New albumin-free compositions of factor viii

FIELD: medicine, hematology, pharmacy.

SUBSTANCE: invention relates to the composition of factor VIII composed without addition of albumin and comprising the following excipients of composition in addition to factor VIII: from 4% to 10% of filling agent taken among group consisting of mannitol, glycine and alanine; from 1% to 4% of stabilizing agent taken among group consisting of sucrose, trehalose, raffinose, arginine; from 1 mM to 5 mM of calcium salt, from 100 mM to 300 mM of NaCl, and buffer agent for pH value maintenance about between 6 and 8. Alternatively, the composition can comprise from 2% to 6% of hydroxyethylstarch; from 1% to 4% of stabilizing agent taken among group consisting of sucrose, trehalose, raffinose, arginine; from 1 mM to 5 mM of calcium salt, from 100 mM to 300 mM of NaCl, and buffer agent for pH value maintenance between 6 and 8. In additional variant of realization of invention the composition can comprise: from 300 mM to 500 mM of NaCl, from 1% to 4% of stabilizing agent taken among group consisting of sucrose, trehalose, raffinose and arginine; from 1 mM to 5 mM of calcium salt, and buffer agent. The composition provides stability in the absence of albumin or other proteins.

EFFECT: valuable properties of compositions.

35 cl, 11 tbl, 7 ex

 

Factor VIII is a protein found in blood plasma, which acts as a cofactor in the cascade of reactions leading to blood coagulation. The deficit of the quantity of the activity of factor VIII in the blood leads to blood clotting, known as hemophilia And hereditary transmitted painful condition that mainly affects men. Hemophilia a is usually treated with therapeutic preparations of factor VIII obtained from plasma or produced using recombinant DNA technologies. Such drugs are administered either in response to a case of bleeding (treatment-on-demand), or with frequent regular intervals to prevent uncontrolled bleeding (prophylaxis).

It is known that factor VIII is relatively unstable in therapeutic preparations. In plasma factor VIII is usually notorious with another plasma protein, factor von Willebrand's disease (von Willebrand's, vWF), which is present in plasma in a large molar excess relative to factor VIII, and it is assumed that it protects factor VIII from premature decomposition. Other circulating plasma protein, albumin, may also play a role in the stabilization of factor VIII in vivo. Currently offered for sale the preparations of factor VIII, therefore, intended mainly for the use of albumin and/or vWF for when abilitati factor VIII in the process of the method of manufacture and during storage.

However, albumin and vWF used in offered on the market currently, preparations of factor VIII derived from human blood plasma, and the use of such materials has some drawbacks. As to increase the stability of factor VIII in such preparations usually add a large molar excess of albumin compared to factor VIII, it is difficult to characterize protein factor VIII in such preparations. Adding derived from human albumin to factor VIII is also perceived as disadvantageous in respect recombinante get drugs. This is because recombinante received preparations of factor VIII in the absence of added albumin would otherwise not contain derived from human proteins, and theoretical risk of transmission would be reduced.

Described several attempts to make a recipe of factor VIII without albumin or vWF (or at a relatively low level of such excipients). For example, in U.S. patent No. 5565427 (EP 508194) Freudenberg (successor Behringwerke) described the preparations of factor VIII, which contain certain combinations of detergent and amino acids, particularly arginine and glycine, in addition to the excipients, such as sodium chloride and sucrose. Indicates that the detergent, Polysorbate 20 or Polysorbate 80 is present in quantities of from 0.001 to 0.5% (V/V), whereas rginin and glycine are present in quantities of from 0.01 to 1 mol/L. Indicated that sucrose is present in quantities of from 0.1 to 10%. In example 2 of this patent States that the solutions of (1) to 0.75% sucrose, 0,4M glycine and 0,15M NaCl and (2) 0.01 M sodium citrate, 0.08 M glycine, 0,016 M lysine, 0,0025 M calcium chloride and 0.4 M sodium chloride were unstable in solution for more than 16 hours, whereas the solutions of (3) 1% sucrose, 0,14 M arginine, 0.1 M sodium chloride, and (4) 1% sucrose, 0.4 M glycine, 0,14 M arginine, 0.1 M sodium chloride and 0,05% Tween 80 showed stability.

In U.S. patent No. 5763401 (EP 818204) Nayer (successor Bayer) also described therapeutic composition of factor VIII that do not contain albumin, including 15-60 mm sucrose, up to 50 mm NaCl, up to 5 mm calcium chloride, 65-400 mm glycine and up to 50 mm histidine. Indicate how stable the following specific compounds: (1) 150 mm NaCl, 2.5 mm of calcium chloride and 165 mm mannitol; and (2) 1% sucrose, 30 mm sodium chloride, 2.5 mm calcium chloride, 20 mm histidine and 290 mm glycine. It was found that the composition containing a higher amount of sugar (10% maltose, 50 mm NaCl, 2.5 mm calcium chloride and 5 mm histidine), has low stability in liofilizirovannom condition compared with the composition of (2).

In U.S. patent No. 5733873 (EP 627924) Osterberg (successor Pharmacia & decision Upjohn) describes compositions that include from 0.01 to 1 mg/ml surfactant. This patent describes compositions, it is matter of following ranges of excipients: Polysorbate 20 or 80 in number, at least 0.01 mg/ml, preferably of 0.02-1.0 mg/ml, at least 0.1 M NaCl; at least 0.5 mm calcium salt; and at least 1 mm of histidine. More specifically described, the following specific compounds: (1) 14,7-50-65 mm histidine, 0,31-0.6 M NaCl, 4 mm calcium chloride, 0,001-0,02-0,025% Polysorbate 80, in the presence or absence of 0.1% PEG4000 19.9 mm sucrose; and (2) 20 mg/ml mannitol, to 2.67 mg/ml histidine, 18 mg/ml NaCl, 3.7 mm of calcium chloride and 0.23 mg/ml of Polysorbate 80.

Also described other attempts to use low or high concentrations of sodium chloride. In U.S. patent No. 4877608 (EP 315968) Lee (successor Rhone-Poulenc Rorer) are compounds with relatively low concentrations of chloride of sodium, namely compounds containing 0.5 mm-15 mm NaCl, 5 mm calcium chloride, 0.2 mm-5 mm histidine, 0.01 to 10 mm lysine hydrochloride and up to 10% of sugar. “Sugar” can represent up to 10% maltose, 10% sucrose or 5% mannitol.

In U.S. patent No. 5605884 (EP 0314095) Lee (successor Rhone-Poulenc Rorer) describes the use of compositions with relatively high concentrations of sodium chloride. These compositions include a 0.35 M-1.2 M NaCl, 1.5 to 40 mm calcium chloride, 1 mm-50 mm histidine and up to 10% “sugar”such as mannitol, sucrose or maltose. As an example, the composition comprising 0.45 M NaCl, 2.3 mm of calcium chloride and 1.4 mm histidine.

In the published international patent application WO 96/22107 Roser(successor Quadrant Holdings Cambridge Limited) describes formulations including sugar trehalose. These compounds include: (1) 0.1 M NaCl, 15 mm calcium chloride, 15 mm histidine and 1.27 M (48%) trehalose; or (2) to 0.011% of calcium chloride, 0.12% of histidine, 0,002% Tris, 0,002% Tween 80, 0,004% PEG 3350, 7.5% trehalose and or 0.13 per cent, or of 1.03% NaCl.

Other therapeutic compositions of factor VIII from the prior art usually include albumin and/or vWF to stabilize factor VIII and, therefore, not essential to the present invention. For example, in U.S. patent 5328694 (EP 511234) Schwinn (successor Ocrapharma AG) describes a composition that includes 100-650 mm disaccharide and 100 mm-1,0M amino acids. Specifically, disclosed the following compositions: (1) 0.9 M sucrose, 0.25 M glycine, 0,25M lysine and 3 mm calcium chloride; and (2) 0.7 M sucrose, 0.5 M glycine, and 5 mm calcium chloride.

While attempts were made to obtain the composition of factor VIII containing no albumin or vWF, there is still a need in therapeutic compositions of factor VIII, which are stable in the absence of albumin or other proteins.

SUMMARY of the INVENTION

The present invention relates to therapeutic compositions of factor VIII, which are stable in the absence of albumin. In particular, the present invention relates to the composition of factor VIII, which includes, in addition to factor VIII: from 4% to 10% of a filler selected from the gr is PPI, consisting of mannitol, glycine and alanine; 1% to 4% of a stabilizing agent selected from the group consisting of sucrose, trehalose, raffinose, and arginine; 1 mm to 5 mm calcium salt, from 100 mm NaCl to 300, and a buffering agent to maintain pH in the range from about 6 to 8. This composition may further include a surfactant, such as Polysorbate 20, Polysorbate 80, Pluronic F68 or Brij35. When the surfactant is a Polysorbate 80, the latter must be present in an amount of less than 0.1%.

The buffer in the compositions of factor VIII according to the present invention preferably is present in a concentration of from 10 mm to 50 mm and, preferably, it is chosen from the group consisting of histidine, Tris, BIS-TrisPropane, PIPES, MOPS, HEPES, MES and ACES. Mainly, the buffer agent is either a histidine or Tris. The composition of the factor VIII of the present invention may further include an antioxidant.

The composition of factor VIII of the present invention include both a filler and a stabilizer. The filler may be present in amount from about 6% to about 8%, preferably about 8%. The stabilizing agent preferably is present in an amount of about 2%. Sodium chloride is also present in such compositions, preferably in an amount of from 150 to 350 mm, and more PR is doctitle in an amount of about 225 mm. Salt of calcium in the composition also preferably represents calcium chloride, and the composition preferably is in liofilizovannyh form.

In another embodiment, the present invention may include a composition of factor VIII made without the addition of albumin, which includes the following excipients in addition to factor VIII: from 2% to 6% of hydroxyethylamine; from 1% to 4% of a stabilizing agent selected from the group consisting of sucrose, trehalose, raffinose, and arginine; 1 mm to 5 mm calcium salt; 100 mm to 300 mm NaCl and a buffering agent for maintaining a pH in the range from about 6 to 8.

Preferably the composition comprises about 4% of hydroxyethylamine, and NaCl is present in an amount of about 200 mm. The stabilizing agent preferably is present in an amount of about 2%.

In the following embodiment, the present invention includes a composition factor VIII made without albumin and containing: from 300 mm to 500 mm NaCl; 1% to 4% of a stabilizing agent selected from the group consisting of sucrose, trehalose, raffinose, and arginine; 1 mm to 5 mm calcium salt; and a buffering agent to maintain pH in the range from about 6 to 8. Preferably NaCl is present at a concentration of approximately 400 mm.

In another embodiment, the present is sabreena relates to a method of freeze-drying aqueous compositions of factor VIII in the container using lyophilizate, where the method includes the initial stage of freezing and the initial stage of freezing additionally involves the following stages: (a) lowering the temperature in the chamber lyophilizate to at least -45°C; (b) increasing the temperature in the chamber to a temperature in the range of from about -15°C to -25°C; and then (C) lowering the temperature in the chamber to at least -45°C. In this way the temperature in the chamber, preferably, lower or raise with a speed of between about 0.5°and about 1.0°C per minute. At the stage of (a) the temperature is preferably maintained for about 1 hour and lowered to about -55°C. At the stage (b) the temperature is preferably maintained between -15°C and -25°C for from 1 to 3 hours and, more preferably, it is -22°C, and the temperature in stage (C), preferably, is maintained for about 1 hour. The composition of factor VIII used in this method preferably comprises from 4% to 10% of an agent selected from the group consisting of mannitol, glycine and alanine, and also preferably includes from 1% to 4% of an agent selected from the group consisting of sucrose, trehalose, raffinose, and arginine. In addition, the composition of factor VIII used in this way, also preferably contains from 100 mm to 300 mm NaCl.

DETAILED description of the INVENTION

Definition

As IP is alzavano in this description, the terms listed below, and their variations will be determined as follows, unless otherwise stated.

Factor VIII - factor VIII molecule exists in nature and in therapeutic dosage forms in the form of a heterogeneous distribution of polypeptides from a single gene (see Andersson et al., Proc.Natl.Acad.Sci.USA, 83, 2979-2983, may 1986). The term “factor VIII”, as used herein, refers to all such polypeptides, or obtained from plasma or produced using methods of recombinant DNA. Commercially available examples of therapeutic dosage forms containing factor VIII include sold under the trade name HEMOFIL M and RECOMBINATE (available from Baxter Healthcare Corporation, Deerfield, Illinois, USA). Other drugs that are currently in development include primarily, only a subpopulation of molecules of factor VIII lacking the domain part In the molecule.

International unit, ME - international unit or ME represents a unit of activity measurement (capacity) factor VIII coagulation of the blood, measured in standard analysis, such as one of the following.

One-step analysis. In this area known such one-step analysis, as described in Lee, Martin L., et al. An Effect of Predilution on Potency Assays of Factor VIII Concentrates, Thrombosis Research (Pergamon Pres Ltd.) 30, 511-519 (1983).

Chromogenic analysis. Chromogenic assays can be purchased commercially, such as Coatest Factor VIII available from Chromogenix AB, Molndal, Sweden.

Annealing - the term “annealing” is used to indicate the stage of the way lyophilization of pharmaceutical drug exposed to freeze-drying before drying of the drug by freezing, in which the temperature of the drug increase from a lower temperature to a higher temperature and then cooled over a period of time.

Filler - for purposes of this application, the fillers are chemical objects that provide the structure of the “brick” or residual solid mass of a pharmaceutical product after liabilitiy and which protect it from buckling. Able to crystallization filler will mean the filler, as described in this application may crystallise during lyophilization and different from sodium chloride. HES not included in this group are able to crystallization fillers.

Freeze-drying, freezing, freeze-drying “drying freezing”, if not specified in another context, where appears this term will be used to denote a part of the process of freeze-drying, in which the temperature of a pharmaceutical product increase removal bodies of the drug. The stage of the “freezing” method of freeze-drying are the stages that occur before the stage of freeze drying. “Lyophilization”, unless otherwise stated, will relate to a method of freeze-drying as a whole, including as a stage of freezing, and the stage of freeze drying.

Unless otherwise stated, the term interest expresses the weight/volume percent, and temperatures are indicated in degrees Celsius.

Ingredients

The composition of factor VIII of the present invention include fillers, stabilizing agents, buffering agents, sodium chloride, calcium salts and, mainly, other excipients. These excipients were selected to ensure maximum stability of factor VIII in liofilizovannyh drugs. However, the composition of factor VIII of the present invention also exhibit stability in the liquid state.

The fillers used in the present compositions, which form the crystalline part of liofilizirovannogo product (except HES), which are selected from the group consisting of mannitol, glycine, alanine and hydroxyethylamine (HES). Mannitol, glycine or alanine are present in the amount of 4-10%, preferably 6-9%, and more preferably about 8%. When the filler used HES, he is present in the amount of 2-6%, preferably 3-5%, and more site is preferably, approximately 4%.

The stabilizing agents used in the compositions of the present invention, are selected from the group consisting of sucrose, trehalose, raffinose, and arginine. These agents are present in the compositions of the present invention in an amount of from 1 to 4%, preferably 2-3%, more preferably about 2%. Sorbitol and glycerol were evaluated as potential stabilizers, but it was found that they are weak stabilizers in the present compositions.

Sodium chloride is included in the present compositions in an amount of 100-300 mm, preferably 150-250 mm, and most preferably, about 225 mm. In one embodiment, the present invention can be used in the sodium chloride without any of the above fillers, in this case it will be included in the composition in an amount from 300 mm to 500 mm NaCl, preferably from 350 mm to 450 mm NaCl, and, more preferably, about 400 mm NaCl.

In addition, these compounds are present in the buffers, because it is assumed that the factor VIII molecule may additionally be influenced by the pH shift during lyophilization. Preferably, the pH should be maintained between 6 and 8 during lyophilization and, more preferably, the pH is approximately 7. A buffering agent can be any physiologically acceptable chemical object, or combination of chemical j is, who has the ability to act as a buffer, including histidine, Tris, BIS-Tris Propane, PIPES, MOPS, HEPES, MES and ACES. A complete chemical description of the data buffer agents are listed below in table 1. Typically, the buffer agent include at a concentration of 10-50 mm. When compositions add histidine, using concentrations greater than 20 mm and preferably about 25 mm, individually or in combination with other buffers, such as Tris. Histidine is especially preferred for use in compositions of the present invention, as described further below.

Table 1

Buffer agents

TrisTris-(hydroxymethyl)aminomethan
BIS-Tris Propane1,3-bis-[Tris-(hydroxymethyl)methylamino]propane
PIPESpiperazine-N,N'-bis-(2-econsultancy acid)
MOPS3-{N-morpholino)propanesulfonic acid
HEPESN-2-hydroxyethyl-piperazine-N'-2-econsultancy acid
MES2-(N-morpholino)econsultancy acid
ACESN-2-acetamido-2-aminoetansulfonovaya acid

To preserve the activity of factor VIII is important that the compositions of the present invention also includes calcium or another is vocality cation, able to interact with factor VIII and maintain its activity, presumably by maintaining the Association of the heavy and light chains of factor VIII. You can use from 1 mm to 5 mm calcium salt, more preferably 3-4 mm and, most preferably, about 4 mm. The calcium salt is preferably a chloride of calcium, but it can also represent other calcium salts such as calcium gluconate, glubionate calcium or gluceptate calcium.

The composition of factor VIII of the present invention also preferably include a surfactant, preferably in an amount of 0.1% or less and, more preferably, in an amount of about 0.03 percent. Surfactant, for example, may be selected from the group consisting of Polysorbate 20, Polysorbate 80, plutonomy polyhydric alcohols and Brij 35 (lauric simple ester of polyoxyethylene 23). There are several brands plutonomy polyhydric alcohols (sold under the trade name Pluronic, produced by BASF Wyandotte Corporation). Data polyhydric alcohols with various molecular masses (from 1000 to over 16000) and physico-chemical properties are used as surfactants. Pluronic F-38, with a molecular weight of 5000, and Pluronic F-68, with a molecular weight of 9000, both contain (by weight) 80 percent of the hydrophilic policies lenovich groups and 20 percent hydrophobic polyoxypropylene groups. However, preferred in the present compositions is Tween-80, commercially available Polysorbate, in particular Tween-80 vegetable origin.

The compositions of the factor VIII of the present invention also preferably includes an antioxidant. It was found that the addition of antioxidants to liofilizovane compositions of the invention improves the stability of such compositions and thus increases the duration of the shelf life. Applied antioxidants must be compatible for use with the pharmaceutical drug and, in addition, are preferably water-soluble. Adding antioxidants to the composition, it is preferable to add such antioxidants as late as possible before lyophilization, to exclude spontaneous oxidation of the antioxidant. In the following table 2 lists suitable antioxidants are commercially available from companies such as Calbiochem and Sigma.

Table 2

Antioxidants

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)

Bottled hydroxytoluene (EIT)

Among the above-mentioned antioxidants glutathione is preferred. It was found that all the end of the ation in the range from about 0.05 mg/ml to more than 1.0 mg/ml increased the stability of the compositions of factor VIII, and it is assumed that higher concentrations could also be useful (until the occurrence of any toxicity or adverse effect on the receipt, as, for example, reducing the glass transition temperature of liofilizirovannogo product).

In particular, it was found that the combination of histidine and glutathione exerts a synergistic beneficial effect on the stability of compositions of factor VIII. Histidine, though, and acts as a buffer, can also act as a chelator of metal. The extent to which the inactivation of factor VIII is called metal-induced oxidation, histidine, therefore, may act as a stabilizer of factor VIII by linking such oxidizing metal ions. Assume that by linking these metals glutathione (or actually any present antioxidant) can thus provide additional antioxidant protection, as it is considered the oxidizing action of metal ions associated with histidine.

Other chelating agents may also be used in the compositions of the present invention. Such agents should preferably bind metals such as copper and iron, with a greater affinity than calcium, if a salt of calcium is used in the composition. One of these he is atarov represents deferoxamine, chelating agent, which facilitates the removal of A1++ and iron. Deferoxamine mesilate, C25H48N6O8CH4O3S, available from Sigma (Sigma Prod.N D9533). He is a chelator for aluminum and iron(II), which gelaterie iron (in the form of a 1:1 chelate complex) only in the oxidation state +3, and not in the oxidation state +2, and can also bind ions of manganese and other metals. Deferoxamine mainly can be used in amounts of 0.25 mg/L.

Factor VIII used in these compositions can be either highly purified, obtained from human blood plasma factor VIII, or, preferably, it can be recombinante derived factor VIII. Recombinant factor VIII can be produced by cells of the Chinese hamster ovary (Cho), transfectional vector carrying a DNA sequence encoding a factor VIII molecule. How to create such transfection Cho cells is described, inter alia, in U.S. patent 4757006, 'toole Jr., although this region is also known for alternative ways (see, for example, U.S. patent 4868112 also'toole, Jr., and the publication of the International application WO-A-91/09122). The methods used for the cultivation of Cho cells to obtain factor VIII, also known in this field, for example, as described in the application for the European patent No. 0362218, prin is improper Institute of Genetics (Genetics Institute), entitled “Improved methods of obtaining protein type factor VIII:C”. Recombinant factor VIII can also be produced in other cell lines, such as kidney cells of a newborn hamster (KSS). A molecule of factor VIII with recombinant receipt can be either factor VIII full length or deleteregvalue derived, such as the factor VIII molecule with a deletion In the domain.

Although the composition of factor VIII, as described in this application can be liofilizovane and restored in these concentrations, the person skilled in the art it will be clear that the moisture content of these drugs can also be recovered in a more diluted form. For example, the preparation according to the present invention, which liofilizovane and/or normally restored by the moisture content in 2 ml of solution, can also be recovered in a larger volume of diluent, such as 5 ml. It is particularly suitable when the preparation of factor VIII immediately administered as an injection to the patient, as in this case, it is less likely that the factor VIII will lose activity, which may occur more quickly in more dilute solutions of factor VIII.

The composition (formulation) and development LIABILITIE

To achieve maximum stability of the composition of factor VIII of the present invention, preferably life isout. During the lyophilized factor VIII transform from being in the water phase to being in an amorphous solid phase, which is believed to protect the protein from chemical and/or conformational instability. Liofilizovannye the product not only contains an amorphous phase, but also includes a component that crystallizes during lyophilization. This, as expected, allows for fast drying of the composition of factor VIII and education more elegant briquette (i.e. briquettes with minimal shrinkage from the sides of the container in which it lyophilised). In the compositions of the present invention, the stabilizing agents are selected so that they existed mainly in the amorphous phase of liofilizirovannogo product, whereas fillers (except HES) are selected so that they crystallized during freezing.

As factor VIII, and the stabilizer is preferably dispersed in the amorphous phase of liofilizirovannogo briquette. The weight of the stabilizer is also, preferably, to a large extent compatible with other excipients amorphous form. In addition, the apparent glass transition temperature (Tg') amorphous phase, preferably, is relatively high during freeze-drying, and the glass transition temperature (TD) of a solid substance is similarly preferably high during storage. B the lo set, the crystallization of sodium chloride in the product is desirable, since the amorphous sodium chloride will reduce Tg' amorphous phase.

In order to avoid buckling of the briquette for a particular composition, the initial drying is preferably carried out at a product temperature below the apparent glass transition temperature of the frozen concentrate. Increase drying time may also be required to offset the lowering of Tg'. For more information on lyophilization can be found in J.F. Carpenter 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.), pp.199-264 (1996).

Example 1

The effect of the concentration of factor VIII and add stabilizer to the recovery of factor VIII have been investigated in several studies. Data analyses were performed by using mannitol as a model filler and sucrose as a model of the stabilizer. In these studies used the compositions of the three samples described below in table 3. All formulations used in these studies consisted of 10 mm Tris, 200 mm NaCl, 8% mannitol, 4 mm CaCl2and 0.02% Tween-80 and was carried out at pH 7.0.

Table 3

ID manually. sample numberThe initial content of factor is VIII (ME/ml) Sucrose%
IA600-
IB60-
IC602

These samples were liofilizovane using a cycle of freeze-drying, are shown below in table 4, to maintain the product temperature below the apparent glass transition temperature (Tg'). Studies using differential scanning calorimetry (DSC) indicated the presence of a transition at approximately -40°C. in formulations containing mannitol. To maintain the product temperature below this value, the storage temperature was set at -32°C during primary drying. Primary drying under these conditions was carried out for approximately 55 hours, with a total cycle time of approximately 80 hours.

Table 4

The method of freezing/ processingDescription
I (freezing)Cooling to +5°C;

Cooling to -5°C at 1°C/min, holding for 20 minutes;

Cooling to -20±5°C at a rate of 1°C/min, holding for 1 hour (up to 3 hours);

Cooling to -45°C at a rate of 0.5°C/min, holding for 1 hour.
IIFreezing through with the person I. Conditioning at -35°C for 48 hours
IIIFreezing by the way I. Keeping at -35°C for 48 hours
Keeping at -20°C for 48 hours
IV (Drying, freezing)Store at -32°C during primary drying for approximately 55 hours (up to 100 hours);

Product <-40°C during primary drying; Linear rise from -32°C to +40°C at a rate of 0.2°C/min.

Storage at +40°C during secondary drying for 3 hours

The activity of factor VIII data samples defined using the one-stage analysis of the coagulating activity of the blood, compared with control, was kept at -45°C. the Results are shown below in table 5.

Table 5

The way to handle% Loss of activity of factor VIII during each stage
Part IA (600 IU/ml)Part IB (60 IU/ml)The composition of the IC (60 IU/ml, 2% sucrose)
I6,737,541,7
II2,09,3a 3.9
III7,3the 11.65,0
IV (lyophil the nation) 20,024,218/3

These results indicate that the protein concentration affects the recovery of factor VIII during freezing. Compositions containing 60 IU/ml, lost approximately 37-42% of the initial activity of factor VIII during the stage of freezing, while 6.7% of the activity of factor VIII was lost for the composition containing 600 IU/ml these results indicate that a higher concentration of protein has a protective effect during freezing. Although sucrose, providing some protection factor VIII at an intermediate temperature, has the same effect as during freeze-drying, it is not able to protect the protein during the initial stage of freezing.

Example 2

After the development of a method of freeze-drying, as described in General terms in example 1 was taken further optimization of this method. It was found that liofilizovannye composition having a higher glass transition temperature (and, theoretically, the best stability of factor VIII)may be obtained by: (1) lowering the temperature of the freezing initially, up to -45°C or below (as, for example, to about -50°C or -55°C); (2) increasing the temperature to -20°C or -22°C (±5°C); and then (3) lowering the temperature again to -45°C or below. The temperature is reduced or ivysaur, depending on the stage at a speed ranging from about 0.5°to about 1.0°C per minute. Upon reaching the desired temperature, the composition is maintained at this temperature over a period of time from 1 to 3 hours. This improved method of freezing is shown below in table 6.

Table 6

The method of freezingDescription
ICooling to +5°C;

Cooling to -5°C at a rate of 0.5-1°C/min, holding for 20 minutes;

Cooling to a temperature of -55°C to -45°C at a rate of 0.5-1°C/min, holding for 1 hour;

Heated to -22(±5°C) at a rate of 0.5-1°C/min, holding for 1 to 3 hours; Cooling to -45°C at a rate of 0.5-1°C/min, holding for about 1 hour.

Unless otherwise stated, the indicated temperature referenced in this example and other examples, refer to the temperature of the lyophilisate and not the temperature of the product itself. Following the advanced cycle of freezing, the rest of the way lyophilization can be carried out, as described above in example 1, or in other words, as additionally described herein, or as specified by the specialist in this field.

It is established that this usovershenstvovannyi way is ifilesize can be used for trains which include glycine as a filler, as well as for those compounds, which are used mannitol. It is additionally assumed that the method can also be applied to compositions that use other fillers of the present invention.

Example 3

It is assumed that to obtain dried freeze-drying product with acceptable appearance of brick and glass transition temperature, for filler liofilizovannyh pharmaceutical preparations containing sodium chloride, such as glycine or mannitol may require crystallization. So was developed following an improved method for freeze-drying are able to crystallization fillers.

Table 7a

Stage of freezing

Stage methodTemperatureThe duration of stage
The initial freezing-40°C or less1 hour
The first annealingbetween -23°C and -27°C3 hours
The second freezing-55°C1 hour
The second annealing-36°C4 hours
The third freezing-50°C1 hour

Table 7b

Hundred the AI freeze-drying

Stage methodTemperatureThe duration of stage
Primary drying-35°Cup to 100 hours
Secondary drying: the first stage40°C3 hours
Secondary drying: second stage45°C3 hours
Secondary drying: the third stage50°C3 hours

At the stages of freezing temperature changes took place with the velocity of from about 0.5°C./min to 1°C/minute. It is assumed that the stage of a longer duration would also be effective.

Before the first stage of the freezing temperature adjusted to 2-8°C for about one hour in order to bring all vials to approximately the same temperature. After that lyophilizator cooled to -5°C. the First stage of freezing should be carried out at temperatures below -30°C, preferably below -35°C, and more preferably at about -40°C. In accordance with this first stage of annealing should be performed at a temperature between -30°C and -19°C, or more preferably between about -25°C and -28°C (if glycine is a filler), or between -21°C and -24°C (if mannitol is a filler), when temperature is round between -23°C and -26°With, which is the most preferable, it is assumed that at a given temperature crystallizes, at least partially, the filler is able to crystallize. However, the lower range in the field of -27°C is not recommended for formulations containing mannitol and arginine. This stage is preferably carried out for about 3 hours.

After the first stage of the annealing temperature is reduced, preferably to a temperature below about -50°C. and, more preferably, below -55°C. for about 1 hour. Assume that the sodium chloride in the product at this time forms the germ of crystals.

During the second stage of the annealing temperature of a pharmaceutical product increases to a temperature between about -30°C and -39°C. and preferably to about -33°C to magnisoderjasimi compositions and -36°C for glicenstein compositions. Assume that at this time there is growth of NaCl crystals, at least partially. This stage is preferably carried out for 4 hours. After that, the temperature of lyophilizate reduced to about -50°C., preferably for about 1 hour to reduce the temperature of the drug.

In subsequent stages of the freeze-drying temperature changes carried out with a speed of between about 0.1°C./min and 0.5°C/minute. After reducing the pressure in liofilizadora to about 65 mtorr is the temperature increase to a temperature between from about -32°C to -35°C for primary drying. At this temperature ice crystals in the product will sublimate (wagonette). This stage is carried out for up to 100 hours, or until, until most of the ice sublimates from the drug. The point at which much of the ice sublimated, can be determined, for example, by using a dew point indicator, which indicates the end of the sublimation of ice, when the indications disappear (the inflection point).

After primary drying, the temperature was raised to +40°C, preferably at a rate of 0.2°C/minute to start secondary drying for further removal of water from the drug. This temperature is preferably maintained for about three hours. For this first stage is followed by the second and third stages of secondary drying, during which the temperature was raised to about +45°C for approximately three hours, and then to about +50°C for three hours to remove moisture in liofilizirovannom cake to less than 2% (weight/weight).

Example 4

Conducted additional studies to test specific steps of histidine on liofilizovannye composition of factor VIII containing glycine or mannitol as fillers. Irreversible heat flow (modulated DSC, mdsc) was used to detect crystallization of fillers during cooling. The endotherm Cree is tallization was defined as the crystallization temperature, and the total heat of crystallization. The appearance of endothermy NaCl eutectic melt during heating was used for the discovery of crystallization of NaCl. When mdsc determined the degree of crystallization, as the ratio of the enthalpy of fusion of the composition to the enthalpy of fusion of pure NaCl solution using the total heat flow signal. In addition, conducted x-ray diffraction analysis to determine the degree of crystallization liofilizovannyh compositions.

While the concentration of histidine is less than 20 mm had no significant influence on the crystallization of glycine, 50 mm histidine reduced the degree of crystallization of glycine. Ectothermy crystallization finely ground NaCl was not observed during the cooling compositions containing glycine. However, endothermy eutectic melting during heating indicated that NaCl crystallized (>50%) after cooling below -50°C and annealing at -30°C, -35°C and -40°C. the Inclusion of 50 mm histidine in containing glycine composition was slow crystallization of NaCl. Therefore, for these compositions, the annealing time was increased to 3 times to achieve the equivalent of crystallinity.

However, the effect of 20 mm histidine on crystallization of NaCl in containing glycine structures was minimal. Studies of freeze drying buckling of liofilizirovannogo briquette was observed VI is wlno in containing glycine compounds, comprising 50 mm histidine. These x-ray diffraction studies of the powders indicated a decrease in the crystallinity of NaCl in the samples containing histidine. In containing mannitol formulations usually 83-90% sodium chloride crystallized during cooling to a temperature of between -40°C and -50°C without the need for annealing. While the inclusion of 20 mm histidine in the composition inhibited the crystallization of NaCl during cooling, annealing resulted in approximately 40%of NaCl crystallization.

Therefore, compositions containing capable of crystallization filler, such as glycine or mannitol, and NaCl, the inclusion of histidine can reduce Stepney crystallization of NaCl. Although in some cases this can lead to buckling of the briquette, which is formed during lyophilization, the use of relatively low concentrations of histidine in such compositions may weaken this effect. However, suitable briquettes were obtained at concentrations of histidine 35 mm and 50 mm. Histidine may also be preferred before HEPES as buffer compositions on the basis of mannitol and glycine, as it has been observed that the use of HEPES lowers Tg' to a greater extent than a similar amount of histidine.

Example 5

The physical characteristics of a number of potential compositions of factor VIII, including seven display is the R stabilizers and five fillers, was evaluated in another study. In addition to the filler and stabilizer all the compositions listed below in Table 8 (with the exception of part 11)contained 10 mm Tris· HCl, 200 mm NaCI, 0.02% of Tween-80, 4 mm CaCl2and had mm CaCl2also at pH 7.0. All pH measurements were carried out at ambient temperature.

Table 8

Identification number sampleFillerThe stabilizer protein
18% mannitol2% sucrose
28% mannitol2% trehalose
38% mannitol2% raffinose
48% mannitol2% arginine
58% mannitol2% lysine
68% mannitol2% sorbitol
78% mannitol2% glycerol
84% of hydroxyethylamine2% sucrose
98% glycine2% sucrose
108% glycine2% trehalose
11400 mm NaCl2% sucrose
128% alanine2% sucrose

Temperature measurement buckling using sublimation microscopy and measurement of thermal transitions using DSC was used to predict behavior when blow-drying or freezing. Also used DSC, x-ray powder diffraction and polarized light microscopy to determine the crystallinity liofilizovannyh powders. Also estimated the time of reconstitution, and the appearance of the samples. The results of all these measurements are summarized below in table 9.

Table 9

Sample

No.
TRS

(C)
Tc

(C)
Tg(C)Restore moisture (seconds)Water content (%)Appearance
1-14-105464n/sgraceful
2-20-1553621,4partial loss of stability on top
3-15-1054771,7graceful
4-----partial loss condition is ascioti
5-----buckling
6n/cn/c<10°C*630,6graceful
7--<10°C*--graceful
8--86490,7fine, but compressed from the sides
9--54220,8graceful
10--6318-graceful
11--66110,4fine (the layer at the bottom)
12---570,5graceful

*Sorbitol and glycerin are transitions vitrification at <10°C. the DSC scan range does not include the temperature in this interval;

n/C=not clear;

TRS=Temperature at which there is a partial loss of stability under freeze microscope;

the with=Temperature at which there is a General loss of stability under freeze microscope;

Tg=the glass transition temperature.

With the exception of the composition with the use of mannitol:lysine was found that all other cars have adequate physical appearance. Lysine prevented crystallization in combination with mannitol and glycine, which caused the decrease of glass transition temperature and the loss of stability of liofilizirovannogo briquette.

Example 6

The composition of factor VIII described in table 8 above, were placed in storage at -70°C, 25°C, 40°C and 50°C at different time intervals to evaluate their stability. The activity levels of factor VIII were evaluated at 2 weeks, 1 month, 2 months and 3 months, and the results are summarized below in table 10. Two samples, one using mannitol as filler and sorbitol as a stabilizer, and the other using mannitol as filler and glycerol as stabilizer, showed low stability. All the remaining compounds have demonstrated the ability to stabilize factor VIII.

Table 10

Composition descriptionTemperature (°C)% of the original through the number of months

< / br>  
00,5123
Glycine:Sucrose-70100,0097,43101,7199.89 perthe 97.97
25100,00



85,44
40100,0079,8771,5263,06
50100,0076,3467,9952,1447,64
Glycine:Trehalose-70100,0089,2296,0095,9094,64
25100,00



83,17
40100,0079,9372,4268,03
50100,0080,9764,2857,6050,92
Mannitol:Trehalose-70100,0091,3297,7296,1098,26
25100,00



85,79
40100,0082,5470,7259,44
50100,0066,1665,5148,8152,06
Mannitol:Sucrose-70100,00100,45100,56105,4799,22
25100,00



87,04
40100,0085,5980,78to 55.42
50100,0081,6875,5357,8843,46
Mannitol:Arginine-70100,00102,26105,53103,72105,08
25100,00



95,15
40100,0091,5380,9369,19
50100,0082,2868,0656,3245,94
Mannitol:Raffinose-70100,0093,8898,41100,68103,62
25100,00



83,13
40100,0081,0973,6167,16
50100,0071,6968,5254,2547,11
Mannitol:Glycerin-70100,00







25100,00







40100,00







50 100,00





Mannitol:Sorbitol-70100,00104,06





25100,00







40100,00







50100,0032,73



HES:Sucrose-70100,00102,74103,03100,90


25100,00







40100/0076,8977,47


50100,0071,4767,4030,02
NaCl:Sucrose-70100,0088,5488,4495,58


25100,00







40100,0071,5658,30


50100,0052,7137,9030,34
Alanine:Sucrose-70100,00109,78109,67108,96


25100,00







40100,0092,9973,03


50100,0083,2574,9157,65
Glycine:the RAF is inosa -70100,00111,57114,51105,25


25100,00







40100,0089,2082,10


50100,0093,2172,2253,24

Example 7

On the basis of information obtained during the studies described in examples 5 and 6, it was decided that a candidate for further development will serve compositions with excipients, are presented below in table 11.

Table 11

ExcipientConcentration
Mannitol or glycine6-9%
Arginine or trehalose1-3%
Tween 80of 0.005 to 0.04%
NaCl200-250 mm
CaCl23-5 mm
TRIS20-30 mm
Histidine or HEPES10-50 mm
Glutathione of 0.15-0.25 mg/ml

On the basis of these parameters were developed following specific compounds:

Composition No. 1Composition No. 2Composition No. 3
10 mm HEPES10 mm HEPES10 mm HEPES
20 mm Tris20 mm Tris20 mm Tris
225 mm NaCl225 mm NaCl225 mm NaCl
0,03% (V/V) Tween-800,03% (V/V) Tween-800,03% (V/V) Tween-80
8% (wt/V) mannitol8% (wt/V) glycine8% (wt/V) mannitol
2% (wt/V) trehalose2% (wt/V) trehalose2% (wt/V) arginine
0.2 mg/ml limited glutathione0.2 mg/ml limited glutathione0.2 mg/ml limited glutathione
4 mm CaCl24 mm CaCl24 mm CaCl2

Composition No. 4Composition No. 5
25 mm histidine25 mm histidine
20 mm Tris20 mm Tris
225 mm NaCl225 mm NaCl
0,03% (V/V) Tween-800,03% (V/V) Tween-80
8% (in the C/V) mannitol 8% (wt/V) glycine
2% (wt/V) trehalose2% (wt/V) trehalose
0.2 mg/ml limited glutathione0.2 mg/ml limited glutathione
4 mm CaCl24 mm CaCl2

1. The composition of factor VIII made without the addition of albumin to the specified composition, including the following excipients of the formulation in addition to factor VIII: 4 - 10% of a filler selected from the group consisting of mannitol, glycine and alanine; 1% to 4% of a stabilizing agent selected from the group consisting of sucrose, trehalose, raffinose, and arginine; 1 to 5 mm calcium salt; 100 - 300 mm NaCl, and a buffering agent to maintain pH in the range of approximately 6 - 8.

2. The composition of factor VIII according to claim 1, further comprising a surfactant.

3. The composition of factor VIII according to claim 2, where the aforementioned surface-active substance selected from the group consisting of Polysorbate 20, Polysorbate 80, Pluronic F68 or Brij35.

4. The composition of factor VIII according to claim 3, where the aforementioned surfactant is a Polysorbate 80 and where specified Polysorbate 80 is present in amount of less than 0.1%.

5. The composition of factor VIII according to any one of claims 1 to 4, where the aforementioned surfactant is present in an amount of about 0.03 percent.

6. Composition the fact the RA VIII according to any one of claims 1 to 5, where the specified buffer agent selected from the group consisting of Tris, BIS-Tris Propane, histidine, PIPES, MOPS, HEPES, MES and ACES.

7. The composition of factor VIII according to claim 6, where the specified buffer agent is a Tris.

8. The composition of factor VIII according to claim 7, where Tris is present in an amount of about 20 mm.

9. The composition of factor VIII according to claim 6, where the specified buffer agent includes from about 10 to 50 mm histidine.

10. The composition of factor VIII according to claim 9, where the histidine is present in amount of about 25 mm.

11. The composition of factor VIII according to any one of claims 1 to 10, further comprising an antioxidant.

12. The composition of factor VIII according to claim 11, where the specified antioxidant is a glutathione.

13. The composition of factor VIII indicated in paragraph 12, where the specified glutathione is present in an amount of from about 0.05 to 1.0 mg/ml

14. The composition of factor VIII according to any one of claims 1 to 13, where the specified filler is present in an amount of about 8%.

15. The composition of factor VIII according to any one of claims 1 to 14, where the specified filler is a mannitol.

16. The composition of factor VIII according to any one of claims 1 to 14, where the specified filler is a glycine.

17. The composition of factor VIII according to any one of claims 1 to 16, where the specified stabilizing agent is present in an amount of about 2%.

18. The composition of factor VIII according to any one of claims 1 to 17, where the specified stabilizing agent is with the battle sucrose.

19. The composition of factor VIII according to any one of claims 1 to 17, where the specified stabilizing agent is an arginine.

20. The composition of factor VIII according to any one of claims 1 to 17, where the specified stabilizing agent is a trehalose.

21. The composition of factor VIII according to any one of claims 1 to 20, where specified NaCl is present in an amount of about 200 mm to 250 mm.

22. The composition of factor VIII according to item 21, where the specified NaCl is present in an amount of about 225 mm.

23. The composition of factor VIII according to any one of claims 1 to 22, where the specified salt of calcium is the calcium chloride.

24. The composition of factor VIII according to any one of claims 1 to 23, where this composition is in liofilizovannyh form.

25. The composition of factor VIII made without the addition of albumin to the specified composition, including the following excipients of the formulation in addition to factor VIII: 2 - 6% of hydroxyethylamine; 1 - 4% of a stabilizing agent selected from the group consisting of sucrose, trehalose, raffinose, and arginine; 1 to 5 mm calcium salt; 100 - 300 mm NaCl, and a buffering agent to maintain pH in the range of approximately 6 - 8.

26. The composition of factor VIII by A.25, comprising approximately 4% of hydroxyethylamine.

27. The composition of factor VIII according to any one of p and 26, which includes about 200 mm NaCl.

28. The composition of factor VIII according to any one of p-27, where the specified stabilizing agent is present in the count is the amount of approximately 2%.

29. The composition of factor VIII according to any one of PP-28, where the specified stabilizing agent is a sucrose.

30. The composition of factor VIII according to any one of PP-28, where the specified stabilizing agent is an arginine.

31. The composition of factor VIII according to any one of PP-28, where the specified stabilizing agent is a trehalose.

32. The composition of factor VIII made without the addition of albumin to the specified composition, including the following excipients of the formulation in addition to factor VIII: 300 - 500 mm NaCl; 1 - 48% of a stabilizing agent selected from the group consisting of sucrose, trehalose, raffinose, and arginine; 1 to 5 mm calcium salt; a buffering agent to maintain pH in the range of approximately 6 - 8.

33. The composition according to p, where NaCl is present in an amount of about 400 mm.

34. The composition of factor VIII according to any one of claims 1 to 33, for the treatment of hemophilia.

35. The method of freeze-drying aqueous compositions of factor VIII containing kristallizuetsya filler and NaCl, where the method involves the following stages:

(a) freezing the aqueous pharmaceutical composition at a temperature of less than about -35°C;

(b) annealing the pharmaceutical composition at a temperature in the range of between about -30°and -19°C;

(C) lowering the temperature of the pharmaceutical composition to less than about -50°C;

(d annealing the pharmaceutical composition at a temperature in the range of between about -30° C and -39°; and then

(e) freeze-drying of pharmaceutical composition.

Priority items:

22.02.1999 - according to claims 1-7, 11, 12, 14-35;

01.12.1999 - PP, 9, 10, 13.



 

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