STERILISATION OF S-NITROSOTHIOLS

Abstract

This invention provides a method of sterilising an S-nitrosothiol, for example S-nitrosoglutathione, without reduction of purity by more than about 5.0% through degradation. The invention allows sterile S-nitrosothiol or a sterile pharmaceutical pre-composition comprising S-nitrosothiol, wherein the S-nitrosothiol is in dry solid form, to be produced. The sterile pharmaceutical pre-composition is mixed with one or more diluents, excipients, carriers, additional active agents, or any combination thereof, for example sterile saline, to prepare a pharmaceutical composition of S-nitrosothiol for use.

Claims

1. A sterile S-nitrosothiol or a sterile pharmaceutical pre-composition comprising S-nitrosothiol, wherein the S-nitrosothiol is in dry solid form and the sterile S-nitrosothiol or sterile pharmaceutical pre-composition has an S-nitrosothiol purity of at least about 95% and a sterility assurance level (SAL) of 10.sup.6 or lower.

2. The sterile S-nitrosothiol or sterile pharmaceutical pre-composition comprising S-nitrosothiol of claim 1, wherein the sterile S-nitrosothiol or the sterile pharmaceutical pre-composition comprising S-nitrosothiol is in a dry sterile environment in a sealed container.

3. The sterile S-nitrosothiol or sterile pharmaceutical pre-composition comprising S-nitrosothiol of claim 2, wherein the seal is arranged so that the container can be opened, for example pierced, under sterile conditions to allow the sterile S-nitrosothiol or a sterile pharmaceutical pre-composition comprising S-nitrosothiol to be formulated with other desired ingredients to make a final pharmaceutical composition for administration.

4. The sterile S-nitrosothiol or sterile pharmaceutical pre-composition comprising S-nitrosothiol of claim 2, wherein the seal is arranged so that a sterile liquid can be introduced into the container, for example using a syringe provided with a hollow needle, to dissolve or suspend the sterile dry solid S-nitrosothiol or the sterile pharmaceutical pre-composition in situ within the container.

5. The sterile S-nitrosothiol or sterile pharmaceutical pre-composition comprising S-nitrosothiol of claim 1, wherein the said material has been sterilised by exposure to a sterilising dose of ionising radiation in an environment sealed from external contamination.

6. The sterile S-nitrosothiol or sterile pharmaceutical pre-composition comprising S-nitrosothiol of claim 2, wherein the said material has been sterilised by exposure to a sterilising dose of ionising radiation in the said sealed container.

7. The sterile S-nitrosothiol or sterile pharmaceutical pre-composition comprising S-nitrosothiol of claim 1, wherein the S-nitrosothiol has an S-nitrosothiol purity of at least about 98.0%, for example at least about 98.5%.

8. The sterile S-nitrosothiol or sterile pharmaceutical pre-composition comprising S-nitrosothiol of claim 1, wherein the amount of the S-nitrosothiol or the pharmaceutical pre-composition comprising S-nitrosothiol contains a known weight of S-nitrosothiol selected according to an ultimate desired medical use.

9. The sterile S-nitrosothiol or sterile pharmaceutical pre-composition comprising S-nitrosothiol of claim 1, wherein the S-nitrosothiol is selected from: S-nitrosoglutathione (GSNO); S-nitroso-L-cysteine (CySNO); S-nitroso-N-acetyl-cysteine (SNAC); S-nitroso-L-cysteinemethyl-ester (CMESNO); S-nitroso-D,L-penicillamine (PSNO); S-nitroso-N-acetyl-D,L-penicillamine (SNAP); S-nitroso-N-acetylcysteamine (ACSNO); S-nitroso-beta-mercaptosuccinic acid; 1-S-nitrosothio-beta-D-galactopyranose; S-nitrosothioglycerol; S-nitrosohomocysteine; S-nitrosocysteinylglycine; S-nitrosocaptopril; alkyl, cycloalkyl or aryl thionitrites, such as, for example, methyl thionitrite, ethyl thionitrite, n-propyl thionitrite, s-propyl thionitrite, n-butyl thionitrite, s-butyl thionitrite, tert-butyl thionitrite, n-pentyl thionitrite, n-hexyl thionitrite, cyclohexyl thionitrite, phenyl thionitrite; S-nitroso derivatives of cysteine-containing proteins, oligo- and poly-peptides, for example S-nitrosoalbumin, poly-S-nitrosoalbumin or S-nitrosohemoglobin; and any derivative thereof; and a salt of any of the foregoing.

10. The sterile S-nitrosothiol or sterile pharmaceutical pre-composition comprising S-nitrosothiol of claim 1, wherein the S-nitrosothiol is S-nitrosoglutathione.

11. A kit comprising a container or containers containing sterile the sterile S-nitrosothiol or sterile pharmaceutical pre-composition comprising S-nitrosothiol of claim 1, together with instructions for mixing the sterile S-nitrosothiol or a sterile pharmaceutical pre-composition comprising S-nitrosothiol with one or more sterile pharmaceutical diluents, excipients, carriers, additional active agents, or any combination thereof, to obtain a pharmaceutical composition for a pharmaceutical use containing S-nitrosothiol in a desired concentration.

12. A method for preparing a sterile pharmaceutical composition for human or veterinary use, comprising mixing the sterile S-nitrosothiol or sterile pre-composition containing S-nitrosothiol of claim 1 with one or more sterile pharmaceutical diluents, excipients, carriers, additional active agents, or any combination thereof, to obtain a pharmaceutical composition for a pharmaceutical use containing S-nitrosothiol in a desired concentration.

13. A pharmaceutical composition comprising a desired concentration of the sterile S-nitrosothiol or sterile pharmaceutical pre-composition comprising S-nitrosothiol of claim 1 mixed with one or more sterile pharmaceutical diluents, excipients, carriers, additional active agents, or any combination thereof.

14. A method of treating or preventing a disease or disorder of a human or animal subject which responds to S-nitrosothiol or NO therapy, said method comprising administering the sterile S-nitrosothiol or sterile pharmaceutical pre-composition comprising S-nitrosothiol of claim 1, to a human or animal subject in need thereof.

15. The method of claim 14, the method comprising administering a pharmaceutical composition comprising the sterile S-nitrosothiol or sterile pre-composition containing S-nitrosothiol mixed with one or more sterile pharmaceutical diluents, excipients, carriers, additional active agents, or any combination thereof.

16. The method of claim 14, wherein the sterile S-nitrosothiol or a sterile pharmaceutical pre-composition comprising S-nitrosothiol results in inducing arterial or venous smooth muscle relaxation, reducing augmentation index, reducing augmentation pressure, reducing arterial stiffness, inhibiting platelet aggregation, inducing T cell apoptosis or activating guanylate cyclase in the human or animal subject in need thereof.

17. The method of claim 14, wherein the disease or disorder which responds to S-nitrosothiol or NO therapy is selected from pre-eclampsia, severe pre-eclampsia, eclampsia, HELLP syndrome, organ transplantation perfusion, organ dialysis, post-operative conditions of balloon angioplasty, acute myocardial infarction, unstable angina, cerebral embolism, hypertension, atherosclerosis, restenosis, ischemia and heart failure, other cardiovascular proliferative, inflammatory, contractile and hypertensive disorders, and pre-conditioning related disorders of the heart and brain, oesophageal spasm, biliary spasm, colic and other motility and smooth muscle disorders of the gastrointestinal tract, erectile dysfunction, stroke, bronchial constriction, cystic fibrosis, pneumonia, asthma, pulmonary fibrosis, and other pulmonary disorders involving diminished gas exchange or inflammation, as well as infectious diseases of viral, bacterial and other origin, disorders of red blood cells characterised by S-nitrosothiol deficiency, abnormal rheology or impaired vasodilation, and thrombotic disorders.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0115] The present invention will be described in more detail below, but without limitation, by reference to the accompanying drawings, in which:

[0116] FIG. 1 shows the effects of the (a) e-beam and (b) gamma sterilisation treatment at 35 kGy dose and 15 kGy dose of Example 1 on the purity of the S-nitrosoglutathione (GSNO; % decrease in purity compared with non-irradiated control sample). The temperature reached approximately equal to () 27.5 C. for e-beam and less than or equal to (<) 60 C. or 40 C. for gamma radiation;

[0117] FIG. 2 shows the effects of the (a) e-beam and (b) gamma sterilisation treatment at 35 kGy dose and 15 kGy dose of Example 2 on the purity of the S-nitrosoglutathione (GSNO; % decrease in purity compared with non-irradiated control sample). The temperature was less than (<) 27.5 C. or was held at 80 C. for 35 kGy and 15 kGy for e-beam radiation. The temperature reached less than or equal to (<) 32.5 C. or was held at 80 C. for 35 kGy or less than or equal to (<) 27.5 C. or was held at 80 C. for 15 kGy for gamma radiation.

[0118] FIG. 3 is a composite of FIGS. 1 and 2 and shows the effects of the (a) e-beam and (b) gamma sterilisation treatments of Examples 1 and 2 on the purity of the S-nitrosoglutathione (GSNO; % decrease in purity compared with non-irradiated control sample) at various temperatures;

[0119] FIG. 4 is a schematic representation of the apparatus for e-beam irradiating vials containing S-nitrosoglutathione (GSNO) in Example 2, the arrows marked e show the direction of the e-beam radiation;

[0120] FIG. 5 is a schematic representation of the apparatus for gamma irradiating vials containing S-nitrosoglutathione (GSNO) in Example 2. The arrows marked show the direction of the gamma radiation.

EXAMPLES

[0121] The following non-limiting examples are provided for further illustration of the present invention.

Example 1

Sterilisation of S-nitrosoglutathione Using E-beam and Gamma Radiation at Unconstrained Temperature Settings

[0122] S-nitrosoglutathione granule samples from a single manufacturing batch, contained in glass vials (up to 250 mg/vial), were stored before sterilisation surrounded by dry ice (solid CO.sub.2) at about 80 C. The matching controls were not irradiated but underwent similar conditions of storage and thermal management before, during and after irradiation, as compared with the S-nitrosoglutathione vials that were irradiated. The control purity levels of the S-nitrosoglutathione, and the level of specific impurities glutathione (GSH) and oxidised glutathione (GSSG) content and total impurities content, were analysed. Vials were exposed to either electron beam (e-beam) irradiations or to gamma irradiations. The vials were removed from the dry ice container and sterilised via two radiation dose conditions (15 kGy and 35 kGy) described below under higher temperature conditions (with surrounding air temperature in the range about 18 to 24 C. and with the temperature of the vials free to fluctuate to higher temperature). After irradiation, the vials were immediately surrounded by dry ice at about 80 C. and analysed for post-sterilisation determination of any changes in purity against the control.

[0123] The e-beam radiation was performed using a moving carrier system set up for delivering a 15 kGy dose and a 35 kGy dose. The 15 kGy radiation dose lasted about 10-15 minutes and the temperature reached approximately equal to 27.5 C. (designated 27.5 C. in the Tables and Figs.). The 35 kGy radiation dose lasted about 30 minutes and the temperature reached approximately equal to 27.5 C. (designated 27.5 C. in the Tables and the Figs.). The radiation doses were measured by conventional dosimeters.

[0124] The gamma radiation was performed using a moving carrier system set up for delivering a 15 kGy dose and a 35 kGy dose. The 15 kGy dose radiation lasted about 8-10 hours and the temperature reached up to 40 C. The 35 kGy dose radiation lasted about 18-24 hours, with a temperature reaching up to 60 C. The radiation doses received were measured by conventional dosimeters.

[0125] The results are shown in FIGS. 1 and 3 and Table 1.

TABLE-US-00001 TABLE 1 Summary of results for e-beam and gamma radiation (Example 1) e-beam gamma Dose level 35 kGy 15 kGy 35 kGy 15 kGy Time of irradiation ~30 10-15 18-24 8-10 minutes minutes hours hours Temperature ( C.) ~27.5 ~27.5 <60 <40 during irradiation GSNO purity (decrease 1.37 0.55 9.45 4.47 % of control) GSH impurity 0.36 0.17 0.15 0.34 (% unit change) GSSH impurity 0.32 0.11 6.63 1.25 (% unit change) Total impurities 1.26 0.53 9.12 4.25 (% unit changes)

[0126] The unconstrained temperature e-beam irradiation of Example 1 at 15 kGy and 35 kGy resulted in a dose dependent decrease in the HPLC purity of S-nitrosoglutathione. The unconstrained temperature gamma irradiation of Example 1 at 15 kGy and 35 kGy resulted in a significant decrease in the HPLC purity of S-nitrosoglutathione dependent to temperature, time and radiation dose level. The use of ionising radiation (e-beam and gamma radiation) at unconstrained temperature leads to degradation of S-nitrosoglutathione as shown by the decrease in purity compared to control. However, the degradation is worse in the case of gamma radiation than in the case of e-beam radiation.

[0127] The data show that the level of the specific impurities GSH and GSSG and total impurities increased after e-beam and gamma irradiations in a temperature, time and radiation dose level dependent manner.

[0128] Example 2

Sterilisation of S-nitrosoglutathione Using E-beam and Gamma Radiation at Constrained Temperatures

[0129] The experimental protocol for this Example aims to test in a controlled manner the effects on purity of a sterilizing irradiation temperature (a) at about 80 C. and (b) under positive cooling (with surrounding air temperature in the range about 18 to 24 C. and with the temperature of the vials controlled). The same radiation doses as described for Example 1 were applied.

[0130] For this Example, S-nitrosoglutathione granule samples, from the same single manufacturing batch as in Example 1, contained in amber glass vials (100 mg/vial) were stored before sterilisation at about 80 C. The irradiation process at 80 C. was achieved by the presence of dry ice, which was placed to ensure low temperature of the vials but without influencing the radiation dose delivery characteristics. The matching controls were not irradiated but underwent similar conditions of thermal management before, during and after irradiation, as compared with the S-nitrosoglutathione vials that were irradiated.

[0131] The method employed for the e-beam arm of this experiment was as follows:

[0132] The e-beam radiation was performed using a moving carrier system. The S-nitrosoglutathione vials were stored at 80 C. freezer and transferred to a dry ice container before irradiation. Before the start of irradiation the S-nitrosoglutathione vials destined for irradiation at ambient temperature (and control vials) were removed from the 80 C. freezer and transferred to a location where the surrounding air temperature is controlled at 18 C.

[0133] Individual low-temperature containers were used for the vials designated for each target dose and condition of thermal management. A secondary packaging box was placed in the lateral centre at the bottom of the first low-temperature container, with the vials oriented upright. The position of each vial in the configuration was documented. The vials were always irradiated from the same side. FIG. 4 shows a possible arrangement of vials in a container for e-beam irradiation. The arrows marked e show the direction of the radiation. For the S-nitrosoglutathione vials requiring cold chain management, dry ice was placed at each lateral short side of the container to ensure a cold chain was maintained during the irradiation process. Each container was placed in an irradiation container.

[0134] Both the 15 kGy e-beam and 35 kGy radiation doses lasted a few seconds.

[0135] Following completion of the desired irradiation process, the irradiated S-nitrosoglutathione containing vials and correspondent controls were stored at 80 C. in a freezer for subsequent analysis.

[0136] The method employed for the gamma arm of this experiment was as follows:

[0137] The gamma radiation was performed using gamma radiation emitted from cobalt-60 radioactive sources. The S-nitrosoglutathione vials were stored at 80 C. in a freezer and transferred to a dry ice container before irradiation. Before the start of irradiation, the S-nitrosoglutathione vials destined for irradiation at ambient temperature (and control vials) were removed from the 80 C. freezer and transferred to a location where the surrounding temperature was controlled at 18 C.

[0138] Individual low-temperature containers were used for the samples designated for each target dose and condition of thermal management, as described above in relation to the e-beam arm of this Example.

[0139] The secondary packaging was placed in the lateral centre at the bottom of first low-temperature container, with the vials oriented upright. FIG. 5 shows a possible arrangement of vials in a container for gamma irradiation. The arrows marked show the direction of the radiation. For the S-nitrosoglutathione product requiring cold chain management, dry ice was placed at each lateral short side of the low-temperature container to ensure a cold chain was maintained during the irradiation process.

[0140] Each low-temperature container was placed in the lateral centre of an irradiation container.

[0141] The 15 kGy radiation dose lasted 2 hours and 8 minutes whereas the 35 kGy dose lasted about 5 hours and 8 minutes.

[0142] When necessary, the dry ice was replenished before execution of the second irradiation.

[0143] Following completion of the irradiation process, the irradiated S-nitrosoglutathione products were placed in the designated 80 C. freezer. The control samples were moved from the 18 C. controlled location to the designated 80 C. freezer upon completion of the second irradiation. The time of transfer was recorded.

[0144] The results are shown in FIG. 2 and FIG. 3, and Table 2.

TABLE-US-00002 TABLE 2 Summary of results for e-beam and gamma radiation (Example 2) e-beam gamma Dose level 35 kGy 15 kGy 35 kGy 15 kGy Time of Few sec. Few sec. ~5 h ~2 h irradiation Temperature <27.5 80 <27.5 80 <32.5 80 <27.5 80 ( C.) during irradiation GSNO purity 1.12 0.55 0.28 0.16 2.2 1.01 0.67 0.26 (decrease % of control) GSH impurity 0.29 0.23 0.13 0.10 0.43 0.29 0.17 0.11 (% unit change) GSSH 0.23 0.07 0.13 0.10 0.36 0.36 0.14 0.11 impurity (% unit change) Total 1.08 0.54 0.27 0.16 2.11 0.96 0.63 0.25 impurities (% unit changes)

[0145] The e-beam irradiations of Example 2 at 15 kGy and 35 kGy resulted in a small, temperature, time and dose level dependent decrease in the HPLC purity of S-nitrosoglutathione (GSNO); FIGS. 2 and 3 (labeled (2) for Example 2) and Table 2.

[0146] The gamma irradiations of Example 2 at 15 kGy and 35 kGy resulted in a small, temperature, time and dose level dependent decrease in the HPLC purity of S-nitrosoglutathione (GSNO); FIGS. 2 and 3 labeled (2) for Example 2) and Table 6.

[0147] In all the irradiations of Example 2, the level of specific impurity GSH and GSSG and total impurities increased in a small temperature, time and dose level dependent manner, after e-beam and gamma radiation Table 2.

Discussion

[0148] The results of Examples 1 and 2 are summarized in Tables 3 and 4:

TABLE-US-00003 TABLE 3 Summary of results for e-beam radiation [(1) = Example 1; (2) = Example 2] Conditions Example 1 Example 2 Example 1 Example 2 Dose level 35 kGy 15 kGy Vial type Clear Amber Clear Amber Time of ~30 minutes Few seconds 10-15 Few seconds irradiation minutes Temperature ~27.5 80 <27.5 ~27.5 80 <27.5 ( C.) during irradiation GSNO purity 1.37 0.55 1.12 0.55 0.16 0.28 (decrease % of control)

TABLE-US-00004 TABLE 4 Summary of results for gamma radiation [(1) = Example 1; (2) = Example 2] Conditions Example 1 Example 2 Example 1 Example 2 Dose level 35 kGy 15 kGy Time of 18-24 ~5 8-10 ~2 irradiation (hours) Temperature <60 80 <32.5 <40 80 <27.5 ( C.) during irradiation GSNO purity 9.45 1.01 2.2 4.47 0.26 0.67 (decrease % of control)

[0149] The above results show that optimised specific conditions exist to sterilise pharmaceutical S-nitrosothiols such as, for example, S-nitrosoglutathione. The decomposition of S-nitrosoglutathione in vials was examined following irradiation (by e-beam and gamma) at two dose levels (15 kGy and 35 kGy) using different conditions (different vials, different duration and temperature of irradiation) in two different experiments. These studies confirm that ionising radiation can decompose S-nitrosoglutathione; however they demonstrate, for the first time, that specific conditions of temperature and irradiation time and dose can minimise its decomposition, enabling the fulfilment of pharmaceutical regulatory requirements. E-beam radiation appears to decompose S-nitrosoglutathione less than gamma radiation at a parity of dosage. Decomposition is minimised by lowering the temperature and shortening the duration of the irradiation.

[0150] The foregoing broadly describes the present invention without limitation. Variations and modifications as will be readily apparent to those skilled in the art are intended to be covered. The scope of protection of the present invention shall be determined by reference to the appended claims as properly construed according to law.

REFERENCES

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