FIELD OF THE INVENTION

A method and system to facilitate the growth of desired bacteria in a human's mouth by using a bioadhesive strip that has an encapsulated feature containing at least one bacteria and that has one of a pathogenic or toxic element excised using a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas) system or a CRISPR fromPrevotellaandFrancisella1 (Cpf1) system, including oral strips that include at least one of xylitol,Lachnospira, Veillonella, Faecalibacteriumand/orRothiabacteria.

BACKGROUND OF THE INVENTION

All primary headaches are in need of better treatments. The most important primary headaches (i.e. independent disorders that are not caused by another disease) are migraine, tension-type headache and cluster headache. Migraine has a prevalence of 10% in the general population with a lifetime prevalence of 13% in men and 33% in women. Migraine is a highly disabling disease with high personal and social costs. To date, the precise mechanisms underlying the pathophysiology of migraine have remained elusive. Migraine strikes people during what are expected to be their most productive years: between ages 20 and 40 for most women, with a slightly higher age range for men. Migraine is typically characterized by unilateral onset of head pain, severe progressive intensity of pain, throbbing or pounding, and interference with the person's routine activities. Accompanying symptoms of photophobia (sensitivity to light) or phonosensitivity (intolerance to noise), as well as nausea and/or vomiting, are common, and often leads to the inability to perform daily tasks. A large portion of people with migraine often have no accompanying pain, their predominant symptom instead being vertigo (a spinning sensation) or dizziness/disequilibrium (balance loss), mental confusion, disorientation, dysarthria, visual distortion or altered visual clarity, or extremity paresis. Patients with migraine associated vertigo (MAV) are often seen by audiologists and vestibular rehabilitation therapists for evaluation and treatment. Because the exact mechanisms of migraine are still not completely understood, the management of migraine dizziness presently includes a combination of medications, vestibular rehabilitation, and lifestyle modifications that include limitation of risk factors associated with migraine (those related to diet, sleep, stress, exercise, and environmental factors).

Migraine is a disease associated with increased synthesis and release of calcitonin gene related peptide (CGRP) and a migraine attack can be blocked with CGRP antagonists. The actual pain is generated by nociceptors of trigeminal nerve endings in the dura. Low serotonin levels may sensitize the nociceptors of trigeminal neurons. Triptans and ergotamins, which decrease serotonin, are associated with relief of acute pain. In contrast, tricyclic antidepressants and selective serotonin and noradrenaline reuptake inhibitors, which are associated with increases in serotonin, are utilized for migraine prevention.

Migraine attacks can be triggered by intrinsic cerebral factors (e.g. calcitonin gene related peptide (CGRP) release), nitric oxide like tri-nitroglycerine, corticotrophin releasing hormone (stress), pro-inflammatory cytokines, and degranulation of mast cells located in the dura. While migraine has a genetic background, twin studies reveal that the cause of a majority of migraines appears to be due to environmental factors. The mechanism of triggering migraine is, however, still not understood. The cause of both migraines and chronic dizziness has eluded investigators for centuries and it therefore presents a truly long felt but unsolved mystery as to its causation and treatment.

SUMMARY OF THE INVENTION

One aspect of the present invention is therefore directed to addressing the causative agents involved in migraines and dizziness, instead of merely addressing the symptoms thereof. As can be seen from the prior art as discussed herein, the diagnosis and treatment of both migraines and dizziness have continued to be directed to symptoms, rather than to an underlying treatable cause. The present invention changes that focus and thus, provides both an understanding of causative agents and how best to treat individuals so that their maladies are addressed. The present inventors believe that there is an association between the occurrence of migraines and gastrointestinal (GI) disorders, including irritable bowel syndrome (IBS). People who regularly experience GI symptoms have a higher prevalence of headaches, with a stronger association with increasing headache frequency.

While not bound by theory, it is believed that the gut microbiota is an independent factor that contributes to systemic diseases, often involving the migration of stimulated immune cells, by systemic diffusion of microbial products or metabolites, or by bacterial translocation as a result of decreased intestinal barrier function. The brain and the GI tract are strongly connected via neural, endocrine, and immune pathways. The gut microbiota, as well as the oral microbiota, is associated with brain functions and neurological diseases like migraine.

Empirical data exists to reveal the association of migraines with disruptions of a person's microbiome. For example, children with a mother with a history of migraine are more likely to have infantile colic. Children with migraine are more likely to have experienced infantile colic compared to controls. Several studies demonstrated significant associations between migraine and celiac disease, inflammatory bowel disease, and IBS.

It is believed that the underlying mechanisms of migraine and GI diseases are both related to increased gut permeability and inflammation. In addition, it is believed that a person's oral microbiome is also responsible for migraines. Thus in several embodiments of the present invention, modification of the oral as well as (or solely) the gut microbiome is achieved to combat and address migraine occurrences.

The biological nitrogen cycle involves step-wise reduction of nitrogen oxides to ammonium salts and oxidation of ammonia back to nitrites and nitrates by plants and bacteria. The salivary bacterial reduction of nitrate to nitrite has been recognized as an important metabolic conversion in humans. Several enteric bacteria have also shown the ability of catalytic reduction of nitrate to ammonia via nitrite during dissimilatory respiration. Although to date the importance of this pathway in bacterial species colonizing the human intestine has been little studied, the present inventors submit that it is a major factor involved in the occurrence of migraines.

In addition to the connection between the gut microbiome and migraines, there is also believed to be a connection with the oral microbiome of an individual with various health conditions, including an association with dysbiosis of the oral microbiome and migraines.

Many biochemical, pharmacological, neuropathological, and experimental data suggest a role of nitric oxide in the pathogenesis of migraine. Nitric oxide (NO) is a very important molecule in the regulation of cerebral and extra cerebral cranial blood flow and arterial diameters. It is also involved in nociceptive processing. Glyceryl trinitrate (GTN), a pro-drug for NO, causes headache in normal volunteers and a so called delayed headache that fulfils criteria for migraine without aura in migraine sufferers. One aspect of the present invention is to prevent migraines via the inhibition of NO production; the blockade of steps in the NO-cGMP pathway; or the scavenging of NO. The pain signaling molecules, nitric oxide synthase (NOS) and calcitonin gene-related peptide (CGRP) are implicated in the pathophysiology of migraines.

Non-specific NOS inhibition and a specific neuronal NOS inhibitor have been found to attenuate neurogenic dural vasodilation. Interestingly, specific inducible and endothelial NOS inhibitors had no effect. Non-vascular inducible NOS inhibitors have been shown unable to abort or prevent migraine. The present inventors submit that this leaves neuronal NOS inhibition and bacterial NOS inhibition (bNOS) as candidates for therapy, with the latter being considered the primary causative agents in preventing migraines.

Neurons in the trigeminocervical complex are the major relay neurons and are the neural substrates of head pain. NO production in the TCC and potentially other areas of the brain may be involved in triggering migraines, and therefore blocking NO production is believed to be therapeutic.

The nitric oxide synthase inhibitor NG-monomethyi-L-arginine (L-NMMA) may be employed in several embodiments of the present invention. Blockade of nitric oxide synthases (NOS) by L-NMMA effectively treats attacks of migraine without aura. Similar results have been obtained for chronic tension-type headache and cluster headache.

Inhibition of the breakdown of cGMP also provokes migraine in sufferers, indicating that cGMP is the effector of NO-induced migraine. Several relationships exist between NO, calcitonin gene-related peptide and other molecules important in migraine. Also, ion channels, particularly the K(ATP) channels, are important for the action of NO.

NO donors and their ability to trigger migraine in patients is well known and so nitric oxide synthase (NOS) inhibitors have been explored for the treatment of migraine. While NO donors are known to cause vasodilation, they also cause activation of neurons in the trigeminocervical complex of the brain that are not vascular related, as well as other areas of the brain related to migraine. Thus, the present inventors submit that non-vascular mechanisms are involved and that non-vascular NOS inhibitors are efficacious in the treatment of migraines.

Nitric oxide synthases (NOSs) are multidomain metalloproteins first identified in mammals as being responsible for the synthesis of the wide-spread signaling and protective agent nitric oxide (NO). Nitric oxide synthases are heme-based monooxygenases that oxidize L-arginine to nitric oxide (NO), a signaling molecule and cytotoxic agent in higher organisms. NO is one of the main inflammatory mediators involved in both inflammation and angiogenesis. NO can be synthesized by three different isoforms of NO synthase: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) synthases. NO production due to cytokine-induced expression of inducible nitric oxide synthase (iNOS) is largely involved in the pathophysiology of inflammation.

Although NOS-like activity has been reported in many bacteria, only a few bacterial homologs of mammalian NOSs (mNOSs) have been characterized to date. Nitric oxide synthases (NOSs) play an essential role in synthesizing nitric oxide (NO) by oxidizing I-arginine. NO is a significant mediator in cellular signaling pathways. It serves as a crucial regulator in insulin secretion, vascular tone, peristalsis, angiogenesis, neural development and inflammation. Due to its important role, the inhibition of these vital enzymes provides therapeutic applications that target NOSs.

Over the past 10 years, prokaryotic proteins that are homologous to animal NOSs have been identified and characterized, both in terms of enzymology and biological function. In contrast to mNOSs, which possess both a catalytic and a reductase domain, the bacterial enzymes lack reductase domains and require the supply of suitable reductants to produce NO. A notable exception is a NOS from a gram-negative bacterium that contains a new type of reductase module.

Bacterial NOSs seem to have functions that differ from those of mNOSs, including nitration of different metabolites and protection against oxidative stress. Bacterial NOSs provide a better understanding of the mechanism of NO synthesis and unveil a variety of new functions for NO in microbes.

In one embodiment, the lactic acid bacteriumLactobacillus reuteriis employed as it is believed that by doing so, one is able to induce oxytocin, preferably in a manner that offers a sustained induction of oxytocin, unlike the short effects achieved using intranasal oxytocin sprays, etc. Thus, one aspect of the present invention relates to the employment of probiotics-induced oxytocin to reduce migraine symptoms, especially in the form of an oral adhesive strip as further described herein.

Other embodiments relate to the employment of calcitonin gene-related peptide (CGRP) as a major player in treating migraines. In various embodiments, CGRP triggers a chain of events such that increased amounts of CGRP released at the start of a migraine sensitize the trigeminal nerve to what are normally innocuous signals, resulting in inflammation in the nerves that is relayed to the brain as a pain signal and in such a manner, stop headaches from outside the central nervous system, believed to be active on the trigeminal connections into the brain rather than the brain itself. It is further believed that there is often a fairly massive change in the permeability of the blood-brain barrier during a migraine attack and may have an effect on certain sections of the brainstem that are not believed to be as well protected by the blood-brain barrier. As is the case with autism, it is believed that a gut brain inter-relationship dysbiosis is caused by a loweredLactobacillusspp. and decreased number ofClostridiumspp. populations in an individual's gut microbiome. Thus, restoring the gut microbiome with appropriate levels of particular bacteria species as set forth herein is one aspect of the present invention. In addition, in various embodiments, the maintenance of desired populations in the gut is achieved via the employment of fibre that is essential for the fostering of a healthy gut microbiome once established so that the beneficial bacteria have a food source other than the mucosa proteins that protect the tissue of the gut. In preferred embodiments approximately 6 g/fibre/day is administered to an individual as it is believed that by doing so one is able to increase the numbers of bifidobacteria in the gut microbiome and thus, increase desired fermentation by increasing the number and maintenance of both Bifidobacteriaceae and Lactobacteriaceae families. In preferred embodiments, a diet rich in plant polysaccharides (i.e., fiber) is employed to confer protection against various ailments associated with the gut microbiome dysbiosis, including not only migraines, but also cardiovascular disease (CVD). Such benefits are believed to be derived from the interactions between carbohydrates that reach the distal gut and microbes via production of short chain fatty acids (SOFA, e.g., acetate, propionate and butyrate). Thus, in various embodiments, in addition to the administration of beneficial bacteria as described herein wither to the gut or the oral microbiome of an individual (or both) the consumption of a diet high in fibre is employed to increase microbiota populations to generate short chain fatty acids (SCFAs) such as acetate, which has a protective role in inflammatory diseases. Such a diet is further believed to attenuate the development of cardiac fibrosis due to the belief that inflammation is also implicated in cardiac remodelling. Thus, a high fibre diet, especially one that includes increased levels of acetate, is believed to not only reduce the incidences of migraine, but to further result in significantly less cardiac hypertrophy, perivascular and interstitial cardiac fibrosis, and improved cardiac function. It is further believed that the protective effects of high fibre and acetate are accompanied by a decrease in the ratio of bacteria from the phylum Firmicutes compared to Bacteroidetes. In addition to the association of the gut and the brain as it relates to the incidence of migraines, there is also a gut-heart connection and by employing similar efforts to address the dysbiosis of an individuals' gut and oral microbiomes, another aspect of the present invention is directed to addressing and significantly preventing the occurrence of heart disease.

In situations where there is insufficient fiber for the beneficial bacteria to consume, the bacteria end up eroding the mucus of the gut and leads to epithelial access by mucosal pathogens. It is believed that there is an increase in the amount of calcitonin gene-related peptide released at the start of a migraine that sensitizes the trigeminal nerve to what are normally innocuous signals, resulting in inflammation in the nerves that is relayed to the brain as a pain signal. Thus, one aspect of the present invention relates to the ability to affect the trigeminal connections into the brain, rather than the brain itself, and to do so by modifying the type, number and maintenance of desired bacteria in an individual's gut and oral microbiome. In addition to migraine headaches, other aspects of the invention are directed more generally to addressing other pain related conditions, including but not limited to fibromyalgia, cluster headaches, etc.

Therefore, certain embodiments of the present invention are directed to a method of reducing the likelihood of migraine headaches by providing to an individual in need thereof a buccal bioadhesive strip, with such strip having a first and second side and having a surface comprising a pattern defined by a plurality of spaced apart features each having at least one microscale dimension and having at least one neighboring feature having a substantially different geometry. Preferably the average spacing between adjacent ones of such features is between 0.5 and 5 .mu.m.FIG.2(a)-(d)illustrate some exemplary surface architectural patterns according to the invention.

A bioadhesive is employed that is adapted to bind to a mucosal membrane for at least 1 hour while inside an individual's mouth. Preferably the strip includes xylitol. In other embodiments, the strip includes an encapsulated feature containing a desired bacteria, preferably selected from the group consisting ofLachnospira, Veillonella, FaecalibacteriumandRothia. Still other embodiments include an antibody on the strip to a calcitonin gene-related peptide. The strip(s) may further includeLactobacillusspp.

Preferably, methods of the present invention further include administering a diet of at least 6 g/fibre/day to an individual so as to increase the numbers ofbifidobacteriain the gut microbiome of the individual. Such a diet should preferably include a high fibre diet that includes acetate, especially in an amount sufficient to decrease the ratio of bacteria in the individual's oral cavity from the phylum Firmicutes compared to Bacteroidetes. It is preferred to increase the number of both Bifidobacteriaceae and Lactobacteriaceae bacteria. Other embodiments increase the population of desired bacteria by includingEnterococcus faeciumon the strip. It is preferred to remove from the oral cavity of the individual gram negative bacteria associated with periodontitis, and within 2 hours thereof, to provide the individual with the strip. Still other embodiments include a strip that includesLactobacillus reuteribacteria to induce a sustained induction of oxytocin, and providing the individual with an amount of antibiotic sufficient to reduce the number of undesired bacteria in the oral cavity. Prior to the use of the strip, an antibiotic selected from the group consisting of tetracycline hydrochloride, doxycycline, and minocycline is used to reduce the number of undesired bacteria in the oral cavity. One objective is to retard the growth conditions for spirochetes andP. gingivalis, believed to be associated with migraine headaches. In still other embodiments,Veillonellaand/orPrevotellabacteria is provided on the strip. As one of skill in the art will appreciate, the strip may be made to include at least one polymer selected from the group consisting of pullulan, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, polyvinyl pyrrolidone, carboxymethyl cellulose, polyvinyl alcohol, sodium alginate, polyethylene glycol, tragacanth gum, guar gum, acacia gum, arabic gum, polyacrylic acid, methylmethacrylate copolymer, carboxyvinyl polymer, amylose, high amylose starch, hydroxypropylated high amylose starch, dextrin, pectin, chitin, chitosan, levan, elsinan, collagen, gelatin, zein, gluten, soy protein isolate, whey protein isolate, casein and mixtures thereof. Preferably, the strip includes at least 0.2% xylitol by weight. In certain embodiments, the strip comprises bioluminescent material. Preferably the strip is dissolvable in a person's mouth within a period of 1 hour. In certain preferred embodiments, the strip has least one encapsulated feature that contains an agent selected from the group consisting of an antibiotic; lactic acid bacteria; and xylitol, e.g. at least 200 mg of xylitol. Such a frangible capsule may be constructed so that it may be broken by the individual pressing against said strip with the individual's tongue.

Still other embodiments of the present invention are directed to addressing the incidence of migraines and cluster headaches using bacteria of the oral cavity and gut of an individual. Microbes, among others in the digestive tract, are capable of oxidizing alcohol to acetaldehyde. Compositions containing one or more cysteines as active agents have been shown to bind acetaldehyde. These active agents have been found to also be capable of breaking down biofilms formed by some microbes, particularly in the stomach. One aspect of the present invention is therefore directed to reducing the likelihood of headaches, including not only migraines, but also cluster headaches. Thus, several embodiments employ microbes, preferably particular bacterial species as set forth herein, to assist in decreasing the amount of acetaldehyde in the body of an individual who suffers from such headaches. This can be achieved by employing cysteine generating microbes that are effective in reducing the amount of acetaldehydes. Other microbes are also effective in degrading acetaldehydes.

While migraines are diagnosed more often in women, cluster headaches are more prevalent in men. Cluster headache is a neurological disease that involves, as its most prominent feature, excruciating unilateral headaches of extreme intensity. “Cluster” refers to the tendency of these headaches to occur periodically, with active periods interrupted by spontaneous remissions. The cause of the disease is currently unknown. Cluster headaches are sometimes classified as vascular headaches. The intense pain has been suggested to be linked with the dilation of blood vessels which creates pressure on the trigeminal nerve. Cluster headache episodes are known to be triggered by factors, such as alcohol consumption. Patients who are sensitive to alcohol note that attacks are triggered within 5 to 45 minutes after the ingestion of modest amounts of alcohol, usually being less than a single cocktail or glass of wine. Alcohol triggers attacks in 70 to 80% of exposures. Microbial metabolism may contribute to the toxicity of alcohol, especially in the gastrointestinal tract, where aerobic and facultative anaerobic bacteria convert ethanol to acetaldehyde. Indeed, acetaldehyde is known to be a highly toxic and pro-carcinogenic compound with various negative effects, ranging from DNA damage and impaired DNA excision repair to the degradation of folate. Thus, one aspect of various embodiments of the present invention is directed to providing particular bacteria to a person who consumes alcohol in a manner that such bacteria may ameliorate the accumulation of acetaldehyde. In certain embodiments, bacteria are employed to degrade acetaldehyde, such bacteria preferably having an aldehyde dehydrogenase, such as bacteria of the genusSaccharomycesand/or a threonine aldolase derived fromEscherichia coli. Employment of CRISPR-Cas systems to incorporate the genes of such bacteria into other bacteria as set forth herein forms various other embodiments of the present invention. Thus, use of bacteria in the oral cavity, as well as in the gut microbiome of an individual, are employed to degrade acetaldehyde and thus reduce the incidence of migraines and cluster headaches. While many of the embodiments described herein are principally directed to a method and system for addressing the terrible pain occasioned by headaches caused by migraines and cluster headaches, one may further appreciate other aspects of the invention as being directed to hangover remedies as the ability to degrade alcohol and acetaldehyde to remedy the effects of consuming too much alcohol: thus introducing “Hangover Strips” in addition to Headache Strips as described herein.

One will appreciate that this Summary of the Invention is not intended to be all encompassing and that the scope of the invention nor its various embodiments, let alone the most important ones, are necessarily encompassed by the above description. One of skill in the art will appreciate that the entire disclosure, as well as the incorporated references, figures, etc. will provide a basis for the scope of the present invention as it may be claimed now and in future applications.

