Patent Application
20230115784
The present invention is related to compositions and methods for the treatment of acne vulgaris and, in particular, to topical compositions and methods based upon compositions that are combinations of succinic acid and one or more of topical API including retinoid, salicylic acid, azelaic acid, picolinic acid, benzoyl peroxide, or antibiotic, in a pharmaceutically acceptable preparation.
Acne is a significant inflammatory skin disorder and it is considered the most common skin disorder. There are four disease factors associated with acne vulgaris. They include sebum overproduction, follicular hyperkeratinization, Propionibacterium acnes (P. acnes) proliferation in hair follicles, and inflammation. Acne affects about 50 million people in the US. It is highly common among adolescents, affecting up to 95% of men and 83% of women in that age group. In approximately 10% of cases, acne has been reported to become severe nodulocystic acne with the potential of producing lifelong disfiguring scars. Acne lesions typically recur for years. Therefore, acne is considered a chronic disease.
The psychosocial morbidity associated with acne is important and includes depression and suicidal thoughts. In addition to impact on patient's quality of life, the economic burden of acne treatment is significant.
Currently available treatments for acne are insufficient. Treatment options against acne vulgaris typically only address one or two disease factors—P. acnes proliferation and follicular hyperkeratinization. Treatment options for acne vulgaris suffer from significant side effects and none provides complete protection. Potential side effects include skin irritation including burning, erythema, peeling, dryness, and contact allergy. Antibiotics pose various side effects that range from colitis, vaginal candidiasis, and photosensitivity to the development of bacterial resistance and cross-resistance.
Thus, there is a need to develop new therapies with reduced side effects and therapies that address multiple acne vulgaris disease factors—P. acnes proliferation, follicular hyperkeratinization, and inflammation.
Accordingly, the applicants herein have succeeded in devising new formulations and methods for treating acne vulgaris.
Thus, in various embodiments, the present invention includes a topical composition for treating acne vulgaris in which the composition includes a combination of succinic acid and salicylic acid in a pharmaceutically acceptable preparation. The combination of succinic acid and salicylic acid acts in a synergistic manner in killing Propionibacterium acnes (P. acnes) and treating acne vulgaris, requires ¼ of the dose of succinic acid and salicylic acid to be effective in killing P. acnes and treat acne vulgaris and it is faster acting compared to succinic acid or salicylic acid alone. In various other embodiments, the present invention includes a topical composition for treating acne vulgaris in which the composition includes a combination of succinic acid and an API that may be topical retinoid, salicylic acid, azelaic acid, picolinic acid, benzoyl peroxide, antibiotics or combinations thereof, in a pharmaceutically acceptable preparation. The combination of succinic acid and BPO, succinic acid and azelaic acid, and succinic acid and picolinic acid acts in additive manner against P. acnes and requires ½ of the dose of individual components to kill P. acnes and treat acne vulgaris. The combination of succinic acid and antibiotic prevents antibiotic resistance development and is effective in treating acne vulgaris associated with antibiotic resistant P. acnes. Any combinations containing succinic acid prevent P. acnes biofilm development in follicles.
In various other embodiments, the present invention includes a method of treating acne vulgaris. The method includes administering to a subject in need thereof, a topical composition comprising a combination of succinic acid and another API, such as retinoid, salicylic acid, azelaic acid, picolinic acid, benzoyl peroxide, or antibiotic, in a pharmaceutically acceptable preparation.
In various additional embodiments, the present invention includes a method of preparing a topical formulation for treatment of acne vulgaris. The method includes combining succinic acid with another API in a pharmaceutically acceptable preparation, wherein the combination of succinic acid and another API produces additional effect in treating acne vulgaris, including synergistic or additive effect against P. acnes, prevention of antibiotic resistance development, prevention of P. acnes biofilm formation, or faster acting pharmaceutical composition.
In various embodiments, the present invention includes a topical composition for treating acne vulgaris. The composition may include succinic acid and an API that may be topical retinoid, salicylic acid, azelaic acid, picolinic acid, benzoyl peroxide, antibiotics or combinations thereof, in a pharmaceutically acceptable preparation.
In various other embodiments, the present invention includes a method of treating acne vulgaris. The method includes administer to a subject in need thereof, a topical composition that may include succinic acid and an API selected from the group consisting of topical retinoids, salicylic acid, azelaic acid, picolinic acid, benzoyl peroxide, clindamycin and combinations thereof in a pharmaceutically acceptable preparation.
In various other embodiments, the present invention includes a method of treating inflammatory acne vulgaris that contains a topical composition that includes succinic acid and another component API selected from the group consisting of topical retinoids, salicylic acid, azelaic acid, picolinic acid, benzoyl peroxide, clindamycin and combinations thereof in a pharmaceutically acceptable preparation.
In various additional embodiments, the present invention includes a method of preparing a topical formulation for treatment of acne vulgaris. The method includes combining succinic acid and an API selected from the group consisting of retinoids, salicylic acid, azelaic acid, picolinic acid, benzoyl peroxide, antibiotics, and combinations thereof in a pharmaceutically acceptable preparation.
In various embodiments, the compositions and preparations above can be provided to a subject in a pad, tube, and any container suitable for the delivery of the compositions and preparations so a subject.
These and other features, aspects and advantages of the present teachings will become better understood with reference to the following description, examples and appended claims.
Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
The present invention includes compositions and methods directed to the treatment of skin diseases, and in particular, inflammatory acne vulgaris. The compositions as well as treatment and formulation methods are based upon combinations of agents with antimicrobial, anti-hyperkeratinization, anti-comedogenic, and anti-inflammatory effects that are effective in treating acne vulgaris.
Abbreviations and Definitions
To facilitate understanding of the invention, a number of terms and abbreviations as used herein are defined below as follows:
As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a formulation” includes a plurality of such formulations and reference to “the method” includes reference to one or more methods and equivalents thereof known to those skilled in the art, and so forth.
As used herein, the term “about” is intended to refer to a range of values above and below a stated value such as for example, values encompassing 10% below up to 10% above a stated value.
The term “and/or” is intended to mean either or both of two recited elements.
“Active pharmaceutical ingredient” (“API”) refers to a substance, in particular an antimicrobial agent, in a pharmaceutical composition that is delivered for a desired effect.
As used herein, the term “combination” with respect to active agents refers to a composition of two or more active agents, in particular, agent with antimicrobial and/or anti-inflammatory and/or anti-hyperkeratinization and anti-commedogenic effects. In the present invention, a combination of active agents may include succinic acid and an API that may include retinoid, salicylic acid, azelaic acid, picolinic acid, benzoyl peroxide, antibiotic, and combinations of two or more API's.
Reference herein to an API including, but not limited to succinic acid, retinoids, salicylic acid, azelaic acid, picolinic acid, benzoyl peroxide and antibiotics is intended to include pharmaceutically acceptable solvates, salts, hydrates or hydrated salts, their optical isomers, racemates, diastereomers, enantiomers or the polymorphic crystalline structures of the compounds.
In the Examples below, DRM1 refers to succinic acid.
The term “pharmaceutical composition” or ‘pharmaceutical preparation” refers to a composition that combines one or more API's with a pharmaceutically acceptable carrier such that the composition is suitable for therapeutic use in vitro, in vivo or ex vivo.
As used herein, the term “pharmaceutically acceptable carrier” encompasses any suitable pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, various types of wetting agents and the like. The compositions also can include stabilizers and preservatives. Examples of carriers, stabilizers and adjuvants, can be found in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, Twenty-First edition (May 19, 2005).
A number of carrier systems have been developed, in particular for topical application, including by way of non-limiting examples, vesicular systems such as liposomes, niosomes, ethosomes and transfersomes. (Arora, S. et al., Dermal delivery of drugs using different, vesicular carriers: A comparative review, J Pharm. 2012;6:237-44).
One such vesicular carrier system is the niosome. As used herein, the term noisome refers to unilamellar or multilamellar vesicles in which an aqueous phase is encapsulated in highly ordered bilayer made up of nonionic surfactant (Vyas, J. et al., “Development of topical niosomal gel of benzoyl peroxide,” International Journal of Nanotechnology, vol. 2011, Article ID 503158, 6 pages, 2011.).They are nonionic surfactant vesicles by which skin penetration and accumulation are increased in the superficial skin strata (Manconi, M. et al., Niosomes as carriers for tretinoin: III. A study into the in vitro cutaneous delivery of vesicle-incorporated tretinoin, International Journal of Pharmaceutics, 2006 311(1-2):11-19.); (Vyas, A. et al., Carrier-Based Drug Delivery System for Treatment of Acne, The Scientific World Journal, 2014 Feb 9;2014:276260. doi: 10.1155/2014/276260. eCollection 2014.)
The term “synergy” as used herein is intended to refer the interaction of discrete antimicrobial agents in which the total antimicrobial effect is greater than the sum of the effects of the antimicrobial agent alone. Conversely, the term “antagonism” is intended to refer to an interaction of antimicrobial agents in which the total antimicrobial effect is less than the sum of the effects of the antimicrobial agents alone.
The term “checkerboard experimental design”, is intended to refer to an in vitro two-dimensional array of serial concentrations of test compounds evaluated in paired or triple combinations for their affect in inhibiting the growth of a target microorganism, measured as a “minimal inhibitory concentration” (MIC), i.e. the lowest concentration that prevents of inhibits microbial growth. It is used as the basis for calculation of a “fractional inhibitory concentration index”.
The term “fractional inhibitory concentration index” (FICI) refers to a value that compares the ratio of inhibitory concentration of a combination with that of individual agents separately. The formula used to calculate FICI is as follows:
FICI=MICA/B/MICA alone+MICB/A/MICB alone
The FIC Index (FICI) value is used to determine whether paired combinations of antimicrobial agents can exert inhibitory effects against tested organism that are more or less than the sum of their effects alone. In theory, FICI<1.0 indicates synergy and FICI>1.0) indicates antagonism. However, a more conservative calculation has been recommended such that FICI data may be interpreted as “synergy” for an FICI≤0.5, “antagonism” for an FICI>4.0 and “no interaction” interaction for an FICI>0.5-4.0. (Odds, F.C., Synergy, antagonism, and what the chequerboard puts between them, Journal of Antimicrobial Chemotherapy (2003) 52, 1. As used herein, the FICI is interpreted as follows:
FICI≤0.5=synergy
FICI>0.5−≤1=additive effect
FICI>1−≤4 =indifference
FICI>4=antagonism
Unless otherwise indicated, concentrations are given as mass weight percentages, i.e. w/w %. Mass weight percentages (w/w %) for combination formulations are calculated as follows:
mass % a=mass(a)÷(mass(a)+mass(b)+mass(c)+ . . . )×100 (w/w %).
Compositions
The present invention includes compositions and treatment methods based upon compositions that are combinations of agents having antimicrobial, anti-inflammatory, anti-hyperkeratinization, and anti-commedogenic activities. Such compositions include succinic acid and an API selected from the group consisting of topical retinoids, salicylic acid, azelaic acid, picolinic acid, benzoyl peroxide, antibiotics and combinations thereof.
In various embodiments, the amount of succinic acid in the composition may be from about 0.1 to about 10 w/w % and in particular, from about 0.1, about 0.2, about 0.5 or about 0.75, about 1 w/w % to about 2, about 3, about 4, about 5, about 7.5 or about 10 w/w %. In various embodiments, the amount of succinic acid in the composition may be about 0.1, about 0.2, about 0.5, about 0.75, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10 w/w %. The succinic acid is combined with one or more API's.
In various embodiments, salicylic acid may be included in the composition. Salicylic may be present in the composition in an amount of from about 0.05 to about 5 w/w % and in particular, from about 0.05, about 0.1, about 0.2, about 0.5 or about 0.75 w/w % to about 1, about 2, about 3, about 4 or about 5 w/w %. In various embodiments, the amount of salicylic acid in the composition may be about 0.05, about 0.1, about 0.2, about 0.5, about 0.75, about 1, about 2, about 3, about 4 or about 5 w/w %.
In various embodiments, azelaic acid may be included in the composition. Azelaic acid may be present in the composition in an amount of from about 0.05 to about 10 w/w % and in particular, from about 0.05, about 0.1, about 0.2, about 0.5 or about 0.75, about 1 w/w % up to about 2, about 3, about 4, about 5, about 7.5 or about 10 w/w %. In various embodiments, the amount of azelaic acid in the composition may be about 0.05, about 0.1, about 0.2, about 0.5, about 0.75, about 1, about 2, about 3, about 4 about 5, about 7.5 or about 10 w/w %
In various embodiments, picolinic acid may be included in the composition. Picolinic acid may be present in the composition in an amount of from about 0.01 to about 10 w/w % and in particular, from about 0.01, about 0,02, about 0.05, about 0.1, about 0.2 or about 0.5 w/w % to about 0.75, about 1, about 2, about 3, about 4, about 5, about 7.5 or about 10 w/w %. In various embodiments, the amount of picolinic acid in the composition may be about 0.01, about 0.02, about, about 0.05, about 0.1, about 0.2, about 0.5, about 0.75, about 1, about 2, about 3, about 4, about 5, about 7.5 or about 10 w/w %
In various embodiments, benzoyl peroxide may be included in the composition. Benzoyl peroxide may present in the composition in an amount of from about 0.01 to about 5 w/w % and in particular, from about 0.01, about 0.02, about 0.05, about 0.1, about 0.2 or about 0.5 w/w % to about 0.75, about 1, about 2, about 3, about 4 or about 5 w/w %. In various embodiments, the amount of benzoyl peroxide in the composition may be about 0.01, about 0.02, about, about 0.05, about 0.1, about 0.2, about 0.5, about 0.75, about 1, about 2, about 3, about 4 or about 5 w/w %
In various embodiments, antibiotic may be included in the composition. The antibiotic includes erythromycin, clindamycin, minocycline and doxycycline, or any other antibiotic with activity against P. acnes. Antibiotic may be present in the composition in an amount of from about 0.001 up to about 2 w/w % and in particular, from about 0.001, about 0.002, about 0.005 or about 0.0075, about 0.01, about 0.02, about 0.03, about 0.04, about 0.05 w/w % up to about 0.075, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5 about 1 or about 2% w/w %. In various embodiments, the amount of antibiotic in the composition may be about 0.001, about 0.002, about 0.005, about 0.0075, about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 1% or about 2 w/w %.
The compositions of the present invention may be incorporated into a pharmaceutically acceptable carrier system which may include creams, gels, lotions or other types of suspensions that can stabilize the combination of agents and deliver them to the affected area of skin by topical applications. Pharmaceutically acceptable carrier systems may also contain ingredients that include, but are not limited to, saline, aqueous electrolyte solutions, ethanol, dimethyl sulfoxide, dimethyl isosorbide, isopropyl myristate, lauryl lactate, diisopropyl adipate, sodium lauryl sulfoacetate; ionic and nonionic osmotic agents such as sodium chloride, potassium chloride, glycerol, propylene glycol and dextrose; pH adjusters and buffers such as salts of hydroxide, phosphate, citrate, acetate, borate; and trolamine; antioxidants such as salts, acids and/or bases of bisulfite, sulfite, metabisulfite, thiosulfite, ascorbic acid, acetyl cysteine, cystein, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherols, and ascorbyl palmitate; compounds such as lecithin, phospholipids; petroleum derivatives such as mineral oil and white petrolatum; fats such as lanolin, peanut oil, palm oil, soybean oil; mono-, di-, and triglycerides; polymers of acrylic acid such as carboxypolymethylene gel, and hydrophobically modified cross-linked acrylate copolymer; polysaccharides such as dextrans and glycosaminoglycans such as sodium hyaluronate. Such pharmaceutically acceptable carriers may be preserved against bacterial contamination using preservatives, including, but are not limited to, benzalkonium chloride, ethylene diamine tetra-acetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal, and phenylethyl alcohol, or may be formulated as a non-preserved formulation for either single or multiple use.
Also provided is a composition suitable for oral administration, the composition comprising probiotic bacteria Bacillus subtilis DE111®, Bifidobacterium longum, Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus paracesei and Lactobacillus rhamnosus. In some embodiments, 6.25 billion colony forming units (CFUs) of the probiotic bacteria is present in the composition.
In various embodiments, these compositions are capable of reducing the severity of acne in a subject with mild to moderate acne administered the composition at least once per day. See, e.g., Example 21 below.
These compositions can further comprise any additional ingredients, including but not limited to any ingredients described above. In some embodiments, the composition further comprises 15 mg of prebiotic phages LH01, LLS, T4D and LL12. See Gindin et al., 2019.
In other embodiments, the composition further comprises any or all of vitamin A, vitamin C, vitamin E, zinc and/or spinach leaf extract.
The composition may be taken orally by itself or with other medications, including but not limited to applying a topical composition comprising succinic acid to the acne, for example at least once per day.
Methods of Treatment
The methods of treatment of the present invention are useful for the treatment of skin diseases including acne vulgaris and inflammatory acne vulgaris. Treatment of acne vulgaris may be by topically administering to a subject a composition that includes a combination of active agents.
The compositions of the present invention can be administered at a variety of intervals. In some instances, administration may be once a day. In other instances, administration can be less or more frequently, such as 1, 2, 3, or 4 times a day, 1 time every 2 days, or once a week.
The treatment methods may be monitored by following any of the pathogenic aspects of acne vulgaris including number of acne lesions, sebum excretion, follicular keratinization, comedone formation, bacterial colonization of the follicle, P. acnes proliferation and/or inflammation.
In some embodiments, a method of treating mild to moderate acne in a subject is provided. The method comprises administering the above-described probiotic composition to the subject at least once per day.
This method can be applied alone or with any other treatment, including but not limited to applying a topical composition comprising succinic acid to the acne at least once per day or more often, e.g., twice, three times, four times, five times or more.
In various embodiments, the method causes improvement of acne severity more than improvement due to administration of the topical composition alone.
In other embodiments, the number of acne lesions on the subject is decreased within one week of starting the method.
In additional embodiments, skin tone of the subject is improved within one week of starting the method.
In further embodiments, skin redness of the subject is improved within one week of starting the method.
In additional embodiments, the method causes additional improvements to the skin of the subject, as described in Example 21.
Aspects of the present teachings may be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way. In the examples below, DRM1 refers to succinic acid.
Bacterial Cultivation
P. acnes (ATCC 6919) was cultured on Brucella plates supplemented with 0.1 g/L hemin, 0.01 g/L vitamin K, and 5% (v/v) defibrinated sheep's blood at 37° C. under anaerobic conditions using a Gas-Pak (BD). For each assay, an inoculum of P. acnes was prepared by suspending a colony of P. acnes in 5 mL Reinforced Clostridium medium (RCM) and growing at 37° C. and 250 RPM under anaerobic conditions for 30 hours. Cultures were harvested, washed with PBS and resuspended in fresh, sterile RCM broth to a concentration of 2×106 CFU/ml.
Minimum Inhibitory Concentration (MIC) Determination
Individual MIC values were determined for each drug according to the microbroth dilution method. Briefly, 2-fold serial dilutions of each drug (50 μl) were added to wells in a 96-well plate followed by 50 p1 of the prepared P. acnes inoculum as described in Example 1. Media without P. acnes and medium with P. acnes only were used for negative and positive controls, respectively, and contained the highest concentration of solvent used in each drug analysis. Plates were incubated under anaerobic conditions using a Gas-Pak (BD) for 48 hours at 37° C. Following incubation, each well was resuspended by pipetting and the optical density at 600 nm (0D600) was determined on a plate reader. The MIC value was defined as the first well showing ≥90% reduction in growth compared to controls.
In Vitro Checkerboard Assay
A checkerboard assay was used to determine if combining DRM1 (succinic acid) with commonly used topical treatments for acne showed synergy, indifference or antagonism between the drugs. Checkerboard assays were performed in 96 well plates as an 8 ×8 grid. Two-fold serial dilutions of API A (25 μl) were added at 4× the desired final concentration across 8 rows of the microplate. Two-fold serial dilutions of API B (25 μl) were added at 4× the desired final concentration to 8 columns on the microplate so that the highest concentration of API A and the highest concentration of API B were in the same well at one corner of the 8×8 grid diagonal from the corner containing the lowest concentration of each drug. For each drug, a set of concentrations greater than and less than the MIC as calculated above was used. An inoculum of P. acnes (50 μl) as described in example 1 was added to each well containing API and the plate was incubated at 37° C. for 48 hours under anaerobic conditions and the individual and combined MIC values were determined as stated in example 2 for the MIC assay. The FICI for each combination was determined using the following equation:
FICI=MICA/B/MICA alone+MICB/A/MICB alone
Synergy was defined as an FICI≤0.5, additivity was defined as an FICI>0.5≤1.0, indifference was defined as an FICI>1≤4 and antagonism was defined as an FICI >4.0.
Checkerboard Combination of DRM1 (succinic acid) and Salicylic acid
Table 1, below, shows the MIC values for Salicylic Acid.
As shown in
DRM1 (succinic acid) and salicylic acid were used individually at concentrations 2× their MIC values (30 mM DRM1 (succinic acid) and 15 mM salicylic acid) and in combination at 0.5× MIC for DRM1 (succinic acid, 7.5 mM) and 1× MIC for salicylic acid (7.5 mM) Assays were performed in RCM broth with 12% propylene glycol in 96-well plates. A final concentration of 5×105 CFU/ml P. acnes 6919 was used for each reaction and each assay was performed under anaerobic conditions using a Gas-Pak at 37° C. At various time points (0, 15, 30, 60 and 120 minutes) 10 μl of each sample was removed and diluted 1:10 to 1:104 in sterile PBS and 20 μl of each dilution was spread on a Brucella broth agar plate supplemented with 0.1 μg/ml hemin and 0.01 μg/ml vitamin K using the track plate method.
Bacterial cultivation, MIC calculation and Checkerboard Assay design were as described in Example 1. Table 2, below, shows MIC values for Azelaic Acid.
As shown in
Bacterial cultivation, MIC calculation and Checkerboard Assay design were as described in Example 1. Table 3, below, shows MIC values for picolinic acid.
As shown in
Bacterial cultivation, MIC calculation and Checkerboard Assay design were as described in Example 1. Table 4, below shows MIC values for BPO.
As shown in
Examples 1-5 Illustrate DRM1 (Succinic Acid) Combinations that Maximize Efficacy and Minimize Side Effects:
DRM1 (succinic acid) increases efficacy of other acne treatments including salicylic acid, benzoyl peroxide, azelaic acid, picolinic acid, retinoids, etc. while reducing side effects of the second agents on the skin. This means that combinations can be used in which the amount of a side effect causing agent (such as benzoyl peroxide (BPO), salicylic acid, azelaic acid, retinoids, etc.) can be reduced while maximizing efficacy. We have identified DRM1 (succinic acid) containing combinations and the ratios between DRM1 (succinic acid) and the second agent that are optimal for acne treatment. All combinations improve the performance of the second agent without increasing side effects—they reduce side effects.). The most efficacious combination seems to be DRM1 (succinic acid)+salicylic acid at ratio 2:1. This combination shows the highest synergistic effects against P. acnes, reduces inflammation (provided by DRM1), and has anti-hyperkeratinization (provided by salicylic acid) and anti-commedogenic (provided by salicylic acid) properties. This combination addresses three acne vulgaris diseases factors. In addition, the combinations result in faster killing of P. acnes (time kill assays in Example 2) and prevents biofilm formation in DRM1 concentration above 7.5 mM (Example 8). The combinations of DRM1 (succinic acid) with a second agent can be optimized using specific molar ratios to achieve optimal efficacy while minimizing side effects. In addition to treating acne vulgaris in all patients including inflammatory acne vulgaris, DRM1 (succinic acid) containing combinations could be particularly useful in patients who are prone to side effects on the skin and would benefit from suing topical treatments that contain reduced amount of BPO, salicylic acid, azelaic acid, retinoids, etc.
Bacterial cultivation, MIC calculation and Checkerboard Assay design were as described in Example 1. Table 5, below, shows MIC values for BPO.
As shown in
Table 6 summarizes the MIC values from Examples 1, 3, 4, 5 and 6.
The MIC values for DRM1 (succinic acid) against multi-resistant P. acnes strains were determined as described in example 1. Briefly, P. acnes strains (RMA #20660 and RMA #20661) with low susceptibility to erythromycin, clindamycin, minocycline, and doxycycline were incubated with DRM1 (succinic acid, 0-120 mM) and the minimum inhibitory concentration was determined. As illustrated in
DRM1 (succinic acid) prevented biofilm formation in biofilm-forming strains at a concentration of 7.5 mM and above. Biofilm formation is an important factor in P. acnes pathogenicity and biofilm is detected in approximately half of hair follicles of acne patients.
Resistance development may be measured by two methods: single step for the determination of spontaneous frequency of resistance development and serial passage for the determination of progressive resistance development. Single step resistance frequency was determined by spreading a large inoculum of P. acnes (109 to 1010 CFU) onto a blood agar plate containing 4×, 8×, 16×, or 32× the MIC for DMR1 (succinic acid) as described in Example 2. The spontaneous resistance frequency was calculated from the number of colonies that grow on plates containing DRM1 vs. the number of colonies that grow on DRM1-free agar. As shown in
P. acnes strains did not develop spontaneous or progressive resistance to DMR1 (succinic acid) and including DRM1 (succinic acid) in combination with antibiotics for acne treatment prevents antibiotic resistance emergence and development.
Examples 6-9 Illustrate that Combinations with Antibiotics Prevents Resistance Development while Reducing Side Effects
DRM1 (succinic acid) combined with topical antibiotics can be used to replace side effect causing BPO in combinations containing antibiotics and BPO. DRM1 (succinic acid) is bactericidal. Many antibiotics are bacteriostatic which promotes resistance development. DRM1 (succinic acid) is effective against antibiotic resistant P. acnes strains and prevents emergence of antibiotic resistant P. acnes strains when used in combinations with antibiotics. In addition, DRM1 prevents biofilm formation.
A synthetic SE Microbiome Complex (the “SE Microbiome Complex”) containing a defined combination of S. epidermis metabolites and prebiotics was prepared by combining at least the following in a concentration of 0-10%:
The components of the synthetic SE Microbiome Complex were produced by chemical synthesis or by microbial fermentation using wild-type or engineered microorganisms. The SE Microbiome Complex was optimized for bactericidal properties against P. acnes, skin tolerance, and cosmetic acceptance. The SE Microbiome Complex was formulated for use for the treatment of acne vulgaris.
Acne treatment pads formulation (Formulation 1): The formulation was prepared that contained salicylic acid (1%), organic aloe vera (50%), propanediol (15%), organic grain alcohol (10%) and SE Microbiome Complex. The SE Microbiome complex consisted of 2% succinic acid, 1% Brij L4, 1% dimethyl isosorbide ether, and 1% glycerin (prebiotic compound). Optionally, organic lemon essential oil was added to a final concentration of 0-1% and grapefruit seed extract to a final concentration of 0-1%. Sodium hydroxide was used to adjust the solution pH to 3.9-4.5. Succinic acid was prepared by microbial fermentation or by chemical synthesis. Alternatively, succinic acid in the formulation was replaced by conditioned media prepared by fermenting S. epidermidis as follows: S. epidermidis was incubated in phenol red-free rich medium with glycerol (20 g/l) for one to six days. After that, microorganisms were discarded by centrifugation at 5,000 g for 30 min Fermented media were then passed through 0.2-μm-pore-size filters. Conditioned media contained succinic acid and other S. epidermidis fermentation metabolites. Alternatively, conditioned media was used in addition to succinic acid. Alternatively, glycerin was replaced by other prebiotic compound (such as sucrose, maltose, lactose or other carbohydrates and other prebiotic compounds that support the fermentation of S. epidermidis). Alternatively, organic components in the formulation were replaced by non-organic equivalent components. The formulated solution was poured over pads (material-rayon, Lyocell, Polyester or similar) in a container. The pads were used for applying the topical treatment for acne vulgaris.
Acne spot treatment formulation (Formulation 2) was prepared containing: Salicylic acid (2%), propanediol (15%), organic grain alcohol (10-20%) and SE Microbiome Complex—super strength (consisting of 4% succinic acid, 3% Brij L4, 5% dimethyl isosorbide). Sodium hydroxide was used to adjust the solution pH to 3.9-4.5. Hydroxyethyl cellulose or eco-gel was added to gel the solution at a concentration of 1-3%. Optionally, organic tee tree essential oil (0-10%) and lemon essential oil (0-1%) was added. Optionally, organic aloe vera (0-50%) was added. Optionally, glycerin or other prebiotic compounds (0-5%) were added (such as sucrose or other carbohydrates or prebiotic compounds that support the growth/fermentation of S. epidermidis). Alternatively, conditioned media of fermenting S. epidermidis (prepared as described in Example 1) was used instead of or in addition to succinic acid. Alternatively, organic components in the formulation were replaced by non-organic equivalent components. Gel was filled into tubes for application. Gel was used for topical treatment of acne vulgaris.
P. acnes (ATCC 6919) was cultured on reinforced Clostridial medium (RCM) agar plates at 37° C. under anaerobic conditions using a Gas-Pak (BD). For each assay, an inoculum of P. acnes was prepared by suspending a colony of P. acnes in 5 mL RCM and growing at 37° C. under anaerobic conditions for 72 hours. Cultures were harvested, washed with PBS and resuspended in fresh, sterile RCM broth to a concentration of 2×106 CFU/ml.
Formulation 1 (Example 11) and Formulation 2 (Example 12) was incubated in 96-well plate with P. acnes prepared as described in Example 13. Control sample contained P. acnes in media only. Plates were incubated under anaerobic conditions using a Gas-Pak (BD) for 72 hours at 37° C. Following incubation, the plate was examined visually. Control samples exhibited growth as expected. No growth was observed in wells containing Formulation 1 and Formulation 2 (
The minimal bactericidal concentration for each formulated drug was determined according to the Clinical Laboratory Standards Institute (CLSI) document M26-A. Sample wells from the MIC assay in Example 14 were serially diluted 1:10 to 1:104 in PBS and each dilution (5 μl) was plated on RCM agar plates.
The plates were incubated at 37° C. in a Gas-Pak. Following incubation, Formulation 1 (from Example 11) and Formulation 2 (from Example 12) exhibited no growth (
This example illustrates the growth inhibitory effects of S. epidermidis conditioned media prepared with various prebiotics against P. acnes.
Preparation of Prebiotics: Glycerol, sucrose, 13-lactose, and D-(+)-maltose were purchased from Sigma-Aldrich. Stock solutions of each prebiotic (4% w/v) were prepared in water and filtered through a 0.22 μm filter for sterilization.
Preparation of S. epidermidis conditioned media: S. epidermidis (ATCC 12228) was cultured on trypic soy broth (TSB) agar plates at 37° C. For each assay, an inoculum of S. epidermidis was prepared by suspending one colony of S. epidermidis from a TSB agar plate into 7 ml of TSB broth and growing at 37° C., 215 RPM for 16 hours. The overnight culture was subcultured by adding 100 μl of overnight culture to 10 ml of fresh TSB. Once mid-log phase was reached, the cells were pelleted by centrifugation, washed with PBS and resuspended in fresh PBS. In 15 ml culture tubes, 4.9 ml of Rich Broth (20 g/L yeast extract, 6 g/L TSB) was combined with 40 μl 0.5% phenol red indicator, 5 ml of 4% prebiotic (glycerol, sucrose, (-lactose, or D-(+)-maltose) or PBS as a control, and S. epidermidis (105 CFU/ml) and placed in an anaerobic Gas-Pak (BD). The cultures were incubated at 37° C., 215 RPM for 6 days. Following fermentation, the cells were pelleted and the supernatants were filtered through a 0.22 μm filter for sterilization.
Minimum Inhibitory Concentration (MIC) Determination: Individual MIC values were determined for each conditioned medium according to the microbroth dilution method. P. acnes (ATCC 6919) was cultured in RCM broth and harvested during mid-log phase of growth. The bacterial pellet was washed and resuspended in PBS. A stock of P. acnes (107 CFU/ml) was prepared using the estimation that an OD600 nm of 1.0 corresponds to about 107 CFU/ml. Two-fold serial dilutions of each conditioned medium (90 μl) in PBS were added to wells in a 96-well plate followed by 10 μl of the prepared P. acnes inoculum. Conditioned media without P. acnes and RCM medium with P. acnes only were used for negative and positive controls, respectively. Plates were incubated under anaerobic conditions using a Gas-Pak (BD) for 48 hours at 37° C. Following incubation, each well was resuspended by pipetting and the optical density at 600 nm (OD600) was determined on a plate reader. The MIC value was defined as the first well showing ≥90% reduction in growth compared to controls.
Fermentation of prebiotics by S. epidermidis:
P. acnes growth inhibition by S. epidermidis conditioned media: Conditioned media prepared by fermentation in the presence of prebiotics—glycerol, sucrose, D-(+)-maltose and β-lactose under anaerobic condition were able to inhibit P. acnes growth (undiluted media was the MIC). RCM control media prepared in the absence of prebiotics were not inhibitory.
Formulation 1 and Formulation 2 with and without aloe vera was tested in human volunteers for skin tolerability and cosmetic acceptance. Evaluation was performed by using questionnaire and a scoring system. Score of 1-5 was used. Score 1 for skin tolerability corresponded to irritation, skin reddening. Score 5 for skin tolerability corresponded to no adverse reaction and excellent tolerability. Score 1 for cosmetic acceptability corresponded to unacceptable product due to smell and feel. Score 5 for cosmetic acceptability represented excellent cosmetic acceptability (smell and feel). Commercially available products were used as controls. Results are recorded in table 1. Results indicate that addition of aloe vera significantly improves skin tolerability and that Formulation 1 and Formulation 2 have significantly improved cosmetic acceptance (smell and feel) compared to commercially available products.
Formulation 1 (Example 11) and Formulation 2 (Example 12) were tested in human subjects. The Formulation 1 was applied using Acne Treatment Pads one or two times per day. Improvement in acne symptoms was observed (
The five step treatment system combined the topical treatment containing SE Microbiome Complex with oral probiotics and oral supplements (such as zinc). Oral probiotics contained a diverse combination of probiotic strains of 500 million-30 billion CFUs/capsule. The combination of probiotic strains included any combination of Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus casei, Lactococcus lactis, or additional probiotic strains and optionally contained prebiotics (such as FOS or Arabinogalactan).
Five step treatment system consisted of:
Step 1: Washing face in the morning and evening.
Step 2: Applying Acne Treatment Pads (Formulation 1 from Example 11) to affected areas and Acne Spot Treatment (Formulation 2 from Example 12) to individual pimples.
Step 3: Covering whiteheads and inflamed pimples with Acne Pimple Patches (containing hydrocolloid) to treat blemishes overnight.
Step 4: Taking Probiotics (1-3 times daily) and 25-50 mg Zinc (1 time daily with a meal).
Step 5: Repeating for at least 2 months.
Compared to subjects using only topical products, the subjects who used topical products in combination with oral probiotics and zinc achieved improvement in acne symptoms.
This example will be performed to screen combinations of DRM1 and retinoids (tretinoin, adapalene, tazarotene, isotretinoin, retinyl palmitate, retinol, or retinaldehyde) for antimicrobial and anti-inflammatory properties, and to determine if the compounds can decrease the amount of acne lesions in acne.
The minimal inhibitory concentration (MIC) and minimum bactericidal concentration of combinations of DRM1 and retinoids will be determined to monitor the growth inhibitory effects of the combination. To determine the MIC values, DRM1 will be analyzed in concentrations ranging from 1-100 mM and the retinoids will vary in concentrations from 0.025%-0.1%. Two-fold serial dilutions of the DRM1-retinoid combination will be made in PBS (50 μl). An inoculum of 2×106 CFU/ml P. acnes in RCM will be prepared and 50 μl will be added to the wells containing the DRM1-retinoid combinations.
Bactericidal properties of the DRM1 and retinoid combinations will be analyzed using the time-kill assay. Briefly, 0.4 ml of 10× the desired final concentration of DRM1 and retinoid will be added to 2.8 ml RCM broth along with 0.4 ml of 107 CFU/ml P. acnes. Media with P. acnes only will be used for a positive control. A separate tube will be prepared for each time point for all measured concentrations of the DRM1-retinoid combination. The assay will be performed under anaerobic conditions at 37° C. The reaction will be analyzed after 0, 0.5, 1, 2, 4, 8, and 24 hours. At each time point the corresponding tube will be removed and the sample will be diluted 1:10 to 1:105 in PBS. Each dilution will be spotted (10 μl) in triplicate on RCM agar plates and incubated under anaerobic conditions using a Gas-Pak (BD) for 4-7 days at 37° C. Following incubation, the plate will be visually examined and the bacterial counts for each time point will be determined.
The best in vitro performing DRM1-retinoid combination, as determined by the MIC assay and time-kill assay, will be formulated into a topical cream, gel, or solution applied with pads. The topical formulation will be applied to acne infected areas twice a day (morning and night) following cleansing of the skin.
It is expected that the application of a DRM1-retinoid combination to acne infected skin will decrease the acne lesion counts when compared to controls—placebo, DRM1 alone or retinoid alone. It is expected that the count of both inflammatory and non-inflammatory lesions will decrease compared to controls.
Materials and Methods
A single site blinded study was conducted to evaluate the effect of a topical regimen and an oral supplement on acne.
25 males and 42 females 13-35 years of age were enrolled, consisting of 28 Caucasian, 29 African American, 8 Hispanic, and 2 Asian subjects. All Fitzpatrick skin types were represented.
The subjects possessed mild to moderate acne with an IGA score of 2, 2.5, 3, 3.5 based on the Modified Cook's scale. In addition, subjects possessed 10-100 total non-inflammatory lesions (open comedones and closed comedones), 10-50 total inflammatory lesions, no cysts, and up to 2 nodules on the face.
The subjects were randomized into two groups:
Group 1: Subjects were provided with a cleanser, a moisturizer, a topical acne treatment product, and an oral supplement. One capsule of the supplement was taken once per day; the cleanser, moisturizer and topical acne treatment was used once in the morning and once in the evening.
Group 2: Subjects were provided with a cleanser, a moisturizer, and a topical acne treatment product, each used once in the morning and once in the evening.
The topical acne treatment product consisted of 2% succinic acid, 1% salicylic acid, 50% aloe vera; 10% organic grain alcohol, polyethylene glycol dodecyl ether, glycerol, cocamidopropyl dimethylamine, sodium hydroxide and organic lemon essential oil.
The oral supplement (each dose) consisted of the following:
probiotic bacteria (10.07 billion colony forming units (CFUs)) consisting of 27.8 mg Bacillus subtilis DE111® (8.33 billion CFUs) (Trotter et al., 2020, Benef. Microbes 11:621-630), 3.1 mg Bifidobacterium longum (0.31 billion CFUs), 0.9 mg Lactobacillus paracesei (0.36 billion CFUs), 0.9 mg Lactobacillus plantarum (0.36 billion CFUs), 0.8 mg Lactobacillus rhamnosis HN001 (0.36 billion CFUs), 0.6 mg Bifidobacterium animalis lactis (0.36 billion CFUs), guaranteed to have 6.25 CFUs after 24 months storage;
a prebiotic bacteriophage mix (15 mg) consisting of phages LH01, LLS, T4D and LL12 (Gindin et al., 2019, J. Am. Coll. Nutr. 38:68-75);
zinc (fom zinc gluconate)—15 mg;
vitamin C (ascorbic acid)—30 mg;
vitamin E (d-α-tocopherol)—10 mg;
vitamin A—600 μg;
spinach leaf extract—100 mg;
other ingredients—delayed release capsule (HPMC, pectin, water); microcrystalline cellulose, medium chain triglycerides, silica.
The cleanser consisted of 2% succinic acid, polyethylene glycol dodecyl ether, glycerol, water, lauryl glucoside, laureth-7 citrate, glyceryl cocoate, propanediol, PEG-7 glyceryl cocoate, Citrus sinensis (sweet orange), Citrus limonium, citric acid, glycerin, phosphatidylcholine, panthenol (provitamin B5), arginine, proline, glycine, alanine, phenoxyethanol, potassium sorbate, ethylhexylglycerin and tetrasodium EDTA.
The moisturizer consisted of 2% succinic acid, polyethylene glycol dodecyl ether, glycerol, water, glycerin, glyceryl stearate se, caprylic/capric triglyceride, Butyrospermum parkii, cetearyl alcohol, squalane, cetyl alcohol, dimethicone, avocado oil, colloidal oatmeal, acrylates/C10-30 alkyl acrylate crosspolymer, propanediol, phosphatidylcholine, sodium hyaluronate, niacinamide, sodium hydroxide, camellia sinensis leaf extract, glycyrrhiza glabra root extract, ethylhexylglycerin, potassium sorbate, sodium phytate, allantoin, tocopherol acetate, Vitis vinifera fruit cell extract, Argania spinosa sprout cell extract, lactose, sucrose, maltose and ceramide NG.
Assessments
Dermatologist Investigator lesion counts were made at baseline (visit 1), week 1 (visit 2), week 4 (visit 3), and week 8 (visit 4), and week 12 (visit 5). The lesion counts were as follows:
Dermatologist Investigator skin assessments were also made at each visit. Assessments were made on a 0-9 scale (0=none, 1-3=mild, 4-6=moderate, 7-9=severe) by evaluating redness, uneven skin tone, acne marks and scars.
Dermatologist Investigator also assessed tolerability parameters at each visit. All assessments were made on a 5-point ordinal scale (0=none, 1=minimal, 2=mild, 3=moderate, 4=severe) by evaluating peeling, dryness, redness and swelling.
Subject assessed parameters were also made at each visit using a self-assessment/treatment perception questionnaire.
The following study endpoints were assessed:
Along with descriptive statistics (means, standard deviations and percentages), Modified Cook's Scale, investigator, and subject ordinal nonparametric data were analyzed using the Wilcoxon signed rank test. Change were considered significant at a p value less than or equal to 0.05. Numerical acne lesion counts (inflammatory, noninflammatory, and total lesions) were assessed with a Student's t test again with significance at a p value less than or equal to 0.05.
Results
Tolerability
As shown in Table 8, the treatments were well tolerated, as there were no incidences of peeling, dryness, redness or swelling at any time point in either group.
Lesion Counts
As shown in Tables 9 and 10, there were statistically significant improvements in both inflammatory and noninflammatory lesions in both groups at all assessment time points. At week 1, there was a statistically significant superior inflammatory (p=0.022) and noninflammatory (p=0.035) lesion count reduction favoring the supplement group. At week 12 there was a statistically significant superior reduction in inflammatory lesion count in the probiotic supplement group. (p=0.010).
Global Acne Assessment (IGA)
As shown in Tables 11 and 12, both groups produced statistically significant improvement in acne as measured by IGA (see, e.g., Alsulaimani et al., 2020, Clin. Cosmet. Invest. Dermatol. 13:711-716). The improvement was always superior in group 1 consuming the supplement. This benefit was statistically significant at week 12 (p=0.004).
Investigator Skin Assessment
Tables 13 and 14 provide the results of the investigator skin assessments. The blinded dermatologist investigator assessed redness, uneven skin tone, acne marks and scars at each visit using the 0-9 scale. A lower score is a superior score. Both treatment groups produced statistically significant improvement over time and the improvement was superior for group 1 consuming the supplement. Table 13 provides the results vs. baseline; Table 14 provides the comparisons between the groups. In those comparisons, the supplement Group 1 outperformed the skin care alone Group 2. The earliest statistically significant point occurred at week 1 in terms of skin tone. By week 12, there was statistically significant improvement favoring the supplement in all skin assessments—redness (p=0.043), skin tone (p=0.006), acne marks (p=0.027), and scars (p=0.026). These findings are consistent with the IGA (Table 12) and other findings and support the added benefit of the probiotic supplement after 12 weeks of consumption.
Subject Questionnaires
Table 15 (supplement treatment, Group 1) and Table 16 (topicals only treatment, Group 2) provide results of subject questionnaires where subjects were asked to agree/disagree with a variety of statements. The following scale was used: 5=agree completely, 4=agree somewhat, 3=neither agree nor disagree, 2=disagree somewhat, 1=disagree completely. A higher score was a superior score. The percent of subjects in the top box (score 4 and 5) are listed for each week. Both groups noted improvement and satisfaction with their assigned study products.
Table 17 (supplement treatment, Group 1) and Table 18 (topicals only treatment, Group 2) provide results of subject questionnaires where subjects were asked about a variety of skin appearance issues on a score of 1 to 10. A higher score was a more favorable score. The percent of subjects in the top box (favorable score 7-10) are listed for each week the assessment was completed by the subjects. Both groups noted improvement in their acne assessments.
Discussion
As demonstrated by the above data, the study endpoints were met as follows.
Safety Endpoint: The safety endpoint was met for both the supplement and the topical skin care products. No adverse experiences or adverse events occurred during the conduct of the study.
Tolerability Endpoint: The tolerability endpoint was met for both the supplement and the topical skin care products. No irritation or side effects were observed.
Primary Efficacy Endpoint: The primary efficacy endpoint was met for both the supplement and topical skin care products. The additional effect of the oral supplement was also evaluated as compared to the group having only a topical regimen. Statistically significant superior improvement was seen with the supplement group over the topical treatment only group.
The detailed description set-forth above is provided to aid those skilled in the art in practicing the present invention. However, the invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed because these embodiments are intended as illustration of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description which do not depart from the spirit or scope of the present inventive discovery. Such modifications are also intended to fall within the scope of the appended claims.
All publications, patents, patent applications and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention.
Other publications incorporated herein by reference in their entirety include:
Wang Y(1), Kuo S, Shu M, Yu J, Huang S, Dai A, Two A, Gallo R L, Huang C M. Staphylococcus epidermidis in the human skin microbiome mediates fermentation to inhibit the growth of Propionibacterium acnes: implications of probiotics in acne vulgaris. Appl Microbiol Biotechnol. 2014 January ;98(1):411-24. doi: 10.1007/s00253-013-5394-8. Epub 2013 Nov. 22.
This application is a continuation-in-part application of U.S. application Ser. No. 17/327,559, filed May 21, 2021, which is a continuation of U.S. application Ser. No. 16/302,637, filed Nov. 17, 2018, now U.S. Pat. No. 11,040,046, which is a National Stage Entry of International Application No. PCT/US2017/033346, filed May 18, 2017, which claims the benefit of U.S. Provisional Application No. 62/338,334 filed May 18, 2016 and U.S. Provisional Application No. 62/470,130 filed Mar. 10, 2017, all of which are incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62470130 | Mar 2017 | US | |
| 62338334 | May 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16302637 | Nov 2018 | US |
| Child | 17327559 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17327559 | May 2021 | US |
| Child | 18064676 | US |
