Bioactive protein fraction DLBS1033 containing lumbrokinase isolated from Lumbricus rubellus: ex vivo, in vivo, and pharmaceutic studies

Raymond R Tjandrawinata, Jessica Trisina, Puji Rahayu, Lorentius Agung Prasetya, Aang Hanafiah, Heni Rachmawati, Raymond R Tjandrawinata, Jessica Trisina, Puji Rahayu, Lorentius Agung Prasetya, Aang Hanafiah, Heni Rachmawati

Abstract

DLBS1033 is a bioactive protein fraction isolated from Lumbricus rubellus that tends to be unstable when exposed to the gastrointestinal environment. Accordingly, appropriate pharmaceutical development is needed to maximize absorption of the protein fraction in the gastrointestinal tract. In vitro, ex vivo, and in vivo stability assays were performed to study the stability of the bioactive protein fraction in gastric conditions. The bioactive protein fraction DLBS1033 was found to be unstable at low pH and in gastric fluid. The "enteric coating" formulation showed no leakage in gastric fluid-like medium and possessed a good release profile in simulated intestinal medium. DLBS1033 was absorbed through the small intestine in an intact protein form, confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) analysis. This result confirmed that an enteric coating formula using methacrylic acid copolymer could protect DLBS1033 from the acidic condition of the stomach by preventing the release of DLBS1033 in the stomach, while promoting its release when reaching the intestine. From the blood concentration-versus-time curve, (99m)Tc-DLBS1033 showed a circulation half-life of 70 minutes. This relatively long biological half-life supports its function as a thrombolytic protein. Thus, an enteric delivery system is considered the best approach for DLBS1033 as an oral thrombolytic agent.

Keywords: bioactive protein fraction; enteric coated tablet; pharmacodynamic.

Figures

Figure 1
Figure 1
Zymogram and SDS PAGE of DLBS1033 in solutions with different pH. Notes: (A) DLBS1033 in purified water; (B) DLBS1033 in phosphate-buffered saline; and (C) DLBS1033 in sodium acetate buffer. Abbreviation: SDS PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Figure 2
Figure 2
Zymogram and SDS PAGE of (A) negative control (gastric fluid); (B) DLBS1033 from the ex vivo experiment; and (C) DLBS1033 from the in vivo experiment. Abbreviation: SDS PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Figure 3
Figure 3
Zymogram of permeated DLBS1033 at different time points.
Figure 4
Figure 4
Fibrin plate of samples from intestinal permeability assay. Notes: Saline blank = phosphate-buffered saline solution before the experiment (not concentrated); DLBS1033 T0 = solution before experiment (not concentrated); DLBS1033 T210 min = inside intestine (not concentrated). Abbreviation: PBS, phosphate-buffered saline; min; minutes.
Figure 5
Figure 5
Fibrin plate result of DLBS1033 after intestinal permeability assay.
Figure 6
Figure 6
Paper chromatography profile of 99mTc-DLBS1033. Abbreviation: RF, retardation factor.
Figure 7
Figure 7
Blood clearance of 99mTc-DLBS1033 in rats (n=3).
Figure 8
Figure 8
Drug-release profile of DLBS1033 enteric coated tablet in acidic medium (pH 2.8).
Figure 9
Figure 9
Drug-release profile of DLBS1033 enteric coated tablet in phosphate buffer medium (PBS) (pH 7).

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