NO containing compositions

Abstract

Disclosed are metal organic framework materials (MOFs), comprising an extra-framework NO releasing compound within the internal pores and/or channels of the MOF, the NO-releasing compounds and their preparation and uses. The MOFs and NO-releasing compounds are capable of releasing NO on application of an external stimulus and may provide materials with multiple modes of antibacterial and/or drug action.

Claims

1. A medical article comprising a nitric oxide (NO) complexed compound or porous framework material or metal organic framework (MOF) comprising an extra framework NO complexed compound or salt thereof, wherein the NO complexed compound or salt thereof comprises a functional group of the general structure D: ##STR00013## wherein R.sub.1-R.sub.12 independently comprise a substituted or unsubstituted C1-C10 alkyl-, aryl-, aldehyde-, carboxylic acid-, ester-, thiol-, phosphonate-, phosphinyl-, sulfonate-, boron-, or amine-based moiety, H or halogen, or two or more R groups together form part of a heterocyclic ring structure comprising one or more substituted or unsubstituted rings; wherein the substituents are selected from the group consisting of OH, halogen, NH.sub.3, oxo, C.sub.1-C.sub.6 alkyl, and phenyl; wherein at least one of R.sub.3-R.sub.7 is NO; and wherein R.sub.8-R.sub.12 are each optional, but when present result in the N atom to which they are bound becoming positively charged.

2. The medical article of claim 1, wherein only R.sub.6, R.sub.7, or both R.sub.6 and R.sub.7 are NO.

3. The medical article according to claim 1, selected from the group consisting of a stent, catheter, wound dressing, bandage, self-adhesive plaster and patch.

4. The medical article according to claim 1, wherein the NO complexed compound is formed from a precursor biologically active agent.

5. The medical article of claim 4, wherein the biologically active agent is an antibiotic, a biocidal agent, a fungicidal agent, or a sporicidal agent.

6. The medical article of claim 4, wherein the biologically active agent is selected from the group consisting of ciprofloxacin, a biguanide, and a complex or salt thereof, such that the medical article comprises a NO complexed quinolone compound or porous framework material or MOF comprising an extra framework NO complexed quinolone compound.

7. The medical article of claim 4, wherein the biologically active agent is selected from the group consisting of an NO complexed biguanide compound or a complex or salt thereof, a sulfonamide or a complex or salt thereof, a porous framework material, and an MOF comprising an extra framework NO complexed biguanide compound.

8. The medical article of claim 4, comprising a further biologically active agent, as a guest species within the pores and/or channels of the porous framework material or MOF.

9. The medical article of claim 8, wherein the further biologically active agent is NO.

10. The medical article of claim 9, wherein the further biologically active agent is irreversibly or releasably adsorbed NO.

Description

DESCRIPTION OF THE DRAWINGS

(1) Non-limiting example embodiments will now be described with reference to the following figures in which:

(2) FIG. 1A and FIG. 1B show UV-visible spectra of chlorhexidine dichloride and chlorhexidine diacetate before and after NO loading;

(3) FIG. 2A and FIG. 2B show adsorption (▬.square-solid.▬) and desorption (▬□▬) isotherms of NO at 298K (measured using gravimetric analysis) for each of two samples of chlorhexidine diacetate plotted as (FIG. 2A) nitric oxide concentration (mmol/g) and (FIG. 2B) molecules of nitric oxide per molecule of chlorhexidine;

(4) FIG. 3A and FIG. 3B show chemiluminescence analysis of nitric oxide delivery from chlorhexidine diacetate on contact with humid atmosphere (11% RH). Plotted data of concentration of NO over time (FIG. 3A) and total NO release over time (FIG. 3B);

(5) FIG. 4A and FIG. 4B show chemiluminescence analysis of total Nitric Oxide delivery from chlorhexidine diacetate, in contact with humid atmosphere (11% RH), triggered by UV light. The sample was kept in a vial for 48 hours at room temperature and expose to air prior to irradiation;

(6) FIG. 5A and FIG. 5B show chemiluminescence analysis of nitric oxide delivery from chlorhexidine NO complex in water (100% RH) (FIG. 5A) and triggered by UV light (FIG. 5B);

(7) FIG. 6 shows chemiluminescence analysis of Nitric Oxide delivery from chlorhexidine dichloride, in contact with humid atmosphere (11% RH), triggered by UV light;

(8) FIG. 7A and FIG. 7B show chemiluminescence analysis of total nitric oxide delivery from chlorhexidine diacetate silver metal complex, in contact with humid atmosphere (11% RH), triggered by UV light;

(9) FIG. 8A and FIG. 8B show total NO release (measured using chemiluminescence analysis) on contact with humid atmosphere (11% RH), plotted as (FIG. 8A) mmole per gram and (FIG. 8B) molecules of NO per molecule of chlorhexidine (CHX) over time, from polymer cast chlorhexidine loaded with NO at 4 bar (blue), and of polymer cast chlorhexidine loaded with NO at 1 bar (red);

(10) FIG. 9A and FIG. 9B show chemiluminescence analysis of nitric oxide delivery from (FIG. 9A) polymer cast chlorhexidine loaded with NO at 4 bar and (FIG. 9B) polymer casted chlorhexidine loaded with NO at 1 bar, each in contact with humid atmosphere (11% RH), triggered by UV light;

(11) FIG. 10 shows chemiluminescence analysis of nitric oxide delivery triggered by UV light from polymer cast chlorhexidine loaded with NO at 4 bar (upper, blue plot) and at 1 bar (lower, red plot) in contact with humid atmosphere (11% RH);

(12) FIG. 11A and FIG. 11B show chemiluminescence analysis of Nitric Oxide delivery from ciprofloxacin, in contact with humid atmosphere (11% RH), triggered by UV light;

(13) FIG. 12A and FIG. 12B show FT-IR spectra of (FIG. 12A) chlorhexidine diacetate before (blue) and after (red) exposure to NO and of (FIG. 12B) ciprofloxacin before (blue) and after (red) exposure to NO;

(14) FIG. 13A and FIG. 13B show total NO release (measured using chemiluminescence analysis) from furosemide on contact with humid atmosphere (11% RH) (plotted as mmole per gram);

(15) FIG. 14A and FIG. 14B show chemiluminescence analysis of nitric oxide release from furosemide in contact with humid atmosphere (11% RH), triggered by UV light;

(16) FIGS. 15A and 15B show chemiluminescence analysis of total nitric oxide delivery from CPO 27 Mg on contact with humid atmosphere (11% RH) FIG. 15A. NO release from CPO 27 Mg, in contact with humid atmosphere (11% RH), triggered by UV light FIG. 15B. The sample was stored on the bench at room temperature exposed to air and humidity for over 68 hours in between the two analyses;

(17) FIG. 16A and FIG. 16B show chemiluminescence analysis of total nitric oxide delivery from CPO 27 Ni on contact with humid atmosphere (11% RH) FIG. 16A. NO release from CPO 27 Ni, in contact with humid atmosphere (11% RH), triggered by UV light FIG. 16B. The sample was stored on the bench at room temperature exposed to air and humidity for over 48 hours in between the two analyses;

(18) FIG. 17A1 and FIG. 17B shows chemiluminescence analysis of total nitric oxide delivery from HKUST-1 on contact with humid atmosphere (11% RH) FIG. 17A. NO release from HKUST-1, in contact with humid atmosphere (11% RH), triggered by UV light FIG. 17B. The sample was stored on the bench at room temperature exposed to air and humidity for over 64 hours in between the two analyses;

(19) FIG. 18A and FIG. 18B shows (FIG. 18A) FTIR analysis of chlorhexidine, CPO-27 Mg and chlorhexidine loaded CPO-27 Mg and (FIG. 18B) TGA analysis of chlorhexidine, CPO-27 Mg and chlorhexidine loaded CPO-27 Mg;

(20) FIG. 19 shows chlorhexidine release from CPO 27 Ni and CPO 27 Mg;

(21) FIG. 20A and FIG. 20B show chemiluminescence analysis of total nitric oxide delivery from chlorhexidine loaded CPO 27 Mg on contact with humid atmosphere (11% RH): concentration of NO over time (FIG. 20A) and total NO release over time (FIG. 20B);

(22) FIG. 21 shows total NO release over time of chlorhexidine acetate, CPO 27 Mg and chlorhexidine-loaded CPO 27 Mg;

(23) FIG. 22A and FIG. 22B show chemiluminescence analysis of nitric oxide delivery from chlorhexidine-loaded CPO 27 Mg, in contact with humid atmosphere (11% RH), triggered by UV light. The sample was stored on the bench at room temperature exposed to air and humidity for over 50 hours in between the two analyses; and

(24) FIG. 23A shows chlorhexidine release from CPO 27 Ni cast in a polyurethane film.

(25) FIG. 23B chemiluminescence analysis of total nitric oxide delivery from chlorhexidine-loaded CPO 27 Ni cast in a polyurethane film, triggered by humid atmosphere (11% RH).

DETAILED DESCRIPTION OF EXAMPLE EMBODIMENTS

(26) Preparation and release of NO from chlorhexidine NONOate and/or N-nitroso compounds

(27) Chlorhexidine is reported on the World Health Organization's List of Essential Medicines. This pharmaceutical product is widely used in disinfectants (for external use on skin and hands) and topical use (preservative in eye drops, active substance in wound dressings and antiseptic mouthwashes). Furthermore this biomolecule can also be found in cosmetics (additive to creams, toothpaste and deodorants). This drug is primarily sold as salts (dihydrochloride, diacetate and digluconate). Recently, different chlorhexidine-metal complexes have been reported; the drug binds to specific metals (like copper and silver) providing a system for the controlled release of chlorhexidine, while maintaining the drug performance [1].

(28) Chlorhexidine contains primary and secondary amine groups. The inventors have found that these amine groups are capable of binding NO when exposed to nitric oxide gas under high pressure.

(29) In addition, the release of NO from chlorhexidine may be triggered by ultra-violet light (UV) or exposure to humidity. Indeed, NO release can be triggered by either or both of these external stimuli. As detailed below, after an initial burst caused by exposure to humidity, the NO release can be repeatedly triggered and stopped by switching the source of UV light on and off.

(30) Advantageously and unexpectedly, the chlorhexidine NONOate and/or N-nitroso compounds have been found to be stable in air, meaning no special storage conditions are necessary.

(31) Light-controlled release is possible from a number of different chlorhexidine salts. The combination of chlorhexidine and NO has a synergistic effect, which reduces the risk of potential bacterial resistance and can be useful in combatting already resistant strains of microbes.

(32) A benefit of a chlorhexidine-NONOate/N-nitroso compound is that the chlorhexidine precursor which is regenerated after NO release is a well understood and beneficially biologically active agent. Moreover, appropriate dosages, side effects and toxicity are well understood.

(33) (1) Formation of and NO Release from Chlorhexidine-NONOate and/or N-Nitroso and Complexes and M-Chlorhexidine-NONOate and/or N-Nitroso Salts

(34) Chlorhexidine, its salts and complexes (precursor compounds) may be converted to their NONOate and/or N-nitroso compound using the high temperature dehydration and NO loading technique previously reported by Morris [2], in relation to MOF and zeolite materials.

(35) The chlorhexidine-NONOate and/or N-nitroso compounds may also be prepared generally as outlined by Lowe at al. [17] in which the material is subjected to high vacuum at approximately room temperature before being exposed to NO atmosphere.

(36) These techniques have previously only be considered for use in loading MOF and other molecular sieve materials with NO, so that the NO adsorbs to framework ions or ligands. Such methods have not previously been applied to “free” NONOate and/or N-nitroso precursor compounds.

(37) Any chlorhexidine salt can be employed as a starting material, as demonstrated in relation to chlorhexidine diacetate, chlorhexidine dihydrochloride and chlorhexidine digluconate.

(38) The identity of the chlorhexidine precursor may be selected in for a preferred NO release profile. The inventors have observed that the release profile on exposure of the NONOate and/or N-nitroso compound to humidity is particularly sensitive to the particular precursor which has been selected.

(39) For example, if a large initial “burst” of NO on contact with humidity or moisture is desired, then chlorhexidine dihydrochloride salt may be appropriate, for example. Whereas, the chlorhexidine diacetate salt has a more gradual release profile on exposure to humidity/moisture.

(40) The formation of NONOates and/or N-nitroso compounds has also been demonstrated for metal-chlorhexidine complexes. M-chlorhexidine NONOate and/or N-nitroso complexes have been formed by solvothermal/hydrothermal synthesis and mechanochemical synthesis. A preferred method is generally via the low temperature process reported by Morris at al. [23]. Again, this low temperature process has only previously been used to prepare MOF materials.

(41) The metal employed can be any metal but preferably those with antimicrobial properties such as Ag, Ni, Zn and Cu. These metals themselves have biological (e.g. antibacterial) activity and provide the NONOate and/or N-nitroso salt/complex with a still further mode of action (in addition to the activity and/or release profile of the NO and the chlorhexidine anion).

(42) In addition to release of NO by exposure to humid air, moisture or UV-light, NO release may also be initiated or stimulated by heating the chlorhexidine NONOate compound.

(43) It is a particular feature of these materials that a combined release trigger can be used to afford an initial burst followed by sustained release.

Example 1—Chlorhexidine Diacetate and Dihydrochloride NO Loading

(44) A sample of 50 mg of chlorhexidine diacetate hydrate and chlorhexidine dihydrochloride were exposed to high vacuum (10.sup.−4 Torr) for 1 hour at room temperature. Using a schlenk line, 4 atmospheres of NO gas were introduced into the schlenk tube over 2 hours allowing the dehydrated chlorhexidine to adsorb the radical gas. The samples were then exposed to vacuum and flushed with argon for 30 minutes. The glass vials containing the samples were then sealed.

(45) FIG. 1 and FIG. 1B show the detected UV Vis-spectra of the pure chlorhexidine compared to the NO containing complex. The exposure of the drug to the nitric oxide gas changes the colour of the material from white to pale yellow. The UV data indicates a change in the absorbance band at −370 cm.sup.−1 for both the chlorhexidine dihydrochloride and diacetate NO complexes. This is consistent with literature reports for other NO containing materials[16].

(46) The nitric oxide adsorption/desorption profiles for two further chlorhexidine diacetate samples (25 mg) were collected using a bespoke gravimetric adsorption system. Each sample was exposed to high vacuum at a pressure of 1×10.sup.−4 mbar overnight until no further mass loss was observed. The samples were cooled to 298K using a water bath (temperature accuracy of 0.02K).

(47) For one sample, shown in the outer plot of FIGS. 2A-2B, NO was introduced in increasing increments. After each dose of NO, the mass of the sample was allowed to stabilise (indicating completion of adsorption) before the next addition was made. This process was continued until the introduced pressure of NO was equal to atmospheric pressure. The second sample was exposed to one atmosphere of NO in a single step, and its mass allowed to equilibrate. Data is shown in the inner plot of FIGS. 2A-2B. The desorption profiles of both samples, shown in FIGS. 2A-2B, were measured by reducing the pressure in increments to a final value of 2×10.sup.−2 mbar.

(48) The gravimetric analyses show that a maximum of ˜0.9 mmole of NO per gram are adsorbed (FIG. 2A) which equates to 0.55 molecules of NO per molecule of chlorhexidine (FIG. 2B).

(49) The shape of the adsorption curve shows a dependence between the applied pressure of NO and the quantity of NO bonding to the molecule. The pressure of NO normally used for the loading on the schlenk line is 4 times the level obtainable during the gravimetric isotherm analysis, so we would expect an even higher quantity of radical gas coordinating to chlorhexidine. Through reapplication of vacuum the NO levels reduce progressively, and both samples reach a level of stored NO of ˜0.4 mmole per gram, circa 0.25 molecules per molecule of chlorhexidine. These data indicate that a significant proportion of the NO initially stored has been adsorbed by the chlorhexidine precursor.

(50) The release of NO from the sample was first triggered by passing a constant flow of humid nitrogen gas (11% RH) over it. The amount of NO released over time was detected using a Sievers NOA 280i chemiluminescence nitric oxide analyser until the emission of NO reached a level lower than 20 ppb.

(51) The initial burst of release of NO reached 512 PPM (FIG. 3). The sample released up to 0.042 mmol/g of nitric oxide in 19 hr.

(52) After the NO release was completed the sample was kept on the bench at room temperature, exposed to air and humidity for over 48 hr. The sample was exposed to UV light from two Ritek Electronics UV tube lamps each containing 4×15 W bulbs with an emission of 300-400 nm and total power of 50-200 W. These parameters should not be viewed as limiting with regards to the invention. The light triggered NO release that immediately burst from 30 to 105 ppb. A maximum of around 120 ppb was reached on continuous exposure for about 5 mins.

(53) The emission immediately stopped when the source of UV light was switched off as shown in FIG. 4A. This on-off process can be repeated and controlled over time. Continuous exposure to UV light triggers a release of nitric oxide over 70 ppb for more than 4 hrs, as shown in FIG. 4B. The release of NO over time was recorded using the same analyser described above.

Example 2—NO Release from Chlorhexidine NO Complex Suspended in Water

(54) A 100 mg sample of chlorhexidine diacetate was exposed to high vacuum (10.sup.−4 Torr) for 12 hours at room temperature. Using a schlenk line, 4 atmospheres of NO gas were introduced into the schlenk tube and maintained for 2 hours allowing the dehydrated chlorhexidine to adsorb the gas. The sample was then exposed to vacuum and flushed with argon for 30 minutes. The glass vials containing the sample were then sealed.

(55) The NO-loaded sample was submersed under 5 ml of deionized water in a sealed chamber connected to an NO analyser. A constant flow of nitrogen was bubbled through the suspension while measuring the concentration of NO present in the chamber atmosphere.

(56) The release of NO from the sample triggered by the water was measured in ppm and ppb over time until the level of NO dropped below 20 ppb. The initial burst release of NO reached 40 ppm. The sample released up to 0.035 mmol/g of nitric oxide in 7 hrs (FIG. 5A).

(57) Immediately after the NO release was completed the sample was exposed to UV light, which triggered further release of NO. A maximum of around 1000 ppb was recorded during continuous exposure for about 10 minutes. The emission immediately stopped when the source of UV light was switched off as shown in FIG. 5B. This on-off process can be repeated and controlled over time.

Example 3—Chlorhexidine Dihydrochloride NO Loading and Release

(58) The same general process described above was followed using chlorhexidine dihydrochloride as starting material. An initial burst release of NO was obtained on exposing the sample to a constant flow of humid nitrogen gas (11% RH). The material released a small amount of gas for a couple of minutes and then stopped. After storing the sample on the bench, exposed to humid air for 2 days, an additional release of NO was triggered using UV light. Chlorhexidine dihydrochloride releases a burst of NO up to 150 ppb dropping to 75 ppb over 1 hr. This trigger mechanism can be repeated and controlled over time as shown in FIG. 6.

Example 4—NO Loading and Release from a Silver-Chlorhexidine Complex

(59) Following the procedure of Song a sample of silver-chlorhexidine was prepared using silver nitrate and chlorhexidine diacetate. After characterisation (XRD, UV Vis, SEM and EDX) 50 mg of the sample were loaded with NO following the high pressure procedure previously reported [17].

(60) The initial burst release of NO, triggered by exposing the sample to humidity, lasted for a couple of minutes. The sample was stored exposed to humid air for over 60 hrs before an additional release of nitric oxide was triggered using UV light as shown in FIG. 7.

(61) The silver chlorhexidine complex released a burst of NO (up to 175 ppb) that slowly dropped to 100 ppb over the course of an hour. As in the cases above, the release of NO stopped abruptly when the UV light was switched off. This trigger mechanism can be repeated and controlled over time as shown in FIG. 7A. The NO release can be switched on and off multiple times even after 85 hrs as shown in FIG. 7B.

Example 5—NO Release from Polymer Film Containing NO-Complexed Chlorhexidine Diacetate

(62) A polyurethane polymer was chosen as a casting material as it is commonly used in medical devices. A sample of chlorhexidine diacetate (1.5 g) was dispersed in a pre-dissolved mixture of polyurethane (3 g) and THF (40 ml). The mixture was solvent cast using doctor blade techniques, to produce a ˜100 μm thick film, which was set by evaporation of the solvent.

(63) Samples of the polymer film were exposed to vacuum overnight and NO loaded using two different pressures (1 bar and 4 bar) of nitric oxide.

(64) FIG. 8 shows NO release profiles (chemiluminescence analysis), using humid nitrogen only (humidity controlled 11% RH), of 100 mg samples of the NO-loaded chlorhexidine-containing films, loaded at 4 bar and 1 bar. The data show that the sample loaded at 4 bar releases up to 0.1 mmole per gram of nitric oxide within a 30 hour time period (FIG. 8A), which equates to 0.06 molecules of NO per molecule of chlorhexidine (FIG. 8B).

(65) It has also been found that polymer-cast chlorhexidine loaded with NO at 1 bar does not release any NO under analogous conditions (as also shown FIG. 8).

Example 6—UV Triggered NO Release from Polymer-Cast NO-Complexed Chlorhexidine Diacetate

(66) After the initial NO release was completed by exposing the samples to humid nitrogen, the samples were stored on the bench at room temperature, exposed to air and humidity for over 48 hrs. Both samples were then exposed to UV light in a flow of humid nitrogen gas (humidity controlled 11% RH).

(67) This triggered additional release of NO from the samples, including the film that had been loaded with NO at 1 bar, in direct contrast to its performance in solely humid nitrogen. A maximum of around 180 ppb was reached on continuous exposure for about 10 minutes from this sample.

(68) The emission immediately stopped when the source of UV light was switched off as shown in FIG. 9A and FIG. 9B. This on-off process can be repeated and controlled over time. Continuous exposure to UV light triggered a release of nitric oxide over 20 ppb for more than 14 hrs, as shown in FIG. 9A and FIG. 9B.

(69) The chlorhexidine-containing polymer sample that had been loaded with NO at 4 bar released almost twice the amount obtained from the 1 bar counterpart as shown in FIG. 10. The pressure of NO used during the gas-loading process therefore significantly influences the final release performance obtained from the sample, using only water and/or UV-light as a trigger. A maximum of around 300 ppb was reached from this sample on continuous exposure for about 10 minutes as shown in FIG. 9B. The emission immediately stopped when the source of UV light was switched off. This on-off process can be repeated and controlled over time.

(70) (2) Formation of and NO Release from NO-Complexed Ciprofloxacin Compound

(71) It has been found that the process described above can be used on different drugs containing secondary amines in their structure, such as ciprofloxacin. Ciprofloxacin is an antibiotic useful for the treatment of different bacterial infections.

Example 7

(72) 50 mg of ciprofloxacin was NO loaded following the high pressure method reported above. A small initial burst of nitric oxide, lasting a couple of minutes, was obtained after exposure to a constant flow of humid nitrogen gas (11% RH). However, even after storing the sample on the bench exposed to humid air for 24 hrs, an additional release of NO was triggered using UV light. Ciprofloxacin released a burst of NO up to 500 ppb dropping to around 100 ppb over 1 hr. The mechanism can be repeated over time and controlled as shown in FIG. 11A. After keeping the sample exposed to humid air for over 50 hrs additional NO was still released using UV light as a trigger as shown in FIG. 11B. The burst of NO release reached 320 ppb dropping to 50 ppb over 1.5 hrs.

(73) FT-Ir-Analyses

(74) Evidence for the attachment of NO to the ciprofloxacin molecule is provided by the appearance of new stretching frequencies in the FT-IR spectra for both samples after exposure to NO (FIG. 12); for example, the stretching frequencies at 1040-1043 cm.sup.−1 (N—O) (FIG. 12A), 1310-1320 cm.sup.−1 (N—O) (FIGS. 12A-12B) and 1550-1500 cm.sup.−1 (N—O) (FIGS. 12A-12B). There is also an additional stretching band; 2270-2275 cm.sup.−1 present in all NO-modified compounds, which is most likely due to an N—N stretch.

(75) There is also a small stretch above 1700 cm.sup.−1 present in each of the NO complexed compound samples. Although the origin of this stretch is not fully understood, it has been found to be present in other literature-reported spectra of NO-containing compounds (see for example J. G. Nguyen, Kristine K. Tanabe and S. M. Cohen Cryst. Eng. Comm, 2010, 12, 2335-2338). Furthermore, FIG. 11B also shows the disappearance of the NH stretch at 3300 cm.sup.−1 when ciprofloxacin is exposed to NO.

(76) (3) Furosemide

(77) Furosemide is a loop diuretic used in the treatment of congestive heart failure and edema. Along with some other diuretics, furosemide is also included on the World Anti-Doping Agency's banned drug list due to its alleged use as a masking agent for other drugs. It is also on the World Health Organization's List of Essential Medicines, a list of the most important medication needed in a basic health system.

(78) Furosemide is primarily used for the treatment of hypertension and edema. It is the first-line agent for most people with edema caused by congestive heart failure. It is also used for hepatic cirrhosis, renal impairment, nephrotic syndrome, and in the management of severe hyperkalemia in combination with adequate rehydration

Example 8—Furosemide NO Loading and Release

(79) 25 mg of furosemide was exposed to high vacuum (10.sup.−4 Torr) for 1 hour at room temperature. Using a schlenk-line, 4 atmospheres of NO gas were introduced into the schlenk tube and maintained for 2 hours allowing the dehydrated furosemide to adsorb the gas. The sample was then exposed to vacuum and flushed with argon for 30 minutes. The glass vials containing the samples were then sealed.

(80) The release of NO was first triggered by passing a constant flow of humid nitrogen gas (11% RH) from the sample. The amount of NO released over time was detected in ppm and ppb until the emission of NO dropped below 20 ppb.

(81) The initial burst of release of NO reached 512 ppm (see FIG. 13A and FIG. 13B). The sample released up to 0.042 mmol/g of nitric oxide in 19 hrs.

Example 9—UV Triggered NO Release from Furosemide NO Complex

(82) After the initial NO release by humid nitrogen was completed the sample was stored on the bench at room temperature, exposed to air and humidity for over 24 hrs. The sample was then exposed to UV light in a flow of humid nitrogen gas (humidity controlled 11% RH).

(83) A maximum of around 55 ppb was reached on continuous exposure for about 10 minutes from the furosemide NONOate sample. The emission immediately stopped when the source of UV light was switched off as shown in FIG. 14A and FIG. 14B.

(84) This on-off process can be repeated and controlled over time. Continuous exposure to UV light triggered a release of nitric oxide over 20 ppb for more than 7 hrs.

(85) (4) Light Triggered NO Release from MOFs

(86) UV light-triggered NO release from MOFs has been demonstrated for CPO-27 and HKUST-1 type structures amongst others (in particular other MOFs having coordinatively unsaturated framework metal sites). However, the technique can be applied to any MOF that shows affinity for NO.

(87) The MOFs were prepared following the method previously reported by Morris [23]. The activation and NO loading was carried out in accordance with the high temperature dehydration method previously reported by Morris [2,8]. However, NO-loading may be performed by any suitable method, for example as described by Lowe [17], in which the material is subject to a vacuum at room temperature before being exposed to a high pressure of nitric oxide.

(88) The MOF may be selected for a desired NO release profile. For example Mg and Ni-CPO-27 tend to release higher quantities of NO than HKUST-1.

(89) Release of the adsorbed NO is triggered by exposing the material to UV light. Alternatively, or in addition, NO release can also be achieved on exposure to humid air and/or heat.

(90) For example, in some case an initial NO burst can be triggered by contact with moisture and, once the release of nitric oxide dissipated, UV light can be used to selectively trigger the release of additional NO by switching the UV light source on and off.

(91) In this particular method may provide for release of a greater amount or proportion of the stored NO than has been previously possible. Although not wishing to be bound by theory, this may be a consequence of the UV light triggering the release of more strongly bonded (high energy) NO, which would not ordinarily be released by being displaced by water, or under thermal conditions conventionally applied. The UV triggered release of NO has particular use with MOFs that show poor NO release when exposed exclusively to humidity (eg. CPO-27 Mg and HKUST-1). Such materials are known to have relatively high NO storage capabilities, which it has not previously been possible to readily release.

Example 10—CPO-27 Mg

(92) A sample of 50 mg CPO-27 Mg prepared following the procedure reported by Morris [23] was exposed to high vacuum (10.sup.−4 Torr) for 1 hr at room temperature. The sample was then exposed to 4 atm of NO gas for 2 hr before being evacuated and flushed with argon for 30 minutes and sealed in glass vials.

(93) Total NO release: The sample was exposed to a constant flow of humid nitrogen gas (11% RH) and the NO released was monitored over time. The analysis was carried out until the NO gas levels detected were lower than 20 ppb. CPO-27 Mg only released up to 0.05 mmol/g over 25 hrs as shown in FIG. 15A. After the NO release was finished the sample was kept on the bench at room temperature exposed to air and humidity of over 68 hours. Subsequent exposure to UV light resulted in further release of nitric oxide. The light triggered a slow burst of NO release from 80 to over 500 ppb. The emission immediately stopped when the source of UV light was switched off. The process can be repeated as shown in FIG. 15B.

Example 11—CPO-27 Ni

(94) A sample of 50 mg of CPO-27 Ni prepared following the procedure reported by Morris [23] was activated and NO loaded following the same high pressure technique described above.

(95) Total NO release—After exposing the sample to a constant flow of humid nitrogen gas (11% RH) CPO-27 Ni released a total of 2.8 mmol/g of NO over 40 hrs as shown in FIG. 16A. The sample was kept on the bench at room temperature exposed to air and humidity for over 2 days. Further NO release was then triggered by UV light, as shown in FIG. 16B, giving a burst release up to 85 ppb and sustained release over the duration of irradiation. The release of nitric oxide from the framework can be repeatedly triggered by switching the UV light on and off.

Example 12—HKUST-1

(96) A sample of 50 mg of HKUST-1 prepared following the procedure reported by Morris [23] was activated and NO loaded following the same high pressure technique described above.

(97) Total NO release—An initial release of NO was obtained by exposing the sample to a constant flow of humid nitrogen gas (11% RH). The framework released up to 0.2 mmol/g over 7 hrs as shown in FIG. 17A. After storing the sample on the bench, exposed to humid air for 64 hours, an additional release of NO was triggered using UV light. HKUST-1 released a burst of NO up to 65 ppb with a plateau of 60 ppb for over 1 hr. This trigger mechanism can be repeated and controlled over time (FIG. 17B).

(98) (5) Chlorhexidine-Loaded MOFs

(99) Chlorhexidine and NO-complexed chlorhexidine have been successfully incorporated into and released from MOFs. Moreover, the MOFs have been demonstrated to be capable of releasing NO over time with exposure to UV light and/or a combination of humidity and light.

(100) Chlorhexidine and NO loaded MOFs may be prepared in accordance with the methods of Morris [23] or Lowe [17].

(101) Where the MOF is first loaded with a precursor compound such as a chlorhexidine compound, the exposure of the MOF to nitric oxide may have a double effect; the NO gas is bonded to the MOF and also to the chlorhexidine compound, so as to form a NO and chlorhexidine-NO complex loaded MOF.

(102) NO may be released from the chlorhexidine-NO complex loaded MOF by commonly employed methods including but not limited to exposure to humid air, heat or UV light.

(103) Light triggered release of NO is analogous to that reported above for the NO complexes themselves and for the NO-loaded MOFs. The presence of two different types of NO binding sites may provide for in an increase in the total NO release.

Example 13—Loading of Chlorhexidine into CPO-27 Mg and CPO-27 Ni

(104) A sample of 100 mg of MOF (CPO-27 Mg or CPO-27 Ni) was mixed with 100 mg of chlorhexidine diacetate. The mixture was dehydrated in an oven at 110° C. overnight. The sample vial was then sealed and cooled to room temperature before anhydrous ethanol (100 ml) was introduced through a rubber septum. After 4 days, the suspension was then filtered and washed with ethanol. FT-IR and TGA analysis confirm the presence of chlorhexidine in the framework as shown in FIG. 18A and FIG. 18B.

Example 14—Release of Chlorhexidine from CPO-27 Mg and CPO-27 Ni

(105) 50 mg of drug-loaded MOF (CPO-27 Mg or CPO-27 Ni) was suspended in 50 ml of methanol. The solution was sampled over time and the concentration of chlorhexidine was detected using UV Vis. FIG. 19 shows the effective release of the drug from the frameworks over time. The drug-loaded CPO-27 Ni shows a burst release reaching 1.3 μg/ml over the first 40 hrs, whereas CPO-27 Mg offers a higher affinity and higher potential release of chlorhexidine over time reaching a plateau of 2.5 μg/ml over 80 hrs.

Example 15—NO Loading and Release from Drug-Loaded CPO-27 Mg

(106) A sample of 50 mg of drug-loaded CPO-27 Mg was activated and NO loaded following the high pressure procedure reported above at room temperature. The sample was then exposed to a constant flow of humid nitrogen (11% RH). The quantity of chlorhexidine CPO-27 complex used has a burst release that peaks at 512 PPM. The drug loaded MOF achieves a total NO release of 0.15 mmol/g over 45 hrs as shown in FIG. 20A and FIG. 20B.

(107) Comparison of total NO release from the pure chlorhexidine NO complex, the pure MOF and drug-loaded MOF shows the advantage of the bifunctional material over the two separate moieties, as shown in FIG. 21. The total NO release from NO-complexed chlorhexidine e reaches a maximum of 0.03 mmol/g in 18 hrs and the total NO release from CPO-27 Mg reaches 0.05 mmol/g in 25 hrs. However, the chlorhexidine CPO-27 Mg complex achieves a total NO release of 0.15 mmol/g over 40 hrs due to the combined effect of both moieties.

(108) The sample of chlorhexidine CPO-27 Mg was kept on the bench at room temperature exposed to air and humidity for over 40 hrs. An additional quantity of NO release was then triggered using UV light, as shown in FIG. 22A. The data shows that the NO dose can be tuned by manipulating the length of exposure to UV-light, over at least 2 hrs (FIG. 22B). The light triggered mechanism repeatedly controls the release of NO gas over time. After initial exposure to UV light, the sample was stored again at room temperature, open to air and humidity for 50 hrs. Further exposure of the sample to UV light triggered an additional release of the gas as shown in FIG. 22B. The NO release from the studied quantity of material reached a plateau of 0.5 PPM over 1 hr.

(109) (6) Incorporation of NO Complexed Materials into Matrices for Applications

(110) Each of the above materials, including drug NONOates and/or N-nitroso compounds (chlorhexidine salts and ciprofloxacin), MOFs, and drug loaded MOFs can be incorporated into different matrices. These matrices include but are not restricted to resins and binders (such as those used in paints, inks and coatings for example), creams, ointments, polymers, ceramics and glasses, particularly those employed in healthcare and medical applications (e.g. devices, dressings and topical treatments), or where antiseptic/antimicrobial performance is required (e.g. coatings on surfaces).

(111) The materials can be introduced into these matrices by any appropriate means such as, but not limited to, milling, high speed/sheer mixing, extrusion, electrospinning, casting and moulding. The materials may be employed in a coating on, for example, textiles, plastic, metal, wooden and glass surfaces. This could be achieved by any appropriate means, for example dispersing the material in a resin to be applied by painting, dip coating, spray coating, printing etc. Powder coating can also be employed where appropriate. Additional agents such as dispersion and rheology modifiers may be employed as appropriate and as necessary to aid formulation.

Example 16—Chlorhexidine Release from Polymer Containing Chlorhexidine-Loaded CPO-27 Ni

(112) A polyurethane polymer was chosen as a casting material as it is commonly used in catheters. A sample of CPO-27 Ni was drug loaded following the procedure previously reported by Morris et al [23]. The drug loaded MOF was suspended in THF using a high sheer homogeniser and dispersed in predissolved polyurethane. The mixture was solvent cast using doctor blade techniques to produce a ˜100 μm thick film.

(113) The films were suspended in an appropriated volume of methanol. The solution was sampled over time and the concentration of chlorhexidine was detected using UV spectroscopy. FIG. 23A shows the drug release from the loaded polymer. The release reached a maximum of 0.12 μg/ml in 70 hrs.

Example 17—NO Release from Polyurethane Containing Chlorhexidine NO Complexloaded CPO-27 Ni

(114) MOF-loaded films (prepared as outlined above) were dehydrated and NO loaded following the previously reported procedure. FIG. 23B shows the total NO release from chlorhexidine loaded CPO-27 Ni cast in a polyurethane polymer when exposed to humid air. The sample delivered a maximum of 0.25 mmol/g after 8 hrs.

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