Nanoemulsion compositions of taxoid drugs, and methods for the use thereof to target cancer cells and cancer stem cells

Abstract

A composition of an omega-3 polyunsaturated fatty acid (PUFA)-taxoid conjugate encapsulated in an oil-in-water nanoemulsion (NE) drug delivery system. A method of treating cancer by administering an effective amount of a pharmaceutical composition including a PUFA-taxoid conjugate encapsulated in an oil-in-water NE drug delivery system to a subject in need of treatment, and treating cancer. A method of overcoming multidrug resistance by exposing a multidrug resistant cell to an effective amount of a pharmaceutical composition including an omega-3 polyunsaturated fatty acid (PUFA)-taxoid conjugate encapsulated in an oil-in-water NE drug delivery system, and inducing the death of the multidrug resistant cell. A method of eliminating a cancer stem cell. Methods of reducing stemness of a cancer stem cell, retaining drug in the body, and providing a slower release profile.

What is claimed is: 
     
       1. A composition comprising an omega-3 polyunsaturated fatty acid (PUFA)-taxoid conjugate of DHA-SBT-1214 encapsulated in an oil-in-water nanoemulsion (NE) drug delivery system, wherein the oil comprises one or more omega fatty acids and wherein said composition is formulated for parenteral administration. 
     
     
       2. The composition of  claim 1 , wherein said PUFA is chosen from the group consisting of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and alpha-linolenic acid (LNA). 
     
     
       3. The composition of  claim 1 , wherein said PUFA-taxoid conjugate is DHA-SBT-1214. 
     
     
       4. The composition of  claim 1 , wherein a taxoid in said PUFA-taxoid conjugate is chosen from the group consisting of paclitaxel, docetaxel, SBT-1213, SBT-12854, SBT-121303, SBT-1216, SBT-11033, SBT-121313, SBT-121602, cabazitaxel, SBT-1212, SBT-1217, SBT-1102, SBT-1103, SBT-1104, SBT-1106, SBT-1107, SBT-121301, SBT-121302, SBT-121304, SBT-121403, SBT-11031, SBT-11032, SBT-11034, SBT-12851, SBT-12852, SBT-12853, SBT-12855, SBT-12851-1, SBT-12851-3, SBT-12852-1, SBT-12852-3, SBT-12853-1, SBT-12853-3, SBT-12854-1, SBT-12854-3, SBT-12855-1, and SBT-12855-3. 
     
     
       5. The composition of  claim 1 , wherein said PUFA-taxoid conjugate is chosen from the group consisting of DHA-paclitaxel, DHA-docetaxel, DHA-SBT-1213, DHA-SBT-1103, DHA-SBT-1104, DHA-SBT-1216, LNA-SBT-1213, LNA-paclitaxel, LNA-docetaxel, DHA-cabazitaxel, and LNA-cabazitaxel. 
     
     
       6. The composition of  claim 4 , wherein said PUFA-taxoid conjugate is a DHA or LNA ester of said taxoid defined in  claim 4 . 
     
     
       7. The composition of  claim 1 , wherein said oil-in-water NE includes mean droplet diameters ranging from 50 to 1000 nm. 
     
     
       8. The composition of  claim 1 , wherein said oil-in-water NE includes mean droplet diameters less than 200 nm. 
     
     
       9. The composition of  claim 1 , wherein an oil in said oil-in-water NE is an omega-3 fatty acid-rich edible oil chosen from the group consisting of fish oil, pine nut oil, flax-seed oil, safflower oil, primrose oil, black currant oil, borage oil, wheat germ oil, chia oil, hemp oil, perilla oil, grape oil, squalene oil, and fungal oil. 
     
     
       10. The composition of  claim 1 , wherein said oil is modified with a substance chosen from the group consisting of surfactants, targeting agents, image contrast agents, and combinations thereof. 
     
     
       11. The composition of  claim 1 , wherein said PUFA-taxoid conjugate is encapsulated in nanoparticles. 
     
     
       12. The composition of  claim 1 , wherein said composition is a pharmaceutical composition including pharmaceutically acceptable carriers. 
     
     
       13. The composition of  claim 1 , wherein said composition is physically stable at 4° C. for up to 6 months. 
     
     
       14. The composition of  claim 13 , wherein the composition has a stable particle size for up to 6 months. 
     
     
       15. The composition of  claim 1 , wherein said composition has increased retention times in the body than a solution form of said PUFA-taxoid conjugate. 
     
     
       16. The composition of  claim 1 , wherein said composition has a release profile that is at least three times slower in the body than a solution form of said PUFA-taxoid conjugate. 
     
     
       17. A method of treating cancer, including the steps of: administering parenterally an effective amount of a pharmaceutical composition including an omega-3 polyunsaturated fatty acid (PUFA)-taxoid conjugate of DHA-SBT-1214 encapsulated in an oil-in-water NE drug delivery system, wherein the oil comprises one or more omega fatty acids and wherein the composition is formulated for parenteral administration, to a subject in need of treatment; and treating cancer chosen from the group consisting of colon, pancreatic, non-small cell lung, and prostate. 
     
     
       18. The method of  claim 17 , wherein the PUFA is chosen from the group consisting of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and alpha-linolenic acid (LNA). 
     
     
       19. The method of  claim 18 , wherein the PUFA-taxoid conjugate is DHA-SBT-1214. 
     
     
       20. The method of  claim 18 , wherein a taxoid in the PUFA-taxoid conjugate is chosen from the group consisting of paclitaxel, docetaxel, SBT-1213, SBT-12854, SBT-121303, SBT-1216, SBT-11033, SBT-121313, SBT-121602, cabazitaxel, SBT-1212, SBT-1217, SBT-1102, SBT-1103, SBT-1104, SBT-1106, SBT-1107, SBT-121301, SBT-121302, SBT-121304, SBT-121403, SBT-11031, SBT-11032, SBT-11034, SBT-12851, SBT-12852, SBT-12853, SBT-12855, SBT-12851-1, SBT-12851-3, SBT-12852-1, SBT-12852-3, SBT-12853-1, SBT-12853-3, SBT-12854-1, SBT-12854-3, SBT-12855-1, and SBT-12855-3. 
     
     
       21. The method of  claim 18 , wherein the PUFA-taxoid conjugate is chosen from the group consisting of DHA-paclitaxel, DHA-docetaxel, DHA-SBT-1213, DHA-SBT-1103, DHA-SBT-1104, DHA-SBT-1216, LNA-SBT-1213, LNA-paclitaxel, LNA-docetaxel, DHA-cabazitaxel, and LNA-cabazitaxel. 
     
     
       22. The method of  claim 20 , wherein said PUFA-taxoid conjugate is a DHA or LNA ester of the taxoid defined in  claim 20 . 
     
     
       23. The method of claim  19   17 , further including the step of reducing the expression of stemness-promoting genes and transcription factors in cancer stem cells. 
     
     
       24. The method of  claim 23 , wherein the stemness-promoting genes are chosen from the group consisting of ABCG2, ACAN, ACTB, AIN1, ALDH1A1, ALPI, ASCL2, BMP1, BMP3, CCND1, CD3D, CD4, CD8A, CD8B, CD8B1, CDH2, COL1A1, COL2A1, COL9A1, CTNNA1, DHH, DLL1, DLL3, DTX1, DVL1, FGF1, FGF3, FGFR1, FZD1, GDF2, GDF3, GJA1, GJB1, IGF1, ISL1, JAG1, KRT15, MME, MSX1, MYOD, NEUROG2, NCAM1, NOTCH1, NUMB, PARD6A, PPARD, RB1, RPL13A, S100B, SOX1, SOX2, TERT and combinations thereof. 
     
     
       25. The method of  claim 23 , wherein the transcription factors are chosen from the group consisting of Sox-2, Oct3/4, c-Myc, Klf4, and combinations thereof. 
     
     
       26. The method of claim  19   17 , further including the steps of reducing or eliminating a cancer stem cell component of a tumor, and rendering the tumor more susceptible to therapy. 
     
     
       27. The method of claim  19   17 , further including the steps of rapidly polymerizing tubulin and inducing cell death. 
     
     
       28. The method of claim  19   17 , further including the step of administering a non-conjugated version of the PUFA-taxoid taxoid in combination with said (PUFA)-taxoid conjugate to the subject. 
     
     
       29. The method of claim  19   17 , wherein the subject has paclitaxel-sensitive or paclitaxel-resistant tumors. 
     
     
       30. The method of claim  19   17 , further including the step of down-regulating expression of a gene selected from the group consisting of CDX2, DLX2, DNMT3B, EGR, FOXP3, GLI2, HOX family TFs, IRX4, JUN, KLF2, NFATC1, NR2F2, PCNA, PITX3, POU4F1, SIX2, SOX9, WT1, and combinations thereof. 
     
     
       31. The method of claim  19   17 , further including the steps of suppressing tumor growth, inducing tumor shrinkage, reducing production of adherent holoclones, and reducing vascularization of the tumor. 
     
     
       32. The method of claim  19   17 , wherein said administering step further includes the step of providing tumor-specific accumulation of composition through gp60-mediated transcytosis into tumor interstitium due to an affinity of the composition to human serum albumin. 
     
     
       33. The method of claim  19   17 , further including the step of retaining the pharmaceutical composition in the subject for a longer period of time than a solution form of the pharmaceutical composition. 
     
     
       34. The method of  claim 33 , wherein the pharmaceutical composition is retained in a tumor in the subject for a longer period of time than a solution form of the pharmaceutical composition. 
     
     
       35. The method of claim  19   17 , further including the step of providing a release profile of the pharmaceutical composition that is at least three times slower than a release profile of a solution form of the pharmaceutical composition. 
     
     
       36. A method of overcoming multi-drug resistance, including the steps of: exposing a multi-drug resistant cell selected from the group consisting of colon cancer cells and prostate cancer cells to an effective amount of a pharmaceutical composition including an omega-3 polyunsaturated fatty acid (PUFA)-taxoid conjugate of DHA-SBT-1214 encapsulated in an oil-in-water NE drug delivery system, wherein the oil comprises one or more omega fatty acids and wherein the composition is formulated for parenteral administration; and inducing the death of the multi-drug resistant cell. 
     
     
       37. The method of  claim 36 , wherein the PUFA-taxoid conjugate is DHA-SBT-1214. 
     
     
       38. The method of  claim 36 , further including the step of reducing the expression of stemness-promoting genes and transcription factors in cancer stem cells. 
     
     
       39. The method of  claim 38 , wherein the stemness-promoting genes are chosen from the group consisting of ABCG2, ACAN, ACTB, AIN1, ALDH1A1, ALPI, ASCL2, BMP1, BMP3, CCND1, CD3D, CD4, CD8A, CD8B, CD8B1, CDH2, COL1A1, COL2A1, COL9A1, CTNNA1, DHH, DLL1, DLL3, DTX1, DVL1, FGF1, FGF3, FGFR1, FZD1, GDF2, GDF3, GJA1, GJB1, IGF1, ISL1, JAG1, KRT15, MME, MSX1, MYOD, NEUROG2, NCAM1, NOTCH1, NUMB, PARD6A, PPARD, RB1, RPL13A, S100B, SOX1, SOX2, TERT and combinations thereof. 
     
     
       40. The method of  claim 38 , wherein the transcription factors are chosen from the group consisting of Sox-2, Oct3/4, c-Myc, Klf4, and combinations thereof. 
     
     
       41. The method of  claim 36 , further including the steps of reducing or eliminating a cancer stem cell component of a tumor, and rendering the tumor more susceptible to therapy. 
     
     
       42. The method of  claim 36 , further including the steps of rapidly polymerizing tubulin and inducing cell death. 
     
     
       43. The method of  claim 36 , wherein the subject has paclitaxel-sensitive or paclitaxel-resistant tumors. 
     
     
       44. The method of  claim 36 , further including the step of down-regulating expression of a gene selected from the group consisting of CDX2, DLX2, DNMT3B, EGR, FOXP3, GLI2, HOX family TFs, IRX4, JUN, KLF2, NFATC1, NR2F2, PCNA, PITX3, POU4F1, SIX2, SOX9, WT1, and combinations thereof. 
     
     
       45. The method of  claim 36 , further including the steps of suppressing tumor growth, inducing tumor shrinkage, reducing production of adherent holoclones, and reducing vascularization of the tumor. 
     
     
       46. The method of  claim 36 , wherein said administering step further includes the step of providing tumor-specific accumulation of composition through gp60-mediated transcytosis into tumor interstitium due to an affinity of the composition to human serum albumin. 
     
     
       47. A method of eliminating a cancer stem cell, including the steps of: exposing a cancer stem cell selected from the group consisting of colon cancer stem cells and prostate cancer stem cells to an effective amount of a pharmaceutical composition including an omega-3 polyunsaturated fatty acid (PUFA)-taxoid conjugate of DHA-SBT-1214 encapsulated in an oil-in-water NE drug delivery system, wherein the oil comprises one or more omega fatty acids and wherein the composition is formulated for parenteral administration; and inducing the death of the cancer stem cell. 
     
     
       48. The method of  claim 47 , wherein the PUFA-taxoid conjugate is DHA-SBT-1214. 
     
     
       49. A method of reducing the stemness of a cancer stem cell, including the steps of: exposing a cancer stem cell selected from the group consisting of colon cancer stem cells and prostate cancer stem cells to an effective amount of a pharmaceutical composition including an omega-3 polyunsaturated fatty acid (PUFA)-taxoid conjugate of DHA-SBT-1214 encapsulated in an oil-in-water NE drug delivery system, wherein the oil comprises one or more omega fatty acids and wherein the composition is formulated for parenteral administration; and reducing the expression of stemness-promoting genes in the cancer stem cell. 
     
     
       50. The method of  claim 49 , wherein the PUFA-taxoid conjugate is DHA-SBT-1214. 
     
     
       51. A method of increasing retention times of an omega-3 polyunsaturated fatty acid (PUFA)-taxoid conjugate in the body of a subject, including the steps of: administering parenterally an effective amount of a pharmaceutical composition including an omega-3 polyunsaturated fatty acid (PUFA)-taxoid conjugate of DHA-SBT-1214 encapsulated in an NE drug delivery system, wherein the oil comprises one or more omega fatty acids and wherein the composition is formulated for parenteral administration; and retaining the pharmaceutical composition in the body for a longer period of time than a solution form of the pharmaceutical composition. 
     
     
       52. The method of  claim 51 , wherein said retaining step is further defined as retaining the pharmaceutical composition in an area of the body chosen from the group consisting of plasma and a tumor for a longer period of time than a solution form of the pharmaceutical composition. 
     
     
       53. A method of providing a slower release profile of an omega-3 polyunsaturated fatty acid (PUFA)-taxoid conjugate in the body of a subject, including the steps of: administering parenterally an effective amount of a pharmaceutical composition including an omega-3 polyunsaturated fatty acid (PUFA)-taxoid conjugate of DHA-SBT-1214 encapsulated in an NE drug delivery system, wherein the oil comprises one or more omega fatty acids and wherein the composition is formulated for parenteral administration; and releasing the pharmaceutical composition in the body at least three times slower than a solution form of the pharmaceutical composition. 
     
     
       54. A composition having a taxoid-conjugate encapsulated in a nanoemulsion (NE) drug delivery system,
 wherein the taxoid-conjugate comprises a SBT-1214;   wherein the taxoid-conjugate comprises an omega-3 polyunsaturated fatty acid (PUFA);   wherein the taxoid-conjugate is encapsulated in the NE drug delivery system and formulated for parenteral administration;   wherein the NE drug delivery system comprises an aqueous phase and an oil/lipid phase; and   wherein the taxoid-conjugate is released from the NE delivery system in a formulation more slowly than the taxoid-conjugate is released from the formulation lacking the NE delivery system.    
     
     
       55. The composition of  claim 54 , wherein said NE drug delivery system has an oil/lipid phase comprising droplets having a mean diameter ranging from 50 to 1000 nm.  
     
     
       56. The composition of  claim 54 , wherein said NE drug delivery system has an oil/lipid phase comprising droplets having a mean diameter of 200 nm.  
     
     
       57. The composition of  claim 54 , wherein said oil/lipid phase is an omega-3 fatty acid-rich edible oil chosen from the group consisting of fish oil, pine nut oil, flax-seed oil, primrose oil, black currant oil, borage oil, wheat germ oil, chia oil, hemp oil, perilla oil, grape oil, squalene oil, and fungal oil.  
     
     
       58. The composition of  claim 54 , wherein said NE drug delivery system is modified with a substance chosen from the group consisting of surfactants, targeting agents, and image contrast agents, or combinations thereof.  
     
     
       59. The composition of  claim 58 , wherein said surfactant is polysorbate 80.  
     
     
       60. The composition of  claim 54 , wherein said composition is a pharmaceutical composition including pharmaceutically acceptable carriers.  
     
     
       61. The composition of  claim 54 , wherein said composition is physically stable at 4° C. for up to 6 months, and has a stable particle size for up to 6 months.  
     
     
       62. The composition of  claim 54 , wherein said composition has increased retention times in the body than a solution form of said taxoid-conjugate.  
     
     
       63. The composition of  claim 54 , wherein said taxoid-conjugate is released from the NE delivery system in a formulation at least three times more slowly than the taxoid-conjugate is released from the formulation lacking the NE delivery system.  
     
     
       64. A method of treating a subject having cancer, said method comprising administering parenterally to the subject a therapeutically effective amount of the composition of  claim 54 .  
     
     
       65. The method of  claim 64 , wherein the cancer is breast, ovary, lung, head and neck, colon, rectal, pancreatic, melanoma, brain, prostate, leukemia, sarcomas, thyroid, Non-Hodgkin Lymphoma, bladder, gliomas, endometrial, or renal cancer.  
     
     
       66. The method of  claim 64 , wherein expression of stemness-promoting genes and transcription factors in a stem cell in the cancer is reduced, wherein the stemness-promoting genes are chosen from the group consisting of ABCG2, ACAN, ACTB, AIN1, ALDH1A1, ALPI, ASCL2, BMP1, BMP3, CCND1, CD3D, CD4, CD8A, CD8B, CD8B1, CDH2, COL1A1, COL2A1, COL9A1, CTNNA1, DHH, DLL1, DLL3, DTX1, DVL1, FGF1, FGF3, FGFR1, FZD1, GDF2, GDF3, GJA1, GJB1, IGF1, ISL1, JAG1, KRT15, MME, MSX1, MYOD, NEUROG2, NCAM1, NOTCH1, NUMB, PARD6A, PPARD, RB1, RPL13A, S100B, SOX1, SOX2, and TERT, or combinations thereof, and wherein the transcription factors are chosen from the group consisting of Sox-2, Oct3/4, and c-Myc, Klf4, or combinations thereof.  
     
     
       67. The method of  claim 64 , wherein cancer stem cells of the cancer are reduced or eliminated rendering the tumor more susceptible to therapy, and wherein the tubulin of the cancer rapidly polymerizes, inducing cell death.  
     
     
       68. The method of  claim 64 , wherein the subject has a paclitaxel-sensitive tumor.  
     
     
       69. The method of  claim 64 , wherein a gene chosen from the group consisting of CDX2, DLX2, DNMT3B, EGR, FOXP3, GLI2, HOX family TFs, IRX4, JUN, KLF2, NFATC1, NR2F2, PCNA, PITX3, POU4F1, SIX2, SOX9, and WT1, and combinations thereof of a cancer cell of the cancer are down-regulated.  
     
     
       70. The method of  claim 64 , wherein the method results in suppression of tumor growth, induction of tumor shrinkage, reduction production of adherent holoclones, or reduction vascularization of the tumor and wherein the taxoid-conjugate is DHA-SBT-1214.  
     
     
       71. The method of  claim 64 , wherein the taxoid-conjugate is released from the NE delivery system in a formulation more slowly than the taxoid-conjugate is released from the formulation lacking the NE delivery system.  
     
     
       72. The method of  claim 64 , wherein the taxoid-conjugate is retained in the tumor for a longer period of time than a solution form of the pharmaceutical composition and wherein the taxoid-conjugate is released from the NE delivery system in a formulation at least three times more slowly than the taxoid-conjugate is released from a formulation lacking the NE delivery system.  
     
     
       73. A method of  claim 64 , wherein the cancer is a multi-drug resistant cancer.  
     
     
       74. The method of  claim 73 , wherein expression of stemness-promoting genes and transcription factors in a stem cell in the cancer is reduced, wherein the stemness-promoting genes are chosen from the group consisting of ABCG2, ACAN, ACTB, AIN1, ALDH1A1, ALPI, ASCL2, BMP1, BMP3, CCND1, CD3D, CD4, CD8A, CD8B, CD8B1, CDH2, COL1A1, COL2A1, COL9A1, CTNNA1, DHH, DLL1, DLL3, DTX1, DVL1, FGF1, FGF3, FGFR1, FZD1, GDF2, GDF3, GJA1, GJB1, IGF1, ISL1, JAG1, KRT15, MME, MSX1, MYOD, NEUROG2, NCAM1, NOTCH1, NUMB, PARD6A, PPARD, RB1, RPL13A, S100B, SOX1, SOX2, and TERT, and combinations thereof, and wherein the transcription factors are chosen from the group consisting of Sox-2, Oct3/4, c-Myc, and Klf4, or combinations thereof, and wherein the cancer stem cells of the cancer are reduced or eliminated rendering the tumor more susceptible to therapy and wherein the tubulin of the cancer rapidly polymerizes, inducing cell death.  
     
     
       75. The method of  claim 64 , wherein the subject has a paclitaxel-resistant tumor or paclitaxel-sensitive tumor.  
     
     
       76. The method of  claim 73 , wherein a gene chosen from the group consisting of CDX2, DLX2, DNMT3B, EGR, FOXP3, GLI2, HOX family TFs, IRX4, JUN, KLF2, NFATC1, NR2F2, PCNA, PITX3, POU4F1, SIX2, SOX9, and WT1, and combinations thereof of a cancer cell of the cancer is down-regulated, and wherein the method results in suppression of tumor growth, induction tumor shrinkage, reduction production of adherent holoclones, or reduction vascularization of the tumor.  
     
     
       77. The method of  claim 73 , wherein the cancer is breast, pancreatic, prostate, colon, or rectal, and wherein the composition is the composition of  claim 54 .  
     
     
       78. A method of reducing the stemness of a cancer stem cell, comprising the steps of:
 exposing a cancer stem cell selected from the group consisting of colon cancer stem cells and prostate cancer stem cells to an effective amount of the composition of claim 54.    
     
     
       79. The composition of  claim 54 , wherein the aqueous phase comprises egg phosphatidylcholine (E-80), polysorbate 80, DSPE-PEG2000 and water.  
     
     
       80. The composition of  claim 54 , wherein the aqueous phase comprises egg phosphatidylcholine (E-80), polysorbate 80, DSPE-PEG2000 and water.  
     
     
       81. The composition of  claim 80 , wherein the oil/lipid phase comprises an omega-3 fatty acid-rich oil selected from the group consisting of fish oil, pine nut oil, flax-seed oil, primrose oil, black currant oil, borage oil, wheat germ oil, chia oil, hemp oil, perilla oil, grape oil, squalene oil, and fungal oil.  
     
     
       82. The composition of  claim 54 , wherein the NE delivery system further comprises a modifier selected from the group consisting of surfactants, targeting agents, and image contrast agents.  
     
     
       83. The composition of  claim 81 , wherein the NE delivery system further comprises a modifier selected from the group consisting of surfactants, targeting agents, and image contrast agents.  
     
     
       84. The composition of  claim 54 , wherein the taxoid-conjugate is released from the NE delivery system in an amount chosen from the group consisting of less than 10% in the course of 5 hours, less than 10% in the course of 10 hours, and less than 10% in the course of 48 hours.  
     
     
       85. The composition of  claim 80 , wherein the DHA-SBT-1214 is released from the NE delivery system in an amount chosen from the group consisting of less than 10% in the course of 5 hours, less than 10% in the course of 10 hours, and less than 10% in the course of 48 hours.  
     
     
       86. The composition of  claim 54 , wherein the NE delivery system maintains at least 80% of its original concentration of the taxoid-conjugate upon storage under conditions of 4° C. for an amount chosen from the group consisting of at least 2 months, at least 3 months, and at least 6 months.  
     
     
       87. The composition of  claim 80 , wherein the NE delivery system maintains at least 80% of its original concentration of DHA-SBT-1214 upon storage under conditions of 4° C. for an amount chosen from the group consisting of at least 2 months, at least 3 months, and at least 6 months.  
     
     
       88. A pharmaceutical composition comprising the composition of  claim 54 .  
     
     
       89. A pharmaceutical composition comprising the composition of  claim 80 .  
     
     
       90. The method of  claim 64 , wherein the cancer is resistant to 5-FU, oxaliplatin, irinotecan, docetaxel, or cabazitaxel.