EP0726762A1 - Preparation aux liposomes charges electriquement - Google Patents

Preparation aux liposomes charges electriquement

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Publication number
EP0726762A1
EP0726762A1 EP94931585A EP94931585A EP0726762A1 EP 0726762 A1 EP0726762 A1 EP 0726762A1 EP 94931585 A EP94931585 A EP 94931585A EP 94931585 A EP94931585 A EP 94931585A EP 0726762 A1 EP0726762 A1 EP 0726762A1
Authority
EP
European Patent Office
Prior art keywords
liposome
amount
electrically charged
charged component
component
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94931585A
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German (de)
English (en)
Inventor
Werner Krause
Andreas Sachse
Mark Sullivan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer Pharma AG
Original Assignee
Schering AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Schering AG filed Critical Schering AG
Publication of EP0726762A1 publication Critical patent/EP0726762A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes

Definitions

  • This invention relates to liposomes and their administration to host organisms.
  • Liposomes have been used as carriers for active agents, including therapeutic, diagnostic, and prophylactic agents.
  • Liposomes generally comprise amphipathic compounds, having a hydrophilic head and a hydrophobic tail, which react under certain conditions to form a bimolecular leaf structure of at least two layers of lipid, in which the polar head groups are at the interface between the aqueous medium and the lipid and the hydrophobic tails interact to form an environment that excludes water.
  • the lipid bilayers are stable structures held together by the non-covalent interaction of the hydrocarbon groups of the acyl groups. When the lipid bilayer closes in on itself, it forms a spherical vesicle called a liposome having an internal space separated from the external environment. Desired agents may be encapsulated in this space, or within the bilayer, itself.
  • the lipids which comprise the liposome are typically phosphoglycerides, such as phosphatidyl choline (lecithin) , and sphingolipids.
  • Liposomes may be used as carriers for, e.g., thera ⁇ Guideic, diagnostic, and prophylactic agents. They are especially advantageous in that they may protect suscep ⁇ tible agents from degradation, thereby increasing the time during which the agent is active in the body.
  • tissue or cell specific targeting of the lipo ⁇ some may be achieved by incorporating a component into the liposome, e.g., coupling, conjugating, absorbing or adsorbing it to the liposome bilayer surface, which is selective for a specific tissue or cell type.
  • lipids, antibodies, lectins, receptors, ligands, and other such components may be incorporated into liposomes for the purpose of achieving tissue specific targeting.
  • liposome technology is in the treatment, prophylaxis, and diagnosis of liver disorders.
  • diagnostic difficulties are the low contrast difference between normal and tumor liver parenchyma, which only allows delineation of lesions above 1 to 2 cm. Accordingly, it would be desirable to be able to concentrate a radiographic or MRI contrast medium in the liver in order to increase the contrast for a time period that permits thorough examination of the entire organ.
  • liposomes may be taken up by the phagocytic cells of the reticulo-endothelial system. Thus, they are chiefly concentrated in the Kupffer-cells of the liver and macrophages in the spleen. Since phagocytic activity is not displayed by tumor tissue, the liposomal contrast agent can be concentrated selectively in the healthy tissue. This results in an increased density difference ( ⁇ HU for X-ray imaging, ⁇ T1 or ⁇ T2 for MRI) .
  • contrast-carrying liposomes produced by this method, opacification of the liver and spleen has been achieved.
  • iopromide was chosen as liposomal contrast agent because its 7-day retention values in the liver and spleen are significantly lower than those of iotrolan.
  • liposomes are potentially an important advance in drug delivery systems, such as for carrying contrast media as described above, their use in living organisms is limited owing to adverse systemic side effects which may be observed upon liposome administration. These systemic effects include, e.g., transient hemodynamic depression, affecting blood pressure, heart rate, and ECG; depression of blood counts.
  • liposomes may have adverse hemodynamic effects when administered intravenously to animals, posing serious hazards to the safety of the host animal.
  • liposome preparations for drug delivery.
  • Figure 1 is a schematic diagram of the ethanol- evaporation method of making liposomes.
  • EDP enddiastolic pressure
  • a component having a negative charge e.g., a fatty acid such as stearic acid
  • a component having a negative charge e.g., a fatty acid such as stearic acid
  • the improved tolerance may be reflected in the reduction or elimination of adverse systemic effects produced by a liposome preparation in which the electric charge component is absent.
  • hemodynamic effects such as hemodynamic depression, changes in blood pressure, heart rate, systolic pressure, diastolic pressure, or ECG intervals (PR, QRS, QT, QTc) , reflex tachycardia, prema ⁇ ture ventricular arrhythmias, increased respiration, sedation, and also changes in the blood cell and platelet counts, and death.
  • hemodynamic effects such as hemodynamic depression, changes in blood pressure, heart rate, systolic pressure, diastolic pressure, or ECG intervals (PR, QRS, QT, QTc)
  • reflex tachycardia prema ⁇ ture ventricular arrhythmias
  • increased respiration sedation
  • sedation changes in the blood cell and platelet counts, and death.
  • this basic principle of improving tolerance to an otherwise deleterious structure by electrical-charge modification may be applicable to reagents other than liposomes.
  • the present invention relates to an amount of electrical charge which is effective in, e.g
  • the undesired or adverse effects of a liposome preparation upon administration may be reduced or eliminated by the addition of an electrical charge, regardless of the composition of the administered liposomes.
  • a liposome preparation having systemic effects may already contain electrically-charged components.
  • the introduction of an effective amount of an electrically-charged component may be in addition to the charged components which are already present in the liposome.
  • the charged component introduced may be of the same type or charge already present in the liposome or of an altogether different type or charge.
  • the present invention relates to the addition of a charge to a liposome already having charged components.
  • the charged component may be incorporated into the liposome in various ways.
  • the charged component may become, e.g. , a structural component of the lipid bilayer, and/or conjugated or non- covalently attached to a constituent of the liposome.
  • the stearic acid may be incorporated into the lipid bilayer of the liposome.
  • an electrically-charged component may also be added to an already formed liposome preparation, or an incompletely formed preparation, e.g., by conjugating or non-covalently attaching a charged component to the surface of the lipid bilayer.
  • the electrically-charged component is present in the liposome in an amount which is effective, e.g., to improve host tolerance, reduce adverse effects, and/or reduce the hemodynamic effects produced by the administration of a liposome preparation.
  • the electrical-charge can therefore be carried and incorporated into the liposome in a variety of ways, including, e.g., by the addition of charged components such as acids, e.g., stearic acid, and salts of these components such as ammonium salts; by coupling charged moieties to components of the lipid bilayer; and by the addition of bilayer-forming substances with charges, either coupled covalently or adhering by any other force. While the invention is not bound by any theory, typically a liposome according to the present invention presents an electrical-charge on the outer surface of its bilayer.
  • a liposome preparation comprising phosphatidyl choline is known to have an adverse hemodynamic effect. Its tolerance by the host organism may be improved by preparing a new batch of liposomes comprising an effective amount of an electrically-charged component in addition to the phosphatidyl choline, e.g., the effective amount of electrical charge may be carried by stearic acid. Alternatively, the charge may be introduced into liposomes which are already formed. For example, an electrical-charge may be introduced to the outer (exterior) surface of the lipid bilayer by chemical modification, or by covalently or noncovalently adding a charged moiety to its surface.
  • the addition of a charge to a liposome preparation may also permit higher amounts of the preparation to be administered to the host organism without producing dele- terious effects normally attributed to liposome admini ⁇ stration. This is significant in that larger dosages of the agent encapsulated in the liposome may be attained.
  • the addition of a charge to a liposome may also improve the stability and storage of the liposome preparation, e.g. , at temperatures greater than room temperature, such as 30° or 40°C.
  • the nature of the effective amount of electrically charge component may be either positive or negative, as long as the desired effect is achieved, e.g., to improve tolerance, reduce adverse effects, and/or reduce the hemodynamic effects produced by administration of a liposome preparation.
  • a negative charge is preferred.
  • the charge may be, e.g., ionic or electronegative.
  • a charged component may be selected and then incorporated into the liposome preparation for the purpose of reducing or eliminating the side effects associated with its administration.
  • the amount of effective electrical charge may be carried by a molecule or a charge carrier.
  • the molecule may have one or more charged moieties, e.g., a zwitterion.
  • the charge may be located at any position on the charge carrier.
  • the choice of the charge carrier will depend on various factors; e.g., availability, biocompatability, and desired effect.
  • the charge carrier may be selected for other properties in addition to its ability to carry the desired charge.
  • the charge carrier may also function as a targeting agent, i.e., directing the liposome selectively to a desired tissue or cell type in the host organism.
  • the charge carrier may be, e.g., biomolecule, a polymer, a lipid, a protein, a nucleic acid, a carbohydrate, a synthetic molecule, or any molecule or molecular structure which may be incorporated into the liposome bilayer and which possesses an effective amount of electrical charge.
  • the molecule may become structurally part of the liposome vesicle, e.g., integrated into the lipid bilayer, or peripherally attached to the liposome surface, or as a lipid constituent, itself.
  • small compounds with an additional acidic group can be coupled to an "anchor" such as cholesterol, phosphatidyl choline, fatty acid etc. sitting in the bilayer.
  • the charge preferably negative, can also be carried by amino acids or sugar acids coupled to any moiety able to adhere to the bilayer (cholesterol, phosphatidyl choline, fatty acid, etc.).
  • the charge carrier may also be any lipid which carries a charge, e.g., including, sterols, fatty acids, glycerol esters, sphingosine, terpenes, or generally lipids, e.g., see TEXTBOOK OF CLINICAL CHEMISTRY (1986) , N.W. Tietz, editor, W.B. Saunders Co., Chapter 7, pp. 829-900.
  • Stearic acid is a preferred example of a fatty acid having a negative electrical charge which may be employed in the present invention in an effective amount, e.g., to reduce hemodynamic effects of a parenterally administered liposome preparation.
  • Other fatty acids may include, e.g., palmitic acid, arachidonic acid, linolic acid, linoleic acid, and/or mixtures thereof.
  • the charge carrier may also be a protein which, e.g., is capable of integrating into the lipid bilayer or attaching peripherally to the surface.
  • the charge of the protein may be produced by amino acids, carbohydrate linkages, or non-protein groups linked or joined to the protein.
  • the protein may also be a protein having a lipid moiety.
  • a protein may be chosen because of its desired charge and also because of its ability to target liposomes to a specific tissue.
  • the amount of charge to be incorporated into the liposome may be that amount which is effective to reduce the undesired systemic effects such as hemodynamic depression and/or tolerance.
  • the amount of charge may be varied by changing the molar ratio between the charged component and the other constituents of the liposome.
  • a charged component may be added to a liposome comprising phosphatidyl choline by the addition of stearic acid.
  • the amount of charge in the liposome may be achieved by adjusting the molar ratio between the two constituents to a desired amount; e.g. , phosphatidyl choline/stearic acid, 9:1.
  • the improved tolerance by the host to a liposome preparation and the consequent elimination or reduction in systemic affects may be produced by the incorporation of a charged component into the liposome vesicle.
  • This improvement when mediated by changes to the cell surface caused by the charged component, may also be produced by using agents or methods which modify surfaces.
  • the already formed liposomes having a deleterious systemic affect may be treated enzymatically or chemically to alter the surface properties of the liposome and improve its tolerance when administered.
  • Chemical modification may include e.g. , reacting the liposome reducing agents, oxidizing agents, or coupling charged components to its surface. These methods are accomplished conventionally but to achieve the reduction or elimination of adverse effects as revealed here.
  • the liposomes may be prepared according to methods which are conventional in the art, e.g., as reviewed in W086/00238. Additionally, liposomes may be prepared according to EP69307; U.S. Pat. No. 5,110,475 which, e.g., describes a process for the production of an aqueous dispersion involving removal of liquid(s) from an optionally multiphase liquid mixture by means of membrane distillation; and W086/00238 which is described as an extrusion techniques for producing liposomes having substantially a unimodal and defined size distribution.
  • the electrically- charged moiety may be oriented in the liposome in several different directions; e.g., facing the interior of the liposome vesicle and facing the exterior at the interface between aqueous medium and the lipid.
  • Liposomes having the electrical charge on the exterior are preferred, e.g., the exterior surface. It may be desirable to separate, e.g., the "interior” charged from the "exterior” charged liposomes.
  • liposome preparations containing the electrically-charged component may be subjected to conditions which eliminate orientations which are not desirable for the purposes of the present invention.
  • an active agent may be encapsulated in the liposomes according to conventional methodology.
  • a liposome preparation having an effective amount of an electrically charged component may comprise or encapsulate an active agent.
  • Encapsulation may be accomplished by methods which are known, e.g., by forming the liposome in the presence of an amount of an active agent under conditions in which the lipid bilayers form around and encapsulate the active agent, by introducing an active agent to an already formed liposome by fusion with a liposome, cell, or other structure comprising the active agent, e.g., such fusion may be carried out by PEG, electric current, lectins, and other well known methods in the art.
  • liposomes having an active agent may be prepared according to WO/89593, e.g., by treating the liposomes in an aqueous medium under pressure conditions effective to reduce the order of the lipid arrangement therein to permit entry of the active agent and performing a pressure drop.
  • An active agent may be encapsulated into a liposome preparation having an effective amount of an electrically charged component, including e.g. , conventional active agents such those used for therapy, including agents as listed in the Physicians Desk References, Medical Economics Data, 44th Edition, 1993 or Remington ' s Pharmaceutical Sciences , Mack Publishing Co., 18th Edition, 1990, antibodies, peptides, DNA, RNA, ribozymes, and oligonucleotides, prophylaxis, and diagnostics, e.g., contrast agents, such as iopromide, paramagnetic entities, ferromagnetic entities, radioisotopes, and other conventional structures or compounds.
  • an active agent may be one that is used and recognized by the ordinary skilled worker in the field.
  • the liposomes may be administered according to conventional methods.
  • a liposome preparation may be administered parenterally, e.g., intravenously by arterial or venous catheter, by injection with a syringe, however they may also be administered orally or through the intestines.
  • a typical amount of liposome preparation is, e.g., 100-200 mg lipid/kg, this amount being dependent on how much of the agent is intended for administration and how much of it is encapsulated per liposome.
  • the amount of liposome preparation which is to be administered may be determined routinely, according to methods which are known in the art.
  • the liposomes may be administered in a single injection or it may be administered or infused over a period of time at a constant or variable rate, e.g., 2 ml/min for 30 minutes. Because the liposomes comprising the charged component are well tolerated by the host, infusion rates and amounts of liposome to be administered may be increased over the amounts that are typically used for liposome preparations which have adverse effects.
  • the liposomes may be administered to any organism, e.g., mammals, such as humans, rats, mice, dogs, cats, rabbits, cows, horses, but also birds, amphibians, and reptiles.
  • the purpose of administration may be to diagnose, treat, medicate, sedate, or prevent disorders but it may also be used in conjunction with the development of animal models to evaluate the safety and efficacy of drugs, contrast media, antibodies, and other agents for their eventual use in humans.
  • the method of the present invention of e.g., reducing hemodynamic effects, improving host tolerance, or reducing adverse effects, associated with liposome administration, may be accomplished in any organism, such as those listed above.
  • the liposomes can be used for any diagnostic or therapeutic use, including e.g., MRI, Ultrasound, and Nuclear Medicine.
  • FIG. 1 The preparation process for iopromide liposomes by the "ethanol vaporation method" is depicted in Figure 1.
  • ethanol is removed from the mixture under reduced pressure until a white liposomal suspension is obtained.
  • This suspension is then filtered (5 ⁇ m and 1.2 ⁇ m) , filled into vials and lyophilized to produce a stable form for storage.
  • Such lyophilizates Prior to application, such lyophilizates are first rehydrated with 4 ml 135 mM mannitol solution per g of dry substance. The resulting liposomal suspension is then filtered (20 ⁇ m glass-fiber pre-filter and 5 ⁇ m CN main filter) to remove non-liposomal structures. These structures are sling- or loop-shaped bilayer formations in part larger than 10 ⁇ m. It has been shown that these structures are removed by the above filtration step.
  • the process is potentially suited to performance under aseptic conditions.
  • the solutions employed to produce the liposomes can be ultra- or sterile-filtered.
  • the composition of a resuspended lyophilizate (4 ml 135 mM mannitol solution per g lyophilizate) is listed below (without ranges) : iopromide: phosphatidylcholine (PC) DL- ⁇ -tocopherole: cholesterol (CH) : stearic acid (SS) : tromethamine: 1 N HC1: disodium edetate: mannitol: water f.i. :
  • liposome lyophilizates For an initial stability study, a batch of liposome lyophilizate was produced under aseptic laboratory conditions. The resuspended liposomes were characterized regarding size (QUELS) , iodine encapsulation, pH and microscopic appearance. The microbial status was not determined. According to the results obtained in this study (six months' storage), liposomal lyophilizates
  • PC/CH/SS 4:5:1 can be stored at temperatures up to 30°C without perceivable quality changes. After 6 months at 40°C, however, a significant increase in size and a decrease in pH were observed in the resuspended material. These changes were accompanied by an unpleasant odor coming from the resuspended material.
  • the catheter was secured with 2-0 silk suture and exteriorized via a subdermal tunnel to the nape of the neck.
  • the right jugular vein was catheterized using Tygon microbore tubing (0.04" ID, #S- 54-HL) and exteriorized to the nape of the neck also.
  • Both arterial and venous catheters were filled with sterile, heparinized (600 U/mL) saline, sutured and painted with betadine to reduce infection.
  • the exposed catheters were secured in a small plastic pouch and enclosed in a cotton-mesh collar reinforced with adhesive tape. Dogs were administered standard intramuscular injections of Combiotic to reduce the risk of infection and allowed to recover from surgery.
  • dogs were administered an infusion of DMPC/DPPC, prepared by the interdigitation/fusion method (See WO 91/10422; EP 510096) , or DPPC at 2 mL/minute for "25 minutes. Parameters monitored were direct arterial blood pressure and lead II surface ECG.
  • DMPC/DPPC or DPPC liposome vesicles were admini ⁇ stered intravenously as an opaque suspension at a rate of 2 mL/minute in a concentration of 1 mg/kg/ L until a total dose of 50 mg/kg was administered. All doses were loaded into sterile 60 mL syringes and infused using a Razel infusion pump. Any remaining suspension in the catheter was flushed into the dog using sterile saline at the same infusion rate.
  • Intravenous administration of DMPC/DPPC or DPPC vesicles caused a marked decrease in systolic, diastolic, and mean arterial pressure.
  • the catheter was secured with 2-0 silk suture and exteriorized via a subdermal tunnel to the nape of the neck.
  • the right jugular vein was catheterized using Tygon microbore tubing (0.04 inch ID, #S-54-HL) and also exteriorized to the nape of the neck.
  • Both arterial and venous catheters were filled with sterile, heparinized (600 U/mL) saline (1 mL) and capped with sterile caps. The 2 incisions were closed with silk suture and painted with Betadine® to reduce the risk of infection. The exposed catheters were secured in a small plastic pouch and enclosed in a cotton-mesh collar reinforced with adhesive tape. Dogs were administered standard intramuscular injections of Combiotic to further reduce the risk of infection and allowed to recover from surgery.
  • EPC liposome vesicles were administered intraven ⁇ ously as an opaque suspension at a rate of 2 mL/minute in a concentration of 1 mg/kg/mL until a total dose of 50 mg/kg was administered. All doses were loaded into sterile 60 mL syringes and infused using a Razel infusion pump. Any remaining suspension in the catheter was flushed into the dog using sterile saline at the same infusion rate to assure that the total dose was administered to each dog.
  • EPC liposome vesicle infusions The effects of EPC liposome vesicle infusions on heart rate (HR) , mean blood pressure (MAP) , systolic pressure (SP) , diastolic pressure (DP) , ECG intervals
  • Intravenous administration of EPC liposome vesicles caused a marked decrease in systolic, diastolic, and mean arterial pressure in 3 of the 5 dogs tested.
  • EPC liposome vesicles had no ob- served effect in 2 of the 5 dogs tested; however, 3 of the 5 dogs tested demonstrated marked falls in mean arterial pressure. The average fall in mean pressure was 35%, but those dogs with marked hemodynamic depression experienced mean pressure drops ranging from 57% to 61% below basal levels. While these drops in pressure were severe, they were also transient as pressure values returned to near basal levels by the end of the infusion of the EPC vesicles.
  • EPC liposome vesicles had no physio ⁇ logically significant effect on ECG intervals. No changes were observed in QRS duration. Changes occurring in PR, QT and QTc intervals were related to heart rate changes that occurred in the 3 dogs overtly affected by the EPC infusion. None of these changes were determined to be of physiologic significance due to their transient nature. In one dog, spontaneous arrhythmias (in the form of isolated premature ventricular contractions) occurred during the EPC infusion. Examples are shown in Figure 2.
  • Intravenous administration of EPC liposome vesicles in conscious dogs resulted in hemodynamic depression.
  • the platelet depletion observed in this study paralleled a similar effect noted in the previous study.
  • platelet depletion seemed closely related to severe falls in blood pressure resulting from liposome infusion.
  • the overt reactions observed during liposome infusion are most likely related to an anaphylactic response.
  • the spontaneous arrhythmias observed in one dog receiving EPC vesicles may be a response to the sudden hypotensive effect with a reflex activation of catecholamines.
  • Liposomes according to the present invention comprising the electrically-charged component stearic acid (preparations SA 504/02079 and SA 504/300591) , were administered to healthy dogs.
  • the right omocervical artery and right jugular vein were isolated for cannulation.
  • the artery was cannulated first using Tygon microbore tubing (0.05" ID, #S-54-HL) .
  • the catheter was secured with 2-0 silk suture and exteriorized via a subdermal tunnel to the nape of the neck.
  • the right jugular vein was catheterized using Tygon microbore tubing (0.05" ID, /S-54-HL) and also exteriorized to the nape of the neck.
  • Both arterial and venous catheters were filled with sterile, heparinized (600 U/mL) saline (1 mL) and capped with sterile painted with Betadine® to reduce infection.
  • the exposed catheters were secured in a small plastic pouch and enclosed in a cotton-mesh col ⁇ lar reinforced with adhesive tape. Dogs were admini ⁇ stered standard intramuscular injections of Combiotic to reduce the risk of infection and allowed to recover from surgery.
  • dogs were administered an infusion of either SA 504/020791 or SA 504/300591 at approximately 2 mL/minute for exactly 30 minutes.
  • Parameters monitored were arterial blood pressure and lead II surface ECG.
  • Heart rate was determined from the ECG tracing.
  • SA 504/020791 or SA 504/300591 iodine-containing liposome vesicles were administered intravenously as an opaque suspension at a rate of approximately 2 mL/minute at a final volume of 4.2 mL/kg and 4.5 mL/kg, respec- tively.
  • the infusion rate was adjusted such that the total infusion time was 30 minutes. All doses were loaded into sterile syringes and infused using a Razel infusion pump. Any remaining suspension in the catheter was flushed into the dog using sterile saline at the same infusion rate.
  • test substances were prepared according to the following procedure: SA 504/020791: 17.6 mLs of mannitol solution (135 mM) was added to each vial of the test sub ⁇ stance and allowed to sit for at least 10 minutes. Vials were then shaken vigorously and allowed to sit for ano ⁇ ther 10 minutes. After repeating the shaking procedure, the suspensions were checked visually for agglomerates (if agglomerates were present, the above procedure of shaking the vials was repeated) . Suspensions were then drawn into 60 mL syringes and filtered through a sterile filter apparatus supplied by Schering AG. Each animal received 4.5 mL/kg.
  • SA 504/300591 The procedure is exactly as outlined above except that 18.0 mLs mannitol solution (135 mMO were used per vial and each animal received 4.2 mL/kg.
  • SA 504/020791 and SA 5041/300591 were prepared according to the following instructions.
  • a solution 1 was prepared by dissolving 54.6 gms of lipoid S100, 33.0 gms of cholesterol, and 4.9 gms of stearic acid in 9.5 ml of 96.5% ethanol.
  • 2000 ml of a solution 2 was prepared by combining 250 ml of iopromide solution with 9.5 ml of 20 mM Tris HCl, pH 7..5, and 9.5 ml of 96% ethanol. While stirring, solution 1 was mixed with solution 2. The ethanol was removed under vacuum and the remaining solution was freeze-dried. 5 gm of freezed-dried material was resuspended in 8.8 ml of 135 mmol mannitol. The resuspended liposome preparation was typically injected at 300 mg iodine/kg.
  • Intravenous administration of SA 504/020791 or SA 504/300591 vesicles produced no significant changes in systolic, diastolic or mean arterial pressures.
  • control mean arterial pressure for dogs treated with SA 504/300591 was 104 ⁇ 6 and remained relatively unchanged throughout the duration of the experiment. Similar results were obtained with the test substance SA 504/020791.
  • the liposome vesicle preparations had no significant effect on heart rate in conscious dogs. Of the five dogs treated with SA 504/300591, three dogs had a decrease in heart rate during the infusion and two had an increase. Thus, no consistent pattern was seen. In one animal receiving SA 504/020791 (Dog #10003) , the control heart was particularly high (164 bpm) . This animal was highly reactive to any stimuli in the room (i.e., laboratory personnel) , but eventually seemed to quiet down as the experiment progressed. This was most likely due to acclimatization and not a drug-induced effect.
  • Preparation l Liposomes which only contained phos ⁇ phatidyl choline at a concentration of ca. 25 mg/ml.
  • Preparation 2 Liposomes with phosphatidyl choline and stearic acid. The molar content was ca. 9:1 at a total lipid concentration of ca. 70 mg/hl.
  • preparations were infused intravenously into five dogs each at a dose level of ca. 50 mg lipid per kg body weight (preparation 1) and ca. 30 mg lipid per kg (preparation 2) .
  • Preparation l Transient fall in blood pressure associated with a significant reflex tachycardia in 3 of the 5 animals tested. Other observed effects in these 3 animals included: premature ventricular arrhythmias dur ⁇ ing the infusion and briefly following the administration in 1 dog and increased respiration in the form of panting in the 2 other dogs. There were no physiologically sig ⁇ nificant changes in the ECG intervals in any of the dogs. Platelet counts fell in 4 of 5 dogs receiving the prepa ⁇ ration. Those dogs experiencing hemodynamic depression seemed somewhat sedated and had the most consistent platelet depletion when compared with those dogs who did not experience decreases in blood pressure.
  • Preparation 2 Intravenous administration of this formulation had no adverse hemodynamic effects in any of the five animals treated.
  • the liposomes which were used in this study were prepared by a continuous high pressure extrusion method. Briefly, an ethanolic solution of the respective lipid or lipid mixtures was deposited on the wall of a round bottom flask. The resulting lipid film was dispersed in 300 mM mannitol solution to give a lipid concentration of approximately 100 mg/ml. The resulting MLV dispersions were subsequently extruded using a high pressure extrusion apparatus (Maximator® model HPE 10.0 - 250, Schmidt, Kranz & Co., Zorge, Germany).
  • Each batch was sequentially extruded 10 times over two stacked polycarbonate membranes (Nucleopore, Tiibingen, Germany) of decreasing pore sizes (1.0 - 0.4 and 0.1 ⁇ m) to give a total of 30 filter passages for each preparation.
  • the obtained liposomal suspensions were filtered through sterile filter holders (0.4 or 0.2 ⁇ m pore size cellulose acetate, Sartorius, G ⁇ ttingen, Germany) into sterile glass vials which were stoppered under aseptic conditions.
  • Distearoylphosphatidylcholine (DSPC- batches LP-04-013- 114219 and -116298) and Distearoylphosphatidyl-glycerol (DSPG - batch LP 04-017-115751) were obtained from Sygena, Liestal, Switzerland. Liposome size was determined by photon correlation spectroscopy (PCS) using a submicron particle-sizer, autodiluteTM model 370, Nicomp Instr. Corp., Santa Barbara, CA, USA.
  • PCS photon correlation spectroscopy
  • the rats were anaesthetized with pentobarbital, 60 mg/kg intraperitoneally.
  • the trachea was cannulated to facilitate spontaneous respiration.
  • Body temperature was maintained at 38 + 0.5 C by means of a heated operating table and a heating lamp.
  • Rats were infused with a liposome dose containing a total amount of 300 mg lipid/kg body weight and at an injection rate of 100 mg lipid/kg/min into the left femoral vein. Controls received an identical volume of 300 mM mannitol solution.
  • the investigated liposome formulations were made from DSPC or DSPC/DSPG.
  • BP and TPR decreased after a short transient increase to -33.3% and -35.5% of prevalue, respectively (Figs. 2 and 3).

Abstract

L'invention se rapporte à un procédé d'administration d'une préparation aux liposomes contenant une quantité efficace d'un composant chargé électriquement qui réduit de manière efficace les effets indésirables d'une préparation à base de liposomes, par exemple, en réduisant les effets hémodynamiques, et qui améliore la tolérance de l'hôte.
EP94931585A 1993-11-04 1994-11-04 Preparation aux liposomes charges electriquement Withdrawn EP0726762A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US14554193A 1993-11-04 1993-11-04
US145541 1993-11-04
PCT/EP1994/003668 WO1995012386A1 (fr) 1993-11-04 1994-11-04 Preparation aux liposomes charges electriquement

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EP0726762A1 true EP0726762A1 (fr) 1996-08-21

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US5834025A (en) * 1995-09-29 1998-11-10 Nanosystems L.L.C. Reduction of intravenously administered nanoparticulate-formulation-induced adverse physiological reactions
JP5846711B2 (ja) * 2005-06-09 2016-01-20 メダ アーベー 炎症性疾患の治療のための方法及び組成物
KR100761706B1 (ko) * 2006-09-06 2007-09-28 삼성전기주식회사 인쇄회로기판 제조방법

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JPS60155109A (ja) * 1984-01-23 1985-08-15 Terumo Corp リポソ−ム製剤
DE3934656A1 (de) * 1989-10-13 1991-04-18 Schering Ag Verfahren zur herstellung von waessrigen dispersionen
CA2046997C (fr) * 1990-07-16 2000-12-12 Hiroshi Kikuchi Liposomes
WO1994027580A1 (fr) * 1993-05-21 1994-12-08 The Liposome Company, Inc. Reduction de reactions physiologiques indesirables induites par des liposomes

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NO961826L (no) 1996-05-06
FI961893A0 (fi) 1996-05-03
PL314132A1 (en) 1996-08-19
KR960705545A (ko) 1996-11-08
JPH09506084A (ja) 1997-06-17
HUT74517A (en) 1997-01-28
AU8061494A (en) 1995-05-23
CZ128096A3 (en) 1997-08-13
CA2174326A1 (fr) 1995-05-11
NO961826D0 (no) 1996-05-06
FI961893A (fi) 1996-05-03
WO1995012386A1 (fr) 1995-05-11
HU9601190D0 (en) 1996-07-29

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