| 9-Nitrocamptothecin (9-NC), a novel analogue of camptothecin, has been identified to be a very promising anti-tumor drug with high potency against a wide spectrum of human tumors in preclinical evaluation. In addition,9-NC has been proved to be efficacious as first-line therapy in the treatment of advanced pancreatic cancer. However, the clinical application of 9-NC was largely hampered because of its poor solubility and stability, which led to low therapeutic efficacy and a number of side effects owing to the conversion of 9-NC from active lactone form to the inactive carboxylate form under physiologic conditions. As a result, up to now, none of the 9-NC formulations could be applied for clinical therapy. Therefore, the development of a novel parenteral formulation should be taken into consideration.Hydroxypropyl-β-cyclodextrin (HP-(3-CD) is a hydroxyalkylated-β-CD derivative that combines relatively high water solubility with low toxicity and satisfactory inclusion ability. Several commercial formulations are composed of cyclodextrin inclusion complexes, illustrating the usefulness of this approach. The main objective of this study was to develop a HP-β-CD-based formulation with high solubility and stability for 9-NC using a unique but simple and highly reproducible method and evaluate the property in vitro and in vivo.Phase-solubility techniques were used to assess the interaction of 9-NC and HP-P-CD. The solubilization profile suggested AL-tpye relationship, indicating the formation of a 1:1 complex (Kc= 184.4 M-1). Thermodynamic parameters showed that in the presence of HP-β-CD, the solubility of 9-NC increased with increasing temperature.The solubility of 9-NC increased slightly with increasing pH. However, the apparent Kc values were found to decrease with increasing pH.9-NC/HP-β-CD complex was prepared by suspend and freeze-drying method. After adjusting temperature and pH, the 9-NC concentration in water was 0.52mg/ml (40%HP-β-CD, approximately 150-fold higher than that of free 9-NC), which could satisfy the clinical therapeutic dose. The light yellow powder displayed a fast dissolution and stability when dissolved by saline. In addition, hydrolysis of 9-NC following pseudo-first-order kinetics was decelerated significantly in physiologic condition in the presence of HP-β-CD.The in vitro anti-tumor activity was tested by MTT assay. Three kinds of adherent tumor cell lines (Skov-3, MCF-7 and Hela) and one kind of suspended tumor cell line (SI80) were studied. Our study revealed that the 9-NC/HP-β-CD complex showed significant anti-tumor activity towards Skov-3, MCF-7, Hela and S180 cell lines with IC50 values of 0.24±0.09,0.59±0.20,0.83±0.11, and 6.30±2.42μg/ml, respectively, significantly superior to the free 9-NC.The stability tests of 9-NC/HP-β-CD complex were conducted. The studies suggested that the complex should be sealed and stored in dark. The accelerating test indicated, in the condition of 40℃and RH 75%, the complex were stable in 6 months. Long-term test indicated that the complex were stable after stored at room temperature for 12 months.Pharmacokinetic studies of 9-NC formulations in rats indicated that the complex had higher bioavailability and ratio of lactone form in plasma compared to free 9-NC, which suggested that the complex may exhibit better therapeutic efficacy.The in vivo therapeutic efficacy was investigated in ICR mice bearing mouse sarcoma S180. Both the high (3 mg/kg) and low (1 mg/kg) doses of 9-NC/HP-β-CD complex demonstrated high inhibition ratio of tumor growth (>75%).Physiological compatibility tests were also conducted. The results indicated that the complex could be administrated via muscle injection or infusion after dissolved by saline.The subacute toxicity test was performed by measuring the body weight, histopathology, blood cell counts and clinical chemistry parameters (total bilirubin, alanine transferase, aspartate transferase, blood urea nitrogen and creatinine), and the results indicated the good safety profile of the complex. |
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