Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1997-05-21
2004-01-20
Borin, Michael (Department: 1631)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S002600, C530S324000, C435S007100, C435S007210, C435S063000
Reexamination Certificate
active
06680295
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for the prevention and treatment of neuronal cell damage in the brain of mammals, including, for example, damage induced by ischemia followed by reperfusion or that induced by toxic substances.
BACKGROUND OF THE INVENTION
Neuronal cell damage results from many causes. The most common cause of neuronal cell damage is ischemia/reperfusion of the brain following vascular occlusion by thrombosis or embolism, and cardiac failure, which occasionally occurs during heart surgery. Neuronal cell damage or death can be caused both by the ischemia and by reperfusion of blood after the transient ischemia has subsided. See W. D. Dietrich, “Morphological Manifestations of Reperfusion Injury in Brain,” Ann. N.Y. Acad. Sci. 723:15 (1994). Transient ischemia of the brain sometimes also occurs in newborn babies during complicated deliveries. Neuronal cell damage or death can also be induced by various toxic substances, including, for example, the gp120 envelope glycoprotein of the HIV virus, other viral toxins, bacterial toxins, animal toxins, e.g., snake venom, toxic waste, e.g., methylated mercury, and others. The methods and compositions of the present invention can be used to protect and/or attenuate neuronal cells in the brain, spinal cord or elsewhere in the body, caused by trauma, infection e.g., encephalitis, AIDS or degenerative diseases such as Parkinson's disease, Alzheimer's disease, etc.
Brain cell damage frequently results in permanent impairment of brain function. Memory loss, emotional instability, impairment of speech, impairment of movement, recognition, learning, sight, hearing, sensation or other body functions are typical symptoms. At present, no effective method for prevention or treatment of brain cell damage in mammals is available.
Pituitary adenylate cyclase activating polypeptide (PACAP) was originally isolated from ovine hypothalamic tissues based on its ability to activate adenylate cyclase in rat pituitary cell cultures. A. Miyata, et al.,
Biochem Biophys Res Commun
164: 567-574 (1989). As published, PACAP exists in two basic forms: the complete form with 38 amino acids (PACAP38) and a truncated form with 27 amino acids (PACAP27). A. Miyata, A. et al.,
Biochem Biophys Res Commun
170: 643-648 (1990). The amino acid sequences for those two versions are shown in
FIG. 1
(SEQ ID NO:1 and SEQ ID NO:2). PACAP is a new member of the secretin/glucagon/vasoactive intestinal peptide (VIP) family, being the most homologous to VIP, but its adenylate cyclase stimulating activity in cultured pituitary cells, neurons and astrocytes is about 1,000-10,000 times greater than VIP, A. Miyata et al., supra. PACAP is a pleiotropic neuropeptide, exhibiting a number of neurotropic actions in different organs and tissues. For example, PACAP enhances proliferation and differentiation of sympathetic neuroblasts, stimulates neurite outgrowth of an adrenal chromaffin cell line, PC12 cells, and stimulates growth of astrocytes, E. DiCicco-Bloom et al.,
Reg. Pep.
37: 219 (1992); Okazaki et al.,
FEBS
29F: 49-566 (1992). The in vivo cytoprotective action of PACAP has been investigated in rats with transient forebrain ischemia. Uchida et al.,
Soc. Neurosci. Abst.,
Vol. 20, 1994 (Abstract No. 193.10).
There are two types of PACAP receptors (Gottschall et al.,
Endocrinology
127/1:272-277 (1990); Shivers et al.,
Endocrinology,
128/6:3055-3056 (1991); A. Arimura, Trends in Endocrinology and Metabolism, 3/8: 288-294 (1992). The Type I PACAP receptor specifically binds to PACAP with high affinity, but do not bind to VIP. The Type II PACAP receptors bind to both PACAP and VIP with similar high affinities, and may be very similar to or identical with the VIP receptor.
SUMMARY OF THE INVENTION
The present invention relates to a method and pharmaceutical preparations for treating or preventing neuronal cell damage in mammals, comprising administering a effective amount of a PACAP, or an agonist, analog or derivative thereof having PACAP neurotrophic activity, in a pharmaceutically acceptable carrier, in a concentration which is effective for protection of neuronal nerve cells in vivo.
It has now been discovered that, unexpectedly, although PACAP is extremely effective in protecting and/or resuscitating neuronal cells, there is a rather narrow window of concentrations of PACAP which provide such results, i.e., the effectiveness of the treatment falls off rapidly both above and below that concentration range. Thus the present invention involves a method of treatment of mammalian neuronal cells in which the concentration of the PACAP compound is between about 10
−15
and 10
−12
M in the tissues. Even more unexpectedly, it has been discovered that within the generally effective concentration range of the PACAP pharmaceuticals of this invention, there are two sub ranges of concentration, in each of which there is a peak effectiveness, above and below which the effectiveness of the composition falls off to a significant degree. As shown in
FIG. 8
, the preferred concentration range for treatment with the PACAP compounds of the present invention lies between about 10
−14
and about 10
−12
M and another range of concentration lies between about 10
−11
and about 10
−9
M. The preferred concentration range for treatment is the range between about 10
−14
and about 10
−12
M in the tissue, which permits treatment of the subject with minimal risks of side effects from the treatment. The present discovery makes possible the use of such PACAP pharmaceuticals in extremely low concentrations to provide very substantial protection of neuronal cells, such as brain cells, from death due to transient ischemia, reperfusion, toxic substances, trauma, or other causes.
Pharmaceutical compositions in accordance with the present invention include PACAP, in either of its forms, commonly referred to as PACAP38 and PACAP27, as well as any peptide or non-peptide agonist for PACAP receptors, especially agonists for the Type I PACAP receptor. Preferably the PACAP compound is a polypeptide, or a salt or derivative thereof, which contains at least twelve amino acids joined in a sequence corresponding to a part of the sequence shown for PACAP38 in
FIG. 1
, and which binds to at least one receptor which binds to PACAP. As used herein a “PACAP
12
agonist” refers to a polypeptide, or salt or derivative thereof, which has at least 12 amino acids corresponding in sequence to some part of the amino acid sequence of PACAP38, as shown in
FIG. 1
, and which binds to at least one PACAP receptor. Similarly, the terms “PACAP
23
agonist” and “PACAP
27
agonist” refer to polypeptides, or salts or derivatives thereof, which has at least 23 and 27 amino acids, respectively, corresponding in sequence to some part of the amino acid sequence of PACAP38, as shown in
FIG. 1
, and which binds to at least one PACAP receptor. Determination of the amino acid sequence of the polypeptide, and determination as to whether it binds to a PACAP receptor, are both well within the skill in the art. The ability to treat neuronal cells at such low concentrations also makes possible the administration of the PACAP compounds intravenously or otherwise into the blood, in concentrations sufficient to provide PACAP compound transfers across the blood/brain barrier sufficient to provide concentrations of the PACAP compound in contact with the neuronal cells which are effective to protect and/or resuscitate the traumatized neuronal cells.
It has also been discovered that administration of the PACAP compound is effective in protecting and/or resuscitating traumatized neuron cells up to at least 24 hours after injury.
Preferably the PACAP compound has the general formula:
X-PACAP
[a−b]
-Y
wherein X is H or a solubility effecting group such as C
1-20
carboxylic acid moiety, such as formyl, acetyl, etc.; a and b are N and C terminus amino acids taken in the sequence of PACAP38 as shown in
FIG. 1
, and Y is H, NH
2
, OH, or C
1-
Borin Michael
Pennie & Edmonds LLP
The Administrators of the Tulane Educational Fund
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