Patent Application: US-201615016224-A

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 .

Description:
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 . fig1 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 . 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 ). 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 st hour ), or at each hour ( from the 2 nd to 5 th hour ). 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 ) ( fig2 ). 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 . 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 5 cells are seeded onto each well of the tissue - culture treated polycarbonate costar trans - well 6 wells / plates ( growth area 4 . 7 cm 2 , corning costar corp ., n . y .). the caco - 2 monolayer culture is kept in an atmosphere of 95 % air and 5 % co 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 2 to 500 ohm / cm 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 . fig3 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 ( fig3 ). it is confirmed that the hemoglobin encapsulated multiparticulates can be absorbed with the presence of absorption enhancers and protease inhibitors . 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 . 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 . 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 . the following references relate to various aspects of the present invention and are incorporated by reference herein : artursson , p ., et al . “ effect of chitosan on the permeability of monolayers of intestinal epithelial cells ( caco - 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