Numerous polypeptides and proteins regulate the growth or survival of cells; such molecules are termed "growth factors". Examples of growth factors include epidermal growth factor (EGF), acidic and basic fibroblast growth factor (aFGF and bFGF), platelet derived growth factor (PDGF), ciliary neurotrophic factor (CNTF), and nerve growth factor (NGF). Of these, NGF was the first to be identified and characterized (Levi-Montalcini, R., et al., J. Exp. Zool., 116:321, 1951).
NGF promotes the survival and activity of certain types of neuronal cells. In addition, NGF promotes the differentiation of premature neuronal cells into post-mitotic mature neurons.
Purification of NGF from mouse submaxillary gland resulted in the identification of a complex comprising three subunits, .varies., .beta., and .gamma.. All of the neurotrophic activity of NGF is presumed to reside in the .beta. subunit, a 118 amino acid protein having a molecular weight of about 13,000 Da (Varon, S., et al., Proc. Natl. Acad. Sci. USA, 57:1782-1789, 1967; Greene, L. A., et al., Neurobiol., 1:37-48, 1971). In solution, .beta. subunits form dimers of molecular weight about 26,500 Da.
NGF has been suggested to be effective for treating certain degenerative diseases of both the peripheral and central nervous systems. It has been suggested that the administration of NGF may be beneficial in treating diseases in which a deficiency of NGF, abnormalities of its receptor, or changes in its transport or intracellular processing lead to a decrease in neuronal function, atrophy or even cell death. Such diseases include hereditary sensory and motor neuropathies, hereditary and sporadically occurring system degeneration, amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease (Goedert, M., et al., Mol. Brain Res., 1:85-92, 1986; Mobley, W. C., et al., Soc. Neurosci. Abstr., 13:186, 1987; Mobley, W. C., et al., Soc. Neurosci. Abstr., 4:302, 1988; Hefti, F., et al., Ann. Neurol., 20:275-281, 1986). NGF is also thought to decrease neuron cell death after exposure to certain toxins, such as 6-hydroxy-dopamine, (Aloe, L., Arch. Ital. Biol., 113:326-353, 1975), vinblastine and colchicine (Menesini-Chen, M. G., et al., Proc. Natl. Acad. Sci. USA, 74:5559-5563, 1977; Johnson, E. M., Brain Res., 141:105-118, 1978) and capsaicin (Otten, U., Nature, 301:515-577, 1983).
The high expression of NGF mRNA in the hippocampus, an area associated with memory and learning, suggests that clinical application of NGF may be effective for the treatment of dementia (Kaisho, Y., et al., Biochem. Biophys. Res, Comm., 174:379-385, 1991). The intraventricular administration of NGF has been reported to prevent the death of basal forebrain cholinergic neurons after axotomy suggesting that NGF may be effective in promoting cell survival 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 for therapy poses significant problems. These problems are associated with 1) maintaining the bioactivity of the NGF, which may be altered during manufacturing, purification, or storage; and 2) administering NGF, a relatively large, hydrophilic molecule, so it reaches the active site in sufficient amounts to be effective. The bioactivity of NGF, like other proteins, is dependent on its secondary and tertiary structure. The .beta. subunit of NGF has three internal disulfide bonds, which are thought to be 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, K. E., Gene, 70:57-65, 1988). In addition, to the extent that any of the protein is denatured, the effective amount of biologically active NGF is diminished. Protein integrity must therefore be maintained during manufacture and storage as well as during administration. Proteins are particularly prone to degradation at elevated temperatures.
Lower temperatures generally decrease protein degradation. However, it is more economical to store the protein at room temperature, i.e., about 25.degree. C., rather than at refrigerated temperatures of about 4.degree. C. Therefore, formulation stability is desirable for storage at either room temperature or refrigeration at approximately 4.degree. C.
In addition to problems of stability, NGF, like many other proteins, binds nonspecifically to surfaces. Such nonspecific binding may occur to a variety of materials including glass and plastics, for example polyethylene or polypropylene. These materials may be in the form of vials, tubing, syringes, implantable infusion devices or any other surface which may come in contact with NGF during its manufacture, storage or administration.
Other difficulties in administering proteins such as NGF as therapeutics are poor absorption by the body and degradation by stomach acids. Oral administration is therefore generally unsuitable. Injections and infusion of such proteins may be necessary to overcome such absorption barriers.
Injection is useful when the site of treatment is readily accessible. However, if the site is relatively inaccessible such as the CNS, continuous infusion may be more practical for long term administration. Such administration has been impractical due to various complications. For example, continuous infusion may be achieved by implanting NGF pumps into the brain, but long term exposure of a protein to body temperature often causes degradation of the protein. Also, there may be additional losses due to protein adsorption to the pump chamber over time.
In addition to the problems associated with the administration of NGF, there are also problems associated with its long term storage from the time of manufacture to administration. Lyophilization is one method of long term storage of biological proteins, impeding degradation, aggregation, and/or nonspecific adsorption. However, the lyophilization process itself presents difficulties. As the volume of liquid decreases during the freezing process, the effective salt concentration increases dramatically, which may denature the protein, reducing effective therapeutic activity upon reconstitution. In addition, formation of ice crystals during the freezing process may cause denaturation and also decrease the effective amount of bioactive NGF available. The formulation then must be such as to prevent salt concentration fluctuations and minimize formation of ice crystals.
One object of this invention is to provide aqueous formulations of NGF which retain bioactivity for at least one month over a temperature range from about 4.degree. C. to about 40.degree. C.
Still another object of the present invention is to provide formulations of NGF in which bioactivity is maintained after lyophilization and reconstitution.
It is still another object of the invention to provide methods of storing biologically active NGF in solution.