Recycling of hyoscyamine 6ß-hydroxylase for the in vitro production of anisodamine and scopolamine.

The tropane alkaloids hyoscyamine, anisodamine, and scopolamine are extensively used medicines. In particular, scopolamine has the greatest value in the market. Hence, strategies to enhance its production have been explored as an alternative to traditional field-plant cultivation. In this work, we developed biocatalytic strategies for the transformation of hyoscyamine into its products utilizing a recombinant Hyoscyamine 6ß-hydroxylase (H6H) fusion protein to the chitin-binding domain of the chitinase A1 from Bacillus subtilis (ChBD-H6H). Catalysis was carried out in batch, and recycling of H6H constructions was performed via affinity-immobilization, glutaraldehyde crosslinking, and adsorption-desorption of the enzyme to different chitin matrices. ChBD-H6H utilized as free enzyme achieved complete conversion of hyoscyamine in 3- and 22-h bioprocesses. Chitin particles demonstrated to be the most convenient support for ChBD-H6H immobilization and recycling. Affinity-immobilized ChBD-H6H operated in a three-cycle bioprocess (3 h/cycle, 30 °C) yielded in the first and third reaction cycle 49.8% and 22.2% of anisodamine and 0.7% and 0.3% of scopolamine, respectively. However, glutaraldehyde crosslinking decreased enzymatic activity in a broad range of concentrations. Instead, the adsorption-desorption approach equaled the maximal conversion of the free enzyme in the first cycle and retained higher enzymatic activity than the carrier-bound strategy along the consecutive cycles. The adsorption-desorption strategy permitted the reutilization of the enzyme in a simple and economical manner while exploiting the maximal conversion activity displayed by the free enzyme. This approach is valid since other enzymes present in the E. coli lysate do not interfere with the reaction. KEY POINTS: • A biocatalytic system for anisodamine and scopolamine production was developed. • Affinity-immobilized ChBD-H6H in ChP retained catalytic activity. • Enzyme-recycling by adsorption-desorption strategies improves product yields.

Fully Automated Screening of a Combinatorial Library to Avoid False Positives: Application to Tetanus Toxoid Ligand Identification.

Peptide ligands are widely used in protein purification by affinity chromatography. Here, we applied a fully automated two-stage library screening method that avoids false positive peptidyl-bead selection and applied it to tetanus toxoid purification. The first library screening was performed using only sulforhodamine (a fluorescent dye), and fluorescent beads were isolated automatically by flow cytometry and discarded. A second screening was then performed with the rest of the library, using the target protein (tetanus toxoid)-rhodamine conjugate. This time, fluorescent beads were isolated, and peptide sequences were identified by matrix-assisted laser desorption/ionization tandem mass spectrometry. Those appearing with greater frequency were synthesized and immobilized on agarose to evaluate a range of chromatographic purification conditions. The affinity matrix PTx1-agarose (Ac-Leu-Arg-Val-Tyr-His-Gly-Gly-Ala-Gly-Lys-agarose) showed the best performance when 20 mM sodium phosphate, 0.05% Tween 20, pH 5.9 as adsorption buffer and 100 mM Tris-HCl, 100 mM NaCl, pH 8.0 as elution buffer were used. A pure tetanus toxoid (Ttx) was loaded on a chromatographic column filled with the PTx1 matrix, and 96% adsorption was achieved, with a K d of 9.18 ± 0.07 nmol/L and a q m of 1.31 ± 0.029 µmol Ttx/mL matrix. Next, a Clostridium tetani culture supernatant treated with formaldehyde (to obtain the toxoid) was applied as a sample. The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed a band, identified by electrospray ionization mass spectrometry as the Ttx, that appeared only in the elution fraction, where an S-layer protein was also detected.

Pharmacokinetics of a novel spot-on formulation of praziquantel for dogs.

Praziquantel (PZQ) is an anthelmintic drug used both in humans and animals that can be administered through various routes. There are transdermal formulations for cats, but only oral or subcutaneous dosage forms for dogs. Given the fact that the cat's skin and the dog's skin have different characteristics, which in turn affect bioavailability, we developed a PZQ spot-on formulation for dogs. This study was aimed at determining the plasmatic behavior of topically administered PZQ (Labyes®) in adult dogs. Dogs were administered PZQ (14.5mg/kg PZQ, from a solution of 100mg/ml). Blood samples were drawn before treatment onset and at the following time points after PZQ administration: 1, 2, 4, 6, 12, 24 and 48h. PZQ plasma concentration was determined by ultra-high performance liquid chromatography (UPLC) coupled to tandem mass spectrometry (MS/MS). Observed maximum concentration (Cmax), area under the concentration-time curve from the time of drug administration to infinity (AUCinf) and time to maximum concentration (Tmax) were calculated for each animal, and mean±SD for each parameter was obtained. Results were as follows: Cmax=56.0±15ng/ml; AUCinf=910.2±220ng*h/ml, Tmax=5.0±1.1h. This is the first study to provide pharmacokinetic data of a praziquantel spot-on formulation for dogs.

Tetronic® 904-containing polymeric micelles overcome the overexpression of ABCG2 in the blood-brain barrier of rats and boost the penetration of the antiretroviral efavirenz into the CNS.

AIM: To assess the involvement of ABCG2 in the pharmacokinetics of efavirenz in the blood-brain barrier (BBB) and investigate a nanotechnology strategy to overcome its overexpression under a model of chronic oral administration. Materials & methods A model of chronic efavirenz (EFV) administration was established in male Sprague-Dawley rats treated with a daily oral dose over 5 days. Then, different treatments were conducted and drug concentrations in plasma and brain measured. RESULTS: Chronic treatment with oral EFV led to the overexpression of ABCG2 in the BBB that was reverted after a brief washout period. Moreover, gefitinib and the polymeric amphiphile Tetronic(®) 904 significantly inhibited the activity of the pump and potentiated the accumulation of EFV in CNS. The same effect was observed when the drug was administered within mixed micelles containing TetronicT904 as the main component. CONCLUSION: Tetronic 904-containing polymeric micelles overcame the overexpression of ABCG2 in the BBB caused by chronic administration of EFV then boosting its penetration into the CNS.