EFFECT OF KIDNEY TARGETED THYMOQUINONE NANOSUSPENSION

ON LPS AND CLP-INDUCED ACUTE KIDNEY INJURY

Introduction to DessertationBy

Sanket GandhiM Pharm (Pharmacology)

R.C.Patel Institute Of Pharmaceutical Education and ResearchShirpur, 425405. 1

Acute kidney injury (AKI) is common in ICU and may be associated with mortality.

There are many of treatments like Continuous renal replacement therapy,

Intermittent hemodialysis, Treatment with diuretics and antibiotics etc. but

Currently, there is no effective treatment except renal replacement therapy (RRT).

(Yu et al,2016).

About 2 million people die because of AKI every year with estimated mortality of

>70%. (Philip Li et al,2013).

LPS, a surface component of gram-negative bacteria is a common cause of AKI.

LPS activates NF-kB resulting into increased proinflammatory cytokine release

and oxidative stress, nitric oxide in kidney tubules. (Cunningham et al.,2002).2

BACKGROUND INFORMATION

The another most frequently used model is cecal ligation and puncture (CLP)

model in rodents.

In this model, sepsis originates from a polymicrobial infectious focus within the

abdominal cavity, followed by bacterial translocation into the blood

compartment, which then triggers a systemic inflammatory response.

A requirement of this model is that it is performed with high consistency to

obtain reproducible results.3

BACKGROUND INFORMATION (Rittirsch et al.,2008)

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The therapeutic properties of thymoquinone include antioxidant (Mansoor

et al.,2002), anti-inflammatory (Umar et al., 2012), hepato- protective (Abdel-Wahab,

2013),neuroprotective (Al-Majed et al.,2006), anticancer (Muhtasib et al., 2006) etc.

It was found that thymoquinone exhibit up-regulating activity on PPAR-ϒ) (Woo et

al,2011), Nrf-2 (Gore et al.,2016),and down-regulating activity on PI3K/AKT(Zheng et

al.,2016), p53, p73, NF-Kβ ( sethi et al.,2008) etc.

TQ-loaded nanoparticles proved more effective compared to TQ solution mainly in

case of targted therapies like cancer (Dehghani et al,2014).

BACKGROUND INFORMATION

PHARMACOKINETICS OF THYMOQUINONE (alkharfy et al.,2014)

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• Clearance rate: 7.9±0.83ml/kg/min

• Volume of distribution at steady state:

700.90±55.01ml/kg

• Half life: 63.43±10.69 min

Intravenous

• Clearance rate: 12.30±0.30ml/kg/min

• Volume of distribution at steady state:

5109.4±196.08ml/kg

• Half life: 274.61±8.48 mins

oral

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WHY TO MAKE SUCH FORMULATION? (Yuan et al,. 2011)

Nanoparticles cause increase in dissolution rate, absorption and biavailability

and decrease in renal clearance.

Drug bound with N-acetylated low molecular weight chitosan shows higher

acummulation of drug in the kidney when given by i.v. route of drug

administration.

It is slowly excreted in urine i.e. 6.34% of nano drug was excreted after 2hrs of

Intravenous injection. 15% of the dose injected was retained in the kidney after

48hrs.

kidney

Thymoquinone nanoparticles

Low mol.wt. chitosan

Chitosan loaded thy.nanosuspension

LPSTNFα

TLR 2/4

CD40L

IKBα

Rel B/P65

Rel A/P50

IKKαIKKβIKKγ

IKBαP P P

NUCLEAS

NFkB

Increase in level of ,Cytokines(TNFα,IL-1,IL-6),ROS(Oxidative stress),Tissue Apoptosis

THYMO.NANO.

Nrf2keap1

Increase in Nitric oxide

MOLECULAR PATHWY OF DRUGS REACTION FOR LPS INDUCE SEPSIS 7

keap1

Nrf2

(Zoja et al.,2013),(Edward Abraham.,2003),(Itoh et al.,1997)

ARE

Maf

NQO1

GST

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LPS THYMO.PI3K

AKT

GSK3β

NF-kβ

PPARγ

Proinflammatory cytokine release

(TNF-α,IL-1β,IL-12)

OTHER MECHANISMS

(sumokhwalov et al.,2014),(woo et al.,2011),(zheng et al.,2016)

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Polymicrobial contamination

Systemic circulationInflammatory

response

Bacterial Transloca

tion

CYTOKINES REALISE

INTERFERON-γ

IL-6

IL-1βTNFα SEPTIC

SHOCK

MULTI ORGANE DYSFUNCTION

DEATH

MECHANISM FOR CLP MODEL INDUCED AKI MECHANISM OF CLP INDUCED AKI BY SEPSIS

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HYPOTHESIS

Lipopolysaccharide induced acute kidney injury by sepsis (LPS and CLP induced) via

activating NFKβ activity, protected by kidney targeted thymoquinone

nanosuspension by downregulation of NFkβ and upregulation of Nrf2 signaling

pathways.

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OBJECTIVES Primary objectives :

1) To prepare and standardised kidney targeted thymoquinone nanosuspension

formulation.

2) To evaluate protective effect of prepared formulation on lipopolysaccharide and

cecum ligation puncher(CLP) induced acute kidney injury by sepsis.

Secondary objectives :

3) To check inhibitory and enhancing effect of thymoquinone on NFkB and Nrf2

respectively.

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EVALUATION AND STANARDIZATION OF THYMOQUINONE NANOSUSPENSION

Scanning electron microscope Differential scanning calorimetry

Preparation of FITC labelled NPs Biodistribution of TQ-NPs

Pharmacokinetics of TQ-NPs

(Yuan et al.,2011),(El-najjzar et al.,2011)

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saline/vehicle solution,i.p.(Normal group)

LPS induced group (Diseased control group): 10mg/kg,ip.

Thymo. Nanoparticles (1mg/kg, i.v.)+LPS(10mg/kg,i.p.) at 8th day Thymo. Nanoparticles (2mg/kg ,i.v.)+LPS(10mg/kg,i.p.) at 8th day Thymo. Nanoparticles (5mg/kg ,i.v.)+LPS(10mg/kg,i.p.) at 8th day Thymo. Without nanoparticle (5mg/kg,i.v.)+LPS(10mg/kg,i.p.) at 8th day

Per se Group (Thymoquinone Nanoparticles (5mg/kg,i.v.))

EXPERIMENTAL PROTOCOLTotal 42 male Wistar rats weighing 250-300gm are used in the study as follows:(Total 7

group of animals each contains 6 animals). ( For LPS induced AKI)

(Yu et al.,2015),(Lie et al.,2015)

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saline/vehicle solution

LPS 10mg/kg

Thymo.nano.1mg/kg

Thymo.nano. 2mg/kg

Thymo.nano.5mg/kg

Thymo.withoutnano.5mg/kg

Perse(Thymo.Nano. 5mg/kg)

1 dose

1 dose

Day 8

Day 1 Day 7

LPS 10mg/kgAll parameters related to Acute kidney injury

will be perform with various statastical analysis .

After 12hrs.

( For LPS induced AKI) STUDY PROCEDURE(Yu et al.,2015)

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EXPERIMENTAL PROTOCOL

Sham operated sham control group(vehicle/saline solution oral)

CLP surgical group (Diseased control group)

Total 36 male wistar rats, weighing 200-250 gm are used in the study as follows:(Total 6 group of animals each contains 6 animals).

(For CLP induced AKI)

CLP + Thymo. Nano.(1mg/kg I.V.)

CLP + Thymo. Nano.(2mg/kg I.V.)

CLP + Thymo. Nano.(5mg/kg I.V.)

Per se group Thymo. Nano.(5mg/kg I.V.)

(Yu et al.,2015)

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( For CLP induced AKI) STUDY PROCEDURE

PROTOCOL OF CLP MODEL FOR SEPSIS INDUCED AKI (Toscano et al.,2011)

PROTOCOL OF CLP MODEL FOR SEPSIS INDUCED AKI (Rittirsch et al.,2008)

CLP MODEL FOR SEPSIS INDUCED AKI –VIDEO PART-1

CLP MODEL FOR SEPSIS INDUCED AKI –VIDEO PART-2

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PARAMETERS TO BE PERFORMED

Biochemical measurement- (Oxidative stress(Catalase,GSH,SOD,LPO),BUN,SCr,TNFα,IL-1β)

Histopathological examination-(Tissue necrosis level)

Immunohistochemical study-( proteins like NRf2)

Western blot analysis-(Protein expression eg. Ikβα,IKKβ,.NF-kB )

TUNEL assay-(Apoptosis)

Comet Assay-(DNA damage)

(Yu et al.,2015),(Stoyanoff et al.,2014)

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STATISTICS TO BE APPLIED

Data is expressed as mean ± SEM

Analyzed by one way ANOVA

Followed by Dunnett’s post hoc test.

PARAMETERS UNDER THIS STATASTICS ARE,Oxidative stress (SOD, GSH ,Catalase) , Cytokine

level( TNFα, IL-1β), Comet Assay

(Yu et al.,2015),(Stoyanoff et al.,2014)

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CHEMICALS REQUIREMENTSSr.

No. List of chemicals required Availability Cost

(Rs.)1 Lipopolysaccharide Available 5,010

2 Thymoquinone Available 18,500

3 chitosan Available 4,900

4 Acetic acid , Acetic anhydride , Acetone

Available 750

5 TNF-α and IL-1β ELISA KIT Available 57,000

6 0.1M methanolic KOH, 5M NaOH Available 1,250

7 TPP, FITC. Available 2,080

8 TUNEL KIT Available 5,000

9 Westen blot KIT Available 30,000

10 IHC KIT Available 80,000

TOTAL 2,04,490

FOR LPS INDUCED AKI

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CHEMICALS REQUIREMENTS

Sr. No.

List of chemicals required Availability Cost (Rs.)

1 ketamine Available 4,340

2 Xylazine Not available 6,370

3 70% alcohol Available 6,580

4 Buprenorphine Not available 10,780

5 6.0 silk suture Not available 1,200

6 25G needle Not available 5,00

TOTAL 29,770

6.0 silk suture 25G needle

FOR CLP INDUCED AKI

SEP. OCT. NOV. DEC. JAN. FEB. MAR.

Completion of protocol requirement

protocol set up

Checking results and errors

Main experimental study

Thesis writting

GANTT CHART FOR PROJECT PLANNING

Correction of errors

Paper writting

Thesis submmision

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Abraham, E., 2003. Nuclear Factor—κB and Its Role in Sepsis-Associated Organ Failure. J

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Al-Majed, A.A., Al-Omar, F.A. and Nagi, M.N., 2006. Neuroprotective effects of thymoquinone against transient forebrain ischemia in the rat hippocampus.European journal of pharmacology, 543(1), pp.40-47. Cunningham, P.N., Dyanov, H.M., Park, P., Wang, J., Newell, K.A. and Quigg, R.J., 2002. Acute renal failure in endotoxemia is caused by TNF acting directly on TNF receptor-1 in kidney. The Journal of Immunology,168(11), pp.5817-5823. Dehghani, H., Hashemi, M., Entezari, M. and Mohsenifar, A., 2015. The Comparison of Anticancer Activity of Thymoquinone and Nanothymoquinone on Human Breast Adenocarcinoma. Iranian journal of pharmaceutical research: IJPR, 14(2), p.539.

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