ACNE: ETIOPATHOGENESIS, PROPIONIBACTERIUM ACNES AND BIOFILM

Transcription

1 ACNE: ETIOPATHOGENESIS, PROPIONIBACTERIUM ACNES AND BIOFILM Phone:

2 INTRODUCTION It was traditionally claimed that acne lesions develop due to an increased activity of the sebaceous glands leading to seborrhea, abnormal follicular differentiation and increased cornification as well as microbial colonization with inflammatory reaction and the subsequent immunological processes.* Results of recent research have significantly changed the classical view of acne pathogenesis by identifying upstream mechanisms that lead to the development of acne lesions. Androgens, skin lipids and regulatory neuropeptides (ie substance P, SP) appear to be involved in this multifactorial process.* *New concepts in acne pathogenesis, Christos C. Zouboulis, December 2013

3 PATHOGENESIS According to the current knowledge, the biological events recognized as crucial in the development of acne lesions are: 1.Outlet obstruction of the sebaceous follicle; 2.Hyperactivity and hyper-responsiveness of the sebaceous gland; 3.Pro-inflammatory hyperactivity of Propionibacterium acnes; 4.Intra-and peri-follicular inflammation.

4 1. OUTLET OBSTRUCTION OF THE SEBACEOUS FOLLICLE An hyperproliferation of the follicular epithelium decreases the opening of the duct even up to complete closure, leading to the formation of microcomedons. The latter represents the initial acne lesions but they can also be found in normal-looking skin.

5 This early stage of development of acne lesions is associated with the activation of the vascular endothelium under the participation of inflammatory processes. This confirms the hypothesis that the very beginning of acne is an inflammatory procedure. The results of Ingham et al. point out in the same direction: they found bioactive IL-1 in open acne comedons in untreated patients (Fig.)*. *New concepts in acne pathogenesis, Christos C. Zouboulis, December 2013 Phone:

6 2. HYPERACTIVITY AND HYPER-RESPONSIVENESS OF THE SEBACEOUS GLAND Sebaceous glands of acneic patients are much more active than those of non acneic ones. In fact, in sebaceous glands of acneic patients are more pronounced: 1.The sensitivity to androgens action; 2.The number and activity of cellular receptors for various other hormones, vitamins, drugs, neurotransmitters...; 3.The response to the stress of such receptors, with production of substances with pro-inflammatory activity.

7 The image of the sebaceous gland as a structure exclusively intended for the production of sebum is to be considered obsolete. According to recent studies, the sebocyte seems to be a protean cell, able to perform several activities and regulate various biological effects. In addition to the sebum production, this cell expresses the ability to release pro-inflammatory substances contributing to the development of papules, pustules and nodules. It has long been known that the sebaceous gland is able to synthesize cholesterol starting from acetate. Recent studies have demonstrated that sebocytes contain everything necessary to independently produce androgens from cholesterol. Therefore, sebaceous glands activity is not exclusively regulated by hematic androgens but also by those they produce. Phone: info@general-topics.com

8 The substantial production of sebum, blocked in the output phase by the occlusion of the follicular channel, is responsible for the progressive dilatation of the follicular duct walls until their bulging and consequent breaking. The content of the channel, consisting mainly of sebum, keratin and bacteria, is poured into the surrounding dermis with further increase of the pro-inflammatory thrust. Phone:

9 3. PRO-INFLAMMATORY HYPERACTIVITY OF PROPIONIBACTERIUM ACNES Within the channel of sebaceous follicles, mixed with sebum, proliferates Propionibacterium acnes (P. acnes). This happens both in healthy and acneic skin. The blocking of follicular sebum outflow to the skin surface creates a microenvironment rich of sebum and poor of oxygen, ideal conditions for the growth and viability of P.acnes.

10 4. INTRA-AND PERI-FOLLICULAR INFLAMMATION For the formation of acne lesions an adequate inflammatory reaction by the host/patient to the pro-inflammatory stimulus promoted by hormones, vitamins, sebaceous lipids, stress, P.acnes, etc., is necessary. If these pro-inflammatory stresses are not followed by this reaction, papules, pustules and nodules do not appear.

11 WHICH FACTORS ARE INVOLVED IN THE DEVELOPMENT OF ACNE?

12 1. Hereditary factors It seems that hereditary factors play an important but indirect role in the development of acne. The positive correlation of familial incidence and severity of acne has long been known. Recent studies have shown a correlation between the acne severity grade with a history of severe acne in the parents, especially the mother*. There are interesting new insights into a direct genetic association of acne with androgen and lipid-associated disorders*. Currently, genetic predisposition does not indicate a clear causal link to acne. It is only noticed a genetic predisposition but it is not known if it is linked to similar hormonal balances or similar sebum composition*. *New concepts in acne pathogenesis, Christos C. Zouboulis, December 2013

13 2. Gender As suggested through clinical observations, androgens play a key role in acne pathogenesis both in the increase of sebaceous gland volume as well as in the production of sebum. Gender-related genes are involved in the development of acne.* Androgens stimulate the proliferation of keratinocytes of the ductus seboglandularis and the acroinfundibulum.* The acne-affected skin expresses higher levels of androgen receptors and higher 5αreductase activity than the non-affected skin.* In vitro experiments with sebaceous gland-like cells of the rat and human sebocytes have shown that sebaceous lipid synthesis is up-regulated in the presence of androgens and certain fatty acids.* The relationship between androgens, proliferative pathways and acne shows lots of confirmations but hyper seborrhea does not necessarily lead to acne.* *New concepts in acne pathogenesis, Christos C. Zouboulis, December 2013

14 3. Neuropeptides Current experimental data indicate the involvement of neuropeptides (with hormonal or non-hormonal activity) in the development of sebaceous follicle inflammatory processes. In the last years have been discovered new nuclear and cell surface receptors on sebocytes. Among the various receptors discovered it is interesting to mention those for substance P, neuropeptide produced by peripheral nerve endings located around the sebaceous glands. It has been proven the role of substance P in cutaneous responses to stress. The activation of these systems by stressful events, of both local and systemic origin, induces the release of pro-inflammatory cytokines and lymphokines and an overproduction of sebum and can result in onset or worsening of acne.

15 5. Propionibacterium acnes Gram-positive, anaerobic but oxygen-tolerant, normally present on human skin, hair and mucous membranes. This bacterium is known to be one of the main inducers of inflammation in acne, acting through various mechanisms such as: 1. TOLL-LIKE RECEPTORS 2 ACTIVATION: TLR-2 are expressed in human monocytes and keratinocytes. The chemokine/cytokine synthesis is stimulated in keratinocytes by activation of TLR-2 via P.acnes. (this activation is P.acnes subtype-dependent). However, P.acnes was found incapable to stimulate IL-1 release from human keratinocytes in vitro.* 2. RELEASE OF LIPASE AND RELEASE OF FATTY ACIDS BY TRIGLYCERIDES HYDROLISIS: it uses sebum as a source of energy through specialized enzymes (lipases) which hydrolyze triglycerides releasing proinflammatory fatty acids and small molecules as propionic acid. 3. PRODUCTION OF PORPHYRINS, ROS, METALLOPROTEASES, LEUKOTRIENES AND PROSTAGLANDINS, able to contribute to the follicular channel walls damage up to rupture. Moreover, it directly induces infundibular hyperkeratosis and seborrhea. *New concepts in acne pathogenesis, Christos C. Zouboulis, December 2013

16 FOCUS ON: Toll-like Receptors Toll-Like Receptors (TLR) represent a family of cell surface receptors pattern recognition receptors (PRRs), expressed by different cell types. They are able to recognize specific structures typical of pathogens and microbes, named pathogen-associated molecular patterns (PAMPs). The binding of TLRs with microbial structures activates these receptors, triggering an inflammatory response. Such a PAMP is the peptidoglycan and lipoproteins of gram-positive bacteria. TLR2 is among the better characterized and studied Toll-like receptors, and has an essential role in detection of invading pathogens. TLR2's primary function is LP (lipoprotein) mediated signaling. Specifically, TLR2 recognizes peptidoglycans (PGN) and lipoproteins associated with Gram-positive bacteria (Propionibacterium Acne), as well as a host of other microbial and endogenous ligands including peptidoglycan

17 FOCUS ON: Toll-like Receptors The common downstream signaling pathway of TLR2 leads to the activation of NF-kB through the myeloid differentiation protein (MyD88) and IL-1-receptor-associated kinase in various cell types.

18 FOCUS ON: Toll-like Receptors TLR2 can be activated by lipoproteins, released by Gram +, including P. Acnes.

19 TLR2 and inflammation Kinetics of TLR2 expression in acne according to the evolution of the lesion. In early acne lesions, up to 6 h, few rare TLR2 cells were detected. In lesions obtained between 12 and 24 h, TLR2 cells were much more numerous around the pilo-sebaceous follicles. In later older lesions obtained between 48 and 72 h, greater numbers of TLR2 cells were detected. 6 hours hours hours

20 TLR-2 and inflammation TLR2 mediated the ability of P.acnes to activate monocyte-release of IL-12 and IL-8. The detection of TLR2 cells in the peri-follicular region provides indirect evidence that TLR2 activation contributes to the pathogenesis of acne, suggesting that these cells promote inflammatory responses at the site of the disease activity. IL-8 attracts neutrophils to the site of active lesion, and release of lysosomal enzymes by neutrophils leads to rupture of follicular epithelium and further inflammation. In contrast, IL-12 promotes development of Th1-mediated immune responses. Overproduction of Th1 cytokines such as IL-12 has been implicated in the development of tissue injury in certain autoimmune and inflammatory diseases. In this manner, the activation of TLR2 on monocytes and other TLRs as well as other inflammatory cells are likely involved in the pathogenesis of acne.

21 TLR-2 and inflammation PAMP treatment led to a pattern of hypercornification similar to that created by IL-1α exposure (and, indeed, that are seen in comedons in situ), via TLR2- and dependent mechanisms.

22 TLR-2 and pro-il 1β release TLR 2 activation induces pro-il 1β synthesis, NLRP3 activation induces its proteolytic activation via inflammasome.

23 TLR-2 and pro-il 1β release Keratinocytes express the protein NLRP3, which belongs to the NLR family (nucleotidebinding domain), a family of PPRs recently identified, which is able to trigger a response against external agents in the cytoplasm and activate the inflammosome complex.

24 TLR-2 and pro-il 1β release Inflammosome represents a multimeric complex formed by NLRP3, an adapter protein called ASC and the pro-caspase-1; its activation ends in the production of caspase 1, which transforms pro-il-1β in the active form IL-1β.

25 IL-1β Drives Inflammatory Responses to Propionibacterium acnes In Vitro and In Vivo IL-1β mrna and the active processed form of IL-1β are abundant in inflammatory acne lesions. Moreover, P.acnes is a trigger of monocyte macrophage NLRP3-inflammasome activation, IL-1β processing and secretion that is dependent on phagocytosis, lysosomal destabilization, reactive oxygen species, and cellular K+ efflux. In mice, inflammation induced by P. acnes is critically dependent on IL-1β and the NLRP3 inflammasome of myeloid cells. These findings show that the commensal P. acnes by activating the inflammasome can trigger an innate immune response in the skin, thus establishing the NLRP3-inflammasome and IL-1β as possible therapeutic targets in acne.

26 Gt-peptide-10 and Interleukins down modulation A reconstructed artificial human skin model comprising normal human epidermal keratinocytes, growing as an integrated three-dimensional cell culture model, perfectly mimicking the human skin in vitro. The model exhibits normal barrier functions (presence of a differentiated stratum corneum). It has been proven that GT-peptide-10 can down modulate the release of IL-1, IL-1, IL-8 and IL-12, induced by SLS, from keratinocytes.

27 Gt-peptide-10 and Interleukins down modulation Interleukins release inhibition from keratinocytes after 24 hours of exposure to the irritant SLS.

28 5. Propionibacterium acnes It exists in both the planktonic and biofilm form.

29 BACTERIAL BIOFILMS BACTERIAL BIOFILMS GROWTH AND DEVELOPMENT PROCESSES Phone:

30 WHAT IS A BACTERIAL BIOFILM? A bacterial biofilm is a structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface. Phone: info@general-topics.com

31 For a long time, in the history of microbiology, microorganisms were considered suspended cells, namely planktonic organisms, and classified according to the type of their growth in a rich culture medium. Towards the end of 1600 Anton van Leeuwenhoek, scraping the surface of the teeth, remarked, with his primitive microscope, the "animaluculae" which constituted a microbial community (biofilm). Phone: info@general-topics.com

32 Unicellular organisms show two different ways of behavior: 1. The first is the familiar floating form, or planktonic, in which the separated cells float or swim independently in a liquid carrier. 2. The second is the aggregated state (biofilm), or sessile, in which the cells are tightly bound and firmly attached to one another and also, usually, to a solid surface. Phone: info@general-topics.com

33 The change in behavior (from planktonic to sessile) is activated by a mechanism of chemical communication that differs between species. Biofilm formation seems to be regulated by the secretion of particular molecules, a process that takes the name of QUORUM SENSING. Phone: info@general-topics.com

34 It is a process of communication between bacterial cells. It is a self-induced process: when the molecules begin to accumulate in the area surrounding the microorganisms, the latter undergo a series of physiological changes that allow the formation of extracellular biofilm. WHAT IS QUORUM SENSING? Phone: info@general-topics.com

35 In the biofilm, we can distinguish two parts: 1- EPS (Extracellular Polymeric Substances): gel consisting of water, polymers of uronic acids or teichoic acids, proteins and salts. The EPS can represent up to 80% by weight of the biofilm. Because of its high water content, the matrix can contribute to resistance to antibiotics preventing the diffusion of these substances through the biofilm, probably by binding these molecules. 2- Planktonic bacteria represent the remaining part of the biofilm. WHAT IS A MICROBIAL BIOFILM MADE OF? Phone: info@general-topics.com

36 WHY BACTERIA FORM THE BIOFILM? Bacteria form biofilms to protect themselves in hostile environments. Phone:

37 It allows bacteria to show a coordinated behavior, giving them biological advantages such as resistance to antibiotics and to the host immune system. It is structured to enable breathing, the exchange of fluids and nutrients, and to prevent access of immune cells, such as phagocytes. It reduces the concentration of antimicrobial inhibitory or lytic factors, preventing them to reach the microorganisms. WHY BACTERIA FORM THE BIOFILM? Phone: info@general-topics.com

38 Biofilms release antigens and stimulate the immune response and thus the production of antibodies but antibodies are not effective in killing the bacteria within the biofilm and they may cause damage to the surrounding tissues. Biofilms can release free single cells, planktonic, which quickly multiply and disperse. The planktonic bacterial populations, however, are more exposed to a variety of factors deleterious for their survival (such as the host immune system). BIOFILM AND HUMAN INFECTIONS Phone: info@general-topics.com

39 BIOFILM AND HUMAN INFECTIONS

40 Antibiotic therapy typically resolves symptoms caused by planktonic cells released from the biofilm, but it s unable to kill and eradicate bacteria in the biofilm. The bacteria, in the form of biofilm, are times more resistant to antibiotic treatment compared to the planktonic phenotype. BIOFILMS AND ANTIBIOTIC RESISTANCE BIOFILM MICROBICI E PATOLOGIE Phone: info@general-topics.com

41 ACNE: PROPIONIBACTERIUM ACNES AND BIOFILM Phone:

42 PROPIONIBACTERIUM ACNES AND BIOFILM Evidence for the presence of biofilms in acne is predominantly derived from the ability of Propionibacterium acnes (P.acnes) to form biofilms both in vitro and on implanted medical devices. In addition, sequencing of the P.acnes genome reveals the presence of genes involved in the production of EPS and Quorum Sensing molecules.

43 PROPIONIBACTERIUM ACNES AND BIOFILM P.acnes strains isolated from acne patients form biofilms in vitro that are characterized by increased lipase activity as compared to planktonic organisms. This may explain a pathogenic role for P. acnes biofilms in acne, as lipase is not only a well known virulence factor, but it also produces irritant fatty acids that promote inflammation and enhance P. acnes adhesion to the sebaceous follicle.* Keratin plugging has long been considered a key component of acne pathogenesis, and the adhesive properties of the EPS produced by P. acnes biofilms in sebum may be responsible for the tenacious binding of keratinocytes to the infundibular epithelium.** *Jappe U. Pathological mechanisms of acne with special emphasis on Propionibacterium acnes and related therapy. Acta Derm Venereol. 2003; 83(4): *Gribbon EM, Cunliffe WJ, Holland KT. Interaction of Propionibacterium acnes with skin lipids in vitro. J Gen Microbiol Aug;139(8): **Burkhart CG, Burkhart CN. Expanding the microcomedone theory and acne therapeutics: Propionibacterium acnes biofilm produces biological glue that holds corneocytes together to form plug. J Am Acad Dermatol Oct;57(4):722-4.

44 ANTI-ACNE THERAPY AGAINST P.ACNES BIOFILM Phone:

45 ANTI-ACNE THERAPY AGAINST P.ACNES BIOFILM An in vitro study * evaluating multiple anti-acne agents, alone or in combination, found that only: 0.1% triclosan 5% benzoyl peroxide + 0.5% erythromycin 5% benzoyl peroxide + 1% clindamycin were effective in both reducing biofilm mass and killing >99% of biofilm-associated P.acnes. Interestingly, 5% benzoyl peroxide alone was ineffective unless combined with erythromycin or clindamycin, possibly as a result of antibiotics inhibiting protein synthesis and therefore making P.acnes cells vulnerable to benzoyl peroxide generated radicals. *Coenye T, Peeters E, Nelis HJ. Biofilm formation by Propionibacterium acnes is associated with increased resistance to antimicrobial General agents Topics and srl increased production of AKNICARE putative virulence Rev. factors Res Microbiol May;158(4):

46 ANTI-ACNE THERAPY AGAINST P.ACNES BIOFILM It should be noted that although 30mM azelaic acid was bactericidal, it did not reduce biofilm mass. Minocycline was the only agent in its class that removed biofilm and displayed the greatest bactericidal effect of all the tetracyclines tested. It has been postulated that the success of isotretinoin therapy may be related to reduction of sebaceous gland size with a subsequent decrease in sebum production, thus depleting the nutrient source for P.acnes biofilm. * The effectiveness of photodynamic therapy may also be due to an indirect effect on biofilms mediated by decreased sebaceous gland activity.** *Burkhart CG, Burkhart CN. Expanding the microcomedone theory and acne therapeutics: Propionibacterium acnes biofilm produces biological glue that holds corneocytes together to form plug. J Am Acad Dermatol Oct;57(4): **Hongcharu W, Taylor CR, Chang Y, et al. Topical ALA-photodynamic therapy for the treatment of acne vulgaris. J Invest Dermatol Aug;115(2):

47 &BIOFILM Phone:

48 PROPIONIBACTERIUM ACNES AND BIOFILM Propionibacteria and especially Propionibacterium acnes (P. acnes) are regarded as common members of the skin bacterial landscape. P. acnes inhabits pilosebaceous units and occurs often as biofilms. Biofilms are recognized as the default evolutionary mode of microbial growth. An important feature of biofilms is an increased antibiotic tolerance and the ability to persist over long periods of time. Figure 1. Confocal microscopic images of transversely sectioned skin biopsies highlighting different patterns of P. acnes colonization in hair follicles; P. acnes labelling in green and cytokeratin labeling in red. a. Attachment of P. acnes to the follicle wall; scale bar 2 µm. b. Attachment of P. acnes to the hair shaft; scale bar 2 µm. c. P. acnes biofilm spreading over nearly the entire lumen of the hair follicle; scale bar 10 µm. d. Matrix-encased P. acnes biofilm without obvious attachment to the follicle wall; scale bar 20 µm.

49 A NEW APPROACH TO ACT ON THE BACTERIAL BIOFILM WITHOUT DESTROYING IT Destroying biofilm releases planktonic bacteria

50 ANTI-ACNE THERAPY AGAINST P.ACNES BIOFILM 1 2 P.ACNES BIOFILM Bactericidal activity on Propionibacterium acnes bacteria inside the biofilm Biofilm disruption and release of Propionibacterium acnes in form of planktonic bacteria Delay in formation of new biofilm Bactericidal activity on planktonic bacteria

51 &BIOFILM In vitro evaluation of the effectiveness of Triethylcitrate and GT Peptide-10 on Propionibacterium acnes, Propionibacterium granulosum, Propionibacterium avidum and on P.acnes biofilm formation and dispersal. Prof. Oleg Alexeyev, Researcher, Department of Medical Biosciences/Pathology, Umeå University

52 &BIOFILM Introduction Propionibacterium species are non sporulating, gram-positive anaerobic bacilli that are considered commensal bacteria on the skin. They are usually non pathogenic and are common contaminants of blood and body fluid cultures. These species are slow-growing and require at least 6 days for growth in culture. Propionibacterium species belong to the genera of coryneforms and are the best studied because of their association with acne vulgaris. Propionibacterium species, however, can also cause numerous other types of infections, including endocarditis, postoperative shoulder infections and neurosurgical shunt infections. Propionibacterium Infections, Author: Sajeev Handa, MBBCh, BAO, LRCSI, LRCPI; Chief Editor: Burke A Cunha, MD

53 &BIOFILM Introduction Propionibacterium acnes is found briefly on the skin of neonates, but true colonization begins during the 1-3 years prior to sexual maturity. During this time, numbers of P.acnes rise from fewer than 10/cm² to about 106/cm², chiefly on the face and upper thorax. P.acnes grows in the lipid-rich microenvironment of the hair follicle. In acne vulgaris, P.acnes produces inflammatory mediators that result in acne papules, pustules, and nodulocystic lesions. Propionibacterium Infections, Author: Sajeev Handa, MBBCh, BAO, LRCSI, LRCPI; Chief Editor: Burke A Cunha, MD

54 &BIOFILM Introduction Propionibacterium granulosum is found in the same areas but at numbers about one hundredth of those of P.acnes. Both P.acnes and P.granulosum may be isolated from the gastrointestinal tract. Propionibacterium avidum is found in the axilla rather than on exposed areas and increases in numbers at puberty. Propionibacterium Infections, Author: Sajeev Handa, MBBCh, BAO, LRCSI, LRCPI; Chief Editor: Burke A Cunha, MD

55 &BIOFILM Aim of the study The aim of the performed experiments was to study the effects of two antimicrobial substances, triethylcitrate and a small peptide (GT-Peptide-10), on three bacterial strains commonly found on the human skin, namely Propionibacterium acnes, Propionibacterium granulosum and Propionibacterium avidum. Moreover, the effect of both substances on P.acnes biofilm formation and dispersal, a characteristic growth state putatively involved in the development of acne vulgaris, was studied.

56 &BIOFILM Materials and Methods The MIC (the lowest concentration of an antimicrobial that inhibits the growth of a microorganism) of both single substances and as a mixture for all three bacteria was determined by growth experiments and measuring the optic density of cultures at 590 nm. The effect of the substances on pre-existing P.acnes biofilms was studied by a modified crystal violet assay as described previously. Viability assays were performed using the LIVE/DEAD BacLight Bacterial Viability Kit (LifeSciences) and microscopy analysis of biofilms treated with triethyl citrate and the peptide.

57 &BIOFILM Evaluation results of the effectiveness of Triethylcitrate and GT Peptide-10 on Propionibacterium acnes, Propionibacterium granulosum and Propionibacterium avidum In each experiment, the three bacteria showed specific sensitivities against both antimicrobial substances. The MIC for the GT-Peptide-10 for P.acnes and P.granulosum was 2.5μl/ml, whereas growth of P.avidum was inhibited at concentrations of 6-7μl/ml (Fig.1). In contrast, the MIC for triethylcitrate for P.acnes, P.granulosum and P.avidum was 3%, 2% and 3%, respectively (Fig. 2).

58 &BIOFILM Evaluation results of the effectiveness of Triethylcitrate and GT Peptide-10 on Propionibacterium acnes, Propionibacterium granulosum and Propionibacterium avidum Prof. Oleg Alexeyev, Researcher, Department of Medical Biosciences/Pathology, Umeå University

59 &BIOFILM Evaluation results of the effectiveness of Triethylcitrate and GT Peptide-10 on Propionibacterium acnes, Propionibacterium granulosum and Propionibacterium avidum Prof. Oleg Alexeyev, Researcher, Department of Medical Biosciences/Pathology, Umeå University

60 &BIOFILM Evaluation results of the effectiveness of Triethylcitrate and GT Peptide-10 on Propionibacterium acnes, Propionibacterium granulosum and Propionibacterium avidum A combination of both substances (1% TEC with increasing concentrations of the peptide) showed a synergistic effect that lowered the MIC of the peptide for P.acnes, P.granulosum and P.avidum to1.5μl/ml, 1μl/ml and 5μl/ml, respectively (Fig. 3, a-c). Prof. Oleg Alexeyev, Researcher, Department of Medical Biosciences/Pathology, Umeå University

61 &BIOFILM Evaluation results of the effectiveness of Triethylcitrate and GT Peptide-10 on Propionibacterium acnes, Propionibacterium granulosum and Propionibacterium avidum Prof. Oleg Alexeyev, Researcher, Department of Medical Biosciences/Pathology, Umeå University

62 &BIOFILM Evaluation results of the effectiveness of Triethylcitrate and GT Peptide-10 on Propionibacterium acnes, Propionibacterium granulosum and Propionibacterium avidum Prof. Oleg Alexeyev, Researcher, Department of Medical Biosciences/Pathology, Umeå University

63 &BIOFILM Evaluation results of the effectiveness of Triethylcitrate and GT Peptide-10 on Propionibacterium acnes, Propionibacterium granulosum and Propionibacterium avidum Prof. Oleg Alexeyev, Researcher, Department of Medical Biosciences/Pathology, Umeå University

64 &BIOFILM Evaluation results of the effectiveness of Triethylcitrate and GT Peptide-10 on P.acnes biofilm formation and dispersal As the formation of biofilm by P.acnes requires growth of the bacterial culture, the formation of the biofilm has been inhibited in the same way as the growth of the bacterial culture. To study the effects of both substances on pre-existing P.acnes biofilms, 6 day old biofilms were treated with different concentrations for 24h and dispersal documented by crystal violet staining. Neither the single substances (C max Triethylcitrate: 4%, C max peptide: 2,5 μl/ml), nor the combination of both substances (4% Triethylcitrate, C max peptide: 50μl/ml = 20x MIC) affected biofilm integrity or resulted in biofilm dispersal (Fig. 4, a-c). In contrast, DNase I (deoxyribonuclease I, enzyme), which served as a positive control in this experiment, led to almost complete biofilm dispersal (Fig. 4, b and c). Prof. Oleg Alexeyev, Researcher, Department of Medical Biosciences/Pathology, Umeå University

65 &BIOFILM Evaluation results of the effectiveness of Triethylcitrate and GT Peptide-10 on P.acnes biofilm formation and dispersal Prof. Oleg Alexeyev, Researcher, Department of Medical Biosciences/Pathology, Umeå University

66 &BIOFILM Evaluation results of the effectiveness of Triethylcitrate and GT Peptide-10 on P.acnes biofilm formation and dispersal Prof. Oleg Alexeyev, Researcher, Department of Medical Biosciences/Pathology, Umeå University

67 &BIOFILM Evaluation results of the effectiveness of Triethylcitrate and GT Peptide-10 on P.acnes biofilm formation and dispersal Prof. Oleg Alexeyev, Researcher, Department of Medical Biosciences/Pathology, Umeå University

68 &BIOFILM Evaluation results of the effectiveness of Triethylcitrate and GT Peptide-10 on P.acnes biofilm formation and dispersal Viability of single bacterial cells in biofilms treated with both substances for 24h decreased in a dose-dependent manner, although lethality varied from experiment to experiment. 4% Triethyl citrate killed 67-92% of all bacteria, whereas peptide concentrations of 50μl/ml led only to 50% dead cells. In good agreement with the previous results, a combination of both compounds (C max. Triethyl citrate: 4%, C max. peptide: 50μl/ml = 20x MIC) showed a synergistic effect that led to 96% dead bacteria in P.acnes biofilms (Fig. 5). Prof. Oleg Alexeyev, Researcher, Department of Medical Biosciences/Pathology, Umeå University

69 &BIOFILM Evaluation results of the effectiveness of Triethylcitrate and GT Peptide-10 on P.acnes biofilm formation and dispersal Prof. Oleg Alexeyev, Researcher, Department of Medical Biosciences/Pathology, Umeå University

70 &BIOFILM Evaluation of anti-bacterial effect of different compounds inside biofilm Active Dead bacteria Alive bacteria Clindamycin 30% 70% Gt-peptide-10 50% 50% Triethyl citrate 60% 40% Triethyl citrate + GT-peptide-10 92% 8% Prof. Oleg Alexeyev, Researcher, Department of Medical Biosciences/Pathology, Umeå University

71 &BIOFILM Conclusion Both triethylcitrate and the small peptide successfully inhibited growth of three distinct Propionibacterium species commonly found on the human skin, as well as biofilm formation by P.acnes, in a dose dependent manner. The integrity of pre-existing biofilms of P.acnes was neither affected by the single compounds nor by a combination of both, but both compounds, especially when combined, killed significant numbers of bacterial cells in the biofilm. Thus, the combination of triethylcitrate and the GT Peptide-10 is effectively preventing de novo formation or growth of existing P.acnes biofilms. Moreover, we conclude from our data that high concentrations of triethyl citrate (>3%) are bactericidal, whereas the GT Peptide-10 has bacteriostatic properties and presumably acts on dividing cells. Prof. Oleg Alexeyev, Researcher, Department of Medical Biosciences/Pathology, Umeå University

72 CLINICAL EVALUATION short term (4 weeks) evaluation of AKNICARE Lotion vs Antibiotic + Benzoyl peroxide in comedonic and inflammatory acne. Department of Dermatology Cracovia Hospital - Polonia Dr. Monika Kapinska, 2008

73 &BIOFILM The study enrolled 45 patients (15 patients treated with AKNICARE lotion, 15 patients treated with clindamycin 1% + benzoyl peroxide 5%, 15 patients treated with hydro-alcoholic solution-placebo). The products where applied two times per day. In the short term evaluation the analysis was made at time 0 (t0) and time 4 weeks (t4). Comedonal and inflammatory lesions were counted at t0 and t4.

74 OPEN COMEDONES Statistical evaluation T-Student: Aknicare t0 - t4 weeks p<0.005, antibiotic + BPO t0 t4 weeks p<0.05

75 CLOSED COMEDONES Statistical evaluation T-Student: Aknicare t0 - t4 weeks p<0.005, antibiotic + BPO t0 t4 weeks p<0.05

76 PAPULES Statistical evaluation T-Student: Aknicare t0 - t4 weeks p<0.005, antibiotic + BPO t0 t4 weeks p<0.05

77 PUSTULES Statistical evaluation T-Student: Aknicare t0 - t4 weeks p<0.005, antibiotic + BPO t0 t4 weeks p<0.05

78 CONSIDERATIONS OF THE INVESTIGATOR After a 4 weeks treatment with Aknicare, the amount of open comedones is reduced to %, where the antibiotic treatment s reduction is %. The clindamycin 1%+benzoyl peroxide 5% treatment seems then to be more effective on this kind of lesion. Even the T student s results confirm this hypothesis (clindamycin 1% + benzoyl peroxide 5% value: p<0,005; Aknicare s value: p<0,5) even if the AKNICARE treatment becomes active sooner in reducing open comedones as the T test results confirm (p<0,05 between the 1st and the 2nd week of treatment).

79 CONSIDERATIONS OF THE INVESTIGATOR After 4 weeks of treatment with Aknicare the amount of closed comedones is reduced to %. This date refers to the total amount lesions counted on the face of all the 15 patients. It s also very interesting to note the high reduction in the number of closed comedones between the first and the second week of treatment (-15,18%) and between the second and the third (-12,16%). The same calculation on the clindamycin 1%+benzoyl peroxide 5% treatment evidences -4,11% between the first and the second week and -3,74% between the second and the third week of treatment. The clindamycin 1%+benzoyl peroxide 5% treatment s reduction is %. This date refers to the total amount lesions counted on the face of the 15 patients treated. The Aknicare results reached with the T student test, suggest high efficacy mainly between 1st 3rd and 1st 4th weeks ( p<0,0005 means that there is a 0.05% possibility that the reduction in the number of papules after 4 weeks of treatment it s due to the chance). Even the results reached between 1st 2nd and 2nd 3rd are extremely interesting.

80 CONSIDERATIONS OF THE INVESTIGATOR After 4 weeks of treatment with Aknicare the amount of papules reduced to %, where the antibiotic treatment reduction is %. There is a big difference between the results reached after the 1st week with Aknicare (average of the number of papules of all the subjects involved: %) and the 4th one (average of the number of papules of all the subjects involved: 11.33%). This proves the Aknicare treatment efficacy. Moreover, the result obtained with the T student test, which compares these two average data (p<0,005), demonstrates that this reduction is not accidently: there is a 5% possibility, that the reduction in the number of papules after 4 weeks treatment it s due to chance. Comparing the T student test s results reached with AKNICARE to those reached with clindamycin 1%+benzoyl peroxide 5% treatment, we can assume to be able to reach sooner (from the first week) better results with AKNICARE.

81 CONSIDERATIONS OF THE INVESTIGATOR After a 4 weeks treatment with Aknicare, the amount of pustules reduced to %, where the antibiotic treatment reduction is %. The T test confirms in a highly significant way the AKNICARE s results. Again, the results show a big difference between the 1st week with Aknicare (average of the number of papules of all the subjects involved: 23.00%) and the 4th one (average of the number of papules of all the subjects involved: 5.27%). Moreover, the result obtained with the T student test, which compares these two average data (p<0,005), demonstrates once more that this reduction is not accidently: there is a 0.5% possibility, that the reduction in the number of papules after 4 weeks treatment is due to chance. Comparing the T student test s results reached with AKNICARE to those reached with clindamycin 1%+benzoyl peroxide 5% treatment, we can assume to be able to reach sooner (from the first week) better results with AKNICARE.

82 CONCLUSIONS The combination of triethyl citrate and GT-peptide-10 (antimicrobial peptide) is effective in treating comedonal and papulo-pustular acne.

83 &BIOFILM RANDOMIZED CLINICAL TRIAL ON PATIENTS AFFECTED BY MILD TO MODERATE ACNE OF THE FACE Evaluation of differences in efficacy and tolerability between Aknicare cream and Duac gel 1%+5% Dott.ssa Nevena Skroza, Dott.ssa Ersilia Tolino, Prof.ssa Patrizia Potenza, UOC di Dermatologia Università Sapienza di Roma, Polo Pontino

84 &BIOFILM Primary endpoints Endpoints To evaluate differences in efficacy between Duac gel and Aknicare cream through GAGs score and the lesions count, measured at time 0, after 4 (t1) and 12 weeks (t2). To evaluate differences in tolerability between Duac gel and Aknicare cream through a questionnaire administered at time 0, after 4 (t1) and 12 weeks (t2). Secondary endpoints To evaluate differences in efficacy between Duac gel and Aknicare cream, detected by biopsy follicular (number and size of microcomedones). To compare with Wood's light the presence of P.acnes in two parallel therapeutic regimens.

85 &BIOFILM Design of the study Randomized clinical trial in patients with mild-moderate acne on the face. The patients will be visited at time 0 (baseline), after 4 weeks (t1) and after 12 weeks (t2). The evaluation of clinical efficacy, by GAGs scale and lesions count, will be assessed at baseline, after 4 and 12 weeks.

86 &BIOFILM Design of the study At the baseline visit each patient will receive both Aknicare cream and Duac gel; they will apply Aknicare cream twice a day on a selected area of the left hemi-face and Duac gel once a day on a specific area of the contro-lateral hemi-face. The areas identified will be of 25 cm². Subsequent measurement of GAGs, lesions count and the reading of follicular biopsy will be performed by another physician. At baseline visit and during the following checkups each patient will receive two questionnaires: for the assessment of quality of life and for the assessment of products tolerability. Photographs will be taken with a digital camera, follicular biopsy by tape stripping and observation by Wood's light.

87 &BIOFILM Inclusion criteria Age between 14 and 30 years old. Mild/moderate acne on the face. Wash out of 2 weeks for topical/systemic therapies. Women of childbearing potential with a negative pregnancy test. Patients who accepted the study protocol and signed the informed consent form.

88 &BIOFILM Exclusion Criteria Severe acne. Subjects with diseases and/or dermatological conditions that require the use of interfering topical and/or systemic therapies. Subjects who participated in clinical trials over the past 30 days. Pregnant or breastfeeding women.

89 MEDICAL DEVICES EFFECTIVE AS DRUGS, SAFE AS COSMETICS AKNICARE LOTION CLINDAMYCIN+BENZOYL PEROXIDE AKNICARE CREAM

90 &BIOFILM July 2014 Preliminary results in 10 patients

91 &BIOFILM To determine the degree of acne severity, the Global Acne Grading System (GAGS) has been used. This system incorporates the benefits of the most elaborated systems of definition of acne severity preserving a remarkable simplicity. ACNE SEVERITY GLOBAL SCORE NONE 0 MILD 1-18 MODERATE SEVERE VERY SEVERE >39

92 &BIOFILM The study enrolled 10 patients. At the baseline visit, each patient received both Aknicare cream and Clyndamicin 1% + benzoyl peroxide 5%. The patients applied: on the right cheek: Clindamycin 1% + benzoyl peroxide 5%, once a day for 12 weeks. on the left cheek: Aknicare cream, twice a day for 12 weeks. Subsequent measurement of GAGs, lesions count and the reading of follicular biopsy were performed by another physician. At baseline visit and during the following checkups, each patient received two questionnaires: for the assessment of quality of life and for the assessment of products tolerability. Photographs were taken with a digital camera, follicular biopsy was done by tape stripping and observation by Wood's light.

93 GAGs &BIOFILM Comparison between global GAGs, Aknicare cream GAGs and Duac gel GAGs at t0, t1 and t t0 t1 t2 GAGs DUAC GEL GAGs AKNICARE CREAM GAGs GLOBALE

94 &BIOFILM Investigator considerations Patients did not develop adverse events with both products, however they reported a better tolerability following the application of Aknicare cream. The most significant data is a faster improvement achieved with Duac gel 1% +5% but the study showed a comparable outcome after the use of the two products, in terms of lesions and seborrhea reduction, at the end of therapy, after 12 weeks of treatment.