Polymer systems and their applications in diagnostics and drug delivery

Abstract

The present invention provides a biodegradable precision polymer system (poly system) and methods for preparing pharmaceutically effective precision polymer nanosystems (polymer nanosystem) for delivery and/or targeting of drugs or drug like compounds.

Claims

1. A composition comprising: one or more biodegradable block copolymer drug carriers comprising A-B, A-B-A or B-A-B block copolymers, wherein A is a biodegradable polyester block and B is a polyethylene glycol (PEG) block; and a plurality of pendent carboxyl groups derived from aromatic or aliphatic dianhydrides incorporated within the backbone of the one or more biodegradable block copolymer drug carriers, wherein at least one pendant carboxyl group of the plurality of carboxyl groups is covalently bonded to a diamine linker.

2. The composition according to claim 1, wherein the diamine linker comprises a first amine that is conjugated to the at least one pendant carboxyl group and a second amine that is covalently connected to a ligand with carboxyl functionality.

3. The composition according to claim 1, wherein at least one pendant carboxyl group of the plurality of carboxyl groups is covalently bonded to a drug, a ligand, a contrast agent, or a near IR-fluorescent agent, directly or via a linker.

4. The composition according to claim 1, wherein at least one pendant carboxyl group of the plurality of carboxyl groups is covalently bonded to a single drug, multiple drugs, or a drug and a ligand.

5. The composition according to claim 3, wherein the drug has at least one of limited aqueous solubility or limited tissue penetration.

6. The composition of claim 1, wherein the composition comprises an aqueous carrier and the composition is a uniform colloidal system.

7. A method of administration comprising: reconstituting the composition of claim 1 by addition of water or an aqueous solution to form a uniform colloidal system; and administering the uniform colloidal system to a subject.

8. The method of administration of claim 7, wherein the administering is carried out orally or perorally.

9. The method of administration of claim 7, wherein the administering is by nose, skin, or injection.

10. The composition according to claim 1, wherein the biodegradable polyester block is synthesized from monomers selected from the group consisting of D,L-lactide, D-lactide, L-lactide, D,L-lactic acid, D-lactic acid, L-lactic acid, glycolide, glycolic acid, -caprolactone, 1,4-dioxan-2-one, -hydroxy hexanoic acid, -butyrolactone, -hydroxy butyric acid, -valerolactone, -hydroxy valeric acid, hydroxybutyric acids, malic acid, and a mixture thereof.

11. The composition according to claim 1, wherein the dianhydrides are tetracarboxylic anhydrides.

12. The composition according to claim 11, wherein the tetracarboxylic anhydrides are either pyromellitic dianhydride or cyclohexane tetracarboxylic dianhydride.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A shows the decrease in molecular weight of hP2-400 over a period of 96 hours in PBS at 37 C.;

(2) FIG. 1B shows the degradation profile of hP2-400 over a period of 96 hours in PBS at 37 C.;

(3) FIG. 1C shows the decrease in molecular weight of hP2-1000H over a period of 96 hours in PBS at 37 C.;

(4) FIG. 1D shows the degradation profile of hP2-1000H over a period of 96 hours in PBS at 37 C.;

(5) FIG. 1E shows the decrease in molecular weight of hP2-2000H over a period of 96 hours in PBS at 37 C.;

(6) FIG. 1F shows the degradation profile of hP2-2000H over a period of 96 hours in PBS at 37 C.;

(7) FIG. 2A shows the mean fluorescent intensity of fluorescent P2Ns of hP2-400 in RBCs; and

(8) FIG. 2B shows the mean fluorescent intensity of fluorescent P2Ns of hP2-400 in plasma.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

(9) Biodegradable means that the block copolymer or oligomer can chemically break down or degrade within the body to form nontoxic components. The rate of degradation can be the same or different from the rate of drug release.

(10) Drug shall mean any organic or inorganic compound or substance having biological or pharmacological activity that can be adapted or used for a therapeutic purpose.

(11) Peptide, polypeptide, oligopeptide and protein shall be used interchangeably when referring to peptide or protein drugs and shall not be limited as to any particular molecular weight, peptide sequence or length, field of bioactivity or therapeutic use unless specifically stated.

(12) PLA shall mean a polymer derived from the poly-condensation of lactic acid or by the ring opening polymerization of lactide.

(13) Biodegradable polyester refers to any polymer preferably synthesized from monomers selected from the group consisting of D,L-lactide, D-lactide, L-lactide, D,L-lactic acid, D-lactic acid, L-lactic acid, glycolide, glycolic acid, -caprolactone, 1,4-dioxan-2-one, -hydroxy hexanoic acid, -butyrolactone, -hydroxy butyric acid, -valerolactone, -hydroxy valeric acid, hydroxybutyric acid, malic acid, ethyleneglycol and mixtures thereof.

(14) In one embodiment, the biodegradable polymer system comprises ABA-type or BAB-type triblock copolymers, AB-type diblock copolymers or mixtures thereof, where the A-blocks are relatively hydrophobic and comprises a biodegradable polyester, and the B-blocks are relatively hydrophilic and comprises polyethylene glycol (PEG).

(15) In another embodiment, the biodegradable polymer system comprises multiblock ABA- or BAB-type copolymers of lactic acid and ethylene glycol with periodically spaced side chain carboxyl groups. Multiple pendent carboxyl groups are imparted into the PLA-PEG backbone via the incorporation of an aromatic or aliphatic tetracarboxylic anhydride moiety in the chain extension step. In certain embodiments of the invention, the tetracarboxylic dianhydrides that are used are pyromellitic dianhydride and cyclohexane tetracarboxylic dianhydride.

(16) In addition, an embodiment of the present invention is directed to a variation of polymer system that is synthesized with a diamine linker conjugated to the carboxyl groups, such that one of the amine moieties of the linker is covalently bonded to the carboxyl of the polymer while the other remains free for conjugating ligands. The two variations of the polymer, with free carboxyl and amine unit, provides the flexibility to attach the ligands with either a free hydroxyl, amine or carboxyl functionality respectively, without the need of any further alteration of either the polymer or ligand.

(17) As proof of concept some embodiments of the present invention, have been synthesized with PLA and PEG blocks and aromatic or aliphatic linkers imparting multiple pendent carboxyl groups in the polymer backbone. The polymers are characterized using nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC) to verify their chemistry and evaluate their molecular weight. As described above, carboxyl groups for certain embodiments of invention are capped with diamine moieties. Both the variations of polymer nanosystem, with pendent carboxyl and amine groups are further conjugated to ligands with free reactive hydroxyl, amine or carboxyl functionalities. Polymers thus obtained can be used to form polymer nanosystem by appropriate methods. The X-ray photoelectron spectroscopy (XPS) analysis performed on the polymer nanosystem shows that the ligands attached to the polymer system are displayed on the surface of the polymer nanosystem, which is imperative for their availability for binding to the respective receptors on the cells.

(18) The PEG, PEG derivatives or mixtures thereof used in the present invention dissolves or uniformly mixes with the biodegradable block copolymer and so reduces the viscosity and increases the fluidity of the composition. The compositions of the present invention are flowable liquids or can be easily formulated with an aqueous vehicle to afford a homogeneous uniform colloidal system. The composition can be easily administered as is or reconstituted with an aqueous or appropriate vehicle. After the administration, the block copolymer drug carrier may or may not form a drug depot. Therefore, the liquid PEG, PEG derivative or mixtures thereof of the present invention should be a material that does not cause loss of activity of the physiologically active substance.

(19) Drugs that may be incorporated with the drug delivery compositions of the present invention can be any bioactive agent, but particular advantage is achieved with bioactive agents having limited solubility or dispersibility in an aqueous or hydrophilic environment, or any bioactive agent that requires enhanced solubility or permeability. In addition, the choice of ligand conjugated to the polymer system will play an important role in the absorption and targetability of the polymer nanosystem and encapsulated drugs, thus enhancing their site/target specificity.

(20) The present invention thus provides compositions comprising biodegradable block copolymer drug carriers and PEG, PEG derivatives or mixtures thereof that are powders or can be rapidly reconstituted in an aqueous vehicle to afford useful forms that are uniform colloidal systems. The drug solution formed with the drug delivery compositions of the present invention has desirable physical stability, therapeutic efficacy, and toxicology. The PEG, PEG derivatives or mixtures thereof of the present invention can be used for water soluble or water insoluble block copolymeric drug carriers, particularly for biodegradable di- or triblock copolymers that have reverse gelation properties and/or polymers that can enhance the solubility of drugs, especially hydrophobic drugs.

WORKING EXAMPLES

Abbreviations

(21) CH.sub.2Cl.sub.2 Methylene Chloride

(22) DIEA N,N-diisoethylpropylamine

(23) DMAB Didodecyldimethylammonium bromide

(24) DMAP 4-Dimethylaminopyridine

(25) DMF N,N-dimethylformamide

(26) EDA Ethylenediamine

(27) EDC 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide

(28) GA Gambogic acid

(29) GaL Galactose

(30) RGD Arginylglycylaspartic acid

(31) PVA Poly(vinyl alcohol)

(32) TFA Trifluoroacetic acid

(33) TfR Transferrin receptor

(34) TPGS D--Tocopheryl polyethylene glycol 1000 succinate

(35) NS Nanosystems

(36) P2s Precision polymers

(37) PLGA Polylactide-co-glycolide

(38) P2Ns Ligand functional precision polymer nanosystems

Example 1

(39) This example describes the synthesis of PLA-PEG400 block pre-polymer from 1-lactide and 7% (w/w) PEG-400.

(40) A 250 ml three necked round-bottomed flask fitted with a reflux condenser, magnetic stirrer, heat-on-block and under a gentle purge of nitrogen, was charged with 1-Lactide (18.0 g; 0.125 mol), PEG400 (3.52 g; 2.65 ml; 0.0088 mol), Stannous Octoate (21.52 mg; 17.20 l; 0.1% w/w), Toluene (50 ml). The reaction mixture was heated to a temperature of 115 C. while stirring to allow the solvent to reflux. The reaction was carried out under these conditions for 24 h, after which reaction mixture was cooled and solvent was evaporated under reduced pressure. The residue left upon evaporation of solvent was dissolved in 12 ml methylene chloride and precipitated in 400 ml cold diethyl ether under vigorous stirring. The white polymer was obtained by filtration under suction and dried under vacuum to a constant dry weight (16 mg).

Example 2

(41) This example describes the chain extension of the pre-polymer from example 1 with the incorporation of an aromatic linker to impart multifunctionality.

(42) A 250 ml three necked round-bottomed flask fitted with a reflux condenser, magnetic stirrer, heat-on-block and under a gentle purge of nitrogen was charged with PLA-PEG400 (7 g; 0.6364 mmol), Pyromellitic dianhydride (166.56 mg; 0.7636 mmol), Triethylamine (128.81 mg; 177.42 l; 1.2728 mol), Stannous Octotate (7 mg; 5.6 l; 0.1 w/w) and Toluene (40 ml). The reaction mixture was heated to a temperature of 115 C. while stirring to allow the solvent to reflux. The reaction was carried out under these conditions for 24 h, after which reaction mixture was cooled and solvent was evaporated under reduced pressure. The residue dissolved in 10 ml methylene chloride and precipitated in 500 ml cold diethyl ether under vigorous stirring. The white polymer was collected by filtration under suction and dried under vacuum to a constant dry weight (6.2 g).

Example 3

(43) This example describes the chain extension of the pre-polymer from example 1 with the incorporation of an aliphatic linker to impart multifunctionality.

(44) A 250 ml three necked round-bottomed flask fitted with a reflux condenser, magnetic stirrer, heat-on-block and under a gentle purge of nitrogen, was charged with PLA-PEG400 (8 g; 0.7273 mmol), Cyclohexane tetracarboxylic dianhydride (195.66 mg; 0.8728 mmol), Triethylamine (147.25 mg; 202.82 l; 1.455 mmol), Stannous Octoate (8 mg; 6.4 l; 0.1% w/w) and Toluene (50 ml). The reaction mixture was heated to a temperature of 115 C. while stirring to allow the solvent to reflux. The reaction was carried out under these conditions for 24 h, after which reaction mixture was cooled and solvent was evaporated under reduced pressure. The residue dissolved in 10 ml methylene chloride and precipitated in 400 ml cold diethyl ether under vigorous stirring. The white polymer was collected by filtration under suction and dried under vacuum to a constant dry weight (6.1 g).

Example 4

(45) This examples describes the synthetic process of the aminated version of hP2-400 (hP2-400-EDA) described in example 3 using EDA.

(46) A 50 ml three necked round bottomed flask equipped with magnetic stirrer and under a gentle purge of nitrogen was charged with hP2-400 (250 mg; 0.0142 mmol; 11 COOH/mol), EDC (40.83 mg; 0.2131 mmol) and DMF (3 ml) and stirred at room temperature for 30 min. n-boc-EDA (34.12 mg; 33.72 l; 0.213 mmol) and DIEA (27.53 mg; 37.1 l) were subsequently added to the sitting solution and the reaction was continued for 18 h. The polymer was obtained by precipitation after pouting the reaction mixture in cold water. The precipitate was washed twice with water and dried under vacuum. The dried polymer was dissolved in 2.2 ml of 10:1 mixture of CH2Cl2:TFA and stirred at room temperature for 1 h under nitrogen atmosphere. The solvent was then stripped off on a rotary evaporator and product washed with water to get a white polymer. The hP2-400-EDA polymer was dried under vacuum to a constant dry weight (195 mg).

Example 5

(47) This examples describes synthesis process of the thioester version of pP2-400 described in example 2 using 2-mercaptoethanol.

(48) A 50 ml three necked round bottomed flask equipped with magnetic stirrer, condenser and a heat-on block, and under a gentle purge of nitrogen was charged with pP2-400 (100 mg; 0.011 mmol; 13 COOH/mol), 2-mercaptoethanol (12.89 mg; 11.61 l; 0.165 mmol), methanesulfonic acid (15.86 mg; 10.71 l; 0.165 mmol) and DMF (2 ml). The reaction mixture was stirred at 100 C. for 24 h, after which the product was obtained by precipitation in cold water. The product was further washed four times with water until the sulfur smell was eliminated and dried under vacuum to a constant dry weight (57.7 mg).

Example 6

(49) This examples describes the conjugation of Gambogic acid to the aminated hP2-400 described in example 4.

(50) A 50 ml three necked round bottomed flask equipped with magnetic stirrer and under a gentle purge of nitrogen was charged with gambogic acid (65.2 mg; 0.1037 mmol), EDC (29.83 mg; 0.1556 mmol) and CH2Cl2 (1 ml) and stirred at ambient temperature for 30 min hP2-400-EDA (125 mg; 0.0069 mmol) pre-dissolved in CH2Cl2 (2 ml) and DIEA (20.11 mg; 27.1 l; 0.1556 mmol) were subsequently added to the stirring solution and the reaction was carried out at these conditions for another 18 h. The product was obtained by precipitation in cold ether. The polymer was further washed one with ether and another time with water and dried under vacuum to a constant dry weight (104 mg).

Example 7

(51) This example describes the conjugation of Galactose to hP2-400 described in example 3.

(52) A 50 ml three necked round bottomed flask equipped with magnetic stirrer and a heat-on block, and under a gentle purge of nitrogen was charged with hP2-400 (250 mg; 0.0142 mmol; 11 COOH/mol), D-galactose (38.39 mg; 0.2131 mmol), methanesulfonic acid (20.48 mg; 13.83 l; 0.2131 mmol) and DMF (3 ml). The reaction mixture was stirred at 60 C. for 24 h, after which the product was obtained by precipitation in cold water. The product was further washed twice with water and dried under vacuum to a constant dry weight (242.1 mg).

Example 8

(53) This example describes the conjugation of arginylglycylaspartic acid (RGD peptide) to hP2-400 described in example 3.

(54) A 50 ml three necked round bottomed flask equipped with magnetic stirrer and under a gentle purge of nitrogen was charged with hP2-400 (150 mg; 0.0085 mmol; 11 COOH/mol), EDC (24.44 mg; 0.1275 mmol) and DMF (2 ml) and stirred at room temperature for 30 min RGD (44.16 mg; 0.1275 mmol) pre-dissolved in 1 ml DMF and DIEA (16.48 mg; 22.21 l; 0.1275 mmol) were subsequently added to the stirring solution and the reaction was continued at these conditions for 18 h. The product was obtained by precipitation in cold water. The polymer was further washed twice in water and dried under vacuum to a constant dry weight (122 mg).

Example 9

(55) This example describes the conjugation of fluorescent probe, Fluorescein to aminated hP2-400 described in example 4.

(56) A 50 ml three necked round bottomed flask equipped with magnetic stirrer and under a gentle purge of nitrogen was charged with hP2-400-EDA (25 mg; 0.00128 mmol; 9 NH.sub.2/mol), NHS-Fluorescein (7.24 mg; 0.0153 mmol), DIEA (1.98 mg; 2.7 l; 0.0153 mmol) and DMF (1 ml). The reaction mixture was stirred in dark under inert atmosphere for 18 h. The product was obtained by precipitation in cold water. The polymer was further washed twice in water and dried under vacuum to a constant dry weight (12.5 mg).

Example 10

(57) This example describes the conjugation of Hesperetin to pP2-400 described in example 2.

(58) A 50 ml three necked round bottomed flask equipped with magnetic stirrer and under a gentle purge of nitrogen was charged with pP2-400 (100 mg; 0.0105 mmol; 11 COOH/mol), EDC (30.19 mg; 0.1575 mmol) and DMF (1 ml) and stirred at room temperature for 30 min. Hesperetin (47.61 mg; 0.1575 mmol) and DMAP (1.92 mg; 0.01575 mmol) pre-dissolved in 1 ml DMF were subsequently added to the stirring solution and the reaction was continued at these conditions for 18 h. The product was obtained by precipitation in cold water. The polymer was further washed twice and dried under vacuum to a constant dry weight.

Example 11

(59) This example describes the conjugation of 1-Lysine to pP2-400 described in example 2.

(60) A 50 ml three necked round bottom flask equipped with a magnetic stirrer and under a gentle purge of nitrogen was charged with pP2-400 (100 mg; 0.1 mmol; 11 COOH/mol), EDC (28.75 mg; 0.15 mmol) and DMF (0.5 ml) and stirred at ambient temperature for 30 min 1-Lysine (21.93 mg; 0.15 mmol) predissolved in DMF (0.5 ml) and DIEA (19.39 mg; 26.13 l; 0.15 mmol) were subsequently added to the reaction flask and the reaction was carried out under nitrogen atmosphere and room temperature for 18 h. A pale yellow product was obtained by precipitation in water. The polymer was further washed twice with water and dried under vacuum to a constant dry weight (82.9 mg).

Example 12

(61) This example describes the conjugation of Cysteine to pP2-400 described in example 2.

(62) A 50 ml three necked round bottom flask equipped with a magnetic stirrer and under a gentle purge of nitrogen was charged with pP2-400 (100 mg; 0.1 mmol; 11 COOH/mol), EDC (28.75 mg; 0.15 mmol) and DMF (0.5 ml) and stirred at ambient temperature for 30 min. Cysteine (18.17 mg; 0.15 mmol) predissolved in DMF (0.5 ml) and DIEA (19.39 mg; 26.13 l; 0.15 mmol) were subsequently added to the reaction flask and the reaction was carried out under nitrogen atmosphere and room temperature for 18 h. A pale yellow polymer was obtained by precipitation in water. The polymer was further washed twice with water and dried under vacuum to a constant dry weight (65.1 mg).

Example 13

(63) This example describes the conjugation of Arginine to pP2-400 described in example 2.

(64) A 50 ml three necked round bottom flask equipped with a magnetic stirrer and under a gentle purge of nitrogen was charged with pP2-400 (100 mg; 0.1 mmol; 11 COOH/mol), EDC (28.75 mg; 0.15 mmol) and DMF (0.5 ml) and stirred at ambient temperature for 30 min 1-Arginine (26.13 mg; 0.15 mmol) predissolved in DMF (0.5 ml) and DIEA (19.39 mg; 26.13 l; 0.15 mmol) were subsequently added to the reaction flask and the reaction was carried out under nitrogen atmosphere and room temperature for 18 h. A pale yellow polymer was obtained by precipitation in water. The polymer was further washed twice with water and dried under vacuum to a constant dry weight (87.6 mg).

Example 14

(65) This example describes the conjugation of Paclitaxel to pP2-400 described in example 2.

(66) A 50 ml three necked round bottomed flask equipped with magnetic stirrer and under a gentle purge of nitrogen was charged with pP2-400 (100 mg; 0.0072 mmol; 10 COOH/mol), EDC (20.70 mg; 0.108 mmol) and DMF (0.5 ml) and stirred at room temperature for 30 min. Paclitaxel (92.2 mg; 0.108 mmol) and DMAP (1.32 mg; 0.0108 mmol) pre-dissolved in 0.5 ml DMF were subsequently added to the stirring solution and the reaction was continued at these conditions for 18 h. The product was obtained by precipitation in cold 1:1 mixture of ethanol and ethyl ether. The polymer was further washed twice and dried under vacuum to a constant dry weight (75 mg).

Example 15

(67) This example describes the conjugation of Paclitaxel to hP2-400 described in example 3.

(68) A 50 ml three necked round bottomed flask equipped with magnetic stirrer and under a gentle purge of nitrogen was charged with hP2-400 (100 mg; 0.00521 mmol; 9 COOH/mol), EDC (11.98 mg; 0.0625 mmol) and DMF (0.5 ml) and stirred at room temperature for 30 min. Paclitaxel (53.37 mg; 0.0625 mmol) and DMAP (0.76 mg; 0.00625 mmol) pre-dissolved in 0.5 ml DMF were subsequently added to the stirring solution and the reaction was continued at these conditions for 18 h. The product was obtained by precipitation in cold 1:1 mixture of ethanol and ethyl ether. The polymer was further washed twice and dried under vacuum to a constant dry weight (75 mg).

Example 16

(69) This example describes the conjugation of Cisplatin to hP2-400 described in example 2.

(70) A 50 ml three necked round bottom flask equipped with a magnetic stirrer and under a gentle purge of nitrogen was charged with pP2-400 (100 mg; 0.1 mmol; 8 COOH/mol), EDC (19.71 mg; 0.1 mmol) and DMF (1 ml) and stirred at ambient temperature for 30 min. Cisplatin (30 mg; 0.1 mmol) predissolved in DMF (1 ml) and DIEA (12.92 mg; 17.42 l; 0.1 mmol) were subsequently added to the reaction flask and the reaction was carried out in the dark under nitrogen atmosphere and room temperature for 24 h. A yellow conjugated polymer was obtained by precipitation in water. The polymer was further washed twice with water and dried under vacuum to a constant dry weight (84.3 mg).

Example 17

(71) This example describes the conjugation of Gemcitabine to pP2-400 described in example 2.

(72) A 50 ml three necked round bottom flask equipped with a magnetic stirrer and under a gentle purge of nitrogen was charged with pP2-400 (100 mg; 0.1 mmol; 8 COOH/mol), EDC (19.71 mg; 0.1 mmol) and DMF (1 ml) and stirred at ambient temperature for 30 min. Gemcitabine (26.32 mg; 0.1 mmol) predissolved in DMF (1 ml) and DIEA (12.92 mg; 17.42 l; 0.1 mmol) were subsequently added to the reaction flask and the reaction was carried out in the dark under nitrogen atmosphere and room temperature for 24 h. A yellow conjugated polymer was obtained by precipitation in water. The polymer further washed twice with water and dried under vacuum to a constant dry weight (80.6 mg).

Example 18

(73) This example describes the conjugation of Withaferin A to pP2-2000H (A variation of pP2-400 described in example 2 with 2000 g/mol PEG chains and higher lactide concentration per chain of final polymer).

(74) A 50 ml three necked round bottomed flask equipped with magnetic stirrer and under a gentle purge of nitrogen was charged with pP2-2000H (50 mg; 0.0017 mmol; 7 COOH/mol), EDC (3.26 mg; 0.017 mmol) and DMF (1 ml) and stirred at room temperature for 30 min. Withaferin A (8.0 mg; 0.017 mmol) and DMAP (0.21 mg; 0.0017 mmol) pre-dissolved in 1 ml DMF were subsequently added to the stirring solution and the reaction was continued at these conditions for 18 h. The product was obtained by precipitation in cold water. The polymer was further washed twice and dried under vacuum to a constant dry weight.

Example 19

(75) This example describes the conjugation of diagnostic agents to P2s.

(76) P2s can be functionalized with diagnostic agents e.g. MRI and ultrasound contrast agents, Near-IR fluorescent agents etc. by coupling them to the carboxyl or amine functionality of the P2s via ester or amide bonds based on the available functional unit of the agent. Both the processes involve activation of carboxyl on either the polymer or the diagnostic moiety by EDC followed by either amination in the presence of DIEA or esterification in the presence of DMAP. Alternatively, Fischer esterification can be employed to conjugate a moiety with hydroxyl functionality to the carboxylated P2, if the said moiety can withstand higher temperatures and strong acid catalyst.

Example 20

(77) This example describes the use of P2s for coupling moieties of different functionalities.

(78) The methods described in examples 4-19 can be employed on any and all P2s listed in table 3. All P2s can be aminated or thiolated and coupled to ligands, labeling and diagnostic agents, drug molecules etc. as described for the model P2s in the above mentioned examples. The choice of polymer is subject to the requirements for the end product in terms of level of multifunctionality, molecular weight and degradation profile. FIG. 1A shows the decrease in molecular weight of hP2-400 and FIG. 1B shows the degradation profile of hP2-400 over a period of 96 hours. FIG. 1C shows the decrease in molecular weight of hP2-1000H and FIG. 1D shows the degradation profile of hP2-1000H over a period of 96 hours. FIG. 1E shows the decrease in molecular weight of hP2-2000H and FIG. 1F shows the degradation profile of hP2-2000H over a period of 96 hours.

Example 21

(79) This example describes the general preparation of NS of P2s.

(80) Due to the multifunctional nature of P2s, large amount of ligand molecules can be attached to the polymer chains resulting in an increase in weight without the increase in chain length. To compensate for the lower level of backbone polymer available in conjugated polymers, additional plain P2s are added equal to the amount of ligand available on the polymer. To prepare NS, 25 mg of P2s or conjugated P2s (with additional plain P2s equal to cumulative ligand weight) were solubilized in 1.25 ml (2% w/v) mixture of CH2Cl2 and Ethyl acetate (based on their solubility; tablet) and emulsified in a 2.5 ml (0.1-2% w/v) solution of a stabilizer like DMAB, PVA, TPGS or a combination of the stabilizers. The emulsion was stirred at 1500 rpm for 5 min followed by homogenization at 15000 rpm for 7 min. The final emulation was diluted with 10 ml water and stirred at 1000 rpm for 3 h. The NS were collected by centrifugation at 15000-20000g at 4 C. for 30 minutes. The pellets of NS were suspended in 0.5 ml 5% (w/v) solution of trehalose dehydrate in water and freeze dried. The emulsification process can be modified based on the physico-chemistry of different drugs to be encapsulated to maximize entrapment efficiency.

Example 22

(81) This example described the preparation of fluorescent P2Ns from hP2-400, hP2-400-GA, hP2-400-Gal and hP2-400-RGD described in examples 3, 6, 7 and 8 with incorporation of hP2-400-fluoroscein described in example 9.

(82) Due to the multifunctional nature of hP2-400, large amount of ligand molecules can be attached to the polymer chains resulting in an increase in weight without the increase in chain length. To compensate for the lower level of backbone polymer available in conjugated hP2-400, additional plain hP2-400 was added equal to the amount of ligand available on the polymer. To prepare NS, 25 mg of hP2-400 or conjugated hP2-400 (with additional plain polymer equal to cumulative ligand weight) were mixed with 1 mg hP2-400-fluorescein and the mixture was solubilized in 1.25 ml (2% w/v) in a 1:4 mixture of CH2Cl2 and ethyl acetate. The organic phase was emulsified in a 2.5 ml (1% or 0.5% w/v) solution of DMAB. The emulsion was stirred at 1500 rpm for 5 min followed by homogenization at 15000 rpm for 7 min. The final emulation was diluted with 10 ml water and stirred at 100 rpm for 3 h. The NS were collected by centrifugation at 15000-20000g at 4 C. for 30 minutes. The pellets of NS were suspended in 0.5 ml 5% (w/v) solution of trehalose dehydrate in water and freeze dried.

Example 23

(83) This example describes the encapsulation of Paclitaxel in NS of P2s.

(84) To prepare paclitaxel loaded NS, 50 mg P2s and paclitaxel (5, 10, 15 or 20% w/w based on loading amount) were solubilized in 5 ml mixture of CH2Cl2 and ethyl acetate (based on their solubility; table 1) and emulsified in a 5 ml (1% w/v) solution of DMAB. The emulsion was stirred at 1500 rpm for 5 min followed by homogenization at 15000 rpm for 5 min. The final emulation was diluted with 20 ml water and stirred at 1000 rpm for 3 h. The NS were collected by centrifugation at 15000g at 4 C. for 30 minutes. The pellets of NS were suspended in 0.5 ml 5% (w/v) solution of trehalose dehydrate in water and freeze dried.

Example 24

(85) This example describes the encapsulation of Insulin in NS of P2s.

(86) Insulin loaded P2s-NS were prepared using a double emulsion technique. 50 mg P2s were dissolved in 3 ml mixture of CH2Cl2 and ethyl acetate (based on their solubility; tablet). Insulin was separately dissolved in 0.5 ml 1.5% (w/v) solution of PVA in 0.01M HCl in an amount equal to 5, 10 or 15% (w/w) of the polymer content based on intended loading. The aqueous phase containing insulin was emulsified in the organic phase containing polymer and stirred at 1500 rpm for 3 min this primary emulsion was then added to 10.5 ml 1.5% (w.v) PVA solution in water to form a secondary emulsion which was further stirred for 3 min at 1500 rpm. The emulsion was homogenized at 15000 rpm for 7 min followed by dilution in 20 ml water. The final suspension was stirred for 3 h at 1000 rpm to allow the organic solvent to diffuse out and evaporate. The NS were collected by centrifugation at 15000g at 4 C. for 30 minutes. The pellets of NS were suspended in 0.5 ml 5% (w/v) solution of trehalose dehydrate in water and freeze dried.

Example 25

(87) This example describes the encapsulation of Cyclosporine A in P2s_NS.

(88) To prepare cyclosporine A loaded NS, 50 mg P2s and cyclosporine A (5, 10 or 15% w/w based on loading amount) were solubilized in 2.5 ml mixture of CH2Cl2 and Ethyl acetate (based on their solubility; table 1) and emulsified in a 5 ml (0.1% w/v) solution of DMAB. The emulsion was stirred at 1500 rpm for 1 min followed by probe sonication at 20% amplitude for 30 s. The final emulation was diluted with 20 ml water and stirred at 1000 rpm for 3 h. The NS were collected by centrifugation at 15000g at 4 C. for 30 minutes. The pellets of NS were suspended in 0.5 ml 5% (w/v) solution of trehalose dehydrate in water and freeze dried.

Example 26

(89) This example describes the uptake of fluorescent P2Ns of hP2-400 described in example 22 in Caco-2 cells monolayers.

(90) Freeze-dried fluorescent P2NS and PLGA NS (plain or conjugated to GA, Gal or RGD) were suspended in dH20 at a concentration of 50 and 250 g/ml respectively. Caco-2 cells were incubated with the 200 l of F-NS suspension for 5 min at 37 C./5% CO2, followed by addition serum free medium and further incubation for 1 h at 37 C./5% CO2. The cells were washed three times with phosphate buffered saline (PBS, pH 7.4) and further processed for fluorescent microscopy and FACS for uptake.

Example 27

(91) This example describes the evaluation of non-competitive binding of fluorescent P2Ns-GA of hP2-400 described in example 22 to transferrin receptor (TfR) in Caco-2 cells monolayers.

(92) The TfR on Caco-2 monolayers were blocked using human specific TfR primary antibody by incubating for 30 min at 37 C. in CO.sub.2 incubator. After three consecutive washings with PBS, P2Ns-GA were added and incubated for further 1 h. These were then washed with PBS 3 times and trypsinized and subjected to FACS analysis.

Example 28

(93) This example describes the uptake of fluorescent P2Ns of hP2-400 described in example 22 in ex vivo intestinal tissue.

(94) To study the uptake of P2Ns in intestinal tissue, 4 m cryosections of previously excised small intestine were treated with fluorescent plain or ligand conjugated P2Ns suspension in PBS. After 1 h incubation at 37 C., the sections were washed multiple times to remove unbound un-internalized NS followed by staining of actin and mounting with mountant containing DAPI.

Example 29

(95) This example describes the evaluation of non-competitive binding of fluorescent P2Ns-GA of hP2-400 described in example 22 to transferrin receptor (TfR) in ex vivo small intestine tissue sections.

(96) The TfR on 4 m cryosections of previously excised small intestine were blocked using human/mouse specific TfR primary antibody by incubating for 30 min at 37 C. in CO.sub.2 incubator. After three consecutive washings with PBS, P2Ns-GA were added and incubated for further 1 h. These were then washed with PBS 3 times and processed for microscopic evaluation.

Example 30

(97) This example describes the kinetics of fluorescent P2Ns of hP2-400 described in example 22 in vivo in Sprague Dawley rats.

(98) Male Sprague Dawley rats (300-320 g) were dosed perorally with fluorescent plain and conjugated P2Ns (16 mg/rat). Blood was collected via heart puncture at terminal sacrifice at 2, 12 and 24 h. Plasma and RBCs were processed for fluorescence quantification. The plasma kinetics shows ligand specific variable absorption, where those with no ligands were absorbed over extended periods of times suggesting they are lodged in the intestine. FIG. 2A shows the mean fluorescent intensity of fluorescent P2Ns of hP2-400 in RBCs. FIG. 2B shows the mean fluorescent intensity of fluorescent P2Ns of hP2-400 in plasma.

(99) TABLE-US-00001 TABLE 1 Solubility of hP2s in mixture of methylene chloride and ethyl acetate Ratio Polymer (methylene chloride:ethyl acetate) hP2-400 1:4 hP2-400LP 1:4 hP2-1000 2:3 hP2-2000 3:2 hP2-1000H 1:4 hP2-2000H 1:4

(100) TABLE-US-00002 TABLE 2 Molecular weighs and composition of different PLA-PEG prepolymers with varying chain lengths of PEG and ratios of lactide or lactic acid to ethylene glycol and the constituent PEG molecules PEG Chain Prepolymer MW Lactide:EG LA:EG Lactide:PEG LA:PEG MWn MWw PDI PLA-PEG400 400 1.59 3.17 14.29 28.57 11000 13000 1.2 PLA- 400 3.17 6.35 28.57 57.14 16000 18200 1.1 PEG400LP PLA-PEG1000 1000 0.65 1.30 14.29 28.57 5300 6100 1.2 PLA-PEG2000 2000 0.32 0.63 14.29 28.57 8600 9500 1.1 PLA- 1000 1.59 3.18 34.98 69.96 16100 18300 1.1 PEG1000H PLA- 2000 1.59 3.18 71.55 143.10 18600 22000 1.2 PEG2000H

(101) TABLE-US-00003 TABLE 3 Range of P2s prepared by chain extending prepolymers from table 1 with their respective molecular weight profiles and mol/mol carboxyl side groups perdiosically placed along the polymer chains. mol/ Prepolymer Polymer Linker MWn MWw PDI mol carboxyls PLA-PEG400 pP2-400 PMDA 15700 22300 1.4 7 hP2-400 HCDA 19200 30900 1.6 9 PLA-PEG400LP pP2-400LP PMDA 27700 44300 1.6 10 hP2-400LP HCDA 24500 37500 1.5 10 PLA-PEG1000 pP2-1000 PMDA 6000 7300 1.2 5 hP2-1000 HCDA 9700 13800 1.4 6 PLA-PEG2000 pP2-2000 PMDA 11000 12700 1.2 8 hP2-2000 HCDA 11700 14400 1.2 6 PLA-PEG1000H pP2-1000H PMDA 18100 22300 1.2 12 hP2-1000H HCDA 19700 26100 1.3 12 PLA-PEG2000H pP2-2000H PMDA 28800 38100 1.3 7 hP2-2000H HCDA 33000 44600 1.4 11 *PMDA: Pyromellitic dianhydride; HCDA: Cyclohexanetetracarboxylic dianhydride

(102) TABLE-US-00004 TABLE 4 Particle size and entrapment efficiency of Paclitaxel in P2s-NS and conventional PLGA (for comparison) 5% 10% 15% 20% Polymer Size % EE Size % EE Size % EE Size % EE PLGA 172 15 51 5 The method failed to produce particles beyond 5% paclitaxel load hP2-400 146 7 39 3 117 8 33 4 145 16 44 3 124 7 44 4 hP2-1000H 114 8 25 5 The method failed to produce particles beyond 5% paclitaxel load hP2-2000H 170 2 33 6 The method failed to produce particles beyond 5% paclitaxel load

(103) TABLE-US-00005 TABLE 5 Particle size and entrapment efficiency of Insulin in P2s-NS and conventional PLGA (for comparison) 5% 10% 15% Polymer Size % EE Size % EE Size % EE PLGA 249 15 47 5 264 9 46 2 262 12 46 6 hP2-400 351 25 37 4 340 23 48 5 276 12 62 4 hP2-2000H 305 56 10 0.2 343 10 12 2 363 10 7 1

(104) TABLE-US-00006 TABLE 6 Particle size and entrapment efficiency of Cyclosporine A in hP2- 2000H 5% 10% 15% Polymer Size % EE Size % EE Size % EE hP2-2000H 208 7 49 2 211 9 49 3 206 3 50 1

(105) While the invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.