This study demonstrates the selective tumor targeting and the antitumor efficacy of the N-(2-hydroxypropyl)methacrylamide (HPMA)copolymer-bound mesochlorin e6 monoethylenediamine(Mce6) and HPMA copolymer-bound Adriamycin (ADR) in combination photodynamic therapy (PDT) and chemotherapy against human ovarian OVCAR-3 carcinoma xenografted in female athymic mice. The concentrations of Mce6 and ADR in blood and tissues, in free or HPMA copolymer-bound form, were determined by fluorescence and high-performance liquid chromatography fluorescence assays,respectively. Xenograft responses to single and combination therapies were recorded. The peak concentration of HPMA copolymer-Mce6 conjugate in tumor was achieved 18 h after administration. For HPMA copolymer-bound drugs, the concentration ratios of liver and spleen versus muscle were significantly higher than those of free drugs. The HPMA copolymer-bound drugs demonstrated selective targeting and accumulation in the tumor,probably attributed to the enhanced permeability and retention effect. In vivo studies revealed that all tumors in the treatment groups showed significant responses after receiving any of the various types of therapy as compared with controls(P < 0.001). PDT with HPMA copolymer-Mce6 conjugate (PDTMC) at a dose of 13.4 mg/kg(1.5 mg/kg of Mce6 equivalent) and light doses of 110 J/cm2 at 12 and 18 h, respectively, resulted in significant suppression of the growth of OVCAR-3 tumors. Three courses of chemotherapy using 35 mg/kg (2.2 mg/kg of ADR equivalent) of HPMA copolymer-ADR conjugate (CHEMO) were effective in suppressing the growth of tumors. Single PDTMC plus multiple CHEMO exhibited significantly greater therapeutic efficacy than multiple CHEMO. In the group of mice receiving multiple PDTMC, tumor recurrence became obvious after day 20. However, 10 of 12 tumors exhibited complete responses in the group of mice receiving multiple PDTMC plus multiple CHEMO. The least to most effective treatments were ranked as follows: multiple CHEMO < single PDTMC plus multiple CHEMO < multiple PDTMC < multiple PDTMC plus multiple CHEMO. The results clearly demonstrate that: (a) HPMA copolymer-bound drugs exhibited selective tumor accumulation contrary to free drugs;(b) PDT using HPMA copolymer-Mce6 conjugate with multiple light irradiations was a better therapy than that with single light irradiation; and (c) combination chemotherapy and photodynamic therapy with HPMA copolymer-ADR and HPMA copolymer-Mce6 conjugates was the most effective regimen.

The use of polymeric drug delivery systems is rapidly becoming an established approach for improvement of cancer chemotherapy. The covalent binding of low molecular weight drugs to water-soluble polymer carriers offers a potential mechanism to enhance the specificity of drug action. The major distinction between low molecular weight anticancer drugs and their macromolecular conjugates is the mechanism of cell entry. Although low molecular weight drugs may penetrate into all cell types by diffusion, their attachment to macromolecules limits cellular uptake of polymer-drug conjugates to the endocytotic route. Internalized macromolecules are transferred via endosomes into the lysosomal compartment of the cell (1).

We have designed, synthesized, and evaluated HPMA3 copolymers as anticancer drug carriers (reviewed in Ref. 2).4The scientific evidence as well as the results of clinical trials seem to indicate the potential of macromolecular therapeutics in cancer treatment. The fact, that the maximum tolerated dose of HPMA copolymer-ADR conjugate in Phase I clinical trials (320 mg/m2) was several times higher when compared with free ADR (3, 4) bodes well for their potential to treat some forms of resistant cancers (5, 6). Moreover,because of decreased immunotoxicity and myelotoxicity of macromolecular therapeutics (7), cancer patients may not suffer from recurrent viral and fungal infections, which are common after intensive conventional chemotherapy.

The EPR effect is the predominant mechanism by which macromolecules preferentially accumulate in solid tumors (8). This results in the increased efficacy of soluble macromolecular anticancer drugs in the treatment of solid tumors when compared with low molecular weight drugs (6). The phenomenon is attributed to high vascular density of the tumor, increased permeability of tumor vessels,defective tumor vasculature, and defective or suppressed lymphatic drainage in the tumor interstitium (8, 9).

A number of drug carriers such as antibodies have been evaluated to enhance the EPR effect with biorecognition (10, 11). The conjugation of a photosensitizer, Mce6, or a chemotherapeutic agent, ADR, to an antibody carrier demonstrated bioactivity both in vitro and in vivo(12, 13, 14). Targetable conjugates (15) have great potential; however, cross-reactivity during clinical trials was observed, leading to a decrease in the maximum tolerated dose of bound ADR (16).

To improve the outcome of therapy, drugs may be administered in sequences and/or in combination (17). Chemotherapy can combine with radiotherapy (18), immunotherapy (19), or photodynamic therapy (20) to accomplish the highest cell destruction after surgery (17). For example, the combination tumoricidal efficacy of ADR and photodynamic therapy with Photofrin II as the photosensitizer on the human malignant mesothelioma (H-MESO-1) was better than the efficacy of either chemotherapy using ADR or Photofrin II-PDT alone (21).

A novel concept of combination chemotherapy and PDT using HPMA copolymer-bound drugs (ADR and Mce6) was developed. On two cancer models, Neuro 2A neuroblastoma (22) and human ovarian carcinoma heterotransplanted in the nude mice (23), it was revealed that combination therapy with P-ADR and P-Mce6 showed tumor cures that could not be obtained with either chemotherapy or PDT alone. Cooperativity of the action of both drugs contributed to the observed effect (24). In addition, the use of a macromolecular photosensitizer (such as P-Mce6) has the potential to decrease skin accumulation and resulting light sensitivity after treatment (25).

This study was designed to address the issue of antitumor activity of HPMA copolymer-drug conjugates in combination therapy. We hypothesize that multiple combination therapies of P-Mce6 and P-ADR may acquire low effective doses without sacrificing the therapeutic efficacy. This study investigates the biodistribution and enhanced tumor accumulation of P-Mce6 and P-ADR,the enhancement of therapeutic efficacy of P-Mce6at low drug doses, and the combined efficacy of P-Mce6 and P-ADR against s.c. human ovarian carcinoma OVCAR-3 xenografts in nude mice.

Materials.

Mce6, obtained from Porphyrin Products(Logan, UT), was purified on an LH-20 column (2 × 50 cm) that had been equilibrated with methanol before use. ADR was a generous gift from Dr. A. Suarato (Pharmacia-Upjohn, Milan, Italy), and it was used as received without further purification. Bacteriostatic saline buffer was from Sigma Chemical Co. (St. Louis, MO).

Syntheses of Conjugates.

The conjugates were synthesized by a two-step procedure as described previously (25, 26). The polymer precursor was prepared by radical precipitation copolymerization of HPMA and N-methacryloylglycylphenyl-alanylleucylglycine p-nitrophenyl ester. The polymer precursor contained 7.5 mol% of p-nitrophenoxy groups at a molecular weight of∼22,000 and polydispersity of 1.3. In the second step,Mce6 or ADR was bound to the polymer precursor by aminolysis. The drug conjugates were purified by precipitating the reaction mixtures into acetone:ether (4:1, v/v), filtered, and desiccated. The product was further purified on an LH-20 column (2 × 50 cm) equilibrated with methanol. The P-Mce6and P-ADR conjugates fractions were collected, evaporated to dryness,redissolved in de-ionized water, and lyophilized. The drug content in P-Mce6 and P-ADR conjugates, as determined by UV spectrophotometry (ε394 =1.58 × 105m−1cm−1 for Mce6 andε 488 = 1.19 × 104m−1cm−1 for ADR in methanol), was 3.0 and 2.1 mol% (10.8 and 6.3 weight %) of Mce6 and ADR,respectively. The chemical structures of HPMA copolymer conjugates,P-Mce6 and P-ADR, are shown in Fig. 1.

Animal Model.

Female nude mice (athymic nu/nu; 5–6 weeks of age; 15–17 g) were purchased from Simonsen Laboratories (Gilroy, CA). All animal experiments were performed in accordance with the approved protocol and guidance of the Institutional Animal Care and Use Committee, University of Utah. They were accommodated in a pathogen-free mouse colony for 2 weeks before receiving any drug solution or treatment. The OVCAR-3 carcinoma was obtained by inoculating 3 ×106 viable OVCAR-3 cells in both flanks of the mice s.c. (25). The stock tumor was then dissected and s.c. xenografted to nude mice for experiments. No treatment was initiated until a consistent tumor growth rate and a minimum tumor volume of 20 mm3 was attained in ∼3 weeks. P-Mce6 and P-ADR were dissolved in appropriate amounts of bacteriostatic 0.9% NaCl solution to give solutions of the desired concentrations before i.v. injection into the caudal vein of nude mice. The concentrations of the solutions were confirmed by UV/VIS spectroscopy.

Biodistribution.

The concentrations of free Mce6 and P-Mce6 in blood and tissues were determined by a fluorescence assay (25). The mice were sacrificed at chosen time intervals, tumors were harvested, lyophilized for 48 h, homogenized in 2 m NaOH (containing 1 weight % hexadecyl trimethylammonium bromide) at 55°C for 20 h in the dark to make 10 mg/ml solutions, cooled, incubated in a shaker for 1 h at room temperature, and then centrifuged at 4500 rpm(∼2000 × g) for 15 min. The fluorescence intensity of the supernatant solution was then recorded(λexc = 394 nm, λemi =650 nm) using an ISS/PC-1 photon-counting spectrofluorometer (ISS,Champaign, IL). Calibration curves were established for each tissue by adding known concentrations of free Mce6 or P-Mce6 solution to the blood or respective tissues harvested from mice receiving bacteriostatic 0.9% NaCl solution.

An HPLC assay was used to determine the ADR concentration in tissues,as described previously (25). In brief, lyophilized tissues were homogenized in a 0.2 m sodium phosphate buffer(pH 7.4) for 18 h in an incubator at a concentration of 15 mg/ml. Each sample (0.7 ml) was transferred to a tube, an appropriate amount of daunomycin as the internal standard was added, and silver nitrate (100 μl, 0.1 n) was added to each tube before vortex mixing. Double extractions were performed by adding 0.7 ml of chloroform:isopropanol (3:1, v/v) extraction medium before vigorous vortex mixing three times in a 15-min period. The organic layers were collected after centrifugation at 16,000 × g for 15 min, combined, and filtered to remove all particulate matter. The solutions were concentrated by evaporating to dryness under vacuum and redissolved in 50 μl of mobile phase solution(methanol:isopropanol:Sorensen’s buffer, 10:20:70, v/v/v). The sample was then applied to a Dionex HPLC system equipped with a C18 column and a fluorescence detector(λexc. = 480 nm, λemi.= 560 nm) under isocratic condition. The ADR concentrations in blood and tissues were determined using a calibration curve that was established by adding ADR stock solution of known concentration to the blood or respective tissues harvested from mice receiving bacteriostatic 0.9% NaCl solution.

A protocol similar to the assay for free ADR was established for the quantitative determination of P-ADR concentrations in blood and tissues except for an additional thermal acid hydrolysis (25)before extraction. The homogenized tissue samples were hydrolyzed in 2 m HCl at 85°C for a period of 10 min while ∼100% of the adriamycinone, the aglycone moiety of ADR, was detached from the P-ADR conjugate, followed by adding appropriate amounts of NaOH solution to neutralize the acid. The subsequent procedures were the same as those for free ADR as described above. Calibration curves were established the same way as for free ADR, except that P-ADR was used as the stock solution.

The thigh muscle, obtained from the mice sacrificed at chosen time intervals after administration of drug solution, was the standard reference for calculating the concentration ratio between tissues. The fluorescence intensity from precipitated samples after centrifugation only resulted in minimal or background signal. The errors in the results were estimated within 5% in all cases (25).

Instrumentation.

An KTP dye laser (600 series Dye Module, Laserscope Surgical Systems,San Jose, CA) was used as the light source to irradiate the tumor in PDT. The wavelength of the laser light was adjusted to 650 nm,corresponding to the absorption spectrum of Mce6. The light (200 mW) was delivered to the tumor by a flat cut silicone optical fiber with a diameter of 400 μm. The beam size ranged from 7 to 8.6 mm in diameter as measured with a caliper. Depending on the experiment, the tumors were irradiated for an appropriate time to receive a light dose of 110 or 220 J/cm2. The power density used does not lead to a thermal effect that could enhance the photodynamic effect (27).

Bioactivity Evaluation.

The growth of the tumors was monitored every 2–4 days by measuring the tumor volume with a digital caliper. Tumor volumes were calculated using the formula:(1/6)πD12D2,where D1 is the smaller diameter measured. The day that mice received drug solutions was set as day 0,and the tumor volume was normalized to 100%. All subsequent tumor volumes were then expressed as the percentage relative to those at day 0, and a mean ± SD was calculated for each day measured.

The dependence of bioactivity of P-Mce6 on light dose and irradiation schedule was evaluated after administration of P-Mce6 at a dose of 13.4 mg/kg (1.5 mg/kg Mce6 equivalent) to mice. The treatment protocols for mice receiving different light doses at different time intervals are summarized in Table 1: group A, 110 J/cm2 at 18 and 24 h; group B, 110 J/cm2 at 12 and 18 h; and group C, 220 J/cm2 at 12 and 18 h. The control mice received saline buffer instead of drug solution. There were six mice in each group.

Two therapeutic modalities, chemotherapy and PDT, were used in combination therapy. To evaluate the bioactivity, the mice received injections i.v. with a 35-mg/kg (2.2 mg/kg of ADR equivalent) dose of P-ADR in chemotherapy (CHEMO). PDT was performed after i.v. injection of a 13.4 mg/kg (1.5 mg/kg of Mce6 equivalent)dose of P-Mce6; this was followed by two 110 J/cm2 light doses from a 650-nm laser 12 and 18 h after the drug solution injection (PDTMC). Four groups of mice were given various combinations of the two treatment modalities over a 10-day period, as shown in Table 1: group D, one CHEMO on days 0, 5, and 10, respectively; group E, one CHEMO plus one PDTMC on day 0 and one CHEMO on days 5 and 10, respectively; group F, one PDTMC on days 0, 5, and 10, respectively; group G, one CHEMO plus one PDTMC on days 0, 5, and 10, respectively. The control mice received saline buffer instead of drug solution. There were six mice in each group, and the tumor size was recorded every 3–5 days for up to 40 days after the first treatment on day 0.

Less than 0.5% TAD of free Mce6 was accumulated in tumor at 2 h after i.v. administration, and it gradually decreased to almost 0% after 24 h, as demonstrated in Fig. 2. In contrast, the percentage of TAD of P-Mce6 in tumor increased with time and reached a maximum of 1.1% at 18 h after administration and decreased to <0.2% after 48 h. The samples from free Mce6 at 24 h and P-Mce6 at day 7 in tumor yielded background signals, and the concentrations were determined close to the detection limit (∼0.5 ng).

The concentration ratio of blood versus muscle decreased with time before 12 and 24 h for free and HPMA copolymer-bound drugs, respectively, and thereafter the ratios did not change significantly, as shown in Figs. 3 and 4. No significant changes of concentration ratio were found for heart, intestine, kidney, lung,skin, and uterus for all free drugs and copolymer-bound drug conjugates during 2–48 h. However, significant higher concentration ratios of liver and spleen versus muscle were observed after administration of HPMA copolymer-bound drugs; the ratios at 18 h after drug solution administration increased >10 times when compared with other tissues (except tumor) and increased >20 times when compared with those that administered with free Mce6 or ADR solution. The concentration ratios of tumor versus muscle gradually increased with time and reached a peak value 18 h after administration of HPMA copolymer-bound drug solutions. The ratios at 18 h after the mice received the HPMA copolymer-bound drug solutions were >10 times higher when compared with those that received free drug solutions and were more than 6 times higher when compared with those of other tissues(except liver and spleen).

All tumors in the group of mice receiving PDT with P-Mce6 and multiple light irradiations showed significant responses when compared with the controls, as shown in Fig. 5. The mice in group A showed significant inhibition of OVCAR-3 growth for 4 weeks after treatment,whereas the mice in group B exhibited better therapeutic efficacy than those in group A (P < 0.002). The mice in group C showed the best therapeutic efficacy; however, no complete tumor regressions were found. There were no toxic deaths or changes in activity levels in any treatment groups.

The therapeutic efficacies of the various combinations of CHEMO and PDTMC outlined in Table 1 are shown in Fig. 6. All tumor volumes in the treatment groups were significantly less than those in the controls (all P < 0.003). The mice in group D exhibited therapeutic efficacy when compared with the controls, whereas the mice in group F exhibited better therapeutic efficacy than those in group D. The mice in group E showed enhanced therapeutic efficacy over those in group D but less than those in group F. The mice in group G demonstrated the best offset in tumor suppression in this experiment. Tumor recurrence in the mice of group F was observed after day 21. The percentages of complete tumor regression were approximately 20 and 80% for the mice in group F and in group G, respectively. No tumor cures were found for mice in groups D and E. The weight loss of mice in any experimental groups was within 8% below the average weight at the initiation of experiment on day 0, and the mice appeared to regain the weight within 3 weeks after drug administration. Drug toxicity-induced death or loss of activity level did not occur in any experimental groups.

We demonstrated that the biodistribution of the HPMA copolymer-bound drugs in mice was significantly different from that of the free drugs. An enhanced tumor accumulation of HPMA copolymer-bound drugs was observed in accordance with our previous data (25) and results from other laboratories (9, 28).

The concentration ratio of blood versus muscle decreased with time after administration of drug solutions, because the drug in either free or HPMA copolymer-bound form may be redistributed to other tissues or excluded from the body through renal clearance. The values of concentration ratios of liver/spleen versus muscle were over 130:120 and 60:18 for P-Mce6 and P-ADR conjugates, respectively, 18 h after administration; this corresponds to approximately 30:2 and 21:2% of TAD of P-Mce6 and P-ADR conjugates in the tissues. This is consistent with previous results using an 123I-labeled HPMA copolymer-ADR conjugate (29). Discontinuous vascular walls are characteristic of the liver. This allows substances circulating in the plasma to extravasate, permitting an increased permeation and accumulation of the HPMA copolymer-bound drugs in the liver than in other organs. It was reported that the clearance rate of HPMA copolymer-bound drugs was much slower than that of free drugs (25, 30), and this is also demonstrated in Figs. 3 and 4. The half-life of copolymer conjugate in blood is 3–15 times longer than that of free drug (25, 30); this was dependent on the animal model and molecular weight of the conjugate. The prolonged circulation time of copolymer conjugates is also believed to facilitate the high accumulation in the liver. The spleen has physiological properties similar to that of liver. However, because of its smaller size, it accumulated a smaller percentage of TAD. It is important to note that perfusion of organs was not performed, so that the real concentration of P-ADR and P-Mce6 in liver and spleen may be lower than shown in Figs. 3 and 4.

The results clearly indicate that macromolecular therapeutics (P-ADR and P-Mce6) are preferentially accumulated in the tumor (9, 25). A 4-fold larger area under the curve for P-Mce6 was achieved when compared with free Mce6 in the tumor (Fig. 2). For HPMA copolymer-bound drug, the peak concentration ratio in tumor was reached 18 h after injection, as shown in Figs. 3 and 4, and the concentration ratios were always higher than those of the free drug in the tumor. The prolonged retention of HPMA conjugates in tumor, as indicated by the enhanced accumulation and greater area under the curve when compared with those of free drugs, is attributable to the EPR. Several factors may contribute to the enhanced accumulation of polymer conjugates in tumors (9, 28, 30). The tumor accumulation of a drug is dependent on the balance of input from the blood and drainage to the lymphatics. However, the latter is slower or impaired in tumor tissue (31). The angiogenesis supports the abnormal tumor growth by the generation of neovasculatures (31, 32, 33). In addition, the tumor cells secret vascular permeability factors, which make the tumor vasculature abnormally leaky to macromolecules (32). Macromolecular therapeutics diffuse out of the tumor tissue more slowly than low molecular weight drugs because of the size/molecular weight dependence of the diffusion rate. Despite the outward convective flow in the interstitium, which may drain the HPMA copolymer-drug conjugates from the tumor (32), the contributions from the absence of lymphatics,angiogenesis, vascular permeability factors, and slower diffusion rates in the tumor result in the higher concentration and accumulation of the HPMA copolymer-bound drugs in the interstitial space of the tumor (6).

Nonspecific accumulation in solid tumor (Figs. 2,3,4) is a general phenomenon of macromolecules, including antibodies (11, 34). Although conjugation of specific antibody to the conjugate may potentially increase the biorecognition between the specific cell and the drug conjugate (11, 34), problems with cross-reactivity, immunogenicity, and drug loading prevail (10). Bogdanov et al.(35)recently compared accumulation of a nontargeted, long-circulating polymer-drug conjugate with an antibody-targeted conjugate. Although the kinetics of accumulation was different, the antibody-targeted conjugate accumulated faster at early stages, the amounts accumulated at a longer time interval were similar. It appears that both pathways,antibody targeted and nonspecific, long-circulating macromolecular therapeutics, have a potential to be developed into effective anticancer drugs.

It has been pointed out that P-ADR at an ADR equivalent dose of 2.2 mg/kg achieved similar therapeutic efficacy as free ADR at a dose of 1 mg/kg, which is approximately the maximum tolerated dose of free ADR for athymic mice in this study (23). We have also reported that PDT of P-Mce6 at a dose of 12.5 mg/kg (1.5 mg/kg of free Mce6 equivalent) and light dose of 220 J/cm2 was not effective in inhibiting the growth of OVCAR-3 in vivo(23). To explore the possibility of enhancing the therapeutic efficacy of P-Mce6 in PDT without increasing the drug dose,the same drug dose was therefore adopted in this study.

Because the therapeutic effect of PDT is dependent on the concentration of photosensitizer and oxygen in the specific tissue simultaneously,the time lag to irradiate the tumor after administration of photosensitizer and oxygen supply from the capillaries may play important roles in optimal efficacy (36). The prolonged retention time of P-Mce6 in the tumor (Fig. 2)makes multiple irradiation PDT feasible. The PDT with a low drug dose of P-Mce6 (1.5 mg/kg Mce6equivalent) and a light dose of 220 J/cm2 was not effective in the inhibition of OVCAR-3 growth (23). However, PDT with double light doses (two light doses of 110 J/cm2) in groups A and B at the same drug equivalent dose seem to be a more effective treatment than that with a single light dose (220 J/cm2) PDT (Fig. 5). After elevated concentrations of Mce6 in the tumor,significant amounts of oxygen could be consumed during illumination impairing the PDT efficacy. Double irradiation PDT, on the other hand,permits the tumor a recovery interval in which more oxygen is reaccumulated, thus improving the subsequent photodynamic efficacy(Fig. 2). Because multiple irradiation PDT can achieve better therapeutic efficacy than single irradiation PDT at the same drug and light dose, a lower drug dose with multiple irradiation PDT could be used in the treatment to achieve similar therapeutic effect as the single irradiation PDT. Moreover, lower drug doses potentially alleviate nonspecific side effects of the treatment. The 5-day time gap between each treatment course allows the saturation of oxygen in the tumors for the readiness of next PDT treatment; however, adjustment of the treatment protocol, e.g., increasing the drug or light dose, optimized treatment courses, and others, may be necessary to achieve maximal tumor regression.

A synergistic and/or additive effect of the combination efficacy of Mce6 and ADR has been demonstrated in vitro(24, 37); this suggests that a facilitated therapeutic efficacy could potentially be achieved in vivo. A significant difference in tumor responses was found between the mice in groups D and E (P = 0.004). The only difference between groups D and E was one additional PDTMC treatment on day 0;however, this provided a significant reduction in tumor volumes. It appears that the combination therapy of P-Mce6(with multiple irradiation) and P-ADR on day 0 may significantly reduce the survival rate of the tumor cell and therefore inhibit tumor growth. Ten of 12 tumors were cured (40 days) in group G. The combination therapy of multiple CHEMO and PDTMC at such low doses may not be able to completely eradicate the tumor cells, but this treatment definitely reduces the growth rate of the OVCAR-3 carcinoma.

Different approaches have been developed to evaluate the tumor responses in various tumor models. Although i.p. tumor models, e.g., an ascitic tumor model (35) and tumor nodule model (38), have been described, the s.c. xenograft tumor model provides a direct and accessible tumor response after treatment (23, 39).

This study and our previous data (40) suggest the advantages of HPMA copolymer-bound anticancer drugs when compared with free (low molecular weight) drugs. The oligopeptide (GFLG) side chain,used as a drug attachment/release site, is stable in the bloodstream (41) but cleavable by lysosomal enzymes (42). The release of the drug from the carrier is advantageous for both ADR and Mce6. We have shown that the release of Mce6 from the HPMA copolymer carrier results in increased quantum yield of singlet oxygen formation (22). Moreover, the release of ADR from the carrier in the lysosomes occurs in the perinuclear region (43, 44). This results in the inefficient efflux of ADR by the ATP-driven efflux pump and increased efficacy of P-ADR toward multidrug-resistant human ovarian carcinoma xenografts (6). Long-term skin responsiveness from PDT was not observed, and all of the skin photosensitization was recovered within 1 week after irradiation in all experimental groups. This may be attributed to the lower level of drug conjugate accumulated in the skin when compared with free drug (25).

In conclusion, the study of biodistribution facilitates the optimization of treatment protocol. Combination chemotherapy and PDT of P-ADR and P-Mce6 obviously exhibits therapeutic efficacy against human ovarian OVCAR-3 carcinoma. Additional studies will be required to fully characterize the preferential tumor accumulation of HPMA copolymer-bound drugs, take advantage of antibody targeting, and optimize the treatment regimen. The antitumor activity of P-ADR and P-Mce6 demonstrated in this study eloquently warrants clinical interrogation of the polymeric prodrugs as a potential remedy against human tumors.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

        
1

Supported in part by NIH Grant CA 51578 from the National Cancer Institute and a grant from the Research and Development Program, Department of Veterans Affairs.

                
3

The abbreviations used are: HPMA, N-(2-hydroxypropyl)methacrylamide; ADR, Adriamycin(doxorubicin); CHEMO, chemotherapy with P-ADR; EPR, enhanced permeability and retention; Mce6, mesochlorin e6 monoethylenediamine; P, N-(2-hydroxypropyl)methacrylamide copolymer backbone with glycylphenylalanylleucylglycine side chains; P-ADR, HPMA copolymer-ADR conjugate; PDT, photodynamic therapy; PDTMC, PDT with P-Mce6; P-Mce6, HPMA copolymer-Mce6conjugate; TAD, total administered dose.

        
4

J. Kopeček, P. Kopeǩová, T. Minko, and Z-R. Lu. HPMA copolymer–anticancer drug conjugates. Design,activity, and mechanism of action, Eur. J. Pharm. Biopharm, in press,2000.

Fig. 1.

Structure of HPMA copolymer-Mce6and HPMA copolymer-ADR conjugates. R1, Mce6; R2, ADR.

Fig. 1.

Structure of HPMA copolymer-Mce6and HPMA copolymer-ADR conjugates. R1, Mce6; R2, ADR.

Close modal
Fig. 2.

Percentage of TAD of free Mce6 and P-Mce6 after i.v. injection to nude mice bearing s.c. OVCAR-3 carcinoma xenografts. Bars, SD.

Fig. 2.

Percentage of TAD of free Mce6 and P-Mce6 after i.v. injection to nude mice bearing s.c. OVCAR-3 carcinoma xenografts. Bars, SD.

Close modal
Fig. 3.

Concentration ratio of tissues and blood versus muscle after the nude mice bearing s.c. OVCAR-3 carcinoma xenografts received i.v. injections of free Mce6(A) or P-Mce6 (B): 2 h(free Mce6 only; ▨); 6 h (□); 12 h(▪); 18 h ; 24 h (); and 48 h(P-Mce6 only; ⊞). Bars, SD.

Fig. 3.

Concentration ratio of tissues and blood versus muscle after the nude mice bearing s.c. OVCAR-3 carcinoma xenografts received i.v. injections of free Mce6(A) or P-Mce6 (B): 2 h(free Mce6 only; ▨); 6 h (□); 12 h(▪); 18 h ; 24 h (); and 48 h(P-Mce6 only; ⊞). Bars, SD.

Close modal
Fig. 4.

Concentration ratio of tissues and blood versus muscle after the nude mice bearing s.c. OVCAR-3 carcinoma xenografts received i.v. injections of free ADR(A) or P-ADR (B): 2 h (free ADR only; ▨); 6 h (□); 12 h (▪); 18 h; and 24 h (). Bars, SD.

Fig. 4.

Concentration ratio of tissues and blood versus muscle after the nude mice bearing s.c. OVCAR-3 carcinoma xenografts received i.v. injections of free ADR(A) or P-ADR (B): 2 h (free ADR only; ▨); 6 h (□); 12 h (▪); 18 h; and 24 h (). Bars, SD.

Close modal
Fig. 5.

Growth inhibition of s.c. human ovarian OVCAR-3 carcinoma heterotransplanted in nude mice by PDT with P-Mce6 at a dose of 13.4 mg/kg (1.5 mg/kg of Mce6 equivalent) and various light doses. The tumors were irradiated with light (650 nm) at two doses of 110 J/cm2 at 18 h and 24 h (▴; group A); two doses of 110 J/cm2 at 12 h and 18 h (▪; group B); and at two doses of 220 J/cm2 at 12 and 18 h (♦; group C). The mice administered with saline buffer were the controls (•); n = 12 tumors in each group. ∗, mice received PDT with P-Mce6 at 1.5 mg/kg Mce6 equivalent and light (220 J/cm2, 650 nm; ▾; data from 23). Bars, SD.

Fig. 5.

Growth inhibition of s.c. human ovarian OVCAR-3 carcinoma heterotransplanted in nude mice by PDT with P-Mce6 at a dose of 13.4 mg/kg (1.5 mg/kg of Mce6 equivalent) and various light doses. The tumors were irradiated with light (650 nm) at two doses of 110 J/cm2 at 18 h and 24 h (▴; group A); two doses of 110 J/cm2 at 12 h and 18 h (▪; group B); and at two doses of 220 J/cm2 at 12 and 18 h (♦; group C). The mice administered with saline buffer were the controls (•); n = 12 tumors in each group. ∗, mice received PDT with P-Mce6 at 1.5 mg/kg Mce6 equivalent and light (220 J/cm2, 650 nm; ▾; data from 23). Bars, SD.

Close modal
Fig. 6.

Growth inhibition of human ovarian OVCAR-3 carcinoma heterotransplanted in nude mice by multiple combination therapies of P-ADR and/or PDT with P-Mce6. The solutions were given to mice on days 0, 5, and 10. In PDT, the tumor was irradiated with laser light (110 J/cm2, 650 nm) at 12 and 18 h, respectively, after i.v. administration of drug solution:control (•); multiple CHEMO on days 0, 5, and 10 (▴; group D);single PDTMC on day 0 plus multiple CHEMO on days 0, 5, and 10 (▪;group E); multiple PDTMC on days 0, 5, and 10 (♦; group F); and multiple PDTMC on days 0, 5, and 10 plus multiple CHEMO on days 0, 5,and 10 (▾; group G). The control group received saline buffer. n = 12 tumors in each group. Bars,SD.

Fig. 6.

Growth inhibition of human ovarian OVCAR-3 carcinoma heterotransplanted in nude mice by multiple combination therapies of P-ADR and/or PDT with P-Mce6. The solutions were given to mice on days 0, 5, and 10. In PDT, the tumor was irradiated with laser light (110 J/cm2, 650 nm) at 12 and 18 h, respectively, after i.v. administration of drug solution:control (•); multiple CHEMO on days 0, 5, and 10 (▴; group D);single PDTMC on day 0 plus multiple CHEMO on days 0, 5, and 10 (▪;group E); multiple PDTMC on days 0, 5, and 10 (♦; group F); and multiple PDTMC on days 0, 5, and 10 plus multiple CHEMO on days 0, 5,and 10 (▾; group G). The control group received saline buffer. n = 12 tumors in each group. Bars,SD.

Close modal
Table 1

Therapeutic treatment protocols of P-Mce6 and/or P-ADR conjugates

GroupTreatmenta
Day 0Day 5Day 10
PDTMCb NTc NTc 
PDTMC NTc NTc 
PDTMCd NTc NTc 
CHEMO CHEMO CHEMO 
CHEMO+ PDTMC CHEMO CHEMO 
PDTMC PDTMC PDTMC 
CHEMO + PDTMC CHEMO+ PDTMC CHEMO+ PDTMC 
GroupTreatmenta
Day 0Day 5Day 10
PDTMCb NTc NTc 
PDTMC NTc NTc 
PDTMCd NTc NTc 
CHEMO CHEMO CHEMO 
CHEMO+ PDTMC CHEMO CHEMO 
PDTMC PDTMC PDTMC 
CHEMO + PDTMC CHEMO+ PDTMC CHEMO+ PDTMC 
a

CHEMO, P-ADR at a dose of 35 mg/kg (2.2 mg/kg of ADR equivalent); PDTMC, P-Mce6 at a dose of 13.4 mg/kg (1.5 mg/kg of Mce6 equivalent); the tumor was irradiated with laser light (650 nm, 110 J/cm2) at 12 and 18 h, respectively, after administration of drug solution.

b

The tumor was irradiated at 18 and 24 h.

c

NT, no treatment.

d

The tumor was irradiated with laser light(220 J/cm2) at 12 and 18 h, respectively.

We are indebted to Dr. A. Suarato (Pharmacia-Upjohn, Milano,Italy) for the generous gift of Adriamycin.

1
De Duve C., De Barsy T., Poole B., Trouet A., Tulkens P., van Hoof F. Lysosomotropic agents.
Biochem. Pharmacol.
,
23
:
2495
-2531,  
1974
.
2
Putnam D., Kopeček J. Polymer conjugates with anticancer activity.
Adv. Polym. Sci.
,
122
:
55
-123,  
1995
.
3
Vasey P. A., Kaye S. B., Morrison R., Twelves C., Wilson P., Duncan R., Thomson A. H., Murray L. S., Hilditch T. E., Murray T., Burtles S., Fraier D., Frigerio E., Cassidy J. Phase I clinical and pharmacokinetic study of PK1 [N-(2-hydroxypropyl)methacrylamide copolymer doxorubicin]: first member of a new class of chemotherapeutic agents-drug-polymer conjugates.
Clin. Cancer Res.
,
5
:
83
-94,  
1999
.
4
Thompson A. H., Vasey P. A., Murray L. S., Cassidy J., Fraier D., Frigerio E., Twelves C. Population pharmacokinetics in Phase I drug development: a Phase I study of PK1 in patients with solid tumors.
Br. J. Cancer
,
81
:
99
-107,  
1999
.
5
Minko T., Kopečková P., Kopeček J. Chronic exposure to HPMA copolymer-bound Adriamycin does not induce multidrug resistance in a human ovarian carcinoma cell line.
J. Controlled Release
,
59
:
133
-148,  
1999
.
6
Minko, T., Kopečková, P., and Kopeček, J., Efficacy of chemotherapeutic action of HPMA copolymer-bound doxorubicin in a solid tumor model of ovarian carcinoma. Int. J. Cancer, in press, 2000.
7
Říhová B. Antibody-targeted polymer-bound drugs.
Folia Microbiol.
,
40
:
367
-384,  
1995
.
8
Maeda H., Seymour L. M., Miyamoto Y. Conjugates of anticancer agents and polymers: advantages of macromolecular therapeutics in vivo.
Bioconjugate Chem.
,
3
:
351
-362,  
1992
.
9
Noguchi Y., Wu J., Duncan R., Strohalm J., Ulbrich K., Alaike T., Maeda H. Early phase tumor accumulation of macromolecules: a great difference in clearance rate between tumor and normal tissues.
Jpn. J. Cancer Res.
,
89
:
307
-314,  
1998
.
10
Fritzberg A. R. Biorecognition of antibodies in vivo: potential in drug targeting.
J. Mol. Recognit.
,
9
:
309
-315,  
1996
.
11
Sjogren H. O., Isaksson M., Willner D., Hellstrom I., Hellstrom K. E., Trail P. A. Antitumor activity of carcinoma-reactive BR96-doxorubicin conjugate against human carcinomas in athymic mice and rats and syngeneic rat carcinomas in immunocompetent rats.
Cancer Res.
,
57
:
4530
-4536,  
1997
.
12
Rakestraw S. L., Ford W. E., Tompkins R. G., Rodgers M. A., Thorpe W. P., Yarmush M. L. Antibody-targeted photolysis: in vitro immunological, photophysical, and cytotoxic properties of monoclonal antibody-dextran-Sn(IV) chlorin e6 immunoconjugates.
Biotechnol. Prog.
,
8
:
30
-39,  
1992
.
13
Yarmush M. L., Thorpe W. P., Strong L., Rakestraw S. L., Toner M., Tompkins R. G. Antibody targeted photolysis.
Crit. Rev. Ther. Drug Carrier Syst.
,
10
:
197
-252,  
1993
.
14
Trail P. A., Willner D., Lasch S. J., Henderson A. J., Hofstead S., Casazza A. M., Firestone R. A., Hellstrom I., Hellstrom K. E. Cure of xenografted human carcinomas by BR96-doxorubicin immunoconjugates.
Science (Washington DC)
,
261
:
212
-215,  
1993
.
15
Lu Z-R., Kopečková P., Kopeček J. Polymerizable Fab′ antibody fragments for targeting of anticancer drugs.
Nat. Biotechnol.
,
17
:
1101
-1104,  
1999
.
16
Tolcher A. W., Sugarman S., Gelmon K. A., Cohen R., Saleh M., Isaacs C., Young L., Healey D., Onetto N., Slichenmyer W. Randomized Phase II study of BR96-doxorubicin conjugate in patients with metastatic breast cancer.
J. Clin. Oncol.
,
17
:
478
-484,  
1999
.
17
Markman, M. Basic Cancer Medicine. Philadelphia, PA: W. B. Saunders Company, 1997.
18
Meirow D. Ovarian injury and modern options to preserve fertility in female cancer patients treated with high dose radio-chemotherapy for hemato-oncological neoplasias and other cancers.
Leuk. Lymphoma
,
33
:
65
-76,  
1999
.
19
Lidor Y. L., O’Briant K. C., Xu F. J., Hamilton T. C., Ozols R. F., Bast R. C., Jr. Alkylating agents and immunotoxins exert synergistic cytotoxicity against ovarian cancer cells.
J. Clin. Investig.
,
92
:
2440
-2447,  
1993
.
20
Canti G., Nicolin A., Cubeddu R., Taroni P., Bandieramonte G., Valentini G. Antitumor efficacy of the combination of photodynamic therapy and chemotherapy in murine tumors.
Cancer Lett.
,
125
:
39
-44,  
1998
.
21
Brophy P. F., Keller S. M. Adriamycin enhanced in vitro and in vivo photodynamic therapy of mesothelioma.
J. Surg. Res.
,
52
:
631
-634,  
1992
.
22
Krinick N. L., Sun Y., Joyner D., Spikes J. D., Straight R. C., Kopeček J. A polymeric drug delivery system for the simultaneous delivery of drugs activatable by enzymes and/or light.
J. Biomater. Sci. Polym. Ed.
,
5
:
303
-324,  
1994
.
23
Peterson C. M., Lu J. M., Sun Y., Peterson C. A., Shiah J. G., Straight R. C., Kopeček J. Combination chemotherapy and photodynamic therapy with N-(2-hydroxypropyl)methacrylamide copolymer-bound anticancer drugs inhibit human ovarian carcinoma heterotransplanted in nude mice.
Cancer Res.
,
56
:
3980
-3985,  
1996
.
24
Peterson C. M., Lu J. M., Gu Z. W., Shiah J. G., Lythgoe K., Peterson C. A., Straight R. C., Kopeček J. Cooperativity between free and N-(2-hydroxypropyl)methacrylamide copolymer bound Adriamycin and mesochlorin e6 monoethylene diamine induced photodynamic therapy in human epithelial ovarian carcinoma in vitro.
Int. J. Oncol.
,
15
:
5
-16,  
1999
.
25
Shiah J-G., Sun Y., Peterson C. M., Kopeček J. Biodistribution of free and N-(2-hydroxypropyl)methacrylamide copolymer-bound mesochlorin e6 and Adriamycin in nude mice bearing human ovarian carcinoma OVCAR-3 xenografts.
J. Controlled Release
,
61
:
145
-157,  
1999
.
26
Omelyanenko V., Kopečková P., Gentry C., Shiah J. G., Kopeček J. HPMA copolymer-anticancer drug-OV-TL 16 antibody conjugates. I. Influence of the method of synthesis on the binding affinity to OVCAR-3 ovarian carcinoma cells in vitro.
J. Drug Targeting
,
3
:
357
-373,  
1996
.
27
Hashimoto Y., Hirano T., Yamaguchi N. Novel after-loading interstitial photodynamic therapy of canine transmissible sarcoma with photofrin II and excimer dye laser.
Jpn. J. Cancer Res.
,
86
:
239
-244,  
1995
.
28
Takakura Y., Hashida M. Macromolecular carrier systems for targeted drug delivery: pharmacokinetic considerations on biodistribution.
Pharm. Res. (NY)
,
13
:
820
-831,  
1996
.
29
Pimm M. V., Perkins A. C., Strohalm J., Ulbrich K., Duncan R. γ scintigraphy of the biodistribution of 123I-labelled N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin conjugates in mice with transplanted melanoma and mammary carcinoma.
J. Drug Targeting
,
3
:
375
-383,  
1996
.
30
Seymour L. W., Ulbrich K., Strohalm J., Kopeček J., Duncan R. The pharmacokinetics of polymer-bound Adriamycin.
Biochem. Pharmacol.
,
39
:
1125
-1131,  
1990
.
31
Jain R. K. Delivery of molecular and cellular medicine to solid tumors.
Microcirculation
,
4
:
1
-23,  
1997
.
32
Feng D., Nagy J. A., Hipp J., Dvorak H. F., Dvorak A. M. Vesiculo-vacuolar organelles and the regulation of venule permeability to macromolecules by vascular permeability factor, histamine, and serotonin.
J. Exp. Med.
,
183
:
1981
-1986,  
1996
.
33
Holash J., Maisonpierre P. C., Compton D., Boland P., Alexander C. R., Zagzag D., Yancopoulos G. D., Wiegand S. J. Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF.
Science (Washington DC)
,
284
:
1994
-1998,  
1999
.
34
Goff B. A., Blake J., Bamberg M. P., Hasan T. Treatment of ovarian cancer with photodynamic therapy and immunoconjugates in a murine ovarian cancer model.
Br. J. Cancer
,
74
:
1194
-1198,  
1996
.
35
Marecos E., Weissleder R., Bogdanov A., Jr. Antibody-mediated versus nontargeted delivery in a human small cell lung carcinoma model.
Bioconjugate Chem.
,
9
:
184
-191,  
1998
.
36
Andrejevic-Blant, S., Hadjur, C., Ballini, J. P., Wagnieres, G., Fontolliet, C., van-den-Bergh, H., and Monnier, P. Photodynamic therapy of early squamous cell carcinoma with tetra(m-hydroxyphenyl)chlorin: optimal drug-light interval. Br. J. Cancer, 76: 1021–1028, 1997.
37
Peterson C. M., Lu J. M., Gu Z. W., Shiah J. G., Lythgoe K., Peterson C. A., Straight R. C., Kopeček J. Isobolographic assessment of the interaction between Adriamycin and photodynamic therapy with mesochlorin e6 monoethylene diamine in human epithelial ovarian carcinoma (OVCAR-3) in vitro.
J. Soc. Gynecol. Invest.
,
2
:
772
-777,  
1995
.
38
Molpus K. L., Kato D., Hamblin M. R., Lilge L., Bamberg M., Hasan T. Intraperitoneal photodynamic therapy of human epithelial ovarian carcinomatosis in a xenograft murine model.
Cancer Res.
,
56
:
1075
-1082,  
1996
.
39
Colussi V. C., Feyes D. K., Mulvihill J. W., Li Y. S., Kenney M. E., Elmets C. A., Oleinick N. L., Mukhtar H. Phthalocyanine 4 (Pc 4) photodynamic therapy of human OVCAR-3 tumor xenografts.
Photochem. Photobiol.
,
69
:
236
-241,  
1999
.
40
Minko T., Kopečková P., Kopeček J. Comparison of the anticancer effect of free and HPMA copolymer-bound Adriamycin in human ovarian carcinoma cells.
Pharm. Res. (NY)
,
16
:
986
-996,  
1999
.
41
Rejmanová P., Kopeček J., Duncan R., Lloyd J. B. Stability in rat plasma and serum of lysosomally degradable oligopeptide sequences in N-(2-hydroxypropyl)methacrylamide copolymers.
Biomaterials
,
6
:
45
-48,  
1985
.
42
Rejmanová P., Pohl J., Baudys M., Kostka V., Kopeček J. Polymers containing enzymatically degradable bonds. 8. Degradation of oligopeptide sequences in N-(2-hydroxypropyl)methacrylamide copolymers by bovine spleen cathepsin B.
Makromol. Chem.
,
184
:
2009
-2020,  
1983
.
43
Omelyanenko V., Gentry C., Kopečková P., Kopeček J. HPMA copolymer-anticancer drug-OV-TL 16 antibody conjugates. II. Processing in epithelial ovarian carcinoma cells in vitro.
Int. J. Cancer
,
75
:
600
-608,  
1998
.
44
Omelyanenko V., Kopečková P., Gentry C., Kopeček J. Targetable HPMA copolymer-Adriamycin conjugates. Recognition, internalization, and subcellular fate.
J. Controlled Release
,
53
:
25
-37,  
1998
.