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WO1996001106A1 - Emploi d'agents pharmaceutiques pour attenuer ou traiter la dysfonction immunitaire liee a l'infection par le vih ou des virus associes - Google Patents

Emploi d'agents pharmaceutiques pour attenuer ou traiter la dysfonction immunitaire liee a l'infection par le vih ou des virus associes Download PDF

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Publication number
WO1996001106A1
WO1996001106A1 PCT/DK1995/000285 DK9500285W WO9601106A1 WO 1996001106 A1 WO1996001106 A1 WO 1996001106A1 DK 9500285 W DK9500285 W DK 9500285W WO 9601106 A1 WO9601106 A1 WO 9601106A1
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Prior art keywords
cells
hiv
agent
viruses
serotonin
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PCT/DK1995/000285
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English (en)
Inventor
Bo Arne Hofmann
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Bo Arne Hofmann
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Priority to AU28804/95A priority Critical patent/AU2880495A/en
Publication of WO1996001106A1 publication Critical patent/WO1996001106A1/fr

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    • 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
    • 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
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • 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
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/138—Aryloxyalkylamines, e.g. propranolol, tamoxifen, phenoxybenzamine
    • 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
    • A61K31/13—Amines
    • A61K31/15—Oximes (>C=N—O—); Hydrazines (>N—N<); Hydrazones (>N—N=) ; Imines (C—N=C)
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • 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
    • A61K31/403—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 condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4525—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with oxygen as a ring hetero atom
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings

Definitions

  • HIV Human Immunodeficiency Viruses
  • This invention relates to the use of an agent which interacts with 5HT receptors as defined below for increa ⁇ sing the proliferative capacity of immune cells from individuals infected with HIV or related viruses.
  • the invention relates to an agent for increas- ing the CD4 T cell count in such individuals.
  • the agent is also for the preparation of a medicament for alleviation or treatment of the immune dysfunction related to infec ⁇ tion with Human Immunodeficiency Viruses (HIV) or related viruses.
  • HIV Human Immunodeficiency Viruses
  • the invention relates to the immune dysfunction seen in pre-AIDS and AIDS.
  • the medicaments are to be used in human or veterinary medicine.
  • the actions of the agents are considered to be related to a stimulation of receptor sites of cells of the immune system. Said stimulation results in the maintenance and/or reestablishment of a balanced function of the immune cells affected by the virus or virus components.
  • the present invention provides means for the prevention and/or restoration of the impaired immune function - observed in HIV infection, pre-AIDS, and AIDS. This enables the subject treated to better cope with infections and/or avoid development of various forms of neoplasia.
  • retroviruses induces immune deficiencies in the subject infected by said viruses.
  • retroviruses in particular the Human Immunodeficiency Virus (HIV) which causes AIDS character ⁇ ized by severe cellular immune defects.
  • HIV Human Immunodeficiency Virus
  • medicaments directed to interfere with virus repli- cation e.g. azidothymidine (AZT) described in DE patent 3 608 606, have not yet proven effective in restoring the immune capacity of the treated subject.
  • ZHT azidothymidine
  • medicaments for the treatment of functional deficiencies due to HIV infection are described.
  • medicaments inhibiting the cAMP/PKA pathway are the Chinese herb extract "dan-shen", antagonists of adenylate cyclase, antibodies immunologically reactive with adenylate cyclase, dideoxyadenosine, and polyadeny- lates.
  • agents according to the present invention should enable the employment of substances which - like all medicaments - have certain side effects - but which nevertheless - when administered in dosages carefully adapted to the individual subject - should provide for an increase in the proliferative capacity of immune cells and an increase in the CD4 T cell count in such individuals and prevention or alleviation of immune dysfunction without causing side effects so devastating that adminis ⁇ tering to humans or animals is dissuaded.
  • agents according to the present invention is related to the fact that HIV infected patients do not primarily suffer from the HIV infection itself, but from the impact of HIV on cells of the immune system.
  • HIV very little HIV is present in the body of infected indi ⁇ viduals. Most of the HIV is located in the lymph nodes and other lymphatic organs, and most of the T cells in the peripheral blood in HIV seropositive subjects have a decreased function as demonstrated by decreased prolifera ⁇ tion and cytotoxicity (1). The infection alone cannot explain this general functional deficiency, since it is estimated that less than 1% of the CD4 T cells in the peripheral blood is infected with HIV (2, 3).
  • T cells circulate through the lymph nodes and other lymphatic organs. T cells are, during such passage, exposed to infectious HIV and HIV proteins from degraded or defective HIV particles. Viral proteins are produced in such a high concentration that they also can be measured in serum (4). It has been demonstrated that addition of HIV proteins to normal T lymphocytes in test tubes induced a cellular deficiency in these cells resul ⁇ ting in functional anergy i.e. decreased proliferation and cytotoxicity (5, 6, 7, 8).
  • CNS active pharmaceuticals e.g. antimigraine agents, anxiolytica, antidepressiva, and certain neurotransmitters
  • antimigraine agents e.g. antimigraine agents, anxiolytica, antidepressiva, and certain neurotransmitters
  • certain neurotransmitters when used according to the present invention are effective for the increase in proliferative capacity of immune cells as well as an increase in the CD4 T cell count and an alleviation or treatment of the immune dysfunction related to infec ⁇ tion with Human Immunodeficiency Viruses (HIV) or related viruses when an infected individual is exposed to said agents.
  • HIV Human Immunodeficiency Viruses
  • Sumatriptan is an antimigraine agent which is described in e.g. US patent 4 816 470.
  • DPAT 8-hydroxy-2-(di-N-propylamino)tetralin
  • HIV infected individuals should be able to fight common infection and possibly also the development of neoplasia.
  • a main aspect of the present invention is the use of an agent which interacts with 5HT receptors as defined herein for the preparation of a medicament for alleviation or treatment of the immune dysfunction related to infection with Human Immunodeficiency Viruses (HIV) or related viruses.
  • HIV Human Immunodeficiency Viruses
  • 5HT receptors means receptors which interact with 5HT (serotonin). Said inter ⁇ acting may be of the nature of an agonistic or a partially agonistic action, or an antagonistic or a partially antag ⁇ onistic action. Said 5HT receptors comprise the 5HT receptors and subtypes thereof already identified in research.
  • treatment means providing a subject with the agent in question in an amount which is sufficient to prophylactically slow or prevent the development of an unhealthy state due to the infection with Human Immunodeficiency virus or related viruses; or sufficient to alleviate the symptoms or the diseased state; or sufficient to permanently eliminate the symptoms or treat the infection itself.
  • Human Immunodeficiency viruses or related viruses means HIV or other retroviruses causing immune dysfunction similar to the dysfunction seen in HIV infec- tion, pre-AIDS and AIDS.
  • Other examples may be infections caused by HTLV1 and HTLV2, and some of the feline and bovine retroviruses.
  • the alleviation or treatment of the immune dysfunction is mediated via a direct interaction between said agent and a responsive site present on some of the immune cells in the virus infected individual.
  • the aim is to some degree to restore the proliferative capacity and cytotoxicity of said dysfunctional cells.
  • immune cells means cells present in the blood and/or in the hemopoietic tissue of the individual infected with HIV or related viruses. Methods of determining the responsiveness of said immune cells - the target cells - are described below.
  • certain subpopulations of such responsive cells may be determined and "targeted", e.g. cells present in lymph nodes, thymus (e.g. thymocytes) and/or spleen.
  • thymus e.g. thymocytes
  • spleen e.g. IL-4 T cells
  • One par ⁇ ticular subpopulation of lymphocytes may be responsive forms of CD4 T cells and CD8 T cells.
  • the medicaments according to the present invention can be used to provide a targeted restoration of the cellular functional capabilities.
  • One measure for this restoration is an increased CD4 T cell count and/or an increased CD4/CD8 T cell ratio.
  • the responsive site present on some of the immune cells in the virus infected individual may be a cellular receptor site which is structurally or functionally related to the neuronal 5HT receptors or subtypes thereof, i.e. the 5HT receptors present on cells in the nervous system.
  • the response obtained by the use according to the present invention may be due to various molecular actions at the site of a receptor or near to a receptor present on the immune cells. Many of said actions are considered to be initiated and/or to prevail outside the cell exhibiting the receptor, i.e. primarily at the "exterior" side of the cell membrane.
  • buspirone 8- ⁇ 4-[4-(2-pyrimidinyl)-l-piperazi- nyl]butyl ⁇ -8-azaspiro[4,5]decane-7,9-dione, or the class of related compounds as defined below;
  • gepirone 4,4-dimethyl-l- ⁇ 4-[4-(2-pyrimidinyl)-l- piperazinyl]butyl ⁇ -2,6-piperidinedione, or the class of related compounds as defined below;
  • ipsapirone 2- ⁇ 4-[4-(2-pyrimidinyl)-l-piperazi- nyl]butyl ⁇ -l,2-benzisothiazolin-3(2H)-one 1,1 di ⁇ oxide, or the class of related compounds as defined below;
  • suitable members from the class considered to interact with the 5HT1 receptors are selected to be used according the present invention.
  • One important class of subtypes of the 5HT1 receptors is the class comprising the 5HTld receptors.
  • sumatriptan and related compounds are considered to first of all interact with said 5HTld receptors.
  • Another important class of subtypes of the 5HT1 receptors is the class comprising the 5HTla receptors.
  • buspirone and related compounds are considered to first of all interact with said 5HTla receptors.
  • a further aspect of the invention is a method for in ⁇ creasing the proliferative capacity of immune cells of an individual comprising exposing said immune cells to an effective amount of an agent selected from the group consisting of agonists and antagonists of the 5HT receptors of said cells, said individual being infected with HIV or a related retrovirus.
  • Another aspect of the invention is a method for increasing the CD4 T cell count in an individual comprising administering to the individual an effective amount of an agent selected from the group consisting of agonists and antagonists of the 5HT receptors of said cells, said individual being infected with HIV or a related retrovirus.
  • a still further aspect of the invention is a method of alleviating or treating the immune dysfunction related to infection with Human Immunodeficiency Viruses (HIV) or related viruses, which method comprises administering an effective amount of an agent which interacts with 5HT receptors as defined above.
  • HIV Human Immunodeficiency Viruses
  • a particular aspect of the invention therefore relates to a method of alleviating or treating individuals infected with HIV and a related retrovisus which does not also suffer from one or more CNS diseases usually treated by the agents described above.
  • Another main aspect of the invention is the use of a serotonin uptake inhibitor as defined below for the preparation of a medicament for alleviation or treatment of the immune dysfunction related to infection with Human Immunodeficiency Viruses (HIV) or related viruses.
  • said serotonin uptake inhibitor is selected from
  • citalopram 1-[3-(dimethylamino)propyl]-l-(4- fluorophenyl)-1,3-dihydro-5-isobenzofurancarboni- trile;
  • Another aspect of the invention is a method of alleviating or treating the immune dysfunction related to infection with Human Immunodeficiency Viruses (HIV) or related viruses, which method comprises administering to subject an agent which is a serotonin uptake inhibitor.
  • Said serotonin inhibitor may be selected from the group men ⁇ tioned just above.
  • Said administration may be conducted extracorporally.
  • T cells being primed against antigens e.g. micro ⁇ organisms such as Candida albicans, Cytomegalo virus, etc. will - when they encounter such microorganisms - become activated. The cells will undergo a clonal expansion ( roliferation) and provide a large number of T cells directed against the microorganism. This army of T cells will perform a direct killing (cytotoxicity) of the microorganism.
  • antigens e.g. micro ⁇ organisms such as Candida albicans, Cytomegalo virus, etc.
  • One aspect of the invention is the use of the agent
  • HIV Human Immunodeficiency Virus
  • sumatriptan or the class of related compounds means the compounds described in US patent 4 816 470, in so far as the biochemical actions of said compounds on an individual are similar to that of sumatriptan with respect to increase of the proliferative capacity of immune cells, increase of the CD4 T cell count and/or alleviation or treatment of the immune dysfunction related to infection with HIV or related viruses.
  • agents used according to the present invention may also be chosen from physiologically acceptable derivatives of sumatriptan formulated as depot compositions or sustained release preparations.
  • the compounds may be produced by a number of processes described in said US patent, which is incorporated herein by reference.
  • a further aspect of the present invention is the use of the agent
  • buspirone 8- ⁇ 4-[4-(2-pyrimidinyl)-l-piperazi- nyl]butyl ⁇ -8-azaspiro[4,5]decane-7,9-dione,
  • HIV Human Immunodeficiency Virus
  • buspirone or the class of related pharmaceuti ⁇ cal compounds, as defined herein
  • buspirone means the compounds described in US patent 3 717 634 - in so far as the physiological actions of said compounds on an individual are similar to that of buspirone with respect to increase of the proliferative capacity of immune cells, increase of the CD4 T cell count and alleviation or treatment of the immune dysfunction related to infection with HIV or related viruses.
  • the compounds may be produced by a number of processes disclosed in said US patent, which is incorporated herein by reference.
  • a further aspect of the present invention is the use of the agent
  • gepirone or the class of related compounds as defined herein;
  • HIV Human Immunodeficiency Virus
  • gepirone or the class of related pharmaceutical compounds as defined herein means the compounds described in US patent 4 423 049 - in so far as the physiological actions of said compounds are similar to that of buspirone with respect to increase of the proliferative response in immune cells, increase in the CD4 T cell count and/or alleviation or treatment of the immune dysfunction related to infection with HIV or related viruses.
  • the compounds may be produced by a number of processes described in said US patent which is incorporated by reference.
  • ipsapirone or the class of related pharmaceuti ⁇ cal compounds as defined herein means the compounds described in US patent 4 818 756 - in so far as the physiological actions of said compounds are similar to that of buspirone with respect to increase of the proliferative response in immune cells, increase in the CD4 T cell count and alleviation or treatment of the immune dysfunction related to infection with HIV or related viruses.
  • the compounds may be produced by a number of processes described in said US patent which is incorporated by reference.
  • a further aspect of the invention is the use of
  • HIV Human Immunodeficiency Virus
  • derivative thereof and "precursor thereof”, as defined herein, means any of the physiologically accept ⁇ able compounds having related structure and equivalent function to that of serotonin.
  • One of the preferred serotonin derivatives to be used according to the present invention is serotonin-creatinine sulphate.
  • An example of a precursor for serotonin is 5-hydroxy- tryptophane.
  • a further aspect of the invention is the use of the agent
  • HIV Human Immunodeficiency Virus
  • derivative thereof means any of the physiologically acceptable compounds having similar structure and equivalent function to that of DPAT.
  • physiologically acceptable derivatives of DPAT formulated as depot compositions or sustained release preparations are included.
  • the present invention relates to the use of agents which interact with 5HT receptors. Said interacting has to be adapted so as to result in - or to promote - the restoration of a balanced immune function in the infected individuals.
  • Yet another overall aspect is the use of a serotonin uptake inhibitor as defined herein for the preparation of a medicament for increasing the proliferative capacity of immune cells from individuals infected with HIV or related viruses, and/or an increase in the CD4 T cell count, thereby to provide for an alleviation or treatment of the immune dysfunction related to infection with Human Immunodeficiency Viruses (HIV) or related viruses.
  • HIV Human Immunodeficiency Viruses
  • statin uptake inhibitor means an agent which has been shown to increase the amount of 5HT at the site of or near to the site of a 5HT receptor as defined herein.
  • inhibitors at least par ⁇ tially prevent serotonin from being removed from an environment close to a 5HT receptor or a subtype thereof.
  • Said serotonin uptake mechanism can be described as follows: After release from serotonergic neurons, much of the released serotonin is recaptured by an active reuptake mechanism. It is well known that certain inhibitors may interfere with said mechanism.
  • the agents known to increase the availability of serotonin by inhibiting serotonin uptake may be considered to also promote said restoration of the immune function. Accordingly, said agents promote the wanted physiological response by making more serotonin available at appropriate sites in the body.
  • Citalopram 1-[3-(dimethylamino)propyl]-l-(4-fluo ⁇ rophenyl)-l,3-dihydro-5-isobenzofurancarbonitrile, or a precursor or a derivative thereof.
  • Fluoxetine ( ⁇ )-N-methyl-Y-[4-(trifluoromethyl)- phenoxy]benzenepropanamine, or a precursor or a derivative thereof.
  • Paroxetine trans-(-)-3-[(1,3-benzodioxol-5-yl- oxy)methyl]-4(4-fluorophenyl)piperidine; or a precursor or a derivative thereof.
  • the combinations may be combinations of
  • Said combinations may be formulated so as to provide com ⁇ bined pharmaceutical effects or even synergistic effects.
  • Said combinations may also - or alternatively - be formulated with the purpose of trying to minimize the side effects induced by the individual medicaments.
  • the following regimen may be employed: Regardless of which agents are selected to be used according to the present invention, the use may further comprise the use in combination with a specific antiviral medicament.
  • Said combination may be formulated so as to obtain com ⁇ bined effects or even synergistic effects. Said combination may also - or alternatively - be formulated so as to minimize the side effects induced by the individual medicaments.
  • the present invention provides for a method of alleviation or treatment of the immune dysfunction related to infection with Human Immunodeficiency Viruses (HIV) or related viruses, which method comprises adminis ⁇ tering an effective amount of an agent capable of stimu ⁇ lating a site present on some of the immune cells as defined herein.
  • HIV Human Immunodeficiency Viruses
  • the administering is conducted extracorporally in order to specifically target the action of the agent employed to the cells in question.
  • treatment of the cells may also be performed using extracorporal circulation of the blood using a technique similar to regular plasmapheresis. In this way it is possible to use higher concentrations of the active ingredients.
  • the active ingredient is removed, e.g. by filtration or active binding to specific membranes.
  • Another advantage of such administration is that the total amount of medicament to be administered "internally” may be reduced as compared to systemic administration. "Surplus” of medicament may also be eliminated extracorporally - this also reducing the total amount administered to the patient. DETERMINATION OF DOSAGE FORMS AND AMOUNTS OF THE AGENTS
  • An aspect of the invention is to employ tests on living cells obtained from infected individuals in order to assess the effect of the agents on cellular immunity. Whenever appropriate or necessary, the dosage forms and amount of the agents may be determined according to the procedures described herein.
  • the effect of immunorestorative agents on cells from virus infected individuals, in particular pre-AIDS or AIDS patients may be determined in the following way:
  • the starting material may be blood or other cell contain ⁇ ing body fluids, and also samples containing hemopoietic tissue, e.g. obtained by biopsy/aspiration procedures. Important material is also fluids containing bone marrow cells.
  • Examples of cells to be examined are in particular white blood cells, leucocytes, such as lymphocytes and prede ⁇ cessors thereof, including blast cells and other immature cells representing various stages in the maturation proces starting from stem cells.
  • Measurements based on blood cells or various other cells from healthy donors may be used as standard/reference values.
  • the starting material is peri ⁇ pheral blood
  • the first step is the isolation of a fraction enriched in mononuclear cells.
  • peripheral blood mononuclear cells containing about 70% T lymphocytes are isolated from blood.
  • the method involves a centrifugation step, such as density gradient centrifugation.
  • An important aspect is to perform preliminary experiments designed to determine suitable ranges of cell concentra ⁇ tion in relation to various concentrations of agents. Such experiments provide for reference curves applicable for deciding the optimal ratios between cell amount and agent so as to ensure a suitable biochemical response.
  • a predetermined amount of these cells e.g. 5 000 to 100 000 cells, such as from 25 000 to 75 000 cells, typically approximately 50 000 of these cells are added to each well in a microtitre plate.
  • said wells are designed to each contain a maximal volume of 200 1.
  • a standard "activating" stimulus is added to the cultures.
  • This stimulus may be selected from the following:
  • mitogenic agents e.g. poke weed mitogen (PWM) or phytohaemagglutinin (PHA); non-specific activators of most T cells; or recall antigens, i.e. antigens from microorganisms e.g. tetanus toxoid or Candida albicans.
  • PWM poke weed mitogen
  • PHA phytohaemagglutinin
  • non-specific activators of most T cells i.e. antigens from microorganisms e.g. tetanus toxoid or Candida albicans.
  • a suitable culture medium containing appro- priate nutrients and buffers e.g. RPMI supplemented with 10% fetal calf serum and antibiotics
  • a suitable culture medium containing appro- priate nutrients and buffers e.g. RPMI supplemented with 10% fetal calf serum and antibiotics
  • a known total volume e.g. 200 1.
  • microplate cultures are incubated in an incubator uunnddeerr ssuuiittaabbllee ccoonnddiittiioonnss ((ssuucchh as with 5% C0 « and 100% humidity at 37C) for e.g. 6 days. 3
  • a labelling agent such as H-thymidine (for instance 1 ⁇ Ci) is added to all cultures.
  • cultures are harvested on glass filter plates, transferred to counting vials, and a suitable developer is added (e.g. 5 ml of scintillation fluid).
  • the amount of label incorporated e.g. the amount of 3 H-thymidine incorporated in the DNA of the dividing cells, is measured according to methods known in the art.
  • cAMP concentrations in various cells from infected individuals may also be determined during the testing of agents and during treatment in order to elucidate the possible role of cAMP as an endogenous compound useful for monitoring of various cellular functional states.
  • CD4 T cells and CD8 T cells may be determined by means of flow cytometry. Said cell determinations may be used to ident ⁇ ify individuals susceptible to treatment, and said cell determinations may also be one of the parameters of a system designed to monitor the effect of treatments.
  • the agent is added to the blood or other cell contain- ing body fluids before the step of cell isolation is performed;
  • the agent is added to the cell enriched fraction placed in the microtitre plate prior to the addition of the mitogenic stimulus (e.g. 10 - 30 minutes earlier). Afterwards, the reaction media containing the agent to be tested is kept under suitable conditions including a suitable temperature until the addition of stimulus.
  • a commonly employed temperature is room temperature.
  • agents used according to the present invention should be administered in amounts effective to attain the above mentioned restoration of immune function without inducing unbalanced effects or severe side effects.
  • the concentrations relate to amount of active compound.
  • the agents are used to prepare medicaments containing physiologically acceptable additives known in the art. During test periods and treatment periods careful moni ⁇ toring of side effects should be performed.
  • Administration on a daily basis in a dose of 100-300 mg, e.g. administered as the only single initial treatment which may enable determination of the degree of response.
  • results obtained are used as the basis for the choice of further dosages and for the set-up of an individually adapted regimen.
  • a suitable protocol for a re-treatment or maintenance treatment would be the following:
  • the maximal therapeutic non-toxic dose of buspirone is considered to be approximately 2400 mg per day.
  • e.g. six tablets of 10 mg (total 60 mg) are administered initially as the only single initial treatment which may enable determination of the degree of response.
  • Side effects such as nausea, dizziness, headache, anxiety, are monitored.
  • the medicament "Buspar”. Bristol-Myers Squibb, may be employed.
  • results obtained are used as the basis for the choice of further dosages and for the set-up of an individually adapted regimen.
  • a suitable protocol for a re-treatment or maintenance treatment would be the same or a somewhat higher dosaging - with regular intervals adapted individually.
  • Serotonin is preferably administered i.v., e.g. by infu ⁇ sion to patients admitted to hospitals, or out patients with catheters placed intravenously.
  • serotonin has a systemic effect when administered in a concentration of 15 nM/min/kg. If serotonin is administered in this concentration for 2-4 hours most lymphoid cells in the peripheral blood and possibly also in the tissues have been reached. This treatment must be repeated with regular intervals, e.g. every day. Other preparations of serotonin may be employed, e.g. sustained release preparations for sub ⁇ cutaneous injection of depot preparations.
  • This drug is a specific agonist of the 5HTla receptor.
  • a suitable dosage for this drug would be in the range of 10-1000 mg, e.g. 50 mg, given daily as tablets administered orally 1-3 times daily. Alternatively parenteral administration may be employed.
  • Another approach is to increase the circulating concen ⁇ tration of serotonin by preventing the removal of seroto ⁇ nin produced in the body.
  • Such drugs may be given as tablets on a daily basis.
  • FIG. 1 A first figure.
  • Fig. 1 illustrates the effect of Imigran (sumatriptan) on lymphocytes isolated from blood from HIV seropositive subjects.
  • peripheral blood mononuclear cells were incubated in wells of microtitre plates. Imigran was added in 8 different ten fold dilutions starting with 10 ⁇ g/ml. After 10 minutes the activator poke weed mitogen (PWM) was added. The measurements are expressed in "cpm" (counts per minute) indicating the amount of labelled DNA incorporated into the dividing cells. The results are given as medians of triplicate determinations.
  • PWM poke weed mitogen
  • the first column in the diagram represents cultures to which no agents had been added.
  • the second and third column represents the control cultures to which Imigran - but not PWM - had been added in different concentrations.
  • the fourth column represents cultures to which PWM had been added, and the fifth and sixth column represents cultures to which both PWM and Imigran had been added.
  • Fig. 2 illustrates the effect of Buspar (buspirone) on lymphocytes isolated from blood from HIV seropositive subjects.
  • peripheral blood mononuclear cells were incubated in wells of microtitre plates.
  • Buspar was added in 8 different ten fold dilutions starting with 10 ⁇ g/ml. After 10 minutes the activator poke weed mitogen (PWM) was added.
  • PWM poke weed mitogen
  • the measurements are expressed in "cpm" (counts per minute) indicating the amount of labelled DNA incorporated into the dividing cells.
  • the results are given as medians of triplicate determinations.
  • the first column in the diagram represents cultures to which no agents had been added.
  • the second and third column represents the control cultures to which Buspar - but not PWM - had been added in different concentrations.
  • the fourth column represents cultures to which PWM had been added, and the fifth and sixth column represents cultures to which both PWM and Buspar had been added.
  • Fig. 3 illustrates the effect of serotonin (5HT) on lymphocytes isolated from blood from HIV seropositive subjects.
  • peripheral blood mononuclear cells were incubated in wells of microtitre plates. Serotonin was added in 8 different ten fold dilutions starting with 50 ⁇ g/ml. After 10 minutes the activator poke weed mitogen (PWM) was added.
  • PWM poke weed mitogen
  • the first column in the diagram represents cultures to which no agents had been added.
  • the second and third column represents the control cultures to which serotonin - but not PWM - had been added in different concentra ⁇ tions.
  • the fourth column represents cultures to which PWM had been added, and the fifth and sixth column represents cultures to which both PWM and serotonin had been added.
  • Fig. 3 illustrates the effect of 8-hydroxy-2-(di-n-propyl- amino)tetralin (DPAT) on lymphocytes isolated from blood from HIV seropositive subjects.
  • DPAT 8-hydroxy-2-(di-n-propyl- amino)tetralin
  • peripheral blood mononuclear cells were incubated in wells of microtitre plates.
  • DPAT was added in 8 different ten fold dilutions starting with 50 ⁇ g/ml. After 10 minutes the activator poke weed mitogen (PWM) was added.
  • PWM poke weed mitogen
  • the first column in the diagram represents cultures to which no agents had been added.
  • the second and third column represents the control cultures to which DPAT - but not PWM - had been added in different concentrations.
  • the fourth column represents cultures to which PWM had been added, and the fifth and sixth column represents cultures to which both PWM and DPAT had been added.
  • Samples of peripheral whole blood were obtained from HIV infected individuals. Blood mononuclear cells containing about 70% T lymphocytes were separated from whole blood by centrifugation on "Histopac” (density gradient separ ⁇ ation). 50 000 of these cells were added to each well in a microtitre plate for further cultivation and testing.
  • test agent Imigran (sumatriptan) was added to each well in various concentrations.
  • the employed concentra- tions of Imigran were 8 different ten fold dilutions starting with 10 ⁇ g/ml. After 10 minutes the activator PWM was added. Cultures without addition of PWM but with addition of Imigran were used as controls.
  • RPMI culture medium supplemented with 10% fetal calf serum and antibiotics was added to a final volume of 200 1.
  • the microplate cultures were incubated in an incubator with 5% CO.-, and 100% humidity at 37C for 6 3 days. At day 5, 1 ⁇ Ci H-thymidine was added to all cultures. At day 6, cultures were harvested on glass filter plates and transferred to counting vials. Scintil-
  • Samples of whole blood were obtained from HIV infected individuals.
  • Peripheral blood mononuclear cells containing about 70% T lymphocytes were separated from blood by centrifugation on "Histopac” (density gradient separ- ation). 50 000 of these cells were added to each well in a microtitre plate for further cultivation and testing.
  • test agent Buspar buspiron
  • the test agent Buspar was added to each well in various concentrations.
  • the employed concentrations of Buspar were 8 different ten fold dilutions starting with 10 ⁇ g/ml. After 10 minutes the activator PWM was added. Cultures without addition of PWM but with addition of Buspar were used as controls.
  • RPMI culture medium supplemented with 10% fetal calf serum and antibiotics was added to a final volume of 200 1. Then the microplate cultures were incubated, labelled, and harvested as described in Example
  • Buspar was able to increase/restore the proliferative response of blood lymphocytes obtained from HIV seropositive subjects.
  • Samples of whole blood were obtained from HIV infected individuals.
  • Peripheral blood mononuclear cells containing about 70% T lymphocytes were separated from blood by centrifugation on "Histopac” (density gradient separ ⁇ ation). 50 000 of these cells were added to each well in a microtitre plate for further cultivation and testing.
  • test agent serotonin (5HT) was added to each well in various concentrations.
  • concentrations of serotonin were 8 different ten fold dilutions starting with 50 ⁇ g/ml. After 10 minutes the activator PWM was added. Cultures without addition of PWM but with addition of serotonin were used as controls.
  • RPMI culture medium supplemented with 10% fetal calf serum and antibiotics was added to a final volume of 200 1. Then the microplate cultures were incubated, labelled, and harvested as described in Example
  • Patient 1 from 4900 cpm to 7500
  • Patient 2 from 2000 cpm to 3100
  • Samples of whole blood were obtained from HIV infected individuals.
  • Peripheral blood mononuclear cells containing about 70% T lymphocytes were separated from blood by centrifugation on "Histopac” (density gradient separ ⁇ ation). 50 000 of these cells were added to each well in a microtitre plate for further cultivation and testing.
  • test agent 8-hydroxy-2-(di-n-propylamino)tetralin was added to each well in various concentrations.
  • concentrations of DPAT were 8 different ten fold dilutions starting with 50 ⁇ g/ml.
  • RPMI culture medium supplemented with 10% fetal calf serum and antibiotics was added to a final volume of 200 1. Then the microplate cultures were incubated, labelled, and harvested as described in Example
  • Stone et al. (1993) describe how 5HT is administrated i.v.
  • the sole side effect was an increase in the diastolic blood pressure of a few mm Hg.
  • the procedure of adminis ⁇ tration described by Stone can be followed step by step.
  • the procedures as described herein aim at con ⁇ tinuing the infusion of serotonin over a period of 4 hours.
  • Serotonin is administered according to the following schedule:
  • the administration of serotonin is started by mixing this to the saline i.v. drop using an electronic drop counter.
  • the serotonin solution is 5 mM serotonin dissolved in 0.15 mM saline.
  • the infusion is kept at 8 ml/min using electronic drop counters.
  • the blood serotonin concentration is measured before the start of the study and after 1, 4, and 20 hours.
  • Serotonin infusion is able to affect peripheral T cells and induce a homing.
  • a steady state resulting in enhanced proliferative capacity of peripheral T cells was not achieved during this short experiment, but must be expected with continuous treatment. The strongest effect was achieved in individuals with high CD4 T cell counts.
  • CD4 increase in two patients ending more than 50 CD4 cells higher after 1 week to 1 month.
  • Buspiron is able to affect peripheral T cells and induce a homing to the lymphatic tissues.
  • a steady state resulting in enhanced proliferative capacity of peripheral T cells was not achieved during this short experiment, but an enhancement of the immune system by continuous treatment is expected.
  • Fig. 5 The cell activation. From T cell activation to cell proliferation, the cell is guided by two pathways.
  • the activation pathway which includes the T cell receptor, inositol phospholipid metabolism (PIP2 to IP3 and DAG), calcium influx, and activation of PKC.
  • the inhibitory pathway that balance the other, is the PKA pathway, which only is active in the presence of cAMP.
  • cAMP is generated by the enzyme adenylate cyclase.
  • T lymphocytes from HIV seropositive subjects there is an increased activity of the inhibitory PKA/cAMP pathway. Inhibition of this pathway restores the immune function in T cells from HIV seropositive subjects.
  • Fig. 6 The normal immune system.
  • T cells circulate through the lymph nodes and other lymphatic tissues into the peripheral blood and back. Only about 2% or all lymphocytes are in the peripheral blood. The rest are in the tissues.
  • a T lymphocyte on its passage encounters a macrophage presenting a microbial antigen (virus, fungus etc. ) with the right specificity for the T cell, the cell will start to proliferate.
  • This proliferation generates an army of effector cells that will fight the foreign microbial organism (first generating an expansion of CD4 T helper cells which in turn provide help for B cells and CD8 cytotoxic cells in a cascade).
  • CD4 T helper cells which in turn provide help for B cells and CD8 cytotoxic cells in a cascade.
  • these final steps are blocked because T cells have decreased proliferative capacity and decreased cytotoxic capacity.
  • Fig. 7 Buspiron increases the proliferative response of lymphocytes from HIV seropositive subjects in vitro. Buspiron increases the T cells' capacity to proliferate in vitro experiments as shown above with relation to Fig. 2.
  • the proliferative capacity of the T cells will increase, first in the tissues, because in vivo the immune reactions will not take place in the peripheral blood, and later, when a steady state is reached, an increased proliferative capacity should also be detected in the peripheral blood. Also an increased proliferative capacity may result in a higher number of newly generated CD4 T cells.
  • PBMC peripheral blood nononuclear cells
  • Fig. 8 Buspiron induces a homing of reactive peripheral lymphocytes to the lymphatic tissues within a few hours when given in a single dose of 60 mg perorally.
  • the lymphocyte proliferative capacity to stimulation with PWM was measured at time 0, 1 h, 4 h, 24 h, 1 week and 1 month (data not ready yet) as described above. Because the drop in reactive lymphocytes happens within one hour, a redistribution is evident.
  • Fig. 9 Two or three patients seem to increase in CD4 T cells. In two patients with decreased CD4 T cell numbers an increase of more than 50 was seen after 1 week/1 month. An increase after one week or more indicates a production more than a redistribution. No changes were seen in the patient who had the highest CD4 T cell count from the beginning. Normal level is above 500 CD4 T cells per. ⁇ l.
  • Fig. 10 Two of three patients increase in their CD4/CD8 T cell ratio. In two patients an increase in CD4/CD8 ratio was seen after one week. Normal ratios are above 1.0. References

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Abstract

L'invention porte sur l'emploi d'agents pharmaceutiques interagissant avec les récepteurs de 5HT pour atténuer ou traiter la dysfonction immunitaire liée à l'infection par le VIH ou des virus assoicés tels que ceux rencontrés dans le pré-SIDA ou le SIDA. L'intéraction peut se produire par l'intermédiaire d'un récepteur de cellule immunitaire, p. ex. présent dans les lymphocytes T, et lié structurellement ou fonctionnellement aux récepteurs de 5HT ou à leurs sous-types présents dans les cellules du système nerveux. Les agents préférés sont: le 3-[2-(diméthylamino)éthyl]-n-méthyl-1H-indole-5-méthansulfonamide (sumatriptane), le 8-{4-[4-(2-pyrimidinyl)-1-pipérazinyl]butyl}-8-azaspiro[4,5]décane-7,9-dione (buspirone), le 4,4-diméthyl-1-{4-[4-(2-pyrimidinyl)-1-pipérazinyl]butyl}-2,6-pipéridinedione (gépirone), le 2-{4-[4-(2-pyrimidinyl)-1-pipérazinyl]butyl}-1,2-benzisothiazolin-3(2H)-one 1,1 dioxide (ipsapirone), la 5-hydroxytryptamine (5HT, sérotonine), et la 8-hydroxy-2-(di-N-propylamino)tétraline (DPAT); ou leurs dérivés ou précurseurs.
PCT/DK1995/000285 1994-07-06 1995-07-05 Emploi d'agents pharmaceutiques pour attenuer ou traiter la dysfonction immunitaire liee a l'infection par le vih ou des virus associes WO1996001106A1 (fr)

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AU28804/95A AU2880495A (en) 1994-07-06 1995-07-05 Use of pharmaceutical agents for alleviation or treatment of the immune dysfunction related to infection with human immunodeficiency viruses (hiv) or related viruses

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AT409084B (de) * 2000-07-13 2002-05-27 Ledochowski Maximilian Dr Antimykotisch wirkendes arzneimittel

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992004015A2 (fr) * 1990-09-04 1992-03-19 Miles Inc. REGULATION DE LA PROLIFERATION DE LYMPHOCYTES T VIA UN NOUVEAU RECEPTEUR, LE 5HT1a

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992004015A2 (fr) * 1990-09-04 1992-03-19 Miles Inc. REGULATION DE LA PROLIFERATION DE LYMPHOCYTES T VIA UN NOUVEAU RECEPTEUR, LE 5HT1a

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AT409084B (de) * 2000-07-13 2002-05-27 Ledochowski Maximilian Dr Antimykotisch wirkendes arzneimittel

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