HK1205685B - Lyophilization of synthetic liposomal pulmonary surfactant - Google Patents

Description

Lyophilization of synthetic liposomal lung surfactant

Background

1. Field of the invention

The present invention relates to a solid synthesized lung surfactant and a manufacturing method thereof.

2. Description of related Art

Pulmonary surfactant (also referred to as "pulmonary surfactant") is a complex mixture of lipids and proteins that promotes the formation of a monolayer at the air-water interface of the alveoli, and, by reducing surface tension, collapse of the alveoli upon exhalation can be prevented. Pulmonary surfactant is distributed in the alveolar epithelial cells of the lungs of mature mammals. The natural lung surfactant has been described as a "lipoprotein complex" because it contains both phospholipids and apoproteins, which interact to reduce the surface tension at the lung gas-liquid interface. Four proteins have been found to be associated with lung surfactant, namely SP-A, SP-B, SP-C and SP-D. In particular, SP-B appears to be essential for the biophysical effects of lung surfactant. It is accepted therapy for the treatment of various respiratory disorders to administer a pulmonary surfactant to the lungs of a patient.

From a pharmacological point of view, the best exogenous lung surfactant to be used in therapy is completely synthesized in the laboratory. In this regard, one mimetic of SP-B that has been found to be useful is KL4, which is a 21 amino acid cationic polypeptide.

One method for manufacturing lung surfactants for medical use on a commercial scale is by operation using a Thin Film Evaporator (TFE) unit. A process suitable for the production of KL4 lung surfactant, consisting of the steps of: 1) solubilization of four major formulation components, Dipalmitoylphosphatidylcholine (DPPC), Palmitoyl Oleoyl Phosphatidylglycerol (POPG) and Palmitic Acid (PA) and KL4, in ethanol; 2) removing ethanol by using TFE; and 3) dispensing the final dispersion into vials. The operation of the TFE unit is inherently complex and has size limitations. Specifically, 1 square foot of TFE yields a 40 liter batch, and the maximum available unit is 10 square feet of TFE. This limits the batch size, which is undesirable because other indications that have been shown to be associated with KL4 surfactant require ever increasing amounts of surfactant. Furthermore, the process is performed under sterile conditions, which contributes significantly to the cost, flexibility of scheduling, and complexity of the product.

In addition to the cost and complexity of using TFE, there is a further complication in that the composition is stored in a liquid state. Because the polypeptide and lipid components of the composition are subject to degradation, the solution must be kept refrigerated to retard any degradation and achieve long-term stability.

Lyophilization or freeze-drying is an important process in the manufacture of solid pharmaceutical formulations. Solid formulations have longer stability and are easier to transport and store than liquid formulations. During the freeze-drying process, the pharmaceutical formulation can be dried to a residual moisture content of 2% or less without raising the temperature above 30 ℃. Thus, this process is less likely to result in thermal degradation of the formulation than a high temperature process, such as spray drying.

The freeze-drying process involves freezing a liquid formulation and removing the solvent associated with the formulation by sublimation directly from the solid phase to the gas phase, without passing through an intermediate liquid phase. Generally, the freeze-drying process consists of three stages, a freezing stage, a first drying and a second drying.

Freezing is the process of solidifying the starting liquid by cooling the material below a given temperature of less than or equal to 0 ℃. The first drying is part of a freeze-drying cycle in which most of the sublimation of the frozen solvent is removed while maintaining the material below a threshold temperature to maintain the structure established during freezing. The second drying is a desorption process of a part of the residual moisture, and is generally performed at a temperature of 25 ℃ or higher. The key process parameters associated with each of these three steps are shelf temperature (shelf time), chamber pressure and time.

The freeze-drying process has been continuously developed over decades. Despite the full knowledge developed in this field, there remains the challenge of producing uniformly distributed masses with mechanically stable structures on a commercial scale at reasonable cost and time.

U.S. patent No. 5952303 to bernstein describes a freeze-dried synthetic lung surfactant obtained by freeze-drying an aqueous suspension of a combination of phospholipids, palmitic acid and peptides.

U.S. patent No. 7582312 to johnson describes a process for preparing a freeze-dried synthetic lung surfactant by freeze-drying a liquid formulation of phospholipids, palmitic acid and peptides in a solvent system containing 20% or more of an organic solvent.

The use of the freeze-drying process described in these above patents for freeze-drying liquid synthetic lung surfactant with organic solvents ranging from 5% or more to less than 20% results in brittle and collapsed materials that are unacceptable for commercial distribution. The manufacture of synthetic lung surfactant using the freeze-drying cycle described previously, results in the lifting of the substance from the vial bottom ("suspension"), which results in reduced heat transfer, uneven heat distribution and production of products with different quality attributes, such as physical morphology and residual moisture.

Thus, there is a need for an improved method of preparing a lung surfactant composition and an improved lung surfactant composition. The present invention proposes a solution to the problem of manufacturing dry-synthesized lung surfactants that are chemically and mechanically stable during the freeze-drying process without compromising their biological activity.

All references cited herein are incorporated by reference in their entirety.

Summary of the invention

One aspect of the invention features a method of making a freeze-dried synthetic lung surfactant having cake suspension (cake suspension) reduced or eliminated in the method. The method comprises the following steps: providing a pre-freeze-dried mixture to a freeze-drying chamber, the pre-freeze-dried mixture comprising at least one phospholipid and a synthetic peptide dispersed in a solvent having an organic solvent and the remainder being water and/or a buffer, the organic solvent being in the range of 3% (v/v) to 20% or less (v/v) of the pre-freeze-dried mixture, wherein the pre-freeze-dried mixture is filled into a container and wherein the synthetic peptide has at least 10 amino acid residues and is represented by the formula:

(Z_aU_b)_cZ_d

wherein Z represents a hydrophilic amino acid residue and U represents a hydrophobic amino acid residue; wherein each Z is independently R, D, E or K; each U is independently V, I, L, C, Y or F; and wherein a is an integer having an average value of about 1 to about 5; b is an integer having an average value of about 3 to about 20; c is an integer from about 1 to about 10; d is an integer from about 1 to about 3; reducing the temperature in the freeze-drying chamber, initiating cooling, and solidifying the pre-freeze-dried mixture during the freezing phase; and an annealing stage prior to the first drying stage, whereby cake suspension in the freeze-dried synthetic lung surfactant is reduced or eliminated.

In one embodiment, further comprising performing a freeze-drying phase in a freeze-drying chamber during the reduction of temperature, wherein the pre-freeze-dried mixture is cooled to a first temperature of less than-45 ℃ using a rate of 0.1 to 1.0 ℃/minute and held at the first temperature for a first period of time sufficient to solidify at least 76% of the solvent to form a first solidified mixture; an annealing stage is performed whereby lump suspension of the first curing mixture is reduced or eliminated, wherein, said first curing mixture is (i) heated to a second temperature, said second temperature being selected to reduce or eliminate suspension of said first curing mixture, (ii) maintained at said second temperature for a second period of time, said second period of time being sufficient to reduce or eliminate suspension of said first curing mixture, and (iii) cooling at a rate of 0.1 to 1.0 ℃/minute to a third temperature below-45 ℃ to form a second solidified mixture, wherein the second solidified mixture is maintained at the third temperature for a third time period sufficient to facilitate separation of unfrozen organic solvent from the second solidified mixture, thereby allowing the unfrozen organic solvent to migrate to the interface between the container and the second solidified mixture; performing a first drying phase at a reduced pressure of 30mT or more, wherein the second solidified mixture is held at a fourth temperature for a fourth time period sufficient to remove at least 5% of the organic solvent, followed by heating to a fourth temperature sufficient to keep the second solidified mixture out of suspension in the vessel and to maintain the structure established in the annealing phase, and held at the fourth temperature for a fifth time period sufficient to remove at least 70% of the solvent and thereby form a third solidified mixture; and performing a second drying phase at reduced pressure for a sixth time period sufficient to produce a freeze-dried synthetic pulmonary surfactant having a residual solvent content of at most 2%.

In certain embodiments of the method, the ratio of the volume of the pre-freeze-dried mixture in the container to the volume of the container is from about 28% to about 68%.

In certain embodiments of the method, the ratio of the height of the pre-freeze dried mixture in the vessel to the diameter of the vessel is in the range of about 0.3 to about 0.8.

In certain embodiments, the method comprises: providing a pre-freeze dried mixture, wherein the organic solvent is in the range of about 3% to about 15%. More specifically, the organic solvent is in the range of about 5% to about 10%. Even more specifically, the organic solvent is in the range of about 7% to about 10%.

Any of the above-described variations on the method may be practiced by: (1) performing a freezing phase such that the pre-freeze dried mixture is cooled to a first temperature of-50 ℃ ± 5 ℃ at a rate of 0.1 to and 1.0 ℃/minute; (2) performing an annealing stage such that the first cured mixture is (i) heated at a rate of 0.1 to 1.0 ℃/minute to a second temperature of-22 ℃ ± 5 ℃, (ii) held at the second temperature for a second period of time of 4 hours to 8 hours, (iii) cooled at a rate of 0.1 to 1.0 ℃/minute to a third temperature of-50 ℃ ± 5 ℃; and (iv) maintained at said third temperature for a third time period of about 3 to 8 hours; (3) the first drying stage is carried out at a pressure selected from about 30mT to about 200mT and a first drying temperature selected from about-25 ℃ to 0 ℃ ramped from-50 ℃ ± 5 ℃ (rampedup) and is further maintained at the first drying temperature for at least 10 hours.

In certain embodiments of the summarized process described above, the second drying stage is carried out at a pressure selected from the range of about 30mT to about 200mT and a temperature of at most 46 ℃. + -. 5 ℃.

In various embodiments of the above-described methodThe pre-freeze dried mixture comprises a peptide having SEQ ID NO: 1 (KL4 polypeptide), Dipalmitoylphosphatidylcholine (DPPC), Palmitoyl Oleoyl Phosphatidylglycerol (POPG) and palmitic acid, and which process results in a freeze-dried synthetic lung surfactant having at least 2.2m²Specific surface area in g. In a specific embodiment, the specific surface area is about 3.7m²G to about 2.2m²In the range of/g. In certain embodiments, the freeze-dried synthetic pulmonary surfactant has a porosity of more than 40% by volume of the total area of the freeze-dried synthetic pulmonary surfactant.

Another aspect of the invention features a freeze-dried synthetic lung surfactant composition including one or more phospholipids and a synthetic polypeptide having at least 10 amino acid residues and represented by the formula:

(Z_aU_b)_cZ_d

wherein Z represents a hydrophilic amino acid residue and U represents a hydrophobic amino acid residue; wherein each Z is independently R, D, E or K; each U is independently V, I, L, C, Y or F; and wherein a is an integer having an average value of about 1 to about 5; b is an integer having an average value of about 3 to about 20; c is an integer from about 1 to about 10; d is an integer from about 1 to about 3, wherein the freeze-dried synthetic lung surfactant composition has a specific surface area of at least 2.7m²/g。

In certain embodiments, the freeze-dried synthetic pulmonary surfactant has a specific surface area ranging from about 3.7m²G to about 2.7m²/g。

In certain embodiments, the freeze-dried synthetic pulmonary surfactant has a porosity of more than 40% by volume of the total area of the freeze-dried synthetic pulmonary surfactant.

In certain embodiments, the freeze-dried synthetic pulmonary surfactant comprises: has the sequence shown in SEQ ID NO: 1 (KL4 polypeptide), Dipalmitoylphosphatidylcholine (DPPC), Palmitoyl Oleoyl Phosphatidylglycerol (POPG), and palmitic acid.

Brief description of the several views of the drawings

Figure 1 is a bar graph showing movement or lack of movement of the freeze-dried substance in the vials after inversion, the number of vials containing the freeze-dried substance that moved after inversion (shown as black bars) and the number of vials containing the freeze-dried substance that did not move after inversion (shown as shaded bars) (see example 5).

FIG. 2 is a graph showing the suspension of a substance during lyophilization (see example 5).

Fig. 3A and 3B are Scanning Electron Microscope (SEM) images of a lyophilized pulmonary surfactant of the invention produced in a 30ml vial, at X20 magnification, surface a and surface B, respectively.

Fig. 4A and 4B are SEM images of freeze-dried lung surfactant preparation II made from born lysine Cycle in a 30ml vial, as described in example 3, using X20 magnification, surface a and surface B, respectively.

Fig. 5A and 5B are SEM images of a freeze-dried lung surfactant of the invention made in a 30ml vial, surface a and surface B, respectively, using X100 magnification.

Fig. 6A and 6B are SEM images of freeze-dried lung surfactant preparation II made from born lysine Cycle in a 30ml vial, as described in example 3, using X100 magnification, surface a and surface B, respectively.

Detailed description of preferred embodiments of the invention

It has been found that dry synthetic lung surfactant with evenly distributed solids can be produced by an improved freeze-drying cycle, said lung surfactant being provided in a mechanically stable, rigid formulation, said formulation being capable of withstanding transport and handling through air.

A freeze-dried pharmaceutical formulation is expected to have a uniform appearance in structure and texture and good physical strength (e.g., able to withstand shipping and handling) to be suitable for commercial distribution. Uniform appearance has been associated with improved stability and less variability in drug activity, drug aesthetics, residual moisture and reconstitution time.

Freeze drying of pharmaceutical formulations containing a suspension of a liposome composition and an organic solvent is a complex task because the organic solvent is trapped as a non-frozen liquid and vaporized at a different rate than other frozen liquids, such as ice, thus creating variations in the solvent composition, resulting in loss of process control, difficulty in maintaining critical process parameters of the chamber pressure, and difficulty in controlling the presence of residual solvent in the dried product.

Attempts to produce dry synthetic lung surfactant from pre-freeze dried mixtures containing organic solvents in the range of 3% to 20% using the freeze drying cycle described in us patent nos. 5952303 and 7582312 did not provide a commercially acceptable product. The resulting product had solids that were non-uniformly distributed along the surface of the freeze-dried vial; the solid appeared to have been suspended in the vial during the process and had a collapsed powdery surface.

Conventional modifications of shelf temperature, pressure and time do not result in the desired product having a uniform appearance in terms of structure and texture and good physical strength (e.g., a freeze-dried product that is able to retain its shape and hold in place after inversion of a vial in which the freeze-dried product is freeze-dried). The initial step in developing the lyophilization process of the present invention is to determine the thermal analysis data associated with the material that is inherent to the particular ingredients and ratios of ingredients contained in the formulation (most amorphous excipients also contain salts in buffers and organic solvents). This thermal analysis was performed by performing freeze-drying microscopy (FDM), Electrical Resistance (ER) and low temperature differential scanning calorimetry (LT-DSC) measurements. Thermal analysis provides important information, such as sufficient solidification temperature and threshold temperature data, in which the material is simultaneously and safely dried in the presence of ice in a first drying process to ensure retention of the structure established during the freezing step. Based on knowledge of the nature and behavior of the organic components during the process, it is expected that this complete solidification cannot be achieved under the conditions used in conventional freeze-drying processes. This fact poses a further significant handling challenge, since the bulk solution (i.e. the pre-freeze dried mixture) comprises a solvent system with a volatile component, such as an organic solvent, in the range of 3% to 20% or less, preferably 3% to 15%, more preferably 5% to 10%, more preferably 7% to 10% (v/v) of the total volume of the pre-freeze dried mixture, the remainder being water and/or buffer. The volatile components have a melting point below the temperature that can be reached by the condenser. Therefore, the organic solvent cannot be solidified by the condenser and is generally not efficiently collected by the condenser throughout the drying process, which is the case in the conventional method. In this process, during the drying phase of freeze drying, organic vapors removed from the material with reduced chamber pressure are temporarily collected on the condenser surface due to the temperature difference between the solvent in the product and the condenser. These vapors, when on the surface of the condenser, are present in a liquid state. The vapor pressure associated with each organic solvent collected as a liquid when reduced to the temperature of the condenser is sufficiently higher than the pressure of the chamber to cause subsequent organic liquid to switch back to a vapor state. This series of events is called reflux (vapor > liquid > vapor) and is repeated continuously throughout the process until, over time, the organic vapor is freed from the condenser and removed from the chamber by the vacuum pump. At the same time, the material in the reflux state is constantly releasing thermal energy (vapor to liquid) and consuming thermal energy (liquid to vapor) to transfer from one phase to another. The material collected in the condenser, as a result, is subjected to the absorption and release of heat associated with the reflux, and is no longer able to maintain steady state conditions. If the sublimation of ice occurs at a higher level during the reflux of the organic solvent, the constant temperature fluctuations and the amount of free surface area of the condenser, without refluxed organic solvent, all have an effect on: condensation of water vapor to ice, controlled chamber pressure, and ultimately the effect on ice sublimation from the product. The traditional method of freeze-drying is sublimation of ice to form water vapor and the conversion of the water vapor back to ice upon collection in a condenser, and is considered to be prior art in the art. To successfully overcome these effects, a specific two-step first drying application was implemented, based on the challenges posed by the presence of organic solvents, and in combination with the convention of lump suspension during processing. The purpose of the two first drying stages is to separate from the sublimation of the frozen water the evaporation of any peripheral organic solvent from the matrix/vial interface. A mass spectrometer was used to measure the residual gas content in the freeze drying chamber. These data indicate that organic solvent was removed during the initial portion of the first drying and that the target cycle parameters for that particular portion resulted in a reduced level of organic solvent, indicating that the process may proceed to the second portion of the first drying. Using this technique, the first elimination of the suspension of the lumps in the target presentation is caused by the implementation of a section dedicated to the removal of the free solvent by evaporation. It is then elaborated that the removal of non-frozen organic solvent in this initial stage can reduce the phenomenon of lump suspension, if the parameters of the first drying-related stage, such as freezing, annealing and temperature rise rate, can be further controlled to circumvent the mobility of the product associated with suspension in vials.

The present inventors have determined that one of the reasons for suspension is that insufficient solidification was achieved by previously employing freezing temperatures of-30 ℃ and-40 ℃. Attempts to reduce the suspension by lowering the shelf temperature to-45 c and increasing the chamber pressure during the first drying step did not yield significant improvements. The presence of alcohol in the freeze-dried mixture creates a "lubricating" effect; the presence of alcohol along the sides of the vial caused the mass to become suspended when it was placed at warmer temperatures. The suspension is eliminated by reducing the final freezing temperature to-45 ℃ or less in the freezing stage, including a heat treatment of annealing prior to the first drying stage, to separate more organic solvent from the mixture. Preferred parameters for the freezing phase are as follows: stepwise cooling to a shelf temperature of-45 ℃ or less, preferably-50 ℃ to-40 ℃ ("final freezing temperature"), followed by holding the shelf temperature for 1-10 hours, preferably 2-8 hours, more preferably 3-5 hours, all at atmospheric pressure. After the pre-freeze-dried mixture was equilibrated at 2-8 ℃ on a shelf for several hours, gradual cooling began, and then the temperature was initially lowered at an approximate rate of 0.1 to 1 ℃ per minute.

Further, to improve processing time and still maintain the desired uniformity and appearance, the inventors analyzed the ratio between the volume of the sample and the size of the vial. Since cake suspension is a rare phenomenon, it is new to evaluate the handling behavior in case of different vial sizes based only on the larger surface area to which frozen cakes adhere, rather than the conventional method of evaluating individual vial sizes to obtain a minimum filling height for the vial ratio. Surprisingly, it was found that varying vial size from 20ml capacity to 50ml capacity, while having the same initial fill volume (13.7 ml), did not provide the associated improvement in appearance of freeze-dried cakes for vials from smaller vials to the largest size. While 30mL vial samples had more evenly distributed chunks than 20mL vial samples, 50mL vial samples were less uniform than 20mL vial samples. The lack of improvement in the material processed in the 50ml capacity vials is likely related to the elevated level of cake suspension observed in the container, which is highly related to the resulting cake, being a higher level than the cake height of the 20 cc and 30 cc vials. In scale-up filling, the fill volume (volume of the pre-freeze dried mixture) and the volume of the vial (or other type of container used to hold the fill) and/or the ratio between the pre-freeze dried mixture fill height of the pre-freeze dried mixture in the vial and the vial diameter should be observed to eliminate suspension of the fill material in the vial. The ratio of the filling height in the vial to the vial diameter should be in the following range: from about 0.3 to about 0.8, preferably from about 0.4 to about 0.7, and more preferably from about 0.5 to about 0.6. The ratio of fill volume to vial volume should be about 28% to about 68%, with a preferred range being from about 35% to about 55%. It should be understood, however, that the "listed" volume of the vial used in these calculations (i.e., the volume listed in catalogues and trade pamphlets) is not the actual internal volume, where the listed amount may differ from the actual volume by about 10%.

The inventors have determined that adding an annealing phase after the freezing phase, or as an intermediate step before reaching the final freezing temperature, helps to create a more rigid mass with an expanded skeleton that will adhere close to the sides and bottom of the vial and thus eliminate solid suspension. Another goal achieved by increasing the annealing stage is to obtain an increase in the sublimation rate, thus reducing the overall processing time without jeopardizing the appearance of the cake.

The annealing stage will be described in detail below. The term "annealing stage" refers to heat treatment conditions that promote separation of components of a mixture and/or crystallization of components. The heat treatment may involve (a) cooling to an intermediate temperature followed by further cooling to a final low temperature ("final freezing temperature"), or (B) cooling to a low temperature, then heating to an intermediate temperature, then re-cooling to a final low temperature, or (C) cooling to a low temperature, then warming to an intermediate temperature. The "annealing stage" occurs during the freezing stage of the freeze-drying process. Annealing is performed to increase the likelihood of preventing clumps from floating from the bottom of the vial during processing by creating a rigid frozen matrix. Furthermore, annealing facilitates migration of unfrozen solvent to the vial/matrix interface and further facilitates subsequent separation of solvent from the product during the initial conditions for the first drying, effectively reducing the amount of solvent and making the solutes, i.e., API and buffer salts, more rigid, while any unfrozen water is converted to ice. The intermediate temperature is selected based on low temperature thermal analysis and visual characterization of the substance in which the glass transition temperature (Tg') or a temperature corresponding to the observed physical change is determined. The low temperature is selected based on thermal analysis and visual characterization of the material, in which characterization the temperature of full cure is measured. Tg' or other observed physical changes, such as changes in opacity or liquid-like movement, determine the intermediate temperature at which the product should be annealed, and the temperature at which it fully solidifies determines what low temperature set point should be used to promote the desired frozen substance properties for primary drying. In particular, the material should be annealed at a temperature a few degrees above Tg', or the physical change observed, and at a minimum, the material should be cooled to a temperature at or below the temperature at which it fully solidifies. For the substance described in this document as formulation I, the physical changes in liquid-like motion observed during thermal analysis were observed at a temperature of-28 ℃ (the temperature at which liquid-like motion was observed under a freeze-drying microscope). In certain embodiments, a complete cure temperature is observed at-45 ℃. Nucleation must first be confirmed before intermediate or low hold. The temperature of each of the a, B and C variants is the same or similar, however, the order in which the temperatures are performed will vary. The selection of temperature may be based on formulation composition or threshold temperature. The threshold temperature is defined as the following temperature: the temperature of the product needs to be kept below the aforementioned threshold temperature during the first drying, in the presence of ice, to avoid collapse, melting, or in some cases, suspension of the cake.

In certain embodiments, the annealing step is performed as described in variation (B) above. In these embodiments, the annealing stage comprises the following steps: warming the frozen cake from-45 ℃ +/-5 ℃ (final freezing temperature) to-22 ℃ +/-5 ℃ (intermediate temperature) at a rate selected from about 0.1 ℃/minute to about 1 ℃/minute (warming step), and maintained at-22 deg.C +/-5 deg.C for a time sufficient to facilitate separation of the organic solvent from the solute mixture, and a more rigid solid to reduce or eliminate suspension, preferably for 3-8 hours (holding step), then re-cooled at a rate of about 0.1 deg.C/min to about 1 deg.C/min to-45 deg.C +/-5 deg.C (cooling step) and held at-45 deg.C +/-5 deg.C for a period of time to ensure adequate curing, for example, 3 to 8 hours, preferably 4 hours, wherein all four steps are carried out at atmospheric pressure. In certain embodiments, the annealing step is performed as described in example 2, and 9-13.

Alternatively, sufficient curing may be achieved as follows: cooling directly to the final cryogenic temperature without a final hold step by cooling the vial to the final cryogenic temperature at a constant rate selected from the range of about 0.1 ℃/minute to about 1 ℃/minute until the desired temperature is reached. In another embodiment (variant (a) above), an annealing stage is performed as an intermediate step before the freezing stage reaches the final low temperature. In this example, the annealing phase begins when the temperature of the shelf reaches the temperature at which the liquid product (about-15 ℃ +/-5 ℃.) (a "nucleation temperature") nucleates, and includes holding at the nucleation temperature for a period of time sufficient to convert the water to ice and separate the solutes in the mixture, the period of time being between 45 minutes and 4 hours (intermediate holding step), and then continuing to cool the mixture at a rate of 0.1 ℃/minute to 1.0 ℃/minute until the desired final freezing temperature (-50 ℃ +/-5 ℃) is reached and the holding step is performed at this temperature until the freezing phase is complete.

In one embodiment, the annealing stage comprises raising the frozen mass from-50 ℃ +/-5 ℃ to-22 ℃ +/-5 ℃ at a rate of 0.1 to 1 ℃/min and holding at-22 ℃ +/-5 ℃ for 3-8 hours, then cooling again to-50 ℃ +/-5 ℃ at a rate of 0.1 to 1 ℃/min and holding at-50 ℃ +/-5 ℃ for 3-8 hours, preferably 4 hours, wherein all four steps are carried out at atmospheric pressure.

In certain embodiments, the annealing step is performed as described in variation (a) above. The intermediate temperature may be selected from-10 ℃ to-35 ℃. The holding at the intermediate temperature is for a period of time sufficient to convert the water to ice and separate the solutes in the mixture, the period of time being between 45 minutes and 4 hours. Ramping up to the final freezing temperature at an approximate rate of 0.1 to 1 deg.c/minute followed by holding at that temperature for about 0.5 to 5 hours until the curing stage is complete. A first drying step is then carried out as described. An example of this process is described in example 14, see table 16.

The first drying stage is carried out at reduced pressure (vacuum) and comprises the following steps: the temperature of the previous step is optionally maintained for a period of time sufficient to vaporize at least 5%, preferably, 5 to 10%, of the organic solvent, gradually ramped from the temperature of the previous step-50 ℃ +/-5 ℃ to a temperature in the range of about-25 ℃ to about 0 ℃ (the first drying temperature) (the heating step) at a rate of 0.1 ℃/minute to 2 ℃/minute and a pressure of +/-10mT of 30mT to 200mT, preferably 40mT, followed by a maintenance step of the first drying temperature for a period of time sufficient to sublimate at least 70%, preferably at least 80%, of the solvent, preferably 5 to 10% of the organic solvent, or for a period of time sufficient to sublimate at least 80% of the solvent if the optional maintenance step is not performed. The maintaining step may last for 10 hours or more. In certain embodiments, the holding step is from 18 to 140 hours, preferably from 18 to 100 hours. The temperature of the first drying is chosen to be between 0 ℃ and-25 ℃ based on the desire to keep the sample in the vial out of suspension, dry and retain the structure established during freezing. The ability to optimize the duration of the first drying stage is enhanced by the design of the annealing stage.

Next, the second drying stage will be explained. The purpose of the second drying stage is to reduce the residual moisture content in the product obtained in the first drying stage by increasing the temperature of the product obtained in the first drying stage, typically to super-ambient temperature, to remove any residual water and holding at the selected temperature for a period of time sufficient to produce a freeze-dried synthetic lung surfactant having a residual solvent content of at most 2%. The secondary drying temperature typically ranges from 20 ℃ to 30 ℃. However, the second drying temperature range may be as low as-7 ℃ to as high as +60 ℃. The second drying temperature set point should be selected based on: 1) maintaining the product stable in the second drying stage by implementing a shelf temperature that maintains the product temperature at least 5 ℃ below the observed Tg, and 2) promoting desorption by achieving a temperature that is warm enough to effectively reduce residual moisture to within specifications that employ commercially reasonable rates. The observed Tg of formulation I was between 45 deg.C and 51 deg.C, so the temperature during secondary drying could be as high as 46 deg.C. The residual moisture results indicate that the residual moisture results are effectively achieved at the second drying temperature set point of 25 ℃. The product is then held at the selected second drying temperature for a period of time sufficient to produce a final product having a residual solvent content of at most 2%. One way to assess whether drying is complete is to perform a pressure rise test (reward test) in which a pressure of 10 microns indicates that residual moisture is within specification. In certain embodiments, the second drying stage is carried out by heating the shelf temperature in the previous step to 25 ℃ +/-3 ℃ at a rate of 0.1 to 1 ℃/minute, preferably at a rate of 0.2 to 0.5 ℃/minute. This stage is also performed in vacuum as the previous stage. The preferred pressure range is from 30mT to 500mT, more preferably from 40mT to 150 mT. After the product reaches the selected shelf temperature, the holding step is carried out at 25 deg.C +/-3 deg.C for 4-10 hours, preferably 6 hours.

The freeze-dried material is then purged with 0.5 bar of an inert gas, e.g., nitrogen, before finally fully inserting the stopper and sealing for storage.

To determine the reproducibility of the target cycle parameters, important product attributes such as temperature profile, sublimation temperature (break temperature) and product characteristics were determined after processing with the same freeze-drying cycle. The sublimation "break" temperature is the temperature just before the point in time at which the product temperature is extremely close to the shelf temperature during the first drying. The product temperature "break" indicates that sublimation is complete in a given vessel, while taking into account the placement of the measuring thermocouple being positioned in a location (bottom center) where ice may be found last. For each meaningful step in the process (e.g., annealing, freezing, primary drying, sublimation break, and secondary drying), the product temperature ranges studied were within 0.5 ℃ for both annealing and freezing, within 1 ℃ for primary drying and sublimation break, and within 1.5 ℃ for secondary drying. Based on this nominal variation, the thermal behavior during processing is considered reproducible. Evaluation of the sublimation end time range showed that in different studies sublimation was completed within 6 hours, also suggesting sufficient reproducibility. Finally, evaluations of the finished product, such as physical appearance, residual moisture, reconstitution time, clarity of the solution, reconstituted pH, thermogravimetric mass loss (TGA) and high temperature differential scanning calorimetry (HT-DSC) all gave similar results, further supporting the ability to reproduce consistent product quality in larger scale different studies. The conclusion drawn from these study evaluations is that the freeze-drying cycle parameters implemented to freeze-dry the synthetic pulmonary surfactant containing organic solvent at 3 to 20% or less, preferably 3 to 15%, more preferably from 5 to 10%, and more preferably from 7 to 10% (v/v) of the total volume of the pre-freeze dried mixture are stable and sufficient to obtain a consistent material with acceptable product quality attributes without the phenomenon of cake suspension.

Freeze drying was carried out in a four shelf freeze dryer apparatus providing 8 square feet of shelf space with a 15 kg internal ice condenser capacity. The apparatus is constructed of type 304L stainless steel, which is identified as a pressure vessel operating at up to 20psig for steam sterilization. A typical freeze-drying apparatus includes a nominal pressure chamber, a condenser, a vacuum system with pressure control features, and a circulating heat transfer fluid cycle capable of achieving a temperature range of about-55 ℃ to 50 ℃.

The lyophilized substance is white in color, uniformly dispersed in the vial, cylindrical in shape (i.e., mimicking the shape of the vial), compact in appearance, has minimal shrinkage compared to the initial fill, has a rigid structure such that it does not move upon inversion of the vial, and has a top surface free of traces of the substance or edges. The material had matte sides along the top, sides and bottom of the block. The freeze-dried material achieved the specifications for residual moisture, DSC, reconstitution, pH and viscosity.

The pre-freeze-dried mixture and its preparation will be described in detail below. Major components-the Active Pharmaceutical Ingredient (API) are phospholipids (e.g., Dipalmitoylphosphatidylcholine (DPPC) and Palmitoyl Oleoyl Phosphatidylglycerol (POPG)), Palmitic Acid (PA) and synthetic lung peptide (preferably KL 4).

In certain embodiments, a peptidomimetic of a pulmonary surfactant refers to a polypeptide having a sequence of amino acid residues with a complex hydrophobicity (complex hydrophilicity) of less than zero, preferably less than or equal to-1, more preferably less than or equal to-2. By e.g. Hopp et al proc.natl.acad.sci.78: 3824-3829, 1981, the disclosure of which is incorporated herein by reference, the composite hydrophobicity value of a peptide is determined by assigning a corresponding hydrophilicity value to each amino acid residue of the peptide. For a given peptide, the hydrophobicity values are summed, and the sum represents the composite hydrophobicity value.

In certain embodiments, the polypeptides of the lung surfactant mimic the amino acid sequence, mimic the pattern of hydrophobic and hydrophilic residues of SP18, and perform the function of the hydrophobic region of SP18, as described in US patent No. 3789381 (which is incorporated herein in its entirety). In certain embodiments, SP-B mimetics useful herein include polypeptides having regions of alternating hydrophobic and hydrophilic amino acid residues characterized as having at least 10 amino acid residues represented by the formula:

(Z_aU_b)_cZ_d

z and U are amino acid residues such that each occurrence of Z and U is independently selected. Z is a hydrophilic amino acid residue, preferably selected from the group consisting of R, D, E and K. U is a hydrophobic amino acid residue, which is preferably selected from the group consisting of V, I, L, C, Y and F. The letters "a", "b", "c" and "d" are numbers, which indicate the number of hydrophilic or hydrophobic residues. The letter "a" has an average value of about 1 to about 5, preferably about 1 to about 3. The letter "b" has an average value of from about 3 to about 20, preferably from about 3 to about 12, and most preferably from about 3 to about 10. The letter "c" is 1 to 10, preferably 2 to 10, most preferably 3 to 6. The letter "d" is 1 to 3, preferably 1 to 2.

By specifying that the amino acid residues represented by Z and U are independently selected, it is meant that, at each occurrence, a residue is selected from the specified group. That is, when "a" is 2, for example, each hydrophilic residue represented by Z will be independently selected and thus may include RR, RD, RE, RK, DR, DD, DE, DK, etc. By specifying that "a" and "b" have an average value, it is meant that, although the sequence (Z) is repeated_aU_b) The number of residues within may vary somewhat in the peptide sequence, but the average values of "a" and "b" are from about 1 to about 5 and from about 3 to about 20, respectively.

In certain embodiments, exemplary SP-B peptide mimetics that can be used in the present invention include, but are not limited to, those shown in the table of lung surfactant mimetic peptides.

Table of lung surfactant mimetic peptides

¹The name is an abbreviation for the indicated amino acid residue sequence.

Examples of phospholipids useful in the compositions delivered by the present invention include natural and/or synthetic phospholipids. Phospholipids that may be used include, but are not limited to, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, sphingolipids, diacylglycerides, cardiolipin, ceramides, cerebrosides, and the like. Exemplary phospholipids include, but are not limited to, Dipalmitoylphosphatidylcholine (DPPC), Dilaurylphosphatidylcholine (DLPC) (C12: 0), Dimyristoylphosphatidylcholine (DMPC) (C14: 0), Distearoylphosphatidylcholine (DSPC), diphytanoylphosphatidylcholine, nonadecylphosphatidylcholine, arachidoylphosphatidylcholine, Dioleoylphosphatidylcholine (DOPC) (C18: 1), dipalmitoylphosphatidylcholine (C16: 1), linoleoylphosphatidylcholine (C18: 2), Myristoylphosphatidylcholine (MPPC), Stearoylphosphatidylcholine (SMPC), Stearoylpalmitoylphosphatidylcholine (SPC), palmitoylphosphatidylcholine (POPC), palmitoylphosphatidylethanolamine (PPoPC), Dipalmitoylphosphatidylethanolamine (DPPE), palmitoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine (DOPE), Dimyristoylphosphatidylethanolamine (DMPE), Distearoylphosphatidylethanolamine (DSPE), Dioleoylphosphatidylglycerol (DOPG), palmitoyloleoylphosphatidylglycerol (POPG), Dipalmitoylphosphatidylglycerol (DPPG), Dimyristoylphosphatidylglycerol (DMPG), Distearoylphosphatidylserine (DSPS), palmitoyloleoylphosphatidylserine (POPS), soybean lecithin, egg lecithin, sphingomyelin, phosphatidylinositol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidic acid, and Egg Phosphatidylcholine (EPC).

Examples of fatty acids and fatty alcohols useful in these mixtures include, but are not limited to, sterols, palmitic acid, cetyl alcohol, lauric acid, myristic acid, stearic acid, phytanic acid, dipalmitic acid (dipalmitic acid), and the like. Preferably, the fatty acid is palmitic acid and the preferred fatty alcohol is cetyl alcohol.

Examples of fatty acid esters suitable for use in these mixtures include, but are not limited to, methyl palmitate, ethyl palmitate, isopropyl palmitate, cholesterol palmitate, sodium palmitate, potassium palmitate, tripalmitin and the like.

An example of a semi-synthetic or modified natural lipid is any of the lipids described above that have been chemically modified. Chemical modifications may include several modifications; however, a preferred modification is the conjugation of one or more polyethylene glycol (PEG) groups to the desired moiety of the lipid. Polyethylene glycol (PEG) has been widely used in biomaterials, biotechnology and medicine, primarily because PEG is a biocompatible, non-toxic, non-immunogenic and water-soluble polymer. In the field of drug delivery, PEG derivatives have been widely used for covalent attachment to proteins (i.e., "pegylation") to reduce immunogenicity, proteolysis, and renal clearance, and to improve solubility.

Lipids that have been conjugated to PEG are collectively referred to herein as "PEG-lipids". Preferably, when PEG-lipids are used in the methods and compositions of the present invention, they are present in alcohols and/or aldehydes.

Other excipients may be combined with the lung surfactant polypeptide, one or more lipids, and organic solvent system prior to lyophilization, including, but not limited to, various sugars, such as glucose, fructose, lactose, maltose, mannitol, sucrose, sorbitol, trehalose, and the like, surfactants such as, for example, polysorbate 80, polysorbate 20, sorbitan trioleate, tyloxapol, and the like, polymers such as polyethylene glycol, dextran, and the like, salts such as NaCl, CaCl, and the like₂Etc., alcohols such as cetyl alcohol, and buffering agents.

Preferably, the lung surfactant peptide binds to phospholipids and free fatty acids or fatty alcohols, e.g., DPPC (dipalmitoylphosphatidylcholine), POPG (palmitoyl oleoylphosphatidylglycerol) and Palmitic Acid (PA). See, for example, U.S. patent No. 5789381, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

The first step in preparing the pre-freeze-dried mixture is to obtain a substantially homogeneous liquid mixture of lung surfactant peptide, one or more lipids in an organic solvent system containing 93-100% organic solvent, preferably 95% ethanol. By the term "substantially homogeneous" is meant that the components are uniformly dispersed in each other, e.g., as in a solution. The API was mixed in an organic solvent system heated to 45 ℃ ± 5 ℃ until a solution was obtained. The resulting solution is then filtered through a sterile filter (0.22 micron) into a buffer, preferably a TRIS (hydroxymethyl) aminomethane (TRIS) buffer solution heated to 45 ℃ ± 5 ℃, and stirred to produce a pre-freeze dried mixture in the form of a liposome suspension having an organic solvent concentration in the range of 3% to 20% or less (v/v), preferably 3% to 15%, more preferably from 5% to 10%, still more preferably 7% to 10%, of the total volume of the pre-freeze dried mixture, with the remainder being water and/or buffer.

In certain preferred embodiments, the lung surfactant peptide, phospholipid and free fatty acid or fatty alcohol, e.g., a mixture of DPPC (dipalmitoylphosphatidylcholine) and POPG (palmitoyl oleoylphosphatidylglycerol) and Palmitic Acid (PA), are mixed with an organic solvent system to form a substantially homogeneous liquid mixture. The individual components may be present in the mixture in any concentration. The total concentration of phospholipids in the dispersion may range, for example, from about 1 to over about 80 mg-total phospholipid content/ml. Suitable buffering agents include, but are not limited to, tris acetate, tris hydrochloride, sodium phosphate, potassium phosphate, and the like. Buffers are generally commercially available.

In a preferred embodiment, the liposome suspension used in the method of the invention comprises DPPC, POPG, PA and KL4 (in a weight ratio of about 7.5: 2.5: 1.35: 0.267) in a physiologically acceptable solvent having an organic solvent concentration in the range of 3% to less than 20% of the total volume of the pre-freeze dried mixture (v/v), preferably 3% to 15%, more preferably 5% to 10%, and more preferably 7% to 10%, the remainder being water and/or buffer.

In certain embodiments, the organic solvent system further comprises additional excipients including, but not limited to, various sugars such as glucose, fructose, lactose, maltose, mannitol, sucrose, sorbitol, trehalose, and the like, surface active agents such as, for example, polysorbate 80, polysorbate 20, sorbitan trioleate, tyloxapol, and the like, polymers such as polyethylene glycol, dextran, and the like,salts, e.g. NaCl, CaCl₂And a buffer. In certain preferred embodiments, the organic solvent system is substantially salt-free. In certain preferred embodiments, the organic solvent system is substantially free of NaCl.

In certain embodiments, the organic solvent system may be prepared by combining all of the system components. For example, in certain embodiments, wherein the organic solvent comprises an organic solvent and an aqueous medium at room temperature, the aqueous medium and organic solvent may be combined to complement the organic solvent system. Preferably, the organic solvent system is an emulsion or a miscible solution.

The organic solvent selected is preferably compatible with sterile filtration and freeze-drying. Preferably, the organic solvent of the present invention is selected from the group consisting of lower oxyhydrocarbon, lower halogenated hydrocarbons, lower halogenated oxyhydrocarbons, lower sulfonylhydrocarbons (sulfinyl, sulfoxy hydro carbons), lower cyclic hydrocarbons and combinations thereof.

Suitable organic solvents for use in the present invention include, but are not limited to, isopropanol, methanol, ethanol, acetone, acetonitrile, cyclohexane, chlorobutanol, dimethyl sulfoxide, t-butanol, hexanol, benzyl alcohol, acetic acid, pentanol (1-pentanol), n-butanol, n-propanol, methyl acetate, dimethyl carbonate, methyl ethyl ketone, methyl isobutyl ketone, carbon tetrachloride, hexafluoroacetone, chlorot-butanol, dimethyl sulfone, cyclohexane, and combinations thereof. Preferred solvents include lower alkanols such as t-butanol, ethanol, isopropanol, and the like. A particularly preferred solvent for the present invention is ethanol.

In certain preferred embodiments, the lung surfactant composition is lucinactant or other lung surfactant preparation comprising synthetic surfactant peptide KLLLLKLLLLKLLLLKLLLLK (KL 4; SEQ ID NO: 1). In certain preferred embodiments, the freeze-dried lung surfactant of the present invention, when reconstituted, results in a combination of APIs: a weight ratio of about 7.5: 2.5: 1.35: DPPC, POPG, PA and KL4 peptides of 0.267), or with DOf iscovery laboratories, Inc. (Warrington, Pa., USA)(lucinactant) liquid synthesis of DPPC, POPG, PA and KL4 peptides in the same weight ratio in lung surfactant. In certain embodiments, the lung surfactant composition is formulated to a phospholipid content of, for example, 10, 20, and 30 mg/ml. In certain other embodiments, the lung surfactant composition is formulated at higher concentrations, such as a phospholipid content of 60, 90, 120 or more mg/ml, with an increase in KL4 concentration.

In certain exemplary embodiments of the invention, the relative amounts of lung surfactant peptide, phospholipid and free fatty acid or fatty alcohol are about 1 part by weight of synthetic surfactant peptide; from about 20 to about 150 parts by weight of a phospholipid per part by weight of synthetic surfactant peptide; from about 0 to about 25 parts by weight of free fatty acid or fatty acid alcohol per part by weight of synthetic surfactant peptide. In certain embodiments, the relative amount of the organic solvent system is in the range of greater than 62.5 and less than 250 parts by weight per part by weight of lung surfactant peptide. In certain embodiments, the organic solvent system is present in a range of 80 to 125 parts by weight per part by weight of lung surfactant peptide. In certain exemplary embodiments, the relative amount of lung surfactant peptide, phospholipid and free fatty acid or fatty alcohol is about 1 part by weight of lung surfactant peptide, such as, for example, KL 4; about 20 to about 100 parts by weight of DPPC; 0 to about 50 parts by weight of POPG; and about 0 to about 25 parts by weight palmitic acid.

The lyophilized substance of the present invention is white in color, uniformly dispersed in the vial, cylindrical in shape (i.e., mimicking the shape of the vial), dense, has minimal shrinkage compared to the initial fill, has a rigid structure such that it does not move upon inversion of the vial, and has a top surface free of traces of material or edges. The lyophilized compositions of the present invention prepared by the above method are comparable to pulmonary surfactants prepared by other lyophilization methodsThe lung surfactant has large specific surface area (at least 2.2 m)²/g), and a larger total pore area (at least 40%). Preferably, the specific surface area of the freeze-dried synthetic lung surfactant of the present invention ranges from about 3.7m²G to about 2.2m²(ii) in terms of/g. It has also been found that, in addition to using annealing for creating a more rigid bulk structure, changes in vacuum during the first drying affect the specific surface area. The BET data presented in example 15 shows that the specific surface area is greatest at 40 microns compared to the specific surface area of the sample prepared at 150 microns. This finding will further contribute to the improved design of the structure of the lyophilized synthetic lung surfactant cake.

The freeze-dried material passed a check for residual moisture, reconstitution time, pH, viscosity, and its biological activity the freeze-dried material was reconstituted with 10ml sterile water for injection (WFI) and the solids were dispersed within 20-30 seconds the pH was in the range of 7.6-7.9 the viscosity was measured at 37 ℃ (Brookfield engineering laboratories, Inc., Middleboro, MA) with a Brookfield viscometer, Model LVDV-II was in the range of 77-105 centipoise (0.077-0.105Pa s.) the ability of the sample to reduce surface tension (e.g., biological activity) was analyzed by a Pulsing Bubble Surfactometer (PBS) Model EC-PBS-B (electronics Corporation, U.S.A., non General inco, Largo, FL) at 37 ℃; the average surface tension was measured at 2-7 cm/352 (2 cm 2/2 × 10)^-3To 7x10^-3N/m). Acceptable values for effective lung surfactant are below 10 dynes/cm (0.01N/m).

One way to characterize a lyophilized material is to measure its specific surface area. Specific surface area is a measure of the exposed surface of a solid sample at the molecular scale. BET (Brunauer Emmet and Teller) surface area analysis is an established model for determining the specific surface area of a solid by physical adsorption of gas molecules (see united states pharmacopeia title:<846>specific surface area (Http:// www.pharmacopeia.cn/v29240/usp29nf24s0_ c846. html)). The samples were typically prepared as follows: while heating, evacuating or passing a gas throughOn the sample to remove free impurities. The prepared samples were then cooled with liquid nitrogen or krypton and analyzed by measuring the volume of gas adsorbed at a particular pressure. Used by micromeritics pharmaceutical Services (Norcross, GA)2420 Accelerated Surface Area and porosity System (Accelerated Surface Area and porosity System) (Mike Instruments, Inc., Norcross, Ga.) selected samples were tested at BET11 points under vacuum at 25 ℃ for 16 hours 100% Krypton was used as the adsorbate analysis bath temperature was about 77 K.the following parameters were measured, sample mass (grams), cold free space (cubic centimeters), warm free space (milliliters), saturation pressure (Po) (mmHg), absolute pressure (P) (mmHg), and elapsed time.A linear plot of isotherms was calculated for each sample, where the Y axis is the amount adsorbed (cubic centimeters per gram STP) and the X axis is the relative pressure (P/Po). STP is referred to as the standard temperature and pressure, i.e., a temperature and atmospheric pressure of 273.15K (1.36013 10K) (1. 1.013 × 10K)⁵Pascal). The data are presented in the examples below.

Another parameter useful in the morphological characterization of a freeze-dried substance is its porosity, which is defined as the ratio of the volume of all pores in the substance to the volume of the substance as a whole. The porosity of the freeze-dried material was determined with a Scanning Electron Microscope (SEM) JEOL6480 scanning electron microscope (JEOL, japan). The sample was removed from the vial by cutting the vial top with a diamond saw and cutting it in half across its width. The cross-cut sections of the cut samples were placed on a scanning electron microscope device and visualized at magnifications (X) of 20 and 100. Analysis was performed at room temperature under vacuum. The surface area of the magnification X20 was approximately 6.4 mm X5.1 mm. The surface area of the magnification X100 is about 1200 microns X965 microns.

SEM micrographs of the transected scaffolds revealed microchannels or porous structures throughout the cross-section of each sample. Two good spots were selected for imaging: the top (uncut area) of the block is "surface a" and the interior of the block is "surface B". Fig. 3A and 3B show magnified images of the freeze-dried lung surfactant of the invention prepared in 30ml vials, surface a and surface B, respectively, at X20 magnification. Fig. 4A and 4B show magnified images of freeze-dried lung surfactant preparation II made from born lysine Cycle as described in example 3 in a 30ml vial, using X20 magnification, surface a and surface B respectively. Fig. 5A and 5B show magnified images of the freeze-dried lung surfactant of the invention prepared in 30ml vials, surface a and surface B, respectively, at X100 magnification. Fig. 6A and 6B show magnified images of freeze-dried lung surfactant preparation II made by Bornstein Lyo Cycle in a 30ml vial as in example 3, using X100 magnification, surface a and surface B respectively.

Notably, formulation III (A0490-62), prepared as described in example 4 using "Johnson Lyo Cycle", was described as not being amenable to analysis by scanning electron microscopy due to its fragility. The blocks do not even withstand the slight pressure of the saw and collapse. Comparing the total pore area of formulation I (A0490-55) made using Novel Lyo Cycle as described in example 2 with the total pore area of formulation II (A0490-58) made using "Bornstein Lyo Cycle" as described in example 3, it was observed that formulation I was more porous with an absolute difference of at least 11%.

Using microscopesImages Plus 2.0 software (mcondi group ltd, mansion, china) calculated the open area of the selected image. A relief filter (relief filter) is applied to highlight the open area and then the open surface area is calculated using the automatic segmentation and automatic calculation functions. This approach minimizes manual manipulation of the data and removes bias between comparing images. For formulation I, the well region constituted 49.1% of the measured area of the top of the sample (surface a) and 50.5% of the measured area of the interior of the cake (B-side). For the preparationII, the hole region constituted 37.3% of the measured area of the sample top (surface A) and 36.7% of the measured area of the interior of the cake (B-side). The corresponding differences are 11.8% and 13.8%.

Manual pore size calculations were performed to test the accuracy of the above method on the samples, which are shown in fig. 5A (formulation I, surface a, X100) and fig. 6A (formulation ii, surface a, X100). Each micrograph is trimmed to dimensions of 6.4 mm (width) and 3.9 mm (height), and 20 "holes" in each image are measured for height and width and compared.

The freeze-dried synthetic lung surfactant composition of the present invention has a unique combination of: larger surface area (at least 2.7 m)²/g), greater porosity (above 40% by volume) and exhibit rigidity, for example, resistance to movement when inverted and also to movement when the vial containing the substance is tapped. A more rigid mass would be associated with reduced molecular mobility, precursors to chemical reactions, and therefore more stable products.

It was previously observed that of the four APIs, the cinasopeptide or KL4 peptide degraded in a liquid environment faster than in the solid state as a lyophilized substance (see U.S. patent No. 5952303, born). As it is believed that the homogeneous appearance of the freeze-dried formulation is also an embodiment of a more stable product, the inventors expect that the freeze-dried formulation obtained by the inventive method described herein will exhibit an increased stability of at least the KL4 peptide during storage at 25 ℃ for at least 3 months. It is expected that the stability of KL4 and other APIs of the freeze-dried formulation prepared by the method of the invention will be statistically superior to freeze-dried formulations obtained by other freeze-drying methods over longer shelf-life, e.g. 6 and 9 months, at 25 ℃ and if stored at higher temperatures, such as 30 ℃ and 40 ℃. Stability is expected to increase by at least 2% or more, at least 5% or more, or at least 10% or more.

The freeze-dried lung surfactant of the present invention may be reconstituted with water or other pharmaceutically acceptable diluents. The use of liquid or freeze-dried lung surfactants has been described previously. The new freeze-dried lung surfactant exhibits excellent bulk and resistance to movement and transport, which are essential attributes of pharmaceutical products.

The present invention will be described in more detail with reference to the following examples, but it should be understood that the present invention is not construed as being limited thereto.

Examples

Example 1

Lyophilization was performed using the novel process of the present invention and the methods described in previously published U.S. patent nos. 5952303 and 7582312 to demonstrate the differences each method imparts to the resulting lyophilized product.

Materials: the components in the lyophilized formulations from these three methods are summarized in Table 1 below. The actual amount is adjusted for the purity of the starting material.

TABLE 1 starting materials for formulations I, II and III (3000g batch size)

The procedure is as follows: two 3000 gram batches were prepared for each of the three methods. A standard fill weight of 13.7 grams was added to each vial using a syringe. Preparation of the pre-freeze-dried mixture: the API was dissolved in 95% ethanol at 46 ℃ ± 1 ℃ to give a solution. The resulting solution was sterile filtered through a 0.22 micron 33 mm filter using pressure into a stirred TRIS (hydroxymethyl) aminomethane (TRIS) buffer solution at 45 ℃ ± 2 ℃ to yield a liposomal formulation with a final ethanol concentration of 10% (w/w). After cooling to a temperature below 30 ℃, the resulting liposome preparation, i.e. the pre-freeze dried mixture, was transferred to 20, 30 and 50mL borosilicate glass freeze dried vials with a fill volume of 13.7 grams per vial. The resulting lyophilizate was stored at 5 ℃.

Example 2

Formulation i. the pre-freeze-dried mixture from table 1 was used as a fill in 20, 30 and 50mL glass bottles and freeze-dried using the novel freeze-drying method described above. Table 2 summarizes the parameters of the freeze-drying process.

TABLE 2 parameters of the novel lyophilization process

The stages are carried out at atmospheric pressure. The stages were performed under vacuum.

Example 3

Preparation II the pre-freeze dried mixture from Table 1 was used as a fill in 20, 30 and 50mL glass vials and freeze dried using the method described in Bornstein, US patent 5952303 ("Bornstein Lyo Cycle"). Table 3 summarizes the parameters of the process.

TABLE 3 Freeze-drying parameters of Bornstein Lyo Cycle

Initial shelf temperature:	25℃.
		cooling step	At-40 deg.C, 1.0 deg.C/min
Maintaining step	At-40 ℃ for 2 hours
		Vacuum:	100mT
heating step	0.5 ℃/min at 0 DEG C
		Maintaining step	0 ℃ for 48 hours
Heating step	At 26 deg.C, at 0.5 deg.C/min
		Maintaining step	At 26 ℃ for 12 hours
Backwashing:	N2closing the vial up to 1/2 bar

The stages are carried out at atmospheric pressure. The stages were performed under vacuum.

Example 4

Formulation iii pre-freeze dried mixture from table 1 was used as a fill in 20, 30 and 50mL glass bottles and freeze dried using the method described in Johnson Lyo Cycle 7582312. Table 4 summarizes the parameters of the process.

TABLE 4 Freeze-drying parameters for Johnson Lyo Cycle

Initial shelf temperature:	25℃.
		cooling step	At-30 deg.C, 1.0 deg.C/min
Maintaining step	-30 ℃ until the vial reaches temperature
		Vacuum:	500mT
heating step	At 0 deg.C, at 1 deg.C/min
		Maintaining step	Kept until the vials reach temperature
Backwashing:	N2closing the vial up to 1/2 bar

The stages are carried out at atmospheric pressure. The stages were performed under vacuum conditions.

Example 5

The physical appearance of the freeze-dried material was evaluated. 20 vials were randomly selected. Production runs 55-20, 55-30 and 55-50 correspond to formulation I in 20, 30 and 50ml vials, respectively. Production runs 58-20, 58-30 and 58-50 correspond to formulation II in 20, 30 and 50ml vials, respectively. Production runs 62-20, 62-30 and 62-50 correspond to formulation III in 20, 30 and 50ml vials, respectively. Frequency of use, and where appropriate,% of use, summarizes all categorical variables. All continuous variables were summarized using mean and Standard Deviation (SD), with median and range (min, max) used for selected evaluations. Lyophilized formulations I, II and III in 20, 30 and 50ml vials were visually inspected to determine signs of suspension, such as white rings above the initial fill height. The liquid fill height was 25mm for 20ml vials, 20mm for 30ml vials and 15mm for 50ml vials.

Measurements of the actual suspension distance and white circles from the bottom of the vial to above the initial fill height minus the initial fill height were taken and are given in table 5 and fig. 2.

TABLE 5

Samples of formulation I (production runs 55-30 and 55-50) (lyophilized lung surfactant of the invention) did not show any signs of suspension in the 30 and 50ml vials, whereas in the 20ml vial sample (production run 55-20) there was some slight suspension of up to 2 mm in 7 out of 20 samples. The formulation II samples (58-20, 58-30 and 58-50) and the formulation III samples (62-20, 62-30 and 62-50) in all three sizes showed signs of suspension during the freeze-drying process, with the formulation III sample in the 20ml vial being the most severe. Clearly, with the new freeze-drying process, the problem of suspension has been significantly reduced or eliminated.

20 randomly selected vials of freeze-dried formulations I, II and III in 20, 30 and 50ml vials were examined to check to determine if there was evidence of movement caused by inverting the vial once. Figure 1 is a bar graph showing some vials containing the lyophilized substance that has moved after inversion (shown as black bars) and some vials containing the lyophilized substance that has not moved after inversion (shown as shaded bars). None of the formulation I samples (freeze-dried lung surfactant according to the invention) moved after inversion, whereas all of the formulation III samples and most of the formulation ii samples moved. This experiment shows that the freeze-dried lung surfactant according to the invention has a superior position in the vial compared to other samples.

Example 6

Studies were presented for stability and API efficacy. For completeness, four APIs, KL4 (sinapultide), DPPC, POPG and Palmitic Acid (PA) were tested with HLPC at 25 ℃, 30 ℃ or 40 ℃ over a 3 to 12 month storage at selected time intervals. The HPLC parameters are listed in Table 6.

TABLE 6 chromatographic conditions

A standard comprising 4 APIs each was run to determine the elution pattern. The sample was loaded on the column and the amount of API was calculated.

Example 7

Formulations I, II and III in 20, 30 and 50ml vials were simultaneously subjected to BET testing. Notably, when formulations I, II and III were fed to Micromeritics overnight via FEDEX for BET testing, formulation III was not delivered perfectly, which resulted in the cake having significantly collapsed and therefore not being available for testing. The foregoing was shipped in a safer package, but the samples (62-50) in 50ml vials still collapsed. Therefore, the test results for samples 62-50 will not represent the true values.

The BET test is performed as follows: analysis of the adsorbent: krypton; thermal correction: (ii) present; balancing interval: 10 seconds; ambient temperature: 22.00 ℃; automatic degassing: are present. This result supports the results for the lyophilized material of the invention (formulation i) in 20ml, 30ml and 50ml vials as shown in table 7. The results for formulations II and III are shown in Table 8.

TABLE 7

TABLE 8

Sample specific surface area of formulation I was about 3.7m²G to about 2.7m²(ii) in terms of/g. The specific surface area of the sample of formulation II was about 1.7m²(ii) in terms of/g. The specific surface area of the formulation III sample was about 0.6m²A/g to about 0.9m²(ii) a range of/g. It is clear that the specific surface area of the formulation I sample is significantly larger than that of the other samples.

Example 8

And (4) preparation IV.

Upon reconstitution with 10ml sterile water for injection, the lyophilized formulation IV will provide the following concentrations of API, as shown in table 9:

TABLE 9

API	(mg/mL)
		Sinapsin (KL)4)	0.862
Palmitic acid	4.05
		DPPC	22.50

API	(mg/mL)
		POPG	7.50

TABLE 10 raw materials for formulation IV (8000g batch size)

Several batches were prepared according to table 10. Preparation of the pre-freeze-dried mixture: API was dissolved in 95% ethanol at 46 ℃. + -. 1 ℃ to give a solution. The resulting solution was filtered under pressure through a 0.22 micron 33 millimeter (PVDF) Millipore Millex GV cat no SLGV033NS filter at 45 ℃ ± 2 ℃ into stirred TRIS buffer to produce a liposome formulation with a final ethanol concentration (w/w) of 7%. After cooling to a temperature below 30 ℃, the resulting liposome formulation, i.e., the pre-freeze dried mixture, was transferred to a 30ml borosilicate glass freeze dried vial using a peristaltic pump at a fill volume of 13.7 grams per vial and freeze dried as described in examples 9-14 for 1-6 runs.

Example 9

Run 2

TABLE 11

Example 10

Run No. 3

TABLE 12

Example 11

Run 4

Watch 13

Example 12

Run 5

TABLE 14

Example 13

Run No. 6

Watch 15

Example 14

Run No. 1

TABLE 16

Example 15

The freeze-dried products from runs 1-6 were simultaneously BET tested.

The BET test was performed using the same parameters as described in example 7 above. Three vials were tested per run. The results are shown in Table 17.

TABLE 17

The method comprises the following steps: and (6) reconstructing. The requirement for the reconstituted solution is that there are no visible insolubles, and the solution is not less clear than the diluent after a predetermined amount of time. The volume used for reconstitution may be such that the product returns to the same volume and concentration as the starting product for filling with bulk solution (bulk solution), and may be the same volume as used for patient delivery in a clinical setting. Purified water, USP as diluent, was drawn into the syringe in a volume of 10 ml. The diluent is then squeezed into the center of the dried cake and a timer is started. The product was then examined in a light box at time intervals of about 5 seconds to verify the absence of any insoluble material and the clarity of the solution. The solution, once fully reconstituted, is characterized as clear, colorless, hazy, opaque and/or cloudy. Particles, if present, are classified as micro-insoluble to coarse fibers. The insoluble excipients or APIs were all recorded as received.

pH measurement-measurements of reconstituted solutions (see above) to determine pH values per USP <791>, pH normalization was performed prior to use using two or three pH buffers (which included the desired sample range). The pH buffer is selected to be no greater than 3 pH units, but no less than 2 pH unit intervals (e.g., 4.01, 7.00, and 10.01). The ATC probe is used for automatic temperature compensation. The amount of sample solution is sufficient to cover the pH probe sensor, and any reference junctions on the side of the probe are dispensed into a suitable container. The solution was gently swirled and then allowed to stand and stabilize to a constant value over a period of at least 15 seconds, at which point the pH value shown was recorded.

Coulometric karl fischer titration-moisture testing follows the widely accepted traditional method outlined in the united states pharmacopoeia <921>, the determination of water. The initially dried sample and container were weighed. The solvent extraction method uses absolute methanol, a special reagent, and A.C.S to inject into a container for suspending and dissolving dried substances. For each study, the methanol extraction volume for the blanket dried material was 13.0 ml to 13.7 ml. The sample is then allowed to soak for a predetermined time to extract the water from the product. Aliquots were then removed, the volume measured, and then injected into the reaction vessel of the KF instrument. After the end point of the titration is reached, the results are reported. KF instruments resolve water content to micrograms. The empty containers were then weighed and the percent humidity was calculated for the initial container contents.

High temperature differential scanning calorimetry (DSC HT) -is used as a means of determining the glass transition of a solid material, which provides useful information for evaluating formulations and evaluating behavior in the dry state. HT DSC complies with the current United States Pharmacopeia (USP)<891>Thermal analysis and HTDSC with Perkin Elmer DSC 7 interfaced to TAC 7/7 instrument controller. Test parameters and data analysis were performed using PYRIS software version 4.0, on a PC interface. Approximately 10-15mg of solid material was placed in an aluminum sample pan with a crimped vent lid. Nitrogen, NF, was used to continuously purge the sample at a flow rate of 20 ml/min. The freeze-dried material was heated to evaluate thermal behavior at higher temperatures. The evolution or uptake of heat by the sample during the warming period reflects the difference in energy source at which the sample undergoes the thermal event. The scan data is recorded and used simultaneously4.0 software drawing. Once the scan is complete, the temperature is calculated which determines the onset and peak of the thermal event. Based on the results of the scan, the temperature of the thermal event, such as glass transition (Tg), crystallization, melting point (Tm), and the associated heat of fusion, and/or specific heat, of the dried end product is determined using the method.

The average residual moisture value of the freeze-dried samples of formulation I was close to 0%. The average reconstitution time is 8 seconds to 10 seconds. High temperature DSC scans of the material at 2 ℃ per minute showed that consistent significant endothermic peaks were observed at temperatures between 49.0 ℃ and 51.0 ℃.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Claims (14)

1. A method of preparing a freeze-dried synthetic pulmonary surfactant having reduced or eliminated cake suspension in the method, comprising:

providing a pre-freeze-dried mixture to a freeze-drying chamber, the pre-freeze-dried mixture comprising dipalmitoylphosphatidylcholine DPPC, palmitoyl oleoyl phosphatidylglycerol POPG, palmitic acid, and SEQ ID NO: 1 synthetic peptide KL4 polypeptide, the phospholipid and synthetic peptide being dispersed in a solvent, the solvent having an organic solvent, and the remainder being water and/or a buffer, the organic solvent being in the range of 3% v/v to 20% v/v or less of the total volume of the pre-freeze dried mixture, wherein the pre-freeze dried mixture is filled into a container,

reducing the temperature in the freeze-drying chamber to initiate cooling and solidifying the pre-freeze-dried mixture during the freezing phase; and

an annealing stage is carried out prior to the first drying stage to reduce or eliminate lump suspension in the freeze-dried synthetic pulmonary surfactant having a specific surface area of at least 2.2m²/g。

2. The method of claim 1, comprising:

performing a freezing phase in a freeze drying chamber during which the pre-freeze dried mixture is cooled to a first temperature of-45 ℃ or below-45 ℃ at a rate of 0.1 to 1.0 ℃/min and holding the pre-freeze dried mixture at the first temperature for a first period of time sufficient to solidify at least 76% of the solvent to form a first solidified mixture;

an annealing stage is performed whereby lump suspension of the first curing mixture is reduced or eliminated, wherein, said first curing mixture (i) being heated to a second temperature, said second temperature being selected to reduce or eliminate suspension of said first curing mixture, (ii) being maintained at said second temperature for a second period of time, said second period of time being sufficient to reduce or eliminate suspension of said first curing mixture, and (iii) cooling at a rate of from 0.1 to 1.0 ℃/min to a third temperature of-45 ℃ or less than-45 ℃ to form a second solidified mixture, wherein the second solidified mixture is maintained at the third temperature for a third time period sufficient to facilitate separation of unfrozen organic solvent from the second solidified mixture, thereby allowing the unfrozen organic solvent to migrate to the interface between the container and the second solidified mixture;

performing a first drying phase at a reduced pressure of 30mT or more, wherein the second solidified mixture is held at a third temperature for a fourth period of time sufficient to remove at least 5% of the organic solvent, followed by heating to a fourth temperature sufficient to keep the second solidified mixture out of suspension in the vessel and to maintain the structure established in the annealing phase, and further held at the fourth temperature for a fifth period of time sufficient to remove at least 70% of the solvent and thereby form a third solidified mixture; and

performing a second drying phase at reduced pressure for a sixth time period sufficient to produce a freeze-dried synthetic pulmonary surfactant having a residual solvent content of at most 2%.

3. The method of claim 2, wherein the ratio of the volume of the pre-freeze dried mixture in the container to the volume of the container is from 28% to 68%.

4. The method of claim 2, wherein the ratio of the height of the pre-freeze dried mixture in the vessel to the vessel diameter ranges from 0.3 to 0.8.

5. The method of claim 2, comprising: providing a pre-freeze dried mixture, wherein the organic solvent is in the range of 3% to 15%.

6. The method of claim 2, comprising: providing a pre-freeze dried mixture wherein the organic solvent is in the range of 5% to 10%.

7. The method of claim 2, comprising: providing a pre-freeze dried mixture wherein the organic solvent is in the range of 7% to 10%.

8. The method of any one of claims 2-7, comprising:

performing a freezing phase in which the pre-freeze dried mixture is cooled to a first temperature of-50 ℃ ± 5 ℃ at a rate of 0.1 to 1.0 ℃/min;

performing an annealing phase wherein said first cured mixture is (i) heated at a rate of 0.1 to 1.0 ℃/min to a second temperature of-22 ℃ ± 5 ℃, (ii) held at the second temperature for a second period of time of 3 hours to 8 hours, (iii) cooled at a rate of 0.1 to 1.0 ℃/min to a third temperature of-50 ℃ ± 5 ℃; and (iv) held at said third temperature for a third time period of 3 to 8 hours;

the first drying stage is carried out at a pressure selected from the range of 30mT to 200mT and at a fourth temperature selected from the range of-25 ℃ to 0 ℃ and ramped from-50 ℃ ± 5 ℃ and further held at the fourth temperature for a fifth time period of at least 10 hours.

9. The process of any one of claims 2 to 7, which comprises carrying out the second drying stage at a pressure selected from the range of 30mT to 200mT and a temperature of at most 46 ℃ ± 5 ℃.

10. The process of claim 1, wherein the specific surface area is 3.7m²G to 2.2m²In the range of/g.

11. The method of claim 1, wherein the freeze-dried synthetic lung surfactant has a porosity of greater than 40% by volume of the total area of the freeze-dried synthetic lung surfactant.

12. A freeze-dried synthetic lung surfactant composition comprising:

SEQ ID NO: 1 synthetic peptide KL4 polypeptide, dipalmitoylphosphatidylcholine DPPC, palmitoyl oleoyl phosphatidylglycerol POPG and palmitic acid, wherein the freeze-dried composition of synthetic lung surfactant has a specific surface area of at least 2.2m²/g, wherein the freeze-dried synthetic lung surfactant is according to any of claims 1 to 11The method of (1).

13. The lyophilized synthetic lung surfactant composition of claim 12, wherein the specific surface area is at 3.7m²G to 2.2m²In the range of/g.

14. The lyophilized synthetic pulmonary surfactant composition of claim 12, wherein the lyophilized synthetic pulmonary surfactant has a porosity of greater than 40% by volume of the total area of the lyophilized synthetic pulmonary surfactant.