INTRODUCTION:

Risedronate sodium is a pyridinyl bisphosphonate that inhibits osteoclast arbitrate bone desorption and alters bone metabolism. Risedronate tablet for oral administration encloses the corresponding of 5, 30 and 35 mg of anhydrous risedronate sodium in the form of the hemi-pentahydrate with little quantities of monohydrate. Risedronate is rapidly absorbed¹⁰ from the GI tract subsequent an oral dose within 1 hour. Its absorption is self-determining of dose in excess of the range studied (2.5 to 30mg). Mean oral bioavailability of the tablet is 0.63%. It was establish in one study that intra-subject variability of intravenous Risedronate was a smaller amount than 20%, at the same time as oral supervision resulted in elevated intra-subject variability (45-83%) for parameters predisposed by absorption.

Bioavailability diminishes when Risedronate is specified with food. Risedronate is indicated for the conduct and deterrence of osteoporosis in postmenopausal women, for the treatment of glucocorticoid osteoporosis and for the treatment of paget’s disease of the bone¹.

Majority of unpleasant events detected all through the proven trials were moreover mild or moderate in severity. On the whole frequency of undesirable events was originated to be analogous in the midst of Risedronate and placebo-treated patients most commonly reported adverse events related to drug were indigestion, queasiness, abdominal pain, constipation, pimples, musculoskeletal pain and nuisance. A quantity of bisphosphonates has been connected with oesophagitis and esophageal or gastric ulceration.

The main objective of this study was to develop new formulation and evaluate the pharmacokinetics and the comparative oral bioavailability by using the simple, fast, sensitive and precise method was developed for the pharmacokinetics study of Risedronate was employed. Compared with the published LCMS/MS method developed a sensitive procedure.

MATERIALS AND METHODS:

Chemicals and reagents:

Acetonitrile of gradient grade, Water HPLC grade was obtained from a Milli-Q water system. Ammonium formate HPLC grade was procured from CDH. Formic acid analytical reagent grade and Trimethyl silyl diazomethane were procured from Merck. Fresh frozen human plasma (K3-EDTA as anticoagulant) used during validation was prepared in-house.

Investigational Study Design:

Equal allocation of treatment was done as per the randomization schedule. Serial blood samples were collected before dosing and up to 24 hours after the drug administration in each period. The subjects were checked out 24 hours after dosing. Ethical committee clearance obtained and study conducted in accordance with declaration of Helsinki². Risedronate in plasma was quantified independently by a validated LC-MS/MS method. The pharmacokinetic parameters were calculated from the drug concentration-time profile by non-compartmental model using Winonlin Software. Statistical comparisons of three formulation pharmacokinetic parameters of were carried out by using statistical analysis software to assess the bioequivalence of the test and reference drug. Descriptive statistics were computed and reported for three formulations.

Clinical Phase:

Nine healthy male volunteers with age group ranges from 18 and 45 years and having a Body Mass Index involving 18kg/ m2 and 27kg/m², having no major ailment or important unusual laboratory values throughout selection, medical history, clinical examination, chest X-ray, ECG recordings, able to exchange a few words efficiently with the study staff and capable to provide written consent for participation in the trial were registered in the study^3-4.

The study was an open, randomized, three-period, three-group crossover trial with an 7-day washout interval^5-8. During the first period, volunteers from group A received a single dose of Risedronate 35mg of test 1 and 2, while volunteers from group B and C received a single Risedronate 35mg reference drug. During the second and third period, the procedure was repeated on the groups in reverse. The tablets were administered to the volunteers after an overnight fast, with 250ml of water. Subjects received standard lunch and supper, respectively, 4 and 10 h post drug administration. Subjects did not consume any alcoholic drink coffee or other xanthine-containing drinks during the trial. In addition, they did not take any other drug and during the execution. Blood samples were taken at pre-dose (0 h) and 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 6.0, 8.0, 10.0, 12.0, 18.0 and 24.0 hours. After blood collection, the tubes were centrifuged for 10 min at 4000rpm and stored in appropriately labeled freeze resistant bags at −70^◦C until sent to the analytical laboratory.

Bioanalytical Method:

The plasma concentrations of Risedronate were analyzed using a suitably validated LCMS/MS assay method^9-11. A calibration curve extending over the range 0.1974 ng/mL to 29.6121ng/mL with a LLOQ of 0.1974ng/mL was used in subject sample analysis of Risedronate. During the validation following parameters as per the USFDA guidelines i.e. specificity / selectivity, carry over, linearity, precision and accuracy, recovery, dilution integrity, ruggedness, stabilities like freeze thaw stability, bench top stability, long term stability, stock solution stability^12-13.

Fig 1: A Representative Chromatogram of a Blank Plasma

Fig. 2: A Representative Chromatogram of a QCLLQ

Pharmacokinetics and Statistical Phase:

All pharmacokinetic parameters of Risedronate were calculated using non-compartment methods^14-17 C_max (maximum plasmatic concentration) and T_max (the time to reach C_max) were obtained directly from the concentration– time curve. Area under the plasma concentration– time curve of Risedronate from zero to the last measurable sample time was calculated using the linear trapezoidal method. K_el was calculated by applying a log–linear regression analysis to at least the last three concentrations of Risedronate. the terminal half-life was calculated as 0.693/K_el^18-19.

RESULTS AND DISCUSSION:

The bioavailability study was performed in nine healthy subjects after administration of single oral dose of Risedronate 35 mg^20-28. Subjects completed the entire duration study were included in the pharmacokinetic and statistical analysis.

On oral administration of the Reference product and the Test product in the fed state exhibited Measurable Risedronate blood levels in all the volunteers from 0.50 hr onwards. Measurable Risedronate blood levels were noticed in all subjects up to 25.00h for both Reference and Test Products. The mean peak plasma concentration (C_max) for Risedronate after administration of the Test 1, 2 and Reference drug 23.6023±8.6157, 24.0037± 12.9547 and 24.4272±11.6229 (ng/mL). The time to peak concentration (t_max) for Risedronate after administration of the Test 1, 2 and Reference drug was 1.21±0.40, 1.19±0.65and 1.04±0.56 hrs. The area under the plasma concentration-time curve (AUC0-t) for Risedronate after administration of the Test 1, 2 and Reference drug was 86.8677±34.2885, 82.1357± 39.4217 and 87.0428±46.7592ng.h/ml. The elimination rate constant for Risedronate after administration of the Test 1, 2 and Reference drug was 0.07514 ± 0.01657, 0.08424±0.02887 and 0.07921±0.03009 h-1. No significant period, treatment and sequence effects observed.

The mean pharmacokinetic parameters of Risedronate for Reference drug C Test 1 and 2 drugs are summarized in the following in Table 1, 2 and 3. Graphical presentation of linear and Semi log of plasma concentration-time curves of Risedronate are depict in Fig. 3 and 4. Further verified the equivalence of the test 1, test 2 and reference drug and found as good as in terms of degree and absorption. During the study there were 5 adverse event observed 2 moderate and 3 mild in nature respectively are tabulated in Table no: 4. No serious adverse event observed throughout the study. Verified the quantifiable laboratory values after completion of the study and found within the acceptable ranges.


Fig. 4: Linear Plot of Mean Plasma Concentration of Risedronate Versus Time Curves After Administration of Reference drug and Test 1 and 2	Fig. 5: Semi logarithmic Plot of Mean Plasma Concentrations of Risedronate Versus Time Curves After Administration of Reference drug and Test 1 and 2.

Table No. 1: Mean Pharmacokinetic Parameters of Test 1 Drug (Risedronate 35mg)

Parameter	Unit	Mean	SD	CV (%)	Min	Median	Max
AUC_t	ng*h/mL	86.8677	34.2885	39.47	31.3187	90.6287	155.8211
AUC_inf	ng*h/mL	92.2055	35.6435	38.66	34.0926	95.9564	163.3447
C_max	ng/mL	23.6023	8.6157	36.50	10.2676	23.8374	41.0332
T_max	h	1.21	0.40	33.06	0.75	1.25	2.50
T_half	h	9.69	2.28	23.50	6.28	8.79	15.04
K_el	1/h	0.07514	0.01657	22.05	0.04608	0.07885	0.11042

Table No. 2: Mean Pharmacokinetic Parameters of Test 2 Drug (Risedronate 35mg)

Parameter	Unit	Mean	SD	CV (%)	Min	Median	Max
AUC_t	ng*h/mL	82.1357	39.4217	48.00	35.6068	73.2562	186.0795
AUC_inf	ng*h/mL	86.9556	41.4799	47.70	38.1922	76.4888	195.0442
C_max	ng/mL	24.0037	12.9547	53.97	8.7280	20.1083	65.6048
T_max	h	1.19	0.65	54.50	0.50	1.00	2.50
T_half	h	8.94	2.30	25.69	4.39	9.09	12.50
K_el	1/h	0.08424	0.02887	34.27	0.05543	0.07624	0.15793

Table No. 3: Mean Pharmacokinetic Parameters of Reference Drug (Risedronate 35mg)

Parameter	Unit	Mean	SD	CV (%)	Min	Median	Max
AUC_t	ng*h/mL	87.0428	46.7592	53.72	37.0928	72.0902	254.1166
AUC_inf	ng*h/mL	92.1808	48.2551	52.35	40.7743	77.4919	263.4115
C_max	ng/mL	24.4272	11.6229	47.58	12.8722	21.9002	66.3496
T_max	H	1.04	0.56	53.59	0.25	0.75	2.50
T_half	H	9.65	2.65	27.47	3.98	10.27	13.30
K_el	1/h	0.07921	0.03009	37.99	0.05212	0.06750	0.17430

Table No. 4: List of Adverse Events

Treatment	Adverse Event	Severity
Test 1	Generalized weakness with mild body ache	Mild
Reference drug	Fever and body ache	Moderate
Test 2	Emesis	Mild
Test 2	Fever	Moderate
Reference drug	Belching	Mild

CONCLUSION:

The intention of the present study was to appraise the pharmacokinetic variables and investigate the pharmacokinetic of newly developed and reference drug. Pharmacokinetic parameters C_max, AUC_tand AUC_inf,t_half, K_el were calculated for Risedronate were calculated using the non-compartmental representation of pharmacokinetic software. The confidence intervals were calculated for the Test 1 vs. Reference drug t-C and Test 2 vs. Reference drug -C was observed similar. Newly developed and reference drug was well tolerated by all subjects. No casualty or severe unsympathetic events occurred during the course of the trial. In conclusion, this newly developed test drugs has appropriate pharmacokinetic uniqueness to reference drug when administered once-a-day during the study.

ACKNOWLEDGEMENT:

The authors articulate the gratefulness to the management of GITAM University and Srinivas College of Pharmacy, for providing necessary support to carry out the work.

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Received on 19.01.2020 Modified on 14.03.2020

Research J. Pharm. and Tech. 2020; 13(12):5876-5880.

DOI: 10.5958/0974-360X.2020.01024.0