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From: Cancer Drug Discovery and Development:
Combination Cancer Therapy: Modulators and Potentiators Edited by: G. K. Schwartz © Humana Press Inc., Totowa, NJ
10
INTRODUCTION
Cancer cells survive and thrive because they are very similar to normal cells expressing growth and invasion “programs” that many cell types recognize and respond to in “programmed” patterns (1). As the understanding of cancer has increased, the complexity of the molecular events that comprise malignant dis- ease has become evident (2). Interactions involved in intertwining signaling networks including membrane receptors, enzymes along with activators, deac- tivators and regulators, protein–protein interactions, protein–nucleic acid inter- actions, and small-molecule effectors in multiple cell types are all recognized targets for therapeutic attack. Agents are targeted to specific abnormalities in the sequence and expression of genes/proteins that operate in a stepwise, combina- torial manner to permit the malignant disease to progress (3). Cell growth, motility, differentiation, and death are regulated by signals received from the environment (4). Signals may come from interactions with other cells or compo- nents of the extracellular matrix, or from binding of soluble signaling molecules to specific receptors at the cell membrane, thereby initiating different signaling pathways inside of the cell. Cancer may be visualized as a permanent perturba-
Use of Animal Models to Evaluate Signal Transduction Inhibitors
As Modulators of Cytotoxic Therapy
Beverly A. Teicher,
PhDCONTENTS
INTRODUCTION
RECEPTOR TYROSINE KINASES
PROTEIN SERINE-THREONINE KINASE INHIBITORS
INHIBITORS OF CELL-CYCLE SIGNAL TRANSDUCTION
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tion of signaling pathways. Over the last 10 yr, numerous compounds have been rationally targeted toward components in signaling pathways shown to be abnor- mal in cancer (5). Some of these rationally targeted agents have progressed through clinical trials, and some are now approved clinical treatment agents.
Genomics and proteomics technologies have been applied to many cancers to identify markers of the disease and to elucidate the intracellular and intercellular signaling pathways that support the malignant disease (6–14).
Cancer cure requires eradication of all malignant cells. Cancer growth, how- ever, requires proliferation of malignant cells and normal cells. The several anticancer treatment modalities currently available, including surgery, chemo- therapy, radiation therapy, and immunotherapy, have been envisioned to target primarily the malignant cells. Research over the past 35 yr has reinforced the hypothesis put forth by Folkman that, without the proliferation of normal cells, especially endothelial cells, a tumor cannot grow beyond the size of a colony (15). The consequence of this finding is that both the normal cells and the malignant cells involved in tumor growth, as well as the chemical and mechani- cal signaling pathways that interconnect them, are valid targets for therapeutic intervention. The integration of therapeutics directed toward the vascular com- ponents, extracellular matrix components, and stromal and infiltrating cells, with classical cytotoxic anticancer therapies, may be regarded as a systems approach to cancer treatment (16).
Although new noncytotoxic agents directed toward normal cells and extracel- lular enzymatic activities target processes critical to tumor growth, it is highly unlikely that treatment with these new agents alone will lead to tumor cure. The question arises of how to integrate these new therapeutic agents into existing cancer treatment regimens. Thus, by choosing multiple cellular and process targets for therapeutic attack, a systems approach to anticancer therapy regimen development may lead to the cure of systemic malignant disease (17).
RECEPTOR TYROSINE KINASES
In complex multicellular organisms, intercellular signal transduction networks mediating growth, differentiation, migration, and death are regulated, in part, by polypeptide growth factors that activate cell-surface receptors acting in either an autocrine or paracrine manner (18). Receptor tyrosine kinases (RTKs) are key mediators of many normal cellular processes and are often deregulated in human cancers. Several signaling pathways controlled by tyrosine kinases have been selected as important targets for anticancer therapeutic intervention.
The epidermal growth factor receptor (EGFR) autocrine pathway contributes to several processes important to the deregulated invasion growth that is the hallmark of malignant disease (19). The family of ligands for EGFR include epidermal growth factor, transforming growth factor (TGF)-_, amphiregulin,
heparin-binding epidermal growth factor, and betacellulin. TGF-_ is a key modu- lator in cell proliferation in both normal and malignant cells. The receptor family includes EGFR (ErbB-1), HER-2/neu (ErbB-2), HER-3 (ErbB-3), and HER-4 (ErbB-4) (20). The TGF-_-EGFR autocrine pathway is activated in cancer cells by mechanisms ranging from overexpression of EGFR and increased concentra- tion of ligand(s) to decreased phosphatase activity, decreased receptor turnover, or the presence of mutant receptors such as EGRFvIII, which lacks domains I and II of the extracellular domain and cannot bind ligand but has a constitutively activated kinase domain (21,22). EGFR is being targeted both by monoclonal antibodies (MAbs) to prevent ligand binding and small-molecule inhibitors of the tyrosine kinase enzymatic activity to inhibit auto-phosphorylation and down- stream intracellular signaling.
Mendelsohn first proposed the blockade of EGFR via a specific MAb as a cancer therapy in the 1980s (23,24). Mendelsohn’s group isolated a mouse MAb to EGFR designated MAb 225. The murine MAb 225 antibody was shown to have antitumor activity against human A431 epidermoid carcinoma and human MDA-MB-468 breast carcinoma grown as xenografts in nude mice, in combina- tion with doxorubicin or cisplatin (25,26). For administration to patients, the MAb 225 antibody was humanized and designated C225 (IMC-C225). The hu- manized antibody C225 has been studied in combination with gemcitabine, topotecan, paclitaxel, and radiation therapy in several human tumor xenograft models (27–30). The combination of C225 with cytotoxic therapies has demon- strated greater antitumor activity than the cytotoxic therapy alone. In the fast- growing GEO human colon carcinoma, C225 (10 mg/kg, intraperitoneally [ip], twice weekly for 5 wk) produced a tumor growth delay of 24 d; topotecan (2 mg/
kg, ip, twice weekly for 5 wk), a camptothecin analog, produced a tumor growth delay of 14 d; and the combination regimen produced a tumor growth delay of 86 d (Fig. 1) (27). At least part of the activity of C225 can be attributed to antiangiogenic activity (31,32). Bruns et al. (28) implanted L3.6pl human pan- creatic carcinoma cells into the pancreas of nude mice and beginning on d 7 after tumor cell implantation began treatment with C225 (40 mg/kg, ip, twice weekly for 4 wk), gemcitabine (250 mg/kg, ip, twice weekly for 4 wk) or the combina- tion. The animals were sacrificed on d 32 just at completion of the treatment regimens. Gemcitabine appeared to be most effective against the liver and lymph- node metastasis, and C225 appeared to be most effective against the primary disease. The combination regimen appeared to be more effective than either treatment alone. Combination treatment regimens including C225 with radiation therapy appeared to produce at least additive tumor growth delay in two head and neck squamous carcinoma xenograft models (30). C225 has undergone three consecutive phase I clinical trials, a phase Ib clinical trial, several single-agent and combination phase II trials, and is currently in phase III clinical trial (23,32).
Among the several small-molecule ATP-binding-site competitive inhibitors of EGFR kinase activity currently in development, ZD1839 (Iressa) has pro-
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gressed the furthest toward clinical approval. ZD1839 is [4-(3-chloro-4- fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline] (MW 447) and is an orally active antitumor agent in mice at daily doses of 12.5–200.0 mg/
kg (33). ZD1839 has been studied in combination with cisplatin, carboplatin, oxaliplatin, paclitaxel, docetaxel, doxorubicin, etoposide, ralitrexed, and radia- tion therapy in human tumor xenograft models (34–39). It has been shown that the contribution of ZD1839 to anticancer activity of combination treatment regi- mens is due, at least in part, to its activity as an antiangiogenic agent (37,40).
When nude mice bearing the fast-growing human GEO colon carcinoma were treated with ZD1839 daily for 5 d per wk for 4 wk by intraperitoneal injection for doses of 50, 100, or 200 mg/kg, tumor growth delays of 4, 6, and 18 d, respec- Fig. 1. Antitumor activity of topotecan and MAb C225 on established GEO human colon carcinoma xenografts. Mice were injected subcutaneously in the dorsal flank with 107 human GEO colon carcinoma cells. After 7 d (average tumor size, 0.2 cm3), mice were treated ip with topotecan alone (2 mg/kg/dose, twice weekly on d 1 and 2 of each wk for 2 wk) or with MAb C225 alone (0.25 mg/dose, twice weekly on d 3 and 6 of each wk for 5 wk) or with both drugs with the same sequential schedule. Each group consisted of 10 mice. The experiment was repeated three times. Data represent the average on a total of 30 mice for each group; bars, standard deviation. Student’s t test was used to compare tumor sizes among different treatment groups at d 29 after tumor-cell implantation. MAb C225 vs control (p < 0.001); topotecan vs control (p < 0.001); topotecan followed by MAb C225 vs control (p < 0.001); topotecan followed by MAb C225 vs MAb C225 (p < 0.001);
topotecan followed by MAb C225 vs topotecan (p < 0.001).
tively, resulted (34). The 100 mg/kg dose of ZD1839 was selected for combina- tion studies. Using the GEO colon xenograft tumor model, Ciardiello et al. (34) found that ZD1839 administered daily by intraperitoneal injection for 5 d per week for 4 wk produced 6 to 10 d for tumor growth delay, whereas standard regimens for paclitaxel (20 mg/kg), topotecan (2 mg/kg), and tomudex (12.5 mg/
kg) resulted in 9, 7, and 10 d of tumor growth delay. The combination treatment regimens of ZD1839 with each cytotoxic agent resulted in 33, 27, and 25 d of tumor growth delay, respectively. Sirotnak et al. (35) administered ZD1839 (150 mg/kg) orally daily for 5 d for 2 wk to nude mice bearing A431 human vulvar epidermoid carcinoma, A549, SK-LC-16, or LX-1 human non-small-cell lung carcinomas, or PC-3 or TSU-PR1 human prostate carcinomas as a single agent or along with cisplatin, carboplatin, paclitaxel, docetaxel, doxorubicin, edatexate, gemcitabine, or vinorelbine. ZD1839 was a positive addition to all of the treat- ment combinations except gemcitabine, where it did not alter the antitumor activity compared with gemcitabine alone, and vinorelbine, where the combina- tion regimen was toxic. For example, in the LX-1 non-small-cell lung carcinoma xenograft, ZD1839 (150 mg/kg, po) produced a tumor growth delay of 8 d, paclitaxel (25 mg/kg, ip) produced a tumor growth delay of 16 d, and the com- bination treatment regimens resulted in a tumor growth delay of 26 d. Working in the human GEO colon carcinoma, Ciardiello et al. (37) found that ZD1839 (150 mg/kg, ip, daily for 5 d per week for 3 wk) was a more powerful antiangiogenic therapy than paclitaxel (20 mg/kg, ip, once per week for 3 wk) and that the combination treatment regimen was most effective. Given these results, it is unlikely that ZD1839 would be a highly effective single agent in the clinic, but it could be a useful component in combination treatment reigmens. Expand- ing on these studies, Tortora et al. (41) examined combinations of an antisense oligonucleotide targeting protein kinase A, a taxane, and ZD1839 in the fast- growing human GEO colon carcinoma xenograft. The tumor growth delays were 8 d with the taxane IDN5109 (60 mg/kg, po), 20 d with ZD1839 (150 mg/kg, po), 23 d with the antisense AS-PKAI (10 mg/kg, po), and 61 d with the three-agent combination treatment regimen. Recently, Naruse et al. (42) found that a subline of human K562 leukemia made resistant to the phorbol ester TPA and designated K562/TPA was more sensitive to ZD1839 administered intravenously or subcu- taneously to nude mice bearing subcutaneously implanted tumors than was the parental K562 line. The mechanism by which the K562/TPA line multidrug resistance occurs is unknown. The K562/TPA line does not express GP170 P- glycoportein or MDR-1 protein. ZD1839 has been evaluated in five phase I clinical trials including 254 patients, and it appears that response to ZD1839 does not correspond to EGFR expression (43). A pilot clinical study of 24 non-small- cell lung cancer patients showed that ZD1839 could be combined with caboplatin/
paclitaxel. A phase I study of 26 colorectal cancer patients showed that ZD1839 could be combined with 5-fluorouracil and leucovorin safely (44). Two large
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multicenter phase III clinical trials of ZD1839 (250 or 500 mg daily) in combi- nation with carboplatin/paclitaxel or cisplatin/gemcitabine as first-line treat- ment in nonoperable stage III and stage IV non-small-cell lung cancer patients are underway (37,43,45). Other small-molecule inhibitors of EGFR that are progressing through development are OSI-774, PD183805/CI-1033, PKI-1033, PKI166, and GW2016 (46,47).
The deregulated tyrosine kinase activity of the BCR-ABL fusion protein has been established as the causative molecular event in chronic myelogenous leu- kemia. The BCR-ABL has proven to be an ideal protein receptor kinase for pharmacological inhibition. SRI571 (Gleevec; Glivec; CGP57148B), a phenyla- mino-pyrimidine derivative, is a potent inhibitor of the Abl tyrosine kinase that is present on the malignant cells in 95% of patients with chronic myelogenous leukemia (CML). The compound selectively inhibits proliferation of v-Abl and Bcr-Abl expressing cells and has antitumor activity as a single agent in animal models at well-tolerated doses (48–57). Unlike many other tyrosine kinase in- hibitors that are cytostatic, STI571 is cytotoxic toward chronic myeloid leuke- mia-derived cell lines, as demonstrated in colony-formation assays using the surviving fraction endpoint (58). In cell culture, STI571 adds to the cytotoxicity of other cytotoxic agents such as etoposide in cells that express the BCR-ABL mutation (58,59). In cell-culture studies modeling combinations that may be used for bone-marrow pretransplantation conditioning regimens, using the BV173 and EM-3 BCR-ABL-positive cell lines with an MTT growth inhibition endpoint, Topaly et al. (60) found that STI571 produced greater-than-additive growth inhibition in combination with radiation therapy and produced additive to less-than-additive growth inhibition with busulfan and treosulfan by the com- bination index method. Mice reconstituted with P210(BCR/ABL)-transduced bone-marrow cells succumb to a rapidly fatal leukemia (61). When these animals were treated with STI571, survival time was markedly increased. In contrast to the polyclonal leukemia in control mice, STI571-treated mice develop a CML- like leukemia that is generally oligoclonal, suggesting that STI571 eliminated or severely suppressed certain leukemic clones. None of the STI571-treated mice were cured of the CML-like myeloproliferative disorder, and STI571-treated murine CML transplanted with high efficiency to fresh recipient animals.
Progression of chronic myelogenous leukemia to acute leukemia (blast crisis) in humans has been associated with acquisition of secondary chromosomal trans- locations frequently resulting in the NUP98/HOXA9 fusion protein. Dash et al.
(62) developed a murine model expressing BCR/ABL and NUP98/HOXA9 to cause blast crisis. The phenotype depends upon expression of both mutant pro- teins, and the tumor retains sensitivity to STI571. Despite the success of STI571, resistance can develop to this agent in the clinic (63,64).
It has been recognized for some time that STI571 is not a specific inhibitor of BCR/ABL and is, indeed, a potent inhibitor of other tyrosine kinases, including
the receptor tyrosine kinase KIT and the platelet-derived growth factor receptor (PDGFR). These other tyrosine kinases are involved in many malignant diseases.
About 90% of malignant gastrointestinal stromal tumors (GISTs) have a muta- tion in c-kit leading to KIT receptor autophosphorylation and ligand-indepen- dent activation. Initial clinical studies have found that about 50% of GISTs respond to STI571 (65–72). PDGFR is expressed in several other human cancers and is also expressed by tumor endothelial cells, thus, enabling STI571 as an antiangiogenic agent for a wide variety of malignant diseases.
Receptor tyrosine kinases have also been a focus for drug development in the area of antiangiogenic therapy (73–76). The therapeutically revolutionary con- cept of therapy directed toward the process of angiogenesis has been to focus the therapeutic attack of cancer away from the malignant cell and toward a “normal”
cell, one of several types of stromal cell, the endothelial cell (1). The validation for this concept is the recognition that cancer is a disease process directed by the malignant cells but critically requiring the active involvement of a variety of normal cells to enable tumor growth, invasion, and metastasis (77–86). Numer- ous receptor tyrosine kinases have been identified that are directly or indirectly involved in angiogenesis, including VEGFR2 (Flk-1, KDR), VEGFR1 (Flt-1), PDGFR, bFGFRs, Tie-1, and Tie-2 (87). SU5416 has been under development as a selective inhibitor of VEGFR2 (Flk-1, KDR) kinase activity, and SU6668 is under development as a more broad-spectrum receptor kinase inhibitor block- ing VEGFR2, bFGFRs, and PDGFR kinase activity. Early in vivo work with SU5416 suffered from the use of DMSO as a vehicle for the compound, admin- istered by intraperitoneal injection to mice once daily beginning 1 d after tumor- cell implantation (88). Using the DMSO vehicle, tumor growth delays of 0.5, 3, 6, 8, and 13 d were obtained in the human A375 melanoma xenograft with daily doses of SU5416 of 1, 3, 6, 12.5, and 25 mg/kg, respectively. The murine CT-26 colon carcinoma was used to assess the effect of SU5416 and SU6668 on the growth of liver metastases (89). For the study, 104 CT-26 cell were implanted beneath the capsule for the spleens of male Balb/c mice. Four days later, treat- ment with SU5416 or SU6668 began. SU5416 (12 mg/kg) was administered in 99% PEG-300/1% Tween-80, and SU668 (60 mg/kg) was administered in 30%
PEG-300/phosphate-buffered saline (pH 8.2). The compounds were injected once daily until the end of the experiment on d 22 after tumor cell implantation.
The mean number of liver nodules was decreased to about 9 with SU5416 treat- ment, and about 8 with SU6668 treatment, from about 19 in the control animals.
SU5416 has a plasma half-life of 30 min in mice (90). The activity of SU5416, despite the short circulating half-life, led to cell-culture studies, which indicated that exposure to 5 μM SU5416 for 3 h inhibited the proliferation for HUVEC for 72 h. These studies also showed that SU5416 accumulated intracellularly. Geng et al. (91) found that SU5416 increased the sensitivity of the murine B16 mela- noma and the murine GL261 glioma to radiation therapy. When the GL261
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glioma was grown subcutaneously in C57BL mice, administration of SU5416 (30 mg/kg, ip, twice weekly for 2 wk) produced a tumor-growth delay of 4.5 d.
Fractionated radiation therapy (3 Gy × 8) resulted in 8.5 d of tumor-growth delay.
The combination regimen involving SU5416 administration along with and after completion of the radiation resulted in 16 d of tumor-growth delay. SU5416 and SU6668 have been tested as single agents and in combination with fractionated radiation therapy in C3H mice bearing SCC VII squamous carcinomas (92–94).
SU5416 (25 mg/kg, daily for 5 d) or SU6668 (75 mg/kg, daily for 5 d) were administered before or after radiation (2 Gy daily for 5 d). The tumor growth delay with SU5416 was 2 d, which increased to 6.5 d when combined with radiation therapy. The tumor-growth delay with SU6668 was 3.3 d, which in- creased to 11.9 d when combined with radiation therapy. Administration of the compounds before or after radiation delivery did not affect the tumor response.
SU5416 and SU6668 are undergoing clinical trial (69–72,95).
Like STI571, SU5416 and SU6668 have been found to inhibit c-kit (KIT), the stem-cell factor receptor tyrosine kinase (96–98). C-kit is essential for the devel- opment of normal hematopoietic cells and has been proposed to play a functional role in acute myeloid leukemia (AML). Among the six indoline-family tyrosine kinase inhibitors tested, including SU5416, SU6668, and SU6597, SU5416 was the most potent growth inhibitor of a series of human small-cell lung cancer cells expressing c-kit. Mesters et al. (98) reported a 4-mo response in a patient with acute myeloid leukemia after treatment with SU5416. SU5416 and similar agents may also be useful for the treatment of von Hippel–Lindau syndrome patients (99). While SU5416 and similar agents appear to be quite tolerable as single agents, SU5416 was difficult to administer in combination with cisplatin and gemcitabine owing to the incidence of thromboembolic events (100–103). Other small-molecule tyrosine kinase inhibitors showing promise in early clinical trial include PTK787/ZK222584 and ZD6474. PTK787/ZK222584 has shown activ- ity in several solid-tumor models (104–108). When the RENCA murine renal- cell carcinoma was grown in the subrenal capsule of Balb/c mice, the animals developed a primary tumor and metastases to the lung and to the abdominal lymph nodes. Daily oral treatment with PTK787/ZK222584 (50 mg/kg) resulted in a significant decrease of 61 to 67% in primary tumors after 14 and 21 d, respectively. The occurrence of lung metastases was reuced 98% and 78% on d 14 and 21, respectively; and lymph-node metastases appeared only on d 21 (Fig.
2) (105). A similar cohort of animals were treated with TNP-470, with treatment administered alternate days at 30 mg/kg, subcutaneously. While similar tumor response was seen, the compound was toxic, and therapy was discontinued early.
The major alternative therapeutic methodology being developed to inhibit the vascular endothelial growth factor (VEGF) signaling pathway is anti-VEGF neutralizing MAbs (109,110). Bevacizumab, an anti-VEGF antibody, is show- ing promise in clinical trial (111).
PROTEIN SERINE-THREONINE KINASE INHIBITORS Protein kinase C isoforms are centrally involved in signaling transduction path- ways related to regulation of the cell cycle, apoptosis, angiogenesis, differentiation, invasiveness, senescence, and drug efflux (112–114). Protein kinase C is a gene family consisting of at least 12 isoforms (115–117). Based on differing substrate Fig. 2. (A) Effect of PTK787/ZK 222584 on tumor volume and number of metastases in murine renal cell carcinoma. PTK787/ZK 222584 was administered daily at 50 mg/kg orally. Therapy was initiated 1 d after inoculation of RENCA cells into the subcapsular space of the left kidney of syngeneic BALB/c mice. Animals were sacrificed after either 14 (n = 12) or 21 (n = 20) d, and primary tumor volume, number of lung metastases, and number of visible lymph nodes were assessed. (B) Effects of TNP-470 on tumor volume and number of metastases. BALB/c mice were sacrificed 14 (n = 10) or 21 (n = 10) d after inoculation of RENCA with TNP-470 (30 mg/kg administered sc every other day) was initiated 1 d after inoculation of RENCA cells. The control group received vehicle only.
In the group that was sacrificed after 21 d, TNP-470 treatment had to be discontinued in all animals on d 13 because of strong side effects, such as weight loss > 20%, and ataxia.
Values are means; bars, SE. Ps were calculated by comparing means of the treated group and means of the control group using the Mann Whitney t test. *, significant.
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specificity, activator requirements, and subcellular compartmentalization, it is hypothesized that activation of individual protein kinase C isoforms preferentially elicits specific cellular responses (116,117). Treatment of adrenal cortex endothe- lial cells with VEGF resulted in protein kinase C activation and elevated endothelial nitric oxide synthase (eNOS) expression. Inhibition of protein kinase C with isoform-specific inhibitors abolished VEGF-induced eNOS upregulation (118).
When protein kinase C pathways were activated in human glioblastoma U973 cells by phorbol 12-myristate 13-acetate (PMA), VEGF mRNA expression was upregulated via a posttranscriptional mRNA stabilization mechanism (119). PMA increased VEGF mRNA half-life from 0.8 to 3.6 h, which was blocked by protein kinase C inhibitors (staurosporine or calphostin C) but not by protein kinase A or cyclic nucleotide-dependent protein kinase inhibitors. Recent results provide evi- dence for the involvement of protein kinase C in the invasiveness of breast cancer cells through regulation of urokinase plasminogen activator (120,121). In cervical cancer cells in culture, protein kinase C inhibitors or high concentrations of a phorbol ester decreased telomerase activity in the cells (122). Several studies have associated specific isoforms of protein kinase C with metabolic pathways in pros- tate cancer cells (123–126). Protein kinase C ¡ may be involved in the induction of P-glycoprotein-mediated drug resistance in LNCaP human prostate carcinoma cells. Protein kinase C 6 may be involved in activating anticancer drug-induced apoptosis signaling by amplifying the ceramide-mediated death pathway. Protein kinase C c may be involved in the proliferation and responsiveness to apoptotic signals in murine TRAMP prostate cancer cells in culture. Activation of protein kinase C by exposure of LNCaP prostate carcinoma cells to a phorbol ester in- creased the secretion of prostatic acid phosphatase by the cells in culture. Protein kinase C has also been identified as an interesting therapeutic target for the treat- ment of malignant gliomas (127,128). Inhibitors of protein kinase C have been shown to decrease proliferation and invasion in preclinical glioma models.
The most clear-cut, direct-acting, most frequently found angiogenic factor in cancer patients is VEGF (77,128). The signal transduction pathways of the KDR/
Flk-1 and Flt-1 receptors include tyrosine phosphorylation, activation of PLC, diacylglycerol generation, and PI-3 kinase, with downstream activation of pro- tein kinase C and activation of the MAP kinase pathway (130–133) or, possibly, by translocation of protein kinase C into the cell nucleus (134,135).
To assess the contribution of protein kinase C activation to VEGF signal transduction leading to neovascularization and enhanced vascular permeability, the effects of a protein kinase C` selective inhibitor that blocks the protein phosphorylation activity of conventional and novel protein kinase C isoforms via an interaction at the ATP binding site was studied (136–140). At concentrations predicted to selectively inhibit protein kinase C completely, the compound abrogated VEGF-stimulated growth of bovine aortic endothelial cells (136).
Oral administration of the inhibitor decreased neovascularization in an ischemia-
dependent model of in vivo retinal angiogenesis and blocked increases in retinal vascular permeability stimulated by the intravitreal instillation of VEGF (137–
139). Administration of LY333531 to animals bearing BNL-HCC hepatocellular carcinoma xenografts transfected with the VEGF gene under tetracycline control markedly decreased tumor growth subcutaneously and orthotopically, and decreased VEGF overexpression in the tumors (140). This protein kinase C`- selective inhibitor has also demonstrated antitumor activity alone and in combi- nation with standard cancer therapies in the murine Lewis lung carcinoma and in several human tumor xenografts (141).
The National Cancer Institute 60-cell line identified the protein kinase C inhibitor UCN-01, 7-hydroxy-staurosporine, as of interest. UCN-01 has under- gone phase I clinical trial (142–144). UCN-01 has been shown to inhibit the in vitro and in vivo growth of many types of tumor cells including breast, lung and colon cancer (145–147). However, the growth inhibitory properties of UCN-01 may correlate more closely with its ability to block the activity of cell-cycle progression by inhibition of chk1 rather than with inhibition of protein kinase C (148–153).
Kruger et al. (154) showed that the protein kinase C inhibitor UCN-01, at concen- trations lower than those necessary to inhibit cancer cell growth, inhibited prolif- eration of human endothelial cells in vitro, prevented microvessel outgrowth from explant cultures of rat aortic rings, and abrogated hypoxia-mediated transactivation of hypoxia-inducible factor (HIF-1)-responsive promoters.
Based upon this background, a search was made for the optimal protein kinase C` inhibitor for application in oncology. The compound LY317615 is a potent and selective inhibitor of protein kinase C`. When compared in a panel of kinases with the well-known kinase inhibitor staurosporine, the compound LY317615 demon- strated marked selectivity for inhibition of the protein kinase C` isoforms vs other protein kinase C isoforms as well as protein kinase A, calcium calmodulin kinase, casein kinase src-tyrosine kinase, and rat brain protein kinase C (Table 1) (155).
In monolayer culture, human umbilical vein endothelial cells (HUVEC) in basal medium were stimulated to proliferate by exposure to human VEGF (20 ng/
mL). When various concentrations of LY317615 were added to the cultures for 72 h, the proliferation of the VEGF-stimulated HUVEC was profoundly inhib- ited by 600 nM of the compound (Fig. 3). The IC50 for the assay was 150 nM of LY317615. In a similar experiment, when human SW2 small-cell lung carci- noma cells were exposed to various concentrations of LY317615 for 72 h, a potency differential in the effect of the compound on the malignant cells vs the HUVEC was apparent. The IC50 for LY317615 in the SW2 cells was 3.5 μM; thus there was a 23-fold selectivity in the concentration of LY317165 for the growth inhibition of HUVEC compared to human SW-2 small-cell lung carcinoma cells in culture (155).
The cornea is normally an avascular tissue. Surgical implantation of a small filter disc impregnated with vascular endothelial growth factor (VEGF) into the
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Table 1 Protein Kinase C Isozyme and Other Kinase IC5 Values (μM) for LY317615 and Staurosporine PKC isozymeCaCaseinsrc-rat-brain Compound_`I`IIa6¡cdPKAcalmoulinkinaseTKPKC LY3176150.80.030.03210.380.4>10010>100>1001 Staurosporine0.0450.0230.0190.110.0280.018>1.50.0050.10.004140.0010.19
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cornea of a rat will result in robust neoangiogenesis that is quantifiable in 7 to 10 d. Administration of LY317615 orally twice per day on d 1 through 10 after surgical implant of VEGF-impregnated filters resulted in markedly decreased vascular growth in the cornea of Fisher 344 female rats. A dose of 10 mg/kg of LY317615 decreased vascular growth to about one-half that of the VEGF-stimu- lated controls, whereas a dose of 30 mg/kg of LY317615 decreased vascular growth to the level of the unstimulated surgical control (Fig. 4) (155). Basic fibroblast growth factor (bFGF) is another major angiogenic factor in tumors and is known to be a substrate for phosphorylation by protein kinase C. Surgical implantation of a small filter disc impregnated with bFGF into the cornea of Fisher 344 female rats resulted in robust neoangiogenesis that was quantifiable in 7 to 10 d. Administration of LY317615 (30 mg/kg) orally twice per day on d Fig. 3. Concentration-dependent growth inhibition of human umbilical vein endothelial cells and human SW2 small-cell lung carcinoma cells after 72 h exposure to various concentrations of LY317615 as determined by WST-1 assay. Points are the means of three determinations; bars are SEM.
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Fig. 4. Photographic image of VEGF-induced corneal neovascularization in a rat eye taken at 10 d postimplantation of the stimulus after no treatment or after treatment of the animals with LY317615 (10 or 30 mg/kg) orally twice daily for 10 d. Images are represen- tative of each treatment group.
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1 through 10 after surgical implantation of bFGF resulted in decreased vascular growth to a level of 26% of that of the bFGF control.
Nude mice bearing human SW2 small-cell lung carcinoma growing as a sub- cutaneous xenograft on the thigh of the animals were treated with LY317615 orally twice daily on d 14 through 30 after tumor cell implantation. On d 31, tumors were collected, preserved in 10% phosphate-buffered formalin, and 5- mm-thick sections were immunohistochemically stained for expression of endothelial specific markers, either factor VIII or CD31. The number of intratumoral vessels in the samples was quantified by counting stained regions in 10 high-power microscope fields (×200). There was a LY317615 dose-depen- dent decrease in the number of countable intratumoral vessels in the human SW2 xenograft tumors. The number of intratumoral vessels stained by factor VIII was decreased to one-half that of the controls in animals treated with LY317615 (30 mg/kg), and the number of vessels stained by CD31 was decreased to one-quarter that of the controls in animals treated with LY317615 (30 mg/kg) (Fig. 5) (155).
The plasma levels of VEGF in mice bearing the human SW2 SCLC, HCT116 colon, and Caki-1 renal-cell carcinomas treated or untreated with LY317615 were measured by the Luminex assay (156). For studies using the SW2 human SCLC, plasma samples were obtained every 3 d starting on d 7 after implantation and carried through treatment, as well as after the termination of treatment. Three individual mice were obtained from the control group and the treatment group at each time point. Plasma VEGF levels were undetectable in both treatment groups in SW2 tumor-bearing mice until d 17, when tumor volumes were near 600 mm3 and plasma VEGF levels reached 20 pg/mL (Fig. 6). Plasma VEGF levels were similar between the treated and untreated groups through d 20, when plasma VEGF levels reached 75 pg/mL. Plasma VEGF levels in the SW2 control group continued to increase throughout the study, reaching values of 400 pg/mL on d 40 after implantation. Beginning on d 23, 9 d after beginning therapy, plasma VEGF levels in the LY317615-treated SW2-bearing animals were similar through the duration of therapy. Upon termination of treatment, plasma VEGF levels slightly increased to 100 ng/mL, which were still significantly decreased compared to the untreated control group. Plasma VEGF in Caki-1-bearing mice was not detectable in the untreated animals until the tumor volume reached near 500 mm3 on d 27 (35 pg/mL), 5 d after beginning treatment. At this time, plasma VEGF levels in the LY317615 treatment group were already decreased com- pared to control values. The VEGF levels in the control Caki-1 group continued to increase through the study and peaked at 225 pg/mL on d 49 after tumor implantation. In the treatment group, the plasma levels remained suppressed compared to those of controls throughout the treatment period (d 21–39). The plasma VEGF levels, reaching a maximum of 37 pg/mL, remained suppressed out to d 53, which was 14 d after terminating treatment. Plasma VEGF levels in HCT116-bearing animals were not detectable until d 21 after tumor implantation
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(10 pg/mL), when tumor volumes averaged 900 mm3. The highest plasma VEGF levels in the untreated control group reached 50 pg/mL, and remained at this level for the duration of the study. However, treatment with LY317615 did not decrease plasma VEGF levels in HCT116-bearing animals (156). In patients, serum VEGF levels have also been found to change during therapy and to be related to treat- ment response (157–160).
A sequential treatment regimen was used to examine the efficacy of LY317615 in the SW2 small-cell lung cancer xenograft. The compound LY317615 was effective in this tumor model. Administration of LY317615 alone on d 14 through 30 after tumor implantation over a dosage range from 3 to 30 mg/kg produced tumor-growth delays between 7.4 and 9.7 d (Fig. 7). The SW2 tumor is quite responsive to paclitaxel, and treatment with that drug alone produced a 25-d tumor-growth delay. Treatment with paclitaxel followed by LY317615 (30 mg/
kg) resulted in over 60 d of tumor-growth delay, a 2.5-fold increase in the dura- tion of tumor response. The SW2 small-cell lung cancer was less responsive to carboplatin, which produced a tumor-growth delay of 4.5 d. Sequential treatment with carboplatin followed by LY317615 resulted in 13.1 d of tumor-growth delay (155).
The antitumor activity of LY317615 alone and in combination with cytotoxic antitumor agents has been explored in several human tumor xenografts growing subcutaneously in nude mice (155,156,161–164). While in most of the tumor models, the tumor-growth delay produced by treatment with LY317615 as a single agent was not sufficient to predict single-agent activity in the clinic, in combination regimens LY317615 was a useful addition to the therapeutic regi- men. Unexpectedly, administration of LY317615 to animals bearing human tumor xenografts that secrete measurable VEGF into circulating blood, mark- edly decreased the VEGF in the plasma of the animals (156). LY317615 is currently in phase I clinical trial (165).
TNP-470, a synthetic derivative of fumagillin, an antibiotic that has little antibacterial or antifungal activity, but does have marked amebicidal activity (166), is a potent inhibitor of endothelial cell migration (167), endothelial cell proliferation (168), and capillary tube formation (169). TNP-470 also inhibits angiogenesis as demonstrated in chick CAM, the rabbit and rodent cornea (169).
TNP-470 has been shown to inhibit the growth of primary and metastatic murine tumors, as well as human tumor xenografts (170–179). When administered to animals bearing the Lewis lung carcinoma, subcutaneously on alternate days beginning on d 4 after tumor implantation and continuing until day 18 after tumor implantation, TNP-470 was a moderately effective modulator of the cytotoxic therapies (Table 2). TNP-470 was most effective with melphalan, BCNU, and radiation, increasing the tumor-growth delay produced by these treatments 1.8- to 2.4-fold. TNP-470, along with minocycline, administered intraperitoneally daily on d 4 through d 18, comprised a highly effective antiangiogenic agent
Fig. 5. Countable intratumoral vessels in human SW2 small-cell lung carcinoma xenograft tumors after treatment of the tumor-bearing animals with LY317615 (3, 10, or 30 mg/kg) orally twice per day on d 14 through 30 after tumor implantation. Tumors were immunohistochemically stained for Factor VIII or CD31. Intratumoral vessels were counted manually. Data are the means of 10 determinations; bars are SEM.
combination. The increases in tumor-growth delay produced by the modulator combination TNP-470/minocycline, along with the cytotoxic therapies, ranged from twofold to fourfold. In the treatment group receiving TNP-470/minocycline and cyclophosphamide, approx 40% of the animals were long-term (>120 d) survivors. Each of the cytotoxic therapies (including radiation, which was deliv- ered locally to the tumor-bearing limb) produced a reduction in the number of lung metastases found on d 20 (Table 3). Neither TNP-470/minocycline nor the combination of antiangiogenic agents altered the number of lung metastases or the percentage of large (vascularized) lung metastases on d 20 after tumor im- plantation. The modulators did not alter the number of lung metastases from
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Fig. 6. Plasma VEGF levels in nude mice bearing human SW2 SCLC, Caki-1 renal cell carcinoma, or HCT116 colon carcinoma xenograft tumors, either untreated controls or treated with LY317615 orally twice daily, d 14–30 (21–39 for Caki-1-bearing mice). The data represent the average results for three trials, with each point being the average of nine individual tumors. Bars represent SEM. Asterisk indicates statistically significant differences (p < 0.05).
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those obtained with the cytotoxic therapies, except in the case of cyclophospha- mide, in which many animals treated with the drug and antiangiogenic agent combination had very few metastases on d 20, and most of those were very small (180–188).
The efficacy of the modulator combination of TNP-470/minocycline against the primary Lewis lung tumor is compared with that of other potential antiangiogenic modulator combinations in Table 4. The most effective combina- tion with cisplatin was 14(SO4)`-cyclodextrin/tetrahydrocortisone/minocycline;
with the other cytotoxic therapies, the three antiangiogenic agent combinations, along with cyclophosphamide, were highly effective therapies, resulting in 40–
50% long-term survivors. None of the antiangiogenic agent combinations alone was effective against metastatic disease, although in each case the percentage of large metastases on d 20 was reduced (Table 5). There was a trend toward the combination of 14(SO4)`-cyclodextrin/tetrahydrocortisone/minocycline being Fig. 7. Growth delay of the human SW2 small-cell lung carcinoma after treatment with LY317615 (3, 10, or 30 mg/kg) orally twice per day on d 14 through 30 alone or along with paclitaxel (24 mg/kg, intravenously) on d 7, 9, 11, 13, or carboplatin (50 mg/kg, intraperitoneally) on d 7. Points are the means of five animals; bars are SEM.
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Table 2
Growth Delay of Lewis Lung Tumor Produced by Various Anticancer Treatments Alone or in Combination With Potential Antiangiogenic Modulators
Tumor growth delay, daysa
Treatment group Alone +Minocyclineb +TNP-470 +TNP-470-MINO
— — 1.2 ± 0.4 2.1 ± 0.4 1.8 ± 0.4
CDDP (10 mg/kg) 4.5 ± 0.3 5.1 ± 0.3 6.0 ± 0.5 10.9 ± 0.8 Cyclophosphamide 21.5 ± 1.7 32.4 ± 1.8 25.3 ± 2.2 44.8 ± 2.8c
(3× 150 mg/kg)
Melphalan (10 mg/kg) 2.7 ± 0.3 4.3 ± 0.3 6.0 ± 0.5 8.5 ± 0.6 BCNU (3 × 15 mg/kg 3.6 ± 0.4 5.2 ± 0.4 6.3 ± 0.5 14.6 ± 1.0 X-rays (5 × 3 Gy 4.4 ± 0.3 7.8 ± 0.6 10.6 ± 1.1 15.3 ± 1.2
aTumor-growth delay is the difference in days for treated tumors to reach 500 mm3, compared with untreated control tumors. Untreated control tumors reach 500 mm3 in about 14 d. Mean ± SE of 15 animals.
bMinocycline (10 mg/kg) was administered ip daily on d 4–18. TNP-470 (32Pøg/kg) was administered subcutaneously on alternate days for eight injections, beginning on d 4. CDDP and melphalan were administered intraperitoneally (ip) on d 7. Cyclophosphamide and BCNU were administered ip on d 7, 9, and 11. X-rays were delivered daily on d 7–11 locally to the tumor- bearing limb.
cFive of 12 long-term survivors (>180 d).
the most effective antiangiogenic agents along with cytotoxic therapies against metastatic disease (180–182).
Given the relatively modest impact of established combination chemotherapy regimens on survival in advanced non-small-cell lung cancer, the development of new treatments for this very common malignancy is imperative. Among the newer chemotherapeutic agents, the taxane paclitaxel has demonstrated signifi- cant activity against metastatic non-small-cell lung cancer as a single agent, with much improved median survival (189,190). Since platinum-based therapeutic combinations have been historically important in the treatment of non-small-cell lung cancer, several phase II studies were conducted combining administration of paclitaxel and carboplatin (191–195). These phase II studies produced prom- ising results, showing that the combination of paclitaxel and carboplatin is an active and generally well tolerated regimen for non-small-cell lung cancer. This two-drug regimen produced response rates between 30 and 50%, and prolonged median survival >1 yr. Paclitaxel/carboplatin is not curative in advanced non- small-cell lung cancer, and complete responses are rare. Paclitaxel administered by intravenous injection on d 7 through 11 after tumor-cell implantation pro- duced 4.6 d of tumor-growth delay, which was increased 1.4-fold to 6.4 d of tumor-growth delay when administered along with TNP-470 and minocycline (Table 6) (196). A single intraperitoneal injection of carboplatin on d 7 after
tumor-cell implantation produced a tumor-growth delay of 4.2 d. When carboplatin was administered along with TNP-470 and minocycline, a tumor- growth delay of 7.8 d resulted, a 1.9-fold increase compared with carboplatin alone. The combination of the cytotoxic anticancer drugs paclitaxel and carboplatin was well tolerated by the animals and produced a tumor-growth delay of 6.6 d. The complete regimen, including TNP-470 and minocycline along with paclitaxel and carboplatin, produced a tumor-growth delay of 10.5 d, a 1.6- fold increase compared with the cytotoxic drug combination alone. Treatment with the antiangiogenic agent combination decreased the number of lung me- tastases on d 20 after Lewis lung tumor implantation to 68% of the number found in untreated control animals (Table 6) (196). Both of the cytotoxic chemothera- peutic agents also decreased the number of lung metastases on d 20. Paclitaxel administration decreased the number of lung metastases to 55% of the control number, which was not significantly altered by the addition of co-administration of TNP-470/minocycline. Treatment with carboplatin decreased the number of lung metastases to 63% of the number in the untreated control animals. Addition of TNP-470/minocycline administration to treatment with carboplatin did not significantly alter the number of lung metastases compared with carboplatin alone. The combination of the cytotoxic drugs reduced the number of lung metastases to 33% of the number in the control animals. With the addition of treatment with TNP-470/minocycline to the combination of cytotoxic anticancer drugs, the number of lung metastases was reduced to 20% of the number in the untreated control animals.
The antiangiogenic combination of TNP-470 and minocycline administered for 2 wk did not alter the growth of the Lewis lung carcinoma, the EMT-6 mammary carcinoma, the 9L gliosarcoma, or the FsaII fibrosarcoma (181–188).
Table 3
Number of Lung Metastases on Day 20 From Subcutaneous Lewis Lung Tumors, After Various Anticancer Therapies Alone or in Combination With Potential Antiangiogenic Modulators
Mean number of lung metastases (% large)
Treatment group Alone +Minocycline +TNP-470 +TNP-470-MINO
— 20 (62) 20 (50) 21 (51) 18 (54)
CDDP (10 mg/kg) 13 (58) 11 (48) 14.5 (34) 14 (50)
Cyclophosphamide
(3× 150 mg/kg) 12 (40) 6 (33) 6 (30) 2 (25) Melphalan (10 mg/kg) 13 (48) 11 (50) 15 (47) 15 (45) BCNU (3 × 15 mg/kg) 16 (53) 15 (38) 15.5 (45) 13 (38)
X-rays (5 × 3 Gy) 15 (40) 13 (30) 10 (40) 12 (42)
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However, when TNP-470 and minocycline were added to treatment with cyto- toxic anticancer therapies, tumor response was markedly increased. When C3H mice bearing the FSaIIC fibrosarcoma were treated with TNP-470/minocycline for 5 d prior to intravenous injection of the fluorescent dye Hoechst 33342, there was a shift toward greater brightness of the entire tumor-cell population, so that the 10% brightest and the 20% dimmest cell subpopulations in the control tumor were fivefold dimmer than the same populations in the TNP-470-treated tumors (Fig. 8) (181,183). The TNP-470/minocycline treated tumors were more easily
Table 4
Growth Delay of Lewis Lung Tumor Produced by Various Anticancer Treatments Alone or in Combination With Potential Antiangiogenic Modulators
Tumor growth delay, days
+ 14 (SO4)bCD- + 14 (SO4)bCD- Treatment group Alone THC-MINO THC-TNP-470 + MINO/TNP-470
— — 1.2 ± 0.4 1.5 ± 0.3 1.8 ± 0.4
CDDP (10 mg/kg) 4.5 ± 0.3 26.2 ± 2.5 10.6 ± 0.7 10.9 ± 0.8 Cyclophosphamide 21.5 ± 1.7 48.8 ± 3.3 49.2 ± 3.4 44.8 ± 2.8
(3× 150 mg/kg) (5/12)a (6/12)a (5/12)a
Melphalan (10 mg/kg) 2.7 ± 0.3 10.5 ± 0.9 12.2 ± 1.4 8.5 ± 0.6 BCNU (3 × 15 mg/kg 3.6 ± 0.4 9.8 ± 0.8 10.6 ± 1.1 14.6 ± 1.0 X-rays (5 × 3 Gy) 4.4 ± 0.3 12.6 ± 1.2 10.3 ± 0.9 15.3 ± 1.2
aLived a normal lifespan (approx 2 yr).
Table 5
Number of Lung Metastases on Day 20 From Subcutaneous Lewis Lung Tumors After Various Anticancer Therapies Alone or in Combination With Potential Antiangiogenic Modulators
Mean number of lung metastases (% large)
+ 14 (SO4)bCD- + 14 (SO4)bCD- Treatment group Alone THC-MINO THC-TNP-470 +MINO-TNP-470
— 20 (62) 17 (46) 18 (50) 18 (54)
CDDP (10 mg/kg) 13 (58) 8 (42) 15 (40) 14 (50)
Cyclophosphamide 12 (40) 1 (0) 2 (50) 2 (25)
(3× 150 mg/kg)
Melphalan (10 mg/kg) 13 (48) 7 (50) 15 (40) 15 (45) BCNU (3 × 15 mg/kg) 16 (53) 14 (45) 14 (43) 13 (58)
X-rays (5 × 3 Gy) 15 (40) 9 (43) 11 (36) 12 (42)
penetrated by the lipophilic dye. This was the first indication that TNP-470 and minocycline treatment might allow greater distribution of small molecules into tumors. To determine whether the TNP-470/minocycline affected cyclophos- phamide tissue distribution, animals were injected intraperitoneally with [14C]- cyclophosphamide on d 8; they were killed 6 h later and tissue levels of 14C were determined. There was an increased level of 14C in all of the tissues from TNP- 470/minocycline treated animals except blood, compared with levels in [14C]- cyclophsophamide only treated animals. The largest increases were 2.6-fold in the tumor, 2.3-fold in the kidney, 3.2-fold in the heart, 5.6-fold in the gut, and 7.9- fold in skeletal muscle (181,183).
In a similar study, Lewis lung tumor-bearing mice pretreated with TNP-470/
minocycline, or untreated, were injected intraperitoneally with a single dose of cisplatin on d 8, then killed 6 h later, and tissue levels of platinum were deter- mined. There were increased levels of platinum in all of the tissues taken from animals treated with TNP-470/minocycline (except blood) compared with ani-
Table 6
Growth Delay of Lewis Lung Carcinamo, and Number of Lung Metastases on Day 20 After Treatment of the Animals With Paclitaxel and/or Carboplatin With or Without Antiangiogenic Agents
Treatment group Tumor growth delaya (d) Number of lung metastases
Controls — 40 ± 7
TNP-470 (30 mg/kg) 1.0 ± 0.3 27 ± 5
sc, alt. d 4–18
+ minocycline (10 mg/kg) ip, d 4–18
Paclitaxel (36 mg/kg) 4.6 ± 0.3 22 ± 4
iv d 7–11
TNP-mino-paclitaxel 6.4 ±0.4b 20± 4
Carboplatin (50 mg/kg) 4.2 ± 0.3 25 ± 4
ip, d 7
TNP-mino-carboplatin 7.8 ± 0.5c 21± 3
Paclitaxel/Carboplatin 6.6 ± 0.4 13 ± 2
TNP/MINO/Paclitaxel/ 10.5 ± 0.6b 8 ± 1
Carboplatin
sc, subcutaneously; ip, intraperitoneally; iv, intravenously.
aTumor growth delay is the difference in days for treated tumors to reach 500 mm3, compared with untreated control tumors. Untreated control tumors reach 500 mm3 in about 12.4 ± 0.3 d. Mean
± SE of 15 animals.
bSignificantly increased tumor growth delay, compared with the cytotoxic therapy alone, p <
0.01.
cp < 0.005.
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mals that were treated with cisplatin only. The largest increases were 5.2-fold in the tumor, 3.8-fold in the gut, 3.0-fold in the skin, and 2.5-fold in the skeletal muscle (181,183).
Both cyclophosphamide and cisplatin are cytotoxic through the formation of crosslinks in cellular DNA. DNA alkaline elution from tumors treated in vivo showed that there was increasing DNA crosslinking with increasing dose of cyclophosphamide (Table 7). Treatment with cyclophosphamide alone resulted in a crosslinking factor of 4.7; treatment with the same dose of cyclophospha- mide in animals pretreated with TNP-470/minocycline resulted in a crosslinking factor of 6.2, which extrapolates to an equivalency of about 650 mg/kg of cyclo- phosphamide. Increased DNA crosslinking also was detected with an increasing dose of cisplatin. Treatment with cisplatin alone resulted in a crosslinking factor of 2.0; treatment with the same dose of cisplatin in animals pretreated with TNP- 470/minocuycline resulted in a crosslink (181,183).
[14C]paclitaxel was administered to Lewis lung carcinoma-bearing animals pretreated with TNP-470/minocycline, or not pretreated, on d 8 after tumor implantation, and tissues were collected over 24 h (196). At early time points (1 and 15 min) after intravenous administration of the [14C]paclitaxel, there was a fivefold higher concentration of the drug in the tumors of animals that had been pretreated with TNP-470/minocycline; however, by 24 h, there was a twofold greater concentration of [14C]paclitaxel in the tumors of the animals pretreated with TNP-470/minocycline compared with those that had not received the antiangiogenic therapy. The pattern of [14C]paclitaxel distribution into the other tissues was similar, with greater peak levels of [14C]paclitaxel in the tissues of animals pretreated with TNP-470/minocycline. In the liver, however, there was a prolonged increased level of [14C]paclitaxel of the pretreated animals. By far, the highest levels of [14C]paclitaxel were found in the lungs of the animals, in which the peak level in the pretreated animals reached 4800 μg/g tissue. Other tissues with relatively high paclitaxel concentrations were gut and heart.
Concentrations of platinum from carboplatin were two- and threefold higher in the tumors of animals pretreated with TNP-470/minocycline at 15 and 30 min after drug administration. Between 6 and 24 h after carboplatin administration, platinum levels in the tumors of pretreated animals remained about twofold greater than in animals that did not receive the antiangiogenic therapy. Overall, the tissues of the animals pretreated with TNP-470/minocuyline had higher plati- num levels, with the greatest differentials being in kidney, brain, muscle, and liver. The highest platinum levels overall were in kidney, gut, liver, and brain.
To determine whether pretreatment with TNP-470/minocycline might also alter the tissue distribution of large molecules into tumor and tissues, [14C]albumin was administered to TNP-470/minocycline pretreated and nonpretreated animals.
There was a two- to threefold higher concentration of [14C]albumin in the tumors of TNP-470/minocyline-treated animals over the first hour after protein injection
and a concentration differential with higher concentrations in those tumors. This effect persisted over the 24 h examined. A similar pattern pertained for the other tissues. The highest peak levels of [14C]albumin were in liver and lung (196).
There has been much discussion regarding the best designs for early clinical trials for antiangiogenic agents and cytostatic agents (197–199). TNP-470 has undergone clinical trial both as a single agent and in combination with cytotoxic anticancer therapies, with results warranting further investigation (200–205).
INHIBITORS OF CELL-CYCLE SIGNAL TRANSDUCTION The most frequent alteration in human malignant disease thus far recognized is the overexpression, mutation, and/or disregulation of cyclin D (206–208). The cyclin D1 gene CCND1 is amplified in about 20% of breast cancers, and the protein cyclin D1 is overexpressed in about 50% of breast cancers (209–213).
Overexpression of cyclin D1 has been reported in proliferative breast disease and in ductal carcinoma in situ, indicating that this change is important at the earliest stages of breast oncogenesis (211,213). Kamalati et al. (210) overexpressed cyclin D1 in normal human epithelial cells and found that the transfected cells had reduced growth-factor dependency, a shortened cell-cycle time, thus provid- Fig. 8. Fluorescence distribution in FSaIIC tumor cells after iv injection of tumor-bearing animals with Hoechst 33342 (2 mg/kg). The data shown are for an untreated control tumor and a tumor treated with TNP-470 (3 × 30 mg/kg, subcutaneously) and minocycline (5× 10 mg/kg, intraperitoneally).
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ing the cells with a growth advantage. In 123 colorectal carcinoma specimens, those staining strongly for cyclin D1 corresponded to patients with a 5-yr sur- vival rate of 53.3%, while those that were negative or weakly staining had 5-yr survival rates of 96.2 and 78.8% (214,215). Amplification of CCND1 was found in 25% of dysplastic head-and-neck lesions, 22% of head-and-neck carcinomas, and overexpression of cyclin D1 was found in 53% of head-and-neck carcino- mas, indicating that in this disease, like breast cancer, alterations in cyclin D1 occur at the very earliest stages of tumorigenesis (216,217). In a study of 218 specimens of esophageal squamous-cell carcinoma, patients with cyclin D1- positive tumors had significantly worse survival than patients with cyclin D1- negative tumors (218). In eight human esophageal carcinoma cell lines, seven (87.5%) and six (75%) cell lines had homozygous deletions of the p16 and p15 genes, respectively (219). All of the p16-negative cell lines express high levels of cyclin D1 and cdk4. In a transgenic mouse in which the Epstein–Barr virus ED-L2 promoter was linked to human cyclin D1 cDNA, the transgene protein localizes to squamous epithelium in the tongue and esophagus, resulting in a dysplastic phenotype associated with increased cell proliferation and indicating that cyclin D1 overexpression may be a tumor-initiating event (220,221). In a series of 84 specimens of soft-tissue sarcomas, there was no amplification of the
Table 7
DNA Crosslinking Factors From Lewis Lung Tumors by Alkaline Elutiona
Treatment group DNA crosslinking factorb
TNP-470-minocycline 1.2
Cyclophosphamide
150 mg/kg 3.9
300 mg/kg 4.7
500 mg/kg 5.6
TNP-470-minocycline -
cyclophosphamide (300 mg/kg) 6.2
CDDP
10 mg/kg 1.7
20 mg/kg 2.0
30 mg/kg 2.8
TNP-470-minocycline-CDDP
(20 mg/kg) 8.9
aFor DNA alkaline elution studies, Lewis Lung Carcinoma-bearing animals were treated with TNP-470 (30 mg/kg) subcutaneously on d 4, 6, and 8, with minocycline (10 mg/kg) intraperitoneally (ip) daily on d 4–8; and/or with cyclophosphamide (150, 300, or 500 mg/kg) ip or CDDP (10, 20, or 30 mg/kg) ip on d 8 after tumor cell implantation. [14C]-thymidine was administered ip on d 7 and 8. The animals were sacrificed on d 9.
bA DNA crosslinking factor of 1.0 indicates no crosslinks.
CCND1 gene, but there was overexpression of cyclin D1 in 29% of cases, and the overexpression of cyclin D1 was significantly associated with worse overall survival (222,223). Marchetti et al. (224) found that abnormalities of cyclin D1 and/or Rb at the gene and/or expression level were present in more than 90% of of a series of non-small-cell lung cancer specimens, indicating that cyclin D1 and/or Rb alterations represent an important step in lung tumorigenesis. In 49 out of 50 pancreatic carcinomas (98%), the Rb/p16 pathway was abrogated exclu- sively through inactivation of the p16 gene (225). Mantle-cell lymphoma is defined as a subentity of malignant lymphomas characterized by the chromo- somal translocation t(11;14)(q13;q32) resulting in overexpression of cyclin D1 and, in addition, about 50% of these tumors have deletion of the p16 gene (226,227). In a series of 17 hepatoblastomas, 76% showed overexpression of cyclin D1 and 88% showed overexpression of cdk4 (222). There was a correla- tion between high-level cyclin D1 expression and tumor recurrence.
Six distinct classes of small molecules from natural products have been idenitified as inhibitors of cdks: the purine-based compound olomoucine and analogs, butyrolactone, flavopiridol, staurosporine, UCN-01, suramin, and 9- hydroxyellipticine (228–235). All of these molecules bind at the ATP-binding site of the enzyme and are competitive with ATP. Olomoucine is an inhibitor of Cdc2, cdk2, cdk5, and MAP kinase in micromolar concentrations and has much weaker effects toward cdk4 and cdk6 (234).
Flavopiridol, a novel synthetic flavone, potently inhibits several cyclin-de- pendent kinases, including cdk1, cdk2, cdk4, and cdk7 (236–243). Exposure to flavopiridol can cause cells to arrest in both the G1 and G2 phases of the cell cycle, at concentrations similar to those required for cell-growth inhibition (236,243). Flavopiridol inhibits the cdks in a manner competitive with ATP and noncompetitive with the substrate. Flavopiridol also inhibits other protein ki- nases, such as protein kinase C, protein kinase A, and EGFR, but at concentra- tions of 10 μM/L or greater. Flavopiridol is an active antitumor agent in several human tumor xenograft models, including Colo-205 colon carcinoma and DU- 145 and LNCaP prostate carcinomas, MCF-7 and MB-468 breast carcinomas, and SW-2 and H82 small-cell lung carcinomas (206–208,236,240). Tumor growth delay was used to determine the sensitivity of human tumor xenografts to the cell-cycle agents flavopiridol and olomoucine. The human breast carinoma cell lines MCF-7 and MB-468 were grown as xenograft tumors in female nude mice (Fig. 10). Flavopiridol was administered orally to the mice over a dosage range once daily for 5 d per wk for 3 wk beginning on d 7 after tumor cell implantation. The MB-468 tumor was more responsive to treatment with flavopiridol than was the MCF-7 tumor. The tumor-growth delay at the dose of 10 mg/kg of flavopiridol in the MCF-7 tumor was 6.2 d, while the tumor-growth delay for the same treatment in the MB-468 tumor was 10.9 d. Olomoucine was administered to animals bearing the MCF-7 tumor or the MB-468 tumor by
