POLYMER COMPOSED OF REPEAT UNITS HAVING A BIOLOGICALLY ACTIVE MOLECULE ATTACHED THERETO VIA A PH-SENSITIVE BOND

Abstract

There is provided a polymer comprising: (i) a repeat unit derived from a compound of formula (I) (Formula (I)) wherein, R.sup.1 and R.sup.2 are each independently selected from OH, OR′, SH, SR′, NH.sub.2, NHR′ and NR′.sub.2; R′ is C.sub.1-20 hydrocarbyl; each n is independently 0 or an integer between 1 and 6; each m is independently 0 or an integer between 1 and 4, and preferably at least one m is 1; and q is an integer between 1 and 8; and; (ii) a biologically active molecule, wherein said biologically active molecule is covalently bonded to said repeat unit; as well as methods for preparing such polymers, particles comprising said polymers and uses of said polymers and particles including use in the treatment of disease.

##STR00001##

Claims

1-58. (canceled)

59. A particle comprising a polymer, wherein said polymer comprises a repeat unit of formula (III): ##STR00042## wherein: each n is independently 0 or an integer between 1 and 6; each m is independently 0 or an integer between 1 and 4, and at least one m is 1; ---- is a bond which may be absent or present; each D is a moiety which is a biologically active molecule, or a derivative thereof, when the C to D bond(s) is broken; each q is an integer between 1 and 8; X is selected from O, S, NH and NR′; Y is selected from O, S, NH and NR′; R′ is C.sub.1-20 hydrocarbyl; Q is selected from —(CH.sub.2).sub.p—, —(CH.sub.2CH.sub.2O).sub.sCH.sub.2CH.sub.2—, —(CH.sub.2).sub.rNR′—(CH.sub.2).sub.r— and —(CH.sub.2CH.sub.2CH.sub.2O).sub.sCH.sub.2CH.sub.2CH.sub.2—; and each of p, r and s is independently an integer between 1 and 16.

60. A particle as claimed in claim 59, wherein in said repeat unit of formula (III), q is 1.

61. A particle as claimed in claim 59, wherein in said repeat unit of formula (III), m is 1 or 2.

62. A particle as claimed in claim 59, wherein in said repeat unit of formula (III), n is 1, 2 or 3.

63. A particle as claimed in claim 59, wherein said repeat unit of formula (III) is derived from dimethyl-2-oxo-glutarate or dimethyl-3-oxo-glutarate.

64. A particle as claimed in claim 59, wherein the unit of formula (II) within the repeat unit (III) ##STR00043## is 1,8-octanediol, triethylene glycol or N-methyldiethanolamine.

65. A particle according to claim 59, wherein said biologically active molecule is selected from the group consisting of small molecule drugs, peptides, proteins, peptide mimetics, antibodies, antigens, DMA, mRNA, small interfering RNA, small hairpin RNA, microRNA, PNA, foldamers, carbohydrates, carbohydrate derivatives, non-Lipinski molecules, synthetic peptides and synthetic oligonucleotides.

66. A particle as claimed in claim 59, wherein said biologically active molecule comprises a functional group that is able to form a covalent bond with a keto group.

67. A particle as claimed in claim 66, wherein said biologically active molecule comprises at least one hydrazine group, at least one hydrazide group, at least one amine group, at least one aminooxy group, at least one hydroxyl group or at least one thiol group.

68. A particle as claimed in claim 59, wherein the polymer comprises a repeat unit of formula (IIIa): ##STR00044## wherein: each n is independently 0 or an integer between 1 and 6; each m is independently 0 or an integer between 1 and 4, and at least one m is 1; each B is the remainder of D; each q is an integer between 1 and 8; X is selected from O, S, NH and NR′; Y is selected from O, S, NH and NR′; R′ is C.sub.1-20 hydrocarbyl; Q is selected from —(CH.sub.2).sub.p—, —(CH.sub.2CH.sub.2O).sub.sCH.sub.2CH.sub.2—, —(CH.sub.2).sub.rNR′—(CH.sub.2).sub.r— and —(CH.sub.2CH.sub.2CH.sub.2O).sub.sCH.sub.2CH.sub.2CH.sub.2—; and each of p, r and s is independently an integer between 1 and 16.

69. A particle as claimed in claim 59, wherein the polymer comprises a repeat unit of formula (IIIb): ##STR00045## wherein: each n is independently 0 or an integer between 1 and 6; each m is independently 0 or an integer between 1 and 4, and at least one m is 1; each B is the remainder of D; each q is an integer between 1 and 8; X is selected from O, S, NH and NR′; Y is selected from O, S, NH and NR′; R′ is C.sub.1-20 hydrocarbyl; Q is selected from —(CH.sub.2).sub.p—, —(CH.sub.2CH.sub.2O).sub.sCH.sub.2CH.sub.2—, —(CH.sub.2).sub.rNR′—(CH.sub.2).sub.r— and —(CH.sub.2CH.sub.2CH.sub.2O).sub.sCH.sub.2CH.sub.2CH.sub.2—; and each of p, r and s is independently an integer between 1 and 16.

70. A particle as claimed in claim 59, wherein the polymer comprises a repeat unit of formula (IIIc): ##STR00046## wherein: each n is independently 0 or an integer between 1 and 6; each m is independently 0 or an integer between 1 and 4, and at least one m is 1; each B is the remainder of D; each q is an integer between 1 and 8; X is selected from O, S, NH and NR′; Y is selected from O, S, NH and NR′; R′ is C.sub.1-20 hydrocarbyl; Q is selected from —(CH.sub.2).sub.p—, —(CH.sub.2CH.sub.2O).sub.sCH.sub.2CH.sub.2—, —(CH.sub.2).sub.rNR′—(CH.sub.2).sub.r— and —(CH.sub.2CH.sub.2CH.sub.2O).sub.sCH.sub.2CH.sub.2CH.sub.2—; and each of p, r and s is independently an integer between 1 and 16.

71. A particle as claimed in claim 59, wherein the polymer comprises a repeat unit of formula (IIId): ##STR00047## wherein: each n is independently 0 or an integer between 1 and 6; each m is independently 0 or an integer between 1 and 4, and at least one m is 1; each B is the remainder of D; each q is an integer between 1 and 8; X is selected from O, S, NH and NR′; Y is selected from O, S, NH and NR′; R′ is C.sub.1-20 hydrocarbyl; Q is selected from —(CH.sub.2).sub.p—, —(CH.sub.2CH.sub.2O).sub.sCH.sub.2CH.sub.2—, —(CH.sub.2).sub.rNR′—(CH.sub.2).sub.r— and —(CH.sub.2CH.sub.2CH.sub.2O).sub.sCH.sub.2CH.sub.2CH.sub.2—; and each of p, r and s is independently an integer between 1 and 16.

72. A particle as claimed in claim 59, wherein the polymer comprises a repeat unit of formula (IIIe): ##STR00048## wherein: each n is independently 0 or an integer between 1 and 6; each m is independently 0 or an integer between 1 and 4, and at least one m is 1; each B is the remainder of D; each q is an integer between 1 and 8; X is selected from O, S, NH and NR′; Y is selected from O, S, NH and NR′; R′ is C.sub.1-20 hydrocarbyl; Q is selected from —(CH.sub.2).sub.p—, —(CH.sub.2CH.sub.2O).sub.sCH.sub.2CH.sub.2—, —(CH.sub.2).sub.rNR′—(CH.sub.2).sub.r— and —(CH.sub.2CH.sub.2CH.sub.2O).sub.sCH.sub.2CH.sub.2CH.sub.2—; and each of p, r and s is independently an integer between 1 and 16.

73. A particle as claimed in claim 59, wherein the polymer comprises a unit of formula (V): ##STR00049## wherein: each n is independently 0 or an integer between 1 and 6; each m is independently 0 or an integer between 1 and 4, and at least one m is 1; each D.sup.1 is a moiety which is a biologically active molecule, or a derivative thereof, when the C to D.sup.1 bond is broken; D.sup.2 is a moiety which is a biologically active molecule, or a derivative thereof, when the Y to D.sup.2 bond is broken or is a targeting agent; each q is an integer between 1 and 8; X is selected from O, S, NH and NR′; Y is selected from O, S, NH and NR′; R′ is C.sub.1-20 hydrocarbyl; Q is selected from —(CH.sub.2).sub.p—, —(CH.sub.2CH.sub.2O).sub.sCH.sub.2CH.sub.2—, —(CH.sub.2).sub.rNR′—(CH.sub.2).sub.r— and —(CH.sub.2CH.sub.2CH.sub.2O).sub.sCH.sub.2CH.sub.2CH.sub.2—; and each of p, r and s is independently an integer between 1 and 16.

74. A particle as claimed in claim 73, wherein said biologically active molecule from which D1 derives comprises at least one hydrazine group, at least one hydrazide group, at least one amine group, at least one aminooxy group, at least one hydroxyl or at least one thiol group.

75. A particle as claimed in claim 73, wherein the biologically active molecule from which D.sup.2 derives comprises a functional group selected from a carboxylic acid group, a carboxylic ester group, a carboxylate group, a carboxyl thioester group, an acyl phosphate group, a carboxylic acid anhydride group, a hydroxyl group, an acyl halide group, an amine group and a thiol group.

76. A particle as claimed in claim 59, wherein the polymer further comprises a repeat unit derived from a biologically active molecule.

77. A particle comprising a polymer, wherein said polymer comprises a repeat unit of formula (VI): ##STR00050## wherein: each n is independently 0 or an integer between 1 and 6; each m is independently 0 or an integer between 1 and 4, and at least one m is 1; each q is an integer between 1 and 8; p is an integer between 1 and 16; and D is a moiety which is a biologically active molecule, or a derivative thereof, when the polymer backbone is degraded.

78. A method for making a particle as claimed in claim 59, wherein said method involves a method for making a polymer, comprising: reacting a compound of formula (I) ##STR00051## wherein: R.sup.1 and R.sup.2 are each independently selected from OH, OR′, SH, SR′, NH.sub.2, NHR′ and NR′.sub.2; R′ is C.sub.1-20 hydrocarbyl; each n is independently 0 or an integer between 1 and 6; each m is independently 0 or an integer between 1 and 4, and at least one m is 1; and q is an integer between 1 and 8; with a compound of formula (II) ##STR00052## wherein: X is selected from OH, OR′, SH, SR′, NH.sub.2, NHR′ and NR′.sub.2; Y is selected from OH, OR′, SH, SR′, NH.sub.2, NHR′ and NR′.sub.2; R′ is C.sub.1-20 hydrocarbyl; Q is selected from —(CH.sub.2).sub.p—, —(CH.sub.2CH.sub.2O).sub.sCH.sub.2CH.sub.2—, —(CH.sub.2).sub.rNR′—(CH.sub.2).sub.r— and —(CH.sub.2CH.sub.2CH.sub.2O).sub.sCH.sub.2CH.sub.2CH.sub.2—; and each of p, r and s is independently an integer between 1 and 16; and with a biologically active molecule.

79. A method as claimed in claim 78, which is carried out enzymatically.

80. A particle as claimed in claim 59 having an average diameter of 50 to 800 nm.

81. A particle as claimed in claim 59, further comprising an agent selected from the group consisting of a biologically active molecule, a molecular probe and a diagnostic agent which is non-covalently bound to said polymer.

82. A particle as claimed in claim 81, wherein said non-covalently bound agent is encapsulated in a structure formed by said polymer.

83. A particle as claimed in claim 81, wherein said non-covalently bound agent is a biologically active molecule, which may be the same as or different from the biologically active molecule(s) covalently bound to said polymer.

84. A method for making a particle as claimed in claim 59, wherein said method is selected from nanoprecipitation, emulsion-diffusion, double emulsification, emulsion-coacervation, polymer-coating and layer-by-layer method.

85. A method as claimed in claim 84, wherein said method is nanoprecipitating and a further biologically active molecule is present during said nanoprecipitation process.

86. A pharmaceutical composition comprising a particle as claimed in claim 59.

87. A dosage form comprising a particle as claimed in claim 59.

88. A method of treating a patient in need thereof, comprising administering to said patient a therapeutically effective amount of a particle as claimed in claim 59.

89. A method of treating a disease selected from inflammatory diseases (e.g., inflammatory bowel disease, rheumatoid arthritis and artherosclerosis), metabolic disorders (e.g., diabetes, insulin resistance, obesity), cancer, bacterial infections (e.g., tuberculosis, pneumonia, endocarditis, septicaemia, salmonellosis, typhoid fever, cystic fibrosis, chronic obstructive pulmonary diseases), viral infections, cardiovascular diseases, neurodegenerative diseases, neurological disorders, behavioral and mental disorders, blood diseases, chromosome disorders, congenital and genetic diseases, connective tissue diseases, digestive diseases, ear, nose, and throat diseases, endocrine diseases, environmental diseases, eye diseases, female reproductive diseases, fungal infections, heart diseases, hereditary cancer syndromes, immune system diseases, kidney and urinary diseases, lung diseases, male reproductive diseases, mouth diseases, musculoskeletal diseases, myelodysplastic syndromes, nervous system diseases, newborn screening, nutritional diseases, parasitic diseases, rare cancers and skin diseases; wherein said method comprises administering to a patient in need thereof a therapeutically effective amount of a particle as claimed in claim 1.

90. A particle as claimed in claim 59, wherein release of said biologically active molecule from the polymer is pH sensitive and is dependent upon the nature of the bond between said biologically active molecule and the repeat unit of the polymer to which it is covalently bound.

91. A polymer comprising a repeat unit of formula (III): ##STR00053## wherein: each n is independently 0 or an integer between 1 and 6; each m is independently 0 or an integer between 1 and 4, and at least one m is 1; ---- is a bond which may be absent or present; each D is a moiety which is a biologically active molecule, or a derivative thereof, when the C to D bond(s) is broken; and each q is an integer between 1 and 8; and further wherein: (a) X is selected from O, S, NH and NR′; Y is selected from O, S, NH and NR′; R′ is C.sub.1-20 hydrocarbyl; Q is selected from —(CH.sub.2).sub.p—, —(CH.sub.2CH.sub.2O).sub.sCH.sub.2CH.sub.2—, —(CH.sub.2).sub.rNR′—(CH.sub.2).sub.r— and —(CH.sub.2CH.sub.2CH.sub.2O).sub.sCH.sub.2CH.sub.2CH.sub.2—; and each of p and s is independently an integer between 4 and 12, and r is an integer between 2 and 8; or (b) the unit of formula (II) within the repeat unit (III) ##STR00054## is 1,8-octanediol, triethylene glycol or N-methyldiethanolamine.

92. A polymer as claimed in claim 91, wherein said repeat unit of formula (III) is derived from dimethyl-2-oxo-glutarate or dimethyl-3-oxo-glutarate, and wherein the unit of formula (II) within the repeat unit (III) ##STR00055## is 1,8-octanediol, triethylene glycol or N-methyldiethanolamine.

Description

BRIEF DESCRIPTION OF FIGURES

[0160] These and other aspects of the invention will now be further described, by way of example only, with reference to the accompanying figures in which:

[0161] FIG. 1 is a graph showing Gel Permeation Chromatography (GPC) analysis of blank polymer E55 after a 22 h polymerization time;

[0162] FIG. 2 is a .sup.1H-NMR spectrum of blank polymer 1 in DMSO-d6;

[0163] FIG. 3 is a bar graph showing the number-mean size distribution of E60 nanoparticles at pH 7.4;

[0164] FIG. 4 is an Atomic Force Microscopy (AFM) image of E60 nanoparticles;

[0165] FIG. 5 is a fluorescent image of human monocyte-derived macrophages incubated for 1 h with a 0.5 mg mL.sup.−1 E60 nanoparticle suspension (encapsulating the dye Nile Red™) in Opti-MEM cell culture medium. The left-hand image shows fluorescence at 50% cell height; the central image shows a light microscopy image; and the right-hand image shows an overlay of the two images. Bars: 10 μm;

[0166] FIG. 6 is a graph showing Gel Permeation Chromatography (GPC) analysis of blank polymer 2 after a 2 h polymerization time;

[0167] FIG. 7 is a .sup.1H-NMR spectrum of blank polymer 2 in CDCl.sub.3;

[0168] FIG. 8 is a graph showing Gel Permeation Chromatography (GPC) analysis of blank polymer 3 after a 36 h polymerization time;

[0169] FIG. 9 is a graph showing Gel Permeation Chromatography (GPC) analysis of blank polymer 4 after a 4 h polymerization time;

[0170] FIG. 10 is a graph showing GPC analysis of polymer E67 after a 30 min incubation time;

[0171] FIG. 11 is a graph showing GPC analysis of blank polymer 1 incorporating INH after a 72 h incubation time;

[0172] FIG. 12 is a .sup.1H-NMR spectrum of blank polymer 1 incorporating INH in DMSO-d6;

[0173] FIG. 13 is an AFM image of E67 nanoparticles;

[0174] FIG. 14 is an AFM image of E80 nanoparticles;

[0175] FIG. 15 is a graph showing the GPC analysis of polymer E65;

[0176] FIG. 16 is graph showing the HPLC analysis used for the characterisation of ciprofloxacin-containing polymer;

[0177] FIG. 17 is a .sup.1H NMR spectrum of memantine-containing polymer 1 in DMSO-d6;

[0178] FIG. 18 is a graph showing GPC analysis of o-benzylhydroxylamine-containing polymer 1 after 48 h incubation time;

[0179] FIG. 19 is a .sup.1H-NMR spectrum of o-benzylhydroxylamine-containing polymer 1 in DMSO-d6;

[0180] FIG. 20 is a graph showing GPC analysis of prednisone-containing polymer 1 after 48 h incubation time;

[0181] FIG. 21 is a .sup.1H-NMR spectrum of prednisone-containing polymer 1 in DMSO-d6;

[0182] FIG. 22 is a graph showing the GPC analysis of polymer E52 after a 49 h polymerization time;

[0183] FIG. 23 is a .sup.1H-NMR spectrum of blank polymer 2 incorporating INH in CDCl.sub.3;

[0184] FIG. 24 is a graph showing the cumulative release of isoniazid from nanoparticles formed from blank polymer 1 incorporating INH;

[0185] FIG. 25 is a bar graph showing the bioluminescence of Mycobacterium bovis BCG-lux grown in human monocyte-derived macrophages of the INH-containing nanoparticles E67 and the corresponding blank nanoparticles E55 on BCG-lux viability after 72 h incubation;

[0186] FIG. 26 is a bar graph showing the bioluminescence of Mycobacterium bovis BCG-lux grown in human monocyte-derived macrophages in the presence of E67 nanoparticles for 24 h;

[0187] FIG. 27 is a bar graph showing the bioluminescence of Mycobacterium bovis BCG-lux grown in human monocyte-derived macrophages in the presence of E67 nanoparticles for 72 h;

[0188] FIG. 28 is a bar graph showing the bioluminescence of Mycobacterium bovis BCG-lux grown in human monocyte-derived macrophages in the presence of E67 nanoparticles for 120 h;

[0189] FIG. 29 is a bar graph showing the bioluminescence of Mycobacterium bovis BCG-lux grown in human monocyte-derived macrophages in the presence of free INH (grey bars) compared to the respective control groups (striped bars);

[0190] FIG. 30 is a bar graph showing the bioluminescence of Mycobacterium bovis BCG-lux grown in human monocyte-derived macrophages in the presence of E72 or E80 nanoparticles for 24 h;

[0191] FIG. 31 is a bar graph showing the bioluminescence of Mycobacterium bovis BCG-lux grown in human monocyte-derived macrophages in the presence of E72 or E80 nanoparticles for 72 h;

[0192] FIG. 32 is a bar graph showing the bioluminescence of Mycobacterium bovis BCG-lux grown in human monocyte-derived macrophages in the presence of E72 or E80 INH RIF nanoparticles for 72 h;

[0193] FIG. 33 is a bar graph showing the bioluminescence of Mycobacterium bovis BCG-lux grown in human monocyte-derived macrophages in the presence of E66 nanoparticles for 24 h;

[0194] FIG. 34 is a bar graph showing the bioluminescence of Mycobacterium bovis BCG-lux grown in human monocyte-derived macrophages in the presence of E66 nanoparticles for 72 h;

[0195] FIG. 35 is a bar graph showing the cytotoxicity of E80 INH and E72 nanoparticles; and

[0196] FIG. 36 is a graph showing RP-HPLC analysis of blank polymer 1 (A), Insulin (B), and the polymer after reaction with insulin and after treatment (precipitation and washes) (C).

EXAMPLES

Materials

[0197] All starting materials employed are commercially available. 1,8-octanediol, dimethyl-2-oxoglutarate, dimethyl-3-oxoglutarate, triethylene glycol, N-methyldiethanolamine, N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), Novozym™ 435, isoniazid (INH), rifampicin (RIF), ciprofloxacin, memantine, prednisone, insulin, ethambutol (EMB) were obtained from Sigma Aldrich.

[0198] Other commercially available chemicals were also employed. Chloroform, diphenyl ether, hexane, diisopropylethylamine (DIPEA), dichloromethane (DCM), magnesium sulfate, sodium hydroxide, toluene, o-benzylhydroxylamine, acetonitrile (MeCN), High Performance Liquid Chromatography (HPLC) water, Nile Red™ fluorescent dye, dimethyl sulfoxide (DMSO), p-tolunesulfonic acid (PTSA), ethanol, mica substrate, phosphate buffer, citrate buffer, acetate buffer, trifluoroacetic acid (TFA), diethyl ether, sodium-1-heptanesulfonate, modified Eagle's Minimum Essential Media (Opti-MEM) cell culture medium, Dulbecco's Modified Eagle Medium (DMEM), Fetal Calf Serum (FCS), penicillin (PEN), fluorescein isothiocyanate (FITC), propidium iodide (PI) were obtained from Sigma Aldrich, Fisher scientific, Life technologies, Lonza and Agar Scientific.

Analysis Methods

[0199] Gel Permeation Chromatography (GPC) analysis was performed to determine polymer molecular weight, polydispersity and degree of polymerization using a GPC system PL-GPC-50 (Polymer laboratories, Agilent Technology). Analytical grade chloroform was used as eluent at a flow rate of 0.3 mL min.sup.−1. The run time for one analysis was 30 min. The injection loop was purged with 3 mL chloroform prior to each injection. A 10 point mass calibration was done with polystyrene standards (EasyCal PS-2, Polymer Laboratories, Agilent). Samples of the bulk phase of polymerization were diluted in 1 mL chloroform and directly injected into the GPC.

[0200] Further characterization of polymers was performed using Nuclear Magnetic Resonance (NMR) on 400 and 500 MHz NMR spectrometers. Note that complete assignment of peaks in .sup.1H NMR spectra requires further NMR analysis.

[0201] Dynamic Light Scattering (DLS) analysis was performed using a Zetasizer Nano series (Malvern Instruments, UK). DLS size and zeta potential analysis was performed at 25° C. at 173° backscatter angle in low volume sizing cuvettes (1.5 mL semi-micro, PMMA; Brand, UK) or in DTS1060c clear disposable zeta cells (Malvern, UK). Water or PBS was set as dispersants for all measurements, and PLGA was used as a model for the unknown RI of the sample. One analysis comprised 10 measurements consisting of 10 sub-runs per measurement with 10 s analysis per run. For the determination of zeta potential, 10 measurements with 20 sub-runs were used. Results were calculated internally from Smoluchowski's model.

[0202] Atomic Force Microscopy (AFM) was performed using a PicoPlus AFM™ with a PicoSPM II controller from Molecular Imaging, Agilent. A drop of nanoparticle suspension was deposited on a cover slip with a mica surface. After drying, the samples were analyzed in intermittent Contact Mode AFM.

[0203] Fluorescent Imaging was performed using a Zeiss LSM 780 Confocal Laser Scanning Microscope.

[0204] High Performance Liquid Chromatography (HPLC) was performed using a HPLC system (Waters) consisting of a 1525 Binary HPLC Pump, a 2487 Dual A Absorbance Detector, and an Ascentis® Express Peptide ES-C18 column with a length of 50 mm. The eluent was 99.9% water and 0.1% Trifluoroacetic acid for INH quantification. A nine-point standard curve was made by serial dilution of INH in water, various buffers and 0.5 M NaOH (linear regression y=36.005 x+0.1685, R.sup.2=99.4%.) A gradient of eluent A (99.9% water and 0.1% Trifluoroacetic acid) and eluent B (90% acetonitrile, 9.9% water and 0.1% and Trifluoroacetic acid) was used for the analysis of insulin, blank polymer 1 and insulin-containing polymer.

[0205] Ion-Pair High Performance Liquid Chromatography (HPLC) was performed using a HPLC system (Waters) consisting of a 1525 Binary HPLC Pump, a 2487 Dual A Absorbance Detector, and an Ascentis® Express Peptide ES-C18 column with a length of 50 mm. HPLC analysis was performed with a mobile phase containing cupric ions, which are known to form UV-absorbing complexes with EMB. The mobile phase consisted of 4 g Sodium-1-heptanesulfonate and 0.16 g copper sulfate dissolved in 750 mL deionized (DI) water and 250 mL Tetrahydrofuran. The apparent pH was adjusted to pH 4.5 with 1 M NaOH before each analysis. A seven-point standard curve was made by preparing solutions of EMB in the mobile phase (linear regression: y=57.93 x−0.036, R.sup.2=99.9%.)

Ultraviolet (UV) Spectrometry was performed using a UV-Vis spectrometer (Varian Cary® 300 UV-Vis Spectrometer, Agilent Technologies, UK). An UV spectrum of a 25 μg mL.sup.−1 RIF solution in acetonitrile was recorded, and the absorption of five standard concentrations of RIF were measured at the wavelength of 475 nm. A linear correlation function of RIF concentration and absorbance was derived.

[0206] Flow cytometry was performed using conventional apparatus.

[0207] Bioassay against Mycobacterium bovis BCG-lux (Bacille Calmette Guerin) grown in human monocyte-derived macrophages was performed as follows: Macrophage cell culture: Human peripheral blood monocyte-derived macrophages. Venal blood from healthy blood donors mixed 10:1 (v:v) with PBS containing 4% citrate. 15 mL Lympholyte-Human (Cedarlane, Canada) peripheral blood mononuclear cell (PBMC) separation medium was filled into 50 mL Falcon tubes. The fluid was overlayered without mixing with 25 mL blood/citrate per tube. The blood was fractionated by centrifugation for 22 min at 1900 rpm without braking in a Beckman & Coulter Alegra X-15R centrifuge. PBMCs accumulated in the layer between blood serum (top) and Lympholyte medium (bottom), with red blood cells and thrombocytes in the pellet. The PBMC layers of all Falcon tubes were pipetted to a fresh Falcon tube. Cells were washed twice by suspending in 50 mL Roswell Park Memorial Institute Medium 1640 (RPMI 1640) and pelleting at 1450 rpm for 10 min.

A 5 μL aliquot of the cell pellet was mixed with 5 μL Trypan Blue, and the cell concentration was measured in a Countess™ Automated Cell Counter (Life Technologies, California, USA).

[0208] CD14.sup.+ cells were selected and concentrated by a magnetic assisted cell sorting (MACS®) technique. The pellet was resuspended in 80 μL PBS 0.4% citrate per 10.sup.7 cells. 20 μL ferromagnetic MACS MicroBeads covered with CD14 specific monoclonal antibodies were added per 10.sup.7 cells. The mixture was incubated at 4° C. for 30 min to allow antibody-antigen binding. The resulting PBMC-bead adducts were washed in 50 mL PBS 4% citrate and collected by centrifugation at 1300 rpm for 10 min. The pellet was resuspended in 0.5 mL PBS 0.4% citrate per 10.sup.8 cells. LS columns with 8 mL reservoir volume (Miltenyi Biotech) were mounted in a MidiMACS™ separator and rinsed with 3 mL PBS 0.4% citrate. 500 μL PBMC-bead adduct suspension was pipetted to each column. Unbound cells were washed away by thrice rinsing with 3 mL PBS 0.4% citrate. The column was then removed from the magnetic source, and bound cells were eluated by two-fold rinsing with 5 mL PBS 0.4% citrate. Elution was supported by firm application of a plunger that was supplied with the columns. CD14.sup.+ cells were concentrated by centrifugation at 1300 rpm for 10 min. The pellet was resuspended in Dublecco's Modified Eagle Medium (DMEM medium, Lonza, Switzerland) containing 20 vol.-% fetal calf serum (FCS). The cell concentration of an aliquot was determined as described above, and the cell suspension was diluted to a concentration of 10.sup.6 cells mL.sup.−1 with DMEM FCS.

[0209] 200 μg macrophage colony stimulating factor (PeproTech Inc., New Jersey, USA, in aqueous 100 μg mL.sup.−1 stock) was added per mL cell culture. 2*10.sup.5 cells were seeded in 24 well culture plates (Greiner BioOne, Austria) and incubated for 5 days at 37° C. in a 5% CO.sub.2 incubator for differentiation to macrophages.

[0210] BCG-lux cell culture: A cryo conservation vial with BCG lux cells in glycerol was defrosted at room temperature. The inoculum was poured into a 250 mL single-use Erlenmeyer flask equipped with 100 mL growth medium, and incubated at 37° C. and 220 rpm for 48 h. The growth medium contained 4.7 g L.sup.−1 7H9 broth (Middlebrook, UK), 10 vol. % ADC supplement (Becton Dickinson and Co.), 50 mg L.sup.−1 Hygromycin B (Roche, UK), 2 vol. % glycerol and 2 vol. % Tween 80 in ultrapure water.

[0211] BCG lux cells were harvested by centrifugation at 2000 rpm for 10 min. The pellet was redispersed in 20 mL DMEM medium with 10% FCS and 60 mg mL.sup.−1 penicillin (Roche, UK) (DMEM FCS PEN medium). The concentration of luminescent cells was measured in triplicate in a Glomax™ 96 microplate luminometer (Promega, UK). 200 μL cell suspension were pipetted into 3 wells of an opaque 96 well luminometer plate (Greiner BioOne, Austria). The luminometer measured the relative light units (RLU) emitted from luminescent bacteria upon automated injection of 25 μL 1% aqueous decanol solution to each vial. The method was calibrated for the equivalence of 1 RLU per 1 BCG-lux bacteria.

[0212] For infection of human macrophages, the BCG-lux concentration was diluted to 250000 RLU mL.sup.−1.

[0213] Macrophage infection: The growth medium was pipetted from the confluent macrophage culture, and non-adherent cells were removed by two-fold washing with 0.5 mL sterile PBS. Macrophages were incubated with 200 μL BCG lux dispersion per well (50000 bacteria) for 1 h at 37° C. and 5% CO.sub.2. The fluid was removed, and the cells were washed twice with sterile PBS. To eliminate extracellular BCG lux, the cells were incubated for 10 min with DMEM FCS PEN medium containing 60 mg mL.sup.−1 streptomycin. The streptomycin medium was removed, and the cells were washed twice with sterile PBS.

[0214] Determination of nanoparticle efficacy: The infected cells were incubated for 1, 3 or 5 days with 200 or 300 μL of nanoparticle dispersion in DMEM FCS PEN medium. As a positive control, pure drug was used. The free drug concentration was equivalent to the quantified drug release from nanoparticles. In some experiments, free drugs were additionally tested at the minimum inhibitory concentrations (MIC) given in literature. Pure medium was used as a negative control. All conditions were applied in 6 wells.

[0215] After incubation, the medium was removed, and the cells were washed twice with sterile PBS. The macrophages were lysed in 250 μL sterile destilled water for 10 min. During the lysis time, the wells were scraped with a sterile cell scraper. A new cell scraper was used for every condition. 200 μL cell lysate was transferred to a luminometer plate, and the luminescence RLU was measured as described above.

[0216] Significant reduction of BCG-lux RLU in comparison to untreated cells was tested with unpaired Student t-test at independent variance (α=0.05, n=6).

Preparative Example for the Synthesis of a Blank Polymer 1

[0217] ##STR00029##

Blank polymers 1 including E55, E60, E72, E73 were synthesized as following. 1,8-Octanediol and dimethyl-2-oxo-glutarate were combined in a round-bottomed flask, which was heated to 75° C. on a hot plate equipped with an oil bath. Candida Antarctica Lipase B (CALB) as immobilized enzyme (Novozym™ 435, N435. N435 contains 10% w/w CALB and 90% w/w acrylic resin) beads were added and the bulk was stirred magnetically at 200 rpm for 1.5 h. A chemo-resistant diaphragm vacuum pump then was connected to the reaction vessel and run continuously. Diphenyl ether (1 mL) was then added to reduce viscosity and the temperature was increased to 90° C. Gel Permeation Chromatography (GPC) analysis of molecular weight was performed until sufficient size and monodispersity was achieved.

[0218] The GPC analysis for blank polymer 1 E55 after a 22 h polymerization time is shown in FIG. 1. The product was purified by two-fold precipitation in hexane. The dashed lines indicate the high and low limits of molecular weight calibration. The square data points represent calibration data.

[0219] The reaction was quenched by addition of chloroform (3 mL). The enzyme beads were filtered off and the reaction vessel and filtering unit were washed with additional chloroform (3 mL). The filtrates were precipitated with hexane (50 mL) and washed by centrifugation. The pellets were dried under nitrogen for 15 min and left in a vacuum desiccator overnight. Table 1 indicates the polymer size and dispersity for each of the blank polymers synthesised. The yield of polymerization Y.sub.P was estimated from the dry mass of polymer and the theoretic mass of an ideal (monodisperse) polymer.

[0220] The .sup.1H-NMR spectrum of blank polymer 1 in DMSO-d6 is shown in FIG. 2.

Polymer Size and Dispersity

[0221]

TABLE-US-00001 TABLE 1 Blank polymer 1 time, h M.sub.p, Da M.sub.n, Da PD Y.sub.p, % P.sub.n E55 22 5973 4014 1.68 64 15.6 E60 24 3302 2567 1.55 58 9.9 E72 24 26654 16462 2.23 89 64.2 E73 6 10675 7654 2.10 80 29.8 [M.sub.p: molecular weight at peak maximum; M.sub.n: number-mean molecular weight; PD: polydispersity; Y.sub.p: yield of polymerisation; P.sub.n: number-mean degree of polymerization].

Preparative Example for the Preparation of Nanoparticles of a Blank Polymer 1

[0222] The nanoparticles were prepared by nanoprecipitation. The polymer was dissolved in acetonitrile (20 mg/mL) at room temperature. The polymer solution was slowly dropped into a 20-fold volume of HPLC grade water, which was being stirred continuously. The nanoparticles were washed with HPLC grade water and collected by centrifugation. The resulting pellets were dispersed in water and stored at 4° C.

[0223] The particle size and zeta potential of the polymer nanoparticles were measured by DLS as shown in Table 2. A first aliquot of the final dispersion was diluted to 1:20 in 10 mM citrate buffer at pH 5 to mimic phagolysosomal pH. A second aliquot was diluted to 1:20 in 10 mM phosphate buffer at pH 7.4 to simulate physiological extracellular pH conditions.

DLS Characterization of Polymeric Nanoparticles

[0224]

TABLE-US-00002 TABLE 2 Blank Z-average ± d.sub.Number, zeta polymer 1 PDI width d, nm nm PDI potential, mV E55 BLANK pH 5 169 ± 36 145 ± 4 0.05 ± 0.02 −54 ± 4 pH 7.4 169 ± 52 137 ± 4 0.10 ± 0.02 −66 ± 3 E60 BLANK pH 5 175 ± 65 135 ± 5 0.14 ± 0.03 −70 ± 6 pH 7.4 176 ± 66 135 ± 9 0.14 ± 0.03 −65 ± 4 E72 BLANK pH 5 150 ± 45 118 ± 6 0.09 ± 0.03 −40 ± 3 pH 7.4 146 ± 29 123 ± 3 0.04 ± 0.02 −71 ± 2 Z-average: cumulants mean; d.sub.Number: number-mean size; values ± standard deviation of DLS measurement runs; PDI: polydispersity index.

[0225] FIG. 3 shows a number-mean size distribution of E60 nanoparticles at pH 7.4. DLS was used to measure the size of the nanoparticles at scattering angle of 173° in 10 mM phosphate buffer pH 7.4. 10 measurements were performed, each having 10 sub-runs. The error bars represent ±standard deviation of the runs.

[0226] Nanoparticle morphology was determined by AFM. FIG. 4 shows nanoparticles of E60 with heights of ˜15 nm and widths of ˜300 nm. Deformation of the nanoparticles on the mica substrate was observed, which was attributed to the drying of the nanoparticles for imaging.

[0227] To assess the uptake of nanoparticles by macrophages, the fluorescent dye Nile Red™ was encapsulated in nanoparticles of blank polymer E60. Encapsulation was achieved by nanoprecipitation as previously described with 0.05 mg mL.sup.−1 Nile Red™ in the acetonitrile phase.

[0228] FIG. 5 shows human monocyte-derived macrophages incubated for 1 h with a 0.5 mg mL.sup.−1 nanoparticle suspension in Opti-MEM cell culture medium. Nanoparticles encapsulated the dye Nile Red™ for fluorescent staining. The left-hand image of FIG. 5 shows fluorescence at 50% cell height; the central image shows a light microscopy image; and the right-hand image shows an overlay of the two images.

Preparative Example for the Synthesis of a Blank Polymer 2 Made from Dimethyl-3-Oxoglutarate and 1,8-Octanediol

[0229] ##STR00030##

[0230] Blank polymer 2 was prepared by reacting dimethyl-3-oxoglutarate and 1,8-octanediol using the same polymerisation process as that described for the preparation of blank polymer 1 above.

[0231] The GPC analysis for blank polymer 2 after a 2 h polymerization time is shown in FIG. 6. The dashed lines indicate the high and low limits of molecular weight calibration. The square data points represent calibration data.

Table 3 indicates the polymer size and dispersity for the blank polymer 2 synthesised by reaction of dimethyl-3-oxoglutarate and 1,8-octanediol. The .sup.1H-NMR spectrum of blank polymer 2 in CDCl.sub.3 is shown in FIG. 7.

Polymer Size and Dispersity of Blank Polymer 2

[0232]

TABLE-US-00003 TABLE 3 time, h M.sub.p, Da M.sub.n, Da PD Y.sub.p, % P.sub.n 2 8585 5234 2.02 65 20 [M.sub.p: molecular weight at peak maximum; M.sub.n: number-mean molecular weight; PD: polydispersity; Y.sub.p: yield of polymerisation; P.sub.n: number-mean degree of polymerization].

Preparative Example for the Preparation of Nanoparticles of a Blank Polymer 2 Prepared from Dimethyl-3-Oxoglutarate and 1,8-Octanediol

[0233] Nanoparticles were prepared from blank polymer 2 prepared above in the same manner as the nanoparticles prepared from blank polymer 1 above.

[0234] The particle size and zeta potential of the polymer nanoparticles were measured by DLS as shown in Table 4. A first aliquot of the final dispersion was diluted in water. A second aliquot was centrifugated and resuspended in 10 mM citrate buffer at pH 5 to mimic phagolysosomal pH. A third aliquot was centrifugated and resuspended in 10 mM phosphate buffer at pH 7.4 to simulate physiological extracellular pH conditions.

DLS Characterization of Polymeric Nanoparticles

[0235]

TABLE-US-00004 TABLE 4 Blank Z-average ± d.sub.Number, zeta potential ± polymer 2 PDI width d, nm nm PDI zeta deviation, mV water 236 ± 118 180 0.248 −32 ± 10 pH 5 281 ± 142 187 0.252 −18 ± 28 pH 7.4 287 ± 20  194 0.174 −25 ± 56 Z-average: cumulants mean; d.sub.Number: number-mean size; PDI: polydispersity index.

Preparative Example for the Synthesis of a Blank Polymer 3 Made from Dimethyl-2-Oxoglutarate and Triethylene Glycol

[0236] ##STR00031##

[0237] Blank polymer 3 was prepared by reacting dimethyl-2-oxoglutarate and triethylene glycol using the same polymerisation process as that described for the preparation of blank polymer 1 above.

[0238] The GPC analysis for blank polymer 3 prepared from reaction of dimethyl-2-oxoglutarate and triethylene glycol after a 36 h polymerization time is shown in FIG. 8. The dashed lines indicate the high and low limits of molecular weight calibration. The square data points represent calibration data.

Table 5 indicates the polymer size and dispersity for the blank polymer 3 synthesised.

Polymer Size and Dispersity of Blank Polymer 3

[0239]

TABLE-US-00005 TABLE 5 Blank polymer 3 time, h M.sub.n, Da PD Y.sub.p, % P.sub.n 3 1500 1.3 52 5 22 3500 1.8 77 12 36 5500 3.7 57 19 [M.sub.p: molecular weight at peak maximum; M.sub.n: number-mean molecular weight; PD: polydispersity; Y.sub.p: yield of polymerisation; P.sub.n: number-mean degree of polymerization].

Preparative Example for the Preparation of Nanoparticles of a Blank Polymer 3 Made from Dimethyl-2-Oxoglutarate and Triethylene Glycol

[0240] Nanoparticles were prepared from blank polymer 3 prepared above in the same manner as the nanoparticles prepared from blank polymer 1 above.

[0241] The particle size and zeta potential of the polymer nanoparticles were measured by DLS as shown in Table 6. An aliquot of the final dispersion was diluted in water.

DLS Characterization of Polymeric Nanoparticles

[0242]

TABLE-US-00006 TABLE 6 Blank Z-average ± d.sub.Number, zeta potential ± polymer 3 PDI width d, nm nm PDI zeta deviation, mV water 630 ± 462 304 0.527 −19 ± 12 Z-average: cumulants mean; d.sub.Number: number-mean size; PDI: polydispersity index.

Preparative Example for the Synthesis of a Blank Polymer 4 Made from Dimethyl-2-Oxoglutarate and N-Methyldiethanolamine

[0243] ##STR00032##

Blank polymer 4 was prepared by reacting dimethyl-2-oxoglutarate and N-methyldiethanolamine using the same polymerisation process as that described for the preparation of blank polymer 1 above.

[0244] The GPC analysis for blank polymer 4 after a 4 h polymerization time is shown in FIG. 9. The dashed lines indicate the high and low limits of molecular weight calibration. The square data points represent calibration data.

Table 7 indicates the polymer size and dispersity for the blank polymer 4 synthesised.

Polymer Size and Dispersity of Blank Polymer 4

[0245]

TABLE-US-00007 TABLE 7 Blank polymer 4 time, h M.sub.n, Da PD Y.sub.p, % P.sub.n 4 11242 1.75 34 46 [M.sub.p: molecular weight at peak maximum; M.sub.n: number-mean molecular weight; PD: polydispersity; Y.sub.p: yield of polymerisation; P.sub.n: number-mean degree of polymerization].

Example 1: Synthesis of Isoniazid-Containing Polymer Nanoparticles from Blank Polymer 1

[0246] ##STR00033##

[0247] INH-containing polymers including E67 (E67 INH), E74A, E74B, E80 (E80 INH), E90 (E90 INH), E91 (E91 INH) and E92 (E92 INH) were prepared from blank polymers 1 including E55 (E55 blank), E72 (E72 blank), E73 and BP1 as shown in Table 8. Blank polymers E55, E72, E73 and BP1 were prepared as described above. INH was then covalently linked to the carbonyl groups of the blank polymers by solubilizing the polymer in DMSO. INH (1.2 eq. molar amounts of estimated carbonyl groups in the polymer) was then added and the resultant mixture was stirred at 200 rpm at room temperature (or 37° C.) for the reaction time. The solution was then added dropwise to ethanol (50 mL) and the precipitated polymers were sedimented by centrifugation and dried. The size of each of the polymers was measured by GPC analysis as described above. Table 8 gives data for the characterisation of each of the INH-containing polymers synthesized.

Characterisation of INH-Containing Polymers

[0248]

TABLE-US-00008 TABLE 8 yield Name Name bp time temp. (%) M.sub.p, Da M.sub.n, Da PD ΔM.sub.n, % E67 E55 30 min room temp. N/A 8491 7121 1.35 77.4 E74A E72 30 min room temp. N/A 27668 22594 1.93 37.2 E74B E73 30 min room temp. N/A 13356 10178 1.61 33.0 E80 E72 35 min room temp. N/A 17026 15987 2.40 −2.9 E90 BP1 30 min room temp. 30-60 N/A  5500-15041 1.1-1.5 N/A E91 BP1 24 h room temp. 47 N/A 2300 1.2  N/A E92 BP1 72 h 37° C. 70 N/A 1300-2100 1.7-1.8 N/A [bp: blank polymer; M.sub.p: molecular weight at peak maximum; M.sub.n: number-mean molecular weight; PD: polydispersity; ΔM.sub.n: procentual increase of M.sub.n in respect to blank polymer]. BP1 = blank polymer 1

[0249] FIG. 10 shows the GPC analysis of polymer E67 after 30 min incubation. Dashed lines indicate the high and low limit of molecular weight calibration. The square data points represent calibration data.

[0250] FIG. 11 shows the GPC analysis of blank polymer 1 after 72 h incubation with INH. Dashed lines indicate the high and low limit of molecular weight calibration. The square data points represent calibration data.

[0251] FIG. 12 shows the .sup.1H NMR spectrum of blank polymer 1 incorporating INH in DMSO-d6.

[0252] The polymers were formulated into nanoparticles and characterised as described above. The DLS characterisation results are detailed in Table 9.

DLS Characterization of Polymeric Nanoparticles

[0253]

TABLE-US-00009 TABLE 9 Z-average ± d.sub.Number, zeta polymer PDI width d, nm nm PDI potential, mV E67 INH pH 5 158 ± 34 133 ± 6 0.05 ± 0.02 −55 ± 2 pH 7.4 156 ± 36 131 ± 7 0.06 ± 0.03 −72 ± 5 E80 INH pH 5 195 ± 45 170 ± 6 0.06 ± 0.03 −37 ± 2 pH 7.4 190 ± 51 159 ± 7 0.07 ± 0.02 −71 ± 4 Z-average: cumulants mean;; d.sub.Number: number-mean size; values ± standard deviation of DLS measurement runs; PDI: polydispersity index.

[0254] FIG. 13 shows nanoparticles of E67 with heights ranging from 20-35 nm and widths ranging from 120-400 nm. Deformation of the nanoparticles on the mica substrate was observed, which was attributed to the drying of nanoparticles for imaging.

[0255] FIG. 14 shows nanoparticles of E80 with heights ranging from 70-90 nm and widths ranging from 300-400 nm. Deformation of the nanoparticles was observed on the mica substrate, which was attributed to the drying of nanoparticles for imaging.

[0256] The INH loading of each of the polymers was then assessed. Quantification of INH was assessed via HPLC as described above in the analysis methods. Table 10 shows the results of drug release from nanoparticles E67, E80, E90, E91 and E92. INH release was measured by HPLC after incubation of the nanoparticles in 0.5 and 1 M NaOH for 24 h at 60° C. The drug content is specified in respect to the dry mass of nanoparticles.

Drug Loading

[0257]

TABLE-US-00010 TABLE 10 nanoparticles c.sub.drug, mg/mL c.sub.np, mg/mL L.sub.drug, wt % E67 INH 0.080 1.4  6 E80 INH 1.046 2.5 42 E90 INH 0.02-0.045 N/A .sup. 2-4.5 E91 INH 0.47  N/A 47 E92 INH 0.28-0.64  N/A 19-26 [c.sub.drug = drug concentration; c.sub.np = nanoparticle concentration, L.sub.drug, drug loading wt %].

[0258] The E67 INH nanoparticles were also degraded in 10 mM citrate buffer pH 5 at 37° C. After 24 h, a drug release of 0.4 wt. % was quantified.

Example 2: Synthesis of Rifampicin-Containing Polymer Nanoparticles from Blank Polymer 1

[0259] ##STR00034##

[0260] Rifampicin (RIF) polymer E65 was produced by enzymatic linkage of RIF to blank polymer 1 E60. Blank polymer E60 was synthesized as described above. The blank polymer 1 was then dissolved in diphenyl ether (3 mL) at 60° C. A molar amount of RIF was then added to the solution followed by Candida Antarctica Lipase B (CALB) as immobilized enzyme (Novozym™ 435, N435. N435 contains 10% w/w CALB and 90% w/w acrylic resin). The reaction mixture was stirred under vacuum for 75 min at 60° C. Molecular weight increase was confirmed by GPC analysis. The enzyme was filtered off and the fluid was washed in diethyl ether (3×45 ml). Residual diethyl ether was evaporated by flushing with nitrogen followed by incubation in a desiccator overnight.

[0261] FIG. 15 shows the GPC analysis of polymer E65. The dashed lines indicate the high and low limits of molecular weight calibration. The square data points represent calibration data. After 75 mins, the reaction was terminated and a polymer with M.sub.p/M.sub.n=7188/6467 Da and PD=1.25 was obtained.

[0262] The polymers were formulated into nanoparticles and characterised as described above. The DLS characterisation results are detailed in Table 11.

DLS Characterization of Polymeric Nanoparticles

[0263]

TABLE-US-00011 TABLE 11 Z-average ± d.sub.Number, zeta polymer PDI width d, nm nm PDI potential, mV E65 RIF pH 5 177 ± 57 143 ± 4 0.11 ± 0.04 −77 ± 5 pH 7.4 173 ± 53 139 ± 5 0.10 ± 0.02 −61 ± 8 Z-average: cumulants mean;; d.sub.Number: number-mean size; values ± standard deviation of DLS measurement runs; PDI: polydispersity index.

Example 3: Synthesis of Ciprofloxacin-Containing Polymer Nanoparticles from Blank Polymer 1

[0264] ##STR00035##

This illustrates the preparation of a biologically active molecule containing polymer of the present invention which is able to bind the biologically active molecule via the formation of an ester bond.
Ciprofloxacin-containing polymer was prepared from blank polymer 1. Blank polymer 1 was prepared as described above. Ciprofloxacin (10.7 mg, 32.4 μmol) and blank polymer 1 (100 mg, 27.0 μmol) were combined with DIPEA (282 μL, 162 μmol) and HBTU (12.3 mg, 32.4 μmol) in 1 mL of DCM. The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with DCM and poured in water in an extraction vessel. The water phase was back-extracted twice with DCM. Organic layers were combined, washed with water and brine then dried over MgSO.sub.4. Solvent was evaporated and the orange solid (94 mg, 86%) was dried overnight in a dessicator.

[0265] The polymers were formulated into nanoparticles in the same manner as described in Example 1 and characterised as described above. The DLS characterisation results are detailed in Table 12.

DLS Characterization of Polymeric Nanoparticles

[0266]

TABLE-US-00012 TABLE 12 ciprofloxacin- containing Z-average ± d.sub.Number, zeta potential ± polymer PDI width d, nm nm PDI zeta deviation, mV water 161 ± 40 134 0.062 N/A pH 5 178 ± 50 152 0.065 −54 ± 10 pH 7.4 176 ± 44 148 0.065 −70 ± 10 Z-average: cumulants mean;; d.sub.Number: number-mean size; PDI: polydispersity index.

[0267] The drug loading of the polymer was then assessed. 100 μL of nanoparticles were incubated at 60° C. with 100 μL NaOH 1 M. After 48 h the suspension was centrifuged for 2 min at 13000 rpm then 20 μL of the supernatant was collected and analysed by HPLC. A ten-point standard curve was made by serial dilution of the required drug in NaOH 0.5 M. Nanoparticle dry mass was determined by lyophilisation of a 200 μL sample.

[0268] FIG. 16 is a graph showing RP-HPLC analysis of ciprofloxacin polymer (A), sample from supernatant after incubation of particles made with the polymer incorporating ciprofloxacin in 0.5 M NaOH (B); sample from supernatant after incubation of particles made with the polymer incorporating ciprofloxacin in 0.5 M NaOH with ciprofloxacin reference (C).

[0269] Table 13 shows the results of drug release from ciprofloxacin-containing polymer nanoparticles. Drug release was measured by HPLC after incubation of the nanoparticles in 0.5 M NaOH for 48 h at 60° C. The drug content is specified in respect to the dry mass of nanoparticles.

Drug Loading

[0270]

TABLE-US-00013 TABLE 13 nanoparticles c.sub.drug, mg/mL c.sub.np, mg/mL L.sub.drug, wt % ciprofloxacin- 0.014 1.0 1.4 containing polymer [c.sub.drug = drug concentration; c.sub.np = nanoparticle concentration, L.sub.drug, drug loading wt %].

Example 4: Synthesis of Memantine-Containing Polymer from Blank Polymer 1

[0271] This illustrates the preparation of a biologically active molecule containing polymer of the present invention which is able to bind the biologically active molecule via the formation of an imine bond.

##STR00036##

Memantine-containing polymer 1 was prepared from blank polymer 1. Blank polymer 1 was prepared as described above. Blank polymer 1 (50 mg, 8 μmol) was solubilized in anhydrous toluene. p-Toluenesulfonic acid was added in catalytic amount, and the free base of memantine (28 mg, 158 μmol) was added to the toluene mixture. A Dean-Stark set up was adapted to a round-bottom flask, and the reaction mixture was heated up to 84° C. The reaction was stirred for 30 minutes. Reaction product was retrieved by evaporation of toluene and the resulting product was dried in a desiccator.
FIG. 17 shows the .sup.1H NMR spectrum of the memantine-containing polymer in DMSO-d6. The .sup.1H-NMR spectroscopy analysis in FIG. 17 shows a singlet at 0.79 ppm which is attributed to the two methyl groups of memantine. The other protons of memantine appear in the range of 1 to 2.1 ppm together with the protons of the backbone of the blank polymer 1 as shown in FIG. 2.

Example 5: Synthesis of o-Benzylhydroxamine-Containing Polymer from Blank Polymer 1

[0272] This illustrates the preparation of a biologically active molecule containing polymer of the present invention which is able to bind the biologically active molecule via the formation of an oxime bond.

##STR00037##

o-Benzylhydroxylamine-containing polymer was prepared from blank polymer 1. Blank polymer 1 was prepared as described above. Blank polymer 1 (30 mg, 5 μmol) and o-benzylhydroxylamine (19 mg, 119 μmol) were solubilized separately in anhydrous DMSO. Both solutions were mixed in a round-bottom flask with mechanical stirring, p-Toluenesulfonic acid was added in catalytic amount. Vacuum was applied, and reaction was stirred during 48 h. Reaction product was retrieved by precipitation of the reaction mixture in aqueous buffer and water (pH 9).
FIG. 18 shows the GPC analysis of the o-benzylhydroxylamine-containing polymer after 48 h incubation time. Dashed lines indicate the high and low limit of molecular weight calibration. The square data points represent calibration data.
FIG. 19 shows the .sup.1H-NMR spectrum of the o-benzylhydroxylamine-containing polymer 1 in DMSO-d6.

Example 6: Synthesis of Prednisone-Containing Polymer from Blank Polymer 1

[0273] ##STR00038##

This illustrates the preparation of a biologically active molecule containing polymer of the present invention which is able to bind the biologically active molecule via the formation of a ketal bond.
Prednisone-containing polymer 1 was prepared from blank polymer 1. Blank polymer 1 was prepared as described above. Blank polymer 1 (50 mg, 8 μmol) and prednisone (56 mg, 158 μmol) were solubilized separately in anhydrous DMSO. Both solutions were mixed in a round-bottom flask with mechanical stirring. p-Toluenesulfonic acid was added in catalytic amount. Vacuum was applied, and reaction was stirred during 72 h. Reaction product was retrieved by precipitation of the reaction mixture and washes in aqueous buffer (pH 9) and water.
FIG. 20 shows the GPC analysis of the prednisone-containing polymer after 72 h incubation time. Dashed lines indicate the high and low limit of molecular weight calibration. The square data points represent calibration data.
FIG. 21 shows the .sup.1H-NMR spectrum of the prednisone-containing polymer 1 in DMSO-d6. The .sup.1H-NMR spectroscopy analysis in FIG. 21 shows peaks which are attributable to the protons of prednisone and to the protons of blank polymer 1 as shown in FIG. 2.

Example 7: Synthesis of Ethambutol-Containing Polymer Nanoparticles

[0274] ##STR00039##

[0275] Polymers containing ethambutol (EMB) were synthesized using the same procedure described above for the preparation of a blank polymer.

[0276] The EMB polymers E52 (E52 EMB), E66 (E66 EMB) and E79 (E79 EMB) contained diethyl adipate as the diester component. The diol components consisted of 77 mol % ethambutol and 23 mol % 1,8-octanediol to increase molecular weight by addition of a favorable substrate for the enzyme. EMB polymers were purified by two-fold precipitation in hexane in an analogous fashion to the purification of the blank polymers.

[0277] FIG. 22 shows the GPC analysis of polymer E52 after reaction for 49 h in diphenyl ether at 90° C. and purification by precipitation in hexane. The dashed lines indicate the high and low limits of molecular weight calibration. The square data points represent calibration data. After 49 h, the reaction was terminated and a polymer with M.sub.p/M.sub.n=2569/1858 Da and PD=1.60 was obtained.

[0278] The polymers were formulated into nanoparticles and characterised as described above. The DLS characterisation results are detailed in Table 14.

DLS Characterization of Polymeric Nanoparticles

[0279]

TABLE-US-00014 TABLE 14 Z-average ± d.sub.Number, zeta polymer PDI width d, nm nm PDI potential, mV E52 EMB pH 5 156 ± 53  112 ± 12 0.12 ± 0.02 +16 ± 1 pH 7.4 167 ± 57  124 ± 8  0.12 ± 0.02 −40 ± 4 E66 EMB pH 5 442 ± 223 343 ± 91 0.25 ± 0.06 +14 ± 1 pH 7.4 352 ± 179 249 ± 12 0.26 ± 0.06 −25 ± 3 E79 EMB pH 5 286 ± 129 223 ± 32 0.20 ± 0.03 +14 ± 1 pH 7.4 356 ± 237 183 ± 20 0.44 ± 0.06 +11 ± 2 Z-average: cumulants mean;; d.sub.Number: number-mean size; values ± standard deviation of DLS measurement runs; PDI: polydispersity index.

[0280] The EMB loading of each of the polymers was then assessed. Quantification of EMB was initially assessed as described in the analysis methods above.

[0281] Table 15 shows the results of drug release from nanoparticles E52 and E66. EMB release was measured by HPLC after incubation of the nanoparticles in 1 M NaOH for 24 h at 60° C. The drug content is specified in respect to the dry mass of nanoparticles.

Drug Loading

[0282]

TABLE-US-00015 TABLE 15 nanoparticles c.sub.drug, mg/mL c.sub.np, mg/mL L.sub.drug, wt % E52 EMB 0.797 5.0 16 E66 EMB 0.519 3.0 17 [c.sub.drug = drug concentration; c.sub.np = nanoparticle concentration, L.sub.drug, drug loading wt %].

Example 8: Preparation of Polymer Nanoparticles with Encapsulated Rifampicin

[0283] Nanoparticles encapsulating rifampicin (RIF) were prepared using blank polymer E72 and INH-containing polymer E80. The nanoparticles were prepared as described above for blank polymer 1 with several concentrations of RIF from 2 to 40 mg.Math.mL.sup.−1 in the acetonitrile/polymer phase.

[0284] RIF quantification was determined by UV spectrometry at 475 nm. A five-point standard curve was made by preparing solutions of rifampicin in acetonitrile (linear regression: Abs.=16.64*c.sub.RIF, R.sup.2=99.8%).

[0285] Table 16 shows the results of drug release from nanoparticles encapsulating RIF. Drug release was measured by UV spectroscopy after dissolution of the nanoparticles in acetonitrile. The drug content is specified in respect to the dry mass of nanoparticles.

Drug Loading

[0286]

TABLE-US-00016 TABLE 16 nanoparticles c.sub.drug, mg/mL c.sub.np, mg/mL L.sub.drug, wt % E72 blank RIF 0.012 1.0 1.2 E80 INH RIF 0.009 1.0 0.9 [c.sub.drug = drug concentration; c.sub.np = nanoparticle concentration, L.sub.drug, drug loading wt %].

Example 9: Synthesis of INH-Containing Polymer Nanoparticles from Blank Polymer 2

[0287] ##STR00040##

INH-containing polymer 2 nanoparticles were prepared from blank polymer 2. Blank polymer 2 was prepared as described above. The INH-containing polymer 2 was prepared from blank polymer 2 and INH using the same method described above for the preparation of INH-containing polymer 1 in Example 1.

[0288] FIG. 23 shows the .sup.1H NMR spectrum of blank polymer 2 incorporating INH in CDCl.sub.3.

[0289] The INH-containing polymer 2 prepared from blank polymer 2 was formulated into nanoparticles and characterised as described in Example 1 above. The DLS characterisation results are detailed in Table 17.

DLS Characterization of Polymeric Nanoparticles

[0290]

TABLE-US-00017 TABLE 17 INH-containing polymer (from blank Z-average ± d.sub.Number, zeta potential ± polymer 2) PDI width d, nm nm PDI zeta deviation, mV water 152 ± 58 114 0.146 N/A pH 5  677 ± 400 493 0.363 −24 ± 16 pH 7.4 154 ± 35 128 0.056 −37 ± 31 Z-average: cumulants mean; d.sub.Number: number-mean size; PDI: polydispersity index.

[0291] Table 18 shows the results of drug release from the INH-containing polymer nanoparticles. Drug release was measured by HPLC after incubation of the nanoparticles in 0.5 M NaOH for overnight at 60° C. The drug content is specified in respect to the dry mass of nanoparticles.

Drug Loading

[0292]

TABLE-US-00018 TABLE 18 nanoparticles c.sub.drug, mg/mL c.sub.np, mg/mL L.sub.drug, wt % INH-containing polymer 0.009 1.0 0.9 (from blank polymer 2) [c.sub.drug = drug concentration; c.sub.np = nanoparticle concentration, L.sub.drug, drug loading wt %].

Example 10: Synthesis of INH-Containing Polymer Nanoparticles from Blank Polymer 3

[0293] ##STR00041##

INH-containing polymer 3 nanoparticles were prepared from blank polymer 3. Blank polymer 3 was prepared as described above. The INH-containing polymer was prepared from blank polymer 3 and INH using the same method described above for the preparation of INH-containing polymer 1 in Example 1.

[0294] The polymers were formulated into nanoparticles and characterised as described above in Example 1. The DLS characterisation results are detailed in Table 19.

DLS Characterization of Polymeric Nanoparticles

[0295]

TABLE-US-00019 TABLE 19 INH-containing polymer (from blank Z-average ± d.sub.Number, zeta potential ± polymer 3) PDI width d, nm nm PDI zeta deviation, mV water 532 ± 365 134 0.471 −26 ± 10 Z-average: cumulants mean;; d.sub.Number: number-mean size; PDI: polydispersity index.

[0296] Table 20 shows the results of drug release from the INH-containing polymer nanoparticles. Drug release was measured by HPLC after incubation of the nanoparticles in 0.5 M NaOH overnight at 60° C. The drug content is specified in respect to the dry mass of nanoparticles.

Drug Loading

[0297]

TABLE-US-00020 TABLE 20 nanoparticles c.sub.drug, mg/mL c.sub.np, mg/mL L.sub.drug, wt % INH-containing polymer 0.04 N/A 4 (from blank polymer 3) [c.sub.drug = drug concentration; c.sub.np = nanoparticle concentration, L.sub.drug, drug loading wt %].

Example 11: In Vitro Release of INH from Nanoparticles

[0298] The in vitro release of INH from nanoparticles of INH-containing polymer 1 was assayed as follows. 200 μL of a suspension containing INH-containing polymer 1 formulated into nanoparticles was centrifuged for 2 min at 13 000 rpm. The supernatant was discarded and the pellet was resuspended in various buffers: 10 mM HCl adjusted to pH 2;

25 mM acetate buffer adjusted to pH 4;
25 mM phosphate buffer adjusted to pH 7.4;
and incubated at 37° C. under orbital shaking at 150 rpm. At regular time intervals the suspensions were centrifuged 2 min at 13 000 rpm then 200 μL of the supernatant was pipetted and replaced by 200 μL of fresh buffer. Pellets were resuspended and suspensions were further incubated while the amount of isoniazid in the supernatant was measured by HPLC as described previously.
FIG. 24 is a graph showing the cumulative release of isoniazid from the nanoparticles formulated with INH-containing polymer 1 incubated in various buffers at 37° C. INH is released at different rate depending on the pH. After a month more than 50% of INH is released at pH 2 and 15% at pH 4 whereas less than 1% release is observed at pH 7.4. This graph shows the effect of pH on the stability of the bond between INH and the polymer. INH formulated with the blank polymer is stable at physiological pH (pH 7.4) and is released in acidic conditions found in cell compartments such as lysosome, endosome, phagosome, phagolysosome and autophagosome found in various cells such as macrophages.

Example 12: In Vitro Evaluation of Nanoparticles on Tuberculosis-Infected Macrophages

[0299] The antibiotic efficacy of the synthesized nanoparticles was next evaluated and compared to that of the free drugs against Mycobacterium bovis BCG-lux (Bacille Calmette Guerin) grown in human monocyte-derived macrophages. The bioluminescence of BCG-lux after lysis of macrophages was used as an indicator of BCG-lux viability and therefore as an experimental cell model of tuberculosis. Results are presented against the BCG-lux viability of corresponding untreated (control) cells.

[0300] FIG. 25 shows the effect of the INH-containing nanoparticles E67 and the corresponding blank nanoparticles E55 on BCG-lux viability. The mass concentration of blank nanoparticles E55 corresponds to equimolar polymer concentrations of a 0.25 mg mL.sup.−1 suspension of E67 nanoparticles. In this test, the bioluminescence of Mycobacterium bovis BCG-lux grown in human monocyte-derived macrophages was investigated for 72 h. Cells were grown in 24 well cell culture plates at 37 C and 5% CO.sub.2 in 200 μL DMEM containing 10% FCS, 60 mg mL.sup.−1 PEN and containing nanoparticles. Error bars indicate ±standard deviation (n=6). The asterisks indicates a significant difference to the control group, determined by unpaired Student t-test at α=0.05.

[0301] The results show that the bioluminescence of BCG-lux was not significantly affected when co-incubated with E55 blank nanoparticles. INH nanoparticles E67 containing the same backbone polymer reduced the viability of BCG-lux by 70% and 74% at nanoparticle concentrations of 0.1 and 0.25 mg mL.sup.−1, respectively.

[0302] The effect of concentration and time of incubation of the INH-containing nanoparticles E67 and free INH on BCG-lux viability was also investigated and the results are shown in FIGS. 26-29. Macrophages were cultivated in 24 well cell culture plates at 37° C. and 5% CO.sub.2 in 300 μL DMEM containing 10% FCS, 60 mg mL.sup.−1 PEN and the nanoparticles and free drug. Free INH was used as a positive control. The INH concentration corresponded to the experimental release from a 0.25 mg mL.sup.−1 E67 nanoparticle suspension. The error bars indicate ±standard deviation (n=6). The asterisks indicate a significant difference to the control group, determined by unpaired Student t-test at α=0.05.

[0303] The results show that co-incubation of intracellular BCG-lux with E67 INH nanoparticles for 24 h resulted in an optimum antimycobacterial efficacy at a nanoparticle concentration of 0.25 mg mL.sup.−1. The BCG-lux viability decreased significantly between 24 h and 72 h in all of the tests in the presence of E67 nanoparticles.

[0304] 120 h efficacy was only determined at a nanoparticle concentration of 0.25 mg mL.sup.−1. The results show that the antibiotic efficacy compared to untreated cells increased from 61% after 72 h to 74% after 120 h.

[0305] The results also show an initial 32% increase in presence of free INH of BCG-lux bioluminescence after 24 h when compared to untreated cells. After 72 h, the viability of cells incubated with INH was 15% lower than that for control cells. After 120 h, the 53% reduction of BCG-lux bioluminescence was observed. Thus, the efficacy of free INH was lower than the efficacy of the corresponding nanoparticle dispersion at all times.

[0306] The effect of polymer chain length on the antimycobacterial efficacy was also investigated and the results are shown in FIGS. 30 and 31. Nanoparticles of the 16.5 kDa blank polymer E72 and the 16 kDa INH polymer E80 were investigated in the experimental tuberculosis model. Nanoparticle concentrations were kept at the E67 optimum concentration of 0.25 mg mL.sup.−1. Macrophages were cultivated in 24 well cell culture plates at 37° C. and 5% CO.sub.2 in 300 μL DMEM containing 10% FCS, 60 mg mL.sup.−1 PEN and the nanoparticles and free drug. Free INH was used as a positive control. The error bars indicate ±standard deviation (n=6). The asterisks indicate a significant difference to control group, determined by unpaired Student t-test at α=0.05.

[0307] The results show that after 24 h, the BCG-lux viability was statistically identical for blank and INH nanoparticles. The antibiotic efficacies of the INH nanoparticles compared to the control group increased from 25% to 41% after 72 h. These effects were significantly lower than those noticed for the shorter polymer length E67 nanoparticles and it was also noted that the blank polymer E72 had no significant efficacy after 72 h. The standard deviation was comparably high.

[0308] Free INH was used as a positive control in a concentration corresponding to the experimental release of a 0.25 mg mL.sup.−1 E80 INH nanoparticle suspension. After 24 h, no antimycobacterial activity of free INH was measured, as was also noted in earlier experiments described above. After 72 h, the antibiotic efficacy was equivalent to the antibiotic efficacy of E80 nanoparticles.

[0309] The antibiotic efficacy of E72 and E80 polymers encapsulating RIF were also investigated. FIG. 32 shows the bioluminescence of Mycobacterium bovis BCG-lux grown in human monocyte-derived macrophages in the presence of E72 or E80 INH RIF nanoparticles for 72 h. Free RIF was used as a positive control in a concentration corresponding to the experimental release of a 0.25 mg mL.sup.−1 E80 INH RIF nanoparticle suspension.

[0310] The results show that after 72 h, efficacies of the INH nanoparticles E80, E80 INH RIF were not distinguishable. In contrast to the E72 blank nanoparticles, RIF encapsulating E72 nanoparticles induced a 41% reduction of BCG-lux viability. Free RIF did not induce a significant antimycobacterial effect in this experiment.

[0311] The efficacy of ethambutol-containing polymer nanoparticles E66 was analyzed as described above and the results are shown in FIGS. 33 and 34. EMB was used as a positive control. Macrophages were cultivated in 24 well cell culture plates at 37 C and 5% CO.sub.2 in 300 μL DMEM containing 10% FCS, 60 mg mL.sup.−1 PEN and the drug formulation. The error bars indicate ±standard deviation (n=6). The asterisks indicate a significant difference to the control group, determined by unpaired Student t-test at α=0.05.

[0312] The results show that a 0.8 mg mL.sup.−1 suspension of EMB nanoparticles E66 induced a 28% reduction of BCG-lux luminescence after 24 h and a 40% reduction after 72 h. E66 efficacy was also tested at a concentration of 0.4 mg mL.sup.−1. EMB nanoparticles E66 at a concentration of 0.4 mg mL.sup.−1 did not induce a significant effect on BCG-lux viability after 72 h.

[0313] Free EMB was used as a positive control in a concentration corresponding to a 15 wt % release of a 0.8 mg mL.sup.−1 E66 nanoparticle suspension. The free drug did not reduce BCG-lux viability.

Example 13: Cytotoxicity of Nanoparticles

[0314] The cytotoxicity of the nanoparticle formulations E72 blank and E80 was tested using an Dead cell apoptosis kit with Annexin V FITC and PI, for flow cytometry (MolecularProbes®, UK) and the results are shown in FIG. 35. 2*10.sup.5 cells per well were seeded in 24 well cell culture plates, differentiated for 5 days, and incubated with nanoparticles as described previously. The kit was used according to manufacturer's instructions. Live cells do not bind the human anticoagulant protein Annexin V and are therefore not stained by the conjugated fluorescent dyes Fluorescein thiocyanate (FITC) and Propidium iodide (PI). Apoptotic cells bind Annexin V, but only show FITC fluorescence because the intact membranes are impermeable for PI. Dead cells are permeable for PI and show both FITC and PI fluorescence in a Fortessa 4 laserflow cytometer (Becton, Dickinson and Company, UK).

[0315] For the purposes of this experiment, dead cells are FITC/PI+, live cells are FITC/PI−, and apoptotic cells are FITC+, PI−. In FIG. 35, the striped bars represent a control group without treatment, the grey bars represent the INH nanoparticles and the dotted bars represent the blank nanoparticles. The error bars indicate ±standard deviation.

[0316] The results show that macrophages treated with E72 and E80 nanoparticles did not contain significantly increased percentages of dead or apoptotic cells.

Example 14: Synthesis of Insulin-Containing Polymer

[0317] Blank polymer 1 (30 mg, 5 μmol) and insulin (15 mg, 2.6 μmol) were solubilized separately in anhydrous DMSO. Both solutions were mixed in a round-bottom flask with mechanical stirring, p-Toluenesulfonic acid was added in catalytic amount. Vacuum was applied, and the reaction was left for 48 h. The reaction product was retrieved by precipitation of the reaction mixture in aqueous buffer (pH 9). The precipitate was spun down, washed with buffer and water, and then retrieved in DCM which was then evaporated. The reaction product was left to dry overnight in a desiccator before RP-HPLC analysis.
FIG. 36 is a graph showing RP-HPLC analysis of blank polymer 1 (A), Insulin (B), and the polymer 1 after reaction with insulin and after treatment (precipitation and washes) (C).
FIG. 36 shows that after the treatment of the reaction mixture another product is formed as seen by the peak at t.sub.R=6.82 min. The blank polymer 1 (t.sub.R=9.11 min) and some residual Insulin (t.sub.R=6.01) are also observed.