughout the 7 day length of the study. There was no difference in structural viability between black and white skin.
• dopa oxidase activity of cultured skin melanocytes was examined by dopa staining, a similar staining pattern between day 0 and day 7 foreskin cultures was observed, showing that the culture conditions maintain the viability of the skin for many days.
• Foreskins placed in culture within 3-4 hours of surgical removal showed normal structural integrity throughout the culture period whereas samples placed in culture more than 4 hours after removal did not maintain structural integrity.
• Foreskins treated with ⁇ -MSH (melanocyte stimulating hormone from Sigma, St. Louis, Mo.) showed an increase in tyrosinase activity whereas foreskins treated with
• D-phe-MSH (Sigma, St. Louis, Mo.) showed a marked stimulation of tyrosinase. This shows that the organ culture system can respond to hormones. A stronger stimulation of tyrosinase was found with theophylline. Human foreskins were treated with the compounds shown in Table 1, placed in culture, and incubated for 72 hours. The skins were then assayed for tyrosinase activity with the tyrosine hydroxylase assay method as described herein. As shown in Table 1, dibutyryl cAMP was less effective in stimulating tyrosinase activity.
• Table 2 shows the activity of theophylline in a pharmaceutical topical carrier. Organ cultures were incubated with the compounds shown in Table 2 for 72 hours. The medium and compounds were changed daily. Tyrosinase activity was determined by measuring the tyrosine hydroxylase activity of the enzyme.
• dbcAMP is dibutyryl cAMP
• Values are the averages of triplicate determinations ⁇ s.d.
• Values are the averages of triplicate assays ⁇ s.d.
• 20 _Values are the averages triplicate determinations ⁇ s.d.

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• 1993
  • 1993-12-21 WO PCT/US1993/012521 patent/WO1995017161A1/en not_active Application Discontinuation
  • 1993-12-21 AU AU59590/94A patent/AU5959094A/en not_active Abandoned
  • 1993-12-21 EP EP94905498A patent/EP0735857A1/de not_active Withdrawn

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