Combination Therapy with Controlled-Release CNP Agonists

Abstract

The present invention relates to a combination of a CNP agonist and at least one further biologically active moiety or drug for use in a method for the treatment or prevention of disorders that benefit from stimulating growth, pharmaceutical compositions comprising at least one CNP agonist, preferably controlled-release CNP agonist, wherein the pharmaceutical composition comprises at least one further biologically active moiety or drug, to using these pharmaceutical compositions as a medicament, to their use in the treatment of disorders that benefit from stimulating growth and to methods of preventing or treating a patient having a disorder that benefits from stimulating growth.

Claims

1-16. (canceled)

17. A pharmaceutical composition comprising at least one controlled-release CNP agonist, wherein the pharmaceutical composition comprises at least one further biologically active moiety or drug.

18. The pharmaceutical composition of claim 17, wherein the CNP agonist is or comprises a CNP agonist selected from the group consisting of small molecules, natural products, oligonucleotides, polypeptides and proteins.

19. The pharmaceutical composition of claim 17, wherein the CNP agonist is a CNP having the sequence of SEQ ID NO:2, 19, 20, 21, 22, 23, 24, 25, 26, 30, 32, 38, 39, 40, 41, 42, 43, 91 or 92.

20. The pharmaceutical composition of claim 17, wherein the CNP agonist is a controlled-release CNP agonist.

21. The pharmaceutical composition of claim 17, wherein the at least one further biologically active moiety or drug is selected from the group consisting of antihistamins; human anti-FGFR3 antibodies; soluble forms of human fibroblast growth factor receptor 3; tyrosine kinase inhibitors; statins; CNP agonists; growth hormone; IGF-1; ANP; BNP; inhibitors of peptidases and proteases; and inhibitors of NPR-C.

22. The pharmaceutical composition of claim 17, wherein the at least one further biologically active moiety or drug is human growth hormone.

23. The pharmaceutical composition of claim 17, wherein the at least one further biologically active moiety or drug is a drug in its free form.

24. The pharmaceutical composition of claim 17, wherein the at least one further biologically active moiety or drug is in the form of a stable conjugate.

25. The pharmaceutical composition of claim 17, wherein the at least one further biologically active moiety or drug is in the form of a controlled-release compound.

26. The pharmaceutical composition of claim 17, wherein the CNP agonist and the at least one further biologically active moiety or drug are formulated for simultaneous, separate or sequential administration.

27. The pharmaceutical composition of claim 17, wherein the CNP agonist and the at least one further biologically active moiety or drug are formulated in one pharmaceutical composition for simultaneous administration.

28. The pharmaceutical composition of claim 17, wherein the CNP agonist is a water-insoluble controlled-release CNP agonist.

29. The pharmaceutical composition of claim 17, wherein CNP agonist is a water-soluble controlled-release CNP agonist.

30. The pharmaceutical composition of claim 17, the CNP agonist is a controlled-release CNP agonist of 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 agonist moiety; -L.sup.1- is a reversible prodrug linker moiety; -L.sup.2- is a single chemical bond or a spacer moiety; Z is a water-soluble carrier 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 or 16; and y is an integer selected from the group consisting of 1, 2, 3, 4 and 5.

31. The pharmaceutical composition of claim 17, wherein the CNP agonist is a controlled-release CNP agonist of formula (IIf a) ##STR00165## wherein the unmarked dashed line indicates the attachment to the nitrogen provided by the side chain of the lysine at position 26 of the CNP moiety of SEQ ID NO:24 by forming an amide bond; and the dashed line marked with the asterisk indicates attachment to Z having the structure ##STR00166## wherein k is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12; each Z.sup.a is ##STR00167## wherein each c1 is an integer independently ranging from 200 to 250.

32-35. (canceled)

36. A method of treating or preventing a patient having a disorder that benefits from stimulating growth, the method comprising administering to the patient an effective amount of a combination of a CNP agonist and at least one further biologically active moiety or drug.

37. The method of claim 36, wherein the CNP agonist is or comprises a CNP agonist selected from the group consisting of small molecules, natural products, oligonucleotides, polypeptides and proteins.

38. The method of claim 36, wherein the CNP agonist is a CNP having the sequence of SEQ ID NO:2, 19, 20, 21, 22, 23, 24, 25, 26, 30, 32, 38, 39, 40, 41, 42, 43, 91 or 92.

39. The method of claim 36, wherein the CNP agonist is a controlled-release CNP agonist.

40. The method of claim 36, wherein the at least one further biologically active moiety or drug is selected from the group consisting of antihistamins; human anti-FGFR3 antibodies; soluble forms of human fibroblast growth factor receptor 3; tyrosine kinase inhibitors; statins; CNP agonists; growth hormone; IGF-1; ANP; BNP; inhibitors of peptidases and proteases; and inhibitors of NPR-C.

41. The method of claim 36, wherein the at least one further biologically active moiety or drug is human growth hormone.

42. The method of claim 36, wherein the at least one further biologically active moiety or drug is a drug in its free form.

43. The method of claim 36, wherein the at least one further biologically active moiety or drug is in the form of a stable conjugate.

44. The method of claim 36, wherein the at least one further biologically active moiety or drug is a controlled-release compound.

45. The method of claim 36, wherein the CNP agonist and the at least one further biologically active moiety or drug are formulated for simultaneous, separate or sequential administration.

46. The method of claim 36, wherein the CNP agonist and the at least one further biologically active moiety or drug are formulated in one pharmaceutical composition for simultaneous administration.

47. The method of claim 36, wherein the disorder that benefits from stimulating growth are selected from the group comprising achondroplasia, hypochondroplasia, short stature, dwarfism, osteochondrodysplasias, thanatophoric dysplasia, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozygous achondroplasia, camptomelic dysplasia, congenital lethal hypophosphatasia, perinatal lethal type of osteogenesis imperfecta, short-rib polydactyly syndromes, rhizomelic type of chondrodysplasia punctata, Jansen-type metaphyseal dysplasia, spondyloepiphyseal dysplasia congenita, atelosteogenesis, diastrophic dysplasia, congenital short femur, Langer-type mesomelic dysplasia, Nievergelt-type mesomelic dysplasia, Robinow syndrome, Reinhardt syndrome, acrodysostosis, peripheral dysostosis, Kniest dysplasia, fibrochondrogenesis, Roberts syndrome, acromesomelic dysplasia, micromelia, Morquio syndrome, Kniest syndrome, metatrophic dysplasia, and spondyl oepimetaphyseal dysplasia.

48. The method of claim 36, wherein the disorder that benefits from stimulating growth is achondroplasia.

49. The method of claim 36, wherein the controlled-release CNP agonist is water-soluble.

50. The method of claim 36, wherein CNP agonist is a controlled-release CNP agonist of 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 agonist moiety; -L1- is a reversible prodrug linker moiety; -L2- is a single chemical bond or a spacer moiety; Z is a water-soluble carrier 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 or 16; and y is an integer selected from the group consisting of 1, 2, 3, 4 and 5.

51. The method of claim 36, wherein the CNP agonist is a controlled-release CNP agonist of formula (IIf a) ##STR00168## wherein the unmarked dashed line indicates the attachment to the nitrogen provided by the side chain of the lysine at position 26 of the CNP moiety of SEQ ID NO:24 by forming an amide bond; and the dashed line marked with the asterisk indicates attachment to Z having the structure ##STR00169## wherein k is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12; each Za is ##STR00170## wherein each c1 is an integer independently ranging from 200 to 250.

Description

[1315] FIG. 1: Structure of CNP according to SEQ ID NO:1.

[1316] FIG. 2: Proliferative zone width (Pz.Wi; m) of right tibia measured using histomorphometry.

METHODS

[1317] Reversible Lys26 CNP-38 PEG410 kDa conjugate 1 was synthesized as described in WO2016/110577, example 11 (compound 11i).

EXAMPLES

Example 1

[1318] Administration of Reversible Lys26 CNP-38 PEG410 kDa Conjugate in Combination with Rosuvastatin is More Effective in Ameliorating the Achondroplasia Phenotype than Either Agent Alone

Method:

[1319] Rosuvastatin and the reversible Lys26 CNP-38 PEG410 kDa conjugate are administered by intraperitoneal or subcutaneous injection, respectively, to Fgfr3Y367C/+ mice from day 1 of birth and for a total of 15 days. Animals are sacrificed on day 16 and tissues fixed for histologic or immunohistochemical analysis. Either compound is administered alone or in combination. The dose levels employed for Rosuvastatin are 0.25, 0.5, 1.0 and 2.0 mg/kg. The reversible Lys26 CNP-38 PEG410 kDa conjugate is employed at dose levels of 1.5 3.0 and 6.0 mg/kg CNP equivalents. The reversible Lys26 CNP-38 PEG410 kDa conjugate/Rosuvastatin ratios are based on doses administered in mg/kg for Rosuvastatin or CNP equivalents for the reversible Lys26 CNP-38 PEG410 kDa conjugate/Rosuvastatin. The ratios ranges from 1:1-24:1

[1320] Reversion of phenotypical features are assessed using whole body autoradiography. The animals are placed on their right side, with the left hind leg more forward than the right, to allow both hind legs to be visible on the X-ray. Bones obtained at necropsy include femur, tibia, humerus, ulna, lumbar vertebra segment L4-6 and are measured with a caliper.

Results:

[1321] The combination of Rosuvastatin and the reversible Lys26 CNP-38 PEG410 kDa conjugate are found to increase body length and length of extremities. Amelioration of key relevant achondroplasia clinical features including bowed femur and tibia, anterior crossbite and domed skull are observed in treated animals. The beneficial effect on reversal of the achondroplasic phenotype is observed in both animals treated with a single agent and in combination. Combination of reversible Lys26 CNP-38 PEG410 kDa conjugate and Rosuvastatin are found to be superior in efficacy to an equivalent dose of the reversible Lys26 CNP-38 PEG410 kDa conjugate or Rosuvastatin administered alone.

Example 2

[1322] Administration of Lys26 CNP-38 PEG410 kDa Conjugate (TransCon CNP, ACP-015) in Combination with Somatropin is More Effective in Growth Induction in Hypophysectomized Rats than Either Agent Alone

Method:

[1323] This study was performed in order to test and compare the effect of Lys26 CNP-38 PEG410 kDa conjugate, Somatropin and combinations of the two compounds in an animal model relevant for investigating treatment of growth deficiency. The Somatropin (human growth hormone, hGH) was a United States Pharmacopeia (USP) Reference Standard and was prepared according to supplier instructions. Lys26 CNP-38 PEG410 kDa conjugate and Somatropin were administered alone or in combination via subcutaneous injection to hypophysectomized (HYPOX) SPF Sprague Dawley rats for a total of 29 days. The animals were dosed daily with Somatropin or vehicle, and weekly with Lys26 CNP-38 PEG410 kDa conjugate (at two different sites in animals receiving both test items on days 1, 8, 15, 22, and 29). The dose levels employed for Lys26 CNP-38 PEG410 kDa conjugate were 0.3, 1.0, and 2.0 mg/kg. Somatropin was employed at 10 L/animal with a concentration of 10 g/mL (8-g/kg with animal weights between 100-120 g during the study period). The animals were weighed predose and once daily during the dosing period. Body weight gain was calculated from these data. Animals were sacrificed on day 30, and femur and tibia were trimmed and their length measured using a caliper. Hereafter, right tibia was fixed and processed for histologic and histomorphometric analysis.

[1324] Results:

[1325] Significant body weight gains were observed for HYPOX rats treated with Somatropin or Lys26 CNP-38 PEG410 kDa conjugate alone compared to vehicle treated group. Administration of Lys26 CNP-38 PEG410 kDa conjugate and Somatropin in combination were found to increase body weight in an additive manner (Table 2).

TABLE-US-00011 TABLE 2 Body weight gain (BWG) from day 0 (predose) to day 28. Low dose = 0.3 mg/mL, mid dose = 1.0 mg/mL, high dose = 2.0 mg/mL. Average BWG (g) ( SE) Vehicle 0.78 (0.64) Somatropin 4.44 (0.94) Lys26 CNP-38 PEG4 10 kDa conjugate low dose 7.89 (1.53) Lys26 CNP-38 PEG4 10 kDa conjugate mid dose 8.44 (0.93) Lys26 CNP-38 PEG4 10 kDa conjugate high dose 8.89 (1.24) Somatropin + Lys26 CNP-38 PEG4 10 kDa conjugate 7.78 (1.17) low dose Somatropin + Lys26 CNP-38 PEG4 10 kDa conjugate 11.89 (1.23) mid dose Somatropin + Lys26 CNP-38 PEG4 10 kDa conjugate 13.00 (0.75) high dose

[1326] A dose related increase in mean longitudinal growth of long bones were found in Lys26 CNP-38 PEG410 kDa conjugate treated animals (femur: 0.950.20, 1.290.16, 1.810.17 mm, tibia: 1.430.28, 1.850.18, 2.060.18 mm for 0.3, 1.0, and 2.0 mg/kg Lys26 CNP-38 PEG410 kDa conjugate, respectively). Only a small increase in bone length was observed on tibia (0.550.17 mm) and no increase on femur (0.040.08 mm) in Somatropin treated animals relative to the vehicle group. Hence, it was a surprise to find that Somatropin enhanced the mean effect (30%) of 2.0 mg/Lys26 CNP-38 PEG410 kDa conjugate on femur length when the two compounds were administered in combination (2.350.24 mm).

[1327] A histologic evaluation examining the width of the endochondral/ossification zone after treatment with vehicle, Somatropin, Lys26 CNP-38 PEG410 kDa conjugate, or Somatropin+Lys26 CNP-38 PEG410 kDa conjugate demonstrated superior efficacy (growth potential) in the combination groups compared to groups that received Lys26 CNP-38 PEG410 kDa conjugate alone. The endochondral/ossification zone width in Somatropin treated animals did not differ from background (vehicle).

TABLE-US-00012 TABLE 3 Histologic evaluation of HE stained right tibia with focus on growth zone morphology. Decrease Increase Sum of Minimal Normal Minimal Mild Moderate grades Vehicle 2 7 Somatropin 9 Lys26 CNP-38 0 9 18 PEG4x10 kDa conjugate Low dose Lys26 CNP-38 0 9 18 PEG4x10 kDa conjugate Mid dose Lys26 CNP-38 0 7 2 20 PEG4x10 kDa conjugate High dose Somatropin + 0 9 18 Lys26 CNP-38 PEG4x10 kDa conjugate Low dose Somatropin + 0 5 4 22 Lys26 CNP-38 PEG4x10 kDa conjugate Mid dose Somatropin + 0 1 8 26 Lys26 CNP-38 PEG4x10 kDa conjugate High dose Width of the endochondral/ossification zone was classified using the following 5-grade system: normal = 0 (within normal range), minimal = 1, mild = 2, moderate = 3, marked = 4, and massive = 5. Number of animals observed in each group are listed within the categories. Sum of grades = (n animals .Math. grade 1) + (n animals .Math. grade 2) + (n animals .Math. grade 3).

[1328] Hence, it was a surprise to find the width of the endochondral/ossification zone increased by 30% in animals treated with the combination of Somatropin and 2.0 mg/mL Lys26 CNP-38 PEG410 kDa conjugate compared to animals treated with solely 2.0 mg/mL Lys26 CNP-38 PEG410 kDa conjugate (ACP-015) (Table 3).

[1329] These observations of surprising benefits from combining treatment of Lys26 CNP-38 PEG410 kDa conjugate and Somatropin were supported by histomorphometric measurements of the proliferative zone width in the same tibial growth plates. The width of the proliferative zone in Somatropin treated animals was not different from control (vehicle) animals. A dose-related increase in mean width (SE) of the proliferative zone was found in Lys26 CNP-38 PEG410 kDa conjugate treated animals (4.942.69, 17.696.57, 30.614.06 m for 0.3, 1.0, and 2.0 mg/kg Lys26 CNP-38 PEG410 kDa conjugate, respectively). A similar but further augmented dose related increase in mean width of the proliferative zone was found in animals treated with the combination of Somatropin and Lys26 CNP-38 PEG410 kDa conjugate (13.452.16, 30.054.27, 41.602.50 m for 0.3, 1.0, and 2.0 mg/kg Lys26 CNP-38 PEG410 kDa conjugate (ACP-015)+Somatropin, respectively). It was a surprise to find this increase in width in combination-treated animals compared to animals treated with only Lys26 CNP-38 PEG410 kDa conjugate since the tested dose of Somatropin on its own failed to stimulate the proliferative zone. Moreover, it was surprising to find that Lys26 CNP-38 PEG410 kDa conjugate and Somatropin in combination stimulated a response in all dosed animals compared to animals treated only with Lys26 CNP-38 PEG410 kDa conjugate where non- and low-responders were observed within the groups (FIG. 2). This was also reflected in the reduced distribution of individual responses within the combination treated groups compared to groups of animals treated only with Lys26 CNP-38 PEG410 kDa conjugate.

CONCLUSION

[1330] Combination of Lys26 CNP-38 PEG410 kDa conjugate and Somatropin were found to be superior in efficacy compared to Lys26 CNP-38 PEG410 kDa conjugate or Somatropin administered alone. The presented data shows a combinatorial effect from hGH and Lys26 CNP-38 PEG410 kDa conjugate treatment with both additive and surprising synergistic effects (above 25% an additive effect). Importantly, the combination of Lys26 CNP-38 PEG410 kDa conjugate and Somatropin were found to eliminate the non- and low-responders observed in groups of animals dosed only with Lys26 CNP-38 PEG410 kDa conjugate.