Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-11-10
2003-12-30
Carlson, Karen Cochrane (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
53
Reexamination Certificate
active
06670323
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to novel hemoglobin compositions, particularly novel recombinant mutant hemoglobin compositions, which eliminate or substantially reduce 1) the creation of heart lesions, 2) gastrointestinal discomfort, 3) pressor effects, and 4) endotoxin hypersensitivity associated with the administration of extracellular hemoglobin compositions in various therapeutic applications. Applications described include use as a blood substitute, volume expander and/or oxygen carrier in various therapeutic treatments.
BACKGROUND OF THE INVENTION
Hemoglobin (Hb) is the oxygen-carrying protein of blood, comprised of four associated polypeptide chains that bear prosthetic groups known as hemes. About 92% of adult human hemoglobin is composed of two alpha globin subunits (&agr;1, &agr;2) and two beta, globin subunits (&bgr;1, &bgr;2) that associate noncovalently to form &agr;2 &bgr;2, commonly known as hemoglobin A
0
(WO 93/09143). However, adult hemoglobin may also comprise delta globin subunits. The delta globin subunit replaces beta globin and pairs with alpha globin as alpha2delta2 to form hemoglobin A2. In addition to the globin subunits of adult hemoglobin, a number of hemoglobin subunits are expressed in nature only during embryonic and fetal development including, gamma globin, zeta globin, and epsilon globin. To form embryonic or fetal hemoglobin, zeta globin may replace alpha globin and epsilon and gamma globin may replace beta globin (e.g. to form tetrameric hemoglobin such as alpha2epsilon2, alpha2gamma2, zeta2epsilon2, and zeta2gamma2). Embryonic hemoglobin confers a biological advantage to the developing fetus because it generally has a higher oxygen affinity relative to adult hemoglobin and thus, facilitates fetal oxygen uptake from the maternal blood stream. The structure of hemoglobin is well known and described in Bunn & Forget, eds.,
Hemoglobin: Molecular, Genetic and Clinical Aspects
(W. B. Saunders Co., Philadelphia, Pa.: 1986) and Fermi & Perutz “Hemoglobin and Myoglobin,” in Phillips and Richards,
Atlas of Molecular Structures in Biology
(Clarendon Press: 1981).
Expression of various recombinant hemoglobins containing naturally-occurring and non-naturally occurring globin mutants has been achieved. Such methods include the expression of individual globins in recombinant cells, as described, for example, in U.S. Pat. No. 5,028,588, and co-expression of alpha and beta globins in the same cell, as described in U.S. Pat. No. 5,545,727. In addition, di-alpha globin expression, wherein two alpha globins are joined with a short polypeptide linker through genetic fusion and are later coupled with two beta globins to produce a pseudotetrameric hemoglobin molecule, has been described in U.S. Pat. No. 5,545,727 and Looker et al.,
Nature
356:258-260 (1992). Other modified recombinant hemoglobins are disclosed, e.g., in U.S. Pat. No. 5,844,090.
Solutions of extracellular hemoglobin have been demonstrated to have many therapeutic uses. U.S. Pat. Nos. 5,658,879 and 5,679,638 describe the administration of stroma-free purified wildtype hemoglobin to cancer patients in order to enhance the effects of chemotherapy or radiation therapy. U.S. Pat. No. 5,614,490 describes the use of stroma-free diaspirin crosslinked hemoglobin to increase the perfusion of tissues to treat stroke and ischemia, and to treat hypovolemic, cardiogenic, and septic shock. U.S. Pat. No. 5,428,007 describes the use of recombinant mutant hemoglobin with altered oxygen affinity to increase tissue oxygenation in order to treat burn victims. U.S. Pat. No. 5,631,219 teaches the use of recombinant mutant hemoglobin with altered oxygen affinity to treat anemias, cytopenias, and cachexia, and to stimulate hematopoiesis. And, WO 98/17289 describes the use of stroma-free diaspirin crosslinked hemoglobin to treat head injuries in mammals. The use of crosslinked oxyhemoglobin to treat sickle cell disease is described by Walder et al., J. Mol. Bio., vol. 141, 195-216 (1980).
Nitric oxide acts as a chemical messenger in the control of many important processes in vivo, including neurotransmission, inflammation, platelet aggregation, and regulation of gastrointestinal and vascular smooth muscle tone. The biological actions of nitric oxide are mediated by binding to and activation of soluble guanylyl cyclase, which initiates a biochemical cascade resulting in a variety of tissue-specific responses (Feldman et al.,
Chem. Eng. News
Dec. 26-38 (1993)).
Elucidating the functions of nitric oxide has depended largely on inhibition of the nitric oxide-generating enzyme, nitric oxide synthase. Most conclusions about the effects of cell-free hemoglobin have been drawn based on experiments involving nitric oxide synthase inhibitors and/or nitric oxide donors. While the rapid, high-affinity binding of nitric oxide to deoxyhemoglobin is well known, the importance of the oxidative reaction between nitric oxide and oxyhemoglobin is not as widely appreciated. In this reaction, the nitric oxide molecule does not bind to the heme, but reacts directly with the bound oxygen of the oxyhemoglobin complex to form methemoglobin and nitrate (Doyle et al.,
J. Inorg. Biochem.
14: 351-358 (1981)). The chemistry is analogous to the rapid reaction of nitric oxide with free superoxide in solution (Huie et al.,
Free Rad. Res. Comms.
18: 195-199 (1993)). Both the heme iron and nitric oxide become oxidized by the bound oxygen atoms, and the reaction occurs so rapidly that no replacement of oxygen by nitric oxide is observed (Eich et al., infra.).
Since nitric oxide is produced and consumed on a continuous basis, there is a natural turnover of nitric oxide in vivo. When a cell-free hemoglobin is administered, the balance between nitric oxide production and consumption is altered by reactions with hemoglobin. The most relevant parameter for nitric oxide scavenging by hemoglobin is the rate of reaction with nitric oxide, not the position of the hemoglobin allosteric (R/T) equilibrium. The oxidative reaction is irreversible, and nitric oxide binding to deoxyhemoglobin is effectively irreversible on physiologic timescales since the half-life for dissociation of nitrosylhemoglobin is 5-6 hours (Moore et al.,
J. Biol. Chem.
251: 2788-2794 (1976).
When nitric oxide molecules react with oxyhemoglobin or deoxyhemoglobin, they are eliminated from the pool of signal molecules. Once sufficient nitric oxide molecules are eliminated, it is believed, certain adverse conditions are created. For example, hemoglobin can bind nitric oxide causing the prevention of vascular relaxation and potentially leading to hypertension that is sometimes observed after administration of certain extracellular hemoglobin solutions. In addition, the ability of nitric oxide to oxidize oxyhemoglobin producing nitrate and methemoglobin could also lower free concentrations of nitric oxide and lead to hypertension.
Nitric oxide is also needed to mediate certain inflammatory responses. For example, nitric oxide produced by the endothelium inhibits platelet aggregation. Consequently, as nitric oxide is bound by cell-free hemoglobin, platelet aggregation may be increased. As platelets aggregate, they release potent vasoconstrictor compounds such as thromboxane A
2
and serotinin. These compounds may act synergistically with the reduced nitric oxide levels caused by hemoglobin scavenging resulting in significant vasoconstriction. In addition to inhibiting platelet aggregation, nitric oxide also inhibits neutrophil attachment to cell walls, which in turn may lead to cell wall damage.
Several undesirable side effects have been observed by applicants upon administering solutions of extracellular wild-type human hemoglobin to test subjects. Applicants' experimental results in sensitive test animals, as described in detail below, demonstrate that extracellular hemoglobin compositions such as those including wild-type adult human hemoglobin molecules administered at therapeutic dosages result in the formation of myocardial necrosis in heart tissue. While not bei
Apostol Izydor Z.
Brucker Eric A.
Burhop Kenneth E.
Doyle Michael P.
Foster David L.
Baxter International Inc.
Carlson Karen Cochrane
Senniger Powers Leavitt & Roedel
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