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  Pharmaceutical Patents  

 

Title:  Therapy for cerebral vasospasm
United States Patent: 
7,498,332
Issued: 
March 3, 2009

Inventors:
 Niklason; Laura E. (Hillsborough, NC), McKee; Andy (Durham, NC), Borel; Cecil (Chapel Hill, NC)
Assignee:
  Duke University (Durham, NC)
Appl. No.: 
10/074,250
Filed: 
February 14, 2002


 

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Abstract

The present invention relates to a method of treating or preventing cerebral vasospasm that accompanies sub-arachnoid hemorrhage.

Description of the Invention

TECHNICAL FIELD

The present invention relates, in general, to cerebral vasospasm and, in particular, to a method of treating or preventing cerebral vasospasm that accompanies subarachnoid hemorrhage (SAH).

BACKGROUND

Subarachnoid hemorrhage (SAH), resulting from intracerebral bleeding or from trauma, is a serious neurologic event. Frequent complications of SAH include cerebral infarction (stroke), cerebral edema with increased intracranial pressure, and death.

If a patient survives the initial insult of SAH, a further complication is that of cerebral vasospasm. Cerebral vasospasm is a syndrome that accompanies SAH and generally has peak clinical manifestations at 7-10 days following SAH. The syndrome is characterized by diffuse narrowing of cerebral arteries in the general region of the hemorrhage. Clinically, this arterial narrowing is correlated with the amount of blood that is present in the subarachnoid space. The arterial narrowing can become sufficiently severe that blood flow to previously undamaged brain is compromised, resulting in risk of subsequent stroke without adequate treatment.

Current treatment for vasospasm includes increasing systemic blood pressure and expanding the intravascular space, both of which are correlated clinically with improving symptoms of cerebral ischemia in vasospasm. This therapy necessitates an intensive care unit setting, is not universally successful, and can lead to complications in some patients.

Despite several decades of research, no clear etiology for vasospasm has been elucidated. Multiple general approaches have been taken, including investigation of vasoconstrictors, cytokines, and other pro-inflammatory modulators. None of these putative agents, however, has emerged as clearly causative in the syndrome of vasospasm.

Histologic analyses of cerebral vessels suffering from vasospasm has revealed evidence of vascular cell and adventitial cell proliferation at times of peak arterial narrowing. Vascular cell proliferation is often attributable to locally high concentrations of relevant mitogenic agents, such as growth factors. Intriguingly, blood that is outside the vascular space, such as blood in the subarachnoid space, spontaneously forms clots with concomitant platelet activation. Platelets are known to be repositories of several growth factors, including platelet-derived growth factors, that are potent mitogens for cells in the vascular wall.

The present invention results from the realization that the narrowing of cerebral arteries that is characteristic of cerebral vasospasm is in fact due to proliferation of cells in the vascular wall and/or accumulation of extracellular matrix under the influence of growth factors.

The extracellular matrix contains cross-linked collagen and elastin fibers. Collagen and elastin fibers in the extracellular matrix of blood vessels bear the tensile load in response to pressure from blood flow within the blood vessel. After their synthesis, collagen molecules are processed in the Golgi and endoplasmic recticulum. 4-Prolyl hydroxylase (PH) is the enzyme responsible for hydroxylating residues in collagen molecules (Kivirikko et al, Matrix Biology 16:357 (1998)). This modification permits collagen molecules to associate strongly in small helical fibers. Following production of helically-wound collagen fibers, collagen is excreted from the cell into the extracellular space, where it can be strengthened by spontaneously forming larger fibers ("fibrils") and by being cross-linked. Lysyl oxidase (LO) is the enzyme primarily responsible for cross-linking collagen and elastin fibers once these molecules have been secreted into the extracellular space. (Rucker et al, Am. J. Clin. Nutr. 67(suppl.):9965 (1998).) Collagen fibrils are substantially strengthened with the addition of cross-links between and within fibers that restrict movement of the fibers under tension.

The present invention provides a method of treating or preventing cerebral vasospasm by inhibiting vascular proliferation and/or extracellular matrix synthesis, secretion or strengthening (e.g., collagen fiber formation and self-assembly, as well as molecular cross-linking).

SUMMARY OF THE INVENTION

The present invention relates to a method of preventing or treating narrowing of cerebral arteries ("cerebral vasospasm") that accompanies SAH. The method comprises administering to a patient in need of such prevention or treatment an agent that inhibits vascular cell proliferation and/or extracellular matrix synthesis, secretion or strengthening.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of preventing or treating narrowing of cerebral arteries that accompanies SAH. The method comprises administering to a patient in need of such prevention or treatment an agent that inhibits vascular cell proliferation and/or extracellular matrix synthesis, secretion or strengthening.

Agents suitable for use in the present invention include compounds that inhibit mitogens of cells of the vascular wall, including smooth muscle cells and fibroblasts and pericytes. Suitable agents also include compounds that inhibit stimulators of extracellular matrix production in vascular walls and compounds that otherwise reduce the amount or strength of extracellular matrix in vascular walls. Examples of mitogens targeted in accordance with the invention include growth factors, for example, insulin-like growth factor (IGF), platelet-derived growth factors, endothelial growth factors, fibroblast growth factors (FGF), transforming growth factors (TGF), thrombin and other products of the coagulation and fibrinolytic system that are mitogenic. Examples of extracellular matrix production stimulators include TGF.beta., ascorbate and other growth factors, including connective tissue growth factor (CTGF).

Preferred agents suitable for use in the invention include compounds that neutralize the effect of growth factors responsible for vascular cell proliferation and/or extracellular matrix production by binding to the growth factor, or the receptor therefor, or by otherwise inhibiting the binding of the growth factor to its receptor. Antibodies (e.g., monoclonal antibodies) specific for growth factors that are involved in proliferation of cells of the vascular wall (e.g., growth factors that are elaborated from clots during the first several days following SAH) represent one type of such compounds. Such antibodies are available from a variety of sources, including Chemicon, Inc. and R&D Systems, Inc (e.g., anti-TGF-.beta. receptor antibodies, anti PDGF-AA and -BB antibodies, anti PDGF receptor .alpha. and receptor .beta. antibodies). Retinoids can also be used to inhibit vascular cell growth (see Chen et al, J. Clin. Invest. 102(3):653 (1998); Braunhut et al, J. Biol. Chem. 269(18):13472 (1994); Davidson et al, J. Biol. Chem. 272(1):345 (1997)).

In addition to the above, interruption of the cell cycle/cell proliferation can be effected using chemotherapeutic agents that are safe and efficacious when administered to the CSF. Chemotherapeutic agents that are given orally or intravenously but distribute into the CSF can also be used. Examples of such agents include BCNU (bis(chloroethyl)nitrosourea, which distributes into the CSF following IV administration), methotrexate (which can be admininistered IV or directly into CSF), and 5-fluorouracil (distributes into the CSF). (See also Goodman et al, eds. Goodman & Gilman's The Pharmacological Basis of Therapeutics. New York, McGraw-Hill, Health Professions Division, 1996; Gumerlock and Neuwelt, Blood-brain Barrier Modification in the Delivery of Antitumor Agents. In: Wilkins and Rengachary, eds. Neurosurgery. McGraw-Hill, N.Y. (1996), pages 1967-19071.)

Preferred agents for use in the invention also include compounds that inhibit the synthesis, secretion or strengthening (e.g., via cross-linking) of extracellular matrix, as well as compounds that weaken or degrade extracellular matrix. PH (prolyl-4-hydroxylase) can be inhibited specifically and irreversibly by peptides containing 5-oxaproline (such as benzyloxycarbonyl-Phe-Oxaproline-Gly-benzylester) and by antraacyclines (such as doxorubicin and daunorubicin). PH can also be inhibited using weaker inhibitors (such as competitive inhibitors), including derivatives of 2-oxoglutarate (e.g., coumalic acid, pyridine 2,4 dicarboxylate, pyridine 2,5 dicarboxylate, N-oxalylglycine and 3,4-dihydroxybenzoate). (See Kivirikko et al, Matrix Biology 16:357 (1998).) LO can be inhibited specifically and strongly by small molecules such as .beta.-aminopropionontrile (BAPN), .beta.-bromoethylamine, p-halobenzylamines, ethylenediamine and homocysteine thiolactone. Weaker inhibitors of LO (e.g. competitive inhibitors) can also be used such as hydrazine, dipyridyl, phenylhydrazine and semicarbazide. (See Kagan, Acta Tropica 77:147 (2000).) Examples of compounds that weaken or degrade extracellular matrix include matrix metalloproteinase (MMP) and serine proteases, or mimetics thereof or derivatives thereof.

Agents that are candidates for use in the present methods can be identified using a simple binding assay. For example, a test compound can be contacted with a growth factor that stimulates proliferation or extracellular matrix production (or receptor therefor) and the ability of the test compound to bind the growth factor (or receptor) determined. For example, ELISAs (enzyme-linked immunosorbant assays) can be used. Alternatively, binding assays can take the form of competitive assays in which the ability of a test compound to compete with a receptor for binding to a growth factor is determined.

A candidate compound that is identified as being capable of binding to a growth factor (or its receptor) (or otherwise inhibiting binding of a growth factor to its receptor) can then be further assayed (bio-assayed) for its ability to inhibit cell proliferation and/or extracellular matrix production. Such assays can be conducted, for example, by measuring cell proliferation and/or extracellular matrix production in vitro using segments of excised cerebral arteries exposed to the growth factor in the presence and absence (control) of the test compound. Compounds that inhibit cell proliferation and/or extracellular matrix production can be expected to be useful as active agents in the present prevention/treatment method.

Alternatively, bio-assays can be used as the primary screen, rather than subsequent to a binding assay. As indicated above, bio-assays can be carried out by bringing cells in a blood vessel wall into contact with a growth factor and the test compound to determine if cell growth and/or extracellular matrix production is inhibited in the presence of the test compound.

Assays suitable for determining whether a test compound (proteinaceous or non-proteinaceous) can degrade/weaken extracellular matrix can be carried out using, for example, the Human Type I Collagenase Activity Assay Kit provided by Chemicon International, Inc. Temecula, Calif. 92590. In accordance with the basic principle of this assay, a test compound can be incubated with collagen bound to a solid support under conditions such that degradation of the collagen can occur (when the test compound has collagen degrading activity). A determination can made as to whether degradation of the collagen occurs in the presence of the test compound (as compared to in its absence), for example, by washing the solid support following incubation with the test compound and measuring the amount of collagen that remains attached to the solid support.

Blood vessel cells are capable of producing compounds that degrade/weaken extracellular matrix, MMP-1 being one such compound. Blood vessels cultured in vitro can be exposed to a test compound and the determination made as to whether the test compound stimulates the release of a compound(s) (e.g., MMP-1) that degrades/weakens extracellular matrix. Culture medium levels of release compound (e.g., MMP-1) can be semi quantified using commercial assays such as the MMMP-1 Immunoassy Kit provided by Chemicon International, Inc.

Agents of the invention, including those identifiable using one or more of the above-described assays, can be formulated into pharmaceutical compositions with a suitable carrier and at a strength effective for administration by various means to a patient. Pharmaceutical compositions that contain agents of the invention can be prepared using standard techniques. Such compositions can be prepared in dosage unit form and as oral consumables or injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The agent can be mixed with excipients that are pharmaceutically acceptable. Suitable excipients include water, saline, dextrose, glycerol, or the like, and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, and pH buffers that enhance the effectiveness of the agent. Where appropriate, the composition can be sterile.

The compositions of the invention can be administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the patient, the agent and the effect sought. Suitable regimes can be readily determined by one skilled in the art.

A variety of administrative techniques can be used, depending on the agent, including oral administration, parenteral techniques such as subcutaneous, intravenous, intramuscular and intraperitoneal injections, catheterizations and the like. In the case of antibodies, administration is preferably through a catheter placed into the subarachnoid space (Mapstone et al, Techniques of Ventricular Puncture. In: Wilkins and Rengachary, eds. Neurosurgery, McGraw-Hill, N.Y. (1996), pages 179-183). This technique can be used with other agents of the invention. Antibodies (as well as other agents of the invention) can also be injected through a needle placed in the cerebrospinal fluid in the lumbar spine using a "single-shot" technique (Gaiser, Spinal, Epidural and Caudal Anesthesia. In: Longnecker and Murphy (eds) Introduction to Anesthesia, W B Saunders Co., Philadelphia, 1997; Miller, ed., Anesthesia, Churchill Livingstone, Inc., New York). A potential advantage of catheter-based administration of agents with relatively short half lives is that doses can be given at frequent (e.g., daily) intervals, allowing for re-dosing and continued efficacy. Alternatively, delivery of the agents of the invention to the CSF can be accomplished using an Ommaya reservior such as described by Collins (J. Neuro-Onc. 1:283 (1983)), Sandberg et al (Neurosurgery 47:49 (2000)) and Laske et al (Neurosurgery 41:1039 (1997)).
 

Claim 1 of 2 Claims

1. A method of treating cerebral vasospasm that follows subarachnoid hemorrhage (SAH) comprising administering to a patient in need of such treatment an amount of an agent that inhibits vascular cell proliferation sufficient to effect said treatment, wherein said agent is a chemotherapeutic agent.
 

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