CA2052164A1 - Lipid membrance structures for oral administration - Google Patents
Lipid membrance structures for oral administrationInfo
- Publication number
- CA2052164A1 CA2052164A1 CA 2052164 CA2052164A CA2052164A1 CA 2052164 A1 CA2052164 A1 CA 2052164A1 CA 2052164 CA2052164 CA 2052164 CA 2052164 A CA2052164 A CA 2052164A CA 2052164 A1 CA2052164 A1 CA 2052164A1
- Authority
- CA
- Canada
- Prior art keywords
- membrane
- structures
- lipid membrane
- lipid
- mannan
- 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
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
-
- 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
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
- A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
Abstract
Abstract of the Disclosure Lipid membrane structures for oral administration contain phosphatidylserine, a mannan derivative or a mannose derivative as ingredient of the membrane. In particular, the lipid mambrane structures contain a drag therein, the drug being surrounded with the membrane. It is in the form of liposomes, emulsions or micelles and capable of efficiently delivering a drug contained therein to Peyer's patches in the small intestines. Therefore, the lipid membrane structures of the present invention are excellent as oral dosage lipid membrane structures.
Description
21~5~16~
Specification Title of the Invention Lipid Membrane Structures for Oral Administration Background of the Invention The present invention relates to lipid membrane structures capable of efficiently reaching Peyer's patches in a digestive tract after oral administration. The lipid membrane structures prepared by 10the present invention are capable of efficiently delivering a drug contained therein to Peyer's patches in a digestive tract.
Recently intensive investigations are made on a drug delivery system (DDS) for the purpose of improving the effect, safety and usage of a drug. DDS is classified to a contral-release type (the first 15generation DDS) and a targeting contral type (the second generation DDS) in the scientific field.
As for the drugs for oral administration, the investigations ~; hàve been made heretofore on mainly DDS of the former type (control-;; release type) but the DDS~ of the latter type (targeting type) has been~scarcely~inves;t1gated~. The eeyer's patch in a~digestive tract is~a~protrus1on~1n a~mucous~membrane of~the small intestine and it is an aggregate of limph follicIes. It is also a tissue recently attracting attention~in this technical field and investigations are made~for the~purpose;~of eluoidating the~lmmunological coles thereof.
25 ~Inve;~t1gations;are~made~on~the mechanism of uptake of latex beads thccugh Peyer'~patahe~ ICIinicAI Experimental Immunology, Vol. 76, p.
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144 (1989)] and uptake of a drug enclosed in a biodegradable polymer through Peyer's patches (European Patent Application No. 87309286.0).
However, according to these conventional techniques, it is impossible for a drug to be selectively taken up through Peyer's patches in a di.gestive tract after oral administration.
Investigations were made also on the use of liposomes for an oral drug. Namely, investigations were made on encapsulating, in liposomes, a high-molecular drug such as insulin [Biochimica et Biophysica Acta, Vol. 716, p; 188 (1982)], blood coagulation factor VIII ~ ~ancet, i, p. 70 (1980)] or heparin [Chemical and Pharmaceutical Bulletin, Vol. 30, p. 2245 (1982)]; a hardly water-soluble drug such as vitamin K, [Journal of Pharmacy and Pharmacology, Vol. 36, p. 527 (1984)] or griseofulvin [Journal of Pharmaceutical Sciences, Vol. 73, p. 757, (1984)1; or~a Streptococcus cell wall antigen [Immunology, vol. 54, P. 189(1985)] (for the purpose of production of IgA antibody). ~owever, an idea of modifying the liposomal membrane with an additlve so as to impart a selectivity toward Peyer's patches in the digestive tract has never been reported Further, as~for the liposomes used as an injection, it was 20 ~ reported that when the membrane thereof was modified with phosphatidylserine [Cancer Research;, Vol. 40, p. 4460 (1980)], alveolar macrophages were activated to lmprove the antitumor effect afte~r; intraveno~s injection and that when~ it was modified with a mannose derivatlve [~iochimica et Biophysica Acta, Vol. 632, p. 562 ~5;~ (1980)] or a mànnan~derlvative l"~yotai Seiri", Vol. 6, p. 771 l985)l~,: the liposomes were distributed to the liver and the lung.
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Specification Title of the Invention Lipid Membrane Structures for Oral Administration Background of the Invention The present invention relates to lipid membrane structures capable of efficiently reaching Peyer's patches in a digestive tract after oral administration. The lipid membrane structures prepared by 10the present invention are capable of efficiently delivering a drug contained therein to Peyer's patches in a digestive tract.
Recently intensive investigations are made on a drug delivery system (DDS) for the purpose of improving the effect, safety and usage of a drug. DDS is classified to a contral-release type (the first 15generation DDS) and a targeting contral type (the second generation DDS) in the scientific field.
As for the drugs for oral administration, the investigations ~; hàve been made heretofore on mainly DDS of the former type (control-;; release type) but the DDS~ of the latter type (targeting type) has been~scarcely~inves;t1gated~. The eeyer's patch in a~digestive tract is~a~protrus1on~1n a~mucous~membrane of~the small intestine and it is an aggregate of limph follicIes. It is also a tissue recently attracting attention~in this technical field and investigations are made~for the~purpose;~of eluoidating the~lmmunological coles thereof.
25 ~Inve;~t1gations;are~made~on~the mechanism of uptake of latex beads thccugh Peyer'~patahe~ ICIinicAI Experimental Immunology, Vol. 76, p.
:: :
.~ : , - ~- - . :
21~S~16~
144 (1989)] and uptake of a drug enclosed in a biodegradable polymer through Peyer's patches (European Patent Application No. 87309286.0).
However, according to these conventional techniques, it is impossible for a drug to be selectively taken up through Peyer's patches in a di.gestive tract after oral administration.
Investigations were made also on the use of liposomes for an oral drug. Namely, investigations were made on encapsulating, in liposomes, a high-molecular drug such as insulin [Biochimica et Biophysica Acta, Vol. 716, p; 188 (1982)], blood coagulation factor VIII ~ ~ancet, i, p. 70 (1980)] or heparin [Chemical and Pharmaceutical Bulletin, Vol. 30, p. 2245 (1982)]; a hardly water-soluble drug such as vitamin K, [Journal of Pharmacy and Pharmacology, Vol. 36, p. 527 (1984)] or griseofulvin [Journal of Pharmaceutical Sciences, Vol. 73, p. 757, (1984)1; or~a Streptococcus cell wall antigen [Immunology, vol. 54, P. 189(1985)] (for the purpose of production of IgA antibody). ~owever, an idea of modifying the liposomal membrane with an additlve so as to impart a selectivity toward Peyer's patches in the digestive tract has never been reported Further, as~for the liposomes used as an injection, it was 20 ~ reported that when the membrane thereof was modified with phosphatidylserine [Cancer Research;, Vol. 40, p. 4460 (1980)], alveolar macrophages were activated to lmprove the antitumor effect afte~r; intraveno~s injection and that when~ it was modified with a mannose derivatlve [~iochimica et Biophysica Acta, Vol. 632, p. 562 ~5;~ (1980)] or a mànnan~derlvative l"~yotai Seiri", Vol. 6, p. 771 l985)l~,: the liposomes were distributed to the liver and the lung.
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Thus the efficient delivery of a drug to Peyer's patches in a digestive tract after oral administration has not yet been proposed in the prior art.
5Summary of the Invention A primary object of the present invention is to provide lipid membrane structures capable of efficiently delivering a drug therein to Peyer's patches in a digestive tract after oral administration.
This and other objects of the'present invention will be 10apparent from the following description and examples.
The present invention has been comple,ted on the basis of a finding that the above-described objects can be attained by using at least one of phosphatidylserines, mannose derivatives and mannan derivatives as a component of a membrane of lipid membrane structures Brief Description of the Drawings Fig. 1 shows the results of the distribution of PS-liposomes of the present invention and SA-liposomes to Peyer's patches.
Fig. 2 shows the effect of the amount of~PS content of PS-20~liposomes of the present invention on the distribution to Peyer's , patGhes.
Fig. 3 shows changes ~of the distribution of PS-liposomes of t~he'~present invention to Peyer's patches with time after admlnistratlon of~the~test disperslon, 5~Flg. 4 shows~the`specif1city~of PS-liposomes of the present invention for~the portions of the intestinal tract in the distribution 3~~
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to Peyer's patches and non-Peyer's patches.
Fig. 5 shows an improvement of the distribution of PS-liposomes of the present invention to Peyer's patches by modifying it with mannan derivatives.
S Fig. 6 shows an improvement of the distribution of PS-liposomes of the present inventicn to Peyer's patches by modifying with mannan derivatives.
The abbreviations in the above brief description are referred in Example hereinafter.
Description of the Preferred Embodiments The lipid membrane structures of the present invention indicate lipid particles having such membrane structures wherein polar head groups of amphiphilic lipids are arranged at the interface toward an aqueous phase. Examples of the lipid membrane structures include liposomes, emulsions and water-soluble micelles. The lipid membrane structures are characterized in that phosphatidylserines andlor mannose derivatives and/or mannan derivatives are incorporated 20~ therein as a component of the membrane so that the lipid membrane structures ~can be efficiently distributed to Peyer's patches in a ~ : -dlgestlve tract~after oral administration.
he phosphatldylserines used in the present invention include natural soybean or egg-yolk phosphatidylserine, hydrogenated 25~ phosph~at1~dy1serlne~abtalned~ by hydrogenation of the natural soybean o~r~eg~g-~yolk~phosphatldy1ser1ne and semi-synthetic dimyristoyl : ~ : `
~ 4 -.. . .
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phosphatidylserine, dipalmitoyl phosphatidylserine and distearoyl phosphatidylserine. Among them, preferred are hydrogenated phosphatidylserine, dipalmitoyl phosphatidylserine and distearoyl phosphatidylserine.
The mannose derivatives used in the present invention include phosphatidylethanolamine derivatives having a mannose residue [Biochimica et siophysica Acta, Vol. 632, p. 562 (1980)], cholesterol derivatives having a mannose residue [Proceeding of National Academic Scienses U.S.A. Vol. 77, p. 4430 (1980)], dimannosyl diglyceride [Biochemical and Biophysical Research Communications, Vol. 110, p.
140 (1983)~, mannobiose fatty acid esters and amides (preferably, fatty acid residue having 12 to 30 carbon atoms)[Japanese Patent Unexamined Published Application (hereinafter referredto as "J.P.
KOKAI") No. Hei 1-104088], phosphatidyl mannose, etc.
The mannan derivatives in the present invention include, for example, mannan (a polysaccharide) partially substituted with a fatty acid or cholesterol (preferably, fatty acid residue having 12 to 30 carbon atoms)["Byotai Seiri", Vol. 6, p. 771 (1985)].
These additives used as a component of the membrane are used : ~:
either singly or in the form of a mixture of them and they usually `~ ~ can be incorporated into the membrane of the lipid membrane structures mainly~comprising lecithin (i.e., phosphatidylcholine) or the like to modify the membrane of the lipid membrane structures. Alternatively, they can singly form the lipid membrane structures. Namely, the .::
~ phosphatidylserines per se can form liposomes or emulsions, and mannose derivatives such as mannobiose fatty acid esters and amides ~ 5 -,.. : . . : , ; -. : , . . : "
205~
thereof can singly form emulsions or micelles. Therefore, the upper limit of the relative amount of the phosphatidylserines, mannose derivatives or mannan derivatives used as the component of the lipid membrane structures is not limited at all in the present invention.
The lower limit of the molar ratio is 1 % based on the total membrane components of the lipid membrane structures. Preferably, the amount of them used is 5 to 50 %.
The drugs which can be contained in the lipid membrane structures of the present invention vary depending on the kind of the lipid membrane structures. For example, drugs which can be encapsulated in the liposomes are not particu,larly limited and they include high-molecular compounds such as insulin, hormones and polysaccharides; water-soluble drugs such as immunopotentiators and immunosuppressants; and fat-soluble drugs. The drugs which can be contained in the emulsions include fat-soluble drugs and those which can be contained in the micelles include hardly water-soluble drugs.
They can be kept in the lipid membrane structures prepared by ordinary processes which are described below.
The description will be made on processes for preparing the 20~ l1pid membrane structures of the present invention containing phosphatidylserines and/or mannonose derivatives and/or mannan derivatlves added thereto.
a) Process for preparing liposomes An aqueous dispersion of liposomes is prepared from membrane-25 ;~ ~ forming components such as lecithin or sphingomyelin and additive(s),e.~phosphatidylserines and/or mannose derivatives and/or mannan ~ .
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derivatives by a known process [Annual Review of Biophysics and Bioengineering, Vol. 9, p. 467 (1980)]. The thus-prepared liposomes may contain stabilizers such as sterols, e.g. cholesterol, charged lipids such as dialkyl phosphate, diacylphosphatidic acid or stearylamine, or antioxidants such as a -tocopherol.
b) Process for preparing emulsions An aqueous dispersion of emulsions can be prepared from amphiphilic substances such as lecithins or polyoxyethylene sorbitan fatty acid esters (Tween's), additive(s), i.e. phosphatidyl serines and/or mannose derivatives and/or mannan derivatives, and oil such as soybean oil by a known emulsion-preparation process.
c) Process for preparing micelles An aqueous dispersion of micelles can be prepared from micelle-forming surfactants (to be added to an aqueous solvent at a lS concentration not lower than a critical concentration of micelle formation) such as polyoxyethylene sorbitan fatty acid esters (Tween's), sodium salt of fatty acid or polyoxyethylene hydrogenated castor oil and additive(s), i.e. posphatidylserines and/or mannose derivatives andior mannan derivatives by a known micelle-preparation process.
The lipid membrane strùctures of the present invention are capable of efficiently delivering a drug contained therein to Peyer's patches after oral administration. The Peyer's patch is an aggregate of lymph follicles in the mucus membrane of the small intestine and : ~ "
~ ~ it is one of tissues concerned with absorption of drugs. In particular, it is a quite important tissue for absorption of .~ .
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particularly high-molecular drugs or drugs relating to control of immunological activity.
Therefore, in case where a drug which could not be absorbed well when it was given by oral administration is encapsulated in the lipid membrane structures of the present invention, its absorption can be improved.
Therefore, the lipid membrane structures of the present invention are very unigue and excellent as oral lipid membrane structures for drugs which have been hardly able to be administrated orally by conventional techniques.
The following Examples and Test Examples will further llustrate the present invention, which by no means limit the invention. The basic method for preparation of the liposomes and experiments of the distribution of the liposomes to the Peyer's patches are summarized below.
Preparation of llPosomes (Examples l to 4 and Control Examples 3 and 4) Lipids in a molar ratio given below were placed in a short-neck flask in a total amount of S0 ~ mol. They were thoroughly 20~ dissolved in a mixture of chloroform and methanol and then the o;rganic solvents~were dist1lled off m an evaporator under reduced pressure.~ The organic;solvents were completely removed under reduced pressUre in a` desiccator to dry the lipid mixture. Then 2 ~1 of~
tris-hydrochlorio acid-buffered physiological saline (pH 7.4) 2~S~ oontaining lO~mg¦ml~of~6-carboxyfluorescein (6-CF) or lO mg/ml of phenol red;wh1ch was~an~inner ~gu~ou~phase marker was added thereto :
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and they were stirred and shaken in a vortex mixer at 50C . Then the dispersion was passed through a polycarbonate membrane filter having a pore diameter of 0.4 ~ m at 50~C to obtain a liposomal di.spersion having a particle diameter of not larger than 0.4~ m.
The inner aqueous phase marker which remained not encapsulated in the liposomes was removed by applying the thus-obtained liposomal dispersion through a column of Sephadex G-100 (3 cm diameter x 45 cm;
elution buffer: tris-hydrochloric acid-buffered physiological saline having pH of 7.4). The liposomal fractions containing the inner agueous phase marker were finally diluted with the tris-hydrochloric acid-buffered physiological saline (pH 7.4) to 6-CF concentration of 4 0 ~ g/ml or phenol red concentration of 400 ~ g/ml (final total lipid concentration: about 1 mM = 1 ~ mol/ml).
The liposomes modified with mannan derivative in Example 4 were prepared by first preparing liposomes having PC/PS/CHOL molar ratio of 7/3/2 in the same manner as that of Example 2 and then reacting the liposomes with a mannan-cholesterol derivative [the amount of the mannan-cholesterol derivative was 53.1 ~ g (in terms of :~: mannan) per ~ mol of the phospholipid in the liposome] at 4C for 2 ~20 h.
Control Ex. 3 : PC~PS/CHOL molar ratio = 7~0/2 - Example 1 : PC~PS~CHOL molar ratio = 7~1/2 :~ ~
Example 2 : PC~PS/CHOL molar ratio = 7~3~2 Example 3 : PC~PS/CHOL molar ratio = 7/5~2 25~ Example 4 : PC~PS/CHOL/Man-CHOL
Control Ex. 4 : PC~SA~CHOL molar ratio = 7/3/2 - ~
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[Symbols]
PC : egg yolk phosphatidylcholine PS : hydrogenated soybean phosphatidylserine CHOL : cholesterol Man-CHOL : mannan-cholesterol derivative [N-(2-N-cholesterylcarboxyaminoethyl)carbamylmannan, trade name: Chol-AECM-Mannan (a product of Wako Jun'yaku Co.)]
SA : stearylamine Experiment on distribution of liposomes to Peyer's patches (Test Examples 1 to 6) The distribution of the liposomes to the Peyer's patches was investigated by the in situ loop method. In the test, male Wistar rats weighing around 250 g were made to fast for 18 hours. After the celiotomy conducted under anesthesia with urethane, the small intestine was ligatured to divide it into four parts each having a ~length of about l5 cm to form loops. Then 1.0~ml of a test solution or dispersion (contalning 4.0 ~ g~of 6-CF, 400~ U g~of phenol red or about~l~ mol of ~total liplds) was~injected ln each~loop. After a 2~0~ given~period of~time,~the intestinal tract was taken ~ut and washedwith a~physiolog~ical~saline solution. Twenty mm' of the tissue of the~Pèyer's patches~and a tlssue around the Peyer's patcb (non-Peyer's patch~es)~were~cut~and taken~ and the inner aqueous phase marker dellvered~to the~both~tissues was~extracted with isoamyl alcohol and 25 ~ d~etermlned.
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Test Example l The test solution and dispersions of Control Example l (4.0 u g/ml of free 6-CF), Control Example 3 (PC-liposomes) and ~xample 2 (PS-liposomes) were used in the test of delivery of the markers to the Peyer's patches. The results obtained two hours after injection are shown in Fig. l (left) and Table l. It is evident that the delivery of 6-CF encapsulated in the PC-liposomes or PS-liposomes to the Peyer's patches and non-Peyer's patches was improved as compared with that of free 6-CF. It is also evident that the distribution of PS-liposome to the Peyer's patches was significantly improved as compared with that to the non-Peyer's patches.
Table 1 Dispersion Control j Control Example 2 ¦ Significant Example 1 Example 3 difference . (level of Free PC- PS- significance Tissue 6-CF liposomes liposomes '::
;~ Peyer's patch tissue 3.53 13.67 50.81 ~(number of ~ + 0.34 + 1.67 + 3.52 ~2~0~ times of ~n=21) (n=13) (n=19) ~repetition ~ Significant ....... ~............ ....................... .............................. ~.......... difference ~Significance~ , , ;~l ~ was found in ~difference ~ Yes ~ Yes Example 2 level of ~ (1%) (1%) (1~) significance) 25~ ~Non-Peyer's~ ~ ~
2 ~patch tissue~ 3.35~ ~ 12.46 30.71 ~number Qf~~+ 0.41~ ~+ 1.62 + 2.66 ~times~of ~~(n=20) ~ (n-16) (n=17) ~repeti~tion~) ~ ~
nit: ng/mm'X 10-', mean + standard error : : : . . : .
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Test Example 2 The distribution test to the Peyer's patches was conducted with the test solution and dispersion of Control Example 2 (400~ g/ml of free phenol red) and Control Example 4 (SA-liposome). The results obtained two hours after injection are shown in Fig. l (right) and Table 2. The distribution to the Peyer's patches and non-Peyer's patches was similar to that observed when free phenol red was given, and no significant difference was recognized.
Table 2 Dispersion Control Control Example 2 Example 4 Free SA- ~
Tissue phenal red liposomes Peyer's patch tissue 4.35 5.27 (number of + 0.55 + 0.73 times of(n=2) (n=6) ~ repetition :~ ~ Non-Peye~r's ~ 20 ~ patçh tissue 4.60 3.77 :: (number of : + 0.44 + 0.13 : times of(n=3) (n-3) : ~repetltion) Unit: ng/mm2x 10-', mean + standard error :
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Test Example 3 The influence of the amount of PS on the delivery of 6-CF to the Peyer's patches was examined. In the experiment of the distribution to the Peyer's patches, PC-liposomes of Control Example 3 and PS-liposomes of Examples l, 2 and 3 were used as the test dispersions. The results obtained two hours after injection are shown in Fig. 2 and Table 3. As compared with PC-liposomes, PS-liposomes exhibited an improved effect of delivering the enclosed 6-CF to the Peyer's patches as the PS content was increased. No significant difference was observed in the three PS-liposomes having different PS
contents (Examples 1, 2 and 3).
Table 3 Dispersion ¦ Control Example 1 Example 2 I Example 3 Example 3 i PC/PS/CHOL PC/PS/CHOL PC/PS/CHOL PC/PS/CHOL
Tissue = 7/0/2 = 7/1/2 = 7/3/2 = 7/5/2 Peyer's patch tissue ~number13.67 31.31 50.81 44.22 of times of + 1.67 + 4.39 + 3.52 + 9.35 repetition ~(n=13) (n=10) (n=19) (n=6) ...... ... ... ...... . . ..... ..... ... ................ .......
Significance l l ~difference ' ' Yes ~(level of ' ' Yes (1%) significance) Yes (5%) (1%) I
Non-Peyer's ; patch tissue 12.46 19.76 30.71 17.60 (number of + 1.62 + 2.72 + 2.65 + 2.83 2~5 ~ times of (n=16) (n=9) (n=17) (n=6) ~r~epetition) Unit: ng/mm2x 10-', mean + standard error :
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Test Example 4 The effect of incubation time on the distribution of PS-liposomes to the Peyer's patches and non-Peyer's patches was examined.
In the experiment of the distribution to the Peyer's patches, the test dispersion of Example 2 (PS-liposome) was used. Changes with time after injection of the test dispersion are shown in Fig. 3 and Table 4. It was found that the distribution to the Peyer's patches was accelerated with the incubation time and reached the saturation after about two hours. The distribution to the non-Peyer's patches was not so remarkable and was substantially constant.
Table 4 tnimUebati Peyer's patch tissue (number 14.85 21.81 54.60 48.61 of times of + 0.95 + 1.91+ 5.07 + 4.90 repetition (n=13) (n=10) (n-8) (n=ll) ............................... ....................... ....................... ....................... ......................
Non-Peyer's patch tissue 13.84 11.51 12.60 6.29 (number of + 1.19+ 1.35 + 2.07 + 0.99 ~20 times of (n=ll) (n=10) (n=6) (n=5) repètition) : :
*Significant ~difference No Yes Yes Yes (level of (1%) (1%) (1~) significance) :
~25 ~ ~ Unit: nglmmZX 10- 2, mean + standard error * Significant difference between movement to the Peyer's patch tissue and distribution to the non-Payer'~s patch tissue '-' :: ' :
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Test Example 5 Specificity of the test dispersion of Example 2 ~PS-liposomes) for the position of the intestinal t~act to which the PS-liposomes were distributed was examined in the distribution test to the Peyer's patches and non-Peyer's patches. The results are shown in Fig. 4. No particular difference in the position was found.
Test Example 6 The test dispersions of Example 2 (PS-liposomes) and Example ~
(PS-liposomes modified with mannan derivative) were used in the test of distribution to the Peyer's patches. The results are shown in Fig.
Thus the efficient delivery of a drug to Peyer's patches in a digestive tract after oral administration has not yet been proposed in the prior art.
5Summary of the Invention A primary object of the present invention is to provide lipid membrane structures capable of efficiently delivering a drug therein to Peyer's patches in a digestive tract after oral administration.
This and other objects of the'present invention will be 10apparent from the following description and examples.
The present invention has been comple,ted on the basis of a finding that the above-described objects can be attained by using at least one of phosphatidylserines, mannose derivatives and mannan derivatives as a component of a membrane of lipid membrane structures Brief Description of the Drawings Fig. 1 shows the results of the distribution of PS-liposomes of the present invention and SA-liposomes to Peyer's patches.
Fig. 2 shows the effect of the amount of~PS content of PS-20~liposomes of the present invention on the distribution to Peyer's , patGhes.
Fig. 3 shows changes ~of the distribution of PS-liposomes of t~he'~present invention to Peyer's patches with time after admlnistratlon of~the~test disperslon, 5~Flg. 4 shows~the`specif1city~of PS-liposomes of the present invention for~the portions of the intestinal tract in the distribution 3~~
: .
- 21DS~6~
to Peyer's patches and non-Peyer's patches.
Fig. 5 shows an improvement of the distribution of PS-liposomes of the present invention to Peyer's patches by modifying it with mannan derivatives.
S Fig. 6 shows an improvement of the distribution of PS-liposomes of the present inventicn to Peyer's patches by modifying with mannan derivatives.
The abbreviations in the above brief description are referred in Example hereinafter.
Description of the Preferred Embodiments The lipid membrane structures of the present invention indicate lipid particles having such membrane structures wherein polar head groups of amphiphilic lipids are arranged at the interface toward an aqueous phase. Examples of the lipid membrane structures include liposomes, emulsions and water-soluble micelles. The lipid membrane structures are characterized in that phosphatidylserines andlor mannose derivatives and/or mannan derivatives are incorporated 20~ therein as a component of the membrane so that the lipid membrane structures ~can be efficiently distributed to Peyer's patches in a ~ : -dlgestlve tract~after oral administration.
he phosphatldylserines used in the present invention include natural soybean or egg-yolk phosphatidylserine, hydrogenated 25~ phosph~at1~dy1serlne~abtalned~ by hydrogenation of the natural soybean o~r~eg~g-~yolk~phosphatldy1ser1ne and semi-synthetic dimyristoyl : ~ : `
~ 4 -.. . .
- Z6)5~ 6~
phosphatidylserine, dipalmitoyl phosphatidylserine and distearoyl phosphatidylserine. Among them, preferred are hydrogenated phosphatidylserine, dipalmitoyl phosphatidylserine and distearoyl phosphatidylserine.
The mannose derivatives used in the present invention include phosphatidylethanolamine derivatives having a mannose residue [Biochimica et siophysica Acta, Vol. 632, p. 562 (1980)], cholesterol derivatives having a mannose residue [Proceeding of National Academic Scienses U.S.A. Vol. 77, p. 4430 (1980)], dimannosyl diglyceride [Biochemical and Biophysical Research Communications, Vol. 110, p.
140 (1983)~, mannobiose fatty acid esters and amides (preferably, fatty acid residue having 12 to 30 carbon atoms)[Japanese Patent Unexamined Published Application (hereinafter referredto as "J.P.
KOKAI") No. Hei 1-104088], phosphatidyl mannose, etc.
The mannan derivatives in the present invention include, for example, mannan (a polysaccharide) partially substituted with a fatty acid or cholesterol (preferably, fatty acid residue having 12 to 30 carbon atoms)["Byotai Seiri", Vol. 6, p. 771 (1985)].
These additives used as a component of the membrane are used : ~:
either singly or in the form of a mixture of them and they usually `~ ~ can be incorporated into the membrane of the lipid membrane structures mainly~comprising lecithin (i.e., phosphatidylcholine) or the like to modify the membrane of the lipid membrane structures. Alternatively, they can singly form the lipid membrane structures. Namely, the .::
~ phosphatidylserines per se can form liposomes or emulsions, and mannose derivatives such as mannobiose fatty acid esters and amides ~ 5 -,.. : . . : , ; -. : , . . : "
205~
thereof can singly form emulsions or micelles. Therefore, the upper limit of the relative amount of the phosphatidylserines, mannose derivatives or mannan derivatives used as the component of the lipid membrane structures is not limited at all in the present invention.
The lower limit of the molar ratio is 1 % based on the total membrane components of the lipid membrane structures. Preferably, the amount of them used is 5 to 50 %.
The drugs which can be contained in the lipid membrane structures of the present invention vary depending on the kind of the lipid membrane structures. For example, drugs which can be encapsulated in the liposomes are not particu,larly limited and they include high-molecular compounds such as insulin, hormones and polysaccharides; water-soluble drugs such as immunopotentiators and immunosuppressants; and fat-soluble drugs. The drugs which can be contained in the emulsions include fat-soluble drugs and those which can be contained in the micelles include hardly water-soluble drugs.
They can be kept in the lipid membrane structures prepared by ordinary processes which are described below.
The description will be made on processes for preparing the 20~ l1pid membrane structures of the present invention containing phosphatidylserines and/or mannonose derivatives and/or mannan derivatlves added thereto.
a) Process for preparing liposomes An aqueous dispersion of liposomes is prepared from membrane-25 ;~ ~ forming components such as lecithin or sphingomyelin and additive(s),e.~phosphatidylserines and/or mannose derivatives and/or mannan ~ .
.: :
: ' ' ~' ` , ~. '' ' :
.
., ~ . .
- .: .
2~S~
derivatives by a known process [Annual Review of Biophysics and Bioengineering, Vol. 9, p. 467 (1980)]. The thus-prepared liposomes may contain stabilizers such as sterols, e.g. cholesterol, charged lipids such as dialkyl phosphate, diacylphosphatidic acid or stearylamine, or antioxidants such as a -tocopherol.
b) Process for preparing emulsions An aqueous dispersion of emulsions can be prepared from amphiphilic substances such as lecithins or polyoxyethylene sorbitan fatty acid esters (Tween's), additive(s), i.e. phosphatidyl serines and/or mannose derivatives and/or mannan derivatives, and oil such as soybean oil by a known emulsion-preparation process.
c) Process for preparing micelles An aqueous dispersion of micelles can be prepared from micelle-forming surfactants (to be added to an aqueous solvent at a lS concentration not lower than a critical concentration of micelle formation) such as polyoxyethylene sorbitan fatty acid esters (Tween's), sodium salt of fatty acid or polyoxyethylene hydrogenated castor oil and additive(s), i.e. posphatidylserines and/or mannose derivatives andior mannan derivatives by a known micelle-preparation process.
The lipid membrane strùctures of the present invention are capable of efficiently delivering a drug contained therein to Peyer's patches after oral administration. The Peyer's patch is an aggregate of lymph follicles in the mucus membrane of the small intestine and : ~ "
~ ~ it is one of tissues concerned with absorption of drugs. In particular, it is a quite important tissue for absorption of .~ .
:: ~ ::
: , ~ - .
. : , . , . - :
21[~5~6~
particularly high-molecular drugs or drugs relating to control of immunological activity.
Therefore, in case where a drug which could not be absorbed well when it was given by oral administration is encapsulated in the lipid membrane structures of the present invention, its absorption can be improved.
Therefore, the lipid membrane structures of the present invention are very unigue and excellent as oral lipid membrane structures for drugs which have been hardly able to be administrated orally by conventional techniques.
The following Examples and Test Examples will further llustrate the present invention, which by no means limit the invention. The basic method for preparation of the liposomes and experiments of the distribution of the liposomes to the Peyer's patches are summarized below.
Preparation of llPosomes (Examples l to 4 and Control Examples 3 and 4) Lipids in a molar ratio given below were placed in a short-neck flask in a total amount of S0 ~ mol. They were thoroughly 20~ dissolved in a mixture of chloroform and methanol and then the o;rganic solvents~were dist1lled off m an evaporator under reduced pressure.~ The organic;solvents were completely removed under reduced pressUre in a` desiccator to dry the lipid mixture. Then 2 ~1 of~
tris-hydrochlorio acid-buffered physiological saline (pH 7.4) 2~S~ oontaining lO~mg¦ml~of~6-carboxyfluorescein (6-CF) or lO mg/ml of phenol red;wh1ch was~an~inner ~gu~ou~phase marker was added thereto :
. : .: . . - . ,:
:
, , . - ~.
' 2~
and they were stirred and shaken in a vortex mixer at 50C . Then the dispersion was passed through a polycarbonate membrane filter having a pore diameter of 0.4 ~ m at 50~C to obtain a liposomal di.spersion having a particle diameter of not larger than 0.4~ m.
The inner aqueous phase marker which remained not encapsulated in the liposomes was removed by applying the thus-obtained liposomal dispersion through a column of Sephadex G-100 (3 cm diameter x 45 cm;
elution buffer: tris-hydrochloric acid-buffered physiological saline having pH of 7.4). The liposomal fractions containing the inner agueous phase marker were finally diluted with the tris-hydrochloric acid-buffered physiological saline (pH 7.4) to 6-CF concentration of 4 0 ~ g/ml or phenol red concentration of 400 ~ g/ml (final total lipid concentration: about 1 mM = 1 ~ mol/ml).
The liposomes modified with mannan derivative in Example 4 were prepared by first preparing liposomes having PC/PS/CHOL molar ratio of 7/3/2 in the same manner as that of Example 2 and then reacting the liposomes with a mannan-cholesterol derivative [the amount of the mannan-cholesterol derivative was 53.1 ~ g (in terms of :~: mannan) per ~ mol of the phospholipid in the liposome] at 4C for 2 ~20 h.
Control Ex. 3 : PC~PS/CHOL molar ratio = 7~0/2 - Example 1 : PC~PS~CHOL molar ratio = 7~1/2 :~ ~
Example 2 : PC~PS/CHOL molar ratio = 7~3~2 Example 3 : PC~PS/CHOL molar ratio = 7/5~2 25~ Example 4 : PC~PS/CHOL/Man-CHOL
Control Ex. 4 : PC~SA~CHOL molar ratio = 7/3/2 - ~
: ;: :~ : :
; ~ .
:~ :
_ g _ ;.: , , - . .
: .
,..... : : :
-, , :
21~5~
[Symbols]
PC : egg yolk phosphatidylcholine PS : hydrogenated soybean phosphatidylserine CHOL : cholesterol Man-CHOL : mannan-cholesterol derivative [N-(2-N-cholesterylcarboxyaminoethyl)carbamylmannan, trade name: Chol-AECM-Mannan (a product of Wako Jun'yaku Co.)]
SA : stearylamine Experiment on distribution of liposomes to Peyer's patches (Test Examples 1 to 6) The distribution of the liposomes to the Peyer's patches was investigated by the in situ loop method. In the test, male Wistar rats weighing around 250 g were made to fast for 18 hours. After the celiotomy conducted under anesthesia with urethane, the small intestine was ligatured to divide it into four parts each having a ~length of about l5 cm to form loops. Then 1.0~ml of a test solution or dispersion (contalning 4.0 ~ g~of 6-CF, 400~ U g~of phenol red or about~l~ mol of ~total liplds) was~injected ln each~loop. After a 2~0~ given~period of~time,~the intestinal tract was taken ~ut and washedwith a~physiolog~ical~saline solution. Twenty mm' of the tissue of the~Pèyer's patches~and a tlssue around the Peyer's patcb (non-Peyer's patch~es)~were~cut~and taken~ and the inner aqueous phase marker dellvered~to the~both~tissues was~extracted with isoamyl alcohol and 25 ~ d~etermlned.
.:.
, 2~5~
Test Example l The test solution and dispersions of Control Example l (4.0 u g/ml of free 6-CF), Control Example 3 (PC-liposomes) and ~xample 2 (PS-liposomes) were used in the test of delivery of the markers to the Peyer's patches. The results obtained two hours after injection are shown in Fig. l (left) and Table l. It is evident that the delivery of 6-CF encapsulated in the PC-liposomes or PS-liposomes to the Peyer's patches and non-Peyer's patches was improved as compared with that of free 6-CF. It is also evident that the distribution of PS-liposome to the Peyer's patches was significantly improved as compared with that to the non-Peyer's patches.
Table 1 Dispersion Control j Control Example 2 ¦ Significant Example 1 Example 3 difference . (level of Free PC- PS- significance Tissue 6-CF liposomes liposomes '::
;~ Peyer's patch tissue 3.53 13.67 50.81 ~(number of ~ + 0.34 + 1.67 + 3.52 ~2~0~ times of ~n=21) (n=13) (n=19) ~repetition ~ Significant ....... ~............ ....................... .............................. ~.......... difference ~Significance~ , , ;~l ~ was found in ~difference ~ Yes ~ Yes Example 2 level of ~ (1%) (1%) (1~) significance) 25~ ~Non-Peyer's~ ~ ~
2 ~patch tissue~ 3.35~ ~ 12.46 30.71 ~number Qf~~+ 0.41~ ~+ 1.62 + 2.66 ~times~of ~~(n=20) ~ (n-16) (n=17) ~repeti~tion~) ~ ~
nit: ng/mm'X 10-', mean + standard error : : : . . : .
,. : -, . : , ~` `. .: ' : ~' ;~
Z~5~ 6~
Test Example 2 The distribution test to the Peyer's patches was conducted with the test solution and dispersion of Control Example 2 (400~ g/ml of free phenol red) and Control Example 4 (SA-liposome). The results obtained two hours after injection are shown in Fig. l (right) and Table 2. The distribution to the Peyer's patches and non-Peyer's patches was similar to that observed when free phenol red was given, and no significant difference was recognized.
Table 2 Dispersion Control Control Example 2 Example 4 Free SA- ~
Tissue phenal red liposomes Peyer's patch tissue 4.35 5.27 (number of + 0.55 + 0.73 times of(n=2) (n=6) ~ repetition :~ ~ Non-Peye~r's ~ 20 ~ patçh tissue 4.60 3.77 :: (number of : + 0.44 + 0.13 : times of(n=3) (n-3) : ~repetltion) Unit: ng/mm2x 10-', mean + standard error :
2~s~
Test Example 3 The influence of the amount of PS on the delivery of 6-CF to the Peyer's patches was examined. In the experiment of the distribution to the Peyer's patches, PC-liposomes of Control Example 3 and PS-liposomes of Examples l, 2 and 3 were used as the test dispersions. The results obtained two hours after injection are shown in Fig. 2 and Table 3. As compared with PC-liposomes, PS-liposomes exhibited an improved effect of delivering the enclosed 6-CF to the Peyer's patches as the PS content was increased. No significant difference was observed in the three PS-liposomes having different PS
contents (Examples 1, 2 and 3).
Table 3 Dispersion ¦ Control Example 1 Example 2 I Example 3 Example 3 i PC/PS/CHOL PC/PS/CHOL PC/PS/CHOL PC/PS/CHOL
Tissue = 7/0/2 = 7/1/2 = 7/3/2 = 7/5/2 Peyer's patch tissue ~number13.67 31.31 50.81 44.22 of times of + 1.67 + 4.39 + 3.52 + 9.35 repetition ~(n=13) (n=10) (n=19) (n=6) ...... ... ... ...... . . ..... ..... ... ................ .......
Significance l l ~difference ' ' Yes ~(level of ' ' Yes (1%) significance) Yes (5%) (1%) I
Non-Peyer's ; patch tissue 12.46 19.76 30.71 17.60 (number of + 1.62 + 2.72 + 2.65 + 2.83 2~5 ~ times of (n=16) (n=9) (n=17) (n=6) ~r~epetition) Unit: ng/mm2x 10-', mean + standard error :
: . . . ~ . : :
:; ,. ' : .
: ~ :
:
~5~6~
Test Example 4 The effect of incubation time on the distribution of PS-liposomes to the Peyer's patches and non-Peyer's patches was examined.
In the experiment of the distribution to the Peyer's patches, the test dispersion of Example 2 (PS-liposome) was used. Changes with time after injection of the test dispersion are shown in Fig. 3 and Table 4. It was found that the distribution to the Peyer's patches was accelerated with the incubation time and reached the saturation after about two hours. The distribution to the non-Peyer's patches was not so remarkable and was substantially constant.
Table 4 tnimUebati Peyer's patch tissue (number 14.85 21.81 54.60 48.61 of times of + 0.95 + 1.91+ 5.07 + 4.90 repetition (n=13) (n=10) (n-8) (n=ll) ............................... ....................... ....................... ....................... ......................
Non-Peyer's patch tissue 13.84 11.51 12.60 6.29 (number of + 1.19+ 1.35 + 2.07 + 0.99 ~20 times of (n=ll) (n=10) (n=6) (n=5) repètition) : :
*Significant ~difference No Yes Yes Yes (level of (1%) (1%) (1~) significance) :
~25 ~ ~ Unit: nglmmZX 10- 2, mean + standard error * Significant difference between movement to the Peyer's patch tissue and distribution to the non-Payer'~s patch tissue '-' :: ' :
z~s~
Test Example 5 Specificity of the test dispersion of Example 2 ~PS-liposomes) for the position of the intestinal t~act to which the PS-liposomes were distributed was examined in the distribution test to the Peyer's patches and non-Peyer's patches. The results are shown in Fig. 4. No particular difference in the position was found.
Test Example 6 The test dispersions of Example 2 (PS-liposomes) and Example ~
(PS-liposomes modified with mannan derivative) were used in the test of distribution to the Peyer's patches. The results are shown in Fig.
5 and Table 5 (left). It is understood that when the membranes of PS
liposomes were further modified with the mannan derivative, the amount thereof distribution to the Peyer's patches was significantly increased and that distribution to the non-Peyer's patches was significantly decreased.
The results obtained in Control Example 5 wherein the PS-liposomes modified with mannan derivative was injected together with an excess amount of the mannan derivative(100 parts per part of the mannan derivative added to the liposomes) and Control Example 6 ~20 wherein PS-liposomes modified with the mannan derivative was injected together with empty PS-liposomes free from the inner aqueous phase marker [the membrane composition was the same as that of Example 2 but 100 ~ mol (100 times as much as that used in Example 2) of the pids were usedl are shown in Fig. 6 (right) and Table 5 (right).
I~t~was proved that the liposomes of the present invention has properties Gf selectively distribution to the Peyer's patches from :
: : :
,, , ~ .
. .
2~5~
the facts that the distribution of the PS-liposomes modified with mannan derivative to the Peyer's patches was significantly inhibited by the use of the above-described combination but the distribution to the non-Peyer's patches was not particularly influenced.
Table 5 Dispersion Example 2 Example 4 Control Control Example 5 Example 6 mannan Example 4 Example 4 PS- modified + excess + empty Liposomes PS- amount of PS-Tissue Liposomes mannan Liposomes Peyer's patch tissue (number 50.81 73.09 19.97 7.99 of times of+ 3.52+ 7.52 + 1.72 + 0.98 repetition (n=19)(n=20) (n=57) (n=35) ........ ,........ ....................... ....................... .............................................
Significance ~
difference Yes ' ' Yes (level of (5%) Yes ~1%) , ~ significance) ~ ~(1%) ~;Non-Peyer's ~ ~ ~
~patch tissue 30.71 7.8413.65 8.03 2~0;~ ; (number of+ 2.65+ 0.98 ~ 1.84+ 1.00 ~;timès of ~(n=17j(n=24) (n=27)(n=23) repetltiQn) .................. ~.. ..................... ................. ,.. ......................
~Significance~ ~
difference Yes ' I No evel Qf ~ (1%) No ~ignificance) 2~5~ ~
Unit: ng/mm'~x 10-', mean ~ standard error .. ~ : ' . "
: : ,
liposomes were further modified with the mannan derivative, the amount thereof distribution to the Peyer's patches was significantly increased and that distribution to the non-Peyer's patches was significantly decreased.
The results obtained in Control Example 5 wherein the PS-liposomes modified with mannan derivative was injected together with an excess amount of the mannan derivative(100 parts per part of the mannan derivative added to the liposomes) and Control Example 6 ~20 wherein PS-liposomes modified with the mannan derivative was injected together with empty PS-liposomes free from the inner aqueous phase marker [the membrane composition was the same as that of Example 2 but 100 ~ mol (100 times as much as that used in Example 2) of the pids were usedl are shown in Fig. 6 (right) and Table 5 (right).
I~t~was proved that the liposomes of the present invention has properties Gf selectively distribution to the Peyer's patches from :
: : :
,, , ~ .
. .
2~5~
the facts that the distribution of the PS-liposomes modified with mannan derivative to the Peyer's patches was significantly inhibited by the use of the above-described combination but the distribution to the non-Peyer's patches was not particularly influenced.
Table 5 Dispersion Example 2 Example 4 Control Control Example 5 Example 6 mannan Example 4 Example 4 PS- modified + excess + empty Liposomes PS- amount of PS-Tissue Liposomes mannan Liposomes Peyer's patch tissue (number 50.81 73.09 19.97 7.99 of times of+ 3.52+ 7.52 + 1.72 + 0.98 repetition (n=19)(n=20) (n=57) (n=35) ........ ,........ ....................... ....................... .............................................
Significance ~
difference Yes ' ' Yes (level of (5%) Yes ~1%) , ~ significance) ~ ~(1%) ~;Non-Peyer's ~ ~ ~
~patch tissue 30.71 7.8413.65 8.03 2~0;~ ; (number of+ 2.65+ 0.98 ~ 1.84+ 1.00 ~;timès of ~(n=17j(n=24) (n=27)(n=23) repetltiQn) .................. ~.. ..................... ................. ,.. ......................
~Significance~ ~
difference Yes ' I No evel Qf ~ (1%) No ~ignificance) 2~5~ ~
Unit: ng/mm'~x 10-', mean ~ standard error .. ~ : ' . "
: : ,
Claims (15)
1. Lipid membrane structures for oral administration containing phosphatidylserine as an ingredient of the membrane.
2. Lipid membrane structures of claim 1 wherein the membrane comprises phosphatidylserine at least 1 mole % based on the total components of the membrane.
3. Lipid membrane structures of claim 2 wherein the membrane comprises phosphatidylserine in an amount of 5 to 50 mole % based on the total components of the membrane.
4. Lipid membrane structures of claim 3 wherein a drug is contained therein and the drug is surrounded with the membrane which comprises phosphatidylserine and a component selected from the group consisting of an amphiphilic substance and a micelle-forming surfactant.
5. Lipid membrane structures of claim 4 wherein the component is lacithin or sphingomyelin.
6. Lipid membrane structures of claim 5 wherein the membrane comprises a stabilizer.
7. Lipid membrane structures of claim 1 wherein a mannose derivative or a mannan derivative is incorporated in the membrane thereof.
,
,
8. Lipid membrane structures of claim 4 wherein a mannose derivative or a mannan derivative is incorporated in the membrane thereof.
9. Lipid membrane structures of claim 4 wherein a mannan derivative is incorporated in the membrane thereof.
10. Lipid membrane structures of claim 7 wherein the mannose derivative is selected from the group consisting of phosphatidylethanolamine derivatives having a mannose residue, cholesterol derivatives having a mannose residue, dimannosyl diglyceride, mannobiose fatty acid esters and amides and phosphatidyl mannose.
11. Lipid membrane structures of claim 7 wherein the mannan derivative is selected from the group consisting of mannan partially substituted with a fatty acid and mannan partially substituted with cholesterol.
12. Lipid membrane structures for oral administration wherein a mannose derivative or a mannan derivative is incorporated in the membrane thereof.
13. A method for delivering a drug to Peyer's patches in a digestive tract comprising orally administering lipid membrane structures in which the drug is contained therein and the drug is surrounded with a membrane which comprises phosphatidylserine and a component selected from the group consisting of an amphiphilic substance and a micelle-forming surfactant.
14. A method of claim 13 wherein the component is lecithin or sphingomyelin.
15. A method of claim 13 wherein the lipid membrane structures contains a mannose derivative or a mannan derivative in the membrane thereof.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP132385/1991 | 1991-03-25 | ||
| JP3132385A JP2922017B2 (en) | 1991-03-25 | 1991-03-25 | Oral lipid membrane structure |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2052164A1 true CA2052164A1 (en) | 1992-09-26 |
Family
ID=15080154
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2052164 Abandoned CA2052164A1 (en) | 1991-03-25 | 1991-09-24 | Lipid membrance structures for oral administration |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JP2922017B2 (en) |
| CA (1) | CA2052164A1 (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000024377A1 (en) * | 1998-10-23 | 2000-05-04 | Idea Innovative Dermale Applikationen Gmbh | Method for developing, testing and using associates of macromolecules and complex aggregates for improved payload and controllable de/association rates |
| US7175850B2 (en) | 1998-12-23 | 2007-02-13 | Idea Ag | Formulation for topical non-invasive application in vivo |
| US7459171B2 (en) | 1999-07-05 | 2008-12-02 | Idea Ag | Method for the improvement of transport across adaptable semi-permeable barriers |
| US7473432B2 (en) | 2002-10-11 | 2009-01-06 | Idea Ag | NSAID formulations, based on highly adaptable aggregates, for improved transport through barriers and topical drug delivery |
| US7867480B1 (en) | 1999-01-27 | 2011-01-11 | Gregor Cevc | Non-invasive vaccination through the skin |
| US7927622B1 (en) | 1999-01-27 | 2011-04-19 | Gregor Cevc | Methods of transnasal transport/immunization with highly adaptable carriers |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005119986A (en) * | 2003-10-15 | 2005-05-12 | Japan Science & Technology Agency | Method for administering physiologically active substance, physiologically active substance-containing emulsion, and method for producing physiologically active substance-containing emulsion |
| US9757332B2 (en) | 2013-05-20 | 2017-09-12 | Uha Mikakuto Co., Ltd. | Gel-like composition having high ubiquinol content |
-
1991
- 1991-03-25 JP JP3132385A patent/JP2922017B2/en not_active Expired - Fee Related
- 1991-09-24 CA CA 2052164 patent/CA2052164A1/en not_active Abandoned
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000024377A1 (en) * | 1998-10-23 | 2000-05-04 | Idea Innovative Dermale Applikationen Gmbh | Method for developing, testing and using associates of macromolecules and complex aggregates for improved payload and controllable de/association rates |
| AU765385B2 (en) * | 1998-10-23 | 2003-09-18 | Idea Ag | Method for developing, testing and using associates of macromolecules and complex aggregates for improved payload and controllable de/association rates |
| AU765385C (en) * | 1998-10-23 | 2004-05-20 | Idea Ag | Method for developing, testing and using associates of macromolecules and complex aggregates for improved payload and controllable de/association rates |
| US7175850B2 (en) | 1998-12-23 | 2007-02-13 | Idea Ag | Formulation for topical non-invasive application in vivo |
| US7867480B1 (en) | 1999-01-27 | 2011-01-11 | Gregor Cevc | Non-invasive vaccination through the skin |
| US7927622B1 (en) | 1999-01-27 | 2011-04-19 | Gregor Cevc | Methods of transnasal transport/immunization with highly adaptable carriers |
| US7459171B2 (en) | 1999-07-05 | 2008-12-02 | Idea Ag | Method for the improvement of transport across adaptable semi-permeable barriers |
| US7591949B2 (en) | 1999-07-05 | 2009-09-22 | Idea Ag | Method for the improvement of transport across adaptable semi-permeable barriers |
| US7473432B2 (en) | 2002-10-11 | 2009-01-06 | Idea Ag | NSAID formulations, based on highly adaptable aggregates, for improved transport through barriers and topical drug delivery |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2922017B2 (en) | 1999-07-19 |
| JPH0517344A (en) | 1993-01-26 |
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