Methods, compositions and sets for lyophilisation

FIELD: chemistry.

SUBSTANCE: invention relates to a method of lyophilisation of a composition, which contains purified antithrombin III (AT III) and a crystallised substance, selected from alanine, mannitol, glycine or NaCl. The claimed method includes freezing the composition at a temperature from -52°C to -60°C for 6-15 hours, annealing the composition at -30°C for 1 hour, re-freezing the composition at a temperature from -52°C to -60°C for 2-15 hours at keeping the product temperature between -48°C and -52.7°C for 4-10 before lyophilisation and drying the composition with obtaining a lyophilised cake. The invention also relates to a pharmaceutical set, which contains the said lyophilised cake and a liquid reagent.

EFFECT: invention provides obtaining the lyophilised composition, containing AT III, which preserves its activity and stability.

14 cl, 24 dwg, 5 tbl, 2 ex

 

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to application U.S. No. 61/264014, filed November 24, 2009, which is incorporated into this description by reference in its entirety.

The technical FIELD TO WHICH the INVENTION RELATES

The present invention relates to a method of freeze-drying compositions, in particular aqueous pharmaceutical compositions containing at least one active ingredient, and compositions derived from them, in particular to compositions, kits and methods of lyophilization of anti-thrombin III (AT III).

The prior art of the PRESENT INVENTION

Lyophilization is a widely used way to obtain the active ingredients in the form of a more solid forms of pharmaceutical preparations. For example, it was shown that the active ingredient, such as AT III, which is an alpha-glycoprotein normally present in plasma, and is a plasma inhibitor of thrombin, has a relatively low stability in solution. Accordingly, AT III processed in lyophilized preparations.

It is assumed that the lyophilization reduces or inhibits the degradation of the active ingredient by removing components of the solvent in the composition to such quantities, which is not supported any chemical reaction or biological is systematic growth. In addition, I believe that the removal of solvent reduces molecular mobility, reducing the possibility of destructive reactions. In addition, it is desirable that the crystallizing excipients (for example, amino acids and salts), which are widely used in freeze-dried products, crystallized as completely as possible during freezing, in order to provide a solid matrix to support the structure of the cake. However, in some previous attempts to liofilizirovanny aqueous pharmaceutical compositions failed to achieve a satisfactory degree of crystallization. For example, it was shown that different stages of freezing and/or annealing of a typical lyophilization Protocol per se ineffective support crystallization. Moreover, it is assumed that the presence of certain crystallizing excipients (e.g., alanine and sodium chloride) may inhibit or decrease the crystallization of any excipients, thereby limiting the degree of crystallization.

Despite the fact that there have been several attempts to liofilizirovanny aqueous pharmaceutical compositions, there remains a need for methods of freeze-drying and the compositions obtained with their help.

The INVENTION

In one aspect the present invention provides STRs what about freeze-drying of the composition, containing at least one active ingredient and at least one crystallizing excipient. This method includes: maintaining the composition at a first temperature for a first period of time sufficient to obtain a first composition comprising at least one partially or fully crystallized crystallizing excipient.

In another aspect the present invention provides a method of freeze-drying a liquid composition containing plasma-derived AT III, NaCl and alanine. This method includes:

(a) maintaining the composition at about 54°C or below, so that the temperature of the composition is equal to approximately 48°C or below for about 5 hours or more to ensure complete or almost complete crystallization of the first composition that includes one or more components; and

(b) drying the first composition, in order to obtain a lyophilized cake.

In some aspects, the present invention provides compositions comprising freeze-dried cake obtained in accordance with methods disclosed in the present description.

In other aspects the present invention provides a set containing one or more of the compositions and/or liability avannah Chekov, obtained in accordance with methods disclosed in the present description.

BRIEF DESCRIPTION of DRAWINGS

Figure 1 - DSC thermogram during freezing and heating of NaCl (0.15 M).

Figure 2 - DSC thermograms during freezing (A) and heating (B) solution of alanine (0.1 M).

Figure 3 DSC thermograms during freezing (A) and heating (B) recovered AT III.

Figure 4 - optimal conditions of crystallization analysis DOE.

Figure 5: A - the change of heat capacity (Cp) during freezing and annealing cycle ETP-5807; B - change the Cp during the first freezing; C - change Cp during annealing; D - change of Cp during the second freeze.

Figure 6 - change in heat flow with temperature in the cycle ETP-5807. Was determined the heat of fusion for melting peak, which amounted to 5.5 j/g

Figure 7: A - change of Cp during freezing and annealing with increasing the exposure time when freezing up to 5 hours; B - change the Cp during the first freezing; C - change Cp during linear heating from -52°C to -30°C; D - change of Cp during annealing; E - change the Cp during the second freeze.

Figure 8 - change in heat flow with temperature when increasing the exposure time by freezing from 2 hours to 5 hours. Was determined the heat of fusion for melting peak, which amounted to 6.4 j/g

Phi is ur 9 - profile lyophilization AT III in the cycle ETP-5807, made in the apparatus lyostar II FTS.

Figure 10 - data on the temperature of the product during the freezing cycle ETP-5807 apparatus FTS.

Figure 11 - profile lyophilization AT III by freezing at -54°C within 2 hours.

Figure 12 - data on the temperature of the product during freezing at -54°C within 2 hours.

Figure 13 - profile lyophilization AT III by freezing at -54°C for 6 hours in the apparatus of the FTS.

Figure 14 - data on the temperature of the product during freezing at -54°C for 6 hours in the apparatus of the FTS.

Figure 15 - profile lyophilization AT III by freezing at -50°C for 6 hours in the apparatus of the FTS.

Figure 16 - data on the temperature of the product during freezing at -50°C for 6 hours in the apparatus of the FTS.

Figure 17 - profile lyophilization AT III by freezing at -60°C for 6 hours in a Usifroid.

Figure 18 - data on the temperature of the product during freezing AT III at -60°C for 6 hours in a Usifroid.

Figure 19 - profile lyophilization AT III by freezing at -52°C for 15 hours.

Figure 20 - data on the temperature of the product during freezing at -52°C for 15 hours.

Figure 21 - raster-electron micrograph of Chekov (200x magnification). Scale labels equal to 100 μm. A: the cake with the broken structure. B: solid cake.

Figure 22: scanning-electron micrograph of NaCl, 200x increase the s on the left and 1500x magnification on the right. Scale label equal to 100 μm (A) and 10 µm (B).

Figure 23 - raster-electron micrograph of alanine, 50x magnification on the left and 200x magnification on the right. Scale label equal to 500 μm (A) and 100 µm (B).

Figure 24 is a powder x-ray diffraction (RD) charts with the use of the diffractometer for NaCl, alanine, ETP 5807 (cake with disturbed structure and material from the second passage ETP 5807 (solid cake).

DETAILED description of the INVENTION

The present invention provides an unexpected discovery that a single stage low-temperature freezing before drying is sufficient to cause crystallization capable of crystallizing excipients in the formulations containing the active ingredient, and therefore, these methods offer a reliable crystallization of excipients, at the same time offering more efficient, convenient and/or reliable Protocol lyophilization. These methods allow an increased amount of crystalline obamaobama agents compared with previous methods, while maintaining the stability and activity of the active ingredient present in the compositions.

In one aspect the present invention provides a method of freeze-drying a composition containing at least one active ingredient and at least one of crystallis the different excipients. This method includes curing the composition at a first temperature for a first period of time sufficient to obtain a first composition comprising at least one partially or fully crystallized crystallizing excipient.

The composition may be a liquid or semi-liquid composition. For example, the composition may be an aqueous pharmaceutical solution or suspension containing at least one active ingredient and at least one crystallizing excipient.

In one embodiment, the composition is a liquid composition, preferably an aqueous solution. In another embodiment, the composition is a suitable for pharmaceutical applications, for example, a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent.

In one embodiment, the composition is a pharmaceutical composition comprising at least one active ingredient, at least one crystallizing excipient and a pharmaceutically acceptable carrier. In the context of the present description "pharmaceutically acceptable carrier" includes any and all solvents, dispersion medium, dormancy is itia and the like, which are physiologically compatible. The media type can be selected based on the intended route of administration. In some embodiments, the implementation of media suitable for injection through, without limitation, intravenous, inhalation, parenteral, subcutaneous, intramuscular, intra-articular, intrabronchial, intraabdominal, intracapsular, wnoutrefresh, intracavitary, intracellular, vnutriposelkovyh, intracerebroventricularly entered inside the colon, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardially, intraperitoneal, intrapleural, vnutriploschadnykh, intra-lungs, intrarectal, vnutripochechnykh, intraretinal, intraspinal, intra-articular, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal means. Pharmaceutically acceptable carriers include, without limitation, sterile aqueous solutions or dispersions to obtain sterile injectable solutions or dispersions.

The active ingredient

In some embodiments, the implementation of at least one active ingredient may be any active ingredient, including, without limitation, proteins, nuclein what s acid, and combinations thereof. Proteins may include, without limitation, glycoproteins (e.g., AT III), coagulation factors, growth factors, cytokines, antibodies, and chimeric constructs. The term "protein" in the present description is considered as broad and refers to individual or collective native proteins of human or other mammal; and/or homogeneous or heterogeneous distribution of polypeptides originating from one or more gene products; and/or fragments of proteins exhibiting a specific activity; and/or such proteins and/or their active fragments produced by recombinant methods, including transgenic technology.

In some embodiments, the implementation of at least one active ingredient is a protein. In one embodiment, the protein represents AT III. In other embodiments, the implementation of the composition contains only one active ingredient, and this active ingredient is an AT III. In another embodiment, AT III is the only active ingredient in the composition, but the composition contains other proteins, including proteins that are not AT III, and/or an inactive form of AT III. For example, functional AT-III can be part of the total protein in the composition.

The term "AT III in the context of the present description discusses how wide the cue, unless otherwise explicitly specified. For example, this term refers to all naturally occurring the polymorphs AT III. This term also includes functional fragments of AT III, chimeric proteins containing AT III, or their functional fragments, homologues obtained by substitution analogs of one or several amino acids AT III, and species homologs. This term also applies to all polypeptides AT III, which are the product of recombinant DNA technology, including AT III, which is a product of transgenic technology. For example, the gene encoding the AT III, can be inserted into a gene of a mammal, encoding whey protein, so that the DNA sequence is expressed in the mammary gland, as described, for example, in U.S. patent No. 5322775, which is included in the present description by reference for the sake of the idea of method of obtaining protein compounds. This term also refers to all proteins AT III, chemically synthesized by methods known in the prior art, such as, for example, solid-phase peptide synthesis. This term also refers to AT III, obtained from plasma. This term also refers to AT III, which can be obtained commercially. AT III can match human or not human AT III.

In one embodiment, AT III made the focus of a plasma-derived AT III. In another embodiment, AT III is obtained from the suspension fractions of plasma. In other embodiments, the implementation of AT III is obtained from depleted albumin fraction of plasma or pre-purified fractions to obtain AT III. U.S. patent No. 5561115, Tenold, included in the present description by reference for the sake of the idea of method of obtaining AT III from serum or plasma.

In other embodiments, the implementation of AT III is a recombinant AT III. The preparation of recombinant proteins, including recombinant AT III, is described, for example, in U.S. patents№№ 4517294, 4632981, 4873316, 5420252, 5618713, 5700663, 5843705, 6441145, 6878813, 7019193, Fan et al., JBC, 268:17588 (1993), Garone et al., Biochemistry, 35:8881 (1996), international publication No. WO02/02793; publications U.S. No. US2003/096974 and US2006/0024793 and Gillespie et al., JBC, 266:3995 (1991), all of them are included in the present description by reference for the sake of the idea of production of recombinant proteins, including recombinant AT III.

In one embodiment, the composition is characterized as containing AT III, having a purity of more than 90%. In other embodiments, the implementation of AT III has a purity of more than 95%, preferably at least about 99%. In some embodiments, the implementation of at least about 50%, for illustration, from about 50% to about 100%, from about 60% to about 90%, from about 70% to about 80% of AT III in HDMI is tion is active AT III.

In other embodiments, implementation liabilitiesa composition contains at least about 0.1 mg/ml AT III, to illustrate from about 0.1 to about 100 mg/ml, from about 0.5 to about 50 mg/ml, from about 1 to about 30 mg/ml and from about 5 to about 15 mg ml AT III, and AT III is partially or completely total protein present in the composition.

In one embodiment, the composition comprises a therapeutically effective amount of AT III. "Therapeutically effective amount" refers to an amount, effective for the required dosages and periods of time to achieve the desired therapeutic result, such as, for example, anticoagulation associated with hereditary deficiency of anti-thrombin. A therapeutically effective amount of AT III may vary according to factors such as the disease state, age, sex and weight of the individual subject and the ability of AT III to achieve the desired response of the subject. Therapeutically effective amount may also be that in which any toxic or detrimental effects of AT III were outweighed by therapeutically favorable effects.

In other embodiments, the implementation of the composition comprises a prophylactically effective amount of AT II. "Prophylactically effective amount" refers to an amount, effective for the required dosages and periods of time to achieve the desired prophylactic result, such as, for example, prevention or inhibition of thromboembolic episodes in subjects who had several thromboembolic episodes, or patients who are at risk of future episodes. Prophylactically effective amount can be determined, as described above, for a therapeutically effective amount.

Crystallizing excipient

In one embodiment, at least one crystallizing excipient selected from the group consisting of alanine, mannitol, glycine and NaCl.

In some embodiments, the implementation of at least one crystallizing excipient is present in the composition with the total number of crystallizing excipients comprising at least about 0.01% (wt./vol.), to illustrate from about 0.01% to about 10%, from about 0.1% to about 5% and from approximately 0.7% to approximately 1.8% (wt./vol.).

In other embodiments, the implementation of the freeze-dried product contains at least about 20% (wt./about.) all crystallizing vspomogatelny substances, to illustrate from approximately 20 to approximately 80%, from about 30 to about 70%, and from about 36 to about 60% (wt./about.) all crystallizing excipients.

In some embodiments, the implementation of at least one crystallizing excipient is an alanine and NaCl. In one embodiment, the NaCl is present in amount equal to from about 50 mm to about 300 mm, preferably from about 100 mm to about 250 mm. In one embodiment, the sodium chloride as such can be used without any other of the above-mentioned crystallizing excipients, in which case it can be included in the composition in an amount of approximately 300 mm or more. In other embodiments, the implementation of the composition (for example, aqueous pharmaceutical composition) is a hypertonic solution.

In addition to the at least one active ingredient and at least one crystallizable auxiliary substance, the composition can additionally contain one or more other excipients, i.e., one or more other substances used in combination with the active ingredient, in order to make a composition. Some non-limiting approx the market of one or more other excipients include stabilizing agents, buffer agents, divalent cations (e.g. calcium salts), binders, lubricants, disintegrating agents, diluents, dyes, corrigentov, sliding substances, surface-active agents, absorbents and sweeteners.

The combination of active ingredients and excipients in accordance with the present invention can ensure the stability of the active ingredient in lyophilised preparations; however, the composition of the present invention can also show a certain stability in liquid or semi-liquid state.

In other embodiments implement the composition further comprises a stabilizing agent. For example, the stabilizing agent can be selected from the group consisting of sucrose, mannitol and trehalose. Before lyophilization stabilizing agents may be present in the composition with the total number of stabilizing agents comprising at least about 1%, for illustration from approximately 1% to approximately 4% and approximately 2% to approximately 3%. In some embodiments, the implementation of the stabilizing agent is present in the composition in amounts of approximately 2%.

The buffer may also be present in the compositions of the present invention, in particular, when the active ingredient is exposed to blagopri temu the effects of pH shifts during lyophilization. the pH should preferably be maintained in the range from approximately 6 to 8 during lyophilization and more preferably at a pH of approximately 7. A buffering agent can be any physiologically acceptable chemical compound or combination of chemical compounds that have the ability to act as buffers, including, without limitation, phosphate buffer, citrate buffer, acetate buffer, the buffer is citric acid/phosphate, histidine, Tris(hydroxymethyl)aminomethan (Tris), 13-bis-[Tris-(hydroxymethyl)methylamino]propane (Bis-Tris propane), piperazine-N,N'-bis-(2-econsultancy acid) (PIPES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-2-hydroxyethylpiperazine-N'-2-econsultancy acid (HEPES), 2-(N-morpholino)acanalonia acid (MES) and N-2-acetamido-2-aminoethanesulfonic acid (ACES).

In one embodiment, the buffer agent included in the composition at a concentration equal to from about 10 to about 50 mm. When the composition add histidine, can be used in concentrations equal to at least about 20 mm, preferably about 25 mm, separately or in combination with other buffers, such as Tris.

In other embodiments implement the composition further comprises a divalent cation, for example a salt of calcium. In one embodiment, the implementation of the population is a salt of calcium is present in a quantity equal to from about 1 mm to about 5 mm.

In one embodiment, the composition further comprises a surfactant. Surfactant may be present in a quantity of approximately 0.1% or less. Non-limiting examples of surfactants include POLYSORBATE 20 (e.g., TWEEN® 20), POLYSORBATE 80 (e.g., TWEEN® 80), polyoxyethylene (80) ether of sorbitol and fatty acids, plutonomy polyols (for example, F-38, F-68) and dodecylamine esters of polyoxyethyleneglycol (e.g., Brij-35).

In accordance with the present invention the composition may also optionally contain an antioxidant. The antioxidant may be present in the composition in a total amount equal to at least 0.05 mg/ml, for illustration, from about 0.05 to about 50 mg/ml, from about 0.1 to about 10 mg/ml and from about 1 to about 5 mg/ml non-limiting examples of antioxidants include N-acetyl-L-cysteine/homocysteine, glutathione, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), lipoic acid, methionine, sodium thiosulfate, platinum, glycine-glycine-histidine (Tripeptide) and bottled hydroxytoluene (BHT). In some embodiments, the implementation of the composition additionally contains glutathione in an amount equal to from about 0.05 mg/ml to p is blithedale 5 mg/ml

Compositions of the present invention may also contain calcium or other divalent cations, in particular when the cation interacts with the active ingredient, in order to maintain its activity. In one embodiment, the composition further comprises a divalent cation. In another embodiment, the divalent cation is offered in the form of calcium salts such as calcium chloride, but may also represent other calcium salts such as calcium gluconate, glubionate calcium or gluceptate calcium. In some embodiments, the implementation of the calcium salt is present in amount equal to from about 1 mm to about 5 mm. In other embodiments, implementation of the calcium salt is present in amount equal to from about 3 mm to about 4 mm, preferably approximately 4 mm.

In some embodiments, the implementation of a combination of histidine and glutathione can have a synergistically beneficial effect on the stability of a particular active ingredient present in the composition. For example, histidine, acting as a buffer, can also act as a chelator of metal. In those cases, when considered on the level of activity of the active ingredient is the effect caused by metal oxidation, histidine, for example, may, sledovatel is but to do so, in order to stabilize the binding of oxidizing metal ions. I believe that by linking these metals glutathione (or any other present antioxidant) thereby able to provide further antioxidant protection, because of the oxidizing effect of metal ions associated histidine, is suppressed. Other chelating agents can also be included in the compositions/formulations of the present invention. Such chelating agents are preferably bind metals such as copper and iron, with greater affinity than calcium, such as when the composition is used is a salt of calcium. One example of this chelator is deferoxamine, which is a chelating agent that facilitates the removal of Al++and iron.

Lyophilization

Typically certain temperature and/or temperature ranges way lyophilization related to the storage temperature liabilitiesare equipment, unless otherwise noted. Storage temperature refers to the temperature control of the coolant flowing through the storage lyophilizate, which typically operate as temperature during lyophilization. The sample temperature (i.e. the temperature of the product) is dependent on the storage temperature, the pressure in the chamber and/or the rate of evaporation/s is bimali during primary drying (with evaporative cooling, the temperature of the ingredients below, than the temperature of storage).

A. Freezing

In one embodiment, the first temperature is approximately -48°C or below. In another embodiment, the first temperature is approximately equal to -54°C or below. In other embodiments, the implementation period is at least about 30 minutes, to illustrate from about 30 minutes to about 20 hours, from about 1 to about 18 hours, from about 2 to about 16 hours, from about 3 to about 14 hours, from about 4 to about 10 hours, from about 5 to about 8 hours, and from about 6 to about 7 hours. In one embodiment, the period of time is approximately 6 hours.

The temperature and time period may depend on such factors as the volume of solution in the bottle, regardless of liofilizirovannoe composition.

The present invention in some cases refers to the goal of full or 100% crystallization of excipients, and a specialist in the art understands that full crystallization may be difficult to verify, in particular, when the sensitivity of the method is insufficient for full confidence in the fact that the auxiliary substance is fully or 100% crystallized. Therefore, in practice the ICA the present invention provides methods of freeze-drying, which at least improve the crystallization of excipients in comparison with the previous methods. Accordingly, in the context of the present description, "a fully crystallized products can be determined, for example, by differential scanning calorimetry (DSC), the specialist in the art determines that an irreversible exothermic act when the first scan is the act of crystallization, which indicates that the crystallizing excipient is not fully crystallized during lyophilization. In some embodiments, the implementation of at least one crystallizing excipient partially crystallized, and partial crystallization is characterized as the degree of crystallization of approximately 50% or more, to illustrate at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%at least about 99.5% pure, at least about 99.8 per cent and less than 100%.

B. Annealing

In other variants of the Ah implementation the method further comprises maintaining the first composition at the second temperature for a second period of time, in order to obtain a second composition, the second temperature higher than the first temperature.

In one embodiment, the second temperature is at least about 5°C higher than the first temperature, to illustrate from approximately 5°to approximately 30°C and from about 10°to about 20°C higher than the first temperature. For example, in some embodiments, the implementation of the second temperature of approximately -30°C, and the first temperature is about -50°C.

In some embodiments of the second period of time is at least 10 minutes, to illustrate from about 10 minutes to about 10 hours, from about 30 minutes to about 8 hours, from about 1 hour to about 6 hours, and from about 2 hours to about 4 hours. In other embodiments, implementation of the second time period is less than, more than or approximately equal to the first period of time.

Without limitation to any specific theory, it is assumed that such recovery stages can help to improve the speed of sublimation and/or to reduce the heterogeneity within the party, depending on conditions and specific compositions.

In one embodiment, stage annealing is optional.

In other embodiments, the implementation after vtoro what about the period of time the second composition is subjected to a third temperature for a third period of time, moreover, the third temperature below the second temperature. For example, in some embodiments, the implementation of the third temperature is approximately the same as the first temperature. In other embodiments, implementation of the third temperature at least 5°C lower than the second temperature, to illustrate from approximately 5°to approximately 30°C and from about 10°to about 20°C lower than the second temperature. For example, in some embodiments, the implementation of the second temperature of approximately -30°C, and the second temperature is about -50°C.

In some embodiments, the implementation of the present invention provides a method of freeze-drying a composition containing at least one active ingredient and at least one crystallizing excipient. This method includes:

(a) maintaining the composition at a first temperature for a first period of time sufficient to obtain a first composition comprising at least one partially or fully crystallized crystallizing excipient;

(b) curing the first composition at the second temperature for a second period of time, in order to obtain a second composition, the second temperature higher than the first temperature; and

(c) wederive the e second composition at a third temperature for a third period of time, in order to obtain a third composition, and the third temperature below the second temperature.

In one embodiment, the time period is at least about 30 minutes, to illustrate from about 30 minutes to about 20 hours, from about 1 to about 18 hours, from about 2 to about 16 hours, from about 3 to about 14 hours, from about 4 to about 10 hours, from about 5 to about 8 hours, and from about 6 to about 7 hours. In another embodiment, the period of time is approximately 6 hours. In other embodiments, the implementation period of time is approximately 3 hours. In other embodiments, implementation of the third time period is less than, more than or approximately equal to the first period of time. In other embodiments, implementation of the conditions (e.g. temperature and time) on the stage (a) and (b) are the same or essentially the same.

C. Drying

In other embodiments, implementation of the methods of the present invention additionally include a phase of drying. Phase drying may include phase primary drying and secondary phase drying.

Accordingly, in some embodiments, the implementation of the present invented the e proposes a method of freeze-drying of the composition, containing at least one active ingredient and at least one crystallizing excipient. This method includes:

(a) maintaining the composition at a first temperature for a first period of time sufficient to obtain a first composition comprising at least one partially or fully crystallized crystallizing excipient; and

(b) drying the first composition to form a dried cake.

In other embodiments, implementation of the present invention provides a method of freeze-drying a composition containing at least one active ingredient and at least one crystallizing excipient, and this method includes:

(a) maintaining the composition at a first temperature for a first period of time sufficient to obtain a first composition comprising at least one partially or fully crystallized crystallizing excipient;

(b) curing the first composition at the second temperature for a second period of time, in order to obtain a second composition, the second temperature higher than the first temperature;

(c) curing the second composition at the third temperature is re during the third time period, in order to obtain a third composition, and the third temperature is lower than the second temperature; and

(d) drying the third composition to form a dried cake.

In one embodiment, the drying stage contains primary drying. Primary drying can remove frozen water (sublimation of ice). Preferably, the unbound or easily removable ice is removed from the sample by the primary drying. Unbound water in the early stage of primary drying may preferably be in the form of free ice, which can be removed by sublimation, i.e., turning directly from a solid state into vapor.

In some embodiments, the implementation stage of the primary drying can be conducted at a temperature equal to from about -35°C to about 20°C, or from about -25°C to about 10°C, or from about -20°C to about 0°C. In one embodiment, the stage of primary drying is carried out at approximately 0°C. In other embodiments, the implementation stage of the primary drying can be carried out within a total time of at least about 1 hour, to illustrate from about 1 hour to about 1 week, from about 10 hours to about 4 is it, and from about 20 hours to about 40 hours. In another embodiment, initial drying includes drying the first or the third composition at a pressure of from about 0 to about 200 mtorr, preferably approximately 100 mtorr at a temperature of approximately -50°C, for about 1 hour and then at 0°C for about 35 hours.

Optional step line changes before the initial drying" (i.e., increasing the temperature from the stage preceding the primary drying, to a temperature of primary drying) may be carried out in accordance with the methods of the present invention at a speed equal to from about 0.1°to about 10°C per minute.

Initial drying can be carried out in a period of time sufficient to insure that essentially all of the frozen water is removed from the sample. Specialist in the art understands that time is of primary drying varies with the configuration, since the length of the primary drying may depend on the filled volume and geometry (surface area of the cake - the resistance/flow). In one embodiment, the length of the primary drying is at least about 5 hours, to illustrate from approximately 5 hours prior to when listello 100 hours, from about 10 hours to about 80 hours, from about 30 hours to about 60 hours, and from about 40 to about 50 hours.

During the primary drying can be monitored using any number of methods, including observation of changes in the temperature of the products during freeze-drying. Another way is to monitor the pressure changes in the chamber, because when sublimation ends, the camera is no more water molecules, contributing to changes in pressure. The end stage of primary drying can be defined as the moment when the temperature of the product (sample) is close to the storage temperature, as, for example, shows a significant change in the slope of the temperature curve of the product due to the reduced speed of sublimation; when sublimation ends, ends evaporative cooling. In order to prevent a premature end, in some embodiments, the implementation of the duration of primary drying can be increased by an additional 2-3 hours. Another way to monitor the completion of primary drying is checking ascending pressure when disconnecting a source of vacuum pressure in the chamber should increase with speed, depending upon the amount of moisture in the product. In one the m of the embodiment, the end of primary drying can be installed as a moment, when the speed increases, the pressure becomes lower than a certain value. Another way to determine the end stage of primary drying is the measurement of the rate of heat transfer.

In other embodiments, directly in front of the primary drying, the composition may be placed in vacuum at a temperature of stage preceding a primary drying. Once enabled, the vacuum may be present in the remaining part of the lyophilization process, although the level of vacuum may vary.

For more information on drying during lyophilization can be found in Carpenter, J. F. and Chang, B. S., Lyophilization of Protein Pharmaceuticals, Biotechnology and Biopharmaceutical Manufacturing, Processing and Preservation, K. E. Avis and V. L. Wu, eds. (Buffalo Grove, IL Interpharm Press, Inc.) (1996), which is incorporated into this description by reference for the sake of the idea of drying.

In one embodiment, the drying optionally includes one or more stages of secondary drying to reduce the moisture level, preferably to a level that provides the necessary biological and/or structural properties of the final product.

In some embodiments, the implementation of each of the one or more stages of secondary drying is carried out at a temperature of approximately 0°C or above, to illustrate from the roughly 0°C to about 100°C, from approximately 10°to approximately 90°C, from about 20°C to about 80°C, from about 30°C to about 70°C, from about 40°C to about 60°C and about 45°C to about 50°C. In one embodiment, stage secondary drying includes first, second and third stage of secondary drying carried out at approximately 40°C, about 45°C and about 50°C, respectively. In one embodiment, stage secondary drying temperature is approximately 35°C, over a period of time of approximately 16 hours.

Stage temperature increase towards one or more stages of secondary drying, which may be optional in this description referred to as "linear change before the secondary drying". Linear change before the secondary drying can be carried out at a speed of temperature increase equal to from about 0.1°to about 10°C per minute.

Each of the one or more stages of secondary drying can be carried out in a period of time sufficient to reduce the residual moisture level in the dried product to the final level. In some embodiments, the final residual moisture level equal to bring the flax 10% or less, to illustrate approximately 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, about 0.8% or less, about 0.6% or less, about 0.5% or less, about 0.2% or less, and approximately 0.1% or less.

In one embodiment, stage of secondary drying is carried out at about 35°C. In other embodiments, the implementation stage of secondary drying can be carried out within a total time equal to at least about 1 hour, to illustrate from about 1 hour to about 1 week, from about 10 hours to about 4 days, and from about 16 hours to about 40 hours. In another embodiment, secondary stage of drying includes drying under a pressure of from about 0 to about 200 mtorr, preferably approximately 100 mtorr at a temperature of approximately 35°C for approximately 16 hours.

In order to determine the residual moisture level in the sample, can be used, for example, the method of Karl Fischer. In addition, checking ascending pressure or velocity measurement before the Chi heat can also be used for to determine the end of each of the one or more stages of secondary drying. Alternatively, you can also use electronic hygrometer or a residual gas analyzer. The minimum duration of one or more stages of secondary drying can be determined using various combinations of storage temperature (when the temperature storing one or more stages of secondary drying is the same or less than the temperature applied to the high-temperature phase) and length. The residual moisture content can be determined using several methods, including loss on drying, titration according to Karl Fischer, thermal gravimetric analysis (TGA), gas chromatography (GC) or infrared spectroscopy.

Without limitation to any specific theory believe that during the lyophilization of the active ingredient is converted from staying in the aqueous phase in staying in the amorphous solid phase, which is believed to protect the active ingredient from chemical and/or conformational instability. Dried product not only contains an amorphous phase, but also includes a component that crystallizes during lyophilization. This can provide the lyophilization of the active ingredient and education over chestvennogo cake (for example, cake with minimal reduction relative to the walls of the container in which it was dried).

In one embodiment, the lyophilized cake is characterized as having a structure, broken less than 50%. In another embodiment, the lyophilized cake is characterized as having the structure disturbed from approximately 0% to approximately 24%.

In another aspect the present invention provides a method of freeze-drying the aqueous pharmaceutical composition containing AT III, and this method includes:

(a) the keeping of this composition at a temperature below about -45°C over a period of time sufficient to obtain a first composition comprising at least one partially or fully crystallized crystallizing excipient; and

(b) drying the first composition to form a dried cake.

In other aspects the present invention provides a method of freeze-drying the aqueous pharmaceutical composition containing AT III, and this method includes:

(a) the keeping of this composition at a temperature below freezing approximately -50°C over a period of time sufficient to obtain a first composition comprising at least one partially or fully the capacity of crystallized crystallizing excipient; and

(b) drying the first composition to form a dried cake. In some embodiments, the implementation of this method optionally further comprises the stage of annealing, in which the composition is maintained at a temperature of annealing, which is above freezing temperature.

In another aspect the present invention provides a method of freeze-drying the aqueous pharmaceutical composition containing AT III, and this method includes:

(a) maintaining the composition at a temperature below about -60°C over a period of time sufficient to obtain a first composition comprising at least one partially or fully crystallized crystallizing excipient; and

(b) drying the first composition to form a dried cake.

Also provides compositions (e.g., crystallized and/or lyophilized pharmaceutical compositions and cake) obtained in accordance with the methods of the present invention.

Accordingly, in some embodiments, the implementation of the present invention provides lyophilized composition AT III or cake obtained in accordance with the present invention.

In other embodiments, implementation of the methods of the present invention offer products that m is Nisha least retain or essentially retain the activity of the active ingredient(s) after storage of lyophilized product. In one embodiment, the activity of the active ingredient(s) is maintained or substantially maintained after storage of the product is lyophilized at approximately 5°C, about 25°C. or about 40°C for about 1, about 2, about 3, or about 6 or more months. In another embodiment, after storage of the lyophilized product of the activity of the active ingredient is at least approximately 70%, 80%, 90%, 95%, 99% and 100% relative to the activity before lyophilization.

Sets

In other aspects also provides kits containing a pharmaceutical composition of the present invention, the kit further comprises a dry and a liquid component, and dry and liquid components may be present in separate containers in the kit, or some of the components can be combined in one container, such as in the set in which the dry ingredients are present in the first container, and the liquid components are present in the second container, where the data containers may be present or not be present in the merged configuration. Optionally, the kits may further comprise a number of additional reagents. Optionally, the kits can further include instructions for use of components n is boron, including, for example, how to restore dried composition with an appropriate diluent. These instructions may be present in the kits in the form of a liner in the package, the label of the container of the kit or its components.

The present invention will be illustrated in more detail using examples, but it should be noted that the present invention is not limited to these examples.

EXAMPLES

Example 1

In order to determine the freezing conditions which promote crystallization of the components in the solution AT III and improves the physical properties of the final product, has been lyophilization formulations with AT III, containing human plasma-derived AT III (6,88 mg/ml), alanine (100 mm) and NaCl (150 mm). As alanine, and NaCl are crystalline excipients. For this composition, physical properties can be directly related to the degree of crystallization of excipients. It is necessary to crystallize NaCl and alanine as completely as possible during freezing, in order to provide a solid matrix to support the structure of the cake.

Differential scanning calorimetry: investigated thermal acts of freezing-thawing composition with AT III using a differential scanning calorimeter (Model 292, TA instruments, Inc., New Castle, DE). The temperature and the cell constant DSK changed in accordance with the standard method using high-purity indium. Modulated DSC was used to study the change in heat flow and heat capacity (Cp) is maximally concentrated by freezing solutions. The experiments were carried out with an amplitude of 0.5°C, with a period equal to 80 seconds. Sample, 20 microliters, corked in aluminum sealed crucible and scanned in the negative temperature range.

Thermal acts in NaCl, alanine and restored the solution AT III: investigated acts of crystallization and melting in NaCl, alanine and restored the solution AT III.

The planning of the experiment DSK using E-CHIP: completed DOE developed Echip, in order to determine the influence of freezing temperature, the exposure time by freezing and exposure time of the annealing on the crystallization of excipients. The linear speed changes during freezing (from 5°C to freezing temperature) was set to 2°C/min After annealing product was frozen from -30°C to freezing temperature at 5°C/min linear Speed of the heat set in 1°C/min

The effect of the rate of linear change on crystallization: compared the different cooling rate (2°C/min versus 0.2°C/min) in order to investigate the influence of the SC the grow linear change on crystallization. Also compared different linear speed changes during annealing (5°C/min versus 0.2°C/min, 1°C/min versus 0.2°C/min).

The formation of the condensed phase: during hypothermia molecular conformations and configurations that are available in the liquid phase, but not in the crystalline solid phase, freezes. This process of 'capturing' conformations and configurations during cooling occurs when the rate of increasing viscosity exceeds the rate of change of orientation of molecules. 'Marzenie' conformational States leads to a condensed phase, which has a certain degree the middle of molecular order, but, similar to the liquid, no signs of distant molecular order crystalline solid PI.

The formation of the condensed phase was observed by modulated DSC, in which the heat capacity (Cp) concentrated by freezing amorphous phase decreased continuously until, until it reached equilibrium. Cp is an intrinsic property and is directly related to molecular mobility. Large Cp means greater mobility and less Cp indicates a lower mobility. The material in the liquid state has a higher Cp than its solid counterpart. The reduction of Cp is caused by the physical transformation of a material from a liquid state to a solid state is.

Cp tracked using Protocol freezing and annealing, are shown in table 1.

The solution was frozen from 0 to -52°C and maintained for 120 minutes and Then it was heated up to -30°C and maintained for 1 hour. Finally, the product was again frozen to -52°C for the other 2 hours and then linearly heated up to 0°C. the velocity of the first freeze was 0.2°C/min Also compared the influence of the exposure time by freezing (2 hours, 5 hours and 10 hours) and temperature (-46°C -48°C and -52°C) on Cp.

Freeze drying: a large part of the experiments was conducted in the Lyostar II FTS system (SP Industries). Some spent in the freeze-dryer Minilyo (Usifroid). Methods of freezing are listed in table 2.

Table 2
Methods of freezing
MethodThe freezing temperature (°C)The time when freezing (hours)
1-522
2-542
3-54 6
4-506
5-606
6-5215

Methods 1-5 are different storage temperature and exposure time for the first stage of freezing. The second freezing temperature was set equal to the first temperature freezing. And the exposure time for the second freeze was 2 hours. Method 6 describes the mode in which the product was frozen at -52°C for 2 hours, annealed at -30°C for 1 hour and froze again to -52°C for another 15 hours. Annealing, primary and secondary drying were the same for all cycles listed in table 1.

Scanning electron microscope (SEM): in order to study the structure sublimated Chekov used SEM (Hitachi, model S-3200, NCSU). Sample images from surfaces or lower surfaces depicted with an increase from 50 to 5000 times. Because freeze-dried cakes are good electrical insulators, they were charged under the influence of the electron beam. This led to the loss of resolution. In order to reduce the charging effect of the electron beam on the sample, all about Ozzy coated with a thin layer of gold by sputtering with the use of table Denton Vacuum. Got a picture of the cake with the broken structure, solid cake of NaCl crystal and crystal alanine.

Powder x-ray diffraction: in order to characterize the degree of crystallization of the cake with the broken structure (ETP 5807) and solid oxide (ETP 5807 26N9540) used powder x-ray diffraction (RD). RD diagrams were recorded using diffractometer (Rigaku, model Multiflex) with radiation of copper cathode at 40 kV and 40 mA. Scanning was performed in a 20 range from 10° to 90°. The scan rate was 1°/min for sample NaCl and 0.125°/min for alanine, ETP 5807.

Results and discussion

The purpose of DSK was to characterize the critical factors that govern the properties of the crystallization of excipients in the composition with AT III, determined the crystallization temperature (Tx), the temperature of the eutectic melting (Te) and the degree of crystallization.

Crystallization and melting of NaCl, alanine and mortar AT III: for pure NaCl exothermic peak of crystallization is located at approximately -38°C during freezing, and the endothermic melting peak appeared at -19°C during heating (Fig. 1). Determined the heat of fusion for melting peak, amounting to 7.4 j/, thermogram for the solution of alanine showed an exothermic peak at -45°C during freezing, including crystallization. In EMA heating, however, there has been a group of small peaks located at approximately -44°C. the Origin of these peaks were difficult to identify (Fig. 2).

In the analysis of the recovered solution AT III evidence exothermic activity during freezing is not observed. However, observed eutectic melting peak at -22° C, most likely due to NaCl (Fig.3). The heat of fusion (of 2.0 j/g) was less than that of pure NaCl. Reduce heat melting can be attributed to fractional crystallization of NaCl in the composition with AT III. On the basis of the correlation calculated the degree of crystallization by dividing the heat of fusion obtained from the compositions at a constant of 7.4 j/g, which represents the heat of fusion for pure NaCl solution.

The DOE results: completed DOE developed ECHIP using the model of the Central compound of the cube, in order to determine the influence of freezing temperature, the exposure time by freezing and exposure time of the annealing on the crystallization solution AT III (table 3).

The DOE results indicate that all studied variables conditions have a significant influence on the crystallization solution. Freezing at -52°C leads to a greater amount of crystalline NaCl compared to freezing at those whom perature, equal to -44°C and -60°C. the Decrease of crystallization at lower temperatures (-60°C) can be explained by the inverse relationship between the degree of crystallization and crystallization rate. The degree of crystallization was greater at lower temperatures. However, the solution becomes so viscous that the crystallization rate is significantly decreased. Data analysis DOE gave the optimum temperature freezing at -54°C (Fig. 4).

A study of exposure times shows that increasing the exposure time by freezing and exposure time of the annealing leads to an increase in the degree of crystallization. The DOE results indicate that the optimal exposure time is 10 hours for freezing and annealing (Fig. 4).

The data analysis also gives a message about the discrepancy, indicating that the model created E-CHIP, may not fully reflect the process of crystallization. Therefore performed an additional experiment DSK, in order to better understand the change in the physical properties accompanying the process of crystallization during freezing and annealing.

The effect of ramp rate on crystallization: cooling rate: as a plasticizer, water acts as a natural thinner, which increases the free volume and molecular mobility. The property of water is increased the e molecular mobility, that can facilitate a controlled diffusion processes, such as crystallization. Rapid cooling captures more water in the amorphous phase, whereas slow cooling allows water to drain from the system. Accordingly, rapid cooling promotes the formation of crystals. When the rate of freezing was reduced from 2°C/min to 0.2°C/min, the degree of crystallization of NaCl was reduced by 82% (from 17% to 3%).

Linear speed change during annealing: in a linear change from the freezing temperature to a warmer molecular mobility increases to such an extent that the formation of active centers and crystallization. Linear speed changes at this stage should be sufficiently slow in order to obtain a satisfactory crystals. Reducing the linear speed changes from 1°C to 0.2°C/min increased the degree of crystallization of from 38% to 95%. Further reduction of the linear velocity change of 0.1°C/min did not lead to significant difference.

When the linear change from -30°C to -52°C reduction rate of 5°C/min to 0.2°C/min increased the crystallization 1.35 times (from 17% to 39%). These results indicate that a linear change in velocity equal to 0.2°C/min were enough to crystallization occurred.

Condensed phase and crystallization: to anitelea study was devoted to the condensed phase and crystallization. Fig. 5A shows the change in Cp over time in the cycle ETP-5807 (table 1). A small change in Cp takes place during the first freezing (Fig. 5B), annealing (Fig. 5C) and the second freezing (Fig. 5D). The formation of the condensed phase can be seen on the fall of the Cp. A small change in Cp indicates that during freezing and annealing, there is a small phase change. Using these parameters received crystallization only at 75%. The degree of crystallization was calculated by dividing the heat of fusion, which is 5.5 j/g (Fig. 6), the constant of 7.4 j/g, which represents the heat of fusion for pure NaCl solution.

The formation of the condensed phase is observed when increasing the exposure time by freezing. Fig. 7A shows the whole picture of Cp changes during freezing and annealing. When the first time when freezing was increased from 2 hours to 5 hours, Cp fell to the lowest equilibrium value that indicates a change from a liquid phase to a more condensed phase (Fig. 7B). Further increasing the exposure time from 5 hours to 10 hours showed no further reduction in Cp (data not shown). Peak crystallization was observed during linear heating (Fig. 7C). This unique peak was absent when the exposure time when the freeze was tolko hours. If the solution is fully crystallized during the first freezing and linear heating, it can be assumed that the additional annealing or freezing will have a small effect on Cp or no. This was demonstrated by the fact that there were no changes in Cp during annealing and the second freezing (Fig. 7D and 7E). The degree of crystallization was increased to 87% when increasing time freeze 2 hours to 5 hours. The degree of crystallization was again calculated by dividing the heat of fusion, which is 6.4 j/g (Fig. 8), the constant of 7.4 j/,

These results show that it is necessary 5 hours at -52°C, in order to complete the physical transformation of the amorphous phase AT III. Condensed phase only begins when freezing during the entire 2 hours. Enough time when freezing is one prerequisite for crystallization.

A similar study conducted at temperatures higher than -52°C. these results show that the crystallization lack of activity, when the temperature of the product is equal to -46°C. At a temperature equal to -48°C, when the exposure time was increased from 4 hours to 5 hours, the degree of crystallization increased from 36% to 84%. Therefore, it is preferable that the solution AT III frozen below -48°C for at least 5 hours, the La is also sufficient to cause crystallization.

The development of the lyophilization process: in order to confirm the results of the study DSK in the macroscopic scale, four freezing temperature (-50°C -52°C -54°C and -60°C) and two exposure times (2 hours and 6 hours) were compared in a laboratory freeze-dryer.

Freezing at -52°C for 2 hours: the initial assessment of options table 1 current cycle used during the run was carried out with application of the device Lyostar II FTS. The profile of temperature and pressure in the chamber shown in Fig. 9. Higher product temperature measured by thermocouples during the freezing process was approximately -49°C (Fig. 10.). After processing the physical examination showed that only 2% Chekov were acceptable, 17% had small holes, 57% were partially damaged structure, and 23% were destroyed. Based on the results of the DSC temperature of the product (-49°C) was low enough to cause crystallization, however, the time when the freeze should be at least 5 hours in order before crystallization formed condensed phase. The duration of freezing equal to 2 hours, was too small to get enough crystals.

Freezing at -54°C within 2 hours: in this cycle the solution AT III was frozen at -54°C at the tip is of 2 hours, annealed at -30°C for 1 hour and again froze at -54°C for 2 hours. Freezing was carried out in the apparatus Lyostar II FTS. Primary and secondary drying was carried out in CSl0-0.5 (Serail 14L03). Graphs in Fig. 11 and 12 demonstrate that the product temperature remained below -50°C during freezing. Physical examination showed that 74% Chekov were acceptable, and 26% had small holes. Although we see improvement in the properties of the cake with decreasing product temperature from -49°C to -50°C, the result is still unsatisfactory. These results show that only one freezing at low temperature is insufficient to cause complete crystallization.

Freezing at -54°C within 6 hours: in this cycle the solution AT III was frozen at -54°C for 6 hours, annealed at -30°C for 1 hour and again froze at -54°C for 2 hours. The cycle was carried out in the apparatus FTS Freeze-dry. The product temperature was maintained below -50°C during freezing (Fig. 13 and 14). Physical examination showed that all the cakes were acceptable. These results show that the temperature of the product and the time when freezing equally important to ensure optimal crystallization. This result is consistent with the data of the DSC.

Freezing at -50°C for 6 hours: in this C the glue solution AT III was frozen at -50°C for 6 hours, annealed at -30°C for 1 hour and was again frozen at -50°C for 2 hours. The cycle was carried out in the apparatus Lyostar II FTS. Using these parameters, the product temperature was maintained below -47°C and above -48°C during freezing (Fig. 15 and 16). Physical examination showed that only 18% were acceptable, 23% had small holes and 59% showed damage to the structure. This study confirms the previous conclusion on the basis that the product temperature should be below -48°C in order to start the crystallization. It also shows that merely increasing the exposure time by freezing is not enough, in order to form a satisfactory crystals.

Freezing at -60°C for 6 hours: in this cycle the solution AT III were frozen at -60°C for 6 hours, annealed at -30°C for 1 hour and was again frozen at -60°C for 2 hours. The cycle was performed in a freeze-dryer Minilyo (Usifroid). The temperature of the product during freezing was -51,6°C on the top shelf and -52,7°C on the bottom shelf (Fig. 17 and 18). Physical examination showed that all the cakes were acceptable. This experiment additionally demonstrates that lowering the storage temperature and increasing the exposure time by freezing are important approaches to obtain pharmaceutically acceptable Chekov.

Zamora is ivanie at -52°C for 15 hours: in this cycle the solution AT III was frozen at -52°C for 2 hours, annealed at -30°C for 1 hour and froze again to -52°C for 15 hours. Freezing was carried out in the apparatus Lyostar II FTS. Primary and secondary drying was carried out in the Serail, because isolation valve in FTS stuck during primary drying. Higher product temperature measured by thermocouples during freezing were below -48°C (Fig. 19 and 20). The physical properties of all Chekov were acceptable. Based on the results of DSC -48°C is the highest temperature of the product required, in order to cause crystallization. And the waiting time is equal to 15 hours, turned out to be large enough to ensure complete crystallization.

The result: table 4 shows the reaction temperature at various setpoints temperature storage.

Since the temperature of the product tends to be higher at 4-6°C, than the preset value of the target temperature storage target storage temperature is preferably set at -54°C, in order to have confidence that all of the product temperature remains below -48°C throughout liofilizadora. Based on the results of these studies (table 5) you can choose the target storage temperature during freezing in order to have confidence that the product temperature is significantly below -48°C.

In addition, sufficient time can be allocated, to ensure complete crystallization. The data show that the storage temperature equal to -54°C, with a 6-hour exposure can provide these conditions and to promote proper crystallization. So as to increase the exposure time by freezing from 2 hours to 6 hours, and lowering the setpoint target storage temperatures from -52 to -54°C will improve the physical properties of the final product.

Structure sublimated Chekov: patterns sublimated Chekov observed by scanning electron microscope. Cake with partially broken structure was used as a control for firm and durable cake. Cake with disturbed structure contains many thin and porous flakes (Fig. 21A). Solid cake (Fig. 21B) consists mainly of lamellar crystals with a number of rounded crystals distributed throughout the cake. NaCl itself forms a small rounded crystals (Fig. 22). Alanine by itself (Fig. 23) forms a continuous plate with a certain number of holes, probably due to ice sublimation. We can assume that the lamellar crystals in Fig. 21B are primarily alanine and crystals round shape NaCl.

X-ray powder diffraction: on the basis of the data is, from studies of DSC and freezing, made a second pass ETP-5807 with maximum load. This passage was included low temperature freezing during the first stage of freezing, as well as increased exposure time. In a modified cycle received a product with acceptable physical properties.

In order to characterize the degree of crystallization of the cake with the broken structure after the first pass, and the solid cake after the second passage, compared KMG chart NaCl, alanine, ETP 5807 (cake with disturbed structure and material from the second passage ETP 5807 (solid oxide) (Fig. 24). The main peaks of the crystalline diffraction NaCl located at 31,7° and 45.5°. The main peak of the crystalline diffraction alanine is at 20.5° C. A broad peak in the samples alanine, refers to the amorphous part. Cake ETP 5807 (first pass) and ETP 5807 (second pass) demonstrate a combination of peaks NaCl and alanine. A broad peak is also observed for both samples.

The degree of crystallization calculated by dividing the square of the crystalline peak to the sum of the areas of amorphous and crystalline peaks. The degree of crystallization of NaCl, alanine, ETP 5807 and ETP 5807 (second pass), respectively, correspond to 99±20%, 50±1%, 66±2% and 60±1%. For cake with disturbed structure and the solid cake was not marked differences in the diffraction diagram

Conclusions: composition with AT III described, paying special attention to the degree of crystallization, Protocol design freeze when freeze-drying. The results show that the freezing temperature and time are equally important preconditions complete crystallization. In some embodiments, the implementation of the lyophilization may include a freezing temperature of approximately -54°C and increased the exposure time of approximately 6 hours. In the trials received a pharmaceutically acceptable end products.

Example 2

Molded bottles thirty ml filled with ten milliliters of sterile filtered solution containing AT III (~6,88 mg/ml), alanine (100 mm (~8,91 mg/ml) and NaCl (150 mm (~8,7 mg/ml)). Samples of AT III were first frozen to -25°C, incubated for 2 hours and then frozen for up to -54°C, followed by aging for 6 hours. Then, the storage temperature is slowly raised to -30°C at a rate of 0.2°C/min, and kept at this temperature for 2 hours, and then slowly lowered at 0.2°C/min back to -54°C. the Products stood at -54°C for 2 hours prior to primary drying. Primary drying was conducted at a storage temperature of 0°C, and a given pressure in the chamber equal to 100 mtorr. The primary was vassilakopoulos for about 32 hours before the secondary drying. Secondary drying was performed at a storage temperature of 35°C and the pressure in the chamber 100 mtorr within 14 hours.

After drying has received approximately 100% of pharmaceutically acceptable lyophilized cake. Share pharmaceutically acceptable cake was calculated by dividing the number of acceptable cake on the number Chekov throughout the party. In addition, the applied modulated DSC and observed the formation of the condensed phase during the stage of freezing and crystallization during linear heating.

1. The method of freeze-drying a composition containing a purified antithrombin III (AT III) and one or more crystallizable substances selected from the group consisting of alanine, mannitol, glycine and NaCl, and this method includes:
(a) freezing the composition at a temperature in the range from -52°C to -60°C for 6-15 hours;
(b) annealing the composition at -30°C for 1 hour;
(C) re-freezing of the composition at a temperature in the range from -52°C to -60°C for 2-15 hours; maintaining the temperature of the product between -48°C and -52,7°C for from about 4 to about 10 hours before lyophilization;
(g) drying the composition to obtain liofilizirovannogo cake.

2. The method according to p. 1, in which the time period is at least about 5 hours.

3. The method according to p. 1, to which m is at least one crystallizing excipient is an alanine and NaCl.

4. The method according to p. 3, in which alanine and NaCl are present in the composition at about 100 mm each.

5. The method according to p. 1, in which temperature and time sufficient to at least one crystallizing excipient completely or almost completely crystallized.

6. The method according to p. 1, in which the composition additionally contains one or more auxiliary substances, each selected from the group consisting of a stabilizing agent, a buffer agent, a surfactant, an antioxidant and a divalent cation.

7. The method according to p. 1, in which the composition further comprises a buffer selected from the group consisting of phosphate buffer, acetate buffer, citrate buffer and the buffer is citric acid/phosphate, histidine, Tris(hydroxymethyl)-aminomethane, 1,3-bis-[Tris-(hydroxymethyl)methylamino]propane, histidine, piperazine-N,N'-bis-(2-econsultancy acid), 3-(N-morpholino)propanesulfonic acid, N-2-hydroxyethylpiperazine-N'-2-econsultancy acid, 2-(N-morpholino)econsultancy acid and N-2-acetamido-2-aminoethanesulfonic acid.

8. The method according to p. 1, in which the lyophilized cake represents at least 50% solid cake.

9. The method according to p. 1, in which the composition is a liquid pharmaceutical composition comprising a pharmaceutical is Ki acceptable carrier.

10. Pharmaceutical kit containing lyophilized cake obtained by the method of p. 1, and an additional liquid component.

11. The method of freeze-drying a liquid composition containing plasma-derived AT III, NaCl and alanine, and this method includes:
a) freezing the specified composition at a temperature in the range from -52°C to -60°C for 6-15 hours;
(b) annealing the composition at -30°C for 1 hour;
(C) re-freezing of the composition at a temperature in the range from -52°C to -60°C for 2-15 hours; and
(g) drying the composition to obtain liofilizirovannogo cake.

12. The method according to p. 11, in which the activity of AT III is maintained or substantially maintained after storage of lyophilized cake at from about 25°C to about 40°C for from about 1 to about 6 months.

13. The method according to p. 11, in which alanine and NaCl are present in the composition in an amount of about 100 mm each.

14. The method according to p. 11, in which the composition additionally contains one or more auxiliary substances, each of which is selected from the group consisting of stabilizing agents, buffering agents, divalent cations, surfactants and antioxidants.



 

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2 cl, 10 dwg, 3 tbl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of pharmaceutics and represents method of treating prostate cancer, which includes introduction to patient of composition, which contains degarelix lyophilisate or its pharmaceutically acceptable salt and excipient, dissolved in solvent, in initial dose 200-300 mg of degarelix in concentration 20-80 mg of degarelix per ml of solvent with the following after 14-56 days after initial dose supporting dose 320-55 mg of degarelix in concentration 50-80 mg of degarelix per ml of solvent, possibly with one or more than one following additional supporting dose 320-550 mg of degarelix in concentration 50-80 mg of degarelix per ml of solvent, introduced with interval from 56 days to 112 days between each supporting dose.

EFFECT: invention provides long release of degarelix from obtained depot of medication without increase of occurrence of side effects.

11 cl, 1 ex, 2 dwg, 4 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to pharmaceutical compositions based on botulinum toxin and is intended for diagnostic or therapeutic introduction to a subject. A lyophilised or dried in vacuum composition contains botulinum toxin, stabilised with a non-protein excipient; a compound, selected from the group, consisting of the first monosaccharide, the first disaccharide, the first trisaccharide and the first alcohol, obtained by the reduction of the first monosaccharide; and a compound, selected from the group, consisting of the second monosaccharide, the second disaccharide, the second trisaccharide, the second alcohol and amino acid. In the other aspect the pharmaceutical composition contains botulinum toxin, stabilised with the non-protein excipient; polyethyleneglycol and a compound, selected from the group, consisting of monosaccharide, disaccharide, trisaccharide and amino acid. The pharmaceutical composition can contain botulinum toxin, stabilised with the non-protein excipient, polyvinylpyrrolidone; and disaccharide. The pharmaceutical composition of botulinum toxin, which does not contain an animal protein, includes botulinum toxin; a compound, selected from the group, consisting of the first monosaccharide, the first disaccharide, the first trisaccharide and amino acid.

EFFECT: application of the group of inventions provides the stable pharmaceutical composition for diagnostic or therapeutic introduction to a subject.

6 cl, 8 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention represents a medicinal preparation storage system comprising a chamber (3) accommodating at least two lyophilised active and/or additive substances (W1, W3, W5, W7) together in at least one chamber (3).

EFFECT: improving the system.

6 cl, 2 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics and represents an injectable form of 5α-androstane-3β,5,6β-triol containing a liquid injectable form, containing a solvent, or a solid injectable form containing at least one soluble additive with the above at least one soluble additive containing hydroxypropyl-β-cyclodextrine.

EFFECT: invention provides preparing the stable injectable form of 5α-androstane-3β,5,6β-triol.

10 cl, 7 ex, 4 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: storage-stable pharmaceutical composition represents a liquid formulation containing bortezomib and a system of anhydrous solvents and applicable for injection. A primary ingredient of the system of anhydrous solvents is propylene glycol. Bortezomib is found in the concentration of at least 1 mg/ml. The pharmaceutical composition contains a total amount of aqueous buffer of 10 vol. % or more.

EFFECT: stable pharmaceutical composition according to invention maintains bortezomib degradation at the level of not less than 10 wt % when keeping the liquid formulation for at least three months in the ambient environment.

9 cl, 10 tbl

FIELD: medicine.

SUBSTANCE: microspheres contain diclofenac in the form of an acid included in a matrix of a biodegradable polymer specified in a group consisting of polylactide and polylactide-co-glycolide; an average size of microspheres falls within the range of 5 to 150 mcm; a degree of diclofenac inclusion makes 5-50%, and the said microspheres release 60 to 95% diclofenac for 14 days.

EFFECT: extended range of methods for preparing injectable agents for treating inflammatory conditions, such as rheumatoid arthritis, osteoarthritis and rheumatoid spondylitis characterised by the prolonged diclofenac release.

4 cl, 3 dwg, 1 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to methods of obtaining a lyophilised preparation of tetrodotoxin and to a tetrodotoxin preparation for relief of the drug withdrawal in case of addiction to opiates. The method of obtaining the lyophilised preparation of tetrodoxin includes the following stages: 0.1-20 mcg/dose of tetrodotoxin is dissolved with 0.1% solution of citric acid to regulate pH within the range of 3.5-4.5 in injection water and filtered to remove pyrogen; separately dissolved are: a stabiliser - dextran or trehalose - and a filling agent, representing an isotonic solution of sodium chloride or mannit in injection water. After that, 0.1% solution of citric acid is added to regulate pH within 3.5-4.5, then, activated carbon is added with keeping at a temperature of 60°C and mixing for more than 30 minutes, filtering to remove pyrogen and cooling to room temperature. After homogeneous mixing of the obtained solutions and realisation of ultrafiltration, lyophilic drying is carried out. Lyophilic drying consists in preliminary freezing, drying under vacuum at reduced temperature, drying under vacuum at increased temperature, with each drying being performed at a certain temperature for the specified time period. After that, filling with inert gas is performed with control of water content at 3% level, with further sealing. Another version includes addition of additional solution of lidocaine chloride to the solution of tetrodotoxin and citric acid at the first stage. Also disclosed is the tetrodotoxin preparation for relief of the drug withdrawal in case of addiction to opiates, obtained by the said method, which is characterised by the weight ratio tetrodotoxin:filling agent:stabiliser, equal to 1:(150-3000):(50-6000).

EFFECT: claimed group of inventions ensures obtaining the stable tetrodotoxin solution with accurate dosage, which is used for introduction into the human organism.

25 cl, 9 tbl, 14 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to medicine. Described is a composition for the transdermal introduction of medications (M) into a human and animal organism, including cases of urgent drug treatment and prevention of acute pathological conditions. The composition consists of a solution of one or several M in a mixture of at least two solvents: dialkylamide of lower carboxylic acid and (or) alkylpyrrolidone and monoterpenes and (or) monoterpenoids. The total volume concentration of N,N-dialkylamides of lower carboxylic acids or N-alkylpyrrolidones constitutes from 1% to 99% of the mixture volume.

EFFECT: composition makes it possible to realise urgent drug treatment and prevention of acute pathological conditions of a human and animal organism by a simple in implementation, safe and cheap method.

12 cl, 6 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to compounds or their pharmaceutically acceptable salts which can be applicable for biologically active substances delivery.

EFFECT: invention refers to pharmaceutical formulations containing the above compounds, and to a method for administering the biologically active substances.

17 cl, 10 tbl, 19 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to medicine, namely to pharmacology and describes a histidine-free pharmaceutical composition containing high-purity factor VIII; arginine and saccharose, a surfactant for the prevention or at least the inhibition of a surface adsorption of factor VIII; 0.5 to 10 mM calcium chloride for the specific stabilisation of factor VIII, and sodium citrate or maleic acid as a pH buffer.

EFFECT: invention provides the protective function for preserve high-yield factor VIII over the whole cycle of pharmaceutical processing, long storage and end recovery and administration into the patient.

18 cl, 16 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to dermatooncology, and can be used in treatment of actinic keratosis. For this purpose foci of disease are identified with further carrying out their US-examination and derma analysis. In case if hypoechogenic zone, occupying from 5% to 30% of entire derma thickness, is present in derma, applications with liquid nitrogen with textile tip are performed. In case if hypoechogenic zone, occupying from 30% to 70% of entire derma thickness, is detected, applications with liquid nitrogen with copper tip or photodynamic therapy with application of photosensitiser are performed. In case if hypoechogenic zone, occupying from 70% to entire derma thickness, is detected, photodynamic therapy with application of photosensitiser is carried out.

EFFECT: method makes it possible to select tactics of treating said pathology in the most accurate way basing not only on clinical manifestations of disease, but also due to taking into account proliferative abilities of cells, which excludes necessity of carrying out tissue biopsy.

3 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely rheumatology, and is applicable for preventing and treating rheumatoid arthritis in a patient. That is ensured by the oral administration of a therapeutically effective amount of calcitonin in the free form or in the form of a salt or a delivery agent specified in a group specified in N-(5-chlorosalicyloyl)-8-aminocaprylic acid (5-CNAC), N-(10-[2- hydroxybenzoyl]amino)decanoic acid (SNAD), N-(8-[2-hydroxybenzoyl]amino)caprylic acid (SNAC) and their pharmaceutically acceptable salts.

EFFECT: oral introduction promotes improved absorption, more acceptable pharmacokinetic and pharmacodynamic profile and a smaller degree of calcitonin variability, as compared with the other methods of delivery that provides reduced chondrolysis.

8 cl, 2 dwg, 2 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to pharmacy and medicine. Pharmaceutical composition includes mixture of: (a) active macromolecular substance; (b) aromatic alcohol, absoption amplifier, selected from propylgallate, butylated hydroxytotuol (BHT), butylated hydroxyanisole (BHA) and their analogues and derivatives, or their mixtures; and (c) biguanide or its pharmaceutically acceptable salt, capable of increasing solubility of aromatic alcohol, absorption amplifier, in water medium, where aromatic alcohol, absorption amplifier, is present in amount (by weight) greater or equal to amount of active substance. Application in pharmaceutical composition of aromatic alcohol, selected from propylgallate, BHT, BHA and their analogues and derivatives, together with biguanide or its pharmaceutically acceptable salt, capable of increasing solubility of aromatic alcohol in water medium, as amplifier of macromolecule absorption through intestine wall. Method of increasing absorption of active macromolecular substance and method of patient treatment include introduction of claimed composition to patient. Application of active macromolecular substance, selected from insulin, C-peptide, GLP-1 or their mixture; aromatic alcohol, absorption amplifier, selected from propylgallate, butylated hydroxytotuol (BHT), butylated hydroxyanisole (BHA) and their analogues and derivatives, or their mixtures; and biguanide or its pharmaceutically acceptable salt, capable of increasing solubility of aromatic alcohol, absorption amplifier, in water medium, in manufacturing medications for treatment of diabetes, osteoporosis, obesity, cancer, osteoarthritis.

EFFECT: group of inventions ensures facilitation of passage of peptides, proteins and other macromolecular substances through intestinal wall for improvements of their bioavailability.

27 cl, 3 tbl, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to method of obtaining composition, including lyophilised one, containing epoprostenol, which includes obtaining solution of epoprostenol or its salt and arginine and bringing solution pH to more than 12 by addition of potassium hydroxide or sodium hydroxide. Invention also relates to pharmaceutical composition and stable solution for treatment of cardio-vascular diseases, which contain epoprostenol or its salt and arginin and have pH higher than 12. Also described is method of treating patient with cardio-vascular disease, which includes introduction of effective amount of said composition.

EFFECT: invention ensures obtaining hemocompatible, stable to microorganisms and stable epoprostenol compositions.

46 cl, 30 tbl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, namely to pharmaceutical compositions containing a peptide and propylene glycol, to methods of preparing such compositions, and also concerns methods of reducing contamination of injection devices by a peptide composition and reducing sediment formation in the production equipment during manufacturing of the peptide composition. The agonist GLP-1 is used as said peptide in the composition.

EFFECT: reduced contamination of the injection devices during injection.

23 cl, 6 ex, 7 dwg

FIELD: medicine.

SUBSTANCE: offered invention refers to medicine, namely to surgery, and can be used for prevention of inconsistency of anastomoses of hollow organs in gastrointestinal tract surgery. That is ensured by the oral introduction of 5-oxymethyluracil pills of the weight 1.5 g and diameter 7-7.5 mm on the 2nd postoperative day in the following proportions: 5-oxymethyluracil - 0.5 g, 5 % spirit-acetone acetylphthalylcellulose - 3 layers, 10 % hexane solution of low-molecular polyethylene - 1-3 layers, biological adhesive Sulphacrylate - 1-3 layers.

EFFECT: introduction of 5-oxymethyluracil in said dosage form provides stimulation of repair regeneration ensured by selective maximum concentration of the preparation in gastrointestinal segments where the anastomosis has been created, and reduced ischemic disorders.

1 ex, 1 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to a chrysophanol conjugate or its derivative, characterised by general formula (I), in which R1-R8 is a group selected from -H, -OH, -OCH3, -CH3, provided that not less than two groups from R1-R8 denote -H or provided that one or two of groups R2, R3, R6 and R7 are a -COOH group, M is a nitrogen organic base selected from a group comprising chitosamin, glucosamin, or a basic amino acid selected from a group comprising arginine, lysine, carnitine, and group M is bonded to the chrysophanol part in the conjugate.

EFFECT: invention relates to use of the conjugate as a medicinal agent for preventing or treating diabetic nephropathy, osteoarthritis, rheumatic or rheumatoid arthritis, intestinal adhesion, intestinal peristalsis restoration, and a medicinal agent based on the conjugate.

8 cl, 4 dwg, 57 ex, 18 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to a method of producing paramagnetic iron oxide nanoparticles. The disclosed method includes reacting an iron complex having iron as a central atom and a carboxylate group having 10 to 22 carbon atoms which is bonded to the central atom in a ligand form; a C10-C22 fatty acid and a C10-C22 aliphatic alcohol or C10-C22 aliphatic amine to obtain iron oxide nanoparticles. The iron oxide nanoparticles are obtained by raising the temperature from room temperature to 200 to 250°C with at a rate of 5°C/min or higher, and conducting the reaction at 200 to 250°C for 5 to 60 min.

EFFECT: invention enables to obtain iron oxide nanoparticles having a size of 4nm or less, which can be used as a MRI T1 contrast agent.

7 cl, 1 tbl, 25 dwg, 31 ex

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