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WO2009076449A1 - Procédés et compositions de traitement des virus du groupe pox - Google Patents

Procédés et compositions de traitement des virus du groupe pox Download PDF

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Publication number
WO2009076449A1
WO2009076449A1 PCT/US2008/086246 US2008086246W WO2009076449A1 WO 2009076449 A1 WO2009076449 A1 WO 2009076449A1 US 2008086246 W US2008086246 W US 2008086246W WO 2009076449 A1 WO2009076449 A1 WO 2009076449A1
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virus
pharmaceutically acceptable
acceptable salt
subject
catecholic
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PCT/US2008/086246
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English (en)
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Ian Timothy Donald Petty
Justin Joseph Pollara
Scott M. Laster
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North Carolina State University
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Publication of WO2009076449A1 publication Critical patent/WO2009076449A1/fr

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    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group

Definitions

  • the present invention relates to compositions for use in treating or preventing poxviral infections, preferably for poxviral infections that are caused by a poxvirus that is a member of the orthopoxvirus genus.
  • the present invention also relates to methods of using the anti-poxvirus compositions.
  • Poxviruses are large DNA viruses that replicate in the cytoplasm of host cells. Poxviruses are the largest animal viruses and can even exceed the size of some bacteria. As currently recognized by the International Committee on Taxonomy of Viruses (ICTV), there are eleven genera of poxviruses. The orthopoxvirus genus is included among the subfamily of eight currently recognized genera by ICTV representing viruses that affect vertebrates. The orthopoxvirus genus includes members such as variola virus, monkeypox virus, cowpox virus (CPXV), and vaccinia virus (VAC). Variola virus is responsible for the devastating human disorder known as smallpox.
  • Smallpox was the most destructive disease in recorded history, estimated to have killed, crippled, or disfigured nearly 1/10 of all humankind. More than 300 million people succumbed to the disease in the 20 th century alone.
  • the variola virus is extremely infectious and leads to smallpox in more than 30% of individuals that become exposed to the virus. Smallpox is usually contracted through the respiratory tract by inhalation of aerosol droplets spread by individuals that have become infected with the virus. Once contracted, the virus spreads to the lymphoid tissues, such as lymph nodes, spleen, and bone marrow where large scale replication begins. The virus then migrates towards the epithelium cells where pustule formation occurs.
  • Pustular lesions result in extreme levels of epidermal shedding resulting in a systemic rash that can last anywhere from 2 to 3 weeks.
  • Variola major the more virulent form of smallpox, leads to the death of as many as 30% of individuals it infects. Those individuals that recover remain scarred with severely deformed skin for life.
  • Smallpox is a human-specific disease with no other known animal reservoirs. While smallpox has been eradicated in the human population through successful vaccination campaigns, there is concern that the virus could reemerge as an agent of bioterrorism. Routine vaccination of the population stopped after the World Health Organization officially declared smallpox eradicated in 1980. It is estimated that even those vaccinated against the disease retained immunity for only 10 years after receiving the vaccine. Therefore, the worldwide population at large would be vulnerable in the event the virus was reintroduced to the natural environment through an intentional act. While the natural transmission of the variola virus has been eradicated, virulent strains of the related monkeypox virus still exist causing outbreaks of similarly severe disease in humans. However, human-to-human transmission of the virus is rare. The virus is more commonly transmitted through direct contact with wild animals killed for food such as squirrels and monkeys.
  • CPXV While mostly found in Europe, the reservoir hosts of CPXV are rodents from which it can occasionally spread to cats, cows, humans, and zoo animals. Transmission to humans has traditionally occurred through contact with the infected teats of dairy cows. Currently, human infections are more commonly transmitted through domestic cats. Infection with CPXV in humans produces a localized, pustular lesion at the site of the introduction of the virus into the skin. Secondary lesions can occur in individuals that suffer immunological deficiencies.
  • CPXV and VAC gained notoriety for their ability to confer immunity to variola virus.
  • CPXV was the original vaccine for smallpox. After becoming infected with CPXV, the body usually gained the ability to recognize the similar variola virus from its antigens and became able to fight the smallpox disease more effectively.
  • attenuated strains of VAC replaced CPXV as the virus in smallpox vaccine preparations.
  • the precise origin of VAC remains uncertain with some theorizing it to be a product of genetic recombination and others suspecting it to be a species derived from either CPXV or variola virus.
  • VAC also produces epidermal lesions, but additionally may trigger more severe complications, such as progressive vaccinia, eczemic vaccinia, generalized vaccinia, postvaccinial encephalitis, and general encephalopathy, all of which could prove to be potentially fatal following contraction of VAC. These severe conditions are more typically seen in newborns and immuno- compromised individuals. Research interest in VAC has recently focused on the possible use of the virus as vectors for other viral and bacterial antigens.
  • orthopoxvirus genus examples include buffalopox virus, camelpox virus, ectromelia virus, rabbitpox virus, raccoonpox virus, taterapox virus, and volepox virus and tentative species skunkpox virus, and Uasin Gishu disease virus.
  • the orthopoxvirus genus is one of the eight genera of chordopoxvirinae subfamily of viruses that, during their lifecycle, infect a vertebrate host.
  • the chordopoxvirinae subfamily also includes the parapoxvirus genus that includes the bovine papular stomatitis, orf, parapo, pseudocowpox, and squirrel parapo viruses and tentative species Auzduk disease, chamois contagious ecthyma, and seal pox viruses;
  • the avipoxvirus genus that includes the canarypox, fowlpox, juncopox, mynahpox, pigeonpox, psittacinepox, quailpox, sparrowpox, starlingpox, and turkeypox viruses and tentative species peacockpox and penguinpox viruses;
  • the capripoxvirus genus that includes the goatpox, lumpy
  • the orthopoxviruses are highly related at the DNA level, making it likely that any antiviral agent effective against one member would inhibit the replication of this entire group of viruses.
  • the only well-known drug that is effective at inhibiting orthopoxvirus infections in cultured cells is methisazone or IBT (N-methylisatin ⁇ -thiosemicarbazone), but it has demonstrated only limited value in treating infected humans.
  • Cidofovir has been demonstrated to be a potent orthopoxvirus inhibitor, which must be delivered by injection, but it is known to cause severe side effects in humans. There remains a need in the art for a safe, effective, anti-orthopoxvirus drug.
  • the present invention relates to methods of treating poxviral infections (e.g., poxviruses of the orthopoxvirus genus) by the administration of a catecholic butane or a pharmaceutically acceptable salt thereof. While not wishing to be bound by theory, it is believed the methods of the present invention act to decrease replication or growth of a poxvirus in a host and decrease and/or prevent the occurrence of various diseases or disorders accompanying poxviral infection.
  • poxviral infections e.g., poxviruses of the orthopoxvirus genus
  • One embodiment of the invention includes a method of treating or preventing a poxviral infection in a subject.
  • the method comprises administering to the subject a therapeutically effective amount of catecholic butane of the following general formula (I) or a pharmaceutically acceptable salt thereof:
  • Rj and R 2 each independently represents a hydrogen, a lower alkyl, a lower acyl, an alkylene, or -OR] and -OR 2 each independently alternatively represents an unsubstituted or substituted amino acid residue or salt thereof;
  • R 3 , R 4 , R 5 , R 6 , Rio, Rn, R 12 , and R] 3 each independently represents a hydrogen or a lower alkyl;
  • R 7 , R 8 , and R 9 each independently represents a hydrogen, -OH, a lower alkoxy, a lower acyloxy, an unsubstituted or substituted amino acid residue or pharmaceutically acceptable salt thereof, or any two adjacent groups together may be an alkylene dioxy, except that if any one of R 7 , R 8 , and R 9 represents a hydrogen, then -ORi, -OR 2 , and the other two of R 7 , R 8 , and R 9 cannot all be represented by -OH.
  • Another embodiment of the present invention includes a method of treating a poxviral infection in a subject by administering to the subject a therapeutically effective amount of a nordihydroguaiaretic acid (NDGA) derivative of the following general formula (II) or a pharmaceutically acceptable salt thereof:
  • NDGA nordihydroguaiaretic acid
  • Unsubstituted or substituted amino acid residues and pharmaceutically acceptable salts thereof are preferably bonded to the aromatic ring at their carboxy terminus.
  • Another embodiment of the present invention includes a method of treating a poxviral infection in a subject by administering to the subject a therapeutically effective amount of a NDGA derivative of the following general formula (III) or a pharmaceutically acceptable salt thereof:
  • Unsubstituted or substituted amino acid residues and pharmaceutically acceptable salts thereof are preferably bonded to the aromatic ring at their carboxy terminus.
  • Another embodiment of the present invention includes a method of treating a poxvirus of the orthopoxvirus genus in a subject by administering to the subject a therapeutically effective amount of a tetra-O-methyl NDGA, also known as meso- ⁇ ,4- bis(3,4-dimethoxyphenyl)-(2i?,3£)-dimethylbutane, terameprocol, or M 4 N, of the following formula (IV) or a pharmaceutically acceptable salt thereof:
  • the invention relates to methods of vaccination to immunize a subject against a poxviral infection caused by a poxvirus by the administration of a catecholic butane or a pharmaceutically acceptable salt thereof of the present invention in combination with at least one vaccine.
  • the catecholic butane or pharmaceutically acceptable salt thereof of this aspect is selected from the group consisting of the catecholic butane of the general formula (I) or a pharmaceutically acceptable salt thereof, the NDGA derivative of the general formula (II) or a pharmaceutically acceptable salt thereof, the NDGA derivative of the general formula (III) or a pharmaceutically acceptable salt thereof, and M 4 N of formula (IV) or a pharmaceutically acceptable salt thereof.
  • the method of vaccination to immunize a subject against a poxviral infection includes administering to the subject a therapeutically effective amount of catecholic butane or a pharmaceutically acceptable salt thereof of the present invention and administering to the subject a therapeutically effective amount of at least one of CPXV and VAC.
  • the method of vaccination to immunize a subject against a poxviral infection includes administering to the subject a therapeutically effective amount of catecholic butane or a pharmaceutically acceptable salt thereof of the present invention substantially contemporaneously with administering to the subject a therapeutically effective amount of at least one vaccine.
  • the method of vaccination to immunize a subject against a poxviral infection includes administering to the subject a therapeutically effective amount of catecholic butane or a pharmaceutically acceptable salt thereof of the present invention followed by, after a therapeutically effective amount of time, administering to the subject a therapeutically effective amount of at least one vaccine.
  • the method of vaccination to immunize a subject against a poxviral infection includes administering to the subject a therapeutically effective amount of at least one vaccine followed by, after an incubation period, administering to the subject a therapeutically effective amount of catecholic butane or a pharmaceutically acceptable salt thereof of the present invention.
  • the method of vaccination to immunize a subject against a poxviral infection includes administering to the subject a therapeutically effective amount of at least one vaccine and administering to the subject a therapeutically effective amount of catecholic butane or a pharmaceutically acceptable salt thereof of the present invention at least one of prior to for a therapeutically effective amount of time before, substantially contemporaneously with, and following an incubation period from administration of the at least one vaccine.
  • Another aspect of the invention includes a kit comprising a catecholic butane of any one of general formulas (I)-(IV) or a pharmaceutically acceptable salt thereof, and instructions for treating a poxviral infection in a subject by using the catecholic butane or the pharmaceutically acceptable salt thereof.
  • FIG. IA is an image of plaque formation using negative staining of a cell monolayer of 143B human osteosarcoma cells infected with CPXV without M 4 N dosing and incubated for 24 hours in the growth medium DMEM with 10% fetal bovine serum (FBS) using the control vehicle dimethyl sulfide (DMSO);
  • FIG. 1 B is an image of plaque formation using negative staining of a cell monolayer of 143B human osteosarcoma cells infected with CPXV dosed with a concentration of 6.25 ⁇ M M 4 N and incubated for 24 hours in the growth medium DMEM with 10% FBS;
  • FIG. 2 A is an image of plaque formation using negative staining of a cell monolayer of 143B human osteosarcoma cells infected with VAC without M 4 N dosing and incubated for 24 hours in the growth medium DMEM with 10% FBS using the control vehicle DMSO;
  • FIG. 2B is an image of plaque formation using negative staining of a cell monolayer of 143 B human osteosarcoma cells infected with VAC dosed with a concentration of 6.25 ⁇ M M 4 N and incubated for 24 hours in the growth medium DMEM with 10% FBS;
  • FIG. 3 A is an image of plaque formation using negative staining of a cell monolayer of MRC-5 human lung fibroblast cells infected with CPXV without M 4 N dosing and incubated for 24 hours in the growth medium DMEM with 10% FBS using the control vehicle DMSO;
  • FIG. 3B is an image of plaque formation using negative staining of a cell monolayer of MRC-5 human lung fibroblast cells infected with CPXV dosed with a concentration of 25 ⁇ M M 4 N and incubated for 24 hours in the growth medium DMEM with 10% FBS;
  • FIG. 4A is an image of plaque formation using negative staining of a cell monolayer of MRC-5 human lung fibroblast cells infected with VAC without M 4 N dosing and incubated for 24 hours in the growth medium DMEM with 10% FBS using the control vehicle DMSO;
  • FIG. 4B is an image of plaque formation using negative staining of a cell monolayer of MRC-5 human lung fibroblast cells infected with VAC dosed with a concentration of 25 ⁇ M M 4 N and incubated for 24 hours in the growth medium DMEM with 10% FBS.
  • active agent refers to one or more catecholic butanes, including NDGA derivatives, and the pharmaceutically acceptable salts thereof.
  • alkoxy as used herein, either alone or in combination with another substituent, means an alkyl group that is bound through a single, terminal ether linkage that is represented by the structure -O-alkyl.
  • alkyl as used herein, either alone or in combination with another substituent, means acyclic, straight, or branched chain alkyl substituents containing any number of carbon atoms and which may optionally include one or more unsaturated carbon-carbon bonds.
  • alkylene as used herein, either alone or in combination with another substituent, means a divalent group derived from a straight or branched chain saturated hydrocarbon by the removal of two hydrogen atoms.
  • An “alkylene” can include substituted alkylenes.
  • Non-limiting examples of an “alkylene” include methylene, ethylene, propylene and any isomer thereof, and the like.
  • alkylene dioxy as used herein, either alone or in combination with another substituent, means a divalent group with the structure -0-R-O- wherein R is an alkylene.
  • Non-limiting examples of an “alkylene dioxy” include methylene (or substituted methylene) dioxy or ethylene (or substituted ethylene) dioxy.
  • buffer or “buffering agent” as used herein means an agent used to resist changes in pH in a compound of which it is part.
  • Buffer or “buffering agent” suitable for use herein includes any buffer or buffering agent conventional in the art, such as, for example, Tris, phosphate, imidazole, and bicarbonate.
  • a “carrier” as used herein refers to a non-toxic solid, semisolid or liquid filler, diluent, vehicle, excipient, solubilizing agent, encapsulating material or formulation auxiliary of any conventional type, and encompasses all of the components of the composition other than the active pharmaceutical ingredient.
  • the carrier may contain additional agents such as wetting or emulsifying agents, or pH buffering agents. Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary.
  • CPXV cowpox virus
  • a "cyclodextrin” as used herein means an unmodified cyclodextrin or a modified cyclodextrin, and includes without limitation ⁇ -cyclodextrin, ⁇ -cyclodextrin, ⁇ - cyclodextrin and any modified cyclodextrins containing modifications thereto, such as hydoxypropyl- ⁇ -cyclodextrin ("HP- ⁇ -CD”) or sulfobutyl ether ⁇ -cyclodextrin ("SBE- ⁇ - CD").
  • HP- ⁇ -CD hydoxypropyl- ⁇ -cyclodextrin
  • SBE- ⁇ - CD sulfobutyl ether ⁇ -cyclodextrin
  • Cyclodextrin typically has 6 ( ⁇ -cyclodextrin), 7 ( ⁇ -cyclodextrin), and 8 ( ⁇ - cyclodextrin) sugars, up to three substitutions per sugar, and 0 to 24 primary substitutions are therefore possible (primary substitutions are defined as substitutions connected directly to the cyclodextrin ring).
  • the modified or unmodified cyclodextrins used in the present invention may have any appropriate number and location of primary substitutions or other modifications.
  • the term "immunize” or “immunization” as used herein means to stimulate the immune system in such a way that the immune system recognizes an invading poxvirus and produces at least one antibody to destroy the invading poxvirus.
  • incubation period means the amount of time between when a vaccine is administered to a subject and when a therapeutically effective amount of the virus of the vaccine has been developed within the subject.
  • lower acyl as used herein means a Ci - C 6 acyl, which may be linear or branched and which may optionally include one or more unsaturated carbon-carbon bonds.
  • lower alkoxy as used herein means an alkoxy having an alkyl group that is a lower alkyl.
  • lower alkyl as used herein means a Ci - C 6 alkyl, which may be linear or branched and which may optionally include one or more unsaturated carbon-carbon bonds.
  • Lower alkyl includes, but is not limited to, methyl, ethyl, n-propyl, n-butyl, 1- methylethyl (i-propyl), 1 -methylpropyl, 2-methylpropyl, l,l-dimethylethyl(tert-butyl), pentyl, and hexyl.
  • M 4 N refers to the compound otherwise known as tetra-O-methyl NDGA, mer ⁇ -l,4-bis(3,4-dimethoxyphenyl)-(2i?,35)-dimethylbutane, terameprocol, or EM- 1421 of formula (IV) or a pharmaceutically acceptable salt thereof.
  • NDGA as used herein refers to nordihydroguaiaretic acid.
  • NDGA derivative refers to one or more compounds each having the general formula (II), or a pharmaceutically acceptable salt thereof, wherein Ri 4 , Ri5, Ri 6 , and Ri 7 each independently represents -OH, lower alkoxy, lower acyloxy, or an unsubstituted or substituted amino acid residue or pharmaceutically acceptable salt thereof, except that Ri 4 , Ri 5 , Ri 6 , and Ri 7 are not all represented by -OH, and Ris and R 19 each independently represents -H or an alkyl such as a lower alkyl.
  • Rig and R 19 are each -H or a lower alkyl.
  • Ri 8 and R, 9 are each -CH 3 or -CH 2 CH 3 .
  • orthopoxvirus refers to one of the eleven recognized genera of poxviruses and among the eight recognized genera that affect vertebrates.
  • the "orthopoxvirus” genus includes, but is not limited to, the following species: variola virus, monkeypox virus, cowpox virus (CPXV), vaccinia virus (VAC), buffalopox virus, camelpox virus, ectromelia virus, rabbitpox virus, raccoonpox virus, taterapox virus, and volepox virus and tentative species skunkpox virus, and Uasin Gishu disease virus.
  • the "orthopoxvirus" genus is one of the eight genera of chordopoxvirinae subfamily of viruses that, during their lifecycle, infect a vertebrate host.
  • the chordopoxvirinae subfamily also includes the parapoxvirus genus that includes the bovine papular stomatitis, orf, parapo, pseudocowpox, and squirrel parapo viruses and tentative species Auzduk disease, chamois contagious ecthyma, and sealpox viruses; the avipoxvirus genus that includes the canarypox, fowlpox, juncopox, mynahpox, pigeonpox, psittacinepox, quailpox, sparrowpox, starlingpox, and turkeypox viruses and tentative species peacockpox and penguinpox viruses; the capripoxvirus genus that includes the goatpox, lumpy
  • a “pharmaceutically acceptable carrier” as used herein refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any conventional type.
  • a “pharmaceutically acceptable carrier” is non-toxic to recipients at the dosages and concentrations employed, and is compatible with other ingredients of the formulation.
  • the carrier for a formulation containing the present catecholic butane preferably does not include oxidizing agents and other compounds that are known to be deleterious to such.
  • Suitable carriers include, but are not limited to, water, dextrose, glycerol, saline, ethanol, buffer, dimethyl sulfoxide, Cremaphor EL, and combinations thereof.
  • the carrier may contain additional agents such as solubilizing, wetting or emulsifying agents, or pH buffering agents. Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary.
  • pharmaceutically acceptable excipient includes vehicles, adjuvants, or diluents or other auxiliary substances, such as those conventional in the art, which are readily available to the public.
  • pharmaceutically acceptable auxiliary substances include pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like.
  • “Pharmaceutically acceptable salts” as used herein include the acid addition salts which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, mandelic, oxalic, and tartaric acids. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, and histidine.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, mandelic, oxalic, and tartaric acids.
  • Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-eth
  • “Pharmaceutically acceptable salts” means the aforementioned salts, within the scope of sound medical judgment, that are suitable for use in contact with tissues of a subject without causing undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio and are effective for their intended use.
  • a "poxvirus” as used herein refers to any group of viruses containing DNA that are responsible for a wide range of pox diseases in humans and other animals.
  • a "poxvirus” refers to a member of the orthopoxvirus genus. More preferably, a "poxvirus” refers to at least one of variola virus, monkeypox virus, cowpox virus (CPXV), and vaccinia virus (VAC).
  • subject refers to an animal being treated with the present compositions, including, but not limited to, simians, humans, avians, felines, canines, equines, rodents, bovines, porcines, ovines, caprines, mammalian farm animals, mammalian sport animals, and mammalian pets.
  • substantially purified as used herein in reference to the catecholic butanes is one that is substantially free of compounds that are not the catecholic butane of the present invention (hereafter, “non-NDGA materials”).
  • substantially free means at least 50%, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% free of non-NDGA materials.
  • terapéuticaally effective amount or “effective amount” as used herein when referring to the active agent, compound, or drug of the invention, means that amount of an active agent, a compound, or a drug, that elicits a desired biological or medicinal response in a tissue system of a subject, or in a subject, that is being sought by a researcher, veterinarian, medical doctor or other clinician.
  • the desired response includes interdicting, preventing, palliating, or alleviating an existing viral infection in the subject that is being treated.
  • the desired response includes at least a reduction in one or more symptoms, disorders, or diseases of a poxviral infection in the subject under treatment.
  • the desired response includes a reduction in the count of virus, or an inhibition of the replication or growth of a poxvirus in the subject under treatment.
  • the "therapeutically effective amount" of an active agent to be used in the instant invention can vary with factors, such as the particular subject, e.g., age, weight, diet, health, etc., severity and complications of viral infection condition sought to be treated or prevented, the mode of administration of the active agent, the particular active agent used, etc. Standard procedures can be performed to evaluate the effect of the administration of an active agent to a subject, thus allowing a skilled artisan to determine the effective amount of the active agent to be administered to the subject.
  • the syndrome of viral infection such as fever or inflammation, etc., or the count of virus, can be measured from the subject prior to or after the administration of the active agent.
  • techniques such as surveys or animal models, can also be used to evaluate the effectiveness of an active agent in treating or preventing a viral infection.
  • the term "therapeutically effective amount of time” or “effective amount of time” as used herein means that amount of time that elicits a desired biological or medicinal response in a tissue system of a subject, or in a subject, that is being sought by a researcher, veterinarian, medical doctor or other clinician.
  • the desired response includes interdicting, preventing, palliating, or alleviating an existing viral infection in the subject that is being treated.
  • the desired response includes at least a reduction in one or more symptoms, disorders, or diseases of a poxviral infection in the subject under treatment.
  • the desired response includes a reduction in the count of virus, or a inhibition of the replication or growth of a poxvirus in the subject under treatment.
  • the desired response includes an increase in the count of virus, or an increase in the replication or growth of a poxvirus in the subject under treatment.
  • the "therapeutically effective amount of time" to be used in the instant invention can vary with factors, such as the particular subject, e.g., age, weight, diet, health, etc., severity and complications of viral infection condition sought to be treated or prevented, the mode of administration of the active agent, the particular active agent used, and the like.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a condition or disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a condition or disease and/or adverse affect attributable to the condition or disease.
  • Treatment covers any treatment of a condition or disease in a mammal, particularly in a human, and includes: (a) preventing the condition or disease or symptom thereof from occurring in a subject which may be predisposed to the condition or disease but has not yet been diagnosed as having it; (b) inhibiting the condition or disease or symptom thereof, such as, arresting its development; and (c) relieving, alleviating or ameliorating the condition or disease or symptom thereof, such as, for example, causing regression of the condition or disease or symptom thereof.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of at least one compound of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • the specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
  • unsubstituted or substituted amino acid residue or salt thereof as used herein in reference to one of the -ORi, -OR 2 or other R groups as appropriate, in the formulas for the catecholic butanes herein is an amino acid residue or a substituted amino acid residue or salt of an amino acid residue or salt of a substituted amino acid residue including but not limited to: alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, 5-hydroxylysine, 4-hydroxyproline, thyroxine, 3-methylhistidine, ⁇ -N-methyllysine, ⁇ -N,N,N-trimethyllysine, aminoadipic acid, ⁇ -carboxyglutamic acid, phosphoser
  • VAC as used herein refers to vaccinia virus, which is a species of the orthopoxvirus genus.
  • the term "vaccine” means a preparation comprising at least one of an epitope, peptide, antigen, and virus that, when administered to a subject, produces or artificially increases immunity to a poxvirus.
  • Catecholic Butanes The invention described herein relates to catecholic butanes and the use of the compounds for the treatment of a poxvirus. Catecholic butanes have the following general formula (I) or a pharmaceutically acceptable salt thereof:
  • Ri and R 2 each independently represents a hydrogen, a lower alkyl, a lower acyl, an alkylene, or -ORi and -OR 2 each independently alternatively represents an unsubstituted or substituted amino acid residue or salt thereof;
  • R 3 , R 4 , R 5 , R 6 , Rio, Rn, Ri 2 , and Ri 3 each independently represents a hydrogen or a lower alkyl;
  • R 7 , R 8 , and R9 each independently represents a hydrogen, -OH, a lower alkoxy, a lower acyloxy, an unsubstituted or substituted amino acid residue or pharmaceutically acceptable salt thereof, or any two adjacent groups together may be an alkylene dioxy, except that if any one of R 7 , R 8 , and R 9 represents a hydrogen, then -ORi, -OR 2 , and the other two of R 7 , R 8 , and R 9 cannot all be represented by -OH.
  • Unsubstituted or substituted amino acid residues and pharmaceutically acceptable salts thereof are preferably bonded to the aromatic ring at their carboxy terminus.
  • the catecholic butanes of the invention can be combined with pharmaceutically acceptable carriers or excipients to produce pharmaceutical compositions that can be formulated for a wide variety of routes of delivery.
  • the catecholic butane has the general formula (I), wherein Ri and R 2 are independently -H, a lower alkyl, a lower acyl, or -ORi and -OR 2 each independently alternatively represents an unsubstituted or substituted amino acid residue or salt thereof; R 3 and R 4 , are independently a lower alkyl; R 5 , R 6 , Ri 0 , Rn, Ri 2 , and Ri 3 are independently -H; and R 7 , R 8 , and R 9 are independently -H, -OH, a lower alkoxy, a lower acyloxy, or an unsubstituted or substituted amino acid residue or pharmaceutically acceptable salt thereof, except that the catecholic butane is not NDGA.
  • the catecholic butane has the general formula (I), wherein Rj and R 2 are independently -H, a lower alkyl, a lower acyl, or -ORi and -OR 2 each independently alternatively represents an unsubstituted or substituted amino acid residue or salt thereof; R 3 and R 4 , are independently a lower alkyl; R 5 , R 6 , R 7 , Rio, Ri i, Ri 2 , and Rj 3 are independently -H; and R 8 and R 9 are independently -OH, a lower alkoxy, a lower acyloxy, or an unsubstituted or substituted amino acid residue or pharmaceutically acceptable salt thereof, except that the catecholic butane is not NDGA.
  • the catecholic butane has
  • catecholic butane has the formula (I), wherein Ri and R 2 are each -CH 3 and R 8 and R 9 are each -OCH 3 .
  • Unsubstituted or substituted amino acid residues and pharmaceutically acceptable salts thereof are
  • composition containing a substantially pure preparation of at least one NDGA derivative is effective for the prevention and treatment of poxvirus infections.
  • Ri 8 and R 19 can both be -H, -CH 3 , or -CH 2 CH 3 .
  • one or more of Rj 4 , Ri 5 , Ri 6 , and Ri 7 represents an unsubstituted or a substituted amino acid residue or salt thereof, the residue is bonded to the aromatic ring at the carboxy terminus.
  • the catecholic butane used in methods according to embodiments of the present invention is a tetra-O-methyl NDGA, also known as mes ⁇ -l ,4-bis(3,4-dimethoxyphenyl)-(2/?,3S)-dimethylbutane, terameprocol, EM- 1421, or M 4 N, of the following formula (IV) or a pharmaceutically acceptable salt thereof:
  • the compounds disclosed herein may contain chiral centers, which may be either of the (R) or (S) configuration, or may comprise a mixture thereof. Accordingly, the present invention also includes stereoisomers of the compounds described herein, where applicable, either individually or admixed in any proportions.
  • Stereoisomers may include, but are not limited to, enantiomers, diastereomers, racemic mixtures, and combinations thereof. Such stereoisomers can be prepared and separated using conventional techniques, either by reacting enantiomeric starting materials, or by separating isomers of compounds of the present invention.
  • Isomers may include geometric isomers. Examples of geometric isomers include, but are not limited to, cis isomers or trans isomers across a double bond.
  • the isomers are contemplated among the compounds of the present invention.
  • the isomers may be used either in pure form or in admixture with other isomers of the compounds described herein.
  • the catecholic butane and pharmaceutically acceptable salts thereof of the present invention in a suitable formulation, with a pharmaceutically acceptable carrier or excipient where appropriate, can be safely administered as a prophylactic or therapeutic treatment to a subject in need of such treatment by one or more routes of administration selected from the group consisting of intranasal administration; oral administration; inhalation administration; subcutaneous administration; transdermal administration; intravenous administration; buccal administration; intraperitoneal administration; intraocular administration; peri-ocular administration; intramuscular administration; implantation administration; infusion, and central venous administration.
  • the catecholic butanes and pharmaceutically acceptable salts thereof can be safely administered as a prophylactic or therapeutic treatment to a subject in need of such treatment in solution, suspension, semisolid or solid forms as appropriate, or in liposomal formulations, nanoparticle formulations, or micellar formulations for administration via one or more routes mentioned above.
  • the catecholic butanes and pharmaceutical acceptable salts thereof in liposomal formulations, nanoparticles formulations, or micellar formulations can be embedded in a biodegradable polymer formulation and safely administered, such as by subcutaneous implantation.
  • the route of administration for purposes herein is other than by parenteral administration, where parenteral administration herein means intravenous, intramuscular, subcutaneous, transdermal and intraperitoneal administration.
  • a catecholic butane can be given in combination with one or more other agents or drugs.
  • the catecholic butane of the present invention can be administered by at least one of simultaneously with, prior to, and following the administration of one or more other agents or drugs.
  • a catecholic butane can be administered in combination with one or more additional anti-inflammation agents.
  • the additional anti-inflammation agents are selected from the group consisting of: (1) serotonin receptor antagonists; (2) serotonin receptor agonists; (3) histamine receptor antagonists; (4) bradykinin receptor antagonists; (5) kallikrein inhibitors; (6) tachykinin receptor antagonists, including neurokinin] and neurokinin receptor subtype antagonists; (7) calcitonin gene-related peptide (CGRP) receptor antagonists; (8) interleukin receptor antagonists; (9) inhibitors of enzymes active in the synthetic pathway for arachidonic acid metabolites, including (a) phospholipase inhibitors, including PLA 2 isoform inhibitors and PLC ⁇ isoform inhibitors (b) cyclooxygenase inhibitors, and (c) lipooxygenase inhibitors; (10) prostanoid receptor antagonists including eicosanoid EP-I and EP-4 receptor subtype antagonists and thromboxane receptor subtype antagonists; (11) leukotriene receptor antagonist
  • a catecholic butane or a pharmaceutical acceptable salt thereof of the present invention can be administered in combination with one or more other anti -poxvirus agents, such as a second catecholic butane of the general formula (I) or a pharmaceutically acceptable salt thereof, Methisazone, or Cidofovir.
  • a catecholic butane or a pharmaceutical acceptable salt thereof of the present invention can be administered in combination with at least one vaccine for the purpose preventing a poxviral infection caused by a poxvirus.
  • Non- limiting examples of such vaccines can include CPXV and VAC.
  • the catecholic butane or a pharmaceutical acceptable salt thereof of the present invention is administered with the at least one vaccine allowing a virus of the at least one vaccine to survive for a therapeutically effective amount of time to allow the immune system to recognize the virus administered as a vaccine and begin to develop antibodies to act against the virus and perhaps even other poxviruses.
  • the catecholic butane of this embodiment is selected from the group consisting of the catecholic butane of the general formula (I) or a pharmaceutically acceptable salt thereof, the NDGA derivative of the general formula (II) or a pharmaceutically acceptable salt thereof, the NDGA derivative of the general formula (III) or a pharmaceutically acceptable salt thereof, and M 4 N of formula (IV) or a pharmaceutically acceptable salt thereof.
  • a method of vaccination to immunize a subject against a poxviral infection caused by a poxvirus includes first administering to the subject at least one vaccine followed by, after an incubation period, administering to the subject a catecholic butane or a pharmaceutical acceptable salt thereof of the present invention.
  • the catecholic butane of this embodiment is selected from the group consisting of the catecholic butane of the general formula (I) or a pharmaceutically acceptable salt thereof, the NDGA derivative of the general formula (II) or a pharmaceutically acceptable salt thereof, the NDGA derivative of the general formula (III) or a pharmaceutically acceptable salt thereof, and M 4 N of formula (IV) or a pharmaceutically acceptable salt thereof.
  • a method of vaccination to immunize a subject against a poxviral infection caused by a poxvirus includes first administering to the subject a catecholic butane or a pharmaceutical acceptable salt thereof of the present invention and, after a therapeutically effective amount of time, administering to the subject at least one vaccine.
  • the catecholic butane of this embodiment is selected from the group consisting of the catecholic butane of the general formula (I) or a pharmaceutically acceptable salt thereof, the NDGA derivative of the general formula (II) or a pharmaceutically acceptable salt thereof, the NDGA derivative of the general formula (III) or a pharmaceutically acceptable salt thereof, and M 4 N of formula (IV) or a pharmaceutically acceptable salt thereof.
  • the method of vaccination to immunize a subject against a poxviral infection caused by a poxvirus include administering to the subject at least one vaccine and administering to the subject a catecholic butane or a pharmaceutically acceptable salt thereof of the present invention at least one of prior to for a therapeutically effective amount of time before, substantially contemporaneously with, and following an incubation period from administration of the at least one vaccine.
  • the catecholic butane of this embodiment is selected from the group consisting of the catecholic butane of the general formula (I) or a pharmaceutically acceptable salt thereof, the NDGA derivative of the general formula (II) or a pharmaceutically acceptable salt thereof, the NDGA derivative of the general formula (III) or a pharmaceutically acceptable salt thereof, and M 4 N of formula (IV) or a pharmaceutically acceptable salt thereof.
  • the present invention further includes a method of producing the pharmaceutical composition of the present invention, the method having the steps of making or providing a catecholic butane composition in a substantially purified form, combining the composition with a pharmaceutically acceptable carrier or excipient, and formulating the composition in a manner that is compatible with the mode of desired administration.
  • kits comprising compositions or formulations as above to be used as for the treatment of a poxvirus or for the purpose of preventing a poxviral infection caused by a poxvirus
  • the compositions are formulated for delivery as above, including but not limited to intranasal administration, inhalation, oral administration, topical administration, intravenous administration, intraperitoneal administration and other parenteral administration, optionally, including delivery device for such administration, and instructions for such administration.
  • the catecholic butanes of the present invention can be prepared by any conventional methodologies.
  • such compounds can be made as described in U.S. Pat. No. 5,008,294 (Jordan et al, issued Apr 16, 1991); U.S. Pat. No. 6,291,524 (Huang et al, issued Sep 18, 2001); Hwu, et al. (Hwu, J. R. et al, "Antiviral activities of methylated nordihydroguaiaretic acids. 1. Synthesis, structure identification, and inhibition of Tat-regulated HIV transactivation. J. Med. Chem., 41(16): 2994-3000" (1998)); or McDonald, et al. (McDonald, R. W.
  • a catecholic butane, tetra-O-methyl NDGA also known as me.r ⁇ -l,4-bis(3,4-dimethoxyphenyl)-(2i?,3 ⁇ S)-dimethylbutane, terameprocol, EM- 1421, or M 4 N, of formula (IV)
  • NDGA tetra-O-methyl NDGA
  • EM- 1421 terameprocol
  • M 4 N of formula (IV)
  • a solution was made containing NDGA and potassium hydroxide in methanol in a reaction flask. Dimethyl sulfate was then added to the reaction flask and the reaction was allowed to proceed. The reaction was finally quenched with water, causing the product to precipitate. The precipitate was isolated by filtration and dried in a vacuum oven.
  • the compound was then dissolved in a solution of methylene chloride and toluene and subsequently purified through an alumina column.
  • the solvents were removed by rotary evaporation and the solid was resuspended in isopropanol and isolated by filtration.
  • the filter cake was dried in a vacuum oven.
  • the resulting tetra-O-methyl NDGA (M 4 N) was crystallized by refluxing the filter cake in isopropanol and re-isolating the crystals by filtration.
  • certain catecholic butanes of the present invention such as G 4 N, also known as meso-l,4-bis[3,4- (dimethylaminoacetoxy)phenyl]-(2i?,3iS)-dimethylbutane or tetra-dimethyl glycinyl NDGA, of the following formula (V) or a hydrochloride salt thereof, or similar compounds having amino acid substituents, can be prepared according to conventional methods, as described in, for example, U.S. Pat. No. 6,417,234, which is incorporated herein by reference in its entirety.
  • compositions comprising the catecholic butanes and pharmaceutically acceptable carriers or excipients.
  • These compositions may include a buffer, which is selected according to the desired use of the catecholic butanes, and may also include other substances appropriate for the intended use. Those skilled in the art can readily select an appropriate buffer, a wide variety of which are known in the art, suitable for an intended use.
  • the composition can comprise a pharmaceutically acceptable excipient, a variety of which are known in the art.
  • compositions herein are formulated in accordance to the mode of potential administration.
  • the composition may be a converted to a powder or aerosol form, as conventional in the art, for such purposes.
  • Other formulations, such as for oral or parenteral delivery, are also used as conventional in the art.
  • Compositions for administration herein may form solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
  • compositions for use in the invention include those wherein the composition is formulated for delivery or administration as described above such as, for example, in the form of a tablet, a capsule, a liquid that is either hydrophilic or hydrophobic, a powder such as one resulting from lyophilization, an aerosol, or in the form of an aqueous water soluble composition, a hydrophobic composition, a liposomal composition, a micellar composition, such as that based on polysorbate 80 or diblock copolymers, a nanoparticle composition, a polymer composition, a cyclodextrin complex composition, emulsions, or lipid based nanoparticles termed "lipocores.”
  • compositions or formulations suitable for oral or injectable delivery additionally includes a pharmaceutical composition containing a catecholic butane for treatment of a poxvirus where the composition is formulated with a pharmaceutically acceptable carrier, wherein the carrier comprises at least one of a solubilizing agent and an excipient selected from the group consisting of: (a) a water-soluble organic solvent; (b) a cyclodextrin (including a modified cyclodextrin); (c) an ionic, non-ionic or amphipathic surfactant, (d) a modified cellulose; (e) a water-insoluble lipid; and a combination of any of the carriers (a) - (e).
  • a solubilizing agent selected from the group consisting of: (a) a water-soluble organic solvent; (b) a cyclodextrin (including a modified cyclodextrin); (c) an ionic, non-ionic or amphipathic surfactant, (d) a modified
  • the water-soluble organic solvent may be preferably, but not necessarily, other than dimethyl sulfoxide.
  • Non-limiting exemplary water-soluble organic insolvents include polyethylene glycol (“PEG”), for example, PEG 300, PEG 400 or PEG 400 monolaurate, propylene glycol (“PG”), polyvinyl pyrrolidone (“PVP”), ethanol, benzyl alcohol or dimethylacetamide.
  • PEG polyethylene glycol
  • PG propylene glycol
  • PVP polyvinyl pyrrolidone
  • PG polyvinyl pyrrolidone
  • ethanol benzyl alcohol or dimethylacetamide
  • benzyl alcohol or dimethylacetamide benzyl alcohol or dimethylacetamide.
  • the water-soluble organic solvent is PG
  • the PG is in the absence of white petrolatum, in the absence of xanthan gum (also known as xantham gum and xanthum gum) and in the absence of at least one of
  • the water-soluble organic solvent is PEG
  • the PEG is present in the absence of ascorbic acid or butylated hydroxytoluene ("BHT"), and for certain embodiments, when the PEG is polyethylene glycol 400, the polyethylene glycol 400 preferably is present in the absence of polyethylene glycol 8000.
  • BHT butylated hydroxytoluene
  • the cyclodextrin or modified cyclodextrin may be, without limitation, ⁇ - cyclodextrin, ⁇ -cyclodextrin, ⁇ -cyclodextrin, HP- ⁇ -CD or SBE- ⁇ -CD.
  • the ionic, non-ionic or amphipathic surfactant may include, for example without limitation, a surfactant such as polyoxyethylene sorbitan monolaurate (also known as polysorbate), which is a non-ionic surfactant, for example, polysorbate 20 and polysorbate 80, commercially available as Tween® 20 or Tween® 80, d-alpha-tocopheryl polyethylene glycol 1000 succinate ("TPGS”), glycerol monooleate (also known as glyceryl monooleate), an esterified fatty acid or a reaction product between ethylene oxide and castor oil in a molar ratio of 35:1, commercially available as Cremophor® EL.
  • a surfactant such as polyoxyethylene sorbitan monolaurate (also known as polysorbate), which is a non-ionic surfactant, for example, polysorbate 20 and polysorbate 80, commercially available as Tween® 20 or Tween® 80, d-alpha
  • the surfactant when the surfactant is a non-ionic surfactant, the non-ionic surfactant is present in the absence of xanthan gum.
  • a modified cellulose include ethyl cellulose ("EC"), hydroxylpropyl methylcellulose (“HPMC”), methylcellulose (“MC”) or carboxy methylcellulose (“CMC”).
  • the catecholic butane may be solubilized in modified celluloses that can be diluted in ethanol (“EtOH”) prior to use.
  • the water-insoluble lipids include, for example, an oil or oils, such as castor oil, sesame oil or peppermint oil, a wax or waxes, such as beeswax or carnauba wax, and mixed fat emulsion compositions such as Intralipid® (Pharmacia & Upjohn, now Pfizer), used as per the manufacturer's recommendation.
  • an oil or oils such as castor oil, sesame oil or peppermint oil, a wax or waxes, such as beeswax or carnauba wax
  • mixed fat emulsion compositions such as Intralipid® (Pharmacia & Upjohn, now Pfizer), used as per the manufacturer's recommendation.
  • adult dosage is recommended to be not exceeding 2 g of fat/kg body weight/day (20 mL, 10 mL and 6.7 mL/kg of Intralipid® 10%, 20% and 30%, respectively).
  • Intralipid® 10% is believed to contain in 1,000 mL: purified soybean oil 10Og, purified egg phospholipids 12 g, glycerol anhydrous 22 g, water for injection q.s. ad 1,000 mL. pH is adjusted with sodium hydroxide to pH approximately 8.
  • Intralipid® 20% contains in 1,000 mL: purified soybean oil 200 g, purified egg phospholipids 12 g, glycerol anhydrous 22 g, water for injection q.s. ad 1,000 mL. pH is adjusted with sodium hydroxide to pH approximately 8.
  • Intralipid® 30% contains in 1,000 mL: purified soybean oil 300 g, purified egg phospholipids 12 g, glycerol anhydrous 16.7 g, water for injection q.s. ad 1,000 mL. pH is adjusted with sodium hydroxide to pH approximately 7.5. These Intralipid® products are stored at controlled room temperature below 25 0 C and should not be frozen.
  • the oil is an oil other than castor oil, and for certain embodiments of oral formulations, the castor oil is present in the absence of beeswax or carnauba wax.
  • the catecholic butane is dissolved or dissolved and diluted in different carriers to form a liquid composition for oral administration into animals, including humans.
  • the catecholic butane is dissolved in a water-soluble organic solvent such as a PEG 300, PEG 400 or a PEG 400 monolaurate (the "PEG compounds") or in PG.
  • the compounds herein are dissolved in a modified cyclodextrin, such as HP- ⁇ -CD or SBE- ⁇ - CD.
  • the present compounds are solubilized and/or diluted in a combination formulation containing a PEG compound and HP- ⁇ -CD.
  • the compounds herein are dissolved in a modified cellulose such as HPMC, CMC or EC.
  • the compounds herein are dissolved in another combination formulation containing both a modified cyclodextrin and modified cellulose, such as, for example, HP- ⁇ -CD and HPMC or HP- ⁇ -CD and CMC.
  • the compounds herein are dissolved in ionic, non-ionic or amphipathic surfactants such as Tween® 20, Tween® 80, TPGS or an esterified fatty acid.
  • the present compounds can be dissolved in TPGS alone, or Tween® 20 alone, or in combinations such as TPGS and PEG 400, or Tween® 20 and PEG 400.
  • the present compounds are dissolved in a water-insoluble lipid such as a wax, fat emulsion, for example Intralipid®, or oil.
  • a water-insoluble lipid such as a wax, fat emulsion, for example Intralipid®, or oil.
  • the present compounds can be dissolved in peppermint oil alone, or in combinations of peppermint oil with Tween® 20 and PEG 400, or peppermint oil with PEG 400, or peppermint oil with Tween® 20, or peppermint oil with sesame oil.
  • EC may be substituted or added in place of the HPMC or CMC in the foregoing examples
  • PEG 300 or PEG 400 monolaurate can be substituted or added in place of PEG 400 in the foregoing examples
  • Tween® 80 may be substituted or added in place of Tween® 20 in the foregoing examples
  • other oils such as corn oil, olive oil, soybean oil, mineral oil or glycerol, may be substituted or added in place of the peppermint oil or sesame oil in the foregoing examples.
  • heating may be applied, for example, heating to a temperature of about
  • the catecholic butane may be administered orally as solids either without any accompanying carrier or with the use of carriers.
  • the compounds herein are first dissolved in a liquid carrier as in the foregoing examples, and subsequently made into a solid composition for administration as an oral composition.
  • the present compounds are dissolved in a modified cyclodextrin such as HP- ⁇ -CD, and the composition is lyophilized to yield a powder that is suitable for oral administration.
  • the present compounds are dissolved or suspended in a TPGS solution, with heating as appropriate to obtain an evenly distributed solution or suspension.
  • the composition Upon cooling, the composition becomes creamy and is suitable for oral administration.
  • the present compounds are dissolved in oil and beeswax is added to produce a waxy solid composition.
  • the compounds herein are first solubilized before other excipients are added so as to produce compositions of higher stability.
  • Unstable formulations are not desirable.
  • Unstable liquid formulations frequently form crystalline precipitates or biphasic solutions.
  • Unstable solid formulations frequently appear grainy and clumpy and sometimes contain runny liquids.
  • An optimal solid formulation appears smooth, homogenous, and has a small melting temperature range.
  • the proportions of excipients in the formulation may influence stability. For example, too little stiffening agent such as beeswax may leave the formulation too runny for an elegant oral formulation.
  • the excipients used should be good solvents of the catecholic butane compounds herein, such as M 4 N, for example.
  • the excipients should be able to dissolve the catecholic butane without heating.
  • the excipients should also be compatible with each other independent of the catecholic butane such that they can form a stable solution, suspension or emulsion.
  • the excipients used should also be good solvents of the catecholic butane to avoid clumps and non-uniform formulations. To avoid solid formulations that are too runny or heterogeneous in texture, which are undesirable, the excipients should be compatible with each other such that they form a smooth homogeneous solid, even in the absence of the catecholic butane.
  • the active agents can be used alone or in combination with appropriate additives as liquids in the form of solutions or suspensions or as solids in the form of tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
  • conventional additives such as lactose, mannitol, corn starch or potato starch
  • binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins
  • disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose
  • the pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents, are conventional in the art.
  • Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the vehicle may contain minor amounts of auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents or emulsifying agents.
  • auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents or emulsifying agents.
  • Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985.
  • the composition or formulation to be administered will, in any event, contain a quantity of the agent adequate to achieve the desired state in the subject being treated.
  • the active agents can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable or other similar oils, including corn oil, castor oil, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • Suitable therapeutic formulations for parenteral delivery of a catecholic butane in accordance with the present invention also include the various injectable carrier/excipient formulations disclosed in U.S. Provisional Patent Application No.
  • the active agents can be utilized in aerosol formulation to be administered via inhalation.
  • the compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
  • the active agents can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases.
  • the compounds of the present invention can be administered rectally via a suppository.
  • the suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
  • the present invention includes formulations of catecholic butanes in a nanoparticle ("NP") preparation.
  • NP nanoparticle
  • a number of different NP formulations suitable for use herein can be made depending on the method of delivery.
  • the NP formulation can differ based on the drug release profile desired, by controlling the molecular weight, the copolymer ratio, the drug loading, the microparticle size and porosity and the fabrication conditions.
  • the NP formulations can also differ on the basis of polymers, stabilizers, and surfactants used in the production process. Different excipients may also have different effects on drug uptake, drug distribution throughout the body and persistence of the drug in plasma.
  • a person having skills conventional in the art will be able to determine the desired properties or characteristics, and accordingly determine the appropriate NP formulation to use.
  • the polymeric matrix of the NP must meet the criteria of biocompatibility, bioavailability, mechanical strength and ease of processing.
  • the best known polymers for this purpose is the biodegradable poly(lactide-co-glycolide)s ("PLGAs").
  • the NP herein can be made by any process conventional in the art.
  • the NP can be made as described in, for example, Lockman, et al. (Lockman, P. R. et al. , "Nanoparticle Technology for Drug Delivery Across the Blood-Brain Barrier.”, Drug Development Indus. Pharmacy, 28(1): 1-13, (2002)).
  • the types of manufacturing process include, for example, emulsion polymerization, interfacial polymerization, desolvation evaporation and solvent deposition.
  • the polymerization process consists of building a chain of polymers from a single monomer unit, as described in, for example, Kreuter (Kreuter, J., "Nanoparticles, Encyclopedia of Pharmaceutical Technology, Swarbick, J.; Boylan, J. C. Eds.; Marcel Dekker (New York, 1994), pp. 165-190, (1994)). Polymerization occurs spontaneously at room temperature after initiation by either free radical or ion formation, such as by use of high-energy radiation, UV light, or hydroxyl ions. Once polymerization is complete the solution is filtered and neutralized. The polymers form micelles and droplets consisting of from about 100 to 10 7 polymer molecules.
  • the NP herein can also be made by an interfacial polymerization process as described in, for example, Khouri (Khouri, A.I. et al., "Development of a new process for the manufacture of polyisobutyl-cyanoacrylate nanoparticles," Int. J. Pharm., 28: 125 (1986)).
  • Khouri Khouri, A.I. et al., "Development of a new process for the manufacture of polyisobutyl-cyanoacrylate nanoparticles," Int. J. Pharm., 28: 125 (1986)
  • monomers are used to create the polymer and polymerization occurs when an aqueous and organic phase are brought together by homogenization, emulsification, or micro-fluidization under high-torque mechanical stirring.
  • polyalkylcyanoacrylate nanocapsules containing the catecholic butanes can be made by combining the lipophilic catecholic butanes and the monomer in an organic phase, dissolving the combination in oil, and slowly adding the mixture through a small tube to an aqueous phase with constant stirring.
  • the monomer can then spontaneously form 200- 300 nm capsules by anionic polymerization.
  • a variation of this process involves adding a solvent mixture of benzyl benzoate, acetone, and phospholipids to the organic phase containing the monomer and the drug, as described in, for example, Fessi, et al. (Fessi, H. et al.
  • Macromolecules such as albumin and gelatin can be used in oil denaturation and desolvation processes in the production of NPs.
  • oil emulsion denaturation process large macromolecules are trapped in an organic phase by homogenization. Once trapped, the macromolecule is slowly introduced to an aqueous phase undergoing constant stirring.
  • the nanoparticles formed by the introduction of the two immiscible phases can then be hardened by crosslinking, such as with an aldehyde or by heat denaturation.
  • macromolecules can form NPs by "desolvation.”
  • desolvation In the desolvation process, macromolecules are dissolved in a solvent in which the macromolecules reside in a swollen, coiled configuration. The swollen macromolecule is then induced to coil tightly by changing the environment, such as pH, charge, or by use of a desolvating agent such as ethanol. The macromolecule may then be fixed and hardened by crosslinking to an aldehyde. The NDGA Compounds can be adsorbed or bound to the macromolecule before crosslinking such that the derivatives become entrapped in the newly formed particle.
  • Solid lipid NP can be created by high-pressure homogenization. Solid lipid NPs have the advantage that they can be sterilized and autoclaved and possess a solid matrix that provides a controlled release.
  • the present invention further includes NP with different methods of drug loading.
  • the NP can be solid colloidal NP with homogeneous dispersion of the drug therein.
  • the NP can be solid NP with the drug associated on the exterior of the NP, such as by adsorption.
  • the NP can be a nanocapsule with the drug entrapped therein.
  • the NP can further be solid colloidal NP with homogeneous dispersion of the drug therein together with a cell surface ligand for targeting delivery to the appropriate tissue.
  • the size of the NPs may be relevant to their effectiveness for a given mode of delivery.
  • the NPs typically are about 10 nm to about 1000 nm; optionally, the NPs can be about 30 nm to about 800 nm; further typically, about 60 nm to about 270 nm; even further typically, about 80 nm to about 260 nm; or about 90 nm to about 230 nm, or about 100 nm to about 195 nm.
  • factors influence the size of the NPs, all of which can be adjusted by a person of ordinary skill in the art, such as, for example, pH of the solution used during polymerization, amount of initiation triggers (such as heat or radiation, etc.) and the concentration of the monomer unit.
  • the NPs herein such as polysaccharide NPs or albumin NPs, may optionally be coated with a lipid coating.
  • polysaccharide NPs can be crosslinked with phosphate (anionic) and quarternary ammonium (cationic) ligands, with or without a lipid bilayer, such as one containing dipalmitoyl phosphatidyl choline and cholesterol coating.
  • polymers/stabilizers include, but are not limited to: soybean oil; maltodextrin; polybutylcyanoacrylate; butylcayanoacrylate/dextran 70 kDa, Polysorbate-85; polybutylcyanoacrylate/dextran 7OkDa, Polysorbate-85; stearic acid; poly- methylmethylacrylate.
  • the NP preparations containing the catecholic butanes such as by adsorption to the NPs, can be administered intravenously for treatment of a poxvirus.
  • the NPs may be coated with a surfactant or manufactured with a magnetically responsive material.
  • a surfactant may be used in conjunction with the NP.
  • polybutylcyanoacrylate NPs can be used with a dextran-70,000 stabilizer and Polysorbate- 80 as a surfactant.
  • Other surfactants include, but not limited to: Polysorbate-20, 40, or 60; Poloxamer 188; lipid coating-dipalmitoyl phosphatidylcholine; Epikuron 200; Poloxamer 338; Polaxamine 908; Polaxamer 407.
  • Polyaxamine 908 may be used as a surfactant to decrease uptake of NPs into the RES of the liver, spleen, lungs, and bone marrow.
  • the magnetically responsive material can be magnetite (Fe 3 O 4 ) which can be incorporated into the composition for making the NP. These magnetically responsive NPs can be externally guided by a magnet.
  • the NPs herein can be made as described in Mu and Feng using a blend of poly(lactide-co-glycolide)s (“PLGAs”) and d- ⁇ -tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS or TPGS) (Mu, L. and Feng, S.S., "A novel controlled release formulation for the anticancer drug paclitaxel (Taxol®): PLGA nanoparticles containing vitamin E TPGS.” J. Control. ReI. 86: 33-48 (2003)).
  • the latter can also act as an emulsifier, in addition to being a matrix material.
  • the present invention includes catecholic butanes formulated in micelle forming carriers, where the micelles are produced by processes conventional in the art. Examples of such are described in, for example, Liggins (Liggins, R.T. and Burt, H.M., "Polyether- polyester diblock copolymers for the preparation of paclitaxel loaded polymeric micelle formulations.” Adv. Drug Del. Rev. 54: 191-202, (2002)); Zhang, et al. (Zhang, X. et al, "Development of amphiphilic diblock copolymers as micellar carriers of taxol.” Int. J. Pharm. 132: 195-206, (1996)); and Churchill (Churchill, J.R., and Hutchinson, F.G.,
  • polyether-polyester block copolymers which are amphipathic polymers having hydrophilic (polyether) and hydrophobic (polyester) segments, are used as micelle forming carriers.
  • Another type of micelle is, for example, that formed by the AB-type block copolymers having both hydrophilic and hydrophobic segments, which are known to form micellar structures in aqueous media due to their amphiphilic character, as described in, for example, Tuzar (Tuzar, Z. and Kratochvil, P., "Block and graft copolymer micelles in solution.”, Adv.
  • poly(D,L-lactide)- ⁇ -methoxypolyethylene glycol (MePEG: PDLLA) diblock copolymers can be made using MePEG 1900 and 5000.
  • the reaction can be allowed to proceed for about 3 hr at about 16O 0 C, using stannous octoate (0.25%) as a catalyst.
  • a temperature as low as about 13O 0 C can be used if the reaction is allowed to proceed for about 6 hr, or a temperature as high as about 19O 0 C can be used if the reaction is carried out for only about 2 hr.
  • N-isopropylacrylamide (“IPAAm”) (Kohjin, Tokyo, Japan) and dimethylacrylamide (“DMAAm”) (Wako Pure Chemicals, Tokyo, Japan) can be used to make hydroxyl -terminated poly(IPAAm-co-DMAAm) in a radical polymerization process, using the method of Kohori, F. et al. (1998). (Kohori, F. et al, "Preparation and characterization of thermally Responsive block copolymer micelles comprising poly(N- isopropylacrylamide-b-D,L-lactide)." J. Control. ReI, 55: 87-98, (1998)).
  • the obtained copolymer can be dissolved in cold water and filtered through two ultrafiltration membranes with a 10,000 and 20,000 molecular weight cut-off.
  • the polymer solution is filtered through a 20,000 molecular weight cut-off membrane.
  • the filtrate is filtered again through a 10,000 molecular weight cut-off membrane.
  • Three molecular weight fractions can be obtained as a result, a low molecular weight, a middle molecular weight, and a high molecular weight fraction.
  • a block copolymer can then be synthesized by a ring opening polymerization of D,L-lactide from the terminal hydroxyl group of the poly(IPAAm- co- DMAAm) of the middle molecular weight fraction.
  • the resulting poly(IPAAm-co- DMAAm)- ⁇ -poly(D,L-lactide) copolymer can be purified as described in Kohori, F. et al. (1999). (Kohori, F. et al., "Control of adriamycin cytotoxic activity using thermally responsive polymeric micelles composed of poly(N-isopropylacrylamide-co-jV,jV- dimethylacrylamide)-b-poly(D,L-lacide).", Colloids Surfaces B: Biointerfaces 16: 195- 205, (1999)).
  • the catecholic butanes can be loaded into the inner cores of micelles and the micelles prepared simultaneously by a dialysis method.
  • a chloride salt of the catecholic butanes can be dissolved in N,N-dimethylacetamide (“DMAC”) and added by triethylamine (“TEA”).
  • DMAC N,N-dimethylacetamide
  • TAA triethylamine
  • the poly(IPAAm-co-DMAAm)-6-poly(D,L-lactide) block copolymer can be dissolved in DMAC, and distilled water can be added.
  • the solution of catecholic butanes and the block copolymer solution can be mixed at room temperature, followed by dialysis against distilled water using a dialysis membrane with 12,000-14,000 molecular weight cut-off (Spectra/Por®2, spectrum Medical Indus., CA. U.S.A.) at 25°C.
  • PoIy(IP AAm-co-DMAAm)-6-poly(D,L-lactide) micelles incorporating catecholic butanes can be purified by filtration with a 20 nm pore sized microfiltration membrane (ANODISCTM, Whatman International), as described in Kohori, F., et al. (1999), supra.
  • Multivesicular liposomes can be produced by any method conventional in the art, such as, for example, the double emulsification process as described in Mantriprgada (Mantriprgada, S., "A lipid based depot (DepoFoam® technology) for sustained release drug delivery.”, Prog Lipid Res. 41: 392-406, (2002)).
  • a "water-in-oil" emulsion is first made by dissolving amphipathic lipids, such as a phospholipid containing at least one neutral lipid, such as a triglyceride, in one or more volatile organic solvents, and adding to this lipid component an immiscible first aqueous component and a hydrophobic catecholic butane, such as a hydrophobic catecholic butane.
  • amphipathic lipids such as a phospholipid containing at least one neutral lipid, such as a triglyceride
  • the mixture is then emulsified to form a water-in-oil emulsion, and then mixed with a second immiscible aqueous component followed by mechanical mixing to form solvent spherules suspended in the second aqueous component, forming a water-in-oil-in-water emulsion.
  • the solvent spherules will contain multiple aqueous droplets with the catecholic butane dissolved in them.
  • the organic solvent is then removed from the spherules, generally by evaporation, by reduced pressure or by passing a stream of gas over or through the suspension. When the solvent is completely removed, the spherules become MVL, such as DepoFoam particles.
  • the neutral lipid is omitted in this process, the conventional multilamellar vesicles or unilamellar vesicles will be formed instead of the MVL.
  • catecholic butanes are water-soluble, hydrophilic compounds, such as G 4 N.
  • This invention includes formulation of hydrophilic compounds in a pharmaceutically acceptable carrier or excipient and delivery of such as oral formulations, such as in the form of an aqueous liquid solution of the compound, or the compounds can be lyophilized and delivered as a powder, or made into a tablet, or the compounds can be encapsulated.
  • the tablets herein can be enteric coated tablets.
  • the formulations herein can be sustained release and/or controlled release including either slow release or rapid release formulations.
  • the amount of the catecholic butanes to be included in the oral formulations can be adjusted depending on the desired dose to be administered to a subject. Such an adjustment is conventional and within the skill of persons familiar with the art.
  • Some catecholic butanes are hydrophobic or lipophilic compounds, such as M 4 N.
  • In vivo absorption of lipophilic compounds can be improved by using pharmaceutically acceptable carriers that can enhance the rate or extent of solubilization of the compound into the aqueous intestinal fluid.
  • Lipidic carriers are known in the art, such as, for example, as described in Stuchlik (Stuchlik, M. and Zak, S., "Lipid-Based Vehicle for Oral Delivery, Biomed. Papers 145(2): 17-26, (2001)).
  • the formulations herein can be delivered as oral liquids or can be encapsulated into various types of capsules.
  • the present invention includes, in one embodiment, a formulation containing the lipophilic catecholic butanes that are formulated for oral delivery by dissolution of such compounds in triacylglycerols, and the formulation is then encapsulated for oral delivery.
  • Triacyglycerols are molecules with long chain and/or medium chain fatty acids linked to a glycerol molecule.
  • the long chain fatty acids range from about Ci 4 to C 24 , and can be found in common fat.
  • the medium chain fatty acids range from about C 6 to Q 2 , and can be found in coconut oil or palm kernel oil.
  • Triacylglycerols suitable for use herein include structured lipids that contain mixtures of either short-chain or medium chain fatty acids or both, esterified on the same glycerol molecule.
  • one or more surfactants can be added to a mixture of catecholic butanes and lipidic carrier such that the drug is present in fine droplets of oil/surfactant mix.
  • the surfactants can act to disperse the oily formulation on dilution in the gastrointestinal fluid.
  • the present invention also includes a formulation for oral delivery of the catecholic butanes in the form of a micro-emulsion consisting of hydrophilic surfactant and oil.
  • the micro-emulsion particles can be surfactant micelles containing solubilized oil and drug.
  • Solid lipid nanoparticles can be prepared in any manner conventional in the art, such as, for example, as described in Stuchlik, M. and Zak, S. (2001), supra.
  • the solid lipid nanoparticle can be prepared in a hot homogenization process by homogenization of melted lipids at elevated temperature.
  • the solid lipid is melted and the catecholic butane is dissolved in the melted lipid.
  • a pre-heated dispersion medium is then mixed with the drug-loaded lipid melt, and the combination is mixed with a homogenisator to form a coarse pre-emulsion.
  • High pressure homogenization is then performed at a temperature above the lipids melting point to produce a oil/water-nanoemulsion.
  • the nanoemulsion is cooled down to room temperature to form solid lipid nanoparticles.
  • the solid lipid nanoparticles can be prepared in a cold homogenization process.
  • the lipid is melted and the catecholic butane is dissolved in the melted lipid.
  • the drug-loaded lipid is then solidified in liquid nitrogen or dry ice.
  • the solid drug-lipid is ground in a powder mill to form 50- 100 ⁇ m particles.
  • the lipid particles are then dispersed in cold aqueous dispersion medium and homogenized at room temperature or below to form solid lipid nanoparticles.
  • the present invention also includes formulation of the lipophilic catecholic butanes in liposomes or micelles for oral delivery. These formulations can be made in any manner conventional in the art.
  • Micelles are typically lipid monolayer vesicles in which the hydrophobic drug associates with the hydrophobic regions on the monolayer.
  • Liposomes are typically phospholipids bilayer vesicles.
  • the lipophilic catecholic butane will typically reside in the center of these vesicles.
  • the present invention includes formulations of catecholic butanes for intranasal delivery and intranasal delivery thereof.
  • Intranasal delivery may advantageously build up a higher concentration of the active agents in the brain than can be achieved by intravenous administration. Also, this mode of delivery avoids the problem of first pass metabolism in the liver and gut of the subject receiving the drug.
  • hydrophilic catecholic butanes can be dissolved in a pharmaceutically acceptable carrier such as saline, phosphate buffer, or phosphate buffered saline.
  • a pharmaceutically acceptable carrier such as saline, phosphate buffer, or phosphate buffered saline.
  • a 0.05 M phosphate buffer at pH 7.4 can be used as the carrier, as described in, for example, Kao, et a (Kao, H. D. et ah, "Enhancement of the Systemic and CNS
  • Intranasal delivery of the present agents may be optimized by adjusting the position of the subject when administering the agents.
  • the head of the patient may be variously positioned upright-90 o , supine-90°, supine-45°, or supine-70° to obtain maximal effect.
  • the carrier of the composition of catecholic butanes may be any material that is pharmaceutically acceptable and compatible with the active agents of the composition.
  • the carrier is a liquid, it can be hypotonic or isotonic with nasal fluids and within the pH of about 4.5 to about 7.5.
  • the carrier is in powdered form it is also within an acceptable pH range.
  • the carrier composition for intranasal delivery may optionally contain lipophilic substances that may enhance absorption of the active agents across the nasal membrane and into the brain via the olfactory neural pathway.
  • lipophilic substances include, but are not limited to, gangliosides and phosphatidylserine.
  • One or several lipophilic adjuvants may be included in the composition, such as, in the form of micelles.
  • compositions of active agents for intranasal delivery to a subject for treatment of a poxvirus can be formulated in the manner conventional in the art as described in, for example, U.S. Pat. No. 6,180,603.
  • the composition herein can be formulated as a powder, granules, solution, aerosol, drops, nanoparticles, or liposomes.
  • the composition may contain appropriate adjuvants, buffers, preservatives, salts. Solutions such as nose drops may contain anti- oxidants, buffers, and the like.
  • the catecholic butanes herein may be delivered to a subject for treatment by surgical implantation, such as subcutaneous implantation of a biodegradable polymer containing the catecholic butanes.
  • This treatment may be combined with other conventional therapy besides or in addition to surgery.
  • the biodegradable polymer herein can be any polymer or copolymer that would dissolve in the interstitial fluid, without any toxicity or adverse effect on host tissues.
  • the polymer or monomers from which the polymer is synthesized is approved by the U.S. Food and Drug Administration, or other equivalent regulatory body outside the United States, for administration into humans.
  • a copolymer having monomers of different dissolution properties is preferred so as to control the dynamics of degradation, such as increasing the proportion of one monomer over the other to control rate of dissolution.
  • the polymer is a copolymer of l,3-bis-(p- carboxyphenoxy)propane and sebacic acid, as described in Fleming A. B. and Saltzman, W.M., Pharmacokinetics of the Carmustine Implant, Clin. Pharmacokinet, 41(6): 403-419 (2002); and Brem, H., and Gabikian, P., "Biodegradable polymer implants to treat brain tumors.”, J. Control. ReI. 74: 63-67, (2001)).
  • the polymer is a copolymer of polyethylene glycol ("PEG”) and sebacic acid, as described in Fu, et al. (Fu, J. et al., "New Polymeric Carriers for Controlled Drug Delivery Following Inhalation or Injection.”, Biomaterials, 23: 4425-4433, (2002)).
  • Polymer delivery systems are applicable to delivery of both hydrophobic and hydrophilic catecholic butanes herein.
  • the catecholic butanes are combined with the biodegradable polymers and surgically implanted.
  • Some polymer compositions are also usable for intravenous or inhalation therapy herein.
  • the catecholic butanes herein may be delivered systemically and/or locally by administration to the lungs through inhalation.
  • Inhalation delivery of drugs has been well accepted as a method of achieving high drug concentration in the pulmonary tissues without triggering substantial systemic toxicity, as well as a method of accomplishing systemic circulation of the drug.
  • the techniques for producing such formulations are conventional in the art. Efficacy against pulmonary diseases may be seen with either hydrophobic or hydrophilic catecholic butanes delivered in this manner.
  • the catecholic butanes herein may be formulated into dry powders, aqueous solutions, liposomes, nanoparticles, or polymers and administered, for example, as aerosols.
  • Hydrophilic formulations may also be taken up through the alveolar surfaces and into the bloodstream for systemic applications.
  • the polymers containing the active agents herein are made and used as described in Fu, J. et al. (2002), supra.
  • the polymers herein can be polymers of sebacic acid and polyethylene glycol (“PEG”), or can be poly(lactic-co- glycolic) acid (“PLGA”), or polymers of polyethyleneimine (“PEI”) and poly-L-lysine (“PLL”).
  • the catecholic butanes for inhalation delivery may be dissolved in saline or ethanol before nebulization and administered, as described in Choi, et al. (Choi, ⁇ .S. et al., "Inhalation delivery of proteins from ethanol suspensions.”, Proc. Natl. Acad. Sci. USA, 98(20): 11103-11107, (2001)).
  • the agents herein are also effective when delivered as a dry powder, prepared in the manner conventional in the art, as described in, for example, Patton, et al. (Patton, J.S. et al., "Inhaled Insulin,”, Adv. Drug Deliv. Rev., 35: 235-247 (1999) (2001)).
  • the present invention includes delivery of the catecholic butanes with the aid of microprocessors embedded into drug delivery devices, such as, for example, SmartMistTM and AERxTM, as described in, for example, Gonda, I. et al. (1998), "Inhalation delivery systems with compliance and disease management capabilities.” J. Control. ReI. 53: 269- 274.
  • Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more active agents.
  • unit dosage forms for injection or intravenous administration may comprise the active agent(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
  • Kits with multiple or unit doses of the active agent are included in the present invention.
  • kits in addition to the containers containing the multiple or unit doses of the compositions containing the NDGA derivatives will be an informational package insert with instructions describing the use and attendant benefits of the drugs in treating the pathological condition of interest, in this case, a poxvirus.
  • catecholic butanes and compositions of the subject invention find use as prophylactic or therapeutic agents in situations where one wishes to provide a treatment to a subject for purposes of preventing a poxviral infection caused by a poxvirus or treatment of a subject suffering from a poxviral infection.
  • a variety of animal hosts are treatable according to the subject methods, including human and non-human animals, such as cows in the case of CPXV, where there is concern about trans-species infection from cows to mammals in general and humans in particular.
  • such hosts are "mammals" or “mammalian,” where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., guinea pigs, and rats), and other mammals, including cattle, goats, horses, sheep, rabbits, pigs, and primates (e.g., humans, chimpanzees, and monkeys).
  • the hosts will be humans.
  • animal models are also of interest for experimental investigations, such as providing a model for treatment of human disease. Further, the present invention is applicable to veterinary care as well.
  • compositions of the instant invention will contain from less than about 1% up to about 99% of the active ingredient, that is, the catecholic butanes herein; optionally, the instant invention will contain about 5% to about 90% of the active ingredient.
  • the present invention additionally provides compositions in which the active agents, such as the catecholic butanes, including the NDGA derivatives, for example, M 4 N, are administered to subjects, such as humans, at an oral dose of about less than 0.1 mg/kg to about 400 mg/kg or more based on the weight of the animals, such as humans, for example.
  • the subjects may be treated via any suitable route of administration, with a range from about 0.01 to about 400 mg/kg of body weight per dose, such as less than about 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.5 mg/kg, 5.0 mg/kg, 10 mg/kg, 15 mg/kg, 25 mg/kg, 50 mg/kg, 100 mg/kg, 150 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, or 400 mg/kg, or more.
  • body weight per dose such as less than about 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.5 mg/kg, 5.0 mg/kg, 10 mg/kg, 15 mg/kg, 25 mg/kg, 50 mg/kg, 100 mg/kg, 150 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, or 400 mg/kg, or more.
  • the appropriate dose to be administered depends on the subject to be treated, such as the general health of the subject, the age of the subject, the state of the disease or condition, the weight of the subject, for example. Generally, about 0.1 mg to about 500 mg may be administered to a child and about 0.1 mg to about 5 grams may be administered to an adult.
  • the active agent can be administered in a single or, more typically, multiple doses. Preferred dosages for a given agent are readily determinable by those of skill in the art by a variety of means. Other effective dosages can be readily determined by one of ordinary skill in the art through routine trials establishing dose response curves. The amount of agent will, of course, vary depending upon the particular agent used.
  • the care giver When administering the active agent as a prophylactic agent to prevent a poxviral infection caused by a poxvirus, the care giver will consider such factors as age, weight, disease status, health status, amount of time since a previous administration in determining the amount and number of doses that are needed as well as other factors that are well known by those having ordinary skill in the art.
  • the frequency of administration of the active agent will be determined by the care giver based on age, weight, disease status, health status and patient responsiveness.
  • the agents may be administered continuously, intermittently, one or more times daily or in other periods as appropriate for as long as needed as conventionally determined.
  • the catecholic butanes or active agents of the present invention can be incorporated into a variety of formulations for therapeutic administration. More particularly, the catecholic butanes of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semisolid, liquid or gaseous forms, such as tablets, capsules, powders, aerosols, liposomes, nanoparticles, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.
  • administration of the active agents can be achieved in various ways, such as oral, buccal, rectal, intranasal, intravenous, subcutaneous, intramuscular, intra-tracheal, topical, interstitial, transdermal, etc., or by inhalation or implantation.
  • nanoparticle, micelle and liposomal preparation can be administered systemically, including parenterally and intranasally, as well as interstitially, orally, topically, transdermally, via inhalation or implantation, such as for drug targeting, enhancement of drug bioavailability and protection of drug bioactivity and stability.
  • Nanoparticle bound drugs herein are expected to achieve prolonged drug retention in vivo.
  • the active agents may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds.
  • the following methods and excipients are merely exemplary and are in no way limiting.
  • the catecholic butanes and compositions of the present invention are administered to a human subject for the purpose of preventing a poxviral infection caused by a poxvirus.
  • the catecholic butanes and compositions are administered to a human subject as a prophylactic treatment to prevent a poxviral infection caused by an orthopoxvirus strain of poxvirus.
  • the catecholic butanes and compositions are administered to a human subject as a prophylactic treatment to prevent a poxviral infection caused by variola virus.
  • the catecholic butane and compositions are administered to a human subject as a prophylactic treatment to prevent a poxviral infection caused by monkeypox virus.
  • the catecholic butane and compositions are administered to a human subject as a prophylactic treatment to prevent a poxviral infection caused by CPXV. In certain preferred embodiments, the catecholic butane and compositions are administered to a human subject as a prophylactic treatment to prevent a poxviral infection caused by VAC.
  • the plaque reduction assay is formed by infecting a cell monolayer with a poxvirus.
  • a plaque is formed when the virus spreads radially to surrounding cells from a single, initially infected cell. Following infection, the inoculum is removed and replaced with a growth medium in the well of the assay. Varying amounts of catecholic butane is added to the growth medium of some of the wells.
  • MOI multiplicity of infection
  • Plaques were visualized by negative staining the incubated plates with a solution containing 0.1% crystal violet and 20% ethanol.
  • the number of plaques per well was determined by automated counting with GEL DOCTM XR plaque counting software.
  • the catecholic butane and poxvirus are added to cell monolayers. Since it is not necessary to visualize discrete plaques in these cultures, a higher MOI — on the order of 1 virion for each of 1-10 cells — is used.
  • Virus replication was allowed to proceed for 24 hours, then supernatants and cell lysates were collected and the relative accumulation of virus determined by plaque assay using 143B cells.
  • FIG. IB The plaque growth in the assay having a concentration of 6.25 ⁇ M of M 4 N is illustrated in FIG. IB. As these figures illustrate, plaque formation by CPXV was almost completely inhibited with this concentration of M 4 N. Table 1 shows the effect of varying M 4 N concentrations on CPXV growth in the 143B cells. TABLE 1
  • Example 2 Cell monolayers of 143B human osteosarcoma cells were infected as in Example 1 , except the poxvirus VAC was used instead of CPXV. Following infection, the inoculum was removed and replaced with a growth medium of DMEM with 10% FBS. The growth media added to the wells of the plates were each individually supplemented with 3.125 ⁇ M, 6.25 ⁇ M, and 12.5 ⁇ M of M 4 N or the control vehicle DMSO. The plates were incubated for 24 hours at 37 0 C in the presence of 5% CO 2 to allow for sufficient plaque development. The plaque growth in the control group is illustrated in FIG. 2A. The plaque growth in the assay having a concentration of 6.25 ⁇ M of M 4 N is illustrated in FIG. 2B.
  • FIG. 3A The assays having lower concentrations Of M 4 N showed no observable effects on either plaque number or size.
  • the assay having a concentration of 25 ⁇ M Of M 4 N shows a slight reduction in the number of plaques formed by CPXV but shows a substantial reduction in the size of plaques formed by the poxvirus.
  • Example 4 Cell monolayers of MRC-5 human lung fibroblast cells were infected as in Example 3 using VAC. Following infection, the inoculum was removed and replaced with a growth medium of DMEM with 10% FBS. The growth media added to the wells of the plates were each individually supplemented with varying doses of M 4 N up to a concentration of 25 ⁇ M or the control vehicle DMSO. The plates were incubated for 24 hours at 37 0 C in the presence of 5% CO 2 to allow for sufficient plaque development. The plaque growth in the control group is illustrated in FIG. 4A. The assays having lower concentrations of M 4 N showed no observable effects on either plaque number or size. The assay having a concentration of 25 ⁇ M of M 4 N, as illustrated in FIG. 4B, shows a slight reduction in the number of plaques formed by VAC but shows a substantial reduction in the size of plaques formed by the poxvirus.
  • Example 5 Example 4
  • the stringency of the virus challenge was increased by inoculating the assay at an MOI of 1 — i.e., at a ratio of approximately 1 CPXV virion for each RAW 264.7 cell.
  • MOI i.e. 1 — i.e. 1 CPXV virion for each RAW 264.7 cell.
  • the effect of pre-inoculation treatment with a concentration of 25 ⁇ M of M 4 N reduced CPXV growth by about 99% compared to about 98% for post-inoculation dosing at the same concentration of M 4 N (see Table 2).
  • Example 8 Cell monolayers of RAW 264.7 cells were pretreated with either the control vehicle DMSO or 25 ⁇ M M 4 N for 1 hour at 37 0 C in the presence of 5% CO 2 and then infected with VAC for 45 minutes at 37 0 C. Following the inoculation period, the inoculum was removed and replaced with the growth medium DMEM with 10% FBS and allowed to incubate for 24 hours at 37°C in the presence of 5% CO 2 . For the assay containing M 4 N, a constant concentration of 25 ⁇ M of M 4 N was maintained by dosing throughout the inoculation and growth periods.
  • the stringency of the virus challenge was increased by inoculating the assay at an MOI of 1 — i.e., at a ratio of approximately 1 VAC virion for each RAW 264.7 cell.
  • MOI i.e., at a ratio of approximately 1 VAC virion for each RAW 264.7 cell.
  • the effect of pre-inoculation treatment with a concentration of 25 ⁇ M of M 4 N reduced VAC growth by about 88% compared to about 86% for post-inoculation dosing at the same concentration of M 4 N (see Table 2).
  • Low MOI virus growth assays were used to evaluate the effect of M 4 N treatment on cowpox replication in vitro. Growth assays were conducted in a variety of cell types including human, mouse, and monkey tissues, chosen to serve as representatives for biologically relevant host organ systems. For all experiments, the MOI was 0.01, or one infectious virion for every 100 cells in culture.
  • the cells Prior to infection, the cells were treated with 25 ⁇ M of M 4 N or 0.05% DMSO, the vehicle control, for 1 hour at 37 0 C and 5% CO 2 . Cells were then infected with CPXV at MOI of 0.01 for 1 hour at 37 0 C in serum free media supplemented with 25 ⁇ M of M 4 N or 0.05% DMSO. After infection, the growth media supplemented with 10% FBS and 25 ⁇ M of M 4 N or 0.05% DMSO was added. Using this methodology, cells were in the presence of M 4 N or the control vehicle before, during, and following infection with CPXV. The cells were then incubated at 37 0 C and 5% CO 2 for 5, 12, 24 and 48 hours.
  • Freeze-thaw lysis and sonication was used in preparing samples of the virus replications from each tissue culture flask for each of the incubation periods.
  • the titer of infectious CPXV was determined by conducting standard plaque assay on human 143B osteosarcoma cells. Plaque counts were performed using Bio-Rad QUANTITY ONE® GEL DOC XR counting software.
  • Example 10 Example 11 Example 12 Example 13 Example 14 Example 15 Example 16

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Abstract

Cette invention concerne des procédés de prévention et de traitement d'une infection à virus du groupe pox causée, de préférence, par un virus du genre orthopoxvirus, qui comprend l'administration chez un sujet d'une quantité thérapeutiquement efficace d'un butane de structure catécholique de formule générale suivante (I) ou de son sel pharmaceutiquement acceptable. Des modes de réalisation spécifiques des procédés de traitement d'une infection à virus du groupe pox comprennent l'administration chez un sujet d'une quantité thérapeutiquement efficace d'acide tétra-O-méthyl nordihydroguaïarétique. Des procédés de vaccination permettant d'immuniser un sujet contre l'infection à virus du groupe pox sont décrits et comprennent l'administration au sujet d'un vaccin et d'un butane de structure catécholique ou de son sel pharmaceutiquement acceptable avant et/ou pendant une durée thérapeutiquement efficace, sensiblement en même temps que, et après une durée d'incubation suivant l'administration du vaccin.
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EP3492079A1 (fr) * 2017-11-29 2019-06-05 Université de Bourgogne Corroles permettant de traiter une infection par le poxvirus

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Cited By (6)

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Publication number Priority date Publication date Assignee Title
CN105688184A (zh) * 2016-03-11 2016-06-22 杜峰 一种用于治疗骨肉瘤的皮下注射用原位凝胶
EP3492079A1 (fr) * 2017-11-29 2019-06-05 Université de Bourgogne Corroles permettant de traiter une infection par le poxvirus
WO2019105940A1 (fr) * 2017-11-29 2019-06-06 Universite De Bourgogne Corroles pour le traitement d'une infection à poxvirus
JP2021504486A (ja) * 2017-11-29 2021-02-15 ユニヴェルシテ ドゥ ブルゴーニュＵｎｉｖｅｒｓｉｔｅ Ｄｅ Ｂｏｕｒｇｏｇｎｅ ポックスウイルス感染症を処置するためのコロール
US11529332B2 (en) 2017-11-29 2022-12-20 Universite De Bourgogne Corroles for treating poxvirus infection
JP7229265B2 (ja) 2017-11-29 2023-02-27 ユニヴェルシテ ドゥ ブルゴーニュ ポックスウイルス感染症を処置するためのコロール

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