CA1116518A - Method for purifying a liposomic suspension - Google Patents
Method for purifying a liposomic suspensionInfo
- Publication number
- CA1116518A CA1116518A CA000312035A CA312035A CA1116518A CA 1116518 A CA1116518 A CA 1116518A CA 000312035 A CA000312035 A CA 000312035A CA 312035 A CA312035 A CA 312035A CA 1116518 A CA1116518 A CA 1116518A
- Authority
- CA
- Canada
- Prior art keywords
- resin
- suspension
- purified
- lipidic
- liposomic
- 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.)
- Expired
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
- A61K9/1277—Processes for preparing; Proliposomes
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Dispersion Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Preparation (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
A method is disclosed for purifying a liposomic suspen-sion from a non-entrapped free drug by contacting same with an ion exchange resin either in the activated or the non-activated form. The resin may be of the anioinic type or the cationic type, and either strong or weak. The liposomic suspension, thus purified, may thereafter be lyophilized for the purpose of stabilizing same.
The purified liposomic suspension and the stabilized purified liposomic suspension are also part of the invention. An adsorbent resin may also be used.
A method is disclosed for purifying a liposomic suspen-sion from a non-entrapped free drug by contacting same with an ion exchange resin either in the activated or the non-activated form. The resin may be of the anioinic type or the cationic type, and either strong or weak. The liposomic suspension, thus purified, may thereafter be lyophilized for the purpose of stabilizing same.
The purified liposomic suspension and the stabilized purified liposomic suspension are also part of the invention. An adsorbent resin may also be used.
Description
i5~F~
1 This invention relates to a new method ~or preparing injectable liposomes.
Liposomes are a pharmaceu-tical composition in which the drug is contained in corpuscles or globules consisting of aqueous and lipidic concentric layers. The drug may be present in either or both of the aqueous and/or lipidic layers. The lipidic layer comprises or consists generally of a phospholipid, such as lecithin and sphyngomyelin; a steroid, for example cholesterol;
and an ionic tensioactive or surface active substance, such as dicetylphosphate, stearylamine and phosphatidic acid. The dia-meter ofliposomes ranges from 0.01 to 5 ~. The procedure usually employed for preparing liposomes comprises two main stepso 1. Preparation of liposomes. The lipidic components are dissolved in chloroform, which is then evaporated under vacuum.
In the flask containing the residue as a thin layer, the drug solution is added and the whole is subjected to ultrasonic shaking for a time ranging from 30 seconds to 12 hours. The liposomic suspension so obtained contains an important fraction of non-entrapped drug which must be separated ~rom the liposomes.
1 This invention relates to a new method ~or preparing injectable liposomes.
Liposomes are a pharmaceu-tical composition in which the drug is contained in corpuscles or globules consisting of aqueous and lipidic concentric layers. The drug may be present in either or both of the aqueous and/or lipidic layers. The lipidic layer comprises or consists generally of a phospholipid, such as lecithin and sphyngomyelin; a steroid, for example cholesterol;
and an ionic tensioactive or surface active substance, such as dicetylphosphate, stearylamine and phosphatidic acid. The dia-meter ofliposomes ranges from 0.01 to 5 ~. The procedure usually employed for preparing liposomes comprises two main stepso 1. Preparation of liposomes. The lipidic components are dissolved in chloroform, which is then evaporated under vacuum.
In the flask containing the residue as a thin layer, the drug solution is added and the whole is subjected to ultrasonic shaking for a time ranging from 30 seconds to 12 hours. The liposomic suspension so obtained contains an important fraction of non-entrapped drug which must be separated ~rom the liposomes.
2. Separation of the liposomec, from the non-entrapped drug. This procedure is carried out by column chromatograph~ with `
e.g. Sepharose 6B as a molecular sieve material. Sepharose is a registered trademark for spherical agarose gel beads manufactured by Pharmacia Fine Chemicals AB. The "6B" refexs to an agarose concentration of about 6% and a particle size in the wet state of about 40 to 210,u. The liposomes eluate first, whereas the free drug is retained by the Sepharose.
Another suitable procedure is ultracentrifuga-tion at lOO,OOOg and subsequent washing, always by unltracentrifugation, with buffered solution.
~lfiS~
1 The liposomes have demons-trated, in the animal, an abil-ity to direct antitumoral drugs selectively toward neoplastic cells.
The presen-t invention relates to a new procedure for separatiny liposomes from non-entrapped drug. It utilizes ion-exchange and adsorbent resins without the necessity of employing chromatography. The ion-exchange resin is added to the liposomic suspension of the drug which is to be purified, and the whole shaken for 1-120 minutes.
The free liposomic suspension is ob-tained after filtra-tion through porous sheet that retains the resin on which the free drug has been adsorbed. A suitable porous sheet is one made e.g.
of a sin-tered glass filter material, such as manufactured by the German firm Jena er Glaswerk Schott 8 Gen. Mainz that produces a wide range o~ suitable glasses. These glasses are named with different alphabetical letters and numbers (e.g. Gl : in this case number 1 reEers to the broader pores generally used in filtration).
The pore size abviously changes. The alphabetical letter refers to the kind of sintered glass that also changes depending on the eventual desired transfer o~ residue due to filtration.
This new purification procedure, utilizing ion-exchange resins, shows the great advantage of giving very concentrated liposomic suspensions (up to 4 mg/ml of Adriamycin hydrochloride) which cannot be achieved by chromatography with molecular sieves ~max 200 r/ml). The liposomic suspension so obtained is very stable and is not inclined to sedimentation in contrast to the suspensions obtained by ultracentrifugation at high r.p.m.'s.
The desired chemical stabilization is achieved by lyophilization of the liposomic suspension.
The following examples are provided to still further illustrate the invention but without limiting it:
L6~
In a saponiEication flask, l.5 y of egg lecithin, 0.4 g of cholesterol, and 0.2 g of dicetylphosphate were dissolved in chloroform and the solvent evaporated to dryness. An adriamycin hydrochloride solution (concentration 20 mg/ml) in 0.007 N buffer phosphate was then added and the resulting solution was subjected to ultrasonic shaking for one minute.
The suspension was then allowed to stand at room temper-ature under a nitrogen atmosphere for 30 minutes, then 2 g of BDH IR-50 resin (a granular-polymeric resin manufactured by R~hm Haas), previously activated in the sodium form, were added (weight refers to dry weight and is equivalent to 5 ml of inflated resin. By "inflated resin" is meant that the resin has been activated by two successive treatments, first in a 4% HCl solution and then in a 4~ NaOH solution).
The flask was subjected for 30 minutes to shaking; the suspension was then filtered over a porous sintered glass sheet Gl.
Liposomes of size varying from 0.5 to 2~1 and containing ~O 20% of the starting amount of adriamycin have been obtained. They were made stable by lyophilization.
Operating as described in Example l, and employing the same amounts of adriamycin and lipids, the ultrasonic shaking time was extended to lQ minutes in order to obtain liposomes of size less than l,u. A non-granular resin, having also the function of a sieve, was selected in view of the fact that the whole body of the liposomes is not perfectly homogeneous in size.
Accordingly, l0 ml of DOWEX 50W-X 4 100-200 mesh resin (a macroreticular resin available from Dow), and previously Trade Marks -3-,,~ j ~L65~
1 a~tivated in sodium form, were introduced into the flask. After filtration, a suspension was ob-tained containing liposomes ranging from 0.2 and ~.8~ in size and containing 12% of the starting adriamycin. The liposomes were then made sta~le by lyophilization.
A solution of 5-fluorouracil at a concentration of 10 mg/ml in 0.007N buffer phosphate and pH 8, was poured into the saponification flask containing the lipidic phase prepared as previously described. The suspension was treated as in Example 1, using as filtering resin 10 ml of Amberlite IRA-400 (Cl), a granular polymeric resin produced by Rohm & Haas, previously activated as the hydrochloride. The liposomes so obtained were made sta~le by lyophilization.
Liposomes of 5-fluorouracil were prepared as prevlously described utilizing 1.5 g of egg lecithin, 0.4 g of cholesterol, and 0.2 g of stearylamine. As the purification agent 10 ml of a previously activated DOWEX 1 resin (50-100 mesh), a granular poly-meric resin available from Dow, were used. The liposomes so ob-tained were stabilized by lyophilization.
Operating under the same conditions and with the same amounts as in Example 2, an adriamycin liposomic suspension was prepared, purified by usiny 15 g of adsorbing Rohm h Haas XAD 7 resin (a granular polymeric resin), directly placed into the pre-paration flask.
A~ter shaking for 40 minutes and filtration through sintered filter glass Gl, a suspension of liposomes containing about 50% of the startiny quanti-ty of adriamycin was obtained, which was then stabilized as before by lyophilization.
Trade Marks -4-~ 1
e.g. Sepharose 6B as a molecular sieve material. Sepharose is a registered trademark for spherical agarose gel beads manufactured by Pharmacia Fine Chemicals AB. The "6B" refexs to an agarose concentration of about 6% and a particle size in the wet state of about 40 to 210,u. The liposomes eluate first, whereas the free drug is retained by the Sepharose.
Another suitable procedure is ultracentrifuga-tion at lOO,OOOg and subsequent washing, always by unltracentrifugation, with buffered solution.
~lfiS~
1 The liposomes have demons-trated, in the animal, an abil-ity to direct antitumoral drugs selectively toward neoplastic cells.
The presen-t invention relates to a new procedure for separatiny liposomes from non-entrapped drug. It utilizes ion-exchange and adsorbent resins without the necessity of employing chromatography. The ion-exchange resin is added to the liposomic suspension of the drug which is to be purified, and the whole shaken for 1-120 minutes.
The free liposomic suspension is ob-tained after filtra-tion through porous sheet that retains the resin on which the free drug has been adsorbed. A suitable porous sheet is one made e.g.
of a sin-tered glass filter material, such as manufactured by the German firm Jena er Glaswerk Schott 8 Gen. Mainz that produces a wide range o~ suitable glasses. These glasses are named with different alphabetical letters and numbers (e.g. Gl : in this case number 1 reEers to the broader pores generally used in filtration).
The pore size abviously changes. The alphabetical letter refers to the kind of sintered glass that also changes depending on the eventual desired transfer o~ residue due to filtration.
This new purification procedure, utilizing ion-exchange resins, shows the great advantage of giving very concentrated liposomic suspensions (up to 4 mg/ml of Adriamycin hydrochloride) which cannot be achieved by chromatography with molecular sieves ~max 200 r/ml). The liposomic suspension so obtained is very stable and is not inclined to sedimentation in contrast to the suspensions obtained by ultracentrifugation at high r.p.m.'s.
The desired chemical stabilization is achieved by lyophilization of the liposomic suspension.
The following examples are provided to still further illustrate the invention but without limiting it:
L6~
In a saponiEication flask, l.5 y of egg lecithin, 0.4 g of cholesterol, and 0.2 g of dicetylphosphate were dissolved in chloroform and the solvent evaporated to dryness. An adriamycin hydrochloride solution (concentration 20 mg/ml) in 0.007 N buffer phosphate was then added and the resulting solution was subjected to ultrasonic shaking for one minute.
The suspension was then allowed to stand at room temper-ature under a nitrogen atmosphere for 30 minutes, then 2 g of BDH IR-50 resin (a granular-polymeric resin manufactured by R~hm Haas), previously activated in the sodium form, were added (weight refers to dry weight and is equivalent to 5 ml of inflated resin. By "inflated resin" is meant that the resin has been activated by two successive treatments, first in a 4% HCl solution and then in a 4~ NaOH solution).
The flask was subjected for 30 minutes to shaking; the suspension was then filtered over a porous sintered glass sheet Gl.
Liposomes of size varying from 0.5 to 2~1 and containing ~O 20% of the starting amount of adriamycin have been obtained. They were made stable by lyophilization.
Operating as described in Example l, and employing the same amounts of adriamycin and lipids, the ultrasonic shaking time was extended to lQ minutes in order to obtain liposomes of size less than l,u. A non-granular resin, having also the function of a sieve, was selected in view of the fact that the whole body of the liposomes is not perfectly homogeneous in size.
Accordingly, l0 ml of DOWEX 50W-X 4 100-200 mesh resin (a macroreticular resin available from Dow), and previously Trade Marks -3-,,~ j ~L65~
1 a~tivated in sodium form, were introduced into the flask. After filtration, a suspension was ob-tained containing liposomes ranging from 0.2 and ~.8~ in size and containing 12% of the starting adriamycin. The liposomes were then made sta~le by lyophilization.
A solution of 5-fluorouracil at a concentration of 10 mg/ml in 0.007N buffer phosphate and pH 8, was poured into the saponification flask containing the lipidic phase prepared as previously described. The suspension was treated as in Example 1, using as filtering resin 10 ml of Amberlite IRA-400 (Cl), a granular polymeric resin produced by Rohm & Haas, previously activated as the hydrochloride. The liposomes so obtained were made sta~le by lyophilization.
Liposomes of 5-fluorouracil were prepared as prevlously described utilizing 1.5 g of egg lecithin, 0.4 g of cholesterol, and 0.2 g of stearylamine. As the purification agent 10 ml of a previously activated DOWEX 1 resin (50-100 mesh), a granular poly-meric resin available from Dow, were used. The liposomes so ob-tained were stabilized by lyophilization.
Operating under the same conditions and with the same amounts as in Example 2, an adriamycin liposomic suspension was prepared, purified by usiny 15 g of adsorbing Rohm h Haas XAD 7 resin (a granular polymeric resin), directly placed into the pre-paration flask.
A~ter shaking for 40 minutes and filtration through sintered filter glass Gl, a suspension of liposomes containing about 50% of the startiny quanti-ty of adriamycin was obtained, which was then stabilized as before by lyophilization.
Trade Marks -4-~ 1
Claims (19)
1. A method for purifying a liposomic suspension which consists of a drug contained in corpuscles or globules consisting of aqueous and lipidic concentric layers, said method comprising contacting said suspension with a compound selected from the group consisting of an activated ion exchange resin, a non activated ion exchange resin and an adsorbent polymeric substance.
2. A method as claimed in claim 1 wherein said resin is a strong anionic resin.
3. A method as claimed in claim 1 wherein said resin is a weak anionic resin.
4. A method as claimed in claim 1 wherein said resin is a strong cationic resin.
5. A method as claimed in claim 1 wherein said resin is a weak cationic resin.
6. A method as claimed in claim 1 further including the step of lyophilizing said purified suspension.
7. A method as claimed in claim 1 wherein said lipidic layer consists of a phospholipid, a steroid and an ionic tensio-active or surface active substance.
8. A method as claimed in claim 7 wherein said phosphol-ipid is lecithin or sphyngomyelin.
9. A method as claimed in claim 7 wherein said steroid is cholesterol.
10. A method as claimed in claim 7 wherein said ionic tensioactive or surface active substance is selected from the group consisting of dicetylphosphate, stearylaminee and phosphatidic acid.
11. A method as claimed in claim 1 wherein said drug is contained in globules of aqueous and lipidic concentric layers.
12. A method as claimed in claim 1 wherein said compound is added to said suspension, shaken and then filtered.
13. A method as claimed in claim 12 wherein said filtration is carried out through a porous sheet which retains said compound on which the free drug has been adsorbed.
14. A method as claimed in claim 13 wherein said porous sheet is made of sintered glass filter material.
15. A method as claimed in claim 11 wherein the diameter of said globules is from about 0.01/µ to about 5/µ.
16. A purified liposomic suspension which consists of layers of aqueous and lipidic concentric layers whenever prepared by the process as claimed in claim 1.
17. A stabilized and purified liposomic suspension which consists of layers of aqueous and lipidic concentric layers whenever prepared by the process as claimed in claim 6.
18. A purified liposomic suspension as claimed in claim 16 wherein said lipidic layer consists of a phospholipid, a steroid and an ionic tensioactive or surface active substance.
19. A stabilized and purified liposomic suspension as claimed in claim 17 wherein said lipid layer consist of a phospholip, a steroid and an ionic tensioactive or surface active substance.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IT28147A/77 | 1977-09-30 | ||
| IT2814777 | 1977-09-30 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1116518A true CA1116518A (en) | 1982-01-19 |
Family
ID=11223032
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000312035A Expired CA1116518A (en) | 1977-09-30 | 1978-09-25 | Method for purifying a liposomic suspension |
Country Status (13)
| Country | Link |
|---|---|
| JP (1) | JPS5459314A (en) |
| AT (1) | AT368879B (en) |
| AU (1) | AU528260B2 (en) |
| BE (1) | BE870882A (en) |
| CA (1) | CA1116518A (en) |
| CH (1) | CH642540A5 (en) |
| DE (1) | DE2842608C2 (en) |
| DK (1) | DK154048C (en) |
| FR (1) | FR2404454A1 (en) |
| GB (1) | GB2004745B (en) |
| NL (1) | NL7809709A (en) |
| SE (1) | SE433038B (en) |
| ZA (1) | ZA785510B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IT1110989B (en) * | 1979-01-19 | 1986-01-13 | Erba Farmitalia | PHARMACEUTICAL FORMS CONSTITUTED BY LIPOSOMES AND RELATED PROCEDURES |
| JPS5775916A (en) * | 1980-10-29 | 1982-05-12 | Nippon Chemiphar Co Ltd | Coenzyme q pharmaceutical and its preparation |
| JPS5782310A (en) * | 1980-11-11 | 1982-05-22 | Tanabe Seiyaku Co Ltd | Production of liposome preparation |
| CA1270198C (en) * | 1984-08-08 | 1990-06-12 | Marcel B Bally | Encapsulation of antineoplastic agents in liposomes |
| US4619795A (en) * | 1984-12-24 | 1986-10-28 | Technicon Instruments Corp. | Method for preparing lipid vesicles |
| DE4038075C1 (en) * | 1990-11-29 | 1992-03-19 | B. Braun Melsungen Ag, 3508 Melsungen, De | Encapsulating solid or liq. lipophilic agents - comprises mixing hydration medium with phospholipid increasing temp. to above soln. phase change temp. and adding remaining medium |
| DE10137515A1 (en) * | 2001-07-26 | 2003-02-13 | Diagnostikforschung Inst | Production of pharmaceutical preparations in charged particle dispersion form, e.g. contrast agent dispersion, including separation of particles using ion exchangers or by electrophoresis |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2249552A1 (en) * | 1971-10-12 | 1973-05-30 | Inchema S A | PROCESS FOR THE INCAPSULATION OF IN PARTICULAR WATER-SOLUBLE COMPOUNDS |
| GB1523965A (en) * | 1976-03-19 | 1978-09-06 | Ici Ltd | Pharmaceutical compositions containing steroids |
-
1978
- 1978-09-25 CA CA000312035A patent/CA1116518A/en not_active Expired
- 1978-09-25 NL NL7809709A patent/NL7809709A/en not_active Application Discontinuation
- 1978-09-25 AU AU40174/78A patent/AU528260B2/en not_active Expired
- 1978-09-26 DK DK425478A patent/DK154048C/en not_active IP Right Cessation
- 1978-09-26 AT AT0693178A patent/AT368879B/en active
- 1978-09-26 FR FR7827488A patent/FR2404454A1/en active Granted
- 1978-09-26 SE SE7810109A patent/SE433038B/en not_active IP Right Cessation
- 1978-09-27 JP JP11809678A patent/JPS5459314A/en active Granted
- 1978-09-28 ZA ZA00785510A patent/ZA785510B/en unknown
- 1978-09-28 GB GB7838495A patent/GB2004745B/en not_active Expired
- 1978-09-29 DE DE2842608A patent/DE2842608C2/en not_active Expired
- 1978-09-29 CH CH1016078A patent/CH642540A5/en not_active IP Right Cessation
- 1978-09-29 BE BE190800A patent/BE870882A/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| ATA693178A (en) | 1982-04-15 |
| AT368879B (en) | 1982-11-25 |
| BE870882A (en) | 1979-03-29 |
| JPS6134402B2 (en) | 1986-08-07 |
| NL7809709A (en) | 1979-04-03 |
| ZA785510B (en) | 1979-09-26 |
| DK425478A (en) | 1979-03-31 |
| SE7810109L (en) | 1979-03-31 |
| FR2404454A1 (en) | 1979-04-27 |
| AU4017478A (en) | 1980-04-03 |
| DE2842608A1 (en) | 1979-04-12 |
| AU528260B2 (en) | 1983-04-21 |
| FR2404454B1 (en) | 1980-06-20 |
| DE2842608C2 (en) | 1986-02-06 |
| GB2004745A (en) | 1979-04-11 |
| JPS5459314A (en) | 1979-05-12 |
| DK154048C (en) | 1989-03-13 |
| CH642540A5 (en) | 1984-04-30 |
| SE433038B (en) | 1984-05-07 |
| DK154048B (en) | 1988-10-10 |
| GB2004745B (en) | 1982-04-07 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |