Pharmacokinetics of prolonged-release CPT-11-loaded microspheres in rats

Journal of Controlled Release 66 (2000) 159–175 www.elsevier.com / locate / jconrel

Pharmacokinetics of prolonged-release CPT-11-loaded microspheres in rats a b, c b Yoshiaki Machida , Hiraku Onishi *, Akinobu Kurita , Harumi Hata , Akinobu Morikawa a , Yoshiharu Machida b a

Department of Pharmacy, Cancer Institute Hospital, 1 -37 -1, Kami-ikebukuro, Toshima-ku, Tokyo 170 -0012, Japan b Department of Clinical Pharmacy, Hoshi University, 2 -4 -41, Ebara, Shinagawa-ku, Tokyo 142 -8501, Japan c Yakult Central Institute for Microbiological Research, 1796, Yaho Kunitachi, Tokyo 186 -8650, Japan Received 18 February 1999; accepted 12 November 1999

Abstract CPT-11-containing microspheres composed of poly-D,L-lactic acid or poly ( D,L-lactic acid-co-glycolic acid) copolymers were prepared by an oil-in-water evaporation method. The size and shape of the microspheres were examined, and the drug release rates were analyzed from the in vitro release profiles. CPT-11 aqueous solution was intravenously or intraperitoneally injected at 10 mg / kg, and microspheres were intraperitoneally administered at 50 mg eq CPT-11 / kg in rats. The microspheres had an average diameter of around 10 mm and their shape was spherical. All the microspheres contained CPT-11 in a lactone form, and their drug contents and release profiles were basically similar to those of previous microspheres. After i.v. injection of CPT-11 solution, the CPT-11 plasma concentration decreased quickly, SN-38 decreased slowly at a much lower level, and SN-38 glucuronide (SN-38G) declined very slowly at a higher level than SN-38. The plasma concentration of CPT-11 reached a maximum at 30 min after i.p. administration of CPT-11 solution. The area under the plasma concentration–time curve (AUC) of CPT-11 after i.p. administration was somewhat lower compared with that after i.v. administration, but the plasma concentration–time profiles of SN-38 and SN-38G were nearly identical between i.v. and i.p. administration. An i.p. administration of the microspheres resulted in gradually increasing or almost constant CPT-11 levels. The levels of SN-38 were also stable during the observation period (4 days) except for the slowest releasing microsphere in which SN-38 was not detected after 24 h following administration. Intraperitoneal administration of any of the microspheres resulted in stable and similar levels of SN-38G after 24 h following administration. When judging from apparent simple pharmacokinetic analysis, an inconsistency was found between the in vitro drug release and the plasma level to a fair extent, but overall the in vivo drug release rate from microspheres was considered parallel to the in vitro one. The microspheres showing a faster release of CPT-11 exhibited higher plasma levels of CPT-11 and SN-38, explaining the previous results that efficacy was better when the in vitro release rate was higher. That the SN-38 level could be attained to a certain extent even at the range of modest or low plasma concentration of CPT-11 in each administration may be related to the non-linear metabolic conversion from CPT-11 to SN-38.  2000 Elsevier Science B.V. All rights reserved. Keywords: Prolonged-release CPT-11-loaded microspheres; Pharmacokinetics; CPT-11; SN-38; Drug release rate

*Corresponding author. 0168-3659 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 99 )00267-9

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1. Introduction 7 - Ethyl - 10 - [4 - (1 - piperidino) - 1 - piperidino]carbonyloxycamptothecin (CPT-11) is a water-soluble semi-synthetic derivative of CPT (camptothecin) which was isolated from Camptotheca acuminata [1]. CPT-11 has marked antitumor effects on many types of tumors [2–4], and it is the prodrug of 7-ethyl-10-hydroxycamptothecin (SN-38) which strongly inhibits DNA topoisomerase I [5–7]. Previously, the microspheres that exhibited prolonged release of CPT-11 were prepared using poly-D,Llactic acid or poly( D,L-lactic acid-co-glycolic acid) copolymers as matrix [8]. These polymers were selected due to their good biodegradability and biocompatibility [9] and because many reports have shown that microspheres made from them release drugs for periods from several days to several weeks [10–15]. The microspheres obtained were previously tested for antitumor characteristics against P388 ascitic tumor and Sarcoma 180 solid tumor, and exhibited marked antitumor activity against P388 ascitic tumor via i.p. administration. However, the microspheres administered intraperitoneally were not significantly effective against Sarcoma 180 solid tumor implanted subcutaneously. Prolonged release of CPT-11 was effective in the i.p.–i.p. system but not in the i.p.–s.c. system. Some reports have shown the in vivo release profile of PIA or PLGA microsphere parallels the in vitro release one [13,14]. Therefore, the in vivo release rate of CPT-11 from microspheres may be predictable to a fair extent from the in vitro release ones. However, since CPT11 is a prodrug, it is necessary to examine the in vivo level of the active metabolite SN-38 and its in vivo behavior. CPT-11 is primarily converted to SN-38 in vivo by carboxyesterase [16], and SN-38 is metabolized mainly to its glucuronide SN-38G [17]. All three compounds are preferentially excreted into bile and urine [17–21]. A correlation between the plasma level of SN-38 and the efficacy of CPT-11 has been demonstrated in effective concentration analysis [5]. Plasma levels of SN-38 affect antitumor efficacy and the presence of toxic side effects [5,18]. Delayed diarrhea is thought to be a dose-limiting toxicity at administration of CPT-11. The relationship among the plasma levels of CPT-11, SN-38 and SN-38G has been studied in terms of CPT-11-in-

duced intestinal toxicities [18,22,23]. Thus, the relation among those three compounds is essential for efficacy and toxic side effect. However, the in vivo behaviors of CPT-11, SN-38 and SN-38G have not been performed very much for the prolonged-release systems of CPT-11. In this study, prolonged-release CPT-11-loaded microspheres were examined in terms of the pharmacokinetics of CPT-11, SN-38 and SN-38G, and their in vivo CPT-11 release rate and the plasma appearance of SN-38 were investigated.

2. Materials and methods

2.1. Materials Irinotecan hydrochloride (CPT-11), 7-ethyl-10-hyroxycamptothecin (SN-38), SN38 glucuronide (SN38G) and camptothecin (CPT) were supplied by Yakult Honsha (Tokyo, Japan). Poly-D,L-lactic acid (MW 10 000), poly( D,L-lactic acid–coglycolic acid), (3:1, mol / mol) copolymer (MW 10 000) and poly( D,L-lactic acid–co-glycolic acid), (1:1, mol / mol) copolymer (MW 10 000) were purchased from Wako Pure Chemical Industries, and used as matrix polymers for microspheres. All other chemicals were of reagent grade.

2.2. Preparation and in vitro characterization of microspheres The microspheres were prepared according to the previous study [8] except that the final temperature was raised to 508C in the evaporation process. CPT11 was suspended in a weakly basic aqueous solution of pH 8.5 and extracted with chloroform, then solidified by solvent evaporation. The obtained CPT11 powder became easily chloroform-soluble, and this powder was used for preparation of microspheres. This CPT-11 (300 mg) and the polymer (1.2 g) were dissolved in chloroform (30 ml), and the solution was dropped into 450 ml of 2% (w / v) gelatin aqueous solution stirred at 600 rpm. The resultant emulsion was gradually heated to 508C and stirred under reduced pressure using an aspirator, and the microspheres formed were separated by centrifugation at 3000 rpm for 10 min. After washing them with sufficient water three times repeatedly by

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centrifugation at 3000 rpm for 10 min, they were dried in a desiccator containing silica gel at room temperature. CPT-11-containing microspheres, prepared using poly-D,L-lactic acid, poly( D,L-lactic acid– coglycolic acid), (1:1, mol / mol) copolymer and poly( D,L-lactic acid–co-glycolic acid) (3:1, mol / mol) copolymer as matrix polymers, were named M0010, M5010 and M7510, respectively. The microspheres were characterized by drug content, particle size and shape by the following procedures. Namely, the microsphere with a certain weight was taken and dissolved in chloroform, and then the drug content was determined by measuring the solution spectrophotometrically at 365 nm.The size and shape of the microspheres were examined using a JEOL JSM T200 scanning electron microscope after the samples ˚ thick. In were coated with a gold layer about 200 A vitro release was examined using a method previously reported [8]. Briefly, the microspheres (10 mg) were suspended in 25 ml of 1 / 15 M phosphatebuffered solution, pH 7.4, and incubated at 60 strokes per min and 378C (n53). At appropriate times, aliquot samples (1 ml) were withdrawn and centrifuged, and the supernatant was diluted with the same buffer and measured spectrophotometrically at 365 nm to determine the amount of released CPT-11. Fresh buffer (1 ml) was added to the centrifuged pellet and the mixture was returned to the incubation mixture. The in vitro release profiles were analyzed using a bi- or monophasic release model, that is, a bi- or mono-exponential release model (see Fig. 5B), in which curve fitting was executed by the nonlinear least-squares program MULTI [24].

2.3. Animal experiments Male Wistar rats (7 weeks old; 180–200 g) were purchased from Tokyo Laboratory Animals Science. They were anesthetized by i.p. injection of urethane at a dose of 0.9 g per 4 ml of saline per kg, and restrained on their backs until the blood sampling time at 8 h in the experiments, when food and water were not supplied. After blood sampling at 8 h, they were kept on the breeding diet MF (Oriental Yeast, Japan) with water ad libitum and blood sampling was executed under light ether anesthesia. The administration, sampling and sample treatment were executed as follows. CPT-11 aqueous solution was

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administered intravenously into the jugular vein at a dose of 10 mg / ml water per kg. Further, CPT-11 aqueous solution was administered intraperitoneally at a dose of 10 mg / ml water per kg. CPT-11containing microspheres were administered into the peritoneal cavity at a dose of 50 mg eq CPT-11 / kg by the following procedure. Namely, an incision of approximately 5-mm length was made in the abdominal skin and then opened, and the microspheres were put into the peritoneal cavity, and then the incision was sewn up with surgical string. Blood samples (0.5 ml) were withdrawn from the jugular vein with a heparinized syringe at appropriate times and centrifuged at 15 000 rpm for 30 s. One-tenth ml of the plasma was transferred to a sample tube, and 0.1 ml of 0.15 M phosphoric acid was added to that plasma sample (0.1 ml), and the mixture was thoroughly vortexed to prevent the conversion of CPT-11 to SN-38 and to change a carboxylate form to a lactone one entirely (see Fig. 5A). The final sample was analyzed by high performance liquid chromatography (HPLC) for the determination of the concentrations of CPT-11, SN-38 and SN-38G.

2.4. HPLC assay To the final sample mixture was added the same volume of 0.15 M phosphoric acid solution containing a known amount of CPT (internal standard). The mixture was injected on a cartridge-C 18 analytichem in the automated solid-phase extraction system (Prospekt; Spark Holland, Emmen, The Netherlands) linked to the HPLC system. After the cartridge was washed with 10 mM phosphoric acid, the compounds, CPT-11, SN-38 and SN-38G in the cartridge were eluted directly from the cartridge to the analytical column with the mobile phase. The HPLC system included a Waters 616 pump and Waters 470 scanning fluorescence detector, and the chromatogram was analyzed with the software program, WATERS MILLENIUM 2010J CHROMATOGRAPHY MANAGER. A Waters symmetry C 18 column P/ N 45905 (4.6mm I.D.3150 mm) with a Waters symmetry C 18 guard column P/ N 54225 (3.9 mm I.D.320 mm) was used at 508C by employing a Waters column heater. The mobile phase was a mixture of 50 mM KH 2 PO 4 – H 3 PO 4 buffer (pH 3.5) and acetonitrile (7:3, v / v) containing 4 mM sodium 1-decanesulfonate. The

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flow-rate was 1.5 ml / min. CPT-11 and SN-38G were detected fluorometrically at 428 nm with excitation at 373 nm. SN-38 was detected fluorometrically at 540 nm with excitation at 380 nm. In these analytical conditions, the lowest detection limits of CPT-11, SN-38 and SN-38G were 1.0 ng / ml except for when especially mentioned.

t-test, and the result was evaluated to be significantly different when p,0.05.

2.5. Pharmacokinetics

The preparation of the microspheres was accomplished faster by raising the evaporation temperature to 508C, which was 58C higher than that in the previous preparation. This temperature increase did not affect the formation of the microspheres, and it caused faster and more complete evaporation of chloroform. The size and shape of the microspheres are shown in Fig. 1. For each microsphere, the mean particle diameter in number was approximately 10 mm and the diameter of the particles ranged from 5 to 40 mm. The microspheres were mostly spherically shaped. The drug contents of M0010, M5010 and M7510 were 7.2, 7.5 and 7.2% (w / w), respectively. These microspheres may be slightly larger than the previous ones [8], but the drug content was similar. Further, the CPT-11 extracted with chloroform and contained in each microsphere was recognized to exist in a lactone form from HPLC study (data not shown). The release profiles of CPT-11 from the microspheres are shown in Fig. 2. The release rates were faster in the order of M5010.M7510.M0010. The released amounts were significantly different ( p, 0.05) except for the difference between M5010 and M7510 at 2 and 7 days. M5010 and M7510 released 65% and 40% (w / w) of their CPT-11 during the first 4 days of incubation, respectively. M0010 had the slowest release at 6% (w / w) after the first 4 days of incubation.The release profile of each microsphere was similar to the previous one [8]. The in vitro release profiles were analyzed using bi- and monoexponential models, which were expressed by the following equations, respectively.

Plasma concentration–time data was used to calculate the area under the plasma concentration– time curve (AUC), mean residence time (MRT) and variance of residence time (VRT). Each value was calculated by the trapezoidal method unless otherwise indicated. The plasma concentration profile at i.v. administration of aqueous CPT-11 solution was analyzed using a tri-exponential model, and the apparent pharmacokinetic parameters were calculated. The plasma concentration profile at i.p. administration of aqueous CPT-11 solution was analyzed by simple linear kinetics using the apparent parameters of absorption efficiency (Fip ) and firstorder absorption rate constant (k a ) linked to transfer from the administration part to the blood circulation with the i.v. parameters to be fixed. The in vivo CPT-11 release from the microspheres was estimated using apparent simple linear kinetics. Each estimated parameter was obtained by curve fitting using the nonlinear least-squares program MULTI [24]. The plasma level of SN-38 and the conversion from CPT-11 to SN-38 for each formulation were also discussed using apparent simple linear kinetics.

2.6. Data analysis The release amounts were compared with one another at each sampling time. The plasma concentrations of CPT-11, SN-38 and SN-38G were compared with one another from 24 h after i.p. administration of the microspheres, and their concentrations were also compared with those given by i.v. and i.p. administration. Further, the moment data (AUC, MRT, VRT ) were compared among each formulation. Every comparison was performed based on the significant difference between the mean values, which were analyzed using the Student’s

3. Results and discussion

3.1. In vitro characteristics

Mb (t) 5 M1 3 [1 2 exp(2k r1 t)] 1 M2 3 [1 2 exp(2k r2 t)] Mm (t) 5 M 3 [1 2 exp(2k r t)],

(1) (2)

where Mb (t) and Mm (t) were release ratios to the

Y. Machida et al. / Journal of Controlled Release 66 (2000) 159 – 175

Fig. 1. Scanning electron photomicrographs of the microspheres, M5010 (A), M7510 (B) and M0010 (C). The length of a white scale bar corresponds to 10 mm in each photomicrograph.

total content at time t for the bi-exponential model and mono-exponential one, respectively, and M1 , M2 and M were the ratios of the released amount to the

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total content, and k r1 , k r2 and k r were first-order rate constants. The bi-exponential model was better fitted for M5010 and M7510, and each model fitted well for M0010 (Fig. 2). The release rate constants were converged according to each analytical mode as shown in Table 1. As the molar ratio of glycolic acid to lactic acid in the polymer nears the maximum hydration state ratio of 7 / 3, a drug generally gets released more easily [9,12–14], possibly explaining the drug release rate order of M5010.M7510. M0010. Drug release kinetics are affected by hydration, degradation and erosion of the matrix polymer. Release of leuprolide acetate (MW 1269) from PLA or PLGA microspheres was controlled mainly by the polymer erosion rate and the release approximated the zero-order release kinetics [13]. The release of such a rather large molecule from PLA or PLGA microspheres may be ruled by the polymer erosion rate probably due to the slow diffusion. The release kinetics of cisplatin (MW 300)-containing PLA microspheres is quite different from that of leuprolide-containing PLA or PLGA microspheres [15]. In the case of cisplatin-containing PLA microspheres,the diffusion from the matrix seems important for the drug release. As to the PLA or PLGA microspheres containing CPT-11 (MW 587), the release could be expressed approximately by the pseudo-first order kinetics or the summation of pseudo-first order kinetics (Fig. 2, Table 1). Since the molecular weight of CPT-11 is much smaller than leuprolide but larger than cisplatin, the diffusion as well as the polymer erosion may be related to the release. Since each microsphere contains CPT-11 in a lactone form, the CPT-11 was exposed to the medium in a lactone form. When the contained CPT11 is exposed in the medium and possibly retained for some period in the microspheres, it may undergo the exchange between lactone and carboxylate. Therefore, the form of CPT-11 released out from the microspheres may not be equivalent completely. This situation will be noted in the following Sections 3.2 and 3.3. However, the release results have demonstrated that the better-swelled microspheres can release CPT-11 faster even if such exchange between lactone and carboxylate is possibly caused. Also, the present microspheres showed release profiles similar to previous ones prepared at a temperature a little lower.

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Fig. 2. In vitro release profiles of CPT-11 from the microspheres, M5010 (A), M7510 (B) and M0010 (C). s: observed concentration of CPT-11; each point represents the mean6S.D. (n53). The dotted or broken lines are profiles calculated using Eqs. (1) or (2) based on parameters in Table 1; - - -: bi-exponential model, – ? – ?: mono-exponential (mode 1) model, – – –: mono-exponential (mode 2) model.

3.2. Plasma concentration After CPT-11 was administered intravenously and intraperitoneally, the levels of CPT-11, SN-38 and SN-38G were examined for 72 and 96 h, respectively. The plasma concentration–time courses are shown in Fig. 3. At i.v. administration, the levels of CPT-11 and SN-38G were higher than that of SN-38. The higher concentration of SN-38G indicates the rapid glucuronidation of SN-38. The level of SN38G was higher than those of CPT-11 and SN-38

from 24 h after administration. Maximum levels of CPT-11 and SN-38G were found at 30 min after i.p. administration of CPT-1 1, and after these peaks their plasma levels declined in a similar fashion as i.v. administration, suggesting rapid transfer of CPT11 from the injection site into the blood circulation. The concentration of SN-38 was a little higher than that of SN-38G at 15 min after i.p. administration but was then lower after that. The level of SN-38G was higher than those of CPT-11 and SN-38 from 24 h after administration as was the case for i.v. adminis-

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Table 1 Release parameters of microspheres using release models based on in vitro and in vivo release of CTP-11 a Release model

Parameter

Microsphere M5010

M7510

M0010

Bi-exponential

M1 k r1 (h 21 ) M2 k r2 (h 21 )

0.5401 0.0410 0.4599 0.00201

0.3654 0.0332 0.6346 0.00115

0.7702 0.000923 0.0462 0.000198

Mono-exponential (model 1)

M k r (h 21 )

0.7537 0.0231

0.5842 0.0146

0.7940 0.000906

Mono-exponential (mode 2)

M k r (h 21 )

1.0 0.00823

1.0 0.00306

1.0 0.000691

a

M1 1M2 was fixed to 1.0 if M1 1M2 was far from 1.0; M was fixed to 1.0 in the mono-exponential (mode 2). The models were handled by Eqs. (1) and (2) in Section 3.1.

tration. As to CPT-11, the AUC0 – 72 h value at i.p. administration was smaller to that at i.v. administration (Table 2), though not significantly. This suggests the transfer of CPT-11 to the blood circulation after i.p. administration may not be complete. The levels of SN-38 during the observation were not significantly different between i.p. and i.v. administrations. The levels of SN-38G were also not significantly different between i.v. and i.p. administrations, except for the initial time from 0 to 30 min after administration. The MRT 0 – 72 h and VRT 0 – 72 h

values of CPT-11, SN-38 and SN-38G were not significantly different between i.p and i.v. administrations (Table 2). Fig. 4 shows the plasma levels of CPT-11, SN-38 and SN-38G, respectively, after i.p. administration of the microspheres. The plasma levels of CPT-11 were significantly higher ( p,0.05) in the order of M5010.M7510.M0010 from 24 h after administration, probably due to the difference in the drug release rates. The order was parallel with the in vitro release result. The AUC0 – 72 h values were also

Fig. 3. Plasma concentration–time profiles of CPT-11 (d), SN-38 (m) and SN-38G (j) after i.v. (A) or i.p. (B) administration of CPT-11 aqueous solution at 10 mg / kg. Each point represents the mean6S.E. (n55 except for n54 at 0.25 h of i.v. administration). a p,0.001 vs. SN-38. b p,0.01 vs. SN-38. c p,0.001 vs. CPT-11 and SN-38. d p,0.05 vs. CPT-11 and SN-38. e p,0.05 vs. SN-38.

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Table 2 Moment values of CTP-11, SN-38 and SN-38G at 0–72 h after i.v. and i.p. administration of CTP-11 aqueous solution at 10 mg / kg to rats and after i.p. administration of the microspheres at 50 mg eq CTP-11 / kg to rats a Moment

Species

Formulation CPT-11 solution (i.v.) (10 mg / kg)

CTP-11 solution (i.p.) (10 mg / kg)

M5010 (50 mg eq CPT-11 / kg)

M7510 (50 mg eq CPT-11 / kg)

M0010 (50 mg eq CPT-11 / kg)

AUC 0 – 72 h b (ng h / ml)

CPT-11 SN-38 SN-38G

10 40061700 423668 43206510

68506700 373688 45206880

47006430 163628 22106130

17406200 143614 19206310

675646 24.864.7 18306450

c

CPT-11 SN-38 SN-38G

4.6160.55 10.461.0 28.462.4

8.8760.72 8.1662.37 27.464.4

44.560.6 37.261.4 43.26130

32.860.8 30.861.4 42.560.6

25.462.4 3.5360.64 42.162.1

d

CPT-11 SN-38 SN-38G

74.1615.1 123637 407645

242632 102671 354651

475665 687662 307620

559617 587612 454624

744683 5.0061.82 346638

MRT 0 – 72 (h)

h

VRT 0 – 72 (h 2 )

h

a

Each value is expressed as the mean6S.E. (n55 except that n54 for M0010). Significantly different: (CPT-11) i.v., i.p..each microsphere; M5010.M7510, M0010; M7510.M0010: (SN-38) i.v., i.p..each microsphere; M5010, M7510.M0010: (SN-38G) i.v..each microsphere; i.p..M7510, M0010. c Significantly different (CPT-11) i.v.,i.p., each microsphere; i.p.,each microsphere; M5010.M7510, M0010; M7510.M0010: (SN-38) i.v..M0010; i.v., i.p.,M5010, M7510; M5010.M7510, M0010; M7510.M5010: (SN-38G) i.v., i.p..each microsphere. d Significantly different (CPT-11) i.v.,i.p., each microsphere; i.p.,each microsphere; M0010.M5010: (SN-38) i.v..M0010; i.v., i.p.,M5010, M7510; M0010,M5010, M7510: (SN-38G) M7510.M5010, M0010. b

significantly larger ( p,0.01) in the order of M5010.M7510.M0010. Some reports have shown that the in vivo release profiles of PLA and PLGA microspheres parallels their in vitro release profiles supporting our observations. From 24 h after administration, M5010 showed the significantly higher ( p,0.05) concentration of CPt-11 than intravenously and intraperitoneally injected CPT-11 solution. M7510 showed the significantly higher ( p0.05) concentration of CTP-11 than intravenously injected CPT-11 solution at 48 and 72 h after administration. The level of CPT-11 tended to be higher in the injection of M0010 than in the i.v. injection of the CPT-11 solution at 72 h after administration, while the CTP-11 level tended to be lower in the injection of M0010 than in the i.p. injection of the CPT-11 solution from 24 h after administration. These results indicate all the microspheres supply CPT-11 continuously in the systemic circulation. This thought is further supported by the large values of MRT 0 – 72 h and VRT 0 – 72 h (Table 2). M5010 and M7510 maintained detectable levels of SN-38 in the blood circulation throughout the ob-

servation period. After 48 h following administration, they showed the higher level of SN-38 than intravenously administered CPT-11 solution. However, administration of M0010 resulted in undetectable levels of SN-38 after 24 h following administration. The AUC0 – 72 h value of SN-38 for M0010 was 1 / 7 and 1 / 6 of those for SN-38 for M5010 and M7510, respectively (both significantly different, p,0.01). The release of CPT-11 from M0010 may be too slow to give sufficient levels of SN-38 due to fast metabolic reaction from SN-38 to SN-38G. This suggests M5010 and M7510 should be more available than M0010 against tumors localized apart from the administration site. Although M5010 and M7510 showed significantly smaller AUC0 – 72 h values of SN-38 than intravenously and intraperitoneally administered CPT-11 solution ( p,0.05), they exhibited significantly larger MRT 0 – 72 h and VRT 0 – 72 h values of SN-38 ( p,0.01), indicating M5010 and M7510 could supply SN-38 to the systemic circulation for a prolonged period. All the microspheres showed similar and constant levels of SN-38G during the observation period. The

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Fig. 4. Plasma concentration profiles of CPT-11 (A), SN-38 (B) and SN-38G (C) after i.v. (s) and i.p. (n) administration of CPT-11 at 10 mg / kg to rats and after i.p. administration of M5010 (d) or M7510 (j) M0010 (m) at 50 mg eq CPT-11 / kg to rats. Each point represents the mean6S.E. (n55 for M5010 and M7510; n54 for M0010 except for n53 at 24 h of M0010). In A, a p,0.05 vs. M7510, M0010 and CPT solution (i.v.) and CPT-11 solution (i.p.); b p,0.01 vs. M0010; c p,0.05 vs. M0010 and CPT-11 solution (i.v.); d p,0.01 (lower) vs. CPT-11 solution (i.v.) and CPT-11 solution (i.p.); e p,0.05 vs. CPT-11 solution (i.v.) and M0010. In B, a p,0.05 (lower) vs. CPT-11 solution (i.v.); b p,0.05 vs. CPT-11 solution (i.v.); c p,0.05 vs. CPT-11 solution (i.p.). In C, a p,0.01 vs. each microsphere; b p,0.05 vs. M5010. c p,0.05 vs. M7510.

moment values of SN-38G were also similar among all the microspheres (Table 2). All the microspheres showed significantly lower levels of SN-38G until 24 h after administration ( p,0.05) when compared with intravenously and intraperitoneally administered CPT-11 solution, but exhibited similar levels of SN38G from 48 h. As for M5010 and M7510, the ratio

of SN-38 concentration to SN-38G one was maintained around 1 / 10–1 / 20 after 24 h following administration and the ratio of the AUC0 – 72 h value of SN-38 to that of SN-38G was also about 1 / 14. On the other hand, the ratio of the AUC0 – 72 h value of SN-38 to that of SN-38G for M0010 was much lower (1 / 74), because the concentration of SN-38

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was lower than the detectable level after 24 h following administration. The plasma levels of CPT-11, SN-38 and SN-38G in each microsphere were higher in the order of CPT-11.SN-38G.SN-38 for M5010, of SN-38G. CPT-11.SN-38 for M7510, and of SN-38G.CPT11.SN-38 for M0010. The plasma levels of CPT11, SN-38 and SN-38G tended to be higher in M5010 and M7510 than in M0010, which suggests that the in vivo release should be parallel with the in vitro release. Antitumor effect is decisively dependent on the concentration of SN-38; for example, the ED 50 value of SN-38 against L1210 cells is around 1 ng / ml, and it has been reported to be important for good efficacy to maintain the plasma level of SN-38 above the ED 50 value [5]. Also, as stated in the biliary index theory by Gupta et al. [18] in which the index is given by (AUCCPT-11 3 AUCSN-38 /AUCSN-38G ), diarrhea gets more severe when the plasma levels of SN-38 increases compared to SN-38G. Therefore, M0010 should cause the least toxicity; however, it is probably not effective against distal tumors due to the very low SN-38 concentration. On the contrary, the i.p. administration of M5010 and M7510 maintained plasma levels of SN-38 above or near 1 ng / ml longer than the i.v. and i.p. administration of CPT-11 solution. Further, the biliary index is smaller in i.p. administration of M5010 and M7510 than in i.v. and i.p. administration of CPT-11 solution. Therefore, M5010 and M7510 (50 mg eq CPT-11 / kg) may exhibit good antitumor characteristics. However, since the microspheres supply the drug for long periods, the biliary index for a long-term drug exposure will have to be examined. Diarrhea is related mainly to biliarily excreted SN-38 and SN38G [18,23]. Their prolonged biliary excretion may suppress the recovery of intestinal damage and cause more severe diarrhea. Actually, although acute lethal toxicity was lower in the microspheres than in CPT11 solution, toxic side effects such as diarrhea and non-lethal decreases in body weight were observed much longer in the microspheres [8]. Also, the discussion stated above has been stated based on the total amount of SN-38, but the problem that the possible exchange between lactone and carboxylate [25] may make their balance different from the balance in i.v. and i.p. administration of CPT-11

solution where CPT-11 exist in a lactone form (data not shown). Namely, it implies that the drug released from microspheres may not be equivalent to that observed from the CPT-11 injected intravenously and intraperitoneally from the viewpoint of the structural form, i.e. lactone or carboxylate. Therefore, the problem of the kind of their structural forms may restrict the direct comparison of the plasma data from the microspheres with those from i.v. or i.p. injection of the CPT-11 solution. These unknown features may have to be analyzed clearly for more exact evaluation of plasma concentration from prolonged-release systems such as these microspheres.

3.3. Apparent pharmacokinetics Relatively short (612 h) plasma concentration– time profiles of CPT-11 after administration have been analyzed by mono-exponential and bi-exponential equations corresponding to the one- and twocompartment model, respectively [5,17,26]. Since a long-term plasma concentration–time course obtained after i.v. administration of CPT-11 had hardly been investigated, it was examined. As a result, a tri-exponential equation apparently fitted well (see Fig. 6A). When aqueous CPT-11 solution was injected intravenously, the plasma concentration equation was obtained as follows: CCTP-11 (t) 5 4450 3 exp(20.761t) 1 443 3 exp(20.135t) 1 6.44 3 exp(20.00813t)

(3)

where CCPT-11 (t) was the concentration (ng / ml) at time t (h) and where CPT-11 was introduced in a lactone form (data not shown). CPT-11 is excreted to a fair extent with no metabolism, and some pa0rt undergoes enzymatic cleavage by carboxyesterase to give SN-38. By assumption of an apparent simple linear conversion from CPT-11 to SN-38, the plasma profiles of CPT-11 and SN-38 after i.v. injection of CPT-11 can give that conversion rate by considering their individual pharmacokinetics. The SN-38 plasma concentration–time course after i.v. injection of SN38 solution in DMSO / H 2 O at the dose of 4 mg / kg was apparently expressed as follows (data not shown).

Y. Machida et al. / Journal of Controlled Release 66 (2000) 159 – 175

CSN-38 (t) 5 16 400 3 exp(216.07t) 1 276 3 exp(21.38t) 1 1.23 3 exp(20.163t) (4) where CSN-38 (t) was the concentration (ng / ml) at time t (h) and where SN-38 was introduced in a lactone form (data not shown). The scheme on translocation and conversion may be provided as shown in Fig. 5B. The excretion rate constant of CPT-11 with no metabolism is expressed as k b2 and the metabolic rate constant is shown with k b1 in an apparent linear kinetic model (Fig. 5B). An apparent conversion rate constant (k b1 ) was calculated by regression using Eqs. (3) and (4). The result is described in Table 3. The pharmacokinetics after i.p. administration of CPT-11 solution were analyzed using the apparent parameters of the absorption efficiency (Fip ) and absorption rate constant (k a ) from the administration site to the blood circulation (Fig. 5). The other parameters were obtained by handling tri-exponential Eq. (3) with a 3-compartment model. As a result, Fip and k a were obtained as shown in Table 3. When only k a was used as a variable parameter without considering Fip , the calculated profile could not be fitted well to the observed one. Fip is considered to reflect the transfer efficiency of CPT-11 from the administration site to systemic circulation. CPT-11 was introduced intrapenitoneally in a lactone form, but the exchange between lactone and carboxylate may occur intraperitoneally to some extent before transfer into blood circulation, and it may affect the plasma concentration profile in addition to the transfer efficiency. In i.v. injection of CPT-11 (Fig. 6A), the CPT-11 profile given by Eq. (3) fitted well to the observed one. The SN-38 profile calculated using k b1 of 0.02928 fitted well at the initial stage, but lower than the observed profile at the moderate and low concentration range in the latter stage. As regards i.v. administration of CPT-11, the conversion rate (R c , ng / h) from CPT-11 to SN-38 could be calculated by the deconvolution technique as shown in Fig. 7A. The observed conversion rate (solid line) was initially high, which was considered to be due to rapid generation of SN-38 occurring prior to reaching the enzymatic equilibrium [27].

169

When CCPT-11 was the CPT-11 concentration at the conversion rate of R c , the value of R c /(CCTP-11 3 Vd ) corresponding to the conversion rate constant in the linear system was compared with k b1 of 0.02928. The result is shown in Fig. 7B. At the moderate and low concentration range of CPT-11, i.e. in the latter stage after administration, the R c /(CCTP-11 3Vd ) was near 0.077 (h 21 ), which was larger than k b1 of 0.02928 (h 21 ). The metabolic conversion rate was approximated to the Michaelis–Menten kinetics equation as follows. R c 5 (Vm 3 CCPT-11 3Vd ) /(Km 1 CCTP-11 )

(5)

where Vd was the distribution volume of CPT-11 and where Vm and Km were constants. Eq. (5) could be rewritten to the following form. CCPT-11 5Vm 3 [(CCPT-11 3Vd ) /R c ] 2 Km

(6)

The plots of [(CCPT-11 3Vd ) /R c ] vs. CCPT-11 presented Vm as a slope of the curve and Km as an intercept. As a result, Vm and Km were determined as 92.7 (ng / ml / h) and 822.8 (ng / ml), respectively. The R c /(CCPT-11 3Vd ) values were obtained for the observed R c values and those calculated by the Michaelis–Menten kinetics [Eq. (5)] as described in Fig. 7B. The discrepancies between the calculated and observed profiles of R c or R c /(CCPT-11 3Vd ) are supposed due to rapid generation of SN-38 prior to the enzymatic equilibrium [27]. When the data at 0–0.5 h were removed at the calculation, the better profile was obtained (Fig. 7), when Vm and Km were determined as 71.5 (ng / ml / h) and 844.6 (ng / ml), respectively. In i.p. administration of CPT-11 (Fig. 6B), the plasma concentration of CPT-11 was simulated well using the calculated parameters. As regards SN-38, the profile calculated by k b1 of 0.02928 was lower than the observed one at the low concentration range in the latter stage as was the case of i.v. injection. The latter curve fitted better when k b1 was changed to 0.077 which was close to the R c /(CCPT-11 3Vd ) value observed at the moderate and low concentration range of CPT-11. This is considered to reflect the non-linear metabolic conversion from CPT-11 to SN-38 [17,27]. The in vivo release from the microspheres was analyzed with a bi- or mono-exponential model as

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Fig. 5. Exchange scheme between a lactone form and a carboxylate one (A) and the apparent pharmacokinetic model for translocation and conversion (B). k r , k r1 , k r2 , k a , k b1 and k b2 : first order rate constants. Fip : absorption efficiency from the i.p. administration site to blood circulation. M, M1 and M2 : mass fraction contributing to a first order release of CPT-11 in the microspheres. The broken arrow means the minor pathway.

Y. Machida et al. / Journal of Controlled Release 66 (2000) 159 – 175 Table 3 Estimated parameters on i.p. absorption of CPT-11, conversion from CPT-11 to SN-38, and in vivo release of CPT-11 from microspheres based on the compartment model Parameter k a (h 21 ) Fip k b1 (h 21 ) Ma

k r (h 21 )a

Microsphere species

Estimated value

M5010 M7510 M0010 M5010 M7510 M0010

2.880 0.5201 0.02928 1.0 1.0 1.0 0.003913 0.001691 0.000914

a

The parameters were well converged only when the monoexponential (mode 2) model, described in the in vitro model analysis (Table 1), was used for fitting.

was the case of in vitro release. Assuming that the process from drug release to elimination in the central compartment followed simple linear kinetics, a good conversion was obtained only in the case of using a mono-exponential model with the parameter M fixed to 1.0 for each microsphere. At that time, the calculated in vivo release rate constant (k r ) is shown in Table 3, and the equation for the CPT-11 plasma concentration [CCPT-11 (t), (ng / ml)] at i.p. administration of the microsphere is expressed as follows.

O [A 3 exp(2l t)], 5

CCPT-11 (t) 5

i

i

constant (solid line) was near the curve calculated from the in vitro release rate constant (broken line). As stated above in Section 3.2., in this approach, the form of CPT-11, the exchange between lactone and carboxylate possibly occurs before release out of the microspheres to some extent [25]. However,the exclusive existence of CPT-11 in the lactone form in the microspheres is favorable for analysis using Eq. (3) because that equation was obtained after the i.v. injection of CPT-11 in a lactone form, and also it may enable the simulation using Eq. (7) to be applicable. Thus, the direct comparison of the plasma data from the microspheres with those from i.v. and i.p. injections of the CPT-11 solution may not necessarily lead to exact evaluation; though the plasma level of CPT-11 is considered to reflect the in vivo release rate as a whole. Namely, since the plasma levels were higher overall in the order of M5010.M7510.M0010, the in vivo release rates are considered faster in the same order. By assuming the simple linear kinetics, the plasma concentration profile of SN-38 for each microsphere was simulated from CPT-11 plasma concentration Eq. (7) for the microsphere, its conversion rate constant (k b1 ) and the pharmocokinetic parameters of SN-38 itself. At that time, the equation of the plasma concentration of SN-38 [CSN-38 (t), (ng / ml)] for each microsphere is expressed as follows.

SO 8

(7)

i 51

where (A 1 , A 2 , A 3 , A 4 , A 5 , l1 , l2 , l3 , l4 , l5 )5 (281.28, 236.10, 215.58, 109.88, 23.08, 0.761, 0.135, 0.00813, 0.00391, 2.880), (235.02, 215.34, 24.41, 44.8, 9.96, 0.761, 0.135, 0.00813, 0.00169, 2.880) and (218.92, 28.25, 22.13, 23.91, 5.39, 0.761, 0.135, 0.00813, 0.000914, 2.880) for M5010, M7510 and M0010, respectively, and where t is time (h); for units, A i : ng / ml, li : h 21 . The curves simulated by Eq. (7) did not fit well to the observed ones in the initial phase but fitted to the observed ones to a fair extent in the latter phase (Fig. 6C). As regards M5010 and M7510, the curve simulated by in vitro release rate constant (broken line) exhibited considerably higher levels than the observed ones. On the other hand, concerning M0010, the profile simulated by Eq. (7) with in vivo release rate

171

CSN-38 (t) 5 k b1 3

i 51

D

[Bi 3 exp(2ui t)] ,

(8)

where (B1 , B2 , B3 , B4 , B5 , B6 , B7 , B8 , u1 , u2 , u3 , u4 , u5 , u6 , u7 , u8 )5(20.369, 15.150, 0.360, 262.889, 223.854, 29.779, 68.917, 12.464, 16.068, 1.379, 0.163, 0.761, 0.135, 0.00813, 0.00391, 2.880), (20.159, 6.536, 0.154, 227.097, 210.136, 22.767, 28.088, 5.382, 16.068, 1.379, 0.163, 0.761, 0.135, 0.00813, 0.00169, 2.880) and (20.086, 3.532, 0.083, 214.638, 25.449, 21.335, 14.984, 2.909, 16.068, 1.379, 0.163, 0.761, 0.135, 0.00813, 0.00169, 2.880) for M5010, M7510 and M0010, respectively, and where k b1 is a conversion rate constant from CPT-11 to SN-38 and t is time (h); for units, k b1 : h 21 , Bi : ng?h / ml, ui : h 21 . The curves simulated by Eq. (8) are described in Fig. 6D. As a result, the level of SN-38 seems to be underestimated. When the conversion rate constant was changed to k b1 of 0.077

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Fig. 6. Plasma concentration–time profiles of CPT-11 and SN-38 simulated based on the apparent pharmacokinetics. (A) I.v. administration of CPT-11 aqueous solution at 10 mg / kg, (B) i.p. administration of CPT-11 aqueous solution at 10 mg / kg, (C) CPT in i.v. administrations of microspheres at 50 mg eq CTP-11 / kg, D: SN-38 in i.v. administrations of microspheres at 50 mg eq CTP-11 / kg. d: observed concentration of CPT-11 (mean6S.E.), m: observed concentration of SN-38 (mean6S.E.). ———: CPT-11 concentration profile simulated by Eq. (3) or (7), - - -: SN-38 concentration profile simulated using k b1 of 0.02928 (h 21 ), – ? – ?: SN-38 concentration profile simulated using k b1 of 0.077 (h 21 ). As regards the profiles simulated based on in vitro release of CPT-11 in C, s: biexponential, h: mono-exponential (mode 1), n: mono-exponential (mode 2).

Y. Machida et al. / Journal of Controlled Release 66 (2000) 159 – 175

173

Fig. 7. Conversion rate from CPT-11 to SN-38 (A) and its coefficient (B) after i.v. administration of CPT-11 solution at 10 mg / kg to rat. ———: observed, - - -: calculated by k b1 of 0.02928 (h 21 ), – – –: calculated by Michaelis–Menten equation at Vm of 92.7 (ng / ml / h) and Km of 822.8 (ng / ml), – ? – ?: calculated by Michaelis–Menten equation at Vm of 71.5 (ng / ml / h) and Km of 844.6 (ng / ml). In A, the fine dots (? ? ? ? ? ? ? ? ?) express the step function of observed rate.

which was close to the R c /(CCPT-11 3Vd ) value observed at the moderate and low concentration range of CPT-11, the simulated curves fitted better to the observed profiles. This is considered to be due to the non-linear conversion from CPT-11 to SN-38 as stated in apparent kinetic analysis for the i.v. and i.p. injections of CPT-11 solution. Since the question whether the form of CPT-11 released is lactone or carboxylate remains as stated above, this approach is considered to present the apparent kinetics. The kinetics suggest that the conversion rate from CPT11 to SN-38 after i.p. administration of each microsphere should be apparently similar to that after the i.v. and i.p. injections of CPT-11 solution. As a result, the long residence of CPT-11 at a modest plasma concentration leads to maintaining the plasma concentration of SN-38 to a certain level. The conversion rate from CPT-11 to SN-38 after i.p. administration of the microsphere is apparently similar to that after the i.v. and i.p. injections of CPT-11 solution; though the CPT-11 released out possibly undergoes the exchange between lactone and carboxylate as stated above. Fig. 8 shows the amounts of CPT-11 in the absorption compartment after i.p. administration of CPT-11 at 50 mg eq CPT-11 / kg, which are simulated from the apparent i.p. absorption rate constant

of CPT-11 and the apparent in vivo release rate constant from the microspheres. As regards the CPT11 solution, CPT-11 is quickly eliminated from the absorption compartment, while the fair amount of

Fig. 8. Free CPT-11 amount–time profiles in the absorption compartment simulated based on apparent pharmacokinetics. The profiles were simulated for i.p. administrations at the dose of 50 mg eq CPT-11 / kg using the parameters described in Table 3. - - -: CPT-11 solution, – – –: M5010, — — — : M7510, – ? – ?: M0010.

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free CPT-11 is maintained for a long time in the absorption compartment for each microsphere. Thus, the prolonged-release system seems to maintain the amount of free CPT-11 to a certain level in the absorption compartment much longer than the bolus injection of CPT-11 solution. Further, it suggests that the prolonged drug release of active drugs from the microspheres that M5010 and M7510 exhibited, resulted in the good antitumor effect against intraperitoneal P388 leukemia as previously reported [8]. Since each microsphere contains CPT-11 in the lactone form as stated above, the active form, lactone, is primarily exposed to the physiological solution permeating the microspheres. Therefore, more amounts of CPT-11 should be released in the active form than expected from the equilibrium state of the CPT-11 forms [25]. Thus, these microspheres are considered to exhibit better efficacy against intraperitoneal tumor by maintaining a higher level of free CPT-11 in the active form in the absorption compartment.

4. Conclusion M5010 exhibited the highest plasma levels of CPT-11 and SN-38, being consistent with the previous results showing M5010 had the greatest antitumor effect [8]. The plasma level of an active metabolite SN-38 is considered most importantly related to the efficacy of CPT-11-containing formulations [5]. M0010 did not provide a sufficient concentration of SN-38. Intraperitoneal administration of CPT-11 resulted in lower plasma levels of CPT-11 than i.v. administration of CPT-11. This could be treated by an apparent parameter of absorption efficiency from the administration site to systemic circulation. The order of the plasma levels for CPT11, SN-38 and SN-38G was varied among M5010, M7510 and M0010. Although M5010 and M7510 showed lower AUC0 – 72 h of SN-38 than CPT-11 solution, they maintained the levels of SN-38 longer. The apparent pharmacokinetic approach suggested that the in vivo release rate of CPT-11 should be higher in the order of M5010.M7510.M0010 as observed in the in vitro release. The long residence of CPT-11 at a modest plasma concentration was found to lead to maintenance of the plasma con-

centration of SN-38 to a certain level. These results propose that M5010 and M7510 may have a good therapeutic potency as prolonged-release CPT-11loaded microparticulate systems. Each microsphere contained CPT-11 exclusively in a lactone form. Since the exchange between lactone and carboxylate is possibly caused before release out from the microspheres, the present pharmacokinetic approach may be apparent and present the apparent parameters. However, the present approach handling the total concentrations of lactone and carboxylate for CPT-11 or SN-38 can give the qualitative characteristics on translocation and conversion. The pharmacokinetics of both lactone and carboxylate forms will be required to be analyzed in order to more exactly evaluate these prolonged-release systems in terms of plasma concentrations.

References [1] K. Nitta, T. Yokokura, S. Sawada, T. Kunimoto, T. Tanaka, N. Uehara et al., Antitumor activity of novel derivatives of camptothecin, Jpn. J. Cancer Chemother. Part II 14 (1987) 850–857. [2] T. Tsuruo, T. Matsuzaki, M. Matsushita, H. Saito, T. Yokokura, Antitumor effect of CPT-11, a new derivative of camptothecin, against pleiotropic drug-resistant tumors in vitro and in vivo, Cancer Chemother. Pharmacol. 21 (1988) 71–74. [3] T. Furuta, T. Yokokura, M. Mutai, Antitumor activity of CPT-11 against rat Walker 256 carcinoma, Jpn. J. Cancer Chemother. 15 (1988) 2757–2760. [4] T. Furuta, T. Yokokura, Effect of administration schedules on antitumor activity of CPT-11, a camptothecin derivative, Jpn. J. Cancer Chemother. 17 (1990) 121–130. [5] N. Kaneda, H. Nagata, T. Furuta, T. Yokokura, Metabolism and pharmacokinetics of the camptothecin analogue CPT-11 in the mouse, Cancer Res. 50 (1990) 1715–1720. [6] Y. Kawato, M. Aonuma, Y. Hirota, H. Kuga, K. Sato, Intracellular roles of SN-38, a metabolite of the camptothecin derivative CPT-11, in the antitumor effect of CPT-11, Cancer Res. 51 (1991) 4187–4191. [7] K. Okada, T. Ando, Cancer chemotherapy: Topoisomerases I and II, and the inhibitors to them, Antibiot. Chemother. 9 (1993) 221–228. [8] Y. Machida, H. Onishi, A. Morikawa, Y. Machida, Antitumor characteristics of irinotecan-containing microspheres of polyD,L-lactic acid or poly( D,L-lactic acid-co–glycolic acid) copolymers, S.T.P. Pharma Sci. 8 (1998) 175–181. [9] D.K. Gilding, A.M. Reed, Biodegradable polymers for use in surgery, polyglycolic / poly(lactic acid) homo- and copolymers: 1, Polymer 20 (1979) 1459–1464.

Y. Machida et al. / Journal of Controlled Release 66 (2000) 159 – 175 [10] N. Wakiyama, K. Juni, M. Nakano, Preparation and evaluation in vitro of polylactic acid microspheres containing local anesthetics, Chem. Pharm. Bull. 29 (1981) 3363–3368. [11] K. Juni, J. Ogata, M. Nakano, T. Ichihara, K. Mon, M. Akagi, Preparation and evaluation in vitro and in vivo of polylactic acid microspheres containing doxorubicin, Chem. Pharm. Bull. 33 (1985) 313–318. [12] Y. Ogawa, M. Yamamoto, S. Takada, H. Okada, T. Shimamoto, Controlled-release of leuprolide acetate from polylactic acid or copoly(lactic / glycolic) acid microcapsules: Influence of molecular weight and copolymer ratio of polymer, Chem. Pharm. Bull. 36 (1988) 1502–1507. [13] Y. Ogawa, H. Okada, M. Yamamoto, T. Shimamoto, In vivo release profiles of leuprolide acetate from microcapsules prepared with polylactic acids or copoly(lactic / glycolic) acids and in vivo degradation of these polymers, Chem. Pharm. Bull. 36 (1988) 2576–2581. [14] T. Heya, H. Okada, Y. Ogawa, H. Toguchi, Factors influencing the profiles of TRH release from copoly( D,L-lactic / glycolic acid) microspheres, Int. J. Pharm. 72 (1991) 199–205. [15] T. Sasabe, An experimental study of lactic acid oligomer microspheres incorporating cisplatin administered intraperitoneally, J. Kyoto Prefectual Univ. Med. 103 (1994) 339– 348. [16] T. Satoh, M. Hosokawa, R. Atsumi, W. Suzuki, H. Hakusui, E. Nagai, Metabolic activation of CPT-11, 7-ethyl-10-[4-(1piperidino)-1-piperidino]carbonyloxycamptothecin, a novel antitumor agent, by carboxyesterase, Biol. Pharm. Bull. 17 (1994) 662–664. [17] N. Kaneda, T. Yokokura, Nonlinear pharmacokinetics of CPT-11 in rats, Cancer Res. 50 (1990) 1721–1725. [18] E. Guputa, T.M. Lestingi, R. Mick, J. Ramirez, E.E. Vokes, M.J. Ratain, Metabolic fate of irinotecan in humans: correlation of glucuronidation with diarrhea, Cancer Res. 54 (1994) 3723–3725. [19] L.P. Rivory, J. Robert, Identification and kinetics of a bglucuronide metabolite of SN-38 in human plasma after

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

175

administration of the camptothecin derivative irinotecan, Cancer Chemother. Pharmacol. 36 (1995) 176–179. E. Gupta, X. Wang, J. Ramirez, M.J. Ratain, Modulation of glucuronidation of SN-38, the active metabolite of irinotecan, by valproic acid and phenobarbital, Cancer Chemother. Pharmacol. 39 (1995) 440–444. K. Takasuna, T. Hagiwara, M. Hirohashi, M. Kato, M. Nomura, E. Nagai et al., Involvement of b-glucuronidase in intestinal microflora in the intestinal toxicity of the antitumor camptothecin derivative irinotecan hydrochloride (CPT-11) in rats, Cancer Res. 56 (1996) 3752–3757. R. Mick, E. Gupta, E.E. Vokes, M.J. Ratain, Limited-sampling models for irinotecan pharmacokinetics-pharmacodynamics: Prediction of biliary index and intestinal toxicity, J. Clin. Oncol. 14 (1996) 2012–2019. E. Araki, M. Ishikawa, M. Iigo, T. Koide, M. Itabashi, A. Hoshi, Relationship between development of diarrhea and the concentration of SN-38, an active metabolite of CPT-11, in the intestine and the blood plasma of athymic following intraperitoneal administration of CPT-11, Jpn. J. Cancer Res. 84 (1993) 697–702. K. Yamaoka, Y. Tanigawara, Y. Nakagawa, T. Uno, A pharmacokinetic analysis program ( MULTI ) for microcomputer, J. Pharmacobio-Dyn. 4 (1981) 879–885. J. Fassberg, V.J. Stella, A kinetic and mechanistic study of the hydrolysis of camptothecin and some analogues, J. Pharm. Sci. 54 (1992) 676–684. W. Suzuki, R. Atsumi, H. Hakusui, Y. Esumi, Y. Jin, Studies on the metabolic fate of CPT-11 (2): Pharmacokinetics in rats following a dose — metabolites pattern in serum, liver, kidney and intestinal tissue, Xenobio. Metabol. and Dispos. 6 (1991) 97–104. T. Tsuji, N. Kaneda, K. Kado, T. Yokokura, T. Yoshimoto, D. Tsuru, CPT-11 converting enzyme from rat serum: Purification and some properties, J. Pharmacobio-Dyn. 14 (1991) 341–349.