Exploring the transmucosal permeability of cyclobenzaprine: A comparative preformulation by standardized and controlled ex vivo and in vitro permeation studies
Graphical abstract
Introduction
Advantages such as the ability to achieve higher bioavailability, reduced dosages, and improved patient adherence render the oral mucosa a beneficial alternative to the oral and intravascular routes of drug administration. However, transmucosal diffusion primarily depends on the physicochemical properties of active pharmaceutical ingredients (APIs) and the given environmental conditions at the site of administration. Since the environment of a drug can be substantially affected by the drug formulation, which subsequently affects drug permeability, knowledge of influencing factors and additives are essential to support and customize galenical development. The latter aspect can be of particular importance when developing dosage forms for alternative sites of application, such as the oral cavity. Therefore, permeation studies are considered helpful for drug preformulation and oromucosal drug delivery (Kottke et al., 2020, Wang et al., 2020).
However, current permeation studies often have limited predictivity stemming from insufficient adaptation to clinical conditions, lack of monitoring, and inhomogeneous designs (Kolli and Pather, 2015, Patel et al., 2012). These mandatory aspects for decisive insights into drug permeability were considered in the standardization of an innovative ex vivo permeation process incorporating the Kerski diffusion cell (Kerski et al., 2020) coupled to fully automated sampling and sample preparation with validated drug quantification via LC-MS/MS (Majid et al., 2020). The Kerski diffusion cell is a U-shaped diffusion cell consisting of an acceptor, isofill and donor chamber with horizontal orientation of the inserted membrane. Unlike the common Franz cell, this model allows for automation of the sampling process, standardized throughput, and high-volume exchange on the acceptor side to maintain sink conditions. Despite the exchange of 45% of the acceptor volume per sampling, the isofill chamber prevents air bubbles from collecting under the membrane, which remains continuously wetted with medium, thus reducing the risk of a negative influence on permeation. Due to the design of the Kerski cell, an additional hydrodynamic pressure gradient occurs resulting from the volume difference between donor and acceptor volumes. An alteration of the hydrodynamic pressure gradient is immediately compensated by refilling the acceptor chamber to initial condition after sampling. Additionally, this allowed the adaption of the experimental study design on clinical conditions (i.e., first sampling point after ≤ 5 min, human saliva collection, therapeutic doses, sink conditions). Furthermore, routine monitoring via innovative tissue integrity and viability tests were integrated into a comprehensive analytical control system. Nevertheless, due to the need for time-effective preparation, the reduction of biological variability, and high reliability, in vitro studies using artificial membranes based on phospholipids offer a valid alternative to mucosal tissue-based ex vivo studies (Brandl and Bauer-Brandl, 2019). However, artificial membranes are generally adapted to simulate the passive transcellular diffusion of the intestine; thus, their broad applicability for oromucosal permeability is presently restricted to certain drugs. Therefore, the beneficial and suitable applications in drug development must be explored for both models, starting at the preformulation stage.
Cyclobenzaprine hydrochloride (CBP-HCl) (Fig. 1) is a centrally-acting skeletal muscle relaxant used as an oral formulation for the treatment of pain-associated muscle spasms (Chou et al., 2004). Recent studies concluded that cyclobenzaprine offers therapeutic potential for the treatment of posttraumatic stress disorder (PTSD), which is characterized by hyperarousal symptoms such as sleep disturbances (Bestha et al., 2018, Sartori and Singewald, 2019). The use of a sublingual CBC-HCl tablet appears beneficial due to its indication-appropriate rapid onset and discussed safety improvements such as fewer daytime side effects (Davidson, 2015). This has been correlated to decreased formation of the active metabolite norcyclobenzaprine by avoiding first-pass metabolism (Daugherty et al., 2016). However, to the best of our knowledge, no work has yet been published that provides a detailed investigation of the transmucosal permeability of cyclobenzaprine via either ex vivo or in vitro studies.
Hence, this work aimed to comprehensively characterize the permeability of cyclobenzaprine in a preformulation study to obtain valuable insights for targeted, lean, and time-efficient formulation development. This work included an investigation of the transmucosal diffusion pathway and affecting factors such as the type and quantity of excipients as well as environmental/experimental conditions (i.e., pH, membrane thickness, storage condition, and dose). Moreover, a direct comparison between tissue-based ex vivo studies versus artificial in vitro studies was conducted to assess their respective predictivity for preformulation.
Section snippets
Chemicals and material
Cyclobenzaprine hydrochloride (≥98%) (Hetero drugs Ltd, Hyderabad, India), melatonin (100%) and metronidazole (101%) (Caesar & Loretz GmbH, Hilden, Germany) were used as active pharmaceutical ingredients (API). Caffeine anhydride (99.9%) was supplied from Siegfried AG (Zofingen, Switzerland). Cyclobenzaprine-d3 (98%, internal standard), formic acid (≥98%, p.a.), alizarin yellow (dye content 50%), fluorescein isothiocyanate-dextran (average molecular weight 20000), blue dextran 20 (average
Effects of pH, phosphate salt type, and quantity on permeability
As an essential part of the preformulation study, the effect of pH alteration on the transmucosal permeability of cyclobenzaprine hydrochloride was investigated and evaluated using two approaches (ex vivo/in vitro correlation). Within this study, six different solutions of cyclobenzaprine—representing various phosphate compositions and their subsequent pH values—were used. The changes in pH due to different phosphate salts (from pH 5.5 to 8.9) influenced the protolysis equilibrium of the active
Conclusion
The oromucosal permeability of cyclobenzaprine was comprehensively investigated and characterized for the first time in terms of impacting factors and various study conditions using esophageal mucosa as surrogate membrane. The standardized and controlled ex vivo permeation process proved to be suitable for predictive evaluations during preformulation studies and was purposefully superior to the cell-free artificial in vitro approach. Thus, successful permeation enhancement was achieved by
CRediT authorship contribution statement
Haidara Majid: Conceptualization, Methodology, Investigation, Formal analysis, Writing - original draft, Visualization. Andreas Puzik: Conceptualization, Writing - review & editing. Tanja Maier: Conceptualization, Writing - review & editing. Daniel Eberhard: Investigation, Writing - review & editing, Visualization. Anke Bartel: Methodology, Investigation. Hans-Christian Mueller: Conceptualization, Writing - review & editing. Bjoern B. Burckhardt: Conceptualization, Methodology, Investigation,
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
The authors thank Raphaela Merk for the knowledge exchange and providing valuable advice concerning experimental design as well as Dina Kottke for her scientific support in terms of permeation experiments.
Funding
Hexal AG, Industriestrasse 25, 83607 Holzkirchen, Germany.
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