ENTERIC-COATED HEMOGLOBIN MULTIPARTICULATE FOR ORAL DELIVERY OF HEMOGLOBIN BASED OXYGEN CARRIERS

Abstract

The present invention provides an enteric-coated hemoglobin multiparticulate comprising a core, a hemoglobin formulation coating, an inner or sub-coating, and an enteric coating. The present invention also provides a method of preparing said enteric-coated hemoglobin multiparticulate. The present invention further provides a method for treating various diseases caused by oxygen deficiency comprising administering to a subject said enteric-coated hemoglobin multiparticulate in order to orally deliver the encapsulated hemoglobin-based oxygen carriers to a specific target of said subject in a controlled release manner.

Claims

1. An enteric-coated hemoglobin multiparticulate for oral delivery of hemoglobin-based oxygen carrier to a subject in need thereof, said multiparticulate comprising a core, a hemoglobin formulation coating surrounding said core, an inner or sub-coating surrounding said hemoglobin formulation coating, and an enteric coating surrounding said inner or sub-coating.

2. The multiparticulate of claim 1, wherein said core is made of starch to form a starch pellet core.

3. The multiparticualte of claim 1, wherein said hemoglobin formulation coating is formed by a hemoglobin formulation comprising hemoglobin-based oxygen carriers, one or more stabilizers, and an aqueous solution.

4. The multiparticulate of claim 1, wherein said inner or sub-coating comprises hydroxypropyl methylcellulose.

5. The mutliparticulate of claim 1, wherein said enteric coating comprises EUDRAGIT® L30 D-55, Triethyl citrate and Talc.

6. The multiparticulate of claim 3, wherein said hemoglobin formulation further comprises one or more absorption enhancers and a protease inhibitor.

7. The multiparticulate of claim 3, wherein said hemoglobin-based oxygen carriers are at a concentration of 10 g/dL in said hemoglobin formulation.

8. The multiparticulate of claim 3, wherein said one or more stabilizers comprise 6% w/v sucrose and 4% w/v hydroxypropyl-β-cyclodextrin in said hemoglobin formulation.

9. The multiparticualte of claim 6, wherein said one or more absorption enhancers comprise ethylene glycol tetraacetic acid and palmitoyl dimethyl ammonio propane- sulfonate at 70 mg/mL and 4.5 mg/mL, respectively, in said hemoglobin formulation.

10. The mutliparticulate of claim 6, wherein said protease inhibitor comprises soybean trypsin inhibitor at 25 mg/mL in said hemoglobin formulation.

11. The multiparticulate of claim 1, wherein said core has a weight percentage of 43.7% w/w to total weight of said multiparticulate.

12. The multiparticulate of claim 1, wherein said hemoglobin formulation coating has a weight percentage of 26.3% w/w to total weight of said multiparticulate.

13. The multiparticulate of claim 1, wherein said inner or sub-coating has a weight percentage of 10% w/w to total weight of said multiparticulate.

14. The multiparticulate of claim 1, wherein said enteric coating has a weight percentage of 20% w/w to total weight of said multiparticulate.

15. The multiparticulate of claim 1, wherein said mutliparticulate has an average size of about 395 μm.

16. A method of preparing the multiparticulate of claim 1 comprising: a) Providing a core; b) Providing a hemoglobin formulation; c) Coating said hemoglobin formulation on said core by spray coating to form a hemoglobin formulation coating surrounding said core; d) Coating a solution of hydroxypropyl methylcellulose on said hemoglobin formulation coating by spray coating to form an inner or sub-coating surrounding said hemoglobin formulation coating; e) Coating on said inner or sub-coating with an enteric coating by spray coating.

17. The method of claim 16, wherein said core is a starch pellet core.

18. The method of claim 16, wherein said hemoglobin formulation comprises hemoglobin-based oxygen carriers at 10 g/dL, 6% w/v sucrose and 4% w/v hydroxypropyl-β-cyclodextrin.

19. The method of claim 18, wherein said hemoglobin formulation further comprises 70 mg/mL ethylene glycol tetraacetic acid, 4.5 mg/mL palmitoyl dimethyl ammonio propanesulfonate and 25 mg/mL soybean trypsin inhibitor.

20. The method of claim 16, wherein said enteric coating comprises 12.5% w/v EUDRAGIT® L30 D-55, 1.25% w/v Triethyl citrate and 6.25% w/v Talc.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 shows the structure of enteric-coated hemoglobin multiparticulate.

[0017] FIG. 2 shows the dissolution profiles and HPLC analysis of enteric-coated hemoglobin multiparticulates in simulated gastric fluid and simulated intestinal fluid.

[0018] FIG. 3 TEER change and permeability of enteric-coated hemoglobin multiparticulates across Caco-2 cell layer.

DETAILED DESCRIPTION OF INVENTION

[0019] The present invention is directed to an improved oral delivery formulations for HBOCs which deliver oxygen to the vasculature via oral administration. In an embodiment, enteric-coated hemoglobin multiparticulates are provided for effectively delivering the hemoglobin-based oxygen carrier to a specific site. Said enteric-coated hemoglobin multiparticulates are administered via oral administration. The hemoglobin-based oxygen carrier may include but not limited to purified hemoglobin, cross-linked hemoglobin, non-polymeric tetrameric hemoglobin, polymeric hemoglobin, and conjugated hemoglobin of various molecular weights. Examples of hemoglobin that can be used in the oral pharmaceutical compositions of the present invention are set forth in U.S. Pat. Nos. 7,932,356, 7,989,593, 8,048,856, 8,084,581, 8,106,011, the disclosures of which are incorporated by reference herein.

[0020] FIG. 1 is a schematic diagram showing the structure of two-layer enteric-coated hemoglobin spray-dried encapsulated multiparticulate. Hemoglobin encaupsulated multiparticulates are prepared by Mini-Glatt Fluid Bed System. Fluidized bed coater was invented by Dr. D. Wurster at 1959, and it is applied in the field of pharmaceutical industry [U.S. Pat. No. 2,799,241]. The advantage of this method is to result in uniform coatings within a short operating time and with large-scale production. In this example, 10 g/dL bovine hemoglobin with 6% w/v sucrose and 4% w/v HPβCD in a hemoglobin formulation (or 26.3% w/w of the multiparticulate) (102) is sprayed on top of the starch pellet core (101) (43.7% w/w to total weight of the multiparticulate), followed by coating a layer of inner/sub-coating (103) comprising 10% w/w hydroxypropyl methylcellulose (HPMC), and a layer of enteric coating (104) (20% w/w to total weight of the multiparticulate) comprising 12.5% w/v EUDRAGIT® L30 D-55, 1.25% w/v Triethyl citrate and 6.25% w/v Talc, with the size of the multiparticulate of about 395 μm in diameter. Inside the multiparticulate, the diameter of the core is about 300 μm and the diameter of the spray-dried hemoglobin on top of the starch pellet core (or hemoglobin pellet) is about 350 μm.

[0021] Surcose and hydroxypropyl-β-cyclodextrin (HPβCD) are added into the present multiparticulate as stabilizer and cyroprotectant, while N-acetyl cysteine can serve as an antioxidant or an alternative to sucrose. HPβCD is a cyclic oligosaccharide with 7-membered sugar ring molecule. This molecule is approved by FDA as an oral drug stabilizer, and is commonly used in pharmaceutical applications for drug delivery. Its spatial arrangement of toroid structure (hydrophobic inside and hydrophilic outside) allows it to penetrate body tissues and forms complexes with hydrophobic pharmaceutical active ingredients. Thus, this stabilizer, HPβCD, enhances the solubility and bioavailability of the active ingredients (Becket et al., 1999).

[0022] Release study of the multiparticulates is performed in the simulated gastric fluid (pH 1.2 HCl solution, without pepsin) and the simulated intestinal fluid (pH 6.8 PBS solution, without pancreatin) at 37° C. Multiparticulate (0.05 g/mL) is placed into 50 mL of dissolution medium in two scenarios under peddle stiffing speed at 100 rpm: (1) acidic stage: simulated gastric fluid for 2 hours; (2) buffer stage: simulated intestinal fluid for 5 hours. Amount of hemoglobin released at different sampling time is determined by HPLC-UV measurement at 410 nm: (1) 2 mL of simulated gastric medium is aliquot in every 30 minutes, for hemoglobin quantitative analysis; (2) 2 mL of simulated intestinal fluid is aliquot at each 15 minutes (during the 1.sup.st hour), or at each hour (from the 2.sup.nd to 5.sup.th hour).

[0023] In the hemoglobin quantitative measurement of dissolution test, the release of hemoglobin from the multiparticulates is compared with the one in hemoglobin pellet without enteric coating (NB-01) and pellet with one-layer inner coating (NB-02) (FIG. 2). For the multiparticulates without enteric coating (NB-01), it is seen that 12% of hemoglobin releases in pH 1.2 dissolution HCl solution at 37° C. within two hours, whereas no hemoglobin releases from both one-layer inner coating multiparticulates (NB-02) and enteric-coated multiparticulates (NB-03) in the simulating gastric fluid. It indicates that the enteric coating of the multiparticulates can well-protect the hemoglobin in the acidic simulated gastric fluid. At the same time, the enteric-coated multiparticulates (NB-03) has more satisfy hemoglobin release in simulated intestinal fluid (30% hemoglobin released), to compare with the one without coating (NB-01) (12% hemoglobin released) and with one-layer inner coating (NB-02) (0% hemoglobin released). HPLC result also supports the presence of hemoglobin tetramer in the simulated intestinal fluid mixed with enteric-coated multiparticulates (NB-03) for 2 hours.

[0024] TEER of hemoglobin enteric-coated multiparticulates is studied by using the Caco-2 cells monolayer trans-well culturing setup. Caco-2 cell culture model, which is culture of the human epithelial colorectal adenocarcinoma cell line, is a well-recognized method to the study of human intestine function and thereby drug intestinal absorption mechanism. Firstly, Caco-2 cells are grown in the T75 flask. DMEM (high glucose, Gibco) supplemented with 10% Fetal bovine serum (FBS), 1% Non-essential amino acids (NEAA), and antibiotics (50 U/ml penicillin and 50 μg/ml streptomycin) is used as the culture medium. The grown Caco-2 cells are trypsinized and 6×10.sup.5 cells are seeded onto each well of the tissue-culture treated polycarbonate Costar Trans-well 6 wells/plates (growth area 4.7 cm.sup.2, Corning Costar Corp., N.Y.). The Caco-2 monolayer culture is kept in an atmosphere of 95% air and 5% CO.sub.2 at 37° C. The medium is replaced every other day in the both apical and basolateral compartments. Millicell-Electrical Resistance System (Millipore Corp.) connected to a pair of chopstick electrodes is used to monitor the transepithelial electric resistance (TEER) which reveals the tightness of the tight junction between cells. The Caco-2 monolayer culture is used for the trans-epithelial transport study 19-21 days after seeding. The difference in TEER between the blank and the cell monolayer should be in the range of 400 ohm/cm.sup.2 to 500 ohm/cm.sup.2. The cells are fed with fresh medium 24 hours prior to the trans-epithelial transport study. Hemoglobin encapsulated multiparticulates with or without enteric coating, additional absorption enhancers (e.g. EGTA and PPS) and soybeans trypsin inhibitor (e.g. SBTI) is optionally loaded into the apical compartment. In one embodiment, 70 mg/mL EGTA, 4.5 mg/mL PPS, and/or 25 mg/mL SBTI can be loaded into the apical compartment. Cells are incubated at 37° C. with orbital shaking at 50 r.p.m. for 3 hours after the loading. The initial and time point TEER values are measured. FIG. 3 shows the result that TEER values rebounded after treating with multiparticulates alone, which indicates that the multipaticulates are not invasive. After the experiment, 2 mL of HBSS at the basolateral compartment are collected for the permeability measurement by HPLC analysis. The permeability is calculated by comparing the amount of multiparticulates in the basolateral compartment at the end and the initial multiparticulates amount in the apical compartment. A 30-fold (with enteric coating) and 40-fold (without enteric coating) enhancement of hemoglobin encapsulated multiparticulates permeability is achieved by the addition of EGTA, PPS and SBTI (FIG. 3). It is confirmed that the hemoglobin encapsulated multiparticulates can be absorbed with the presence of absorption enhancers and protease inhibitors.

[0025] Compared to intravenous delivery of peptides or proteins, oral delivery has an advantage in pharmacokinetics because an oral delivery system enables controlled release of peptide or protein from the carriers. Such a controlled release mode of delivery of peptide or protein drug is unavailable in direct intravenous delivery. For hemoglobin being introduced into the vascular system, a controlled release and sustained elevation of the hemoglobin concentration in the blood has a greater physiological benefit than that from a sudden substantial increase of free hemoglobin in the injection site from direct injection. A rapid increase in the hemoglobin level increases the chance of developing side effects such as extravasation, myocardial infarction and renal toxicity.

[0026] The heme group of hemoglobin in HBOC consists of an iron (Fe) ion (charged atom) held in a heterocyclic ring. In addition to delivering oxygen to the human body by HBOC, the heme group can provide heme iron to the body to aid in the production of more red blood cells. Acetazolamide, steroids and Rhodiola cannot provide heme iron to the body.

[0027] Oral delivery of HBOCs is a non-invasive, convenient and efficient way to prevent or treat HAS, and therefore, it is favorable for people to take before or during travel from a sea level region to a high altitude region. Absorption of undegraded hemoglobin in intestinal tract, skipping de novo synthesis of hemoglobin, increases the oxygen-carrying capacity of blood thus increasing the rate of acclimatization. The orally-deliverable HBOCs can also be used to treat acute anemia due to blood loss or to prepare individuals for physically-demanding activities in normal or low oxygen supply atmosphere, e.g. for athletes, astronauts, divers, or navy personnel stationed in submarines. Improving tissue oxygenation by HBOCs is further useful for preventing/treating tissue ischemia, and promotes wound healing, such as diabetic foot ulcers. While the foregoing invention has been described with respect to various embodiments, such embodiments are not limiting. Numerous variations and modifications would be understood by those of ordinary skill in the art. Such variations and modifications are considered to be included within the scope of the following claims.

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