Polymer Gels - American Chemical Society

Chapter 20

Controlled Drug Delivery from Injectable Biodegradable Triblock Copolymer Downloaded by OHIO STATE UNIV LIBRARIES on June 23, 2013 | http://pubs.acs.org Publication Date: October 15, 2002 | doi: 10.1021/bk-2002-0833.ch020

Young Jin Kim and Sung Wan Kim Center for Controlled Chemical Delivery,

University

of

Utah,

30 So. 2000 E., Room 201, Salt Lake City, UT 84112-5820

The A B A and BAB triblock copolymers composed of poly(DL-lactide-co-glycolide) (PLGA) and poly(ethylene glycol) (PEG) were used in this study. It is a new biodegradable and injectable implant system, which has sol to gel transition behavior. It is a sol between 5 and 30 °C but forms a gel at the body temperature in an aqueous solution. Two model drugs, ketoprofen and spironolactone, which have different hydrophobicities, were released from the PEG -PLGA-PEG triblock copolymer hydrogel. Ketoprofen was released over 2 weeks while spironolactone was released over more than 2 months with a sigmoid release profile. Human insulin was released from the PLGA-PEG-PLGA triblock copolymer hydrogel in a sink condition of phosphate buffer saline solution. We tried to modify the association states of insulin by zinc in order to inhibit the initial burst effect and obtain a constant release rate. Insulin associated from monomer and dimer to hexamer with increasing zinc concentration. The insulin release profile showed the constant release rate over more than 2 weeks.

300

© 2003 American Chemical Society In Polymer Gels; Bohidar, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

Downloaded by OHIO STATE UNIV LIBRARIES on June 23, 2013 | http://pubs.acs.org Publication Date: October 15, 2002 | doi: 10.1021/bk-2002-0833.ch020

301 Thermosensitive polymers have been developed extensively over the past years [1-4]. In particular, polymers showing a sol-gel transition by temperature change have been proposed for an injectable drug delivery depot [5,6]. As an example of temperature sensitive polymers, aqueous solutions of commercially available poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO; Pluronics (BASF) or Poloxamers (ICI)) demonstrated sol-gel phase transitions with increasing temperature. Hydrogel of triblock copolymers consisting of poly(DL-lactic acid-coglycolic acid) (PLGA) and poly(ethylene glycol) (PEG) were used in this study. It is a new injectable implant system, which has thermosensitivity and biodegradability. It is a free flowing sol below 15 °C but it forms a high viscosity gel at body temperature in an aqueous solution. In addition to biodegradability of the polymers, the in-situ formed gel maintains for more than one month, where as the known gelling polymer, Poloxamer, is not biodegradable and the formed gel is dissolved out in a days at best. Hydrogels are useful in biomedical and pharmaceutical applications because of their biocompatibility, high water content, and rubbery state. Additionally, as carriers of bioactive agents, they can also provide protection for the proteins or drugs [7]. Generally, protein and peptide drugs have different properties like biological half-life and conformational stability from the conventional ones. One way to increase the therapeutic efficiency of these polypeptides is encapsulating them in a sustained dosage form that is capable of releasing the macromolecule continuously, at a controlled rate [8]. Most hydrogel-based drug delivery systems are designed as implants that release drug locally at a predetermined rate. Drug release from a hydrogel can be affected by several factors, such as pore size, degradability of the hydrogel, size, hydrophobicity, concentration of a drug, and presence of specific interactions between hydrogels and the incorporated drugs. Typically, the release mechanism from a biodegradable hydrogel follows the diffusion at an initial stage and then a combination of diffusion and degradation at a later stage [9].

A B A triblock copolymer An ABA (hydrophihc-hydrophobic-hydrophilic) triblock copolymer has been studied extensively because of its ability to form a hydrogel. As an example, aqueous solutions of commercially available PEO-PPO-PEO polymers (Pluronics (BASF) or Poloxamers (ICI)) demonstrate phase transitions from sol to gel (low temperature sol-gel boundary) and gel to sol (high temperature gelsol boundary) as monotonically increasing temperature when the polymer concentration is above a critical value [4,10]. Continuous heating the polymer solutions in a temperature range above the high temperature boundary makes the

In Polymer Gels; Bohidar, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

302


solution opaque. The sol-gel transition behavior of PEO-PPO-PEO triblock copolymers have been utilized for the delivery of labile drugs such as polypeptides and proteins because such drugs can be formulated in an aqueous solution [5,77]. The formulation forms a gel depot in situ when exposed to body temperature via subcutaneous injection. The incorporated drug is then released into the body in a controlled manner. The gel depot of PEO-PPO-PEO triblock copolymer dissolved from its surface within 1 day into soluble unimers, which may cause a harmful or toxic effect in the body, thus resulting in difficulties in sustained release in a long-term base. In this report, we use PEG-PLGA-PEG triblock copolymers as an ABA type phase transition polymer. It also showed sol-gel phase transition by increasing the temperature of aqueous solutions. The transition temperature monotonically increased as concentration increased. The formed gels of PEG-PLGA-PEG in rats maintained their integrity longer than 1 month [72,73]. In addition to their biodegradability, longer duration of PEG-PLGA-PEG gels is clearly distinguished from PEO-PPO-PEO gels. PEG-PLGA-PEG gels are not erodible by dilution and are more beneficial for the carriers of polypeptide, proteins, and other pharmaceuticals for long-term delivery. The ABA triblock copolymer is good for hydrophilic drugs. In this study, we discuss the effect of the hydrophilicity on the drug release in PEG-PLGA-PEG triblock copolymers .

Sol-gel transition Ring-opening polymerization of lactide and glycolide onto monomethoxy poly(ethylene glycol) was performed to synthesize PEG-PLGA diblock copolymers. Diblock copolymers were then coupled using hexamethylene diisocyanate to produce the PEG-PLGA-PEG (Mn:550-2810-550) triblock copolymers. The sol-gel transition temperature was determined by a test tube inverting method with temperature increments of 1 °C per each step [12,14,15]. Each sample with a given concentration was prepared by dissolving the polymer in distilled water in a 4 ml vial. After equilibration at 4 °C for 12 h, the vials containing samples were immersed in a water bath at a constant designated temperature for 20 min. Inverting the vial determined a gel state when no fluidity in 1 min was visually observed. A minimum shear stress of 62 Pa is needed for the system to flow in vial [75]. The sol-gel transition temperature determined by this method has a precision of ±0.5 °C. Figure 1 shows the sol-gel transition of PEG-PLGA-PEG triblock copolymer in aqueous solution. The viscosity of PEG-PLGA-PEG triblock aqueous solution at 33 wt% concentration at room temperature was 10 cP, which makes it easy to formulate and inject through a syringe needle. The viscosity abruptly increased at the sol-gel


303 transition temperature. With further increasing temperature, the transparent gel became turbid. - n - r r r r r r r r

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20

f1 25

30

35

40

45

Concentration fwt, %)

Figure 1. Sol-gel transition ofPEG-PLGA-PEG triblock copolymer aqueous solution. Cross bar indicates the temperature at which the gel becomes turbid. (Reproduced with permission from reference 13. Copyright 2000 Elsevier.)

Drug delivery PEG-PLGA-PEG (550-2810-550) triblock copolymers were studied for drug release of ketoprofen (Sigma) and spironolactone (Sigma). Ketoprofen or spironolactone was dissolved in a PEG-PLGA-PEG triblock copolymer solution at a concentration of 10 mg/ml (ketoprofen) or 2.5 mg/ml (spironolactone), respectively. An aqueous polymer solution containing the model drug (0.4 ml) was injected into 4 ml vials, which were thermostated in a shaking water bath (16 strokes/rnin) at 37 °C, to form a clear gel. After 2 min, 3.5 ml of release medium at 37 °C was added to the formed gel. A 10 mM phosphate buffer (pH 7.4) was used as a drug release medium to improve drug solubility. All release medium was replaced with the same amount of fresh medium at designated sampling intervals to mimic a sink condition and subjected to reversed-phase high performance liquid chromatographic (RP-HPLC; Shimadzu) analysis with a C reversed-phase column (Phenomenex). Mobile phases were acetonitrile and water with a feed ratio of 50:50 for Ketoprofen and 70:30 for spironolactone with a total flow rate of 1.0 ml/rnin. UV-Vis detection at 260 nm and 254 nm was used for the analysis of Ketoprofen and spironolactone, respectively. 1 8


304

o

"SOCCH3

O'


(a)

(b)

Figure 2. Structure of the model drugs, (a) ketoprofen and (b) spironolactone. (fromRef. 13). The PEG-PLGA-PEG triblock copolymer hydrogel system is thought to have a domain (core-shell micelle) structure in aqueous environments. The hydrophilic PEGs occupy the outer shell region and hydrophobic PLGAs constitute the inner core in order to decrease free energy of hydration. Two drugs differing in hydrophobicity were studied as model drugs. Figure 2 shows the structure of the two model drugs. Ketoprofen (KP) has a carboxyl group (pKa=5.0) which is ionized at pH 7.4, making it hydrophilic [76]. Spironolactone is relatively hydrophobic [77]. The difference in hydrophobicity may force the drug to partition into different polymer domains, resulting in different release profiles. The more hydrophobic the drug, the more it will partition into the PLGA micellar core in the hydrogel, and consequently have a sustained drug release profile. Figure 3 shows KP release from the PEG-PLGAPEG (550-2810-550) triblock copolymer hydrogel. The ketoprofen release rate was controlled by changing initial polymer solution concentration. The release rate decreased to near zero after 5 days. The higher initial polymer solution concentration, the slower was the drug release rate observed, due to tighter polymer-polymer contacts among the gel at higher concentrations of the polymer. In case of spironolactone, the release profile (Figure 4) shows an sigmoid release profile typical of a biodegradable polymer, with diffusion followed by a degradation/diffusion mechanism [9,18]. The more hydrophobic and the smaller the water content in the gel, the slower the degradation rate, resulting in a slower drug release rate in the degradation dominant stage. Drug release from the hydrophobic domain mainly comesfromthe degradation. With degradation of the core polymers, the number of end groups increases, and free volume increases.



3

0

2

4

6

S Days

K)

32

14

F/gwre 5. Ketoprofen release from PEG-PLGA-PEG triblock copolymer hydrogel The legend indicates initial concentration of the polymers in PBS buffer (Reproduced with permission from reference 13. Copyright 2000 Elsevier.) When drug hydrophobicity and release profile are compared, important conclusions can be drawn about the gel structure. The more hydrophilic drug (ketoprofen) tends to be more partitioned into the hydrophilic domain, with easier diffusion out of the hydrogel system into the release medium. There is continuous dynamic repartitioning between the two polymer phases during the release of the drug from the hydrogel, resulting in a single stage-like drug release profile for the hydrophilic drug. In this case, the major mechanism of drug release is diffusion. Hydrophobic drugs (spironolactone) tend to be more partitioned into the PLGA core of the micelles. However, there is some small release of drug by diffusion at the first stage, which comes predominantly from the drug partitioned into the hydrophilic region. At a later stage, the drug is released by the core degradation and diffusion from the hydrophobic component [75].

:A

0

W

20

30 Days

40

S»

W

Figure 4. Spironolactone release from PEG-PLGA-PEG triblock copolymer hydrogel The legend indicates initial concentration of the polymers in PBS buffer. (Reproduced with permissionfromreference 13. Copyright 2000 Elsevier.)


306


B A B triblock copolymer A BAB (hydrophobic-hydrophilic-hydrophobic) triblock copolymer was also studied because it could form the hydrogel in certain conditions. It shows the similar properties of the ABA triblock copolymer that is water soluble, biodegradable, and thermosensitive. In this study, we used the PLGA-PEGPLGA (1500-1000-1500) triblock copolymer that also showed the sol-gel transition in an aqueous solution. The gelation mechanism is somewhat different and the gelation temperature (15 - 25 °C) is lower than PEG-PLGA-PEG triblock copolymer (30 °C). The BAB triblock copolymer was good for the hydrophobic drugs as a new injectable implant system that possesses both thermosensitivity and biodegradability [19]. Insulin is a hydrophobic peptide drug for diabetes. Diabetes mellitus is a serious pathologic condition responsible for major health care problems all around the world costing billions of dollars annually. In the United States, it represents the fourth leading cause of death. Diabetes also leads to severe complications such as kidney disease, retinopathy, neuropathy, leg or foot amputations and heart disease [20]. As a consequence of poor oral bioavailability and current lack of alternative delivery routes, insulin is presently administered parentally. The subcutaneous route, requiring single or multiple daily injections, is the main stay of conventional insulin therapy [21]. In this study, we designed the sustained release system, which provides basal line insulin release for duration of over several weeks by one injection. Human insulin was entrapped in the hydrogel in order to sustain its release as a subcutaneous insulin delivery system. We tried to modify the association states of insulin by zinc in order to inhibit the initial burst effect and obtain constant release rate. At otherwise equivalent conditions, insulin associated from monomer and dimer to hexamer with increasing zinc concentration [22]. Insulin samples with different zinc contents exhibited different release profiles due to association-state differences within the hydrogel.

Sol-gel transition PLGA-PEG-PLGA (1500-1000-1500) triblock copolymers (ReGel™) were made by ring-opening polymerization. The composition of the PLGA block was 75/25 (LA/GA) in molar ratio. ReGel™ has hydrophihc-hydrophobic groups inside the copolymer. Considering the enhanced hydrophobic interactions at elevated temperatures, the hydrophobic domain of the copolymer forms a physical crosslink (or aggregate) which makes the gelation state (Figure 5).



307

Figure 5. Schematic diagram of hydrogel formation in PLGA-PEG-PLGA triblock copolymer.

0

10

20

30

40

50

temperature(°C)

Figure 6. Sol-gel transition curve of ReGel™ aqueous solution by UV spectrometer. (Reproduced with permission from reference 19. Copyright 2001 Kluwer Aca demic.)



308 The aqueous solution of ReGel was investigated by U V spectroscopy to determine the sol-gel transition temperature. The triblock copolymer aqueous solutions were prepared by dissolving the polymers in the cold water at 4°C to make 15 and 23 wt% solutions. The sol-gel transition temperature was measured by increasing the temperature at 2°C increments. The UV cuvet was immersed in a water/glycerol bath at each temperature for 5 min. The sol-gel transition was monitored by U V spectrophotometer (Lambda 19, Perkin Elmer) at 500 nm. Figure 6 shows the sol-gel transition temperature at 15 - 20 °C with the concentration variation of ReGel™ solution. The ReGel™ solution is a free flowing sol below 15°C and forms a high viscosity gel at body temperature in aqueous solutions. At low temperature (< 15 °C), the solution can be formulated with a labile drug such as a bioactive protein, and the formulated solution can be injected into the body for the controlled release of macromolecular drugs.

Drug delivery The PLGA-PEG-PLGA triblock copolymer was dissolved in the cold water at 5 °C to make a 23 wt% solution. Insulin solutions were prepared in buffer (isotonic 10 mM PBS, pH 7.4) to a concentration of 5.04 mg/ml and zinc was added (0.0, 0.2 wt%) to the hydrogel solution. Then 2 ml of each formulation were placed in vials, incubated at 37 °C until forming gels, and 10 ml of PBS solution was added as a release medium. Release medium samples were withdrawn and replaced immediately to keep the sink condition. They were analyzed by reversed-phase high performance liquid chromatography (RP-

- zinc 0.0% - zinc 0.2%

0.15

12

15

Figure 7. Daily release amount of insulin from ReGel™ formulation in vitro test. In Polymer Gels; Bohidar, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

309 HPLC) to measure the concentration of insulin. RP-HPLC (SCL-lOAvp, Shimadzu) was equipped with a C column (Vydac), which was previously equilibrated. The mobile phases were water and acetonitrile with a gradient flow and the flow rate is 1.2 rrd/min. Figures 7 and 8 show the result of insulin releasefromthe hydrogel in an in vitro test. Different associations of insulin were entrapped in the gelatinized hydrogel with various zinc contents. First of all, there were no initial burst effects of the insulin release from ReGel™ formulation. Two release stages were shown in the release profile. The ReGel™ hydrogel system is thought to have a core-shell structure in an aqueous environment. The hydrophilic PEG occupies the shell region and hydrophobic PLGA hides into the core in order to decrease the free energy. Insulin is a hydrophobic drug and may locate inside the hydrogel network. The release mechanism from the hydrogel follows the diffusion at an initial stage and then a combination of diffusion and degradation at a later stage. Generally, insulin forms a hexamer with zinc. The insulin association resulted from the zinc content in the insulin [22]. However, without zinc, insulin formed various association states such as monomer, dimer, hexamer, and aggregate. It is thought that insulin without zinc formed the aggregation state inside the gel. The aggregated insulin may not diffuse fast from the ReGel™ formulation, which presented a slower release (60% after 15 days). Insulin with 0.2 wt% zinc formed the hexameric state. The release profile of the insulin with


4

100

time(day) 1M

Figure 8. Cumulative amount of released insulin from ReG el formulation in vitro test. > (Reproduced with permission from reference 19. Copyright 2001 Kluwer Aca demic.)


310

zinc showed a constant release rate and almost 90 % of the initial amount was released over 15 days. We verified the result with an animal study using Sprague-Dawley rats with insulin (0.2 wt% zinc)/ReGel™ formulation.


Conclusions ABA and BAB triblock copolymers composed of PLGA and PEG were used as a drug delivery carriers for the continuous release of drugs. The triblock copolymers in aqueous solutions arefree-flowingsols at room temperature and become gels at body temperature. These are water soluble, biodegradable, thermosensitive polymers for an injectable drug delivery depot. The release of hydrophilic drug (ketoprofen) shows 5 day release profile from a PEG-PLGAPEG triblock copolymer. Comparing a more hydrophobic drug (spitonolactone) shows an sigmoid release profile from a PEG-PLGA-PEG triblock copolymers because the drug located inside the hydrophobic part of the hydrogel and showed two release stages, diffusion and diffusion/degradation. The release of human insulin from PLGA-PEG-PLGA triblock copolymers showed no initial burst and a constant release (zero-order) rate in vitro. It was necessary to modify the insulin's zinc content to 0.2 wt% in order to get a maximum release rate. Insulin with 0.2 wt% zinc showed a constant release rate over more than 2 weeks. The PLGA-PEG-PLGA triblock copolymer is the ideal injectable biodegradable phase transition polymer.

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311 (10) Malmsten, M.; Lindman, B. Macromolecules 1992, 25, 5440. (11) Miyazaki, S.; Ohkawa, Y.; Takada, M.; Attwood, D. Chem. Pharm. Bull. 1992, 40(8), 2224. (12) Jeong, B.; Bae, Y. H.; Kim, S. W. Macromolecules 1999, 32, 7064. (13) Jeong, B.; Bae, Y. H.; Kim, S. W. J. Control. Rel. 2000, 63, 155. (14) Gilbert, J.C.;Richardson, J. L.; Davies, M. C.; Palin, K. J.; Hadgraft, J. J. Control. Rel. 1987, 5, 113. (15) Tanodekaew, S.; Godward, J.; Heatley, F.; Booth, C.Macromol.Chem. Phys. 1997, 198, 3385. (16) PDR Generics, 3 edn.; Medical Economics: New Jersey, 1997, 1845. (17) British Pharmacopoeia, Vol. 1, Constable & Co.: London, 1988, 531. (18) Youxin, L.; Volland,C.;Kissel, T. J. Control Rel. 1994, 32, 121. (19) Kim, Y.J.; Choi, S.; Koh, J.J.; Ko, K.S.; Kim, S.W. Pharm. Res. 2001, 18, 548. (20) Baudys, M.; Kim, S.W.Adv. Drug Delivery Rev. 1999, 35, 141. (21) Hinchcliffe, M.; Illum, L. Adv. Drug Delivery Rev. 1999, 35, 199. (22) Brange, J.; Langkaer, L. in: Stability and Characterization of Protein and Peptide Drugs: Case Histories; Wang, Y. J.; Pearlman R.; Ed.; Plenum Press: New York, 1993, 315.


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Polymer Gels - American Chemical Society

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