WO2021071846A1

WO2021071846A1 - Methods and materials for treating eosinophilic esophagitis and other eosinophilic disorders

Info

Publication number: WO2021071846A1
Application number: PCT/US2020/054418
Authority: WO
Inventors: Benjamin MITLYNG; Robert A. Ganz
Original assignee: Mg Labs, Inc.
Priority date: 2019-10-07
Filing date: 2020-10-06
Publication date: 2021-04-15

Abstract

Methods and materials are described for treating eosinophilic disorders such as eosinophilic esophagitis using helminthic compositions (e.g., compositions comprising helminths, or any portion, product, extract, or components of a helminth thereof such as eggs or larvae).

Description

METHODS AND MATERIALS FOR TREATING EOSINOPHILIC ESOPHAGITIS AND OTHER EOSINOPHILIC

DISORDERS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No. 63/019,730, filed May 4, 2020, U.S. Provisional Application Serial No. 63/008,327, filed April 10, 2020, and U.S. Provisional Application Serial No. 62/911,759, filed October 7, 2019. The disclosures of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.

TECHNICAL FIELD

This document relates to methods and materials for treating eosinophilic disorders such as eosinophilic esophagitis and more particularly, to using a helminthic composition (e.g., a composition comprising helminths, helminth eggs, helminth larvae, or any portion, product, extract, or component of a helminth, helminth egg, or helminth larva) to treat eosinophilic disorders.

BACKGROUND

Eosinophilic esophagitis is an important disease of the esophagus that appears to be increasing in incidence and importance. A recent study from Mayo Clinic reported an incidence of 9 cases per 100,000 population as of January 2007, and an approximate prevalence of 105 cases per 100,000. However, a subsequent multicenter analysis from 2006-2013 suggests the incidence continues to rise and indeed may be accelerating (see abstract at end). See, Menard-Katcher el al ., American Journal of Gastroenterology, 109:S31-S32, 2014. The disease is a chronic allergic inflammatory disease unique to the esophagus and occurs when an elevated number of eosinophils accumulates in that organ, causing injury and inflammation to the esophagus including scarring, fibrosis, and inability to swallow normally. In general, eosinophils are not present in esophageal tissue. Patients with eosinophilic esophagitis present with esophageal symptoms, and, in the absence of other known causes of tissue eosinophilia, the diagnosis is made via compatible clinical findings (e.g., dysphagia and/or food impaction), compatible endoscopic findings (e.g., rings, furrows and exudate), and a minimum of 15/eosinophils per high-power microscopic field (a normal esophagus has zero eosinophils). The disease is formally defined by a consensus statement: “Eosinophilic esophagitis represents a chronic immune and antigen mediated esophageal disease characterized clinically by esophageal dysfunction and histologically by eosinophil predominant inflammation.”

The disease is important clinically, as it affects swallowing, inhibits the ability to eat normally, and can cause chronic pain. Due to the eosinophilic infiltrate, the esophagus can become significantly inflamed, with exudate, edema of the mucosa and/or wall of the esophagus, and potentially fibrosis and stricturing or scarring of the esophagus, resulting in the inability to eat and food impactions. Eosinophilic esophagitis, is the most common cause of food impaction and emergency food removal in the United States. Treatment involves either using chronic medication on a daily basis, which is expensive and inconvenient, or a food elimination diet, which also is inconvenient and potentially disruptive. Approximately one third of patients do not respond to medical management, and often times the identifying food responsible for the allergy cannot be identified.

Eosinophilic esophagitis is thought to be predominately an allergic disease with an inflammatory response. The etiology of eosinophilic esophagitis is hypothesized to be an abnormal immune response to antigenic exposure, typically antigens in food. The esophagus is unique in the gastrointestinal tract since as noted above, eosinophils are not normally seen in the esophagus. In other parts of the gastrointestinal tract, eosinophils are normally present even in the absence of any disease. A recent study by Straumann et al.

( Gastroenterology , 154(2)346-359, 2018), demonstrated that eosinophilic esophagitis induces a selective inflammatory response limited to the esophagus, including infiltration of esophageal mucosa (and possibly deeper layers) by interleukin-5 (IL-5) expressing T cells, B cells, eosinophils, and immunoglobulin E expressing mast cells. Other studies have demonstrated that eotaxin, an eosinophil chemoattractant chemokine, plays an important role in directing eosinophils to the mucosal surface of the gastrointestinal tract in general, and more specifically to the esophagus. Subsequently it has been reported that IL-5, a T helper 2 cytokine (Th2), also plays an important role in directing eosinophils to the esophagus. Other studies have demonstrated that another Th2 to cytokine, interleukin 13 (IL-13) helps induce eosinophilic esophagitis by inducing IL-5 and eotaxin 1. Other studies have implicated other chemical messengers in trafficking and directing eosinophils to the esophagus including IL-4 and eotaxin 3, and several others. The result of driving so many eosinophils to the esophagus as a result of food allergies, is eosinophil-associated esophageal remodeling and subepithelial fibrosis, potentially resulting in scarring and narrowing of the esophagus, resulting in difficulty swallowing and potentially food impactions. See, Straumann et al. 2018, supra.

Thus, treatment of the disease involves reducing eosinophilic infiltration of the esophagus, either by removing a specific food allergen, such as eliminating dairy, wheat, soy, eggs, peanuts, tree nuts, fish and/or seafood, etc., which is laborious and involves numerous endoscopic exams, versus using medication to reduce eosinophilia or inhibit eosinophil recruitment or function. Medications used to treat the disease include, among others, proton pump inhibitors, which inhibit eotaxin (among other effects), or topical swallowed steroids, which inhibit eosinophil function. Experimental therapies involve specific monoclonal antibodies directed at specific interleukins, chemokines or eotaxin. There is no therapy that works on all patients, and many patients fail all therapies leaving them at risk for repeated food impactions, however any therapy that reduces the number of eosinophils, inhibits eosinophil recruitment or inhibits eosinophil function would be expected to be beneficial. Thus, the field is in need of novel therapies for reducing eosinophils or eosinophil function in the esophagus that are safe, effective, less expensive and less intrusive than existing therapies.

SUMMARY

This document provides methods and materials for treating eosinophilic disorders such as eosinophilic esophagitis using a helminthic composition. The word helminth is a general scientific term meaning “worm” and there are three major groupings of worms that are recognized. Nematodes are roundworms that have long unsegmented tube-like bodies with anterior mouths and longitudinal digestive tracts. Cestodes or tapeworms have long flat ribbon-like bodies with a single anterior organ (or scolex), that can fasten to another organism. Trematodes or flukes have small flat leaf-like bodies with oral and ventral suckers and a blind sac-like gut.

Helminths, or worms, have co-evolved with mammals for hundreds of millions of years, and co-evolved with proto-humans and humans for hundreds of thousands of years. The human immune response to worm infections is unlike the response to other pathogenic organisms such as viruses, bacteria, protozoa, and fungi. Unlike bacteria or viruses, helminths are complex multicellular organisms and cannot reproduce quickly enough to overwhelm the mammalian or human immune system, so worms rely instead on down-regulating the host immune response. The worm infection-reaction, and subsequent human immune response to worm infection, is extraordinarily complex and involves, among other things, but not limited to, production of eosinophils (along with other agents such as immunoglobulin E and IgG₄), worm products that alter or interfere with epithelial cell alarmins and dendritic cell activation, macrophage function, T-and B- cell responsiveness via promotion of an immunoregulatory environment, i.e. upregulation of CD4+ Tregs (abolished by Treg depletion), chemokine, cytokine, enzyme and metabolite production with enzymes such as glutamate dehydrogenase, eicosanoid function, lipoxygenase and cyclooxygenase pathways, prostaglandins, prostacyclins, neutrophil and granulocyte function, mast cells, plasma cells, and many other immune factors. It is widely believed that the eosinophil cell evolved in humans and other mammals in large part to fight worm infections (although eosinophils can also be present in non-worm disease states such as allergies and eosinophilic esophagitis). During worm infections, the number of eosinophils in the blood increases dramatically from chemical messengers such as Th2-derived IL-4, IL-5, IL-13 and GM-CSF, and eosinophils move from the circulation into worm-infected tissues from eotaxin signaling. Eotaxin is a potent eosinophil chemoattractant, acting through the cell receptor, CCR3. Eotaxin acts in concert with IL-5, to stimulate the release of eosinophils from bone marrow and to recruit eosinophils to sites of inflammation (worm or otherwise).

Just as the human eosinophilic immune response has evolved to combat worm infestation, in turn, the helminths have evolved to ward off said response in a complex co-evolution sort of arms race. Helminths, in all forms of development, from eggs, to free-living larval stages L1-L3, tissue migratory stage, L4, and juvenile or adult stage,

L5, harbor and secrete specific substances and molecules to help evade or thwart the human eosinophil-associated immune response; these substances help worms survive in humans in vivo. In fact, helminths typically induce chronic infections in humans that may last months or even years, despite an active human response. Helminth-derived products harbor specific components that lead to up- or down regulation of inflammatory cytokines such as the non-limiting examples of IL-4, IL-5, L-l, IL-12, IL-18, TNF-alpha, interferon gamma, and GM-CSF; up-regulation of anti-inflammatory cytokines such as the non-limiting examples of IL-10, IL-4, IL-11, and IL-13; alterations in other immune modulators such as chemicals, cells etc., that can be impacted; or down regulation of eosinophil release, eosinophil recruitment, and/or the eosinophil allergic response. This allows the worms to evade host immune responses in certain circumstances. For example, certain proteins from Toxascaris leonina inhibit the general human Th2 immune response, Anisakis simplex manufactures proteins that inhibit the eosinophil-mediated immune response, Heligmosomoides polygrus down regulates eotaxin and CCR3 expression, Toxocara canis secretes proteins that impair eosinophils, P. westermani manufactures proteases that attenuate the effective function of eosinophils, and the liver fluke F hepatica secretes proteinases that prevent eosinophil antibody-mediated attachment to cells. Additional non-limiting examples of helminth-derived products include, metalloproteases, e.g., secreted by certain worms such as the hookworm,

Necator americanus , that can cleave eotaxin and prevent its function in recruiting eosinophils; cystatin, e.g., a product of Acanthocheilonema viteae , that can inhibit IL-4 and promote IL-10; and a venom, e.g., secreted by Steinernema carpocapsae , that can down-regulate the host immune system.

The liver fluke Fasciola hepatica and eggs from S. mansoni can suppress the Thl/Thl7 mediated autoimmunity. Multiple helminth species can inhibit the development of allergic reactions, like H. polygyrus , S. mansoni , L sigmodontis , the pig whipworms Trichurus trichiura , and Trichuris suis, and various other hookworms and roundworms.

In addition, various worm infestations, for example with H. polygyrus can cause alterations in the microbiome with altered unfavorable or favorable immune effects. Some worms, such as H. polygyrus , H. muris or N brasiliensis can secrete Hp-ARI (alarmin release inhibitor), Hp-TGM, block IL-33, secrete glutamate dehydrogenase and have other immune effects. H. polygyrus glutamate dehydrogenase can have important immune effects in macrophages, in eicosanoid function and varied other cellular and chemical arms of the immune response. Other hookworms such as A. duodenale , can produce substances and some hookworms can produce HIP-2 with important immune effects.

Thus, as noted, helminthic worms have evolved numerous and complex mechanisms and substances to affect and attenuate mammalian and human immune responses, more specifically, affecting eosinophils, eosinophil recruitment and the eosinophil-mediated tissue inflammatory response. Thus, helminthic-human immune responses can be manipulated to treat human diseases manifest by an abnormal or excess eosinophilic immune response such as eosinophilic esophagitis or other eosinophilic disorders such as, eosinophilic gastritis, eosinophilic colitis, asthma, or other human allergic diseases. Helminths, helminth eggs, or helminth larvae, or associated components, extracts or helminth-associated substances, can be purposely introduced into humans, or other mammals, afflicted with certain allergic or eosinophilic-associated diseases, for the express purpose of altering, affecting, changing, or down-regulating the human or mammalian immune response, to favorably impact or affect said disease.

In some embodiments, a helminthic composition is provided. In some embodiments, a method for treating and/or preventing an eosinophilic disorder in a mammal is provided. The method can include administering the helminthic composition to the mammal (e.g., a human). The helminthic composition can include a helminth organism or a portion thereof. The helminthic composition can include a juvenile helminth organism and/or an adult helminth organism. The helminth organism can be viable, attenuated, or non- viable. The composition can include from about 10 to about 1000 adult or juvenile helminth organisms. The helminthic composition can include helminth eggs (e.g., from about 50 to about 10,000 eggs) and/or helminth larvae (e.g., from about 5 to about 2500 helminth larvae). The eggs and/or larvae can be viable or non-viable. The larvae can be at stage LI, L2, L3, L4, L5, or a combination thereof. For example, the larvae can be at stage L3. The helminthic composition can be from a human pathogen or can be from a helminth that is non-pathogenic to a human. The helminthic composition can include a helminth-derived product or helminthic extract from a helminth organism, helminth eggs, and/or helminth larvae (e.g., the helminth-derived product or helminthic extract can include tegument or cuticle, or any fragment or portion of the tegument or cuticle, or can include cell membrane or any fragment or portion of the cell membrane). The helminthic composition can include supernatant, filtrate, a cytoplasmic product, one or more proteins, one or more carbohydrates, or any other portion or product of the worm, larvae or egg, either as an intrinsic part of the physical structure of the organism, larvae and egg, or as any product produced internally or secreted or emitted externally to the organism, larvae or egg itself.

In some embodiments, the helminth-derived product or extract can include a helminth enzyme or combination of different helminth enzymes. In some embodiments, the enzyme can be a protease (e.g., a metalloprotease, a cysteine protease, an aspartic protease, or a serine protease). For example, the helminthic composition can include a helminth metalloprotease such as a Necator americanus metalloprotease. The metalloprotease can inhibit, cleave, or retard the function of eotaxin. In some embodiments, the helminthic composition can include a helminth cystatin (e.g., cystatin C, E, or S). The cystatin can be a filarial nematode cystatin (e.g., from Acanthocheilonema viteae). The helminthic composition can include a glutamate dehydrogenase.

In some embodiments, the helminthic composition includes a roundworm (nematode) composition. For example, the roundworm composition can be selected from the group consisting of a Trichuris suis, Trichuris trichiuris , Toxascaris leonina , Anisakis simplex , Heligomosomoides polygyrus , Heligomosomoides polygyrus bakeri , Toxocara canis, Necator americanus , A. duodenale , Acanthocheilonema viteae , L. sigmodontis , H. muris , and N brasiliensis composition.

In some embodiments, the helminthic composition includes a fluke (trematode) composition. For example, the fluke composition can be selected from the group consisting of Fasciola hepatica and Schistosoma mansoni. In some embodiments, the helminthic composition includes a tapeworm (cestode) composition. For example, the tapeworm composition can be selected from the group consisting of Taenia solium , Taenia saginata, Diphyllobotherium latum , I) nihonkaiense , I) dendriticum , and Hymenolepsis nana.

In any of the embodiments, the helminthic composition can alter, affect, change, or manipulate the immune response of said mammal. For example, production of one or more cytokines (one or more of IL-4, IL-5, IL-10, and IL-13) can be altered in the mammal. For example, immunoglobulin production can be altered. For example, eosinophil immune response can be altered in the mammal (e.g., circulating eosinophil numbers, eosinophil recruitment, eosinophil function, eosinophil bone marrow production, eosinophil tissue function, and/or eosinophil migration can be altered in the mammal).

In any of the embodiments, the helminthic composition can be administered orally, trans-dermally, topically, intravenously, trans-dermally, subcutaneously, intramuscularly, sublingually, trans-nasally or by inhalation.

In any of the embodiments, the helminth composition can be a formulation, substance, pill, capsule, liquid, semi-solid, solid, drink, ointment, paste, cream, lotion, emollient, compound, soap, powder, crystal, effervescence, foam, enema, injection, infusion, inhalant, aerosol, or any other type of product to be delivered internally or externally to said patient to treat and/or prevent said eosinophilic disease.

In some embodiments, the composition can include two or more different types of helminths and/or eggs, larvae, or extracts from two or more different types of helminths.

In some embodiments, the composition includes H. muris and/or// polygyrus helminths, eggs, larvae, and/or extracts.

In some embodiments, the composition includes a component, a secretion, an extract, or a product of H. muris or H. polygyrus or their eggs or larvae.

In some embodiments, the composition includes a H. polygyrus glutamate dehydrogenase.

In any of the embodiments, the eosinophilic disorder can be eosinophilic esophagitis, eosinophilic gastritis, eosinophilic colitis, asthma, atopic dermatitis, chronic rhino sinusitis, or any human or mammalian allergic disorder that involves an eosinophil immune response.

This document also features a method for treating a patient in need thereof. The method includes determining if the patient has an eosinophilic disorder based on one or more diagnostic criteria, and administering a helminthic composition to the patient identified as having the eosinophilic disorder. The eosinophilic disorder can be eosinophilic esophagitis, eosinophilic gastritis, eosinophilic colitis, asthma, atopic dermatitis, chronic rhinosinusitis, or any human or mammalian allergic disorder that involves an eosinophil immune response. The eosinophilic disorder can be eosinophilic esophagitis. The diagnostic criteria can include one or more of tissue eosinophilia, dysphagia, food impaction, and endoscopic findings. The method further can include monitoring the patient for improvement in one or more diagnostic criteria. The method further can inlcude administering an adjunct therapy to the patient identified as having the eosinophilic disorder. The adjunct therapy can be selected from the group consisting of pain medication, a steroid, a proton pump inhibitor (PPI), or an immune modulator.

In another aspect, this document features use of a helminthic composition described herein in the preparation of a medicament for the treatment and/or prevention of an eosinophilic disorder such as eosinophilic esophagitis, eosinophilic gastritis, eosinophilic colitis, asthma, atopic dermatitis, chronic rhinosinusitis, or any human or mammalian allergic disorder that involves an eosinophil immune response.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used he materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DETAILED DESCRIPTION

This document provides materials and methods for treating or preventing an eosinophilic disorder using helminths (worms), or components or products of helminths. As described herein, suitable helminth compositions can include any portion or derivative of any helminth, including but not limited to, an entire organism (including, viable, attenuated or non- viable organisms, of juvenile or adult helminths), larvae from stages 1- 5 (viable or non-viable larvae, infective or non-infective larvae, from stages LI, L2, L3, L4, or L5, ), eggs of the organism (including viable or non-viable eggs), any helminth- derived product (e.g., any product secreted or excreted from a helminth), an extract of helminths, larvae, or eggs (e.g., an extract that includes one or more components found in the supernatant and/or filtrate and/or pellet after mechanical homogenization and/or biochemical extraction of helminths, larvae, or eggs), or any other portion or product of the worm either as an intrinsic part of the physical structure or the worm or as any product produced internally or secreted or emitted externally to the worm itself. For example, an extract can include any portion of a helminth organism, larva, or egg, including, for example, the tegument or cuticle, or any fragment or portion of the tegument or cuticle; the cell membrane or any fragment or portion of the cell membrane; any cell product or cytoplasmic product, including carbohydrates, proteins such as cystatin, alarmin release inhibitor, or TGF-b Mimic (TGM), or enzymes such as a protease (e.g., a metalloprotease, cysteine protease, aspartic protease, or serine protease) or a glutamate dehydrogenase. The extracts can be cell free extracts.

Any eosinophilic disorder characterized by abnormal or excessive eosinophil activity can be treated using a helminthic composition described herein, including eosinophilic esophagitis, eosinophilic gastritis, eosinophilic colitis, asthma, atopic dermatitis, chronic rhinosinusitis, or asthma. For example, the methods can be used to treat eosinophilic esophagitis, eosinophilic gastritis, or eosinophilic colitis. In addition, any appropriate mammal can be treated as described herein, including humans, monkeys, dogs, horses, sheep, pigs, goats, rabbits, rats or mice.

Treatments as described herein can be effective to reduce one or more symptoms of an eosinophilic disorder in a mammal (e.g., a human). For example, treatments as described herein can be administered to mammal identified as having an eosinophilic disorder (e.g., a human patient), wherein a symptom of the eosinophilic disorder is reduced in the mammal after treatment. For example, for eosinophilic esophagitis, treatment with a helminthic composition can be used to reduce inflammation (e.g., reduce exudate, edema of the mucosa and/or wall of the esophagus), or improve dysphagia (swallowing), in the mammal. Without being bound to a particular mechanism, helminthic compositions described herein can improve one or more symptoms of eosinophilic disorders by, for example, altering the immune response of the mammal, e.g., by altering production of one or more cytokines (e.g., one or more of IL-4, IL-5, IL- 10, and IL-13), altering production of immunoglobulins (e.g., immunoglobulin E and IgG₄), or altering the eosinophil immune response (e.g., by altering circulating eosinophil numbers, eosinophil recruitment, eosinophil function, eosinophil bone marrow production, eosinophil tissue function, and/or eosinophil migration). In some embodiments, one or more symptoms of an eosinophilic disorder can be improved by reducing eosinophilic infiltration, reducing the number of eosinophils, inhibiting eosinophil recruitment, or inhibiting eosinophil function.

In any of the compositions described herein, the helminthic composition can be from a helminth that naturally colonizes humans or can be from a helminth that naturally colonizes a non-human animal, but can be used to alter an immune response in a human. In some embodiments, the helminthic composition can be from a human pathogen or can be from a non-pathogenic helminth. In some embodiments, the helminth composition can be from a symbiont, a colonizer, a helminth that transiently passes through a human, a helminth that completes its lifecycle in a human, a helminth that completes part of its lifecycle in a human. In some embodiments, the helminthic composition can be from a helminth that naturally colonizes a non-human animal, and such a helminth can be pathogenic or non-pathogenic, a colonizer, or a symbiont in any mammalian species. For example, in some embodiments, a helminthic composition used in the methods described herein can be a nematode (roundworm) composition. For example, the roundworm composition can be from one or more genera selected from Trichuris , Toxascaris, Anisakis, Heligomosomoides , Toxocara, Ancylostoma, Necator, Ascaris, Enterobius , Litomosoides , Nippostrongylus , Strongyloides, Trichinella , and Acanthocheilonema , In some cases, a roundworm composition can be a whipworm composition (e.g., Trichuris suis (pig whipworm), Trichuris trichiuris (human whipworm), or Trichuris muris (mouse whipworm)). In some cases, a roundworm composition can be a Toxascaris leonina , Anisakis simplex , Heligomosomoides polygyrus , Heligomosomoides polygyrus baker i, Toxocara canis, Necator americanus , Ascaris lumbricoides , Enterobius vermicularis , Ancylostoma duodenale , Acanthocheilonema viteae , Litomosoides sigmodontis , Strongyloides stercoralis , Trichinella spiralis , or Nippostrongylus brasiliensis (also known as Heligmosomum muris and Nippostrongylus muris) composition.

In some embodiments, a helminthic composition used in the methods can be a platyhelminth composition (e.g., atrematode or a cestode composition). In some embodiments, a helminthic composition used in the methods described herein can be a trematode (fluke) composition. For example, a fluke composition can be from one or more genera selected from Fasciola and Schistosoma. In some embodiments, a fluke composition can be a Fasciola hepatica or a Schistosoma mansoni composition.

In some embodiments, a helminthic composition used in the methods described herein is a cestode (tapeworm) composition. For example, a tapeworm composition can be from one or more genera selected from Taenia, Diphyllobotherium , or Hymenolepsis. In some cases, a tapeworm composition can be a Taenia solium , Taenia saginata, Diphyllobotherium latum , I) nihonkaiense , /A dendriticum , or Hymenolepsis nana composition.

In some embodiments, a helminth composition can include a combination of two or more different adult helminths, larvae, eggs, and/or extracts from different helminths (e.g., two or more different genuses of helminths or two or more different species of helminths). For example, in some embodiments, a helminthic composition can include a adult roundworms and/or larvae or eggs from a flatworm.

Methods for isolating, maintaining, and propagating helminths, larvae, and eggs can be found, for example, in Cambris et al, Current Protocols in Immunology (2003) 19.12.1-19.12.27. For example, viable helminths, larvae, and/or eggs can be isolated from a host animal (e.g., a mammal such as a rat, a mouse, a hamster, a guinea pig, a goat, a pig, a dog, a cat, a ferret, or a weasel) such as a host animal raised in a specific pathogen- free environment. To improve yield of the helminths, a host animal can be treated with an immunosuppressant. Depending on the helminth and its life cycle, an intermediate host animal also can be used for maintaining and propagating helminths, larvae, and eggs. In embodiments in which attenuated helminths are used, the isolated helminths can be attenuated using, for example, radiation or chemical adjuvants such as soaps, oils, bacterial factors, natural predators, or heat or cold. Helminths, eggs, and/or larvae can be harvested from fecal matter (e.g., by culturing fecal pellets for about one to three weeks in a dark, humid environment), the intestines, livers, or lungs of an infected host animal, depending on the helminth. Harvested helminths, eggs, and/or larvae can be repeatedly washed in buffer (e.g., phosphate buffered saline) to remove debris. Larvae can be isolated at the desired stage (e.g., L3 for N brasiliensis). In some embodiments, viable helminths, eggs, and/or larvae can be used. In some embodiments, non-viable helminths, eggs and/or larvae can be used. Non-viable helminths, eggs, and/or larvae can be produced, for example, by cryopreserving in the absence of cryoprotectants such as dimethylsulfoxide (DMSO) and glucose or trehalose.

Helminth-derived products (e.g., any product secreted or excreted from a helminth) can be obtained by culturing helminths, larvae, or eggs and isolating the products from the medium. For example, helminths, larvae, or eggs can be cultured for one to five days at 37°C in a medium such as RPMI medium (e.g., from Life Technologies) containing glucose (e.g., 1% w/v) and glutamine, and optionally containing one or more antibiotics. The supernatant from the culture can be collected and filtered through a membrane, and concentrated (e.g., by ultrafiltration). Protein concentration can be assessed for preparing formulations. In some embodiments, helminth-derived products can be obtained from helminths or larvae by homogenizing the helminths or larvae (e.g., mechanically homogenizing the helminths or larvae in buffer).

Extracts of helminths, larvae, and/or eggs (e.g., cell free extracts) can be produced by any method, including, for example, mechanical homogenization, osmotic shock, sonication, and/or a repeated freeze-thaw cycle followed by filtration and/or centrifugation. In some embodiments, the helminth-derived products or extracts can be from, for example, infective larvae harvested from naturally infected mammals such as pigs, mice or rats, or organs of mammals. In some embodiments, the helminth-derived products or extracts can be from, for example, Heligmosomoides polygyrus bakeri, H. muris , or H. polygyrus helminths, eggs, larvae (e.g., infective larvae), and/or extracts.

In some embodiments, helminth-derived products or extracts can be subjected to one or more purification techniques such as gel filtration, size exclusion chromatography, anion exchange chromatography, and/or affinity chromatography, and the resulting products can be analyzed, for example, by liquid chromatography and mass spectrometry. Protein containing fractions can be assessed in vitro to determine if the protein fraction alters an immune response, e.g., increases or decreases cytokine production, or proteolyzes a chemokine.

For example, a helminth-derived product or extract can include an alarmin release inhibitor, a 26 kDa protein that contains three predicted complement control protein modules, and that can bind to IL-33 and prevent its release, suppressing type 2 immune responses. See, Osbourn, et al, Immunity, 47(4):739-751 (2017). The amino acid sequence of the alarmin release inhibitor is found in the WormBase Parasite database under Accession No. HPBE 0000813301.

For example, a helminth-derived product or extract can include a helminth cystatin (a cysteine protease inhibitor), e.g., a type 1 cystatin or stefins (cystatin A and B), a type 2 cystatin (cystatin C, E, or S), or a type 3 cystatin or kininogen. For example, a cystatin can be from a cestode or nematode, such as the roundworm Ascaris lumbricoides , or a filarial nematode, Acanthocheilonema viteae). Cystatins can range in size from about 100 amino acids (type 1) or 120 amino acids (type 2) in length to about 350 amino acids (type 3) in length. For example, Type 2 cystatins can interfere with antigen presentation by reducing surface expression of HLA-DR, CD40, and CD86 co stimulatory molecules on human monocytes. Cystatins also can increase nitric oxide production, induce anti-inflammatory cytokines such as IL-10 and TGF-b, and decrease expression or production of IFN-g, TNF-a, IL-5, IL-6, and IL-17. See, e.g., Khatri, et al. , Pathogens , 9(6):431 (2020); and Coronado, et al, Front Immunol., 10:2280 (2019).

For example, a helminth-derived product or extract (e.g., from// polygyrus ) can include a TGF-b Mimic (TGM). The TGM from// polygyrus is a 404 amino acid secreted protein that can bind to TGF-b receptors and activate signaling, inducingFoxp3⁺ immunosuppressive regulatory (Treg) cells. See, e.g., Johnston, et al, Nat. Commun ., 8:1741 (2017).

For example, a helminth-derived product or extract can include a metalloprotease such as a metalloprotease that can inhibit, cleave or retard the function of eotaxin, an eosinophil chemoattractant. Helminthic metalloproteases range in size from about 15 to 50 kDa. In some embodiments, the metalloprotease can be a N. americanus metalloprotease. See, e.g., Culley, etal., J. Immunol., 165(ll):6447-6453 (2000).

For example, a helminth-derived product or extract can include a glutamate dehydrogenase, e.g. a glutamate dehydrogenase from// polygyrus or H. polygyrus bakeri that induces production of anti-inflammatory cytokines such as IL-10 and IL-Ib. The amino acid sequence of a glutamate dehydrogenase from H. polygyrus bakeri can be found under UniProtKB Accession Number A0A183FP08.

This document also provides methods for administering a helminthic composition to a mammal (e.g., a human). For example, a helminthic composition provided herein can be administered to a mammal (e.g., a human) in need thereof to treat an eosinophilic disorder, disease, or condition. For example, it can be determined if a patient has an eosinophilic disorder based on one or more diagnostic criteria (e.g., one or more of tissue eosinophilia such as 15/eosinophils per high-power microscopic field in esophageal tissue, dysphagia, food impaction, and endoscopic findings such as rings, furrows and exudate), and then a helminthic composition can be administered to the patient identified as having the eosinophilic disorder. Any appropriate method can be used to administer a helminthic composition provided herein to a mammal (e.g., a human). For example, a helminthic composition provided herein can be administered to a mammal (e.g., a human) orally, trans-dermally, intravenously (e.g., via an intravenous injection or infusion), subcutaneously (e.g., via a subcutaneous injection), intraperitoneally (e.g., via an intraperitoneal injection), intranasally, via inhalation, or intramuscularly (e.g., via intramuscular injection). In some embodiments, the helminthic composition is in a food (e.g., a drink, shake, or smoothie) that is ingested by the mammal (e.g., a human). In some cases, the route and/or mode of administration of a helminthic composition can be adjusted for the mammal being treated and the type of helminth or helminth product within the composition. The patient can be monitored for improvement in one or more diagnostic criteria (e.g., reduced number of eosinophils in tissue, improved swallowing, and/or less food impactions).

A helminthic composition provided herein can be in any appropriate form. For example, a helminthic composition provided herein can be formulated as a liquid, a semi solid, or a solid. In some embodiments, a helminthic composition provided herein can be formulated as a liquid solution (e.g., an injectable and/or infusible solution), a dispersion, a suspension, a tablet, a pill, a capsule, a powder, a microemulsion, a liposome, a foam, or a suppository. In some embodiments, a helminthic composition can be formulated as a drink, ointment, paste, cream, lotion, emollient, a soap, powder, crystal, effervescence, enema, or any other type of product that can be delivered internally or externally to a patient to treat and/or prevent an eosinophilic disorder. In some embodiments, a helminthic composition provided herein can be lyophilized. In some embodiments, a helminthic composition provided herein can be formulated with a carrier or coating designed to protect against rapid release. For example, a helminthic composition provided herein can be formulated as a controlled release formulation. See, for example, Bayer, Molecules, 25(11):2649 (2020); and Tran and Tran, Pharmaceutics , 11(6):290 (2019).

In some embodiments, a helminthic composition provided herein can include a pharmaceutically acceptable carrier such as a buffer, a salt, a surfactant, a sugar, a tonicity modifier, or combinations thereof. See, for example, Gervasi, el al ., Eur. ./. Pharmaceutics and Biopharmaceutics, Volume 131, pages 8-24 (2018); and Jain, et al.,. Drug Dev. Ind. Pharm ., 45(9):1403-1420 (2019). Examples of pharmaceutically acceptable carriers that can be used to make a pharmaceutical composition provided herein include, without limitation, water, lactic acid, citric acid, sodium chloride, sodium citrate, sodium succinate, sodium phosphate, a surfactant (e.g., polysorbate 20, polysorbate 80, or poloxamer 188), dextran 40, or a sugar (e.g., sorbitol, mannitol, sucrose, dextrose, or trehalose), or combinations thereof. For example, a helminthic composition provided herein can be formulated to include a buffer (e.g., an acetate, citrate, histidine, succinate, phosphate, or hydroxymethylaminomethane (Tris) buffer), a surfactant (e.g., polysorbate 20, polysorbate 80, or poloxamer 188), and a sugar such as sucrose. Other ingredients that can be included within a helminthic composition can include, without limitation, an amino acid such as glycine or arginine, or an antioxidant such as ascorbic acid, methionine, or ethylenediaminetetraacetic acid (EDTA) or combinations thereof.

Effective amounts of a helminthic composition can vary depending on the severity of the disorder, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents, and the judgment of the treating physician.

A helminthic composition can include an amount of helminth, larvae, eggs, helminth-derived product or extract that is effective to reduce the excess eosinophil activity within a mammal without producing significant toxicity to the mammal.

For example, when a helminthic composition includes an entire organism (e.g., live, attenuated or non-viable organisms, of juvenile or adult helminths), the composition can include from about 10 to about 1000 helminths (e.g., 10 to 950, 50 to 900, 50 to 750, 50 to 500, 50 to 400, 50 to 350, 50 to 300, 50 to 250, 50 to 200, 100 to 850, 150 to 800, 200 to 750, 200 to 700, 200 to 650, 200 to 600, 250 to 550, 250 to 500, 250 to 700, 300 to 650, 350 to 600, 400 to 550, 400 to 500, 500 to 1000, 550 to 950, 600 to 900, 650 to 850, or 700 to 800 helminths).

For example, when a helminthic composition includes larvae, e.g., from stages 1- 5 (LI, L2, L3, L4, or L5), including viable or non-viable larvae, the composition can include from about 5 to about 2500 larvae (e.g., about 5 to 2400, 5 to 1000, 5 to 100, 10 to 90, 20 to 80, 10 to 2300, 25 to 2250, 50 to 2500, 50 to 2400, 50 to 2300, 50 to 2200, 50 to 2100, 50 to 2000, 50 to 1900, 50 to 1800, 100 to 1700, 100 to 1600, 100 to 1500, 100 to 1400, 100 to 1300, 100 to 1200, 100 to 1100, 100 to 1000, 200 to 900, 200 to 850, 200 to 800, 200 to 750, 200 to 700, 200 to 650, 200 to 600, 200 to 550, 300 to 2400, 400 to 2400, 500 to 2400, 600 to 2400, 700 to 2400, 800 to 2400, 900 to 2400, 1000 to 2400,

1100 to 2400, 1000 to 2500, 1500 to 2500, 1750 to 2500, or 2000 to 2500 larvae).

For example, when a helminthic composition include eggs (including viable or non- viable eggs), the composition can include from about 50 to about 10,000 eggs (e.g., 50 to 100, 75 to 125, 100 to 150, 125 to 175, 150 to 225, 200 to 300, 250 to 350, 300 to 400, 350 to 450, 400 to 500, 450 to 550, 500 to 600, 650 to 750, 700 to 800, 750 to 850, 800 to 900, 950 to 1050, 1000 to 10000, 1000 to 9000, 1100 to 8000, 1200 to 7000, 1300 to 6000, 1400 to 5000, 1500 to 4000, 2000 to 3000, 3000 to 10000, 4000 to 10000, 5000 to 10000, 6000 to 10000, 7000 to 1000, 8000 to 10000, or 9000 tolOOOO eggs).

For example, when a helminthic composition includes a helminth-derived product or extract, the composition can include from about 0.5 mg to about 500 mg (e.g., from about 1 mg to about 500 mg, from about 10 mg to about 500 mg, from about 50 mg to about 500 mg, from about 100 mg to about 500 mg, from about 0.5 mg to about 250 mg, from about 0.5 mg to about 150 mg, from about 0.5 mg to about 100 mg, from about 0.5 mg to about 50 mg, from about 1 mg to about 300 mg, from about 2 mg to about 200 mg, from about 10 mg to about 300 mg, from about 25 mg to about 300 mg, from about 50 mg to about 150 mg, or from about 150 mg to about 300 mg) of the helminth-derived product or extract. In some embodiments, when a helminthic composition includes a helminth-derived product or extract, the composition can include from about 0.01 to about 30 mg/kg (e.g., about 0.01 to about 25 mg/kg, about 0.1 to about 30 mg/kg, about 0.15 to about 25 mg/kg, about 0.2 to about 20 mg/kg, about 0.5 to about 20 mg/kg, about 1 to about 30 mg/kg, about 1 to about 25 mg/kg, about 1 to about 20 mg/kg, about 2 to about 20 mg/kg, about 5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, or about 3 to about 30 mg/kg) of the helminth-derived product or extract. The effective amount of helminths, eggs, larvae, helminth-derived products, or extract can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition may require an increase or decrease in the actual effective amount administered.

The frequency of administration of helminthic composition can be any amount that reduces symptoms of excess eosinophil activity within a mammal without producing significant toxicity to the mammal. For example, the frequency of administration can be from about once daily, once a week, once every two weeks, or once every three weeks for up to about 52 weeks. The frequency of administration can remain constant or can be variable during the duration of treatment. A course of treatment with a helminthic composition can include rest periods. For example, a helminthic composition can be administered once a week for three weeks, followed by a one to three week rest period, and such a regimen can be repeated multiple times. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition may require an increase or decrease in administration frequency.

An effective duration for administering a helminthic composition can be any appropriate duration that reduces the excess eosinophilic activity within a mammal without producing significant toxicity to the mammal. In some cases, the effective duration can vary from several weeks to several years (e.g., three weeks to three or more years). Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, and severity of the condition being treated.

In some cases, a helminthic composition described herein can be used in combination with adjunct therapies such as medications to control pain, steroids, proton pump inhibitors (PPIs) (e.g., esomeprazole, omeprazole, or lansoprazole), other drugs, or immune modulators. The helminthic composition and other treatments can be administered together (e.g., formulated together), or the helminthic composition can be administered concurrently with, prior to, or subsequent to, one or more other treatments (e.g., medications to control pain, steroids, PPIs such as esomeprazole, omeprazole, or lansoprazole, other drugs, or immune modulators). The particular combination of therapies to employ in a combination regimen will take into account compatibility of the desired therapeutics and the desired therapeutic effect to be achieved. It also will be appreciated that the therapies employed may achieve a desired effect for the same disorder or they may achieve different effects (e.g., control of any adverse effects).

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for treating and/or preventing an eosinophilic disorder in a mammal, said method comprising administering a helminthic composition to said mammal.

2. The method of claim 1, wherein said mammal is a human.

3. The method of claim 1 or claim 2, wherein said helminthic composition comprises a helminth organism or a portion thereof.

4. The method of claim 3, wherein said helminthic composition comprises a juvenile helminth organism.

5. The method of claim 3, wherein said helminthic composition comprises an adult helminth organism.

6. The method of claim 5, wherein said composition comprises from about 10 to about 1000 adult or juvenile helminth organisms.

7. The method of any one of claims 3-6, wherein said helminth organism is a viable organism.

8. The method of any one of claims 3-6, wherein said helminth organism is an attenuated organism.

9. The method of any one of claims 3-6, wherein said helminth organism is non-viable organism.

10. The method of claim 1 or claim 2, wherein said helminthic composition comprises helminth eggs.

11. The method of claim 7, wherein said helminthic composition comprises from about 50 to about 10,000 eggs.

12. The method of claim 10 or claim 11, wherein said eggs are viable.

13. The method of claim 10 or claim 11, wherein said eggs are non-viable.

14. The method of claim 1 or claim 2, wherein said helminthic composition comprises helminth larvae.

15. The method of claim 14, wherein said helminth larvae are at stage LI, L2, L3, L4, L5, or combinations thereof.

16. The method of claim 14, wherein said helminth larvae are at stage L3.

17. The method of any one of claims 14-16, wherein said helminthic composition comprises from about 5 to about 2500 helminth larvae.

18. The method of any one of claims 14-17, wherein said larvae are viable.

19. The method of any one of claims 14-17, wherein said larvae are non-viable.

20. The method of any one of claims 1-19, wherein said helminthic composition is from a human pathogen.

21. The method of any one of claims 1-19, wherein said helminthic composition is non- pathogenic to a human.

22. The method of any one of claims 1-21, wherein said helminthic composition comprises a helminth-derived product or helminthic extract from a helminth organism, helminth eggs, and/or helminth larvae.

23. The method of claim 22, wherein said helminth-derived product or helminthic extract comprises tegument or cuticle, or any fragment or portion of the tegument or cuticle.

24. The method of claim 22, wherein said helminthic extract comprises cell membrane or any fragment or portion of the cell membrane.

25. The method of any one of claims 1-24, wherein said helminthic composition comprises supernatant, filtrate, a cytoplasmic product, one or more proteins, one or more carbohydrates, or any other portion or product of the worm, larvae or egg, either as an intrinsic part of the physical structure of the organism, larvae and egg, or as any product produced internally or secreted or emitted externally to the organism, larvae or egg itself.

26. The method of any one of claims 22-25, wherein said helminth-derived product or extract comprises a helminth enzyme or combination of different helminth enzymes.

27. The method of claim 26, wherein said enzyme is a protease.

28. The method of claim 27, wherein said protease is a metalloprotease, a cysteine protease, an aspartic protease, or a serine protease.

29. The method of claim 28, wherein said helminthic composition comprises a helminth metalloprotease.

30. The method of claim 29 wherein said metalloprotease is a Necator americanus metalloprotease.

31. The method of claims 29 or claim 30, wherein said metalloprotease inhibits, cleaves or retards the function of eotaxin.

32. The method of any one of claims 22-31, wherein said helminthic composition comprises a helminth cystatin.

33. The method of claim 32, wherein said helminth cystatin is Cystatin C, E, or S.

34. The method of claim 32 or claim 33, wherein said helminth cystatin is a filarial nematode cystatin.

35. The method of claim 34, wherein said filarial nematode is Acanthocheilonema viteae.

36. The method of any one of claims 22-35, wherein said helminthic composition comprises a glutamate dehydrogenase.

37. The method of any one of claims 1-36, wherein said helminthic composition comprises a roundworm (nematode) composition.

38. The method of claim 37, wherein said roundworm composition is selected from the group consisting of a Trichuris suis, Trichuris trichiuris , Toxascaris leonina , Anisakis simplex , Heligomosomoides polygyrus , Heligomosomoides polygyrus baker i, Toxocara canis, Necator americanus , A. duodenale , Acanthocheilonema viteae , L. sigmodontis , H. muris , and N brasiliensis composition.

39. The method of any one of claims 1-38, wherein said helminthic composition comprises a fluke (trematode) composition.

40. The method of claim 39, wherein said fluke composition is selected from the group consisting of Fasciola hepatica and Schistosoma mansoni.

41. The method of any one of claims 1-40, wherein said helminthic composition comprises a tapeworm (cestode) composition.

42. The method of claim 41, wherein said tapeworm composition is selected from the group consisting of Taenia solium , Taenia saginata, Diphyllobotherium latum , I) nihonkaiense , I) dendriticum , and Hymenolepsis nana.

43. The method of any one of claims 1-42, wherein said helminthic composition alters, affects, changes, or manipulates the immune response of said mammal.

44. The method of claim 43, wherein production of one or more cytokines are altered in said mammal.

45. The method of claim 44, wherein said cytokines are selected from the group consisting of IL-4, IL-5, IL-10, and IL-13.

46. The method of any one of claims 41-45, wherein immunoglobulin production is altered.

47. The method of any one of claims 43-46, wherein said eosinophil immune response is altered in said mammal.

48. The method of any one of claims 1-47, wherein circulating eosinophil numbers, eosinophil recruitment, eosinophil function, eosinophil bone marrow production, eosinophil tissue function, and/or eosinophil migration is altered in said mammal.

49. The method of any one of claims 1-48, wherein said helminthic composition is administered topically, intravenously, trans-dermally, subcutaneously, intramuscularly, sublingually, trans-nasally or by inhalation.

50. The method of any one of claim 1-48, wherein said helminthic composition is administered orally.

51. The method of any one of claims 1-48, wherein said helminthic composition is administered trans-dermally.

52. The method of any one of claims 1-48, wherein said helminthic composition is administered topically.

53. The method of any one of claims 1-52, wherein said helminth composition is a formulation, substance, pill, capsule, liquid, semi-solid, solid, drink, ointment, paste, cream, lotion, emollient, compound, soap, powder, crystal, effervescence, foam, enema, injection, infusion, inhalant, aerosol, or any other type of product to be delivered internally or externally to said patient to treat and/or prevent said eosinophilic disease.

54. The method of any one of claims 1-53, wherein said composition comprises two or more different types of helminths and/or eggs, larvae, or extracts from two or more different types of helminths.

55. The method of any one of claims 1-54, wherein said composition comprises H. muris and/or H. polygyrus helminths, eggs, larvae, and/or extracts.

56. The method of claim 55, wherein said composition comprises a component, a secretion, an extract, or a product of H. muris or H. polygyrus or their eggs or larvae.

57. The method of claim 55 or claim 56, wherein said composition comprises a H. polygyrus glutamate dehydrogenase.

58. The method of any one of claims 1-57, wherein said eosinophilic disorder is eosinophilic esophagitis, eosinophilic gastritis, eosinophilic colitis, asthma, atopic dermatitis, chronic rhinosinusitis, or any human or mammalian allergic disorder that involves an eosinophil immune response.

59. A method for treating a patient in need thereof, said method comprising a) determining if said patient has an eosinophilic disorder based on one or more diagnostic criteria, and b) administering a helminthic composition to said patient identified as having said eosinophilic disorder.

60. The method of claim 59, wherein said eosinophilic disorder is eosinophilic esophagitis, eosinophilic gastritis, eosinophilic colitis, asthma, atopic dermatitis, chronic rhinosinusitis, or any human or mammalian allergic disorder that involves an eosinophil immune response.

61. The method of claim 60, wherein said eosinophilic disorder is eosinophilic esophagitis.

62. The method of claim 61, wherein said diagnostic criteria comprises one or more of tissue eosinophilia, dysphagia, food impaction, and endoscopic findings.

63. The method of any one of claims 59-63, said method further comprising monitoring said patient for improvement in one or more diagnostic criteria.

64. The method of any one of claims 59-63, said method further comprising administering an adjunct therapy to said patient identified as having said eosinophilic disorder.

65. The method of claim 64, wherein said adjunct therapy is selected from the group consisting of pain medication, a steroid, a proton pump inhibitor (PPI), or an immune modulator.