AU734807B2

AU734807B2 - Dhea combination therapy

Info

Publication number: AU734807B2
Application number: AU25741/97A
Authority: AU
Inventors: Patrick T. Prendergast
Original assignee: Individual
Current assignee: Individual
Priority date: 1996-04-17
Filing date: 1997-04-17
Publication date: 2001-06-21
Anticipated expiration: 2017-04-17
Also published as: NO984851D0; IL126623A0; CA2251733A1; KR20000005539A; JP2000508654A; AU2574197A; NO984851L; CN1216470A; EP0901375A1; WO1997038695A1

Description

ZJ/U4 U1 MUN 14:18 FAA tl J bi i 94J 1 6M1I1ITH HAUK IgL UUb 1 DHEA COMBINATION THERAPY SUMMARY OF THE INVENTION According to the invention, there are provided combinations of compounds for use in restoring normal levels of Interleukin 10 (IL-10) by enhancing or curtailing synthesis of effect of Interleukin 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. Louis, Mo., USA), secalonic acid D, isoflavonoids, 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 is a 17-ketosteroid compound having the general formula (I) o

C

R

3 (I)

R,

\\melbfiles\home \WendyS\Keep\species\25741-97 Prendergapt.doc 23/04/01 23/04 '01 MON 14:17 [TX/RX NO 9253] 23/04 '01 MON 14:18 FAX 61 3 9243 8333 lHIrI'TH1 HACK 0JUU7 2 in which R is a hydrogen atom, and Ri is a chemical group selected from the group consisting of a hydrogen atom, an

SO

2 0M group wherein M is selected from the group consisting of a hydrogen atom, sodium atom, a sulphatide group -S0 2 0-CH 2

CH.CH

2

O.CO.R

3 O.CO.

R

2 wherein each of R 2 and R 3 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 0

II

-P-O.CH

2

.CH.CH

2 .0.CO.R 3 II I O O.CO.R 2 wherein each of R 2 and R 3 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

S. HO of both configurations.: When Risotherthanahydrogen wherein the broken line represents an optional double bond, and the hydrogen atom at position 5 is present in the or P-configuration, or the compound comprises a mixture of both configurations. When Ri is other than a hydrogen atom, the compounds are conjugated compounds.

In another aspect of the present invention, there is provided a method of enhancing the Thl immune protective response when using one or more 17-ketosteroid compound as an anti-viral, anti-bacterial, anti-mycoplasma or antiintra cellular parasitic agent by combining it with antielbfiles\home$\WendyS\Keep\species\2574-97 Prendergast.doc 23/04/01 23/04 '01 MON 14:17 [TX/RX NO 9253] 23/04 '01 MON 14:18 FAX 61 3 9243 8333 GRIFFITH HACK A008 3 serum either poly or monoclonal to Interleukin 10 (cytokine inhibitory factor) and/or with any compound which can effectively inhibit synthesis or the biological function of this specific cytokine Interleukin 10 and/or an Interleukin (cytokine inhibitory factor) receptor molecule blocking agent.

For example, Thl immune protective response is required by patients 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 administered locally or systemically. By systemic administration is meant any mode 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 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, 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 also be formulated for transdermal administration, for example, in the form of \\mai.bEiles\home$\WendyS\Keep\pecies\25141-97 Prendergat.doc 23/04/01 23/04 '01 MON 14:17 [TX/RX NO 9253] U UL MUi lq _L14 rtA O X a 4J OJ.U unim in nrt~n Lg uu" 4 transdermal patches so as to achieve systemic administration.

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 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 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 R and RI are each hydrogen. An especially preferred compound is dehydroepiandrosterone (DHEA) wherein R and RI are each hydrogen and the double bond is present.

In a further embodiment of the invention, the compound is epiandrosterone wherein R and RI are each hydrogen and the double bond is absent. This saturated 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 further embodiment of the invention, the compound is 16(-bromoepiandrosterone, wherein R is Br, RI 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, RI is H and the double bond is not present \\melbfiles\hom$\WandyS\Keep\species\25741-97 Prendergast.doc 23/04/01 23/04 '01 MON 14:17 [TX/RX NO 9253] ZJ/U4 U1 MUN 14:1I VAA bi 3 HZ43 53JJU HUKIM11H lHAUl I UiU where the dotted line is shown in formula I, there is a single bond).

Other preferred compounds are dehydroepiandrosterone sulphate, wherein R is H, RI is S0 2 -OM and M is as hereinbefore defined and the double bond is present, and 5-androstan-3p-ol-17-one.

Alternatively, the compound is selected from dehydroepiandrosterone sulphatides, phosphatides or glucuronide wherein R is H, and RI is a sulphatide, phosphatide, or glucuronide group as hereinabove defined, and the double bond is present. In particular, when RI is not hydrogen, the compounds are DHEA conjugates such as hexyl sulfate, dodecyl sulfate, octadecyl sulfate, octadecanoylglycol sulfate, O-dihexadecylglycero sulfate, hexadecane sulfonate, dioctadecanoylglycero phosphate, O-hexadecylglycero phosphate.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art.

The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

S\AL .melbfilea\home$S\W dyS\Keep\species\25 -97 Prendargast.doc 23/04/01

LU

23/04 '01 MON 14:17 [TX/RX NO 9253] Ult WX LUU11 r11A 01 J Uz4J 544a bxKlMrrllu HACE I0011 Houston Study Experimental evidence using DHEA therapy in HIV+ patients has demonstrated that IL-12 levels, as measured by antibody ELISA methods, are elevated, natural killer cell levels increased together with the synthesis and presence of y (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, Th, immune improvement did not occur.

In fact, due to the elevated levels of Interleukin 12 generated by this monotherapy with DHEA, Interleukin levels increased, which caused a subsequent decline in T4 (helper) cell numbers and the disappearance of the Thl (Delayed Type Hypersensitivity Response). Skin reaction in patients as evidenced by patient data was down-regulated by 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 open-label dose-escalation trial or oral DHEA (Dehydroepiandrosterone) tolerance and pharmacokinetics in patients with HIV disease. In the Phase o o• oo 23/04 '01 MON 14:17 [TX/RX NO 9253] WO 97/38695 PCT/IB97/00414 6 I DHEA trial (early symptomatic HIV disease and 200 to 500 CD4+ lymphocytes/pL), 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 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 using Combination Therapy To counteract this Th 1 suppressive immune side effect of DHEA monotherapy this anti-viral steroid had to be combined with an agent to inhibit 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 I) wherein the anti-viral agents are allowed to generate a Th, response. The component of the combination therapy which counteracts the Th 1 suppressive Interleukin 10 immune side 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 reference, which may be used in the combination therapy to counteract the Th 1 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 bloodstream was enhanced by 3 (three) logs, relative to the monotherapy, while simultaneously enhancing by over 80% the Th 1 (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 allows for substantial therapeutic benefit to be achieved which previously could not be accomplished by the use of DHEA alone as a monotherapy. The WO 97/38695 PCT/IB97/00414 7 beneficial action of up-regulation of the immune system together with the antiviral action of compounds of formula I 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 Th 1 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 14/7/1983 RD is a patient under my care. He has Acute Myeloid Leukaemia M3 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 WO 97/38695 PCT/IB97/00414 8 whereas patients who have been administered DHEA, but who, due to other cytokine and immune factors, have not experienced an Interleukin elevation have not demonstrated relief of symptoms. Therefore, we have identified that a more direct means of causing immediate relief in these autoimmune conditions is to administer exogenously recombinant Interleukin 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 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 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 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 Lupus, MS and HIV 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 DHEA therapy. When the steroid is first administered the immune therapeutic response is very much patient specific and cannot be relied upon to produce WO 97/38695 PCT/IB97/00414 9 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 enhancement without the general negative effects of Interleukin 10 whereas Thi 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 Th 1 auto-immune condition and requires Interleukin 10 to down regulate the Th 1 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, great patient variability to treatment. Any remission of symptoms in Multiple Sclerosis was identified by patients who experienced significant elevation of their endogenous levels of Interleukin 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 HIV-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 WO 97/38695 PCT/IB97/00414 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 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 demonstrated that in vivo administration of murine Interleukin 12 (IL-12) to mice results in augmentation of cytotoxic natural killer (NK)/lymphocytesactivated 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.

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 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, 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 the antitumor effects against subcutaneous tumors.

Los Angeles Patient Study WO 97/38695 PCT/IB97/00414 11 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 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 Form: Liquid Concentration: 2.7 mg/ml Stabilizers: None Preservative: None Sterility: Sterile filtered Host Species: Rabbit Antibody Class: IgG Antigen Used: Recombinant human Method of Purification: Ion Exchange chromatography Method of Quantification: Pierce BCA Protein Assay Specificity: Human Cross-Reactivity: No cross reactivity with WHO standards: IL-la, IL-1B, IL-2, IL-3, IL-4, IL-6, IL-7 IL-8, MIP-la, TNFa and GM-CSF done by EIA.

Storage: Short term 4 0 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, (#219-IL) 5 fg should be sufficient for the entire experiment.

WO 97/38695 PCT/IB97/00414 12 4. Antibody to human IL-2 receptor, R&D Systems (AB-233-NA), 1 mg lyophilized, 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 5nM/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 g should be sufficient for the entire experiment.

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 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 106 cells or more are present, seed the cells into two 96 well plates. At 10 x 106 we will end up with 0.5 x 10 5 cells/well or 2.5 x 105 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).

UJ.^ A A J.JJ J-mVj'J.k Wjd U..

13 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 or RIO 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 RIO for each sample to be used for the IL-2 ELISA, add 2 fg/mL of antibody to the IL-2 receptor.

*oo$: 7. Preparation of DHEA: ee 7.1 Dissolve 1 g of DHEA of 1 mL of absolute ethanol Incubate in a 37°C water bath. Additional ethanol .may be added up to 3.47 mL. This will give a 1 M solution.

S" If the entire 3.47 mL is not needed for it to go into solution the difference can be made up with RIO medium.

7.2 For each sample, we will need with medium DHEA at the t: following concentrations: 10- 4 10 6 10-10, 10- 1 2 7.3 At each dilution prepare 2 mL of media (already containin 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 10- 8 6 mL of each will be needed.

At each dilution prepare 2 x 1.5 mL of RIO medium without gp 120 and with and without antibody from step 6.

For 10- 8 3 mL of each will be needed.

S\\melb-iles \homr\WendyS\Kerp\species\25741-97 Prendargast.doc 23/04/01 23/04 '01 MON 14:17 [TX/RX NO 9253] tj/u4 UI MUn 1i;Ll rAA 01 0 VZ0 ojJa UNiriln nAUi Lula.

7.4 Making dilutio A. Take 20 fL of 2

M.

B. Take 20 fL of 10-4M.

Take 15 fL of C. Take 20 fL of 10-6M.

Take 15 fL of D. Take 40 fL of Take 30 fL of E. Take 20 fL of 10-1 0

M.

Take 15 mL of F. Take 20 fL of 10- 12

M.

Take 15 fL of )ns Use 5 mL tubes.

1 M DHEA into 2 mL of R10 medium 10-2M DHEA into 2 mL of step 7.3 medium 10-2M DHEA into 1.5 mL of R10 10-4M.

10- M DHEA into 2mL of step 7.3 medium 10-4M DHEA into 1.5mL of R10 10-6M.

10-M DHEA into 4 mL of step 7.3 medium 10-6M DHEA into 3 mL of R10 10- M.

10-1M DHEA into 2 mL of step 7.3 medium 10-M DHEA into 1.5 mL of R10 10-1M.

10-1M DHEA into 2 mL of step 7.3 medium 10- 12

M.

9 0

S

S..

OSSO

i or S a a 5

S

4 a *5* a.

a i'"2" 7.5 To half of the 10-M DHEA media types add antibody to human IL-12.

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 EDso=1U. The EDso of the IL-12 will be in the literature received with this reagent.

\\melb_ ilea\home$\WendyS\Keep\species\2'741-97 Prendergast.doc 23/0I/01 23/04 '01 MON 14:17 [TX/RX NO 9253] J. US. uIll. tt.G rni u. aGr a 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 0 C, 5% COz.

9. If it is a two plate assay, 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 4h incubation.

After 7 days using the plate with the antibody to the IL-2 receptor: Take 100 fL per well and use to perform 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.

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 Specially Formulated for Persons with HIV infection who have developed resistance to protease and RT inhibitors.

DHEA combined with Isopentenyl adenosine monophosphate herein referred to as Compound \\mlb-iles\humeS \WendyS\ eep\species\25741-97 Prendergast.doc 23/04/01 23/04 '01 MON 14:17 [TX/RX NO 9253] 2./04 '01 MON 14:21 FAX 61 3 9243 8333 GRIFFITH HACK 11015 Indication: Treatment of HIV-1 infection.

Type of Study: Phase I/II Clinical Trial.

Study Objectives: a. Determine the safety and tolerance of administered Compound in persons with advanced HIV diseases.

b. Determine the effect of administration of Compound on measures of HIV Viral Load. Serum PCR (RNA) levels together wit HIV p24 antigen (by acid dissociation method).

c. Determine the immune and toxicological effects of administered Compound d. Determine the pharmacokinetics of administered Compound Inclusion Criteria a. Age 18 years or older; Sb. HIV-1 seropositive; S. c. A CD4+ -T-lymphocyte count of 50 to 300 cells/mm 3 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 cells/pl Neutrophils 1000 cells/gl Platelets 25,000 cells/pl Bilirubin <2.Omg/dl \\melb_files\hume$\W ndyS\Keep\species\25741-97 Prendergast.doc 23/04/01 23/04 '01 MON 14:17 [TX/RX NO 9253] WO 97/38695 PCT/IB97/00414 16 AST, ALT, Alkaline Phosphatase <5x upper limit of normal Creatinine <1.5 mg/dl; 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 AIDS-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 v. Assessment of PCR (RNA) and DNA measures alteration with therapy.

Effectiveness Measures: WO 97/38695 PCT/IB97/00414 17 Measures of viral load will include HIV-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 lymphocyte counts, percent alterations in WBC, in Interleukin 10 levels which would demonstrate the ability of Compound 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 particle size distribution, 87%: 5 pm, 100%: <15 pm, administration in gelatine capsules of 200 mg per capsule.

Each capsule contains: 600 mg of DHEA and 600 mg of Isopentenyl adenosine Control Drug: None Placebo: None Patient data required before and after administration of Compound (D+I) WO 97/38695 WO 9738695PCT/lEB97/00414 Screen Week Base- 1 2 3 4 8 Physical Exam Medical History 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 p24 Antigen X X X X X Creati nine x X X X X SGOT X X X X X X SGPT X X X X X X JgG X X-X X X IgA X X X X X 1gM X X X X X DHEA X X X X X X DHEAS X X X X X X Testosterone x x x I 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 PCIR (RNA) (Cell free, serum) X X X X X X PCIR (DNA) X X X X C4X X X X X

Claims

1. A method of enhancing a Thl immune protective response, comprising the step of administering an effective amount of one or more 17-ketosteroid compounds in conjunction with one or more agents selected from the group consisting of a) an inhibitory antibody directed against b) a compound which has the ability to inhibit the synthesis or biological function of IL-10, and c) a compound which has the ability to inhibit the function of receptors for to a subject in need of such treatment.

2. A method according to claim 1, in which the subject is suffering from a condition selected from the group consisting of a viral, bacterial, mycoplasma or intracellular parasitic infection, an immune dysfunction and cancer.

3. A method of treating or arresting the progression of a viral, bacterial, mycoplasma or intracellular parasitic S" infection or an immune dysfunction or cancer, comprising the step of administering an effective amount of one or more 17-ketosteroid compounds in conjunction with one or more agents selected from the group consisting of a) an inhibitory antibody directed against b) a compound which has the ability to inhibit the synthesis or biological function of IL-10, and g c) a compound which has the ability to inhibit the function of receptors for to a subject in need of such treatment. l _files\hom$ \WendyS\KXep\species\25 P41-97 rendergast.doc 23/04/01 23/04 '01 MON 14:17 [TX/RX NO 9253]

4. A method according to claim 2 or claim 3, in which the viral infection is HIV infection. A method according to claim 2 or claim 3, in which the cancer is metastatic and/or multi-drug resistant.

6. A method according to any one of claims 1 to 3, in which the inhibitory antibody is directed against the portion of the Epstein-Barr virus open reading frame BCRF1 with amino acid sequence homology to

7. A method according to any one of claims 1 to 6, in which the compound which has the ability to inhibit the synthesis of IL-10 is selected from the group consisting of canavanine sulfate, L-canavanine sulfate, herbimycin A, genistein, secalonic acid D, isoflavonoids, cytokinins, amphiphilic triterpenoids, and analogs of any of these compounds.

8. A method according to claim 7, in which the cytokinin is selected from the group having the formula: S S S S. S in which: a) RI -H,R 2 -CH 3 R 3 -CH 3 and R 4 or \\melbtiles\homeS\WendyS\Keep\spcies\25741-97 Prendergast.doc 23/04/01 23/04 '01 MON 14:17 [TX/RX NO 9253] 1.3 Uq 4 UI MUIn Z 1'.tA 01 a3 vq r- J q.x.r ai 0.3.3.1 1 lt unirrxin n^ n 'L'J 21 b) R, -H or -CH 3 S and R 4 CH 2 R in which R-3 -CH 3 -Cl, -OH or a monophosphate group, R= -CH 3 -CH 2 OH or -Cl, R 7 -H or -Br; or C) R, -H and \/X2 X, R4 C. C C. C C C. C C. C* C C .C C C .C C in which X, and X 2 are independently selected from the group consisting of methyl, ethyl, hydroxyl, the halogens and carboxyl; or ;or R 4 or R 4 (0)-NH-Ra, \\ek-ie~oa\adSXe~pca\5219 Prendcrqa3t.doc 23/04/01 23/04 '01 MON 14:17 [TX/RX NO 9253] "i UtL MUIA 14.L VZ4 A UA a vrI. DoG. -l U\ 22 in which R 8 is CI or -(CH 2 7 CH 3 and in which R 2 is -OH and R 3 is selected from the group consisting of -OH, monophosphate group, diphosphate group and triphosphate group; or R 2 and R 3 are linked to form a monophosphate derivative, or a metabolite of said compound, said metabolite selected from the group consisting of N6- (A2-isopentenyl)adenine, 6-N-(3-methyl-3- hydroxybutylamino)purine, adenine, hypoxanthine, uric acid and methylated xanthines.

9. A method according to any one of claims 1 to 8, in which the 17-ketosteroid has the formula: C CH3 0 RIO v in which the broken line represents an optional double CH3 bond, and the hydrogen atom at position 5 is present in the i a-or p-configuration or the compound comprises a mixture of both configurations and in which R is a hydrogen atom, and RI is a chemical group selected from the group consisting of a) a hydrogen atom; \\-mel -Eile \homa\WendyS\Keep\specics\25741-97 Prendergast.doc 23/04/01 23/04 '01 MON 14:17 [TX/RX NO 9253] 23 b) an SO20M group, in which M is a hydrogen atom or a sodium atom; c) a sulphatide group -SO 2 0-CH 2 CH.CH 2 O.CO.R 3 I O.CO. R 2 d) a phosphatide group 0 II -P-O.CH 2 .CH.CH 2 .0.CO.R 3 II I 0 O.CO.R 2 in which for c) and d) each of R 2 and R 3 is independently selected from the group consisting of straight and branched chain alkyl radicals of 1 to 14 carbon atoms, and e) a glucuronide group *OH HO A composition, comprising: a) one or more 17-ketosteroid compounds, and b) one or more compounds selected from the group consisting of: i) an inhibitory antibody directed against ii) a compound which has the ability to inhibit the synthesis or biological function of IL-10, and \\melbfiles\homeS\janel\Keep\Speci\25741-97.doc 26/04/01 iii) a compound which has the ability to inhibit the function of receptors for together with a pharmaceutically acceptable carrier.

11. A kit when used for the treatment of viral, bacterial, mycoplasma or intracellular parasitic infections, immune dysfunction or cancer, comprising: a) at least one unit-dosage of one or more 17- ketosteroid compounds, and b) at least one unit-dosage of one or more compounds selected from the group consisting of: i) an inhibitory antibody directed against ii) a compound which has the ability to inhibit the synthesis or biological function of IL-10, and g iii) a compound which has the ability to inhibit the function of receptors for

12. Use of a composition, comprising *eo a) one or more 17-ketosteroid compounds, and b) one or more compounds selected from the group consisting of: i) an inhibitory antibody directed against ii) a compound which has the ability to inhibit the synthesis or biological function of IL-10, and iii) a compound which has the ability to inhibit the function of receptors for for the preparation of a medicament for use in the treatment of viral, bacterial, mycoplasma or intracellular parasitic infections, immune dysfunction or cancer. \\melbfiles\home$\janel\Keep\Speci\ 257

41-97.doc 26/04/01 23/04 '01 MON 14:35 FAX 61 3 9243 8333 GRIFFITH HACK b 009 13. A method according to claim 1, substantially as herein described with reference to the examples. 14. A method according to claim 3, substantially as herein described with reference to the examples. A composition according to claim 10, substantially as herein described with reference to the examples. DATED'this 2 3 rd day of April 2001 P.T. PRENDERGAST By Their Patent Attorneys: GRIFFITH HACK Fellows Institute of Patent Attorneys of Australia e e" o e \\melbfilea\hom$S\WendyS\KeeB\species\25741-97 Prendergast.doc 23/04/01 23/04 '01 MON 14:34 [TX/RX NO 9254]