(19) T ZZ _T
(11) EP 2 448 577 B1
(12) EUROPEAN PATENT SPECIFICATION
(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A61K 31/4375 (2006.01) A61K 9/00 (2006.01) 14.09.2016 Bulletin 2016/37 A61K 9/06 (2006.01) A61K 9/08 (2006.01) A61P 17/00 (2006.01) A61P 17/06 (2006.01) (2006.01) (2006.01) (21) Application number: 10793513.2 A61P 17/08 A61K 45/06
(22) Date of filing: 30.06.2010 (86) International application number: PCT/CN2010/000983
(87) International publication number: WO 2011/000218 (06.01.2011 Gazette 2011/01)
(54) COMPOSITIONS CONTAINING BERBERINE OR ANALOGS THEREOF FOR TREATING ROSACEA OR RED FACE RELATED SKIN DISORDERS ZUSAMMENSETZUNGEN MIT BERBERIN ODER ANALOGEN DAVON ZUR BEHANDLUNG VON MIT ROSACEA ODER ANDEREN GESICHTSRÖTUNGEN ZUSAMMENHÄNGENDEN HAUTERKRANKUNGEN COMPOSITIONS CONTENANT DE LA BERBÉRINE OU DES ANALOGUES DE CELLE-CI POUR TRAITER L ACNÉ ROSACÉE OU DES TROUBLES CUTANÉS ASSOCIÉS À UNE ROUGEUR FACIALE
(84) Designated Contracting States: (56) References cited: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB CN-A- 1 085 600 CN-A- 1 182 788 GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO CN-A- 1 923 199 DE-U1-202010 004 750 PL PT RO SE SI SK SM TR JP-A- 63 179 812 KR-A- 20030 082 200 US-A1- 2006 286 054 US-B1- 6 440 465 (30) Priority: 30.06.2009 US 221725 P US-B1- 6 482 839
(43) Date of publication of application: • REUTER JULIANE ET AL: "Botanicals in 09.05.2012 Bulletin 2012/19 dermatology: an evidence-based review.", AMERICAN JOURNAL OF CLINICAL (73) Proprietor: Derman Biomedicine Co. Ltd. DERMATOLOGY 2010, vol. 11, no. 4, 2010, pages Taoyuan County 33380 (TW) 247-267, ISSN: 1175-0561 • LÍVIA SLOBODNÍKOVÁ ET AL: ’Antimicrobial (72) Inventors: activity of Mahonia aquifolium crude extract and • HUNG, Shuen-lu its major isolated alkaloids’, [Online] vol. 18, no. Zhanghua City 500 (TW) 8, 01 August 2004, pages 674 - 676, XP008158021 • CHUNG, Wen-Hung DOI: DOI:10.1002/PTR.1517 ISSN: 0951-418X Nantong County 541 (TW) PHYTOTHERAPY RESEARCH, JOHN WILEY & • CHANG, Tse-Wen SONS LTD. CHICHESTER, GB Retrieved from the Taipei City 103 (TW) Internet:
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• JUQIANG ET AL: "In vitro effects of the traditional • SEKI T ET AL: "Effect of some alkaloids, chinese medicine, berberine, matrine and flavonoids and triterpenoids, contents of baicalin, on the proliferation and lipid synthesis Japanese-Chinese traditional herbal medicines, of immortalized human sebocyte SZ95", on the lipogenesis of sebaceous glands.", SKIN ZHONGHUAPIFUKE ZAZHI - CHINESEJOURNAL PHARMACOLOGY :THE OFFICIALJOURNAL OF OF DERMATOLOGY, ZHONGGUO YIXUE THE SKIN PHARMACOLOGY SOCIETY 1993, vol. KEXUEYUAN PIFUBING YANJIUSUO, NANJING, 6, no. 1, 1993, pages 56-60, XP008176201, ISSN: CN, vol. 38, no. 11, 1 November 2005 (2005-11-01), 1011-0283 pages 662-664, XP008158075, ISSN: 0412-4030
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Description 3.1. Genetic contribution
BACKGROUND OF THE INVENTION [0005] Earlier studies have indicated genetic predis- position to flushing, the earliest manifestation of facial 1. Rosasea and its major symptoms 5 rosacea10. Additionally, glutathione S-transferase MU-1 (GSTM1) and glutathione S-transferase theta 1 (GSTT1) [0001] Rosacea is a chronic skin disease that mani- null genotype has been reported to be associated with fests redness and swelling, primarily on the face, espe- an increased risk of rosacea11. cially on the central facial area. Other areas affected in- clude the scalp, neck, ears, chest, back and the eyes. 10 3.2. Inflammation and innate immune system Rosacea is characterized by facial flushing, erythema, telangiectasia, and inflammatory episodes with papules [0006] As rosacea progresses, inflammatory lesions and pustules and, in severe cases, rhinophyma. Come- become evident. Unlike acne vulgaris, inflammatory ro- dones are notably absent1. sacea is not a bacterial disease of the pilosebaceous [0002] Patients with rosacea mostly have increased 15 unit. Comedones are usually not present, and only nor- sensitivity of the facial skin and dry, flaking facial derma- mal bacterial flora is identified in skin samples taken from titis, edema of the face, and persistent granulomatous rosacea patients12. The inflammatory stage of rosacea papulonodules2. According to clinical and histopatholog- can be regarded as a form of chronic sterile cellulitis13. ic features, the disease can be classified into 4 subtypes: While the presence of microorganisms has been exam- (a) erythematotelangiectatic, (b) papulopustular, (c) phy- 20 ined as a potential contributing factor to rosacea, results matous, and (d) ocular, each with 3 grades of severity have been inconclusive1. Demodex folliculorum mites (mild, moderate, severe)3. The course of the disease is are considered as commensal and do not play a signifi- typically chronic, with recurrent remissions and relapses. cant pathogenic role in rosacea, although an inflamma- tory reaction to the mites may aggravate symptoms 14. 2. Other red face related skin disorders 25 [0007] Yamasaki et al found an abnormally high level of cathelicidins by histopathological staining in skin le- [0003] Rosacea is the most common red face skin dis- sions from patients with rosacea. Human epidermal ke- order. Other red face related skin disorders, which share ratinocytes stimulated by cathelicidin peptides were symptomatic similarities and probably pathological caus- found to increase the release of IL-8. Injection of cathe- es, include acne vulgaris, seborrheic dermatitis, photo- 30 licidin peptides into the skin of mice caused inflammatory dermatitis and contact dermatitis. These red face related changes with increased neutrophil infiltration and micro- conditions may range from feelings of heat and sensitivity vessels characteristic of the skin disorder of rosacea in to flushing or burning with intense sensitivity4. Patients humans15. Cathelicdins possibly have dual roles in im- with rosacea and other red face related skin disorders munity because it can both kill microorganisms and stim- often exhibit extreme sensitivity to environmental and 35 ulate host inflammatory responses such as inducing IL- topical factors5. Steroid-induced rosacealike dermatitis 8 release16. Other inflammatory cytokines found to be (or steroid rosacea) is a papular or pustular lesions with increased in rosacea include IL-1alpha and transforming erythematous and edematous base with or without tel- growth factor beta-217,18. angiectasia., which is caused by prolonged application of topical steroids to the face or as a rebound condition 40 3.3. Vascular mediators after discontinuation of topical steroids 6. 7 (Chen AY Zir- was MJ, 2009; Lee DH, Li K, Suh DH 2008). EGFR in- [0008] Inflammatory mediators may be responsible for hibitors, such as cetuximab, erlotinib, gefitinib, cause ac- the vasodilation seen in rosacea patients. For example, neiform dermatitis on face or other skin area, including substance P, histamine, serotonin, bradykinin, or pros- papulopustular reaction, erythema, telangiectasias, and 45 taglandins have been suggested19 . Smith et al has re- flushing in 30 to 90 % of patients and may also superin- ported an increased expression of vascular endothelial fected with bacteria, such as staphylococcus aureus8,9 growth factor and its receptors in rosacea20. (Wollenberg A, Kroth J et al, 2010; Lacouture ME, Mait- land ML et al, 2010). 4. Current management of rosasea 50 3. Pathogenesis of rosacea [0009] A number of antibiotics, such as tetracycline and doxycycline have been used in treating rosacea. It [0004] The etiology of rosacea is not well understood. has been suggested that such antibiotics render anti-in- Various factors have been suggested to contribute to the flammatory rather than antimicrobacterial effects. How- development and manifestation of rosacea. None of55 ever, other anti-inflammatory agents are not effective in them, however, has been definitely confirmed1. treating rosacea. Immunosuppressive agents such as corticosteroids often worsen the inflammatory condition of rosacea1.
3 3 EP 2 448 577 B1 4
[0010] Topical metronidazole and certain systemic an- sacea and other skin problems 31. The composition of this tibiotics are often used as first-line therapy for rosacea. invention includes antimicrobial agents selected from Oral tetracycline, doxycycline, and minocycline are com- several agents including berberine. In these neutraceu- monly used for treating rosacea. The efficacy of oral an- tical compositions, berberine is included as one of the tibiotics is probably due more to anti-inflammatory rather 5 many ingredients and its concentration is not specified. than to antibiotic effects 21. Azelaic acid 15% gel was ap- There has been a 10% Mahonia aquifolium cream (Re- proved by FDA of USA in 2002 for the topical treatment lieva™, Apollo Pharmaceutical Canada Inc) containing of mild to moderate rosacea22. Other traditional topical 0.1% berberine for the treatment of psoriasis 32. US pat- agents that have been used in a "off label" fashion include ent application 2006/0286054 describes the treatment of clindamycin, sulfacetamide and sulfur, but their mecha- 10 skin disorders and discloses compositions comprising nism is not well understood. psorberine, which is an undefined extract from the Ma- honia aquifolium plant39. 5. The use of berberine in non-skin disorders [0015] The therapeutic effect of berberine in treating rosacea and other red face-related skin disorder is un- [0011] Berberine (Natural Yellow 18, 6-dihydro-9,10- 15 known. Until now, there is no direct evidence suggesting dimethoxybenzo(g)-1,3-benzodioxolo (5,6-a) quinolizin- that berberine can improve the symptoms of rosacea. ium) is an isoquinoline alkaloid present in herb plants, such as coptis ( Coptidis rhizome), phellodenron, Scutel- DESCRIPTION OF THE INVENTION laria baicalensis, Mahonia aquifolium and berberis23. Berberine and its derivatives have been found to have 20 [0016] There is a need for an effective therapy for the antimicrobial and antimalarial activities. It can act against treatmentof rosaceaand other related skin disorders with various kinds of pathogens such as fungi, saccharomyc- minimal side effects. The present invention pertains to ete, parasite, bacterium and virus 24. Berberine has been topical pharmaceutical formulations that are effective found to have other potential benefits. For example, it and safe in treating rosacea and other red face related may have potential to treat high blood cholesterol, car- 25 skin disorders, selected from the group consisting of se- diovascular disease, diabetes, and tumor25. borrheic dermatitis, contact dermatitis photodermatitis [0012] Berberine also has anti-inflammatory function, steroid-induced rosacea-like dermatitis, and EGFR in- yet the exact mechanism is unknown. Recently, some hibitors-induced acneiform dermatitis. In particular, the researcher reported that the anti-inflammatory mecha- present inventionprovides a topicalpharmaceutical com- nism of berberine is mediated through cyclooxygenase- 30 position for use in treating red face related skin disorders, 2 (COX-2) pathway, since COX-2 plays a key role in the comprising at least 0.02% w/w of berberine or a biolog- synthesis of prostaglandins, which is elevated inically equivalent analogue of berberine, wherein the bi- inflammation26. Berberine is used as an ingredient in ologically equivalent analogue of berberine is selected some eye drop solution or eye ointment for the treatment from the group consisting of jatrorrhizine, palmatine, cop- of tracoma27. 35 tisine, 9-demethylberberine, 9-demethylpalmatine, 13- hydroyberberine, berberrubine, palmatrubine, 9-O-ethyl- 6. The use of berberine in skin disorders befberrubine, 9-O-ethyl-13-ethylberberrubine, 13-meth- yldihydroberberine N-methyl salt, tetrahyprotoberber- [0013] US patent #6440465 pertains to topical skin for- ines and N-methyl salts thereof, and 9-lauroylberberru- mulations of glucosamine in an emollient base which con- 40 bine chloride, wherein berberine or the biologically equiv- tains berberine for the treatment of psoriasis28. Patent alent analogue of berberine is the only pharmaceutically application #20050158404 pertains to a nutritional prod- active component, and wherein the red face related skin uct, dietary supplement or pharmaceutical composition disorder is selected from the group consisting of rosacea, which contains vitamin A, vitamin E, selenium, vitamin seborrheic dermatitis, photodermatitis, contact dermati- B6, zinc, chromium, and a herbal source of berberine for 45 tis, steroid-induced rosacea-like dermatitis, and EGFR the treatment of acne in oral administration 29. US patent inhibitors-induced acneiform dermatitis. Preferably, the #6974799 relates to topical compositions comprising a red face related skin disorder is rosacea. In various em- tripeptide (N-palmitoyl-Gly-His-Lys) and a tetrapeptide bodiments, the concentration of berberine or the biolog- (N-palmitoyl-Gly-Gln-Pro-Arg) for the treatment of visible ically equivalent analogue of berberine is between about signs of aging including wrinkles, stretch marks, dark 50 0.1% and about 2% w/w. circles30. The formulation may contain additional ingre- [0017] This invention recognizes the deficiency in cur- dients, including berberine. In these inventions, berber- rently available topical pharmaceutical formulations or ine is included as one of the many ingredients and its experimented formulations that contain berberine as a concentration is not specified. component, and improves over this deficiency. [0014] US patent application #20040146539 relates to 55 [0018] There are lines of evidence that indicate that topical neutraceutical compositions with body slimming berberine is a drug active ingredient in animal studies and tone-firming anti-aging benefits that may be used to and human clinical trials of berberine, either with purified treat skin aging, skin wrinkle, skin exfoliating, acne, ro- berberine or formulations containing berberine herbal ex-
4 5 EP 2 448 577 B1 6 tract. In many disease indications, such as in the treat- trations on biological pathways that may be involved in ment of bacterial and fungal infections and cardiovascu- the pathogenesis of rosacea. Based on those results and lar diseases, statistically significant efficacy results of therationale described above, we have developed chem- berberine have sometimes been obtained. In the trials ically defined topical pharmaceutical formulations that on psoriasis with formulations containing berberine-rich 5 contain berberine at defined percentages that are higher extract, efficacious results were also obtained, although than the concentrations of berberine in traditional herbal the efficacy of berberine in psoriasis has not been ac- berberine-containing pharmaceutical formulations. We cepted. These results suggest that berberine can act on then tested those formulations on affected skin area on molecular targets and cause modifications in certain mo- patients with rosacea. Our findings indicate that topical lecular pathways and cellular functions, such as de-10 pharmaceutical formulations containing berberine above scribed in the background section in this patent applica- 0.1% (w/w) can achieve efficacious and tolerable results tion. in treating rosacea and related sensitive red face disor- [0019] It has been clearly shown in the pharmacolog- ders. ical studies of numerous pharmaceutical compounds that a pharmaceutically active compound must be present in 15 Analogs of berberine the body or affected tissues above certain threshold con- centrations for the drug to achieve meaningful biological [0023] The structure of berberine (5,6-dihydro-9,10- and pharmacological effects and hence therapeutic ef- dimethoxybenzo(g)-1,3-benzodioxolo (5,6-a) quinolizin- fects in the treated subject. In herbal medicinal prepara- ium) is shown below: tions that contain the extracts of one or multiple plant(s), 20 many active drug ingredients are present. In most treat- ments using herbal preparations either in an oral or top- ical route, the individual drug ingredients are present at sub-threshold concentrations in the body or affected tis- sues of a treated subject. However, several compounds 25 from the same or different plants may act on the same molecular target or several compounds from the same plant or different plants may act on different molecular targets in the same biological pathway. As a result, the various compounds acted concertedly to cause a mean- 30 ingful biological and pharmacological effect and hence [0024] Several protoberberine alkaloids can be pre- therapeutic effect. pared with variable biological activity similar to berberine, [0020] When a herbal pharmaceutical preparation fails such as: jatrorrhizine, palmatine, coptisine , 9-demethyl- to cause a therapeutic effect in a treated subject, it is berberine, 9-demethylpalmatine, 13-hydroyberberine, likely that an otherwise pharmacologically active com- 35 berberrubine, palmatrubine, 9-O-ethylberberrubine , 9- pound contained therein is present at too low a concen- O-ethyl-13- ethylberberrubine, 13-methyldihydroberber- tration in the treated subject and that the compound by ine N-methyl salt, tetrahydroprotoberberines, and their itselfor in combinationwith compounds in thepreparation N-methyl salts, 13-Hexylberberine, 13-hexylpalmatine fail to cause a meaningful biological and pharmacological and 9-lauroylberberrubine chloride33,34. effect. In fact, many important drug compounds (single 40 [0025] Palmatine is present in plants of various fami- chemical entities) have been identified and isolated from lies, most notably in the rhizomes of Fibrarurea Tinctoria plants that are used in herbal preparations. With these Lour. Palmatine is an isoquinoline alkaloid and formula- pure compounds, therapeutic efficacy often exceed that tions containing palmitine have been broadly used in Chi- is achievable with the herbal preparations that contain na for the treatment of gynecological inflammation, bac- the compounds. 45 illary dysentery, enteritis, respiratory tract infection, uri- [0021] Topical herbal pharmaceutical formulations nary infection. Additionally, palmatine has the function of that include berberine-rich plant extracts have been used anti- arrhythmia, antisepticise, bacteriostasis, and anti- for centuries in the treatment of various ailments, includ- viral activities. Palmatine can be also used as a com- ing a variety of skin disorders, such as psoriasis, acne, pound in anti-tumor drug screening 35. There has been a eczema, etc. These topical herbal preparations have50 palmatine-containing pharmaceutical as topical hair achieved variable results. In some of those preparations, growth inhibitor (Keramene, Divine Skin Solutions D S berberine -containing extract consists of about 10% of Laboratories Keramene Body Hair Minimizer). the various components used to constitute the formula- [0026] Coptisine is an alkaloid found in Chinese tion. It has been estimated that the berberine compound goldthread (Coptis chinensis). It is used in Chinese herb- in those topical total preparations accounts approximate- 55 al medicine along with the related compound berberine ly 0.1 % (w/w) of the finished formulations 32. for treating digestive disorders caused by bacterial infec- [0022] Based on the above rationale, we have inves- tions. Coptisine also exhibits some significant inhibition tigated in vitro the effects of berberine at various concen- on tumor growth. Coptisine has been shownin vitro to
5 7 EP 2 448 577 B1 8 be cytotoxic on human tumor colon cell line36, human Example 2: Preparation of topical pharmaceutical hepatoma and leukaemia cell lines37. formulations containing purified berberine and pal- [0027] In our studies, we have also investigated in vitro matine at defined percentages and in vivo the effects of palmatine, and coptisine at var- ious concentrations on biological pathways that may be 5 [0030] Based on the rationale described above, the involved in the pathogenesis of rosacea. Based on those topical berberine-containing pharmaceutical formula- results and the rationale described above, we have also tions of this invention have one key feature: it contains developedchemically defined topical pharmaceutical for- purified berberine at defined percentages that are higher mulations that contain palmatine or coptisine at defined than can be obtained in previous formulations using ex- concentrations. These formulations could achieve effica- 10 tracts of berberine-rich plants. The ranges of concentra- cious and tolerable results in treating rosacea and related tions were subjected to tests in animal model studies and sensitive red face disorders. human clinical studies. [0031] For our studies on animal models and human Example 1: Effects of berberine on inhibiting cathe- patients, purified berberine was dissolved in 100% eth- licdin peptides-induced cytokine secretion by hu- 15 anol, and then water was added to reach a desired con- man keratinocytes (in vitro assay) centration of berberine in the final solution. In the gel formulation, for example, 0.1 % or 0.2% berberine was [0028] For our in vitro study, berberine (Sigma, St. Lou- prepared in 10% ethanol. The solution or gel formulation is, MO, USA) was dissolved in water, methanol, ethanol were capped and stored at 4°C until use. The results of or dimethyl sulfoxide (DMSO). Normal human keratino- 20 our studies in animal models and human patients with cytes (Invitrogen, CA, USA) were grown in EpiLife me- rosacea indicate that the concentration of berberine in dium (Invitrogen, CA, USA) supplemented with 0.06 mM the formulation should be 0.1% or higher, in order to Ca+2, 1% EpiLife defined growth supplement, and 1% achieve consistently satisfactory results. These concen- penicillin/streptomycin (Invitrogen, CA, USA). Cells were trations are higher than previously prepared topical ber- 25 grown at 37°C in a humidified atmosphere of 5% CO2 berine-containing formulations using berberine-rich plant and 95% air. The human keratinocytes were cultured to extract. confluence and treated with synthetic cathelicidin pep- [0032] Experiments are on going to prepare formula- tides (LL-37) (6.4 mM) for 16 h to induce inflammatory tions in the form of an ointment, gel, cream, lotion, or response similar to that observed in rosacea. Some of spray, which are more suitable for use for clinicians and the cathelicidin-treated keratinocyte cultures were co-in- 30 patients. In the topical pharmaceutical formulations of cubated with berberine of concentrations from 1.25 mg/ml our invention, berberine or a biologically equivalent an- to 12.5 mg/ml. The keratinocytes cultures treated with alog of berberine (e.g. palmatine and coptisine) is the cathrlicidin or cathrlicidin with 1% ethanol and without only or primary active drug compound. The purified pal- berberine were used as negative controls. Supernatants matine used for our studies is dissolved in 100% water, were collected and placed in a sterile 96-well plate for 35 and then diluted to reach in the final solution or gel for- ELISA of interleukin-8 (IL-8), interleukin-1 alpha (IL-1 al- mulation with defined palmatine concentrations, for ex- pha), and venous epithelial cell growth factor (VEGF) in ample, 0.02%, 0.1 %, or 0.2% of palmitine. accordance with the manufacturer’s instructions (R&D [0033] However, improved or modified formulations Systems, MN, USA). may include additional ingredients for increased solubility [0029] The result showed that cathelicidin can induce 40 of berberine or its analogue, emulsification, lubrication, IL-8, IL-1 alpha and VEGF release from cultured human antibiotic activity, or hydration. keratinocytes. The inhibitory effect of berberine on the [0034] One preferred embodiment of our invention to release of IL-8 (Figure 1A), IL-1 alpha (Figure 1B) and increase the solubility of berberine or a biological equiv- VEGF (Figure 1C) was examined by adding different con- alent analog of berberine is to add glycerol into the for- centrations (0∼12.5 mg/ml) of berberine in the culture me- 45 mulation. One embodiment of our invention to increase dium. There was 31.4%, 24.9 % and 29.1% decrease of the antibiotic activity of the formulation is to add plant the release of IL-8, IL-1 alpha and VEGF respectively, extract that has been shown to have antibiotic activity. when cathelicidin-stimulated keratinocytes treated with One embodiment to enhance the hydration property of 1.25 mg/ml berberine comparing to cathelicidin peptide- the topical formulation of our invention is to add hyaluron- treated with 1% ethanol control (P < 0.05). These results 50 ic acid. showed that berberine can significant inhibit cathelicidin induced inflammatory response in a dose-dependent Example 3: The effects of the topical pharmaceutical manner, especially when the concentration of berberine preparation of this invention on a mouse model of was larger than 6.25 mg/ml, indicating that berberine has rosacea anti-inflammatory activity against cathelicidin-induced 55 release of cytokines, which were related to rosacea. [0035] The animal model of rosacea: the animal model of rosacea was adopted from previous reported 18. Brief- ly, BALB/c and C57BL/6 mice, shaved 24 h before treat-
6 9 EP 2 448 577 B1 10 ments, were injected subcutaneously on the back with ment was 4 (1-24) years. Among the 20 patients with 40 ml of cathelicdin peptide (320 mM) twice a day. Forty- rosacea, 13 cases were of erythematotelangiectatic type eight hours after the initial injection (four injections in to- (65%), 7 cases papulopustular type (35%), and 5 cases tal), erythema and edema were observed on the injected (25%) phymatous type. site mimicking the clinical features of rosacea. 5 [0040] According to the 7-point score system, IGA [0036] In our experiments, cathelicidin-injected mice score of rosacea at baseline (initiation of treatment) was were treated with or without topical berberine twice a day 4.16 1.3. This score decreased to 2.6 6 0.9 at week 2, to observe the effect of berberine on reducing inflamma- then 1.6 6 0.8 at week 6. The difference of IGA scores tion. The results showed that mice given subcutaneous between week 0, week 2, and week 6 was statistically injections of cathelicidin peptides induced erythema and 10 significant (W2 vs W0: paired t test P < 0.0001; W6 vs vascular dilatation in the skin, which resembled clinical W0: paired t test P < 0.0001). At the beginning of treat- features of rosacea after 48 h. The cathelicidin-injected ment, the majority of patients (95%) had grading from mice were then divided into 2 groups, which were treated mild to moderate (3) to severe (6). By the end of treat- with berberine (n=3) or not treated with berberine (n=3; ment, 19 of the 20 patients (95%) had a mild (2) to clear as controls), respectively, for a subsequent 2 days. The 15 (0) rating. topical formulation containing 0.1% berberine was ap- [0041] The overall erythema severity evaluated by the plied on the cathelicidin-induced lesions twice a day. The investigator was 2.35 6 0.6 at the beginning of treatment, erythematous or inflammatory lesions lasted for more 1.5 6 0.5 at week 2, and 0.956 0.4 at week 6. The than 7 days in the control group. At the 4 th day, erythema improvement at week 2 or week 6 was statistically sig- and vascular dilatation were significant reduced in the 20 nificant (W2 vs W0: paired t test P < 0.0001; W6 vs W0: berberine treated group comparing to controls. These paired t test P < 0.0001). At the beginning of treatment, results indicate that topical berberine can reduce the in- the majority of patients (95%) had erythema rating from flammatory reaction induced by cathelicidin in vivo. moderate (2) to severe (3). By the end of treatment, 19 of the 20 patients (95%) had a mild (1) to none (0) ery- Example 4: A human clinical study investigating the 25 thema rating. efficacy of the topical pharmaceutical formulation of [0042] Safety and tolerability: There was no serious this invention on patients with rosacea adverse event during the study. Only 2 cases (10%) had transient itchy/stinging sensation in the area of topical [0037] Method: an open-label clinical study was car- medication, but were tolerable without discontinuation of ried out to determine the efficacy of the topical berberine 30 study. formulations of this invention for the treatment of rosacea and related skin disorders. Patients included in this study Example5: Topicalberberine is effective for thetreat- were diagnosed by dermatologists to have clinically de- ment of steroid-induced rosacealike dermatitis, as fined rosacea. All patients were given 0.1% berberine gel well as EGFR inhibitors induced acneiform dermati- twice a day for 6 weeks. At the time points of treatment 35 tis initiation, and 2-weeks and 6-weeks after treatment, the patients were evaluated for their rosasea symptoms. The [0043] We also studied the 0.1% berberine gel on 10 patients were not allowed to use other medications, in- patients with steroid-induced rosacealike dermatitis, 5 cluding antibiotics, for their skin conditions. Only oral an- patients with EGFR inhibitors induced acneiform derma- tihistamines were allowed for relief of pruritus symptoms. 40 titis, used in twice a day for 6 weeks. All 15 patients [0038] To evaluate the efficacy of treatment, the stand- showed similar effective and tolerated response as ob- ard grading system for rosacea developed by the Nation- served with rosacea. al Rosacea Society Expert Committee on the Classifica- tion and Staging of Rosacea was used 3. Additionally, the Example 6: Palmitine showed efficacy for the treat- investigator’s global assessment (IGA) and overall ery- 45 ment of rosacea or red face disorders thema severity of the patients were scored at week 0, week 2, and week 6 of berberine therapy. The IGA was [0044] We also studied topical formulation containing expressed according to a 7-point scoring system with a palmatine at 0.02% (w/w) on 10 patients with rosacea range of 0 (clear) to 6 (severe). The severity of overall and related red face disorders. All 10 patients showed facial erythema and telangiectasia, respectively, was50 similar effective and tolerated response as observed with graded as ’none’, ’mild’, ’moderate’, or ’severe’ with berberine. scores from 0 to 3. The grading system used to assess overall facial erythema severity was describedConclusion made from the examples: previously38. [0039] Results: a total of 20 patients with rosacea (18 55 [0045] In vitro culture studies have demonstrated that females and 2 males) were enrolled in this study. The berberine exhibits anti-inflammatory effects by inhibiting mean age of the study population was 43.3 (19-85) years. cathelicidin-induced IL-8, IL-1 alpha and VEGF produc- The mean duration of rosacea prior to berberine treat- tion by human keratinocytes. Since inflammation is in-
7 11 EP 2 448 577 B1 12 volved in the pathogenesis of rosacea and related skin 6.Chen AY, Zirwas MJ. Steroid-induced rosacealike disorders, the anti-inflammatory effects of berberine may dermatitis: case report and review of the litera- account for its clinically beneficial effect in rosacea and ture.Cutis. 2009 83(4):198-204. related inflammatory skin disorders. [0046] The results of our clinical studies have shown 5 7. Lee DH, Li K, Suh DH. Pimecrolimus 1% cream that the topical pharmaceutical formulations of this inven- for the treatment of steroid-induced rosacea: an 8- tion containing purified berberine at concentrations high- week split-face clinical trial. Br J Dermatol. 2008; er than 0.1% or palmatine at concentrations higher than 158(5):1069-76. 0.02% can be efficacious, safe and well tolerable for the treatment of rosacea and red skin related disorders, such 10 8. Wollenberg A, Kroth J, Hauschild A, Dirschka T. as acne, contact dermatitis, seborrheic dermatitis and Cutaneous side effects of EGFR inhibitors--appear- photodermatitis, steroid-induced rosacealike dermatitis, ance and management. Dtsch Med Wochenschr. and EGFR inhibitors induced acneiform dermatitis. 2010;135(4):149-54.
BRIEF DESCRIPTION OF THE FIGURES 15 9. Lacouture ME, Maitland ML, Segaert S, et al. A proposed EGFR inhibitor dermatologic adverse [0047] event-specific grading scale from the MASCC skin toxicity study group.Support Care Cancer. Figure 1. Berberine inhibited cathelicidin peptide 2010;18(4):509-22. (LL-37)-induced IL-8, IL-1alpha and VEGF release 20 from human keratinocytes. Keratinocytes were stim- 10. Palleschi GM, Torchia D. Rosacea in a monozy- ulated by cathelicidin peptide (LL-37), and the re- gotic twin. Australas J Dermatol. 2007; 48:132-133. lease of IL-8 (Figure 1A), IL-1 alpha (Figure 1B) and VEGF (Figure 1C) by the keratinocytes was evalu- 11. Yazici AC, Tamer L, Ikizoglu G, Kaya TI, Api H, ated by ELISA assay. 25 Yildirim H, Adiguzel A. GSTM1 and GSTT1 null gen- otypes as possible heritable factors of rosacea. Pho- Figure 2. A. Investigator’s global assessment scores todermatol Photoimmunol Photomed. 2006; at the beginning of berberine treatment and at 2 22:208-210. weeks and 6 weeks of treatment. B. Overall ery- thema severity scores at the beginning of topical ber- 30 12. Jansen T, Plewig G. Rosacea: classification and berine treatment and at week 2 and week 6 of treat- treatment. J R Soc Med. 1997; 90:144-150. ment 13. Wilkin JK. Rosacea. Pathophysiology and treat- REFERENCES CITED ment. Arch Dermatol. 1994; 130:359-362. 35 [0048] 14. Forton F, Seys B: density of Demodex folliculo- rum in rosacea: A case- control study using stand- 1. Plewig G, Jansen T. Rosacea. In: Freedberg IM, ardized skin-surface biopsy. Br J Dermatol. 1993; Eisen AZ, Wolff K, et al., eds. Dermatology in Gen- 128:650. eral Medicine. 6th ed. New York, NY: McGraw-Hill 40 Health Professions Division (2003) pp.688-696. 15. Yamasaki K, Di Nardo A, Bardan A, Murakami M, Ohtake T, Coda A, Dorschner RA, Bonnart C, 2. Lonne-Rahm SB, Fischer T, Berg M. Stinging and Descargues P, Hovnanian A, Morhenn VB, Gallo RL. rosacea. Acta Derm Venereol 1999; 79:460-461. Increased serine protease activity and cathelicidin 45 promotes skin inflammation in rosacea. Nat Med. 3. Wilkin J, Dahl M, Detmar M, et al. Standard grading 2007; 13:975-980. system for rosacea: report of the National Rosacea Society Expert Committee on the Classification and 16. Zuyderduyn S, Ninaber DK, Hiemstra PS, Rabe Staging of Rosacea. J Am Acad Dermatol 2004; KF. The antimicrobial peptide LL-37 enhances IL-8 50:907-912. 50 release by human airway smooth muscle cells. Al- lergy Clin Immunol. 2006; 117:1328-1335. 4. Griffiths WA. The red face-an overview and delin- eation of the MARSH syndrome. Clin Exp Dermatol. 17. Afonso AA, Sobrin L, Monroy DC, Selzer M, 1999; 24:42-47. Lokeshwar B, Pflugfelder SC. Tear fluid gelatinase 55 B activity correlates with IL-1alpha concentration 5. Draelos ZD. Assessment of skin barrier function and fluorescein clearance in ocular rosacea. Invest in rosacea patients with a novel 1% metronidazole Ophthalmol Vis Sci. 1999; 40:2506-2512. gel.J Drugs Dermatol. 2005; 4:557-562.
8 13 EP 2 448 577 B1 14
18. Pu LL, Smith PD, Payne WG, Kuhn MA, Wang antiaging benefits. United States Patent Application X, Ko F, Robson MC. Overexpression of transform- 20040146539 (2004). ing growth factor beta-2 and its receptor in rhinophy- ma: an alternative mechanism of pathobiology. Ann 32. Gulliver WP, Donsky HJ. A report on three recent Plast Surg. 2000; 45:515-519. 5 clinical trials using Mahonia aquifolium 10% topical cream and a review of the worldwide clinical expe- 19. Guarrera M, Parodi A, Cipriani C, et al. Flushing rience with Mahonia aquifolium for the treatment of in rosacea: a possible mechanism. Arch Dermatol plaque psoriasis. Am J Ther. 2005; 12:398-406. Res. 1982; 272:311-316. 10 33. Iwasa K, et al. Fungicidal and herbicidal activities 20. Smith JR, Lanier VB, Braziel RM, Falkenhagen of berberine related alkaloids. Biosci. Biotechnol. Bi- KM, White C, Rosenbaum JT. Expression of vascu- ochem. 2000; 64:1998-2000. lar endothelial growth factor and its receptors in ro- sacea. Br J Ophthalmol. 2007; 91:226-229. 34. Iwasa K, Nanba H, Lee DU, Kang SI. Structure- 15 activity relationships of protoberberines having anti- 21. McDonnell JK, Tomecki KJ. Rosacea: an update. microbial activity. Planta Med. 1998; 64:748-751. Clev Clinic J Med. 2000; 67:587-590. 35. Prabal Giri, Maidul Hossain and Gopinatha 22. Gupta AK, Gover MD. Azelaic acid (15% gel) in Suresh Kumar. RNA specific molecules: Cytotoxic the treatment of acne rosacea. Int J Dermatol. 2007; 20 plant alkaloid palmatine binds strongly to poly(A). 46:533-538. Bioorganic & Medicinal Chemistry Letters. 2006; 16:2364-2368. 23. Berberine (2000). Altern Med Rev. 5:175-177 36. Colombo M.L. et al. Cytotoxicity evaluation of 24. Yu HH, Kim KJ, Cha JD, et al. Antimicrobial ac- 25 natural coptisine and synthesis of coptisine from Ber- tivityof berberinealone and in combinationwith amp- berine. Farmaco 2001; 56:403-409. icillin or oxacillin against methicillin-resistant Staphy- lococcus aureus. J Med Food. 2005; 8:454-461. 37. Chun-Ching Lin et al. Cytotoxic effects of Coptis chinensis and Epimedium sagittatum extracts and 25. Mantena SK, Sharma SD, Katiyar SK. Berberine, 30 their major constituents (berberine, coptisine and a natural product, induces G1-phase cell cycle arrest icariin)on hepatoma andleukaemia cell growth. Clin- and caspase-3-dependent apoptosis in human pros- ical and Experimental Pharmacology and Physiolo- tate carcinoma cells. Mol. Cancer Ther. 2006; gy 2004; 31:65-69. 5:296-308. 35 38. Thiboutot D, Thieroff-Ekerdt R, Graupe K. Effi- 26. Kuo CL, Chi CW, Liu TY. The anti-inflammatory cacy and safety of azelaic acid (15%) gel as a new potential of berberine in vitro and in vivo. Cancer treatment for papulopustular rosacea: results from Lett. 2004; 203:127-137. twovehicle-controlled, randomized phase III studies. J Am Acad Dermatol 2003; 48:836-845. 27. Khosla PK, Neeraj VI, Gupta SK, Satpathy G 40 Berberine, a potential drug for trachoma. Rev Int 39. Gomez HJ. Pharmaceutical compositions for the Trach Pathol Ocul Trop Subtrop Sante Publique. treatment of psoriasis. United States patent applica- 1992; 69:147-65. tion 2006/0286054 (2006).
28. Meisner; Lorraine Faxon. Topical composition 45 for the treatment of psoriasis and related skin disor- Claims ders. United States Patent 6440465 (2002). 1. A topicalpharmaceutical composition for use in treat- 29. Goodless, Dean R. Composition and method for ing red face related skin disorders, comprising at treatment of acne. United States Patent Application 50 least 0.02% w/w of berberine or a biologically equiv- 20050158404 (2005). alent analogue of berberine, wherein the biologically equivalent analogue of ber- 30. Lintner; Karl. Compositions containing mixtures berine is selected from the group consisting of jatror- of tetrapeptides and tripeptides. United States Pat- rhizine, palmatine, coptisine, 9-demethylberberine, ent 6974799 (2005). 55 9-demethylpalmatine, 13-hydroyberberine, berber- rubine, palmatrubine, 9-O-ethylberberrubine, 9-O- 31. Gupta, Shyam K. Topical nutraceutical compo- ethyl-13-ethylberberrubine, 13-methyldihydrober- sitions with selective body slimming and tone firming berine N-methyl salt, tetrahydroprotoberberines and
9 15 EP 2 448 577 B1 16
N-methyl salts thereof, and 9-lauroylberberrubine Revendications chloride, wherein berberine or the biologically equiv- alent analogue of berberine is the only pharmaceu- 1. Composition pharmaceutique topique pour utilisa- tically active component, and tiondans letraitement destroubles cutanés associés wherein the red face related skin disorder is selected 5 à une rougeur faciale, comprenant au moins 0,02 % fromthe groupconsisting of rosacea, seborrheic der- p/p de berbérine ou d’un analogue biologiquement matitis, photodermatitis, contact dermatitis, steroid- équivalent de la berbérine, induced rosacea-like dermatitis, and EGFR inhibi- dans laquelle l’analogue biologiquement équivalent tors-induced acneiform dermatitis. de la berbérine est sélectionné dans le groupe cons- 10 titué par la jatrorrhizine, la palmatine, la coptisine, la 2. The topical pharmaceutical composition for use ac- 9-déméthylberbérine, la 9-déméthylpalmatine, la cording to claim 1, wherein the red face related skin 13-hydroyberbérine, la berberrubine, la palmatrubi- disorder is rosacea. ne, la 9-O-éthylberberrubine, la 9-O-éthyl-13-éthyl- berberrubine, le sel N-méthyle de la 13-méthyldihy- 3. The topical pharmaceutical composition for use ac- 15 droberbérine, les tétrahydroprotoberbérines et leurs cording to claim 1, wherein the concentration of ber- sels N-méthyle, et le chlorure de 9-lauroylberberru- berine or the biologically equivalent analogue of ber- bine, la berbérine ou l’analogue biologiquement berine is between about 0.1% and about 2% w/w. équivalent de la berbérine étant le seul constituant pharmaceutiquement actif, et 20 dans laquelle le trouble cutané associé à une rou- Patentansprüche geur faciale est sélectionné dans le groupe constitué par la rosacée, la dermatite séborrhéique, la photo- 1. Topische pharmazeutische Zusammensetzung für dermatite, la dermatite de contact, la dermatite de die Verwendung bei der Behandlung von rotem Ge- type rosacée induite par les stéroïdes, et la dermatite sicht-bezogenen Hauterkrankungen, umfassend25 acnéiforme induite par les inhibiteurs de l’EGFR. mindestes 0,02% w/w Berberin oder ein biologisch äquivalentes Analogon von Berberin, 2. Composition pharmaceutique topique pour utilisa- wobei das biologisch äquivalente Analogon von Ber- tion selon la revendication 1, dans laquelle le trouble berin ausgewählt ist aus der Gruppe bestehend aus cutané associé à une rougeur faciale est la rosacée. Jatrorrhizin, Palmatin, Coptisin, 9-Demethylberbe- 30 rin, 9-Demethylpalmatin, 13-Hydroberberin, Berber- 3. Composition pharmaceutique topique pour utilisa- rubin, Palmatrubin, 9-O-ethylberberrubin, 9-O-ethyl- tion selon la revendication 1, dans laquelle la con- 13-ethylberberrubin, 13-Methyldihydroberberin N- centration de berbérine ou de l’analogue biologique- methyl-Salz, Tetrahydroprotoberberine und N-me- ment équivalent de la berbérine est comprise entre thyl-Salze davon, und 9-Lauroylberberrubinchlorid, 35 environ 0,1 % et environ 2 % p/p. wobeiBerberin oder dasbiologisch äquivalente Ana- logon von Berberin der einzige pharmazeutisch wirk- same Bestandteil ist, und wobei die rotes Gesicht-bezogene Hauterkrankung ausgewählt ist aus der Gruppe bestehend aus Ro- 40 sacea, seborrhoische Dermatitis, Photodermatitis, Kontaktdermatitis, Steroid-induzierte Rosacea-ähn- liche Dermatitis, und EGFR-Inhibitor-induzierte Ak- ne-ähnliche Dermatitis. 45 2. Topische pharmazeutische Zusammensetzung für die Verwendung nach Anspruch 1, wobei die rotes Gesicht-bezogene Hauterkrankung Rosacea ist.
3. Topische pharmazeutische Zusammensetzung für 50 die Verwendung nach Anspruch 1, wobei die Kon- zentration von Berberin oder dem biologisch äqui- valenten Analogon von Berberin zwischen ungefähr 0,1% und ungefähr 2% w/w liegt. 55
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REFERENCES CITED IN THE DESCRIPTION
This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.
Patent documents cited in the description
• US 20040146539 A [0014] • US 69747992005 A [0048] • US 20060286054 A [0014] • US 200401465392004 A [0048] • US 64404652002 A [0048] • US 200602860542006 A [0048] • US 200501584042005 A [0048]
Non-patent literature cited in the description
•PLEWIGG; JANSEN T. ROSACEA. et al. Derma- • JANSEN T ; PLEWIG G. Rosacea: classification and tology in General Medicine. McGraw-Hill Health Pro- treatment. J R Soc Med., 1997, vol. 90, 144-150 fessions Division, 2003, 688-696 [0048] [0048] • LONNE-RAHMSB ; FISCHERT ; BERG M.Stinging • WILKIN JK. Rosacea. Pathophysiology and treat- and rosacea. Acta Derm Venereol, 1999, vol. 79, ment. Arch Dermatol., 1994, vol. 130, 359-362 [0048] 460-461 [0048] •FORTONF; SEYS B. density of Demodex folliculo- • WILKIN J ; DAHL M ; DETMAR M et al. Standard rum in rosacea: A case- control study using stand- grading system for rosacea: report of the National ardized skin-surface biopsy. Br J Dermatol., 1993, Rosacea Society Expert Committee on the Classifi- vol. 128, 650 [0048] cation and Staging of Rosacea. J Am Acad Dermatol, • YAMASAKI K ; DI NARDO A; BARDAN A ; MU- 2004, vol. 50, 907-912 [0048] RAKAMI M ; OHTAKE T ; CODA A ; DORSCHNER • GRIFFITHS WA. The red face-an overview and de- RA ; BONNART C ; DESCARGUES P ; HOVNANI- lineation of the MARSH syndrome. Clin Exp Derma- AN A. Increased serine protease activity and cathe- tol., 1999, vol. 24, 42-47 [0048] licidin promotes skin inflammation in rosacea.Nat • DRAELOS ZD. Assessment of skin barrier function Med., 2007, vol. 13, 975-980 [0048] in rosacea patients with a novel 1% metronidazole • ZUYDERDUYN S ; NINABER DK ; HIEMSTRA PS ; gel. J Drugs Dermatol., 2005, vol. 4, 557-562 [0048] RABEKF. The antimicrobial peptide LL-37 enhances • CHEN AY ; ZIRWAS MJ. Steroid-induced rosacea- IL-8 release by human airway smooth muscle cells. like dermatitis: case report and review of the litera- Allergy Clin Immunol., 2006, vol. 117, 1328-1335 ture. Cutis., 2009, vol. 83 (4), 198-204 [0048] [0048] • LEE DH ; LI K ; SUH DH. Pimecrolimus 1% cream • AFONSO AA ; SOBRIN L ; MONROY DC ; SELZER for the treatment of steroid-induced rosacea: an M; LOKESHWAR B ; PFLUGFELDER SC. Tear flu- 8-week split-face clinical trial. Br J Dermatol., 2008, id gelatinase B activity correlates with IL-1alpha con- vol. 158 (5), 1069-76 [0048] centration and fluorescein clearance in ocular rosa- • WOLLENBERG A ; KROTH J; HAUSCHILD A ; cea. Invest Ophthalmol Vis Sci., 1999, vol. 40, DIRSCHKA T. Cutaneous side effects of EGFR in- 2506-2512 [0048] hibitors--appearance and management. Dtsch Med •PULL; SMITH PD; PAYNE WG; KUHN MA ; Wochenschr., 2010, vol. 135 (4), 149-54 [0048] WANG X ; KO F ; ROBSON MC. Overexpression of • LACOUTURE ME ; MAITLAND ML ; SEGAERT S transforming growth factor beta-2 and its receptor in et al. A proposed EGFR inhibitor dermatologic ad- rhinophyma: an alternative mechanism of pathobiol- verse event-specific grading scale from the MASCC ogy. Ann Plast Surg., 2000, vol. 45, 515-519 [0048] skin toxicity study group. Support Care Cancer, 2010, • GUARRERA M ; PARODI A ; CIPRIANI C et al. vol. 18 (4), 509-22 [0048] Flushing in rosacea: a possible mechanism.Arch • PALLESCHI GM ; TORCHIA D. Rosacea in a Dermatol Res., 1982, vol. 272, 311-316 [0048] monozygotic twin. Australas J Dermatol., 2007, vol. •SMITHJR; LANIER VB ; BRAZIEL RM ; FALKEN- 48, 132-133 [0048] HAGEN KM ; WHITE C ; ROSENBAUM JT. Expres- • YAZICI AC ; TAMER L ; IKIZOGLU G ; KAYA TI ; sion of vascular endothelial growth factor and its re- API H ; YILDIRIM H ; ADIGUZEL A. GSTM1 and ceptors in rosacea. Br J Ophthalmol., 2007, vol. 91, GSTT1 null genotypes as possible heritable factors 226-229 [0048] of rosacea. Photodermatol Photoimmunol Pho- tomed., 2006, vol. 22, 208-210 [0048]
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• MCDONNELL JK ; TOMECKI KJ. Rosacea: an up- • IWASA K et al. Fungicidal and herbicidal activities date. Clev Clinic J Med., 2000, vol. 67, 587-590 of berberine related alkaloids. Biosci. Biotechnol. Bi- [0048] ochem., 2000, vol. 64, 1998-2000 [0048] • GUPTA AK ; GOVER MD. Azelaic acid (15% gel) in •IWASAK; NANBA H ; LEE DU ; KANG SI. Struc- the treatment of acne rosacea. Int J Dermatol., 2007, ture-activity relationships of protoberberines having vol. 46, 533-538 [0048] antimicrobial activity. Planta Med., 1998, vol. 64, • BERBERINE. Altern Med Rev., 2000, vol. 5, 175-177 748-751 [0048] [0048] • PRABAL GIRI ; MAIDUL HOSSAIN ; GOPINATHA • YU HH ; KIM KJ ; CHA JD et al. Antimicrobial activity SURESHKUMAR. RNA specific molecules: Cytotox- of berberine alone and in combination with ampicillin ic plant alkaloid palmatine binds strongly to poly(A). or oxacillin against methicillin-resistant Staphylococ- Bioorganic & Medicinal Chemistry Letters, 2006, vol. cus aureus. J Med Food., 2005, vol. 8, 454-461 16, 2364-2368 [0048] [0048] • COLOMBO M.L. et al. Cytotoxicity evaluation of nat- • MANTENA SK ; SHARMA SD ; KATIYAR SK. Ber- ural coptisine and synthesis of coptisine from Berber- berine, a natural product, induces G1-phase cell cy- ine. Farmaco, 2001, vol. 56, 403-409 [0048] cle arrest and caspase-3-dependent apoptosis in hu- • CHUN-CHING LIN et al. Cytotoxic effects of Coptis man prostate carcinoma cells.Mol. Cancer Ther., chinensis and Epimedium sagittatum extracts and 2006, vol. 5, 296-308 [0048] their major constituents (berberine, coptisine and • KUO CL ; CHI CW ; LIU TY. The anti-inflammatory icariin) on hepatoma and leukaemia cell growth. Clin- potential of berberine in vitro and in vivo. Cancer Lett., ical and Experimental Pharmacology and Physiolo- 2004, vol. 203, 127-137 [0048] gy, 2004, vol. 31, 65-69 [0048] • KHOSLA PK ; NEERAJ VI ; GUPTA SK ; SATPA- • THIBOUTOT D ; THIEROFF-EKERDT R ; GRAUPE THY G BERBERINE. a potential drug for trachoma. K. Efficacy and safety of azelaic acid (15%) gel as a Rev Int Trach Pathol Ocul Trop Subtrop Sante Pub- new treatment for papulopustular rosacea: results lique, 1992, vol. 69, 147-65 [0048] from two vehicle-controlled, randomized phase III • GULLIVER WP ; DONSKY HJ. A report on three re- studies. J Am Acad Dermatol, 2003, vol. 48, 836-845 cent clinical trials using Mahonia aquifolium 10% top- [0048] ical cream and a review of the worldwide clinical ex- perience with Mahonia aquifolium for the treatment of plaque psoriasis.Am J Ther., 2005, vol. 12, 398-406 [0048]
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