IMPROVED ORAL BIOAVAILABILITY OF

BREVISCAPINE VIA A PLURONIC P85-

MODIFIED LIPOSOMAL DELIVERY

SYSTEM

PRESENTED BY:JYOTI YADAV

M.PHARM Ist YEARDEPT. OF P’CEUTICS

Breviscapine is a hydrophobic drug obtained from ERIGERON BREVISCAPUS used for treating cerebrovascular disease such as paralysis due to cerebral infraction,hypertension etc

Main chemical constituent is SCUTELLARIN

But the problem with this drug is it’s low bioavailability due to it’s efflux by P-gp present in intestinal epithelium.

So to overcome this problem it is encapsulated in Pluronic P85-modified liposomes.

These are non-ionic triblock co-polymers composed of central hydrophobic chain of polypropylene oxide(PPO) Flanked by two hydrophilic polyethylene oxide (PEO).

P85 is made up of 26 ethylene oxide monomers in each of the outside blocks and 40 propylene oxide monomers in middle block.

In P85

•P-PASTE• 8-8x300=2400(approx molecular weight of hydrophobe)•5x10=50(percentage of polyethylene content)

The multidrug resistance-associated protein transport breviscapine from intestinal epithelium to the

intestinal cavity

Thus reducing the absorption of it and decreases it’s bioavailability

Pluronic P85 inhibits this efflux system by

ATP DEPLEATION as well as by INHIBITION OF MRP ATPase ACTIVITY

Thus increases the bioavailability of the drug

•Vesicles composed of phospholipid bilayer enclosing aqueous compartment in alternate fashion.

•Biodegradable,non-toxic in nature

• Types: MLV

ULV-SUV(up to 100 nm)

• LUV(more than 100 nm)

• Polar drugs are incorporated in aqueous layer while lipophilic drugs are intercalated in liposome membrane.

They are prepared by THIN FILM HYDRTION TECHNIQUE

Accurately weighed quantities of P85(0,10,25 and 50

micrograms) and 100 mg of lipids HPSC:CHOL in a ratio of 7:3 were dissolved in choloform-methanol mixture(2:1, v/v)

Organic solvents were removed under vaccum in a rotary evaporater at 50°c for 15 min

A thin film is formed on the wall of flask which is further kept in dessicator for 24 hr to ensure total

removal of trace solvents

Resultant film was hydrated using 10 ml of 5mM EDTA buffer soln containing 20mg of drug at 55°c

Resulting liposomal dispersion was allowed to mature overnight at 4°c,then were extruded 6 times through a

200nm polycarbonate filter

It is determined by Dialysis.

0.5-ml suspension of drug was added into dialysis membrane bag with 0.9% Nacl as dialysis medium and

process is conducted for 4 hr at 37°c

EE%=W(entrapped drug)/{W(total drug)x100}

DETERMINATION OF PARTICLE SIZE AND ZETA POTENTIAL

It is done by using ZETASIZER NANO ZS after diluting the preparation with ultrapure water to final conc of 0.04mg/ml.

DETERMINATION OF MORPHOLOGY

Done by TRANSMISSION ELECTRON MICROSCOPY

Formulation Mean diameter(nm)

EE%

Bre-soln 106.3± 8.5 90.3± 2.9

Bre-PLs(1oµ m) 110.4± 7.6 92.8± 1.7

Bre-PLs(25µ m) 118.8± 4.9 95.2 ±3.1

Bre-PLs(50µ m) 124.2± 7.2 96.0 ±2.9

TRANSPORT EXPERIMENT IN THE Caco-2 CELLS CULTURE MEDIUM

Caco-2 cells was cultured in Dulbecco’s modified Eagle’s minimal essential medium which was supplemented with L-

glutamine,10% fetal bovine serum and 1%penicillin and streptomycin mix

cells were maintained at 37° c in 5% carbon dioxide and 95%

humidity

Cells were grown in 12-well plates and seeded,at approx 50000 cells per insert.cells were used for experiment b/w 14 and 21

days

Toxicity of Bre-soln,Bre-Ls and Bre-PLs were determined by 3-(4,5-dimethylthiazole-2-yl)-2-5-diphenyltetrazolium bromide

assay to determined the safe conc of these chemicals for Caco-2 cells which was found to be 50 micromol/for each soln.

They were cultured for 21 days to achieve differentiation.Cells having chemical resistance of 500 ohms/cm2

After washing the monolayer cells twice with HBSS(TO adjust ph to 7.7) these 3 soln were loaded in cell inserts.And

receiving chamber contained the corresponding volumes of HBSS

Each sample was added into 3 wells for parallel exp

An oscillation incubator was used to mimic intestinal motility at 37°c and 50 rpm.

At 0,20,40,60,90 and 120 min,a 100 microliter sample was collected at receiver

chamber and equivalent volume of HBSS was subsequently added.

The sample was then immediately lyophilized and preserved below -20° c for

subsequent HPLC

It is calculated by equn:

Papp=dQ/dt × 1/A×Co

WHERE,

Papp=appearent permeability coefficient

A = surface area of monolayer

dQ/dt =rate of appearance of drug in the

basolateral side.

Done by DIALYSIS in a phosphate buffer solution(150

ml) at 37° c±0.2°c by using a rotational speed of 100 rpm

Sample was removed at scheduled time

intervals(0.5,1,2,4,6,8,10,12 and 24 h)

The same amount of release medium was added.Residual

drug

Content of the liposome in the bag was determined by

HPLC.

The liposomal solution and Bre-sol showed an

initial fast release followed by a slow release.

There is no significant difference b/w the scutellarin

release from Bre-Ls and from Bre-PLs.

The initial fast release of drug from the uncoated liposome and the P85-coated liposome is because of

adsorption of drug on the surface of liposome or

dispersion in the outer layers of liposome vesicles..

Similar release profile is because P85 could only form

a relatively loose layer that coats the lipiosome

surface but could not sufficiently prevent yhe release

of drug from liposome.

18 Rats were randomly divided in to 3 groups orally

administered a dose of 90mg/kg of Bre-solution,Bre-Ls and

Bre-PLs

After administration ,blood sample were collected at

0.25,0,5,1,2,4,6,8,10,12and 24 h.

The heparinized blood was immediately centrifuged for 15

min.And rat plasma was obtained and stored at -20°c

Data of the plasma concentration versus time were analysed.

Parameter Bre-solution Bre-Ls Bre-PLs

Cmax(ng/ml) 181.78 ± 49.77 608.8418 ± 113.69 970.70 ± 178.60

Tmax(h) 4.62 ± 0.65 6.83 ± 1.27 7.01 ± 1.39

AUC(h-ng/ml) 1545.61 ± 313.69 4746.81 ± 778.39 8711.0518 ± 890.59

MRT/h8.22 ± 1.35 11.30 ± 1.85 11.73 ± 1.67

Relativebioavailability

-307.12 563.60

P85-coated breviscapine liposome were successfully formulated.

The in-vitro release study indicated that P85-cpated loaded with breviscapine had sustained release property.

In-vitro transport studies in Caco-2 models revealed that P85-coated liposome successfully enhanced the absorption of breviscapine since P85 could inhibit ATPase activity and thereby reduces the MRP efflux transport.

In conclusion ,the encapsulation of breviscapine into P85-coated liposome may be a promising way for improving the oral bioavailability of breviscapine.