Takehiko Suzuki

Preparation of gelatin microparticles by co-lyophilization with poly(ethylene glycol): characterization and application to entrapment into biodegradable microspheres

Gelatin microparticles were prepared by co-lyophilization with poly(ethylene glycol) (PEG) as a protein micronization adjuvant. Aqueous solutions containing gelatin and PEG at various mixing ratios were freeze-dried. The lyophilizates were dispersed in methylene chloride and subjected to particle size analysis. The particle size decreased as the PEG/gelatin ratio increased. The microparticles isolated from the suspension had spherical microdomains with sizes ranging from 1 to 10 microm, which indicated that phase separation between PEG and gelatin during freezing was involved in the formation mechanism of gelatin microparticles. By using this technology, gelatin microparticles with an average size of less than 10 microm, with high purity of more than 90% and with good dispersibility could be obtained with high yield. The gelatin microparticles with average sizes from 5 to 20 microm were applied to encapsulation into biodegradable PLGA/PLA microspheres via a solid-in-oil-in-water emulsion process. The entrapment efficiency was highly dependent on the particle size and the size distribution, signifying that solid microparticles with an average diameter of less than 5 m and an maximal diameter of less than 10 microm would be required for effective encapsulation. These gelatin microparticles would be useful for studying and developing various drug delivery systems.

Anti-tumor effect of intraperitoneal administration of cisplatin-loaded microspheres to human tumor xenografted nude mice

This study evaluates the anti-tumor effect of cisplatin-loaded microspheres (CDDP-MS) against peritoneal carcinomatosis using human tumor xenografts. The incorporated CDDP was released from CDDP-MS for 3 weeks in vivo as well as in vitro. CDDP-MS at a dose of 35 mg/kg (at maximal tolerable dose (MTD)) showed effective anti-tumor activity (tumor growth inhibition rate (IR)=70.3%) against Li-7 (human liver cancer) xenografts transplanted into the peritoneal cavity. This procedure also resulted in increased life span (ILS (%)=47.2%), whereas CDDP dissolved in saline solution (CDDP-SOL) at a dose of 8 mg/kg (at MTD) was ineffective (IR=15.7%, ILS=2.6%). Likewise, CDDP-MS (35 mg/kg) significantly prolonged the mean survival time (ILS=50.8%) compared with a CDDP-SOL group (8 mg/kg) (ILS=13.1%) in the mice with Li-7 xenografts transplanted into the spleen. Furthermore, CDDP-MS showed markedly effective anti-tumor activity (IR=82.2%) against H-154 (human stomach cancer) xenografts, in which CDDP-SOL was effective (IR=69.5%) at the MTDs. The suppressive effect of CDDP-MS on accumulation of malignant ascites was intimately related to unchanged CDDP concentration in ascites. These results demonstrated that the administration of CDDP-MS resulted in an unchanged CDDP concentration in ascites, and induced a sustained tumor growth inhibition along with a prolonged survival time.

Organ distribution of cisplatin after intraperitoneal administration of cisplatin-loaded microspheres

The aim of this study was to clarify the organ distribution of cisplatin (CDDP) after intraperitoneal (i.p.) administration of cisplatin-loaded microspheres (CDDP-MS). The distribution of CDDP to normal organs lying in the peritoneal cavity after i.p. administration of CDDP-MS was assessed by comparing with subcutaneous administration to non-cancerous mice. The organ distribution of CDDP after i.p. administration of CDDP-MS shows that CDDP released from microspheres was distributed to the organs lying in the peritoneal cavity and in the retroperitoneum. These are mainly from the systemic circulation, but are not directly from the organ surface. The distribution of CDDP to tumors was evaluated in sarcoma180 tumor-bearing mice by comparing with a bolus injection. The CDDP-MS delivered CDDP to tumors more effectively than did bolus injection. The distribution of CDDP-MS in the peritoneal cavity was in accord with the tumor distribution. This concordance and sustained exposure of CDDP to the tumors might play a critical role in enhancing the CDDP accumulation in tumors. It is concluded that CDDP-MS have a distinct regional pharmacokinetic advantage for peritoneal carcinomatosis, and that i.p. administration of CDDP-MS is an effective treatment for peritoneal carcinomatosis.

Prejunctional inhibition mediated via α2-adrenoceptors in excitatory transmission of canine small intestine

Noradrenaline(NA) inhibited the contractile responses of longitudinal and circular muscles of canine small intestine to field stimulation (20 Hz, 1 ms, supramaximal voltage for 5 s). The purpose of this study was to see if NA‐induced inhibitory action is mediated via α1‐ or α2‐receptors and whether the inhibitory action involved prostaglandins. In both muscle layers the α2‐agonist, clonidine inhibited the contractile response to field stimulation more potently than did the α1‐agonists phenylephrine or methoxamine. It is suggested that NA‐mediated prejunctional inhibition is mediated via α2‐receptors. Yohimbine, an α2‐adrenoceptor blocking drug antagonized NA‐induced prejunctional inhibition, while E‐643, an α1‐blocker, did not affect the inhibition. These results suggest that the inhibitory action of NA is mediated via α2‐receptors in both muscle layers. Indomethacin, an inhibitor of prostaglandin synthesis, did not affect NA‐induced prejunctional inhibition in circular muscle, while augmenting it in longitudinal muscle. It is thought that NA‐induced prejunctional inhibition is not mediated via prostaglandin release.

Effects of prostaglandin E1, I2 and isoproterenol on the tissue cyclic AMP content in longitudinal muscle of rabbit intestine

Effects of prostaglandin E1(PGE1) and prostaglandin I2(PGI2) on the mechanical activity and tissue cyclic AMP content of the longitudinal muscle of rabbit intestine were examined, comparing that of the tissue cyclic AMP content. Isoproterenol caused a relaxation and increased tissue cyclic AMP content.

Relation between dissolution profiles and toxicity of cisplatin-loaded microspheres.

The aim of this study is to evaluate and compare the dissolution profiles of cisplatin-loaded microspheres (CDDP-MS) in vitro and in vivo, and to determine the relationship between the dissolution profiles in vitro and systemic toxicity. For this purpose, three types of CDDP-MS that release the CDDP for 1, 2 and 5 weeks without a large amount of initial release in phosphate buffered saline (pH 7.4) were prepared. The dissolution profiles of these formulations in vivo were well correlated with in vitro studies, and resulted in well-controlled plasma platinum concentration. The systemic toxicity of the CDDP-MS and CDDP dissolved in saline (CDDP-SOL) were assessed by intraperitoneal administration in mice. The maximal tolerable dose (MTD) of CDDP-SOL was 13.4 mg/kg, whereas the CDDP-MS of 1, 2 and 5-week types were 34.6, 44.2, 62.6 mg/kg, respectively. The MTD of CDDP increased proportionally when 50% of CDDP had been released from MS in vitro (MTD (mg/kg)=5.22 x T(50(day)) + 13.2, R(2)=0.9935). We demonstrate that the systemic toxicity of CDDP-MS can be predicted by evaluation of the dissolution rate in vitro since in vivo dissolution was correlated with the in vitro.

Difference between the inhibitory actions of prostaglandin E1 and verapamil in canine small intestine

The effect of prostaglandin (PG) E1 on the contractile responses of canine separated longitudinal and circular muscles to field stimulation, which produced the release of acetylcholine (ACh), was examined and compared with the effect of verapamil. PGE1 inhibited the contractile response of circular but not of longitudinal muscle to field stimulation. However, it did not affect or only slightly inhibited ACh-induced contraction in both muscles. Verapamil inhibited the contractile responses of both muscles to field stimulation, and it also inhibited ACh-induced contraction in both muscles. These results suggest that PGE1 mainly acts on the cholinergic nerve terminals of the circular muscle layer, whereas verapamil influences the smooth muscle cells of both muscle layers. The inhibitory effects of PGE1 and verapamil were partly neutralized by an increase in the external calcium concentration.

Influences of prostaglandins on electrophoretic mobility and aggregation of rabbit platelets.

Influences of prostaglandin (PG)s on electrophoretic mobilities and aggregation of rabbit platelets were studied. The PGs studied (PGI2, PGE1, PGD2, PGF2 alpha, PGA2 and PGA1) had no effect on platelet electrophoretic mobility. However, both PGE1 and PGI2 in 0.3 and 3.0 muM inhibited ADP-induced aggregation and ADP-induced decrease in the mobility. PGD2 in 0.3 and 3.0, and PGE2 in 30 microM inhibited the aggregation but did not depress the ADP-induced decrease in the mobility. PGF2 alpha, PGA2 and PGA1 had no effect on the decrease in electrophoretic mobility and on the aggregation caused by ADP.

Pharmacokinetic behaviour of cisplatin in peritoneal fluid after intraperitoneal administration of cisplatin-loaded microspheres

The objective of this study was to establish a pharmacokinetic model for the estimation of unchanged cis‐dichlorodiammine‐platinum (II) (CDDP) concentration in peritoneal fluid after intraperitoneal administration of cisplatin‐loaded microspheres (CDDP‐MS) and to elucidate the accuracy of this model by comparisons between actual and simulated values after intraperitoneal administration of CDDP‐MS. We developed a method enabling the precise and quick assessment of the drug concentration in the peritoneal cavity. The pharmacokinetic parameters obtained after intravenous bolus injection at a dose of 2 mg kg−1 were total body clearance (1026 mL h−1 kg−1), elimination rate constant (3.24 h−1) and distribution volume of systemic circulation (316.7 mL kg−1). After an intraperitoneal bolus injection at a dose of 5 mg kg−1, the absorption rate constant from the peritoneal cavity (3.64 h−1) and the distribution volume of the peritoneal cavity (13.5 mL kg−1) were determined. The protein‐binding rate constant in ascites was 0.58 h−1. Using these pharmacokinetic parameters, we established a pharmacokinetic model consisting of two compartments. Administration of CDDP‐MS at a dose of 10 mg kg−1, which released CDDP over 7 days in‐vitro, yielded sustained concentrations of unchanged CDDP (1–2 mg mL−1) in the peritoneal cavity that persisted for 7 days, and that were predictable by applying the in‐vitro dissolution profile to the pharmacokinetic model. The findings obtained from this study are useful for understanding the basic pharmacokinetic characteristics of unchanged CDDP in the peritoneal cavity and may also be important in the development of optimized CDDP‐MS formulations.