Quantification of Plasmodium falciparum HRP-2 as an alternative method to [3H]hypoxanthine incorporation to measure the parasite reduction ratio in vitro.

In the absence of a highly efficacious vaccine, chemotherapy remains the cornerstone to control malaria morbidity and mortality. The threat of the emergence of parasites resistant to artemisinin-based combination therapies highlights the need for new antimalarial drugs ideally with superior properties. The killing rate reflects the speed of action of antimalarial drugs, which can be measured in vitro through the parasite reduction ratio (PRR) assay to shortlist interesting candidates. As a standard, the in vitro PRR assay is performed by measuring [3H]hypoxanthine incorporation of Plasmodium falciparum. This methodology is restricted to specialised laboratories owing to the handling of radioactive material. In this work, we describe a sandwich enzyme-linked immunosorbent assay to detect P. falciparum histidine-rich protein 2 (HRP-2) as an alternative methodology to assess the PRR. We first validated the methodology with established antimalarial drugs (artesunate, chloroquine, pyrimethamine and atovaquone) by comparing our results with previous results of the [3H]hypoxanthine incorporation readout provided by an expert laboratory, and subsequently assessed the speed of action of four new antimalarial candidates (compound 22, chlorotonil A, boromycin and ivermectin). The HRP-2 PRR assay achieved comparable results to the [3H]hypoxanthine incorporation readout in terms of parasite growth rate over time, lag phase and parasite clearance time. In addition, parasite growth following drug exposure was quantified after 7, 14, 21 and 28 days of recovery time. In conclusion, the PRR assay based on HRP-2 is similar to [3H]hypoxanthine in determining a drug's parasite killing rate and can be widely used in all research laboratories.

Dysregulation of the kallikrein-kinin system in bronchoalveolar lavage fluid of patients with severe COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to the angiotensin-converting enzyme 2 (ACE2) receptor, a critical component of the kallikrein-kinin system. Its dysregulation may lead to increased vascular permeability and release of inflammatory chemokines. Interactions between the kallikrein-kinin and the coagulation system might further contribute to thromboembolic complications in COVID-19. METHODS: In this observational study, we measured plasma and tissue kallikrein hydrolytic activity, levels of kinin peptides, and myeloperoxidase (MPO)-DNA complexes as a biomarker for neutrophil extracellular traps (NETs), in bronchoalveolar lavage (BAL) fluid from patients with and without COVID-19. FINDINGS: In BAL fluid from patients with severe COVID-19 (n = 21, of which 19 were mechanically ventilated), we observed higher tissue kallikrein activity (18·2 pM [1·2-1535·0], median [range], n = 9 vs 3·8 [0·0-22·0], n = 11; p = 0·030), higher levels of the kinin peptide bradykinin-(1-5) (89·6 [0·0-2425·0], n = 21 vs 0·0 [0·0-374·0], n = 19, p = 0·001), and higher levels of MPO-DNA complexes (699·0 ng/mL [66·0-142621·0], n = 21 vs 70·5 [9·9-960·0], n = 19, p < 0·001) compared to patients without COVID-19. INTERPRETATION: Our observations support the hypothesis that dysregulation of the kallikrein-kinin system might occur in mechanically ventilated patients with severe pulmonary disease, which might help to explain the clinical presentation of patients with severe COVID-19 developing pulmonary oedema and thromboembolic complications. Therefore, targeting the kallikrein-kinin system should be further explored as a potential treatment option for patients with severe COVID-19. FUNDING: Research Foundation-Flanders (G0G4720N, 1843418N), KU Leuven COVID research fund.

Total Synthesis of the Antimycobacterial Natural Product Chlorflavonin and Analogs via a Late-Stage Ruthenium(II)-Catalyzed ortho-C(sp2)-H-Hydroxylation.

The continuous, worldwide spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) endanger the World Health Organization's (WHO) goal to end the global TB pandemic by the year 2035. During the past 50 years, very few new drugs have been approved by medical agencies to treat drug-resistant TB. Therefore, the development of novel antimycobacterial drug candidates to combat the threat of drug-resistant TB is urgent. In this work, we developed and optimized a total synthesis of the antimycobacterial natural flavonoid chlorflavonin by selective ruthenium(II)-catalyzed ortho-C(sp2)-H-hydroxylation of a substituted 3'-methoxyflavonoid skeleton. We extended our methodology to synthesize a small compound library of 14 structural analogs. The new analogs were tested for their antimycobacterial in vitro activity against Mycobacterium tuberculosis (Mtb) and their cytotoxicity against various human cell lines. The most promising new analog bromflavonin exhibited improved antimycobacterial in vitro activity against the virulent H37Rv strain of Mtb (Minimal Inhibitory Concentrations (MIC90) = 0.78 µm). In addition, we determined the chemical and metabolic stability as well as the pKa values of chlorflavonin and bromflavonin. Furthermore, we established a quantitative structure-activity relationship model using a thermodynamic integration approach. Our computations may be used for suggesting further structural changes to develop improved derivatives.

Enalapril and Enalaprilat Pharmacokinetics in Children with Heart Failure Due to Dilated Cardiomyopathy and Congestive Heart Failure after Administration of an Orodispersible Enalapril Minitablet (LENA-Studies).

Angiotensin-converting enzyme inhibitors (ACEI), such as enalapril, are a cornerstone of treatment for pediatric heart failure which is still used off-label. Using a novel age-appropriate formulation of enalapril orodispersible minitablets (ODMTs), phase II/III open-label, multicenter pharmacokinetic (PK) bridging studies were performed in pediatric patients with heart failure due to dilated cardiomyopathy (DCM) and congenital heart disease (CHD) in five participating European countries. Children were treated for 8 weeks with ODMTs according to an age-appropriate dosing schedule. The primary objective was to describe PK parameters (area under the curve (AUC), maximal concentration (Cmax), time to reach maximal concentration (t-max)) of enalapril and its active metabolite enalaprilat. Of 102 patients, 89 patients (n = 26, DCM; n = 63 CHD) were included in the primary PK endpoint analysis. Rate and extent of enalapril and its active metabolite enalaprilat were described and etiology and age could be identified as potential PK modifying factors. The dosing schedule appeared to be tolerated well and did not result in any significant drug-related serious adverse events. The PK analysis and the lack of severe safety events supports the applied age-appropriate dosing schedule for the enalapril ODMTs.

Picomolar Sensitivity Analysis of Multiple Bradykinin-Related Peptides in the Blood Plasma of Patients With Hereditary Angioedema in Remission: A Pilot Study.

Background: Hereditary angioedema (HAE) is a rare autosomal dominant disease; the most well understood forms concern the haplodeficiency of C1 esterase inhibitor (C1INH) and a gain of function mutation of factor XII (FXII). The acute forms of these conditions are mediated by an excessive bradykinin (BK) formation by plasma kallikrein. Methods: A validated LC-MS/MS platform of picomolar sensitivity developed for the analysis of eleven bradykinin-related peptides was applied to the plasma of HAE-C1INH and HAE-FXII sampled during remission. Results: In HAE-C1INH plasma, the concentrations of the relatively stable BK1-5 fragment (mean ± S.E.M.: 12.0 ± 4.2 pmol/L), of BK2-9 (0.7 ± 0.2 pmol/L) and of the sums of BK and its tested fragments (18.0 ± 6.4 pmol/L) are significantly greater than those recorded in the plasma of healthy volunteers (1.9 ± 0.6, 0.03 ± 0.03 and 4.3 ± 0.8 pmol/L, respectively), consistent with the previous evidence of permanent plasma kallikrein activity in this disease. Kinin levels in the plasma of HAE-FXII patients did not differ from controls, suggesting that triggering factors for contact system activation are not active during remission. Conclusion: BK1-5, BK2-9 and the sum of BK and its fragments determined by the sensitive LC-MS/MS technique are proposed as potential biomarkers of HAE-C1INH in remission while this was not applicable to HAE-FXII patients.

Mass spectrometric study of variation in kinin peptide profiles in nasal fluids and plasma of adult healthy individuals.

BACKGROUND: The kallikrein-kinin system is assumed to have a multifunctional role in health and disease, but its in vivo role in humans currently remains unclear owing to the divergence of plasma kinin level data published ranging from the low picomolar to high nanomolar range, even in healthy volunteers. Moreover, existing data are often restricted on reporting levels of single kinins, thus neglecting the distinct effects of active kinins on bradykinin (BK) receptors considering diverse metabolic pathways. A well-characterized and comprehensively evaluated healthy cohort is imperative for a better understanding of the biological variability of kinin profiles to enable reliable differentiation concerning disease-specific kinin profiles. METHODS: To study biological levels and variability of kinin profiles comprehensively, 28 healthy adult volunteers were enrolled. Nasal lavage fluid and plasma were sampled in customized protease inhibitor prespiked tubes using standardized protocols, proven to limit inter-day and interindividual variability significantly. Nine kinins were quantitatively assessed using validated LC-MS/MS platforms: kallidin (KD), Hyp4-KD, KD1-9, BK, Hyp3-BK, BK1-8, BK1-7, BK1-5, and BK2-9. Kinin concentrations in nasal epithelial lining fluid were estimated by correlation using urea. RESULTS: Circulating plasma kinin levels were confirmed in the very low picomolar range with levels below 4.2 pM for BK and even lower levels for the other kinins. Endogenous kinin levels in nasal epithelial lining fluids were substantially higher, including median levels of 80.0 pM for KD and 139.1 pM for BK. Hydroxylated BK levels were higher than mean BK concentrations (Hyp3-BK/BK = 1.6), but hydroxylated KD levels were substantially lower than KD (Hyp4-KD/KD = 0.37). No gender-specific differences on endogenous kinin levels were found. CONCLUSIONS: This well-characterized healthy cohort enables investigation of the potential of kinins as biomarkers and would provide a valid control group to study alterations of kinin profiles in diseases, such as angioedema, sepsis, stroke, Alzheimer's disease, and COVID-19.

Embedding a Sensitive Liquid-Core Waveguide UV Detector into an HPLC-UV System for Simultaneous Quantification of Differently Dosed Active Ingredients during Drug Release.

Individual dosing of pharmaceutics and personalized medicine have become important with regard to therapeutic safety. Dose adjustments, biorelevant drug release and combination of multiple active substances in one dosage form for the reduction in polymedication are essential aspects that increase the safety and acceptance of the patient's pharmacotherapy. Therefore, not only innovative drug products but also new analytical methods are needed during the drug development phase and for quality control that can simultaneously determine different active ingredients and cover wide concentration ranges. We investigated a liquid-core waveguide UV absorbance flow cell detector coupled to an existing HPLC-UV system. A Teflon AF 2400 capillary tubing of 20 cm length was connected in series to the HPLC flow line and enabled a lower limit of quantification of 1 ng/mL pramipexole (increase in sensitivity by 20 compared to common 0.9 cm flow cells). This allowed the low-concentration of pramipexole and the higher concentrations of levodopa and benserazide occurring during drug release to be determined in a single chromatographic run within 22.5 min.

Reliable measurement of plasma kinin peptides: Importance of preanalytical variables.

BACKGROUND: The kallikrein-kinin system is involved in many (patho)physiological processes and kinin peptides are considered potential clinical biomarkers. Variance in blood specimen collection and processing, artificial ex vivo bradykinin formation, and rapid degradation of kinins have contributed to divergence in published plasma levels, therefore limiting their significance. Thus, reliable preanalytical settings are highly required. OBJECTIVES: This study aimed to develop and evaluate a standardized preanalytical procedure for reliable kinin quantification. The procedure was based on identification of the most impactful variables on ex vivo plasma level alterations. METHODS: Suitable protease inhibitors and blood specimen collection and handling conditions were systematically investigated. Their influence on plasma levels of seven kinins was monitored using an established in-house liquid chromatography-tandem mass spectrometry platform. RESULTS: In nonstandardized settings, ex vivo rise of bradykinin was found to already occur 30 seconds after blood sampling with high interindividual variation. The screening of 17 protease inhibitors resulted in a customized seven-component protease inhibitor, which efficiently stabilized ex vivo kinin levels. The reliability of kinin levels was substantially jeopardized by prolonged rest time until centrifugation, phlebotomy methodology (eg, straight needles, catheters), vacuum sampling technique, or any time delays during venipuncture. The subsequently developed standardized procedure was applied to healthy volunteers and proved it significantly limited interday and interindividual kinin level variability. CONCLUSION: The developed procedure for blood specimen collection and handling is feasible in clinical settings and allows for determination of reliable kinin levels. It may contribute to further elucidating the role of the kallikrein-kinin system in diseases like angioedema, sepsis, or coronavirus disease 2019.

Predictivity of standardized and controlled permeation studies: Ex vivo - In vitro - In vivo correlation for sublingual absorption of propranolol.

In preclinical drug development, ex vivo and in vitro permeability studies are a decisive element for specifying subsequent development steps. In this context, reliability, physiological alignment and appropriate in vivo correlation are mandatory for predictivity regarding drug absorption. Especially in oromucosal drug delivery, these prerequisites are not adequately met, which hinders its progressive development and results in the continuous need for animal experiments. To address current limitations, an innovative, standardized, and controlled ex vivo permeation model was applied. It is based on Kerski diffusion cells embedded in automated sampling and coupled to mass spectrometric quantification under physiologically relevant conditions. This study aimed to evaluate the predictivity of the developed model using porcine mucosa (ex vivo) in relation to data of sublingual propranolol absorption (in vivo). In addition, the usefulness of biomimetic barriers (in vitro) as a replacement for porcine mucosa was investigated. Therefore, solubility and permeability studies considering microenvironmental conditions were conducted and achieved good predictivity (R2 = 0.997) for pH-dependent permeability. A multiple level C correlation (R2 ≥ 0.860) between obtained permeability and reported pharmacokinetic animal data (AUC, Cmax) was revealed. Furthermore, a point-to-point correlation was demonstrated for several sublingual formulations. The successful IVIVC confirms the standardized ex vivo model as a viable alternative to animal testing for estimating the in vivo absorption behavior of oromucosal pharmaceuticals.

Formulation Development of Sublingual Cyclobenzaprine Tablets Empowered by Standardized and Physiologically Relevant Ex Vivo Permeation Studies.

Suitable ex vivo models are required as predictive tools of oromucosal permeability between in vitro characterizations and in vivo studies in order to support the development of novel intraoral formulations. To counter a lack of clinical relevance and observed method heterogenicity, a standardized, controlled and physiologically relevant ex vivo permeation model was established. This model combined the Kerski diffusion cell, process automation, novel assays for tissue integrity and viability, and sensitive LC-MS/MS analysis. The study aimed to assess the effectiveness of the permeation model in the sublingual formulation development of cyclobenzaprine, a promising agent for the treatment of psychological disorders. A 4.68-fold enhancement was achieved through permeation model-led focused formulation development. Here, findings from the preformulation with regard to pH and microenvironment-modulating excipients proved supportive. Moreover, monitoring of drug metabolism during transmucosal permeation was incorporated into the model. In addition, it was feasible to assess the impact of dosage form alterations under stress conditions, with the detection of a 33.85% lower permeation due to salt disproportionation. Integrating the coherent processes of disintegration, dissolution, permeation, and metabolization within a physiological study design, the model enabled successful formulation development for cyclobenzaprine sublingual tablets and targeted development of patient-oriented drugs for the oral cavity.

Development and evaluation of a composite dosage form containing desmopressin acetate for buccal administration.

Desmopressin acetate (DDAVP) is an oligopeptide indicated for the treatment of primary nocturnal enuresis, for example. The poor oral bioavailability of DDAVP accelerated a shift to alternative routes of administration like nasal and oromucosal, whereby nasal administration results in high fluctuations increasing the risk of undesirable side effects. Aim of the study was to use a new composite dosage form (solid matrix attached to a bilayer mucoadhesive film) to make DDAVP available via oromucosal route, reducing the risk of undesirable side effects through precise dosing. DDAVP was incorporated into a solid matrix in the form of a minitablet, and both direct tableting (AV > 30) and granulation followed by tableting (AV = 17.86) were compared. Minitablets with content uniformity could only be obtained by granulation and loss supplementation (AV = 11.27) with immediate drug release (>80% after 7-8 min) and rapid disintegration (<49 s). Permeation studies were performed with a clinically relevant dose (200 µg) in a time interval of up to one hour, resulting in apparent permeation coefficients of 4.90 × 10-6 cm/s (minitablet) and 2.04 × 10-6 cm/s (composite). Comparable fluctuations showed no inferiority of composite and minitablet regarding dosing accuracy. Thus, a step towards controlled and dose-accurate transmucosal delivery of systemically active DDAVP could be achieved.

The Development of an Educational Video on Blood Pressure Measurement for Pharmacy Students.

INTRODUCTION: High blood pressure is an important worldwide health issue. Pharmacists can perform multifaceted tasks in hypertension management such as measuring blood pressure. In a time where the use of educational videos in health professions education has increased, an educational video might be an option for teaching blood pressure measurement skills to pharmacy students. This project aimed to develop an educational video tailored to pharmacy students on oscillometric blood pressure measurement in a community pharmacy setting that can be used as a self-instruction video. METHODS: The video was created with support from the university's multimedia center. The video development was roughly divided into pre-production, production, and post-production. Students' satisfaction with and perception of the video was surveyed. RESULTS: An 11-minute 33-second self-instruction video in the German language on proper oscillometric blood pressure measurement tailored for pharmacy students was created. Along with descriptive slides, the video delineates the necessary steps of blood pressure measurement in a community pharmacy setting in a role-play, to support students in communication with the patient. Results of a survey on the satisfaction and perception of the video from thirty-seven pharmacy students were included in the analysis and revealed that the video was well accepted by pharmacy students. Moreover, approximately 95% responded that instructional videos should be included in future pharmacy education. CONCLUSION: We successfully developed an educational video on oscillometric blood pressure measurement for a community pharmacy setting. This work is a valuable form of support for faculty members, who intend to develop educational videos. This might be of interest especially during the coronavirus disease 2019 (COVID-19) pandemic, where distance learning has become highly relevant.

Exploring the transmucosal permeability of cyclobenzaprine: A comparative preformulation by standardized and controlled ex vivo and in vitro permeation studies.

As part of early drug development, preformulation studies are used to comprehensively explore the properties of new drugs. In particular, this includes the biopharmaceutical characterization and evaluation of impacting factors (e.g. excipients, microenvironmental conditions etc.) by permeation studies. To overcome the limitations of current studies, a novel standardized ex vivo procedure using esophageal mucosa as surrogate has been established successfully and applied to preformulation studies for oromucosal delivery of cyclobenzaprine hydrochloride, a tricyclic muscle relaxant with potential for psychopharmacotherapeutic use. By using the standardized ex vivo permeation process, a twofold enhancement of permeability (0.98 ± 0.16 to 1.96 ± 0.10 * 10-5 cm/s) was observed by adjustment and controlling of microenvironmental pH, empowering a targeted and effective development of sublingual formulations. Predictivity and suitability were superior compared to in vitro experiments using artificial biomimetic membranes, revealing a determination coefficient (R2) of 0.995 vs. 0.322 concerning pH-dependent permeability of cyclobenzaprine. In addition, diffusion properties were extensively examined (e.g. influence of mucosal thicknesses, tissue freezing etc.). The alignment of the study design regarding physiologically/clinically relevant conditions resulted in ex vivo data that allowed for the estimation of plasma AUC levels in the extend of reported in vivo ranges.

3-Hydroxy-propanamidines, a New Class of Orally Active Antimalarials Targeting Plasmodium falciparum.

3-Hydroxypropanamidines are a new promising class of highly active antiplasmodial agents. The most active compound 22 exhibited excellent antiplasmodial in vitro activity with nanomolar inhibition of chloroquine-sensitive and multidrug-resistant parasite strains ofPlasmodium falciparum (with IC50 values of 5 and 12 nM against 3D7 and Dd2 strains, respectively) as well as low cytotoxicity in human cells. In addition, 22 showed strong in vivo activity in thePlasmodium berghei mouse model with a cure rate of 66% at 50 mg/kg and a cure rate of 33% at 30 mg/kg in the Peters test after once daily oral administration for 4 consecutive days. A quick onset of action was indicated by the fast drug absorption shown in mice. The new lead compound was also characterized by a high barrier to resistance and inhibited the heme detoxification machinery in P. falciparum.

Application and validation of a coaxial liquid core waveguide fluorescence detector for the permeation analysis of desmopressin acetate.

In recent years, the development of peptide drugs and alternative routes of administration, such as buccal and sublingual routes, has become increasingly important to the pharmaceutical industry. Performing experiments under physiologically relevant conditions is still a challenge that has not yet been fully mastered. The requirements associated with these alternative administration routes (e.g. permeation testing for buccal administration) push common analytical detection systems in pharmaceutical technology to their limits, especially with regard to large molecules and peptides. An HPLC-coupled coaxial liquid-core waveguide fluorescence detector has been developed and evaluated within this study to overcome these limits by achieving a more sensitive detection. Desmopressin acetate was selected as the peptide drug with the aim of investigating its permeation behavior during the clinically relevant application period of one hour. Based on the detector system, a complete validation according to the requirements of international guidelines was successfully performed. The results of the validation showed an increase in sensitivity resulting in a limit of detection of 4.7 ng/mL and a lower limit of quantification of 9.5 ng/mL. Moreover, it has been demonstrated that the permeation of desmopressin can be observed in clinically relevant dosages and time periods of up to one hour using this innovative detector system.

Targeted LC-MS/MS platform for the comprehensive determination of peptides in the kallikrein-kinin system.

The kallikrein-kinin system (KKS) is involved in many physiological and pathophysiological processes and is assumed to be connected to the development of clinical symptoms of angioedema or COVID-19, among other diseases. However, despite its diverse role in the regulation of physiological and pathophysiological functions, knowledge about the KKS in vivo remains limited. The short half-lives of kinins, their low abundance and structural similarities and the artificial generation of the kinin bradykinin greatly hinder reliable and accurate determination of kinin levels in plasma. To address these issues, a sensitive LC-MS/MS platform for the comprehensive and simultaneous determination of the four active kinins bradykinin, kallidin, des-Arg(9)-bradykinin and des-Arg(10)-kallidin and their major metabolites bradykinin 2-9, bradykinin 1-7 and bradykinin 1-5 was developed. This platform was validated according to the bioanalytical guideline of the US Food and Drug Administration regarding linearity, accuracy, precision, sensitivity, carry-over, recovery, parallelism, matrix effects and stability in plasma of healthy volunteers. The validated platform encompassed a broad calibration curve range from 2.0-15.3 pg/mL (depending on the kinin) up to 1000 pg/mL, covering the expected concentrations in disease states. No source-dependent matrix effects were identified, and suitable stability of the analytes in plasma was observed. The applicability of the developed platform was proven by the determination of endogenous levels in healthy volunteers, whose plasma kinin levels were successfully detected in the low pg/mL range. The established platform facilitates the investigation of kinin-mediated diseases (e.g. angioedema, COVID-19) and enables the assessment of the impact of altered enzyme activities on the formation or degradation of kinins.

Sensitive mass spectrometric determination of kinin-kallikrein system peptides in light of COVID-19.

The outbreak of COVID-19 has raised interest in the kinin-kallikrein system. Viral blockade of the angiotensin-converting enzyme 2 impedes degradation of the active kinin des-Arg(9)-bradykinin, which thus increasingly activates bradykinin receptors known to promote inflammation, cough, and edema-symptoms that are commonly observed in COVID-19. However, lean and reliable investigation of the postulated alterations is currently hindered by non-specific peptide adsorption, lacking sensitivity, and cross-reactivity of applicable assays. Here, an LC-MS/MS method was established to determine the following kinins in respiratory lavage fluids: kallidin, bradykinin, des-Arg(10)-kallidin, des-Arg(9)-bradykinin, bradykinin 1-7, bradykinin 2-9 and bradykinin 1-5. This method was fully validated according to regulatory bioanalytical guidelines of the European Medicine Agency and the US Food and Drug Administration and has a broad calibration curve range (up to a factor of 103), encompassing low quantification limits of 4.4-22.8 pg/mL (depending on the individual kinin). The application of the developed LC-MS/MS method to nasal lavage fluid allowed for the rapid (~ 2 h), comprehensive and low-volume (100 µL) determination of kinins. Hence, this novel assay may support current efforts to investigate the pathophysiology of COVID-19, but can also be extended to other diseases.

Mass spectrometric studies on the peptide integrity of substance P and related human tachykinins in human biofluids.

The neurokinin-1 receptor plays a profound role in inflammatory processes and is involved in immune cell differentiation, cytokine release, and mast cell activation. Due to their similar peptide structures, the neurokinin-1 receptor does not discriminate between the endogenous ligands substance P (SP) and human hemokinin-1 (hHK-1), which both demonstrate biological receptor affinity. In addition, due to cross-reactivity, the current bioanalytical method of choice-immunoassays-also displays limitations in differentiating between these peptides. Thus, a recently developed mass spectrometric assay was utilized for the selective quantification of SP and hHK-1 in various biofluids and tissue. By applying the sample processing protocols developed, SP was quantified in porcine brain tissue (4.49 ± 0.53 nM), human saliva (113.3 ± 67.0 pM), and human seminal fluid (0.52 ± 0.15 nM) by mass spectrometric analysis. As previously reported, neither SP nor hHK-1 could be detected in human plasma by mass spectrometry. Comparison with analysis using a commercial immunoassay of the same plasma sample revealed SP like-immunoreactivity concentrations of 37.1-178.0 pM. The previously reported carboxylic acid of SP, whose identity was confirmed by high-resolution mass spectrometric analysis, did not show cross-reactivity in the applied immunoassay and did not contribute to SP-like immunoreactivity results. Subsequent compound discovery of the immunocaptured substance indicated the presence of a precursor of SP as possible cross-reactor in human plasma samples. The found cross-reactivity might be the cause for the high variance of SP plasma levels in former determinations.

Development, validation and standardization of oromucosal ex-vivo permeation studies for implementation in quality-controlled environments.

Tissue-based ex-vivo studies on the oromucosal permeability of drugs are often insufficiently adapted to physiological and clinical conditions, which limits their predictivity. Moreover, the scientific community demands for the standardization of ex-vivo studies, since conceptual limitations (e.g. low sensitivity of analytical methods, insufficient monitoring, different designs) restrict the wide implementation in preclinical drug development. Therefore, an innovative ex-vivo permeation process consisting of novel Kerski diffusion cell coupled to fully automated sampling and sample preparation with LC-MS/MS quantification was developed and standardized. Novel assays for routine examination of tissue integrity and viability were developed and embedded in a comprehensive analytical control system. The high level of standardization and automation reduced the differences of between-run to within-run precision to ≤ 0.27 % CV. Successful validation proved a broad calibration range of 0.93-952.38 ng/mL of the model drug cyclobenzaprine with guideline-compliant relative errors from -7.9-12.6 % (between-run accuracy). Consequently, the method allowed the physiological-clinical alignment of the study conditions to therapeutic doses and the short residence time of intraoral drugs (sampling times 1-60 min). Applicability was demonstrated by assessing the oromucosal permeability for different sublingual cyclobenzaprine hydrochloride formulations representing the excipient selection as a common aspect during galenic development. Thereby, expressive evaluation of the dosage forms was achieved resulting in an improved permeation by replacing croscarmellose into polyvinylpyrrolidone (cumulative amount of 42.6 vs. 112.6 µg/cm²). Thus, the automated permeation process ensured lean, standardized and reproducible assessment of oromucosal permeability within quality-controlled academic and regulatory environments. Simultaneously, the improved ex-vivo predictivity through physiological-clinical adjustments facilitates the reduction of costly in-vivo studies.