Modulation of the Immune System: Treatment Options and new Developments

PD Dr. Chris Rundfeldt

DrugConsult.Net

Magdeburg and Dresden, Germany

Diseases and conditions with (pathological) involvement of the immune system

Inflammatory conditions, associated with trauma, infection or tissue destruction

Chronic perpetuating inflammations: COPD, arthritis Allergic diseases: type I-IV Atopic diseases: atopic dermatitis, atopic rhinitis,

asthma Autoimmune related diseases: rheumatoid arthritis,

inflammatory bowl disease, colitis ulcerosa, morbus Crohn, psoriasis, multiple sclerosis, coeliac disease, Rasmussen's encephalitis, myasthenia gravis, ……

Organ / tissue transplantation and rejection reactions

Localized effects of the ubiquitous organ “Immune system”

The immune system is involved in numerous physiological and pathological processes

Each organ can be a disease target (CNS, skin, gut, muscle, soft tissue…..)

Often targeted tissues are limited and reasons for localized effects are not known

Potential reasons for localization are hidden in the complex nature of such multifactor diseases

A drug with immune modulator activity can be useful for diverse diseases

For example glucocorticoids are used for many conditions

Intervention options: well established drugs

Anti-inflammatory drugs Inhibitors of cyclooxygenases (Ibuprofen...) Inhibitors of lipoxygenases (Zileuton…) Leukotriene receptor antagonists ( Montelukast…)

Immune modulators Glucocorticoids

Selective cytokine blockers (Biologics) TNF inhibitor antibody / fusion protein (Infliximab,

Ethernacept…) IL-1 receptor antagonist (Ankarinra) Anti-T-cell fusion protein (Alefacept) …….

Intervention options: Immunosuppressives

Antimetabolites Purine synthesis inhibitors (Azathioprine …) Purine analog mimics adenosine, DNA synth. Inhibit. (Cladribine) Pyrimidine synthesis inhibitors (Leflunomide …) Antifolate (Methotrexate)

Calcineurin inhibitors (IL2 synthesis inhibitors) Cyclosporin Tacrolimus, Pimecrolimus

mTOR, kinase inhibitor, activation of T-cells inhibited Sirolimus (=Rapamycin)

TNFa-synthesis inhibitor (Thalidomide, Lenalidomide)(Thalidomide: teratogenic effect phocomelia, now used for Erythema nodosum leprosum, cancer, etc.

Limitations of pharmacological intervention

Immune suppressive agents can facilitate potentially life threatening infections Invasive aspergillosis Tuberculosis General infections

Unexpected adverse events include disturbance of learning and memory (Rapamycin)

Inhibition of rapidly dividing tissues Glucocorticoids lack a good separation of

pharmacologically active and toxic doses

Limitations of pharmacological intervention: Biologics

Biologics are thought to be selective but can induce: Flu-like symptoms, nausea, fatigue, loss of appetite Redness, rash, and/or pain at injection site Allergic reaction to mouse protein with monoclonal antibodies Increased risk of Hodgkin’s and non-Hodgkin’s lymphoma and

other types of cancer in children and teens taking TNF inhibitors Lupus-like syndrome Possible reactivation of latent tuberculosis infections with TNF

inhibitors Progressive Multifocal Leukoencephalopathy (PML), see

Natalizumab case (Tysabri) Induction of antibodies against therapeutics results in

loss of effect

New treatment options?

Novel treatment options should be:

Selective for pathological conditions (reduce over-activation of the immune system component)

Have a good separation of effects and adverse reactions

Be easy to handle, at best oral treatment

While no such drugs are readily available, a few interesting candidates can be discussed

Examples for new treatment options

Inhibitors of Phosphodiesterase 4

Stabilized cAMP analogues

Dimethylfumarate

Raft-modulators

Phosphodiesterase 4 inhibitors

Inhibitors of Phosphodiesterase 4 (PDE4)

Prototype: Rolipram Initial development as antidepressant Early investigation in multiple sclerosis indicate

pharmacological effect, but GI related side effects

Numerous development projects worldwide for diverse diseases

Currently only roflumilast marketed in EU for treatment of COPD

Other potential indications for roflumilast: Asthma, atopic dermatitis, psoriasis

Roflumilast (PDE4i)

Initial studies in patients with asthma

Roflumilast (PDE4i)

Significant effects in patients with COPD

But effect size lower than known for Theophyllin!

Roflumilast (PDE4i)

Significant effects: reduction of exacerbations

Novel PDE4i: how to test preclinically

In vitro tests: Cytokine release in PBMCs or whole blood Induction with LPS or anti-CD3/CD28 or similar

Ex vivo tests: Cytokine release from whole blood

In vivo tests: Ovalbumin induced eosinophilia in sensitized BN rats (asthma model)

LPS-induced neutrophilia in Lewis rats and in ferrets (COPD model)

Emesis in ferrets and domestic pigs

Example development strategy for PDE4i

CYP3A4 Induction

Stable in plasma,no or low turnover in liver preps.

Expl. kinetics in rat and dog(i.v. vs p.o.)

Dog: t 1/2 > 2 hBV > 30%

Side effect profile in- ferrets (emesis and CNS)- pigs (cardiovascular)- mice, rats (CNS)

TI > 10

Inhibition of cytokine release - PBMC‘s ; Maph(IL2, 4, 5, 13, IFN)(IL1, 6, 8, 12, 15, 17, MCP1)

Metabolic stability inplasma and liver prepar.,species comparison, metabolite profile

Quality Lead

IC50 < 100 nM

Effect on- LPS-induced neutrophilia in rats and ferrets- late phase eosinophilia in rats- bronchial hyperreactivity in mice

Equal or better than RoflumilastLTB4

(neutrophils)LTC(eosinophils)

IC50 < 100 nM

no induction

Preselection preclinical candidate

Example development strategy for PDE4i

Clinical Program

Identification of - major metabolites - metabolic pathways- involved enzymes

acceptable

Non-respiratory inflammation models- skin (mice)- other models (CRO)CNS diseases

Indication extension

PK / ME Safety

14C Pharmacokineticand metabolicprofiling

Tolerability- Irwin test (rat)- acute toxicity (rat, mice, dog)

Respiratory models- allergic rhinitis (rat)- LPS-induced neutrophilia ( ferrets and pigs)- allergen-induced broncho- constriction (guinea pigs)

Efficacy

Full 4-week tox package

Preclinical Candidate

Receptor screening

Selectivity > 100

Genotoxicity- Ames test - chromosome aberration test- micronucleus test

No effect

initiatedinitiated

Example data: Ovalbumin induced eosinophilia

Novel compound

Example data: LPS induced neutrophilia

Novel compound

Example data: Safety profile in experimenta animals

Rationale: clinical dose of roflumilast is limited by CNS side effects,not by emesis

Test Novel Roflumilast

LPS neutrophilia >50% 0.01mg/kg

0.01 mg/kg

Highest dose testedwithout emesis

3 mg/kg 0.1 mg/kg

Highest dose testedwithout CNS effects

0.3 mg/kg 0.01 mg/kgnot free of CNS effects

TI in relation to emesis p.o. ~300 ~10

TI in relation toCNS effects p.o.

~30 < 1, no separation

Compounds were administered to ferrets with gastric tube as a solutionor suspension

Translation of preclinical data in phase I

Phase I is dedicated to determination of safety Options to get insight in efficacy in phase I

LPS induced TNFa release from whole blood i.v. LPS induced hyperthermia and flu-like reaction in volunteers Intratracheal LPS administration followed by lung lobe lavage Local non-allergic or allergic skin irritation in sensitive patients,

with skin prick test or with Balsam of Peru Allergen induced late phase bronchoconstriction in volunteer-

patients with mild stable asthma Methacholine induced bronchoconstriction Local non-allergic or allergic skin irritation in sensitive patients,

with skin prick test or with Balsam of Peru

PDE4 inhibition, other options

Increased cAMP levels: cAMP substitution instead of PDE4 inhibition

Stable cAMP analogues can be active but activate also protein kinases

Clinically known stable analogue: Dibuturyl-cAMP (bucladesine) Bucladesine is a safe drug, no emesis Was developed as cardiotonic for i.v. administration Was developed as skin healing cream Failed for different reasons: Cardiotonic function

difficult to test, skin cream has strong odor (butyric acid is released)

Clinical data showing a homogenous clinical effect of cAMP level modulation

Goyarts et al., Skin Pharmacol Appl Skin Physiol 2000:13, 86-92

Dibuturyl-cAMP is active in arachidonic acid induced ear swelling in mice

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Drugs were administered onto the outer surface of the mouse ear 3 h before arachidonic acid challenge. Measurement of ear thickness. 60 min later the ear thickness was measured.

DB-cAMP, water free ointment

Ketoprofen, commercial cream

2-deoxy-cAMP may be an interesting candidate for a topical anti-inflammatory ointment, but safety of this compound is not tested

No patent protection possible

Fumaric acid esters: old drugs with novel mode of action

Dimethyl fumarate: A novel old drug for oral treatment of immune diseases

Dimethyl fumarate (DMF) is marketed in Germany as main component of a first line oral drug for treatment of moderate to severe psoriasis (Fumaderm®)

Fumaric acid esters are an effective therapy in patient with moderate to severe psoriasis, even in those who have previously been intolerant of systemic therapy

The mode of action of DMF is not fully understood Problem: tolerability, GI irritation and flush, skin irritation Problem: Papilloma / carcinoma in forestomach rats Biogen Idec has developed a novel micro-tablet formulation

(immediate release) and tested positive for psoriasis and MS. Now in phase III for MS

Forward Pharma has solved the challenge of developing a modified release formulation. Now in phase II for Psoriasis

Fumaric acid ester (FAE) and Dimethylfumarate: Pharmacology

FAEs cause an increase of the intracellular calcium concentration FAEs inhibit the expression of the ICAM-1 T-cell receptor ligands The treatment of psoriatic patients with Fumaderm® results in a reduced

infiltration of the skin with granulocytes and T helper cells, followed by a reduction of the acanthosis and hyperkeratosis

Monomethyl fumarate increases the IL-4 and IL-5 production of activated T cells, in a dose-dependent manner

Dimethyl fumarate inhibits cytokine-induced E-selectin, VCAM-1 expression in human endothelial cells

Dimethyl fumarate inhibits cytokine-induced nuclear translocation of NF-kB. Dimethyl fumarate induces apoptosis in human monocyte-derived dendritic

cells. Monomethyl fumarate suppresses the FMLP-stimulated respiratory burst in

granulocytes

Fumaric acid ester (FAE) and Dimethylfumarate: Pharmacology (2)

In in vitro tests DMF is more potent than MMF In plasma samples, only MMF can be found (rapid

demethylation) In urine, DMF-conjugates with glutathione were

found Proposed MOA: Intracellular uptake in leukocytes Rapid conjugation with glutathione Dimethyl fumarate induces immune suppression

via glutathione depletion and subsequent induction of heme oxigenase

DMF pathways

Lumen -Gut wall- Blood streem

DMFTablet

ERY

ERY no

metabol.

ERYERY ERY

Plasma EsterasesDMF MMF

WBC:DMF

DMF-GSHActive Transp.

DMF-GSH Plasma EsterasesMMF-GSH

WBC:MMF

MMF-GSHActive Transp.

Mercapturic acid derivatives, excreted in urine

1

2

Glutathione depletion is immune suppressive

Top:PBMCs were stimulated with LPS/IFNg (1 mg/ml, 10 ng/ml) or PHA (100 mg/ml) in thepresence of DMFBottom: Mixed lymphocyte reaction to quantify dose response and reversibility

Substitution of reduced GSH abolishes the inhibition ofinflammatory cytokine secretion and alloreactive lymphocyte proliferation by DMF and DEF

FAEs are active in moderate to severe psoriasis

Mrowietz et al. Br. J. Dermatol. 1998138: 456-460

Effect of DMF in MS patients (Biogen)

Tabel from: Lee et al., The International MS Journal 2008; 15: 12–18

Figure from: Kappos et al., Lancet 2008; 372: 1463–72

Raft modulation as novel lipid chemistry target

Raft modulation: Innovation ahead?

Lipid Rafts are specific clusters of lipids and proteins regulating a wide range of biological and pathological processes

Lipid Rafts concentrate proteins to increase process efficacy

Rafts coordinate disease events at specific sites and times Signal transduction, virus entry & budding, bacterial entry,

receptor recycling, protein maturation are dependent on Lipid Rafts

Each process occurs in a Raft of unique composition

Rafts are novel pharmaceutical targets

Example: IgE receptor RAFT as drug target

IgE-receptor clustering drives RAFT formation Protein-driven (specificity) reaction is combined

with novel lipid chemistry target

RAFTlipids

IgE

FcRI kinase unrelatedRAFT receptor

multivalent allergen

Receptor clustering and RAFT coalescence

Signal cascade, release of immune modulators

Miltefosine is a clinically effective RAFT modulator

Skin-prick test on human volunteers; IgE-dependent allergic response Pretreatment with miltefosine (Miltex) or saline Skin-prick with allergen & positive control (histamine)

Weller et al. 2008 Miltefosine inhibits human mast cell activation in-vitro and in-vivo. J Invest Dermatol

Clinical effect back-translated in preclinical model: TDI induced ear swelling in mice

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Conclusion

Immune modulator drugs represent a diverse family of drugs

Despite the large number of marketed drugs, there is need for better drugs

Even among old drugs there are interesting new approaches to immune modulation

Recent data indicate the reduction in glutathione concentration can induce immune modulation

Raft modulation is a 2nd novel approach to immune modulation

Thank you for your attention

I am ready to take your questions