WO1999048526A1 - Methode pour rendre ineffective in vivo une replication de population virale vih - Google Patents

Methode pour rendre ineffective in vivo une replication de population virale vih Download PDF

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WO1999048526A1
WO1999048526A1 PCT/US1999/003454 US9903454W WO9948526A1 WO 1999048526 A1 WO1999048526 A1 WO 1999048526A1 US 9903454 W US9903454 W US 9903454W WO 9948526 A1 WO9948526 A1 WO 9948526A1
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hiv
virus
cells
combination
therapy
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Franco Lori
Julianna Lisziewicz
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Franco Lori
Julianna Lisziewicz
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Priority to AU26853/99A priority Critical patent/AU2685399A/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • A61K39/13—Poliovirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32411—Hepatovirus, i.e. hepatitis A virus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates generally to the field of treatment of human beings with Human Immunodeficiency Virus (HIV) infections.
  • HIV Human Immunodeficiency Virus
  • the inventors have found that the combination of hydroxyurea (HU) and a reverse transcriptase inhibitor can be used for long-term therapy (years) in human beings without provoking viral rebound, even in cases where patients have developed genotypic resistance to the reverse transcriptase inhibitor.
  • This combination can be used, particularly in conjunction with a more potent combination employing a protease inhibitor, and a method of activating quiescent cells harboring the virus, in a general method for rendering an HIV population replication incompetent in vivo, thereby allowing long-term, perhaps very long-tern, remission of the disease.
  • Viruses are microorganisms that depend, to some degree, on host cell components for their growth and replication. Viral infection and replication in host cells generally results in disease, whether the host is an animal or plant. Human diseases caused by viral infections include the acquired immunodeficiency syndrome (AIDS) and hepatitis. A general discussion of this field is presented in Fundamental Virology, Second Edition, (ed. B. N. Fields, D. M. Knipe, R. M. Chanock, M. S. Hirsh, J. L. Melnick, T. P. Monath, and B. Roizman, Raven Press, Ltd., New York, N.Y. 1991 ).
  • AIDS acquired immunodeficiency syndrome
  • Retroviruses comprise a large family of viruses that primarily infect vertebrates. Many diseases, including the induction of some tumors, are associated with retroviral infection (see Fundamental Virology, supra, pp.645- 708). All retroviruses, regardless of their clinical manifestations, have related structures and modes of replication.
  • Retroviruses contain an RNA genome that is replicated through a DNA intermediate. Inside the cell, the viral genome serves as a template for the synthesis of a double-stranded deoxyribonucleic acid (DNA) molecule that subsequently integrates into the genome of the host cell. This integration occasionally results in the induction of a tumor in the infected host organism. Following integration, a complex sequence of events leads to the production of progeny virions which are released from the infected cell.
  • DNA deoxyribonucleic acid
  • RNA genome is copied into DNA by the virally encoded reverse transcriptase (RT).
  • This enzyme can use both RNA and DNA templates, thereby producing the first strand of DNA (the negative strand) from the infecting RNA genome and a complementary second strand (the positive strand) of DNA using the first DNA strand as a template.
  • the RT utilizes cellular substrates called deoxynucleoside t phosphates (dNTP).
  • HTLV type viruses human retroviruses can be grouped into the leukemia viruses (HTLV type viruses) and the immunodeficiency viruses (HIV type viruses).
  • HIV infection may lead to one form of leukemia.
  • Acquired immunodeficiency syndrome (AIDS) is caused by a form of HIV, with HIV-1 being more virulent than HIV-2.
  • AIDS HIV infect peripheral blood lymphocytes (PBL).
  • HIV Infection HIV-1 was first identified as the causative agent of AIDS in 1983. The
  • AIDS pandemic is now one of the most serious health problems worldwide. Catastrophic medical and social consequences are likely to extend into the next century.
  • the World Health Organization (WHO) has estimated that between eight and ten million people are currently infected with HIV, and that approximately ten times as many individuals will be affected in the next decade.
  • the large pool of HIV carriers makes the development of effective antiviral treatments a medical priority.
  • the initial HIV-1 infection may occurwithout accompanying symptoms, but most of the patients experience an acute HIV syndrome within 2 to 6 weeks of exposure to the virus.
  • This syndrome is characterized by fever, headaches, sore throat with pharyngitis, generalized lymphadenopathy and rashes.
  • the virus is replicating abundantly and is detectable in the blood and the CD4+ T-cell number falls from a normal amount of 1000/mm 3 to about 500/mm 3 .
  • Antibdies to HIV-1 proteins appear in the serum between 2-12 weeks after primary infection. The sequence of appearance of these antibodies can be followed by the Western blot test, which detects the serum antibodies that bind to specific viral proteins.
  • a positive Western blot response to gp160, gp120, p65, p55, gp41 , p32, p24 and p18 proteins demonstrates that antibodies to various HIV-1 proteins are being produced.
  • the process of change from negative for all the proteins to positive for the entire set is referred to as seroconversion. It has recently been demonstrated that during seroconversion there is a high level of virus present in the blood. The cellular arm of the immune response is also activated during seroconversion. (Borrow et al. Nature Medicine 3:(2) 212- 217, 1997; Goulder et al. Nature Medicine 3:(2) 205-211 , 1997).
  • an individual may have undetectable levels of virus as measured by viral load in plasma and biopsy of lymph nodes during treatment, and yet remain infected: once treatment is stopped, the viral rate of replication increases, and the viral load rebounds.
  • the present inventors have used the most sensitive test methods available. Further, testing of lymph nodes is done by extracting an entire node as opposed to a biopsy sample.
  • escape mutants play such a significant role in the development of the disease
  • a major focus in current efforts to find a mode of treatment for AIDS is to develop strategies that feature multiple, highly effective, concurrent attacks on HIV in an effort to completely eradicate the virus from an individual's system.
  • the only conclusive proof of effectiveness will be lack of rebound of the viral load in the individual's tissues over time.
  • a triple drug combination involving the use of AZT, 3TC and protease inhibitors has been suggested for the treatment of HIV-1 infection and eradication of the virus.
  • the efficacy of this combination is thought to originate from the potency of the protease inhibitors and the mechanism of action of the AZT/3TC combination in inhibiting the rebound of resistant mutants.
  • neither the protease inhibitors nor 3TC easily penetrate to certain organs such as lymph nodes and the brain, and the combination of protease inhibitor, AZT and 3TC apparently does not completely eradicate
  • HIV-1 in macrophages or in quiescent cells which are major reservoirs of HIV-1.
  • patients who have interrupted therapy using AZT, 3TC and protease inhibitors and then rebounded cannot be as effectively treated with the same combination because they develop resistant mutants.
  • protease-containing combinations without hydroxyurea have shown at best, response rates of 80-90% and 53% "failure" - a combined figure including people who never responded to therapy, those who could not tolerate side effects, those who responded initially but later saw a return of detectable virus, and those who had difficulty adhering to the strict dosing regimens required by the drugs.
  • Hydroxyurea as an Inhibitor of Human Immunodeficiency Virus-Type 1 Replication, F. Lori, et al., Science 266:801-805 (1994); possibly in combination with a nucleoside analog such as AZT, ddl, or ddC, although it has been admitted that clinical trials using hydroxyurea alone or in combination with nucleoside analogs will be essential to assess the actual impact of use of hydroxyurea in HIV-1 impacted patients. Hydroxyurea and AIDS: An Old Drug Finds a New Application? F. Lori and R. Gallo, Aids Research and Human Retroviruses Vol. 11 , No. 10 Mary Ann Liebert, Inc. (1995). EPO patent publication
  • Hydroxyurea and nucleoside analogs such as ddl have potent effects on resting cells and macrophages (ref. Lori, PNAS 93 and Science 94; Goa-
  • the inventors have reported their discovery that a combination of hydroxyurea, a nucleoside analog, and a protease inhibitor can be used to inhibit HIV in human beings, with greatly improved results in that viral rebound may be delayed for at least three to eight weeks or more. These results indicate that the triple combination which includes hydroxyurea may be used for the treatment of HIV infection. Again, this combination takes advantage of the potency of the protease inhibitors, especially Indinavir.
  • the inventors now have found that the double combination of hydroxyurea and a reverse transcriptase inhibitor can also be used, without the addition of a protease inhibitor, for long-term treatment of HIV infections, without provoking viral rebound. See Atty Docket no.
  • a further object of this invention is to provide a treatment for HIV infections that reduces the presence of the virus in both plasma and the lymphoid system, and which inhibits viral rebound after cessation of treatment.
  • Yet another object of this invention is to provide a treatment for HIV which relatively less expensive and has relatively low toxicity, therefore increasing its suitability for widespread use in a large population.
  • An even further object of this invention is to provide a method of activating quiescent cells harboring integrated viral DNA under controlled conditions for the purpose of eliminating the integrated viral DNA.
  • the present inventors have found that the hydroxyurea (HU) in combination with a reverse transcriptase inhibitor such as 2', 3'-dideoxyinosine (ddl) alone can be used to reduce the level of viral load in the blood to undetectable levels (less than 500 copies per mL), and that such treatment can be sustained over long periods of time.
  • a reverse transcriptase inhibitor such as 2', 3'-dideoxyinosine (ddl) alone
  • ddl reverse transcriptase inhibitor
  • An advantage of the present invention is that it can be used very early after infection to prevent seroconversion of a person infected with HIV, as well as after seroconversion.
  • a further advantage is that it includes combinations that have relatively low toxicity, and may be suitable as a long-term treatment for chronic infection for a wide range of individuals. Yet another advantage is that, in addition to reducing the viral load in plasma and in the lymph nodes to undetectable levels, the present invention has been shown to inhibit viral rebound after treatment is stopped. Yet another advantage is that an alternative mode of treatment may be made available to individuals who are failing protease inhibitor therapy.
  • Figs. 1-3 are the original Western Blot test results corresponding to the data in Tables 1-3, respectively.
  • Fig. 4 is a timeline showing viral load for one patient. Detailed Description of the Invention
  • Hydroxyurea is one of many inhibitors of ribonucleotide reductase, an enzyme known for catalyzing the reduction of ribonucleoside diphosphates to their deoxyribonucleoside counterparts for DNA synthesis.
  • Hydroxyurea inhibits viral replication, and also acts to down- modulate the immune system.
  • Another material which inhibits viral replication and down-modulates the immune system is cyclosporine, a cyclophilin inhibitor.
  • ribonucleotide reductase inhibitors include guanazole, 3,4- dihydroxybenzo-hydroxamic acid, N,3,4,5-tetrahydroxybenzimidamide HCI, 3,4-dihydroxybenzamidoxime HCI , 5-hydroxy-2-formylpyridine thiosemicarbazones, and ⁇ -(N)-heterocyclic carboxaldehyde th iosemicarbazones , 4-methyl-5-amino- 1 -formyl isoqu i nol ine thiosemicarbazone, N-hydroxy-N'-amino-guanidine (HAG) derivatives, 5- methyl-4-aminoisoquinolinethiosemicarbazone, diaziquone, doxorubicin, 2,3- dihydroxylbenzoyl-dipeptides and 3,4-dihydroxylbenzoyl-dipeptides, iron- complexed 2-acetylpyridine 5-[(
  • acylclonucleoside hydroxamic acids e.g., N- hydroxy- ⁇ -(2-hydroxyethoxy)-1 (2H)-pyrimidineacetamides 1-3
  • Hydroxyurea has been widely used in cancer therapy as a broad spectrum antineoplastic drug (R. C. Donehower, Seminars in Oncology 19 (Suppl. 9), 11 (1992)). Hydroxyurea is readily absorbed after oral ingestion, rapidly distributed in the body fluids, including the cerebrospinal fluid, and enters cells efficiently by passive diffusion (Id.). Its toxic effects are less profound and easier to control than other chemotherapeutic drugs ⁇ Id.).
  • hydroxyurea is currently administered using two basic schedules: (a) a continuous daily oral dose of 20-40 mg per kg per day, or (b) an intermittent dose of 80 mg per kg per every third day. Either schedule could be used in the treatment of viral infections.
  • lower dosages of hydroxyurea may also be effective in treating HIV infections.
  • the presently preferred dosage range for use of hydroxyurea in treating HIV infections is 800-1500 mg per day, which can be devided over a 24 hour period, for example as 300-500 mg three times a day (TID), assuming an adult weighing about 70 kg.
  • TID three times a day
  • Hydroxyurea is classified as a mildly toxic drug and does not cause immunosupression. Myelotoxicity is hydroxyurea's dose-dependent toxicity. However, such toxicity can be easily monitored and it is constantly and rapidly reversible after decreasing the dose or suspending the treatment
  • hydroxyurea can be administered for years, and sometimes for decades.
  • a second member of the combination of the present invention is a reverse transcriptase inhibitor.
  • examples include nucleoside analogs, such as the 2',3'-dideoxyinosine (ddl)(available as Videx® from Bristol Myers- Squibb) used in the Examples. Nucleoside analogs are a class of compoounds known to inhibit HIV, and ddl is one of a handful of agents that have received formal approval in the United States for clinical use in the treatment of AIDS. See Clinical Microbiology Reviews, Supra, p. 200.
  • zidovudine (3'-azido-2',3' -dideoxythymidine or azidothymidine [ ⁇ ZT] available from Glaxo Wellcome)
  • zalcitabine (2', 3' - dideoxycytidine [ddC] available as Hivid® from Hoffman-La Roche)
  • stavudine (2',3' -didehydro-2',3'-dideoxythimidine [D4T] available as Zerit® from Bristol
  • ddl belongs to the class of compounds known as 2', 3' -
  • nucleoside reverse transcriptase inhibitors include adefovir (Preveon® an adenine nucleotide analog from Gilead Sciences), abacavir (1592U89 available from Glaxo Wellcome), and lubocavir (a guanosine analog available from Bristol
  • Non-nucleoside reverse transcription inhibitors include nevirapine (ViramuneTM available from Boehringer Ingelheim Pharmaceuticals, Inc.), delaviridine (Rescriptor® available from Pharmacia & Upjohn) and efavirenz (available as Sustiva®, from DuPont Merck)
  • nevirapine Virtually Ingelheim Pharmaceuticals, Inc.
  • delaviridine Rescriptor® available from Pharmacia & Upjohn
  • efavirenz available as Sustiva®, from DuPont Merck
  • antiviral therapy requires doses of ddl at 200 mg per day BID for an adult human. Similar dosages may be used in the present invention.
  • use of the combination drugs may increase the effectiveness of these nucleoside phosphate analogs so that they can be used at lower dosages or less frequently.
  • the presently preferred range for ddl is 100-300 mg twice a day (BID), assuming an adult weighing 70 kg.
  • BID twice a day
  • the preferred range is 40 mg BID.
  • protease inhibitors compounds such as hydroxyethylamine derivatives, hydroxyethylene derivatives, (hydroxyethyl)urea derivatives, norstantine derivatives, symmetric dihydroxyethylene derivatives, and other dihydroxyethylene derivatives have been suggested, along with protease inhibitors containing the dihydroxyethylene transition state isostere and its derivatives having various novel and high-affinity ligands at the P 2 position, including 3-tetrahydrofuran and pyran urethanes, cyclic sulfolanes and tetrahydrofuranylglucines, as well as the P 3 position, including pyrazine amides.
  • constrained "reduced amide"-type inhibitors have been constructed in which three amino acid residues of the polypeptide chain were locked into a ⁇ -tum conformation and designated ⁇ -turn mimetics.
  • Other alternatives include penicillin-derived compounds and non-peptide cyclic ureas. At present, the inventors have no
  • protease inhibitors used in the present invention include Indinavir sulfate, (available as CrixivanTM capsules from Merck & Co., Inc, West Point, PA.), saquinavir (Invirase® and Fortovase® available from Hoffman- LaRoche), ritonavir (Norvir® available from Abort Laboratories) ABT-378
  • the present invention may utilise integrase inhibitors such as AR177 (Zintenvir® available from Aronex); fusion inhibitors such as pentafuside, (T-
  • cytokine inhibitors available from Chiron
  • chemokine inhibitors available from Chiron
  • Suitable human dosages for these compounds can vary widely.
  • dosages to adult humans of from about 0.1 mg to about 1 g or even 10 g are contemplated.
  • the combination of compounds of the present invention may be administered by any conventional route. Administration may be oral, intravenous, intraperitoneal, intramuscular, subcutaneous, transdermal, transmucosal (e.g., by inhalation or by means of a suppository), or by any other suitable route. Administration orally in a physiologically acceptable buffered solution is preferred.
  • the buffered solution may be used for one or more members of the combination, while the other member or members may be administered in another form.
  • the particular dosage, toxicity, and mechanism for delivery of the individual of drugs of the present invention are either already known, or can be readily determined by conventional empirical techniques, as can dosages for the combination.
  • the combination may result in the ability to use lower amounts of one or more of the constituents.
  • This aspect of the invention may be particularly valuable with respect to the protease inhibitors, which generally are poorly soluble in water and have poor bioavailability.
  • the present invention may address this problem in part by allowing lower dosages.
  • the present invention may be used before and after acute infection, before seroconversion, and after seroconversion.
  • the data presented herein demonstrates an early treatment of the infection that may result in a profound modification of the natural evolution of the HIV-1 infection.
  • the hydroxyurea-containing combinations of the present inventions might be administered prophylactically to high-risk individuals, orto individuals failing protease inhibitor therapy.
  • the present invention allows for variation in the mode of treatment over time.
  • the protease inhibitors are known known to be most useful in certain types of activated T-cells that are actively producing virus.
  • the protease inhibitor-containing combinations could be used only in the initial phase of therapy until the viral load is reduced in the plasma (less than 500 copies per milliliter) for longer than 2 months. At this point, the protease inhibitors have very likely accessed all the virus producing cells in the reservoirs they can access and have blocked active replication of the virus. Following this phase, the HU/reverse transcriptase inhibitor combination can be used for therapy until the virus is completely eliminated from the body. In the alternative, where the initial viral load in the blood is 50,000-100,000 or less, or when the patient is failing other therapies, the hydroxyurea/reverse transcriptase inhibitor combinations without the protease inhibitor can be used. Depending on the status of the patient, the time of the treatment can be from several months to lifelong.
  • Another mode of treatment would be to deliberately activate certain types of quiescent cells during intensive triple combination therapy. Certain quiescent cells do not express HIV-1 proteins, and act as particularly stubborn reservoirs for the virus. In these cells, the HIV-1 DNA is integrated and both
  • the cells may remain dormant for years before they spontaneously activate, and begin producing virus particles with the same ferocious reproductive rate and mutation rate as the original, acute infection. None of the presently known drugs can eliminate integrated viral DNA. This difficulty could be overcome if these cells were activated during effective combination therapy.
  • the cells could be activated by vaccination against any of a number of diseases known to activate such cells, including, for example, HIV-1 , Hepatitis B, Influenza, and Polio vaccination. HIV-1 genetic immunization is preferred, as disclosed in USSN 60/604,627, filed February 21 , 1996. Such activation should preferably take place after the elimination of active virus production (that is, after the patient's viral load is undetectable for at least 2 months). Repeated activation would be helpful to ensure that all quiescent cells harboring HIV-1 DNA had been activated.
  • CD4 + helper T cells are crippled very early in the course of HIV disease. See Vigorous HIV-1 -Specific CD4 + T Cell responses Associated with Control of Viremia, Rosenberg, et al., Science Magazine 278:5342 (Nov. 1977) 1447-1450.
  • the patient BM who has stopped therapy without rebound has a high level of CD4 + helper activity, unlike both untreated patients and those treated with conventional therapies including non-hydroxyurea drug combinations containing protease inhibitors.
  • the patient's immune system may now play a pivotal role in reducing the replication competence of the HIV viral population. This situation may have been induced by the drug combination, and possibly by prime-and-boost stimulation of the immune system.
  • Classic vaccination techniques often utilize a prime-and-boost method of enabling the immune system to successfully recognize and control pathogens. That is, the immune system receives a stimulus, has time to
  • Example 3 where four out of five patients treated with hydroxy urea-based triple combination therapy tested negative for HIV competent virus using the standard test, and two tested negative when the test sensitivity was increased ten-fold. Significantly, the only patient to test positive had no history of either vaccination or exposure to hepatitis in some form.
  • hydroxyurea may be essential to the use of this prime-and- boost technique because of its function. Hydroxyurea modulates the immune system. That is, it reduces the rate of immune system cells somewhat, or has a mild cytostatic effect.
  • the CD8 (killer) cells attack. Normally this would lead to control of a pathogen. But this activity does not eliminate the HIV infection, so the CD8 cells kill the CD4+ helper cells, and more CD8 cells are activated, which results in the killing of more CD4+ helper cells until the net result is a loss of the CD4 + helper cells and exhaustion of CD8 cells. This overexpansion of the immune system is ineffective to control the virus, which simply keeps replicating.
  • a key step of HIV-1 infection of lymphocytes is the conversion of the viral RNA genome into double-stranded DNA by the action of HIV-1 RT.
  • DNA synthesis differs in different states of infected lymphocytes. In quiescent cells, viral DNA synthesis can be initiated as efficiently as in activated cells. However, in contrast to the activated cells, DNA synthesis in quiescent lymphocytes may terminate prematurely (J. A. Zack, et al., Ce//61 :213 (1990); J. A. Zack, etal., V/ro/ogy 66:1717 (1992)) producing no HIV-1 progeny (Zack, et al, supra; M. Stevenson, et al., EMBO J. 9:1551 (1990); M. I. Bukrinsky, et al., Science 254:423 (1991 )).
  • Example 1 illustrates the various methods that can be used to quantitate the replication of the HIV-1.
  • a variety of different tests with different sensitivities are currently in use, particularly since researchers have found that older screening methods with a sensitivity of ⁇ 400 copies per milliliter plasma are simply not sensitive enough to tell whether a dangerous infection continues to exist in the individual. It has also been demonstrated
  • lymphoid tissues are the major reservoirs of HIV-1 .
  • Inguinal lymph nodes were surgically removed and cut in half along a longitudinal axis. One part was fixed in formalin for in situ hybridization and the other part was frozen in liquid nitrogen. The frozen tissue was homogenized and its DNA was extracted. HIV-1 DNA was amplified by a highly sensitive poiymerase chain reaction (PCR assay), described in detail in Methods in Molecular Biology, Vol. 15: PCR Protocols.
  • PCR assay highly sensitive poiymerase chain reaction
  • PCR SK primers SK38 and SK39 are available from Perkin-Elmer, Norwalk, CT.
  • the PCR-reaction mixture contained the following: 500 ng of genomic DNA, 0.2 mM of each primer, 100 ⁇ M of each nucleoside triphosphate, 1.5 mM MgCI 2 , 20 mM Tris-HCI (pH 8.4), 50 mM KCI and 1 units ofTaq DNA poiymerase (BoheringherManheim Corporation, Indianapolis, IN) in a final volume of 100 ⁇ l.
  • the cycle conditions were 95 °C for 3 minutes, 50 times (94 °C for 1.30 min, 56 °C for 1.00 min and 72 °C for 1.00 min) and 72
  • PCR RT primers were designed and used by the inventors: sense-primer RT-F1 (5-GGACCTACACCTGTCAACAT-3, nucieotides 127 to 146 of HXB2 pol gene) and antisense-primer RT-R8 (5- CATTTATCAGGATGGAGTTCATA-3, nucieotides 886 to 908 of HXB2 pol gene)
  • the PCR-reaction mixture contained the following: 500 ng of genomic DNA, 0.2 ⁇ M of each primer, 100 ⁇ M of each nucleoside triphosphate, 2 mM MgCI 2 , 20 mM Tris-HCI (pH 8.4), 50 mM KCI and 1 units of Taq DNA poiymerase (Boheringher Manheim Corporation, Indianapolis, IN) in a final volume of 100 ⁇ l.
  • the cycle conditions were 95 °C for 3 minutes, 50 times (94 °C for 1.30 min, 56 °C for 1.30 min and 72 °C for 1.30 min) and 72 °C for 10 minutes.
  • Hybridization primers RT - F7 GGATGGAAAGGATCACCAGC
  • Sense-primer RT-F5 (5-CAGGAATGGATGGCCCAAAAGT-3, nucieotides 233 to 254 of HXB2 pol gene)
  • antisense-primer RT-R12 (5- TTCATAACCCATCCAAAG-3, nucieotides 874 to 891 of HXB2 pol gene).
  • PCR conditions were 1 ⁇ l from the first PCR reaction, 0.4 ⁇ M of each primer, 200 ⁇ M of each nucleoside triphosphate, 1.5 mM MgCI 2 , 10 mM Tris-
  • the cycle conditions were 95° C for 3 minutes, 45 times (94° C for 30 sec, 55 °C for 30 sec. and 72 °C for 30 sec), and 72 °C for 10 minutes.
  • the DNA from PCR reaction was separated on an agarose gel and visualized by Ethidium Bromide staining. Polaroid pictures were taken. To increase the sensitivity at least 100 fold, the DNA was blotted to nitrocellulose paper and hybridized with a fluorecents labeled oligonucleotide according to the manufacturer protocol (ECL 3-oligolabelling and detection systems. Amersham Life Science, Little Chalfont, England).
  • Primer F1 was previously described by Xiping W, Ghosh S, Taylor M, Johnson V, Emini E, Deutusch P, Lifson J, Bonhoeffer S, Nowak M, Hahn B, Saag M, Shaw G. Viral dynamics in human immunodeficiency virus type 1 infection. Nature 1995;373: 117-122; Primer F5 was described by Saag, M.S., Emini, E.A., Laskin, O.L., Douglas, J., Lapidus, W.I., Schleif, W.A., Whitley,
  • Quantitation of HIV-1 RNA in semen was performed by using a NASBATM HIV-1 RNA QT kit available from Organon Teknika, Netherlands, according the the manufacturer's protocol. Briefly, 200 ⁇ l of semen were mixed with 1.8 ml of lysis buffer and frozen until use. Nucleic acids were extracted using a guanidine thiocyanate-silica based method (Boom, R., Sol, C.J.A., Salimans, M.M.M., Jansen, C.L., Wertheim-van Dillen, P.M.E., van der Noordaa , J . ( 1990) A rapid and simple method for purification of nucleic acids.
  • HIV-1 genome (Kievits, T., van Gemen, B., van Strijp, D., Schukkink, R., Dircks, M., Adriaanse, H., Malek, L, Sooknanan, R., Lens, P (1990) NASBATM isothermal enzymatic in vitro nucleic acid amplification optimized for the diagnosis of HIV-1 infection. J. Virol. Meth. 35: 273-286, and van Gemen, B., van Beuningen, R., Nabbe, A., van Strijp, D., Jurriaans, S., Lens, P., Kievits,
  • Figs. 1-3 contain the corresponding Western Blot information for patients.
  • HIV-1 RNA by in situ hybridization.
  • the first node of FC was analyzed 8 weeks after beginning treatment, while the virus was still detectable in the plasma, and HIV-1 RNA was mainly associated to the follicular dendritic cells. At this time, the CD4 count and CD4/CD8 ratio was normal. Later, when HIV-1 was no longer detectable in the plasma, another inguinal lymph node was obtained and analyzed as before. No HIV-1 RNA was detected at that time. Again, DNA was extracted from the half of the lymph node which was frozen and tested by PCR analysis using 2 different primers and also a nested primer (sensitivity of this test is to one copy of viral DNA per sample). The PCR was positive, indicating that FC had at least one copy of HIV-1 DNA in the lymph node. See Table 1.
  • the third patient (BM, see Table 3) was treated starting 7 weeks after the probable date of infection. Between 19 and 22 days after starting the therapy, he interrupted the treatment for three days, concomitantly with an episode of orchitis. A rebound of plasma viremia was monitored immediately after the three day suspension. Therapy was started again, and at about week 5 after initiating treatment, HIV-1 was undetectable in the plasma. At week 16, an inguinal lymph node was analyzed and 2 RNA producing cells were found out of 44 million cells screened. At week 17, treatment was again interrupted, this time due to an episode of acute hepatitis A.
  • LJ The patient who had been seropositive for the longest period of time, LJ, (See Table 6) had low but detectable levels of HIV-1 RNA and proteins in the lymph node after 27 weeks of treatment.
  • Patient TD had been seroconverted for approximately 6 months before treatment had begun.
  • Patient TD currently shows no traces of viral DNA or RNA in the sperm, serum, or lymph nodes. This patient has had a history of hepatitis infection in March, 1996.
  • These data indicate that the combination of hydroxyurea, ddl and a protease inhibitor present a potent new combination that can rapidly clear the virus from plasma and lymph nodes, and inhibit viral rebound after cessation of treatment. Further, this combination blocks HIV-1 replication in the lymphoid system and, at least in one case, shows hope for HIV-1 eradication.
  • these data indicate that HIV-1 infection is treatable as an emergency disease.
  • Patients should be tested not for seroconversion, but for free virus particles in the blood if there are any symptoms or suspicion of infection, and treated immediately with the combination therapy before seroconversion takes place.
  • the present invention will eliminate the free virus, block new infections, restore the immune system, and may eliminated virus integration in millions of cells. This method would also be economical, as treatment would be begun earlier and be of shorter duration than treatment for people with chronic infections.
  • the present results show that the patients treated early restored the normal lymphocyte status in short periods of time (see CD4+cells and CD4/CD8 ratio). However, the patient that had been infected for 5-9 years (LJ, see Table 6) could not as rapidly restore the
  • the present results indicate a method of eliminating quiescent cells, that is, cells which have integrated viral DNA, but do not currently express the genes or produce virus.
  • HIV-1 DNA was measured in the lymphocytes of the patients with a highly sensitive nested PCR able to detect as little as 1 copy of viral DNA. Two patients in this group had no detectable HIV-1 DNA in the lymphoid organ and 3 had detectable DNA. Other investigators have also reported (in all cases) detectable DNA in the lymphoid organs even in the absence of virus producing cells (Markovitz, Retrovirus Conference, 1997) The present inventors are not aware of any other patients other than the two in the present study (TD, BM) who have undetectable viral DNA in the lymphoid organs.
  • BM had Hepatitis A and TD had hepatitis B. Both infections are characterized by activation of cells which can harbor HIV-1 DNA. After activation, these cells can produce viral particles which will be mainly defective in the presence of protease inhibitors. HU and ddl will work at the early phase, inhibiting reverse transcription with two different mechanisms, consequently blocking both new infection and new
  • lack of a full seroconversion in 3 of these patients treated prior to seroconversion suggests that the replication of the virus has at least been reduced to a minimum.
  • all of the above considerations hold true even after the treatment has been suspended in one of the patients.
  • DNA was repeatedly undetectable in the lymph nodes, even with a methodology able to detect a single copy of viral DNA.
  • Example 3 Samples from five patients were analyzed according to the method described in Identification of a Reservoirfor HIV-1 in Patients on Highly Active Antiretroviral Therapy, Finzi et al., Science Magazine 278 (5341 ): 1295-1300. This is a test for replication-competent virus that may persist in the resting CD4+T cells of patients who have no evidence of active virus replication.
  • the test sensitivity was increased by a factor of 10, and the patient registered as positive on the more sensitive test.
  • Each table summarizes viral load, lymphocyte and, where applicable, seroconversion analyses for a single patient over time.
  • Viral load was measured on the dates indicated by a contract laboratory ("bDNA/PCR”) or by the inventor's laboratory ("PCR/RIGHT”).
  • HIV-RNA in situ measured by nucleotide binding to lymph node tissue.
  • HIV DNA PCR standard test for viral load. "Not detected” means not tested on that date.
  • FDC Follicular Dendritic Cells
  • Cells Lymph node cells generally.
  • Lymphocyte population analysis shows the complement of the patient's lymphocytes at the given dates.
  • Lymph Total number of lymphocytes.
  • B.Ly Total number of B-lymphocytes.
  • T-Ly Total number of T-Lymphocytes.
  • CD4 Total number of CD4+ lymphocytes.
  • CD4% The percentage of total lymphocytes that are CD4+ lymphocytes.
  • CD8 Total number of CD8+ lymphocytes.
  • CD8% The percentage of total lymphocytes that are CD8+ lymphocytes.
  • Seroconversion was shown by Western Blot analysis. The patient's blood was screened for antibodies to the listed HIV proteins at the given dates.
  • Acute hepatitis A infection stop therapy 26 Oct. 96 HU 3 00 mg TID

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Abstract

L'invention concerne une méthode pour rendre ineffective in vivo une réplication de population virale dépendante de la transcriptase inverse. Cette méthode consiste à administrer aux cellules concernées une combinaison de composés choisis dans le groupe comprenant l'hydroxyurée, un analogue de nucléoside et, éventuellement un inhibiteur de protéase, puis à administrer un agent pour activer les cellules quiescentes qui abritent le virus.
PCT/US1999/003454 1998-03-26 1999-02-18 Methode pour rendre ineffective in vivo une replication de population virale vih WO1999048526A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001089509A2 (fr) * 2000-05-19 2001-11-29 Thomas Jefferson University Procede pour traiter une maladie due au vih-1 residuel
US6579521B2 (en) 2000-10-20 2003-06-17 Chiron Corporation Methods of therapy for HIV infection
WO2018208826A1 (fr) * 2017-05-08 2018-11-15 Meharry Medical College Hydroxyurée servant à améliorer les cellules spermatiques

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001089509A2 (fr) * 2000-05-19 2001-11-29 Thomas Jefferson University Procede pour traiter une maladie due au vih-1 residuel
WO2001089509A3 (fr) * 2000-05-19 2002-06-27 Univ Jefferson Procede pour traiter une maladie due au vih-1 residuel
US6579521B2 (en) 2000-10-20 2003-06-17 Chiron Corporation Methods of therapy for HIV infection
US7048924B2 (en) 2000-10-20 2006-05-23 Chiron Corporation Combination antiviral and interleukin-2 therapy for HIV infection
WO2018208826A1 (fr) * 2017-05-08 2018-11-15 Meharry Medical College Hydroxyurée servant à améliorer les cellules spermatiques
US11926846B2 (en) 2017-05-08 2024-03-12 Meharry Medical College Hydroxyurea to enhance sperm cells

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