ISSN 0003-6838, Applied Biochemistry and Microbiology, 2006, Vol. 42, No. 6, pp. 625–630. MAIK “Nauka /Interperiodica” (Russia), 2006.
Original Russian Text A.P. Bonartsev, G.A. Bonartseva, T.K. Makhina, V.L. Myshkina, E.S. Luchinina, V.A. Livshits, A.P. Boskhomdzhiev, V.S. Markin, A.L. Iordanskii, 2006,published in Prikladnaya Biokhimiya i Mikrobiologiya, 2006, Vol. 42, No. 6, pp. 710–715.
New Poly-(3-hydroxybutyrate)-Based Systems for Controlled
Release of Dipyridamole and Indomethacin
A. P. Bonartsevc, G. A. Bonartsevaa, T. K. Makhinaa, V. L. Myshkinaa, E. S. Luchininaa,
V. A. Livshitsa, A. P. Boskhomdzhieva, V. S. Markinb, and A. L. Iordanskiic
a Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, 119071 Russia b Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow, 119991 Russia c Faculty of Biology, Moscow State University, Moscow, 119899 Abstract—New poly-(3-hydroxybutyrate)-based systems for controlled release of anti-inflammatory and anti-
thrombogenic drugs have been studied. The release occurs via two mechanisms (diffusion and degradation)
operating simultaneously. Dipyridamole and indomethacin diffusion processes determine the rate of the release
at the early stages of the contact of the system with the environment (the first 6–8 h). The coefficient of the
release diffusion of a drug depends on its nature, the thickness of the poly-(3-hydroxybutyrate) films containing
the drug, the concentrations of dipyridamole and indomethacin, and the molecular weight of the poly-(3-
hydroxybutyrate). The results obtained are critical for developing systems of release of diverse drugs, thus,
enabling the attainment of the requisite physiological effects on tissues and organs of humans.
DOI: 10.1134/S0003683806060159
Poly-(-3-hydroxybutyrate) (PHB) and its copoly- enclosing the implant and, if the polymer contacts vas- mers obtained using biotechnological methods have cular tissues, the extent of the hypertrophy of the blood become the subject of increasing interest due to their vessel walls. As is clear from the above, the processes biodegradability and biocompatibility, which make it constituting the organism’s response to implantation of possible to use these polymers in medicine. The physi- biodegradable polymers (i.e., inflammation, thrombus cochemical and biological properties of PHB allow this formation, and cell proliferation) need to be regulated polymer to be used as a material for implantable medi- cal devices (e.g., membranes for treatment of periodon- The buildup of inflammatory and thrombogenic pro- tal disease and prevention of adhesions) and coatings cesses may be regulated by systemic administration of (to be applied onto the surface of net endoprostheses, antiaggregant and anti-inflammatory preparations. In pacemakers, stents, vascular prostheses, etc.) [1].
certain cases, however, this approach is not efficient, Implantation of devices made from biodegradable because the local concentrations of the drugs within the materials, including PHB and its copolymers, into tis- region of the implantation are either not sufficient for sues of an organism may be associated with a series of attaining the pharmacological effect or lack stability, undesirable processes; these include pathological whereas any further increase in the dose administered inflammatory reactions, formation of thrombi, and the systemically entails side effects [3].
lack of correspondence between the rates of the implant Systems of controlled release of drugs, based on replacement by the surrounding body tissues and the polymer materials, make it possible to regulate the pro- rate of its biodegradation (which may be higher or cesses of inflammation, thrombus formation, and devel- lower). The character of the inflammatory process opment of new tissue within the immediate vicinity of accompanying polymer implantation determines to a implantation of medical devices. In designing such sys- considerable extent the intensity of the biodegradation tems, it is important to make the right choice of the of this polymer. The success of the integration of an drug. Dipyridamole (DP), a widely used antithrombo- implant into the surrounding tissues (if there is a con- genic drug, is a phosphodiesterase inhibitor promoting tact between the implant and blood or intraperitoneal intracellular accumulation of cGMP and cAMP, which fluid) depends in its resistance to thrombus formation.
inhibits both platelet aggregation and cell proliferation The increased coagulability of peritoneal fluid favors [4]. Indomethacin (IM), a nonsteroidal anti-inflamma- the development of peritoneal adhesions, which is a tory drug (NSAID), inhibits cyclooxygenase, thereby serious pathology. The intensity of the cell proliferation preventing the synthesis of prostaglandins (which are associated with polymer implantation determines both major mediators of inflammation), and cell prolifera- the rate of formation of a connective tissue capsule tion [5]. It is noteworthy that DP and IM, as well as PHB, are soluble in organic solvents (chloroform and added to 2 ml of the suspension, and the mixture was methylene chloride), which simplifies the technology heated at 100°ë for 2 h (water bath); the insoluble res- of creating polymer systems of controlled release.
idue (PHB granules) was separated by centrifugation at The molecular weight (MW) of a polymer consider- 8000 g for 20 min. Following the addition of 5 ml of ably affects the kinetics of the release of drugs intro- chloroform to the residue, the tube was hermetically duced into its matrix [4]; for this reason, development sealed and incubated overnight (28°ë) under continu- of controlled release systems for drugs that have pre- ous shaking (shaker). Thereafter, the tube was centri- defined characteristics requires a technology for syn- fuged and the chloroform extract was dried in an air thesizing polymers with a particular MW. When PHB is flow. Following the addition of concentrated sulfuric obtained using biotechnological methods, the condi- acid (5 ml per each 0.1 ml of extract), the mixture was tions of culturing of the PHB producer strains may heated at 100°ë for 10 min (water bath) and allowed to influence the molecular weight of the polymer [6].
cool. The amount of crotonic acid (formed as a result of Thus, a technology for the biosynthesis of PHB with a acidic hydrolysis of PHB and subsequent hydroxybu- defined MW is prerequisite to creating controlled tyrate dehydration) was measured at 235 nm (against release systems for the requisite characteristics of the concentrated sulfuric acid) on a Beckman DU-650 kinetics of the drug release from the polymer matrix.
spectrophotometer (Germany) in 1-cm cuvettes [8].
The use of such systems for controlled release of The MW of the polymer was determined viscomet- antithrombogenic and anti-inflammatory drugs is rically. Measurement of the changes in the viscosity of expected to (a) increase the resistance of medical the PHB solution in chloroform were performed at devices contacting blood (e.g., coatings of stents and 30°C. The MW was calculated using the Mark–Hou- vascular prostheses) to thrombus formation, (b) regu- wink–Kuhn equation; the value of the coefficient [η] late inflammatory processes and the rate of the implant biodegradation and capsulation (e.g., in the case of The chemical structure of the polymer, the type of reticular endoprostheses for hernioplasty and mem- its crystal lattice, and the extent of its crystallinity branes for treatment of periodontal disease), and (0.74) were previously determined using the methods (c) prevent the formation of adhesions (endoprostheses of differential scanning calorimetry, IR Fourier spec- for hernioplasty and anti-adhesion membranes).
troscopy, and crystal X-ray structure analysis [10].
In this work, we sought to obtain and study PHB- The traces of residual solvent were controlled by based films incorporating DP and IM.
measuring the IR spectra on a Brucker IFS-48 IR spec-trometer (Germany). The extent of the weight lossresulting from degradation was determined gravimetri- The PHB producer strain used in this work (Azoto- In experiments aimed at studying the kinetic charac- bacter chroococcum 7B) was capable of synthesizing teristics of the drug release from the PHB matrix, two PHB in an amount of up to 80% of the dry weight of the PHB batches were used differing in their MWs: bacterial cells. The strains were isolated from the rhizo- 320 kDa (low-molecular-weight PHB) and 1470 kDa sphere of wheat (sod-podzol soil). A collection of (high-molecular-weight PHB). The PHB films were 10, strains of the genus Azotobacter were maintained on 20, or 40 µm thick, containing 3.3, 10, or 30 wt %, Ashbey’s medium [6]. To achieve cellular PHB hyper- respectively, of DP or IM. Systems with a predefined production, the culture of the Azotobacter strain was content of the drugs were prepared by evaporating chlo- grown on Burke’s medium under conditions of an roform on a glass substratum. In addition to films, a excess content of the source of carbon (g/l): polypropylene net was studied, which was modified by ågSO4 · 7H2O, 0.4; FeSO4 · 7H2O, 0.01; Na2MoO4 · applying onto its surface a polymer composition con- 2H2O, 0.006; trisodium citrate, 0.5; CaCl2, 0.1; taining PHB (320 kDa) and DP (10 wt %).
K2HPO4 · 3H2O, 1.05; KH2PO4, 0.2; and sucrose, 40 [6, 7].
The rate of the drug release was recorded by UV The process of isolation and purification of the poly- spectrometry (DU-650) within the region of maximum mer from the biomass of A. chroococcum 7B included absorption of aqueous solutions of DP and IM (at 293 the following stages: dissolution of PHB in chloroform and 256 nm, respectively). The release was performed by shaking at 37°ë for 12 h (shaker), separation of the in phosphate-buffered saline (pH 7.4) at 37°ë for 18 h.
PHB solution from the cell residue by filtration, isola-tion of the PHB by isopropanol precipitation, andrepeated dissolution of the PHB in chloroform followed by isopropanol precipitation and drying at 60°ë.
Effect of the conditions of culturing on the molec-
The content of PHB in the cells was determined ular weight of the poly-(3-hydroxybutyrate) synthe-
using the method of Zevenhuisen [8]. A suspension of sized. In experiments addressing the effects of the con-
the cells (20–100 mg of dry biomass) was centrifuged ditions of the culturing on the MW of the polymer syn- at 5000 g for 20 min. Thereafter, the cells were resus- thesized, we varied the concentration of the pended in 10 ml water and homogenized. 2M HCl was APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 42 No. 6 2006 NEW POLY-(3-HYDROXYBUTYRATE)-BASED SYSTEMS As demonstrated previously, the addition of organic Table 1. Effect of a supplemental carbon source (sodium ac-
acids to a sucrose-containing medium decreases the etate) on the molecular weight (MW) of PHB synthesized by MW of the polymer synthesized [6]. For this reason, we designed experiments in which we varied the concen-tration of sodium acetate in the culture medium. The results obtained are summed up in Table 1. On increas- ing the concentration of sodium acetate in the mediumfrom 0 to 5 g/l (the content of sucrose, which served asthe primary source of carbon, remained constant and was equal to 40 g/l), we observed a decrease in theMW of the PHB synthesized by the cells of A. chroo- It is conceivable that an increase in the intracellular concentration of the acetyl groups stimulates the activ-ity of acetoacetyl-CoA reductase (EC, which,in turn, elevates the content of hydroxybutyryl-CoA. At high concentrations of acetate, the numbers of poly-merization centers and initial fragments of polymer * Molecular weights of the PHB batches used for creating systems chains increase, which results in the synthesis of PHB for controlled release of dipyridamole and indomethacin.
Thus, the method used in this work makes it possi- ble to synthesize PHB with a defined MW.
rate of release is near-constant. Our analysis of the Studies of the kinetics of drug release from a
curves presented in Fig. 1 demonstrates that the mech- poly-(3-hydroxybutyrate) matrix. Figure 1 shows
anism of release is determined by a superposition of typical kinetic curves of DP and IM release from PHB two processes: (1) DP and IM desorption proper (diffu- films (each graph is a time dependence of the relative sion mechanism) and (2) hydrolytic PHB degradation amount (%) of the drug released). As is evident from (which becomes most obvious when the first, diffusion- the figure, most of the systems lack constant limiting related stage has been completed). As a result of this values of the concentrations, which would be observed degradation, the release of the drugs is linear over the if the release were underlain solely by diffusion mech- anisms. These kinetic curves are characterized by the To analyze the kinetics of the release, we subtracted presence of an initial nonlinear (with respect to time) the linear input of the hydrolytic degradation from the segment and a terminal linear segment within which the common current values of the concentration of the drug Fig. 1. Kinetic curves of drug release: IM 10% (1–3) and
DP 10% (4, 5) and 30% (1–4) from PHB (MW = 320 kDa)
Fig. 2. Kinetic desorption curves of indomethacin (1–3) and
films with a thickness of 10 µm (1–4) and 20 µm (6) or a dipyridamole (46) following the diffusion mechanism.
polypropylene surgical net coated with a 20 µ layer of PHB The samples of PHB (MW = 320 kDa) used had a thickness of 10 (1, 4), 20 (2, 5), and 40 (3, 6) µm.
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 42 No. 6 2006 ues of the freely diffusing component (DP or IM) than their thicker counterparts. This result may be accounted for by the observation that thin films preclude organiza-tion of perfect crystalline structures, this being the rea- son why the sorption capacity of the low-molecular- weight component in such polymer systems increases The analysis of the kinetic curves in the diffusion equation 1 plots makes it possible to calculate the dif- fusion coefficients of the drugs and, consequently, givea quantitative characterization of the systems for con- ∂G/∂t = D[∂2G/∂x2] + k, (1) where D is the diffusion coefficient (of DP or IM), cm2/s; k is the constant of the polymer hydrolysis, s–1; G is the concentration (of DP or IM), %; and x and t are Fig. 3. Graphical solution of the diffusion equation for
the coordinate position (cm) and time (s) of the diffu- determining the coefficient of dipyridamole diffusion in PHB (MW = 320 kDa) films with a thickness of 10 (1), The solution of this equation for the condition 20 (2), and 40 (3) µm.
Gt/Goo > 0.5 has the classic appearance released (such as those shown in Fig. 1). The result of Gt/Goo = 1 – (8/π2exp(–Dt/L2), this data processing characterizing the diffusion pro-cess proper is depicted in Fig. 2. Figure 2 shows that where L is the thickness of the PHB film, cm (the other thin PHB films (10 µm thick) have higher limiting val- designations being the same as in Eq. 1).
If the logarithm of this equation is taken, the diffu- sion coefficients may be determined by solving the Table 2. Diffusion parameters of the system PHB–drug (DP
graphical equation in log (1 – G /G ) Examples of such solutions are shown in Fig. 3 for DP diffusion from films of variable thickness. The val- ues of the diffusion coefficient, calculated using equa-tion (3), are listed in Table 2.
Diffusion coefficients are known to characterize the mobility of polymeric segments, the morphology of PHB, and the intensity of the interactions of the drugwith functional groups (in this case, ester groups) of the polymer. The rate of the diffusion-mediated release is higher for IM than DP, all other conditions (i.e., the filmthickness and drug concentration) being the same. The maximum sorption capacity of PHB is also higher for IM than DP, regardless of the film thickness, as Fig. 4demonstrates. It is exactly this amount of the drug that is contained within PHB in a nonimmobilized form capable of free diffusion from the matrix. Thus, thenature of the drug considerably affects the rate of its release, which is particularly important in the case of combined systems releasing two or more drugs.
The rate of the drug release also depends on the MW diffusion coefficient of DP was two times greater in the case of the low-molecular-weight PHB (320 kDa) ascompared to the high-molecular-weight species (1470 kDa). It is conceivable that the higher rate of the drug release from the matrix of the low-molecular- APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 42 No. 6 2006 NEW POLY-(3-HYDROXYBUTYRATE)-BASED SYSTEMS Fig. 4. Dependence of the maximum concentration of the
Fig. 5. Rate of release (R, µg/day per cm2) of dipy-
freely diffusing component (Goo) on the thickness (L, µm) ridamole (1, 2) and indomethacin (3) from a matrix of of a PHB (MW = 320 kDa) film for dipyridamole (1) and PHB (MW = 320 kDa): 1, 30 wt % DP; 2, 10 wt % DP; and weight PHB is accounted for by the greater mobility of PHB films containing the drug, the concentrations of its polymeric segments. However, the relationship was DP and IM, and on the MW of the PHB. The results reversed when we examined 10-µm films. This obser- obtained are critical for developing systems of release vation may be underlain by the fact that the organiza- of diverse drugs enabling the attainment of the requisite tion of the polymer molecules in thin films is lower than physiological effects on tissues and organs of human In recent years, systems for controlled release of DP and IM based on other biodegradable polymers (e.g., polylactides and copolymers thereof) have been thesubject of active development and investigation [13, This work was supported in part by state contract 14]. Judging by the reported evidence, these systems no. 02.467.11.3004 of March 30, 2005, which was con- are pharmacologically efficient. The results of our stud- cluded within the framework of an integrated project of ies (the kinetics of drug release from PHB matrices and the Federal Targeted Scientific and Technological Pro- the underlying mechanisms) are comparable with these gram “Live Systems” for the years 2005–2006, and by data. Moreover, our observation that the release of DP the Russian Foundation for Basic Research (project and IM from the PHB matrix occurs at a uniform rate and for a considerable period of time (Fig. 5) makes itpossible to use these systems for long-term regulationof processes involving inflammation, thrombus forma- tion, and tissue proliferation in the immediate vicinity 1. Chen, G.-Q. and Wu, Q., Biomaterials, 2005, vol. 26, of the implantation zone. The possibility to regulate the rate of drug release from the matrix by changing theMW of the PHB offers an opportunity to design PHB 2. Shtilman, M.I., Polymeric Biomaterials. Part I: Polymer systems for controlled drug release with predefined Implants, Utrecht: VSP. Science Press, 1993, pp. 3–28.
3. Controlled Drug Delivery: Fundamentals and Applica- In conclusion, we propose new polymer systems tions, Robinson, J.R. and Lee, V.H.L., Eds., New York: (PHB-based) for controlled release of anti-inflamma- tory and antithrombogenic drugs. The release occurs 4. Aktas, B., Utz, A., Hoenig-Liedl, P., Walter, U., and Gei- via two mechanisms (diffusion and degradation) oper- ger, J., Stroke, 2003, vol. 34, pp. 764–769.
ating simultaneously. The diffusion of dipyridamoleand indomethacin, which determines the rate of the 5. Fosslien, E., Crit. Rev. Clin. Lab. Sci., 2000, vol. 37, release at the early stages of contact of the system with the environment (the first 6–8 days), is examined in 6. RF Patent No. 2 194 759, Byull. Izobret., 2001.
detail. The coefficients of diffusion are shown todepend on the nature of the drug, the thickness of the 7. RF Patent No. 2 201 453, Byull. Izobret., 2001.
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 42 No. 6 2006 8. Zevenhuizen, L.P., Antonie van Leeuwenhoek, 1981, 12. Iordanskii, A.L., Rudakova, T.E., and Zaikov, G.E., Interaction of Polymers with Bioactive and Corrosive 9. Akita, S., Einada, Y., Miyaki, Y., and Fugita, H., Macro- Media. Ser. New Concepts in Polymer Science, Utrecht: mol., 1976, vol. 9, no. 2, pp. 774–780.
10. Rebrov, A.V., Dubinskii, V.A., Nekrasov, Yu.P., Bonart- 13. Puebla, P., Pastoriza, P., Barcia, E., and Fernandez-Car- seva, G.A., Shtamm, M., and Antipov, E.M., Vysokomol. ballido, A.J., Microencapsul., 2005, vol. 22, no. 7, Soedin., 2002, vol. 44, no. 2, pp. 347–351.
11. Suzuki, T., Deguchi, H., Yamane, T., Shimizu, S., and 14. Zhu, W., Masaki, T., Bae, Y.H., Rathi, R., Cheung, A.K., Gekko, K., Appl. Microbiol. Biotechnol., 1988, vol. 27, and Kern, S.E., J. Biomed. Mater. Res., Ser. B: App. Bio- mater., 2006, vol. 77, no. 1, pp. 135–143.


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