CN100371018C - Calcitonin composition - Google Patents
Calcitonin composition Download PDFInfo
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- CN100371018C CN100371018C CNB031428266A CN03142826A CN100371018C CN 100371018 C CN100371018 C CN 100371018C CN B031428266 A CNB031428266 A CN B031428266A CN 03142826 A CN03142826 A CN 03142826A CN 100371018 C CN100371018 C CN 100371018C
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Abstract
The present invention relates to a composition of calcitonin and liposome. The composition comprises calcitonin and liposome composed of natural and/or synthetic phosphatide, cholesterol, adjuvant and other surfactants. The composition can increase the bioavailability of the administration of the calcitonin on the mucosa and the skin, is nearly nonirritant to the mucosa, and the composition can also be used for administration by injection.
Description
Invention field the present invention relates to the calcitonin liposome composition of the higher bioavailability of having of a kind of through mucous membrane and percutaneous drug delivery, and said composition also can be used for drug administration by injection.
Background technology is along with the genetically engineered drug progress of research, and very potential albumen and polypeptide drug seem more and more important in recent years on clinical treatment.Calcitonin is the hormone of excretory a kind of 32 peptides of parafollicular cell in the thyroid, is the polypeptide with disulfide bridge bond, and molecular weight is about 3400-3600.At present, calcitonin has shown good curative effect at aspects such as treatment osteoporosis, hypercalcemia, hyperparathyroidism, scleromalacia and bone healings.Therefore, calcitonin has good application prospects.
As everyone knows, because the hydrolysis of gastrointestinal tract protease, albumen and polypeptide drug are oral invalid substantially, therefore the general parenteral route administration that adopts, administering mode commonly used is a drug administration by injection, and as intravenous administration and subcutaneous administration, but this administering mode brings the side effect of whole body and causes pain because of the rapid rising and the decline subsequently of blood level, and the half-life is shorter, effects limit such as patient's compliance difference need the clinical practice of the genetically engineered drug of long-term, repeat administration.Therefore select suitable non-injection administration approach, or the half-life of prolong drug, be subjected to vast pharmaceutics worker's concern.Through mucous membrane and percutaneous drug delivery, medicine can be directly sees through that mucosa and skin absorbs enter in the body and the advantages such as degraded of avoiding first pass effect of hepar and gastrointestinal mucosal metabolism and gastro-intestinal Fluid, so mucosa and percutaneous drug delivery are considered to the drug systemic administration route of albumen and the comparatively ideal replacement drug administration by injection of polypeptide drug.Yet calcitonin is the macromole water soluble drug, is not having under the situation of absorption enhancer, and light water solution through mucous membrane and percutaneous drug delivery exist the low-down shortcoming of bioavailability.Therefore the direction of many researchs is to seek absorption enhancer and carrier effective and that toxicity is low.
The calcitonin nasal mucosa medicine administration is morning and comparatively successful a kind of route of administration of research, but do not having under the situation of absorption enhancer, the regular solution nasal-cavity administration exists the low shortcoming of bioavailability, compare with intravenous injection, abroad the bioavailability of the calcitonin salmon nasal spray (Calcitonin) that has gone on the market only is 3%.[Seiya Kagatani such as Kagatani, Tatsuki Shinoda, Muneo Fukui, et al., Enhancement ofnasal salmon calcitonin absorption by lauroylcarmitine chloride in rats.Pharmaceutical Research 13 (1996), 739～743] adopt and to fall the blood calcium method effect of the short salmon calcitonin see calcimar nasal absorption of carnitine hydrochloric acid salt penetrating agent is studied.The result shows that the short effect of oozing of the lauroyl carnitine (LCC) with 12 carbon atom chains is best, has increased the salmon calcitonin see calcimar per nasal significantly and has absorbed.The ratio of LCC and salmon calcitonin see calcimar is short to ooze best results at 5: 1, and is not subjected to the influence of osmotic pressure, but its short effect of oozing is relevant with pH value, and to urge to ooze effect during for 3.1-4.0 best as pH, but at this moment very big to the zest of nasal mucosa, limited its application.[Kazuhiro Morimoto such as Morimoto, Hideyuki Katsumata, etal., Evaluation of gelatin microspheres for nasal and intramuscular administrations ofsalmon calcitonin, European Journal of Pharmaceutical Science 13 (2001), 179-185] with salmon calcitonin see calcimar gelatine microsphere nasal-cavity administration with different electric charges.Discover that the mucosal adhesive ability of falling blood calcium effect and microsphere behind the salmon calcitonin see calcimar nose administration is electrically charged relevant with institute.Because the mucosal adhesive effect of positively charged gelatine microsphere is apparently higher than electronegative group, correspondingly, positively charged salmon calcitonin see calcimar gelatine microsphere to fall the blood calcium effect more effective.But no matter be positively charged or electronegative gelatine microsphere owing to have mucosa-adherent, so the absorption of salmon calcitonin see calcimar gelatine microsphere is all than the height of PBS (pH7.0) solution group, but the very easy foreign bodies removal that is taken as of salmon calcitonin see calcimar gelatine microsphere.[Akira Yamamoto, Tomoya Iseki, et al., Absorption of water-soluble compounds with different molecularweights and[Asu such as Yamamoto
1.7]-eel calcitonin from various mucosal administration sites, Journalof controlled Release 76 (2001) 363-374] Anguillar japonica calcitonin solution is carried out different mucosa deliveries, the result shows that the absorption of pulmonary is the highest, and next coming in order are nasal cavity, large intestine, small intestinal and oral cavity.Studies show that salmon and human calcitonin can obtain 17% absolute bioavailability after the administration in the rat trachea; Compare with subcutaneous injection, the absolute bioavailability of human calcitonin pulmonary administration is 68%[J.S.Patton, P.Trinchero, R.M.Plats, Bioavailability of pulmonary delivery of peptides and proteins:alpha-interferon, calcitonins, and parathyroid hormone, J Control.Release 28 (1994) 79～85].[L.J.Deffos such as Deffos, J.J.Nolan, B.L.Seely et al., Intrapulmonary drug delivery of salmoncalcitonin, Calcif.Tissue Int.61 (1997) 345～347] estimated different salmon calcitonin see calcimar powder formulations, compare with injection, pulmonary sucks the biological activity of experiment demonstration 66% and 28% bioavailability, but patient can not autonomous easily medication.
[Juie L.Richardson such as Richardson, Piera Angela Ramires, et al., Novel vaginaldelivery systems for calcitonin:I.Evaluation of HYAFF/calcitonin microspheres inrats, International Journal of Pharmaceutics 115 (1995) 9-15] to salmon calcitonin see calcimar aqueous solution and salmon calcitonin see calcimar HYAFF microsphere the absorption after the rat vagina administration study.The result shows that salmon calcitonin see calcimar HYAFF microsphere has shown good adhesiveness and can reduce serum calcium ion concentration significantly, and it is suitable with subcutaneous injection salmon calcitonin see calcimar solution (10IU/kg) effect to fall the blood calcium effect behind the vagina administration 100IU/kg salmon calcitonin see calcimar HYAFF microsphere.[Nakada Y such as Nakada, Miyake M, Awata N, Some factorsaffecting the vaginal absorption of human calcitonin in rats, International Journal ofPharmaceutics 89 (1993) 169-175] human calcitonin is discovered through the rat vagina administration absorption of human calcitonin is subjected to the influence of pH and has nothing to do with osmotic pressure.It is very low that human calcitonin does not add any additives vagina administration artifact availability under the pH7.4 condition, in solution, add sodium deoxycholate, when fixed, good Copeptin of protease inhibitor beta and Pepstatin A, the absorption of human calcitonin is improved, and the facilitation of human calcitonin transvaginal administration post-absorption is relevant with the inhibitory action of protease inhibitor.[Golomb G such as Golomb, Avramoff A, Hoffman A, Anew route of drug administration:Intrauterine delivery ofinsulin and calcitonin.Pharmaceutical Research 10 (1993) 828～833] healthy rat of oophorectomize after one month is divided into salmon calcitonin see calcimar subcutaneous injection group at random, in utero splashes into administration group and normal saline and in utero splash into three groups.Dosage is 0.8IU/kg (0.2ml).The result in utero with the subcutaneous injection administration after, obtain similar calcium ion concentration-time graph; Serum calcium ion concentration all significantly reduces.Low blood calcium is reflected at behind two kinds of administrations and reaches minimum in the 4hr, reduce respectively 29% and the AUC meansigma methods difference of 28.3%, two kind of approach not obvious, in utero administration and subcutaneous injection administration are effective equally to have proved calcitonin.
[Byran H.P.Li such as Byran, George C Y Chiou, Systemic administration of calcitoninthrough ocular route.Life of Science, 50:359-354] calcitonin has been done research through the feasibility that the eye administration carries out whole body therapeutic.When only with simple calcitonin aqueous solution (0.05%) dosing eyes, the blood drug level peak value of gained is compared low (about 1%) very with intravenous injection; And after in eye drop, adding 0.5%BL-9, calcitonin solution (0.5%) has been improved 16 times through the blood drug level peak value that eye absorbs; The short assimilation effect of Brij-78 is better, and the peak value of blood drug level has been improved 24 times, and its blood drug level reaches peak value at 45min, and the blood drug level when 30min has been higher than the intravenous blood drug level of 0.05% calcitonin solution.Result of study shows that under the condition of accelerant B L-9 and Brij-78 existence, calcitonin solution is behind dosing eyes, calcitonin can be absorbed and enter systemic circulation system, and obtain to reach the blood drug level of treatment level, but promoter is too big to the zest of eyes, is unsuitable for using.
Calcitonin can not enter in the body by spontaneous transdermal originally as macromole polypeptide, water soluble drug, and needs finish percutaneous dosing by other active promotion mechanism.Mainly be the calcitonin percutaneous to be imported in the body at present by iontophoresis.[Patrizia Santi such as Santi, Paolo Colombo, et al., Drug reservoircomposition and transport of salmon calcitonin in transdermal Iontophoresis, Pharmaceutical Research 14 (1997) 63-66] by comparing salmon calcitonin see calcimar iontophoresis transport through skin ability size under pH4.2 and pH7.4 condition, proved that salmon calcitonin see calcimar solution is more because of institute's positively charged under the pH4.2 condition, the blood calcium effect of falling that iontophoresis causes wants much remarkable.People in order to eliminate the skin polarization phenomena, reduce the zest to skin in application, adopted the pulse current with certain frequency and dutycycle morely.[Katsuhiro Nakamura such as Nakamura, Kazuya Katagai, et al., Transdermaladministration of salmon calcitonin by pulse depolarization-iontophoresis in rats, International Journal ofPharmaceutics 218 (2001) 93-102] use depolarization pulse ion introductory technique (PDP-IP).Studies show that frequency is that 30kHz, dutycycle are at 30% o'clock, the best electric current that imports salmon calcitonin see calcimar is 0.1-1.0mA, can import the salmon calcitonin see calcimar of 0.2-4 μ g effectively; When electric current increases again, make the absorption of medicine descend on the contrary, infer that its former because excessive electric current causes due to the destruction of skin texture.
[Sophie Thysman such as Thysman, Cecile Hanchard, Veronique Preat, Human calcitonindelivery in rats by Iontophoresis, Journal of Pharm.Pharmacol.46 (1994) 725-730] at research salmon calcitonin see calcimar solution (pH4,50 μ g/ml) during iontophoresis, adopt electric current 0.33mA.cm
-2, 2.5kHz, dutycycle be 1: 1, administration time extends to 1 hour from 20min, the importing flow of finding salmon calcitonin see calcimar does not raise, and thinks that this may be that salmon calcitonin see calcimar gets clogged at the skin pore place of transhipment medicine or elder generation accumulates in skin, thereafter the reason that constantly therefrom discharges.What experimental results show that in the body that this method can cause rat falls the blood calcium effect.Penetration enhancer and iontophoresis merge to use and may produce synergism, for greatly reducing drug dose and the penetration enhancer consumption provides possibility.[Yuso Tomohira such as Tomohira, Yoshiharu Machida, et al., Iontophoretic transdermal absorption of insulin andcalcitonin in rats with newly-devised switching technique and addition of urea, International Journal of Pharmaceutics 155 (1997) 231-239] carbamide and iontophoresis use in conjunction have just been obtained good effect, and reduced zest to skin.In addition, in preparation, add enzyme inhibitor such as aprotinin [Kazuhiro Morimoto, Yasushi Iwakura, et al., Effects of proteolyticenzyme inhibitors as absorption enhancers on the transdermal iontophoresis deliveryof calcitonin in rats, Journal of Pharm.Pharmcol.44 (1992) 216-218] etc. the degraded of the enzyme that existed in the skin of reduction, also improved the transport through skin efficient of salmon calcitonin see calcimar effectively.
Prior art shows that absorption enhancer and other technology also cause the bigger zest to mucosa and skin in the permeability that increases mucosa and skin, and bioavailability can't be satisfactory.
Therefore, still need to develop a kind of nonirritant and toxicity, good stability, through mucous membrane and percutaneous drug delivery with higher bioavailability, or the calcitonin composition of the half-life of prolong drug injection.
Summary of the invention the invention provides a kind of calcitonin liposome composition on the one hand.
On the other hand, the invention provides the purposes that the calcitonin liposome composition is used for through mucous membrane and percutaneous drug delivery, also can be used for drug administration by injection.
According to an aspect of the present invention, it provides a kind of liposome composition that is used for through mucous membrane and skin and drug administration by injection, the liposome that it comprises calcitonin and is made of phospholipid, cholesterol, adjuvant and other surfactant.
The invention still further relates to the method for preparing liposome composition, this method is selected from membrane process, reverse phase evaporation, injection method, multi-emulsion method, fusion method, freeze-drying, centrifuging, calcium fusion method, ammonium sulphate gradient, pressurization extrusion molding, pro-liposome method, aerosol method and dialysis.In each preparation method, all earlier calcitonin is dissolved in aqueous phase.
The present composition can be mixed with various preparations according to any conventional method, for example nasal drop, spray, aerosol, gel, cream, ointment, injection.
The present composition can be used for preparing treatment osteoporosis, scleromalacia, hypercalcemia and hypercalcemia crisis, trophesy, acute pancreatitis disease, hyperparathyroidism, bone healing, osteodynia and cataract and with the medicine of the unusual diseases associated of blood calcium.
The present composition is used to prepare the medicine for the treatment of above-mentioned disease can be by following administration: nasal mucosa, oral mucosa, vaginal mucosa, uterine mucosa, lung mucosa, intestinal mucosa, eye and skin, injection.Preferably by nasal mucosa and/or mouth mucosa drug administration.More preferably pass through nasal mucosa medicine administration.
Liposome composition of the present invention is stable homodisperse Emulsion form, and comprises the calcitonin as active component, also comprises the liposome that is made of phospholipid, cholesterol, adjuvant and other surfactant.
Compositions of the present invention improves the interior bioavailability of the body of calcitonin through mucous membrane and percutaneous drug delivery greatly.The representative example of the calcitonin in the present composition is salmon calcitonin, human calcitonin, clcatonin, chicken calcitonin, pig calcitonin, cattle calcitonin, sheep calcitonin, Mus calcitonin and Oncorhynchi calcitonin.Calcitonin in the present composition is wherein a kind of at least, and the content of calcitonin is 0.0001-1% in the compositions, preferred 0.0005-0.01%, and more preferably 0.0005-0.005% is in the gross weight of compositions.
Phospholipid is the main component that constitutes the liposome in the present composition.Can use phospholipid in the present invention is natural and/or synthetic phospholipid, and its representative example comprises soybean phospholipid, lecithin, Polyethylene Glycol-DSPE, PHOSPHATIDYL ETHANOLAMINE, cephalin, dipalmitoyl phosphatidyl choline, sphingomyelin, sphingo, two myristoyl lecithin or their derivant or their mixture.Contain phospholipid 2-10% in the present composition, preferred 2-6%, more preferably 3-5% is in the gross weight of compositions.The preferred phospholipid that uses in the present invention is phospholipid of natural soybean and lecithin.
Cholesterol also is the composition of the formation liposome used always, and its adding can be regulated the flowability of double-layer of lipoid.The present composition contains cholesterol 0-5%, preferred 0-3%, and more preferably 0.5-2% is in the gross weight of compositions.
Adjuvant makes liposome positively charged, with the mucosa or the skin adherence performance of the increase present composition, and helps the stable of liposome when storing.The adjuvant that can use in the present invention is cholesterol derivative, 18-amine., dimethyl bromine ammonium, chitosan, the arabic gum of lotus positive electricity, and their mixture.Contain adjuvant 0-5% in the present composition, preferred 0-2%, more preferably 0-1% is in the gross weight of compositions.The preferred adjuvant that uses in the present invention is a cholesterol derivative.
The ability that other surfactant helps to strengthen the morphotropism of liposome and sees through mucosa and skin improves transmitance and penetration speed that medicine sees through mucosa and skin.Contain other surfactant 0-6% in the present composition, preferred 0-4%, more preferably 0-2% is in the gross weight of compositions.Representational other surfactant that can use in the present invention comprises:
(1) cholate and deoxidation cholate are as sodium cholate, NaGC;
(2) polyoxyethylene sorbitan fatty acid ester (Tween) is as Tween-80;
(3) sorbitan fatty acid ester (Span) is as Span80;
(4) polyoxyethylene fatty acid ester (Myrj) is as Myrj59;
(5) polyoxyethylene aliphatic alcohol ether (Brij) is as Brij35;
(6) the acidifying natural or hydrogenated vegetable oil (Cremophor) of polyoxyethylene glycol is as CremophorEL;
(7) ethylene oxide-propylene oxide copolymer (Pluronic) is as Pluronic F68;
(8) quaternary ammonium salt that is the straight chained alkyl of 10-20 with a carbon atom quantity, and their mixture.
In above-mentioned surfactant, being preferred for of the present invention is sodium deoxycholate and sodium cholate.
In addition, compositions of the present invention also comprises pharmaceutically acceptable antioxidant and/or antiseptic.
Liposome composition of the present invention can prepare by conventional method, and these methods are selected from membrane process, reverse phase evaporation, injection method, multi-emulsion method, fusion method, freeze-drying, centrifuging, calcium fusion method, ammonium sulphate gradient, pressurization extrusion molding, pro-liposome method, aerosol method and dialysis.Concrete operations are referring to " novel pharmaceutical formulation and new technique ", and 115-120 page or leaf (People's Health Publisher) is hereby incorporated by.In each preparation method, all earlier calcitonin is dissolved in aqueous phase.Wherein the effective grain size of liposome is below 200nm.
The present composition can be mixed with various preparations with pharmaceutically useful additive and adjuvant, for example nasal drop, spray, aerosol, gel, cream, ointment and injection according to any conventional method.
The present composition can be used for preparing treatment osteoporosis, scleromalacia, hypercalcemia and hypercalcemia crisis, trophesy, acute pancreatitis disease, hyperparathyroidism, bone healing, osteodynia and cataract and with the medicine of the unusual diseases associated of blood calcium.Therefore, can select to add other adjuvant and additive according to the difference of route of administration.
The present composition is used to prepare the medicine for the treatment of above-mentioned disease can be by following administration: nasal mucosa, oral mucosa, vaginal mucosa, uterine mucosa, lung mucosa, intestinal mucosa, eye and skin and injection.Preferably by nasal mucosa and/or mouth mucosa drug administration.
In order to reach purpose of the present invention, the present invention carried out the local irritation test, fall the blood calcium Effect Evaluation and through the pharmacokinetics test of the rat nasal administration present composition.
Description of drawings with reference to the accompanying drawings 1 and 2, by the following description of the present invention, above-mentioned and other purpose of the present invention and feature will be conspicuous.Accompanying drawing 1 has shown that calcitonin aqueous solution subcutaneous injection and nasal mucosa medicine administration and calcitonin liposome composition of the present invention fall the blood calcium effect through nasal mucosa medicine administration.Accompanying drawing 2 has shown the bioavailability of calcitonin liposome composition of the present invention through nasal mucosa medicine administration and calcitonin aqueous solution intravenous administration.
As mentioned above, compositions of the present invention significantly improves the bioavailability of calcitonin through mucous membrane and percutaneous drug delivery.
Following examples are used to further describe the present invention, but limit the scope of the present invention anything but.
The specific embodiment
Embodiment 1: the preparation of nasal drop
Phospholipid of natural soybean 525.0mg, cholesterol 175.0mg, 18-amine. 27.0mg, Brij35 166.0mg and an amount of vitamin E are put in the eggplant-shape bottle, (1: 1v/v) mixed solvent makes its dissolving to add 200ml chloroform-methanol, rotary evaporation is removed organic solvent, forms even class membrane of lipoprotein at the eggplant-shape bottle wall.Film is washed in phosphate-buffered aqueous solution (pH7.0) the aquation rotation that adds the calcitonin of 10ml30 μ g/ml then, again the vortex vibration make film disperse liposome turbid liquor.Promptly get homodisperse calcitonin liposome composition through 0.2 μ m filtering with microporous membrane.
Then, add benzalkonium chloride in resulting composition, mix homogeneously is divided in the calcitonin liposome composition in the drop bottle again.
Embodiment 2: the preparation of nasal drop
Phospholipid of natural soybean 90.0mg, 18-amine. 10.8mg, Cremophor EL 4.8mg and an amount of vitamin E are dissolved in the 20ml ether altogether, this diethyl ether solution is splashed into 2ml at the uniform velocity lentamente to be contained in the phosphate buffered solution (pH7.0) of 700 μ g/ml calcitonins (30 ℃-35 ℃), stir while dripping, add the back and continue to stir 2 hours, again vortex vibrate liposome turbid liquor.Promptly get homodisperse calcitonin liposome composition through 0.2 μ m filtering with microporous membrane.
Then, add benzalkonium chloride in resulting composition, mix homogeneously is divided in the calcitonin liposome composition in the drop bottle again.
Embodiment 3: the preparation of nasal drop
Phospholipid of natural soybean 37.5mg, cholesterol 17.7mg, Polyethylene Glycol-DSPE 0.38mg, 18-amine. 2.7mg, Tween-80 8.3mg and an amount of vitamin E are put in the eggplant-shape bottle, add the 20ml chloroform and make its dissolving, the phosphate buffered solution (pH7.0) that adds the calcitonin that contains 25 μ g/ml again, the vortex concussion, 25 ℃ of ultrasonic one-tenth breasts of bath formula, room temperature decompression rotary evaporation is removed organic solvent, become after the colloidal state again that reduction vaporization gets even suspension, promptly get homodisperse calcitonin liposome composition through 0.2 μ m filtering with microporous membrane.
Then, add benzalkonium chloride in resulting composition, mix homogeneously is divided in the calcitonin liposome composition in the drop bottle again.
Embodiment 4: the preparation of spray
With natural phosphatidyl choline 525.0mg, cholesterol 77.0mg, 3 β-[N-(N ', N '-dimethylaminoethyl) carboxyl] cholesterol 54.0mg, sodium cholate 64.0mg be dispersed in the isoosmotic phosphate buffer (pH7.0), ultrasonic 5min intermittently, add an amount of mannitol mixing, with the freeze drying rate quick freezing of 7 ℃/min to-30 ℃.After the lyophilizing it is dispersed in the phosphate-buffered aqueous solution (pH7.0) of calcitonin of the 10ml 50 μ g/ml that contain appropriate vitamin C, vortex vibrate liposome turbid liquor.Promptly get homodisperse calcitonin liposome composition through 0.2 μ m filtering with microporous membrane.
Then, add benzalkonium chloride in resulting composition, mix homogeneously is in being divided in the calcitonin liposome composition in spray spray bottle.
Embodiment 5: the preparation of aerosol
With phospholipid of natural soybean 105.0mg, cholesterol 30.8mg, 3 β-[N-(N ', N '-dimethylaminoethyl) carboxyl] cholesterol 1.3mg, Myrj59 40.0mg and an amount of vitamin E put in the eggplant-shape bottle, (1: 1v/v) mixed solvent makes its dissolving to add 15ml chloroform-methanol, rotary evaporation is removed organic solvent in 35 ℃ of waters bath with thermostatic control, forms even class membrane of lipoprotein at the eggplant-shape bottle wall.Film is washed in phosphate-buffered aqueous solution (pH7.0) the aquation rotation that adds the calcitonin of 2ml 30 μ g/ml then, again the vortex vibration make film disperse liposome turbid liquor.Promptly get homodisperse calcitonin liposome composition through 0.2 μ m filtering with microporous membrane.
Then, gained calcitonin liposome composition is divided in the aerosol pressure vessel, adds valve, after the deadend, in container, pour into propellant (F
1211) promptly.
Embodiment 6: the preparation of gel
Natural phosphatidyl choline 450.0mg, cholesterol 154.0mg, dimethyl bromine ammonium 27.0mg, Brij3557.0mg are put in the eggplant-shape bottle, (1: 1v/v) mixed solvent makes its dissolving to add 200ml chloroform-methanol, rotary evaporation is removed organic solvent in 35 ℃ of waters bath with thermostatic control, forms even class membrane of lipoprotein at the eggplant-shape bottle wall.Film is washed in phosphate-buffered aqueous solution (pH7.0) the aquation rotation of calcitonin that adds the 50 μ g/ml of the 10ml contain appropriate vitamin C then, again the vortex vibration make film disperse liposome turbid liquor.Promptly get homodisperse calcitonin liposome composition through 0.2 μ m filtering with microporous membrane.
Then, add an amount of benzalkonium chloride and isotonic agent in resulting composition, mix homogeneously is dispersed in the calcitonin liposome composition in the hydroxypropyl emthylcellulose gel-type vehicle again, is divided in the drop bottle then.
Embodiment 7: the preparation of gel
The 1g sorbitol is put in the eggplant-shape bottle, phospholipid of natural soybean 1050mg, cholesterol 154mg, Polyethylene Glycol-DSPE 10mg, sodium deoxycholate 30mg and an amount of vitamin E are dissolved in 200ml chloroform-methanol (1: 1v/v) in the mixed solvent altogether, the gradation of lipoids solution is added in the eggplant-shape bottle, rotary evaporation is removed organic solvent in 35 ℃ of waters bath with thermostatic control, the product that will make were put in 4 ℃ of vacuum desiccators dry 24 hours, take out, cross 40 mesh sieves, promptly get blank liposome.The phosphate-buffered aqueous solution (pH7.0) that adds the calcitonin of 20ml 50 μ g/ml then, vortex vibration make it to disperse liposome turbid liquor.Behind 0.2 μ m filtering with microporous membrane, it is dispersed in the aqueous solution of chitosan promptly gets homodisperse calcitonin liposome composition.
Then, add an amount of benzalkonium chloride in resulting composition, mix homogeneously is dispersed in the calcitonin liposome composition of gained in Ka Baimu and the glycerol substrate and promptly gets gel.
Embodiment 8: the preparation of cream
Phospholipid of natural soybean 750mg, cholesterol 350mg, 18-amine. 54mg, NaGC 130mg and an amount of vitamin E are put in the eggplant-shape bottle, (1: 1v/v) mixed solvent makes its dissolving to add 400ml chloroform-methanol, rotary evaporation is removed organic solvent in 35 ℃ of waters bath with thermostatic control, forms even class membrane of lipoprotein at the eggplant-shape bottle wall.Film is washed in phosphate-buffered aqueous solution (pH7.0) the aquation rotation that adds the calcitonin of 20ml 20 μ g/ml then, again the vortex vibration make film disperse liposome turbid liquor.Promptly get homodisperse calcitonin liposome composition through 0.2 μ m filtering with microporous membrane.
Then,, under agitation slowly add triethanolamine, ethyl hydroxybenzoate and sodium lauryl sulphate and the present composition, be stirred to condensation promptly liquid paraffin, stearic acid, octadecanol, vaseline heating and melting.
Embodiment 9: the preparation of ointment
Phospholipid of natural soybean 190mg, cholesterol 38mg, 18-amine. 14mg, Pluronic F68 60mg and an amount of vitamin E are put in the eggplant-shape bottle, (1: 1v/v) mixed solvent makes its dissolving to add 200ml chloroform-methanol, rotary evaporation is removed organic solvent in 35 ℃ of waters bath with thermostatic control, forms even class membrane of lipoprotein at the eggplant-shape bottle wall.Film is washed in phosphate-buffered aqueous solution (pH7.0) the aquation rotation that adds the calcitonin of 5ml 50 μ g/ml then, again the vortex vibration make film disperse liposome turbid liquor.Promptly get homodisperse calcitonin liposome composition through 0.2 μ m filtering with microporous membrane.
Then, the present composition is added benzalkonium chloride and lanoline grinds well, add fused vaseline again, make it mixing and promptly get ointment.
Embodiment 10: the preparation of injection
Phospholipid of natural soybean 260mg, cholesterol 77mg, Polyethylene Glycol-DSPE 3.4mg and an amount of vitamin E are put in the eggplant-shape bottle, (1: 1v/v) mixed solvent makes its dissolving to add 150ml chloroform-methanol, rotary evaporation is removed organic solvent in 35 ℃ of waters bath with thermostatic control, forms even class membrane of lipoprotein at the eggplant-shape bottle wall.Film is washed in phosphate-buffered aqueous solution (pH7.0) the aquation rotation that adds the calcitonin of 5ml 50 μ g/ml then, again the vortex vibration make film disperse liposome turbid liquor.Promptly get homodisperse calcitonin liposome composition through 0.2 μ m filtering with microporous membrane.
Add an amount of antibacterial benzyl alcohol aqueous solution in resulting composition, mix homogeneously filters, and embedding, radiation sterilization are promptly.
Embodiment 11: the preparation of freeze-dried powder
Phospholipid of natural soybean 260mg, cholesterol 77mg, Tween-80 2mg are put in the eggplant-shape bottle, (1: 1v/v) mixed solvent makes its dissolving to add 15ml chloroform-methanol, rotary evaporation is removed organic solvent in 35 ℃ of waters bath with thermostatic control, forms even class membrane of lipoprotein at the eggplant-shape bottle wall.Film is washed in phosphate-buffered aqueous solution (pH7.0) the aquation rotation that adds the calcitonin of 5ml 50 μ g/ml then, again the vortex vibration make film disperse liposome turbid liquor.Promptly get homodisperse calcitonin liposome composition through 0.2 μ m filtering with microporous membrane.
In the liposome composition of gained, add an amount of mannitol, mix homogeneously, lyophilizing, sealing, radiation sterilization is promptly.
Test example 1: nasal ciliary toxicity test
Employing is in body toad palate modelling.Concrete operations are as follows: it is fixing that Bufo siccus is lain on the back, the oral cavity is opened and fixing, dropping is subjected to reagent liquid 0.5ml in last palatine mucosa place, make complete submergence palate, clean with normal saline behind the contact 30min and be subjected to reagent liquid, separate upward palatine mucosa with operating scissors, get about 3mm * 3mm size, clean clot and foreign material with normal saline immediately, mucosal surface upwards is tiled on the microscope slide, drip normal saline in mucomembranous surface, covered in the motion conditions that 40 * 10 times optical microscopes are observed down mucomembranous cilium, is held on microscope slide subsequently and is added with in the water saturated chromatography cylinder of a small amount of distillation, airtight, ambient temperature is 20～25 ℃.After this take out specimen at regular intervals, put microscopically and observe, continue motion as cilium and then put back in the chromatography cylinder, stop until ciliary movement.Record is from stopping the time to be continued to ciliary movement after the administration.Other gets Bufo siccus and as above operates, and drips normal saline 0.5ml in contrast.Cilium persistent movement time (t with the administration group
i) divided by the cilium persistent movement time (t of normal saline matched group
0), obtain the relative percentage of cilium persistent movement time, promptly
P＝t
i/t
0×100％
Percentage rate is high more, and expression is subjected to reagent liquid more little to effect on ciliary movement, and is promptly more little to cilium toxicity.
The cilium toxicity of the suspension of table 1 surfactant solution and embodiment 1-4 liposome (
, n=5)
| Drugs | T | P(％) |
| Normal saline 0.25%Cremophor EL 0.86% sodium cholate solution 1.0%Brij35 solution 1.0% |
734±99 521±43 440±50 204±59 541±112 709±50 706±94 719±79 723±88 | 100.0 70.9 59.9 27.8 73.7 96.6 96.2 98.0 98.6 |
The cilium sustained oscillation time sees Table 1 behind the compositions Bufo siccus maxillary administration 30min of each surfactant solution and embodiment 1.
Employing is estimated different surfaces activating agent and liposome composition to the nasal mucosa effect on ciliary movement in body toad palate modelling.The result shows that liposome does not all have obvious influence to ciliary movement; The different surfaces activating agent has certain influence to ciliary movement, so liposome has significantly reduced the toxicity of surfactant to the nasal mucosa cilium.
Test example 2: fall the blood calcium effect test
Get the male SD rat of body weight 250g-300g, 3 groupings at random, 5 every group.24hr fasting before the experiment can freely be drunk water.The dosage of salmon calcitonin see calcimar is 5.0 μ g/kg, route of administration: 1. subcutaneous injection medication: the rat back subcutaneous injection; 2. nasal-cavity administration method: rat lies on the back, the nostril up, with the administration of collunarium mode.Earlier gets blood as blank from rat tails before the administration, after the administration 0.5,1,1.5,2,3,4,5,6, regularly got the about 0.5ml of blood from rat tails in 8 hours, the centrifugal 10min of 6000rpm gets supernatant employing OCPC colorimetric method for determining serum calcium ion concentration.Record and the results are shown in Table 1 and Fig. 1.
After table 2 nasal-cavity administration or the subcutaneous injection administration different time points fall the blood calcium percentage rate (
N=5)
| Time (hour) | Calcitonin aqueous solution (nasal-cavity administration) | Embodiment 1 (nasal-cavity administration) | Calcitonin aqueous solution (subcutaneous injection) |
| 0.0 | 100±0.0 | 100±0.0 | 100±0.0 |
| 0.5 | 100.2±3.2 | 93.7±2.4 | 89.3±2.7 |
| 1.0 | 96.5±2.3 | 81.6±3.9 | 80.7±3.8 |
| 1.5 | 93.8±4.2 | 77.1±3 5 | 79.1±3.5 |
| 2.0 | 95.1±3.8 | 72.5±3.0 | 73.9±2.4 |
| 3.0 | 96.2±2.1 | 67.2±7 9 | 70.1±4.5 |
| 4.0 | 98.7±2.1 | 72.5±7.7 | 66.0±5.5 |
| 5.0 | 96.3±3.6 | 74.3±8.7 | 61.0±6.2 |
| 6.0 | 95.8±4.6 | 80.9±9 9 | 58.8±5.7 |
| 8.0 | 99.5±5.4 | 84.6±6.8 | 58.7±7.7 |
The result of table 2 and Fig. 1 shows that the calcitonin aqueous solution has only slightly reduced the serum calcium ion level through nasal mucosa medicine administration, compares with the subcutaneous injection of calcitonin aqueous solution, and the absolute bioavailability of its pharmacology is 13.6%.And the compositions of embodiment 1 is through nasal mucosa medicine administration, then reduced calcium ion level in the serum significantly, subcutaneous injection basic and the calcitonin aqueous solution is suitable in preceding 3 hours after administration, compare with the subcutaneous injection of calcitonin aqueous solution, the absolute bioavailability of its pharmacology is 69.4%, be 5.1 times of nasal mucosa medicine administration of calcitonin aqueous solution, shown the superiority of the present composition.
Test example 3: pharmacokinetics test
For investigation is included in salmon calcitonin see calcimar in the present composition through the bioavailability of nasal mucosa medicine administration, following to the preparation of the present invention among the embodiment 1 through nasal mucosa medicine administration, salmon calcitonin see calcimar aqueous pharmaceutical through intravenous injection, carry out pharmacokinetic study respectively, relatively the salmon calcitonin see calcimar absorption in vivo.(FITC) carries out labelling to salmon calcitonin see calcimar with fluorescein isothiocyanate (fitc), with the content of fluorophotometric methods analyst calcitonin.
Get the male SD rat of body weight 250g-300g, divide 3 groups at random, 5 every group.Test fasting in preceding 24 hours, can freely drink water.Weigh the back by the FITC-calcitonin dosage administration of 200 μ g/kg, route of administration: 1. intravenous administration method: the rat vena femoralis injection; 2. nasal-cavity administration method: rat lies on the back, and the nostril gives preparation of the present invention among the embodiment 1 in the collunarium mode up.Earlier gets blood as blank before the administration, after the administration 0,0.25,0.5,1,1.5,2,3,4, regularly got the about 0.6ml of blood in 6,8 hours from rat tails from rat tails, and separation of serum.
Respectively get serum 150 μ l, behind the adding pH7.0 phosphate buffer 2.5ml mix homogeneously, carry out fluorophotometric analysis.Record result such as table 2 and Fig. 2.
The blood drug level value of the nasal-cavity administration of aqueous solution intravenous injection of table 3 calcitonin and embodiment 1
| Time (hour) | Calcitonin aqueous solution intravenous injection C (ng/ml) | |
| 0 | 172.651±33.248 | 4.561±0.000 |
| 0.25 | 93.661±10.410 | 7.493±1.472 |
| 0.5 | 73.300±10.372 | 9.749±0.915 |
| 1 | 63.956±6.237 | 12.863±1.618 |
| 1.5 | 61.102±6.855 | 14.260±1.082 |
| 2 | 63.214±5.089 | 16.960±3.071 |
| 3 | 57.219±12.827 | 12.885±1.445 |
| 4 | 47.329±5.860 | 11.478±1.998 |
| 6 | 37.791±6.293 | 10.408±1.636 |
| 8 | 32.133±7.134 | 8.976±1.466 |
The result of table 3 and Fig. 2 shows, compares with the intravenous injection of calcitonin aqueous solution, and embodiments of the invention 1 are through nasal mucosa medicine administration, and its absolute bioavailability is 28.4%, obviously exceeds nearly 10 times than external commercially available product.
Invention has been described though utilized above-mentioned specific embodiment, it should be understood that those skilled in the art also can carry out various improvement and change, and within their scopes of the present invention that also should limit as claims.
Claims (7)
1. compositions that is used for mucosa or percutaneous dosing, it comprises calcitonin, natural and/or liposome that synthetic phospholipid, cholesterol, adjuvant and other surfactant constitute.
2. compositions as claimed in claim 1, wherein adjuvant is selected from cholesterol derivative, 18-amine., dimethyl bromine ammonium, chitosan, the arabic gum of lotus positive electricity, and their mixture.
3. compositions as claimed in claim 1, wherein other surfactant be selected from the acidifying natural or hydrogenated vegetable oil of cholate, polyoxyethylene sorbitan fatty acid ester, sorbitan fatty acid ester, polyoxyethylene fatty acid ester, polyoxyethylene aliphatic alcohol ether, polyoxyethylene glycol, ethylene oxide-propylene oxide copolymer, with the carbon atom quantity quaternary ammonium salt that is the straight chained alkyl of 10-20, and their mixture.
4. pharmaceutical composition, it comprises each compositions and pharmaceutically useful adjuvant and additive of claim 1-3.
5. be used to prepare the purposes of the medicine of treatment osteoporosis, scleromalacia, hypercalcemia and hypercalcemia crisis, trophesy, acute pancreatitis, hyperparathyroidism, bone healing, osteodynia and cataract disease as each compositions of claim 1-3.
6. purposes as claimed in claim 5 is characterized in that by nasal mucosa, oral mucosa, vaginal mucosa, uterine mucosa, lung mucosa, intestinal mucosa, eye and percutaneous drug delivery.
7. purposes as claimed in claim 5 is characterized in that by nasal mucosa and/or mouth mucosa drug administration.
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| CNB031428266A CN100371018C (en) | 2003-06-12 | 2003-06-12 | Calcitonin composition |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1055483A (en) * | 1990-04-12 | 1991-10-23 | 赫彻斯特股份公司 | The long-acting liposome preparation and the preparation thereof of peptide medicine |
| CN1185974A (en) * | 1996-11-29 | 1998-07-01 | 沃维汉 | Human calcium degrading gene concerned peptide fatty composite and preparation thereof |
| WO2002013782A1 (en) * | 2000-08-11 | 2002-02-21 | Hyundai Pharmaceutical Ind. Co., Ltd | Oral delivery of peptide |
| WO2003030865A1 (en) * | 2001-10-11 | 2003-04-17 | Imi Biomed, Inc. | Pro-micelle pharmaceutical compositions |
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2003
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1055483A (en) * | 1990-04-12 | 1991-10-23 | 赫彻斯特股份公司 | The long-acting liposome preparation and the preparation thereof of peptide medicine |
| CN1185974A (en) * | 1996-11-29 | 1998-07-01 | 沃维汉 | Human calcium degrading gene concerned peptide fatty composite and preparation thereof |
| WO2002013782A1 (en) * | 2000-08-11 | 2002-02-21 | Hyundai Pharmaceutical Ind. Co., Ltd | Oral delivery of peptide |
| WO2003030865A1 (en) * | 2001-10-11 | 2003-04-17 | Imi Biomed, Inc. | Pro-micelle pharmaceutical compositions |
Non-Patent Citations (1)
| Title |
|---|
| Promoting Effect of the Ultradeformable Liposomes onTransdermal Delivery. Hu Xin，et al.Journal of Chinese Pharmaceutial Sciences,Vol.11 No.1. 2002 * |
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