EP0256119A1 - Systeme d'administration liposomique a liberation entretenue - Google Patents

Systeme d'administration liposomique a liberation entretenue

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Publication number
EP0256119A1
EP0256119A1 EP87901859A EP87901859A EP0256119A1 EP 0256119 A1 EP0256119 A1 EP 0256119A1 EP 87901859 A EP87901859 A EP 87901859A EP 87901859 A EP87901859 A EP 87901859A EP 0256119 A1 EP0256119 A1 EP 0256119A1
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EP
European Patent Office
Prior art keywords
liposomes
liposome
compound
composition
site
Prior art date
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EP87901859A
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German (de)
English (en)
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EP0256119A4 (fr
Inventor
Annie Yau-Young
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Liposome Technology Inc
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Liposome Technology Inc
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Publication of EP0256119A1 publication Critical patent/EP0256119A1/fr
Publication of EP0256119A4 publication Critical patent/EP0256119A4/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes

Definitions

  • the present invention relates to a liposome-based system and method for delivering a pharmacologically active compound to the bloodstream at a controlled rate.
  • Liposorae delivery systems have been proposed for a variety of pharmacologically active compounds. such as drugs and peptide hormones.
  • liposomes For compounds which are administered parenterally. liposomes have the potential of providing controlled "depot” release over an extended time period, and of reducing toxic side effects, by limiting the plasma peak level of the free compound in the bloodstream.
  • liposomes administered by this route are generally cleared by the reticuloendothelial system (RES), and as a consequence, the liposomes tend to concentrate in organs, such as liver, spleen, and lung, which are rich in RES cells.
  • RES reticuloendothelial system
  • the ability to direct liposomes somewhat specifically to RES-rich tissue is advantageous, for example, in treating diseases of the liver, spleen, or lungs. This approach is described, for example, in U.S. Patent Application for "Liposome/Anthraquinone Drug Composition and Method.” Serial No. 806.084. filed 6 December 1985.
  • Intramuscular (IM) or subcutaneous (SQ) administration of liposome-entrapped compounds have also been proposed.
  • This approach has the advantage that, as long as the liposomes are contained at the site of injection, rapid uptake and clearance by the RES cannot occur.
  • the liposomes immobilized at the site of injection can then release the entrapped compound into the bloodstream over an extended period.
  • U.K. Patent Application No. 2.050.287 describes a liposome system which is intended for slow release of insulin from an SQ injection site. More recently, a system for releasing liposome-encapsulated calcitonin from an IM site of administration has been proposed (Fukunaga) .
  • the invention includes a method for selectively increasing the rate of release of a liposome-impermeable compound into the bloodstream.
  • the compound is administered by forming a suspension of liposomes which contain the compound in entrapped form, and injecting the suspension into an IM or SQ site.
  • the rate of release of the agent from the site is increased selectively by increasing the average size of the liposomes and the total amount of Iiposome lipid injected into the site.
  • the encapsulated agent may be a peptide. such as insulin, growth hormone, or calcitonin, interferon. or interleukin-2, which is advantageously released into the bloodstream at a controlled rate over a several day period.
  • the rate of release of the liposome-encapsu- lated agent can also be controlled by changes in the lipid composition of the liposomes.
  • Negatively charged phospholipids such as phosphatidylglycerol (PG) . act to decrease the release rate, and cholesterol, to increase the rate.
  • PG phosphatidylglycerol
  • the liposomes are prepared by first forming a suspension of small. compound-encapsulating liposomes, removing the non-encapsulated compound from the suspension, filter sterilizing the suspension, then adding sterilely prepared empty liposomes to the suspension until the desired Iiposome average size and concentration are reached.
  • the small liposomes are preferably less than about 0.3 microns in size, and the empty liposomes. 0.5 micron or more.
  • the invention also includes a Iiposome composition for administering a liposome-impermeable compound to the bloodstream from an IM or SQ site of injection.
  • the composition is composed of an aqueous suspension of liposomes containing the substance in entrapped form, and having average particle sizes less than about 0.3 microns, and a quantity of empty liposomes. in an amount effective to increase the half life of clearance of the substance from such injection site to a desired half life between about 1-14 days.
  • the size, lipid composition, and relative quantity of empty liposomes are selected to produce a desired rate of release of the encapsulated agent from the site of i jection.
  • the invention includes a liposome/calcitonin (CT) composition
  • CT calcium calcitonin
  • the composition may be further stabilized by the presence of ferrioxamine. in molar excess of the amount of ferric iron in the suspension.
  • Figure 1 is a flow diagram of a processing method used in preparing a Iiposome composition containing both empty and peptide-encapsulating lipids;
  • Figure 2 shows the kinetics of release of a Iiposome tracer lipid from the site of an IM injection (circles) and the accumulation of tracer (triangles) excreted from the injected animal;
  • Figure 3 is a semi-log plot of the tracer release data from Figure 2 (dotted line) and analogous tracer release data from Iiposome composition with smaller Iiposome sizes (solid line);
  • Figure 4 shows the kinetics of release of encapsulated radioactive calcitonin (CT) from the site of an IM injection (circles), the accumulation of hormone (triangles) excreted from the animal, and release of free CT from the site of injection (squares); and
  • Figure 5 is a semi-log plot of CT release data from Figure 4.
  • the liposomes in the composition are formed from standard vesicle-forming lipids. which generally include neutral and negatively charged phospholipids and a sterol. such as cholesterol.
  • the selection of lipids is generally guided by considerations of (a) desired liposome size and ease of liposome sizing, and (b) lipid and hormone release rates from the site of liposome injection.
  • the major lipid component in the liposomes is phosphatidylcholine (PC).
  • PCs having a variety of acyl chain groups of varying chain length and degree of saturation are available, or may be isolated or synthesized by well-known technigues.
  • acyl chain composition of the phospholipid may also affect the rate of clearance of liposome lipids and entrapped compound from the site injection, although acyl-chain saturation effects appear to have less of an effect on drug release rates than when liposomes are administered intravenously.
  • One preferred PC is egg PC (EPC) which is derived from egg lipids. and contains a mixture of both saturated and unsaturated acyl chain groups.
  • the lipophilic free radical scavenger used in the composition is preferably ⁇ -T, or a pharmacologically acceptable analog or ester thereof, such as ⁇ -T succinate.
  • suitable free radical scavengers include butylated hydroxytoluene (BHT) , propyl gallate (Augustin). and their pharmacologically acceptable salts and analogs.
  • BHT butylated hydroxytoluene
  • Additional lipophilic free radical quenchers which are acceptable for parenteral administration in humans, at an effective level in liposomes. may also be used.
  • the free radical quencher is typically included in the lipid components used in preparing the liposomes, according to conventional procedures.
  • Preferred concentrations of the protective compound are between about 0.2 and 2 mole percent of the total lipid components making up the liposomes; however, higher levels of the compound, par icularly ⁇ -T or its succinate analog, are compatible with liposome stability and are pharmacologically acceptable.
  • the water soluble iron-specific chelating agent is selected from the class of natural and synthetic trihydroxamic acids and characterized by a very high binding constant for ferric iron (on the order of
  • 2-valence cations such as calcium and magnesium.
  • a variety of trihydroxamic acids of natural origin are known, including compounds in the ferrichrome class. such as ferrichrome, ferrichrome A, and albomycin; compounds in the ferrioxamine class, including the ferrioxamines and ferriomycines; and compounds in the fusaramine class.
  • ferrioxamine B also known variously as ferrioxamine, desferrioxamine, desferrioxamine B, and DesferalTM 1 . This compound shows exceptional iron binding affinity and has been proven safe for parenteral use in humans in treating iron-storage disease and iron-poisoning.
  • the chelating agent is present in the composition at a concentration which is in molar excess of the ferric iron in the liposome suspension.
  • aqueous media used in liposome preparation contains at least about 1-2 uM ferric iron, and may contain up to 100 yM or more ferric iron.
  • concentrations of chelating agent of about 50 ⁇ M are preferred.
  • the chelating agent is preferably added to vesicle-forming lipids at the time of liposome formation, so that the lipids are protected against drug-promoted lipid oxidation damage during liposome preparation.
  • Methods for preparing liposomes by addition of an aqueous solution of chelating agent are described below.
  • the liposome suspension formed by this method contains chelating agent both in the bulk aqueous phase and in the encapsulated form, i.e., within the aqueous internal liposome region.
  • the chelating agent may be included in the suspension after liposome formation.
  • the compound entrapped in the liposomes is a liposome-impermeable drug or peptide whose rate of diffusion out of liposomes is not significantly greater than the rate of breakdown of liposomes at an IM site of injection.
  • the agent may be either a lipophilic drug or hormone whose oil/water partitioning strongly favors the liposome bilayer phase, or a water-soluble drug or peptide which is capable of diffusing across the liposomal bilayer slowly, if at all.
  • water-soluble drugs which can freely diffuse out of liposomes with a half life of less than a few hours.
  • Peptide hormones and immunological activators are one important class of compounds for use in the invention.
  • Representative peptide hormones include insulin, growth hormone, and calcitonin (CT), which regulates calcium blood levels.
  • CT calcitonin
  • Interferon and interleukin-2 are representative of immunological activators.
  • THe present invention allows the selected peptide compound to be released into the bloodstream at a slow, controlled rate over a several hour to several day period, thus avoiding the large fluctuations in blood peptide levels that are characteristic of free hormone administration.
  • the carrier liposome may significantly enhance the stability of the peptide on storage, when the peptide is present at a relatively low concentration.
  • the study reported in Example II shows that the stability of CT in free form is enhanced severalfold when the CT concentration is raised from 0.010 to 1.0 mg/ml.
  • the invention allows peptide hormones such as CT which are more stable at high concentration to be stored and delivered in a relatively dilute form in which the high-concentration microenvironment of the hormone promotes good stability on storage.
  • peptide hormones such as CT which are more stable at high concentration
  • the preparation of liposomes which encapsulate water-soluble peptides, such as CT. at a selected internal volume concentration are considered below.
  • Steroid hormones and anti-inflammatory agents are another important class of compounds which are useful in the present invention.
  • Representative steroids include hydrocortisone, estradiol, and testosterone.
  • Liposomes containing entrapped steroids are readily formulated by including the compounds in vesicle-forming lipids. The rate of release of the steroids from the site of IM or SQ injections would be controlled by the partition function of the drug, as well as by liposome stability in and migration from the site of injection.
  • antibiotics such as as amphoteracin B
  • immunosuppressives such as cyclosporin.
  • anti-tumor agents such as doxorubicin.
  • One feature of the present invention which is particularly advantageous for antibiotic or anti-tumor drug delivery is the ability to select the site of drug release and highest drug concentration.
  • vesicle-forming lipids including a lipophilic free radical protecting agent, and if suitable, a lipophilic drug compound, are dissolved in a suitable organic solvent or solvent system and dried under vacuum or an inert gas to form a thin lipid film.
  • the film may be redissolved in a suitable solvent, such as tertiary butanol, and then lyophilized to form a more homogeneous lipid mixture which is in a more easily hydrated powder-like form.
  • a suitable solvent such as tertiary butanol
  • This film is covered with hydration medium and allowed to hydrate, typically over a 15-60 minute period with agitation.
  • the size distribution of the resulting MLVs can be shifted toward smaller sizes by hydrating the lipids under more vigorous agitation conditions.
  • the compounds is dissolved in the hydration medium at a concentration which is desired in the interior volume of the liposomes in the final liposome suspension.
  • the hydration medium would contain 1 mg/ml CT.
  • the hydration medium may also be prepared to include an iron-specific chelater. at a preferred concentration of between about 10-50 mM.
  • Example I describes a method of producing a suspension of MLVs encapsulating CT. Another advantageous method of producing liposomes is the reverse-phase evaporation method described by Szoka (1978).
  • a solution of vesicle-forming lipids in an organic solvent or solvent system is added to an aqueous solution of the material to be encapsulated, at relative volume amounts which are compatible with a water-in-oil emulsion.
  • the mixture is then emulsified, and the organic phase removed to produce a reverse-phase lipid gel composed of lipid monolayer structures encapsulating aqueous droplets.
  • This gel when resuspended in an aqueous solution, forms a suspension of relatively large oligolamellar vesicles, commonly referred to as reverse-evaporation vesicles (REVs).
  • REVs reverse-evaporation vesicles
  • the method produces high encapsulation efficiencies. typically between about 30-40% of the total water-soluble material added, and is thus useful for encapsulating expensive drug or peptide compounds, such as peptide hormones.
  • liposome preparation is preferably carried out under conditions which lead to a sterile liposome suspension. This is accomplished by employing conventional sterile techniques throughout the procedure.
  • the method of choice for liposome sterilization and the only method available for sterilizing liposomes containing heat-sensitive encapsulated material, is by filtration through a conventional depth filter, typically a 0.22 micron filter. This method can be carried out on a practical. high through-put basis only if the liposomes have first been sized down to about 0.2-0.3 microns or less.
  • Extrusion of liposome through a small-pore polycarbonate membrane is an effective method for reducing liposome sizes down to a relatively well-defined size distribution (Szoka 1978).
  • the suspension is cycled through the membrane several times until the desired liposome size distribution is achieved.
  • the liposomes may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in liposome size.
  • the maximum encapsulation efficiency for a water-soluble compound is about 50%. so that the initial liposome suspension will contain 50% or more of the compound in free (non-entrapped) form. The amount of non-encapsulated material is still higher in a liposome suspension formed by the above MLV method, where encapsulation efficiencies between about 10-20% are typical.
  • the studies reported in Example V show that a free (non-encapsulated) peptide is cleared from the site of an IM injection in less than an hour, as compared with several hours to several days for liposomes encapsulated compound.
  • the liposomes in the suspension are pelleted by high-speed centrifugation. leaving free compound and very small liposomes in the supernatant.
  • Another method involves concentrating the suspension by ultrafiltration. the resuspending the concentrated liposomes in a drug-free replacement medium.
  • gel filtration can be used to separate larger liposome particles from solute molecules.
  • Yet another approach for removing free compound utilizes ion-exchange, molecular sieve, or affinity chromatography.
  • the liposome suspension is passed through a column containing a resin capable of binding the compound in free, but not entrapped, form, or a support having attached binding molecules, such as antibodies, which bind specifically to the non-encapsulated compound.
  • the approach may also be effective in removing free pyrogens, with proper selection of resin(s).
  • the liposome suspension is brought to a desired concentration for use in IM or SQ administration. This may involve resuspending the liposomes in a suitable volume of injection medium, where the liposomes have been concentrated, for example by centrifugation or ultrafiltration. or concentrating the suspension, where the drug removal step has increased total suspension volume. The suspension is then sterilized by filtration as described above.
  • FIG. 1 is a flow diagram of a liposorae- processing scheme suitable for preparing the liposome composition of the invention.
  • THe flow scheme at the left in the figure shows steps in forming a sterile suspension of liposomes which have an entrapped compound.
  • THese steps which have been detailed above. involve first preparaing a suspension of liposomes containing the compound at a selected entrapped concentration. The suspension is then sized down to 0.2-0.3 or less, to allow for eventual sterile filtration. After removing non-entrapped drug, the material is brought to a desired lipid concentration and sterilized by filtration.
  • the righthand column in Figure 1 illustrates parallel steps used in preparing empty liposomes.
  • the rationale for preparing empty liposomes is based on the discovery herein that the release rate of liposome- encapsulated material from an IM or SQ injection site can be selectively varied by changing (a) the lipid composition, (b) average liposome size, or (c) total lipid amount of the injected liposomes.
  • the relationship between these parameters and compound release rate will be detailed in Section II below, and in Example III-VI. At this point, it is sufficient to note that these effects are achieved even when a large portion of the liposomes injected into the site do not contain entrapped compound. That is.
  • empty liposomes as a method of varying lipid composition, size, and amount, is that they can be prepared in sterile form without the need of a final sterile filtration step. It will be recalled that the sterility of a liposome preparation is compromised primarily because of the need for remove free compound from the suspension. By forming liposomes in the absence of the compound, or in the presence of an amount which does not produce an appreciable amount of free compound, the final sterile filtration step can be avoided.
  • An important advantage here is that since the empty liposomes do not have to be sized down for filtration, relatively large liposomes can be added. allowing for greater size-related effects in compound-release rate to be achieved.
  • the empty liposomes may be treated, if desired, to remove smaller liposomes. This can be done, for example, by allowing larger liposomes to settle in a vessel, and aseptically removing non-settled material.
  • the liposomes may be added directly to the filter-sterilized liposomes, without further treatment, to produce a final composition having a selected average lipid composition, size, and concentration, or the liposomes may be sized, such as by extrusion through a 1 micron polycarbonate membrane under sterile conditions, before addition to the filter-sterilized liposomes.
  • the processing method described above thus allows for the preparation of a sterile liposome composition having (a) a selected concentration of entrapped compound, (b) little or no free compound, and (c) an average liposome size, concentration and composition which allows the entrapped compound to be released from an injection site at a selected release rate.
  • One advantageous feature of the present invention is the increased stability of a pharmacological compound, such as a peptide hormone, which is achieved with liposome encapsulation.
  • a pharmacological compound such as a peptide hormone
  • liposome encapsulation allows the compound to be stored in stable form in a suspension in which the compound has a high localized concentration, but at relatively low overall concentration.
  • the study reported in Example II shows that free CT is substantially more stable at 1 mg/ml than at 0.01 mg/ml. even in the presence of a carrier protein.
  • the peptide shows the stability characteristics of the high concentration of CT.
  • composition stability is enhanced by including a lipophilic free radical quencher, such as ⁇ -T, at a mole ratio of at least about 0.2. Further. composition stability may be achieved by addition of an iron-specific chelator. such as ferrioxamine, in molar excess of the amount of free ferric iron in the liposome suspension.
  • the CT/liposomes can be sized, treated to remove free CT. and sterile filtered, as above.
  • the resulting composition includes (a) a sterile, aqueous suspension of liposomes containing at least about 0.2 mole percent ⁇ -T. and (b) calcitonin entrapped in the liposomes at a concentration of at least about 1 mg/ml.
  • the concentration of free CT is preferably less than about 10 mole percent of the encapsulated peptide.
  • I-PE I-labeled phosphatidylethanolamine
  • FIG. 4 shows plots of the loss of tracer from the sites of injection (circles) and accumulation of excreted tracer (triangles) from a typical experiment.
  • the linear relationship between the log of CT retained and time indicates that lipid is cleared from the site of injection with first order kinetics. Also shown in the figure is the rapid clearance of free CT from the sites of injection into the bloodstream.
  • Example IV The effect of liposome size on lipid and CT clearance rates is apparent from the data in Example IV comparing liposomes with sizes ranging from 0.2 microns to about 5 microns, with larger liposomes showing 60-100% longer lipid clearance rates.
  • the data in Example VI show that the clearance of lipid tracer from small liposomes (about 0.2 microns) can be increased by addition to the injected liposomes of larger unlabeled liposomes (about 1 micron).
  • the latter results indicate that lipid clearance is governed by bulk effects related to average liposome sizes, and forms the basis, according to one aspect of the invention, of controlling release characteristics of smaller liposomes by the addition of larger, empty ones.
  • Example IV The effect of liposome dose on lipid clearance is also reported in Example IV.
  • lipid clearance half life can be increased more than twofold with increased lipid dose, independent of lipid size or composition.
  • the same twofold increase in clearance rate with increased dose is seen for encapsulated CT (Example V) .
  • a comparison of lipid and CT release rates from liposomes having the same size and dose properties shows that CT clearance from the site of injection is about twice as fast as that of the corresponding lipid tracer. This finding suggests that liposomes are destabilized and release their encapsulated contents predominantly at the site of injection, with lipid clearance from the site being handled by a different, slower mechanism.
  • lipid composition on lipid and CT clearance from an IM site was also examined.
  • addition to the liposomes of a negatively charged phospholipid, such as PG significantly increases the rate of clearance of lipid and encapsulated CT from an IM site.
  • the PG effect may be related in part to the reduced liposome aggregation which is seen in PG-containing liposomes.
  • Cholesterol at a mole ratio of about 40%. had little effect on lipid clearance from PG-containing liposomes. but showed a significant stabilizing effect — that is. longer clearance half life -- on CT release from neutral, PC liposomes.
  • CT was cleared about twice as rapidly as lipid tracer from liposomes having the same size and lipid composition properties, confirming that liposome lipid is cleared from the injection site by a different, slower mechanism than that acting on the encapsulated compound.
  • the results above show that release of an encapsulated compound from liposomes at an injection site can be controlled selectively by changes in liposome size, dose, and lipid composition, specifically, in practicing the method of the invention.
  • the rate of release of an encapsulated compound from an injection site is controlled according to an average size and total amount of liposome injected into the site.
  • average size and lipid amount are selectively increased by adding larger, empty liposomes to smaller, filter sterilized liposome encapsulating the compound of interest.
  • the liposomes can be formulated to include progressively more negatively charged lipid, such as PG.
  • the liposome composition of the invention is useful for administering a variety of liposome-impermeable compounds parenterally.
  • One important application is for use in administering a peptide hormone or immunostimulator to bloodstream in a controlled fashion over a several day period.
  • the composition allows the half life of peptide release to be selectively varied, to provide release for a selected period of up to several days.
  • the peptide can then be given less often and without the sharp fluctuations seen when free peptide injections are used. Further, a greater degree of control can be achieved than with liposome formulations which have been proposed heretofore.
  • Insulin and CT are examples of peptides which are now routinely administered in free form. Both of these peptides are readily incorporated into the liposome composition of the invention, and both can be delivered over a several day period by IM injection of the composition. The rate of hormone delivery is controlled, according to the method of the invention, by use of selected average liposome size, amount, and composition. For some peptides, such as CT, an added benefit of the liposome composition is the increased stability which is achieved, allowing the material to be stored in relatively dilute form over an extended period.
  • EPC Egg phosphatidylcholine
  • ⁇ -T ⁇ -tocopherol
  • 125 Pharmaceuticals. Kankakee, IL. and sCT was T- radiolabeled with 125I by the chloramme T method
  • Multilamellar vesicles containing one of the lipid compositions A-E indicated in Table I were prepared.
  • the molar ratios of the lipid components in the four vesicle preparations are shown in Table I.
  • the values in the table indicate the ⁇ molar ratios of each lipid component that were used in forming the various vesicle preparations.
  • the vesicle-forming lipids also included a radioactive iodinated derivative of
  • I-PE 125 phosphatidylethanol- amme
  • the iodinated lipid was incorporated at 1 x 10 cpm per injection in the range of 0.2 to 10 ⁇ mole total lipid.
  • the lipid components in chloroform stock solution were mixed in a tube or round bottom flask.
  • the chloroform was removed by rotaevaporation and the lipid mixture was dissolved in tert-butanol.
  • the butanol solution was then frozen in dry-ice/acetone and lyophilized overnight.
  • the dry lipids were hydrated in several ml of phosphate-buffered saline (PBS). pH 7.4
  • the hydration buffer contained 0.2-5 mg/ml of
  • the lipid film was hydrated with vortexing for about 15 minutes at room temperature to form an MLV suspension having heterogeneous sizes ranging from about
  • the vesicle preparations were sized by extrusion through a polycarbonate membrane having selected pore sizes. The entire preparation was extruded through a 1.0 micron polycarbonate membrane, producing vesicles which have an initial vesicle size (before aggregation) of about 1 micron. In forming smaller-size vesicles, the sized vesicles were further extruded successively through 0.4 and 0.2 micron pore size membranes, to produce vesicles with sizes initially in the 0.2 micron size range. MLVs containing encapsulated sCT were freed of non-liposome-associated free sCT by three washes with PBS. The formulations were tested for pyrogen by the Limulus Amoebic Lysate assay (Haemachem. Inc.. St. Louis, MO).
  • the stability of the F-sCT and L-sCT preparations was tested after incubating the vials at either 4°C, room temperature (about 24°C), and 37°C, and assaying the biological activity of the sCT after a given period of incubation.
  • the samples were lysed in 0.5% Triton-X, obtained from Sigma (St. Louis. MO), and diluted to 40 and 120 mU per ml of PBS containing 0.5% BSA. An aliquot of frozen standard F-sCT was similarly treated.
  • Each sample was tested in eight rats, with four rats receiving 10 mU of sCT (0.25 ml of the 40 mU/ml solution), and four rats receiving 30 mU (0.25 ml of the 120 mU/ml solution), administered subcutaneously.
  • Control rats received 0.25 ml of PBS/BSA.
  • blood samples were drawn from each rat. The blood samples were allowed to clot, and serum samples collected by centrifugation. The calcium levels in the serum were determined by a dye binding assay, using a kit supplied by Sigma Chemical Co. (St. Louis, MO).
  • the hypocalcemic activity of the samples, 60 minutes after injection, was determined from a standard curve. The results, expressed as the average of four rats for each data value, are given in Table II.
  • Example III Lipid Clearance From the Site of IM Injection
  • the study reported here examines the rate of clearance of liposome lipids. as measured by a radioactive lipid tracer, from the site of intramuscular (IM) injection in laboratory rats.
  • the animals were injected with MLVs hawing one of the lipid compositions A-D from Example I, at a selected dose -of 0.2. 2, or 10 ⁇ mole lipid.
  • mice Male Spragu ⁇ -Dawley rats weighing 95-110 g were lightly anesthetized with ether, and 20 ⁇ l containing 0.2 ⁇ mole lipid was injected into the forelimbs or 100 ⁇ l containing 2 to 10 ⁇ mole lipid was injected into the hindlimbs of eactt. rat. Sixteen rats were used in each experiment. Two of the rats were placed in metabolic cages to monitor the excretion of radiolabel in urine and feces. Eight time points were taken for each experiment over a test period of 70 hours to 6 days.
  • the rats were then killed by asp ixiation in a CO_ chamber, and the injected limbs were dissected and counted for radioactivity.
  • Urine and feces were collected from the two animals in the metabolic cages throughout the experiment. These two animals were also sacrificed, serving as the last time point, and were also dissected for their thyroid, lungs, heart, liver, spleen, stomach, intestines, and genitals for counting. The remaining carcass was digested prior to counting. Radioactivity levels were measured to determine the disposition of residual lipid tracer in the animals at the end of the experimental period.
  • the accumulated level of radioactivity measured in the urine and feces is shown by the closed triangles in the figure.
  • the total excreted radioactivity, at the end of a 70-hour test period in this experiment, is about 72% of the original dose administered.
  • the remaining counts are distributed among the limbs (about 5%), carcass (about 20%). and organs (about 3%). Of the roughly 3% remaining radioactivity which is localized in the organs, about 55% is found in the gut, about 34% in the liver, and less than about 5% each in the spleen, trachea, kidneys, stomach, heart, lungs, and genitals.
  • the upper dotted line in Figure 3 shows the same data expressed as a semi-log plot as a function of time. From this plot, the clearance half life of lipid from the sites of injection can be readily determined. In the particular example, the half life (at which the lipid is 50% retained) is 48 hours. The half life data. determined from the slope of the semi-log plot for each experiment, is shown in Table III below.
  • Liposome sizes are expressed in Table III both in terras of the pore size of the polycarbonate membrane used in sizing the MLVs. and the actual average size of the MLVs, as determined by a Nicomp Particle Sizer (Model 200), calibrated with standardized latex beads.
  • the MLVs injected were prepared by extrusion through a 1 micron polycarbonate membrane, but showed an average measured particle size of about 3.2 microns.
  • the discrepancy between extruded and measured size presumable reflects liposome aggregations, which tends to occur more at higher liposome concentrations, and primarily only in composition A and E MLVs, which do not contain negatively charged lipid (PG) .
  • the dose which is administered to the rat is measured in terms of ⁇ mole lipid per 20 or 100 ⁇ l per injection.
  • the values shown in the dose column in the table are total doses per injection.
  • the data in rows 1-3 illustrate the effect of total lipid dose on lipid clearance from the IM site.
  • increasing the amount of injected lipid from about 0.2 to 10 ⁇ mole lipid produces about a twofold increase in clearance half life.
  • the same effect is seen with Composition B MLVs (rows 5 and 6) and Composition C MLVs (rows 8 and 9).
  • the half life data also demonstrate that the lipid clearance rate is increased significantly with larger liposome sizes. This effect is seen from rows 4 and 5. and from rows 7 and 8. both of which compare clearance rates for MLVs extruded through 0.2 and 1.0 micron filters, respectively.
  • the larger liposomes increased the clearance half lives by about 60-100%.
  • the relatively high clearance rates observed with the Composition A MLVs may also reflect, at least in part, the relative large sizes of these MLVs as measured by laser particle sizing.
  • Figure 2 shows semi-log plots from which the half lives in row 1 (dotted line) and row 4 (solid line) were calculated.
  • MLVs having one of the lipid compositions A. B. D. and E described in Example I. at a selected dose of 0.2. 2. or
  • Free sCT was removed from the liposome suspension by centrifugation. Following injection, pairs of animals were sacrificed at period in the intervals over a 70-hour to 6-day test period, and the retention of radioactive sCT in the injected limbs of the animals, and in a number of organs, were measured as described in Example III above. Two of the injected animals were monitored for excretion of sCT by urine and feces throughout the test period.
  • half life is about 8 hours.
  • the half life data determined in this manner for the four lipid compositions and the two lipid doses which were examined are given in Table IV below.
  • the MLVs had been previously sized by extrusion through 1.0 pore polycarbonate membranes, but not further extruded through smaller-pore membranes.
  • compositions B and D The data also show a marked effect of lipid composition on sCT clearance rates.
  • PG in the MLVs
  • compositions A and E the two non-PG-containing MLVs
  • the addition of cholesterol to non-PG-containing lipids increases the sCT clearance rates noticeably. This is seen from a comparison of rows 2 and 6. which relate to a 2/100 dose and rows 3 and 7, which relate to a 10/100 dose.
  • Table V below compares a few of the sCT clearance rates from Table IV and the corresponding rates from Table III, for the lipid tracer.
  • the sCT rates are about half those of the lipid tracer, for all of the lipid composition and dose for which comparative data are available. This finding suggests that liposomes are destabilized and release their encapsulated contents predominantly at the site of injection, with lipid clearance from the injection site being handled by a different, and slower, mechanism.
  • 125 EPC: ⁇ -T at 99:1 and encapsulated I-sCT were prepared according to a standard REV procedure (Szoka).
  • the vesicles were extruded through 0.4 ⁇ m and 0.2 ⁇ m polycarbonate filters, and non-encapsulated sCT was removed from the vesicle suspension by centrifugation and washing three times in PBS. The preparation was then filtered through a sterile. 0.22 micron filter. Empty MLVs of the same composition were prepared and extruded through a 1 ⁇ ra filter as described in Example I under sterile conditions. The REVs were mixed with the MLVs in a mole ratio of 1:50 such that each IM injection contained about 1 x 10

Abstract

Un procédé permet de régler sélectivement la vitesse de libération d'un composé enfermé dans un liposome à partir d'un site d'injection intra-musculaire ou sous-cutanée. Le procédé comprend la sélection des dimensions, de la quantité et de la composition lipidique moyennes des liposomes injectés dans un site de façon à obtenir une demi-vie voulue de libération du composé. Une composition préférée comprend une suspension aqueuse de liposomes renfermant le composé et ayant des particules dont les dimensions moyennes sont inférieures à 0,3 microns environ, et des liposomes plus grands, vides, en quantité suffisante pour augmenter la demi-vie de libération du composé depuis le site de l'injection jusqu'à une demi-vie voulue longue d'environ 1 à 14 jours. L'invention concerne également une composition stable de liposome/calcitonine.
EP19870901859 1986-02-10 1987-02-09 Systeme d'administration liposomique a liberation entretenue. Withdrawn EP0256119A4 (fr)

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US828153 1986-02-10

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JPS63502117A (ja) 1988-08-18
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WO1987004592A1 (fr) 1987-08-13

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