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WO1996041634A2 - Utilisation d'un inhibiteur de transcriptase inverse non-nucleosidique en association avec des inhibiteurs de nucleosides pour le traitement d'infections a vih - Google Patents

Utilisation d'un inhibiteur de transcriptase inverse non-nucleosidique en association avec des inhibiteurs de nucleosides pour le traitement d'infections a vih Download PDF

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Publication number
WO1996041634A2
WO1996041634A2 PCT/EP1996/002540 EP9602540W WO9641634A2 WO 1996041634 A2 WO1996041634 A2 WO 1996041634A2 EP 9602540 W EP9602540 W EP 9602540W WO 9641634 A2 WO9641634 A2 WO 9641634A2
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WO
WIPO (PCT)
Prior art keywords
equal
nucleoside
dose
azt
methyl
Prior art date
Application number
PCT/EP1996/002540
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English (en)
Other versions
WO1996041634A3 (fr
Inventor
Paolo La Colla
Maria Elena Marongiu
Giovanna Piras
Original Assignee
Sardinian Antiviral Research Consortium - Sarc S.C.R.L.
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 Sardinian Antiviral Research Consortium - Sarc S.C.R.L. filed Critical Sardinian Antiviral Research Consortium - Sarc S.C.R.L.
Priority to AU63552/96A priority Critical patent/AU6355296A/en
Publication of WO1996041634A2 publication Critical patent/WO1996041634A2/fr
Publication of WO1996041634A3 publication Critical patent/WO1996041634A3/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof

Definitions

  • the present invention refers to the use of a non-nucleoside reverse transcriptase inhibitor, and in particular of derivatives of 6-benzyl-
  • X is selected from the group consisting of 0 , S ;
  • R is selected from the group consisting of alkyl from C- ⁇ to C ⁇ , cycloalkyl from Cc to Cg ;
  • R' , R" and Z, equal or different among them may be H, alkyl from C- ⁇ to C ⁇ considering that when X is equal to 0, R and R' cannot be both equal to H, their pharmaceutically acceptable salts and their soluble derivatives , in association with nucleoside inhibitors selected from the group consisting of 3 ' ⁇ azido-2 ' , 3 ' -dideoxythymidine (AZT) , 2 * , 3 ' dideoxy-2 ' , 3 ' didehydro thymidine ( d*IT) and their phosphodiester or phosphotriester derivatives , if necessary associated also to dideoxy inosine ( ddl ) or dideoxy cytosine ( ddC ) , for the preparation of
  • compositions useful for the treatment of HIV infection consisting of the association of the two or three components are also described.
  • AIDS acquired immunodeficiency syndrome
  • Human Immunodeficiency Virus - HIV Human Immunodeficiency virus
  • it is characterized by the progressive compromising of immunologic defences.
  • the search for new therapeutic strategies more efficacious than those at present available in slowing the disease progression in the affected subjects and the infection diffusion in the population has been given a substantial boost.
  • the process of retrotranscription of the genome from RNA to double-chain DNA by means of the "reverse transcriptase" enzyme is, probably, the step studied in greatest detail.
  • the reverse transcriptase has three functions: the DNA polymerase RNA-dependent one, the DNA polymerase DNA-dependent one and the RNAse-H one.
  • NRTI nucleoside reverse transcriptase inhibitors
  • NRTI non-nucleoside reverse transcriptase inhibitors
  • To the first class in particular belong the AZT, d4T, ddl and ddC compounds, all already known and used in the clinic in the therapy of AIDS. Once phosphorylated, they inhibit the synthesis of the viral DNA competing with the physiologic nucleosides for the specific site in the reverse transcriptases of HIV-1 and HIV-2 and they act as terminators of the chain of the DNA on the way to be synthesized as they lack the hydroxyl in the C3' of the sugar.
  • NNRTI NNRTI
  • HEPT High Efficiency Polymorphonucleic acid
  • TIBO TIBO
  • Nevirapine BHAP
  • TSAO TSAO
  • all specific inhibitors of the reverse transcriptase of HIV-1 but not of HIV-2 which are defined in detail in the publications shown below.
  • 3'-Spiro nucleosides a new class of specific human immunodeficiency virus type 1 inhibitors synthesis and antiviral activity of [2',5' ⁇ bis-0-(tert-butyldimethylsilyl)-beta-D-xylo-and-ribofuranosyl]-3'- spiro-5"-[4"-amino-l", 2"-oxathiole-2", 2"-dioxide](TSA0)pyrimidine nucleosides. J Med Chem 1992; 35:2721-7.
  • the present invention allows to overcome the above described drawbacks as it allows to obtain the extinction of the HIV infection not only as a result of prolonged treatments, but also as a result of limited duration treatments, and therefore not much toxic.
  • the present invention refers to the use of a non-nucleoside reverse transcriptase inhibitor, and in particular of a derivative of 6- benzyl-4-oxopyrimidine (DABO) having general formula (I)
  • X is selected from the group consisting of 0, S; R is selected from the group consisting of alkyl from C- ⁇ to C ⁇ , cycloalkyl from Cc to Cg; R' , R" and Z, equal or different among them may be H, alkyl from C- ⁇ to C ⁇ considering that when X is equal to 0, R and R' cannot be both equal to H, their pharmaceutically acceptable salts and their soluble derivatives, in association with a nucleoside inhibitor selected from the group consisting of AZT, d4T and their phosphodiester or phosphotriester derivatives, if necessary associated also to ddl or ddC, for the preparation of pharmaceutical compositions active in the treatment of HIV infection.
  • the present invention refers also to the pharmaceutical compositions useful for the treatment of HIV infection constituted of the association of a non-nucleoside inhibitor with one or two nucleoside inhibitors, as defined above.
  • Figure 1 represents the long term cytotoxicity of a high DABO and AZT concentration
  • Figure 2A represents the p24 antigen levels in the treatments with DABO (5 uM) or ATZ (0.16 ⁇ M) alone in comparison with DABO + ATZ association;
  • Figure 2B represents the cytopathic effect in the treatments with DABO or AZT alone in comparison with DABO + AZT assciation
  • Figure 3A represents the p24 antigen levels in the treatments with DABO (5 uM) or AZT (0.08 ⁇ M) alone in comparison with DABO + AZT association;
  • Figure 3B represents the cytopathic effect in the treatments with DABO or AZT alone in comparison with DABO + AZT association
  • Figure 4A represents the p24 antigen levels in the treatments with DABO (50 ⁇ M) or AZT (2.5 ⁇ M) alone in comparison with DABO + AZT association;
  • Figure 4B represents the cytopathic effect in the treatments with DABO or AZT alone in comparision with DABO + AZT association
  • Figure 5 represents the viral DNA sequences in the treatment with AZT (2.5 ⁇ M) in comparison with DABO (10 ⁇ M) + AZT (2.5 ⁇ M) association.
  • compositions according to the present invention against the HIV infections, we report for illustrative purpose some of the results obtained by us, relating to cytotoxicity and anti-HIV activity of a compound representative of the class of the substituted 6-benzyl-4-oxopyrimidines , i.e. 2- cicloexylthio-6-(3'-methyl)benzyl-3,4-dihydro-4-oxo-5-methyl- pyrimidine, and a compound representative of the class of nucleoside inhibitors, i.e.
  • 3'-azido-2', 3'-dideoxythymidine (AZT), individually used or, according to the invention, in association in different ratios, in models of in vitro infection able to estimate either the cytotoxicity or the efficacy of the long term anti-HIV action.
  • the evaluation of cytotoxicity and antiviral activity has been carried out in MT-4 cells, line of T4 lymphocytes permissive for the replication of the HIV viruses.
  • the cells have been cultivated in RPMI 1640 added with 10% fetal calf serum (FCS) , penicillin 100 U/ml and streptomycin 100 ⁇ g/ml.
  • FCS fetal calf serum
  • the cultures have been incubated at 37 °C in a % CO2 atmosphere and periodically controlled in order to verify the absence of mycoplasmas contamination.
  • the virus used in antiviral activity tests (HIV-1, strain Hlg) has been obtained from supernatant of H9/III B cronically infected cells.
  • the stock virus solutions have been titrated in C8166 and maintained at -80 °C until the moment of use.
  • cytotoxicity 50 ⁇ l of RPMI containing 1x10 MT- 4 cells have been added, in 96 well multiplates, to 50 ⁇ l of RPMI containing or not scalar dilutions of the compounds under examination, alone and in association. Then the cultures have been incubated at 37 'C . Each 4 days the whole culture has been suspended again in a new medium, containing the same concentrations of inhibitors, in order to take again the cultures to a density (lxlO-'/ml) such that their exponential growth is allowed.
  • the cells viability has been determined by a colorimetric method based on the use of a tetrazolium salt, the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium-bromide (MTT) , which is transformed by the succinic dehydrogenase mitocondrial enzyme in a product having blue color (formazan) whose amount in our experimental conditions, turns out to be proportional to the number of vital cells.
  • MTT 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium-bromide
  • the first one with the aim to verify the possibility to block an acute infection process in progress, if necessary extinguishing the infection; the second one predictive of a treatment aimed to prevent the spreading of the infection from an infected cell to healthy bystander cells. Therefore, MT-4 cells, seeded at a density equal to lxl0 6 /ml, have been infected for an hour at 20 °C with lxlO 6 CCID 5Q (1 CCIDJ-Q 2 - 50 infecting viruses). After the removal of the inoculum, the cells have been washed three times and then suspended again at a density equal to 2xl0- > /ml as such or after dilution 1:100 in normal cells.
  • RPMI containing 1x10 MT-4 cells have been added, in 96 well multiplates, to 0 ml of RPMI containing or not scalar dilutions of the compounds under examination, alone or in association. Then the cells have been incubated at 37 °C. In case of survival, each 4 days the whole culture has been suspended again in a new medium containing the same concentrations of inhibitors, in order to take again the cells to a density (lxlO- 5 /ml) such that their exponential growth is allowed. If necessary, after a determined treatment period, specified case by case, duplicate cultures have been suspended again lacking the inhibitors and propagated till the thirty-second day in an inhibitor free culture medium (reverted cultures) .
  • PCR polymerase chain reaction
  • This procedure allows to assure density conditions optimal for the exponential growth of the cells (10 initial cells multiply till producing, on the twelfth day, 4.1x10' cells) and to maintain in the culture all the cells originally infected for a period of time of twelve days.
  • Cytotoxicity The long term cytotoxicity of a high DABO and AZT concentration is shown in Fig. 1.
  • the maximum not cytotoxical DABO and AZT concentrations as a result of the prolonged treatment with the single compounds for 32 days are, respectively, >100 ⁇ M and 2.5 ⁇ M.
  • the two compounds are without cytotoxicity when are used in association for 32 days at the doses of 50 ⁇ M (DABO) and 2.5 ⁇ M (AZT)
  • HIV infection spreads producing, on the fourth day after the infection, p24 antigen levels greater than 950 ng/ml
  • 0.16 ⁇ M AZT individually used, inhibits the viral replication only for the first 4 days. Whereupon the viral replication starts again and one has an increase in the p24 antigen levels and development of the cytopathic effect within the next 4-8 days. On the contrary, in the cultures treated with 5 uM DABO in association with 0.16 ⁇ M AZT, the p24 antigen amount quickly decreases and it is no more detectable for the duration of the experiment (Fig. 2A) . In this case one has a complete protection from the development of the cytopathic effect and the cells multiply exponentially as in the non infected controls (Fig. 2B).
  • the results shown in the figures 1, 2 and 3 demonstrate that associations of DABO with AZT according to the present invention, which allow to reach an hematic concentration of DABO ranging from 0.6 to 20 ⁇ M, preferably ranging from 0.6 to 10 uM, and of AZT ranging from 0.08 to 5 ⁇ M, preferably varying from 0.08 to 0.32 ⁇ M, are not cytotoxical and are efficacious in inhibiting the HIV multiplication.
  • DABO + AZT associations are able to extinguish the HIV infection in cultures having a high density of infected cells as a result of a treatment limited in time. In therapy this may reflect in discontinuous treatments able to reduce the toxicity of the drugs and their selective pressure towards pharmaco-resistant mutants.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (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)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne l'utilisation, pour le traitement d'infections à VIH, d'un inhibiteur de transcriptase inverse no n-nucléosidique, notamment des dérivés de 6-benzyl-4-oxopyrimidine de formule générale (I), dans laquelle X est choisi dans le groupe constitué de O, S; R est choisi dans le groupe comprenant alkyle de C1 à C4, cycloalkyle C5 et C6; R', R' et Z sont identiques ou différents et représentent H, alkyle de C1 à C4, sous réserve que lorsque X vaut 0, R et R' ne peuvent être tous les deux égaux à H, leurs sels pharmaceutiquement acceptables et leurs dérivés solubles, en association avec des inhibiteurs de nucléoside choisis dans le groupe constitué de AZT, d4T et leurs dérivés phosphodiesters ou phosphotriesters. L'invention concerne également des compositions pharmaceutiques destinées au traitement d'infections à VIH comprenant une association de différents composants.
PCT/EP1996/002540 1995-06-13 1996-06-12 Utilisation d'un inhibiteur de transcriptase inverse non-nucleosidique en association avec des inhibiteurs de nucleosides pour le traitement d'infections a vih WO1996041634A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU63552/96A AU6355296A (en) 1995-06-13 1996-06-12 Use of a non-nucleoside reverse transcriptase inhibitor in a ssociation with nucleoside inhibitors for the treatment of h iv infection

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT95CA000009A IT1281502B1 (it) 1995-06-13 1995-06-13 Impiego di un inibitore non nucleosidico della trascrittasi inversa in associazione con inibitori nucleosidici per il trattamento della
ITCA95A000009 1995-06-13

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WO1996041634A2 true WO1996041634A2 (fr) 1996-12-27
WO1996041634A3 WO1996041634A3 (fr) 1997-01-23

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999051239A1 (fr) * 1998-04-07 1999-10-14 Du Pont Pharmaceuticals Company Procede utilisant des super-desintegrants dans la preparation de capsules ou de comprimes d'efavirenz a dissolution rapide
WO2001007027A3 (fr) * 1999-07-22 2001-08-09 Vertex Pharma Inhibiteurs d'helicase virale
AU2004268390B2 (en) * 2003-09-03 2011-03-31 Janssen Sciences Ireland Uc Combinations of a pyrimidine containing NNRTI with RT inhibitors
WO2014093553A1 (fr) * 2012-12-11 2014-06-19 Vanderbilt University Méthodes et compositions pour le traitement d'une infection par le vih
US9127005B2 (en) 2009-07-24 2015-09-08 Vanderbilt University Isoform selective phospholipase D inhibitors
US9453017B2 (en) 2011-09-30 2016-09-27 Vanderbilt University Antiviral therapies with phospholipase D inhibitors

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MICROBIOLOGICA (PAVIA), 17 (4). 1994. 269-279., XP000609825 TRAMONTANO E ET AL: "Characterization of the anti-HIV-1 activity of 3,4-dihydro-2-alkoxy-6-benzyl-4-oxopyrimid ines (DABOs), new non-nucleoside reverse transcriptase inhibitors" *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999051239A1 (fr) * 1998-04-07 1999-10-14 Du Pont Pharmaceuticals Company Procede utilisant des super-desintegrants dans la preparation de capsules ou de comprimes d'efavirenz a dissolution rapide
US6238695B1 (en) 1998-04-07 2001-05-29 Dupont Pharmaceuticals Company Formulation of fast-dissolving efavirenz capsules or tablets using super-disintegrants
US6555133B2 (en) 1998-04-07 2003-04-29 Bristol-Myers Squibb Company Formulation of fast-dissolving efavirenz capsules or tablets using super-disintegrants
WO2001007027A3 (fr) * 1999-07-22 2001-08-09 Vertex Pharma Inhibiteurs d'helicase virale
AU2004268390B2 (en) * 2003-09-03 2011-03-31 Janssen Sciences Ireland Uc Combinations of a pyrimidine containing NNRTI with RT inhibitors
US9127005B2 (en) 2009-07-24 2015-09-08 Vanderbilt University Isoform selective phospholipase D inhibitors
US9453017B2 (en) 2011-09-30 2016-09-27 Vanderbilt University Antiviral therapies with phospholipase D inhibitors
WO2014093553A1 (fr) * 2012-12-11 2014-06-19 Vanderbilt University Méthodes et compositions pour le traitement d'une infection par le vih

Also Published As

Publication number Publication date
AU6355296A (en) 1997-01-09
IT1281502B1 (it) 1998-02-18
ITCA950009A0 (it) 1995-06-13
ITCA950009A1 (it) 1996-12-13
WO1996041634A3 (fr) 1997-01-23

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