Efficient and rapid digestion of proteins with a dual-enzyme microreactor featuring 3-D pores formed by dopamine/polyethyleneimine/acrylamide-coated KIT-6 molecular sieve.

The microreactor with two types of immobilized enzymes, exhibiting excellent orthogonal performance, represents an effective approach to counteract the reduced digestion efficiency resulting from the absence of a single enzyme cleavage site, thereby impacting protein identification. In this study, we developed a hydrophilic dual-enzyme microreactor characterized by rapid mass transfer and superior enzymatic activity. Initially, we selected KIT-6 molecular sieve as the carrier for the dual-IMER due to its three-dimensional network pore structure. Modification involved co-deposition of polyethyleneimine (PEI) and acrylamide (AM) as amine donors, along with dopamine to enhance material hydrophilicity. Remaining amino and double bond functional groups facilitated stepwise immobilization of trypsin and Glu-C. Digestion times for bovine serum albumin (BSA) and bovine hemoglobin (BHb) on the dual-IMER were significantly reduced compared to solution-based digestion (1 min vs. 36 h), resulting in improved sequence coverage (91.30% vs. 82.7% for BSA; 90.24% vs. 89.20% for BHb). Additionally, the dual-IMER demonstrated excellent durability, retaining 96.08% relative activity after 29 reuse cycles. Enhanced protein digestion efficiency can be attributed to several factors: (1) KIT-6's large specific surface area, enabling higher enzyme loading capacity; (2) Its three-dimensional network pore structure, facilitating faster mass transfer and substance diffusion; (3) Orthogonality of trypsin and Glu-C enzyme cleavage sites; (4) The spatial effect introduced by the chain structure of PEI and glutaraldehyde's spacing arm, reducing spatial hindrance and enhancing enzyme-substrate interactions; (5) Mild and stable enzyme immobilization. The KIT-6-based dual-IMER offers a promising technical tool for protein digestion, while the PDA/PEI/AM-KIT-6 platform holds potential for immobilizing other proteins or active substances.

[The pharmacokinetics and bioequivalence of acipimox sustained-release tablets after a single and multiple oral administration in healthy dogs].

AIM: To study the pharmacokinetics and bioequivalence of acipimox sustained-release tablets (SRT) after a single and multiple oral dose in healthy dogs. METHODS: The plasma concentrations of of SRT and reference capsules with a single and multiple oral doses. RESULTS: The drug concentration-time profiles fitted to a noncompartment model. After a single dose administration of sustained-release tablets and capsules, the pharmacokinetic parameters were as follows: AUC were (158 +/- 30) and (147 +/- 37) microg x h x mL(-1); Tmax were (4.3 +/- 0.8) and (2.6 +/- 1.3) h; Cmax were (29 +/- 6) and (42 +/- 10) microg x mL(-1); T(1/2) were (2.3 +/- 0.7) and (1.60 +/- 0.10) h; MRT were (6.0 +/- 0.8) and (3.9 +/- 0.7) h, respectively. The relative bioavailability of the sustained-release tablet was (108 +/- 16) %. After a multiple oral administration of sustained-release tablets and capsules, the pharmacokinetic parameters were as follows: AUC were (209 +/- 23) and (195 +/- 26) microg x h x mL(-1); Tmax were (6.3 +/- 0.8) and (3.4 +/- 1.5) h; Cmax were (27 +/- 4) and (36 +/- 5) microg x mL(-1); Cmmin were (2.2 +/- 1.0) and (0.20 +/- 0.20) microg x mL(-1); Cav were (8.7 +/- 1.0) and (8.1 +/- 1.1) micro x mL(-1); FI were (293 +/- 73) % and (448 +/- 91) % , respectively. The relative bioavailability of the sustained-release tablet was (114 +/- 19) %. CONCLUSION: The results of two one-side test from single dose administration shown that two preparations were bioequivalent. The Cmax of sustained-release tablet was lower than that of capsules, while the Tmax and MRT of sustained-release tablet were higher than that of capsule, which indicating a good retarding effect. The results from multiple dose administration also shown that two preparations were bioequivalent and the DF of sustained-release tablet was significant lower than that of capsule.