Improve Bioequivalence predictions with PAMPA Dissolution using Etoricoxib and five other Drug Formulations

 

Joe Chou¹*, Roger Lai², Jason Chou¹, Shelly Fu², Li Chuan Chung¹

¹YQ Biotech Ltd., Taiwan.

²Isuzu Optics Ltd., Taiwan.

 

ABSTRACT:

In the generic drug formulation development, pilot bioequivalence (BE) study with a small group of subjects is the current practice for oral formulation prediction. However, due to the difficulty in differentiating the variation between subject and drug formulation with the current BE practice，a new instrument called PAMPA Dissolution is proposed to eliminate the subjects variation and to enhance the correlation between in vitro to in vivo absorption in BE study. PAMPA Dissolution simultaneously measures drug dissolution (Cb) and permeation (Pe), following the validated oral drug absorption equation F (drug absorbed) = Cb*Pe*Area. The use of biorelevant media further allows this device to mimic in vivo conditions closely. Formulations of 60 mg etoricoxib tablets were studied to verify system reproducibility and BE prediction to demonstrate the potential of PAMPA Dissolution in generic drug development. The BE predictions between generic and brand etoricoxib tablets (test/reference) from this system produced a Cmax value of 99.0% and AUC value of 99.1%, indicating that PAMPA Dissolution predictions conform with bioequivalence results. Other oral formulations of valsartan/hydrochlorothiazide, ezetimibe, telmisartan, and amlodipine were also tested for their permeation (Pe) by PAMPA Dissolution. Results of drug permeation compared to the literature values indicates that the PAMPA Dissolution is reliable and precise in formulation development.

 

 

 

INTRODUCTION:

Bioequivalence (BE) is a crucial parameter of generic drug development. Traditionally, dissolution experiments are used to examine the performance of drug formulations following FDA (U.S Food and Drug Administration) guidance, and finally the pilot bio studies were conducted to verify generic formulations.

 

However, traditional pilot BE study has the following deficiencies: Firstly, small group of human subjects absorption variations makes it difficult to meet BE limits. There is no definite way to differentiate variation between subject and formulation from current pilot bio study. Another issue is that buffer solutions used in dissolution tests as required per the (FDA) regulation, which often falls short in mimicking conditions in the gastrointestinal (GI) tract such as the various enzymes from bile and pancreatic secretions, result in poor correlation between in vitro to in vivo correlation (IVIVC)¹. This is especially true with BCS II drugs that have low aqueous solubility since solubility and permeability often influence one another⁹.

 

Figure 1: PAMPA Dissolution apparatus

 

To improve oral drug development, a better IVIVC techniques² is needed. A popular method involves the use of Caco-2 cells, however, Caco-2 cells have to be incubated for up to twenty to thirty days before testing; it is not a time-effective method. An alternative that can be used to improve IVIVC for BE prediction is Parallel Artificial Membrane Permeability Assay (PAMPA), which uses a chemically-based membrane instead of live cells and has been proven to be able to accurately mimic the human small intestine^3,6,7,8. PAMPA Dissolution system (Figure 1) is a newly designed instrument that combines dissolution and permeation in a way closely mimic in vivo conditions of human gastro-intestinal tract (GIT). It also measures the two necessary parameters: dissolution (Cb) and permeation (Pe) following oral drug absorption by the previous validated equation F (drug absorbed) = Cb*Pe*Area^2,3. This PAMPA Dissolution system also produces real-time graphs for dissolution and permeation simultaneously. From these graphs, Area Under Curve (AUC) and maximum plasma concentration (Cmax) values can be calculated to predict bioequivalence. Instead of traditional buffer solutions, PAMPA Dissolution uses biorelevant media, gastric (FaSSGF) and intestinal (FaSSIF) fluids with the respective pH values^4,5, further giving it more potential to mimic IVIVC and lead to better BE prediction^{10, 11,12}.

 

In this study, etoricoxib, a poorly water soluble drug used to treat osteoarthritis and rheumatoid arthritis, was selected to validate PAMPA Dissolution as a feasible way to predict BE study. In addition, five other drug formulations were also studied for their permeation as a proof that the PAMPA Dissolution system is feasible in bioequivalence prediction.

 

Materials and Methods:

Generic etoricoxib (Etor) and brand (Arcoxia) 60mg tablets were purchased from a local market. Reagents including NaH₂PO_4·2H₂O, NaOH, NaCl, HCl, DMSO, phospholipids and n-dodecane were purchased from First Chemicals (Taiwan). FaSSIF/FeSSIF/FaSSGF powder was purchased from Biorelevant (London, UK). Other oral formulations of valsartan/hydrochlorothiazide, ezetimibe, telmisartan, and amlodipine tablets were purchased from a local market.

 

PAMPA Dissolution (YQ Biotech Co.) was used to study Arcoxia tablets and generic etoricoxib tablets. The dissolution consists of six standard vessels of USP 2 apparatus (TDTF, China). Fiber optic UV probes at 285 nm are inserted to record concentrations of etoricoxib in real-time^13,14,15. On the PAMPA side of the apparatus, Vessels have inner compartments filled with buffer solution at pH=6.5 (with 2% DMSO) and divided from the outer compartments by a 0.45um hydrophobic permeation membrane impregnated with 160μL of 3.3% (w/v) phospholipids solution in n-dodecane.

 

Vessels on the dissolution side are each filled with 400 mL of pH 1.6 fasted state simulated gastric fluid (FaSSGF) made with FaSSIF/FeSSIF/FaSSGF           powder^{16, 17,18}, NaCl, and water. After 30 minutes, 100mL of pH 7.5 concentrated fluid consisting of FaSSIF/ FeSSIF/ FaSSGF powder, NaH₂PO_4·2H₂O, and NaOH is added in each vessel to turn the original fluid into fasted state simulated intestinal fluid (FaSSIF) of pH 6.5. Stirring on the dissolution side is set to 50rpm; while the PAMPA magnetic stirrer is set to 200rpm to minimize the unstirred water layer in order to mimic actual intestinal conditions as closely as possible. Experimental data were automatically collected and calculated against freshly prepared calibration curves of UV probes at 285nm.

Table 1: PAMPA Dissolution of Arcoxia 60 mg tablets - dissolution data

Time (min)	CH1	CH2	CH3	Red line: Arcoxia-60mg (average of 1-3)	Time (min)	CH4	CH5	CH6	Blue Line: Arcoxia-60mg (average of 4-6)	Average of 6 channels (mg)	Red and Blue line-- %RSD	%RSD of 6 Channels
11.14	44.47	40.29	41.09	41.95	11.28	44.17	52.05	40.83	45.69	43.82	1.80	10.06
22.39	52.89	51.99	48.60	51.16	22.53	50.09	54.49	41.98	48.86	50.01	12.98	8.89
33.64	51.19	54.89	18.19	41.42	33.78	51.97	56.14	38.62	48.91	45.17	4.38	32.36
44.89	72.53	71.82	68.77	71.04	45.03	72.23	76.07	45.29	64.53	67.78	26.87	16.62
56.16	64.84	62.81	62.43	63.36	56.30	61.76	64.48	35.60	53.94	58.65	33.47	19.37
67.42	60.93	61.69	60.29	60.97	67.57	61.83	62.87	52.14	58.95	59.96	10.41	6.55
78.69	61.38	62.08	60.57	61.34	78.84	62.20	62.53	52.00	58.91	60.12	10.98	6.72
89.96	61.75	62.30	61.17	61.74	90.11	62.65	63.40	53.11	59.72	60.73	10.05	6.27

 

Table 2: PAMPA Dissolution of Arcoxia 60 mg tablets - permeation data

Time (min)	CH7	CH8	CH9	Red Line: Arcoxia-60mg (average of CH 7-9)
18.82575	-0.00168	0.00175	0.00441	0.00150
30.07617	0.00007	0.00337	0.01131	0.00492
41.32630	0.00193	0.00295	0.00946	0.00478
52.57693	0.02891	0.03225	0.03820	0.03312
63.84343	0.08956	0.09184	0.09766	0.09302
75.11035	0.15179	0.15220	0.15794	0.15398
86.37648	0.21363	0.21145	0.21954	0.21487
97.64412	0.27173	0.26883	0.27890	0.27315
108.91132	0.32936	0.32665	0.33634	0.33078
120.17825	0.38711	0.38107	0.39275	0.38697
131.44532	0.44028	0.43463	0.44735	0.44075
142.71147	0.49447	0.48568	0.50147	0.49387
153.97907	0.54579	0.53603	0.55354	0.54512
165.24557	0.59654	0.58479	0.60401	0.59511
176.51238	0.64682	0.63069	0.65487	0.64413
187.77942	0.69464	0.67415	0.70044	0.68974
199.04632	0.73942	0.71576	0.74523	0.73347

 

Table 2: Cont…..

Time(min)	CH10	CH11	CH12	Blue Line: Arcoxia-60mg (average of CH 10-12)	Average of 6 Channels (mg)	Red and Blue line %RSD	%RSD of 6 Channels
18.99605	-0.00496	0.00745	0.03912	0.01387	0.00768	113.87	208.43
30.24698	-0.00238	0.00553	0.04211	0.01509	0.01000	71.90	164.20
41.49782	-0.00453	0.01356	0.05637	0.02180	0.01329	90.56	165.68
52.74835	0.02619	0.04488	0.14430	0.07179	0.05246	52.13	86.73
64.01550	0.09834	0.10730	0.12877	0.11147	0.10225	12.76	14.07
75.28285	0.16476	0.17115	0.18718	0.17436	0.16417	8.78	8.24
86.54932	0.22979	0.23416	0.25510	0.23968	0.22728	7.72	7.16
97.81708	0.29459	0.29763	0.31405	0.30209	0.28762	7.11	6.08
109.08487	0.35627	0.35687	0.38099	0.36471	0.34775	6.90	6.00
120.35267	0.41845	0.41202	0.43661	0.42236	0.40467	6.18	5.27
131.61973	0.48094	0.46718	0.48969	0.47927	0.46001	5.92	4.92
142.88590	0.53659	0.52260	0.54327	0.53415	0.51401	5.54	4.59
154.15445	0.59485	0.57380	0.59451	0.58772	0.56642	5.32	4.44
165.42080	0.64892	0.62497	0.64407	0.63932	0.61722	5.06	4.25
176.68827	0.70076	0.67320	0.69278	0.68891	0.66652	4.75	4.09
187.95590	0.75040	0.71948	0.74009	0.73666	0.71320	4.65	4.05
199.22290	0.79808	0.76356	0.77674	0.77946	0.75647	4.30	3.86

 

 

Results and Discussion:

Reproducibility of Arcoxia 60 mg tablets:

Figure 2 show the results of PAMPA Dissolution for 60 mg of Arcoxia tablets. The red line represents the average value of three brand data; the blue line also represents the average value of three brand formulation. As seen from Figure 2, the difference between the red and blue lines are minimal. Drug absorption changes after the addition of concentrated FaSSIF solution at 30 minutes, the rate of absorption raised in a faster manner. Furthermore, the amount of etoricoxib absorbed at 120 minutes (Tmax) is around 0.4mg. Dissolution and absorption data specific to individual chambers are shown in Table 1 and Table 2, respectively. Table 2 shows that the % RSD of the amount of Arcoxia absorbed is around 6% RSD beyond 90 minutes, which is within an acceptable range of less than 10% RSD. `The minimal difference of about 5% RSD between the red and blue lines for absorption further supports the reliability of PAMPA Dissolution¹⁹.

 

Figure 2: PAMPA Dissolution of 60 mg Arcoxia tablets

 

Bioequivalence prediction on Etor 60mg and Arcoxia 60mg tablets:

Since one of the primary purposes of PAMPA Dissolution is to facilitate the drug formulation and particularly on BE prediction, a study was conducted between 60mg tablets of Etor (generic formulation) and 60mg of Arcoxia to demonstrate its prediction in BE study (Figure 3). The results for permeation between Etor and Arcoxia tablets (test/reference) reaches bioequivalence limits of 80.0 to 125.0% with the calculated Cmax value (slope ratio) of 99.0% and AUC value (AUC ratio) of 99.1%, respectively. This system produces a valid results in BE prediction which conform with its marketed status.

 

Figure 3: Results of PAMPA Dissolution with 60 mg of Arcoxia and 60 mg of Etor tablets

 

PAMPA Dissolution predictions of five other drugs formulations:

In addition to etoricoxib tablets formulation, five other formulations of valsartan/hydrochlorothiazide, ezetimibe, telmisartan, and amlodipine tablets were tested by PAMPA Dissolution system. Results are indicated in Table 3. The permeation data were calculated at Tmax and were compared to the literature data^{20,21,22,23,24,25}, which indicates the feasibility of PAMPA Dissolution system in BE prediction.

 

Table 3: Permeation data from PAMPA Dissolution for six drugs formulations

(1) HCT 12.5 mg			(2) Valsartan 80 mg			(3) Telmisartan 40 mg
F(mg) at 200 min	Cb (mg/ml)	Area (cm2)	F(mg) at 200 min	Cb(mg/ml)	Area (cm2)	F (mg) at 120 min	Cb (mg/ml)	Area (cm2)
0.003	0.025	14.522046	0.16	0.16	14.522046	0.17	0.08	14.522046
Pe (PAMPA)	Pe (Human)	Tmax (min)	Pe (PAMPA)	Pe (Human)	Tmax (min)	Pe (PAMPA)	Pe (Human)	Tmax (min)
6.88608E-07	4.80E-07	200	5.7384E-06	6.60E-06	200	2.03235E-05	2.70E-05	120
Ref: Pharmaceutics 2019, 11, 638			Ref：Mol. Pharmaceutics 2021, 18, 2947−2958			Ref：J of Pharmaco. and Exp. Therap. 2014
(4)Ezetimibe 10 mg			(5) Etoricoxib 60 mg			(6) Amlodipine 10 mg
F (mg) at 120 min	Cb (mg/ml)	Area (cm2)	F (mg) at 120 min	Cb (mg/ml)	Area (cm2)	F (mg) at 255 min	Cb (mg/ml)	Area (cm2)
0.04	0.02	14.522046	0.37	0.12	14.522046	0.035	0.02	14.522046
Pe (PAMPA)	Pe (Human	Tmax (min)	Pe (PAMPA)	Pe (Human)	Tmax (min)	Pe (PAMPA)	Pe (Human)	Tmax (min)
1.14768E-05	4.80E-05	200	2.9489E-05	5.23E-05	120	7.87624E-06	7.60E-06	255
Ref：Colloidal and Surface B: Biointerfaces 100, 50-61			Ref：AAPS PharmSciTech, Vol. 16, No. 1			Ref：Asian J Pharm Clin Res Vol 11,6, 204

 

Conclusion:

The PAMPA Dissolution system that measures drug dissolution and absorption simultaneously produces reproducible and reliable results. This system has a potential in making bioequivalence predictions by excluding subject variation in pilot bio studies, and for the same reason, effectively distinguishes formulation-induced drug absorption. The characteristics of PAMPA dissolution improve formulation study in finding out the effects of excipients and manufacturing procedures within a shorter time and at a relatively lower cost. Overall, PAMPA Dissolution appears to be a feasible tool for bioequivalence predictions in the field of oral drug formulation development.

 

Acknowledgments:

This work is supported by YQ Biotech Ltd., Taiwan and Isuzu Optics Ltd., Taiwan.

 

Conflicts of Interest:

All authors declare they have no conflicts of interest.

 

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Received on 04.04.2023         Modified on 01.07.2023

Accepted on 09.09.2023   ©AandV Publications All Right Reserved