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)
4
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
6
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
8
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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