Functionalized phage for acne treatment

Abstract

The present disclosure relates to phages functionalized with pharmacologically active molecules for the treatment of acne. Such phages are useful in the treatment of infectious diseases, as they target pharmacological substances to tissue colonized by bacteria. In an embodiment herein, the functionalized phages of the present invention correspond to lytic phages of the Caudoviricetes class having activity against Cutibacterium acnes with pharmacologically active molecules, specifically with isotretinoin, having properties of interest for the treatment of acne caused by Cutibacterium acnes, because it acts at the sebaceous gland level, reducing inflammation, hyperproduction of sebum, and keratosis.

Description

[0001] FUNCTIONALIZED PHAGE FOR ACNE TREATMENT

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to the general field of phage therapy, particularly to phage therapy applied for the treatment of acne and more particularly to the use of a phage functionalized with pharmacologically active molecules, with properties of interest for the target tissue for the treatment of acne. The functionalized phage of the present disclosure has the additional advantage of targeting active molecules to the specific tissue colonized by bacteria.

[0004] BACKGROUND

[0005] Acne is a skin condition in which hair follicle obstruction occurs due to 4 pathophysiological factors: infection by Cutibacterium acnes. inflammation, sebum overproduction, and hyperkeratosis. Disease treatment involves using oral antibiotics (tetracyclines, erythromycin), systemic retinoids (isotretinoin) or antiandrogens (spironolactone) in women with hormonal acne. It may also include the use of topical medicines, such as benzoyl peroxide, salicylic acid, topical retinoids, such as tretinoin or adapalene, as well as topical antibiotics, such as clindamycin or erythromycin. In some cases, however, the effectiveness of this treatment may vary due to disease complexity and individual differences in each person, considering that one of the key factors in treatment is compliance. The latter is mainly affected by the adverse effects of the medicines employed.

[0006] There are publications describing different functionalized phages, e.g., the paper "Phage- Guided Targeting, Discriminated Imaging and Synergistic Killing of Bacteria by AIE Bioconjugates" (He X, etal. 2020. Phage-Guided Targeting, Discriminative Imaging, and Synergistic Killing of Bacteria by AIE Bioconjugates. J. Am. Chem. Soc. 2020; 142(8):3959-3969 hdps- / / pubs acs.org / doi / 10 102i / iacs.9bl 2936) discloses luminogen-functionalized phages with aggregation-induced emission property that are active in photodynamic inactivation to generate a type of bioconjugate capable of targeted imaging and synergistic destruction of certain bacterial species. The bioconjugates disclosed herein have the ability to generate an inherited orientation of the bacteriophage that allows them to specifically recognize the target bacteria. They are also described as potent photosensitizers in situ producing high-efficiency reactive oxygen species under white light irradiation, achieving selective selection and elimination of antibioticsensitive multi-resistant bacteria.

[0007] Moreover, patent US2014 / 0030697A1 refers to methods, reagents, and kits for the functionalization of proteins present on the surface of viral particles, e.g., bacteriophages, using sortase-mediated transpeptidation reactions. It also discloses methods for conjugating an agent, e.g., a detectable tag, a binding agent, a click chemistry identifier, or a small molecule to a surface protein of a viral particle. Furthermore, this patent mentions kits comprising reagents useful for the generation of functionalized viral particles, as well as precursor proteins comprising a sortase recognition fragment and viral particles comprising such precursor proteins, as well as nucleic acids encoding viral proteins comprising a sortase recognition fragment, and expression vectors comprising such nucleic acids.

[0008] However, there remains a need to develop a therapeutic alternative for the treatment of infectious diseases with advantages over traditional treatments, i.e., that does not generate adverse effects at a systemic level, which may compromise the patients’ quality of life, and that also achieves bacterial load reduction without antimicrobial resistance. Specifically, there is still a need to generate alternatives for the treatment of infectious diseases such as acne, whose current treatment is based on oral retinoid-type molecules, which have various side effects, despite having good activity in reducing seborrhea and limiting the proliferation of sebocytes and keratinocytes. Therefore, it is necessary to develop new approaches for acne treatment which can be based on phages functionalized with pharmacologically active molecules, ensuring that the bacteriophages reach the target bacteria and enhance the effect of the active molecules in the infected tissue, reducing the risks of side effects.

[0009] SUMMARY

[0010] The present disclosure refers to a biological-pharmacological formulation consisting of functionalized phages that allows targeting pharmacologically active molecules to a specific tissue colonized by bacteria. The functionalized phages of the present disclosure correspond to lytic bacteriophages capable of infecting acne-causing bacteria, which are bound to one or more anti-acne molecules.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 Absorbance spectra showing bacteriophage-isotretinoin complex formation. A. Spectral scanning of bacteriophage and isotretinoin molecules. The line identified by a triangle shows the curve corresponding to the mixture of bacteriophage and isotretinoin in a single sample without the process of isotretinoin-free molecule removal. The line identified by a dot shows the spectrum curve corresponding to an isotretinoin solution. The curve identified by a square shows the spectrum of the bacteriophage alone. B. The curve shows the spectral scan corresponding to the formation of the bacteriophageisotretinoin complex, after being subjected to the processes of isotretinoin-free molecule removal.

[0013] FIG. 2. Results of the RT-qPCR technique to measure IL-ip relative expression of the experimental groups compared between points zero and 24 hours after inoculation with each treatment. Wherein C corresponds to the group inoculated with C. acnes, CF corresponds to the group that was exposed to C. acnes and to the phage, CFI corresponds to the group inoculated with bacteria, phage-isotretinoin, and F to the group inoculated with bacteriophage. Moreover, the statistical significance values for each experimental group are shown, wherein NS corresponds to non-significant, *P<0.05 and **P<0.01.

[0014] DETAILED DESCRIPTION

[0015] The present disclosure relates to a functionalized phage comprising a lytic bacteriophage against acne-associated bacteria, and an active molecule against acne having a carboxylic acid terminal group in its structure; wherein the bacteriophage and the active molecule are bound by a covalent bond without an additional spacer or ligand. For the purposes of the present disclosure, functionalized phage is understood to be a bacteriophage to which active molecules / medications are coupled, and wherein the bacteriophage acts as a vehicle for said medicines targeting them to the target tissue, thus producing high specificity against the pathogenic bacteria of each tissue. Functionalized phages may be useful in the treatment of various infectious diseases that also benefit from the synergistic effect of medicines, e.g., acne, gastritis secondary to Helicobacter pylori, wound infections by Pseudomonas spp., pneumonia of bacterial origin, among others. In an embodiment, the present invention relates to functionalized phages for the treatment of acne, comprising lytic bacteriophages against acne-causing bacteria bound to molecules active against acne having a carboxylic acid terminal group in their structure.

[0016] For the purposes of the present disclosure, a lytic bacteriophage is a bacteriophage capable of infecting and generating lysis in a bacteria of interest, i.e., they are phages that use the machinery of the bacterial cell after infecting the bacteria to manufacture their viral progeny and subsequently lyse the cell for the release of new phage particles. Lytic bacteriophages have high host specificity and are harmless to adjacent tissues, as well as to other microorganisms that are part of the microbiota of these tissues. Therefore, lytic phages are considered an alternative and safe solution to combat bacterial pathogens, even those resistant to antibiotics. According to the present disclosure, lytic bacteriophages do not insert into the bacteria genome and have proteins with hydroxyl and amine residues exposed on their surface. Moreover, these bacteriophages are resistant to a pH range between 4 and 8 and are stable in a solution of dimethyl sulfoxide (DMSO).

[0017] In a particular embodiment of the present disclosure, the lytic bacteriophages are specific to bacteria involved in the development of acne, they specifically have the ability to infect the bacteria Cutibacterium acnes and more specifically strains of phylotypes IA1, IA2, IB and II of C. acnes. For the purposes of the present disclosure such phages have species specificity and are unable to lyse any bacteria other than C. acnes, even though they have the ability to infect a wide range of bacterial strains within the species C. acnes.

[0018] In an embodiment, the bacteriophages of the present disclosure have a genome with a nucleotide sequence that is selected from the sequences of SEQ ID NOs: 1, 2 and 3. For the purposes of the present disclosure, functionalize a bacteriophage is understood to be the process by which the surface of the bacteriophage is modified by binding to one or more molecules with exposed hydroxyl or amine residues. In various embodiments of the invention, the binding of the molecules to the phage surface is mediated by covalent bonds. In an embodiment, binding of lytic bacteriophage to the active molecule is performed by means of a covalent bond between the carboxylic acid residues of the molecule of interest and the amine or hydroxyl residues of the bacteriophage. In a particular embodiment of the present invention said covalent bond does not require an additional spacer or ligand or a catalyst to form and is stable at pH 8.

[0019] In a particular embodiment of the present disclosure the covalent link is an ester or amide bond.

[0020] For the purposes of the present disclosure, active molecules against acne correspond to drugs whose action in the treatment of acne corresponds to regulating sebum production, reducing follicle obstruction, and decreasing inflammation. For the purposes of the present disclosure, the active molecule against acne has carboxylic acid terminal groups in its structure and is stable when dissolved in a lytic phage-tolerating solvent.

[0021] For the purposes of the present disclosure, drugs such as retinoid drugs, are considered active molecules against acne. They are synthetic derivatives of vitamin A that bind to specific receptors and exert an action that regulates the proliferation and differentiation of keratinocytes while acting on the sebaceous gland, decreasing its size and activity. Other drugs active against acne are alpha hydroxy acids, glycolic acid, salicylic acid, lactic acid, adapalene, azelaic acid, niacinamide, and antibiotics such as amoxicillin and lymecycline. These drugs have a terminal carboxylic acid group that makes them suitable candidates for active molecules to functionalize bacteriophages in the treatment of acne.

[0022] In an embodiment of the present invention, the following are considered active molecules against acne: retinoids, such as retinoic acid, acitretin, tretinoin, isotretinoin, and bexarotene. In an embodiment of the present invention the active retinoid against acne is isotretinoin. In an embodiment of the present invention the bacteriophage is functionalized with isotretinoin. In a particular embodiment, a lytic phage of C. acnes is functionalized in a concentration of approximately 2.2xlO5PFU / mL, with a solution of isotretinoin inDMSO at a 1 mM concentration.

[0023] The present invention will be disclosed in detail through the following examples, which are provided for illustrative purposes only and not intended to limit its scope.

[0024] EXAMPLES

[0025] EXAMPLE 1. Functionalization of bacteriophage with isotretinoin.

[0026] Functionalization of bacteriophage was carried out through the following steps:

[0027] Obtaining phases

[0028] The phages were obtained from a collection of the C. J. Marinkelle Natural History Museum of the University of the Andes, and were cultured using the double agar layer technique, as follows: 200 pL of an inoculum of the host bacteria (C. acnes), in a concentration of between 108- 109CFU / mL, were taken and mixed by vortex with 100 pL of the initial bacteriophage stock. This bacteria-phage mixture was placed in a test tube with 2 mL New Soft agar, mixed again by vortex and poured onto a Petri dish with New Agar until the mixture was homogeneously distributed, and it was allowed to solidify for a few minutes. The Petri dish was then incubated for 24 hours at 36 °C in an atmosphere of 10 to 15% CO2. Thereafter, the agar surface was scraped with a glass slide to collect only the superficial layer of New Soft agar containing the mixture of bacteria and bacteriophage. Then, the New Soft agar was mixed with 2 mL SM buffer for each Petri dish that was scraped into a conical centrifuge tube and allowed to stand for 24 hours. Finally, the mixture was centrifuged to separate agar from the solution containing the bacteriophages and was filtered using a 0.22 pm filter to obtain a new stock of the desired bacteriophage.

[0029] Functionalization

[0030] From the previous step, bacteriophages were obtained in a titer of between 1.0 x 1011and 1.0 x 1013PFU / mL and mixed with the active molecule in DMSO solution, at a concentration between 1.0 and 10 mM. The mixture was made in a solution in SM buffer without gelatin with a pH between 7.8 and 8.

[0031] Activation of functionalized phase (Incubation)

[0032] The above solution was incubated for a period between 24 and 48 hours at a temperature between 4 and 20 °C.

[0033] Ultracentrifugation or dialysis to obtain functionalized phase

[0034] Active molecules that did not bind to the bacteriophage proteins were removed by means of ultracentrifugation, resuspending the functionalized bacteriophage and discarding the supernatant with the free active molecules. Alternatively, removal was done by means of dialysis, using a membrane with a pore size of 14 kDa to retain the functionalized bacteriophage inside the membrane, while the active molecule was diluted in a washing solution. This process was carried out at a temperature of 37 °C.

[0035] Functionalized phage characterization

[0036] It was determined that the molecule was bound to the bacteriophage by means of UV- VIS spectrophotometry, wherein the spectra of each element separately and of the binding process were analyzed, before and after the removal of free molecules. The isotretinoin spectral scan that was experimentally obtained shows a single absorbance band at 344 nm, which is consistent with the spectrum that has been previously reported in other experimental studies. Moreover, the spectrum of the bacteriophage and drug mixture indicates the presence of three bands of higher absorbance at 220, 260, and 344 nm, which coincide with the absorbance bands identified in the individual spectra of both compounds (Figure 1A). On the other hand, when performing the spectral scanning of the solution containing the bacteriophageisotretinoin complex, after the process of medicine-free molecule removal, the presence of a single peak at 214 nm is observed (Figure IB).

[0037] EXAMPLE 2. Inhibition assays with functionalized phage

[0038] The lytic activity of the bacteriophage was evaluated after functionalization by spot plating assays of the bacteriophage. The bacteriophage viral titer was obtained with 2.2xl05PFU / mL bound isotretinoin molecules, which is evidence that the bacteriophage retains its lytic activity after the functionalization process.

[0039] Finally, the anti-inflammatory capacity of the active molecule was evaluated after binding to the bacteriophage by adding this complex to a keratinocyte cell culture, and measuring the expression levels of an inflammation tag, interleukin ip (IL-ip), at 0 and 24 hours of exposure. The results of this assay revealed that the group of keratinocytes that was exposed to phage-isotretinoin treatment shows significant differences between the two time points, with IL-ip significantly decreased after 24 hours of inoculation, compared to expression levels at zero hours of inoculation (FIG. 2), which means that phage-bound isotretinoin preserves its pharmacological activity.

[0040] According to the results obtained, the functionalized bacteriophages proved to have a synergistic effect since both the bacteriophage and the active molecule retained their activity after functionalization, which means that the results obtained are greater than those obtained by using each of the elements separately.

Claims

CLAIMS1. A functionalized phage consisting of:(a) a lytic bacteriophage against acne-associated bacteria, and(b) an active anti-acne molecule having a carboxylic acid terminal group in its structure; wherein the bacteriophage and the active molecule are bound by a covalent bond without an additional spacer.

2. The functionalized phage according to Claim 1, wherein the covalent bond is an ester or amide bond.

3. The functionalized phage according to Claim 1, wherein the bacteriophage is from the Class Caudoviricetes and infects Cutibacterium acnes.

4. The functionalized phage according to Claim 1, wherein the bacteriophage infects strains of phylotypes IA1, IA2, IB and II of Cutibacterium acnes.

5. The functionalized phage according to Claim 2, wherein the ester or amide bond is formed between carboxylic acid residues of the active molecule and hydroxyl or amine residues of the bacteriophage, respectively.

6. The functionalized phage according to Claim 1, wherein the active molecule is selected from the group consisting of retinoids, such as, retinoic acid, acitretin, tretinoin, isotretinoin, and bexarotene; alpha hydroxy acids, glycolic acid, salicylic acid, lactic acid, adapalene, azelaic acid, niacinamide, and antibiotics, such as, amoxicillin and lymecycline.

7. The functionalized phage according to Claim 1, wherein the active molecule is isotretinoin.

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