Potentiation of High Linear Energy Transfer Radiation with Paclitaxel for the Treatment of Disseminated Peritoneal Disease Diane E

Cancer Therapy: Preclinical

MultimodalityTherapy: Potentiation of High Linear Energy Transfer Radiation with Paclitaxel for the Treatment of Disseminated Peritoneal Disease Diane E. Milenic,1, 2 Kayhan Garmestani,1Erik D. Brady,1Kwamena E. Baidoo,1Paul S. Albert,3 Karen J.Wong,2 Joseph Flynn,4 and Martin W. Brechbiel1

Abstract Purpose: Studies herein explore paclitaxel enhancement of the therapeutic efficacy of a-particle-targeted radiation therapy. Experimental Design: Athymic mice bearing 3day i.p. LS-174T xenografts were treated with 300 or 600 Ag paclitaxel at 24 h before, concurrently, or 24 h after [213Bi] or [212Pb]trastuzumab. Results: Paclitaxel (300 or 600 Ag) followed 24 h later with [213Bi]trastuzumab (500 ACi) provided no therapeutic enhancement. Paclitaxel (300 Ag) administered concurrently with [213Bi]trastuzumab or [213Bi]HuIgG resulted in median survival of 93and 37days, respectively; no difference was observed with 600 Ag paclitaxel. Mice receiving just [213Bi]trastuzumab or [213Bi]HuIgG or left untreated had a median survival of 31, 21, and 15 days, respectively, 23 days for just either paclitaxel dose alone. Paclitaxel (300 or 600 Ag) given 24 h after [213Bi]trastuzumab increased median survival to 100 and 135 days, respectively. The greatest improvement in median survival (198 days) was obtained with two weekly doses of paclitaxel (600 Ag) followed by [213Bi]trastuzumab. Studies were also conducted investigating paclitaxel administered 24 h before, concurrently, or 24 h after [212Pb]trastuzumab (10 ACi). The 300 Ag paclitaxel 24 h before radioimmunotherapy (RIT) failed to provide benefit, whereas 600 Agextendedthe median survival from 44 to 171days. Conclusions:These results suggest that regimens combining chemotherapeutics and high linear energy transfer (LET) RIT may have tremendous potential in the management and treatment of cancer patients. Dose dependency and administration order appear to be critical factors requiring careful investigation.

In the past year in the United States, it was estimated that percentage points, although that seems imperceptible in the f37,170 people would be diagnosed with pancreatic adeno- 5-year relative survival rates for both diseases. Clearly, carcinoma and f33,370 will die (1). The 5-year relative development of new tactics for the treatment and management survival rate is only 5% for all stages of pancreatic cancer. The of patients with pancreatic or ovarian cancer remains a high outlook for patients with ovarian cancer is not appreciably priority (1). better. In 2007, it was estimated that f22,430 women were Targeted radiation therapy is one such strategy that has been diagnosed with ovarian cancer, 67% to 69% of whom reinvigorated in recent years with the Food and Drug presented with distant, metastatic disease. The overall 5-year Administration approval of Zevalin and Bexxar. In fact, relative survival rate is 40% to 45%. Over the years, there has numerous preclinical and clinical studies have appeared in been a trend toward improvement in this situation by a few the literature that focus on the application of radioimmunotherapy (RIT) as a treatment modality for i.p. disease (2–9). The majority of these studies have used monoclonal antibodies (mAb) conjugated with h-emitting radionuclides (e.g., 131I, 90Y, 1 Authors’ Affiliations: Radioimmune and Inorganic Chemistry Section, Radiation and 177Lu). The strategy followed by this laboratory has been Oncology Branch, and 2Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, NIH; 3Biometric Research Branch, National Cancer to exploit the superb cytotoxicity of a-particle radiation using Institute, Bethesda, Maryland; and 4Hematology and Oncology, College of a mAb as the targeting vehicle of s.c. and i.p. xenografts Medicine, Ohio State University, Columbus, Ohio (6–8, 10). Only three to six transversals of a cell’s nucleus by Received 1/30/08; revised 5/13/08; accepted 5/14/08. a-particles delivers a dose of 70 to 100 cGy; a-particle radiation Grant support: Intramural Research Program of the NIH, National Cancer Institute, is cytotoxic at a dose rate as low as 1 cGy/h (11–13). The Center for Cancer Research. The costs of publication of this article were defrayed in part by the payment of page characteristic short path lengths of a-particles render this charges. This article must therefore be hereby marked advertisement in accordance radiation ideal for the treatment of small tumor burdens, with 18 U.S.C. Section 1734 solely to indicate this fact. disseminated disease, and micrometastatic disease and for the Requests for reprints: Diane E. Milenic, NIH, 10 Center Drive, MSC-1088, elimination of malignant single cells. The short path length is Building 10, Room 1B40, Bethesda, MD 20892. Phone: 301-496-9086; Fax: 301- 402-1923; E-mail: [email protected]. also hypothesized to limit normal tissue toxicity. This F 2008 American Association for Cancer Research. laboratory has recently reported on the therapeutic potential doi:10.1158/1078-0432.CCR-08-0256 of two a-emitting radionuclides, 213Bi and 212Pb (the latter as

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causes BCL2 dysfunction, and activates apoptosis (20–22). Translational Relevance Paclitaxel has activity against ovarian and pancreatic cancer and is a recognized radiosensitizer, which has been reported to These investigations reported herein show the potential sensitize ovarian and pancreatic cancer cells/tumors to the of combining chemotherapeutics with high-LET RIT for the cytotoxic effects of radiation (23–25). The drug has been management and treatment of cancer patients who present evaluated in combination with radiotherapy and radiolabeled with disseminated peritoneal disease at the time of their antibodies in animal models and in patients and has been diagnosis. These studies are a natural progression to prior shown to provide additional therapeutic benefit (9, 26–31). studies that established the efficacy of paclitaxel adminis- The mechanism of action by paclitaxel distinctly differs from tered in conjunction with h-radiation RIT for the treatment that of gemcitabine and as such would add another class of of ovarian patients. Chemotherapy in conjunction with chemotherapeutics to the armamentarium to be combined with a-particle RITusing the appropriate targeting vehicle would radiolabeled antibodies. The studies described herein are an be an effective adjuvant therapy following procedures such evaluation of the ability of paclitaxel to potentiate the as cytoreductive surgery or peritoneal external beam radia- therapeutic efficacy of HER-2 targeting a-emitting high-LET tion therapy. The intent of developing a treatment regimen 213Bi- and 212Pb-labeled trastuzumab in a multimodality using a-targeted radiation is to expand the repertoire to regimen for the management of disseminated i.p. disease. patient populations. Such a strategy would be potentially beneficial for not only those with pancreatic or ovarian cancer but also those with cancers of the colon, stomach, Materials and Methods and small intestine, which result in peritoneal carcinomatosis as well as those with peritoneal mesothelioma. In gener- Cell lines. All therapy studies were conducted using the LS-174T, a al, the results obtained define a potentiating interaction human colon carcinoma cell line. SKOV-3, a human ovarian carcinoma between paclitaxel and the high-LET a-radiation-labeled cell line that expresses f1 106 HER-2 molecules per cell, was used for trastuzumab. The studies also illustrate the necessity of in vitro analysis (32). LS-174Twas grown in supplemented DMEM as establishing the optimal administration sequence of the described previously (33). SKOV-3 cells were maintained in McCoy’s 5a treatment components and that dose dependency and medium supplemented with 10% fetal bovine serum and 1 mmol/L administration order are critical factors that require careful nonessential amino acids. Media and supplements were obtained from investigation. Quality Biologicals, Invitrogen, or Lonza. Chelate synthesis and mAb conjugation. The synthesis, characterization, and purification of the bifunctional ligands, TCMC and CHX-A00- DTPA, have been described previously (34, 35). Conjugation of an in vivo generator of 212Bi), in a peritoneal murine model trastuzumab with either CHX-A00-DTPA or TCMC has been described (6–8). The delivery vector in these studies was trastuzumab previously (34, 35). The final concentration of trastuzumab was 00 targeting HER-2. HER-2 is expressed in an array of epithelial quantified by the method of Lowry (36). The number of CHX-A - tumors: 35% to 45% of all pancreatic adenocarcinomas, 25% to DTPA or TCMC molecules linked to the mAb was determined using 30% of ovarian cancers, and 4% to 83% of colorectal spectrophotometric assays based on the titration of either yttrium or lead-Arsenazo (III) complex, respectively (37, 38). adenocarcinomas as well as other diseases (14–16). A unique Radiolabeling. A 5 to 10 mCi 224Ra/212Pb generator (AlphaMed) advantage of RITversus monotherapy with trastuzumab is that was washed with 2 mol/L HCl (3 mL) to remove impurities, unbound neither high expression nor homogeneous expression of HER-2 224Ra, daughter isotopes, and any damaged resin or organic residue. On throughout the tumor is required to affect therapy. This the following day, 212Pb was eluted from the generator with 1 mol/L laboratory has taken a systematic approach in the development HCl (3 mL). The eluate was heated to dryness; the residue was dissolved 213 212 of treatment regimens using either Bi- or Pb-labeled in 0.1 mol/L HNO3 (2 0.2 mL) and used for radiolabeling of mAb trastuzumab. Trastuzumab with each of the radionuclides was as detailed elsewhere (8). The radiolabeled mAb was purified using effective in increasing the median survival of mice bearing i.p. a desalting column (PD-10; GE Healthcare) with PBS as the eluant. HuIgG (ICN) was similarly conjugated with TCMC and radiolabeled tumor xenografts after a single administration and subsequently 212 in a multidosing scheme. In the management of oncology with Pb as described above to serve as a negative control in these studies. A calibrated Ge(Li) detector (model GEM10185-P; EG&G patients, combinations of radiotherapy and chemotherapy Ortec) coupled to a multichannel analyzer Gamma Vision version are common practice to derive a greater therapeutic response 5.2 software (EG&G Ortec) was used for radioactivity measurements. (14, 17–19). To improve the results, many preclinical and 212Pb activity was determined by measurement of the 238.6 keV g-ray clinical studies have been conducted to identify more effective (43.6%). cytotoxic agents or radiosensitizers and combinations thereof. 213Bi was obtained from a 225Ac/213Bi generator (Oak Ridge National Within this context, studies in this laboratory have been Laboratories or Actinium Pharmaceuticals). Elution of the 213Bi extended to explore whether high linear energy transfer (LET) and radiolabeling of trastuzumab-CHX-A’’ was done as described radiation therapy could be potentiated by incorporating previously (10). chemotherapeutics into the regimen. Gemcitabine was the first Radioimmunoassay. Immunoreactivities of the radiolabeled prepa- chemotherapeutic drug that was evaluated in conjunction rations were assessed in a radioimmunoassay using methanol-fixed cells. Briefly, SKOV-3 cells were trypsinized, pelleted, and resuspended with [212Pb]trastuzumab (7). In the study, it was shown that in PBS (pH 7.2) containing 1% bovine serum albumin. Serial dilutions therapeutic responses were greatly improved in a one- or of radiolabeled trastuzumab (f140 to 8 nCi) were added in duplicate two-cycle regimen. to cells (1 106)in50AL of 1% bovine serum albumin in PBS. The Paclitaxel (Taxol) is another chemotherapeutic under intense cells were washed with 4 mL of 1% bovine serum albumin in PBS scrutiny. Paclitaxel stabilizes microtubule formation, which following a 2 h (213Bi) or 18 h (212Pb) incubation at room temperature, results in cells being arrested in the G2-M phase of the cell cycle, pelleted at 1,000 g for 5 min, and the supernatant was decanted. The

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Progression of disease, especially during the first weeks of the Table 1. Effect of paclitaxel administration experiment, was observed as an extension of the abdomen, develop- 213 schedule on the therapeutic response to Bi RIT ment of ascites, and with noticeable, palpable nodules in the abdomen. with trastuzumab: median survival Weight loss would occur as an acute response following treatment or much later as the experiments progressed. Mice were monitored and RIT Paclitaxel euthanized if found to be in distress, moribund, or cachectic. Mice were 0 Concurrent 24 h after RIT also euthanized when 10% to 20% weight loss occurred or if bloating or tumor nodules were apparent. All animal protocols were approved M M M M 300 g 600 g 300 g 600 g by the National Cancer Institute Animal Care and Use Committee. None 15 (1.0) 23 (1.5) 23 (1.5) Statistical analyses. A Cox proportional hazards model was used to Trastuzumab 31 (2.1) 93 (6.2) 53 (3.5) 100 (6.7) 135 (9.0) test for a dose-response relationship between the dose of [212Pb]tras- HuIgG 21 (1.4) 37 (2.5) 53 (3.5) 45 (3.0) 29 (1.9) tuzumab or [213Bi]trastuzumab and survival (time to sacrifice or natural death). Groups were compared using a log-rank test. A dose-response NOTE: Median survival in days. Mice bearing 3-d tumor burden relationship was examined by treating the dose level as a linear were injected i.p. with 300 or 600 Ag paclitaxel concurrent with or covariate in the Cox model and tested whether the corresponding 24 h after i.p. injection with 500 ACi 213Bi RIT. Therapeutic index, regression variable was zero using a likelihood ratio test. treatment group median survival divided by the untreated group For the animal weight data, the maximum percent reduction from median survival. baseline was estimated for each mouse. This was calculated as the ratio of the maximum reduction in weight from baseline during the initial 4-week period divided by the baseline weight of the mouse. Box 212Pb was detected using the Ge(Li) detector, whereas the 213Bi was plots were constructed for each treatment group that show the median, measured in a g-counter (WizardOne; Perkin-Elmer). The binding upper, and lower quartiles as well as identifying outliers. Differences percentage was calculated for each dilution. The specificity of the between treatment groups were tested using a Kruskal-Walis test radiolabeled trastuzumab was confirmed by incubating one set of cells (nonparametric ANOVA) for comparison of multiple groups and the with f200,000 counts/min of radiolabeled trastuzumab with an excess Wilcoxon rank-sum test was applied when comparing two groups. All (5 Ag) of unlabeled trastuzumab. reported P values correspond to two-sided tests. Therapy studies. Therapy studies were done using 19 to 21 g female athymic (nu/nu) mice (Charles River Laboratories). The mice were injected i.p. with 1 108 cells LS-174Tin 1 mL medium or PBS as Results reported previously (39). Radiolabeled trastuzumab, 500 ACi [213Bi]CHX-A00- trastuzumab, or 10 ACi [212Pb]TCMC-trastuzumab Previous studies from this laboratory showed potentiation of 212 was administered to the mice (n = 8-10) 3 days after implantation of high-LETradiation ([ Pb]trastuzumab) by gemcitabine (7). 213 212 tumor in 0.5 mL PBS. HuIgG labeled with Bi or Pb served as a Induction of G2 arrest via microtubule stabilization by paclitaxel nonspecific control. In all of the experiments outlined below, radiation provides for an alternate strategy for enhancing the therapeutic therapy doses were administered 3 days after tumor implantation. The response to a-particle radiation. The first study exploring the choice of 500 and 10 ACi for [213Bi] and [212Pb]trastuzumab, combination of paclitaxel with high-LETradiation was done respectively, is based on previous studies that determined these with [213Bi]trastuzumab in the LS-174Ti.p. model (6–8). doses were the lower range of the maximum effective therapeutic dose Athymic mice (n = 10) bearing a 2-day tumor burden were (10, 11, 16). When combining a-particle RITwith chemotherapeutics, treated i.p. with either 300 or 600 Ag paclitaxel followed 24 it was considered that alterations in tumor sensitivity to radiation 213 would be more discernible if the lower effective doses of [213Bi] or to 30 h thereafter with i.p. injection of [ Bi]trastuzumab A [212Pb]trastuzumab were administered and that any incipient toxicity (500 Ci). Control groups included mice given no treatment, 213 that might be incurred by doing these studies at higher doses would not treatment with paclitaxel alone, or treatment with [ Bi]HuIgG. obscure observation of enhanced therapy. The decision to administer the paclitaxel before RIT was based In study 1, mice (n = 9-10) with 2 days i.p. LS-174Txenografts on the 45.6 min half-life of the 213Bi. It was reasoned that the were injected (i.p.) with 300 or 600 Ag paclitaxel. On the following 213Bi would no longer be present and most likely not be effective day (24-30 h), mice received 213Bi-labeled trastuzumab or HuIgG if the paclitaxel were given afterwards. The data, detailed as 213 (500 ACi). Additional groups of mice received paclitaxel, [ Bi]trastu- follows, effectively illustrate that this line of reasoning was 213 zumab, or [ Bi]HuIgG or were left untreated. incorrect. Mice injected with the [213Bi]trastuzumab realized a Study 2 involved reevaluating the sequence of administration of 213 median survival of 43 days (data not shown), whereas mice that paclitaxel and Bi RIT. In this experiment, mice (n = 10) received 213 an i.p. injection of paclitaxel (300 or 600 Ag) concurrently or 24 to received the [ Bi]HuIgG had a median survival of 25 days 30 h after the 213Bi RIT. Control groups were the same as outlined in compared with 19 days for the untreated group (P = 0.09). The study 1. median survival for the mice that were injected with the In study 3, multiple doses of paclitaxel (600 Ag/wk for up to 3 weeks) paclitaxel alone was 31 days for the 300 Ag dose and 43 days for were given to mice (n = 8-10) bearing LS-174Ti.p. xenografts following the 600 Ag dose. In those animals given the combination, no a single treatment with either 500 ACi [213Bi]trastuzumab or further improvement in the median survival was observed at [213Bi]HuIgG. Additional groups of mice included those injected with either 300 Ag (42 days) or 600 Ag (46 days) dose of paclitaxel one, two, or three doses of paclitaxel at weekly intervals, with either (P = 0.84). The two groups that were injected with 300 and 213 213 [ Bi]trastuzumab or [ Bi]HuIgG, or no radiation treatment. 600 Ag paclitaxel followed by treatment the next day with Study 4 assessed the combination of paclitaxel with 212Pb RIT. [213Bi]HuIgG had a median survival of 42 versus 25 days for Tumor-bearing mice (n = 10) were injected with paclitaxel (300 or 213 600 Ag) 24 h before, concurrently, or 24 h after treatment with the group that received [ Bi]HuIgG alone. The increase from 10 ACi [212Pb]trastuzumab or [212Pb]HuIgG. These treatment 25 to 42 days observed in these groups was found to be groups were compared with sets of mice that received only statistically insignificant (P = 0.07). paclitaxel, [212Pb]trastuzumab, or [212Pb]HuIgG or those without any Based on these results, the timing of the paclitaxel treatment. administration was reexamined and a study was conducted in

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A study was then designed to investigate the effectiveness of multiple doses of paclitaxel with [213Bi]trastuzumab. Groups of mice were treated with 500 ACi [213Bi]trastuzumab followed by one, two, or three 600 Ag doses of paclitaxel. (illustrated in Fig. 1A–C, respectively). The first dose was given 24 to 30 h after the [213Bi]trastuzumab with subsequent doses being given at 7-day intervals. Additional groups received paclitaxel only, [213Bi]HuIgG only, or no treatment. As shown in Table 2, the median survival of the untreated group was 25 days. There was no increase (P = 0.82) in median survival for those mice receiving one, two, or three doses of paclitaxel only; median survival was 45, 63, and 46 days in these three groups, respectively. Mice injected with [213Bi]trastuzumab experienced a 5-fold improvement over controls with a median survival of 126 days. When one, two, or three doses of paclitaxel (600Ag) were administered after the [213Bi]trastuzumab, the median survivals were 182, 198, or 140 days, respectively. There was an observed but not statistically significant (P = 0.17) shortening of survival following administration of the third dose of paclitaxel. A comparison of the three dose schedules of paclitaxel with [213Bi]trastuzumab was not statistically different (P = 0.17). Once again, this therapeutic response was specific to [213Bi]trastuzumab. The corresponding groups of mice treated with [213Bi]HuIgG and paclitaxel had a median survival of only 36, 38, 43, and 63 days for zero, one, two, or three doses of paclitaxel. The three dose schedules of paclitaxel with HuIgG were not statistically significant (P = 0.13) among themselves. The weights of the mice were recorded over a 4-week period monitoring the potential toxicity of the treatment regimens. Some weight loss was observed among the various groups in Fig. 1. Effect of weekly paclitaxel administration following 213Bi RITon survival. Athymic mice bearing 3d i.p. LS-174T xenografts were injected i.p. with paclitaxel this particular RITstudy (Fig. 2). Thedifferences were (600 Ag) 24 h after receiving 500 ACi (i.p.) 213Bi-labeled trastuzumab or HuIgG significant among the groups that were untreated or injected (A). Groups were then given additional doses of paclitaxel at weekly intervals for 213 213 atotaloftwo(B)orthree(C) doses. (.), untreated; (r), 6 00 Ag paclitaxel; with [ Bi]trastuzumab and [ Bi]HuIgG (P = 0.02) with 213 (n), [213Bi]trastuzumab; ( w ), paclitaxel and [213Bi]trastuzumab; (E), 213HuIgG; those animals treated with [ Bi]HuIgG showing the greatest (o), paclitaxel and 213HuIgG. amount of weight reduction. A lack of difference was also noted for the groups that received paclitaxel only (P = 0.30) or those that received paclitaxel and the [213Bi]trastuzumab (P = 0.93). which mice received the paclitaxel at the time of 213Bi RITor A significant difference was found in the groups that were f24 to 30 h afterwards. The resulting data presented in Table 1 injected with [213Bi]HuIgG, with toxicity decreasing with were encouraging. The median survival of the group injected number of weekly doses (P = 0.05). with [213Bi]trastuzumab was 31 days, a 2-fold increase The potential of combining [212Pb]RITwith paclitaxel was compared with 15 days for the untreated mice. When 300 Ag also investigated. A therapy study was again conducted in paclitaxel was administered concurrently with [213Bi]trastuzumab, median survival increased to 93 days, which translates to a therapeutic index of 6.2. The response was specific to Table 2. Effect of weekly paclitaxel administration trastuzumab in that the median survival of mice receiving following a single injection of [213Bi]trastuzumab: A 213 300 g paclitaxel at the time of injection with [ Bi]HuIgG was median survival 37 days. The benefit and specificity appeared to be negated when the paclitaxel dosage was increased to 600 Ag. The RIT No. paclitaxel treatments 213 median survival of the mice injected with either Bi-labeled 01 2 3 trastuzumab or HuIgG was 53 days. Therapeutic efficacy of combining paclitaxel with 213Bi RITbecame evident when the None 25(1.0) 45(1.8) 63 (2.5) 46 (1.8) A Trastuzumab 126 (5.0) 182 (7.3) 198 (7.9) 140 (5.6) paclitaxel, at either 300 or 600 g, was administered the day HuIgG 36 (1.4) 38 (1.5) 43 (1.7) 63 (2.5) after 213Bi-labeled trastuzumab. In this administration scenario, 213 median survival of mice injected with [ Bi]trastuzumab or NOTE: Median survival in days. Mice bearing 3-d tumor burden 213 [ Bi]HuIgG and 300 Ag paclitaxel was 100 or 45 days, were injected i.p. with 300 or 600 Ag paclitaxel. Twenty-four hours respectively. The benefit was even greater when 600 Ag later, they were injected i.p. with 500 ACi 213Bi RIT. The indicated paclitaxel was injected after [213Bi]trastuzumab. A therapeutic groups then received an additional injection of 600 Ag paclitaxel at index of 9.0 (135 days) was obtained, whereas only a 1.9 7-d intervals for a total of one, two, or three doses administered to the mice. Therapeutic index, treatment group median survival therapeutic index was observed with the combination of divided by the untreated group median survival. [213Bi]HuIgG followed by 600 Ag paclitaxel (P = 0.05).

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therapeutic efficacy of [212Pb]trastuzumab becomes evident in the group that received 600 Ag paclitaxel 24 h before the RIT. Under these conditions, there is a 3.9-fold increase in the median survival compared with the group that received [212Pb]trastuzumab alone resulting in an increase from 44 to 171 days. This response appears to be specific to the [212Pb]trastuzumab because the median survival of the corresponding group receiving the [212Pb]HuIgG remained at 44 days. There is an increase in median survival (86 days) of the mice that were given the paclitaxel the day following the [212Pb]trastuzumab, which translates to a 1.9-fold increase over the [212Pb]trastuzumab alone, whereas no effect is observed when the chemotherapeutic is given at the same time as the [212Pb]trastuzumab. A box plot showing the distribution of maximum percent reduction in weight over follow-up across the 17 groups is given in Fig. 3. Toxicity was low in 13 of the groups. Changes in Fig. 2. Effect of multiple paclitaxel administrations combined with 213Bi RIT on weight were observed in those groups that were injected with 212 animal weight.The maximum percent reduction in weight was calculated for each of paclitaxel, at either 300 or 600 Ag dose, 24 h after the Pb RIT the treatment groups and presented as box plots. Light line, median; top region, was administered. A comparison of paclitaxel after [212Pb]tras- third quartile; bottom region, first quartile; brackets, 1.5 times the interquartile range; lines outside of the brackets, outlying observations. Alterations in the weights tuzumab with trastuzumab alone showed a statistically of the mice during and 4 wk following treatment were monitored as an indicator significant increase in toxicity with paclitaxel at the 300 Ag of toxicity. dose (P = 0.05). There is a statistical trend of an increase for the 600 Ag dose (P = 0.10). Similar differences for HuIgG were also noted with a statistically significant increase in toxicity at the which paclitaxel was administered at two dose levels (300 and 600 Ag paclitaxel 24 h after [212Pb]HuIgG compared with 600 Ag) 24 h before, concurrently, or 24 h after the injection of HuIgG alone (P = 0.03). [212Pb]trastuzumab or [212Pb]HuIgG. Additional groups of 212 mice were treated with each of the Pb-labeled antibodies Discussion alone, at each of the paclitaxel doses alone, and one group was left untreated. Consistent with prior studies (Table 3), a modest Food and Drug Administration approval of ‘‘naked’’ mAbs but significant therapeutic effect due to the paclitaxel alone was such as trastuzumab (Herceptin), cetuximab (Erbitux), and observed. Median survival of the untreated group was 16 days, panitumumab (Vectibix) and of the radiolabeled mAbs, Zevalin whereas for those receiving either 300 or 600 Ag paclitaxel this and Bexxar, are ample evidence of the burgeoning interest and was extended to 31 and 29 days, respectively (P = 0.007). The ongoing efforts to incorporate mAbs into cancer patient 300 Ag dose of paclitaxel appears to only have a modest affect treatments. The strategy of molecular targeting, in conjunction on median survival irrespective of the timing of the adminis- with the realization that treatments will likely be tailored to tration of the [212Pb]trastuzumab. The median survival of the individual patients, provides the impetus for development and mice injected with [212Pb]trastuzumab is 44 days, whereas it is refinement of mAb-based therapies. 49, 53, or 51 days when paclitaxel (300 Ag) is injected 24 h The efficacy of targeted a-particle radiation has been shown before, concurrently, or 24 h after the [212Pb]trastuzumab. At with increasing frequency in both preclinical and clinical this dose level of paclitaxel, there is a 2-fold increase in the settings (2, 5–8). As hypothesized, targeted a-particle radiation median survival of animals that are injected with the chemo- is effective in the treatment of low disease burden, espe- therapeutic 24 h before [212Pb]HuIgG. An enhancement of the cially when administered locoregionally or when applied for

Table 3. Effect of paclitaxel administration schedule on the therapeutic response to 212Pb RIT with trastuzumab: median survival

RIT Paclitaxel treatment 0 24 h before RIT Concurrent 24 h after RIT 300 Mg 600 Mg 300 Mg 600 Mg 300 Mg 600 Mg None 16 (1.0) 31 (1.9) 29 (1.8) Trastuzumab 44 (2.8) 49 (3.1) 171 (10.7) 53 (3.3) 51 (3.2) 51 (3.2) 86 (5.4) HuIgG 22 (1.4) 44 (2.8) 44 (2.8) 25(1.6) 44 (2.8) 29 (1.8) 29 (1.8)

NOTE: Median survival in days. Mice bearing 3-d tumor burden were injected i.p. with 300 or 600 Ag paclitaxel either 24 h before RIT, concurrent, or24hafterRITwith10ACi 212Pb RIT. Therapeutic index, treatment group median survival divided by the untreated group median survival.

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The 500 and 10 ACi doses of [213Bi] and [212Pb]trastuzumab used in these experiments had been determined to be the lower range of the maximum effective therapeutic dose for each radioimmunoconjugate (6–8, 14). The decision to use these doses in subsequent experiments was based on the reasoning that the lower effective dose ranges were less likely to mask alterations in sensitivity conferred by other modalities such as chemotherapeutics during combination therapy. The first study evaluating paclitaxel with targeted a-particle was conducted with trastuzumab radiolabeled with 213Bi. Based on the 46 min half-life of the radionuclide, administration of paclitaxel to the mice was executed 24 h before administration of [213Bi]trastuzumab. Paclitaxel has been shown to persist in the peritoneal cavity (t1/2 =73F 18 h), which would allow time for radiosensitization to occur (44). In this situation, the assumption was incorrect. No improvement in therapeutic efficacy of [213Bi]trastuzumab was observed in this scenario with this radionuclide. A subsequent experiment evaluated the effect of administering paclitaxel, at two dose levels, concurrently with [213Bi]trastuzumab as well as on the day after [213Bi]trastuzumab. Consistent with the literature, the greatest therapeutic response was obtained when 600 Ag paclitaxel was given f24 h after RIT, with a therapeutic index of 9 being realized. It should be noted that a therapeutic index of 6.2 was also obtained for the group of animals that were treated with 300 Ag paclitaxel given with the [213Bi]trastuzumab. Fig. 3. Effect of paclitaxel administration schedule with 212Pb RITon animal weight as an indicator of toxicity. Athymic mice bearing i.p. LS-174T xenografts were At the time of designing this first study, there were two injected i.p. with paclitaxel (600 Ag) either 24 h before, concurrently, or 24 h after areas of experience to draw upon in the literature. One was receiving 10 ACi (i.p.) 212Pb-labeled trastuzumab or HuIgG. Data presentation is as described for Fig. 2. the combination of paclitaxel with conventional external beam radiotherapy. Paclitaxel had been shown to enhance in vivo the radiation response of murine mammary carcinoma treatment of single-cell disease. There remains, however, a great to radiotherapy when given up to 24 h before radiotherapy deal to improve upon with this therapeutic mode. (45). The combination of paclitaxel and radiotherapy resulted As with other modalities, expectations with RITare that this in an increase in mitotically arrested tumor cells and with a would not be a stand-alone treatment but would become corresponding increase in apoptosis. In vitro RITstudies by incorporated as an element of a multimodality treatment Supiot et al. showed that paclitaxel added to three different scheme. Chemotherapeutics (e.g., gemcitabine, paclitaxel, multiple myeloma cell lines 24 h before 213Bi RITusing mAb cisplatin, 5-fluoruracil, and doxorubicin) in combination with B-B4 resulted in a decrease in survival in all three cell lines RITare under evaluation (7, 9, 22, 26–29, 40–42). This (22). When examined further, the investigators found that laboratory has recently reported on the potentiation of high-LET only one of the cell lines responded to the combined radiation using gemcitabine in combination with [212Pb]tras- treatment with a modest increase in apoptosis. In these same tuzumab targeting HER-2 (7, 8). Paclitaxel as the next studies, the potential of doxorubicin was also evaluated for its chemotherapeutic for investigation was an attractive candidate ability to sensitize cells to targeted a-particle radiation. In this based on several factors including established usage and case, doxorubicin not only inhibited cell growth but also was radiosensitization properties. The studies reported here were found to increase the amount of DNA strand breaks and designed to explore conditions for the i.p. administration of frequency of apoptosis. The first RIT study combining paclitaxel with a-particle RITwith the objective of developing a paclitaxel with 90Y-ChL6 therapy from DeNardo et al. showed multicycle treatment scheme for both 213Bi and 212Pb RIT. potentiation of therapy by paclitaxel (29). Scheduling of In the design of the experiments, it was felt that i.p. paclitaxel with the RITwas examined and was found to affect administration of the paclitaxel would result a higher local a response rate of 100% when started 24 h before RIT. The concentration of the paclitaxel being achieved. The adsorption greatest number of cures, however, was obtained when rate of paclitaxel from the peritoneum into the peripheral paclitaxel was administered 24 h after the 90Y-ChL6 (29). circulation is slow, which in turn prolongs the exposure of Clinical trials have also been conducted in which the tumor to the drug and minimizes systemic exposure and feasibility of i.p. radioimmunochemotherapy was evaluated. resultant toxicities (43, 44). Paclitaxel given by i.p. administra- Patients with ovarian carcinoma were treated with paclitaxel tion, particularly in combination with RIT, is not unprecedent- (i.p.) 2 days before receiving an i.p. injection of 177Lu-CC49 ed. Ovarian cancer patients tolerated 177Lu RITin combination or 90Y-CC49 (9). It should be noted that all of these studies with paclitaxel and IFN with only a transient neutropenia noted were conducted using h – emitting radionuclides, 90Y, 177Lu, 2 to 3 weeks after the paclitaxel administration (9). Similar and 131I, which have half-lives that are appreciably longer findings were observed in ovarian cancer patients receiving (2.7, 6.7, and 8 days, respectively) than that of 213Bi (46 min) paclitaxel, IFN, and 90Y-CC49 (9). or 212Pb (10.2 h). One also must note that the h – emitters are

www.aacrjournals.org 5113 Clin Cancer Res 2008;14(16) August 15, 2008 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2008 American Association for Cancer Research. Cancer Therapy: Preclinical low-LETradiation as opposed to the high-LETradiation RITwith a chemotherapeutic. Paclitaxel was chosen due to its associated with a-emitters. (a) reported radiosensitization properties, (b) reported syner- Because therapeutic efficacy was observed with paclitaxel gism with trastuzumab, and (c) proven effectiveness in treating administered concurrently with the [213Bi]trastuzumab, the pancreatic cancer patients (6, 47, 48). Interestingly, the LS-174T experiment combining paclitaxel and [212Pb]trastuzumab xenografts used in these studies are modestly responsive to evaluated the effects of paclitaxel given before, concomitantly, paclitaxel with increases in the therapeutic index ranging from and after [212Pb]trastuzumab. Consistent with studies from 1.5 to 2.5, comparable with the therapeutic responses observed other laboratories, an enhancement of therapeutic efficacy was for the radiolabeled HuIgG combined with paclitaxel. In stark observed when tumor-bearing mice were treated with 600 Ag contrast, potentiation of [213Bi] and [212Pb]trastuzumab was paclitaxel 24 h after administration of [212Pb]trastuzumab realized, with therapeutic indices reaching values as high as 9. (26–29, 40, 46). In fact, an increase in the median survival of To understand how paclitaxel enhances the therapeutic the mice in this group was observed compared with those that response to RIT, a study was conducted by Miers et al. to received only [212Pb]trastuzumab (86 versus 44 days, respec- answer whether there is an effect on the cumulative activity in tively). However, the therapeutic efficacy of [212Pb]trastuzumab the tumor. The authors compared patients receiving 90Y- RIT was even more impressive when paclitaxel was given 24 h before (m170) with (n = 5) and without (n = 7) paclitaxel (49). When the [212Pb]trastuzumab. The median survival realized was 171 patients were given paclitaxel 2 days after the RIT, an increase in days, a nearly 4-fold increase. This is actually in accordance the cumulated tumor activity was observed. No differences were with the literature (26, 29). In the original study, a 100% observed in normal tissue. Similar results were obtained when response rate and responses that included partial and complete mice bearing breast cancer xenografts were injected with 111In- regression with cures were reported when paclitaxel was given DOTA-Gly3-Phe-ChL6 and then treated with paclitaxel 24 h before the RIT(29). Paclitaxel (600 Ag) given 24 h after RIT later (49). Similar studies are planned to test whether the same generated the highest number of cures (29). Clearly, empirical phenomenon occurs with the LS-174Ti.p. xenograft model studies evaluating the scheduling of a chemotherapeutic with following paclitaxel therapy. RITis warranted. As shown by the studies described here, the These investigations suggest that regimens combining sequence of administration is directly dependent on the chemotherapeutics and high-LETRITmay have tremendous radionuclide being targeted. One probably should not be potential in the management and treatment of pancreatic and surprised then to discover that the administration sequence ovarian cancer patients who present with peritoneal disease at might also be dependent on the targeting vehicle and the target the time of diagnosis. This treatment regimen may also prove as well. beneficial to other patient populations with cancers, such as With the objective of developing a multicycle chemo-RIT the colon, stomach, and small intestine, which result in treatment regimen, studies were done assessing a single peritoneal carcinomatosis as well as those with peritoneal injection of [213Bi]trastuzumab with paclitaxel being given up mesothelioma (50). to three times at 1-week intervals. Paclitaxel was found to Chemotherapy in conjunction with a-particle RITusing potentiate the therapeutic effectiveness of [213Bi]trastuzumab the appropriate targeting vehicle would be an effective with the greatest benefit being derived from two doses of adjuvant therapy following procedures such as cytoreductive paclitaxel given after the RIT. To date, one other study has surgery or peritoneal external beam radiation therapy. reported results of administering multiple doses of paclitaxel Although some of the observed effects are difficult to explain with RIT(28). In this particular study, 300 Ag paclitaxel was and warrant further study, on the whole, the general results administered 48 and 72 h after 90Y RITwith two different mAb, obtained point in the direction of a potentiating interaction one targeting carcinoembyronic antigen (T84.66) and the other between paclitaxel and the high-LET a-radiation-labeled targeting HER-2 (trastuzumab). The fractionated paclitaxel dose trastuzumab. The studies also illustrate the necessity of estab- was found to be effective in inhibiting tumor growth; however, lishing the optimal administration sequence of the treatment the investigators did not include a direct comparison with a components and that dose dependency and administration single dose of paclitaxel, nor did they include a nonspecific order are critical factors that require careful investigation. mAb making it challenging to objectively deconvolve their results versus the therapeutic components. Disclosure of Potential Conflicts of Interest The overall objective of these studies was to investigate the conditions for a treatment regimen that incorporated a-particle No potential conflicts of interest were disclosed.

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Diane E. Milenic, Kayhan Garmestani, Erik D. Brady, et al.

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