Oral administration of nano-emulsion curcumin in mice suppresses inflammatory-induced NFkB signaling and macrophage migration

Nicholas Young, PhD

Financial disclosure:none

Competing interests declaration:Care Trition LLC

patent- US 20130143969 "CURCUMIN COMPOSITIONS AND USES THEREOF”

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Curcumin• a yellow powder derived from

the turmeric plant• Native to India and Southeast Asia

• turmeric is widely used as a spice and yellow food coloring agent

• curry

Therapeutic agent in traditional Indian medicine has been used extensively for hundreds of years to treat

inflammatory problems: allergies asthma arthritis sinusitis ulcers trauma diabetes inflammatory skin diseases

Traditional use of Curcumin

Recent interest in the clinical application of curcuminEpidemiology

Epidemiological studies from this region have shown a decreased prevalence and milder onset of rheumatoid arthritis and other connective tissue disorders when compared to the West

Safety for human use No dose-limiting toxicity in phase I clinical trialsWell tolerated at dose as high as 12g/day for 3-4 month

Molecular targets of curcuminMany well-characterized molecular targets

The Curcumin Challenge – poor systemic bioavailablity Curcumin has yet to be translated clinically to treat

inflammatory disease Low solubility in aqueous solution

poor absorption is attained from luminal epithelial cells in the gastrointestinal (GI) tract

rats given a dose (1 g/kg) of curcumin excreted 75% in the feces unchanged

Rapid metabolic elimination by reduction and conjugation C3-complex, Sabinsa Corporation- human toxicity study

only small plasma levels of curcumin or its metabolites detected after oral administration in spite of very high doses

Attempts to increase oral bioavailability of curcumin Adjuvant Phospholipid complexes Nano-particle formulation Microemulsion Novel curcumin analogsNano-emulsion [polyethylene glycol and castor oil

(stabilize emulsions of nonpolar materials in aqueous systems)]

Preparing and testing various formulation of Nano-emulsion curcumin (NEC)

  PEG 400

PEG400/Cremophor EL

(v/v)

PEG400/Cremophor EL /Tween 80 (v/v)

PEG600/Cremophor EL (v/v)

1 + -    

2 + 4:1    

3 + 2:1    

4 + 4:3    

5 + 1:1    

6     4:2:1  

7       2:1

Plasma concentration of curcumin (determined by LC-MS) 1-h post-oral administration after single dose administration (1,000 mg/kg; n = 2)

PEG, polyethylene glycol

Cancer Chemother Pharmacol. 2012 69, 679-689

Curcumin is significantly more soluble in NEC

Curcumin in water

Curcumin in NEC

0.5 mg/ml 100 mg/ml

NEC 7 is stable

Cancer Chemother Pharmacol. 2012 69, 679-689

>5% CUR decompse in 60 days at RT

NEC has 10.5-fold more bioavailability than Suspension curcumin

Cancer Chemother Pharmacol. 2012 69, 679-689

Time course of plasma level of curcumin after oral administration (1.8g/kg) of NEC or SC (1% methylcellulose)

24 hours

in vivo BALB/C-Tg(NFkB-RE-luc)-Xen mouse

Transgenic NFκB-RE-luc mouse

p65 p50

B site

pNFB

Luciferase

NF-kB is a transcription factor- controls expression of many genes- plays a crucial role in activation of genes associated with inflammation and an immune response

in vivo imaging system (IVIS) 200

14

Xenogen

No

Yes

NF-κB-RE-luc mouse Bioluminescent signal

Luciferin 150 mg/kg IP:

Experimental set-up

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Inject 100µg LPS IPTreatments

1. SC (suspension curcumin; 1 g/kg) 2. NEC (1 g/kg) n = 13

luciferin injection 0

2 hrs4hrs6hrs

IVIS

Bioluminescence in IVIS

10 min

LPS-induced NF-kB reporter gene expression is suppressed with nano-emulsion curcumin (NEC)

FACS analysis shows that NEC selectively diminishes levels of blood monocytes

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NEC suppresses LPS-induced blood monocyte accumulation

IB

p65 p50

IB

p65 p50

Genes

TLR4

IKK complex

degradation

B site

pNFB

RAGE

LPS

TLR2

p

p

NEC suppresses LPS-induced TLR4 and RAGE expression

MCP-1 recombinant protein (pg/mL)2000 1000 500 250 125 62.5

Mouse serum

Vehicle

SCNEC

Baseline 4 hr post-LPS treatment

* #

NEC suppresses LPS-induced MCP-1 secretion

Curcumin inhibits mouse macrophage-migration both in vivo

Chemiluminescence of curcumin

Curcumin labels monocytes in humans

Curcumin inhibits macrophage migration in human cells – primary macrophages

IB

p65

p50

IBp65

p50

Genes

TLR4

IKK complex

RAGEMCP-1

p

degradation

B site

Curcumin

p NF-B

RAGE

LPS

Curcumin

p

p

Next step---Clinical studies

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Healthy Volunteers

Single dose 2g NEC (n = 4)Daily dose 200mg NEC for 2 wks (n = 6)Daily dose 100mg NEC 3 mo (n = 10)

Initial resultsWhen compared to C3 complex:

Curcumin plasma peak level (Cmax)- 70-fold increase

No adverse events noted following oral ingestion by clinical assessment

vital signsmetabolic profilesblood chemistry28

Results / Conclusions Establish novel application of luciferase mouse model to

monitor/test/validate curcuminn mediated therapies Curcumin can suppress inflammation by inhibiting

macrophage migration via NF-B and MCP-1 inhibition Establish NEC as an effective therapeutic formulation to

modulate this response

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Future work NEC was shown here to preferentially suppress

macrophage recruitment and migration inflammatory diseases whose pathology is most strongly

associated with macrophages may be optimal candidates for future NEC therapeutic studies glomerulonephritis, Crohn’s disease, rheumatoid arthritis,

inflammatory bowel disease, cardiovascular disease, diabetes, and obesity.

Future work in our laboratory will be in testing the therapeutic potential of NEC in various models of these chronic inflammatory conditions

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Acknowledgements

Wael Jarjour, MDLai-Chu Wu, PhDWilliam Willis, PhDBenjamin Kaffenberger, MDAlexandra FriedmanMichael BrussMark GardnerAmanda Kibler

The Ohio State University Wexner Medical Center, Columbus, OHDepartment of Internal MedicineDivision of Rheumatology and Immunology

Department of PharmacologyComprehensive Cancer Center

Department of Microbial Infection and ImmunityCenter for Microbial Interface BiologyZhongfa Liu, PhD

Yu Cao, PhD Larry S. Schlesinger, MDMurugesan V.S. Rajaram, PhD

Center for Biostatistics

Xiaokui Mo, PhD