CZ128096A3 - Charged liposome preparation - Google Patents

Abstract

The invention relates to a method of administering a liposome preparation having an effective amount of an electrically charged component which is effective in reducing adverse effects of a liposome preparation, e.g., in reducing hemodynamic effects, and improving host tolerance.

Description

organism.

Prior art. Liposomes are used as carriers for active agents, including therapeutic, diagnostic and prophylactic agents.

In general, liposomes include amphipathic compounds having a hydrophilic head and a hydrophobic end that react under certain conditions to form a bimolecular leaf structure of at least two lipid layers in which polar head groups at the aqueous medium-lipid interface and hydrophobic ends interact to form an environment that excretes water. Lipid bilayers are stable structures that are held together by a non-covalent interaction of hydrocarbon groups and acyl groups. When the lipid bilayer encloses, it forms a spherical vesicle called a liposome, having an internal space separate from the external environment. The desired agents can be encapsulated in this space or in the bilayer itself.

Lipids that contain liposomes are typically phosphoglycerides such as phosphatidylcholine (lecithin) and sphingolipids.

Liposomes can be used as carriers for, for example, therapeutic, diagnostic and prophylactic agents. They are particularly useful because they can protect sensitive agents from degradation and thereby increase the time the agent is active in the body. In addition, tissues or cells specifically targeted by a liposome can be achieved by incorporating a component into the liposome, e.g., by coupling, conjugating, absorbing, or adsorbing the component to the surface of a liposome bilayer that is selective for a specific tissue or cell type. For example, lipids, antibodies, lectins, receptors, ligands, and other such components may be incorporated into liposomes to achieve tissue-specific targeting.

Another application of liposome technology is the treatment, prophylaxis and diagnosis of liver disorders. Despite the many different imaging techniques and methods currently available, accurate assessment of liver lesions remains a difficult diagnostic problem. One of the reasons for the difficulties of the diagnosis is the low contrast difference between the normal and the tumor liver parenchyma, which allows to identify only lesions over 1 to 2 cm. Accordingly, it would be desirable to have a concentrated radiographic or MRI contrast medium available in the liver in order to increase the contrast for a period of time that allows for the evaluation of the whole organ.

One way to achieve the uptake of hydrophilic substances into the liver is to encapsulate them in liposomes. After i.v. administration, the liposomes can be taken up by the reticuloendothelial system. They are thus mainly concentrated in Kupffer cells of the liver and macrophages in the spleen. Because phagocytic activity is not imaged by tumor tissue, the liposomal contrast agent can be selectively concentrated in healthy tissue. This leads to an increased density difference (^ HU for X-ray imaging, ^ T1 or T2 for MRI).

Various researchers are able to demonstrate in principle the effectiveness of such a solution. In most cases, however, they are unable to achieve a sufficient difference in density due to k

ΐ small capacity of liposomes to load iodine; nor are there any methods for preparing a liposome-bearing contrast agent that are suitable for the reproducible production of pharmaceutically acceptable liposome preparations in bulk (adequate viability, sterility, pyrogenicity, high iodine encapsulation).

A method for the preparation of contrast liposomes has been developed which i

overcomes all the above limitations. See WO 91/1603; P 40 135802. This method, called the ethanol-evaporation method, allows the reproducible production of liposomes in bulk under aseptic conditions. The original liposome suspension produced by this method is lyophilized to obtain a product with adequate shelf life. Prior to administration, the lyophilisate is resuspended in 135 mM mannitol solution to obtain a liposomal suspension which is infused after the filtration step.

Using contrast liposomes produced by this method, opacification of the liver and spleen is achieved. Based on initial biodistribution studies, iopromide was chosen as the liposomal contrast agent because its 7-day retention values in liver and spleen are significantly lower than those for iotrolan.

Although liposomes are a potentially important benefit in drug delivery systems such as contrast media, how? ............ are described above, the use of which in living organisms is limited by their own undesirable systemic side effects that may be observed after administration of liposomes. These systemic effects include, for example, transient hemodynamic depression, effects on blood pressure, heart rate, and ECG; depression in the number of blood cells. For example, it is known that liposomes can have undesirable hemodynamic effects when administered intravenously to animals, which is dangerous for the host animal.

In view of the occurrence of such systemic effects, it is not possible to use such liposome preparations for drug delivery. Surprisingly, it has been found that when the same liposomes are prepared but with the incorporation of an electrically charged component, systemic side effects are reduced or eliminated after administration.

Description of the figures in the accompanying drawings

Fig. 1 is a schematic diagram of an ethanol evaporation method for liposome production.

Figure 2 is the mean blood pressure (percentage of previous value) after intravenous infusion of liposomes made from different lipids (300 mg lipid / kg, 100 mg lipid / kg / min) and mannitol (300 mM), n = 6, means + SEM.

«Mannitol o DSPC / DSPG Q DSPC Fig. 3 is the total peripheral resistance (percentage of previous value) after intravenous infusion of liposomes made of different lipids (300 mg lipid / kg, 100 mg lipid / kg / min) and mannitol (300 mM), n = 6, means + SEM.

• mannitol or DSPC / DSPG Π DSPC

Figure 4 is the final diastolic pressure (EDP) after intravenous infusion of liposomes made from different lipids (300 mg lipid / kg, 100 mg lipid / kg / min) and mannitol (300 mM), n = 6, means + SEM.

* mannitol or DSPC / DSPG Q DSPC

The essence of the invention

When incorporating an electrically charged component into liposomes that have an undesirable systemic effect, the resulting liposomes have been found to reduce or eliminate the occurrence of unexpected or detrimental systemic effects that are otherwise observed when administered liposomes formed from the same lipid components but with electrically charged discharge. folders.

In particular, the addition of a negatively charged component, e.g., fatty acids such as stearic acid, to the liposome formulation significantly improves host tolerance to the formulation compared to when the same formulation is administered without the negatively charged component. Improved tolerance may be reflected in the reduction or elimination of systemic side effects produced by a liposome preparation in which no electrically charged component is present. These systemic effects include, eg, haemodynamic effects such as haemodynamic depression, changes in blood pressure, heart rate, systolic blood pressure, diastolic blood pressure or ECG intervals (PR, QRS, QT, Qtc), reflex tachycardia i, prematurated ventricular arrhythmias, increased respiration , sedation as well as changes in blood cell and platelet counts and deaths. It is recognized that these basic principles of improving tolerance to other detrimental structures by electric charge modifications can be applied to agents other than liposomes.

The present invention relates to an amount of electric charge that is effective to, e.g., reduce hemodynamic effects, improve tolerance or reduce side effects, in a liposome preparation. An effective amount of electric charge can be added to a liposome preparation by forming a liposome in the presence of an effective amount of electrically charged components or by adding an effective amount of charge to already formed liposomes.

Adverse or detrimental effects of the liposome preparation after administration can be reduced or eliminated by the addition of an electric charge, regardless of the composition of the liposomes administered. For example, a liposome preparation having systemic effects may already contain electrically charged components. In this case, the introduction of an effective amount of the electrically charged component can be performed in addition to the charged components that are already present in the liposomes. The charged introduced component may be of the same type or charge already present in the liposomes or of a completely different type or charge. The present invention thus relates to the addition of a charge to liposomes having charged components.

The charged component can be incorporated into liposomes in a variety of ways. Upon incorporation of the electrically charged component into liposomes, the charged component may become, e.g., a structural component of the lipid bilayer and / or be conjugated or non-covalently attached to the liposome component. For example, if the liposomes are formed from a mixture containing an effective amount of negatively charged stearic acid, the stearic acid can be incorporated into the lipid bilayer of the liposomes. However, an effective amount of the electrically charged component can also be added to an already formed liposome preparation or incompletely formed preparation, e.g., by conjugation or non-covalent attachment of the charged component to the surface of the lipid bilayer.

How the addition of the electrically charged component to the liposomes is made is not critical. A crucial feature is that the electrically charged component is present in the liposomes in an amount that is effective, e.g., to improve host tolerance, reduction

- the deleterious effects and / or the reduction of the haemodynamic effects produced by the administration of the liposome preparation. The electric charge can therefore be carried and incorporated into liposomes in a variety of ways, including, e.g., the addition of charged components such as acids, e.g., stearic acid, and salts of these components such as ammonium salts; coupling the charged groups to the components of the lipid bilayer; and the addition of bilayer-forming components with charges, either covalently coupled or adhered by any other force. Although not wishing to be bound by theory, the liposome of the present invention carries an electric charge on the outer surface of its bilayer.

For example, a liposome preparation containing phosphatidylcholine is known to have a deleterious hemodynamic effect. Its tolerance by the host organism can be improved by preparing a new batch of liposomes containing an effective amount of an electrically charged component added to phosphatidylcholine, e.g., an effective amount of electric charge can be carried by stearic acid. Alternatively, the charge can be introduced into liposomes that have already been formed. For example, an electric charge can be introduced to the outer surface of the lipid bilayer by chemical modification or by covalent or non-covalent addition of a charged group to its surface.

The addition of charge to the liposome preparation can also be accomplished by higher amounts of the preparation, which is administered to the host organism without the detrimental effects normally attributed to liposome administration. This is significant in that larger doses of the agent encapsulated in liposomes can be maintained. The addition of a charge to the liposomes can also improve the stability and storage of the liposome preparation, e.g., at temperatures above room temperature, such as 30 or 40 ° C.

The nature of the effective amount of the electrically charged component can be either positive or negative as long as the desired effect is achieved, e.g., to improve tolerance, reduce adverse effects, and / or reduce hemodynamic effects produced by administration of the liposome preparation. Negative charge is preferred. The charge can be, for example, ionic or electronegative. In principle, once a liposome preparation is known not to be tolerated by a host animal, e.g., as manifested by systemic side effects, the charged component may be selected and then incorporated into the liposome preparation to reduce or eliminate side effects associated with its administration. When preparing a series of liposome formulations having different amounts and types of charge, it should be routine to administer and determine how much and what type of charge is effective in, e.g., reducing hemodynamic effects and improving host tolerance.

The amount of effective electric charge can be carried by the molecule or charge carrier. The molecule must have one or more charged groups, eg a zwitterion. The charge can be placed in any placed charge carrier. The choice of charge carrier will depend on various factors, such as availability, bioavailability, and the desired effect. The charge carrier may be selected for other properties in addition to its ability to carry the desired charge. For example, the charge carrier may also have the function of a targeting agent, i.e., directing liposomes selectively to the desired type of tissue or cell in the host organism. The charge carrier can be, e.g., a biomolecule, polymer, lipid, protein, nucleic acid, carbohydrate, synthetic molecule, or any molecule or molecular structure that can be incorporated into a liposome bilayer and that exhibits an effective amount of electric charge. When a molecule is incorporated into a liposome bilayer, the molecule can become

-.l ,. .. a structural part of the liposome vesicle, e.g., to be integrated into the lipid bilayer or to be circumferentially attached to the liposome surface or as a component of the lipid itself. Furthermore, small compounds with another acidic group can be coupled to an anchor such as cholesterol, phosphatidylcholine, fatty acid, etc. located on the bilayer. The charge, preferably negative, can also be carried by amino acids or sugar acids coupled to any group capable of adhering to the bilayer (cholesterol, phosphatidylcholine, fatty acid, etc.).

The charge carrier can also be any charge-carrying lipid, e.g., including sterols, fatty acids, glycerol esters, sphingosine, terpenes, or lipids in general, e.g., TEEXTBOOK OF CLINICAL CHEMISTFY (1986), N.W. Tietz, publisher, W. B. Saunders Co., Chapter 7, pp. 829-900. A preferred example of a fatty acid having a negative electric charge that can be used in the present invention in an effective amount, e.g., to reduce the hemodynamic effects of a parenterally administered liposome preparation. Other fatty acids may include, for example, palmitic acid, arachidonic acid, linoleic acid, linoleic acid, and / or mixtures thereof.

The charge carrier may also be a protein which is, for example, capable of integrating into a lipid bilayer and attaching circumferentially to a surface. The charge of a protein can be produced by amino acids by carbohydrate bonds, non-protein groups attached to or attached to the protein. The protein may also be a protein having a lipid group. The protein may be selected for its desired charge and also for its ability to direct liposomes to a specific tissue.

AND

The amount of charge incorporated into the liposome must be

i. ¹ m » ¹ ·· * 'u í ¹¹¹ such that this amount is effective in reducing systemic side effects such as hemodynamic depression and / or tolerance. The amount of charge can be varied by changing the molar ratio between the charged component and the other components of the liposome. For example, the charged component can be added to the phosphatidylcholine-containing liposome by the addition of stearic acid.

The amount of charge in the liposome can be achieved by adjusting the molar ratio between the two components to the desired amount; eg phosphatidylcholine / stearic acid, 9: 1.

Improved host tolerance to the liposome preparation and subsequent elimination or reduction of systemic effects can be achieved by incorporating a charged component into the liposome vesicle. This improvement, when mediated by cell surface changes induced by the charged component, can also be produced using agents or methods that modify the surfaces.

For example, already formed liposomes having a deleterious systemic effect can be enzymatically or chemically processed to alter the surface properties of the liposome and to improve its tolerance upon administration. Chemical modification may include, for example, the reaction of liposome reducing agents, oxidizing agents, or coupling charged components to its surface. These methods are performed in a conventional manner, but the procedure set forth herein is to achieve the reduction or elimination of adverse effects.

Liposomes can be prepared according to methods common in the art, e.g., which are reviewed in WO86 / 00238. In addition, liposomes can be prepared according to EP69307; U.S. Pat.

5110475, which, for example, describes a process for the production of an aqueous dispersion, comprising removing the liquid (s) from an optional multiphase liquid mixture by membrane distillation; and WO86 / 00238, which describes extrusion techniques for liposome production having a substantially unimodal and defined size distribution.

If the liposome is prepared with an electrically charged component, it is possible that the electrically charged group may be oriented in the liposome in several different directions; for example, by fitting the interior of the liposome vesicle and fitting the exterior at the interface between the aqueous medium and the lipid. Liposomes having an electrical charge on the outside are preferred, e.g., the outer surface. It may be desirable to separate, for example, internally charged liposomes from externally charged liposomes. This can be done by any method known in the art, including column chromatography using, for example, charged or otherwise modified supports, electrophoresis, electrical separation processes, and other processes that separate structures based on charge, charge density, or charge orientation. In general, liposome formulations containing an electrically charged component may be subjected to conditions that eliminate orientations that are not desirable for the purposes of the present invention.

The active agent can be encapsulated in liposomes by conventional procedures. A liposome preparation having an effective amount of an electrically charged component may contain or encapsulate an active agent. Encapsulation can be performed by methods known in the art, e.g., by forming liposomes in the presence of an active agent under conditions in which lipid bilayers form around and encapsulating the active agent, introducing the active agent to the already formed liposomes by fusion with a liposome, cell, or other structure. containing the active agent, e.g., such fusion can be performed using PEG, electric current, lectin, and other methods well known in the art. Furthermore, liposomes having an active agent can 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 lipid rearrangement, thereby allowing the active agent to enter and effect a pressure drop.

the active agent may be encapsulated in a liposome preparation having an effective amount of an electrically charged component, including, for example, conventional active agents such as those used for therapy, including agents as disclosed in Physicians Desk References, Medical Economics Data,

X,

44th edition 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 units, ferromagnetic units, radioisotopes and other common structures or compounds. Such an active agent may be one that is used and recognized by those skilled in the art.

Liposomes can be administered by conventional methods. For example, the liposome preparation may be administered parenterally, e.g., intravenously through an arterial or venous catheter, by syringe injection, but may also be administered orally or via the gastrointestinal tract. A typical amount of liposome preparation is, e.g., 100 to 200 mg lipid / kg, depending on how much agent is intended for administration and how much is encapsulated in the liposome. The amount of liposome preparation to be administered can be determined routinely by methods known in the art. Liposomes may be administered in a single injection or 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 liposomes containing a charged component are well tolerated by the host, the infusion rates and amounts of liposomes administered may be increased over the amounts typically used for liposome formulations that have deleterious effects.

Liposomes can be administered to any organism, e.g., mammals such as humans, rats, mice, dogs, rabbits, cows, horses, but also birds, amphibians and reptiles, the purpose of which may be to diagnose, treat, medicate, sedate or prevent disorders, but they can 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 potential use in humans. The method of the present invention, e.g., the reduction of hemodynamic effects, the improvement of host tolerance, or the reduction of the deleterious effects associated with liposome administration, can be performed in any organism, such as the above-mentioned organisms. Liposomes can be used for any diagnostic or therapeutic use, including, for example, MRI, ultrasound, and nuclear medicine.

Without further elaboration, it is expected that one skilled in the art can, using the preceding description, utilize the invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as illustrative and not restrictive.

In the preceding and following examples, all temperatures are given uncorrected in degrees Celsius and, unless otherwise indicated, all parts and percentages are by weight.

The entire disclosures of all applications, patents, and publications already cited and cited below are incorporated herein by reference.

Examples of embodiments of the invention

Example 1

The method of preparation of iopromide liposomes by the ethanol evaporation method is shown in Fig. 1. An ethanolic solution of membrane lipids (standard = 92.5 mg / ml phosphatidylcholine / cholesterol / stearic acid 4: 5: 1 molar) is mixed with the iopropide solution obtained by dilution aliquots of Ultravist ^R 370 solution with 20 mM tromethamine buffer, pH 7.4 to give a final iodine concentration of 46.25 mg / ml. After the stirring period, the ethanol is removed from the mixture under reduced pressure until a liposomal suspension is obtained. This suspension is then filtered (5 μm and 1.2 μm), filled into vials and lyophilized to obtain a stable storage form.

Prior to administration, such lyophilizates are first rehydrated with 4 ml of a 135 mM mannitol solution per g of dry substance. The resulting liposomal suspension is then filtered (20 μm glass fiber pre-filter and 5 μm CN main filter) to remove non-liposomal structures. These structures are loop-shaped or spoon-shaped bilayer formations partially larger than 10 μm. These structures have been shown to be removed by the above filtration step.

The suitability of this method of preparation for the reproducible production of larger amounts of liposomes has also been demonstrated. Preparation of 16 batches (each 1000 ml of liposomal suspension) resulted in encapsulation of 42.1 + 4.9% and an average diameter of 458 + 55 nm in the resuspended material (2 ml of 135 mM mannitol solution per g of lyophilisate).

The method is potentially suitable for performance under aseptic conditions. The residues used to make the liposomes can be ultra- or sterile filtered. \

Composition of the resuspended lyophilisate (4 ml 135 mM

mannitol solution and g lyof i 1izátu) is given below range): iopromide 145.5 mg phosphatidylcholine (PC) 41.3 mg DL-α-tocopherol 0.04 mg cholesterol (CH) 25.0 mg stearic acid <SS) 3.7 mg

(without tromethamine IN HCl disodium edetate mannitol water for inj.

3.7 mg 25.4 mg 0.15 mg 21.8 mg d 1 ml

The molar lipid mixture PC / CH / SS 4: 5: 1 leads to the highest degree of encapsulation (42.5 + 3.7%, n = 6) as well as the smallest mean diameter (439 ± 87 nm) in the resuspended material. These values are in close agreement with the values obtained when the batch sizes were increased 10-fold (see 3.1). The molar ratio of these membrane components as well as the type and quality of each lipid component were optimized with respect to these target parameters.

The use of other nonionic monomeric contrast agents (iopamidol, iohexol and ZK 119095) rather than iopromide did not provide an improvement in iodine encapsulation. When the nonionic dimer iotrolan was encapsulated, higher weight ratios (mg iodine / mg lipid) were achieved. This compound was excluded from further studies due to unacceptable long retention of liposomal iotrolan in the liver and spleen of the test animal (biodistribution study) and current pathological changes in the liver.

In the ethanol-evaporation process, the iodine / lipid ratio of 1: 1 in the original solutions (see Fig. 1) proved to be the most suitable for the optimal use of lipid (i.e. with the highest possible weight ratio of iodine to lipid) in the final preparation. In the resuspension medium tested, 135 mM mannitol leads to the highest encapsulation values compared to double distilled water, various buffer solutions and lower concentrations of mannitol solutions.

Example 2

For the initial stability study, batches of liposome lyophilizates were prepared under aseptic preparation conditions. Resuspended liposomes were characterized in terms of size (QUELS), iodine encapsulation, pH and microscopic appearance. Microbial status has not been determined. According to the results obtained in this study (six months of storage), liposomal lyophilisates (PC / CH / SS 4: 5: 1) can be stored at temperatures up to 30 ° C without noticeable changes in quality. However, after 6 months at 40 ° C, a significant increase in size and decrease in pH was observed in the resuspended material. These changes were associated with an unpleasant odor coming from the resuspended material.

In a stability study of (unfiltered) resuspended material, no significant changes in size (QUELS), encapsulation or pH were observed during the 24 h period. However, possible changes in the microscopic appearance of the product were not observed in this study; thus, the microscopic appearance of the resuspended lyophilizates was monitored separately both before and after filtration during a time period of 8 hours. No significant changes were observed in any of the tested materials.

Example 3 (A) Methods (i) Surgical Procedures

Six to seven days before the study, 6 normal healthy adult canine hybrids were selected for similar weight (14.3 - 15.6 kg) and anesthetized with 35 mg / kg iv sodium pentobarbital, shaved and prepared for sterile surgery by washing the area of the intended incision. twice with a BetadineR surgical brush and then by staining with Betadine ^R solution. Dogs were intubated using a manned endotracheal tube and mechanically respirated with room air using a Harvard animal respirator (model 607), placed supine on a surgical table and covered for surgery using sterile techniques. A small (about 1 inch) incision was made between the sternal manuber and the larger right humerus tubercle. Using blunt dissection, the right omocervical artery and the right jugular vein were isolated for cannulation. The artery was cannulated using a Tygom microtube (0.05 ID, # S-54-HL). The catheter was secured by suturing 2-0 silk and exteriorized in front of the subcutaneous tunnel to the back of the neck. In a similar manner, the right jugular vein was catheterized using a Tygon microtube (0.04 ID, # S-54-HL) and also exteriorized to the back of the neck. Both arterial and venous catheters were filled with sterile, heparinized (600 U / ml) saline, sutured, and stained with betadine to reduce infection. The exposed catheters were secured with small plastic pouches and sealed in a cotton collar reinforced with adhesive tape. Dogs were given standard intamuscular injections of Combiotic to reduce the risk of infection and allowed to recover from surgery.

(ii) Experimental procedures

On the day of the study, the dogs were introduced into two individual mobile loops in the laboratory. The dogs were shaved on their ankles and instrumented with II electrocardiographic (ECG) leads using surface electrodes and conductive connections. Special collars were removed, blood was drawn from each catheter, and the catheters were washed with sterile saline. The arterial catheter was connected to a pressure transducer and calibrated using a mercury manometer. 10 ml venous blood samples were taken from the venous catheter using a sterile Sarsdtet monovette tube containing EDTA. After an equilibration period of 20-40 minutes, the dogs were infused with DMPC / DPPC, prepared by the interdigitation / fusion method (see WO 91/10422; EP 510096), or DPPC at 2 ml / minute for about 25 minutes. The monitored parameters were direct arterial blood pressure and surface ECG leads II.

(iii) Drug administration

DMPC / DPPC or DPPC liposome vesicles were administered intravenously as an opaque suspension at a rate of 2 ml / minute at a concentration of 1 mg / kg / ml until a total dose of 50 mg / kg was administered. All drugs were placed in a sterile 60 ml syringe and infused using a Razel infusion pump. Any remaining suspension in the catheter was washed into the dog using sterile saline at the same infusion rate.

(iv) Statistical analysis

Selected CBC counts after vesicle administration were compared to baseline using a paired sample t-test. The differences were considered significant at ý 0.05.

(B) Results

The effects of DMPC / DPPC or DPPC infusions on heart rate (HR), mean blood pressure (MAP), systolic pressure (SP), diastolic pressure (DP), ECG intervals (PR, QRS, QT, Qtc) and selected blood cell count listed in Tables 1-4. complete blood cell count (CBC) values are included in the appendix.

(i) Blood pressure

Intravenous administration of DMPC / DPPC or DPPC vesicles causes a significant reduction in systolic, diastolic and mean arterial pressure.

(ii) Systolic pressure (Table 1)

Systolic blood pressure decreases by an average of 37% during the DMPC / DPPC infusion. A greater decrease in systolic blood pressure was also observed during the infusion of DPPC vesicles with a 55% decrease in systolic blood pressure. Because systolic blood pressure returned close to baseline levels in only one dog from each group during the observation period, this decrease was determined to be transient. levels within 30 minutes after administration of the vesicles.

(iii) Diastolic pressure (Table 2)

Diastolic pressure decreases by 39% in the DMPC / DPPC group by 48% in the DPPC group during vesicle infusion. One dog in the DMPC / DPPC group maintains control diastolic blood pressure levels during the infusion and 1 dog in the DPPC group demonstrates minimal effects on diastolic blood pressure. On average, the effect of iposomal vesicles on diastolic pressure was transient, as mean diastolic pressure returned to levels close to control within 30 minutes of vesicle administration.

(iv) Mean pressure (Table 3)

The effect of liposome vesicles on mean arterial pressures was much more pronounced in the DPPC group with lower and much longer lasting pressure drops. Dogs in the DMPC / DPPC group showed a mean mean pressure drop of 38% during the infusion, but returned to values close to control at the end of the infusion. However, dogs receiving DPPC vesicles experienced a 52% decrease in mean blood pressure and did not return to near control levels within 30 minutes of the end of the infusion.

to

1) (v) Heart rate (Table 4)

The effect of liposome vesicles on heart rate in conscious dogs was variable. Although mean heart rate values increased in both groups, its manifestation as marked tachycardia was observed in only one dog in each group. The other 2 dogs in each group maintained a heart rate equal to or lower than the control during the infusion and for 45 minutes following the follow-up period.

(vi) Electrocardiographic intervals (Table 5)

Infusion of DMPC / DPPC or DPPC liposome vesicles has no physiologically significant effect on ECG intervals. No changes in PR and QRS intervals were observed (Table 3A and 3B, p. 13). Small increases in QT (Table 3C, p. 14) have been reported, but do not represent a major physiological effect sufficient to be significant.

(vii) CBC values (Table 6)

Infusion of DMPC / DPPC liposome vesicles.lu significantly increases white blood cell counts and slightly reduces circulating platelets, red blood cell counts and hematocrit were not affected (Table 4A, p.15). DPPV vesicle infusion also had little effect on red blood cell count and hematocrit, however, it significantly reduced platelet count and slightly reduced white blood cell count (Table 4B, p.15).

. (C) Discussion

The present study, in which awake dogs were monitored for acute hemodynamic and ECG changes, shows that intravenous infusions of DMPC / DPPC and DPPC liposome vesicles cause a sudden and significant drop in blood pressure leading to many physiological reactions including lethargy and mild sedation in all dogs with vomiting. , appearing in only one dog. No physiologically significant changes in ECG parameters were observed, but white blood cell and circulating platelet counts were affected.

Most of these overt reactions could be attributed to a transient but significant reduction in blood pressure resulting from a possible anaphylactic reaction to the test substance. Changes in white blood cell count and circulating platelet count could be related to such a reaction.

Example 4 (A) Methods (i) Surgical Procedures

Six to seven days before the study, 5 normal healthy adult canine hybrids of both sexes were selected for similar weight (13.7 - 14.1 kg) and anesthetized with 35 mg / kg iv sodium pentobarbital, shaved, and prepared for sterile surgery. washing the area of the intended incision twice with Betadine ^R surgical brush and then staining with Betadine ^R solution. Dogs were intubated using a manned endotracheal tube and mechanically respirated with room air using a Harvard animal respirator (model 607), placed supine on a surgical table and covered for surgery using sterile techniques. A small (about 1 inch) incision was made between the sternal manuber and the larger right humerus tubercle. Using blunt dissection, the right omocervical artery and the right jugular vein were isolated for cannulation. The artery was cannulated using a Tygom microtube (0.05 ID, # S-54-HL). The card was secured with 2-0 silk sutures and exteriorized in front of the subcutaneous tunnel to the back of the neck. In a similar manner, the right jugular vein was catheterized using a microtube

Tygon (0.04 ID, # S-54-HL) and also exteriorized to the back of the neck. Both arterial and venous catheters were filled with sterile, heparinized (600 U / ml) saline and sealed with sterile caps. 2 incisions were closed with silk sutures and stained with betadine to reduce the risk of infection. The exposed catheters were secured with small plastic pouches and sealed in a cotton collar reinforced with adhesive tape. Dogs were given standard intamuscular injections of Combiotic to further reduce the risk of infection and allowed to recover from surgery.

(ii) Experimental procedures

On the day of the study, the dogs were introduced into two individual mobile loops in the laboratory. The dogs were shaved on their ankles and instrumented with II electrocardiographic (ECG) leads using surface electrodes and conductive connections. Special collars were removed, blood was drawn from each catheter, and the catheters were washed with sterile saline. The arterial catheter was connected to a pressure transducer and calibrated (0 - 200 mmHg) using a mercury manometer. 10 ml venous blood samples were taken from a venous catheter using sterile Sarstedt monovette tubes containing EDTA. After an equilibration period of 20-40 minutes, the dogs were infused with EPC at a rate of 2 ml / minute for 25 minutes. The monitored parameters were direct arterial blood pressure and surface ECG leads II.

(iii.) Drug administration

EPC liposome vesicles were administered intravenously as an opaque suspension at a rate of 2 ml / minute at a concentration of 1 mg / kg / ml until a total dose of 50 mg / kg was administered. All doses were placed in a sterile 60 ml syringe and infused using a Razel infusion pump. Any remaining suspension in the catheter was washed into the dog using sterile saline at the same infusion rate to ensure that each dog was given the total dose.

(B) Results

The effects of EPC liposome infusions on heart rate (HP), mean blood pressure (MAP), systolic pressure (SP), diastolic pressure (DP), ECG intervals (PR, QRS, QT, Qtc) and total blood cell counts (CBC) are described below. Peak changes in blood pressure and heart rate occur during the infusion of liposome vesicles.

(i) Blood pressure

Intravenous administration of EPC liposome vesicles causes a significant reduction in systolic, diastolic and mean arterial pressure in 3 of 5 dogs tested.

(ii) Systolic pressure (Table 7)

Systolic pressure decreases by an average of 36% in a group of 5 dogs. These dogs showing acute hemodynamic depression (n = 3) had systolic pressures ranging from 50% to 62% below basal levels. These changes in systolic blood pressure were transient as systolic blood pressure values returned to normal levels at the end of the EPC infusion.

(iii) Diastolic pressure (Table 8)

Diastolic pressure is reduced by 36% in 5 tested dogs.

Those 3 dogs that showed a significant reduction in blood pressure had decreases in diastolic pressure ranging from 56% to 61% below basal levels. Although these pressure drops were difficult, they were also transient because the pressure values returned to levels close to basal levels at the end of the EPC vesicle infusion.

(iv) Mean pressure (Table 9)

The effect of infusion of EPC liposome vesicles had no observed effect in 2 of 3 tested dogs; however, 3 of the 5 dogs tested demonstrated significant decreases in mean arterial pressure. The mean decrease in mean pressure was 35%, but those dogs with significant hemodynamic depression showed mean pressure drops ranging from 57% to 61% below basal levels. Although these decreases were severe, they were also transient as the pressure values returned close to basal values at the end of the EPC vesicle infusion.

(v) Heart rate (table Ϊ0)

The effect of EPC liposome vesicles on heart rate in conscious dogs was closely related to the physiological response to a decrease in blood pressure. Those dogs that were monitored hemodynamically during EPC infusion demonstrated marked tachycardia during EPC infusion followed by brief bradycardia with return of heart rate to basal levels in all dogs within 30 minutes after EPC infusion. The 2 dogs that did not show hemodynamic adjustments during the EPC infusion maintained relatively constant heart rates during the study.

(vi) Electrocardiographic intervals (Table 11, A-D)

Infusion of EPC liposome vesicles has no physiologically significant effect on ECG intervals. No changes in QRS duration were observed.

The changes occurring in the PR, QT, and QTc intervals were related to the changes in heart rate that occur in 3 psu, clearly due to EPC infusion. None of these changes were determined to be physiologically significant due to their transient nature.

One dog developed spontaneous arrhythmias (in the form of ₍ isolated prematurated ventricular contractions) occurring during EPC infusion, examples of which are shown in Figure 2.

(vii) Mean CBC values (Table 12)

Infusion of EPC liposome vesicles does not significantly increase blood cell count or hematocrit. White blood cell counts were inconsistent. Platelet counts were clearly suppressed in those 3 dogs that developed hemodynamic depression (USDA # 97684) showed platelet depletion but not below the normal range.

(C) Discussion

Intravenous administration of EPC liposome vesicles to awake dogs leads to hemodynamic depression. The platelet depletion observed in this study corresponds to a similar effect observed in the previous study. In both studies, platelet depletion appeared to be closely related to the severe decreases in blood pressure resulting from liposome infusion. The apparent reactions observed during liposome infusion (platelet depletion, significant hypotension and overt tachycardia) are most likely related to the anaphylactic response. Spontaneous arrhythmias observed in one dog given EPC vesicles may be a response to a sudden hypotensive effect with reflex catecholamine activation.

Example 5

The liposomes of the present invention containing the electrically charged stearic acid component (SA 504/02079 and SA 504/300591) were administered to healthy dogs.

(A) Methods (i) Surgical procedures

Six to seven days before the study, 6 normal healthy adult canine hybrids of both sexes were selected for similar weight (12.3 - 15.1 kg) and anesthetized with 35 mg / kg iv sodium pentobarbital, shaved, and prepared for sterile surgery by washing the area. intended incision twice with a Betadine ^R surgical brush and then by staining the endotracheal tube area and mechanically respirating with room air using a Harvard animal respirator (model 607), placed in a supine position on a surgical table and covered for surgery using sterile techniques. A small (1 inch) incision was made between the sternal manuber and the larger tubercle of the right humerus. Using blunt dissection, the right omocervical artery and the right jugular vein were isolated for cannulation. The artery was cannulated using a Tygom microtube (0.05 ID, # S-54-HL). The card was secured with 2-0 silk sutures and exteriorized in front of the subcutaneous tunnel to the back of the neck. In a similar manner, the right jugular vein was catheterized using a Tygon microtube (0.04 ID, # S-54-HL) and also exteriorized to the back of the neck. Both arterial and venous catheters were filled with sterile, heparinized (600 U / ml) saline (1 ml) and sealed with sterile Betadin ^R staining to reduce infection. The exposed catheters were secured with small plastic pouches and sealed in a cotton collar reinforced with adhesive tape. Dogs were given standard intamuscular injections of Combiotic to reduce the risk of infection and were allowed to recover from surgery.

(ii) Experimental procedures

On the day of the study, the dogs were introduced into individual mobile loops in the laboratory. The dogs were shaved on their ankles and instrumented with II electrocardiographic (ECG) leads using surface electrodes and conductive connections. Special collars were removed, blood was drawn from each catheter, and the catheters were washed with sterile saline. The arterial catheter was connected to a pressure transducer and calibrated using a mercury manometer. 10 ml venous blood samples were taken from a venous catheter using a sterile Sarstedt monovette tube containing EDTA. After an equilibration period of 20-40 minutes, the psum was infused with SA 504/020791 or SA 504/300591 at a rate of approximately 2 ml / minute for exactly 30 minutes. The monitored parameters were direct arterial blood pressure and surface ECG leads II. Heart rate was determined from ECG articulation.

(iii) Drug administration

SA 504/020791 or SA 504/300591 iodine-containing liposome vesicles were administered intravenously as an opaque suspension at a rate of 2 ml / minute to a final volume of 4.2 ml / kg and 4.5 ml / kg. The infusion rate was adjusted so that the total infusion time was 30 minutes. All doses were placed in sterile syringes and infused using a Razel infusion pump. Any remaining suspension in the catheter was washed into the dog using sterile saline at the same infusion rate.

Test substances were prepared as follows: SA .504 / 020791: 17.6 ml of mannitol solution (135 mM) was added to each test substance vial and allowed to settle for at least 10 minutes. The vials were then shaken vigorously and allowed to settle for an additional 10 minutes. After repeating the shaking procedure, the suspensions were visually evaluated for agglomerates (if agglomerates are present, the above procedure was repeated). The suspensions were then drawn into 60 ml syringes and filtered through a sterile Schering AG filter device. Each animal received 4.5 ml / kg.

SA 504/300591: The procedure was exactly the same as above, except that 18.0 ml of mannitol solution (135 mM was used per vial and each animal received 4.2 * ml / kg.

(iv) Statistical analysis

Hemodynamic parameters, ECG intervals, and selected complete blood cell counts were compared to baseline values prior to treatment using a paired sample t-test.

Differences were considered significant at <. 0.05.

(v) Liposome preparation

SA 504/020791 and SA 5041/300591 were prepared according to the following instructions.

!

Solution 1 was prepared by dissolving 54.6 g of lipoid S100, 33.0 g of cholesterol and 4.9 g of stearic acid in 9.5 ml of 96.5% ethanol. 2000 ml of solution 2 was prepared by combining 250 ml of iopropid solution with 9.5 ml of 20 mM Tris HCl, pH 7.5 and 9.5 ml of 96% ethanol. With stirring, solution 1 was mixed with solution 2. The ethanol was removed in vacuo and the remaining solution was frozen. 5 g of frozen material was resuspended in 8.8 ml of 135 mM mannitol. The resuspended liposome preparation was typically injected at a dose of 300 mg iodine / kg.

(B) Results

Effects of SA 504/02091 or SA 504/300591 infusion on heart rate (HR), mean blood pressure (MAP), systolic pressure (SP), diastolic pressure (DP), ECG intervals (PR, QRS, QT, Qtc) and selected the values of the number of blood cells are given in Tables 13-16.

I * (i) Blood · pressure (Table 13)

Intravenous administration of SA 504/020791 or SA 504/300591 vesicle does not cause any significant changes in systolic, diastolic and mean arterial pressure. As can be seen from Table 13C, the control mean arterial pressure in dogs treated with SA 504/300591 104 + 6 was relatively constant for the duration of the test. Similar results were obtained with test substance SA 504/020791.

(v) Heart rate

Liposome vesicle preparations did not have a significant effect on heart rate in conscious dogs. Of the five dogs treated with SA 504/300591, three dogs had a reduced heart rate during the infusion and two had an increased heart rate. In one animal that received SA 504/020791 (dog # 10003), heart control was particularly high. This animal responded highly to each stimulus in the room (i.e., laboratory staff), but calmed down as the experiment progressed. This was more likely due to acclimatization and was not a drug effect.

(vi) Electrocardiographic intervals (Table 5)

Infusion of either test agent has no physiologically significant effect on ECG intervals. No changes in PR, QRS, QT or QTC intervals were observed, (vii) CBC values

Infusion of either test agent had no significant effect on RBC or WBC counts. Although WBC levels were not significantly affected, there appears to be a tendency to increase WBC after administration of test agents. If data from both groups are combined (ie, n = 8), then static significance is achieved to increase WBC (p = 0.026). In 5 experiments, platelet values before and after the test substances were categorized as bud

JU adequate, increased or decreased. In all 5, platelet values were characterized as adequate. In the remaining 3 experiments, the values of the platelet counts (tice / ml) were as follows:

treatment dog # control 45 ' SA 504/300591 10174 435 376 SA 504/020791 10003 198 243 SA 504/020791 98701 210 298

(C) Discussion

The present studies of liposome preparations performed on awake dogs with DMPC / DPPC, DPPC and EPC liposome vesicles demonstrate a dramatic, transient hypotensive response in about 40-60% of the animals tested. The present study, in which awake dogs were monitored for acute hemodynamic and ECG changes, shows that intravenous infusions of SA 504/300591 and SA 504/020791 liposome preparations do not produce any significant changes in hemodynamic variables (blood pressure and heart rate), ECG intervals or EBC and WBC values and are therefore well tolerated and free from adverse haemodynamic effects.

Example 6

Two liposome preparations were prepared and tested in a dog model for hemodynamic effects.

Formulation 1: Liposomes containing only phosphatidinylcholine at a concentration of about 25 mg / ml.

Preparation 2: Liposomes with phosphatidylcholine and stearic acid. The molar content was about 9: 1 at the total concentration

1ipidu as at 70 mg / ml.

These preparations were infused intravenously into five dogs, each at a dose of about 50 mg lipid per kg body weight (Preparation 1) and about 30 mg lipid per kg (Preparation 2).

The following effects were observed in dogs.

Formulation 1: Transient decrease in blood pressure associated with significant reflex tachycardia in 3 of 5 tested animals. Other effects observed in these 3 animals include: prematurated ventricular arrhythmias during infusion and shortly after administration in 1 dog and increased respiratory respiration in 2 additional dogs. There were no physiologically significant changes in ECG intervals in any of the dogs. Platelet counts decreased in 4 of 5 dogs who received the product. These dogs exhibiting hemodynamic depression appeared to be attenuated and had the most consistent platelet depletion compared to dogs that did not show a decrease in blood pressure.

Formulation 2: Intravenous administration of this preparation had no adverse effects on any of the five treated animals.

Example 7

In this example, the cardio-hemodynamic effects of liposomes prepared from saturated uncharged DSPC phospholipid alone or in combination with negatively charged DSPG phospholipid (9: 1) were tested.

Preparation of liposomes: The liposomes used in this study were prepared by continuous high pressure extrusion.

Briefly, an ethanolic solution of the appropriate lipid or lipid mixture would be placed on the wall of a round bottom flask. The resulting lipid film was dispersed in a 300 mM mannitol solution to obtain a lipid concentration of approximately 100 mg / ml. The resulting MLV dispersions were subsequently extruded using a high pressure ^{extruder (Maximator R} model HPE 10.0-250, Schmidt, Kranz & Co. Zorge, Germany). Each batch was then extruded 10 times through two superimposed polycarbonate membranes (Nucleopore, Tlibingen, Germany) of decreasing orifice size (1.0 - 0.4 and 0.1 μm) to give a total of 30 filter passages for each formulation. At the end of the extrusion process, the obtained liposomal preparations were filtered through sterile filter holders (0.4 or 0.2 μm pore size, cellulose acetate, Sartorius Gottingen, Germany) into sterile glass vials which were sealed under aseptic conditions.

Lipid substances

All phospholipids were stored below -20 ° C and used without further purification.

Distearoylphosphatidylcholine (DSPC - batch LP-04-013-114219 and -116298) and distearoylphosphatidyl-glycerol (DSPG - batch LP-04-017-115751) were obtained from Sygen, Liestal, Switzerland.

Liposome size was determined by photon correlation spectroscopy (PCS) using a submicron part ic1e-sizer, autodilute model 370, Nicomp Instr. Corp., Santa Barbara, CA, USA.

The osmolality (mOsm / kg) of the sample and the pH values were determined with an automatic freezing point osmometer (Knauer, Berlin, Germany) and a pH meter of 761.

Calimatic (Knick, Berlin, Germany).

Animal experiments

Eighteen male rats (strain: Han-Wistar, cov: Schering SPF, standard diet and breeding conditions), weighing 360 to 430 g, were randomly divided into three groups with n = 6 animals per group.

Rats were anesthetized with pentobarbital 60 mg / kg intraperitoneally. The trachea was cannulated to facilitate spontaneous respiration. Body temperature was maintained at 38 +

0.5 ° C using a heated operating table and a heating lamp.

Left ventricular pressure and femoral artery blood pressure were recorded via polyethylene-filled catheters connected to Statham pressure transducers. Cardiac output was determined by thermodilution (thermistor catheter in the abdominal aorta, injection of ice-cold saline solution into the right atrium). Animals were heparinized i.v. bolus 400 IU / kg.

Rats were monitored to record / calculate the following parameters (BPsyst. (Systolic blood pressure, mmHg), BPmedium (mean blood pressure, mmHg), BPdiast. (Diastolic blood pressure, mmHg) HR (heart rate, 1 / min), CO ( minute output / 100 g body weight, ml / min / 100 g), LVEDP (left ventricular final diastolic pressure, mmHg), TPR (total peripheral resistance, dyn * s * cm ^-5 10 ³ ), ECG (electrocardiogram, ms) and dP / dt max (increase in left ventricular pressure rate maximum, mmHg / s).

Surgery and instrumentation were followed by a 30-min equilibration period, at the end of which were pre-treatment values. Data were recorded 45 min after liposome or mannitol administration (volume control). Each animal was treated only once.

Rats were infused with a liposome dose containing a total of 300 mg lipid / kg body weight and at an injection rate of 100 mg lipis / kg / min into the left femoral vein. Controls received an equal volume of 300 mM mannitol solution. The liposome preparations evaluated were made of DSPC or DSPC / DSPG.

Statistics

Mean percentages versus baseline values were calculated (except for LVEDP, which was expressed as the absolute change from baseline values before treatment). The data were compared with a control group. The Bartlett test was performed to test the same variations and one-way analysis of variance (ANOVA) was used to demonstrate statistical differences. Calculations of statistical significance were performed using Student's t-test. a p value below 0.05 was considered statistically significant. The values in the figures are expressed as the mean percentage of changes from the original value ± SEM.

Results

Control experiments

Mannitol 300 mM (volume control) does not cause a significant deviation of the cardio-hemodynamic parameters from the original values. bMedium increases by a maximum of 1.8% (Fig.2),

BPsyst. by 2.3% and BP diast. decreases by -2.2% in the first 10 minutes after administration. HR decreases by -3.4%. TPR (Fig.3) and LVEDP

J J (Fig. 4) responds with a short-term increase of 5.5% and 2.8 mmHg (Table 17). Contract i 1ita decreases by -3.3 X.

DSPC liposomes

The use of saturated DSPCs leads to neutral, gel-like liposomes that lead to significant cardiac and hemodynamic disorders when infused at a dose of 300 mg lippid / kg into rats. Bmedium and TRP were significantly reduced by -53.7% and -46.3% (Figures 2 and 3). The contract was reduced by -34.7 X 5 minutes after the start of the infusion. During this time, LVEDP (Fig. 4) was significantly increased (p <0.05). All of these effects have been associated with severe cardiac arrhythmias. These were ventricular extrasystole, ectopic beats, AV conduction delay, and ventricular conduction failure. However, HR and CO were only weakly affected during the first 10 minutes after administration. At the end of these 10 min periods, BP and contract returned to baseline. However, HR begins to increase slightly above the baseline level (about 10 X) as well as the contract and 1. By the end of the 45 min observation period p.a. contractility was increased by +66.3 X above baseline (p <0.01). However, TPR does not return to baseline at the end of the observation period.

DSPC / DSPG liposomes

The addition of a negatively charged phospholipid (10 X mol) to DSPC significantly reduces the risk of cardio-hemodynamic side effects of DSPC. PB and TPR decrease after a short transient increase by -33.3 X and -35.5 X of the original value (Figures 2 and 3). The contract i 1 i ta decreases by -14.3 X during the first 10 minutes after application. It returns to normal values and was increased in 30 min p.a. HR is also consistent with increases at later time points. Electrocardiographic changes occur in four of six rats during liposome infusion. the effects were interpreted as conductive and non-conductive atrial premature contractions in three animals and in one animal as a complex arrhythmia produced by paired atrial premature contractions. All ECG effects occur after the infusion.

The addition of a negative charge to the DSPC liposomes significantly improves the cardio-hemodynamic side effects of the preparation in rats.

The foregoing examples may be repeated with similar success, substituting the generically or specifically described reactants and / or operating conditions of the invention for those used in the preceding examples.

From the foregoing description, one skilled in the art can readily recognize the essential characteristics of the present invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.

Table 1

A.Systolic pressure

Treatment dog# basis infusion 0 ' 15 ' 30 ' 45 ' 'DMPC / DPPC 97547 137 140 145 135 135 145 ir 97481 145 60 115 125 125 130 you 97557 120 55 85 115 108 100 av. + S.E. 134 ± 7 85 + 28 115 + 17 125 + 6 123 + 8 125 + 13 DPPC 9751 1 125 85 95 100 120 120 II 97454 140 55 70 115 115 120 > 1 97502 150 45 50 100 125 115 av. + S.E. 138 + 7 62 + 12 72 ± 13 105 + 5 120 + 3 118 + 2

Table 2

Iast.Diastolic pressure

Treatment dog# basis infusion 0 ' 15 ' 30 ' 45 ' DMPC / DPPC 97547 120 140 120 115 105 120 you 97481 100 35 90 90 80 80 II 97557 90 35 60 85 65 75 av. + S.E. 103 + 9 . 63 + 28 90 + 17 97 + 9 83 + 12 92 + 14 DPPC 9751 1 90 70 70 80 85 85 II 97454 80 25 45 75 75 75 H 97502 80 35 ^; 40 75 85 75 prúm. + S.E. 83 ± 3 43 + 14 52 + 9 82 + 3 120 + 3 78 + 3

Table 3 C, Mean pressure

Treatment dog# basis infusion 0 ' 15 ' 30 ' 45 ' DMPC / DPPC 97547 126 127 128 122 115 128 II 97481 115 43 98 102 95 97 II 97557 120 42 68 95 79 83 av. + S.E. 114 + 8 71 + 28 98 + 17 106 + 8 96 + 10 103 + 13 DPPC 9751 1 102 75 78 87 97 120 97 n 97454 100 35 53 88 88 90 r # 97502 103 38 43 83 98 88 prúm. + S.E. 102 + 1 49 + 13 58 + 10 86 + 2 94 + 3 92 + 3

Table 4

Treatment dog# basis infusion 0 ' 15 ' 30 ' 45 ' DMPC / DPPC 97547 120 115 HIM 120 110 110 II 97481 110 180 170 160 180 180 rr 97557 100 60 80 70 100 100 av. + S.E. 110 ± 6 122 + 35 12O ± 26 117 + 26 130 + 25 130 + 25 DPPC 9751 1 100 100 60 70 70 80 you 97454 85 180 220 190 210 160 II 97502 100 110 110 65 100 75 prúm. + S. E. 95 + 5 130 + 25 130 + 47 108 + 41 127 + 43 105 + 28

Table 5 A. PR interval Dog treatment # basis 0 ' 15 ' 30 ' 45 ' DMPC / DPPC 97547 103 115 105 118 105 II 97481 94 90 88 83 80 II 97557 92 92 95 80 77 av. + S.E. 96 + 3 99 + 8 96 + 5 94 + 12 87 + 9 DPPC 97511 102 113 118 117 107 II 97454 90 78 78 75 80 II 97502 103 90 110 105 107 prúm. + S.E. 98 + 4 94 + 10 102 + 12 99 + 12 98 + 9

B. QRS duration

Treatment dog# basis 0 ‘ 15 ' 30 ' 45 ' DMPC / DPPC 97547 40 40 40 40 40 f t 97481 44 45 47 43 43 II 97557 41 43 43 40 40 av. + S.E. 42 + 1 43 + 1 43 + 2 41 ± 1 41 ± 1 DPPC 9751 1 41 40 40 43 43 ii 97454 45 36 42 42 40 n 97502 42 38 39 40 40 av. + S.E. 43 + 1 38 + 1 ^: 40 ± l 42 ± 1 41 + 1

C. QT interval

Treatment dog# basis 0 '15' 30 ' 45 ' DMPC / DPPC 97547 197 203 213 217 213 H 97481 173 195 198 188 187 H 97557 227 248 247 232 220 av. + S.E. 199 ± 16 215 + 17 219 + 15 212 + 13 207 ± 10 DPPC 9751 1 205 228 228 222 207 n 97454 223 177 188 195 202 II 97502 220 209 222 227 233 av. + S.E. 210 + 6 205 + 15 213 + 12 215 ± 10 214 ± 10

D. QTc interval

Treatment dog# basis 0 ' 15 ' 30 ’45 ' DMPC / DPPC 97547 292 309 272 244 253 II 97481 255 298 290 302 313 ir 97557 248 272 285 285 333 av. + S.E. 249 + 3 293 + 11 282 + 5 277 + 17 300 + 24 DPPC 9751 1 245 281 255 254 240 ir 97454 262 340 335 287 264 If 97502 285 275 215 242 258 prúm. + S.E. 264 + 12 299 + 21 '· 268 ± 35 261 + 13 254 + 7

Table 6 A DMPC / DPPC

Parameter basic 45'po p-value (paired-t) @ 24 h number of whites 12.5 ± 4.0 16.3 ± 4.0 0.009 16.4 ± 3.2 blood cells number of Reds 5.96 ± 0.08 5.97 ± 0.27 0.974 6.12 ± 0.13 blood cells hematocrit 42.9 ± 1.0 43.3 ± 2.1 0.818 42.0 ± 0.4 plates 252 + 87 140 + 47 0.203 227 + 58

Β.DPPC

Basic parameter 45'po p-value @ 24 h (paired-t)

number of whites 8.95 ± 1.6 7.1 ± 1.3 0.444 9, 6 + 1.0 blood cells number of reds 5.66 ± 0.31 5.84 ± 0.36 0.335 6.11 ± 0.4 blood cells hematocrit 41.2 ± 1.4 42.2 ± 1.7 0.433 42.8 ± 2.0 plates 244 + 38 69 ± 7 0.041 173 ± 27 Table 7 Systolic pressure (mmHG) u EPC-treated dogs USDA # basic infusion 0 ' 15 ' 30 ' 45 ' 97513 140 70 130 140 140 145 97682 150 57 in 05 140 145 150 97492 165 65 145 180 185 190 97804 140 143 145 130 143 143 97684 150 145 140 140 143 140 average + SEM 149 + 5 96 + 20 133 + 8 146 + 8 151 + 8 154 + 9 Diastolic pressure (mmHG) u EPC vesicles treated dog USDA # basic infusion 0 ' 15 ' 30 ’ 45 ' 97513 95 35 100 90 100 90 97682 85 35 85 75 75 90 97492 95 42 90 115 105 110 97804 90 92 93 80 80 90 97684 92 90 88 80 75 70 average + SEM 92 + 2 59 + 13 91 + 3 88 + 7 87 + 6 91 + 6

Table 9

Average arterial pressure values (mmHG) in EPC-treated dogs USDA # basic infusion 0 ' 15 ' 30 ' 45 ' 97513 110 47 110 107 113 116 97682 107 42 92 97 98 110 97492 118 50 108 137 132 137 97804 107 110 110 97 101 108 97684 113 110 105 100 98 93 mean + SEM 111 + 2 72 ± 16 105 + 3 108 + 8 108 + 7 113 + 7

Table 10

Heart rate values (beats / minute) in EPC-treated dogs

USDA # basic infusion 0 ' 15 ' 30 ' 45 ' 97513 135 310 130 120 120 130 97682 90 220 50 65 100 130 97492 166 273 76 132 112 105 97804 113 115 139 109 94 118 97684 100 100 111 88 85 92 mean + SEM 121 ± 14 204 + 42 101 + 17 1O3 ± 12 102 ± 6 115 + 7 Table 11 And PR (ms) values in EPC treated dogs USDA # basic 0 ' 15 '30' 45 ' 97513 85 83 87 93 92 97682 ⁹³ 135 128 105 88 97492 87 101 87 85 81 97804 119 115 116 120 119 97684 107 101 96 100 97 avg + SEM 98 ± 5 107 ± 9. 103 ± 8 101 ± 6 95 + 6

and

B QRS (ms) values in EPC treated dogs

USDA # basic 0 ' 15 ' 30 ' 45 ' 97513 35 40 37 39 40 97682 30 30 33 33 33 97492 35 35 35 35 35 97804 33 33 34 34 35 97684 34 34 35 35 33 average < + SEM 33 + 1 34 + 2 34 + 2 35 + 1 35 + 1 C. QT (ms) values u EPC treated psu USDA # basic 0 ' 15 ' 30 ' 45 ' 97513 195 197 200 209 197 97682 210 260 247 215 200 97492 163 228 188 170 180 97804 163 228 188 170 180 97684 210 211 235 232 226 prúm .; + SEM 196 + 9 219 + 11 217 ± 11 209 + 11 202 + 7 D. QT _C (ms) values in EPC treated and dogs USDA # basic 0 ' 15 ' 30 ' 45 ' 97513 289 293 276 299 285 97682 270 233 298 320 283 97492 266 260 227 231 224 97804 267 296 261 289 302 97684 292 298 275 287 273 prúm .; + SEM 277 + 6 276 ± 13 267 + 12 285 + 15 273 + 13

Table 12

Blood cell counts in EPC-treated dogs

Basic 45 ' 24h USDA # WBC WBC WBC 97513 10 13 13 97682 18 21 22 97492 20 19 18 97804 29 26 26 97864 9 9 11 avg + SEM 17 + 4 18 + 3 18 + 3 USDA # RBC RBC RBC 97513 6 6 6 97682 6 6 6 97492 6 6 7 97804 6 6 6 97864 6 6 7 avg + SEM 6 + 1 6 ± 1 6 + 2 USDA # hematocrit hematocrit hematocrit

97513 44 42 39

97682 44 42 38

97492 47 46 46

97804 41 41 38

97864 43 41 46 avg + SEM 44 + 1

43 + 1 41 + 2 f

ilka 13 ^r stool pressure

: ření dog# basis infusion »4 / 98661 150 170 ! 91 H 9841 1 130 140 ir 98741 140 140 II 98783 145 135 tr 10174 162 155 i. + S.E. 145 + 5 148 ± 6 ; 04 / 98682 205 195 '91 10003 150 145 98701 155 155 i. + S. E. 170 + 18 165 + 15

0 ' 15 ' 30 ’ 45 ' 175 165 155 155 145 145 150 140 140 145 140 140 160 140 145 170 145 ¹⁴⁵ 145 145 153 + 6 148 + 4 149 + 4 152 ± 6 210 190 200 195 152 150 155 155 145 145 140 145 169 ± 21 162 ± 14 165 ± 18 165 + 15

astolic pressure

solution dog# basis inf uze 0 ' 15 ' 30 ' 45 ' 4 / 98661 100 100 125 100 95 90 91 11 9841 1 65 70 80 80 80 70 11 98741 90 · 90 90 90 85 95 II 98783 70 75 90 70 70 90 ri 10174 90 95 85 85 85 85 i. + S.E. 83 + 7 86 + 6 94 ± 8 85 + 5 83 + 4 86 + 4 04 / 98682 105 130 115 105 105 110 91 10003 103 100 105 98 98 103 98701 85 90 80 98 107 109 i. + S.E. 98 + 6 107 + 12 100 ± l0 98 + 4 96 + 6 101 + 6

C.Medium pressure

Treatment dog# basis infusion 0 ' 15 ' 30 ' 45 ' SA504 / 300591 98661 117 123 142 122 118 115 and" 9841 1 87 93 102 102 103 93 II 98741 107 107 107 108 103 110 II 98783 95 95 113 93 95 117 II 10174 114 115 105 105 105 105 av. + S.E. 104 + 6 107 + 6 114 + 7 106 ± 5 105 + 4 108 + 4 SA 504 / 020791 98682 138 152 ^: 147 133 137 138 II 10003 119 115 121 115 117 120 II 98701 108 112 102 108 103 108 av. + S.E. 122 + 9 126 + 13 123 + 13 119 + 7 119 + 10 122 + 9 Table 14 Treatment dog# basis infusion 0 ' 15 ' 30 ' 45 ' SA504 / 300591 98661 90 102 120 110 83 75 II 9841 1 75 61 67 ⁷⁷ 103 60 II 98741 120 142 120 105 138 127 kl 98783 80 48 * 140 ** 96 66 84 '1 10174 88 80 79 89 90 76 av. + S.E. 91 + 8 87 ± 17 105 + 14 95 + 6 96 + 12 84 + 11 SA 504 / 98682 127 120 127 98 138 113 020791 n 10003 164 136 149 102 108 90 II 98701 101 106 125 112 113 120 av. + S.E. 131 ± 1 121 + 9 134 + 8 1O4 ± 4 120 + 9 108 + 9

* animals asleep during infusion ** entry of personnel into the room. ⁽¹

Table 15

A. PR interval

Treatment dog# basis 0 ' 15 ' 30 ' 45 ' SA 504 / 9S661 97 111 107 105 104 300591 H 9841 1 103 109 101 103 103 , 1 98741 99 104 109 104 105 fl 98783 85 95 88 93 85 H 10174 97 107 111 105 107 av. + S. E. 96 + 3 105 ± 3 102 + 4 102 + 2 101 ± 4 SA 504 / 98682 89 85 91 92 92 020791 1, 10003 104 109 121 108 119 11 98701 116 125 116 120 115 av. + S.E. 103 + 8 106 + 12 109 + 9 107 ± 8 109 ± 8 B.QRS duration . .... ;;-with. Treatment dog# basis 0 ' 15 ' 30 ' 45 ' SA 504 / 98661 32 32 32 29 29 300591 1 » 98411 31 31 33 31 31 řř 98741 33 33 29 29 31 II 98783 28 28 25 28 28 It 10174 24 23 24 25 24 av. + S.E. 30 ± 2 29 + 2 29 + 2 28 + 1 29 + 1 SA 504 / 98682 27 27 28 28 31 020791 11 10003 29 31 28 29 29 1 ' 98701 24 25 23 25 25 av. + S.E. 27 ± 1 28 ± 2 26 + 2 27 + 1 28 ± 2

Table 15 (continued)

C. QT interval

Treatment dog# basis 0 ' 15 ' 30 ' 45 ' SA 504 / 98661 172 160 171 175 187 300591 It 9841 1 195 199 193 181 197 you 98741 181 187 185 192 184 μ 98783 183 189 191 197 195 you 10174 176 184 176 173 167 av. + S.E. 181 ± 4 184 ± 6 183 + 4 184 ± 5 186 ± 5 SA 504 / 98682 160 159 164 164 163 020791 H 10003 189 196 199 201 205 11 98701 183 187 185 185 188 av. + S.E. 177 ± 9 181 ± 11 183 + 10 183 ± 11 185 ± 12

D. QTc interval

Treatment dog# basis 0 ' 15 ' 30 ' 45 ' SA 504 / 98661 233 235 251 229 252 300591 ir 9841 1 228 236 234 248 245 M 98741 252 279 230 256 254 ir 98783 218 289 232 233 234 TI 10174 234 195 226 216 217 av. + S.E. 233 ± 6 247 + 17 235 + 4 236 + 7 240 + 7 SA 504 / 98682 232 238 202 241 215 020791 tr 10003 270 278 277 239 266 II 98701 262 244 251 267 229 prúm. + S.E. 255 + 12 253 ± 12 243 + 22 249 + 9 237 ± 1

Table 16

A. RBC (million / ml)

Treatment dog# control 45 ' SA 504/300591 98661 6.5 6.4 1 ' 9841 1 6.1 6.2 H 98741 6.7 6.7 M 98783 6.6 6.7 »Ř 10174 7.3 6.6 prúm, + S.E. 6.6 ± 0.2 6.5 ± 0.1 SA 504/020791 98682 6.9 6.9 10003 5.7 5.7 98701 6.8 6.6 avg. + S.E. 6.5 +0.4 6.4 ± 0.4 B. WBC (thousand / ml) .... . - Treatment dog# control 45 ' SA 504/300591 98661 9.2 12.8 at 9841 1 13.9 19.2 H 98741 9.0 10.1 11 98783 9.8 8.6 11 10174 13.8 27.3 prúm, + S.E. 11.1 ± 1.1 15.6 ± 3.4 SA 504/020791 98682 9.8 13.6 10003 10.8 17.3 98701 6.8 9.1 avg. + S.E. 9.1 + 1.2 13.3 ± 2.4

Table 17

BP prúm. (%) HR (%) LVEDP dP / dt CO TPR (%) (%) ( mmHg) max (%) vehicle + 1.8 -3.4 + 2.8 -3.3 -4.5 +6.5 (mannitol 300 DSPC vs. mM) -53.7 *** -7.2 + 12.5 * -34.7 * * -13.2 +15.3 control DSPC / DSPG vs. control + 12.3 (2 min) * -3.6 +5.3 -14.3 ** (lmin) * -48.3 (7min) *** +11.8 (2min) -4.8-35.5 (7min) DSPCvs. -33.3 (6 min) *** * * * n. s. * n. s. * n. s.

DSPC / DSPG n = 7, * p <0.05, ** p <0.01, *** p <0.001, Student's t-test

Maximum percentage change in hemodynamic and cardiac parameters during the first 10 min after the start of the infusion of liposomes made from different lipids (300 mg lipid / kg, 100 mg lipid / kg / min) into the femoral vein.

Claims (14)

PATENTOVÉ Claims A liposome formulation which does not otherwise contain an electrically charged component and which has deleterious systemic effects when administered to a mammal, comprising in said liposome an incorporated fraction of an electrically charged component effective to reduce said systemic effects, wherein said fraction is additionally to any amount of said component in said liposome. Liposome formulation according to claim 1, characterized in that the electrically charged component has a negative charge. that The liposome formulation of claim an electrically charged component is fatty 1, characterized by an acid. se The liposome formulation of claim 1, wherein the electrically charged component is stearic acid. The liposome formulation of claim 1, further comprising an active agent 6. The liposome formulation of claim 1, further comprising a contrast agent. ^! ' 7. ' Liposome preparation containing electrically> a charged component and having deleterious systemic effects when administered to a mammal, comprising in said liposome incorporated a portion of an electrically charged component effective to reduce said systemic effects, said portion being in addition to any amount of said component in said &quot; liposomes. Liposome formulation according to claim 7, characterized in that the electrically charged component has a negative charge. The liposome formulation of claim 7, wherein the electrically charged component is a fatty acid. 10. The liposome formulation of claim 7, wherein the electrically charged component is stearic acid. The liposome preparation of claim 7, further comprising an active agent. The liposome formulation of claim 7, further comprising a contrast agent. 13. The liposome formulation of claim 1 or 7, wherein the electrically charged component incorporated into said liposome formulation is stearic acid and the component is incorporated in an amount such that the liposome comprises phosphatidylcholine, cholesterol and stearic acid in a molar ratio of about 4: It was 5 - 1 at the break. 14. The liposome formulation of claim 1 or 7, wherein the electrically charged component incorporated into said liposome formulation is stearic acid, and the component is incorporated in an amount such that the liposome comprises phosphatidylcholine and stearic acid in a molar ratio of about 9: 1. Liposome formulation according to claim 1 or 7, characterized in that the electrically charged component 0 .. incorporated into said liposome preparation is DSPG and it is incorporated in an amount such that the liposome contains DSPC and DSPG in a molar ratio of about 9: 1.