Compounds and methods for diagnostic imaging and therapy

• US 20050085417A1
• Filed: 10/16/2003
• Published: 04/21/2005
• Est. Priority Date: 10/16/2003
• Status: Abandoned Application

• Current Assignee: Geneseen LLC
• Original Assignee: Thomas Jefferson University (Main Line Health System)
• Inventors: Wickstrom, Eric, Thakur, Mathew L.
• Application Number:
  US10/688,821
• Publication Number:
  US 20050085417A1
• Time in Patent Office: Days
• US Class Current: 514/44.R
• CPC Class Codes: A61K 51/088 conjugates with carriers being peptides, polyamino acids or proteins antibodies A61K51/10
• CPC Class Codes: A61K 51/1268 host-guest, closed hollow molecules, inclusion complexes, e.g. with cyclodextrins, clathrates, cavitates, fullerenes
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 15/1138 against receptors or cell surface proteins
• CPC Class Codes: C12N 2310/3181 Peptide nucleic acid, PNA
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2310/3517 Marker; Tag
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific

Abstract

Compounds comprising a diagnostic or therapeutic moiety can be retained inside a cell by conjugating the moiety to at least one PNA that is targeted to the transcripts from a gene of interest. The diagnostic or therapeutic moiety is also conjugated to at least one targeting moiety specific for an extracellular receptor or other cell surface molecule. The targeting moiety binds to the surface of a cell, and the entire compound is then internalized. Once inside the cell, the PNA portion of the diagnostic or therapeutic compound binds to RNA transcripts in a sequence specific manner. Binding of the PNA to its target RNA transcript retains the compound within the cell. The PNA can be designed to bind to a predetermined nucleic acid sequence from an RNA transcript, for example a mutated or overexpressed sequence that is characteristic of a pathological state.

Claims

1. A compound comprising a polymeric diagnostic or therapeutic moiety conjugated to at least one PNA and at least one targeting moiety, wherein the PNA comprises a base sequence that is complementary to a target nucleic acid sequence, or pharmaceutically acceptable salts thereof.

2. The compound of claim 1, comprising the formula

X-L_i—

Y

or pharmaceutically acceptable salts thereof, wherein;

X is a polymeric diagnostic or therapeutic moiety;

L_i is a chemical bond or at least one linking moiety; and

Y is P-L₂-T or T-L₂-P, in which

P is at least one peptide nucleic acid comprising a base sequence that is complementary to a target nucleic acid sequence;

L₂ is a chemical bond or at least one linking moiety; and

T is at least one targeting moiety.

3. The compound of claim 2, wherein L₁ and L₂ can be the same or different, and are independently selected from the group consisting of —

NH(O)C—

CH₂CH₂—

C(O)O— and

—

HN—

CH₂CH₂—

O—

CH₂CH₂—

O—

CH₂C(O)O, (Gly)₄ and 4-amino butyric acid.

4. The compound of claim 1, comprising a diagnostic moiety.

5. The compound of claim 1, comprising a therapeutic moiety.

6. The compound of claim 1, wherein the diagnostic or therapeutic moiety comprises a linear oligomeric polychelant.

7. The compound of claim 1, wherein the diagnostic or therapeutic moiety comprises a branched oligomeric polychelant.

8. The compound of claim 1, wherein the diagnostic or therapeutic moiety comprises a dendrimer.

9. The compound of claim 8, wherein the dendrimer is selected from the group consisting of starburst dendrimers, cascade dendrimers controlled hyperbranched dendrimers and random hyperbranched dendrimers.

10. The compound of claim 8, wherein the dendrimer is a polyamidoamine (PAMAM) dendrimer, a polypropylamine (POPAM) dendrimer, a polyether (PE) dendrimer or a polyethyleneimine (PEI) dendrimer.

11. The compound of claim 1, wherein the diagnostic or therapeutic moiety comprises at least one biodegradation cleavage site.

12. The compound of claim 1, wherein the diagnostic or therapeutic moiety comprises a bridged dendrimeric or polymeric moiety.

13. The compound of claim 4, wherein the diagnostic moiety comprises a plurality of chelants optionally complexed to one or more diagnostic metal ions.

14. The compound of claim 13, wherein the diagnostic metal ion is a paramagnetic metal ion, a heavy metal ion or an ion of a radioactive metal isotope.

15. The compound of claim 14, wherein the paramagnetic metal ion is selected from the group consisting of Eu, Ho, Gd, Dy, Mn, Cr and Fe.

16. The compound of claim 14, wherein the paramagnetic metal ion is selected from the group consisting of Gd(III), Mn(II) and Dy(III).

17. The compound of claim 14, wherein the heavy metal ion is selected from the group consisting of Hf, La, Yb, Dy and Gd.

18. The compound of claim 14, wherein the ion of radioactive metal isotopes is selected from the group consisting of ⁹⁹Tc, ⁸⁷Y, ⁶⁷Ga, ⁶⁸Ga, ⁶⁴Cu, and ¹¹¹In.

19. The compound of claim 4, wherein the diagnostic moiety comprises a diagnostic metal ion suitable for use in PET imaging.

20. The compound of claim 4, wherein the diagnostic moiety comprises a radioactive halogen.

21. The compound of claim 5, wherein the therapeutic moiety comprises a plurality of chelants optionally complexed to one or more therapeutic metal ions.

22. The compound of claim 21, wherein the therapeutic metal ion is an ion of a radioactive metal isotope.

23. The compound of claim 22, wherein the ion of a radioactive metal isotope is selected from the group consisting of ⁶⁴Cu, ⁹⁰Y, ¹⁰⁵Rh, ¹¹¹In, ^117mSn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁵Yb, ¹⁷⁷Lu, ^186/188Re, ¹⁹⁹Au, ⁴⁷Sc, ⁶⁷Cu, ⁶⁷Ga, ²¹²Pb ⁶⁸ Ga, ²¹²Bi, ²¹⁰At, and ²¹¹ At.

24. A compound of claim 1, wherein the polymeric diagnostic or therapeutic moiety comprises a plurality of NxSy chelants.

25. The compound of claim 24, wherein the NxSy chelants are N2S2 chelants, N3 chelants, N2S3 chelants, N2S4 chelants, N3S3 chelants, N4 chelants or NS3 chelants.

26. A compound of claim 1, wherein the polymeric diagnostic or therapeutic moiety comprises a plurality of linear, cyclic or branched polyamino-polycarboxylic acid chelants or their phosphorous oxyacid equivalents.

27. The compound of claim 26, wherein the linear, cyclic or branched polyamino-polycarboxylic acid chelants are selected from the group consisting of ethylenediamine-N,N,N′

,N′

-tetraacetic acid (EDTA);

N,N,N′

,N″

,N″

-diethylene-triaminepentaacetic acid (DTPA);

1,4,7,10-tetraazocyclododecane-N,N′

N”

,N′

″

-tetraacetic acid (DOTA);

1,4,7,10-tetraazo-cyclododecane-N,N′

N″

-triacetic acid (DO3A);

1-oxa-4,7,10-triazacyclododecane-N,N′

N″

-triacetic acid (OTTA);

trans(1,2)-cyclohexanodiethylene-triamine-pentaacetic acid (CDTPA);

1-oxa-4,7,10-triazacyclododecantriaacetic acid (DOXA);

1,4,7-triazacyclononanetriacetic acid (NOTA);

and 1,4,8,11-tetraazacyclotetradecanetetraacetic acid (TETA), and phosphorous oxyacid equivalents thereof.

28. The compound of claim 1, wherein the PNA comprises N-ethylaminoglycine backbone units, and the bases are covalently bound to the backbone units by methylene-carbonyl groups.

29. The compound of claim 1, wherein the PNA is about 8 to about 60 bases in length.

30. The compound of claim 1, wherein the target nucleic acid sequence comprises some or all of a consecutive sequence of bases in an RNA transcript.

31. The compound of claim 30, wherein the RNA transcript is heteronuclear RNA or messenger RNA.

32. The compound of claim 30, wherein the RNA transcript is produced from an oncogene or proto-oncogene.

33. The compound of claim 32, wherein the oncogene or proto-oncogene is selected from the group consisting of K-RAS, c-MYB, BCR-ABL, p53, CCND1, HER2, MYC, c-fms, c-kit, c-met, c-trk, c-neu, c-src, c-fes, c-abl, c-fgr, c-yes, c-erbA, c-evi-1, c-gli-1, c-maf, c-lyl-1, c-ets, c-fos, c-jun, c-myb, b-myb, N-myc, L-myc, c-rel, c-vav, c-ski, and c-spi.

34. The compound of claim 1, wherein the targeting moiety is a protein, a glycoprotein, a peptide, a steroid, a carbohydrate, a lipid or a vitamin.

35. The compound of claim 34, wherein the protein-targeting moiety is selected from the group consisting of peptide hormones, antigens, antibodies, growth factors, cytokines, and peptide toxins.

36. The compound of claim 35, wherein the antibody-targeting moiety is selected from the group consisting of monoclonal antibodies, chimeric antibodies, single chain antibodies, humanized antibodies, and antibody fragments.

37. The compound of claim 1, wherein the targeting moiety is selected from the group consisting of folate, transferrin and fragments and homologs thereof, epidermal growth factor (EGF) and fragments and homologs thereof;

platelet-derived growth factors and fragments and homologs thereof;

urogastrone and analogs thereof;

thyrotrypsin releasing hormone (TRH) and fragments and homologs thereof;

nerve-growth factor (NGF) and fragments and homologs thereof;

an HIV viral antigen;

α

₂-macroglobulin;

thiodothyronine;

thrombine;

arachidonic acid;

transforming growth factor-α

(TGF-α

) and fragments and homologs thereof;

heregulins (HRGs) and fragments and homologs thereof; and

alpha fetoprotein (AFP) and fragments and homologs thereof.

38. The compound of claim 1, wherein the targeting moiety is IGF1, ST, or fragments or homologs thereof.

39. The compound of claim 1, wherein the targeting moiety is the disulfide-bonded D-peptide Gly-Cys-Ser-Lys-Ala-Pro-Lys-Leu-Pro-Ala-Ala-Leu-Cys or the disulfide-bonded D-peptide Cys-Ser-Lys-Ala-Pro-Lys-Leu-Pro-Ala-Ala-Tyr-Cys.

40. The compound of claim 1, wherein the polymeric diagnostic agent comprises an ultrasound contrast agent.

41. A diagnostic imaging method, comprising:

(1) contacting cells of a subject that contain transcripts comprising a target nucleic acid sequence with a compound of claim 4, such that the compound binds to the cells via the targeting moiety and is internalized by the cell;

(2) allowing the PNA to bind to the target nucleic acid sequence and retain the compound inside the cell; and

(3) detecting the compound within the cells.

42. The method of claim 41, wherein the presence of the compound within the cells indicates a pathological state.

43. The method of claim 41, wherein the diagnostic moiety comprises a dendrimer.

44. The method of claim 41, wherein the diagnostic moiety comprises a plurality of chelants optionally complexed to one or more diagnostic metal ions.

45. The method of claim 44, wherein the diagnostic metal ion is a paramagnetic metal ion, a heavy metal ion or an ion of a radioactive metal isotope.

46. The method of claim 41, wherein the diagnostic moiety comprises a diagnostic metal ion suitable for use in PET imaging.

47. The method of claim 41, wherein the diagnostic moiety comprises a radioactive halogen.

48. The method of claim 42, wherein the pathological state is cancer.

49. The method of claim 48, wherein the cancer is pancreatic or breast cancer.

50. The method of claim 41, wherein the target nucleic acid sequence comprises some or all of a consecutive sequence of bases in an RNA transcript.

51. The method of claim 41, wherein the RNA transcript is produced from an oncogene or proto-oncogene.

52. The method of claim 41, wherein the targeting moiety is a protein, a glycoprotein, a peptide, a steroid, a carbohydrate, a lipid or a vitamin.

53. The method of claim 41, wherein the targeting moiety is IGF1, ST, or fragments or homologs thereof.

54. The method of claim 41, wherein the compound is detected within the cells by magnetic resonance imaging (MRI), scintigriphic imaging, X-ray, gamma camera imaging, ultrasound, or detection of fluorescent or visible light.

55. The method of claim 41, wherein the cells are contacted with the compound by an enteral or parenteral route of administration.

56. The method of claim 55, wherein the parenteral administration routes are selected from the group consisting of intravascular administration;

peri- and intra-tissue injection;

subcutaneous injection;

subcutaneous deposition;

subcutaneous infusion; and

direct application to the tumor or to tissue surrounding a tumor.

57. A therapeutic method, comprising:

(1) contacting cells of a subject that contain transcripts comprising a target nucleic acid sequence indicative of a pathological state with a compound of claim 5, such that the compound binds to the cells via the targeting moiety and is internalized by the cell;

(2) allowing the PNA to bind to the target nucleic acid sequence and retain the compound inside the cell, wherein the presence of the compound within the cell inhibits cell growth or causes death of the cell.

58. The method of claim 57, wherein the therapeutic moiety comprises a dendrimer.

59. The method of claim 57, wherein the therapeutic moiety comprises a plurality of chelants optionally complexed to one or more therapeutic metal ions.

60. The method of claim 59, wherein the therapeutic metal ion is an ion of a radioactive metal isotope.

61. The method of claim 57, wherein the pathological state is cancer.

62. The method of claim 61, wherein the cancer is pancreatic or breast cancer.

63. The method of claim 57, wherein the target nucleic acid sequence comprises some or all of a consecutive sequence of bases in an RNA transcript.

64. The method of claim 57, wherein the RNA transcript is produced from an oncogene or proto-oncogene.

65. The method of claim 57, wherein the targeting moiety is a protein, a glycoprotein, a peptide, a steroid, a carbohydrate, a lipid or a vitamin.

66. The method of claim 57, wherein the targeting moiety is IGF 1, ST, or fragments or homologs thereof.

67. The method of claim 57, wherein the cells are contacted with the compound by an enteral or parenteral route of administration.

68. The method of claim 67, wherein the parenteral administration routes are selected from the group consisting of intravascular administration;

peri- and intra-tissue injection;

subcutaneous injection;

subcutaneous deposition;

subcutaneous infusion; and

direct application to the tumor or to tissue surrounding a tumor.

69. A method of retaining a compound inside a cell, comprising:

(I) contacting a cell that contains transcripts comprising a target nucleic acid sequence with a compound of claim 1, such that the compound binds to the cell via the targeting moiety and is internalized by the cell;

(2) allowing the PNA to bind to the target nucleic acid sequence and retain the compound inside the cell.

70. The method of claim 69, wherein the diagnostic moiety comprises a dendrimer.

71. The method of claim 69, wherein the target nucleic acid sequence comprises some or all of a consecutive sequence of bases in an RNA transcript.

72. The method of claim 71, wherein the RNA transcript is produced from an oncogene or proto-oncogene.

73. The method of claim 69, wherein the targeting moiety is a protein, a glycoprotein, a peptide, a steroid, a carbohydrate, a lipid or a vitamin.

74. The method of claim 69, wherein the targeting moiety is IGF 1, ST, or fragments or homologs thereof.

75. The method of claim 69, wherein the cell is a cancer cell.

76. A method for detecting the overexpression of an RNA transcript comprising a target nucleic acid sequence within a cell, comprising:

(1) contacting a cell suspected of overexpressing the transcript with a compound of claim 4, such that the compound binds to the cells via the targeting moiety and is internalized by the cell;

(2) allowing the PNA to bind to the target nucleic acid sequence and retain the compound inside the cell; and

(3) detecting the compound within the cells, wherein the presence of the compound within the cells indicates overexpression of the RNA transcript.

77. The method of claim 76, wherein the diagnostic moiety comprises a dendrimer.

78. The method of claim 76, wherein the diagnostic moiety comprises a plurality of chelants optionally complexed to one or more diagnostic metal ions.

79. The method of claim 78, wherein the diagnostic metal ion is a paramagnetic metal ion, a heavy metal ion or an ion of a radioactive metal isotope.

80. The method of claim 76, wherein the RNA transcript is heteronuclear RNA or messenger RNA.

81. The method of claim 76, wherein the RNA transcript is produced from an oncogene or proto-oncogene.

82. The method of claim 81, wherein the oncogene or proto-oncogene is selected from the group consisting of MYC, K-RAS, c-myb, bcr-abl, p53, CCND1, HER2, c-fms, c-kit, c-met, c-trk, c-neu, c-src, c-fes, c-abl, c-fgr, c-yes, c-erbA, c-evi-1, c-gli-1, c-maf, c-lyl-1, c-ets, c-fos, c-jun, c-myb, b-myb, N-myc, L-myc, c-rel, c-vav, c-ski, and c-spi.

83. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier.

85. A pharmaceutical composition comprising the compound of claim 2 and a pharmaceutically acceptable carrier.

86. A pharmaceutical composition comprising the compound of claim 4 and a pharmaceutically acceptable carrier.

87. A pharmaceutical composition comprising the compound of claim 5 and a pharmaceutically acceptable carrier.