FORMULATION AND METHOD OF PREPARATION

Abstract

There is described formulations for human or animal administration and to a method of preparation thereof. In particular, there is described more stable pharmaceutical formulations, such as those for intravenous administration, and to a method of preparation thereof. There are also described lyophilised formulations having an active pharmaceutical ingredient (API), a buffering agent and a lyoprotectant, wherein the API is an imaging agent comprising at least one cMet binding peptide, suitable for optically imaging the mammalian body in vivo. Also described are a method of preparing a lyophilised formulation, a pharmaceutical composition, and a kit for the preparation of the pharmaceutical composition. Further described are methods of imaging using the formulation or pharmaceutical composition, such as in detection, diagnosis, surgery, staging, treatment, monitoring of treatment, monitoring of disease progression or monitoring therapy of conditions such as cancer.

Claims

1. A lyophilised formulation comprising: (i) an active pharmaceutical ingredient (API); (ii) a buffering agent; and (iii) a lyoprotectant; wherein the API is an imaging agent comprising at least one cMet binding peptide, suitable for optically imaging the mammalian body.

2. The lyophilised formulation of claim 1, wherein the imaging agent comprises at least one optical reporter suitable for imaging using light of green to near-infrared wavelength (400 to 1,200 nm).

3. The lyophilised formulation of claim 1 or claim 2, wherein imaging agent comprises a compound of Formula I: ##STR00018## wherein: Z.sup.1 is attached to the N-terminus of cMBP, and is H or M.sup.IG; Z.sup.2 is attached to the C-terminus of cMBP, and is OH, NH.sub.2, OB.sup.c or M.sup.IG; wherein B.sup.c is a biocompatible cation; cMBP is a cMet binding cyclic peptide of 17 to 30 amino acids, which comprises the amino acid sequence (SEQ-1): TABLE-US-00038 Cys.sup.a-Xaa.sup.1-Cys.sup.c-Xaa.sup.2-Gly-Pro-Pro-Xaa.sup.3-Phe-Glu-Cys.sup.d- Trp-Cys.sup.b-Tyr-Xaa.sup.4-Xaa.sup.5-Xaa.sup.6; wherein: Xaa.sup.1 is Asn, His or Tyr; Xaa.sup.2 is Gly, Ser, Thr or Asn; Xaa.sup.3 is Thr or Arg; Xaa.sup.4 is Ala, Asp, Glu, Gly or Ser; Xaa.sup.5 is Ser or Thr; Xaa.sup.6 is Asp or Glu; and Cys.sup.a-d are each cysteine residues such that residues a and b as well as c and d are cyclised to form two separate disulphide bonds; M.sup.IG is a metabolism inhibiting group, which is a biocompatible group that inhibits or suppresses in vivo metabolism of the peptide; L is a synthetic linker group of formula -(A)m- wherein each A is independently CR.sub.2, CRCR, CC, CR.sub.2CO.sub.2, CO.sub.2CR.sub.2, NRCO, CONR, NR(CO)NR, NR(CS)NR, SO.sub.2NR, NRSO.sub.2, CR.sub.2OCR.sub.2, CR.sub.2SCR.sub.2, CR.sub.2NRCR.sub.2, a C.sub.4-8 cycloheteroalkylene group, a C.sub.4-8 cycloalkylene group, a C.sub.5-12 arylene group, a C.sub.3-12 heteroarylene group, an amino acid, a sugar or a monodisperse polyethyleneglycol (PEG) building block; each R is independently chosen from H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.1-4 alkoxyalkyl or C.sub.1-4 hydroxyalkyl; m is an integer of value 1 to 20; n is an integer of value 0 or 1; IM is an optical reporter imaging moiety suitable for imaging the mammalian body using light of violet to near-infrared wavelength (400 to 1,200 nm).

4. The lyophilised formulation of claim 3, wherein in addition to SEQ-1, the cMBP further comprises an Asp or Glu residue, or an analogue thereof, within 4 amino acid residues of either C- or N-cMBP peptide terminus, and -(L).sub.nIM is functionalised with an amine group, which is conjugated to the carboxyl side chain of said Asp or Glu residue, or analogue thereof, to give an amide bond.

5. The lyophilised formulation of claim 3 or claim 4, wherein in addition to SEQ-1, the cMBP comprises a Lys residue, or an analogue thereof, within 4 amino acid residues of either C- or N-cMBP peptide terminus, and -(L).sub.nIM is functionalised with a carboxyl group, which is conjugated to the epsilon amine side chain of said Lys residue, or analogue thereof, to give an amide bond.

6. The lyophilised formulation of any one of claims 3 to 5, wherein cMBP comprises the amino acid sequence of either SEQ-2 or SEQ-3: TABLE-US-00039 (SEQ-2) Ser-Cys.sup.a-Xaa.sup.1-Cys.sup.c-Xaa.sup.2-Gly-Pro-Pro-Xaa.sup.3-Phe-Glu- Cys.sup.d-Trp-Cys.sup.b-Tyr-Xaa.sup.4-Xaa.sup.5-Xaa.sup.6; (SEQ-3) Ala-Gly-Ser-Cys.sup.a-Xaa.sup.1-Cys.sup.c-Xaa.sup.2-Gly-Pro-Pro-Xaa.sup.3- Phe-Glu-Cys.sup.d-Trp-Cysb-Tyr-Xaa.sup.4-Xaa.sup.5-Xaa.sup.6-Gly-Thr.

7. The lyophilised formulation of any one of claims 3 to 6, wherein Xaa.sup.3 is Arg.

8. The lyophilised formulation of any one of claims 3 to 7, wherein in addition to SEQ-1, SEQ-2 or SEQ-3, cMBP further comprises at either the N- or C-terminus a linker peptide, which is chosen from -Gly-Gly-Gly-Lys (SEQ-4), -Gly-Ser-Gly-Lys-(SEQ-5) and -Gly-Ser-Gly-Ser-Lys (SEQ-6).

9. The lyophilised formulation of any one of claims 3 to 8, wherein cMBP comprises the amino acid sequence (SEQ-7): TABLE-US-00040 Ala-Gly-Ser-Cys.sup.a-Tyr-Cys.sup.c-Ser-Gly-Pro-Pro-Arg-Phe- Glu-Cys.sup.d-Trp-Cys.sup.b-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly- Gly-Lys.

10. The lyophilised formulation of any one of claims 3 to 9, wherein both Z.sup.1 and Z.sup.2 are independently M.sup.IG.

11. The lyophilised formulation of any one of claims 3 to 10, wherein Z.sup.1 is acetyl and Z.sup.2 is a primary amide.

12. The lyophilised formulation of any one of claims 3 to 11, wherein n is 0.

13. The lyophilised formulation of any one of claims 3 to 12, wherein IM is a dye having an absorbance maximum in the range 600 to 1,000 nm.

14. The lyophilised formulation of claim 13, wherein IM is a cyanine dye.

15. The lyophilised formulation of claim 14, wherein the cyanine dye has Formula III: ##STR00019## wherein: R.sup.1 and R.sup.2 are independently H or SO.sub.3M.sup.1, and at least one of R.sup.1 and R.sup.2 is SO.sub.3M.sup.1, where M.sup.1 is H or B; R.sup.3 and R.sup.4 are independently C.sub.1-4 alkyl or C.sub.1-6 carboxyalkyl; R.sup.5, R.sup.6, R.sup.7 and R.sup.8 are independently R.sup.a groups; wherein R.sup.a is C.sub.1-4 alkyl, C.sub.1-6 carboxyalkyl or (CH.sub.2).sub.kSO.sub.3M.sup.1, where k is an integer of value 3 or 4; with the proviso that the cyanine dye has a total of 1 to 4 SO.sub.3M.sup.1 substituents in the R.sup.1, R.sup.2 and R.sup.a groups.

16. The lyophilised formulation of any preceding claim, wherein the formulation, when reconstituted, has a pH of between approximately pH 6.3 and approximately pH 9, optionally between approximately pH 6.3 and approximately pH 9, optionally between approximately pH 6.5 and approximately pH 8.5, optionally between approximately pH 6.8 and approximately pH 8.2, optionally between approximately pH 6.8 and approximately pH 8, optionally between approximately pH 7 and approximately pH 8.

17. The lyophilised formulation of any preceding claim, wherein the buffering agent is present in an amount to provide, when reconstituted, a solution having a pH of between approximately pH 6.3 and approximately pH 9, optionally between approximately pH 6.3 and approximately pH 9, optionally between approximately pH 6.5 and approximately pH 8.5, optionally between approximately pH 6.8 and approximately pH 8.2, optionally between approximately pH 6.8 and approximately pH 8, optionally between approximately pH 7 and approximately pH 8.

18. The lyophilised formulation of any preceding claim, wherein the mole ratio of the API:buffering agent is approximately 1 mole of API:approximately 17 to approximately 47 moles of buffering agent, optionally approximately 1 mole of API:approximately 27 to approximately 47 moles of buffering agent, optionally approximately 1 mole of API:approximately 30 to approximately 47 moles of buffering agent, optionally approximately 1 mole of API:approximately 34 to approximately 47 moles of buffering agent, optionally approximately 1 mole of API:approximately 27 to approximately 38 moles of buffering agent, optionally approximately 1 mole of API:approximately 30 to approximately 38 moles of buffering agent, optionally approximately 1 mole of API:approximately 34 to approximately 38 moles of buffering agent.

19. The lyophilised formulation of any preceding claim, wherein the mole ratio of API:lyoprotectant is approximately 1 mole API:approximately 105 to approximately 216 moles of lyoprotectant, optionally approximately 1 mole API:approximately 105 to approximately 163 moles of lyoprotectant, optionally approximately 1 mole API:approximately 105 to approximately 154 moles of lyoprotectant, optionally approximately 1 mole API:approximately 105 to approximately 145 moles of lyoprotectant, optionally approximately 1 mole API:approximately 105 to approximately 141 moles of lyoprotectant, optionally approximately 1 mole API:approximately 105 to approximately 132 moles of lyoprotectant, optionally approximately 1 mole API:approximately 105 to approximately 129 moles of lyoprotectant, optionally approximately 1 mole API:approximately 129 to approximately 216 moles of lyoprotectant, optionally approximately 1 mole API:approximately 129 to approximately 163 moles of lyoprotectant, optionally approximately 1 mole API:approximately 129 to approximately 154 moles of lyoprotectant, optionally approximately 1 mole API:approximately 129 to approximately 145 moles of lyoprotectant, optionally approximately 1 mole API:approximately 129 to approximately 141 moles of lyoprotectant, optionally approximately 1 mole API:approximately 129 to approximately 132 moles of lyoprotectant.

20. The lyophilised formulation of any preceding claim, wherein the formulation comprises from approximately 4 to approximately 12% by weight API, optionally approximately 8 to approximately 10% by weight API, optionally approximately 9% by weight API.

21. The lyophilised formulation of any preceding claim, wherein the formulation comprises from approximately 3 to approximately 35% by weight buffering agent, optionally from approximately 7 to approximately 35% by weight buffering agent, optionally from approximately 12 to approximately 35% by weight buffering agent, optionally from approximately 15 to approximately 35% by weight buffering agent, optionally from approximately 18 to approximately 35% by weight buffering agent, optionally from approximately 25 to approximately 35% by weight buffering agent, optionally from approximately 32 to approximately 35% by weight buffering agent, optionally approximately 32% by weight buffering agent.

22. The lyophilised formulation of any preceding claim, wherein the formulation comprises from approximately 56 to approximately 91% by weight lyoprotectant, optionally from approximately 56 to approximately 82% by weight lyoprotectant, optionally from approximately 56 to approximately 77% by weight lyoprotectant, optionally from approximately 56 to approximately 75% by weight lyoprotectant, optionally from approximately 56 to approximately 72% by weight lyoprotectant, optionally from approximately 56 to approximately 66% by weight lyoprotectant, optionally from approximately 56 to approximately 58% by weight lyoprotectant, optionally approximately 58% by weight lyoprotectant.

23. The lyophilised formulation of any preceding claim, wherein the buffering agent is at least one of a phosphate buffer and an alkanolamine buffer.

24. The lyophilised formulation of claim 23, wherein the phosphate buffer comprises hydrogen phosphate and dihydrogen phosphate, optionally wherein the phosphate buffer is hydrated.

25. The lyophilised formulation of claim 23 or claim 24, wherein the alkanolamine buffer is tris(hydroxymethyl)aminomethane.

26. The lyophilised formulation of any preceding claim, wherein the lyoprotectant is at least one of sucrose and mannitol, and derivatives thereof.

27. The lyophilised formulation of any preceding claim, wherein the lyoprotectant is mannitol, or a derivative thereof.

28. The lyophilised formulation of any preceding claim, wherein the formulation further comprises a tonicity regulator.

29. The lyophilised formulation of any preceding claim, wherein the lyoprotectant also acts as a tonicity regulator.

30. A method of preparing a lyophilised formulation, the method comprising the steps of: a) providing an active pharmaceutical ingredient (API), a buffering agent, and a lyoprotectant to a lyophilisation vessel; b) performing a first water removal step; and c) performing a second water removal step; wherein the lyoprotectant and the buffering agent are added before the lyophilisation is carried out.

31. The method of claim 30, wherein the first water removal step is carried out at a temperature of approximately 30 C. or lower, optionally approximately 35 C. or lower, optionally approximately 38 C. or lower.

32. The method of claim 30 or claim 31, wherein the second water removal step is carried out at a temperature of approximately 10 C. or higher, optionally approximately 15 C. or higher, optionally approximately 20 C. or higher.

33. The method of any one of claims 30 to 32, wherein at least one of the first and second water removal steps is carried out at a pressure of 50 bar or less.

34. The method of any one of claims 30 to 33, wherein both of the first and second water removal steps are carried out at a pressure of 50 bar or less.

35. The method of any one of claims 30 to 34, wherein the lyophilised formulation is the lyophilised formulation any one of claim 1 to 29.

36. A lyophilised formulation prepared by the method of any one of claims 30 to 34.

37. A pharmaceutical composition comprising the formulation of any one of claims 1 to 29 and a biocompatible carrier, in a form suitable for mammalian administration.

38. The pharmaceutical composition of claim 37, wherein the pharmaceutical composition has a pH of between approximately pH 6.3 and approximately pH 9, optionally between approximately pH 6.3 and approximately pH 9, optionally between approximately pH 6.5 and approximately pH 8.5, optionally between approximately pH 6.8 and approximately pH 8.2, optionally between approximately pH 7 and approximately pH 8.

39. A kit for the preparation of the pharmaceutical composition of claim 37 or claim 38, the kit comprising the formulation of any one of claim 1 to 29 in sterile, solid form such that upon reconstitution with a sterile supply of the biocompatible carrier of claim 37 or claim 38, dissolution occurs to give the desired pharmaceutical composition.

40. A method of imaging of the mammalian body comprising use of at least one of the formulation of any one of claim 1 to 29 and the pharmaceutical composition of claim 37 or claim 38.

41. The method of claim 40, wherein the imaging is in vivo.

42. The method of claim 40 or claim 41, wherein the imaging is optical imaging.

43. The method of any one of claims 40 to 42, wherein the imaging is to obtain images of sites of cMet over-expression or localisation

44. The method of any one of claims 40 to 43, wherein the formulation of any one of claim 1 to 29 or the pharmaceutical composition of claim 37 or claim 38 has been previously administered to the mammalian body.

45. The method of any one of claims 40 to 44, wherein the method comprises the steps of: a) illuminating a tissue surface of interest with an excitation light; b) detecting fluorescence from the imaging agent, which is generated by excitation of the imaging agent; c) optionally filtering the light detected by the fluorescence detector to separate out the fluorescent component; and d) forming an image of the tissue surface of interest from the fluorescent light of steps (b) or (c).

46. The method of claim 45, wherein the excitation light of step (a) is continuous wave (CW) in nature.

47. The method of any one of claims 40 to 44, wherein the method comprises the steps of: a) exposing light-scattering biologic tissue of said mammalian body having a heterogeneous composition to light from a light source with a pre-determined time varying intensity to excite the imaging agent, the tissue multiply-scattering the excitation light; b) detecting a multiply-scattered light emission from the tissue in response to said exposing; c) quantifying a fluorescence characteristic throughout the tissue from the emission by establishing a number of values with a processor, the values each corresponding to a level of the fluorescence characteristic at a different position within the tissue, the level of the fluorescence characteristic varying with heterogeneous composition of the tissue; and d) generating an image of the tissue by mapping the heterogeneous composition of the tissue in accordance with the values of step (c).

48. The method of any one of claims 40 to 47, wherein the optical imaging method comprises fluorescence imaging, optionally fluorescence endoscopy.

49. The method of any one of claims 40 to 48, wherein the method is used to assist in detection, diagnosis, surgery, staging, treatment, monitoring of treatment, monitoring of disease progression or monitoring therapy.

50. The method of any one of claims 40 to 49, wherein the method is used to assist in detection, diagnosis, surgery, staging, treatment, monitoring of treatment, monitoring of disease progression or monitoring therapy of one or more of a precancerous condition and cancer, optionally one or more of colorectal cancer, oesophageal cancer, breast cancer, prostate cancer, head cancer, neck cancer, ovarian cancer, rectal cancer, pancreatic cancer, thyroid cancer, gastric cancer and sarcoma.

51. A method of detection, diagnosis, surgery, staging, treatment, monitoring of treatment, monitoring of disease progression or monitoring therapy comprising the imaging method of any one of claims 40 to 50.

52. The formulation of any one of claims 1 to 29, or the pharmaceutical composition of claim 37 or claim 38, for use as an imaging agent in imaging of the mammalian body.

53. The formulation of any one of claims 1 to 29, or the pharmaceutical composition of claim 37 or claim 38, for use as a medicament.

54. The formulation of any one of claims 1 to 29, or the pharmaceutical composition of claim 37 or claim 38, for use in detection, diagnosis, surgery, staging, treatment, monitoring of treatment, monitoring of disease progression or monitoring therapy.

55. The formulation of any one of claims 1 to 29, or the pharmaceutical composition of claim 37 or claim 38, for use in the detection, diagnosis, surgery, staging, treatment, monitoring of treatment, monitoring of disease progression or monitoring therapy of one or more of a precancerous condition and cancer, optionally one or more of colorectal cancer, oesophageal cancer, breast cancer, prostate cancer, head cancer, neck cancer, ovarian cancer, rectal cancer, pancreatic cancer, thyroid cancer, gastric cancer and sarcoma.

56. The formulation of any one of claims 1 to 29, or the pharmaceutical composition of claim 37 or claim 38, for use in the detection, diagnosis, surgery, staging, treatment, monitoring of treatment, monitoring of disease progression or monitoring therapy of sites of cMet over-expression or localisation.

57. The formulation of any one of claims 1 to 29, or the pharmaceutical composition of claim 37 or claim 38, for use in obtaining an image of sites of cMet over-expression or localisation, optionally in vivo.

58. A method of detection, diagnosis, surgery, staging, treatment, monitoring of treatment, monitoring of disease progression or monitoring therapy using at least one of the formulation of any one of claims 1 to 29, and the pharmaceutical composition of claim 37 or claim 38.

59. A method of imaging the mammalian body using at least one of the formulation of any one of claims 1 to 29, and the pharmaceutical composition of claim 37 or claim 38.

60. A method of detection, diagnosis, surgery, staging, treatment, monitoring of treatment, monitoring of disease progression or monitoring therapy of one or more of a precancerous condition and cancer, optionally one or more of colorectal cancer, oesophageal cancer, breast cancer, prostate cancer, head cancer, neck cancer, ovarian cancer, rectal cancer, pancreatic cancer, thyroid cancer, gastric cancer and sarcoma, using at least one of the formulation of any one of claims 1 to 29, and the pharmaceutical composition of claim 37 or claim 38.

61. A method of detection, diagnosis, surgery, staging, treatment, monitoring of treatment, monitoring of disease progression or monitoring therapy of sites of cMet over-expression or localisation using at least one of the formulation of any one of claims 1 to 29, and the pharmaceutical composition of claim 37 or claim 38.

62. A method of obtaining an image of sites of cMet over-expression or localisation, optionally in vivo, using at least one of the formulation of any one of claims 1 to 29, and the pharmaceutical composition of claim 37 or claim 38.

63. The use of at least one of the formulation of any one of claims 1 to 29, and the pharmaceutical composition of claim 37 or claim 38.

64. The use of at least one of the formulation of any one of claims 1 to 29, and the pharmaceutical composition of claim 37 or claim 38, in detection, diagnosis, surgery, staging, treatment, monitoring of treatment, monitoring of disease progression or monitoring therapy.

65. The use of at least one of the formulation of any one of claims 1 to 29, and the pharmaceutical composition of claim 37 or claim 38, as an imaging agent.

66. The use of at least one of the formulation of any one of claims 1 to 29, and the pharmaceutical composition of claim 37 or claim 38, in the detection, diagnosis, surgery, staging, treatment, monitoring of treatment, monitoring of disease progression or monitoring therapy of one or more of a precancerous condition and cancer, optionally one or more of colorectal cancer, oesophageal cancer, breast cancer, prostate cancer, head cancer, neck cancer, ovarian cancer, rectal cancer, pancreatic cancer, thyroid cancer, gastric cancer and sarcoma.

67. The use of at least one of the formulation of any one of claims 1 to 29, and the pharmaceutical composition of claim 37 or claim 38, in the detection, diagnosis, surgery, staging, treatment, monitoring of treatment, monitoring of disease progression or monitoring therapy of sites of cMet over-expression or localisation.

68. The use of at least one of the formulation of any one of claims 1 to 29, and the pharmaceutical composition of claim 37 or claim 38, in obtaining an image of sites of cMet over-expression or localisation, optionally in vivo.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0229] Embodiments of the invention will now be described, by way of example, with reference to the drawings, in which:

[0230] FIG. 1 depicts an API (EMI-137).

DETAILED DESCRIPTION

[0231] An optical imaging agent as described in WO2008/139207 was used to run a series of experiments to find a more stable formulation that illustrated better reconstitution, in particular at an appropriate pH and/or tonicity for intravenous use. The compound used was EMI-137 as shown in FIG. 1.

[0232] The experiments discussed below used the acetate salt of EMI-137, but all weights and calculations were made using the free ion.

[0233] After reconstitution, an intravenous drug product should be an isotonic solution and have a physiological pH to make it suitable for intravenous administration. It should also be homogenous and should solubilise without formation of agglomerates. However, when dissolved in water EMI-137 has a pH of 4.6, and shows a significant degree of formation of larger structures or agglomerates. This agglomeration is only partly reversible upon increasing the pH of the solution, thus making it unsuitable for intravenous administration. Therefore, a formulation is required that on reconstitution will be a pH suitable for intravenous administration, and that is substantially homogeneous and substantially free from agglomerates or particles. Furthermore, EMI-137 has a reasonably short shelf-life, and therefore for practical and safety purposes is not very useable in its isolated form. Therefore, a formulation is required that will extend the stability and shelf-life of EMI-137.

[0234] Preparation of and Imaging Using EMI-137

[0235] Example 1 provides the synthesis of a cMBP peptide (Compound 1 (comprising SEQ-7)). Example 2 provides the synthesis of a related peptide as a negative control, in which the peptide sequence of Compound 1 is scrambled, to give compound 2, which has the peptide sequence:

TABLE-US-00010 (SEQ-8) Thr-Gly-Glu-Cys-Thr-Cys-Pro-Tyr-Trp-Glu-Phe-Arg- Pro-Cys-Glu-Cys-Gly-Ser-Tyr-Ser-Gly-Ala-Gly-Gly- Gly-Lys.

[0236] Example 3 provides the synthesis of cyanine dye Cy5**. Example 4 provides the synthesis of an active ester of Cy5**. Example 5 provides the conjugation of cyanine dyes to peptides (cMBP peptide and control). Compounds 3 (comprising SEQ-7), 4 (comprising SEQ-8), 5 (comprising SEQ-7), 6 (comprising SEQ-7) and 7 (comprising SEQ-8) were compared in this way. Example 6 provides a method of determination of the affinity of the peptides to cMet in vitro. The results show that the binding is selective, even when an optical reporter imaging moiety (a cyanine dye) is attached. Example 7 provides data on the in vivo testing of Compounds 5 and 7 in an animal model of cancer. Superior tumour: background ratios were seen with Compound 5, whereas Compound 7 (negative control) did not discriminate between tumour and background.

[0237] Structures of the Compounds are provided in Table 2 below.

TABLE-US-00011 TABLE 2 Structures of Compounds No. Structure of Compounds. 1 Ac-AGSCYCSGPPRFECWCYETEGTGGGK-NH.sub.2 2 Ac-TGECTCPYWEFRPCECGSYSGAGGGK-NH.sub.2 (negative control) 3 [00010] 4 [00011] 5 [00012] 6 Ac-AGSCYCSGPPRFECWCYETEGTGGGK(-Alexa647)-NH.sub.2 7 Ac-TGECTCPYWEFRPCECGSYSGAGGGK(-Cy5**)-NH.sub.2 (negative control).

Example 1: Synthesis of Compound 1

[0238] Step (a): Synthesis of Protected Precursor Linear Peptide.

[0239] The precursor linear peptide has the structure:

TABLE-US-00012 (comprisingSEQ-7) Ac-Ala-Gly-Ser-Cys-Tyr-Cys(Acm)-Ser-Gly-Pro-Pro- Arg-Phe-Glu-Cys(Acm)-Trp-Cys-Tyr-Glu-Thr-Glu-Gly- Thr-Gly-Gly-Gly-Lys-NH.sub.2.

[0240] The peptidyl resin H-Ala-Gly-Ser(tBu)-Cys(Trt)-Tyr(tBu)-Cys(Acm)-Ser(tBu)-Gly-Pro-Pro-Arg(Pbf)-Phe-Glu(OtBu)-Cys(Acm)-Trp(Boc)-Cys(Trt)-Tyr(tBu)-Glu(OtBu)-Thr(.sup.Me,Mepro)-Glu(OtBu)-Gly-Thr(tBu)-Gly-Gly-Gly-Lys(Boc)-Polymer (comprising SEQ-7) was assembled on an Applied Biosystems 433A peptide synthesizer using Fmoc chemistry starting with 0.1 mmol Rink Amide Novagel resin. An excess of 1 mmol pre-activated amino acids (using HBTU) was applied in the coupling steps.

[0241] Glu-Thr pseudoproline (Novabiochem 05-20-1122) was incorporated in the sequence. The resin was transferred to a nitrogen bubbler apparatus and treated with a solution of acetic anhydride (1 mmol) and NMM (1 mmol) dissolved in DCM (5 mL) for 60 min. The anhydride solution was removed by filtration and the resin washed with DCM and dried under a stream of nitrogen.

[0242] The simultaneous removal of the side-chain protecting groups and cleavage of the peptide from the resin was carried out in TFA (10 mL) containing 2.5% TIS, 2.5% 4-thiocresol and 2.5% water for 2 hours and 30 min. The resin was removed by filtration, TFA removed in vacuo and diethyl ether added to the residue. The formed precipitate was washed with diethyl ether and air-dried affording 264 mg of crude peptide.

[0243] Purification by preparative HPLC (gradient: 20-30% B over 40 min where A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 10 mL/min, column: Phenomenex Luna 5 C.sub.18 (2) 5021.20 mm, detection: UV 214 nm, product retention time: 30 min) of the crude peptide afforded 100 mg of pure Compound 1 linear precursor. The pure product was analysed by analytical HPLC (gradient: 10-40% B over 10 min where A=H2CVO.1% TFA and B=ACN/0.1% TFA, flow rate: 0.3 mL/min, column: Phenomenex Luna 3 C.sub.18 (2) 502 mm, detection: UV 214 nm, product retention time: 6.54 min). Further product characterisation was carried out using electrospray mass spectrometry (MH.sub.2.sup.2+ calculated: 1464.6, MH.sub.2.sup.2+ found: 1465.1).

Step (b): Formation of Monocyclic Cys4-16 Disulfide Bridge

[0244] Cys4-16; Ac-Ala-Gly-Ser-Cys-Tyr-Cys(Acm)-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys(Acm)-Trp-Cys-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys-NH.sub.2 (comprising SEQ-7).

[0245] The linear precursor from step (a) (100 mg) was dissolved in 5% DMSO/water (200 mL) and the solution adjusted to pH 6 using ammonia. The reaction mixture was stirred for 5 days. The solution was then adjusted to pH 2 using TFA and most of the solvent removed by evaporation in vacuo. The residue (40 mL) was injected in portions onto a preparative HPLC column for product purification.

[0246] Purification by preparative HPLC (gradient: 0% B for 10 min, then 0-40% B over 40 min where A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 10 mL/min, column: Phenomenex Luna 5 C.sub.18 (2) 25021.20 mm, detection: UV 214 nm, product retention time: 44 min) of the residue afforded 72 mg of pure Compound 1 monocyclic precursor. The pure product (as a mixture of isomers P1 to P3) was analysed by analytical HPLC (gradient: 10-40% B over 10 min where A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 0.3 mL/min, column: Phenomenex Luna 3 C.sub.18 (2) 502 mm, detection: UV 214 nm, product retention time: 5.37 min (P1); 5.61 min (P2); 6.05 min (P3)). Further product characterisation was carried out using electrospray mass spectrometry (MH.sub.2.sup.2+ calculated: 1463.6, MH.sub.2.sup.2+ found: 1464.1 (P1); 1464.4 (P2); 1464.3 (P3)).

Step (c): Formation of Second Cys6-14 disulfide bridge (Compound 1)

[0247] The monocyclic precursor from step (b) (72 mg) was dissolved in 75% AcOH/water (72 mL) under a blanket of nitrogen. 1 M HCl (7.2 mL) and 0.05 M I.sub.2 in AcOH (4.8 mL) were added in that order and the mixture stirred for 45 min. 1 M ascorbic acid (1 mL) was added giving a colourless mixture. Most of the solvents were evaporated in vacuo and the residue (18 mL) diluted with water/0.1% TFA (4 mL) and the product purified using preparative HPLC.

[0248] Purification by preparative HPLC (gradient: 0% B for 10 min, then 20-30% B over 40 min where A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 10 mL/min, column: Phenomenex Luna 5 C.sub.18 (2) 25021.20 mm, detection: UV 214 nm, product retention time: 43-53 min) of the residue afforded 52 mg of pure Compound 1. The pure product was analysed by analytical HPLC (gradient: 10-40% B over 10 min where A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 0.3 mL/min, column: Phenomenex Luna 3 C.sub.18 (2) 502 mm, detection: UV 214 nm, product retention time: 6.54 min). Further product characterisation was carried out using electrospray mass spectrometry (MH.sub.2.sup.2+ calculated: 1391.5, MH.sub.2.sup.2+ found: 1392.5).

Example 2: Synthesis of Compound 2

[0249] Ac-Thr-Gly-Glu-Cys-Thr-Cys(Acm)-Pro-Tyr-Trp-Glu-Phe-Arg-Pro-Cys(Acm)-Glu-Cys-Gly-Ser-Tyr-Ser-Gly-Aa-Gly-Gy-Gly-Lys-NH.sub.2 Compound 2 (comprising SEQ-8) is a negative control, where the peptide sequence of Compound 1 has been scrambled.

[0250] Step (a): Synthesis of Protected Precursor Linear Peptide

[0251] The peptidyl resin H-Thr(tBu)-Gly-Glu(OtBu)-Cys(Trt)-Thr(tBu)-Cys(Acm)-Pro-Tyr(tBu)-Trp(Boc)-Glu(OtBu)-Phe-Arg(Pbf)-Pro-Cys(Acm)-Glu(OtBu)-Cys(Trt)-Gly-Ser(tBu)-Tyr(tBu)-Ser(.sup.Me,Mepro)-Gly-Ala-Gly-Gly-Gly-Lys(Boc)-Polymer (comprising SEQ-8) was assembled on an Applied Biosystems 433A peptide synthesizer using Fmoc chemistry starting with 0.1 mmol Rink Amide Novagel resin. An excess of 1 mmol pre-activated amino acids (using HBTU) was applied in the coupling steps. Tyr-Ser pseudoproline (Novabiochem 05-20-1014) was incorporated in the sequence. The resin was transferred to a nitrogen bubbler apparatus and treated with a solution of acetic anhydride (1 mmol) and NMM (1 mmol) dissolved in DCM (5 mL) for 60 min. The anhydride solution was removed by filtration and the resin washed with DCM and dried under a stream of nitrogen.

[0252] The simultaneous removal of the side-chain protecting groups and cleavage of the peptide from the resin was carried out in TFA (10 mL) containing 2.5% TIS, 2.5% 4-thiocresol and 2.5% water for 2 hours and 10 min. The resin was removed by filtration, TFA removed in vacuo and diethyl ether added to the residue. The formed precipitate was washed with diethyl ether and air-dried affording 216 mg of crude peptide.

[0253] Purification by preparative HPLC (gradient: 20-30% B over 40 min where A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 50 mL/min, column: Phenomenex Luna 5 C.sub.18 (2) 25050 mm, detection: UV 214 nm, product retention time: 34.1 min) of the crude peptide afforded pure DX-1662 negative control linear precursor dissolved in 200 mL of ACN/water. The pure product was analysed by analytical HPLC (gradient: 10-40% B over 5 min where A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 0.6 mL/min, column: Phenomenex Luna 3 C.sub.18 (2) 202 mm, detection: UV 214 nm, product retention time: 3.52 min). Further product characterisation was carried out using electrospray mass spectrometry (MH.sub.2.sup.2+ calculated: 1464.6, MH.sub.2.sup.2+ found: 1464.9).

Step (b): Formation of Monocyclic Cys4-16 Disulfide Bridge

[0254] Cys4-16; Ac-Thr-Gly-Glu-Cys-Thr-Cys(Acm)-Pro-Tyr-Trp-Glu-Phe-Arg-Pro-Cys(Acm)-Glu-Cys-Gly-Ser-Tyr-Ser-Gly-Aa-Gly-Gy-Gly-Lys-NH.sub.2 (comprising SEQ-8).

[0255] DMSO (10 mL) was added to the negative control linear precursor solution from step (a) (200 mL, see 4.3.1) and the solution adjusted to pH 7 using ammonia. The reaction mixture was heated at 40 C. for 18 hours, then at 60 C. for 60 min. The solution was adjusted to pH 2 using TFA and ACN removed by evaporation in vacuo. The residue was subjected to preparative HPLC purification.

[0256] Purification by preparative HPLC (gradient: 0% B for 5 min, then 20-30% B over 60 min where A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 50 mL/min, column: Phenomenex Luna 5 C.sub.18 (2) 25050 mm, detection: UV 214 ran, product retention time: 29.6 min) of the residue afforded pure negative control monocyclic precursor in 100 mL of ACN/water. The pure product was analysed by analytical HPLC (gradient: 10-40% B over 5 min where A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 0.6 mL/min, column: Phenomenex Luna 3 C.sub.18 (2) 202 mm, detection: UV 214 nm, product retention time: 3.46 min). Further product characterisation was carried out using electrospray mass spectrometry (MH.sub.2.sup.2+ calculated: 1463.6, MH.sub.2.sup.2+ found: 1463.7).

Step (c): Formation of Second Cys6-14 disulfide bridge (Compound 2)

[0257] Cys4-16, 6-14; Ac-Thr-Gly-Glu-Cys-Thr-Cys-Pro-Tyr-Trp-Glu-Phe-Arg-Pro-Cys-Glu-Cys-Gly-Ser-Tyr-Ser-Gly-Ala-Gly-Gly-Gly-Lys-NH.sub.2 (comprising SEQ-8).

[0258] The negative control monocyclic precursor solution from step (b) (100 mL) was diluted with AcOH (100 mL). 1 M HCl (5 mL) and 0.05 M I.sub.2 in AcOH (7 mL) were added in that order under a blanket of argon and the mixture stirred for 20 min. 1 M ascorbic acid (1 mL) was added giving a colourless mixture. Most of the solvents were evaporated in vacuo and the residue (30 mL) diluted with water/0.1% TFA (100 mL) and the product purified using preparative HPLC. Purification by preparative HPLC (gradient: 0% B for 10 min, then 20-30% B over 60 min where A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 50 mL/min, column: Phenomenex Luna 5 C.sub.18 (2) 25050 mm, detection: UV 214 nm, product retention time: 32.8 min) of the residue afforded 30 mg of pure Compound 2. The pure product was analysed by analytical HPLC (gradient: 10-40% B over 10 min where A=H.sub.2O/0.1% TFA and B=ACN/0.1% TFA, flow rate: 0.3 mL/min, column: Phenomenex Luna 3 C.sub.18 (2) 502 mm, detection: UV 214 nm, product retention time: 6.54 min). Further product characterisation was carried out using electrospray mass spectrometry (MH.sub.2.sup.2+ calculated: 1391.5, MH.sub.2.sup.2+ found: 1392.5).

Example 3: Synthesis of the Cyanine Dye 2-{(1E,3E,5E)-5-[1-(5-carboxypentyl)-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium-5-sulfonate (Cy5**)

[0259] ##STR00013##

(3a) 5-Methyl-6-oxoheptane-1-sulfonic acid

[0260] ##STR00014##

[0261] Ethyl 2-methylacetoacetate (50 g) in DMF (25 ml) was added to a suspension of sodium hydride (12.0 g of 60% NaH in mineral oil) in DMF (100 ml), dropwise with ice-bath cooling over 1 hour, (internal temperature 0-4 C.). This mixture was allowed to warm to ambient temperature for 45 mins with stirring before re-cooling. A solution of 1,4-butanesultone (45 g) in DMF (25 ml) was then added dropwise over 15 minutes. The final mixture was heated at 60 C. for 18 hours. The solvent was removed by rotary evaporation and the residue partitioned between water and diethyl ether. The aqueous layer was collected, washed with fresh diethyl ether and rotary evaporated to yield a sticky foam. This intermediate was dissolved in water (100 ml) and sodium hydroxide (17.8 g) added over 15 minutes with stirring. The mixture was heated at 90 C. for 18 hours. The cooled reaction mixture was adjusted to -pH2 by the addition of concentrated hydrochloric acid (40 ml). The solution was rotary evaporated and dried under vacuum. The yellow solid was washed with ethanol containing 2% hydrochloric acid (3150 ml). The ethanolic solution was filtered, rotary evaporated and dried under vacuum to yield a yellow solid. Yield 70 g.

(3b) 2,3-Dimethyl-3-(4-sulfobutyl)-3H-indole-5-sulfonic acid, dipotassium salt

[0262] ##STR00015##

[0263] 4-Hydrazinobenzenesulfonic acid (40 g), 5-methyl-6-oxoheptane-1-sulfonic acid (from 3a; 60 g) and acetic acid (500 ml) were mixed and heated under reflux for 6 hours. The solvent was filtered, rotary evaporated and dried under vacuum. The solid was dissolved in methanol (1 L). To this was added 2M methanolic potassium hydroxide (300 ml). The mixture was stirred for 3 hours and then the volume of solvent reduced by 50% using rotary evaporation. The resulting precipitate was filtered, washed with methanol and dried under vacuum. Yield 60 g. MS (LCMS): MH.sup.+362. Acc. Mass: Found, 362.0729. MH.sup.+=C.sub.14H.sub.2ONO.sub.6S.sub.2 requires m/z 362.0732 (0.8 ppm).

(3c) 2,3-Dimethyl-13-bis(4-sulfobutyl)-3H-indolium-5-sulfonate, dipotassium salt

[0264] ##STR00016##

[0265] 2,3-Dimethyl-3-(4-sulfobutyl)-3H-indole-5-sulfonic acid (from 3b; 60 g) was heated with 1,4 butane sultone (180 g) and tetramethylene sulfone (146 ml) at 140 C. for 16 hours. The resulting red solid was washed with diethyl ether, ground into a powder and dried under vacuum. Yield 60 g.

3d) Cy5**, as TFA salt

[0266] 1-(5-Carboxypentyl)-2,3,3-trimethyl-indolenium bromide-5-sulfonic acid, K.sup.+ salt (2.7 g), malonaldehyde bis(phenylimine) monohydrochloride (960 mg), acetic anhydride (36 ml) and acetic acid (18 ml) were heated at 120 C. for 1 hour to give a dark brown-red solution. The reaction mixture was cooled to ambient temperature. 2,3-Dimethyl-1,3-bis(4-sulfobutyl)-3H-indolium-5-sulfonate (from 3c; 8.1 g) and potassium acetate (4.5 g) were added to the mixture, which was stirred for 18 hours at ambient temperature. The resulting blue solution was precipitated using ethyl acetate and dried under vacuum. The crude dye was purified by liquid chromatography (RPC.sub.18. Water+0.1% TFA/MeCN+0.1% TFA gradient). Fractions containing the principal dye peak were collected, pooled and evaporated under vacuum to give the title dye, 2 g. UV/Vis (Water+0.1% TFA): 650 nm. MS (MALDI-TOF): MH.sup.+887.1. MH.sup.+=C.sub.38H.sub.50N.sub.2O.sub.14S.sub.4 requires m/z 887.1.

Example 4: Synthesis of the 2-[(1E,3E,5E)-5-(1-{6-[2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl]-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium-5-sulfonate, diisopropylethylamine salt (NHS Ester of Cy5**)

[0267] ##STR00017##

[0268] Cy5** (Example 3; 10 mg) was dissolved in anhydrous DMSO (3 ml); to this were added HSPyU (20 mg) and N,N-diisopropylethylamine (80 l). The resulting solution was mixed for 3 hours, whereupon TLC (RPC.sub.18. Water/MeCN) revealed complete reaction. The dye was isolated by precipitation in ethyl acetate/diethyl ether, filtered, washed with ethyl acetate and dried under vacuum. UV/Vis (Water) 650 nm. MS (MALDI-TOF) MH.sup.+983.5. MH.sup.+=C.sub.42H.sub.53N.sub.3O.sub.16S.sub.4 requires m/z 984.16.

Example 5: Conjugation of Dyes, Synthesis of Compounds 3 to 7

[0269] Cys4-16, 6-14; Ac-Ala-Gly-Ser-Cys-Tyr-Cys-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys-Trp-Cys-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys(Cy5)-NH.sub.2 (Compound 3) (comprising SEQ-7).

[0270] Compound 1 (10 mg), NMM (4 L) and Cy5 NHS ester (5.7 mg; GE Healthcare PAI 5104) were dissolved in NMP (1 mL) and the reaction mixture stirred for 7 hours. The reaction mixture was then diluted with 5% ACN/water (8 mL) and the product purified using preparative HPLC.

[0271] Purification by preparative HPLC (gradient: 5-50% B over 40 min where A=H.sub.2O/0.1% HCOOH and B=ACN/0.1% HCOOH, flow rate: 10 mL/min, column: Phenomenex Luna 5 C18 (2) 25021.20 mm, detection: UV 214 nm, product retention time: 35.5 min) of the crude peptide afforded 8.1 mg of pure Compound 3. The pure product was analysed by analytical HPLC (gradient: 5-50% B over 10 min where A=H.sub.2O/0.1% HCOOH and B=ACN/0.1% HCOOH, flow rate: 0.3 mL/min, column: Phenomenex Luna 3 C.sub.18 (2) 502 mm, detection: UV 214 nm, product retention time: 8.15 min). Further product characterisation was carried out using electrospray mass spectrometry (MH.sub.2.sup.2+ calculated: 1710.6, MH.sub.2.sup.2+ found: 1711.0).

[0272] Compound 4 was prepared in a similar mannerelectrospray mass spectrometry (MH.sub.2.sup.2+ calculated: 1710.6, MH.sub.2.sup.2+ found: 1710.9).

[0273] Other dye-peptide conjugates (Compounds 5 to 7) were prepared by analogous methods. Alexa647 was purchased from Molecular Probes (A20106):

[0274] Compound 5 (MH.sub.2.sup.2+ calculated: 1825.7, MH.sub.2.sup.2+ found: 1825.9),

[0275] Compound 6 (MH.sub.2.sup.2+ calculated: 1811.7, MH.sub.2.sup.2+ found: 1812.0),

[0276] Compound 7 (MH.sub.2.sup.2+ calculated: 1825.7, MH.sub.2.sup.2+ found: 1826.2).

Example 6: In Vitro Fluorescence Polarisation Assay

[0277] The principle of the fluorescence polarisation method can briefly be described as follows:

[0278] Monochromatic light passes through a horizontal polarizing filter and excites fluorescent molecules in the sample. Only those molecules that oriented properly in the vertically polarized plane adsorb light, become excited, and subsequently emit light. The emitted light is measured in both horizontal and vertical planes. The anisotropy value (A), is the ratio between the light intensities following the equation:

A=(Intensity with horizontal polarizerIntensity with vertical polarizer)/(Intensity with horizontal polarizer+2* Intensity with vertical polarizer).

[0279] The fluorescence anisotropy measurements were performed in 384-well microplates in a volume of 10 L in binding buffer (PBS, 0.01% Tween-20, pH 7.5) using a Tecan Safire fluorescence polarisation plate reader (Tecan, US) at ex646/em678 nm. The concentration of dye-labelled peptide was held constant (20 nM) and the concentrations of the human or mouse cMet/Fc chimera (R&D Systems) or Semaphorin 6A (R&D Systems) were varied from 0-150 nM. Binding mixtures were equilibrated in the microplate for 10 min at 30 C. The observed change in anisotropy was fit to the equation:

[00001]$r_{\mathrm{obs}}=r_{\mathrm{free}}+\left(r_{\mathrm{bound}}-r_{\mathrm{free}}\right).Math.\frac{\begin{array}{c}\left(K_D+\mathrm{cMet}+P\right)-\\ \sqrt{\left(K_D+\mathrm{cMet}+P\right).Math.2-4.Math.\mathrm{cMet}.Math.P}\end{array}}{2.Math.P}$

where r.sub.Dbs is the observed anisotropy, r.sub.free is the anisotropy of the free peptide, r.sub.bound is the anisotropy of the bound peptide, K.sub.D is the dissociation constant, cMet is the total cMet concentration, and P is the total dye-labelled peptide concentration. The equation assumes that the synthetic peptide and the receptor form a reversible complex in solution with 1:1 stoichiometry. Data fitting was done via nonlinear regression using GraphPad Prism software to obtain the K.sub.D value (one-site binding).

[0280] Compounds 3 and 4 were tested for binding towards human and mouse cMet (Fc chimera). The results showed a K.sub.D of 3+/1 nM for the binding of Compound 3 to human c-Met. There was no binding of Compound 4 to human cMet. Furthermore, Compounds 3 and 4 showed no binding to mouse cMet in the tested range.

[0281] Using the same method, Compound 5 was found to have a K.sub.D for human cMet of 1.1 nM.

Example 7: In Vivo Testing of Compounds 5 and 7

[0282] (a) Animal Model

[0283] 54 Female BALB c/A nude (Bom) mice were used in the study. The use of the animals was approved by the local ethics committee. BALB c/A nude is an inbred immunocompromised mouse strain with a high take rate for human tumours as compared to other nude mice strains. The mice were 4 weeks old upon arrival and with a body weight of approx. 20 grams at the start of the study. The animals were housed in individually ventilated cages (IVC, Scanbur BK) with HEPA filtered air. The animals had ad libitum access to Rat and Mouse nr. 3 Breeding diet (Scanbur BK) and tap water acidified by addition of HCl to a molar concentration of 1 mM (pH 3.0).

[0284] The colon cancer cell HCT-15 is derived from human colon carcinomas and is reported to express cMet according to Zeng et al [Clin. Exp. Metastasis, 21, 409-417. (2004)]. The cell line was proven to be tumorigenic when inoculated subcutaneously into nude mice [Flatmark et al, Eur. J. Cancer 40, 1593-1598 (2004)].

[0285] HCT-15 cells were grown and prepared for subcutaneous inoculation in RPMI (Sigma Cat # R0883) with 10% serum and penicillin/streptomycin. Stocks were made at passage number four (P4) and frozen down for storage in liquid nitrogen at 310.sup.7 cells/vial in the culture media containing 5% DMSO. On the day of the transplantation, the cells were thawed quickly in a 37 C. water bath (approx. 2 min), washed and resuspended in PBS/2% serum (centrifugation at 1200 rpm for 10 min). Thorough mixing of cells in the vials was ensured every time cells were aspirated into the dosing syringe. Volumes of 0.1 ml of cell suspension were injected s.c. at the shoulder and at the back using a fine bore needle (25 G) while the animals were under light gas anaesthesia. The animals were then returned to their cages and the tumours were allowed to grow for 13-17 days. The animals were allowed an acclimatisation period of at least 5 days before the inoculation procedure.

[0286] (b) Procedure

[0287] All test substances were reconstituted with PBS from freeze-dried powder. A small stack of white printer paper was imaged to obtain a flat field image which was used to correct for illumination inhomogeneities. The test substances were injected intravenously in the lateral tail vein during physical fixation. The injection volume was 0.1 ml, which corresponds to a dose of 1 nmol test substance per animal. After injection the animals were returned to their cages. The animals were sacrificed immediately before imaging by cervical dislocation. The optimal imaging time point for each test substance was estimated based on comparison of wash out rates in the skin and in muscle tissue in a limited number of animals (n=1-6). The imaging time point for Compounds 3 and 4 was 120 minutes post injection.

[0288] For each animal the subcutaneously grown tumours were excised post mortem. A thin slice, approximately 1.6 mm thick and 3-4 mm in diameter, was cut off the edge of one of the tumours. The tumour slice was then imaged against an area of normal colon from the same animal.

[0289] (c) Imaging

[0290] Imaging was performed through a clinical laparoscope adapted to use a light source to excite the reporter and a filtering system to extract the fluorescence component. A 635 nm laser was used for excitation of the reporter molecule. A Hamamatsu ORCA ERG CCD camera was used as the detector. The camera was operated in 22 binning mode with 0 gain.

[0291] Standard exposure time for colon imaging was 10 s. System calibration measurements indicate that the 10 s exposure time with the animal imaging system corresponds to 40 ms exposure with a clinically relevant light source, field of view, and distance to the tissue surface. The intensity distribution in the image was corrected for illumination inhomogeneities through system calibration data. A target to background ratio was computed from regions of interest placed over the tumour, and normal colon background. The images were visually scored using the standard scoring system employed for receiver operating characteristic analysis.

[0292] (d) Results

[0293] Compound 5 had a tumour to normal ratio of 1.46:1 and the corresponding scrambled control peptide with the same dye (Compound 7) had a ratio of 1.04:1. Compound 5 had a readily identifiable tumour, whereas nothing was discernible against background with Compound 7.

[0294] Preparation of Lyophilised EMI-137 Formulation

[0295] The formulation needs to freeze dry effectively (e.g., without bumping or collapse) to provide a homogenous solid that is stable. As noted previously, the pH of EMI-137 in water is approximately 4.6, which is unsuitable for intravenous administration. Therefore, further requirements of the formulation are that on reconstitution it is a pH that is suitable for intravenous administration. Furthermore, the tonicity of the formulation on reconstitution must be suitable for intravenous administration. A further benefit would be for the formulation to be storable at room temperature, albeit refrigerated storage would also be acceptable.

[0296] Initial formulation studies showed that formulations that had a pH of less than approximately pH 6.8 tended to agglomerate. Therefore, any formulation that provided a pH below 6.8 was deemed unacceptable. Furthermore, it is known at a pH of more than approximately pH 8 (in some instances up to approximately pH 9) there is a risk of degradation of disulphide bridges (of which there are two in EMI-137). Therefore, it would be of benefit to arrive at a formulation that on reconstitution provides a pH that is high enough to avoid agglomeration, but is low enough to mitigate the risk of degradation of disulphide bridges.

[0297] Thus, to maintain the pH of the EMI-137 solution within an acceptable range on reconstitution, it is necessary to include a buffer prior to freeze-drying. Studies have shown that the viscosity of the EMI-137 solution increases in the presence of potassium chloride enriched phosphate buffered saline (PBS), and that a substantial gel formation develops. This gelling effect was not observed when EMI-137 was dissolved in sodium phosphate buffer at pH 7.4. Therefore, the use of NaCl and KCl was discounted (due to gelling issues).

[0298] The lyophilisation process was carried out as highlighted in Table 3.

TABLE-US-00013 TABLE 3 Lyophilisation Parameters Batch No. FFF169/096-904 Freeze dryer Lyomax 2 MTE 1500, Development Pharmaceuticals Oslo Shelf Chamber temp. Time pressure Temperature Phase C. Hr:min bar ramp rate Note Freeze drying Loading 40 Uses time needed to load freeze dryer Freezing 55 0:10 1.5 C./min Temperature ramp to reach freezing temperature 55 02:05 Steady state freezing conditions Primary drying 55 00:45 50 Time necessary to prepare freeze dryer for vacuum phase 38 00:15 50 1 C./min Prepare primary drying 38 211:00 50 Steady state primary drying conditions Primary to secondary drying 30 10:00 50 6.8 C./hour transition phase (ramp phase) Secondary drying 30 6:03 50 Steady state secondary drying conditions 20 00:10 50 1 C./min Bring chamber to ambient temperature before vial headspace gas is added 20 00:30 50 Storage/unloading 5 n/a n/a Store product at cold temperature during unloading and capping Special functions Pre-aeration Yes Pressure setpoint 0.80 bara Gas Nitrogen Stoppering Semi-auto/Auto Semi- Stoppering pressure Auto 80 BarA Aeration Air/nitrogen Air Vacuum control Nitrogen bleed Vacuum gauge MKS Pressure deviation 20 bar Product protection 80 bar Moisture test No Nominal time 230:58 (Real 239:29 including loading)

[0299] The lyophilisation was carried out as follows.

[0300] The constituents of the formulations as highlighted in Tables 4A to 4F below were added to a suitable lyophilisation vessel. Note that all components listed (including the buffer) were added prior to lyophilisation.

[0301] The loading phase is carried out at approximately 40 C. The temperature is then reduced to approximately 55 C. The rate of cooling may be approximately 1.5 C. per minute. The mixture is then held at steady state freezing conditions for at least two hours. The pressure is then reduced to 50 pBar, which takes approximately 45 minutes. The temperature is then increased to approximately 38 C. for the primary drying phase. The rate of heating may be approximately 1 C. per minute. The mixture is then held at steady state freezing conditions for approximately nine days. The mixture is then heated to approximately 30 C. at a rate of approximately 6.8 C. per hour over at least a ten-hour period (primary to secondary drying transition phase, or ramp phase). The mixture is then held at steady state secondary drying conditions for approximately six hours. The mixture is then cooled to approximately 20 C. at a rate of approximately 1 C. per minute, before addition of headspace gas (i.e., and inert gas such as argon or nitrogen), thus releasing the vacuum in the lyophilisation vessel. The product is then unloaded/stored at 5 C.

[0302] The primary drying step, the ramp phase and the secondary drying step are all carried out by placing the mixture under vacuum of approximately 50 bar. This pressure may be introduced in the primary drying phase and then maintained until the storage/unloading phase.

[0303] The use of a buffer such as sodium phosphate is known to cause a pH shift towards low pH during freezing and thus the formulation was expected to be challenging to freeze dry without collapse, as the glass transition temperatures of the freeze concentrates would be low. As such, the current invention includes a combined tonicity regulator and lyoprotectant. Mannitol is one of the most widely used and documented bulking agents in freeze drying, but several thermal issues and crystallization complications have been reported, which could potentially affect production and product stability. As such, the use of mannitol alone is not normally thought to be advantageous on lyophilisation and is known to give rise to several problems.

[0304] As the combination of buffer and lyoprotectants in the present invention was difficult to freeze-dry without experiencing collapse of the freeze-dried cake, a tailored freeze-drying cycle was required. The freeze-drying cycle was selected to have sufficiently low vacuum and shelf temperature to achieve a low ice temperature to prevent this melt-back/collapse of the freeze-dried cake. The collapse of the freeze-dried cake must be avoided to prevent EMI-137 from forming larger structures/agglomerates during the drying step of the freeze-drying process (this is found to happen at pH less than 6.8 and is not entirely reversible).

[0305] After some initial formulation studies, and in view of the above, the following variables were chosen for further testing: [0306] Buffer: sodium phosphate or Tris HCl (25, 40 and 55 mM); [0307] Lyoprotectant: sucrose or mannitol.

[0308] Examples of Lyophilised Formulations

[0309] The formulation candidates were investigated using a factorial design with four variables combined with a mixed level design as shown in Tables 4A to 4F. The design was made up of a fractional two-level design with four variables (samples 1 to 8), four centerpoints (samples 10 to 13), and a mixed level design with three levels for the active pharmaceutical ingredient (API) and two levels for the categorical variables of buffer type and lyoprotectant type (samples 14 to 21). In addition, various reference samples were included (samples 22 to 24). Two additional samples (samples 5 and 26) were also studied. Sample 25 refers to the composition of the solution before freeze drying in 1 mL of water, and sample 26 refers to a further typical formulation that was prepared based on the results of the previous samples.

TABLE-US-00014 TABLE 4A Formulation Screening Design (mg/ml) Water Buffer API conc. Cryo conc. Buffer conc. Sample (ml) Buffer type conc. Cryo type (mg/ml) (mg/ml) (mg/ml) 1 5 Tris HCl 25 mM Mannitol 4.3 46.2 3.03 2 5 Na Phosphate (1) 25 mM Mannitol 5.3 40.76 3.5 3 5 Tris HCl 55 mM Mannitol 5.3 40.79 6.66 4 5 Na Phosphate (2) 55 mM Mannitol 4.3 30.17 7.7 5 5 Tris HCl 25 mM Sucrose 5.3 84.29 3.03 6 5 Na Phosphate (1) 25 mM Sucrose 4.3 74.36 3.5 7 5 Tris HCl 55 mM Sucrose 4.3 74.43 6.66 8 5 Na Phosphate (2) 55 mM Sucrose 5.3 52.59 7.7 10 5 Tris HCl 40 mM Mannitol 4.8 43.5 4.85 11 5 Na Phosphate (3) 40 mM Mannitol 4.8 34.8 5.6 12 5 Tris HCl 40 mM Sucrose 4.8 79.36 4.85 13 5 Na Phosphate (3) 40 mM Sucrose 4.8 65.25 5.6 14 5 Tris HCl 40 mM Mannitol 4.3 43.5 4.85 15 5 Tris HCl 40 mM Mannitol 5.3 43.5 4.85 16 5 Tris HCl 40 mM Sucrose 4.3 79.36 4.85 17 5 Tris HCl 40 mM Sucrose 4.3 79.36 4.85 18 5 Na Phosphate (3) 40 mM Mannitol 4.3 34.8 5.6 19 5 Na Phosphate (3) 40 mM Mannitol 5.3 34.8 5.6 20 5 Na Phosphate (3) 40 mM Sucrose 4.3 65.25 5.6 21 5 Na Phosphate (3) 40 mM Sucrose 4.3 65.25 5.6 22 5 Tris HCl 40 mM None 4.8 0 4.85 23 5 Na Phosphate (3) 40 mM None 4.8 0 5.6 24 5 None 0 None 4.8 0 0 25* 1 Na Phosphate (4) 50 mM Mannitol 4.80 30.82 7.00 26** 5 Na Phosphate (4) 50 mM Mannitol 4.80 30.82 7.00

TABLE-US-00015 TABLE 4B Formulation Screening Design (mg) Total Total Water Total API Cryo Anhydrous Total Hydrated Sample (ml) Buffer type Cryo type (mg) (mg) Buffer (mg) Buffer (mg) 1 5 Tris HCl Mannitol 21.5 231 15.15 15.15 2 5 Na Phosphate (1) Mannitol 26.5 203.8 17.5 42.61 3 5 Tris HCl Mannitol 26.5 203.95 33.3 33.30 4 5 Na Phosphate (2) Mannitol 21.5 150.85 38.5 93.72 5 5 Tris HCl Sucrose 26.5 421.45 15.15 15.15 6 5 Na Phosphate (1) Sucrose 21.5 371.8 17.5 42.61 7 5 Tris HCl Sucrose 21.5 372.15 33.3 33.30 8 5 Na Phosphate (2) Sucrose 26.5 262.95 38.5 93.72 10 5 Tris HCl Mannitol 24 217.5 24.25 24.25 11 5 Na Phosphate (3) Mannitol 24 174 28 68.14 12 5 Tris HCl Sucrose 24 396.8 24.25 24.25 13 5 Na Phosphate (3) Sucrose 24 326.25 28 68.14 14 5 Tris HCl Mannitol 21.5 217.5 24.25 24.25 15 5 Tris HCl Mannitol 26.5 217.5 24.25 24.25 16 5 Tris HCl Sucrose 21.5 396.8 24.25 24.25 17 5 Tris HCl Sucrose 21.5 396.8 24.25 24.25 18 5 Na Phosphate (3) Mannitol 21.5 174 28 68.14 19 5 Na Phosphate (3) Mannitol 26.5 174 28 68.14 20 5 Na Phosphate (3) Sucrose 21.5 326.25 28 68.14 21 5 Na Phosphate (3) Sucrose 21.5 326.25 28 68.14 22 5 Tris HCl None 24 0 24.25 24.25 23 5 Na Phosphate (3) None 24 0 28 68.14 24 5 None None 24 0 0 0.00 25* 1 Na Phosphate (4) Mannitol 24.00 154.10 35.02 85.24 26** 5 Na Phosphate (4) Mannitol 24.00 154.10 35.02 85.24

TABLE-US-00016 TABLE 4C Formulation Screening Design (mmol) Water API Cryo Buffer Sample (ml) Buffer type Cryo type (mmol) (mmol) (mmol) 1 5 Tris HCl Mannitol 0.01 1.27 0.13 2 5 Na Phosphate (1) Mannitol 0.01 1.12 0.12 3 5 Tris HCl Mannitol 0.01 1.12 0.27 4 5 Na Phosphate (2) Mannitol 0.01 0.83 0.27 5 5 Tris HCl Sucrose 0.01 1.23 0.13 6 5 Na Phosphate (1) Sucrose 0.01 1.09 0.12 7 5 Tris HCl Sucrose 0.01 1.09 0.27 8 5 Na Phosphate (2) Sucrose 0.01 0.77 0.27 10 5 Tris HCl Mannitol 0.01 1.19 0.20 11 5 Na Phosphate (3) Mannitol 0.01 0.96 0.20 12 5 Tris HCl Sucrose 0.01 1.16 0.20 13 5 Na Phosphate (3) Sucrose 0.01 0.95 0.20 14 5 Tris HCl Mannitol 0.01 1.19 0.20 15 5 Tris HCl Mannitol 0.01 1.19 0.20 16 5 Tris HCl Sucrose 0.01 1.16 0.20 17 5 Tris HCl Sucrose 0.01 1.16 0.20 18 5 Na Phosphate (3) Mannitol 0.01 0.96 0.20 19 5 Na Phosphate (3) Mannitol 0.01 0.96 0.20 20 5 Na Phosphate (3) Sucrose 0.01 0.95 0.20 21 5 Na Phosphate (3) Sucrose 0.01 0.95 0.20 22 5 Tris HCl None 0.01 0.00 0.20 23 5 Na Phosphate (3) None 0.01 0.00 0.20 24 5 None None 0.01 0.00 0.00 25* 1 Na Phosphate (4) Mannitol 0.00 0.17 0.05 26** 5 Na Phosphate (4) Mannitol 0.01 0.85 0.25

TABLE-US-00017 TABLE 4D Formulation Screening Design (mole ratio) API Cryo Buffer Water (mole (mole (mole Sample (ml) Buffer type Cryo type ratio) ratio) ratio) 1 5 Tris HCl Mannitol 1.00 215.29 21.23 2 5 Na Phosphate (1) Mannitol 1.00 154.10 17.20 3 5 Tris HCl Mannitol 1.00 154.22 37.87 4 5 Na Phosphate (2) Mannitol 1.00 140.59 46.66 5 5 Tris HCl Sucrose 1.00 169.60 17.23 6 5 Na Phosphate (1) Sucrose 1.00 184.42 21.20 7 5 Tris HCl Sucrose 1.00 184.59 46.67 8 5 Na Phosphate (2) Sucrose 1.00 105.82 37.86 10 5 Tris HCl Mannitol 1.00 181.59 30.45 11 5 Na Phosphate (3) Mannitol 1.00 145.27 30.40 12 5 Tris HCl Sucrose 1.00 176.32 30.45 13 5 Na Phosphate (3) Sucrose 1.00 144.97 30.40 14 5 Tris HCl Mannitol 1.00 202.71 33.99 15 5 Tris HCl Mannitol 1.00 164.46 27.57 16 5 Tris HCl Sucrose 1.00 196.82 33.99 17 5 Tris HCl Sucrose 1.00 196.82 33.99 18 5 Na Phosphate (3) Mannitol 1.00 162.17 33.94 19 5 Na Phosphate (3) Mannitol 1.00 131.57 27.54 20 5 Na Phosphate (3) Sucrose 1.00 161.83 33.94 21 5 Na Phosphate (3) Sucrose 1.00 161.83 33.94 22 5 Tris HCl None 1.00 0.00 30.45 23 5 Na Phosphate (3) None 1.00 0.00 30.40 24 5 None None 1.00 0.00 0.00 25* 1 Na Phosphate (4) Mannitol 1.00 128.66 38.02 26** 5 Na Phosphate (4) Mannitol 1.00 128.66 38.02

TABLE-US-00018 TABLE 4E Formulation Screening Design (% w/w before freeze drying) Total Mass of Components (mg)- before Water freeze API Cryo Buffer Sample (ml) Buffer type Cryo type drying (% w/w) (% w/w) (% w/w) 1 5 Tris HCl Mannitol 267.65 8.03 86.31 5.66 2 5 Na Phosphate (1) Mannitol 247.80 10.69 82.24 7.06 3 5 Tris HCl Mannitol 263.75 10.05 77.33 12.63 4 5 Na Phosphate (2) Mannitol 210.85 10.20 71.54 18.26 5 5 Tris HCl Sucrose 463.10 5.72 91.01 3.27 6 5 Na Phosphate (1) Sucrose 410.80 5.23 90.51 4.26 7 5 Tris HCl Sucrose 426.95 5.04 87.16 7.80 8 5 Na Phosphate (2) Sucrose 327.95 8.08 80.18 11.74 10 5 Tris HCl Mannitol 265.75 9.03 81.84 9.13 11 5 Na Phosphate (3) Mannitol 226.00 10.62 76.99 12.39 12 5 Tris HCl Sucrose 445.05 5.39 89.16 5.45 13 5 Na Phosphate (3) Sucrose 378.25 6.35 86.25 7.40 14 5 Tris HCl Mannitol 263.25 8.17 82.62 9.21 15 5 Tris HCl Mannitol 268.25 9.88 81.08 9.04 16 5 Tris HCl Sucrose 442.55 4.86 89.66 5.48 17 5 Tris HCl Sucrose 442.55 4.86 89.66 5.48 18 5 Na Phosphate (3) Mannitol 223.50 9.62 77.85 12.53 19 5 Na Phosphate (3) Mannitol 228.50 11.60 76.15 12.25 20 5 Na Phosphate (3) Sucrose 375.75 5.72 86.83 7.45 21 5 Na Phosphate (3) Sucrose 375.75 5.72 86.83 7.45 22 5 Tris HCl None 48.25 49.74 0.00 50.26 23 5 Na Phosphate (3) None 52.00 46.15 0.00 53.85 24 5 None None 24.00 100.00 0.00 0.00 25* 1 Na Phosphate (4) Mannitol 213.12 11.26 72.31 16.43 26** 5 Na Phosphate (4) Mannitol

TABLE-US-00019 TABLE 4F Formulation Screening Design (% w/w after freeze drying) Total Mass of Components (mg)-per Water freeze API Cryo Buffer Sample (ml) Buffer type Cryo type dried vial (% w/w) (% w/w) (% w/w) 1 5 Tris HCl Mannitol 267.65 8.03 86.31 5.66 2 5 Na Phosphate (1) Mannitol 272.91 9.71 74.68 15.61 3 5 Tris HCl Mannitol 263.75 10.05 77.33 12.63 4 5 Na Phosphate (2) Mannitol 266.07 8.08 56.70 35.22 5 5 Tris HCl Sucrose 463.10 5.72 91.01 3.27 6 5 Na Phosphate (1) Sucrose 435.91 4.93 85.29 9.78 7 5 Tris HCl Sucrose 426.95 5.04 87.16 7.80 8 5 Na Phosphate (2) Sucrose 383.17 6.92 68.63 24.46 10 5 Tris HCl Mannitol 265.75 9.03 81.84 9.13 11 5 Na Phosphate (3) Mannitol 266.14 9.02 65.38 25.60 12 5 Tris HCl Sucrose 445.05 5.39 89.16 5.45 13 5 Na Phosphate (3) Sucrose 418.39 5.74 77.98 16.29 14 5 Tris HCl Mannitol 263.25 8.17 82.62 9.21 15 5 Tris HCl Mannitol 268.25 9.88 81.08 9.04 16 5 Tris HCl Sucrose 442.55 4.86 89.66 5.48 17 5 Tris HCl Sucrose 442.55 4.86 89.66 5.48 18 5 Na Phosphate (3) Mannitol 263.64 8.16 66.00 25.84 19 5 Na Phosphate (3) Mannitol 268.64 9.86 64.77 25.36 20 5 Na Phosphate (3) Sucrose 415.89 5.17 78.45 16.38 21 5 Na Phosphate (3) Sucrose 415.89 5.17 78.45 16.38 22 5 Tris HCl None 48.25 49.74 0.00 50.26 23 5 Na Phosphate (3) None 92.14 26.05 0.00 73.95 24 5 None None 24.00 100.00 0.00 0.00 25* 1 Na Phosphate (4) Mannitol 26** 5 Na Phosphate (4) Mannitol 263.34 9.11 58.52 32.37

[0310] Reference to (1), (2), (3) and (4) in Tables 4A to 4F above are as follows:

[0311] (1) 0.25 mg/mL anhydr NaH.sub.2PO.sub.4+3.25 mg/mL anhydr Na.sub.2HPO.sub.4

[0312] (2) 0.56 mg/mL anhydr NaH.sub.2PO.sub.4+7.14 mg/mL anhydr Na.sub.2HPO.sub.4

[0313] (3) 0.41 mg/mL anhydr NaH.sub.2PO.sub.4+5.19 mg/mL anhydr Na.sub.2HPO.sub.4

[0314] (4) 0.50877 mg/mL anhydr NaH.sub.2PO.sub.4+6.49467 mg/mL anhydr Na.sub.2HPO.sub.4

[0315] The amounts of anhydrous and hydrated sodium phosphate used are illustrated below in Table 5.

TABLE-US-00020 TABLE 5 Equivalent Amount of Hydrated Sodium Phosphate Total Total NaH.sub.2PO.sub.4 NaH.sub.2PO.sub.4 Na.sub.2HPO.sub.4 Na.sub.2HPO.sub.4 Total NaH.sub.2PO.sub.4 Na.sub.2HPO.sub.4 Buffer Buffer 2H.sub.2O 2H.sub.2O 12H.sub.2O 12H.sub.2O Buffer (mmol/mL) (mmol/mL) (mmol/mL) (mmol) (mmol) (mg) (mmol) (mg) (mg) (1) 0.00208 0.02289 0.02497 0.12485 0.0104 1.62 0.11445 40.99 42.61 (2) 0.00467 0.05030 0.05497 0.27485 0.02335 3.64 0.2515 90.07 93.72 (3) 0.00342 0.03656 0.03998 0.19990 0.0171 2.67 0.1828 65.47 68.14 (4) 0.00425 0.04575 0.05000 0.05000 0.02125 3.32 0.22875 81.92 85.24

[0316] The molar masses used in the formulations were as follows: [0317] Sodium dihydrogen phosphate (anhydr)=119.98 g/mol [0318] Disodium hydrogen phosphate (anhydr)=141.96 g/mol [0319] Tris HCl=121.14 g/mol [0320] Mannitol=182.17 g/mol [0321] Sucrose=342.29 g/mol [0322] EMI-137=3,650.3 g/mol

[0323] From the above, it can be seen that the sodium phosphate buffer range=4.26-18.26% w/w before freeze drying and 9.78-35.22% w/w after freeze drying, whereas the Tris HCL buffer range=3.27-12.63% w/w before freeze drying and 3.27-12.63% w/w after freeze drying.

[0324] The materials used in the formulations are listed in Table 6 below.

TABLE-US-00021 TABLE 6 Materials Used in the Formulations for Screening Material Vendor / article no. Na.sub.2HPO.sub.4 12 H.sub.2O Merck 1.06573 NaH.sub.2PO.sub.4 2 H.sub.2O Merck 1.06345 Mannitol Merck 1.05980 Sucrose Merck 1.07653 Tris(hydroxymerthyl)aminomethane Merck 1.08386 Water for injection GEHC in-house bulk WFI EMI-137 ammonium acetate GEHC in-house

[0325] The screening study was followed by a 4 week pre-stability study at 25 C. and 40 C. The set of experiments was designed to achieve both screening of selected buffers and lyoprotectants, and to test robustness around production relevant API concentrations (target 4.8 mg/ml). A10% range around the target concentration was studied. The design would also uncover potential interactions between EMI-137, buffer and lyoprotectants. As all of the samples were made isotonic, the buffer concentration and lyoprotectant concentration were covariables. The test parameters are shown in Table 7. In addition to in-process testing of the bulk solution before freeze drying, the testing also included samples from the freezing step to uncover whether a potential instability could be ascribed to the process of freezing or drying.

TABLE-US-00022 TABLE 7 Testing Parameters Sample from Bulk freezing step, not Freeze Parameters solution dried dried Visual appearance of the n.a. n.a. X freeze dried cake Reconstitution n.a. n.a. X pH X n.a. X HPLC purity X X X Agglomeration as X X X measured by PCS n.a. = not applicable

[0326] Lyophilised Formulation Results

[0327] Stability data for up to 102 months at 2 to 8 C., 61 months at 30 C. and 6 months at 40 C. is illustrated in Tables 8A to 8C, 9A to 9C and 10 respectively (the following key/abbreviations are used: Amb=ambient humidity, not regulated; NMT=not more than; ND=not detected; ---: not performed; *24 months sampling point postponed due to summer vacation; **additional sampling point).

TABLE-US-00023 TABLE 8A Stability of EMI-137 Formulation Stored at 5 C. for 102 Months SAMPLING POINT (MONTHS) 0 6 12 DETERMINATION SPECIFICATION July 2009 January 2010 July 2010 Appearance of dry product Heterogeneous blue Blue cake or Blue cake or Blue cake or lyophilisate powder powder powder Appearance of reconstituted Clear, dark blue Clear, dark blue Clear, dark Clear, dark product solution, practically solution, blue solution, blue solution, free from visible practically free practically free practically free particles from particles from particles from particles Identification by HPLC, retention Conforms to reference Conforms to Conforms to Conforms to time reference reference reference Content of EMI-137 20.0-26.5 mg/vial 24.0, 24.7 pH 6.8 to 8.0 7.5, 7.5, 7.5 7.5 7.5 Osmolality 250-330 mOsm/kg 304, 278, 278 291 Related Each single Report value, x.xx% RRT 0.24 substances by related area 0.27 HPLC substance RRT 0.12 0.86 RRT 0.11 0.92 RRT 0.14 0.94 RRT 0.25 1.08 RRT 0.22 1.15 RRT 0.15 1.17 RRT 0.14 1.20 RRT 0.71 1.69 RRT 0.12 1.70 Sum of NMT 8.00% area 2.19 2.15, 2.16 1.74, 1.72 related substances Oxygen in head space Report value x.x l/vial 26.5, 10.7, 54.7, 13.0, 2.9, ND or NMT 2.0 Water NMT 5.0% m/m 2 1.4, 1.5, 1.3 1.7, 1.8, 2.0 Bacterial Endotoxins NMT 110 EU/vial NMT 0.5 Sterility Passes Ph. Eur./USP Passes Ph. Eur./USP Particulate 10 um NMT 6000 NOT IN SPECS contamination particles/container FOR PRECLINICAL 25 um NMT 600 NOT IN SPECS particles/container FOR PRECLINICAL Uniformity of mass of single- Passes Ph. Eur./USP dose preparation Diastereomers, ratio by HPLC Report value, x.xx 1.01, 1.01 1.01, 1.01 1.01, 1.01 Stopper

TABLE-US-00024 TABLE 8B Stability of EMI-137 Formulation Stored at 5 C. for 102 Months SAMPLING POINT (MONTHS) 25* 39** 61** DETERMINATION SPECIFICATION August 2011 October 2012 August 2014 Appearance of dry product Heterogeneous blue Blue cake or Heterogeneous Heterogeneous blue lyophilisate powder blue lyophilisate lyophilisate Appearance of reconstituted Clear, dark blue Clear, dark Clear, dark blue Clear, dark blue product solution, practically blue solution, solution, practically free from visible solution, practically free free from particles particles practically from particles free from particles Identification by HPLC, Conforms to Conforms to Conforms to retention time reference reference reference Content of EMI-137 20.0-26.5 mg/vial 22.9, 23.8 23.9, 23.9 22.1, 23.2 pH 6.8 to 8.0 7.5 7.6 7.4 Osmolality 250-330 mOsm/kg Related Each single Report value, x.xx% RRT 0.27 0.24 substances by related area HPLC substance RRT 0.94 0.13 RRT 1.08 0.21 RRT 1.15 0.19 RRT 1.17 0.13 RRT 1.20 0.13 RRT 1.67 0.71 Sum of NMT 8.00% area 1.90, 1.89 1.86, 1.84 1.74 related substances Oxygen in head space Report value x.x 58.6, 62.4, 76.8, 71.0, 73.9 , 150.4, 138.7 l/vial 48.6 Water NMT 5.0% m/m 1.7, 1.7, 1.8 1.2, 1.4, 1.6 1.5, 1.5, 1.6 Bacterial Endotoxins NMT 110 EU/vial Sterility Passes Ph.Eur./USP Particulate 10 um NMT 6000 contamination particles/container 25 um NMT 600 particles/container Uniformity of mass of single- Passes Ph. Eur./USP dose preparation Diastereomers, ratio by HPLC Report value, x.xx 1.01, 1.01 Stopper Passes Fragmentation and Penetrability according to Ph. Eur 1.5 years after expiry date (5 years)

TABLE-US-00025 TABLE 8C Stability of EMI-137 Formulation Stored at 5 C. for 102 Months SAMPLING POINT (MONTHS) 91 95 102 DETERMINATION SPECIFICATION February 2017 June 2017 January 2018 Appearance of dry product Heterogeneous blue Complies Complies lyophilisate Appearance of reconstituted Clear, dark blue solution, Complies Complies product practically free from visible particles Identification by HPLC, Conforms to reference Complies Complies retention time Content of EMI-137 20.0-26.5 mg/vial 23 22.7 pH 6.8 to 8.0 7.5 7.6 Osmolality 250-330 mOsm/kg 299 292 Related Each single Report value, x.xx % area substances by related HPLC substance Sum of NMT 8.00% area 0.41 2.01 related substances Oxygen in head space Report value x.x l/vial Water NMT 5.0% m/m 1.1 0.9 Bacterial Endotoxins NMT 110 EU/vial Passes Passes Sterility Passes Ph. Eur./USP Passes Passes Particulate 10 um NMT 6000 particles/container Passes Passes contamination 25 um NMT 600 particles/container Passes Passes Uniformity of mass of single- Passes Ph. Eur./USP Complies Complies dose preparation Diastereomers, ratio by HPLC Report value, x.xx Stopper

TABLE-US-00026 TABLE 9A Stability of EMI-137 Formulation Stored at 30 C. for 61 Months SAMPLING POINT (MONTHS) 0 3 6 DETERMINATION SPECIFICATION July 2009 October 2009 January 2010 Appearance of dry product Heterogeneous blue Blue cake or Blue cake or Blue cake or lyophilisate powder powder powder Appearance of reconstituted Clear, dark blue Clear, dark blue Clear, dark Clear, dark product solution, practically solution, blue solution, blue solution, free from visible practically free practically free practically free particles from particles from particles from particles Identification by HPLC, retention Conforms to reference Conforms to time reference Content of EMI-137 20.0-26.5 mg/vial 24.0, 24.7 pH 6.8 to 8.0 7.5, 7.5, 7.5 7.5 7.5 Osmolality 250-330 mOsm/kg 304, 278, 278 Related Each single Report value, x.xx% RRT 0.24 substances by related area 0.27 HPLC substance RRT 0.12 0.86 RRT 0.11 0.92 RRT 0.14 0.94 RRT 0.25 1.08 RRT 0.22 1.15 RRT 0.15 1.17 RRT 0.14 1.20 RRT 0.71 1.69 RRT 0.12 1.70 Sum of NMT 8.00% area 2.19 2.04, 2.05 2.15, 2.14 related substances Oxygen in head space Report value x.x l/vial 26.5, 10.7, 54.7, 13.0, 2.9, ND or NMT 2.0 Water NMT 5.0% m/m 2 2.4, 2.0 1.4, 1.4, 1.3 Bacterial Endotoxins NMT 110 EU/vial NMT 0.5 Sterility Passes Ph. Eur./USP Passes Ph. Eur./USP Particulate 10 um NMT 6000 NOT IN SPECS contamination particles/container FOR PRECLINICAL 25 um NMT 600 NOT IN SPECS particles/container FOR PRECLINICAL Uniformity of mass of single- Passes Ph. Eur./USP dose preparation Diastereomers, ratio by HPLC Report value, x.xx 1.01, 1.01 1.01, 1.01

TABLE-US-00027 TABLE 9B Stability of EMI-137 Formulation Stored at 30 C. for 61 Months SAMPLING POINT (MONTHS) 9 12 18 DETERMINATION SPECIFICATION April 2010 July 2010 October 2010 Appearance of dry product Heterogeneous blue Blue cake or Blue cake or Blue cake or lyophilisate powder powder powder Appearance of reconstituted Clear, dark blue Clear, dark blue Clear, dark blue Clear, dark blue product solution, practically free solution, solution, solution, from visible particles practically free practically free practically free from particles from particles from particles Identification by HPLC, retention Conforms to reference time Content of EMI-137 20.0-26.5 mg/vial 24.6, 23.4 pH 6.8 to 8.0 7.5 7.5 7.5 Osmolality 250-330 mOsm/kg 291 292 Related Each single Report value, x.xx % substances by related area HPLC substance Sum of NMT 8.00% area 2.01, 2.03 1.76, 1.74 2.27, 2.23 related substances Oxygen in head space Report value x.x l/vial Water NMT 5.0% m/m 1.2, 1.4, 1.2 1.5, 1.6, 1.6 1.4, 1.3, 1.5 Bacterial Endotoxins NMT 110 EU/vial Sterility Passes Ph. Eur./USP Particulate 10 um NMT 6000 particles/container contamination 25 um NMT 600 particles/container Uniformity of mass of single- Passes Ph. Eur./USP dose preparation Diastereomers, ratio by HPLC Report value, x.xx 1.01, 1.01

TABLE-US-00028 TABLE 9C Stability of EMI-137 Formulation Stored at 30 C. for 61 Months SAMPLING POINT (MONTHS) 25 39 61 DETERMINATION SPECIFICATION August 2011 October 2012 August 2014 Appearance of dry product Heterogeneous blue Blue cake or Heterogeneous Heterogeneous lyophilisate powder blue lyophilisate blue lyophilisate Appearance of reconstituted Clear, dark blue Clear, dark Clear, dark blue Clear, dark blue product solution, practically blue solution, solution, solution, free from visible practically free practically free practically free particles from particles from particles from particles Identification by HPLC, Conforms to retention time reference Content of EMI-137 20.0-26.5 mg/vial 23.6, 23.5 23.4, 24.7 22.5, 24.3 pH 6.8 to 8.0 7.5 7.6 7.4 Osmolality 250-330 mOsm/kg Related Each single Report value, x.xx% RRT 0.27 0.25 substances by related area RRT 0.86 0.13 HPLC substance RRT 0.94 0.13 RRT 1.08 0.19 RRT 1.15 0.28 RRT 1.17 0.20 RRT 1.20 0.13 RRT 1.23 0.11 RRT 1.68 0.66 RRT 1.68 0.11 Sum of NMT 8.00% area 1.98, 1.95 1.98, 1.95 2.19 related substances Oxygen in head space Report value x.x MT 136.0, MT MT 136.0, MT l/vial 136.0, MT 136.0, MT 136.0 136.0 Water NMT 5.0% m/m 1.7, 1.7, 1.7 1.8, 1.6, 1.4 1.8, 2.3, 2.4 Bacterial Endotoxins NMT 110 EU/vial NMT 0.50 Sterility Passes Ph. Eur./USP Ph. Eur./USP Particulate 10 um NMT 6000 contamination particles/container 25 um NMT 600 particles/container Uniformity of mass of single- Passes Ph. Eur./USP dose preparation Diastereomers, ratio by HPLC Report value, x.xx 1.01, 1.01

TABLE-US-00029 TABLE 10 Stability of EMI-137 Formulation Stored at 40 C. for 6 Months SAMPLING POINT (MONTHS) 0 1 3 6 DETERMINATION SPECIFICATION July 2009 August 2009 October 2009 January 2010 Appearance of dry product Heterogeneous blue Blue cake or Blue cake Blue cake Blue cake lyophilisate powder or powder or powder or powder Appearance of reconstituted Clear, dark blue Clear, dark Clear, dark Clear, dark Clear, dark product solution, practically blue solution, blue blue blue free from visible practically solution, solution, solution, particles free from practically practically practically particles free from free from free from particles particles particles Identification by HPLC, Conforms to Conforms to retention time reference reference Content of EMI-137 20.0-26.5 mg/vial 24.0, 24.7 pH 6.8 to 8.0 7.5, 7.5, 7.5 7.5 7.5 7.5 Osmolality 250-330 mOsm/kg 304, 278, 278 Related Each single Report value, x.xx% RRT 0.24 substances by related area 0.27 HPLC substance RRT 0.12 0.86 RRT 0.11 0.92 RRT 0.14 0.94 RRT 0.25 1.08 RRT 0.22 1.15 RRT 0.15 1.17 RRT 0.14 1.20 RRT 0.71 1.69 RRT 0.12 1.70 Sum of NMT 8.00% area 2.19 2.22, 2.34 2.15, 2.05 2.10, 2.41 related substances Oxygen in head space Report value x.x 26.5, 10.7, l/vial 54.7, 13.0, 2.9, ND or NMT 2.0 Water NMT 5.0% m/m 2 2.4, 2.0 1.5, 1.5, 1.7 2.0, 2.3 Bacterial Endotoxins NMT 110 EU/vial NMT 0.5 Sterility Passes Ph.Eur./USP Passes Ph. Eur./USP Particulate 10 um NMT 6000 NOT IN SPECS contamination particles/container FOR PRECLINICAL 25 um NMT 600 NOT IN SPECS particles/container FOR PRECLINICAL Uniformity of mass of single- Passes Ph. Eur./USP dose preparation Diastereomers, ratio by HPLC Report value, x.xx 1.01, 1.01 1.01, 1.01

[0328] The testing parameters are shown above (Table 7). Purity was tested by HPLG with UV/visual detection at 650 nm and expressed as % area units. The agglomeration was expressed as scattering intensity measured by Photon Correlation Spectroscopy (PCS). Evaluation of the reconstitution and visual appearance after freeze drying followed a rating system developed to describe various characteristics of the freeze-dried cakes.

[0329] Visual Appearance After Freeze Dryinq

[0330] In general, the good bulking properties of the mannitol gave a very good structure to all mannitol containing formulations. The mannitol-phosphate containing cakes appeared to have an inhomogeneous blue/whitish colour on the vial wall as opposed to mannitol-Tris-HCl formulations. Since the drug substance is intensely blue, these colour gradients became very pronounced. The inhomogeneous appearance was expected to originate partly from the inherently random nature of freezing and partly from properties of the formulation. When reconstituted with water the product was a homogeneous solution. The other analytical results, both initially and during the pre-stability period indicated that the inhomogeneous appearance could be considered as purely a cosmetic issue. Sucrose in combination with Tris-HCl tended to collapse/melt to a variable degree during freeze drying. Considerable vial-to-vial variation was seen, as 50% of the batch had freeze dried cakes with no apparent collapse. Upon storage at 40 C., the partly collapsed plugs gradually developed into completely collapsed material, already seen after seven days. This may be due to an elevated water concentration in collapsed material. The massive collapse seen during storage had a negative effect on purity, reconstitution and the tendency to form agglomerates.

[0331] Reconstitution After Freeze Dryinq

[0332] All of the buffered formulations containing lyoprotectant were easily reconstituted using very gentle swirling for a few seconds. The results were unaltered for the samples with non-collapsed and moderately collapsed plugs after four weeks of storage (25 C. and 40 C.). Massively collapsed freeze dried plugs were very difficult to reconstitute. These products also had inferior purity and contained agglomerates. The formulations without lyoprotectants (samples 22 to 24) needed more mixing for reconstitution, but were still considered acceptable.

[0333] pH

[0334] For sodium phosphate buffered formulations the pH was stable during freeze drying but decreased during the four weeks storage at 40 C. The Tris-HCl buffered formulations had a pH decrease during freeze drying but were generally more stable during storage at 40 C. The pH results were difficult to interpret and chemometric analysis did not give a clear picture. No trends could be ascribed to mannitol or sucrose. It was, however, clear that an increase in phosphate buffer concentration would reduce the pH drop during storage. At low buffer concentration the pH drop would increase with higher EMI-137 concentration. The Tris-HCl formulations were even more sensitive to increased EMI-137 drug substance concentration. This indicated that a buffer concentration in the upper test range should be selected.

[0335] Agglomerates (PCS)

[0336] Each formulation was tested by PCS for presence of large structures/agglomerates before and after freeze drying, and at the end of the four weeks pre-stability period. The centerpoint formulations (samples 10 to 13) were also tested after one week and two weeks. Values below approximately 20 ks/s were considered as background and were not judged as agglomerates. No agglomerates were detected in any of the bulk solutions before freeze drying. All of the lyoprotectant containing formulations were judged free of larger structures/agglomerates after freeze drying. After four weeks of storage at 40 C. all samples were still free of agglomerates 4, except for a massively collapsed sample (sample 17) and the non-buffered sample 24 containing EMI-137 in water. The latter formulation had a pH of 4.2. Agglomeration at low pH was consistent with previous initial findings. Phosphate buffered formulation without lyoprotectant (sucrose or mannitol) did, however, contain agglomerates after freeze drying (sample 23). Analysis of the reconstituted samples showed the presence of approximately 100 nm large structures. The agglomerates were formed during the drying process, not during the freezing step, as no agglomerates were found in samples withdrawn after the freezing. Sodium phosphate buffer is known to form pH gradients and shift the pH towards low values during freezing due to difference in solubility and crystallization of the phosphate salts. pH as low as 3.5 has been reported. At this pH agglomerates would be formed in the EMI-137 solution. Though frozen, the prolonged period at this pH during the freeze drying could potentially lead to agglomerate formation. It is believed that mannitol and sucrose prevented this from happening. (No lyoprotectant was needed in the Tris-HCl formulations. The Tris-HCl would shift the pH towards high pH (>9) during freezing.) It was concluded that significant melt back/collapse will affect the agglomerate level of the drug product and should be avoided. It was confirmed that agglomerates would form at low pH. When phosphate buffer was used as constituent during freeze drying, a lyoprotectant was needed to prevent EMI-137 from forming larger structures/agglomerates during the drying step of the freeze drying process.

[0337] Differential Scanning Calorimetry (DSC)

[0338] Formulations representing the centerpoints in the formulation design were analysed by DSC as freeze concentrated samples. Approximately 20 l sample was contained in a sealed aluminum crucible and analyzed using a Perkin Elmer Diamond DSC instrument. The samples were cooled to -80 C. at 5 C./min and held at 80 C. for 10 minutes, then heated to 24 C. at 5 C./min under nitrogen atmosphere. All calculations were performed on data collected upon heating.

[0339] Mannitol Formulations

[0340] The Tris-HCl-mannitol formulation had complex thermograms, showing three different glass transitions and two crystallization peaks. No visual collapse of the freeze dried cake was observed in spite of one glass transition below 60 C. This was probably due to the bulking properties of the crystallized mannitol. The phosphate-mannitol formulation thermogram showed two glass transitions and one crystallization peak. One of the glass transitions occurred between 50 C. and 60 C., which is significantly lower than the practical operating temperature of a normal freeze dryer. The other glass transition took place around 41 C., which is close to the operating limit when designing a freeze drying cycle. Apart from a slight shrinking of the freeze dried cake, there was no macroscopic meltback/collapse of phosphate-mannitol formulations. It was anticipated that mannitol had partly crystallized, and that the crystallized portion of the mannitol was sufficient to give mechanical stability to the freeze dried product. The exotherm crystallization peak observed at 27.6 C. (Tcryst onset) corresponded to the potential devitrification temperature of mannitol, as a recrystallization may occur slightly above the Tg of mannitol (33 C. to 27 C.) upon heating of the formulation.

[0341] Sucrose formulations

[0342] The thermograms of the sucrose containing formulations showed two thermal events consistent with glass transitions well below 50 C. and at approximately 35 C. to 37 C., respectively. Crystallization or eutectic melting peaks were not observed for any of the sucrose containing formulations, consistent with the content of sucrose. However, the heat flow from very small crystallization reactions would not be discernible if overlapping with the much higher heat flow from the low temperature side of the endothermic melting of water. Significant visual collapse was seen in some of the TrisHCI-sucrose containing vials, but not in the phosphate-sucrose containing formulation. Although the DSC data could be interpreted in favour of the phosphate-sucrose containing formulation (sample 13), it was concluded that the batch size of only twelve vials was too small to determine if a similar problem could be expected to occur with phosphate-sucrose formulation.

[0343] Near Infrared Analysis (NIR)

[0344] It is well known that mannitol has a strong tendency to crystallize from frozen aqueous solutions, both during cooling and reheating, and has also been observed to continue to crystallize after freeze drying, as the freeze drying process may produce partially amorphous, partially crystalline material. The crystallization during freeze drying can lead to different anhydrous polymorphs (, , ) and their mixtures, which leaves room for polymorphic transformations during storage.

[0345] Preliminary NIR studies combined with Principal Component Analysis (PCA) indicated the presence of structural differences between vials, that was ascribed to various degree of crystallization.

[0346] HPLC Purity

[0347] The HPLC purity profiles showed that both the phosphate and Tris-HCl formulations had a very similar purity profiles and also a similar degradation patterns compared to the pure EMI-137. Also, sucrose and mannitol containing formulations had similar purity profiles. Some small ratio differences between some of the peaks were found. No new peaks were seen in any of the formulations when compared to the EMI-137 drug substance. The buffer concentration did not seem to affect the. However, during the visual appearance analysis it was observed that sucrose in combination with Tris-HCl gave rise to vial-to-vial variability with regards to collapsed vials, as some vials had a perfect appearance while others had significantly collapsed freeze dried cakes. As both Tris HCl and sodium phosphate are substances with low Tg, the batch size of twelve vials was judged to be too small to uncover potential similar problems with phosphate-sucrose containing formulations.

[0348] Chemometric Analysis Summary

[0349] The chemometric modeling focused on changes in HPLC purity as a function of the various freeze dried formulations over a period of up to four weeks at 40 C., chemical changes in the NIR spectra to identify significant changes in degraded versus non-degraded freeze dried vials, and changes in agglomerates measured by PCS. The hemometric modeling indicated that all formulations containing Tris-HCl would have more degradation compared to the phosphate buffered formulations, though this could not easily be detected from visual comparison of the HPLC purity data. The response surfaces of the chemometric analysis indicated that increase of Tris-HCl concentration lead to increased degradation and chemical interactions. Generally, the Tris HCl containing formulations were concluded to be less predictable than phosphate regarding chemical degradation. This was supported by principal component analysis (PCA) of the NIR spectra obtained from samples 10 to 12 (representing centerpoints in the screening study) and correlation between NIR and HPLC purity.

[0350] The chemometric models made with sodium phosphate containing formulations were precise and reliable. In combination with sucrose the phosphate buffered formulations were chemically very stable within the formulation space tested, as very little degradation took place. However, more testing was needed to confirm that collapse during freeze drying would not be a potential problem. Sodium phosphate in combination with mannitol was concluded to give an adequate formulation judged from all of the analysed parameters.

[0351] Robustness/Design Space

[0352] Though the HPLC traces showed that the buffer concentration did not seem to be crucial for the degradation profile, chemometric analysis indicated that increased phosphate concentration could be expected to give a more chemically stable product when the formulation contained mannitol. The response surface showed a particularly stable area above 40 mM buffer concentration, where degradation was not significantly influenced by the EMI-137 concentration. A design space of 10% around the EMI-137 target concentration was tested. This covers a normal specification range for content of drug substance. It was found that a variation in the EMI-137 concentration within this range hardly affected purity at higher buffer concentrations, giving sufficient robustness to the formulation during manufacturing. The response surface of the pH changes demonstrated that increase in sodium phosphate buffer concentration reduced the changes of pH during storage. It was also confirmed that the formulation would not be robust towards changes in the EMI-137 concentration if the buffer concentration was too low. At buffer concentrations above 40 mM the pH changes were less than 0.2 pH units, when the EMI-137 concentration varied 10% from the target concentration (4.8 mg/ml). Above 50 mM the predicted pH changes during storage were small and stable towards variations of both EMI-137 concentration and buffer concentration, giving good robustness to the formulation during manufacturing. The multivariate analysis showed that the buffer concentration can vary between 40-55 mM with a drug substance concentration varying between 4.3 and 5.3 mg/ml without affecting the quality of the drug product.

[0353] cMet Receptor Affinity In Vitro Test

[0354] Sample 11 was tested for its affinity to c-Met receptor, and showed binding data equivalent to those of the pure EMI-137 drug substance. It was concluded that the mannitol-40 mM phosphate formulation did not adversely affect the binding of the peptide to the target receptor.

[0355] Discussion of Results of Lyophilised Formulation

[0356] As is illustrated in the tables above, the EMI-137 formulation remains stable over a long period of time (102 months), even at elevated temperature, thus it has an extended shelf-life. It is also notable that there is no trend of degradation of the product over the timescale of measurement, which is evidence of exceptional stability. Also, it is surprising that practically no related substances (i.e., impurities) form over the time period of measurement, that that there is no trend in the formation of these. Furthermore, on reconstitution, the formulation has a pH suitable for intravenous administration (approximately pH 7.5) and solubilises without agglomeration. In addition, the dry lyophilised solid retains a heterogenous blue appearance. It is also worth noting that the reconstituted formulation remains stable and substantially free from related substances up to 24 hours after reconstitution. Therefore, the EMI-137 formulation has been shown to be very stable in both dry (lyophilised) and reconstituted form.

[0357] Based on the analytical results and chemometric analysis the conclusions below were drawn.

[0358] It is necessary to have buffer in the formulation during preparation and freeze drying to maintain a neutral pH and avoid agglomeration. The sodium phosphate buffered formulations were judged to be more robust than the TrisHCI formulations based on chemometric analysis of all results. The minimum phosphate buffer concentration should be 40 mM. Lyoprotectant was needed in order to freeze dry the phosphate buffered formulation to prevent agglomerate formation during the drying process. Mannitol was shown to have good lyoprotectant activity. Massively collapsed freeze dried cakes were difficult to reconstitute, had inferior purity and a tendency to form agglomerates. This was not seen in vials with moderate but still clearly visible melt back/collapse. Sodium phosphate-sucrose containing formulations were considered the chemically most robust formulations, but were potentially not as robust from a freeze drying point of view. The freeze dried sodium phosphate-mannitol formulations had an inhomogeneous appearance and were less pharmaceutically elegant than Tris HCl-mannitol formulations as colour gradients appear in the freeze dried cakes. This was anticipated to originate from the freezing phase and concluded to be acceptable, as the product formed a homogeneous solution upon reconstitution. The Tris HCl containing formulations had generally less predictive data, and based on chemometric analysis these formulations were considered potentially a riskier (albeit still valid) choice. The overall conclusion from the study was that the sodium phosphate-mannitol containing formulation was considered to provide a satisfactory balance of risk, predictability and acceptability based on the limited pre-stability data available, the chemometric analysis of the analytical data and robustness considerations, albeit Tris HCl and sucrose may also be independently used as alternatives.

SUMMARY

[0359] The present invention teaches that in order to obtain adequate stability of the API, a freeze-dried drug product formulation is required. After reconstitution, the drug product should be an isotonic solution and have a physiological pH to make it suitable for intravenous administration. EMI-137 in water has a pH of 4.6, and shows a significant degree of formation of larger structures or agglomerates. This agglomeration is only partly reversible upon increasing the pH of the solution, thus making it unsuitable for intravenous administration. As such, a pH of greater than 6.8 and less than 9 (or potentially lower) is advantageous, since the formation of agglomerates is not observed in this pH range. This pH range should be maintained at all times, and should also be applied during the manufacturing of the bulk drug product solution. Thus, to maintain the pH of the EMI-137 solution within an acceptable range, it is beneficial to include a buffer to the solution prior to freeze-drying. It is also beneficial to include a lyoprotectant to ensure that EMI-137 does not degrade during freeze-drying, and a tonicity regulator (which may be the same material as the lyoprotectant) to ensure that the reconstituted product is isotonic and/or suitable for intravenous administration.

[0360] The use of the buffer and lyoprotectant/tonicity regulator as described above provides a homogenous solid that is stable and that, on reconstitution, is at a pH and tonicity that is suitable for intravenous administration. In addition, the formulation described retains a sufficient pH during lyophilisation that prevents agglomeration from taking place, and that does not result in the breakdown of the disulphide bridges in EMI-137. Furthermore, the formulation is storable at room temperature for elongated periods of time without notable degradation, and remains stable on and after reconstitution.

[0361] As noted above, to maintain the pH of the EMI-137 solution within an acceptable range on reconstitution, it is necessary to include a buffer prior to freeze-drying.

[0362] Various modifications and variations to the described embodiments of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.

Abbreviations

[0363] Conventional single letter or 3-letter amino acid abbreviations are used.

[0364] Acm: Acetamidomethyl [0365] ACN (or MeCN): Acetonitrile [0366] Boc: tert-Butyloxycarbonyl [0367] DCM: Dichloromethane [0368] DMF: Dimethylformamide DMSO: Dimethylsulfoxide [0369] Fmoc: 9-Fluorenylmethoxycarbonyl [0370] HBTU:O-Benzotriazol-1-yl-N,N,N,N-tetramethyluronium [0371] hexafluorophosphate [0372] HPLC: High performance liquid chromatography [0373] HSPyU: O(N-succinimidyl)-N,N,N,N-tetramethyleneuronium hexafluorophosphate [0374] NHS: N-hydroxy-succinimide [0375] NMM: N-Methylmorpholine [0376] NMP: 1-Methyl-2-pyrrolidinone [0377] Pbf: 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl [0378] PBS: Phosphate-buffered saline [0379] tBu: t-butyl [0380] TFA: Trifluoroacetic acid [0381] TIS: Triisopropylsilane [0382] Trt: Trityl [0383] Tris HCl: 2-Amino-2-(hydroxymethyl)-1,3-propanediolhydrochloride

Sequence Listing Free Text

[0384]

TABLE-US-00030 SEQ-1 Cys.sup.a-Xaa.sup.1-Cys.sup.c-Xaa.sup.2-Gly-Pro-Pro-Xaa.sup.3-Phe-Glu-Cys.sup.d- Trp-Cys.sup.b-Tyr-Xaa.sup.4-Xaa.sup.5-Xaa.sup.6

[0385] Xaa.sup.1 is Asn, His or Tyr;

[0386] Xaa.sup.2 is Gly, Ser, Thr or Asn;

[0387] Xaa.sup.3 is Thr or Arg;

[0388] Xaa.sup.4 is Ala, Asp, Glu, Gly or Ser;

[0389] Xaa.sup.5 is Ser or Thr; and

[0390] Xaa.sup.6 is Asp or Glu.

[0391] 17-mer cMET binding peptide.

TABLE-US-00031 SEQ-2 Ser-Cys.sup.a-Xaa.sup.1-Cys.sup.c-Xaa.sup.2-Gly-Pro-Pro-Xaa.sup.3-Phe-Glu- Cys.sup.d-Trp-Cys.sup.b-Tyr-Xaa.sup.4-Xaa.sup.5-Xaa.sup.6

[0392] Xaa.sup.1 is Asn, His or Tyr;

[0393] Xaa.sup.2 is Gly, Ser, Thr or Asn;

[0394] Xaa.sup.3 is Thr or Arg;

[0395] Xaa.sup.4 is Ala, Asp, Glu, Gly or Ser;

[0396] Xaa.sup.5 is Ser or Thr; and

[0397] Xaa.sup.6 is Asp or Glu.

[0398] 18-mer cMET binding peptide.

TABLE-US-00032 SEQ-3 Ala-Gly-Ser-Cys.sup.a-Xaa.sup.1-Cys.sup.c-Xaa.sup.2-Gly-Pro-Pro-Xaa.sup.3- Phe-Glu-Cys.sup.d-Trp-Cys.sup.b-Tyr-Xaa.sup.4-Xaa.sup.5-Xaa.sup.6-Gly-Thr

[0399] Xaa.sup.1 is Asn, His or Tyr;

[0400] Xaa.sup.2 is Gly, Ser, Thr or Asn;

[0401] Xaa.sup.3 is Thr or Arg;

[0402] Xaa.sup.4 is Ala, Asp, GIu, Gly or Ser;

[0403] Xaa.sup.5 is Ser or Thr; and

[0404] Xaa.sup.6 is Asp or GIu.

[0405] 22-mer cMET binding peptide.

TABLE-US-00033 SEQ-4 Gly-Gly-Gly-Lys

[0406] Tetrapeptide sequence that is part of cMET binding peptide.

TABLE-US-00034 SEQ-5 Gly-Ser-Gly-Lys

[0407] Tetrapeptide sequence that is part of cMET binding peptide.

TABLE-US-00035 SEQ-6 Gly-Ser-Gly-Ser-Lys

[0408] Gly-Ser-Gly-Ser-Lys

[0409] Five peptide sequence that is part of cMET binding peptide.

TABLE-US-00036 SEQ-7 Ala-Gly-Ser-Cys-Tyr-Cys-Ser-Gly-Pro-Pro-Arg-Phe- Glu-Cys-Trp-Cys-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly- Gly-Lys

[0410] 26-mer cMET binding peptide.

TABLE-US-00037 SEQ-8 Thr-Gly-Glu-Cys-Thr-Cys-Pro-Tyr-Trp-Glu-Phe-Arg- Pro-Cys-Glu-Cys-Gly-Ser-Tyr-Ser-Gly-Ala-Gly-Gly- Gly-Lys

[0411] 26-mer srambled cMET binding peptide (negative control).