Method of diagnosing and therapeutically treating a patient for a traumatic brain injury

Abstract

Known or suspected traumatic brain injuries may be treated therapeutically by administering a therapeutically effective dose of resibufogenin. A preferred method for determining if a patient has a traumatic brain injury includes obtaining a body specimen from the patient, determining the concentration of marinobufagenin in the body specimen, comparing the concentration of marinobufagenin to the concentration in such body specimens in normal patients, and if the marinobufagenin concentration is substantially above the concentration of a normal patient, concluding traumatic brain injury exists. In a preferred embodiment, a substantial elevation is deemed to be an increase of about 30 percent above the marinobufagenin concentration of a normal patient. The body specimen may be blood, urine, or cerebrospinal fluid. If a substantial elevation is deemed to exist, the magnitude of the departure from the concentration of a normal patient may be employed in determining the timing and nature of treatment provided to the patient. The method may be repeated at predetermined intervals to monitor changes in the marinobufagenin with time.

Claims

1. A method of reducing the risk of injury to brain tissue in a patient who is suspected to have a traumatic brain injury based on elevated marinobufagenin (MBG) levels, comprising administering to a patient who experienced a direct or indirect shock to the head and has elevated levels of MBG a therapeutically effective amount of a pharmaceutical composition comprising resibufogenin (RBG).

2. The method of claim 1 wherein the patient suspected of having a traumatic brain injury has a marinobufagenin level at least 30 percent higher than a subject that has not experienced an indirect or direct shock to the head.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates the chemical structure for resibufogenin.

(2) FIG. 2 illustrates the chemical structure for marinobufagenin.

(3) FIG. 3 through 5 represent respectively, representative photomicrographs of three groups of ten rats which have been subjected to the development of traumatic brain injury through impact by a weight under experimental conditions described hereinafter. The image shown in FIG. 3 shows the sham operated rat. The image shown in FIG. 4 shows the rat subjected to impact acceleration injury, and the image shown in FIG. 5 shows the animals which received a 120 g bolus of resibufogenin 90 minutes after the imposition of brain trauma.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(4) As employed herein, the term traumatic brain injury shall mean a brain injury resulting from direct or indirect shock load or loads applied to the brain causing it to move rapidly and unnaturally within a patient's skull and shall expressly include, but not be limited to, brain injuries caused by: (a) objects penetrating the skull, such as, bullets, arrows, and other physical objects which pass through the skull and enter the brain, (b) impact loads applied to the head or other portions of the patient's body, (c) surgically induced trauma, (d) explosions, such as might exist in warfare, through impacting of grenades, bombs, and other explosives, which cause substantial tremors in the earth in relatively-close proximity to where an individual is standing, as well as similar tremors created by nonexplosive means, such as vehicular accidents, collapse of buildings and earthquakes, for example.

(5) As employed herein, the term normal patient(s) means a group of non-traumatized subjects matched for age and sex.

(6) The results of traumatic brain injury may be of various types, but in each instance, will involve temporary or permanent reduction in the ability of the brain to function normally and may cause death.

(7) One of the consequences of a traumatic brain injury frequently is the generation of inflammation within the brain as the shock to the brain serves to increase the permeability of the endothelial cells, thereby permitting loss of fluids from the vascular structure into the brain. Such a leakage frequently occurs due to the increased porosity of the blood vessels resulting from the trauma, thereby causing blood serum to leak through the vessels into the brain area. As this builds up, this can generate inflammation and swelling of the brain, which may require surgical intervention.

(8) The diagnostic portion of the present invention may involve measuring a body specimen which may be urine or blood, such as blood serum or blood plasma, or cerebrospinal fluid.

(9) The preferred method involves determining if a patient has a traumatic brain injury by obtaining a body specimen from the patient, determining the concentration of marinobufagenin in the body specimen, and comparing the concentration of marinobufagenin with the marinobufagenin concentration in a similar body specimen in normal patients. If the marinobufagenin concentration is substantially above the concentration of a normal patient, this indicates that a traumatic brain injury exists and therapeutic action is initiated.

(10) The diagnostic means for determining if a traumatic brain injury exists is represented by use of an immuno-fluorescent ELISA assay, for example, to provide the amount of marinobufagenin in the urine, blood or cerebrospinal fluid specimen.

(11) In a preferred embodiment of the present invention, it is determined that a traumatic brain injury exists if the elevation of marinobufagenin is at least about 30 percent over that of a normal patient.

(12) The diagnostic tests and therapeutic treatment may be repeated periodically to determine trends. If the marinobufagenin concentration continues to increase, this reinforces the conclusion that a traumatic brain injury and probably brain cell damage exist. If it decreases, comparison of the concentration with normal patients will facilitate a determination of reduced concern.

(13) As will be seen from a comparison of the chemical structure of resibufogenin in FIG. 1 and chemical structure of marinobufagenin in FIG. 2, the difference between the two compounds is the absence of an hydroxyl group at the -5 position in the resibufogenin structure.

(14) Low and high magnification micrographs of glial fibrillary acidic proteins (GFAP) immuno-fluorescence using a laser-scanning confocal microscope produced FIGS. 3 through 5. The GFAP antibody labels the intermediate filaments of most astrocytes in the brain. These photomicrographs, which were taken 24 hours after the animals were subjected to impact acceleration injury, show areas of cortex slightly lateral to the midline. The pial surface is at the top of the images and the midline is to the left of all images. In the sham animals shown in FIG. 3, a normal distribution of GFAP labeled astrocytes is seen, including their endfeet which surround the neurovasculature. At 24 hours after an impact acceleration injury, as shown in FIG. 4, a noticeable scar is observed as indicated by the plurality of arrows in layers 2 and 3 of the cortex. There is also a patchy loss of GFAP-labeled astrocytes throughout the cortex and the vasculature shown by the arrow heads (as contrasted with the full arrows) appears damaged. It should also be noted that many of the remaining astrocytes appear to be hypertrophied. The loss of all GFAP-immunoreactivity and appearance of astrocyte hypertrophy are indicative of a neuroinflammatory response to injury. As seen in FIG. 5, most of these alterations are ameliorated if the animals are treated with a therapeutically effective dose of resibufogenin at about 90 minutes after the impact acceleration injury. It is important to note that in FIG. 5, there is no observed glial scar. The resibufogenin treatment also functions to reduce the decrease in GFAP immunoreactivity and demonstrates that a greater proportion of GFAP labeled astrocytes have a normal, rather than a hypertrophied appearance.

(15) The resibufogenin may be introduced into the patient by at least one method selected from the group consisting of intravenously, intraperitoneally, intramuscularly, intrathecally, subcutaneously, orally, intraoperatively, topically and during brain surgery by introducing it directly into brain tissue.

(16) Assuming that the initial monitoring of the marinobufagenin indicated that a traumatic brain injury had occurred and treatment with resibufogenin was administered in order to diminish the adverse effect of the traumatic brain injury, one might perform the marinobufagenin test again in an effort to determine the remaining intensity of the traumatic brain injury. This test may be repeated periodically and compared with at least one prior test until the marinobufagenin levels approach or reach the levels of normal patients. Additional doses of resibufogenin may be administered during the course of treatment.

(17) Once the determination that a traumatic brain injury exists has been made, the preferred mode of treatment for a particular patient can be determined. While any suitable means for determining the presence of a traumatic brain injury may be employed, a preferred method is the testing for the marinobufagenin concentration as disclosed herein. The magnitude of increase of marinobufagenin may be employed to influence the timing and nature of the treatment to be provided.

(18) It will be appreciated, therefore, that the present invention provides a method for making a prompt, reliable, and effective determination as to whether an individual is suffering from traumatic brain injury, so as to minimize the risk of an inaccurate diagnosis leading to potentially serious consequences.

(19) It will further be appreciated that the present invention provides a preferred means of treating a patient who has been determined to have a traumatic brain injury. The method of treatment may also be employed for patients suspected of having a traumatic brain injury prior to confirmation that a traumatic brain injury exists. This, in many instances, will serve to reduce the risk of injury to brain tissue. The administration to said patient of a pharmaceutical composition comprising resibufogenin is effective in these circumstances.

EXAMPLE

(20) In order to provide additional information regarding the invention, experiments were performed on animals who were subjected to the impact acceleration injury described hereinbefore, with the animals subsequently being sacrificed and the brains examined 24 hours after the injury. Tests were performed with body specimens which were urinary excretion and also with blood, either blood plasma or blood serum. In each group of ten animals, all had their urine tested and six had blood tested. With the sham animals, the mean urinary excretion values of marinobufagenin were 423130 (SE)pgMBG/mg creatinine. With the impact acceleration injury rats, the mean urinary excretion values of marinobufagenin were 33161654 MBG/mg creatinine. The corresponding measurements from blood serum showed the sham marinobufagenin values were 33.14.2 pg/mL and with impact acceleration injury were 54.510.1 pg/mL. These tests confirm the evaluation of marinobufagenin in both urine and blood when an impact acceleration injury is present.

(21) Whereas particular embodiments of the present invention have been described herein for purpose of illustration, it will be evident to those skilled in the art that numerous variations of the details may be made without departing from the invention, as set forth in the appended claims.