Paclitaxel Delivery from Novel Polyphosphoesters - American

Chapter 4

Paclitaxel Delivery from Novel Polyphosphoesters: From Concept to Clinical Trials *

Downloaded by UNIV OF GUELPH LIBRARY on July 7, 2012 | http://pubs.acs.org Publication Date: March 9, 2006 | doi: 10.1021/bk-2006-0924.ch004

Stephen K. Dordunoo, William C. Vincek, Mahesh Chaubal, Zhong Zhao, Rena Lapidus, Randall Hoover, and Wenbin Dang Guilford Pharmaceuticals, Inc., 6611 Tributary Street, Baltimore, MD 21224 Corresponding author: [email protected]

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Novel and versatile biodegradable copolymers containing both phosphonate and lactide ester linkages in the polymer backbone (Polilactofate), which degrade in vivo into nontoxic residues, were investigated. Synthesis of Polilactofate (PLF) is achieved by bulk or solvent polymerization techniques. P L F is more hydrophilic than corresponding polylactides, takes up more than its own weight of water, swells and degrades with continuous mass loss over periods from weeks to in excess of 3 months. Paclitaxel, incorporated into P L F microspheres by standard methods was continuously released in vitro and in vivo over at least 60 days. Preclinical pharmacology studies have demonstrated that paclitaxel released from P A C L I M E R microspheres has significant anti-tumor activity against various human cancer cells in murine models. The P A C L I M E R formulation was tested for safety and toxicity in mice, rats and dogs. A no-adverse-effect level for PACLIMER microsphere administrations could not be determined in these studies but tolerability was demonstrated and a maximum tolerated dose was identified. Pilot studies in human ovarian patients showed good tolerability of the drug, with continuous release of paclitaxel from P A C L I M E R microspheres over at least 8 weeks. Paclitaxel plasma concentrations were far below those causing systemic toxicity. ®

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© 2006 American Chemical Society

In Polymeric Drug Delivery II; Svenson, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

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Introduction The present study relates to novel biodegradable copolymers, in particular those containing both phosphonate and lactide ester linkages in the polymer backbone (Polilactofate), which degrade in vivo into nontoxic residues. The copolymers are particularly useful as implantable medical devices and drug delivery systems. Biocompatibility of terephthalate polymers (Dacron) is well known (1). However, this polymer is non-biodegradable, which limits its clinical applica bility. Incorporation of phosphoester linkages into the polymer converts this polymer into a biodegradable form. By careful selection of monomers, several novel polymers with different physical properties and various degradation profiles could be synthesized. Poly(lactide-co-ethylphosphate) (Polilactofate) is one class of linear phosphorus-containing copolymers made by extending the chains of low molecular weight polylactide prepolymers (made from lactide and propylene glycol monomers) with ethyl dichlorophosphate or ethyl phosphate monomers. A typical structure of Polilactofate is depicted in Figure 1.

Figure 1. Structure of Polilactofate polymers, with (x+y)/2 =10 and n = number of repeating units.

The versatility of these polymers comes from the phosphorus atom, which is known for a multiplicity of reactions. Its bonding can involve the 3p orbitals or various 3s-3p hybrids; spd hybrids are also possible because of the accessible d orbitals. Thus, the physicochemical properties of poly(phosphoesters) can be readily changed by varying the pendent groups. The biodegradability of the polymer is due primarily to the physiologically labile phosphoester bond in the polymer backbone. B y manipulating the backbone or the side chain, a wide range of biodegradation rates are attainable (1,2). A n additional feature of poly(phosphoesters) is the availability of functional side groups. Because phosphorus can be pentavalent, drug molecules or other biologically active substances can be chemically linked to the polymer. For example, drugs containing carboxy groups may be coupled to phosphorus via a hydrolyzable ester bond. The P-O-C group in the backbone lowers the glass transition temperature of the polymer and, importantly, confers solubility in common organic solvents and a degree of hydrophilicity.


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Drug delivery to solid tumors Locally confined tumors are best treated by surgical excision, unless adjacent vital structures prevent complete removal (3). Tumors in surgically difficult sites may be amenable to radiation therapy; however, tumors of certain histologic types are resistant to the effects of radiation. Current chemotherapeutic agents damage normally dividing cells as well as malignant cells. The dose of a chemotherapeutic agent that can be administered systemically is therefore limited by its toxic side effects to other organs, primarily hemato poietic, gastrointestinal, renal pulmonary and cardiac tissues. Although chemo therapy has achieved significant improvement of the treatment of hematological neoplasms, there has been, at best, marginal improvement in the treatment of solid tumors with chemotherapy alone. The concept of intratumoral administration of antineoplastic agents was designed to circumvent some of the barriers to drug accumulation in the tumor (3). Intratumor delivery of anticancer agents in a suitably designed controlled release system is postulated to optimize drug delivery to solid tumors by maintaining adequate concentration of the drug in the tumor for a considerable period of time while minimizing systemic exposure with concomitant reduction in toxic side effects. Several studies have demonstrated a moderate to high degree of success following the administration of drug directly into or near the tumor. The pharmacological response elicited following intratumor delivery of anticancer agents depends, among other factors, on the nature of the drug, the polymer type, formulation, site of implantation, the tumor type and the degree and rate of necrosis-necrobiosis. Some arguments against the intratumor delivery include tumor inaccessi bility, seeding of needle tracts, escape of fluids from the tumor due to high intra tumor pressure and increased probability of inducing metastasis. Some or all of these arguments can also be made regarding tumor biopsy. However, fineneedle biopsy is an established diagnostic procedure that has been shown to be safe. Paclitaxel - active ingredient Paclitaxel is a novel anti-microtubule agent and the active ingredient in TAXOL®. Paclitaxel is a white to off-white crystalline powder with empirical formula C47H51NO14 and a molecular weight of 853.9. The structure of pacli taxel is provided in Figure 2.



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Figure 2. Structure ofpaclitaxel. Paclitaxel is highly lipophilic, insoluble in water, and melts at 213-217°C. TAXOL® is approved by the U.S. Food and Drug Administration (FDA) for the treatment of (i) metastatic carcinoma of the ovary after failure of first-line or subsequent therapy, (ii) treatment of breast cancer after failure of combination chemotherapy for metastatic disease or relapse within six months of adjuvant therapy, and (iii) second-line treatment of AIDS-related Kaposi's sarcoma. TAXOL® is also approved for first line treatment for non-small cell lung cancer (NSCLC) in combination with cisplatin in patients who are not candidates for potentially curative surgery and/or radiation therapy. We evaluated the use of peri- or intratumoral chemotherapy with controlled release paclitaxel microspheres (PACLIMER®) in various solid tumor models and in ovarian cancer patients.

Materials and Methods General Synthesis of Polyester-polyphosphonate Copolymers The most common general reaction in preparing a poly(phosphonate) is a dehydrochlorination between a phosphonic dichloride and a diol. A FriedelCrafts reaction can also be used to synthesize poly(phosphonates) (4). Polymerization typically is effected by reacting either bis(chloromethyl) com pounds with aromatic hydrocarbons or chloromethylated diphenyl ether with triaryl phosphonates. Poly(phosphonates) can also be obtained by bulk conden sation between phosphorus diimidazolides and aromatic diols, such as resorcinol and quinoline, usually under nitrogen or some other inert gas. A n advantage of bulk polycondensation is that it avoids the use of solvents and large amounts of other additives, thus making purification more



48 straightforward. Somewhat rigorous conditions, however, are often required and can lead to chain acidolysis or hydrolysis in the presence of water. Thermally induced side reactions, such as crosslinking reactions, can also occur i f the polymer backbone is susceptible to hydrogen atom abstraction or oxidation with subsequent macroradical recombination. To minimize these side reactions, the polymerization is preferably carried out in solution. Solution polycondensation requires that both the diol and the phosphorus component be soluble in a common solvent, typically a chlorinated organic solvent such as chloroform, dichloromethane, or dichloroethane. The solution polymerization is preferably run in the presence of equimolar amounts of the reactants and a stoichiometric amount of an acid acceptor, usually a tertiary amine such as pyridine or triethylamine. The product is typically isolated from solution by precipitation with a non-solvent and purified to remove the hydrochloride salt by conven tional techniques such as washing with dilute hydrochloride. Reaction times tend to be longer with solution polymerization than with bulk polymerization. However, because overall milder reaction conditions can be used, side reactions are minimized, and more sensitive functional groups can be incorporated into the polymer. The disadvantage of solution polymerization is that the attainment of high molecular weights, i.e., M W higher than 20,000 Da, is less likely. Interfacial polycondensation can be used when high molecular weight polymers are desired at high reaction rates. M i l d conditions minimize side reactions. In addition, the dependence of high molecular weight on stoichio metric equivalence between diol and dichloride, inherent in solution methods, is avoided. However, hydrolysis of the acid chloride may occur in the alkaline aqueous phase. Sensitive dichlorides that have some solubility in water are generally subject to hydrolysis rather than polymerization. Phase transfer catalysts, such as crown ethers or tertiary ammonium chloride, can be used to bring the ionized diol to the interface to facilitate the polycondensation reaction. Synthesis of Polilactofate The detailed synthesis of the polymer has been discussed by Chaubal et al. (5). Briefly, the lactide-phosphate copolymer was synthesized in two steps as shown in Figure 3. In the first step, a lactide prepolymer was prepared via a ring opening polymerization of D,L-lactide, using propylene glycol (PG) as the initiator and Sn(Oct) as the catalyst. Unreacted lactide was removed by application of a vacuum at 125 °C for 2 hours. In the second step, the lactide prepolymer was reacted with ethyl dichlorophosphate at low temperature (-10 to -15 °C) in a solvent such as chloroform with triethylamine (TEA) and dimethylaminopyridine (DMAP) as acid acceptor and catalyst, respectively. At the end of the reaction, the solvent was removed using a rotary evaporator, and the residue was dissolved in acetone. The insoluble salts were filtered out and the residual base and catalyst were subsequently removed using a combination of 2


49 acidic and neutral ion exchange resins. The final polymer was dissolved in dichloromethane and precipitated in a mixed solvent system of petroleum ether: ethyl ether (3:1, v/v), followed by drying in a vacuum oven to constant weight. The resulting copolymers were white amorphous solids, soluble in common organic solvents such as acetone, chloroform and dichloromethane. The structure of the resulting purified polymer was confirmed using one-dimensional H and P N M R and two-dimensional H - H C O S Y N M R (6).


l

3 1

!

l

Polilactofate

Figure 3. Two-step synthesis of poly(lactide-co-ethyl phosphate). Polymer Hydrophilicity Measurements The hydrophilicity of polymers was estimated using three parameters: water uptake, contact angle and surface polarity. For water uptake, polymer wafers produced by direct compression, films cast from polymer melt or polymer rods were immersed in 0.1 M phosphate buffer and incubated at 37 °C. Samples were retrieved at predetermined intervals from the incubation vials, blotted of excess water, and weighed to determine water uptake. As shown in Figure 4, Polilactofate rods absorb water up to 10-times their weight and swell before breaking up, demonstrating higher hydrophilicity compared to polylactic acid.


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1200

0

5

10

15

20

Time (weeks)

Figure 4. Water uptake and swelling behavior of Polilactofate, compared to polylactic acid (PLA).

Biodegradation Characteristics Microspheres made from Polilactofate were studied under accelerated and normal in vitro degradation conditions (7). Gel permeation chromatography (GPC), H and P - N M R , weight loss measurements, and differential scanning calorimetry (DSC) techniques were used to characterize the change of molecular weight ( M ) , chemical composition, and glass transition temperature (Tg) of the degrading polymers. The results indicated that the copolymers degraded in a two-stage fashion, with cleavage of the phosphate-lactide linkages contributing mostly to the initial, more rapid degradation phase and cleavage of the lactidelactide bonds being responsible for the slower, latter stage degradation (Figures 5 and 6). The decrease in the copolymer molecular weight was accompanied by a continuous mass loss (Figure 6) (6). A two-stage hydrolysis pathway was thus proposed to explain the degrada tion behavior of the copolymers. In vivo degradation studies performed in mice demonstrated a good in vitro and in vivo correlation for the degradation rates. In vivo clearance of the polymer was faster and without any lag phase. These copolymers are potentially advantageous for drug delivery and other biomedical applications, where rapid clearance of the polymer carrier and repeated dosing capability are essential to the success of the treatment. !

3l

w



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Figure 5. Reduction in molecular weight ofPolilactofate (PPE) and poly (lactic acid) (PLA).

Figure 6. Degradation and mass loss behavior of Polilactofate (PPE) and poly (lactic acid) (PLA).


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In vivo Biocompatibility ofPolilactofate About 100 mg polymer wafer was formed from Polilactofate and, as a reference, a copolymer o f lactic and glycolic acid ( P L G A RG755), which is known to be biocompatible. These wafers were inserted between muscle layers of the right limb of adult SPF Sprague-Dawley rats under anesthesia. The wafers were retrieved at specific times, and the surrounding tissues prepared for histopathological analysis by a certified pathologist. Similar tests were done after intramuscular or subcutaneous injection of various amounts of Polilactofate microspheres into S-D rats, tabulating implant site macrophage counts, as well as irritation scores. The phosphoester copolymer, Polilactofate, was shown to have an acceptable biocompatibility, similar to that exhibited by the P L G A reference wafer. Preparation of PACLIMER® microspheres Paclitaxel was incorporated into Polilactofate microspheres at 10-60% (w/w) loading by dissolving both substances in ethyl acetate and pumping the solution through an in-line homogenizer with 0.5% polyvinyl acetate) solution to a container with an overhead stirrer. After the microspheres had hardened, they were filtered and lyophilized to give the PACLIMER® product. Particle size and size distribution of the resultant PACLIMER® microspheres were deter mined by a single particle optical sensing system (Model 770; Particle Sizing Systems, Langhome, PA). The median diameter of the microspheres was 53 um, with a range of 20200 um. Microspheres containing up to 40% paclitaxel are spherical in shape with smooth surfaces, while microspheres containing 50 or 60% drug were spherical but with rough surfaces (Figure 7).

In Vitro Release ofpaclitaxelfromPACLIMER

microspheres

The in vitro release of paclitaxel was performed by incubating P A C L I MER® microspheres in phosphate buffered saline (PBS) (pH 7.4) at 37°C. Paclitaxel has very low solubility and poor stability in PBS, and to maintain sink conditions, an octanol layer was placed on top of PBS to continuously extract the released paclitaxel. Paclitaxel concentration in octanol was analyzed at specific time points. As shown in Figure 10, the release of paclitaxel from PACLIMER® microspheres was slow, with 500 n M . In fact, C was lower than 500 n M for all doses tested intraperitoneally in mouse, rat and dog. While C appears to correlate with dose for PACLIMER® microspheres and the commercial TAXOL®, there is no clear correlation bet ween systemic paditaxel exposure (as expressed by A U C ) and dose. The plasma paditaxel exposure appears to be reasonably dose proportional between 15 and 120 mg/kg in the dog; however, non-linearity was noted at higher doses, i.e., the 180-210 mg/kg dose range. m a x

m a x

m a x


m a x

Table IV. Average plasma pharmacokinetic parameters of paditaxel following intraperitoneal administration of PACLIMER® microspheres or paditaxel to female beagle dogs. AUC500nM), such as bone marrow depression. The average maximal concentration measured in dogs was 11.6 n M at a human-equivalent dose of 2400 mg/m . Therefore, PACLIMER® micro spheres is hypothesized to deliver paditaxel locally (e.g. within the peritoneal 2


67 cavity) at therapeutic concentrations for a prolonged period of time without reaching plasma levels associated with systemic toxicity.

Acknowledgements


The contributions of the following researchers are hereby acknowledged: Thomas Hamilton, Eric Burak, Eric Spicer, Harris Holland, Kory Engelke, Robert Garver, and Jim English.

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68 resistance and cross-resistance to various chemotherapeutic agents in unrelated human ovarian cancer cell lines. Cancer Res. 1997, 57, 850-856. 11. Harper, E.; Dang, W.; Lapidus, R.G.; Garver, R.I., Jr. Enhanced efficacy of a novel controlled release paclitaxel formulation ( P A C L I M E R delivery system) for local-regional therapy of lung cancer tumor nodules in mice. Clinical Cancer Research 1999, 5, 4242-4248. 12. Lapidus, R.G.; Dang, W.; Rosen, D . M . ; Gady,A.M.;Zabelinka, Y . ; O'Meally, R.; DeWeese, T.L.; Denmeade, S.R. Anti-tumor effect of combination therapy with intratumoral controlled-release paclitaxel ( P A C L I M E R microspheres) and radiation. Prostate (New York) 2003, 58, 291-298. 13. Li, K . W . ; Dang, W.; Tyler,B.M.;Troiano, G.; Tihan, T.; Brem, H . ; Walter, K . A . Polilactofate microspheres for paclitaxel delivery to central nervous system malignancies. Clinical Cancer Research 2003, 9, 3441-3447.


Paclitaxel Delivery from Novel Polyphosphoesters - American

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