Antimicrobial irrigation solutions for endourological procedures

Abstract

Irrigation solutions for endourological procedures in accordance with embodiments of the invention are illustrated. One embodiment includes an irrigation solution that includes saline and between 0.001 and 1.0 % w / v chitosan, where chitosan includes chitosan, a chitosan derivative, or a salt thereof. In some embodiments, the irrigation solution uses water or glycine instead of saline. In various embodiments, glycosaminoglycan is included to assist in healing. In many embodiments, chitosan solutions are used for sterilization.

Description

Antimicrobial Irrigation Solutions for Endourological ProceduresSTATEMENT OF FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with Government support under contract DK131776 awarded by the National Institutes of Health. The Government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The current application claims the benefit of and priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 593,205 entitled “Antimicrobial Irrigation Solutions for Endourological Procedures” filed October 25, 2023. The disclosure of U.S. Provisional Patent Application No. 63 / 593,205 is hereby incorporated by reference in its entirety for all purposes.FIELD OF THE INVENTION

[0003] The present invention generally relates to solutions that include chitosan for use in endourological procedures, namely solutions that reduce, prevent or eradicate microorganisms and their related biofilms in order to reduce, prevent, or treat infections following an endourological procedure.BACKGROUND

[0004] The urinary system, also called the urinary tract or renal system, is the system of organs which broadly are responsible for eliminating waste from the body and regulating blood volume and chemistry. The urinary system is split into the upper urinary tract including the kidneys and ureters, and the lower urinary tract including the bladder and urethra. Many urological disorders are treated through surgery. A common factor of urinary tract surgeries is the constant use of irrigation, typically saline at 0.9% sodium chloride. Irrigation is necessary to distend the organs, allowing instrumentation maneuverability at the same time as enhancing visibility by removing blood and debris present in the urine.

[0005] Nephrolithiasis, or “kidney stones” is a urological disorder associated with significant pain, often requiring surgical intervention. Kidney stones are hard deposits of minerals and salts that form, often inside the kidney. When urine becomes concentrated, there is an elevated risk of forming kidney stones. When small, stones can pass on their own and be excreted with the urine. However, larger stones often require surgery for removal which typically involves stone fragmentation, followed by extraction of fragments. The most common procedures for kidney stone removal are urerteroscopic laser lithotripsy (URSLL), and percutaneous nephrolithotomy (PCNL).

[0006] The urinary tract can harbor a wide range of bacteria, some of them pathogenic which can lead to urinary tract infections, the most common bacterial infection worldwide affecting approximately 150 million people annually. Stone formers are at a higher risk of infections since bacteria can form biofilms on the stones. Biofilms are three dimensional bacterial communities that are encased on a self-produced polymeric matrix. The matrix, consisting of extracellular DNA and polysaccharides, renders protection against systemic antibiotic treatments.

[0007] Chitosan is a well-known biopolymer extracted from chitin. Its polymeric chain is composed of 1 ,4-beta-linked D-glucosamine and N-acetyl-D-glucosamine residues. At neutral and acidic pH values, the amino groups of the polymeric backbone get protonated yielding a highly positively charged molecule. The structure of chitosan is:

[0008] Chitosan is a polycation and it can interact with the negatively charged cell walls of bacteria, disrupting their cell viability and killing them. In addition, chitosan can interact with the negatively charged macromolecules of bacterial biofilms, ultimately disrupting them.

[0009] The foregoing examples of the related art and limitations related there with are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.SUMMARY OF THE INVENTION

[0010] Irrigation solutions for endourological procedures in accordance with embodiments of the invention are illustrated. One embodiment includes an irrigation solution that includes saline and between 0.001 and 1.0 % w / v chitosan, where chitosan includes chitosan, a chitosan derivative, or a salt thereof.

[0011] In another embodiment, the solution includes between 0.045 and 0.055 % w / v chitosan.

[0012] In a further embodiment, the solution includes 0.05% chitosan.

[0013] In still another embodiment, the saline is water containing 0.9% sodium chloride.

[0014] In a still further embodiment, the saline is phosphate buffer saline.

[0015] In yet another embodiment, a pH of the irrigation solution is between pH 4 and pH 7.

[0016] In a yet further embodiment, the method further includes steps for at most 1 % of glycosaminoglycan.

[0017] Another embodiment includes a method for reducing infection in a urinary tract of a human subject, the method includes irrigating the urinary tract during an endourological procedure using an irrigation solution includes saline and between 0.001 and 1.0 % w / v chitosan, where chitosan includes chitosan, chitosan derivative, or salt thereof.

[0018] In a further embodiment, the irrigation solution includes between 0.045 and 0.055 % w / v chitosan.

[0019] In another additional embodiment, the irrigation solution includes 0.05% chitosan.

[0020] In a further additional embodiment, the saline includes 0.9% sodium chloride.

[0021] In another embodiment again, the saline is phosphate buffer saline solution.

[0022] In a further embodiment again, the irrigation solution further includes at most 1 % of glycosaminoglycan.

[0023] In still yet another embodiment, a pH of the irrigation solution is adjusted to between pH 4 and pH 7.

[0024] In a still yet further embodiment, the irrigation solution is instilled for durations longer than 1 minute to about 150 minutes.

[0025] In still another additional embodiment, the irrigation solution is instilled through the endoscope from an infusion bag or a syringe.

[0026] In a still further additional embodiment, the irrigation solution is instilled through the endoscope at a rate faster than 1 mL / min to about 30 mL / min.

[0027] In still another embodiment again, the irrigation solution has antimicrobial, cidal or static, activity for durations longer than 1 minute to about 150 minutes.

[0028] One embodiment includes a sterilization solution that includes saline and between 0.001 and 1.0 % w / v chitosan, where chitosan includes chitosan, a chitosan derivative, or a salt thereof.

[0029] In still another embodiment, the solution includes between 0.045 and 0.055 % w / v chitosan.

[0030] In a still further embodiment again, the solution includes 0.05% chitosan.

[0031] In yet another additional embodiment, the saline is water containing 0.9% sodium chloride.

[0032] In a yet further additional embodiment, the saline is phosphate buffer saline solution.

[0033] In yet another embodiment again, a pH of the sterilization solution is between pH 4 and pH 7.

[0034] Yet another embodiment includes an endourological irrigation solution that includes glycine and between 0.001 and 1.0 % w / v chitosan, where chitosan includes chitosan, a chitosan derivative, or a salt thereof.

[0035] A further embodiment includes an endourological irrigation solution that includes water and between 0.001 and 1.0 % w / v chitosan, where chitosan includes chitosan, a chitosan derivative, or a salt thereof.

[0036] A further embodiment again includes a solution for endourological irrigation orsterilization containing only a liquid base and 0.001 and 1.0 % w / v chitosan, where chitosan includes chitosan, a chitosan derivative, or a salt thereof.

[0037] In yet another embodiment, the liquid base is saline.

[0038] In a yet further embodiment, the liquid base is glycine.

[0039] In another additional embodiment, the liquid base is water.

[0040] Additional embodiments and features are set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the specification or may be learned by the practice of the invention. A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and the drawings, which forms a part of this disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The description and claims will be more fully understood with reference to the following figures and data graphs, which are presented as exemplary embodiments of the invention and should not be construed as a complete recitation of the scope of the invention.

[0042] FIG. 1 is a table illustrating chitosan disruption on fresh biofilms in accordance with an embodiment of the invention.

[0043] FIG. 2 is a chart showing cell viability in patient-derived struvite stone fragments treated with different concentrations of chitosan-saline irrigation solutions in accordance with an embodiment of the invention.

[0044] FIG. 3 is a box plot of cell viability of stone fragments treated in saline or chitosan-saline irrigation solution in accordance with an embodiment of the invention.

[0045] FIG. 4 is a chart showing number of viable cells of different pathogen strains at different concentrations of chitosan-saline irrigation solution in accordance with an embodiment of the invention.

[0046] FIG. 5A is a micrograph of a catheter having an Escherichia coli biofilm, and FIG. 5B is a micrograph of the catheter after treatment with a chitosan-saline irrigation solution in accordance with an embodiment of the invention.

[0047] FIG. 6 is a series of four hematoxylin and eosin (H&E) stained slides of human urothelium incubated with different concentrations of chitosan-saline irrigation solution in accordance with an embodiment of the invention.DETAILED DESCRIPTIONDefinitions

[0048] Solutions containing chitosan for use during endourological procedures are discussed herein. The term chitosan include, but is not limited to, chitosan, chitosan hydrochloride (Chitosan HCI), chitosan glutamate, chitosan acetate, chitosan lactate, carboxymethyl chitosan, and trimethyl chitosan. The term “chitosan” encompasses all types of chitosan derivatives regardless of their molecular weight, viscosity, degree of deacetylation, purity, etc.

[0049] The term “saline” as defined herein refers to 0.9% sodium chloride in isotonic solution having a pH 4 - pH 7. However, it is contemplated that saline may have slightly different sodium chloride concentrations, e.g. + / - 10%.

[0050] “Endourological procedures” include any urethroscopy, cystoscopy, ureteroscopy, or nephoscopy procedure. Among them, continuous bladder irrigation, transurethral incision of prostate (TUIP), transurethral resection of prostate (TLIRP), transurethral resection of bladder tumor (TURBT), cystolitholopaxy, ureteroscopy with laser lithotripsy, ureteral stent removals are some common examples, but not all examples of endourological procedures.

[0051] “Preventing” or “prevention” refers to the reduction of risk of acquiring a disease or the display of some of its clinical symptoms.Chitosan Solutions

[0052] Minimally invasive surgeries are the new standard in urology. The urinary tract is most commonly accessed with an endoscope through the urethra, or alternatively through a percutaneous access through the kidney. The urologists can inspect the urethra, the bladder, the ureter, and the kidney via endoscope. A common factor of endourological surgeries is the constant use of irrigation, typically with isotonic saline. Endourological procedures are associated with an increased risk of urinary tract infectionsand bacteremia particularly in patients with catheter, stents or any foreign body that can serve as nidus for bacterial biofilm formation. Infection rates depend on the procedure, but they can go as high as 40% as in the case of kidney stone surgeries.

[0053] Kidney stone removal often involves the intentional (or unintentional) fragmentation of kidney stones. During fragmentation, bacterial biofilms can disperse into the irrigation fluid (typically isotonic saline) used to facilitate endoscopic visibility. The released, now planktonic bacteria, can infect the urothelium that can lead to UTIs and / or flow back into the bloodstream due to the high intrarenal pressures of the procedures causing bacteremia that can lead to sepsis. Following stone surgery, up to 30% of patients develop a UTI and between 10-15% will become septic. Infection is common and a potentially life-threatening postoperative complication that comes with a huge health and economic burden.

[0054] Infections are not exclusive to kidney stone surgeries. Any urological procedure carries risk of infection, either by dispersing bacteria already present in the urinary tract (for example, in catheters or stents) or by introducing new bacteria during the procedure.

[0055] Preoperative prophylaxis treatments are not effective in disrupting these biofilms and currently there are no suitable approaches to reduce bacterial burden and biofilms within the urinary tract during the surgical procedure. Following infection, antibiotic treatments are known to have poor efficacy given low residence time in the urine, and these untreated bacteria can lead to life-threatening complications such as sepsis.

[0056] To reduce the likelihood of infection, irrigation solutions containing chitosan are described. Chitosan-saline irrigation solutions (CSIS) have a number of advantageous properties. At neutral and acidic pH values, the amino groups of the polymeric backbone get protonated yielding a highly positively charged molecule. Chitosan interacts with negatively charged bacterial cell surfaces, ultimately disrupting it, and inducing cell death by leakage of intracellular components. Moreover, chitosan can disrupt biofilms, which are 3D bacterial communities resistant to antibiotics, by interacting with extracellular DNA (e-DNA) and proteins that bacteria utilize for biofilm formation. Additionally, in the urinary tract, chitosan interacts with the negatively charged integrins of the urothelium, leading to its exfoliation which is needed for eradication of highly resistant intracellular bacterialreservoirs. CSIS further has similar fluid properties as conventional saline, meaning that it can be used in with conventional equipment. Irrigation pressure similarly can range from 0 to 300 mmHg, with a flow rate from I mL / min to 60mL / min. CSIS is also very safe and can be used for up to 150 minutes continuously. However, it may be able to be used during longer procedures, even continuously, depending on pressure and / or flow rate, as well as the procedure. Once the CSIS has been irrigated into the surgical site, the composition may be washed away with conventional isotonic saline or left behind and be excreted with normal urine flow.

[0057] In many embodiments, CSIS is a saline solution having 0.05 % w / v chitosan. However, CSIS solutions having between 0.0001 % w / v and 1 % w / v are contemplated. In numerous embodiments, the CSIS is between 0.001 % w / v and 0.5 % w / v. In many embodiments, the CSIS is 0.5 % w / v, + / - 0.05% w / v. In numerous embodiments CSIS includes approximately 1 % or lower of glycosaminoglycan, which can assist with recovery of the urothelium post irrigation. Other molecules may be added without departing from the scope or spirit of the invention.

[0058] In various embodiments, CSIS is used to irrigate endourological procedures via an irrigation line embedded within an endoscope. However, CSIS can further be used as part of sterilization procedures, or irrigation in non-endourological procedures.

[0059] Chitosan is typically provided as a dried solid, flake, or fiber, or in an aqueous solution. Chitosan can be directly dissolved in sterile water, after which sodium chloride is added. In some embodiments, chitosan is directly dissolved in saline. In some embodiments, the chitosan, or derivatives, are dissolved first in a small volume of mild acid (acetic acid or lactic acid with concentrations ranging 1 %-25% v / v). The resulting solution is then mixed with saline. Hydrochloric acid or sodium hydroxide can be used to adjust the pH for the final solution. While saline alone is usable for irrigation and sterilization purposes, other salts can be used. For example, phosphate buffer saline (PBS) is a particular variant of saline which can be used. However, caution should be used when introducing phosphate is introduced to the body depending on the patient. Similarly, glycine or other aminoacids can be used without departing from the scope or spirit of the invention and can be considered derivatives of CSIS. In various embodiments,the CSIS is sterilized before clinical use to improve safety. For example, thermal processes such as autoclaving, or plasma treatment can be used to sterilize CSIS.

[0060] CSIS has particular efficacy in kidney stone removal procedures. During the most common kidney stone removal procedures, the kidney stone is fragmented which results in dispersal of bacterial biofilms into the irrigation solution. The released, now planktonic bacteria can infect the urothelium where they form reservoirs that can lead to recurrent UTIs. The bacteria may also flow back into the bloodstream due to the high intrarenal pressure of the procedure, causing bacteremia that can then lead to sepsis. Additionally, small fragments that still contain bacteria are frequently left behind due to the impossibility of achieving stone-free-status. These fragments are known to contribute to stone recurrence, particularly in the case of infectious struvite stones.

[0061] Despite the known complications associated with bacteria and stones, little effort has been made to address this issue. Standard clinical practice involves testing the urine for infection at least a week prior to surgery. If the culture is positive, patients are treated with appropriate antibiotics. However, urine is not always representative of the bacteria found within stones. Several studies have demonstrated that bladder urine culture positivity rates are very low compared to pelvic and stone culture.

[0062] This lack of correlation can be explained by the different microbiota present in the upper and lower urinary tract. Similarly, studies have found that patients undergoing antibiotic treatment before surgery still have positive stone culture. Because larger and obstructing stones are often required to manifest symptoms, stones can sometimes take months to years to be detected. During this time, bacteria form mature biofilms that get entrapped and grow as the stone increases in size. The short exposure time in the urinary tract of the antibiotic, the shielding effect provided by the stone matrix, and, more recently, the escalating emergence of antibiotic-resistant bacterial strains can explain the poor antibiotic efficacy in eradicating stone biofilms. Further, ubiquitous use of antibiotics to address these issues may lead to even more antibiotic resistance.

[0063] CSIS addresses these issues using at least the properties described above, namely that the antimicrobial and exfoliative properties of CSIS provide a non-antibiotic, one time, solution to mitigate these long-term risks. To demonstrate this data illustrating effectiveness is provided. Turning now to FIG. 1 , a table showing CSIS at differentconcentrations of chitosan having disruptive effects on biofilms of Escherichia coli (EC) and Klebsiella pneumoniae (KP) in accordance with an embodiment of the invention is illustrated. As can be seen, inhibition over 99% at 0.05 % w / v can be seen. Turning now to FIG. 2, a chart showing cell viability in patient-derived struvite stone fragments treated with different concentrations of CSIS for 90 minutes is illustrated in accordance with an embodiment of the invention. As can be seen, a statistically significant drop in viable cells from saline alone is seen with respect to CSIS having 0.05 and 0.5 % w / v chitosan. However, no statistically significant difference is seen between the 0.05 and 0.5 % w / v chitosan CSIS solutions. Brackets with *’s indicate no statistically significant difference p > 0.05.

[0064] A box plot showing cell viability of stone fragments treated in saline or CSIS for 90 minutes in accordance with an embodiment of the invention is illustrated in FIG. 3, providing additional demonstration that CSIS has an antibacterial effect. This can be further seen in FIG. 4, where a chart showing viable cell counts for different bacteria at different concentrations of CSIS after 4 hours of incubation in accordance with an embodiment of the invention is illustrated. All bacteria counts were reduced when CSIS was applied.The antibacterial properties extend to surfaces of medical equipment. FIG. 5A shows a catheter hosting an Escherichia coli biofilm. In contrast, FIG. 5B shows the catheter after treatment with CSIS in accordance with an embodiment of the invention. As can be seen under scanning electron microscopy, the biofilm has been destroyed post-treatment. When used as an irrigation solution during surgery, the instrumentation is constantly awash in solution, leading to continuous disruption of biofilm formation. By killing, and / or neutralizing microorganisms dispersed throughout the urinary tract, this in turn inhibits or prevents microorganisms from transferring and disseminating into the bloodstream, causing sepsis. The microorganisms causing the infection may, for example, be Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, coagulase-negative Staphylococci, Staphylococcus aureus, Proteus mirabilis, and Pseudomonas aeruginosa among others. Notably, the broad-spectrum antimicrobial activity of CSIS can treat infections cause by any microorganism.

[0065] The exfoliating properties of CSIS are demonstrated in FIG. 6. H&E-stained human urothelium slides are shown, where slide A is a normal urothelium, slide B shows exposure with 0.05 % w / v CSIS, slide C shows exposure with 0.005 % w / v CSIS, and slide D shows exposure with 0.0005 % w / v CSIS. Slides B and C demonstrate complete exfoliation of the urothelial barrier, where slide D only demonstrates partial exfoliation.

[0066] Although CSIS formulation are discussed above, different compositions that include other additives can be made and used in accordance with many different embodiments of the invention. Similarly, while CSIS is discussed primarily with respect to endourological procedures such as (but not limited to) kidney stone removal, it can also be used as an irrigation solution for any number of different medical procedures, and / or as part of a sterilization protocol. It is therefore to be understood that the present invention may be practiced in ways other than specifically described, without departing from the scope and spirit of the present invention. Thus, embodiments of the present invention should be considered in all respects as illustrative and not restrictive. Accordingly, the scope of the invention should be determined not by the embodiments illustrated, but by the appended claims and their equivalents.

Claims

WHAT IS CLAIMED IS:1 . An irrigation solution comprising saline and between 0.001 and 1.0 % w / v chitosan, where chitosan comprises chitosan, a chitosan derivative, or a salt thereof.

2. The irrigation solution of claim 1 , where the solution comprises between 0.045 and 0.055 % w / v chitosan.

3. The irrigation solution of claim 1 , wherein the solution comprises 0.05% chitosan.

4. The irrigation solution of claim 1 , wherein the saline is water containing 0.9% sodium chloride.

5. The irrigation solution of claim 1 , wherein the saline is phosphate buffer saline.

6. The irrigation solution of claim 1 , wherein a pH of the irrigation solution is between pH 4 and pH 7.

7. The irrigation solution of claim 1 , further comprising at most 1 % of glycosaminoglycan.

8. A method for reducing infection in a urinary tract of a human subject, comprising irrigating the urinary tract during an endourological procedure using an irrigation solution comprising saline and between 0.001 and 1.0 % w / v chitosan, where chitosan comprises chitosan, chitosan derivative, or salt thereof.

9. The treatment method of claim 8, where the solution comprises between 0.045 and 0.055 % w / v chitosan.

10. The treatment method of claim 8, wherein the saline comprises 0.9% sodium chloride.

11. The treatment method of claim 8, wherein the saline is phosphate buffer saline solution.

12. The treatment method of claim 8, wherein the irrigation solution further comprises at most 1 % of glycosaminoglycan.

13. A sterilization solution comprising saline and between 0.001 and 1.0 % w / v chitosan, where chitosan comprises chitosan, a chitosan derivative, or a salt thereof.

14. The sterilization solution of claim 13, where the solution comprises between 0.045 and 0.055 % w / v chitosan.

15. The sterilization solution of claim 14, wherein the solution comprises 0.05% chitosan.

16. The sterilization solution of claim 13, wherein the saline is water containing 0.9% sodium chloride.

17. The sterilization solution of claim 13, wherein the saline is phosphate buffer saline solution.

18. The sterilization solution of claim 13, wherein a pH of the sterilization solution is between pH 4 and pH 7.

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