Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1993-11-08
2002-06-18
Ulm, John (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S069100, C536S023500
Reexamination Certificate
active
06407207
ABSTRACT:
BACKGROUND OF THE INVENTION
Bradykinin is a hormonal nonapeptide (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg)(SEQ ID NO:1) which mediates pain, vascular permeability, inflammation, gastrointestinal function, and smooth muscle tone in vascular and other tissues. Bradykinin (BK) is one of the key mediators of the body's response to trauma and injury. BK levels are generally low until a traumatic event triggers a cascade of biochemical reactions and a rise in the concentration of BK by proteolytic generation. High molecular weight precursors, the kininogens, are found in blood and tissue. This cascade is initiated by the activation of the Hageman factor which also initiates fibrinolysis and coagulation.
Receptors for BK exist in the nervous system, epithelia, smooth muscle and fibroblasts. In each tissue type BK triggers specific responses including neurotransmitter release, muscle contraction, fluid secretion by epithelia, and the stimulation of cell growth. It can also act as a neurotransmitter.
The initial interaction for biological response occurs at a BK receptor site on a cell. Specific BK antagonists have been developed (Vavrek,
Peptides,
6, 161-165 (1985)). Their potential use includes use as anti-nociceptive and anti-inflammatory agents. Bradykinin activates neurons and produces neurotransmitter release. It also stimulates the production of a number of bioactive intermediates including inositol triphosphate (Ins-1,4,5-P
3
) and diacylglycerol (DAG) and arachidonic acid (AA) and its cyclooxygenase and lipooxygenase products. These substances cause cellular levels of cAMP, cGMP, and Ca
2+
to increase. BK also activates phospholipase C and A
2
. In neurons, the most important points of action for the substances released by BK stimulation are ion channels. Miller, R. J.,
Trends Neurosci.,
10, 226-228 (1987).
Bradykinin released during tissue damage causes vasodilation, increased vascular permeability, altered gut motility and pain. Specific bradykinin receptors exist in intestinal mucosa and muscle. Bradykinin and analogues stimulate C1 secretion in the gut. Specific BK receptor binding sites occur in the mucosa and in muscle. BK has a contractile effect in muscle. Manning et al.,
Nature,
299, 256-259 (1982).
Addition of nanomolar concentrations of BK to the serosal surface of the mucosal later of the guinea pig ileum rapidly increased transepithelial potential difference (p.d.) and the short circuit current (I
SC
). This suggests localization of BK receptors at the serosal surface of the villus and crypt epithelium. The increase in I
SC
is thought to be due to stimulation of anion secretion (C1 out of the cell produces a larger potential difference). Manning et al.,
Nature,
299, 256-259 (1982).
Bradykinin could open calcium channels as indicated by the inhibitory effects of Ca
2+
channel blockers. Calcium may be involved in regulating BK receptor binding. See Innis et al.,
Proc. Natn. Acad. Sci.,
2630-2634 (1981). BK also stimulates sodium intake and DNA synthesis. Owen et al.,
Cell,
32, 979-985 (1983).
Excessive kinin activity may play some role in carcinoid syndrome and in inflammatory bowel disease. Patients with ulcerative colitis have abnormally high levels of active kallikrein, the kinin-releasing enzyme and plasma and tissue levels of peptidiyl dipeptidase which degrades kinins are depressed in patients with regional enteritis. Manning et al.,
Nature,
299, 256-259 (1982).
Autoradiographic studies localize BK receptor binding sites to the substantia gelatinosa, dorsal root, and a subset of small cells in both the dorsal root and trigeminal ganglia of the guinea pig. Binding was also observed over myocardial/coronary visceral afferent fibers. The localization of BK receptors to nociceptive pathways supports a role for BK in pain mediation. Several BK antagonists block BK induced acute vascular pain in the rat. BK antagonists also relieve BK and urate induced hyperalgesia in the rat paw. These results indicate that BK is a physiologic mediator of pain and that BK antagonists have analgesic activity in both acute and chronic pain models. The BK receptor involved in vascular pain may be different from the receptor involved in cutaneous hyperalgesia. Steranka et al.,
Proc. Natl. Acad. Sci. USA.,
85, 3245-3249 (1988).
BK receptors have been classified as two major subtypes—B
1
and B
2
. The BK metabolite des-Arg-bradykinin is a B
1
receptor agonist which has higher potency than BK but it is inactive at B
2
receptors. Steranka et al.,
Proc. Natl. Acad. Sci. USA.,
85, 3245-3249 (1988). BK also binds to G protein-coupled receptors that activate phospholipase C or phospholipase A
2
and increases synthesis of inositol triphosphate or arachidonic acid. Olsen et al.,
J. Bio. Chem.
263, 18030-18035 (1988). G-proteins are a family of membrane proteins that become activated only after binding guanosine triphosphate (GTP). Activated G-proteins in turn activate an amplifier enzyme on the inner face of a membrane; the enzyme then converts precursor molecules into second messengers. For example, an external signal molecule (bradykinin) may bind to its cell-surface receptor (BK-2) and induce a conformational change in the receptor. This change is transmitted through the cell membrane to a G-protein, making it able to bind to GTP. Binding of GTP causes another conformational change in the G-protein that enables it to activate adenylate cyclase (amplifier enzyme) to initiate formation of cAMP (second messenger).
In Swiss 3T3 fibroblasts, BK stimulated phospholipase C mediated InsP formation and PGE-2 synthesis. G proteins were implicated in the mediation of the effects of bradykinin suggesting that the receptor is bound to a G protein which interacts with the particular enzyme. Burch et al.,
Proc. Natl. Aca. Sci. USA,
84, 6374-6377 (1987). Two different G-proteins mediate neuropeptide Y and bradykinin stimulated phospholipid breakdown in cultured rat sensory neurons. Perney et al.,
J Biol. Chem.,
264, 7371-7327 (1991).
It is known that there is a large degree of heterogenicity within the muscarinic, adrenergic, and serotonergic class of receptors. Furthermore, “[s]imple classification of subtypes of BK receptors cannot fully account for the properties of these receptors on cells from a variety of tissues.” Mahan et al.,
Mol. Pharmacol.,
37, 785-789 (1990).
Bradykinin induced increases in InsP formation through the activation of phosphatidylinositol-specific phospholipase C and subsequent mobilization of intracellular Ca
2+
and direct activation of phospholipase A
2
, which causes the release of arachidonate and subsequent synthesis of prostaglandin E
2
have been found to exist in Swiss albino mouse 3T3 cells and BALBc (SV-T2) mouse 3T3 cells and involve receptors coupled to pertussis toxin-insensitive G proteins. These receptors belong to the B
2
subtype. Mahan et al.,
Mol. Pharmacol.,
37, 785-789 (1990).
The effect of bradykinin on the neuroeffector junction of the isolated rat vas deferens has been studied. Llona et al.,
J. Pharmacol. Exp. Ther.,
241, 608-614 (1987). BK potentiated the magnitude of the muscular response to the electrically driven twitches and contracted the smooth muscle generating an increased muscle tone. The former action is referred to as the neurogenic or presynaptic effect and the latter is called the musculotropic or postjunctional action. The rat vas deferens contains bradykinin receptors on the nerve endings and on the smooth muscle membrane. The structural prerequisites for the activation of these receptor sites appear to be slightly different. Their results support the existence of B
2
receptors. des-Arg
9
-BK and des-Arg
9
-[Leu
8
]-BK are inactive in causing either pre- or postsynaptic BK like responses and incubation of des-Arg-9-[Leu
8
]-BK at high concentrations failed to antagonize BK responses in the vas deferens. This peptide is a known B
1
antagonist. The authors suggest that there are several classes of BK-2 receptors. Llona et al.,
J. Pharmacol. Exp. Ther.,
241, 6
Borkowski Joseph A.
Hess John W.
Ransom Richard W.
Strader Catherine D.
Durette Philippe L.
Merck & Co. , Inc.
Ulm John
Winokur Melvin
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