JPH04505616A - Accumulation of drugs into liposomes by proton gradient - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] to liposomes by protons Standing on top of each other The present invention relates to pharmaceutical compositions and Hendersosie Hasselbach. n-Hasselbach) than expected by the relationship defined by the equation The present invention relates to methods of producing liposomes containing compositions that exhibit enhanced uptake properties.

The present invention also provides rapid release of the drug after it is formulated into liposomes. Some membrane stabilizing components, such as cholesterol, that exhibit favorable properties for preventing The present invention relates to liposome compositions containing the present invention.

The present invention also provides bronchodilators, metaproterenol, isoproterenol and The present invention relates to a novel sustained release liposome composition containing terbutaline.

The present invention also provides for liposomal encapsulation of pharmaceuticals while maintaining most of the initial pH gradient. It concerns the minimum buffering capacity necessary to achieve this.

Ejro Toeki The therapeutic performance of many drugs is dramatically improved by administration in liposome-encapsulated form. [For example, P, N, 5hek and R,F, Barbe r, ModoMed, Canada, 41.314-382 (1986) Reference. In some cases, for example, administration of amphotericin B and doxorubicin In [Lopez-Bernstein et al., J. Infect, Dis. , 151, 704-710 (1985) and Rahman et al., Cancer Res., 40.1532 (1980)], toxicity decreased, while efficacy decreased. held or even increased. From drugs encapsulated in liposomes The benefits obtained are perhaps surprising and depend on the pharmacokinetics and This appears to be due to changes in biodistribution (Ostr.

et al., Amer, J., Ho5p, Pharm, in press).

The pharmacokinetics and biodistribution of incorporated drugs depend primarily on the nature of the carrier system. There will be. Optimizing liposomal drugs requires determining vesicle size, lipid composition, and Testing of a number of variables is required, including drug to lipid ratios. but , most drug loading protocols do not allow varying these parameters alone. I can't do it. Drugs that are passively incorporated into liposomes are Changing the size of the liposomes due to changes in It will show that

Some biogenic amines and anti-tumor agents are 'remotely loaded'. accumulation within liposomes by applying a proton gradient known as [e.g., Mayer et al., Biochim, Biop] hys, Acta, 857.123 (1986), Mayer et al., Bio chemistry, 27.2053 (1988) and M, B, Ba1l See y et al., C:hem, Phys, Lipds, 47.97 (1988) ]. This loading technique allows for independent alteration of any liposome parameter. Wear. Achieving much higher drug to lipid ratios compared to conventional techniques [Mayer et al., Cheffi, Phys, Lipids, 40.333 (1986)], furthermore, the transmembrane distribution of drugs is generally It is determined by the proton gradient that modulates drug leakage by changing the buffering capacity of the medium. Non Proton and other ion gradients are used to incorporate drugs that are zwitterionic weak bases. Adriamycin, local anesthetics dibucaine and dopamine may be used. and other drugs. The advantage of this system is , efficient drug uptake, and slower drug release than passively incorporated drugs. , and higher drug to lipid ratios than can be achieved elsewhere.

Furthermore, since liposomes can be produced in iW in the absence of drugs, drugs during storage can be Problems with drug degradation during release or liposome preparation can be avoided.

Intraliposomal drug accumulation in pH gradients can be achieved by using other weak acids, e.g. pH gradient probes, It is believed to occur in a similar manner to thylamine. Methylamine is uncharged The liposomes penetrate the membrane and equilibrate to the pH of their environment. It reionizes according to the Ssselbach relation. The equilibrium distribution is determined by the transmembrane pH gradient. The redistribution can be used to measure these slopes.

However, it has the ability to be ionized by the Hendersosie-Hasselbach relationship. Not all drugs accumulate within liposomes according to this relationship.

In fact, some drugs do not appear to accumulate at all. Furthermore, according to this relationship Certain drugs that may accumulate in the body are immediately released and must be used immediately after manufacture This results in an impractical pharmaceutical formulation that cannot be practically used as a sustained release product.

Liposomes are completely closed lipid monolayers containing an entrapped aqueous volume. Ru. Liposomes can be unilamellar (one membrane) or multilamellar (multiple membrane-like bimolecules). characterized in that the membranes are separated from each adjacent membrane by an aqueous layer The vesicles may be onion-like structures). The bilayer membrane has a hydrophobic “tail” region and a hydrophilic “tail” region. It is composed of two lipid monolayers with a head region. In this case, the hydrophobic (nonpolar) “tails” of the lipid monolayer are oriented toward the center of the bilayer. However, the hydrophilic (polar) "heads" are oriented toward the aqueous phase. liposome The basic structure of can be made by various known techniques.

Liposome encapsulation offers many beneficial effects for various pharmaceuticals. It may be possible to remotely load technology encapsulated in liposomes. You will find that it helps the medicine to be effective. Furthermore, lipo saw The higher the uptake efficiency during loading into the encapsulation process, the less drug is lost during the encapsulation process. much less, which is an important advantage when dealing with expensive drugs. death However, the use of liposomes to deliver drugs improves drug encapsulation and treatment. Problems arise with both drug release. For example, the current use of “remote loading” systems Also, the uptake efficiency should be adjusted to minimize the lipid burden on patients. There is a constant need to increase rates. Second, regarding increasing uptake efficiency, However, the release properties of the loaded liposomes may provide acceptable sustained release properties. There is no guarantee. Many drugs are rapidly released from liposomes after encapsulation I know that. Such release may contribute to the beneficial effects of liposome encapsulation. reduce

11LL1 loss The object of the present invention is to provide a novel liposome composition and the incorporation of a drug into the liposome. to increase the amount of drug that can be loaded into the liposomes. This drug is designed to reduce the lipid burden on patients during liposome administration. The object of the present invention is to provide a general method for producing such compositions.

Yet another object of the present invention is to provide novel liposome compositions and liposome formulations. A method for producing such compositions that prevents undesirable rapid release of the drug prior to administration is disclosed. It is to provide.

Yet another object of the present invention is to provide novel bronchodilator liposome compositions. That's true.

Brief description of the drawing Figure 1 shows an internal aqueous buffer system containing 300mM citrate, pH 4°0 and 300mM External aqueous buffer system containing NaCl, 20mM HEPES, pH 7.5 Midoxanthro by EPC vesicles exhibiting a proton gradient ne) accumulation. Accumulation is rapid and complete with no release over several hours proved that.

S2A shows the response of timolol uptake into EPC vesicles (partial accumulation, uptake The level of uptake is approximately 100,100 n/umol (stable with available drugs). (approximately 50%).

Figure 2B shows the response of quinacrine, similar to timolol within EPC vesicles.

The level of uptake is approximately 80 nmol/umol lipid after 30 minutes.

Figure 3A shows the response of quinidine uptake into EPC vesicles (complete accumulation, rapid release). out). Approximately 50% of the drug leaks from the vesicles within 30 minutes.

Figure 3B shows the effect of added cholesterol on quinidine uptake and p The stability of the H gradient is shown.

Figure 4 shows the effect of physostigmine on the transmembrane pH gradient. Drug uptake was measured under the conditions used to evaluate physostigmine (200 uM physostigmine). Only a slight decrease in pH was observed.

Figure 5 shows pH gradient using egg phosphatidylcholine 200 nm extruded liposomes. of metaproterenol, terbutaline and isoproterenol. .

Figure 6 shows the effects of methylamine redistribution in the presence and absence of isoproterenol. Figure 3 shows the effect of drug uptake on the residual pH gradient as measured by shown As such, when the internal and external pH is 7°4 or 4.0 (isostatic), methyl acetate Min does not detect pH gradients.

[i! 17 is 21'C metaprotere at pH gradient at 137 °C and 60 °C. The effect of temperature on nol uptake is shown.

Figure 8 shows the effect of cholesterol on metaproterenol accumulation against pH gradient. Show impact.

Figure 9 shows the effect of changing external drug concentration on the level of metaproterenol uptake. Show the impact of

Figure 10 shows the effect of internal buffering capacity on drug uptake.

And Royal 1ri The present invention provides a first internal buffer aqueous solution and a drug concentration in the internal buffer after uptake. Formulate the liposomes using a second external buffer aqueous solution that exceeds the solubility product of the product. than expected from the Hendersozy Hasselbach relationship. Regarding liposomal compositions with pH gradients exhibiting significantly higher drug accumulation, .

Therefore, the drug is dissolved within the liposome at a lower concentration than the final concentration of drug within the liposome. It is preferable to show resolution. Preferably, the solubility of the drug is less than about 20mM. and is particularly preferably lower than about 10mM. Furthermore, the internal buffer solution is about 5 0mMol, preferably about 100 to about 300mMol, most preferably about 300mMol It has a buffering strength of

The present invention also provides rapid release of certain drugs from liposomes that do not contain membrane stabilizing components. membranes, such as cholesterol, among other lipids, to prevent The present invention relates to liposome compositions containing some stabilizing component. this Such liposomes contain phosphatidylcholine and choline in a molar weight ratio of approximately 55:45. Preferably, it contains a mixture of sterols.

Moreover, the present invention also provides for metaproterenol, isoproterenol and terbutaline. A liposome composition with a pH gradient comprising a bronchodilator selected from the group consisting of It is related to. Therefore, it is not formulated into such liposomes. The above drug accumulates in the liposomes to a considerable extent and slows down the bronchodilator. It is possible to create effective and stable liposomal compositions useful for therapeutic conditions requiring release of It is shown. Liposomal compositions containing bronchodilators can be used to treat many conditions including asthma. It can be useful in treating many conditions. Such compositions have the same It is expected to have a longer residence time in the lungs than other drugs, and therefore in the bronchus for a longer period of time than currently available compositions. Obtain the concentration of the dilator. Bronchodilators directly into the lungs to treat acute asthma attacks For administration, such compositions can be administered as an aerosol in a pharmacologically acceptable solution. It may also be formulated as

The bronchodilator composition of the present invention is a mixture of phosphatidylcholine and cholesterol. (55:45, W: W) as well as egg phosphatidylcholine (EPC) It has also been shown to accumulate effectively in liposomes containing . Time required for accumulation is longer for cholesterol-containing liposomes, but for phosphatidylcholine and cholesterol. Liposomes containing a mixture of sterols have approximately the same relative composition as EPC liposomes. bronchodilators accumulate to a large extent.

Generally, the liposome compositions of the present invention contain a fat content ranging from about 0.5% to about 50%. It has a molar ratio of drug to quality. The liposomes of the present invention are particularly suitable for Sphatidylcholine (EPC), hydrogenated soybean phosphatidylcholine, distearo Ilphosphatidylcholine, Simyristoylphosphatidylcholine, Dialatide It may also contain phospholipids such as noyl phosphatidylcholine, and may also contain many steroids. roid compositions and other compositions.

Generally, the size of liposomes ranges from about 0.05 to greater than 2 microns. The preferred range is about 0.05 to about 0.3 microns. most preferably , liposomes are unilamellar and range in size from about 0.1 to about 0.3 microns. It is in. These unilamellar liposomes are homogeneous or monomorphic with respect to size distribution. It may be in a state.

The liposomes of the present invention can be administered orally, parenterally, bucally, topically, among other routes of administration. and can be administered by transdermal routes of administration.

]”B arrow! LIJL sucking The present invention provides a method for forming liposomes into liposomes exhibiting a transmembrane ion gradient, preferably a transmembrane pH gradient. Take advantage of the efficient uptake of drugs in resulting in a much greater accumulation of medicine than would be expected from the Rubatsuha relationship. be able to.

The liposome compositions of the present invention include at least one lipid, a drug accumulated therein, an internal buffer in which the solubility of the drug in the buffer is lower than the concentration of drug in the liposome; and an external buffer in which the solubility of the drug is preferably about 0.2 mM or greater.

The term ``pharmaceutical agent'' and the term ``dru'' The terms g) have the same meaning when used in this specification.

The liposome of the present invention can be formed by any known method, but preferably Baley et al., PCT Application No. US86101102.1986 Published February 27, and Mayer et al., PCT Application No. US88100646 ．． Preferably, it is formed by the method described in Published September 7, 1988. . These technologies allow liposomes to be loaded with ionized drugs. If not, neutral pH and/or passive uptake techniques can than would be expected from the solubility of the drug in aqueous solution at higher concentrations than possible. A higher internal concentration is achieved. In this technique, the transmembrane ion (pH) gradient is The ionic (pH) gradient that occurs between the inner and outer membranes of posomes causes liposomes to The drug is loaded into the some and causes uptake to take place. For example, Na+, C1 -1K+, Li+, 0H-1 preferably H+. Create a concentration gradient of different species across the liposome membrane. As a result, a transmembrane gradient is created, and the ionic gradient causes the Uptake of the ionized drug takes place. In the present invention, transmembrane ions (H+) Gradients are used to create an ionic gradient to lipolyze drugs that tend to have weakly basic nitrogen groups. Preferably, it is loaded intrasomally.

In the present invention, it is preferred that liposomes are first formed in a buffered aqueous solution.

The initial solution is such that the drug to be loaded produces a charged species at a basic pH. It is either acidic or basic, depending on whether it occurs at an acidic pH or at an acidic pH. example For example, when loading a weakly basic drug, the charged species may be present at a low pH of 1 or about 2°. It is produced at a pH of 1 to 5.0, preferably about 4.0. Acidic internal buffer aqueous solution After formation of liposomes with The pH of the solution is significantly higher than the pH of the buffer, preferably at least about 3. 0-4. Change to a higher pH by OpH unit. By changing the external buffer, A pH gradient is created that causes the accumulation of drug within the somes. Internal buffers have different pH It may differ from the external buffer only in this respect. In general, uncharged Drugs are much more tolerable than charged (or, in the case of weakly basic drugs, protonated) drugs. It will easily pass through the lipid layer of the liposome. In this way, inside the external buffer The uncharged drug easily passes through the liposome and enters the internal buffer. “Incorporated” protonation that is rotonized and does not easily pass through the liposome layer The molecule will remain within the liposome as a molecule. In this way, medicines will assemble within the liposomes as a function of the pH gradient between the solution and the external buffer. cormorant.

Although such loading by the above method is effective for some drugs, the maximum loading It often doesn't. the drug is maximally soluble in the internal and external buffers; Even if it easily passes through the liposome layer (an assumption that is not necessarily proven in reality) , the maximum load is generally Hender-Sogy-Hasselbach type [HA+] in/ [HA+] out=[H+] in/[H+] out Reflect relationships. However, it is believed that numerous factors influence a drug's ability to accumulate. It is being These factors include the partitioning of unprotonated drugs into lipid layers; between the protonated species present in the buffer and the membrane-associated species. differences in pKa, buffering capacity of the internal buffer and protonated species within the internal buffer. It has solubility.

in liposomes, more than expected from the Hendersozy-Hasselbach equation. The most important factor influencing drug accumulation in the internal buffer is the drug protocol in the internal buffer. We now know that the solubility of the chemical species is of accumulated medicines The solubility of protonated species is predicted by the Hender-Sogy-Hasselbach equation. It is likely that higher uptake and accumulation levels will be affected than measured. Understood. The solubility of the ionized drug is minimal, preferably ionized. Liposomal compositions that are formulated with an internal buffer that precipitates the drug are produces liposomes with consistently high uptake efficacy approaching 100% when This will cause drug accumulation within the liposomes beyond what would be expected to occur.

In the present invention, surprisingly, the solubility of the drug within the internal buffer is within liposomes to a greater extent than predicted by the Sogy-Hasselpach relationship. It has been discovered that this may ultimately control the ability of drugs to accumulate. Ta. Thus, in the present invention, basic (pH approximately 8 to 10) or acidic (pH approximately 8 to 10) or acidic (pH approximately 8 to 10) a first internal buffer of about 3.0 to 5.0) and a pH of preferably about 6.5 to 8.0. Liposomes formed using a second external buffer that is preferably 7.4 Compositions are preferred. Increase the pH of the internal buffer relative to the neutral pH of the external buffer. It acts to cause the drug to accumulate within the liposomes by increasing or decreasing the A transmembrane gradient is generated. Surprisingly, in the Hendersogie Hatselbach style Using a transmembrane gradient that accumulates more drug into liposomes than predicted. , the most important factor in determining the maximum amount of drug loaded into liposomes is It was found that the solubility of the drug in the buffer solution is

In general, internal buffers useful in embodiments of the invention are such that the drug to be accumulated is so that it has a lower solubility in the internal buffer than the total drug that accumulates within the soma. selected. Generally, the solubility of the drug in the internal buffer is about 65mM or less, preferably Preferably, it is about 20mM or less, most preferably 10mM or less.

Also, the internal buffer is selected to maximize the buffering strength of the internal solution. It will be done. The strength of the internal buffer is also important for the total accumulation of drug within the liposomes. It is believed that the internal buffer is chosen to make this strength as high as possible. It will be done. Of course, the solubility of the drug within the internal buffer is also the most important factor in determining accumulation. This is one of the important factors. Therefore, when choosing a buffer, choose one that is useful. The solubility factor and buffer strength factor must be made as large as possible. In the present invention, the buffer strength of the internal buffer is preferably about 50 mM or more. or less than about 20mM or less than 300mM, most preferably about 300mM. I found out something.

Lipids that can be used in the liposome formulation of the present invention include synthetic or natural phospholipids. High quality, especially phosphatidylcholine (pc), phosphatidyl ethanol amine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylinositol (PI) , sphingomyelin (SPM), and calciolibin alone or in combination. 6 Phospholipids useful in the present invention include dimyristoyl phosphatidyl phospholipids. phosphorus (DMPC) and simyristoylphosphatidylglycerol (DMPG) can also be mentioned. In other examples, distearylphosphatidylcholine ( DSPC), dipalmitoylphosphatidylcholine (DPPC) or hydrogenated soybean Phosphatidylcholine (H3PC) may also be used.

Simiristoyl phosphatidylcholine (DMPC) and dialatidonoylphospha Tidylcholine (DAPC) may be used similarly. DSPC (Tc at approximately 65°C ), DPPC (Tc of about 45℃), DAPC (Tc of about 85℃), To make these liposomes, their Tc# i or up to a slightly higher temperature, e.g. about 5°C above Tc. Preferably, the fat is heated. In a preferred embodiment, egg phosphatidyl Choline is used.

In many embodiments of the invention, a steroid component may be added to the liposomes. . Add any of the above phospholipids to cholesterol, cholestanol, or cholestanol. in combination with at least one other component selected from the group consisting of It may also be used. Furthermore, like cholesterol hemisuccinate (CHS) Along with the organic acid derivatives of sterols, polyethylene glycol of cholesterol PEG-cholesterol derivative (PEG-cholesterol) in combination with any of the above phospholipids. May be used. Alpha-tocopherol hemisuccinate (TH3) organic acid Derivatives may also be used. CH3 and TH3 containing liposomes and their tris The morphology is generally made by any known method for producing liposomes containing sterols. can be done. The resulting phospholipid-styrene mixture is transformed into stable liposomes. Any of the above sterols may be used as long as it is. In particular, Janoff et al. , U.S. Patent No. 4,721,612, issued January 26, 1988, entitled “STEROID” Liposomes” and Janoff et al., PCT Publication No. 87102219.198 Published on April 23, 2017, with the name ``Alpha-tocopherol-based media'' See Law. Relevant parts of these are included herein for reference.

In certain instances, liposomes are designed to prevent rapid release of drug. , an amount of cholesterol equal to about 30 to about 45 mole percent by weight of the lipid containing liposomes. in combination with any of the above phospholipids or phospholipid/steroid combinations. It is preferable to use Generally, such compositions contain accumulated pharmaceutical liposomes. This will surely prevent undesirable rapid release from the membrane. Medicines from liposomes Any combination of membrane-stabilizing components and lipids that prevent the rapid release of the membrane may be used as described in the art. membrane stabilization to formulate liposomes that prevent rapid drug release. The ingredients and phospholipids could be modified. In this aspect of the invention , about 45% by weight of cholesterol and about 55% by weight of phosphatidyl coli. Most preferably, a mixture of ingredients is used. In this aspect of the invention, from liposomes Any number of drugs that exhibit a tendency to release rapidly may be used, but quinine, diphenylene In particular, the drugs hydramine and quinidine tend to be rapidly released from liposomes. Therefore, liposome formulations containing these drugs contain membrane-stabilizing components added to lipids. an amount of cholesterol equal to about 30 to 45 mol%, preferably about 45 mol% of the It has been found that it is preferable to include a roll. A certain drug is made of liposomes. strictly determined based on the chemical structure that it will be rapidly released from the drug. Although it is difficult, those skilled in the art can determine the degree of drug release! ! The teachings of the present invention It would be possible to formulate liposome products consistent with the instructions. Others of the present invention As in the examples, the internal and external buffers in this embodiment of the invention include Any buffer can be used regardless of the solubility of the drug. However, good Preferred buffers, as in other embodiments of the invention, are suitable for drug solubility in liposomes. preferably less than 20mM, most preferably less than the concentration of the drug in the or less than 10mM.

Several methods can be used to form the liposomes of the invention. example For example, multilamellar vesicles (MLV), stable multilamellar vesicles (SPLV) or reverse phase Evaporative vesicles (REV) can be used. However, pushing MLV out of the filter and large lamellar vesicles (L), the size of which depends on the filter size used. It is preferable to form UV). Generally, 30.5o, 60.100.200 or A polycarbonate filter with pores of 800 nm can be used. C. u1lis et al., PCT publication number W○86100O238, January 16, 1986 The method described in (the relevant parts of which are included here for reference) In this step, the liposome suspension is repeatedly passed through an extruder to achieve uniform size distribution. Population of posomes. For example, straight-through Membrane filter (Nucleopore) polycarbonate membrane filter) or serpentine path filter (e.g., 0.1 um size nucleoboaf). Filter membrane filter (mixed cellulose Filtration may be carried out by stell)) or other methods such as homogenization. Other grinding techniques may also be used. The size of liposomes is approximately 0.03 mm in diameter. may vary from about 2 microns or more, preferably from about 0.05 to 0.3 microns. and most preferably about 0.1 to about 0.2 microns. This size range is It includes liposomes that are MLV, 5PLV or LUV. Smell of the present invention Preferred liposomes are unilamellar liposomes of about 0.1 to about 0.2 microns. It is something that is.

As mentioned above, liposomes with Tc from gel to liquid crystal above room temperature A number of lipids can be used to form the membrane. In such cases, the heating bar Extruders with barrels or thermal jackets can be used. This way This device helps to increase the liposome suspension temperature which allows the extrusion of LUv. .

Lipids used in extruders with thermal jackets include e.g. DSPC , DPPC, DMPC and DAPC or mixtures thereof, which are In some embodiments, cholesterol is added to prevent rapid release of drug from the posomes. May include.

Liposomes containing DSPC are generally kept at about 65°C and those containing DPPC at about 4°C. At 5°C, those containing DAPC were pressed at about 85°C (about 5°C higher than the lipid Tc). Served.

As previously indicated, preferred liposomes for use in the present invention have a size of The LUV is about 0.06 to about 0.3 microns.

As defined in this application, a homogeneous population of vesicles is defined as having substantially identical sized liposomes, even though they have a Gaussian distribution of particle sizes. good. Such a population is said to have a uniform size distribution, and in terms of size, It may be unimodal. The term ``single mode'' means a population with a narrow polydispersity of particle sizes, with particles having a single pamode ”. The population of liposomes is determined by the quasi-elastic light scattering method. The population of approximates a Gaussian distribution, and the quadratic polynomial is the natural pair of autocorrelation numbers of samples. If the number matches, it is unimodal [Koppel, J. Chem, Ph. ys,, 57.4818 (1972)], :: meeting was just right. The higher the number, the better the degree of unimodality. child The exactness of the match is determined by how close the chi-square (chi2) value of the sample is to 1. A claim can be made depending on the circumstances. A chi2 value below 2.0 is a unimodal population. Show the group.

Other comminution techniques may be used in practicing the present invention. For example, homogenization Can successfully use milling and milling techniques. With such technology, Liposomes are obtained that are homogeneous or unimodal in terms of size distribution.

liposol by encapsulating a first buffered aqueous solution with the properties mentioned above. You may also prepare a sample. ”: Typical liposome preparation techniques as detailed in Now, this first buffered aqueous solution surrounds the liposomes as they form. , which serve as the buffer inside and outside the liposome. To create a concentration gradient, the load must be The original external buffer is acidified or Either make the external buffer basic or replace the external buffer with a new external buffer with a different charged species. It may be replaced with a medium. Replacement of external media can be done using various techniques, e.g. Equilibrated gel filtration columns, such as Sephadex columns This can be done by passing the liposomal formulation through the membrane or by dialysis or related techniques. Ru.

Organic solvents may be used to suspend the lipids during liposome preparation. present invention Organic solvents suitable for use in solubilizing lipids have a variety of polar and dielectric properties. For example, chloroform, methanol, ethanol, etc. , demethyl sulfoxide (DMSO), methylene chloride and benzene:methanol Examples include solvent mixtures such as (70:30). As a result, a solution containing lipids ( A mixture (with lipids and other ingredients evenly distributed throughout) is formed. The solvent is , generally selected on the basis of their biocompatibility, low toxicity and solubilization ability.

One preferred embodiment of the invention provides highly effective uptake of the drug in the clinic. It is a three-component liposome-medicinal treatment system. If the drug is acidic inside the liposome, The first component of the system (vial l) is an acidic buffer, for example, citrate buffer (300 mmol, pH about 3.8- 4.2, preferably 4.0) or the ionized form of the drug is minimally incorporated. Only dissolve in other buffers (solubility lower than the final concentration of drug in the liposomes) , preferably about 20 mM or less, most preferably about 10 mM or less) including. The second component of the system (vial 2) is a basic buffer, e.g. 5M, pH about 10-12, preferably about pH 11.5, sodium carbonate or bis Contains a sodium phosphate solution, which becomes part of the external buffer of the liposome formulation.

Dissolution of the drug in an external buffer to maximize drug accumulation within the liposomes Preferably, the degree is about 0.2 mmol or more. Third component (vial 3) is a medicine. The above treatment system comprises a first vial containing liposomes in an acidic medium. a second vial containing al, a base and a third vial containing a medicament as described above. It can be provided as a 3-vial system. The internal buffer of liposomes Drugs that accumulate in membrane gradients that are relatively basic, i.e. with a pH of about 8.5-11.5 A similar processing system can be provided for The first component is, for example, about 8.0 Sodium carbonate or sodium bisphosphate at a pH of ~11.0, preferably about 10 including liposomes in relatively basic buffers such as solutions. The second component is relative a generally acidic or neutral solution, e.g. 150mM NaC] buffer with a pH of approximately 7.4 /150mM HEPES buffer is included as a buffer for the liposomes. third ingredient does not ionize much at the pH of the external buffer, but ionizes at the pH of the internal buffer. Contains drugs that

Formation of a pH gradient across the liposomes (mixing the first and second vials) The liposomes may be heated before mixing with the medicament. under certain circumstances and at least about 30 mol% to minimize rapid release of the drug. If you want to accumulate a drug in liposomes containing cholesterol, Preferably, the somes are heated to about 60°C to promote accumulation.

In order to accumulate drugs in liposomes using the above processing system, Mayer et al. PCT publication number W○88106442. September 7, 1988 (the relevant parts are (herein incorporated by reference) may be applied to the medicaments of the invention. May be modified for use.

In liposome-drug delivery systems, drugs are incorporated into liposomes or and then administered to the patient to be treated. throughout the statement When used as a pharmaceutical agent, Drug and agent are used interchangeably. I can stay. For example, Rahman et al., U.S. Pat. No. 3,993,754; ars, U.S. Patent No. 4,145,410; Papahadjopoulos et al., U.S. Pat. No. 4,235,871: 5Chneider et al., U.S. Pat. No. 114,179; Renke et al., U.S. Pat. No. 4,522,803; and F. See U.S. Pat. No. 4,588,578 to Ountain et al. In the present invention, Any number of different drugs and drug types can be incorporated into liposomes or can be made to meet with the team. For example, pharmaceuticals useful in the present invention include: The ion concentration or At pH, some drugs have low solubility in the inner buffer for liposomes. can be lost. Such medicines include, among many other medicines, Antitumor agents such as Santron; lidocaine, dibucaine, chlorpromazine, etc. Local anesthetics; such as metaproterenol, terbutaline, isoproterenol, etc. Bronchodilators; beta-ads such as propranolol, timolol, labetolol Renarin blockers; antihypertensive drugs such as pronisin and hydralazine; imibramine, Antidepressants such as amitributyline and doxepin; anticonvulsants such as phenytoin Medicines; antiemetics such as procainamide and prochlorperazine; diphenhydrami Antihistamines such as quinidine, chloropheniramine, and promethacin; Antiarrhythmic drugs such as viramide; antimalarial drugs such as chloroquine, quinacrine, and quinine drugs; and analgesics.

In general, the internal buffer used in the liposome compositions of the present invention will meet several criteria. The most important of these is the buffer strength, as mentioned above. Next to the solubility characteristics of the drug to be loaded into the buffer. internal buffer The buffer system used as 00mM, most preferably about 300mM. preferable. Accordingly, one of the most preferred buffers for use as the internal buffer system of the present invention is: characterized in that it does not solubilize ionized, preferably protonated, drugs. do. That is, the ionized drug will generally be about 65 mmol or less, preferably in the buffer to an extent of about 20 mmol or less, most preferably about 10 mmol or less. lysed, they are about 50mM or more, preferably about 100 to about 300mM, most preferably Preferably, it also has a buffering strength of about 300mM. Most preferably, the internal buffer is The ionized species of the drug is precipitated from solution.

The choice of buffer to use as the internal buffer will depend on the drug chosen for loading. It will change. A person skilled in the art will determine the buffer to be used as the internal buffer. Assessing the relative solubility and buffering strength of ionized species in pharmaceuticals You will be able to do that.

In the present invention, if the solution is pharmaceutically compatible, i.e., it does not have an adverse effect. Any buffer having the properties generally described above may be used, provided that it can be administered to the patient without hesitation. can be done. Typical internal buffers include citric acid and oxalic acid, among others. , succinic acid and other preferred organic acid salts. Approximately 100mM to approximately 3 Citric acid at concentrations ranging up to 0.000 mM is preferred. The concentration of citrate buffer is approximately Most preferably the range is 100mM to about 300mM. Typical external Examples of buffer solutions include N a C1, KCI, potassium phosphate, sodium bicarbonate, among others. sodium carbonate, sodium bisphosphate, potassium sulfate and HEPES , as well as mixtures thereof.

Pharmaceutical loading efficiencies utilizing the present invention generally range from about 20% to about 100%. 50% or more, preferably about 50% or more. In general, the loading effect of the medicament according to the invention The rate is higher than you would expect from a Hendersosie-Hasselbach relationship. Of course Well, all drugs are contained within liposomes according to the Hendersozy-Hasselbach relationship. Some drugs (see Example 1, Table 1) do not readily accumulate in does not seem to accumulate at all. This phenomenon occurs because the polarity of the drug is too large and the liposomal This may be the result of an inability to penetrate the system or other factors. of course , those skilled in the art will understand that in order to load as much drug into liposomes as possible, In some cases, changing the lipid composition of liposomes or practicing the present invention may be In some cases, ionophores or other agents can be used to increase the penetration of liposomes by drugs. You will realize that this is necessary but not overwhelming.

Liposomes formed by the method of the invention can be lyophilized or lyophilized at various stages of formation. may be dehydrated. For example, after removing the solvent and before adding the drug, the lipid film can be frozen. May be dried by freezing. On the other hand, before hydrating the liposomes, the lipid-drug film is frozen. May be dried by freezing. Such dehydration involves placing the lipids or liposomes under reduced pressure. This can be carried out by removing all suspended solvent. B a11y, etc., PCT Publication Number 8610Ii02. Published February 27, 1986, Name ``Encapsulation of antitumor agents within liposomes''; Janoff et al., PCT Publication Release number 86101103. Published on February 27, 1986, name: “Dehydrated Liposome” 5chneider et al., U.S. Patent No. 4,229,360, January 1980 Published on the 29th; and Mayer et al., PCT Publication No. 88106442.1988. Published September 7th (relevant parts of these are included here for reference) The liposomes may be dehydrated by the method in the presence of a hydrophilic agent. on the other hand, or in addition Additionally, the dehydrated liposomal formulation is placed in the surrounding medium in liquid nitrogen and subjected to the dehydration process. Hydrated liposome formulations may be dehydrated by freezing them. in advance Dehydration with freezing is described by Ba11y et al., PCT Publication No. 86101103.1986. Published February 27, 2015 (the relevant parts of which are included here for reference) The chemical process can be carried out in the presence of one or more protective agents such as sugars. Wear. Such techniques enhance long-term storage and stability of the formulation. For example, about 0. Sugar:lipid weight/weight ratio ranging from 5:1 to about 100:1, preferably about 20=1. without affecting the ability of the liposomes to retain the loaded agent during dehydration. Can be mixed with sugar solution. In this aspect of the invention, liposomes is preferably in the range of about 0.1 to 0.2 microns in size.

In one preferred embodiment, the sugar is mannitol or mannitol in a 2:l:) weight ratio. Glucose: Lactose. Rehydration in distilled water followed by elevated temperature, e.g. Preferably, the formulation is heated, for example, at 60° C. for 10 minutes. Desorption of the above liposome formulation Other suitable methods for water can also be used. Liposo, without pre-freezing You can also dehydrate the mucus.

Once liposomes are dehydrated, they can be stored for long periods of time until used. . The appropriate temperature for storage depends on the lipid composition of the liposome and the temperature of the encapsulated material. It will depend on your sensitivity. For example, various antitumor agents are heat-labile and Therefore, dehydrated liposomes containing such agents should be cooled to prevent the efficacy of the agent from being lost. It must be stored under frozen conditions, for example at about 4°C. Also, regarding such agents, Therefore, it is preferable to perform the dehydration step at a low temperature rather than room temperature.

When using dehydrated liposomes, simply add an aqueous solution, e.g. distilled water or a suitable buffer. Rehydration is carried out by adding it to liposomes and hydrating it. Vortex the solution gently. By rolling, the liposomes can be resuspended in an aqueous solution. Rehydration at room temperature or at other temperatures appropriate to the composition of the liposomes and their internal contents. Wear. If the anti-tumor drug to be administered has a high drug to lipid ratio, Liposomes are mixed before dehydration and do not require any other changes in composition. Rehydrated liposomes can be directly applied to cancer treatment according to known methods of administering encapsulated drugs. Can be used directly. On the other hand, using the above-mentioned transmembrane pH gradient method, immediately before administration, Ionizable anti-tumor agents can be incorporated into rehydrated liposomes. this In connection with the method, the concentration gradient used to generate the transmembrane pH gradient is before or after rehydration using the external media exchange techniques described above. Can be done. For example, liposomes with a high drug-to-lipid ratio can be placed on a transmembrane pH gradient. For example, they can be first dehydrated from the external medium before setting up.

Upon rehydration, mixing the liposomes with a second external medium of relatively acidic or basic pH By doing so, a pH gradient can be set.

Anti-tumor agents can be mixed with liposomes at the same time or after setting up the pH gradient. Wear.

When IJ posomes are dehydrated after having a transgenic pH gradient, they are placed in a neutral DH For example, liposomes may be rehydrated by mixing with an aqueous solution of In the above case using liposomes containing enoic acid buffer, the rehydration step is performed using sodium carbonate. This will proceed by adding a drug such as propranolol.

Liposomes already contain a base (e.g. sodium carbonate) and therefore already have membranes. If you have an internal and external pH gradient, add water or other aqueous solution and doxorubicin. , rehydrate. Finally, the drug-containing liposomes are released with a transmembrane pH gradient. In the case of hydration, rehydration proceeds using water or other aqueous solutions. On the other hand, whatever is necessary Ba.

A second medicament may also be added.

For the treatment of numerous medical conditions or pharmacological conditions that require repeated administration and sustained release formulations. Liposomes containing the pharmaceutical formulation of the present invention can be used to treat mammals, especially humans. can be done.

Organs to which the compound is delivered by the dosage form of the drug-containing liposome of the present invention The site and cells will be determined. The liposomes of the present invention can be administered alone. Yes, but generally the choice is made with respect to the intended route of administration and standard pharmaceutical practice. It will be administered in admixture with a pharmaceutical carrier. The formulation can be administered parenterally, e.g. May be injected intravenously. For parenteral administration, make them isotonic, e.g. A sterile aqueous solution that may contain sufficient salt or other solutes such as glucose to make the Can be used in liquid form.

The liposomes of the invention may be used subcutaneously or intramuscularly. Other uses include Depending on the properties of the agent, one skilled in the art would be able to predict it.

For oral dosage forms, the liposomal formulations of the invention can be formulated into tablets, capsules, lozenges, etc. Used in the form of diuretics, troches, powders, syrups, elixirs, aqueous solutions, suspensions, etc. can do. For tablets, carriers that can be used include ragdose and citric acid. Mention may be made of the salts of sodium and phosphoric acids. starch, lubricant, talc A variety of disintegrants are commonly used in tablets, such as. Oral administration in capsule form Useful diluents include ragdose and high molecular weight polyethylene glycols. It is. When aqueous suspensions are required for oral use, the active ingredient can be combined with emulsifying or suspending agents. Let me do it. Sweetening and/or flavoring agents can be added if desired.

For topical administration forms, the liposomal formulations of the present invention can be used in gels, oils, emulsions, etc. It may also be incorporated into dosage forms such as versions and the like. These preparations include creams, paces It can be administered by direct application, such as in gels, ointments, gels, and lotions.

For administration to humans in the treatment of medical or pharmacological conditions, the prescribing physician must will ultimately determine the appropriate dosage of the antitumor agent for a given human subject. So This will depend on the age, weight and response of the individual, as well as the pharmacokinetics of the agent used. It can be predicted that this will change. Also, the nature and severity of the patient's medical condition or Pharmacological status will also affect dosage. Drug administration in liposome form The amount is generally expected to be on the order of that used for the free drug, but in some cases patients may need to administer doses outside these limits.

The following examples are given for illustrative purposes only and do not limit the scope of the invention. It should not be thought of as something that should be done.

Example 1 Preparation of loaded liposomes The following medicines, egg phosphatidylcholine (EPC, Avanti Po1ar) Lipids, Inc., Birmingham, Alabama) Or EPC and cholesterol 55:45 (molar ratio) (Sigma Chemi cals, St. Louis.

Loaded or attempted to load liposomes containing cholesterol from MO, Propranolol, Timolol, Dibucaine, Glorpromazine, Lidocaine, quinidine, pilocarbin, physostigmine, dopamine, imipramine, dife hydramine, quinine, chloroquine, quinacrine, daunorubicin, vincris Chin and Vinblastine (Sigma Chemical, s, St, Lo uis, No.), doxorubicin, evilubicin (Adria L. laboratories.

Midoxant (obtained from Mississauga, Ont., Canada) Ron (Cyanamid Canada Inc, Montreal Qu e, obtained from Canada), codine and pethidine (Abbot Labo rations, Ltd, Downsview, Ont.

(obtained from Canada). Radioactive label, “H-dipalmitoylphosphatidylco Phosphorus, ``4C-dipalmitoylphosphatidylcholine, +4cm dopamine and'' 4C-imipramine was obtained from Amersham, while 'H-glorpro Mazin, °H-propranolol, "4C-pyrocarbin, 'C-glorproma" gin, "CC-methylamine" 4C-lidocaine, New England Obtained from Nuclear.

IC-Timolol is available from The Liposome Company, Inc. (Princeton.

N.J.). The salts and reagents used were containing phosphorus) or in an EPC:cholesterol mixture (55:45 molar ratio). It was. The EPC:cholesterol mixture was prepared using benzene/methanol (95:5v/ Prepared by co-lyophilization from v). 300 m with dry lipid as internal buffer Hydrated with M citrate pH 4.0, Mayer et al., Bioehim, Biop M obtained according to hys, Acta, 817, 193 (1986) LV was subjected to freeze-thaw using liquid nitrogen five times to enhance solute distribution. Then H ope etc., Bioehim, Biophys, Acta, 812.55 (+985) using a method such as that described by (+985) Extract with a carbonate filter (NucLeopore, Inc.) (Lipex Biomembranes, Vancouver, Ca. Enlarged lamellar vesicles were prepared using (Nada).

Then, to set up the pH gradient, add 300mM NaCl, 20mM HEPE 5 ephadex G-50 (fine) column pre-equilibrated with S, pH 7.5 The vesicles were passed through (1,5xlOcm).

Enlarged lamellar vesicles in 300mM NaCl, 20mM-HEPES% pH 7.5 (~1mM lipid) was incubated with drug (0.2mM). 2 hours At various times, take a portion (100 u1.) of the mixture and , Biochem, Biophys, 212.186 (1981). As described above, 1ml of 5ephadex G-50 (medium) Separate iJz cells from unincorporated drugs by centrifugation using a “Mini Column” did. Lipids and drugs were quantified by the following method.

Lipid concentration was determined using a Packard 2000 CA instrument. or 14C-DPPC liquid scintillation counting. Similarly, pin locarbin, glolbromazine, timolol, propranolol, imipramine , lidocaine, and dopamine in a fixed amount of “H- or “C-radioactively labeled tracer. It was quantified using

Physostigmine was fluoresced after solubilizing the vesicles in 60% ethanol (V/V). Measured by spectroscopy. The excitation and emission wavelengths used were 305 and 305, respectively. It was 350 nm. Quinacrine, chloroquine and quinine are also each 420 nm, 505 nm; 335 nm, 375 nm; and 335 nm, 365 nm excitation. It was quantified from the fluorescence using the emission and emission wavelengths.

Vinblastine and vincristine were added after solubilizing the vesicles in 80% ethanol. , their at 262 nm and 297 nm, respectively, by ultraviolet spectroscopy. Measured from absorption. Codin is also present, in this case 40mM Ogutyluvator D- Measured by ultraviolet spectroscopy at 220 nm after solubilization in glucopyranoside. .

Midoxantrone was vesiclesized in 2% Triton-X100. After solubilization, it was quantified from its absorption at 670 nm.

Diphenhydramine is Chromatographic 5special HP with es DB-225 (25% cyanopropylphenyl) capillary column Measured by gas-liquid chromatography using a 985o gas chromatograph did. The flow rate of the helium carrier was 1 ml/min, and detection was by flame ionization. It was. Extraction from aqueous samples in diethyl ether and thin layer chromatography using an internal standard, methylbentadecate, after separation from EPC by , diphenhydramine was quantified. Ba1ly etc., Chem, Phys, L Weak bases, as previously described by ipids, 47°97 (1988) The pH gradient inside and outside the bilayer membrane was quantified using methylamine (C-labeled).

The results of the loading experiments are shown in Table 1 below. Basically, liposol with the above agents The loading of the systems can be limited based on their uptake characteristics. Them Based on their properties, four categories of drugs can be defined.

Drugs in the first category showed complete and stable uptake.

propranolol, dopamine, daunorubicin, evirubicin, dibucaine, Mibramine and doxorubicin exhibited characteristics of this drug category. This card All drugs within the category are as predicted by the Henderson/Hasselbach equation. It also showed a high uptake. MID by EPC liposomes exhibiting a proton gradient The accumulation of xanthrone is shown in FIG.

The second category is 1, indicating stable, albeit partial, uptake. Chimorow Lidocaine, Chlorpromazine, Serotonin and Chloroquine are The characteristics of the category were shown. Timolol is approximately 100 nmol 1/umo l Accumulates to the extent of lipids (approximately 50% of the available drug, see Figure 2A) and Figure 2B reference). Although the accumulation is lower than with the first group of agents, nevertheless The amount of effort involved is actually considerable. For timolol, at an external concentration of 1100 u In contrast, an internal concentration of approximately 65 mM is achieved.

The third category shows partial uptake and then release of the agent from liposomes. Rapid release occurs. Figure 3 shows that quinidine rapidly and virtually completely accumulates within the vesicles. and within 30 minutes, approximately 50% of the agent leaked from the vesicles (Figure 3A). . Other agents that leak from EPC vesicles include quinine, diphenhydramine, and vinine. Examples include blastin and vingristin. The leakage rate is vincristine and The vinblastine-loaded vesicles significantly altered the initial only 27% of the drug taken up by the patient is lost. This loss can be reduced using methylamine. associated with a corresponding decrease in the residual change in pH when measured at Kinin and When diphenhydramine and diphenhydramine are released from EPC vesicles, a similar profile of the proj. A significant decrease is observed.

The fourth category of drugs, physostigmine, codin and pilocarbin, It showed no measurable response to internal and external pH gradients. These agents create an ionic gradient. The suggestion that this would result in a large increase in membrane permeability leading to a loss of Not confirmed by data from stigmine (Figure 4). Physostigmine ( Under the conditions used to assess the uptake of 200 uM), the pH measurement Only a slight decrease in the change was observed.

Table 1 Degree of accumulation and stability of various drug vesicles showing pH gradient Uptake 15 minutes Uptake 2 time Midoxanrone Anti-tumor agent 200 198 Epirubicin Anti-tumor agent 201 200 Daunorubicin Anti-tumor agent 200 204 Doxorubicin Anti-tumor agent 202 203 Dibucaine Local anesthetic 194 176 Dopamine Biological amine 1901 177 Lidocaine Local anesthesia #J 87 87 Chlorpromazine Local anesthetic 98 96 Serotonin Biological amine 802 78 Chloroquine Anti Malaria drug 1043 88 Quinacrine Antiprotozoal drug 731 71 From left to left Quinidine Antiarrhythmic drug 203 74 Kinin Antimalarial drug 1483 81 Vinblastine Antitumor drug 1753 127 Vincristine Anti-tumor drug 178 130 Left second base 22nd Codin painkiller <1<1 Pilocarbin Cholinergic agonist <1 <1 Footnote = 1. Maximum uptake at 30 minutes, 2 ．． Maximum uptake in 90 minutes, maximum uptake in 3.5 minutes 1 mile above Comparison between the concentration of drug uptake and the drug's octatool/water partition coefficient Compare the concentration of drug uptake with the drug's octatool/water partition coefficient to determine the partition coefficient. and the possibility that the drug is distributed within the liposome bilayer membrane. I decided on a range that might determine the degree. From Table 2 below, drug uptake and There seems to be no clear relationship between the distribution coefficient of The numbers shown are Although they do not accurately reflect membrane/water partition coefficients, they are used merely for comparison. It's just that. For example, chlorpromazine and doxorubicin have different (98 vs. 202) but have the same partition coefficient. On the other hand, Chimo Even though their partition coefficients are significantly different, roll and glolbromazine , accumulates in vesicles to a similar extent. The partition coefficient for ionized drugs is may influence drug absorption, but may not be used to explain differences between the drugs studied. I can't.

Table 2 Comparison between the concentration of drug uptake and its octanol:water partition coefficient Maximum uptake logarithmic octatool: water Drug (nmol/umol lipid) Partition coefficient: Daunorubicin 200 3. 5 Doxorubicin 202 1.1 Vincristine 178 2.8 Glorpromazine 98 1゜5 Jibucain 194 4.4 Propranolol” 198 1.3 Timolol”95-0.1 Physostigmine OO32 Imibramine 182 4.6 Diphenhydramine 176 3.4 Footnotes = 1 - All data except propranolol and timolol are from A, Le et al., Chem, Rev., 71.525 (1971).

2-L, G., Merbate et al., Biophys, J., 49.91 ( +986) from.

11th 11th Domino and of and of and of. Besides buffer strength, the factor that most significantly influences the concentration of drug uptake is internal relaxation. It is the solubility of a protonated species in a buffer solution. Protonated drug in vesicles If the concentration of an agent exceeds its solubility product and precipitation occurs, this effectively reducing the membrane concentration gradient for the fraction, thus allowing further accumulation by vesicles. Ru.

For most of the drugs tested for proton gradient-dependent uptake, 300 mM citric acid The maximum apparent solubility in acid buffer, pH 5.0, is shown in Table 3. complete and stable take Midoxantrone, Evirubicin, Doxorubicin, Daunorubicin Drugs such as drugs are relatively insoluble in the intravesicular medium. This means that most savings The loaded drug is in the form of a precipitate, creating a concentration gradient of soluble protonated species. This shows that the uptake does not contribute to There is. Additionally, the precipitation of most intravesicular drugs leaves free material available for partition into the membrane. The concentration of the drug decreases accordingly, leading to the observed stability of the transmembrane proton gradient. will contribute. As expected, the Hender Sogy Hatselbach style and well Timolol, lidocaine, quinacrine, and chloroquine showing consistent uptake. Such agents have an apparent solubility that exceeds the vesicle white concentration achieved (see the table below). (See 3). Without being bound by any theory, the solubility data are can explain most of the observed differences in uptake properties.

The data indicate that solubility data is most important in determining drug incorporation into liposomes. It shows that it is important.

We also use dibucaine, propranolol and dopamine. accumulated within the liposomes in an amount significantly greater than predicted by the Lubatzha equation. Focus on what you can do.

This indicates that the apparent solubility of the three agents is less than the final internal concentration of that agent within the liposome. This is a surprising result considering that the difference is also large.

Apparent maximum drug solubility in 300mM citrate buffer, pH 5.0 Midoxantrone (0,01 Evilubicin 0.26 Daunorubicin 9.10 Doxorubicin 0.24 Propranolol Example 4 Isoproterenol, metabrolenol and lubricant of phosphate liposomes Rabbit phosph yf di) Lt choline (EPC) is Avanti Po1ar Lip c-methyl Amine was purchased from New England Nuclear. all other Chemicals and buffers were purchased from Sigma (St. Louis, MO). and used without purification.

Hope et al., Biochim, Biophys, Acta, 817.19 3 (1985) to produce lamellar vesicles (LUV) by extrusion. Ta. Briefly, frozen and thawed samples prepared in 300mM citrate, pH 4.0, The obtained lipid dispersion was transferred to an extrusion device (Lipex Biomembranes, V 0.1 or 0.2um polycarbonate using an ancouver (Canada). -By extruding it through a carbonate filter (Nucleopore), LU V was manufactured. 0. Vesicles prepared by this technique using a lum filter. is 1.5 uL/umol of phospholipid when measured using 4C or Na. It has an uptake capacity and has an average diameter of 90 nm. The phospholipid concentration was previously determined by Fi ske and Subbarrow, J., Biol, Chem., 66, 37 5 (1925) by determination of lipid phosphorus. Intramembrane The external pH gradient was set up according to Example 1, and the internal buffer that was not incorporated was External buffer [150mM NaCl, 20mM HEPES, pH 7, 4 Removed by passing LUV through an equilibrated 5ephadex G-50 column. was removed. The resulting pH gradient is as described by Hope et al., supra. This was determined by measuring the transmembrane distribution of 4C-methylamine. In short, the final Add methylamine to the vesicle system so that the concentration is 0.5 uCi/mL. I got it. At an appropriate time, remove a portion (100 uL) and add lmL as described above. It was passed through a 5ephadex G-50 mini column. The captured probe Using a Packard 2000C liquid scintillation counter, liquid Measured by scintillation counting. The transmembrane pH gradient is formula 9 formula %] Calculated by.

Bronchodilators, isoproterenol, metaproterenol and terbutaline, 150mM Nacl, containing 500uM bronchodilator and 6mM phospholipid; in 20mM HEPES, pH 7.4 buffer at the indicated temperature. were incubated with liposomes with a transmembrane pH gradient prepared as follows. .

Control samples with no transmembrane pH gradient were prepared at pH 4.0 or 7.4 (both inside and outside the vesicle). and the degree of gradient-independent membrane binding was determined. pH 4,0 pair The buffer consisted of vesicles prepared as described above, but the external buffer was 150mM NaCl, 20mM citrate, pH 4.0. Regarding the pH 7.4 control Both inside and outside, vesicles were prepared with 150mM NaCl, 20mM HEPESS pH 7°4. was prepared.

As described above, using a 1 m L 5 ephadex G-50 mini column. , free drug was separated from the vesicles and measured (photometrically). Figure 5 shows (EPC) Bronchodilator, isoprotease in pH gradient using 200 nm extruded liposomes The uptake of lenol, metaproterenol and terbutaline is shown. 300m Vesicles containing M citrate, pH 4, were incubated with a 500 uM drug solution at pH 7.4. Incubated with. Liposomes are higher than 60 nmol/umol lipids Allow the drug to accumulate in concentration. This is approximately 190% with an uptake efficiency of approximately 70%: Corresponds to the inner:outer drug concentration ratio of 1, presenting an internal volume of 2.2 uL/umol lipid. do. If all the drug is in the aqueous space of the vesicle, the internal drug concentration is approximately 30 Calculated as mM. Uptake is stable for more than 4 hours and is complete for several minutes. be. When there is no transmembrane pH gradient, pH 4.0 (inside and outside the vesicle) and pH 7.4 The background binding of these drugs for both (internal and external) was 15 nmol. 1/umo less than 1 lipid, and non-specific binding to lipids is rather than causing association with liposomes, the association is greater than the absolute pH. This shows that the effect is due to the proton gradient.

1 upright The appearance of the buttons made with methylmine is even more impressive! The methylamine probe quenches the internal proton pool at these concentrations. Therefore, the logarithm of the ratio of the internal and external concentrations of radioactive methylamine can be calculated as It can be used to measure changes. Internal and external pH 7.4 or 4.0 (no gradient), methylamine does not detect any gradient (Figure 6). internal If the cells were incubated at a pH of 7.4, the methyl acetate The min distribution shows a pH slope of 3.0 units, which is in good agreement with the slope of 3.4 units. most Metaproterenol was added to the external buffer to a final concentration of 500 uM. As drug accumulates, the gradient disappears to about 2.3 pH units. This data is The 190-fold achieved by the drug is close to the residual proton gradient (a few units of pH). It shows that they are similar.

1 upright The rate of uptake of metaproterenol in 1. The temperature increases as the temperature rises (see Figure 7), and reaches a steady level after 2 hours at 11'C. However, when incubated at 60°C, it is faster than 15 minutes. Drug uptake The extent of this is not dramatically affected by the temperature of incubation.

31 giant canter of C-cholesterol Incorporation of cholesterol into liposomes during bronchodilator uptake was investigated. Beta. Cholesterol is determined by the rate of uptake and the uptake per umol of lipid. and the total amount of drug administered.

EPC cholesterol (5545 mol) measured using C inulin and Na %) is approximately 30% lower than in vesicles consisting of EPC alone. Therefore, the actual concentration gradient of metaproterenol achieved is similar in both cases. be. See Figure 8.

1151 The amount of drug taken up in the ratio of It depends on the ratio of drug to quality (see Figure 9). When the ratio of drug to lipid is low is a transmembrane pH gradient in which the amount of drug taken up by the vesicle is greater than 3 units. resulting in the generation of At sufficiently high drug concentrations, re-ionization of the drug inside the vesicles is possible. oxidation lowers the internal pH (Figure 6). Therefore, the ionized drug Internal buffering capacity is overcome so that the equilibrium level of drug uptake is greater than the initial change in pH. It would be expected to result in a final change in pH.

1 Hogoko 6 on the number The degree of buffering capacity (buffer strength) is the ability of liposomes to take up ionized drugs. It is a factor that affects Low internal buffering capacity affects the extent of drug accumulation.

Below 100mM citrate, the extent of drug accumulation actually depends on the internal citrate concentration. (Figure 10). Under these conditions, reionization of the drug is Overcomes the internal buffering capacity inside the vesicle, increases the pH of the vesicle, and eliminates pH changes ( (insert graph). In these cases, there is still a large residual pH gradient and the internal buffering capacity is To not limit drug uptake, vesicles were loaded with citrate at concentrations higher than 300mM. Inclusion does not dramatically increase the concentration of drug incorporated.

The foregoing description and examples are illustrative of practicing the invention and are in no way limiting. It will be understood by those skilled in the art that this is not the case. The details shown here are Changes may be made without departing from the spirit and scope of the invention.

Figure 1 Incubation time (min) ・-pH within 4.0/pH outside of 7.5 〇-pH within 7.5/pH outside of 7.5 △-pH within 4,0/pHa, outside hours minutes Ill 2B Quinacrine Incubation time (min) ・-pH within 4.0/pH outside of 7.5 0-p) within 17.5/pH outside 7.5 △-pH4.0 inside/pH4.0 outside Times 3 Figure 4 - EPC vesicles Q-100 uM physostinium incubated in the presence of drug EPC vesicles incubated with gamine - incubated with 200 uM physostigmine Incubated EPC vesicles Figure 5 hours minutes Terbutaline Δ incubated in the presence of a mu pH gradient - absence of a pH gradient Incubated in the presence of terbutaline-pH gradient Metaproterenol incubated in the absence of a 0-pH gradient Taproteretool - isoproterenoincubated in the presence of a pH gradient Isoproterenol incubated in the absence of a Roullow pH gradient Figure 6 ・-pH gradient, no drugs 0-pH gradient, drug present △-control without pH gradient) Figure 7 ・At -60℃ Low at 37℃ at -21℃ Figure 8 ・-No cholesterol ■-Cholesterol tomorrow Figure 9 Drug concentration (mM) Figure 10 Citrate concentration (mM) international search report

Claims (42)

[Claims] 1. Local anesthetics, bronchodilators, beta-adrenergic blockers, antihypertensives, Depressants, anticonvulsants, antiemetics, antihistamines, antiarrhythmics, antimalarials and a transmembrane ionic gradient comprising at least one drug selected from the group consisting of analgesics and analgesics; a ribosome having at least one lipid; a first internal buffer aqueous solution; and a second external buffer aqueous solution, wherein the medicament has a concentration of the medicament in the internal buffer. drug with a lower solubility than would be predicted from the transmembrane ion gradient. A liposome composition, wherein the liposome composition is produced in the liposome. 2. 2. The composition of claim 1, wherein said ionic gradient is a pH gradient. 3. The drug has a solubility in the internal buffer of less than about 20 mM, and the drug has a solubility in the internal buffer of less than about 0. The composition according to claim 1, having a solubility in the external buffer of 2mM or more. 4. Claim 3, wherein said medicament has a solubility in said internal buffer of less than about 10 mM. composition. 5. The drug is dopamine, dibucaine, chlorpromazine, lidocaine, serotonin. Nin, quinacrine, metaproterenol, terbutaline, isoproterenol, selected from the group consisting of quinidine, quinine, diphenhydramine and chloroquine The composition according to claim 2. 6. If the drug contains dibucaine, dopamine, quinidine, imipramine and diphenhyde 4. The composition according to claim 3, wherein the composition is selected from the group consisting of doramine. 7. The lipids include phosphatidylcholine, phosphatidylserine, phosphatidyl Inositol, sphingomyelin, cardiolipin and phosphatidyl ethano Phosphatide for phosphatidylcholine and phosphatidylcholine From a mixture in which the weight ratio of diethanolamine is about 30:70 to about 45:55. The composition according to claim 2, which is selected from the group consisting of: 8. The liposome composition according to claim 1, which is dehydrated. 9. The liposome composition according to claim 1 and a pharmacologically acceptable carrier or diluted A pharmaceutical composition comprising a diluent. 10. The group consisting of metaproterenol, terbutaline and isoproterenol a liposome having a transmembrane pH gradient containing at least one drug selected from; at least one lipid; and at least one aqueous buffer solution, wherein the medicament has an uptake efficiency of about 70%. accumulated within the liposome in an amount equal to . 11. The lipids include phosphatidylcholine, phosphatidylserine, phosphatidyl Luinositol, sphingomyelin, cardiolipin and phosphatidyletha Phosphate for phosphatidylcholine of nolamine and phosphatidylcholine A mixture having a weight ratio of tidylethanolamine of about 30:70 to about 45:55. 11. The composition according to claim 10, which is selected from the group consisting of: 12. 11. The composition according to claim 10, wherein the lipid is phosphatidylcholine. 13. The buffer solution is a buffer solution combination containing an internal buffer aqueous solution and an external buffer aqueous solution. 11. The composition according to claim 10, which is 14. The first buffer consists of citric acid, oxalic acid, succinic acid, and an organic acid salt. selected from one or more groups, wherein the second buffer is sodium chloride, potassium chloride, potassium phosphate, sodium bicarbonate, sodium carbonate, sodium bisphosphate selected from the group consisting of potassium phosphate, sodium phosphate, potassium sulfate and HEPES. The composition according to claim 10. 15. The buffer includes a first internal buffer comprising a citrate buffer, NaCl and H and a second external buffer containing a mixture of EPES. The composition according to claim 10. 16. The first internal buffer has a concentration ranging from about 100 mM to about 300 mM. a citrate buffer solution, the second buffer solution containing NaCl and HEPES; the concentration of NaCl is in the range of about 100 to about 400 mM; 16. The composition of claim 15, wherein the amount is in the range of about 10mM to about 30mM. 17. The liposome composition according to claim 10, which is dehydrated. 18. The liposome composition according to claim 10 and a pharmacologically acceptable carrier or is a pharmaceutical composition containing a diluent. 19. Kinin, diphenhydramine and A transmembrane ion containing at least one drug selected from the group consisting of quinidine. liposomes with a gradient; after accumulation, the drug is rapidly released from the liposomes; at least one lipid that prevents and a liposome composition containing an aqueous buffer solution. 20. 20. The composition of claim 19, wherein said ionic gradient is a pH gradient. 21. The lipids include phosphatidylcholine, phosphatidylserine, phosphatidyl Luinositol, sphingomyelin, cardiolipin and phosphatidyletha Phosphatide for phosphatidylcholine of nolamine and phosphatidylcholine A mixture having a weight ratio of tidylethanolamine of about 30:70 to about 45:55. Contains at least one first lipid selected from the group consisting of: and a membrane stabilizing lipid component. Claim 19 is a lipid combination consisting of at least one second lipid that Compositions as described. 22. 22. The composition of claim 21, wherein the membrane stabilizing component is cholesterol. 23. The first lipid is phosphatidylcholine, and the second lipid is cholesterol. The weight ratio of phosphatidylcholine to cholesterol is about 70. 23. The composition of claim 22, wherein the composition is from: 30:30 to about 55:45. 24. The weight ratio of phosphatidylcholine to cholesterol is about 55:45. 24. The composition according to claim 23. 25. 20. The liposome composition of claim 19, which is dehydrated. 26. The liposome composition according to claim 19 and a pharmacologically acceptable carrier or is a pharmaceutical composition containing a diluent. 27. After accumulation, quinine, diphenhydramine and a transmembrane ionic gradient comprising at least one drug selected from the group consisting of quinidine; liposomes having the following properties; preventing the drug from being rapidly released from the liposomes after accumulation; at least one lipid; a first internal buffer aqueous solution; and a second external buffer aqueous solution, wherein the medicament has a concentration of the medicament in the internal buffer. drug with a lower solubility than would be predicted from the transmembrane ion gradient. A liposome composition that produces within the liposome. 28. 28. The composition of claim 27, wherein the ionic gradient is a pH gradient. 29. The lipids include phosphatidylcholine, phosphatidylserine, phosphatidyl Luinositol, sphingomyelin, cardiolipin and phosphatidyletha Phosphatide for phosphatidylcholine of nolamine and phosphatidylcholine A mixture having a weight ratio of tidylethanolamine of about 30:70 to about 45:55. at least one lipid selected from the group consisting of: and at least one membrane stabilizing lipid. 28. The composition according to claim 27, which is a combination of lipids containing the ingredients. 30. 29. The composition of claim 28, wherein the membrane stabilizing lipid component is cholesterol. . 31. The first lipid is phosphatidylcholine, and the membrane stabilizing lipid component is a collector. It is a sterol, and the weight ratio of phosphatidylcholine to cholesterol is 29. The composition of claim 28, wherein the ratio is about 70:30 to about 55:45. 32. The weight ratio of phosphatidylcholine to cholesterol is about 55:45. The composition according to claim 29. 33. the drug has a solubility in the internal buffer of less than about 20 mM; ．． 28. The composition of claim 27, having a solubility in said external buffer of 2mM or more. 34. Claim 2 wherein said medicament has a solubility in said internal buffer of less than about 10 mM. 7. The composition according to 7. 35. 27. The internal buffer has a buffer strength of at least about 50 mM. Compositions as described. 36. The internal buffer has a buffer strength ranging from about 100 to about 300 mM. The composition according to claim 27. 37. 28. The set of claim 27, wherein said internal buffer has a buffer strength of about 300mM. A product. 38. 28. The liposome composition according to claim 27, which is dehydrated. 39. The liposome composition according to claim 27 and a pharmacologically acceptable carrier or is a pharmaceutical composition containing a diluent. 40. A small amount selected from the group consisting of dibucaine, propranolol and dopamine. Liposomes with transmembrane ion gradients containing at least one drug; at least one lipid; a first internal buffer aqueous solution; and a second external buffer aqueous solution, wherein the drug is predicted from the transmembrane ion gradient. A liposome composition that accumulates within the liposome in a larger amount than that of the liposome. 41. The liposome according to claim 40, wherein the lipid is egg phosphatidylcholine. Composition. 42. The buffer contains citric acid at a concentration between about 100 and about 300 mM. 42. The liposome composition according to claim 41, which is a buffer solution.