Drug – bio-affecting and body treating compositions – Lymphokine
Reexamination Certificate
1993-03-08
2003-05-20
Achutamurthy, Ponnathapu (Department: 1652)
Drug, bio-affecting and body treating compositions
Lymphokine
C514S012200
Reexamination Certificate
active
06565841
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the pulmonary administration of a therapeutic protein and, more particularly, to the systemic administration via the respiratory system of therapeutically effective amounts of granulocyte colony stimulating factor (G-CSF) or chemically modified G-CSF. In another aspect, the present invention relates to the pulmonary administration of a pegylated protein.
BACKGROUND OF THE INVENTION
G-CSF is a hormone-like glycoprotein which regulates hematopoiesis and is required for the clonal growth and maturation of normal hematopoietic precursor cells found in the bone marrow; Welte et al.,
Proc. Natl. Acad. Sci.,
Vol. 82, pp. 1526-1530 (1985). More specifically, G-CSF, when present in low concentrations, is known to stimulate the production of neutrophil granulocytic colonies when used in vitro. G-CSF is also known to enhance neutrophil migration; Gabrilove, J.,
Seminars in Hematology,
Vol. 26, No. 2, pp. 1-4 (1989). Moreover, G-CSF can significantly increase the ability of neutrophils to kill tumor cells in vitro through antibody mediated cellular cytotoxicity; Souza et al.,
Science,
Vol. 232, pp. 61-65 (1986).
In humans, endogenous G-CSF is detectable in blood plasma; Jones et al.,
Bailliere's Clinical Hematology,
Vol. 2, No. 1, pp.83-111. G-CSF is produced by fibroblasts, macrophages, T cells, trophoblasts, endothelial cells and epithelial cells and is the expression product of a single copy gene comprised of four exons and five introns located on chromosome seventeen. Transcription of this locus produces a mRNA species which is differentially processed, resulting in the expression of two forms of G-CSF, one version having a mature length of 177 amino acids, the other having a mature length of 174 amino acids. The form comprised of 174 amino acids has been found to have the greatest specific in vivo biological activity. G-CSF is species cross-reactive, such that when human G-CSF is administered to another mammal such as a mouse, canine or monkey, sustained neutrophil leukocytosis is elicited; Moore et al.,
Proc. Natl. Acad. Sci.,
Vol. 84, pp. 7134-7138 (1987).
Human G-CSF can be obtained and purified from a number of sources. Natural human G-CSF (nhG-CSF) can be isolated from the supernatants of cultured human tumor cell lines. The development of recombinant DNA technology, see, for instance, U.S. Pat. No. 4,810,643 (Souza), incorporated herein by reference, has enabled the production of commercial scale quantities of G-CSF in glycosylated form as a product of eukaryotic host cell expression, and of G-CSF in non-glycosylated form as a product of prokaryotic host cell expression.
Chemically modified G-CSF may also be obtained in numerous ways. Chemical modification may provide additional advantages, such as increasing the stability and clearance time of the therapeutic protein. A review article describing protein modification and fusion proteins is Francis,
Focus on Growth Factors
3: 4-10 (May 1992)(published by Mediscript, Mountview Court, Friern Barnet Lane, London N20 OLD, UK). For example, see EP 0 401 384, entitled, “
Chemically Modified Granulocyte Colony Stimulating Factor
,” which describes materials and methods for preparing G-CSF to which polyethylene glycol molecules are attached. (Such modified G-CSF is referred to herein as “pegylated G-CSF” or “PEG-G-CSF.”). Such chemically modified G-CSF may be obtained by modifying nhG-CSF or G-CSF obtained as a product of prokaryotic or eukaryotic host cell expression.
G-CSF has been found to be useful in the treatment of cancer, as a means of stimulating neutrophil production to compensate for hematopoietic deficits resulting from chemotherapy or radiation therapy. The effective use of G-CSF as a therapeutic agent requires that patients be administered systemic doses of the protein. Currently, parenteral administration via intravenous, intramuscular or subcutaneous injection is the preferred route of administration to humans and has heretofore appeared to be the only practical way to deliver therapeutically significant amounts of G-CSF to the bloodstream, although attempts have been made at oral delivery; see, for example, Takada et al.,
Chem. Pharm. Bull.,
Vol. 37, No. 3, pp. 838-839 (1989). Pulmonary delivery of chemically modified G-CSF has not been demonstrated previously, nor has pulmonary delivery of protein to which one or more polyethylene glycol molecules has been attached.
The pulmonary delivery of relatively large molecules is not unknown, although there are only a few examples which have been quantitatively substantiated. Leuprolide acetate is a nonapeptide with luteinizing hormone releasing hormone (LHRH) agonist activity having low oral availability. Studies with animals indicate that inhalation of an aerosol formulation of leuprolide acetate results in meaningful levels in the blood; Adjei et al., Pharmaceutical Research, Vol. 7, No. 6, pp. 565-569 (1990); Adjei et al.,
International Journal of Pharmaceutics,
Vol. 63, pp. 135-144 (1990).
Endothelin-1 (ET-1), a 21 amino acid vasoconstrictor peptide produced by endothelial cells, has been found to decrease arterial blood pressure when administered by aerosol to guinea pigs; Braquet et al.,
Journal of Cardiovascular Pharmacology,
Vol. 13, suppl. 5, s. 143-146 (1989).
The feasibility of delivering human plasma &agr;1-antitrypsin to the pulmonary system using aerosol administration, with some of the drug gaining access to the systemic circulation, is reported by Hubbard et al.,
Annals of Internal Medicine,
Vol. III, No. 3, pp. 206-212(1989)
Pulmonary administration of &agr;-1-proteinase inhibitor to dogs and sheep has been found to result in passage of some of that substance into the bloodstream; Smith et al.,
J. Clin. Invest.,
Vol. 84, pp. 1145-1146 (1989).
Experiments with test animals have shown that recombinant human growth hormone, when delivered by aerosol, is rapidly absorbed from the lung and produces faster growth comparable to that seen with subcutaneous injection; Oswein et al., “
Aerosolization of Proteins
”, Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colo., March, 1990. Recombinant versions of the cytokines gamma interferon (IFN-&ggr;) and tumor necrosis factor alpha (TNF-&agr;) have also been observed in the bloodstream after aerosol administration to the lung; Debs et al.,
The Journal of Immunology,
Vol. 140, pp. 3482-3488 (1988).
Pulmonary administration of pegylated proteins has not been demonstrated previously, although, as noted above, chemical modification of proteins, including pegylation, has been demonstrated for a variety of proteins, including G-CSF.
SUMMARY OF THE INVENTION
The present invention is based on the discovery that G-CSF can be administered systemically to a mammal via the pulmonary route. Typically, this is accomplished by directing a stream of a therapeutically effective amount of G-CSF into the oral or nasal cavity of the inhaling mammal. Importantly, and surprisingly, substantial amounts of G-CSF are thereby deposited in the lung and absorbed from the lung into the bloodstream, resulting in elevated blood neutrophil levels. Moreover, this is accomplished without the necessity to resort to special measures such as the use of absorption enhancing agents or protein derivatives specifically designed to improve absorption. Pulmonary administration of G-CSF thus provides an effective non-invasive alternative to the systemic delivery of G-CSF by injection.
In another aspect, the present invention is based on the discovery that chemically modified G-CSF may be absorbed from the lung into the bloodstream. In addition to the advantages of pulmonary delivery as described above, this provides additional advantages. Chemical modification may lengthen the circulation time of the protein in the body, alter immunoreactivity, reduce toxicity, alter bioactivity, and alter certain physical properties of the therapeutic peptide.
In yet another aspect, the present invention is based on the broad discovery that a protein to which a polyethylene gly
Niven Ralph
Pitt Colin G
Achutamurthy Ponnathapu
Amgen Inc.
Crandall Craig A.
Kerr Kathleen
Levy Ron K.
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