OA17683A - Deoxynojirimycin derivatives and methods of their using. - Google Patents

Abstract

The present application provide novel iminosugars and their use in treatment of viral infections, such as Dengue infection and Influenza A infection. The present inventors discovered certain deoxynojirimycin derivatives may be effective against one or more viruses, which may be. For example, a Dengue virus and/or a virus belonging to the Orthomyxoviridae family, such as an Influenza A virus. In particular, such deoxynojirimycin derivatives may be useful for treating a disease or condition caused by or associated with one or more viruses. In certain embodiments, the deoxynojirimycin derivatives may increase a survival rate or probability for a subject infected with one or more viruses, which may be, for example, a Dengue virus and/or a virus belonging to the Orthomyxoviridae family, such as an Influenza A virus.

Description

[0001] The présent invention relates generally to iminosugars and their methods of use and in particular, to JV-substituted deoxynojirimycin compounds and their use for treating 5 and/or preventing viral infections.

SUMMARY [0002]

One embodiment is a compound of formula (I):

or a pharmaceutically acceptable sait thereof, wherein Wm and R1.3 are each independently selected from hydrogen-and Ci-3-alkyl groups and wherein at least one of

R1-3 is not hydrogen.

-2[0003] Yet another embodiment is a method of treating a disease or condition caused by or associated with a virus belonging to the Orthomyxoviridae family comprising administering to a subject in need thereof the compound of formula (I) or a pharmaceutically acceptable sait thereof.

[0004] And yet another embodiment is a method of treating a disease or condition caused by or associated with Dengue virus comprising administering to a subject in need thereof the compound of formula (I) or a pharmaceutically acceptable sait thereof.

FIGURES [0005] Figure 1 : Survival of infected mice grouped by treatment. Groups of mice (n=T0) received the treatment TID starting 1 hour prior to infection; Mice were infected intranasally with influenza at a dose of ~ 1 LD90. Survival data is plotted as percent survival against days post infection. Graph shows survival of animais in each group.

[0006] Figure 2: Weights of infected mice grouped by treatment. Groups of mice (n=T 0) received the treatment TID starting 1 hour prior to infection; Mice were infected intranasally with influenza at a dose of ~ 1 LD90. Weight data is plotted as percent of original weight against days post infection.

[0007] Figure 3 : Températures of infected mice grouped by treatment. Groups of mice (n=10) received the treatment TID starting 1 hour prior to infection; Mice were infected intranasally with influenza at a dose of ~ 1 LD90. Température data is plotted as percent of original température against days post infection.

[0008] Figure 4: Health scores of infected mice grouped by treatment. Groups of mice (n=10) received the treatment TID starting 1 hour prior to infection; Mice were infected intranasally with influenza at a dose of ~ 1 LD90. Health data is plotted as health score vs days post infection.

[0009] Figure 5: présents a plot demonstrating in vivo efficacy of UV-12 against dengue virus.

[0010] Figure 6: Survival of mice grouped by treatment delivery route. Groups of mice (n=10) received the first treatment dose of compound in water 1 h before an intranasal infection with Influenza A/Texas/36/91 (H1N1) at a dose of ~ 1 LD90. Survival data is plotted as percent survival against days post infection. (A) Treatment of 100 mg/kg of

-3 UV-4B, UV-8, UV-9, UV-10, UV-11, and UV-12, assuming a starting weight of ~20g per mouse. Graph includes survival curve of vehicle only control group.

[0011] Figure 7: Statistical analysis of survival of infected mice. The survival data plotted in Figure 6 were analyzed using the Mantel-Cox and Gehan-Breslow-Wilcoxon tests. (A) Statistical analysis is by comparing the treated groups to the H2O vehicle control. Statistical significance is indicated by a p value < 0.05.

[0012] Figures 8A-F: Analysis of Weights. Mice received the first dose of compound in

H2O at 1 hour prior to the intranasal infection with ~ 1 LD90 of Influenza A/Texas/36/91 (H1N1). The mean weights for each group are plotted as percent of the weight on day 0 (baseline) with the standard déviations. (A) Treatment of infected mice with UV-4B or UV-8, (B) treatment of infected mice with UV-4B or UV-9, (C) treatment of infected mice with UV-4B or UV-10, (D) treatment of infected mice with UV-4B or UV-11, (E) treatment of infected mice with UV-4B or UV-12, and (F) treatment of uninfected mice with UV-8, -9, - 10, -11, or -12, without standard déviations.

[0013] Figure 9: Statistical analysis of weight data. The weight data for the influenza infected mice plotted in Figure 8 were analyzed using a repeated-measures 2 -way ANOVA (GraphPad Prism) against the vehicle control. Data was only analyzed through day 7 post-infection (p.i.) due to deaths at later time points. Compounds UV-9, UV-10, and UV-11 had both 0% survival and a MTD < 9 days and were thus omitted from further statistical analysis. Statistical significance is indicated by a p value lower than 0.05 (p<0.05).

[0014] Figure 10: Analysis of Températures. Mice received the first dose of compound in

H2O at 1 hour prior to the intranasal infection with ~ 1 LD90 of Influenza A/Texas/36/91 (H1N1). The mean températures for each group are plotted as percent of the weight on day 0 (baseline) with the standard déviations. (A) Treatment of infected mice with UV-4B or UV-8, (B) treatment of infected mice with UV-4B or UV-9, (C) treatment of infected mice with UV-4B or UV-10, (D) treatment of infected mice with UV-4B or UV-11, (E) treatment of infected mice with UV-4B or UV-12, and (F) treatment of uninfected mice with UV-8, -9, -10, -11, or -12, without standard déviations.

[0015] Figure 11 : Statistical analysis of température data. The température data for the influenza infected mice plotted in Figure 10 were analyzed using a repeated-measures 2way ANOVA (GraphPad Prism) against the vehicle control. Data was only analyzed

-4through day 7 post-infection (p.i.) due to deaths at later time points. Compounds UV-9, UV-10, and UV-11 had both 0% survival and a MTD < 9 days and were thus omitted from further statistical analysis. Statistical significance is indicated by a p value lower than 0.05 (p<0.05) [0016] Figures 12 présent results of the study performed in Example 4. This study was determining survival of dengue virus infected mice. The survival data and animal body weight are plotted in Figure 12. Ail compounds were given in water by the oral route (3x per day intragastric via oral gavage - IG) for a total number of 7 days after the start of dosing. The treatment dose was 50 mg/kg of UV-4B, UV-8, UV-9, UV-10, UV-11, and UV-12. Groups of mice received the first treatment dose of compound 0.5-1 h before an intravenous infection with dengue virus at a dose of ~ 1 LD90.· Survival and body weights were measured until 3 days after dosing.

[0017] Figure 13 présents chemical formulae of UV-8, UV-9, UV-10, UV-11 and UV-12 compounds.

[0018] Figure 14 présents chemical formula of UV-28.

[0019] Figure 15 provides a synthesis scheme for UV-8.

[0020] Figure 16 provides a synthesis scheme for UV-9.

[0021] Figure 17 provides a synthesis scheme for UV-10.

[0022] Figure 18 provides a synthesis scheme for UV-11.

[0023] Figure 19 provides a synthesis scheme for UV-12.

[0024] Figure 20 provides a synthesis scheme for UV-28.

[0025] Figure 21 provides an alternative synthesis scheme for UV-28.

DETAILED DESCRIPTION

Related Documents [0026] The following patent documents, each of which is incorporated herein by reference in its entirety, may be useful for understanding the présent disclosure: US patents nos. 6,545,021; 6,809,803; 6,689,759; 6,465,487; 5,622,972; 7,816,650; 7,256,005; 8,450,345; 7,612,093; and 8,426,445; US patent application publications nos. 20110184019; 20130150405; 20100222384; 20110065754; 20110065753; 20110065752; and 2007-0275998; and US patent application no. 13/870,341 filed April 25, 2013.

-5Definition of terms [0027] Unless otherwise specified, a or an means one or more.

[0028] As used herein, the term viral infection describes a diseased state, in which a virus invades a healthy cell, uses the cell's reproductive machinery to multiply or replicate and ultimately lyse the cell resulting in cell death, release of viral particles and the infection of other cells by the newly produced progeny viruses. Latent infection by certain viruses is also a possible resuit of viral infection.

[0029] As used herein, the term treating or preventing viral infection means to inhibit the réplication of the particular virus, to inhibit viral transmission, or to prevent the virus from establishing itself in its host, and to ameliorate or alleviate the symptoms of the disease caused by the viral infection. The treatment is considered therapeutic if there is a réduction in viral load, decrease in mortality and/or morbidity.

[0030] IC50 or IC90 (inhibitory concentration 50 or 90) is a concentration of a therapeutic agent, such as an iminosugar, used to achieve 50% or 90% réduction of viral load, respectively.

[0031] LD90 stands for (léthal dose 90%) is an estimated dose of an agent at which 90% of the population is expected to die.

[0032] DENV stands for Dengue virus.

[0033] INFV stands for influenza virus.

[0034] IV stands for intravenous.

[0035] IG stands for intragastric.

[0036] IP stands for intraperitoneal.

[0037] PFU stands for a plaque-forming unit.

[0038] PBS stands for phosphate buffered saline.

[0039] ANOVA stands for an analysis of variance.

DISCLOSURE [0040] The présent inventors discovered certain deoxynojirimycin dérivatives may be effective against one or more viruses, which may be, for example, a Dengue virus and/or a virus belonging to the Orthomyxoviridae family, such as an Influenza A virus.

[0041] In particular, such deoxynojirimycin dérivatives may be useful for treating a disease or condition caused by or associated with one or more viruses, which may be, for

-6example, a Dengue virus and/or a virus belonging to the Orthomyxoviridae family, such as an Influenza A virus. In certain embodiments, the deoxynojirimycin dérivatives may increase a survival rate or probability for a subject infected with one or more viruses, which may be, for example, a Dengue virus and/or a virus belonging to the Orthomyxoviridae family, such as an Influenza A virus.

Dengue viruses [0042] Dengue virus belongs to the genus Flavivirus of the Flaviridae family and causes dengue hémorrhagie fever (DHF). Dengue virus includes four closely related serotypes, usually referred to as Dengue 1, Dengue 2, Dengue 3 and Dengue 4. Recovery from infection by one provides lifelong immunity against that serotype but confers only partial and transient protection against infection by the other three. A good evidence exists that sequential infection increases the risk of more serious disease, resulting in DHF. Emerging DHF épidémies are causing increasing concem in the Americas and in Asia, where ail four dengue viruses are endemic. DHF has become a leading cause of hospitalization and death among children in several countries. In 2007, there were more than 890,000 reported cases of dengue in the Americas, of which 26,000 cases were DHF.

[0043] Dengue is transmitted primarily by the Aedes aegypti mosquito and is the most cornmon mosquito-bome viral disease of humans. Globally, 2.5 billion people - 40% of the world's population - live in the warm areas where Aedes aegypti is cornmon and dengue can be transmitted. The rapid growth of tropical cities and their human and mosquito populations is bringing ever greater numbers of people into contact with this vector. The geographical spread of both the mosquito vectors and the virus has led to a global resurgence of épidémie dengue fever and the emergence of dengue hémorrhagie fever (DHF).

Orthomyxoviridae family [0044] The Orthomyxoviridae family is a family of RNA viruses that includes five généra:

[0045] Influenzavirus A, Influenzavirus B, Influenzavirus C, Isavirus and Thogotovirus.

The fîrst three généra contain viruses that can cause influenza in vertebrates, including birds, humans and other mammals.

-7 [0046] The Influenzavirus A genus includes a single species, which can causes influenza in birds and certain mammals, including humans, pigs, felines, canines and equines.

[0047] Influenza A viruses are négative sense, single-stranded, segmented RNA viruses.

Several subtypes of Influenza A virus exist, labeled according to an H number (for the type of hemagglutinin) and an N number (for the type of neuraminidase). Currently known 16 different H antigens (H1 to H16) and nine different N antigens (NI to N9). Serotypes and subtypes of Influenza A include H1N1 Influenza A; H1N2 Influenza A; H2N2 Influenza A; H3N1 Influenza A; H3N2 Influenza A; H3N8 Influenza A; H5N1 Influenza A; H5N2 Influenza A; H5N3 Influenza A; H5N8 Influenza A; H5N9 Influenza

A; H5N9 Influenza A; H7N1 Influenza A; H7N2 Influenza A; H7N3 Influenza A; H7N4

Influenza A; H7N7 Influenza A; H9N2 Influenza A; H10N7 Influenza A.

[0048] The Influenzavirus B genus includes a single species, which can cause influenza in humans and seals.

[0049] The Influenzavirus C genus includes a single species, which can cause influenza in humans and pigs.

Deoxynojirimycin dérivatives [0050] In some embodiments, the deoxynojirimycin dérivative may be a compound belonging to a genus defined by formula (I):

such that Wi_4 and R1.3 are each independently selected from hydrogen and Ci_₃ alkyl groups, where at least one of Ri_₃ is not hydrogen. Ci_₃ alkyl groups include methyl, ethyl and propyl. In some embodiments, R₂ and R₃ may be such that they form together one of the following groups: -CH₂-, -CH₂-CH₂- or -CH₂-CH₂-CH₂-.

[0051] A compound of formula (I) with each of W1.4 and Ri_₃ being hydrogen is N-(9Methoxynonyl) deoxynojirimycin, which is also known as N9-DNJ or UV-4. The compounds of the defined above genus may be viewed as dérivatives of UV-4. The compounds of the defined above genus, such as compounds UV-12 and UV-28 (see Figures 13-14) may hâve one or more advantages compared to other dérivatives of UV-4, such as, for example, compounds UV-8, UV-9, UV-10 and UV-11 (see Figure 13). For example, the compounds of the defined above genus, such as compounds UV-12 and UV28 may be more efficient compared to other dérivatives of UV-4, such as compounds UV-8, UV-9, UV-10 and UV-11, against one or more viruses, which may be, for

-9example, a Dengue virus and/or a virus belonging to the Orthomyxoviridae family, such as an Influenza A virus.

[0052] UV-4 dérivatives, such as UV-8, UV-9, UV-10, UV-11, UV-12 and UV-28, may be synthesized as depicted in Figures 15-21.

[0053] Methods of synthesizing deoxynojirimycin dérivatives are also disclosed, for example, in U.S. Patent nos. 5,622,972, 5,200,523, 5,043,273, 4,994,572, 4,246,345, 4,266,025, 4,405,714, and 4,806,650 and U.S. Patent application publication no. 2007/0275998, which are ail incorporated herein by reference.

[0054] In some embodiments, the deoxynojirimycin dérivative may be in a form of a sait derived from an inorganic or organic acid. Pharmaceutically acceptable salts and methods for preparing sait forms are disclosed, for example, in Berge et al. (J. Pharm. Sci. 66: 118, 1977). Examples of appropriate salts include but are not limited to the following salts: acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptanoate, glycérophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydiobromide, hydroiodide, 2hydroxyethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2naphthalcncsulfonate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, mesylate, and undecanoate.

[0055] In some embodiments, the deoxynojirimycin dérivative may be also used in a form of a prodrug. Prodrugs of DNJ dérivatives, such as the 6-phosphorylated DNJ dérivatives, are disclosed in U.S. Patents nos. 5,043,273 and 5,103,008.

[0056] In some embodiments, the deoxynojirimycin dérivative may be used as a part of a composition, which further comprises a pharmaceutically acceptable carrier and/ or a component useful for delivering the composition to an animal. Numerous pharmaceutically acceptable carriers useful for delivering the compositions to a human and components useful for delivering the composition to other animais such as cattle are known in the art. Addition of such carriers and components to the composition of the invention is well within the level of ordinary skill in the art.

- 10[0057] In some embodiments, the pharmaceutical composition may consist essentially of //-substituted deoxynojirimycin, which may mean that the 7V-substituted deoxynojirimycin is the only active ingrédient in the composition.

[0058] Yet in some embodiments, /V-substituted deoxynojirimycin may be administered with one or more additional antiviral compounds.

[0059] In some embodiments, the deoxynojirimycin dérivative may be used in a liposome composition, such as those disclosed in US publications nos. 2008/0138351, 2009/0252785 and 2010/0266678.

[0060] The DNJ dérivative may be administered to a cell or an animal affected by a virus.

The DNJ dérivative may inhibit morphogenesis of the virus, or it may treat the individual. The treatment may reduce, abate, or diminish the virus infection in the animal.

[0061] In some embodiments, the animal may be an animal infected with a Dengue virus which may be a vertebrate, such as a mammal, which may be, for example, a rodent or a primate, such as a human.

[0062] In some embodiments, the amount of the DNJ dérivative administered to an animal or to an animal cell to the methods of the invention may be an amount effective to inhibit the morphogenesis of Dengue virus from the cell. The term inhibit as used herein may refer to the détectable réduction and/or élimination of a biological activity exhibited in the absence of the iminosugar. The term effective amount may refer to that amount of the DNJ dérivative necessary to achieve the indicated effect. The term treatment as used herein may refer to reducing or alleviating symptoms in a subject, preventing symptoms from worsening or progressing, inhibition or élimination of the causative agent, or prévention of the infection or disorder related to the Dengue virus in a subject who is free therefrom.

[0063] In some embodiments, the animais may be an animal infected with a virus that belongs to the Orthomyxoviridae family, which may be a vertebrate, such as a bird or a mammal, including primates, such as humans; felines; equines, and canines.

[0064] In some embodiments, the amount of the DNJ dérivative administered to an animal or to an animal cell to the methods of the invention may be an amount effective to inhibit the morphogenesis of a virus belonging to the Orthomyxoviridae family from the cell. The term inhibit as used herein may refer to the détectable réduction and/or élimination of a biological activity exhibited in the absence of the DNJ dérivative. The

- 11 term effective amount may refer to that amount of the DNJ dérivative necessary to achieve the indicated effect. The term treatment as used herein may refer to reducing or alleviating symptoms in a subject, preventing symptoms from worsening or progressing, inhibition or élimination of the causative agent, or prévention of the infection or disorder related to the virus belonging to the Orthomyxoviridae family in a subject who is free therefrom.

[0065] Treatment of the disease caused by or associated with a virus, which may be, for example, a Dengue virus or a virus belonging to the Orthomyxoviridae family, such as Influenza A virus, may include destruction of the infecting agent, inhibition of or interférence with its growth or maturation, and neutralization of its pathological effects. The amount of the DNJ dérivative, which may be administered to the cell or animal is preferably an amount that does not induce toxic effects which outweigh the advantages which accompany its administration.

[0066] Actual dosage levels of active ingrédients in the pharmaceutical compositions may vary so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular patient.

[0067] The selected dose level may dépend on the activity of the DNJ dérivative, the route of administration, the severity of the condition being treated, and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound(s) at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration, for example, two to four doses per day. It will be understood, however, that the spécifie dose level for any particular patient may dépend on a variety of factors, including the body weight, general health, diet, time and route of administration and combination with other therapeutic agents and the severity of the condition or disease being treated. The adult human daily dosage may range from between about one microgram to about one gram, or from between about 10 mg and 100 mg, of the DNJ dérivative per 10 kilogram body weight. In some embodiments, a total daily dose may be from 0.1 mg/kg body weight to 100 mg/kg body weight or from 1 mg/kg body weight to 60 mg/kg body weight or from 2 mg/kg body weight to 50 mg/kg body weight or from 3 mg/kg body weight to 30 mg/kg body weight. The daily dose may be administered over

- 12one or more administering events over day. For example, in some embodiments, the daily dose may be distributed over two (BID) administering events per day, three administering events per day (TID) or four administering events (QID). In certain embodiments, a single administering event dose ranging from 1 mg/kg body weight to 10 mg/kg body weight may be administered BID or TID to a human making a total daily dose from 2 mg/kg body weight to 20 mg/kg body weight or from 3 mg/kg body weight to 30 mg/kg body weight. Of course, the amount of the DNJ dérivative which should be administered to a cell or animal may dépend upon numerous factors well understood by one of skill in the art, such as the molecular weight of the DNJ dérivative and the route of administration.

[0068] Phamiaceutical compositions that are useful in the methods of the invention may be administered systemically in oral solid formulations, ophthalmic, suppository, aérosol, topical or other similar formulations. For example, it may be in the physical form of a powder, tablet, capsule, lozenge, gel, solution, suspension, syrup, or the like. In addition to the DNJ dérivative, such pharmaceutical compositions may contain pharmaceuticallyacceptable carriers and other ingrédients known to enhance and facilitate drug administration. Other possible formulations, such as nanoparticles, liposomes, resealed érythrocytes, and immunologically based Systems may also be used to administer the DNJ dérivative. Such pharmaceutical compositions may be administered by a number of routes. The term parentéral used herein includes subcutaneous, intravenous, intraarterial, intrathecal, and injection and infusion techniques, without limitation. By way of example, the pharmaceutical compositions may be administered orally, topically, parenterally, systemically, or by a pulmonary route.

[0069] These compositions may be administered a in a single dose or in multiple doses which are administered at different times. Because the inhibitory effect of the composition upon a virus may persist, the dosing regimen may be adjusted such that virus propagation is retarded while the host cell is minimally effected. By way of example, an animal may be administered a dose of the composition of the invention once per week, whereby virus propagation is retarded for the entire week, while host cell fonctions are inhibited only for a short period once per week.

[0070] Embodiments described herein are forther illustrated by, though in no way limited to, the following working examples.

- 13 WORKING EXAMPLES

EXAMPLE 1

Efficacy of UV-12 and UV-28 against INFV A/Texas/36/91 (H1N1) Challenge in Mice [0071] Study Summary: This study tested the ability of iminosugars UV-12 and UV-28 to protect mice from léthal influenza infection (~1 LDgo of influenza A/Texas/36/91 (H1N1) administered intranasally). Compounds were delivered 30-60 minutes prior to viral challenge via the oral (IG) route at 100 or 40 mg/kg and continued three times daily for 10 days. The mice used were ~20 gram, 6-8 week old female BALB/c mice in groups of 10 for efficacy (a summary of the study groups is shown in Table 1). Température and weights were taken daily. Endpoint was day 14 post infection, death, or euthanasia.

Animais displaying severe illness as determined by >30% weight loss, extreme lethargy, or paralysis were euthanized.

I. Introduction [0072] Purposed: This study aimed to détermine the efficacy of small molécules UV-12 and UV-28 in vivo against a léthal influenza A/Texas/36/91 (H1N1) infection.

II. Materials and Methods

Materials

Table 1 : Test articles

Name	Amount (mg)	Solvent
UV-12	20 or 8 mg/ml	water
UV-28	20 or 8 mg/ml	water

Table 2: Viruses for challenge

Name	Strain	Stock titer	Additional Info
Influenza	A/Texas/36/91 (H1N1)	2.6xl05 PFU/ml	100 ul of a 1:500 viral dilution was given to mice for ~52 PFU/mouse

- 14Table 3. Animais used

Species	Strain	Age	S ex	Vendor	Additional Info
Mouse	BALB/c	6-8 weeks	F	Charles River	n=10 per group

Table 4: Equipment

Item	Vendor
Syringes	BD
Animal Housing	Inno Vive
Biodata chips and scanner	Bio Medic Data Systems
Ohause scale	Ohause

Study design [0073] This study tested the ability of UV-12 and UV-28 to protect mice from léthal influenza infection. The mice used were 6-8 week old female BALB/c mice in groups of (see Table 5 below). Mice were treated with 100 or 40 mg/kg three times daily (TID) via IG route starting approximately 30-60 minutes prior to challenge. Mice were challenged with ~1 LD90 of influenza A/Texas/36/91 (H1N1) administered intranasally (IN). Endpoint was day 14 post infection, death, or euthanasia. Animais displaying severe illness as determined by >30% weight loss, extreme lethargy, or paralysis were euthanized. Température and weights were taken daily.

Table 5: Mouse groups for the study.

[0074] The time point of initial dosing (treatment start relative to the infection), dosing regimen and the dose levels/routes are listed.

Group (n=10)	Test Article	Dosage, TID	Readouts (After ~ 1 LD90 of influenza A/Texas/36/91 (H1N1) administered intranasally)
1	Water	Vehicle Only	• Endpoint is day 14 post infection, server morbidity, death, or >30% weight loss. • Animais displaying severe illness (as determined
2	UV-12	100 mg/kg
3	40 mg/kg
4	UV-28	100 mg/kg

5

40 mg/kg

by >30% weight loss, extreme lethargy, or paralysis) will be euthanized. • Health assessments, weights and température to be taken daily for 15 days total (days 0-14 post infection)

STANDARD PROTOCOLS

Standard Protocol for intranasal infection of mice [0075] 1. Female 6-8 week old BALB/c mice were housed in groups of 5 mice. Mice were quarantined at the study site (Noble Life Sciences, Gaithersburg, MD) for at least 3 days prior to the start of the study.

[0076] 2. Food and water was provided ad libitum.

[0077] 3. The groups of mice challenged with INFV were infected via intranasal (IN) inoculation with -lxLDço in 100 pL of a 1:500 dilution of INFV in PBS under light anesthésia (Isoflurane).

[0078] 4. After the infection, mice were placed back into their cages for observation and subséquent dosing.

Protocol for oral gavage of mice for test article delivery [0079] 1. Mice were treated with 100 or 40 mg/kg of test article given by the IG route in 100 pL of water (see Table 5 for dosing regimens) three times daily for 10 days.

[0080] 2. After dosing, mice were retumed to their cages and monitored for any distress related to dosing.

Observation of mice [0081] 1. Mice were observed through 13 days post infection (14 days total, 0-13 days post infection).

[0082] 2. Mice were weighed daily on an Ohause scale and the weights were recorded.

[0083] 3. Ail animais had chips implanted at least 3 days prior to virus challenge that monitored the body température. The températures were recorded daily.

-16[0084] 4. Survival and health of each mouse was evaluated once time a day using a scoring System of 1-7.

Table 6. Scoring System

Score	Initiais	Description	Appearance	Mobility	Attitude
1	H	Healthy	Smooth Coat, Bright Eyes	Active, Scurrying, Burrowing	Alert
2	SR	Slightly Ruffled	Slightly Ruffled coat (usually only around head and neck)	Active, Scurrying, Burrowing	Alert
3	R	Ruffled	Ruffled Coat throughout body. A “wet” appearance.	Active, Scurrying, Burrowing	Alert
4	S	Sick	Very Ruffled Coat. Slightly closed, inset eyes.	Waling, but no scurrying.	Mildly Léthargie
5	VS	Very Sick	Very Ruffled Coat. Closed, inset eyes.	Slow to no movement. Will retum to upright position if put on its side.	Extremely Léthargie
6	E	Euthanized	-	-	-
7	FD	Found Dead	-	—	—

III. RESULTS

Survival [0085] Mice were infected with a ~1 LD90 of influenza A/Texas/36/91 (H1N1) and treated with 100 or 40 mg/kg of UV-12 or UV-28 three times daily for 10 days. Survival in each infected treatment group, calculated as percent survival versus days postinfection, is shown in Figure 1 and Table 7. The infected groups which were treated with 10 100 mg/kg of both UV-12 and -28 showed 100% survival. The infected groups that were treated with 40 mg/kg of UV-12 and -28 displayed 20 and 60% survival, respectively.

The untreated control group showed 0% survival and was ail 'found-dead' or 'euthanized' by day 7 post-infection.

- 17[0086] Figure 1 is a plot presenting survival data of infected mice grouped by treatment.

Table 7 présents results of analysis of survival of infected mice. The survival data plotted in Figure 1 were analyzed using the Mantel-Cox (Log rank) test in GraphPad Prism.

Table 7

Group	Mean Survival (Days)	% Survival	P value to water control
Water	7	0	N/A
UV-12 100 mg/kg	>13	100	<0.0001
UV-12 40 mg/kg	9	20	<0.0001
UV-28 100 mg/kg	>13	100	<0.0001
UV-28 40 mg/kg	>13	60	<0.0001

Biométrie analysis [0087] During the course of this study, individual weights, health scores, and températures were monitored daily for each group. The average weights for each group of mice are shown in Figure 2. Every animal was tagged with a chip to perform daily température readings using a BMDS scanner; the average températures are shown in Figure 3. The health scores are shown is Figure 4.

CONCLUSIONS [0088] Influenza-infected mice were treated with 100 or 40 mg/kg of UV-12 or UV-28 via oral gavage three times daily for 10 days. Both groups that were treated with 100 mg/kg showed 100% survival, and groups that were treated with 40 mg/kg showed 60 and 20% survival for UV-28 and UV-12, respectively. While UV-28 did appear to show better efficacy at 40 mg/kg, it appears to be more toxic than UV-12 (Figure 4). While mice that were treated with 100 mg/kg of UV-12 fully recovered from the infection and retumed to normal, mice that were treated with 100 mg/kg of UV-28 never recovered their health score and remained 'ruffled' for the entire course of the study. In combination with a higher health score, indicating morbidity, the mice that were treated with UV-28 did not recover their weight as well, while mice treated with UV-12 almost completely

- 18recovered. Mice that were treated only with vehicle ail succumbed to infection by day 7 post-infection, displaying 0% survival.

EXAMPLE 2

Survival Analysis of UV-12 in A129 ADE Model [0089] Purpose: This study determined the efficacy of UV-12 in promoting survival of mice challenged with dengue virus. Compound were given by the oral route (3x per day intragastric via oral gavage - IG) for a total number of 7 days after the start of dosing. The experiment used the A129 ADE model of infection. (Prestwood TR, Morar MM, Zellweger RM, Miller R, May MM, Yauch LE, Lada SM, Shresta S.Gamma interferon (IFN-γ) receptor restricts systemic dengue virus réplication and prevents paralysis in IFNα/β receptor-deficient mice. J Virol. 2012 Dec;86(23): 12561-70.) Animais received the virus challenge dose ~1 LD90 on day 0. The first dose was given 0.5-1 hr pre-virus challenge. Survival was measured until 30 days after infection.

Iminosugar candidate: UV-12.

Experimental Design for the Study:

[0090] Control, H₂O DENV (S221 ) [ 10 mice] [0091] UV-12, (100 mg/kg/dose) + DENV (S221) [10 mice] [0092] Mice: Sex matched 5-6 weeks old A129 (129/SV IFN-α, -β receptor'⁷')

Administration Route:

[0093] Iminosugar: Orally 3 x day, (gavage (IG)) every 8 hours

Antibody: IP

Virus: IV [0094] Antibody and Iminosugar Compound were given simultaneously, then virus within 30 minutes Virus Challenge:

[0095] Antibody: 100 pg 2H2 (IgG2a anti-DENVl-4 prM) from ATCC, day 0 and day 1 in 40 ul [0096] Virus: DENV2 Strain S221 (v512) (Zellweger RM, Prestwood TR, Shresta S.

-19[0097] Enhanced infection of liver sinusoïdal endothélial cells in a mouse model of antibody-induced severe dengue disease. Cell Host Microbe, 2010 Feb 18;7(2): 128-39) [0098] Dose: 1E11 GE (genomic équivalents) per animal

Read-out:

[0099] Animal survival. Animais displaying severe illness (as determined by 20% weight loss, extreme lethargy, ruffled coat, or paralysis) are euthanized.

Resources:

[0100] A129 mice, 2H2 antibody, and S221 virus were supplied by Sujan Shresta, La

Jolla Institute for Allergy and Immunology

Supplied by Unither Virology: UV-12 [0101] Figure 5 présents results of this study. Mice were treated with UV-12 diluted in 50 uL water orally, three times per day for a total of 7 days. Initiation of treatment was 1 hr before virus intravenous challenge. UV-12 treated A129 mice displayed an increased survival compared to control animais that was only orally administered 50 uL of water three times per day.

EXAMPLE 3

Efficacy of selected iminosugars in Mice: Influenza A/Texas/36/91 (H1N1) Challenge [0102] Study Summary: This study analyzed the toxicity and efficacy of UV-8, UV-9,

UV-10, UV-11, and UV-12 in mice during an H1N1 influenza infection. Previously, studies that were performed with oral delivery of UV-4B ter in die (TID) at 100 mg/kg resulted in efficacy against H1N1 and did not show any discemible signs of toxicity. In the current study, we examined delivery doses of UV-4B, UV-8, UV-9, UV-10, UV-11, and UV-12 at 100 mg/kg, delivered ter in die by the oral route (intragastric via oral gavage or IG ). Small groups (n=3) that were included to examine gross toxicity received 100 mg/kg of the iminosugars but without a viral challenge. UV-4B, UV-8, UV-9, UV10, UV-11, or UV-12 was delivered to the animais starting at 1 hour before intranasal infection (IN ) with ~1 LD90 of influenza A/Texas/36/91 (H1N1). Animais were then treated TID for 10 days total (days 0-9 post infection). Efficacy was evaluated by

-20comparing survival, température changes, and weight gain/loss to an infected untreated control group. Mice dosed with UV-8, -9, -10, or -11 did not show any improvement in survival over the untreated control group, while the group dosed with UV-12 and the positive control group dosed with UV-4B both showed a significant increase in survival.

I. Introduction [0103] This study aimed to détermine the efficacy of UV-8, UV-9, UV-10, UV-11, and

UV-12 when administered orally at 100 mg/kg TID against a léthal intranasal infection with influenza virus A/Texas/36/91 (H1N1) in the BALB/c mouse model. In addition to the efficacy arm, each of the iminosugars was tested in a small group of animais with the 10 same treatment regimen (100 mg/kg, oral gavage, TID) in the absence of a viral infection to examine gross toxicity of each analog (general health, weight, température, and mortality évaluations).

II. Materials and Methods

Materials

Table 8. Test articles

Name	Concentration	Solvent	Additional Info
UV-4B	100 mg/kg (2mg/dose)	H2O	HCl sait
UV-8	100 mg/kg (2mg/dose)
UV-9	100 mg/kg (2mg/dose)
UV-10	100 mg/kg (2mg/dose)
UV-11	100 mg/kg (2mg/dose)
UV-12	100 mg/kg (2mg/dose)

Table 9. Viruses for Challenge

Name	Strain	Stock titer
Influenza A virus	A/Texas/36/91 (H1N1)	2.8x105 PFU/mL

-21 Table 10. Animais used

Species	Strain	Age	S ex	Vendor	Additional Info
Mouse	BALB/c	4-6 weeks	F	Charles River	7 groups n=10 5 groups n=3

Table 11 : Equipment

Item	Vendor
Syringes	BD
Animal Housing	Inno Vive
Plastic Feeding Tubes	Instech Solomon
Biodata chips and scanner	Bio Medic Data Systems
Ohause scale	Ohause

Study design [0104] UV-8, -9, -10, -11, -12, and UV-4B were prepared in H₂O at 20 mg/ml for a delivery dose of 100 mg/kg, assuming ~20g mice given 0.1 mL of compound, Groups of BALB/c mice were treated at one hour before (-1 h) intranasal infection with ~1 LD90 of INFV A/Texas/36/91 (H1N1) and then compounds were administered three times daily for a total of 10 days (see Table 12 for study design summary). Weights, température, and survival were monitored and used for évaluation of protective efficacy and toxicity of each analog.

[0105] Table 12: Mouse groups for the study. The time point of initial dosing (treatment start relative to the infection), dosing regimen and the dose levels/routes are listed. Mice were dosed once or twice per day for total of 10 days.

Group	Mouse Strain	N	Treatment	Delivery route/frequency on days 0-9 (10 days total starting at lh before infection)	Challenge	Readouts
1	Female BALB/C 4-6 weeks of âge	10	Vehicle	IG lOOul, TID	1 LDqq of Influenza A/Texas/36/91 (H1N1)	• Endpoint is day 14, death, or >30% weight loss. • Animais displaying severe illness (as determined by >30% weight loss, extreme lethargy, or paralysis) will be euthanized. • Température and weights to be taken daily for 14 days.
2	10	100 mg/kg UV-4B
3	10	100 mg/kg UV-8
4	10	100 mg/kg UV-9
5	10	100 mg/kg UV-10
6	10	100 mg/kg UV-11
7	10	100 mg/kg UV-12
8	3	100 mg/kg UV-8	None (Toxicity assessment)
9	3	100 mg/kg UV-9
10	3	100 mg/kg UV-10
11	3	100 mg/kg UV-11
12	3	100 mg/kg UV-12

STANDARD PROTOCOLS

Standard Protocol for intranasal infection of mice [0106] 1. Female 4-6 week old BALB/c mice were housed in groups of 3-5 mice.

Mice were quarantined at the study site (Noble Life Sciences, Gaithersburg. MD) for at least 3 days prior to the start of the study.

-23 [0107] 2. Food and water was provided ad libitum.

[0108] 3. The groups of mice challenged with influenza were anesthetized with 5%

Isofluorene and maintained at 2.5% prior to intranasal inoculation with ~1 LDgo of INFV in 100 pL PBS.

[0109] 4. After the infection mice were placed back into their cages for observation and dosing.

Protocol for oral gavage or injection of mice for compound delivery [0110] 1. Mice were treated starting at 1 hour before infection with 100 pL of compound in H2O (see Table 12 for dosing regimens) three times a day for 10 days total with 100 mg/kg of UV-4B, UV-8, UV-9, UV-10, UV-11, or UV-12 compound given by the oral route (intragastric via oral gavage).

[0111] 2. After dosing, mice were retumed to their cages and monitored for any distress related to dosing.

Observation of mice [0112] 1. Mice were observed through 13 days post infection ( 14 days total, 0-13 days post infection).

[0113] 2. Mice were weighed daily on an Ohause scale and the weights were recorded.

[0114] 3. Ail animais had chips implanted that monitored the body température. The températures were recorded daily.

[0115] 4. Survival and health of each mouse was evaluated three times a day using a scoring System of 1-7.

[0116] 5. Mice were euthanized when scored at 5 or above (Very Sick; Very Ruffled

Coat; Closed, inset eyes; Slow to no movement; Will retum to upright position if put on its side; extremely léthargie).

III. RESULTS

Survival [0117] Mice were infected with a ~1 LD90 of Influenza virus A/Texas/36/91 (H1N1 ) one hour after their first dose of UV-4B or UV-4 analogs, as outlined above. Survival tables, calculated as percent survival versus days post-infection, are shown in Figure 6. As

-24expected based on previous studies, groups that were dosed orally TID with UV-4B at 100 mg/kg showed a survival rate of 100% and a MTD of >13 days (Figure 7). Mice that were dosed orally with UV-12 at 100 mg/kg showed a survival rate of 90%, and a MTD of > 13 days (Figure 6, 7). Mice treated with UV-8, UV-9, UV-10, or UV-11 displayed a MTD of 10.5, 7, 7.5, and 8 days, respectively, and survival rates of 0% with the exception of UV-8 (30%) (Figure 6, 7). Négative control mice dosed with vehicle (H2O) demonstrated 30% survival and a MTD of 9 days (Figures 6, 7).

[0118] Mice which received UV-8, -9, -10, -11, or -12 at 100 mg/kg without a viral challenge to examine gross toxicity displayed 100% survival (data not shown).

Biométrie Analysis [0119] During the course of this study, individual weights and températures were monitored daily for each group. The average weights for each group of mice are shown in Figure 8 with statistical analysis shown in Figure 9. The average températures are shown in Figure 10 with statistical analysis shown in Figure 11.

[0120] As a second biométries the animais' températures were evaluated. Every animal was tagged with a chip to perform daily température readings using a scanner. The graphs in Figure 10 show the body températures for each test group. Significance shown against the vehicle control is indicated in Figure 11.

CONCLUSIONS [0121] The group of infected mice dosed orally TID with 100 mg/kg of UV-4B exhibited

100% survival, where the groups which were orally dosed with 100 mg/kg of UV-9, UV10, and UV-11 exhibited 0% survival. Statistical analysis on weights and températures for these groups was not performed due to a lower survival rate and MTD than the vehicle control. Mice dosed orally with UV-8 exhibited 30% survival, and thus no significant différence from the vehicle control group. Mice dosed orally with 100 mg/kg of UV-12 exhibited 90% survival, as well as significant increases in overall température and weight. UV-12 also showed mild toxicity with steady weight loss in the uninfected group, but uninfected mice dosed with UV-12 did not lose more than 10% weight overall and they were able to fully recover after the dosing regimen had been completed. Statistical analysis was not performed on any of the parameters examined for the uninfected groups

-25of mice (gross toxicity) as the number of mice per group was limited (n=3) there was no uninfected, undosed control group for comparison.

EXAMPLE 4

Survival Analysis of UV-4 and UV-12 in ADE Model [0122] Purpose: This study determined the efficacy of UV-4 and UV-12 in promoting survival of mice challenged with dengue virus. Ail compounds were given by the oral route (3x per day intragastric via oral gavage - IG) for a total number of 7 days after the start of dosing. The experiment used the ADE model of infection developed in the lab (Zellweger et al. CellHostMicrob 7; ppl-12 (2010)). Animais received the virus challenge dose ~1 LDgo on day 0. The first dose was given 0.5-1 hr pre-virus challenge. Survival was measured until 3 days after dosing was completed. UV-4 HCl sait was used in this study, which is equal to U V-4B.

[0123] Iminosugar candidates:

1. N-9-methoxynonyl-deoxynojirimycin (UV-4) (HCl Sait), UV-4B

2. UV-12

3. Control, H₂O [0124] Experimental Design for the Study

1. Control, H₂O + DENV (S221 ) [7 mice]

2. UV-4, 1 mg (50 mg/kg/dose) + DENV (S221) [5 mice]

3. UV-12, 1 mg (50 mg/kg/dose) + DENV (S221) [5 mice] [0125] Mice: Sex matched 5-6 weeks old [0126] AG129 (129/SV IFN-α, β, and y-receptor-/-) breeded at LIAI by Sujan Shresta (also publically available by The Jackson Laboratory, Bar Harbor, Maine) [0127] Route:

[0128] Iminosugar: Orally 3 x day, (gavage (IG)) every 8 hours [0129] Antibody: IP (Intraperitoneal) [0130] Virus: IV [0131] Antibody and Compound were given simultaneously, then virus within 30 minutes [0132] Virus Challenge:

[0133] Antibody: 5pg 2112 (anti-prM) available from ATCC [0134] Virus: DENV2 Strain S221 (v476) (Zellweger R M, Prestwood TR, Shresta S.

-26[0135] Enhanced infection of liver sinusoïdal endothélial cells in a mouse model of antibody-induced severe dengue disease. Cell Host Microbe, 2010 Feb 18;7(2): 128-39) Dose: 1E9 GE (genomic équivalents) per animal

Read-out:

[0136] Animal survival. Animais displaying severe illness (as determined by 20% weight loss, extreme lethargy, ruffled coat, or paralysis) were euthanized.

[0137] Figure 12 présents results of this study. Ail groups were treated with the same dose of compounds:

[0138] Experimental Design for the Study

1. Control, H2O + DENV (S221 ) [7 mice]

2. UV-4,1 mg (50 mg/kg/dose) 1 DENV (S221) [5 mice]

3. UV-8, 1 mg (50 mg/kg/dose) + DENV (S221) [5 mice]

4. UV-9, 1 mg (50 mg/kg/dose) + DENV (S221) [5 mice]

5. UV-10, 1 mg (50 mg/kg/dose) + DENV (S221) [5 mice]

6. UV-11, 1 mg (50 mg kg/dose) + DENV (S221 ) [5 mice]

7. UV-12, 1 mg (50 mg/kg/dose) + DENV (S221) [5 mice] [0139] This study was to détermine the efficacy of UV-4 and its analogues in promoting survival of mice challenged with dengue virus. Ail compounds were given by the oral route (3x per day intragastric via oral gavage - IG) for a total number of 7 days after the start of dosing. The experiment used the ADE model of infection (Zellweger et al. CellHostMicrob 7; ppl-12 (2010)). Animais received the virus challenge dose ~1 LD90 on day 0. The first compound dose began 0.5- 1 hr pre-virus challenge. Survival were measured until 3 days after dosing.

[0140] Although the foregoing refers to particular preferred embodiments, it will be understood that the présent invention is not so limited. It will occur to those of ordinary skill in the art that various modifications may be made to the disclosed embodiments and that such modifications are intended to be within the scope of the présent invention.

[0141] Ail of the publications, patent applications and patents cited in this spécification are incorporated herein by reference in their entirety.

Claims (16)

1. -27WHAT IS CLAIMED IS:

   1. A compound of formula (I):

   R₂ r₃or a pharmaceutically acceptable sait thereof, wherein Wm and R4.3 are each independently selected from hydrogen and C1-3 alkyl groups and wherein at least one of R4.3 is not hydrogen

   10 or wherein R₂ and R3, when taken together, are -CH₂-, -CH₂CH₂-, or -CH₂-CH₂-CH₂-,
2. 2.

   The compound of formula (I) according to claim 1, wherein each of Wm is hydrogen.

   -283. The compound of formula (I) according to claim, wherein the compound is:

   or a pharmaceutically acceptable sait thereof.
3. 4. The compound of formula (I) according to claim 1 wherein the compound is:

   or a pharmaceutically acceptable sait thereof.
4. 5. A pharmaceutical composition comprising a) a pharmaceutically effective amount of the compound of formula (I) according to claim 1 and b) a pharmaceutically acceptable carrier.

   10 6. Use of the compound of formula (I) according to claim 1 or a pharmaceutically acceptable sait thereof in the manufacture of a médicament for treating or preventing a Dengue viral infection

   -297. The use of claim 6, wherein the dengue viral infection is by or associated with a Dengue 2 virus.
5. 8. The use of claim 6, wherein the compound of formula (I) is

   5 or a pharmaceutically acceptable sait thereof.
6. 9. The use of claim 6, wherein the subject is a mammal.
7. 10. The use of claim 9, wherein the subject is a human.
8. 11. Use of the compound of formula (I) according to claim 1 or a pharmaceutically acceptable sait thereof, in the manufacture of a médicament for treating a disease or

   10 condition caused by or associated with a virus belonging to the Orthomyxoviridae family
9. 12. The use of claim 11, wherein the virus is an Influenza virus.
10. 13. The use of claim 12, wherein the virus is an Influenza A virus.
11. 14. The use of claim 13, wherein the virus is a H3N2 subtype of the Influenza A virus.
12. 15. The use of claim 13, wherein the virus is a H1N1 subtype ofthe Influenza A virus.
13. 16. The use of claim 11, wherein the compound of formula (I) is or a pharmaceutically acceptable sait thereof.

    5
14. 17. The use of claim 11, wherein the compound of formula (I) is the compound or a pharmaceutically acceptable sait thereof.
15. 18. The use of claim 11, wherein the subject is a mammal.
16. 19. The use of claim 18, wherein the subject is a human being.

    1/19 %, of Original Weight Percent survival