WO2001002009A1 - Method and composition for inhibition of vasospasm - Google Patents

Method and composition for inhibition of vasospasm Download PDF

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Publication number
WO2001002009A1
WO2001002009A1 PCT/US2000/018323 US0018323W WO0102009A1 WO 2001002009 A1 WO2001002009 A1 WO 2001002009A1 US 0018323 W US0018323 W US 0018323W WO 0102009 A1 WO0102009 A1 WO 0102009A1
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cdl
anti
antibody
vasospasm
method according
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PCT/US2000/018323
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French (fr)
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Rafael J. Tamargo
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Johns Hopkins University
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2845Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta2-subunit-containing molecules, e.g. CD11, CD18
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/395Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/505Medicinal preparations containing antigens or antibodies comprising antibodies

Abstract

A method for inhibiting cerebral vasospasm in a mammal is provided, wherein the mammal is administered an amount of an anti-CD11/CD18 antibody sufficient to inhibit onset or progression of vasospasm in the mammal.

Description

METHOD AND COMPOSITION FOR INHIBITION OF VASOSPASM

FIELD OF THE INVENTION

The present invention relates generally to methods for preventing or treating vasospasm in mammals More specifically, the invention relates to methods for inhibiting intercellular adhesion processes associated with \ asospasm in mammals

BACKGROUND OF THE INVENTION

Chronic vasospasm is a gradual and delayed narrowing of arterial vasculature that is associated with contact of blood with the adventitial surface, i.e.. the external coveπng, of a blood vessel The decrease in the absolute diameter of the blood vessel causes a decrease m the lumenal (internal) diameter of the vessel, which in turn causes a decrease in the rate of blood flow through the vessel. The resulting ischemia and oxygen deficit causes injury or infarction in tissues distal to the site of the vasospasm Thus, vasospasm is a major cause of morbidity and mortality following hemorrhage in the brain, e.g , subarachnoid hemorrhage due to aneurysmal rupture, but is also implicated with hemorrhage elsewhere in the body. While certain authors have postulated an inflammatory component to v asospasm (Pelletieπ et al , Experientia, 37 1 170-1 171 (1981 ), Peterson et al , J Neiirosurg , 72 161 -11 ( 1990), Handa et al Neurosurgen 28 542-549 ( 1991 )), the exact mechanisms of its genesis and progression remain unclear

Numerous attempts have been made to develop methods for preventing or treating vasospasm Once it was realized that vasospasm produced a substantial decrease in blood flow, efforts were directed to increasing the mtravascular volume or pressure to increase perfusion rates Kassell et al , Neurosurgen 11 337-343 ( 1982), Kassell et al J Neiirosurg 77 848-852 (1992) Such hypervolemic-hypertensive therapy has proven partially effective for reducing the effects of vasospasm. but ineffectiv e to reduce its onset or shorten its duration

Certain groups have attempted to treat v asospasm by l elaxing the contractile elements (e g , smooth muscle) in the blood v essel w alls Vasodilatois and vasoconstπction antagonists hav e been used, w ith limited success In particular, papa eπne, a potent non-specific v asodilator and phosphodiesterase inhibitor, has manifested v ariable v asodilatory effects with respect to cerebral vasospasm This compound does not appear to improve the outcome of post-subarachnoid hemoπhage patients Kassell et al supra ( 1992) Calcium antagonists such as nimodipine and mcardipme have also failed to provide clear evidence of efficacy Nimodipine, for example has failed to show anv effect on vasospasm as measured by angiography Haley et al J Neiirosurg 78 537-547 (1993), Meyer, Neiirosurg Chn N Am 1(2) 361-316 ( 1990) Nicardipme has shown modest anti-vasospastic effect, but has not demonstrated improved outcome in patient as compared to hypervolemic-hypertensive therapy Haley et al supra ( 1993)

Efforts have also been directed to combating vasospasm by treating its putative inflammatory characteπstics Thus, lbuprofen and methylpredmsolone have been employed to prevent vasospasm in dogs Chyatte et al J Neiirosurg 59 925-932

( 1983), Chyatte et al Stroke 20 1021-1026 (1989) However, systemic administration of these compounds has resulted in severe side effects Chyatte et al supra ( 1983)

Immunosuppressive therapy is another approach that has been tπed for treating vasospasm Systemic administration of cyclospoπn and dexamethasone has resulted in significant reduction of cerebral vasospasm However, the use of compounds such as cyclospoπn causes profound systemic immunosuppression, which can lead to death Peterson et al supra ( 1990)

Sills et al Neurosw ger\ 41 453-461 (1997), report that, using a rat temoral model of vasospasm, expression of ICAM-1 by endothe al cells is increased following contact of blood with the adventitial surface of the artery, and that this increase m

ICAM-1 expression correlates with incidence of vasospasm

Oshiro et al Str oke 28( 10) 2031 -2037 (1997), have described a rat model of vasospasm in which intrapeπtoneal injection of an antι-ICAM-1 antibody was observed to inhibit arterial narrow ing and inflammatory cell infiltrates following exposure of femoral artery to autologous blood The authors conclude that inflammatory changes mediated by ICAM-1 play a role in development of v asospasm in this rat model Oshiro et al Stroke ϊHA23 d Int'l Joint Co/if Stroke Cei ebi al Cu e ( 1998), have further described antι-ICAM- 1 and antι-LFA- 1 antibodies as inhibiting post-hemorrhagic vasospasm in a similar rat model Hirashima et al I Neiirosurg 84(5) 826-830 ( 1996) describe inhibition of vasospasm by intravenous administration ot an inhibitor of platelet-activating factor (PAF) following subarachnoid injection of blood into rabbits The authors conclude that the model provides evidence that PAF may play a role in the pathogenesis of vasospasm This group has previously identified increased levels of PAF and PAF acetylhydrolase (PAF-AH) m cerebrospinal fluid m humans following cerebral infarct associated with vasospasm Hirashima et al J Neurosurg , 80(1) 31 -36 (1994)

Other miscellaneous methods and compositions have been used m an effort to inhibit vasospasm For example. U S Patent No 4,792.564 to Harder et al descπbes a method for inhibiting vasospasm m an animal by administering to the animal an effective amount of nicorandil. a K7 channel activator In another approach, U S Patent No 5,621.079 to Cascieπ et al discloses a mammalian neuropeptide Y receptor and methods of using the receptor to identify specific modulators for the receptor This document discloses that such modulatory compounds are useful in treating a vaπety of disease conditions including cardiac and cerebral vasospasm, obesity, diabetes, congestive heart failure, Alzheimer's disease, and others As a further example, U S Patent No 4,103,687 to Ishn descπbes a method for treating cerebral vasospasm in a patient by admmisteπng haptoglobm (α2 globulin) or a haptoglobin-hemoglobin complex

Various types of blood cells are involved m inflammatory processes in vivo Leukocytes, the major cellular component of the inflammatory response, circulate throughout the body and interact with a number of tissues to achieve their myriad effects The interactions of leukocvtes with target tissues and many of the functional effects associated with such interactions are mediated by adherence of the leukocytes to cells and extracellular matπx of those tissues To do so, the leukocytes express a vaπety of cell surface adhesion molecules, which interact wtth complementary adhesion molecules expressed bv the target tissues Harlan et al Adhesion Its Role in Inflammaton Disease W H Freeman & Co . New York ( 1992)

One family of leukocyte adhesion molecules, v ariously designated "leukocyte integπns," "leukomtegπns " CD1 1/CD18 mtegrms." and "β. mtegnns," are involved in cell-cell and cell-protem interactions of all leukocytes For reviews, see Gahmberg, Curr Opin Cell Biol 9 643-650 ( 1997), Hynes. Cell 48 549-554 (1987), Beatty et al J Immunol 757 2913-2918 ( 1983) The members of the leukocyte integπn family are heterodimers. each consisting of a unique (alpha) chain (CD1 l a. CD1 lb. CD1 l c. α t) and a common β (beta) chain (CD 18, also known as β2). The CDl la/CD 18 integrin is referred to as "LFA-1 " (alternatively, " , β2"); the CDl lb/CD18 integrin is referred to as "Mac-1 " (alternatively, "CR3," "Mo-1 ," or "αMβ2"); and the CDl lc/CD18 integrin is referred to as "pi 50,95" (alternatively, "CR4" or "α β2"). The fourth member of the family, defined by the αd subunit, is described in U.S. Patent No. 5.437,958. See also,

El-Gabalawy et α/., Arthritis and Rheumatism, 39: 1913-1921 (1996).

Numerous murine hybridomas have been generated which produce monoclonal antibodies (mAbs) of varying isotype characterized as binding specifically to the common β chain of the leukocyte integrins. These include, for example, mAb 1B4 (IgG2a; Wright et al., Proc. Nat'l. Acad. Sci. USA, 80:5699-5103 (1983)); mAb 60.3 (IgG2a; Beatty et al.. J. Immunol., 737:2913-2918 (1983)); mAb TS1/18 (IgGl; Sanchez-Madπd et al., J. Exp. Med., 755: 1785-1803 (1983)); mAb H52 (IgGl. K; Hildreth et al.. Science, 244: 1015-1018 (1989); and ATCC TIB 218 (IgG2a, K; Springer et al., J. Exp. Med., 755:586-602 (1983). Production of chimeric and humanized monoclonal antibodies characterized as binding specifically to human CD 18 is referred to in European Patent

Application 440 351 A2.

Monoclonal antibodies against the common β-chain of the leukocyte integrins completely inhibit adherence of polymorphonuclear cells (PMNs) to un-activated endothelial cells and certain matrix proteins in vitro. Harlan et al.. Blood, 66:167-178 (1985); Zimmerman et al., J. Clin. Invest., 81:531-531 (1988); Bohnsack et al. J. Exp.

Med.. 777:1221-1237 (1990); Luscmskas et al.. J. Immunol, 142:2251-2263 (1989). Moreover, the systemic administration of anti-leukocyte integπn antibodies inhibits tissue accumulation of PMNs. Springer et al.. Nature. 346:425-434 ( 1990); Jutila et al.. Transplantation, 48:121-131 (1989); Arfors et al.. Blood, 60:338-340 (1987); Price et al., J. Immunol., 139:4X 14-4X 11 ( 1987); Doerschuk et al., J. Immunol., 144:2321-2333

( 1990).

Hernandez et al.. Am. J. Physwl, 5 :H699 ( 1987) investigated whether PMNs mediate the increase in microvascular permeability produced by ischemia-reperfusion (I/R) by treating cats with either saline, anti-neutrophil serum (ANS). or mAb 60.3. The results indicated that both PMN depletion and prevention of PMN adherence significantly attenuated the increase in microvascular permeability induced by I/R. and that prevention of neutrophil adherence with mAb 60.3 afforded protection against I/R-induced microvascular injury. See also. U S. Patent No. 4,191.211. Similarly, Vedder et al . J Clin Invest . 81:939 (1988) concluded that increased leukocyte adhesiveness plays an important role in the development of multiple organ injury and death after general lschemia-reperfusion and that this injury may be significantly reduced by blocking leukocyte adherence functions with the mAb 60 3

In addition to U.S. Patent No 4,797.277, which addresses methods for lschemia-reperfusion therapy with anti-leukocyte-integπn antibody preparations, a number of quite generalized proposals for therapeutic uses of such antibodies have been made. U.S Patent No 4,935,234 proposes use of an antibody to Mol (CDl lb/CD18) to reduce tissue injury mediated by inflammatory phagocytic leukocytes duπng myocardial infarction or myocardial reperfusion caused by thrombolytic treatment or balloon catheteπzation PCT WO 91/19511, published December 26, 1991, proposes use of an "LFA-1 antagonist" in the treatment of a number of inflammatory responses, including atherosclerosis. PCT WO 94/12214 projects utility for humanized anti— CD 18 antibodies m the treatment of a vaπety of conditions, including lschemia-reperfusion injury caused by myocardial infarction, coronary artery bypass surgery, elective angioplasty, and the like; sepsis; autoimmune diseases, transplant rejection and neoplasia, among others U.S Patent No. 5,147,637 proposes use of anti-CDl 8 antibodies to inhibit leukocyte influx into organs duπng sepsis or other infectious or non-infectious trauma.

European Patent Application No. EP 440 451 illustrates usefulness of recombmant humanized antι-CD18 antibodies in the treatment of rabbit dermal inflammation but projects utility in treatment of inflammation in certain other tissues including endocardium U S Patent No 4,797,277 to Arfors discloses a method of reperfusion therapy using an antibody preparation having specificity for a "leukocyte adhesion complex (LAC)" epitope responsible for leukocyte-endothehal adherence The LAC according to Arfors consists of three subumts. LFA-1, Mac-1 , and pi 50,95 This method is said to be useful in treating conditions that involve lschemia/reperfusion-mduced tissue damage, such as coronary infarction, organ transplantation, and shock This document does not appear to disclose or suggest any method for treating vasospasm Arfors et al , supra, descπbes a study of the effect of the antibody mAb 60.3 on induced PMN accumulation in vivo It was found that both PMN accumulation and PMN-dependent plasma leakage were abolished in the inflammatory skm lesions of rabbits treated with mAb 60.3 pπor to intradermal injection with the chemotactic factors f-Met-Leu-Phe (fMLP), leukotπene (LTB4), and C5a. Doerschuk et al , supra, reported that mAb 60.3 inhibited PMN emigration in response to peπtoneal inflammation in rabbits induced by S pneumoniae, hydrochloπc acid, E coli endotoxm or PMA, but that this antibody exhibited different effects at pulmonary sites of inflammation.

U.S. Patent No 5,147,637 to Wπght et al discloses a method of inhibiting leukocyte infiltration into lung and other organs duπng sepsis or other infectious or non-mfectious trauma The method includes administeπng to a patient a therapeutic amount of antι-CD18 antibody in conjunction with an anti-mfective agent This document discloses the method as being useful to treat several inflammatory disease states, but does not disclose or suggest any method for treating vasospasm U.S. Patent No 5,002,869 to Schlossman et al discloses a monoclonal antibody that is specific to an epitope of LFA-1. This antibody is said to permit differential identification of killer effector cells and suppressor effector cells m human T8 lymphocyte populations

Kling et al , Arteriosclerosis and Thrombosis, 72:997-1007 (1992) reported that, in rabbits subjected to electπcal stimulation of the carotid arteries, treatment with the monoclonal antibody 60 3 completely abolished neutrophil emigration into the stimulated artenes but only partially inhibited mononuclear leukocyte accumulation in the intima, and did not prevent SMC migration into the mtima. Khng et al , Circ Re , 9 77-1121-1 128 (1995) also reported that treatment of these rabbits with antιVLA-4 antibody HP 1/2 inhibited mononuclear leukocyte invasion by about 70% without affecting neutrophil infiltration, and resulted m an approximately 50% reduction in SMC migration, the addition of 60.3 antibody to the HP1/2 antibody treatment completely abolished the influx of monocytes, lymphocytes and all types of granulocytes into the intima and resulted in a 70% reduction in intimal SMC accumulation Guzman et al Coronan Arten Dis , 6-693-701 (1995) report that, m an atherosclerotic rabbit arteπal injury model, administration of antι-CD18 monoclonal antibody, R15 7, after balloon angioplasty resulted in no observed difference in lumen diameter and percentage stenosis on angiography, and no observed difference in the cross-sectional areas of vascular mtima and media

In an approach relying on another class of adhesion molecule, U S Patent No 5,632,991 to Gimbrone, Jr discloses a use of an immunoconjugate of anti-E-selectin

(antι-ELAM-1) antibody with a therapeutic agent m a method to target the agent to activated endothehum It is said that the method may be useful in the treatment of a vascular smooth muscle disorder, vasculitis, inflammation, post-reperfusion injury, and the like Thus, while it appears that inflammatory processes may be involved in the pathogenesis of post-hemorrhagic vasospasm, it is not at all clear which elements if any are centrally involved, or how such pathogenesis can be effectively ameliorated Furthermore, Applicants are not aware of any descπption of the use of an antι-CDl l/CD18 antibody in the inhibition of vasospasm in mammals, especially humans Accordingly, there is a continuing need for safe and effective methods and mateπals for preventing and treating vasospasm, such as that associated with subarachnoid hemorrhage

SUMMARY OF THE INVENTION The present invention provtdes novel methods for prophylactically or therapeutically treating mammals at risk of vasospasm such as that which occurs association with sub-arachnoid hemorrhage The mammal may be treated with a therapeutically effective amount of an anti-CDl 1/CD18 antibody substance effective to reduce vasospasm. such as an amount of an anti-CDl 1/CD18 antibody substance effective to inhibit adhesion of monocytes or macrophages to vascular endothehum, or an amount of an anti-CDl 1/CD18 antibody substance effective to inhibit transmigration of monocytes or macrophages through vascular endothehum Alternatively stated, the present invention also addresses use of an anti-CDl 1/CD18 antibody substance in the manufacture of a medicament for the prophylactic or therapeutic treatment of vasospasm. resulting m reduction of vasospasm

Thus, the invention is a method for prophylactically or therapeutically treating vasospasm in a mammal, compπsing admimsteπng to the mammal an amount of an antι-CDl l/CD18 antibody substance sufficient to inhibit onset or progression of vasospasm in the mammal. The method is useful in treating, inter alia, cerebral vasospasm, and is useful m treating vasospasm associated with subarachnoid hemorrhage. Vasospastic events involving blood vessels in other organs or tissues may also be treated. The method can be practiced in any mammal, and is preferably practiced humans.

The antι-CDl l/CD18 antibody substance can comprise a complementaπty determining region of an antibody secreted by hybπdoma cell line 23F2G (ATCC No. HB 1 1081). Alternatively, the anti-CDl 1/CD18 antibody substance is an antibody secreted by hybπdoma cell line 23F2G (ATCC No. HB 11081). Preferably, the antibody substance compπses a human or humanized antibody A highly prefeπed antibody substance compπses the humanized antibody produced by a Hu23F2G cell line (ATCC Nos. CRL 1 1397 and CRL 1 1398). The antι-CDl l/CD18 antibody substance may compπse and antibody substance that competes for binding with a monoclonal antibody produced by a cell line selected from the group consisting of cell line ATCC No. HB 11081, cell line ATCC No. CRL 1 1397, and cell line ATCC No. CRL 1 1398.

According to the invention, the anti-CDl 1/CD18 antibody substance can be administered to the mammal either prior to or subsequent to onset of the vasospasm. The method may further compπse admimsteπng to the mammal an adjuvant substance selected from the group consisting of vasodilators, anti-mflammatoπes, calcium antagonists, lmmunosuppressants, potassium channel activators. PAF inhibitors, neuropeptide Y receptor inhibitors, complement-depleting substances, haptoglobm-contaming substances, substances that interfere with leukocyte binding to endothehal tissue, substances that interfere with leukocyte infiltration of endothehal tissue, and the like. Thus, prefeπed adjuvant substance can be selected from the group consisting of anti-ICAM antibodies, antι-LFA-1 antibodies. anti-Mac- 1 antibodies, anti-p 150,95 antibodies, and antι- dβ2 antibodies

The admmisteπng step can compπse admmisteπng the anti-CDl 1/CDl 8 antibody substance by intravascular miection Alternatively, the administering step can compnse regionally delivering the anti-CDl 1/CD18 antibody substance to a site subject to vasospasm by selective catheteπzation of the involved vessel and injection or infusion of the anti-CDl 1/CD18 antibody substance through the catheter

The anti-CDl 1/CD18 antibody substance can be administered at a dose between about 1 μg kg to about 100 mg/kg of body weight Preferably, the anti-CDl 1/CD18 antibody substance is administered at a dose between about 1 mg/kg to about 4 mg/kg of body weight

In another embodiment, the invention is a method of prophylactically or therapeutically treating vasospasm in a mammal, comprising admmisteπng to the mammal a prophylactically or therapeutically effective amount of an anti-CDl 1/CD18 antibody substance that competes for binding with monoclonal antibody 23F2G produced by hybπdoma cell line 23F2G (ATCC No. HB 1 1081 )

In another embodiment, the invention is a method of prophylactically or therapeutically treating vasospasm in a mammal, compπsmg admmisteπng to the mammal an amount of an antι-CDl l/CD18 antibody substance effective to inhibit adhesion of leukocytes to vascular endothehum

In still another embodiment, the invention is a method of prophylactically or therapeutically treating vasospasm m a mammal, compπsmg admmisteπng to the mammal an amount of an anti-CDl 1/CD18 antibody substance effective to inhibit transmigration of leukocytes through vascular endothehum Numerous additional aspects and advantages of the invention will become apparent to those skilled m the art upon consideration of the following detailed descπption of the invention which descπbes presently prefeπed embodiments thereof

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In accordance with the present invention, there is provided a method for prophylactically or therapeutically treating a mammal at risk for or suffering from vasospasm The method compπses admmisteπng an anti-CDl 1/CD18 antibody substance to the mammal in an amount sufficient to achieve significant reduction in vasospasm or its clinical indicia By "antι-CDl l/CD18 antibody substance" is meant a protein, preferably an lmmunoglobu n or fragment thereof, that has significant binding affinity for (lmmunoreactivity with) an epitope common among the CDl 1/CD18 complexes or an epitope common among the heterodimeπc human leukocyte integπns The anti-CDl 1/CDl 8 antibody substance should have no substantial immunoreactivity with the monomers of the integπns except when the monomers are combined into their heterodimeπc state Thus, an anti-CDl 1/CD18 antibody substance according to the invention is substantially differentiated from substances that bind CD11 or CDl 8 monomers Alternatively, an antι-CDl l/CD18 antibody substance according to the present invention has substantial binding affinity for a CD 1 1 /CD 18 heterodimer irrespective of which CDl 1 isoform is involved For example, an antibody that uniquely binds only one of LFA-1, Mac-1, and pi 50,95 would not constitute an anti-CDl 1/CD18 antibody as defined herein

An exemplary anti-CDl 1/CD18 antibody substance is the 23F2G antibody (IgG2a isotype) produced by the hybπdoma cell line 23F2G deposited with the Ameπcan Type Culture Collection (ATCC), 12301 Parklawn Dπve, Rockville, M D 20852 U S A on June 30, 1992 under Accession No HB 11081 The 23F2G antibody is known to immunoprecipitate LFA-1 , Mac-1, pl50,95, and αdβ2, making this antibody and engineered vaπants of this antibody highly prefeπed for use in the method of the invention

Prefeπed antibodies specifically contemplated for practice of the invention are monoclonal antibody 23F2G of the, monoclonal antibodies which compete with monoclonal antibody 23F2G for binding to LFA-1 or other leukocyte integπns, and antibodies compπsmg one or more CDRs from the 23F2G antibody For administration m humans, it is contemplated that the anti-CDl 1/CD18 antibodies will be "humanized" (I e , be made more human by generating counterpart chimeπc or CDR-grafted antibodies, with or without accompanying framework ammo acid modifications) The

23F2G antibody produced by ATCC No HB 1 1081 has been "humanized." and transformed mammalian cells expressing humanized forms of the mouse 23F2G antibody (/ e , "Hu23F2G") have been deposited under ATCC Accession Nos CRL 1 1397 and CRL 1 1398 Accordingly, prefeπed antibody substances according to the invention also include the humanized Hu23F2G antibodies The 60 3 antibody may also be employed

The anti-CDl 1/CDl 8 antibody substances according to the methods of the present mvention may be selected from among, for example, monoclonal and polyclonal antibodies, antigen-binding antibody fragments, single chain antibodies, chimeπc and/or complementaπty determining region (CDR)-grafted antibodies with or without accompanying framework ammo acid modifications, and the like Antibodies may be of any class or subclass including IgG, IgA, IgD, IgE, and/or IgM

"Vasospasm" may be defined as delayed chronic lumenal narrowing of blood v essels m response to a stimulus such as peπ-adventitial contact with blood Two phases of vasospasm are recognized, a short term phase, designated "acute vasospasm," and a longer term phase designated "chronic vasospasm " Acute vasospasm appears to be mediated by contraction of the smooth muscle coat of the vessel By contrast, the mechanisms underlying chronic vasospasm are not well understood, but may involve inflammatory events "Cerebral vasospasm" refers to vasospasm occurπng m the bram Cerebral hemoπhage typically occurs m response to subarachnoid hemorrhage such as that following rupture of an aneurysm, and is a major cause of morbidity and mortality following such events

In one embodiment, the invention is a method of treating a mammal subject to hemoπhage, compπsmg steps of treating the individual surgically or pharmaceutically to ameliorate the immediate effects of the hemoπhage and subsequently admmisteπng an effective amount of an anti-CDl 1/CDl 8 antibody as descπbed herein For example, in the case of hemoπhagic stroke or aneurysmal rupture incidental to surgery, immediate management of the hemoπhagic event may be required, with subsequent administration of the antι-CDl l/CD18 antibody employed as a prophylactic measure to stabilize the individual and to prevent the delaved phenomena characteπstic of vasospasm Alternatively, in management of cerebral aneurysm, an anti-aneurysmal agent (e g , a matπx metalloproteinase inhibitor) may be administered together with or followed by administration an anti-vasospastic amount of an antι-CDl l/CD18 antibody

While the invention is of particular benefit m ameliorating injury associated with vasospasm attendant upon hemoπhage in the bram, vasospasm may occur elsewhere in the body and in response to other stimuli such as traumatic hemoπhage, and may have significant clinical consequences Accordingly, the invention can be expected to have utility in treatment of vasospasm in other regions of the body The invention is also expected to be beneficial in treatment of related vascular conditions, such as conditions characteπzed by vaπcosities, e g , vaπcose veins and the like.

The method of the invention compπses admimsteπng an anti-CDl 1/CDl 8 antibody substance to a mammal Mammals include, for example, humans and other primates, farm animals such as cows, pigs, horses, goats and sheep, pet or companion animals such as dogs and cats, as well as animals commonly studied m the laboratory, such as rats, mice, and rabbits, and the like. Preferably, the mammal is a human.

Administration of antι-CDl l/CD18 antibody substances may be expected to reduce vasospasm by, e g , decreasing the incidence of vasospasm, reducing the physical extent of vasospasm, decreasing the seventy of vasospasm, and/or slowing the progression of vasospasm Thus, the method can compπse admimsteπng to the mammal an amount of an antι-CDl l/CD18 antibody substance that will show efficacy in inhibiting adhesion of leukocytes to vascular endothehum. Alternatively, the method can comprise admmisteπng to the mammal an amount of an antι-CDl l/CD18 antibody substance effective to inhibit transmigration of leukocytes through vascular endothehum.

Pharmaceutical compositions suitable for administration according to the invention compπse antι-CDl l/CD18 antibody substances with a physiologically acceptable excipient, diluent, or earner, and may optionally include other appropπate therapeutic agents, e g , antibodies to other cellular adhesion molecules, antibodies to growth factors, or chemokines or cytokmes involved m macrophage/monocyte migration and activation

It is also contemplated that the administration of pharmaceutical compositions compπsing antι-CDl l/CD18 antibody substances as the sole active agent may be accompanied by the concuπent administration of other (adjuvant) therapeutic substances. Adjuvant substances may be employed that provide complementary effects, such as substances that have direct or indirect effect on the onset or progression of vasospasm. or substances that have a direct or indirect effect on symptoms or sequelae of vasospasm Such substances may contπbute to improvement of the outcome or status of the mammal treated according to the invention For example, such adjuvant substances may be selected from among vasodilators (e g , mtπc oxide donors), phosphodiesterase inhibitors

(e g , papaveπne, zapπnast, sildenafil. the compounds disclosed m PCT publication WO 95/19978 published 7/27/95, WO 96/32003 published 10/17/96, WO 97/03985 published 2/6/97, and WO 97/43287 published 1 1/20/97), anti-inflammatones (e g , lbuprofen, methylpredmsolone), calcium antagonists {e g , nimodipine, mcardipme), immunosuppressants (e g , cyclospoπn A, dexamethasone), potassium channel activators (e g , nicorandil), PAF inhibitors, neuropeptide Y receptor inhibitors, complement-depleting substances (e g , cobra venom factor), matnx metalloprotemase inhibitors, and haptoglobin-contammg substances Suitable adjuvant substances also include substances that that interfere with leukocyte binding to endothehal tissue and substances that interfere with leukocyte infiltration of endothehal tissue Prefeπed adjuvant substances include anti-ICAM antibodies, antι-LFA-1 antibodies, anti-Mac- 1 antibodies, anti-p 150,95 antibodies, and antι-αdβ2 antibodies Appropπate doses and modes of administenng the adjuvant substance(s) in combination with an anti-CDl 1/CDl 8 antibody substance are readily appreciated by those skilled in the art

Pharmaceutical compositions compπsmg anti-CDl 1/CDl 8 antibody substances may be administered by any route suitable for the condition to be treated Thus, administration can be accomplished via subcutaneous, intramuscular, intravenous, intraarteπal, lntracoronary, intraatπal, intrapulmonary, intraocular, intrapeπtoneal, intradermal, transdermal, mtrathecal, oral, topical, suppository, or other route of administration A prefeπed route of administration is intravenous, which may be via continuous infusion or via intermittent injection or infusion In some instances, it is advantageous to deliver the drug controllably to a selected region, i e , to the site of mechanical vascular injury, by catheteπzation of the involved vessel and injection or infusion of the drug through the catheter

Dosages of anti-CDl 1/CDl 8 antibody substance will vary between about 1 μg/kg and about 100 mg/kg body weight, and preferably between about 1 mg/kg and about 4 mg/kg body weight Administration of anti-CDl 1/CDl 8 antibody substances may be administered pπor to or after mechanical vascular injury and may be repeated at 48 hours or other intervals for a week or more to obtain the desired effect Preferably, high levels of antibody substance (e g , 80% leukocyte cell surface CDl 1/CDl 8 saturation) will be maintained for about one to about six weeks Optimal dosages for different anti-CDl 1/CDl 8 antibody substances may be determined bv the duration of high levels of leukocyte saturation as determined by conventional assay methods, e g , a flow cytometry assay descπbed m Example 1 below One exemplary dosing schedule for the andCDl 1/CDl 8 antibody substance is 1 5 mg/kg given every three days for two weeks The doses of the anti-CDl 1/CDl 8 antibody substance may be increased or decreased, and the duration of treatment may be shortened or lengthened as determined by the treating physician The frequency of dosmg will depend on the pharmacokmetic parameters of the agents and the route of administration The optimal pharmaceutical formulation will be determined by one skilled in the art depending upon the route of administration and desired dosage See, for example, Remington 's Pharmaceutical Sciences. X 8th Ed , Mack Publishing Co , Easton, PA (1990), at pages 1435-1712 Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered agents

Those of ordinary skill m the art will readily optimize effective dosages and concuπent administration regimens as determined by good medical practice and the clinical condition of the individual patient For example, the optimal duration of therapy may be shortened in patients with significant clinical signs that contramdicate continued administration of the anti-CD 11 /CD 18 antibody substance Regardless of the manner of administration, the specific dose may be calculated according to body weight, body surface area, and/or organ size Further refinement of the calculations necessary to determine the appropπate dosage for treatment involving each of the above mentioned formulations is routinely made by those of ordinary skill in the art without undue experimentation, especially in light of the dosage information and assays disclosed herein, as well as the pharmacokmetic data observed in in vitro and in vivo studies Appropπate dosages may be ascertained through use of established assays for determining blood levels dosages in conjunction with appropriate dose-response data

The final dosage regimen will be determined by the attending physician, consideπng various factors which modify the action of drugs, e g the drug's specific activity, the seventy of the damage and the responsiveness of the patient, the age, condition, body weight, sex, and diet of the patient, other clinical conditions, time of administration and other clinical factors As studies are conducted, further information will emerge regarding the appropnate dosage levels for the treatment of vaπous diseases and conditions.

The examples provided herein below illustrate experimental methods that are useful in demonstrating the method of the invention.

EXAMPLES

Example 1

The ability of anti-CD 11 /CD 18 antibody to inhibit vasospasm is demonstrated using a rat surgical model of vasospasm as adapted from procedures described by Oshiro et al., Stroke, 28(1 :2031 -2037 (1997).

Surgical Procedure

Blood exposure of rat femoral arteries is performed according to the method described by Okada et al, Neurosurgery, 27:349-356 (1990). Femoral arteries are isolated by microsurgical dissection in the inguinal region of adult male Sprague-Dawley rats (200-250 g), anesthetized by intraperitoneal injection (3 mg/kg) of a stock solution of ketamine (25 mg/mL), xylazine (2.5 mg/mL), and ethanol (14.2%) in 0.9%) saline. The isolated arteries are enclosed in an 8 x 8 mm latex pouch, sealed loosely with cyanoacrylate glue. Autologous whole blood is harvested from the adjacent femoral vein, allowed to clot partially, and injected into the pouch. In the same animal, the contralateral femoral artery is prepared similarly, except that normal saline is injected into the pouch instead of the blood, thus serving as an internal control.

Antibody Treatment

The group of rats is randomly divided into three subgroups; Group 1 receives intraperitoneal injections of anti-CDl 1/CDl 8 antibody (2 mg/kg/dose); Group 2 receives isotype-matched control monoclonal antibody (2 mg/kg/dose); and Group 3 receives saline solution. Each group receives doses administered at 3 hr, 3 d, and 6 d, and 9 d following surgery. The animals are euthanized 12 d after surgery. Blood Vessel Preparation

In selected animals, the femoral arteries are harvested for routine histology and for measurement of arterial lumen cross-section areas. Femoral arteries are fixed in situ with 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS, pH 7.4), via cannulation of the aorta. The arteries are then dissected out of the latex pouches, and immersion fixed in 4% paraformaldehyde for an additional 24 hr at 4°C. Following fixation, the vessels are placed in 20%> sucrose at 4°C for 24 hr for cryoprotection, and then frozen in tissue-embedding matrix by dry-ice equilibrated isopentane at -50°C, and stored at -40°C until sectioning.

Measurement of Blood Vessel Cross-Sectional Areas

The vessels are sectioned (10 μm) in a transverse orientation using a cryostat, and then stained with hematoxylin and eosin. Lumen cross-sectional areas are determined by computerized image analysis using publicly available software. To coπect for vessel deformation and off-transverse sectioning, the areas are determined by measuring the circumference of the vessel lumen and calculating the area as an idealized circle based on the measured circumference. For each vessel, five separate sections at least 200 μm apart are measured and averaged. For comparisons among treatment groups, the ratios of lumenal cross-section areas of blood-exposed arteries to saline-exposed arteries in the same animal are calculated. Comparison of group mean ratios is performed using one-way ANOVA.

The mean ratios of blood-exposed to saline-exposed lumen cross-sectional area, (expressed as percent lumen patency) are substantially greater for the animals treated with anti-CDl 1/CDl 8 antibody, as compared with the ratios for the animals treated with isotype-matched control antibody and the saline control animals. This difference in lumen patency represents an inhibition of vasospasm that is statistically significant by one-way ANOVA. No significant systemic toxicity is attributable to the anti-CDl 1/CDl 8 antibody treatment, and there is no apparent inhibition of surgical wound healing or increase in wound infections in the animals treated with this antibody. Histologic examination of the blood vessels with hematoxyhn/eosin staining does not reveal any significant deleteπous effects of anti-CDl 1/CDl 8 treatment compared with either of the control treatments

Immunohistochemical Staining

Infiltration of macrophages and granulocytes into the blood-exposed and control femoral arteπes is evaluated using conventional immunohistochemical techniques The vessels are sectioned (10 μm) in a transverse oπentation using a cryostat, with at least 100 μm between sections The slides are hydrated in PBS, then incubated m 3% normal horse serum for 20 mm Commercially available mouse anti-granulocyte/macrophage antibody, diluted in PBS with 1 0% bovine serum albumin (BSA) and 3% normal horse serum, is applied to each section and incubated for 45 mm in a humidified chamber After washing with PBS, the slides are incubated with biotmylated, horse anti-mouse IgG in PBS with 1% BSA and 3% normal horse serum for 45 mm The slides are washed in PBS, then incubated with avidin and biotmylated horseradish peroxidase macromolecular complex for 30 mm The slides are then washed in PBS and incubated for 8 mm with peroxidase substrate Finally the slides are washed in tap water, counterstained with nuclear fast red, and dry mounted Macrophage/granulocyte counts are obtained by identifying all positive staining cells per representative high-powered field (hpf) m a region adjacent to the adventitia of blood-exposed arteπes For each animal. 3 hpf sections of the blood-exposed artery are counted Cell counts for the anti-CDl 1/CDl 8-trated and isotype matched control groups are compared using a two-group t-test

Immunohistochemical staining for macrophages and granulocytes reveals a substantial decrease in inflammatory cell infiltrate in the region of the pen-adventitial blood clot in the animals treated with the anti-CDl 1/CDl 8 antibody compared to that of control animals 24 hr after blood exposure This difference is statistically significant by two-group t-test CD1 1/CDl 8 Saturation Determination

To determine whether in vivo anti-CDl 1/CD18 mAb injections are saturating all CDl 1/CDl 8 sites, splenocytes from antι-CDl l/CD18-treated and control antibody-treated animals are exposed to antιCDl l/CD18 mAb in vitro followed by fluorescent-labeled secondary antibody. If all CDl 1/CDl 8 sites are saturated by the in vivo antι-CDl l/CD18 treatment, then no additional staining should be seen when antι-CDl l/CD18 mAb is added.

Spleens from anti-CDl 1/CDl 8-treated animals and isotype-matched control animals are harvested at 12 d after surgery and mmced in a steπle petπ dish with nutπent medium The resulting splenocyte suspension is washed with medium and pelleted by centπfugation at 1500 rpm at 4°C The pellet is then resuspended in a lysis buffer (0.15 M NH4C1, 1.0 M KHCO3, 0 1 mM Na2EDTA, pH 7 4) and incubated at room temperature for 5 mm to lyse the red blood cells. The cells are washed 3 times with medium and resuspended in flow cytometry wash buffer (2% calf serum, 10 mM HEPES, 0.1%) NaN3, in Hanks' Balanced Salt Solution). For each animal, two a quots of 1 x 106 cells are pelleted by centπfugation at 1000 rpm for 5 mm at 4°C. One of these ahquots from each animal is incubated with additional antι-CDl l/CD18 antibody (1 μL; 3.2 mg/mL) for 20 mm on ice to fully saturate CDl 1/CDl 8 sites pπor to flow cytometry Both ahquots of cells are then incubated with phycoerythπn (PE)-conjugated anti-human IgG for 20 mm on ice. After incubated the cells are washed and resuspended in 0 5 mL of wash buffer, and sampled by flow cytometry CDl 1/CDl 8 saturation is determined by compaπson of flow cytometry histograms for cell samples with and without additional anti-CDl 1/CDl 8 antibody

Flow cytometry results show that the CDl 1/CDl 8 sites are saturated in the animals treated with the anti-CDl 1/CDl 8 antibody Specifically, splenocytes harvested from isotype-matched control antibody-treated animals demonstrate a shift in the flow histograms after the addition of in vitro anti-CDl 1/CDl 8 antibody, indicating that the CDl 1/CDl 8 sites are not saturated in these animals No comparable shift is observed in the samples acquired from the anti-CDl 1/CDl 8-treated animals when anti-CDl 1/CDl 8 antibodv is added m vitro Example 2

A primate surgery model of vasospasm can be used to demonstrate the effectiveness of anti-CDl 1/CDl 8 therapy in reducing vasospasm according to the invention. Adult male cynomologous monkeys (Macaca fascicularis) are obtained from a commercial source. The animals are quarantined for 45 days, and tested for tuberculosis several times. The animals are assigned into two groups; half are assigned as controls, and half are assigned to receive the experimental treatment. Experiments are conducted using pairs of animals (one control and one test animal). Prior to surgery each animal is anesthetized and an blood is drawn to establish baseline status, including baseline levels of ICAM-1. Cerebral angiography is then performed to established baseline status. Following the angiography, the animal is removed to the operating room. A right fronto-temporal craniotomy is performed, the right Sylvian fissure is opened, and the right carotid, middle cerebral, and anterior arteries are microsurgically exposed. Venous blood is then injected around the exposed arteries, and the craniotomy is closed. The animal is then removed to its cage to recover. All animals are checked twice daily to assess general health and wound healing.

Following surgery each animal in the treatment group is administered monoclonal anti-CDl 1/CDl 8 antibody (e.g., Hu23F2G) via intravascular injections at three hours, three days, and six days post-operation. Each animal in the control group is administered nonspecific antibody via intravenous injection at the same three times. Venous blood is drawn from the animals at three and six days post-operation to check serum ICAM-1 levels.

All animals are subjected to follow-up angiography at eight days post-operation, to assess development of vasospasm, and the animals are euthanized following angiography. Cerebral vessels are harvested for histological and immunological study.

Angiography indicates that animals treated with anti-CDl 1/CDl 8 antibody have statistically significant greater vessel lumenal diameter than animals in the isotype-matched control group. Histologic examination reveals that macrophage and granulocyte infiltration of the vessels is significantly decreased in the anti-CDl 1/CDl 8-treated animals relative to the control animals. The experimental results described in the Examples demonstrate that administration of anti-CDl 1/CDl 8 antibody according to the invention is effective in reducing onset or progression of vasospasm in vivo

All publications and patent documents mentioned heremabov e are incorporated herein by reference for all that they disclose

While the present invention has been descπbed with specific reference to certain prefeπed embodiments for purposes of claπty and understanding, it will be apparent to the skilled artisan that certain changes and modifications may be practiced withm the scope of the invention as it is defined m the claims presented below Accordingly, no limitations should be placed on the invention other than those recited in the claims

Claims

WHAT IS CLAIMED IS:
1. A method for prophylactically or therapeutically treating vasospasm in a mammal, compπsing admmisteπng to the mammal an amount of an anti-CDl 1/CDl 8 antibody substance sufficient to inhibit onset or progression of vasospasm in the mammal.
2. A method according to Claim 1, wherein the anti-CDl 1/CDl 8 antibody substance comprises a complementaπty determining region of an antibody secreted by hybπdoma cell line 23F2G (ATCC No. HB 1 1081).
3. A method according to Claim 2, wherein the anti-CDl 1/CDl 8 antibody substance is an antibody secreted by hybπdoma cell line 23F2G (ATCC No. HB 1 1081).
4. A method according to Claim 2, wherein the anti-CDl 1/CDl 8 antibody substance is a humanized antibody secreted by cell line ATCC No CRL 1 1397 or cell
Figure imgf000022_0001
5 A method according to Claim 1, wherein the anti-CDl 1/CDl 8 antibody substance that competes for binding with a monoclonal antibody produced by a cell line selected from the group consisting of cell line ATCC No. HB 1 1081, cell line ATCC No. CRL 1 1397, and cell line ATCC No. CRL 1 1398.
6 A method according to Claim 1, wherein the anti-CDl 1/CDl 8 antibody substance is administered to the mammal pπor to onset of the vasospasm
7 A method according to Claim 1 , wherein the anti-CDl 1/CDl 8 antibody substance is administered to the mammal subsequent to onset of the vasospasm
8 A method according to Claim 1. wherein the vasospasm compπses cerebral vasospasm
9 A method according to Claim 8, wherein the vasospasm is associated with subarachnoid hemoπhage
10 A method according to Claim 1 , wherein the mammal is a human
11 A method according to Claim 1. further comprising admi steπng to the mammal an adjuvant substance selected from the group consisting of vasodilators, anti-inflammatones, calcium antagonists, immunosuppressants, potassium channel activators, PAF inhibitors, neuropeptide Y receptor inhibitors, complement-depleting substances, haptoglobm-contam g substances, substances that interfere with leukocyte binding to endothehal tissue, and substances that interfere with leukocyte infiltration of endothehal tissue
12 A method according to Claim 11. wherein the adjuvant substance is selected from the group consisting of anti-ICAM antibodies, antι-LFA-1 antibodies, antι-Mac-1 antibodies, anti-p 150,95 antibodies, and antι-adb2 antibodies
13 A method according to Claim 1 , wherein the admmistenng step compnses admimstenng the anti-CDl 1/CDl 8 antibody substance by mtravascular injection
14 A method according to Claim 13. wherein the admmistenng step compπses regionally dehveπng the anti-CDl 1/CDl 8 antibody substance to a site subject to vasospasm by selective catheteπzation of the involved vessel and injection or infusion of the anti-CDl 1/CDl 8 antibody substance through the catheter
15 A method according to Claim 1. wherein the anti-CDl 1/CDl 8 antibodv substance is administered at a dose between about 1 μg/kg to about 100 mg/kg of body weight
16. A method according to Claim 15, wherein the anti-CDl 1/CDl 8 antibody substance is administered at a dose between about 1 mg/kg to about 4 mg kg of body weight.
PCT/US2000/018323 1999-07-06 2000-06-30 Method and composition for inhibition of vasospasm WO2001002009A1 (en)

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EP1323976A2 (en) 2001-12-23 2003-07-02 Der Kluth: Decke und Licht GmbH Lighting apparatus

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BAVBEK ET AL.: "Monoclonal antibodies against ICAM-1 and CD18 attenuate cerebral vasospasm after experimental subarachnoid hemorrhage in rabbits", STROKE, vol. 29, 1998, pages 1930 - 1936, XP002932946 *
HANDA ET AL.: "Expression of intercellular adhesion molecule 1 (ICAM-1) on the cerebral artery following subarachnoid haemorrhage in rats", ACTA NEUROCHIR., vol. 132, 1995, (WIEN), pages 92 - 97, XP002932944 *
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EP1323976A2 (en) 2001-12-23 2003-07-02 Der Kluth: Decke und Licht GmbH Lighting apparatus

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