WO2000041547A2

WO2000041547A2 - Methods of promoting or enhancing interleukin-12 production through administration of thalidomide

Info

Publication number: WO2000041547A2
Application number: PCT/US2000/000914
Authority: WO
Inventors: Gilla Kaplan; Stephen J. Oliver; Patrick A. J. Haslett
Original assignee: The Rockefeller University
Priority date: 1999-01-13
Filing date: 2000-01-13
Publication date: 2000-07-20
Also published as: WO2000041547A3; AU2612100A

Abstract

Methods of promoting or enhancing IL-12 production in patients through administration of thalidomide, or pharmaceutically acceptable salts thereof. The methods are useful in promoting or enhancing IL-12 production in patients suffering from HIV and other infections, tuberculosis, and autoimmune disorders such as sarcoidosis and scleroderma.

Description

METHODS OF PROMOTING OR ENHANCING INTERLEUKIN-12 PRODUCTION THROUGH ADMINISTRATION OF THALIDOMIDE

BACKGROUND OF THE INVENTION

Interleukin-12 (IL-12) is a heterodimeric cytokine that plays a central role in promoting type-1 T-helper cell (Thl) responses, and thus, cell-mediated immurrity. It is produced in the body by both macrophages and B-lymphocytes, and plays an important role in the normal host defense against infection by a variety of pathogens. For example, IL-12 is required for T-cell-independent triggering of NK cells by intracellular parasites (Gazzinelli et al, PNAS, 1993,90:6115-6119), and has been found to stimulate the production of interferon-γ from T and natural killer (NK) cells (Scott, Science, 1993, 260:496-497). IL-12 is a pivotal cytokine in the regulation of cellular immunity, steering primary T cell responses toward a Th-1 type cytokine (IL-2 and IFN-γ) secreting profile, and away from a Th-2 type cytokine (IL-4 and IL-5) secreting profile (Trinchieri et al, Immunol. Res., 1998,17:269-278). IL-12 has also been found to have an effect on the treatment of cancer in patients.

Retro viruses are a group of RNA viruses that, during replication, employ reverse transcriptase to convert RNA messages to DNA. The human immunodeficiency virus (HIV-1) or human T-Cell lymphotropic virus (HTLV-III) which causes Acquired Immune Deficiency Syndrome (AIDS), AIDS related complex (ARC), and other AIDS related disease, is a retrovirus. Tumor Necrosis Factor-alpha (TNF-α) is one of several cytokines released by mononuclear phagocytes together with several other cytokines in response to stimuli to the immune system. TNF-α is associated with the destruction of tumor cells, and is required for cell-mediated immune response in order to overcome infections. A correlation has been shown between levels of circulating HIN and TΝF-α, (Klausner et al, J. AIDS., 1996, 11 :247-257), or TΝF-α receptor in the plasma of HIV infected patients (Bilello et al, J. Infect. Dis., 1996, 173:464-467). TΝF-α induces viral replication via a shared ΝFk-B-dependent transcriptional control mechanism (Pomerantz et al, J. Exp. Med., 1990, 172:253-261).

Sarcoidosis (also known as sarcoid of Boeck) is a long-term disease of unknown origin marked by small, round bumps in the tissue around the organs of the body, typically the lungs, spleen, liver, skin, mucous membranes, and tear and salivary glands. The sores usually go away, but lead to widespread grainy swelling and fibrosis.

Scleroderma (also known as Progressive Systemic Sclerosis) is a somewhat rare disease affecting the blood vessels and connecting tissue. It is characterized by hardening of the skin of the face and hands, and is most common in middle-aged women.

Thalidomide (α-phthalimidoglutarimide) is an immunomodulatory compound of the formula:

Although thalidomide was in the past marketed as a sedative, it was later found to be teratogenic and withdrawn from the market. Despite its teratogencity, however, thalidomide has been found in recent years to have various therapeutic applications. For example, it has been shown that thalidomide is capable of inhibiting production of TΝF-α in vivo ( ampaio et al, J. Exp. Med., 1991, 173:699-703), and in vitro (Sampaio et al, J. Infect. Dis., 1993, 168:408-414). Thalidomide can also inhibit HIN replication in cultures of infected monocytes and PBMC (Moreira, et al, AIDS Res. Hum. Retroviruses., 1997, 13:857-863; Makonkawkeyoon et al, PΝAS USA., 1993, 90:5974-5978). However, clinical studies with thalidomide have not shown consistent inhibition of TΝF-α or HIV in vitro (αacobson, et al, New. Engl. J. Med., 1991, 336:1487-1493; Klausner et al, J. AIDS., 1996, 11:247-257).

Recently, studies have revealed an additional and unexpected property of thalidomide in acting as a costimulator of T cells in vitro. Thus, thalidomide treatment of T cells simultaneously receiving a primary stimulus through the T cell receptor results in enhancement of Interleukin-2 (IL-2) and IFN-γ production (Haslett et al, J. Exp. Med., 1998, 187:1885-1892). Thalidomide thus appears to have at least two irnmunomodulatory properties: (i) inhibition of monocyte TNF-α production, and (ii) T cell costimulation.

In some diseases, it is desirable to stimulate or enhance IL-12 production in patients. Due to its role in cell-mediated immunity, and in the defense against infection, stimulation or enhancement of IL-12 production in the body is also desirable in a variety of infections, such as viral infections such as HIV and bacterial infections such as tuberculosis, and autoimmune disorders such as sarcoidosis and scleroderma.

SUMMARY OF THE INVENTION

The invention relates to methods of promoting or enhancing IL-12 production in patients through the administration of effective amounts of thalidomide. The methods are useful in promoting or enhancing IL-12 production in patients suffering from HIV and other infections, tuberculosis, and autoimmune disorders such as sarcoidosis and scleroderma. BRIEF DESCRIPTION OF THE FIGURES Figures 1A, IB, 1C and ID show changes in plasma immunologic markers from the time of randomization of HIV infected patients with thalidomide or placebo (day 12). Treatment with thalidomide or placebo was stopped on day 42. Solid symbols represent patients treated with thalidomide. Open symbols represent patients treated with the placebo. (A) Plasma TNF-α levels; (B) plasma SIL-2R levels; (C) plasma soluble CD8 levels; (D) plasma IL-12 levels.

Figure 2 sets forth responses to thalidomide (1 mg/ml and 10 mg/ml) of PBMC and purified CD4⁺ and CD8⁺ T cells from HIV-infected subjects stimulated in vitro with immobilized anti-CD3 antibody. Proliferative responses were measured after 5 days in culture. Data are expressed as the ratio of cpm of thalidomide-treated cultures to DMSO (control)-treated cultures. Circles represent CD8⁺ T cells; squares represent CD4⁺ T cells; diamonds represent PBMC. Group B sets forth results of IL-2 secreted into culture supernatants assayed at 24 hours. The Data is expressed as increase over DMSO control (normalized to 100%). Clear bars represent the DMSO control. Pale gray bars represent thalidomide 1 mg/ml. Dark gray bars represent thalidomide 10 mg/ml. Group C sets forth results of IFN-γ secreted into culture supernatants assayed at 72 hours. Data is expressed as increase over DMSO control (normalized to 100%). The key is the same as in Group B above. All data represent means of 5 independent experiments.

Figure 3 sets forth the correlation of thalidomide (1 mg/ml) induced increases (over DMSO control) in IFN-γ production and thalidomide-induced changes in IL-12 secretion by anti-CD3 -stimulated PBMC obtained from 9 HIV-infected individuals. Supernatants for ELISAs were harvested at 48 hours (time of peak IL-12 production established in preliminary experiments).

Figure 4 A, 4B and 4C: (A) shows the effects of thalidomide on CD40L expression by CD4⁺, CD8⁺, and total CD3⁺ T cells in PBMCs stimulated by anti-CD3. Data are mean results from 7 HIV-infected donors. Error bars represent SEMs. Cells were harvested for 3-color flow-cytometric analysis at 48 hours. Clear bars represent the DMSO control; pale gray bars represent thalidomide 1 mg/ml; dark gray bars represent thalidomide lOmg/ml. Statistically significant (p<0.05) increases over DMSO control are indicated by an asterisk (*) (B). Effect of antibody blockade of CD40L (lOmg/ml) on IL-12 (ρ40 and p70) production by anti-CD3 stimulated PBMC from HIV-infected subjects in the presence or absence of thalidomide (lOmg/ml). Supernatants were assayed after harvest at 48 hours. Pale gray bars represent the DMSO control; dark gray bars represent thalidomide lOmg/ml. Data are normalized to results for the DMSO treated controls exposed to control mouse IgG, and are the mean of experiments performed with PBMC from 4 HIV-infected individuals. (C) similar to (B), but the supernatants were assayed for the IL-12 p70 heterodimer only. The key is the same as in (B), above. Data shown are means (±SEM) of experiments with PBMC from 2 HIV infected individuals.

Figures 5 A and 5B: (A) shows plasma IL-12 levels in sarcoid patients treated with thalidomide; (B) shows percent increase over base line of IL-12 plasma levels in sarcoid patients.

Figures 6 A and 6B: (A) shows plasma soluble IL-2 receptor levels in sarcoid patients; (B) shows percent increase over base line of plasma sIL-2R in sarcoid patients.

Figures 7 A, 7B, 7C, and 7D: (A) shows HLA-DR surface expression on CD3⁺ lymphocytes; (B) shows CD40 ligand expression on T lymphocytes; (C) shows CD80 expression on T lymphocytes; D) shows CD86 expression on T lymphocytes.

Figures 8 A and 8B: (A) shows plasma IL-12 levels in scleroderma patients; (B) shows percent increase of IL-12 over base line levels in scleroderma patients.

DETAILED DESCRIPTION OF THE INVENTION

Definitions of Terms Used Herein sIL2R: Soluble IL-2 receptor sCD8: soluble CD8 antigen sCD4: Soluble CD4 antigen

DTH: Delay ed-type hypersensitivity PPD: Purified protein derivative of tuberculin

TCR: T cell receptor

APC: Antigen-presenting cell

ZDV/LMV: Zidovudine/lamivudine antiretro viral therapy

The invention relates to methods of stimulating or enhancing IL-12 production in a patient through administration of a compound of the formula:

pharmaceutically acceptable salts thereof, or compositions thereof. The compounds and methods are useful in promoting or enhancing IL-12 production in patients suffering from HIV and other infections, tuberculosis, and autoimmune disorders such as sarcoidosis and scleroderma. A placebo-controlled study was conducted to evaluate the effects of immune modulation with thalidomide on HIV levels, TNF-α levels, and immune status in 31 HIV-infected individuals. Thalidomide therapy, 200mg/day for 4 weeks did not affect plasma TNF-α or HIV levels. However, thalidomide treatment did result in significant immune stimulation. Increased levels of plasma soluble IL-2 receptor, soluble CD8 antigen, and IL-12 (p=0.037, 0.007, and 0.0004, respectively) was observed. In addition, thalidomide treatment increased cutaneous delayed-type hypersensitivity reactions to recall antigens (p=0.008). Further studies were then performed in vitro to elucidate the mechanism of thalidomide-induced immune stimulation. When purified T-cells from HIV-infected individuals were stimulated by immobilized anti-CD3 in the presence of thalidomide, a co-stimulatory effect of the drug was observed, resulting in increased production of IL-2 and IFN-γ, and increased T-cell proliferative responses. Thalidomide thus can be used as a novel immune adjuvant in HIV disease.

Thalidomide-induced IFN-γ secretion correlated with increases in IL-12 production (R=0.83, p=0.0056) by anti-CD3 stimulated peripheral blood mononuclear cells. The drug-induced increase of IL-12 production was abrogated by antibodies against CD40 ligand, suggesting that thalidomide induces IL-12 through a T-cell-dependent pathway. Thalidomide can thus be administered to a patient to stimulate or enhance IL-12 production in a patient suffering from HIV infection. The compounds of the invention, or their pharmaceutically acceptable salts, may be administered alone, but will normally be employed in a composition containing a pharmaceutically acceptable carrier. This includes tablets, pills, lozenges, dragees, and similar shaped or compressed preparations. Further suitable variations include emulsions, or suspensions of the compound in water or aqueous media, or in the form of powders filled into gelatin capsules or the like. Such powders or mixtures for use in the preparation of tablets and other shaped and/or compressed preparations may be diluted by mixing and milling with a solid pulverulent extending agent to the desired degree of firmness or by impregnating the already milled, finely powdered, solid carrier with a suspension of the compounds in water or with a solution thereof in an organic solvent and then removing the water or solvent.

When preparing tablets, pills, dragees, and the like shaped and/or compressed preparations, the commonly used diluting, binding, and disintegrating agents, lubricants, and other tableting adjuvants are employed, provided they are compatible with the agent to be administered. Such diluting agents and other excipients are, for example, sugar, lactose, levulose, starch, bolus alba; as disintegrating and binding agents, gelatin, gum arabic, yeast extract, agar, tragacanth, methyl cellulose, pectin; and as lubricants stearic acid, talc, magnesium stearate, and others.

The compounds and compositions can be administered orally, topically, rectally, vaginally, by pulmonary route by use of an aerosol, or parenterally, i.e., intramuscularly, subcutaneously, intraperitoneallly or intravenously. They can be administered alone, or can be combined with a pharmaceutically-acceptable carrier or excipient according to standard pharmaceutical practice. For oral administration, the compositions can be used in the form of tablets, capsules, lozenges, troches, powders, syrups, elixirs, aqueous solutions and suspensions, and the like. For tablets, suitable carriers include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents include lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can also be added. For parenteral administration, sterile solutions of the polymer networks and compositions thereof are usually prepared, and the pH of the solutions suitably adjusted and buffered. For intravenous use, the total concentration of solutes is controlled to render the preparation isotonic. For ocular administration, ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers. Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers. For pulmonary administration, diluents and/or carriers can be selected to allow the formation of an aerosol. The preferred route of administration is oral.

Dosage amounts will vary depending upon the patient's age, weight, health, and severity of condition. The specific dosage will be determined by the attending physician or caretaker, based upon the individual circumstances of the patient.

The following examples will serve to further typify the nature of the invention but should not be construed as a limitation on the scope thereof, which is defined solely by the appended claims.

The effect of thalidomide treatment on TNF-α and HIV levels of HIV-infected individuals, and T cell activation by thalidomide administration was investigated below. To that end, a double-blind, placebo-controlled study of thalidomide treatment following temporary suppression of viremia with antiretro viral drugs was carried out. The effect of the drug on the "rebound" of viral titers to baseline levels was then evaluated. In addition, the effects of thalidomide on parameters of immune activation in the patients were momtored. Finally, the effects of thalidomide on PBMC and purified T cells from HIV-infected subjects stimulated ex-vivo with immobilized anti-CD3 antibodies was investigated.

EXAMPLE 1: Changes in Plasma Immunologic Markers

Immunologic Assessments. Blood samples collected in the same way and at the same time points as described above were used for these studies. The following cytokines and soluble markers of T cell activation were measured in plasma by ELISA, in accordance with each manufacturer's instructions: TNF-α (Medgenix, Fleurus, Belgium; this assay measures both free and receptor-complexed TNF-α); IFN-γ (Genzyme, Cambridge, MA); interleukin-12 (IL-12) (Endogen Inc., Woburn, MA: this assay measures both p40 and p70 subunits of IL-12); soluble interleukin-2 receptor (sIL-2R) (Genzyme); soluble CD4 antigen (sCD4) and soluble CD8 antigen (sCD8) (T Cell Diagnostics Inc., Woburn, MA). The two initial values for each parameter were averaged to provide a mean starting value for the analysis. T lymphocyte subsets (CD³⁺ CD⁴⁺ and CD³⁺ CD⁸⁺) were enumerated by flow cytometry (FACSTAR, Becton Dickinson, San Jose, CA), at baseline (day 0) and on study days 12 and 42.

Skin tests for delayed-type hypersensitivity (DTH) were conducted on study days 0, 42, and 56. Purified protein derivative of tuberculin (PPD), 5 tuberculin units (Connaught Laboratories Limited, Willowdale, Ontario, Canada), Candida antigen, 0.1ml of undiluted reagent (ALK Laboratories Inc., Berekely, CA) and trichophyton, 0.1ml of undiluted reagent (ALK Laboratories Inc.), were injected intradermally into the volar aspect of the forearms, and induration in two peφendicular diameters measured 48 hours later. A positive response was defined as a mean diameter of induration of greater than or equal to 5mm. The results are set forth in Figure 1.

EXAMPLE 2: In Vitro Study

Study subjects. For in vitro studies, blood was drawn from clinically stable HIV-infected patients recruited through the outpatient clinic of the Rockefeller University Hospital.

Preparation of cells. PBMC were freshly isolated from whole blood by ficoll density gradient separation. CD⁴⁺ and CD⁸⁺ T cell subsets were purified by positive selection with magnetic beads coated with the appropriate monoclonal antibodies, in accordance with the manufacturer's instructions (Dynal AS, Oslo,

Norway). Selected cells were subsequently separated from the beads by incubation with a second antibody that competes for the binding site on the primary antibody ("Detachabead", Dynal AS). This procedure routinely recovered cells which were >99% pure CD³⁺ CD⁴⁺ or CD³⁺ CD⁸⁺. Cells were suspended in RPMI medium (Gibco, Grand Island, NY) supplemented with 10% pooled human AB serum and penicillin (50m/ml)/streptomycin (0.05mg/ml). Cultures were set up in a humidified 37°C incubator with a 5% CO₂ atmosphere.

Stimulation assays. T cells were stimulated by cross-linking CD3, a component of the T cell receptor (TCR) complex. Forty eight- or 96- well flat-bottomed tissue culture plates were coated with mouse anti-human CD3 monoclonal antibody (Orthobiotech, Raritan, NJ) at a coating concentration of lOmg IgG/ml as previously described (Haslett et al, J. Exp. Med., 1998, 187:1885-1892). For each experimental condition, duplicate cultures were set up at 10° cells (either purified T cells or PBMC) in 1ml per well in the 48-well plates, and triplicates of 10⁵ cells in 0.2 ml per well in the 96-well plates. Supernatant culture medium from the 48-well plates was harvested at the time points indicated in the results and stored at -70°C until assayed by ELISA for IL-2, IFN-γ, IL-12 (p40 and p70) (Endogen, Inc.) and IL-12 (p70 subunit only) (R&D Systems Minneapolis, MN).

To estimate lymphocyte proliferation, DNA synthesis was assayed in the 96-well plates by measuring the incorporation of 3H-thymidine over the last 12 hours of 120 hour cultures as described previously. Proliferation data are expressed as "stimulation ratios" of thalidomide treated cultures over DMSO treated controls (cpm_ttøu_do-n_jde, cpm_contr0|).

In some experiments, CD40 ligand (CD40L) on T cells was blocked with mouse monoclonal anti-human CD40L IgG (clone TRAP1, Pharmingen). Surface expression of CD40L was assessed on CD3⁺ CD8⁺ and CD3⁺ CD4⁺ lymphocyte subsets by three-color flow cytometry (anti-CD40L, Pharmingen; anti-CD3, anti-CD4 and anti-CD8, Becton-Dickinson).

Thalidomide. Thalidomide (a-phthalimido-glutarimide, Celgene Corporation) was dissolved in dimethyl sulfoxide (DMSO) to achieve stock concentrations of 20mg/ml. Thalidomide at various concentrations or DMSO (control) was added at culture initiation so that the concentration of DMSO was identical in all culture conditions. Culture media refreshed daily with 50% volume exchanges containing fresh thalidomide/DMSO or medium/DMSO.

Statistical analysis. For longitudinal within group comparisons, data were compared by Student's paired t test. For data acquired following randomization (day 12), changes from the time of randomization were calculated for each individual at each subsequent time point. Data from the treatment groups were then compared employing Student's 2-tailed independent t test. Chi-square analysis was used for comparing proportions, and simple linear regression analysis for evaluating relationships between variables. Data were entered into Excel spreadsheets, and Excel data analysis software used for these calculations (Microsoft Corporation, Redmond, WA).

Clinical Study Results Tolet ability ofZDV/LMV. Thirty-six patients started treatment with ZDV/LMV. Five of these dropped out of the study before or at day 12 for the following reasons: opportunistic infection with Penicillium marnffei (n=2, on days 6 and 12, respectively), acute respiratory tract infection (n=l, on day 12), drug rash (n=l, on day 7), nausea and vomiting (n=l, on day 7). The data from these patients are not included in the analysis.

Tolerability of Thalidomide. The 31 patients who completed 12 days of antiviral treatment were randomized to receive thalidomide (n=l 8), or placebo (n=13). There were no significant differences in clinical characteristics and immunologic markers between the two groups at baseline (see Table 1).

Table 1 Patient Baseline Clinical Parameters by Subsequent Randomization

Parameter (units) Placebo Thalidomide p val

Number of patients 13 18

Age (years) 32 (29-35) 31 (28-36) 0.96

CD4 count (cells/MM3) 86 (34-199) 126 (44-399) 0.17

Weight (lbs) 116 (99-130) 117 (107-135) 0.38

Karnofsky score 100 (90-100) 100 (80-100) 0.54

Data expressed as median values (interquartile range).

* Data for thalidomide and placebo treatment groups compared by independent t test. All subjects completed at least two weeks on thalidomide or placebo (up to study day 28). Five patients in the thalidomide treatment subsequently discontinued the study drug: 4 because of skin rash, and one lost to follow-up. Data for these individuals are included up to the time of study drug discontinuation for the purpose of analysis. One individual who was randomized to the placebo arm of the study exhibited markedly anomalous indicators of immune activation from day 28 of the study period, but in the absence of any clinical changes. To avoid severe skewing of data in such a small sample by the results of this one patient, values from this individual were not included in the analysis. Study drugs were otherwise well tolerated. In particular, no signs or symptoms of peripheral neuropathy, a known toxicity of thalidomide treatment, were detected.

Effect ofZD V/LMV and thalidomide on plasma HIV levels. At baseline, the overall mean plasma HIV titer (±SEM) was 4.80 (±0.106) log₁₀ RNA equivalents/ml (see Table 2). After 12 days of ZDV/LMV, 19/31 (61%) of subjects had undetectable plasma HIV, with a mean decline in viremia of 1.93 log₁₀ RNA equivalents/ml (see Table 2). Following randomization to study drug and withdrawal of antiviral treatment, patients in both treatment groups exhibited a return to baseline viral titers by study day 28 (i.e., 14 days after stopping antiviral drugs). Analysis of the changes in viral titers subsequent to the day 12 nadir showed no significant differences between the thalidomide and placebo treated groups at any time point. Effect of ZDV/LMV and thalidomide on plasma immunologic markers. Plasma levels of TNF-α were not remarkably elevated at the start of the study (Day 0) (see Table 2). In response to antiviral therapy, there was a slight, but statistically significant decline in circulating TNF-α levels (Table 2). After withdrawal of antiviral therapy and randomization to thalidomide or placebo, TNF-α levels returned to baseline levels by day 28. There was no difference in the changes in TNF-α levels throughout the study between the thalidomide and placebo treated patients (see Figure 1A). Table 2

Patient Virologic and Immunologic Parameters at Baseline and After

12 days of Antiretroviral Therapy

Mean values measured (SEM) Parameter (units) Day 0 Day 12 p value*

HIV (loglO RNA units/ml) 4.80 (0.11) 2.87 ( 0.06) <10-14 TNF-α (pg/ml) 52.5 (4.2) 41.3 (5.3) 0.004 sIL2R (pg/ml) 3433 (299) 3273 (456) 0.44 sCD4 (U/ml) 26.1 (6.6) 27.8 (6.7) 0.30 sCD8 (U/ml) 722 (53) 654 (48) 0.10

IL-12 (pg/ml) 343 (35) 295 (29) 0.06

CD4+ T cells (cells/ml) 210 (45) 277 (62) 0.02

CD8+ T cells (cells/ml) 1204 (125) 1158 (121) 0.40

* Values for study cohort completing 12 days of antiretroviral therapy (n=31), at baseline

(mean of two measurements) compared with day 12 by paired t test.

Levels of sIL-2R, sCD4 and sCD8 were not significantly affected by the antiretroviral therapy (Table 2). However, sIL-2R and sCD8 levels in plasma increased significantly by day 28 in the thalidomide treatment group compared to the placebo group, and remained elevated until withdrawal of thalidomide on day 42.

Subsequently, sIL-2 and sCD8 levels returned to baseline levels (Figures IB and 1C). In contrast to sCD8 levels, plasma sCD4 did not change in response to thalidomide treatment (not shown). IFN-γ was undetectable in the plasma of the majority of subjects and remained extremely low throughout the study (maximum level 1.44 pg/ml), with no measurable effect due to thalidomide treatment (data not shown). A modest decline in circulating IL-12 was observed in response to antiretroviral therapy (Table 2) which almost achieved statistical significance (p=0.06). Following randomization, thalidomide treatment induced an increase in circulating levels of IL-12, which peaked on day 42. This increase was the most robust difference from the placebo group that was observed (p=0.0004) (Figure ID). The peak in plasma IL-12 levels was delayed relative to the increases in the T cell activation markers, sIL2R and sCD8, suggesting that the increase in IL-12 was secondary to T cell activation. There was a prompt return of IL-12 to baseline levels following cessation of thalidomide treatment (Figure ID).

Thus, of the soluble immunologic parameters assayed, short-term antiretroviral therapy caused modest declines in the monokines TNF-α and IL-12 only. In contrast, subsequent treatment with thalidomide induced marked increases in sCD8, sIL-2R and IL-12 levels, while having no effect on TNF-α or sCD4 levels. Delayed type hypersensitivity. The results of DTH skin testing are summarized in Table 3. At baseline, only 1 out of a possible maximum of 36 positive tests (2.8%) was observed in the group subsequently randomized to placebo. Baseline results for the thalidomide group were 3/45 (6.7%) positive skin tests. Following study drug treatment, there was a significant increase in skin test responses in the thalidomide treatment group, with 10/39 (25.6%) positives compared with 1/33 (3.0%) in the placebo recipients (p<0.01). One individual exhibited a positive response to PPD at baseline and then had a blistering response after 4 weeks (study day 42) of thalidomide treatment, and so was not subsequently re-tested with this antigen. However, for the purpose of analysis, this individual was assumed to have a positive PPD response on day 56. At the final time point, following withdrawal of study drug (day 56), the proportions of responders were equivalent in the thalidomide and placebo groups (17.9% versus 15.4%, respectively, p=0.83). Of note, at this time point all 6 of the responses in the placebo group and 5/7 of the responses in the thalidomide group were to Candida antigen, suggesting possible sensitization by serial testing with this antigen. Table 3 - Patient Delayed Type Hypersensitivity Responses

Treatment Skin Test Total Number of Tests > 5mm in

Diameter

Assignment Antigen Day O Day 42 Day 56

Placebo PPD 0 0 0

Trichophyton 0 0 0

Candida 1 1 6

(n=12)^a (n=l l) (n=10)

Response index⁰ 1/36 1/33 6/30

Thalidomide PPD 1 4 2

Trichophyton 0 10

Candida 2 5 5

(n=15) (n=13) (n=13)

Response index 3/45 10/39 7/39

p value* 0.42 0.0079 0.83

a n = number of patients in group. b Response index = number of positive tests/total number of possible positive tests

(48h).

* Response indices at each time point for thalidomide and placebo treatment groups compared by chi-square analysis.

T cell subsets. For the whole cohort completing 12 days of antiretroviral therapy, the mean absolute peripheral blood CD4 T cell counts increased modestly (from 210 ± 45 cells/ml on day 0 to 277 ±62 cells/1 on day 12, p=0.02), while CD8 counts did not change significantly (from 1204 ± 125 cells/ml on day 0 to 1158 ± 121 cells/ml on day 12; p=0.40) (Table 2). Subsequently, during treatment with thalidomide, CD8 counts increased significantly (from 1199 ± 176 cells/ml on day 12 to 1600 ± 207 cells/ml on day 42; p=0.03), while CD4 counts did not change significantly (311 ± 96 cells/ml on day 12 to 216 ± 51 cell/ml on day 42; p=0.36). In the placebo treatment group, there were no significant changes in either CD4 (from 227 ± 57 cells/ml on day 12 to 162 ± 50 cells/ml on day 42; p=0.14) or CD8 T cell counts (from 1096 ± 153 cells/ml on day 12 to 1113 ± 240 cells/ml on day 42; p=0.90).

The results from this patient study suggested that thalidomide exerted an immunostimulatory effect in vitro in this cohort of HIV-infected patients, reflected not only in increases in soluble immunologic markers, but also in the increased DTH responses and CD8⁺ T cell counts.

Lymphocyte proliferation assays. To investigate the cellular mechanisms underlying the immune stimulation observed in HIV-infected patients treated with thalidomide, the ex vivo stimulatory effects of thalidomide on T cell and PBMC responses from HIV-infected patients were studied. Highly purified CD4⁺ and CD8⁺ T cells from HIV-infected individuals were stimulated by cross-linking the

TCR with immobilized anti-CD3 mAbs in the presence of varying concentrations of thalidomide. Figure 2A shows the mean data from 5 experiments. Thalidomide induced a concentration-dependent increase in proliferation of CD4⁺ and CD8⁺ T cells. For some individuals, the thalidomide induced augmentation of CD4⁺ T cell proliferative responses was identical to that of the CD8⁺ T cells, but on average, the CD8⁺ response was greater. In contrast, thalidomide caused very modest increases in proliferation of anti-CD3 stimulated, unsorted PBMCs (see Figure 2A).

Cytokine Production. Next the effects of thalidomide on cytokine production by T cells and PBMC from HIV-infected donors was examined. Thalidomide consistently induced concentration-dependent increases in IL-2 and

IFN-γ production by PBMC and by purified CD4⁺ and CD8⁺ T cells stimulated with immobilized anti-CD3 antibodies. Figures 2B and 2C show the mean results of 5 experiments. In contrast to the proliferative responses, the proportional increases in IL-2 and IFN-γ production induced by thalidomide were similar in PBMC and purified T cells. These findings indicated that thalidomide can costimulate primary T cells from HIV-infected individuals in vitro.

To study the effect of thalidomide on IL-12 production in vitro, in a system where the primary stimulus is delivered to the T cell, PBMC from HIV-infected donors with immobilized anti-CD3 was stimulated. The production of IL-12 and IFN-γ, as well as the expression of CD40L, were evaluated by ELISA and flow cytometry, respectively. Preliminary experiments revealed that maximum levels of IL-12, IFN-γ, Y and CD40L expression occurred 48 hours after stimulation with anti-CD3. Thalidomide-induced increases in IL-12 (p40 and p70) production were observed at 48 hours in 7/9 subjects. There was a significant positive correlation between changes in IL-12 secretion and increases in IFN-γ production in response to thalidomide at lmg/ml (R=0.83, p= 0.006), see Figure 3, and at lOmg/ml (R=0.74, p=0.02) (data not shown). The two subjects who did not show increased IL-12 secretion in response to thalidomide were those with the smallest increments in IFN-γ secretion.

In the same experiments, modest thalidomide-induced increases in CD40L expression on T cells, were observed, which were statistically significant only when total CD3⁺, or CD3⁺ CD4⁺ T cell responses to lOmg/ml of thalidomide were analyzed (see Figure 4A). There was therefore an association between thalidomide-induced increases in T cell activation (indicated by increased IFN-γ production and CD40L expression) and IL-12 production. No IL-12 was detected in thalidomide-treated or control cultures in the absence of anti-CD3 (data not shown).

Next, the effects of antibody blockade of CD40L on the production of IL-12 by PBMC from HIV-infected subjects treated with thalidomide was studied (Figures 4B and 4C).

Blockade of the CD40L-CD40 pathway resulted in inhibition of IL-12 production, and abrogated thalidomide-induced increases in the levels of this cytokine. Similar results were observed whether total IL-12 (p40 and p70) (Figure 4B), or the biologically active IL-12 (p70) heterodimeric form only (Figure 4C) was measured. Together, these results show that thalidomide alone has no direct effect in increasing IL-12 production by PBMC. However, when T cells are stimulated via the TCR, and the CD40-CD40L pathway is intact, thalidomide can stimulate production of IL-12. These in vitro experiments are therefore consistent with our interpretation of the clinical data, suggesting that thalidomide exerts a primary effect in activating (costimulating) T cells, resulting in secondary increases in IL-12 production by antigen-presenting cells.

To heighten the sensitivity of a short term study, thalidomide was administered during a "rebound" in HIV levels following temporary suppression of HIV replication with ZDV/LMV therapy. No effects of thalidomide in retarding or stimulating the return of viral titers to baseline levels, evaluated 14 days after cessation of antiretroviral treatment were observed. As with viremia, thalidomide did not appear to effect plasma TNF-α levels. Levels of circulating TNF-α were not remarkably elevated at study baseline, but did fall modestly during exposure to antiretroviral drugs. Similarly to viremia, TNF-α levels returned to baseline within 14 days of withdrawal of antiretrovirals, independently of the presence of thalidomide or placebo. However, a correspondence between changes in HIV levels and TNF-α levels in patient plasma was observed in this study, suggesting a biological relationship between TNF-α and HIV replication. In contrast with the lack of effect on HIV and TNF-α plasma levels, a remarkable immunostimulatory effect of thalidomide in HIV-infected patients was observed. This was reflected in increases in DTH responses, increased plasma sIL-2R and sCD8 levels, increased absolute CD8⁺ T cell numbers and elevated plasma IL-12 levels. These observations are consistent with preliminary observations made in two prior studies in HIV and/or tuberculosis-infected patients, where thalidomide treatment appeared to induce increases in sIL-2R and CD8⁺ T cell numbers (see Haslett et al, AIDS Res. Hum. Retrovir., 1997, 13:1047-1054), and increased plasma levels of INF-γ (Tramontana, et al, Molec. Med., 1995, 1:384-397).

Optimal activation of T cells requires the delivery of a costimulus to the T cell in addition to primary antigenic stimulation via the TCR. Physiologically, such costimulation is afforded by the interaction of specific pairs of ligands on the APC and the T cell (Mueller et al, Annul. Rev. Immunol., 1989, 7:445-480; Linsley et al, Annul. Rev. Immunol., 1993, 11:191-212). Because antigenic stimulation in the absence of costimulation results in T cell anergy or apoptosis, the presence or absence of costimulation is critically important in the induction and regulation of cellular immunity (Mueller et al, Annul. Rev. Immunol., 1989, 7:445-480).

Moller et al. recently observed that thalidomide inhibits IL-12 production in a T cell-independent system in vitro, where the bacterial products, lipopolysaccharide and Staphylococcus aureus Cowan strain 1 were employed as direct stimuli of IL-12 production by APCs (Moller et al, J. Immunol., 1997, 159:5157-5161). However, it has now been found that in vitro, PBMCs from 7/9 HIV-infected subjects stimulated by immobilized anti-CD3 produced increased IL-12 in response to thalidomide, which correlated with increases in production of IFN-γ. In this system, both IL-12 production and its augmentation by thalidomide were T cell-dependent, since blockade of the CD40L on activated T cells abrogated these responses. In experiments with PBMCs from healthy HIV-uninfected donors, it has been shown that for the same donor, there is a thalidomide-concentration-dependent inhibition or increase in IL-12 in response to lipopolysaccharide or anti-CD3, respectively. Thus, in a purely T cell-dependent system, thalidomide increases IL-12 production, while in a purely T cell-independent system, the drug can inhibit production of this cytokine. It follows that the immune-modulating effects of thalidomide in the complex in vivo situation may be dictated by the relative importance of T cell and macrophage activation in different immunopatho logic states. Indeed, this explains the dichotomous effect of thalidomide as an in vivo inhibitor of TNF-α in erythema nodosum leprosum (where the cellular target of the drug may be M. leprae stimulated macrophages) (Sampaio et al, J. Infect. Dis., 1993, 168:408-414), while here thalidomide did not inhibit TNF-α production, but was instead primarily an activator of T cells. Defective IL-12 responses have been suggested to play an important role in the progressive immune deficiency of HIV disease (Clerici et al. , Science, 19943, 262:1721-1724). It has been shown that deficient IL-12 responses in HIV infected patients can be restored in vitro by the same T cell-dependent stimuli that thalidomide induces, namely CD40 ligand and IFN-γ (Chougnet et al, Eur. J.

Immunol., 1998, 28:646-656). Thus, thalidomide therapy may achieve a restoration of IL-12 production in HIV-infected patients in vivo.

EXAMPLE 3: Thalidomide in Sarcoidosis

A 16 week, open label, dose escalation study with 7 patients was performed. Thalidomide starting dose was 50 mg/day from weeks 0-4, 100 mg/d for weeks 5-8, and 200 mg/day for weeks 9-16.

Disease Activity Measurements. In cases with skin lesions, a serial photographic record of lesions was kept throughout the protocol. Skin punch biopsies in these patients were done at weeks 0 and 12. Chest X-rays were done on all patients at weeks 0 and 12.

Skin lesion histology. All patients with sarcoid skin lesions were biopsied at week 0 and week 12. Comparison of H&E stained sections from the pre- and post-treatment skin punch biopsy specimens was then performed.

Results Initial clinical response of sarcoid patients typically occurs by one month of daily thalidomide treatment. Lesions continued to improve over the entire 16 weeks of the protocol. Pulmonary symptoms of dyspnea on exertion and cough also improved during this time period. Patients on systemic corticosteroids were able to taper their dose while on thalidomide without experiencing a disease flare. The results are set forth in Table 4. Table 4 - Sarcoid Patient Responses to Thalidomide Protocol at Week 16

Patient Skin lesions Corticosteroids Pulmonary Function Chest X-ray

1 Responded Tapered Off NA NA

2 Responded Decreased Improved No change

3 NA NA Improved Improved

4 Responded NA Improved Improved

5 Responded NA Improved Improved

6 Responded Decreased NA NA

7 Responded Decreased NA NA

N.A. = not applicable in these patients.

Pre-treatment. Diffuse granulomatous inflammation infiltrating the dermis with thinning of the epidermis and loss of epidermal ridges.

Post-treatment. Marked reduction and consolidation of overall inflammatory cell infiltration, maturation of granulomas with increased presence of large giant cells, repigmentation of melanocytes, and thickened epidermal layer with return of epidermal ridges.

Plasma cytokine levels. Serial blood samples were obtained at weeks 0, 4, 8, 12, and 16. Plasma levels of IL-12 and soluble IL-2 receptor were determined using an enzyme-linked immunosorbent assay (Endogen, Inc., Woburn, MA).

Increased plasma levels of IL-12 were observed in sarcoidosis patients in response to daily thalidomide treatment (see Figures 5A and 5B).

Mean plasma IL-12 levels in above patients at base line and serial time points during thalidomide treatment are shown (Figure 5 A). Mean plasma IL-12 from normal controls (n=13) are also shown. Elevated IL-12 levels in sarcoid patients prior to treatment increased further during thalidomide treatment. Initial increase in plasma IL-12 occurred as early as week 1, and peak mean levels occurred at week 12. Plasma IL-12 levels were significantly higher at weeks 4, 8, and 12 of thalidomide treatment when compared to week 0 in sarcoid patients (paired T-test for two sample means).

Figure 5B shows percent increase over base line IL-12 plasma levels in sarcoid patients. The initial peak in Plasma IL-12 levels occurred at week 4 with a second, higher peak mean level occurring at week 12 of thalidomide therapy.

IL-12 is a critical inducer of the cell mediated immune response. Plasma sIL-2R is an indirect measure of T-lymphocyte activation during the cell mediated immune response. Thalidomide treatment resulted in an initial elevation of sIL-2R in sarcoid patients (see Figures 6A and 6B) at week 4, followed by a gradual decline in sIL-2R levels.

Figure 6A shows plasma soluble IL-2 receptor levels in sarcoid patients, and in particular the mean plasma sIL-2R levels in sarcoid patient at base line and serial time points during thalidomide treatment. Plasma sIL-2R is an indirect measure of systemic T-lymphocyte activation. Mean plasma sIL-2R levels from normal controls (n=13) are also shown. Elevated sIL-2R levels in sarcoid patients increased further with daily thalidomide therapy, peaking at week 4. Plasma sIL-2R levels were significantly increased at week 4 of thalidomide treatment when compared to week 0 in sarcoid patients (paired T-test for two sample means). Figure 6B shows percent increase over base line of plasma sIL-2R in sarcoid patients. This graph shows the initial rise in sIL-2R by 4 weeks of daily thalidomide treatment, followed by a gradual decline during the remainder of the protocol to below base line levels.

Flow cytometry. Peripheral blood mononuclear cells were isolated at week 0 and week 12 from six sarcoid patients and evaluated for activation marker expression using flow cytometry (see Figure 7). T-lymphocyte surface expression of HLA-DR, CD40 ligand, CD80, and CD86 was upregulated at week 12 compared to week 0 in these patients. HLA-DR surface expression is upregulated in immunologically activated cells. CD80 and CD86 are involved in the co-stimulatory pathway that is critical for antigen-specific clonal expansion of T-cells. CD40 ligand is expressed on activated T-cells and is one of two pathways leading to monocyte/macrophage production of IL-12.

Figure 7A shows HLA-DR surface expression on CD3⁺ lymphocytes PBMCs isolated from sarcoid patients at week 0 (pre-tx) and week 12 (post-tx) were stained with labeled monoclonal antibodies (BDIS, San Jose, CA) and analyzed by three color flow cytometry and CellQuest software (BDIS). HLA-DR surface expression was significantly increased in post treatment PBMCs compared to pre-treatment PBMCs (p=0.012, paired T test for two sample means).

Figure 7B shows CD40 ligand expression on T lymphocytes. PBMCs were prepared as described in Figure 7A. CD40 ligand was upregulated in post-treatment PBMCs compared to pre-treatment PBMCs (p=0.086, paired T test for two sample means).

Figure 7C shows CD80 expression on T lymphocytes. PBMCs were prepared as described in Figure 7A. CD80 (B7.1) cell surface expression was significantly increased in post-treatment PBMCs compared to pre-treatment PBMCs (p=0.036, paired T test for two sample means). Figure 7D shows CD86 expression on T lymphoctyes. PBMCs were prepared as described in Figure 7A. CD86 (B 7.2) cell surface expression was significantly increased in post-treatment PBMCs compared to pre-treatment PBMCs (p=0.006, paired T test for two sample means).

EXAMPLE 4: Thalidomide Treatment of Scleroderma Patients A 12 Week, open label, dose escalation study with 6 patients was conducted. Thalidomide starting dose was 50 mg/day for weeks 0-2, 100 mg/d for week 3-4, 200 mg/day for week 5-8 and 400 mg/d for week 9-12.

Disease Activity Measurements. Physical exams at base line and at each dose escalation were performed. Skin punch biopsies were done at weeks 0 and 12.

Skin lesion histology. Skin punch biopsies were obtained at week 0 and week 12 from 4 patients. Comparison of H&E stained sections from pre- and post-treatment biopsy specimens was done.

Results

Clinical changes observed in scleroderma patients treated with thalidomide included resolution of gastroesophageal reflux, improvement of depigmentated skin areas, healing of chronic extremity ulcers, and the development of dry skin.

Pre-treatment. Heavy infiltration of tightly packed collagen fibers throughout the dermal layer of skin was seen. Hypertrophy of vessel walls was present with resulting concentric narrowing of vessel lumens.

Post-treatment. Reduction of dermal collagen fiber density was seen, with repigmentation of the melanocyte layer.

Immunologic markers. Serial blood samples were obtained at weeks 0, 2, 4, 8, and 12. Plasma levels of IL-12 were determined using an enzyme-linked immunosorbent assay (Endogen, Inc., Woburn, MA). Increased plasma levels of IL-12 were observed in scleroderma patients in response to daily thalidomide treatment (see Figures 8 A, 8B). Mean plasma IL-12 levels peaked at week 8 in these patients.

Figure 8 A shows plasma IL-12 levels in scleroderma patients. This graph shows mean plasma IL-12 levels in scleroderma patients at base line and serial time points during treatment with thalidomide. Mean plasma IL-12 levels in normal controls (n=13) are also shown. Plasma levels increase as early as week 2 of daily thalidomide treatment, with peak mean levels present at week 8. Plasma IL-12 levels were significantly elevated at weeks 2 and 4 of thalidomide treatment compared to week 0 (paired T-Test for two sample means). Figure 8B shows percent increase of IL-12 over base line levels in scleroderma patients. This graphs shows the progressive increase in plasma IL-12 levels, peaking at week 8 of daily thalidomide treatment.

Thalidomide administration induced increased IL-12 plasma levels in patients with autoimmune diseases, including sarcoidosis and scleroderma. The increased IL-12 plasma levels were associated with enhanced activation of the cell mediated immune response (elevated sIL-2R plasma levels, increased surface expression of HLA-DR, CD40L, CD80 and CD86).

Claims

WHAT IS CLAIMED IS:

1. A method of stimulating or enhancing IL- 12 production in an HIV-infected patient comprising administering to the patient an effective amount of a composition comprising thalidomide or a pharmaceutically acceptable salt thereof.

2. An immune adjuvant for treating an HIV-infected mammal comprising thalidomide or a pharmaceutically acceptable salt thereof.

3. A method of stimulating or enhancing LL- 12 production in a patient suffering from an autoimmune disorder comprising administering to the patient an effective amount of a composition comprising thalidomide or a pharmaceutically acceptable salt thereof.

4. The method according to claim 3 wherein said autoimmune disorder is sarcoidosis.

5. The method according to claim 3 wherein said autoimmune disorder is scleroderma.

6. A method of stimulating or enhancing IL- 12 production in a patient suffering from tuberculosis comprising administering to the patient an effective amount of a composition comprising thalidomide or a pharmaceutically acceptable salt thereof.

7. Use of thalidomide or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for stimulating or enhancing IL- 12 production in an HIN-infected patient.

8. Use of thalidomide or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for stimulating or enhancing IL-12 production in a patient suffering from an autoimmune disorder.

9. The method according to claim 8 wherein said autoimmune disorder is sarcoidosis.

10. The method according to claim 8 wherein said autoimmune disorder is scleroderma.

11. Use of thalidomide or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for stimulating or enhancing IL-12 production in a patient suffering from tuberculosis.