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Discovery Research Oncology [M. Z., M. F., A. F., E. P., A. S.] and Global Drug Metabolism Research [A. G., M. G. C., E. F., R. d.], Pharmacia Corporation, 20014 Nerviano (MI), Italy; ISS, Champaign, Illinois 61822 [M. v.]; Laboratory for Fluorescence Dynamics, University of Illinois, Urbana-Champaign, Illinois 61801 [E. G.]; and Fondazione Centro San Raffaele, Scientific Institute, 20132 Milan, Italy [V. R. C.]

2 To whom requests for reprints should be addressed, at Discovery Research Oncology, Pharmacia Corp., V.le Pasteur 10, 20014 Nerviano (MI), Italy. Phone: 39-0248385318; Fax: 3-0248383750; E-mail: moreno.zamai{at}Pharmacia.com (to M. Z.) and Fondazione Centro San Raffaele, DIBIT Scientific Institute, Via Olgettina 58, 4A1 20132 Milan, Italy. Phone: 39-0226434780; Fax: 39-0226434861; E-mail: valeria.caiolfa{at}hsr.it (to V. R. C.)

	Abstract

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Soluble copolymers of camptothecin (CPT), based on poly[N-(2-hydroxypropyl) methacrylamide] (pHPMA), were obtained by conjugation through the degradable spacers -Gly-Phe-Leu-Gly- or -Gly-6-aminohexanoyl-Gly-. We investigated to what extent passive accumulation and retention of hydroxypropyl methacrylamide copolymer of CPT (pHPMA-CPT) in tumors and modulation of the drug release influence efficacy. Release of CPT in vivo was detected by time-resolved phase-shift fluorescence imaging on tumor specimens, based on the evidence that free and bound drug had different fluorescence lifetimes in solution. HT-29 murine specimens, obtained at several times after treatment with ³H-labeled free CPT, pHPMA-Gly-Phe-Leu-Gly-CPT, or pHPMA-Gly-6-aminohexanoyl-Gly-CPT, were either imaged for time-resolved phase-shift fluorescence or subjected to autoradiography. Phase shifts of CPT conjugates were equal or longer than those of free CPT, indicating the presence of both free and polymer-bound drug in the tumor, in agreement with autoradiograms. pHPMA-Gly-Phe-Leu-Gly-CPT underwent relevant intratumor hydrolysis during the first 24 h, whereas the hydrolysis of pHPMA-Gly-6-aminohexanoyl-Gly-CPT was slow. The latter showed antitumor activity at doses from 10 to 22.5 mg/kg/day against s.c. HT-29, A2780, M14, and A549 s.c. xenografts. Moreover, inhibition of tumor growth lasted for up to 73–88 days, and cures were observed on mice with orthotopic implanted HT-29; pHPMA-Gly-Phe-Leu-Gly-CPT was 2-fold more potent than pHPMA-Gly-6-aminohexanoyl-Gly-CPT but less tolerated. Our data suggest that the efficacy of pHPMA-CPT copolymers is related to their intratumor accumulation, and in vivo properties of releasing CPT by esterolytic and proteolytic degradation.

	Introduction

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The antitumor activity of CPT,⁵ was established in experimental models in the early 1960s (1, 2). Because of the limited water solubility, a water-soluble sodium salt of the drug was formulated for use in clinical trials (3). Antitumor activity of CPT was noted in patients with GI tumors (4); however, patients experienced neutropenia, thrombocytopenia, hemorrhagic cystitis, and GI symptoms with significant diarrhea (3, 4). Therefore, clinical studies with CPT sodium were halted. During the next 20 years, the discovery of topoisomerase-I as the cellular target of CPT and its analogues (5–7) renewed the interest on this class of compounds. Topoisomerase-I is a nuclear enzyme involved in relaxation of DNA supercoils during transcription, replication, and other vital cell processes (8). This enzyme is overexpressed in advanced stages of human colon adenocarcinoma and other malignancies but not in normal tissue (9, 10). CPTs inhibit the breakage-rejoining reaction of DNA-topoisomerase I, and drug-induced accumulation of topoisomerase I-DNA complexes was identified as an essential step, which ultimately led to cell death by apoptosis (11). It was found that CPT acted mostly in the S phase of the cell cycle with little toxicity toward normal resting cells (11). A structure-activity relationship was determined for semisynthetic and synthetic CPT derivatives (12, 13). These studies confirmed the importance of the lactone, closed-ring molecule, for drug activity and, in retrospect, recognized that the carboxylated form of CPT results in insufficient and erratic amounts of the active form being available. The synthetic analogues, 9-amino camptothecin and 10,11-methylenedioxy camptothecin, showed unprecedented effectiveness against human colon adenocarcinoma in immunodeficient mice (14). At that time, CPT-11 (irinotecan) and topotecan were prepared and tested in experimental and clinical settings. Both of these drugs underwent broad testing in the early 1990s (15, 16). Numerous trials have been conducted in an attempt to establish the efficacy in various tumor types, to determine the dose-limiting toxicity and to define the optimal schedule of administration. It seems that large doses of these drugs given on intermittent schedules are not effective. O’Leary and Muggia (17) observed that, to be most effective, the CPTs require a prolonged schedule of administration given continuously at low doses. With these schedules, normal hematopoietic cells and mucosal progenitor cells with low topoisomerase-I levels may be spared, whereas efficacy is preserved. A variety of different strategies are being used to modulate the systemic delivery of this class of agents, frequently to increase antitumor activity and/or reduce experienced side effects. Approaches including formulation vehicles search for more water-soluble prodrugs, modulation of routes of administration, and both pharmacodynamic and pharmacokinetic biomodulation have been recently reviewed by Kehrer et al. (18).

We have regarded the conjugation to soluble pHPMA copolymers as a strategy to increase the therapeutic index of this class of compounds and considered CPT as the ideal candidate for testing our hypothesis. The use of polymeric drug carriers is an established approach for improvement of cancer chemotherapy (19, 20). The covalent binding of low Mw drugs to water-soluble polymer carriers offers a potential mechanism to enhance the specificity of drug action. Specificity might be achieved either by an active targeting of polymer-drug conjugates to cancer cells or passive accumulation of conjugates within solid tumors (19, 20). The latter is because of unique aspects of tumor structural biology. The abnormal vasculature of tumors with an increased vessel permeability makes them take up large molecules more efficiently than normal tissues. At the same time, the poor lymphatic drainage of tumors allows high concentrations of polymeric drug to build up in these tissues (21–24). This phenomenon was referred to as the enhanced permeability and retention effect (24–26). Soluble copolymers based on pHPMA bearing doxorubicin on pendent oligopeptide spacers have been synthesized previously and widely characterized (19, 26, 27). Careful design of the oligopeptide spacer can tailor the conjugate as a prodrug for activation by selected proteases, such as lysosomal cathepsin enzymes (27). This leads to very low levels of free drug released in the circulation and results in substantial decrease of systemic toxicities compared with free doxorubicin (27, 28). The polymeric doxorubicin is also known, from animal studies, to exhibit a tumor tropism thought to result from the enhanced permeability and retention effect in many solid tumors (24, 25). The resulting high levels of drug associated with solid tumors in vivo coincide with impressive anticancer efficacy against a range of model tumors (25, 28). A Phase I clinical study was performed in the United Kingdom by the Phase I/II Trials Committee of the Cancer Research Campaign on PK1, a doxorubicin covalently bound to N-(2-hydroxypropyl)methacrylamide copolymer by a peptide linker (29). PK1, which is not actively targeted, is now undergoing Phase II studies.

We have described previously the synthesis of the first pHPMA conjugates of CPT (30). The lactone form of CPT was covalently linked at its -hydroxyl group to the polymer through the classical -Gly-Phe-Leu-Gly- peptide (19, 27). By this arrangement, we aimed at modulating the release of CPT by proteolytic cleavage at the tumor site but also obtaining water-soluble derivatives that partly protect the active lactone form of CPT in plasma. In preliminary studies on nude mice with s.c. HT-29 human colon carcinoma, pHPMA-Gly-Phe-Leu-Gly-CPT was more active than CPT by the i.v. route (30).

In this paper, we describe a novel HPMA copolymer of CPT in which the cleavable peptide -Gly-Phe-Leu-Gly- was substituted by the spacer -Gly-C6-Gly-. By comparing in vivo hydrolysis and efficacy of conjugates having different drug content (5% or 10% CPT by weight) and hydrolytic properties (-Gly-Phe-Leu-Gly- versus -Gly-C6-Gly-), we investigated to what extent the release of CPT was modulated at the tumor site and influenced antitumor activity. To this end, we have succeeded in monitoring simultaneously free and polymer-bound CPT at several times after administration by direct tumor imaging. We have taken advantage of the fluorescence properties of free and polymer-bound CPT, first investigated in solution, to develop time-resolved fluorescence phase-shift imaging of CPT in tumor specimens from nude mice with s.c. HT-29. We have compared the distribution of free and bound CPT as acquired by phase-shift imaging with autoradiograms on ³H-labeled CPT and CPT-conjugates, which, however do not allow us to distinguish between free and bound drug, and only yield the total CPT content in tissues.

Antitumor efficacy and tolerability of CPT conjugates were investigated in multiple dose studies on several human tumors s.c. implanted and on HT-29 orthotopically implanted, and discussed in relation to the hydrolytic properties of the new drugs.

	Materials and Methods

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Compounds
Chemicals were reagent grade or better. HSC, HAS, and proteases were either from Sigma-Aldrich or from Calbiochem. CPT was purchased from Sigma Chemical Co. (St. Louis, MO). HPMA copolymers of CPT with either 5% or 10% drug content (w/w) were synthesized by the Pharmacia Corp. (Milan, Italy) using a three-step procedure described previously (30), which yields conjugates free of contaminants (Fig. 1). Weight-average (Mw) and polydispersity (Mw/Mn) were determined by size exclusion chromatography (31, 32). Drug content (w/w %) was quantified by total hydrolysis (30). Concentrations and doses of pHPMA-CPT conjugates were expressed as CPT equivalents according to the drug content of each conjugate. pHPMA-Gly-C₆-Gly-[³H]CPT and pHPMA-Gly-Phe-Leu-Gly-[³H]CPT were synthesized and characterized by the Pharmacia Corp. using commercially available [³H]CPT. For in vivo experiments, CPT was formulated as suspension in 10% Tween 80/0.9% NaCl solution (9:1, v/v). pHPMA-CPT conjugates, freely soluble in water, were administered in sterile 0.9% NaCl solution. All of the drug solutions were prepared immediately before use. To determine the concentration of CPT in solution precisely and avoid errors because of the low solubility of the drug, preparations were checked photometrically at 360 nm.

View larger version (20K): [in this window] [in a new window]   Fig 1. Chemical characteristics of CPT-conjugates.

Time-resolved Fluorescence Spectroscopy.   Fluorescence lifetime decays in buffer solution were measured by harmonic spectrofluorimetry (33, 34) at room temperature. We used the K2 multifrequency modulation fluorometer (ISS Inc.) at 18A of lamp current and 2 nm excitation slits. Excitation was set at 350 nm, and the fluorescence signal was collected at 90° through a Schott KV 408 filter to cut Rayleigh and Raman scattering. The cross-correlation frequency was 80 Hz. A dilute glycogen suspension (Sigma; Type II from Oyster) in double-distilled water was taken as the reference (33, 34). CPT was dissolved in DMSO and then diluted to 2 µM in buffer up to a final DMSO content of 1% (v/v). CPT conjugates were dissolved in double-distilled water just before use and diluted to 2 µM CPT equivalent in buffer. Human serum albumin crystallized powder was dissolved in buffer. The buffer was PBS (pH 7.4) or PBS + KCl/HCl (pH 2.3). Data were collected with an accuracy of 0.2° for the phase values and 0.004 for the modulation. For <10 min/sample with an ISS A2D, two-channel digital acquisition card and FFT data acquisition were used. No bleaching of the sample was observed during this time interval of 10 min.

In Vivo Experiments
Animals.   Female nude HSD- nu/nu mice, supplied by Harlan Italy (San Pietro al Natisone, Udine, Italy), 4–6 weeks of age and weighing 20–25 g, were used in experiments with human tumors. They were maintained under specific pathogen-free conditions, and provided sterile food and water ad libitum. Animal health was routinely tested for the absence of antibodies to a panel of pathogens.

Tumor Models.   MX1 human mammary carcinoma was from the National Cancer Institute (Frederick, MD); Human lung (A549), colon (HT-29), and prostate (DU 145) carcinomas were from American Type Culture Collection (Rockville, MD). Human pancreatic carcinoma (CAPAN 1) was obtained from the Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia (Brescia, Italy). Human ovarian (A2780) carcinoma was provided by Dr. Robert F. Ozols, initially developed at the National Cancer Institute. Human melanoma (M-14) was given by Dr. Gabriella Pezzoni (Novuspharma SpA, Monza, Italy; Tables 2–4).

The orthotopic HT-29 transplantation was performed after a modification of the procedure described by Grosios et al. (35). HT-29 cells were maintained in vitro, and 1 x 10⁶ cells/mice were injected into the cecal wall of a nude mice. Treatment started 1 week after transplant. Drugs were administered by the i.v. route according to the dose and frequency schedule indicated in Table 5. Mice treated with vehicle or drug were sacrificed at day 41 after implantation for assessing primary tumor growth and the presence of distant metastasis.

WBA and Time-resolved Imaging.   HT-29 human colon carcinoma was maintained by serial s.c. transplantation in athymic mice using 15–20 mg of tumor brei. For the experiment, tumors were excised, and 15–20-mg fragments were implanted s.c. into the left flank. Treatment started 10 days after transplantation when tumor reached the size of about 0.3–0.4 g. Animals were i.v. administered in the tail vein with 20 mg/kg of [³H]CPT in PEG 400/water/10 mM H₃PO₄ or with 20 mg CPT equivalent/kg of either pHPMA-Gly-Phe-Leu-Gly-[³H]CPT or pHPMA-Gly-C6-Gly-[³H]CPT in sterile saline. The radioactivity dose was 2.5 mCi/kg. WBA was carried out by a modification of the method of Ullberg (38). Animals were sacrificed deeply anesthetized at 20 min, and 1, 4, 24, and 48 h after administration. Carcasses were immediately embedded in a gel of carboxymethyl cellulose, frozen in hexane and dry ice at -70°C, and stored at -20°C. Multiple sagittal sections of 20-µm thickness were cut by a cryomicrotome at -20°C and caught on tape. Sections were left for freeze-drying for at least 24 h. For autoradiography, sections were placed on image plates and processed at different exposure times from 24 to 72 h. The imaging results were transformed into concentration units (nCi/mg) by ³H standards. For in vivo fluorescence imaging, sections were kept at room temperature and in the dark until analysis. In this case, we have used a synchronously pumped DCM dye laser, Coherent model 700, set at 690-nm excitation wavelength. This dye laser was pumped by a Coherent model Antares 76-YAG Nd-Yag laser. The dye laser output power was 50 mW, and the laser beam diameter was 0.442 mm. A Spectra Physics model 390 external frequency doubler generated UV light at 345 nm. The optical pulse train repetition rate was 76.2 MHz. Via a set of mirrors and intensity adjusting Glan-Taylor air-spaced polarizers, the laser excitation light entered an ISS Koala automated sample compartment. For these studies, the standard sample compartment was replaced by a stepper-motor driven X-Y scanner with a range of 6.35 cm in the vertical and 3.81 cm in the horizontal direction. Step size resolution was 0.5 mm. The mouse section caught on tape was attached to the scanner, and the normal of the whole assembly was oriented at 40° with respect to the illumination beam to prevent reflections and scattered light to enter directly into the emission optical path. The X-Y stepper motors used the monochromator motor parameters. For scanner control we used the otherwise not used ports 5 and 9 of the right emission channel, because there were physically no emission monochromators attached to this Koala unit. The left emission photomultiplier monitored the emission through a set of interference emission filters. The reference lifetime 0.00 ns was taken on the tape background of the murine specimen. To this end the scanner started in a corner of the scan field. The bidirectional scan mode was activated in the ISS software routines to use also the return track.

	Results

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The Nature of the Spacer Affects the Efficacy and the Releasing Properties of CPT Conjugates in Mice with HT-29 Human ca
Efficacy.   Enzymatic degradation or hydrolysis in buffer of pHPMA-Gly-Phe-Leu-Gly-CPT at either low (5%) or high (10%) CPT content by cathepsins and human leukocyte elastase, and in the presence of human serum complement was linear for a large extent 3–5 h, and was shown in details previously (30). Here we report the hydrolysis of the new conjugates bearing the -Gly-C₆-Gly- linker (Table 1). Regardless of the CPT content, hydrolysis in buffer of pHPMA-Gly-C₆-Gly-CPT conjugates to free CPT were always <1 nmol/hour of CPT from pH 6.0 to pH 7.5, at 37°C, and it did not increase in the presence of human serum (Table 1). Only cathepsins cleaved pHPMA-Gly-C₆-Gly-CPT in a measurable extent above the buffer background. The rate of release of CPT from pHPMA-Gly-C₆-Gly-CPT was 1.7–3.4-fold higher in the presence of cathepsin B than in buffer at pH 6.0 (Table 1). Clearly, release of CPT from -Gly-C₆-Gly-conjugates was mostly because of pH-driven hydrolysis, and it was distinctly slower (20–50-fold less by cathepsin B) than pHPMA-Gly-Phe-Leu-Gly-CPT copolymers, as reported previously (30). Thus, these new conjugates, which are i.v. administered, might be expected to be even more stable into circulation than pHPMA-Gly-Phe-Leu-Gly-CPTs.

By the i.v. route (6 doses, every 4 days), pHPMA-Gly-C₆-Gly-CPT (5% CPT) had no effect at doses of 2.5–5.0 mg/kg/day (T/C% > 42), whereas tumor growth inhibition was observed at 10 mg/kg/day with 10 < T/C% < 42 for native CPT. The activity of pHPMA-Gly-C₆-Gly-CPT (10% CPT) was observed starting at the dose of 5 mg/kg/day. At the dose of 10 mg/kg/day, T/C% values were those of native CPT. Nevertheless, the conjugate was tolerated for up to 22.5 mg/kg/day with no deaths because of toxicity and a number of long-term survivors (Table 2). Modification of the schedule, from 20 mg/kg/day x 6 doses every 4 days to 20 mg/kg/day x 8 doses every 7 days, did not affect efficacy of the conjugate, nor did prolonged treatments (20 mg/kg/day x 8 doses every 4 days), increase toxicity. The batch to batch slight variation on CPT content in pHPMA conjugates (0.15%) was evaluated in three repeated studies on HT-29 xenografts and did not affect the efficacy. A comparison of these results with our previous ones, obtained for pHPMA-Gly-Phe-Leu-Gly-CPTs on the same tumor model and at identical schedule (30), indicates that the new -Gly-C₆-Gly- conjugates are not as potent as pHPMA-Gly-Phe-Leu-Gly-CPTs, but their tolerability might allow for larger therapeutic windows.

Intratumor Accumulation and Release.   Obvious differences of distribution of radioactivity were observed in HT-29-bearing mice after a single i.v. injection of pHPMA-Gly-C₆-Gly-[³H]CPT, pHPMA-Gly-Phe-Leu-Gly-[³H]CPT (both at 10% CPT content), and [³H]CPT (Fig. 2).

View larger version (64K): [in this window] [in a new window]   Fig 2. Whole body autoradiograms showing the distribution of radioactivity as a function of the time after single i.v. injection (20 mg/kg) of pHPMA-Gly-C6-Gly-[3H]CPT (top), pHPMA-Gly-Phe-Leu-Gly-[3H]CPT (middle), and [3H]CPT (bottom) to mice with HT-29 s.c. xenografts. CPT conjugates were at 10% CPT by weight. These are representative sections obtained from a 2-mice/time study. Plates were exposed for 72 h. In the figures, dark areas represent sites of high radioactivity uptake. The gray to white areas are sites of medium to low levels of radioactivity. Tumor areas are circled. Insets, time at which mice were sacrificed after administration of the drugs.

View larger version (21K): [in this window] [in a new window]   Fig 3. Tissue to muscle radioactivity ratios as measured by radioimaging after a single i.v. administration (20 mg/kg) of pHPMA-Gly-C6-Gly-[3H]CPT (10% CPT content by weight) to mice with HT-29 s.c. xenografts. Data are the mean of three mice/time ± mean error. --, tumor mass; -•-, tumor periphery; -*-, blood; --, intestinal mucosa; --, spleen; --, liver; -x-, kidney; --, bone marrow; --, lung.

WBA only shows [³H]CPT-derived radioactivity, giving a limited understanding of organ uptake and kinetics of the active drug in the case of pHPMA-CPT conjugates. Alternatively, by applying harmonic spectrofluorimetry, we have been able to distinguish free to copolymer-bound CPT at the tumor site in murine specimens. Excitation of the sample by sinusoidally modulated light yielded the distribution of fluorescence phase shifts of CPT, which is proportional to the distribution of fluorescence lifetimes (33, 34) of the free and bound drug. The experiment was based on previous in vitro observations on the fluorescence decay of native CPT and CPT conjugates, as measured in buffer (Fig. 4, A–C). The phase shift and the demodulation of the fluorescence signal with respect to the incident signal were acquired as a function of the modulation frequency in the range 2–200 MHz. These quantities are related to the fluorescence lifetime of the fluorescent species by expressions involving the Fourier transformation of the fluorescence intensity (33, 34). Thereby, a fit of phase shifts and demodulations versus modulation frequency gives the parameters of the fluorescence decay reported in Fig. 4 (associated table). CPT in buffer had a single fluorescence lifetime ranging from the 3.65 ns of the lactone to the 4.03 ns of the carboxylate form (Fig. 4, bottom table). To mimic the noncovalent interactions that CPT engages with carrier plasma proteins (40), we have measured the decays also in the presence of human serum albumin (Fig. 4, bottom table). At equimolar concentrations, CPT had two discrete lifetimes 4.05 ns. In contrast, the main lifetime of both pHPMA-Gly-Phe-Leu-Gly-CPT and pHPMA-Gly-C₆-Gly-CPT clustered above 6 ns, with the presence of a short component of about 2 ns. Although not very large, there was a distinct difference in the fluorescence lifetime of CPT conjugates and free CPT, even when the latter was noncovalently bound to a carrier protein such as albumin.

View larger version (38K): [in this window] [in a new window]   Fig 4. Fluorescence lifetimes analysis of pHPMA-CPT conjugates and native CPT in solution as measured by harmonic spectrofluorimetry. Top, increase of phase (degrees) and decrease of modulation as a function of the modulation frequency from 2 MHz to 200 MHz for CPT (A), pHPMA-Gly-Phe-Leu-Gly-CPT (B), and pHPMA-Gly-C6-Gly-CPT (C) in buffer at pH 7.40. Solid lines are the mathematical fit (residuals are shown above) that yields the decay parameters reported in the bottom table. Notice that the frequency of 76.2 MHz falls around the phase and modulation crossing points for both conjugates and native CPT. This frequency was chosen for phase shift imaging in vivo. Table, parameters of the fluorescence decays, where fi is the fractional intensity of the decay time i; 2 is the goodness of fit. CPT conjugates were at 10% CPT content by weight.

View larger version (55K): [in this window] [in a new window]   Fig 5. Phase-shift fluorescence imaging of HT-29 s.c. tumor sections from mice treated with pHPMA-Gly-Phe-Leu-Gly-CPT, pHPMA-Gly-C6-Gly-CPT, and CPT. These are representative scans obtained from a 2 mice/time study. Phase-shift fluorescence distribution is represented by a pseudocolor scale in which phase-shift values increase from blue to red. In the background, black and white photographs of the tumor sections are reproduced for an easier localization of the scanned areas (red circles). The insets show the time at which mice were sacrificed after single i.v. administration (20 mg/kg) of the drugs. CPT conjugates were at 10% CPT blue areas identify phase-shift values given by the tumor autofluorescence (from 28.0° to 28.5°), measured on nontreated sections. The light-blue to white areas represent values measured after treatment with native CPT (from 29.5° to 30.0°) and, therefore, are attributed to the signal of the free drug in the tumor. After treatment with pHPMA-CPT conjugates, higher phase shifts were measured together with those given by the drug alone, indicating the presence of both free and polymer-bound CPT. Notice that higher phase shifts were persistently measured for up 48 h after treatment with pHPMA-Gly-C6-Gly-CPT, suggesting a slow intratumor hydrolysis of this conjugate.

Finally, we have tested pHPMA-Gly-C₆-Gly-CPT, at 10% CPT content, on mice with orthotopically implanted HT-29 human colon carcinoma (Table 5). Native CPT at the dose of 10 mg/kg/day was highly toxic to these mice. Strikingly different was the effect of pHPMA-Gly-C₆-Gly-CPT, which at 20 mg/kg/day, given i.v. every 4 days for six times, not only induced the regression of the primary tumors but also of distant metastases in liver, spleen, and diaphragm with no detectable signs of toxicity.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
CPT failed clinical development mainly because of the water insolubility of the active lactone form and the toxicity of the carboxylate form, which predominates at physiological pH. Efforts have been spent to search for more soluble and less toxic analogues (reviewed in Ref. 18). Alternatively, HPMA copolymers of CPT are novel water-soluble polymer conjugates, which, whereas stabilizing the lactone form of CPT in plasma (30), take advantage of the well-documented permeability and retention effect of macromolecules in tumors (20, 21, 23, 41). Here we have compared two new conjugates for their antitumor activity against s.c. HT-29 xenografts to the pHPMA-Gly-Phe-Leu-Gly-CPT reported previously at 5% and 10% CPT content (30). The latter were designed using the well-known lysosomal cleavable peptide -Gly-Phe-Leu-Gly- (19, 27), whereas in the new ones CPT was linked through a -Gly-6-aminohexanoyl-Gly- (-Gly-C₆-Gly-CPT) spacer for which we expected higher stability to proteolytic attack and limited hydrolysis at neutral pH.

We have investigated to what extent the retention of HPMA copolymers of CPT in tumors and the passive modulation of CPT release influence efficacy. To this end, we have carried out both intratumor distribution and efficacy studies on a single tumor model, the HT-29 human colon carcinoma, which is known to respond to CPTs.

The antitumor activity profile of conjugates with 5% CPT content was comparable with that of native CPT given by i.v. route, although they were somewhat better tolerated. In contrast, the conjugates with 10% CPT content had a more interesting profile of activity. At the given schedule of six doses every 4 days, pHPMA-Gly-C₆-Gly-CPT was as potent as CPT, whereas pHPMA-Gly-Phe-Leu-Gly-CPT was 2-fold more potent in inhibiting tumor growth (30). In contrast to CPT, for which the onset of toxicity was observed sharply above the active dose, both conjugates at 10% CPT content showed at least 2-fold increase in the active dose range. However, the efficacy of pHPMA-Gly-C₆-Gly-CPT (10% CPT) was by far more impressive on this tumor model. A relevant number of cures were observed with this conjugate but not with pHPMA-Gly-Phe-Leu-Gly-CPT (10% CPT; Ref. 30). The latter was clearly more toxic, as the sharp inversion from activity at 10 mg/kg/day to major toxicity at 12.5 mg/kg/day (5/7 toxic deaths) indicated (30). Conversely, prolonged treatment with pHPMA-Gly-C₆-Gly-CPT (10% CPT) at its maximum tolerated dose of 20 mg/kg/day (eight doses) was still well tolerated, and the delayed treatment (every 7 days) was effective. At variance with the majority of low Mw topoisomerase-I inhibitors, the efficacy of pHPMA-Gly-C₆-Gly-CPT (10% CPT) was, therefore, less dependent on treatment schedule by the i.v. route.

WBA (Fig. 2) has been extensively applied for after the distribution of native CPT in vivo as function of the formulation and the site of administration (39). However, WBA only shows [³H]CPT-derived radioactivity; therefore, it gives a limited understanding of organ uptake and kinetics of the active drug in the case of pHPMA-CPT conjugates. Because the weight-average Mw and polydispersity of the two conjugates were very similar, the different radioactivity levels (Fig. 2; Ref 30) resulted from dissimilar rates of hydrolysis in vivo, as suggested by the in vitro stability to hydrolytic and proteolytic cleavage (Table 1; Ref 30). Yet, WBA of conjugates labeled with [³H]CPT gives no direct information on the distribution of pHPMA-bound/free CPT forms. On the other hand, conjugates radiolabeled at the polymer backbone or spacer could not be of any use for comparing CPT conjugates to native CPT, neither would they yield any information on the in vivo hydrolysis kinetics of the macromolecules. Furthermore, laborious biochemical approaches such as extraction of free and polymer-bound CPT from tumor and organs can suffer from a series of technical difficulties resulting in an improper analysis of the two forms. For these reasons, we have obtained time-resolved fluorescence phase-shift images of CPT in tumor sections (Fig. 5) after observing that CPT conjugates had distinctly longer fluorescence lifetime decay in solution (Fig. 4). The fluorescence excitation and emission maxima of free CPT and CPT conjugates (data not shown) were equally broad bands peaking at 370 nm excitation and 430 nm emission. Although the fluorescence emission of CPT conjugates was 50% less intense, we did not observe any emission shift that could have been used for measuring the bound versus the free drug by more conventional methods. Alternatively, we have observed different fluorescence lifetime decays (Fig. 4). Autoradiography (Figs. 2 and 3) showed that retention of [³H]CPT-derived radioactivity mainly occurred at the most vascularized periphery of the tumor. However, although quantitative, this approach does not allow us to distinguish the radioactivity derived from copolymer bound or free CPT. Alternatively, fluorescence imaging (Fig. 5) detects the distribution of the different forms of the drug, but it does not quantify their relative amounts. In fact, the fluorescence imaging in Fig. 5 is a map of the fluorescence lifetimes in situ, and it is independent on concentration (33, 34). Therefore, any direct comparison between color intensity in Fig. 5 and autoradiograms (Figs. 2 and 3) would be misleading: autoradiography shows how much CPT is in the tumor, in any of its forms, whereas fluorescence imaging detects different signals for the free and the bound drug, just on the base of their different structure. Our data are also not meant to differentiate among extra-, intracellular release, or vascular space, although previous work on cultured cells demonstrated that HPMA copolymers undergo pinocytosis and intracellular release of the active drug by lysosomal degradation of the -Gly-Phe-Leu-Gly- peptide (19, 29). However, fluorescence showed how both forms were present in the tumor mass for at least up to 2 days after injection and that in situ hydrolysis was definitely slower for pHPMA-Gly-C₆-Gly-CPT. The combined WBA and time resolved fluorescence images indicated that both pHPMA-Gly-Phe-Leu-Gly-CPT and pHPMA-Gly-C₆-Gly-CPT were retained in tumor as they had never been observed for native CPT (39). The drug was then released in situ by a sum of hydrolytic and proteolytic mechanisms, following kinetics that agreed with the different hydrolytic properties of the two cleavable spacers. Clearly, tumor accumulation and retention play a role in the efficacy of pHPMA-CPT conjugates in terms of higher tolerability for pHPMA-Gly-C₆-Gly-CPT (10% CPT) or even enhanced potency as for pHPMA-Gly-Phe-Leu-Gly-CPT (10% CPT). Nevertheless, the overall in vivo efficacy is a delicate balance of tumor versus organ exposure to the active (free) CPT. The 2-fold higher potency of pHPMA-Gly-Phe-Leu-Gly-CPT, as it was seen against all of the tested tumors, could be because of higher intratumor exposure to free CPT shortly after administration (Fig. 5) because of the faster hydrolysis of the -Gly-Phe-Leu-Gly- peptide. However, this might result in a less-effective treatment probably because of two main reasons. Firstly, effectiveness of topoisomerase-I inhibitors is improved more by long exposure times than by high drug concentration (18). Secondly, because the uptake pHPMA-CPT was not limited to tumors (Fig. 2), hydrolysis could occur in other organs leading to toxicity. The superior efficacy profile of pHPMA-Gly-C₆-Gly-CPT (10% CPT), also confirmed on the orthotopic tumor model (Table 5), might, in turn, be ascribed to the very slow hydrolysis (Fig. 5) of this conjugate that exposed the tumor to steady but low amounts of CPT. CPT released from the latter conjugate was enough for a cure, yet not relevant for toxicity. This peculiar property of pHPMA-Gly-C₆-Gly-CPT (10% CPT) also allowed delayed treatment schedules (Table 2) that preserved efficacy and contributed to reduce toxicity. Our study strongly suggests that the efficacy of CPT can be remarkably enhanced when long exposure to the drug, rather than higher local concentrations, is achieved locally by conjugation to HPMA copolymers. Delayed release may also reduce the dependence of efficacy on the treatment schedule, as always observed with CPT and analogues. Future studies comparing pHPMA-Gly-C₆-Gly-CPT (10% CPT) to the most common CPT analogues of clinical use, at different treatment schedules, will fully evaluate the spectrum and degree of the antitumor efficacy of this new conjugate and its suitability to clinical development programs.

	Acknowledgments

 
We thank Dr. Theodore Hazlett from the Laboratory for Fluorescence Dynamics and Dr. Beniamino Barbieri from ISS, Inc. for precious support during the fluorescence imaging experiments at the Laboratory for Fluorescence Dynamics.

	Footnotes

 
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 The Laboratory for Fluorescence Dynamics is funded by the NIH, Grant PH55P41RR03155, and by the University of Illinois.

³ Present address: Department of Biology, Limburgs Universitair Centrum, Universitaire Campus, gebouw D B-3590 Diepenbeek, Belgium.

⁴ Present address: Department of Pharmacokinetics and Metabolism, Active Biotech Research AB, Lund, SE 220 07, Sweden.

⁵ The abbreviations used are: CPT: camptothecin; pHPMA, poly[N-(2-hydroxypropyl) methacrylamide]; -Gly-C6-Gly-, Gly-6-aminohexanoyl-Gly-; HSC: human serum complement; GI, gastrointestinal; HPMA: hydroxypropyl methacrylamide; Mw, molecular weight; Mn, molecular number; RTV, relative tumor volume; T/C%, percentage of tumor growth inhibition; WBA, whole body autoradiography.

Received 7/ 8/02; revised 10/31/02; accepted 11/ 6/02.

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