Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Particulate form
Reexamination Certificate
1998-06-11
2003-12-16
Kunz, Gary (Department: 1647)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Particulate form
C424S773000, C424S641000, C424S625000, C424S630000, C424S646000, C424S650000, C424S611000, C424S602000, C424S682000
Reexamination Certificate
active
06663899
ABSTRACT:
BACKGROUND
1. Field of the Invention
This invention relates to formulations of nerve growth factor (“NGF”) and their use to induce nerve cell growth, differentiation, survival, repair, maturation, or function in vitro, in vivo or ex vivo. More particularly, this invention relates to microencapsulation compositions having controlled release characteristics, preferably with increased stability, for the NGF component, particularly human recombinant NGF (“rhNGF”). Methods for making and using such compositions are provided.
2. Description of Related Disclosures
Nerve growth factor (NGF) is a neurotrophic factor required for the growth and survival of sympathetic and sensory neurons during development and in mature animals (Thoenen, et al.,
Physiol. Rev.
60:1284-1335 (1980)). Clinical indications for recombinant human NGF include peripheral sensory neuropathy and Alzheimer's disease. For example, the systemic administration of NGF has been shown to reduce the sensory neuropathy induced by administration of cisplatin and taxol to mice (Apfel, et al.,
Ann. Neurol.
28:87-90 (1991); Apfel, et al.,
Ann. Neurol.
31:76-80 (1992)). In recent clinical trials, NGF has been administered to humans to improve sensory function in diabetic neuropathies (Petty, et al.,
Ann. Neurol.
36:244-246 (1994)). Although non-toxic and efficacious, administration of NGF in liquid parenteral formulation has been reported as associated with injection-site hyperalgesia and, particularly, myalgesia in current clinical trials.
Intracerebroventricular (ICV) administration of rhNGF can prevent degeneration of basal forebrain cholinergic neurons in rats and monkeys with fimbria-fornex lesions (Koliatsos, et al., Exp. Neurol. 112:161-73 (1991); Koliatsos, et al. Ann. Neurol. 30:831-40 (19991); Tuszynski, et al. Ann. Neurol. 30:626-36 (1991)). Studies have shown that ICV administration of rhNGF can enhance choline acetyltransferase activity and improve spatial memory in aging rats (Williams,
Neurobiol. Aging
12:39-46 (1991); Fisher, et al.,
J. Neurosci
11(7):1889-1906 (1991)). The ICV delivery of rhNGF across the blood-brain barrier has been accomplished by syringe, Ommaya® reservoir or Alzet pump. For the treatment of Alzheimer's disease, the use of implantable infusion pumps with catheters to deliver rhNGF continuously and directly to the ventricle of the brain has been considered. However, rhNGF has been observed to degrade via deamidation and iso-aspartate formation in some implantable pumps at 37° C. (physiological condition), as determined by RP-HPLC. The stability of NGF, particularly in liquid, is complicated beyond the usual chemical and physical degradation pathways as a result of the dimeric structure of NGF. Protein stability may be further complicated when recombinant protein is a mixture of C-terminally clipped NGF variants. While NGF normally exists as a dimer (the crystal structure of murine NGF shows 3 antiparallel pairs of b-strands forming a flat surface through which the monomers dimerize (McDonald, et al.
Nature
354:411-414 (1991)); the dimer dissociation constant is <10
−13
M (Bothwell et al.,
J. Biol. Chem.
252:8532-8536 (1977); Timm, et al.,
Biochem.
33:4667-4676 (1994)), higher order aggregation of NGF has been observed.
Thus, there exists a need for formulations containing NGF that maintain NGF stability and activity while providing a means for treating a variety of conditions, being effective for therapeutic administration to mammals, particularly human subjects, and particularly for intracerebral administration. The advantages of the present invention meet these needs and others as well.
SUMMARY
The present invention is based on the finding of formulation conditions and methods for controlled sustained release of NGF with low initial release rates and enhanced stability of NGF during the release period. A controlled sustained release formulation of NGF which provides a low initial release rate and retains enhanced stability of NGF is provided to effectively induce nerve cell growth, survival, differentiation, maturation, repair, or function, in vitro, in vivo or ex vivo. Provided is a controlled release formulation containing polymeric microspheres containing NGF, or its genetically engineered forms, especially human NGF, preferably the 118 form, that demonstrate very little loss of activity or aggregation during the release period. In preferred embodiments the formulations contain zinc-complexed NGF. In various embodiments the formulations, which have a controlled sustained release characteristic, have enhanced stability to agitation, freezing, thawing, light, or storage.
NGF polymeric controlled release systems are described wherein the NGF retains useful biological activity and is released over an extended period of time of at least one day, more typically one to two weeks, following administration. In the preferred embodiment, the NGF polymeric microspheres are made using very cold temperatures to freeze the polymer-NGF mixtures into polymeric microspheres with very high retention of biological activity and material. NGF is first preferably complexed in solution with a metal and, optionally, mixed with a stabilizing (and NGF-load-increasing) polyol, such as trehalose or mannitol, and dried, preferably spray freeze dried. The dried powder is mixed with a polymer, preferably a poly(lactide), or co-polymer, dissolved in a solvent such as ethyl acetate or methylene chloride. The polymer/NGF mixture is atomized into a vessel containing a frozen non-solvent such as ethanol, overlayed with a liquefied gas such as nitrogen, at a temperature below the freezing point of the polymer/active agent solution or suspension. The atomized particles freeze into microspheres upon contacting the cold liquefied gas, then sink onto the frozen non-solvent layer. The frozen non-solvent is then thawed. As the non-solvent thaws, the microspheres are still frozen and sink into the liquid non-solvent. The solvent in the microspheres also thaws and is slowly extracted into the non-solvent, resulting in hardened microspheres containing NGF.
Embodiments are provided in which sustained release of biologically active NGF from the microspheres, in vitro, ex vivo, or in vivo, extends over a period of one week to three months. The release profile can be achieved by inclusion of polymer degradation modifiers, pore forming agents, polymer-copolymer ratios, and stabilizers of NGF, particularly zinc.
Also provided is a method for making controlled-release microspheres containing NGF or its genetically engineered forms, preferably metal-complexed, preferably with zinc, with very little loss of activity or material during the formulation process. Provided are a method for making microspheres formed from a broad range of polymers which contain active NGF releasable in a controlled fashion, and the microspheres produced by such a process.
Provided herein is an NGF formulation with enhanced consistency for improved application to the neuron or mammal. A stable NGF formulation for use in treating a mammal, preferably human, in need of NGF treatment so as to provide a therapeutically effective amount of NGF, is provided. The microencapsulated devices also find use in cell culture methods, for example, with primary neuron cultures or neuronal cell lines. These and other aspects will become apparent to those skilled in the art in view of the present specification and drawings.
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Cleland Jeffrey L.
Duenas Eileen T.
Lam Xanthe M.
Dreger Ginger R.
Genentech Inc.
Hayes Robert C.
Heller Ehrman White & McAuliffe LLP
Kunz Gary
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