Nanoencapsulation of hydrophilic active compounds

Abstract

Provided is a nanoparticle including a water-soluble protein, a glucan and a hydrophilic active agent, the glucan being at least partially cross-linked by a metaphosphate.

Claims

1. A nanoparticle comprising a matrix of albumin and a glucan, the matrix encapsulating a hydrophilic active agent, the glucan being at least partially cross-linked by sodium trimetaphosphate (STMP), wherein the albumin, at least one glucan and at least one hydrophilic active agent are uniformly distributed.

2. The nanoparticle of claim 1, wherein said albumin is selected from human serum albumin (HSA) and bovine serum albumin (BSA).

3. The nanoparticle of claim 1, wherein the glucan is an α-glucan.

4. The nanoparticle of claim 3, wherein said a-glucan is selected from dextran, glycogen, pullulan, starch, lichenin, mannan, galactomannan, arabinoxylan, and galacton.

5. The nanoparticle of claim 1, wherein said glucan has a molecular weight of between about 5 KDa and 2,000 KDa.

6. The nanoparticle of claim 1, wherein said hydrophilic active agent comprises —NH.sub.2 moieties.

7. The nanoparticle of claim 1, wherein said hydrophilic active agent is selected from a vitamin, a protein, an anti-oxidant, a peptide, a polypeptide, a carbohydrate, a hormone, an antibody, a monoclonal antibody, a vaccine, a prophylactic agent, a diagnostic agent, a contrasting agent, a nucleic acid, a nutraceutical agent, a small molecule of a molecular weight of less than about 1,000 Da, an electrolyte, a drug, an immunological agent, and combinations thereof.

8. The nanoparticle of claim 7, wherein said hydrophilic active agent is selected from the group consisting of insulin, exenatide, growth hormone, octreotide acetate, lanreotide acetate, goserelin acetate, copaxone, etanercept, and monoclonal antibodies.

9. The nanoparticle of claim 7, wherein said hydrophilic active agent is selected from insulin and exenatide.

10. The nanoparticle of claim 1, having a diameter of at most 500 nm.

11. The nanoparticle of claim 10, having a diameter between about 50 nm and 250 nm.

12. The nanoparticle of claim 1, being a nanosphere or a nanocapsule.

13. A carrier comprising a hydrophobic polymer and a plurality of nanoparticles as claimed in claim 1, the plurality of nanoparticles being (i) encapsulated by said hydrophobic polymer or (ii) embedded in a matrix formed of said hydrophobic polymer.

14. The carrier of claim 13, wherein said hydrophobic polymer is selected from poly(lactic glycolic) acid (PLGA), polymethyl-methacrylate (PMMA), hydroxypropyl methylcellulose (HPMC), poly(lactic acid) (PLA), poly(lacto-co-glycolide) (PLG), poly(lactide), polyglycolic acid (PGA), and poly(hydroxybutyrate).

15. The carrier of claim 13, wherein the nanoparticles are encapsulated in microcapsules or embedded in microparticles, the microcapsules or microparticles having a diameter of between 1 and 30 microns.

16. A pharmaceutical composition comprising the nanoparticle of claim 1 and further comprising a carrier.

17. The pharmaceutical composition of claim 16, being adapted for topical, oral, inhalation, nasal, transdermal, ocular or parenteral administration of said hydrophilic active agent.

18. The delivery system of claim 13, further encapsulated within a biodegradable capsule, wherein said biodegradable capsule is optionally in the form of an entero-coated capsule.

19. A process for the preparation of a nanoparticle comprising albumin, a glucan and a hydrophilic active agent, the glucan being at least partially cross-linked by sodium trimetaphosphate (STMP), the process comprising: mixing a first aqueous solution comprising albumin and said glucan with a second aqueous solution comprising said hydrophilic active agent to form a mixture; delivering an organic solvent into said mixture; and adding sodium trimetaphosphate to said mixture to thereby at least partially cross-link said glucan for obtaining said nanoparticle.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

(2) FIG. 1A is a schematic description of a cross-linking mechanism of dextran by STMP; FIG. 1B is a schematic depiction of a cross-linked particle.

(3) FIG. 2 shows the cross-linking reaction of albumin, rendering the albumin insoluble in aqueous solutions.

(4) FIG. 3 shows body weight results for different formulation of Exenatide (FI-V) and Byetta.

(5) FIG. 4 shows the blood glucose levels from preliminary experiments with Byetta.

(6) FIG. 5 shows the blood glucose levels from different formulation of Exenatide (FI-V) and Byetta.

(7) FIG. 6 shows the plasma insulin levels of the different groups at different days.

(8) FIG. 7A shows the glycosylate hemoglobin levels of the different groups at different days; FIG. 7B shows the total mean of glycosylate hemoglobin levels in 10 days treatment.

(9) FIGS. 8A-8B present Cryo-TEM images of (FIG. 8A) DX-50 NPs and (FIG. 8B) DX-150 NP formulations.

(10) FIGS. 9A-9C show XHR-SEM images of (FIG. 9A) Glut-1 MPs, (FIG. 9B) DX-50 MPs and (FIG. 9C) DX-150 MPs formulations.

(11) FIG. 10 shows the pharmacokinetic profile of exenatide in rats following subcutaneous injection of Byetta™ and exenatide solution at a dose of 20 μg/rat (56 μg/kg) and oral administration of various formulations at a dose of 50 μg/rat (165 and 65 μg/kg for DEX50 and DEX150 respectively).

(12) FIGS. 11A-11C are freeze-fractured SEM images of double encapsulated BSA/insulin NPs following a cryo-protection process.

(13) FIG. 12 shows the blood glucose levels following subcutaneous administration of the various insulin loaded nanoparticles formulations in fasting conditions (N=3).

(14) FIG. 13 shows blood glucose levels following subcutaneous administration of h-insulin loaded nanoparticles formulation in non-fasting conditions (N=3).

DETAILED DESCRIPTION OF EMBODIMENTS

(15) Exenatide is a synthetic version of exendin-4, a hormone found in the saliva of the Gila monster that displays biological properties similar to human glucagon-like peptide-1 (GLP-1), a regulator of glucose metabolism and insulin secretion. The incretin hormones, GLP-1 and glucosedependent insulinotropic peptide (GIP), are produced by the L and K endocrine cells of the intestine following ingestion of food. GLP-1 and GIP stimulate insulin secretion from the beta cells of the islets of Langerhans in the pancreas. Although only GLP-1 causes insulin secretion in the diabetic state, it is ineffective as a clinical treatment for diabetes as it has a very short half-life (a few minutes) in vivo.

(16) Exenatide is a 39-amino-acid peptide, an insulin secretagogue, with glucoregulatory effects. The medication is injected subcutaneously twice a day using a filled pen device. Exenatide bears a 50% amino acid homology to GLP-1, is structurally analogous to GLP-1, and has a longer half-life (2.4 h) in vivo. Thus, it was tested for its ability to stimulate insulin secretion and lower blood glucose in mammals, and was found to be effective in the diabetic state. In studies on rodents, it has also been shown to increase the number of beta cells in the pancreas.

(17) Exenatide raises insulin levels quickly (within about ten minutes of administration) with the insulin levels subsiding substantially over the next hour or two. Exenatide has been approved as an adjunctive therapy for patients with type 2 diabetes failing to achieve glycemic control with oral antidiabetic agents. A dose taken after meals has a much smaller effect on blood sugar than one taken beforehand. The effects on blood sugar diminish after six to eight hours. The medicine is available in two doses: 5 μg and 10 μg. Treatment often begins with the 5 μg dosage, which is increased if adverse effects are insignificant. According to the manufacturer, the autoinjector must be stored in a refrigerator between 2 and 8° C. before first use, and then at a temperature between 2 and 25° C. In hot weather, therefore, it should be continuously refrigerated. It should be emphasized that a potential disadvantage in exenatide clinical applications is the frequent subcutaneous (SC) injections required. SC injections can cause pain, side effects and possible infections at the sites of injection that could adversely affect patient compliance.

(18) A long-acting release form of exenatide has been developed for use as a once-weekly injection. This sustained-release formulation consists of injectable microspheres of exenatide and poly (D,L lactic-co-glycolic acid), a common biodegradable polymer with established use in absorbable sutures and extended-release pharmaceuticals, that allows gradual drug delivery at a controlled rate. Thus, exenatide extended release is a useful option for the treatment of type 2 diabetes, particularly in patients where bodyweight loss is an essential aspect of the individual patient's management. However, it is still an injection and need to be injected once weekly.

(19) Exenatide and insulin are hydrophilic biomacromolecules which exhibit low oral human bioavailability (estimated at less than 2%) following extrapolation from data regenerated in animals, which has been attributed to proteolytic instability and limited ability to permeate through biological membranes.

(20) In the present invention, encapsulation of hydrophilic macromolecules is demonstrated for exenatide and insulin. The first objective is entrapment of exenatide in the matrix of the nanoparticles at a reasonable level, with an aim at increasing the loading of the nanoparticles in the microparticles for oral administration, in order to ensure that the drug content in the final powdered formulation is the highest possible.

(21) The present invention further provides the incorporation of peptidic drugs into primary nanocapsules or nanoparticles that are further embedded in larger nanocapsules, resulting in the formation of double-coated nanoparticulate delivery systems that are designed to protect the peptide from the detrimental effects of the external environmental for prolonged release using parenteral route of administration. Peptide loaded primary nanocapsules are encapsulated within larger secondary nanocapsules. It should be noted that in a nanocapsule having a diameter of 400 or 600 nm, it is theoretically possible to incorporate at least 64 and 216 nanocapsules of 100 nm diameter respectively based on volume calculations.

(22) The secondary microparticles, i.e. carriers encapsulating the primary nanoparticles of the invention, were obtained by a spray drying technique. Spray drying is a process that converts liquids or suspensions into dry powders at a continuous single step process. Spray drying was carried out by using Buchi laboratory scale spray dryers that can generate microparticles in the size range of 1000 nm to 20 μm for small samples quantities (few milligrams or milliliters) at high yields (>70%), thereby forming microparticles at a relatively high yield. The secondary microparticles generally have a size (diameter) of between 1 and 30 microns.

(23) Materials

(24) Bovine serum albumin (BSA) and Dextran 12 KDa were purchased from Sigma-Aldrich (Rehovot, Israel). Exenatide was kindly donated by Teva Pharmaceuticals (Jerusalem, Israel). Glutaraldehyde 8% in water was purchased from Sigma-Aldrich (Rehovot, Israel). Sodium trimetaphosphate (STMP) was purchased from Alfa Aesar (Haverther chemicals and hill, MA, USA). Poly(methacrylic acid, Ethyl acrylate 1:1 (Eudragit® L100-55) was obtained from Rohm (Dramstadt. GmbH, Germany). Hydroxypropylmethylcellulose (Methocel E4M Premium) was purchased from Dow Chemical Company (Midland, Mich., USA). Sodium phosphate monobasic, monohydrate was purchased from Mallinckrodt chemicals (Phillipsburg, N.J., USA). All organic solvents were HPLC grade and purchased from J.T. Baker (Deventer, Holland).

(25) Preparation of Primary NPs

(26) The first line of protection on the sensitive biomacromolecule, exenatide, was achieved by loading the peptide into primary BSA NPs. Two different types of NPs were prepared: BSA NPs cross-linked with glutaraldehyde 8% and BSA combined with dextran 12 KDa NPs cross-linked with STMP.

(27) BSA NPs Cross-Linked with Glutaraldehyde

(28) The BSA NPs cross-linked with glutaraldehyde, were prepared by an established desolvation method as previously described by Weber et. al [ref-Desolvation process and surface characterisation of protein nanoparticles]. 200 mg of BSA and 4 or 8 mg of exenatide were dissolved in 20 ml of bi-distilled water (DDW). After 0.5 hour, the pH of the solution was adjusted to 8.5 by 0.1M NaOH. Then, 40 ml of acetone were slowly added to the aqueous phase. An o/w emulsion was formed as evidenced by the rapid formation of opalescence in the dispersion medium. BSA NPs were then cross-linked using 12.5 μl of glutaraldehyde 8% solution over 2 hours. Following cross-linking reaction completion, the acetone was evaporated under laminar air flow. This formulation was denominated Glut-1.

(29) BSA/Dextran NPs Cross-Linked with STMP

(30) The BSA/dextran NPs were similarly prepared by dissolving in 20 ml DDW, the following compounds: 200 mg of BSA, 50 mg of dextran 12 KDa and 4 or 8 mg of exenatide when needed. After 0.5 hour, the pH of the solution was adjusted to 8.5 by 0.1M NaOH to make sure that the adjacent hydroxyl groups on dextran are available for the reaction with the STMP cross-linker. Then, 20 ml of acetone were slowly added to the aqueous phase. BSA/dextran NPs were then cross linked using 50 mg of STMP over 3 hours and acetone was evaporated as described above. Preliminary formulations were prepared and evaluated by varying the process parameters. Two formulations that differ in the dextran amount were selected for further animal studies: 50 and 150 mg. The formulation with 50 mg was denominated as DX-50- and 150 mg as DX-150.

(31) Microspheres (MPs) Preparation

(32) The microspheres (MPs) were formed by microencapsulating the exenatide containing NPs using the spray drying technique. For the purpose of microencapsulation, 100 ml of NaH.sub.2PO.sub.4 buffer was prepared. pH of the buffer was adjusted to 6.5 by 1M NaOH solution. An amount of 750 mg of Eudragit was dissolved in that solution maintaining pH at 6.5. In addition, 1% w/v hydroxypropylmethylcellulose (HPMC) solution was prepared by adding 1000 mg of HPMC to 100 ml of pre-heated (˜80° C.) DDW. Then, the Eudragit solution was added via funnel with a gaza band (to filter Eudragit particles that might have not dissolved) to the HPMC solution.

(33) Once the acetone was evaporated from the NPs suspension, the combined solution of the microparticle polymers was added to the NPs suspension. The suspension was then spray-dried with a Buchi mini spray-drier B-190 apparatus (Flawil, Switzerland) under the following conditions: inlet temperature 160° C.; outlet temperature 85° C.; aspiration 100%; feeding rate of the suspension was 7 ml/min; the powder was collected in the cyclone separator and the outlet yield was calculated.

(34) Physicochemical characterization of the NPs and subsequent MPs

(35) NPs Characterization

(36) The mean diameter and zeta potential of the various NPs were characterized using Malvern's Zetasizer (Nano series, Nanos-ZS, UK) at 25° C. and using water as diluent. Morphological evaluation was performed using cryo-transmission electron microscopy (Cryo-TEM). In the Cryo-TEM method, a drop of the solution is placed on a carbon-coated holey polymer film supported on a 300-mesh Cu grid (Ted Pella Ltd., Redding, Calif., USA), and the specimen is automatically vitrified using Vitrobot (FEI) by means of a fast quench in liquid ethane to −170° C. The samples were studied using an FEI Tecnai 12 G2 TEM, at 120 kV with a Gatan cryo-holder maintained at −180° C., and images were recorded on a slow scan cooled charge-coupled device camera.

(37) Extra High Resolution Scanning Electronic Microscopy (SEM) Studies of MPs

(38) Morphological and size evaluation of spray dried MPs were carried out using Extra High Resolution Scanning Electron Microscopy (model: Magellan 400 L, FEI, Germany). The samples were fixed on a SEM-stub using double-sided adhesive tape and then made electrically conductive following standard coating by gold spattering (Pilaron E5100) procedure under vacuum.

(39) Drug Content

(40) The total amount of exenatide in the powder was analyzed by dissolving the sample in 2 ml of water overnight. Afterwards, the mixture was centrifuged at 14000 rpm for 2 min 1 ml from the supernatant was injected into HPLC under the following conditions: Column Restek Viva C4 (5 μm), 250/4.6 mm. Column temperature was kept at 45° C. Mobile phase A was acetonitrile (ACN), and mobile phase B was potassium dihydrogen phosphate (KH.sub.2PO.sub.4, 20 mmol/L) adjusted to pH 2.5 by phosphoric acid. The KH.sub.2PO.sub.4 buffer was filtered through a 0.2 μm membrane filter prior to use. The following gradient conditions were used for exenatide: from 30% to 45% mobile phase A in 15 min, and re-equilibrated back to 30% mobile phase A for 3 min. Flow rate was 1.5 mL/min. Injection volume was 20 μL. UV signal was detected at 215 nm. The exenatide content was calculated using a calibration curve constructed from exenatide concentrations ranging between 0 to 20 μg/ml that yielded a linear correlation (r.sup.2=0.999).

(41) Pharmacokinetic Studies in Rats

(42) All the animal studies were approved by the local Ethical Committee of Laboratory Animal Care at The Hebrew University of Jerusalem (MD-13575-4). Sprague Dawley male rats (300-350 g) were used in this study. The animals were housed in SPF conditions, fasted and had free access to drinking water. Seven groups of 3 rats were randomly divided to evaluate the oral absorption and exenatide plasma levels over time. Exenatide was injected subcutaneously as a solution or formulated in Byetta® at a dose of 65 μg/kg (20 μg/rat). The third and fourth groups of rats were orally administrated with either 31 mg of blank MPs spiked externally with exenatide or exenatide solution at a dose of 165 μg/kg (50 μg/rat) to determine whether the blank formulation has an effect. Finally, 35, 33 and 32 mg of Glut-1, DX-50 and DX-150, respectively, were orally administrated at a dose of 165 μg/kg (50 μg/rat). All oral suspensions were dispersed in 2 mL DDW, while the volume of subcutaneous injection was 200 μl.

(43) Blood samples (500 μl) were taken from the rat tail at 0, 0.5, 1, 2, 4, 6, 8 and 24 h. The blood samples were collected in EDTA and aprotinin containing tubes. The samples were centrifuged at 10,000 rpm, 4° C. for 10 min, after which 250 μl of plasma samples were transferred to new tubes and stored at −80° C. until analyzed. Exenatide levels were determined using CEK-0130-01 ELISA Kit (AB Biolabs, USA) following the protocol suggested by the company.

(44) Bioavailability Calculations

(45) The pharmacokinetic parameters were calculated using WinNonlin software, applying the trapezoid rule for calculation of AUC. The AUC values were adjusted following size dose corrections.

(46) The relative bioavailability of the different oral formulations compared to the standard marketed formulation Byetta® injected subcutaneously was calculated using the following equation:

(47) $\mathrm{Relative}\mathrm{bioavailability}=\frac{\left[\mathrm{AUC}\mathrm{oral}\right]}{\left[\mathrm{AUC}\mathrm{sc}\right]}*100$
Exenatide Formulations
Formulation F-1: Exenatide Primary Nanoencapsulation with BSA and Cross-Linked Dextran

(48) 200 mg BSA (Sigma-A7906) and 50 mg of Dextran 12 KDa (Sigma-31418) were dissolved in 10 ml DDW. 4 mg of Exenatide were separately dissolved in 10 ml DDW. Albumin/Dextran solution was added to the Exenatide solution to complete peptide dissolution (solution A). The pH 6.8 was adjusted with NaOH 0.1M to reach pH of ˜8.5. 20 ml acetone were injected to solution-A during strong stirring to elicit formation of BSA-Dextran NPs comprising most of Exenatide. Dextran cross-linking was obtained by addition of 5% (1 ml) of sodium trimetaphosphate (STMP) and the solution was agitated at 900 rpm at room temperature over 3 hours. A schematic representation of the synthesis of formulation F-1 is shown in FIGS. 1A-1B.

(49) Formulation F-2: Exenatide Primary Nanoencapsulation with BSA and Crosslinked Glutaraldehyde

(50) 200 mg BSA (Sigma-A7906) were dissolved in 10 ml DDW. 4 mg of Exenatide were separately dissolved in 10 ml DDW. Albumin solution was added to the Exenatide solution to complete peptide dissolution. pH was adjusted with NaOH 0.1M to reach pH between of 8.5. 15 ml acetone were injected to solution-A during strong vortex to elicit formation of BSA-Exenatide NPs comprising most of Exenatide. BSA cross-linking was obtained by addition of 25 μl glutaraldehyde 4% and the solution was agitated at 900 rpm at room temperature over 3 hours. A schematic representation of cross linking of formulation F-2 is shown in FIG. 2.

(51) Formulation F-3: Conjugation of Exenatide to Nanoparticles Surface

(52) Exenatide activation was carried out by reacting 4 mg of Exenatide with the spacer sulfo-SMCC (Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) for 2 hours at room temperature.

(53) For preparing the nanoparticles, 150 mg PLGA (50K) (Resomer RG 504H, Boehringen Ingelheim), 150 mg PLGA-co-PEG (45K and 5K) (Resomer RGP d 50105) and 10 mg oleyl cysteinamide were dissolved in 50 ml acetone. 100 mg Solutol HS 15 (BASF) was dissolved in 100 ml DDW while stirring. The organic phase was added to the aqueous phase under stirring (900 rpm) and allowed to mix over 15 min. The formulation was evaporated to less that 10 ml on 37° C. using Rotor evaporator under reduced pressure. pH was adjusted to 6.7-6.8 using NaOH 0.1M and volume was adjusted to 10 ml. The formulation was centrifuged at 4000 rpm to sediment large particles (3-4%). Only the colloidal supernatant was used for final formulation.

(54) The activated exenatide was incubated immediately with the preformed formulation. Incubation was performed at room temperature, overnight on magnetic stirrer. The maleimide groups of the LC-SMCC reacted with the sulfhydryl groups of the oleyl cysteinamide at pH=6.5-7.5 to form stable thioether bonds.

(55) Formulation F-4: Exenatide Double Encapsulation

(56) 100 mg BSA (Sigma-A7906) were dissolved in 2 ml DDW. 5 mg of Exenatide were separately dissolved in 3 ml DDW. Albumin solution was added to the Exenatide solution to complete peptide dissolution. pH was adjusted with NaOH 1M to 7.4-8. 10 ml acetone was injected into the solution during strong stirring to elicit formation of BSA nanoparticles comprising most of Exenatide.

(57) 128 mg PLGA (50K) was dissolved in 93 ml acetonitrile. 6 ml of BSA nanoparticles formulation was added during stirring to form double encapsulation (nanospray conditions: 4 μm mesh, in temperature: 50° C.).

(58) Microencapsulation of Formulations F1-F4 with Eudragit L and HPMC

(59) 500 mg of HPMC were dissolved in 100 ml preheated DDW, and 500 mg of Eudragit (anionic) were dissolved in 100 ml PBS.

(60) To the nanoparticles solution, Eudragit solution and HPMC solution were added. The combined solutions were stirred for 30 minutes at 500 rpm in room temperature; the combined dispersion was evaporated by spray drier at the following conditions: inlet Temp=160° C.; Outlet Temp=100° C. Microparticles comprising drug-loaded nanoparticles were obtained.

(61) The physicochemical characterization of the drug-loaded nanoparticles is shown in Table 1.

(62) TABLE-US-00001 TABLE 1 Physicochemical properties of Exenatide formulations. % Size Zeta Yield Formulation NPs formation method (nm) potential PDI (dry) EXA-12 BSA:Dextran 173.4 −38.4 0.565 45.9 (cross-linked) F-1* BSA:Dextran 83 −28.3 0.51 25 (cross-linked) 359 −33.5 0.306 44 F-2 BSA (cross-linked 132.5 −45 0.1 45 with glutaraldehyde) F-3 Peptide conjugated to 142.8 −45 0.026 40 PLGA (50K NPs) F-4** Double encapsulation 163.1 −48.6 0.093 43 88.29 −54.4 0.048 63 *2 batches were united. **for the unified formulation
Evaluation of Glucose Lowering Effect of Exenatide in Various Formulations (F1-F5) on OB/OB Mice

(63) Acclimation of the mice for few days was carried out by giving glucose and monitored glucose levels over 6 hours. Afterwards, all mice were injected with Byetta (commercial exenatide injectible formulation) at a dose of 20 μg/kg and monitoring blood glucose levels over 6 hours.

(64) Evaluation of the glucose lowering effect of Exenatide in various formulations on OB/OB mice was carried out for a group size of n=8 per group of animals. Each group of OB/OB mice was provided with one of the following:

(65) i) Saline+empty vehicle (F-5)

(66) ii) Byetta-injection

(67) iii) Exenatide (F-1)

(68) iv) Exenatide (F-2)

(69) v) Exenatide (F-3)

(70) vi) Exenatide (F-4)

(71) On 1st day, all animals were fasted 18 hours prior to the experiments then after fasting, blood glucose was monitored. Glucose was injected (18 mm/kg) by i.p. to all animals; 60 min after glucose injection the following parameters were examined: blood glucose measurement, blood collection for Elisa, body weight measurement. Then, Formulations F1-F5 were orally administered (Byetta was administered by s.c.). After administration, blood glucose levels were monitored at 30 min, 1 hr, 1.5 hr, 2 hr, 3 hr and 6 hr from administration.

(72) From 2nd day to 9th day, body weight was measured at time zero and blood was collected for ELISA. This was followed by administration of the formulations, and measuring blood glucose levels within 1 hr from drug administration (as well as blood collection for ELISA).

(73) On 10th day, prior to administration the last doses of the tested formulations, glucose was given again followed by body weight measurement and blood collection of for ELISA. After this, the formulations were administered in double dose (to check the potentiation of these formulations) to the respective animals and then after 1 hr of drug administration, blood glucose levels were measured and blood was collected for ELISA.

(74) Body Weight Measurements

(75) Body weight measurements were carried out at the same time for each mouse. The results of the different formulation of Exenatide (F1-F5) and Byetta are shown in FIG. 3.

(76) Blood Glucose Levels Measurements

(77) Blood samples were collected on day 1 to 10. Animals at 15-19 weeks of age were fasted overnight (up to 16 h) prior to blood glucose measurement procedures, by transferring mice to a clean cage base with clean nesting material and a small amount of soiled bedding and environmental enrichment from their old cage. The change of cage and bedding obviated the possibility that mice may access spilled food. Water remained freely available throughout the entire fasting period. Food was returned following collection of the final blood sample. A drop of blood was obtained from unrestrained mice by nicking the tail tip with a blade. Measurements were taken using a handheld blood glucose meter (Accu-chek Aviva, Roche Diagnostics, UK). The blood glucose levels from preliminary experiments with Byetta are shown in FIG. 4, while the blood glucose levels for different formulation of Exenatide (F1-F5) and Byetta are shown in FIG. 5.

(78) Plasma Insulin Levels

(79) For insulin determination, 150 mL of blood was sampled from the tail vein (blood was processed in a centrifuge at 3000 cycles/min for 10 and 5 min). Blood plasma was separated into two heparin-coated tubes for blood parameters and insulin measurements (30 mL each). Plasma insulin levels were measured using a mouse insulin enzyme-linked immunosorbent assay kit (Mercodia, Sylveniusgatan, Sweden). The plasma insulin levels of the different groups at different days are shown in FIG. 6.

(80) Glycosylate Hemoglobin Levels

(81) Collect plasma using EDTA as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at −20° C. or −80° C. Glycosylate hemoglobin levels were measured using a mouse insulin enzyme-linked immunosorbent assay kit (Life Science, Inc, Florida, USA). The glycosylate hemoglobin levels of the different groups at different days are shown in FIG. 7.

(82) Physicochemical Characterization of the NPs and Subsequent MPs

(83) Bovine serum albumin (BSA) is a well-known and abundant protein carrier for oral drug delivery (except peptides and proteins). Its major advantages are biodegradability, biocompatibility, safety, non-antigenicity, well tolerability and availability. Furthermore, incorporation of peptides and proteins in primary NPs is challenging as most of the coating polymers are soluble in water and need to be cross-linked to elicit in-vitro prolonged release of the peptides under sink conditions. In the case of BSA NPs, widely accepted as nanocarriers, the issue is even more complicated. Any denaturation process of albumin, including cross linking with glutaraldehyde, denaturation by heat or use of organic solvents will obviously affect the chemical integrity of the peptide or protein as observed also in the present work.

(84) To avoid such a drawback, the BSA matrix was combined with the polysaccharide dextran which can be cross-linked via its reactive hydroxyl groups as shown in FIG. 8 [**Polysaccharides as building blocks for nanotherapeutics]. To achieve a stable nanoparticulate system, the reactive hydroxyl groups of dextran were cross-linked using TSMP, under suitable pH conditions.

(85) Various formulations were prepared by varying different parameters, such as pH, type of cross-linking molecule, TSMP amount, dextran amount. The BSA NPs containing exenatide prepared with glutatraldehyde as cross-linker exhibited a mean diameter size of 59.34±0.32 nm based on triplicate measurement with a poly dispersity index (PDI) value of 0.138 reflecting a narrow size range and a zeta potential value of −50.3±3.03 mV. The properties of the NPs formulations composed of BSA:dextran blend are presented in Table 2. Based on the data depicted in Table 2, two formulations differing in dextran amount: 50 mg and 150 mg were selected as previously mentioned. The mean diameter of the NPs, irrespective of the formulation, ranged from 190 to 210 nm, with a relative narrow distribution range as reflected by the relative low PDI values observed.

(86) TABLE-US-00002 TABLE 2 Composition and properties of the different NPs formulations prepared from a blend of BSA and Dextran Dextran 12 KDa Mean Zeta amount TSMP diameter Potential (mg) pH (mg) (nm) PDI (mV) 50 8.5 50 192.7 ± 3.5 0.370 −39.5 50 10 50  91.4 ± 1.3 0.429 −45.7 50 12 50  38.6 ± 5.5 0.699 −44.7 50 8.5 5 NA NA NA 50 8.5 75 504.9 ± 1.8 0.399 −38.9 50 8.5 100 NA NA NA 100 8.5 50 337 ± 6 0.316 −41.3 150 8.5 50 355.4 ± 6.4 0.259 −40.5 200 8.5 50  691.4 ± 17.3 0.364 −42.65 50 8.5 50 189.9 ± 2.4 0.335 −40.5 5 8.5 50 NA NA NA 5 8.5 50 NA NA NA 5 8.5 50 NA NA NA 5 8.5 50 NA NA NA

(87) Visualization of the primary NPs composed of the BSA/dextran blend was carried out using Cryo TEM (FIG. 1A-B). The images show a spherical morphology of the NPs regardless the difference in composition with a diameter size similar in range value to the range observed with Zetasizer measurements.

(88) The various NP-loaded MPs characterization was mainly visualized by XHR SEM analysis (FIG. 9A, B, C). The microencapsulated NPs, showed that the coating of the MPs is smooth, ranging qualitatively in size from 1 to 15 μm and the MPs are deflated as a result of the vacuum applied for SEM visualization.

(89) The final exenatide content in Glut-1, DX-50 and DX-150 was 0.147, 0.153 and 0.158% w/w respectively, leading to an encapsulation yield efficiency of approximately 40% irrespective of the formulation.

(90) Rats Pharmacokinetics and Absorption Studies

(91) Exenatide plasma levels from 3 rats, for each treatment are presented in FIG. 10. At 8 and 24 hours, exenatide plasma concentrations were below the detection limit of the kit irrespective of the formulation, hence data are not shown. Furthermore, it can noted from the results presented in FIG. 10 that the blank MPs formulation (prepared with DX-50 NPs with no exenatide) spiked with exenatide solution, the Glut-1 formulation and the free exenatide solution administered orally did not elicit any detectable exenatide plasma level. It was also observed that the actual exenatide elicited a plasma profile close to the profile of the commercial Byetta™ product whereas the formulation DX-50 elicited a higher pharmacokinetic profile than DX-150 but both were administered at much higher dose that the injectable preparations (165 μg/kg versus 65 μg/kg respectively).

(92) Indeed, following calculations of the pharmacokinetic parameters it can clearly be noted following normalization of the dose that the highest AUC value was elicited by the Byetta injection, followed by the exenatide injected solution, the DX-50 and DX-150 formulation. Furthermore, irrespective of the dose all the formulations elicited C.sub.max values higher than 1,000 ng/ml and no difference in the T.sub.max values. More importantly, ANOVA analysis pointed out that there is no significant difference between the normalized AUC values between Byeatt and Exenatide solution and the DEX-50 oral suspension. The only significant difference was with DEX10-50 which elicited a relative oral bioavailability of 46.5%.

(93) TABLE-US-00003 TABLE 3 Average PK parameter values (mean ± SE) following S.C injection of Byetta and exenatide solution (Exe. Sol.) at a dose of 65 μg/kg or oral administration of DX-50 and DX-150 at a dose of 165 μg/kg, N = 3 T ½ T max C max CL AUC all F relative Formulation (h) (h) (ng/mL) (mL/h/kg) (h*ng/mL) AUC/D (%) Byetta SC 0.76 ± 0.09 1.33 ± 0.33 1216.56 ± 5.91  18.70 ± 2.12 3101.11 ± 369.67 0.16 ± 0.02 Exe. Sol. SC 0.21 ± 0.00 0.83 ± 0.17 1057.29 ± 146.36 23.43 ± 4.48 2637.10 ± 544.43 0.13 ± 0.03  85.04 ± 17.56 DX-50 PO 1.64 ± 0.13 1.33 ± 0.33 2089.53 ± 324.45 27.25 ± 1.72 5944.55 ± 394.88 0.12 ± 0.01 76.68 ± 5.10 DX-150 PO 0.23 ± 0.00 1.00 ± 0.00 1800.65 ± 222.27 48.50 ± 6.90 3599.25 ± 575.02 0.07 ± 0.01 46.43 ± 7.42
Observations

(94) All the procedures related to animal handling, care, and the treatment in this study were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

(95) All values in the figures and text are expressed as mean standard error (s.e.m.) of the mean of n observations. For the in vivo studies, n represents the number of animals studied. In the experiments involving histology the figures shown are representative at least three experiments (histological coloration) performed on different experimental days on the tissues section collected from all the animals in each group. Data sets were examined by one- or two-way analysis of variance, and individual group means were then compared with Student's unpaired t test. A p-value less than 0.05 was considered significant.

(96) Discussion

(97) The therapeutic efficacy and safety of different formulations of Exenatide (F1-F5) was evaluated in OB/OB mice. In particular, there were no significant differences in body weight after administration of Exenatide (F1-F5) and Byetta (FIG. 3).

(98) In the preliminary experiments with Byetta, after 1 h of glucose injection, mice were administered with Byetta, as shown in FIG. 4; a reduction in blood glucose levels is demonstrated until 6 h. This result supports the idea that Byetta represent a goal standard in the treatment of diabetes. In addition, different formulations of Exenatide (F1-F4) were compared with Byetta treatment. It is of note that the Exenatide formulations (F1-F4) were orally administered, while the Byetta composition was administered by injection. The results showed that, daily oral administration of F-1 and F-2 are able to reduce the increase of blood glucose levels in OB/OB mice (FIG. 5). On this basis, it is speculated that these two formulations show the most interesting results.

(99) The data in FIG. 6 demonstrates the plasma insulin levels of the different groups at different days. As can be observed, the treatment with the different formulations of Exenatide (F1-F5) is able to increase the insulin levels as well as treatment with Byetta.

(100) Moreover, during the 10 days treatment, it was found that Byetta injection reduces the levels of glycosylated hemoglobin (HbA1c) in OB/OB mice (FIGS. 7-11). The rate of glycosylated hemoglobin clarified, as compared to FIG. 5, shows that only two formulations of Exenatide F-1 and F-2 show an effect in the reduction of glycosylated hemoglobin levels.

(101) From this it was concluded that daily oral administration of Exenatide F-1 (and to a much lesser extent oral administration of F-2, because the peptide is cross-linked by glutaraldehyde and loss part of its activity), may have a beneficial effect in the symptomatic treatment of diabetes.

(102) Double Nanoencapsulation of h-Insulin

(103) Double nanoencapsulated samples comprising h-insulin were prepared for obtaining an injectable dry powder, for eliciting prolonged release of the peptide in vivo.

(104) h-Insulin Primary Nanoencapsulation with HSA:

(105) 200 mg of human serum albumin (HSA) were dissolved in 5 ml of DDW under stirring. Separately, 20 mg of human or bovine insulin were dissolved in 5 ml of DDW and vortexed for 30 seconds. The h-insulin solution was then added to the HSA solution and stirred for 30 minutes to complete peptide dissolution. The pH of the resulted solution was adjusted to 7.4-8 with NaOH 0.1M. Then, 20 ml of acetone were injected quickly (within 20 seconds) under vigorous stirring (900 rpm) to elicit formation of HSA nanocapsules load with h-insulin (solution A). Solution A was covered with aluminum foil to avoid acetone evaporation and was stirred over 1 hour at 900 rpm. Samples (50-100 μl) were withdrawn following one hour stirring for Zeta potential and size measurements. Separately, a solution of PLGA 100K (50:50) was dissolved in 80 ml of acetonitrile and added to solution A.

(106) Nanoencapsulation of HSA/h-Insulin Nanoparticles into by PLGA Using Nanospray Dryer—Organic Mode

(107) Nanocapsules were prepared via spray drying on the NSD B-90 operating at ‘closed loop’ mode, hence, N.sub.2 (g) and CO.sub.2 (g) were flowed in the system instead of air. In all experiments, gas flow was about 120 l/min. The air was soaked with volatile vapors and humidity transferred to a Dehumidifier unit for drying and condensation, then was returned dry to the system in a circular path. Spray drying was carried out at low temperatures (T.sub.m=30°-60° C.) with mesh size membrane 4 μm. Various formulations of different HSA/h-insulin/PLGA ratios were prepared. Unlike conventional spray dryers that operate on turbulent flow, the NSD B-90 operates on a laminar flow; hence gentle heating is achievable, thus making the system compatible for heat-sensitive biopharmaceutical products.

(108) Physicochemical Characterization of Drug-Loaded Primary Nanocapsules

(109) Insulin Content within the Primary Nanocapsules

(110) An appropriate analytical method by HPLC was developed at the following conditions:

(111) A c4 column was used for separation and analyzing h-insulin (RESTEK viva 4.6 mm×250 mm, i.d., 5 μm particles, Bellefonte, Pa. USA). Column temperature was kept at 45° C. Mobile phase A was acetonitrile (ACN), and mobile phase B was potassium di-hydrogen phosphate (KH.sub.2PO.sub.4, 20 mmol/L) adjusted to pH 2.5 with phosphoric acid. The mobile phase was filtered through a 0.45 μm membrane filter and degassed via vacuum prior to use.

(112) The following gradient conditions were used for h-insulin: from 30% to 45% mobile phase A in 15 min, and re-equilibrated back to 30% mobile phase A for 3 min. Flow rate was 1.5 mL/min. Injection volume was 20 UV signal was detected at 215 nm.

(113) TABLE-US-00004 TABLE 4 h-insulin content in various formulations Encap- sulation Content efficiency (Insulin/mg Formulation Nanoparticles method (%) formulation) DEHI-002 PLGA 100K (3X), 69 15.01 insulin (3X) nanoencapsulation DEHI-003 PLGA 100K (3X), insulin (1X) 145 12.36 nanoencapsulation DEHI-004 PLGA 100K (1X), 25 13.09 insulin (1X) nanoencapsulation

(114) TABLE-US-00005 TABLE 5 The zeta potential and particle size of the primary nanocapsules of various formulations Zeta Size potential Formulation Nanoparticles method (nm) PDI (mV) DEHI-002 PLGA 100K (3X), 151.1 0.17 −59.6 insulin (3X) double nanoencapsulation DEHI-003 PLGA 100K (3X), 150.1 0.17 −61.9 insulin (1X) nanoencapsulation DEHI-004 PLGA 100K (1X), 160.9 0.10 insulin (1X) nanoencapsulation
Freeze-Fractured SEM Images of Nanocapsules at Fluid Interfaces

(115) Samples were suspended in ultra pure water+vortex, and then were shaken for at least 30 minutes, prior to freezing. A suspension volume of 1.5 μm was sandwiched between two flat aluminum platelets with a 200 mesh TEM grid used as a spacer between them. The sample was then high-pressure frozen in a HPM010 high-pressure freezing machine (Bal-Tec, Liechtenstein). The frozen samples were mounted on a holder and transferred to a BAF 60 freeze fracture device (Bal-Tec) using a VCT 100 Vacuum Cryo Transfer device (Bal-Tec). After fracturing at a temperature of −120° C. samples were etched at −110° C. for 5 minutes and coated with 3 nm Pt/C by double axis rotary shadowing. Samples were transferred to an Ultra 55 SEM (Zeiss, Germany) using a VCT 100 and were observed using a secondary electrons in-lens detector at 1.5 kV at a temperature of −120° C. The SEM images are shown in FIGS. 11A-11C.

(116) Induction of Diabetes Mellitus (DM)

(117) The DM was induced in the rats by intravenous injection of streptozotocin (STZ) diluted in 0.05M citrate buffer (50 mg/kg body weight). Two weeks afterwards, animals selected as diabetic were those that exhibited fasting glycemia above 250 mg/dL. Glycemia was measured by the glucose oxidase method (Bergmeyer and Bernt, 1974) using a clinical glucometer (Contour™, BAYER).

(118) The diabetic rats were then used to evaluate the hypoglycemic effects of different formulations containing insulin nanocapsules via oral feeding at 5; 10 IU/(175; 350 μg) and subcutaneous injection at 5 IU (175 μg) per animal in different conditions (fasted and non fasted).

(119) FIGS. 12-13 show the blood glucose levels following subcutaneous administration of the various insulin loaded nanoparticles formulations in fasting and non-fasting conditions (N=3), respectively.

(120) It can be seen from the SEM images that primary HSA nanocapsules of h-insulin are nanoencapsulated in larger nanocapsules of PLGA. These primary nanocapsules are also coated internally by the polymer PLA suggesting that the insulin release may be controlled upon i.m. or s.c. injection. This assumption was verified (as can be seen in FIGS. 12-13), as the injection of both types of insulin in double nanocapsules elicit a marked prolonged decrease in blood glucose over 24 h in fasting conditions, whereas in non fasting conditions the effect is shorter. It can be concluded that the novel technique does not affect at least markedly the pharmacological activity of the insulin.