An epileptic seizure strikes without warning, making rapid intervention essential[1]. Every second counts, yet not every patient has immediate access to an injection or another rapid drug delivery method. Imagine a simple, safe, and fast alternative – a thin film that dissolves in the mouth and releases a drug with anti-seizure effects. Swipp brought this concept from idea to clinical trials, and Quinta-Analytica played a key role in assessing the bioavailability of this product and contributed to its future market approval.
Building on a successful previous collaboration with Quinta-Analytica, Dr. Bengt Westrin, co-founder of Swipp, approached Quinta-Analytica with a new project: to help Swipp with a novel midazolam product, the first of its kind and first midazolam buccal film ever to enter clinical phase. This product was aimed to offer lower manufacturing costs, longer shelf life, and fewer steps in administration. However, the bioavailability would be the key feature and based on Dr. Westrin’s previous good experiences of Quinta-Analytica, Swipp wanted Quinta-Analytica to do the job.
Midazolam is a widely used benzodiazepine with indications ranging from acute epileptic seizures to moderate sedation and pre-sedation[2]. Due to its rapid onset of action, it plays a crucial role as medicine for treating seizures. In addition, midazolam syrup is commonly administered to children before dental procedures to prevent pain and, thanks to its short-term memory-blocking effect, avoid the development of a fear of doctors — an essential aspect of pediatric medicine[3].
However, developing an active substance into a new dosage form requires a precise characterization of bioavailability and pharmacokinetic properties, as these may vary depending on formulation and mode of administration.
Accurate determination of midazolam and its metabolites is essential not only for regulatory approval but also for optimizing dosing and minimizing adverse effects. Innovative dosage forms, such as buccal films, can offer more reliable and faster absorption, yet they introduce new analytical and clinical challenges that must be addressed.
GMP Support Before the Clinical Study
Before the clinical trials could begin, Swipp commissioned Quinta-Analytica to conduct a Good Manufacturing Practice (GMP) audit of the European manufacturing site (CMO) producing the oral films for this study. The audit focused not only on verifying compliance with GMP standards, but also on assessing whether additional responsibilities could be delegated—since the CMO was not authorized to perform final packaging and blinding of the investigational medicinal product (IMP).
As a result, Quinta-Analytica assumed responsibility for secondary packaging, blinding, and final Qualified Person (QP) release, ensuring that the IMP was fully compliant and ready for clinical use.
With these tasks completed, the investigational product was ready for administration in the first clinical trial. Or was it?
The Clinical Study: Overcoming Challenges in Real-World Testing
The goal of the clinical program was to compare the bioavailability of the buccal film with established reference products and to evaluate pharmacokinetic parameters under different conditions.
Securing the appropriate reference products, however, proved more complex than initially expected. Clinical Managers at Quinta-Analytica played a critical role in sourcing the necessary comparators from both EU and US markets. In parallel, they collaborated closely with key regulatory authorities, including the Ministry of Health of the Czech Republic and the State Institute for Drug Control (SÚKL), to obtain all required study approvals. Because midazolam is a strictly controlled substance, managing the import and export permits involved extensive coordination with agencies in US, France and the Czech Republic.
Once these regulatory and logistical challenges were resolved, clinical study could begin. It ultimately consisted of three separate trials, with a total of 60 healthy male volunteers receiving single doses of 5 or 10 mg of the investigational film in crossover designs ranging from two to four periods.
A key challenge throughout the studies was ensuring consistent conditions for drug absorption in the studies. Since buccal films rely on mucosal uptake, factors like salivary flow, mucosal condition, and study subject compliance could significantly influence bioavailability. To mitigate variability in these conditions, strict oral cavity control was introduced, including a standardized pre-administration period to stabilize the environment before dosing and including clear written instructions to study nurses and study subjects.
Given midazolam’s sedative properties, extensive safety precautions were required. Emergency airway equipment and staff trained in intubation were always available. Following administration, subjects remained in a supine position under continuous observation, with frequent monitoring of vital signs and pharmacodynamic parameters for up to eight hours post-dose. To maintain safety and comfort, additional support measures were implemented, including the provision of urinals and staff assistance for restroom visits up to 8 hours after administration. These operational demands required detailed planning, robust documentation, and increased clinical staffing throughout the study.
To further assess absorption, in the initial study, saliva collection was part of the procedure, with excess saliva being expelled at specific time intervals with a precision of 30 seconds. The saliva samples were then analyzed for coloration and the presence of residual active ingredients. This helped determine whether the drug was effectively retained and absorbed through the buccal mucosa or if any loss occurred due to excessive saliva production.
Another technical challenge involved the administration of half doses. Although the film was manufactured with uniform distribution of the active ingredient, physical splitting a film into two equal parts required high precision. Quinta-Analytica provided specialized training to clinical staff and implemented strict size control protocols, ensuring that deviations remained within ±5%. These steps were essential for preserving dose accuracy and generating reliable pharmacokinetic data for the bioanalytical department.
Bioanalytical Determination: Precision in Midazolam and Metabolite Measurement
Following the completion of each of the three clinical studies, pharmacokinetic samples were analysed in Quinta-Analytica’s bioanalytical laboratories. The quantification of MDZ and its active metabolite, 1-hydroxy midazolam (1-OH MDZ), was performed using a highly sensitive LC/MS-triple quadrupole method.
To ensure accurate measurement, analytes were extracted from plasma samples using a protein precipitation method with a solution containing internal standards. The supernatant was then injected into an HPLC system utilizing a two-dimensional chromatography setup. This system incorporated two HPLC columns with a switching valve: the first, shorter column separated major co-eluting substances, while the second, longer analytical column finalized the separation of the analytes from interfering components. This approach significantly improved the specificity and robustness of the method.
During method development, it was essential to account for midazolam’s metabolic pathways. In addition to the primary metabolite 1-hydroxy midazolam (1-OH MDZ), several glucuronide conjugates of midazolam and hydroxy midazolam are also formed. These glucuronides have a weak bond to their parent compounds and may undergo back conversion to MDZ or 1-OH MDZ during sample processing or storage, potentially leading to overestimated concentrations. To mitigate this risk, several precautionary steps were implemented: cooling of all samples to 0–4 °C throughout preparation and autosampler storage, optimizing the solvent composition and plasma-to-precipitant ratio, and using robust chromatographic separation to prevent thermal decomposition in the ion source of the mass spectrometer. The method’s selectivity and separation efficiency were confirmed by monitoring the mass-to-charge (m/z) transitions for MDZ, 1-OH MDZ, and their respective glucuronides.
However, because commercial standards of these glucuronide metabolites were not available—and custom synthesis would have been prohibitively expensive—direct validation of back conversion prevention was not possible using reference materials. To address this, the effectiveness of the applied precautions was further verified through stability testing on real study samples. These samples were analysed under various conditions, including processing in a water-ice bath versus room temperature and storage in a cooled autosampler versus laboratory temperature. The results demonstrated that no back conversion occurred, confirming that the measured concentrations of MDZ and 1-OH MDZ were stable and could be reliably used for bioequivalence evaluation.
Tomáš Hosnedl, Deputy Head of the Bioanalytical Department, commented on the significance of these precautions:
“Ensuring the stability of MDZ and its metabolites was a major concern. Given the potential for back conversion, we implemented rigorous validation steps, including real sample testing under controlled conditions. The data confirmed that our approach effectively preserved analyte integrity, allowing us to generate precise and reproducible pharmacokinetic results.”
With these validated methodologies in place, Quinta-Analytica successfully delivered high-quality bioanalytical data, supporting the overall assessment of bioavailability and bioequivalence between the investigational and reference midazolam products.
Innovation in Bioanalysis and Pharmacokinetics
After overcoming all challenges, results from the clinical study and bioanalytical analysis eventually became available and revealed a very beneficial bioavailability and pharmacodynamic response for the buccal film compared with reference products. In addition, groundbreaking insights were gained about how to achieve consistent conditions when studying absorption from buccal films in general and benzodiazepines in particular.
Through close collaboration between Swipp and Quinta-Analytica, numerous technical, regulatory, and logistical challenges were thus successfully overcome, paving the way for future advancements in this field, which will benefit patients and their caretakers.
Quinta-Analytica’s role extended beyond analytical work, encompassing study design, regulatory support, and the resolution of complex administrative hurdles. The successful execution of this project demonstrated that innovation in pharmaceutical research is not just about technology—it is also about effective teamwork, scientific expertise, good communication and strategic problem-solving.
As the development of this midazolam buccal film progresses, we look forward to the day when it reaches the market and will improve patient outcomes by offering a faster, more reliable, and more patient-friendly treatment option for acute seizures and moderate sedation needs. The most recent news from our client Swipp is that outlicensing negotiations are now ongoing with a European pharma company.
Conscio group is a scientific service provider committed to improving the safety and quality of pharmaceuticals, food and our environment. Beyond clinical and analytical expertise, the Group offers GMP manufacturing solutions for investigational medicinal products (IMPs), as well as formulation development and technology transfer across multiple dosage forms. Learn more at www.conscio.group
