Biorelevant dissolution profiles of a BCS Class IIA free acid and acidic salt using USP2 dissolution apparatus and their relationship with in vivo pharmacokinetics

PURPOSE

To establish and compare the dissolution behaviour of an immediate release formulation of an acid and a salt form of the same drug in biorelevant media. Both chemical forms were first tested in the three biorelevant dissolution media (FaSSGF, FaSSIF and FeSSIF) using a standardized USP Dissolution Apparatus 2 method. As a second step, to understand the behaviour of the two forms in the fed state in more depth, the two forms were subsequently tested in the three fed gastric media (early, intermediate and late).

Such studies are of importance to developers because they can help them  select the most appropriate chemical form for a drug, enabling the development of products that are better suited to patients.

DRUG BACKGROUND

The molecule's structure is a lipophilic carboxylic acid-based drug. Two chemical forms of the insoluble drug were examined: free acid and an acidic salt.

The drug has a wide therapeutic index and is used for treating inflammation and acute pain, so fast onset is desirable. It can be taken with or without food: The manufacturer recommends the product should be taken with a glass of water. However, prescribing/formulary administration guidance instructs patients to take the drug (both the acid and salt form) with or just after food to reduce stomach irritation.

To explore exposure of both forms, a crossover clinical trial was carried out on both forms in the fasted and fed condition. In fasted patients taking the drug with a glass of water, the Tmax for the salt form was x3 faster than the free acid with the Cmax of the salt was 50% higher than the free acid. The AUC for both forms was bioequivalent.

In contrast, when taken with food (reflecting the formulary guidance), the Tmax, Cmax and AUC of free acid and the acid salt form of the drug were found to be bioequivalent.
 
A therapeutic dose of 200 mg of the free acid and the acidic salt were tested in the in vitro biorelevant dissolution tests.

METHOD

Media preparation

Three biorelevant dissolution media simulating the fasted stomach (FaSSGF), fasted intestine (FaSSIF) and fed intestine (FeSSIF) were prepared first using FaSSIF/FeSSIF/FaSSGF Powder and the three corresponding biorelevant buffer concentrates according to the relevant online Media Preparation Tool.

The three biorelevant dissolution media simulating the fed stomach fluids at the early, intermediate and late-stage residency after a meal were subsequently prepared using FEDGAS gel in combination with the three corresponding biorelevant buffer concentrates according to the usage instructions supplied by biorelevant.com.

HPLC method

Preliminary assessment

Preliminary assessment studies were carried out to ensure the HPLC-UV analysis (to check the media did not interfere with drug product analysis) and dissolution set up (including filter adsorption) were satisfactory. A summary of the method used is provided in Table 1.

Table 1 HPLC summary

Parameter

Value

Mobile phase

60% Acetonitrile 40% H2O 2.65mL/L triethylamine, 1.5mL/L orthophosphoric acid

pH

3

Column

C18HS-3 75mm x 4.6mm x 3µm

Wavelength (nm)

254

Retention time (min)

2.6

Temperature (°C)

22

Analysis time (min)

3.5

Flow rate (mL/min)

1.5

Filter adsorption 

Filter adsorption was checked by passing each medium containing drug substance through the 0.45 µm polypropylene filter (n = 3) and establishing the minimum volume of each medium trequired to pre-saturate each filter.

Dissolution conditions

The conditions provided in Table 2 were taken from the existing QC method as the basis for this biorelevant dissolution study in all six media. The following dissolution parameters were used for all three media tested.

Table 2 Dissolution conditions

Parameter (unit)

Value

USP Dissolution apparatus

2

Volume (mL)

900

Rotation speed (RPM)

75

Temperature (°C)

37

Vessels for each media

6

The only deviation from the QC method was the sampling points were extended to up to 2 hours to make sure a plateau had been reached in media tested and included 10, 20, 30, 45, 60, 90 and 120 minute sampling points.

Tablets of the two chemical forms were tested in six different biorelevant media (FaSSGF, FaSSIF and FeSSIF followed by FEDGAS Early, Intermediate and Late). The dissolution runs in each of the six media were carried out for 2 hours with n=6 vessels, according to a standardized biorelevant dissolution method (available upon request) which is based around the dissolution procedure and methodology as described in USP Chapter <1092>. The average % drug released at each time point was determined by HPLC and plotted against the time when the sample was removed from the dissolution vessel.

RESULTS

Preliminary assessment

HPLC analysis

HPLC samples were diluted x10 fold prior to injection. For FaSSIF, FaSSGF and FeSSIF samples, the sample diluent used was a mixture of acetonitrile and water. For FEDGAS samples, the sample diluent was a mixture of methanol and water.

None of the media interfered with drug analysis. No significant modifications to the method were necessary.

Filter adsorption

To pre-saturate the filter it was found that at least 4 mL of each dissolution medium with drug had to be passed through the filter just prior to removing the first sample from each vessel at the 10 minute time point.

Figure 1 Dissolution in simulated fasted state gastric medium

The free acid clearly has lower and slower dissolution than the salt form in simulated gastric medium (FaSSGF). Less than 20% of the dose of the free acid dissolves after 2 hours. In contrast, almost double the amount of acid salt dissolves in FaSSGF.

Figure 2 Dissolution in simulated fasted state intestinal fluid

The free acid dissolved quite rapidly, more than 90% within 20 minutes, in the fasted state intestinal medium. However, the salt form dissolved even more rapidly and almost completely after 10 minutes. This suggests the drug in both the free form and acid salt have a high solubility in the fasted simulated intestinal fluid.

Figure 3 Dissolution in simulated fed state intestinal fluid

In the simulated fed intestinal fluid, the drug in the free and salt form had a slightly slower dissolution rate compared to the fasted state. However, after 30 minutes the drug even in the free form had fully dissolved.

Figure 4 Dissolution in simulated fed state gastric fluid (early)

Figure 5 Dissolution in simulated fed state gastric fluid (intermediate)

Figure 6 Dissolution in simulated fed state gastric fluids (late)

DISCUSSION

In vitro dissolution

Fasted stomach fluid

The relatively slow dissolution of both forms in simulated fasted stomach fluid in relation to the shorter time typically required for the quick emptying of a fasted stomach suggest that the emptying of the stomach fluid followed by the dissolution in the intestinal fluids will dictate the dissolution of the drug. The free form showed around only 1% drug dissolved in 20 minutes in FaSSGF, indicating that even under the homogenous mixing conditions of an in vitro dissolution experiment, the free acid will not significantly dissolve in stomach fluids. In contrast, the salt form appeared to disintegrate faster and dissolve to a greater extent but dissolution was still slow and less than 30% at the 20 minute time point.

Fasted and fed intestinal fluids

In sharp contrast, compared to the fasted stomach fluids, the drug in both the free acid and acid salt form dissolved to a much greater extent and was faster in both simulated fasted (FaSSIF) and fed intestinal (FeSSIF) fluids. The slightly slower dissolution in the simulated fed state intestinal medium is attributable to the slightly lower pH than the corresponding simulated fasted intestinal medium.

Fed stomach fluids

The fed state gastric media showed significant difference compared to the fasted state gastric media. All three fed state media had faster and greater dissolution rates than the fasted state medium (FaSSGF). The dissolution of the salt was equally rapid in all three media: after 20 minutes: all of the drug dose dissolved. In contrast, the free acid was markedly slower compared to the salt form and had three distinct profiles in the three fed gastric media reflecting the pH sensitivity of the free drug: The dissolution was most rapid in FEDGAS Early, moderate in FEDGAS Intermediate and slowest in FEDGAS Late. This indicates that drug dissolution is likely to be fastest just after eating a meal and will be slower as the stomach fluid re-establishes its basal fasting state.

Stable 

In all chromatograms, no new impurity peaks appeared in any of the media even after two hours of dissolution. The molecule in both forms was chemically stable in all three media under the conditions tested. 

Understanding the impact of the in vitro dissolution results

The slow and low solubility of the free drug in simulated fasted gastric medium is highly indicative that the dissolution within the fasted intestinal fluid will dictate its absorption once the drug is transferred into the small intestine. Even though there is higher solubility of the acid salt form in the simulated fasted gastric medium which suggests more drug would be dissolved when it is transferred to the small intestine, because of the relatively short residence time in the fasted stomach, drug dissolution in the small intestine fluids would still be needed for complete absorption.

It is clear from the dissolution profiles that the salt form dissolves in simulated fasted state and fed state intestinal media much quicker than the free acid form. This quicker in vitro dissolution profile is likely to reflect the in vivo performance of the salt form in the fasted state, which results in a shorter Tmax and higher Cmax indicating a quicker absorption of the acid salt than the free acid in the fasted state.  

It is not just about dissolution

Although the dissolution of the drug was quicker in all three simulated fed stomach media for salt form compared to the simulated fasted stomach in vitro, this is not directly reflected in vivo in terms of the Tmax and Cmax. This is because food in the fed stomach greatly delays the emptying of the contents (along with drug) into the small intestine and results in the loss of the benefit of enhanced relatively rapid dissolution of the salt. This emphasises that other physiological factors of the gastrointestinal tract, not just dissolution have to be taken into account when understanding your results.

Impact for selection of salt

Selection of the appropriate chemical and physical form can be a complicated, multi-faceted decision involving many commercial (for example IP cost) as well as technical factors (such as stability, hygroscopicity, developability and toxicity). However, these simple and straightforward dissolution experiments can be carried out cost effectively and at almost any stage of development. This grants commercial and technical teams rapid insight into the potential benefits that a different chemical form, for example a salt, may yield compared to a free form. These invaluable insights into dissolution in simulated fluids in turn assist with how this can impact on the magnitude of improvement a salt form is likely to have on drug dissolution and subsequent absorption. The data can be used to support the development of alternative chemical forms of a molecule. Even without modelling, biorelevant data generated from simple dissolution experiments coupled with a basic understanding of biopharmaceutics can help you understand how the solubility and dissolution can potentially impact on in vivo pharmacokinetics. In this case, the in vitro fasted state dissolution results reflect the in vivo pharmacokinetic benefits of a salt form can yield when the salt form is administered in fasted state. However, if the drug is administered in the fed state, the benefit of faster in vivo dissolution is likely to be negated because the slow emptying of the fed stomach will retard the onset of the Tmax and lower the Cmax.

CONCLUSION

Dissolution tests in biorelevant media can help developers examine the potential advantages of one form over another in the fasted and fed state using simple in vitro experiments. The standard dissolution conditions remove variability due to differences in human physiology.

The shorter in vivo Tmax and higher Cmax of the salt form compared to the free acid in the fasted state (without a meal) are reflected by the more rapid in vitro dissolution characteristics in fasted gastric and fasted intestinal fluids.

However, as prescribers generally recommend this drug should be administered with food, these potential therapeutic benefits of quicker Tmax and higher Cmax in the fasted state do not translate in vivo in the fed state. If administered in the fed state, even though the dissolution of the salt may be more extensive in the fed stomach than the fasted stomach, this does not translate into a faster Tmax of the salt because the fed stomach delays the entry of the contents into the small intestine which in this case will delay the onset of the Tmax and reduce Cmax.