MXPA00006299A - Preparation of a lipid blend and a phospholipid suspension containing the lipid blend - Google Patents

Abstract

The present invention describes processes for the preparation of a lipid blend and a uniform filterable phospholipid suspension containing the lipid blend, such suspension being useful as an ultrasound contrast agent.

Description

PREPARATION OF A COMBINATION OF LIPIDS AND A SUSPENSION OF PHOSPHOLIPIDS CONTAINING THE COMBINATION OF LIPIDS Field of the Invention The present invention relates generally to processes for the preparation of a combination of lipids and a uniform filterable phospholipid suspension containing the combination of lipids, such a suspension being useful as an ultrasonic contrast agent.

Background of the Invention The manufacture of a phospholipid contrast agent can be divided into the following steps: (1) preparation of the lipid combination; (2) mixing the volumetric solution, which comprises the hydration and dispersion of the combination of lipids in an aqueous medium essentially to produce a lipid suspension; (3) filtration of a volumetric solution through a sterilization filter (s) to produce the suspension free of microbial contaminants; (4) distributing the sterile suspension in the individual vials in a controlled aseptic area; (5) load the bottles distributed in a lyophilizing chamber to replace the gas in the space left of the bottle with perfluoropropane gas (PFP); (ß) transfer the Ref. 0120691 sealed bottles, after the gas change to an autoclave for the terminal sterilization. There are three main obstacles in this process: (1) uniformity of the lipid combination; (2) hydration of the lipid combination; (3) uniformity and particle size of the suspension; and, (4) sterile filtration of the suspension through a sterilization filter (s).

Phospholipid combinations are typically produced by dissolving or suspending the required lipids in a suitable aqueous or non-aqueous solvent system, and then reducing the volume either by lyophilization or distillation. Ideally, this process produces mixed solids with uniformity and high content purity. However, while they work well on a small laboratory scale, this simple approach is often problematic with scaling up to quantities of industrial capacity. The difficulties include: (1) maintaining content uniformity during the solvent removal step (due to differential solubilities); (2) maintain purity (often a problem when water is used due to hydrolytic side reactions); (3) increase the purity; (4) minimize the volume of solvent; and (5) recovery of final solids (For example, it is not practical to scrape the solids from a large reactor).

After the manufacture of a combination of lipids, the final composition typically comprises introducing the combination into an aqueous medium. Since phospholipids are hydrophobic and are not readily soluble in water, the addition of phospholipids or a combination of lipids directly in an aqueous solution causes the lipid powder to form lumps which are very difficult to disperse. In this way, the hydration process can not be controlled within a reasonable process time. The direct hydration of the phospholipids or a combination of lipids in an aqueous medium produces a turbid suspension with particles ranging from 0.6 μm to 100 μm. Due to the relatively large particle size distribution, the suspension can not be filtered at room temperature when the temperature of the suspension solution is below the gel phase to liquid crystal transition temperatures of the lipids. The lipids would accumulate in the filters causing a restriction in the flow rate, and in most cases, the filters would be completely blocked shortly thereafter. Furthermore, the reduction in particle size of the suspension can not be achieved through a conventional batch process, even after prolonged mixing (eg, 6 hours) at elevated temperatures. (for example, 40 ° C to 80 ° C) with a commonly used marine propeller.

Although filtration at elevated temperatures, i.e., higher than the phase transition temperatures of the lipids, it is possible, a significant amount of larger lipid particles would be excluded when a normal filtration pressure is used. In turn, the concentrations of the sterile filtrate would have a variable lipid content from batch to batch depending on how the lipids are hydrated initially, which is in turn determined by the physical characteristics, eg, morphology, of the starting materials .

The process of directly hydrating the lipids or combination of lipids to produce a uniform suspension and filtration of the suspension through a sterilization filter (s) can be difficult and costly to be scaled up to any reasonable commercial scale, eg, > 20L.

Thus, the processes presently claimed for the manufacture of a combination of lipids and the subsequent phospholipid suspension are directed to solving the above problems by providing a practical process that can be scaled up and easily adopted by various manufacturing equipment without a modification or as customary by existing equipment specifications.

Brief Description of the Invention Accordingly, an object of the present invention is to provide a novel process for preparing a combination of lipids.

Another object of the present invention is to provide a novel process for preparing a phospholipid suspension from the lipid combination.

These and other objects, which will become apparent during the following detailed description, have been achieved by the discovery of the inventors that dissolving a combination of lipids in a suitable non-aqueous solvent prior to the introduction of an aqueous solution takes into account the production of a phospholipid suspension.

Detailed description of the invention [1] Thus, in a first embodiment, the present invention provides a novel process for preparing a phospholipid suspension, comprising: (1) contacting a combination of lipids with a non-aqueous solvent, whereby the Lipid combination dissolves substantially in the non-aqueous solvent; Y, (2) contacting the solution of step (1) with an aqueous solution to form a lipid suspension. [2] In a preferred embodiment, the non-aqueous solvent is selected from propylene glycol, ethylene glycol, and polyethylene glycol 300. [3] In a more preferred embodiment, the non-aqueous solvent is propylene glycol. [4] In another preferred embodiment, the lipid combination comprises: (a) 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine; (b) monosodium salt of 1,2-dipalmitoyl-sn-glycero-3-phosphotidic acid; Y (c) monosodium salt of N- (methoxypolyethylene glycol carbamoyl 5000) -1, 2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine. [5] In another preferred embodiment, in step (1), the non-aqueous solvent is heated to a temperature of about 30 to 70 ° C before contacting the lipid combination. [6] In another more preferred embodiment, the non-aqueous solvent is heated to a temperature of about 50 to 55 ° C before contacting the lipid combination. [7] In another preferred embodiment, the ratio of the combination of lipids to non-aqueous solvent is from about 5 mg of lipid combination per mL of non-aqueous solvent to approximately 15 mg / mL. [8] In another more preferred embodiment, the ratio of the combination of lipids to non-aqueous solvent is approximately 10 mg / mL. [9] In another preferred embodiment, in step (2), the aqueous solution is selected from water, metal salt, a metal salt / glycerin mixture, and a metal salt / glycerin / non-aqueous solvent mixture.

[10] In another more preferred embodiment, the aqueous solution is a mixture of metal salt and glycerin.

[11] In another more preferred embodiment, the aqueous solution is a mixture of metal salt, glycerin, and propylene glycol.

[12] In another more preferred embodiment, 6.8 mg / mL of sodium chloride are present, 0.1 mL / mL of glycerin are present, 0.1 mL / mL of propylene glycol are present, and approximately 0.75 to 1.0 mg / mL are present. of the combination of lipids.

[13] In an even more preferred embodiment, 0.75 mg / mL of lipid combination are present.

[14] In another more preferred embodiment, 1.0 mg / mL of lipid combination is present.

[15] In another preferred embodiment, in step (2), the aqueous solution is heated to a temperature of about 45 to 60 ° C before being contacted with the solution of step (1).

[16] In another more preferred embodiment, the aqueous solution is heated to a temperature of about 50 to 55 ° C before contacting with the solution of step (1).

[17] In another preferred modality, the process also includes: (3) heating the lipid suspension from step (2) to a temperature approximately equal to or higher than the gel phase to liquid crystal transition temperature higher than the lipids present in the suspension.

[18] In another more preferred embodiment, in step (3), the lipid suspension is heated to a temperature of at least about 67 ° C.

[19] In another more preferred embodiment, the process also comprises: (4) Filter the lipid suspension through a sterilization filter.

[20] In another even more preferred embodiment, in step (4), the filtration is carried out using two sterilization filter cartridges.

[21] In a further preferred embodiment, in step (4), the sterilization filter cartridges are at a temperature of from about 70 to 80 ° C.

[22] In another, more preferred embodiment, in step (4), 0.2 μm hydrophilic filters are used.

[23] In another even more preferred modality, the process also includes: (5) distributing the filtered solution of step (4) in a flask.

[24] In another, more preferred embodiment, the process further comprises: (6) change the gas from the space left of the vial of step (5) with a perfluorocarbon gas.

[25] In another even more preferred embodiment, the perfluorocarbon gas is perfluoropropane.

[26] In another even more preferred embodiment, the gas change of the space left is carried out using a lyophilizing chamber.

[27] In another even more preferred embodiment, the process also comprises: (7) sterilize the vial of step (6).

[28] In a yet still preferred embodiment, in step (7), the bottle is sterilized at about 126-130 ° C for 1 to 10 minutes.

[29] In a second embodiment, the present invention provides a novel process for preparing a combination of lipids, comprising: (a) contacting at least two lipids with a first non-aqueous solvent; (b) concentrating the solution to a thick gel; (c) contacting the thick gel with a second non-aqueous solvent; and, (d) collect the resulting solids.

[30] In a preferred embodiment, in step (a), the lipids are: (a) 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine; (b) monosodium salt of 1,2-dipalmitoyl-sn-glycero-3-fssphotidic acid; Y (c) monosodium salt of N- (methoxypolyethylene glycol carbamoyl 5000) -1, 2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine.

[31] In another preferred embodiment, in step (a), the first non-aqueous solvent is a mixture of methanol and toluene.

[32] In another preferred embodiment, in step (c), the second non-aqueous solvent is a methyl t-butyl ether.

[33] In another preferred embodiment, in step (a), the solution is heated to a temperature sufficient to complete the dissolution of the lipids in the solvent.

[34] In another more preferred embodiment, in step (a), the solution is heated to about 25 to 75 ° C.

[35] In another preferred embodiment, in step (d), the collected solids are washed with methyl t-butyl ether and dried under vacuum.

[36] In a third embodiment, the present invention provides a novel phospholipid suspension, comprising: (a) a combination of lipids in an amount of about 0.75-1.0 mg / mL of suspension; (b) sodium chloride in an amount of about 6.8 mg / mL of suspension; (c) glycerin in an amount of about 0.1 mL / mL of suspension; (d) propylene glycol in an amount of about 0.1 mL / mL of suspension; Y (e) water; wherein the suspension is prepared by the process, comprising: (1) contacting a combination of lipids with a non-aqueous solvent, whereby the lipid combination dissolves substantially in the non-aqueous solvent; (2) contacting the solution of step (1) with an aqueous solution to form a lipid suspension; (3) heating the lipid suspension from step (2) to a temperature approximately equal to or greater than the highest gel-to-liquid crystal transition temperature of the lipids present in the suspension; Y, (4) Filter the lipid suspension through a sterilization filter.

[37] In another preferred embodiment, the lipid combination comprises: (a) 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine; (b) monosodium salt of 1,2-dipalmitoyl-sn-glycero-3-phosphotidic acid; and (c) monosodium salt of N- (carbamoyl of ethoxypolyethylene glycol 5000) -1, 2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine.

[38] In another more preferred embodiment, the non-aqueous solvent is heated to a temperature of about 50 to 55 ° C before contacting the lipid combination.

[39] In another more preferred embodiment, the ratio of the combination of lipids to non-aqueous solvent is approximately 10 mg / mL.

[40] In another more preferred embodiment, the aqueous solution is a mixture of metal salt, glycerin, and propylene glycol.

[41] In an even more preferred embodiment, 0.75 mg / mL of lipid combination are present.

[42] In another more preferred embodiment, the aqueous solution is heated to a temperature of about 50 to 55 ° C before being contacted with the solution of step (1).

[43] In another more preferred embodiment, in step (3), the lipid suspension is heated to a temperature of at least about 67 ° C.

[44] In another, more preferred embodiment, in step (4), two 0.2 μm hydrophilic filters are used.

Formulation The present invention is contemplated to be practical in at least one multigram scale, kilogram scale, multikilogram scale, or industrial scale. The multigram scale, as used herein, is preferably the scale wherein at least one starting material is presented in 10 grams or more, more preferably at least 50 grams or more, even more preferably at least 100 grams. or more. The multikilogram scale, as used herein, means that it means the scale where more than one kilogram of at least one starting material is used. The industrial scale as used here means that it means a scale which is different from a laboratory scale and which is sufficient to provide a sufficient product for either clinical testing or distribution to consumers.

The combination of lipids or combination of phospholipids, as used herein, means that it represents two or more lipids which have been combined. The combination of lipids is generally in powder form. Preferably, at least one of the lipids is a phospholipid. Preferably, the lipid combination contains 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), monosodium salt of 1,2-dipalmitoyl-sn-glycero-3-phosphotidic acid (DPPA), and monosodium salt of N- (methoxypolyethylene glycol carbamoyl 5000) -1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (MPEG500-DPPE). The amount of each lipid present in the mixture will depend on the desired final product. The preferred proportions of each lipid are described in the Examples section. A wide variety of other lipids, similar to those described in Unger et al, U.S. Pat. No. 5,469,854, the contents of which are therefore incorporated by reference, can be used in the present process.

The phospholipid, as used herein, is a fatty substance that contains an oily hydrocarbon (hydrophobic) chain (s) with a polar (hydrophilic) phosphoric main group. Phospholipids are amphiphilic. These spontaneously form closed boundaries and vesicles in an aqueous medium. Phospholipids constitute approximately 50% of the mass of animal cellular plasma membrane.

Preparation of the combination of lipids The combination of lipids can be prepared via an aqueous suspension-lyophilization process or a dissolution-precipitation process with organic solvent using organic solvents. In the aqueous suspension-lyophilization process, the desired lipids are dispersed in water at an elevated temperature and then concentrated by lyophilization. Preferably a dissolution process is used.

Step (a): The dissolution-precipitation method with organic solvent comprises contacting the desired lipids (e.g., DPPA, DPPC, and MPEG5000-DPPE) with a first non-aqueous solvent system. This system is typically a combination of solvents, for example CHCl3 / MeOH, CH2Cl2 / ieOH, and toluene / MeOH. Preferably, the first non-aqueous solvent is a mixture of toluene and methanol. It may be desirable to heat the lipid solution to a temperature sufficient to achieve complete dissolution. Such a temperature is preferably about 25 to 75 ° C, preferably about 35 to 65 ° C.

After dissolution, it may be desirable to remove the above-mentioned undissolved material by hot filtration or cooling at room temperature and then filtration. Known filtration methods can be used (eg, gravity, filtration, vacuum filtration, or pressure filtration).

Step (b); Then the solution is concentrated to a thick / semi-solid gel. The concentration is preferably done by vacuum distillation. Other methods of concentration of the solution, such as rotary evaporation may also be used. The temperature of this step is preferably about 20 to 60 ° C, more preferably 30 to 50 ° C.

Step (c): Then the thick / semi-solid gel is dispersed in a second non-aqueous solvent. The mixture becomes a suspension, preferably close to room temperature (eg, 15-30 ° C). The second useful non-aqueous solvents are those that cause the lipids to precipitate from the filtered solution. The second non-aqueous solvent is preferably methyl t-butyl ether (MTBE). Other ethers and alcohols can be used.

Step (d): The solids produced are then collected with the addition of the second non-aqueous solvent. Preferably the collected solids are washed with another portion of the second non-aqueous solvent (e.g., MTBE). The collection can be carried out via vacuum filtration or centrifugation, preferably at room temperature. After the collection, it is preferred that the solids are dried under vacuum at a temperature of about 20-60 ° C.

For the following reasons, the dissolution-precipitation process with organic solvent is preferred over the aqueous suspension / lyophilization process: (1) Because the lipids are completely soluble in toluene / methanol, the volumes of solvent are significantly reduced (relative to the aqueous process). (2) because of this increased solubility, the process temperature is also lower in relation to the aqueous process, thereby avoiding the hydrolytic instability of fatty acid esters. (3) When cooled again to room temperature, the toluene / methanol solution of lipids remains homogeneous, allowing a filtration at room temperature to remove the aforementioned solid matter. (4) Precipitation with MTBE allows rapid and easy isolation of the solids from the Lipid Combination. With the aqueous process, a lyophilization process is used that requires a lot of time to isolate the material. (5) Precipitation with MTBE also takes into account the elimination of any of the soluble impurities in the MTBE, which go in the waste stream of the filtrate. This potential for removing impurities is not realized when the solution is concentrated or lyophilized directly to a solid. (6) The present process gives uniform solids.

Preparation of the lipid suspension In step one, a combination of lipids is contacted with a non-aqueous solvent, whereby the lipid combination dissolves substantially in the non-aqueous solvent. Alternatively, the individual lipids may be contacted with the non-aqueous solvent periodically in the order: DPPC, DPPA, and MPEG5000-DPPE; DPPC, MPEG5000-DPPE, and DPPA; MPEG500-DPPE, DPPA, and DPPC; or MPEG5000-DPPE, DPPC, and DPPA. The DPPA, which is the least soluble and the least abundant of the lipids, is not added first. Addition of one of the other lipids before or concurrently with adding DPPA facilitates the dissolution of DPPA. In another alternative, the individual lipids can be combined in their solid forms and the combination of the solids put in contact with the non-aqueous solvent.

The substantial dissolution is usually indicated when the mixture of the combination of lipids and the non-aqueous solvent becomes clear. As previously observed, phospholipids are not generally soluble in water. Thus, the direct introduction of a combination mixture of phospholipids into an aqueous medium causes the lipid combination to form lumps which are very difficult to disperse. The present invention overcomes this limitation by dissolving the combination of lipids in a non-aqueous solvent prior to the introduction of the aqueous solution. This also makes it possible to disperse the combination of lipids in a liquid. Then the liquid dispersion can be introduced into a desired aqueous medium.

Non-aqueous means that it means a solvent or mixture of solvents in which the amount of water present is sufficiently low as to not prevent the dissolution of the lipid combination. The amount of non-aqueous solvent required will depend on the solubility of the lipid combination and also on the final desired concentration of each component. As one of ordinary skill in the art would appreciate, the level of water present in the non-aqueous solvent, which can be tolerated will vary based on the solubilities in water of the individual lipids in the lipid combination. The more soluble in water are the individual phospholipids, the more water can be present in step (1). Preferably, the propylene glycol is used as the non-aqueous solvent. However, other members of the polyol family, such as ethylene glycol, and polyethylene glycol 300, can be used.

It may be necessary to mechanically mix the combination of lipids and the non-aqueous solvent to achieve complete dissolution. One of ordinary skill in the art will recognize that a variety of mixing forms are available. It is preferred that a high cut homogenizer be used.

One of ordinary skill in the art would recognize that increasing the temperature of the solvent would aid in the dissolution of the lipid combination. The temperature at which step (1) can be carried out can fluctuate from room temperature to the boiling temperature of the chosen solvent. Preferably the temperature is from about 30 to about 70 ° C, preferably about 45 to about 60 ° C, and even more preferably about 50, 51, 52, 53, 54, or 55 ° C. When using ethylene glycol or polyethylene glycol 300, it is preferred that the temperature be from about 50 to about 60 ° C and more preferably about 55 ° C. Maintaining the solution at an elevated temperature would reduce the viscosity of the solution and facilitate the preparation of the formulation.

A preferred method for dissolving the lipid combination is as follows: (a) adding propylene glycol to a suitable weighing vessel; (b) heating the propylene glycol to about 40-80 ° C in a heating bath; (c) weigh the combination of lipids in a separate container; (d) when the propylene glycol has reached the desired temperature range, transfer the solution into the container containing the lipid combination; (e) placing the container again in the heating bath until the solution is clear; (f) mechanically mixing the Lipid / Glycol Combination solution of Propylene to further ensure complete dissolution and uniform dispersion of the lipid combination.

The ratio of the combination of lipids to non-aqueous solvent, of course, will be limited by the solubility of the lipid combination. This ratio will also be influenced by the desired amount of lipid combination in the final formulation. Preferably, the ratio is from about 1 mg of lipid combination per mL of solvent (mg / mL) to about 100 mg / mL. Most preferably, the lipid combination occurs at about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg / mL. Even more preferably, the lipid combination occurs at about 10 mg / mL.

Step 2) : The second step comprises contacting the solution of step (1) with an aqueous solution to form a lipid suspension. The aqueous solution can be water, metal salt, a metal salt / glycerin mixture or a metal salt / glycerin / non-aqueous solvent mixture. The non-aqueous solvent is as previously defined, preferably propylene glycol. The suspension, as used herein, means that it indicates a dispersion in which the insoluble particles are dispersed in a liquid medium.

Once the complete dissolution of the lipid combination has been achieved (step (1)), the resulting solution can then be introduced into an aqueous solution. The aqueous solution may contain one or more components selected from sodium chloride, glycerin, and a non-aqueous solvent. Preferably the aqueous solution contains glycerin and sodium chloride. Preferably, a sufficient amount of propylene glycol is present in the aqueous solution, before the addition of the solution from step 1, to achieve the final desired concentration of propylene glycol.

The order of addition of the desired components is not expected to have a serious impact on the resulting lipid suspension. However, it is preferred that the lipid combination solution be added to water, which may already contain the additional components noted above. Then additional desired components can be added. It is more preferred that the lipid combination solution be added to a solution of water and sodium chloride (i.e., metal salt). It is further preferred that the lipid combination solution is added to a solution of water, sodium chloride, and glycerin. It is still further preferred that the lipid combination solution is added to a solution of water, sodium chloride, glycerin, and propylene glycol.

It is preferred that 6.8 mg of NaCl per mL of formulation be present. Preferably, 0.1 mL of glycerin per mL of formulation is presented. A final concentration of 0.1 mL of Propylene Glycol per mL of formulation is preferred. The final pH of the formulation is preferably about 5.5-7.0. The lipid combination is preferably presented in an amount of 0.75-1.0 mg / mL of formulation.

The temperature of the aqueous solution can fluctuate from room temperature to 70 ° C. Preferably, the temperature is about 45 to 60 ° C, with 50, 51, 52, 53, 54, or 55 ° C being even more preferred.To obtain a complete solution, the mixture will need to be stirred, preferably wagged. Also, the pH of the solution may need to be adjusted, depending on the desired final formulation.Either acid (eg, HCl) or base (eg, NaOH) can be added to make such an adjustment.

The lipid suspension will contain liquid particles of varying sizes. One of the benefits of the present invention is the ability to consistently obtain small particles of an almost uniform size. Thus, it is preferred that the majority of particles obtained be less than 100 nm in diameter, more preferably less than 50 nm.

A preferred method for dissolving the lipid combination is as follows: (a) adding Water for Injection (FI) in a composition vessel; (b) start mixing and ensure that the temperature is from 50-55 ° C; (c) adding sodium chloride to the composition vessel, wait until the solid has completely dissolved before proceeding to the next step; (d) adding glycerin to the composition vessel, leaving sufficient time for complete mixing; (e) add the remaining propylene glycol that is not in the combination of Lipid / Glycol Propylene, leave some time for complete mixing; (f) reducing the mixing ratio to reduce turbulence in the composition vessel; (g) adding the Lipid Combination / Propylene Glycol solution to the composition vessel; (h) readjust the mixing to the original ratio; (i) adding Water for Injection (WFI) if necessary; (j) continue mixing for approximately 25 minutes and ensure complete mixing; (k) verify and adjust the solution to an objective pH.

Step 3) : Step three comprises heating the lipid suspension obtained from step (2) to a temperature approximately equal to or greater than the highest gel-to-liquid crystal transition temperature of the lipids present in the solution.

One of the objectives of this step is to provide a filterable suspension. A solution / suspension is considered filterable if there is no significant reduction in the flow rate within a normal process, and if there is no significant increase in the pressure drop in the filtration system.

Experimental data indicates that the lipids in the formulation would be outside their transition from gel phase to crystalline liquid to simplify sterile filtration.

When the lipids are below the phase transition temperature, the particles in the suspension are rigid. However, when they are above their gel-to-liquid phase transition temperatures, they are in a more or less organized configuration, and thus, they are more easily filtered.

The DPPC and DPPA show phase transitions of 41 ° C and 67 ° C respectively. MPEG5000-DPPE is soluble in water, therefore it does not show a transition from gel phase to liquid crystal, which is a characteristic of most hydrated lipid suspensions. Because the lipids in the preferred formulation all show different gel-to-liquid phase transitions, the higher phase transition temperature, 67 ° C, is preferably used to filter the solution. Maintaining the temperature at or above 67 ° C, all the lipids are out of their respective phase transition, ensuring free configuration, while passing through the filters.

Heating can be accomplished by capping the composition vessel with a heat exchange coil. The hot water stream from a controlled source, e.g., a hot water bath, or a water heater, would transmit sufficient heat to maintain the composition solution at a fixed temperature. Other heat sources known to those skilled in the art could also be used.

Step 4) : Step 4 is carried out by filtering the lipid suspension through a sterilization filter. The purpose behind this step is to provide a bacteria-free suspension substantially. A filtrate is considered substantially free of bacteria when the probability of the filtrate to contain at least one colony forming microorganism is less than 10"6.

Filtration is preferably done using sterilization filter cartridges. Also, a means of forcing the solution through the filters (eg, pumping or pressurization) may be required. Since the solution is filtered, it needs to be maintained at a temperature at or higher than the gel-to-liquid phase transition temperature higher than the lipids present in the solution, the filtration should be carried out at about this same temperature. To achieve this, the filter (eg, sterilization filter cartridges) is preferably enclosed in jacketed filter housings, which are heated continuously, for example, by a stream of hot water from a temperature-controlled water bath, to ensure that the suspension is above the lipid phase transition temperatures. The temperature of the sterilization filter is preferably from 50 to 100 ° C, more preferably from 60 to 90 ° C, and even more preferably 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 ° C.

One or more sterilization filters can be used to filter the suspension. The number required will be based on its effectiveness in eliminating bacteria. It is preferred that two filters be used. The pore size of the filter will be limited by the need to provide a bacteria-free suspension. Preferably, 0.2 μm hydrophilic filters are used.

A volumetric solution of the preferred formulation was continuously filtered through two 0.2 μm hydrophilic filters for up to 3 hours at a rate of about 1 liter per minute (1 L / minute), i.e., passing a total of 180 liters of the solution in suspension through the filters. The experimental results show that there is no apparent blockage of the filters. Lipid assays indicate that there is no measurable loss during the filtration process (due to accumulation in the filter material).

A volumetric solution of the preferred formulation was made at 40 ° C-80 ° C, and the suspension was cooled to room temperature before sterile filtration. No apparent clogging of the filters was observed, indicating that the particle size distribution of the suspension is convenient below 0.2 μm of the filter pore size. It is desirable to use heat during filtration to ensure maximum recovery of the lipid combination in the sterile filtrate (i.e., to minimize the potential retention of the lipid particles in the filter material).

A preferred method for filtering the lipid suspension is as follows: (a) to ensure that all jacketed filters are at 70 ° C - 80 ° C; (b) ensure that all valves in the filtration unit are closed; (s) connecting the filtration inlet hose to the outlet of the composition vessel; (d) open the valves to let the solution pass through the filters; (e) flushing three liters of solution with water through the filters before collecting the filtrate; (f) continue the filtration until it is completed.

Step (5): Distributing the filtered solution in a bottle completes step five. Preferably, this step is carried out in a controlled aseptic area. One of ordinary skill in the art would recognize that the selected bottle and the amount of suspension delivered to the bottle would depend on the final use considered for the lipid suspension. The distribution can be achieved via a variety of methods, including pipette, manual syringe dispenser (for example, Filamatic® syringe dispensing machine), or industrial automatic dispensing machine (for example, Cozzoli or TL automatic filling machine).

Faso (6); Step six is carried out by changing the gas from the space left of the flasks of step five with a perfluorocarbon gas. A preferred method of change is to charge the dispensed bottles in a lyophilizing chamber and replace the gas in the space left of the bottle with a perfluorocarbon gas. A preferred gas is perfluoropropane (PFP). Other methods of space gas exchange left known to those skilled in the art may be employed.

The bottles are sealed at the end of the gas change cycle of the space left on the bottle. When pressure is produced in the lyophilizing chamber, return to atmospheric pressure by charging the chamber with PFP. The bottle caps are fixed to seal the bottles.

Step (7); Step seven comprises periodically sterilizing a bottle after step six. A method of terminal sterilization is through the use of an autoclave. Also, sealed bottles can be periodically sterilized in a steam sterilizer to further increase the sterility assurance of the product. Care must be taken in the sterilization process since some degradation of the lipids can be observed as a result of sterilization in an autoclave. Preferably, the bottle is sterilized at about 126-130 ° C for 1 to 10 minutes.

Other features of the invention will become apparent in the course of the following descriptions of the exemplary embodiments, which are given for illustration of the invention and are not intended to be limiting thereof.

EXAMPLES Table 1: Objective Composition of the Lipid Combination Lipid Compound Manufacturing Procedure of MPBGSO8DFFE A flask is charged with toluene (3.3 L), methanol (1.2 L), DPPA (50.6 g), DPPC (535 g), and MPEG5000-DPPE (450 g). After rinsing the solid contact surfaces with 0.9 L of methanol, the suspension is heated to 45-55 ° C until the dissolution is complete.

The solution is filtered and then concentrated in vacuo to -. 35-45 ° C until a thick gel. Methyl t-butyl ether (MTBE, 5.4 L) is added and the mixture is converted, in suspension at 15-30 ° C.

White solids are collected by centrifugation or vacuum filtration, and washed with MTBE (0.9 L). The solids are then placed in a vacuum oven and dried to a constant weight at 40-50 ° C. The dry Lipid Combination is transferred to a bottle and stored at 15 to 25 ° C.

In another embodiment of the manufacturing process of the lipid combination of the present invention, the following procedure can also be used.

Manufacturing Procedure of the Alternative Lipid Combination from The amounts of phospholipid are adjusted to a purity based on a "Use As" value from the certificates of analysis. The lot size (combined phospholipid weight) of this experiment was 2 kg.

A rotary evaporation flask is charged consecutively with toluene (3,300 mL), methanol (1,200 mL), DPPA (122.9 g, corrected for a "use as" purity of 97.0%), DPPC (1.098.5 g total, 500.8 g of a batch with 98.4% purity of "use as" and 597.7 g of a batch with 96.7% purity of "use as"), and MPEG5000-DPPE (815.7 g, corrected for a purity of "use as" of 99.3%) . After rinsing the residual solids in the flask with methanol (900 mL), the flask is placed in a rotary evaporator (without vacuum) and the suspension is heated to between 45 and 55 ° C (external temperature). After the dissolution is complete, the external temperature is reduced to between 35 and 45 ° C, a vacuum is applied, and the solution is concentrated to a white semi-solid. The flask is removed from the evaporator and the solids are separated with a spatula. The flask is readapted to the evaporator and the concentration is continued. After reaching the final temperature (final vacuum pressure 20 mbar, white solid, granular, coarse), MTBE (5,400 mL) is added through the addition tube of the rotary evaporator, the vacuum is interrupted, and the mixture becomes suspension for 15 to 45 minutes at 15 to 30CC. The solids are isolated by either centrifugal or vacuum filtration, rinsed with MTBE (3,800 mL), and dried to a constant weight in a vacuum oven (40 to 505C). Before transferring to polyethylene bottles with polypropylene lids, the solids are desagrumed through a sieve (0.079 inch mesh), giving 1,966.7 grams (98%) of lipid combination (SG896) as a white solid.

The preferred lipid suspension contains: monosodium salt of 1,2-dipalmitoyl-sn-glycero-3-phosphotidic acid (DPPA); 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC); monosodium salt of N- (methoxypolyethylene glycol carbamoyl 5000) -1, 2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (MPEG5000-DPPE); Propylene glycol, USP; Glycerin, USP; Sodium Chloride, USP; and, Water for Injection, USP.

Table 2. Preferred Contrast Agent Formulations * The formula A had mg mL e com nac n ce p os. Formulation B had a concentration of the lipid combination of 0.75 mg / mL.

** The lipid combination consists of 53.5% by weight of DPPC, 6.0% by weight of DPPA and 40.5% by weight of MPEG5000-DPPE.

Table 3: Recipient and Closing Preferred The filling volume of the finished product can be from 1.0 - 2.0 mL / bottle.

In the preparation of the preferred formulation, when the combination of lipids is hydrated directly with the aqueous matrix solution containing water for injection, sodium chloride, glycerin and propylene glycol, the filtrates have less lipids compared to the prefiltration volumetric solution . Lipid loss varies from 12% to 48%. These results show that the sterile filtration process is not effectively controlled, and therefore, the lipid content of the final product is highly variable.

In contrast, using the process described herein, the lipid test results show the complete recovery of the lipids during the filtration process. The variability of the test results around the theoretical reference values are within the variability of the normal test method. The particle size distribution by number, by volume and by reflective intensity of a suspension prepared by first solubilizing the combination of lipids in propylene glycol indicates that most of the particles are less than 50 nm in the volumetric solution prefiltered at 55 ° C , also at 70 ° C. The particle distribution profile does not change after filtering.

UTILITY SECTION The currently claimed process is useful for preparing ultrasonic contrast agents. Such agents would be useful for a variety of imaging applications, including increasing the contrast in echocardiographic and radiological ultrasonic images.

Obviously, numerous modifications and variations of the present invention are possible in the clarity of the above teachings. It is therefore understood that within the scope of the amended claims, the invention may be practiced otherwise than as specifically described herein.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following is claimed as property.

Claims (44)

1. A process for preparing a phospholipid suspension, characterized in that it comprises: (1) contacting a combination of lipids with a non-aqueous solvent, whereby the lipid mixture dissolves substantially in the non-aqueous solvent; Y, (2) contacting the solution of step (1) with an aqueous solution to form a lipid suspension.

2. A process according to claim 1, characterized in that the non-aqueous solvent is selected from propylene glycol, ethylene glycol, and polyethylene glycol 300.

3. A process according to claim 2, characterized in that the non-aqueous solvent is propylene glycol.

4. A process according to claim 2, characterized in that the lipid combination comprises: (a) 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine; (c) monosodium salt of 1,2-dipalmitoyl-sn-glycero-3-phosphotidic acid; Y (c) monosodium salt of N- (methoxypolyethylene glycol carbamoyl 5000) -1, 2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine.

5. A process according to claim 2, characterized in that the non-aqueous solvent is heated to a temperature of about 30 to 70 ° C before contacting the lipid combination.

6. A process according to claim 5, characterized in that the non-aqueous solvent is heated to a temperature of about 50 to 55 ° C before contacting the lipid combination.

7. A process according to claim 2, characterized in that the ratio of the combination of lipids to non-aqueous solvent is from about 5 mg of lipid combination per mL of non-aqueous solvent to approximately 15 mg / mL.

8. A process according to claim 7, characterized in that the ratio of the combination of lipids to non-aqueous solvent is approximately 10 mg / mL.

9. A process according to claim 2, characterized in that in step (2), the aqueous solution is selected from water, metal salt, a metal salt / glycerin mixture, and a mixture of ethyl salt / glycerin / non-aqueous solvent.

10. A process according to claim 9, characterized in that the aqueous solution is a mixture of metal salt and glycerin.

11. A process according to claim 9, characterized in that the aqueous solution is a mixture of metal salt, glycerin, and propylene glycol.

12. A process according to claim 11, characterized in that 6.8 mg / mL of sodium chloride are present, 0.1 mL / mL of glycerin are present, 0.1 mL / mL of propylene glycol are present, and approximately 0.75 to 1.0 mg are present. / mL of the lipid combination.

13. A process according to claim 12, characterized in that 0.75 mg / mL of the lipid combination is present.

14. A process according to claim 12, characterized in that 1.0 mg / mL of the lipid combination are present.

15. A process according to claim 2, characterized in that in step (2), the aqueous solution is heated to a temperature of about 45 to 60 ° C before contacting the solution of step (1).

16. A process according to claim 15, characterized in that the aqueous solution is heated to a temperature of about 50 to 55 ° C before contacting the solution of step (1).

17. A process according to claim 1, characterized in that the process further comprises: (3) heating the lipid suspension from step (2) to a temperature approximately equal to or higher than the gel phase to liquid crystal transition temperature higher than the lipids present in the suspension.

18. A process according to claim 17, characterized in that in step (3), the lipid suspension is heated to a temperature of at least about 67 ° C.

19. A process according to claim 17, characterized in that the process also comprises: (4) Filter the lipid suspension through a sterilization filter.

20. A process according to claim 19, characterized in that in step (4), the filtration is carried out using two sterilization filter cartridges.

21. A process according to claim 20, characterized in that in step (4), the sterilization filter cartridges are at a temperature of about 70 to 80 ° C.

• 22. A process according to claim 21, characterized in that in step (4), 0.2 μm hydrophilic filters are used.

23. A process according to claim 19, characterized in that the process further comprises: (5) distributing the filtered solution of step (4), in a bottle.

24. A process according to claim 23, characterized in that the process also comprises: (6) change the gas from the space left of the vial of step (5) with a perfluorocarbon gas.

25. A process according to claim 24, characterized in that the perfluorocarbon gas is perfluoropropane.

26. A process according to claim 25, characterized in that the gas change of the space left is carried out using a lyophilization chamber.

27. A process according to claim 24, characterized in that the process also comprises: (7) sterilize the step bottle (6)

28. A process according to claim 27, characterized in that in step (7), the bottle is sterilized at approximately 126-130 ° C for 1 to 10 minutes.

29. A process for preparing a combination of lipids, characterized in that it comprises: (a) contacting at least two lipids with a first non-aqueous solvent; (b) concentrating the solution to a thick gel; (c) contacting the thick gel with a second non-aqueous solvent; Y, (d) Collect the resulting solids.

30. A process according to claim 29, characterized in that in step (a), the lipids are: (i) 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine; (ii) monosodium salt of 1,2-dipalmitoyl-sn-glycero-3-phosphophide acid; Y, (iii) monosodium salt of N- (carbamoyl of methoxypolyethylene glycol 5000) -1, 2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine.

31. A process according to claim 30, characterized in that in step (a), the first non-aqueous solvent is a mixture of methanol and ethanol.

32. A process according to claim 30, characterized in that in step (c), the second non-aqueous solvent is a methyl t-butyl ether.

33. A process according to claim 30, characterized in that in step (a), the solution is heated to a temperature sufficient to complete the dissolution of the lipids in the solvent.

34. A process according to claim 33, characterized in that in step (a), the solution is heated to about 25 to 75 ° C.

35. A process according to claim 33, characterized in that in step (d), the collected solids are washed with methyl t-butyl ether and dried under vacuum.

36. A phospholipid suspension, comprising: (a) a combination of lipids in an amount of about 0.75-1.0 mg / mL of suspension; (b) sodium chloride in an amount of about 6.8 mg / mL of suspension; (s) glycerin in an amount of about 0.1 mL / mL of suspension; (d) propylene glycol in an amount of about 0.1 mL / mL of suspension; Y (e) water; characterized in that the suspension is prepared by the process, which comprises: (1) contacting a combination of lipids with a non-aqueous solvent, whereby the lipid combination dissolves substantially in the non-aqueous solvent; (2) contacting the solution of step (1) with an aqueous solution to form a lipid suspension; (3) heating the lipid suspension from step (2) to a temperature approximately equal to or greater than the highest gel-to-liquid crystal transition temperature of the lipids present in the suspension; Y, (4) Filter the lipid suspension through a sterilization filter.

37. A phospholipid suspension according to claim 36, characterized in that the lipid combination comprises: (a) 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine; (b) monosodium salt of 1,2-dipalmitoyl-sn-glycero-3-phosphotidic acid; Y, (c) monosodium salt of N- (methoxypolyethylene glycol carbamoyl 5000) -1, 2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine.

38. A phospholipid suspension according to claim 37, characterized in that the non-aqueous solvent is heated to a temperature of about 50 to 55 ° C before contacting the lipid combination.

39. A phospholipid suspension according to claim 37, characterized in that the ratio of the combination of lipids to non-aqueous solvent is approximately 10 mg / mL.

40. A phospholipid suspension according to claim 37, characterized in that the aqueous solution is a mixture of metal salt, glycerin, and propylene glycol.

41. A phospholipid suspension according to claim 40, characterized in that 0.75 mg / mL of the lipid combination is present.

42. A phospholipid suspension according to claim 37, characterized in that the aqueous solution is heated to a temperature of about 50 to 55 ° C before contacting with the solution of step (1).

43. A phospholipid suspension according to claim 37, characterized in that in step (3), the lipid suspension is heated to a temperature of at least about 67 ° C.

44. A phospholipid suspension according to claim 43, characterized in that in step (4), two 0.2 μm hydrophilic filters are used.