Formulation Development of Metformin HCl and Repaglinide Bilayer Tablets: A Novel approach to improve Therapeutic Efficacy

 

M. Sujatha Kumari^*, M. Kishore Babu, K. Rajesh, G. Jyothsna, G. Jeevan Reddy

Department of Pharmaceutics, Sri Siddhartha Pharmacy College, Nuzvid, Andhra Pradesh-521201, India

 

ABSTRACT:

Many antidiabetic drugs including sulfonyl ureas, Rosiglitazone and even Insulin have been suspected to have adverse cardiovascular effects. Lactic acidosis is the feared adverse effect of the biguanide drugs with high doses. We suggest that the mean plasma concentrations of Metformin over a dosage interval be maintained below 2.5 mg/L in order to minimize the development of this adverse effect. The present research work was an attempt to design a novel formulation to improve the oral therapeutic efficacy with optimal control of plasma drug level which contains two antidiabetic drugs Metformin HCl and Repaglinide. Bilayer tablet formulation has been developed consisting of two drug containing layers which comprises Metformin sustained mucoadhesive release layer and an immediate release layer of Repaglinide was optimised separately and constituted in bilayer tablet, a common analytical method for quantitative combined drug estimation was employed and evaluated by HPLC.

 

 

 

INTRODUCTION:

Metformin HCl is an (N,N-dimethylimidodicarbonimidic diamide)  oral anti diabetic agent in the treatment of type 2 diabetes. It is a first line drug of choice in type 2 diabetes. The biological half life was 1.5 to 4.5hrs and approximately 90% of the drug is absorbed in the duodenum and eliminated through the kidney. Metformin HCl increases the risk of lactic acidosis when given to patients with renal impaired function. Bioavailability of Metformin HCl is 50 to 60% due to its site specific absorption limitation.

The obstacle the more successful use of Metformin HCl therapy is the high incidence of concomitant gastro intestinal symptoms such as abdominal discomfort, nausea, diarrhoea that especially occur during initial weeks of treatment and also it have short plasma elimination half life 1.5 to 4.5hrs and there is a need for administration 2 or 3 times per day but when larger doses are required can decrease patient compliance.

 

To maintain blood levels of Metformin HCl can be held at constant therapeutic level for improved drug delivery, accuracy, safety and reduced side effects. Reduction of adverse effects can be accompanished by the drug release at the absorption site as well as sustaining the rate of release enabling total drug content to be reduced.^1,2,3

 

The progressive nature of  type 2 diabetes was combination therapy with current oral anti diabetic drugs was proved side effects. The major Metformin HCl combination therapy was with Sulfonyl ureas and the literature proved that the major side effect of sulfonyl ureas was cardiovascular side effects so, to minimise these side effects the alternative drug of choice was Repaglinide ( Non Sulfonyl urea derivative).^4,5

 

Repaglinide is an meglitinide antidiabetic drug and is chemically S(+)2-ethoxy-4(2((3-methyl-1-(2-(1-piperidinyl) phenyl)-butyl) amino)-2-oxoethyl) benzoic acid with a mechanism of action different  from that of other insulin secretagogues. It lowers blood glucose levels by stimulating the release of insulin from the pancreas by closing adenine tri phosphate dependent potassium channels in the beta cell membrane by binding at characterisable sites. Repaglinide is completely and rapidly absorbed from the GIT after oral administration with mean absolute bioavailability of 56% and peak plasma drug levels occur within 1 hr. Since the plasma half life is about 1hr this makes necessary of frequent dosing to maintain the therapeutic blood level of the drug for a long term treatment.⁶

 

Progressive nature of type 2 diabetes monotherapy with current oral anti diabetic drugs was difficult because the limitation making combination therapy is needed alternatively for achieving recommending glycemic control. Repaglinide and Metformin have separate but complimentary mechanism of action targeting different defects in type 2 diabetes that makes these drugs ideal choice for the use in combinational therapy. Metformin as extended release form and Repaglinide as immediate release form were separately developed by wet granulation process and combined in a Bilayer tablet form. Metformin extended release form was prepared by mucoadhesive system to target duodenum. The present research work is an attempt made for formulation development of Metformin HCL and Repaglinide a novel approach to improve therapeutic efficacy and to improve bioavailability and to reduce the major side effect of lactic acidosis for Metformin HCl by reducing of dose.^17-21 For this purpose different hydrophilic polymers like HPMC K4, K15, K100 and different superdisintegrants like SSG, Crospovidone and CCS were used to enhance poor solubility of Repaglinide. Among different techniques, to fulfil my object i have selected Bilayer tablet technique for treatment of type 2 diabetes.^7,8,9

 

MATERIAL AND METHODS :

Metformin HCl and Repaglinide was a gift sample from Aurabindo pharma pvt.ltd, Hyderabad. HPMC K4, HPMC K15, HPMC K100, SSG, CCS, Crospovidone, PVP K30, MCC were obtained from Otto chemika-biochemica reagents. Other ingredient lactose and sodium CMC were obtained from Finar chemicals limited, Ahmedabed. Magnesium stearate from Otto chemika-biochemica reagents and talc was obtained from Karnataka finar chemicals. The other entire chemical used was of high analytical grade.^10,11,12

 

 

Preparation of  Bilayer tablet :                                                                                       

Step 1: Preparation of SR Metformin HCl granules with HPMC K4, HPMC K15 and HPMC K100 by Wet Granulation method:

Metformin HCl granules were prepared by wet granulation method. The composition of the formulation used in the study is given in Table 1. In  this formulation the required quantities of Metformin HCl and HPMC K4, K15 and K100 were weighed and sieved in 40mm separately and then mixed with required quantities of other exicipients such as Sodium CMC,  Lactose. The powders were blended and granulated with granulating agents such as Isopropyl alcohol: water (1:1) containing 10 mg of PVP K30. The wet mass was passed through sieve 10 and granules were dried in oven at 50ºc for 30 min and the dried granules were again passed through sieve 14 and added required quantities of Mg stearate and talc. Weighed the granules for individual quantities. ^13,14

 

Step 2: Preparation of IR Repaglinide granules with SSG by Wet Granulation method:

Repaglinide granules were prepared by wet granulation method. The composition of the formulation used in the study is given in Table 2. In this formulation the required quantities of Repaglinide and SSG, Crospovidone and CCS were weighed and sieved in 40mm separately and then mixed with required quantities of other exicipients such as MCC, Lactose. The powders were blended and granulated with granulating agents such as Isopropyl alcohol: water (1:1) containing 5 mg of PVP K30. The wet mass was passed through sieve 10 and granules were dried in oven at 50ºc for 30 min and the dried granules were again passed through sieve 14 and added required quantities of Mg stearate and talc. Weighed the granules for individual quantities.¹⁵

 

Step 3: Pre Compression of SR granules and addition of IR granules and compression

Bilayer  tablets were compressed as one layer only for Metformin HCl and second d layer for Repaglinide by using 9mm concave tooling set in 16 stationary rotary tablet punching machine. The tablet was compressed as a bilayer tablet using both Metformin HCl and Repaglinide granules. In this, Metformin HCl granules were introduced first in to the die cavity and a slight pre compression was made so that the layer was uniformly distributed after that Repaglinide granules were added and a final compression was made.^16,17

 

 

 

 

 

 

 

Table 1: Composition of Metformin HCl granules  by Wet Granulation method

INGREDIENTS	F1	F2	F3	F4	F5	F6	F7	F8	F9	F10	F11	F12	F13	F14	F15
Metformin HCl	350	350	350	350	350	350	350	350	350	350	350	350	350	350	350
HPMC K4	10%	20%	30%	40%	50%	---	---	---	---	---	---	---	---	---	---
HPMC K15	---	---	---	---	---	10%	20%	30%	40%	50%	---	---	---	---	---
HPMC K100	---	---	---	---	---	---	---	---	---	---	10%	20%	30%	40%	50%
Sodium CMC	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
PVP K30	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
Lactose	140	105	70	35	---	140	105	70	35	---	140	105	70	35	---
Mg stearate	2%	2%	2%	2%	2%	2%	2%	2%	2%	2%	2%	2%	2%	2%	2%
Talc	2%	2%	2%	2%	2%	2%	2%	2%	2%	2%	2%	2%	2%	2%	2%
Total tablet wt	565	565	565	565	565	565	565	565	565	565	565	565	565	565	565

 

Table 2: Composition of Repaglinide granules with by Wet Granulation method:

INGREDIENTS	F1	F2	F3	F4	F5	F6	F7	F8	F9	F10	F11	F12
Repaglinide	10	10	10	10	10	10	10	10	10	10	10	10
SSG	1%	2%	3%	4%	---	---	---	---	---	---	---	---
Crosspovidone	---	---	---	---	1%	2%	3%	4%	---	---	---	---
CCS	---	---	---	---	---	---	---	---	1%	2%	3%	4%
MCC	10	10	10	10	10	10	10	10	10	10	10	10
PVP K30	5	5	5	5	5	5	5	5	5	5	5	5
Lactose	168	166	164	162	168	166	164	162	168	166	164	162
Mg stearate	3	3	3	3	3	3	3	3	3	3	3	3
Talc	2	2	2	2	2	2	2	2	2	2	2	2
Total tablet wt	200	200	200	200	200	200	200	200	200	200	200	200

 

EVALUATION OF TABLETS:

Weight variation test:

To study weight variation, 20 tablets of each formulation were weighed using an electronic balance (Citizen, India), and the test was performed according to the official method18

 

Hardness and friability:

For each formulation, the hardness and friability of 6 tablets were determined using the Hardness tester (Monsanto, India) and the friabilator (Roche Friabilator India), respectively¹⁹

 

In Vitro release studies:

Dissolution studies were carried out using USP, Paddle method (apparatus II). The stirring rate was 100 rpm. The ability of the release of the drug from dosage form  was assessed by conducting in vitro drug release studies in simulated gastric fluid (SGF-P^H 1.2) for 2 hr, simulated intestinal fluid (SIF P^H 6). Sampling was done at predetermined time intervals and the samples were estimated for drug content after suitable dilution by UV method under specific nm. ²⁰

 

Drug content:

Ten tablets were weighed and powdered and required mg equivalent weight of drug was accurately weighed and transferred into a 100 ml volumetric flask. It was dissolved and made up the volume with medium. Subsequently the solution in volumetric flask was filtered and suitable dilutions were made and analyzed at particular nm using UV-Visible spectrophotometer. The drug content of each sample was estimated from standard curve of drug using particular medium. ²¹

 

Drug content = (Actual drug content / theoretical drug content) X100

 

Swelling index:

Muco adhesive dosage forms were weighed individually (W1) and placed separately in petridishes containing 4 ml of medium at regular intervals (.5,1,2,3,4,5...hrs) the dosagr forms were removed from the petri dishes and excess surface water was removed using filter paper. The dosage form were re weighed and swelling index (SI) was calculated²²

 

SI=(W2-W1)/W1

W1 = Initial Weight, W2 = Final Weight

 

Releasekinetics:

In order to examine the release mechanism of drug sample from the prepared matrix tablets of the optimized formulation (FG₃), the results of the dissolution study was examined in accordance to the kinetic models such as zero-order, first order, Higuchi equation, Korsemeyer–Peppas equation and Hixson–Crowell equation. ²³

 

 

Stabilitystudies:

Stability of a drug can be defined as the time from the date of manufacture and the packaging of the formulation, until its chemical or biological activity is not less than a predetermined level of labeled potency and its physical characteristics have not changed appreciably or deleteriously. The selected formulations were packed in yellow-color PVDC/ALU, Blister. They were then stored at 40⁰c / 75 % RH for 3 months and evaluated. ²⁴

 

 

Reports:

The present research work is an attempt made to formulation development of  Metformin HCL and Repaglinide A novel approach to improve therapeutic efficacy and to improve bioavailability and to reduce the major side effect of lactic acidosis for Metformin HCl due to reducing of dose. For this purpose different hydrophilic polymers like HPMC K4, K15, K100 and different superdisintegrants like SSg, Crospovidone, CCS were used to enhance poor solubility of Repaglinide. Among different techniques to fulfil my object i have selected bilayer tablet technique for treatment of type 2 diabetes. Based on the above investigational reports i concluded following results and discussions.

 

 

Fig 1: FTIR Spectra for Metformin HCl and Repaglinide optimised formulation

 

 

Characterisation of Micromeritic parameters of Metformin HCl and Repaglinide:

Characterisation of Metformin HCl and Repaglinide was conducted by different parameters and the reports were shown in the table.

 

 

 

Table 3: Characterization Of Metformin HCl Granules Formulated With HPMC K4, K15, K100

Formulation	F1	F2	F3	F4	F5	F6	F7	F8	F9	F10	F11	F12	F13	F14	F15
Angle of repose	21.5º	21.15º	22.2º	23.5º	22.8º	24.5º	25.5º	25.2º	24.5º	24.8º	22.9º	23.5º	24.2º	22.5º	23.8º
Bulkdensity(g/cc)	0.215	0.239	0.223	0.248	0.219	0.285	0.289	0.283	0.278	0.269	0.206	0.217	0.223	0.243	0.229
Tapped density (g/cc)	0.310	0.315	0.322	0.320	0.316	0.331	0.335	0.332	0.328	0.321	0.291	0.305	0.282	0.298	0.311
Compressibility index (%)	12.5	11.13	12.9	11.5	12.2	13.2	11.5	12.2	12.5	13.2	14.7	12.5	13.0	10.4	12.2
Total porosity (%)	32.5	31.2	33.2	31.9	30.5	36.5	37.2	36.2	35.1	36.5	33.5	34.2	35.2	34.1	35.5
Drug content (%)	97.5	96.3	95.7	98.6	97	97.5	95.5	96.5	98.2	98	96.5	95.9	96	97.2	98

 

 

 

 

Table 4: Characterization Of Repaglinide Granules Formulated With SSG,CRSPVD,CCS

Formulation	F1	F2	F3	F4	F5	F6	F7	F8	F9	F10	F11	F12
Angle of repose	21.9º	22.5º	24.2º	23.5º	22.9º	23.5º	24.2º	23.7º	22.9º	23.5º	24.2º	22.5º
Bulk density(g/cc)	0.217	0.209	0.229	0.213	0.206	0.217	0.223	0.243	0.206	0.217	0.223	0.243
Tapped  density(g/cc)	0.285	0.297	0.291	0.289	0.291	0.305	0.282	0.298	0.291	0.305	0.282	0.298
Compressibility index (%)	13.7	12	13.9	11.17	11.7	10.5	13	12.4	13.7	11.5	12.0	9.4
Total porosity (%)	32.5	36.2	33.2	34.1	33.5	34.2	35.2	34.1	33.5	34.2	35.2	34.1
Drug content (%)	95.9	94.7	96	97.2	96.5	95.9	96	97.2	96.5	95.9	96	97.2

 

 

EVALUATION OF POST COMPRESSION  PARAMETERS:

Table 5: Post  compression  Parameters Of Metformin HCl tablets Formulated With HPMC K4, K15, K100

Formulation

F1

F2

F3

F4

F5

F6

F7

F8

F9

F10

F11

F12

F13

F14

F15

Weight variation (mg

564 ±0.79

564 ±0.98

564 ±0.69

565 ±0.78

565 ±0.91

565 ±0.89

565 ±0.98

564 ±0.67

566 ±0.43

565 ±0.89

563 ±0.89

565 ±0.78

564 ±0.67

565 ±0.43

565 ±0.89

Hardness (kg/cm2)

6.0 ±1.02

6.0 ±1.02

6.0 ±1.02

6.0 ±1.02

6.0 ±1.02

6.0 ±1.02

6.0 ±1.02

6.0 ±1.02

6.0 ±1.02

6.0 ±1.02

6.0 ±1.02

6.0 ±1.02

6.0 ±1.02

6.0 ±1.02

6.0 ±1.02

Friability (%)

0.89 ±0.01

0.75 ±0.01

0.79 ±0.01

0.82 ±0.01

0.89 ±0.01

0.9 ±0.01

0.86 ±0.01

0.75 ±0.01

0.79 ±0.01

0.94 ±0.01

0.85 ±0.01

0.76 ±0.01

0.85 ±0.01

0.86 ±0.01

0.9 ±0.01

Surface pH

6.21 ±0.09

6.06 ±0.09

6.03 ±0.09

5.93 ±0.09

6.33 ±0.09

6.13 ±0.09

6.56 ±0.09

6.63 ±0.09

6.53 ±0.09

5.93 ±0.09

6.03 ±0.09

6.24 ±0.09

6.33 ±0.09

6.03 ±0.09

5.83 ±0.09

Thickness (mm)

3.43 ±0.06

3.87 ±0.06

3.76 ±0.06

3.87 ±0.06

3.63 ±0.06

3.63 ±0.06

3.78 ±0.06

3.62 ±0.06

3.91 ±0.06

3.53 ±0.06

3.67 ±0.06

3.78 ±0.06

3.82 ±0.06

3.91 ±0.06

3.93 ±0.06

Mucoadhesive strength (gm)

6.05 ±0.07

6.24 ±0.07

6.35 ±0.07

6.17 ±0.07

6.32 ±0.07

6.43 ±0.07

6.34 ±0.07

6.75 ±0.07

6.05 ±0.07

6.22 ±0.07

6.43 ±0.07

6.54 ±0.07

6.65 ±0.07

6.71 ±0.07

6.82 ±0.07

Mucoadhesive time(hr)

6

7

8

10

12

8

9

10

12

16

7

8

9

11

14

Drug content (%)

97.23 ±0.23

98.23 ±0.23

95.87 ±0.23

96.45 ±0.23

99.03 ±0.23

97.63 ±0.23

98.73 ±0.23

95.07 ±0.23

97.95 ±0.23

99.17 ±0.23

98.63 ±0.23

96.73 ±0.23

95.07 ±0.23

97.95 ±0.23

99.48 ±0.23

 

 

Table 6: Post compression studies of Repaglinide tablets formulated with SSG, CRSPVD, CCS

Formulation

F1

F2

F3

F4

F5

F6

F7

F8

F9

F10

F11

F12

Weight variation (mg

200 ±0.79

198 ±0.98

201 ±0.69

199 ±0.78

201 ±0.54

198 ±0.65

201 ±0.43

200 ±0.96

201 ±0.79

199 ±0.75

201 ±0.47

200 ±0.96

Hardness (kg/cm2)

5.0 ±1.02

5.0 ±1.02

5.0 ±1.02

5.0 ±1.02

5.0 ±1.02

5.0 ±1.02

5.0 ±1.02

5.0 ±1.02

5.0 ±1.02

5.0 ±1.02

5.0 ±1.02

5.0 ±1.02

Friability (%)

0.79 ±0.01

0.85 ±0.01

0.69 ±0.01

0.72 ±0.01

0.56 ±0.01

0.43 ±0.01

0.75 ±0.01

0.63 ±0.01

0.87 ±0.01

0.77 ±0.01

0.64 ±0.01

0.84 ±0.01

Disintegration (min)

5 ±0.3

4 ±0.3

4 ±0.3

3 ±0.3

4 ±0.3

3 ±0.3

3 ±0.3

1 ±0.3

4.5 ±0.3

4 ±0.3

3.3 ±0.3

2 ±0.3

Drug content (%)

96 ±0.06

94 ±0.06

98 ±0.06

97 ±0.06

98 ±0.56

94 ±0.06

95 ±0.96

97 ±0.74

98 ±0.76

96 ±0.78

95 ±0.54

98 ±0.93

 

 

 

 

 

 

 

RELEASE PROFILE GRAPH OF BILAYER TABLET OF METFORMIN HCL and REPAGLINIDE IN HPLC:

 

Fig 2: % Drug release of Repaglinide

 

Fig 3: % Drug release of Metformin HCl

CONCLUSION:

The approach of the present study was  carried out to develop Metfirmin HCl and Repaglinide bilayer tablets for SR and IR to improve therapeutic efficacy by uing different polymers with different ratios. Metformin HCl tablets prepared with HPMC K4, K15, K100 in different percentages. Among all the formulations formulated,  K15 (F10) sustained the release for 16hr and fulfilled my objective of this work. The ability of the used polymers to sustain the release is HPMC K15 > HPMC K100 > HPMC K4 . Repaglinide tablets prepared with SSG, Crospovidone, CCS in different percentages. Among all the formulations formulated,  F8 CRSPVD 8% releases drug with in 20min and fulfilled my objective of this work. The ability of the used superdisintegrants  release is Crosspovidone < CCS < SSG. The study shows that the release of Metformin HCl in the physiological environment of duodenum is due to the use of HPMC K15. Because the absorption is more in duodenum, the use of this polymer can overcome lactic acidosis in gastric environment. The bilayer tablet is the  best dosage form to treat type 2 diabetes. Because it was reported the combination of these two drugs with reduced dose increases the therapeutic efficacy. 

 

FUTURE:

In future, the combination of three drugs with reduced dose are formulated to increase the therapeutic efficacy.

 

ACKNOWLEDGEMENTS:

We are sincerely thankful to Aurabindo Pharma Pvt. Ltd for providing gift sample of Metfirmin HCl and Repaglinide drugs and we would also like to thank the management and staff of Vikas College of Pharmacy, Vissannapeta and Sri Siddhartha Pharmacy College, Nuzvid  for encouraging us and providing all facilities to proceed on the work

 

REFERENCES:

1.     Lucile Packard childrens hospital at Stanford:Overview of Diabetes from healthy library Over view on Diabetes :National Diabetes Information Clearinghouse (NDIC) Over view on Diabetes : WebMD

2.     Drugs for diabetes: Diabetic Mediterranean Diet

3.     Hope for diabetics: New drug increases body’s ability to lower blood sugar: Express India.com From Medscape Diabetes and Endocrinology Major Milestones in Diabetes: 2010 June 29, 2006 Pharmaceuticals Diabetes: A 21st Century Epidemic by Morgan Stanley Overview on Type 2 Diabetes: About drugs.com

4.     Type 2 Diabetes Mellitus : Romesh Khardori, MD, PhD, FACP; Chief Editor: George T Griffing from web MD

5.     Type 2 Diabetes methods and protocol by clairey stocker

6.     Type 2 Diabetes overview: Diabetic Health professional Web MD

7.     International Journal on Pharmaceutical and Biological Research Vol. 1(1), 2010, 30-41Stomach Specific Mucoadhesive Tablets As Controlled Drug Delivery System – AReview Work by G.C.Rajput, Dr. F.D. Majmudar1, Dr. J.K. Patel, K.N. Patel, R.S.Thakor,B.P.Patel, Rajgor NB Updated Review: Improved Glycemic Control with Repaglinide-Metformin in Fixed Combination for Patients with Type 2 Diabetes: John W. Richard III and Philip Raskin

8.     Biguanide: From Wikipedia, the free encyclopedia Clinical Pharmacokinetics of metformin.Graham GG, Punt J: pubmed articles.An overview of metformin in the treatment of type 2 diabetes mellitus,david son MB,Peters AL.: pubmed articles

9.     Combination Therapy for Patients With Type 2 Diabetes: Overview of Oral Antidiabetic Medications from webmd International journal of pharma and bio sciences mucoadhesive drug delivery: mechanism and methods of evaluation corresponding author pranshu tangri Indian Journal of Ph. Scs.2009. vol.1(6): A Review on Bilayer Tablet Technology

10.  International Journal of Biopharmaceutical and Toxicological Research Review article: immediate release drug delivery systems: a review by syed azeem, Shaweta, Sharma

11.  Metformin drug information From Wikipedia, the free encyclopedia

12.  Repaglinide drug information From Wikipedia, the free encyclopedia

13.  HPMC K4 polymer information From Wikipedia, the free encyclopedia

14.  HPMC K15 polymer information From Wikipedia, the free encyclopedia

15.  HPMC K100 polymer information From Wikipedia, the free encyclopedia

16.  Sodium starch glycolate information From Wikipedia, the free encyclopedia

17.  Crospovidone  information From Wikipedia, the free encyclopedia

18.  Croscarmelose drug information From Wikipedia, the free encyclopedia

19.  International Journal of Pharmaceutical Scs. 2012; 4(1): 21-25;John W. Richard III and Philip Raskin et.al Updated Review: Improved Glycemic Control with Repaglinide-Metformin in Fixed Combination for Patients with Type 2 Diabetes.

20.  International Journal of Biopharmaceutics. 2011; 2(1): 25-29; Jayaprakash.S, Mohamed Halith.S et al Developed Formulation and Evaluation of Bilayer Tablets of Metformin HCl .

21.  International Journal of  Research in Pharmaceutics. 2012; 6(1): 14-18; Durga Prasad Pattanayak and Subash C. Dinda et al developed Bilayer tablet Formulation of Metformin Hydrochloride and Glimepiride.

 

 

Accepted on 20.06.2018          © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(9): 4119-4125.