HYDROGEL IRRADIATION

Abstract

The present invention relates to a process for the irradiation of a water-insoluble conjugate comprising a polymer Z to which a plurality of moieties -L.sup.2-L.sup.1-D is covalently conjugated or of a water-insoluble complex comprising a plurality of releasably and non-covalently bound drug molecules D-H or D-OH embedded in a polymer Z′, wherein the process comprises the steps of (a) providing said conjugate or complex; and (b) exposing the conjugate or complex to ionizing radiation; wherein each -L.sup.2- is independently a chemical bond or is a spacer moiety; each -L.sup.1- is independently a linker moiety covalently and reversibly attached to -D; and each -D is independently a drug moiety.

Claims

1. A process for the irradiation of a water-insoluble conjugate comprising a polymer Z to which a plurality of moieties -L.sup.2-L.sup.1-D- is covalently conjugated or of a water-insoluble complex comprising a plurality of releasably and non-covalently bound drug molecules D-H or D-OH embedded in a polymer Z′, wherein the process comprises the steps of (a) providing said conjugate or complex; and (b) exposing the conjugate or complex to ionizing radiation; wherein each -L.sup.2- is independently a chemical bond or is a spacer moiety; each -L.sup.1- is independently a linker moiety covalently and reversibly attached to -D; and each -D is independently a drug moiety.

2. The process of claim 1, wherein the process is performed with the water-insoluble conjugate.

3. The process of claim 1, wherein the process is performed with the water-insoluble complex.

4. The process of claim 1, wherein the ionizing radiation is selected from the group consisting of electron beam radiation, X-ray radiation, gamma radiation, proton beam radiation, neutron beam radiation, positron beam radiation, alpha particle radiation, UV radiation and any combination thereof.

5. The process of claim 1, wherein the ionizing radiation is electron beam.

6. The process of claim 1, wherein the ionizing radiation is X-ray radiation.

7. The process of claim 1, wherein the ionizing radiation is gamma radiation.

8. The process of claim 1, wherein the ionizing radiation is proton beam radiation.

9. The process of claim 1, wherein the ionizing radiation is neutron beam radiation.

10. The process of claim 1, wherein the ionizing radiation is positron beam radiation.

11. The process of claim 1, wherein the ionizing radiation is alpha particle radiation.

12. The process of claim 1, wherein the ionizing radiation is UV radiation.

13. The process of claim 1, wherein the ionizing radiation is a combination of one or more selected from the group consisting of electron beam radiation, X-ray radiation, gamma radiation, proton beam radiation, neutron beam radiation, positron beam radiation, alpha particle radiation and UV radiation.

14. The process of claim 1, wherein irradiation is performed as a continuous irradiation or as multiple irradiation exposures.

15. The process of claim 1, wherein the exposing to ionizing radiation in step (b) is performed with a total absorbed radiation dose ranging from 10 to 80 kGy.

16. The process of claim 1, wherein the exposing to ionizing radiation in step (b) is performed with a total absorbed radiation dose ranging from 17.5 to 35 kGy.

17. The process of claim 1, wherein the conjugate or complex is in a liquid formulation in step (b).

18. The process of claim 1, wherein the conjugate or complex is in a dry formulation in step (b).

19. The process of claim 1, wherein step (b) is performed at a temperature ranging from −196° C. to +45° C.

20. The process of claim 1, wherein each -D is independently selected from the group consisting of small molecule drug moieties, large molecule drug moieties, oligonucleotide moieties, peptide nucleic acid moieties, peptide moieties and protein moieties.

21. The process of claim 1, wherein all moieties -D of the conjugate or complex are identical.

22. The process of claim 1, wherein the conjugate or complex comprises more than one type of -D.

23. The process of claim 1, wherein the conjugate or complex comprises two, three, four or five different types of -D.

24. An irradiated water-insoluble conjugate comprising a polymer Z to which a plurality of moieties -L.sup.2-L.sup.1-D is covalently and reversibly conjugated or an irradiated water-insoluble complex comprising a plurality of releasably and non-covalently bound drug molecules D-H or D-OH embedded in a polymer Z′ obtainable from the process of claim 1.

Description

EXAMPLE 1

[0829] Transient Daptomycin-Linker PEG-Hydrogel Conjugate 1

[0830] Transient daptomycin-linker PEG-hydrogel conjugate 1 was synthesized as described for compound 9b in WO 2020/064844 A1, example 9. The obtained material had a daptomycin content of 470 mg/g dry material.

EXAMPLE 2

[0831] Transient Daptomycin-Linker HA-Hydrogel Conjugate 2

[0832] Transient daptomycin-linker HA-hydrogel conjugate was synthesized as described for compound 5 in WO 2020/064847 A1, example 15. The representative material 2 that was used for all experiments had a daptomycin content of 459 mg/g dry material.

EXAMPLE 3

[0833] X-Ray Irradiation of Transient Daptomycin-Linker Hydrogel Conjugates

[0834] Two 11-15 mg samples of transient daptomycin-linker PEG-hydrogel conjugate 1 and two 15-17 mg samples of a representative transient daptomycin-linker HA-hydrogel conjugate (equal to compound 2) were separately filled into 2 mL plastic vials under an argon atmosphere. A set of one sample tube of compound 1 and one sample tube of representative compound 2 was irradiated with X-rays at 78° C. in a bed of dry ice and a total dose of 32.1-33.6 kGy to form sample set A consisting of compound 1a (−78° C. irradiated transient daptomycin-linker PEG-hydrogel conjugate) and compound 2a (−78° C. irradiated transient daptomycin-linker HA-hydrogel conjugate).

[0835] A second set of one sample tube of compound 1 and one sample tube of representative compound 2 was irradiated with X-rays at room temperature and a total dose of 32.1-33.6 kGy to form sample set B consisting of compound 1b (r.t. irradiated transient daptomycin-linker PEG-hydrogel conjugate) and compound 2b (r.t. irradiated transient daptomycin-linker HA-hydrogel conjugate).

EXAMPLE 4

[0836] Purity of Released Daptomycin from Irradiated and Non-Irradiated Transient Daptomycin-Linker Hydrogel Conjugates

[0837] To investigate the influence of X-rays on the purity of the transiently bound daptomycin in the carriers, a time limited cumulative release of daptomycin from compounds 1, 2, 1a, 2a, 1b and 2b was carried out. For that purpose, the materials were incubated under physiological conditions in concentrations of 4 mg/mL for exactly 42 hours. After this time, samples were taken from the supernatants and analyzed by UPLC-MS at 215 nm. Daptomycin itself is prone to degrade under physiological conditions via different pathways. Therefore, intrinsic degradation of daptomycin in the cumulative release solution to a reduced purity of 88-89% was expected and observed. It was additionally found that the purity of the released daptomycin from all irradiated samples was at least 96% of the purity of the released daptomycin from the respective non-irradiated samples.

TABLE-US-00001 Purity of daptomycin in the supernatant after Relative Compared to Compound 42 hours cumulative release purity compound 1 88.0% / / 2 88.8% / / 1a 85.6% 97.3% 1 2a 87.1% 98.1% 2 1b 84.5% 96.0% 1 2b 85.3% 96.1% 2

EXAMPLE 5

[0838] Linker Release Kinetics for Irradiated and Non-Irradiated Transient Daptomycin-Linker Hydrogel Conjugates

[0839] The linker kinetics with respect to the daptomycin species release from irradiated and non-irradiated transient daptomycin-linker hydrogel conjugates 1, 2, 1a, 2a, 1b and 2b were investigated by incubation of the materials under physiological conditions. Daptomycin is prone to hydrolytic degradation and some minor different degradation pathways upon aqueous incubation. For determination of the linker kinetics on the carriers, the supernatants of the incubated suspensions were analyzed by UPLC-MS at 215 nm and all daptomycin-related peaks were taken into account for the calculation of the linker kinetics. The linker kinetics did not differ between the irradiated and the respective non-irradiated samples with respect to half-life, release rate and the finally reached plateau. Linker half-lives have been determined to be twelve days for the transient daptomycin-linker PEG-hydrogel conjugates 1, 1a and 1b and ten days for the transient daptomycin-linker HA-conjugates 2, 2a and 2b.

EXAMPLE 6

[0840] Degradation Studies of Irradiated and Non-Irradiated Transient Daptomycin-Linker Hydrogel Conjugates

[0841] Irradiated and non-irradiated transient daptomycin-linker hydrogel conjugates 1, 2, 1a, 2a, 1b and 2b were analyzed regarding carrier degradation. For that purpose, the materials were incubated under physiological conditions. The samples were visually checked for the presence of the solid carrier particles daily. As soon as no particles could be detected in the sample anymore, the material was deemed to be fully degraded to soluble products. It was found that the degradation time for the HA-hydrogel conjugates 2, 2a and 2b was 52-56 days and did not differ significantly between the non-irradiated and the irradiated samples. The degradation of the irradiated PEG-hydrogel samples 1a and 1b occurred with 27-29 days slightly faster than it was observed for the non-irradiated sample 1 with 40 days. A slight prolongation of the degradation time could be observed for the samples 1b and 2b, which were irradiated at r.t. compared to the samples 1a and 2a, which were treated at −78° C.

TABLE-US-00002 Compound 1 1a 1b 2 2a 2b Irradiation not −78° C. r.t. not −78° C. r.t. temperature irradiated irradiated Time for full 40 d 27 d 29 d 55 d 52 d 56 d degradation

EXAMPLE 7

[0842] Transient Resiquimod-Linker PEG-Hydrogel Conjugate 3

[0843] Transient resiquimod-linker PEG-hydrogel conjugate 3 was synthesized as described for compound 14 in PCT/EP2020/050093, example 8. The obtained material had a resiquimod content of 17.2 mg/g dry material. It was formulated to 0.5 mg resiquimod/mL in phosphate-buffered saline containing 137 mM sodium chloride, 2.68 mM potassium chloride, 4.3 mM sodium phosphate, dibasic, and 1.4 mM potassium phosphate, monobasic, pH 7.4.

EXAMPLE 8

[0844] X-Ray Irradiation of Transient Resiquimod-Linker Hydrogel Conjugates

[0845] 0.5-mg samples (1-mL fill) of transient resiquimod-linker PEG-hydrogel conjugate 3 are separately filled into 5-mL glass vials under atmospheric gas. Vials with 1.0-mL fill of only aqueous buffer were separately filled into 5-mL glass vials under atmospheric gas.

[0846] A set of multiple sample vials of compound 3 are irradiated with x-rays in a vessel containing dry ice, a total absorbed dose of 25-40 kGy with a temperature inside the vials of −50° C.±10° C. during irradiation to form a sample set of compound 3a.

EXAMPLE 9

[0847] E-Beam Irradiation of Transient Resiquimod-Linker Hydrogel Conjugates

[0848] 0.5-mg samples (1-mL fill) of transient resiquimod-linker PEG-hydrogel conjugate 3 were separately filled into 5-mL glass vials under atmospheric gas. Vials with 1.0-mL fill of only aqueous buffer were separately filled into 5-mL glass vials under atmospheric gas.

[0849] A set of multiple sample vials of compound 3 were irradiated with electron beam in a vessel containing dry ice and a target absorbed dose of 25-40 kGy with an executed total absorbed dose of 29-32 kGy to form a sample set of compound 3b. Temperature mapping data for e-beam irradiation demonstrated the temperatures inside vials were colder than −25° C.±10° C.

EXAMPLE 10

[0850] Drug Purity for Irradiated and Non-Irradiated Resiquimod-Linker Hydrogel Conjugates

[0851] Irradiated and non-irradiated resiquimod-linker hydrogel conjugates 3 and 3b were tested for resiquimod purity by releasing all or nearly all transiently bound resiquimod from the hydrogel under basic conditions. The samples were incubated under basic conditions overnight, after which they were pH adjusted and the supernatants were analyzed by UPLC-MS at 320 nm. The measured purity values of conjugates 3 and 3b were 99.7% and 98.1%, respectively.

EXAMPLE 11

[0852] Linker Release Kinetics for Irradiated and Non-Irradiated Resiquimod-Linker Hydrogel Conjugates

[0853] The resiquimod release rate under physiological conditions of irradiated and non-irradiated resiquimod-linker hydrogel conjugates 3 and 3b was measured. The supernatants of incubated samples were analyzed by UPLC-MS at 320 nm; as resiquimod is stable to the incubation conditions, only the resiquimod peak was considered for the release kinetics determination. The rate of resiquimod release was fit using a first order exponential equation to estimate the half-life of drug release. The resulting half-life parameter fits for conjugates 3 and 3b were 18.1 and 20.8 days, respectively.

EXAMPLE 12

[0854] Accelerated Degradation Studies of Irradiated and Non-Irradiated Resiquimod-Linker Hydrogel Conjugates

[0855] The carrier degradation profile under accelerated conditions of irradiated and non-irradiated resiquimod-linker hydrogel conjugates 3 and 3b were measured. Samples were incubated under basic conditions and the supernatants were sampled over the course of one week. After the final timepoint was collected, the supernatant samples were further hydrolyzed overnight to ensure that all transiently bound resiquimod was released from solubilized carrier fragments. The samples were pH adjusted and analyzed by SEC to measure soluble carrier fragments. The degradation rate of the carrier was fit using a five parameter asymmetric sigmoidal equation, which was used to estimate the time to 50% degradation (T50). The T50 values for conjugates 3 and 3b were 73.7 and 75.1 hours, respectively.

Abbreviations

[0856] HA Hyaluronic Acid [0857] HPLC High Performance Liquid Chromatography [0858] PEG Poly(ethylene glycol) [0859] r.t. Room Temperature [0860] SEC Size Exclusion Chromatography [0861] T50 Time to 50% hydrogel degradation [0862] TFA Trifluoroacetic Acid [0863] UPLC-MS Mass Spectrometry Coupled Ultra Performance Liquid Chromatography