THERAPIE ASSOCIEE REPOSANT SUR L'UTILISATION DE LA DEHYDROEPIANDROSTERONE (DHEA)

DHEA COMBINATION THERAPY

Abrégé français

L'invention a trait à des médicaments, à leurs procédés de production ainsi qu'à des nécessaires comprenant, (1), un composé de 17-cétostéroïde et/ou (2), un antisérum, polyclonal ou monoclonal, dirigé contre l'interleukine 10, l'interleukine 2 ou l'interleukine 12, ou en combinaison, soit avec n'importe quel composé en mesure d'inhiber réellement une synthèse ou la fonction biologique de l'interleukine 10, de l'interleukine 12 ou de l'interleukine 2, soit avec un agent bloquant de molécule réceptrice de l'interleukine 10, de l'interleukine 12 ou de l'interleukine 2, soit avec un antisérum, qu'il soit polyclonal ou monoclonal, dirigé contre l'alpha-foetoprotéine. Cette invention se rapporte également à des méthodes thérapeutiques faisant intervenir ces composés ou des combinaisons de ces composés, à des méthodes renforçant notamment une réponse immunitaire de protection de Th¿1? par l'utilisation du composé de 17-cétostéroïde en tant qu'agent antiviral, antibactérien, anti-mycoplasme ou en tant qu'agent antiparasite intracellulaire. L'invention a, en outre, trait à des traitements concernant divers composés et leurs combinaisons.

Abrégé anglais

There are provided medicaments, methods of making them, and kits, which
include (1) a 17-ketosteroid compound and/or (2) anti-serum either poly- or
monoclonal to Interleukin 10, Interleukin 2 or Interleukin 12, or with any
compound which can effectively inhibit synthesis or the biological function of
Interleukin 10, Interleukin 12 or Interleukin 2, or with an Interleukin 10,
Interleukin 12, or Interleukin 2 receptor molecule blocking agent, or with
anti-serum, either polyclonal or monoclonal to human alpha-fetoprotein. There
are also provided methods of treatment involving such compounds or
combinations of compounds, including enhancing Th1 immune protective response
when using the 17-ketosteroid compound as an anti-viral, anti-bacterial, anti-
mycoplasm or anti-intra cellular parasitic agent, and other treatments for
various compounds and combinations as described.

Revendications

19
I Claim
1. A method of enhancing the Th1 immune protective response
when using one or more 17-ketosteroid compound as an anti-viral,
anti-bacterial, anti-mycoplasm or anti-intra cellular parasitic agent by combining
it with anti-serum either poly or monoclonal to Interleukin 10 (cytokine
inhibitory factor), or with any compound which can effectively inhibit synthesisor the biological function of this specific cytokine Interleukin 10 whose
synthesis is detrimentally enhanced by DHEA monotherapy, or with an
Interleukin 10 (cytokine inhibitory factor) receptor molecule blocking agent.
2. A method of enhancing the Th2 immune response when using
one or more 17-ketosteroid compound as an anti-viral, anti-bacterial or anti-intra
cellular parasitic agent by combining it with anti-serum either poly or
monoclonal to Interleukin 12, or with any compound which can effectively
inhibit synthesis or the biological function of this specific cytokine Interleukin
12 whose synthesis is detrimentally enhanced by DHEA monotherapy during
the course of Th1 activated disease state, or with an Interleukin 12 receptor
molecule blocking agent.
3. A method of enhancing the Th2 immune response when using
one or more 17-ketosteroid compound as an anti-viral, anti-bacterial or anti-intra
cellular parasitic agent by combining it with anti-serum either poly or
monoclonal to Interleukin 2, or with any compound which can effectively
inhibit synthesis or the biological function of this specific cytokine Interleukin
2 whose synthesis is detrimentally enhanced by DHEA monotherapy during
the course of a Th1 activated disease state, or with an Interleukin 2 receptor
molecule blocking agent.
4. A method of enhancing the Th1 immune protective response
when using one or more 17-ketosteroid as an anti-viral, anti-bacterial, anti-mycoplasm
or anti-parasitic agent by combining it with anti-serum either
polyclonal or monoclonal to human alpha-fetoprotein or similarly
immunosuppressive peptides of human bacterial, viral or synthetic origin.

5. A method as recited in any one of claims 1-4, wherein the Th1
immune protective response is required by a patient in need of anti-cancer,
anti-viral, anti-metastatic, anti-multi drug resistant cancer cell and/or bacterial,
non-resistant bacterial infection therapy.
6. A method as recited in claim 1, wherein polyclonal and/or
monoclonal anti-serum is used to remove or neutralize Interleukin 10 and is
directed to the Epstein-Barr virus open reading frame BCRFI which has
amino acid sequence homology to human cytokine synthesis inhibitory factor
(Interleukin 10).
7. A process of administering to a patient a combination therapy
of at least one 17-Ketosteroid with an Interleukin 10 inhibitor or an Interleukin
10 receptor molecule blocking agent.
8. A process of administering to a patient a combination therapy
of at least one 17-Ketosteroid with an Interleukin 12 inhibitor or an Interleukin
12 receptor molecule blocking agent.
9. A process of administering to a patient a combination therapy
of at least one 17-Ketosteroid with an Interleukin 2 inhibitor or an Interleukin2 receptor molecule blocking agent.
10. A method of treating or arresting the progression of an immune
dysfunction in a patient in need of such treatment which comprises
administering a combination therapy of at least one 17-Ketosteroid with an
Interleukin 10 Inhibitor or an Interleukin 10 receptor molecule blocking agent.
11. A method of treating or arresting the progression of an immune
dysfunction in a patient, in need of such treatment, which comprises
administering a combination therapy of at least one 17-Ketosteroid with an
Interleukin 12 inhibitor or an Interleukin 12 receptor molecule blocking agent.
12. A method of treating or arresting the progression of an immune
dysfunction in a patient in need of such treatment which comprises
administering a combination therapy of at least one 17-Ketosteroid with an
Interleukin 2 inhibitor or an Interleukin 2 receptor molecule blocking agent.

21
13. A process of administering to a patient recombinant or cloned
Interleukin 10 for the therapy of Lupus and/or Graft versus host disease.
14. A process of administering to a patient recombinant or cloned
Interleukin 10 as adjuvant in vaccine therapy.
15. A process of treating multiple sclerosis in a patient in need of
such treatment, comprising administering recombinant or cloned Interleukin
10 to said patient.
16. A method of treating Lupus or graft versus host disease in a
patient in need of such treatment, comprising administering recombinant or
cloned Interleukin 10 to said patient.
17. A method as recited in claim 1, 7 or 10 wherein the synthesis of
Interleukin 10 is inhibited by any one or a combination of the following
compounds: - Canavanine Sulphate, L-Canavanine Sulphate, Herbimycin A,
Genistein, secalonic acid D, isoflavinoids, cytokinins, amphiphilic
triterpenoids, or analogues to any of the above.
18. A method as recited in claim 17 wherein the cytokinin is
selected from the group having the formula
<IMG>
wherein:
R1 = H, R2 = CH3, R3 = CH3 and R4 = H, or
R1 = H or CH3S and

22
<IMG>
and
R5 = CH3, Cl, OH or a monophosphate group
R6 = CH3, CH2OH or Cl
R7 = H or Br
or R1 = H and
<IMG>
and X1 and X2 are independently selected from H, methyl, ethyl, hydroxyl, the
halogens and carboxyl
<IMG>
or
or
<IMG>
<IMG>
or
and
<IMG>

23
or R8 = (CH2)7CH3;
and R2 = OH and R3 = OH, monophosphate, diphosphate or triphosphate
group or R2 and R3 are linked to form a 3', 5'-cyclic monophosphate
derivative, or a metabolite of said compound, said metabolite being a member
of the group consisting of:
N6 (.DELTA. 2-isopentenyl)adenine:
6-N-(3-methyl-3-hydroxybutylamino) purine;
Adenine;
Hypoxanthine;
Uric Acid; and
Methylated xanthines.
19. A method as recited in any one of claims 1-12, 17 and 18, wherein
said 17-ketosteroid has the formula
<IMG>
in which R is a hydrogen atom, and R1 is a chemical group selected from the
group consisting of a hydrogen atom, an SO2OM group wherein M is selected
from the group consisting of a hydrogen atom, a sodium atom, a sulphatide
group
<IMG>

24
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
<IMG>
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 radical of 1 to
14 carbon atoms, and a glucuronide group
<IMG>
atom at position 5 is present in the .alpha.- or .beta.- configuration, or the compound
comprises a mixture of both configurations.
20. A method of treating a viral infection, a bacterial infection, a
mycoplasm infection or a parasitic infection in a patient in need of such
treatment, comprising administering to said patient:
(1) one or more 17-ketosteroid compound, and
(2) one or more member selected from the group consisting of:
anti-serum, either poly or monoclonal, to Interleukin 10, compounds which can
effectively inhibit synthesis or the biological function of Interleukin 10, and
Interleukin 10 receptor molecule blocking agents,
in respective amounts which are effective to provide said treatment.

21. A method as recited in claim 20, wherein said patient is suffering
from HIV infection.
22. A method of treating cancer, viral infection, metastasis, multi
drug resistant cancer and/or bacterial, non-resistant bacteria in a patient in
need of such treatment, comprising administering to said patient:
(1) one or more 17-ketosteroid compound, and
(2) one or more member selected from the group consisting of: anti-serum,
either poly or monoclonal, to Interleukin 10, compounds which can
effectively inhibit synthesis or the biological function of Interleukin 10, and
Interleukin 10 receptor molecule blocking agents,
in respective amounts which are effective to provide said treatment.
23. A method of treating Lupus or Multiple Sclerosis and/or Graft
versus Host disease in a patient in need of such treatment, comprising
administering to said patient recombinant or cloned Interleukin 10.
24. A composition comprising:
(1) one or more 17-ketosteroid compound, and
(2) one or more member selected from the group consisting of:
anti-serum, either poly or monoclonal, to Interleukin 10, compounds which can
effectively inhibit synthesis or the biological function of Interleukin 10, and
Interleukin 10 receptor molecule blocking agents.
25. A kit comprising:
(1) at least one unit-dosage of a 17-ketosteroid compound, and
(2) at least one unit-dosage of one or more member selected from the
group consisting of: anti-serum, either poly or monoclonal, to Interleukin 10,
compounds which can effectively inhibit synthesis or the biological function
of Interleukin 10, and Interleukin 10 receptor molecule blocking agents.
26. A method of treating Lupus or Multiple Sclerosis and/or Graft
versus Host disease in a patient in need of such treatment, comprising
administering to said patient recombinant or cloned sequences derived from
sequences present in the Interleukin-10 molecule or sequences which mimic
Interleukin-10's immunosuppressive action.

26
27. A method of treating cancer, viral infection, metastasis, multi drug
resistant cancer and/or bacterial, non-resistant bacteria in a patient in need
of such treatment, comprising administering to said patient a combination
comprising:
(1) recombinant or cloned Interleukin-12 and
(2) one or more member selected from the group consisting of
anti-serum, either poly or monoclonal, to interleukin 10 compounds which can
effectively inhibit synthesis or the biological function of Interleukin 10,
Interleukin 10 receptor molecule blocking agents, in respective amounts
which are effective to provide said treatment.
28. A method as recited in claim 1, 7 or 10 wherein the synthesis of
Interleukin 10 is inhibited by any one or a combination of the following
compounds: - Canavanine Sulphate, L-Canavanine Sulphate, Herbimycih A,
Genistein, secalonic acid D, isoflavinoids, amphiphilic triterpenoids, or
analogues to any of the above.
29. A method as recited in claim 1, 7 or 10 wherein the synthesis of
Interleukin 10 is inhibited by at least one cytokinin, with the proviso that said
cytokinin is other than a compound selected from the group having the
formula
<IMG>
wherein:
R1 = H, R2 = CH3, R3= CH3 and R4= H, or

27
R1 = H or CH3S and
<IMG>
and
R5 = CH3, Cl, OH or a monophosphate group
R6 = CH3, CH2OH or Cl
R7 = H or Br
or R1 = H and
<IMG>
and X1 and X2 are independently selected from H, methyl, ethyl, hydroxyl, the
halogens and carboxyl
or R4 = <IMG>
or R4 = <IMG>
or R4 = <IMG>

28
and R8 =
<IMG>
or R8 = (CH2)7CH3;
and R2 = OH and R3 = OH, monophosphate, diphosphate or triphosphate
group or R2 and R3 are linked to form a 3', 5'-cyclic monophosphate
derivative, or a metabolite of said compound, said metabolite being a member
of the group consisting of:
N6 (~2-isopentenyl)adenine:
6-N-(3-methyl-3-hydroxybutylamino) purine;
Adenine;
Hypoxanthine;
Uric Acid; and
Methylated xanthines.
30. A method of treating lupus or graft versus host disease in a patient
in need of such treatment, comprising administering to said patient:
(1) one or more 17-ketosteroid compound, and
(2) one or more member selected from the group consisting of: anti-serum,
either poly or monoclonal, to Interleukin 12, compounds which can
effectively inhibit synthesis or the biological function of Interleukin 12, or
Interleukin 12 receptor molecule blocking agents,
in respective amounts which are effective to provide said treatment.
31. A method of treating lupus or graft versus host disease in a patient
in need of such treatment, comprising administering to said patient:
(1) one or more 17-ketosteroid compound, and
(2) one or more member selected from the group consisting of: anti-serum,
either poly or monoclonal, to Interleukin 2, compounds which can
effectively inhibit synthesis or the biological function of Interleukin 2, or
Interleukin 2 receptor molecule blocking agents,
in respective amounts which are effective to provide said treatment.

29
32. A method of treating a bacterial infection, a mycoplasm infection
or a parasitic infection in a patient in need of such treatment, comprising
administering to said patient:
(1) one or more 17-ketosteroid compound, and
(2) one or more member selected from the group consisting of: anti-serum,
either poly or monoclonal, to Interleukin 12, compounds which can
effectively inhibit synthesis or the biological function of Interleukin 12, or
Interleukin 12 receptor molecule blocking agents,
in respective amounts which are effective to provide said treatment.
33. A method of treating a bacterial infection, a mycoplasm infection
or a parasitic infection in a patient in need of such treatment, comprising
administering to said patient:
(1) one or more 17-ketosteroid compound, and
(2) one or more member selected from the group consisting of: anti-serum,
either poly or monoclonal, to Interleukin 2, compounds which can
effectively inhibit synthesis or the biological function of Interleukin 2, or
Interleukin 2 receptor molecule blocking agents,
in respective amounts which are effective to provide said treatment.
34. A method for preventing or reducing bacterial translocation in a
patient in need of such treatment, comprising administering to said patient:
(1) one or more 17-ketosteroid compound, and
(2) one or more member selected from the group consisting of: anti-serum,
either poly or monoclonal, to Interleukin 10, compounds which can
effectively inhibit synthesis or the biological function of Interleukin 10, or
Interleukin 10 receptor molecule blocking agents,
in respective amounts which are effective to provide said treatment.
35. A composition comprising:
(1) one or more 17-ketosteroid compound, and
(2) one or more member selected from the group consisting of:
anti-serum, either poly or monoclonal, to Interleukin 12, compounds which can

effectively inhibit synthesis or the biological function of Interleukin 12, and
Interleukin 12 receptor molecule blocking agents.
36. A kit comprising:
(1) at least one unit-dosage of a 17-ketosteroid compound, and
(2) at least one unit-dosage of one or more member selected from the
group consisting of: anti-serum, either poly or monoclonal, to Interleukin 12,
compounds which can effectively inhibit synthesis or the biological function
of Interleukin 12, and Interleukin 12 receptor molecule blocking agents.
37. A composition comprising:
(1) one or more 17-ketosteroid compound, and
(2) one or more member selected from the group consisting of: anti-serum,
either poly or monoclonal, to Interleukin 2, compounds which can
effectively inhibit synthesis or the biological function of Interleukin 2, and
Interleukin 2 receptor molecule blocking agents.
38. A kit comprising:
(1) at least one unit-dosage of a 17-ketosteroid compound, and
(2) at least one unit-dosage of one or more member selected from the
group consisting of: anti-serum, either poly or monoclonal, to Interleukin 2,
compounds which can effectively inhibit synthesis or the biological function
of Interleukin 2, and Interleukin 2 receptor molecule blocking agents.
39. A method as recited in claim 1, wherein said Interleukin-10
inhibitor is selected from:
1) NG-monomethyl-I-arginine (L-NMMA), and
1) sodium nitroprusside (SNP).

Description

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WO 97/38695 PCT/IB97/00414 -
DHEA Combination Therapy
SUMMARY OF THE INVENTION
According to the invention, there are provided combinations of
~ 5 compounds for use in restoring normal levels of Interleukin 12 (IL-12) and/or
Interleukin 10 (IL-10) by enhancing or curtailing synthesis or effect of
Interleukin 12 and/or Interleukin 10.
In one aspect of the present invention, the present inventor has found
that the anti-viral agents (general formula I herein) as disclosed in U.S.
Patent No. 4,956,355 (Prendergast) have additional beneficial therapeutic
effects when used in a combination therapy with agents that inhibit Interleukin
10 synthesis and/or action. Agents which inhibit Interleukin 10 can be
identified by identifying those compounds which have the ability to inhibit
cyclic AMP activity in addition to agents which demonstrate Interleukin 10
inhibition when employed in the screening protocol (Screening IL-10) as
herein described. The synthesis of Interleukin 10 can be inhibited by any of
a variety of compounds, including one or a combination of the following
compounds: - Canavanine Sulphate, L-Canavanine Sulphate, Herbimycin A
- (Wako Pure Chemicals Industries, Ltd., Japan), Genistein (Sigma Chemicals
Co., St. Lous, Mo., USA), secalonic acid D, isoflavinoids, cytokinins,
amphiphilic triterpenoids, or analogues to the above together with polyclonal
or monoclonal antiserum to Interleukin 10 or any of its peptide sequences.
The anti-viral agent is a 17-ketosteroid compound having the general
formula (I)
~Ç
1~~1
CONFlRM~llON ~OPt

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WO 97138695 PCT/IB97/00414 -
in which R is a hydrogen atom, and R1 is a chemical group selected from the
group consisting of a hydrogen atom, an SO20M group wherein M is selected
from the group consisting of a hydrogen atom, a sodium atom,
a sulphatide group
-so2o-cH2 ~ I H ~ CH2 O ~ CO ~ R3
O . CO . R2
10 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
- ~-O.CH2- IH.CH2-0-CO-R3
~ O.CO.R2
- 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 radical of 1 to
14 carbon atoms, and a glucuronide group
COOH
~o
,~
wherein the broken line represents an optical double bond, and the hydrogen
atom at position 5 is present in the a- or ~- configuration, or the compound
comprises a mixture of both configurations. When R1 is other than a
30 hydrogen atom, the compounds are conjugated compounds.

CA 022~l733 l998- lO- l~
WO 97138695 PCT/IB97/00414
In another aspect of the present invention, there is provided a method
of enhancing the Th1 immune protective response when using one or more
17-ketosteroid compound as an anti-viral, anti-bacterial, anti-mycoplasm or
anti-intra cellular parasitic agent by combining it with anti-serum either poly
5 or monoclonal to Interleukin 10 (cytokine inhibitory factor) and/or with any
compound which can eflectively inhibit synthesis or the biological function of
this specific cytokine Interleukin 10 and/or an Interleukin 10 (cytokine
inhibitory factor) receptor molecule blocking agent.
For example, Th1 immune protective response is required by patients
10 in need of anti-cancer, anti-viral, anti-metastatic, anti-multi drug resistant
cancer cell and/or bacterial, non-resistant bacterial infection therapy.
The present invention is also directed to the use of such compounds
in the manufacture of a medicament for providing any such treatment.
The pharmaceutical formulation according to the invention may be
15 adminisler~d locally or systemically. By systemic administration is meant anymode or route of administration which 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.
~ The pharmaceutical formulation for systemic administration according
20 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.
Suitable formulations for oral administration include hard or soft gelatin
capsules, dragees, pills, tablets, including coated tablets, elixirs,
25 suspensions, syrups or inhalations and controlled release forms thereof.
Solid dosage forms in addition to those formulated for oral
administration include rectal suppositories.
Suitable formulations for topical administration include creams, gels,
jellies, mucilages, pastes and ointments. The compounds may be also be
30 formulated for transdermal administration, for example, in the form of
transdermal patches so as to achieve systemic administration.

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Suitable injectable solutions include intravenous, subcutaneous and
intramuscular injectable solutions. The compounds may also be administered
in the form of an infusion solution or as a nasal inhalation or spray.
The pharmaceutical formulation according to the invention is
administered in unit doses comprising from 10 to 1000 mg of active
ingredient. Preferably, each unit dose comprises from 5 to 500 mg of each
active ingredient. As per this invention the pharmaceutical formulation
contains at least two active ingredients.
According to one embodiment of the invention, the combination
therapy is administered at a rate of from 1 unit dose to 10 unit doses per day.
Administration of the therapy in accordance with the invention is continued
for a period of at least one day and in certain cases may be given for the life
of the individual.
Compounds according to general formula (I) are disclosed in U.S.
Patent No. 4,956,355 (Prendergast) the entirety of which is hereby
incorporated by reference.
Preferably in the compound of formula (I), R and R1 are each
hydrogen. An especially preferred compoùnd is dehydroepiandrosterone
(DHEA) wherein R and R1 are each hydrogen and the double bond is
present.
In a further embodiment of the invention, the compound is
epiandrosterone wherein R and R1 are each hydrogen and the double bond
is absent. 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, iodine).
In a fùrther embodiment of the invention, the compuond is 16a-
bromoepiandrosterone, wherein R is Br, R1 is H and the double bond is
present. In a still further embodiment of the invention, the compound is
according to formula 1, wherein R is Br, R1 is 11 and the double bond is not
present (i.e., where the dotted line is shown in formula 1, there is a single
bond).

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Other preferred compounds are dehydroepiandrosterone sulphate,
wherein R is H, R1 is SO2-OM and M is as hereinbefore defined and the
double bond is present, and 5~-androstan-3~-ol-1 7-one.
Alternatively, the compound is selected from dehydroepiandrosterone
5 sulphatides, phosphatides or glucuronide wherein R is H, and R1 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-dihexadecylglycero sulfate,
10 hexadecane sulfonate, dioctadecanoylglycero phosphate, O-
hexadecylglycero phosphate.
Houston Study
Experimental evidence using DHEA therapy in HIV+ patients has
demonstrated that IL-12 levels, as measured by antibody ELISA methods, are
15 elevated, natural killer cell levels increased together with the synthesis and
presence of V (gamma) interferon, HIV viral loads as measured by HIV PCR
(RNA) measurement and quantitative culturing techniques demonstrated
more than one log reduction after four weeks of DHEA monotherapy.
~ However, while viral load levels were considerably reduced, Th1 immune
20 improvement did not occur. In fact, due to the elevated levels of Interleukin12 generated by this monotherapy with DHEA, Interleukin 10 levels
increased, which caused a subsequent decline in T4 (helper) cell numbers
and the disappea, a"ce of the Th1 (Delayed Type Hypersensitivity Response).
Skin reaction in patients as evidenced by patient data was down-regulated by
25 DHEA monotherapy, contrary to previous beliefs of some. Skin reaction is
only restored by the removal of Interleukin 10 which is elevated by the DHEA
monotherapy.
San Francisco Study (In-vivo)
The following is a summary of using DHEA as a monotherapy in an
30 open-label dose-escalation trial of oral DHEA (Dehydroepiandrosterone)
tolerance and pharmacokinetics in patients with HIV disease. In the Phase

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I DHEA trial (early symptomatic HIV disease and 200 to 500 CD4+
Iymphocytes/,uL), absolute CD4 counts in the control, placebo-assigned
patients declined by a median 5 cell/month. In contrast, patients in the
lowest-dose group studied in the Phase I DHEA trial (whose immune system
5 would not be expected to decline faster than that of placebo-treated patients
in the other trial) had a median CD4+ decline of 31 cells/month.
In-Vivo Trial usin~ Combination Therapy
To counteract this Th1 suppressive immune side effect of DHEA
monoll ,el ~,uy this anti-viral steroid had to be combined with an agent to inhibit
10 or interrupt the synthesis and/or action of Interleukin 10. This combination
therapy is the preferred embodiment of using the said anti-viral agents
(compounds according to general formula 1) wherein the anti-viral agents are
allowed to generate a Th1 response. The component of the combination
therapy which counteracts the Th1 suppressive Interleukin 10 immune side
15 effect of the anti-viral therapy may be anti-serum either polyclonal or
monoclonal in origin to Interleukin 10 and/or compounds to inhibit or interrupt
the synthesis or effectiveness of the unwanted Interleukin 10. Representative
compounds which inhibit Interleukin 10 are disclosed in U.S. Patent No.
~ 5,292,725 (Prendergast), the entirety of which is hereby incorporated by
20 reference, which may be used in the combination therapy to counteract the
Th1 suppressive immune side effect of the anti-viral monotherapy.
When the combination therapy was administered to HIV+ patients, the
removal of viral particles from each patient's bioodstream was enhanced by
3 (three) logs, relative to the monotherapy, while simultaneously enhancing
25 by over 80% the Th1 (T4 helper cell count). The Delayed Type
Hypersensitivity response lost at sero-conversion was also restored. This
combination therapy using DHEA as the non-toxic, non-resistant strain
development anti-viral agent, combined with antiserum and/or compounds
necessary to inhibit Interleukin 10 synthesis, and/or effect of Interleukin 10,
30 allows for substantial therapeutic benefit to be achieved which previously
could not be accomplished by the use of DHEA alone as a monotherapy. The

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beneficial action of up-regulation of the immune system together with the anti-
viral action of compounds of formula I (U.S. Patent No. 4,956,355--
Prendergast) would have wider therapeutic usefulness than in HIV treatment.
Discussion
The aspect of cytokine production following the administration of DHEA
has allowed us to review the therapeutic benefits previously ascribed to
DHEA therapy. We now know that the therapeutic benefit of DHEA therapy
to lupus patients and to other Th1 auto-immune conditions is directly related
to the increase of endogenous Interleukin 10 levels achieved in the patient
by the administration of DHEA. Bone marrow transplant rejection was put into
remission by DHEA administration to enhance IL-10 levels.
Doctor's Report
Patient: RD - DOB 14/711983
RD is a patient under my care. He has Acute Myeloid Leukaemia M3
~5 in remission following allogenic bone marrow transplant. His major active
problems have been GUT Graft Versus Host Disease and severe lung
disease. RD's general health has improved over the last 3 months. This has
coincided with him taking the therapy 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 other drug regime and has been taking this
medication for nearly 3 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 Pulmicort four times per
day, with oral steroids when necessary. Now he nebulizes only twice a day.
I have never seen such a vast improvement in his health with no apparent
side effects.
Patients in our experiments who have achieved elevations of their
endogenous Interleukin 10 levels have experienced remission in lupus

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whereas patients who have been administered DHEA, but who, due to other
cytokine and immune factors, have not experienced an Interleukin 10
elevation have not demonstrated relief of symptoms. Therefore, we have
identified that a more direct means of causing immediate relief in these
5 autoimmune conditions is to administer exogenously recombinant Interleukin
10 to facilitate the remission of symptoms of lupus and graft versus host
disease.
Another area of potential therapeutic benefit previously ascribed to
DHEA is the enhancement of vaccine antigen recognition by the immune
10 system in the elderly. This has now been identified and verified by our
analysis of the Interleukin 10 levels achieved versus the DHEA therapies
efficacy as vaccine adjuvant. We have identified that the administration of
recombinant Interleukin 10 to the elderly in association with or in advance of
treatment with an antigen vaccine created an enhanced adjuvant effect, which
15 enhanced the antibody response directly. Whereas with DHEA there is a
chance of not producing elevated levels of Interleukin 10. The effectiveness
of DHEA therapy really depends on the metabolism, blood levels achieved
and timing of the DHEA administration to the patient with or prior to the
vaccine antigen. The co-administration of recombinant Interleukin 10 is a
20 more direct means of achieving enhanced antibody response in the elderly
or very young patient and removes the uncertainties of steroid metabolism
and cytokine responses associated with DHEA monotherapy. This for the
first time explains why DHEA has very often produced contradictory
responses in the therapy of specific conditions e.g., Lupus, MS and HIV
25 because the real therapeutic effect is dependent upon cytokine profiles and
immune reactions which are generated upon the administration of the steroid
or its analogues. Therefore, the therapeutic effectiveness of DHEA is
unpredicatable as an immune modulator and dependent upon both the
steroids metabolism and the cytokine profile of the patient during and prior to
30 DHEA therapy. When the steroid is first administered the immune therapeutic
response is very much patient specific and cannot be relied upon to produce

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consistent therapeutic benefit. Desired immune therapeutic response can
only be achieved by utilizing directly the Interleukin 10 cytokine required or
by co-administering Interleukin 10 inhibitors and/or specific antisera to same.
DHEA thus administered in a combination therapy will facilitate Interleukin 12
S enhancement without the general negative effects of Interleukin 10 whereas
Th1 response is desired for therapeutic benefit. Experiments with DHEA and
the cytokines profile of patients who respond to DHEA therapy and those who
do not respond to DHEA therapy have led to my discovery that elevated
Interleukin 10 is the active agent responsible for the therapeutic response
observed to alleviate the clinical symptoms of lupus. Other experiments with
DHEA and cytokine profiles of senior patients, who responded to a vaccine
antigen with enhanced antibody production, has led to the discovery that
Interleukin 10 is the active agent responsible for creating the enhanced
vaccine response. In general this patient profile would normally, due to age,
have reduced antigen vaccine take or immune response. Multiple Sclerosis
is a Th1 auto-immune condition and requires Interleukin 10 to down regulate
the Th1 immune response and cause remission of the condition. We have
found a similar response to DHEA therapy for this condition, as we
discovered with lupus, i.e., great patient variability to treatment. Any
remission of symptoms in Multiple Sclerosis was identified by patients who
experienced siy"iricant elevation of their endogenous levels of Interleukin 10.
Therefore, direct admission of recombinant Interleukin 10 to a multiple
sclerosis model in the Lewis rat demonstrated remission of symptoms. If
Interleukin 10 is administered prior to the onset of myelin damage the
symptoms would be prevented altogether.
Washington in-vitro DHEA IL-12 Study with HIV+ blood
Protocol to demonstrate that DHEA enhances endogenous levels of
Interleukin 12
Restoration of HlV-Specific Cell-Mediated Immune Responses by DHEA
One HIV-1 negative control (E9B) and three HIV-1 positive specimens
(E9C, E9E and E9F) were stimulated by the addition of DHEA or IL-12 in the

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presence of gp120. The stimulation caused by the DHEA in each of these
cases was equal to or greater than that caused by the IL-12, although the
concentration of DHEA causing the stimulation varied from sample to sample.
In the remaining blood samples, E9A (HIV-1 negative) as well as E9D and
5 E9G (HIV-1 positive) proliferation in the presence of gp120 was suppressed
by the addition of DHEA or IL-12.
Human IL-12 is a disulfide-bonded heterodimeric cytokine consisting
of a 40- and a 35-kD subunit. The genes for this cytokine have been cloned
and purified recombinant protein has been produced. It has recently been
10 demonstrated that in vivo administration of murine Interleukin 12 (IL-12) to
mice results in augmentation of cytotoxic natural killer (NK)/lymphocytes-
activated killer cell activity, enhancement of cytolytic T cell generation, and
induction of interferon gamma secretion. In this study, the in vivo activity of
murine IL-12 against a number of murine tumors has been evaluated.
15 Experimental pulmonary metastases or subcutaneous growth of the B16F10
melanoma were markedly reduced in mice treated intraperitoneally with IL-12,
resulting in an increase in survival time. The therapeutic effectiveness of IL-
12 was dose dependent and treatment of subcutaneous tumors were
- effectively treated by IL-12 at doses which resulted in no gross toxicity. Local
20 peritumoral 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 mice depleted of NK cell
activity by treatment with antiasialo GM1, suggesting that NK cells are not the
primary cell type mediating the antitumor effects of this cytokine. However,
25 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 antitumor and antimetastatic effects against murine
tumors and demonstrate as well the critical role of CD8+ T cells in mediating
30 the antitumor effects against subcutaneous tumors.
Los Angeles Patient Study

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The involvement of Interleukin 12 with CD8+ cell generated was
demonstrated in a HIV+ patient study conducted for this patent. Patients with
a CDE8+ cell population showed an 84% increase above baseline values and
IIIV viral load was reduced to zero by the administration of polyclonal
~ 5 antibodies to human Interleukin 10. The removal of Interleukin 10 allowed
CD8+ cell increase and allowed for HIV viral clearance by restoring HIV
specific cell mediated Immune response.
Specification of Polyclonal Antiserum
PRODUCTION 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: lon Exchange chromatography
20 Method of Quantification: Pierce BCA Protein Assay
Specificity: Human IL-10
Cross-Reactivity: No cross reactivity with WHO standards:
IL-1a, IL-1B, IL-2, IL-3, IL-4, IL-6, IL-7
IL-8, MlP-1a, TNFa and GM-CSF done by EIA.
Storage: Short term 4~C and -20~C for long term
Material.and Reagents: used to demonstrate DHEA's ability to enhance
Interleukin 12 synthesis.
1. IL-2 ELISA, available in house, minimum of six plates.
2. MTS assay, Promega, minimum of 7 plates.
3. IL-12 R&D Systems, (#21 9-lL) 5 fg should be sufficient for the entire
experiment.

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12
4. Antibody to human iL-2 receptor, R&D Systems (AB-233-NA), 1 mg
Iyophilized, goat human.
~. Rabbit polyclonal antibody to p40 chain of human IL-2, Genetics
Institute, Cambridge, MA (617-498-8647).
6. Native gp 120, available in house (50 fg/vial, about 1 mg/mL).
Need SnM/mL. 50 fg is enough for two assays with two plates each.
7 Normal human (HIV-1 negative) PBMC unstimulated.
8. 5 HIV+ samples of blood from which to obtain non-responsive
PBMC. 5 mL per sample.
9. DHEA(dehydroisoandrosterone), Sigma D4000. 1 9 ~Idbe
sufficient for the entire experiment.
10. 100% Ethanol to solubilize the DHEA.
11. R10 Medium: RPMI, 10% FBS, 50 fg/mL gentamicin.
12. 96 well flat bottom tissue culture grade cluster dishes, 2 per blood
1 5 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 10 x 1 o6 cells or
more are present, seed the cells into two 96 well plates. At 10 x 1 o6 we will
end up with 0.5 x 105 cells/well or 2.5 x 1 05 cells/mL. If fewer use only one
plate. Record the number actually plated per well. If two plates are used one
will be for IL-2 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, that plate will receive antibody.
3. If using one plate resuspend the cells in 20 mL R10; if two plates
resuspend in 40 mL. Aliquot 200 fL per well. Allow to settle overnight. If
natural settling is not practical, 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 needed
for the IL-2 ELISA not all replicates grown up will be used in the ELISA).

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5. Each experiment will need 16 or 32 mL of medium with native gp
120 at 5 nM/mL. FW = 120,000. Amount to add per 16 mL; 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 120
and not without gp 120 these cells are normal reactive cells. If they
behave the same vis-a-vis proliferation and IL-2 production regardless
of whether gp 120 has been added, the cells are non-reactive. It is
only in the non-reactive cells that we should see the effect of IL-12 and
DHEA.
6. To the 16 mL with gp 120 and the 12 mL of R10 for each sample to
be used for the IL-2 ELISA, add 2 fg/mL of antibody to the IL-2 receptor.
7. Preparation of DHEA:
7.1 Dissolve 1 g of DHEA in 1 mL 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 1 M solution. If the entire 3.47 mL is not needed for it togo into solution the difference can be made up with R10 medium.
7.2 For each sample, we will need with medium DHEA at the following
concentrations: 10-4 10-6 10-1~ 1012
7.3 At each dilution prepare 2 mL of media (already containing gp 120
and antibody from step 6) and another 2 ml of media with gp 120 but no
antibody if a second plate is used. For 1 o-8, 6 mL of each will be needed.
At each dilution prepare 2 x 1.5 mL of R10 medium without gp 120 and
with and without antibody from step 6. For 10-8, 3 mL of each will be needed.
7.4 Making dilutions. Use 5 mL tubes.
A. Take 20 fL of 1 M DHEA into 2 mL of R10 medium = 1 0-2M.
B. Take 20 fL of 1 o-2 M DHEA into 2 mL of step 4.3 medium = 1 o-4M.
Take15fLof10~2MDHEAinto1.5mLofR10=10~4M.
C. Take 20 fL of 104 M DHEA into 2 mL of step 4.3 medium = 10-6M.
Take 15 fL of 10-4M DHEA into 1.5 mL of R10 = 10-6M.

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14
D. Take 40 fL of 10~ M DHEA into 4 mL of step 4.3 medium = 1 o-8M.
Take 30 fL of 1 0-6M DHEA into 3 mL of R10 = 1 o-8M.
E. Take 20 fL of 1o-8 M DHEA into 2 mL of step 4.3 medium = 10-1~M.
Take 15 fL of 1 o-8 M DHEA into 1 .5 mL of R10 = 1 0-1~M.
F. Take 20 fL of 10-1~ M DHEA into 2 mL of step 4.3 medium = 1 0-12M.
Take 15 fL of 10-12M.
7.5 To half of the 10-8 M DHEA media types add antibody to human
IL-1 2.
7.6 IL-12 medium for each plate:
A. To 2 ml of gp 120 medium with and without antibody for IL-2 add
10 U/mL of recombinant IL-12.
B. To 1.5 mL of R10 with and without antibody to IL-2 add 10 U/mL of
recombinant IL-12.
C. 1 ED50=1 U. The ED50 ~f the IL-12 will be in the literature received
with this 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% CO2.
9. If it is a two plate assay, afler 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 4h 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 proliferation 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 HIV+ samples.
13. Another HIV- sample should be run after all the HIV+ samples
have been completed.

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14. Compile and analyze data.
Protocol Summary
Title: A Clinical Trial of Administered DHEA combined with Isopentenyl
adenosine 5'-monophosphate as a specific inhibitor of Interleukin 10
- 5 Specially Formulated for Persons with HIV infection who have developed
resistance to protease and RT inhibitors.
DHEA combined with Isopentenyl adenosine 5'-monophosphate herein
referred to as Compound (D+l).
Indication: Treatment of HIV-1 infection.
Type of Study: Phase l/ll Clinical Trail.
Study Objectives:
a. Determine the safety and tolerance of administered Compound
(D+l) in persons with advanced HIV diseases.
b. Determine the effect of administration of Compound (D+l) on
measures of HIV Viral Load. Serum PCR (RNA) levels together with HIV p24
antigen (by acid dissociation method).
c. Determine the immune and toxicological effects of administered
Compound (D+l).
~ d. Determine the pharmokinetics of administered Compound (D+l).
20 Inclusion Criteria
a. Age 18 years or older;
b. HIV-1 seropositive;
c. A CD4+ -T-lymphocyte count of 50 to 300 cellslmm3 within one
month prior to study entry, measured on two separate occasions 72 hours to
28 days apart;
d. The following baseline laboratory values:
Hemoglobin > 9g/dl
WBCs > 1500 cellsl,ul
Neutrophils > 1000 cells/,ul
Platelets 25,000 cellsl~l
Bilirubin <2.0mgldl

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16
AST, ALT, Alkaline Phosphatase c5x upper limit of normal
Creatinine <1.5 mgldl;
e. A history of prior anti-retroviral therapy as follows:
i. In patients with a prior history of anti-retroviral therapy using
AZT, ddl, ddC or d4T alone or in combination with protease inhibitors who are
not receiving such therapy at study entry, these patients must have
discontinued this medication at study entry.
g. Use of suitable contraception by women of childbearing potential
(requires one negative serum pregnancy test, beta-HCG, within one week
prior to study entry in women of childbearing potential).
h. Medium to high PR HIV RNA titre at entry to study.
Exclusion Criteria:
a. Previous treatment with chemotherapeutic agents within eight
weeks of enrollment;
b. Active, major infection, including AlDS-defining opportunistic
infection, or other life-threatening medical crisis;
c. Pregnant or breast-feeding;
d. Any condition which, in the investigator's opinion places the patient
at undue risk or jeopardized the objectives of the trial;
e. 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:
i. Documentation and assessment of adverse events.
ii. Hematology.
iii. Clinical chemistries and urinalysis.
iv. Assessment of the immune responses resultant from Compound
(D+l).
v. Assessment of PCR (RNA) and DNA measures alteration with
therapy.
Effectiveness Measures:

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Measures of viral load will include HlV-p24 antigenemia, 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 Iymphocyte counts, percent alterations
in WBC, in Interleukin 10 levels which would demonstrate the ability of
Compound (D+l) to cause the patients' immune system to move to TH-1
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 of new opportunistic infection will be
summarized.
Study Design:
Open-label, daily administration of a dose per patient of 1200 mg/day, with
review and assessment of the dosage schedules and efficacy after therapy
for 4 weeks.
Study Size: 5 Patients (total) - 5 patients ~ 1200 mg/day for 30 days.
Test Articles:
Test Drug: Compound (D+l) particle size distribution, 87%: < 5 ~m,
100%: <15 ,um, administration in gelatine capsules of 200 mg per capsule.
Each capsule contains: 600 mg of DHEA and 600 mg of Isopentenyl
adenosine 5'-monophosphate
Control Drug: None
Placebo: None
Patient data required before and after administration of Compound (D+l)

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Screen Week
? Base- 1 2 3 4 8
line
... .. . ~ .. f
Physical Exam & Medical History X X X X X
Urinalysis X X X X X
Glucose X X X X X
Neopterin X X X X X
Beta2-microglobulin X X X X X
RBC X X X X X
hb X X X X X
WBC X X X X X X
Platelet X X X X X X
T Cell Panel X X X X X X
p24Antigen X X X X X
Creatinine X X X X X
SGOT X X X X X X
SGPT X X X X X X
IgG X X X X X
IgA X X X X X
IgM X X X X X
DHEA X X X X X X
DHEAS X X X X X X
Testosterone X X X X X X
17 Ketosteroids X X X X X X
Interleukin 10 X X X X X X
Interleukin 2 X X X X X X
PCR (RNA) (Cellfree, serum) X X X X X X
PCR (DNA) X X X X
CD4 X X X X X X