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WO1994015621A1 - Procede d'utilisation de polynucleotides, d'oligonucleotides et de leurs derives pour traiter divers etats pathologiques - Google Patents

Procede d'utilisation de polynucleotides, d'oligonucleotides et de leurs derives pour traiter divers etats pathologiques Download PDF

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Publication number
WO1994015621A1
WO1994015621A1 PCT/US1994/000638 US9400638W WO9415621A1 WO 1994015621 A1 WO1994015621 A1 WO 1994015621A1 US 9400638 W US9400638 W US 9400638W WO 9415621 A1 WO9415621 A1 WO 9415621A1
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Prior art keywords
dose
disease
marker
therapeutic compound
markers
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PCT/US1994/000638
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English (en)
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Arsinur Burcoglu
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Arsinur Burcoglu
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Priority to AU60904/94A priority Critical patent/AU692433B2/en
Priority to EP94907246A priority patent/EP0678029A1/fr
Priority to CA002153778A priority patent/CA2153778C/fr
Publication of WO1994015621A1 publication Critical patent/WO1994015621A1/fr
Priority to US09/754,066 priority patent/US6699985B2/en
Priority to US10/768,089 priority patent/US7049430B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/18—Type of nucleic acid acting by a non-sequence specific mechanism

Definitions

  • the invention relates to a method of using sequence non-specific polydeoxyribonucleotides, oligodeoxyribonucleotides and/or derivatives thereof, such as defibrotide, to treat various human disease states.
  • Defibrotide is a polyanion salt of a deoxyribonucleic acid obtained from mammalian tissue.
  • Defibrotide is a single-stranded polydeoxyribonucleotide with molecular weight of approximately 20 kDa (low molecular weight form) which may be obtained from bovine lung DNA by controlled hydrolysis.
  • Patents related to its manufacture include U.S. Patent 3,770,720 directed to a process for extracting DNA from mammalian tissue, and U.S. Patent 3,899,481 directed to a process for the controlled partial degradation of DNA extracted from animal organs.
  • Defibrotide is noted primarily for its profibrinolytic effects (Pescador et al. , 1985, Thromb. Res. , 30: 1-11).
  • Defibrotide increases the release of tissue-type plasminogen activator (t-PA) and decreases plasminogen activator inhibitor (PAH) activity.
  • t-PA tissue-type plasminogen activator
  • PAH plasminogen activator inhibitor
  • the increase in t-PA is in conjunction with the decrease in PAI1, the latter being the more prominent action.
  • the profibrinolytic activity of defibrotide is likely due to a decrease in PAH levels rather than to an increase in t-PA level (Pogliani et al. , 1987, Farmaci E Therapia IV, (2): 1-5; Ulutin et al. , International Scientific .Symposium on Fib ⁇ nogen, Thrombosis, Coagulation and Fib ⁇ nolysis, August 30 - September 1, 1989, Taipei, Taiwan, ROC).
  • U.S. Patent 3,829,567 is directed to the use of an alkali metal salt of a polynucleotide or an oligonucleotide of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA) as a fibrinolytic agent.
  • U.S. Patent 4,649,134 is directed to a method of treating acute renal insufficiencies accompanied by thrombotic microangiopathy with defibrotide. Such pathologies include hemolytic ure ic syndrome (HUS), collagenopathies ⁇ e.g.
  • HUS hemolytic ure ic syndrome
  • collagenopathies ⁇ e.g.
  • U.S. Patent 4,693,995 is directed to a method of treating acute states of myocardial ischemia and infarction with defibrotide.
  • Defibrotide has been found to be a prostaglandin I 2 (P y secretory agent (Niada et al. , 1982, Pharmacological Res. Comm. , 14:949-957). Defibrotide also induces synthesis of other prostanoid metabolites, such as prostaglandin E 2 (Pg ⁇ ). The increase in secretion of the prostanoid metabolites, in particular Pgl 2 and PgEz, from vascular endothelial cells seems to involve interaction with arachidonic acid metabolites (Costantini et al. , 1989, Eur. J. Int. Med. , 1:115-120).
  • Defibrotide induces an in vivo increase in platelet cyclic adenosine monophosphate (cAMP) levels resulting in deaggregation of platelets and plasma prostanoid levels, as shown in humans (Cizmeci, 1986, Haemostasis, 16 (suppl. l):31-35). Defibrotide does not increase levels of malonylaldehyde, thromboxane A 2 , thromboxane B 2 , ⁇ 2 -antiplasmin, or ⁇ 2 -macroglobulin activities.
  • cAMP platelet cyclic adenosine monophosphate
  • Defibrotide is also known to exhibit antithrombotic actions (Niada et al. , 1981, Thromb. Res. , 23:233-246). Defibrotide has been shown to elevate Protein CA and Protein Cl levels, which affects antithrombotic action. The reported elevations in the levels of Protein CA and Protein Cl are proposed to be via defibrotide' s modulatory effects on the vascular endothelial cell-thrombomodulin levels. At the dose levels utilized thus far, it is devoid of anti-coagulant effects (Coccheri et al. , 1982, Int. J. Clin. Pharm. Reg. , ll(3):227-245), and no clinical applicability as an anti-coagulant agent has been taught heretofore.
  • Defibrotide exhibits a synergistic action with, and potentiates the effect of heparin (Ulutin et al. , International Scientific Symposium on Fibrinogen, Thrombosis, Coagulation and Fibrinolysis, August 30 - September 1, 1989, Taipei, Taiwan, ROC).
  • the synergistic mechanism between defibrotide and heparin is not totally clear.
  • One proposed theory is that defibrotide competitively binds with heparin receptors, promoting prolonged circulation of endogenous heparin.
  • Ulutin et al. reported an increase in anti-Factor Xa activity (Ulutin et al. , International Scientific Symposium on Fibrinogen, Thrombosis, Coagulation and Fibrinolysis, August 30 - September 1, 1989, Taipei, Taiwan, ROC). This effect may add to its antithrombotic action.
  • defibrotide was shown to inhibit endothelin-induced contraction of the vascular smooth muscle (Fareed et al. , 1990, In: Advances in vascular pathology, Elsevier Science Publishers B.V. , pp. 171-177). This implies that factors other than impaired fibrinolysis were being treated by defibrotide, such as defibrotide-induced suppression in the levels of the vasoactive amines secreted from the vascular endothelial cells in response to injury.
  • defibrotide was shown to increase cell number and protein content in the culture supernatant, implying a greater role in translation than in the induction of mitotic activity (Bilsel et al. , 1990, Thromb. Res. , 58:455-460).
  • defibrotide can modulate lipid peroxidation of membrane phospholipids and oxygen radical induced inhibition of the cyclooxygenase pathway, two major mechanisms in the process of vascular endothelial cell injury. Analogous to these are defibrotide-induced inhibition of superoxide generation by neutrophils induced from platelet activating factor (PAF) (Cirillo et al. , 1991 , Haemostasis, 21:98-105). Defibrotide has shown protective effect in mice against pulmonary embolism, analogous to the free radical scavenging enzymes superoxide dismutase and catalase (Niada et al.
  • PAF platelet activating factor
  • Defibrotide-based antithrombotic action in pulmonary embolism may be analogous to the antioxidant effects of cardiovascular drugs.
  • defibrotide acts via modulation of vascular endothelial cells
  • its recently formally adopted pharmaceutical classification as a "polypharmaceutical agent” is uniformly ascribed to defibrotide' s Pgl 2 secretory action.
  • POVD Peripheral obliterative vascular disease
  • Defibrotide is manufactured by CRINOS Farmacobiologica S.p.A., Villa Guardia (Como), Italy, and is currently marketed only in Italy.
  • Defibrotide obtained from CRINOS for investigational purposes and clinical trials in the United States, is available in ampules containing 200 milligrams for parenteral administration and in tablets containing 400 milligrams for oral administration.
  • defibrotide as a nucleic acid, is not toxic, mutagenic or harmful to fetal or embryonic development
  • maximum dosages of defibrotide administered to humans have been limited to either a body weight- dependent dose of 10 - 30 milligrams per kilogram, to an empirically established dose of 5.6 grams per day, intravenously, or a fixed dose of 800 milligrams per day, intravenously or by mouth.
  • Coccoheri and Biagi (Cardiovascular Drug Reviews, 1991, 9(2): 172-196) report the highest dose of 2.4 to 5.6 grams daily which was given for three days only. These doses, which were based on previous animal studies, were administered empirically. Defibrotide was administered in conjunction with conventional therapy and produced modest advantage.
  • the pharmacodynamic effect obtained with oral administration of defibrotide is approximately one-half that of parenteral dosing (Fareed et al. , 1988, Seminars in Thrombosis and Hemostatis - Supplement, 14:27-37).
  • the maximum dosages of oral defibrotide reported was 1600 milligrams per day. Even at these dosages, clinical improvement was much slower with the oral form of defibrotide than with the parenteral form.
  • the pro-fibrinolytic effect in healthy volunteers was confirmed by the administration of a single 400 - 600 mg dose with shortening only in euglobulin lysis time.
  • POVD peripheral obstructive vascular disease
  • a repeat study of POVD patients confirm significant defibrotide induced declines in PAH levels (Coccoheri and Biagi, 1991 , Cardiovascular Drug Reviews, 9(2): 172-196).
  • the fibrinolytic effects in healthy volunteers were in general not reproducible, but in patients fibrinolytic effects were reproducible.
  • defibrotide has been shown in the art to have antithrombotic, thrombolytic, cytotropic, nephroprotective, platelet deaggregatory and anti-shock properties. These properties have been ascribed to its capacity to release Pgl 2 or its stable analogues from vascular endothelium. Defibrotide has also been shown to increase t-PA, decrease PAH, increase protein S and C levels, increase ATIII (unconfirmed reports), increase platelet cAMP levels and, more recently, decrease endothelin-I levels and increase EDRF (endothelium derived relaxing factor) in in vitro models.
  • EDRF endothelium derived relaxing factor
  • alkali salts of deoxyribonucleic acid such as defibrotide
  • defibrotide may be useful in the treatment of viral infections, including HIV infection.
  • Another object of the invention is directed to a method of gene therapy for treating viral infections, such as the human immunodeficiency virus (HIV) without employing retrovirus, adenovirus or other viral vector.
  • viral infections such as the human immunodeficiency virus (HIV)
  • retrovirus such as the human immunodeficiency virus (HIV)
  • retrovirus such as the human immunodeficiency virus (HIV)
  • this invention provides a method of treatment comprising the steps of:
  • a therapeutic compound comprising one or more sequence non-specific nucleic acids selected from the group consisting of polydeoxyribonucleotides, oligodeoxyribonucleotides and derivatives thereof,
  • step (d) administering the therapeutic compound at a dose level incrementally higher than the previous dose, (e) repeating step (d) each time the intensity of the repair marker increases following an incrementally higher dose,
  • step (f) repeating steps (d) and (e) until the intensity of the repair marker in step (c) no longer increases
  • step (g) administering the therapeutic compound at the highest dose level attained in step (f) until the intensity of the repair marker returns to the normal condition
  • the patient is monitored weekly for three or more weeks. If relapse occurs, as indicated by a deviation of one or more disease and/or repair markers from the normal level, therapy is reinitiated at the highest dose level of the prior course of therapy until normalization is again reached.
  • the method of treatment comprises the steps of:
  • a therapeutic compound comprising one or more sequence non-specific nucleic acids selected from the group consisting of polydeoxyribonucleotides, oligodeoxyribonucleotides and derivatives thereof, wherein the dose of the therapeutic compound is at a level which raises a universal marker to at least five times its normal level, the universal marker being a constitutively expressed molecule which is transcriptionally activated by the therapeutic compound in all disease status, and
  • the treatment of viral diseases involves the administration of the therapeutic agent in the same marker dependent manner as indicated above.
  • levels of those molecules misproduced by the normal cell but also molecules produced by infected cells are assayed, e.g. , viral encodal proteins.
  • Cell cultures and other conventional tests are performed to detect the presence and activity level of virus and viral related components.
  • the method includes titration of the dose to concurrently reestablish normal cell functions and neutralize and eliminate infectious viral particles.
  • the invention provides a method of gene therapy which comprises administration of combinations of (1) sequence specific nucleic acids corresponding specifically to selected parts of the viral genome or transcriptional factors and (2) sequence non-specific nucleotides, such as defibrotide and analogues thereof, in incrementally higher dose levels in a marker dependent manner.
  • the method of treating HIV infection or other viral disease comprises the steps of administering an effective dose of one or more sequence non-specific nucleic acids selected from the group consisting of polynucleotides, oligonucleotides and derivatives thereof.
  • the method is practiced in a marker dependent manner comprising:
  • a therapeutic compound comprising one or more sequence non-specific nucleic acids selected from the group consisting of polynucleotides, oligonucleotides and derivatives thereof, optionally in combination with one or more sequence specific nucleic acids,
  • step (e) repeating step (d) each time the intensity of the repair marker increases following an incrementally higher dose
  • step (f) repeating steps (d) and (e) until the intensity of the repair marker in step (c) no longer increases
  • the invention also provides a method of treating HIV infection in which HIV is not expressed and wherein the concentration of at least one immunological molecule is elevated above the normal level.
  • Immunological molecules include CD4, CD25, IL-1 , IL-3, IL-4, IL-6, TNF and sIL2R. The method comprises:
  • a therapeutic compound comprising one or more nucleic acids selected from the group consisting of polynucleotides, oligonucleotides, and derivatives thereof, wherein the effective amount is the amount which causes a universal marker to rise at least five times its normal level, the universal marker being the concentration of a constitutively expressed molecule which is transcriptionally activated by the therapeutic compound in all disease states, and
  • the invention is directed to the universal treatment of disease states characterized by the compromise and/or absence of normal cell functions.
  • Treatment with therapeutic nucleic acid compounds, such as defibrotide is universal in its application with respect to any disease condition wherein the diseased cell preserves the biological capacity for the physiologically predefined events of the recovery process.
  • the decisive factor in the success of this therapeutic approach is not only the pharmaceutical agent itself, but how it is utilized.
  • Treatment in accordance with the invention results in the activation of the inherent reparative cellular activity which will lead to the reinstitution of normal cellular functions. Revival or restoration of normal cellular functions is, by definition, a state of cure.
  • Figure 1 is a diagram schematically illustrating the method of the invention.
  • Figure 2 is a graph showing normal peripheral blood cells labelled with 0, 10, 20 and 40 ⁇ g defibrotide-biotin combination.
  • Figure 3 shows the data of Figure 18 on a linear scale.
  • Figure 4 is a graph showing the lymphocyte uptake of defibrotide without biotin and labelled with Cy5.18.
  • Figure 5 is a graph showing the monocyte uptake of defibrotide without biotin and labelled with Cy5.18.
  • Figure 6 is a graph showing the granulocyte uptake of defibrotide without biotin and labelled with Cy5.18.
  • Figure 7 is a graph showing the percent expression of HIV viral proteins remaining when blood lymphocytes of an HIV infected individual were exposed to various doses of defibrotide with and without Con-A stimulation.
  • Figure 8 is a graph showing the laboratory response expressed in terms of mean linear fluorescence intensity of the peripheral blood mononuclear cells of an HIV infected individual, the cells being subjected in vitro to varying levels of defibrotide using a cell culture assay technique with and without Con-A stimulation.
  • This invention provides a method for the therapeutic multiphase application of therapeutic compounds which are polydeoxyribonucleotides, oligodeoxyribo ⁇ nucleotides, and/or derivatives thereof, including but not restricted to defibrotide, in the treatment of disease states, including HIV. It has now been discovered that therapeutic nucleic acids such as defibrotide can be used in a manner not heretofore employed in the art to manage vascular, cardiac, renal, viral, autoimmune, inflammatory, and other disease states in which the reparative cellular mechanisms and modulation of cellular genomes are induced.
  • Opportunistic secondary infections include herpes, cryptococcial diarrhea, scleroderma, arthralgia and tuberculosis.
  • HIV infection is characterized by a progressive decline in immune system function, suppressing the infected host's ability to overcome other, secondary infections. No cure has been found for HIV infection.
  • the pathogenetic process in HIV infection is never unidimensional but, rather, extremely complex and multifactorial. The pathogenic progression may be only tangentially related to the direct infection of a given target cell. Fauci, 1993, Science 262: 1011-1018. Death is inevitable, usually from an overwhelming secondary infection and/or HIV related neoplasm.
  • TB tuberculosis
  • TB tuberculosis
  • TB remains the leading cause of death in the world from a single infectious agent. While the risk of TB infection in the United States had been declining over the past century, the incidence of TB is now increasing. The increased number of TB cases is attributable in part to the result of HIV and.TB co-infection. Science, 1992, 257:1055-1064. AIDS patients often exhibit TB as a presenting opportunistic infection. TB occurring with HIV infection is a serious and growing public health problem. The Lancet, 1993, 342:268-272.
  • sequence non-specific DNA decreases IL-1, IL-2, TNF- ⁇ and IL-6, all of which are known inducers of HIV proliferation.
  • Administration of such an agent also increases total lymphocyte and T-lymphocyte counts, CD4 counts, and CD8 counts, as well as increasing cGMP/cAMP ratios to proportions seen in normal laboratory controls. An anabolic effect has also been observed.
  • the deoxyribonucleic acid is administered in a marker dependent manner.
  • a "marker” is an observable characteristics of the patient which may be observed directly by the clinician or determined by diagnostic procedures. The state of individual markers is correlatable with the status of disease or repair processes in the patient. Dosing of the therapeutic nucleic acids according to the method of this invention is based on changes in the status of these markers as taught herein.
  • Treatment of a disease state in accordance with this invention involves the administration of the therapeutic nucleic acids or derivative at a daily dose level sufficient to increase the intensity, determined as concentration or clinical observation, of a marker of cellular repair processes ("repair marker") to a plateau level (i.e., where the intensity of the marker is not changed by continued administration of the therapeutic compound).
  • This daily dose level is the "maximum efficacious dose" for the particular disease and repair marker.
  • Administration of the therapeutic compound is continued at the same dose level until the repair marker stabilizes by returning to the normal level.
  • the daily dose level of the therapeutic compound is increased. At least one other repair marker will increase in intensity, and the daily dose level is increased until the intensity of the new marker reaches a plateau level. Administration of the therapeutic compound is continued at this new maximum efficacious level until the respective repair marker stabilizes at the normal level.
  • defibrotide and other nucleic acids are cell modulators.
  • nucleic acids such as defibrotide and derivatives thereof have now been recognized by the inventor as the only class of agents committed to restoration of normal cell function. In itself, this effect of the therapeutic compound is incompatible (in a state of cure) with an "over-functional" state, i.e. any complications caused by the therapy.
  • defibrotide has now been recognized to be a member of a new class of pharmaceutical agents that will act via activation of respective cell functions with varying polypharmacological actions that are disease specific, induced by varying dose levels of defibrotide.
  • this class of agents have to be administered at escalating dose levels until and when maximum clinical benefit is observed.
  • Pleiotropism as used herein, is defined as the tissue and cell specific expression of cellular genomes via phosphorylation of the enzymes, via reinstitution of cell-surface receptor ligand interactions, protein kinase A and protein kinase C pathways as well as second messengers of cAMP, Ca 2+ , diacylglycerol (DAG), and 1 - 3 phosphoinositol which may under- or over- regulate the genomic transcriptional/translational activities.
  • DAG diacylglycerol
  • maximum efficacious dose is defined herein as the daily dose rate, that will elicit, in nearly 100% of treated patients, the reversal of the respective disease markers to the uniformly normal level, and establishment of normal cellular markers.
  • the maximum efficacious dose is usually expressed as amount of therapeutic compound administered per kilogram body weight per day (DKGD).
  • DKGD body weight per day
  • maximum therapeutic dose is defined herein as the total cumulative dose (the daily dose summed over the duration of administration) that will elicit in nearly 100% of treated patients the irreversible and complete normalization of the respective disease markers and resumption of normal cellular functions, i.e. , the state of cure.
  • minimum efficacious dose is used herein to refer to the dose used in the heretofore universal practiced method of administering a pharmaceutical agent.
  • the minimum efficacious dose is the dose (daily dose or steady state level) that will elicit a particular pharmaceutical action in a certain percentage of patients, without inducing the pleiotropism of the whole repair process.
  • Pharmacological agents have heretofore been administered at set dose levels (i.e. , the "minimum efficacious dose") to treat the gross pathology and discontinued when complete or partial remission of the gross pathology was achieved.
  • the object of the invention is to achieve complete remission of each disease stage.
  • Treatment according to this invention begins at the gross pathology stage which has one or more associated markers. Normalization or improvement of those markers indicate that the treatment is beneficial.
  • treatment is stopped after a certain period of time at a set dose level, usually corresponding to complete or partial remission of the disease indicators of gross pathology.
  • such a remission is not the event which causes discontinuation of therapy in accordance with this invention.
  • maximum tolerable dose is defined as the highest daily dose that can be administered without any complications, e.g. , no bleeding complications or thrombopathy, etc. This in fact has been the sole and primary side effect of the high-molecular weight nucleic acid (defibrotide) utilized in the studies reported herein, i.e., the antithrombotic effect inducing bleeding complications at 300 mg/kg/day dose or above. If the maximum efficacious dose should be higher than the maximum tolerable dose, chemical modification of the nucleotide for more efficacious transmembrane transport and cellular entry would be necessary.
  • the inventor has determined that the therapeutic nucleic acid will not indefinitely increase transcriptional activity with increasing doses.
  • transcriptional activity will shut off when the repair molecules are no longer needed, i.e. , when no more "injury signal" is transmitted via stimulation of adenylate cyclase, second messengers, etc.
  • no matter how high the dose range in the normal individual may be there is no induction of transcriptional activity (as indicated by, e.g. , elevation in the von Willebrandt antigen (vWAg) levels). This supports the fact that no complications are seen with therapy using nucleotides which modulate cellular repair mechanisms for a therapeutic effect.
  • vWAg von Willebrandt antigen
  • tissue plasminogen activator antigen AgTPA
  • AgTPA tissue plasminogen activator antigen
  • the disease states treatable by the method of this invention are those characterized by injury-based alteration in the production, expression or activity of compounds whose production, expression or activity is regulated by the cell at least in part through cell surface receptors such as Adenosine A, and A 2 , collagen, thrombin, epinephrin and norepinephrine receptors, through the protein kinase A or protein kinase C pathways, or by protein factors whose phosphorylation affects genomic translation and transcription.
  • the disease states treated by the method of this invention may have an abnormal genomic component.
  • Diseases with an abnormal genomic component are characterized in that one factor in the disease process is the expression of an abnormal DNA sequence related to the disease.
  • Such sequences may include viral genomes, transformed genes such as activated oncogenes, or heritable genetic defects associated with genetic diseases.
  • the method of this invention will result in the return of normal cellular function in the diseases with an abnormal genomic component, a complete cure as defined herein will not remove the abnormal DNA sequence solely by administration of the therapeutic nucleotide compound.
  • the preferred method for treatment of disease states with an abnormal genomic component contemplates adding to administration of the therapeutic nucleotide compound a coupled treatment directed at eliminating disease-related DNA sequences, for instance by removing the genomic defect.
  • Diseases with no abnormal genomic component are those diseases within the broad definition of diseases treatable by the method which do not involve expression of an abnormal DNA sequence. Complete cure can be achieved in diseases with no abnormal genomic component solely by administration of the therapeutic compound as described herein. It is within the contemplation of this invention to administer the therapeutic compound either alone or contemporaneously with other therapy directed to the particular disease process. It should be recognized that the other therapies are intended to force non-normal cellular function, and therefore they are likely to extend the time required for therapy with the therapeutic nucleotide compounds to achieve a return to normal cellular function.
  • Disease states involving cell malfunction of, for example, vascular endothelial cells, the cellular components of parenchymal tissue damage involving the kidney, heart, liver, etc., any disease state involving malfunctioning of the blood vessels, circulating blood cells, disease states of the central, peripheral and autonomic nervous systems and viral diseases involving reversible genetic modulation of the immune cells not excluding infestation of the tissue cells of the involved organs may be treated with defibrotide and its derivatives.
  • diseases include, but are not limited to POVD, DVT, DIC, thrombotic microangiopathies (e.g.
  • Kawasaki's disease polyarteritis nodosa, midline granuloma
  • scleroderma Raynaud's disease
  • inflammatory (autoimmune or otherwise) myositis inflammatory dermatitis
  • inflammatory symptoms of systemic lupus erythematosus (SLE) and other collagen vascular diseases Sjogren's disease
  • IBD inflammatory bowel disease
  • thyroiditis stasis ulcers
  • sickle cell disease pulmonary fibrosis
  • hypersensitivity pneumonitis burns
  • peripheral obstructive arterial disease septic shock
  • eclampsia graft vs.
  • nucleotide defibrotide and its derivatives may also be therapeutically beneficial in treating diseases of the nervous system.
  • the methodology described herein has universal application within the scope of disease states characterized by the absence or inadequacy of one or more of those cell functions which are normally regulated through the cell surface receptors listed above, through the protein kinase A or C pathways or by protein factors whose phosphorylation affects gene transcription, so long as the abnormalities in these cell functions are yet still reversible.
  • the methodology is also applicable to the disease states characterized by acquired or genetic dismodulation, and/or transformation. Revival, institution or reinstitution of the normal state of those functions is, by definition, a state of cure.
  • Revival of the normal cell functions can occur where the diseased cell preserves the biological capacity for the physiologically predefined events of the cellular repair functions of the recovery process, if those events are pharmacologically induced by the correct use of the therapeutic nucleic acids.
  • Complete cure is the therapeutic objective.
  • the decisive factor in the success of this therapeutic approach is not only the pharmaceutical agent, but how it is utilized. If the biological capacity for regaining normalcy is there, therapeutic failure is eliminated.
  • This biologically predetermined potential for cure is reproducibly and predictably obtainable, however, only by the correctly determined iatrogenetically controlled dose levels, and duration of therapy. Incorrect dose administration leads to the therapeutically missed event of complete cure. Complete cure, however, is not possible if necessary dose levels cannot be attained without complications such as bleeding or thrombopathy.
  • the invention uses polypharmacological medical therapy components which are defined by the predetermined biological events of the cellular repair process.
  • the invention defines a previously unidentified class of drugs whose pharmaceutical actions modulate selectively and most effectively the injury specific cellular functions.
  • This class of drugs include defibrotide as well as other nucleic acid derivatives.
  • the pharmaceutical action is guided by the intrinsic biological material of the cell matching specifically and completely the cellular events of the recovery to the exact components of the injury.
  • the therapeutic compound contemplated for treatment by the method of this invention is an oligomer or a polymer of deoxyribonucleotides or ribonucleotides, or a derivative thereof.
  • the compound may be native or chemically synthesized, or a fragment of a native polynucleotide.
  • the compound has at least three nucleotide residues, and may have up to about 250 residues.
  • the nucleotide compound will have from about 15 to about 200 residues, more preferably from about 20 to about 150 residues, most preferably from about 50 to about 75 residues.
  • the sequence of the nucleotide residues in the polymer is not critical, and may include sense, anti-sense, non-sense or missense sequences.
  • a therapeutic composition may contain polynucleotide molecules with varying numbers of residues within the range described above.
  • the skilled worker will be able to select an appropriate length (degree of polymerization) based on the ability of the compound to penetrate the cell and on the ability of the compound to cause a change in the level of various repair markers in accordance with the method of this invention.
  • the nucleic acid compound will preferably be relatively resistant to ecto- and endonucleases.
  • the 3' OH of the terminal residue of the therapeutic compound according to this invention may be phosphorylated or not, and the compound will still function without the need for intracellular phosphorylation.
  • the therapeutic compound according to this invention is a polyanion, and the negative charge is balanced by counter ions.
  • the counter ions may be alkali metal ions or alkaline earth ions, biologic amines or other suitable counter ions which do not interfere with treatment according to the method of this invention.
  • at least some of the counter ions are zinc ions.
  • Commercially available defibrotide contains about 150 - 350 ⁇ g of zinc and about 25 - 45 ⁇ g of iodine per gram of iodine.
  • Zinc may be present in the nucleotide compound at a ratio of from 2 - 20 zinc atoms per phosphate group or iodine atom. Zinc containing compounds may be co-administered with the defibrotide to reach any desired ratio.
  • Defibrotide is chemically characterized as a polyanionic derivative of deoxyribonucleic acid. While further discussion will be directed to the use of defibrotide as the prototype drug, it is to be understood that other nucleic acids and derivatives are included in the use thereof. Derivatives included within the scope of the invention include nucleic acid, i.e.
  • DNA and/or RNA which is conjugated with poly(L-lysine) or which is modified by, for example, the addition of amino acids such as lysine, histidine and arginine, the addition of optimum concentrations of folate and/or biotin, the addition of the optimum ratios of metals and ions including zinc, manganese and iodine, by the addition of 5'-polyalkyl moieties, cholesterol, vitamin E, l-2-di-O-hexadecyl-3-glyceryland other lipophilic moieties and/or modified by the replacement of phosphodiester bonds with phosphothiotate bonds, and/or modified nucleotide sequences of the prototype nucleic acid, defibrotide.
  • amino acids such as lysine, histidine and arginine
  • the addition of optimum concentrations of folate and/or biotin the addition of the optimum ratios of metals and ions including zinc, manganese and iodine
  • Verifications of the relative therapeutic unimportance of defibrotide' s molecular structure in directly producing its various polypharmacological actions was provided by various means. These included non-uniformity of its various actions in different disease states; differences between the types of various "disease markers” reproducibly and effectively treated by defibrotide; lack of uniformity in the states of responsiveness of the same "disease markers” to the same "dose levels” at different stages of the disease process confirming greater responsiveness of the disease markers to lower dose levels with higher disease activity states and vice versa. This supports the concept that defibrotide' s polypharmacological profile is a function of the injury state. The efficacy and the emergence of selected polypharmacological spectrum of actions, in turn, is a direct function of adequately administered dose levels.
  • derivatives of nucleic acids act upon malfunctioning cells to restore normal cell function.
  • certain cell malfunctions may be caused by the disease while other cell functions are normal. Only the malfunctions of the cell will be affected by treatment with defibrotide. Normal functions will not be hindered and/or increased during treatment.
  • the claimed method involves the use of a "marker dependent dose assessment" methodology for determining the therapeutically most efficacious use of the respective pharmaceutical agents.
  • the use of incremental marker stratification reflects the concept that "maximum efficacious dose” is redefined through the different stages of treatment, each time adjusted to the respective specific marker most representative of the respective pathogenic/clinical picture of the disease state. Treatment at respectively higher doses corresponding to the progressively lower disease activity levels are continued until a state of total cure is reached.
  • Intrinsic to the claimed method is the total elimination of empirically assessed doses or constant therapy doses, arrived at by the universal pharmaceutical principal of "minimum efficacious dose" for a class of drugs, which, until the present time, has been the standard for the definition of the "effective therapeutic dose.”
  • the respective doses thereof are defined to elicit a response corresponding to different disease functions of the treated cell and revival of the respective disease parameters, in a stratified fashion.
  • the method of treating various diseases uses specific clinical and laboratory markers to assess dosages to be administered.
  • the markers vary from gross clinical observations of pathology to the progressively subclinical yet valid detection of certain laboratory levels associated with a particular disease.
  • the preferred markers are the clinical parameters as well as the molecular products produced, or inhibited, present or absent when cellular events associated with a particular disease occur.
  • Certain laboratory assays are used to assure that the dosages are safe for the patient being treated.
  • these may include prothrombin time, activated partial prothromboplastin time, thrombin time, reptilase time, bleeding time, platelet function assays, and coagulation factors.
  • a second set of laboratory assays i.e. , "disease markers" are utilized to indicate the efficacy of the doses.
  • Disease markers are utilized to assess clinical adequacy of dose escalation and duration of therapy.
  • normal cellular markers are molecules of normal cellular function. They are tissue and cell specific and may share common pathways of second messengers or signal transduction pathways and normal cellular genomes. At the genome level, normal cell markers are genes that are constitutively expressed, transcribed, translated and transduced. Establishing dose and duration of therapy based on second messengers, signal transduction pathways and induction of genomic transcription is a novel modality of administering a pharmaceutical agent.
  • disease markers are markers which are induced and defined by the type of disease process. Disease markers are clinical or laboratory parameters that deviate from normalcy. A disease marker may be absent or present, decreased or increased. At the genome level, disease markers are genomes of genetic dismodulation (e.g. viral genome, transcribed oncogenes, mistranscribed genomes); nontranscribed genomes (e.g. , familial/genetically absent genomes, under-regulated/ suppressed genomes), and/or over-expressed, not appropriately shut off transcriptions of genomes (e.g. activated repair molecules, second messengers and molecules of signal transduction pathways). Preferred disease markers for specific disease states are described herein.
  • Disease markers are observable characteristics of the organism whose status in a disease state differs from the status in the normal (non-disease) state. Such characteristics and their association with their respective disease states are well known to the skilled practitioner. In the practice of the method of this invention, it is contemplated that the practitioner will monitor the status of multiple disease markers related to the disease being treated, either simultaneously or sequentially.
  • the disease markers include both clinical markers, which are observed directly by clinician, and laboratory markers, which represent quantitative values determined by support staff. These characteristics include, but are not limited to, the concentration of compounds whose production or expression is affected by injury-based alteration of cell surface receptors such as Adenosine A, and A 2 , collagen, thrombin, epinephrin and norepinephrine receptors, of protein kinase A or protein kinase C pathways, or of protein factors whose phosphorylation affects genomic translation and transcription, or hybridization of genomic enhancers/inhibitors infusion or excess enhancers, infusion of excess genomes to deplete viral/cellular transactivation transcription factors, etc. where the concentration in the disease state differs from the concentration in the normal state.
  • the normal state concentration of these markers will be known to the skilled practitioner, and usually represents a range of concentration values determined by measurement of the concentration of the compound in a large number of individuals who are not in a disease state, by the respective laboratories).
  • Repair markers are compounds that participate in the regulatory pathways which include protein kinase A or protein kinase C.
  • Adenylate cyclase is known to be activated by G-proteins (see Ross, 1992, Current Biology, 2(10):517-519, the disclosure of which is incorporated herein by reference) with eventual production of cAMP and cAMP-dependent activation of protein kinase A, leading to phosphorylation of the respective transcription factors, until 100% of the cell membrane receptors are taken up by the ligands.
  • these receptors are 0-adrenergic receptors, collagen receptors, adenosine Aj/A 2 receptors, ADP receptors, thrombin receptors, collagen receptors, etc).
  • a parallel pathway operates through activation of protein kinase C, in response to intracellular calcium ion level, inositol triphosphate and diacylglycerol, responsive to ligand binding to another set of receptors and similarly controlling transcription/translation of respective proteins.
  • repair markers are molecules in the pathways of the respective cellular repair processes defined by the type of injury. Repair markers are transcribed or shut off genes, second messengers and/or molecules of the signal transduction pathways that may be increased, decreased, or absent in response to cellular injury.
  • the term “repair marker” may refer to the compound or its concentration or the measurement value of an assay associated with the concentration of the compound.
  • the level of a repair marker may deviate from the level present in the cell during normal function, and when it does so deviate, cellular repair processes are activated. This deviation may be positive or negative, depending on the disease state and the precise state of cellular repair currently in progress. As discussed herein, the "intensity" of the repair marker will refer to the degree of deviation from the level during normal cellular function, without regard to whether the deviation is positive or negative.
  • the use of repair markers in establishing dose and duration of therapy is a novel mode of administering a pharmaceutical agent.
  • a “universal marker” is a constitutively expressed molecule transcriptionally activated by the respective nucleic acid universally in all disease states for which the nucleic acid is specific.
  • “Universal markers” are specific for each nucleic acid employed. While the universal marker is the only molecule that is not injury specific and has no therapeutic value, it is expressive of the event and duration of the ongoing repair process. Transcriptional activation gets shut off with the establishment of the state of cure. As such, the universal marker does not get modulated unless there is a disease state and the respective nucleic acid has therapeutic specificity.
  • the universal marker carries a direct quantitative relationship to the daily per kilogram body weight dose (DKGD) of the nucleic acid employed.
  • the universal marker defined for the prototype nucleic acid (defibrotide) is vWAg.
  • Other "housekeeping genes” related to particular nucleic acids can be selected as per the target cell involved from the respective "housekeeping genes.”
  • Clinical and clinical laboratory markers may be determined through blood tests, urine tests, clinical observation or identification of blood clots by any of several conventional techniques, as well as the more novel techniques of determining genomic transcriptional and translational activity by DNA finger printing, PCR and the like. To evaluate the markers, the laboratory analyses measure levels of certain proteins, lymphokines, enzymes and relevant molecules. Clinical markers include blood pressure, visible tissue damage, signs of inflammation, ecchymoses, and the like. Clinical markers vary from one disease to another. Moreover, many diseases progress through several clinical stages during the process of recovery. The clinical markers of one stage of a disease are frequently different from the clinical markers in other stages of the disease, befitting different stages of the pathogenic picture.
  • markers relevant to the particular disease, stage of that disease, and as baseline for dose escalation must first be identified. Any observable characteristic generally accepted by the skilled practitioner as being associated with a specific disease state may be employed as a clinical marker. See, e.g. , Harrison's PRINCIPLES OF INTERNAL MEDICINE, 10th Edition, Petersdorf et al. Eds., McGraw Hill.
  • the normal state concentration of these markers will be known to the skilled practitioner, and usually represents a range of concentration values determined by measurement of the concentration of the compound in a large number of individuals who are not in a disease state, by the respective laboratory.
  • APT T Activated partial prothromboplastin time
  • ATR Activated partial prothromboplastin time
  • TRR Thrombin time
  • Reptilase time Reptilase time
  • dRVV/dRVV Mix Reptilase time
  • Serum prothrombin time (SER PT); Bleed time;
  • Panel 2 Repair Marker (Signal Transduction Pathway Panel (Intracellular in the target cell, such as platelets, peripheral blood mononuclear cells)
  • Cyclic AMP endogenous and PHA stimulated
  • assay is to be run in all the disease states along with the coagulation panel, along with vWAg (preferably messenger RNA of pro vWAg by PCR);
  • cGMP endogenous and PHA stimulated
  • GTP/GDP (7-quinosine triphosphate/ ⁇ -glutanyl diphosphate);
  • RpAI small G-protein
  • Gs 1 Alpha/Gi 1 Alpha Activated G-protein/Inactivated G-protein ratio); oxygen radicals;
  • FAD/FADH 2 ratio intracellular Ca 2+ (endogenous, Interleukin 2 stimulated, PHA stimulated); intracellular 1,2 diacylglycerol (endogenous, IL-2 stimulated, PHA stimulated); intracellular inositol triphosphate (IP 3 ) (endogenous, IL-2 stimulated, PHA stimulated); adenylyl cyclase; protein kinase A; protein kinase C;
  • Panel 5 Pro-platelet Panel (for "Antiplatelet Therapy” with Concurrent
  • Panel 6 Anti-inflammatory Panel (Rheumatic, Autoimmune, Burns,
  • IL-6 soluble Interleukin 2 receptor
  • sIL2R soluble Interleukin 2 receptor
  • ⁇ lFN ⁇ -interferon
  • TNF TNF
  • ⁇ -antichy motripsin haptoglobin
  • fibrinogen albumin
  • lactoferrin metallothionein (hepatocyte); catalase (liver enzyme); fatty acid synthetase; sialyltransferase; glycosyltransferase;
  • SAP serum amyloid protein
  • Panel 7 Anti-ischemic/Cytotropic/Lipid Peroxydation Panel
  • NADP/NADPH FAD/FADH 2
  • O 2 -radicals intracellular glutathione
  • RBC-2,3 DPG whole blood viscosity
  • plasma viscosity intracellular ADP.
  • IL-1 whole blood viscosity
  • plasma viscosity intracellular ADP.
  • Panel 9 Vascular Endothelial Cell Injury. Glomerular Tubular Injury.
  • IL-l ⁇ IL-l ⁇
  • EDRF endothelial cell relaxing factor
  • PB/lymphocytes cell differentiation marker
  • creatinine creatinine clearance.
  • Genomic Disease Panel (Lymphoma/Castleman's Disease (Translocation of c-myc Oncogene), Auto-immune Disease (Rheumatoid Arthritis), Chronic Fatigue Syndrome, HIV induced B-cell Differentiation, Atrial Myeloma)
  • IL-6 protein Kinase A type II catalytic subunit; protein Kinase A type II regulatory subunit; 1 , 2-diacylglycerol ; phosphatidyl inositol bi-phosphate; inositol tri-phosphate; intracellular Ca 2+ ; protein Kinase C; phospholipase C; cGMP; cGMP phosphodiesterase;
  • One critical marker is chosen at each respective stage of the repair process and the maximum efficacious dose for that marker established. Administration of that dose induces correction of other stage-specific markers not necessarily identified or aimed at during therapy (i.e. , "stage specific pleiotropism"). Following normalization of the first chosen marker, a second marker which continues to deviate from the- normal condition is chosen. The dose that normalizes the second marker (i.e. , the higher dose) is likely to further improve the first marker incrementally.
  • the in ⁇ efficiency score can thereafter be used along with the maximum highest levels of the last day to assess how long therapy should be continued to complete the repair process, i.e. , when the maximum efficacious dose is continued when m-efficiency score is less than 1.0, the nucleotide no longer exerts any further therapeutic effect.
  • maximum therapeutic dose "i.e. , the time slot of the total administered dose beyond which further repair of the selected marker would not take place at that particular dose level.
  • FIG. 1 A preferred embodiment of the treatment method applicable to all disease states treated according to this invention is diagramed in Figure 1.
  • An initial laboratory test panel (box 1) is first run which would consist of the respective set of “disease markers” and the universal panel of "repair markers” consisting of signal transduction/second messenger panel molecules. Additionally certain laboratory assays are used to assure that the dosages are safe for the patient being treated. For defibrotide these may include prothrombin time, activated partial prothromboplastin time, thrombin time, reptilase time, bleeding time, platelet function assays and coagulation factors (see baseline coagulation panel). "Disease markers" are utilized to indicate the overall therapeutic efficacy of the doses.
  • markers may be identified through blood tests, urine tests, clinical observation or identification of blood clots by any of several conventional techniques, or by the more refined techniques such as DNA fingerprinting and PCR.
  • Disease markers the laboratory analyses measure levels of certain proteins, lymphokines, enzymes and relevant molecules.
  • Clinical markers may include blood pressure, visible tissue damage, signs of inflammation, ecchymoses, and the like.
  • An initial bolus of defibrotide (box 2) is given intravenously over 15 to 30 minutes. Immediately thereafter the patient is given the daily dose of 40 - 400 mg/kg by continuous infusion. Preferably, the initial dose is a bolus (25 - 50 g/kg) followed by 24-hour dose which is increased in 50 mg/kg/day increments every 1 - 3 days.
  • the starting base-line dose may be from 40 - 400 mg/kg/day depending upon physician preference and the respective disease state treated. Lower initial doses are preferred for those therapeutic compounds which enter the cell nucleus more readily and are thus effective at lower doses.
  • the bolus and daily dose for chemical derivatives of the nucleic acids may be calculated as a proportion of the defibrotide dose based on the relative cell-entry rate. It is preferred to administer this dose intravenously using two IV bags of 50 ml D5W, each bag infused over 12 hours. If for any reason the infusion is interrupted, the rate of infusion would be thereafter adjusted so that the patient will have received the calculated 12 hour dosage at the completion of the specified time period. This 24 hour dose range can also be administered in 2 - 4 bolus injections or per oral administration.
  • Defibrotide or other selected nucleic acid derivative may be administered parenterally or orally.
  • Parenteral administration is in the form of continuous intravenous infusion or intravenous bolus injection.
  • Intravenous infusion may be accomplished by gravity feed, pump delivery or other clinically accepted methods.
  • Oral administration may include the use of capsules, tablets or powders for any method of enteric administration.
  • materials for delivery of the agent optionally comprise 2 x 50 ml D5W IV bags each containing one-half of the calculated total 24 hour dose in milligrams of defibrotide, each bag infused over 12 hours for the IV-continuous infusion at the maximum tolerable doses.
  • the total 24-hour dose can be administered by bolus injection every 8 - 12 hours.
  • the initial bolus injection and the subsequent outpatient bolus maintenance infusions are given, for example, in 3 x 25 ml D5W bags, each bolus to be infused over fifteen to thirty minutes.
  • the oral dosage outpatient maintenance therapy in milligrams given daily would be the multiples of 2X the maximum tolerable IV dose.
  • defibrotide when administered to humans, decreases AgPAIl (plasminogen activator inhibitor antigen) production and increases AgTPA production most efficiently starting at a dose range of 40 - 80 mg/kg/day, optimum effects being reached at 300 - 400 mg/kg/day dose range and above.
  • AgPAIl plasmaogen activator inhibitor antigen
  • defibrotide can be used as an alternate to, or as an adjunctive thrombolytic agent with t-PA, t-PA analogues, urokinases, streptokinases and their derivatives, when used in dose ranges of 300 mg/kg/day or above in the treatment of venous thrombosis and arterial thrombosis.
  • defibrotide is also an effective adjunctive antiplatelet and cytotropic agent to t-PA, t-PA analogues, urokinases, streptokinases and their derivatives.
  • Defibrotide is the drug of choice over aspirin as an adjunctive therapeutic agent to thrombolytic therapy at dose ranges starting at 40 - 80 mg/kg/day, most preferably 200 mg/kg/day and above.
  • Defibrotide can also be used with vasopressin as an adjunctive therapeutic agent in thrombolytic therapy at dose ranges starting with 40 - 80 mg/kg/day, preferably 300 - 400 mg/kg/day or above.
  • the drug of choice either alone or as an adjunctive, to agents that elevate cGMP at dose ranges starting with 40 - 80 mg/kg/day with optimum effects being obtained at 200 mg/kg/day and above.
  • vWAg is greater than five times the initial baseline level.
  • the upper limit may vary among the various derivative nucleic acids depending on their respective cell entry rates.
  • the safe upper limit is at least about 400 mg/kg/day and may reach 600 mg/kg/day.
  • the high molecular weight (45 - 50 kDA) defibrotide is about 350 mg/kg/day.
  • the dose ranges with the chemical derivatives of the nucleotide would correspond to dose ranges that elevate the base line vWAg antigen level to 5 times the initial value.
  • the correct identification of markers are based on the identification of the pathways of disease pathogenesis and the respective repair processes and pathways.
  • the mechanism of efficacy of the therapeutic nucleic acid simulate or are superimposed on the cellular pathways of the respective repair process they induce. For example, using defibrotide as the clinical agent, one would (1) identify the known signal transduction systems and second messengers of the repair process, (2) define the most probable nucleic acid-induced repair markers of the known cellular repair pathway, and (3) define markers of the disease process related to disease pathogenesis.
  • a compound whose intracellular concentration can be a repair marker in one disease state can be a disease marker in another disease state.
  • the marker would usually be under-regulated by defibrotide instead of induced.
  • a marker of normal cellular function if deficient, may be a disease marker.
  • the paralysis of cellular function of CD4 cells by the HIV retrovirus is secondary to the compromise of normal cellular markers of transduction pathways and second messengers.
  • G-proteins instrumental in the activation of adenylyl cyclase are likely to be deficient in their active form with a low dose threshold level.
  • the deficiency of the normal cellular marker of G-proteins would be a disease marker. Since defibrotide affects the adenylate cyclase pathway (increased cAMP by defibrotide), defibrotide would restore the second messenger of cAMP, which therefore would be a repair marker.
  • disease specific markers may be present in patients in need of treatment.
  • disease specific markers include blood pressure, creatinine clearance, creatinine, fPAI, and fPAI/ AgTPA.
  • disease specific markers include protein CA, protein Cl, protein SI, F-IX, F-XI and F-XII and APTT.
  • prothrombotic states and thrombotic diseases include protein CA, protein Cl, protein SI, F-IX, F-XI and F-XII and APTT.
  • specific disease markers include fPAI/ AgTPA, protein CA, protein Cl and protein SI whenever there are no concurrent changes in F-IX, F-XI and F-XII.
  • acquired thrombopathy disease markers include bleeding time and platelet aggregation with arachidonic acid and other platelet aggregation assays.
  • HIV disease markers include arthralgia, Herpes labialis, Herpes genitalis, and cryptococcal diarrhea.
  • endothelial cells play a prominent role in heart disease, liver disease and kidney disease.
  • Defibrotide has cyto-protective effects in myocardial cells and hepatic cells, as verified in patients with tissue necrosis; marker dependent doses of defibrotide can effectively treat these diseases.
  • protein CA, protein Cl and protein SI will be additional, yet optional, disease markers.
  • Disease specific markers for liver diseases include SGOT, SGPT and GGPT.
  • Disease specific markers for kidney disease include creatinine, creatinine clearance, blood pressure, 24-hour urine volume, and 24-hour urine protein.
  • vWAg and platelet aggregation by arachidonic acid will also be markers for these diseases.
  • the maximum efficacious dose is the dose which will normalize the platelet aggregation assays, so as to established normal levels of 60 - 150% in all cases.
  • Defibrotide and derivative nucleic acids are the first side-effect free platelet deaggregatory agents which induce this effect by restoring the integrity of the platelet functions (which by definition inhibit aggregation), and not by creating further defects in platelet functions like, for example, aspirin would.
  • this study shows the therapeutic nucleic acid to be the first proplatelet agent to be used within the arena of antiplatelet therapy.
  • the latter is cAMP dependent initiator of the transcription of factor of the CRE which in turn is the portion of the DNA enhancer sequence responsive to cAMP and cAMP associated transcription factors, such as c-myc products, c-fos products, ATP Activation Factor, Serum Responsive Element (SRE), API transcription factor (ATF), HIV-Long Terminal Repeat (LTR), leucine zipper transcription factors of c-fos/c-jun.
  • SRE Serum Responsive Element
  • ATF API transcription factor
  • LTR HIV-Long Terminal Repeat
  • ATF, SRE, API sites in c-fos promoter/enhancer all respond to cAMP without the requirement of SRE.
  • Protein Kinase A activates endogenous CREB activity and will enhance viral transactivation).
  • protein Cl and protein SI are representative markers for both transcriptional and redox state effects of this class of nucleotides.
  • protein Cl and protein SI are representative markers for both transcriptional and redox state effects of this class of nucleotides.
  • platelet aggregation markers they represent pharmacologically the antithrombotic actions of the nucleotide and its derivatives at the respective dose levels.
  • These representative dose levels for the antithrombotic effect of this nucleotide is ideally to start at 120 mg/kg/day dose level or above.
  • the projected dose values show, unlike the purely transcriptionally activated molecules like AgTPA and vWAg, progressive potentiation of the antithrombotic effects of the nucleotide without any slowing down or leveling off at higher dose levels.
  • antithrombotic action also depends on the "heparin-like" activity of the nucleotide measured in the laboratory with anti-factor Xa activity, PT/APTT, thrombin time, reptilase time markers.
  • the prototype nucleotide and derivatives proposed are alternative pharmaceutical agents with dose ranges starting with 120 milligrams per kilogram per day.
  • Disease states involving the modulation of the anti-phospholipid antibodies include clinical states such as myocardial infarction, valvular cardiac lesions, glomerular tubular renal lesions, leak proteinuria, nephrotic syndrome, skin and soft tissue necrosis, deep vein thrombosis, and post phlebitic syndrome related to the presence of lupus anti ⁇ coagulant and anti-phospholipid antibodies.
  • the nucleic acid also induced complete disappearance of the circulating anti-phospholipid antibody for the duration of drug administration.
  • the clinical beneficial effects were irreversibly established after cessation of drug administration, circulating anti- phospholipid antibody levels returned to abnormal values, albeit without any clinical abnormalities accompanying reappearance of circulating antibodies.
  • Endothelin-I is constitutively released from the vascular and the endothelial cell, secretion of endothelin-I being regulated at the transcriptional/translational level.
  • Gene expression of endothelin-I depends on protein kinase C pathway which has been shown in this study to be suppressed by the prototype nucleotide, i.e., high molecular weight defibrotide.
  • the dose level that suppress transcriptional/translational activity involving protein kinase C pathway is also representative of the dose level at which the prototype nucleotide will be able to suppress or inhibit the protein kinase C pathway; including the receptor-ligand interaction, inhibition of inositol triphosphate and diacylglycerol and inhibition of release of Ca 2+ intracellular levels.
  • Action by this nucleotide has been qualitatively mentioned and shown in the prior art.
  • doses representing the corresponding protein kinase C pathway inhibition have not been outlined in humans until this study.
  • High molecular weight defibrotide will suppress endothelin-I levels starting at dose levels of 40 - 80 milligrams per kilograms per day ideally at dose ranges of 160 - 200 milligram per kilogram per day or above. Dose levels of 275 mg/kg/day depress interleukin levels. High molecular weight defibrotide will furthermore balance activation of phosphoinositol/diacylglycerol signal transduction pathway also at the same dose levels.
  • PMN Intact polymorphonuclear leukocytes
  • PMN metabolize linoleic acid into 13-HODE under basal conditions.
  • Down regulation of platelet adhesion to endothelial cells following vascular wall injury in vivo was in part due to the presence of 13-HODE in PMN at the site of injury.
  • Prior art has shown in the annular flow chamber that adhesion of platelet to the thrombogenic surface was inversely co ⁇ elated with PMN 13-HODE levels.
  • no therapeutic agent increasing PgE, generation and 13-HODE generation has heretofore been identified.
  • the pertinent markers for proplatelet effect are: intracellular Ca 2+ , intracellular cAMP, platelet factor 4, platelet jS-TG, glycoprotein lb, collagen/arachidonic acid stimulated ATP secretion by platelets, platelet activating factor (PAF), 6-keto PgE, (Pgl 2 metabolite in plasma), 6-keto Pgl, ⁇ (Pgl 2 metabolite in plasma), 13-HODE (PMN), 5-HETE (PMN), 6 PgE 1/5 HETE ratio.
  • NADPH oxidase exists in some membranes of inflammatory phagocytes. Prior art has shown increased NADP ratio with the prototype nucleotide defibrotide.
  • Markers AgPAIl and endothelin-I are representative of existing endothelial cell injury involving the vascular endothelial cell glomerular basal membrane and nephrogenic microvascular vascular spasm. Hence, markers for endothelial cell injury are AgPAIl, endothelin-I, TNF- ⁇ , IL-6 and IL-1.
  • These genes include vasoactive intestinal peptide (VIP), somatostatin, human chorionic gonadotropin, phosphoenolpyruvate carboxylkinase, tyrosine hydroxylase, fibronectin, prolactin, ornithine decarboxylase, interleukin-6 gene, c-fos oncogene, haptoglobin, hemopexin, C- reactive protein (CRP), as well as other cellular genes which are regulated by cAMP responsive element (CRE), transcriptional factors interacting with CREB (which is 43 kd protein that interacts with CRE via leucine zipper, such as c-myc products, c-fos products, ATP (Activating Protein), SRE (serum responsive element), API.
  • CRE
  • the selected nucleic acid e.g. , defibrotide
  • For the nucleotide transcriptionally-activated parameters analysis is made for the highest values in each dose range.
  • For the nucleotide transcriptionally shut-off parameters analysis is made for the lowest value in each dose range. Therapy Based on Universal Markers
  • markers have now been shown to reflect transcriptional genomic activity by nucleotides which increase cAMP, adenylate cyclase via the interaction of G-proteins, and phosphorylate transcriptional factors via protein kinase A. These markers are vWAg, AgTPA and 3 2 -microglobulin. While vWAg and AgTPA are representative markers, any molecules which are initiated by nucleotides, or derivatives such as defibrotide, to induce transcriptional activity are included.
  • vWAg may be employed as a universal marker to guide the assessment of the duration of therapy, i.e. , the most therapeutic dose, as well as the most efficacious daily dose. It has been discovered, as reported herein, that vWAg is transcriptionally activated by defibrotide irrespective of the type of injury. Analysis of patient data has led to the unexpected finding that with the onset of cure, vWAg levels decline. The production of vWAg will be activated by defibrotide only for the duration of the injury and the repair process. In this regard, defibrotide will not effect vWAg levels in healthy individuals or following the establishment of cure, i.e. , vWAg level will decline to baseline regardless of ongoing therapy.
  • vWAg is classified according to this invention as being a universal dose marker.
  • vWAg can be utilized as the universal marker for all nucleotides that induce activation of cAMP and protein kinase A enzymes.
  • vWAg is a plasma glycoprotein having a molecular weight of approximately 200,000 which is constitutively secreted by the endothelial cell.
  • vWAg levels go up with increasing defibrotide dose levels when the dose is adequate to stimulate vascular endothelial function.
  • an increase in the vWAg level co ⁇ esponds to the induction of transcriptional activity of this gene by the nucleic acid. Elevation of vWAg is representative of the ongoing repair process. The decline in the level and eventual normalization of vWAg during therapy is representative of the cure process. Plateau in the level of vWAg co ⁇ elates with the application of the maximum efficacious dose.
  • the elevation in the level of vWAg is concu ⁇ ent with modulation of the disease marker and activation of the repair marker.
  • the maximum efficacious dose is determined along with vWAg, so as to normalize the levels of these molecules between 65 - 150% , and eliminate the intracellular oxygen radicals (measured by chemiluminescence, normal state being negative).
  • the maximum efficacy in inducing transcriptional activation of vWAg occurs at doses of 40 DKGD and above, ideally within the DKGD range of 40 - 400.
  • the universal marker vWAg dose levels are representative dose levels by the prototype's transcriptional/translational modulatory effects. Fitting the definition of universal marker, vWAg does not contribute to the expected correction of bleeding time but acts as a functionally dormant molecule.
  • Another option is to empirically repeat therapy after three weeks following cessation of therapy on the above principles.
  • the half life of the nucleic acid appears to be about three weeks, based on the observation that the universal marker vWAg requires 2 - 3 weeks to come down to baseline levels with cessation of therapy. If the universal marker vWAg is elevated during therapy with the previous maximum efficacious dose, there is still a lesion to treat, irrespective of the fact that there are no known or visible clinical, and/or documented biochemical repair or disease markers.
  • vWAg is normalized while on established maximum effective dose. Thereafter therapy is discontinued and the same cycles are repeated until the maximum efficacious dose therapeutically initiated no longer induces any elevation in vWAg, as would be observed in a normal healthy individual.
  • platelet aggregation with arachidonic acid, ADP 20, ADP 10, ADP 5 and collagen also are therapy increasing markers, they are not representative markers for the transcriptional activity of the nucleotide. Rather, they constitute markers for the various receptors the co ⁇ esponding statistics of which are to be interpreted as for vWAg and AgTPA.
  • the dose ranges are representative of the dose ranges affecting receptor ligand interactions as well as stimulation of prostanoid secretion by the prototype nucleotide, since prior art has shown that defibrotide up-regulates Adenosine A, and Adenosine A 2 receptors (represented in this work by the up-regulation/normalization of the arachidonic acid and ADP induced platelet aggregations). Prior art also has shown that up-regulation of Adenosine A, and Adenosine A 2 receptors is inductive of up-regulation of Pgl 2 secretion, independent of cyclooxygenase/arachidonic acid pathway.
  • vWAg level be raised 5 times (or 500%) and cAMP to 100% of the baseline levels, respectively, since 100% occupation of epinephrine/norepinephrine receptors (prototype receptor/ligand interaction for defibrotide and derivative nucleotides) would translate into 100% increase in intracellular levels of cAMP via activation of adenyl cyclase enzyme.
  • the maximum efficacious dose has to be increased to reach these numerical values.
  • the maximum therapeutic dose is determined by when the vWAg and/or intracellular cAMP levels decline to baseline levels during therapy with maximum efficacious dose.
  • the maximum therapeutic dose (until universal marker or vWAg returns to the baseline level during therapy) (established normal is 0.5 - 2.00 U/ml) is administered.
  • Markers representative of shutting off transcriptional activity by the nucleotide are AgPAIl and endothelin-I molecules.
  • the interpretation has to be made in the opposite direction of induction of transcription/translation, i.e. , maximum values of the lowest level (worst performance) on the last day should be followed to determine the maximum efficacious dose.
  • vWAg will increase whether the nucleotide is used for transcription induction, maintenance of the redox state, or modulation of second messengers of cAMP, directly or indirectly of opposing signal transduction pathways of intracellular Ca 2+ , diacylglycerol and/or phosphoinositol, or with prostaglandin Pgl 2 or PgE,.
  • the maximum therapeutic dose with declining molecules will still be the duration and/or dose of therapy where vWAg becomes normal on therapy.
  • the best parameter to follow the dose related induction of transcriptional activity is the "minimum values of the increasing levels" obtained on the first day of the initiation of each dose range.
  • “Highest or increasing levels” represent the increase in level of a molecule whose production (transcription) is turned on with increasing dose levels. Choosing the minimum increase in the level in the transcribed genome among all patients treated in any dose range enables the prediction of the worst performance with that dose of the therapeutic compound. This enables the treatment of the worst performer, which allows turning on the genomic transcriptional activity in the greatest number of patients within each respective dose range.
  • Increase in the marker as shown by "minimum highest value” represents that the repair process is ongoing, that is, repair molecules are being produced and transcriptional activity is ongoing.
  • vWAg level The quantitative relationship between vWAg level and daily dose of the therapeutic compound is best visualized when the minimum value of the increasing vWAg levels in the population are analyzed (i.e. , the worst performance levels in any one patient at any one dose range, "worst performance” implying that increasing the dose will incrementally continue to elevate the vWAg, which is biologically interpreted as meaning that there are more repair events to go through.
  • Minimum increasing value is the parameter to use to confirm the event of ongoing cellular repair.
  • Maximum increasing value is the statistical parameter to use to follow the completion of the repair event.
  • Maximum therapeutic dose is the dose at which vWAg on continued therapy will decline to a normal level.
  • the "maximum values of the lowest levels" obtained among all patient data on the last days of treatment at each dose range are similarly used to analyze how increasing dose ranges affect the transcriptional activities involving the "turned on" molecules. The levels will show progressive declines, i.e. , the progressive turning off of the repair process with the onset of cure, in spite of the higher doses.
  • the m-efficiency value is the ratio of the respective elevated level over the time taken for elevation to occur. The higher the dose, the higher the value of the numerator and the higher the m-efficiency level. Alternatively, the shorter the time (denominator), the higher is the m-efficiency value.
  • non-sequence specific DNA such as defibrotide decreases II- 1, U-2, TNF- ⁇ and 11-6, all of which are known inducer of HIV proliferation.
  • Administration of such an agent also increases total lymphocyte and T-lymphocyte counts, CD4 counts, and CD8 counts, as well as increasing cGMP/cAMP ratios. An anabolic effect has also been observed.
  • Defibrotide' s mechanism of efficacy relates to modulation of cell functions at the nuclear genomic level through one or more pathways by modulation of the cell's genetic material, i.e. , DNA itself or translation or transcription of the genetic information.
  • Defibrotide-induced cellular modulation restores the normal functions of the cell such as the production of normal proteins needed by the cell and, in the case of HIV, the co ⁇ ection of the effects of the abnormal, viral encoded genetic material by inhibiting its further production at the expense of the normal, virus-free genetic material.
  • defibrotide is administered at dosages much greater than previously described in the literature.
  • an initial bolus dose of 100 mg/kg in 50 ml DSW is infused over a period of 30 - 60 minutes followed by 200 mg/kg/day infused in 250 - 500 ml DSW over a period of 3 - 24 hours.
  • dose is escalated to maximum tolerable dose, maximum efficacious dose and maximum therapeutic dose levels.
  • the dosages and durations of the phases of therapy are adjusted according to the results of laboratory studies performed on the patient's infected cells. In this way, the HIV virus may be inactivated and its proliferation a ⁇ ested. Therefore, the progress of the disease may be a ⁇ ested or ameliorated.
  • defibrotide can effectively treat HIV infection as long as the carrier CD4 + cell and/or the monocyte harboring the virus preserves the physiological ability to revive itself. Therapeutic success with defibrotide, however, is strictly dependent upon the assessment of the co ⁇ ect treatment doses for the respective disease states. Moreover, since the optimum function of the normal cell, by definition, would not be compatible with any complications, defibrotide at any defined maximum efficacious dose, specific for any patient and disease state would be complication-free.
  • Defibrotide may provide anti-sense neutralization of the viral proteins.
  • Defibrotide's mechanism of efficacy may be at the nuclear level by modulation of genetic functions via other pathways as well.
  • Defibrotide's actions may be more apparent during viral phases which involve translation and/or transcription of the DNA message, so as to revive the normal function of the cell at the expense of the disease-specific molecules. This action may be analogous to anti-viral effects of Ampligen (a mismatched double stranded RNA-molecule. However, whereas Ampligen exhibits immunostimulating effects, agents such as defibrotide are both immunostimulants and immunosuppressants.
  • Defibrotide may modulate viral penetration into the cell via its known action of inhibiting intracellular calcium mobilization. Also, defibrotide may directly inhibit viral enzyme reverse transcriptase via inducing ATP production analogous to ddl (dedeoxyinosine), by virtue of its known action of inducing high energy metabolites (ATP, ADP, NADP/NADPH), possibly via modulation of Complex-I respiratory molecule. Defibrotide may inhibit protein kinase C analogous to Hypericin. Additionally, defibrotide decreases Tissue Necrosis Factor (TNF), a cytokine known to promote HIV-I activation, by its known effect on increasing cAMP levels at the correct defibrotide dose level.
  • TNF Tissue Necrosis Factor
  • Defibrotide may jointly and/or selectively modulate one or several pathways. This modulation will be, only to the appropriate degree thus surpassing all of the other anti-HIV agents in its lack of side effects, yet presence of proven efficacy. Defibrotide will achieve this result only when the dose levels are tailored to the patient, stage of disease activity and/or reigning stage of viral activity,
  • AIDS virus can manifest itself in four basic stages which are well known in the art and described in the medical literature: 1) HIV positive blood test but otherwise no abnormal laboratory findings or clinical symptoms; 2) pre-AIDS Related Complex (pre- ARC); 3) AIDS Related Complex (ARC); and 4) Active AIDS.
  • the last three stages may be present in combination with other infections, with central and/or peripheral nervous system disease, with associated autoimmune disease or with related neoplasm. All four stages are treated using the same basic method described herein. There are no patient inclusion or exclusion criteria for therapy. Patients in any or all of the four clinical stages of HIV-infection including history of exposure (i.e. , HIV + , Pre-ARC, ARC, and AIDS) are candidates for therapy.
  • the method of treating the HIV-infected patient begins with a panel of laboratory studies which include the quantitative evaluation of the activated peripheral blood mononuclear cell subsets, circulating viral proteins, cytokinases and soluble cell-surface receptors.
  • the initial administration of a selected dosage of defibrotide is followed by incrementally increasing the dosage of defibrotide until a maximum tolerable dose is reached.
  • the laboratory panel is repeated weekly during this therapy.
  • peripheral blood mononuclear cell subsets by two-color flow cytometry, lymphokines and soluble cell surface receptors by ELISA, and HIV-viral proteins by Western blot analysis.
  • the peripheral blood mononuclear cell subset analysis will usually include either CD4 + , CD8 + , CD19 + , CD25 + , CD56 + , and HLA-DR alone, combined with one another, or combined with the quantification of monocytes.
  • the Western blot protein tests include gp-24, gp-17, gp-120 and gp- 160.
  • the ELISA test measures TNF, sIL2R, sILl and soluble CD8. Every third week, it is prefe ⁇ ed that cell cultures for HIV antibody neutralization, PCR and reverse transcriptase determinations be made. HIV-I Gene Therapy in Accordance with the Invention
  • Gene delivery thus far has been a method by which foreign genetic material is introduced into a suitable target cell usually via viral vectors.
  • Such strategy generally consisted of an e vivo and an in vivo phase.
  • the foreign gene is inserted into target cells derived from the recipient.
  • the engineered cells containing the newly inserted gene are expanded ex vivo.
  • the expanded engineered cells are transplanted into the recipient.
  • This modulatory therapy is the first of its kind which manages therapy from cell surface signaling to genomic modulation utilizing the oral and/or intravenous administration of nucleotides, without utilizing retrovirus, adenovirus or other gene viral vectors traditionally employed in gene therapy.
  • Gene therapy has not, heretofore, been utilized without cellular transfection with viral vectors, and never before by oral or intravenous administration of nucleotides to humans.
  • Gene therapy has not, heretofore, been tried without the interaction of viral vectors, i.e. , by the administration of nucleic acid-based pharmaceutical agents orally and/or by intravenous route.
  • the prototype drug defibrotide although administered to patients over the past 5-6 years, has never heretofore been contemplated for gene therapy.
  • dosage has never been assessed by molecule markers.
  • Molecule markers have never been defined within the system of secondary messengers, signal transduction systems, promoters (DNA sites which are on the same chromosome as the gene transcribed and to which RNA polymerase binds), enhancers (DNA regions that control a promotor from a great distance, sometimes as much as 30,000 bases), and transcription factors (diffusible regulatory proteins which bind to DNA transcription activation domains and regulate the rate of transcription by RNA polymerase).
  • HIV-disease has not been previously interpreted as a disease of dismodulation involving the genomes, cellular secondary messengers and cellular signal transduction systems. The specific pathways affected by the HIV-retrovirus have not been clearly delineated. Therapy of HIV-disease has not previously attempted to reclaim the affected cellular function systems from the virus by reversing the dismodulation at the various levels by using exogenous therapy involving various modulators of these systems.
  • the therapeutic approach of the invention disengages itself from the common practice of planning therapy based on clinical staging.
  • the planning of therapy is based on the identified mismodulations of (a) membrane lipids and cytoskeleton; (b) cell-surface receptor/ligand interactions; (c) secondary messengers; (d) signal transducers; (e) cellular transcription factors utilized in viral replication: as well as based on the identified (f) oncogenes; (g) viral transcription factors; and (h) viral genomes.
  • the method of therapy disclosed herein for HIV may also be used in treatment of other viral infections and neoplasms.
  • the object of therapy in accordance with the invention is to (i) reestablish repair markers at the constitutively expressed tissue levels; and (ii) eliminate disease markers (in case of the oncogenes to reverse the transformation).
  • repair markers reflect the underlying logic of transcriptional regulation. Therapy is aimed to concu ⁇ ently induce some markers and suppress other markers.
  • the prototype nucleotide if used at the co ⁇ ect doses (which are guided by the respective repair markers) can accomplish this goal. Elimination of disease markers by the therapeutic nucleotide compound will occur at various levels. It can be an indirect phenomenon based on modulations of secondary messengers, such as cAMP; it can be a direct phenomenon based on modulations of the phosphorylation events involving genes and transcription factors.
  • cAMP activates protein kinase A enzymes
  • Ca 2+ activates protein kinase C enzymes
  • the prototype nucleotide up-regulates cAMP, and downregulates Ca 2+ , or it can be a direct phenomenon based on modulation of cAMP responsive gene promoters (CREM, as enumerated above).
  • Proposed Mechanism A Induction of sIL2R gene and HIV-I LTR are interdependent phenomena. If the protein kinase C dependent sIL2R gene is turned off by high cAMP levels, activation of HIV-I LTR is concu ⁇ ently suppressed as well.
  • Proposed Mechanism B Increased cAMP levels have been shown to induce viral replication (Nokta and Pollard, 1992, AIDS Research and Human Retroviruses 8(7): 1255-1261). HIV-I REV/ENV genes are both phosphoproteins. There may be other routes for cAMP-induced replication of HIV-I. Although administration of the maximum efficacious dose will increase cAMP levels, prolonged administration of the nucleic acid at the maximum efficacious dose, so as to realize the successful administration of the maximum therapeutic dose would culminate in declining cAMP levels, since vWAg decreases on therapy if and once the maximum therapeutic dose is administered. Hence administration of the maximum therapeutic dose is paramount in overcoming cAMP induced viral replication.
  • This phenomenon may at least partially, be based on the induction of protein kinase C, as a secondary biochemical event (i.e. , protein kinase C induces sILR2 gene, which in turn modulates protein kinase C so as it can directly inhibit cAMP).
  • the transcription factor NF-kB binds to both the HIV-I enhancer, and the sILR2 gene. Protein kinase C phosphorylates its inhibitor IkB and releases active NF-kB. Increased cAMP levels by inhibiting directly the Ca + induced activation of protein kinase C would modulate this phosphorylation event, and downregulate the transcriptional activities related to NF-kB. Since NF- kB binds to both the HIV enhancer and IL2 receptor, increased cAMP levels will downregulate HIV-I replication.
  • Proposed mechanisms B and C show that increased cAMP levels can be both deleterious and beneficial. It can be clearly seen that the prototype nucleotide is an overall "downregulator" of biochemical events, if maximum therapeutic and efficacious dose levels are administered.
  • defibrotide would (1) alleviate the complication of cAMP induced viral replication; (2) induce inhibition of viral replication mediated via modulations of cAMP, protein kinase A, protein kinase C, cellular redox state, G-proteins, or cAMP induced gene promoters (in this regard, defibrotide and other nucleotide derivatives introduce for the first time into anti-HIV therapy nucleotides with no sequence specificity that concurrently modulate the totality of the cellular second messenger/signal systems for rapidly transducing extracellular signals into specific patterns of gene expression in the nucleus); (3) concu ⁇ ently induce inhibition of viral replication with sense, anti- sense, or missense nucleic acids (e.g.
  • gene therapy i.e. , genetic engineering
  • Nucleic acid derivatives having chemical modifications are as described previously (e.g., nucleotides conjugated with poly(L-lysine) or which is modified by, for example, the addition of amino acids such as lysine, histidine and arginine, the addition of optimum concentrations of folate and/or biotin, the addition of the optimum ratios of metals and ions including zinc, manganese and iodine, by the addition of 5'- polyalkyl moieties, cholesterol, vitamin E, l-2-di-O-hexadecyl-3-glyceryland other lipophilic moieties and/or modified by the replacement of phosphodiester bonds with phosphothiotate bonds) and combination nucleic acids would be employed.
  • Combination nucleic acids will, hereinafter, be refe ⁇ ed to as "combo- nucleic acids" .
  • Combination of sequence nonspecific prototype nucleic acids (e.g. , defibrotide and derivatives thereof) with various sequence specific nucleotides has never, heretofore, been proposed for use as a multimodal therapy for HIV.
  • sequence specific nucleotides such as TAR decoy RNA, negative mutants of the viral REV transactivator, synthetic promoters with the consensus sequence for binding of the transcription factor Spl , and the TATA box, TAT mutants, mutations involving the seven cysteine residues, REV mutants, NEF- cDNA sequence with or without U3 region sequence of the 3 'LTR, HIV-I LTR enhancer (-137 to -17) mutant, HIV-I LTR sense sequence of the negative regulatory element (-340 to -185), LTR Spl (GC box) binding site and TATA box mutants and LTR GAG gene sequence mutants, are known in the prior art. However, such sequence specific nucleotides have been used only in the in vitro setting as "single mode" anti-viral agents, the mode of actions being limited to the sequences.
  • combo-nucleic acids examples include a) defibrotide sequence + TAR decoy RNA, b) defibrotide sequence + negative mutants of the viral REV transactivator, c) defibrotide sequence + synthetic promoters with the consensus sequence for binding of the transcription factor Spl , and the TATA box, d) defibrotide sequence -I- TAT mutants, mutations involving the seven cysteine residues, e) defibrotide sequence + sense derivatives of CIS acting negative elements (CRS) present in the integrase gene + REV mutants, 0 defibrotide sequence + transdominant suppressor of REV
  • CRS CIS acting negative elements
  • defibrotide sequence + NEF-cDNA sequence with or without U3 region sequence of the 3 'LTR h) defibrotide sequence + POL reverse transcriptase gene mutants, i) defibrotide sequence + POL viral integrase gene mutant, j) defibrotide sequence + POL viral protease gene mutant, k) defibrotide sequence + HIV-I LTR enhancer (-137 to -17) mutant, 1) defibrotide sequence + HIV-I LTR sense sequence of the negative regulatory element (-340 to -185), m) defibrotide sequence + LTR NFkB mutant (-104 to -80), n) defibrotide sequence + LTR Spl (GC box) binding site and TATA box mutants, o) defibrotide sequence + LTR GAG gene sequence mutants, p) defibrotide sequence + ENV, GAG, POL gene sequences placed
  • sequence nonspecific chemical derivative in the combo-nucleic acids exemplified above are preferably also modified for improved cellular uptake, and enhanced anti-viral activity with the chemical modifications described above.
  • Clinical markers consist of any and all disease markers noted on physical examination, without restriction.
  • Clinical laboratory markers consist of any and all noted clinical laboratory abnormalities, without any restrictions. While one skilled in the art would readily recognize which markers are indicative of a pathological state to follow, the following list is, in addition to those listed above (i.e. , Panels 1-11), provided as a guideline.
  • Panel 11 Peripheral HIV 1 Panel
  • HIV-l rgp 160 glycosylated full length protein
  • HIV-l rgp 120 (Binds to CD4 + cells and inhibits syncytia;
  • HIV-l pl21 immunodominant region of gp41
  • HIV-l pg2 amino acid from core proteins p24 and pl5
  • HIV-l GAG5 amino acids from core protein p24
  • rs CD4 fully glycosylated, binds to gpl2o and inhibits syncytia
  • Anti gp41 MAb 2A2 (IgG 2A antibody reacts with gp41); Anti V3 Loop Mab 50.1 (Binds to principle neutralizing determinant of HIV);
  • HIV antigen p24 reverse transcriptase
  • syncytia formation formation
  • cytopathic effect plaque formation
  • Intracellular platelet PBMNC Intracellular platelet PBMNC; vWAg (preferably mRNA pro-vWAg); cAMP (endogenous and PMA stimulated); cGMP (endogenous and PMA stimulated); glutathione;
  • RpA 1 small G protein
  • p 21 ras G protein group
  • v-nef viral G protein
  • DAG 1,2 diacyglycerol
  • Inositol triphosphate IP 3, endogenous, IL2/PHA stimulated
  • Example 1 To measure the effect of defibrotide on HIV it was first necessary to label the drug and determine whether defibrotide will enter the nucleus of the human cell. Knowing the phosphodiester linkages in defibrotide, its comparative nuclear penetration was assessed by labelling defibrotide with a photo-activatable analogue of biotin. The biological activity of defibrotide after labelling was considered to have been preserved since published data shows that previous oligonucleotide probes have been labelled with conjugates and still remained biologically active. Image analysis utilizing a cold CCD camera revealed that uptake of defibrotide was localized in the nucleus.
  • HIV infected peripheral blood mononuclear cells with varying doses of defibrotide were evaluated by staining for all viral envelope proteins using concanavalin A (Con-A) stimulated and unstimulated cells (Anti-HIV 1 , and Anti- HIV 3 specific Anti-HIV antibody).
  • Con-A concanavalin A
  • Anti-HIV 1 , and Anti- HIV 3 specific Anti-HIV antibody The blood sample was obtained from a patient using an evacuated blood collection tube containing sufficient EDTA to prevent coagulation of the sample.
  • the white cell population was suspended in a solution of the RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum and gentamicin, at the concentration of 5 micrograms/milliliter.
  • the white cells were then concentrated to a level of two million cells per three milliliters (2 x 10 ⁇ cells/3 ml) of the above solution.
  • the white cells were collected in flat-bottomed microtiter containers (Cell Wells, Corning).
  • the cell populations were further divided into two groups.
  • One group received stimulation by Con-A, the other group remained unstimulated by Con-A.
  • Con-A stimulation enhances the uptake of the antibody-dye label by HIV- contaminated cell components, thereby demonstrating an increase in the expression of the HIV protein.
  • Subpopulations of unstimulated and stimulated white cells were then incubated in the presence of discrete concentrations of defibrotide. Each successive assay employed successively greater concentrations.
  • a control sample of incubate containing no defibrotide was also prepared.
  • a labelling antibody solution was prepared by directly conjugating Cy5.18 with human ⁇ -HIV antibody to a final dye/protein ratio of 5.0 ( ⁇ -HIV-Cy5.18).
  • the cell subpopulations were again divided into two groups, one group for intracellular antibody labelling, and one group for surface antibody labelling.
  • Cells reserved for intracellular labelling were fixed with 70% ETOH, washed twice with monoclonal wash, and then resuspended into a solution containing 200 microliters of Hank's balanced salt solution (HBSS), supplemented with 2% FCS and 0.1 % sodium azide (monoclonal wash) and 5 microliters of ⁇ -HIV-Cy5.18 solution.
  • HBSS Hank's balanced salt solution
  • FCS 2% FCS
  • 0.1 % sodium azide monoclonal wash
  • ⁇ -HIV-Cy5.18 solution 5 microliters of ⁇ -HIV-Cy5.18 solution.
  • the entire cell preparation was incubated for 45 minutes at 4°C.
  • the cell preparation was then washed twice with the monoclonal wash, and resuspended in 1 % paraformaldehyde.
  • the surface glycoprotein antibody solution contained CD3-FITC (heterogenous T-cell antibody conjugated with fluorescein isothiocyanate dye) and CD4-RPE (helper T-cell antibody conjugated with phycoerythrin dye) obtained from Becton-Dickinson.
  • Con-A stimulated cells expressed 32% more viral proteins. However, after administration of 20 mg of defibrotide, both stimulated and unstimulated cells express 70% less viral proteins. At 30 mg concentration of defibrotide in both Con-A stimulated and unstimulated cells the expression of viral proteins leveled off. This supports the specificity of defibrotide for HIV-virus as well as the fact that if cells are induced to divide, translating into proliferation of the virus, more HIV virus can be killed, albeit, at higher doses.
  • hematological recovery in thrombotic microangiopathy generally, yet not exclusively, occu ⁇ ed when the patient received doses of defibrotide ranging from 20 to 30 mg/kg/day. These doses however did not cure the renal lesions since creatinine levels remained above normal (or only partially co ⁇ ected) at the dose levels where hematological recovery was complete. Renal recovery evidenced by normalization of creatinine levels occurred between 40 and 250 mg/kg/day.
  • vWAg predicts the transcriptional rate of the respective repair molecules induced by the nucleotide and will guide the assessment of maximum efficacious dose and maximum therapeutic dose.
  • the minimum value of the vWAg level among a group of disease patients (“minimum highest value”) was monitored when defibrotide was administered at a daily dose within the range of 10 - 400 mg defibrotide/kg/day (DKGD). Each increase in DKGD increases the preceding minimum highest value of vWAg by an increment smaller in each successive interval. Using defibrotide, the highest percentages of increments were found to occur at the borderline of 40 DKGD. Increasing DKGD above 400 induces only negligible improvements over levels below this dose. In practice, this is the dose level above which complications of bleeding have been observed by the inventor with high molecular weight defibrotide.
  • escalation to a new DKGD range will induce escalation of transcriptional activity for the universal marker vWAg within a period ranging from 1 - 9 days to 13 - 58 days.
  • a higher dose range will show a more prompt and more significant increase in transcriptional activity if compared to lower dose range, both being initiated on the first day of therapy. If two patients exhibiting the same disease state are treated at different maximum daily dose levels, dose dependent remission would be observed.
  • Examples 5 -7 report the treatment of three HIV infected patients. These patients were all treated in Turkey with defibrotide obtained from CRINOS.
  • arthralgia The effect on arthralgia was observed by the third consecutive day of treatment and was found to be strictly dose dependent. Upon cessation of therapy arthralgia relapsed to original condition and entered remission upon reinitiation of DNA therapy.
  • Herpes labialis lesions were in complete remission. No relapses were seen with temporary cessation of defibrotide.
  • Elevated cAMP/cGMP was observed at the onset of therapy, signifying activation of both protein kinase A and protein kinase C pathways.
  • a progressive rise in absolute and T lymphocyte numbers, CD4 and CD8 was seen.
  • a decrease in IL1, IL-2, IL-6 and TNF- ⁇ was observed during treatment.
  • Complete remissions in accompanying disease states include He ⁇ es labialis, oropharyngeal candidiasis, arthralgia, and He ⁇ es genitalis as well as accompanying tissue damage.
  • a 25-year old white HIV + female was treated with defibrotide. At the onset of therapy, the patient was asymptomatic but had a low CD4 count.
  • DNA therapy was terminated after 29 days secondary to a rise of CD4 percent and absolute counts. DNA therapy was reinitiated 25 days later secondary to a decline in CD4 percent and absolute counts. Therapy was continued on an outpatient basis, intravenous administration being alternated with oral administration.
  • Tables III and IV summarize pertinent laboratory data. In this patient, all tested interleukin levels were normal.
  • Treatment was characterized by increases in CD4, CD8, total lymphocyte, total T-lymphocyte counts accompanied by elevations in cAMP and cGMP, and in therapy related decreases in IL-6 and TNF- ⁇ . A total weight gain of 7 kg was observed.
  • a 33-year old white male with AIDS and opportunistic infections including He ⁇ es labialis associate with necrotic lesions, oral/pharyngeal candidiasis, tuberculosis and crytococcal dia ⁇ hea.
  • defibrotide and other polynucleotides are as pharmaceutical cell modulators committed to revival of normal cell function.
  • the doses and the markers will vary among diseases. Moreover, the markers for any given disease will frequently vary among different stages of the disease.
  • the aim of treatment is the i ⁇ eversible normalization of a marker or markers. Once this is obtained, a subsequent marker for the lower disease activity will be sought and treated. The possibility of cure depends on the identification of appropriate markers.
  • markers to determine doses can be best understood with reference to selected patient data presented hereafter as representative examples of patients suffering from thrombotic microangiopathy (HUS), prothrombotic states of neoplastic chemotherapy induced or immunological (anti-phospholipid antibody) etiologies, deep vein thrombosis, and soft tissue necrosis, as well as acquired platelet function abnormalities.
  • HUS thrombotic microangiopathy
  • prothrombotic states of neoplastic chemotherapy induced or immunological (anti-phospholipid antibody) etiologies etiologies
  • deep vein thrombosis deep vein thrombosis
  • soft tissue necrosis as well as acquired platelet function abnormalities.
  • Incidental therapeutic effects of defibrotide in these patients also involved pulmonary (e.g. , adult respiratory distress syndrome (ARDS)), cardiac (e.g. , subendocardial myocardial infarction) and renal systems (e.g. , glomerular proteinuria).
  • pulmonary
  • Patient No. 4 Initial Pathological State Primary anti-phospholipid antibody syndrome with clinical components of end stage renal disease on dialysis, adrenal insufficiency, mitral insufficiency, immunological prothrombotic state (secondary to presence of the anti-phospholipid antibody), bilateral femoral thrombi, Gl-tract ischemic necrosis (as the terminal event after cessation of the nucleotide therapy), uremic pericarditis, acquired thrombopathy with ecchymoses and soft tissue hematoma, nephrogenic hypertension.
  • the patient showed a transient decline in endothelin-I levels with a rise in DKGD from 60 to 160 mg/kg/day which was concurrent with onset of urine flow.
  • Rise in DKGD to 160 mg/kg/day was also concurrent with rises in AgTPA and increased conversion of protein Cl to protein SI which accompanied the clinical markers of the lysis of left femoral thrombus and amelioration of prothrombotic tendency during dialysis, respectively.
  • the conversion of protein Cl to protein SI was postulated to be inhibited by anti- phospholipid antibody, which inhibition was reversed by the nucleotide therapy.
  • the patient showed the immunosuppressive effect of the nucleotide over inhibition of the protein kinase C pathway and possibly also via hybridization with the sIL2R genomic sequence.
  • activation of the sIL2R is known in the art to occur via protein kinase C pathway induced phosphorylation of transcription/ translation related factors/molecules. Decline in DKGD to 100 mg/kg/day on day 35 was concu ⁇ ent with recu ⁇ ent increase in the sIL2R level while reinitiation of therapy at 150 - 200 mg/kg/day dose on days 41 , 42, 43 was concu ⁇ ent with complete normalization of sIL2R level.
  • Patient No. 5 Initial Pathological State: Rejection, nephrogenic hypertension, recurrent hemolytic uremic syndrome in transplanted kidney microangiopathic hemolysis, consumptive thrombocytopenia.
  • Renal disease specific disease markers were creatinine and endothelin-I, each of which showed concu ⁇ ent normalization with the administration of the nucleotide. With declining dose levels, the more crude marker of creatinine stayed normal; however, the more refined marker of endothelin-I escalated to abnormal levels.
  • Patient No. 7 Initial Pathological State Primary anti-phospholipid antibody syndrome, immunological prothrombotic state,- post-phlebitic syndrome, bilateral femoral thrombi, bilateral soft tissue necrosis of digits, hypertension, nephrotic syndrome, subendocardial myocardial infarction, mitral valve prolapse, acute respiratory distress syndrome, lupus anti-coagulant.
  • nucleotides will not induce uniform pharmacological actions in every patient reproducibly, as shown herein by the nucleotide induced increase in the initially low levels of protein SI in patient No. 4 (same disease state) and nucleotide induced decrease in the initially elevated/normal levels of protein SI in Patient No. 7. Since in both cases an antithrombotic effect was successfully obtained, the nucleotide is not acting in an e ⁇ atic manner. Hence the pharmacological actions of the nucleotide are defined by the presence and type of the abnormality.
  • This patient showed therapeutically successful human doses of defibrotide in the modulation of redox-state, NADP/NADPH, FAD/FADH 2 oxidative phosphorylation pathway, the so-called "cytotropic" action of the drug.
  • the clinical co ⁇ esponding pictures of soft-tissue necrosis and deep vein thrombosis as well as post-phlebitic syndrome were irreversibly, completely cured.
  • Patient No. 8 Initial Pathological State: Microangiopathy hemolysis, consumptive thrombopathy, hemolytic uremic syndrome/clostridium difficile toxin, lupus anti-coagulant, nephrogenic hypertension, anuric renal failure.
  • thrombocytopenia was confirmed as being "consumptive", i.e. secondary to increased destruction via platelets' increased aggregations to the injured endothelial cell, all reversed with the nucleotide. It is noted here that within the concept of "antiplatelet therapy” there is no pharmaceutical agent yet identified until the present text which reverses the "i ⁇ eversible aggregation of platelets” as represented here by the impaired ADP 20 induced platelet aggregations.
  • thrombotic microangiopathy picture developed with elevated parameters of endothelial cell injury markers of AgPAIl, endothelin-I, low levels of protein SI, marked thrombopathy with impaired platelet aggregation of arachidonic acid, ADP 5, and high levels of ongoing cytokine levels of sIL2R and TNF, as well as Natural Killer Cells.
  • Patient No. 10 Initial Pathological State: Idiopathic hemolytic uremic syndrome auric renal failure, nephrogenic hypertension, microangiopathic hemolysis consumptive thrombocytopenia.
  • This patient represents an example for marker-non-dependent dose assessment.
  • Patient No. 10 showed an initial decline of fPAI, with increasing platelet counts on 60 mg/kg/day dose, yet subsequent increase in fPAI in spite of continuing hematological recovery.
  • Patient No. 10 represents failure to increase the dose to push vWAg levels higher.
  • vWAg levels declined by day 20 in spite of an increase in dose from 60 to 80 mg/kg/day from day 8 to day 20.
  • Increasing AgPAIl disease marker levels should have been pursued to increase the dose high as necessary to increase the vWAg universal marker level to nearly 500% (5x) and disease marker AgPAIl to completely normal levels.
  • the decline on therapy of vWAg does not signify in this case completion of the repair event. Since no plateau in 5x elevated levels precedes decline on therapy, the vWAg decline in spite of modest increases in dose signify shifting of the lesion into a subacute/chronic phase.
  • Patient No. 11 Initial Pathological State: Breast cancer/Stage IV prothrombotic state, bilateral femoral thrombi, bilateral lower extremity deep vein thrombosis, bilateral ischemic soft tissue necrosis of toes, acquired thrombopathy.
  • Patient No. 11 represents successful thrombolytic/doses co ⁇ esponding very clearly to increasing AgTPA levels and decreasing AgPAIl levels for the duration of therapy with the clinical events of dissolution of bilateral hemoral thrombi. Cessation of therapy led to recu ⁇ ent pathology of declining AgTPA and increasing fPAI and AgPAIl levels. This represents failure to follow the markers adequately to ensure complete thrombolytic therapy.
  • Patient No. 11 represent adequate doses for i ⁇ eversible control of the prothrombotic state, since antithrombotic molecules remain at normal levels of therapy. This represents a clear example of how the pleiotropic action of the nucleotide is completely dependent on the stratified dose levels, different dose levels governing different actions of the nucleotide ranging from modulation of the redox state, for example to genomic modulation.
  • Patient No. 13 Initial Pathological State: Congenital hepatic vein thrombosis (Banti's Syndrome) prothrombotic state with recu ⁇ ent deep vein thrombosis of left axillary vein and left subclavian vein, thrombosis of sagittal and saphenous sinus, portal hypertension with gastric varices, recu ⁇ ent GI bleeding, acquired thrombopathy, ascending cholangitis, ischemic parenchymal disease, dysfibrinogenemia.
  • This patent illustrates the biological interdependency of protein kinase C/A pathways, cell surface receptor/ligand interactions, and genomic transcriptional/translational activity.
  • the drug i ⁇ espective of dose
  • Patent No. 13 showed, impaired arachidonic acid aggregation with elevated liver functions, high WBC count (IL-6 induced response) and amelioration thereof with nucleotide therapy. Normalization of arachidonic acid aggregation occurred with increasing DKGD. The inhibiting action of the nucleotide upon the protein kinase C pathway was represented by decreased endothelin-I levels and the dose dependency thereof. Nucleotide therapy was found to induce a decline in AgPAIl level without concu ⁇ ent elevation in AgTPA but which is concu ⁇ ent with protein CA level.
  • Patient No. 14 Initial Pathological State: Idiopathic hemolytic uremic syndrome, nephrogenic hypertension, lupus anti-coagulant consumptive thrombopathy, microangiopathic hemolysis.
  • Result of Therapy This patient shows that treatment of a subsequently identified second marker leads to the further amelioration of the first marker. Partial/near complete regaining of renal function was accompanied by the nucleotide induced treatment of nephrogenic hypertension. Dose dependent reproducible variation in vWAg levels confirms that automatic elevation of thrombolytic molecule AgTPA will not take place only by virtue of the on therapy declining AgPAIl levels, if elevation of AgTPA transcriptional activity is not specifically needed for the treatment of the particular lesion.
  • AgPAIl elevation in this patient reflected endothelial cell injury, as proven by the fact that its successful treatment also co ⁇ ected endothelin-I levels.
  • the effect on the endothelin-I levels were dose dependent and recu ⁇ ed off therapy, necessitating antihypertensives. If this patient's maximum therapeutic dose level had been adjusted so as to discontinue therapy only when vWAg levels on therapy declined to normal, nephrogenic hypertension and renal function would be irreversible co ⁇ ected.

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Abstract

Des oligodésoxyribonucléotides, des polydésoxyribonucléotides et leurs dérivés, tels que la défibrotide, sont des agents de modulation génétique aux niveaux de la transcription, la traduction, des messagers secondaires et de systèmes de tranduction de signaux cellulaires. L'on peut traiter divers états pathologiques en modifiant la dose de ces agents en réponse aux fluctuations observées (par exemple, augmentation, diminution, apparition, disparition) dans des marqueurs de maladies et de réparations normaux.
PCT/US1994/000638 1991-08-21 1994-01-13 Procede d'utilisation de polynucleotides, d'oligonucleotides et de leurs derives pour traiter divers etats pathologiques WO1994015621A1 (fr)

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AU60904/94A AU692433B2 (en) 1993-01-13 1994-01-13 Method for using polynucleotides, oligonucleotides and derivatives thereof to treat various disease states
EP94907246A EP0678029A1 (fr) 1993-01-13 1994-01-13 Procede d'utilisation de polynucleotides, d'oligonucleotides et de leurs derives pour traiter divers etats pathologiques
CA002153778A CA2153778C (fr) 1993-01-13 1994-01-13 Methode d'utilisation de polynucleotides, d'oligonucleotides et de leurs derives pour traiter divers etats maladifs
US09/754,066 US6699985B2 (en) 1991-08-21 2001-01-05 Method of treating HIV infection and related secondary infections thereof
US10/768,089 US7049430B2 (en) 1991-08-21 2004-02-02 Method of treating HIV infection and related secondary infections thereof

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Cited By (10)

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US5977083A (en) * 1991-08-21 1999-11-02 Burcoglu; Arsinur Method for using polynucleotides, oligonucleotides and derivatives thereof to treat various disease states
EP1202750A4 (fr) * 1997-04-28 2002-10-16 Arsinur Burcoglu Traitement de l'infection par vih et de ses infections opportunistes
US6699985B2 (en) 1991-08-21 2004-03-02 Arsinur Burcoglu Method of treating HIV infection and related secondary infections thereof
US7211659B2 (en) 2001-07-05 2007-05-01 Chiron Corporation Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US7282364B2 (en) 2001-08-31 2007-10-16 Novartis Vaccines And Diagnostics, Inc. Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides and uses thereof
US7348177B2 (en) 1998-12-31 2008-03-25 Novartis Vaccines And Diagnostics, Inc. Expression of HIV polypeptides and production of virus-like particles
US7485666B2 (en) 2004-06-17 2009-02-03 Kimberly-Clark Worldwide, Inc. Vaginal health products
US7608642B2 (en) 2002-12-16 2009-10-27 Kimberly-Clark Worldwide, Inc. Wound and skin care compositions
WO2011036632A1 (fr) 2009-09-23 2011-03-31 Valerio Di Nicola Composition contenant un constituant hématique et son utilisation dans le traitement de l'arthrose
US7943375B2 (en) 1998-12-31 2011-05-17 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof

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EP1310555A3 (fr) * 1992-03-16 2003-08-27 Isis Pharmaceuticals, Inc. Modulation par oligonucleotide de la protéine kinase C

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EP0152148A2 (fr) * 1984-02-16 1985-08-21 Crinos Industria Farmacobiologica S.p.A. Composition pharmaceutique pour le traitement d'ischémie myocardiale accusée

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BILSEL, S. ET AL.: "Interaction of 3H-defibrotide with cultured human umbilical vein endothelial cells", THROMBOSIS RESEARCH, vol. 58, no. 5, 1 June 1990 (1990-06-01), XP022878770, DOI: doi:10.1016/0049-3848(91)90250-Z *
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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5977083A (en) * 1991-08-21 1999-11-02 Burcoglu; Arsinur Method for using polynucleotides, oligonucleotides and derivatives thereof to treat various disease states
US6699985B2 (en) 1991-08-21 2004-03-02 Arsinur Burcoglu Method of treating HIV infection and related secondary infections thereof
US7049430B2 (en) 1991-08-21 2006-05-23 Arsinur Burcoglu Method of treating HIV infection and related secondary infections thereof
EP1202750A4 (fr) * 1997-04-28 2002-10-16 Arsinur Burcoglu Traitement de l'infection par vih et de ses infections opportunistes
US7348177B2 (en) 1998-12-31 2008-03-25 Novartis Vaccines And Diagnostics, Inc. Expression of HIV polypeptides and production of virus-like particles
US7943375B2 (en) 1998-12-31 2011-05-17 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US7211659B2 (en) 2001-07-05 2007-05-01 Chiron Corporation Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US8133494B2 (en) 2001-07-05 2012-03-13 Novartis Vaccine & Diagnostics Inc Expression cassettes endcoding HIV-1 south african subtype C modified ENV proteins with deletions in V1 and V2
US9598469B2 (en) 2001-07-05 2017-03-21 Novartis Vaccines And Diagnostics, Inc. HIV-1 south african subtype C env proteins
US7282364B2 (en) 2001-08-31 2007-10-16 Novartis Vaccines And Diagnostics, Inc. Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides and uses thereof
US7608642B2 (en) 2002-12-16 2009-10-27 Kimberly-Clark Worldwide, Inc. Wound and skin care compositions
US7485666B2 (en) 2004-06-17 2009-02-03 Kimberly-Clark Worldwide, Inc. Vaginal health products
US8344022B2 (en) 2004-06-17 2013-01-01 Kimberly-Clark Worldwide, Inc. Vaginal health products
WO2011036632A1 (fr) 2009-09-23 2011-03-31 Valerio Di Nicola Composition contenant un constituant hématique et son utilisation dans le traitement de l'arthrose

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