TB Host-Directed Therapy (HDT) Candidate Agents

December 2015 – Tuberculosis Clinical Research Branch, DAIDS, NIAID

TB HDT Rationale

TB drug resistance continues to develop and HDT will be effective despite resistance and may help to prevent increases

Few new anti-TB drugs are in or entering clinical evaluation and some have issues with safety and significant PKIs interactions

MANY candidate HDT drugs await clinical evaluation for rapid adaptation for TB – re-purposing avoids agent development costs• If only a small % of potential new classes of HDT drugs are useful, the

number of available TB agents will be greatly expanded

HDT agents for TB may also have therapeutic benefit for other infections and also may serve as vaccine “potentiators”

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Rationale for Specific, Small Molecule Adjunctive Immunomodulators in TB Rx

1. Improving TB-induced immune defects – Particularly macrophages/other innate cells/autophagy

2. *Decreasing inflammatory tissue pathology/damage* AND protected Mtb sanctuaries

– Less inflammation, necrosis, caseation, granulomas, lessened inhibitory molecules

• Improved immune cell access/function• Improved anti-TB drug delivery to bacilli • “NRP” TB may reactivate to be killed more quickly by TB drugs3. Overall Effect Improved TB clearance occurs in models

NIAID RFA - HDT for TB: New approaches

Objective: Identify and characterize adjunctive host-directed therapy agents to improve treatment outcomes• Agents with evidence of therapeutic benefit in animal models and have FDA approval or be in Phase II or III trials

Mechanism: Phased UH2 / UH3 (5 yrs)• UH2 – Pre-clinically identify/optimize specific agent and dosing regimen to

advance to POC clinical trial and trial preparation• UH3 - Phase II POC Trial performance

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Four NIAID UH2/UH3 TB HDT Awards

• Autophagy Inducers (Deretic)

• Statins and other lipid-metabolism modulators (Karakousis)

• Imatinib (Kalman)

• Anakinra (Flynn) interleukin-1 receptor antagonist (IL-1Ra)

[Metformin grant application under review] 5

Simvastatin increases the in vivo activity of the first-line tuberculosis regimenJ Antimicrob Chemother 2014; 69: 2453–2457

Relative to the standard oral regimen of R/H/Z, addition of 25 mg/kg simvastatin in a BALB/c mouse model reduced lung cfu by an additional 1 log10 at Day 28 (P=0.01) and by a further 1.25 log10 at Day 56 (P=0.01)

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• In Mtb-infected mice, adjunctive metformin (MET) reduces the intracellular growth of DS a DR Mtb in an AMPK–dependent manner

• MET increased production of mitochondrial reactive oxygen species and facilitates phagosome-lysosome fusion

• MET reduced inflammation and lung pathology, and enhanced specific immune responses and efficacy of conventional TB drugs

Collectively, these data indicate that MET is a promising candidate host-adjunctive therapy for improving TB treatment

Tyrosine Kinase Inhibition

Imatinib – abl and c-kit targets• Myelomonocytic targets –

– Improved autophagy and phagosomal acidification– Mobilization of myeloid progenitor cells– Decreases the number and function of immune suppressor cells in some

malignancies • Studies in NHP (Rhesus macaques) TB and TB-SIV models with

MDR Rx combinations are ongoing and appear promising

Planned BMGF Sponsored 5-arm Phase II Trial• PDE-4 inhibitor - CC-11050

• Auranofin (complexed gold) • Everolimus (mTOR inhibitor)

• Vitamin D

• Standard DS TB regimen9

First-generation HDT candidates were chosen as “anti-inflammatory agents”, or empirically, e.g., steroids, anti-TNF, statins, imatinib, metformin

Next generation HDT agents will be TARGETED agents chosen to reverse specific abnormalities caused by Mtb in• Cell regulatory pathways of infected/immune reactive cells

- “Precision Medicine”• Immune effector functions of immune reactive cells

- “Immuno-oncology”

For BOTH innate and adaptive TREATMENT AND VACCINE Responses

Next Generation HDT?

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Cell regulatory pathways - Precision Medicine Advances for TB

“Precision Medicine” Therapeutics• Targeted HDTs have revolutionized cancer treatment by reversing effects of

“molecular drivers” subverting core cell regulatory pathways

• Some of the same cell pathways exploited by cancers are also disrupted by Mtb to enhance survival/proliferation, e.g., altered cholesterol metabolism AND to impair reactive immune cell function by disruption of cell metabolism

Many precision medicine agents now approved or in clinical trials can be re-purposed for TB and other infections

Precision Medicine and TB - Approaches

What research is needed?• Identification of therapeutic targets - Precise determination of the

immune cell regulatory pathways disrupted by Mtb to cause immunopathogenesis

• Screening - A wide variety of clinically available precision medicine agents for effect on TB infection in improved in vitro “granuloma model” systems

Animal models (as appropriate) POC clinical Trials

Molecular Targets of Precision Medicine Agentsof Relevance for TB HDT

Evidence for PG role or benefit• AMPK• P13K-AKT-mTOR• MAPKs – JNK, ERK• Protein kinases: abl, c-kit, VEGF,

EGFR, JAK/STAT• Mevalonate pathway• Autophagy inducers• Rho/ROCK• Ras• Cathelicidin/AMPs –

HDACs as inducers• Wnt/beta-catenin • MMPs

NOT YET EXPLORED for PG or benefit• PARPS• Sirtuins – STACs/inhibitors• Hedgehog• Notch• HIF-1α• Other kinases – SIK, FAK-Src, S6…• Inflammasomes• DPP-4• ERS/UPR reduction• Angiotensin II receptors• Bcl-2• GSK-3 • Many more 13

1) Immune checkpoint modifications (Directly on Mtb-reactive cells)Immune cell co-receptor checkpoints

• Prevent inhibitory T-cell co-receptor/ligand signaling – AGENTS - PD-1, CTLA-4, LAG-3, A2aR blockers (OX40* is stimulatory)

Immunometabolic checkpoint modifications • Regulate immune cell differentiation and function by pathway controlled cellular

metabolism, e.g., to enhance Th1/TH17 and memory CD8+ T cell responses - AGENTS - AMPK, sirtuin1/HIF-1α, mTOR inhibition* VACCINE potentiation

* Used in combination for vaccine potentiation - animals

2) Block Suppressor cell induction (Indirect effect on Mtb-reactive cells)• Tregs, MDSCs, M2 polarized macrophages induced by Mtb and BCG vaccination- AGENTS – IDOi, ATRA, TKIs, PDE-5i, bisphosphonates, others

Reversing TB immunosuppressive Mechanisms

Leveraging the exciting advances in precision medicine for the development of innovative next-generation HDTs may lead to entirely new paradigms for treatment and prevention of tuberculosis and other infectious diseases.

BACK-UPS

Targeting Immunometabolic Cell Regulatory Pathways

to improve Vaccine ResponsesAMPK Stimulation

• Regulates CD4 T cell responses to infection by control of a glucose-sensitive metabolic checkpoint for Th1 and Th17 development

• Controls metabolic transition of active/glycolytic effector CD8 T lymphocytes to quiescent T cells to support CD8 T-cell memory development

Sirtuin1/HIF1a signaling • Sirtuin1 inhibition enhances HIF1a activity in DCs to improve Th1 T cell differentiation and to

decrease Tregs• HIF1a activity stabilizing agents improve Th1 responses by inhibiting HIF-PHs (Hypoxia-

Inducible Factor prolyl hydroxylases – PHDs)

mTORC inhibition – including with BCG vaccination • Improves memory CD8+ T cell generation in animal models – see “Beyond Adjuvants:

Immunomodulation Strategies to enhance T-cell immunity” Vaccine 33S (2015) B21-28.

DAMPs

PRR

JNKERK

ROS

OxLDL

SR

ERSTRESS

PARP

OxLDL

NF-κB

AMPK

SIRT

Foamy Macrophage

M2

OxidativeStress

cAMP

PKA

PAMP or DAMP

β-AR

PI3K

AKT

mTOR

RTK

PTEN

Adenylate Cyclase

ATP

PDE

Anti-inflammatory

PRO-inflammatory And Cell Death

Autophagy

Metformin• Decreases glycolation (pyridoxamine and buformin may

be better) reacts with dicarbonyls• Decreased TNF and other inflammatory CKs by inhibiting

ERK/EGR-1 pathway and suppressing scavenger receptors (CD36 and SR-A)

• AMPK activation– Inhibits mTOR – enhances autophagy in TB infected

macrophages mediated by PPAR-gamma– Inhibits PARP activation and activates Bcl-6– Decreases effects of RAGE signaling on some cells and inhibits

HMGB1 release – Decreases NF-kB expression/Suppresses ROS formation 20

StatinsHIGHLY PLEIOTROPHIC EFFECTS• AGE/RAGE effects

– Decrease MPO-dependent AGE generation– Control AGE-mediated histone modification – Displaces RAGE from membrane – Decrease RAGE expression by inhibition of Rac-1

• Decreases LOX-1 effects• Enhance Efflux pumps for lipids• Matrix metalloproteinase down-regulationAnd Others - Will all of these be sufficiently additive to significantly impact TB-DM treatment outcomes?

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Metabolic pathways in T cell fate and functionImmunometabolism: linking metabolism and immune functionTrends in Immunology April 2012, Vol. 33, No. 4

T-cell Activation Immune Checkpoints

Checkpoints for development of effective cellular immunityT cell Co-receptors• Blocking PD-1 and CTLA-4 checkpoints with antibodies improves antigen-

specific T cell functions – reverses senescence• The next generation of immune checkpoint blockade for clinical evaluation will

include oral A2aR antagonists. Some are in trials (e.g., oral istradefylline) to increase immune responses to tumors (by T-cells, macrophages, NK cells)

• TB connection - Journal of Infectious Diseases 2014;210:824–33

“… a role for the purinergic pathway in the host response to M. tuberculosis. Dampening inflammation through signaling via the adenosine A2A receptor may limit tissue damage but may also favor bacterial immune escape.”

Myeloid-derived Suppressor Cells (MDSCs)

MDSCs are immunosuppressive lesion-infiltrating cells • Tumor-associated MDSCs are being targeted in cancer vaccine and other

immunotherapies to improve host immune responses • Large numbers of MDSCs develop during active TB in humans and BCG infection

recruits MDSCs • In a mouse model, BCG-induced MDSCs blocked T cell proliferation and dampened

Ag-specific priming in lymph nodes

Interventions - MDSC suppression or conversion to improve cancer immunoRx by• Sirtuin1 inhibition directs a switch to a M1 lineage by glycolytic activation of

cells through enhanced HIF1a function• All-trans retinoic acid, PDE-5 and c-KIT inhibition (sunitilib), bisphosphonate…

Innovative New Research Tools for Use at Single Cell Level

High through-put assays to define effects of molecular drivers of disease on individual cell regulation and to identify intervention targets • Epigenomic assays – defining the regulome

• ChIP (Chromatin Immunoprecipitation)-based assays (acetyl, methyl, TF binding, etc.)• Chromatin accessibility assays (ATAC-seq, FAIRE-seq, …)• DNA methylation sequencing – bisulfite methylation sequencing (BMS)

• Advanced proteomics, metabolomics… • Combined for simultaneous use – interactomes

• DNA + RNAseq, CHiP + DNAseq, ChIP + BMS, Chip+proteomics, etc.

• Sensitive detection of histone-altering gene polymorphisms (acetylation ChiP-DNAseq to find haQTL SNPs) – on a genome-wide basis using cohorts < 100, MAF ’s < 1%

Results: Correlating changes in specific cell’s signaling (and influence of genetics, epigenetics) gene expression (transcription/translation/post-translational modifications) effects on cell metabolism and defense functions (phenotype)

“Signaling Network Modeling” for OutcomesSingle cell methods are key to this modeling

• With a sufficient signaling database for a specific cell type – develop mathematical modeling for effects of signaling changes on cellular outcomes

-- gene expression cell metabolism/function• Adaptive – modify model as new data becomes available • In silico study of network dynamics to predict “network nodes” of most

vulnerability to intervention • Modeling can predict effect of targeted drug interventions and if combination

therapy would be significantly more effective• After drug intervention – include dosing effects to determine minimal effective

dose for a drug The future of therapeutic development for most diseases

Immune Protection vs. Inflammatory Damage

Immune reaction regulation will need to change over the time course of an evolving immune reaction to pathogensInitially -• Enhance robust and usually highly inflammatory reaction to first control and then kill and

begin to remove the pathogen -- or react to vaccine• HDT’s best use may be to reverse pathogen immune disruptive effects

Later - ? Exactly when• Inflammatory down-regulation must INCREASE as the immune process evolves in order to

limit tissue damage and implement tissue repair• NOW- HDT role may be to help modulate inflammation/damage – also to improve access and

function of immune cells and antibiotics

Timing/context of use of different HDTs may be crucial

Key Cellular Regulatory Signaling Pathways

Core pathways• PARP family• mTOR• Sirtuin family• AMPKOther key pathways – with known links to TB pathogenesis• Cellular Kinase networks (and Phosphatases)

• MAPK and cascades• Kinases – TKs – (e.g., abl, c-kit, VEGF, JAK/STAT) and MANY more classes

• Small molecule GTPases• Wnt/beta-catenin family

• Hedgehog, Notch1/SOCS3…. Implicated in BCG-induced suppression of immune responses

* US FDA-approved or available OTC # Approved in other countries with stringent regulatory authorities Bold: Preclinical testing against MTB being performed

Drug Class/Target Examples Tyrosine kinase inhibitors

c-abl, c-kit JAK/STAT VEGF Src/FAK

Ser-thr Kinase SIK inhibition

Imatinib* and others Tofactinib* Ruxolitinib* Pazopanib* Bevacizumab Dasatinib*, Bosutinib*

AMPK activators Metformin*, AICAR and AZD-769662 Berberine* ,Resveratrol* ASA*

MAPK Cascade inhibitors RAF-B MEK ERK JNK

Vemurafenib*, Dabrafenib Trametinib* SCH772984. VTX11e (pre) CC-930

PI3K-, AKT-, mTOR Pathway mTOR Idelalisib*, Hispidulin, Afuresertib, Perifosine, MK-2206, Triciribine, Ridaforolimus

Small GTPase inhibitors Ras (-RAF-MEK-ERK) Rho/ROCK

Tipifarnib+Statin, Salirasib Fasudil# Statins, metformin

PARP inhibitors NAM*, NR*, NMN*,

Tetracylcines*,

Olaparib* Sirtuin activators Resveratrol*

Statins* Metformin Berberine Disulfiram

Oxidative Stress Reduction Silymarin Tanshinone

Angiotensin II receptors telmisartan*

ERS/UPR Reduction Phenylbutyrate* Ursolic acid* Fasudil# Tauroursodeoxycholic acid

Inflammasome inhibitors Βeta-hydroxybutyrate, MCC950

Dipeptide dipeptiase-4 inhibitors Sitagliptin*

GSK-3β Inhibitors CHIR98014, KICG1338

Cathelicidin Inducers Vitamin D, Phenylbuturate, Lithocholic acid, curcumin, nicotinamide, reseveratrol, pterostilbene

Autophagy Inducers (46) Curcumin*, Imatinib*/TKIs,, Metformin*, Statins*, Verapamil*, SRIs*, Carbamazepine*, Berberine*, rapalogues

Mevalonate metabolism modulation - ERK signaling inhibition through RAS farnesylation γδ T cell activation

Tipifarnib+Statin

Amino-bisphophonates*

Highly Pleiotropic Metformin, statins, phenylbutyrate, telmisartan, fasudil#, berberine, dimethyl fumatate* Benfotiamine

CombinationsVEGFR Inhibitors Fasudil and Statins (ROCK) Vitamin D and Phenylbutyrate (AMPs) Metformin and disulfiramPazopanib

Drug Class/Target Examples

HDT CAVEATS• May not work

– PK/PD issues - delivery to site of action in active form with sufficient exposure

– Extrapolation from different disease models/states – In vitro and animal model (rodent) data not translating– Actions depend on “tissue/cellular context”– Complexity of regulation/signaling – counter-reactions– ETC.

• Could cause harm– Worsen TB disease course– Increase lung damage– Impact on HIV co-infection