Preparation, Characterization and In-Vitro

Evaluation of Metformin HCl and Glipizide

Loaded Microspheres of Different polymers by

Using Emulsion Solvent Evaporation Technique

Submitted by:

Md. Maksud Al- Hasan

Registration no: 14107095

Session: Spring- 2014.

AIM, SCOPE AND OBJECTIVES OF THE

STUDY

AIM: The present work was aim to formulate and

evaluate combination microspheres of Metformin HCl

and Glipizide by emulsion solvent evaporation technique

using EC, HPMC 15CPS, Eudragit RSPO and Eudragit

RLPO polymers. The ultimate aim to use those polymers

is to increase bioavailability and decreasing

gastrointestinal side effects.

SCOPE: Prepared Microspheres of Metformin and

Glipizide are expected to shows synergetic effect in their

action and can be utilized for controlled release of

Metformin HCl and Glipizide for an extend period in the

management of type-II diabetes.

OBJECTIVES: Reduce the dosing frequency

fluctuation in therapeutic blood level is avoid.

Increase patient compliance, decrease in dose

Preparation of Metformin HCl and Glipizide

Microspheres

Ethanol and DCM

Polymer, Ethanol and DCM

Polymer, Ethanol and DCM.

Polymer

Metformin HCl & Glipizide

Homogenization for 4 hrs 30 min.

450 RPM, 50ml Liquid Paraffin with

1% Tween 80.

Settle for few minutes

Washing

Drying at Room Temperature

Store

Standard Curve of Metformin HCl

y = 0.008x + 0.004R² = 0.999

00.10.20.30.40.50.60.70.80.9

0 20 40 60 80 100 120

Ab

sorb

ance

Concentration (µg/ml)

Standard Curve of Metformin HCl

Figure: Standard curve of metformin HCl

Standard Curve of Glipizide

y = 0.023x - 0.005R² = 0.999

-0.1

0

0.1

0.2

0.3

0.4

0.5

0 5 10 15 20 25

Ab

so

rban

ce

Concentration µg/ml

Standard Curve of Glipizide

Figure: Standard curve of glipizide

Overlay Spectra of Metformin HCl and

Glipizide

Figure: Overlay spectra of metformin HCl and glipizide

Metformin

HCl

Glipizide

Isobestic point

In vitro dissolution study of microsphere containing

Metformin HCL & Glipizide Absorption Ratio Method (Q Method):

For Q method, 259.5 nm (isobestic point) and 275 nm (λmax of GD) were

selected as wavelengths of measurements. Concentrations of MH and GD

were determined using following equations.

Cx = (Qm-Qy). A1 / (Qx-Qy). Ax1

Cy = (Qm-Qx). A1 / (Qy-Qx). Ay1

Where ,

Qm = A2/ A1

Qx = ax2/ ax1

Qy = ay2/ ay1

A2= Absorbance of Mixture at 275nm

A1= Absorbance of Mixture at 259.5 nm

ax1= absorptivity of MH at 259.5 nm

ay1= absorptivity of GD at 259.5 nm

ax2= absorptivity of MH at 275 nm

ay2= absorptivity of GD at 275 nm.

Table-1: Composition of drug loaded

microspheresF- Code Drug (mg) Polymers (mg) Ration

(D:P)MET GLI EC HPMC

15CPS

Eudragit

RSPO

Eudragit

RLPO

F1 250 10 260 - - - 1:1

F2 250 10 520 - - - 1:2

F3 250 10 780 - - - 1:3

F4 250 10 - 260 - - 1:1

F5 250 10 - 520 - - 1:2

F6 250 10 - 780 - - 1:3

F7 250 10 - - 260 - 1:1

F8 250 10 - - 520 - 1:2

F9 250 10 - - 780 - 1:3

F10 250 10 - - - 260 1:1

F11 250 10 - - - 520 1:2

F12 250 10 - - - 780 1:3

F13 250 10 - 85 175 - 1:1

F14 250 10 - 170 250 - 1:2

F15 250 10 - 255 525 - 1:3

F16 250 10 175 85 - - 1:1

F17 250 10 250 170 - - 1:2

F18 250 10 525 255 - - 1:3

F19 250 10 130 - 130 - 1:1

F20 250 10 260 - 260 - 1:2

F21 250 10 390 - 390 - 1:3

F22 250 10 130 - - 130 1:1

F23 250 10 260 - - 260 1:2

F24 250 10 390 - - 390 1:3

Where, F= Formulation; Met= Metformin HCl; Gli= Glipizide; EC= Ethyl Cellulose; D= Drug; P- Polymer.

RESULT AND DISCUSSION

0

10

20

30

40

50

60

70

80

90

100

% D

EE

Formulation

Drug Entrapment efficiency (%)

Figure: Drug entrapment efficiency of formulation F1 to F24.

Discussion

It was found in the range of 56.56% to 88.02% Formulation F11 containing eudragit RLPO

showed maximum drug loading about 88.02% Formulation F6 containing HPMC 15CPS shows

minimum drug loading about 56.56% Microspheres of HPMC 15CPS are irregular

shape therefore more drug loss from surface during washing leads to less drug entrapment efficiency

Rank order of % drug loading of various formulations:F11>F4>F16>F17>F19>F14>F13>F20>F10>F5>F7>F21>F22>F12>F24>F15<F8>F1>F2>F18>F9>F23>F3>F6

(a) (b)

(c)

Effect of ethyl cellulose on surface

morphology of F1 microspheres (a)

magnification at X200 SEI (b)

magnification at X200 SEI and (c)

magnification at X1000 SEI

(a)

(b)

(c)

Effects of HPMC 15CPS and Ethyl

Cellulose on surface morphology of F17

microspheres. (a) Magnification at

X100 SEI, (b) Magnification at X100

SEI and (c) Magnification at X400 SEI.

Drug release Study from single polymer loaded

microspheres

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

0.00 5.00 10.00 15.00

Cu

mu

lati

ve %

rele

ase

Time (hr)

F1

F2

F3

F4

F5

F6

F7

F8

F9

F10

F11

F12

Figure: Comparative % drug release of Metformin HCl from formulation F1 to

F12

0.00

5.00

10.00

15.00

20.00

25.00

30.00

0.00 2.00 4.00 6.00 8.00 10.00 12.00

Cu

mu

lati

ve %

Rele

ase

Time (hr)

F1

F2

F3

F4

F5

F6

F7

F8

F9

F10

F11

F12

Figure: Comparative % drug release of Glipizide from formulation F1 to

F12

Figure: Comparative % drug release of Metformin HCl from formulation F13 to

F24

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

0.00 2.00 4.00 6.00 8.00 10.00 12.00

Cu

mu

lati

ve %

rele

ase

Time (hr)

F13

F14

F15

F16

F17

F18

F19

F20

F21

F22

F23

F24

Drug release Study from combination polymer loaded

microspheres

0.00

5.00

10.00

15.00

20.00

25.00

30.00

0.00 2.00 4.00 6.00 8.00 10.00 12.00

Cu

mu

lati

ve %

Rele

ase

Time (hr)

F13

F14

F15

F16

F17

F18

F19

F20

F21

F22

F23

F24

Figure: Comparative % drug release of Glipizide from formulation F13 to

F24

Discussion

Dissolution Medium: Buffer (pH 6.8), Temp: 370C

Drug release from these microspheres were found to be slow, extended and depended in the type and conc. of polymer used.

It was observed that metformin hydrochloride release was higher when compared to that of glipizide at the end of the release study this may be due to reason that release of glipizide from the microspheres depends on the core: coat ratio i.e., drug: polymer ratio. Here the ratio was 1:26; 1:52 and 1:78 for glipizide and 1:1.04; 1: 2.08 and 1: 3.12 for metformin hydrochloride, which resulted in low cumulative percentage drug release of glipizide from the microspheres.

Formulation F10 and F2 containing Eudragit RLPO and Ethyl cellulose showed the max. release of 87.02% and 27.31% for metformin and glipizide respectively for formulation F1 to F12 after 10hrs, due to high swelling property and high dissolution of polymer.

Formulation F20 and F24 containing combination polymers of Ethyl cellulose+ eudragit RSPO and Ethyl cellulose+ eudragit RLPO showed the max. release for metformin and glipizide from formulation F13 to F24 respectively.

Comparative release of Metformin HCl and

Glipizide microspheres with available film coated

tablet (Metglip DS) and pure drug

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

0 2 4 6 8 10 12

Cu

mu

lati

ve

% r

elea

se

Time (hr)

MF10 GF1 MF20 GF21

Metglip-MET Metglip-GLI Pure-MET Pure-GLI

Where, MET= Metformin HCl, GLI= Glipizide

FormulationsZero order First order Higuchi

krosmeyer

K0 R2 K1 R2 KH R2 n R2

MF1 7.963 0.96 -0.074 0.971 27.27 0.986 0.439 0.966

MF2 6.021 0.975 -0.041 0.982 20.37 0.987 0.402 0.954

MF3 5.983 0.934 -0.039 0.965 20.56 0.966 0.428 0.929

MF4 6.664 0.988 -0.048 0.963 22.06 0.948 0.453 0.972

MF5 7.674 0.934 -0.061 0.977 26.44 0.983 0.425 0.981

MF6 5.571 0.988 -0.035 0.974 18.46 0.95 0.419 0.993

MF7 6.873 0.898 -0.052 0.875 22.38 0.968 1.039 0.917

MF8 6.707 0.945 -0.053 0.911 22.72 0.95 0.736 0.92

MF9 7.826 0.992 -0.066 0.963 26 0.959 0.837 0.989

MF10 8.271 0.994 -0.077 0.93 27.47 0.957 0.436 0.992

MF11 6.792 0.985 -0.051 0.991 23.22 0.993 0.416 0.982

MF12 5.322 0.918 -0.035 0.945 18.54 0.976 0.584 0.973

Interpretation of release rate constants and R-squared values for different release kinetics

of (a) Metformin HCl and (b) Glipizide in single polymer based Metformin HCl and

Glipizide microspheres

aWhere, M=Metformin HCl

Formulation

s

Zero order First order Higuchi krosmeyer

K0 R2 K1 R2 KH R2 n R2

GF1 2.355 0.976 -0.011 0.985 7.95 0.973 0.425 0.948

GF2 2.439 0.966 -0.012 0.951 7.876 0.881 0.405 0.975

GF3 2.576 0.989 -0.012 0.984 8.281 0.895 0.421 0.964

GF4 1.499 0.962 -0.007 0.969 5.106 0.977 0.433 0.988

GF5 1.369 0.953 -0.006 0.946 4.371 0.85 0.426 0.926

GF6 2.599 0.988 -0.013 0.988 8.534 0.932 0.419 0.946

GF7 2.153 0.968 -0.01 0.976 7.305 0.976 0.485 0.97

GF8 2.054 0.977 -0.01 0.975 6.659 0.899 0.439 0.855

GF9 1.095 0.981 -0.005 0.984 3.672 0.966 0.352 0.967

GF10 1.678 0.942 -0.008 0.941 5.64 0.929 0.581 0.929

GF11 1.926 0.987 -0.009 0.983 6.494 0.793 0.427 0.973

GF12 2.585 0.967 -0.012 0.975 8.619 0.941 1.052 0.983

Where, G= Glipizide b

Successive fractional dissolution time (hr) of Metformin

HCl from Formulation F1 to F12

0

5

10

15

20

25

Tim

e (

hr)

Formulations

T25 T50 MDT T80

Successive fractional dissolution time (hr) of Glipizide from

Formulation F1 to F12

0

50

100

150

200

250T

ime (

hr)

Formulation

T25 T50 MDT T80

Interpretation of release rate constants and R-squared values for

different release kinetics of (a) Metformin HCl (b) Glipizide in

combined polymer based Metformin HCl and Glipizide

microspheres

Formulatio

ns

Zero order First order Higuchikrosmeyer

K0 R2 K1 R2 KH R2 n R2

MF137.503 0.974 -0.066 0.901

24.9

8

0.94

5

0.37

4

0.96

4

MF146.73 0.942 -0.049 0.904

21.8

5

0.86

9 0.88

0.90

6

MF156.955 0.968 -0.056 0.881

21.6

1

0.89

6

0.25

5

0.96

2

MF167.297 0.979 -0.06 0.958 24.6

0.97

4

0.38

2

0.99

3

MF176.739 0.982 -0.05 0.949

22.0

8

0.92

3

0.40

9

0.96

9

MF186.291 0.94 -0.047 0.9

21.2

8 0.94

0.41

8 0.94

MF197.654 0.995 -0.063 0.93

25.0

3

0.93

1

0.39

2

0.99

4

23.8 0.85 0.42 0.95

a

FormulationsZero order First order Higuchi krosmeyer

K0 R2 K1 R2 KH R2 n R2

GF13 1.687 0.98 -0.008 0.983 5.641 0.959 0.599 0.971

GF14 1.921 0.962 -0.009 0.966 6.414 0.939 1.148 0.965

GF15 288 0.977 -0.011 0.98 7.584 0.939 0.724 0.965

GF16 1.878 0.992 -0.009 0.993 6.192 0.943 0.67 0.951

GF17 1.469 0.961 -0.007 0.964 4.937 0.949 0.507 0.96

GF18 1.468 0.981 -0.007 0.984 4.938 0.971 0.618 0.98

GF19 1.474 0.948 -0.007 0.957 5.1 0.992 0.556 0.992

GF20 1.93 0.959 -0.009 0.972 6.522 0.958 0.787 0.963

GF21 2.345 0.963 -0.011 0.973 7.983 0.976 0.618 0.977

GF22 1.764 0.978 -0.008 0.978 5.854 0.942 0.621 0.951

GF23 1.944 0.982 -0.009 0.979 6.357 0.919 0.726 0.978

GF24 2.317 0.975 -0.013 0.982 8.766 0.957 0.838 0.987

b

0

5

10

15

20

25

MF13 MF14 MF15 MF16 MF17 MF18 MF19 MF20 MF21 MF22 MF23 MF24

Tim

e (

hr)

Formulations

T25 T50 MDT T80

Successive fractional dissolution time (hr) of Metformin

HCl from Formulation F13 to F24

0

50

100

150

200

250

300

350

GF13 GF14 GF15 GF16 GF17 GF18 GF19 GF20 GF21 GF22 GF23 GF24

Tim

e (

hr)

Formulation

T25 T50 MDT T80

Successive fractional dissolution time (hr) of Glipizide from

Formulation F13 to F24

FTIR Spectra of pure Metformin HCL

Primary amine N-

H stretching

3370.66)

Primary amine N-H

bending(1630.84)

Imino compound N-H

stretching (3293.51)

Secondary amine

N-H stretching

(3173.92)

Imino compound / Secondary

amine N-H bending (1571.05)

C-H bending

(1486.18,1447.60,1418.67)

FTIR Spectra of pure Glipizide

3351 for CONH

stretching

3351 for NH-CO-

NH stretching

3030 (aromatic

C-H stretching)

FTIR Spectra of Formulation F1

FTIR Spectra of Formulation F19

DSC of pure Metformin HCl

DSC of pure Glipizide

DSC of Formulation F1

DSC of formulation F19

Formulation

code

Mean

size (µm)

Median

size (µm)

F1 325 320

F2 352 314

F3 365 356

F4 246 356

F5 325 324

F6 321 412

F7 325 243

F8 214 257

F9 352 346

F10 245 256

F11 351 354

F12 235 268

F13 245 312

F14 235 358

F15 244 324

F16 256 324

F17 261 298

F18 225 314

F19 443 387

F20 245 364

F21 354 415

F22 356 289

F23 391 471

F24 412 346

Formulation

code

Mean

size (µm)

Median

size (µm)

Particle size distribution data

Conclusions

• Ethyl Cellulose, HPMC 15CPS, Eudragit RSPO,

and Eudragit RLPO all are retardant polymers. So by

using this polymers optimum sustained release

microsphere can be formed.

•Drug loading & polymer ratio have direct effect on

different properties of the prepared microsphere (

Entrapment efficiency, dissolution, surface

morphology etc).