Thomas Birngruber

Intratumoural drug delivery can achieve high local therapeutic exposure while limiting systemic effects, but heterogeneous distribution may compromise treatment efficacy and precision. This proof-of-concept study investigated open flow microperfusion (OFM) as a novel platform for local delivery of tigilanol tiglate (TT), an intratumoural antineoplastic agent with immunomodulatory and vascular-disrupting effects. Four horses with histologically confirmed cutaneous neoplasia (three sarcoids and one squamous cell carcinoma) underwent OFM using TT concentrations of 0.01–0.04 mg/mL delivered at 9 µL/min for 7–24 h over one to three consecutive days. OFM systems were well tolerated and remained functional for up to three days. Localized vascular disruption, polymorphonuclear cell recruitment, oncosis and tissue sloughing developed in association with perfused regions, consistent with recognized TT-induced tissue responses. Two sarcoids exhibited biologic responses that did not result in complete local tumour ablation. Tumour ablation was achieved initially in one squamous cell carcinoma, although deep-seated disease subsequently progressed, while one sarcoid was ablated and remained tumour-free 497 days after treatment. These preliminary findings demonstrate that OFM allows precise, sustained intratumoural TT challenge, resulting in characteristic tissue responses supporting further investigation of this local delivery platform for tumour-targeted therapies.

The central nervous system represents a major target tissue for therapeutic approach of numerous lysosomal storage disorders. Fabry disease arises from the lack or dysfunction of the lysosomal alpha-galactosidase A (GLA) enzyme, resulting in substrate accumulation and multisystemic clinical manifestations. Current enzyme replacement therapies (ERTs) face limited effectiveness due to poor enzyme biodistribution in target tissues and inability to reach the brain. We present an innovative drug delivery strategy centered on a peptide-targeted nanoliposomal formulation, designated as nanoGLA, engineered to selectively deliver a recombinant human GLA (rhGLA) to target tissues. In a Fabry mouse model, nanoGLA demonstrated improved efficacy, inducing a notable reduction in Gb3 deposits in contrast to non-nanoformulated GLA, even in the brain, highlighting the potential of the nanoGLA to address both systemic and cerebrovascular manifestations of Fabry disease. The EMA has granted the Orphan Drug Designation to this product, underscoring the potential clinical superiority of nanoGLA over authorized ERTs and encouraging to advance it toward clinical translation.

Dupilumab is a monoclonal antibody approved for the treatment of atopic dermatitis (AD); however, its effects on molecular, cellular, and immunological levels remain to be elucidated. In this study, blood and dermal interstitial fluid (ISF) from nonlesional (NL) and lesional (L) skin were collected from eight patients with moderate to severe AD, before (visit 2-v2) and at the end of a 16-week treatment with dupilumab (visit 10-v10). Clinical treatment effect was demonstrated by significantly decreased AD severity scores at the end of treatment. At v10 versus v2, the percentages of CD4+ interleukin-producing cells showed a decreasing trend in ISF L and NL, unbound IL-4 levels in plasma were increased, IL-5 levels in ISF L reduced, and levels of factors involved in anti-inflammatory pathways and re-epithelization increased. At v2, ISF L showed that AD lesions might have altered amino acid pathways and lipid signaling compared to ISF NL. At v10, ISF L exhibited raised levels of long- and very-long-chain fatty acids and lipids compared to v2. Furthermore, dupilumab administration caused reduced expression of miR-155-5p and miR-378a-3p in ISF L. In conclusion, results from the present study provided novel knowledge by linking local immune and metabolic alterations to AD pathogenesis and treatment response.

The increasing relevance of improved therapeutic monoclonal antibodies (mAbs) to treat neurodegenerative diseases has strengthened the need to reliably measure their brain pharmacokinetic (PK) profiles. The aim of this study was, therefore, to absolutely quantify the therapeutic antibody ocrelizumab (OCR) as a model antibody in mouse brain interstitial fluid (ISF), and to record its PK profile by using cerebral open flow microperfusion (cOFM). Further, to monitor the blood–brain barrier (BBB) integrity using an endogenous antibody with a similar molecular size as OCR. The study was conducted on 13 male mice. Direct and absolute OCR quantification was performed with cOFM in combination with zero flow rate, and subsequent bioanalysis of the obtained cerebral ISF samples. For PK profile recording, cerebral ISF samples were collected bi-hourly, and brain tissue and plasma were collected once at the end of the sampling period. The BBB integrity was monitored during the entire PK profile recording by using endogenous mouse immunoglobulin G1. We directly and absolutely quantified OCR and recorded its brain PK profile over 96 h. The BBB remained intact during the PK profile recording. The resulting data provide the basis for reliable PK assessment of therapeutic antibodies in the brain thus favoring the further development of therapeutic monoclonal antibodies.

In vivo investigation of brain pharmacokinetics and pharmacodynamics (PK/PD) is an integral part of neurological drug development. However, drugs intended to act in the brain may reach it at very low concentrations due to the protective effect of the blood-brain barrier (BBB). Consequently, very sensitive measurement methods are required to investigate PK/PD of drugs in the brain. Also, these methods must be capable of continuously assessing cerebral drug concentrations with verifiable intact BBB, as disrupted BBB may lead to compound efflux from blood into brain and to biased results. To date, only a few techniques are available that can sensitively measure drug concentrations in the brain over time; one of which is cerebral open flow microperfusion (cOFM). cOFM's key features are that it enables measurement of cerebral compound concentrations with intact BBB, induces only minor tissue reactions, and that no scar formation occurs around the probe. The membrane-free cOFM probes collect diluted cerebral interstitial fluid (ISF) samples that are containing the whole molecule spectrum of the ISF. Further, combining cOFM with an in vivo calibration protocol (e.g. Zero Flow Rate) enables absolute quantification of compounds in cerebral ISF. In general, three critical aspects have to be considered when measuring cerebral drug concentrations and recording PK/PD profiles with cOFM: (a) the BBB integrity during sampling, (b) the status of the brain tissue next to the cOFM probe during sampling, and (c) the strategy to absolutely quantify drugs in cerebral ISF. This work aims to review recent applications of cOFM for PK/PD assessment with a special focus on these critical aspects.

Background: Pharmacokinetic (PK) assessment of drugs in brain is especially challenging, as the blood-brain-barrier (BBB) impedes the access of substances to the brain. However, rising incidence of monoclonal antibodies as treatment option for diseases of the central nervous system makes continuous measurement of their brain PK profile with verifiably intact BBB increasingly relevant. Such measurements can be performed with continuous brain interstitial fluid (ISF) sampling techniques like e.g., cerebral open flow microperfusion (cOFM). Although cOFM has already provided antibody concentrations in brain ISF in a time-resolved manner, accurate time-resolved quantification of antibody in brain to record the PK profile requires continuous sampling over an extended period of time and knowledge on the BBB integrity for the respective study drug during the entire sampling period. We thus aimed to absolutely quantify the therapeutic antibody ocrelizumab (OCR) in mouse brain ISF over 96 hours, and to record its PK profile. OCR, with a target on human CD20⁺ B-cells, was selected as study drug. We also aimed to monitor the BBB integrity during the entire study duration using an endogenous antibody as tracer with similar molecular size as OCR. Methods: Direct and absolute OCR quantification was performed using cOFM combined with the quantification protocol Zero Flow Rate, and data were corrected with the cOFM probe´s in vivo relative recovery. For PK profile recording the cOFM samples were collected bi-hourly, and brain tissue and plasma at the end of the sampling period. BBB monitoring was performed simultaneously during the entire PK profile recording using the endogenous mIgG1. This study was performed in male C57Bl/6 mice. Results: We directly, absolutely quantified OCR, and reliably recorded its brain PK profile over 96 hours. BBB integrity was sustained during the entire study. Conclusions: Results demonstrated that cOFM is able to accurately, absolutely quantify OCR in brain ISF and to record its brain PK profile over a prolonged duration with verifiably intact BBB. Our data provide the basis for reliable PK assessment of therapeutic antibodies in brain, which is likely to promote the development of therapeutic monoclonal antibodies to treat neurological diseases.

Skin equivalents and skin explants are widely used for dermal penetration studies in the pharmacological development of drugs. Environmental parameters, such as the incubation and culture conditions affect cellular responses and thus the relevance of the experimental outcome. However, available systems such as the Franz diffusion chamber, only measure in the receiving culture medium, rather than assessing the actual conditions for cells in the tissue. We developed a sampling design that combines open flow microperfusion (OFM) sampling technology for continuous concentration measurements directly in the tissue with microfluidic biosensors for online monitoring of culture parameters. We tested our design with real-time measurements of oxygen, glucose, lactate, and pH in full-thickness skin equivalent and skin explants. Furthermore, we compared dermal penetration for acyclovir, lidocaine, and diclofenac in skin equivalents and skin explants. We observed differences in oxygen, glucose, and drug concentrations in skin equivalents compared to the respective culture medium and to skin explants.