SIG1459: A novel phytyl‐cysteine derived TLR2 modulator with in vitro and clinical anti‐acne activity

Cutibacterium (formerly Propionibacterium acnes) is a major contributor to the pathogenesis of acne. C. acnes initiates an innate immune response in keratinocytes via recognition and activation of toll‐like receptor‐2 (TLR2), a key step in comedogenesis. Tetramethyl‐hexadecenyl‐cysteine‐formylprolinate (SIG1459), a novel anti‐acne isoprenylcysteine (IPC) small molecule, is shown in this study to have direct antibacterial activity and inhibit TLR2 inflammatory signalling. In vitro antibacterial activity of SIG1459 against C. acnes was established demonstrating minimal inhibitory concentration (MIC = 8.5 μmol\L), minimal bactericidal concentration (MBC = 16.1 μmol\L) and minimal biofilm eradication concentration (MBEC = 12.5 μmol\L). To assess SIG1459's anti‐inflammatory activity, human keratinocytes were exposed to C. acnes and different TLR2 ligands (peptidoglycan, FSL‐1, Pam3CSK4) that induce pro‐inflammatory cytokine IL‐8 and IL‐1α production. Results demonstrate SIG1459 inhibits TLR2‐induced IL‐8 release from TLR2/TLR2 (IC50 = 0.086 μmol\L), TLR2/6 (IC50 = 0.209 μmol\L) and IL‐1α from TLR2/TLR2 (IC50 = 0.050 μmol\L). To assess the safety and in vivo anti‐acne activity of SIG1459, a vehicle controlled clinical study was conducted applying 1% SIG1459 topically (n = 35 subjects) in a head‐to‐head comparison against 3% BPO (n = 15 subjects). Utilizing the Investigator Global Assessment scale for acne as primary endpoint, results demonstrate 1% SIG1459 significantly outperformed 3% BPO over 8 weeks, resulting in 79% improvement as compared to 56% for BPO. Additionally, 1% SIG1459 was well tolerated. Thus, SIG1459 and phytyl IPC compounds represent a novel anti‐acne technology that provides a safe dual modulating benefit by killing C. acnes and reducing the inflammation it triggers via TLR2 signalling.

capable of inducing hyperkeratinization of the follicular epithelium resulting in comedone formation. [3] The comedone entrapped C. acnes are postulated to undergo a virulent transformation and interact with keratinocytes, initiating the production and release of pro-inflammatory mediators attracting neutrophils and macrophages. [4,5] These events progress to comedone wall rupture, releasing bacteria to surrounding inflammatory cells, further amplifying inflammation. [6,7] Hyperkeratinization and the inflammatory activation of keratinocytes and macrophages have all been shown to be mediated through toll-like receptor-2 (TLR2), whose signalling also plays a key role in the pathogenesis of several dermatological diseases. [8,9] Moreover, it has recently been demonstrated that TLR2 formation of TLR2 homodimers and/or TLR2/6 heterodimers are required for C. acnes recognition in human cells. [10] Current treatments for acne and/or acne prone skin include topical and oral retinoids, antibiotics, and topical antimicrobials, all of which have potential adverse effects. In addition, longterm exposure to antibiotics can result in selection and growth of antibiotic-resistant bacteria. Furthermore, the oxidizing agent benzoyl peroxide (BPO) for which resistance does not occur can result in skin bleaching, dryness and stinging. Thus, development of safe, new compounds which provide relief to individuals with skin prone to acne is desired.
Isoprenylcysteine (IPC) analogs represent a novel class of topical non-steroidal anti-inflammatories, [11] which have also been previously reported to have antibacterial and anti-acne properties. [12] IPC analogs contain a 15-or 20-carbon side chain linked to the amino acid cysteine, thereby mimicking the C-terminus of processed CAAX proteins. [13] Introducing chemical modifications to both the fatty acid tail and cysteine moieties of the IPC backbone, yielded a novel phytyl IPC compound: Tetramethyl-hexadecenyl-cysteineformylprolinate (SIG1459) (Supplemental data, Figure S1). In this study, we report that SIG1459 possesses in vitro antibacterial and anti-inflammatory properties inhibiting two TLR2 family members required for C. acnes recognition, TLR2 homodimer and TLR2/6 heterodimer. Furthermore, clinical results in subjects with acne prone skin demonstrate SIG1459 is well-tolerated and significantly outperforms benzoyl peroxide (BPO) when applied topically for 8 weeks. BPO

| Antimicrobial assays
Cutibacterium acnes strains (ATCC ® 6919 ™ , ATCC ® 11827 ™ ) were cultured in Reinforced Clostridial or Brain Heart Infusion broth media (BD Diagnostics; Sparks, MD) under anaerobic conditions (37°C, under shaking at 175 rpm). Compounds to be tested for antibacterial activity were added dissolved in water or DMSO, diluted by 2-fold serial titrations and then 7.5 μL added to 142.5 μL of a 7.5 × 10 5 CFU/ mL bacteria suspension and incubated for 72 hours. Turbidity (OD 595 ) was measured using a microplate reader (PerkinElmer; Houston, TX, USA). The Minimum Inhibitory concentration (MIC) was defined as the lowest concentration of an agent that achieved ≥90% growth inhibition. The Minimum Bactericidal concentration (MBC) was determined by sub-culturing broth samples after test material incubation from the MIC assay to agar plates. MBC was defined as the lowest antimicrobial concentration that completely inhibited colony formation. For Minimum Biofilm Eradication concentration (MBEC) determination, we followed protocols previously described [14] using the crystal violet staining assay. MBEC was defined as the minimum concentration necessary to achieve ≥80% eradication of attached biofilm compared to vehicle control.
Inhibition activity for each compound was calculated by the difference between the vehicle-only and inducer plus vehicle treated cells.  ing the study (two subjects in the 1% SIG1459 group and two subjects in the vehicle control group were lost to follow-up). The subjects were 18 years and above. Subjects reported to the testing facility for baseline screening at which time Informed Consent and demographics were obtained. Inclusion/Exclusion criteria were verified, using expert clinical grading evaluations to determine eligibility. Subjects had to exhibit mild to moderate acne on their face (mild to moderate acne on the Investigator Global Assessment (IGA) acne scale = 2 or 3, where 2 is mild severity; some non-inflammatory lesions with no more than a few inflammatory lesions (papules/pustules only, no nodular lesions) and 3 is moderate severity; up to many non-inflammatory lesions and may have some inflammatory lesions, but no more than one small nodular lesion). Thirty-five subjects received 1% SIG1459 cream, 15 subjects 3% benzoyl peroxide (BPO) cream and 15 subjects the vehicle cream for facial application (Supplemental data, Table S1 for subject demo-

| Statistical analysis
For all antibacterial measurements and cytokine levels, samples were assayed in triplicates. Statistical significance was determined by ANOVA followed by a Dunnett multiple comparisons test using P-values less than .05 as significant difference. Cytokine concentration-response curves were generated by fitting data with the logistic, four-parameter equation using the Sigma Plot software, from which the IC 50 and maximum inhibition were determined. For the Clinical study, the paired one-way ANOVA was used to assess within-subject improvements over baseline, and the unpaired oneway ANOVA to compare responses between SIG1459, BPO and vehicle treatments. P < .05 was considered significant.

| SIG1459 possesses antibacterial activity against Cutibacterium acnes
Previously, we demonstrated that IPCs possessed antimicrobial activity vs Cutibacterium acnes, exhibiting a significant minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). [12] To investigate the antibacterial properties of the new phytyl IPC compound, identified as SIG1459 and compare its activity to commonly used anti-acne cosmetic actives and drug compounds, we determined MIC and MBC values vs two common commercially available C. acnes strains isolated from facial acne (ATCC ® 6919™ and ATCC ® 11827™). SIG1459 demonstrated antibacterial activity against the two C. acnes strains as did BPO and clindamycin. SIG1459's MIC value of 8.5 μmol\L was ~200 times more potent than BPO (1.75 mmol\L) and it's MBC value of 16.1 μmol\L, was ~800 times more active than BPO (Table 1). Clindamycin demonstrated the strongest antibacterial activity (MIC = 0.3 μmol\L, MBC = 2 μmol\L) ( Table 1). As expected, salicylic acid had a negligible effect against C. acnes.
Recently, it has been suggested that C. acnes cells residing within the follicles grow as a biofilm, which in turn can make them more resistant to antimicrobial agents. [15] Thus, in addition to demonstrating strong MIC and MBC activity, we explored for the first time the potential of IPC compounds such as SIG1459 to specifically eradicate biofilms. Our results demonstrate SIG1459 disrupts C. acnes biofilms with a minimal biofilm eradication concentration (MBEC) of 12.5 μmol\L, once again significantly outperforming BPO and salicylic acid (MBEC > 10 mmol\L) ( Table 1). As was the case with MIC and MBC determination, clindamycin was the most potent with an MBEC = 0.6 μmol\L (Table 1).

| SIG1459 inhibits TLR2-activated cytokine release from human keratinocytes
TLR2 expression is increased in acne lesions, facilitating recognition of Cutibacterium acnes and contributing to the inflammatory response. [6,16] C. acnes activates TLR2 signalling in keratinocytes of the intact comedone wall and macrophages after it is released to the surrounding dermis following comedone rupture. Our results demonstrate that SIG1459 strongly inhibits C. acnes-induced IL-8 levels in NHEKs with an IC 50 = 0.003 μmol\L (Figure 1) of magnitude more potent than BPO and salicylic acid (>1 μmol\L) (Supplemental data, Figure S3, Table S2). No inhibitory activity was found for C. acnes-induced IL-1α levels (Table S2). Furthermore, given SIG1459's IC 50 value for inhibiting C. acnes-induced IL-8 is ~3000 times lower than its MIC, MBC and MBEC values (Table 1), it is unlikely that its anti-inflammatory activity is directly a consequence of bacterial killing.
Recently, it has been demonstrated that TLR2 formation of TLR2/6 heterodimers is required for C. acnes recognition in human cells. [10] To investigate SIG1459's potential anti-inflammatory activity via inhibition of TLR2 signalling in keratinocytes, we uti-   Figure S3), thus validating the experimental conditions ( Figure S3D). Altogether, these results also suggest that while BPO and salicylic acid have been reported to possess anti-inflammatory properties, [17,18] they are unlikely to confer this activity by inhibiting C. acnes-TLR2 signalling.

| SIG1459 is well-tolerated and outperforms BPO in human subjects
In view of SIG1459's in vitro anti-acne potential, we sought to determine its tolerability and activity in human subjects. 1% SIG1459 was first tested clinically in a Human Repeated Insult Patch Test and was found to cause no skin sensitization or irritation (PCR, data not shown). Given this result, 1% SIG1459 in a topical cream was tested in a single-blinded vehicle controlled-study, to determine its tolerability in subjects with acne prone skin and to compare its activity vs 3% BPO, a commonly used anti-acne treatment. No adverse effects were reported for the 1% SIG1459 group, indicating again that it was well tolerated. Visual evaluation data for the 35 subjects completing use of 1% SIG1459 and the 15 subjects using 3% BPO, showed statistically significant improvement in average IGA scores at 2-week, 4-week and 8-week assessments as compared to baseline ( Figure 2).
However, after 8 weeks of use, the average assessment scores demonstrated 1% SIG1459 significantly outperformed the 3% BPO group with a 77% reduction in IGA score compared to baseline vs a 56% reduction in the average score for the BPO group ( Figure 2).
These results were even more impressive given that the 15 subjects in the vehicle control group worsened by ~30% on average over the same 8-week period ( Figure 2). This reduction in IGA scores was confirmed in the photographs taken of the 1% SIG1459 group, where marked visual improvement in the signs and symptoms of acne from baseline to Week 8 (Supplemental data, Figure S2) were observed.
Interestingly, photographs also taken using UV light illumination were utilized to visualize the production of porphyrins by C. acnes  F I G U R E 1 SIG1459 inhibits Cutibacterium acnes induced proinflammatory cytokine production. NHEKs were co-incubated with 1 × 10 7 CFU/mL (ATCC ® 6919™) and SIG1459 in supplementdepleted media for 24 h. Pro-inflammatory cytokine (IL-8) levels in media supernatants were assayed by ELISA. The data represent the mean ± SE of cumulative from three independent experiments. *P ≤ .05; **P ≤ .01 indicates a statistically significant difference compared to C. acnes-only treated cells

| D ISCUSS I ON
Acne is a multifactorial disease caused primarily by increased sebum production, hyperkeratinization, inflammation and Cutibacterium acnes activity within the follicle. C. acnes is an anaerobic gram-positive commensal bacterium known for becoming virulent in the development of acne that affects ~85% of adolescents and in some instances, persists into adulthood. [19] In inflammatory acne lesions, C. acnes activates an innate immune response via TLR2. [6] In vivo studies demonstrate that TLR2 is increased in the epidermis of acne lesions and in vitro studies using human keratinocytes show incubation with C. acnes also results in an increase of TLR2 expression. [16,20] Additional studies performed in keratinocytes demonstrate that TLR2 activation is an initiating step in comedone formation [21] indicating a key role for C. acnes and TLR2 in the pathogenesis of acne.
Previously, we reported that IPC compounds inhibited TLR2and TLR4-induced pro-inflammatory cytokine production of IL-6 and IL-8 and possessed antibacterial activity. [12,22] Figure S1). Phytol has been reported to have antibacterial activity against several organisms including Staphylococcus aureus, [23] Mycobacterium tuberculosis [24] and Pseudomonas aeruginosa. [25] For C. aeruginosa it was proposed that phytol exerts its antibacterial properties by inducing oxidative stress. [25] However, we have previously demonstrated that phytyl-cysteine compounds similar in structure to SIG1459 possess antioxidant properties and reduce oxidative stress, [26] which would contrast with BPO's antibacterial mechanism of action (oxidation and the formation of free radicals decrease C. acnes). Thus, this is an unlikely mechanism for SIG1459 and its class of compounds to confer antibacterial activity.
Phytol has also been reported to induce leakage of potassium ions from S. aureus cell walls, thus damaging cell membranes and killing the bacteria. [23] Yet, in-house antibacterial studies testing SIG1459 and other phytyl IPC compounds against S. aureus show that they do not kill or inhibit the growth of this organism (data not shown). Given its lipophilic nature, SIG1459 may still disrupt C. acnes cell membranes as part of its mechanism of action for antibacterial activity, but results presented here suggest phytyl IPC compounds have a distinct and novel mode of action that is yet to be determined.
Lastly, we recognize that there is strain heterogeneity in C. acnes virulence potency, [27][28][29] but for initial antibacterial screening we used two of the most commonly used commercially available C. acnes strains isolated from facial acne (ATCC ® 6919™, ATCC ® 11827™). In the future, we plan to determine SIG1459's antibacterial activity vs the most virulent C. acnes strains.
To begin to elucidate how SIG1459 successfully reduces C. acnesinduced inflammation in vitro and yielded positive clinical results in subjects with acne prone skin, we utilized specific molecularly defined TLR2 homodimer and heterodimer ligands to induce a proinflammatory response in NHEKs. IL-8 was previously shown to be induced by TLR2 and is one of the highest upregulated genes in acne lesions. [30] IL-1α was previously shown to be induced by C. acnes, [31] but not TLR2/TLR6 ligand. [32] Interestingly, SIG1459 was found to  (Table 2). However, using the TLR2/1 heterodimer ligand Pam 3 CSK 4, SIG1459 tested up to 1 μmol\L did not inhibit IL-8 production. Not surprisingly, these results are consistent with the recently reported findings that C. acnes is mainly recognized by TLR2 and TLR6, but not TLR1. [10] This suggests that TLR2/2 and TLR2/6 dimers share a specific molecular conformation that is recognized by SIG1459 and is required for C. acnes induced inflammation. C. acnes preincubation with TLR2 and TLR6 but not TLR1 antibodies inhibits NFĸB activation, [10] suggesting that SIG1459's target(s) is in these specific TLR-NFĸB signalling modules. SIG1459 blocking PGN, but not C. acnes induced IL-1α is not surprising, as previous published results indicate that stimulation with C. acnes could also activate TLR2independent pathways (ie PAR-2) to induce IL-1α expression. [33] As reported here, we were able to induce IL-1α with peptidoglycan (TLR2 ligand), suggesting that SIG1459 activity is dependent on TLR2 inhibition. IPC compounds have also been reported to modulate MEK/MAPK kinase [34] and ERK signalling. [35] Our next goal will be to elucidate where SIG1459 is targeting along the C. acnes-TLR pathway that leads to NFĸB activation and ultimately cytokine release.  In addition, SIG1459's ability to modulate TLR2 raises the potential of it ameliorating a third important hallmark of acne, hyperkeratinization. C. acnes activated TLR2 signalling has been shown to induce epidermal hypercornification through the release of IL1α, which is a positive feedback stimulator of keratinocyte differentiation, in addition to its pro-inflammatory activity. [21] Oleic acid and other unsaturated fatty acids generated through sebum lipid hydrolysis have also been shown to induce epidermal hyperkeratinization [37] and to be TLR2 receptor ligands. [38,39] Perhaps oleic acid specifically activates TLR2/2 and does not involve direct TLR2/6 activation by C. acnes to initiate hyperkeratinization. Thus, in addition to its antimicrobial and anti-inflammatory properties, SIG1459 and other phytyl IPC derivatives may also inhibit another key factor of acne pathogenesis, hyperkeratinization. Additional studies need to be performed to explore this potential mechanism, though initial results are promising as SIG1459 inhibits TLR2 induced IL-1α production (Table S2). In conclusion, SIG1459 is a novel phytyl-cysteine derived TLR2 modulator that targets acne through its dual acting properties. The data presented here provides in vitro and in vivo proof-of-concept that phytyl IPC derivatives are effective for subjects with acne prone skin. Further research and development will potentially identify more potent new chemical entities that can treat at least two and perhaps three cardinal factors of acne.

CO N FLI C T O F I NTE R E S T
All authors for this manuscript are paid employees or consultants for Signum Biosciences which is where the research was performed.