WO2015184380A1

WO2015184380A1 - Snake venom for use in the treatment prior to a surgical procedure in the bran

Info

Publication number: WO2015184380A1
Application number: PCT/US2015/033365
Authority: WO
Inventors: John H. ZHANG; Cherine KIM; Prativa SHERCHAN; Paul Krafft; Devin MCBRIDE; Tim LEKIC
Original assignee: Loma Linda University; Loma Linda University Medical Center
Priority date: 2014-05-30
Filing date: 2015-05-29
Publication date: 2015-12-03
Also published as: WO2015184380A8

Abstract

A treatment for reducing brain or other injury that can be used before, during, or after a surgical treatment or other injury to the brain to decrease or prevent subsequent damage. Injury to the brain during surgery or from other trauma can create elevations in brain edema and blood-brain barrier disruption thereby decreasing neurological recovery and patient outcomes. Treatment of the brain with venom, including snake venom, prior to or after injury can reduce the bleeding, swelling, and/or inflammation that results from the trauma. These pre-conditioning and/or rapid-conditioning treatments of the brain with venom can reduce brain edema and blood-brain barrier disruption and improve patient outcomes.

Description

SNAKE VENOM CONDITIONING FOR REDUCTION OF HEMORRHAGE

BACKGROUND

[0001] The present claims priority to U.S. Provisional Application No. 62/005,871, filed May 30, 2014, entitled "SNAKE VENOM CONDITIONING FOR REDUCTION OF HEMORRHAGE." The aforementioned application is incorporated by reference herein in its entirety, and is hereby expressly made a part of this specification.

Field of the Invention

[0002] Venom, e.g., snake venom or its components, is utilized for preconditioning or rapid-conditioning of a patient to prevent and/or reduce damage to healthy/non-diseased tissue.

Description of the Related Art

[0003] Normal and routine neurosurgery practice will produce "brain injury" in patients because neurosurgeons have to open the skull to access the injured brain tissue, remove tissues, all of which cause bleeding. This type of brain injury is considered unavoidable and thus no specific efforts are made to prevent or attenuate it. This surgical brain injury, however, not only causes post-surgical complications but also prevents neurosurgeons from adopting a more aggressive or invasive approach to curative options. Surgical brain injury especially causes concern in anatomically confined areas such as the posterior cranial vault and deeper structures such as the brain stem. Brain injury during surgery may cause the brain to swell (e.g., in a manner similar to how a cut on a finger swells). This swelling may kill a patient, because unlike a finger or other unconstrained structure, the brain sits inside a sealed bone structure, such that the swelling will increase the pressure in the brain such that tissue damage results.

[0004] There are over 800,000 brain or spinal cord surgeries per year in the United States, along with multiple millions of general surgeries to these physical structures each year. The American Medical Association has reported that 75% of neurosurgeons do not operate on children due to the potential for damage to healthy tissue. Furthermore, 79% of all physicians practice "defensive medicine" due to liability crisis and fear of lawsuits. This defensive medicine practice results in an estimated excessive expenditure of $70-$ 126 billion per year. In forms of brain injury such as traumatic head injury or stroke, there is a narrow window of opportunity for therapy once an injury has occurred. In contrast, however, surgical brain injury can be scheduled in advance, such that an open window of opportunity for neuroprotection before and/or during surgery is available. This opportunity could improve patient outcome, postoperative care and healthcare costs.

SUMMARY

[0005] The delicate and complex architecture of the brain presents significant challenges for neurosurgery. To date, developing less invasive surgical methods and administering nonspecific postoperative care have been the predominant strategies for limiting surgical brain injury. However, relatively little research exists which focuses on understanding the biochemical pathophysiology of surgical brain injury or therapies targeting specific pathways. The complications of surgical brain injury not only require vigilant postoperative care, but may also hinder therapeutic approach. Additionally, the lack of an effective and specific therapy for surgical brain injury that can be prevent inevitable damage to healthy/non-diseased tissue and/or reduce the impact of surgery and post-operative care can cause physicians and other surgeons to opt against surgical intervention. Therefore, diminishing the perioperative risks of surgical brain injury will not only provide better quality of life and outcome, but may also allow for more aggressive surgical interventions to be utilized.

[0006] Accordingly, there is a need for a technology that can be adopted by physicians and other surgeons to prevent the inevitable damage to healthy/non-diseased tissue. Additionally, the ability to further reduce the impact of surgery and post-operative care on the healthcare system is desirable.

[0007] In accordance with one aspect, a method is provided of administering treatment prior to a surgical procedure in a brain comprising: applying one or more components of a hemorrhagic snake venom to a brain surgery site before a surgery, whereby an immune system response is triggered; and thereafter performing a surgery at the brain surgery site, whereby a primary injury at the brain surgery site is inflicted, wherein a secondary injury at the brain surgery site is reduced through the immune system response. In some embodiments, the one or more components are selected from the group consisting of metalloproteinases, serine proteases, and phospholipase 2. In some embodiments, the secondary injury comprises bleeding, swelling, or inflammation that occurs during or following the surgery. In some embodiments, the method further comprises priming the brain surgery site with the components for responding to the secondary injury. In some embodiments, the hemorrhagic snake venom is Crotalus atrox venom. In some embodiments, the hemorrhagic snake venom increases levels of fibrinogen, platelets, and clotting at the brain surgery site. In some embodiments, the hemorrhagic snake venom is administered in 1 to 6 doses. In some embodiments, a total amount of the doses of the hemorrhagic snake venom administered is from 5-20% of the LD50 value for Crotalus atrox venom in the patient being treated. In some embodiments, the method further comprises administering one or more components of a hemorrhagic snake venom from 0 to 6 days after the primary brain injury. In some embodiments, the doses of the hemorrhagic snake venom are administered from 0 to 6 days before the surgery. In some embodiments, the doses of the hemorrhagic snake venom are administered from 0 to 6 days before the surgery to 0 to 6 days after the surgery.

[0008] In accordance with another aspect, a method is provided of administering treatment after a primary brain injury to a brain comprising: applying one or more components of a hemorrhagic snake venom to a site of a primary brain injury, whereby an immune system response is triggered, wherein a secondary injury is reduced through the immune system response triggered by the application of the one or more components to the site of the primary brain injury. In some embodiments, the one or more components are selected from the group consisting of lectins, serine proteases, vascular endothelial growth factor inhibitor, cysteine-rich secretory proteins, matrix metalloproteinases, and phospholipase A2. In some embodiments, the secondary injury comprises reducing bleeding, swelling, or inflammation that occurs during or following the injury. In some embodiments, the hemorrhagic snake venom is Crotalus atrox venom. In some embodiments, the hemorrhagic snake venom increases levels of fibrinogen, platelets, and clotting at the site of the injury. In some embodiments, the hemorrhagic snake venom is administered in 1 to 6 doses. In some embodiments, a total amount of the doses of the hemorrhagic snake venom administered is from 5-20% of the LD50 value for Crotalus atrox venom in the patient being treated. In some embodiments, the doses of the hemorrhagic snake venom are administered from 0 to 6 days after the primary brain injury. In some embodiments, the primary brain injury is caused by a stroke.

[0009] In accordance with another aspect, a composition is provided to administer treatment after a primary brain injury to a brain comprising one or more components of a hemorrhagic venom to be applied to a site of a primary brain injury, wherein the venom comprises a combination of unfractionated venoms and venom components from one or more species of snake. In accordance with another aspect, a method is provided of treating surgical brain injury as described herein.

[0010] In accordance with another aspect, a method is provided of administering treatment prior to a surgical procedure of a patient comprising: applying one or more components of a hemorrhagic snake venom to a surgery site before a surgery, whereby an immune system response is triggered; and thereafter performing a surgery at the surgery site, whereby a primary injury at the surgery site is inflicted, wherein a secondary injury at the surgery site is reduced through the immune system response.

[0011] In accordance with another aspect, a method is provided of administering treatment after a primary injury to a patient comprising: applying one or more components of a hemorrhagic snake venom to a site of a primary injury, whereby an immune system response is triggered, wherein a secondary injury is reduced through the immune system response triggered by the application of the one or more components to the site of the primary injury.

[0012] In accordance with another aspect, a composition is provided to administer treatment after a primary injury to a patient comprising: one or more components of a hemorrhagic venom to be applied to a site of a primary injury, wherein the venom comprises a combination of unfractionated venoms and venom components from one or more species of snake.

BRIEF DESCRIPTION OF THE DRAWINGS [0013] FIG. 1A illustrates an embodiment of a rat brain cut along the horizontal plane, and frontal lobe surgical injury, known as the surgical brain injury model.

[0014] FIG. IB illustrates a 3D simulated construction of a frontal lobe surgical injury.

[0015] FIG. 2A illustrates embodiments of dose and timing of Crotalus atrox venom pre-conditioning dose regimens.

[0016] FIG. 2B shows photos of tissue injury at the venom injection site (subcutaneous, hindquarter) for Crotalus helleri venom at doses of 50% LD₅₀ (A,B) and 100% LD₅₀ (C, D).

[0017] FIGS. 3A-D illustrates the blood-brain barrier disruption after surgical brain injury through IgG staining.

[0018] FIG. 4 shows how pre-conditioning with Crotalus atrox venom reduces brain swelling after surgical brain injury.

[0019] FIGS. 5A-B show the reduction in bleeding associated with surgical brain injury with the use of Crotalus atrox venom pre-conditioning.

[0020] FIG. 6 illustrates a time course of neurological deficits after surgical brain injury.

[0021] FIGS. 7A-B convey the improvement of functional outcome with Crotalus atrox venom using the results of the Garcia Neuroscore and the Foot Fault Test.

[0022] FIG. 8 illustrates brain water content following surgical brain injury and reduction with Crotalus venom pre-conditioning.

[0023] FIG. 9 shows the intraoperative hemorrhage during surgical brain injury as assessed with a hemoglobin assay.

[0024] FIG. 10 displays the neurological function after surgical brain injury based on the modified Garcia scores.

[0025] FIG. 11 shows the effect of RSG on inflammation and shows the effect of Rosiglitazone (RSG) (1 mg/kg) on myeloperoxidase (MPO) activity at 24 hours after surgical brain injury in the ipsilateral frontal lobe.

[0026] FIG. 12 shows immunohistochemical staining for inflammatory markers IL-Ιβ and TNFa following surgical brain injury. [0027] FIG. 13 demonstrates that hyperbaric oxygen pre-conditioning (HBO- PC) increased COX-2 expression in western blot after pre-conditioning but before surgical brain injury.

[0028] FIG. 14 shows that HBO-PC can reduce COX-2 elevation after global cerebral ischemia and a COX-2 inhibitor administered before HBO-PC can abolish the protective effect of HBO-PC.

[0029] FIG. 15 shows a western blot analysis of COX-2 following surgical brain injury and Cv-PC.

[0030] FIG. 16 shows an immunohistochemical staining for inflammatory marker PGE2 following surgical brain injury which is reduced with Crotalus atrox venom preconditioning (C).

[0031] FIGS. 17A-D illustrate the effects of MMP inhibitor-1 with a single and daily dose treatment regimen after surgical brain injury.

[0032] FIGS. 18 A- J are pictographs showing the colocalization of MMP-9 with different cell markers in brain sections adjoining the surgical brain injury.

[0033] FIG. 19 shows a zymogram and bar graph depicting the MMP-9 and MMP-2 enzymatic activity at different time points after surgical brain injury.

[0034] FIGS. 20A-C illustrate an estimation of brain edema by MRI scans.

[0035] FIG. 21 shows surgical brain injury can cause a significant increase of thrombin activity at 24 hours after operation.

[0036] FIG. 22 illustrates plasma fibrinogen following Cv-PC (Crotalus atrox venom preconditioning).

[0037] FIG. 23 shows plasma fibrinogen degradation products following Cv- PC.

[0038] FIG. 24 shows partial thromboplastin time following Cv-PC.

[0039] FIGS. 25A-F display the blood parameters present during surgical brain injury with and without pre-conditioning with Crotalus atrox venom.

[0040] FIGS. 26A-C are images showing the inflammation in sham surgery, surgical brain injury, and surgical brain injury with venom pre-conditioning.

[0041] FIG. 27 displays the fractionation chromatograph of Crotalus atrox venom with the reverse-phase HPLC. [0042] FIG. 28 displays a PAGE gel of each fraction for Cortalus atrox venom.

[0043] FIGS. 29A-B show the reduction in bleeding or hemorrhage with fractionated Crotalus atrox venom.

[0044] FIGS. 30A-B show the improved outcome of rats treated with preconditioning with fractionated Crotalus atrox venom.

[0045] FIGS. 31A-B show intraoperative hemorrhage (FIG. 31 A) and postoperative bleeding (FIG. 31B) was reduced by subcutaneous Cv-PC in a dose- dependent manner.

[0046] FIGS. 31C shows a Modified Garcia Score at 24 hours post-surgical brain injury.

[0047] FIGS. 32A-B show intraoperative hemorrhage (FIG. 32 A) and postoperative bleeding (FIG. 32B) with Cv-PC, MMP-inhibitor (MMP-I), Cv fractions, and Fibrinogen (Fb).

[0048] FIG. 32C shows a Modified Garcia Score at 24 hours post-SBI.

[0049] FIG. 33 shows a fractionation graph of Crotalus atrox venom.

[0050] FIG. 34 illustrates an embodiment of a proposed pathway for the use of venom for platelet activation and platelet aggregation to reduce or stop hemorrhagic transformation.

[0051] FIG. 35 illustrates an embodiment of a dose response experiment.

[0052] FIG. 36 shows results of the infract volume following middle cerebral artery occlusion in rats. Crotalus atrox venom does not alter infarct volume.

[0053] FIG. 37 shows brain swelling volume results following middle cerebral artery occlusion.

[0054] FIG. 38 shows hemorrhagic transformation results following middle cerebral artery occlusion.

[0055] FIG. 39 shows results of the Modified Garcia Score following middle cerebral artery occlusion.

[0056] FIG. 40 shows results of the corner turn test following middle cerebral artery occlusion. [0057] FIG. 41 shows forelimb placement test following middle cerebral artery occlusion.

[0058] FIG. 42 illustrates an embodiment of the experimental parameters for observing the platelet activation and hemostatic analysis.

[0059] FIG. 43 illustrates an embodiment of the experimental parameters for observing the long term functional outcomes.

[0060] FIG. 44 show an embodiment of a proposed pathway for the use of venom for platelet activation and platelet aggregation to reduce or stop hemorrhagic transformation.

[0061] FIGS. 45 - 47 show results of C. atrox fractionation.

[0062] FIG. 48 shows an embodiment of the experimental parameters to determine active proteins.

[0063] FIG. 49 shows blood analysis for Crotalus atrox venom fraction 3.

[0064] FIG. 50 shows an embodiment of the experimental parameters to determine active proteins.

[0065] FIG. 51 illustrates the B. Jararaca venom components.

[0066] FIG. 52 illustrates an embodiment of a proposed mechanism for decreasing intraoperative bleeding.

[0067] FIG. 53 illustrates an embodiment of a study design for topical administration of the venom and/or venom components.

[0068] FIG. 54 illustrates an embodiment of a study design for intra nasal administration of the venom and/or venom components.

[0069] FIG. 55 shows intraoperative bleeding after topical administration of the venom and/or venom components.

[0070] FIG. 56 shows the brain water content at 24 hours post SBI.

[0071] FIG. 57 shows the results of the Modified Garcia Test at 24 hours post

SBI.

[0072] FIG. 58 shows the results of the beam balance at 24 hours post SBI.

[0073] FIG. 59 shows the intraoperative bleeding after intranasal administration.

DETAILED DESCRIPTION [0074] Collateral damage and bleeding are unavoidable during surgeries, especially neurosurgeries, however preventing this damage with the use of therapeutics will have a significant improvement to patient outcome as well as be beneficial for healthcare.

[0075] Currently, there are no specific efforts or techniques utilized to prevent or attenuate hemorrhage, edema, or inflammation during and after surgical injury, including surgical brain injury. Additionally, current therapies do not target surgeries and hemorrhage following injuries, such as hemorrhagic transformation following stroke. Since the complications of surgical brain injury require vigilant postoperative care and possibly hinder the therapeutic approach, diminishing the perioperative risks of surgical brain injury will not only provide better quality of life and outcome, but may also allow for more aggressive surgical interventions to be utilized.

[0076] While pre-conditioning studies have demonstrated promising neuroprotective effects for other models of brain injury, clinical translation is limited because many injuries occur spontaneously. The elective nature of many neurological procedures makes surgical brain injury a prime candidate for preventative therapy. Currently, hyperbaric oxygen pre-conditioning (HBO-PC) is the only pre-conditioning modality that has been studied in animal models of surgical brain injury. HBO-PC can show a decrease in brain water content and improve neurological function 24 hours following surgical brain injury.

[0077] Pre-conditioning (PC) has emerged as a potential therapeutic strategy. A number of pre-conditioning therapies exist, such as inhaled gas (HBO, xenon) and hypoxic/ischemic-PC, which induce minimal damage in order to enhance the body's innate response to reduce damage from a full-insult. The use of these pre-conditioning therapies, while able to confer protection for a variety of diseases, have specific innate responses. Hypoxic/ischemic-PC is unlikely to be used in the clinical setting due to the possibility of dire outcomes. HBO, which has promising results in models of stroke and brain injury, is limited by the lack of congruity in dosing regimens and possible oxygen toxicity. Furthermore, hemorrhage, the immediate complication of surgical brain injury, is unaffected by either of these PC treatments. [0078] Surgical brain injury develops by both primary and secondary mechanisms of injury. Primary injury, inflicted directly by mechanical manipulation during surgery, is inevitable and irreversible. However, secondary injury, such as hemorrhage, has been shown to be a major postoperative complication of surgical brain injury. Hemorrhage can be another major obstacle in neurosurgery, it lengthens and complicates surgery and can trigger further injury by hypoperfusion of tissue as well as disruption of the blood-brain barrier. Additionally, the use of electrocautery can be beneficial in the field of neurosurgery. However, electrocauterization causes significant thermal injury in surrounding tissues and contributes to the injury involved in neurosurgery. Measures to improve hemostasis during surgery would improve patient outcome in numerous ways. In some embodiments, reducing time under anesthesia, diminishing tissue manipulation, and limiting fluctuations in blood volume can be improved through the use of pre-conditioning or rapid-conditioning therapies described herein.

[0079] Secondary injury by brain edema occurs several hours after surgical brain injury, and loss of blood-brain barrier integrity has been implicated in contributing to edema. Many rodent studies indicate that the brain water content of tissue surrounding the resection site is increased by as much as 3-4% during the first three postoperative days. This 4% increase in brain water content can translates into a 25% increase in tissue volume, leading to high intracranial pressure, hypoperfusion of neurons, and further cell death.

[0080] Even neurosurgical patients without life-threatening complications must be monitored closely in the critical care unit for 3 to 7 days. A preventative therapy which reduces postoperative hemorrhage and brain edema can decrease the need for blood transfusions, shorten hospital stay, and ultimately cut perioperative costs. Additionally, 31% of elective neurosurgeries require blood transfusions and the associated costs of one red blood cell unit can be between $522 to $1183 USD. This is an estimated $1.62 to $6.03 million that is spent on blood and transfusion-related care for surgical patients. Thus, the benefits of limiting the complications of surgical brain injury extend beyond improving morbidity and mortality. [0081] FIG. 1A illustrates an embodiment of a rat brain cut along the horizontal plane, and frontal lobe surgical injury, known as the surgical brain injury model, in relation to the bregma which is marked by an X. In some embodiments, the 2 incisions can be made leading away from the bregma along the sagittal and coronal planes 2 mm lateral and 1 mm proximal to the sagittal and coronal sutures, respectively. FIG. IB illustrates a 3D simulated construction of a frontal lobe surgical injury. This model illustrates the results of surgery on regions of the brain. The red area in the model of FIG. IB represents the surgically-induced brain injury. The 3-dimensional image of the proposed model shows the frontal lobe surgical injury from different angles and planes.

[0082] The complications of brain surgery are typically left to resolve on their own because understanding of the mechanism of injury and treatment options for surgical brain injury are lacking. The molecular mechanisms of surgical brain injury and the ability to manipulate these mechanisms can provide a possible treatment option for surgical brain injury. Additionally, the therapy can provide a possible therapeutic option for other elective surgeries.

[0083] Venom, including snake venom, can be utilized to prevent hemorrhage during surgery and diseases or disorders. In particular, the venom can be used during, before, and/or after general surgery and neurosurgery. In some embodiments, venom components can be used after an injury to the brain or a stroke to reduce collateral damage or bleeding.

[0084] Snakes were used by the ancient civilizations, such as the Greeks and Mayans, to treat a variety of ailments, including skin and blood disorders, even epilepsy. This practice was lost as the poisonous nature of snake venom was recognized. Snake venom has been used more recently in the medical field for its effects on hemostasis. Snake venom toxins have been used to detect coagulative disorders. The anticoagulative properties of snake venom have been studied for their potential in treating patients with hypercoagulative states, and the venoms with coagulative properties have been used to stem bleeding. Due to the hemostatic nature of Crotalus (commonly referred to as rattlesnake) venoms, in some embodiments, Crotalus venoms can be a therapeutic agent utilized in pre-conditioning therapy for preventing hemorrhage. The snake venom can be obtained from rattlesnakes including but not limited to C. intermedius, C. pricei, C. adamanteus, C. aquilus, C. atrox, C. basiliscus, C. catalinensis, C. cerastes, C. durissus, C. enyo, C. horridus, C. intermedius, C. lannomi, C. lepidus, C. mitchellii, C. molossus, C. oreganus, C. polystictus, C. pricei, C. pusillus, C. ruber, C. scutulatus, C. simus, C. stejnegeri, C. tigris, C. tortugensis, C. totonacus, C. transversus, C. triseriatus, C. viridis, C. willardi. Venom from other venomous snakes can be used including venom from Inland taipan, Dubois' seasnake, Eastern brown snake, Coastal Taipan, Yellow bellied sea snake, Peron's sea snake, BlackTiger snake, Many-banded krait, Black-banded sea krait, Mainland Tiger snake, Western Australian Tiger snake, Beaked sea snake, or other snakes from the families tractaspididae, Colubridae, Elapidae, and Viperidae. Additionally, other venoms can be used to provide the hemostatic effect necessary to prevent hemorrhage.

[0085] The therapeutic venom can be used by all surgeons and especially neurosurgeons to prevent or attenuate hemorrhage before, during, or after a surgical procedure. Additionally, venom can be used by physicians for treatment and/or prevention of hemorrhagic transformation following injuries. For example, venom can be used to treat or prevent hemorrhage in a patient following a stroke. Additionally, in some embodiments, the use of venom can reduce the impact of surgery and post-operative care on the healthcare system due to its ability to reduce damage to healthy tissue.

Venom Components

[0086] Venom components have been shown to reduce bleeding, swelling, and inflammation during and following surgery. Additionally, administering venom before surgery reduces bleeding and improves outcome and quality of life after surgery. The proteins in the venom, for example, snake venom, act as a trigger for the body's innate system response which "primes" the body for responding to further damage. The use of venom to prime the body for responding to further damage can be adopted by physicians and other surgeons to prevent the inevitable damage to healthy and/or non- diseased tissue.

[0087] Venoms are generally classified as either neurotoxic or hemorrhagic. While neurotoxic venoms affect the nervous system, hemorrhagic venoms affect the blood. Hemorrhagic venoms affect many components of the blood stream, such as fibrinogen, platelets, and coagulation parameters or clotting. Fibrinogen is the principle protein of blood clotting. During normal blood coagulation, fibrinogen is converted by thrombin into fibrin during blood clot formation. Primary hemostasis involves the formation of a platelet plug at the injury site and during the simultaneous secondary hemostasis process coagulation factors or clotting factors respond during the coagulation cascade to form fibrin stands that strengthen the platelet plug.

[0088] Available pre-conditioning therapies such as HBO-PC can show a decrease in brain water content and improve neurological function 24 hours following surgical brain injury in subjects via cycloogenase-2 (COX-2) inhibition. However, surgical brain injury-induced COX-2 overexpression was altered by HBO-PC. The available pre-conditioning therapies may not provide the optimal outcome following surgical brain injury as they do not have an effect on bleeding. However, the altered expression of COX-2 in the HBO-PC treated subjects highlights the PLA₂/COX-2 pathway as promising target for therapy.

[0089] In some embodiments, hemorrhagic venoms can be used before surgery and/or after surgery and/or injury to reduce bleeding and improve patient outcomes as described herein. For example, snake venom can be used to condition the patient before, during, or after surgery or injury to the brain or other injury or surgery site. In some embodiments, venom from Crotalus atrox (commonly referred to as the Western Diamondback Rattlesnake) can be used. Crotalus atrox venom is hemorrhagic, thereby affecting components of the blood stream including fibrinogen, platelets, and clotting. The Crotalus atrox venom contains twenty-four proteins. These proteins include metalloproteinases (MMPs), serine proteases, phospholipase 2, and neurotoxins. MMPs degrade extracellular matrix proteins and cleave fibrinogen. MMPs' function of cleaving fibrinogen make it an important component in coagulation and hemostasis. Further, serine proteases also play a large role in coagulation and hemostasis. The coagulation factors of the coagulation cascade are generally serine proteases, which act by cleaving downstream proteins.

[0090] The protein or protein mixture present in the venom provides a therapeutic effect shown to reduce bleeding, swelling, and/or inflammation. In some embodiments, snake venom, or other venom, can be used to prevent hemorrhage during general surgery, neurosurgery, and diseases or disorders.

[0091] The toxic properties of snake venom can be harnessed to elicit endogenous protective processes. Specifically, Crotalus atrox venom can provide anti- hemorrhagic effects through fibrinogen production induced by MMPs and anti- inflammation effects through the PLA₂-COX pathway. Therefore, in some embodiments, Crotalus atrox venom can provide a therapeutic benefit following surgical brain injury through reduction of edema, inflammation, and hemorrhage, ultimately improving neurological outcome.

[0092] The venom, venom fraction, or venom proteins will be given as a preconditioning regime or as a rapid-conditioning regime to prevent hemorrhage. In some embodiments, the pre-conditioning and/or rapid-conditioning treatments can speed up the healing process of patients and allow neurosurgeons, or other surgeons, to take a more aggressive approach in surgery. The pre-conditioning and rapid-conditioning therapies can reduce risks and costs for health care providers and insurance companies. The use of MMPs and venom components can be easily utilized in the clinical setting and can be commercialized for the use of pre-conditioning and rapid-conditioning.

[0093] Additionally, in some embodiments, the pre-conditioning and rapid- conditioning can be used for other surgeries, including general surgery. Further, the preconditioning and rapid-conditioning techniques can be used to treat injury as well as dental procedures, veterinary procedures, or other procedures to control bleeding and inflammation that typically results from the procedure.

Pre-conditioning

[0094] In some embodiments, the priming technique can utilize venom, or components of it, for conditioning a patient to prevent and/or reduce damage. In some embodiments, the venom can be utilized in pre-conditioning techniques. The preconditioning technique can include the use of venom prior to surgery by exposing the surgical site to the venom components. The proteins in the venom can act as a trigger for the patient's innate system response thereby priming the patient's body for responding to any future damage due to the surgical procedure. [0095] Surgical brain injury has an open window of opportunity for neuroprotection before and/or during surgery that could improve patient outcome/postoperative care and healthcare costs.

[0096] In some embodiments, the venom treatment can be utilized with a preconditioning strategy. The pre-conditioning strategy can reduce bleeding or damage to the surgery site. Pre-conditioning therapy introduces harmful stimuli, in very low doses, to activate pathways and confer tolerance to injury. For example, Crotalus atrox venom can reduce bleeding from brain surgery. Reducing hemorrhage during surgeries can lower the postoperative care and improve the outcome of surgical patients.

[0097] In some embodiments, Crotalus atrox venom pre-conditioning (Cv- PC) can improve outcome of patients following surgical brain injury by reducing brain edema and intraoperative hemorrhage. With Cv-PC administration a subsequent improvement in neurological deficits following surgical brain injury can be expected. A promising reduction in both brain edema and intraoperative hemorrhage in surgical brain injury following the administration of Cv-PC can improve patient outcomes. In some embodiments, surgical brain injury-induced neurological deficits can also be mitigated by this pre-conditioning treatment.

[0098] The efficacy of Cv-PC in attenuating surgical brain injury can be established by determining optimal dosing and examining toxicity and side-effects, assessing brain water content, blood-brain barrier integrity and hemorrhage volume, and evaluating neurological behavior deficits.

[0099] In some embodiments, the optimal Cv-PC dosing regimen can limit the side effects of Crotalus venom administration while still allowing the patient to benefit from the treatment. The dose and scheduling regime can be administered to precondition the brain surgery site prior to the surgical procedure. The treatment can be administered at various dosages and scheduling. The chosen dose can be dependent on the type of surgical procedure, the time available before the procedure, and the availability of the patient and healthcare provider.

[0100] Different dose concentrations can be chosen based on previous hematologic studies performed to study the effects of Crotalus venoms. These studies establish the median lethal dose or lethal dose, 50% (LD₅₀) values and have demonstrated that it is possible to witness the hemostatic effects of the venom without inflicting undue pain to the animals. The multiple doses can include from between about 5%, 10%, and 20% of published LD₅₀ values in accordance to these studies. In some embodiments, the dose concentrations and dosing schedule can include those outlined in FIG. 2A. FIG. 2A illustrates embodiments of dose and timing of Crotalus atrox venom pre-conditioning dose regimens. In some embodiments, the doses can be administered as 3 doses over 1.5 days, 3 doses over three days, or 6 doses over six days. Additionally, the doses can be administered in divided doses of from about 1 to about 6 doses. In some embodiments, the doses can be administered as from about 1, about 2, about 3, about 4, about 5, or about 6 divided doses over the treatment period. The treatment period can range from about 0 days (immediately prior to or contemporaneous with surgery) to about 6 days before surgery. In some embodiments, the treatment period can involve administration of doses over a time period of about 0 days (immediately prior to or contemporaneous with surgery), about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, and/or about 6 days before surgery.

[0101] In some embodiments, an amount of venom characteristic of 5-20% of LD₅₀ values can be used for pre-conditioning to reduce or avoid local inflammation caused by the injury. For example, about 5%, about 10%, about 15%, about 20%, and about 25% of LD₅₀ values can be used for pre-conditioning as an effective treatment for surgically induced inflammation or hemorrhage, surgical brain injury, and/or other inflammation or hemorrhage.

[0102] FIG. 2B shows photos of tissue injury at the venom injection site (subcutaneous, hindquarter). FIG. 2B shows the injection site 24 hours after Crotalus helleri venom injection. Injection of Crotalus helleri venom of 50% (0.9 mg/kg) of reported LD₅₀ values causes a local reaction and hemorrhage in Sprague Dawley rats as shown in panels A and B. Injection of Crotalus helleri venom of 100% (1.8 mg/kg) of reported LD₅₀ values causes a local reaction and hemorrhage in Sprague Dawley rats as shown in panels C and D. External (panels A and C) and internal (panels B and D) are shown. Based upon these results, about 5 to about 20% of LD₅₀ values for preconditioning can be used to avoid local inflammation. About 5%, about 10%, about 15%, about 20%, and about 25% of LD₅₀ values can be used for pre-conditioning as an effective treatment for surgically induced inflammation or hemorrhage.

Brain Edema and Blood-Brain Barrier Integrity

[0103] In some embodiments, pre-conditioning or rapid-conditioning can be used before or after surgery or injury to reduce brain edema and preserve the blood-brain barrier integrity. For example, in some embodiments, Cv-PC prior to surgical brain injury can reduce brain edema and preserve blood-brain barrier integrity following surgical brain injury. Brain edema is a serious postoperative complication of surgical brain injury and a major determinant of clinical outcome. In surgical brain injury, edema develops from the endogenous inflammatory response to direct trauma during surgery. The generation of inflammatory molecules threatens the integrity of the blood-brain barrier, leading to brain edema. In some embodiments, administering Cv-PC introduces several minor inflammatory challenges in the body prior to injury thereby priming the surgical site and preparing the surgical site for the major insult of surgical brain injury. The reduction in brain edema and integrity of the blood-brain barrier can be examined by the brain water content in surgical brain injury following Cv-PC.

[0104] FIGS. 3A-D illustrates the blood-brain barrier disruption after surgical brain injury through IgG staining. IgG staining denotes blood-brain barrier disruption localized to the surgical brain injury area in different planes. FIG. 3A shows a horizontal section showing the resected area. The asterisk denotes the contralateral hemisphere, which is not stained. FIG. 3B shows the coronal section passing through the injury (bregma+2.20 mm). FIGS. 3C-D shows coronal sections at bregma+0.20 mm and bregma, respectively, just distal to the resection showing IgG staining in the ipsilateral hemisphere. A shift in midline due to the increased size of the ipsilateral hemisphere is marked by the dotted line. Scale bar denotes 1 mm in FIG. 3A and 2 mm in FIGS. 3B-D.

[0105] In some embodiments, a reduction in brain swelling associated with surgical brain injury can result from pre-conditioning with venom as described herein. FIG. 4 shows how pre-conditioning with Crotalus atrox venom reduces brain swelling after surgical brain injury. FIG. 4 shows the reduction in brain swelling in surgical brain injury treated with pre-conditioning therapies using Crotalus atrox venom by comparing the brain water content percentage in patients with surgical brain injury without preconditioning with venom (SBI) and surgical brain injury with pre-conditioning with venom (SBI+Cv-PC). Brain water content was measured at 24 hours and 72 hours after surgery. During sham surgery or placebo surgery the brain water content is about 80%. Surgical brain injury without pre-conditioning (SBI) at 24 hours was about 83% with a slight increase at 72 hours after surgery. Surgical brain injury treated with Crotalus atrox venom pre-conditioning (SBI+Cv-PC) has about 82% brain water content at 24 hours with a light decrease in brain water content at 72 hours.

[0106] The decrease in brain water content with surgical brain injury treated with Crotalus atrox venom pre-conditioning shows a decrease in brain swelling related to surgical brain injury. Additionally, the maintenance or slight reduction in brain water content between 24 hours and 72 hours in surgical brain injury treated with Crotalus atrox venom pre-conditioning shows that the pre-conditioning continues to be effective in decreasing swelling and therefore further damage to the surgery area long after surgery. However, brain swelling in surgical brain injury without pre-conditioning would continue to increase up to at least 72 hours after surgery and therefore continue to further injure healthy or nondiseased tissue that otherwise would be unaffected.

Hemorrhage

[0107] In some embodiments, pre-conditioning or rapid-conditioning can be used before and/or after surgery or injury to reduce intraoperative bleeding associated with injury or surgery. In some embodiments, Cv-PC can reduce intraoperative bleeding by improving endogenous coagulation during surgery. Intraoperative bleeding is a major obstacle in neurosurgery. It obstructs the surgical field, lengthens time under anesthesia, and introduces secondary injury resulting from blood neurotoxicity. Electrocoagulation is vital to neurosurgical procedures, but inflicts thermal injury. Limiting the need for electrocoagulation can reduce the collateral damage to surrounding neural tissue. In some embodiments, the hemorrhagic, fibrinogenolytic effects of Crotalus venom, introduced in sublethal doses, can prepare the endogenous mechanisms coagulation in surgical brain injury-induced hemorrhage.

[0108] In some embodiments, the reduction in bleeding associated with surgical brain injury can be achieved with the use of venom pre-conditioning. FIGS. 5A- B show the reduction in bleeding associated with surgical brain injury with the use of Crotalus atrox venom pre-conditioning. FIG. 5A shows the intraoperative blood loss during sham surgery or placebo surgery, surgical brain injury without pre-conditioning (SBI), and surgical brain injury at a site with Crotalus atrox venom pre-conditioning (SBI+Cv-PC). The surgical brain injury results in about 150( L of blood loss as a result of the injury. The surgical brain injury treated with Crotalus atrox venom preconditioning resulted in 90( L of blood loss. The reduction in blood loss shows that preconditioning the surgical site with Crotalus atrox venom is effective at reducing blood loss in neurosurgical patients. The use of Crotalus atrox venom pre-conditioning in other surgical procedures or at other injury sites can also reduce the blood loss from tissue injury.

[0109] FIG. 5B shows the reduction in postoperative hematoma in surgical brain injury treated with pre-conditioning therapies using Crotalus atrox venom. FIG. 5B shows the postoperative hematoma during sham surgery or placebo surgery, surgical brain injury without pre-conditioning (SBI), surgical brain injury with Crotalus atrox venom pre-conditioning (SBI+Cv-PC), and surgical brain injury with Crotalus atrox venom pre-conditioning and combined fractions (SBI+Cv-PC: Combined Fractions). The postoperative hematoma during surgical brain injury without pre-conditioning is greater than 20μΙ_^. The postoperative hematoma during surgical brain injury with preconditioning and surgical brain injury with pre-conditioning and combined fractions is about 5μΙ_^. The reduction in postoperative hematoma shows the success of preconditioning with Crotalus atrox venom in reducing bleeding during surgical brain injury. The success in reduction of bleeding in the surgical area can be utilized in other surgeries or injuries beyond surgical brain injury as described herein and/or known in the art.

Neurological Deficits

[0110] In some embodiments, pre-conditioning or rapid-conditioning treatments can be effective before or after surgery or injury to improve a patient's behavioral and functional outcomes after injury or surgery. In some embodiments, Cv-PC can limit neurological deficits following surgical brain injury. The behavioral and functional outcomes of the patients can be improved with the therapeutic techniques disclosed herein. [0111] FIG. 6 illustrates a time course of neurological deficits after surgical brain injury. The most pronounced neurological deficits occurred in the first 72 hours after surgical brain injury. FIG. 6 shows the time-course of neurological function on a line graph showing neurological scoring for 10 rats at different time points after surgical brain injury. The neurological deficits were maximal at 24 hours post-surgery and started recovering after day 3 to almost baseline by 1 week.

[0112] In some embodiments, pre-conditioning treatments can improve the functional outcome associated with surgical brain injury. FIG. 7A-B conveys the improvement of functional outcome with Crotalus atrox venom using the results of the Garcia Neuroscore and the Foot Fault Test. FIG. 7A displays the results of the Garcia Neuroscore which scores the results of the Garcia test. The Garcia test is a composite neurological test that evaluates sensorimotor deficits. The score of a sham surgery is about 20. The results after surgical brain injury (SBI) and surgical brain injury treated with Crotalus atrox venom pre-conditioning (SBI+Cv-PC) at 24 hours and 72 hours after surgery were recorded. The results show a higher score of about 18 for subjects treated with pre-conditioning with Crotalus atrox venom while those with surgical brain injury without pre-conditioning have a Garcia Neuroscore of below 18.

[0113] FIG. 7B displays the results of the Foot Fault test. The Foot Fault test assesses locomotor function of subjects. The number of left foot faults was recorded to show the locomotor function in subjects with sham surgery (Sham), surgical brain injury without pre-conditioning (SBI), and surgical brain injury treated with Crotalus atrox venom pre-conditioning (SBI+Cv-PC). The sham surgery showed a low number of left foot faults at below 20 and the surgical brain injury treated with pre-conditioning also produced a low number of left foot faults at lower than 20. The surgical brain injury produced a reduced locomotor function producing a higher number of left foot faults at about 60. Subject treated with pre-conditioning showed a locomotor function at about the same level as subjects without surgical brain injury, sham subjects.

[0114] In some embodiments, the treatment with venom pre-conditioning has been shown to reduce brain water content and intraoperative hemorrhage volume while also improving neurological function after surgery. Crotalus helleri venom dosage of 0.36mg/kg was administered daily for 3 days with the last dose given 24h prior to surgical brain injury-induction. At 24 hours and 72 hours following surgical brain injury, brain water content was evaluated as illustrated in FIG. 8. The intraoperative hemorrhage volume was quantified by spectrophotometric assay for hemoglobin as illustrated in FIG. 9. Further, FIG. 10 illustrates the neurological function assessed at 24 hours and 72 hours following surgical brain injury.

[0115] FIG. 8 illustrates brain water content following surgical brain injury. Brain water content was significantly increased in ipsilateral frontal lobe (RF) in surgical brain injury animals compared to that of sham animals at 24 hours and 72 hours. Cv-PC significantly reduced brain water content in the ipsilateral frontal lobe at both 24 hours and 72 hours in comparison to vehicle treatment. Other brain sections (LF, LP, RP) are shown as controls. The reduction in brain water content shows that Cv-PC reduced brain edema after surgical brain injury.

[0116] FIG. 9 shows the intraoperative hemorrhage during surgical brain injury as assessed with a hemoglobin assay. Intraoperative hemorrhage volume was significantly increased in surgical brain injury animals compared to that of sham animals. Intraoperative hemorrhage volume was significantly reduced in Cv-PC-treated animals compared to that of vehicle-treated animals.

[0117] FIG. 10 displays the neurological function after surgical brain injury based on the modified Garcia scores. Surgical brain injury animals exhibited neurological deficits and scored significantly lower on neurobehavioral tests compared to sham animals at 24 hours and 72 hours. Cv-PC-treated animals scored significantly higher than vehicle-treated animals at 24 hours, but not at 72 hours. Therefore, neurological function after surgical brain injury was improved in Cv-PC treated animals.

[0118] Therefore, based on the Cv-PC data it has been demonstrated that Cv- PC reduced brain edema after surgical brain injury, decreased hemorrhage during surgery, improved neurological function after surgical brain injury, and only at high dosages 50-100% of LD₅₀ Crotalus venom caused local inflammatory reaction as shown in FIG. 2B.

PLA?/CQX-2 Inflammatory Signaling Pathway.

[0119] As described herein, Cv-PC produces neuroprotective effects in surgical brain injury through the PLA₂/COX-2 inflammatory signaling pathway. The expression of inflammatory mediators after surgical brain injury can be reduced with Cv- PC. There is an increase in expression of inflammatory mediators, such as IL-Ιβ and TNFa, following surgical brain injury. Therefore, the inflammatory pathway can be a key player in the surgical brain injury pathophysiology. In some embodiments, the repetitive administrations of Cv-PC doses can down-regulate the response of the PLA₂/COX-2 to injury, thereby reducing the inflammatory response.

[0120] Surgical brain injury will result in the elevation of PLA₂ and COX-2 expression in perilesional brain tissue. COX-2 has been implicated to be an integral player in the pathophysiology of surgical brain injury-induced brain edema. Cv-PC can attenuate the surgical brain injury-induced increase in COX-2 and thereby decrease surgical brain injury induced brain edema.

[0121] It has been demonstrated that COX-2 expression can be increased at 24 hours following surgical brain injury and that the administration of a NS398, a selective COX-2 inhibitor, mitigated this increase of COX-2. A chief component of Crotalus venom is PLA₂, an enzyme directly upstream of COX-2 in the inflammatory cascade. Cv-PC can elevate systemic levels of PLA₂ prior to surgical brain injury induction. This sustained elevation of PLA₂ prior to surgical brain injury can trigger endogenous down-regulation of this inflammatory pathway, ultimately reducing the activity of the PLA₂/COX-2 pathway following the insult introduced by surgery and thereby reducing the inflammatory response.

[0122] FIG. 11 demonstrates inflammation occurring after surgical brain injury as shown by enhanced myeloperoxidase activity. Additionally, FIG. 11 and FIG. 12 illustrate the increased neuronal expression of IL-Ιβ and TNFa that occurs. Rosiglitazone (RSG) reduced Myeloperoxidase activity as well as IL-Ιβ and TNFa expression are shown in FIG. 11 and FIG. 12.

[0123] FIG. 11 shows the effect of Rosiglitazone (RSG) on inflammation and shows the effect of Rosiglitazone (RSG) (1 mg/kg) on myeloperoxidase (MPO) activity at 24 hours after surgical brain injury in the ipsilateral frontal lobe. There is increased MPO activity after surgical brain injury in the vehicle treated group as compared to the sham surgery. This increased MPO activity is indicative of inflammation that was attenuated by RSG treatment (1 mg/kg). [0124] FIG. 12 shows immunohistochemical staining for inflammatory markers IL-Ιβ and TNFa following surgical brain injury. FIG. 12 shows double fluorescent immunohistochemical representative pictographs depicting inflammatory markers IL-Ιβ and TNFa (green color, FITC) co-stained with NeuN, a marker for neuronal cells (red color, Texas Red) in ipsilateral frontal lobe at 24 hours after surgical brain injury. The region of interest (ROI) from the sections used for fluorescent immunostaining was obtained from the ipsilateral frontal lobe, more precisely from the edge of the resection as depicted in A and B for vehicle and RSG treated groups respectively. The distribution of immunoreactivities in the vehicle-treated groups (panels A-C and G-I) suggests that neurons express inflammatory mediators IL-Ιβ and TNFa after surgical brain injury. Qualitatively, these markers are attenuated in the RSG treated (6 mg/kg) group (panels D-F and J-L). The merged images (panels C, F, I and L) show magnified images of the cells in the insets. Arrows depict the merged immunoreactivities of the inflammatory markers and NeuN. The scale bar denotes 100 μπι. The figures are representative of data from 3 animals per group. The expression of inflammatory mediators IL-Ιβ and TNFa after surgical brain injury show that they play a role in inflammation following surgical brain injury.

Inflammatory Mediators TXA? And Prostaglandins

[0125] In some embodiments, surgical brain injury can result in the elevation of inflammatory mediators TXA₂ and prostaglandins in the brain and blood following surgical brain injury. Activation of the PLA₂/COX-2 pathway can induce the production of inflammatory mediators such as TXA₂ and prostaglandins. Therefore, pre-conditioning can be utilized to reduce the levels of inflammatory mediators present after injury. For example, Cv-PC can reduce the levels of these inflammatory mediators following surgical brain injury.

[0126] As discussed herein, the neuroprotective effects of Cv-PC are conferred through the altered expression of PLA₂ and COX-2. Neuroprotection by HBO- PC in surgical brain injury has been shown to be mediated through the COX-2 pathway and inhibition of COX-2 has been shown to reverse the therapeutic effect. Therefore, in some embodiments, the inhibition of PLA₂ or COX-2 can reverse the effects of Cv-PC. Without the elevation of PLA₂ and COX-2 in the days leading up to surgical brain injury and thus the removal of repetitive harmful stimuli, the pre-conditioning effects of Cv-PC are lost.

[0127] In addition, hyperbaric oxygen pre-conditioning (HBO-PC) increased COX-2 expression in western blot after pre-conditioning but before surgical brain injury as shown in FIG. 13. HBO-PC reduced COX-2 elevation induced by surgical brain injury as shown in FIG. 13. FIG. 13 demonstrates the increase in COX-2 levels reduced with HBO-PC. Western blotting data (n=4) demonstrate that the increase in COX-2 levels in the normoxia with surgical brain injury group at 24 hours was reduced with HBO-PC.

[0128] As illustrated in FIG. 14, in a global cerebral ischemia rat model, HBO-PC abolished COX-2 elevation after global cerebral ischemia and a COX-2 inhibitor administered before HBO-PC abolished the protective effect of HBO-PC. This response demonstrated that inflammation is a major pathophysiology, mediated by COX- 2 pathways after surgical brain injury as well as global cerebral ischemia. Therefore, preconditioning can reduce COX-2 expression and brain injury.

[0129] FIG. 14 shows that HBO-PC can reduce COX-2 elevation after global cerebral ischemia and a COX-2 inhibitor administered before HBO-PC can abolish the protective effect of HBO-PC. In a transient global cerebral ischemia rat model (GI), rats were pressurized in a research hyperbaric chamber (1300B; Sechrist) at 2.5 atmospheres absolutes with 100% oxygen (flow of 22 L/min). Compression and decompression were maintained at a rate of 5 psi/min. A 1-hour HBO session was administered daily for 5 consecutive days (HBO-PC) and the last dive was performed 24 hours before ischemia. COX-2 inhibitor NS-398 at a dose of 1 mg/kg suspended in 10% dimethyl sulfoxide in phosphate-buffered saline was intraperitoneally injected 10 minutes before each HBO session (HBO-PCI). The rats in the HBO-PC group received vehicle by itself (10% dimethyl sulf oxide/phosphate buffered saline) according to the same injection regimen. Western blot analysis of cyclooxygenase-2 (COX-2) levels in the hippocampus was performed on day 1 after global cerebral ischemia. NS-398 (HBO-PCI) abolished the effect of HBO on COX-2 expression.

[0130] PLA₂-PC can reduce brain edema and preserve blood-brain barrier integrity following surgical brain injury. PLA₂-PC will mimic the anti-inflammatory effects of Cv-PC. Administering PLA₂-PC, like Cv-PC, introduces minor inflammatory challenges in the body prior to injury, preparing for the major insult of surgical brain injury.

[0131] Cv-PC data demonstrated that COX-2 protein levels increased after surgical brain injury, which is consistent with our previous publications in surgical brain injury and global cerebral ischemia models as shown in FIG. 13 and FIG. 14. Cv-PC decreased COX-2 levels by about 50%.

[0132] Crotalus helleri venom dosage of 0.36mg/kg was administered daily for 3 days with the last dose given 24 hours prior to surgical brain injury-induction. In the Cv-PC+NS398 group, a COX-2 inhibitor (10 mg/kg) was administered 1 hour prior to each Cv-PC dose. At 24 hours, animals were sacrificed and brain samples were collected to measure COX-2 by Western blot analysis as shown in Figurel5. Immunohistochemistry staining was conducted to show effect of Cv-PC on PGE2 expression after surgical brain injury as demonstrated in FIG. 15.

[0133] FIG. 15 shows a western blot analysis of COX-2 following surgical brain injury and Cv-PC. COX-2 expression in the ipsilateral frontal lobe was significantly increased in surgical brain injury animals compared to that of sham animals at 24 hours following surgical brain injury. Cv-PC significantly reduced COX-2 expression compared to that of vehicle-treated animals. Co-administration of Cv-PC and NS398, a COX-2 inhibitor (10 mg/kg), reversed the effect of Cv-PC and restored COX-2 expression to beyond that of vehicle-treated animals.

[0134] FIG. 16 shows an immunohistochemical staining for inflammatory marker PGE2 following surgical brain injury which is reduced with Cortalus atrox venom preconditioning (shown in photo C). FIG. 16 shows fluorescent immunohistochemical representative photomicrographs depicting inflammatory marker PGE2 in ipsilateral frontal lobe at 24 hours after surgical brain injury. The region of interest from the sections used for fluorescent immunostaining was obtained from the ipsilateral frontal lobe, more precisely from the edge of the resection, as depicted in panels A (sham), B (vehicle- treated), and C (Cv-PC-treated). The distribution of immunoreactivity in the vehicle-treated groups (panel B) suggests that PGE2 expression is increased after surgical brain injury compared to that of sham. Additionally, the PGE2 marker is attenuated in the Cv-PC-treated group (panel C). [0135] The effect of Cv-PC is reversed by a COX-2 inhibitor NS398 as shown in FIG. 15, which supports a role of COX-2 in Cv-PC. Furthermore, PGE2 expression is enhanced after surgical brain injury and Cv-PC reduced PGE2 elevation, as illustrated in FIG. 16, supports the role of PLA₂ and its downstream metabolites.

Fibrinogenolytic Activity

[0136] Cv-PC can reduce hemorrhage produced by surgical brain injury by its fibrinogenolytic activity. Cv-PC can upregulate fibrinogen synthesis, thereby facilitating coagulation and limiting intraoperative hemorrhage. Increases in thrombin induced by surgical brain injury have been shown thereby confirming the role of blood toxicity in surgical brain injury. Cv-PC can reduce intraoperative hemorrhage as illustrated in FIG. 1A-B. Coagulative factors following Cv-PC as well as their role in the reduction of intraoperative hemorrhage can be examined.

[0137] Cv-PC can increase plasma fibrinogen. Crotalus venoms contain metalloproteinases (sMMPs) that are fibrinolytic. These sMMP can produce fibrin and fibrinogen degradation products (FDPs) without inducing clotting. In the days leading up to surgical brain injury induction, the FDPs can upregulate the biosynthesis of fibrinogen by hepatocytes.

[0138] In some embodiments, coagulative parameters can remain in normal ranges. Crotalus venoms can have anti-coagulative effects by causing hypofibrinogenemia. However, in some embodiments, at the correct levels of Cv-PC dosing, fibrinogen biosynthesis can be induced without causing hypofibrinogenemia. In addition, since sMMP in Crotalus venom does not induce clotting as it creates FDPs, the levels of D-dimers can be minimal.

[0139] In some embodiments, the improved coagulation of Cv-PC is the result of sMMP activity. Previous studies indicate that sMMPs in Crotalus venom create FDPs. Therefore, in some embodiments, inhibition of sMMP can block the antihemorrhagic effects of Cv-PC.

[0140] FIGS. 17-20 illustrate MMP levels and the effect of MMP inhibitors. Additionally, thrombin activities after surgical brain injury are illustrated in FIG. 21. It has been observed that MMP9 levels are increased in brain tissues after SAH in western blot and immunohistochemistry staining as seen in Fig.17-18. MMP inhibitor-1 reduced MMP9 levels, reduced brain edema, and improved neurological functions as illustrated in FIGS. 17A-D.

[0141] FIGS. 17A-C illustrate the effects of MMP inhibitor-1 with a single and daily dose treatment regimen after surgical brain injury. FIG. 17A is a representative zymogram showing that both single and daily dose regimens inhibit the MMP-9 and MMP-2 enzymatic activity compared with the vehicle-treated at 72 hours post-surgery, which is quantified from four experiences in FIG. 17B.

[0142] FIG. 17C shows a bar graph showing that both treatment regimens attenuate the brain edema indicated by decreased brain water content at 72 hours after surgical injury. FIG. 17D shows a line graph showing the effects of single and daily dose regimens on neurological score over multiple time points after surgical injury. The number of animals is indicated in parentheses.

[0143] FIGS. 18 A- J shows pictographs showing the colocalization of MMP-9 with different cell markers in brain sections adjoining the surgical brain injury. MMP-9 immunoreactivities (green, FITC labeled) were abundantly co-localized with NeuN and MPO immunoreactivities (red, TRITC labeled), the neuronal and neutrophilic markers, respectively, in the ipsilateral hemisphere. All individual pictographs are representative of four experiments each. The scale bar in FIGS. 18A-C, E-G, and I represents 50 μπι. FIGS. 18D and 181 are magnified merged images from FIGS. 18C and 18H, respectively, with the scale bar representing 10 μπι. FIG. 18J is an IgG stained coronal section depicting the regions of interest in the ipsilateral and contralateral hemisphere for the immunohistochemical data presented herein.

[0144] The time course of MMPs after surgical brain injury has shown that MMP9 increased earlier, and was followed by MMP2 increases at 3 days after surgical brain injury as shown in FIG. 19. FIG. 19 shows a zymogram and bar graph depicting the MMP-9 and MMP-2 enzymatic activity at different time points after surgical brain injury. The upper panel shows a representative zymogram showing greatly increased MMP-9 activity from days 1 and 3 after surgical injury, which is quantified in the lower panel from a total of four experiments. Both MMP-9 and MMP- 2 activities are significantly higher than preoperative levels; however, MMP-9 activity is greatly increased. [0145] Additionally, as illustrated in FIGS. 20A-C, MRI studies show blood- brain barrier and brain edema at different times after surgical brain injury and the protective effect of MMP inhibitor- 1. FIGS. 20A-C illustrate an estimation of brain edema by MRI scans. FIG. 20A is a representative contrast-enhanced Tl -weighted imaging scan from vehicle-treated and MMP inhibitor- 1 -treated animals 3 hours post- surgery. In some embodiments, there can be large areas of blood-brain barrier permeability as evidenced by the high ipsilateral signal as shown by the dotted line. There is slightly increased signal at brain regions distant from the original lesion as denoted by the arrows in FIG. 20A. FIG. 20A shows that blood-brain barrier permeability is dramatically muted in the MMP inhibitor- 1- treated animals.

[0146] FIG. 20 B shows a representative T2-weighted MRI scans (bregma + 2.20 mm) illustrating the potential protective effect of MMP inhibitor- 1 at different time points. In the vehicle-treated animals, the T2 signal intensity was dramatically decreased within the surgical lesion at 3 hours, possibly due to cellular swelling. However, at 72 hour, there was a dramatic increase in signal intensity, indicating edema formation. By 7 days, there was a gradual reduction in signal intensity within the lesion and normalization of the surrounding cortex. In the MMP inhibitor- 1 -treated animals, there is a temporal shift in that edema formation that appears within 3 hours after surgery. Residual edema seems to be reduced at 72 hours and is dissipated by 7 days with the cortex appearing normal. Dotted lines indicate the approximate location of the surgical resection.

[0147] FIG. 20C shows Apparent Diffusion Coefficient maps computed for vehicle- and MMP inhibitor- 1 -treated animals. Vehicle-treated animals showed decreased diffusion within the surgery site at 3 hours that slowly increased during the next 7 days. The development of increased diffusion is related to increased edema formation and tissue inflammation. However, in the MMP inhibitor- 1 -treated animal, there is decreased diffusion within the lesion site throughout the 7 days. Dotted lines indicate the approximate location of the surgical resection.

[0148] These studies demonstrated a marked pathological role of MMPs after surgical brain injury and support the fact that MMPs in Crotalus venom can upregulate endogenous protective mechanisms against surgical brain injury, especially intraoperative hemorrhage. Additionally, surgical brain injury can increase thrombin activity as shown in FIG. 21. The increased thrombin activity can further indicate coagulation changes after surgical brain injury. FIGS. 22-23 demonstrate that plasma fibrinogen and degradation products are increased by Cv-PC after surgical brain injury. The level of thromboplastin time remains unchanged by Cv-PC as shown in FIG. 24.

[0149] FIG. 21 shows surgical brain injury can cause a significant increase of thrombin activity at 24 hours after operation. Surgical brain injury increased thrombin activity in the frontal lobe of surgical brain injury animals compared to sham-operated animals.

[0150] FIG. 22 illustrates plasma fibrinogen following Cv-PC (Cortalus atrox venom preconditioning). After 3 daily doses of Cv-PC, plasma fibrinogen was significantly increased compared to that of the vehicle group.

[0151] FIG. 23 shows plasma fibrinogen degradation products following Cv- PC. The plasma of vehicle-treated animals showed negligible (0-10 μΙ7πιΙ_^) amounts of fibrinogen degradation products (FDPs). After 3 daily doses of Cv-PC, the FDP concentration in Cv-PC-treated animals was relatively increased (10-160 μΙ7πιΙ_^) compared to that of vehicle-treated animals. FIG. 24 shows partial thromboplastin time following Cv-PC. Partial thromboplastin time (PTT) does not differ between vehicle- and Cv-PC-treated animals.

[0152] Cv-PC data demonstrates that Cv-PC upregulates fibrinogen and its degradation products which can change coagulation ability and reduce hemorrhage during surgical brain injury. Though these processes Cv-PC can reduce intra-operative bleeding.

Blood Parameters Present During Surgical Brain Injury

[0153] The blood of subjects is tested to determine the effectiveness of the treatment with pre-conditioning with venom on subjects and to ensure effective healing and coagulation and hemostasis activity. FIGS. 25A-F displays the blood parameters present during surgical brain injury with and without pre-conditioning with Crotalus atrox venom. The amount of fibrinogen present was recorded in subjects with surgical brain injury (SBI), subjects with surgical brain injury treated with Crotalus atrox venom pre-conditioning (SBI+Cv-PC), and surgical brain injury with MMP inhibitor (SBI+Inhibitor) as shown in FIG. 25A. The surgical brain injury treated with Crotalus atrox venom pre-conditioning shows the highest level of fibrinogen present and thereby showing a greater level of healing, coagulation, and clot formation. The MMP inhibitors used can include Marimastat and AG-3340. These results show that the MMPs in the venom are at least partially responsible for the beneficial effects.

[0154] FIG. 25B displays the number of fibrin degradation products (FDPs). Fibrin degradation products are the substances left behind from clots that have dissolved. These components in the blood show that the clots are forming and thereby dissolving properly as the injury heals. In FIG. 25B the 8 vehicle rats tested represent the control rats. The vehicle rats have between O-lC^g/mL of FDPs. Rats with Crotalus atrox venom pre-conditioning (Cv-PC) have more than 10 μg/mL. The FDP test shows that the venom at least partially degrades fibrinogen. The degradation of fibrinogen can do two things including: (1) inducing fibrinogen synthesis; and (2) placing fibrinogen into a slightly more "ready" state for clot formation.

[0155] FIG. 25C displays an increase in clumping in subjects with surgical brain injury treated with Crotalus atrox venom pre-conditioning (SBI+Cv-PC). Those subjects with MMP inhibitors have little to no increase in clumping. Therefore, surgical brain injury treated with Crotalus atrox venom pre-conditioning is more successful at clumping than surgical brain injury without pre-conditioning as well as surgical brain injury with inhibitors. The MMP inhibitors used can include Marimastat and AG-3340. These results also show that the MMPs in the venom are at least partially responsible for the beneficial effects.

[0156] The blood is also tested to ensure clotting time is not effective by measuring the partial thromboplastin time. The partial thromboplastin time determines the factors present in the coagulation cascade to ensure proper blood clotting and therefore no excess bleeding. FIG. 25D displays little to no change in the partial thromboplastin time (PTT(s)) between vehicle and surgical brain injury treated with Crotalus atrox venom pre-conditioning. Therefore, there is no loss in partial thromboplastin time with patients treated with venom pre-conditioning.

[0157] Platelet sticking is observed to ensure proper clot formation and healing after injury. FIG. 25E determines the platelet sticking of subjects with surgical brain injury (SBI), subjects with surgical brain injury treated with Crotalus atrox venom pre-conditioning (SBI+Cv-PC), and surgical brain injury with inhibitor (SBI+Inhibitor). Platelet sticking is higher with surgical brain injury treated with Crotalus atrox venom pre-conditioning (SBI+Cv-PC) and surgical brain injury with an MMP inhibitor (SBI+Inhibitor) than with subjects with surgical brain injury without treatment. The increased platelet sticking ensures at least sustained level of clot formation if not increased clot formation. The MMP inhibitors used can include Marimastat and AG- 3340.

[0158] FIG. 25F shows clotting time observed with subjects with surgical brain injury (SBI), subjects with surgical brain injury treated with Crotalus atrox venom pre-conditioning (SBI+Cv-PC), and surgical brain injury with an MMP inhibitor (SBI+Inhibitor). The surgical brain injury treated with Crotalus atrox venom preconditioning and surgical brain injury with inhibitor have a shorter clotting time than surgical brain injury without treatment. A decreased clotting time shows a decrease in the time necessary for blood clotting thereby decreasing blood loss. The MMP inhibitors used can include Marimastat and AG-3340.

[0159] FIGS. 26A-C are images showing the inflammation in sham surgery, surgical brain injury, and surgical brain injury with venom pre-conditioning. The decreased inflammation in subjects with surgical brain injury with venom preconditioning is shown in FIG. 26C with a lesser amount of inflammation visible in white as compared with the inflammation seen in FIG. 26B. The decrease in inflammation shows that the pre-conditioning with venom decreases the inflammation that results from surgical brain injury by priming the surgical site with venom pre-conditioning.

Pre-Conditioning With Fractionated Venom For Surgical Brain Injury

[0160] Fractionation of Crotalus atrox venom was reviewed to determine the fractions of the components of the venom. FIG. 27 displays the fractionation of Crotalus atrox venom with the reverse-phase HPLC chromatograph of Crotalus atrox venom. FIG. 28 displays a PAGE gel of each fraction for Cortalus atrox venom. The protein ladder on the left-most side indicates the molecular weights of each band. The PAGE gel shows which proteins are in each fraction showing the fractionation and the proteins present in the peaks. [0161] FIGS. 29A-B show the reduction in bleeding or hemorrhage with fractionated Crotalus atrox venom. The venom can be fractionated into Fractions 1-9. Fraction 1 contains high molecular weight proteins and some MMPs. Fractions 2 and 3 contain MMPs. Fraction 4 contains MMPs and serine proteases. Fraction 5 contains PLA₂. Fractions 6-9 contain neurotoxins and peptides. Fractions 1-4 can have an effect on reducing blood loss and fractions 5-9 can have an effect on inflammation. FIG. 29 A displays the results of pre-conditioning with venom combined fractions (SBI+Cv-PC: Combined Fractions) as well as pre-conditioning with separate venom fractions. All preconditioning with fractions 1-4 showed a reduction in blood loss or hemorrhage regardless of the fraction used. Fractions 5-9 can have an effect on inflammation. The highest reduction in blood loss was observed with SBI+Cv-PC: Fraction 4 IV. Additionally, FIG. 29B displays the postoperative hematoma for patients treated with venom pre-conditioning. A lower level of postoperative hematoma was observed with patients treated with pre-conditioning with venom (SBI+Cv-PC) and pre-conditioning with venom combined fractions (SBI+Cv-PC: Combined Fractions). The combined fractions test proves that the fractionation process did not alter venom/protein activity. The venom was fractionated and then all venom fractions were combined back into a single injection.

[0162] FIGS. 30A-B show the improved outcome of rats treated with preconditioning with fractionated Crotalus atrox venom. FIGS. 30A-B show the observed outcomes of pre-conditioning with venom combined fractions (SBI+Cv-PC: Combined Fractions) as well as pre-conditioning with separate venom fractions. The outcomes of subjects treated with pre-conditioning resulted in higher neuroscores and substantially less foot faults and thereby improved outcomes of subjects treated with pre-conditioning fractions.

[0163] FIGS. 31 A-C and 32A-C show preconditioning with CV fraction. This surgical brain injury model can mimic neurosurgery procedures. Preconditioning was performed for all treatments (except the Fibrinogen (Fb) group). The intraoperative bleeding represents bleeding during the surgery. The postoperative hematoma represents the blood remaining in the brain 24 hours after surgery. The Modified Garcia Score is a test for neurobehavior and function (sensorimotor tests). [0164] FIGS. 31A-C shows Cv-PC dose-dependent intraoperative hemorrhage and postoperative hematoma in SB I. FIGS. 31A-B show intraoperative hemorrhage (FIG. 31 A) and postoperative bleeding (FIG. 3 IB) was reduced by subcutaneous Cv-PC in a dose-dependent manner. FIGS. 31C shows the Modified Garcia Score at 24 hours post surgical brain injury.

[0165] FIGS. 32A-C shows Cv-PC intraoperative hemorrhage and postoperative hematoma in SBI. FIGS. 32A-B show the intraoperative hemorrhage (FIG. 32A) and postoperative bleeding (FIG. 32B) with Cv-PC, MMP-inhibitor (MMP-I), Cv fractions, and Fibrinogen (Fb). FIG. 32C shows the Modified Garcia Score at 24 hours post-SBI.

[0166] Crotalus atrox venom was fractionated via size exclusion chromatography using a Superdex gel filtration column (HiLoad 16-/60 Superdex 75PG, 17-1068-02, GE Healthcare) and Amersham Biosciences AKTAFPLC (18-1900-26, GE Healthcare). Crude venom (4 mg/mL in 0.15 ammonium bicarbonate) was injected into the column (500 μΕ) and separated in 0.15 M ammonium bicarbonate at a flowrate of 1 mL/min. Individual fractions were collected manually at the local minimums of each peak (based on absorbance at 214 nm). All fractions collected were lyophilized and stored at -20°C until use. FIG. 33 shows the fractionation graph. A2 corresponds to Fraction 1 (i.e. all fractions = A#-l).

[0167] In other embodiments, this preconditioning method can also be used for other applications. In some embodiments, the preconditioning methods as described herein can be used for veterinary surgeries similar to the methods and use described herein for humans. Bleeding can occur in numerous surgeries. In some embodiments, the preconditioning treatment can be used for other surgeries in which bleeding occurs including but not limited to use for cardiac surgeries, muscle reconstruction, back surgery, and/or other surgeries or procedures described herein. The preconditioning treatment can be administered for other bleeding using methods and treatment protocols as described herein with reference to surgical brain injury.

Rapid-conditioning [0168] In some embodiments, the venom application and exposure can occur during a small window to prevent hemorrhage during surgery or injury and/or after a trauma to the patient with rapid-conditioning techniques. In some embodiments, rapid- conditioning techniques are utilized following a stroke to prevent hemorrhage. This can prevent further damage to the brain due to hemorrhage and thereby reduce the overall damage to the brain due to the stroke and/or other injury. In some embodiments, rapid- conditioning techniques can be utilized during surgery or injury to prevent and/or reduce hemorrhage. In some embodiments, the venom can be used to stop bleeding during and/or after surgery. This treatment of bleeding during or after surgery is similar to treatment of bleeding after stroke and can be done in addition to or instead of the preconditioning treatment discussed previously. The hemorrhagic venom components and hemorrhagic venom types used for rapid-conditioning can be similar to those described previously with reference to preconditioning treatments. In some embodiments, hemorrhagic venom components and hemorrhagic venom types used for rapid- conditioning can be different from those previously discussed with reference to preconditioning.

[0169] As with other forms of brain injury such as traumatic head injury or stroke, there is a narrow window of opportunity for therapy once injury has occurred. Reducing hemorrhage during or after traumatic head injury, surgery, stroke, or other injury can lower the postoperative care and improve the outcome of surgical patients similar to improvements discussed herein with reference to pre-conditioning.

[0170] In some embodiments involving rapid-conditioning with a venom, Crotalus atrox venom or Crotalus helleri venom can be administered at the doses and a schedule as described herein with reference to pre-conditioning treatments. In some embodiments, the application of rapid-conditioning treatment can be altered for the particular condition to be treated. For example, because rapid-conditioning is meant to be applied to an injury after the occurrence of the injury or during the occurrence of an injury, rapid-conditioning can be more effective when applied in closer intervals than are used for pre-conditioning therapies. Rapid-conditioning can be administered at about three doses over 1.5 days, three doses over three days, or six doses over six days after injury. Additionally, the doses can be administered in from about 1 to about 6 divided doses. In some embodiments, the doses can be administered in from about 1, about 2, about 3, about 4, about 5, or about 6 divided doses over the treatment period. The treatment period can range from about 0 days to about 6 days after injury. In some embodiments, the treatment period can involve administering doses over about 0 days, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, and/or about 6 days or more after injury. In addition to administration before surgery or injury, administration during surgery or injury, or administration after injury or surgery, the administration of venom can be conducted both before, during, and after surgery or injury, as outlined above.

[0171] In some embodiments, an amount of venom characteristic of 5-20% of LD₅₀ values can be used for rapid-conditioning to reduce or avoid local inflammation caused by the injury. For example, about 5%, about 10%, about 15%, about 20%, and about 25% of LD₅₀ values can be used for rapid-conditioning as an effective treatment for surgically induced inflammation or hemorrhage, stroke, and/or other inflammation or hemorrhage.

[0172] The venom can be used as a treatment for stroke (hemorrhagic) and/or other injury. Hemorrhagic transformation occurs in up to 48% of stroke patients. Typically hemorrhagic transformation occurs 2-3 days after ischemic insult in patients, and less than 24 hours in rats. In hyperglycemic rats there is up to 100% occurrence. Crotalus atrox venom is a hemorrhagic venom. Crotalus atrox venom can increases fibrinogen and its degradation products and activate platelets. Therefore, Crotalus atrox venom and/or other hemorrhagic venoms can be used to treat hemorrhagic transformation and/or other bleeding due to injury by activating platelets and increasing fibrinogen and its degradation products thereby promoting blood clotting and healing at the injury site.

[0173] Crotalus atrox venom has been shown to reduce hemorrhagic transformation following middle cerebral artery occlusion (MCAO) in hyperglycemic rats. Treatment with Crotalus atrox (C atrox) venom can reduce hemorrhagic transformation following MCAO in hyperglycemic rats through platelet activation. For example, a proposed pathway for the use of venom for platelet activation and platelet aggregation to reduce or stop hemorrhagic transformation is shown in FIG. 34. The lectin and/or serine protease can be responsible for and/or used to activate platelets. [0174] The treatment regime was identified for reduction of hemorrhagic transformation and neurological deficits. This rat model is of an ischemic stroke which then begins to have bleeding in the infarction (known as hemorrhagic transformation). Currently no therapy can prevent or reduce hemorrhagic transformation (rate or amount). Currently only stabilizing measures are used (i.e. stabilize a patient's BP, HR, glucose, etc.).

[0175] The dose response, platelet activation and hemostatic analysis, and long term outcomes for treatment with venom are reviewed. For example, a dose response experiment is outlined in FIG. 35. The mortality results for this experiment include: Sham: 0/13 (0%); MCAO + Vehicle: 3/15 (20%); MCAO + C atrox (20% LD50): 5/19 (26%); and MCAO + C atrox (30% LD50): 1/12 (8%). As shown, the mortality decreased with MCAO + C atrox (30% LD50). FIG. 36 shows the results of the infract volume following middle cerebral artery occlusion in rats. Crotalus atrox venom does not alter infract volume. FIG. 37 shows the swelling volume results following middle cerebral artery occlusion. FIG. 37 shows that treatment can reduce brain swelling. FIG. 38 shows the hemorrhagic transformation results following middle cerebral artery occlusion. As shown in FIG. 38 bleeding is greatly reduced after stroke when treated with 20% LD₅₀ and 30% LD₅₀. As can be seen, treatment with C. atrox venom can reduce ipsilesional hemisphere swelling and reduce hemorrhagic transformation.

[0176] FIG. 39 shows the results of the Modified Garcia Score following middle cerebral artery occlusion. FIG. 40 shows the results of the corner turn test following middle cerebral artery occlusion. FIG. 41 shows the forelimb placement test following middle cerebral artery occlusion. In some embodiments, the low dose (20% LD50) can improve neurobehavior. In some embodiments, the high dose (30% LD50) can have a tendency to improve neurobehavior.

[0177] Platelet activation and hemostatic parameters and number of thrombi formed and occluded vessels can be measured using immunohistochemistry (IHC). Additionally, the long term functional outcome can be observed (water maze, foot fault, open field, rotorod). FIG. 42 illustrates an embodiment of the experimental parameters for observing the platelet activation and hemostatic analysis. FIG. 43 illustrates an embodiment of the experimental parameters for observing the long term functional outcomes.

[0178] The active proteins in C. atrox responsible for platelet activation have also been studied. Active fractions of C. atrox can be determined by studying in vitro testing on human blood of venom fractions and hemostatic parameters. The active protein(s) can be determined with in vitro testing on human blood with inhibitors of proteins within the active fractions and in vivo treatment with active fractions, with and without inhibitors of proteins. FIG. 44 show an embodiment of a proposed pathway for the use of venom for platelet activation and platelet aggregation to reduce or stop hemorrhagic transformation as shown in FIG. 34, however, the focus of the fractionation is noted as reviewing lectin and/or serine protease which can activate platelets.

[0179] FIGS. 45 - 47 show the results of C. atrox fractionation. FIG. 48 shows an embodiment of the experimental parameters to determine active proteins. In vitro hemostatic parameter analysis can be used to determine active fractions. In some embodiments, human blood samples can be treated with venom and evaluated over 30 minutes. Clumping, clotting, and sticking tests can be performed. Soluble fibrin can be observed. FIG. 49 shows the blood analysis for Crotalus atrox venom fraction 3.

[0180] FIG. 50 shows an embodiment of the experimental parameters to determine active proteins. Active fractions and proteins can be studied in vivo. Six protein families which may be responsible for platelet activation can include: lectins; serine proteases; vascular endothelial growth factor (VEGF) inhibitor; cysteine-rich secretory proteins (CRISP); matrix metalloproteinases (MMP); and phospholipase A2 (PLA2). It has been shown that fractions 3-6 can affect platelet function. In some embodiments, fractions 3-6 can be used for the treatment of and/or reduction of hemorrhagic transformation and neurological deficits.

[0181] MCAO models can be treated with fractions 3-6 and/or can use inhibitors of primary targets. In vitro hemostatic analysis of fractions, with and without protein inhibitors, can be performed. High Performance Liquid Chromatography (HPLC) of fractions can verify proteins contained within each fraction.

[0182] In some embodiments, the hemorrhagic snake venom can be used as a treatment for hemorrhagic stroke after tissue plasminogen activator (tPA) administration. Currently tPA is used to remove blood clots, but it can also increases hemorrhagic transformation. Hemorrhagic venom can be used after administration of tissue plasminogen activator to reduce hemorrhagic transformation.

[0183] Additionally, intraoperative bleeding in surgical brain injury as well as other bleeding during or after injury can be treated using Bothrops Jararaca (B. Jararaca) venom and/or Bothrops Jararaca venom components. It has been shown that treatment decreases intraoperative bleeding in surgical brain injury rats by increasing platelet and vWF interaction. In some embodiments, botrocetin treatment has been shown to decreases intraoperative bleeding in surgical brain injury rats by increasing platelet and vWF interaction. Botrocetin is a protein in the whole venom which targets C-type lectins. FIG. 51 illustrates the B. Jararaca venom components. FIG. 52 illustrates an embodiment of a proposed mechanism for decreasing intraoperative bleeding. In some embodiments, B. Jararaca can be used to reduce perioperative bleeding by increasing vWF and platelet interaction at the resection site in SBI rats. FIG. 53 illustrates an embodiment of a study design for topical administration of the venom and/or venom components. FIG. 54 illustrates an embodiment of a study design for intra nasal administration of the venom and/or venom components. FIG. 55 shows intraoperative bleeding after topical administration of the venom and/or venom components. FIG. 56 shows the brain water content at 24 hours post SBI. As shown, intraoperative bleeding and brain water content were reduced in some subjects treated with the venom and/or venom components. FIG. 57 shows the results of the Modified Garcia Test at 24 hours post SBI. The results of the beam balance at 24 hours post SBI is shown in FIG. 58.

[0184] FIG. 59 shows the intraoperative bleeding after intranasal administration. As shown, the intraoperative bleeding was reduced in subjects treated with the venom and/or venom components.

[0185] In other embodiments, this treatment method can also be used for other applications. In some embodiments, the treatment methods as described herein can be used for veterinary surgeries similar to the methods and use described herein for humans. Bleeding can occur in numerous surgeries and/or injuries. In some embodiments, the treatment can be used for other surgeries and/or injuries in which bleeding occurs including but not limited to use for cardiac surgeries, muscle reconstruction, back surgery, and/or other surgeries or procedures described herein. The venom treatment can be administered for other bleeding using methods and treatment protocols as described herein with reference to stroke, hemorrhagic transformation, and/or surgical brain injury.

Production of Fibrinogen

[0186] Additionally, preconditioning treatments and/or administration of venom can be utilized for the production of fibrinogen and/or purified fibrinogen. In some embodiments, for example, purified fibrinogen from the blood of different animals can be produced utilizing the methods described herein for administration of venom, hemorrhagic venom, and/or hemorrhagic venom fractions. In some embodiments, these methods and techniques described herein can be used to increase the amount of fibrinogen in an animal pre-sacrifice and the ultimate yield of fibrinogen from purification can be greatly increased (-50-100% increased yield). For example, for this application, the dosing can be longer than the few days utilized for preconditioning. In some embodiments, similar to preconditioning the hemorrhagic venom administration can be utilized on a healthy or uninjured animal. In some embodiments, the dosing can be for a few days, a few weeks, or up to one or two months.

[0187] As used herein, the secondary injury can be any type of injury that occurs after the initial insult which stems from the initial insult. As used herein, reducing the secondary injury or bleeding by preconditioning, post injury treatments, and/or rapid conditioning treatments includes lowering and/or reducing the inflammation, swelling, and/or bleeding. This reduction in inflammation, swelling, and/or bleeding can improve patient outcome. Therefore, a successful preconditioning, post injury, and/or rapid conditioning treatment includes administering venom to the patient such that the immune cells are activated and the immune response increased to reduce inflammation, swelling, and bleeding at an injury site.

EXAMPLE 1: CV-PC WILL REDUCE BRAIN EDEMA, HEMORRHAGE, AND NEUROLOGICAL DEFICITS AFTER SURGICAL BRAIN INJURY

[0188] Dose regimen is established through experiments and toxicity and side effects of Cv-PC are examined. An optimal Cv-PC dosing regimen can limit the side effects of Crotalus venom administration. 3 different dose concentrations in accordance to previous hematologic studies performed to study the effects of Crotalus venoms are selected. The studies have established LD₅₀ values and have demonstrated that it is possible to witness the hemostatic effects of the venom without inflicting undue pain to the animals. Experiments with multiple doses (5%, 10%, and 20% of published LD₅₀ values) in accordance to these studies are preformed to establish an ideal dose.

[0189] Male Sprague-Dawley (SO) rats are used. Animal groups are given Crotalus venom at 3 different doses according to the 3 regimens outlined in FIG. 2A. Animals are monitored for behavioral changes indicating pain and bleeding. Efficacy is assessed by neurobehavioral exam and brain water content. Experimental Groups include surgical brain injury with vehicle (SBI+ Vehicle), surgical brain injury with Crotalus venom pre-conditioning (SBI+Cv-PC (9 different groups)).

[0190] Brain edema and blood-brain barrier integrity are evaluated by brain water content in surgical brain injury following Cv-PC. Brain water content is measured to measure brain edema and blood-brain barrier integrity. Animals are given s.c. injections of Crotalus venom according to the optimal dosing regimen determined. At 24 hours or 72 hours after surgical brain injury induction, animals are euthanized and brain samples are collected of brain water content analysis. The experimental groups include sham, surgical brain injury with vehicle (SBI+ Vehicle), and surgical brain injury with Crotalus venom pre-conditioning (SBI+Cv-PC).

[0191] For these experiments, sham and surgical brain injury Sprague Dawley rats (n=90) are used. The surgical brain injury procedure involves partial resection of the right frontal lobe under volatile anesthesia. 4% isoflurane and 2.5% isoflurane, respectively, are used to induce and maintain anesthesia. Sham animals receive only a craniotomy. Cv-PC-treated animals receive s.c. injections of Crotalus atrox venom for 3 consecutive days, with the last treatment administered 24 hours before induction of surgical brain injury. Vehicle-treated animals receive s.c. injections of normal saline.

[0192] At 24h or 72h following surgical brain injury, brain water content and neurobehavioral scoring are performed by an independent researcher blinded to the experimental conditions. Brain water content is evaluated using the wet/dry method. Animals are sacrificed under deep isoflurane anesthesia and the brains are immediately divided on ice into frontal ipsilateral (right), frontal contralateral (left), parietal ipsilateral, parietal contralateral, cerebellum, and brain stem to be weighed immediately (wet weight) and weighed again after drying at 95 °C for 48h (dry weight). The percent of water content is calculated as [(wet weight - dry weight) / wet weight] x 100%.

[0193] Intraoperative hemorrhage volume is measured in surgical brain injury following Cv-PC. Animals are given s.c. injections of Crotalus venom according to the optimal dosing regimen determined. During surgical brain injury surgery, intraoperative hemorrhage volume is collected. 24 hours after surgical brain injury, brains are collected for histological analysis of the hemorrhage volume using 1mm slices of tissue. These animals then have the brain tissue homogenized for spectrophotometnc assay of hemoglobin. The hemorrhage volume is determined by spectrophotometric assay. The experimental groups include sham, surgical brain injury with vehicle (SBI+ Vehicle), and surgical brain injury with Crotalus venom pre-conditioning (SBI+Cv-PC).

[0194] To quantify intraoperative hemorrhage volume, a standard spectroscopic assay for measuring hemoglobin content is used. Blood is collected during surgery, sonicated, and centrifuged. Aliquoted supernatants are incubated with Drabkin's reagent (Sigma- Aldrich) for 15 min and absorbance will be measured at 540nm and hemorrhage volume will be quantified using known standards.

[0195] Additionally, neurological deficits of surgical brain injury following Cv-PC is evaluated. Animals will be given s.c. injections of Crotalus venom according to the optimal dosing regimen determined. At 24 hours or 72 hours after surgical brain injury induction, neurobehavior is evaluated using a modified Garcia scoring system sensorimotor (modified Garcia test, foot fault, beam balance, sticker test, and water maze) and anxiety (elevated zero maze and open field test) behavior tests are performed. The experimental groups include sham, surgical brain injury with vehicle at 24 hours (SBI+ Vehicle (24 hours)), surgical brain injury with vehicle at 72 hours (SBI+ Vehicle (72h)), surgical brain injury with Crotalus venom pre-conditioning at 24 hours (SBI+Cv- PC (24h)), surgical brain injury with Crotalus venom pre-conditioning at 72 hours (SBI+Cv-PC (72h)).

[0196] Neurobehavioral scoring utilizes a modified Garcia 21-pt sensorimotor exam. The sensorimotor testing is graded on a scale of 0 to 3 in a battery of 7 tests: spontaneous activity, side stroking response, vibrissae response, limb symmetry, lateral turning, symmetry of forelimb walking, climbing. Scores are assigned per test as follows: 0 = complete deficit, 1 = definite deficit with some function, 2 = mild deficit or decreased response, and 3 = no evidence of deficit/symmetrical responses. Long term neurological function test are not studied because previous studies shown neurological functions recovered in a week as illustrated in FIG. 6.

[0197] Data is represented as mean + SEM. Statistical differences among more than two groups is analyzed using between- and within- subjects ANOVAs followed by Tukey post-hoc analysis. P values of less than 0.05 are considered statistically significant. All data collection and analysis is conducted blindly. A power analysis (using G*Power3) suggests that a power of 0.8 for detecting a medium effect size (0.3) requires a sample size of 8 per group. Previous experiments using similar models suggest that this sample size is sufficient for detecting brain edema, hemorrhage and relatively subtle behavioral differences among different treatment groups.

[0198] Cv-PC is expected to mitigate the increase in brain water content observed following surgical brain injury and a dose-dependent response is expected. Hemorrhage volume during surgical brain injury surgery is reduced by Cv-PC. Neurological deficits observed following surgical brain injury are expected to improve in animals treated with Cv-PC compared to those of vehicle-treated animals.

[0199] The surgical brain injury model shows significant increases in brain water content that develops in the ipsilateral frontal lobe within 24 hours and persists for at least 72 hours as shown by the wet-dry method. This method is not a clinical relevant method for the measurement of brain water content. If deemed necessary, a T2-weighted and diffusion-weighted imaging (DWI) MRI methods to track the progression of brain edema can be employed.

[0200] The brain water content method does not distinguish between cytotoxic and vasogenic edema. Therefore, an Evans blue extravasation assay and IgG staining can be performed to demonstrate changes in the integrity of the blood-brain barrier.

[0201] The effective therapeutic window of Cv-PC can overlap with unacceptable levels of toxicity. If this is the case, the venom may be purified by liquid chromatography separate out unnecessary enzymes to reduce toxicities. None of the animals with Cv-PC showed any signs of irritation or withdrawn, but all eat and sleep normally.

EXAMPLE 2: ROLE OF PLA2 AND COX-2 IN NEUROPROTECTION BY CV-PC

[0202] The expression of inflammatory mediators after Cv-PC and surgical brain injury is examined. The role of PLA₂ and COX-2 in neuroprotection by Cv-PC is also determined. The effects of antagonizing PLA₂ and COX-2 while administering Cv- PC is evaluated. PLA₂ is administered as a pre-conditioning treatment, instead of Cv-PC, in surgical brain injury and also evaluated.

[0203] A reduction in surgical brain injury-induced overexpression of COX-2 in peri-lesional brain tissue following Cv-PC can be accomplished. The overexpression of COX-2 can be shown by administering each Cv-PC dose in conjunction with NS398, a selective COX-2 inhibitor, which can reverse the effect and thereby suggesting the pivotal role COX-2 plays in Cv-PC. The expression of PLA₂ and COX-2 after surgical brain injury is characterized and their role in the neuroprotection provided by Cv-PC is examined. The expression of PLA₂ and COX-2 in the brain following surgical brain injury with or without Cv-PC is measured. Then, the inflammatory mediators, TXA2 and PGE2, in the brain and blood following surgical brain injury and Cv-PC is measured. The role of PLA₂ and COX-2 in Cv-PC and the effects of PLA₂ pre-conditioning (PLA₂-PC) are examined.

[0204] The expression of PLA₂ and COX-2 in the brain following surgical brain injury with or without Cv-PC is measured. Surgical brain injury results in the elevation of PLA₂ and COX-2 expression in perilesional brain tissue. Cv-PC attenuates the surgical brain injury-induced increase in COX-2. COX-2 has been implicated to be an integral player in the pathophysiology of surgical brain injury-induced brain edema. It has been demonstrated that COX-2 expression increased at 24 hours following surgical brain injury and that the administration of a NS398, a selective COX-2 inhibitor, mitigated this increase of COX-2. A chief component of Crotalus venom is PLA₂, an enzyme directly upstream of COX-2 in the inflammatory cascade. Cv-PC elevates systemic levels of PLA₂ prior to surgical brain injury induction. The sustained elevation of PLA₂ prior to surgical brain injury triggers endogenous down-regulation of this inflammatory pathway, ultimately reducing the activity of the PLA₂/COX-2 pathway following the insult introduced by surgical brain injury.

[0205] Animals are given s.c. injections of Crotalus venom according to the optimal dosing regimen determined. Animals are sacrificed at 24 hours after surgery and brain tissues are collected for Western blot analysis to measure the expression of PLA₂ and COX-2. The experimental groups include sham, surgical brain injury with vehicle (SBI+ Vehicle), surgical brain injury with Crotalus venom pre-conditioning (SBI+Cv- PC).

[0206] Additionally, inflammatory mediators TXB2 and prostaglandins in the brain and blood following surgical brain injury and Cv-PC are measured.

[0207] Surgical brain injury can result in the elevation of inflammatory mediators TXA2 and prostaglandins in the brain and blood following surgical brain injury. Cv-PC can reduce the levels of these inflammatory mediators following surgical brain injury compared to vehicle-treated animals. Activation of the PLA₂/COX-2 pathway can induce the production of inflammatory mediators such as TXA2 and prostaglandins. Cv-PC-treated animals show elevation of such mediators, but at tempered levels compared to that of vehicle-treated animals.

[0208] Animals will be given s.c. injections of Crotalus venom according to the optimal dosing regimen determined. Animals will be sacrificed at 24 hours after surgery. Brain tissues and blood are collected for Western blot analysis to measure the expression of TXB2, a metabolite of TXA2, and PGE2. The experimental groups include sham, surgical brain injury with vehicle (SBI+Vehicle), surgical brain injury with Crotalus venom pre-conditioning at 72 hours (SBI+Cv-PC).

[0209] For the experiments, sham and surgical brain injury animals are used. PLA₂, COX-2, TXB2, and PGE2 expression is evaluated at 24 hours after surgical brain injury. To measure PLA₂, COX-2, TXA2, and PGE2 expressions, ispsilateral frontal lobes are processed and subjected to SDS-PAGE as described. Western blotting protocol is performed with anti-PLA₂, anti-COX-2, anti-TXB2, and anti-PGE2, and anti- -actin goat polyclonal antibodies. Bands are measured by densitometry on a Versadoc System with Quantity One 4.4.2 software (Bio-Rad) and compared to loading controls. [0210] The role of PLA₂ and COX-2 in Cv-PC can be established. The neuroprotective effects of Cv-PC are conferred through the altered expression of PLA₂ and COX-2. Previous reports indicate that neuroprotection by HBO-PC in surgical brain injury is mediated through the COX-2 pathway. The inhibition of COX-2 is shown to reverse the therapeutic effect. The inhibition of PLA₂ or COX-2 reverses the effects of Cv-PC. Without the elevation of PLA₂ and COX-2 in the days leading up to surgical brain injury and thus the removal of repetitive harmful stimuli, the pre-conditioning effects of Cv-PC should be lost.

[0211] Animals are given s.c. injections of Crotalus venom according to the optimal dosing regimen determined in Aim 1A. One hour prior to each Cv-PC dose, quinacrine (PLA₂ inhibitor, 5 or 10 mg/kg) or NS398 (COX-2 inhibitor, 3 or 10 mg/kg) are administered i.p. Animals are sacrificed at 24 hours after surgery. Brain tissues are collected for Western blot analysis to measure the expression of PLA₂ and COX-2. The experimental groups include sham, surgical brain injury with vehicle (SBI+ Vehicle), surgical brain injury with Crotalus venom pre-conditioning (SBI+Cv-PC), surgical brain injury with Crotalus venom pre-conditioning and quinacrine (5 mg/kg) (SBI+Cv-PC+ quinacrine (5 mg/kg)), surgical brain injury with Crotalus venom pre-conditioning and quinacrine (10 mg/kg) (SBI+Cv-PC+quinacrine (10 mg/kg)), surgical brain injury with Crotalus venom pre-conditioning and NS398 (3 mg/kg) (SBI+Cv-PC+NS398 (3 mg/kg)), surgical brain injury with Crotalus venom pre-conditioning and NS398 (10 mg/kg) (SBI+Cv-PC+NS398 (10 mg/kg)).

[0212] For the experiments, sham and surgical brain injury animals are used. Cv-PC doses are preceded 1 hour by PLA₂ antagonist quinacrine (5 or 10 mg/kg, Abeam) COX-2 antagonist NS398 (3 or 10 mg/kg, Abeam) administered i.p. Western blotting is performed as described herein.

[0213] Additionally, the effects of PLA₂-PC is examined. PLA₂-PC can reduce brain edema and preserve BBB integrity following surgical brain injury. PLA₂-PC mimics the anti-inflammatory effects of Cv-PC. Administering PLA₂-PC, like Cv-PC, introduces minor inflammatory challenges in the body prior to injury, preparing for the major insult of surgical brain injury. [0214] Animals are given s.c. injections of PLA₂ (15, 45, and 90 μg kg) according to the dose timing determined. Animals are sacrificed at 24 hour after surgery. Brain water content and neurological function are assessed. Brain tissues are collected for Western blot analysis to measure the expression of COX-2. The experimental groups include sham, surgical brain injury with vehicle (SBI+ Vehicle), surgical brain injury with PLA₂ pre-conditioning with 15 g kg (SBI+ PLA₂-PC (15 μg/kg)), surgical brain injury with PLA₂ pre-conditioning with 45 g kg (SBI+ PLA₂-PC (45 μg/kg)), surgical brain injury with PLA₂ pre-conditioning with 90 μg/kg (SBI+ PLA₂-PC (90 μg/kg)).

[0215] For the experiments, sham and surgical brain injury animals are used. PLA₂ purified from Crotalus durissus terrificus is administered in doses (15, 45, and 90 μg/kg, Sigma Aldrich) according to the optimal dose timing and dose concentrations as determined. Brain water content and neurobehavioral scoring is performed by an independent research blinded to the experimental conditions.

[0216] Endogenous PLA₂ and COX-2 can increase following surgical brain injury. Treatment with Cv-PC introduces PLA₂ and initially activate the PLA₂/COX-2 pathway prior to surgical brain injury onset. The sustained elevation of PLA₂/COX-2 confers tolerance to a later inflammatory insult by surgical brain injury. PLA₂ and COX-2 expression are increased following Cv-PC and surgical brain injury in comparison to sham. However, the expression is significantly less than that of vehicle-treated animals. Similar trends with the downstream inflammatory markers, TXA2 and PGE2, are expected. With COX-2 inhibition, this improvement is eradicated. PLA₂ pre-conditioning is anticipated to mimic the anti-inflammatory effects of Cv-PC.

[0217] 3 and 10 mg/kg NS398 are elected as the selective COX-2 inhibitor in our mechanistic studies because of previous surgical brain injury studies. Other COX-2 inhibitors are available, such as celecoxib. If the potency and half-life of NS398 prove unacceptable, celecoxib would serve as an alternative inhibitor.

[0218] There are two other isozymes of COX (COX-1 and COX- 1 -variant). Although COX-2 has been discussed, COX-1 can be used if necessary to contrast the effects of Cv-PC on COX-1 and COX-2 or to focus on the mechanism of COX-1, indomethacin (selective COX-1 inhibitor) as an alternative inhibitor. [0219] Since TXA2 is labile, its stable metabolite TXB2 is the usual target for measurement. Anti-TXB2 is available for Western blot assays. However, if this proves inadequate in detecting the levels of TXB2, ELISA can be used as an alternative approach. ELISA, with its improved limit of detection allows the levels of TXB2 in Cv- PC- or vehicle-treated groups to be compared.

[0220] The surgical brain injury model is mostly conducted by observing changes within 24 hours after surgery. However, the model can be characterized for up to 7 days following surgical brain injury as shown in FIG. 6. The time of observation to study outcomes if deemed necessary can be expanded.

EXAMPLE 3: CV-PC REDUCES HEMORRHAGE IN SURGICAL BRAIN INJURY

BY ITS FIBRINOGENOLYTIC ACTIVITY

[0221] The role of sMMP and fibrinogen in Cv-PC reduction of hemorrhage volume is evaluated by determining coagulative parameters. Cv-PC can upregulate fibrinogen synthesis, thereby facilitating coagulation and limiting intraoperative hemorrhage. Surgical brain injury increases thrombin, confirming the role of blood toxicity in surgical brain injury. Cv-PC reduces intraoperative hemorrhage as illustrated in FIG. 3A-D. Coagulative factors following Cv-PC are evaluated as well as their role in the reduction of intraoperative hemorrhage. These are evaluated by measuring plasma fibrinogen and fibrinogen degradation product concentrations following surgical brain injury with or without Cv-PC, determining coagulative parameters after surgical brain injury and Cv-PC, and establishing the role of sMMP in Cv-PC.

[0222] Plasma fibrinogen and fibrinogen degradation product concentrations following surgical brain injury with or without Cv-PC is measured. Cv-PC can increase plasma fibrinogen. Previous studies have reported that Crotalus venoms contain metalloproteinases (sMMPs) that are fibrinolytic. These sMMP are able to produce fibrin and fibrinogen degradation products (FOPs) without inducing clotting. In the days before surgical brain injury induction, these FOPs upregulate the biosynthesis of fibrinogen by hepatocytes.

[0223] Animals are given s.c. injections of Crotalus venom according to the optimal dosing regimen determined. Some animals have blood drawn at 24h following the final dose of the treatment regimen, without surgical brain injury-induction. Two groups (Cv-PC- or vehicle-treated) receive surgical brain injury modeling, blood is drawn at 6 hours post-surgery. The experimental groups include sham, surgical brain injury with vehicle (SBI+ Vehicle), surgical brain injury with Crotalus venom pre-conditioning (SBI+Cv-PC).

[0224] Additionally, coagulative parameters, PTT, PT, D-dimer, after surgical and Cv-PC are determined. Coagulative parameters remain in normal ranges. Crotalus venoms are known to have anti-coagulative effects by causing hypofibrinogenemia. At the correct levels of Cv-PC dosing, fibrinogen biosynthesis is induced without causing hypofibrinogenemia. In addition, since sMMP in Crotalus venom does not induce clotting as it creates FOPs, the levels of D-dimers are expected to be minimal.

[0225] Animals are given s.c. injections of Crotalus venom according to the optimal dosing regimen determined. Some animals have blood drawn at 24 hours following the final dose of the treatment regimen, without surgical brain injury-induction. Two groups (Cv-PC- or vehicle-treated) receive surgical brain injury-inducting surgery, blood is drawn at 6 hours post-surgery. The experimental groups include sham, vehicle, Crotalus venom pre-conditioning, surgical brain injury with vehicle (SBI+ Vehicle), surgical brain injury with Crotalus venom pre-conditioning (SBI+Cv-PC).

[0226] Blood is collected from animals from the heart by the terminal draw method. Whole blood is collected using an evacuated sample tube containing a fixed amount of citrate as anticoagulant. Ratio is one part citrate solution to nine parts of whole blood. Anticoagulated blood is mixed gently by inversion and sent to the clinical laboratory to be tested within 2 hours. For PTT, platelet poor plasma is incubated with a platelet membrane substitute and a factor XII activator, and is recalcified. Clotting time is measured in seconds. Plasma fibrinogen is measured by a modified Clauss method. The concentration of fibrinogen is inversely proportional to the clotting time when a known amount of thrombin in excess is added to dilate patient plasma. To assay D-dimers, plasma is mixed with latex beads coated with an antibody to the Fragment D-dimer domain of fibrin. For FDPs assays, plasma is mixed with latex beads coated with an antibody to degradation products D and E of fibrinogen. Agglutination occurs in the presence of either fibrin or fibrinogen degradation products. [0227] The role of sMMP in Cv-PC is established. The improved coagulation of Cv-PC is the result of sMMP activity. Previous studies indicate that sMMPs in Crotalus venom create FOPs. The inhibition of sMMP block the antihemorrhagic effects of Cv-PC.

[0228] Animals are given s.c. injections of Cv-PC according to the dosing regimen determined. One hour prior to each Cv-PC dose, Marimastat (MMP inhibitor, 10 or 40 mg/kg) is administered i.p. Animals are sacrificed at 24 hours after surgery. During surgical brain injury-induced surgery, intraoperative hemorrhage volume is collected. The hemorrhage volume is determined by spectrophotometric assay. The experimental groups include sham, surgical brain injury with vehicle (SBI+ Vehicle), surgical brain injury with Crotalus venom pre-conditioning (SBI+Cv-PC), surgical brain injury with Crotalus venom pre-conditioning with 10 mg/kg Marimastat (SBI+Cv-PC+Marimastat (10 mg/kg)), and surgical brain injury with Crotalus venom pre-conditioning with 40 mg/kg Marimastat (SBI+Cv-PC+Marimastat (40 mg/kg)).

[0229] Cv-PC doses are preceded 1 hour by MMP antagonist Marimastat (10 or 40 mg/kg, Sigma) administered i.p. Intraoperative bleeding is assessed as described.

[0230] Increased plasma fibrinogen following Cv-PC is expected. Treatment with Cv-PC introduces sMMP, which produce FDPs that upregulates fibrinogen biosynthesis. Following Cv-PC, coagulative parameters such as PTT and PT remain with normal ranges, indicating that Cv-PC does not disrupt coagulation. D-dimers are not present in plasma, suggesting that Cv-PC s effects result without the induction of clot formations. The inhibition of sMMP by Marimastat reverse the beneficial effects of Cv- PC on intraoperative blood loss.

[0231] The FDPs are measured in ranges of concentration, instead of values of concentration. A potential alternative is to measure FDPs by ELISA. Coagulation Factor I (fibrinogen) is monitored. However, the levels of other coagulation factors can be used to achieve a broader understand of Cv-PC and its mechanism if necessary.

[0232] The broad-spectrum MMP inhibitors are used; however, specific inhibitors of the sMMPs from Crotalus venoms are yet to be found. Marimastat may not sufficiently inhibit the sMMP from Cv-PC. Other broad-spectrum MMP inhibitors (such as Batimastat, CL-82198) are commercially available to serve as alternatives in the event that Marimastat is not efficacious or is not well-tolerated.

[0233] All references cited herein are incorporated herein by reference in their entirety. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

[0234] A "patient" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to a human or a non-human mammal, e.g. ., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate.

[0235] An "effective amount" or a "therapeutically effective amount" as used herein are broad terms, and are to be given their ordinary and customary meaning to a person of ordinary skill in the art (and are not to be limited to a special or customized meaning), and refer without limitation to an amount of a therapeutic agent that is effective to relieve, to some extent, or to reduce the likelihood of onset of, one or more of the symptoms of a disease or condition (e.g., bleeding), and includes curing the disease or condition. A "prophylactically effective amount" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to an amount that is effective to prevent or delay the onset of one or more symptoms of a disease or condition (e.g., bleeding), or otherwise reduce the severity of said one or more symptoms, when administered to a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition.

[0236] "Treat," "treatment," or "treating," as used herein are broad terms, and are to be given their ordinary and customary meaning to a person of ordinary skill in the art (and are not to be limited to a special or customized meaning), and refer without limitation to administering a compound or pharmaceutical composition to a subject for prophylactic and/or therapeutic purposes. The term "prophylactic treatment" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to treating a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition. The term "therapeutic treatment" as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to administering treatment to a subject already suffering from a disease or condition.

[0237] Administration of the compounds disclosed herein or the pharmaceutically acceptable salts thereof can be via any of the accepted modes of administration for agents that serve similar utilities including, but not limited to, orally, subcutaneously, intravenously, intranasally, topically, transdermally, intraperitoneally, intramuscularly, intrapulmonarilly, vaginally, rectally, or intraocularly.

[0238] The compounds useful as described above can be formulated into pharmaceutical compositions for use in treatment of these conditions. Standard pharmaceutical formulation techniques are used, such as those disclosed in Remington's The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins (2005), incorporated herein by reference in its entirety. Accordingly, some embodiments include pharmaceutical compositions comprising: (a) a safe and therapeutically effective amount of a compound described herein (including enantiomers, diastereoisomers, tautomers, polymorphs, and solvates thereof), or pharmaceutically acceptable salts thereof; and (b) a pharmaceutically acceptable carrier, diluent, excipient or combination thereof.

[0239] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" as used herein are broad terms, and are to be given their ordinary and customary meaning to a person of ordinary skill in the art (and are not to be limited to a special or customized meaning), and refer without limitation to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. In addition, various adjuvants such as are commonly used in the art may be included. Considerations for the inclusion of various components in pharmaceutical compositions are described, e.g., in Oilman et al. (Eds.) (1990); Goodman and Oilman's: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press, which is incorporated herein by reference in its entirety.

[0240] Some examples of substances, which can serve as pharmaceutically- acceptable carriers or components thereof, are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerine, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as the TWEENS; wetting agents, such sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; and phosphate buffer solutions.

[0241] The choice of a pharmaceutically-acceptable carrier to be used in conjunction with the subject compound is basically determined by the way the compound is to be administered.

[0242] The compositions described herein are preferably provided in unit dosage form. As used herein, a "unit dosage form" is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to a composition containing an amount of a compound that is suitable for administration to an animal, preferably mammal subject, in a single dose, according to good medical practice. The preparation of a single or unit dosage form however, does not imply that the dosage form is administered once per day or once per course of therapy. Such dosage forms are contemplated to be administered once, twice, thrice or more per day and may be administered as infusion over a period of time (e.g., from about 30 minutes to about 2-6 hours), or administered as a continuous infusion, and may be given more than once during a course of therapy, though a single administration is not specifically excluded. The skilled artisan will recognize that the formulation does not specifically contemplate the entire course of therapy and such decisions are left for those skilled in the art of treatment rather than formulation.

[0243] The compositions useful as described above may be in any of a variety of suitable forms for a variety of routes for administration, for example, for oral, nasal, rectal, topical (including transdermal), ocular, intracerebral, intracranial, intrathecal, intra-arterial, intravenous, intramuscular, or other parental routes of administration. The skilled artisan will appreciate that oral and nasal compositions include compositions that are administered by inhalation, and made using available methodologies. Depending upon the particular route of administration desired, a variety of pharmaceutically- acceptable carriers well-known in the art may be used. Pharmaceutically-acceptable carriers include, for example, solid or liquid fillers, diluents, hydrotropies, surface-active agents, and encapsulating substances. Optional pharmaceutically-active materials may be included, which do not substantially interfere with the inhibitory activity of the compound. The amount of carrier employed in conjunction with the compound is sufficient to provide a practical quantity of material for administration per unit dose of the compound. Techniques and compositions for making dosage forms useful in the methods described herein are described in the following references, all incorporated by reference herein: Modern Pharmaceutics, 4th Ed., Chapters 9 and 10 (Banker & Rhodes, editors, 2002); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1989); and Ansel, Introduction to Pharmaceutical Dosage Forms 8th Edition (2004).

[0244] Various oral dosage forms can be used, including such solid forms as tablets, capsules, granules and bulk powders. Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple-compressed, containing suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules, containing suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, melting agents, coloring agents and flavoring agents.

[0245] The pharmaceutically- acceptable carriers suitable for the preparation of unit dosage forms for peroral administration is well-known in the art. Tablets typically comprise conventional pharmaceutically-compatible adjuvants as inert diluents, such as calcium carbonate, sodium carbonate, mannitol, lactose and cellulose; binders such as starch, gelatin and sucrose; disintegrants such as starch, alginic acid and croscarmelose; lubricants such as magnesium stearate, stearic acid and talc. Glidants such as silicon dioxide can be used to improve flow characteristics of the powder mixture. Coloring agents, such as the FD&C dyes, can be added for appearance. Sweeteners and flavoring agents, such as aspartame, saccharin, menthol, peppermint, and fruit flavors, are useful adjuvants for chewable tablets. Capsules typically comprise one or more solid diluents disclosed above. The selection of carrier components depends on secondary considerations like taste, cost, and shelf stability, which are not critical, and can be readily made by a person skilled in the art.

[0246] Peroral compositions also include liquid solutions, emulsions, suspensions, and the like. The pharmaceutically-acceptable carriers suitable for preparation of such compositions are well known in the art. Typical components of carriers for syrups, elixirs, emulsions and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water. For a suspension, typical suspending agents include methyl cellulose, sodium carboxymethyl cellulose, AVICEL RC-591, tragacanth and sodium alginate; typical wetting agents include lecithin and polysorbate 80; and typical preservatives include methyl paraben and sodium benzoate. Peroral liquid compositions may also contain one or more components such as sweeteners, flavoring agents and colorants disclosed above.

[0247] Such compositions may also be coated by conventional methods, typically with pH or time-dependent coatings, such that the subject compound is released in the gastrointestinal tract in the vicinity of the desired topical application, or at various times to extend the desired action. Such dosage forms typically include, but are not limited to, one or more of cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl cellulose, Eudragit coatings, waxes and shellac.

[0248] Compositions described herein may optionally include other drug actives.

[0249] Other compositions useful for attaining systemic delivery of the subject compounds include sublingual, buccal and nasal dosage forms. Such compositions typically comprise one or more of soluble filler substances such as sucrose, sorbitol and mannitol; and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose and hydroxypropyl methyl cellulose. Glidants, lubricants, sweeteners, colorants, antioxidants and flavoring agents disclosed above may also be included.

[0250] A liquid composition, which is formulated for topical ophthalmic use, is formulated such that it can be administered topically to the eye. The comfort may be maximized as much as possible, although sometimes formulation considerations (e.g. drug stability) may necessitate less than optimal comfort. In the case that comfort cannot be maximized, the liquid may be formulated such that the liquid is tolerable to the patient for topical ophthalmic use. Additionally, an ophthalmically acceptable liquid may either be packaged for single use, or contain a preservative to prevent contamination over multiple uses.

[0251] For ophthalmic application, solutions or medicaments are often prepared using a physiological saline solution as a major vehicle. Ophthalmic solutions may preferably be maintained at a comfortable pH with an appropriate buffer system. The formulations may also contain conventional, pharmaceutically acceptable preservatives, stabilizers and surfactants.

[0252] Preservatives that may be used in the pharmaceutical compositions disclosed herein include, but are not limited to, benzalkonium chloride, PHMB, chlorobutanol, thimerosal, phenylmercuric, acetate and phenylmercuric nitrate. A useful surfactant is, for example, Tween 80. Likewise, various useful vehicles may be used in the ophthalmic preparations disclosed herein. These vehicles include, but are not limited to, polyvinyl alcohol, povidone, hydroxypropyl methyl cellulose, poloxamers, carboxymethyl cellulose, hydroxyethyl cellulose and purified water. [0253] Tonicity adjustors may be added as needed or convenient. They include, but are not limited to, salts, particularly sodium chloride, potassium chloride, mannitol and glycerin, or any other suitable ophthalmically acceptable tonicity adjustor.

[0254] Various buffers and means for adjusting pH may be used so long as the resulting preparation is ophthalmically acceptable. For many compositions, the pH will be between 4 and 9. Accordingly, buffers include acetate buffers, citrate buffers, phosphate buffers and borate buffers. Acids or bases may be used to adjust the pH of these formulations as needed.

[0255] In a similar vein, an ophthalmically acceptable antioxidant includes, but is not limited to, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole and butylated hydroxytoluene.

[0256] Other excipient components, which may be included in the ophthalmic preparations, are chelating agents. A useful chelating agent is edetate disodium, although other chelating agents may also be used in place or in conjunction with it.

[0257] For topical use, creams, ointments, gels, solutions or suspensions, etc., containing the compound disclosed herein are employed. Topical formulations may generally be comprised of a pharmaceutical carrier, co-solvent, emulsifier, penetration enhancer, preservative system, and emollient.

[0258] For intravenous administration, the compounds and compositions described herein may be dissolved or dispersed in a pharmaceutically acceptable diluent, such as a saline or dextrose solution. Suitable excipients may be included to achieve the desired pH, including but not limited to NaOH, sodium carbonate, sodium acetate, HC1, and citric acid. In various embodiments, the pH of the final composition ranges from 2 to 8, or preferably from 4 to 7. Antioxidant excipients may include sodium bisulfite, acetone sodium bisulfite, sodium formaldehyde, sulfoxylate, thiourea, and EDTA. Other non- limiting examples of suitable excipients found in the final intravenous composition may include sodium or potassium phosphates, citric acid, tartaric acid, gelatin, and carbohydrates such as dextrose, mannitol, and dextran. Further acceptable excipients are described in Powell, et al., Compendium of Excipients for Parenteral Formulations, PDA J Pharm Sci and Tech 1998, 52 238-311 and Nema et al., Excipients and Their Role in Approved Injectable Products: Current Usage and Future Directions, PDA J Pharm Sci and Tech 2011, 65 287-332, both of which are incorporated herein by reference in their entirety. Antimicrobial agents may also be included to achieve a bacteriostatic or fungistatic solution, including but not limited to phenylmercuric nitrate, thimerosal, benzethonium chloride, benzalkonium chloride, phenol, cresol, and chlorobutanol.

[0259] The compositions for intravenous administration may be provided to caregivers in the form of one more solids that are reconstituted with a suitable diluent such as sterile water, saline or dextrose in water shortly prior to administration. In other embodiments, the compositions are provided in solution ready to administer parenterally. In still other embodiments, the compositions are provided in a solution that is further diluted prior to administration.

[0260] In embodiments that include administering a combination of a compound described herein and another agent, the combination may be provided to caregivers as a mixture, or the caregivers may mix the two agents prior to administration, or the two agents may be administered separately.

[0261] The actual dose of the active compounds described herein depends on the specific compound, and on the condition to be treated; the selection of the appropriate dose is well within the knowledge of the skilled artisan.

[0262] Some embodiments include the combination of compounds, therapeutic agents, and/or pharmaceutical compositions described herein. In such embodiments, the two or more agents may be administered at the same time or substantially the same time. In other embodiments, the two or more agents are administered sequentially. In some embodiments, the agents are administered through the same route (e.g. orally) and in yet other embodiments, the agents are administered through different routes (e.g. one agent is administered orally while another agent is administered intravenously).

[0263] Unless otherwise defined, all terms (including technical and scientific terms) are to be given their ordinary and customary meaning to a person of ordinary skill in the art, and are not to be limited to a special or customized meaning unless expressly so defined herein. It should be noted that the use of particular terminology when describing certain features or aspects of the disclosure should not be taken to imply that the terminology is being re-defined herein to be restricted to include any specific characteristics of the features or aspects of the disclosure with which that terminology is associated. Terms and phrases used in this application, and variations thereof, especially in the appended claims, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing, the term 'including' should be read to mean 'including, without limitation,' 'including but not limited to,' or the like; the term 'comprising' as used herein is synonymous with 'including,' 'containing,' or 'characterized by,' and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; the term 'having' should be interpreted as 'having at least;' the term 'includes' should be interpreted as 'includes but is not limited to;' the term 'example' is used to provide exemplary instances of the item in discussion, not an exhaustive or limiting list thereof; adjectives such as 'known', 'normal', 'standard', and terms of similar meaning should not be construed as limiting the item described to a given time period or to an item available as of a given time, but instead should be read to encompass known, normal, or standard technologies that may be available or known now or at any time in the future; and use of terms like 'preferably,' 'preferred,' 'desired,' or 'desirable,' and words of similar meaning should not be understood as implying that certain features are critical, essential, or even important to the structure or function of the invention, but instead as merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the invention. Likewise, a group of items linked with the conjunction 'and' should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as 'and/or' unless expressly stated otherwise. Similarly, a group of items linked with the conjunction 'or' should not be read as requiring mutual exclusivity among that group, but rather should be read as 'and/or' unless expressly stated otherwise.

[0264] Where a range of values is provided, it is understood that the upper and lower limit, and each intervening value between the upper and lower limit of the range is encompassed within the embodiments.

[0265] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. The indefinite article "a" or "an" does not exclude a plurality. A single processor or other unit may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0266] It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and/or "an" should typically be interpreted to mean "at least one" or "one or more"); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should typically be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, typically means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to "at least one of A, B, or C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, or C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0267] All numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification are to be understood as being modified in all instances by the term 'about.' Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims in any application claiming priority to the present application, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0268] Although this invention has been disclosed in the context of certain preferred embodiments and examples, it will be understood by those skilled in the art that the present invention extends beyond the specifically disclosed embodiments to other alternative embodiments and/or uses of the invention and obvious modifications and equivalents thereof. In addition, while a number of variations of the invention have been shown and described in detail, other modifications, which are within the scope of this invention, will be readily apparent to those of skill in the art based upon this disclosure. It is also contemplated that various combinations or subcombinations of the specific features and aspects of the embodiments may be made and still fall within the scope of the invention. Accordingly, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form varying modes of the disclosed invention. Thus, it is intended that the scope of the present invention herein disclosed should not be limited by the particular disclosed embodiments described above, but should be determined only by a fair reading of the claims that follow.

Claims

WHAT IS CLAIMED IS:

1. A method of administering treatment prior to a surgical procedure in a brain comprising:

applying one or more components of a hemorrhagic snake venom to a brain surgery site before a surgery, whereby an immune system response is triggered; and thereafter

performing a surgery at the brain surgery site, whereby a primary injury at the brain surgery site is inflicted,

wherein a secondary injury at the brain surgery site is reduced through the immune system response.

2. The method of Claim 1 , wherein the one or more components are selected from the group consisting of metalloproteinases, serine proteases, and phospholipase 2.

3. The method of Claim 1, wherein the secondary injury comprises bleeding, swelling, or inflammation that occurs during or following the surgery.

4. The method of Claim 1 , further comprising priming the brain surgery site with the components for responding to the secondary injury.

5. The method of Claim 1, wherein the hemorrhagic snake venom is Crotalus atrox venom.

6. The method of Claim 1 wherein the hemorrhagic snake venom increases levels of fibrinogen, platelets, and clotting at the brain surgery site.

7. The method of Claim 5, wherein the hemorrhagic snake venom is administered in 1 to 6 doses.

8. The method of Claim 7, wherein a total amount of the doses of the hemorrhagic snake venom administered is from 5-20% of the LD₅₀ value for Crotalus atrox venom in the patient being treated.

9. The method of Claim 1, further comprising administering one or more components of a hemorrhagic snake venom from 0 to 6 days after the primary brain injury.

10. The method of Claim 9, wherein the doses of the hemorrhagic snake venom are administered from 0 to 6 days before the surgery.

11. The method of Claim 9, wherein the doses of the hemorrhagic snake venom are administered from 0 to 6 days before the surgery to 0 to 6 days after the surgery.

12. A method of administering treatment after a primary brain injury to a brain comprising:

applying one or more components of a hemorrhagic snake venom to a site of a primary brain injury, whereby an immune system response is triggered, wherein a secondary injury is reduced through the immune system response triggered by the application of the one or more components to the site of the primary brain injury.

13. The method of Claim 12, wherein the one or more components are selected from the group consisting of lectins, serine proteases, vascular endothelial growth factor inhibitor, cysteine-rich secretory proteins, matrix metalloproteinases, and phospholipase A2.

14. The method of Claim 12, wherein the secondary injury comprises reducing bleeding, swelling, or inflammation that occurs during or following the injury.

15. The method of Claim 12, wherein the hemorrhagic snake venom is Crotalus atrox venom.

16. The method of Claim 12, wherein the hemorrhagic snake venom increases levels of fibrinogen, platelets, and clotting at the site of the injury.

17. The method of Claim 15, wherein the hemorrhagic snake venom is administered in 1 to 6 doses.

18. The method of Claim 17, wherein a total amount of the doses of the hemorrhagic snake venom administered is from 5-20% of the LD₅₀ value for Crotalus atrox venom in the patient being treated.

19. The method of Claim 18, wherein the doses of the hemorrhagic snake venom are administered from 0 to 6 days after the primary brain injury.

20. The method of Claim 12, wherein the primary brain injury is caused by a stroke.

21. A composition to administer treatment after a primary brain injury to a brain comprising: one or more components of a hemorrhagic venom to be applied to a site of a primary brain injury, wherein the venom comprises a combination of unfractionated venoms and venom components from one or more species of snake.

22. A method of treating surgical brain injury as described herein.

23. A method of administering treatment prior to a surgical procedure of a patient comprising:

applying one or more components of a hemorrhagic snake venom to a surgery site before a surgery, whereby an immune system response is triggered; and thereafter

performing a surgery at the surgery site, whereby a primary injury at the surgery site is inflicted,

wherein a secondary injury at the surgery site is reduced through the immune system response.

24. A method of administering treatment after a primary injury to a patient comprising:

applying one or more components of a hemorrhagic snake venom to a site of a primary injury, whereby an immune system response is triggered, wherein a secondary injury is reduced through the immune system response triggered by the application of the one or more components to the site of the primary injury.

25. A composition to administer treatment after a primary injury to a patient comprising:

one or more components of a hemorrhagic venom to be applied to a site of a primary injury, wherein the venom comprises a combination of unfractionated venoms and venom components from one or more species of snake.