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Thermosensitive MicellesHydrogel Hybrid System Based onPoloxamer 407 for Localized Delivery of PaclitaxelSURAJ CHODHARYDept. of PharmaceuticsAL AMEEN COLLEGE OF PHARMACYBangalore

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1REFERENCE JOURNALVolume:102Issue: 8Page:2409 - 88Year: Aug. 2013Edited By: Dr. Ronald T.B.Impact Factor: 3.13

Caoyun J, Juan S, Peng Z, Xiang J, Can Z.Center of Drug Discovery,China Pharmaceutical University, Nanjing 210009, Peoples Republic of China.

INTRODUCTION

HYDROGEL

2HYDROGELWidely used for tissue scaffolds, and drug and cell delivery systems in regenerative medicine because of its unique characteristics.There into, in situ gel has attracted more attention because of..its transition of sol to a gel state by the variation of environment Thereby, hydrogel as accurate dosage, ready to use, good compliance for patients in sol state;prolonged drug action, reduced side effects, and Low frequency

3HYDROGELIn particular,Thermosensitive hydrogel is one of the most frequently applied carriers because of the conveniently controllable adjustment of temperature.In addition, in situ gel via intratumoral (i.t.) injection has been explored in cancer therapy because of the distinguished advantages ofLonger exposure time in tumor mass and Less systemic exposure

POLOXAMER 407P407

4POLYMER (P-407)Hydrophilic linear tri-block polymer.

Can be transformed from sol to a gel through micellization and gelation at a certain concentration and temperature.

P407 gels have been widely used in drug delivery systems, especially the local delivery, because Can be administered in liquid form and serve on sustained release depot at body temperature

5POLYMER (P-407)Drawbacks:rapid erosion in the physiological environment.low loading capacity for poor soluble drugs.

1. inducedby the macrodilution of body fluid, making the concentrationof P407 to drop below the critical gelationconcentration level

2. P407 gels narrowsthe application of drug carriers as well9

Carboxymethyl ChitosanCMCS

6POLYMER (CMCS)Water soluble derivative of chitosan (CS) with excellent biocompatibility.Investigated for a wide range of biomedical applications, such as tissue engineering scaffolds and drug-delivery carriers.Has abundant 2-NH2 groups that can be cross-linked by glutaraldehyde (GA) to generate a network, which promotes the mechanical intensities &prevents it from the fast erosion of P407 gels.

PACLITAXELPTX

7DRUG (PACLITAXEL)Chemotherapeutic agent in Tumors.Acts by stabilizing microtubules and imparting mitotic arrest to the tumorous cells..Broad-spectrum activity in several solid tumors.DRAWBACKS:low therapeutic index (extremely hydrophobic property)

OBJECTIVE

8OBJECTIVETo overcome the fast dissolution,Improve the mechanical strength of P407 gels,Enlarge the loading capability &Extend the release period of PTX.

RAW MATERIALS

9MATERIALS REQUIREDPoloxamer 407 (P407, Mw = 12, 600, PEO99-PPO67-PEO99)CMCS (carboxylation degree of 45%)Drug - PaclitaxelHuman hepatocyte cell line L02 Mice hepatoma solidity cell (Heps)RPMI-1640 medium (Hyclone), Fetal bovine serum, Penicillinstreptomycin solution (Hyclone),Phosphate buffered saline (PBS, Hyclone), 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazoliumbromide (MTT)Trypsin (Gibco)NOTE: All other chemicals and reagents were of analytical grade

METHOD

10METHODS

PREPARATION:Unloaded CMCS/GA-Modified P407 Hydrogels (P407-CMCS/GA)Preparation of PTX-M-MG

Unloaded CMCS/GA-Modified P407 Hydrogels

11Unloaded CMCS/GA-Modified P407 Hydrogels

(P407 19%, CMCS 1.5%, w/v), Overnight at 4oC

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12Lower Critical Solution Temperature (LCST) and Gelation TimeMethod: Tube Inverting (for sol-gel transition temp. & time) LCST: The temperature when the liquid did not flow for 30 s was recorded as LCST.

1 mL sample5mL tubes at 4oCHeated in temp. controlled water bath

added18 to 40C at a heatingrate of 1C/min

13Lower Critical Solution Temperature (LCST) and Gelation TimeMethod: Tube Inverting (for sol-gel transition temp. & time) Gelation time:The time at which the liquid did not flow for 30 s was recorded as gelation time.

1 mL sampleWater bathInverting the tube every 0.5 min

incubateConstant temp.

14Rheological StudiesApparatus: By a Rheometer (with plate geometry in oscillation mode).

PROCESS:Measurements were performed at a fixed frequency of 1 Hz and a strain of 0.1% with a gap size of 0.5 mm. NOTE: To prevent the evaporation of samples, a solvent trap was used in conjunction with liquid paraffin.The viscosities of P407 and P407-CMCS solutions + storage modulus (G) + the loss modulus (G)NOTE: The gelation temperature was got when G and G were equivalent in value.

15SWELLING BEHAVIOURSwelling Ratio (SR):(Mixed)For 2 mins.To form gel(immersed)Weighed at predetermined timeReloaded in a

16MECHANICAL PROPERTYApparatus: Texture analyzer TA-XT2 (SMS, Stable Micro Systems)

NOTE: At least five replicate analyses of each sample were performed.To allow complete gelationAt defined rate (1 mm/s) & defined depth (3 mm)

17INVITRO CYTOTOXICITYPerformed on human hepatocyte cell line L02 as normal cells by MTT assay.About 1 105 cells per well were seeded in 96-well plates.

After 24 h culturing, the cells were treated with diluted P407-CMCS/GA solution.(for 24 h, 48 h, and 72 h, respectively)

Subsequently, 20 L of MTT PBS solution (5 mg/mL) was added into each well

After incubating for 4 h at 37C, the medium was removed, and 150 L of dimethyl sulfoxidewas added and shaken thrice.

Readings were taken at 570 nm using Eliasa (Thermo Scientific)

PTX-M

18PTX-MPreparation of PTX-M

Mixed Solution

subjected to dialysis against deionized water overnight

micelle solution was filtrated through a 0.45 m pore-sized membrane lyophilized by a freeze dryer system to obtainthe dried powers of PTX-MPTX (16 mg)

0.355 mL of dehydrated ethanol and 4 mL of distilled waterNOSC (16 mg).

0.355 mL of dehydrated ethanol and 4 mL of distilled water

19PTX-M EvaluationThe drug-loading content (LC%) was calculated using the following equations:

LC% = (WPTX in PTX-M/WPTX-M) 100%

Where,WPTX in PTX-M: the weight of PTX loaded in the micelles,WPTX-M : the weight of micelles. The particle size and polydispersity index (PDI) of the PTX-M were assayed by Dynamic Light Scattering.PTX-M was dispersed in deionized water with 0.05% (w/v) GA or 1.5% (w/v) CMCS for different times, respectively.

PTX-M-MG

20PTX-M-MGPreparation of PTX-M-MG

Mixed Solution (in a solution of P407)

GA was added in (0.05% GA in final, w/v)

Agitated intenselycertain concentration of CMCS (1.5%, w/v)

PTX-M solutions(4 mg/mL, 5 mL)

21PTX-M-MG EVALUATION1. Dispersibility of PTX-M in PX-M-MGCross-sectional images of hydrogels were examined with Field Emission Scanning Electron Microscope (FE-SEM, Sirion 200, Holland).Samples obtained by freeze drying, and the cross-sectional area was sputter coated with platinum before examination. P407-modified hydrogels loaded with same amount of PTX (PTX-MG) were used as a control.

22PTX-M-MG EVALUATION2. In vitro drug release:Membrane-less model is used to assay the release behaviour.SHY-2A thermostatic shaker under 37oC at 50 10 rpmIncubated37oC, 30 minsRelease medium(sink condition)Replaced with fresh release mediumHPLCColumn: C18 column (250 4.6 mm, Diamonsil)M.P. : CH3OH + H2O (75:25 v/v)Temp. : 35oC Flow rate: 1.0 mL/minDetector: UV

23PTX-M-MG EVALUATION3. Heps Tumor-Bearing Mice Model:For tumor formation,Heps cells from ascites of tumor bearing mice were harvested.Cell suspended in PBS.inoculated subcutaneously cell suspension in the same flank of each mouse. The Tumor volume (V) was calculated:

V = [length (width)2]/2NOTE: Once the solid tumor reached about 100mm3, used for Tissue distribution & anti-tumor activity study in rats.

mice hepatoma solidity cell (heps)35

24PTX-M-MG EVALUATION4. Tissue Distribution:Animals divided in 4 grps,Taxol PTX-M, PTX-M-P407, and PTX-M-MGBlood sample collection at predefined time points by: Retro-orbital plexus puncture into heparinized tubes.Plasma obtained by: Centrifugation.Animal sacrificed tumor & liver harvested HPLC estimation.

Given intratumorally,20mg/kg

HPLC EstimationConc. Of PTX determined:100 L or 200 L of tissue homogenate

Mixed with 200 L of Acetone (5 min)

Centrifuged 12,000 rpm, 10 min

20 L supernatant introduced in HPLC system


25PTX-M-MG EVALUATION5. Anti Tumor Activity:Selected infected six groups (n =15)Negative control group (sterile normal saline, i.t.); Positive control group one (Taxol intravenous (i.v.), every other day for four times); Positive control group two (Taxol i.t.); PTX-M (i.t.); PTX-M-P407 (i.t.); PTX-M-MG (i.t.).NOTE: Single Dose administration, 20mg/kg of PTX formulations


26PTX-M-MG EVALUATION5. Anti Tumor Activity:To evaluate the antitumor efficacy, the change oftumor volume, in vivo toxicity, survival rate, andbody weightTumor size and body weight (day for eight days) after administration.Sacrificed by cervical dislocation and tumors were harvested Followed by photographing. NOTE: The survival time of six groups treated was calculated by GraphPad Prism 5.


RESULTS &DISCUSSION

27SolGel Transition Behaviour

LCST: P407- (29.96 0.25OC) and P407-CMCS (28.03 0.15OC).(Indicates that they are reversibly thermo sensitive.)

LCST: P407-CMCS GA (0.025%0.10%, w/v)(Converts them into non-thermo-sensitive after certain period of time due to cross linking.)

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28PTX-M-MG EVALUATION5. SolGel Transition Behaviour:

The crosslinkedCMCS network interpenetrated the P407-CMCS/GA gels might become more serried as thecross-linking time increased, avoiding the occurrenceof disaggregation of collective P407 micelles or P407gels when the temperature was below LCST, eventuallylosing the thermosensitivity of the hydrogels.41

29Rheology StudiesWithout GAWith GA

Gelation time > by 2 degrees% GA incre, visco incre42

30Swelling Behaviour & Mechanical Propertiesc) % of GAd) Hardness/integrity based on % GA

Swelling: 0.05%Hardness data - brittle43

31In vitro CytotoxicityL02 cells treated with different conc. Of P407 CMCS/GA

cell survival ratios were all above 80% for conc. Below 360 mciroG/ml.Thus, hardly any toxicity Thus appropriate carrier.44

32Dispersibility of Micelles in P407-M-MG HydrogelsEffect of CMCS or GA on Micelles

GA : no effectCMCS: incre in size due to etanglemnt of CMCS with micelles.

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33Dispersibility of Micelles in P407-M-MG HydrogelsFE-SEM (Cross Sectional Images)

a) PTX onlyb) PTX-Micelles

Crystallization of PTX in a)No crystallization, but uniform PTX-M (no leakage of PTX)

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34In Vitro Drug Release

Burst release faster from PTX-M in 2 hr sustained for only 6 hrs.P407-M-MG is extended for 15 days - suitable for drug depot.47

35In Vitro Drug ReleaseTEM - Images

Micellar drug loaded PTXSize: 200nm

1-route: from micelle to poly compart then diffuse 2 dissolution 2-route: micelle loaded frm poly to disso-thn frm micelle2nd type followedbut., from micelles is future.48

36Tissue DistributionIntratumoral administration in Heps tumor bearing micemice

High conc of PTX f taxol & PTX-M initialVery low conc pf PTX f blue n purple- initialBut it extends for 15-18days49

37Tissue DistributionIntratumoral administration in Heps tumor bearing mice

PTX Far below other formulations, due to restricted polymer, thus low side effects and less hepatic metabolism.

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38Tissue DistributionIntratumoral administration in Heps tumor bearing mice

PTX-M-MG High conc at site, still 26.44 mcg/g even at 20th dayWhereas in other undetected

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39In Vivo Antitumor Efficacy Assay

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2.38-, 1.71-, 1.91- , 1.70-, and 1.50-fold53


64.27%, followed by PTX-M-P407(56.80%), PTX-M (28.76%), Taxol(i.v.) (26.17%), andTaxolR (i.t.) (21.06%)

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42In Vivo Antitumor Efficacy AssaySurvival Percentage

Anti-tumor effective vs survivalmedian survival time prolonged to 18.5 daysTaxol iv 10days

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43In Vivo Antitumor Efficacy AssayI.V. vs Intramural route

30% weight loss existed in intravenous injection due to incre toxicity & severe side effectsGroup than other, so intramural route safer56

CONCLUSION

44In shortNew intratumoral injectable PTX-M-MG hydrogels were constructed and characterized.Incorporation of CMCS-GA cross-linked networks finally transformed the thermosensitive P407 gels into nonthermosensitive hydrogels thereby increasing:higher swell ratios, stronger mechanical property, and Improved drug-release profile.P407-CMCS/GA hydrogels containing 0.05% (w/v) GA could be a remarkable drug carrier for in situ injection.


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45In shortPTX-M-MG hydrogels had excellent performance as a drug depot, including extending retention time at tumor sites, reducing hepatic metabolism,improving antitumor efficiency, and weakening side effects of PTX NOTE: when compared with Taxol, PTXM, and PTX-M-P407In a word, the INJECTABLE MICELLESHYDROGEL SYSTEM may provide a platform for the insoluble drug to achieve -enhanced localized delivery and treatment efficacy with minimal side effects.


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