Treatment of stress-induced migraine headache with a corticotropin releasing hormone blocker

Non-traumatic immobilization (restrain) stress causes rapid degranulation of rat dura mast cells, as shown both by light and electron microscopy. These morphologic findings were accompanied by elevation of rat mast cell protease cerebrospinal fluid. Mast cell activation due to stress was abolished in animals that had been treated neonatally with capsaicin, indicating that neuropeptides in sensory nerve endings are involved in this response. Complete inhibition was also achieved by pretreating the animals intraperitoneally with antiserum to corticotropin releasing hormone (CRH). Mast cells in the dura were localized close to nerve processes containing substance P, but no CRH-fibers were identified even though these were found close to mast cells elsewhere in the brain, i.e., in the median eminence. This is the first time that stress is shown to activate intracranial mast cells, apparently through the sequential actions of CRH and one or more sensory neuropeptides such as Substance P. These findings suggest that therapy of neuroinflammatory disorders such as stress-induced migraine headaches, can be achieved with blockers of the physiological actions of CRH or inhibitors of CRH production or secretion.

FIELD OF THE INVENTION 
This invention relates to a method of treatment of migraine headaches. More 
particularly, it relates to the treatment of stress-induced migraine 
headaches with agents that inhibit the action or actions of the 
neuroendocrine polypeptide, corticotropin releasing hormone. 
BACKGROUND OF THE INVENTION 
Migraine headaches are known to produce the most intense headaches 
reported. The pathophysiology of migraine headaches involve 
vasoconstriction and vasodilation. A variety of stress stimuli, including 
intense light, noise, anxiety, exertion, extremes of temperature, 
hormones, exhaustion, infection and trauma result in constriction of 
extracranial blood vessels. The vasoconstriction is followed by reflexive 
or sequential vasodilation, which subsequently spreads to intracranial 
vessels. It is during this latter phase that the patient feels the 
intense, throbbing headache characteristic of migraines. Increased levels 
of norepinephrine, serotonin, histamine, and the neuropeptides bradykinin 
and substance P, in addition to products of tissue anoxia, are considered 
to be the main endogenous pain producing molecules, accompanied by direct 
sensory nerve stimulation because of the stretching that accompanies 
vasoconstriction and dilation. 
Mast cells, normal components of connective tissues, are thought to play an 
important role in the development of migraine headaches (see, e.g., 
Theoharides, U.S. Pat. No. 5,250,529). Each mast cell contains as many as 
500 secretory granules, each storing over 20 different kinds of 
biochemicals, including histamine, neuropeptide kinins, prostagandims 
PGP.sub.2, PGD.sub.4, leukotriene C.sub.4, and serotonin which are 
vasoconstrictive, as well as vasodilatory, vasoactive intestinal 
polypeptide, tumor necrosis factor and nitric oxide. Degranulation of mast 
cells, which is defined herein as the release of any or all biochemicals 
from secretory granules to the local tissue area or circulation, for 
whatever reason in whatever sequence, occurs in response to interaction of 
various agents with specific mast cell surface receptors or other binding 
proteins. The best known of these receptors is the one for immunoglobulin 
E (IgE). There is also evidence that neurotransmitters such as 
acetylcholine and neuropeptides, released from neurons, and female sex 
hormones (estradiol) may also trigger mast cell degranulation through 
specific receptors, especially in response to stress. Other known triggers 
include viruses, bacterial toxins, drugs such as aspirin, morphine and 
curare, radiological contrast media, extremes of temperature, solar 
radiation, etc. A method of alleviating or preventing a migraine headache 
comprising the administration of a direct mast cell degranulation blocking 
agent during the prodromal phase of the migraine and in the absence of an 
analgesic is the subject of the aforementioned U.S. Pat. No. 5,250,529. 
Stress is long known to activate the hypothalamic-pituitary-adrenal axis 
and can affect illness, especially autoimmune and neuroinflammatory 
syndromes. These effects may be mediated both through psychoneuroimmune 
and neuroendocrine-immune interactions which contribute to inflammation 
and inflammatory diseases. Stress precipitates or worsens certain 
neuroinflammatory conditions such as migraines (Theoharides, Prospect. 
Biol. Med., 26:672 (1983)), and interstitial cystitis (Sant et al., Urol. 
Clin. North Amer., 21:41-53, (1994)), both of which have been associated 
with mast cell activation. Mast cells are necessary for the development of 
allergic and late phase reactions, but may be involved in inflammation as 
they release numerous cytokines (Galli, N. Eng. J. Med., 328:257 (1993)). 
Mast cells have also been found in close apposition to neurons and, as 
noted above, are activated by neuropeptides, as well as by antidromic 
nerve stimulation in the dura. Moreover, mast cell secretion of histamine 
occurs after repetitive exposure to odors, after Pavlovian conditioning 
and in response to isolation stress (Bugajski et al., Agents Actions, 
41:C75-76 (1994)). These findings have raised speculations that mast cells 
may be involved in neuroimmunoendocrine physiology (Stead et al., in 
Burger et al., eds. Cell to Cell Interaction, Karger, Basel, 1990, pp 
170-187) and pathology (Theoharides, Life Sci., 46:607 (1990)). 
It is, therefore, an object of this invention to describe the biochemical 
and anatomical link between stress and the development of migraine 
headaches, and to provide pharmaceutical means for disrupting this link. 
SUMMARY OF THE INVENTION 
This object has been achieved, using an animal model of nontraumatic 
restrain stress previously known to activate the hypothalamic-pituitary 
axis, by the unexpected discoveries that: stress activates mast cells in 
the brain dura where they have been implicated in the pathophysiology of 
migraine headaches; corticotropin releasing hormone (CRH) indirectly 
mediates stress-induced intracranial mast cell degranulation and 
consequent migraine headaches; and agents that interfere with the 
physiological actions of CRH, such as anti-CRH and anti-CRH mast cell 
receptor antibodies and competitive inhibitors of the binding of CRH to 
active sites, unexpectedly inhibit stress-induced migraine headaches 
indirectly through the regulation of neuropeptides and neurotransmitters 
that trigger the degranulation of intracranial mast cells. 
It is an aspect of this invention, therefore, to provide pharmaceutical 
compositions containing one or more CRH blockers for the treatment of 
stress-induced migraine headaches.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
As noted above, stress is known to precipitate or worsen a number of 
disorders, such as migraines, in which mast cells are suspected of being 
involved, by indirectly releasing vasoactive, nociceptive and 
proinflammatory mediators. However, no functional association has 
previously been demonstrated between a migraine trigger and brain mast 
cell activation. Non-traumatic immobilization (restrain) stress is known 
to stimulate the hypothalamic-pituitary-adrenal axis, and cause 
redistribution of immune cells. Here, restrain stress caused degranulation 
in 70% of rat dura mast cells within 30 minutes (which is the timeframe 
during which a migraine typically develops), as shown both by light and 
electron microscopy. These morphologic findings were accompanied by 
cerebrospinal fluid elevation of rat mast cell protease I, but not II, 
indicating secretion from connective tissue type mast cells, such as those 
found in the dura, the meningeal membrane found closest to the skull. 
Mast cell activation due to stress was abolished in animals that had been 
treated neonatally with capsaicin, indicating that such neuropeptides in 
sensory nerve endings mediate this response to stress. Capsaicin, found in 
hot peppers, causes depletion of neuropeptides contained in sensory nerve 
endings. 
We have found that inhibition of the stress response is achieved by 
pretreating laboratory animals with a blocker of the physiological 
action(s) of CRH, for example, by an antiserum to this neuropeptide 
hormone. Although applicant need not be bound by any particular theory of 
mechanism of action, it is likely that the antibody, by binding to CRH 
blocks the binding of CRH to its physiological receptor(s). 
Other CRH blockers are expected to have the same beneficial effects as the 
anti-CRH antibody. Examples include an anti-CRH receptor antibody that 
blocks the binding of CRH to its physiological receptor(s), a competitive 
or non-competitive CRH antagonist (peptide and neuropeptide) such as the 
CRH analog Neurocrine Biochemicals, Inc.'s D-Phe12, Nle Ala32 21,38 hCRH 
(12-41)NH2 (cat. no. 1P-36-41, mw 3474.1) and Pfizer CP-154,526-1 that 
will compete with CRH for binding to CRH receptor(s), and inhibitors of 
CRH secretion such as agents that block interleukin-6 and histamine-3 
receptors, are also expected to relieve stress-induced migraine headaches. 
Although all of the aforementioned CRH blocking agents are expected to be 
therapeutic against stress-induced migraine headaches, they will be 
operating by different mechanisms. Therefore, the dosage required of each 
of these agents to achieve a therapeutically effective concentration in 
brain must be determined on a case-by-case basis. Such a determination 
will not require undue experimentation for one skilled in the art. 
In the present animal experiments, mast cells in the dura were found to be 
localized close to nerve processes containing the neuropeptide substance 
P, but no CRH-positive fibers were identified even though these were found 
close to mast cells elsewhere in the brain, i.e., in the median eminence. 
It is suggested that the effect of CRH on the activation of intracranial 
mast cells is an indirect one, with the primary action of CRH being on the 
release of sensory neuropeptides such as substance P that then activate 
brain mast cells. 
Several of the techniques used in making the present invention and in 
determining anti-CRH drug dosages are described below generically: 
1. Immobilization restrain stress 
Male Sprague/Dawley rats, each weighing approximately 150 g (Taconic, 
Germantown, N.Y.), are kept in their cage in the laboratory (control) or 
are restrained for 30 min in a plexiglass immobilizer (Harvard Apparatus, 
Cambridge) located on a bench top at room temperature (stressed). Each 
animal is equilibrated to the laboratory environment for 60 min prior to 
handling. Each rat is then taken through the entire procedure separately 
and the next rat is not brought in until 60 min after completion of the 
dissection of the previous one. Consequently, no rat should be present or 
in close proximity while another is stressed or dissected. At the end of 
this 60 minute equilibration period, each animal is anesthetized with a 
single intraperitoneal injection containing 0.5 ml ketamine (20 mg/ml) and 
0.5 ml xylazine HCl (20 mg/ml), following which cerebrospinal fluid (CSF) 
is removed by entering the cisternum magnum with a tuberculin syringe. The 
brain is then rapidly removed and the dura attached to the skull is fixed 
by immersion of the skull in 4% paraformaldehyde for 2 hr at room 
temperature. The dura is then removed carefully en bloc, and fixed in 4% 
paraformaldehyde overnight at 4.degree. C. It is then frozen using Tissue 
Freezing Medium (Triangle Biomedical Sciences, Durham, N.C.) and thin 
sections (7.mu.) are cut using a cryostat (Jung CM 3000, Leica, Luc. 
Deerfield, Ill.). The sections are stained with acidified (pH&lt;2.5) 
toluidine blue (Sigma, St. Louis). Mast cells are counted (by at least two 
different researchers blinded to the experimental conditions) at 
400.times. magnification, for example, by using a Diaphot inverted Nikon 
microscope (Don Santo, Natick, Mass.). 
2. Immunohistochemistry 
All specimens are treated with 0.3% H.sub.2 O.sub.2 in methanol for 30 min 
to block endogenous peroxidase. After briefly rinsing in 
phosphate-buffered saline (PBS), sections are incubated in 5% normal goat 
serum in PBS for 30 min, and then exposed to rabbit anti-substance P (SP) 
polyclonal serum (Shimonaka et al., J. Neurochem., 59:81 (1992)) at 1:4000 
or to anti-CRH polyclonal serum at 1:100 in PBS containing 5% normal goat 
serum for 48 hr at 4.degree. C. Visualization of the location for 
immobilized antigen may be made using the avidin-biotin-peroxidase system 
(Vector Laboratories, Burlingame, Calif.) and 3',3'-diaminobenzidine as 
the peroxidase substrate. Negative controls are performed by using anti-SP 
and anti-CRH serum preabsorbed respectively with 1 .mu.M SP or CRH as 
primary antibody (Pang et al., Br. J. Urol. 75:744 (1995)). 
3. Electron microscopy 
Tissue is fixed in modified Karnovsky's medium containing 0.5% tannic acid 
and processed as previously described (Demitriadou et al., Neuroscience, 
44:97 (1991)). 
4. Capsaicin treatment 
Rats are treated within the first 3 days after birth with capsaicin as 
previously described (Demitriadou et al. above), and are used seven weeks 
later. 
5. Treatment with CRH blockers 
Rats are injected parenterally with a CRH blocking agent. For example, with 
a single 1 ml dose (1 mg protein/ml) of anti-CRH polyclonal serum 
intraperitoneally one hour prior to being stressed. Companion animals are 
sham-injected with an equal amount of 0.9% NaCl and are then handled 
similarly. 
The present data clearly demonstrate that stress induces intracranial mast 
cell activation that appears to result mostly in intragranular changes 
accompanied by secretion of at least RMCP-I, rather than the massive 
degranulation by compound exocytosis seen in anaphylactic reactions. Such 
intragranular changes, often seen in mast cells found in close 
juxtaposition to neuronal processes, have previously been noted both in 
the gastrointestinal tract of patients with inflammatory bowel disease 
(Dvorak et al., Int. Arch. Allerg. Immunol., 98:158 (1992)) and in the 
urinary bladder of patients with interstitial cystitis (Letourneau et al., 
Br. J. Urol., in press, 1995). Similar intragranular activation had 
previously been reported in mast cells undergoing differential release of 
mediators (Kops et al., Cell, 262:415 (1990)) without exocytosis. This 
type of activation may represent either a unique process and/or an effect 
due to small concentrations of neuropeptides released at a distance close 
to that of a typical synapse. In fact, it was recently shown that 
application of picomolar concentrations of Substance P to mast cells 
resulted in electrical responses that induced degranulation upon 
re-exposure (Janiszewski et al., Am. J. Physiol., 267:C138 (1994)). 
In the present study, the antiserum to CRH was able to reach the dura 
because, even though it is intracranial, it is located outside the 
blood-brain barrier. As no CRH positive nerve processes were present in 
the dura, it must be concluded that CRH released during stress induces 
subsequent release of neuropeptides, such as Substance P and calcitonin 
gene-related peptide (CGRP) stored in sensory nerve endings, which then 
activate dura mast cells. These neuropeptides have previously been shown 
to stimulate intracranial mast cell secretion directly (Demitriadou et 
al., Pain, Suppl. 5: Abstr. 14, 1990), and to be partially responsible for 
neurogenic inflammation in response to antidromic trigeminal ganglion 
stimulation. In fact, dura mast cell activation by trigeminal ganglion 
stimulation is abolished by neonatal animal treatment with capsaicin which 
destroys peptidergic nerve endings (Demitriadou et al., 1991 above). 
Peripheral mast cells are known to be activated by many neuropeptides, 
especially Substance P. Substance P has been shown to induce granulocyte 
infiltration through mast cell degranulation, thus contributing to 
neurogenic inflammation. It should be noted that the secretory effect of 
Substance P is augmented by estradiol, which may partially explain the 
higher incidence of migraines in women, especially at the time of 
ovulation. In fact, interstitial cystitis, which also occurs more often in 
women and is worse at midcycle, is associated with a higher incidence of 
migraines and is characterized by an increased number of activated mast 
cells expressing high affinity estrogen receptors. Parasympathetic nerve 
stimulation can augment or trigger mast cell secretion, while mast 
cell-derived histamine can then stimulate peripheral neurons, suggesting 
that mast cell-neuron interactions may be involved in pathophysiology and 
pathology. Such results have led to the hypothesis that sensory 
neuropeptides regulate hypersensitivity reactions. In fact, such 
neuropeptides were shown to have different effects on lymphocyte function 
and proliferation suggesting that these are specific effects. 
CRH has recently been shown to degranulate skin mast cells (Boucher et al., 
FASEB J. Abstract issue, 1995) indicating that there may also be such an 
effect in tissues where mast cells have access to products of CRH action. 
This may be true especially in the hypothalamus where mast cell proximity 
to CRH-positive nerve processes was observed. The facts that mast cells 
are activated by somatostatin and mast cells secrete interleukin-6 which 
has been implicated in the control of CRH (Navarra et al., Endocrinology 
128:37 (1991); Mastorakos et al., J. Clin. End. Metab. 77:1690 (1993)), 
suggests that a functional, albeit indirect, interaction may exist between 
CRH and mast cells in the hypothalamus. The possibility, therefore, exists 
that hypothalamic mast cells may also be affected by stress leading to 
changes in mood and cognitive function. It is, therefore, of interest that 
atopic diseases occur more frequently in children born to women with 
migraines and there is a higher incidence of atopic disorders in affective 
patients. 
The present results may explain the pathophysiology of neuroimmunoendocrine 
disorders, such as migraines, which are clearly exacerbated by stress. 
Mast cells have been proposed to play a key role in migraines 
(Theoharides, Perspect. Biol. Med. 26:672 (1983)) through the release of 
vasoactive, nocioceptive and pro-inflammatory molecules (Scutieri, 
Headache, 86 (1963). For instance, histamine elevations have been 
documented in the serum of patients during migraine. Nitric oxide (NO) has 
been proposed as a key molecule in the pathogenesis of migraines, and mast 
cells have been shown to release NO upon stimulation. Dura mast cell 
degranulation in response to antidromic trigeminal ganglion stimulation is 
accompanied by vascular changes which are similar to those seen in 
migraines. Moreover, the clinical efficacy of 5-hydroxytryptamine receptor 
agonists used to treat migraines corresponds to their ability to block 
dura mast cell degranulation and neurogenic inflammation (Buzzi et al., 
Brain Res. 583:137 (1992)). Brain mast cell activation in response to 
stress may also be involved in other neuroinflammatory disorders. For 
instance, migraines occur more frequently in multiple sclerosis (MS) 
patients, and the mast cell specific enzyme tryptase was shown to be 
elevated in the CSF of MS patients. 
Stress-induced brain mast cell degranulation, resulting sequentially and 
functionally from the release of CRH and certain neuropeptides, may prove 
to be a useful model to further investigate the pathophysiology of 
migraines and screen for more effective anti-migraine drugs, such as novel 
CRH receptor antagonists 
EXAMPLES 
Example 1 
Stress and Mast Cell Activation 
Mast cell activation, judged by granule content extrusion and loss of 
cellular staining was present in 69.9.+-.5.3% (n=501) of mast cells in 
stressed animals (FIG. 1B), as compared to 38.7.+-.5.0% (n=683) in 
controls (FIG. 1A and Table 1). The data of Table 1 show that this 
difference was statistically significant (Mann Whitney U-test, P=0.0018; 
t-test, P&lt;0.05). 
TABLE 1 
______________________________________ 
Brain Dura Mast Cell Activation During Stress 
Variable n Activation (% total) 
P.sup.a 
P.sup.b 
______________________________________ 
Control 683 38.7 .+-. 5.0 
Stressed 501 69.9 .+-. 5.3 0.0018 
&lt;0.05 
Anti-CRH 460 33.1 .+-. 4.0 0.0007 
&lt;0.05 
Capsaicin 415 25.8 .+-. 8.5 0.0016 
&lt;0.05 
______________________________________ 
.sup.a Mann-Whitney Utest comparing the results with the stressed animal 
to every other condition. 
.sup.b Unpaired ttest 
n = Total no. of mast cells studied in a total of 12 rats for each 
condition tested 
Example 2 
Effect of Capsaicin Plus CRH Blocker on Mast Cell Activation 
In animals that had been pretreated neonatally with capsaicin to destroy 
sensory nerve termini, stress-induced dura mast cell activation was 
reduced (Mann Whitney U-test, P&lt;0.0016; t-test, P&lt;0.05) to 25.8.+-.8.5% 
(n=415) which was below control levels (Table 1). 
Pretreatment of animals intraperitoneally with 1 mg/ml of a polyclonal 
antiserum to CRH for 60 min prior to stress also reduced dura mast cell 
activation ((Mann Whitney U-test, P&lt;0.0007; t-test, P&lt;0.05) to 
33.1.+-.4.0% (n=460) which was slightly below control (Table 1). 
The amount of rat mast cell protease (RMCP)-I recovered in the CSF of 
stressed animals was 5.3.+-.3.1 (n=22) and was significantly higher (Mann 
Whitney U-test, P=0.009) than that in control animals: 3.8.+-.1.3 (n=11). 
RMCP-I was undetectable in animals treated with capsaicin (n=3) or with 
anti-CRH serum (n=4). RMCP II was undetectable in all groups studied. 
Example 3 
Immunochemistry 
Immunohistochemistry, along with metachromatic staining with toluidine 
blue, showed localization of mast cells adjacent to substance 
P-immunoreactive nerve fibers in the dura (FIG. 2A stained brown), but no 
CRH-immunoreactive nerve processes were present in the dura (FIG. 2). 
However, numerous CRH-positive nerve processes stained golden brown were 
present close to mast cells in the median eminence (FIG. 2B). 
Example 4 
Electron Microscopy 
Electron microscopy also captured images of mast cells with signs of subtle 
intragranular changes surrounding terminal nerve processes (N) containing 
synaptic vesicles (small solid arrows) (FIG. 3). The ultrastructural 
appearance of dura mast cells from stressed animals was characterized by 
extensive alterations of secretory granule (g) electron dense content 
consistent with secretion. These included partially filled or empty 
granules, as well as others the content of which had entirely different 
texture with distinct crystalline or amorphous, nonhomogeneous patterns 
(FIG. 4).