Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/075—Ethers or acetals
  • A61K31/085—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals

Definitions

• This invention relates to use of 2-chloro-l-[[4-[(2,6-dichlorophenoxy)methyl]- phe ⁇ yl]methoxy]-4-methoxybenzene for the preparation of a medicament for the selective treatment and/or prevention of enteroviral-caused infections in humans.
• Enteroviruses are members of the RNA-containing Picomavirus Family listed i Table 1 and are known to cause a wide variety of diseases and deaths in humans .
• drug may exhibit in vitro and in vivo activity against enteroviruses.
• such a drug must have broad activity within the enterovirus genus since the same disease may be caused by a variety of different enteroviruses (see Table 2).
• enteroviruses Although about 70 immunotypes of enteroviruses are known, surveillance data from the Centers for Disease Control (CDC) on nonpolio-enteroviruses from 197 to 1983 show that enteroviruses from the following 15 common immunologic groups account for 65-89% of the isolates for a given year in the United States: echoviruses (echo) 3,4,5,6,7,9,11,24 and 30; coxsackievirus A (CVA) 9; and coxsackieviruses B (CVB) 1 ,2,3,4, and 5.
• echoviruses echo
• CVA coxsackievirus A
• CVB coxsackieviruses B
• This invention provides the use of 2-chloro-l-[[4-[(2,6-dichlorophenoxy)methyl]- phenyl]methoxy]-4-methoxybenzene represented by formula I for the preparation of a medicament for the of treatment and/or prevention of enteroviral infections in humans.
• the present invention also provides a pharmaceutical composition for selectively treating and/or preventing enteroviral infections in humans which comprises an anti-enterovirally effective amount of 2-chloro-1-[[4-[(2,6- dichlorophenoxy)methyl]phenyl]methoxy]-4-methoxybenzene represented by formula I suspended in a vegetable oil and optionally containing at least one pharmaceutically acceptable excipient.
• This invention also provides a method of selectively treating and/or preventing enteroviral infections in humans caused by an enterovirus selected from the group consisting of coxsackieviruses A and B, echoviruses, enteroviruses and polioviruses comprising administering to a human in need of such treating and/or preventing an anti-enterovirally effective amount of 2-chloro-l-[[4-[(2,6-dichlorophenoxy)methyl]- phenyl]methoxy]-4-methoxybenzene represented by formula I
• This invention further provides a method of selectively treating and/or preventing an enteroviral infection in humans selected from the group consisting of enteroviral respiratory diseases, enteroviral aseptic meningitis, enteroviral meningoencephalitis, chronic enteroviral infections in a gammaglobulinemics and acute hemorrhagic conjunctivitis caused by enterovirus-70 which comprises administering to humans in need of such treating and/or preventing an antienterovirally effective amount of 2-chloro-l-[[4-[(2,6- dichlorophenoxy)methyl]methoxy]-4-methoxybenzene and represented by formula I
• the compound of formula I is a potent, broad spectrum, orally-active anti-enteroviral agent which has significant activity against enteroviruses believed to be responsible for mos symptomatic enteroviral infections in the United States. Similar activity is expected against enteroviral infections found in other countries.
• CNS central nervous system
• the antiviral activities of the compound of formula I were determined against laboratory strains of enteroviruses and less frequently isolated enteroviruses as well as hepatitis A virus (HAV), which is classified in its own genus.
• HAV hepatitis A virus
• the average IC5 0 of the compound of formula I against 30 enteroviruses (including polio 1 and polio 2) w 0.97 ⁇ g/ml (2.29 ⁇ M) and an EC ⁇ o 0.8 ⁇ g/ml (1.89 ⁇ M) was obtained for these viruses
• some of the viruses are less frequently isolated in the United States, severa of these viruses are of considerable importance to other countries and especially those in tropical climates. For example, poliomyelitis in populations not receiving the poliovirus vaccine remains a major health problem.
• enterovirus 70 a major cause of acute hemorrhagic conjunctivitis.
• enterovirus 70 a major cause of acute hemorrhagic conjunctivitis.
• the compound of formula I is inhibitory: five strains of polio 1 (including recent human isolates) had IC5 0 values ranging from 0.009 to 0.04 mg/ml (0.021-0.094 ⁇ M); the average IC5 0 for the polio 2 EFI strain in HeLa cells was 0.02 ⁇ g/ml (0.047 ⁇ M); and 4 strains of entero 70 were inhibited in a range of 0J to 0.4 ⁇ g/ml (0.24-0.9 ⁇ M).
• the compound of formula I was inactive against 19% (7/37) of less frequent isolates and laboratory strains at the maximum concentration tested. This result is largely due to the inactivity of the compound of formula I against isolates of coxsackievirus A. Without testing additional coxsackievirus A imunotypes, it is not possible to establish whether the activity of the compound of formula I against these viruses is representative of other viruses in these immunotypes.
• cytotoxicity IC value divided by the antiviral IC5 0 value.
• cytotoxicity IC value divided by the antiviral IC5 0 value.
• enteroviruses exhibiting measurabl activity in the plaque assay only 8.7% had Tls ⁇ 10 and 5.4% had Tls > 11 and ⁇ 25, while 75J% has Tls > 51. Therefore, for the majority of enteroviruses tested, the antiviral activity of the compound of formula I is independent of possible adverse effects of the compound of formula I on cells and cytotoxicity does not contribute to th antiviral activity of the compound of the formula I.
• the compound of formula I is efficacious against both echovirus-4 ("echo 4") and coxsackievirus A9 ("CVA9”) viral infections when orally administered to mice in a therapeutic (+3.5 hr.) regimen at 20 mg/kg/day (4 doses of 5 mg/kg).
• mice treated with the compound of the formula I in a therapeutic regimen infected intracranially with CVA9 at 1,200 PFU have an AUC activity of 71.7%.
• the AUC activity decreases from 75.2% to 34.4% as the infectious challenge increases 3 orders of magnitude from 980 PFU to 980,000 PFU.
• the AUC activity decreases from 71.7% to 35.8% as the infection challenge increase 3 orders of magnitude from 1,200 PFU to 1,200,000 PFU
• the compound of formula I was also effacious against the polio-2-viral infectio in the murine model in a therapeutic regimen when oral administration of the compound of formula I is 3.5, 6, and 24 hours after the mice were infected intracranially with polio-2 virus.
• mice infected with 98,000 PFU of Echo-4 and 1,200,000 PFU of CVA9 exhibited AUC activity of 59.0% and 50%, respectively.
• Serum levels after oral administration of the compound of formula I in corn oil dosages of 1 , 5, 20 and 30 mg/kg were measured in mice, rats, dogs, and monkeys. All species showed a dose-related increase in AUC, although the AUC did not increase at the higher dose. Similarly, the CM AX increases with the dose of the compound of formula I in all species. Among the four species, the highest serum levels of the compound of formula I occurred in beagles. The serum half-life of the compound of formula I was 4.7 hours in mice, but was dramatically greater in the higher species: 26 hours and > 24 hours in beagles and monkeys, respectively.
• Th time to CMAX had a similar value in mice, rates and dogs (i.e., 2-4 hrs.), but it was longer in monkeys (8-12 hrs.).
• the shorter TMAX in dogs suggests that th serum life of 26 hrs. is independent of absorption in this species.
• the expected human levels of the compound of formula I should achieve antiviral levels (1 mg/ml) for 24 hours at a single dose of 10 mg/kg administered orally to humans.
• Enteroviruses representing the 15 most commonly isolated immunotypes in the USA were obtained from Centers for Disease Control (CDC), Atlanta, Georgia. Normally, five viruses from each immunotype were obtained. Other, less frequently isolated enteroviruses and the rhinoviruses were obtained from the American Type Culture Collection (ATCC), Rockville, Maryland. Enteroviruses and rhinoviruses were propagated in an appropriate cell line, eg HeLa, (human), BGMK (Buffalo greemh monkey), or RD (human rhabdosarcoma) cells and working stocks were prepared as cellular lysates. The standard procedures used for quantitating antiviral activity of the compoundd of formula I in vitro and in vivo are given herein below.
• Pre-mix plaque assay 150 plaque forming units (PFU; infectious particles) were mixed with test compound (0.0001-50 mg/ml) in Eagles' modified minimal essential medium (EMEM) for 45 min. and added to monolayers of appropriate cells. After 45 min. at 33°C, the inoculum was aspirated, the cells were washed, and plaque analysis was performed.
• PFU plaque forming units
• EMEM Eagles' modified minimal essential medium
• the number PFU was determined at each concentration and plotted as a percentage of control PFU using Cricket Graph ® or Wingz ® graphics programs and the individual values were connected by a line through each value.
• a perpendicular line from the intersection of the PFU curve and a horizontal line representing 50% inhibition were connected to the abscissa.
• the value on the abscissa which intersects the perpendicular line represents the IC50 value. Values indicative of percentage inhibition (ordinate) are represented linearly, while the concentrations of test molecule (abscissa) are represented logarithmically.
• CPE cytopathic effect
• CPE cytotoxicity assay using MTT The 50% inhibitory concentration (IC50), referred to as the cytotoxic IC 50 was determined as follows: Uninfected monolayers of cells in 96-well microelisa plates were treated with the compound of formula I (0.025-50 mg/ml) for prescribed periods of time prior to lysis of the monolaye and treatment with MTT. The IC50 value is determined as described for the pre-mix plaque assay
• Therapeutic index The therapeutic index "Tl" of the compound of formula I, which is dependent on the enterovirus tested as well as the type of cell in which the test was performed, was determined by two procedures: (i) The antiviral IC5 0 was determined by pre-mix plaque assay and the cytotoxic IC50 was determined by the MTT assay. In the latter, test molecule was present for 45 min. prior to removal by aspiration and the period of incubation prior to MTT treatment was identical with that o cells used in the plaque assay, (ii) The antiviral IC 50 and the cytotoxic IC50 values were determined by CPE assay using MTT to determine the endpoints.
• the MTT assay is suitable for quantitating the antiviral effect of a test agent by indirectly assessing cellular metabolic functions.
• concentrations in which an antiviral agent inhibits viral replication relatively more cells remain viable as measured by the MTT assay compared to a less effective antiviral agent that permits comparatively more viral replication and subsequent killin of cells.
• the MTT assay is particularly advantageous for determination of Tl since the virus and the antiviral molecule are tested under identical conditions with an equivalent end-point methodology.
• mice Studies with mice (as well as other animals) were performed as recomended in the Guide for the Care and Use of Laboratory Animals (NIH Publications 85-23). Groups of 15-25 male mice (16-20 g; Harland Sprague Dawley) were infected intracranially with poliovirus 2, echovirus 4, or coxsackievirus A9 as described in McKinlay and Steinberg (1986 Antimicrob. Agents Chemothen. 29: 30-32). Treatment with the compound of formula I was by oral gavage (0.3 ml in corn oil) or other oleogacious materials such as peanut oil could also be used. Survival was monitered daily for 21 days.
• AUC activity [(experimental AUC-placebo AUC) divided by (maximum AUC-placebo AUC) x 100].
• Dosing Formulation in a preferred aspect of the present invention, a 2 mg/ml stock of the compound of formula I was prepared as a suspension in com oil for studies in which the compound was administered orally. On each day of the study, the stock was diluted with the correct amount of corn oil to give the appropriate mg/kg dosage based on the average weight of the mice for that day.
• the amount of the compound of formula I which may be suspended in one milliliter of com or peanut oil to form a pharmaceutical compositions is in the range of about 100mg to about 1 gram
• the pharmaceutical compositions of the present invention may also contain the typical pharmaceutically acceptable excipients, e.g., stabilizers, surfactants and antioxidants.
• typical antioxidants include butylated hydroxyanisole “BHA” and butylated hydroxytoluene "BHT" which are used to prevent vegetable oils from becoming rancid.
• Stabilizers e.g., lecithin and surfactants, e.g., polysorbate 60 or Arlacel 186 (a mixture of glyceryl oleate and propylene glycol available from ICI Americas, Inc., Wilimington, Delaware) may also be added.
• surfactants e.g., polysorbate 60 or Arlacel 186 (a mixture of glyceryl oleate and propylene glycol available from ICI Americas, Inc., Wilimington, Delaware) may also be added.
• Food matrix was prepared by mixing the compound of formula I in 1% lechitin and then adding it to a hot solution of 2x Jello ® cherry flavored gelatin (but any gelatin could be used) plus sifted ground food (Purina® Mouse chow). The final concentratio of the compound of formula I in the matrix for a 10 mg/kg dose was calculated based on a retriction of 10 grams of food a day per mouse (a 1 mg/kg dosage was present in each g of food matrix). In the efficacy studies, food matrix was made every four days and corrected for the weight change in the groups. Food was placed in trays at the bottom of cages for consumption.
• the compound of formula I was prepared by the below-described Williamson ether synthesis as described in Example 15a of Orally Active Antiviral Compounds, International Publication No. WO 92/22520, published 23 Dec. 1992, International Appln. No. PCT/US92/04961.
• A. Add 5g of 2-chloro-4-methoxyphenol to a mixture of 17g of ⁇ , ⁇ '-dibrom p-xylene to a stirred mixture of 2.5g of 50:50 (w/w) NaOH:H2 ⁇ and 50mL of DMF. Sti the so-formed mixture for 4 hours. Partition the reaction mixture with methylene chloride and water. Separate the organic layer and remove the solvent by rotary evaporation to obtain a solid residue.
• the compounds of formula I may be administered by any conventional mode o administration by employing an antienterovirally effective amount of a compound of formula I for such mode.
• the dosages may be varied depending upon the requirements of the patient in the judgement of the attending clinician, the severity of the condition being treated and the particular mode of administration being employe Determination of the proper dosage for a particular situation is within the skill of the a Treatment can be initiated with similar dosages which are less than the optimum dos of the compound. Thereafter, the dosage should be increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired.
• the compound of formula I may be administered for example, orally, intravenously, intraventricularly, subcutaneously, intramuscularly, or via rectal suppository; intravenous dosage should preferably comprise continuous drip infusion
• the pharmaceutical composition of this invention contains an anti-enterovirall effective amount of the compound of formula I suspended in a vegetable oil, e.g., cor oil or peanut oil and optionally a pharmaceutically aceptable excipient.
• a vegetable oil e.g., cor oil or peanut oil
• a pharmaceutically aceptable excipient e.g., a pharmaceutically acceptable excipient.
• the preferred carrier is corn oil which may be used alone or in combination with stabilizer e.g., lecithin, or surfactants, e.g., polysorbate 60 or Arlacel 186.
• the anti-enterovirally effective amount of the compound of formula I is in the range of about 1 to 90 mg/kg of body weight per day. A dose of about 10 to 15 mg/kg given one to six times a day, in equal or divided doses is preferred. A dose of 10 mg/kg day is more preferred.
• the exact dosage of the compounds of this invention which is administered is dependent, in the judgement of the attending clinician, upon a variety of factors, e.g. the age and weight of the individual being treated, the mode of administration, the potency of the administered compound, the indication for which the durg is administered and the severity of the ailment being treated.
• the compound of formula I Because of the broad anti-enteroviral activity of the compound of formula I, the high degree of potency of the molecule against enteroviruses, the efficacy afforded to enterovirus-infected mice following oral administration of the molecule, and measurable blood levels in dogs and primates following oral dosage of the molecule, we propose that the compound of formula I will have utility in treating and/or preventing human enterovirus infections, including the diseases listed in Table 2.
• enteroviral respiratory disease ie., summer cold, summer grippe
• enteroviral aseptic meningitis enteroviral meningoencephalitis
• enteroviral meningoencephalitis chronic enterovirus infection in agammaglobulinemics
• acute hemorrhagic conjunctivitis caused by enterovirus-70.
• Patient Population Selection of a patient population experiencing enterovirus disease would be based on: (i) pathognomic symptomatology associated with some enterovirus diseases (e.g., acute rhinitis/cold symptoms during the summer, skin lesions in hand-foot-and-mouth diseases, vesicle in pharynx, or myalgia associated with pleurodynia); (ii) confirmation of enterovirus infection by isolation of virus using cell culture techniques ; (iii) PCR (polymerase chain reaction) diagnosis of viral genetic material (e.g., in cerebral spinal fluid for enteroviral aseptic meningitis); immunological techniques such as fluorescent antibody enzyme-linked imunoabsorbant technolgy; or a combination of the above.
• enterovirus diseases e.g., acute rhinitis/cold symptoms during the summer, skin lesions in hand-foot-and-mouth diseases, vesicle in pharynx, or myalgia associated with pleurodynia
• Efficacy Evaluation Evaluation of the efficacy of the compound of formula I in humans affected with enteroviral disease is measured by reduction of symptomatology and/or the viral tissue load associated with a particular enteroviral illness.
• parameters that could be measured to assess efficacy include: (i) decrease of symptoms associated with summer cold; decrease in shed virus, decrease in mucous weight, decrease in fever, and decrease in myalgia compared t placebo-treated individuals; (ii) for aseptic meningitis: reduction of time in hospital, reduction of headache & fev «r, decrease in time to normal neurological findings compared to placebo patients; (iii) for hemorrhagic conjunctivitis: improvement in sight, clearing of conjunctiva more rapidly compared to placebos.
• Other parameters t assess the efficacy of the compound of formula I may need to be established for each enteroviral disease under clinical testing, in the opinion of the attending clinician.
• the compound of formula I Against 154 enteroviruses representing 15 common isolated enteroviral immunotypes, the compound of formula I exhibits an average IC5 0 (50% inhibitory concentration) of 0.98 ⁇ g/ml (2.31 ⁇ M) in the plaque assay (Table 3).
• the MTT assay as a measure of adverse effects produced by the compound of formula I in uninfected cells, no adverse effects were observed in cell cultures at concentrations of the compound of formula I equal to or greater than 51 times the antiviral IC5 0 concentrations for 75% of the enteroviruses tested (138 of 184 viruses, including common clinical isolates and laboratory strains of enteroviruses).
• the antiviral activity of the compound of formula I is not due to cytotoxic effects of the compound.
• the compound of formula I exhibited an activity (IC 50 ) equal to or less than 10 ⁇ g/ml (23.6 ⁇ M) agains 38% of rhinoviruses which were surveyed.
• RNA viruses or DNA viruses comprising ten viral families which were tested, including influenza virus, human immunodeficiency virus, herpes simplex virus, and adenovirus.
• the compound of formula I was orally active prophylactically (24 hrs. before infection) and therapeutically (3.5 hrs. after infection) against Echovirus 4, Coxsackievirus A9, and poliovirus 2 infections in murine models.
• the compound of formula I also exhibited increased activity in vivo as the size of the viral inoculum decreased and it reduced viral tissue titers in mice in both therapeutic and proprophylactic regimens.
• the compound of formula I is orally bioavailable after a single oral dose: In mice receiving 1 mg/kg, oral absorption was 78% and absolute oral bioavailability wa 50%. After oral administration to mice, the compound of formula I reaches inhibitory levels in the brain, the primary site of viral infection in the murine models. Blood level of the compound of formula I are also achieved in mice, rats, dogs, and monkeys following oral dosage. The half-life of the molecule in the serum increases from 4.7 hours in mice, to 26 hours in dogs.
• Hepatitis A hepatitis A virus ..d 1 Hepatitis A
• a Number refer to the number of viruses or number of viral immunotypes in a subgroup. D Abbreviations for the names of viruses are shown. In instances where numbers or viruses are missing (e.g.. CVA23), viruses having these numbers originally were incorrectly classified in the enterovirus genus. Upon reclassification, the numbers were dropped from the classification system foe enteroviruses
• Vilyuisk virus is associated with a neurodegenerative disease occurring in humans in Siberia (Casals, 1963; Lipton et al., 1983). Legend to Table 1. (continued)
• mice Lipton & Rozhon, 1986.
• CVB 1-6 echo 1-4, 6, 7, 9, 11, 16, 18, 19. 30; entero 70, 71 myocarditis, pericarditis, dilated CVB 1-5 cardiomyopathy exanthems (rubelliform, roseolifom, CVA 2, 4, 5, 9, 16; CVB 1 , 3, 4, 5; echo 9, herpetiform) 16; other less frequent echo types sepsis, systemic infection in neonates & CVB 1-5; echo 6, 9, 11, 14, 19, 31; CVA 2, infants 9, 16 less frequently
• Viruses shown in this column represent the most common enteroviruses isolated from individuals diagnosed with each disease, however other enteroviruses, which are not listed as being associated with the disease, may be capable of producing the same disease.
• CA Coxsackievirus A Immunotype.
• CB Coxsackievirus B Immunotype.
• Antiviral Activity was determined by per-mix plaque assay (see Materials and Methods Section).
• EC ⁇ o represents the concentration of the compund formula I that inhibits 80% of the viruses tested based on their IC50 values.
• c The 15 common enteroviral isolates are listed in Table 3.
• d Includes polioviruses 1-3 and enterovirus 70.

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• 1994
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