Sterilisation of S-nitrosothiols

Abstract

This invention provides a method of sterilizing 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 method of preparing sterile dry solid S-nitrosothiol or a sterile dry solid pharmaceutical pre-composition comprising S-nitrosothiol, said method comprising: exposing dry solid S-nitrosothiol or a dry solid pharmaceutical pre-composition comprising S-nitrosothiol to a sterilising dose of ionising radiation in an environment sealed from external contamination to yield a sterile product of said sterile dry solid S-nitrosothiol or sterile dry solid pharmaceutical pre-composition comprising S-nitrosothiol, wherein said sterile product has an S-nitrosothiol purity of at least about 95% and a sterility assurance level (SAL) of 10.sup.6 or lower.

2. A method as claimed in claim 1, wherein the ionising radiation is selected from electron beam (e-beam) radiation, gamma radiation and X-rays.

3. A method as claimed in claim 1, wherein the exposure to the ionising radiation is performed under conditions such that there is no reduction of the purity of the S-nitrosothiol or that its purity is reduced by not more than about 2.0%, through degradation.

4. A method as claimed in claim 1, wherein the dry solid S-nitrosothiol or dry solid pharmaceutical pre-composition comprising S-nitrosothiol is maintained at a temperature not greater than about 40 C. during the sterilising exposure to the ionising radiation.

5. A method as claimed in claim 1, wherein an absorbed dose of ionising radiation up to about 50 kGy is used to sterilise the dry solid S-nitrosothiol or dry solid pharmaceutical pre-composition comprising S-nitrosothiol and (a) electron beam (e-beam) radiation is used for an exposure time which is less than about 1 hour; or (b) gamma radiation is used for an exposure time which is less than about 24 hours.

6. A method as claimed in claim 1, wherein: (a) the ionising radiation is electron beam (e-beam) radiation and the dry solid S-nitrosothiol or dry solid pharmaceutical pre-composition comprising S-nitrosothiol is maintained during the sterilising exposure to the ionising radiation at a temperature not greater than about 35 C.; or (b) the ionising radiation is gamma radiation and the dry solid S-nitrosothiol or dry solid pharmaceutical pre-composition comprising S-nitrosothiol is maintained during the sterilising exposure to the ionising radiation at not a temperature greater than about 35 C.

7. A method as claimed in claim 1, wherein the ionising radiation is electron beam radiation at an absorbed dose of up to about 50 kGy, the dry solid S-nitrosothiol or a dry solid pharmaceutical pre-composition comprising S-nitrosothiol to be sterilised has a starting temperature at room temperature conditions with freedom to fluctuate higher during said exposing, and the exposure to e-beam radiation taking place over up to about 1 hour.

8. A method as claimed in claim 1, wherein the ionising radiation is electron beam radiation at an absorbed dose of up to about 50 kGy, the dry solid S-nitrosothiol or a dry solid pharmaceutical pre-composition comprising S-nitrosothiol to be sterilised is maintained at a temperature below about 35 C., and the exposure to the radiation taking place over up to about 1 hour.

9. A method as claimed in claim 1, wherein the ionising radiation is gamma radiation at an absorbed dose of up to about 50 kGy, the dry solid S-nitrosothiol or a dry solid pharmaceutical pre-composition comprising S-nitrosothiol to be sterilised is maintained at a temperature below about 35 C., and the exposure to the radiation taking place over up to about 24 hours.

10. A method as claimed in claim 1, wherein the environment sealed from external contamination is in a dry sealed container in which the dry solid S-nitrosothiol or a dry solid pharmaceutical pre-composition comprising S-nitrosothiol is contained prior to said exposing.

11. A method as claimed in claim 10, wherein the environment after said exposing is a dry sterile environment within the sealed container.

12. A method as claimed in claim 10, wherein the container has walls that are tinted or opaque to light.

13. A method as claimed in claim 10, wherein the container is oxygen-impermeable and moisture-impermeable.

14. A method as claimed in claim 1, wherein prior to said exposing, said method further comprising: placing said dry solid S-nitrosothiol or a dry solid pharmaceutical pre-composition comprising S-nitrosothiol into a dry container; and sealing said container to yield said environment sealed from external contamination.

15. A method as claimed in claim 14, wherein after said exposing said container holds said sterile product for storage and transportation until used for a final pharmaceutical composition for therapeutic or prophylactic use.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present invention will be described in more detail below, but without limitation, by reference to the accompanying drawings, in which:

(2) FIG. 1 shows the effects of the (a) e-beam and (b) gamma sterilisation treatment of Example 1 on the purity of the S-nitrosoglutathione (GSNO; % decrease in purity compared with non-irradiated control sample);

(3) FIG. 2 shows the effects of the (a) e-beam and (b) gamma sterilisation treatment of Example 2 on the purity of the S-nitrosoglutathione (GSNO; % decrease in purity compared with non-irradiated control sample);

(4) 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; data from Example 1 identified by (1); data from Example 2 identified by (2));

(5) FIG. 4 is a schematic representation of the apparatus for e-beam irradiating vials containing S-nitrosoglutathione (GSNO) in Example 2; and

(6) FIG. 5 is a schematic representation of the apparatus for gamma irradiating vials containing S-nitrosoglutathione (GSNO) in Example 2.

EXAMPLES

(7) The following non-limiting examples are provided for further illustration of the present invention.

Example 1

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

(9) 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.

(10) 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 1015 minutes and the temperature reached approximately equal to 27.5 C. (designated 27.5 C. in the Tables and Figures). 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 Figures). The radiation doses were measured by conventional dosimeters.

(11) 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.

(12) The results are shown in FIGS. 1 and 3 (bars labeled (1) for Example 1) and Table 1.

(13) 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 10-15 ~30 minutes minutes 18-24 hours 8-10 hours Temperature ( C.) ~27.5 ~27.5 60 40 during irradiation GSNO purity 1.37 0.55 9.45 4.47 (decrease % 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)

(14) 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.

(15) 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.

Example 2

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

(17) 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.

(18) 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.

(19) The method employed for the e-beam arm of this experiment was as follows:

(20) 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.

(21) 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.

(22) Both the 15 kGy e-beam and 35 kGy radiation doses lasted a few seconds.

(23) 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.

(24) The method employed for the gamma arm of this experiment was as follows:

(25) 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.

(26) 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.

(27) 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.

(28) Each low-temperature container was placed in the lateral centre of an irradiation container.

(29) The 15 kGy radiation dose lasted 2 hours and 8 minutes whereas the 35 kGy dose lasted about 5 hours and 8 minutes.

(30) When necessary, the dry ice was replenished before execution of the second irradiation.

(31) 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.

(32) The results are shown in FIG. 2 and FIG. 3 (labeled (2) for Example 2), and Table 2.

(33) 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 impurity 0.23 0.07 0.13 0.10 0.36 0.36 0.14 0.11 (% unit change) Total impurities 1.08 0.54 0.27 0.16 2.11 0.96 0.63 0.25 (% unit changes)

(34) 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.

(35) 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.

(36) 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.

(37) Discussion

(38) The results of Examples 1 and 2 are summarized in Tables 3 and 4:

(39) 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 minutes Few seconds irradiation Temperature ( C.) ~27.5 80 <27.5 ~27.5 80 <27.5 during irradiation GSNO purity 1.37 0.55 1.12 0.55 0.16 0.28 (decrease % of control)

(40) 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 Vial type Clear Amber Clear Amber Time of 18-24 ~5 8-10 ~2 irradiation (hours) Temperature ( C.) 60 80 32.5 40 80 27.5 during irradiation GSNO purity 9.45 1.01 2.2 4.47 0.26 0.67 (decrease % of control)

(41) 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.

(42) 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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