Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-05-19
2004-06-08
Saoud, Christine J. (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069400, C435S243000, C435S320100, C435S325000, C514S003100, C530S303000, C536S023500
Reexamination Certificate
active
06746853
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to novel insulin activity (IA) proteins and nucleic acids. The Invention further relates to the use of the IA proteins in the treatment of Insulin-related disorders such as type 1 diabetes and type 2 diabetes.
BACKGROUND OF THE INVENTION
Insulin is a hormone that plays a major role in the regulation of growth and metabolism in vertebrates. A deficiency of insulin is the most important factor in diabetic disease states. Absence of insulin leads to severe metabolic disorders resulting from the failure to normally metabolize carbohydrate, fat and proteins at a normal rate. These disorders include, for example diabetes mellitus (DM), a complex chronic metabolic disorder. Diabetes mellitus is characterized in two broad groups based on clinical manifestations, namely the non-insulin-dependent diabetes (NIDDM) or maturity onset form, also known as Type 2 and the insulin-dependent diabetes (IDDM) or juvenile onset form, also known as Type 1. In the general population, diabetes mellitus occurs with a prevalence of approximately 1%, with one fourth of these being the Type1. In its most fully expressed clinical form, diabetes mellitus manifests itself as a series of hormone-induced metabolic abnormalities which eventually lead to serious, long-term and debilitating complications such as glucosuria, ketonuria, growth arrest, and negative nitrogen balance. These conditions can ultimately lead to death. Familial hyperproinsulinemla is a genetic disorder characterized by a marked increase in serum proinsulin-like molecules. The cause of this disease is an amino acid substitution which results in incomplete cleavage of proinsulin by the proteases which form insulin.
Type 1 diabetes arises for example, when patients lack beta-cells producing insulin in their pancreatic glands or when the produced insulin is inactive due to mutation(s). Type 2 diabetes occurs in patients with an impaired beta cell function. Type 1 diabetic patients are currently treated with insulin, while the majority of type 2 diabetic patients are treated either with sulfonylureas that stimulate beta cell function or with agents that enhance the tissue sensitivity of the patients towards insulin (e.g., metformin) or with insulin.
Today, insulin administration to diabetic patients is the primary therapeutic means for controlling the disease. In the treatment of diabetes mellitus, many varieties of insulin preparations have been suggested and used. Some of the preparations are fast acting and other preparations have more or less prolonged actions. Such a prolonged action may be obtained by administering the insulin as a suspension of insulin crystal which can be obtained by crystallization of insulin in the presence of zinc (such as LENTE; Novo Terapeutisk Laboratorium) or by crystallization of insulin in the presence of zinc and protamine (such as NPH-insulin).
The human insulin monomer, a 6000 dalton protein, is composed of two chains, the 21 amino acid A-chain and the 30 amino acid B-chain. Insulin is synthesized in pancreatic beta cells located within the islets of Langerhans as a precursor form that is post-translationally processed to the mature two polypeptide chain active hormone. In the biologically active human insulin, the A and B chains are linked with one another via two cysteine bridges, and a further cysteine bridge occurs within the A-chain. The following cysteine residues are linked with one another in human insulin: A6-A-11, A7-B7, and A20B-19 (the letters A and B stand for the amino acid chain, and the numbers for the position of the cysteine residues counted from the amino to carboxyl end of each chain; see FIG.
1
).
Based on the functional analysis of insulins from various species and several insulin analogs or mutants, some pertinent properties of insulin have emerged with respect to the amino acid sequence (see also FIG.
1
): biologically active insulin has three disulfide bonds; B1-Phe is present in all known mammalian insulins; A1-Gly; a terminal tripeptide sequence (A19-21) Tyr-Cys-Asn (removal of A21-Asn by carboxypeptidase digestion results in a loss of activity of>90%); an invariant sequence at B24-26, Phe-Phe-Tyr; B12-Val is highly conserved; A2-A3, Ile-Val is highly conserved; B5-His and B22-Arg are invariant in insulins of high potency; invariant surface residues (highly conserved) A1-Gly, A4-Glu, A5-Gln, A7-Cys, A19-Tyr, A21-Asn, and B7-Cys.
To this end, variants of insulin sequences, applications, production procedures and assays are known;
see for example U.S. Pat. No. 4,421,685 (reports process for producing insulin); U.S. Pat. No. 4,992,417 (reports superactive insulin analogues); U.S. Pat. No. 5,008,241 (reports insulin analogs characterized by amino acid residue substitutions of N21 in the A-chain, with a resulting improvement in stability of insulin solutions at acidic pH levels); U.S. Pat. No. 5,506,202 (reports preparation and use of insulin derivatives comprising various amino acid substitutions); U.S. Pat. No. 5,514,646 (reports analogs of human insulin modified at position 29 of the B-chain that have modified physico-chemical and pharmacokinetic properties and are useful in the treatment of hyperglycemia); U.S. Pat. No. 5,559,094(reports analogs of human insulin containing an aspartic acid at position 1 of the B-chain and U.S. Pat. No. 5,618,913 (reports rapid acting human insulin analogues having amino acid residue substitution and less tendency to self-associate into dimers, tetramers, hexamers or polymers); U.S. Pat. No. 5,621,073 (reports a process for purification of insulin or an insulin acetylated at position A9); U.S. Pat. No. 5,663,291 (reports a process for obtaining insulin having correctly linked cysteine bridges from a corresponding proinsulin amino acid chain); U.S. Pat. No. 5,700,662 (reports process for preparing insulin analogs comprising a modification of position 29 of the B-chain); U.S. Pat. No. 6,034,054 (reports a monomeric insulin analog formulation stabilized against aggregation); all of which are expressly incorporated by reference and further by Marki et al. [Hoppe Seylets Z. Phsiol. Chem. 360(11):1619-32 (1979)]; Hu et al. [Biochemistry 32(10):2631-5 (1993)]; Schwartz et al. [Proc. Natl. Acad. Sci. U.S.A. 84(18):6408-11(1987)]; Kitagawa et al. [Biochemistry 23(7):1405-13 (1984)]; Kobayashi et al. [Biochem. Biophs. Res. Commun. 107(1):329-36 (1982)]; Shoelson et al. [Biochemistry 31(6):1757-67 (1992]; and references cited therein, all of which are expressly incorporated as reference.
Human insulin in solution is known to exist in many molecular forms, such as the monomer, the dimer, the tetramer and the hexamer [Blundell et al., in Advances in Protein Chemistry, Academic Press, New York and London, Vol.26, pp279-330 (1972)], with the oligomer forms being favored at high insulin concentrations and the monomer being the active form of insulin. Insulin in the bloodstream is highly dilute, being 10
−11
to 10
−8
M and is primarily in monomer form. The much more concentrated insulin stored in the beta cell of the pancreas and in the usual administerable solution is largely in the non-active hexamer form, as the well-known 2 zinc hexamer (see below). The delayed absorption phenomena [Binder, Diabetes Care 7(2):188-99 (1984)] is in some large part attributable to the required for the insulin to disassociate from hexamer, tetramer and dimer form into the active monomer form.
in the presence of zinc (Zn), natural human insulin associates to a 2 Zn-hexamer that functions as an allosteric protein. Phenolic ligands or certain salts are capable of inducing a conformational transition, resulting in the N-terminal 8 amino acids of the B-chain converting from an extended conformation to an Q-helix. This conformational state induced by phenolic ligands has been referred to as the R state and the apoinsulin form as the T state. The R state is more compact, less flexible, and the Zn exchange is retarded compared to the T state [D
Dahiyat Bassil I.
Morton Andrew G.
Dahiyat Bassil I.
Dorsey & Whitney LLP
Kosslak Renee M.
Saoud Christine J.
Silva Robin M.
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