In vivo comparison of enteric capsule shells composed of different HPMCAS grades after simultaneous intake by dual MRI labeling and pharmacokinetics

Abstract

Different grades of hypromellose acetate succinate (HPMCAS) differ in their pH dependent dissolution behavior, which is of key relevance when used for enteric coating or gastroresistant capsule shells. By use of specific grades it could be possible to tailor intestinal release in terms of timing and localization. A crossover design might be the gold standard for comparison of two dosage forms but intraindividual variability in gastrointestinal transit conditions still limit robust comparability. In the present study, two prototype capsule shells made of HPMCAS grade L and M, were compared with reference Capsugel® Enprotect® capsules made of grade H.

For avoidance of intraindividual variability affecting the comparison of two dosage forms with a limited group of subjects, an approach with an internal standard should be used. Thus, reference capsules and the two types of test capsules were administered by healthy young volunteers in fasted state in a 2-arm crossover study. Test capsule and reference capsule were always administered together allowing for an internal reference. To enable differentiation in magnetic resonance imaging (MRI), the reference and test capsules contained different amounts of black iron oxide. The test capsules additionally contained manganese gluconate. The capsules also contained different types of ¹³C-labeled caffeine to act as distinguishable pharmacokinetic markers.

Different amounts of iron oxide led to distinguishable artifacts in TRUFI sequences. Manganese gluconate led to a bright cloud in VIBE sequences when test capsule disintegrated, allowing for very sensitive detection of opening and interesting visualization of small intestinal spreading after release. Reference HPMCAS grade H capsules mainly disintegrated in ileum, while test capsules disintegrated in more proximal regions of small intestine or in terms of HPMCAS grade L even in stomach already. Utilization of an internal standard with dual labeling by dosing test and reference simultaneously was a relevant improvement in study design. HPMCAS grade M is a robust alternative to HPMCAS grade H if faster and more proximal disintegration is desirable.

Introduction

Ready-to-fill enteric hard capsules gained more and more attention during the last years. Several gastro-resistant hard capsule products are available utilizing different enteric polymers such as hypromellose (HPMC) in combination with methacrylic acid (Eudracap® enteric), HPMC in combination with hypromellose phthalate (AR-CAPS® Acid Resistant capsules) and HPMC combined with hypromellose acetate succinate (Capsugel® Enprotect® capsules) (Rump et al., 2022; Grimm et al., 2023). Hypromellose acetate succinate (HPMCAS) is available in specific grades referring to high (H), medium (M) and low (L) substitution grade of succinate groups on the HPMC chain. These different substitution grades of acidic groups lead to differences in pH dependent solubility. According to literature, HPMCAS grade H dissolves at pH ≥ 6.8, grade M at pH ≥ 6.0 and grade L at pH ≥ 5.5 (Sarabu et al., 2020). Keeping in mind the physiological pH profiles in small intestines, it should be possible to tailor the release of an enteric dosage form containing HPMCAS at different regions of the small intestine. This way, release site in the intestines could be adjusted to the needs of the respective API using the same enteric capsule platform, by variegating the grade of HPMCAS.

Considering the intraindividual variability in gastrointestinal transit of such monolithic dosage forms, it could be beneficial to compare such tailored vehicles after simultaneous administration, using an imaging technique to assure site-specific release and discrimination of co-administered dosage forms. Although, data on intraindividual variability of dosage form transit are scarce and effects remain speculative, it is very likely that intraindividual variability could interfere the comparability in a typical crossover design. Therefore, it would be statistically more robust, if more than one system could be investigated in parallel. Thus, when comparing the in vivo performance of two dosage forms with each other, an internal standard is desirable. Since in vivo evaluations of dosage forms often require costly techniques, the simultaneous administration can further increase efficiency of studies.

The in vivo behavior of dosage forms can be evaluated by several methods including imaging and other diagnostic tools like pharmacokinetic marker substances. The investigation of disintegration time points or release from dosage forms using pharmacokinetic labels can be performed by blood sampling, but salivary sampling might be beneficial by being noninvasive, mostly inexpensive and being able to safely allow for higher temporal resolution in repetitive measurements. For this salivary tracer technique mainly paracetamol (acetaminophen) or caffeine are reported as labels (Senekowitsch et al., 2022).

Caffeine can be used in various isotope labeled versions and is already known to allow for simultaneous and distinguishable evaluation of beginning release from simultaneously taken oral dosage forms, since chemically they behave the same but remaining distinguishable in LC-MS/MS (Mark et al., 2024; Schiller et al., 2005). Although pharmacokinetic labels offer distinguishability of release and pharmacokinetics of simultaneously administered dosage forms, they do not allow for information about localization of disintegration and release, which is of main interest in terms of enteric or other site specific dosage forms. Information about localization in GI tract needs an imaging technique to evaluate transit and disintegration, opening or release. To increase robustness and sensitivity the imaging can also be combined with pharmacokinetic labels.

One of the methods that has been frequently used in the past to track dosage forms and their disintegration is gamma-scintigraphy (Großmann et al., 2025). A variety of tracers can be used for the scintigraphic labeling of pharmaceutical dosage forms. These include compounds of 99ᵐTc, 153Sm, 171Er, and 111In (Steingoetter et al., 2003; Akbar et al., 2024b). Due to the different radiation energies of these elements, they enable the simultaneous tracking of multiple dosage forms or the concurrent tracking of liquids and solid dosage forms. But availability and stability of radioactive labels, necessary imaging equipment, risks associated with ionizing radiation and regulatory hurdles limit the applicability of scintigraphy for dosage form tracking. Moreover, an anatomic reference image for orientation in GI tract is missing in scintigraphy. This is crucial for robust investigation of the disintegration site of an enteric dosage form due to high morphological variability which cannot be estimated from outside (Grimm et al., 2024).

Another option for dosage form evaluation in vivo is Magnetic Resonance Imaging (MRI) which offers good soft tissue contrast and anatomical orientation in high resolution, various contrast options, while avoiding ionizing radiation. Thus, MRI has been frequently utilized for dosage form evaluation in the past (Senekowitsch et al., 2022; Weitschies and Wilson, 2011; Akbar et al., 2024a). Nonetheless, studies and methodologies for the tracking of two or more simultaneously administered distinguishable pharmaceutical dosage forms in parallel are scarce. Chaddock et al. and Mark et al. reported tracking of several non-disintegrating capsules in parallel, but these have been no pharmaceutical dosage forms aimed to deliver their filling. These capsules were made out of an insoluble polymer and filled with gadolinium contrast agent to investigate object transit for evaluation of motility patterns (Chaddock et al., 2014; Mark et al., 2024). Moreover the method seemed not to allow for distinguishability of the individual capsules.

There are only a few other studies which investigated the transit of more than one distinguishable and simultaneously administered dosage form using MRI. In the study by Schiller et al. several capsules which were administered simultaneously in a specific sequence could be distinguished in T2 weighted MRI by a unique pattern and number of watery gel spheres inside a solid triglyceride matrix (Schiller et al., 2005). But intravenous application of butylscopolamine hydrobromide was necessary to sufficiently paralyze intestinal motility for avoidance of movement artifacts. Such small internal structures inside the capsules would not have been distinguishable during natural movement and another method would be needed if undisturbed transit and dosage form performance should be investigated.

A combination of common tracking labels e.g. black iron oxide labeled powder for T2* sensitive imaging and dried pineapple pieces for T1 weighted imaging (Grimm et al., 2019) in two dosage forms should allow for distinguishable evaluation after simultaneous intake. In a study by Großmann et al. an oil filled capsule and a tablet labeled with black iron oxide were successfully investigated in parallel (Großmann et al., 2025). Nonetheless the method required a 3 T MRI scanner with sufficient spatial resolution and capsules were filled with a liquid filling in a capsule-in-capsule design. This limits applicability for other more representative dosage forms and combination with other necessary excipients. Dried pineapple pieces could also be utilized as contrasting filling, but large pieces are necessary, leaving not much space for other fillings of interest and their residual moisture as well as their batch-dependent signal also limit their applicability (Grimm et al., 2019).

Gadolinium based contrast agents would theoretically work well and have been used in the past (Steingoetter et al., 2003), but for representative dosage forms which should release their payload, these substances seem inappropriate nowadays. Gadolinium should only be used after thorough risk-benefit assessment and as a drug substance it also provokes ethical and regulatory issues.

Recently the approved food additive manganese gluconate gained more attention as MRI contrast agent for evaluation of dosage form performance (Akbar et al., 2024b; Grimm et al., 2024). It is a safe excipient and can be included in powder formulations facilitating representative solid oral dosage forms and combination with other excipients. Manganese gluconate acts as a positive T1-weighted MRI contrast agent. Upon dissolution in aqueous media, Mn2+ ions shorten the longitudinal relaxation time (T1) of surrounding water protons. As these water protons and their preceding spins are mainly responsible for MRI signal in intestinal lumen, this results in increased signal intensity and a bright contrast enhancement on T1-weighted images in a concentration dependent manner. For this mechanism to be effective, manganese gluconate must dissolve and release Mn2+ into an aqueous environment, so that the unpaired electron spins of these ions can interact with the excited spins of the water protons in the hydration shell of the respective ions. The local water availability therefore strongly influences the detectability of the contrast effect. Thus, additional labels like black iron oxide are still needed for robust detectability of the intact dosage form in the intestines before opening due to insufficient amount of water protons inside the dry dosage form. Additionally, a salivary tracer like caffeine can be added to the mixture allowing for simultaneous pharmacokinetic evaluation of different dosage forms in parallel if isotope labeled versions are used (Akbar et al., 2024b; Grimm et al., 2024).

For the aim to compare two simultaneously administered dosage forms, the combination of black iron oxide, manganese gluconate and different caffeine variants would therefore allow for internal standard and reduce the necessary amount of study arms.

Thus, this study had two objectives. First, an in vivo comparison of enteric capsule shells made out of HPMCAS grade H as reference, and HPMCAS grade M and HPMCAS grade L test capsules should be made. Moreover, a novel dual labeling together with pharmacokinetic marker should be established, to allow for simultaneous and distinguishable evaluation of at least two dosage forms by imaging and pharmacokinetics in parallel.

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Materials

The administered capsules were filled comparable to previous studies with caffeine, iron oxide, superdisintegrant and mannitol based filling powder. But in this study, the amounts of black iron oxide were varied and a novel marker was included to allow simultaneous administration and subsequent evaluation of transit and disintegration of test and reference capsules in parallel. Details on used materials and excipients can be found in Table 1. Powder mixtures were prepared by use of a Turbula shaker mixer T2F (Willy A. Bachhofen AG, Germany) and filled manually and individually on a precision balance Sartorius AX623 (Sartorius Lab Instruments GmbH & Co. KG, Germany).

Table 1. Materials and excipients used for study capsules.

The resulting reference Capsugel® Enprotect® capsules (HPMCAS grade H) of size 0 were filled with a mixture of 17.9 mg black iron oxide, 25 mg ¹³C₃ labeled caffeine, croscarmellose and standard filling powder of mannitol and colloidal silicon dioxide. Test capsules (HPMCAS grades L or M) of size 0 were filled with 2.2 mg black iron oxide, 25 mg ¹³C₁ labeled caffeine, 125 mg manganese gluconate, croscarmellose and standard filling powder of mannitol and colloidal silicon dioxide as well. Capsule weight amounted to 390 ± 2 mg for HPMCAS grade H reference capsules, 435 ± 1 mg for HPMCAS grade L test capsules and 436 ± 1 mg for HPMCAS grade M test capsules. Test capsules have been slightly heavier due to higher bulk density of included manganese gluconate.

Simultaneously administered test and reference capsule have been distinguishable due to different amounts of iron oxide, which led to differently sized artifacts in TRUFI sequences (True Fast Imaging with Steady-State Precession). Manganese gluconate led to a bright “cloud” in T1 weighted VIBE sequences (Volumetric Interpolated Breath-hold Examination) when test capsule disintegrated. This way, further differentiation of opening was possible, since only test capsules would produce this specific signal in T1 weighted sequences. Details on imaging aspects can be found in the subsequent chapters Experimental design and Magnetic Resonance Imaging sequences. Furthermore, the different isotope labeled caffeine species offered distinguishable and sensitive evaluation of release.

Michael Grimm, Ruben Lau, Fiona Mankertz, Robin Bülow, Felix Morof, Delphine Nombret, Camille Dumont, Vincent Jannin, Mladen Vassilev Tzvetkov, Werner Weitschies, In vivo comparison of enteric capsule shells composed of different HPMCAS grades after simultaneous intake by dual MRI labeling and pharmacokinetics, International Journal of Pharmaceutics: X, Volume 12, 2026, 100666, ISSN 2590-1567, https://doi.org/10.1016/j.ijpx.2026.100666.

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