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polyaspartic acid microspheres as carriers for the cytotoxic drug adriamycin. II. in vitro drug release rate
Journal
of Controlled
103-109 B.V., Amsterdam - Printed
Release, 8 (1988)
Elsevier Science Publishers
in The Netherlands
HAEMOGtOBIN, TRANSFERRIN AND ALBUMlN/P~LYASPARTlC ACID MICROSPHERES AS CARRIERS FOR THE CYTOTOXIC DRUG ADRIAMYCIN. IN VW?0 DRUG RELEASE RATE N. Willmott*, Department
Il.
Yan Chen and A.T. Florence
of Pharmacy,
School of Pharmacy
and Pharmacology,
University
of Strathclyde,
Glasgow G 1 IXW,
Scotland
(Great Britain) (Recetved June 18, 1987; accepted
in revised form February 22, 1988)
In vitro release rates of the unto-cancer drug adriamycin have been st~ied followi~ its incor~orut~o~ into m~crospheres prepared from albumin, transferr~n, haemoglob~n and an albumin/ po~yaspartic acid mixture. Drug release profiles from albumin, transferrin and haemog~obin microspheres were similar, but significant differences from the albumin/po~yasparti~acid (195 mg/5 mg) system were observed. Mathematical modelling, combined with theoretical considerations, led to the conclusion that the release process is best described as triphasic zero order. Using this model it was observed that initial release rates for a particular system were variable (albumin K, = - 1.34 to - 0.44 nglmg/h; albumin/polyaspartic acid k, = - 1.52 to - 0.41 pg/mg/h)whereas the terminal release rates were more reproducible and appeared characteristic of that system (albumin kCi = - 0.10 t- 0.02 pg/mglh; albuminlpolyaspartic acid k3= - 0.65 2 0.13 uglmglh). The amounts of adriamycin available for release from this compartment were: albumin 3.5 -t I.1 ug; a~bum~nlpolyasparti~acid 18.1 k3.5 pg. The marked difference in release rates of these closely related systems may prove useful in investigations of the relation between release rate and drug potency.
INTRODUCTION
MATERIALS
We have previously identified drug-content, drug release rate and rate of matrix biodegradation as potentially important in the antitumour effect of adriamycin (Adx) -loaded protein microspheres [ 1,2]. In the previous communication we examined drug-content of microspheres prepared from a range of proteins and poiyamino acids: here we investigate in uitro drug release rate from these systems.
Microspheres were prepared and Adx quantified by the techniques previously described [ 1,2] and used in the previous communication.
*To whom correspondence
0168"3659/88/$03.50
should be addressed.
0 1988 Elsevier Science Publishers
AND METHODS
Assay for in vitro drug release The methodused to assess rate of Adx release from microspheres is based on a continuous flow system described by Chien [3]. A sample of dru#-loaded microspheres was applied to a glass column containing glass wool as support material and maintained at 37°C by means of a water jacket. Microspheres were subjected to a
B.V.
104
constant flow of 60 mM phosphate buffer with 0.1% benzalkonium chloride as preservative. Using a fraction collector, samples were collected every hour for 20 hours by which time Adx-content of fractions was near the limit of detection. For systems prepared from pure proteins, measurements of Adx in fractions from the drug release assay and in trypsin digests (to assess drug content) were performed using HPLC with fiuorescence detection. Because HPLC values agreed closely with total fluorescence values for Adx incorporated or released from albumin/polyaspartic acid microspheres, either method was used for Adx measurements involving this system. To promote reproducibility, column packing and sample application were ostensibly identical for each run and flow rat,e was always 5.5 ml/h.
whether curves were biphasic or triphasic was made on the basis of which had the least total sum of squares computed from individual sums of squares of each regression line. RESULTS Release rate of Adx from micraspheres pared from pure proteins
pre-
Release af Adx from microspheres prepared from albumin, haemoglobin and transferrin was measured at. int,ervals of 1 h and release profiles derived. The first point, examined was the influence of amount, (mg) of drug-loaded microspheres applied to the column. To investigate this, five experiments were performed in which drug release was assessed with different amounts of albumin microspheres (22-78.4 mg
Data analysis
The drug release profiles generated from our experiments have been fitted to a model described by Gupt.a et al. [ 41. This model assumes compartmentalisat,ion of Adx within microsphere matrix, a proportion being superficially sit.uated and released rapidly and the remainder being entrapped within the protein matrix and released more slowly. Thus, release curves are multiphasic. Because our release data were equally well described by a series of zero order, first order or Higuchi equations, the principle of adopting the least complex mechanism for a phenomenon (i.e. zero order model) was invoked. Consequently, all release profiles were plotted in the form pg incorporated Adx remaining in microspheres (A) versus time (t). To determine the number of phases a FORTRAN program was used that involved fitting either two or three linear regression lines of the form A =k,t+ C,x (k, is zero order rate constant, and C,, is zero t.ime intercept for that phase. k and C for t,he first two phases were obtained by the method of residuals [5 J_ The judgement as to
Fig. 1. Plot of incorporated Adx remaining in microspheres (/tg/mg) versus time (h) for the albumin system. Symbols correspond to the different experiments listed in Table 1. ~i~rospheres were immobilised on the column and Adx release assessed when subjected to a continuous flow of buffer. Drug content of microspheres and individual fractions from release assay, collected at hourly intervals, were assessed by HPLC with fluorescence detection or measurements of total fluorescence.
105
Fig. 2. Example of use of method of residuals to calculate release rates of first and second phases. Rate constants k, and k, of first and second phases are given by slopes of second residual and first residual respectively.
microspheres, 209-517 pg Adx). The individual drug release profiles are shown in Fig. 1. An example of the use of the method of residuals [ 51 is shown in Fig. 2 and zero order rate constants derived from each experiment in Table 1. In each case drug release was most closely described as triphasic.
It can be seen from Table 1 that the rate constant for the terminal phase (h,) was similar in all five experiments ( - 0.1 ( k 0.02) ,ug/mg/h) and zero time intercept was 3.5 ( 5 1.1) ,ug. On the contrary, the highest initial rate constant (h, ) was observed when drug content was highest. Release of Adx from haemoglobin and transferrin microspheres was similar to albumin (data not shown ) . Elution of Adx solution (150-500 pg) from the column was rapid but had a finite time span. The profiles closely approximated a monophasic first order process [ 21 with a half-time of 1.5 h (h=0.46 h-‘). Release rate of Adx from albumin/polyaspartic acid microspheres
As described by Goldberg with polyglutamic acid [ 61 and ourselves (see previous communication) with polyaspartic acid the use of polyamino acids that are negatively charged at neutral pH can increase drug content of Adxloaded albumin microspheres. It was therefore
TABLE 1 Rate of Adx release from albumin microspheres Column loading’ (,ng Adx on column ) [Symbol in Fig. 1 ]
Adx content
Microsphere
Cm Adx/mg)
(pm)
(&w/h) k, k, k,i k, k, k:, k, k, k,, k, k, k:, k, k2 k:,
209 [o]
7.9
41
292 [WI
8.3
23
13.5
24
8.9
19
6.6
19
297 [.I
456
101
517 [A]
size
Release rate constants”
-0.66 -0.20 -0.11 -0.44 -0.28 -0.11 -1.34 -0.34 -0.10 -0.64 -0.19 -0.12 -0.52 -0.22 -0.08
% Adx released in 20 h
84
80
76
94
87
‘Microspheres prepared in an ostensibly identical manner were applied to the column at different loadings Adx). 200 mg albumin and 10 mg Adx were used in microsphere preparation. ‘Release rate constants were derived from profiles in Fig. 1 by fitting the data to a triphasic zero order model.
(209-517 pg
106 LO,
in Table 3 it appears that increasing the proportion of polyaspartic acid relative to albumin had no marked effect on release rate (compare Table 3 with Table 2).
DISCUSSION
Fig. 3. Plot of incorporated Adx remaining in microspheres (pg/mg) versus time (h) for the albumin/polyaspartic acid system. Symbols correspond to the different experiments in Table 2. Conditions as for Fig. 1.
of interest
to examine the release rate of the albumin/polyaspartic acid system. As in the section describing Adx release from
microspheres prepared with pure proteins the first parameter examined was the effect of changing the amount of drug-loaded microsPeres on the column, i.e. the column loading. In these experiments column loading varied from 31.5 mg to 70.3 mg microspheres (1,109 pg to 2,095 ,ug Adx ) . Individual release profiles are shown in Fig. 3 and zero order rate constants derived from each experiment in Table 2. As for albumin microspheres, drug release was most closely described as triphasic. Terminal release rate constants (h,) computed from these experiments were in fairly close agreement (0.65k 0.13 pg/mg/h) as was the zero time intercept of this phase (18.1535 pg). Wider variation was encountered with k, values that appeared to depend on volumn loading. The proportions of albumin and polyaspartic acid used were varied in an attempt to change the rate of Adx release. However, from the data
The rate at which therapeutic agents are delivered to biological systems can be an important determinant of their pharmacodynamic effect. However, it is impossible to predict at present in which direction dose-response curves will be shifted and each system must be empirically investigated [ 7-101. In animal models cancer chemotherapy appears to follow this paradigm, either when administered systematically [11,12] or at the tumour site [13]. The importance of duration of exposure to drug is supported by observations showing a correlation between response to methotrexate and slow systemic clearance in patients [ 141. In our own studies, protracted release of Adx from albumin microspheres led to higher drug levels at the tumour site and increased anti-tumour effect [151. Because response to a wide range of drugs, chemotherapeutic including some cancer agents, depends on the rate of drug input we have investigated in detail the release of Adx in vitro from the different types of microsphere. By this means we hoped to assign to each system an intrinsic release rate that could be correlated with drug activity in duo. Preliminary results indicate that slow release rate correlates with increased anti-tumour effect [ 161. The flow-through system used for our investigations was chosen because simpler systems were found to have serious deficiencies. For example, merely maintaining Adx-loaded microspheres in suspension by stirring resulted in maximal drug release within minutes and a subsequent apparent loss of drug from the supernatant fraction, presumably due to readsorption to microspheres. The use of dialysis tubing also had to be abandoned because of drug
107 TABLE 2 Rate of Adx release from albumin/polyaspartic
acid microspheres
Column loading’ (pug Adx on column) [Symbol in Fig. 3 ]
Adx content ( pg Adx per mg )
Microsphere (pm)
1,109 [Bl
35.2
27.4
1,345 101
35.3
26.3
1,502”
35.6
32.5
1,925 [A ]
28.9
29.4
(I) size
Release rate constants’ (&w/h)
% Adx released in 20 h
k, -1.52 k, -0.85 k, -0.48 k, -1.26 k2 -0.82 k:, -0.63
89
k, -0.93 k2 -0.79
84
83
k:l -0.73 12, -0.72
84
k, -0.49
2,095 [A]
29.8
k:%-0.61 k, -0.41
18.1
79
k, -0.31 kc3-0.82
‘Microspheres prepared in an ostensibly identical manner were applied to the column at different loadings (1,109-2,095 fig Adx). Albumin/polyaspartic acid ratio used in microsphere preparation was 195/5, total 200 mg. 10 mg Adx used in each case. “Release rate constants were derived from profiles in Fig. 3 by fitting the data to a triphasic zero order model. “For reasons of clarity these data are not shown in Fig. 3. TABLE 3 Rate of Adx release from albumin/polyaspartic Ratio of albumin/PAA’
acid microspheres
(II)
Adx content
Microsphere
( pLgAWmg )
(pm)
(I*dmdh)
200126
22.6
25.4
k, -0.53 k, -0.46
190/10
32.2
28.8
180/20
31.1
27.5
size
Release rate constan&
% Adx eluted in 20 h
(mg/mg) 91
k:, -0.51 k, -0.97 k, -0.60
77
k:, -0.67 k, -0.55 k, -0.37
85
k:, -0.85
‘Microspheres prepared at different ratios of albumin/polyaspartic acid (PAA) were applied to the column and release rate assessed as in Tables 1 and 2.10 mg Adx was used in each case. ‘Release rate constants were derived from release rate profiles by fitting the data to a triphasic zero order model.
adsorption. The flow through system in which all components were made of glass avoided both of these difficulties.
Previous work from this laboratory [2] has shown that the major determinant of in vitro Adx release rate from albumin microspheres
108
was the concentration of cross-linking agent (glutaraldehyde ) used in stabilisation of the albumin dispersion/emulsion. Part.icle size was of minor importance. It was also observed that, over the 20 h bime span, drug release was multiphasic, with portions of the curve fitting eit,her zero order, first, order or Higuchi equations. Similar observations have been reported by Gupta et al. [ 41 who concluded, on t.he basis of theoretical considerations, t,hat Adx release was best described by a series of zero order equations. Invoking the principle of adopting the least, complex model for a given phenomenon, the zero order model was applied t.o our drug release data. In all cases the release profiles (Figs. 1 and 3 1 were best described as triphasic, rather than biphasic, zero order. Thus, it is not possible Tao characterise each microsphere type by a single n~lmerical coefficient for purposes of comparison. The question then arises, which rate constant will be most useful in this regard? I?, is not a good candidat.e because of variation from experiment to experiment. Moreover, it. has been proposed [ 171 that. initial release of Adx is due to ‘loosely’ attached surface drug, the biological effect of which might not be expected to differ from drug in solut.ion, whereas the terminal release rate is a genuine sustained release process. This being sot variation of k, in our system will assume decreased importance whereas comparison of k:% will be most inst,ructive. Although variable, t.he h, values for albumin microsperes ( - 1.34 to -0.44) and albumin/ polyaspartic acid microspheres ( - 1.52 to -0.41) were of a comparable range, perhaps suggesting a similar underlying mechanism of release. On the contrary, h,, values were reproducible within a particular system (h:, for albumin= -0.12 to -0.08 /Lg/mg/h; k,, for the albumin/polyaspartic acid mixture = - 0.82 to - 0.48 /cg/mg/h 1 and therefore could serve as the intrinsic release rate of the system. The amount,s of drug available for release from this compartment, obtained by extrapolating t)be
terminal release phase to t=O, were albumin, 3.5 pg/mg and albumin/polyaspartic acid, 18.1 /lg/mg. The postulated underlying mechanism described by the mathematical model requires drug release from the different compartments to take place in a series of concurrent (not sequential) phases. It has been suggested [4,17] that rapid release in the initial phase(s) is due to drug dissolmion or desorption from superficial sites or pores, or passage through channels, and that slow release is due to drug entrapped within the matrix and distant from the particle surface. Slow release from this compartment. is explained by the increased path length through which drug must diffuse. Whilst our results are not. at variance with this suggestion, we have never observed pores in albumin microspheres prepared in our laboratory and therefore the explanation for rapid initial drug release in our system may be different from Gupta et al. ]4,17]. In our system the differences in reiease rate between initial and final phases may simply reflect the dist,ance that adriamycin has to diffuse through the albumin matrix (it is assumed that after the ~~ashingprocedure involving aqueous buffers, microspheres are fully hydrated prior to their use in the release assay). Increasing the proportion ofpolyaspartic acid relative to albumin had no marked effect on release rate constants or on amount of Adx available for release from the compartment providing slowest release. This may be because it has been estimated in the previous communication that incorporation of 5 mg of polyaspartic acid and 10 mg of Adx represents a stoichiometry of 5 aspartic acid residues to one molecule of Adx. Thus, aspartic acid residues are already in excess and an increase would not be expected to have any effect. In summary, by incorporating polyaspartic acid into albumin microspheres, the in vitro release of Adx has been markedly altered. A comparison of the in uiuo anti-tumour effects and tissue pharmacokinetics of these two systems
109
may be of value in elucidating rate in drug potency.
the role of release
8
9
ACKNOWLEDGEMENTS
The authors thank Mrs. Agnes Hughes for expert technical assistance, Mrs. Elizabeth Carruthers who typed the manuscript and Mr. Don Evans of the Computer Centre, University of Strathclyde who wrote the FORTRAN programme. NW is grateful to the MRC of Great Britain and YC to the British Council for financial support.
10
11
12
13
REFERENCES N. Willmott, J. Cummings, J.F.B. Stuart and A.T. Florence, Adriamycin-loaded albumin microspheres: Preparation, in uiuo distribution and release in the rat, Biopharm. Drug Disposition, 6 (1985) 91-104. N. Willmott, J. Cummings and A.T. Florence, In vitro release of adriamycin from drug-loaded albumin and haemoglobin microspheres, J. Microencapsulation, 2 (1985) 293-304. Y.W. Chien, Novel Drug Delivery Systems, Marcel Dekker, New York, 1982, p. 547. P.K. Gupta, C.T. Hung and D.G. Perrier, Albumin microspheres I. Release characteristics of adriamycin, Int. J. Pharm., 33 (1986) 137-146. M. Gibaldi and D.G. Perrier, Pharmacokinetics, 2nd edn., Marcel Dekker, New York, 1982, p. 433. E.P. Golderg, H. Iwata and W. Longo, Hydrophilic albumin and dextran ion-exchange microspheres for localised chemotherapy, in: S.S. Davis, L. Illum, J.G. McVie and E. Tomlinson (Eds.), Microspheres and Drug Therapy, Elsevier, Amsterdam, 1984, pp. 309325. P. Goldman, Rate controlled drug delivery, N.Eng. J. Med., 307 (1982) 286-290.
14
15
16
17
H.A.J. Struyker Boudier, Rate controlled drug delivery: Pharmacological, therapeutic and industrial perspectives, Trends Pharmacol. Sci., 3 (1982) 162-164. J: Fara, Recent advances in parenteral drug delivery systems, J. Parent. Sci. Technol., 37 (1983) 20-25. J. Urquhart, J.W. Fara and K.L. Willis, Rate-controlled delivery systems in drug and hormone research, Ann. Rev. Pharmacol. Toxicol., 24 (1984) 199236. B.I. Sikic, J.M. Collins, E.G. Mimnaugh and T.E. Gram, Improved therapeutic index of bleomycin when administered by continuous infusion, Cancer Treatment Rep., 62 (1978) 2011-2017. Y.M. Peng, D.S. Alberts, H.S.G. Chen, N. Masor and T.E. Moon, Antitumour activity and plasma kinetics of bleomycin by continuous and intermittent administration, Br. J. Cancer, 41 (1980) 644-647. D.M.Long,L.R. Sehga1,M.E. Derios,M.V. Rios,P.B. Szanto and R. Forrest, Depot cancer chemotherapy through polymer membranes, Rev. Surgery, 30 (1973) 229-240. W.E. Evans, W.R. Crum, C.F. Stewart, W.P. Bowman, C. Chen, M. Abromowitch and J.V. Simone, Methotrexate systemic clearance influences probability of relapse in children with standard-risk acute lymphocytic leukaemia, Lancet, 1 (1984) 359-362. N. Willmott and J. Cummings, Increased anti-tumour effect of adriamycin-loaded albumin microspheres is associated with anaerobic bioreduction of drug in tumour tissue, Biochem. Pharmacol., 36 (1987) 521526. Yan Chen, N. Willmott and A.T. Florence, Adriamytin-loaded protein microspheres: correlation of in uitro release rate and in vivo potency, in: Proc. Intern. Symp. Control. Rel. Bioact. Mater., Vol. 14, Controlled Release Society, Lincolnshire, IL, 1987, pp. 239240. P.K. Gupta, C.T. Hung and D.G. Perrier, Albumin microspheres II. Effect of stabilisation temperature on the release of adriamycin, Int. J. Pharm., 33 (1986) 147-153.
of Controlled
103-109 B.V., Amsterdam - Printed
Release, 8 (1988)
Elsevier Science Publishers
in The Netherlands
HAEMOGtOBIN, TRANSFERRIN AND ALBUMlN/P~LYASPARTlC ACID MICROSPHERES AS CARRIERS FOR THE CYTOTOXIC DRUG ADRIAMYCIN. IN VW?0 DRUG RELEASE RATE N. Willmott*, Department
Il.
Yan Chen and A.T. Florence
of Pharmacy,
School of Pharmacy
and Pharmacology,
University
of Strathclyde,
Glasgow G 1 IXW,
Scotland
(Great Britain) (Recetved June 18, 1987; accepted
in revised form February 22, 1988)
In vitro release rates of the unto-cancer drug adriamycin have been st~ied followi~ its incor~orut~o~ into m~crospheres prepared from albumin, transferr~n, haemoglob~n and an albumin/ po~yaspartic acid mixture. Drug release profiles from albumin, transferrin and haemog~obin microspheres were similar, but significant differences from the albumin/po~yasparti~acid (195 mg/5 mg) system were observed. Mathematical modelling, combined with theoretical considerations, led to the conclusion that the release process is best described as triphasic zero order. Using this model it was observed that initial release rates for a particular system were variable (albumin K, = - 1.34 to - 0.44 nglmg/h; albumin/polyaspartic acid k, = - 1.52 to - 0.41 pg/mg/h)whereas the terminal release rates were more reproducible and appeared characteristic of that system (albumin kCi = - 0.10 t- 0.02 pg/mglh; albuminlpolyaspartic acid k3= - 0.65 2 0.13 uglmglh). The amounts of adriamycin available for release from this compartment were: albumin 3.5 -t I.1 ug; a~bum~nlpolyasparti~acid 18.1 k3.5 pg. The marked difference in release rates of these closely related systems may prove useful in investigations of the relation between release rate and drug potency.
INTRODUCTION
MATERIALS
We have previously identified drug-content, drug release rate and rate of matrix biodegradation as potentially important in the antitumour effect of adriamycin (Adx) -loaded protein microspheres [ 1,2]. In the previous communication we examined drug-content of microspheres prepared from a range of proteins and poiyamino acids: here we investigate in uitro drug release rate from these systems.
Microspheres were prepared and Adx quantified by the techniques previously described [ 1,2] and used in the previous communication.
*To whom correspondence
0168"3659/88/$03.50
should be addressed.
0 1988 Elsevier Science Publishers
AND METHODS
Assay for in vitro drug release The methodused to assess rate of Adx release from microspheres is based on a continuous flow system described by Chien [3]. A sample of dru#-loaded microspheres was applied to a glass column containing glass wool as support material and maintained at 37°C by means of a water jacket. Microspheres were subjected to a
B.V.
104
constant flow of 60 mM phosphate buffer with 0.1% benzalkonium chloride as preservative. Using a fraction collector, samples were collected every hour for 20 hours by which time Adx-content of fractions was near the limit of detection. For systems prepared from pure proteins, measurements of Adx in fractions from the drug release assay and in trypsin digests (to assess drug content) were performed using HPLC with fiuorescence detection. Because HPLC values agreed closely with total fluorescence values for Adx incorporated or released from albumin/polyaspartic acid microspheres, either method was used for Adx measurements involving this system. To promote reproducibility, column packing and sample application were ostensibly identical for each run and flow rat,e was always 5.5 ml/h.
whether curves were biphasic or triphasic was made on the basis of which had the least total sum of squares computed from individual sums of squares of each regression line. RESULTS Release rate of Adx from micraspheres pared from pure proteins
pre-
Release af Adx from microspheres prepared from albumin, haemoglobin and transferrin was measured at. int,ervals of 1 h and release profiles derived. The first point, examined was the influence of amount, (mg) of drug-loaded microspheres applied to the column. To investigate this, five experiments were performed in which drug release was assessed with different amounts of albumin microspheres (22-78.4 mg
Data analysis
The drug release profiles generated from our experiments have been fitted to a model described by Gupt.a et al. [ 41. This model assumes compartmentalisat,ion of Adx within microsphere matrix, a proportion being superficially sit.uated and released rapidly and the remainder being entrapped within the protein matrix and released more slowly. Thus, release curves are multiphasic. Because our release data were equally well described by a series of zero order, first order or Higuchi equations, the principle of adopting the least complex mechanism for a phenomenon (i.e. zero order model) was invoked. Consequently, all release profiles were plotted in the form pg incorporated Adx remaining in microspheres (A) versus time (t). To determine the number of phases a FORTRAN program was used that involved fitting either two or three linear regression lines of the form A =k,t+ C,x (k, is zero order rate constant, and C,, is zero t.ime intercept for that phase. k and C for t,he first two phases were obtained by the method of residuals [5 J_ The judgement as to
Fig. 1. Plot of incorporated Adx remaining in microspheres (/tg/mg) versus time (h) for the albumin system. Symbols correspond to the different experiments listed in Table 1. ~i~rospheres were immobilised on the column and Adx release assessed when subjected to a continuous flow of buffer. Drug content of microspheres and individual fractions from release assay, collected at hourly intervals, were assessed by HPLC with fluorescence detection or measurements of total fluorescence.
105
Fig. 2. Example of use of method of residuals to calculate release rates of first and second phases. Rate constants k, and k, of first and second phases are given by slopes of second residual and first residual respectively.
microspheres, 209-517 pg Adx). The individual drug release profiles are shown in Fig. 1. An example of the use of the method of residuals [ 51 is shown in Fig. 2 and zero order rate constants derived from each experiment in Table 1. In each case drug release was most closely described as triphasic.
It can be seen from Table 1 that the rate constant for the terminal phase (h,) was similar in all five experiments ( - 0.1 ( k 0.02) ,ug/mg/h) and zero time intercept was 3.5 ( 5 1.1) ,ug. On the contrary, the highest initial rate constant (h, ) was observed when drug content was highest. Release of Adx from haemoglobin and transferrin microspheres was similar to albumin (data not shown ) . Elution of Adx solution (150-500 pg) from the column was rapid but had a finite time span. The profiles closely approximated a monophasic first order process [ 21 with a half-time of 1.5 h (h=0.46 h-‘). Release rate of Adx from albumin/polyaspartic acid microspheres
As described by Goldberg with polyglutamic acid [ 61 and ourselves (see previous communication) with polyaspartic acid the use of polyamino acids that are negatively charged at neutral pH can increase drug content of Adxloaded albumin microspheres. It was therefore
TABLE 1 Rate of Adx release from albumin microspheres Column loading’ (,ng Adx on column ) [Symbol in Fig. 1 ]
Adx content
Microsphere
Cm Adx/mg)
(pm)
(&w/h) k, k, k,i k, k, k:, k, k, k,, k, k, k:, k, k2 k:,
209 [o]
7.9
41
292 [WI
8.3
23
13.5
24
8.9
19
6.6
19
297 [.I
456
101
517 [A]
size
Release rate constants”
-0.66 -0.20 -0.11 -0.44 -0.28 -0.11 -1.34 -0.34 -0.10 -0.64 -0.19 -0.12 -0.52 -0.22 -0.08
% Adx released in 20 h
84
80
76
94
87
‘Microspheres prepared in an ostensibly identical manner were applied to the column at different loadings Adx). 200 mg albumin and 10 mg Adx were used in microsphere preparation. ‘Release rate constants were derived from profiles in Fig. 1 by fitting the data to a triphasic zero order model.
(209-517 pg
106 LO,
in Table 3 it appears that increasing the proportion of polyaspartic acid relative to albumin had no marked effect on release rate (compare Table 3 with Table 2).
DISCUSSION
Fig. 3. Plot of incorporated Adx remaining in microspheres (pg/mg) versus time (h) for the albumin/polyaspartic acid system. Symbols correspond to the different experiments in Table 2. Conditions as for Fig. 1.
of interest
to examine the release rate of the albumin/polyaspartic acid system. As in the section describing Adx release from
microspheres prepared with pure proteins the first parameter examined was the effect of changing the amount of drug-loaded microsPeres on the column, i.e. the column loading. In these experiments column loading varied from 31.5 mg to 70.3 mg microspheres (1,109 pg to 2,095 ,ug Adx ) . Individual release profiles are shown in Fig. 3 and zero order rate constants derived from each experiment in Table 2. As for albumin microspheres, drug release was most closely described as triphasic. Terminal release rate constants (h,) computed from these experiments were in fairly close agreement (0.65k 0.13 pg/mg/h) as was the zero time intercept of this phase (18.1535 pg). Wider variation was encountered with k, values that appeared to depend on volumn loading. The proportions of albumin and polyaspartic acid used were varied in an attempt to change the rate of Adx release. However, from the data
The rate at which therapeutic agents are delivered to biological systems can be an important determinant of their pharmacodynamic effect. However, it is impossible to predict at present in which direction dose-response curves will be shifted and each system must be empirically investigated [ 7-101. In animal models cancer chemotherapy appears to follow this paradigm, either when administered systematically [11,12] or at the tumour site [13]. The importance of duration of exposure to drug is supported by observations showing a correlation between response to methotrexate and slow systemic clearance in patients [ 141. In our own studies, protracted release of Adx from albumin microspheres led to higher drug levels at the tumour site and increased anti-tumour effect [151. Because response to a wide range of drugs, chemotherapeutic including some cancer agents, depends on the rate of drug input we have investigated in detail the release of Adx in vitro from the different types of microsphere. By this means we hoped to assign to each system an intrinsic release rate that could be correlated with drug activity in duo. Preliminary results indicate that slow release rate correlates with increased anti-tumour effect [ 161. The flow-through system used for our investigations was chosen because simpler systems were found to have serious deficiencies. For example, merely maintaining Adx-loaded microspheres in suspension by stirring resulted in maximal drug release within minutes and a subsequent apparent loss of drug from the supernatant fraction, presumably due to readsorption to microspheres. The use of dialysis tubing also had to be abandoned because of drug
107 TABLE 2 Rate of Adx release from albumin/polyaspartic
acid microspheres
Column loading’ (pug Adx on column) [Symbol in Fig. 3 ]
Adx content ( pg Adx per mg )
Microsphere (pm)
1,109 [Bl
35.2
27.4
1,345 101
35.3
26.3
1,502”
35.6
32.5
1,925 [A ]
28.9
29.4
(I) size
Release rate constants’ (&w/h)
% Adx released in 20 h
k, -1.52 k, -0.85 k, -0.48 k, -1.26 k2 -0.82 k:, -0.63
89
k, -0.93 k2 -0.79
84
83
k:l -0.73 12, -0.72
84
k, -0.49
2,095 [A]
29.8
k:%-0.61 k, -0.41
18.1
79
k, -0.31 kc3-0.82
‘Microspheres prepared in an ostensibly identical manner were applied to the column at different loadings (1,109-2,095 fig Adx). Albumin/polyaspartic acid ratio used in microsphere preparation was 195/5, total 200 mg. 10 mg Adx used in each case. “Release rate constants were derived from profiles in Fig. 3 by fitting the data to a triphasic zero order model. “For reasons of clarity these data are not shown in Fig. 3. TABLE 3 Rate of Adx release from albumin/polyaspartic Ratio of albumin/PAA’
acid microspheres
(II)
Adx content
Microsphere
( pLgAWmg )
(pm)
(I*dmdh)
200126
22.6
25.4
k, -0.53 k, -0.46
190/10
32.2
28.8
180/20
31.1
27.5
size
Release rate constan&
% Adx eluted in 20 h
(mg/mg) 91
k:, -0.51 k, -0.97 k, -0.60
77
k:, -0.67 k, -0.55 k, -0.37
85
k:, -0.85
‘Microspheres prepared at different ratios of albumin/polyaspartic acid (PAA) were applied to the column and release rate assessed as in Tables 1 and 2.10 mg Adx was used in each case. ‘Release rate constants were derived from release rate profiles by fitting the data to a triphasic zero order model.
adsorption. The flow through system in which all components were made of glass avoided both of these difficulties.
Previous work from this laboratory [2] has shown that the major determinant of in vitro Adx release rate from albumin microspheres
108
was the concentration of cross-linking agent (glutaraldehyde ) used in stabilisation of the albumin dispersion/emulsion. Part.icle size was of minor importance. It was also observed that, over the 20 h bime span, drug release was multiphasic, with portions of the curve fitting eit,her zero order, first, order or Higuchi equations. Similar observations have been reported by Gupta et al. [ 41 who concluded, on t.he basis of theoretical considerations, t,hat Adx release was best described by a series of zero order equations. Invoking the principle of adopting the least, complex model for a given phenomenon, the zero order model was applied t.o our drug release data. In all cases the release profiles (Figs. 1 and 3 1 were best described as triphasic, rather than biphasic, zero order. Thus, it is not possible Tao characterise each microsphere type by a single n~lmerical coefficient for purposes of comparison. The question then arises, which rate constant will be most useful in this regard? I?, is not a good candidat.e because of variation from experiment to experiment. Moreover, it. has been proposed [ 171 that. initial release of Adx is due to ‘loosely’ attached surface drug, the biological effect of which might not be expected to differ from drug in solut.ion, whereas the terminal release rate is a genuine sustained release process. This being sot variation of k, in our system will assume decreased importance whereas comparison of k:% will be most inst,ructive. Although variable, t.he h, values for albumin microsperes ( - 1.34 to -0.44) and albumin/ polyaspartic acid microspheres ( - 1.52 to -0.41) were of a comparable range, perhaps suggesting a similar underlying mechanism of release. On the contrary, h,, values were reproducible within a particular system (h:, for albumin= -0.12 to -0.08 /Lg/mg/h; k,, for the albumin/polyaspartic acid mixture = - 0.82 to - 0.48 /cg/mg/h 1 and therefore could serve as the intrinsic release rate of the system. The amount,s of drug available for release from this compartment, obtained by extrapolating t)be
terminal release phase to t=O, were albumin, 3.5 pg/mg and albumin/polyaspartic acid, 18.1 /lg/mg. The postulated underlying mechanism described by the mathematical model requires drug release from the different compartments to take place in a series of concurrent (not sequential) phases. It has been suggested [4,17] that rapid release in the initial phase(s) is due to drug dissolmion or desorption from superficial sites or pores, or passage through channels, and that slow release is due to drug entrapped within the matrix and distant from the particle surface. Slow release from this compartment. is explained by the increased path length through which drug must diffuse. Whilst our results are not. at variance with this suggestion, we have never observed pores in albumin microspheres prepared in our laboratory and therefore the explanation for rapid initial drug release in our system may be different from Gupta et al. ]4,17]. In our system the differences in reiease rate between initial and final phases may simply reflect the dist,ance that adriamycin has to diffuse through the albumin matrix (it is assumed that after the ~~ashingprocedure involving aqueous buffers, microspheres are fully hydrated prior to their use in the release assay). Increasing the proportion ofpolyaspartic acid relative to albumin had no marked effect on release rate constants or on amount of Adx available for release from the compartment providing slowest release. This may be because it has been estimated in the previous communication that incorporation of 5 mg of polyaspartic acid and 10 mg of Adx represents a stoichiometry of 5 aspartic acid residues to one molecule of Adx. Thus, aspartic acid residues are already in excess and an increase would not be expected to have any effect. In summary, by incorporating polyaspartic acid into albumin microspheres, the in vitro release of Adx has been markedly altered. A comparison of the in uiuo anti-tumour effects and tissue pharmacokinetics of these two systems
109
may be of value in elucidating rate in drug potency.
the role of release
8
9
ACKNOWLEDGEMENTS
The authors thank Mrs. Agnes Hughes for expert technical assistance, Mrs. Elizabeth Carruthers who typed the manuscript and Mr. Don Evans of the Computer Centre, University of Strathclyde who wrote the FORTRAN programme. NW is grateful to the MRC of Great Britain and YC to the British Council for financial support.
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