WO2016172205A1 - Managing ebola viral infections - Google Patents

Abstract

This disclosure relates to managing Ebola viral infections. In certain embodiments, the disclosure relates to methods of treating or preventing a chronic or persistent Ebola virus infection comprising administering an effective amount of an anti-retroviral agent, 6-fluoro-3-hydroxy-2-pyrazinecarboxamide or 6-fluoro-3-hydroxypyrazine-5-d-2-carboxamide (5-deuterium substitution), favipiravir, prodrug, derivative, or salt thereof to a subject in need thereof. In certain embodiments, the subject is at risk of, exhibiting symptoms of, or diagnosed with an Ebola virus infection.

Description

MANAGING EBOLA VIRAL INFECTIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 62/150,020 filed April 20, 2015. The entirety of this application is hereby incorporated by reference for all purposes.

ACKNOWLEDGEMENTS

This invention was made with government support under Grants UL1TR000454 and P30- EY06360 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Ebola, also known as Ebola hemorrhagic fever or Ebola virus disease (EVD), is a rare and deadly disease caused by one of four Ebola virus species known to cause disease in humans. Following exposure to Ebola, symptoms may appear anywhere between 2 and 21 days and include fever, severe headache, myalgias, fatigue, weakness, diarrhea, abdominal pain, and bleeding diathesis. Once an acute infection has been established, patients may develop rapidly progressive symptoms leading to multiple organ failure with severe nausea, vomiting, electrolyte disturbances, altered mental status, and ultimately death from septic shock. Patients who develop significant antibodies to Ebola virus may survive the acute infection although following acute Ebola infection, survivors may develop systemic inflammatory symptoms and signs which may include myalgias, arthritis, and eye disease including uveitis, an inflammatory process that can lead to pain, photophobia, blurred vision, and ultimately blindness if not properly treated.

The 2014 EVD outbreak in West Africa is the largest outbreak in history with over 22,000 persons infected according to the most recent World Health Organization Situation Report. Of 22,000 cases of EVD, over 8900 people have died with the highest numbers of patients in Sierra Leone, Liberia and Guinea. The fatality rate ranges between 57-60% in the three highest transmission countries where Ebola has been identified. Thus there is a need to identify improved treatment and prevention methods. Reports from the World Health Organization have described ocular symptoms and signs in up to 50% of Ebola survivors although the precise burden of disease and anatomic areas of the eye (i.e. cornea, lens, retina, optic nerve) affected are not known at this time.

Favipiravir has been studied in adult patients in anti-influenza trials and has been approved for the treatment of novel or resistant influenza in Japan.

Oestereich et al. report the successful treatment of advanced Ebola virus infection with T- 705 (favipiravir) in a small animal model. Antiviral Research 105 (2014) 17-21. See also WO 2013180149, WO/2000/010569, and De Clercq, Ebola virus (EBOV) infection: Therapeutic strategies, Biochem Pharmacol, 2015, 93(1): 1-10

References cited herein are not an admission of prior art.

SUMMARY

This disclosure relates to managing Ebola viral infections. In certain embodiments, the disclosure relates to methods of treating or preventing a chronic or persistent Ebola virus infection comprising administering an effective amount of an anti -retroviral agent, 6-fluoro-3- hydroxy-2-pyrazinecarboxamide or 6-fluoro-3-hydroxypyrazine-5-d-2-carboxamide (5- deuterium substitution), favipiravir, prodrug, derivative, or salt thereof to a subject in need thereof. In certain embodiments, the subject is at risk of, exhibiting symptoms of, or diagnosed with an Ebola virus infection.

In certain embodiments, the anti-retroviral agent, 6-fluoro-3 -hydroxy -2- pyrazinecarboxamide or 6-fluoro-3-hydroxypyrazine-5-d-2-carboxamide (5-deuterium substitution), favipiravir, prodrug, derivative, or salt thereof is administered to a subject with a chronic or persistent Ebola infection, or other chronic or persistent viral infection.

In certain embodiments, the anti-retroviral agent is administered to the subject after the virus is not detected in the blood of the subject.

In certain embodiments, the subject is diagnosed with uveitis, chorioretinitis, subconjunctival hemorrhage, iritis, choroiditis, retinitis, anterior uveitis, posterior uveitis, intermediate uveitis, panuveitis, scleritis, episcleritis, optic neuritis, low (hypotony) or elevated intraocular pressure (ocular hypertension), less than 9mmHg or above 22mmHg. In certain embodiments, the subject is diagnosed with the virus from a sample of aqueous humor, vitreous humor, iris, choroid, or retina tissue, tear film, conjunctiva, sclera, or seminal fluid.

In certain embodiments, the anti -retroviral agent is administered orally, into aqueous humor, vitreous humor, or suprachoroidal space of an eye or into a gonad of the subject. In certain embodiments, the disclosure contemplates a kit comprising an aqueous solution of favipiravir and a device for delivering the solution into the vitreous humor, e.g., eyedropper or needle containing device, e.g., a 30 gauge or narrower bore needle and smaller than 20 mm in length, e.g., 15mm to 12 mm or microneedles with a length of 400 to 700 microns. In certain embodiments, the kit further comprises an antibiotic agent, e.g., to apply to the eye after an injection, e.g., through the sclera.

In certain embodiments, the anti-retroviral agent is administered in combination with an anti-inflammatory agent such as prednisone, triamcinolone or prodrugs thereof, triamcinolone acetonide.

In certain embodiments, the anti-retroviral agent is selected from a reverse-transcriptase inhibitor, nucleoside derivative, nucleotide derivative, entry inhibitor, protease inhibitor, integrase inhibitor, zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, entecavir, tenofovir, adefovir, efavirenz, nevirapine, delavirdine, etravirine, rilpivirine, loviride, lersivirine, dolutegravir, cobicistat, lopinavir, ritonavir, indinavir, nelfinavir, maraviroc, enfuvirtide, amprenavir, darunavir, atazanavir or prodrug thereof. With regard to favipiravir in the combinations, the compound may be 6-fluoro-3 -hydroxy -2- pyrazinecarboxamide, 6-fluoro-3-hydroxypyrazine-5-d-2-carboxamide (5-deuterium substitution), or combinations thereof.

In certain embodiments, favipiravir is administered in combination with another retroviral agent, entry inhibitor, protease inhibitor, and/or integrase inhibitor.

In certain embodiments, a combination of retroviral agents are administered selected from: favipiravir and lamivudine; favipiravir and zidovudine; favipiravir and zidovudine and lamivudine; zidovudine and lamivudine; favipiravir and lopinavir; favipiravir and ritonavir; favipiravir and lopinavir and ritonavir; lopinavir and ritonavir; favipiravir and lamivudine; favipiravir and abacavir; favipiravir and abacavir and lamivudine; abacavir and lamivudine; favipiravir and abacavir and zidovudine; favipiravir and abacavir and lamivudine; favipiravir and abacavir and zidovudine and lamivudine; abacavir and zidovudine and lamivudine; favipiravir and emtricitabine; favipiravir and tenofovir; favipiravir and tenofovir and emtricitabine; tenofovir and emtricitabine; favipiravir and efavirenz; favipiravir and efavirenz and tenofovir; favipiravir and efavirenz and emtricitabine; favipiravir and efavirenz and tenofovir and emtricitabine; efavirenz and tenofovir and emtricitabine; favipiravir and rilpivirine; favipiravir and rilpivirine and tenofovir; favipiravir and rilpivirine and emtricitabine; favipiravir and rilpivirine and tenofovir and emtricitabine; rilpivirine and tenofovir and emtricitabine; favipiravir and elvitegravir; favipiravir and elvitegravir and tenofovir; favipiravir and elvitegravir and emtricitabine; favipiravir and elvitegravir and tenofovir and emtricitabine; elvitegravir and tenofovir and emtricitabine; favipiravir and cobicistat; favipiravir and cobicistat and tenofovir; favipiravir and cobicistat and emtricitabine; favipiravir and cobicistat and tenofovir and emtricitabine; cobicistat and tenofovir and emtricitabine; favipiravir and elvitegravir and cobicistat; favipiravir and elvitegravir and cobicistat and tenofovir; favipiravir and elvitegravir and cobicistat and emtricitabine; favipiravir and elvitegravir and cobicistat and tenofovir and emtricitabine; elvitegravir and cobicistat and tenofovir and emtricitabine; favipiravir and dolutegravir; favipiravir and dolutegravir and lamivudine; favipiravir and abacavir and lamivudine; favipiravir and dolutegravir and abacavir; favipiravir and dolutegravir and abacavir and lamivudine; and dolutegravir and abacavir and lamivudine.

In certain embodiments, the disclosure contemplates unit dose formulations in the form of a pill, capsule, tablet, or aqueous solution, buffered, optionally comprising a preservative of the anti-retroviral drugs and combinations reported herein for uses reported herein. In certain embodiments, the disclosure relates to a medicament or production of a medicament comprising an anti-retroviral agent or combination thereof for use in the treatment or prevention of an Ebola viral infection.

In certain embodiments, the treatment contemplates 1800 mg of favipiravir twice daily

(loading doses) followed by 800 mg of favipiravir twice daily.

In certain embodiments, the disclosure contemplates methods of treating a persistent Ebola virus infection comprising administering an effective amount of an anti-retroviral agent, 6- fluoro-3-hydroxy-2-pyrazinecarboxamide or 6-fluoro-3-hydroxypyrazine-5-d-2-carboxamide (5- deuterium substitution), favipiravir, prodrug, derivative, or salt thereof to a subject after the subject is diagnosed with the Ebola infection, and the anti-retroviral agent is administered to the subject after Ebola virus is not detected in the blood of the subject.

In certain embodiments, the retroviral agent is administered when the body temperature of the subject is below 100 degrees Fahrenheit after the subject experiences an fever of greater than 101 degree Fahrenheit.

In certain embodiments, the disclosure contemplates methods of treating a persistent Ebola virus infection comprising administering an effective amount of an anti-retroviral agent, 6- fluoro-3-hydroxy-2-pyrazinecarboxamide or 6-fluoro-3-hydroxypyrazine-5-d-2-carboxamide (5- deuterium substitution), favipiravir, prodrug, derivative, or salt thereof to a subject after the subject is diagnosed with Ebola virus from a sample of aqueous humor, vitreous humor, iris, choroid, or retina tissue, tear film, conjunctiva, sclera, or seminal fluid.t In certain embodiments, the anti-retroviral agent is administered orally.

In certain embodiments, the disclosure contemplates methods of treating a persistent Ebola virus infection comprising administering an effective amount of an anti-retroviral agent, 6- fluoro-3-hydroxy-2-pyrazinecarboxamide or 6-fluoro-3-hydroxypyrazine-5-d-2-carboxamide (5- deuterium substitution), favipiravir, prodrug, derivative, or salt thereof to a subject after the subject is diagnosed with Ebola virus wherein administration is into aqueous humor, vitreous humor, or suprachoroidal space of an eye.

In certain embodiments, the anti-retroviral agent is administered in combination with an anti-inflammatory agent such as prednisone, triamcinolone or prodrugs thereof, triamcinolone acetonide.

In certain embodiments, the anti-retroviral agent is selected from a reverse-transcriptase inhibitor, nucleoside derivative, nucleotide derivative, entry inhibitor, protease inhibitor, integrase inhibitor, zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, entecavir, tenofovir, adefovir, efavirenz, nevirapine, delavirdine, etravirine, rilpivirine, loviride, lersivirine, dolutegravir, cobicistat, lopinavir, ritonavir, indinavir, nelfinavir, maraviroc, enfuvirtide, amprenavir, darunavir, atazanavir or prodrug thereof.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows data on visual activity for a subject without detectable levels of Ebola virus in circulating blood previously infected with the virus after oral treatment with favipiravir. Figure 2 shows data on intraocular pressure for a subject without detectable levels of Ebola virus in circulating blood previously infected with the virus after oral treatment with favipiravir as reported in figure 1. DETAILED DISCUSSION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Unless defined otherwise, 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 belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.

The term "substituted" refers to a molecule wherein at least one hydrogen atom is replaced with a substituent. When substituted, one or more of the groups are "substituents. " The molecule may be multiply substituted. In the case of an oxo substituent ("=0"), two hydrogen atoms are replaced. Example substituents within this context may include halogen, hydroxy, alkyl, alkoxy, nitro, cyano, oxo, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, - RaRb, - RaC(=0)Rb, - RaC(=0) Ra Rb, - RaC(=0)ORb, - RaSOzRb, -C(=0)Ra, -C(=0)ORa, -C(=0) RaRb, - OC(=0) RaRb, -ORa, -SRa, -SORa, - S(=0)₂Ra, -OS(=0)₂Ra and -S(=0)₂ORa. Ra and Rb in this context may be the same or different and independently hydrogen, halogen hydroxyl, alkyl, alkoxy, alkyl, amino, alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl.

The term "optionally substituted," as used herein, means that substitution is optional and therefore it is possible for the designated atom to be unsubstituted.

As used herein, "salts" refer to derivatives of the disclosed compounds where the parent compound is modified making acid or base salts thereof. Examples of salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkylamines, or dialkylamines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. In typical embodiments, the salts are conventional nontoxic pharmaceutically acceptable salts including the quaternary ammonium salts of the parent compound formed, and non-toxic inorganic or organic acids. Preferred salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, gly colic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.

"Subject" refers any animal, preferably a human patient, livestock, rodent, monkey or domestic pet.

The term "prodrug" refers to an agent that is converted into a biologically active form in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent compound. They may, for instance, be bioavailable by oral administration whereas the parent compound is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. A prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis.

As used herein, the term "derivative" refers to a structurally similar compound that retains sufficient functional attributes of the identified analogue. The derivative may be structurally similar because it is lacking one or more atoms, substituted with one or more substituents, e.g., substituting a hydrogen for a deuterium, a salt, in different hydration/oxidation states, e.g., substituting a single or double bond, substituting a hydroxy group for a ketone, or because one or more atoms within the molecule are switched, such as, but not limited to, replacing an oxygen atom with a sulfur or nitrogen atom or replacing an amino group with a hydroxyl group or vice versa. Replacing a carbon with nitrogen in an aromatic ring is a contemplated derivative. The derivative may be a prodrug. Derivatives may be prepared by any variety of synthetic methods or appropriate adaptations presented in the chemical literature or as in synthetic or organic chemistry text books, such as those provide in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Wiley, 6th Edition (2007) Michael B. Smith or Domino Reactions in Organic Synthesis, Wiley (2006) Lutz F. Tietze hereby incorporated by reference.

As used herein, the terms "prevent" and "preventing" include the prevention of the recurrence, spread or onset. It is not intended that the present disclosure be limited to complete prevention. In some embodiments, the onset is delayed, or the severity of the disease is reduced.

As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e.g., patient) is cured and the disease is eradicated. Rather, embodiments, of the present disclosure also contemplate treatment that merely reduces symptoms, and/or delays disease progression. As used herein, the term "combination with" when used to describe administration with an additional treatment means that the agent may be administered prior to, together with, or after the additional treatment, or a combination thereof. Use of oral favipiravir for acute panuveitis associated with viable Ebola virus

Described herein is the use of oral favipiravir for acute panuveitis associated with viable Ebola virus, which has persisted in the eye of a patient who recovered from EVD. The disease developed 100 days following the EVD diagnosis while the patient was convalescent with serum and urine that was negative for Ebola RNA.

Once patients have survived the acute viral illness, it is believed that they can no longer develop acute infection and moreover, are not prone to develop systemic disease directly related to infection. However, we have recently reported the finding of viable Ebola virus in a patient with panuveitis (i.e. uveitis involving the entire uveal tract including the iris, ciliary body and choroid). In a report of an EVD survivor who had been treated at Emory University Hospital, the patient developed acute anterior uveitis 100 days after EVD diagnosis during the convalescent phase of EVD. An aqueous humor sample obtained via an anterior chamber paracentesis showed Ebola virus RNA using quantitative reverse transcriptase polymerase chain reaction testing with a cycle threshold of 18.7 and a subsequent culture showed viable Ebola virus. The low cycling threshold was suggestive of a high inoculum of viable Ebola virus.

During the patient's extremely aggressive, sight-threatening disease course, the patient was initially treated with topical corticosteroids and high-dose oral corticosteroids. Despite these measures, the patient developed progressive disease with the development of a hypopyon (i.e. dense layering of inflammatory white blood cells in the anterior chamber), vitritis, optic disc swelling, and choroidal swelling. The patient also developed profound hypotony (i.e. low intraocular pressure) with loss of the normal globe contour, findings which were consistent with a severe, aggressive infection. The visual acuity had declined from a baseline of 20/15 vision to hand motions vision only (i.e. poorer than legal blindness in a 14-day time period, Exhibit A). Because of the rapid visual acuity loss and severity of disease, all metrics demonstrated that the eye was extremely high-risk for permanent vision loss if the infection and inflammation could not b e cl eared rapi dly . Because the clinical signs were thought to be due to acute viral replication in combination with tissue inflammation, the decision was made to proceed with the administration of the antiviral medication favipiravir (T-705; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide), an antiviral drug that selectively inhibits RNA-dependent RNA polymerase. Three days following the administration of favipiravir, a periocular corticosteroid injection was administered (i.e. triamcinolone acetone 40 mg/ml) to treat ongoing active inflammation and tissue edema.

Favipiravir has demonstrated efficacy for the treatment of Ebola virus disease in small animal models of EVD. In a murine model of Zaire Ebola virus, mice lacking the type I interferon receptor (IFNAR(-)/(-), the initiation of T-705 6 days post infection induced rapid viral clearance, reduced biochemical parameters of disease activity, and prevented a lethal outcome in 100% of animals studied.

The patient was seen in a follow-up 15 days after the initiation of favipiravir, and the visual acuity had improved to 20/50 in the involved left eye. In addition, the profound hypotony, which had been indicative of ciliary body inflammation and possibly Ebola virus infection, had resolved with intraocular pressures >/= 9 mmHg and remained in the physiologic range after 10 days of severe hypotony < 5 mmHg (Figure 1 and Figure 2). The inflammation continued to improve and the visual acuity improved to 20/20 at 27 days following the initiation of favipiravir (46 days after his acute uveitis was diagnosed). The ocular inflammation both within the anterior chamber and vitreous cavity, were improved.

Currently, there are no antiviral therapies to treat EVD, and chronic infection due to EVD leading to organ-threatening disease has not been described previously (i.e. eye-threatening and vision-threatening disease in our patient). Because the eye is considered an immune privileged organ, it is likely that the persistence of Ebola virus was on the basis of the tissue specific privilege. Other organs which are considered immune privileged include the gonads, parts of the central nervous system, and articular cartilage. Therefore, other immune privileged organs may also be chronically infected with Ebola virus and could be treated with antivirals including favipiravir. Because of the sight-threatening process complication observed in our patient in whom favipiravir was administered, the eye is likely the most relevant organ for evaluation and treatment of chronic infection due to Ebola virus in immune privileged organs.

Acute EVD care measures are supportive and include IV or oral hydration, electrolyte monitoring due to severe vomiting and diarrhea, and pain medication. Potential antiviral therapies which have been assessed include convalescent plasma, small inhibitory RNA (siRNA), and monoclonal or polyclonal antibodies.

Favipiravir is a promising therapeutic option for the treatment of acute EVD. Formulations

Pharmaceutical compositions disclosed herein may be in the form of pharmaceutically acceptable salts, as generally described below. Some preferred, but non-limiting examples of suitable pharmaceutically acceptable organic and/or inorganic acids are hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, acetic acid and citric acid, as well as other pharmaceutically acceptable acids known per se (for which reference is made to the references referred to below).

When the compounds of the disclosure contain an acidic group as well as a basic group, the compounds of the disclosure may also form internal salts, and such compounds are within the scope of the disclosure. When a compound of the disclosure contains a hydrogen-donating heteroatom (e.g., H), the disclosure also covers salts and/or isomers formed by the transfer of the hydrogen atom to a basic group or atom within the molecule.

Pharmaceutically acceptable salts of the compounds include the acid addition and base salts thereof. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methyl sulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts. Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts. Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts. For a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002), incorporated herein by reference. The compounds described herein may be administered in the form of prodrugs. A prodrug can include a covalently bonded carrier which releases the active parent drug when administered to a mammalian subject. Prodrugs can be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compounds. Prodrugs include, for example, compounds wherein a hydroxyl group is bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxyl group. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol functional groups in the compounds. Methods of structuring a compound as prodrugs can be found in the book of Testa and Mayer, Hydrolysis in Drug and Prodrug Metabolism, Wiley (2006). Typical prodrugs form the active metabolite by transformation of the prodrug by hydrolytic enzymes, the hydrolysis of amide, lactams, peptides, carboxylic acid esters, epoxides or the cleavage of esters of inorganic acids. It is well within the ordinary skill of the art to make an ester prodrug, e.g., acetyl ester of a free hydroxyl group. It is well known that ester prodrugs are readily degraded in the body to release the corresponding alcohol. See e.g., Imai, Drug Metab Pharmacokinet. (2006) 21(3): 173-85, entitled "Human carboxylesterase isozymes: catalytic properties and rational drug design."

Pharmaceutical compositions for use in the present disclosure typically comprise an effective amount of a compound and a suitable pharmaceutical acceptable carrier. The preparations may be prepared in a manner known per se, which usually involves mixing the at least one compound according to the disclosure with the one or more pharmaceutically acceptable carriers, and, if desired, in combination with other pharmaceutical active compounds, when necessary under aseptic conditions. Reference is made to U.S. Pat. No. 6,372,778, U.S. Pat. No. 6,369,086, U.S. Pat. No. 6,369,087 and U.S. Pat. No. 6,372,733 and the further references mentioned above, as well as to the standard handbooks, such as the latest edition of Remington's Pharmaceutical Sciences.

Generally, for pharmaceutical use, the compounds may be formulated as a pharmaceutical preparation comprising at least one compound and at least one pharmaceutically acceptable carrier, diluent or excipient, and optionally one or more further pharmaceutically active compounds. The pharmaceutical preparations of the disclosure are preferably in a unit dosage form, and may be suitably packaged, for example in a box, blister, vial, bottle, sachet, ampoule or in any other suitable single-dose or multi-dose holder or container (which may be properly labeled); optionally with one or more leaflets containing product information and/or instructions for use. Generally, such unit dosages will contain between 1 and 1000 mg, and usually between 5 and 500 mg, of the at least one compound of the disclosure, e.g., about 10, 25, 50, 100, 200, 300 or 400 mg per unit dosage.

The compounds can be administered by a variety of routes including the oral, ocular, rectal, transdermal, subcutaneous, intravenous, intramuscular or intranasal routes, depending mainly on the specific preparation used. The compound will generally be administered in an "effective amount", by which is meant any amount of a compound that, upon suitable administration, is sufficient to achieve the desired therapeutic or prophylactic effect in the subject to which it is administered. Usually, depending on the condition to be prevented or treated and the route of administration, such an effective amount will usually be between 0.01 to 1000 mg per kilogram body weight of the patient per day, more often between 0.1 and 500 mg, such as between 1 and 250 mg, for example about 5, 10, 20, 50, 100, 150, 200 or 250 mg, per kilogram body weight of the patient per day, which may be administered as a single daily dose, divided over one or more daily doses. The amount(s) to be administered, the route of administration and the further treatment regimen may be determined by the treating clinician, depending on factors such as the age, gender and general condition of the patient and the nature and severity of the disease/symptoms to be treated. Reference is made to U.S. Pat. No. 6,372,778, U.S. Pat. No. 6,369,086, U.S. Pat. No. 6,369,087 and U.S. Pat. No. 6,372,733 and the further references mentioned above, as well as to the standard handbooks, such as the latest edition of Remington's Pharmaceutical Sciences.

For an oral administration form, the compound can be mixed with suitable additives, such as excipients, stabilizers or inert diluents, and brought by means of the customary methods into the suitable administration forms, such as tablets, coated tablets, hard capsules, aqueous, alcoholic, or oily solutions. Examples of suitable inert carriers are gum arabic, magnesia, magnesium carbonate, potassium phosphate, lactose, glucose, or starch, in particular, corn starch. In this case, the preparation can be carried out both as dry and as moist granules. Suitable oily excipients or solvents are vegetable or animal oils, such as sunflower oil or cod liver oil. Suitable solvents for aqueous or alcoholic solutions are water, ethanol, sugar solutions, or mixtures thereof. Polyethylene glycols and polypropylene glycols are also useful as further auxiliaries for other administration forms. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.

When administered by nasal aerosol or inhalation, the compositions may be prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. Suitable pharmaceutical formulations for administration in the form of aerosols or sprays are, for example, solutions, suspensions or emulsions of the compounds of the disclosure or their physiologically tolerable salts in a pharmaceutically acceptable solvent, such as ethanol or water, or a mixture of such solvents. If required, the formulation may additionally contain other pharmaceutical auxiliaries such as surfactants, emulsifiers and stabilizers as well as a propellant.

For subcutaneous or intravenous administration, the compounds, if desired with the substances customary therefore such as solubilizers, emulsifiers or further auxiliaries are brought into solution, suspension, or emulsion. The compounds may also be lyophilized and the lyophilizates obtained used, for example, for the production of injection or infusion preparations. Suitable solvents are, for example, water, physiological saline solution or alcohols, e.g. ethanol, propanol, glycerol, sugar solutions such as glucose or mannitol solutions, or mixtures of the various solvents mentioned. The injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.

When rectally administered in the form of suppositories, the formulations may be prepared by mixing the compounds of formula I with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug. In certain embodiments, it is contemplated that these compositions can be extended release formulations. Typical extended release formations utilize an enteric coating. Typically, a barrier is applied to oral medication that controls the location in the digestive system where it is absorbed. Enteric coatings prevent release of medication before it reaches the small intestine. Enteric coatings may contain polymers of polysaccharides, such as maltodextrin, xanthan, scleroglucan dextran, starch, alginates, pullulan, hyaloronic acid, chitin, chitosan and the like; other natural polymers, such as proteins (albumin, gelatin etc.), poly-L-lysine; sodium poly(acrylic acid); poly(hydroxyalkylmethacrylates) (for example poly(hydroxyethyl methacrylate)); carboxypolymethylene (for example CarbopolTM); carbomer; polyvinyl pyrrolidone; gums, such as guar gum, gum arabic, gum karaya, gum ghatti, locust bean gum, tamarind gum, gellan gum, gum tragacanth, agar, pectin, gluten and the like; poly(vinyl alcohol); ethylene vinyl alcohol; polyethylene glycol (PEG); and cellulose ethers, such as hydroxymethyl cellulose (HMC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), methylcellulose (MC), ethylcellulose (EC), carboxyethylcellulose (CEC), ethylhydroxy ethylcellulose (EHEC), carboxymethylhydroxy ethylcellulose (CMHEC), hydroxypropylmethyl-cellulose (HPMC), hydroxypropylethylcellulose (HPEC) and sodium carboxymethylcellulose (Na CMC); as well as copolymers and/or (simple) mixtures of any of the above polymers.

Certain of the above-mentioned polymers may further be crosslinked by way of standard techniques. The choice of polymer will be determined by the nature of the active ingredient/drug that is employed in the composition of the disclosure as well as the desired rate of release. In particular, it will be appreciated by the skilled person, for example in the case of HPMC, that a higher molecular weight will, in general, provide a slower rate of release of drug from the composition. Furthermore, in the case of HPMC, different degrees of substitution of methoxyl groups and hydroxypropoxyl groups will give rise to changes in the rate of release of drug from the composition. In this respect, and as stated above, it may be desirable to provide compositions of the disclosure in the form of coatings in which the polymer carrier is provided by way of a blend of two or more polymers of, for example, different molecular weights in order to produce a particular required or desired release profile.

Microspheres of polylactide, polyglycolide, and their copolymers poly(lactide-co- glycolide) may be used to form sustained-release protein delivery systems. Proteins can be entrapped in the poly(lactide-co-glycolide) microsphere depot by a number of methods, including formation of a water-in-oil emulsion with water-borne protein and organic solvent- borne polymer (emulsion method), formation of a solid-in-oil suspension with solid protein dispersed in a solvent-based polymer solution (suspension method), or by dissolving the protein in a solvent-based polymer solution (dissolution method). One can attach poly(ethylene glycol) to proteins (PEGylation) to increase the in vivo half-life of circulating therapeutic proteins and decrease the chance of an immune response.

EXAMPLES

Investigate imaging modalities in a patient with viable EBOV panuveitis during convalescence.

In order to analyze and describe multimodality diagnostic imaging and clinical features during the disease course, a patient with panuveitis associated with viable Ebola virus (EBOV) during convalescence was evaluated. Specifically fluorescein angiography (FA), indocyanine green angiography (ICG), spectral domain optical coherence tomography (OCT) of the macula and anterior segment, ultrasound biomicroscopy, and B-scan ultrasound throughout the disease course were explore as it relates to pathogenesis of ocular EVD.

Fourteen weeks after the patient's initial EVD diagnosis, an anterior uveitis developed that rapidly progressed into a unilateral panuveitis. The findings improved with corticosteroids and systemic antiviral therapy. Diagnostic multimodality imaging from the patient's course were reviewed in detail and included FA, ICG, SD-OCT of the macula, ultrasound biomicroscopy, and B-scan ultrasound. Anterior segment OCT was also used to qualitatively and quantitatively evaluate iris thickness. Paired and unpaired t-tests were used for within and between eye comparisons of iris thickening changes.

A baseline exam before active uveitis developed showed peripheral chorioretinal scarring and a FA with hyperfluorescent staining of the scars and several separate areas of punctate hyperfluorescence. SD-OCT of the areas of hyperfluorescence was significant for mild disruption of the inner retina. ICG, GVF, and full-field ERG were normal. At day 15, iris heterochromia developed in the affected eye. Anterior segment OCT of the iris at day 15 showed increased thickening in the left eye (506 μπι) when compared to the right eye (376 μπι, p=.0007). Following treatment, the iris decreased to 452 μ at day 32 (p=0.0147) with resolution of the iris heterochromia. UBM displayed ciliary body edema during the hypotonous phase. B-scan at this time was significant for vitreous opacity, shallow peripheral choroidals with thickening, and optic nerve edema. At day 32, B-scan showed complete restoration of globe architecture with choroidal resolution, and decreased optic nerve edema.

Clinical, laboratory, and diagnostic imaging features showed that the pathogenesis of the active EVD panuveitis involves local viral replication and inflammation of the ciliary body, iris, choroid, retina, and optic nerve. Iris and ciliary body epithelium were affected suggestive of active viral replication.

Clinical Management and Detection of Live Virus in Aqueous Humor During Convalescence

Reported herein is the detection of live Ebola virus in the aqueous humor and the management of sight-threatening panuveitis during Ebola virus disease convalescence. A 43- year-old male patient developed EVD while working in a treatment center in Kenema, Sierra Leone. Following a 40-day hospital inpatient course complicated by multi-organ failure requiring mechanical ventilation and hemodialysis and 100 days after the initial EVD diagnosis, the patient developed unilateral, anterior uveitis with ocular hypertension, progressing to severe, sight-threatening panuveitis. The patient's clinical course, diagnostic imaging, therapeutic interventions, and molecular analysis/quantitative RT-PCR for EBOV in the aqueous, conjunctival, tear film, and serum specimens for EBOV are reported.

Ophthalmic exam at 10 weeks after EVD diagnosis showed VA of 20/15 OU and hyperpigmented retinal scars with hypopigmented haloes. Four weeks later, the patient presented with severe pain and photophobia OS. Exam showed VA of 20/20 OS, an IOP of 44 mmHg and anterior uveitis. Topical steroids and ocular hypotensives were started. An anterior chamber tap was performed and qRT-PCR for EBOV was positive with a cycle threshold of 18.7 indicating a high inoculum of viable virus, confirmed by positive EBOV culture. qRT-PCR of serum, conjunctiva and tear film specimens tested negative for EBOV. Diffuse scleritis, hypopyon panuveitis, iris heterochromia, optic neuropathy and severe hypotony developed rapidly despite oral prednisone from days 5 through 19 with VA nadir measured at hand motions. Emergent antiviral favipiravir and a periocular triamcinolone injection (40 mg/1 ml) were given. VA improved to 20/30 at 45-day follow-up with durable hypotony resolution and residual vitreous opacity.

Claims

1. A method of treating a persistent Ebola virus infection comprising administering an effective amount of an anti-retroviral agent, 6-fluoro-3-hydroxy-2-pyrazinecarboxamide or 6- fluoro-3-hydroxypyrazine-5-d-2-carboxamide (5-deuterium substitution), favipiravir, prodrug, derivative, or salt thereof to a subject after the subject is diagnosed with the Ebola infection, and the anti-retroviral agent is administered to the subject after Ebola virus is not detected in the blood of the subject.

2. The method of Claim 1, wherein the subject is diagnosed with uveitis, chorioretinitis, subconjunctival hemorrhage, iritis, choroiditis, retinitis, anterior uveitis, posterior uveitis, intermediate uveitis, panuveitis, scleritis, episcleritis, optic neuritis, low (hypotony) or elevated intraocular pressure (ocular hypertension), less than 9mmHg or above 22mmHg.

3. The method of Claim 1, wherein the retroviral agent is administered when the body temperature of the subject is below 100 degrees Fahrenheit after the subject experiences an fever of greater than 101 degree Fahrenheit.

4. A method of treating a persistent Ebola virus infection comprising administering an effective amount of an anti-retroviral agent, 6-fluoro-3-hydroxy-2-pyrazinecarboxamide or 6- fluoro-3-hydroxypyrazine-5-d-2-carboxamide (5-deuterium substitution), favipiravir, prodrug, derivative, or salt thereof to a subject after the subject is diagnosed with Ebola virus from a sample of aqueous humor, vitreous humor, iris, choroid, or retina tissue, tear film, conjunctiva, sclera, or seminal fluid.

5. The method of Claim 4, wherein the anti-retroviral agent is administered orally.

6. A method of treating a persistent Ebola virus infection comprising administering an effective amount of an anti-retroviral agent, 6-fluoro-3-hydroxy-2-pyrazinecarboxamide or 6- fluoro-3-hydroxypyrazine-5-d-2-carboxamide (5-deuterium substitution), favipiravir, prodrug, derivative, or salt thereof to a subject after the subject is diagnosed with Ebola virus wherein administration is into aqueous humor, vitreous humor, or suprachoroidal space of an eye.

7. The method of Claim 6, wherein the anti-retroviral agent is administered in combination with an anti-inflammatory agent such as prednisone, triamcinolone or prodrugs thereof, triamcinolone acetonide.

8. The method of Claim 7, wherein the anti-retroviral agent is selected from a reverse- transcriptase inhibitor, nucleoside derivative, nucleotide derivative, entry inhibitor, protease inhibitor, integrase inhibitor, zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, entecavir, tenofovir, adefovir, efavirenz, nevirapine, delavirdine, etravirine, rilpivirine, loviride, lersivirine, dolutegravir, cobicistat, lopinavir, ritonavir, indinavir, nelfinavir, maraviroc, enfuvirtide, amprenavir, darunavir, atazanavir or prodrug thereof.