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In vitro release of cytotoxic agents from ion exchange resins
251
of Controlled Release, 8 (1989) 251-257 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
Journal
IN VITRO RELEASE OF CYTOTOXIC AGENTS FROM ION EXCHANGE RESINS C. Jones, M.A. Burton, B.N. Gray* University
Department
of Surgery,
Royal
Perth Hospital,
Box X22
13, G. P.O., Perth,
W.A.
600
1 (Australia)
and J. Hodgkin C.S.I.R.O.,
Division
of Applied
Organic
Chemisrry,
Box 433
1, G.P.O.,
Melbourne,
Vie. 300
1 (Australia)
(Received April 7, 1988; accepted in revised form October 3, 1988)
Polystyrene-based ion exchange resins were evaluated as a carrier system for the sustained delivery of cytotoxic agents. A batch ion exchange procedure was developed for loading of adriamycin (Adr) and novantrone (Nov) onto cation exchange resins and ~-f~~roura~iL (5FU) and floxuridine (FUDR) onto anion exchange resins. Loading proceeded to near resin capacity in the case of Adr, 5FU and FUDR. A closed circuit in vitro drug release system was developed to measure rates at which resins released entrapped drug and reached equilibrium drug concentrations. A large degree of variation in the equilibrium drug concentrations was observed between the different drug systems (6x 10e7 M-1.7~ lop3 M) but the rate at which equilibrium was attained was similar for all drugs. Adr resinate demon&rated equilibrium drug concentrations close to blood levels associated with conventional therapeutic doses. Thus Adr resinate is regarded as having potential for tumour-targeted drug delivery. INTRODUCTION Systemic side effects arising from conventional chemotherapeutic treatment of cancer are undesirable and restrict treatment efficacy. This is a result of the nonspecificity of cytotoxic drugs for tumour tissue. Consequently, it would be advantageous to target these drugs to the tumour bearing organ, reducing systemic exposure to the drug. Much recent work has centered on the entrapment of drugs within particulate carriers. Carriers investigated include polyglutaraldehyde [ 11, poly (lactic acid) microspheres [ 23 and polyalkylcyanoacrylate nanoparticles [ 31. Mitomycin C, entrapped within albumin microspheres, has been used in a recent clinical *To whom all correspondence should be addressed.
0168-3659/89/$03.50
trial [4]. These microspheres were targeted to hepatic tumours and improved patient survival was observed over conventional treatment. Any particulate drug carrier should fulfill certain design criteria before it can be applied to a theraDeutic situation. These include: The-carrier matrix must be nontoxic. The particle should be able to entrap large quantities of drug within its matrix. Release of entrapped drug should be sustained over predetermined time periods. Drug-carrying particles should be easily (d) manufactued with a high final product yield. A monodisperse aqueous suspension of the (e) carrier needs to be readily attainable. Carrier particles need to be sized to em(0 bolise within the arterial tumour microvasculature.
0 1989 Elsevier Science Publishers B.V.
252
Ion exchange resins have been widely utilized for the controlled release of many drugs for oral administration [ 51. A major problem in this type of application is the large volume and very variable ionic environment found in the gastrointestinal tract. The potential for ion exchange release from resin microspheres placed in the bloodstream is considerably greater because of its more constant ionic properties and lower contact volumes. Radioisotopes have also been inco~orated into ion exchange microspheres and used to internally irradiate cancers in patients via arterial delivery f6]. The latter demonstrated that the targeting of ion exchange resin microspheres to a tumour site is a practical technique. Goldberg et al. [7] examined the use of hydrophilic albumin and dextran microspheres containing weak acid (mainly carboxylic) ion exchange groups for drug attachment. This study confirmed the benefits of particulate ion exchange systems as drug carriers. We have examined the in ~~~~0release characteristics of hydrophobic, strong-acid and strong-base resins loaded with cytotoxic drugs. These macroporous, particulate drug carriers show potential for the targeting of cytotoxic drugs to specific sites within the body and for releasing them by ion exchange mechanisms alone. The basic drugs adriamycin (I) and novantrone (II) were attached to the sulphonate groups of a cation exchange resin and the acidic drugs Ei-fluorouracil (III) and floxuridine (IV) were attached to the quaternary ammonium groups of an anion exchange resin.
EXPERIMENTAL PROCEDURES Resin preparation
Aminex A-6 resin (Bio-Rad) was used for the attachment of the drugs adriamycin ( Adr, Farmitalia) and novantrone (Nov, Lederle ) . Resin microspheres with a binding capacity of 1.8 meq/ml had been closely sized to 17.5 + 2.5 pm
diameter. Resin (1 g) was converted to its II+ form by slurrying with 3 M HCl (250 ml) for 1 hour. Resin was then filtered off and washed extensively with distilled water and subsequently oven dried at 75°C. Resin microspheres can be autoclaved at this stage if a sterile final product is required. A similar technique was used for the preparation of anion exchange resin for drug attachment. AG l-X2 (Bio-Rad, 38-74 pm diameter, 0.6 meq/ml binding capacity) was used for the attachment of the drugs 5-fluorouracil (5-FU, David Bull Labs.) and floxuridine (FUDR, Roche). Resin was converted to its OH- form by slurrying AG l-X2 (1 g) with 2 M NaOH (250 ml) for 1 hour. Resin was then filtered and washed extensively with distilled water and oven dried at 75°C. The loading capacity of drugs was reduced when wet resin was used. Drug attachment
A batch manufacturing procedure was used for the attachment of all drugs. This involved the slurrying of ion exchange resins with concentrated solutions of drug for 12 hours. Drugladen resins were filtered, washed extensively and finally suspended in distilled water. Drugs attached to ion exchange resins were: 1. Adriamycin: Adr (40 mg ) was dissolved in water (1.5 ml) and slurried with 35 mg of Aminex A-6 (dry). The resulting resin suspension was frozen at - 10°C as storage at 4°C and room temperature caused extensive aggregation of resin particles. 2. Novantrone: 11 mg Aminex A-6 was slurried with Nov (15 mg) in 5.5 ml of 0.9% NaCl (commercial preparation ). Resulting resin suspension was stored at 4°C. 3. Floxuridine: FUDR (56 mg ) was dissolved in distilled water (1 ml) and slurried with AG 1-X2 (48 mg, dry). Resulting resin suspension was stored at 4 oC. 4. 5-F~uorour~i~ 5-FU (16 mg) was dissolved in water (1.5 ml) and slurried with AG
253
l-X2 (16.5 mg). The final resin suspension stored at 4’ C.
was
equilibrium with trapped drug reached bulk solution. This counterions in the external for all drug-resin system was standardized combinations. Drug resinate (equivalent to 2 x 10M4 mol drug) was immobilised between membrane filters and washed with a continuous flow of 0.9% NaCl, at 37’ C, and returned to a reservoir (50 ml). A constant flow (1.2 ml/min) of release medium was sustained with a peristaltic pump (Ismatec ). Bulk solution drug concentration was continuously monitored by a UV-visible spectrometer interfaced to a microcomputer for data retrieval and analysis.
Resin drug content
Drug content was determined by two methods. Firstly, samples of concentrated drug solution were taken before and after attachment to resins. These were diluted with 0.9% NaCl and concentration determined by UV-visible spectrometry (L.K.B., Adr - 495 nm, Nov 682 nm, FUDR - 268 nm, 5-FU - 266 nm). The difference in the total drug present between the two solutions was that attached to the resin. This was checked by a drug displacement method. Small samples of resin were slurried with an excess of 10% NaCl solution until the drug was totally displaced from the resin. Samples of this supernatant were taken, diluted and their concentration determined by UV-v,jsible spectrometry. The results obtained for *resin drug contents by the two methods were in good agreement, never differing by more than 2.5%.
RESULTS
Table 1 summarizes the results obtained for the four different drug resinates. Drug contents are expressed as the maximum percentage by weight of drug resinate attained. There were large variations in the maximum microsphere loadings for the four drug systems using the batch manufacture procedure. Large differences were also seen in the equilibrium drug concentrations for the different drug resinates using 0.9% NaCl as a washing medium.
Drug release
A closed continuous flow system was developed to measure the rate at which resin-en2.5
0
20
40
60
60
100
TIME
120
140
160
160
200
(min)
Fig. 1. Equilibrium drug release profile for cation exchange resin loaded with Adr (0)
and Nov ( l).
254 1.8 T
0
20
40
80
80
100
120
140
160
180
200
TIME (min)
Fig. 1. Equilibrium drug release profile for anion exchange resin loaded with FUDR (0)
TABLE 1 Equilibrium drug concentrations and maximum drug loadings for Adr, Nov, 5-FU and FUDR resinates Drug
% Drug content (max.)
Equilib~um drug cone.
Adr Nov 5-FU FUDR
34.9 9.8 23.1 31.1
2.1 x lo-5M 0.6~1O-~M 5.6x 1O-4 A4 1.7x 1O-3 M
Figures 1 and 2 show equilibrium drug release profiles for drugs loaded onto cation and anion exchange resins, respectively. The drug release profiles were similar for all drugs tested. The time taken for the concentration in the bulk solution to reach half that of the equilibrium drug concentration was always from 20 to 30 minutes. Under the experimental conditions used to determine eq~lib~um drug concentrations, the percentage of total drug released from the resin at equilibi~m were: Adr - 0.53%, Nov 0.02%, 5FU - 14%, FUDR - 42%. Repeat experiments using the same microspheres washed with a fresh sample of 0.9% NaCl release medium gave identical release kinetics and equi-
and 5-FU (+).
librium levels until the microspheres were exhausted of drug.
DlSCUSSlON
A low-cost, technically uncomplicated procedure has been described for the manufacture of drug-loaded ion exchange resins. Any drug that is not ionically bound to the resin during manufacture is easily recovered for future use. This is important considering the high cost of cytotoxic drugs. The resins used were strong-acid and strongbasic cation and anion exchange resins, respectively. Although the divinyl benzene crosslinked styrene matrix of these resins is nontoxic, commercial resin preparations can contain impurities that cause severe toxicity. Cation exchange resins can be purified by cycling repeatedly between H+ and Na+ forms followed by extensive washing with distilled water. Resin can be sterilized by autoclaving prior to drug attachment. Anion exchange resins can also be purified by similar treatments although they are not as stable to high temperatures when sterilizing. Even after resin purification, large quantities
of resin have been known to cause toxic effects. For example, children given prolonged oral doses of sulfonic acid ion exchange resins developed tetany, resulting from hypocalcemia [ 81. Due to the high drug loadings we have obtained and the large therapeutic index of most cytotoxic drugs, the amount of drug resinate that would be introduced into the blood stream in the present application would not be sufficient to cause similar problems. The narrow size range of resin microspheres is important for various reasons. Meade et al. [ 91 showed that resin microspheres of varying size distributed differently throughout a tumour-bearing organ of rats. Microspheres greater than 50 pm in diameter did not embolise in tumour tissue to the same extent as their smaller counterparts. Resin microsphere size and pore diameter are rate-limiting factors for the process of diffusion of ions in and out of the resin matrix. Therefore, varying the size range of resin microspheres should vary their drug release characteristics, adding to the importance of a narrow size range. Simple batch manufacturing procedures were used for the attachment of drugs to the ion exchange resins, although it has been reported that column manufactuing processes give higher drug loadings [lo]. In practice, the binding of Adr, 5-FU and FUDR were found to proceed to near the capacity of the resins using the batch method. Nov loads to less than the capacity of the resin (18.3% of capacity) because a commercial 2% drug/O.9% NaCl solution was used. Greater loading would probably have been achieved here if more concentrated, nonionic drug solutions were used. The low equilibrium release levels obtained with this drug did not justify further experiments. The final drug resinate products were easily dispersed in 0.9% NaCl or distilled water without the aid of surfactants. In addition, resulting drug resinates were found to have a long bench life and could be frozen. The closed in vitro drug release system was designed to compare equilibrium drug concen-
trations for the different drug resinates and also to test the rates at which drugs come into equilibrium. All variables were standardised (i.e. flow rate, equivalent amount of drug, temperature and wash medium) to enable a valid comparison of results for the different drug resinates. It might be argued that an open drug release system, where drug resinate is washed with fresh release medium, would more closely imitate the in vitro situation. However, for relatively small drug molecules, little can be learned from such a system as drug release rates would be almost completely dependant on flow rates over the resins. The flow characteristics of plasma over a microsphere embolised in a blood vessel are unknown but are likely to be very much slower than in a vessel free from microsphere blockade. In this static, fixed-volume situation there is equilibrium near the microsphere surface with slow diffusion away as drug is removed by surrounding tissue [ 71. This situation is more in keeping with our in vitro test procedure. The rate at which the resin-bound drugs came into equilibrium with the total bulk release medium was similar for all drugs tested. The half time for equilibrium drug concentration to be reached was between 20 and 30 minutes for the conditions used. This suggests that drug release properties are ionically controlled and mainly dependent on the strength with which drugs are bound to the resin matrix. Diffusion of drugs through the microsphere matrix is not a significant rate-limiting factor. If the release of drug from the resin matrix needs modification, several methods have been described which can vary release characteristics. If an increase in time taken for the system to reach equilibrium is required, the coating of drug resinate particles with other polymers could considerably reduce their drug release rate [ 111. Use of gel (or solid) particles rather than macroporous resins would also greatly increase diffusion rates through the particle. Pore sizes could be changed by altering the polymerization procedures used in the manufactue of the
256
microspheres which can also slow drug diffusion rates [ 121. To vary the equilibrium levels of drug released, changes would have to be made to the ionic strength of the acidic and basic groups on the resin or to the hydrophilic/hydrophobic interactions of the drugs with the resin matrix. Drug resinates may be used as a convenient alternative to arterial infusion of cytotoxic drugs into a tumour-bearing organ. Drug resinates have the advantage that they do not require the use of internal or external pumps for drug delivery. Resin microspheres embolise in the microvasculature of a tumour thus slowing blood flow. This enhances the contact time of tumour with released drug. However, equilibrium levels of released drug need to be in the correct concentration ranges. Garnick et al. [ 131 found that after hepatic arterial infusion of Adr at 40 mg rnp2 day-’ for 3 days, the steady-state plasma concentration of Adr was 2.75 x lo-’ M. The in vitro equilibrium Adr concentration is higher than this. However, if a similar dose of adriamycin-loaded resin was introduced into the hepatic artery, it is likely that plasma blood levels would be of the same order as for drug infusion as Adr is extensively metabolised in the liver. This, together with its high loading ability, make Adr an ideal drug for targeted ion exchange resin chemotherapy. Nov is very strongly bound to the Aminex A6 resin with an equilibrium concentration of 6x low7 M in 0.9% NaCl. After a bolus dose of 10 mg rne2, serum drug levels were found to be 0.9 x 10T7 M [ 141. It is unlikely that levels of this order could be obtained using Nov A-6 resinate. Higher drug equilibrium levels could possibly be achieved for Nov if a weak-acid cation exchange resin was used. Therefore, Nov resinate may also be applied to tumour-targeted chemotherapy. When FUDR is peripherally infused at 5 mg (kg body weight) -’ h-l, the steady-state serum FUDR level is approximately 3 X 10M7M [ 151. The in vitro equilibrium drug concentration for
the FUDR resinate is greater than this by a factor of 6000. Therefore, an equivalent dose of drug resinate would produce much too high serum drug levels to make it suitable for in uiuo use. Also, drug release could not be sustained over useful lengths of time. Similarly, 5-FU resinate is unsuitable as an alternative to arterial infusion. The daily dose rate of 5-FU for arterial infusion is approximately 1 g (day) -l. This is equivalent to approximately 4 g of resinate per day, which would cause complete blockade of the microvasculature. Naturally, the in vitro equilibrium drug concentrations obtained in this experiment are likely to be different to the in uiuo situation. A resin microsphere embolised in an arteriole will be bathed in a complex solution of Na+, K+, Ca2+, Mg2+, organic ions and enzymes. Therefore, an in uiuo study examining the release characteristics of drug-carrying ion exchange resins is being carried out and will be reported in a later communication.
CONCLUSIONS
The present investigation has shown that Adr, Nov, 5-FU and FUDR can be readily loaded onto ion exchange resin microspheres. These drug resinates can be made to meet most of the design criteria for therapeutic situations detailed in the introduction. Large drug loadings were obtained for Adr, 5-FU and FUDR. In uitro equilibrium drug concentrations vary considerably between the drugs tested. Potentially useful concentrations were found for Adr-loaded Aminex A-6 resin and experimental modifications may make the method applicable to other drugs. Studies are required to determine the cytotoxic effectiveness of Adr resinate targeted to tumours as compared to conventional chemotherapeutic treatments.
257
8 9
2.A. Takes, K.E. Rogers and A. Rembaum, Synthesis of ~dr~~yc~ coupled ~1ygIut~dehyde microsphares and evamation of %I%?&q&&ati& a&iv&y, Proc, NatI. Acad, Sci. USA, 79 ( 3982) 2~2~-~~. N. Wakiyama, K. Juni and M. Nakano, Preparation and evahration in uitro ofpoIyla~t~c acid micrtxpheres ~~~~~nj~~ iocaf anaesthetics, Cftem. Phafm. BulL, 29 (1981) 33634368. P. Couvreur, B. Kante, V. Lenaerts, V. Scaiheur, M. RoXand and P. Speiser, Tissue distribution and anti tumour drugs associated with polycyanoacrytate nanoparticles, J. Pharm. Sci., 69 (1980 1 199-202. S. Fujimoto, M, Myazaki, F., Endoh, 0. Takahashi, K. Qkui and Y. Morimoto, Biodegradable mitomyein C microspheres given intraarterially for inoperable hapatic cancer, Cancer, 56 (1985) 2404-2410. E.1-f.Scbaeht, Ionic polymers as drug carriers, in: S.D. Bruck (Ed. 1, Controlled Drug Delivery, Vol. If, CRC., Bcrca Raton, FL, 1983, pp. 149-173. RV.P. Mantravadi, D.G. Spigos, W.S. Tan and EL. F&x, Intraarterial yttrium 90 in the treatment of hepatic malignancy, Radiology, 142 (1982) 733-786. E.P. Goldberg, II. Iwata and W. Longo, Hydrophilic albumin and dextran ion-exchange microspheres for localized chemotherapy, in: S.S. Davis, L. IIlum, J.G.
10
11
12
13
14
15
M&Jia and E. ~orn~i~~~ (~s.~, ~~~r~phar~ and Drug Therapy; Pb~a~~t~~~l, ImmunoIo~~aI and Medical Aspects, Eisavier+ Amsterdam, 1984, Chap. 16. R. Macaulay and G.H. Watson, T&any following cation exchange resin therapy, Lance& 2 f 1954 f 7% V.M. Meade, M.A. Burton, B.N. Gray and G.W. Self, Distribution of different sized microspheres in experimental hap&c turnours, Eur. J. Cane. Clin, C&c.>23 (1987) 37-41. NC. Chaudbry and L. Saunders, Sustained release of drugs from ion exchange resins, J. Pharm. Pharmacoi*, a f 1956 ) 975, J.F. Nash. and RE. Crabtree, Absorption of tritiated ~-d~x~eph~ne in s~~i~~ r&ease dosage forms, J. Pharm. Xci., 50 (1961) 134. 3. SeidI, J. MaImsky, K. Dusek and W, Hietz, Macroporous styrene divinyl bensane Copolymers and their application in chromatography and for the preparation ofian exchange resins, Adv. Polym. Sci., 5 (1967) 113. MB. Carnick, W.D. ~n~rn~~~a~~d M. Israel, A. elinical-pharmacological evaluation ofbepatic arterial infusion of adriamycin, Cancer Res,, 39 (1979) 41054110. J. Robas, P.A. Paciucci, D. Sihdes, J. Greaves and J.F. Holland, Detection and ~~~nt~fi~ation of mitoxantrone in human organs, Cancer ~ha~nother. Pbarmacol., 13 f 1984) 67-68, W.D. Ensminger, A. Rosowsky, V. Raso, DC. Levin, M. Glade, 8. Come, C. Steele and E. Frei, A dinicalpharmacological evaluation of hepatic arterial infusions of 5*fluoro-2’-deoxyuridine and 5-fluorouracil, Cancer Rew, 38 (1978) 3784-3792.
of Controlled Release, 8 (1989) 251-257 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
Journal
IN VITRO RELEASE OF CYTOTOXIC AGENTS FROM ION EXCHANGE RESINS C. Jones, M.A. Burton, B.N. Gray* University
Department
of Surgery,
Royal
Perth Hospital,
Box X22
13, G. P.O., Perth,
W.A.
600
1 (Australia)
and J. Hodgkin C.S.I.R.O.,
Division
of Applied
Organic
Chemisrry,
Box 433
1, G.P.O.,
Melbourne,
Vie. 300
1 (Australia)
(Received April 7, 1988; accepted in revised form October 3, 1988)
Polystyrene-based ion exchange resins were evaluated as a carrier system for the sustained delivery of cytotoxic agents. A batch ion exchange procedure was developed for loading of adriamycin (Adr) and novantrone (Nov) onto cation exchange resins and ~-f~~roura~iL (5FU) and floxuridine (FUDR) onto anion exchange resins. Loading proceeded to near resin capacity in the case of Adr, 5FU and FUDR. A closed circuit in vitro drug release system was developed to measure rates at which resins released entrapped drug and reached equilibrium drug concentrations. A large degree of variation in the equilibrium drug concentrations was observed between the different drug systems (6x 10e7 M-1.7~ lop3 M) but the rate at which equilibrium was attained was similar for all drugs. Adr resinate demon&rated equilibrium drug concentrations close to blood levels associated with conventional therapeutic doses. Thus Adr resinate is regarded as having potential for tumour-targeted drug delivery. INTRODUCTION Systemic side effects arising from conventional chemotherapeutic treatment of cancer are undesirable and restrict treatment efficacy. This is a result of the nonspecificity of cytotoxic drugs for tumour tissue. Consequently, it would be advantageous to target these drugs to the tumour bearing organ, reducing systemic exposure to the drug. Much recent work has centered on the entrapment of drugs within particulate carriers. Carriers investigated include polyglutaraldehyde [ 11, poly (lactic acid) microspheres [ 23 and polyalkylcyanoacrylate nanoparticles [ 31. Mitomycin C, entrapped within albumin microspheres, has been used in a recent clinical *To whom all correspondence should be addressed.
0168-3659/89/$03.50
trial [4]. These microspheres were targeted to hepatic tumours and improved patient survival was observed over conventional treatment. Any particulate drug carrier should fulfill certain design criteria before it can be applied to a theraDeutic situation. These include: The-carrier matrix must be nontoxic. The particle should be able to entrap large quantities of drug within its matrix. Release of entrapped drug should be sustained over predetermined time periods. Drug-carrying particles should be easily (d) manufactued with a high final product yield. A monodisperse aqueous suspension of the (e) carrier needs to be readily attainable. Carrier particles need to be sized to em(0 bolise within the arterial tumour microvasculature.
0 1989 Elsevier Science Publishers B.V.
252
Ion exchange resins have been widely utilized for the controlled release of many drugs for oral administration [ 51. A major problem in this type of application is the large volume and very variable ionic environment found in the gastrointestinal tract. The potential for ion exchange release from resin microspheres placed in the bloodstream is considerably greater because of its more constant ionic properties and lower contact volumes. Radioisotopes have also been inco~orated into ion exchange microspheres and used to internally irradiate cancers in patients via arterial delivery f6]. The latter demonstrated that the targeting of ion exchange resin microspheres to a tumour site is a practical technique. Goldberg et al. [7] examined the use of hydrophilic albumin and dextran microspheres containing weak acid (mainly carboxylic) ion exchange groups for drug attachment. This study confirmed the benefits of particulate ion exchange systems as drug carriers. We have examined the in ~~~~0release characteristics of hydrophobic, strong-acid and strong-base resins loaded with cytotoxic drugs. These macroporous, particulate drug carriers show potential for the targeting of cytotoxic drugs to specific sites within the body and for releasing them by ion exchange mechanisms alone. The basic drugs adriamycin (I) and novantrone (II) were attached to the sulphonate groups of a cation exchange resin and the acidic drugs Ei-fluorouracil (III) and floxuridine (IV) were attached to the quaternary ammonium groups of an anion exchange resin.
EXPERIMENTAL PROCEDURES Resin preparation
Aminex A-6 resin (Bio-Rad) was used for the attachment of the drugs adriamycin ( Adr, Farmitalia) and novantrone (Nov, Lederle ) . Resin microspheres with a binding capacity of 1.8 meq/ml had been closely sized to 17.5 + 2.5 pm
diameter. Resin (1 g) was converted to its II+ form by slurrying with 3 M HCl (250 ml) for 1 hour. Resin was then filtered off and washed extensively with distilled water and subsequently oven dried at 75°C. Resin microspheres can be autoclaved at this stage if a sterile final product is required. A similar technique was used for the preparation of anion exchange resin for drug attachment. AG l-X2 (Bio-Rad, 38-74 pm diameter, 0.6 meq/ml binding capacity) was used for the attachment of the drugs 5-fluorouracil (5-FU, David Bull Labs.) and floxuridine (FUDR, Roche). Resin was converted to its OH- form by slurrying AG l-X2 (1 g) with 2 M NaOH (250 ml) for 1 hour. Resin was then filtered and washed extensively with distilled water and oven dried at 75°C. The loading capacity of drugs was reduced when wet resin was used. Drug attachment
A batch manufacturing procedure was used for the attachment of all drugs. This involved the slurrying of ion exchange resins with concentrated solutions of drug for 12 hours. Drugladen resins were filtered, washed extensively and finally suspended in distilled water. Drugs attached to ion exchange resins were: 1. Adriamycin: Adr (40 mg ) was dissolved in water (1.5 ml) and slurried with 35 mg of Aminex A-6 (dry). The resulting resin suspension was frozen at - 10°C as storage at 4°C and room temperature caused extensive aggregation of resin particles. 2. Novantrone: 11 mg Aminex A-6 was slurried with Nov (15 mg) in 5.5 ml of 0.9% NaCl (commercial preparation ). Resulting resin suspension was stored at 4°C. 3. Floxuridine: FUDR (56 mg ) was dissolved in distilled water (1 ml) and slurried with AG 1-X2 (48 mg, dry). Resulting resin suspension was stored at 4 oC. 4. 5-F~uorour~i~ 5-FU (16 mg) was dissolved in water (1.5 ml) and slurried with AG
253
l-X2 (16.5 mg). The final resin suspension stored at 4’ C.
was
equilibrium with trapped drug reached bulk solution. This counterions in the external for all drug-resin system was standardized combinations. Drug resinate (equivalent to 2 x 10M4 mol drug) was immobilised between membrane filters and washed with a continuous flow of 0.9% NaCl, at 37’ C, and returned to a reservoir (50 ml). A constant flow (1.2 ml/min) of release medium was sustained with a peristaltic pump (Ismatec ). Bulk solution drug concentration was continuously monitored by a UV-visible spectrometer interfaced to a microcomputer for data retrieval and analysis.
Resin drug content
Drug content was determined by two methods. Firstly, samples of concentrated drug solution were taken before and after attachment to resins. These were diluted with 0.9% NaCl and concentration determined by UV-visible spectrometry (L.K.B., Adr - 495 nm, Nov 682 nm, FUDR - 268 nm, 5-FU - 266 nm). The difference in the total drug present between the two solutions was that attached to the resin. This was checked by a drug displacement method. Small samples of resin were slurried with an excess of 10% NaCl solution until the drug was totally displaced from the resin. Samples of this supernatant were taken, diluted and their concentration determined by UV-v,jsible spectrometry. The results obtained for *resin drug contents by the two methods were in good agreement, never differing by more than 2.5%.
RESULTS
Table 1 summarizes the results obtained for the four different drug resinates. Drug contents are expressed as the maximum percentage by weight of drug resinate attained. There were large variations in the maximum microsphere loadings for the four drug systems using the batch manufacture procedure. Large differences were also seen in the equilibrium drug concentrations for the different drug resinates using 0.9% NaCl as a washing medium.
Drug release
A closed continuous flow system was developed to measure the rate at which resin-en2.5
0
20
40
60
60
100
TIME
120
140
160
160
200
(min)
Fig. 1. Equilibrium drug release profile for cation exchange resin loaded with Adr (0)
and Nov ( l).
254 1.8 T
0
20
40
80
80
100
120
140
160
180
200
TIME (min)
Fig. 1. Equilibrium drug release profile for anion exchange resin loaded with FUDR (0)
TABLE 1 Equilibrium drug concentrations and maximum drug loadings for Adr, Nov, 5-FU and FUDR resinates Drug
% Drug content (max.)
Equilib~um drug cone.
Adr Nov 5-FU FUDR
34.9 9.8 23.1 31.1
2.1 x lo-5M 0.6~1O-~M 5.6x 1O-4 A4 1.7x 1O-3 M
Figures 1 and 2 show equilibrium drug release profiles for drugs loaded onto cation and anion exchange resins, respectively. The drug release profiles were similar for all drugs tested. The time taken for the concentration in the bulk solution to reach half that of the equilibrium drug concentration was always from 20 to 30 minutes. Under the experimental conditions used to determine eq~lib~um drug concentrations, the percentage of total drug released from the resin at equilibi~m were: Adr - 0.53%, Nov 0.02%, 5FU - 14%, FUDR - 42%. Repeat experiments using the same microspheres washed with a fresh sample of 0.9% NaCl release medium gave identical release kinetics and equi-
and 5-FU (+).
librium levels until the microspheres were exhausted of drug.
DlSCUSSlON
A low-cost, technically uncomplicated procedure has been described for the manufacture of drug-loaded ion exchange resins. Any drug that is not ionically bound to the resin during manufacture is easily recovered for future use. This is important considering the high cost of cytotoxic drugs. The resins used were strong-acid and strongbasic cation and anion exchange resins, respectively. Although the divinyl benzene crosslinked styrene matrix of these resins is nontoxic, commercial resin preparations can contain impurities that cause severe toxicity. Cation exchange resins can be purified by cycling repeatedly between H+ and Na+ forms followed by extensive washing with distilled water. Resin can be sterilized by autoclaving prior to drug attachment. Anion exchange resins can also be purified by similar treatments although they are not as stable to high temperatures when sterilizing. Even after resin purification, large quantities
of resin have been known to cause toxic effects. For example, children given prolonged oral doses of sulfonic acid ion exchange resins developed tetany, resulting from hypocalcemia [ 81. Due to the high drug loadings we have obtained and the large therapeutic index of most cytotoxic drugs, the amount of drug resinate that would be introduced into the blood stream in the present application would not be sufficient to cause similar problems. The narrow size range of resin microspheres is important for various reasons. Meade et al. [ 91 showed that resin microspheres of varying size distributed differently throughout a tumour-bearing organ of rats. Microspheres greater than 50 pm in diameter did not embolise in tumour tissue to the same extent as their smaller counterparts. Resin microsphere size and pore diameter are rate-limiting factors for the process of diffusion of ions in and out of the resin matrix. Therefore, varying the size range of resin microspheres should vary their drug release characteristics, adding to the importance of a narrow size range. Simple batch manufacturing procedures were used for the attachment of drugs to the ion exchange resins, although it has been reported that column manufactuing processes give higher drug loadings [lo]. In practice, the binding of Adr, 5-FU and FUDR were found to proceed to near the capacity of the resins using the batch method. Nov loads to less than the capacity of the resin (18.3% of capacity) because a commercial 2% drug/O.9% NaCl solution was used. Greater loading would probably have been achieved here if more concentrated, nonionic drug solutions were used. The low equilibrium release levels obtained with this drug did not justify further experiments. The final drug resinate products were easily dispersed in 0.9% NaCl or distilled water without the aid of surfactants. In addition, resulting drug resinates were found to have a long bench life and could be frozen. The closed in vitro drug release system was designed to compare equilibrium drug concen-
trations for the different drug resinates and also to test the rates at which drugs come into equilibrium. All variables were standardised (i.e. flow rate, equivalent amount of drug, temperature and wash medium) to enable a valid comparison of results for the different drug resinates. It might be argued that an open drug release system, where drug resinate is washed with fresh release medium, would more closely imitate the in vitro situation. However, for relatively small drug molecules, little can be learned from such a system as drug release rates would be almost completely dependant on flow rates over the resins. The flow characteristics of plasma over a microsphere embolised in a blood vessel are unknown but are likely to be very much slower than in a vessel free from microsphere blockade. In this static, fixed-volume situation there is equilibrium near the microsphere surface with slow diffusion away as drug is removed by surrounding tissue [ 71. This situation is more in keeping with our in vitro test procedure. The rate at which the resin-bound drugs came into equilibrium with the total bulk release medium was similar for all drugs tested. The half time for equilibrium drug concentration to be reached was between 20 and 30 minutes for the conditions used. This suggests that drug release properties are ionically controlled and mainly dependent on the strength with which drugs are bound to the resin matrix. Diffusion of drugs through the microsphere matrix is not a significant rate-limiting factor. If the release of drug from the resin matrix needs modification, several methods have been described which can vary release characteristics. If an increase in time taken for the system to reach equilibrium is required, the coating of drug resinate particles with other polymers could considerably reduce their drug release rate [ 111. Use of gel (or solid) particles rather than macroporous resins would also greatly increase diffusion rates through the particle. Pore sizes could be changed by altering the polymerization procedures used in the manufactue of the
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microspheres which can also slow drug diffusion rates [ 121. To vary the equilibrium levels of drug released, changes would have to be made to the ionic strength of the acidic and basic groups on the resin or to the hydrophilic/hydrophobic interactions of the drugs with the resin matrix. Drug resinates may be used as a convenient alternative to arterial infusion of cytotoxic drugs into a tumour-bearing organ. Drug resinates have the advantage that they do not require the use of internal or external pumps for drug delivery. Resin microspheres embolise in the microvasculature of a tumour thus slowing blood flow. This enhances the contact time of tumour with released drug. However, equilibrium levels of released drug need to be in the correct concentration ranges. Garnick et al. [ 131 found that after hepatic arterial infusion of Adr at 40 mg rnp2 day-’ for 3 days, the steady-state plasma concentration of Adr was 2.75 x lo-’ M. The in vitro equilibrium Adr concentration is higher than this. However, if a similar dose of adriamycin-loaded resin was introduced into the hepatic artery, it is likely that plasma blood levels would be of the same order as for drug infusion as Adr is extensively metabolised in the liver. This, together with its high loading ability, make Adr an ideal drug for targeted ion exchange resin chemotherapy. Nov is very strongly bound to the Aminex A6 resin with an equilibrium concentration of 6x low7 M in 0.9% NaCl. After a bolus dose of 10 mg rne2, serum drug levels were found to be 0.9 x 10T7 M [ 141. It is unlikely that levels of this order could be obtained using Nov A-6 resinate. Higher drug equilibrium levels could possibly be achieved for Nov if a weak-acid cation exchange resin was used. Therefore, Nov resinate may also be applied to tumour-targeted chemotherapy. When FUDR is peripherally infused at 5 mg (kg body weight) -’ h-l, the steady-state serum FUDR level is approximately 3 X 10M7M [ 151. The in vitro equilibrium drug concentration for
the FUDR resinate is greater than this by a factor of 6000. Therefore, an equivalent dose of drug resinate would produce much too high serum drug levels to make it suitable for in uiuo use. Also, drug release could not be sustained over useful lengths of time. Similarly, 5-FU resinate is unsuitable as an alternative to arterial infusion. The daily dose rate of 5-FU for arterial infusion is approximately 1 g (day) -l. This is equivalent to approximately 4 g of resinate per day, which would cause complete blockade of the microvasculature. Naturally, the in vitro equilibrium drug concentrations obtained in this experiment are likely to be different to the in uiuo situation. A resin microsphere embolised in an arteriole will be bathed in a complex solution of Na+, K+, Ca2+, Mg2+, organic ions and enzymes. Therefore, an in uiuo study examining the release characteristics of drug-carrying ion exchange resins is being carried out and will be reported in a later communication.
CONCLUSIONS
The present investigation has shown that Adr, Nov, 5-FU and FUDR can be readily loaded onto ion exchange resin microspheres. These drug resinates can be made to meet most of the design criteria for therapeutic situations detailed in the introduction. Large drug loadings were obtained for Adr, 5-FU and FUDR. In uitro equilibrium drug concentrations vary considerably between the drugs tested. Potentially useful concentrations were found for Adr-loaded Aminex A-6 resin and experimental modifications may make the method applicable to other drugs. Studies are required to determine the cytotoxic effectiveness of Adr resinate targeted to tumours as compared to conventional chemotherapeutic treatments.
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