NAC derivatives for inhibiting urinary catheter encrustation

Abstract

A derivative of N-acetylcysteine (NAC) comprising a protecting group bonded to a thiol group in said NAC, wherein said protecting group is selected from the group consisting of S-nitrobenzyl, S-carbamimido, S-phosphate, S-citrate, S-carboxylate / itaconate and S- acetyl.

Description

[0001] NAC derivatives for inhibiting Urinary Catheter Encrustation

[0002] Technical Field

[0003] This invention relates to novel derivatives of N-acetylcysteine (NAC), processes of preparing said derivatives, and novel uses of the derivatives in inhibiting bacterially induced urease activity in a mammal and reducing the resultant urinary catheter encrustation in catheterised patients arising from the bacterial infection.

[0004] Background

[0005] The major cause of catheter encrustation resulting from a urinary tract infection is the pH change in urine that is caused by urease production by Proteus mirabilis. The bacterial urease hydrolyses urea, generating ammonia and causing the urine to become alkaline. This shift in pH promotes the precipitation of calcium and magnesium phosphate crystals mainly the formation of struvite deposits, leading to encrustation that progressively obstructs urinary flow through the catheter. This encrustation also structurally supports the biofilms formed by P. mirabilis and other pathogens. N-acetylcysteine (NAC) is bacteriostatic for P. mirabilis, prevents its swarming motility, and prevents catheter occlusion by inhibiting the activity of its urease (Reference 1).

[0006] The thiol group of NAC has been shown to produce urease inhibition. Thiol compounds can potentially inhibit urease by both binding to the active site nickel atoms and forming mixed disulfides with the catalytically important cysteine residue (Cysa319 in K. pneumoniae urease and Cys592 in jack bean urease) in the active site mobile “flap” (Reference 2). Indeed, simple thiols, such as 2-mercaptoethanol, have been shown to inhibit urease from Sporosarcina pasteurii (Reference 3).

[0007] The thiol group of NAC is therefore likely to be the moiety thought to be responsible for the urease inhibitor activity. The use of NAC in aqueous solutions does, however, present difficulties in formulating a stable commercial product as the thiol moiety is easily oxidised by atmospheric oxygen, and that this oxidation is also pH sensitive, with the oxidation rate increasing as the pH of the solution increases. This limits the ability to create a stable formulation of NAC as a therapeutic agent. Whilst NAC solutions in water are often used to treat e.g. paracetamol overdoses, these are typically offered in glass ampoules containing an inert atmosphere such as nitrogen.

[0008] Currently, the most common treatment used clinically to prevent the formation of struvite deposits in patients with P. mirablis bladder infections is to treat the patient with anti-urease drugs in the urinary system, primarily acetohydroxamic acid (AHA), which function by inhibiting the urease enzyme produced by certain bacteria, notably P. mirablis, preventing ammonia buildup and urine alkalization, which stops the formation of struvite bladder stones and catheter blockages. However, AHA has significant side effects, which include gastrointestinal issues (nausea, loss of appetite, vomiting), neurological effects (headache, anxiety, nervousness, tremor, tiredness), and skin / hair problems (rash, hair loss), but more serious risks include haemolytic anaemia (destruction of red blood cells), blood clots, and significant mood changes, requiring close monitoring, especially with alcohol use.

[0009] This invention is directed to novel derivatives of NAC having improved stability in water over NAC itself, for the treatment of bladder and urinary catheter encrustations, whilst avoiding the adverse events often encountered using the current AHA treatment. The inventive NAC derivatives comprise a protecting group on the thiol group of the NAC molecule. The protecting group may be cleaved under physiological conditions or in the urinary bladder to release the NAC. Free NAC has also been shown to have antioxidant and biofilm-disrupting properties.

[0010] References:

[0011] 1. Manoharan A, Farrell J, AldillaVR, Whiteley G, Kriel E, GlasbeyT, Kumar N, Moore KH, Manos J and Das T. N-acetylcysteine prevents catheter occlusion and in ammation in catheter associated-urinary tract infections by suppressing urease activity. Front. Cell. Infect. Microbiol. 13:1216798 (2023).

[0012] 2. Svane, S., Sigurdarson, J. J., Finkenwirth, F. et al. Inhibition of urease activity by different compounds provides insight into the modulation and association of bacterial nickel import and ureolysis. Sci Rep 10, 8503 (2020)

[0013] 3. Benini, S., Rypniewski, W R., Wilson, K. S., Ciurli, S. & Mangani, S. The Complex of Bacillus pasteurii Urease with Beta-mercaptoethanol from X-ray Data at 1.65- angstrom Resolution. J. Biol. Inorg. Chem. 3, 268-273 (1998)

[0014] Summary of the Invention

[0015] According to a broad form of the invention, there are provided derivatives of N-acety I cysteine (NAC), which comprises a protecting group bonded to a thiol group in said NAC.According to another form of the invention, there is provided a process for preparing derivatives of N-acetylcysteine (NAC), which process comprises bonding a protecting group to a thiol group in said NAC.

[0016] According to another form of the invention, there is provided a composition for inhibiting urease production in a mammal, which composition comprises one or more derivative of NAC, of the invention, together with a diluent, excipient and / or adjuvant.

[0017] According to another form of the invention, there is provided a composition for reducing encrustation of a catheter in a mammal, which composition comprises one or more of the derivatives of NAC of the invention, together with a diluent, excipient and / or adjuvant. According to another form of the invention, there is provided a method of inhibiting the production of urease in a mammal, which method comprises administering to said mammal one or more of the derivatives of NAC, or composition, of the invention.

[0018] According to another form of the invention, there is provided a method of reducing encrustation of a catheter in a mammal, which method comprises administering to said mammal one or more of the derivatives of NAC, or composition, of the invention.

[0019] The compounds of the invention comprising the thiol-protected NAC derivatives have more bioavailability of NAC in catheterized environments, are well suited for sustained release and provide for aqueous solutions that will be stable with respect to oxidation for a minimum of 1-2 years.

[0020] Brief description of the Figures

[0021] Figure 1 shows the rate of oxidation of NAC at room temperature at differing pHs.

[0022] Figure 2 shows the chemical structures of six preferred NAC derivatives according to the invention.

[0023] Figure 3 shows a process of synthesis of one of the NAC derivatives of the invention. Figure 4 shows a process of synthesis of another of the NAC derivatives of the invention. Figure 5 shows the percentage urease inhibition by a NAC derivative of the invention.

[0024] Detailed Description

[0025] According to the invention, there are provided one or more derivatives of N-acetylcysteine (NAC), comprising a protecting group bonded to a thiol group in said NAC. The derivatives of the invention are stable in aqueous solution since the protecting group masks the thiolgroup, ensuring stability, enabling controlled release of the NAC under specific biological or environmental conditions. The term “mask” will be understood by those skilled in the art as meaning blocking the reactivity of a specific moiety within a molecule by modifying said moiety with a protecting group until such time as the protecting group can be removed from said moiety so as to regenerate the active form of the initial molecule, and thus unmask the active moiety. In the context of the present invention, the NAC derivative is stable in aqueous solution since the protecting group prevents oxidation of the thiol group, ensuring stability, enabling controlled release of the NAC under specific biological or environmental conditions that lead to deprotection of the NAC derivative. The protecting group is cleaved or released from the thiol group in aqueous solution or in-vivo, leaving NAC in solution. The preferred protecting groups are ester-based, enzyme-triggered, pH-responsive or photo-labile, thereby reducing thiol oxidation of NAC and enabling the development of stable targeted delivery to catheterized environments. The protecting groups are preferably selected from the group consisting of a citrate, a phosphate, a carboxylate (various fatty acids and organic acids), a carbamimido and a nitrobenzyl group. In a preferred embodiment, the protecting group is selected from the group consisting of an S-nitrobenzyl, S-carbamimido, S-phosphate, S-citrate, S-carboxylate / itaconate and S-acetyl.

[0026] If the protecting group is a citrate or carboxylate, the protecting group is cleaved from said thiol group by hydrolysis, forming NAC. A preferred carboxylate protecting group is S-acetyl, a derivative of an organic acid. It is anticipated that the minimum inhibitory concentration (MIC) values and anti-urease activity observed for the S-acetyl derivative against P. mirabiiis will be comparable to those of NAC under the same experimental conditions.

[0027] If the protecting group is a phosphate, the protecting group is cleaved enzymatically from said thiol group, forming NAC.

[0028] If the protecting group is a carbamimido, the protecting group is released from the thiol group in basic conditions, forming NAC.

[0029] If the protecting group is a nitrobenzyl, the protecting group is photo-released from the thiol group, forming NAC.

[0030] The chemical structures of the six preferred derivatives of NAC of the invention are shown in Figure 2.The invention is also directed to a composition for inhibiting urease production in a mammal and reducing encrustation of a catheter in a mammal, which composition comprises the derivative of NAC, of the invention, together with a diluent, excipient and / or adjuvant.

[0031] In a preferred embodiment, the adjuvant sodium metabisulphite has surprisingly been found to enhance the release of NAC from the carbamimido derivative. The invention is also directed to a composition for inhibiting urease production in a mammal and reducing encrustation of a catheter in a mammal, which composition comprises a carbamimido derivative of NAC, of the invention, together with sodium metabisulphite and a diluent and / or excipient.

[0032] In a preferred embodiment, the composition of the invention may also contain carboxylic acids capable of complexation with calcium and magnesium ions. Preferred carboxylic acid include, but are not limited to, citric acid, lactic acid, acetic acid, tartaric acid, gluconic acid, glycolic acid, mandelic acid, benzoic acid and combinations thereof. The carboxylic acid(s) dissolve mineralised deposits within a urinary catheter, or within biofilm found in the urinary bladder, thereby synergistically enhancing the activity of the unmasked NAC derivative.

[0033] The NAC derivatives of the invention provide an aqueous stable version of NAC that retains its pH-lowering and reduction in catheter encrustation applications and therefore is useful in the management of catheter-associated complications. The novel compounds of the invention provide a novel method of reducing catheter encrustation, thereby reducing catheter-related complications, significantly improving patient outcomes and providing lower healthcare costs.

[0034] Also anticipated is an embodiment where two or more of the inventive NAC derivatives are used in combination. This can allow, for example, treatment within the bladder by, for example, hydrolysis of the S-carboxylate derivative within the bladder and external photochemical release with an S-nitrobenzoyl derivative to treat the catheter.

[0035] Examples

[0036] Note: All compound numbers refer to the structures numbered consecutively through Figures 3 and 4.Example 1: Experiment demonstrating that the rate of oxidation of NAC is pH dependant.

[0037] Preparation of NAC solutions

[0038] 1 litre of phosphate buffered saline was prepared by dissolving 8.02g sodium chloride, 0.20g of anhydrous potassium chloride, 5.34g disodium hydrogen phosphate dihydrate and 0.16g of anhydrous dipotassium hydrogen phosphate into approximately 950ml DI water. 3.14g of 85% phosphoric acid was added to bring the solution pH to 7.4. The solution was then made up to 1 litre with additional DI water.

[0039] 2.44g of N-acetylcysteine (NAC) was dissolved into 500ml DI water to give a 30mMol solution of NAC. The pH was measured and found to be 2.45.

[0040] 2.44g of N-acetylcysteine (NAC) was dissolved into 500ml of phosphate buffered saline to give a 30mMol solution of NAC. The pH was measured and found to be 4.15.

[0041] 2.44g of N-acetylcysteine (NAC) was dissolved into 450ml of phosphate buffered saline to give a 30mMol solution of NAC. The pH of the solution was then adjusted to 7.44 with 1M sodium hydroxide solution. The solution was then made up to 500ml by the addition of more deionised water.

[0042] Aliquots of each of the above three NAC solutions were then placed into HPLC vials and analysed by HPLC. The solutions were assayed daily for 5 days.

[0043] HPLC method

[0044] The above three NAC solutions were analysed by HPLC using a gradient elution method. Chromatography was performed on a Shimadzu Nexera HPLC system comprising a SCL-40 system controller, SPD-M40 photodiode array detector, DGU-405 degassing unit, LC-40B XR solvent delivery module, SIL-40C XR autosampler and a CTO-40S column oven. The sample tray of the SI L-40C XR autosampler was held at 25°C. Chromatography was performed using a Shimadzu Shim-pack Scepter C18-1205 m 150x2mm column held at 40°C. The column was initially eluted with 3% acetonitrile and 97% water. 1j.il injections of the test solutions were made, with the elution conditions given in Table 1:Table 1

[0045]

[0046] Data acquisition and analysis was performed with Labsolutions Ver 5.127 SP1, using an external standard. Quantification of N-acetyl cysteine was performed at 220nm, using an external standard method. The retention time of the NAC was 5.25 minutes. As the NAC was consumed over several days, a new peak appeared with a retention time of 10.65 minutes. This was identified as the oxidised dimer of NAC, N,N’-diacetyl-L-cystine. As is seen in Figure 1, a 30mM solution of NAC at pH 7.44 lost approximately 50% of its concentration within 7 days, whereas the same concentration held at pH 2.45 only lost approximately 5% over the same timeframe. As is seen from Figure 1 , the rate of oxidation of NAC is pH dependent.

[0047] Example 2: Processes for synthesis of S-carbamimido NAC derivative of the invention.

[0048] Example 2A

[0049] One process for synthesising the S-carbamimido derivative of NAC is shown in Figure 3. This process of synthesizing the S-carbamimido derivative of NAC is described below, with compound numbers as shown in Figure 3.

[0050] Synthesis of methyl A / -(tert-butoxycarbonyl)-O-(methylsulfonyl)- L-serinate (Compound 2):

[0051] A solution of methyl (tert-butoxycarbonyl)-L-serinate (Compound 1, 1.03 g, 4.69 mmol) in dichloromethane (10 mL) was cooled to 0 °C. Triethylamine (14.09 mmol) was added, followed by the dropwise addition of methane sulfonyl chloride (9.38 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 2 hours. Upon completion, as monitored by TLC, the reaction mixture was diluted with water and extracted with dichloromethane. The combined organic extracts were dried overanhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford the crude product. Purification by flash column chromatography (hexane / ethyl acetate, 0-50%) yielded methyl / \ / -(tert-butoxycarbonyl)-O-(methylsulfonyl)- L-serinate (Compound 2, 820 mg) as a yellow oil:

[0052] 1H NMR (400MHz, CDCh): 55.48 (brs, 1H), 4.41(brs, 1H), 4.16-3.98(m, 2H), 3.80 (s, 3H), 1.46(s, 9H).

[0053] Synthesis of methyl O-(methylsulfonyl)-L-serinate (Compound 3):

[0054] A solution of Compound 2 (119 mg, 0.4 mmol) in dichloromethane (10 mL) was cooled to 0 °C. Trifluoroacetic acid (2.0 mmol) was added, and the reaction mixture was allowed to warm to room temperature and stirred for approximately 4 hours. Reaction progress was monitored by thin layer chromatography. Upon completion, the volatile components were removed under reduced pressure to afford a crude residue methyl O-(methylsulfonyl)-L-serinate (Compound 3) as a brown oil (118 mg) and was used directly in the next step without further purification.

[0055] Synthesis of methyl N-acetyl-O-(methylsulfonyl)-L-serinate (Compound 4):

[0056] To a solution of Compound 3 (200 mg, 1.0 mmol) in dichloromethane (10 mL) at 0 °C was added triethylamine (3.0 mmol), followed by the dropwise addition of acetyl chloride (1.2 mmol). The reaction mixture was allowed to warm to room temperature and stirred for approximately 2 hours. Reaction progress was monitored by thin layer chromatography. Upon completion, the reaction mixture was diluted with water and extracted with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford a crude residue (Compound 4). The residue methyl / V-acetyl-O-(methylsulfonyl)-L-serinate (Compound 4) was obtained as a yellow oil (130 mg) and was used directly in the subsequent step without further purification.

[0057] Synthesis of N-acetyl-S-carbamimidoyl-L-cysteine hydrochloride (Compound 5):

[0058] A solution of Compound 4 (2.0 g, 8.3 mmol) in N, / V-dimethylformamide was treated with triethylamine (16.6 mmol) and thiourea (9.9 mmol) at room temperature. The reaction mixture was then heated to approximately 90 °C and stirred for about 18 hours. Reaction progress was monitored by thin layer chromatography.Upon completion, the reaction mixture was cooled to room temperature, diluted with 1 N hydrochloric acid, and stirred for approximately 5 hours. Completion of the transformation was confirmed by LC-MS analysis. The crude reaction mixture was then purified directly by reverse-phase C18 column chromatography using a water / methanol gradient (10-90%) to afford / V-acetyl-S-carbamimidoyl-L-cysteine hydrochloride (Compound 5) (250 mg) as a colourless solid:

[0059] 1H NMR (400MHz, D2O): 54.48-4.45 (m, 1H), 3.35-3.32 (m, 2H), 1.96 (s, 3H).

[0060] Example 2B

[0061] The S-carbamimido derivative of NAC may also be synthesized by converting the serine hydroxy group into chloro which, on further treatment with thiourea, produces the carbamimido group. The NH-acetyl protected serine may be treated with p-toluenesulfonyl chloride in presence of dichloromethane, as solvent, followed by treatment with lithium chloride to yield the S-carbamimido derivative of NAC.

[0062] In use, the isothiouronium salt of the S-carbamimido derivative of NAC may be cleaved off via aminolysis in urine environment. This carbamimido derivative slowly releases NAC due to instability in basic conditions.

[0063] Example 3: Processes for synthesis of the S-(2-nitrobenzyl) NAC derivative Example 3A

[0064] One process for synthesising the S-nitrobenzyl of NAC is shown in Figure 4. This process of synthesizing the S-nitrobenzyl derivative of NAC is detailed below, with compound numbers as shown in Figure 4.

[0065] Synthesis of N-acetyl-S-(2-nitrobenzyl)-L-cysteine (7):

[0066] To a solution of NAC (Compound 6, 2.0 g) in 1,4-dioxane 10 vol at ~15°C was added NaHCOs (3 equiv) followed by 2-nitrobenzyl bromide (1.2 equiv) and stirred at room temperature for 16h. After completion of reaction (monitored by TLC), the mixture was diluted with water, and extracted with ethyl acetate. The combined organic layer was dried over sodium sulphate, and the solvent was removed under reduced pressure to give crude Compound 7 which was purified by combi-flash column chromatography (hexane / ethyl acetate, 0-50%) to give (1.65 g) as a light yellow solid.1H NMR (400MHz, CDCI3): 6 12.89 (brs, 1H), 8.25(d, 1H), 8.02-8.00 (m, 1H), 7.71-7.67 (m, 1H), 7.60-7.52 (m, 2H), 4.38-4.33 (m, 1H), 4.07 (s, 2H), 2.81-2.77 (m, 1H), 2.70-2.64 (m, 1H), 1.85(s, 3H).

[0067] Example 3B

[0068] The S-(2-nitrobenzyl) derivative may also be prepared by treating NAC with 2-nitrobenzyl chloride in presence of base and THF as solvent. The reaction is carried out at room temperature.

[0069] A catheter-based light source (for example a light emitting diode) emitting light between 320-360nm enables photo-release of the thiol functionality from the S-nitrobenzyl derivative.

[0070] Example 4: Process for synthesis of S- citrate and S-carboxylate NAC derivates The S- citrate and S-carboxylate NAC derivates are produced by treating NAC with corresponding anhydrides. These derivatives are prepared by stirring NAC with different benzotriazole amide derivatives of fatty acids or organic acids, using acetonitrile-water (5:1) as solvent and in the presence of triethylamine at 0-5°C, yielding the corresponding products. An alternative method uses acid coupling reagents such as N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC HCI) or hexafluorophosphate azabenzotriazole tetramethyl uronium (HATLI) with corresponding acid chlorides to synthesize the NAC derivative. These esters hydrolyse under basic urine conditions. Example 5: Process for synthesis of S-phosphate NAC derivatives

[0071] The S-phosphate derivative of NAC is prepared using a mild phosphorylating agent. The S-phosphate derivative is synthesized by treating NAC solution with phosphorous oxychloride and with sodium hydroxide. This phosphorylated compound can be cleaved by phosphatases present in Enterobacteriaceae bacterial family.

[0072] Example 6: Stability studies of S-carbamimido and S-(2-nitrobenzyl) derivatives of NAC.

[0073] The aqueous stability of the S-carbamimido (Compound 5) and S-(2-nitrobenzyl) (Compound 7) derivatives were assessed using high-performance liquid chromatography (HPLC). All compounds were dissolved in purified water, other than Compound 7 which was dissolved in purified water to which a few drops of acetonitrile were added, to obtain a solution of defined concentration 2mg / ml and stored under controlled conditions(ambient temperature). Aliquots were collected at different time points and analysed using a validated HPLC method. Reference NAC compound (Compound 6) was also analysed to compare the stability of NAC versus Compound 5 and Compound 7.

[0074] A chromatographic analysis showed that Compound 7 exhibited a consistent retention time with no significant change in peak area percentage even after 80 days (Table 4), indicating the absence of detectable degradation in aqueous solution. Similarly, Compound 5 also retains stability even after 80 days with only 2-3% different peak observation. In the case of the reference NAC compound, within 7 days a new peak is observed at 67% area. It was confirmed that it is NAC-dimer. Hence Compound 5 and Compound 7 demonstrate excellent long-term aqueous stability compared to NAC. Table 2 provides the elution conditions for the study of Example 6.

[0075] Table 2

[0076]

[0077] Table 3 provides the flow rate conditions of the stability study of Example 6. Amount of sample used for HPLC: 2mg / mL; Injected volume: 10 pL

[0078] Table 3

[0079]

[0080] Under these conditions, NAC was observed to have a retention time of 4.2 minutes, Compound 5 was observed to have a retention time of 2.9 minutes, Compound 7 a retention time of 16.6 minutes, and the NAC-dimer a retention time of 8.8 minutes.

[0081] Table 4 provides the results of the HPLC stability study as described in Example 6. Each column shows the stability of each species tested following a 7 day stand at room temperature

[0082] Table 4

[0083]

[0084] Example 6A: Release of NAC from S-(2-nitrobenzyl) derivative under UV radiation The controlled release behaviour of NAC from the S-nitrobenzyl NAC derivative, Compound 7, was evaluated using ultraviolet (UV) irradiation. Compound 7 was dissolved in an ethanol-water mixture to obtain a solution at a concentration of 2 mg / mL, and aliquots were placed in Eppendorf vials. The samples were irradiated with UV light at 365 nm for 10 and 20 minutes, after which the irradiated solutions were analysed by HPLC. The analysis revealed the presence of multiple peaks in the irradiated samples, indicating the formation of photogenerated species. The observed retention times did not correspond to that of an authentic NAC reference standard analysed under identical conditions, suggesting that UV irradiation of Compound 7 under the tested conditions did not result in the direct release of free NAC but generated other species.

[0085] Example 6B: Release of NAC from S-carbamimido derivative in different pHs The controlled release behaviour of the S-carbamimido derivative of NAC, Compound 5, was evaluated under different pH conditions using HPLC. Compound 5 was dissolved in aqueous media to obtain solutions having a defined concentration of 2 mg / ml. The pH of the solutions was adjusted to the desired values using sodium bicarbonate and / or sodium hydroxide solutions. The prepared solutions were stored under controlled conditions atambient temperature. Aliquots were withdrawn at predetermined time intervals and analysed by HPLC to determine the extent of degradation or cleavage of Compound 5. HPLC analysis was performed by monitoring the peak corresponding to Compound 5 and any degradation products, with stability assessed based on retention of the parent compound peak area relative to the initial time point.

[0086] At the Day 1 time point, the sample maintained at pH 9 exhibited no / minimal cleavage of Compound 5, indicating substantial stability of the compound under mild to strong alkaline aqueous conditions (pH adjusted using 1N and 5N NaOH).

[0087] Table 5 provides the HPLC results at Day 1 for the Example 6B study on Compound 5. From the different pH studies using hydroxide ions as base, the higher pH cleaved the compound 5 to NAC as identified by their retention times.

[0088] Table 5

[0089]

[0090] n.d = not detected

[0091] Example 6C: Release of NAC from S-carbamimido derivative with Proteus mirabilis To a bacterial suspension adjusted to an optical density at 600 nm (ODeoo) of 0.1 in artificial urine (AU) containing 0.2 M urea, Compound 5 was added to obtain a final concentration of 2 mg / mL, and the mixture was incubated at 37 °C with shaking at 100 rpm for 1 hour. Following incubation, the samples were centrifuged, and the supernatant was filtered through a syringe filter and analysed by HPLC alongside untreated Compound 5. Both samples exhibited similar retention times with no additional peaks, indicating no detectable cleavage of Compound 5. A similar result was observed after 24 hours of incubation under the same conditions. To further increase the pH of the medium, artificial urine containing 2 M urea was utilized, and Compound 5 was incubated with the bacterial suspension for 24 hours under identical conditions. HPLC analysis of thecentrifuged supernatant showed the appearance of a minor peak corresponding to approximately 9% cleavage of Compound 5, as confirmed by comparison of retention times. (Note: In all experiments at 24 h the pH of the supernatant solution is greater than 9). Table 6 shows the HPLC results for Example 6C.

[0092] Table 6

[0093]

[0094] Pm = Proteus mirabilis

[0095] Example 7: Release of NAC from S-carbamimido derivative with sodium metabisulphite

[0096] The release of NAC from Compound 5 in combination with sodium metabisulfite was evaluated under different timepoints. Compound 5 was dissolved in different concentrations of sodium metabisulfite and the aliquots were withdrawn at predetermined time intervals and analysed by HPLC to determine the extent of release of NAC.

[0097] Table 7 shows the HPLC analysis results performed by monitoring the peak corresponding to NAC and any degradation products.Table 7

[0098]

[0099] As can be seen from Table 7, a 0.5% solution of sodium metabisulphite enhances the release of NAC from the S-carbamimido derivative.

[0100] Example 8: Minimum inhibitory concentration (MIC) of S-carbamimido NAC derivative on Proteus mirabilis for Compound 5

[0101] Overnight bacterial cultures of Proteus mirabilis (Pm 67) at a density of ODeoo nm 0.1 ± 0.02 in Tryptone Soy broth (TSB) were added to 96-well tissue culture plates containing Compound 5. The final concentrations of Compound 5, 30 mM, 15 mM, 7.5 mM and 3.7 mM, were allowed to incubate for 24 h at 37 °C, 100 rpm in an orbital shaker. ODeoo was recorded after 24 h post-incubation using the plate reader Tecan Infinite M1000 pro, Australia. The percentage bacterial growth was calculated relative to untreated control. The experiment was performed in triplicate. At all concentrations tested Compound 5 did not show any antibacterial activity.Example 9: Urease activity of S-carbamimido NAC derivative for Compound 5 To quantify the urease activity of Compound 5 the overnight-grown bacterial cultures as in Example 8 were centrifuged at 3715g for 10 min. The pellet was washed two times using 15 mM K2HPO4 and centrifugation at 4851g for 10 min. The pellet was then resuspended in 15 mM K2HPO4 to an OD6oonm=0.7± 0.05. Bacterial suspensions were exposed to 7.5, 15, 30 mM Compound 5 in a total volume of 5 mL, and incubated with orbital shaking at 150 rpm, 37°C for 2 h. After incubation, bacteria were centrifuged and washed three times to remove any treatment. The pellet was resuspended in 3 mL of K2HPO4 in a 15 mL falcon tube. The bacteria were then sonicated using an ultrasonic water bath sonicator. The sonicated bacterial lysates were then tested for urease activity as follows: 300 pL of sonicated bacterial lysate was added to 200 pL of 25 mM urea and incubated in a water bath at 37°C for 30 min. 500 pL each of three solutions: one containing 10g / L phenol and 0.5g / L sodium nitroprusside; and two containing 5% (v / v) sodium hypochlorite and 5g / L sodium hydroxide, were added and the lysate incubated for an additional 15-30 min until a colour change was observed. Figure 5 shows the absorbance of samples at 625 nm and used as a measure of urease activity.

Claims

Claims1. A derivative of N-acetylcysteine (NAC) comprising a protecting group bonded to a thiol group in said NAC.

2. A derivative according to claim 1 wherein said protecting group masks the thiol group of the NAC.

3. A derivative according to claim 1 or claim 2 wherein said protecting group is selected from the group consisting of a citrate, a phosphate, a carboxylate, a carbamimido and a nitrobenzyl group.

4. A derivative according to claim 3 wherein said protecting group is a carboxylate of a fatty acid and / or an organic acid.

5. A derivative of N-acetylcysteine (NAC) comprising a protecting group selected from the group consisting of an S-nitrobenzyl, S-carbamimido, S-phosphate, S- citrate, S-carboxylate / itaconate and S-acetyl.

6. A derivative according to any one of claims 1 to 5 which is stable in aqueous solution.

7. A derivative according to any one of claims 3, 4 or claim 5 wherein said citrate or carboxylate protecting group is cleaved from said thiol group by hydrolysis.

8. A derivative according to claim 3 or claim 5 wherein said phosphate protecting group is cleaved enzymatically from said thiol group.

9. A derivative according to claim 3 or claim 5 wherein said carbamimido group protecting group is released from the thiol group in basic conditions.

10. A derivative according to claim 3 or claim 5 wherein said nitrobenzyl protecting group is photo-released from said thiol group.

11. A derivative according to any one of claims 1 to 5 wherein said protecting group is cleaved or released from said thiol group in aqueous solution.

12. A derivative according to any one of claims 1 to 5 wherein said protecting group is cleaved or released from said thiol group in vivo.

13. A derivative according to any one of claim 1 to 5 wherein said protecting group is cleaved or released from said thiol group, leaving NAC in solution.

14. An aqueous solution comprising a derivative of NAC according to any one of claims 1 to 5 in water.

15. A process of producing said aqueous solution of claim 14, which process comprises cleaving or releasing said protecting group from said thiol group in said NAC.

16. A composition for inhibiting urease production in a mammal, which composition comprises one or more derivative of any one of claims 1 to 5, together with a diluent, excipient and / or adjuvant.

17. A composition according to claim 16 comprising one or more carboxylic acid capable of complexation with calcium and magnesium ions.

18. A composition according to claim 17 wherein the carboxylic acid is selected from the group consisting of citric acid, lactic acid, acetic acid, tartaric acid, gluconic acid, glycolic acid, mandelic acid, benzoic acid and combinations thereof.

19. A composition for reducing encrustation of a catheter in a mammal, which composition comprises one or more derivative of any one of claims 1 to 5 together with a diluent, excipient and / or adjuvant.

20. A composition according to claim 19 wherein the adjuvant is sodium metabisulphite.

21. A composition according to claim 19, which composition comprises a carbamimido derivative of NAC, of the invention, together with sodium metabisulphite and a diluent and / or excipient.

22. A composition according to claim 19 comprising one or more carboxylic acids capable of complexation with calcium and magnesium ions.

23. A composition according to claim 22 wherein said carboxylic acid is selected from the group consisting of citric acid, lactic acid, acetic acid, tartaric acid, gluconic acid, glycolic acid, mandelic acid, benzoic acid and combinations thereof.

24. A method of inhibiting urease production in a mammal, which method comprises administering to said mammal one or more of said derivatives according to any one of claims 1 to 13, or a composition according to any one of claims 16 to 18.

25. A method of reducing encrustation of a catheter in a mammal, which method comprises administering to said mammal one or more derivative according to any one of claims 1 to 13, or a composition according to any one of claims 19 to 23.

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