Dry Pharmaceutical Formulations of CNP Conjugates

Abstract

A dry pharmaceutical formulation, wherein the pharmaceutical formulation comprises a CNP conjugate, a buffering agent and a bulking agent and wherein the CNP conjugate comprises a CNP moiety that is covalently and reversibly conjugated to a polymeric moiety.

Claims

1. A dry pharmaceutical formulation, wherein the pharmaceutical formulation comprises a CNP conjugate, a buffering agent and a bulking agent and wherein the CNP conjugate comprises a CNP moiety that is covalently and reversibly conjugated to a polymeric moiety.

2. The dry pharmaceutical formulation of claim 1, wherein the buffering agent is selected from the group consisting of succinic acid, citric acid, lactic acid, acetic acid, glutamic acid, fumaric acid, aspartic acid, glutaric acid, phosphoric acid, histidine, gluconic acid, tartaric acid, malic acid and mixtures thereof.

3. The dry pharmaceutical formulation of claim 1, wherein the buffering agent is succinic acid.

4. The dry pharmaceutical formulation of claim 1, wherein the bulking agent is selected from the group consisting of trehalose, mannitol, sucrose, raffinose, gelatin, lactose, dibasic calcium phosphate, sorbitol, xylitol, glycine, histidine, hydroxy ethyl starch, dextrose, dextran, propylene glycol and mixtures thereof.

5. The dry pharmaceutical formulation of claim 1, wherein the bulking agent is selected from the group consisting of trehalose, sucrose and glycine.

6. The dry pharmaceutical formulation of claim 1, wherein the bulking agent is trehalose.

7. The dry pharmaceutical formulation of claim 1, wherein the dry pharmaceutical formulation comprises one or more further excipients.

8. The dry pharmaceutical formulation of claim 7, wherein the one or more further excipients are selected from the group consisting of a preservative, stabilizer, anti-adsorption agent, cryoprotectant, oxidation protection agent and other auxiliary agents.

9. The dry pharmaceutical formulation of claim 1, wherein the dry pharmaceutical formulation comprises a stabilizer.

10. The dry pharmaceutical formulation of claim 1, wherein the dry pharmaceutical formulation comprises a preservative.

11. The dry pharmaceutical formulation of claim 1, wherein the dry pharmaceutical formulation comprises an anti-absorption agent.

12. The dry pharmaceutical formulation of claim 1, wherein the dry pharmaceutical formulation comprises a cryoprotectant.

13. The dry pharmaceutical formulation of claim 1, wherein the dry pharmaceutical formulation comprises an oxidation protection agent.

14. The dry pharmaceutical formulation of claim 1, wherein the dry pharmaceutical formulation comprises a further excipient selected from the group consisting of wetting agents, viscosity modifiers and antibiotics.

15. The dry pharmaceutical formulation of claim 1, wherein the pharmaceutical formulation comprises a pH-adjusting agent.

16. The dry pharmaceutical formulation of claim 15, wherein the pH-adjusting agent is selected from the group consisting of Tris, sodium hydroxide, potassium hydroxide, lysine and mixtures thereof.

17. The dry pharmaceutical formulation of claim 15, wherein the pH-adjusting agent is Tris.

18. The dry pharmaceutical formulation of claim 1, wherein the pharmaceutical formulation comprises a CNP conjugate, succinic acid, trehalose and Tris.

19. The dry pharmaceutical formulation of claim 1, wherein the CNP moiety has the sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:30.

20. The dry pharmaceutical formulation of claim 1, wherein the CNP moiety has the sequence of SEQ ID NO:24.

21. The dry pharmaceutical formulation of claim 1, wherein the polymeric moiety comprises a polymer selected from the group consisting of 2-methacryloyl-oxyethyl phosphoyl cholins, poly(acrylic acids), poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers, poly(amides), poly(amidoamines), poly(amino acids), poly(anhydrides), poly(aspartamides), poly(butyric acids), poly(glycolic acids), polybutylene terephthalates, poly(caprolactones), poly(carbonates), poly(cyanoacrylates), poly(dimethylacrylamides), poly(esters), poly(ethylenes), poly(ethyleneglycols), polyethylene oxides), poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolic acids), poly(hydroxyethyl acrylates), poly(hydroxyethyl-oxazolines), poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides), poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines), poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolic acids), poly(methacrylamides), poly(methacrylates), poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters), poly(oxazolines), poly(propylene glycols), poly(siloxanes), poly(urethanes), poly(vinyl alcohols), poly(vinyl amines), poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses, carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins, chitosans, dextrans, dextrins, gelatins, hyaluronic acids and derivatives, functionalized hyaluronic acids, mannans, pectins, rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethyl starches and other carbohydrate-based polymers, xylans, and copolymers thereof.

22. The dry pharmaceutical formulation of claim 1, wherein the formulation comprises based on the total weight of the dry pharmaceutical formulation: TABLE-US-00050 CNP conjugate 1.3-45.4% (w/w) succinic acid 0.2-3.2% (w/w) trehalose dihydrate 52.6-98.4% (w/w) Tris 0.1-5.6% (w/w).

23. The dry pharmaceutical formulation of claim 1, wherein the formulation comprises based on the total weight of the dry pharmaceutical formulation: TABLE-US-00051 CNP conjugate 1.3-38.7% (w/w) succinic acid 0.2-3.2% (w/w) trehalose dihydrate 52.6-98.4% (w/w) Tris 0.1-5.6% (w/w).

24. The dry pharmaceutical formulation of claim 1, wherein the CNP conjugate is formula (Ia) or (Ib):
ZL.sup.2-L.sup.1-D).sub.x  (Ia),
DL.sup.1-L.sup.2-Z).sub.y  (Ib), wherein -D is a CNP moiety; -L.sup.1- is a reversible linker moiety; -L.sup.2- is a single chemical bond or a spacer moiety; —Z is a polymeric moiety; x is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16; and y is an integer selected from the group consisting of 1, 2, 3, 4 and 5.

25. The dry pharmaceutical formulation of claim 24, wherein x of formula (Ia) is an integer selected from the group consisting of 1, 2, 3, 4, 6 and 8.

26. The dry pharmaceutical formulation of claim 24, wherein y of formula (Ib) is an integer selected from the group consisting of 2, 3, 4 and 5.

27. The dry pharmaceutical formulation of claim 24, wherein the CNP conjugate is of formula (Ia) and x is 1.

28. The dry pharmaceutical formulation of claim 24, wherein -L1- is connected to -D through an amide linkage.

29. The dry pharmaceutical formulation of claim 24, wherein the linkage between —Z and -L.sup.2- is a stable linkage.

30. The dry pharmaceutical formulation of claim 24, wherein -L.sup.2- is a spacer moiety.

31. The dry pharmaceutical formulation of claim 24, wherein -L.sup.2- has a molecular weight in the range of from 14 g/mol to 750 g/mol.

32. The dry pharmaceutical formulation of claim 24, wherein -L.sup.2- has a chain length of 1 to 20 atoms.

33. The dry pharmaceutical formulation of claim 24, wherein -L.sup.2- is of formula (i): ##STR00133## wherein the dashed line marked with the asterisk indicates attachment to -L.sup.1-; the unmarked dashed line indicates attachment to —Z; —R.sup.1 is selected from the group consisting of —H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl; n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18; and wherein the moiety of formula (i) is optionally further substituted.

34. The dry pharmaceutical formulation of claim 33, wherein n of formula (i) is selected from the group consisting of 3, 4, 5, 6, 7, 8 and 9.

35. The dry pharmaceutical formulation of claim 24, wherein —Z has a molecular weight ranging from 5 to 200 kDa.

36. The dry pharmaceutical formulation of claim 24, wherein —Z comprises a polymer selected from the group consisting of 2-methacryloyl-oxyethyl phosphoyl cholins, poly(acrylic acids), poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers, poly(amides), poly(amidoamines), poly(amino acids), poly(anhydrides), poly(aspartamides), poly(butyric acids), poly(glycolic acids), polybutylene terephthalates, poly(caprolactones), poly(carbonates), poly(cyanoacrylates), poly(dimethylacrylamides), poly(esters), poly(ethylenes), poly(ethyleneglycols), polyethylene oxides), poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolic acids), poly(hydroxyethyl acrylates), poly(hydroxyethyl-oxazolines), poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides), poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines), poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolic acids), poly(methacrylamides), poly(methacrylates), poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters), poly(oxazolines), polypropylene glycols), poly(siloxanes), poly(urethanes), poly(vinyl alcohols), poly(vinyl amines), poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses, carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins, chitosans, dextrans, dextrins, gelatins, hyaluronic acids and derivatives, functionalized hyaluronic acids, mannans, pectins, rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethyl starches and other carbohydrate-based polymers, xylans, and copolymers thereof.

37. A method of manufacturing the dry pharmaceutical formulation of claim 1, wherein the method comprises the steps of (i) admixing the CNP conjugate with at least a buffering agent and a bulking agent; (ii) adjusting the pH of the admixture of step (i); (iii) optionally, filtering the admixture from step (ii); (iv) transferring amounts of the admixture from step (ii) or (iii) equivalent to the desired number of dosages into a container; (v) drying the admixture; (vi) sealing the container; and wherein the order of steps (ii) and (iii) may optionally be reversed.

38. A method of reconstituting the dry pharmaceutical formulation of claim 1, wherein the method comprises the step of (a) contacting the dry pharmaceutical formulation of claim 1, with a reconstitution solution.

39. A reconstituted pharmaceutical formulation obtainable from the method of reconstituting of claim 38.

40-43. (canceled)

44. A method of treating, controlling, delaying or preventing in a patient one or more diseases which can be treated by CNP, the method comprising administering to the patient a therapeutically effective amount of the reconstitution pharmaceutical formulation of claim 39.

45. The method of claim 44, wherein the disease is selected from the group consisting of bone-related disorders such as skeletal dysplasias; cancer; autoimmune diseases; fibrotic diseases; inflammatory diseases; central nervous system diseases such as neurodegenerative diseases; infectious diseases; lung diseases; heart and vascular diseases; metabolic diseases and ophthalmic diseases.

46. The method of claim 44, wherein the disease is achondroplasia.

47. The dry pharmaceutical formulation of claim 1, wherein the pharmaceutical formulation comprises a mixture of one or more acid and based pH-adjusting agents.

48. The dry pharmaceutical formulation of claim 25, wherein -D of formula (Ia) or (Ib) has the sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:30.

49. The dry formulation of claim 25, wherein -D of formula (Ia) or (Ib) has the sequence of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25.

Description

EXAMPLES

Materials and Methods

[1069] All materials were commercially available except where stated otherwise.

[1070] RP-HPLC was used to determine the content and purity of compound (1) and to detect free CNP-38: Mobile phase A was composed of 0.05% aqueous TFA and mobile phase B was composed of 0.04% TFA in acetonitrile. A Waters Acquity CSH C18, 130 Å, 1.7 μm, 2.1×100 mm column was used. Flow rate was set to 0.3 mL/min, detection was at a wavelength of 215 nm, the column running temperature was 60° C. (±1° C.). Samples were diluted with formulation buffer containing 0.5% Tween®-20. The content was determined by peak area comparison to a reference solution.

[1071] SE-HPLC was used to determine the purity of compound (1): Mobile phase was composed of 15 mM sodium phosphate pH 7.40, 135 mM sodium chloride, 0.2% Pluronic F-68 in water. A GE Superdex 200 Increase 10/300 GL column was used. Flow rate was set to 0.75 mL/min, detection was at a wavelength of 215 nm, the column running temperature was room temperature. Samples were diluted with formulation buffer containing 0.05% Pluronic F-68.

[1072] Peptide mapping of compound (1) was used to assess the conversion of aspartate (28) into isoaspartate (28) and the oxidation of methionine (33) into methioninoxide (33) (Met(O). Thermolysin digestion of (1) was performed at pH 7.5 for 7 h at 37° C. at a thermolysin/CNP-38 ratio=1:20 (w/w). The resulting peptide mixture was separated by RP-HPLC on Waters Acquity UPLC HSS T3, 100 Å, 1.8 μm, 2.1×150 mm column with 0.10 vol % aq. TFA as mobile phase A and 0.09% TFA in acetonitrile as mobile phase B and detection at 210 nm. Flow rate was set to 0.28 mL/min, the column running temperature was 45° C. (±1° C.). The thermolysin fragments which contain aspartate (28) and methionine (33) were characterized via LC-MS. In routine analysis, fragments are compared to a reference mixture containing these fragment peptides. The fragments were quantified based on their respective peak area relative to the peak area of the corresponding unmodified fragments. Under the conditions of the thermolysin digest and subsequent RP-HPLC, formation of the isoaspartate product of aspartate (28) and oxidation of the methionine (33) could be quantified for (1).

[1073] Biopotency analysis was carried out by in vitro release of CNP-38 from (1) and subsequent biopotency analysis of released CNP-38 in a cell assay.

[1074] In vitro release of CNP-38 from (1) and RP-HPLC quantification: (1) was incubated at pH 10.0 and 15° C. for 24 h. pH was adjusted by dilution of reconstituted formulation with a release buffer (0.5 M boric acid, 10 mM methionine, 2.383 g/L Pluronic-F68; pH adjusted to 10.0 using 4M aqueous NaOH solution) in a 1:3.5 (v/v) dilution (e.g. 50 μL of a 3.6 mg CNP-38 eq./mL solution of (1) were diluted with 175 μL release buffer). After incubation, the release was quenched by dilution 1:1 (v/v) with 5% (v/v) acetic acid. The amount of released CNP-38 was determined by peak area comparison to a CNP-38 reference solution with known content.

[1075] Determination of biopotency of released CNP-38 by functional cGMP stimulation in HEK293 cells: ANPR-B overexpressing Hek293 cell line was developed as follows: The coding region of the NPR-B reference sequence was cloned into a lentiviral vector under a CMV promoter for constitutive receptor expression. A bicistronic element located on the vector for puromycin resistance was used as eukaryotic selection marker. After transduction, stably growing cell pools were subjected to qRT-PCR for confirmation of receptor mRNA-expression compared to parental Hek293 cells. Stimulation of the NPR-B receptor with CNP-38 leads to intracellular production of the second messenger cGMP which is detected with a commercially available cGMP assay.

[1076] Cells were routinely cultured in DMEM/Glutamax/HEPES medium with 10% FBS and 1% puromycin solution (370 μg/mL) at 37° C. and 5% CO.sub.2. For each assay, cells were suspended in DMEM+2% BSA and incubated 3 hours at 37° C. and 5% CO.sub.2. A dilution series of released CNP mixture in stimulation buffer (DMEM+2% BSA+0.1 mM IBMX+0.1% Tween®-20) was prepared and added to the cells (additional 1:2 dilution of CNP-38 serial dilution). After incubation for 60 min at 37° C. and 5% CO.sub.2, the cells were lyzed and cGMP levels were determined with a commercially available cGMP TR-FRET assay (Cisbio, cGMP kit, Cat. No. 62GM2PEB). Potency was determined using four parameter logistic curve fitting in a validated PLA software by parallel line analysis compared to a CNP-38 reference standard.

[1077] RP-HPLC was used to determine the amount of degraded CNP-38 (deamidated and aspartimide variants). Mobile phase A was composed of 0.05% aqueous TFA and mobile phase B was composed of 0.04% TFA in acetonitrile. A Waters Acquity HSS T3 C18, 100 Å, 1.8 μm, 2.1×100 mm column was used. Flow rate was set to 0.5 mL/min, detection was at a wavelength of 215 nm, the column running temperature was set to 30° C. The deamidated and aspartimide variants were quantified relative to the peak area of unmodified CNP-38.

Example 1: Synthesis of Compound (1)

[1078] ##STR00132##

[1079] Compound (1) was synthesized as described in WO2017/118693 for conjugate 11i.

Example 2: Stability Testing of Lyophilized Formulations Containing Compound (1)

[1080] Eight different formulations (F1-F8) containing compound (1) were prepared as shown by Table 1 and 200 μL of each formulation was filled into vials and lyophilized. The water contents of the lyophilizates were below 0.4% in all formulations, as determined by Karl-Fischer titration. Each formulation contained appropriate amounts of compound (1) to yield a concentration of 3.6 mg CNP-38/ml after reconstitution with 200 μL water. Formulations were placed in an incubator set to maintain 25° C. or 40° C. After 3 months (T3M), the formulations were reconstituted with water and subjected to analysis. Results show a favorable purity profile of compound (1) for the trehalose containing formulations compared to the mannitol containing formulations. Also, it was observed that no additives are required in order to prevent methionine oxidation.

TABLE-US-00038 TABLE 1 Content of (1) (mg pH- Trehalose CNP- adjusting dihydrate Mannitol Anneal- 38/mL) Buffer buffer pH (mg/mL) (mg/mL) ing F1 3.6 Succinic Tris 4.5 94 — No acid (10 mM) F2 3.6 Succinic Tris 5.0 94 — No acid (10 mM) F3 3.6 Succinic Tris 5.5 94 — No acid (10 mM) F4 3.6 Succinic Tris 6.0 94 — No acid (10 mM) F5 3.6 Histidine Tris 5.0 94 — No (10 mM) F6 3.6 Histidine Tris 6.0 94 — No (10 mM) F7 3.6 Succinic Tris 5.0 — 46 Yes acid (10 mM) F8 3.6 Succinic Tris 6.0 — 46 Yes acid (10 mM)

TABLE-US-00039 TABLE 2 Peptide Peptide mapping, mapping, Sample Purity by Free Purity by Met[O] Isoasp storage RP-HPLC CNP SE-HPLC content content conditions (%) (%) (%) (%) (%) F1 T0 98.9 <LOQ.sup.a 95.1 0.9 2.0 T3M, 25° C. 98.8 <LOQ.sup.a 95.4 1.1 2.0 T3M, 40° C. 97.4 0.2 94.0 1.2 2.6 F2 T0 98.8 <LOQ.sup.a 94.9 1.0 2.2 T3M, 25° C. 98.6 <LOQ.sup.a 95.4 1.1 2.0 T3M, 40° C. 97.6 0.1 93.9 1.4 2.4 F3 T0 98.8 <LOQ.sup.a 95.2 0.8 2.3 T3M, 25° C. 98.6 <LOQ.sup.a 95.7 0.9 2.0 T3M, 40° C. 97.6 0.2 94.1 1.3 1.8 F4 T0 98.8 <LOQ.sup.a 95.1 0.9 2.2 T3M, 25° C. 98.6 <LOQ.sup.a 95.1 1.0 2.1 T3M, 40° C. 98.1 0.1 94.2 1.3 1.8 F5 T0 98.7 <LOQ.sup.a 95.2 0.8 2.2 T3M, 25° C. 98.6 <LOQ.sup.a 95.5 1.1 1.6 T3M, 40° C. 98.3 0.1 94.1 1.1 1.7 F6 T0 98.6 <LOQ.sup.a 95.1 0.9 2.2 T3M, 25° C. 98.9 <LOQ.sup.a 94.8 1.1 2.1 T3M, 40° C. 98.2 0.2 93.0 1.4 2.1 F7 T0 98.7 <LOQ.sup.a 95.1 0.8 1.9 T3M, 25° C. 97.3 0.2 94.1 1.0 2.3 T3M, 40° C. 89.7 3.2 91.2 1.5 6.7 F8 T0 98.4 <LOQ.sup.a 94.9 0.8 2.2 T3M, 25° C. 96.4 0.5 93.7 1.0 1.8 T3M, 40° C. 80.4 6.1 85.2 1.5 2.0 .sup.aLOQ = 0.1%

Example 3: Stability Testing of Liquid and Lyophilized Formulations Containing Compound (1)

[1081] A formulation (F9) of (1) was prepared and 200 μL of the formulation was filled into a vial and lyophilized. The lyophilized formulation contained appropriate amounts of to yield a concentration of 3.0 mg CNP-38/ml after reconstitution with 200 μL water. The lyophilizate and the liquid formulation that was used for the preparation of the lyophilizate were placed in an incubator set to maintain 40° C.

[1082] After 21 days (T21D), the lyophilizate was reconstituted with water and the formulations were subjected to analysis. Results show that lyophilization protects (1) from degradation like methionine oxidation and isoaspartate formation.

TABLE-US-00040 TABLE 3 Content Trehalose of (1) dihydrate (mg CNP-38/mL) Buffer Base pH (mg/mL) F9 3.0 Succinic acid (10 mM) Tris 5.0 94

TABLE-US-00041 TABLE 4 Peptide Peptide Sample mapping, mapping, storage Met[O] IsoAsp conditions content (%) content (%) F9 T0 <LOQ.sup.a <LOQ.sup.a T21D, 40° C., lyo <LOQ.sup.a <LOQ.sup.a T21D, 40° C., liquid 10.5 6.9 .sup.aLOQ = 1.2%

Example 4: Stability Testing of Formulations Containing (1) and a Surfactant

[1083] Three different formulations (F10-F12) of (1) were prepared and 1080 μL was filled into vials and lyophilized. Each formulation contained appropriate amounts of (1) to yield a concentration of 3.5 mg CNP-38/ml after reconstitution with 1000 μL water. Some formulations contained Polysorbate 20 (PS 20) or Polysorbate 80 (PS 80) as a surfactant. Lyophilized formulations were placed in an incubator set to maintain 40° C./75% RH. After 1 month (TIM) and 3 months (T3M), formulations were reconstituted with 1 mL water and subjected to analysis.

[1084] The surfactant containing formulations F11 and F12 show a slight increase in methionine oxidation of (1) at stressed conditions and a marked effect on reconstitution time (without shaking) after addition of 1 mL water compared to F10.

TABLE-US-00042 TABLE 5 Content of (1) (mg pH- Trehalose CNP-38/ adjusting dihydrate PS 20 PS 80 mL) Buffer buffer pH (mg/mL) (%) (%) F10 3.5 Succinic acid Tris 5.0 84 — — (10 mM) F11 3.5 Succinic acid Tris 5.0 84 0.2 — (10 mM) F12 3.5 Succinic acid Tris 5.0 84 — 0.2 (10 mM)

TABLE-US-00043 TABLE 6 Purity by Peptide Peptide Relative Sample RP- Free Purity by mapping, mapping, reconstitution storage HPLC CNP SE-HPLC Met[O] Isoasp time.sup.c conditions (%) (%) (%) content (%) content (%) (%) F10 T0 99.4 <LOQ.sup.a 98.9 <LOQ.sup.b <LOQ.sup.b 100 T1M, 40° C. 99.2 <LOQ.sup.a 96.4 <LOQ.sup.b <LOQ.sup.b  95 T3M, 40° C. 99.2 0.14 95.3 <LOQ.sup.b <LOQ.sup.b  80 F11 T0 99.5 <LOQ.sup.a 99.1 <LOQ.sup.b <LOQ.sup.b 190 T1M, 40° C. 99.2 <LOQ.sup.a 96.4 <LOQ.sup.b <LOQ.sup.b 149 T3M, 40° C. 98.3 0.22 95.3 0.82 <LOQ.sup.b 186 F12 T0 99.4 <LOQ.sup.a 99.5 <LOQ.sup.b <LOQ.sup.b 161 T1M, 40° C. 98.4 <LOQ.sup.a 96.5 <LOQ.sup.b <LOQ.sup.b 157 T3M, 40° C. 96.6 0.19 95.8 1.02 <LOQ.sup.b 191 .sup.aLOQ = 0.1% .sup.bLOQ = 0.8-1.1% .sup.cwithout shaking

Example 5: Long Term Stability Testing of a Lyophilized Formulations Containing Compound (1)

[1085] A lyophilized formulation (F13) of compound (1) was prepared, by lyophilizing of 1080 μL in a vial. The formulation contained appropriate-amounts of (1) to yield a nominal concentration of 3.9 mg CNP-38/vial and 3.6 mg CNP-38/ml after reconstitution with 1000 μL of water. Formulations were placed in an incubator set to maintain respectively 5° C. 25° C./60% RH, 30° C./65% RH and 40° C./75% RH. After 3 months (T3M), 4 months (T4M), 6 months (T6M), 9 months (T9M), 12 months (T12M), 18 months (T18M) and 24 months (T24M) formulations were reconstituted with 1000 μL of water and subjected to analysis.

[1086] Results show a high stability of compound (1) in the lyophilized formulation F13, as shown here for content, purity and biopotency. Moreover, the formulation showed a good stability when tested with relevant pharmacopeia methods. The lyophilized formulation was characterized by a white cake and no change in cake appearance was detected irrespective of storage. The residual moisture content was very low after lyophilization (0.03%) and only slightly increased during the study. There was no significant change of reconstitution time during storage. Visual inspection revealed that the samples were practically free of visible particles throughout the study for all samples and a low amount of subvisible particles, i.e. ≥25 μm and 71≥10 μm were observed irrespective of storage temperature and storage time (determined with flow-through microscopy). A slight change in color from B9 to >B8 was observed during 12 months storage at 5° C., whereas no increase in color was observed during 6 months storage at 25° C. and 30° C. or 3 months at 40° C. (color was assessed with a spectral colorimeter and the absolute values of the color determination were conducted in accordance to the European Pharmacopoeia, 8.sup.th Edition, monograph 2.2.2). The turbidity (measured with a turbidimeter) of the lyophilized samples stored for up to twelve months at 2-8° C., 25° C./60% r.h. and 30° C./65% r.h., was unchanged. Only a minimal increase of the turbidity was measured for the sample stored for three months at 40° C./75% r.h. No changes in appearance were observed during storage for up to 12 months at 5° C., with values <1 NTU (Nephelometric turbidity unit). During 6 months at 25° C., 30° C. and 40° C. values <1 NTU were also obtained. pH was unaffected by storage. Finally, the osmolality at T0 was within the physiological range and no change was detected during the study.

TABLE-US-00044 TABLE 7 Content of (1) (nominal, Trehalose mg CNP-38 pH-adjusting dihydrate eq./mL) Buffer buffer pH (mg/mL) F13 3.6 Succinic acid Tris 5.0 84 (10 mM)

TABLE-US-00045 TABLE 8 Content Peptide Peptide of (1) Purity Purity mapping, mapping, Sample (CNP-38 by RP- Free by SE- Met[O] IsoAsp storage eq, HPLC CNP HPLC content content Biopotency conditions mg/mL) (%) (%) (%) (%) (%) (%) T0 3.5 97.6 <LOQ.sup.a 98.3 <LOQ.sup.b <LOQ.sup.b  93  T3M, 5° C. 98.4 <LOQ.sup.b <LOQ.sup.b 107  T4M, 5° C. 3.5 97.3 <LOQ.sup.a  T6M, 5° C. 3.5 97.4 <LOQ.sup.a 98.3 <LOQ.sup.b <LOQ.sup.b  95  T9M, 5° C. 3.5 97.3 <LOQ.sup.a 98.2 <LOQ.sup.b <LOQ.sup.b  T12M, 5° C.  3.5 97.1 <LOQ.sup.a 98.2 <LOQ.sup.b <LOQ.sup.b  90, 100.sup.c  T18M, 5° C.  3.5 97.1 <LOQ.sup.a 98.3 <LOQ.sup.b <LOQb 109, 104.sup.c  T24M, 5° C.  3.5 97.1 <LOQ.sup.a 98.3 <LOQ.sup.b <LOQ.sup.b 100, 110.sup.c T0 3.5 97.6 <LOQ.sup.a 98.3 <LOQ.sup.b <LOQ.sup.b T3M, 25° C. 97.8 <LOQ.sup.b <LOQ.sup.b  91 T4M, 25° C. 3.5 97.2 <LOQ.sup.a  T6M 25° C. 3.5 97.1 <LOQ.sup.a 97.4 <LOQ.sup.b <LOQ.sup.b 106 T0 3.5 97.6 <LOQ.sup.a 98.3 <LOQ.sup.b <LOQ.sup.b T3M, 30° C. 97.3 <LOQ.sup.b <LOQ.sup.b  96 T4M, 30° C. 3.4 97.1 <LOQ.sup.a T6M, 30° C. 3.5 97.0 <LOQ.sup.a 96.9 <LOQ.sup.b <LOQ.sup.b  89 T0 3.5 97.6 <LOQ.sup.a 98.3 <LOQ.sup.b <LOQ.sup.b T3M, 40° C. 96.4 <LOQ.sup.b <LOQ.sup.b  94 T4M, 40° C. 3.5 96.4 0.25 .sup.aLOQ = 0.1% .sup.bLOQ = 1% .sup.canalysis in duplicate

Example 6: Stability Testing of a Low Dose Formulation Containing Compound (1)

[1087] A lyophilized formulation (F14) of (1) was prepared, by lyophilization of 1060 μL in a vial. The formulation contained amounts of (1) to yield a nominal concentration of 0.80 mg CNP-38/vial and 0.75 mg CNP-38/ml after reconstitution. Formulations were placed in an incubator set to maintain 5° C., 25° C./60% RH, 30° C./65% RH and 40° C./75% RH. After 1 month (TIM), 3 months (T3M), 6 months (T6M) and 12 months (T12M), formulations were reconstituted with 1.0 mL of water and subjected to analysis.

[1088] Results show a high stability of compound (1) in the lyophilized formulation F14 as shown here for content, purity and biopotency. Moreover, the formulation showed a good stability when tested with relevant pharmacopeia methods. The lyophilized formulation was characterized by a white cake and no change in cake appearance was detected irrespective of storage. The residual moisture content was very low after lyophilization (<0.31%) and increased only slightly during the study. There was no significant change of reconstitution time during storage. Visual inspection revealed that the reconstituted solution was essentially free of visible particles (assessed in accordance with Ph. Eur. 2.9.20 and USP <790>) at the available time points. Moreover, for all samples and a low amount of subvisible particles, i.e. 0≥25 μm and 5≥10 μm were observed irrespective of storage temperature and storage time (determined with flow-through microscopy). A slight increase in colour from =WFI to <B9 was observed after 3 months storage at 30° C. and 40° C. (determined in accordance with Ph. Eur. 2.2.2 using the b-scale). Clarity was not affected during the 3 months storage and the reconstituted solution was found to be =WFI (determined in accordance with Ph. Eur. 2.2.1). pH was unaffected by storage. Finally, the osmolality at T0 was within the physiological range and no change was detected during the 3 months.

TABLE-US-00046 TABLE 9 Content of (1) (nominal, Trehalose mg CNP-38 pH-adjusting dihydrate eq./mL) Buffer buffer pH (mg/mL) F14 0.75 Succinic acid Tris 5.0 89 (10 mM)

TABLE-US-00047 TABLE 10 Content Peptide Peptide of (1) Purity Purity mapping, mapping, Sample (CNP- by RP- Free by SE- Met[O] IsoAsp storage 38 eq, HPLC CNP HPLC content content conditions mg/mL) (%) (%) (%) (%) (%) T0 0.70 99.2 .sup. ND.sup.a 98.0 <LOQ.sup.c <LOQ.sup.c  T3M, 5° C. 0.74 98.9 ND 97.7 <LOQ.sup.c <LOQ.sup.c  T6M, 5° C. 0.75 98.8 ND 98.0 <LOQ.sup.c <LOQ.sup.c T12M, 5° C. 0.74 98.9 ND 98.0 <LOQ.sup.c <LOQ.sup.c T0 0.70 99.2 ND 98.0 <LOQ.sup.c <LOQ.sup.c T1M, 25° C. 0.74 98.9 ND 97.3 <LOQ.sup.c <LOQ.sup.c T3M, 25° C. 0.74 98.8 ND 97.1 <LOQ.sup.c <LOQ.sup.c T6M, 25° C. 0.73 98.7 ND 97.1 <LOQ.sup.c <LOQ.sup.c T0 0.70 99.2 ND 98.0 <LOQ.sup.c <LOQ.sup.c T1M, 30° C. 0.74 98.8 ND 96.9 <LOQ.sup.c <LOQ.sup.c T3M, 30° C. 0.74 98.7 <LOQ.sup.b 96.7 <LOQ.sup.c <LOQ.sup.c T6M, 30° C. 0.75 98.4 <LOQ.sup.b 96.6 <LOQ.sup.c <LOQ.sup.c T0 0.70 99.2 ND 98.0 <LOQ.sup.c <LOQ.sup.c T1M, 40° C. 0.76 98.3 0.10 96.2 <LOQ.sup.c <LOQ.sup.c T3M, 40° C. 0.73 97.9 0.30 95.6 <LOQ.sup.c <LOQ.sup.c T6M, 40° C. 0.73 97.2 0.47 95.6 <LOQ.sup.c <LOQ.sup.c .sup.aND, not detected .sup.bLOQ = 0.10% .sup.cLOQ = 2.0%

Example 7: Stability of CNP-38 at Different pH Values

[1089] A preformulation study was carried out to assess the stability of CNP-38 at different pH values. CNP-38 (synthesized as described in WO2017/118693) was incubated at approx. 1 mg/mL in solution in succinate buffer at pH 4.0, 4.5, 5.0, 5.5 and 6.0 at 37° C. The amount of degraded CNP-38 (sum of deamidated and aspartimide variants of CNP-38) was assessed after 7 d by RP-HPLC. The amount of degraded CNP-38 was 2.7% at pH 4.0, 1.6% at pH 4.5, 1.0% at pH 5.0, 2.9% at pH 5.5 and 3.3% at pH 6.0, indicating a preferred pH of 5.0 for CNP-38 formulations.

Example 8: Stability Testing of a Formulation Containing Compound (1) Reconstituted with Water Containing an Antioxidant and/or a Preservative

[1090] Lyophilized formulation (F13) of compound (1) was prepared, by lyophilizing of 1080 μL in a vial. The formulation contained appropriate-amounts of (1) to yield a nominal concentration of 3.9 mg CNP-38/vial and 3.6 mg CNP-38/ml after reconstitution with 1000 μL of water. The water contained an antioxidant and/or a preservative (R1-R5, Table 11). The vials containing reconstituted formulations were placed in an incubator set to maintain 5° C. After 4 weeks (T4W), samples were subjected to analysis.

[1091] Results show that compound (1) is stable after reconstitution under the conditions tested and not affected by the presence of an antioxidant and/or preservative (Table 12). The pH of the reconstituted solutions was not affected by antioxidant and/or preservative after reconstitution.

TABLE-US-00048 TABLE 11 Methionine m-Cresol Phenol R1 — — — R2 10 mM 0.3% (w/w) — R3 10 mM — 0.3% (w/w) R4 — 0.3% (w/w) — R5 — — 0.3% (w/w)

TABLE-US-00049 TABLE 12 Peptide Peptide Purity Purity mapping, mapping, Sample by RP- Free by SE- Met[O] Isoasp storage HPLC CNP HPLC content content conditions (%) (%) (%) (%) (%) R1 T0 98.3 <LOQ.sup.a 97.9 <LOQ.sup.b <LOQ.sup.b T4W, 5° C. 98.3 <LOQ.sup.a 98.0 <LOQ.sup.b <LOQ.sup.b R2 T0 98.4 <LOQ.sup.a 98.0 <LOQ.sup.b <LOQ.sup.b T4W, 5° C. 98.4 <LOQ.sup.a 97.8 <LOQ.sup.b <LOQ.sup.b R3 T0 98.4 <LOQ.sup.a 98.1 <LOQ.sup.b <LOQ.sup.b T4W, 5° C. 98.3 <LOQ.sup.a 98.0 <LOQ.sup.b <LOQ.sup.b R4 T0 98.3 <LOQ.sup.a 98.1 <LOQ.sup.b <LOQ.sup.b T4W, 5° C. 98.3 <LOQ.sup.a 98.0 <LOQ.sup.b <LOQ.sup.b R5 T0 98.4 <LOQ.sup.a 98.0 <LOQ.sup.b <LOQ.sup.b T4W, 5° C. 98.4 <LOQ.sup.a 97.7 <LOQ.sup.b <LOQ.sup.b .sup.aLOQ = 0.10% .sup.bLOQ = 1.0%

Abbreviations

[1092] BSA—bovine serum albumin [1093] cGMP—cyclic guanosine monophosphate [1094] CI—Confidence interval [1095] DMEM—Dulbecco's Modified Eagle Medium [1096] FBS—fetal bovine serum [1097] HEPES—4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [1098] IBMX—3-Isobutyl-1-methylxanthin [1099] Isoasp—Isoaspartate [1100] LC-MS—liquid chromatography-coupled mass spectrometry [1101] Met(O)—methionine sulfoxide [1102] PLA—parallel line analysis [1103] PS 20—Polysorbate 20 [1104] PS 80—Polysorbate 80 [1105] RH—relative humidity [1106] RP-HPLC—reversed phase high performance liquid chromatography [1107] SE-HPLC—Size exclusion high performance liquid chromatography [1108] TFA—trifluoroacetic acid [1109] TR-FRET—time-resolved fluorescence energy transfer [1110] UPLC—ultra performance liquid chromatography