Composition and methods for affecting metallocorrinoid uptake

Drug – bio-affecting and body treating compositions – Lymphokine – Interferon

Reexamination Certificate

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C514S002600, C514S052000, C424S085400, C530S351000, C536S026400

Reexamination Certificate

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06752986

ABSTRACT:

BACKGROUND OF THE INVENTION
Metallocorrinoids are corrin rings with a metal-atom center, such as Co, Fe, Ni, or Mn. A corrin ring is four reduced pyrrole rings linked together A subclass of naturally occurring metallocorrinoids is known as cobalamin, that is, a cobalt-centered corrin ring. Naturally occurring vitamin B
12
, for example, is a cobalamin.
Vitamin B
12
compounds are known to have many biological functions. They are required by the enzyme methionine synthase, for example, which is involved in the production of DNA. Pregnant women need increased amounts of vitamin B
12
which is involved in the production of red blood cells. It is also believed that vitamin B
12
enhances the effects of other vitamins and nutrients in tissue repair. Lack of vitamin B
12
leads to megaloblastic anemia (characterized by large and immature red blood cells) and neuropathy in man with insidious onset of symptoms. These symptoms include weakness, tiredness, breathlessness (dyspnea) on exertion, tingling and numbness (paresthesia), sore tongue (glossitis), loss of appetite and weight, loss of sense of taste and smell, impotence, psychiatric disturbances (such as irritability, memory impairment, mild depression, hallucinations) and severe anemia (which may lead to signs of cardiac dysfunction). Deficiency of vitamin B
12
leads to defective DNA synthesis in cells; tissues most affected are those with the greatest rate of cell turnover, e.g. the haematopoietic system. In small children Cbl deficiency can result in developmental delay, hematological disorders, and neurological disorders. There may be irreversible damage to the nervous system with specific demyelination of the spinal cord.
Increased availability of vitamin B
12
, on the other hand, appears to have a very beneficial effect. Cbl analogs and cobalamin drug conjugates have been shown to inhibit the growth of leukemia cells by possibly deactivating methionine synthase, thus preventing DNA synthesis. The cobalamins that are analogous to vitamin B
12
compounds would appear to be potential therapeutic agents. These include hydroxocobalamin, cyanocobalamin, nitrocobalamin, mehtylcobalamin, and 5′-deoxyadenocobalamin, as well as nitrosylcobalamin.
All forms of vitamin B
12
(adenosyl-, cyano-, hydroxo-, or methylcobalamin) are bound by the transport proteins intrinsic factor and transcobalamin II, to be biologically active. Those transport proteins involved in the uptake of vitamin B
12
are referred to herein as cobalamin binding proteins. Specifically, gastrointestinal absorption of vitamin B
12
relies upon the intrinsic factor-vitamin B
12
complex being bound by the intrinsic factor receptors in the terminal ileum. Likewise, intravascular transport and subsequent cellular uptake of vitamin B
12
throughout the body is dependent upon transcobalamin II and the cell membrane transcobalamin II receptors, respectively. After the transcobalamin II-vitamin B
12
complex has been internalized, the transport protein undergoes lysozymal degradation, which releases vitamin B
12
into the cytoplasm.
Cellular utilization of Cbl is preceded by two important receptor-mediated endocytic events. First, the dietary Cbl bound to gastric intrinsic factor (IF), a 50-kDa glycoprotein, is transported across the absorptive enterocyte via an intrinsic factor-cobalamin receptor that is expressed exclusively in the apical or the luminal membranes. The plasma transport of cobalamin to tissues/cells appears to occur via transporter transcobalamin II (TC II), by receptor-mediated endocytosis via transcobalamin II-receptor (TC II-R). Intracellularly released Cbl is then converted to its biologically active forms, (e.g. methyl-Cbl and 5′-deoxyadenosyl-Cbl) which are utilized by the cytoplasmic enzyme methionine synthase (MS) and mitochondrial at enzyme methyl-malonyl-CoA mutase (MMCM), respectively. MS activity is required for folate metabolism and DNA synthesis and presents a promising target to block cell proliferation. TCII and serum Cbl levels are both increased in hepatocarcinomas and leukemias. TCII has been identified as an acute phase reactant in autoimmune disorders and infection. Several studies have shown that high levels of Cbl inhibited L1210, P388D1, CCRF-CEM, and NCTC929 cell proliferation. This is likely due to the activation of an autoimmune response.
Recent studies have shown that TC II-R is expressed as a non-covalent homodimer of molecular mass of 124 kDa in tissue plasma membranes of human, rat, and rabbit. A comprehensive review of transcobalamin II, the transcobalamin II receptor, and the uptake of vitamin B
12
is provided in “Transcobalamin II and Its Cell Surface Receptor Vitamins and Hormones”,
Vitamins and Hormones
, Vol. 59, pgs. 337-366 (2000) which is incorporated herein in its entirety by reference thereto. Plasma membrane expression of TC II-R appears important for the tissue/cellular uptake of Cbl since its functional inactivation in vivo by its circulatory antiserum results in intracellular deficiency of Cbl. This intracellular deficiency in Cbl results in the development of Cbl deficiency of the animal as a whole.
The utilization of vitamin B
12
as a delivery vehicle is known art. The art describes an oral delivery system that delivers active substances (hormones, bio-active peptides or therapeutic agents) by binding these agents to cobalamin or an analog thereof.
U.S. Pat. No. 5,936,082, which is hereby incorporated by reference in its entirety, for example, describes the therapeutic effectiveness of vitamin B
12
based compounds. Nitrosylcobalamin (NO-Cbl), in particular, was evaluated for its chemotherapeutic effect. In five human hematological and eight solid tumor cell lines, NO-Cbl exhibited an ID
50
that was 5-100 fold lower in tumor cell lines compared to benign cells (fibroblasts and endothelial cells). When oxidized from NO-Cbl, the NO free radical functions in a number of capacities. NO is involved in vasodilation, and is known to contribute to increased oxidative stress, inhibition of cellular metabolism and induction of DNA damage leading to apoptosis and/or necrosis.
Radiolabelled vitamin B
12
analogs have also been described in the art as useful in vivo imaging agents. For example, U.S. Pat. No. 6,096,290, which is hereby incorporated herein in its entirety by reference thereto, describes the use of radiolabelled vitamin B
12
analogs as in vivo tumor imaging agents.
U.S. Pat. No. 6,183,723, which is also incorporated herein by reference in its entirety, describes certain other cobalamin-drug conjugates.
SUMMARY OF THE INVENTION
The multiple components of Cbl uptake, enzymes, co-factors, and transport systems present several points of attack for the therapeutic delivery of cobalamins. As is described herein, the interrelationship of TCII-R and cytokines make this an attractive target for the therapeutic delivery of biologically active metallocorrinoids. Cytokines, in particular interferon &bgr;, are shown to enhance the uptake or activity of biologically active metallocorrinoids, including vitamin B
12
analogs, homologs, and derivatives.
Vitamin B
12
analogs can be synthesized in a number of ways. In addition to conjugation of the side chains of the corrin ring, conjugation to the Cbl moiety can also be made, as can conjugation to the ribose moiety, phosphate moiety, and to the benzimidazole moiety. The conjugating agent and the drug to be conjugated depend upon the type of Cbl group that is modified and the nature of the drug. One of skill in the art would understand how to adapt the conjugation method to the particular Cbl group and drug to be coupled.
Preferred methods of attaching the drug to the Cbl molecule include conjugation to Cbl via biotin. Biotin is conjugated to either the propionamide or the acetamide side chains of the corrin ring of the Cbl molecule. The initial biotin-Cbl complex can be prepared according to Pathre, et al (Pathre, P.M., et al., “Synthesis of Cobalamin-Biotin conjugates that vary in the position in cobalamin coupling, Evaluation of cobalamin derivative binding

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