Enhancing Bioequivalence Predictions with PAMPA Dissolution Using Formulations of Telmisartan

 

Joe Chou¹*, Roger Lai², Jason Chou¹, Shelly Fu², and Hsin-Ai Tung¹

¹YQ Biotech Ltd., Taiwan.

²Isuzu Optics Ltd., Taiwan.

 

ABSTRACT:

Traditionally, the pilot bioequivalence study is a generalpractice for generic drug development. However, due to its difficulty in differentiating the variation between subject and drug formulation for bioequivalence results. A new instrument called PAMPA Dissolution is proposed in this study to improve the correlation between in vitro and in vivo absorption forbioequivalence study. PAMPA Dissolution allows for the simultaneous measurement of drug dissolution (Cb) and permeation (Pe), two key parameters in oral drug absorption according to the validated equation F (drug absorbed) = Cb*Pe*Area. The use of Parallel Artificial Membrane Permeability Assay (PAMPA) eliminates the concern of subject variation. Biorelevant media further allows this device to simulate in vivo conditions closely. Brand and generic formulations of 40 mg telmisartan tablets were used in various studies to demonstrate the potential of PAMPA Dissolution in generic drug development. Bioequivalence predictions between brand and generic telmisartan from PAMPA Dissolution produced avalue（test/reference）for Cmax of 86.3% and a value for AUC of 91.4%, indicating that PAMPA Dissolution predictions conform with bioequivalence requirements. Other parameters such as stirring at different speeds and phospholipids concentrations for PAMPA are also included for optimal performance of the system. Based on these data, the PAMPA Dissolution system is reproducible, precise, and can therefore be applied in predicting bioequivalence study.

 

 

 

INTRODUCTION:

Bioequivalence study is a critical aspect of generic drug development. Traditionally, dissolution experiments are used to examine the performance of various drug formulations, and at a later stage, paired with pilot bio studies to verify generic formulations.

 

A number of issues exist to make traditional pilot bioequivalence study an inefficient system. Firstly, the large number of variables that inevitably come with human subjects makes it difficult to meet bioequivalence limits. There is no definite way to differentiate variation between subject and formulation in bioequivalence study. Another issue is that buffer solutions used in dissolution tests as required per the U.S Food and Drug Administration (FDA), which often falls short in mimicking conditions in the gastrointestinal (GI) tract such as the various enzymes from bile and pancreatic secretions, resulting in poor correlation between in vitro and in vivo data. This is especially true with active pharmaceutical ingredients (APIs) that have low aqueous solubility since solubility and permeability often influence one another. The issues that arise with traditional pilot bio study calls for the development of a more accurate and cost-effective drug research strategy.

 

In vitro to in vivo correlation (IVIVC)¹ techniques help reduce the length of drug development processes. A popular method involves the use of Caco-2 cells. However, because Caco-2 cells have to be incubated for up to twenty to thirty days before actual testing, it is not a time-effective way to improve IVIVC. The use of live Caco-2 cells also makes this method costly. An alternative that can be used to improve IVIVC for bioequivalence prediction is Parallel Artificial Membrane Permeability Assay (PAMPA), which uses a chemically-based membrane instead of live cells but has been proven to be able to accurately mimic the human small intestine ^{6, 7, 8}. PAMPA Dissolution (Figure 1) is a newly designed instrument that combines dissolution and permeation that closely simulates in vivo conditions. It measures the two necessary parameters–dissolution (Cb) and permeation (Pe)–for finding oral drug absorption via previously validated equation F (drug absorbed) = Cb*Pe*Area^2,3 and produces real-time graphs for dissolution and absorption. From these graphs, Area Under Curve (AUC) and maximum plasma concentration (Cmax) values can be calculated to predict bioequivalence^11,12,13. Unlike traditional dissolution tests, PAMPA Dissolution measures dissolution and permeation simultaneously similar to what it is like in vivo so that the solution does not undergo significant chemical changes before being transferred to permeation tests. Instead of traditional buffer solutions, PAMPA Dissolution uses biorelevant media, which contains the essential constituents in gastric, intestinal fluid and the needed pH values^4,5, further giving it more potential in bioequivalence prediction^14,15,16.

 

In this study, telmisartan tablets, a poorly water soluble drug used to treat hypertension, was selected in various tests to validate PAMPA Dissolution as a feasible way to predict bioequivalence in generic drug development^17,18,19.

 

Figure 1: PAMPA Dissolution apparatus

 

Materials and Methods:

Generic telmisartan tablets 40 mg were purchased from Fuson (China). Micardis tablets 40 mg were purchased from a local market. Reagents, including NaH₂PO₄, NaOH, NaCl, HCl, sodium lauryl sulfate (SLS), lactose, phospholipids, DSMO, and n-dodecane were purchased from First Chemicals (Taiwan). FaSSIF/FeSSIF/FaSSGF powder (Biorelevant powder) was purchased from Biorelevant (London, UK).

 

PAMPA Dissolution (YQ Biotech Co.) was used to study Micardis tablets and generic telmisartan tablets. Dissolution vessels are of USP 2 apparatus (TDTF, China). Fiber optic UV probes (at 297 nm)²⁰ are inserted to record concentrations of telmisartan in real-time. On the PAMPA side of the apparatus, there are six vessels for permeation each corresponding with the six on the dissolution side. Vessels on the PAMPA side have inner compartments filled with buffer at pH 6.5 with 1% (v/v) DSMO and divided from the outer compartments by a 0.45 μm hydrophobic membrane impregnated with 160 μL of phospholipids. Fiber optic UV probes are also inserted in the inner compartments of the vessels of the PAMPA side.

 

Vessels on the dissolution side are each filled with 400 mL of pH 1.6 fasted state simulated gastric fluid (FaSSGF) made with Biorelevant powder, NaCl, and water. After 30 minutes, 100 mL concentrated FaSSIF of pH 7.5 fluid consisting of Biorelevant powder, NaH₂PO₄, and NaOH is added in each vessel to turn the original fluid into fasted state simulatedintestine fluid (FaSSIF) of pH 6.5. Stirring on the dissolution side is set to 75 rpm while the magnetic stirrer of PAMPA is set to 200 rpm to minimize the unstirred water layer in order to mimic actual intestinal conditions as closely as possible. Dissolution solutions were delivered to PAMPA through six reciprocating pumps for permeation study.

 

Results and Discussion:

Reproducibility study for 40 mg of Micardis tablets:

Figure 2 shows the day-1 results of PAMPA dissolution study for 40 mg tablets of Micardis. On the dissolution side, the red line represents the average value of data in vessels 1 to 3; the blue line represents that of vessels 4 to 6. On the PAMPA side, the red line represents the average value of data in vessels 7 to 9; the blue line represents that of vessels 10 to 12. As seen in Figure 3, the difference between the red and blue lines is minimal. After the addition of concentrated FaSSIF at 30 minutes in order to convert FaSSGF into FaSSIF, the rate of absorption raised in a similar manner. Furthermore, examining the amount of Micardis absorbed at 120 minutes in the study reveals negligible differences, as all data points are around 0.14 mg. Dissolution and absorption data specific to individual vessels on the day-3study are shown in Table 1 and Table 2, respectively. The %RSD of Micardis dissolved is around 2%; the amount absorbed is around 8%, which is within an acceptable range of absorption (less than 10%RSD). The minimal difference of about 1% between the red and blue lines for absorption beyond 60 minutes in day-3study further supports the reliability of PAMPA Dissolution. In Table 3, the Pe value of 40 mg telmisartan is derived from day-1 data and is compared to the Pe value from other literature⁹ with Caco-2 cells. The close Pe values suggest that the PAMPA Dissolution system is reliable.

 

Table 1: Micardis 40 mg day-3 PAMPA Dissolution - dissolution data

Time(min)	Average of channel 1-3 (mg)	Average of channel 4-6 (mg)	Average of channel 1-6 (mg)	%RSD between red (channel 1-3) and blue (channel 4-6) lines	%RSD of 6 channels
11.1367	23.5	25.28	24.39	5.18	11.05
22.3545	30.45	30.55	30.5	0.25	1.53
33.5715	31.39	31.31	31.35	0.17	1.51
44.7881	37.61	37.22	37.41	0.73	1.32
56.0041	36.91	36.18	36.54	1.41	1.74
67.2209	36.78	35.78	36.28	1.96	2.49

 

Table 2: Micardis 40 mg day-3 PAMPA Dissolution - permeation data

Time(min)	Average of channel 7-9 (mg)	Average of channel 10-12 (mg)	Average of channel 7-12 (mg)	%RSD between red (channel 7-9),blue (channel 10-12) lines	%RSD of 6 channels
18.80263	0.00347	0.00483	0.00415	23.17	48.06
30.01935	0.00551	0.00696	0.00624	16.4	39.87
41.23662	0.00695	0.01062	0.00879	29.49	48.43
52.45345	0.01294	0.01646	0.0147	16.96	33.56
63.67063	0.03551	0.03619	0.03585	1.33	14.16
74.88713	0.05588	0.05609	0.05599	0.27	11.33
86.10402	0.07645	0.0761	0.07628	0.33	9.90
97.32153	0.09729	0.09694	0.09712	0.26	9.52
108.53783	0.11792	0.1187	0.11831	0.46	8.83
119.75495	0.13836	1.40966	0.13895	0.6	8.68
130.98112	0.1589	0.16178	0.16034	1.27	8.40

 

 

Figure 2: PAMPA Dissolution of day-1 for 40 mg Micardis tablets

 

Table 3: Comparison of Pe from PAMPA Dissolution on day-1 to Pe from Caco-2

F (mg) at 120 min	Cb(mg/ml)	Area (cm2)
0.15	0.08	14.522046

 

 

Pe (PAMPA) cm/s	Pe（Caco2) cm/s	Tmax(min)
1.79E-05	2.70E-05	120

 

Phospholipidsof 10% versus 3.3% in PAMPA permeation:

In PAMPA Dissolution, phospholipids are impregnated onto the inner side of PAMPA membranes. In order to determine whether a 10% (w/v) or 3.3% (w/v) concentration of phospholipids is more suitable for drug permeation, a PAMPA dissolution was conducted on 40 mg of Micardis with 10% concentrations of phospholipids. Results are shown in Figure 3. Compared to the study that used a 3.3% concentration of phospholipids (Figure 2), which showed absorption of around 0.15 mg at 120 minutes, there was less permeation of Micardis with a 10% concentration of phospholipids as the amount of Micardis absorbed is around 0.11 mg at 120 minutes (Figure 3). Table 3 confirms thata better permeation is observedwith 3.3 % phospholipids so that drugs absorbed can be measured by the system more effectively. In addition, a similar concentration of about 3% phospholipids was reported ³. Therefore, the rest of the studies were conducted using phospholipids of 3.3% concentration.

 

Figure 3: PAMPA Dissolution of 10% phospholipids on 40 mg Micardis tablets

 

Excipients effects of lactose and SLS

The PAMPA Dissolution apparatus was also an efficacious way to study the effects of excipients in new formulations. With poorly water soluble APIs like telmisartan, it becomes more important to study different formulations in a setup that closely mimics in vivo conditions and allows for dissolution and absorption to be measured for the pH-sensitive nature of these APIs. Figure 4 shows the results for 40 mg of Micardis tablets tested with lactose and SLS separately as excipients. The results for dissolution in Figure 4 reveal that lactose has a positive effect on the solubility of Micardis when added as an excipient. SLS appears to have no effects in terms of solubility. However, the data on absorption shows that the addition of SLS to Micardis allows for better permeation while lactose has little effect on permeation for Micardis. These results are reasonable in the context of solubility-permeability interplay¹⁰. It is also apparent through the data that PAMPA Dissolution is able to detect the effects of formulation excipients and therefore be used as a helpful tool for generic drug development.

 

Figure 4: Effects of lactose and SLS in 40 mg of Micardis in PAMPA Dissolution

 

Bioequivalence prediction fortablets of Fosun 40 mg and Micardis 40 mg:

Since one of the primary purposes of PAMPA Dissolution is to facilitate the drug formulation and particularly bioequivalence study, a trial was conducted on 40 mg tablets ofFosun (generic formulation) and 40 mg of Micardis to demonstrate its applicability inbioequivalencestudy (Figure 5). The study for dissolution and permeation reach bioequivalence requirements of test/reference, and with the calculated Cmax value of 86.3% and theAUC value of 91.4%, Fosun is projected to produce valid and successful results in bioequivalence requirements of 80%–125%. This prediction data also conforms with Fosun marketed status.

 

 

Figure 5: PAMPA Dissolution with 40 mg tablets of Micardis and Fosun

 

Conclusion:

Experiments of PAMPA Dissolution on separate days suggests that measuring dissolution and absorption concurrently produces reproducible and reliable results. This system has a vivid potential in making bioequivalence predictions by excluding subject variation in pilot bio study, and for the same reason, effectively distinguishes formulation-induced drug absorption differences. This characteristic of PAMPA Dissolution system would greatly help in formulation study within a short time-frame and at a relatively lower cost. Overall, PAMPA Dissolution appears to be a feasible way to produce reliable bioequivalence predictions in the field of generic drug development.

 

Acknowledgements:

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

 

Conflicts of interest:

All authors declare they have no conflicts of interest.

 

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Received on 24.10.2022         Modified on 12.01.2023

Accepted on 25.03.2023   ©AandV Publications All Right Reserved