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Ditlev Birch

Ditlev Birch

Ph.d. stipendiat

Project background

My area of interest covers delivery of macromolecules with an emphasis on protein based therapeutics. Proteins as pharmaceuticals often show high potency and can be modified for high selectivity for a certain target within the body. Despite these properties as well as high aqueous solubility, proteins often exhibit poor bioavailability. This is primarily due to  poor permeability across the mucosal membranes (e.g. in the gastrointestinal tract), and rapid degradation when the compound is introduced to various peptidases found in the human body.

One way to increase the bioavailability of proteins is the use of cell penetrating peptides (CPPs) as carriers for the protein therapeutic. The CPPs are small peptides (typicall less than 40 amino acids) which are able to translocate across the cell membrane, bringing its cargo across as well. This method potentially increases intracellular drug uptake and transcellular drug delivery. The mechanism for which the CPPs translocate is widely debated, but is though to involve endocytosis/transcytosis and direct translocation, perhaps in combination.

While the efficacy of CPPs as carriers are widely investigated, current assesment methods often rely on fluorophores as labels for analysis purposes (e.g. in vitro cell culture models in combination with flow cytometry or HPLC-Flu for quantification, and confocal laser scanning microscopy for intracellular trafficking). However, attaching a hydrophobic moiety such as the fluorophore to a hydrophilic peptide is likely to affect the physicochemical properties of the CPP, thereby affecting its propensity to translocate across a mucosal barrier and thereby its ability as a drug carrier.

My research concerns the development and characterization of a new analysis method based on 19F-NMR in order to circumvent the problems arising from the use of fluorophores. The method is being assessed in relevant in vitro cell culture models (Caco-2, HeLa and IEC-6) and compared to conventional methods by fluorophores in the same systems.

When thoroughly developed and characterized, the method will be used to shed new light on the translocation mechanism of CPPs with respect to both uptake and intracellular trafficking. Finally, the method will be used to thoroughly asses delivery of protein based therapeutics by CPPs.

Aktuel forskning

I am currently involved in two areas of research which are closely related:

First, i am assessing the translocation propensity of penetratin, a cell-penetrating peptide (CPP) when labeled with various commonly used fluorophores (e.g. carboxyfluorescein, 5(6)-carboxy-tetraemethyl-rhodamine). Furthermore, the effect on the physicochemical properties of the labels on penetratin as well as cytotoxicity of labeled penetratin to various in vitro cell cultures (e.g. Caco-2) are also being assessed.

Second, i am elucidating the metabolic pattern of penetratin when introduced to the enzymes found in the gastrointestinal tract, at the brush border and intracellular in Caco-2 cells. The metabolites are being evaluated with respect to their ability to translocate across in vitro cell membranes, formation rate and toxicity.

 

 

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