Pharmaceutical formulations of nerve growth factor

The present invention relates to pharmaceutical compositions for nerve growth. The present invention relates to compositions of nerve growth factor for lyophilization.

factor as well

Cell growth and viability are regulated by an abundance of polypeptides and proteins; such molecules are called growth factors. Examples of growth factors include epidermal growth factor (EGF), acidic and alkaline fibroblast growth factor (aFGF and bFGF), platelet derived growth factor (PDGF), cilia neurotrophic factor (CNTF), and nerve growth factor (NGF). NGF is the first identified and characterized of these factors (LeviMontalcini, R., et al., J. Exp. Zool., 116: 321, 1951).

NGF promotes the survival and activity of some types of neuronal cells. In addition, NGF accelerates the differentiation of immature neuronal cells into pomitotic mature neurons.

Purification of NGF in the mouse gland is completed by the identification of a complex consisting of three subunits, α, β, and γ. All neurotrophic activity of NGF is believed to depend on the β subunit consisting of a protein of 118 amino acids having a molecular weight of approximately 13,000 Da (Varon, S., et al., Proc. Natl. Acad. Sci.

USA, 57: 1782-1789, 1967; Green, LA, et al., Neurobiol., 1: 37-48, 1971). The β subunits in the solution form dimers with a molecular weight of approximately 26,500 Da.

NGF has been shown to be effective in the treatment of some degenerative disorders of the peripheral and central nervous system. NGF administration has been shown to be useful in the treatment of disorders in which NGF deficiency, its receptor pathology, or changes in its delivery and intracellular processing result in a decrease in neuronal function, atrophy or even cell death. Such diseases include hereditary sensory and motor neuropathies, hereditary and sporadic degradation of the body, atrophic lateral sclerosis, Parkinson's and Alzheimer's disease (Goedert, M., et al., Mol, Brain Res., 1: 85-92, 1986; Mobley, WC, et al., Soc. Neurosci. Abstr., 13: 186, 1987; Mobley, WC, et al., Soc. Neurosci. Abstr., 4: 302, 1988; Hefti, F., et al., Ann. .Nurol .. 20: 275-281, 1986). NGF has been shown to reduce the probability of cell death following some toxins, such as 6-hydroxy-dopamine (Aloe, L., Arch. Ital. Biol. 113: 326-353, 1975), vinblastine and colchicine (Monthly-Chen). , MG, et al., Proc. Natl. Acad. Sci. USA, 74: 5559-5563, 1977; Jonson, EM, Brain Res., 141: 105-118, 1978) and capsaicin (Otten, U., Nature, 1978). , 301: 515-577, 1983).

High expression of NGF mRNA in the hippocampus, an area associated with memory and erudition suggests that clinical use of NGF could be effective in the treatment of dementia (Kaisho, Y., et al., Biochem. Biophys. Res. Com, 174: 379). -385, 1991). Intraventricular administration of NGF is known to prevent the death of anterior brain basal cholinergic neurons after axotomy, suggesting that NGF may be effective in enhancing cell viability after injury (Hefti, F., J. Neurosci .. 6: 2155-2162, 1986; Williams, L., et al., Proc. Natl. Acad. Sci. USA, 83: 9231-9235, 1986; Kromer, L., Science, 235: 214-216, 1987).

The use of NGF in therapy poses major challenges. This involves: 1) maintaining the bioactivity of NGF, which can change during preparation, purification, and storage; 2) introducing NGF as a relatively large hydrophilic molecule such that the amount of this molecule capable of reaching the active centers is effective. Like other proteins, the bioactivity of NGF depends on their secondary and tertiary structure. The β subunit of NGF has three internal disulfide bonds important for bioactivity (Kanaya, E., et al., Gene, 83: 65-74, 1989; Iwane, M., et al., Biochem. Biophys. Res. Comm., 171: 116-122, 1990; Hu, G.-L. and

Neet, KE, Gene, 70: 57-65, 1988). In addition, if any protein is denatured, the amount of biologically active NGF is lower. Therefore, the integrity of the protein during preparation and storage should be maintained in the same manner as during administration. Proteins are particularly prone to degradation at higher temperatures.

Lower temperatures usually · reduce protein breakdown. However, it is more economical to store protein at room temperature, such as in the refrigerator, than to approx.

25 ° C

At 4 ° C. Therefore, it is desirable that the composition be room-stable when stored at temperature.

In addition, NGF, like most other proteins, attaches to surfaces non-specifically, contributing to stability problems. Such nonspecific attachment can occur to many materials, including glass and plastics. such as polyethylene and polypropylene. These materials may be in the form of vials, tubes, syringes, implantable infusion sets, or any other surface that may come into contact with NGF during preparation, storage, or administration.

Other difficulties in administering proteins such as NGF for therapeutic use include poor absorption in the body and degradation by the action of gastric acids. Therefore, oral administration is generally inappropriate.

Injections and infusions of such proteins may be unavoidable to overcome the absorption barriers.

Injection is effective when the site is treated lightly

However, if a site such as the CNS is available, continuous infusion is more convenient for prolonged administration. Such prescribing can be impractical due to various complications.

For example, continuous infusion can be achieved by implanting NGF pumps into the brain, but prolonged storage of the protein at body temperature often results in protein. degradation. Also, additional losses can be or about 4^JC is attainable. relatively inaccessible, may be due to protein adsorption to the pump chamber over time.

In addition to the problems associated with prescribing NGF, there are new problems associated with the long shelf life of NGF from its production to appointment. Lyophilization is one way of long-term storage of biological proteins by stopping their degradation, aggregation and / or nonspecific adsorption. However, the lyophilization process itself also presents difficulties. As the volume decreases during refrigeration, the effective salt concentration increases dramatically, which can denature the protein, reducing effective therapeutic activity even before protein recovery. In addition, the formation of ice crystals during freezing may lead to denaturation and also reduce the effective amount of bioactive NGF obtained. In this case, the composition should be such as to prevent fluctuations in salt concentration and to minimize the formation of ice crystals.

One object of the present invention is to provide aqueous formulations of NGF that retain bioactivity for at least one month at a temperature in the range of 4-40 ° C.

Another object of the present invention is to provide NGF compositions wherein the bioactivity is retained after lyophilization and reconstitution. Yet another object of the present invention is to find ways to maintain biologically active NGF in solution for a long time.

The present invention relates to stable compositions of nerve growth factor which can be stored at lower than ambient, ambient and elevated temperatures without significant loss of protein content or activity. The compositions are aqueous solutions comprising:

(a) nerve growth factor;

(b) a biologically acceptable, water-soluble carrier (optional);

(c) a sufficient amount of the biologically acceptable salt to maintain isotonicity;

(d) a buffer to maintain the pH of the composition in the range of about 4.5 to about 6.0; (e) water.

In another aspect, the present invention provides NGF pharmaceutical compositions suitable for lyophilization.

The following pharmaceutical compositions suitable for lyophilization are aqueous solutions consisting of:

(a) nerve growth factor;

(b) a biologically acceptable excipient;

(c) a buffer maintaining the pH of the composition in the range of about 5.5 to about 6.5;

(d) a biologically acceptable water soluble carrier (optional);

(e) water.

water. These are reversible biologically

Further embodiments of the present invention are lyophilized compositions from which the lyophilized compositions have been substantially removed by reconstitution with a solvent optionally containing

As an acceptable carrier, they are suitable for patients in need of therapy.

The present invention also provides a method of storing NGF in aqueous compositions at temperatures in the range of 4-40 ° C.

Another aspect of the present invention is the use of a NGF composition of the present invention for the treatment of a neuronal dysfunction in a human in a therapeutically effective amount.

The development of stable parenteral dosage forms of NGF requires a multitude of factors to be considered, including the route of administration, adsorption interactions, compatibility of manufacturing facilities and potential delivery devices. Further consideration is given to the stability of NGF in aqueous compositions at lower, ambient and elevated temperatures. One embodiment of the present invention is NGF about a composition in aqueous solution which, over a range of temperatures, especially at higher

40 ° C) solution, salt and buffer at approx

4.5-6.0. Additionally, the composition may have a carrier. This combination of components unexpectedly gives the solution very good performance, especially when it comes to stability at higher temperatures. Also provided is a composition of NGF suitable for lyophilization. The present invention also provides a method of storing NGF.

I

The term "biologically acceptable" is used herein for substances which are characterized as having no harmful physiological effects in vivo. The room temperature is approximately 22-25 ° C. The body temperature is approximately 3640 ° C. The term lyophilizable composition refers to an aqueous composition of NGF that can be freeze-dried to a moisture content of up to 2% and retains at least 70% of the initial bioactivity of NGF after reconstitution. The term isotonic refers to a solution having approximately the same osmotic pressure as serum, that is, approximately 300 mM / Ι. The carrier is a biologically acceptable emulsifier, dispersant, surfactant, or protein that reduces NGF adsorption to the surface.

NGF denotes any form of nerve growth factor, with preference given to the β-subunit of nerve growth factor, which exhibits biological activity and binds to the NGF receptor. The term NGF also encompasses hybridized and modified forms of NGF that bind to the NGF receptor and retain the bioactivity of NGF. Modified forms of NGF include the fusion proteins described in Iwai, S., et al., Chem. Pharm. Bull., 34: 47244730, 1986, and Kanaya, E., et al., Gene, 83: 65-74, 1989, as well as NGF fragments and hybrids in which some amino acids are deleted or substituted but retain sufficient bioactivity of NGF. receptor binding, conferring therapeutic activity.

Human NGF (hNGF) is the preferred form of NGF.

The most preferred form of hNGF is recombinant hNGF (rhNGF).

Methods for preparing NGF suitable for use in the compositions of the present invention are known to those skilled in the art. For example, the corresponding rhNGF may be system-assisted 110: 11-24, 1990;

produced by expression of baculovirus (Barnett, J., et al., Exp. Neurol., EPO 370, 171), a yeast expression system (Kanaya, E., et al., Gene, 83: 65-74).

1989), mammalian cell (CHO) expression system (Iwane, M., et al., Biochem. Biophys. Res. Comm., 171: 116-122, 1990) or COS expression system (Bruce, G., et. et al., Neurobiol. Aguing. 10: 89-94, 1989).

NGF must be at least 65% pure; preferably at least 85% purity; preferably at least 95% pure; and most preferably at least 98% pure. The degree of purity of the isolated NGF used in the compositions can be determined by silver-coated SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) or other methods known in the art.

Aqueous compositions provided contain a therapeutically effective amount of NGF. Preferably, the NGF is from about 0.0001 to about 0.125% by weight of the aqueous composition, which corresponds to about 1 to about 1250 µg / ml. Preferably, the NGF is from about 0.001% to about 0.1% w / w (10-1000 µg / ml). Even more preferably, the NGF is from about 0.01 to about 0.10% w / w (100 to 1000 µg / ml). Preferably, the NGF is about 0.010.05% (100-500 µg / ml).

Aqueous formulations of NGF may contain carriers. The presence of the carrier in the composition reduces or hinders the adsorption of NGF on various surfaces. The carrier requirement depends on the concentration of NGF in the aqueous composition. At sufficiently high concentrations of NGF (greater than about 500 µg / ml), enough NGF remains in solution to compensate for the amount lost due to surface adsorption. Suitable carriers include, but are not limited to, polysorbates such as Tween® 80, poloxamers such as Pluronic F68, and proteins such as serum albumin. The preferred carrier is

Protein I. Human serum albumin (HSA) is particularly suitable. The weight ratio of NGF to carrier is in the range of about 0.0001: 1 to 1: 1. A more suitable weight ratio is in the range of about 0.01: 1 to 1: 1. The most suitable ratio of NGF to carrier is in the range of about 0.01: 10.5: 1. When used as a carrier, HSA is conveniently present at a concentration of about 0.1-1.25% by weight of the aqueous composition (i.e., 1-12.5 mg / ml). More preferably, the composition will contain about 0.3-0.7% HSA by weight of the aqueous composition, more preferably about 0.4-0.6% by weight of the aqueous composition. Most preferably, the composition contains about 0.5% (i.e., 5 mg / ml) HSA by weight of the aqueous composition.

The NGF composition also contains a sufficient amount of a biologically acceptable salt to maintain the tonicity of the fluid. The salt also maintains NGF in solution. Preferably, the NGF composition has sufficient salt to maintain isotonicity within physiologically acceptable limits relative to human blood or spinal fluid. Sodium chloride (NaCl) is the preferred salt, although other biologically acceptable salts such as potassium chloride (KCl), calcium chloride (CaCl₂) and magnesium chloride (MgCl₂). The salt may be in the form of a single salt or a mixture of salts having from about 0.5% to about 1.0% salt by weight of the aqueous composition. More preferably, the composition will contain from about 0.6% to about 0.9% salt by weight of the composition. An even more suitable composition has 0.7-0.9% salt by weight of aqueous solution. The optimum composition is approximately 0.87% (ie 8.7 mg / ml) of salt.

In addition, the NGF formulation has a biologically acceptable buffer to maintain an appropriate pH. NGF was found to be more stable at low pH. A suitable stable formulation of NGF in the form of a biologically acceptable buffer is suitable. Suitable (i.e., 5-10 mg / ml) compositions will have a pH of about 4.5 to about 6.0, preferably about pH 5.05.4. The optimal pH of the composition is about 5.2. The preferred buffer is citric acid as well as other buffers capable of maintaining the pH within the desired range. Other suitable buffers include acetic acid / acetate and maleic acid / maleate. The desired amount of buffer depends on the type of buffer used and its buffer capacity. The composition should contain sufficient buffer to maintain the final pH of the composition within the desired range. A suitable concentration of buffer for stable NGF compositions is from about 0.01 to about 0.3% by weight of the aqueous composition (0.1 to 3.0 mg / ml), more preferably from about 0.1 to about 0.25% by weight of the aqueous composition of buffer. (1.0-2.5 mg / ml), the most appropriate buffer concentration is about 0.2% by weight of the aqueous formulation (2.0 mg / ml).

The composition shall contain a quantity of water sufficient to obtain an appropriate concentration of the components.

Suitable aqueous stable formulations of NGF contain from about 1 to about 1250 µg / ml NGF, from 1 to 12.5 mg / ml HSA, from 5 to 10 mg / ml NaCl, from 0.2 to 3.0 mg / ml citric acid and water, and the pH of the composition is set to be in the range of 4.5 to 6.0, more preferably in the range of 5.05.4. Most suitable stable formulations of NGF contain 10-500 mg / ml NGF, 5 mg / ml HSA, 8.7 mg / ml NaCl, 2.5 mg / ml citric acid and water, and the composition has a pH of approx.

5,2.

The lyophilized compositions of the present invention are particularly useful for providing a long shelf life, especially at elevated temperatures. These lyophilized compositions contain NGF, a biologically acceptable excipient, a buffer that maintains the pH of the composition in the range of about 5.5-6.5, an optionally biologically acceptable salt, an optionally biologically acceptable water soluble carrier, and water.

In lyophilized formulations, NGF is used in the same concentrations as aqueous formulations. The filler usually provides mechanical support to assist the matrix in maintaining the composition structure during and after freeze-drying. One or more sugars may be used as fillers. The sugars used herein include, but are not limited to, monosaccharides, oligosaccharides, and polysaccharides. Examples of suitable sugars include, but are not limited to, fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, and dextran. Sugar also includes sugar alcohols such as mannitol, sorbitol, inositol, dulcitol, xylitol, and arabitol. Mixtures of sugars may be used in accordance with the present invention.

A suitable filler comprises a sugar composition. Such an agent is a combination of sucrose and mannitol. Without being bound by theory, sucrose is believed to form an amorphous glass upon freezing followed by lyophilization, which provides an increase in the potential stability of the protein (e.g., protection against aggregation) by the molecular dispersion of NGF in solid glass. Stability can also be enhanced by sugar, which acts as a substitute for water lost during lyophilization. Sugar molecules bind to protein more rapidly than water molecules through hydrogen bonds. Mannitol mixed with sucrose (which has a glass transition temperature of -36 ° C) in a 1: 1 weight ratio raises the glass transition temperature of the composition by 5 ° C to -31 ° C. This significantly shortens the initial drying time of the composition during lyophilization, in addition to forming an amorphous glass matrix of the composition, which is considered an advantage when produced in bulk. Other excipients having the same characteristics may be substituted for one or both of these sugars.

It is desirable that the lyophilized compositions of this invention have a higher pH than the compositions which are not lyophilized or reconstituted. The fillers (sugars) that are present in the lyophilized compositions of the invention are generally more stable at higher pH. It is good that the pH of the composition prior to lyophilization is approximately 5.5-6.5.

More preferably, the temperature of the components to be lyophilized in the NGF formulation is within the range of about 5.86.2. Most preferably, the pH of the lyophilized NGF composition is about 6.0. When the excipient is sucrose, the preferred pH of the lyophilized composition is about 6.0; at acidic pH, sucrose, an unreduced disaccharide, is hydrolyzed to reduced sugars; D-fructose and D-glucose. Citrate is the preferred buffer for lyophilization NGF compositions, but other biologically acceptable buffers such as maleate may be used. Non-acetate buffers are more suitable due to the evaporation of acetic acid during lyophilization. It should be recognized that an acid or alkaline adjustment of the final pH may be necessary. Any loss of stability of aqueous NGF over long periods due to a pH higher than about 6.0 can be balanced by the increase in stability associated with the lyophilization of NGF.

Ideally, the buffer is selected by evaluating potential pH shifts during lyophilization resulting from sequential crystallization of buffer components. For example, with phosphate buffers, the alkaline component has a higher eutectic point than the acidic component, meaning that it crystallizes first and the pH decreases. A citrate buffer is desirable, since the components are expected to have a roughly very low Others suitable with such eutectic points.

The compositions to be lyophilized may also contain a biologically acceptable salt. The salt, which may be selected from the salts used in aqueous compositions, has the same or both buffers having the same eutectic point in the lyophilizable formulation, the pH variation with decreasing buffers having the same or similar reduced concentrations as with aqueous compositions. Because the salt concentration tends to increase during lyophilization, it is desirable that the salt present in the lyophilizate be reduced to prevent denaturation of the protein in the salt to reduce denaturation in the compositions.

lyophilized compositions may be compensated by reconstituting the compositions so as to be sufficiently lenient for administration.

Preferably, the lyophilized compositions comprise a biologically acceptable water-soluble carrier. Carriers and carrier concentrations that can be used in the lyophilized compositions of the present invention are the same and the same as those suitable for use in the aqueous compositions of the present invention.

More preferred lyophilizable compositions contain about 1-1250 µg / ml NGF, 15-45 mg / ml sucrose, 15-45 mg / ml mannitol, optionally 7-9 mg / ml NaCl, and 0.1-0.7 mg / ml citric acid , at a pH of approximately 5,56,5. Preferred formulations for lyophilisation of NGF include 100-1250 µg / ml NGF, 30 mg / ml sucrose, 30 mg / ml mannitol, 5 mg / ml human serum albumin, optionally 8.7 mg / ml NaCl and 0.3 mg / ml citric acid . The preferred pH for the lyophilized compositions is about 6.0.

The compositions of the invention to be lyophilized are lyophilized to less than 2% moisture; however, compositions that retain the biological activity of NGF at higher or lower moisture levels may be formulated.

A more preferred lyophilized composition contains from 0.001 to 1.25 parts of nerve growth factor, 30 to 90 parts of sugar and less than 1 part of water.

The lyophilized NGF composition is reconstituted with a diluent containing a buffer such as citric acid and salt.

for the final composition to be suitable for an individual such as sodium chloride, since the reconstituted composition is required to be similar to a liquid aqueous formulation, i.e., containing about 1-1250 µg / ml NGF, 1-12.5 mg / ml HSA, 5-10 mg / ml NaCl, 0.2-3.0 mg / ml citric acid, 1.5-30 mg / ml sucrose and 1.5-30 mg / ml mannitol; pH 5.2.

Such a lyophilized NGF formulation can be used as a component in a kit since the lyophilized form of NGF can be quickly and easily reconstituted in an appropriate solvent and is intended for patients in need of treatment; it is convenient and cost effective. In addition to the lyophilized NGF formulation, the kits also contain a reconstituting solvent. The reconstituting solvent consists of sterile water and a sufficient amount of salt to render the final reconstituted composition isotonic. Additionally, the reconstituting solvent may have an additional buffer. The total volume of reconstituting solvent in the kit should be sufficient to provide a final concentration of NGF suitable for use by the individual in need of treatment. A preferred embodiment of the present invention is a kit comprising two vials. One vial contains a sterile lyophilized NGF formulation of the present invention and the other contains a sterile reconstitution solvent. When using the kit, an appropriate volume of reconstituting solvent is added to the vial containing the lyophilized NGF formulation. After reconstitution of the lyophilized composition, the reconstituted composition can be administered immediately to the patient.

Due to the long-term stability of the reconstituted composition, it is possible to prepare a sufficient number of doses of the reconstituted composition.

The use of the compositions of the present invention must meet the requirements of NGF therapy. Such compositions should be sterile and suitable for intravenous, intraruminal, parenteral, and intracerebroventricular administration. Such therapy may be useful in the treatment of neuronal dysfunctions, including neuronal injury or degeneration of NGF-responsive neurons. NGF can be particularly useful in treating a condition associated with the loss of central cholinergic neurons, called Alzheimer's disease. Treatment of Alzheimer's disease and other forms of dementia using NGF is described

5 * EPO 370171.

The compositions of the invention, as antipsychotics, may be administered by any one of a number of conventional routes, depending on the intended use. The most appropriate route will depend on the application and the individual being treated.

To overcome the blood-brain barrier, NGF may be administered to the central nervous system by intraventricular injection or drug-impregnated implants and pumps. Alternatively, continuous infusion through an intracerebroventricular cannula · Alternatively, conjugation of NGF to carrier molecules such as transferrin may be required to cross the blood-brain barrier.

The therapeutically effective amount of NGF is approx

0.001 to 0.5 mg per day, preferably about 0.01 to 0.10 mg per day, preferably about 0.02 to 0.06 mg per day. The exact dose and administration regimen will depend on many factors, such as the route of administration and the degree of ailment of the individual receiving treatment.

TEST PROCEDURES

Identification and quantification of NGF using high performance liquid chromatography reverse phase (RP-HPLC)

NGF was identified and quantified by analyzing 100 μΐ samples using RP-HPLC (Hewlett Packard HP 1090 Liquid Chromatograph) equipped with 4-6 mm x 25 cm L o

Dynamax (Rainin Instrument Co., Woburn, MA, USA) 300 A 5 &

µm Analytical Reversed-Phased Column with Dynamax 300 A 5 µm 4.6mmxl, 5cm Protective Column and Diode Device with UV Detection at 220 nm. Mobile phases: (A) - 0.1% trifluoroacetic acid in water and (B) - 0.1% trifluoroacetic acid in acetonitrile, where 45 min. the gradients change from 25% (B) to 60% (B) at a flow rate of 0.5 mL / min, pressure of 1700-2000 psi, ambient temperature.

The identification of NGF was made by comparing its retention time in the sample with the corresponding retention time of freshly prepared calibrated standard NGF solutions prepared from the same batch of NGF. The amount of NGF in the samples is subtracted from the standard curve obtained with serial dilutions of known concentrations.

Determination of NGF concentration (µg / ml) by ELISA

NGF concentrations were also assayed by ELISA. Standards and examples were tested three times. Each plate had a NGF full standard curve and control samples without NGF.

After 100 μΐ of the adsorbed antibody (murine monoclonal 24C1 induced against rhNGF) was added to each well of a 96-well plate, the plates were covered with a Saran damp tissue paper towel and incubated overnight at 2-8 ° C. The wells are aspirated, washed three times using a Wheaton self-priming syringe to serve 250 μΙ / well wash buffer (containing 500 mM TRIS, 2 M sodium chloride buffered at pH 7) and shaken dry. Then, 200 μΐ blocking buffer (1% bovine serum albumin solution) is added to each well to block non-specific areas, 50 μΐ sample is added to each well and the plates are incubated at room temperature for 1 hour with shaking on a platform shaker. Replace the wells and shake dry, adding 50 μΐ of the standard and sample solutions. The plates are covered and incubated for two hours at room temperature. Drain the plate wells, rinse four times with buffer and shake dry. Add 50 μΐ of biotin-treated antibody (murine monoclonal 8C1 induced by rhNGF) to each well, cover the plates and incubate for two hours. Wash, wash and dry the wells of plates as described above and then add 50 μΐ of streptavidin-conjugated horseradish peroxidase to each well. Cover the plates for 20 minutes. incubate at room temperature with mixing on a platform shaker. Wash the plates five times with wash buffer. 50 μΐ of ortho-phenylenediamine (OPD) substrate buffer is then added to each well and the plates are covered for 1 hour. incubated in the dark.

A Vmax kinetic microplate (Molecular Devices, Mountain View) counter is used to determine the absorbance of each well. To determine the true absorbance, the background absorbance at 650 nm is subtracted from each well at 450 nm. The concentration of NGF in the samples is determined by reference to the standard curve of NGF.

Determination of NGF activity

The bioactivity of NGF is determined by the PC-12 bioassay. The essence of the PC-12 bioassay is the increase in the activity of PC-12 pheochromocytoma cells (Green, Trends Neurosci. 7:91, 1986) after NGF exposure. The metabolic activity of PC-12 cells was measured by expression of 3 - [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (Ci₈Hi₆N₅Br) (MTT) which is converted into insoluble intracellular blue crystals by cellular dehydrogenase.

Each well of a 96-well plate is filled with 30,000

PC-12 cells in 50 μΐ RPMI-1640 medium (Sigma).

Serial dilutions of each sample and standard are made to obtain 0.006-400 ng / ml rhNGF solutions prepared in RPMI-1640 medium containing 0.2% bovine serum albumin (BSA). Then 50 μΐ of each solution (0.003-200 ng / ml NGF concentration) is added to each well and each sample is tested three times. Thermostatic plates for 2 days at 5% CO₂ at 37 ° C, add 10 µg MTT to each well and incubate the plates for 4 hours. Subsequently, 1 volume of 20% SDS (sodium dodecyl sulfate) in 50% dimethylformamide (DMF), pH 4.7 is added, the plates are covered with cellophane and incubated hermetically overnight at temperature.

I sealed plastic tested the next day,

37 ° C in containers. Plates using a plate Vmax counter at 575 nm. Sample curves ED₅₀ratio to ED of standard NGF curve₅₀ shows the relative potency of the two formulations.

example

NGF Composition

Aqueous formulations containing 1.10,100 µl / 100 µg / ml rhNGF, 5 mg / ml HSA, 8.7 mg / ml sodium chloride, 2.1 mg / ml citric acid and water are prepared to give 10 ml volumes of buffer at pH 5.2. Citric acid and salt are dissolved in approximately 70% of the total volume of the composition, pH adjusted with NaOH / HCl, HSA and NGF are dissolved by gentle mixing and making up to volume; the formulation is filtered through a 0.2 μ Millipore Millex-GV filter.

rhNGF used for the preparation of the compositions is expressed in insect cells using a baculovirus expression vector and purified by ion exchange and reverse phase chromatography as described by Barnett, J., et al., Exp. Neurol., 110: 11-24, 1990.

example z

Stability of NGF formulations at 5 ° C and 25 ° C

250 Samples of the μΐ volume of the NGF formulation of Example 1 (100 µg / ml) were stored at 5 ° C and 25 ° C (RT) in polyethylene drop-shaped vials for up to 6 months. Analysis of these samples using RP-HPLC, ELISA and bioassay showed no protein loss after 6 months (Table 1).

table

Stability of NGF at 5 ° C and 25 ° C using RPHPLC, ELISA and PC-12 bioassay

<td>Storage tejnp ('c)</td><td>RP-HPLC 'AR% lsav.</td><td>RP-HPLC * AR% 2 weeks</td><td>RP-HPLC * AR% 3 months</td><td>RP-HPLC • 'AR% 4 months</td><td>RP-HPLC 'AR% 6months</td><td>ELISA 'AR% 6months</td><td>Biotestae Relative activity% 6 months</td>

<td> 5^UC</td><td> 9912</td><td> 9916</td><td> 9614</td><td> 10013</td><td> 10113</td><td> 10016</td><td> 106114</td>

<td>RT (22- 25 ° C)</td><td> 9912</td><td> 9115</td><td> 9515</td><td> 10313</td><td> 9915</td><td> 104113</td><td> 12313</td>

* AR% = Resistance Rate% = Sample [NGF] Control [NGF]

% Relative Activity = Sample Activity Control Activity (RP-HPLC data represents mean 1 to 4 replicates standard deviation. Bioassay data represents mean within 95% confidence limit of 3 determinations.)

Stability of various NGF formulations stored in polyethylene catheters at 37 ° C.

250 Samples of μΐ volume NGF compositions obtained as described in Example 1 and containing 1-1000 µg / ml rhNGF were stored in X-ray impermeable polyethylene catheters (0.076 cm internal diameter, 0.122 cm external diameter) at 37 ° C for 4 weeks. As shown in Table 2, there was no loss of either more significant protein (using RP-HPLC) or more significant NGF activity (using the PC-12 bioassay).

2 table

Stability of NGF formulations stored in polyethylene catheters at 37 ° C by RP-HPLC and PC-12 bioassay.

<td>Initial</td><td>AR%</td><td>* AR%</td><td>* AR%</td><td>Relative</td>

<td>[NGF]</td><td>1 week</td><td rowspan="2">2 weeks RP-HPLC</td><td>4 weeks</td><td>activity% 4 weeks</td>

<td>(Hg / ml)</td><td>RP-HPLC</td><td>RP-HPLC</td><td>PC-12 bioassay</td>

<td> 1,0</td><td> 10413</td><td> 8716</td><td> 8513</td><td> 101134</td>

<td rowspan="2"> 10,0 100,0 1000,0</td><td> 94114</td><td> 9214</td><td> 10011</td><td> 118145</td>

<td> 10214</td><td> 9516</td><td> 9512</td><td> 129134</td>

<td></td><td> 10712</td><td> 9214</td><td> 8613</td><td> 108133</td>

* AR% = Resistance Rate% - Sample [NGF] Control [NGF]

% Relative Activity = Sample Activity Control Activity (RP-HPLC data means mean ± 2-4 replicate standard deviation. Bioassay data represents mean ± 95% confidence limits of 3 determinations.)

Stability of NGF composition in various devices

100 The composition samples of 1 µg / ml are filled with Infusaid 'Model 600 Implantable Infusion Pump (Shiley-Infusaid Ine., Norwood, MA), Medtronics Synchromed Implantable Infusion Pump (Medtronic Ine., Minneapolis, MN) and Alzet Model 2ML 4 miniosmotic infusion pump. (Alza Corp., Palo Alto, CA). The pumps are placed in a 37 ° C water bath and circulation of the composition begins. Weekly samples are collected for 4 weeks and analyzed for protein content and activity using RP-HPLC, ELISA and PC-12 bioassay.

the data in the Table do not show a significant decrease in NGF concentration and activity.

table

Stability of NGF formulations when stored in various devices at 37 ° C for 1 month

<td>Supply</td><td>'AR%</td><td>AR%</td><td>'AR%</td><td>* AR%</td><td>Relative</td>

<td>system</td><td> 1</td><td> 2</td><td> 4</td><td> 4</td><td>activity% 4</td>

<td></td><td>week</td><td>weeks</td><td>weeks</td><td>weeks</td><td>weeks</td>

<td></td><td>RP-HPLC</td><td>RP-HPLC</td><td>RP-HPLC</td><td>ELISA</td><td>Biotest</td>

<td>Infusaids Model 600 implantable pump</td><td> 91±3</td><td> 101±3</td><td> 112±4</td><td>90 ± ll</td><td> 100±36</td>

<td>Medtronics Synchromed im planted pump</td><td> 109±2</td><td> 92±2</td><td> 91±3</td><td> 97±4</td><td> 90±6</td>

<td>Alzet 2ML4 Osmotic mini pump</td><td> 96±3</td><td> 95±7</td><td> 90±5</td><td>not set</td><td>not set</td>

* AR% - Resistance Rate% = sample fNGFl control [NGF]

% Relative Activity = Sample Activity Control Activity (RP-HPLC data means mean ± 2-4 replicate standard deviation. Bioassay data represents mean ± 95% confidence limits of 3 determinations.)

Studies on the stability of NGF formulations in the pH range 4-10

Aqueous formulations containing 100 µg / ml NGF, 1 mg / ml HSA, and 9 mg / ml NaCl were prepared at pH 4-10 and sterile filtered through a 0.2 µ filter (Millex-GV; Millipore Corp.) as described in Example 1. Compositions with a pH range of 4-5 are buffered with acetate, compositions with a pH range of 6-10 are buffered with TRIS. 1 Samples of 1 ml are placed in polyethylene drop-shaped vials and incubated at either room temperature (23-25 ​​° C) or 37 ° C. Samples are taken at various time intervals and analyzed by RP-HPLC. The first order reaction rate constants representing the loss of NGF in solution are denoted as a function of time. Decomposition rates of NGF in solution were found to increase at pH below about 4.5 and above pH 6.0. The highest stability is observed at pH 5.2.

example

Stability of NGF compositions as a function of carrier concentration

The type and amount of carriers were investigated to determine their effect on NGF stability. An aqueous composition of 100 µg / ml was prepared as described in Example 1 and aqueous compositions containing other carriers and listed in Table 4. Each formulation was filtered through a 0.2 μ Millipore Millex-GV filter. The stability of NGF in various formulations was determined by incubation at 37 ° C in polypropylene drop-shaped vials. After 2 weeks, samples are taken and the protein content is analyzed by RP-HPLC.

table

Stability of various NGF formulations incubated at 2 weeks for 37 C

<td>The bearer</td><td>Content (% w / v)</td><td>AR% after 2 weeks</td>

<td>Gelatin</td><td> 1,0</td><td> 64+9</td>

<td>Human serum albumin</td><td> 0,1</td><td> 48±7</td>

<td>Human serum albumin</td><td> 0,5</td><td> 99±2</td>

<td>Human serum albumin</td><td> 1,0</td><td> 31±36</td>

<td>Tween 80</td><td> 0,2</td><td> 77±9</td>

<td>Pluronic F-68</td><td> 0,02</td><td> 65±5</td>

AR% = Resistance Ratio% = Sample [NGF] Control [NGF] (RP-HPLC data means mean ± 2-4 replicate standard deviation.)

NGF formulation for lyophilization

Aqueous NGF formulation containing 100 µg / ml NGF, 30 mg / ml sucrose, 30 mg / ml mannitol, 5 mg / ml HSA and 0.3 mg / ml citric acid is prepared at room temperature with pH 6.0 adjustment with NaOH. After dissolving the citric acid and sugars in a volume of about 70% of the total volume, adjust the pH and add HSA and NGF with gentle stirring and pouring the remaining water.

example

Lyophilization of NGF composition

The lyophilization stability of aqueous NGF Example 7 is investigated. Samples of NGF compositions prepared in accordance with Example 7, in a volume of 1 ml, are placed in 5 ml Type I glass vials closed with lyophilization stoppers. Composition vials are placed in a freeze-drying chamber (FTS Systems Ine.) Calibrated prior to freezing at 5 ° C. The chamber temperature is then lowered to -40 ° C. 2 hours later At -40 ° C, the chamber is evacuated by controlling the pressure with a stream of nitrogen in the range of 80-100 milliTorr. Raise the temperature by 4 ° C every hour until the final drying temperature of 25 ° C is reached. The final moisture content of the product is within 1-2% after about 30 hours. cycle.

The freeze-dried powder was stored at 5 ° C and reconstituted at room temperature after 3 days using 1 ml of dilution containing 8.7 mg / ml NaCl and 1.1 mg / ml citric acid buffered to 5.2. The concentration of NGF samples is analyzed by RP-HPLC. Protein loss was not observed during lyophilization.

example

Stability studies of NGF formulations stored in glass vials at 2-8 ° C

An aqueous formulation containing 100 or 1000 µg / ml rhNGF is prepared according to the procedure of Example 1, wherein the batch size is increased to

1.5 liters. Samples of 4.2 ml are placed in Type I English crystal vials with Teflon-coated butyl rubber stoppers and stored at 2-8 ° C. The results in Table 5 show no significant loss of protein (as measured by RP-HPLC and ELISA) or loss of NGF activity (as measured by the PC-12 bioassay).

table

Stability of NGF formulation at 2-8 ° C (measured by RP-HPLC, ELISA and PC-12 bioassay)

<td>Nominal [NGF]</td><td>RP-HPLC * AR% Home</td><td>RP-HPLC • AR% 12 months</td><td>ELISA • AR% 12 months</td><td>PC-Biotest * AR% 12 months</td>

<td>100 µg / ml</td><td> 10311</td><td>101 ± l</td><td> 10213</td><td> 102115</td>

<td>1000 pg / ml</td><td> 10211</td><td>101 ± l</td><td> 10813</td><td> 102120</td>

* AR% - Resistance Rate% = Sample (NGF in time baseline [NGF]

Identification and quantification of NGF using RP-HPLC

The following procedure is used in this example 9:

NGF is identified and quantified by analyzing 100 μΐ volume samples using RP-HPLC (Hewlett Packard HP 1090 Liquid Chromotograph) equipped with

4-6 mm mm 250 mm column, Baker-linked wide, and pore butyl (C4) 300 A pore size (JTBaker Ine. Phillipsburg NJ, USA) and a diode device detecting UV at 210 nm. Mobile phases: (A) 0.2% trifluoroacetic acid in water and (B) 60% acetonitrile in (A) buffer, where for 65 min. the gradients change from 29% (B) to 75% (B) at 1.0 ml / min. for current velocity and 110 bar pressure at ambient temperature.

io

Accordingly, HPLC utilized the procedure previously described for the TEST PROCEDURE.

DEFINITION OF INVENTION