CURCUMIN NANOCRYSTAL FOR ORAL ADMINISTRATION: PREPARATION, CHARACTERIZATION AND ELECTROLYTES STABILITY

1. CURCUMIN NANOCRYSTAL FOR ORAL ADMINISTRATION: PREPARATION, CHARACTERIZATION AND ELECTROLYTES STABILITY Heni Rachmawatia,b, Loaye Al Shaalb, Cornelia M.Keckb aPharmaceutics Research Group, School of Pharmacy, Bandung Institute of Technology, Ganesha 10 Bandung 40132, Indonesia bFreie Universität Berlin, Department of Pharmacy, Pharmaceutical Technology, Biopharmaceutics & NutriCosmetics, Berlin, Germany Email: h_rachmawati@fa.itb.ac.id INTRODUCTION RESULTS DISCUSSION Curcumin, a naturally occuring polyphenolic phytoconstituent, is isolated from the rhizomes of Curcuma longa Linn. (Zingiberaceae). It is insoluble in water under acidic or neutral conditions but dissolves in alkaline environment. Curcumin is highly unstable undergoing rapid hydrolytic degradation in neutral or alkaline conditions to feruloyl methane and ferulic acid. It is reported to be stable below pH 6.0. Thus, the use of curcumin is limited by its poor aqueous solubility in acidic or neutral conditions and instability in alkaline pH. It has anti-cancer, anti-oxidant, anti-inflammatory, hyperlipidemic, anti bacterial, wound healing and hepatoprotective activities. The pharmacological efficacy of curcumin makes it a potential compound for treatment and prevention of a wide variety of human diseases. In addition, it is extremely safe upon oral administration even at very high doses, as proven in various animal models or human studies. In spite of this, curcumin has not yet been approved as a therapeutic agent, and the relative bioavailability of curcumin has been highlighted as a major problem for this. 1. Particle size (LD) vs homogenization cycles Figure 2 shows the different successful nanocrystal production, seems to be stabilizer dependent. Steric stabilization provided by TPGS, PVP and PVA is suggested to be more efficient in which the smaller the molecular weight the more efficient to produce and to stabilize the nanocrystal. The potential stabilizing of the five different investigated stabilizers was confirmed by short-term stability data (fig. 3 and 4). PVA, PVP, and TPGS preserved the particle agglomeration at both temperature over 30 days, while SDS and Na-CMC failed to do so. The zeta potential values (table 1) is useful to explain that phenomenon. Particle agglomeration in SDS-stabilized nanosuspension (fig. 4) was undetected in the LD measurement (fig. 3). This can be explained that the agglomerates were loose and de-aggregated during LD measurement by stirring mechanism. The agglomerates formed in Na-CMC-stabilized nanosuspension was harder and therefore these size increases were clearly exerted (fig.3) Figure 2. Particle size reduction (LD data presented as d95% and d99%) as a function of homogenization cycles for the five different Curcumin nanosuspensions. 2. Short-term physical stability data Table.1. Zeta potential of the five differently stabilized Curcuminnanosuspensions; left: analyzes in water (conductivity 50 mS/cm, pH 5.8); right: analyzed in the original dispersion medium. Figure 1. Chemical structure of Curcumin Several approaches to enhance the solubility of Curcumin such as chemical derivatisation, complexation or interaction with macromolecules e.g. gelatin, polysaccharides, protein, and cyclodextrin have been reported but not successful yet in practical utility. AIM OF STUDY To develop Curcumin nanocrystal as an innovative solution to overcome the oral biovailability problem of Curcumin. Figure 5 and 6 show the influence of electrolytes on the physical stability of all nanosuspensions. These data again confirm that SDS and Na-CMC-stabilized nanosuspensions (electrostatic stabilization) are more sensitive against electrolytes, particularly to CaCl2 and SGF (low pH). When the electrolytes were added, the potential subsequently dropped faster and the diffuse layer was thinner, leading to the decrease of zeta potential (data not shown) therefore reduced the nanosuspension stability. The agglomeration of nanosuspension with Na-CMC was loose hence the change in d99% was undetected, while this destabilization was obviously appeared in polarized micrograph. In addition to electrostatic stabilization, polymer chain of Na-CMC provides a steric protection as well. This explains why the influence of electrolytes on the destabilization was moderate as compared to SDS-stabilized nanosuspension. Figure 3. Stability profile of five differently stabilized Curcuminnanosuspensions as function of days (0-30) stored at room temperature (RT, left) and 4oC (right). METHODS SDS PVP TPGS PVA Na-CMC • 1. Preparation of nanosuspensions • High-pressure homogenization (HPH), Micron Lab 40 (APV Deutchland GmbH, Germany) was used to produce nanosuspension. Five different stabilizers: polyvinyl alcohol (PVA), polyvinylpyrolidone (PVP), D-α-tocopherol polyethylene glycol 1000 succinate (TPGS), carboxymethylcellulose sodium salt (Na-CMC) and sodium dodecyl sulphate (SDS) were used. The homogenization process was applied for 2 cycles of 300, 500, 1000 bar as pre-milling step and continued with applying high pressure at 1500 bar for 20 cycles. • Measurements: • Particle size distribution: LD (Laser Diffractometry) • Particle charge: zeta potential • 2. Short-term stability study on the nanosuspensions • All nanosuspensions were stored in sealed vials at different temperatures (room temperature, RT and 4oC) for 30 days. Samples were taken on day 0 (day of production), day 7, and day 30. Characterizations were carried out including analysis of particle size (PCS) and polarized light microscopy. • 3. Electrolytes challenge on the nanosuspensions • Electrolytes (CaCl2 150 mM, simulated gastric fluid (SGF, pH 1.2), and simulated intestinal fluid (SIF, pH 6.8)) were admixed to the nanosuspensions with the ratio of electrolyte:nanosuspension [1:3]. The physical stability of the nanosuspensions were examined at 0, 30, 60, 120, and 240 minutes after incubation with the electrolyte. LD, zeta potential, microscopic analysis as well as visual observation were performed to monitor the stability of the nanosuspensions. 7 D 30 D PVA PVP SDS Na-CMC TPGS Figure 4. Polarized light micrograph (magnification 160x) of the nanosuspensions after 7 and 30 days storage at RT. Nanosuspension stabilized with non ionic polymers (PVA, PVP, TPGS) are stable with no agglomeration detected, in contrast with ionic stabilized nanosuspensions (SDS and Na-CMC). D = days + CaCl2 + SGF 3. Electrolytes destabilization + SIF Figure 6. Polarized light micrograph (magnification 160x) of the nanosuspensions after incubation with the electrolytes. Nanosuspension stabilized with non ionic polymers (PVA, PVP, TPGS) are stable with no agglomeration detected, in contrast with ionic stabilized nanosuspensions (SDS and Na-CMC). CONCLUSION Nanocrystal of Curcumin was successfully produced with four stabilizers PVA, PVP, TPGS, and SDS with the particle size in the range of 500-700 nm. Na-CMC resulted in slightly larger PCS diameter (about 800 nm) indicating that this polymer is not able to efficiently stabilize the produced crystals at the end of the homogenization process. PVA, PVP and TPGS showed similar performance in preserving the Curcuminnanosuspension stability, including their potential to maintain physical stability of Curcuminnanosuspensions against electrolytes presence in the gastrointestinal tract. In contrast, SDS and Na-CMC was not successful stabilizer in this study. Curcuminnanocrystal seems to be a promising approach to improve oral bioavailability of this potential natural product to the forefront of therapeutic agents for treatment of human diseases. B = before + electrolyte Figure 5. Change in size d99% of five different nanosuspensions after addition of CaCl2 (left), simulated gastric fluid (SGF, middle) and simulated intestinal fluid (SIF, right). Measurements were performed as a function of time. This project was financially supported by DAAD, under scheme of Indonesian-German Scientists Exchange Program.

2. ONGOING PROJECTS (Dr. Heni Rachmawati) • Pharmacokinetic, biodistribution and activity studies • Of curcuminnanocrystalsvscurcumin • Join project between school of pharmacy itbindonesia and fu berlingermany, supported by itb research grant Curcuminnanocrystals Curcumin Biodistribution Oral administration Pharmacokinetics Oral administration Antiinflammatory Evaluation after oral administration • Tumor targeting of docetaxel-trastuzumabnp Pharmacokinetic and • biodistribution in rats • Join project between school of pharmacy itbindonesia and national university of singapore, supported by islamic development bank AB conjugated NPs containing Docetaxel Docetaxel Pharmacokinetics Iv administration Biodistribution after iv administration