The present invention relates to the field of topical dermal delivery formulation to combat chronic infections. More specifically to a hydrogel based antimicrobial formulation for releasing nitric oxide (NO) under illumination to impacted sites for mitigating infection caused by drug resistant micro-organisms.
BACKGROUND ART

[0002] Due to various specific properties, antimicrobial hydrogels have been proposed as promising materials for wound healing. As a result, various scientists have worked to create and synthesize antibacterial hydrogels. The antimicrobial hydrogel mostly consists of zeolites and an antimicrobial agent such as nitric oxide gas.
[0003] Zeolites are nanoporous aluminosilicates that are employed in a wide range of industrial applications by utilizing their porosity and size/shape selectivity. Acid catalysis for crude oil cracking and chemical conversion is one of the wide application examples. In the past few years, zeolites have been explored for usage other than their conventional use as industrial catalysts. One of such usages is loading zeolite with pharmacological molecules for targeted administration based on the range of pore size and shape.
[0004] “Synthetic zeolites as a new tool for drug delivery” titled paper by Rimoli et al. discusses loaded zeolite X and A with ketoprofen (28.5% medication in the impregnated zeolite) by trapping it within the zeolite's mesopores. This loaded zeolite may be effective in the treatment of gastrointestinal inflammatory diseases.
[0005] Nitric oxide (NO) is involved in a variety of biological activities, including infection, vasodilation, angiogenesis, and wound healing regulation. Various laboratories have firmly established the potential of using nitric oxide (NO) to treat a variety of ailments during the last few decades. In terms of lowering microbial burdens in chronic wounds, both in vitro and in vivo investigations have yielded promising results. NO has been demonstrated to play critical roles in the innate immune response, which is the body's first line of defense against pathogen invasion.
[0006] To date, numerous exogenous NO sources have been used to treat infection (by bacteria, parasites and fungi). In order to entirely inhibit bacterial growth, an average of 200 ppm of NO(g) is required for 5 hours. However, NO is also considered a toxic gas, and thus, its usage in a hospital context poses a significant risk.
[0007] WO2003000088A1 discloses a dosing reservoir for distributing an active compound in controlled amounts onto a target surface comprising a first impermeable layer and a second permeable layer facing and affixed to the first layer. A fluid tight cell with a frangible seal and containing an active compound is disposed between the first and second layers. When the frangible seal is ruptured, the active compound is released from the cell and is controllably released from the reservoir (10) through the permeable layer.
[0008] US20090297634A1 discloses compositions for releasing nitric oxide (NO) comprising a matrix that encapsulates nitric oxide. Nitric oxide is released when the composition is exposed to an aqueous e006Evironment. The invention further provides methods of preparing the compositions and uses of the compositions for treating infections and disorders.
[0009] Conventionally, many antimicrobial formulations for delivering NO have been developed. However, using known NO-generating chemicals for therapeutic purposes has been impractically difficult and time intensive. Furthermore, many formulations have a lot of drawbacks when it comes to topical or transdermal delivery of NO from a NO donor.
[0010] In order to overcome the aforementioned drawbacks, there is a need in the art to provide a topical formulation for the controlled administration of NO to tackle chronic infections without any side effects.
OBJECTS OF THE INVENTION

[0011] The principal object of the present invention is to overcome the disadvantages of the prior art by providing the synergy caused due to consumption of the multiple herbal ingredients together which resulted in production of a combined total effect which being much greater then sum of individual agents.
[0012] An object of the present invention is to develop a topical antimicrobial formulation for controlled distribution of nitric acid on the impacted site.
[0013] Another object of the present invention to increase flexibility, patient friendliness, efficiency of therapy, and/or patient safety.
[0014] Another object of the present invention to minimize leakage of unwanted component from the formulation on the impacted site.
[0015] Another object of the present invention is to improve cost-effectiveness storage time and convenience of use.
[0016] Yet another object of the present invention is to provide a method for delivering nitric oxide without developing any side effects.
[0017] The foregoing and other objects of the present invention will become readily apparent upon further review of the following detailed description of the embodiments as illustrated in the accompanying drawings.
SUMMARY OF THE INVENTION

[0018] The present invention relates to a hydrogel based antimicrobial formulation that readily enable gaseous entities such as Nitric oxide (NO) delivery to impacted physiological sites for mitigation bacterial and fungal origin infection under the control of light.
[0019] According to an embodiment of the present invention, a hydrogel based antimicrobial formulation to deliver nitric oxide on impacted physiological sites in order to mitigate infection. The formulation is prepared by incorporating a photoactive metal nitrosyl into a biocompatible material. The biocompatible material is selected from a group which may include but not limited to silicate-based hydrogels, HEMA hydrogel, modified Al-MCM-41 (modified zeolite). The photoactive metal nitrosyl is preferably manganese nitrosyl. The formulation may be used in the form of gel, patch and alike. The formulation may be used as antibacterial agent, antifungal agent and alike.
[0020] While the invention has been described and shown with reference to the preferred embodiment, it will be apparent that variations might be possible that would fall within the scope of the present invention.
BRIEF DESCRIPTION OF DRAWINGS

[0021] So that the manner in which the above-recited features of the present invention may be understood in detail, a more particular description of the invention, briefly summarized above, may have been referred by embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.
[0022] These and other features, benefits, and advantages of the present invention will become apparent by reference to the following text figure, with like reference numbers referring to like structures across the views, wherein:
[0023] Figure 1 illustrates a structure of the photoactive NO metal-nitrosyl.
[0024] Figure 2 illustrates a schematic description of the nitrosyl-incorporated zeolite {Mn-NO}@Al-MCM-41
[0025] Figure 3 illustrates a light-triggered NO release from Mn-NO}@Al-MCM-41 suspension upon exposure to light pulses (duration time in s)
[0026] Figure 4 illustrates effects of moderate dose of NO (photo released from {Mn-NO}@Al-MCM-41) on a) pure yeast form and pure b) hypahl form of C. albicans. The dead cells are identified by the bluish purple color (stained with Trypan blue)
[0027] Figure 5 illustrates a schematic description of formulation based patches with {Mn-NO} incorporated (top: original product, bottom: patch after light-exposure and NO loss)
[0028] Figure 6 illustrates reduction of bacterial loads upon placement of the patch on the right half of the bacterial lawns; (a) P. aeruginosa, (b) E. coli, (c) S. Aureus, and (d) MRSA after one 1 min of light exposure.
[0029] Figure 7 illustrates a top: the HEMA patch with a Ru-nitrosyl incorporated; bottom: schematics of the polymer matrix
DETAILED DESCRIPTION OF THE INVENTION

[0030] While the present invention is described herein by way of example using embodiments and illustrative drawings, those skilled in the art will recognize that the invention is not limited to the embodiments of drawing or drawings described and are not intended to represent the scale of the various components. Further, some components that may form a part of the invention may not be illustrated in certain figures, for ease of illustration, and such omissions do not limit the embodiments outlined in any way. It should be understood that the drawings and the detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the scope of the present invention as defined by the appended claim.
[0031] As used throughout this description, the word "may" be used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense, (i.e., meaning must). Further, the words "a" or "an" mean "at least one” and the word “plurality” means “one or more” unless otherwise mentioned. Furthermore, the terminology and phraseology used herein are solely used for descriptive purposes and should not be construed as limiting in scope. Language such as "including," "comprising," "having," "containing," or "involving," and variations thereof, is intended to be broad and encompass the subject matter listed thereafter, equivalents, and additional subject matter not recited, and is not intended to exclude other additives, components, integers, or steps. Likewise, the term "comprising" is considered synonymous with the terms "including" or "containing" for applicable legal purposes. Any discussion of documents, acts, materials, devices, articles, and the like are included in the specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention.
[0032] In this disclosure, whenever a composition or an element or a group of elements is preceded with the transitional phrase “comprising”, it is understood that we also contemplate the same composition, element, or group of elements with transitional phrases “consisting of”, “consisting”, “selected from the group of consisting of, “including”, or “is” preceding the recitation of the composition, element or group of elements and vice versa.
[0033] The present invention is described hereinafter by various embodiments with reference to the accompanying drawing, wherein reference numerals used in the accompanying drawing correspond to the like elements throughout the description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art. In the following detailed description, numeric values and ranges are provided for various aspects of the implementations described. These values and ranges are to be treated as examples only and are not intended to limit the scope of the claims. In addition, several materials are identified as suitable for various facets of the implementations. These materials are to be treated as exemplary and are not intended to limit the scope of the invention.
[0034] The present invention pertains to a photoactivated indigenous nitric oxide (no) releasing hydrogel formulations for delivering nitric oxide on impacted site to provide treatment for drug-resistant pathogens. Herein, the developed formulation may be used as gel or patches.
[0035] According to an embodiment, a hydrogel based antimicrobial formulation to deliver nitric oxide on impacted physiological sites in order to mitigate infection. The formulation is prepared by incorporating a photoactive metal nitrosyl into a biocompatible material. The biocompatible material is selected from a group which may include but not limited to silicate-based hydrogels, HEMA hydrogel, modified Al-MCM-41 (modified zeolite). The photoactive metal nitrosyl is preferably manganese nitrosyl. The formulation may be used in the form of gel, patch and alike. The formulation may be used as antibacterial agent, antifungal agent and alike
[0036] Several analysis studies have been conducted on the formulation in order to characterize and determine the efficacy of the formulation. Referring to Figure 1, photoactive NO metal-nitrosyl has been incorporated into the negatively-charged pores of the modified zeolite (Al-MCM-41) host. Thus, the formulation utilizes the biocompatible host Al-MCM-41 and manganese nitrosyl [(PaPy3)Mn(NO)]ClO4. Moreover, {Mn-NO} is a very stable NO donor that exhibits its photoband with band maximum at 635 nm with a high quantum yield of NO release at 500 nm (f500= 0.55).
[0037] As per the IR measurement, it is observed that strong electrostatic interaction between the cationic NO complex with the negatively charged walls allows very effective loading of the NO donor. Also, ICP-OES, inductively coupled plasma optical emission spectroscopy states that the extent of loading of {Mn-NO} depends on the Al-content of the MCM-41 material.
[0038] Such interaction not only entrapped the nitrosyl but also retained the photoproduct ([(PaPy3)Mn(H2O)]2+) within the host resulting in very effective NO delivery without any chance of toxic side effects from the NO-spent material. Depending on the pore size of the Al-MCM-41 material, other nitrosyl complexes may be inserted.
[0039] Power X-ray diffraction (PXRD) studies on the loaded materials afforded patterns typical of an ordered mesoporous aluminosilicate consisting of hexagonal channels (supported by the TEM images). Referring to Figure 2, energy-dispersive X-ray analysis (EDX) elemental mapping of the loaded particles confirmed the incorporation of {Mn-NO} into the porous structure of the material and leaching experiments confirmed less than 3% loss of the nitrosyl over a period of 24 h.
[0040] Referring to Figure 3, it is analyzed from the NO electrode that when suspensions of {Mn-NO}@Al-MCM-41 in saline buffer are exposed to visible light (100 mW/cm2, the power of sunlight on a sunny day in the tropics), rapid release of NO is observed. With continuous exposure, it can attain a strong flux of NO with ~5 mg of material/mL buffer and maintain the flux for a period of 60 min. Furthermore, this powder when sprayed over bacterial lawn of both drug susceptible and drug-resistant Acenatobacter baumannii (a Gram-negative bacteria), rapid diminution of the bacterial load is observed upon illumination.
[0041] Also, the formulations that resolves chronic wound infections caused by several pathogens which may include but not limited to scenarios of battle-field wound infections, diabetic ulcer infections, and in burn wounds. Certain aspects of the invention include the elimination of pathogenic infections by a biocompatible zeolite-nitrosyl composite material triggered by exposure to low-power visible light.
[0042] Further, the powder has exhibited high efficacy against Candida albicans, the most prevalent human fungal pathogen. The commensal yeast form of this fungus exists in equilibrium with the normal microbial flora in the oral and intestinal mucosal layer of a high proportion of general population and often poses no threat to the host. However, in response to external stimuli, the fungus makes a transition to the elongated parallel-sided hyphae form. The hyphal form of C. albicans is entirely invasive and causes mild infections (like oral thrush) or severe infections in immunocompromised patients (patients under chemotherapy, HIV patients) and burn victims.
[0043] The pathogenic hyphal form often penetrates the epithelial layer and enters the blood stream causing sepsis. It has been shown that the invasive hyphal form is tackled with moderate doses of NO derived from {Mn-NO}@Al-MCM-41. When colonies of pure yeast and hyphal form of C. albicans in the SSTI model are sprayed with {Mn-NO}@Al-MCM-41 and exposed to low power (100 mW/cm2 ) visible light, a dose-dependent eradication of the invasive hyphal form is noted.
[0044] Referring to Figure 4a , a complete eradication of the hyphal form is noted when the powder delivered 70 mM of NO for 30 min. Referring to Figure 4b, the yeast form is more NO tolerant and showed temporary stunned growth upon such treatment.
[0045] NO poses no detrimental effects on normal cells up to 400 ppm while doses of 100-200 ppm NO effectively eradicate bacterial colonies in wound model. In addition to its antimicrobial effects, NO helps in wound repair by up-regulating collagenase gene expression. In this regard the powder will efficaciously resolve pathogenic infection via topical application. Additionally, application of non-toxic biocompatible {Mn-NO}@Al-MCM-41 powder to the wounds of burn victims will thwart C. albicans invasion.
[0046] Referring to Figure 5a, the strong antimicrobial activity of NO, nitrosyl-loaded polymer matrices as patches to deliver NO (under the control of light) to bacterial colonies (E. coli, P. aeruginosa, S. aureus) and examined the effect(s) on the bacterial growth (as in Figure 5b).
[0047] In the initial attempts, the NO donors have been incorporated into different silicate-based (Fig. 5) and HEMA-based hydrogel matrices (Fig. 7) in order to retain the photoproducts in the polymer patches (to avoid side effects and toxicity) during and after the NO delivery. The porosity of the gel allows release of NO from the patches. In all cases, drastic reductions of bacterial loads are observed following light triggered NO delivery (Fig. 6)
[0048] These results clearly demonstrate that NO released from the patches could act as effective light-activated antimicrobial agents to combat acute infection. NO is also quite effective in cases where the invading pathogen exhibits resistance to regular antibiotics. Exemplary results have also been achieved in eradication of Methicillin-resistant S. aureus (MRSA) colonies in the preliminary experiments.
[0049] The invention includes a set of biocompatible silicate- or methacrylate-based hydrogels that could be cast as patches (much like band-aids) or bandage inserts in which selective NO donors are incorporated. These patches are well-tolerated on human tissue. A very small amount of leakage of the nitrosyl donors from such patches has been noted.
[0050] Placement of the patches on bacterial wounds and exposure of the infected site to light will allow only the delivery of the NO antibacterial formulation to the targets and result in reduction of bacterial loads. Since only NO diffuses out of the patches under illumination (the other products will all be locked within the hydrogel matrices), no side effects are anticipated. Additional antibacterial agents could also be loaded on the patches for better efficacy.
[0051] This invention includes the formulations (powder and patch) of topical site-specific delivery of NO to infected wounds to resolve microbial infections including drug resistant ones. Exposure to simple day light or a LED lamp will release NO from the materials mentioned yet no other side products will be introduced at the targets. After the light exposure (and NO delivery) the powder may be washed off; the patch may be simply taken off.
[0052] Thus, NO donors that may deliver NO under various physiological conditions and at varying rates, from seconds to days, and offer a vast number of possibilities of application of such exogenous NO donors in delivering NO at infected locales to combat pathogen invasion.
[0053] Further, the operations need not be performed in the disclosed order, although in some examples, an order may be preferred. Also, not all functions need to be performed to achieve the desired advantages of the disclosed system and method, and therefore not all functions are required.
[0054] Various modifications to these embodiments are apparent to those skilled in the art from the description and the accompanying drawings. The principles associated with the various embodiments described herein may be applied to other embodiments. Therefore, the description is not intended to be limited to the 5 embodiments shown along with the accompanying drawings but is to be providing the broadest scope consistent with the principles and the novel and inventive features disclosed or suggested herein. Accordingly, the invention is anticipated to hold on to all other such alternatives, modifications, and variations that fall within the scope of the present invention and appended claims.

I/We Claim:

1) A photoactivated indigenous nitric oxide (no) releasing hydrogel formulation to deliver antimicrobial nitric oxide (NO) under the control of light on impacted physiological sites in order to mitigate infection and combat drug resistance pathogen.

2) The formulation as claimed in claim 1, wherein said formulation is prepared by incorporating a photoactive metal nitrosyl into a biocompatible material.

3) The formulation as claimed in claim 2, wherein said biocompatible material is selected from a group which may include but not limited to silicate-based hydrogels, HEMA hydrogel, modified Al-MCM-41 (modified zeolite).

4) The formulation as claimed in claim 2, wherein said photoactive metal nitrosyl is preferably manganese nitrosyl.

5) The formulation as claimed in claim 1, wherein said formulation may be used in the form of gel, patch and alike.

6) The formulation as claimed in claim 1, wherein said formulation may be used as antibacterial agent, antifungal agent and alike.