GPIb-lipid complex and uses thereof

Details GPIb-lipid complex and uses thereof GPIb-lipid complex and uses thereof

1998-12-03

2001-01-23

Russel, Jeffrey E. (Department: 1653)

Drug, bio-affecting and body treating compositions

Radionuclide or intended radionuclide containing; adjuvant...

Molecular bilayer structure

C424S009321, C424S009340, C424S009370, C424S009510, C424S009600, C424S001210, C514S007600, C514S008100, C514S021800, C530S352000, C530S359000, C530S381000, C530S395000, C530S410000

Reexamination Certificate

active

06177059

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a GPIb-lipid complex and use thereof. More particularly, the present invention relates to a GPIb-lipid complex that can be effectively used for the examination, diagnosis and treatment of vascular lesions and to use thereof.
BACKGROUND ART
Various glycoproteins (GP) are present on the surface of platelet membrane, and are involved in the expression of platelet functions. GPIb is one of the platelet membrane glycoproteins, and functions as a receptor of von Willebrand factor (vWF). GPIb is a heterodimer having a molecular weight of 160,000, wherein &agr; chain and &bgr; chain form a disulfide bond.
When vascular damages are caused, platelets quickly adhere to the lesion and form platelet thrombus by aggregation and the like. In forming the platelet thrombus, vWF plays an important role as an adhesive protein. It is considered that GPIb binds with vWF as a receptor thereof and activates or promotes adhesion and aggregation of platelets via vWF at said vascular lesion. In addition, the binding of vWF and GPIb functions to stop bleeding at the vascular lesion but also forms pathologic thrombus. Thus, GPIb is expected to be effectively used for the examination and diagnosis of vascular lesion, detection of pathologic thrombus, and treatment thereof.
Nevertheless, the use of isolated GPIb has not proved successful in artificial expression of the physiological activity as mentioned above. In other words, some idea in the aspect of formulation of pharmaceutical preparations is needed to practically use GPIb as a pharmaceutical agent or reagent.
DISCLOSURE OF THE INVENTION
Under the circumstances, the present inventors have made intensive studies and first found that the physiological activity can be expressed by preparing a lipid complex comprising a conjugate of GPIb and a certain lipid and a normal lipid, which resulted in the completion of the present invention.
Accordingly, the present invention provides the following.
(1) A complex comprising a lipid and a conjugate of GPIb and a lipid having a functional group.
(2) The complex of above (1), which is in the form of a liposome.
(3) The complex of above (1), wherein the lipid is a phospholipid, glycolipid, cholesterol, fatty acid or derivative thereof.
(4) The complex of above (1), wherein the molar ratio of the GPIb:lipid is 1:10-1:1000.
(5) The complex of above (1), wherein the complex can aggregate in the presence of ristocetin.
(6) A pharmaceutical composition comprising a complex comprising a lipid and a conjugate of GPIb and a lipid having a functional group.
(7) The pharmaceutical composition of above (6), wherein the composition is a platelet substitute.
(8) The pharmaceutical composition of above (6), wherein the composition is an agent for the prophylaxis and treatment of vascular disorders, vascular damages or thrombosis.
(9) A pharmaceutical agent for examination or diagnosis, which comprises, as an active ingredient, a labeling substance and a complex comprising a lipid and a conjugate of GPIb and a lipid having a functional group.
(10) The pharmaceutical agent of above (9), wherein the labeling substance is a radioisotope, paramagnetic metal for MRI, iodide compound for X ray imaging, fluorescent substance or pigment.
(11) A drug-containing composition comprising, as an active ingredient, a drug and a complex comprising a lipid and a conjugate of GPIb and a lipid having a functional group.
(12) The composition of above (11), wherein the drug is a hemostatic agent, vasoconstrictor, antiinflammatory agent, fibrinolytic agent, anti-blood coagulator or anti-platelet agent.
(13) A conjugate of GPIb and a lipid having a functional group.
(14) The conjugate of above (13), wherein the GPIb is a GPIb itself, GPIb &agr; chain or GPIb &agr; chain fragment.
(15) The conjugate of above (13), wherein the GPIb is an analog, mutant, modified compound, derivative or sugar chain adduct, having a von Willebrand factor-binding capability that is almost at the same level as GPIb.
(16) The conjugate of above (13), wherein the GPIb is defective in a transmembrane site.
(17) The conjugate of above (13), wherein the lipid having a functional group is a phospholipid, glycolipid, fatty acid, glyceride, cholesterol or amphipathic lipid.
(18) The conjugate of above (13), wherein the functional group is an amino, carboxyl, thiol or aldehyde.
(19) The conjugate of above (13), wherein the GPIb and the lipid having a functional group are chemically bonded by a crosslinking agent.
(20) The conjugate of above (13), wherein the molar ratio of the GPIb:lipid having a functional group is 1:1-1:20.
(21) The complex of above (1), wherein the complex of a lipid and a conjugate of GPIb and a lipid having a functional group is prepared after preparing said conjugate.
The present invention is described in more detail in the following. (I) Conjugate of GPIb and lipid having functional group (GPIb conjugate)
{circle around (1)} GPIb
The GPIb to be used in the present invention may be GPIb itself, its &agr; chain, vWF binding region, namely, a GPIb fragment such as GPIb &agr; chain [His(1)-Thr(302)] fragment and the like. An analog, a modified compound, a mutant, a derivative and a sugar chain adduct thereof are encompassed in the scope of the present invention, as long as they have almost the same level of vWF binding capability as GPIb. Moreover, they may lack a transmembrane site. In the present invention, those without this transmembrane site are preferably used.
Specific examples thereof include GPIb-related substances disclosed in WO92/ 16225, WO93/ 13784, WO93/ 16712, Japanese Patent Unexamined Publication No. 100196/ 1989, Japanese Patent Unexamined Publication No. 221394/1989 and Japanese Patent Application under PCT laid-open under kohyo No. 503708/1993.
Examples thereof include GPIb, GPIb &agr; chain, GPIb &agr; chain fragment, His(1)-Ala(302), His(1)-Arg(293), Ala(165)-Leu(184), Gln(180)-Phe(199), His(195)-Leu(214), Asn(210)-Val(229), Glu(225)-Ala(244), Thr(240)-Tyr(259), Asn(61)-Thr(75), Gln(71)-Ser(85), Thr(81)-Leu(95), Gln(97)-Arg(111), Leu(136)-Leu(150), Asn(210)-Ala(224), Gln(221)-Asp(235) and Ser(241)-Asp(255).
A substituted compound is exemplified by GPIb &agr; chain fragments consisting of His(1)-Ala(302), wherein Gly(233) and Met(239) are respectively substituted by Val.
The GPIb can be prepared by any method and a method comprising extraction and isolation from platelet membrane, a method using cell culture and a production method using genetic engineering are exemplified.
{circle around (2)} Lipid having functional group
With regard to the lipids having a functional group, a functional group capable of directly or indirectly forming a bond with GPIb is exemplified by amino group (NH
2
), carboxyl group (COOH), thiol group (SH), aldehyde group (CHO) and the like. As long as the functional group can form a direct or indirect bond with a GPIb molecule, it is not limited to those exemplified.
The kind of lipid is free of limitation as long as the lipid is amphipathic, such as phospholipid, glycolipid, fatty acid, glyceride, cholesterol and the like.
The lipid having a functional group is exemplified by phosphatidyl ethanolamine (hereinafter to be referred to as PE) and phosphatidyl thioethanol (e.g., 1,2-dioleoyl-sn-glycero-3-phosphatidylthioethanol) for phospholipid. When it is to be indirectly bound with GPIb, a crosslinking agent (i.e., spacer or linker) may be bonded in advance. Examples of such crosslinking agent include dicarboxylic acid, aminocarboxylic acid, bismaleimide compound, bishalocarbonyl compound, halocarbonylmaleimide compound, dithiomaleimide, dithiocarboxylic acid and maleimidecarboxylic acid. These preferably have 2 to 10 carbon atoms.
The phospholipid bonded with said crosslinking agent is exemplified by PE-N-carbonyl amine (e.g., PE-N-caproyl amine, PE-Ndodecanyl amine, PE-N-glutalyl amine and the like), PE-N-carbonyl (e.g., PE-N-succinyl, PE-N-glutalyl (NGPE), PE-N-dodecanyl (NDPE) and the like), PE-N-dithioacylate (e.g., PE-N-3-(2-pyridyldithio)propionate), PE-N

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