+

WO2003061579A2 - Compositions pharmaceutiques a base de phtalocyanine et de porphyrazine - Google Patents

Compositions pharmaceutiques a base de phtalocyanine et de porphyrazine Download PDF

Info

Publication number
WO2003061579A2
WO2003061579A2 PCT/US2003/001619 US0301619W WO03061579A2 WO 2003061579 A2 WO2003061579 A2 WO 2003061579A2 US 0301619 W US0301619 W US 0301619W WO 03061579 A2 WO03061579 A2 WO 03061579A2
Authority
WO
WIPO (PCT)
Prior art keywords
substituted
compound
group
composition
hiv
Prior art date
Application number
PCT/US2003/001619
Other languages
English (en)
Other versions
WO2003061579A3 (fr
Inventor
Richard W. Compans
Luigi G. Marzilli
Dabney W. Dixon
Original Assignee
Emory University
Georgia State University Research Foundation, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Emory University, Georgia State University Research Foundation, Inc. filed Critical Emory University
Priority to AU2003205227A priority Critical patent/AU2003205227A1/en
Publication of WO2003061579A2 publication Critical patent/WO2003061579A2/fr
Publication of WO2003061579A3 publication Critical patent/WO2003061579A3/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/409—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having four such rings, e.g. porphine derivatives, bilirubin, biliverdine

Definitions

  • This application relates to the field of anti-viral compounds, specifically phthalocyanine compounds, for the prevention of sexually transmitted diseases (STDs) caused by viral pathogens such as human immunodeficiency virus, herpes viruses, hepatitis viruses, and papilloma viruses.
  • STDs sexually transmitted diseases
  • STDs affect men and women of all backgrounds and economic levels. They are most prevalent among teenagers and young adults. Nearly two-thirds of all STDs occur in people younger than 25 years of age. The incidence of STDs is rising, in part because in the last few decades, young people have become sexually active earlier yet are marrying later. In addition, divorce is more common. The net result is that sexually active people today are more likely to have multiple sex partners during their lives and are potentially at risk for developing STDs.
  • STDs Health problems caused by STDs tend to be more severe and more frequent for women than for men, in part because the frequency of asymptomatic infection means that many women do not seek care until serious problems have developed.
  • Some STDs can spread into the uterus (womb) and fallopian tubes to cause pelvic inflammatory disease (PID), which in turn is a major cause of both infertility and ectopic (tubal) pregnancy. The latter can be fatal.
  • STDs in women also may be associated with cervical cancer.
  • PPN human papillomavirus infection
  • STDs can be passed from a mother to her baby before, during, or immediately after birth; some of these infections of the newborn can be cured easily, but others may cause a baby to be permanently disabled or even die.
  • HIV Infection and AIDS HIV Infection and AIDS
  • AIDS immunodeficiency syndrome
  • HIN human immunodeficiency virus
  • An estimated 900,000 people in the United States are currently infected with HIN.
  • People who have AIDS are very susceptible to many life-threatening diseases, called opportunistic infections, and to certain forms of cancer. Transmission of the virus primarily occurs during sexual activity and by sharing needles used to inject intravenous drugs.
  • Genital Herpes Genital herpes affects an estimated 60 million Americans.
  • Herpes infections are caused by herpes simplex virus (HSN).
  • HSN herpes simplex virus
  • the major symptoms of herpes infection are painful blisters or open sores in the genital area. These may be preceded by a tingling or burning sensation in the legs, buttocks, or genital region.
  • the herpes sores usually disappear within two to three weeks, but the virus remains in the body for life and the lesions may recur from time to time.
  • Severe or frequently recurrent genital herpes is treated with one of several antiviral drugs that are available by prescription. These drugs help control the symptoms but do not eliminate the herpes virus from the body.
  • Suppressive antiviral therapy can be used to prevent occurrences and perhaps transmission. Women who acquire genital herpes during pregnancy can transmit the virus to their babies. Untreated HSN infection in newborns can result in mental retardation and death.
  • Genital warts are caused by human papillomavirus, a virus related to the virus that causes common skin warts. Genital warts usually first appear as small, hard painless bumps in the vaginal area, on the penis, or around the anus. If untreated, they may grow and develop a fleshy, cauliflower-like appearance. Genital warts infect an estimated 1 million Americans each year. In addition to genital warts, certain high-risk types of HPN cause cervical cancer and other genital cancers. Genital warts are treated with a topical drug (applied to the skin), by freezing, or if they recur, with injections of a type of interferon. If the warts are very large, they can be removed by surgery.
  • STDs in pregnant women are associated with a number of adverse outcomes, including spontaneous abortion and infection in the newborn. Low birth weight and prematurity appear to be associated with STDs, including chlamydial infection and trichomoniasis.
  • Congenital or perinatal infection infection that occurs around the time of birth) occurs in 30 to 70 percent of infants born to infected mothers, and complications may include pneumonia, eye infections, and permanent neurologic damage. This starts on AIDS again- do you want header here?
  • AIDS or acquired immunodeficiency disease
  • helper/inducer T lymphocytes and suppressor T lymphocytes are responsible for the induction of most of the functions of the human immune system, including the humoral immune response involving the production of antibodies by B lymphocytes and the cell-mediated response involving stimulation of cytotoxic T cells.
  • a condition associated with HIN is AIDS-related complex, or ARC. Most human beings suffering from ARC eventually develop AIDS.
  • HIN human immunodeficiency virus type 1 and type 2
  • the genomes of the two viruses are about 50% homologous at the nucleotide level, contain the same complement of genes, and appear to attack and kill the same human cells by the same mechanism.
  • LAN lymphadenopathy-associated virus
  • HTLN-3 human T-lymphotropic virus- type 3
  • ARN AIDS-related virus
  • HIV-1 was identified in 1983.
  • Virtually all AIDS cases in the U.S. are associated with HIN-1 infection.
  • HIN-2 was isolated in 1986 from West African AIDS patients. Both types of HIN are retroviruses, in which the genetic material is
  • the viruses carry with them a polymerase (reverse transcriptase) that catalyzes transcription of viral R ⁇ A into double-helical D ⁇ A.
  • the viral D ⁇ A can exist as an unintegrated form in the infected cell or be integrated into the genome of the host cell.
  • the HIN enters the T4 lymphocyte where it loses its outer envelope, releasing viral R ⁇ A and reverse transcriptase.
  • the reverse transcriptase catalyzes synthesis of a complementary D ⁇ A strand from the viral R ⁇ A template.
  • the D ⁇ A helix then inserts into the host genome where it is known as the pro virus.
  • the integrated D ⁇ A may persist as a latent infection characterized by little or no production of virus or helper/inducer cell death for an indefinite period of time.
  • new viral R ⁇ A and proteins are produced to form new viruses that bud from the cell membrane and infect other cells.
  • a number of compounds have apparent virucidal activity against this virus, including HPA-23, interferons, ribavirin, phosphonoformate, ansamycin, suramin, imuthiol, penicillamine, carbovir, 3'-azido-3'- deoxythymidine (AZT), and other 2',3'-dideoxynucleosides, such as 2',3'- dideoxycytidine (DDC), 2',3'-dideoxyadenosine (DDA), 2',3'-dideoxyinosine (DDI), (CS-87), 2 , ,3'-dideoxy-2 , ,3*- didehydrocytidine (D4C), 3'-deoxy-2 , ,3'-didehydrothymidine (D4T) and 3'- azido-5-ethyl-2',3'-dideoxyuridine (CS-85).
  • DDC dideoxy
  • Inhibitors of cellular processes will often limit viral replication. Unfortunately, they are also usually toxic for the host and therefore cannot be prescribed for a prolonged period of time because of their toxicity. Efforts to decrease the problem of toxicity have primarily been directed towards finding selective, less toxic drugs. Due to the exorbitant cost of the nucleoside type drugs, research has also been centered around compounds which are relatively easy and economical to manufacture. Herpes Simplex
  • Herpes simplex viruses are common sexually transmitted pathogens. Following transmission of the virus to a susceptible individual, HSV-2 replicates in the epithelial cells of genital mucosal surfaces. This replication is usually asymptomatic, as evidenced by the number of individuals who are seropositive for HSN-2 antibody, but have no history of symptomatic infection. However, particularly in individuals who are seronegative for both HSN-1 and HSN-2, primary infection can result in severe, ulcerative lesions. Following replication in epithelia, the virus infects the peripheral endings of sensory neurons innervating the site of infection, and is transported through the neuronal axons to the nuclei..
  • HSV-2 herpes simplex virus type 2
  • Various stimuli including stress, damage to peripheral tissues near the site of infection, or direct nerve damage cause reactivation of latent virus, and productive viral replication is initiated in the neuron..
  • Virus is transported back through neuronal axons to the epithelial tissue, where it again replicates, is shed into extracellular space, and is available for transmission to a new individual (Whitley, et al. Ann Intern Med. 125(5):376-83 (1996); Roizman and Sears, Annu. Rev. Micrbiol. 41:543-571 (1987)).
  • HSV-2 is the most common cause of neonatal herpes infection, which is most frequently transmitted during delivery of an infant to a mother who is shedding infectious virus (Whitley, 1996). Availability of nontoxic, topical virucidal compounds, and their use during delivery, would reduce or eliminate virus available for transmission and thereby also reduce the level of risk to the infant.
  • Genital herpes infections have also been implicated in the transmission of human immunodeficiency viruses. Epidemiologic studies have suggested that infection by HSV-2, along with other sexually transmitted diseases that cause genital ulcers, increases the risk of acquisition of HIV.
  • HIV-susceptible cells CD4+ T cells and macrophages
  • co-infection of HSV-2 and HIV may result in a higher risk of transmission of HIV: HIV virions have been detected in cells present in genital lesions caused by HSV, leading to the hypothesis that HSV lesions may generated a higher level of HIV in the genital tract available for transmission (Schacker et al. J. Infect. Dis. 178(6): 1616-22 (1998); JAMA. 1998 Jul l;280(l):61-6 (1998)).
  • virucidal drugs available for inactivation of HSV replication, including acyclovir, cidofivir, sorivudine, and foscamet.
  • acyclovir cidofivir
  • sorivudine sorivudine
  • foscamet a number of virucidal drugs available for inactivation of HSV replication
  • all of these drags target replication of the viral DNA following infection of susceptible cells; they cannot prevent the initial infection of epithelial cells.
  • several of the drugs have been shown to be only partially effective at reducing viral replication in genital epithelium when applied topically (see, for example, Bravo, et. al., Antiviral Res 21:59- 72 (1993)).
  • Anti-HS V virucides tested to date include compounds with both specific and nonspecific activity. Many of these compounds are effective virucides when tested in cell culture, including those that inhibit specific interactions between the virus and the cell surface (neutralizing antibodies and polyanionic compounds such as heparan sulfate, heparin, dextran sulfate, and carageenan), and those that disrupt virion architecture (nonoxynol-9) (see, i.e., Zeitlin et al., Contraception 56: 329-335 (1997); Zacharopoulos and Phillips, Clinical and Diagnostic Laboratory Immunology 4:465-468 (1997)). However, use of neutralizing antibodies is cost prohibitive, at least at the present time.
  • Polyanionic compounds have had varying success in inactivation of HSV-2 infection in vivo; in a mouse model of genital infection, heparan sulfate was not particularly effective, and dextran sulfate and carageenan prevented infection only of extremely low doses of viras (10 3 pfu or less ) (Zeitlin et al. 1997). Continual use of nonoxynol-9 has been shown to cause inflammation of vaginal and cervical epithelium (Stafford, et al, J. Acquir. Immune. Defic. Syndr. Hum. Retrovirol.
  • a method for preventing a sexually transmitted disease (STD) or viral infection with a virus such as HIV in a human by administering to a mucosal surface, preferably prior to infection, a composition containing an amount of a neutral or negatively charged compound, a metal complex of the compound, a pharmaceutically acceptable salt of the compound or metal complex, or a mixture thereof to a human in an amount effective to decrease a viral infection, wherein the compound has one of the following structures:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 and R 16 taken independently or together can be hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, nitro, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, heteroarylthio, substituted heteroarylthio, cyano, isocyano, substituted isocyano, carbonyl
  • the compound can form a chelate with a main group or transition metal atom.
  • Representative useful metal atoms are gallium (Ga), aluminum (Al), cadmium (Cd), ruthenium (Ru), rhodium (Rh), platinum (Pt), palladium (Pd), osmium (Os), iridium (Ir), iron (Fe), cobalt (Co), zinc (Zn), molybdenum (Mo), titanium (Ti), manganese (Mn), chromium (Cr), nickel (Ni), magnesium (Mg), copper (Cu), indium (In), vanadium (V) or vanadium oxide (VO), silver (Ag), gold (Au), or tin (Sn).
  • the metal atom can have various leaving ligands.
  • the leaving ligands can be any charged or uncharged ligands.
  • the ligand is Cl " .
  • the ligand is an ether.
  • the metal atom is Cu, Fe, Mn, Ni, Zn, or VO.
  • Typical pharmaceutically acceptable salts include H+, Na+, Li+, K+, Ca++, Mg++ and protonated amines and heterocyclic amines.
  • the compounds are formulated for adminitration to a mucosal surface, such as to the female genital tract or rectum.
  • the composition can also be used in combination with any number of other active agents or drugs, including antibiotics, virucidal or antiviral compounds, antifungals, immunomodulatory compounds, and contraceptives.
  • FIG. 1 shows the structures of phthalocyanines and porphyrazines.
  • PcS is sulfonated phthalocyanine
  • PcC is carboxylated phthalocyanine
  • Alcian Blue, Alec Blue and Reactive Blue 15 are representative cationic phthalocyanine.
  • M can be H2 or a metal in all structures; porphyrazines can also have a central metal.
  • Figure 2 shows the synthesis of sulfonated phthalocyanines via protected sulfonic acids.
  • Figure 3 demonstrates the synthesis of an ABBB phthalocyanine via a subphthalocyanine.
  • Figure 4 shows exemplary phthalocyanines bearing OH groups.
  • Figure 5 shows the activity of sulfonated phthalocyanines against HIV-1 IIIB (percent virus inactivated).
  • Figure 6 shows the kinetics of inactivation of HIV-1 IIIB, percent of virus inactivated over time in minutes. Compounds at a final concentration of 50 ⁇ g/ml were mixed with viras and incubated at various time intervals (5, 15, 30, 45, 60 min), diluted 1:10 with complete medium, and infectivity titers determined.
  • Figure 7 shows the activity (percent virus inactivated) of selected phthalocyanines as a function of concentration. Viras samples were mixed with phthalocyanines at the indicated concentrations, and infectivity titers determined.
  • Figure 8 shows the percent inhibition of gpl 20-CD4 binding by various compounds.
  • a 96-well plate coated with soluble CD4 was incubated with HIV-1 IIIB gpl 20 in the presence or absence of compounds for 1 hr at room temperature. After extensive washes the bound gpl 20 was detected by anti-gpl20 peroxidase-conjugated antibodies. Results are given as % of gp 120 binding compared to untreated gp 120 sample ( 100%) .
  • compositions for preventing or treating viral and sexually transmitted diseases are provided herein.
  • the pharmaceutical composition contains a synthetic phthalocyanine and/or porphyrazine or a metal complex thereof in an amount effective to inactivate a viras, preferably prior to infection.
  • the composition may optionally include one or more pharmaceutically effective agents such as antibiotics, virucidals, antivirals, antifungals, immunostimulants, and substances which are effective in inactivating viruses.
  • STDs sexually transmitted diseases
  • exemplary STDs include HIV infection and AIDS, infections caused by hepatitis B and/or C viruses, infection caused by papilloma viruses, genital herpes, genital warts, and syphilis.
  • Phthalocyanines are a class of compounds having a planar conjugated ⁇ c-stack system ( Figure 1). Phthalocyanines have eight nitrogen atoms and eight carbon atoms forming a 16-member cyclic structure. The eight nitrogen atom provide an excellent environment for the molecule coordination to metal atoms. Porphyrazines are homologues of phthalocyanines in which the benzene rings are missing ( Figure 1) (Kobayashi "meso- Azaporphyrins and their analogs" in: The Porphyrin Handbook, edited by K. M. Kadish and K. M. Smith, San Diego: Academic Press, p. 301-360 (2000)). This planar conjugated system has 26 B electrons.
  • porphyrazines are very stable. They can be sublimed above 350 °C. Many metalloporphyrazines are also very stable. For example, complexes with 3d metals (Cu, Co, Ni, Zn) dissociate only slowly in hot concentrated sulfuric acid (Khelevina, et al., J Porph. Phthalo. 4 (5):555-563 (2000)).
  • the advantages of porphyrazines are that the smaller central core gives a less hydrophobic species, with slightly different placements of the side chains (see, for example, Kobayashi 2000; Khelevina, et al., 2000).
  • Phthalocyanines and Porphyrazines Phthalocyanines (Pcs) are being studied intensively in conjunction with their use as photodynamic agents in chemotherapy and in the phototherapeutic destruction of pathogens.
  • Photodynamic tumor therapy involves injection of a photosensitizing agent with a propensity to localize in tumors, waiting for some hours (up to a few days) for maximum tumor localization, and irradiation of the photosensitizing agent at the tumor site.
  • photodynamic therapy by nature, demands that the phthalocyanine be light sensitive. Light is not required for the method described herein.
  • Second, photodynamic agents are used systemically and microbicides are used topically.
  • VSV vesicular stomatitis viras
  • Sindbis virus Rost al., Photochemicstry and Photobiology 60:165-170 (1994)
  • HIV Horowitz et al., Transfusion 31:102-108 (1991); Margolis-Nunno et al., Transfusion 36:743-750 (1996); Ben-Hur, Oetjen, and Horowitz, Photochemistry and Photobiology 54:703-707 (1997); Zmudzka et al.
  • AlPcS4 The anionic aluminum phthalocyanine tetrasulfonate (AlPcS4) as well as the cationic silicon phthalocyanines HOSiPcOSi(CH 3 ) 2 (CH 2 ) 3 N(CH 3 ) 2 (Pc4), and HOSiPcOSi(CH 3 ) 2 (CH 2 ) 3 N + (CH 3 ) 3 1 " (Pc5) were able to inactivate 10 5 infectious doses of cell-free HIV upon illumination with red light. However, of the three Pcs, only the cationic Pc4 was effective in inactivating actively replicating HIV in infected cells.
  • inhibitors bind to components of the virion and prevent infection.
  • the compounds can bind to certain sites on the surface glycoprotein subunit (SU) gpl 20 and block its functions (Neurath et al, Antiviral Chem. Chemother. 3:55-63 (1992); Neurath et al, J. Molecular Recognition 8:345-357 (1995)).
  • SU surface glycoprotein subunit
  • These studies indicate the presence of non-neutralizing and neutralizing domains of gpl 20 as well as a silent face of the glycoprotein.
  • Another open surface appears to be the receptor-binding region of HIV-1 gpl 20. These sites represent potential targets for the binding of virucidal compounds.
  • the virucidal compounds may also conformationally alter the protein or possibly block membrane fusion mediated by the gpl20/gp41 complexes of the envelope protein.
  • composition disclosed herein contain one or more compounds and/or their pharmaceutically acceptable salt, the compound having the following structure:
  • Formula I Formula II wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 and R 16 taken independently or together can be hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, nitro, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, heteroarylthio, substituted heteroarylthio, cyano, isocyano, substituted isocyan
  • Compounds of Formulae I-II can chelate with any of the main group or transition metals to form a metallocomplex.
  • Metal atoms useful for forming the metallocomplex with any of the compounds of Formulae I-II are generally charged, though sometimes uncharged metal atoms may be useable.
  • Representative metal ions are any ions derived from gallium (Ga), aluminum (Al), cadmium (Cd), ruthenium (Ru), rhodium (Rh), platinum (Pt), palladium (Pd), osmium (Os), iridium (Ir), iron (Fe), cobalt (Co), zinc (Zn), molybdenum (Mo), titanium (Ti), manganese (Mn), chromium (Cr), nickel (Ni), magnesium (Mg), copper (Cu), indium (In), vanadium (V) or vanadium oxide (VO), silver (Ag), gold (Au) or tin (Sn).
  • the metal atom is Cu, Fe, Mn, Ni, Zn, or V.
  • the metal atoms may have neutral or ionic ligands.
  • exemplary neutral ligands include H 2 O, pyridine, imidazoles, NH 3 , alkylamines, ethers, oxygen, amino acid or peptide esters, phosphines, and alcohol.
  • Other neutral ligands commonly used in coordination chemistry may also used.
  • Exemplary ionic ligands can be negative charged ligands such as Cl “ , NO " , CN “ , RS “ , and terminal N-bound amino acids or peptides.
  • the complexes formed of any of the disclosed compounds are more exchange labile than their counterparts with the same metal but with other ligands attached.
  • alkyl or aryl ligands can be used.
  • Representative pharmaceutically acceptable salt is a salt of the compound or metallocomplex of the compound with a cation such as H+, Na+, Li+, K+, Ca++, Mg++, protonated amines and heterocyclic amines, and quaternary ammonium including, but not limited to, alkyl and aryl or alkyl and aryls containing heteroatoms, such as oxygen, nitrogen and halides.
  • the compounds disclosed herein are capable of blocking early stages of the viras life cycle.
  • the disclosed compounds can block the initial interaction of the viral envelope glycoproteins with the CD4 receptor as well as specific chemokine coreceptors for virus entry, the subsequent membrane fusion event which involves the N-terminal hydrophobic domain and heptad repeats of the transmembrane (TM) protein subunit, and the initiation of viral biosynthetic processes which result from the virion reverse transcriptase and integrase.
  • TM transmembrane
  • Formulae I and II and their respective metallocomplexes do not encompass the phthalocyanine and porphyrazine compounds described in U.S. Patent Nos. 5,109,016 and 5,192,788.
  • U.S. Patent Nos. 5,109,016 and 5,192,788 describe the following phthalocyanine and porphyrazine compounds which were tested as effective for inhibition of HIV viruses and/or HSV viruses: copper phthalocyanine tetrasulfonic acid tetrasodium salt; nickel phthalocyanine tetrasulfonic acid; copper phthalocyanine
  • Phthalocyanines (Pcs) and porphyrazines ( Figures 1) the compounds of formulae I and II respectively, can be readily formed following procedures available in the art.
  • the formation of a Pc involves condensation of a phthalic acid derivative, which can be phthalic acid itself, the anhydride, the nitrile, the cyano benzamide, the imide or the 1,3-diiminoisoindoline.
  • Leznoff described 19 synthetic variations approximately a decade ago (Leznoff, "Synthesis of Metal-Free Substituted Phthalocyanines” In Phthalocyanines: Properties and Applications” (C.C. Leznoff and A.B. P. Lever Eds.) pp.
  • Synthesis of nesatively-charsed phthalocyanines - sulfonates Synthesis of sulfonated phthalocyanines can be achieved by three major routes: synthesis from sulfophthalic acids, synthesis from protected sulfophthalic acids, and synthesis from precursors bearing a sulfonate group off the ring periphery. All of this chemistry is well developed in the literature and can be readily extendable to the paramagnetic central ions (see, for example, Bekaroglu, "Synthesis of phthalocyanines and related compounds" in Journal Of Porphyrins And Phthalocyanines 4, 465-473
  • Synthesis from sulfophthalic acids Well-characterized sulfonated Pcs are often made via condensation of sulfophthalic acid derivatives (Weber and Busch, 1965).
  • the sodium salt of 4-sulfophthalic acid can be synthesized from 2-naphthalenesulfonamide (Weber and Busch, 1965).
  • the 1-naphthalenesulfonamide gives the 3- sulfophthalate, which can also be used in condensations (Weber and Busch, 1965).
  • Sulfophthalic acids can also be used in crossed condensations (Margaron et al., J. Photochemistry and Photobiology B-Biology 14:187-199 (1992)). Synthesis from phthalonitriles bearing a sulfonic acid removed from the rins
  • the condensation is also successful when the sulfonic acid is removed from the phthalocyanine ring.
  • Crossed condensation can also be used to make a variety of Pcs with the sulfonic acid removed from the phthalocyanine core, e.g., in work of W ⁇ hrle and co-workers (Kliesch et al., 1995). In related work with thioether derivatives, Tabata et al. J. Porphyrins and Phthalocyanines 4:278-284 (2000) were able to make Pcs with as many as 15-16 sulfonate groups per molecule. Synthesis from protected sulfophthalic acids Protected sulfonic acids offer substantial advantages in the synthesis of phthalocyanines. They allow purification to be performed with silica columns on an organic-soluble material.
  • ABBB-type phthalocyanines Sulfonated derivatives via subphthalocyanines
  • Another preferred technique for the synthesis of ABBB-type phthalocyanines involves the ring expansion of a subphthalocyanine.
  • Subphthalocyanines are homologs of phthalocyanine with three isoindole units and boron as the central atom (Torres, 2000). This is allowed to condense with a fourth ring to give the ABBB product.
  • the products of this method depend on the reaction conditions and structure of the starting materials; scrambling is possible under certain conditions (de la Torre et al., European Journal of Organic Chemistry 2821-2830 (2000)). However, other sets of conditions can give the desired ABBB compound cleanly. Shown in
  • Figure 3 is an example of a water-soluble, unsymmetrical, trisulfonated zinc phthalocyanine (ZnPcS3) as the single product of the ring expansion of boron tri(4-sulfo)subphthalocyanine (SubPc) (Kudrevich et al., J. Med. Chem. 40:3897-3904 (1997)); Kobayashi, J. Porphyrins and Phthalocyanines 3:453-467 (1999); Kobayashi et al, J. Am. Chem. Soc. 121:9096-9110 (1999)).
  • ZnPcS3 water-soluble, unsymmetrical, trisulfonated zinc phthalocyanine
  • Conjugates of tetrapyrroles, including phthalocyanines, with a wide variety of molecules have been studied in conjunction with the use of photosensitizers for anticancer therapy.
  • Conjugates include those with lipid-like molecules, antibodies, and macromolecular intemalizable ligands such as conA, insulin, transferrin, epidermal growth factor and peptide signals for targeting to various intracellular compartments.
  • reaction mixtures were purified in 200 - 400 mg batches by reverse-phase HPLC. Very good separation was achieved for the mono- and bis-derivatives.
  • a wide variety of amines can then be used for conjugation; the derivatives chosen would depend on the desired properties of the final product, with our first efforts concentrated on making derivatives with different hydrophobicities.
  • Figure 4 shows some selected examples of phthalocyanines bearing OH groups.
  • the example with OH groups at the 4(5) positions can be a mixture of four isomers (far left).
  • the example with OH groups at the 3(6) positions is usually formed as a single isomer (from protected OH groups).
  • the OH can also be on one of the side chains. Partially substituted derivatives are also available (e.g., the right- hand structure).
  • the 4(5)-tetrahydroxyPc (left-hand structure of Figure 4) can be synthesized by, for example, deprotection of the Pc with, p-n- BuPhCH 2 or Ph 2 CH protecting groups (Leznoff et al., Canadian J.
  • Unsymmetrical phthalocyanines can also be formed via a palladium- catalyzed coupling reaction between and iodophthalocyanine and acetylenic derivatives.
  • the coupling reaction goes in high yield (Ali, 1997; Tian, et al., 2000).
  • the substituents, as well as the overall structure, of the compounds of formulae I and II disclosed herein can be neutral, positively charged or negatively charged. Charged structures have counterions, and many counterions and combinations of counterions are possible.
  • the disclosed compounds can be covalently attached to other molecules, for example, a nucleobase or oligonucleotide (Koval et al. Nucleosides Nucleotides &
  • Metal atoms useful for forming a complex with any of the compounds of Formulae I-II are generally charged, though sometimes uncharged metal atoms may be useable.
  • Representative metal ions are any ions derived from Ga, Fe, Cu, Co, Ni, Pt, Os, Ru, Rh, Ir, Pd, Mn, V, Cr, Zn, Ag, Au, Cd, Ba, Al, Ti, or Sn.
  • the metal ions are derived from Cu, e.g., Cu(II).
  • the metal ions are derived from Fe, e.g., Fe(III).
  • the metal atoms may have neutral or ionic ligands.
  • neutral ligands include H 2 O, pyridine, imidazoles, NH 3 , alkylamines, ethers, oxygen, amino acid or peptide esters, phosphines, and alcohol.
  • Other neutral ligands commonly used in coordination chemistry may also used.
  • Exemplary ionic ligands can be negative charged ligands such as Cl “ , NO “ , CN “ , RS “ , terminal N-bound amino acids or peptides).
  • the substituents, as well as the overall structure, of the compounds disclosed herein can be neutral, positively charged or negatively charged. Charged structures have counterions, and many counterions and combinations of counterions are possible.
  • the disclosed compounds can be covalently attached to other molecules, for example, a nucleobase or oligonucleotide (Koval et al. Nucleosides Nucleotides & Nucleic Acids. 20:1259-1262 (2001), Li et al. Tetrahedron Lett. 42:305-309 (2001)); a peptide or protein (Brasseur et al. Photochem. Photobiol. 69:345-352 (1999); Carcenac et al. Photochem. Photobiol.
  • the technique involves the inactivation of viral replication in human peripheral blood mononuclear cells.
  • the amount of virus produced is determined by measuring the quantity of virus-coded reverse transcriptase (an enzyme found in retroviruses) which is present in the culture medium.
  • Another technique involves measuring inactivation of purified reverse transcriptase in a cell free system.
  • Hydrophobicity of the compounds The relation between hydrophobicity of po hyrin, a class of compounds closely related to phthalocyanines, and its biological activity has been studied. There is documentation that the hydrophobic interactions of the planar extended aromatic porphyrin ring help stabilize its interactions with biomolecules (see, for example, Stephen J. Lippard and Jeremy M. Berg, Principles of Bioinorganic Chemistry, University Science Books, 1994). Other significant interactions include hydrogen bonding and electrostatic interactions of the peripheral substituents, and axial interactions involving the metal (Lippard & Berg, 1994).
  • the hydrophobicity of the compounds disclosed herein can be evaluated by using, for example, capillary electrophoresis and fluorescence assays. Therefore, by analyzing the hydrophobicities of various compounds disclosed herein, it is possible to establish the relationship between hydrophobicity and a particular structure of the compound disclosed herein, thereby allowing the prediction of highest possible hydrophobic interactions of the compound with a biomolecule.
  • QSAR can be used to provide guidance for the selection of the most effective compounds disclosed herein for the prevention of STDs caused by viral pathogens or AIDs caused by HIVs.
  • QSAR has wide application in guiding the design of new pharmaceutical agents (see for example, Karelson et al., Chem. Rev., 96:1027-1043 (1996); 3D QSAR in Drug Design: Ligand- Protein Interactions and Molecular Similarity, Hugo Kubinyi; Yvonne C. Martin; and Gerd Folkers (Eds.), Kluwer Academic Publishers, Dordrecht, Netherlands, Vol. 2, (1998); 3D Qsar in Drag Design: Recent Advances, Hugo Kubinyi; Yvonne C. Martin; and Gerd Folkers (Eds.), Kluwer
  • the formulation may optionally include, or be administered with, one or more other pharmaceutically active agents.
  • antibiotics include antibiotics, viracidals or virastatics, antivirals, antifungals, immunostimulants, and substances which may have other properties, such as contraceptives.
  • These may be synthetic or natural drugs, natural or synthetic proteins, saccharides or polysaccharides, nucleic acid molecules, or combinations thereof. Examples include proteins such as antibodies, microbicidal polymers such as a cyclodextrin, polyethylene hexamethylene biguanide, a polymer such as Buffergel, a seaweed polymer such as Carraguard, or antimicrobial peptides such as a defmsin.
  • the pharmaceutically effective agent can be a drug that inactivates one or more viruses.
  • the formulations will typically be administered to a mucosal surface to prevent infection with a STD or other viral pathogen and/or AIDs caused by HIV.
  • the compounds have been established as not being useful in preventing infection by HSV-1 and HSV-2.
  • the compounds are formulated to provide an effective amount of the compound described herein, a metallocomplex of the compound, a pharmaceutically acceptable salt of the compound, or a mixture thereof.
  • the pharmaceutically effective amount varies with the type of STD.
  • the composition is used for HIV prevention, for which the effective amount of the compound is a concentration in vitro of less than or equal to 10 ⁇ M in the presence of a pharmaceutically acceptable carrier or diluent.
  • Some of the compounds are water soluble and may be administered in sterile water or physiological saline or phosphate buffered saline (PBS).
  • PBS physiological saline or phosphate buffered saline
  • Many porphyrins are not water soluble and are preferably administered in pharmaceutically acceptable non-aqueous carriers including oils and liposomes. Solubility of the compounds can be increased by techniques known to those skilled in the art including introducing hydroxyl groups and changing the counter ions.
  • HIV inhibitory amount is meant an amount of active ingredient sufficient to exert an HIV inhibitory effect as measured by, for example, an assay such as the ones described herein.
  • compositions described herein can be administered by any route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid or solid form.
  • a preferred mode of administration of the compositions described herein is topical or mucosal administration.
  • a specifically preferred mode of mucosal administration is administration via female genital tract.
  • Another preferred mode of mucosal administration is rectal administration.
  • dosage values also vary with the specific severity of the disease condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. It is to be further understood that the concentration ranges set forth herein are exemplary.
  • the active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time.
  • the phthalocyanine and/or porphyrazine compounds, a metallocomplex of the compound, pharmaceutically acceptable salts of the compound or metallocomplex, or a mixture thereof can be formulated as ointments, creams, gels, lotions, troches, suppositories, vaginal rings, liposomes, nanoparticulates, microspheres, and controlled release formulations.
  • Various polymeric and/or non-polymeric materials can be used as adjuvants for enhancing mucoadhesiveness of the composition disclosed herein.
  • the polymeric material suitable as adjuvants can be natural or synthetic polymers.
  • Representative natural polymers include, for example, starch, chitosan, collagen, sugar, gelatin, pectin, alginate, karya gum, methylcellulose, carboxymethylcellulose, methylethylcellulose, and hydroxypropylcellulose.
  • Representative synthetic polymers include, for example, poly(acrylic acid), tragacanth, poly(methyl vinylether-co-maleic anhydride), poly(ethylene oxide), carbopol, poly(vinyl pyrrolidine), poly(ethylene glycol), poly(vinyl alcohol), poly(hydroxyethylmethylacrylate), and polycarbophil.
  • Other bioadhesive materials available in the art of drug formulation can also be used (see, for example, Bioadhesion - Possibilities and Future Trends, Gurny and
  • the formulation may contain the following ingredients: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, corn starch and the like; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; and a sweetening agent such as sucrose or saccharin or flavoring agent such as peppermint, methyl salicylate, or orange flavoring may be added.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, corn starch and the like
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • a sweetening agent such as sucrose or saccharin or flavoring agent such as pepper
  • dosage unit forms may contain other various materials which modify the physical form of the dosage unit, for example, as coatings.
  • Tablets or pills may be coated with sugar, shellac, or other enteric coating agents. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.
  • Solutions or suspensions may also include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methylparabens
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents such as ethylenediaminetetraacetic acid
  • buffers
  • Controlled release formulation including implants and microencapsulated delivery systems may also be used.
  • Biodegradable, biocompatable polymers can be used, such as polyanhydrides, polyglycolic acid, collagen, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) are also useful as pharmaceutically acceptable carriers. Methods for encapsulation or incorporation of compounds into liposomes are described by Cozzani, et al., Chem. Biol. Interact. 53:131-143 (1985) and by Jori, et al., Br. J. Cancer 48:307-309 (1983). These may also be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
  • the formulations can be used to prevent a viral infection by administering to a human being an effective amount of at least one of phthalocyanines, porphyrazines, metal complexes thereof, pharmaceutically acceptable salts thereof, or a mixture thereof that inactivates a viras prior to an infection caused by the viruses being effected.
  • a pharmaceutically effective amount of one or more of other agents can be used in combination with the one of phthalocyanines, porphyrazines, metal complexes thereof, or a mixture thereof.
  • the compound of formulae I-II or a metal complex thereof has broad- spectrum anti- viral activities.
  • These compounds or metal complexes can be formulated for administration to individuals in need of prevention of STDs.
  • the formulations are preferably for local or regional delivery, for example, to the mucosa of the reproductive tract, or intestinal tract, but may also be formulated for systemic delivery.
  • the formulation is designed to administer an amount of porphyrin effective to prevent infection of the STD.
  • the time of administration is determined based on standard clinical criteria, determined using other antibiotic or virucidal formulations, clearance rates, and STD to be treated.
  • compositions can be used to prevent STDs caused by viral pathogens.
  • viruses include HIV viruses, hepatitis B and C viruses, and papilloma viruses.
  • the pharmaceutically effective amount varies with the type of STD.
  • an effective amount of the porphyrin compound is less than or equal to 10 ⁇ M in the presence of a pharmaceutically acceptable carrier or diluent.
  • the compounds described herein are included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to exert a therapeutically useful inhibitory effect in vivo without exhibiting adverse toxic effects on the user.
  • dosage values also vary with the specific severity of the disease condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.
  • agents which can be used in combination with the compounds or metal complexes described herein include antibiotics, viracidals, antivirals, antifungals, immunostimulants, and substances which are effective in inactivating viruses.
  • the pharmaceutically effective agents include synthetic or natural drags, natural or synthetic polymers, and antibodies.
  • the agent can be a microbicidal polymer such as one of cyclodextrins, polyethylene hexamethylene biguanide, a polymer such as Buffergel, a seaweed polymer such as Carraguard, and antimicrobial peptide such as one of definsins.
  • the pharmaceutically effective agent can be a drug that inactivates one or more viruses. The following examples further illustrate the phthalocyanine and
  • Pporphyrazine compositions disclosed herein and the method of using thereof for inactivation of HIVs and pathogens causing STDs Example 1. Prevention of HIV-1 Infection by Phthalocyanines Materials and Methods Phthalocyanines
  • the cationic phthalocyanines tested were Alec Blue (Aldrich, St. Louis, MO) (PcCat(l)) and Alcian Blue 8GX (PcCat(2)) (Kodak). Patrea - original first sentence is OK - 1 should not have changed it.
  • the Al and Co carboxyphthalocyanines (AlPcC and CoPcC) were from Midcentury Chemicals (Posen, Illinois).
  • CuPcS(3,4',4",4') (Aldrich) is designated as CuPcS(l) and
  • CuPcS(4,4' ,4' ' ,4" ') (Fluka, St. Louis, MO) is designated as CuPcS(2).
  • the free acid [CuPcS(H+)] and lithium salt [CuPcS(Li+)] of CuPcS were from Frontier Scientific (Logan, Utah).
  • NiPcS was from Aldrich; the rest of the phthalocyanines were from Midcentury Chemicals.
  • the aluminum and zinc phthalocyanines were available in two sulfonation levels.
  • AlPcS(l) and ZnPcS(l) are the more highly sulfonated forms (presumably largely the tetrasulfonates).
  • AlPcS(2) and ZnPcS(2) are less sulfonated (listed as the trisulfonated forms).
  • mice NIH/3T3 and human HEp2 cell lines were obtained from the American Type Culture Collection (Manassas, VA).
  • the recombinant cell lines human MAGI, monkey sMAGI, mouse 3T3.T4, 3T3.T4.CCR5, 3T3.T4.CXCR4, and human T-cell lines CEMxl74 and HUT78 were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS (NIH).
  • the human 293T cell line was kindly provided by Dr. S. L. Lydy (Emory University, Atlanta, GA).
  • NIH/3T3, HEp2, 3T3.T4, 3T3.T4.CCR5, 3T3.T4.CXCR4, MAGI, sMAGI, and 293T cells were maintained in Dulbecco's minimal essential medium (DMEM) supplemented with 10% fetal calf serum.
  • DMEM Dulbecco's minimal essential medium
  • HUT78 and CEMxl74 cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum.
  • plasmids pRB21 and vRB12 were kindly provided by Drs. Bernard Moss (National Institutes of Health, Bethesda, MD) and David Steinhauer (National Institute for Medical Research, London, UK). The 3'SHIN-89.6 plasmid was obtained from Dr. J. Sodroski (Harvard Medical School, Boston, MA).
  • Recombinant vaccinia viruses expressing full length (VV-239env) and truncated (W- 239T) SIVmac239 Env proteins were previously described by (Ritter, et al., Virology 197:255-264 (1993)), and VVenvl expressing the BH10 Env protein was described by Owens and Compans (Owens and Compans, J. Virol. 63, 978-982 (1989)).
  • a recombinant vaccinia virus encoding a truncated Env protein of HIN-1 89.6 was constructed as follows. The HIN-1
  • 89.6 truncated env gene was obtained by polymerase chain reaction (PCR) amplification from the HIV-1 89.6 plasmid with the following primers: the 5 '-primer introducing an EcoRI site 5'-
  • the recombinant vaccinia viras was obtained by a plaque selection system using a recipient vaccinia viras vRB 12 described by Blasco and Moss (Blasco and Moss, Gene 158:157-162 (1995)).
  • the plasmid plllenv3-l encoding the Env protein of the HXB2 strain of HIV-1 was obtained from the AIDS Research and Reference Reagent Program, Division of AIDS (NIH).
  • the Tat-responsive HIV-LTR in plllenv3-l was used to promote expression of HXB2 Rev and Env.
  • the helper plasmid pCMVtat was kindly provided by Dr. Steven Bartz (Fred Hutchinson Cancer Research Center, Seattle, WA).
  • Virus-infected H9/HTLV-III B NIH 1983 cells were obtained from the AIDS Research and Reference Reagent Program, and the supernatant was used to infect HUT78 cells. HIV-1 IIIB virus was produced by continued passage of infected HUT78 cells and virus stock was prepared as described previously (Vzorov and Compans, J. Virol. 74:8219-8225 (2000)). To prepare HIN-1 89.6 viras, 293T cells were transfected with p89.6 (from the ⁇ IH AIDS Research and Reference Reagent Program). At 48 hr post transfection, DMEM was removed and the cells were washed once in RPMI.
  • CEMxl 74 cells were added to a plate in 5 ml of RPMI containing 10% fetal calf serum and cocultured overnight. The following day, CEMxl 74 cells were removed from virus-producing 293T cells and placed in T-25 flasks for continued passage. SINmacl Al 1 viras stock was described previously (Vzorov and Compans, 2000). Monoclonal antibodies, antisera, proteins
  • SIM.2 and SIM.4 antibodies recognizing human CD4 and recombinant soluble human CD4 were provided by the ⁇ IH AIDS Research and Reference Reagent Program (NIH).
  • the recombinant IIIB gpl 20 protein (baculovirus-expressed) was obtained from Intracel (Cambridge, MA).
  • Anti- mouse IgG peroxidase conjugate was obtained from Sigma (St. Louis, MO). Screening of phthalocyanines for activity against HIV-1 Phthalocyanine stock solutions were prepared at concentrations of 5 mg/ml, diluted 100-fold in growth medium, and mixed with virus stock. Samples were left in the dark at room temperature for 1 hr.
  • gpl20-CD4 binding assay To investigate the possible effect of phthalocyanine compounds on binding of HIV-1 IIIB gpl 20 to CD4, a gpl20-CD4 binding assay was developed. The assay was developed as a modification of a capture gp 120 ELISA kit (Intracel Corporation). Briefly, a 96-well plate was coated with soluble CD4 and 0.5 ⁇ g of HIV-1 IIIB gpl20 per well was incubated in the presence or absence of test compounds for 1 hr at room temperature. After 4 washes with buffer to remove unbound proteins, the bound gpl20 was detected by anti-gpl20 peroxidase-conjugated antibodies and quantitated by the protocol provided by the manufacturer.
  • Cell fusion assays For cell fusion assays, three different expression systems: (1) a recombinant vaccinia expression system, which is able to express high levels of Env; (2) a plasmid expression system which is able to express Env proteins in the absence of other HIV proteins or vaccinia proteins; and (3) cells persistently infected with HIV-1 IIIB or HIV-1 89.6, were compared.
  • HEp2 cells were infected at a m.o.i. of 5. After 24 hr cells were collected, counted and about 2.5x10 3 cells were added to
  • T cells were transfected by the calcium phosphate precipitation method with the plasmid plllenv3-l expressing the HIV- 1 Env protein (HXB2 Env) with an LTR promoter, and cotransfected with a helper plasmid pCMVTAT at a ratio of 10:1; or with plasmids expressing SIV Env proteins using a CMV promoter as described above.
  • IIIB or CEMxl74 cells persistently infected with HIN-1 89.6.
  • the infected cells were counted and cocultured with uninfected cells as in the previous assays.
  • a trypan blue exclusion test was used (Strober, Trypan blue exclusion test of cell viability, p. A.3.3-A.3.4, in J.E. Coligan and A.M. Kruisbeek (ed.), Current protocols in immunology, Wiley-Greene, New York, N.Y., 1994).
  • Compounds at a concentration of 50 ⁇ g/ml in growth medium were added to a 96-well plate with MAGI cells. After 72 hr cells were detached by 0.25% trypsin - 0.05% versene solution and diluted 1 :10 in growth medium.
  • phthalocyanines with positively charged substituents phthalocyanines with carboxylate substituents
  • porphyrazines phthalocyanines with carboxylate substituents
  • sulfonated phthalocyanines Figure 1
  • the positively charged phthalocyanines PcCat(l) and PcCat(2) had very poor anti-HIN activity; PcCat(l) did not block viral infection and PcCat(2) promoted growth of the virus (185 +/- 5%).
  • the phthalocyanines bearing carboxylate substituents had moderate to low activity.
  • the Al and Co chelates blocked 40 +/- 15 and 19 +/- 2% of viral infection, respectively.
  • Figure 5 shows the activity of sulfonated phthalocyanines against HIN-1 IIB, which was assayed under the following conditions: compounds at a concentration of 50 ⁇ g/ml were incubated with HIN-1 IIIB in the dark for 1 hr, diluted 10-fold and used to inoculate MAGI cells. After three days activity against HIN was measured by removal of the media, fixation and staining with X-gal. The activity against HIV was measured by dividing the number of blue cells in wells infected with compound-treated virus by the number in wells infected with untreated viras. In Figure 5, data are plotted as the mean of three experiments, each replicated twice. Error bars represent standard deviations.
  • the most active compounds are the parent PcS (no metal, 94% blocking of HIV infection) and its copper [CuPcS(l), essentially no binding of axial ligands, 97% blocking of HIV infection] and nickel ( ⁇ iPcS, weak binding of axial ligands, 93% blocking of HIV infection) chelates.
  • the VO derivative which has only a tightly bound oxygen atom as an axial ligand, also displayed good activity (86% blocking of HIV infection).
  • the copper chelate was studied further because it had high activity. In addition, because the copper is paramagnetic, copper phthalocyanines are not sensitive to light (essentially eliminating any possible photodynamic side effects).
  • copper phthalocyanines are not sensitive to light (essentially eliminating any possible photodynamic side effects).
  • Four commercially available samples were studied.
  • CuPcS(l), CuPcS(2), CuPcS(H+) and CuPcS(Li+) had activities of 97, 72, 94 and 85% virus inhibition, respectively. It appears that some components of these mixtures of sulfonated copper phthalocyanines are more active than others.
  • viras samples were mixed with phthalocyanines at defined concentrations. As shown by Figure 7, the most effective concentration was the generally the highest concentration of 50 ⁇ g/ml. However, three of the six compounds studied [CuPcS(l), CuPcS(H+) and NiPcS] also exhibited approximately 50% activity at concentrations of ⁇ 6.25 ⁇ g/ml. CuPcS(H+) had approximately 50% activity at 0.6 ⁇ g/ml. Thus, the most active compound had an EC50 of ⁇ 1 ⁇ g/ml.
  • a potential microbicide may be virucidal, e.g., for inhibition of viral replication to continue even after the concentration of the compounds is substantially decreased.
  • a filtration-dilution protocol was used to show that the virus, once treated, was still rendered non-infectious once the unbound compound had been removed. In this protocol, solutions of the virus and compound were filtered until only about 10% of the original volume remained. The solution that had not gone through the filter was diluted to the original volume and the process repeated four times.
  • Spectroscopic assays showed that four dilutions resulted in the original compound concentrations being reduced by 35- to 500-fold. Three compounds were evaluated, NiPcS, PcS and ZnPcS(l). ZnPcS(l) had moderate activity in the screening assay. Filtration-dilution resulted in more than a two-fold recovery of infectivity. NiPcS and PcS were both very effective in blocking of HIV infection; after filtration dilution there was only minimal recovery of infectivity. Thus, PcS and NiPcS are truly virucidal; that is, virus inhibition continues even after free compound is removed.
  • HIV-1 89.6, SrVmacl Al 1 and influenza viras were extended to HIV-1 89.6, SrVmacl Al 1 and influenza viras (A/PR 8/34[HlNl]).
  • the most active compounds against HIV-1 IIIB CuPcS(l), NiPcS and PcS, were selected. These three compounds inactivated HIV-1 89.6 to the extents of 73, 83 and 83%, respectively.
  • SlVmacl Al 1 was also sensitive to the compounds: 82%, 89%, and 59% activation, respectively.
  • influenza virus A/PR/8/34[HlNlj
  • Influenza virus was less sensitive to these compounds: CuPcS(l), NiPcS, and PcS inactivated 44, 37 and 46% of the infectivity, respectively.
  • CD4 Effect of phthalocyanines on interaction ofgp!20 with CD4
  • CD4 the effect of phthalocyanines on binding of gpl 20 to its primary receptor, CD4 (Dalgleish et al., Nature 312:763-767 (1984)) was investigated.
  • a gpl20- CD4 binding assay was used as described in Materials and Methods.
  • the CuPcS(l), CuPcS(H+), NiPcS, and VOPcS all were very effective in blocking HIV infection and also showed substantial (or complete) inhibition of gpl20-CD4 binding.
  • PcS which also is effective in blocking HIV infection (94%) did not block binding of gpl20 to CD4 effectively.
  • ZnPcS(l) had good activity against HIV (77% blocking of HIV infection) but showed almost no inhibition of binding of gpl 20 to CD4.
  • AlPcC had moderate activity against HIV (40% blocking of HIV infection) but completely inhibited binding of gpl 20 to CD4.
  • More than one mechanism of blocking of HIV infection is apparently operative.
  • phthalocyanines bearing metals expected to bind axial ligands were not very effective at inhibiting fusion.
  • All of AlPcC, AlPcS, Co(II)PcS, Co(III)PcS, CrPcS, ZnPcS(l) and ZnPcS(2) had at least 30% of nuclei in syncytia at 8 hr in one of the tests.
  • inhibition of fusion is apparently most easily achieved with planar phthalocyanines [i.e., those with no, or small (VO), axial ligands].
  • MnPcS is an exception; perhaps other mechanisms are important for this chelate, which is not considered further.
  • a correlation between inhibition of fusion and blocking of viral infection was also observed.
  • Fusion activities were demonstrated by comparing the nuclei in syncytia to the total nuclei. 4+, more than 50% of nuclei are in syncytia; 3+, 30 to 50% of nuclei are in syncytia; 2+, 30 to 10% of nuclei are in syncytia; + less than 10% of nuclei are in syncytia; -, no syncytia were observed.
  • plllenv a plasmid expression system that is able to express Env proteins in the absence of other HIN proteins or vaccinia proteins was used.
  • PcS ⁇ iPcS, MnPcS, VOPcS, and all forms of the copper phthalocyanines [CuPcS(l), CuPcS(2), CuPcS(H+), and CuPcS(Li+)] was observed.
  • PcS did not completely block fusion in the plllenv recombinant (after 21 hr incubation, 10% of the cells were fused), though it did inhibit fusion in the recombinant vaccinia expression system.
  • PcS also blocks gpl20/CD4 binding by only about 35%.
  • cells persistently infected with HIV-1 IIIB or HIV-1 89.6 were used for cocultivation with uninfected target cells in the presence or absence of test compounds.
  • the fusion activity observed in this assay was comparable with that found using plasmids expressing Env, and lower than observed with Env expressed by vaccinia viras.
  • the results of fusion inhibition by the compounds tested correlated well with results observed using both other expression systems.
  • the central goal of this study was to identify compounds with high activity in blocking HIV infection, which could be useful as topical formulations to provide a defense against infection by sexually transmitted virus.
  • the vaginal and gastrointestinal surfaces play a major role in the pathogenesis of infection by HIV-1 as potential routes for viral entry.
  • a MAGI assay which is based on usage of an epithelial cell line was used as an assay for anti-HIV activity of test compounds.
  • HeLa cells stably transfected with human CD4 (HeLa-CD4 cells) are permissive for T cell line-adapted X4 or dual usage R5X4 viruses, because they express the coreceptor CXCR4. All the anionic phthalocyanines studied were active with various degrees of efficacy against HIN-1 IIIB.
  • the sulfonated phthalocyanines studied are small herein polyanionic molecules. They inhibit viral binding and fusion/entry into susceptible cells. In this they are similar to other polyanionic species, including the sulfated polymers (De Clercq, 2002b).
  • the interaction of the phthalocyanines with HIN appears to be very rapid. For PcS and ⁇ iPcS, removal of free compound did not result in significant recovery of infectivity, indicating that these are effective virucidal agents. Viracidal activity is desirable, but is presumably not necessary, in that the compounds when used as microbicides will continue to be present at sites of transmission during exposure to virus in vivo.
  • Sulfonated phthalocyanines therefore are a promising class of compounds for further development as microbicides to prevent HIV transmission.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des compositions pharmaceutiques contenant un composé neutre ou chargé négativement présentant une structure phtalocyanine ou l'une des porphyrazines ou le complexe métallique dérivant de ce composé. Ces compositions sont efficaces pour atténuer une infection par le VIH et d'autres agents pathogènes induisant des maladies sexuellement transmissibles. Lesdites compositions peuvent être adaptées à n'importe quel mode d'administration. De préférence, la composition est adaptée à une administration topique, et notamment à une administration mucosale, la composition préférée étant adaptée à une administration par voie vaginale ou rectale.
PCT/US2003/001619 2002-01-18 2003-01-17 Compositions pharmaceutiques a base de phtalocyanine et de porphyrazine WO2003061579A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003205227A AU2003205227A1 (en) 2002-01-18 2003-01-17 Phthalocyanine and porphyrazine pharmaceutical compositions

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US34994402P 2002-01-18 2002-01-18
US60/349,944 2002-01-18

Publications (2)

Publication Number Publication Date
WO2003061579A2 true WO2003061579A2 (fr) 2003-07-31
WO2003061579A3 WO2003061579A3 (fr) 2003-12-04

Family

ID=27613344

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2003/001619 WO2003061579A2 (fr) 2002-01-18 2003-01-17 Compositions pharmaceutiques a base de phtalocyanine et de porphyrazine

Country Status (2)

Country Link
AU (1) AU2003205227A1 (fr)
WO (1) WO2003061579A2 (fr)

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2426978A (en) * 2005-06-08 2006-12-13 Yen Cheng Tsai Novel Phthalocyanine Derivatives, Synthetic Process thereof and their application in optical recording media
WO2007134055A1 (fr) * 2006-05-10 2007-11-22 Medtronic Xomed, Inc. Système de solvatation de polysaccharides extracellulaires antibactériens
WO2007033444A3 (fr) * 2005-09-21 2007-12-21 Leuven K U Res & Dev Nouveaux traitements antiviraux
EP2243485A1 (fr) * 2008-02-20 2010-10-27 Daiwabo Holdings Co., Ltd. Agents antiviraux, fibres antivirales et structures fibreuses antivirales
US7959943B2 (en) 2006-05-10 2011-06-14 Medtronics Xomed, Inc. Solvating system and sealant for medical use in the middle or inner ear
US7976875B2 (en) 2006-05-10 2011-07-12 Medtronic Xomed, Inc. Biofilm extracellular polysaccharide solvating system
US7993675B2 (en) 2006-05-10 2011-08-09 Medtronic Xomed, Inc. Solvating system and sealant for medical use in the sinuses and nasal passages
US8088095B2 (en) 2007-02-08 2012-01-03 Medtronic Xomed, Inc. Polymeric sealant for medical use
US8580294B2 (en) 2010-10-19 2013-11-12 International Partnership For Microbicides Platinum-catalyzed intravaginal rings
US8784790B2 (en) 2008-06-12 2014-07-22 Medtronic Xomed, Inc. Method for treating chronic wounds with an extracellular polymeric substance solvating system
US8940792B2 (en) 2008-10-06 2015-01-27 Next Science, Llc Antimicrobial composition and methods for using same
US9393250B2 (en) 2012-04-12 2016-07-19 University Of Saskatchewan Phthalocyanine compounds useful as RecA inhibitors and methods of using same
US10137031B2 (en) 2013-11-14 2018-11-27 International Partnership For Microbicides, Inc. Combination therapy intravaginal rings
US10537661B2 (en) 2017-03-28 2020-01-21 DePuy Synthes Products, Inc. Orthopedic implant having a crystalline calcium phosphate coating and methods for making the same
US10537658B2 (en) 2017-03-28 2020-01-21 DePuy Synthes Products, Inc. Orthopedic implant having a crystalline gallium-containing hydroxyapatite coating and methods for making the same
US10653133B2 (en) 2011-05-10 2020-05-19 Next Science IP Holdings Pty Ltd Antimicrobial solid and methods of making and using same

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5109016A (en) * 1988-05-23 1992-04-28 Georgia State University Foundation, Inc. Method for inhibiting infection or replication of human immunodeficiency virus with porphyrin and phthalocyanine antiviral compositions
AU1904492A (en) * 1991-04-03 1992-11-02 Agouron Pharmaceuticals, Inc. Methods and compounds for inhibiting rnase h activity of reverse transcriptase
ATE269711T1 (de) * 1996-05-09 2004-07-15 Infectio Recherche Inc Formulierung zur verwendung in der vorbeugung von pathogen-induzierten erkrankungen, einschliesslich hiv und hsv

Cited By (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2426978A (en) * 2005-06-08 2006-12-13 Yen Cheng Tsai Novel Phthalocyanine Derivatives, Synthetic Process thereof and their application in optical recording media
US8193157B2 (en) 2005-09-21 2012-06-05 K.U.Leuven Research & Development Antiviral therapies
WO2007033444A3 (fr) * 2005-09-21 2007-12-21 Leuven K U Res & Dev Nouveaux traitements antiviraux
WO2007134055A1 (fr) * 2006-05-10 2007-11-22 Medtronic Xomed, Inc. Système de solvatation de polysaccharides extracellulaires antibactériens
US8691288B2 (en) 2006-05-10 2014-04-08 Medtronic, Inc. Gallium-containing sealant for medical use
US7959943B2 (en) 2006-05-10 2011-06-14 Medtronics Xomed, Inc. Solvating system and sealant for medical use in the middle or inner ear
US7976873B2 (en) 2006-05-10 2011-07-12 Medtronic Xomed, Inc. Extracellular polysaccharide solvating system for treatment of bacterial ear conditions
US7976875B2 (en) 2006-05-10 2011-07-12 Medtronic Xomed, Inc. Biofilm extracellular polysaccharide solvating system
AU2007249413B2 (en) * 2006-05-10 2012-08-30 Medtronic Xomed, Inc. Antibacterial extracellular polysaccharide solvating system
US7993675B2 (en) 2006-05-10 2011-08-09 Medtronic Xomed, Inc. Solvating system and sealant for medical use in the sinuses and nasal passages
US8088095B2 (en) 2007-02-08 2012-01-03 Medtronic Xomed, Inc. Polymeric sealant for medical use
US9119896B2 (en) 2007-02-08 2015-09-01 Medtronic Xomed, Inc. Polymeric sealant for medical use
EP2243485A4 (fr) * 2008-02-20 2011-07-20 Daiwabo Holdings Co Ltd Agents antiviraux, fibres antivirales et structures fibreuses antivirales
EP2243485A1 (fr) * 2008-02-20 2010-10-27 Daiwabo Holdings Co., Ltd. Agents antiviraux, fibres antivirales et structures fibreuses antivirales
US8784790B2 (en) 2008-06-12 2014-07-22 Medtronic Xomed, Inc. Method for treating chronic wounds with an extracellular polymeric substance solvating system
US9700344B2 (en) 2008-06-12 2017-07-11 Medtronic Xomed, Inc. Method for treating chronic wounds with an extracellular polymeric substance solvating system
US8940792B2 (en) 2008-10-06 2015-01-27 Next Science, Llc Antimicrobial composition and methods for using same
US8580294B2 (en) 2010-10-19 2013-11-12 International Partnership For Microbicides Platinum-catalyzed intravaginal rings
US9427400B2 (en) 2010-10-19 2016-08-30 International Partnership For Microbicides Platinum-catalyzed intravaginal rings
US10653133B2 (en) 2011-05-10 2020-05-19 Next Science IP Holdings Pty Ltd Antimicrobial solid and methods of making and using same
US9393250B2 (en) 2012-04-12 2016-07-19 University Of Saskatchewan Phthalocyanine compounds useful as RecA inhibitors and methods of using same
US11259956B2 (en) 2013-11-14 2022-03-01 International Partnership For Microbicides, Inc. Combination therapy intravaginal rings
US10137031B2 (en) 2013-11-14 2018-11-27 International Partnership For Microbicides, Inc. Combination therapy intravaginal rings
US11793669B2 (en) 2013-11-14 2023-10-24 The Population Council, Inc. Combination therapy intravaginal rings
US10537661B2 (en) 2017-03-28 2020-01-21 DePuy Synthes Products, Inc. Orthopedic implant having a crystalline calcium phosphate coating and methods for making the same
US11141505B2 (en) 2017-03-28 2021-10-12 DePuy Synthes Products, Inc. Orthopedic implant having a crystalline gallium-containing hydroxyapatite coating and methods for making the same
US11058799B2 (en) 2017-03-28 2021-07-13 DePuy Synthes Products, Inc. Orthopedic implant having a crystalline calcium phosphate coating and methods for making the same
US11793910B2 (en) 2017-03-28 2023-10-24 DePuy Synthes Products, Inc. Orthopedic implant having a crystalline calcium phosphate coating and methods for making the same
US10537658B2 (en) 2017-03-28 2020-01-21 DePuy Synthes Products, Inc. Orthopedic implant having a crystalline gallium-containing hydroxyapatite coating and methods for making the same
US11793907B2 (en) 2017-03-28 2023-10-24 DePuy Synthes Products, Inc. Orthopedic implant having a crystalline gallium-containing hydroxyapatite coating and methods for making the same

Also Published As

Publication number Publication date
WO2003061579A3 (fr) 2003-12-04
AU2003205227A1 (en) 2003-09-02

Similar Documents

Publication Publication Date Title
AU2003212790B2 (en) Porphyrins with virucidal activity
WO2003061579A2 (fr) Compositions pharmaceutiques a base de phtalocyanine et de porphyrazine
US5281616A (en) Porphyrin and phthalocyanine antiviral compositions
US5192788A (en) Porphyrin antiviral compositions
Harakeh et al. Suppression of human immunodeficiency virus replication by ascorbate in chronically and acutely infected cells.
ES2224096T3 (es) Poliamidas ciclicas enlazadas con actividad frente al vih.
Montefiori et al. Selective antiviral activity of synthetic soluble L-tyrosine and L-dopa melanins against human immunodeficiency virus in vitro
US5736527A (en) Method of treating HIV in humans by administration of ddI and hydroxycarbamide
US20030225155A1 (en) Pharmacological agents and methods of treatment that inactivate pathogenic prokaryotic and eukaryotic cells and viruses by attacking highly conserved domains in structural metalloprotein and metalloenzyme targets
Vzorov et al. Prevention of HIV-1 infection by phthalocyanines
ES2244961T3 (es) Polimero de condensacion de un acido sulfonico aromatico y un aldehido para inhibir el poder infeccioso del vih.
TURANO et al. Inhibitory effect of papaverine on HIV replication in vitro
North et al. Photodynamic inactivation of free and cell-associated HIV-1 using the photosensitizer, benzoporphyrin derivative
IL105090A (en) Acid N) phosphonoacetyl (Aspartic L in a broad spectrum antiviral agent
Nakagami et al. Antiviral activity of a bile pigment, biliverdin, against human herpesvirus 6 (HHV‐6) in vitro
JPH11500130A (ja) 抗ウイルス性トリアザ化合物
JPH04502621A (ja) 治療法と免疫応答刺激用ワクチン
US4857514A (en) Virus inactivation
EP0255420B1 (fr) Agent antiviral d'inhibition de la croissance du virus du syndrome immuno-deficitaire acquis (SIDA)
RU2005475C1 (ru) Противовирусное средство
CN105362284B (zh) 阿奇霉素和泰利霉素在抗埃博拉病毒感染中的应用
EP0762878B1 (fr) Inhibition des infections retrovirales par administration de composes de l'acide naphtalene sulfonique
JPH0341030A (ja) 抗ウイルス剤
US5955498A (en) Agent for prophylaxis and therapy of diseases
LAURENT-CRAWFORD et al. Antiviral action of polyadenylic-polyuridylic acid against HIV in cell cultures

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SC SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LU MC NL PT SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载