DE102006054192A1 - Method for loading nucleic acid active substances within amphoteric liposomes, comprises: mixing lipid mixture solution and aqueous solution of nucleic acid active substances; and sterile filtrating the liposomes containing the substances - Google Patents

Abstract

Method for loading of nucleic acid active substances within amphoteric liposomes, comprises: (a) providing a lipid mixture solution; (b) providing an aqueous solution of the nucleic acid active substance; (c) mixing the two solutions by injecting the alcoholic solution of the lipid mixture into the nucleic acid solutions or vice versa; (f) separating the non-included active substance, saturation of the liposome suspension, exchange of the aqueous medium and/or separation of the water-miscible solvent, and (g) optionally sterile filtrating the liposomes containing the active substance. Method for loading of nucleic acid active substances within amphoteric liposomes, comprises: (a) providing a solution of a lipid mixture in a water-miscible solvent, preferably an alcohol that can be optionally acidified; (b) providing an aqueous solution of the nucleic acid active substance to be included that can be optionally acidified, where at least one of the two solutions in (a) and (b) has to be set to a pH value with the help of acids and/or buffer substances; (c) mixing the two solutions by injecting the alcoholic solution of the lipid mixture into the aqueous solution of the nucleic acids or vice versa or by combination of two partial flows of the provided lipid solution and nucleic acid solutions, optionally in one or more mixers; (d) optionally diluting the mixed solutions; (e) breaking up of the interactive forces between the amphoteric liposomes and the nucleic acids through an increase of the pH value to more than 7 or by an increase of the ionic strength and subsequent increase of the pH value to more than 7; (f) separating the non-included active substance and/or saturation of the liposome suspension and/or exchange of the aqueous medium and/or separation of the water-miscible solvent, where each of these steps is independently optional; and (g) optionally sterile filtrating the liposomes containing the active substance, where between the steps (c) and (d) and/or between (d) and (e) and/or between (e) and (f) and/or between (f) and (g), an extruding step can take place and/or between the steps (e) and (f) and/or between (f) and (g), one or more freezing/thawing cycle(s) can take place and/or after step (e), (f) or (g), one or more freezing/thawing cycle(s) and/or a lyophilization of the liposomes containing the active substance can take place. An independent claim is included for the amphoteric liposomes comprising the nucleic acids obtained by the above method. ACTIVITY : None given. MECHANISM OF ACTION : Gene therapy.

Description

liposomes can be prepared by a variety of methods.

These include the long-known lipid film method, which, for example, in US 5648090 (Rahman et al.). The lipids are dissolved in organic solvent, which is then removed again with a rotary evaporator. An aqueous solution of the drug is added to the thin film formed, and the lipid film is rehydrated to form liposomes and include the portion of the drug that corresponds to their internal volume. The multilamellar particles resulting from this process can be reduced to smaller sizes and unilamellarity by subsequent extrusion (Olson et al., Biochim. Biophys. Acta., 1979 Oct 19; 557 (1): 9-23). Especially in the preparation of pharmaceutical preparations, control of liposome size and lamellarity is important. During extrusion, the multilamellar liposomes are forced under high pressure through membranes of defined pore size, thus achieving the desired diameter. In this case, similar to the high-pressure homogenization described below, several cycles have to be run through in order to obtain a narrow size distribution. A disadvantage of this method is the use of a membrane, a sensitive Verscheißteils, which must be renewed at regular intervals. Membrane cracks can lead to quality and product losses. In addition, the provision of a lipid film for larger production approaches is technically complicated.

Known High-pressure homogenization, microfluidizer and ultrasonic methods for the production of liposomes use the resulting in the process shear and cavitation effects to the size and lamellarity of the liposomes to control. However, lead These methods are used locally to produce very high pressures and temperatures sensitive lipids, for example unsaturated fatty acid residues or sensitive drugs such as nucleic acids can be damaged. The Product properties, such as stability and effectiveness are therefore changed adversely.

Other methods described (Papahadjopulos, US 4235871 , 1980; Martin et al. US 4752425 , 1988) achieve the inclusion of drugs in the injection of a water-immiscible, organic solution of lipids into an aqueous solution of the entrapped material. If the organic solvent of the lipid is evaporated according to its supply, then spontaneously form liposomes. This process can be continued up to a high lipid concentration. However, water-immiscible organic solvents are not used, which, because of their toxicity, have to be completely removed at the end of the process in complex and expensive processes.

Liposome production per ethanol injection

With the injection of an ethanolic lipid phase into an aqueous phase, liposomes can also be prepared as described by Batzri and Korn in Biophys. Biochem. Acta 298, 1015-1019 (1973) first described. The lipid phase is introduced into a stirred aqueous phase via a fine needle, whereby liposomes spontaneously form by dilution. This method is very simple and allows the production of liposomes on an industrial scale. In US6843942 and US20040032037A1 describes an apparatus for the continuous or batch production of liposomes. An aqueous, optionally active substance-containing solution and an alcoholic lipid-containing solution are fed into the system in two separate lines. The organic phase conduction is perpendicular to the polar aqueous phase. Through an opening at the junction of the two lines, the solutions are mixed by means of a spray valve, whereby turbulence and shear forces are avoided. Liposomes with a narrow size distribution form, which can be influenced by two process parameters: On the one hand, the amount of lipid and / or the amount of solvent which is added to the polar phase can be varied. On the other hand, the size distribution of the liposomes can be influenced by the variation of the pressure during the process. In the examples described, the device is explained in more detail on the basis of the encapsulation of recombinant human superoxide dismutase in liposomes of defined composition. The inclusion of the protein takes place exclusively by means of the so-called "passive" method in which the active ingredient dissolved in the polar phase does not interact with the lipid membranes.

US 6287591 describes a method for including charged active substances in pH-sensitive PEG-liposomes. As pH-sensitive components, lipids with a protonatable amino group are used, which are positively neutral at acidic pH values of 4, and approximately neutral at physiological pH values of 7.5. The liposomes are prepared at acidic pH (eg, 3.8) by a ethanol injection method. At this pH, the negatively charged oligonucleotide drugs interact with the cationically present pH-sensitive lipids. By using another lipid component with hydrophilic polymers (Eg PEG-lipids) prevents aggregation of the forming lipid particles. It forms an intermediate, which is composed of drug-containing liposomes and free drug, which binds by ionic interactions to the surface of the liposomes. In order to arrive at defined size distributions, the liposomes can be further processed by a method known to a person skilled in the art, for example extrusion or ultrasound treatment. In a final step, the oligonucleotides adhering to the outside of the membrane are replaced by a pH change. However, the described method requires the presence of PEG-lipids in the liposomal membrane to avoid aggregation of the lipid particles during the formation process. However, the use of PEG-lipids in pharmaceutical preparations can trigger immune reactions. This leads eventually to repeated circulation of the PEGylated liposomes to shortened circulation half-lives.

amphoteric Liposomes represent a recent described novel class of liposomes which at a pH of 7.5 are anionically or neutrally charged and at a pH of 4 have a cationic charge. The amphoteric liposomes and the for their preparation suitable lipids are described in detail in WO 02 066490 A2, WO 02 066489 A2, WO 02 066012 A2, WO 03 070735 A2 and WO 03/070220 (all described by Panzner et al.). Show amphoteric liposomes an excellent in vivo biodistribution and are very good in the animal compatible.

WHERE 02 066012 A2 describes about it In addition, a method in which nucleic acid agents are effectively amphoteric Liposomes can be included. This method uses the attractive interactions of the anionically charged nucleic acid agents with the cationically charged liposome membrane at acidic pH out.

task The present invention is a process for the preparation Nucleic acid-loaded to provide amphoteric liposomes which are suitable, nucleic acid-containing to produce amphoteric liposomes on an industrial scale. Another The object of the present invention is to provide process parameters with which the size of the amphoteric Controlled liposomes and the drug / lipid ratio in the final product can be.

Brief description of the invention

amphoteric Liposomes can effective with nucleic acids be loaded by adding an alcoholic lipid solution into a nucleic acid-containing polar watery Phase is injected at acidic pH. Surprisingly, it was found that the particle aggregates forming in this process deteriorate a change the pH or after a change the ionic strength dissolve again and liposomes of defined size become. In a further embodiment The invention uses a two - stage process in which first with a higher one Amount of solvent is working. That concludes a dilution step at. With this step, either the acidic pH can initially be maintained and in another step the pH change respectively. Alternatively, the dilution step may change the pH respectively. It was also found that with process parameters such as lipid concentration, temperature, ionic strength, and alcohol content additions like sucrose a precise Adjustment of both liposome size and drug / lipid ratio possible is. Surprisingly result from the litigation under conditions involving an interaction between nucleic acids and Lipid membrane allow significantly different particle sizes than it is the case in an absence of this interaction. Farther was surprising found that with the process control presented the formation irreversible Aggregates even in the absence of PEG-lipids is avoided.

In an embodiment According to the invention, the liposomes produced are prepared by an extrusion step further processed. This step can be at acidic pH, so immediately before the change of the pH to> 7, or after the pH change to pH> 7. Surprisingly It was found that the extrusion step did not become a substantial one Release of the entrapped nucleic acid drug independently which pH the extrusion is carried out. The advantage of this embodiment is that even narrower size distributions are obtained can. By a narrower size distribution In particular, a sterile filtration of the liposomes is facilitated.

Furthermore, it has surprisingly been found that the liposomes produced by the method according to the invention can be frozen or freeze-dried for better storage in a suitable medium without releasing appreciable amounts of enclosed nucleic acid active substances. Quite surprisingly, it has been found that only by freezing and thawing a narrower size distribution of the liposomes can be obtained. Also on this way can be an optional through guided sterile filtration of the liposomes are facilitated.

Detailed description of the invention

As explained above in US6843942 and US20040032037A1 describes an apparatus for the continuous or batch production of active substance-containing liposomes, with which liposomes can be produced on an industrial scale. The specification describes the "passive" inclusion of recombinant superoxide dismutase in liposomes. "Passive" inclusion means that no interactions take place between the lipids and the drug to be entrapped and that entrapment efficiency is determined by the total internal volume of the liposomes formed.

In WO 02 066012 A2 describes the preparation of DNA-loaded amphoteric Liposomes are described by an extrusion process, with the attractive Interactions of the anionic charged nucleic acid agents with the cationically charged liposome membrane at acidic pH is exploited. Will now be nucleic acid-loaded amphoteric liposomes under the binding conditions described in WO 02 066012 A2 produced by ethanol injection, surprisingly shows that the size of the educated active substance-containing amphoteric liposomes clearly on the size of the does not distinguish binding conditions of amphoteric liposomes (see "passive" inclusion) and to larger diameters is postponed.

Further was found to be surprising by a variation of the process parameters, described in more detail below Effects that can be used to increase the size of the nucleic acid-loaded amphoteric Liposomes and the drug / lipid ratio in the final product too check.

So showed up surprisingly, that the entrapment efficiency in the process according to the invention, in contrast to the "passive" inclusion of active ingredients increased in liposomes, with lower lipid concentrations in the process.

Furthermore was surprisingly a temperature dependence the entrapment efficiency found in the method according to the invention. So be at higher Temperatures higher Inclusion benefits achieved.

Further led the increase the ionic strength or osmolarity in the aqueous drug solution surprising with sodium chloride Results in the process according to the invention. Up to a certain NaCl concentration decreases the entrapment efficiency, while at the same time reducing the size of the formed Liposomes increases. If the NaCl concentration is further increased, it changes the size of the liposomes and the inclusion efficiency first no more. At very high salt concentrations, the interaction becomes finally complete between the amphoteric liposomes and the nucleic acids suppressed so that the inclusion about the "passive" procedure takes place. This is also reflected in the smaller size of the formed amphoteric Reflected liposomes.

Becomes a sugar (sucrose) instead of salt to increase the osmolarity to the aqueous drug solution given, however, showed another effect. Surprisingly, the Size of through the inventive method prepared nucleic acid loaded amphoteric liposomes almost constant, while the inclusion efficiency increased.

The inventive method, with which nucleic acid-loaded amphoteric liposomes can be prepared, is described in more detail below.

amphoteric liposomes

amphoteric Liposomes can are prepared from lipid mixtures which are either an amphoteric Lipid or a mixture of lipids with amphoteric properties and optionally include a neutral lipid.

• (i) mindestens eine der geladenen Gruppen einen pk Wert zwischen 4 und 7,4 aufweist,
• (ii) die kationische Ladung bei pH 4 überwiegt,
• (iii) und die anionische Ladung bei pH 7,4 überwiegt, wobei die Liposomen einen isoelektrischen Punkt zwischen 4 und 7,4 besitzen. Der amphotere Charakter unterscheidet sich dabei vom zwitterionischen Charakter, da Zwitterionen keinen pk-Wert im oben angegebenen pH-Bereich aufweisen. Daraus folgt, dass Zwitterionen über einen weiten pH-Bereich ungeladen vorliegen.

"Amphoteric" means that the liposomes comprise anionic as well as cationic charged functional groups, wherein

• (i) at least one of the charged groups has a pk value between 4 and 7.4,
• (ii) the cationic charge at pH 4 predominates,
• (iii) and the anionic charge predominates at pH 7.4, the liposomes having an isoelectric point between own 4 and 7.4. The amphoteric character differs from the zwitterionic character, since zwitterions have no pk value in the above-mentioned pH range. It follows that zwitterions are uncharged over a wide pH range.

neutral Phospholipids in the amphoteric lipid mixtures can phosphatidylcholines or phosphatidylethanolamines or mixtures of phosphatidylcholines and phosphatidylethanolamines. Phosphatidylcholines and Phosphatidylethanolamines are neutral lipids with zwitterionic Character.

In a preferred embodiment, the phosphatidylcholines are selected from the following group: POPC, natural or hydrogenated soy PC, natural or hydrogenated egg PC, DMPC, DPPC or DOPC. (A list of abbreviations The lipids used and their names can be found below. In many cases the common in the literature Abbreviations used).

Especially preferred phoaphatidylcholines are DMPC, POPC, non-hydrogenated soy PC and non-hydrogenated egg pc.

The Phosphatidylethanolamine can be selected from the group DOPE, DMPE and DPPE.

All most preferably, said neutral lipids comprise DOPE, DMPC, POPC, non-hydrogenated soy PC and non-hydrogenated Egg PC.

In another embodiment The lipid mixture of amphoteric lipids may also be the neutral lipid cholesterol include.

The Lipid mixing of the amphoteric liposomes can be an amphoteric lipid include. Suitable amphoteric lipids are described in WO 02/066489 and WO 03/070735. Preferably, the amphoteric lipid from the following Group selected: HistChol, HistDG, isoHistSuccDG, acylcarnosine and HCCHol.

alternative can the lipid mixture of amphoteric liposomes also pH sensitive anionic and / or contain cationic components as disclosed in WO 02/066012. Cationic pH-sensitive lipids are described in WO 02/066489 and WO 03/070220 described. About that In addition, Budker et al. in Nat Biotechnol. 14 (6): 760-4, 1996 further cationic pH-sensitive lipids. The cationic pH-sensitive Lipids can in combination with strongly anionic lipids or with anionic Lipids that are pH sensitive can be used. Conversely, can the cationic charge is also due to strong cationic lipids, the the person skilled in the art, in combination with a pH-sensitive anionic lipid introduced become.

preferred cationic components are DPIM, CHIM, DORIE, DDAB, DAC-Chol, TC-Chol, DOTMA, DOGS, (C18) 2Gly + N, N-dioctadecylamidoglycine, CTAB, CPyC, DODAP and DOEPC, DMTAP, DPTAP, DOTAP, DC-Chol, MoChol and HisChol.

Especially preferred cationic lipids are DMTAP, DPTAP, DOTAP, DC-Chol, MoChol, Chim and HisChol.

The Amphoteric mixtures also include anionic lipids, either constitutively or under certain conditions according to the pH are charged, and these lipids are known in the art. preferred Lipids for use in this invention are DMGSucc, POGSucc, DMGSucc, DPGSucc, DMPS, DPPS, DOPS, POPS, DMPG, DPPG, DOPG, POPG, DMPA, DPPA, DOPA, POPA, CHEMS and Cetyl-P.

Especially preferred anionic lipids are DMGSucc, DMGSucc, DMPG, DPPG, DOPG, POPG, DMPA, DPPA, DOPA, POPA, CHEMS and CetylP.

In an execution of the invention the said cationic lipids one or more of the following Lipids include: DOTAP, DC-Chol, MoChol and HisChol. The mentioned anionic lipids can or more of the following lipids include: DMGSucc, DOGSucc, DOPA, CHEMS and CetylP.

DMPC

Dimyristoylphosphatidylcholine

DPPC

Dipalmitoylphosphatidylcholine

DSPC

Distearoylphosphatidylcholine

POPC

Palmitoyl-oleoylphosphatidylcholine

DOPC

Dioleoylphosphatidylcholine

DOPE

Dioleoylphosphatidylethanolamine

DMPE

Dimyristoylphosphatidylethanolamine

DPPE

Dipalmitoylphosphatidylethanolamine

DOPG

Dioleoylphosphatidylglycerol

POPG

Palmitoyl-oleoylphosphatidylglycerol

DMPG

Dimyristoylphosphatidylglycerol

DPPG

Dipalmitoylphosphatidylglycerol

DMPS

Dimyristoylphosphatidylserine

DPPS

Dipalmitoylphosphatidylserine

DOPS

Dioleoylphosphatidylserine

POPS

Palmitoyl-oleoylphosphatidylserine

DMPA

Dimyristoylphosphatidic acid

DPPA

Dipalmitoylphosphatidic acid

DOPA

Dioleoylphosphatidic acid

POPA

Palmitoyl-oleoylphosphatidic acid

CHEMS

Cholesterolhemisuccinate

DC-Chol

3-β-[N-(N',N'-dimethylethane) carbamoyl]cholesterol

Cet-P

Cetylphosphate

DODAP

(1,2)-dioleoyloxypropyl)-N,N-dimethylammonium chloride

DOEPC

1,2-dioleoyl-sn-glycero-3-ethylphosphocholine

DAC-Chol

3-β-[N-(N,N'-dimethylethane)carbamoyl]cholesterol

TC-Chol

3-β-(N-(N',N'',N''-trimethylaminoethane)carbamoyl]cholesterol

DOTMA

(1,2-dioleyloxypropyl)-N,N,N-trimethylammoniumchloride) (Lipofectin^®)

DOGS

((C18)2GlySper3+)N,N-dioctadecylamido-glycyl-spermine (Transfectam^®)

CTAB

Cetyl-trimethylammoniumbromide

CPyC

Cetyl-pyridiniumchloride

DOTAP

(1,2-dioleoyloxypropyl)-N,N,N-trimethylammonium salt

DMTAP

(1,2-dimyristoyloxypropyl)-N,N,N-trimethylammonium salt

DPTAP

(1,2-dipalmitoyloxypropyl)-N,N,N-trimethylammonium salt

DOTMA

(1,2-dioleyloxypropyl)-N,N,N-trimethylammonium chloride)

DORIE

(1,2-dioleyloxypropyl)-3 dimethylhydroxyethyl ammoniumbromide)

DDAB

Dimethyldioctadecylammonium bromide

DPIM

4-(2,3-bis-palmitoyloxy-propyl)-1-methyl-1H-imidazole

CHIM

Histaminyl-Cholesterolcarbamate

MoChol

4-(2-Aminoethyl)-Morpholino-Cholesterolhemisuccinate

HisChol

Histaminyl-Cholesterolhemisuccinate

HCChol

Nα-Histidinyl-Cholesterolcarbamate

HistChol

Nα-Histidinyl-Cholesterol-hemisuccinate

AC

Acylcarnosine, Stearyl- & Palmitoylcarnosine

HistDG

1,2-Dipalmitoylglycerol-hemisuccinat-N--Histidinylhemisuccinate, & Distearoyl-, Dimyristoyl, Dioleoyl or palmitoyl-oleoylderivatives

IsoHistSuccDG

1,2-ipalmitoylglycerol-O_-Histidinyl-Na-hemisuccinat, & Distearoyl-, Dimyristoyl, Dioleoyl or palmitoyloleoylderivatives

DGSucc

1,2-Dipalmitoyglycerol-3-hemisuccinate & Distearoyl-, dimyristoyl- Dioleoyl or palmitoyl-oleoylderivatives

EDTA-Chol

cholesterol ester of ethylenediaminetetraacetic acid

Hist-PS

Nα-histidinyl-phosphatidylserine

BGSC

bisguanidinium-spermidine-cholesterol

BGTC

bisguanidinium-tren-cholesterol

DOSPER

(1.3-dioleoyloxy-2-(6-carboxy-spermyl)-propylarnide

DOSC

(1,2-dioleoyl-3-succinyl-sn-glyceryl choline ester)

DOGSDO

(1,2-dioleoyl-sn-glycero-3-succinyl-2-hydroxyethyl disulfide ornithine)

DOGSucc

1,2-Dioleoylglycerol-3-hemisucinate

POGSucc

1,2-Palimtoleoylglycerol-3-hemisuccinate

DMGSucc

1,2-Dimyristoylglycerol-3-hemisuccinate

DPGSucc

1,2-Dipalmitoylglycerol-3-hemisuccinate

The following list shows the abbreviations of the lipids used and their names. In many cases, the abbreviations and names commonly used in the literature are used.

DMPC

Dimyristoylphosphatidylcholine

DPPC

Dipalmitoylphosphatidylcholine

DSPC

Distearoylphosphatidylcholine

POPC

Palmitoyl oleoylphosphatidylcholine

DOPC

Dioleoylphosphatidylcholine

DOPE

Dioleoylphosphatidylethanolamine

DMPE

Dimyristoylphosphatidylethanolamine

DPPE

Dipalmitoylphosphatidylethanolamine

DOPG

dioleoylphosphatidylglycerol

POPG

Palmitoyl-oleoylphosphatidylglycerol

DMPG

dimyristoyl

DPPG

dipalmitoylphosphatidylglycerol

DMPS

Dimyristoylphosphatidylserine

DPPS

Dipalmitoylphosphatidylserine

DOPS

Dioleoylphosphatidylserine

POPS

Palmitoyl-oleoylphosphatidylserine

DMPA

Dimyristoylphosphatidic acid

DPPA

Dipalmitoylphosphatidic acid

DOPA

Dioleoylphosphatidic acid

POPA

Palmitoyl oleoyl phosphatidic acid

CHEMS

Cholesterolhemisuccinate

DC-Chol

3-β- [N- (N ', N'-dimethylethanes) carbamoyl] cholesterol

Cet-P

Cetylphosphate

DODAP

(1,2) -dioleoyloxypropyl) -N, N-dimethylammonium chloride

DOEPC

1,2-dioleoyl-sn-glycero-3-ethylphosphocholine

DAC-Chol

3-β- [N- (N, N'-dimethylethane) carbamoyl] cholesterol

TC-Chol

3-β- (N- (N ', N'',N''- trimethylaminoethane) carbamoyl] cholesterol

DOTMA

(1,2-dioleyloxypropyl) -N, N, N-trimethylammoniumchloride) (Lipofectin ^®)

DOGS

((C18) 2GlySper3 +) N, N-dioctadecylamido-glycyl-spermine (Transfectam ^®)

CTAB

Cetyl trimethylammoniumbromide

CPyC

Cetyl pyridinium chlorides

DOTAP

(1,2-dioleoyloxypropyl) -N, N, N-trimethylammonium salt

DMTAP

(1,2-dimyristoyloxypropyl) -N, N, N-trimethylammonium salt

DPTAP

(1,2-dipalmitoyloxypropyl) -N, N, N-trimethylammonium salt

DOTMA

(1,2-dioleyloxypropyl) -N, N, N-trimethylammonium chloride)

Dorie

(1,2-dioleyloxypropyl) -3-dimethylhydroxyethyl ammonium bromide)

DDAB

Dimethyldioctadecylammonium bromide

DPIM

4- (2,3-bis-palmitoyloxy-propyl) -1-methyl-1H-imidazole

CHIM

Histaminyl-Cholesterolcarbamate

MoChol

4- (2-aminoethyl) -morpholino-Cholesterolhemisuccinate

HisChol

Histaminyl-Cholesterolhemisuccinate

HCChol

Nα-histidinyl Cholesterolcarbamate

HistChol

Nα-histidinyl-cholesterol hemisuccinate

AC

Acylcarnosines, stearyl & palmitoylcarnosines

HistDG

1,2-dipalmitoylglycerol hemisuccinate-N - histidinylhemisuccinate, & distearoyl, dimyristoyl, dioleoyl or palmitoyl-oleoylderivatives

IsoHistSuccDG

1,2-ipalmitoylglycerol O_-histidinyl-Na-hemisuccinate, & distearoyl, dimyristoyl, dioleoyl or palmitoyloleoylderivatives

DGSucc

1,2-dipalmitoyglycerol-3-hemisuccinate & distearoyl, dimyristoyl-dioleoyl or palmitoyl-oleoylderivatives

EDTA-Chol

cholesterol ester of ethylenediaminetetraacetic acid

Hist-PS

Nα-histidinyl phosphatidylserine

BGSC

bisguanidinium spermidine-cholesterol

BGTC

bisguanidinium tren-cholesterol

DOSPER

(1.3-dioleoyloxy-2- (6-carboxy-spermyl) -propylarnide

DOSC

(1,2-dioleoyl-3-succinyl-sn-glyceryl choline ester)

DOGSDO

(1,2-dioleoyl-sn-glycero-3-succinyl-2-hydroxyethyl disulfide ornithine)

DOGSucc

1,2-dioleoylglycerol 3-hemisucinate

POGSucc

1,2-Palimtoleoylglycerol-3-hemisuccinate

DMGSucc

1,2-Dimyristoylglycerol-3-hemisuccinate

DPGSucc

1,2-dipalmitoylglycerol-3-hemisuccinate

The following table shows, by way of example and not limitation, lipids useful to prepare amphoteric liposomes of the present invention. The membrane anchors of the lipids are shown by way of example only and not the lipids are not limited to these membrane anchors.

nucleic acids

The The present invention describes a process with which nucleic acid-loaded amphoteric liposomes can be produced.

Nucleic acids can be classified be in nucleic acids, which one or more specific sequences for proteins, polypeptides or RNAs encode and express in oligonucleotides that are very specific inhibit a protein.

In one embodiment of the invention, the nucleic acids encode one or more sequences for proteins, polypeptides or RNAs and are eventually translated into the corresponding proteins, polypeptides or RNAs. These nucleic acids include circular DNA plasmids, linear DNA constructs, such as MIDGE (Minimalistic Immunogenically Defined Gene Expression) vectors, disclosed in WO9821322A1 and DE19753182A1 and mRNAs for direct translation (eg. EP1392341B1 ).

In another embodiment The invention uses oligonucleotides whose target is nucleic acids, the for encode specific proteins so that the expression of the proteins is modulated. In this invention, the term "target nucleic acid" includes DNA specific for specific Proteins encoded, as well as all RNAs derived from such DNA which are pre-mRNA or mRNA. Specific hybridization between the target nucleic acid and one or more Oligonucleotides directed against such sequences lead to Inhibition of protein expression. To such a specific targeting to achieve that Oligonucleotide have a continuous strand of nucleotides, which are complementary to the sequence of the target nucleic acid.

oligonucleotides those mentioned above Fulfill criteria, can be treated with a number of different chemical properties and build topologies. The oligonucleotides may be single-stranded or double-stranded. To the single-stranded Oligonucleotides belong - without however limited to this to be - DNA based Oligonucleotides, closed nucleic acids, 2'-modified oligonucleotides and others, commonly known as antisense oligonucleotides. To the scaffolding or Base modifications count - without, however limited to this to be - phosphothioate DNA (PTO), 2'-O-methyl RNA (2'Ome), 2'-O-methoxyethyl RNA (2'MOE), peptide nucleic acids (PNA), N3'-P5'-phosphoamidate (NP), 2'-fluoroarabinonucleic acids (FANA), closed nucleic acids (LNA), morpholine phosphoamidate (morpholino), cyclohexene nucleic acids (CeNA), Tricyclo DNA (tcDNA) and others. Moreover, are also mixed chemical entities known in the art, these being more as a single nucleotide species as copolymers, block copolymers or Gapmere or in other arrangements are constructed.

In addition to the above-mentioned oligonucleotides, protein expression can also be inhibited with double-stranded RNA molecules containing the complementary sequence motifs. Such RNA molecules are known in the art as siRNA molecules (eg, WO9932619A1 or WO02055693A2). Again, various chemical properties of this class of oligonucleotides have been adapted. DNA / RNA hybrid systems are also known in the art.

Decoy Oligonucleotides represent another class of oligonucleotides This double-stranded DNA molecules do not target nucleic acids but on transcription factors. That means that Decoy oligonucleotides sequence-specific to the transcription factors (DNA-binding proteins) bind and thereby inhibit transcription (eg, Cho-Chung et al. in Curr Opin Mol Ther., 1999).

The specific oligonucleotides or polynucleotides used for the feasibility of the inventive Method not critical, as well as have special modifications in the nucleobases, the nucleoside sugars or the nucleic acid backbone be or would be the implementation of the inventive Procedure over the measure of one expert Make optimization even more difficult. The nucleic acids need only be an anionic Have total charge.

All described oligonucleotides can in length vary between only 10, preferably 15, and more preferably 18 and 50, preferably 30 and more preferably 25 nucleotides. Preferably, oligonucleotide and target sequence match perfectly, wherein each base of the oligonucleotide has an uninterrupted Strand from the aforementioned Number of nucleotides one base pair with its complementary base forms on the target nucleic acid. It is less preferred if this sequence pair is a mismatch contained within the continuous strand of base pairs.

method

• a) Bereitstellung einer Lösung einer Lipidmischung in einem mit Wasser mischbaren Lösungsmittel, bevorzugt einem Alkohol, das optional angesäuert sein kann
• b) Bereitstellung einer wässrigen Lösung des einzuschließenden Nukleinsäure-Wirkstoffes, die optional angesäuert sein kann wobei mindestens eine der beiden Lösungen in a) und b) durch Säuren und/oder Puffersubstanzen auf einen sauren pH-Wert eingestellt werden muss.
• c) Vermischen definierter Mengen der beiden Lösungen durch Einspritzen der alkoholischen Lösung der Lipidmischung in die wässrige Lösung der Nukleinsäuren oder umgekehrt oder durch Zusammenführung von zwei dosierten Teilströmen der bereitgestellten Lipidlösung und Nukleinsäure-Lösungen, optional in einem oder mehreren Mischern.
• d) Verdünnungsschritt, welcher optional ist
• e) Auflösung der Wechselwirkung zwischen den amphoteren Liposomen und den Nukleinsäuren durch durch eine Erhöhung des pH-Wertes auf >7 oder durch eine Erhöhung der Ionenstärke und anschließender Erhöhung des pH-Wertes auf >7
• f) Abtrennung von nicht eingeschlossenem Wirkstoff und/oder Aufkonzentrierung der Liposomensuspension und/oder Austausch des wässrigen Mediums und/oder Abtrennung des mit Wasser mischbaren Lösungsmittels, wobei jeder dieser Schritte unabhängig voneinander optional ist
• g) Sterilfiltration der wirkstoffhaltigen Liposomen, welche optional ist wobei zwischen den Schritten c) und d) und/oder zwischen d) und e) und/oder zwischen e) und f) und/oder zwischen f) und g) ein Extrusionsschritt erfolgen kann und/oder zwischen den Schritten e) und f) und/oder zwischen f) und g) ein oder mehrere Frier/Tau Zyklen erfolgen können und/oder nach Schritt g) ein oder mehrere Frier/Tau Zyklen und/oder eine Lyophilisation der wirkstoffhaltigen Liposomen erfolgen kann

The method according to the invention comprises the following steps:

• a) providing a solution of a lipid mixture in a water-miscible solvent, preferably an alcohol which may optionally be acidified
• b) providing an aqueous solution of the nucleic acid active substance to be enclosed, which may optionally be acidified, wherein at least one of the two solutions in a) and b) has to be adjusted to an acidic pH by acids and / or buffer substances.
• c) mixing defined amounts of the two solutions by injecting the alcoholic solution of the lipid mixture into the aqueous solution of the nucleic acids or vice versa or by combining two metered partial streams of the provided lipid solution and nucleic acid solutions, optionally in one or more mixers.
• d) dilution step, which is optional
• e) dissolution of the interaction between the amphoteric liposomes and the nucleic acids by increasing the pH to> 7 or by increasing the ionic strength and then increasing the pH to> 7
• f) Separation of non-entrapped drug and / or concentration of the liposome suspension and / or exchange of the aqueous medium and / or separation of the water-miscible solvent, each of these steps being independently optional
• g) sterile filtration of the active substance-containing liposomes, which is optional, wherein an extrusion step can take place between steps c) and d) and / or between d) and e) and / or between e) and f) and / or between f) and g) and / or between steps e) and f) and / or between f) and g) one or more freeze / thaw cycles can take place and / or after step g) one or more freeze / thaw cycles and / or lyophilization of the active ingredient-containing Liposomes can be done

The individual steps are described in detail below.

In a preferred embodiment the invention is the water-miscible solvent for the preparation the lipid solution an alcohol. The alcohols are preferably ethanol, isopropanol, 1,2-propanediol, n-propanol, butanol, pentanol, and ethylene glycol, propylene glycol, Methanol.

In a further preferred embodiment In the invention, the alcohol is ethanol, propanol or iso-propanol. Of course can It is also possible to use mixtures of the abovementioned alcohols, for example for optimal solubility all lipid components of an amphoteric system.

In another embodiment The invention may include the solvent also diluted with water as long as the particular lipid mixture in these mixtures still dissolves.

The solubility the lipids are hanging among other things from the temperature. Generally, the solubility of the Lipids higher, The higher the temperature is. In the design of the process parameters only such Concentrations and process temperatures are used, which is a full solution ensure the lipid mixture.

Of the is below the above selected temperature range between 4 ° C and 100 ° C, preferably between 10 ° C and 70 ° C, more preferably between 20 and 50 ° C.

The alcoholic lipid solutions are in the concentration range between 1 mM and 500 mM, preferably between 5 mM and 250 mM, and more preferably between 10 mM and 150 used in mM.

In an execution The invention is the lipid solution acidified. For acidification the lipid solution can either buffer systems known to those skilled in the art, e.g. Acetate buffer or citrate buffer be used. About that In addition, the pH can be adjusted with an acid (e.g., HCl, acetic acid or Citric acid) be set. Preferred are pharmaceutically acceptable buffers like acetic acid, citric acid or glycine.

In a further embodiment For example, the lipids used are in their undissociated form, i. Not used as salts. This concerns in particular the anionic and cationic lipids used to produce the amphoteric liposomes Find use. It is therefore advantageous not to use CHEMS as the sodium salt, but as free acid use. It is equally advantageous not to use MoChol as the hydrochloride, but to use as a free base. The use of salt-free lipids facilitate in later Process steps a unique addition of certain counterions and thus optimal stabilization of the obtained liposomes.

The aqueous Nucleic acid solution has in one execution the invention has a pH <6, preferably has a pH of between 3 and 5.5, and most preferably a pH between 3.5 and 4.5. For adjusting the pH can either buffer systems known to those skilled in the art, e.g. acetate or citrate buffer. In addition, the pH can be with an acid (e.g., HCl, acetic acid or citric acid) be set. Preferred are pharmaceutically acceptable buffers like acetic acid, citric acid or Glycine. The concentration of the aqueous nucleic acid solution is dependent on the amount of cationic lipids used in the process. In a preferred embodiment The invention relates to the molar ratio of the cationic charges the lipids to the anionic charges of the nucleic acid (N / P ratio for individual or double-stranded Oligonucleotides between 1 and 10, more preferably between 1.5 and 5 and most preferably between 2 and 4. For larger individual or double-stranded nucleic acid molecules such as plasmids, Aptamers or RNA molecules with a chain length of 50 or more nucleobases or base pairs, the N / P ratio is preferred between 1 and 50, more preferably between 2 and 30 and completely more preferably between 3 and 20.

In a particular embodiment of the process becomes the aqueous Nucleic acid solution first just before mixing with the lipid phase by mixing two aqueous Made phases. This is particularly advantageous if the Nucleic acid solution as Concentrate is present and suitable before mixing with the lipid solution dilute must become. This embodiment is particularly advantageous if the dissolved active ingredient sensitive to the Binding conditions is and can not be stored under these long. For example, a longer Irreversibly damage DNA exposure below pH 4; it comes to the replacement of Purine bases from sugar / phosphate backbone.

Excessive exposure of the nucleic acids in an acidic environment of course easiest by resolution reach the active ingredients in a neutral aqueous buffer, the Add the necessary acid for adjusting the process pH is in this case about the Lipid reaches, i. the acid becomes the ethanolic lipid solution added.

The mixing of the two solutions provided is carried out on a laboratory scale, preferably by injecting the alcoholic lipid solution into a stirred aqueous nucleic acid solution or vice versa. For the production of nucleic acid-loaded amphoteric liposomes on an industrial scale, the mixing of the two solutions can be carried out in one device. A suitable apparatus for mixing the solutions is described in more detail below. In addition, the mixing in the in US6843942 and US20040032037A1 disclosed apparatus.

In an execution of the invention the amount of the alcoholic lipid solution which is mixed into the aqueous nucleic acid solution is, preferably between 2 and 25% and more preferably between 5 and 20% of the resulting total volume.

During and after mixing of the alcoholic solution of the lipid mixture and the aqueous nucleic acid solution, the forming or formed amphoteric liposomes are in a cationic charge state. For this reason, the anionically charged nucleic acids interact with the cationic lipid layer, which allows aggregates to form during the process. Surprisingly, it was found that aggregate formation during liposome preparation, unlike in US 6287591 has not been prevented by steric hindrance (eg by the use of PEG-lipids). Rather, the interactions after the formation of the amphoteric liposomes can be redissolved in a further step, whereby nucleic acid-loaded amphoteric liposomes with homogeneous size distribution arise.

In a special design the invention concludes to the mixing of the two solutions a dilution step at. In this case is the amount of the alcoholic lipid solution which is mixed into the aqueous nucleic acid solution is, preferably between 20 and 50% and more preferably between 25 and 45% of the resulting total volume.

It so it can be beneficial during the process higher Use ethanol quantities. It was found that amphoteric liposomes from a critical ethanol concentration of about 20-35% are permeable. For that reason, concludes the dilution process of the two solutions to a dilution step, to arrive at stable amphoteric liposomes, the ethanol concentration then to a value of less than 25%, preferably less than 15% is lowered.

In an execution The invention may include the dilution step with an aqueous solution which corresponds in the composition of the nucleic acid solution, however without nucleic acid and about the pH of the mixture corresponds. Alternatively, too other solutions known to those skilled in the art with appropriate acidic pH for dilution.

In another advantageous embodiment of the invention for dilution aqueous Solutions used which change the pH of the present mixture so that the interaction between the amphoteric liposomes and the nucleic acids. The choice of pH is explained in more detail below. Generally, the expert known, which pH by mixing two solutions defined composition, and how a solution can be changed needs to get to a desired pH to get to the mixture.

The dilution is done like this that the amount of ethanol in the mixture is preferably <20%, especially preferably between 2 and 15% and most preferably between 5 and 12% reduced.

Around the interaction between the amphoteric liposomes and the nucleic acids The formation of liposomes can be resolved by different methods be used.

In an execution the invention, the interactions by a change the pH dissolved. As already explained For example, amphoteric liposomes are anionic at pH 7.5 neutrally charged. If the pH is therefore changed so that the amphoteric liposomes become anionic or neutral, this leads simultaneously to a resolution the interactions between the liposomes and the nucleic acids. Surprisingly it was found that the change in pH suffices for larger aggregate assemblies Liposomes and nucleic acids, which are during of the manufacturing process, so dissolve that in the solution after all Nucleic acid-loaded amphoteric liposomes in addition to free, not included nucleic acid molecules.

Surprisingly it was found that the dilution the ethanol concentration and the neutralization of the solution simultaneously, i.e. by adding a single neutralizing solution in corresponding amount performed can be, without it to a substantial loss of trapped Active ingredient comes or where yields reached a comparable level become.

In another embodiment of the invention, the interactions may also be resolved by increasing the ionic strength in the solution. Preferably, sodium chloride is used for this, but is not limited thereto. It was also found with this method that aggregate assemblies formed during the process dissolve again, although the pH remains constant under these conditions. The level of ionic strength needed to inhibit the interactions between the amphoteric liposomes and the nucleic acids depends on the lipid mixtures and nucleic acids used. For example, tie larger nucleic acids, such as DNA plasmids, more tightly to the liposomal membrane, as the greater number of repeating charges results in greater binding. To resolve the interactions between amphoteric liposomes and oligonucleotides, the final NaCl concentration is preferably between 250 and 1500 mM.

Of course you can too Salts other than NaCl to increase the ionic strength can be used as e.g. Sodium or potassium citrate, sodium or potassium phosphate, Ammonium sulfate or other salts. Particularly preferred are pharmaceutically acceptable salts. An increase the ionic strength is also greater by addition Amounts of buffer substance reached.

In another step of the process may not be included Active substance to be separated. These are known to those skilled in the art Methods such as gel filtration, centrifugation, dialysis or ultrafiltration. About that can out the nucleic acid loaded amphoteric liposomes to be concentrated as needed. Also suitable for this methods known to those skilled in the art, e.g. Centrifugation or Ultrafiltration. Further, through these methods, an exchange of the aqueous Medium or a separation of the water-miscible solvent respectively.

Further can those according to the inventive method prepared active ingredient-containing liposomes in a final step be sterilized by filtration.

In an execution The invention can be used between process steps c) and d) and / or between d) and e) and / or between e) and f) and / or between f) and g) an extrusion step, whereby the size distribution the active substance-containing liposomes can be further improved.

In another embodiment The invention is the extrusion at acidic pH, ie between the process steps c) and d) or d) and e), wherein the forming or formed amphoteric liposomes in a cationic charge state and with the anionically charged nucleic acids interact. For this reason, no or only a small Release of the already enclosed nucleic acid active substances.

Surprisingly was found to also have an extrusion after dissolution of the Interactions between the formed amphoteric liposomes and the nucleic acids ie between method steps d) and e) and / or between e) and f) and / or between f) and g) are not appreciable Release of the entrapped nucleic acids leads. In a further embodiment of the Invention are the drug-containing amphoteric liposomes between the method steps d) and e) and / or between e) and f) and / or extruded between f) and g), preferably not more than 30 % of the entrapped drug, most preferably not more as 20% of the entrapped active ingredient, and most preferably not more than 10% of the trapped drug released become. Next became surprising found that the use of extrusion membranes whose pore size is greater as the mean size of the active ingredient amphoteric liposomes at a pH> 7 to a lower release of the enclosed nucleic acid agents leads.

In another embodiment of the invention the active ingredient-containing amphoteric liposomes between the process steps e) and f) and / or between f) and g) one or more freeze / tow Cycles, surprisingly It has been found that this process leads to a narrower size distribution the liposomes leads, without releasing appreciable amounts of entrapped drug become. The freeze / thaw cycles are preferably carried out in the presence of Cryoprotektiva, preferably using sugar. pharmaceutical acceptable sugars are known to those skilled in the art and include e.g. sucrose, Maltose or trehalose, without being limited thereto. In one execution the cryoprotectants form the active-substance-containing amphoteric liposomes added before the freeze / thaw process. In a preferred embodiment the active substance-containing amphoteric liposomes are prepared in the presence of the cryoprotectants, so that the cryoprotectants after the production of liposomes inside and outside the liposomes is located. The amount of cryoprotectants is preferred between 1 and 25%, more preferably between 5 and 15%. Freezing The liposomes can be in liquid Nitrogen, on dry ice, at -70 ° C or at -20 ° C, without any of these Limited methods to be.

Surprisingly, it has also been found that certain salts can increase the stability of the amphoteric liposomes. Among other things, the cations of these salts contribute to such stabilization. Thus, the release of trapped cargo during a freeze-thaw cycle can be improved by using tris (hydroxymethyl) aminomethane. Other suitable cations include triet hanolamine, ammonia, morpholine, piperazine. When using these cations, a lower release of active ingredient was observed than is possible, for example, with sodium or potassium as a cation.

In an embodiment These cations are used as the sole cations in the buffer system, a Mixture of, for example, sodium ions with tris (hydroxymethyl) aminomethanions therefore less preferred.

In another aspect of the use of these preferred cations It was also observed that the size of the liposomes produced is subject to less fluctuations after a freeze-thaw cycle, as if the preferred cations are not used.

In another aspect of the use of preferred cations an improved colloid stability observed. Liposomes made using the preferred cations prepared can be without changes their colloidal properties such as size or viscosity more concentrate than if these cations are not used. By Using the preferred cations, it is possible, for example, the Concentrations of the liposomes in suspension to more than 100 mM to increase, It is often possible To increase this concentration to more than 130mM and partially succeeds Concentration to 150mM or more lipid.

Surprisingly It was found that the positive effects of the preferred Cations and the above-mentioned cryoprotectants act synergistically. This can further improve process control or Storage of the produced liposomes can be achieved. Such improvements include, for example, a more accurate preservation of the size of the Particles, leaving their mean size before and after a freeze-thaw cycle differs by less than 20%. Often it is through synergistic Use of cryoprotectants and cations possible, a size difference of less than 10% of the mean size and in many make is the difference of the particle sizes below one sure demonstrable difference.

As well is a low release of entrapped drug or Cargo beneficial and such a release can be described by the activities to reduce values to less than 10%, in many cases a release of less than 5% possible and often a release reached less than 2% of the entrapped drug. The preferred cations of the buffer system and the cryoprotectants are freely combinable. This can be, for example Sucrose and tris (hydroxymethyl) aminomethane as a cation with each other combine, but also maltose or trehalose.

In a preferred embodiment This aspect of the invention can be achieved by the addition of the preferred Cations reduce the amount of cryoprotectants used, without the release of active ingredient or the size of the Particles are significantly influenced.

In another embodiment The invention relates to the active ingredient-containing amphoteric liposomes following the process step e) or f) or g) as well one or more cycles of freezing / thawing and / or lyophilisation subjected. As described above, it was surprising that the after the method according to the invention prepared active substance-containing liposomes preferably in the presence frozen by cryoprotectants and optionally the salts mentioned above can wherein after thawing usually a narrower size distribution of the liposomes observed and no appreciable amounts of trapped Drug to be released. For this reason, the after the produced according to the invention active substance-containing liposomes in a preferred embodiment of Invention after the final process step, which e), f) or g) can be frozen and stored in a frozen state. In a further execution of the invention those according to the inventive method prepared active substance-containing liposomes after the final process step, which can be e), f) or g) are lyophilized. Lyophilization is preferably also under the addition of the above-explained Cryoprotectants and salts. The liposomes may then be e.g. at room temperature be stored.

Also the process temperature is an important parameter. It is used for liposome formation always higher than the phase transition temperature selected the lipid mixture. The phase transition temperature can be determined by calorimetric methods, for example the DSC ("differential scanning calorimetry ") be determined. The lipid with the highest phase transition temperature as a pure substance can approximately be used to determine the process temperature. To one To ensure safety distance the process temperature should be at least 5 ° C above the determined or approximated phase transition temperature selected the lipid mixture become.

The Preparation of the nucleic acid-containing amphoteric liposomes can be done without further devices, such as by mixing in a stirred container or in the devices described above.

In a preferred embodiment can the inventive Process also as a continuous process, for example in the following Device, guided become.

1 shows the basic structure of the device. From temperature-controllable storage vessels G1 and G2 with the alcoholic lipid solution and the aqueous nucleic acid solution, the components to be mixed are transported to the mixing chamber M1 via hoses and / or pipe connections L1 and L2 made of chemically inert material by means of the pumps P1 and P2 (which preferably operate with as little pulsation as possible).

These Mixing chamber consists of a V-mixer, which has the advantage of a very possesses small mixing volume. In addition, there are other mixer designs conceivable, such as T-mixers, micromixers, dynamic or static / dynamic Mixer.

Of the Total flow after leaving the mixing chamber M1 is the sum the partial flows educated.

Of the Total current is over another hose and / or pipe connection L3 is directed into a feed vessel G3, which the buffer for the dilution or for the setting of the release conditions contains. The Hose or pipe connection L3 can serve as a timer, in the the formed liposomes for held under the binding conditions for a certain time can, until the binding of the active substance to the lipid membrane reaches its maximum value has reached. The residence time is determined by the tube volume and the total flow rate.

The Timer L3 and the reservoir G3 can also be tempered.

The buffer for dilution and / or adjustment of the separation conditions can also be supplied via a further mixing chamber M2 with the pump P3 from the vessel G3 via a hose and / or pipe connection and the total flow collected after passage of the mixing chamber M2 in another vessel G4. For the mixing chamber M2, the same embodiments are possible, as in M1. This embodiment is in 2 shown.

Furthermore may be the partial flow of the aqueous drug solution before being fed into the system, it consists of two separate partial flows become.

This is particularly advantageous if the drug solution is present as a concentrate and must be suitably diluted before mixing with the lipid solution or if the drug solution must be rebuffered before feeding. This is particularly advantageous if the dissolved active ingredient is sensitive to the binding conditions and can not be stored under them for a long time. This embodiment makes reference to 3 the use of the vessel 1a , a pump P1a and a mixing chamber M1a with the corresponding pipe connections required.

The quality features the produced liposomes, such as size, size distribution and drug content can over a suitable process design of the process parameters reproducible to be controlled. The process parameters, which are varied can, include amount of ethanol, lipid concentration, nucleic acid concentration, N / P ratio, composition the aqueous Nucleic acid solution, temperature, flow rates and pressure.

A Another object of the invention is the provision of process parameters, with which the size of the amphoteric Controlled liposomes and the drug / lipid ratio in the final product can be.

It showed up surprisingly, that the entrapment efficiency in the process according to the invention, in contrast to the "passive" inclusion of active ingredients increased in liposomes, with lower lipid concentrations in the process. The However, lipid concentration in the process also affects the size of the liposomes, which increases with increasing Lipidkonznetration first and then one reached constant value. Preferred lipid working concentrations in the process according to the invention are between 0.2 and 10 mM lipid, more preferably between 0.5 and 5 mM lipid, and more preferably between 0.5 and 3 mM.

In addition, surprisingly, a temperature dependence of entrapment efficiency in the inventive method found. Thus, higher inclusion efficiencies are achieved at higher temperatures. The preferred temperature range for preparing nucleic acid-loaded amphoteric liposomes by the method according to the invention is between 20 and 50 ° C.

Further led the increase the ionic strength or osmolarity in the aqueous drug solution too surprising Results in the process according to the invention. Up to a certain ionic strength decreases the entrapment efficiency, while at the same time reducing the size of the formed Liposomes increases. If the ionic strength is further increased, it changes the size of the liposomes and the inclusion efficiency first no more. At very high ionic strengths, the interaction becomes finally complete between the amphoteric liposomes and the nucleic acids suppressed so that the inclusion about the "passive" procedure takes place. This is also reflected in the smaller size of the formed amphoteric Reflected liposomes. The ionic strength can be increased by the addition of sodium chloride, but is not limited to this. Of course are the skilled worker further suitable pharmaceutically acceptable salts known, with which the ionic strength a solution changed can be. These include e.g. Citrate, phosphate or acetate but not limited to this. Preferably, the inventive method with an ionic strength between 0 and 250 mM, preferably between 0 and 150 mM. In a particular embodiment the process becomes at ionic strengths between 0 and 50mM.

Becomes a sugar (e.g., sucrose) rather than a salt to increase osmolarity to the aqueous drug solution given, however, showed another effect. Surprisingly, the Size of through the inventive method produced nucleic acid loaded amphoteric liposomes almost constant, while the inclusion efficiency increased. The process according to the invention is preferred with a sugar concentration between 0 and 500 mM. pharmaceutical acceptable sugars are known to those skilled in the art and include e.g. sucrose, Glucose or trehalose, without being limited thereto.

With the variation of the parameters of the invention Is it possible, nucleic acid-loaded amphoteric liposomes more desired To produce specifications. For Pharmaceutical applications is the size of the nucleic acid loaded amphoteric liposomes preferably between 50 nm and 500 nm and completely preferably between 80 nm and 250 nm. Further, with the inventive method liposomes produced uni-, oligo- or multilamellar be. The in the final product preferred drug / lipid ratio is for oligonucleotide drugs preferably between 1 mg oligonucleotide / g lipid and 300 mg oligonucleotide / g Lipid. More preferably, the drug / lipid ratio is between 10 and 100 mg oligonucleotide per gram of lipid. For larger nucleic acid agents, like DNA plasmids, the preferred drug / lipid ratio is in between 0.3 mg DNA / g lipid and 30 mg DNA / g lipid.

The The invention will be explained in more detail in the following examples, without reference to these limited to be.

Description of the pictures

1 schematically shows an apparatus for continuously carrying out the method according to the invention

2 schematically shows an apparatus for continuously carrying out the method according to the invention with admixture of the buffer for diluting and / or adjusting the Ablösebedingungen.

3 schematically shows an apparatus for continuously carrying out the method according to the invention with additional treatment of the aqueous drug solution by rebuffering or dilution.

4 : Influence of the lipid concentration on the size of the liposomes formed from Example 1

5 : Influence of the lipid concentration on the entrapment efficiency of the formed liposomes of Example 1

6 : Influence of the temperature on the size of the liposomes formed from Example 1

7 : Influence of the temperature on the entrapment efficiency of the formed liposomes of Example 1

8th : Influence of the NaCl concentration on the size of the liposomes formed from Example 1

9 : Influence of the NaCl concentration on the entrapment efficiency of the liposomes of Example 1 formed

10 : Influence of increasing amounts of ethanol on the permeability of amphoteric liposomes

example 1

Preparation of antisense oligonucleotide loaded amphoteric liposomes variation of various process parameters

In this experiment, an 18-mer antisense oligonucleotide was included in amphoteric liposomes of the following composition:
POPC / DOPE / MoChol / Chems 15: 45: 20: 20

The following process parameters were varied one after the other:
lipid concentration
temperature
NaCl concentration in the aqueous antisense solution

The preparation of the antisense-loaded amphoteric liposomes was carried out by means of in 1 illustrated device.

To combine the two metered partial streams of the provided lipid solution and antisense solution, the following volume flows were selected: Volume flow of lipid: 10 ml / min Volume flow antisense: 90 ml / min

The produced batch size was 40 ml each.

Around the interactions between the amphoteric liposomes and the antisense molecules after the Formation of the liposomes was again 1/20 Voulmen 1M Tris solution, pH 8 with stirring added.

Variation of the lipid concentration

The following process parameters were kept constant during production:

The Concentration of the antisense solution was adapted to the appropriate lipid concentration to the N / P ratio to keep constant.

Variation of temperature

The following process parameters were kept constant during production:

The Temperature was varied as follows: 20 ° C, 30 ° C, 40 ° C, 50 ° C

Variation of the NaCl concentration in the aqueous Antisense solution

The following process parameters were kept constant during production:

The NaCl concentration of the aqueous Antisense solution was gradually increased in the range 0 mM - 400 mM. The buffer composition and the pH were not changed.

Determination of liposome size:

The Size of the liposomes was after dilution of the samples (max 1 ethanol) in PBS by photon correlation spectroscopy (PCS) determined.

Separation from not included Antisense oligonucleotide:

Not included antisense oligonucleotide was by sedimentation reached the liposomes.

Determination of entrapment efficiency the antisense oligonucleotides into the amphoteric liposomes:

The Determination of the included amount of antisense, carried out by OD measurement at 260 nm after the liposomes are dissolved in a mixed solvent (chloroform and methanol = 1 + 4) were.

Determination of the lipid concentration:

The Lipid became inorganic after extraction into chloroform Phosphate (P. Veldhofen and G. Mannaerts (1987) Anal. Biochem. 161: 45-48).

Results:

Variation of the lipid concentration

4 shows that with increasing lipid concentration, the size of the liposomes initially increases, but stabilizes from a final lipid concentration of 1.5 mM. In 5 the entrapment efficiency of antisense oligonucleotides is shown as a function of lipid concentration. With increasing lipid concentration the entrapment efficiency decreases.

variation the temperature

The size of the liposomes does not change with increasing temperature, as in 6 shown. On the other hand shows 7 that better entrapment efficiencies are achieved with increasing temperature.

Variation of the NaCl concentration in the aqueous Antisense solution

8th shows that the size of the liposomes initially increases slightly with increasing NaCl concentration in the aqueous antisense oligonucleotide solution, but then remains relatively constant over a wide range. Inclusion efficiency is best without NaCl addition, such as 9 demonstrated. Addition of NaCl initially reduces the entrapment efficiency by approximately 20%, but then remains relatively constant up to a NaCl concentration of approximately 125 mM. Only at even higher salt concentrations does the entrapment efficiency decrease significantly. At a salt concentration of 400 mM virtually no antisense oligonucleotide is included in the amphoteric liposomes.

Example 2:

Treatment of different amphoteric liposomes with increasing amounts of ethanol

Production of various amphoteric liposomes with entrapped fluorescent dye carboxyfluorescein

Out Lipid stock solutions In chloroform, various lipid mixtures were mixed and the solvent removed on a rotary vacuum evaporator. The resulting lipid films were over Dried overnight in vacuo. Then the lipid films were incubated with 100 mM Carboxyfluorescein (CF) in PBS, pH 7.5 hydrated. The result resulting lipid concentration was 20 mM. The suspensions were for Hydrated at RT for 20 min, for Homogenized for 5 minutes in an ultrasonic bath and finally 3 freeze / thaw cycles subjected. After the last thawing, the liposomal suspensions were Extruded 15 times through 100 nm polycarbonate membranes. not Trapped CF was removed by gel filtration giving the liposomes 3 fold dilute were.

The following formulations were prepared:

Influence rising Ethanol levels on integrity the liposomes

The Liposomes were first Diluted 32 times with PBS and 20 μl this dilution were in a black microtiter plate with 180 ul of a PBS / ethanol mixture mixed with increasing amounts of ethanol and incubated for 30 min. In connection was the CF released from the liposomes by fluorescence measurement measured at 490nm / 530nm. To a reference value for the complete release to get the approaches were with 20 μl 2.5% Triton X-100 solution and the fluorescence is measured again.

Result

In 10 is the release of the fluorescent dye carboxy fluorescein from the amphoteric liposomes shown with increasing externally added amounts of ethanol. From a critical ethanol concentration of about 30% ethanol, the liposomes become permeable and release the entrapped dye.

Example 3:

Production of various nucleic Loaded amphoteric liposomes by 10% or 30% ethanol injection

The lipid mixtures were weighed and dissolved in ethanol so that the lipid concentration was 40 mM or 13.3 mM. 0.5 ml and 1.5 ml of these ethanolic lipid solutions were injected in 4.5 ml and 3.5 ml of an aqueous nucleic acid solution (18 mer antisense oligonucleotide) in 90 mM acetate, 300 mM sucrose, pH 4 with stirring. The amount of antisense oligonucleotide was calculated to give an N / P ratio of 3 in the reaction. Following the preparation, the liposome suspension was diluted in each case with 10 ml of 120 mM Na ₂ HPO ₄ , 90 mM NaCl, pH 9, to give a pH of> 7. To the In order to determine confinement efficiency, unencapsulated antisense oligonucleotide was separated by Centriprep ultrafiltration units (100 kDa MWCO) and determined by OD measurement at 260 nm. The size of the liposomes was determined by diluting the samples (maximum 1% ethanol) in PBS by photon correlation spectroscopy (PCS).

The following formulations were prepared as described:

Result:

The following inclusion efficiencies and liposome sizes are given at the end of the preparation:

Example 4:

Preparation of nucleic acid-loaded amphoteric liposomes by means of the in 1 illustrated device

The lipid mixtures were weighed and dissolved in ethanol so that the lipid concentration was 16.6 mM. The 18 mer antisense oligonucleotide was dissolved in 20 mM NaAcetate, 300 mM sucrose pH 4.0. The amount of antisense oligonucleotide was calculated to give an N / P ratio of 3 and 4, respectively (see below). The two solutions were used with flow rates of 24 ml / min (lipid solution) and 56 ml / min (antisense solution) in a device according to 1 mixed. Following preparation, the liposome suspensions were diluted to 10% ethanol content (20 mM NaAcetate, 300 mM sucrose pH 4.0). Then the suspensions were rebuffered to pH 7.5 with 1/5 volume 1 M Tris, pH 8. Unenclosed oligonucleotides and the ethanol were separated by crossflow dialysis and the liposomes subsequently concentrated.

The Determination of the included amount of antisense, carried out by OD measurement at 260 nm after the liposomes are dissolved in a mixed solvent (chloroform and methanol = 1 + 4) were.

Determination of the lipid concentration:

The Lipid became inorganic after extraction into chloroform Phosphate determined (P. Veldhofen and G. Mannaerts (1987) Anal. Biochem. 161: 45-48).

The size of the liposomes was determined in PBS by photon correlation spectroscopy (PCS). Result:

Example 5:

Influence of different Parameters on the freeze-thaw capability

The Liposomes were as described in Example 4 with the following parameters prepared: 80mM ethanolic lipid solution consisting of 6 mol% POPC, 24 mole% DOPE, 23 mole% MoChol and 47 mole% DMGSucc, 25% Ethanol injection, N / P = 2. The dilution to 15% ethanol and the Buffering to pH 7.5 was performed in one step at a flow rate of 25 ml / min with a solution, consisting of 500mM sodium hydrogen phosphate, pH 9. The diluted suspension was concentrated by cross-flow filtration to 160 mM lipid, at the same time the unenclosed CD40 oligonucleotides and ethanol was separated.

The liposome preparation was aliquoted and according to the parameters in the following Table examined for her freeze-thaw behavior. Unless otherwise noted, the samples were frozen at -70 ° C and thawed at room temperature. The treated liposomes were tested for particle size and particle distribution (as in Example 4). The proportion of released drug was separated by ultrafiltration (Centrisart, 100kD, 2500g, 2 hours, 20 ° C) and determined by OD260nm against a reference solution with CD40.

Result:

The Liposome suspension can be so by adding sugar and / or tris be stabilized that size, distribution and drug content is not significantly different from the starting suspension are different.

Example 6:

Influence of Extrusion step on the quality of liposomes

• 1) Filtration durch ein 0,2μm Sterilfilter
• 2) Extrusion durch eine 400nm Polycarbonat-Membran
• 3) Extrusion durch eine 200nm Polycarbonat-Membran und anschließende Filtration durch ein 0,2μm Sterilfilter
• 4) Extrusion durch eine 100nm Polycarbonat-Membran
• 5) Extrusion durch eine 200nm Polycarbonat-Membran, Filtration durch ein 0,2μm Sterilfilter und anschließende Extrusion durch eine 100nm Polycarbonat-Membran

The liposomes were prepared as described in Example 4 with the following parameters: 33 mM ethanolic lipid solution consisting of 6 mol% POPO, 24 mol% DOPE, 47 mol% MoChol and 23 mol% Chems, 30% ethanol injection, N / P The dilution to 10% ethanol and the Umpufferung to pH 7.5 was carried out in one step with a flow rate of 160 ml / min with a solution consisting of 136mM sodium hydrogen phosphate and 100mM sodium chloride, pH 9. In each case 50ml of this preparation were as follows further processed:

• 1) Filtration through a 0.2μm sterile filter
• 2) extrusion through a 400nm polycarbonate membrane
• 3) Extrusion through a 200nm polycarbonate membrane followed by filtration through a 0.2μm sterile filter
• 4) Extrusion through a 100nm polycarbonate membrane
• 5) Extrusion through a 200nm polycarbonate membrane, filtration through a 0.2μm sterile filter and subsequent extrusion through a 100nm polycarbonate membrane

The processed liposomes were tested for particle size, particle distribution and lipid concentration (as in Example 4). The proportion of non-entrapped drug was separated by ultrafiltration (Centrisart, 100kD, 2500g, 2 hours, 20 ° C) and determined by OD260nm against a reference solution with CD40. Result:

through Extrusion can be the quality the liposomes are improved so that a subsequent sterile filtration without appreciable losses of active substance and particles can.

Claims (42)

Method for loading amphoteric liposomes with nucleic acid active substances, characterized in that it comprises the steps of a) providing a solution of a lipid mixture in a water-miscible solvent, preferably an alcohol which may optionally be acidified b) providing an aqueous solution of the nucleic acid to be entrapped Active ingredient, which may be optionally acidified wherein at least one of the two solutions in a) and b) must be adjusted to an acidic pH by acids and / or buffer substances. c) mixing defined amounts of the two solutions by injecting the alcoholic solution of the lipid mixture into the aqueous solution of the nucleic acids or vice versa or by combining two metered partial streams of the provided lipid solution and nucleic acid solutions, optionally in one or more mixers. d) dilution step, which is optional e) dissolution of the interaction between the amphoteric liposomes and the nucleic acids by increasing the pH to> 7 or by increasing the ionic strength and then increasing f) separation of the active substance not included and / or concentration of the liposome suspension and / or replacement of the aqueous medium and / or separation of the water-miscible solvent, each of these steps being independently optional g) sterile filtration of active substance-containing liposomes, which is optionally comprises wherein between the steps c) and d) and / or between d) and e) and / or between e) and f) and / or between f) and g) an extrusion step can take place and / or between steps e) and f) and / or between f) and g) one or more freeze / thaw cycles can take place and / or after step e), f) or g) one or more freeze / thaw cycles and / or one Lyophilization of the active substance-containing liposomes can take place, Method for loading amphoteric liposomes with Nucleic acid agents according to Claim 1, characterized in that the alcohol is selected from the group of ethanol, isopropanol, 1,2-propanediol, n-propanol, butanol, Pentanol, and ethylene glycol, propylene glycol, methanol. Method for loading amphoteric liposomes with Nucleic acid agents according to Claim 2, characterized in that the alcohol is ethanol, propanol or iso-propanol. Method for loading amphoteric liposomes with Nucleic acid agents according to Claim 1, characterized in that the lipid solutions in Concentration range between 1 mM and 500 mM, preferably between 5 mM and 250 mM and most preferably between 10 mM and 150 mM become. Method for loading amphoteric liposomes with Nucleic acid agents according to Claim 1, characterized in that the pH of the aqueous Nucleic acid solution <6 is preferred between 3 and 5.5, and most preferably between 3.5 and 4,5 is. Method for loading amphoteric liposomes with Nucleic acid agents according to Claim 1, characterized in that the active ingredient is single or double Oligonucleotides comprises and the molar ratio of the cationic charges the lipids to the anionic charges of the nucleic acid (N / P ratio) between 1 and 10, more preferably between 1.5 and 5, and most especially preferably between 2 and 4. Method for loading amphoteric liposomes with Nucleic acid agents according to Claim 1, characterized in that the active substance plasmids, Aptamers or RNA with a chain length of more than 50 bases or Base pairs and the molar ratio of the cationic charges the lipids to the anionic charges of the nucleic acid (N / P ratio) between 1 and 50, more preferably between 2 and 30, and most especially preferably between 3 and 20 lies. Method for loading amphoteric liposomes with Nucleic acid agents according to Claim 1, characterized in that the amount of alcoholic Lipid solution which in the watery Nucleic acid solution mixed is, preferably between 2 and 25% and more preferably between 5% and 20% of the resulting total volume. Method for loading amphoteric liposomes with Nucleic acid agents according to Claim 1, characterized in that the amount of alcoholic Lipid solution which in the watery Nucleic acid solution mixed is, preferably between 20 and 50% and more preferably between 25 and 45% of the resulting total volume is and to the mixing of the two solutions, a dilution step follows the alcohol concentration to less than 25%, preferably less than 15% is lowered. Method for loading amphoteric liposomes with nucleic acid agents according to Claim 9, characterized in that the dilution step with an aqueous solution which corresponds in composition to the nucleic acid solution, but without nucleic acid and about the pH of the mixture corresponds. Method for loading amphoteric liposomes with nucleic acid agents according to Claim 9, characterized in that other known to the expert solutions with appropriate acidic pH for dilution. Method for loading amphoteric liposomes with nucleic acid agents according to Claim 9, characterized in that used for diluting aqueous solutions which neutralize the pH of the present mixture. Method for loading amphoteric liposomes with nucleic acid agents according to Claim 1, characterized in that the interactions between the amphoteric liposomes and the nucleic acids by a change the pH dissolved become Method for loading amphoteric liposomes with nucleic acid agents according to Claim 1, characterized in that the interactions between the amphoteric liposomes and the nucleic acids by increasing the ionic strength in the solution disbanded become. Method for loading amphoteric liposomes with nucleic acid agents according to claim 14, characterized in that the ionic strength by the addition of sodium chloride is increased and the concentration of sodium chloride is between 250 and 1000mM. Method for loading amphoteric liposomes with nucleic acid agents according to Claim 1, characterized in that in a further step the process not entrapped drug are separated can and / or the liposome suspension can be concentrated on and / or the aqueous Medium can be replaced and / or miscible with water solvent can be separated, and the method known in the art such as gel filtration, centrifugation, dialysis or ultrafiltration can be used. Method for loading amphoteric liposomes with nucleic acid agents according to Claim 1, characterized in that between the method steps c) and d) and / or between d) and e) and / or between e) and f) and / or between f) and g) an extrusion step can take place. Method for loading amphoteric liposomes with nucleic acid agents according to Claim 17, characterized in that the extrusion in acid pH value, ie between process steps c) and d) or d) and e) becomes. Method for loading amphoteric liposomes with nucleic acid agents according to Claim 17, characterized in that the extrusion after dissolution of the Interactions between the formed amphoteric liposomes and the nucleic acids ie between method steps d) and e) and / or between e) and f) and / or between f) and g). Method for loading amphoteric liposomes with nucleic acid agents according to Claim 19, characterized in that extrusion membranes whose Pore size is larger as the mean size of the active ingredient amphoteric liposomes are used. Method for loading amphoteric liposomes with nucleic acid agents according to Claim 1, characterized in that the active substance-containing amphoteric Liposomes between steps e) and f) and / or between f) and g) are subjected to one or more cycles of freezing / thawing. Method for loading amphoteric liposomes with nucleic acid agents according to Claim 21, characterized in that the freeze / thaw cycles in Presence of cryoprotectants are performed. Method for loading amphoteric liposomes with nucleic acid agents according to Claim 21, characterized in that preferred as cryoprotectants Sugar, with sucrose, maltose or trehalose being preferred are. Method for loading amphoteric liposomes with nucleic acid agents according to Claims 22 and 23, characterized in that the cryoprotectants the formed active-containing amphoteric liposomes before the freeze / thaw Process be added. Method for loading amphoteric liposomes with nucleic acid agents according to Claims 22 and 23, characterized in that the active ingredient-containing amphoteric liposomes produced in the presence of cryoprotectants become. Method for loading amphoteric liposomes with nucleic acid active substances according to one or more of the following several of the preceding claims, characterized in that certain cations are added. Method for loading amphoteric liposomes with nucleic acid agents according to Claim 26, characterized in that the cations are selected from the group comprising tris (hydroxymethyl) aminomethane, triethanolamine, Ammonia, morpholine, piperazine. Method for loading amphoteric liposomes with nucleic acid agents according to Claims 26 and 27, characterized in that the cation tris (hydroxymethyl) aminomethane is. Method according to one of claims 26 to 28, characterized that only one of the preferred cations is selected and no mixtures of solved Cations present. Process according to claim 21, characterized that advantageous cations of any of claims 26 to 29 in the liposome suspension available. Method for loading amphoteric liposomes with nucleic acid agents according to the claims 21 to 30, characterized in that the freezing / thawing cycles in the presence of cryoprotectants and certain cations. Method for loading amphoteric liposomes with nucleic acid agents according to Claim 1, characterized in that the active substance-containing amphoteric Liposomes following process step e) or f) or g) one or more cycles of freezing / thawing and / or lyophilisation be subjected. Method for loading amphoteric liposomes with nucleic acid agents according to Claim 32, characterized in that the freeze / thaw cycles or lyophilization in the presence of cryoprotectants and / or certain Cations performed become. Method for loading amphoteric liposomes with nucleic acid agents according to Claim 1, characterized in that the mixing of the alcoholic solution the lipid mixture and the aqueous solution can be done in a device. A method for loading amphoteric liposomes with nucleic acid active substances according to claim 33, characterized in that the mixing of the alcoholic solution of the lipid mixture and the aqueous solution in a device according to 1 . 2 or 3 can be done. A method for loading amphoteric liposomes with nucleic acid active substances according to claim 34, characterized in that the mixing of the alcoholic solution of the lipid mixture and the aqueous solution in an in US6843942 and US20040032037A1 disclosed device can be made. Amphoteric liposomes, characterized in that they are nucleic acids and according to a method of the preceding claims 1 to 36 are made. Amphoteric liposomes according to Claim 37, characterized that they have a particle diameter between 50 and 500nm. Amphoteric liposomes according to Claim 38, characterized that they have a particle diameter of 80 to 250nm. Amphoteric liposomes according to claims 37 to 39, characterized characterized in that they comprise oligonucleotides and an active-lipid ratio between 1 mg oligonucleotide / g lipid and 300 mg oligonucleotide / g lipid; prefers between 10 and 100 mg of oligonucleotide per gram of lipid. Amphoteric liposomes according to claims 37 to 39, characterized in that they contain plasmids, aptamers or RNA with a chain length of comprise more than 50 nucleobases and an active-lipid ratio between 0.3 and 30mg drug / g lipid. Amphoteric liposomes according to claims 37 to 39, characterized in that they have the following cat ions include: tris (hydroxymethyl) aminomethane, triethanolamine, ammonia, morpholine, piperazine.