CA2203632A1 - A chitosan polymer having a specific degree of acetylation - Google Patents
A chitosan polymer having a specific degree of acetylationInfo
- Publication number
- CA2203632A1 CA2203632A1 CA 2203632 CA2203632A CA2203632A1 CA 2203632 A1 CA2203632 A1 CA 2203632A1 CA 2203632 CA2203632 CA 2203632 CA 2203632 A CA2203632 A CA 2203632A CA 2203632 A1 CA2203632 A1 CA 2203632A1
- Authority
- CA
- Canada
- Prior art keywords
- chitosan
- acetylation
- chitosans
- degree
- molecular weight
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Nutrition Science (AREA)
- Physiology (AREA)
- Molecular Biology (AREA)
- Inorganic Chemistry (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The present invention relates to a pharmaceutical formulation comprising a chitosan polymer having a degree of acetylation of between 20 % and 45 % and a molecular weight of above 75,000, and a therapeutic active agent, a chitosan polymer for preparing said formulation, and the use of said chitosan polymer and a method of enhancing the absorption of a therapeutic active agent.
Description
A chitosan polymer having a specific degree of acetylation.
Field of the invention The present invention relates to a new pharmaceutical formulation comprising a chitosan polymer having a specific degree of acetylation and a specific molecular weight, the chitosan polymer per se, and the use of the chitosan polymer.
Backqround of the invention Chitin and its derivatives have long been known to have numerous potential beneficial uses, such as for example food ingredient or water purification aid (Chitin and chitosan, Eds.: G. Skjak-Braek, T. Anthonsen and P.
Sandford, Elsevier, Amsterdam, 1989). Chitin is found abundantly in nature in for instance crab and shrimp shells, insects, and mushrooms. The primary units in chitin are 2-deoxy-2-acetylamino glucose units which are combined by (1,4) glycosidic linkages into a linear polymer. N-deacetylation of chitin results in chitosan, a high molecular weight cationic polysaccharide. The degree of N-acetylation is an important characteristic of chitosan, as it determines the positive charge of the molecule and its polarity. Chitosan may be manufactured with different degrees of acetylation as well as molecular weight (Anthonsen et al., Carbohydrate Polymers, 22, 1993, pp 193-201).
Chitosans are considered biocompatible macromolecules due to assumed low toxicity and biodegradability. They are degraded by lysozymes and related enzymes, such as N-acetyl-D-glucosaminidases. The lysozymic digestibility of the polymer increases with the degree of N-acetylation (Aiba, Int. J. Biol. Macromol., 14, 1992, pp 225-228; Bourbouze et al., Clin. Chim. Acta, 199, 1991, pp 185-194). Their biocompatibility has initiated evaluation of the compound for many medical applications, e.g. in JP 0509295 A; Nishimura et al., Mol. Biother., 2, 1990, pp 115-120; Segal et al., Mycopathologia, 102, 1988, pp 157-163; Nishimura et al., Vaccine, 2, 1984, pp 93-99; Lida et al,. Vaccine, 5, 1987, pp 270-274; and JP 5414809 A. They are also useful as sustained or controlled release carriers for drugs as is disclosed e.g. in JP 04264023 A and EP 460921-A.
In the patent US 4873 092 by Azuma et al chitosans are used as carriers for physiologically active substances, such as adriamycin and testosterone, with the aim to slowly release these substances from the carrier and therewith obtain constant therapeutic (but non-toxic) blood concentration of the drug over an extended period of time. Chitosans that could be used as such slow release carriers are stated to have a degree of N-acetylation of 0-100% and a molecular weight of 10000 to 23000. A paper by Imai et al, Int. J. Pharm., 67, 1991, pp 11-20, describes the use of chitosans with low molecular weight (<25.000) and intermediate degrees of acetylation (10-34%) as carriers for the oral absorption of indomethacin. These chitosans increases the in vitro dissolution of the acidic drug. Since dissolution of the poorly water soluble ;n~omethacin is rate limiting for its oral absorption, the increased dissolution results in an increased oral absorption of the drug.
Chitosans have been found to have mucoadhesive properties, and due to their close contact with the CA 02203632 l997-04-24 mucosal epithelium may prove useful in delivery of drugs to mucosal absorption sites such as the small intestine or colon and rectal mucosa (EP 514008-A1;
Lehr et al., Int. J. Pharm., 78, 1992, pp 43-48).
For various hydrophilic drugs such as several antibiotics, peptides and proteins the solubility of the drugs is not rate limiting in the absorption process. Many of these compounds, however, are poorly absorbed following oral administration due to extensive enzymatic degradation and poor epithelial permeability.
In order to overcome the physical barriers that h;n~er drug absorption it is often inevitable to coadminister so called absorption enhancers. Absorption enhancers as used in the present patent application refer to compounds that increase drug absorption by an interaction between the mentioned compound and the mucosal epithelium. They do not include compounds that increase drug absorption by an interaction with the drug itself. Numerous classes of compounds have been - described that improve the intestinal absorption of hydrophilic and high molecular weight compounds. Such absorption enhancing compounds include surfactants, bile salts, chelating agents, and fatty acids. (Lee et al., Crit. Rev. Therapeut. Drug Carrier Syst., 8, 1991, pp 91-192). A major concern, however, related to the use of many of such absorption enhancing agents is potential adverse effects on epithelial integrity, morphology, and function (Swenson and Curatolo, Adv.
Drug Delivery Rev., 8, 1992, pp 39-92). Moreover, the delivery of an effective amount of enhancer and drug at the site of absorption to get reproducable dosing is necessary, but difficult to achieve. In addition, the onset of action of the enhancer and the duration of its activity should coincide with the presence of the drug at the absorption site and the time necessary for its uptake (De Boer and Breimer, In Drug absorption enhancement, Eds.: De Boer, Harwood Ac. Publ., Chur, 1994, pp 155-175). Compounds with optimal absorption enhancement characteristics, thus should have a strong effect on epithelial permeability, an early onset of action, and a low toxicity.
Recently, chitosans have been investigated as potential absorption enhancing compounds to aid in the absorption of hydrophilic, and high molecular weight drugs across mucosal tissues. Chitosans have been found to enhance the mucosal absorption of insulin, a small model peptide, and a hydrophilic model marker molecule. In a published patent application by Illum et al.
(WO 9009780-A), it is described that the chitosans Seacure and Seacure+ improve the absorption of insulin across the nasal mucosa of sheep and rat to a large extent. The concentration of the chitosan used in these studies is 0.1 to 0.5% (w/v). Seacure+ is stated to be a water soluble chitosan glutamate with either a low (Seacure+) or an intermediate viscosity (Seacure+210).
The degree of acetylation or the molecular weight of the chitosan are not clearly stated. Effective polymers, however, are stated to be chitosans in a molecular weight range of 10,000 to S00,000.
The same water soluble chitosan (Seacure+ 210) has been used to study chitosan effects on Caco-2 cell monolayers (Artursson et al., Pharm. Res., 1994, 11, pp 1358-1361). Caco-2 is a human intestinal epithelial cell line that is commonly used as a model for human intestinal epithelium. The Seacure+ chitosan increases the permeability of the epithelium 5 to 10 fold as measured by the transport of a model hydrophilic compound across the epithelium. Seacure+ is used at concentration from 0.1 to 0.5% (w/v) and the pH of the solutions ranges from 4.0 to 6Ø
s Intermediate viscosity Seacure +210 has in another study (Rentel et al., Proc. Intern. Symp. Control. Rel.
Bioact. Mater., 20, 1993, pp 446-447) been found to improve the transport of a small peptide across intestinal tissue in vitro. In the same study a different chitosan was also tested. This chitosan, Daichitosan VH (DA=15-20~; Mw=500000-800000) showed similar improvement of transport of the small peptide across intestinal mucosa at a concentration of 1 10 (w/v).
Although all the presented studies show improvement of mucosal epithelial permeability in the nose as well as in the intestinal tract in several animal species, including sheep and rat as effectuated by chitosans at different concentrations (0.1-2~ (w/v)) and at different pH values, they do not elucidate the influence of the two important characteristics of chitosans on the absorption enhancing effect, namely the degree of acetylation and the molecular weight. In fact in the described papers none of the parameters are very well specified.
Brief description of the invention According to the present invention it has unexpectedly been found that intermediate but not high degrees of acetylation, i.e. a degree of acetylation of between 20 and 45%, and a high molecular weight, i.e. a molecular weight of above 75,000, of chitosans are necessary for increasing epithelial permeability, to achieve an early onset of absorption enhancement, and a low toxicity of the enhancer.
Detailed descriPtion of the invention Studying the transport of the marker molecule, mannitol, as a model for hydrophilic drugs tAnderberg et al., Pharm. Res., 10, 1993, pp 857-864), and the cardiovascular drug, atenolol, across Caco-2 cell monolayers it was found that chitosans with a low to intermediate degree of acetylation and/or a large molecular weight are necessary in order to enhance permeability. That is, chitosans with a degree of acetylation of 1% (DA=1%) and either a smaii moiecuiar weight (Mw=31000) or a large molecular weight (Mw=170000) have a strong concentration dependent effect on mannitol transport. At concentrations as low as 50 ~g/ml (0.005% (w/v)) they both increase mannitol transport 10- to 15-fold. At a concentration of 250 ~g/ml the increase in permeability is 50-fold.
Chitosans with an intermediate (DA=35~) or high degree of acetylation (DA=49%) and a large molecular weight (Mw=170000 and 98000, respectively) also improve m~nn; tol transport. Concentrations of 50 and 250 ~g/ml, respectively (0.005 and 0.025% (w/v)) are necessary to achieve a 10 to 15 fold increase in mannitol per~hility of the Caco-2 cell monolayers. The increase in mannitol perm~hility of the latter chitosans does not have such a steep concentration/effect relation as is observed for the former chitosans with a low degree of acetylation (DA=1%). On the other hand, chitosans with an intermediate or a high degree of acetylation and a small molecular weight (chitosan (DA=35%, Mw=5300), chitsoan (DA=35%, Mw=12000), chitosan (DA=49%, Mw=22000)) do not have an effect on mannitol transport at all. Concentrations upto 250 ~g/ml (0.025% ~w/v)) do not change the permeability of m~nn; tol across Caco-2 cell monolayers significantly.
Although at a concentration of 50 ~g/ml chitosans with a degree of acetylation of 1~ and a molecular weight of 31000 and 170000, and chitosans with a degree of acetylation of 35% and a molecular weight of 170000, and at a concentration of 250 ~g/ml chitosan with a W0~6/20730 PCT/SE95/01342 degree of acetylation of 49% and Mw of 98000 all show similar enhancement of mannitol permeability across Caco-2 cell monolayers, their adverse effects on epithelial cells are surprisingly very different. In a toxicity assay which measures intracellular dehydrogenase activity and is based on the observation that injured cells display a reduced dehydrogenase metabolism, a much stronger toxicity was observed for chitosans with a low degree of acetylation (DA~
Mw=31000 or 170000), than for chitosans with an intermediate and high degree of acetylation and a large molecular weight (chitosan (DA=35%, Mw=170000), chitosan (DA=49~, Mw=98000). These findings were confirmed by visualisation of the adverse effects of chitosans on epithelial Caco-2 cell monolayers with transmission electron microscopy, which showed that the morphological changes following exposure to chitosans with intermediate and high degree of acetylation and high molecular weight (chitosan DA=35%, Mw=170000;
chitosan DA=49~, Mw=98000) were small, while clear changes were observed following exposure to chitosan with a degree of acetylation of only 1%. These changes involved dicontinuities and reduced number of microvilli, and a disorganized terminal web.
In addition, the onset of action of increased epithelial permeability is much quicker for a chitosan with intermediate degree of acetylation (35%) and large molecular weight (170000), than for chitosan with a low degree of acetylation and small or large molecular weight (DA=1%, Mw=31000 or 170000). This gives the former chitosan more controllable and reliable absorption enhancing characteristics.
In contrast to the findings of Imai et al. (Int. J.
Pharm., 67, 1991, pp 11-20) who found that chitosans with low molecular weight were necessary to increase the dissolution of acidic drugs, we have found that, in order to improve drug absorption by means of increased epithelial permeability for hydrophilic drugs, chitosans with high molecular weight and intermediate degree of acetylation are preferable with regard to improved permeability, early onset of action, and low toxicity.
The invention provides a pharmaceutical formulation of a form and composition suitable for mucosal administration, particularly for oral and/or rectal delivery, of therapeutic agents in man. By oral route means e.g. buccal, intragastric and intestinal. This preparation comprises a therapeutic active agent as well as a chitosan with the below described characteristics as an absorption enhancing agent.
The present invention is based on the observation that it is possible to select chitosans with optimal absorption enhancement characteristics, that is chitosans with a strong effect on eptithelial permeability, low toxicity, and an early onset of action. Such a chitosan would, according to the present invention have a degree of acetylation of between 20 and 45%, and preferably between 30 and 40%, while its molecular weight should be above 75,000, more preferably between about 75,000 and 250,000, and most preferably between about 98,000 and 200,000.
Therapeutic active agents include drugs, vaccines, proteins, peptides and fragments thereof. The pharmaceutical formulation according to the invention may be used with drugs from the following non-exlusive list.
Pharmaceutical formulations containing hydrophilic drugs with low membrane permeability and biovailability which are absorbed mainly via the paracellular route such as;
- Blood pressure lowering agent such as hydrophilic ACE-inhibitors and ~-blockers - Anti-coagulation agents such as thrombin inhibitors heparin and low molecular weight heparin - Antidiuretic drugs and vasopressin analogs such as DDAVP, AVP, etc.
- Peptide hormones such as insulin, calcitonin, growth hormone, PTH
- Antibiotics such as penicillin and penicillin derivatives, tetracycline, and macrolides - Steroidal anti-inflammatory agents such as beclomethasome and budesonide - Non-steriodal anti-inflammatory agents such as diclofenac, indomethacin, ibuprofen, naproxen - Coagulation factors such as Factor VIII
- Vaccines and antigens such as polio vaccine - Anti-viral drugs such as Foscarnet - Compounds effecting bone metabolism such as clodronate and other bisfosfonates - Analgesics such as opioid peptides, dextropropoxifene and pentazocine - Local anesthetics with antiinflammatory properties such as lidocaine, bupivacaine and ropivacaine - Local anesthetics with analgetic properties such as bupivacaine and ropivacaine - Immunomodifiers such as Cyclosporin A
- Anti-allergic drugs such as sodium cromoglycate The pharmaceutical formulations according to the invention will in practice contain a ph~rm~cologically active amount of therapeutic agent. As regards the amount of chitosan the formulation will contain an amount thereof that enhances the absorption of the active agent present. Hereto, it will be necessary to scale the dose of absorption ~n~n~er from in-vitro cell culture studies and ~n;m~l experiments to the therapeutic situation in man. Such a dose scaling will be common for anyone skilled in the art. Chitosan amounts of 5 ~g/dose to 1000 mg/dose are possible.
Amounts of 50 ~g to S00 mg/dose are preferable.
If desired, other absorption enhancers may be included in the pharmaceutical formulations of the invention.
Such enhancers can be inhibitors of enzymatic degration, in order to reduce the amount of drug that is metabolized before absorption. Enzyme inhibitors used to stabilize drugs in mucosal epihelia and the gastrointestinal lumen include bile salts, bacitracin, aminoboronic acid derivatives, bestatin, FK-448, soybean trypsin inhibitor, and aprotin (Muranishi and Yamamoto, In Drug absorption enhancement, Eds.: De Boer, Harwood Ac. Publ., Chur, 1994, pp.67-100).
Formulations suitable for the delivery of the drugs to the small intestine or colon can utilize coated solid dosage forms or enteric systems, which will loose their W096l20730 PCT/SE95101342 coating on arrival in the small intestine due to dissolution of the coating polymers at the local pH
conditions. Colon specific delivery systems may involve polymer coatings that typically are degraded by the colonic bacterial flora. Upon degradation of the coating these delivery systems will release drug and additives.Upon degradation of the coating, these delivery systems will release drug and additive (Friend, Advanced Drug Delivery Reviews, 7, 1991, pp 149-201) Such polymer coatings may for instance comprise azopolymers (Saffran et al., Science, 233, 1986, pp 1081-1084), and polysaccharides such as calcium pectinate (Rubinstein, Pharm. Res., 10, 1993, pp 258-265).
Alternatively, a time dependent colon drug delivery system can be used.
Naturally, the formulations according to the invention may also contain one or more additives conventionally used in mucosal drug delivery, such as preservatives, stabilizers, inert excipients, etc. Agents suitable for these and other purposes are known to anyone skilled in the art of mucosal drug delivery.
Preparation of formulations The chitosan and therapeutic active agent containing formulation may be prepared in a buffered saline solution at a neutral pH, i.e. pH 6.5-7.5. A lower pH
may be used for easier dissolvation of the chitosan by addition of HCl to the afore-mentioned buffer or a different buffer system. The person skilled in the art will be able to dete~~~.,ine the optimal pH which may be between 1.0 and 11.0 and preferably between 4.0 and 7.5.
The chitocan and therapeutic agent formulation may be prepared in any forms suitable for mucosal delivery, such as oral or rectal delivery, such as tablets (e.g.
immediate release tablets, enteric coated tablets, extended release tablets), gelatine capsules, suppositories, microspheres, gels, solutions, suspensions, etc.
The invention will now be illustrated but not limited by the following examples.
Bxample 1: ToxicitY of chitosans as assessed with a dehydroqenase activitY assay in Caco-2 cells Preparation of chitosans with different molecular weiqht and deqree of acetYlation The preparation and characterization of chitosans was performed according to procedures described in Anthonsen et al., Carbohydrate Polymers, 22, 1993, pp 193-201. The chitosans used in the example are given in table 1.
Table 1: Chitosan samples used for structure-effect studies.
Chitosan Degree of Molecular acetylation weight (~) W09612~730 9 15 l90000 PreParation of chitosan solutions Chitosan solutions were prepared by dissolving them at concentration of 0.5 and 1.O mg/ml in Hank's balanced salt solution buffered with 25 mM 2N morpholino ethane sulfonic acid to a pH=5.5 (HBSS pH=5.5). This pH value is comparable to the pH of the small intestine microclimate and facilitates the dissolutions of the chitosans. HBSS is a buffer commonly used in cell culture experiments. Phosphate buffered saline (PBS) and similar physiological buffers could also be used as solvents. Chitosans were dissolved by vigorously shaking for 24 to 48 hours. Just prior to experiments the chitosan solutions were diluted to the appropriate concentrations with HBSS pH=5.5.
Dehydroqenase activity assaY
In these studies Caco-2 cells obtained from American Type Culture Collection, Rockville, USA, were used.
Cells of passage number 93-105 were used throughout.
The cells were seeded in 96-well tissue culture plates (~low Laboratories, Irvine, UK). Af~ër 24 h they were exposed to different concentrations of chitosans for 60 min at 37 C in air. Subsequently, the intracellular dehydrogenase activity was determined by the MTT method as described in Anderberg et al., Pharm. Sci., 81, 1992, 879-887. MTT is a tetrazolium salt that is cleaved by mitochondrial dehydrogenases in living but not dead cells to give a dark blue product. Following addition of MTT, the cells were incubated for another 60 min. The developed colour was measured with a multiwell scanning spectro photometer. The dehydrogenase activity was evaluated as the relative absorbance at 590 nm of the solutions in the 96-well plate as compared to control, the latter being 100%.
Figure 1 shows the effect of chitosans on intracellular dehydrogenase activity. The data are given as the mean of 3 experiments. In general, the difference between individual data points is less than 25%. In figure 1 the different curves relate to ( ) chitosan (DA=l~, Mw=31000), ( ~ ) chitosan (DA=1%, Mw=170000); ( 0 ) chitosan (DA=15%, Mw=4700); ( ~ ) chitosan (DA=15%, Mw=l90000); ( ) chitosan (DA=35%, Mw=12000); ( ~ ) chitosan (DA=35%, Mw=170000); ( O ) chitosan (DA=49%, Mw=22000); ( o ) chitosan (DA=49%, Mw=98000); ( ~ ) chitosan (DA=35~, Mw=5300). For chitosans with a low degree of acetylation chitosan (DA=1%, Mw=31000);
chitosan (DA=1%, Mw=170000); chitosan (DA=15%, Mw=4700); and chitosan (DA=15%, Mw=lgO000); a dose dependent effect on intracellular dehydrogenase activity is observed. Chitosans with an int~rm~;ate degree of acetylation and large molecular weight (chitosan (DA=35%, Mw=170000)) decrease enzyme activity only at higher concentrations. Chitosan (DA=35%, Mw=5300), chitosan (DA=35%, Mw=12000), chitosan (DA=49%, Mw=22000), and chitosan (DA=49%, Mw= 98000) do not show an effect in the concentration range studied.
Exam~le 2: ToxicitY of chitosans as assessed bY effects on e~ithelial cell morpholoqY
Caco-2 cells were obtained from American Type Culture Collection, Rockville, USA. The cells were cultivated on polycarbonate filters (Transwell cell culture inserts, Costar, Cambridge) as described by Artursson, Pharm. Sci., 79, 1990, 476-482. Cells of passage 93-105 were used throughout. The cells were used for experiments 21 to 35 days after seeding. After washing the monolayers apically with HBSS pH=5.5 and basolaterally with Hank's balanced salt solution buffered with 25 mM Hepes to pH=7.4 (HBSS pH=7.4), chitosans were added apically, while the basolateral solution was replaced with fresh HBSS pH=7.4. The chitosans described in example l were diluted from stock solutions as described in example l. All chitosans were added at a concentration 50 ~g/ml, except chitosan (DA=49%, Mw=98000) which was used at 250 ~gtml. Following exposure to the different chitosan solutions for 60 min at 37C in air, the cell monolayers were rinsed twice with PBS and fixed in l.5%
glutaraldehyde and l~ osmium tetroxide, and immersed in 1% uranyl acetate. Thin sections were examined with a Philips 420 electron microscope at 60 kV.
In monolayers treated with 50 ~g/ml chitosan (DA=1%, Mw=31000) and chitosan (DA=l~, Mw=170000) discontinuities in and reduced number of microvilli were observed, while the terminal web showed a disorganized pattern. The tight junctions appeared not to be effected. Following treatment with 50 ~g/ml chitosan ( DA= 3 5 %, MW= 17 0 0 0 0 ) , and 250 ~g/ml chitosan (DA=49%, Mw=98000) the structural changes were not as pronounced. Microvilli and terminal web appeared normal. See figure 2 which is a transmission electron micrograph of a) control cells (magnification 1950x), b) cells treated for 60 min with 50 ~g/ml chitosan (DA=1%, MW=31 000) (magnification 2500x) and c) cells treated for 60 min with 50 ~g/ml chitosan (DA=35~, Mw=170 000) (magnification 2500x). Exposure to chitosan (DA=35%, Mw=5300); chitosan (DA=35%, Mw=12000);
chitosan (DA=49~, Mw=22000) did not result in obvious structural changes.
ExamDle 3: Absorption enhancement of chitosans - effect on ePithelial PermeabilitY
Caco-2 cell monolayers were used as a model for mucosal and in particular intestin~l epithelium. Drug transport across Caco-2 cell monolayers has in the past been demonstrated to correlate excellently with in vivo human intestinal drug absorption. In the presence of absorption enhancer, correlations between in vitro cell culture experiments and in vivo drug absorption have also been established, with corrections in the applied dose due to upscaling sometimes being necessary.
Mannitol was used as a model compound for hydrophilic drugs which show a low and incomplete uptake across mucosal epithelium. Transport of mannitol in Caco-2 cell monolayers has previously been shown to correlate with the transport of sparingly absorbed drugs within a molecular weight range of 100-500. All transport experiments were performed in HBSS. Prior to the experiments the cells were washed apically with HBSS
pH=5.5, and basolaterally with HBSS pH=i. 4. Just before the experiments chitosans described in example 1 were diluted from stock solutions with HBSS pH=5.5 to the appropriate concentrations as described in example 1.
14C-mannitol was subsequently added. The experiments were initiated by replacing the apical blank HBSS
pH=5.5 medium for a 14C-mannitol/chitosan solution, and the basolateral solution for fresh HBSS pH=7. 4.
Transport studies were performed during 120 min in air at 37 C and 95% relative humidity. Samples were taken at regular time intervals from the basolateral side, and were counted in a liquid scintillation counter (Tricarb, l900CA). The apparent permeability (Papp) was calculated using the following equation:
Papp = dQ/dt x l/(AxCo), in which dQ/dt is the rate of appearance of mannitol on the basolateral side, Co is the initial m~nn; tol concentration on the apical side, W096/20730 pcTlsEs5lol342 and A is the surface area of the monolayer.
The mannitol permeability of the Caco-2 cell monolayers could be improved by some but not all of the chitosans studied. Table 2 shows the mean permeability over 120 min during exposure to 50 ~g/ml chitosan solution. The effect of the chitosans was dependent on both the degree of acetylation and the molecular weight of the polymer. At a concentration of 50 ~g/ml, chitosans with a low degree of acetylation showed a clear effect on mannitol transport. Chitosan (DA=1%, MW=31000);
chitosan (DA=1%, Mw=170000); chitosan (DA=15%, Mw=4700); chitosan (DA=15%, Mw=l90000); showed a mean Papp between 0 and 120 min that was increased 5 . 5 - to 15 16.1-fold, as co~pared to control. Chitosans with a larger degree of acetylation and a low or intenmediate molecular weight, such as chitosan (DA=3596, Mw=5300), chitosan (DA=35%, MW=12000 ) and chitosan (DA=49%, Mw=22000) did not influence mannitol transport significantly at concentrations of 50 ~lg/~ . Chitosan, however, with a high molecular weight and intermediate degree of acetylation (DA=35%, Mw=170000) strongly increased the Papp of mAnn;tol. Chitosan with a very large degree of acetylation, but also a high molecular weight (DA=4996, Mw=98000), showed a small but significant increase in m~nn; tol transport at 50 ~g/ml.
Table 2: Effect of different chitosans at a concentration of 50 ~gJml on the permeability of m~nn; tol across Caco-2 cell monolayers.
Chitosan Degree of Molecular Papp x lOE7 Ratio (~g/ml) acetylation weight (cm/sec) Papp (~) chitosan /Papp control control - 2.2 + 0.2 1 31000 28.9 + 5.8 13.2 1 170000 24.0 + 0.7 10.9 5300 2.3 ~ 0.8 1.1 12000 2.7 + 0.2 1.3 170000 29.0 + 3.8 13.2 49 22000 4.8 + 4.4 2.2 49 98000 9.0 + 1.4 4.1 4700 12.8 + 3.4 190000 35.5 + 10.4 The mean Papp for 14C-mannitol are calculated over the total 120 min. period of the experiments. The Papp values are given as the mean + s.d. of 3-4 experiments.
RY~m~le 4: Absorption enhancement of chitosans -concentration dependencY
Mannitol transport experiments were performed with different concentrations of chitosans to study the concentration dependency of the absorption enhancing effect. The chitosans used in the experiments are described in example 1. Solutions having a concentration of 10, 50 and 250 ~g/ml were prepared according to example 1. The transport experiments were similarly performed as in example 3.
W096/20730 PCT/SE9~/01342 The chitosan effect on mannitol transport was concentration dependent. The effect of chitosans at 3 different concentrations 10, 50, and 250 ~g/ml on the Papp of m~nn; tol across Caco-2 cell monolayers are shown in Figure 3. The Papp is calculated as the mean Papp during the 120 min time period of the experiment.
The data are given as the mean + s~d. of 3-4 experiments. ( ~ ) chitosan (DA=1%, Mw=31000); ( ~ ) chitosan (DA=1%, Mw=170000); ( 0 ) chitosan (DA=15%, Mw_4700); ( ) chitosan (DA=15%, Mw=l90000); ( ) chitosan (DA=35~, Mw=1200~ ) chitosan (DA=35%, Mw=170000); ( O ) chitosan (DA=49%, Mw=22000); ( o ) chitosan (DA=49%, Mw=98000); ( ~ ) chitosan (DA=35%, Mw=5300).
A 10 ~g/ml dose gave a small increase in ~nn; tol transport for chitosan (DA=l~, Mw=31000), chitosan (DA=1%, Mw=170000), and chitosan (DA=15%, Mw=l90000) while all other chitosans did not have an effect at this concentration. At 250 ~g/ml chitosan (DA=1%, Mw=31000), chitosan (DA=1%, Mw=170000), chitosan (DA=15%, Mw=4700) and chitosan (DA=15~, Mw=l90000) increased the permeability to about 50 to 60 times the control value. For chitosan (DA=35~, Mw=170000) the mean Papp at 250 ~g/ml was not significantly different from the Papp value obtained with 50 ~g/ml. Chitosan (DA=49, Mw=98000) increased the Papp approximately 10 times at 250 ~g/ml.
Example 5: AbsorPtion enhancement of chitosans - onset of action of increased ePithelial DermeabilitY
Similar transport experiments as described in example 3 were done to study the onset of action of increased permeability of chitosans. The chitosans used in the experiments and the preparation of the different solutions are described in example 1.
The onset of action of chitosans that increased . epithelial permeability was not the same for chitosans.
Although the mean Papp measured over 120 min was similar for chitosan (DA=1%, Mw=31000), chitosan (DA=1%, MW=170000), chitosan (DA=15%, MW=19OOOO) at a concentration of 50 ~g/ml, and chitosan (DA=49%, MW=98000) at a concentration of 250 ~g/ml, the permeabilities at 20 min after the start of the experiment were very different. Papp values at 20 min are summarized in table 3. Exposure to chitosan with intermediate degree of acetylation and high molecular weight (DA=35, MW=170000) showed a much stronger increase in permeability after 20 min than all other chitosans (compare figure 3 and table 2 with table 3).
Table 3: Effect of different chitosans on the permeability of mannitol across Caco-2 cell monolayers after 20 min exposure.
Chitosan Degree of Molecular Papp x lOE7 Ratio (~gtml) acetylation weight (cm/sec) Papp (~) chitosan /Papp control control - 2.7 + 0.5 1 31000 4.4 + 0.6 1.6 1 170000 3.9 + 0.4 1.4 5300 3.3 + 0.8 ~.. 2 12000 2.9 + 0.2 1.1 170000 8.4 i 1.8 3.1 49 22000 1.7 i 0.3 0.6 250 49 98000 3.8 i 0.7 1.4 4700 4.3 i 1.0 190000 5.0 + 0.4 The Papp for 14C-mannitol are ca~culated over the first 20 min period of the experiments. The Papp values are given as the mean + s.d. of 3-4 experiments.
Example 6: AbsorPtion enhancement of chitosans - effect on the ePithelial ~erm~hilitY of atenolol Just before the start of the experiments chitosan (DA=1%, Mw=31.000) and chitosan (DA=35, Mw=170.000), as described in example 1, were diluted from stock solutions with HBSS pH=5.5 to a concentration of 50 ~g/ml, as described in example 1. Caco-2 cell transport W 096/20730 PCT/SE9~/01342 experiments were performed, subsequently, as described in example 3. Atenolol concentrations were determined using HPLC consisting of a RP Ultrasphere ODS 5 ~m (4.6 mm x 25 cm) column connected to a W detector.
Absorption was measured at 270 nm. The mobile phase consisted of phosphate buffer (pH=3) and acetonitrile in a ratio of 90:10.
Figure 4 shows the Papp of atenolol during exposure to 50 ~g/ml chitosan solution. The data are given as mean ~ SD of 4 experiments. In figure 4 the different curves relate to (x) control; (d) chitosan (DA=1%, Mw=31000);
and (~) chitosan (DA=35, Mw=170000). The permeability of the Caco-2 cell monolayers was low (0.5-1.0x10-7).
The perme~hility could be improved, however, by chitosan (DA=1%, Mw=31.000) and chitosan (DA=35, Mw=170.000). Similarly, as observed for mannitol p~rm~Ahility the effect of chitosan (DA=35, Mw=170.000) was larger than the effect of chitosan (DA=1%, Mw=31.000) during the early stages of the experiment.
For chitosan (DA=35, Mw=170.000) the atenolol permeability did not increase further after 20 min exposure, while for chitosan (DA=1%, Mw=31.000) atenolol permeability kept increasing until the end of the experiment.
Field of the invention The present invention relates to a new pharmaceutical formulation comprising a chitosan polymer having a specific degree of acetylation and a specific molecular weight, the chitosan polymer per se, and the use of the chitosan polymer.
Backqround of the invention Chitin and its derivatives have long been known to have numerous potential beneficial uses, such as for example food ingredient or water purification aid (Chitin and chitosan, Eds.: G. Skjak-Braek, T. Anthonsen and P.
Sandford, Elsevier, Amsterdam, 1989). Chitin is found abundantly in nature in for instance crab and shrimp shells, insects, and mushrooms. The primary units in chitin are 2-deoxy-2-acetylamino glucose units which are combined by (1,4) glycosidic linkages into a linear polymer. N-deacetylation of chitin results in chitosan, a high molecular weight cationic polysaccharide. The degree of N-acetylation is an important characteristic of chitosan, as it determines the positive charge of the molecule and its polarity. Chitosan may be manufactured with different degrees of acetylation as well as molecular weight (Anthonsen et al., Carbohydrate Polymers, 22, 1993, pp 193-201).
Chitosans are considered biocompatible macromolecules due to assumed low toxicity and biodegradability. They are degraded by lysozymes and related enzymes, such as N-acetyl-D-glucosaminidases. The lysozymic digestibility of the polymer increases with the degree of N-acetylation (Aiba, Int. J. Biol. Macromol., 14, 1992, pp 225-228; Bourbouze et al., Clin. Chim. Acta, 199, 1991, pp 185-194). Their biocompatibility has initiated evaluation of the compound for many medical applications, e.g. in JP 0509295 A; Nishimura et al., Mol. Biother., 2, 1990, pp 115-120; Segal et al., Mycopathologia, 102, 1988, pp 157-163; Nishimura et al., Vaccine, 2, 1984, pp 93-99; Lida et al,. Vaccine, 5, 1987, pp 270-274; and JP 5414809 A. They are also useful as sustained or controlled release carriers for drugs as is disclosed e.g. in JP 04264023 A and EP 460921-A.
In the patent US 4873 092 by Azuma et al chitosans are used as carriers for physiologically active substances, such as adriamycin and testosterone, with the aim to slowly release these substances from the carrier and therewith obtain constant therapeutic (but non-toxic) blood concentration of the drug over an extended period of time. Chitosans that could be used as such slow release carriers are stated to have a degree of N-acetylation of 0-100% and a molecular weight of 10000 to 23000. A paper by Imai et al, Int. J. Pharm., 67, 1991, pp 11-20, describes the use of chitosans with low molecular weight (<25.000) and intermediate degrees of acetylation (10-34%) as carriers for the oral absorption of indomethacin. These chitosans increases the in vitro dissolution of the acidic drug. Since dissolution of the poorly water soluble ;n~omethacin is rate limiting for its oral absorption, the increased dissolution results in an increased oral absorption of the drug.
Chitosans have been found to have mucoadhesive properties, and due to their close contact with the CA 02203632 l997-04-24 mucosal epithelium may prove useful in delivery of drugs to mucosal absorption sites such as the small intestine or colon and rectal mucosa (EP 514008-A1;
Lehr et al., Int. J. Pharm., 78, 1992, pp 43-48).
For various hydrophilic drugs such as several antibiotics, peptides and proteins the solubility of the drugs is not rate limiting in the absorption process. Many of these compounds, however, are poorly absorbed following oral administration due to extensive enzymatic degradation and poor epithelial permeability.
In order to overcome the physical barriers that h;n~er drug absorption it is often inevitable to coadminister so called absorption enhancers. Absorption enhancers as used in the present patent application refer to compounds that increase drug absorption by an interaction between the mentioned compound and the mucosal epithelium. They do not include compounds that increase drug absorption by an interaction with the drug itself. Numerous classes of compounds have been - described that improve the intestinal absorption of hydrophilic and high molecular weight compounds. Such absorption enhancing compounds include surfactants, bile salts, chelating agents, and fatty acids. (Lee et al., Crit. Rev. Therapeut. Drug Carrier Syst., 8, 1991, pp 91-192). A major concern, however, related to the use of many of such absorption enhancing agents is potential adverse effects on epithelial integrity, morphology, and function (Swenson and Curatolo, Adv.
Drug Delivery Rev., 8, 1992, pp 39-92). Moreover, the delivery of an effective amount of enhancer and drug at the site of absorption to get reproducable dosing is necessary, but difficult to achieve. In addition, the onset of action of the enhancer and the duration of its activity should coincide with the presence of the drug at the absorption site and the time necessary for its uptake (De Boer and Breimer, In Drug absorption enhancement, Eds.: De Boer, Harwood Ac. Publ., Chur, 1994, pp 155-175). Compounds with optimal absorption enhancement characteristics, thus should have a strong effect on epithelial permeability, an early onset of action, and a low toxicity.
Recently, chitosans have been investigated as potential absorption enhancing compounds to aid in the absorption of hydrophilic, and high molecular weight drugs across mucosal tissues. Chitosans have been found to enhance the mucosal absorption of insulin, a small model peptide, and a hydrophilic model marker molecule. In a published patent application by Illum et al.
(WO 9009780-A), it is described that the chitosans Seacure and Seacure+ improve the absorption of insulin across the nasal mucosa of sheep and rat to a large extent. The concentration of the chitosan used in these studies is 0.1 to 0.5% (w/v). Seacure+ is stated to be a water soluble chitosan glutamate with either a low (Seacure+) or an intermediate viscosity (Seacure+210).
The degree of acetylation or the molecular weight of the chitosan are not clearly stated. Effective polymers, however, are stated to be chitosans in a molecular weight range of 10,000 to S00,000.
The same water soluble chitosan (Seacure+ 210) has been used to study chitosan effects on Caco-2 cell monolayers (Artursson et al., Pharm. Res., 1994, 11, pp 1358-1361). Caco-2 is a human intestinal epithelial cell line that is commonly used as a model for human intestinal epithelium. The Seacure+ chitosan increases the permeability of the epithelium 5 to 10 fold as measured by the transport of a model hydrophilic compound across the epithelium. Seacure+ is used at concentration from 0.1 to 0.5% (w/v) and the pH of the solutions ranges from 4.0 to 6Ø
s Intermediate viscosity Seacure +210 has in another study (Rentel et al., Proc. Intern. Symp. Control. Rel.
Bioact. Mater., 20, 1993, pp 446-447) been found to improve the transport of a small peptide across intestinal tissue in vitro. In the same study a different chitosan was also tested. This chitosan, Daichitosan VH (DA=15-20~; Mw=500000-800000) showed similar improvement of transport of the small peptide across intestinal mucosa at a concentration of 1 10 (w/v).
Although all the presented studies show improvement of mucosal epithelial permeability in the nose as well as in the intestinal tract in several animal species, including sheep and rat as effectuated by chitosans at different concentrations (0.1-2~ (w/v)) and at different pH values, they do not elucidate the influence of the two important characteristics of chitosans on the absorption enhancing effect, namely the degree of acetylation and the molecular weight. In fact in the described papers none of the parameters are very well specified.
Brief description of the invention According to the present invention it has unexpectedly been found that intermediate but not high degrees of acetylation, i.e. a degree of acetylation of between 20 and 45%, and a high molecular weight, i.e. a molecular weight of above 75,000, of chitosans are necessary for increasing epithelial permeability, to achieve an early onset of absorption enhancement, and a low toxicity of the enhancer.
Detailed descriPtion of the invention Studying the transport of the marker molecule, mannitol, as a model for hydrophilic drugs tAnderberg et al., Pharm. Res., 10, 1993, pp 857-864), and the cardiovascular drug, atenolol, across Caco-2 cell monolayers it was found that chitosans with a low to intermediate degree of acetylation and/or a large molecular weight are necessary in order to enhance permeability. That is, chitosans with a degree of acetylation of 1% (DA=1%) and either a smaii moiecuiar weight (Mw=31000) or a large molecular weight (Mw=170000) have a strong concentration dependent effect on mannitol transport. At concentrations as low as 50 ~g/ml (0.005% (w/v)) they both increase mannitol transport 10- to 15-fold. At a concentration of 250 ~g/ml the increase in permeability is 50-fold.
Chitosans with an intermediate (DA=35~) or high degree of acetylation (DA=49%) and a large molecular weight (Mw=170000 and 98000, respectively) also improve m~nn; tol transport. Concentrations of 50 and 250 ~g/ml, respectively (0.005 and 0.025% (w/v)) are necessary to achieve a 10 to 15 fold increase in mannitol per~hility of the Caco-2 cell monolayers. The increase in mannitol perm~hility of the latter chitosans does not have such a steep concentration/effect relation as is observed for the former chitosans with a low degree of acetylation (DA=1%). On the other hand, chitosans with an intermediate or a high degree of acetylation and a small molecular weight (chitosan (DA=35%, Mw=5300), chitsoan (DA=35%, Mw=12000), chitosan (DA=49%, Mw=22000)) do not have an effect on mannitol transport at all. Concentrations upto 250 ~g/ml (0.025% ~w/v)) do not change the permeability of m~nn; tol across Caco-2 cell monolayers significantly.
Although at a concentration of 50 ~g/ml chitosans with a degree of acetylation of 1~ and a molecular weight of 31000 and 170000, and chitosans with a degree of acetylation of 35% and a molecular weight of 170000, and at a concentration of 250 ~g/ml chitosan with a W0~6/20730 PCT/SE95/01342 degree of acetylation of 49% and Mw of 98000 all show similar enhancement of mannitol permeability across Caco-2 cell monolayers, their adverse effects on epithelial cells are surprisingly very different. In a toxicity assay which measures intracellular dehydrogenase activity and is based on the observation that injured cells display a reduced dehydrogenase metabolism, a much stronger toxicity was observed for chitosans with a low degree of acetylation (DA~
Mw=31000 or 170000), than for chitosans with an intermediate and high degree of acetylation and a large molecular weight (chitosan (DA=35%, Mw=170000), chitosan (DA=49~, Mw=98000). These findings were confirmed by visualisation of the adverse effects of chitosans on epithelial Caco-2 cell monolayers with transmission electron microscopy, which showed that the morphological changes following exposure to chitosans with intermediate and high degree of acetylation and high molecular weight (chitosan DA=35%, Mw=170000;
chitosan DA=49~, Mw=98000) were small, while clear changes were observed following exposure to chitosan with a degree of acetylation of only 1%. These changes involved dicontinuities and reduced number of microvilli, and a disorganized terminal web.
In addition, the onset of action of increased epithelial permeability is much quicker for a chitosan with intermediate degree of acetylation (35%) and large molecular weight (170000), than for chitosan with a low degree of acetylation and small or large molecular weight (DA=1%, Mw=31000 or 170000). This gives the former chitosan more controllable and reliable absorption enhancing characteristics.
In contrast to the findings of Imai et al. (Int. J.
Pharm., 67, 1991, pp 11-20) who found that chitosans with low molecular weight were necessary to increase the dissolution of acidic drugs, we have found that, in order to improve drug absorption by means of increased epithelial permeability for hydrophilic drugs, chitosans with high molecular weight and intermediate degree of acetylation are preferable with regard to improved permeability, early onset of action, and low toxicity.
The invention provides a pharmaceutical formulation of a form and composition suitable for mucosal administration, particularly for oral and/or rectal delivery, of therapeutic agents in man. By oral route means e.g. buccal, intragastric and intestinal. This preparation comprises a therapeutic active agent as well as a chitosan with the below described characteristics as an absorption enhancing agent.
The present invention is based on the observation that it is possible to select chitosans with optimal absorption enhancement characteristics, that is chitosans with a strong effect on eptithelial permeability, low toxicity, and an early onset of action. Such a chitosan would, according to the present invention have a degree of acetylation of between 20 and 45%, and preferably between 30 and 40%, while its molecular weight should be above 75,000, more preferably between about 75,000 and 250,000, and most preferably between about 98,000 and 200,000.
Therapeutic active agents include drugs, vaccines, proteins, peptides and fragments thereof. The pharmaceutical formulation according to the invention may be used with drugs from the following non-exlusive list.
Pharmaceutical formulations containing hydrophilic drugs with low membrane permeability and biovailability which are absorbed mainly via the paracellular route such as;
- Blood pressure lowering agent such as hydrophilic ACE-inhibitors and ~-blockers - Anti-coagulation agents such as thrombin inhibitors heparin and low molecular weight heparin - Antidiuretic drugs and vasopressin analogs such as DDAVP, AVP, etc.
- Peptide hormones such as insulin, calcitonin, growth hormone, PTH
- Antibiotics such as penicillin and penicillin derivatives, tetracycline, and macrolides - Steroidal anti-inflammatory agents such as beclomethasome and budesonide - Non-steriodal anti-inflammatory agents such as diclofenac, indomethacin, ibuprofen, naproxen - Coagulation factors such as Factor VIII
- Vaccines and antigens such as polio vaccine - Anti-viral drugs such as Foscarnet - Compounds effecting bone metabolism such as clodronate and other bisfosfonates - Analgesics such as opioid peptides, dextropropoxifene and pentazocine - Local anesthetics with antiinflammatory properties such as lidocaine, bupivacaine and ropivacaine - Local anesthetics with analgetic properties such as bupivacaine and ropivacaine - Immunomodifiers such as Cyclosporin A
- Anti-allergic drugs such as sodium cromoglycate The pharmaceutical formulations according to the invention will in practice contain a ph~rm~cologically active amount of therapeutic agent. As regards the amount of chitosan the formulation will contain an amount thereof that enhances the absorption of the active agent present. Hereto, it will be necessary to scale the dose of absorption ~n~n~er from in-vitro cell culture studies and ~n;m~l experiments to the therapeutic situation in man. Such a dose scaling will be common for anyone skilled in the art. Chitosan amounts of 5 ~g/dose to 1000 mg/dose are possible.
Amounts of 50 ~g to S00 mg/dose are preferable.
If desired, other absorption enhancers may be included in the pharmaceutical formulations of the invention.
Such enhancers can be inhibitors of enzymatic degration, in order to reduce the amount of drug that is metabolized before absorption. Enzyme inhibitors used to stabilize drugs in mucosal epihelia and the gastrointestinal lumen include bile salts, bacitracin, aminoboronic acid derivatives, bestatin, FK-448, soybean trypsin inhibitor, and aprotin (Muranishi and Yamamoto, In Drug absorption enhancement, Eds.: De Boer, Harwood Ac. Publ., Chur, 1994, pp.67-100).
Formulations suitable for the delivery of the drugs to the small intestine or colon can utilize coated solid dosage forms or enteric systems, which will loose their W096l20730 PCT/SE95101342 coating on arrival in the small intestine due to dissolution of the coating polymers at the local pH
conditions. Colon specific delivery systems may involve polymer coatings that typically are degraded by the colonic bacterial flora. Upon degradation of the coating these delivery systems will release drug and additives.Upon degradation of the coating, these delivery systems will release drug and additive (Friend, Advanced Drug Delivery Reviews, 7, 1991, pp 149-201) Such polymer coatings may for instance comprise azopolymers (Saffran et al., Science, 233, 1986, pp 1081-1084), and polysaccharides such as calcium pectinate (Rubinstein, Pharm. Res., 10, 1993, pp 258-265).
Alternatively, a time dependent colon drug delivery system can be used.
Naturally, the formulations according to the invention may also contain one or more additives conventionally used in mucosal drug delivery, such as preservatives, stabilizers, inert excipients, etc. Agents suitable for these and other purposes are known to anyone skilled in the art of mucosal drug delivery.
Preparation of formulations The chitosan and therapeutic active agent containing formulation may be prepared in a buffered saline solution at a neutral pH, i.e. pH 6.5-7.5. A lower pH
may be used for easier dissolvation of the chitosan by addition of HCl to the afore-mentioned buffer or a different buffer system. The person skilled in the art will be able to dete~~~.,ine the optimal pH which may be between 1.0 and 11.0 and preferably between 4.0 and 7.5.
The chitocan and therapeutic agent formulation may be prepared in any forms suitable for mucosal delivery, such as oral or rectal delivery, such as tablets (e.g.
immediate release tablets, enteric coated tablets, extended release tablets), gelatine capsules, suppositories, microspheres, gels, solutions, suspensions, etc.
The invention will now be illustrated but not limited by the following examples.
Bxample 1: ToxicitY of chitosans as assessed with a dehydroqenase activitY assay in Caco-2 cells Preparation of chitosans with different molecular weiqht and deqree of acetYlation The preparation and characterization of chitosans was performed according to procedures described in Anthonsen et al., Carbohydrate Polymers, 22, 1993, pp 193-201. The chitosans used in the example are given in table 1.
Table 1: Chitosan samples used for structure-effect studies.
Chitosan Degree of Molecular acetylation weight (~) W09612~730 9 15 l90000 PreParation of chitosan solutions Chitosan solutions were prepared by dissolving them at concentration of 0.5 and 1.O mg/ml in Hank's balanced salt solution buffered with 25 mM 2N morpholino ethane sulfonic acid to a pH=5.5 (HBSS pH=5.5). This pH value is comparable to the pH of the small intestine microclimate and facilitates the dissolutions of the chitosans. HBSS is a buffer commonly used in cell culture experiments. Phosphate buffered saline (PBS) and similar physiological buffers could also be used as solvents. Chitosans were dissolved by vigorously shaking for 24 to 48 hours. Just prior to experiments the chitosan solutions were diluted to the appropriate concentrations with HBSS pH=5.5.
Dehydroqenase activity assaY
In these studies Caco-2 cells obtained from American Type Culture Collection, Rockville, USA, were used.
Cells of passage number 93-105 were used throughout.
The cells were seeded in 96-well tissue culture plates (~low Laboratories, Irvine, UK). Af~ër 24 h they were exposed to different concentrations of chitosans for 60 min at 37 C in air. Subsequently, the intracellular dehydrogenase activity was determined by the MTT method as described in Anderberg et al., Pharm. Sci., 81, 1992, 879-887. MTT is a tetrazolium salt that is cleaved by mitochondrial dehydrogenases in living but not dead cells to give a dark blue product. Following addition of MTT, the cells were incubated for another 60 min. The developed colour was measured with a multiwell scanning spectro photometer. The dehydrogenase activity was evaluated as the relative absorbance at 590 nm of the solutions in the 96-well plate as compared to control, the latter being 100%.
Figure 1 shows the effect of chitosans on intracellular dehydrogenase activity. The data are given as the mean of 3 experiments. In general, the difference between individual data points is less than 25%. In figure 1 the different curves relate to ( ) chitosan (DA=l~, Mw=31000), ( ~ ) chitosan (DA=1%, Mw=170000); ( 0 ) chitosan (DA=15%, Mw=4700); ( ~ ) chitosan (DA=15%, Mw=l90000); ( ) chitosan (DA=35%, Mw=12000); ( ~ ) chitosan (DA=35%, Mw=170000); ( O ) chitosan (DA=49%, Mw=22000); ( o ) chitosan (DA=49%, Mw=98000); ( ~ ) chitosan (DA=35~, Mw=5300). For chitosans with a low degree of acetylation chitosan (DA=1%, Mw=31000);
chitosan (DA=1%, Mw=170000); chitosan (DA=15%, Mw=4700); and chitosan (DA=15%, Mw=lgO000); a dose dependent effect on intracellular dehydrogenase activity is observed. Chitosans with an int~rm~;ate degree of acetylation and large molecular weight (chitosan (DA=35%, Mw=170000)) decrease enzyme activity only at higher concentrations. Chitosan (DA=35%, Mw=5300), chitosan (DA=35%, Mw=12000), chitosan (DA=49%, Mw=22000), and chitosan (DA=49%, Mw= 98000) do not show an effect in the concentration range studied.
Exam~le 2: ToxicitY of chitosans as assessed bY effects on e~ithelial cell morpholoqY
Caco-2 cells were obtained from American Type Culture Collection, Rockville, USA. The cells were cultivated on polycarbonate filters (Transwell cell culture inserts, Costar, Cambridge) as described by Artursson, Pharm. Sci., 79, 1990, 476-482. Cells of passage 93-105 were used throughout. The cells were used for experiments 21 to 35 days after seeding. After washing the monolayers apically with HBSS pH=5.5 and basolaterally with Hank's balanced salt solution buffered with 25 mM Hepes to pH=7.4 (HBSS pH=7.4), chitosans were added apically, while the basolateral solution was replaced with fresh HBSS pH=7.4. The chitosans described in example l were diluted from stock solutions as described in example l. All chitosans were added at a concentration 50 ~g/ml, except chitosan (DA=49%, Mw=98000) which was used at 250 ~gtml. Following exposure to the different chitosan solutions for 60 min at 37C in air, the cell monolayers were rinsed twice with PBS and fixed in l.5%
glutaraldehyde and l~ osmium tetroxide, and immersed in 1% uranyl acetate. Thin sections were examined with a Philips 420 electron microscope at 60 kV.
In monolayers treated with 50 ~g/ml chitosan (DA=1%, Mw=31000) and chitosan (DA=l~, Mw=170000) discontinuities in and reduced number of microvilli were observed, while the terminal web showed a disorganized pattern. The tight junctions appeared not to be effected. Following treatment with 50 ~g/ml chitosan ( DA= 3 5 %, MW= 17 0 0 0 0 ) , and 250 ~g/ml chitosan (DA=49%, Mw=98000) the structural changes were not as pronounced. Microvilli and terminal web appeared normal. See figure 2 which is a transmission electron micrograph of a) control cells (magnification 1950x), b) cells treated for 60 min with 50 ~g/ml chitosan (DA=1%, MW=31 000) (magnification 2500x) and c) cells treated for 60 min with 50 ~g/ml chitosan (DA=35~, Mw=170 000) (magnification 2500x). Exposure to chitosan (DA=35%, Mw=5300); chitosan (DA=35%, Mw=12000);
chitosan (DA=49~, Mw=22000) did not result in obvious structural changes.
ExamDle 3: Absorption enhancement of chitosans - effect on ePithelial PermeabilitY
Caco-2 cell monolayers were used as a model for mucosal and in particular intestin~l epithelium. Drug transport across Caco-2 cell monolayers has in the past been demonstrated to correlate excellently with in vivo human intestinal drug absorption. In the presence of absorption enhancer, correlations between in vitro cell culture experiments and in vivo drug absorption have also been established, with corrections in the applied dose due to upscaling sometimes being necessary.
Mannitol was used as a model compound for hydrophilic drugs which show a low and incomplete uptake across mucosal epithelium. Transport of mannitol in Caco-2 cell monolayers has previously been shown to correlate with the transport of sparingly absorbed drugs within a molecular weight range of 100-500. All transport experiments were performed in HBSS. Prior to the experiments the cells were washed apically with HBSS
pH=5.5, and basolaterally with HBSS pH=i. 4. Just before the experiments chitosans described in example 1 were diluted from stock solutions with HBSS pH=5.5 to the appropriate concentrations as described in example 1.
14C-mannitol was subsequently added. The experiments were initiated by replacing the apical blank HBSS
pH=5.5 medium for a 14C-mannitol/chitosan solution, and the basolateral solution for fresh HBSS pH=7. 4.
Transport studies were performed during 120 min in air at 37 C and 95% relative humidity. Samples were taken at regular time intervals from the basolateral side, and were counted in a liquid scintillation counter (Tricarb, l900CA). The apparent permeability (Papp) was calculated using the following equation:
Papp = dQ/dt x l/(AxCo), in which dQ/dt is the rate of appearance of mannitol on the basolateral side, Co is the initial m~nn; tol concentration on the apical side, W096/20730 pcTlsEs5lol342 and A is the surface area of the monolayer.
The mannitol permeability of the Caco-2 cell monolayers could be improved by some but not all of the chitosans studied. Table 2 shows the mean permeability over 120 min during exposure to 50 ~g/ml chitosan solution. The effect of the chitosans was dependent on both the degree of acetylation and the molecular weight of the polymer. At a concentration of 50 ~g/ml, chitosans with a low degree of acetylation showed a clear effect on mannitol transport. Chitosan (DA=1%, MW=31000);
chitosan (DA=1%, Mw=170000); chitosan (DA=15%, Mw=4700); chitosan (DA=15%, Mw=l90000); showed a mean Papp between 0 and 120 min that was increased 5 . 5 - to 15 16.1-fold, as co~pared to control. Chitosans with a larger degree of acetylation and a low or intenmediate molecular weight, such as chitosan (DA=3596, Mw=5300), chitosan (DA=35%, MW=12000 ) and chitosan (DA=49%, Mw=22000) did not influence mannitol transport significantly at concentrations of 50 ~lg/~ . Chitosan, however, with a high molecular weight and intermediate degree of acetylation (DA=35%, Mw=170000) strongly increased the Papp of mAnn;tol. Chitosan with a very large degree of acetylation, but also a high molecular weight (DA=4996, Mw=98000), showed a small but significant increase in m~nn; tol transport at 50 ~g/ml.
Table 2: Effect of different chitosans at a concentration of 50 ~gJml on the permeability of m~nn; tol across Caco-2 cell monolayers.
Chitosan Degree of Molecular Papp x lOE7 Ratio (~g/ml) acetylation weight (cm/sec) Papp (~) chitosan /Papp control control - 2.2 + 0.2 1 31000 28.9 + 5.8 13.2 1 170000 24.0 + 0.7 10.9 5300 2.3 ~ 0.8 1.1 12000 2.7 + 0.2 1.3 170000 29.0 + 3.8 13.2 49 22000 4.8 + 4.4 2.2 49 98000 9.0 + 1.4 4.1 4700 12.8 + 3.4 190000 35.5 + 10.4 The mean Papp for 14C-mannitol are calculated over the total 120 min. period of the experiments. The Papp values are given as the mean + s.d. of 3-4 experiments.
RY~m~le 4: Absorption enhancement of chitosans -concentration dependencY
Mannitol transport experiments were performed with different concentrations of chitosans to study the concentration dependency of the absorption enhancing effect. The chitosans used in the experiments are described in example 1. Solutions having a concentration of 10, 50 and 250 ~g/ml were prepared according to example 1. The transport experiments were similarly performed as in example 3.
W096/20730 PCT/SE9~/01342 The chitosan effect on mannitol transport was concentration dependent. The effect of chitosans at 3 different concentrations 10, 50, and 250 ~g/ml on the Papp of m~nn; tol across Caco-2 cell monolayers are shown in Figure 3. The Papp is calculated as the mean Papp during the 120 min time period of the experiment.
The data are given as the mean + s~d. of 3-4 experiments. ( ~ ) chitosan (DA=1%, Mw=31000); ( ~ ) chitosan (DA=1%, Mw=170000); ( 0 ) chitosan (DA=15%, Mw_4700); ( ) chitosan (DA=15%, Mw=l90000); ( ) chitosan (DA=35~, Mw=1200~ ) chitosan (DA=35%, Mw=170000); ( O ) chitosan (DA=49%, Mw=22000); ( o ) chitosan (DA=49%, Mw=98000); ( ~ ) chitosan (DA=35%, Mw=5300).
A 10 ~g/ml dose gave a small increase in ~nn; tol transport for chitosan (DA=l~, Mw=31000), chitosan (DA=1%, Mw=170000), and chitosan (DA=15%, Mw=l90000) while all other chitosans did not have an effect at this concentration. At 250 ~g/ml chitosan (DA=1%, Mw=31000), chitosan (DA=1%, Mw=170000), chitosan (DA=15%, Mw=4700) and chitosan (DA=15~, Mw=l90000) increased the permeability to about 50 to 60 times the control value. For chitosan (DA=35~, Mw=170000) the mean Papp at 250 ~g/ml was not significantly different from the Papp value obtained with 50 ~g/ml. Chitosan (DA=49, Mw=98000) increased the Papp approximately 10 times at 250 ~g/ml.
Example 5: AbsorPtion enhancement of chitosans - onset of action of increased ePithelial DermeabilitY
Similar transport experiments as described in example 3 were done to study the onset of action of increased permeability of chitosans. The chitosans used in the experiments and the preparation of the different solutions are described in example 1.
The onset of action of chitosans that increased . epithelial permeability was not the same for chitosans.
Although the mean Papp measured over 120 min was similar for chitosan (DA=1%, Mw=31000), chitosan (DA=1%, MW=170000), chitosan (DA=15%, MW=19OOOO) at a concentration of 50 ~g/ml, and chitosan (DA=49%, MW=98000) at a concentration of 250 ~g/ml, the permeabilities at 20 min after the start of the experiment were very different. Papp values at 20 min are summarized in table 3. Exposure to chitosan with intermediate degree of acetylation and high molecular weight (DA=35, MW=170000) showed a much stronger increase in permeability after 20 min than all other chitosans (compare figure 3 and table 2 with table 3).
Table 3: Effect of different chitosans on the permeability of mannitol across Caco-2 cell monolayers after 20 min exposure.
Chitosan Degree of Molecular Papp x lOE7 Ratio (~gtml) acetylation weight (cm/sec) Papp (~) chitosan /Papp control control - 2.7 + 0.5 1 31000 4.4 + 0.6 1.6 1 170000 3.9 + 0.4 1.4 5300 3.3 + 0.8 ~.. 2 12000 2.9 + 0.2 1.1 170000 8.4 i 1.8 3.1 49 22000 1.7 i 0.3 0.6 250 49 98000 3.8 i 0.7 1.4 4700 4.3 i 1.0 190000 5.0 + 0.4 The Papp for 14C-mannitol are ca~culated over the first 20 min period of the experiments. The Papp values are given as the mean + s.d. of 3-4 experiments.
Example 6: AbsorPtion enhancement of chitosans - effect on the ePithelial ~erm~hilitY of atenolol Just before the start of the experiments chitosan (DA=1%, Mw=31.000) and chitosan (DA=35, Mw=170.000), as described in example 1, were diluted from stock solutions with HBSS pH=5.5 to a concentration of 50 ~g/ml, as described in example 1. Caco-2 cell transport W 096/20730 PCT/SE9~/01342 experiments were performed, subsequently, as described in example 3. Atenolol concentrations were determined using HPLC consisting of a RP Ultrasphere ODS 5 ~m (4.6 mm x 25 cm) column connected to a W detector.
Absorption was measured at 270 nm. The mobile phase consisted of phosphate buffer (pH=3) and acetonitrile in a ratio of 90:10.
Figure 4 shows the Papp of atenolol during exposure to 50 ~g/ml chitosan solution. The data are given as mean ~ SD of 4 experiments. In figure 4 the different curves relate to (x) control; (d) chitosan (DA=1%, Mw=31000);
and (~) chitosan (DA=35, Mw=170000). The permeability of the Caco-2 cell monolayers was low (0.5-1.0x10-7).
The perme~hility could be improved, however, by chitosan (DA=1%, Mw=31.000) and chitosan (DA=35, Mw=170.000). Similarly, as observed for mannitol p~rm~Ahility the effect of chitosan (DA=35, Mw=170.000) was larger than the effect of chitosan (DA=1%, Mw=31.000) during the early stages of the experiment.
For chitosan (DA=35, Mw=170.000) the atenolol permeability did not increase further after 20 min exposure, while for chitosan (DA=1%, Mw=31.000) atenolol permeability kept increasing until the end of the experiment.
Claims (9)
1. A pharmaceutical formulation characterized in that it comprises a chitosan polymer having a degree of acetylation of between 20 and 45%
and a molecular weight of above 75,000, and a therapeutic active agent.
and a molecular weight of above 75,000, and a therapeutic active agent.
2. A pharmaceutical formulation according to claim 1 c h a r a c t e r i z e d in that the degree of acetylation of the chitosan polymer is between 30 and 40%
3. A pharmaceutical formulation according to claim 1 c h a r a c t e r i z e d in that the molecular weight of the chitosan polymer is between 75,000 and 250,000, and preferably between 98,000 and 200,000.
4. A pharmaceutical formulation according to claim 1 c h a r a c t e r i z e d in that the therapeutic active agent is selected from the group drugs, vaccines, proteins, peptides and fragments thereof.
5. A chitosan polymer for preparing the formulation according to claim 1 c h a r a c t e r i z e d in that it is a chitosan polymer having a degree of acetylation of between 20 and 45% and a molecular weight of above 75,000.
6. A chitosan polymer according to claim 5 characterized in that the degree of acetylation of between 30 and 40%.
7. A chitosan polymer according to claim 5 characterized in that the molecular weight is between 75,000 and 250,000, and preferably between 98,000 and 200,000.
8. Use of a chitosan polymer according to claim 5 in the manufacture of a pharmaceutical formulation comprising a chitosan polymer having a degree of acetylation of between 20 and 45% and a molecular weight of above 75,000 and a therapeutic active agent.
9. A method for enhancing the absorption of a therapeutic active agent by administering a sufficient amount of a formulation comprising a chitosan polymer having a degree of acetylation of between 20 and 45%
and a molecular weight of above 75,000 and said therapeutic active agent to a host in need of such a therapeutic treatment.
and a molecular weight of above 75,000 and said therapeutic active agent to a host in need of such a therapeutic treatment.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE9403905-4 | 1994-11-14 | ||
| SE9403905A SE9403905D0 (en) | 1994-11-14 | 1994-11-14 | New formulations |
| PCT/SE1995/001342 WO1996020730A1 (en) | 1994-11-14 | 1995-11-13 | A chitosan polymer having a specific degree of acetylation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2203632A1 true CA2203632A1 (en) | 1996-07-11 |
Family
ID=20395954
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2203632 Abandoned CA2203632A1 (en) | 1994-11-14 | 1995-11-13 | A chitosan polymer having a specific degree of acetylation |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP0792330A1 (en) |
| JP (1) | JPH10509207A (en) |
| AU (1) | AU710769B2 (en) |
| CA (1) | CA2203632A1 (en) |
| FI (1) | FI972056A (en) |
| IS (1) | IS4469A (en) |
| NO (1) | NO972172D0 (en) |
| SE (1) | SE9403905D0 (en) |
| WO (1) | WO1996020730A1 (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6716970B2 (en) | 2000-04-21 | 2004-04-06 | Adjuvant Pharmaceuticals, Llc | Water soluble, randomly substituted partial N-partial O-acetylated chitosan, preserving compositions containing chitosan, and processes for making thereof |
| US20020018732A1 (en) * | 2000-04-21 | 2002-02-14 | Hung William M. | Preserving compositions containing chitosan and processes for making water soluble O-acetylated chitosan and chitosan |
| GB0315632D0 (en) | 2003-07-04 | 2003-08-13 | West Pharm Serv Drug Res Ltd | Pharmaceutical formulations |
| US7125967B2 (en) | 2003-10-08 | 2006-10-24 | Adjuvant Pharmaceuticals, Llc | Water-soluble chitosan having low endotoxin concentration and methods for making and using the same |
| US20060287278A1 (en) * | 2005-06-21 | 2006-12-21 | Zhenze Hu | Method for improving water solubility of chitosan |
| FR3016882A1 (en) | 2014-01-30 | 2015-07-31 | Sofradim Production | PROCESS FOR THE PREPARATION OF HIGH-DEGREE ACETYLATION CHITOSAN |
| EP3034612A1 (en) * | 2014-12-16 | 2016-06-22 | Greenaltech, S.L. | Chitin and chitosan producing methods |
| EP3372234B1 (en) * | 2015-11-04 | 2021-09-29 | Stelic Institute & Co., Inc. | Complex comprising rnai molecule and n-acetylated chitosan |
| WO2024054707A2 (en) | 2022-09-08 | 2024-03-14 | Luna Labs Usa, Llc | Layer-by-layer delivery of active agents |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4873092A (en) * | 1987-05-21 | 1989-10-10 | Murata Kikai Kabushiki Kaisha | Slow-releasing preparation |
| GB8904370D0 (en) * | 1989-02-25 | 1989-04-12 | Cosmas Damian Ltd | Liquid delivery compositions |
-
1994
- 1994-11-14 SE SE9403905A patent/SE9403905D0/en unknown
-
1995
- 1995-11-13 JP JP8517163A patent/JPH10509207A/en not_active Ceased
- 1995-11-13 WO PCT/SE1995/001342 patent/WO1996020730A1/en not_active Application Discontinuation
- 1995-11-13 AU AU39433/95A patent/AU710769B2/en not_active Ceased
- 1995-11-13 EP EP95937278A patent/EP0792330A1/en not_active Withdrawn
- 1995-11-13 CA CA 2203632 patent/CA2203632A1/en not_active Abandoned
-
1997
- 1997-04-22 IS IS4469A patent/IS4469A/en unknown
- 1997-05-12 NO NO972172A patent/NO972172D0/en not_active Application Discontinuation
- 1997-05-14 FI FI972056A patent/FI972056A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| JPH10509207A (en) | 1998-09-08 |
| FI972056A0 (en) | 1997-05-14 |
| IS4469A (en) | 1997-04-22 |
| SE9403905D0 (en) | 1994-11-14 |
| FI972056A (en) | 1997-05-14 |
| WO1996020730A1 (en) | 1996-07-11 |
| NO972172L (en) | 1997-05-12 |
| AU710769B2 (en) | 1999-09-30 |
| EP0792330A1 (en) | 1997-09-03 |
| NO972172D0 (en) | 1997-05-12 |
| AU3943395A (en) | 1996-07-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Van der Merwe et al. | Trimethylated chitosan as polymeric absorption enhancer for improved peroral delivery of peptide drugs | |
| Kulkarni et al. | Xyloglucan: A functional biomacromolecule for drug delivery applications | |
| Singhvi et al. | Xanthan gum in drug delivery applications | |
| Cortes et al. | Xanthan gum in drug release | |
| Kumar et al. | Chitosan chemistry and pharmaceutical perspectives | |
| Dodane et al. | Pharmaceutical applications of chitosan | |
| Singla et al. | Chitosan: Some pharmaceutical and biological aspects‐an update | |
| Hejazi et al. | Chitosan-based gastrointestinal delivery systems | |
| Di Colo et al. | Effect of chitosan and of N-carboxymethylchitosan on intraocular penetration of topically applied ofloxacin | |
| US7883723B2 (en) | Water soluble chitosan nanoparticle for delivering an anticancer agent and preparing method thereof | |
| RU2424793C2 (en) | Pharmaceutical composition including diclofenac | |
| JP4648632B2 (en) | New biomaterials, their manufacture and use | |
| EP1126881B1 (en) | Muco-adhesive polymers, use thereof and method for producing the same | |
| EP2360188A1 (en) | Hyaluronic acid derivative and pharmaceutical composition thereof | |
| FR2919188A1 (en) | COMPLEXES BETWEEN AN AMPHIPHILIC POLYMER AND A OSTEOGENIC PROTEIN BELONGING TO THE BMPS FAMILY | |
| Noreen et al. | Terminalia arjuna gum/alginate in situ gel system with prolonged retention time for ophthalmic drug delivery | |
| EA010351B1 (en) | Delivery of physiological agents withgels comprising anionic polysaccharides | |
| AU6219599A (en) | Formulations of fexofenadine | |
| AU710769B2 (en) | A chitosan polymer having a specific degree of acetylation | |
| EP3442510A1 (en) | Anti-infective compositions comprising phytoglycogen nanoparticles | |
| Ricci et al. | Chitosan/sulfobutylether-β-cyclodextrin based nanoparticles coated with thiolated hyaluronic acid for indomethacin ophthalmic delivery | |
| Ugoeze | Bioadhesive polymers for drug delivery applications | |
| Hejazi et al. | Chitosan-based delivery systems: Physicochemical properties and pharmaceutical applications | |
| CN111732675A (en) | Hyaluronic acid-glucosamine graft copolymer, preparation method and application thereof | |
| Vibhooti et al. | Eudragit and chitosan—The two most promising polymers for colon drug delivery |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Dead |