Download Studies of a benzoporphyrin derivative with Pluronics and D. Dolphin

1321
Studies of a benzoporphyrin derivative with
Pluronics
N. Hioka, R.K. Chowdhary, N. Chansarkar, D. Delmarre, E. Sternberg,
and D. Dolphin
Abstract: The synthetic route for the benzoporphyrin derivatives produces two regioisomers in equimolar quantities
(ring A and B isomers). A derivative of the A-ring product, BPD-MA (benzoporphyrin-derivative monoacid ring A,
verteporfin), has recently been approved in North America and Europe for the treatment of age-related macular degeneration. The B-ring isomers, contrary to the A-ring isomers, exhibit high aggregation in many formulations, which results in inadequate drug delivery for clinical uses. To avoid aggregation, a non-ionic surfactant polymer such as a
Pluronic — poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) — may be used as a formulation
excipient. The triblock polymer investigated here is designated P123 (or poloxamer 403). When used to formulate a
monoacid benzoporphyrin B-ring derivative (2), a critical micelle concentration of P123 in water occurred at approximately 0.015 to 0.03%. The apparent pKa of compound 2 was dependent on its concentration in P123, and decreased
as the molar ratio (P123:2) increased. High concentrations of P123 and neutral pH were found to be the best conditions to maintain the drug in its monomeric form. Kinetic studies suggest that the aggregate of 2 contains several molecules, and is formed by a catalyzed self-assembly process. Samples with 1 mg mL–1 of drug, at pH = 7.4, and 4.8%
of Pluronic showed satisfactory capacity to load and keep monomers stable. This formulation has potential PDT applications.
Key words: Pluronic, poloxamers, block copolymers, photosensitizing drug, photodynamic therapy (PDT), formulation,
micelles.
Résumé : La voie de synthèse des dérivés de la benzoporphyrine conduit à deux régioisomères en quantités
équimolaires (isomères des cycles A et B). Un dérivé du produit du cycle A, BPD-MA, (le dérivé monoacide du cycle
A de la benzoporphyrine, la vertéporfine) a récemment été approuvé en Amérique du Nord et en Europe pour le
traitement de la dégénérescence maculaire associée à l’âge. Les isomères du cycle B, contrairement aux isomères du
cycle A, présentent des niveaux d’agrégation élevés dans plusieurs formulations qui les rendent impropres à la
dissémination du médicament pour les utilisations cliniques. On peut d’éviter l’agrégation en utilisant comme additif de
formulation un agent de surface polymère, non ionique, tel qu’un Pluronique — poly(oxyde d’éthylène)-poly(oxyde de
propylène)-poly(oxyde d’éthylène). Le polymère à trois blocs étudié ici est désigné P123 (ou poloxamère 403).
Lorsqu’on l’utilise dans la formulation d’un dérivé monoacide du cycle B de la benzoporphyrine (2), on observe une
concentration micellaire critique de P123 dans l’eau aux environs de 0,015 à 0,03%. Le pKa apparent du composé 2
dépend de sa concentration dans P123 et il diminue avec une augmentation du rapport molaire (P123:2). On a trouvé
que des concentrations élevées de P123 et un pH neutre correspondent aux conditions optimales pour maintenir le
médicament dans sa forme monomère. Des études cinétiques suggèrent que l’agrégat de 2 contient plusieurs molécules
et qu’il se forme par un processus d’autoassemblage autocatalysé. Des échantillons de 1 mg mL–1 de médicament, à un
pH = 7,4 et une concentration de 4,8% de pluronique présentent une capacité satisfaisante à dissoudre et à maintenir
les monomères stables. Cette formulation a du potentiel dans les applications de thérapie photodynamique (TPD).
Mots clés: Pluronique, poloxamères, copolymères à blocs, médicament photosensibilisant, thérapie photodynamique
(TPD), formulation, micelles.
[Traduit par la Rédaction]
Introduction
Photodynamic therapy (PDT) is an approved treatment
against cancer and age related macular degeneration, and has
been shown to be efficient against psoriasis, arthritis and
other diseases (1, 2). The first officially approved drug for
PDT was Photofrin®, which exhibited some side effects such
as prolonged skin photosensitivity, a long waiting time be-
Received 26 June 2002. Published on the NRC Research Press Web site at http://canjchem.nrc.ca on 17 October 2002.
N. Hioka. Departamento de Quimica, Universidade Estadual de Maringa, Brazil.
R.K. Chowdhary, N. Chansarkar, D. Delmarre, E. Sternberg, and D. Dolphin.1 Department of Chemistry, University of British
Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1, Canada.
1
Corresponding author (e-mail address: [email protected]).
Can. J. Chem. 80: 1321–1326 (2002)
DOI: 10.1139/V02-167
© 2002 NRC Canada
1322
tween intravenous injection and photo activation (2 days),
and limited light penetration through tissue (1) of the
630 nm light used to activate it. The photoactive component
of the liposomal formulated verteporfin (recently approved
for the treatment of age-related macular degeneration) is a
benzoporphyrin derivative monoacid ring A (BPD-MA, 1).
The synthetic route to this type of molecule provides, in
equimolar quantities, two reduced porphyrin rings (A and B,
which are ring regioisomers). Both sets of regioisomers
show similar ability to accumulate selectively and be retained by abnormal or hyperproliferative cells, such as cancerous tissue (3–5). Both classes exhibit a tendency to form
aggregates (6, 7), following the typical behavior of many
porphyrin and chlorin compounds (8). The A-ring compounds exhibit less tendency to aggregate, however, and can
be used in liposomal formulations (5, 9). In contrast, the
B-ring derivatives exhibit high aggregation behavior, resulting in nonhomogeneous solutions and diminution of singlet
oxygen formation (6), which is suggested as one of the most
significant species involved in cell death (1, 10).
To avoid aggregation and to improve drug concentration
in target tissues, the formulation of the drug is especially important. We have investigated the use of Pluronic
(poloxamers), water-soluble triblock copolymers containing
ethylene oxide units (PEO = the hydrophilic regions) at the
extremities and propylene oxide units (PPO = the hydrophobic section) in the middle (often denoted PEO-PPO-PEO or
(EO)m(PO)n(EO)m). Because of their synthetic versatility, a
number of copolymers are commercially available for distinct uses in different areas, which include drug solubilization and controlled release (11–13). Many of them are
believed to form micelles with the terminal PEO sections
hydrated at the surface and the PPO section in the hydrophobic core (12, 14).
The basis of this work was the investigation of drug formulations for PDT using Pluronic P123 and a benzoporphyrin B-ring derivative (2). P123 (or poloxamer number
403) has a molecular weight of 5750 with 30% of
hydrophile PEO (m = 21 and n = 67).
Materials and methods
Reagents
Pluronic P123 (paste) was supplied by BASF, U.S.A. The
photosensitizing drugs (BPD-MA 1 and 2) were synthesized
using standard procedures (15). 1,6-Diphenyl-1,3,5-hexatriene (DPH, Sigma) was used as received. The ionic
strength was maintained by adding KCl (0.1 mol L–1).
Can. J. Chem. Vol. 80, 2002
Buffers (0.01 mol L–1) were potassium phosphate (pH range
6.2–8.5) and potassium phthalate (pH range 3.9–6.1). All
solvents were analytical grade.
Procedures
The aggregation process was monitored by UV–vis spectroscopy (Hewlett-Packard 8452 A and Cary-50). The spectra of 2 were obtained using cells with path length 0.01 cm
(cylindrical-shaped) for concentrated solutions (approximately 1 × 10–3 mol L–1) and 1.0 cm for dilute solutions (approximately 1 × 10–5 mol L–1). The term molar ratio refers
to the ratio [P123]:[2] using molar concentrations. For the
stock preparation, P123 (paste, 2 g) was dissolved in
100 mL of water and mixed (Mixer Thermolyne, type
16700) for 2 to 3 h. All Pluronic concentrations are presented by percentage of weight of polymer per water volume
(w/v). All stock solutions of 2 (prepared in dimethyl
sulfoxide (DMSO)) and DPH (prepared in methanol) were
protected from light and were refrigerated.
Critical micelle concentration (cmc)
The cmc determination (duplicate) was performed for
Pluronic P123 by a solubilization method (13) using DPH as
a probe, and by a method based on dye monomer–aggregate
equilibration (16) using 2 as the probe. All Pluronic solutions were kept in a water bath for at least 4 h, to achieve
thermal and mechanical equilibrium. A stock solution of
DPH was prepared at 1.1 × 10–3 mol L–1, and aliquots of
3.0 mL (microsyringe) were added to each 2.0 mL of
Pluronic solution. For 2, 4.0 mL from a stock solution (3 ×
10–3 mol L–1 in DMSO) was added to each 2.0 mL of P123
solution, and mixed for 15 s (mixer). The solutions were
kept in the dark (1 h for DPH and 30 min for 2), before the
visible or fluorescence (Aminco-Bowman Series 2) spectra
were recorded.
Effect of pH
Solutions of 2 (1.14 × 10–5 mol L–1) at different pH and in
several fixed concentrations of P123 were investigated
([P123] from 0 to 0.16% w/v). The ionic strength was kept
constant (0.1 mol L–1 KCl), and phosphate and phthalate potassium buffers (0.01 mol L–1) were used. P123 solutions
(2.25 mL) were previously prepared with KCl and KOH (to
set the initial pH to approximately 7.3) and kept at least 4 h
at 25°C. 14 mL of 2 (stock, c = 2.05 × 10–3 mol L–1 in
DMSO) were added to the sample and mixed (using the
mixer for 15 s). After 1 to 2 min, 0.25 mL (micropipette) of
the buffer solutions, at the desired pH, were added, and the
behaviour of monomer and aggregate equilibrium was monitored with respect to time. For 0, 0.04, and 0.06% P123, experiments were performed in duplicate. To investigate the
effect of [2] on the pH dependence of aggregation, 0.06% of
P123 was used with different concentrations of 2.
Kinetic investigation of the aggregation process
The same procedures used to investigate the effect of pH
on 2–P123 solutions were used to study the aggregation behaviour with respect to time. Only the kinetics of acid-pH
solutions were followed, because at neutral or alkaline pH
the aggregation processes were too slow. The monomer and
aggregate peaks were monitored simultaneously.
© 2002 NRC Canada
Hioka et al.
Fig. 1. Visible spectra of 2 in DMSO. Monomer and aggregate
([2] = 1.1 × 10–5 mol L–1 in DMSO–water).
1323
Table 1. Sample preparation with [2] = 1.4 × 10–3 mol L–1
(1 mg mL–1), pH = 7.4.
Sample
0
1
2
3
4
5
6
7
Micellar size determination using laser light scattering
The micelle size was measured by laser light scattering
(submicron particle sizer model 370, NICOMP, Santa
Barbara, CA). Solutions with 1 mg of 2 and 10% of P123
(pH = 7.4) were analyzed. Solutions at low concentrations of
2 (3.5 × 10–6 mol L–1) and Pluronic (0.06 and 0.2%), with
pH = 7.4, were also examined.
Formulations
Solutions with different P123 concentrations were prepared by a thin film method. Compound 2 and P123 were
codissolved in methylene dichloride; after solvent evaporation via rotary evaporation, the thin film produced was hydrated with an aqueous potassium phosphate solution.
Samples were lyophilized and rehydrated using 1 mL of
pure water, leading to final solutions composed of [buffer] =
0.01 mol L–1, pH 7.4, [2] = 1.4 × 10–3 mol L–1 (1 mg mL–1),
and P123 concentrations as presented in Table 1. After the
solution was well mixed, visible spectra were obtained with
respect to time.
Aliquots of the solutions prepared as described in Table 1
were frozen immediately after rehydration, and were used to
check the stability of the diluted samples. These solutions
were defrosted and aliquots of 40 mL were added to 2.0 mL
(1:50 dilution) of pure water and buffer solution (pH = 7.4, c =
0.01 mol L–1). The stability of their monomeric species was
followed with respect to time.
Results and discussion
For 2 in DMSO (or other organic solvents), the most intense Q-band appeared at 692 nm (e = 32 000 M–1 cm–1),
corresponding to the monomeric species, while the aggregate peak showed a red-shifted band with a more intense
peak at 715 to 738 nm (see Fig. 1). The wavelength of the
aggregate peak was dependent on solution conditions. Samples exhibiting aggregation peaks usually precipitated with
time.
For 2 in Pluronic solutions, the monomer peak had a
molar absorptivity (e) around 27 000 M–1 cm –1 , while
the aggregate peak intensity was higher (approximately
50 000 M–1 cm–1).
P123
(mg)
16.9
26.8
47.7
75.6
100.8
125.8
154.3
199.7
[P123]
(%, w/v)
1.7
2.7
4.8
7.6
10.1
12.6
15.4
20.0
Molar ratio
[P123]:[2]
2
3
6
9
13
16
19
25
Fig. 2. Absorbance at 356 nm as a function of P123 concentration ([DPH] = 1.6 × 10–6 mol L–1, pH = 7.25, [phosphate] =
0.01 mol L–1, 30°C).
Critical micelle concentration of P123
DPH in methanol exhibits an absorption peak at 356 nm
and an emission peak at 428 nm (excitation at 356 nm).
DPH is insoluble in water (or low P123 concentration). As
the P123 concentration increased, the absorbance and fluorescence intensity also increased. A plot of absorbance at
356 nm (or fluorescence intensity at 428 nm) vs. log
(%P123) produced two straight lines whose intercept gave
the cmc (see Fig. 2 as an example) (13).
Cmc determination using 2 (soluble in water at low concentration) as a probe is based upon a monomer
aggregation equilibrium (16). Compound 2 in aqueous solutions, at
low P123 concentration, exhibited an aggregation band. At
higher P123 concentration, this band decreased, while the
monomer peak at 692 nm increased. This aggregation behavior of 2 enabled the determination of the P123 cmc. The absorption ratio of the aggregate and monomer peaks as a
function of log (%P123) is shown in Fig. 3. The cmc value
was obtained from the intercept.
Table 2 presents cmc values obtained from experiments
with DPH and 2 under different conditions.
The cited cmc value for P123 at 25°C (13) is 0.03%,
which agrees with our results using both probes (0.028%
and 0.029% for DPH and 2, respectively). At 30°C and
pH 7.3, the value found with both compounds (0.020%)
demonstrated again the agreement between these two methods. The values obtained with 2 at different buffer concentrations (0.01 and 0.1 mol L–1) are in accordance with the
literature, which predicts that the cmc decreases with salt
© 2002 NRC Canada
1324
Fig. 3. Effect of P123 concentration on monomer–aggregate
equilibrium (25°C) ([2] = 6 × 10–6 mol L–1).
Can. J. Chem. Vol. 80, 2002
Table 2. Average cmc values of P123 using DPH and 2.
Probe
Temp
(°C)
Phosphate
(mol L–1)
pH
cmc
DPH
DPH
2
2
2
25
30
25
30
30
—
0.01
—
0.01
0.1
—
7.3
—
7.3
7.3
0.028
0.020
0.029
0.020
0.015
Table 3. Apparent pKa for 2 and the maximum wavelength of
the aggregate band at different P123 concentrations (25°C).
[P123]
(%, w/v)
Fig. 4. Effect of pH on monomer–aggregate equilibrium ([2] =
1.14 × 10–5 mol L–1, [P123] = 0.04%, [KCl] = 0.1 mol L–1,
[buffer] = 0.01 mol L–1) (25°C). Experimental points were obtained at 90 min after buffer addition.
0.0
0.02
0.04
0.05
0.06
0.07
0.08
0.12
0.16
Molar ratio
[P123]:[2]
0
3
6
8
9
11
12
18
24
pKa
(±0.1)
l max
(nm)
6.8
6.6
6.4
—
5.8
—
5.8
5.6
5.7
718
728
728
734
734
735
734
736
736
Note: [KCl] = 0.1 mol L–1; [buffer] = 0.01 mol L–1; [2] = 1.14 ×
10–5 mol L–1.
addition (13, 17). These experiments with 2 clearly demonstrated that the micelles of P123 stabilize it in the
monomeric form.
Effect of pH on stability
Solutions of 2 in P123 at low pH showed a red-shifted
peak corresponding to aggregates. All samples were, therefore, initially prepared at neutral pH (without buffer). At low
pH, after buffer addition, a decrease of the 692 nm peak and
a simultaneous increase in the aggregate band with respect
to time was observed. At neutral or higher pH, aggregation
was diminished (but not completely avoided), probably resulting from the negative–negative charge repulsion of the
carboxylate group on 2. The ratio between the aggregate and
monomer peaks at an arbitrarily fixed time was taken as a
parameter to estimate the magnitude of the effect. An example is presented in Fig. 4.
The extent of the aggregation process with pH (Fig. 4)
permits an estimate of the pKa value of 2 at that Pluronic
concentration. This method cannot directly measure pKa,
however, since protonation is followed by aggregation. The
pKa value did not change with time of analysis. For the example presented in Fig. 4, the measured pKa was 6.4. For solutions without Pluronic, 2 exhibits only one peak around
718 nm, which decreases with time at lower pH. To estimate
these pKas, absorbance as a function of pH was used. Measurements of these apparent pKa values, obtained using this
indirect technique at different P123 concentrations, are
shown in Table 3.
In Pluronic solutions, the aggregate peak undergoes a red
shift as the Pluronic concentration is increased, probably reflecting the different geometry and size of the micelles. The
pKa values (Table 3) obtained were higher than those expected for a simple carboxylic acid, and probably reflect the
local environment.
Increasing the P123 concentration ([P123]:[2]) lead to a
decrease in pKa; this effect was caused by a Pluronic micelle
forcing molecules of 2 inside the micelle core (away from
the micelle surface). Consequently, the porphyrin is exposed
to a less hydrophilic environment (18). An abrupt change in
pKa was observed between 0.04% and 0.06% (6.4 to 5.8).
This suggested two different micelle populations. Perhaps,
one was a unimolecular micelle (0.04%) and the other one
was a multimolecular micelle (in 0.06% or higher) (11, 12).
These values are more appropriately called the apparent pKa,
because P123 micelles can induce local pH changes (18).
Table 4 provides data for the effect of the concentration of 2
on its apparent pKa.
The apparent pKa increased as the concentration of 2 increased. This effect resulted from the limited capacity of
P123 micelles (at 0.06%) to support high quantities of 2 inside the micelle core (drug loading). As the [P123]:[2] molar
ratio decreased, more drug molecules were exposed to the
micelle surface or bulk solution where they could be
protonated and suffer aggregation.
Kinetic investigation of the aggregation process
Acidic solutions of 2 and P123 were prepared by buffer
(phthalate) addition. Visible absorption vs. time showed a
quasi-isosbestic point at approximately 702 nm that was not
© 2002 NRC Canada
Hioka et al.
1325
Table 4. Apparent pKa values of 2 at different concentrations.
[2] (10
0.33
1.14
2.02
–5
–1
mol L )
Molar ratio [P123]:[2]
Apparent pKa
32
9
5
5.5
5.8
6.2
Fig. 6. Absorption at monomer peak as a function of time ([2] =
1.4 × 10–3 mol L–1 (1 mg mL–1), (25°C), pH = 7.4, [phosphate] =
0.01 mol L–1). Light pathway cell length (0.01 cm). Sample 0 (!);
sample 1 (#); samples 2–7 ( ).
>
Note: [P123] concentration fixed at 0.06% (25oC); [KCl] = 0.1 mol L–1;
[buffer] = 0.01 mol L–1.
Fig. 5. Kinetic behaviour of 2–P123 solutions in acidic media
([2] = 1.14 × 10–5 mol L–1, [P123] = 0.04%, [P123]:[2] = 6,
[phthalate] = 0.01 mol L–1, pH = 4, [KCl] = 0.1 mol L–1, T =
25°C). The line represents the theoretical fitting.
well-defined; the absence of a clear isosbestic point suggests
a multiple-step reaction. Moreover, supposing dimeric aggregation, the kinetics data should obey a second-order rate
equation (19), which is not shown in the present case. Figure 5 illustrates the monomer and aggregate absorption behavior with respect to time. As shown, there is a lag phase,
which is preceded by what has been termed “a sigmoidal
progress curve” indicating an autocatalytic-type reaction
(20). The initial phase is a nucleation step, after which the
nucleus promotes a catalytic process where more molecules
are added, forming a polymeric aggregate. The initial nucleus could be a dimer of 2, as is found in the aggregation
process in homogeneous solutions (DMSO–water) (21). Figure 5 shows one example of the kinetics of the aggregation
process for the drug in Pluronic solution at low pH. The kinetic data has been treated using an approach proposed by
Micali et al. (20) and Pasternack et al. (22).
The basis of this kinetic treatment derives from chaos theory, which predicts that the rate constants are themselves
time-dependent (20). The kinetic equation is shown in
eq. [1]:
[1]
(abs – absi) / (abso – absi) =
1 / (1 + (m–1){kot + (n + 1)–1 (kct)n
+ 1 }) 1/(m–1)
where abs is the absorbance at time t, abso is the absorbance
at t = 0, absi is the absorbance at infinite time, and m, n, ko,
and kc are kinetic parameters.
The theoretical fit with experimental points (600 points)
as a function of time was performed using the software
Kaleidagraph (Synergy Software v 3.0.9), which resulted in
an excellent correlation coefficient (>0.9999) for these absorption profiles at monomer and aggregate peaks.
Micelle size determination
Laser light scattering experiments showed that a solution
of 2 and 10% P123 (pH approximately 7.4) resulted in one
population of micelles whose size ranged from 15 to 20 nm.
After 1 day, these micelles maintained the same size, and the
visible spectra confirmed the presence of the drug in
monomeric form. For solutions with low concentrations of
P123 (0.06%) and 2 (3.5 × 10–6 mol L–1) at pH 7.4, the experiments showed two micelle populations with sizes approximately 20 nm and 100 nm, although with a high
c-square error (cr2 = 5). After 1 day, both micelle populations increased their micelle size (almost double) and the
drug started to exhibit the aggregation band. The presence of
drug aggregation probably promotes micelle destabilization,
inducing an increase in total particle size. Similar micelle (or
colloid) size increase was exhibited in a 0.2% P123 solution.
Because of the high c error these results are not accurate,
but suggest that a low [P123] results in micelles that cannot
adequately sustain monomeric species of the drug.
Drug loading and stability
Samples formulated at pH 7.4 and compositions described
in Table 1 were used to investigated the concentrations of
P123 that can support 1 mg mL–1 of drug (in monomeric
form) and their stability, with respect to time, for prolonged
use. Figure 6 shows the initial capacity of Pluronic solution
that is able to solubilize 2 in monomer form, as well as their
stability over time after rehydration with 1 mL of water.
Samples containing P123 at concentrations of 1.7 and
2.7% (samples 0 and 1) exhibited low capacity to load 2 in
its monomeric form (time zero). Their spectra did not exhibit an aggregation peak; however, undissolved material remained in the flask. Taking [2] = 1.4 × 10–3 mol L–1, e =
27 000 M–1 cm–1, and considering the light path of the cell
(0.01 cm), the expected absorbance at 692 nm should be approximately 0.378, which is close to the value of 0.36 obtained experimentally for sample 2 (4.8%) and those at
higher [P123].
After 48 h, samples 2 (4.8%) to 7 (20.0%) did not exhibit
an aggregate peak, and the absorption at 692 nm did not
change (Fig. 7). Samples 0 (1.7%) and 1 (2.7%) exhibited a
© 2002 NRC Canada
1326
Fig. 7. Ratio of aggregate–monomer absorbances for [P123] solutions at 20 h after dilution (in pure water and in [phosphate] =
0.01 mol L–1, pH = 7.4) ([2] = 2.8 × 10–5 mol L–1, 25°C).
Can. J. Chem. Vol. 80, 2002
Samples with a Pluronic concentration of 4.8% (molar ratio =
6) or higher showed high drug loading and capacity to support the drug in monomeric form. After dilution (1:50) in
buffer solution at pH 7.4, these samples still demonstrated
high stability.
Acknowledgements
We express our gratitude to the Brazilian granting agency
CNPq for the support of Noboru Hioka. This work was supported by the Natural Sciences and Engineering Research
Council of Canada (NSERC).
References
new band at 734–736 nm (not shown), which increased with
time, while the monomer peak decreased. Thus, for drug
loading and stability, samples formulated with P123 at concentrations below 4.8% are not useful for clinical use.
After 20 days (sample solutions were maintained at room
temperature in the dark) the absorbance at 692 nm exhibited
only a small decrease. The absorbance of sample 2 (4.8%)
decreased 5.2%, and sample 4 (10.1%) decreased 4.3%. For
samples 5 (12.6%) to 7 (20.0%), absorbance decreases were
not observed. The pH of all solutions was 7.4, which means
that despite the negative charge, 2 still tended to aggregate.
It is important to note, however, that this tendency is much
reduced when compared with low pH.
These results show that dilute buffered solutions (pH =
7.4) were more stable for all samples. This formulation process should, therefore, use buffer solutions at pH » 7.4,
rather than pure water. Previous buffer (from the cake) cannot adequately sustain the pH for formulations after dilution
(1:50) in pure water. At 20 h, samples diluted in pure water
exhibited aggregation at 0.20% (original 10.1%) of P123 (or
higher), while for dilution made in the buffer, the solution at
0.10% (original 4.8%) of P123 was still stable.
Conclusion
Micelles of P123 in water can support 2 in its monomeric
form. The monomer
aggregate equilibrium of the drug can
provide a reasonable estimation of the pKa for 2, and permits cmc determinations for Pluronic. The apparent pKa of 2
decreased as the molar ratio ([P123]:[2]) increased. Mixtures
of 2 with low [P123] showed unstable micellar systems,
leading to drug aggregation. Acidic solutions of the drug in
Pluronic underwent aggregation via a self-catalytic process,
where the rate constant, or “rate coefficient” (22), is time dependent, and can be analyzed by chaos theory. Prevention of
the nucleation step can slow down the aggregation process.
The best conditions for Pluronic micelles to sustain 2 as a
monomeric species were high molar ratios and neutral pH.
1. E.D. Sternberg, D. Dolphin, and C. Brückner. Tetrahedron, 54,
4151 (1998).
2. J.G. Levy. Trends Biotechnol. 13, 14 (1995).
3. A.M. Richter, E. Waterfield, A.J. Jain, B. Allison, E.D. Sternberg, D. Dolphin, and J.G. Levy. Br. J. Cancer, 63, 87 (1991).
4. A.M. Richter, E. Waterfield, A.K. Jain, E.D. Sternberg, and
J.G. Levy. Photochem. Photobiol. 52, 495 (1990).
5. E.D. Sternberg and D. Dolphin. Curr. Med. Chem. 3, 239
(1996).
6. B.M. Aveline, T. Hasan, and R.W. Redmond. J. Photochem.
Photobiol. B, 30, 161 (1995).
7. B.M. Aveline, T. Hasan, and R.W. Redmond. Photochem.
Photobiol. 59, 328 (1994).
8. W.I. White. In The porphyrins. Vol. V. Edited by D. Dolphin.
Academic Press, Inc., New York, U.S.A., 1990. Chap. 7.
pp. 303–339.
9. N. Oku, N. Saito, Y. Namba, H. Tsukada, D. Dolphin, and S.
Okada. Biol. Pharm. Bull. 20, 670 (1997).
10. V.C. Colussi, D.K. Feyes, J.W. Mulvihill, Y. Li, M.E. Kenney,
C.A. Elmets, N.L. Oleinick, and H. Mukhtar. Photochem.
Photobiol. 69, 236 (1999).
11. I.R. Schmolka. In Polymers for controlled drug delivery.
Edited by P.J. Tarcha. CRC Press, Boca Raton, Florida, U.S.A.
1991. Chap. 10. pp. 189–208.
12. P. Alexandridis and T.A Hatton. Colloids Surf A, 96, 1 (1995).
13. P. Alexandridis, J.F. Holyworth, and T.A. Hatton. Macromolecules, 27, 2414 (1994).
14. I.R. Schmolka. J. Am. Oil Chem. Soc. 54, 110 (1977).
15. E.D. Sternberg, D. Dolphin, A. Tovey, A.M. Richter, and J.G.
Levy. U.S. Patent 5 880 145, 1999; Chem. Abstr. 130:223114.
16. N. Hioka. M.Sc. Thesis, University of Sao Paulo, Brazil, 1986.
17. P. Bahadur, K. Pandya, M. Almgreen, P. Li, and P. Stilbs.
Colloid Polym. Sci. 271, 657 (1993).
18. J.H. Fendler. Membrane mimetic chemistry: characterizations
and applications of micelles, microemulsions, monolayers,
bilayers, vesicles, host-guest systems, and polyions. John
Wiley and Sons, New York, U.S.A. 1982.
19. K.J. Laidler. Chemical kinetics. Harper and Row Publishers,
New York. 1987.
20. M. Micali, L.M. Scolaro, A. Romeo, and F. Mallamace.
Physica A, 249, 501 (1998).
21. D. Delmarre, N. Hioka, R. Boch, E. Sternberg, and D. Dolphin. Can. J. Chem. 79, 1068 (2001).
22. R.F. Pasternack, E.J. Gibbs, P.J. Collings, J.C. dePaula, L.C.
Turzo, and A. Terracina. J. Am. Chem. Soc. 120, 5873 (1998).
© 2002 NRC Canada