Dermatan sulfates express their anticoagulant activity by catalyzing the inhibition of thrombin as it is formed in plasma. They specifically activate heparin cofactor II (HCII), a protease inhibitor in plasma and extracellular tissue which inhibits thrombin but not other proteases involved in hemostasis. HCII is activated by fractions of 12 or more residues in length that contain an octasaccharide sequence required for binding to the inhibitor. A hexasaccharide component with high affinity for HCII has been identified.

Fractionation of a native dermatan sulfate of porcine intestinal mucosa affords a high molecular weight fraction and a lower molecular weight fraction. The low molecular weight fraction is enriched in 4,6-di-O-sulfated N-acetyl-D-galactosamine residues which contribute to its higher HCII-mediated anti-factor IIa activity.

References

Larsen ML, Abildgaard U, Teien AN, Gjesdal K. Assay of plasma heparin using thrombin and the chromogenic substrate H-D-Phe-Pip-Arg-PNa (S2238). Thromb Res 1978; 13: 285-8.
Fernandez JA, Petäjä J, Griffin JH. Dermatan sulfate and low molecular weight heparin enhance the anticoagulant action of activated protein C. Thromb Haemost 1999; 82:1462-8.

INTIMATAN [OD-0313], a complex carbohydrate of the sulfated glycosaminoglycan (GAG) family of macromolecules, is a pre-IND candidate as the preferred anticoagulant for “on/off” pump cardiac surgery. It is a sodium salt comprised of > 75% repeating L-iduronic acid→4,6- O-disulfated N-acetyl-D-galactosamine (GalNac) disaccharide units.

This naturally-occurring, but rare, disaccharide may be prepared by synthesis or enriched in native dermatan sulfate (DS) from <2% to >75% by site-selective 6-O-sulfation of the L-iduronic acid→4-O-sulfated GalNac residues. DS are GAGs that comprise up to 30% of vascular tissue and their depletion correlates with increased vascular thrombogenicity [1]. The distinguishing property of Intimatan is its enhanced targeted inhibition of thrombin attributable to the uniform content of the discrete 4,6-O-disulfated disaccharide structure [2], the function of which had previously remained uncharacterized [3].

Thrombin is the key enzyme in the activation of platelets and in the formation of fibrin, both major components of the thrombo-occlusive clot. Heparin complexed to antithrombin III rapidly neutralizes thrombin in blood but not thrombin that is bound to platelets, fibrin, extracellular matrix and other cells at the injury site [4, 5]. The production of factors Va, VIIIa, and XIa by bound thrombin is enhanced by heparin bound to fibrin and these factors drive thrombin generation through the assembly of the intrinsic tenase (VIIIa/IXa) and prothrombinase (Va/Xa) catalytic complexes to promote clot growth. This feedback loop may lead to a hypercoagulation state involving “thrombin rebound” following the neutralization of heparin with protamine [6, 7]. Protamine use is further associated with hypotension, anaphylactic reactions, postoperative arrhythmia and catastrophic pulmonary vasoconstriction [ 8, 9].

Intimatan blocks both thrombin generation [1, 10, 11] and thrombin activity at the site of vessel injury, the fibrin and platelet clot, on biomaterial surfaces of blood-interacting medical devices, and in the systemic circulation [4, 10, 12-14]. Intimatan catalyzes the heparin cofactor IIdependent inhibition of both soluble and surface-bound thrombin [4, 14]. This action blocks thrombin generation via a sustained suppression of vessel wall Xa/Va activity by inhibiting bound thrombin [10], suppressing the thrombin feedback loop at doses that pose minimal systemic anticoagulation or bleeding [12]. This effect on prothrombinase activity and the inhibition of tenase (VIIIa/IXa) activity by the 4,6-O-disulfated GalNac structure are possibly mediated in part by direct inhibition of factor IXa [15] and by the reported enhancing effects of Intimatan on activated protein C [16].

Preclinical studies, thus far, have shown Intimatan to be effective as an inhibitor of (i) thrombin generation and complement activation in the pig model of CPB [1]; (ii) surface-bound thrombin [4, 12-14, 17]; (iii) acute coronary thrombosis [18], carotid artery and jugular vein thrombosis in a canine model of deep vessel wall arterial injury [19] and (iv) neointimal hyperplasia in rabbit balloon-injured aortae [10, 19] and following injury to the carotid artery [13]. A recent study has demonstrated that Intimatan ameliorates the activation of human platelets induced by immune serum of patients with HIT. Moreover, it acts synergistically with the GPIIb/IIIa receptor antagonist, Integrilin®, enhancing the inhibition of human platelet activation caused by thrombin [23].

Development Status

Intimatan is a pre-IND drug candidate as the primary anticoagulant for CPB. Currently, more than 830,000 cardiac surgeries are performed annually in the U.S. alone. Of these patients, ~2- 5% develop heparin-induced thrombocytopenia (HIT), a syndrome where heparin antibodies cause platelet activation and a potentially fatal thrombosis. Although the current standard of care in the management of HIT is a switch from heparin to an alternative anticoagulant, no agent is presently available that directly abates or modifies this disease. Intimatan inhibits the activation of human platelets by the heparin immune complex [21, 22] as well as the activation of complement [2] that contributes to the whole-body inflammation associated with CPB. The inhibition of bound thrombin by Intimatan should afford greater inhibition of intravascular/ extracorporeal circuit thrombosis, enhance hemostasis in the surgical wound and, potentially shorten the duration of anticoagulant therapy during cardiac surgery and post-procedure. Because of these clear advantages relative to heparin and to the alternative anticoagulants currently approved for HIT, Intimatan will be developed as the preferred anticoagulant for CPB.

Other Indications

Its synergistic action with platelet IIb/IIIa antagonist [23} and activated protein C [24] may represent an improvement in adjunctive therapies involving drug combinations for the treatment of the arterial related diseases and sepsis. Other combinations may include its use with fibrinolytic agents to minimize the ischemic–reperfusion injury associated with complement activation in the myocardium and as an improvement over heparin in anticoagulant coatings for blood interacting biomaterials.

In summary, Intimatan is poised for proof of concept studies as the preferred anticoagulant in cardiac surgery and for its potential drug development in other cardiovascular disease states. By attenuating rebound, suppressing inflammation, minimizing bleeding and thus, the need for protamine, Intimatan offers a potentially significant improvement over heparin for the critical care management of CPB. Fragmatan, a highly bioavailable, low molecular weight Intimatan for DVT, and Intiglo for the targeted delivery of radiopharmaceutical agents for imaging and therapy represent a further areas of collaboration.

References

[1] Van Gorp CL, Brister SJ, Buchanan MR and Linhardt RJ. Dermatan disulfate, an inhibitor of thrombin generation and complement activation. U.S. Patent 5,922,690 (1997).
[2] Shirk RA, Parthasarathy N, San Antonio JD, Church FC and Wagner WD. Altered dermatan sulfate structure and reduced heparin cofactor II-stimulating activity of biglycan and decorin from human atherosclerotic plaque. J. Biol. Chem. 2000; 275(24):18085-18092.
[3] Yamada S, Yamane Y, Sakamoto K, Tsuda H, Sugahare K. Structural determination of sulfated tetrasaccharides and hexasaccharides containing a rare disaccharide sequence, -3Gal- Nac(4,6-disulfate)β1-4IdoAα1-, isolated from porcine intestinal dermatan sulfate. Eur J Biochem 1998; 258(2):775-783.
[4] Buchanan MR, Maclean GA, Brister SJ. Evidence for a conformational change of surfacebound thrombin that promotes vessel wall thrombogenicity: Selective and sustained inhibition of its activity by Intimatan but not by heparin. Thromb Haemost 1999; ISTH Suppl: 413.
[5] Hogg PJ, Jackson CM. Fibrin monomer protects thrombin from inactivation by heparinantithrombin III: Implication for heparin efficacy. Proc Natl Acad Sci USA 1989; 86: 619-623.
[6] Kontny F. Reactivation of the coagulation system: rationale for long-term antithrombotic treatment. Am. J. Cardiol. 1997; 80 (5A): 55E-60
[7] Watkins MW, Leutmer PA, Schneider DJ, Witmer WT, Vaitkus PT, and Sobel BE. Determinants of rebound thrombin after cessation of heparin in patients undergoing coronary interventions. Cathet Cardiovasc Diagn 1998; 44(3): 257-264.
[8] Bruins P, te Velthuis H, Eerenberg-Belmer AJ, Yazdanbakhsh AP, de Beaumont EM, Eijsman L, Trouwborst A and Hack CE. Heparin-protamine complexes and C-reactive protein induce activation of the classical complement pathway: studies in patients undergoing cardiac surgery and in vitro. Thromb. Haemost. 2000; 84(2): 237-243.
[9] Cao Y, Shioi K, Narumiya T, Mase T and Nagata Y. Kyobu Geka 2000; 53(5): 390-395.
[10] Yang LY, Cancel QV, Schwartz D, Abendschein D, Eisenberg PR, Van Gorp CL, Brister SJ, Buchanan MR. Intimatan, a heparin cofactor II catalyst, inhibits vessel wall thrombogenicity and intimal hyperplasia more effectively than heparin. Thromb Haemost 1999; ISTH Suppl: 414.
[11] Brister SJ, Buchanan MR, Griffin CC, Linhardt RJ, Van Gorp CL. Effect of heparin and CL- 0313 on complement activation in vitro and thrombin generation during cardiopulmonary bypass (CPB) in vivo. Haemostasis 1996; 26(3):575.
[12] Brister SJ and Buchanan MR. Inhibition of surface-bound thrombin by Intimatan during cardiopulmonary bypass in pigs: Advantages over heparin. Thromb Haemost 1997; PS-1205.
[13] Buchanan MR, Brister SJ. Surface-bound thrombin inhibition and intimal hyperplasia following vessel wall injury: Relative effects of heparin and Intimatan. Haemostasis 1998: 28 (2):4.
[14] Buchanan MR, Van Gorp CL, Griffin CC, Linhardt RJ, Brister SJ. Systemic and surfacebound thrombin activity: Implications for the assessment of the anticoagulant and antithrombotic effects of heparin, dermatan sulfate and Intimatan. Haemostasis 1998: 28 (2): 99.
[15] McGee MP, Teuschler H, Parthasarathy N and Wagner WD. Specific regulation of procoagulant activity on monocytes. Intrinsic pathway inhibition by chondroitin 4, 6-disulfate. J Biol Chem. 1995; 270: 26109-26115.
[16] Fernández JA, Petäjä J, Griffin JH. Dermatan sulfate and low molecular weight heparin enhance the anticoagulant action of activated protein C. Thromb Haemost 1999; 82: 1462-8.
[17] Buchanan MR, Brister SJ. Anticoagulant and antithrombin effects of Intimatan, a heparin cofactor II agonist. Thromb Res 2000; 99: 603-612.
[18] Cardin AD, Van Gorp CL, Buchanan MR, Eisenberg PR, Bailey BD, Muegge LH, Shetler TJ, Jackson CV. Inhibition by Intimatan of acute coronary thrombosis in a canine model of arterial injury. ISTH XVIIIth Congress, July 2001 (submitted).
[19] Hennan JK, Hong TT, Shergill AK, Driscoll EM, Cardin AD, Lucchesi BR. Intimatan prevents arterial and venous thrombosis in the canine model of deep vessel wall injury. J. Pharmacol Exp There 2001, submitted.
[20] Schwartz D, Cancel QV, Waissbluth AD, Yang LY, Van Gorp CL, Griffin CC, Eisenberg PR. Differential inhibition of neointimal thickening after balloon injury in the rabbit aorta by glycosaminoglycans. J Am Col Cardiol 1998; 31: 21A.
[21] Cardin AD, Prechel M, Jeske WP, Walenga JM. Pharmacologic action of Intimatan: An improved heparin cofactor II agonist that mediates the inhibition of surface-bound thrombin. IBC’s 11^th International Symposium, October, 2000.
[22] Prechel M, Jeske W, Cardin AD. In Vitro Profile of Intimatan in Heparin-Induced Thrombocytopenia. ISTH XVIIIth Congress, July 2001 (submitted)
[23] Buchanan MR, Brister SJ, Cardin AC. Synergistic inhibition of thrombin-induced platelet aggregation by Intimatan + Integrilin®: Implications for their clinical use. ISTH XVIIIth Congress, July 2001 (submitted)
[24] White B, Livingstone W, Murphy C, Hodgson A, Rafferty M and Smith OP. An open-label study of the role of adjuvant hemostatic support with protein C replacement therapy in purpurafulminans-associated meningococcemia. Blood 2000; 96(12): 3719-3724.
[25] Buchana MR, Maclean GA, Brister SJ. Selective and sustained inhibition of surface-bound thrombin activity by Intimatan/Heparin Cofactor II and its relevance to assessing systemic anticoagulation in vivo, ex vivo and in vitro. Thrombo Haemost 2001; 86: 909-13.

PERIODATE-OXIDIZED INTIMATAN [RD-0329] containing aldehyde moieties undergoes reversible Schiff-base reactions with organic amines, and when treated with sodium cyanoborohydride the Schiff base intermediate is reduced to its corresponding amine forming an irreversible bond.

References