Glycerol Trinitrate Acts Downstream of Calcitonin Gene-Related Peptide in Trigeminal Nociception-Evidence from Rodent Experiments with Anti-CGRP Antibody Fremanezumab.

Calcitonin gene-related peptide (CGRP) and nitric oxide (NO) have been recognized as important mediators in migraine but their mechanisms of action and interaction have not been fully elucidated. Monoclonal anti-CGRP antibodies like fremanezumab are successful preventives of frequent migraine and can be used to study CGRP actions in preclinical experiments. Fremanezumab (30 mg/kg) or an isotype control monoclonal antibody was subcutaneously injected to Wistar rats of both sexes. One to several days later, glyceroltrinitrate (GTN, 5 mg/kg) mimicking nitric oxide (NO) was intraperitoneally injected, either once or for three consecutive days. The trigeminal ganglia were removed to determine the concentration of CGRP using an enzyme-linked immunosorbent assay (ELISA). In one series of experiments, the animals were trained to reach an attractive sugar solution, the access to which could be limited by mechanical or thermal barriers. Using a semi-automated registration system, the frequency of approaches to the source, the residence time at the source, and the consumed solution were registered. The results were compared with previous data of rats not treated with GTN. The CGRP concentration in the trigeminal ganglia was generally higher in male rats and tended to be increased in animals treated once with GTN, whereas the CGRP concentration decreased after repetitive GTN treatment. No significant difference in CGRP concentration was observed between animals having received fremanezumab or the control antibody. Animals treated with GTN generally spent less time at the source and consumed less sugar solution. Without barriers, there was no significant difference between animals having received fremanezumab or the control antibody. Under mechanical barrier conditions, all behavioral parameters tended to be reduced but animals that had received fremanezumab tended to be more active, partly compensating for the depressive effect of GTN. In conclusion, GTN treatment seems to increase the production of CGRP in the trigeminal ganglion independently of the antibodies applied, but repetitive GTN administration may deplete CGRP stores. GTN treatment generally tends to suppress the animals' activity and increase facial sensitivity, which is partly compensated by fremanezumab through reduced CGRP signaling. If CGRP and NO signaling share the same pathway in sensitizing trigeminal afferents, GTN and NO may act downstream of CGRP to increase facial sensitivity.

Imiquimod-induced pruritus in female wild-type and knockin Wistar rats: underscoring behavioral scratching in a rat model for antipruritic treatments.

OBJECTIVES: Animal models of skin disease are used to evaluate therapeutics to alleviate disease. One common clinical dermatological complaint is pruritus (itch), but there is a lack of standardization in the characterization of pre-clinical models and scratching behavior, a key itch endpoint, is often neglected. One such model is the widely used imiquimod (IMQ) mouse model of psoriasis. However, it lacks characterized behavioral attributes like scratching, nor has widely expanded to other species like rats. Given these important attributes, this study was designed to broaden the characterization beyond the expected IMQ-induced psoriasis-like skin inflammatory skin changes and to validate the role of a potential therapeutic agent for pruritus in our genetic rat model. The study included female Wistar rats and genetically modified knockin (humanized proteinase-activated receptor 2 (F2RL1) female rats, with the widely used C57BL/6 J mice as a methodology control for typical IMQ dosing. RESULTS: We demonstrate that the IMQ model can be reproduced in rats, including their genetically modified derivatives, and how scratching can be used as a key behavioral endpoint. We systemically delivered an anti-PAR2 antibody (P24E1102) which reversed scratching bouts-validating this behavioral methodology and have shown its feasibility and value in identifying effective antipruritic drugs.

Disease pathology signatures in a mouse model of Mucopolysaccharidosis type IIIB.

Mucopolysaccharidosis type IIIB (MPS IIIB) is a rare and devastating childhood-onset lysosomal storage disease caused by complete loss of function of the lysosomal hydrolase α-N-acetylglucosaminidase. The lack of functional enzyme in MPS IIIB patients leads to the progressive accumulation of heparan sulfate throughout the body and triggers a cascade of neuroinflammatory and other biochemical processes ultimately resulting in severe mental impairment and early death in adolescence or young adulthood. The low prevalence and severity of the disease has necessitated the use of animal models to improve our knowledge of the pathophysiology and for the development of therapeutic treatments. In this study, we took a systematic approach to characterizing a classical mouse model of MPS IIIB. Using a series of histological, biochemical, proteomic and behavioral assays, we tested MPS IIIB mice at two stages: during the pre-symptomatic and early symptomatic phases of disease development, in order to validate previously described phenotypes, explore new mechanisms of disease pathology and uncover biomarkers for MPS IIIB. Along with previous findings, this study helps provide a deeper understanding of the pathology landscape of this rare disease with high unmet medical need and serves as an important resource to the scientific community.

The Anti-Calcitonin Gene-Related Peptide (Anti-CGRP) Antibody Fremanezumab Reduces Trigeminal Neurons Immunoreactive to CGRP and CGRP Receptor Components in Rats.

Treatment with the anti-CGRP antibody fremanezumab is successful in the prevention of chronic and frequent episodic migraine. In preclinical rat experiments, fremanezumab has been shown to reduce calcitonin gene-related peptide (CGRP) release from trigeminal tissues and aversive behaviour to noxious facial stimuli, which are characteristic pathophysiological changes accompanying severe primary headaches. To further decipher the effects of fremanezumab that underlie these antinociceptive effects in rats, immunohistochemistry and ELISA techniques were used to analyse the content and concentration of CGRP in the trigeminal ganglion, as well as the ratio of trigeminal ganglion neurons which are immunoreactive to CGRP and CGRP receptor components, 1-10 days after subcutaneous injection of fremanezumab (30 mg/kg) compared to an isotype control antibody. After fremanezumab treatment, the fraction of trigeminal ganglion neurons which were immunoreactive to CGRP and the CGRP receptor components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) was significantly lowered compared to the control. The content and concentration of CGRP in trigeminal ganglia were not significantly changed. A long-lasting reduction in CGRP receptors expressed in trigeminal afferents may contribute to the attenuation of CGRP signalling and antinociceptive effects of monoclonal anti-CGRP antibodies in rats.

Modifying antibody-FcRn interactions to increase the transport of antibodies through the blood-brain barrier.

The blood-brain barrier (BBB) largely excludes antibodies from entering the central nervous system, thus limiting the potential of therapeutic antibodies to treat conditions such as neurodegenerative diseases and neuro-psychiatric disorders. Here, we demonstrate that the transport of human antibodies across the BBB in mice can be enhanced by modulating their interactions with the neonatal Fc receptor (FcRn). When M252Y/S254T/T246E substitutions are introduced on the antibody Fc domain, immunohistochemical assays reveal widespread distribution of the engineered antibodies throughout the mouse brain. These engineered antibodies remain specific for their antigens and retain pharmacological activity. We propose that novel brain-targeted therapeutic antibodies can be engineered to differentially engage FcRn for receptor-mediated transcytosis across the BBB in order to improve neurological disease therapeutics in the future.

Antinociceptive Effects of an Anti-CGRP Antibody in Rat Models of Colon-Bladder Cross-Organ Sensitization.

Irritable bowel syndrome (IBS) and bladder pain syndrome/interstitial cystitis (BPS/IC) are comorbid visceral pain disorders seen commonly in women with unknown etiology and limited treatment options and can involve visceral organ cross-sensitization. Calcitonin gene-related peptide (CGRP) is a mediator of nociceptive processing and may serve as a target for therapy. In three rodent models, we employed a monoclonal anti-CGRP F(ab')2 to investigate the hypothesis that visceral organ cross-sensitization is mediated by abnormal CGRP signaling. Visceral organ cross-sensitization was induced in adult female rats via transurethral infusion of protamine sulfate (PS) into the urinary bladder or infusion into the colon of trinitrobenzene sulfonic acid (TNBS). Colonic sensitivity was assessed via the visceromotor response to colorectal distension (CRD). Bladder sensitivity was assessed as the frequency of abdominal withdrawal reflexes to von Frey filaments applied to the suprapubic region. PS- or TNBS-induced changes in colonic and bladder permeability were investigated in vitro via quantification of transepithelial electrical resistance (TEER). Peripheral administration of an anti-CGRP F(ab')2 inhibited PS-induced visceral pain behaviors and colon hyperpermeability. Similarly, TNBS-induced pain behaviors and colon and bladder hyperpermeability were attenuated by anti-CGRP F(ab')2 treatment. PS into the bladder or TNBS into the colon significantly increased the visceromotor response to CRD and abdominal withdrawal reflexes to suprapubic stimulation and decreased bladder and colon TEER. These findings suggest an important role of peripheral CGRP in visceral nociception and organ cross-sensitization and support the evaluation of CGRP as a therapeutic target for visceral pain in patients with IBS and/or BPS/IC. SIGNIFICANCE STATEMENT: A monoclonal antibody against calcitonin gene-related peptide (CGRP) was found to reduce concomitant colonic and bladder hypersensitivity and hyperpermeability. The results of this study suggest that CGRP-targeting antibodies, in addition to migraine prevention, may provide a novel treatment strategy for multiorgan abdominopelvic pain following injury or inflammation.

Semi-Automated Recording of Facial Sensitivity in Rat Demonstrates Antinociceptive Effects of the Anti-CGRP Antibody Fremanezumab.

Migraine pain is frequently accompanied by cranial hyperalgesia and allodynia. Calcitonin gene-related peptide (CGRP) is implicated in migraine pathophysiology but its role in facial hypersensitivity is not entirely clear. In this study, we investigated if the anti-CGRP monoclonal antibody fremanezumab, which is therapeutically used in chronic and episodic migraines, can modify facial sensitivity recorded by a semi-automatic system. Rats of both sexes primed to drink from a sweet source had to pass a noxious mechanical or heat barrier to reach the source. Under these experimental conditions, animals of all groups tended to drink longer and more when they had received a subcutaneous injection of 30 mg/kg fremanezumab compared to control animals injected with an isotype control antibody 12-13 days prior to testing, but this was significant only for females. In conclusion, anti-CGRP antibody, fremanezumab, reduces facial sensitivity to noxious mechanical and thermal stimulation for more than one week, especially in female rats. Anti-CGRP antibodies may reduce not only headache but also cranial sensitivity in migraineurs.

Commentary: Constipation caused by anti-calcitonin gene-related peptide migraine therapeutics explained by antagonism of calcitonin gene-related peptide's motor-stimulating and prosecretory function in the intestine.

Calcitonin gene-related peptide (CGRP) pathway-targeted treatments have been shown to be efficacious in the prevention of episodic and chronic migraine. Currently approved therapies include monoclonal antibodies (mAbs) that target CGRP (eptinezumab, fremanezumab, and galcanezumab) and the CGRP receptor (erenumab), and small molecule CGRP receptor antagonists (atogepant and rimegepant). While CGRP pathway-targeted treatments are generally well-tolerated, in a review article by Holzer and Holzer-Petsche published in the January 2022 issue of Frontiers in Physiology the authors discussed the role of the CGRP pathway in gastrointestinal physiology, with a specific focus on constipation associated with the use of CGRP pathway-targeted treatments. The authors state that real-world surveys have shown constipation to be a "major adverse event" reported in "more than 50% of patients treated with erenumab, fremanezumab or galcanezumab." As described in the current commentary, the limited data from the cited references in the review article by Holzer and Holzer-Petsche do not support that statement.

The Anti-CGRP Antibody Fremanezumab Lowers CGRP Release from Rat Dura Mater and Meningeal Blood Flow.

Monoclonal antibodies directed against the neuropeptide calcitonin gene-related peptide (CGRP) belong to a new generation of therapeutics that are effective in the prevention of migraine. CGRP, a potent vasodilator, is strongly implicated in the pathophysiology of migraine, but its role remains to be fully elucidated. The hemisected rat head preparation and laser Doppler flowmetry were used to examine the effects on CGRP release from the dura mater and meningeal blood flow of the subcutaneously injected anti-CGRP monoclonal antibody fremanezumab at 30 mg/kg, when compared to an isotype control antibody. Some rats were administered glycerol trinitrate (GTN) intraperitoneally to produce a migraine-like sensitized state. When compared to the control antibody, the fremanezumab injection was followed by reduced basal and capsaicin-evoked CGRP release from day 3 up to 30 days. The difference was enhanced after 4 h of GTN application. The samples from the female rats showed a higher CGRP release compared to that of the males. The increases in meningeal blood flow induced by acrolein (100 µM) and capsaicin (100 nM) were reduced 13-20 days after the fremanezumab injection, and the direct vasoconstrictor effect of high capsaicin (10 µM) was intensified. In conclusion, fremanezumab lowers the CGRP release and lasts up to four weeks, thereby lowering the CGRP-dependent meningeal blood flow. The antibody may not only prevent the released CGRP from binding but may also influence the CGRP release stimulated by noxious agents relevant for the generation of migraine pain.

An in vivo accelerated developmental myelination model for testing promyelinating therapeutics.

BACKGROUND: Therapeutic agents stimulating the process of myelination could be beneficial for the treatment of demyelinating diseases, such as multiple sclerosis. The efficient translation of compounds promoting myelination in vitro to efficacy in vivo is inherently time-consuming and expensive. Thyroid hormones accelerate the differentiation and maturation of oligodendrocytes, thereby promoting myelination. Systemic administration of the thyroid hormone thyroxine (T4) accelerates brain maturation, including myelination, during early postnatal development. The objective of this study was to validate an animal model for rapid testing of promyelinating therapeutic candidates for their effects on early postnatal development by using T4 as a reference compound. METHODS: Daily subcutaneous injections of T4 were given to Sprague Dawley rat pups from postnatal day (PND) 2 to PND10. Changes in white matter were determined at PND10 using diffusion tensor magnetic resonance imaging (DTI). Temporal changes in myelination from PND3 to PND11 were also assessed by quantifying myelin basic protein (MBP) expression levels in the brain using the resonance Raman spectroscopy/enzyme-linked immunosorbent assay (RRS-ELISA) and quantitative immunohistochemistry. RESULTS: DTI of white matter tracts showed significantly higher fractional anisotropy in the internal capsule of T4-treated rat pups. The distribution of total FA values in the forebrain was significantly shifted towards higher values in the T4-treated group, suggesting increased myelination. In vivo imaging data were supported by in vitro observations, as T4 administration significantly potentiated the developmental increase in MBP levels in brain lysates starting from PND8. MBP levels in the brain of animals that received treatment for 9 days correlated with the FA metric determined in the same pups in vivo a day earlier. Furthermore, accelerated developmental myelination following T4 administration was confirmed by immunohistochemical staining for MBP in coronal brain sections of treated rat pups. CONCLUSIONS: T4-treated rat pups had increased MBP expression levels and higher MRI fractional anisotropy values, both indications of accelerated myelination. This simple developmental myelination model affords a rapid test of promyelinating activity in vivo within several days, which could facilitate in vivo prescreening of candidate therapeutic compounds for developmental hypomyelinating diseases. Further research will be necessary to assess the utility of this platform for screening promyelination compounds in more complex demyelination disease models, such us multiple sclerosis.

Dysregulation of serum prolactin links the hypothalamus with female nociceptors to promote migraine.

Migraine headache results from activation of meningeal nociceptors, however, the hypothalamus is activated many hours before the emergence of pain. How hypothalamic neural mechanisms may influence trigeminal nociceptor function remains unknown. Stress is a common migraine trigger that engages hypothalamic dynorphin/kappa opioid receptor (KOR) signalling and increases circulating prolactin. Prolactin acts at both long and short prolactin receptor isoforms that are expressed in trigeminal afferents. Following downregulation of the prolactin receptor long isoform, prolactin signalling at the prolactin receptor short isoform sensitizes nociceptors selectively in females. We hypothesized that stress may activate the kappa opioid receptor on tuberoinfundibular dopaminergic neurons to increase circulating prolactin leading to female-selective sensitization of trigeminal nociceptors through dysregulation of prolactin receptor isoforms. A mouse two-hit hyperalgesic priming model of migraine was used. Repeated restraint stress promoted vulnerability (i.e. first-hit priming) to a subsequent subthreshold (i.e. second-hit) stimulus from inhalational umbellulone, a TRPA1 agonist. Periorbital cutaneous allodynia served as a surrogate of migraine-like pain. Female and male KORCre; R26lsl-Sun1-GFP mice showed a high percentage of KORCre labelled neurons co-localized in tyrosine hydroxylase-positive cells in the hypothalamic arcuate nucleus. Restraint stress increased circulating prolactin to a greater degree in females. Stress-primed, but not control, mice of both sexes developed periorbital allodynia following inhalational umbellulone. Gi-DREADD activation (i.e. inhibition through Gi-coupled signalling) in KORCre neurons in the arcuate nucleus also increased circulating prolactin and repeated chemogenetic manipulation of these neurons primed mice of both sexes to umbellulone. Clustered regularly interspaced short palindromic repeats-Cas9 deletion of the arcuate nucleus KOR prevented restraint stress-induced prolactin release in female mice and priming from repeated stress episodes in both sexes. Inhibition of circulating prolactin occurred with systemic cabergoline, a dopamine D2 receptor agonist, blocked priming selectively in females. Repeated restraint stress downregulated the prolactin receptor long isoform in the trigeminal ganglia of female mice. Deletion of prolactin receptor in trigeminal ganglia by nasal clustered regularly interspaced short palindromic repeats-Cas9 targeting both prolactin receptor isoforms prevented stress-induced priming in female mice. Stress-induced activation of hypothalamic KOR increases circulating prolactin resulting in trigeminal downregulation of prolactin receptor long and pain responses to a normally innocuous TRPA1 stimulus. These are the first data that provide a mechanistic link between stress-induced hypothalamic activation and the trigeminal nociceptor effectors that produce trigeminal sensitization and migraine-like pain. This sexually dimorphic mechanism may help to explain female prevalence of migraine. KOR antagonists, currently in phase II clinical trials, may be useful as migraine preventives in both sexes, while dopamine agonists and prolactin/ prolactin receptor antibodies may improve therapy for migraine, and other stress-related neurological disorders, in females.

A Monoclonal Anti-Calcitonin Gene-Related Peptide Antibody Decreases Stress-Induced Colonic Hypersensitivity.

Irritable bowel syndrome (IBS) is a brain-gut disorder characterized by abdominal pain and altered bowel habits. Although the etiology of IBS remains unclear, stress in adulthood or in early life has been shown to be a significant factor in the development of IBS symptomatology. Evidence suggests that aberrant calcitonin gene-related peptide (CGRP) signaling may be involved in afferent sensitization and visceral organ hypersensitivity. Here, we used a monoclonal anti-CGRP divalent antigen-binding fragment [F(ab')2] antibody to test the hypothesis that inhibition of peripheral CGRP signaling reverses colonic hypersensitivity induced by either chronic adult stress or early life stress. A cohort of adult male rats was exposed to repeated water avoidance stress. Additionally, a second cohort consisting of female rats was exposed to a female-specific neonatal odor-attachment learning paradigm of unpredictable early life stress. Colonic sensitivity was then assessed in adult animals via behavioral responses to colorectal distension (CRD). To analyze spinal nociceptive signaling in response to CRD, dorsal horn extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was measured via immunohistochemistry. Repeated psychologic stress in adulthood or unpredictable stress in early life induced colonic hypersensitivity and enhanced evoked ERK1/2 phosphorylation in the spinal cord after CRD in rats. These phenotypes were reversed by administration of a monoclonal anti-CGRP F(ab')2 fragment antibody. Stress-induced changes in visceral sensitivity and spinal nociceptive signaling were reversed by inhibition of peripheral CGRP signaling, which suggests a prominent role for CGRP in central sensitization and the development of stress-induced visceral hypersensitivity. SIGNIFICANCE STATEMENT: Targeting peripheral calcitonin gene-related peptide (CGRP) with a monoclonal anti-CGRP divalent antigen-binding fragment antibody reduced central sensitization and attenuated colonic hypersensitivity induced by either chronic adult stress or early life stress. CGRP-targeting antibodies are approved for migraine prevention, and the results of this study suggest that targeting CGRP may provide a novel treatment strategy for irritable bowel syndrome-related, stress-induced visceral pain.

Migraine therapeutics differentially modulate the CGRP pathway.

BACKGROUND: The clinical efficacy of migraine therapeutic agents directed towards the calcitonin-gene related peptide (CGRP) pathway has confirmed the key role of this axis in migraine pathogenesis. Three antibodies against CGRP - fremanezumab, galcanezumab and eptinezumab - and one antibody against the CGRP receptor, erenumab, are clinically approved therapeutics for the prevention of migraine. In addition, two small molecule CGRP receptor antagonists, ubrogepant and rimegepant, are approved for acute migraine treatment. Targeting either the CGRP ligand or receptor is efficacious for migraine treatment; however, a comparison of the mechanism of action of these therapeutic agents is lacking in the literature. METHODS: To gain insights into the potential differences between these CGRP pathway therapeutics, we compared the effect of a CGRP ligand antibody (fremanezumab), a CGRP receptor antibody (erenumab) and a CGRP receptor small molecule antagonist (telcagepant) using a combination of binding, functional and imaging assays. RESULTS: Erenumab and telcagepant antagonized CGRP, adrenomedullin and intermedin cAMP signaling at the canonical human CGRP receptor. In contrast, fremanezumab only antagonized CGRP-induced cAMP signaling at the human CGRP receptor. In addition, erenumab, but not fremanezumab, bound and internalized at the canonical human CGRP receptor. Interestingly, erenumab also bound and internalized at the human AMY1 receptor, a CGRP receptor family member. Both erenumab and telcagepant antagonized amylin-induced cAMP signaling at the AMY1 receptor while fremanezumab did not affect amylin responses. CONCLUSION: The therapeutic effect of agents targeting the CGRP ligand versus receptor for migraine prevention (antibodies) or acute treatment (gepants) may involve distinct mechanisms of action. These findings suggest that differing mechanisms could affect efficacy, safety, and/or tolerability in migraine patients.

Immunogenicity of biologic therapies for migraine: a review of current evidence.

BACKGROUND: Monoclonal antibodies (mAbs) targeting the calcitonin gene-related peptide (CGRP) pathway have been shown to be effective in migraine prevention. Eptinezumab, erenumab, fremanezumab, and galcanezumb have shown efficacy in clinical trials along with favorable safety and tolerability profiles. Although erenumab is a human mAb and the others have been humanized to varying degrees, they all have the capacity to provoke immune reactions. The present review article aims to discuss the current relationship between mAbs targeting the CGRP pathway (CGRP mAbs) and immunogenicity and their potential clinical implications. FINDINGS: The incidence of patients developing anti-drug antibodies (ADAs), their titer, and clinical significance are highly variable and depend on a variety of different drug and patient factors. Neutralizing ADAs (NAbs) bind to and inhibit or reduce the pharmacologic activity of the biologic drug molecule, whereas non-neutralizing antibodies (Non-NAbs) bind to the biologic drug molecule without affecting pharmacologic activity in an in vitro test, although pharmacokinetics and drug clearance may be affected. A direct comparison of immunogenicity data across clinical trials with different biologics is not possible due to a lack of standardized assays. Several phase 2, phase 3, and long-term studies evaluating CGRP mAbs for migraine prevention have reported immunogenicity data (5 studies each for eptinezumab, erenumab, fremanezumab, and galcanezumab). Across these studies, prevalence of ADAs varied, ranging from < 1% to ~ 18%. Neutralizing ADAs were slightly less common, with a prevalence ranging from 0 to 12%. Adverse events related to ADA formation were rare. CONCLUSIONS: As more CGRP mAb studies are conducted and more long-term follow-up data become available, evidence is increasing that immunogenicity rates of biologic therapies for migraine are low, and adverse events related to ADAs are rare. Taken together, these results add to the growing body of evidence for the safety and tolerability of this class of migraine medications.

Increased severity of closed head injury or repetitive subconcussive head impacts enhances post-traumatic headache-like behaviors in a rat model.

INTRODUCTION: Posttraumatic headache is one of the most common, debilitating, and difficult symptoms to manage after a traumatic head injury. The development of novel therapeutic approaches is nevertheless hampered by the paucity of preclinical models and poor understanding of the mechanisms underlying posttraumatic headache. To address these shortcomings, we previously characterized the development of posttraumatic headache-like pain behaviors in rats subjected to a single mild closed head injury using a 250 g weight drop. Here, we conducted a follow-up study to further extend the preclinical research toolbox for studying posttraumatic headache by exploring the development of headache-like pain behaviors in male rats subjected to a single, but more severe head trauma (450 g) as well as following repetitive, subconcussive head impacts (150 g). In addition, we tested whether these behaviors involve peripheral calcitonin gene-related peptide signaling by testing the effect of systemic treatment with an anti-calcitonin gene-related peptide monoclonal antibody (anti-calcitonin gene-related peptide mAb). METHODS: Adult male Sprague Dawley rats (total n = 138) were subjected to diffuse closed head injury using a weight-drop device, or a sham procedure. Three injury paradigms were employed: A single hit, using 450 g or 150 g weight drop, and three successive 150 g weight drop events conducted 72 hours apart. Changes in open field activity and development of cephalic and extracephalic tactile pain hypersensitivity were assessed up to 42 days post head trauma. Systemic administration of the anti-calcitonin gene-related peptide mAb or its control IgG (30 mg/kg) began immediately after the 450 g injury or the third 150 g weight drop with additional doses given every 6 days subsequently. RESULTS: Rats subjected to 450 g closed head injury displayed an acute decrease in rearing and increased thigmotaxis, together with cephalic tactile pain hypersensitivity that resolved by 6 weeks post-injury. Injured animals also displayed delayed and prolonged extracephalic tactile pain hypersensitivity that remained present at 6 weeks post-injury. Repetitive subconcussive head impacts using the 150 g weight drop, but not a single event, led to decreased vertical rearing as well as cephalic and extracephalic tactile pain hypersensitivity that resolved by 6 weeks post-injury. Early and prolonged anti-calcitonin gene-related peptide mAb treatment inhibited the development of the cephalic tactile pain hypersensitivity in both the severe and repetitive subconcussive head impact models. CONCLUSIONS: Severe head injury gives rise to a prolonged state of cephalic and extracephalic tactile pain hypersensitivity. These pain behaviors also develop following repetitive, subconcussive head impacts. Extended cephalic tactile pain hypersensitivity following severe and repetitive mild closed head injury are ameliorated by early and prolonged anti-calcitonin gene-related peptide mAb treatment, suggesting a mechanism linked to calcitonin gene-related peptide signaling, potentially of trigeminal origin.

Enhanced post-traumatic headache-like behaviors and diminished contribution of peripheral CGRP in female rats following a mild closed head injury.

INTRODUCTION: Females are thought to have increased risk of developing post-traumatic headache following a traumatic head injury or concussion. However, the processes underlying this susceptibility remain unclear. We previously demonstrated the development of post-traumatic headache-like pain behaviors in a male rat model of mild closed head injury, along with the ability of sumatriptan and an anti-calcitonin-gene-related peptide monoclonal antibody to ameliorate these behaviors. Here, we conducted a follow-up study to explore the development of post-traumatic headache-like behaviors and the effectiveness of these headache therapies in females subjected to the same head trauma protocol. METHODS: Adult female Sprague Dawley rats were subjected to a mild closed head injury using a weight-drop device (n = 126), or to a sham procedure (n = 28). Characterization of headache and pain related behaviors included assessment of changes in cutaneous cephalic and extracephalic tactile pain sensitivity, using von Frey monofilaments. Sensitivity to headache/migraine triggers was tested by examining the effect of intraperitoneal administration of a low dose of glyceryl trinitrate (100 µg/kg). Treatments included acute systemic administration of sumatriptan (1 mg/kg) and repeated systemic administration of a mouse anti-calcitonin gene-related peptide monoclonal antibody (30 mg/kg). Serum levels of calcitonin gene-related peptide were measured at baseline and at various time points post head injury in new cohorts of females (n = 38) and males (n = 36). RESULTS: Female rats subjected to a mild closed head injury developed cutaneous mechanical hyperalgesia, which was limited to the cephalic region and was resolved 4 weeks later. Cephalic pain hypersensitivity was ameliorated by treatment with sumatriptan but was resistant to an early and prolonged treatment with the anti-calcitonin gene-related peptide monoclonal antibody. Following the resolution of the head injury-evoked cephalic hypersensitivity, administration of glyceryl trinitrate produced a renewed and pronounced cephalic and extracephalic pain hypersensitivity that was inhibited by sumatriptan, but only partially by the anti-calcitonin gene-related peptide treatment. Calcitonin gene-related peptide serum levels were elevated in females but not in males at 7 days post head injury. CONCLUSIONS: Development of post-traumatic headache-like pain behaviors following a mild closed head injury, and responsiveness to treatment in rats is sexually dimorphic. When compared to the data obtained from male rats in the previous study, female rats display a prolonged state of cephalic hyperalgesia, increased responsiveness to a headache trigger, and a poorer effectiveness of an early and prolonged anti-calcitonin gene-related peptide treatment. The increased risk of females to develop post-traumatic headache may be linked to enhanced responsiveness of peripheral and/or central pain pathways and a mechanism independent of peripheral calcitonin gene-related peptide signaling.

Fremanezumab and its isotype slow propagation rate and shorten cortical recovery period but do not prevent occurrence of cortical spreading depression in rats with compromised blood-brain barrier.

Most centrally acting migraine preventive drugs suppress frequency and velocity of cortical spreading depression (CSD). The purpose of the current study was to determine how the new class of peripherally acting migraine preventive drug (ie, the anti-CGRP-mAbs) affect CSD-an established animal model of migraine aura, which affects about 1/3 of people with migraine-when allowed to cross the blood-brain barrier (BBB). Using standard electrocorticogram recording techniques and rats in which the BBB was intentionally compromised, we found that when the BBB was opened, the anti-CGRP-mAb fremanezumab did not prevent the induction, occurrence, or propagation of a single wave of CSD induced by a pinprick, but that both fremanezumab and its isotype were capable of slowing down the propagation velocity of CSD and shortening the period of profound depression of spontaneous cortical activity that followed the spreading depolarization. Fremanezumab's inability to completely block the occurrence of CSD in animals in which the BBB was compromised suggests that calcitonin gene-related peptide (CGRP) may not be involved in the initiation of CSD, at least not to the extent that it can prevent its occurrence. Similarly, we cannot conclude that CGRP is involved in the propagation velocity or the neuronal silencing period (also called cortical recovery period) that follows the CSD because similar effects were observed when the isotype was used. These finding call for caution with interpretations of studies that claim to show direct central nervous system effects of CGRP-mAbs.

Fluorescently-labeled fremanezumab is distributed to sensory and autonomic ganglia and the dura but not to the brain of rats with uncompromised blood brain barrier.

BACKGROUND: The presence of calcitonin gene-related peptide and its receptors in multiple brain areas and peripheral tissues previously implicated in migraine initiation and its many associated symptoms raises the possibility that humanized monoclonal anti-calcitonin gene-related peptide antibodies (CGRP-mAbs) can prevent migraine by modulating neuronal behavior inside and outside the brain. Critical to our ability to conduct a fair discussion over the mechanisms of action of CGRP-mAbs in migraine prevention is data generation that determines which of the many possible peripheral and central sites are accessible to these antibodies - a question raised frequently due to their large size. MATERIAL AND METHODS: Rats with uncompromised and compromised blood-brain barrier (BBB) were injected with Alexa Fluor 594-conjugated fremanezumab (Frema594), sacrificed 4 h or 7 d later, and relevant tissues were examined for the presence of Frema594. RESULTS: In rats with uncompromised BBB, Frema594 was similarly observed at 4 h and 7 d in the dura, dural blood vessels, trigeminal ganglion, C2 dorsal root ganglion, the parasympathetic sphenopalatine ganglion and the sympathetic superior cervical ganglion but not in the spinal trigeminal nucleus, thalamus, hypothalamus or cortex. In rats with compromised BBB, Frema594 was detected in the cortex (100 µm surrounding the compromised BBB site) 4 h but not 7 d after injections. DISCUSSION: Our inability to detect fluorescent (CGRP-mAbs) in the brain supports the conclusion that CGRP-mAbs prevent the headache phase of migraine by acting mostly, if not exclusively, outside the brain as the amount of CGRP-mAbs that enters the brain (if any) is too small to be physiologically meaningful.

CGRP-dependent and independent mechanisms of acute and persistent post-traumatic headache following mild traumatic brain injury in mice.

BACKGROUND: Acute and persistent post-traumatic headache are often debilitating consequences of traumatic brain injury. Underlying physiological mechanisms of post-traumatic headache and its persistence remain unknown, and there are currently no approved therapies for these conditions. Post-traumatic headache often presents with a migraine-like phenotype. As calcitonin-gene related peptide promotes migraine headache, we explored the efficacy and timing of intervention with an anti- calcitonin-gene related peptide monoclonal antibody in novel preclinical models of acute post-traumatic headache and persistent post-traumatic headache following a mild traumatic brain injury event in mice. METHODS: Male, C57Bl/6 J mice received a sham procedure or mild traumatic brain injury resulting from a weight drop that allowed free head rotation while under minimal anesthesia. Periorbital and hindpaw tactile stimulation were used to assess mild traumatic brain injury-induced cutaneous allodynia. Two weeks after the injury, mice were challenged with stress, a common aggravator of migraine and post-traumatic headache, by exposure to bright lights (i.e. bright light stress) and cutaneous allodynia was measured hourly for 5 hours. A murine anti- calcitonin-gene related peptide monoclonal antibody was administered after mild traumatic brain injury at different time points to allow evaluation of the consequences of either early and sustained calcitonin-gene related peptide sequestration or late administration only prior to bright light stress. RESULTS: Mice with mild traumatic brain injury, but not a sham procedure, exhibited both periorbital and hindpaw cutaneous allodynia that resolved by post-injury day 13. Following resolution of injury-induced cutaneous allodynia, exposure to bright light stress re-instated periorbital and hindpaw cutaneous allodynia in injured, but not sham mice. Repeated administration of anti-calcitonin-gene related peptide monoclonal antibody at 2 hours, 7 and 14 days post mild traumatic brain injury significantly attenuated the expression of cutaneous allodynia when evaluated over the 14-day post injury time course and also prevented bright light stress-induced cutaneous allodynia in injured mice. Administration of anti-calcitonin-gene related peptide monoclonal antibody only at 2 hours and 7 days after mild traumatic brain injury blocked injury-induced cutaneous allodynia and partially prevented bright light stress-induced cutaneous allodynia. A single administration of anti-calcitonin-gene related peptide monoclonal antibody after the resolution of the peak injury-induced cutaneous allodynia, but prior to bright light stress challenge, did not prevent bright light stress-induced cutaneous allodynia. CONCLUSIONS: We used a clinically relevant mild traumatic brain injury event in mice along with a provocative stimulus as novel models of acute post-traumatic headache and persistent post-traumatic headache. Following mild traumatic brain injury, mice demonstrated transient periorbital and hindpaw cutaneous allodynia suggestive of post-traumatic headache-related pain and establishment of central sensitization. Following resolution of injury-induced cutaneous allodynia, exposure to bright light stress re-established cutaneous allodynia, suggestive of persistent post-traumatic headache-related pain. Continuous early sequestration of calcitonin-gene related peptide prevented both acute post-traumatic headache and persistent post-traumatic headache. In contrast, delayed anti-calcitonin-gene related peptide monoclonal antibody treatment following establishment of central sensitization was ineffective in preventing persistent post-traumatic headache. These observations suggest that mechanisms involving calcitonin-gene related peptide underlie the expression of acute post-traumatic headache, and drive the development of central sensitization, increasing vulnerability to headache triggers and promoting persistent post-traumatic headache. Early and continuous calcitonin-gene related peptide blockade following mild traumatic brain injury may represent a viable treatment option for post-traumatic headache and for the prevention of post-traumatic headache persistence. ABBREVIATIONS: CA Cutaneous allodynia CGRP Calcitonin gene-related peptide mTBI Mild traumatic brain injury PTH Post-traumatic headache APTH Acute post-traumatic headache PPTH Persistent post-traumatic headache.