[11] Ethylcellulose microcapsules for selective drug delivery

[11]

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[11] E t h y l c e l l u l o s e M i c r o c a p s u l e s for Selective Drug Delivery

By

TETSURO K A T O , KATSUO U N N O ,

and AKIO GOTO

Microencapsulation is designed to protect, separate, or change the diverse function of substances encased within a small particle of a diameter less than approximately 500 /zm. Depending on the materials and structures of the shell, microcapsules can alter the physical properties of the encased substances so that a desired availability is achieved while at the same time protecting the activity of the encased substances from the environment. The release of the internal substances may be achieved by erosion, dissociation, or semipermeability of the shell materials used. Thus, encasing of medicinais within microcapsules is usually employed for the purpose of masking taste, protecting against the environment, and/ or influencing the release rate of the encapsulated substances. In most cases, microencapsulation of therapeutic drugs is simply designed for oral administration as a long-acting dosage form. 1,2 However, when the potential of microencapsulation is considered, this technique must be best applied to selective delivery of drugs with a low therapeutic index such as anticancer drugs, s If a microencapsulated anticancer drug, for example, were selectively distributed in a cancerous lesion, then a controlled release of the cytotoxic agent would lead to total killing of cancer cells in the target area without systemic drug toxicity. In this respect, intravascular administration of microcapsules may be the most acceptable and effective way of site specificity. It has been demonstrated that more than 90% of microparticles smaller than 1.4/zm are removed by the reticuloendothelial system, mainly in the liver and spleen, when they are intravenously injected, and almost 100% of microparticles larger than 10 /xm are entrapped in the lungs. 4-6 The reticuloendothelial clearance and the lung embolization are the major J. R. Nixon, "Microencapsulation." Dekker, New York, 1976. 2 L. A. Luzzi, J. Pharm. Sci. 59, 1367 (1970). 3 T. Kato, in "Controlled Drug Delivery" (S. D. Bruck, ed.), Vol. 1I, p. 189. CRC Press, Boca Raton, Florida, 1983. 4 G. C. Ring, A. S. Blum, T. Kurbatov, and G. L. Nicolson, Am. J. Physiol. 200, 1191 (1961). s G. V. Taplin, D. E. Johnson, E. K. Dote, and H. S. Kaplan, J. Nucl. Med. 5, 259 (1964). 6 M. Kanke, G. H. Simmons, D. L. Weiss, B. A. Bivins, and P. P. deLuca, J. Pharm. Sci. 69, 755 (1980).

METHODS IN ENZYMOLOGY, VOL. 112

Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any fi3rm reserved. ISBN 0-12-182012-2

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problems in the intravascular targeting of colloidal drug carriers. To overcome these problems and achieve targeting at the same time, the authors and associates developed ethylcellulose microencapsulation of anticancer drugs for selective intraarterial use, proposing a concept of "chemoembolization." 3.7-9The microcapsules with a considerably larger size when infused into tumor-supplying arteries embolize the arterioles, with gradual release of the entrapped drugs. Approach to intravascular targeting by arterial chemoembolization is limited to cancer therapy at present, and a variety of polymeric materials other than ethylcellulose can be used as the shell of the microcapsules. This chapter briefly describes the method of ethylcellulose microencapsulation and the experimental as well as clinical results of intraarterially administered microcapsules. Preparation of Ethylcellulose Microcapsules The rationale for selecting ethylcellulose as the shell material is that this substance forms a stable, semipermeable membrane and is commonly used as an additive to foods and drugs because of its inert nature. Under certain conditions, ethylcellulose dissolved in an organic solvent accumulates over particulates of water-soluble substances to make a capsular membrane, the phenomenon being described as coacervation or phase separation. Jo

Original Method Mitomycin C (MMC; Kyowa Hakko Kogyo Co. Ltd., Tokyo) was chosen as a prototype anticancer drug in our early stage of investigations. MMC is a typical cytotoxic antibiotic which does not need hepatic microsomal activation, has a broad spectrum against human solid tumors, and also exhibits good stability against heat and chemicals. Two grams of MMC powder with a mean particle diameter of approximately 1 p~m was dispersed in a solution containing 0.5 g ethylcellulose, 0.5 g polyethylene, and 500 ml cyclohexane at 80°, and the mixture was gradually cooled to room temperature with gentle stirring at 380 rpm. In this process, polyeth.ylene promotes the phase separation of ethylcellulose, and MMC particles are encapsulated with ethylcellulose. The micro7 T. Kato and R. Nemoto, Proc. Jpn. Acad., Ser. B 54, 413 (1978). 8 T. Kato, R. Nemoto, H. Mori, and I. Kumagai, Cancer 46, 14 (1980). 9 T. Kato, R. Nemoto, H. Mori, M. Takahashi, Y. Tamakawa, and M. Harada, J. Am. Med. Assoc. 245, 1123 (1981). 10 H. G. Bungenberg de Jong, in "Colloid Science" (H. R. Kruyt, ed.), Vol. II, p. 339. Elsevier, Amsterdam, 1949.

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FIG. 1. Scanningelectron micrographof ethylcellulosemicrocapsuleencasingpeplomycin. Drug content was 80% (w/w). Bar indicates 100/zm. capsules thus prepared were rinsed with n-hexane several times, and airdried at 45 ° for 6 hr so that the polyethylene, cyciohexane, and n-hexane were completely removed. 7 MMC microcapsules were proved to consist, on average, of 80% (w/w) of biologically active MMC as the core and 20% (w/w) of ethylcellulose as the shell, with a mean particle size of 224/xm ranging from 106 to 441 tzm. The release rate of MMC from the microcapsules dispersed in unstirred physiological saline at 37° was 31% of the total encased MMC at 6-hr incubation, showing a sustained-release property of this preparation. Microencapsulation by this method, in general, forms an irregular particle with a rough, invaginated surface and a particle size greater than approximately 100 /zm (Fig. 1). This indicates that ethylcellulose produces a thin coating over the considerably large particles of core substances aggregated during the process of phase separation. Nevertheless this preparation has proved to be acceptable for the purpose of arterial chemoembolization.

142

MICROENCAPSULATION TECHNIQUES

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Modifications in Microencapsulation Although ethylcellulose microcapsules can be prepared by a rather simple technique as described above, further improvements in respect to particle size, yield of microcapsules, and control of drug release are needed. Unfortunately, however, much of the available information relating to microencapsulation appears in the patent literature and is inadequately described. This section describes data derived from our recent investigations. The results have revealed that the type of chemical and its concentration in the microencapsulation system definitely influence the release rate, particle size, and the resultant yield of the microcapsules. In these studies, methylene blue was used instead of MMC as the entrapped drug to facilitate determining release rates. Two grams of lactose was mixed with 1% methylene blue and was dispersed in 100 ml cyclohexane. The release rate was expressed as the percentage of the total encased drug after 10 min incubation in unstirred physiological saline at room temperature, Only microcapsules with a particle size less than 350 /xm collected through a 42-mesh screen were used.S' It is generally appreciated that an increase of shell materials may rt A. Goto, H. Murota, Y. Katsurada, M. Kondo, K. Unno, and T. Kato, to be published.

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decrease the release rate of encased substances. In fact, when the content of ethylcellulose in the system was increased from 0.5 to 1 g, the release rate was markedly inhibited. But, at the same time, our study demonstrated that the concentration of polyethylene definitely influenced the release rate, the maximum inhibition of release being achieved at the concentration of 0.8% (Fig. 2). It was also found that addition of cholesterol in the solvent raised the yield of the microcapsules smaller than 350/xm in proportion to the cholesterol concentration, of which maximum value was limited to approximately 1% at room temperature. In an experimental system in which 2 g lactose was dispersed in 100 ml cyclohexane dissolving 0.5 g ethylcellulose, 0.2 g polyethylene, and l g cholesterol, the yield of microcapsules was increased up to 78%. The value could be well contrasted with the low yield of 21% in the microencapsulation without cholesterol. On the other hand, it seemed that cholesterol did not significantly affect the lactose content of the microcapsules (Fig. 3). Further investigations confirmed these findings. Referring to the aforementioned experimental results, 2 g lactose was added in 100 ml cyclohexane dissolving l g ethylcellulose and 1 g cholesterol with varying concentrations of polyethylene. For the control, cholesterol was excluded. In the presence of cholesterol, addition of polyethylene gradually increased the yield of microcapsules. The yield was increased from 41% without poly-

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FIG. 3. Effect of cholesterol on release rate (©), encasement of core substance (A), and yield (Q) in microencapsulation. Yield <350/~m. Lactose :ethylcellulose ratio was 2:0.5, and polyethylene concentration was 2% in the solvent.

M I C R O E N C A P S U L A T 1TECHNIQUES ON

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ethylene up to 86% at the optimal polyethylene concentration of 0.8%, while no significant increase in the yield was observed without cholesterol (Fig. 4). Even with the presence of cholesterol, the release rate was shown to be clearly inhibited by adding polyethylene. The release rate was decreased from 68% without polyethylene to the value as low as 2% with 0.8% polyethylene. The experiment also revealed that cholesterol in the solvent significantly inhibited the release rate (Fig. 5). The results may indicate that polyethylene promotes the formation of a stable ethylcellulose membrane, and that cholesterol facilitates making a monodispersion of the core particulates in the solvent, thus leading to an increase in uniform aggregation of the particulates. The fine aggregation of the core particulates may, at the same time, enhance the effect of polyethylene on formation of a stable membrane with uniform thickness. These functions of both polyethylene and cholesterol during the process of phase separation will contribute to increase of the yield and decrease of the release rate.

Ferromagnetic Microcapsules An innovative drug delivery system, magnetic control of drug carrier complexes, has been initiated. The rationale for this approach is to guide the intravascular or intraluminal microcapsules into desired sites and/or

[111

ETHYLCELLULOSEMICROCAPSULES

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to retain them at target lesions by means of external magnetic force. For this purpose, ferromagnetic ethylcellulose microcapsules containing both MMC and zinc ferrite were prepared based on the principle of coacervation. Ethylcellulose (1 g) and polyethylene (0.5 g) were dissolved in cyclohexane (100 ml), and MMC powder (2 g) was dispersed in the solution. By cooling to room temperature with gentle stirring, MMC particles were encapsulated with ethyicellulose. MMC microcapsules thus prepared were then mixed with 100 ml of n-hexane containing 0.5 g zinc ferrite (Zn2~Fes0 • F e 2 0 3 ) with mean particle size of !.6 txm, and heated again to 45°C. The mixture was cooled with gentle stirring, whereby the ferrite particles were attached to the capsular surface (outer-type microcapsules). ~2 The mean particle size was 307.9 m 34.5 (SD) /xm and ferrite particles solidly fixed to the capsular surface. The microcapsules consisted, on average, of 50% (w/w) of biologically active MMC as the core, and 34% of ethylcellulose and 16% of ferrite as the shell. With this method, the ferrite content was limited to within 16%. On the other hand, when ferrite particles were added to the initial solvent, the ferrite particles were shown to be entrapped within the ethylcellulose membrane, thus 12 T. Kato, R. Nemoto, H. Mori, K. Unno, A. Goto, M. Harada, and M. Homma, Proc. Jpn. Acad., Ser. B 55, 470 (1979).

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generating an " i n n e r - t y p e " microcapsule. This method could increase the ferrite content up to 50%, thus enhancing the magnetic responsiveness of the microcapsules. The prototype microcapsules, consisting of 30% (w/w) MMC and 50% ferrite as the core and 20% ethylcellulose as the shell, had a mean particle size of 250 -+ 43/zm.13 In vitro examinations revealed that both types of ferromagnetic MMC microcapsules had a sustained-release property and a sensitive responsiveness to conventional magnetic fields. Animal studies demonstrated that VX2 carcinoma transplanted in the rabbit hind limb was successfully treated with arterial infusion of ferromagnetic MMC microcapsules controlled with an extracorporeal magnet, and that VX2 carcinoma in the rabbit bladder wall responded to intravesically infused microcapsules under a magnetic field. TM Independently of our research, Widder and associates also developed magnetic albumin microspheres with a mean particle size of 1/zm for the purpose of intracapillary targeting of an anticancer drug. ~5.16 Intraarterial Targeting of Microcapsules Experimental results of intraarterial targeting of MMC microcapsules demonstrated the effectiveness of this approach. 3 Additional anticancer agents carl be encapsulated other than MMC. Peplomycin (PEP; Nihon K a y a k u Co. Ltd., Tokyo) microcapsules were prepared and tested in animals. 17 Characteristics o f P E P Microcapsules

PEP, a derivative of bleomycin, was encapsulated into ethylcellulose microcapsules by a phase separation method as mentioned above. The P E P content was 80% and the mean particle size was 235 + 52/zm with a rough, invaginated surface (Fig. 1). Since PEP is highly water soluble, the release of the drug from the microcapsules was considerably more rapid as compared with MMC. The release rate of PEP in 200 ml of physiological saline (37%) stirring at 25 rpm was 50% at 30 rain incubation and 90% at 7 hr incubation, respectively (Fig. 6). ~3T. Kato, R. Nemoto, H. Mori, R. Abe, K. Unno, A. Goto, H. Murota, M. Harada, K. Kawamura, and M. Homma, J. Jpn. Soc. Cancer Ther. 16, 1351 (1981). z4T. Kato, Jpn. J. Cancer Chemother. 8, 698 (1981). t5 K. J. Widder, A. E. Senyei, and D. F. Ranney, Adv. Pharmacol. Chemother. 16, 213 (1979). 16K. J. Widder, R. M. Morris, G. Poore, D. P. Howard, and A. E. Senyei, Proc. Natl. Acad. Sci. U.S.A. 78, 679 (1981). 17T. Kato, H. Mori, R. Abe, K. Etori, K. Unno, A. Goto, H. Murota, M. Harada, M. Sbindo, and R. Chiba, Artif Organs 11, 213 (1982).

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F1G. 6. Release rate of peplomycin from ethylcellulose microcapsules in 37 ° saline stirring at 25 rprn.

Pharmacokinetics and Cytotoxic Effect PEP microcapsules containing 5 mg PEP suspended in physiological saline were infused into the left renal artery of Japanese white rabbits via a catheter threaded from the right femoral artery. For the control, 5 mg PEP in the usual dosage form was infused either alone or in combination with 5 mg placebo microcapsules. PEP levels in the circulating blood of the animals infused with PEP microcapsules were significantly lower than those of the control animals infused with nonencapsulated PEP during the first 60 rain after the infusion (Fig. 7). The peak PEP level, observed 1 rain after the infusion, was 26.5 +- 3.18 ~g/ml (mean +- SE) in the control group, while that in PEP microcapsule group was 12.25 +- 0.33/xg/ml. In terms of bioavailability, calculated from the area under the blood level curve, the amount of PEP released from the kidneys into the venous circulation in the PEP microcapsule group was estimated as 70% of that of the control. Since a considerable amount of PEP might be released from the microcapsules into the saline during the period of both the preparation of suspension and the infusion thereafter, further improvement of the microcapsules to control the initial release rate is needed in order to decrease the blood PEP levels. However, bioassay of the tissue homogenate of the kidneys 3 hr after the infusion revealed that PEP concentration in the microcapsule group was 8.85 + 1.38/zg/g, which is in contrast with the low concentration of 0.58 + 0.07/.~g/g in the control group. It may be that even though a large

148

MICROENCAPSULATION TECHNIQUES

[11]

30

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amount of PEP was lost from the target area during the initial period after the microcapsule infusion, the capsular membrane as well as the blood stasis due to microembolization were responsible for retaining the activity of residual drug in the kidney. Morphological examination proved that intraarterial PEP microcapsules, which were entrapped by the intrarenal arterioles, produced the most extensive coagulation necrosis involving the whole target organ when compared with other kinds of treatments. Nonencapsulated PEP caused only minimal changes, and while the placebo microcapsules even in combination with nonencapsulated PEP induced small foci of infarction, the majority of the kidney mass remained unaffected. These results are in agreement with those obtained from intraarterial infusion of MMC microcapsules, The remarkably enhanced cytotoxic effects of the microencapsulated anticancer drugs must result from both infarction and prolonged drug action. Destruction of the surrounding endothelial lining and stromal tissues in the earlier stage of infarction may increase the extent of intraparenchymal migration of the drugs released from the microcapsules. With regard to the mode of action, this kind of treatment using microencapsulated cytotoxic agents was defined as "arterial chemoembolization." Animal study demonstrated that the decrease

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in drug level in circulating blood was responsible for preventing the systemic drug toxicity. 3 Clinical Application of Microcapsules Recent advances in angiography have permitted arteries in various sites to be readily catheterized under X-ray monitoring. Consequently, the microcapsules are able to be selectively infused into tumor-supplying arteries in a variety of organs. During the period from March 1978 to March 1982, 285 patients with advanced carcinoma in the kidney, liver, urinary bladder, prostate, bone, and lung were subjected to transcatheter arterial chemoembolization with microcapsules. The number of treatments was single in 67% of the patients, 2 in 21%, and 3 or more in 12%. Ninety percent of the patients received MMC microcapsules and the others received PEP microcapsules with a mean total dose of 21 mg expressed as the drug content. 9,18 Of the 211 measurable tumors, 74 (35%) showed a marked tumor reduction greater than 50% in area, 77 (37%) had a tumor reduction less than 50%, and 60 (28%) did not respond. Side effects and complications which required medical care were experienced in approximately 10% of the patients. These included bone marrow depression, local pain, fever, anorexia, or local skin ulceration. Since the majority of the tumors were large and highly invasive, the results should be appraised in a positive light. A preliminary controlled study in the treatment of renal cell carcinoma showed that intraarterial infusion of MMC microcapsules enhances the antineoplastic effect and reduces the systemic toxicity of the drug. These findings are consistent with the previous animal studies. 19 Comment Ethylcellulose microencapsulation by coacervation needs no special apparatus or expensive chemicals except for the drug to be entrapped. If necessary, an expensive core substance such as an anticancer drug can be readily recovered by dissolving the ethylcellulose membrane in organic solvents. Besides the chemicals, other technical factors such as temperature or speed of the stirrer may influence the microencapsulation. Optimal conditions of these factors should be examined for each substance to be encapsulated. Furthermore, various polymeric materials other than 18 T. Kato, Jpn. J. Cancer Chemother. 10, 333 (1983). 19 T. Kato, R. Nemoto, H. Mori, M. Takahashi, and Y. Tamakawa, J. Urol. 125, 19 (1981).

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ethylcellulose could be used as the shell of the microcapsules with certain modifications in this method. The particle size and the release rate should be determined depending on the application. For selective arterial administration, the microcapsules must be small enough to be infused through the catheter but large enough to avoid the undesirable migration into fine nontarget arteries such as those supplying the nerve fibers. In this respect, the size of the ethylcellulose microcapsules is acceptable for the clinical practice. On the other hand, in selective drug delivery, extremely slow drug release does not always satisfy a designed therapeutic effect. It should be realized that the drug release from microcapsules surrounded by compact tissue structures is much more inhibited than that in an aqueous environment, and also that the extent of drug diffusion in parenchymal tissues is proportionally influenced by the concentration gradient of drugs released from microcapsules. In this respect, it is preferable to design the release of the encased drugs at a rapid rate after the microcapsules reach the target tissue. Selective drug delivery can be identified by three stages of targeting: first-, second-, and third-order targeting. 3,15 It is most likely that unless first-order targeting which involves the restricted drug distribution to vascular beds of target lesions is achieved, second- and third-order targeting will fail to provide any fruitful result. Selective arterial infusion of ethylcellulose microcapsules has been developed as a prototype of firstorder targeting of anticancer drugs, and has proved to have practical utility in the treatment of cancer. Problems in second- and third-order targeting still remain to be settled.

[12] P o l y a c r o l e i n M i c r o s p h e r e s : Preparation and Characteristics

By M. CHANG, G. RICHARDS, and A. REMSAUM

Introduction Antibody-coated microspheres or immunomicrospheres react in a highly specific way with target cells, viruses, or other antigenic agents. Microspheres incorporate compounds that are radioactive, fluorescent, magnetic, colored, or pharmacologically active. These various types of METHODS IN ENZYMOLOGY, VOL. 112

Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-182012-2