WO1998047516A1 - Combination therapy utilising 17-ketosteroids and interleukin inhibitors, or interleukin-10 potionally with interleukin inhibitors - Google Patents

Combination therapy utilising 17-ketosteroids and interleukin inhibitors, or interleukin-10 potionally with interleukin inhibitors Download PDF

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Publication number
WO1998047516A1
WO1998047516A1 PCT/EP1997/005716 EP9705716W WO9847516A1 WO 1998047516 A1 WO1998047516 A1 WO 1998047516A1 EP 9705716 W EP9705716 W EP 9705716W WO 9847516 A1 WO9847516 A1 WO 9847516A1
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Prior art keywords
interleukin
patient
inhibitor
compound
effective amount
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PCT/EP1997/005716
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French (fr)
Inventor
Patrick T. Prendergast
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Prendergast Patrick T
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Publication date
Priority claimed from PCT/IB1997/000414 external-priority patent/WO1997038695A1/en
Application filed by Prendergast Patrick T filed Critical Prendergast Patrick T
Priority to AU52219/98A priority Critical patent/AU5221998A/en
Publication of WO1998047516A1 publication Critical patent/WO1998047516A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • Ant i -viral agents which inhibit replication of viruses have been known since the mid 1960 ' s . Several hundred or more of these agents are now known , including those of general Formula I which are discussed in U . S .
  • IPA is a naturally occurring compound.
  • it has been shown to be an ancicodon-adj acent nucleoside in certain t-R As (Biochimica et Biophysica Acta, 281:488-500.
  • IPA has been shown to have cytokinin properties, to inhibit the growth of human leukemic myoblasts, to inhibit the growth of cultured lymphocytes stimulated by phytohemagglutinin (PHA) at certain concentrations, and to stimulate the growth of
  • a method of enhancing the T H 1 immune response in a patient comprising administering to the patient a) an effective amount of a 17-ketostero ⁇ d compound and an effective amount of an Interleu m-10 inhibitor.
  • the Interleuk ⁇ n-10 inhibitor can be antiserum to Interleukm-10, a compound effective for inhibiting synthesis or biological function of Interleukm-10 or an Interleukm-10 receptor molecule blocking agent.
  • the Interleukm-10 inhibitor can also be canavanme sulfate, -canavamne sulfate, herbimycm A, geissem, secalonic acid D, an isoflavonoid, a cytokinin, or an amphiphilic triterpenoid.
  • the Interleukm-10 inhibitor can be selected from the group having the general formula as shown m Formula I herein.
  • a method of enhancing the T H 2 immune response m a patient comprises administering to the patient an effective arro_nt of a 17-ketostero ⁇ d compound and an effective amount of an Interleukm- 12 mnibitor.
  • the Interleukm- 12 mmb tor can be antiserum to Interleukm- 12, a compound effective for inhibiting synthesis or biological function of Interleuk ⁇ n-12 or an Interleu-cm-12 receptor molecule blocking agent
  • the method can be used to treat a patient, e.g., for a bacterial infection or autoimmune condition such as lupus or graft versus host disease.
  • Compos tions and pharmaceutical formulations useful for a combination therapy include a 17-ketostero ⁇ d compound and anot ⁇ er compound as disclosed herein, e.g., an Interleuk ⁇ n-10 inhibitor, an Interleukm-2 inhibitor or an Interleukm- 12 inhibitor.
  • Articles of manufacture are also disclosed, comprising for example, packaging material, at least one unit-dosage of a 17-ketostero ⁇ d compound, and a label or package insert indicating that the 17-ketostero ⁇ d compound can be used in a method disclosed herein.
  • a method of treating a patient for an autoimmune condition comprises administering to the patient an effective amount of Interleuk ⁇ n-10.
  • the method is useful for treating the patient for lupus, multiple sclerosis, graft versus host disease or as an adjuvant m vaccine therapy
  • the antiviral agents generally Formula I herein as disclosed m U.S. Patent No. 4,956,355 (Prendergast) have additional beneficial therapeutic effects when _sed in a combination therapy with agents that mnioit I -10 synthesis and/or action, termed 11-10 inhibitory compounds or 11-10 inhibitors .
  • a method of enhancing tne T (l l immune protective response when using a 17-ketosteroid compound as an anti-viral, anti-bacterial, anti-mycoplasma, or anti-intracellular parasitic agent.
  • An immune response for a given patient is enhanced for a T H 1 immune response (cell -mediated immunity) when T H 1 cytokines are produced.
  • T H 1 cytokines can include but are not limited to gamma- interferon ( ⁇ -IFN) , IL-2, and IL-12.
  • a T H 1 immune protective response is required, for example, by patients in need of therapy for cancer, metastatic cancer, multi- drug resistant cancer, viral infection, parasite infection, multi-drug resistant bacterial infection and bacterial infection.
  • the novel method comprises the administration of a 17-ketosteroid compound and an IL-10 inhibitor to a patient.
  • a patient is a mammal, e.g., a rat, a mouse, a human, a cow, or a dog.
  • a patient is a human.
  • a patient can be in need of treatment for a disease or can be suspected of being in need of such treatment.
  • a 17-ketosteroid compound according to the invention has the following general formula I :
  • the broken line represents an optical double bond and the hydrogen atom at position 5 is present in the ⁇ - or ⁇ -configuration .
  • the compound can also comprise a mixture of both configurations.
  • R represents a hydrogen atom and R ⁇ represents, without limitation, a hydrogen atom or an S0 2 OM group with M representing, without limitation, a hydrogen atom; a sodium atom; a sulphatide group; a phosphatide group; or a glucuronide group.
  • the sulphatide group can be the following:
  • R 2 and R 3 which may be the same or different, being straight or branched chain alkyl radicals of 1 to 14 carbon a oms .
  • the phosphatide group can be the following: O
  • R 2 and R 3 which may be the same or different, being straight or branched chain alkyl radical of 1 to 14 carbon atoms .
  • the glucuronide group can be the following:
  • R and R_ are each hydrogen.
  • An especially preferred compound is dehydroepiandrosterone (DH ⁇ A) wherein R and R_ are each hydrogen and the double bond is present.
  • the compound is epiandrosterone wherein R and R_ are each hydrogen and the double bond is absent (i.e., where the dotted line is shown in formula I, there is a single bond) .
  • This unsaturated 5-position steroid can also be prepared as an anti-viral agent wherein the R position is occupied by any of the following halogens: bromine, chlorine, fluorine, or iodines.
  • the compound is 16 ⁇ - bromoepiandrosterone , wherein R is Br, R . is H, and the double bond is present.
  • the compound is according to formula I, wherein R is Br, R. is H, and the double bond is not present .
  • dehydroepiandrosterone sulphate wherein R is H, R t is S0 2 -OM (M is as hereinbefore defined) , and the double bond is present, as well as 53-androstan-3 ⁇ -ol-17-one .
  • the compound is selected from dehydroepiandrosterone sulphatides, phosphatides, or glucuronide wherein R is H, R x is a sulphatide, phosphatide, or glucuronide group as hereinabove defined, and the double bond is present.
  • the compounds are DHEA conjugates such as hexyl sulfate, dodecyl sulfate, octadecyl sulfate, octadecanoylglycol sulfate, O-dihexadecylglycerol sulfate, hexadecane sulfonate, dioctadecanoylglycerol phosphate, or O-hexadecylglycerol phosphate.
  • DHEA conjugates such as hexyl sulfate, dodecyl sulfate, octadecyl sulfate, octadecanoylglycol sulfate, O-dihexadecylglycerol sulfate, hexadecane sulfonate, dioctadecanoylglycerol phosphate
  • I7-ketosteroids can exist in a poly orpr. form.
  • An Iii- 10 inhibitor can be a compound that inhibits 11-10 activity, for example, antiserum to IL-10.
  • Antiserum to IL-10 can be polyclonal , or monoclonal in origin. Antiserum to IL-10 can comprise intact antibody or can comprise antibody fragments that retain IL-10 binding specificity. Anti-IL-10 antibodies can be directed against IL-10 or peptide fragments possessing epitopes useful in inhibiting IL-10 activity in vivo . Further, 11-10 activity can be inhibited by soluble 11-10 receptor, for example, administered at about 20 microgra s per day to about 200 micrograms per day by intravenous injection.
  • An IL-10 inhibitor can also be a compound that inhibits the synthesis of IL-10 in vi vo .
  • the synthesis of IL-IO can be inhibited by any of a variety of compounds, including one or a combination of the following compounds: canavanine sulfate, L-canavanine sulfate, herbimycin A (Wako Pure Chemicals Industries, Ltd., Japan), genistein (Sigma Chemicals Co., St. Louis, Mo., USA), secalonic acid D, isoflavonoids , cytokinins, amphiphilic triterpenoids , or analogues to the above.
  • An 11-10 inhibitor can also be a compound that blocks the IL-10 receptor in cells of a patient.
  • such compounds can be antibodies against the IL- 10 receptor, anti-idiotype antibodies against an anti-IL- 10 antibody, or antibodies against a portion of IL-10 that binds to the IL-10 receptor.
  • a compound that inhibits IL-10 can be identified, for example, by identifying compounds that have the ability to inhibit cyclic AMP activity.
  • agents that demonstrate IL-10 inhibition as determined by standard assay methods can also serve as IL-IO inhibitors.
  • Compounds that inhibit IL-10 synthesis can be identified, for example, by an IL-10 specific ⁇ LISA. Such compounds also include those that specifically alter transcription of 11-10 DNA, translation of IL-10 RNA, or processing of IL-10 protein.
  • Suitable IL-10 inhibitors also include compounds of the following general formula II:
  • R 5 CH 3 , Cl, OH, or a monophosphate group
  • R CH 3 , CH 2 OH, or Cl
  • R a (CH 2 ) 7 CH 3 ; and R 2 and R 3 are linked to form a 3', 5' -cyclic monophosphate derivative, or a physiologically acceptable salt of any such compound.
  • "Formula II" is used herein to refer to all such compounds and salts.
  • N- ( ⁇ 2 - isopentenyl ) adenosine- 3 ' , 5 ' - cycl ic monophosphate
  • R : H
  • R 2 OK
  • R 3 OH
  • R 4 CH 2 C S H 6
  • R 4 CH 2 C 6 H S N 5 -benzyladenosine-5 ' -monophosphate
  • Ri H
  • R 2 and R 3 are linked to form a 3' , 5-cyclic monophosphate derivative
  • R 4 CH 2 C 6 H 6
  • R t H
  • R : OH
  • R 3 OH
  • R R- OH, R 3 monophosphate and R ;
  • R._ H
  • R 2 and R 3 are linked to form a 3', 5 ' -cyclic monophosphate derivative
  • R 4
  • metabolites of the family of compounds of Formula II include-. N 6 ( ⁇ 2 - lsopentenyl) adenme, 6 -N- ( 3 -methyl -3 -hydroxybutylamino) purine, adenme, hypoxanthine, ur c acid, and methylated xanth es .
  • a 17-ketostero ⁇ d compound and a IL-10 inhibitor can be administered essentially simultaneously, e.g., administration of each compound a few mm tes or a few nours apart, or can be administered sequentially, e.g., several days apart, or more than a week apart.
  • administration of an IL-10 inhibitor can be followed by administration of a 17-ketosteroid . All such variations administration of the co omation therapy are encompassed within the scope of the invention.
  • the invention also includes the use of a 17- ketosteroid and an IL-10 inhibitor m the manufacture of a medicament for use in tne treatment of a condition such as cancer, metastatic cancer, multi-drug resistant cancer, viral infection, parasite infection, multi-drug resistant oacter al infection and bacterial infection. It should be appreciated that more than one l 7 - ketost ⁇ roid and/or more than one IL-10 inhibitor may be used some embodiments of the invention.
  • Compounds used according to this invention are administered by any suitable route, including enteral, parenteral, topical, oral, rectal, nasal, or vaginal routes
  • Parenteral routes include subcutaneous, intramuscular, intravenous, and subl gual administration.
  • Topical routes include buccal and sublmgual administration.
  • the 17- etosterc d compound and tne 11-10 inhibitor can be admmistered ⁇ y the same route or by different routes, recognizing that the route by which a compound is administered depends upon the nature of the compound.
  • both compounds can be administered by mjecticn or both compounds can be administered by mouth.
  • one compound can be administered by injection and the other compound by mouth.
  • compositions prepared according to the invention can include a 17 -ketosteroid compound and an IL-1C inhibitor contained acrophage-specific liposome micells of suitable size to facilitate phagocytosis, tablets (including coated tablets), elixirs, suspensions, syrups, inhalations, gelatine capsules tablet form, dragees, syrups, suspensions, topical creams, suppositories, m ectable solutions (such as a pharmaceutically acceptable solution which may include a carrier) , or k ts for the preparation of a syrup, suspension, topical cream, suppository cr ⁇ jecta-le solution just prior to use.
  • a 17- ketosteroid and an IL-10 inhibitor may be included in a composite which facilitates its slow release into the blood stream, e.g., a silicone disc, polymer beads or a transdermal patch.
  • pharmaceutical preparations prepared according to the invention can utilize conventional excipients, that is, pharmaceutically acceptable organic cr inorganic carrier substances which do not deleter ously react with the compounds.
  • suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arable, vegetable oils, gelatine, carbohydrates, magnesium stearate, talc, silicic-acid, viscous paraffin, fatty acid mono- and di -glycerides .
  • the preparative procedure may include the sterilization of the pharmaceutical preparations.
  • the compounds may be mixed with auxiliary agents such as lubricants, preservatives, stabilizers, salts for influencing osmotic pressure, and the like, which do not react deleteriously with the compounds.
  • This invention includes tne use of physiologically acceptable salts of Formula II, for example, those derived from inorganic acids such as hydrochloric, sulphuric or pnosphoric acid, and organic sulphuric acids, 3_.cn as p- oluenesulphomc acid or methanes lphonic ac d, and organic carboxylic acids such as acetic, oxalic, succmic, tartaric, citric, malic, or maleic acid
  • the present invention is also directed to the use of such compounds in the manufacture of a medicament for providing such treatment.
  • the pharmaceutical formulation according to the invention may be administered locally or systemically .
  • Systemic administration means any mode or route of administration that results in effective levels of active ingredient appearing in the blood or at a site remote from the site of administration of said active ingredient.
  • a pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral, or topical administration. Indeed, all three types of formulation may be used simultaneously to achieve systemic administration of the active ingredient.
  • a pharmaceutical formulation according to the invention is administered in un t dose comprising from about 5 to about 1000 mg of active ingredient for a compound of general Formula I, to achieve an amount of 17-ketcsteroid effective for a clinically effective therapy.
  • each unit dose comprises from about 5 to about 500 mg of the 17 -ketosteroid.
  • a pharmaceutical formulation contains an anti-IL-10 antiserum, it is administered in unit dose comprising from about 0.2 g to about 5 mg of antiserum (monoclonal or polyclonal) per day.
  • a pharmaceutical formulation according to the invention contains a compound cf general Formula II
  • the formulation as administered has a unit dose comprising from about 0.01 mg to about 5,000 mg of active ingredient of general Formula II, at a rate of 1 unit does to 10 unit doses per day, to achieve an amount of 1L-10 inhibitor effective for a clinically effective therapy.
  • Compounds of formula II can be administered m the range of 0 3 mg to about 80 mg per kilogram of body weight.
  • IPA or IPA-like compounds will depend on many factors, including the mode of administration and the organism being treated. Dosages can be determined by known techniques, for example, by means of an appropriate pnarmacological protocol that compares the activities of the subject compounds to the corresponding activity of a known agent.
  • a combination tnerapy is administered at a rate of from 1 unit dose to 10 unit doses each of a 17-ketostero ⁇ d compound and an IL-10 inhibitor per day.
  • Administration of tne tnerapy accordance with the invention is cont ⁇ n_ec for a period of at least one day and m certain cases ma/ ce given for one week, one month, or for the life of tne individual.
  • the therapy may be administered at intervals of once a week or every other day.
  • an article of manufacture comprises packaging material, at least one unit dose of a 17- ⁇ etost ⁇ ro ⁇ d compound and a label or package insert indicating that the 17 -ketosteroid compound can be used a memod as described herein
  • the packaging material can be ⁇ ade from one or more generally known materials, e.g., foam, cardboard, fibrebcard, polystyrene and polypropylene, and is of a size suitable to contain the compound (s) accompanying the packaging material.
  • a label or package insert can be a tag or label secured to the packaging material, a label printed on the packaging material or a label inserted within the packaging material.
  • the label indicates that the 17-ketosteroid can be used in a therapy as disclosed herein, e.g., in combination with an IL-10 inhibitor.
  • the label can also indicate that the compound (s) have received approval from an official agency, for example, the U.S. Food and Drug Administration, for medical or veterinary use according to the -ethod.
  • the label may also indicate suitable administration routes, dosage regimen, and the like. If desired, the article may contain additional components such as at least one unit dose of an 11-10 inhibitor or at least one unit dose of a different 17-ketostero ⁇ d .
  • an article of manufacture comprises packaging material, at least one unit dose of an Interleuk ⁇ n-10 inhibitor and a label or package insert indicating that the Interleukm- 10 inhibitor can be used m a method as disclosed herein.
  • the label indicates that the IL-10 inhibitor can be used in a therapy as disclosed herein, e.g., in combination with a 17-ketesceroid.
  • the label can also indicate that the compoun (s) have received approval from an official agency, for example, the U.S. Food and Drug Administra ion, for medical or veterinary use according to the ⁇ ethod.
  • the article may contain, if desired, additional components, such as at least one unit ⁇ ese of a 17-ketosteroid or at least one unit dose of a different 11-10 inhibitor.
  • a patient suffering from an autoimmune condition is treated by administering a compound effective for increasing the amount or activity of IL-10.
  • IL-10 effectors include, for example, bone marrow transplant rejection, graft versus host disease, lupus, and multiple sclerosis. It has been discovered that administration of IL-10 effectors results in a dramatic improvement in patients suffering from such autoimmune conditions. IL-10 effectors have also been discovered to enhance the antibody response in patients who suffer from a reduced immune response to a vaccine, e.g., elderly patients or very young patients. Elderly patients may have a reduced resDor.se to influenza vaccine, flu vaccine, or hepatitis B vaccine, for example.
  • Suitable IL-10 effectors include purified IL-10 and the polypeptide encoded by the Epstein Barr virus open reading frame (ORF) BCRF1.
  • Purified 11-10, as well as other mterleukins such as 11-12, can be prepared from a native source such as blood. However, purified mterleukins such as 11-10 and 11-12 preferably are prepared from an in vi tro cell culture expressing the desired interleukm at high levels. Interleukms can be expressed from a endogenous gene and/or from a recombmant gene introduced into the cultured cells under the control of a constitutive cr inducible regulatory element Tne interleukm is purified, stored and packaged until use under conditions -cnown in the art.
  • An IL-10 effector can also oe a protein having conservative amino acid substitutions relative to the native 11-10 ammo acid sequence, short deletions, in- frame fusions and other modifications that do not alter the biological function of 11-10.
  • a method of enhancing the ⁇ 2 immune response m a patient comprises the step of administering an effective amount of a 17- ketosteroid compound and an effective amount of a compound that inhibits synthesis or b_olog cal activity of 11-12
  • Compounds that inhibit 11-12 syntnesis can be determined oy, for example, 11-12 specific ⁇ 1ISA and can include compounds that specifically alter transcription of 11-12 DNA, translation of 11-12 RNA, or processing of 11-12 protein
  • Compounds that mr.ioit the b_olcg ⁇ cal activity of 11-12 can include compounds that inhibit cyclic A>'P activity in addition to demonstrating 11-12 inhibition in an 11-12 screening protocol.
  • An 11-12 mnibitor can include, without limitation, antiserum to 11-12 or an 11-12 receptor molecule blocking agent.
  • Antiserum to 11-12 can oe either polyclcnal or monoclonal n origin and can be an antibody fragment having specificity and binding capacity substantially similar to intact ant ⁇ -ll-12 antibody.
  • a method of enhancing the T ⁇ 2 immune response in a patient comprises the step of administering an effective amount of a 17- ketosteroid compound and an effective amount of a compound that inhibits synthesis or biological activity of mterleukm-2 (11-2) .
  • Compounds that mhioit IL-2 synthesis can be determined by, for example, IL-2 specific ⁇ LISA and can include compounds that specifically alter transcription of IL-2 DNA, translation of IL-2 RNA, or processing of IL-2 protein.
  • Compounds that inhibit the biological activity of IL-2 can include compounds that mnib t cyclic AMP activity in addition to demonstrating IL-2 inhibition m a standard IL-2 assay.
  • th s rre r.cd is useful for tncse patients wnose response to 17 -ketosteroid monotherapy is detrimentally affecte ⁇ o ⁇ unwanted enhancement of the T 1 immune response.
  • An 11-2 inhibitor can include, without limitation, antiserum to 11-2 or an 11-2 receptor molecule blocking agent.
  • Antiserum to 11-2 can be either polyclonal or monoclonal.
  • Antibody fragments that retain oindmg specificity for 11-2 are also useful as an 11-2 inhibitor .
  • a method of enhancing the T2 immune response in a patient comprises the step of administering an effective amount of a 17- ketosteroid compound and an effective amount of an T H 1 i munosuppressive peptides of human, bacterial, viral, or synthetic organ, such as alpha-fetoprotein (AFP) .
  • a method of enhancing the T ⁇ l immune protective response in a patient comprises administration of a 17- ketosteroid compound and antiserum to human AFP.
  • a combination therapy of a 17- ketosteroid compound and antiserum against AFP is useful, for example, when it is desired to enhance the patient's response tc 17 -ketosteroid mcnotherapy as an anti-viral, anti-bacterial, anti-mycoplasma, or anti-parasitic agent.
  • - Human alpha-fetoprotein antiserum can be either polyclor.al or monoclonal in origin.
  • Antibody fragments that retain specificity and binding capacity substantially similar to intact anti-AFP antibody are also useful .
  • a method of treating cancer, viral infection, metastasis, multi-drug resistant cancer, multi-drug resistant bacterial infection and non-drug resistant bacterial infection in a patient in need of such treatment comprises the step of administering an interieukin- 12 effector and an 11-10 inhibitor .
  • An 11-12 effector can be, for example, recombmant human 11-12.
  • Human 11-12 is a d sulf ide-bonded heterodi eric cytokine consisting of a 40- and 35 -kD subunit. The genes for this cytok e have been cloned and purified recombmant protein has been produced.
  • An 11-10 mhiD tcr useful in this aspect of the invention is similar to those indicated herein.
  • Another aspect of the invention includes administering a 17-ketostero ⁇ d and a lysosomotropic agent to a patient in need of treatment for a disease.
  • a lysosomotropic agent includes, without limitation, amantadme, tnbutylamme , chloroqu e, methylamme, qumac ⁇ ne and pr aqume.
  • Another aspect of the invention includes administering a 17-ketostero ⁇ d and NG- monomethyl - 1 -argmme (1-NMMA) or sodium nitroprusside .
  • 17-ketostero ⁇ d ⁇ such as DH ⁇ A
  • 17-ketostero ⁇ d ⁇ have sometimes produced variable responses v/hen administered as a monotherapy for specific conditions (e.g , lupus, multiple sclerosis, and HIV"* because of patient- o-patient variation in cytcrcme profiles ana immune reactions t at occur in the patient after administration of the steroid
  • the therapeutic effectiveness of a 17-ketostero ⁇ d depends en the cytokme profile of the patient during and prior to 17-ketosteroid monotherapy. When such a steroid s first administered, the immune therapeutic response is very much patient-specific.
  • the present invention advantageously utilizes a combination therapy, e.g., administration of a 17-ketostero ⁇ d and an 11-10 inhibitor when an ennanced T I response to produce a more consistent therapeutic benefit. It is believed tnat SLC ⁇ .
  • a combination therapy renders patient responses more predictable by reducing or eliminating unwanted elevation of 11-10, a characteristic of Th 2 immune responses.
  • Such a combination therapy facilitates enhancement of 11-12 mediated immune responses without the general negative effects of concomitantly enhancing 11-10 mediated immune responses .
  • T H 2 immune response it is sometimes desirable to enhance the T H 2 immune response, e.g., in an autoimmune condition.
  • a combination therapy comprising a 17-ketosteroid compound and an 11-10 effector can be advantageously utilized. It is believed that such a combination therapy produces a more consistent therapeutic benefit by enhancing 11-10 mediated immune responses without the general negative effects cf concomitantly enhancing 11-12 mediated immune responses .
  • Tne following is a summary of using DHEA as a monotherapy in an open-label dose-escalation trial of oral DHEA tolerance and pharmacokmetics in patients with HIV disease.
  • Phase I DHEA trial (early symptomatic HIV disease and 200 to 500 CD4+ lymphocytes/ ⁇ L N , absolute CD4 counts m the control, placebo-assigned patients declined by a median 5 cell/montn.
  • patients m the lowest-dose group studied the Phase I DHEA trial (whose immune system would net be expected to decline faster than that of placeco- treated patients in tne ctner trial) had a median CD4. decline cf 31 cells/month. Substantial therapeutic benefit could not be accomplished by the use of DHEA alone .
  • Example 2 Effect of Administering DHEA in Combination with an IL-10 Inhibitor to HIV Patients
  • the steroid was combined with an agent to inhibit or interrupt the synthesis and/or action of IL-10.
  • This combination therapy is the preferred embodiment of using compounds according to general formula I when it is desired for the 17- ketosteroid to generate a Th 3 immune response.
  • the component used to counteract the Th_ suppressive 11-10 immune side effect was rabbit polyclcnal antiserum against human 11-10.
  • the combination therapy was administered to HIV-p patients, the removal cf viral particles from each patient's bloodstream was enhanced by three logs relative to DHEA monotherapy.
  • the CD4+ helper T-cell count increased by over 80% with combination therapy.
  • the Delayed Type Hypersensitivity response lost at sero-conversion was also restored with combination therapy.
  • the beneficial action of administering a 17- ketosteroid and an 11-10 inhibitor has wider therapeutic usefulness than solely for treatment of HIV.
  • DH ⁇ A therapy to lupus patients and to other T H 1 autoimmune conditions is directly related to the increase of endogenous 11-10 levels achieved in the patient by the administration of DHEA.
  • Bone marrow transplant rejection was put into remission by DHEA administration to enhance 11-10 levels.
  • a patient (RD, date of birth: 14/7/1983) had Acute Myeloid leukaemia M3 in remission following allogenic bone marrow transplant. His major active problems were GUT Graft Versus Host Disease (GVHD) and severe lung disease.
  • GVHD GUT Graft Versus Host Disease
  • a physician's report indicated that RD's general health had improved over a three month period that coincided with administration of therapy for 14 days and he is now enjoying good health.
  • a means of achieving relief of symptoms for such autoimmune conditions is to administer IL-10 in combination with a 17 -ketosteroid to facilitate the remission of symptoms of autoimmune diseases such as lupus and GVHD .
  • IL-10 in combination with a 17 -ketosteroid to facilitate the remission of symptoms of autoimmune diseases such as lupus and GVHD .
  • MS Multiple Sclerosis
  • DHEA monotherapy results m high patient-to-patient variability, similar to the variability observed with lupus.
  • Remission of symptoms m MS was identified in those patients who experienced significant elevation of their endogenous levels of 11-10.
  • direct admission of recombmant 11-10 to a MS model in the lewis rat demonstrated remission of symptoms. If 11-10 is ad inistered prior to the onset of myelin damage the symptoms of multiple sclerosis are prevented altogether.
  • DHEA Another area of therapeutic benefit ascribed to DHEA is the enhancement of vaccine antigen recognition by the immune system in the elderly.
  • Administration of recombmant IL-10 in conjunction with vaccine administration was a means of achieving enhanced antibody response in patients having a reduced vaccine response (e.g., elderly or very young patients) .
  • Administration of 11-10 or an IL-10 effector removes the uncertainties associated with the use of DHEA.
  • EAE Experimental autoimmune encephalomyelitis
  • Reco binant IL-10 was administered to ⁇ AE-induced lewis rats. After administering II- 10, animals were observed for symptoms of MS. All animals receiving II- 10 showed complete remission of symptoms. All animals receiving mock injections showed no change in symptoms or a deterioration m symptoms.
  • ASSAY PARAMETERS Test Reagent Dehydroisoandrosterone (DHEA) Sigma D-4000 Test Cells: Mononuclear cells from five HIV-1 positive donors Mononuclear cells from two HIV-1 negative donors 200 ⁇ L culture; at least 3 replicates of each experimental condition Basic Medium; RPMI 1640. 10% FBS, 50 ⁇ L gentamicin
  • DHEA 10 A 0 0 0 0 0 0 -10M anti-IL-12 0 0 0 0 0 0
  • the stimulation caused by DHEA in each of these cases was equal to or greater than that caused by IL-12, although the concentration of DHEA causing the stimulation varied from sample to sample In blood samples from three other patients (E9A, HIV-1 negative; E9D and ⁇ 9G, HIV-1 positive), proliferation in the presence of gpl20 was suppressed by addition of DHEA or IL-12.
  • IL-12 murine IL-12
  • NK cytotoxic natural killer
  • enhancement cf cy olytic T cell generation results m augmentation of cytotoxic natural killer (NK) /lympnocytes-activated killer cell activity, enhancement cf cy olytic T cell generation, and induction of ⁇ -IFN secretion.
  • NK cytotoxic natural killer
  • cf cy olytic T cell generation results m vo activity of murine IL-12 against a number of murine tumors.
  • Experimental pulmonary metastases or subcutaneous growtn of the Bl ⁇ FLO melanoma ⁇ .ere markedly reduced m rr ce treated intra pen oneally ⁇ ith I -12, resulting m an increase m survival time.
  • I -12 The therapeutic effectiveness of I -12 was dose dependent and treatment of subcutaneous tumors were effectively treated by IL-12 at doses which resulted in no gross toxicity.
  • Local pentumoral injection of IL-12 into established subcutaneous Renca tumors resulted in regression and complete disappearance of these tumors.
  • IL-12 was as effective in NK cell -deficient beige mice or in m ce depleted of NK cell activity by treatment with anti- asialc GM1, suggesting that NK cells are net the primary cell type mediating the anti tumor effects of this cytokine.
  • the efficacy of IL-12 was greatly reduced in nude mice, suggesting the involvement of T cells.
  • depletion of CD8+ but not CD4+ T cells significantly reduced the efficacy of IL-12.
  • IL-12 has potent in vivo anti- tumor and anti -metastatic effects against murine tumors and demonstrate the critical role of CD8+ T- cells in mediating the anti -tumor effects against subcutaneous tumors .
  • the involvement of IL-12 with the generation of CD8+ cell populations was demonstrated in a study of HIV+ pa_ier._s who were treated with anti-human IL-10 antiserum. HIV viral load was reduced to zero by the administration of rabbit anti-human IL-10 antiserum. Patients showed an 84% increase in CD8+ cell population above baseline values. The decrease in in vi vo IL-10 levels allowed CDS. cell populations to increase and allowed for KIV viral clearance by restoring HIV specific cell mediated immune response.
  • Antibody Class IgG Antigen Used: Recombinant human IL-10
  • IL-la IL-13, IL-2, IL-3, IL- 4, IL-6, IL-7, IL-8, MIP-1A, TNFa and GM-CSF done by EIA.
  • IL-12 (R&D Systems, Minneapolis, Minnesota, #219-IL) 5 fg should be sufficient for the entire experiment .
  • gpl20 available in house (50 fg/vial, about 1 mg/mL) . Need 5nM/m . 50 fg is enough for two assays with two plates each.
  • Normal human HBV-1 negative
  • PBMC Peripheral blood mononuclear cells
  • RIO Medium RPMI , 10% FBS, 50 fg/mL gentamicin.
  • Another HIV- sample should be run after all the HIV- samples have been completed.
  • Ty e cf Study Phase I/II Clinical Trial. Stud y Objectives : determine the safety and tolerance of administered D+I in oersons with advanced HIV diseases, the effect of administration of D+I on measures of HIV viral load (serum PCR (RNA) levels and HIV p24 antigen by the acid dissociation method) , the immune and toxicological effects of administered D + I, and the pharmokmetics of administered D+I .
  • measures of HIV viral load serum PCR (RNA) levels and HIV p24 antigen by the acid dissociation method
  • Inclusion Criteria Age 18 years or older; HIV-1 seropositive; a CD4+ -T-lymphocyte count of 50 to 300 cells/m ⁇ r with one month prior to study entry, measured on two separate occasions 72 hours to 28 days apart; baseline laboratory values of Hemoglobin > 9 g/dl, WBCs > 1500 cells/ ⁇ l, Neutrophils > 1000 cells/ ⁇ l, Platelets 25,000 cells/ ⁇ l, Bilirubm ⁇ 2.0 mg/dl, AST, ALT, Alkaline Phosphatase ⁇ 5x upper limit of normal, and Creatimne ⁇ 1.5 mg/dl.
  • Exclusion Criteria Previous treatment with cne otherapeu ic agents within eight weeks of enrollment, active, major infection, including AIDS-defining opportunistic infection, or other life-threatening medical crisis; pregnant or breast-feeding; any condition which, in the investigator's opinion places the patient at undue risk or jeopardized the objectives of the trial; or receiving immunomodulatory therapies including interferon or pharmacological doses of steroids at entry into the study.
  • Safety Measures Weekly analysis up to week 4 of the study of the following parameters: documentation and assessment of adverse events,- hematology; clinical chemistries and urinalysis; assessment of the immune responses resultant from D+I; and assessment of PCR (RNA) and DNA measures alteration with therapy.
  • Measures of viral load will include HIV-p24 antigene ia, and HIV-RNA PCR (cell- free, serum) and cell HIV-DNA analysis.
  • Improvements in immune response will be measured as changes from baseline in CD4/CD8 ratio.
  • Clinical lymphocyte counts, percent alterations in W3C, and percent alterations in IL-10 levels which would demonstrate the ability of D+I to cause the patients' immune system to move to T1 status.
  • Clinical benefit will be assessed by change in total body weight, Karnofsky performance score, and amelioration of signs and symptoms of disease present at baseline .
  • Study Design Open-label, daily administration of a dose per patient of 1200 mg/day, with review and assess ent of the dosage schedules and efficacy after theraov for 4 weeks.
  • Study Size 5 Patients (total) - 5 patients at 1200 mg/day for 30 days.
  • Test Articles Test Drug: compound Dtl with a particle size distribution in gelatine capsules of 200 mg per capsule: 87% ⁇ 5 ⁇ , 100% ⁇ 15 ⁇ m. Each capsule contains: 600 mg of DHEA and 600 mg of Isopentenyl adenosine 5 ' -monophosphate . No control drug or placebo was used. Measurements were gathered from each patient before and after administration of D+I at weeks 0, 1, 2, 3, 4, and 8.

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Abstract

There are provided medicaments, methods of making them, and kits, which include (1) a 17-ketosteroid compound and/or (2) an Interleukin-10 inhibitor, an Interleukin-12 inhibitor, an Interleukin-2 inhibitor, alpha-fetoprotein, or a lysosomotropic agent. Also provided are methods of treating a patient with Interleukin-10, or with Interleukin-12 and an Interleukin-10 inhibitor. There are also provided methods of treatment involving such compounds or combinations of compounds, including enhancing the TH1 immune protective response when using the 17-ketosteroid compound as an anti-viral, anti-bacterial, anti-mycoplasma or anti-intra cellular parasitic agent, and other treatments for various compounds and combinations as described.

Description

- I -
COMBINATION THERAPY UTILISING 17-KET0STER0IDS AND INTERLEUKIN INHIBITORS, OR INTERLEUKIN-10 POTIONALLY WITH INTERLEUKIN INHIBITORS
5 BACKGROUND OF THE INVENTION
Ant i -viral agents which inhibit replication of viruses have been known since the mid 1960 ' s . Several hundred or more of these agents are now known , including those of general Formula I which are discussed in U . S .
10 Patent No . 4 , 956 , 355 .
The compound N5- (Δ2- isopentenyl) adenosine (IPA), which is of general formula II, illustrated below, has been used previously in clinical trials involving the treatment of cancer. (CYTOKININS AS CHEMOTKERAPEUTIC
15 AGENTS, Annals of _he New York Academy of Science, 25, 225-234 Mittleman, Arnold et al . (1975)). IPA is a naturally occurring compound. For example, it has been shown to be an ancicodon-adj acent nucleoside in certain t-R As (Biochimica et Biophysica Acta, 281:488-500.
20 Gallo, Robert D., et al . (1972). IPA has been shown to have cytokinin properties, to inhibit the growth of human leukemic myoblasts, to inhibit the growth of cultured lymphocytes stimulated by phytohemagglutinin (PHA) at certain concentrations, and to stimulate the growth of
25 cultured lymphocytes stimulated by PHA at lower concentrations (Gallo, et al . ) . Further, IPA has been used in clinical experiments on humans as a chemotherapeutic agent. SUMMARY OF THE INVENTION
A method of enhancing the TH1 immune response in a patient is disclosed, comprising administering to the patient a) an effective amount of a 17-ketosteroιd compound and an effective amount of an Interleu m-10 inhibitor. The Interleukιn-10 inhibitor can be antiserum to Interleukm-10, a compound effective for inhibiting synthesis or biological function of Interleukm-10 or an Interleukm-10 receptor molecule blocking agent. The Interleukm-10 inhibitor can also be canavanme sulfate, -canavamne sulfate, herbimycm A, genistem, secalonic acid D, an isoflavonoid, a cytokinin, or an amphiphilic triterpenoid. In addition, the Interleukm-10 inhibitor can be selected from the group having the general formula as shown m Formula I herein.
A method of enhancing the TH2 immune response m a patient is disclosed The method comprises administering to the patient an effective arro_nt of a 17-ketosteroιd compound and an effective amount of an Interleukm- 12 mnibitor. The Interleukm- 12 mmb tor can be antiserum to Interleukm- 12, a compound effective for inhibiting synthesis or biological function of Interleukιn-12 or an Interleu-cm-12 receptor molecule blocking agent The method can be used to treat a patient, e.g., for a bacterial infection or autoimmune condition such as lupus or graft versus host disease.
Compos tions and pharmaceutical formulations useful for a combination therapy include a 17-ketosteroιd compound and anotπer compound as disclosed herein, e.g., an Interleukιn-10 inhibitor, an Interleukm-2 inhibitor or an Interleukm- 12 inhibitor. Articles of manufacture are also disclosed, comprising for example, packaging material, at least one unit-dosage of a 17-ketosteroιd compound, and a label or package insert indicating that the 17-ketosteroιd compound can be used in a method disclosed herein.
A method of treating a patient for an autoimmune condition comprises administering to the patient an effective amount of Interleukιn-10. The method is useful for treating the patient for lupus, multiple sclerosis, graft versus host disease or as an adjuvant m vaccine therapy
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS -.ccordmg to the invention, there are provided corabιπa:ιons of compounds for use restoring normal levels cf Interieukm-12 (IL-12) ard/or Interleukm-10 (_L-10) (cyto.me inhibitory factor), by enhancing or curtailing the synthesis or effect of IL-12 and/or IL-10. In one aspect of the present invention, the antiviral agents (general Formula I herein) as disclosed m U.S. Patent No. 4,956,355 (Prendergast) have additional beneficial therapeutic effects when _sed in a combination therapy with agents that mnioit I -10 synthesis and/or action, termed 11-10 inhibitory compounds or 11-10 inhibitors .
In another aspect of the present invention, there is provided a method of enhancing tne T(ll immune protective response when using a 17-ketosteroid compound as an anti-viral, anti-bacterial, anti-mycoplasma, or anti-intracellular parasitic agent. An immune response for a given patient is enhanced for a TH1 immune response (cell -mediated immunity) when TH1 cytokines are produced. TH1 cytokines can include but are not limited to gamma- interferon (γ-IFN) , IL-2, and IL-12. A TH1 immune protective response is required, for example, by patients in need of therapy for cancer, metastatic cancer, multi- drug resistant cancer, viral infection, parasite infection, multi-drug resistant bacterial infection and bacterial infection.
In one aspect , the novel method comprises the administration of a 17-ketosteroid compound and an IL-10 inhibitor to a patient. For the purposes of this invention, a patient is a mammal, e.g., a rat, a mouse, a human, a cow, or a dog. In preferred embodiments, a patient is a human. A patient can be in need of treatment for a disease or can be suspected of being in need of such treatment.
A 17-ketosteroid compound according to the invention has the following general formula I :
Figure imgf000006_0001
The broken line represents an optical double bond and the hydrogen atom at position 5 is present in the α- or β-configuration . The compound can also comprise a mixture of both configurations. R represents a hydrogen atom and Rλ represents, without limitation, a hydrogen atom or an S02OM group with M representing, without limitation, a hydrogen atom; a sodium atom; a sulphatide group; a phosphatide group; or a glucuronide group. The sulphatide group can be the following:
Figure imgf000007_0001
with R2 and R3 , which may be the same or different, being straight or branched chain alkyl radicals of 1 to 14 carbon a oms .
The phosphatide group can be the following: O
•OCH .CHCH-.OCORT
O
OCOR,
with R2 and R3, which may be the same or different, being straight or branched chain alkyl radical of 1 to 14 carbon atoms .
The glucuronide group can be the following:
Figure imgf000008_0001
When R: is other than a hydrogen atom, the compounds are conjugated compounds. Compounds according to general formula I are disclosed in U.S. Patent No. 4,956,355.
Preferably n the compound of formula I, R and R_ are each hydrogen. An especially preferred compound is dehydroepiandrosterone (DHΞA) wherein R and R_ are each hydrogen and the double bond is present. In a further embodiment of the invention, the compound is epiandrosterone wherein R and R_ are each hydrogen and the double bond is absent (i.e., where the dotted line is shown in formula I, there is a single bond) . This unsaturated 5-position steroid can also be prepared as an anti-viral agent wherein the R position is occupied by any of the following halogens: bromine, chlorine, fluorine, or iodines.
In a further embodiment of the invention, the compound is 16α- bromoepiandrosterone , wherein R is Br, R. is H, and the double bond is present. In a still further embodiment of the invention, the compound is according to formula I, wherein R is Br, R. is H, and the double bond is not present .
Other preferred compounds are dehydroepiandrosterone sulphate, wherein R is H, Rt is S02-OM (M is as hereinbefore defined) , and the double bond is present, as well as 53-androstan-3β-ol-17-one .
Alternatively, the compound is selected from dehydroepiandrosterone sulphatides, phosphatides, or glucuronide wherein R is H, Rx is a sulphatide, phosphatide, or glucuronide group as hereinabove defined, and the double bond is present. In particular, when R: is not hydrogen, the compounds are DHEA conjugates such as hexyl sulfate, dodecyl sulfate, octadecyl sulfate, octadecanoylglycol sulfate, O-dihexadecylglycerol sulfate, hexadecane sulfonate, dioctadecanoylglycerol phosphate, or O-hexadecylglycerol phosphate.
The above-mentioned I7-ketosteroids can exist in a poly orpr. form.
An Iii- 10 inhibitor can be a compound that inhibits 11-10 activity, for example, antiserum to IL-10.
Antiserum to IL-10 can be polyclonal , or monoclonal in origin. Antiserum to IL-10 can comprise intact antibody or can comprise antibody fragments that retain IL-10 binding specificity. Anti-IL-10 antibodies can be directed against IL-10 or peptide fragments possessing epitopes useful in inhibiting IL-10 activity in vivo . Further, 11-10 activity can be inhibited by soluble 11-10 receptor, for example, administered at about 20 microgra s per day to about 200 micrograms per day by intravenous injection.
An IL-10 inhibitor can also be a compound that inhibits the synthesis of IL-10 in vi vo . The synthesis of IL-IO can be inhibited by any of a variety of compounds, including one or a combination of the following compounds: canavanine sulfate, L-canavanine sulfate, herbimycin A (Wako Pure Chemicals Industries, Ltd., Japan), genistein (Sigma Chemicals Co., St. Louis, Mo., USA), secalonic acid D, isoflavonoids , cytokinins, amphiphilic triterpenoids , or analogues to the above. An 11-10 inhibitor can also be a compound that blocks the IL-10 receptor in cells of a patient. For example, such compounds can be antibodies against the IL- 10 receptor, anti-idiotype antibodies against an anti-IL- 10 antibody, or antibodies against a portion of IL-10 that binds to the IL-10 receptor.
A compound that inhibits IL-10 can be identified, for example, by identifying compounds that have the ability to inhibit cyclic AMP activity. In addition, agents that demonstrate IL-10 inhibition as determined by standard assay methods can also serve as IL-IO inhibitors. Compounds that inhibit IL-10 synthesis can be identified, for example, by an IL-10 specific ΞLISA. Such compounds also include those that specifically alter transcription of 11-10 DNA, translation of IL-10 RNA, or processing of IL-10 protein.
Suitable IL-10 inhibitors also include compounds of the following general formula II:
Figure imgf000011_0001
wherein :
R, = H, R: = CK3, R = Chi and R = K, or
Rx = H or CH,S and R. =
Figure imgf000011_0002
and
R5 = CH3, Cl, OH, or a monophosphate group
R, = CH3, CH2OH, or Cl
R, = H or Br or R, = H and R4 =
Figure imgf000012_0001
and XL and X2 are independently selected from H, methyl, ethyl , hydroxyl , the halogens and carboxyl or R4 =
Figure imgf000012_0002
or RΛ =
Figure imgf000012_0003
or R4 =
Figure imgf000012_0004
or Ra = (CH2)7CH3; and R2 and R3 are linked to form a 3', 5' -cyclic monophosphate derivative, or a physiologically acceptable salt of any such compound. "Formula II" is used herein to refer to all such compounds and salts.
Listed below are chemical groups Rx-R4 for especially preferred compounds Ila-IIu according to this invention.
I la: Rλ = H, R2 = OH, R3 = OH and R4 =
Figure imgf000013_0001
Ns = (Δ2-isopentenyl) adenosine lib: R: = R2 = OK, R3 = monophosphate, and R4
Figure imgf000013_0002
N6 = (Δ2-isopentenyl) adenosine- 5 ' -monophosphate lie: R: = H, R: and R3 are linked to form a 3', 5 '-cyclic monophosphate derivative, and R4 =
Figure imgf000013_0003
N- = (Δ2 - isopentenyl ) adenosine- 3 ' , 5 ' - cycl ic monophosphate Ild: R: = H, R2 = OK, R3 = OH, and R4 = CH2CSH6
Ne =benzyladenosine
Figure imgf000014_0001
R3 = R3 = monophosphate, and R4 = CH2C6HS N5-benzyladenosine-5 ' -monophosphate
Ilf: Ri = H, R2 and R3 are linked to form a 3' , 5-cyclic monophosphate derivative and R4= CH2C6H6
N^-benzyladenosine-S ' , 5 ' -cyclic monophosphate
Ilg: Rt = H, R: = OH, R3 = OH, and R
Figure imgf000014_0002
Furfuryladenosine
Ilh: R R- OH, R3 = monophosphate and R;
Figure imgf000014_0003
Nε-furf uryladenosine-5 ' -monophosphate
Hi: Ri = H, R2 and R3 are linked to form a 3', '-cyclic monophosphate derivative, and R4 =
Figure imgf000014_0004
N6-f urf uryladenosine-3 ' , s ' -cyclic monophosphate II j : RL = H, R2 = OH, R3 = OH and R,
Figure imgf000015_0001
N- (purin- 6 -ylcarbamoyl) -O-chloroaniline ribonucleoside Ilk: Rj. = H, R2 = OH, R3 = monophosphate, and R4 =
Figure imgf000015_0002
N- (purin-6 -ylcarbamoyl) -O-chloroaniline ribonucleoside-
5 ' -monophosphate III : R, = H, R2 = OH, R3 = OH and R4 =
Figure imgf000015_0003
N6-adamantyladenosine
Urn: R_ = H, R2 = OH, R3 = monophosphate and R4 =
Figure imgf000016_0001
N6-adamantyladenosine-5 ' -monophosphate
I In: R: = H, R2 = OH R3 OH and R_
O
CNH(CH2)7CH3
N- (purin-6 -ylcarbamoyl) -n-octylamine ribonucleoside
:io: R, = H, R: = OH, R, = monophosphate and R.
O
CNH(CH,)7CH3
N- (purin-6 -ylcarbamoyl ) -n-octylamme rιbonucleosιde-5 ' monophosphate Up: R._ = H, R2 and R3 are linked to form a 3', 5 ' -cyclic monophosphate derivative, and R4 =
O
CNH(CH2)7CH3
N- (purin-6-ylcarbamoyl) -n-octylamine ribonucleosιde-3 ' ,
5 '-cyclic monophosphate
Ilq: R: = O-LS, R; = OH, R, = OH and R =
Figure imgf000017_0001
N- (Δ2 -isopentenyl) -2 -rr.ethylthioadenosine
Ilr: R; = H, R2 = OH, R3 = OK and R4 =
Figure imgf000017_0002
N'- (4 -hydroxy -3 -methyl - trans-2-butenyl) -adenosine
I Is: R: = H, R; = OH, R3 = OH, and R4 =
Figure imgf000017_0003
N6- (3 -chloro- rans -2 -butenyl) adenosine lit: R: = H, R: = OH, R3 = OH and R =
Figure imgf000017_0004
N≤- (3 -chloro-cis-2 -butenyl) adenosine IIu: R, = K, ; = Kj, R3 = CH3 and R4 = H
Also included in the invention are one or more metabolites of the family of compounds of Formula II. Illustrative examoles of metabolites include-. N6(Δ2- lsopentenyl) adenme, 6 -N- ( 3 -methyl -3 -hydroxybutylamino) purine, adenme, hypoxanthine, ur c acid, and methylated xanth es .
A 17-ketosteroιd compound and a IL-10 inhibitor can be administered essentially simultaneously, e.g., administration of each compound a few mm tes or a few nours apart, or can be administered sequentially, e.g., several days apart, or more than a week apart. For example, administration of an IL-10 inhibitor can be followed by administration of a 17-ketosteroid . All such variations administration of the co omation therapy are encompassed within the scope of the invention.
The invention also includes the use of a 17- ketosteroid and an IL-10 inhibitor m the manufacture of a medicament for use in tne treatment of a condition such as cancer, metastatic cancer, multi-drug resistant cancer, viral infection, parasite infection, multi-drug resistant oacter al infection and bacterial infection. It should be appreciated that more than one l7- ketostεroid and/or more than one IL-10 inhibitor may be used some embodiments of the invention.
Compounds used according to this invention are administered by any suitable route, including enteral, parenteral, topical, oral, rectal, nasal, or vaginal routes Parenteral routes include subcutaneous, intramuscular, intravenous, and subl gual administration. Topical routes include buccal and sublmgual administration. In addition, the 17- etosterc d compound and tne 11-10 inhibitor can be admmistered Ωy the same route or by different routes, recognizing that the route by which a compound is administered depends upon the nature of the compound. For example, both compounds can be administered by mjecticn or both compounds can be administered by mouth. As an alternative one compound can be administered by injection and the other compound by mouth.
Pharmaceutical formulations prepared according to the invention can include a 17 -ketosteroid compound and an IL-1C inhibitor contained acrophage-specific liposome micells of suitable size to facilitate phagocytosis, tablets (including coated tablets), elixirs, suspensions, syrups, inhalations, gelatine capsules tablet form, dragees, syrups, suspensions, topical creams, suppositories, m ectable solutions (such as a pharmaceutically acceptable solution which may include a carrier) , or k ts for the preparation of a syrup, suspension, topical cream, suppository cr ιπjecta-le solution just prior to use. Also, a 17- ketosteroid and an IL-10 inhibitor may be included in a composite which facilitates its slow release into the blood stream, e.g., a silicone disc, polymer beads or a transdermal patch.
If desired, pharmaceutical preparations prepared according to the invention can utilize conventional excipients, that is, pharmaceutically acceptable organic cr inorganic carrier substances which do not deleter ously react with the compounds. Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arable, vegetable oils, gelatine, carbohydrates, magnesium stearate, talc, silicic-acid, viscous paraffin, fatty acid mono- and di -glycerides . The preparative procedure may include the sterilization of the pharmaceutical preparations. The compounds may be mixed with auxiliary agents such as lubricants, preservatives, stabilizers, salts for influencing osmotic pressure, and the like, which do not react deleteriously with the compounds.
Compounds of Formula II have especially low toxicity for children and acceptable toxic ty for adult numans , although it is somewhat higher than for children. IPA and IPA-like compounds can be stored dry almost indefinitely if protected from light and storeα at -75=C. IPA s pnotosensitive and deteriorates at room temperature, whetner n a solid form or aσ eo-s or ethanol ;: solutions. It has oeen found tnat the breakdown rate of IPA is approxirrately 3% per month m a dark container at room temperature.
This invention includes tne use of physiologically acceptable salts of Formula II, for example, those derived from inorganic acids such as hydrochloric, sulphuric or pnosphoric acid, and organic sulphuric acids, 3_.cn as p- oluenesulphomc acid or methanes lphonic ac d, and organic carboxylic acids such as acetic, oxalic, succmic, tartaric, citric, malic, or maleic acid The present invention is also directed to the use of such compounds in the manufacture of a medicament for providing such treatment. The pharmaceutical formulation according to the invention may be administered locally or systemically . Systemic administration means any mode or route of administration that results in effective levels of active ingredient appearing in the blood or at a site remote from the site of administration of said active ingredient. A pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral, or topical administration. Indeed, all three types of formulation may be used simultaneously to achieve systemic administration of the active ingredient. A pharmaceutical formulation according to the invention is administered in un t dose comprising from about 5 to about 1000 mg of active ingredient for a compound of general Formula I, to achieve an amount of 17-ketcsteroid effective for a clinically effective therapy. Preferably, each unit dose comprises from about 5 to about 500 mg of the 17 -ketosteroid. If a pharmaceutical formulation contains an anti-IL-10 antiserum, it is administered in unit dose comprising from about 0.2 g to about 5 mg of antiserum (monoclonal or polyclonal) per day. When a pharmaceutical formulation according to the invention contains a compound cf general Formula II, the formulation as administered has a unit dose comprising from about 0.01 mg to about 5,000 mg of active ingredient of general Formula II, at a rate of 1 unit does to 10 unit doses per day, to achieve an amount of 1L-10 inhibitor effective for a clinically effective therapy. Compounds of formula II can be administered m the range of 0 3 mg to about 80 mg per kilogram of body weight.
The dosages of IPA or IPA-like compounds will depend on many factors, including the mode of administration and the organism being treated. Dosages can be determined by known techniques, for example, by means of an appropriate pnarmacological protocol that compares the activities of the subject compounds to the corresponding activity of a known agent.
According to one emoodiment of t e invention, a combination tnerapy is administered at a rate of from 1 unit dose to 10 unit doses each of a 17-ketosteroιd compound and an IL-10 inhibitor per day. Administration of tne tnerapy accordance with the invention is contιn_ec for a period of at least one day and m certain cases ma/ ce given for one week, one month, or for the life of tne individual. Depending on the patient's medical requirements, the therapy may be administered at intervals of once a week or every other day.
In one embodiment, an article of manufacture comprises packaging material, at least one unit dose of a 17-<etostεroιd compound and a label or package insert indicating that the 17 -ketosteroid compound can be used a memod as described herein The packaging material can be ~ade from one or more generally known materials, e.g., foam, cardboard, fibrebcard, polystyrene and polypropylene, and is of a size suitable to contain the compound (s) accompanying the packaging material. A label or package insert can be a tag or label secured to the packaging material, a label printed on the packaging material or a label inserted within the packaging material. The label indicates that the 17-ketosteroid can be used in a therapy as disclosed herein, e.g., in combination with an IL-10 inhibitor. The label can also indicate that the compound (s) have received approval from an official agency, for example, the U.S. Food and Drug Administration, for medical or veterinary use according to the -ethod. The label may also indicate suitable administration routes, dosage regimen, and the like. If desired, the article may contain additional components such as at least one unit dose of an 11-10 inhibitor or at least one unit dose of a different 17-ketosteroιd .
In another embodiment, an article of manufacture comprises packaging material, at least one unit dose of an Interleukιn-10 inhibitor and a label or package insert indicating that the Interleukm- 10 inhibitor can be used m a method as disclosed herein. In this embodiment, the label indicates that the IL-10 inhibitor can be used in a therapy as disclosed herein, e.g., in combination with a 17-ketesceroid. The label can also indicate that the compoun (s) have received approval from an official agency, for example, the U.S. Food and Drug Administra ion, for medical or veterinary use according to the ~ethod. The article may contain, if desired, additional components, such as at least one unit άese of a 17-ketosteroid or at least one unit dose of a different 11-10 inhibitor.
In another aspect of the invention, a patient suffering from an autoimmune condition is treated by administering a compound effective for increasing the amount or activity of IL-10. Such compounds are referred to herein as IL-10 effectors. Autoimmune conditions include, for example, bone marrow transplant rejection, graft versus host disease, lupus, and multiple sclerosis. It has been discovered that administration of IL-10 effectors results in a dramatic improvement in patients suffering from such autoimmune conditions. IL-10 effectors have also been discovered to enhance the antibody response in patients who suffer from a reduced immune response to a vaccine, e.g., elderly patients or very young patients. Elderly patients may have a reduced resDor.se to influenza vaccine, flu vaccine, or hepatitis B vaccine, for example.
Suitable IL-10 effectors include purified IL-10 and the polypeptide encoded by the Epstein Barr virus open reading frame (ORF) BCRF1. Purified 11-10, as well as other mterleukins such as 11-12, can be prepared from a native source such as blood. However, purified mterleukins such as 11-10 and 11-12 preferably are prepared from an in vi tro cell culture expressing the desired interleukm at high levels. Interleukms can be expressed from a endogenous gene and/or from a recombmant gene introduced into the cultured cells under the control of a constitutive cr inducible regulatory element Tne interleukm is purified, stored and packaged until use under conditions -cnown in the art.
An IL-10 effector can also oe a protein having conservative amino acid substitutions relative to the native 11-10 ammo acid sequence, short deletions, in- frame fusions and other modifications that do not alter the biological function of 11-10.
In another aspect of the invention, a method of enhancing the Λ2 immune response m a patient comprises the step of administering an effective amount of a 17- ketosteroid compound and an effective amount of a compound that inhibits synthesis or b_olog cal activity of 11-12 Compounds that inhibit 11-12 syntnesis can be determined oy, for example, 11-12 specific Ξ1ISA and can include compounds that specifically alter transcription of 11-12 DNA, translation of 11-12 RNA, or processing of 11-12 protein Compounds that mr.ioit the b_olcgιcal activity of 11-12 can include compounds that inhibit cyclic A>'P activity in addition to demonstrating 11-12 inhibition in an 11-12 screening protocol. The method is useful for those patients whose response to 17- ketosteroid onotherapy is detrimentally affected by unwanteα enhancement of a T1 immune response (i.e., production of .l cytokines such as IF , 11-2, and 11-12) An 11-12 mnibitor can include, without limitation, antiserum to 11-12 or an 11-12 receptor molecule blocking agent. Antiserum to 11-12 can oe either polyclcnal or monoclonal n origin and can be an antibody fragment having specificity and binding capacity substantially similar to intact antι-ll-12 antibody.
In another aspect of the invention, a method of enhancing the T^2 immune response in a patient comprises the step of administering an effective amount of a 17- ketosteroid compound and an effective amount of a compound that inhibits synthesis or biological activity of mterleukm-2 (11-2) . Compounds that mhioit IL-2 synthesis can be determined by, for example, IL-2 specific ΞLISA and can include compounds that specifically alter transcription of IL-2 DNA, translation of IL-2 RNA, or processing of IL-2 protein. Compounds that inhibit the biological activity of IL-2 can include compounds that mnib t cyclic AMP activity in addition to demonstrating IL-2 inhibition m a standard IL-2 assay. Similar to the effect observed when an 11-12 inhibitor is used, th s rre r.cd is useful for tncse patients wnose response to 17 -ketosteroid monotherapy is detrimentally affecteα oγ unwanted enhancement of the T 1 immune response.
An 11-2 inhibitor can include, without limitation, antiserum to 11-2 or an 11-2 receptor molecule blocking agent. Antiserum to 11-2 can be either polyclonal or monoclonal. Antibody fragments that retain oindmg specificity for 11-2 are also useful as an 11-2 inhibitor .
Patients who have unwanted enhancement of the T 1 response are, for example, patients suffering from an autoimmune condit on. In another aspect of the invention, a method of enhancing the T2 immune response in a patient comprises the step of administering an effective amount of a 17- ketosteroid compound and an effective amount of an TH1 i munosuppressive peptides of human, bacterial, viral, or synthetic organ, such as alpha-fetoprotein (AFP) . For example, a method of enhancing the Tκl immune protective response in a patient comprises administration of a 17- ketosteroid compound and antiserum to human AFP. Administration of a combination therapy of a 17- ketosteroid compound and antiserum against AFP is useful, for example, when it is desired to enhance the patient's response tc 17 -ketosteroid mcnotherapy as an anti-viral, anti-bacterial, anti-mycoplasma, or anti-parasitic agent. - Human alpha-fetoprotein antiserum can be either polyclor.al or monoclonal in origin. Antibody fragments that retain specificity and binding capacity substantially similar to intact anti-AFP antibody are also useful . In another aspect of the invention, a method of treating cancer, viral infection, metastasis, multi-drug resistant cancer, multi-drug resistant bacterial infection and non-drug resistant bacterial infection in a patient in need of such treatment, comprises the step of administering an interieukin- 12 effector and an 11-10 inhibitor .
An 11-12 effector can be, for example, recombmant human 11-12. Human 11-12 is a d sulf ide-bonded heterodi eric cytokine consisting of a 40- and 35 -kD subunit. The genes for this cytok e have been cloned and purified recombmant protein has been produced. An 11-10 mhiD tcr useful in this aspect of the invention is similar to those indicated herein. Another aspect of the invention includes administering a 17-ketosteroιd and a lysosomotropic agent to a patient in need of treatment for a disease. A lysosomotropic agent includes, without limitation, amantadme, tnbutylamme , chloroqu e, methylamme, qumacπne and pr aqume. Another aspect of the invention includes administering a 17-ketosteroιd and NG- monomethyl - 1 -argmme (1-NMMA) or sodium nitroprusside .
Without being bound by theory, it is believed that 17-ketosteroιdε , such as DHΞA, have sometimes produced variable responses v/hen administered as a monotherapy for specific conditions (e.g , lupus, multiple sclerosis, and HIV"* because of patient- o-patient variation in cytcrcme profiles ana immune reactions t at occur in the patient after administration of the steroid It is now believed that the therapeutic effectiveness of a 17-ketosteroιd depends en the cytokme profile of the patient during and prior to 17-ketosteroid monotherapy. When such a steroid s first administered, the immune therapeutic response is very much patient-specific. The present invention advantageously utilizes a combination therapy, e.g., administration of a 17-ketosteroιd and an 11-10 inhibitor when an ennanced T I response to produce a more consistent therapeutic benefit. It is believed tnat SLCΓ. a combination therapy renders patient responses more predictable by reducing or eliminating unwanted elevation of 11-10, a characteristic of Th2 immune responses. Such a combination therapy facilitates enhancement of 11-12 mediated immune responses without the general negative effects of concomitantly enhancing 11-10 mediated immune responses .
On the other hand, it is sometimes desirable to enhance the TH2 immune response, e.g., in an autoimmune condition. In such situations, a combination therapy comprising a 17-ketosteroid compound and an 11-10 effector can be advantageously utilized. It is believed that such a combination therapy produces a more consistent therapeutic benefit by enhancing 11-10 mediated immune responses without the general negative effects cf concomitantly enhancing 11-12 mediated immune responses .
The invention will be further understood with reference to the following illustrative embodiments, which are purely exemplary and should not be taken as limiting the true scope of the present invention as described in the claims. All publications, patents and other references mentioned herein are incorporated by reference in their entirety.
EXAMPLES Example 1
Effect of Administering DHEA Monotherapy to HIV Patients
Experimental evidence using DHEA therapy in HIV. patients has demonstrated that 11-12 levels, as measured by antibody ELISA methods, are elevated; natural killer cell levels are increased together with the synthesis and presence of gamma- mterferon (γ-lFN) ; and HIV viral loads, as measured by HIV PCR (RNA) measurement and quantitative culturing techniques, more than one log after four weeks of DHEA monotherapy. While viral load levels were considerably reduced, however, TH1 immune response improvement did not occur. In fact, due to the elevated levels of IL-12 generated by this monotherapy with DHEA, IL-10 levels increased causing a subsequent decline m CD4-^ cell numbers and the disappearance of a T_l immune response (Delayed Type Hypersensitivity Response'' 3km reaction m patients as evidenced by patient αata was down-regulated by DHEA monotherapy, contrary to previous beliefs of some Skin reaction is only restored oy the removal of IL-10 which is elevated by the DHΞLA monotherapy.
Tne following is a summary of using DHEA as a monotherapy in an open-label dose-escalation trial of oral DHEA tolerance and pharmacokmetics in patients with HIV disease. In the Phase I DHEA trial (early symptomatic HIV disease and 200 to 500 CD4+ lymphocytes/μLN , absolute CD4 counts m the control, placebo-assigned patients declined by a median 5 cell/montn. In contrast, patients m the lowest-dose group studied the Phase I DHEA trial (whose immune system would net be expected to decline faster than that of placeco- treated patients in tne ctner trial) had a median CD4. decline cf 31 cells/month. Substantial therapeutic benefit could not be accomplished by the use of DHEA alone .
Example 2 Effect of Administering DHEA in Combination with an IL-10 Inhibitor to HIV Patients
In-Vivo Trial using Combination Therapy
To counteract the TH1 suppressive immune side effect of DHEA monotherapy, the steroid was combined with an agent to inhibit or interrupt the synthesis and/or action of IL-10. This combination therapy is the preferred embodiment of using compounds according to general formula I when it is desired for the 17- ketosteroid to generate a Th3 immune response. The component used to counteract the Th_ suppressive 11-10 immune side effect was rabbit polyclcnal antiserum against human 11-10. hen the combination therapy was administered to HIV-p patients, the removal cf viral particles from each patient's bloodstream was enhanced by three logs relative to DHEA monotherapy. At the same time, the CD4+ helper T-cell count increased by over 80% with combination therapy. The Delayed Type Hypersensitivity response lost at sero-conversion was also restored with combination therapy. The beneficial action of administering a 17- ketosteroid and an 11-10 inhibitor has wider therapeutic usefulness than solely for treatment of HIV. Example 3
Adminis ration of DHEA and IL-10 Inhibitor to Patients Having TH1 Autoimmune Conditions
The therapeutic benefit of DHΞA therapy to lupus patients and to other TH1 autoimmune conditions is directly related to the increase of endogenous 11-10 levels achieved in the patient by the administration of DHEA. Bone marrow transplant rejection was put into remission by DHEA administration to enhance 11-10 levels. A patient (RD, date of birth: 14/7/1983) had Acute Myeloid leukaemia M3 in remission following allogenic bone marrow transplant. His major active problems were GUT Graft Versus Host Disease (GVHD) and severe lung disease. A physician's report indicated that RD's general health had improved over a three month period that coincided with administration of therapy for 14 days and he is now enjoying good health. For the first time since his diagnosis he has been able to enjoy full days at school . He no longer needs nasal gastric feeds or suffers with diarrhea. His lung function remains at 30% but his exercise tolerance has improved dramatically. He no longer needs a wheelchair and can tolerate light exercise. As he is on no ether drug regime and has been taking this medication for nearly three months, we must consider that this therapy is influencing these beneficial effects on his body. Before commencing the therapy, he was nebulizing Ventolin, Atrovent, and Pul icort four times per day, with oral steroids when necessary. Now he nebulizes only twice a day. I have never seen such a vast improvement m his health with no apparent side effects.
Example 4 Administration of IL-10 to Patients Having an Autoimmune Condition
In clinical experiments, patients who had elevated endogenous 11-10 levels also experienced remission m lupus, whereas patients who, due to other cytokine and immune factors, have not experienced an IL-10 elevation have not demonstrated relief of symptoms. Therefore, a means of achieving relief of symptoms for such autoimmune conditions is to administer IL-10 in combination with a 17 -ketosteroid to facilitate the remission of symptoms of autoimmune diseases such as lupus and GVHD . Experiments with DHEA and measurement of the cytokine profiles of patients o responc to DHEA therapy and those who do net respond to DHEA tnerapy have led to the discovery that elevated 11-10 is the active agent responsible for the alleviation of the clinical symptoms of lupus. Multiple Sclerosis (MS) is believed to be an autoimmune condition. Treatment of MS patients with DHEA monotherapy results m high patient-to-patient variability, similar to the variability observed with lupus. Remission of symptoms m MS was identified in those patients who experienced significant elevation of their endogenous levels of 11-10. Further, direct admission of recombmant 11-10 to a MS model in the lewis rat demonstrated remission of symptoms. If 11-10 is ad inistered prior to the onset of myelin damage the symptoms of multiple sclerosis are prevented altogether.
Example 5 Administration of IL-10 to Patients Having Reduced Vaccine Response
Another area of therapeutic benefit ascribed to DHEA is the enhancement of vaccine antigen recognition by the immune system in the elderly. Analysis of IL-10 levels in elderly patients receiving DHEA in conjunction with a vaccine indicated that those patients having elevated IL-10 levels had enhanced antigen recognition compared to patients not having elevated IL-10 levels. This result suggested that use of 11-10 as a vaccine adjuvant would be useful. Normally, elderly patients would be expected to have a reduced antigen "vaccine take" or immune response due to age.
Administration of recombmant II- 10 to elderly patients in association with or in advance of treatment with an antigen vaccine created an enhanced adjuvant effect and directly enhanced the antibody response.
Administration of recombmant IL-10 in conjunction with vaccine administration was a means of achieving enhanced antibody response in patients having a reduced vaccine response (e.g., elderly or very young patients) . Administration of 11-10 or an IL-10 effector removes the uncertainties associated with the use of DHEA.
Experimental autoimmune encephalomyelitis (EAE) can be induced m a variety of rodent strains and is widely used as a model for MS. Reco binant IL-10 was administered to ΞAE-induced lewis rats. After administering II- 10, animals were observed for symptoms of MS. All animals receiving II- 10 showed complete remission of symptoms. All animals receiving mock injections showed no change in symptoms or a deterioration m symptoms.
Example 6 Administration of IL-12 to HIV Patients In vi tro DHEA 11-12 Study w th HIV + Blood
Protocol to demonstrate that DHEA enhances endogenous levels of 11-12
Restoration of HIV-Specific Cell-Mediated Immune Responses by DHEA Blood samples from four patients were tested for the effect of DHEA or 11-12 the presence of HIV gpl20. One KIV-1 negative control (E9B) and three HIV-1 positive specimens (E9C, Ξ9E and E9F) were stimulated by the addition of DHEA or 11-12 in tne presence of gpl20. Data for these samples is shewn m Tables 1-18.
Table 1
ASSAY PARAMETERS Test Reagent: Dehydroisoandrosterone (DHEA) Sigma D-4000 Test Cells: Mononuclear cells from five HIV-1 positive donors Mononuclear cells from two HIV-1 negative donors 200 μL culture; at least 3 replicates of each experimental condition Basic Medium; RPMI 1640. 10% FBS, 50 μL gentamicin
Figure imgf000036_0001
Table 2A Sample E9C
Values reported are optical density (O.D.)
7 days; M'l'S Cell Proliferation Assay 2h incubation
Figure imgf000037_0001
Control
2 3 4 5 6 7 8 Avy. gpl 20 no ypl 20
D1IEA 10JM 0.177 0 253 0.319 0 479 0.521 0.750 0 486 0.262 0.407 72.8 80.7
DHEA 10-*M 0 227 0 433 0.538 0 685 0 595 0 679 0.697 0 845 0 588 105 1 76.0
DHHA 10'8M 0.325 0 601 0.608 0.570 0 724 0.739 1 053 0.819 0 680 121.6 1 J 2.5
DHEA 10Λ-8M t 0.302 0 425 0.542 0 554 0.543 0.489 0 787 0.804 0.558 99.4 137.3
DHKA ! 0Λ- 10M 0 336 0 513 0 530 0 572 0 505 0 554 0 726 0.608 0.543 97 1 70 6 anti IL- 12 0.281 0 412 0.51 0 718 0 481 86 0 1 16.5
1L-12 0 282 0.494 0 91 1 0 716 0 644 0.624 0 839 0 735 0.681 121 8 144.6
No Reagent 0.270 0.474 0.395 0 612 0.654 0.576 0.684 0.809 0.559 100.0 67.3
Table 2B
without gp 120 % of
2 3 4 Average gp 120 Control
DIIEA 1()Λ-4M 0 604 0.238 0 245 0 717 0.451 80.7
DUE A 10Λ-6M 0 423 0 426 0 465 0.386 0.425 76 0
DIIF.Λ 10Λ-8M 0 400 0.617 0 523 0.975 0.629 1 12.5
DHEA 10Λ-8M + anti IL- 12 0.745 0.504 0.717 1.104 0.768 137.3
DHEA 10Λ-10M 0.375 0.422 0.368 0.413 0.395 70.6 anti IL-12 0.472 0.619 0.582 0.931 0.851 1 16.5
I -12 0 843 0.700 0 701 0 989 0.808 144.6
No Reagent 0.371 0.386 0.414 0.334 0.376 67.3
PHA 1% 0.306 0.281 0.354 0.300 0.310 55.5
Table 3A
Sample E9D
7 days, M 1 S Cell Prolifeiation Assay 275h incubation
- with gpl20- %of gpl20
Control
1 2 3 4 5 6 7 8 Avg gpl20 no gpl20
DIILΛ 10Λ-4M 0689 0320 0720 0804 0690 0443 0709 0579 0619 945 703
DHΓΛ 10Λ-6M 0705 0542 0512 0498 0478 0472 0499 0704 0551 844 480
DHEA 10Λ-8M 0492 0359 0041 0470 0484 0411 0487 0535 0409 626 460
DΠΓΛ IO"MH anti II 12 0661 0419 0619 0351 0484 0473 0463 0472 0493 755 1003
DHEA 10Λ-K)M 0688 0476 0432 0398 0391 0431 0414 0419 0456 699 460 antιIL-12 0517 0486 0500 0534 0509 780 1040
II 12 0588 0548 0378 0447 0565 0451 0525 0608 0514 787 475
No Reagent 0825 0704 0561 0605 0717 0605 0561 0649 0653 1000 444
Figure imgf000039_0001
Table 3B
— without gp 120 % of
2 3 4 Average gp 120 Control
DI IEΛ 10Λ-4M 0.520 0.292 0.522 0.501 0.459 70.3
DI1F.Λ 10Λ-6 0.291 0.286 0.367 0.310 0.314 48.0
DHEA 10Λ-8M 0.300 0.362 0.244 0.295 0.300 46.0
DHEA 10Λ-8M + anti IL- 12 0.491 0.597 0.642 0.880 0.655 100.3
DHEA 10Λ-10M 0.296 0.256 0.416 0.233 0.300 46.0 anti IL- 12 0.517 0.662 0.759 0.778 0.679 104.0
II. 12 0.264 0.259 0.347 0.370 0.310 47.5
No Reagent 0.328 0.314 0.276 0.241 0.290 44.4
PIIA 1% 0.733 0.642 0.582 0.495 0.613 93.9
Table 4Λ Sample E9E
Report of Oellulai Piohferation MTS Assay
7 days, MTS Cell Proliferation Assay 275h incubation
with gpl20 %of gpl20
Control
3 4 5 6 7 Avg gpl20 nogpl20
DHEA 10Λ-4M 0653 0926 0948 0645 0778 0758 0828 0860 0799 1224 732
DHbA 1()Λ-6M 0919 0870 0 1 1131 1068 0725 0739 0943 0916 1402 398
DHEA 10Λ-8 1001 0820 0860 0614 0827 0703 0739 0890 0784 1201 379
DHEA 10Λ-8M i antι!L-12 0909 0880 1368 0864 0873 1218 1375 0648 1017 1557 847
DHbA 10Λ-10M 0988 0764 061K 0715 0718 1069 0781 0618 0788 1204 467 anti 11-12 0783 1091 0873 0753 0875 1340 977
1L-12 1195 1102 0822 0781 0918 0891 0854 0812 0922 1412 807
No Reagent 1114 1051 0.821 0924 1004 0940 0783 0735 0923 1000 648
Table 4B
without gp 120 % of
2 3 4 Average gp 120 Control
D1 IF.Λ 10Λ-4M 0.562 0.608 0.735 0.800 0.675 73.2
DHEA 10Λ-6M 0.413 0.336 0.400 0.320 0.387 39.8
DHEA 10Λ-8M 0.361 0.385 0.321 0.332 0.350 37.9
Figure imgf000042_0001
DHEA 10M 0M 0.551 0.299 0.473 0.402 0.431 46.7 anti II .- 12 0.787 0.905 0.867 1.047 0.902 97.7
1L- 12 0.416 0.991 1 .102 0.469 0.745 80.7
No Reagent 0.607 0.480 0.579 0.726 0.588 84.3
PHA 1 % 0.37 0.487 0.355 0.45 0.418 45.3
Table 5A
Sample E9F
1 days, M I S Cell Proliferation Assay 4h incubation CD44I%
Figure imgf000043_0001
Control
2 3 4 5 6 Avg gpl20 no gp 120
DHEA 10Λ-4M 1227 1309 1302 1288 1 130 1393 1275 1163 981
DHFA 10Λ- ■8M 1 120 1062 1038 1082 1 163 1 167 1 105 1009 697
D11CΛ I0Λ 8 i aIL-12 1 133 1356 1 145 1204 I 157 1232 1208 1100 862 antill -12 0958 1053 1 106 1042 951 871
IL-12 1085 1071 1038 1 179 1052 1024 1075 981 635
No Reagent 1035 1075 1004 1 195 1136 1132 1096 1000 563
Table 5B
-without gp l 20 % oi
2 3 4 Average gp 120 Control
DHEA 10Λ-4M 1 122 0 865 1 201 0 984 1 046 98.1
DHbA 10Λ-8M 0.659 0.628 1 036 0.647 0 743 69 7
DHEA 10Λ-8M ^ anti IL- 12 1 014 0 914 0 907 0 927 0 941 85 2 anti IL- 12 0 924 0 965 0 897 0 929 0.929 87 1
IL- 12 0 638 0 693 0 833 0 549 0 675 63.6
No Reagent 0 510 0 591 0 69 0 589 0 590 55.3
PIIΛ 1% 0 646 0 716 0 595 0 653 70 7
Table 6A Sample E9G
7 days, MTS Cell Proliferation Assay 4h incubation CD441%
Figure imgf000045_0001
Control
3 4 6 Avg gpl20 no gp!20
DHFA 10Λ-4M 1243 270 1229 1351 230 1268 1265 972 1041
DHCA 10Λ- ■8M 1037 1316 1234 1156 1371 1394 1236 950 576
DHLΛ U)Λ 8M+ anti 11 -12 1125 0692 1092 1 172 1271 1332 1147 86 I 905 anti II -12 0989 1 y.u I 532 1268 989 864
IL-12 1404 1204 1273 1225 1248 1120 I 279 982 293
No Reagent 1.373 1279 1229 1361 1325 1243 1302 1000 466
Table 6B
-without gp! 20- % of
2 Average gp 120 Control
DH liA 10Λ-4M 1.416 1.290 .293 .421 1.355 104.1
DHEA 10Λ- 8M 0.644 0.700 0.967 0.690 0.750 57.6
DHEA 10Λ- 8M +anti IL-12 1.046 1.194 1.410 1.063 1.178 90.6 anti IL- 12 1.031 1.262 1.197 1.009 1.125 86.4
IL-12 0.357 0.421 0 402 0.347 0.382 29.5
No Reagent 0.415 0.424 0.416 1.171 0.607 46.6
PI I A 1% 0.478 0.559 0.571 0.638 41.2
Table 7Λ
Sample E9A
5 days, MTS Cell Proliferation Assay 4h incubation No anti IL-2 receptor in plate
wιth pl 20- - -- — % of g l 20 Control
1 2 3 4 5 6 7 8 Avg gpl 20 no gp l20
DHI A 10Λ-4M 1 125 0 861 0 957 0 974 0 969 0 917 0 918 0 887 0 954 95 5 51 4
DHbA 10Λ-6M 0861 0778 0841 0791 0826 0785 0872 0893 083! 833 515
DHbA 10Λ-SM 0871 0803 0768 0812 0820 0807 0886 0749 0815 816 458
DIITA 10Λ 8Mt JIL 12 1060 0795 0785 0775 0726 0758 0750 0843 0812 813 628
DHbA !0Λ-10M I 008 0820 0782 0747 0805 0746 0850 0747 0788 790 396
1)111 A I0Λ-12M 0944 0740 0682 0740 0891 0731 0714 0868 0766 768 402
1L-12 0941 0877 0842 0872 0798 0848 0873 0797 0856 858 585
No Reagent 0894 I 097 0901 1040 1028 0962 0977 1083 0988 100 428
Table 713
without gp 120 -— % of
1 2 3 4 Average gp 120 Control
DHLΛ 10 M 0487 0488 0515 0582 0.513 514
DHEA 10Λ6M 0643 0575 01 7 0514 0535 538
DHEA 10Λ-8M 0458 0.505 0434 0.432 0457 458
DHEA 10Λ-8M tanti IL-12 0468 0713 0578 0747 0627 628
DHEA 10Λ-10M 0387 0376 0388 0431 0395 396
DHEA 10Λ-12M 0399 0.362 0391 0454 0402 403
IL-12 0666 0576 0660 0434 0584 585
No Reagent 0423 0423 0402 0481 0.427 428
Table 8Λ
Sample G9A
7 days, MTS Cell Proliferation Assay 4h incubation With anti II -2 icceplor in plate
OD Wllll p 120- %of gpl20
Control
Avg gpl20 ng p 120
DHbA 10Λ-4M 1123 0601 0719 0555 0651 0873 0719 0880 0743 802 878
DHbA 10Λ-6M 0969 0449 0763 0791 0767 0800 0378 0536 0882 736 581
DHFA 10Λ-8M 0704 0497 0461 0401 0708 0501 0865 0809 0693 841 734
DHEA 10Λ-8Ml al 12 0769 0307 0821 0541 0567 0419 0701 0924 0634 685 917
DHFA 10Λ-10M 0851 0419 0644 0421 0541 0533 0689 0655 0598 648 577
DHΓΛ 10Λ-12M 0799 0548 0420 0S47 0485 0419 0598 0628 0558 603 585
1L-12 1041 0879 0826 0518 0740 0579 0780 0783 0743 803 746
No Reagent Control 0758 1037 0890 0613 0994 0974 0896 1222 0926 100 659
Table 8B
-OD WITHOUT g l 20~ % of
1 2 3 4 Average gp 120 Control
DHEA 10Λ-4M 0.905 0.71 1 0.956 0.681 0.813 87.6
DI I I-Λ 10Λ6M 0.583 0.431 0.555 0.602 0.538 58.1
DHEA 10Λ-8M 0.696 0.955 0.574 0.495 0.680 73.4
DURA 10Λ-8M 1 anti IL-12 0.691 0.823 0.747 0.935 0.849 91.7
DHEA 10Λ-10M 0.595 0.486 0.476 0.580 0.534 57.7
DHEA 10Λ- 12M 0.562 0.497 0.631 0.475 0.541 58.5
IL-12 0.776 0.692 0.617 0.880 0.691 74.6
No Reagent 0.684 0.603 0.565 0.590 0.611 85.9
Table 9A Sample E9B
3 days, MTS Cell Proliferation assay 3h incubation
% of
OD WITH gp 120 gp 120 control 2 3 4 5 Average gpl20 no gp 120~
DHEA 10Λ 0.663 0.668 0.568 0.661 0.634 0.626 0.637 139.9 120.8 -4 M
DHEA 10A 0.577 0.593 0.708 0.667 0.728 0.676 0.658 144.7 11 1.3 -6 M
DHEA 10 0.650 0.621 0.708 0.685 0.692 0.703 0.677 148.7 110.9 -8 M
DHEA 10Λ 0.549 0.719 0.712 0.674 0.717 0.742 0.702 154.3 173.4 -8 - alL-12
DHEA 10A 0.707 0.683 0.691 0.670 0.654 0.648 0.676 148.5 122.3 -10 M
DHEA 10A 0.655 0.619 0.632 0.654 0.638 0.660 0.643 141.3 110.5 -12 M
IL- 12 0.672 0.652 0.665 0.618 0.669 0.689 0.661 145.2 101.3
No Reagent 0.640 0.415 0.398 0.431 0.427 0.419 0.455 100.0 1 13.6 anti IL-2 Receptor? YES YES YES NO NO NO
Table 9B
OD WITHOUT gp 120 % of gp l20 3 4 Average Control
DHEA 10A 0.581 0.595 0.49 0.532 0.550 120.8 -4M
DHEA 10A 0.494 0.517 0.536 0.478 0.506 1 11.3 -6M
DHEA 10A 0.560 0.493 0.475 0.491 0.505 110.9 -8M
DHEA 10 0.823 0.775 0.775 0.783 0.789 173.4 -8M + anti IL- 12
DHEA 10λ 0.556 0.562 0.559 0.548 0.556 122.3 -10M
DHEA 10A 0.531 0.496 0.466 0.528 0.503 1 10.5 -12M
IL-12 0.328 0.4S5 0.542 0.489 0.461 101.3
No Reagent 0.547 0.485 0.523 0.513 0.517 1 13 6 anti 1L-2 YES YES NO NO Receptor?
PHA 1% 0.610 0.862 0.654 0.625 0.638 140.2 -anti 1L-2 Receptor
PHA 1% 0.601 0.672 0.644 0.689 0.662 143.2
Table 10A Sample E9B
6 days, MTS Cell Proliferation assay 3h incubation
% of
OD WITH gp 120 gp 120 control
2 3 4 5 Average gpl20 no gp 120
DHEA 10A 0.903 0.963 1.318 1.398 0.817 1.080 124.0 68.9 -4 M
DHEA 10A 0.601 0.805 0.797 0.963 0.740 1.004 0.817 93.8 77.5 -6 M
DHEA 10A 0.847 0.748 0.770 0.687 0.815 0.773 88.8 55.4 -8 M
DHEA 10A 0.871 0.976 0.897 0.972 0.956 1.332 1.001 1 14.9 104.9
DHEA 10A 0.938 0.727 0.820 0.823 0.850 0.657 0.803 92.1 60.4 -10 M
DHEA 10A 0.959 0.618 0.678 0.771 0.687 0.865 0.763 87.6 59.6 -12 M
1L-12 0.769 0.898 0.91 1 0.864 0.894 0.755 0.852 97.8 58.9
No Reagent 1.012 0.907 0.694 0.871 0.963 0.777 0.871 100.0 61.3 Control anti 1L-2 Receptor? YES YES YES NO NO NO
Table 10B
OD WITHOUT gp 120 % of gp l20 4 Average Control
DHEA 10A 0.538 0.520 0.605 0.737 0.800 68.9 -4M
DHEA 10Λ 0.962 0.570 0.481 0.687 0.675 77.5 -6M
DHEA 10* 0.566 0.524 0.375 0.454 0.482 55.4 -8M
DHEA 10* 0.996 0.913 1.045 0.700 0.914 104.9
-8M -*- anti lL-12
DHEA 10A 0.566 0.578 0.420 0.541 0.526 60.4 -10M
DHEA 10A 0.584 0.546 0.438 0.510 0.520 59.6 -12M
IL-12 0.580 0.612 0.423 0.436 0.513 58.9
No Reagent 0.761 0.350 0.419 0.605 0.534 61.3 anti 1L-2 YES YES NO NO Receptor?
PHA 1% - antι 0.609 0.553 0.548 0.586 0.574 65.9 1L-2 Receptor
PHA 1% 0.567 0.635 0.546 0.524 0.568 65.2
Table 11A DATA SUMMARY FOR SAMPLES E9B, E9A, E9C AND E9D
HIV-1- HIV-1- HIV-1 + HIV-1 + Blood E9B Blood E9A Blood E9C Blood E9D % of % of % of % of gpl20 control gp!20 control gpl20 control gpl20 control gp!20 no gpl20 gpl20 no gpl20 gpl20 no gpl20 gpl20 no gpl20
DHEA 10A 124.0 88.9 80.2 87.8 72.8 80.7 94.8 70.3
-4M
DHEA 10A 93.8 77.5 73.6 58.1 105.1 76.0 84.4 48.0 -6M
DHEA 10* 88.8 55.4 61.1 73.4 121.6 112.5 62.6 46.0 -8M
DHEA 10Λ 114.9 104.9 68.5 91.7 99.4 137.3 75.5 100.3 -8M + anti 1L-12
DHEA 10A 92.1 60.4 64.6 57.7 97.1 70.6 69.9 46.0 -10M
DHEA 10A 87.6 59.6 60.3 58.5 -12M anti IL- 12 86.0 116.5 78.0 104.0
IL-12 97.8 58.9 80.3 74.6 121.8 144.6 78.7 47.5
NoReagent 100.0 61.3 100 66.9 100.0 67.3 100.0 44.4
PHA 1% 65.2 55.5 93.9
Table 11B DATA SUMMARY FOR SAMPLES E9E, E9F AND E9G
E9E E9F E9G
% of % of % of gpl20 control gpl20 control gpl20 control gpl20 no pl20 gpl20 no gpl20 gp!20 no gp!20
DHEA 10A 122.4 73.2 116.3 98.1 97.2 104.1 -4M
DHEA 10A 140.2 89.8 -6M
DHEA 10* 120.1 37.9 100.9 69.7 95.0 57.6 -8M
DHEA 10* 155.7 84.7 110.0 3.2 88.1 90.5 -8M + anti 1L-12
DHEA 10 120.4 46.7 -10M anti IL- 12 134.0 97.7 95.1 87.1 98.9 86.4
IL-12 141.2 80.7 98.1 63.6 98.2 29.3
NoReagent 100.0 64.8 100.0 55.3 100.0 46.6
PHA 1% 45.3 70.7 41.2
Table 12 IL-2 ELISA for E9C HIV-.
Blood E9C pg mL HTV-1 + pg/mL with gp 120 without gp 120 2 3
DHEA 10A 0 0 0.4 -4M
DHEA 10* 0.4 0 0 0 0 -6M
DHEA 10A 0 0 0.9 0 0 -8M
DHEA K 4 0 0 0 0 0 -8M + anit 1L- 12
DHEA 10A 0 0 0 0 0 -10M anti-IL-12 0 0 0 0 0
IL-12 0.9 0 0 0 0
No Reagent 0 0 0 0 0
PHA 1% 7 days 0 0
PHA 1% 3 days 14.7 10.9
Table 13 IL2-ELISA for E9D HIV+
Blood E9D pg/mL HIV-1 + pg/mL with gp 120 without gp 120
1 2 3 1 2
DHEA 10* 0 0 0 0 0 -4M
DHEA 10* 0 0 0 0 0 -6M
DHEA 10A 0 0 2.5 0 0 -8M
DHEA 10* 0 0 0 0 0 -8M + alL-12
DHEA 10* 0 0 0 0 0 -10M anti-IL-12 0 0 0 0 0
IL-12 0 0 0.1 0 0
No Reagent 0 0 0 0 0
PHA 1% 7 days 0 0
PHA 1% 3 days 34.2 35.1
Table 14 IL-2 ELISA for E9E HIV+
pg/mL with gp 120 pg/mL without gp 120 2 3 1 2
DHEA 10* 0 0 0 0
-4M
DHEA 10* 0 0 1.7 3.3 0 -6M
DHEA 10* 0 0 0 0 0 -8M
DHEA 10A 0 0 0 0 515 -SM + alL-12
DHEA 10* 0 0 0 0 0 -10M anti-IL-12 0 0 0 0 0
IL- 12 0 0 0 0 0
No Reagent 0 0 0 0 0
PHA 1% 7 days 0 0
PHA 1% 3 days 18.4 200.1
Table 15 IL-2 ELISA for E9F HIV-t
— -pg/mL with gp 120- pg/mL without gp 120 1 2
DHEA 10Λ-4 M 0 0.9 1.5 0 0.4
DHEA 10Λ-8M 0 0 0.9 0 0
DHEA 10A-8 M + aIL-12 0 0 0 0 0 anti-IL-I2 0 0 0 0 0
IL-12 0 0 0 0 0
No Reagent Control 0 0 0 0 5.3
PHA 1% 7 days 1.8 0
PHA 1% 1 day 231.8 195.7
Table 16 IL-2 ELISA for E9F HIV-1
- — pg/mL with gp 120- pg/mL without gp 120 1 2 3
DHEA 10Λ-4 M 0.4 0.9 0 0 0.4
DHEA 10Λ-8M 0 0 0.9 0 0
DHEA 10Λ-8 M - aIL-12 2 0 0 0 0 anti-IL-12 0 2.4 0.9 0.6 0
IL-12 0 3.6 2.4 0.6 1.8
No Reagent Control 4.1 5.4 4.8 4.1 0
PHA 1% 7 days 0 0
PHA 1% 1 day 211.7 326.1
Table 17 IL-2 ELISA for E9A HIV-
Figure imgf000062_0001
Table 18 IL-2 ELISA for E9B HTV-
Figure imgf000063_0001
The stimulation caused by DHEA in each of these cases was equal to or greater than that caused by IL-12, although the concentration of DHEA causing the stimulation varied from sample to sample In blood samples from three other patients (E9A, HIV-1 negative; E9D and Ξ9G, HIV-1 positive), proliferation in the presence of gpl20 was suppressed by addition of DHEA or IL-12.
It has recently been demonstrated that m vivo administration of murine IL-12 (IL-12) to m ce results m augmentation of cytotoxic natural killer (NK) /lympnocytes-activated killer cell activity, enhancement cf cy olytic T cell generation, and induction of γ-IFN secretion. In this study, the m vi vo activity of murine IL-12 against a number of murine tumors has been evaluated. Experimental pulmonary metastases or subcutaneous growtn of the BlόFLO melanoma <.ere markedly reduced m rr ce treated intra pen oneally Λith I -12, resulting m an increase m survival time. The therapeutic effectiveness of I -12 was dose dependent and treatment of subcutaneous tumors were effectively treated by IL-12 at doses which resulted in no gross toxicity. Local pentumoral injection of IL-12 into established subcutaneous Renca tumors resulted in regression and complete disappearance of these tumors. IL-12 was as effective in NK cell -deficient beige mice or in m ce depleted of NK cell activity by treatment with anti- asialc GM1, suggesting that NK cells are net the primary cell type mediating the anti tumor effects of this cytokine. However, the efficacy of IL-12 was greatly reduced in nude mice, suggesting the involvement of T cells. Furthermore, depletion of CD8+ but not CD4+ T cells significantly reduced the efficacy of IL-12. These results demonstrate that IL-12 has potent in vivo anti- tumor and anti -metastatic effects against murine tumors and demonstrate the critical role of CD8+ T- cells in mediating the anti -tumor effects against subcutaneous tumors . The involvement of IL-12 with the generation of CD8+ cell populations was demonstrated in a study of HIV+ pa_ier._s who were treated with anti-human IL-10 antiserum. HIV viral load was reduced to zero by the administration of rabbit anti-human IL-10 antiserum. Patients showed an 84% increase in CD8+ cell population above baseline values. The decrease in in vi vo IL-10 levels allowed CDS. cell populations to increase and allowed for KIV viral clearance by restoring HIV specific cell mediated immune response.
PRODUCT SPECIFICATIONS
Description: Rabbit anti-Human IL-10
Form: Liquid
Concentration: 2.7 mg/ml
Stabilizers: None Preservative: None
Sterility Sterile filtered
Host Species: Rabbit
Antibody Class: IgG Antigen Used: Recombinant human IL-10
Method of Purification: Ion Exchange chromatography Method of Quantification: Pierce BCA Protein Assay Specificity: Human IL-10 Cross-Reactivity: No cross reactivity with WHO standards :
IL-la, IL-13, IL-2, IL-3, IL- 4, IL-6, IL-7, IL-8, MIP-1A, TNFa and GM-CSF done by EIA. Storage: Short term, 43C; -20°C, long term.
Materials and Reaσents: used to demonstrate DHEA' s ability to enhance IL-12 synthesis.
1. IL-2 ELISA, available in house, minimum of six plates .
2. MTS assay, Fromega, minimum of 7 plates.
3. IL-12 (R&D Systems, Minneapolis, Minnesota, #219-IL) 5 fg should be sufficient for the entire experiment . 4. Antibody to human IL-2 receptor, (R&D
Systems, AB-233-NA), 1 mg lyophilized, goat human.
5. Rabbit polyclonal antibody to p40 chain of human IL-2 (Genetics Institute, Cambridge,
Massachusetts) . 6. Native gpl20, available in house (50 fg/vial, about 1 mg/mL) . Need 5nM/m . 50 fg is enough for two assays with two plates each. 7. Normal human (HIV-1 negative) . Peripheral blood mononuclear cells (PBMC) unstimulated.
8. 5 HIV+ samples of blood from which to obtain non-responsive PBMC. 5 mL per sample. 9. DHEA (dehydroisoandrosterone; Sigma Chemical,
St. Louis, Missouri, D4000) . 1 g should be sufficient for the entire experiment.
10. 100% Ethanol to solubilize the DHEA.
11. RIO Medium: RPMI , 10% FBS, 50 fg/mL gentamicin.
12. 96 well flat bottom tissue culture grade cluster dishes, 2 per blood sample.
Protocol :
1. For each blood sample, separate out PBMCs and do a cell count .
2. Use all the cells available from patient samples. If 1 x 10" cells or more are present, then seed the cells into two 96 well plates. At 1 x lC , we will end up with 0.5 x 105 cells/well or 2.5 x 105 cells/mL. If fewer, then use only one plate. Record the number actually plated per well. If two plates are used, then one will be for IL-12 detection and will receive antibody to human IL-2 receptor. The other plate will be used for the cell proliferation assay and will not receive this antibody. If only one plate is used, then that plate will receive antibody.
3. If using one plate, then resuspend the cells in 20 mL R10; if two plates, then resuspend in 40 mL. Aliquot 200 fL per well. Allow to settle overnight. If natural settling is not practical, then wrap plates in plastic wrap and use gentle centrifugation.
4. Prepare schema showing which special media will be added to which wells. (Be aware that due to the need for blanks and standards for the IL-2 ELISA, not all replicates grown up will be used in the ELISA) .
5. Each experiment will need 16 or 32 mL of medium with native gpl20 at 5 nM/mL. FW = 120,000. Amount to add per 16 L is 96 fL of a 100 fg/mL stock. (6 fL of 100 fg/mL for each mL of medium) .
Also use 12 or 24 mL of R10 medium for each assay. Note well : This will be the key to whether the PBMC are reactive or not. If the cells proliferate and produce IL-2 in the presence of gp!20 and not without gpl20, then these cells are normal reactive cells. If they behave the same vis-a-vis proliferation and IL-2 production regardless of whether gpl20 has been added or not, then the cells are non-reactive. It is only the non-reactive cells that we should see the effect of IL-12 and DHEA.
6. Add 2 fg/mL of IL-2 receptor specific antibody to the 16 mL with σpl20 and the 12 mL of R10 for each sample to be used for the IL-2 ELISA. 7. Preparation of DHEA:
7.1 Dissolve 1 g of DHEA in 1 L of absolute ethanol (100%) . Incubate in a 37°C water bath. Additional ethanol may be added up to 3.47 mL . This will give a i M solution. If the entire 3.47 mL is not needed for it to go nto solution, then the difference can be made up with RIO medium.
7.2 For each sample, we will need medium with DHEA at the following concentration: 10"1, 10"s, 10"10, 10-12.
7.3 At each dilution prepare 2 L of media (already containing gpl20 and antibody from step 6) and another 2 ml of media with gpl20 but no antibody if a second plate is used. For 10"8, 6 mL of each will be needed.
At each dilution prepare 2 x 1.5 L of RIO without gpl20 and with and without antibody from step 6. For 10"a, 3 L of each will be needed.
7.4 Making dilutions. Use 5 mL tubes. A. Take 20 μL of 1 M DHEA into 2 mL of RIO medium = 10 '2M.
3. Take 20 μL of 10": M DHEA into 2 mL of step 4.3 medium = 10'^M.
Take 15 μL of 10'2 M DHEA into 2 L of step 4.3 medium = 10'5M.
C. Take 20 μL of 10'4 M DHEA into 2 mL of step 4.3 medium = 10"≤M.
Take 15 μL of 10"4 M DHΞA into 1.5 mL of R10 = 10"SM. D. Take 40 μL of 10"* M DHEA into 4 L of step
4.3 medium = 10'aM.
Take 30 μL of 10'5 M DHΞA into 3 L of R10 = 10"aM. E. Take 20 μL of 10'a M DHEA into 2 mL of step 4.3 medium = 10"::)M
Take 15 μL of 10"a M DHEA into 1.5 L of RIO - 10'10M. F. Take 20 μL: of 10"10 M DHEA into 2 mL of step
4.3 medium = I0"12M
Take 15 μL of 10"1M.
7.5 To half of the 10"8 M DHΞA media types, add antibody to human IL-12. Use at 1:1000; dilute stock 1:2, then use 3μl in 1.5 ml.
7.6 IL-12 medium for each plate:
A. To 2 ml of gpl20 medium with and without antibody for IL-2, add 10 U/mL of recombinant IL-12.
B. To 1.5 L of R10 with and without antibody to IL-2, add 10 U/mL cf recombinant IL-12.
C. 1 ED; = 1U. The ED5-, cf the IL-12 will be m the literature received with th s reagent.
8. Aspirate medium off of cells and add 200 fL of appropriate medium to each well according to the schema. Place extra medium in peripheral wells. Wrap plates in plastic wrap and place on tray with water. Incubate at 37°C 5% C02.
9. If it is a two plate assay, then after 5 days aspirate off medium from the plate without antibody to the IL-2 receptor. Replace with 100 fL/well of R10 medium. Perform the cell proliferation assay with a 4 hour incubation. 10. After 7 days using the plate with the antibody to the IL-2 receptor: Take 100 fL per well and use to perform the IL-2 ELISA.
11. If there is only one plate for an assay, remove and freeze the rest of the supernatant from each well, then add 100 fL/well of R10 medium and proceed with the cell proli eration assay at this 7 day point.
12. Run PBMC from HIV- blood first to see if all reagents are performing as expected before proceeding with a HIV+ samples.
13. Another HIV- sample should be run after all the HIV- samples have been completed.
14. Compile and analyze data.
Example 7 Administration of DHEA and Isopentenyl
Adenosine 5 ' -Monophosphate to HIV Patients
Protocol Summary
Title: A Clinical Trial of Administered DHEA combined with isopentenyl adenosine 5 ' -monophosphate as a specific inhibitor of IL-10. Specifically formulated for persons with HIV infection who have developed resistance to protease and reverse trar.scπptase inhibitors. DHEA combined with Isopentenyl adenosine 5 ' -monophosphate is referred to as Compound (D+I) . Indication: Treatment of HIV-1 infection.
Ty e cf Study: Phase I/II Clinical Trial. Study Objectives : determine the safety and tolerance of administered D+I in oersons with advanced HIV diseases, the effect of administration of D+I on measures of HIV viral load (serum PCR (RNA) levels and HIV p24 antigen by the acid dissociation method) , the immune and toxicological effects of administered D+I, and the pharmokmetics of administered D+I .
Inclusion Criteria: Age 18 years or older; HIV-1 seropositive; a CD4+ -T-lymphocyte count of 50 to 300 cells/mπr with one month prior to study entry, measured on two separate occasions 72 hours to 28 days apart; baseline laboratory values of Hemoglobin > 9 g/dl, WBCs > 1500 cells/μl, Neutrophils > 1000 cells/μl, Platelets 25,000 cells/μl, Bilirubm < 2.0 mg/dl, AST, ALT, Alkaline Phosphatase <5x upper limit of normal, and Creatimne < 1.5 mg/dl. In addition, a history of prior anti -retroviral tr.erapy was required as follows: patients with a prior ristory of anti-retroviral therapy using AZT, αl , ddC, or d4T alone or m combination with protease mhioitors who are not receiving such therapy at study entry, must have discontinued tms medication at study entry. Women of childoearmg potential required one negative serum pregnancy test, beta, Δ HCG, within one week prior to study entry. A medium to high PCR HIV RNA titre was required at study entry.
Exclusion Criteria: Previous treatment with cne otherapeu ic agents within eight weeks of enrollment, active, major infection, including AIDS-defining opportunistic infection, or other life-threatening medical crisis; pregnant or breast-feeding; any condition which, in the investigator's opinion places the patient at undue risk or jeopardized the objectives of the trial; or receiving immunomodulatory therapies including interferon or pharmacological doses of steroids at entry into the study. Safety Measures: Weekly analysis up to week 4 of the study of the following parameters: documentation and assessment of adverse events,- hematology; clinical chemistries and urinalysis; assessment of the immune responses resultant from D+I; and assessment of PCR (RNA) and DNA measures alteration with therapy.
Effectiveness Measures: Measures of viral load will include HIV-p24 antigene ia, and HIV-RNA PCR (cell- free, serum) and cell HIV-DNA analysis.
Improvements in immune response will be measured as changes from baseline in CD4/CD8 ratio. Clinical lymphocyte counts, percent alterations in W3C, and percent alterations in IL-10 levels which would demonstrate the ability of D+I to cause the patients' immune system to move to T1 status. Clinical benefit will be assessed by change in total body weight, Karnofsky performance score, and amelioration of signs and symptoms of disease present at baseline .
The remission or incidence cf new opportunistic infection will be summarized.
Study Design: Open-label, daily administration of a dose per patient of 1200 mg/day, with review and assess ent of the dosage schedules and efficacy after theraov for 4 weeks. Study Size: 5 Patients (total) - 5 patients at 1200 mg/day for 30 days.
Test Articles: Test Drug: compound Dtl with a particle size distribution in gelatine capsules of 200 mg per capsule: 87% <5 μ , 100% <15 μm. Each capsule contains: 600 mg of DHEA and 600 mg of Isopentenyl adenosine 5 ' -monophosphate . No control drug or placebo was used. Measurements were gathered from each patient before and after administration of D+I at weeks 0, 1, 2, 3, 4, and 8. Measurements taken were: Physical Ξxa & Medical History, Urinalys s, Glucose, Neopterin, beta 2- microglobulm, RBC, h3, WBC, Platelet, T Cell Panel, p24 antigen, creatinine, SGOT, SGPT, IgG, IgA, IgM, DHEA, DHEAS (DKΞA sulfate),, Testosterone, 17-ketosteroids , II- 10, IL-2, PCR (RNA, cell-free, serum), PCR (DNA) and CD4. Physical examination, urinalysis, glucose, neopterin, beta 2 -microglobulm, R3C, hB, p24 antigen, creatinine, IgG, IgA, and IgM were not taken at week 8. PCR (DNA) was not done at weeks 2 and 3. To the extent not already indicated, it will be understood by those of ordinary skill in the art that any one of the various specific embodiments herein described and illustrated may be further modified to incorporate features shown in other of the specific embodiments. The foregoing detailed description has been provided for a better understanding of the invention only and r.c unnecessary limitation should be understood therefrom as some modifications will be apparent to those skilled in the art without deviating from the spirit and scope of the appended claims.

Claims

I CLAIM:
1. A method of enhancing the TH1 immune response in a patient, comprising the step of administering to said patient : a) an effective amount of a 17-ketosteroid compound; and b) an effective amount of an Interleukin-10 inhibitor.
2. The method of claim i, wherein said Interleukin-10 inhibitor is selected from the group consisting of: antiserum to Interleukin-10 , a compound effective for inhibiting synthesis or biological function of Interleukin- 10 and an Interleukin-10 receptor molecule blocking agent .
3. A method as recited in claim 1, wherein said Interl╬╡u m-10 inhibitor is antiserum against Int╬╡rleukin- 10.
4. The method of claim 3, wherein said antiserum is a monoclonal antiserum.
5. The method of claim 3, wherein said antiserum is an ant - urine IL-10 antiserum.
6. The method of claim 3, wherein said antiserum is an anti -human IL-10 antiserum.
7. The method of claim 3, wherein said antiserum is directed against the Epstein-Barr virus open reading frame BCRFI .
8. A method as recited in claim 1, wherein said Interleukin- 10 inhibitor is selected from the group consisting of: canavanine sulfate, L-canavanine sulfate, herbimycin A, genistein, secalonic acid D, an isoflavonoid, a cytokinin, or an amphiphilic triterpenoid .
9. A method as recited in claim 1, wherein said Interleukin- 10 inhibitor is selected from the group having the formula:
Figure imgf000077_0001
wherein R- H, R2 CH; R3 = CHj, and R., H or R: H or CH3S and R4 =
Figure imgf000078_0001
and
R3 = CH3 , Cl , OH or a monophosphate group R6 = CH, , CH2OH , or Cl
R7 = H or Br or R: = H and R4 =
Figure imgf000078_0002
and XL and X2 are independently selected from H, methyl, ethyl, hydroxyl, the halogens and carboxyl
or R4
Figure imgf000078_0003
or RΛ =
Figure imgf000079_0001
or R4 =
O
CNHRs
and R?
Figure imgf000079_0002
or RΓÇ₧ = (CH2)7CH3; and R5 = OH and R3 = CH, monophosphate, diphosphate, or triphosphate group or R2 and R3 are linked to form a 3', 5 '-cyclic monophosphate derivative.
10. The method of ciaim 1, wherein said Interleukin- 10 inhibitor is selected from the group consisting of: Ns- (Δ2- isopentenyl) adenine, 6-N- (3 -methyl -3- hydroxybutylamino) purine, adenine, hypoxanthine, uric acid, and methylated xanthines .
11. A method as recited in any one of claims 1-10, wherein said 17-ketostero d has the formula
Figure imgf000080_0001
wherein R is a hydrogen atom and R, is a chemical group selected from the group consisting of a hydrogen atom and an SOjOM group wherein M is selected from the group consisting of a hydrogen atom; a sodium atom; a sulphatide group
SO2OCH2CHCH2OCOR3
OCOR,
wherein each of R2 and R3 , which may be the same or different, is selected from the group consisting of straight and branched chain alkyl radicals of 1 to 14 carbon atoms ;
a phosphatide group
Figure imgf000081_0001
wherein each of R2 and R3 , which may be the same or different, is selected from the group consisting of straight and branched alkyl radical of 1 to 14 carbon atoms; and a glucuronide group
COOH
OH
OH
and wherein the atom at position 5 is present in the - or ╬▓-configuration, or the compound comprises a mixture of both configurations.
12 A method of treating a patient for a disease, comprising the step of administering to said patient:
(1) an effective amount of a 17-ketostero╬╣d compound ; and 5 (2) an effective amount of an Interleukm-10 inhibitor.
13. The method of claim 12, wherein said Interleukm- 10 inhibitor is selected from the group consisting of: anti -serum to Interleukin- 10 , a compound effective for
10 inhibiting synthesis or biological function of
Interleukm- 10 , and an Interleukm- 10 receptor molecule blocking agent.
14. The method of claim 12, wherein said disease is a viral infection.
15 15. A method as recited in claim 14, wherein said disease is an HIV infection
16. The method of claim 12, wherein said disease is a cancer.
17. A composition comprising: 0 (a) a 17 -ketosteroid compound, and (bl an Interleukm-10 nib tor.
18. The composition of claim 17, wherein sa d Interleukm- 10 inhibitor is selected from the group consisting of: antiserum to Interleukin-10 , a compound effective for inhibiting synthesis or biological function of Interleukin-10 , and an Interleukin- 10 receptor molecule blocking agent.
19. An article of manufacture, comprising:
(a) packaging material;
(b) at least one unit-dosage of a 17-ketosteroid compound; and
(c) a label or package insert indicating that said 17 -ketosteroid compound can be used in the method of claims 1-16.
20. An article of manufacture, comprising:
(a) packaging material;
(b) at least one unit-dosage of an Interleukin- 1C inhibitor; and
(c) a label or package insert indicating that said Interleukin-10 inhibitor can be used in the method of claims 1-16.
21. A method as recited in claim 1, wherein the synthesis of Interleukin-10 is inhibited by at least one cytokinin, with the proviso that said cytokinin is other than a compound of ciaim 9.
22. A method for preventing cr reducing bacterial translccation in a patient in need of such treatment, comprising the step of administering to said patient: (a) an effective amount of a 17 -ketosteroid compound, and
(b) an effective amount of an Interleukin- 10 inhibitor selected from the group consisting of: anti- serum to Interleukm- 10 , a compound effective for inhibiting synthesis or biological function of Interleukm-10 , and an Interleuk╬╣n-10 receptor molecule blocking agent .
23. A method as recited in claim 1, wherein said Interleukm- 10 inhibitor is selected from the group consisting of:
(a) NG-monomethyl- 1-argmine ; and
(b) sodium nitroprusside .
24. A method of enhancing the T2 immune response in a patient, comprising the step of administering to said pa ien : a) an effective amount of a 17-ketosterc╬╣d compound; and b) an effective amount of an Interleukin-12 inhibitor.
25. The method of claim 24, wherein sa d Interleukin- 12 inhibitor is selected from the group consisting of: antiserum to Interleuk╬╣n-12 , a compound effective for inhibiting synthesis or biological function of Interleukm- 12 and an Interleukm - 12 receptor molecule blocking agent .
26. A method as recited in claim 24, wherein said Interleuk╬╣n-12 inhibitor is antiserum against Interleukm-12.
27. A method of treating a patient for a disease, 5 comprising the step of administering to said patient:
(a) an effective amount of a 17-ketosteroid compoun ; and
(b) an effective amount of an Interleukm- 12 inhibitor .
10 28. The method of claim 27, wherein said Interleukin- 12 inhibitor is selected from tne group consisting of: anti-serum to Interleukm-12 , a compound effective for inhibiting synthesis or biological function of Interleukm- 12 , and an Interleukm- 12 receptor molecule
15 blocking agent
29. The method of claim 27, wherein said disease is a bacterial infection.
30. A method of treating a patient in need of treatment for an autoimmune condition, comprising 0 administering to said patient .
(a) an effective amount of a 17-ketosteroid compound ; and
(b) an effective amount of an Interleukm- 12 inhibitor.
31. The method of claim 30, wherein said autoimmune condition is lupus.
32. The method of claim 30, wherein said autoimmune condition is graft versus host disease.
33. A composition comprising:
(a) a 17 -ketosteroid compound, and
(b) an Interleukin-12 inhibitor.
34. The composition of claim 33, wherein said Interleukin-12 inhibitor is selected from the group consisting of: antiserum to Interleukin-12 , a compound effective for inhibiting synthesis or biological function of Interieukin-12 and an Interleukm-12 receptor molecule blocking agent.
35. The composition of claim 33, wherein said Interleukin-12 inhibitor is antiserum to Interleukin-12.
36. The composition of claim 35, wherein said antiserum is a monoclonal antiserum.
37. An article of manufacture, comprising:
(a) packaging material; (b) at least one unit-dosage of a 17-ketosterc╬╣d compound; and (c) a label or package insert indicating that said 17 -ketosteroid compound can be used in the method of claims 24-32.
38. An article of manufacture, comprising: (a) packaging material;
(b) at least one unit-dosage of an Interleukm- 12 inhibitor; and
(c) a label or package insert indicating that said Interleukm-12 inhibitor can be used m the method of claims 24-32.
39. A method of enhancing the T.,2 immune response in a patient, comprising the step of administering to said patient : a) an effective amount of a 17-ketostero╬╣d compound, and b) an effective amount of an Interleukm-2 mnibitor .
40. The method of claim 39, wherein said Interleukm-2 inhibitor is selected from the group consisting of: antiserum to Interleukm-2 , a compound effective for inhibiting synthesis or biological furction of Interleuk╬╣n-2 and an Interleukm-2 receptor molecule blocking agent.
41. A method as recited in claim 39, wherein said Interleukin-2 inhibitor is antiserum against Interleukin- 2.
42. A method of treating a patient for a disease, comprising the step of administering to said patient:
(a) an effective amount of a 17-ketosteroid compound ; and
(b) an effective amount of an Interleukin-2 inhibitor .
43. The method of claim 42, wherein said Interleukin-2 inhibitor is selected from the group consisting of: antiserum to Interleukin-2 , a compound effective for inhibiting synthesis or biological function of Interleukin-2 , and an Interleukin-2 receptor molecule blocking agent .
44. The method of claim. 42, wherein said disease is a bacterial infection.
45. A method of treating a patient in need of treatment for an autoimmune condition, comprising the step of administering to said patient:
(a) an effective amount of a 17-ketosteroid compound ; and
(b) an effective amount of an Interleukin-2 inhibitor .
46. The method of claim 45, wherein said autoimmune condition is lupus.
47. The method of claim 45, wherein said autoimmune condition is graft versus host disease.
48. A composition comprising:
(a) a 17 -ketosteroid compound, and
(b) an Interleukin-2 inhibitor.
49. The composition of claim 48, wherein said Interieuk╬╣n-2 inhibitor is selected from the group consisting of: antiserum to Interleukin-2 , a compound effective for inhibiting synthesis or biological function of Interleukm-2 , and an Interleukm-2 receptor molecule blocking agent .
50. An article of manufacture, comprising: (a) packaging material;
(b) at least one unit-dosage of a 17-ketostero╬╣d compound; and
(c) a label or package insert indicating that said 17 -ketosteroid compound can be used in the method of claims 39-47.
51 . An art icle of manufacture , comprising -.
( a ) packaging material ;
(b) at least one uni t - dosage of an Interl eukin- 2 inhibi tor ; and (c) a label or package insert indicating that said Interleukin-2 inhibitor can be used in the method of claims 39-47.
52. A method of enhancing the TH1 immune response in a patient comprising the step of administering to said patient : a) an effective amount of a 17-ketosteroid compound; and b) an effective amount of human alpha-fetoprotein or similarly immunosuppressive peptides of human bacterial, viral or synthetic origin.
53. A method of treating a patient for an autoimmune condition, comprising the step of administering to said patient an effective amount of Interleukin- 10.
54. A method of treating a patient for lupus comprising the step of administering to said patient an effective amount of Interleukin-10.
55. A method of treating a patient for graft versus host disease, comprising the step of administering to said patient an effective amount of Interleukin-10.
56. A method of treating a patient for multiple sclerosis, comprising the step of administering to said patient an effective amount of Interleukin- 10.
57. A method of treating a patient receiving vaccine therapy, comprising the step of administering an effective amount of Interleukin-10 as an adjuvant to said patient .
5 58. A method of treating a patient in need of treatment for a disease, comprising the step of administering to said patient a combination comprising:
(a) an effective amount of Interleukin-12 ; and
(b) an effective amount of an Interleukin-10 10 inhibitor.
59. The method of claim 58, wherein said disease is cancer .
60. The method of claim 58, wherein said disease is a bacterial infection.
15 61. A method of enhancing endogenous Interleukin-10 levels in a patient in need of such enhancement, comprising administering to said patient an effective amount of a 17 -ketosteroid compound, or an analogue or metabolite of a 17-ketosteroid compound.
20 62. A method as recited in claim 61, wherein said 17- ketosteroid compound is DHΞA or an analogue or metabolite of DHEA.
63. A method of treating a patient in need of treatment for a disease, comprising the step of administering to said patient a combination comprising:
(a) an effective amount of a 17-ketosteroid compound ; and
(b) an effective amount of a lysosomotropic agent .
64. The method of claim 63, wherein said lysosomotropic agent is selected from the group consisting of: amantadine, tributylamine, chloroquine, methyia ine, quinacrine and primaquine .
PCT/EP1997/005716 1997-04-17 1997-10-16 Combination therapy utilising 17-ketosteroids and interleukin inhibitors, or interleukin-10 potionally with interleukin inhibitors WO1998047516A1 (en)

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