Joseph Clark - US grants

DESCRIPTION (Adapted from applicant's abstract): The long-term objective of this application is to discover the molecule(s) that cause cerebral vasospasm following subarachnoid hemorrhage (SAH). Vasospasm is a frequent cause of delayed ischemic stroke in SAH patients. It is proposed that oxidation of bilirubin following SAH produces compounds that inhibit protein phosphatases, and that this inhibition causes the vasoconstriction and vascular proliferation seen in patients with vasospasm. We have identified candidate molecules that are peroxidized fragments of bilirubin that appear to produce vasoconstriction of carotid vessels and proliferation of vascular smooth muscle cells in vitro. In addition, these molecules produce metabolic effects on vessels in vitro that are identical to those produced by CSF from patients with vasospasm. Lastly, the peroxidized bilirubin molecules are present in the CSF of patients with vasospasm. The first two aims in this application will determine which peroxidized forms of bilirubin are found in the CSF of patients with vasospasm; which fragments correlate with the presence of clinical vasospasm; and which peroxidized forms of bilirubin cause vascular constriction and vascular proliferation in vitro. These studies will employ biochemical purification procedures to isolate the oxidized forms of bilirubin and methods to identify their structures. Identification of the compounds may make it possible to develop a test for vasospasm. An in vitro carotid artery ring assay is used to assess the oxygen consumption, isometric forces, high-energy phosphates, and phosphatase activity of CSF from patients with and without vasospasm. Cultures of smooth muscle cells will be used to determine whether CSF from patients with vasospasm and the peroxidized bilirubin compounds stimulate proliferation of the cells in vitro compared to control solutions, and whether this increase in cell proliferation is related to inhibition of protein phosphatases. The third Aim will test whether the vascular constriction might be due to inhibition of smooth muscle phosphatases by the peroxidized bilirubin fragments, and if so which subcellular compartment this occurs in, and which phosphatases are inhibited. The last Aim will determine whether CSF from patients with vasospasm and purified oxidized bilirubin molecules (when injected into the subarachnoid space of rodents) causes vasospasm and cerebral injury in this in vivo model. This model will be used to screen for possible therapies in future studies. The ultimate long-term goal for this project is to define the molecular causes of vasospasm in order to develop effective diagnostic, therapeutic and preventative approaches for this cerebral vascular disease.

DESCRIPTION (provided by applicant) We nave discovered that bilirubin breakdown products are present in the CSF of subarachnoid hemorrhage (SAH) patients. These breakdown products are produced by the oxidation of bilirubin and we call these Bilirubin OXidation products: BOXes. Because they cause vasospasm in vitro and in vivo, and are not found in the CSF of nonvasospasm patients, we believe that the concentration of BOXes will be an objective and quantitative predictor (or assessor) of cerebral vasospasm in SAH patients. Therefore, we will make antibodies against the BOXes and use these new antibodies to diagnose and/or predict cerebral vasospasm by detecting them in the CSF of SAH patients. Following a subarachnoid hemorrhage caused by the rupture of an aneurysm, as many as 40% of the surviving patients develop cerebral vasospasm. Many patients are diagnosed with cerebral vasospasm, but frequently only after they have developed an untreatable stroke. What is needed is a specific, quick and easy method for predicting vasospasm. Currently, there is no specific test for predicting or assessing subarachnoid hemorrhage (SAH) induced cerebral vasospasm. The diagnosis is generally dependent upon clinical parameters such as focal neurological signs and/or angiographically evident vasospasm, and this diagnosis is usually made after there is vasospasm and stroke. We have identified novel molecules in the CSF of SAH patients that are produced by the oxidation of bilirubin and shown that the BOXes (but not pure bilirubin alone) cause vasospasm in vitro and in vivo. This has led us to hypothesize: that BOXes cause cerebral vasospasm following subarachnoid hemorrhage and that the presence and/or concentration of these compounds will predict and correlate with the occurrence of vasospasm. In this project we will make polyclonal antibodies against the three BOXes we have identified and determine the ability of these antibodies to detect BOXes in; 1) control solutions, 2) CSF spiked with known concentrations of BOXes, 3) CSF from vasospasm patients without spiking with BOXes, and 4) correlate the BOX concentrations in CSF to clinical parameters of vasospasm. These data will provide proof of concept that antibodies against BOXes can be used to diagnose and predict vasospasm in SAH patients.

[unreadable] DESCRIPTION (provided by applicant): Vasospasm is a frequent cause of delayed ischemic stroke in subarachnoid hemorrhage (SAH) patients. In this project we will evaluate the molecule(s) that are responsible for causing SAH-induced cerebral vasospasm. The cause of the vasospasm is largely unknown but it has been suggested to be due to a vasoactive molecule in the hemorrhagic CSF. We have found that bilirubin oxidation products (BOXes) are found in the CSF of SAH patients and propose that the BOXes are phosphatase inhibitors that can cause cerebral vasospasm. There have been three structurally related molecules identified. These molecules produce prolonged contractile effects on the vessels in vivo and in vitro that are strikingly similar to the prolonged vasospasm seen from the CSF of SAH patients with vasospasm. [unreadable] [unreadable] We suggest that smooth muscle protein phosphatase inhibition causes prolonged vasospasm. Moreover it is the BOXes that are the phosphatase inhibitors that produce prolonged vasospasm in patients following subarachnoid hemorrhage. In Aims #1 and #2 using cranial window technique, we will examine the time course of cerebral vasospasm in rats caused by the BOXes, and will assess the potency of the individual BOXes. The degree of vascular constriction will be studied over 14 days and the brain examined for evidence of damage. In Aim #3 we will show that BOXes inhibit phosphatases and that this leads to vasospasm using porcine basilar artery in vitro. [unreadable] [unreadable] The long-term goal for this project is to define the molecular causes of vasospasm (such as bilirubin oxidation products, phosphatase inhibition) in order to develop effective diagnostic, therapeutic and preventative approaches for this cerebral vascular disease. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Recently, we described severe expressive and cognitive delays in a 6-year-old boy, who has a unique creatine (Cr) deficiency in the brain, which was diagnosed by proton magnetic resonance spectroscopy (MRS). Upon further analysis, we found that he has a nonsense mutation in the X-lined Cr transporter gene (CT1;SLC6A8), which resulted in the expression of a truncated (non-functional) Cr transporter protein. Since that study, four additional families have been recognized in Cincinnati with mutations in the X-linked Cr transporter gene and nearly 30 families worldwide. These patients all have mental retardation, severe expressive language disorder and mild epilepsy. Despite a growing body of knowledge about the Cr, Cr kinase and phosphocreatine system in the brain, there is no standardized method for improving brain function when the brain creatine transporter is deficient. What is clearly needed is a suitable animal model of this disease such that methods to get creatine across the blood brain barrier can be developed and tested. In this project we will develop a Cr transporter knockout mouse model, such that the efficacy of new treatment paradigms, drugs, and other therapies can be tested. For this research project, we propose to test the following hypotheses: 1) a mouse knockout of this Cr transport defect can model the human disease, and 2) that therapeutic strategies can be given to normalize brain function in these mice. This mouse knockout will model the Cr transporter defect we have discovered in that the brain will lack the ability to transport creatine across the blood brain barrier. To address Hypothesis 1, we will generate a Cr transporter knockout mouse. Cr levels will be determined in the brains of these mice and we will characterize the functional, and biochemical changes observed in Cr transporter knockout mice. Having an animal model that closely mirrors the human disease will enable adequate testing and development of therapies designed at getting creatine across the blood brain barrier and improving brain function. To address Hypothesis 2, the Cr transporter knockout mice and control mice will be treated with Cr formulations that may be capable of transporting creatine across the blood brain barrier and improve brain metabolism and cognitive function of the mice. The goal for Hypothesis 2 is to develop methods and drugs to improve brain energy metabolism by getting creatine into the brain.

DESCRIPTION (provided by applicant): Intracerebral hemorrhage (ICH) is a type of stroke caused by bleeding into the brain, which leads to significant death and disability. Secondary and delayed pathophysiologic events can contribute to the death and disability of the ICH patient. These events are characterized by a breakdown of the blood brain barrier, edema development and cell death in white and gray matter. We have recently discovered that following hemorrhage in the brain, a new molecular species, bilirubin oxidation species (BOXes), are produced within the hematoma during the first 24 hours following ICH. Importantly, the concentration of BOXes in the hematoma is the highest (approximately 20 \iM) that we have observed in any of our experimental and clinical evaluations thus far. This finding is especially significant since we have previously demonstrated that BOXes are cytotoxic and our preliminary data indicates that they can contribute to the pathophysiological events leading to brain injury following ICH. Our overall goal in this research project is to test the Hypothesis that BOXes are acutely generated within the hematoma and contribute to edema formation and perihematomal brain injury following ICH. To address this hypothesis we will use our porcine ICH model. In Aim #1 we will measure the concentration of BOXes in the hematoma and perihematomal brain tissue to define the time course of production. In Aim #2 we will add BOXes to the infused blood to produce the hematoma and assess the damage and examine the underlying pathogenesis observed following experimental ICH. In Aim #3 we will investigate the mechanism(s) for BOXes production by examining 2 biochemical pathways required to generate BOXes: bilirubin generation and oxidative stress. It is anticipated the inhibition of bilirubin production and/or antioxidants will decrease BOXes production in the hematoma. These studies are important because we believe that strategies designed to prevent BOXes production in the hematoma should provide a novel and beneficial therapeutic option for patients who have suffered an ICH.

Abstract Benzylic amines are commonly found in current drug candidates and FDA approved drugs. The direct installation of a nitrogen into a benzylic C?H bond, generating a benzylic amine, would be significant as aromatics are ubiquitous in bioactive molecules. Additionally, direct functionalization of a prevalent C?H bond eliminates the need for pre-installed functionalities, eliminating synthetic overhead and accelerating drug-diversification. A highly site- and chemoselective, inexpensive first row transition metal-catalyzed benzylic C?H amination is proposed for the rapid diversification of topologically complex and functionally diverse molecules. The method will be optimized and the catalyst reactivity will be evaluated in a variety of common organic scaffolds used in drug design. Selectivity trends will be elaborated in more complex commercially available pharmaceuticals. Once reactivity and selectivity trends are established, the method will be used to diversify complex drug scaffolds and natural products. Nitrogen is abundant in pharmaceuticals and natural products, making nitrogen tolerance under the reaction conditions necessary to significantly broaden the applications of the proposed method. Bioactive molecules containing a tertiary amine or pyridine will undergo complexation to the quaternary salt, to quell unwanted side reactions from detrimental nitrogen binding to reaction intermediates in the benzylic C?H amination reaction. If the proposed research is achieved, many new small molecule drug candidates will be accessible in only one step from existing small molecules, pharmaceuticals, and natural products. Such a method would broadly impact human health and the field of medicine as rapid and inexpensive access to new drug candidates will be possible with an earth-abundant, non-toxic, first-row transition metal catalyst.