Cancer Chemotherapy & Biotherapy: Principles & Practices, 4th Edition

New Targets for Anticancer Therapeutics

A. Dimitrios Colevas

Orit Scharf

Lyudmila A. Vereshchagina

Janet E. Dancey

S. Percy Ivy

Len Neckers

Bennett Kaufman

Richard Swerdlow

This chapter summarizes the molecular basis of oncology interest, preclinical development, and early clinical trials of a selection of investigational agents targeting five cellular processes: the cell cycle via cyclin-dependent kinase inhibition (CDKI), the mitotic apparatus via kinesin spindle protein inhibition (KSPI), cell-cell communication via integrin inhibition, intracellular signal transduction via molecular target of rapamycin (mTOR) inhibition, and posttranslational protein processing via heat shock protein 90 (HSP90) inhibition.

All the molecular targets of the agents discussed here are known to be functional in both normal and cancer cells. The rationale for development is therefore not based on the premise that these targets are uniquely expressed in cancer cells, as was the case, for example, with imatinib in diseases associated with Bcr-Abl expression. Targeting a critical cellular process or molecular pathway at any point in that process or pathway may perturb cells sufficiently to alter their survival or growth. The key, therefore, is to elucidate which process or pathway is uniquely essential to cancer cell growth or differentially sensitive to disruption in cancer cells. Because many cancers harbor abnormalities in the pRb pathway or overexpress cyclins, CDK inhibitors are a logical target in these cancers. Cell division is a sine qua non of cancer, while most somatic cells rest in G0. Targeting of proteins expressed only during cell division, such as kinesin spindle proteins, might selectively perturb cancer cells. Tissue-specific integrin expression, such as exclusive neo-endothelial expression of the αvβ3heterodimer, as well as overexpression of αvβ3 in certain cancers, suggests that an αvβ3 integrin inhibitor would have cancer-specific activity. Both mTOR and Hsp90 inhibition could have cancer-specific activity by virtue of the fact that the targeted pathways in the former and molecular entities in the latter seem to be particularly relevant to growth and/or survival of selected cancers. Therefore, the agents described in this chapter have been chosen for development due to their ability to alter pathways rather than target oncogenic gene products.

The preclinical support for the molecular pathway cancer-specific activity of the above agents is summarized in this chapter. It is important to recognize that these preclinical data are derived from experiments in artificial systems that historically have been poor predictors of clinical efficacy. Therefore, the definitive evaluation of the potential of these agents will depend on the results of the ongoing clinical trials.

CYCLIN-DEPENDENT KINASE INHIBITORS

Introduction

The four phases of the cell cycle (G1, S, G2, and M) are characterized by distinct cellular processes that are required for proper cell division.1 Each phase transition is tightly regulated by cyclin-dependent kinase (CDK) complexes composed of a cyclin and a kinase.2 Mitogen-dependent accumulation of cyclin D–dependent kinases triggers the phosphorylation of the retinoblastoma (Rb) protein by CDK4 and/or CDK6 (G1) as well as by CDK2 (G1/S interphase), and this phosphorylation then enables complexes of the E2F transcription factor to activate transcription of target genes (e.g., enzymes required for DNA synthesis such as thymidine kinase and dihydrofolate reductase), leading to cell division.3, 4 This process is then accelerated by the cyclin E–CDK2 complex, which acts through positive feedback to facilitate progressive rounds of Rb phosphorylation and E2F release. Another regulation point is the G2/M transition, where specific expression of regulators is essential to control the correct sequence of events; CDK1 cooperates with other kinases and phosphatases to regulate the final phases of the cell cycle.3 Most human neoplasms are characterized by dysregulation of components of the Rb pathway. This dysregulation leads to aberrant progression into the S phase while ignoring growth-factor and cell-cycle control signals.5 These observations have stimulated a great interest in development of pharmacological small-molecule CDK inhibitors. Several small-molecule CDK inhibitors that bind at the ATP-binding site of CDKs are being studied in clinical trials (alvocidib or flavopiridol, UCN-01, CYC202 or R-roscovitine, BMS-381032, and E7070 or indisulam) (Table 25.1 and Fig. 25.1).6, 7

Alvocidib (Flavopiridol)

Alvocidib (NSC 649890, Flavopiridol, L86-8275, HMR 1275; Sanofi-Aventis Pharmaceuticals, Inc., Bridgewater, NJ; Fig. 25.1) is a synthetic flavone derived from a natural product, rohitukine, isolated from the stem bark of Dysoxylum binectariferum, a plant indigenous to India.24, 25 The biology and chemistry of alvocidib and its mechanism of action, pharmacology, preclinical studies, and clinical experience have been the subject of several review articles.3, 6, 8, 9, 26The antitumor activity of alvocidib has been linked to CDK inhibition, induction of apoptosis, and inhibition of transcription and angiogenesis. Alvocidib disrupts progression of cells through the cell cycle at G1/S and G2/M.24 Alvocidib directly inhibits CDK 1, 2, 4, 6, and 7 in the 30- to 300-nM range.9 In addition, alvocidib inhibits epidermal growth factor receptor (EGFR) tyrosine kinase, the serine/threonine kinases, PKC and PKA, mitogen-activated protein kinase (MAPK) Erk-1, and src family kinases at concentrations in the micromolar range.9 The efficacy of alvocidib is not solely based on cell cycle arrest.27Alvocidib also inhibits the positive transcription elongation factor (P-TEFb; IC50 < 10 nM).10 Alvocidib down-regulates expression of cyclin D1 by reducing cyclin D1 mRNA and inhibiting transcription of cyclin D1 promoter.11 Alvocidib induces apoptosis in various tumor and normal cells in vitro as well as in tumor xenografts in vivo, particularly those of hematopoietic origin, independent of Bcl-2 and p53 status.9, 28, 29 Thus, available data indicate that alvocidib can target a variety of key regulatory molecules involved in cell cycle progression and apoptosis.

TABLE 25.1 CDK INHIBITORS IN CLINICAL DEVELOPMENT

Agent

CDK/CyclinTarget*

Prominent In Vitro/In Vivo Preclinical Data

Clinical

Alvocidib (3, 6, 8, 9, 10, 11, 12)

CDK1/cyclin BCDK2/cyclin ACDK2/cyclin ECDK4/cyclin DCDK6/cyclin DCDK 7/cyclin H (TFIIH), CDK9/cyclin T (P-TEFb), cyclin D1 protein↓

Average IC50 = 0.066 µM; G1/S, G2/M arrest; apoptosis (↑); angiogenesis (↓)

Phase I andphase II single-agent and combination studies (+ paclitaxel, cisplatin, carboplatin, docetaxel, irinotecan, gemcitabine, ara-C, bortezomib, trastuzumab, doxorubicin, imatinib, dapsipeptide, SAHA). Promising activity in CLL.

R-roscovitine(CYC202) (13, 14, 15, 16)

CDK2/cyclinACDK2/cyclin ECDK7/cyclin HCDK5/p35, CDC2/cyclin B, CDK9/cyclin T, CDK1/cyclin B, cyclin D1 protein ↓

Average IC50 = 15.2 µM; 45–62% tumor growth (↓) in xenograft models;G1/S, G2/M arrest; apoptosis (↑)

Phase I single-agentstudies: oral BID.
Phase II combination studies: + capecitabine in breast cancer;+ gemcitabine/cisplatin in NSCLC.

Indisulam(E7070) (17, 18, 19)

CDK2/cyclin E activity ↓; CDK2, CDK4, CDC2, Cyclin A, B1, H proteins ↓

IC50= 0.1–4.4 µg/mL; T/C <42% and 73–85% tumor volume (↓) in xenograftmodels; G1/S, G2/M arrest; apoptosis (↑)

Phase I andphase II single-agent studies: IV d × 5, q7d, q21d.

BMS-387032 (20, 21, 22, 23)

CDK2/cyclin E, CDK1/cyclin B, CDK4/cyclin D

Cellular cytotoxicityIC50 = 0.095 µM; tumor growth (↓) and 9/16 cured mice in xenograft model experiments; cell cycle arrest; apoptosis (↑)

Phase I single-agentstudies: 1-hr IV q7d, q21d; 24-hr CIV q21d.

*Bold implies IC50 < M.

Figure 25.1 Chemical structure of alvocidib.

Alvocidib has demonstrated a potent antiproliferative activity in the National Cancer Institute's (NCI) 60–cell line drug screen (average IC50 = 66 nM), with no obvious tumor-type selectivity.8 IC50 values ranged from approximately 50 to 200 nM, similar to concentrations required to inhibit CDKs.24 Alvocidib inhibits growth of human tumor xenografts, including head and neck squamous cell carcinoma (HNSCC) and lung, colon, ovary, gastric, and breast cancer, and causes regression in glioma, leukemia, and lymphoma models.30, 31, 32, 33

Alvocidib is being clinically developed by Sanofi-Aventis in collaboration with the NCI. Several different schedules of administration have been explored. The initial phase I studies tested multiple-day continuous infusions (CIVs) based on nonclinical data suggesting that prolonged exposure optimized anticancer activity. With a 72-hour CIV on an every-2-week schedule, the maximum tolerated dose (MTD) and recommended phase II dose (RP2D) is 40 to 50 mg/m2 per day 3 3, with secretory diarrhea as the dose-limiting toxicity (DLT).34, 35 Other prominent adverse events include proinflammatory syndrome and thrombosis. The steady-state levels achieved at MTD (300-nM range) are below what is predicted to be active from several nonclinical models. Subsequent studies of 1-, 3-, or 5-consecutive-day 1-hour infusions of alvocidib have yielded MTDs of 62.5, 50, and 37.5 mg/m2 per day, respectively.36, 37, 38 Neutropenia, fatigue, and hepatotoxicity are the DLTs, and nausea and vomiting have been significant.36, 38 Micromolar plasma concentrations, reached at the MTDs, are short-lived. Hints of activity from these trials included a gastric cancer patient with a complete response (CR) for 48 months and a patient with renal cell cancer (RCC) who achieved a partial response.34, 35 Because minimal anticancer activity has been seen in these single-agent phase II trials on these schedules, additional schedules have been tested, including weekly 1-hour and 24-hour infusions. Alvocidib on these additional schedules is tolerable at doses tested on the above schedules with similar pharmacokinetics (PKs).39, 40, 41

Based on the observation that free alvocidib levels in human plasma are significantly lower than in bovine plasma, two groups have pursued a hybrid schedule of bolus followed by infusional alvocidib in order to achieve micromolar steady-state concentrations for several hours. Alvocidib given weekly by 30-minute intravenous (IV) bolus followed by 4-hour IV in patients with fludarabine-refractory chronic lymphocytic leukemia (CLL) has resulted in micromolar concentrations for several hours, tumor lysis syndrome as a DLT at 80 mg/m2 per dose, and dramatic activity against fludarabine-resistant CLL, including nine sustained partial responses in the first 23 patients treated.12, 42

Based on nonclinical evidence of synergism, alvocidib has been combined with the following agents in clinical trials: paclitaxel, docetaxel, irinotecan, gemcitabine, cisplatin, carboplatin, imatinib, trastuzumab, 5-fluorouracil (5-FU), bortezomib, suberoylanilide hydroxamic acid (SAHA), and doxorubicin and also with radiation.43, 44, 45, 46, 47, 48, 49, 50 Combinations with multiple agents have included gemcitabine plus irinotecan, oxaliplatin plus 5-FU, irinotecan plus 5-FU, leucovorin plus 5-FU, cytosine arabinoside plus mitoxantrone, fludarabine plus rituximab, paclitaxel plus cisplatin or carboplatin, and irinotecan plus cisplatin.51, 52, 53, 54, 55 Generally, the doses of alvocidib achievable when combined with conventional agents are in the same range or slightly lower than the single-agent MTD as a 1-hour, 24-hour, or 72-hour infusion. One notable exception is the combination of alvocidib with taxanes, where the tolerability of the combinations is sensitive to the schedule and administration of these agents, despite no known PK interaction.43, 44, 45, 51, 52 The hybrid bolus followed by continuous infusion schedule has yet to be tested in combination with other agents.

Clinical PK data for alvocidib are summarized in Table 25.2. Mean steady-state alvocidib concentrations achieved using 72-hour CIV were not adequate after correction for the conversion from bovine to human plasma environment.35, 56 Preliminary data suggest that levels greater than 1 mm are sustained for more than 5 hours in patients treated using the hybrid infusion.12 The postinfusional peak concentration (Cmax) of alvocidib appears to be related to enterohepatic recirculation, and nonlinear elimination has been observed at alvocidib doses above 50 mg/m2 per day, but there is evidence of postinfusional Cmax with the 1-hour infusion.26, 56 Dose-corrected areas under the curve (AUCs) are similar with the 72- and 1-hour infusions.26 Alvocidib is highly protein bound, with a mean unbound fraction of 6%.56 Irinotecan increases the metabolism of alvocidib, but no other PK interactions are known for the above combinations.50 The systemic glucuronidation of alvocidib is inversely associated with the risk of developing diarrhea in metastatic renal cancer patients.57

TABLE 25.2 CLINICAL PK PARAMETERS OF ALVOCIDIB UNDER DIFFERENT SCHEDULES

 

Phase II Trials

Alvocidib has been administered as a 1-hour IV infusion daily for 3 days every 3 weeks in patients with mantle cell lymphoma, metastatic malignant melanoma, and advanced RCC. Responses were seen in 3/31, 0/17, and 2/34 patients, respectively. Four RCC patients remained progression free during treatment for over 1 year.58, 59, 60 In single-agent phase II trials using the 72-hour CIV every-2-week schedule, response rates in patients with advanced colorectal cancer (ACRC), metastatic non–small cell lung cancer (NSCLC), metastatic androgen-independent prostate cancer, metastatic gastric cancer, and metastatic renal cancer were 0/18, 0/20, 0/36, 0/14, and 2/34, respectively.61, 62, 62, 63, 64, 65

Future Prospects

Because clinically significant antineoplastic activity using alvocidib as a single agent on the 1-hour and 72-hour infusion schedules has not been seen, further efforts are focused on clinical development in four areas. First, the promising activity in CLL on the hybrid schedule will be the subject of follow-up phase II studies. Second, the utility of combining alvocidib with conventional agents continues to be explored. Third, based on promising preclinical synergy data, NCI is sponsoring combinations of alvocidib with other targeted therapies, including histone deacetylase inhibitors, imatinib, and rituxumab. And finally, there are a number of ongoing clinical trials with embedded correlative studies designed to ask whether or not alvocidib in fact inhibits its purported targets in human tumors.

Other CDK inhibitors currently undergoing clinical trials include oral R-roscovitine (CYC202; Cyclacel, Dundee, UK; phase I single-agent and phase II combination studies with capecitabine in breast cancer and with gemcitabine/ cisplatin in NSCLC), BMS-387032 (Bristol-Myers Squibb in collaboration with the NCI; phase I trials), and indisulam (E7070; Eisai; phase I and II single-agent studies).15, 16, 18, 19, 21, 22, 23

SB-715992 (KINESIN SPINDLE KINASE INHIBITOR)

The kinesin superfamily of proteins consists of motor proteins that utilize energy released by hydrolysis of ATP to produce directed force along microtubules.66Kinesins are divided into two major groups by their function; those involved in vesicle transport and membrane organization and those involved in cell division (mitotic kinesins).67 Expression of mitotic kinesins is increased in tumor tissues relative to normal adjacent tissues.68, 69 Mitotic kinesins are predominantly expressed during cell division, further suggesting that they may be more specific anti-mitotic targets than tubulin, which is present in all cells during all phases of the cell cycle.67 The kinesin spindle protein (KSP; also known as Eg5) is a mitotic kinesin required in early mitosis for the establishment of mitotic spindle bipolarity and for cell cycle progression through mitosis.70, 71 SB-715992 (GlaxoSmithKline) is a 2-(aminomethyl) quinazolinone inhibitor of KSP, with broad antiproliferative activity both in vitro and in vivo, 72 and it is the first mitotic KSP inhibitor to enter clinical trials.73 SB-715992 is 70, 000-fold more selective for KSP than other kinesins and disrupts the assembly of functional mitotic spindles, thereby causing cell cycle arrest and subsequent cell death.73

Preclinical Studies

In vitro studies have shown that SB-715992 has a Ki of 0.6 nM and cytotoxic activity at less than 10 nM in a spectrum of tumor cell lines.74 It caused complete tumor regression in two human colon tumor xenografts and tumor growth delay in another. On the other hand, a mammary tumor xenograft was completely refractory to SB-715992. SB-715992 is growth inhibitory in other pancreatic and colon carcinoma xenograft models.72 Efficacy is dose-related, and in the most sensitive tumor models, regressions were seen at doses as low as one third of the MTD.74 Equivalent efficacy and toxicity were seen regardless of administration method.74

Clinical Trials

GlaxoSmithKline has sponsored two clinical trials with SB-715992. One study administered 1-8 mg/m2 on days 1, 8, and 15 every 28 days.75 Twenty-seven patients with solid tumors were treated, and two patients developed a DLT (grade 3 neutropenia) at a dose of 8 mg/m2. As a result, the 7 mg/m2 dose was expanded and no grade 3/4 adverse events were observed. Increases in AUC and Cmax were dose-related. PK parameters measured on days 1 and 15 of the first cycle were as follows: Cmax 5 349 and 218 ng/mL; Cl 5 1, 596 and 7, 546 mL/hour; and volume of distribution at steady state (Vdss5 235 and 240 L, respectively. The RP2D for this study was 7 mg/m2.

In the second study, SB-715992 was administered intravenously at doses of 1 to 21 mg/m2 every 21 days.76 Forty-two patients with solid tumors were treated, and two DLTs were observed at 21 mg/m2, grade 4 neutropenia lasting more than 5 days and grade 4 neutropenic fever (one patient each). The 18 mg/m2 dose level was expanded, and 12 patients were treated at this dose. Two DLTs have been observed, grade 4 neutropenia in both cases. Non-dose-limiting grade 3/4 neutropenia occurred in seven patients and grade 4 leukopenia occurred in one patient. Common grade 2 drug-related adverse events at doses greater than 6 mg/m2 included fatigue, leukopenia, and anemia. Stable disease was observed in four patients for 5 to 11 cycles. AUC and Cmax increased in a dose-dependent manner, and median PK values were as follows: Cmax = 473 ng/mL; AUC55, 074 ng.h/mL;t½ = 33 hours, Cl = 6, 656 mL/hour; and Vdss = 236 L. Monopolar mitotic spindles were observed in a tumor biopsy from a patient with squamous cell carcinoma of the head and neck (SCCHN), at a dose of 16 mg/m2. The RP2D in this study was 18 mg/m2 every 21 days.

In addition to these studies, the NCI is sponsoring phase I and II studies of SB-715992 in patients with solid tumors, acute leukemia, RCC, colorectal cancer, hepatocellular carcinoma, prostate cancer, SCCHN, and melanoma.

αv ANTAGONISTS AS NOVEL ANTICANCER AGENTS

Introduction

Integrins are a widely expressed family of cell adhesion receptors that recognize extracellular matrix proteins and cell-surface molecules through short peptide sequences.77 The integrins consist of two distinct noncovalently associated subunits; 18 α- and 8 β-subunits combine in a restricted repertoire to form 24 known heterodimers.78 Several integrins share the αv subunit and interact strongly with the Arg-Gly-Asp (RGD) peptide sequence found within specific extracellular matrices and cell surface proteins. The αVβ3 and αVβ5 integrins are overexpressed on endothelial cells during tumor angiogenesis77, 78, 79 and participate in other events promoting tumor progression, such as cell migration and capillary morphogenesis. αVβ3 has a limited tissue distribution. It is not typically expressed on epithelial cells and appears at minimal levels on intestinal and vascular cells.80 In contrast, it is extensively expressed on some tumor cells, including late-stage glioblastoma, 81, 82 ovarian carcinoma, 83 melanoma, 84, 85 pancreatic carcinoma, 86 and prostate cancer.87

The importance of both αVβ3 and αVβ5 in tumor angiogenesis88 has generated an interest in antagonists to these integrins. Both blocking antibodies directed against the extracellular domain (LM609) and RGD peptides disrupt blood vessel formation in several in vitro and in vivo models.80 In tumor models, this inhibition disrupts tumor-associated angiogenesis and in some cases causes tumor regression as well.89 Humanized LM609, known as MEDI-522 (Vitaxin; MedImmune, Inc.), and the cyclic RGD peptide EMD 121974 (Cilengitide; Merck KgaA/EMD Pharmaceuticals) are both currently being evaluated in clinical trials.

MEDI-522 (Vitaxin)

Preclinical Studies

Antibodies directed against αVβ3 integrin cause dose-dependent regression of CAM blood vessels as a result of apoptosis associated with activation of endothelial cell p53 and increased expression of p21waf1/cip1.90, 91

Clinical Trials

MedImmune, Inc. is sponsoring two phase I studies in cancer patients. In one study, MEDI-522 was administered to patients with refractory solid tumors at doses up to 6 mg/kg as a single dose, followed 2 to 5 weeks later by weekly doses for up to 1 year.92 The majority of MEDI-522-related adverse events were mild to moderate in severity, with the most common being infusion-type reactions. No anti–MEDI-522 antibodies were observed. Preliminary pharmacokinetic (PK) results showed a nonlinear increase in terminal half-life with increasing doses, reaching 130 hours following the 4 mg/kg dose level. At 6 mg/kg per week, mean serum trough levels of MEDI-522 ranged from 24 to 37 µg/mL. In the other phase I study, MEDI-522 was administered to patients with irinotecan-refractory colorectal cancer at dose levels of 4 to 10 mg/kg.93 MedImmune is currently enrolling patients into two phase II studies (melanoma and prostate cancer), and the NCI is sponsoring two phase I studies in patients with solid tumors. The NCI studies are being conducted in patients with advanced solid tumors utilizing a drug administration schedule similar to that used in the MedImmune-sponsored studies.

EMD 121974 (Cilengitide)

Preclinical Studies

EMD 121974 (Merck KgaA, Germany, and EMD Pharmaceuticals Inc.; Fig. 25.2) is the inner salt of a cyclized pentapeptide containing the RGD sequence and is a potent and selective antagonist of the αVβ3 and αVβ5 integrins.94 EMD 121974 has been shown to bind to integrins αVβ3 and αVβ5, with IC50 values of 1 nM and 140 nM, respectively, 95 and it inhibits integrin binding to extracellular matrix proteins.96 In addition, EMD 121974 inhibits the growth of WM164 melanoma tumor in vivo.96 EMD 121974 inhibits the M21 melanoma xenograft model at 10 to 15 mg/kg administered once or twice a day or every other day.95EMD 121974 inhibits medulloblastoma (DAOY) and glioblastoma (U87MG) orthotopic xenografts at ~5 mg/kg administered intraperitoneally.97 All treated mice survived without evidence of morbidity, and only residual tumor cells or small clusters could be seen (<1 mm3 in size).

Figure 25.2 Chemical structure of cilengitide (EMD 121974).

Toxicology

Four weeks of daily IV bolus administration of EMD 121974 to mice resulted in no evidence of treatment-related effects up to the highest dose level of 90 mg/kg.95 In cynomolgus monkeys, EMD 121974 administered intravenously daily at doses up to 90 mg/kg showed dose-related anemia and reticulocytosis at the end of the study period, with normalization of both parameters after 4 weeks. No bone marrow abnormality was observed at necropsy.95

Clinical Studies

Merck KgaA/EMD Pharmaceuticals has sponsored several phase I and II studies with EMD 121974. In a phase I study, EMD 121974 was administered by a 1-hour IV infusion at doses of 30 to 1, 600 mg/m2 twice weekly to patients with advanced solid tumors.95 PK parameters were approximately linear, and Cmaxwas generally observed at the end of the infusion period. Mean systemic clearance (CL) was 34 to 66 mL/min per m2, and Vdss was 9 to 17 L/m2. Systemic exposure to EMD 121974 was dose-dependent. The target plasma concentration of 11 to 13 mg/mL, derived from murine PK models, was attained at a dose of 120 mg/m2. Hematologic adverse events consisted of grade 2 anemia. Nonhematologic adverse events were generally mild, never exceeding grade 2, and consisted of nausea, anorexia, vomiting, fatigue, and malaise. No DLT occurred.

The NCI is sponsoring several phase I and II studies with EMD 121974. The New Approaches to Brain Tumor Therapy consortium (NABTT) treated 51 malignant glioma patients with EMD 121974 at a starting dose of 120 mg/m2 twice weekly, escalating up to 2, 400 mg/m2.98 Two complete responses (at 360 and 2, 400 mg/m2) and three partial responses (two at 120 mg/m2 and one at 360 mg/m2) have been confirmed. DLTs included grade 4 arthralgia at 480 mg/m2; thrombocytopenia at 600 mg/m2; and anorexia, hypoglycemia, and hyponatremia at 1, 800 mg/m2. The MTD was not reached. Since no serious adverse events or biologic activity had been observed with lower doses of EMD 121974, patients with solid tumors enrolled in another phase I study were treated at 600, 1, 200, and 2, 400 mg/m2 twice weekly. PK analysis from these two studies demonstrates that the twice-weekly schedule appears to maintain the preclinically effective plasma concentration of 1µM for less than 24 hours. In a study of children with refractory brain tumors in which EMD 121974 is administered at a starting dose of 120 mg/m2 twice weekly for 4 weeks, the study has accrued patients up to the 1, 200 mg/m2 dose level.

Because responses have occurred in patients receiving low as well as high doses of EMD 121974, and because there seems to be no dose-related toxicity in the dose ranges studied, the NCI is sponsoring several phase II studies to be conducted with two doses: 500 mg and 2, 000 mg administered intravenously twice weekly. A phase II study in melanoma patients is currently enrolling patients. In addition, a phase I-II study of EMD 121974 combined with radiation therapy in newly diagnosed glioblastoma multiforme patients has been recently approved. During the phase I portion of this study, patients will be administered EMD 121974 at a starting dose of 500 mg twice weekly, escalating to 2, 000 mg concurrently with conventional radiation therapy. In the phase II portion of the study, patients will receive either 500 mg or 2, 000 mg of EMD 121974 during radiation. Additional phase II studies with EMD 121974 in patients with carcinoma of the prostate and glioblastoma are planned.

INHIBITORS OF THE MAMMALIAN TARGET OF RAPAMYCIN

Introduction

The mammalian target of rapamycin (mTOR) is a downstream protein kinase of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. By targeting mTOR, the immunosuppressant and antiproliferative agent rapamycin inhibits signals required for cell cycle progression, cell growth, and proliferation in normal and malignant cells. Currently, mTOR inhibitors rapamycin (sirolimus, Wyeth) and derivatives temsirolimus (CCI-779, Wyeth), everolimus, (RAD001, Novartis Pharma AG), and AP23573 (Ariad Pharmaceuticals) are being evaluated in cancer clinical trials. An additional agent, TAFA-93 (Isotechnika), has recently entered human trials for the prevention of organ rejection after transplantation.

Biochemistry of the PI3K-Akt-mTOR Pathway

The mTOR is a serine-threonine kinase that regulates both cell growth and cell cycle progression by integrating signals from nutrient and growth factor stimuli.99, 100 mTOR functions in a protein complex that integrates signals from a variety of sources, including growth factors, energy stores, and hypoxia, with the protein translation apparatus.101

Growth factor receptor-stimulated mTOR regulation proceeds through the PI3K and Akt pathways (Fig. 25.3). In response to extracellular stimuli, PI3K phosphorylates phosphatidylinositol-4, 5- bis-phosphate (PIP2) to generate phosphatidylinositol-3, 4, 5- triphosphate (PIP3). The formation of PIP3 leads to the binding and activation of phosphatidylinositol-dependent kinase-1 (PDK1) and Akt at the plasma membrane.100 The tumor suppressor phosphatase PTEN (for phosphatase and tensin homolog deleted on chromosome 10) dephosphorylates PIP3, reversing the action of PI3K. Activated Akt phosphorylates and inhibits the tuberous sclerosis complex (TSC), which is composed of TSC1 (hamartin) and TSC2 (tuberin) heterodimer that inhibits cell cycle progression and cell proliferation.102, 103 TSC2 acts as a GTPase-activating protein (GAP) toward the Ras-related small GTPase Rheb (Ras-homolog-enriched-in-brain), 104 a positive upstream regulator of mTOR. Activation of TSC2 by the tumor suppressor gene product LKB1, as may occur in a nutrient-deprived state, inhibits Rheb and results in the down-regulation of mTOR. In contrast, inhibition of TSC2, as occurs in the presence of amino acids, with Akt phosphorylation, or through loss of TSC2 function through mutation, as occurs in tuberous sclerosis patients, leads to mTOR activation and phosphorylation of the downstream mTOR targets.103 The net result of these signaling interactions suggests a model in which growth factor signaling through PI3K-Akt is coordinated with nutrient availability signaling through LKB1-TSC1/2 to Rheb and mTOR.

Figure 25.3 Growth factor receptor (GFR, integrin and G-protein coupled receptor [GPCR]) stimulation leads to activation of PI3K, phosphorylation of the 3′-OH of phosphatidylinositol-4, 5-bis-phosphate (PI4, 5, P2) to generate phosphatidylinositol-3, 4, 5-triphosphate (PI3, 4, 5P3). PI3, 4, 5P3 then recruits phosphatidylinositol-dependent kinase-1 (PDK1) and Akt, to the plasma membrane to be activated. The tumor suppressor phosphatase PTEN (for phosphatase and tensin homolog deleted on chromosome 10) dephosphorylates PI3, 4, 5, P3at the D-3 position of the inositol ring. Activated Akt phosphorylates and inhibits the tuberous sclerosis complex (TSC), removing its inhibitory effect on Ras-related small GTPase Rheb (Ras homolog enriched in brain). TSC2 is also inhibited by the presence of amino acids, allowing Rheb to activate mTOR through an unknown mechanism. Activation of mTOR in complex with other proteins such as raptor (regulatory associated protein of mTOR) and mammalian ortholog of LST8 (mLST8) leads to phosphorylation of eukaryotic initiation factor 4E (eIF-4E)–binding protein (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). This interaction results in an increase in translation rates of a subset of mRNAs, including those encoding proteins required for cell cycle progression such as cyclin D (CyD). Rapamycin (RAPA) in complex with FK506-binding protein of 12 kd (FKBP12) inhibits mTOR. See text for additional details.

mTOR functions in a complex with at least two other proteins: regulatory associated protein of mTOR (raptor)105, 106 and mammalian ortholog of LST8.106, 107Current evidence suggests that activated mTOR, in complex with raptor and possibly other proteins, leads to phosphorylation of two key proteins: eukaryotic initiation factor 4E (eIF-4E) binding protein-1 (4E-BP1) and protein S6 kinase 1 (S6K1)108, 109 (see Fig. 25.3). Activation of mTOR, leads to phosphorylation of 4E-BP1, release of eIF-4E to bind to cap mRNA transcripts and other initiation complex proteins, and the initiation of cap-dependent translation. This effect on translation of certain regulatory mRNAs may be one means by which mTOR regulates cell growth.110

A second mTOR target is the phosphorylation and activation of S6K1. Previously, activation of S6K1 had been correlated with increased translation of 5′ terminal oligopyrimidine tract (TOP) mRNAs, which encode components of the translational apparatus.111 However, the translation of TOP mRNAs may occur independent of S6K1 function (for recent reviews, see refs. 110, 112). The recent results lead to the conclusion that regulation of TOP mRNA translation is primarily through the PI3K pathway, with little role for mTOR.110 S6K1 has been implicated in glucose homeostasis and regulation of eukaryotic elongation factor 2 kinase.110

Consistent with the role of mTOR as a controller of cellular growth, mTOR activation leads to the phosphorylation of several additional downstream signaling effectors and transcription factors, which in turn influence cell proliferation, survival, and angiogenesis. Many, though not all, of the protean functions of mTOR appear to be sensitive to inhibition by rapamycin. The many cellular signaling processes in which mTOR participates and the inhibition of some of these processes by rapamycins have contributed to interest in mTOR inhibition as a strategy for therapeutic development.

mTOR in Human Cancer

Although mutations of mTOR have not been reported in human cancers, both aberrant PI3K-dependent signaling and aberrant protein translation have been identified in a wide variety of malignancies and may contribute to oncogenesis and malignant progression. For example, components of the PI3K pathway that are mutated in different human tumors include activation mutations of growth factor receptors, amplification and/or overexpression of PI3K and Akt, as well as loss of PTEN. The resultant aberrant pathway signaling not only leads to a growth advantage during carcinogenesis and stimulates cancer cell proliferation but also contributes to treatment resistance due to a high PI3K-Akt–mediated survival threshold.100 If such cancer cells are “addicted” to the growth and survival signaling effects of the PI3K-Akt pathway, it is possible that this dependency will result in cancer cell sensitivity to mTOR inhibition.

In addition to cancer cell dependency on aberrant PI3K signaling for proliferation and survival, endothelial cell proliferation may also be dependent on mTOR signaling. Endothelial cell proliferation is stimulated by vascular endothelial cell growth factor (VEGF) activation of the PI3K-Akt-mTOR pathway. VEGF production may be partly controlled by mTOR signaling through mTOR effects on the expression of hypoxia-inducible factor-α (HIF1α).113, 114, 115

Rapamycin and Derivatives

Rapamycin (sirolimus, Rapamune, Wyeth; Fig. 25.4) is a macrocyclic lactone produced by Streptomyces hygroscopicus, a soil bacterium native to Easter Island (Rapa Nui). Rapamycin is used as an immunosuppressant for the prophylaxis of renal allograft rejection. Rapamycin's immunosuppressant effects are due to its inhibition of the biochemical events required for the progression of interleukin-2 (IL-2) stimulated T cells from the G1 to the S phase of the cell cycle. However, rapamycin and derivatives also inhibit cellular proliferation in a variety of tumor models and are currently under clinical evaluation as potential cancer therapeutics.

Mechanism of Action

Rapamycin targets the ubiquitously expressed FK506-binding protein of 12 kd (FKBP12). The FKBP12-rapamycin complex binds to the FKBP12-rapamycin–binding (FRB) domain adjacent to the kinase domain of mTOR and may inhibit mTOR by modifying the conformation and/or composition of the multiprotein mTOR complexes. By disrupting these protein complexes, rapamycin may impair either upstream signaling leading to mTOR activation or kinase access to downstream substrates.116, 117 Rapamycin and its derivatives share the following features: inhibition of cellular proliferation by inducing G1 phase arrest, induction of apoptosis in selected models, and limited normal tissue toxicity.

Figure 25.4 Chemical structure of rapamycin. (Reproduced with permission from Huang S, Bjornsti MA, Houghton PJ. Rapamycins: mechanism of action and cellular resistance. Cancer Biol Ther 2003;2:222–232.)

The rapamycins may inhibit tumor and endothelial cell proliferation in picomolar to nanomolar concentrations and may add to the cytotoxicity of other chemotherapeutic agents and radiation.118, 119, 120, 121, 122, 123 Rapamycins induce reduction of cyclins, particularly cyclin D, 124, 125 as well as increase cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1.126, 127 In most instances, inhibition of TOR by rapamycin leads to an antiproliferative response. However, there are examples in which rapamycin induces apoptosis.128, 129 Rapamycin-induced apoptosis may depend on the functions of p53, p21Cip1, and p27Kip.128,129, 130 Rapamycin inhibits endothelial cell proliferation in the presence of hypoxia, inhibits endothelial cell proliferation due to VEGF stimulation through inhibition of mTOR, and decreases VEGF synthesis through enhanced HIF1α degradation.131, 132, 133 Therefore, the expected clinical activity of rapamycin would be delayed tumor progression rather than tumor regression in most patients with sensitive disease; however, tumor regression through rapamycin-induced apoptosis could occur.

Determinants of Sensitivity and Resistance to mTOR Inhibition

Genetic mutations and/or compensatory aberrant signal transduction both upstream and downstream of mTOR influence tumor cells sensitivity to rapamycins.134, 135 Expression and function of the ataxia-telangiectasia gene product ATM and of 14-3-3, p53, PI3K-Akt, and PTEN have been reported to correlate with rapamycin sensitivity (for reviews, see refs. 134, 135). Inhibition of phosphorylation of S6K1, its target ribosomal S6 protein, and 4E-BP1 correlates with rapamycin sensitivity but is not sufficient for in vitro sensitivity in all cases.135, 136

Rapamycin and related compounds exert selective cytostatic/cytotoxic effects on PTEN -/- tumors in vivo.137, 138 However, the loss of PTEN function does not correlate with rapamycin sensitivity in all models. Aberrant proliferative and prosurriral signaling through the PI3K-Akt pathway may occur not only through loss of the tumor suppressor PTEN, but also through abnormal stimulation of growth factor receptors, PI3K or Akt. These protein kinases may be activated through abnormal paracrine or antocrine loops or through activating mutations and/or amplification.

Rapamycin may add to the cytotoxic effects of standard cancer drugs; however, tumor cell responsiveness to these combinations will be determined by the molecular phenotype of particular cancer cells. Lymphomas expressing Akt but not those expressing bcl-2 were sensitized by rapamycin to chemotherapy-induced apoptosis. Overexpression of the mTOR downstream target eIF-4E renders cells insensitive to concomitant administration of rapamycin and chemotherapy.139 Cancer cells overexpressing Akt that are exposed to rapamycin may be rendered sensitive to standard cancer drugs and be triggered to undergo apoptosis when exposed to the combination, but tumor cells overexpressing eIF-4E or bcl-2 may be insensitive to rapamycin and remain resistant to standard cancer therapies.

Preclinical Anticancer Activity of mTOR Inhibitors

All the rapamycins under clinical development have antiproliferative activity in a variety of hematological and solid tumor systems as single agents and in combinations with standard cancer therapeutics agents and radiation.119, 120, 121, 123, 140, 141, 142, 143, 144, 145, 146, 147, 148 Rapamycins easily cross the blood-brain barrier.119 Therefore, rapamycins may have a role as single agents and in combination with standard therapies in a variety of malignancies, including malignancies within the CNS. While the data are limited, the antitumor effects of rapamycin and its derivatives temsirolimus and everolimus appear to be similar.149, , 150, 151

Pharmacology of Rapamycin and Derivatives

Rapamycin and its derivative everolimus are orally administered, and the efficiency of absorption is modulated by p-glycoproteins (reviewed in ref. 152). Rapamycin has a terminal half-life of 62 hours in renal transplant recipients and a bioavailability of approximately 15%.152 Everolimus has a bioavailability of approximately 30% and a half-life of 30 hours. Both rapamycin and everolimus have been reported to be metabolized by liver and intestinal cytochrome P450 enzyme CYP3A4, and metabolites are mainly excreted through the gastrointestinal tract. Significant interindividual pharmacokinetic variability has been reported and may be explained by interpatient p-glycoprotein and P450 enzyme system variability. The terminal half-life for intravenous temsirolimus is 13 to 22 hours and is associated with significant metabolism to rapamycin.153 AP23573 has a median half-life of 49 hours and no appreciable conversion to rapamycin.154, 155

Toxicity of Rapamycin and Derivatives

Although rapamycins induce immunosuppression with chronic oral dosing, prolonged immunosuppression is not a desirable effect for a cancer therapeutic, particularly when combined with myelosuppressive agents. Intermittent dosing models of the rapamycins were effective in inducing tumor growth delay without causing prolonged immunosuppression.118, 156 Rapamycin and everolimus have been associated with thrombocytopenia, leukopenia, and elevated serum lipids and creatinine in organ transplant recipients. In addition, there have been rare reports of pneumonitis associated with these agents.157, 158 Temsirolimus, everolimus, and AP23573 all induce reversible mucositis and myelosuppression in cancer patients. Given the structural similarities between rapamycin and its ester derivatives temsirolimus, everolimus, and AP23573, their activity, metabolism, and toxicity profiles would be expected to overlap.

Temsirolimus (CCI-779)

Temsirolimus (Fig. 25.5) is the only rapamycin derivative for which there are both intravenous and oral formulations. Human T-cell leukemia, prostate cancer, breast cancer, small cell lung carcinoma, glioma, melanoma, and rhabdomyosarcoma cell lines were among the most sensitive to temsirolimus.118, 119, 148Results from three phase I studies in cancer patients evaluating increasing doses of temsirolimus on different schedules have been reported. The first study evaluated pharmacokinetics and biological effects of temsirolimus administered as a 30-minute IV infusion daily for 5 days every 2 weeks in doses ranging from 0.75 to 19.1 mg/m2 per day.159, 160 Grade 3 toxicities included hypocalcemia, elevation in hepatic transaminases, vomiting, and thrombocytopenia. Other toxicities were mild to moderate and included neutropenia, rash, mucositis, diarrhea, asthenia, fever, and hyperlipidemia. Hypersensitivity reactions were observed. In heavily pretreated patients, the RP2D was 15 mg/m2 per day but has yet to be determined in minimally pretreated patients. In the second study, 24 patients received temsirolimus weekly as a 30-minute infusion over a dose range of 7.5 to 220 mg/m2 per week.153 No immunosuppressive effect was reported. At 220 mg/m2 per week, dose-limiting manic-depressive syndrome, stomatitis, and asthenia seen in two of nine patients prevented further dose escalation. Mucocutaneous reactions were the most frequent drug-related toxicities. Other grade 3 or 4 toxicities included elevations of total cholesterol, triglycerides, and hepatic enzymes, as well as neutropenia, thrombocytopenia, and hypophosphatemia. Of 11 males who had normal baseline testosterone levels, 9 showed reduction of these levels, along with increased follicle-stimulating hormone and/or luteinizing hormone levels. All toxicities were reversible upon treatment discontinuation.

Figure 25.5 Chemical structure of temsirolimus (CCI-779). (Reproduced with permission from Huang S, Bjornsti MA, Houghton PJ. Rapamycins: mechanism of action and cellular resistance. Cancer Biol Ther 2003;2:222–232.)

The pharmacokinetics of temsirolimus are complex. Following treatment, temsirolimus blood levels decrease in a polyexponential manner. AUCs increase proportionally with doses up to 150 mg. At doses higher than 300 mg, aberrantly high AUCs and low clearance have been seen in some patients. The mean Vdss is large, 127 to 384 L. Clearance of temsirolimus increases with increasing dose, ranging from 19 to 51 L/hour. The mean terminal half-life for temsirolimus appears to decrease with increasing dose, from 22 hours following the 34 mg/m2 dose to 13 hours following the 220 mg/m2 dose. Significant conversion of temsirolimus to rapamycin occurs, with a mean AUC ratio (rapamycin/temsirolimus) of ~2.5 to 3.5. The mean terminal half-life for rapamycin ranges from 61 to 69 hours.

The pharmacokinetic profile and the MTD of temsirolimus administered intravenously once per week in patients with recurrent malignant gliomas taking enzyme-inducing antiepileptic drugs (EIAEDs) has also been evaluated.161 The MTD was determined to be 250 mg administered intravenously once per week. The pharmacokinetic profiles were similar to those previously described, but the AUC of rapamycin was 1.6-fold lower for patients on EIAEDs. A phase II study is ongoing to determine the efficacy of this agent at 250 mg administered intravenously once per week in patients taking EIAEDs.

A phase I study evaluating the safety and tolerability of oral temsirolimus, 25 to 100 mg daily for 5 days every 2 weeks, indicated that at the 100-mg dose level two of six patients experienced dose-limiting toxicity consisting of grade 3 stomatitis, grade 3 AST elevation, or grade 3 solar-plantar desquamative rash.162 The MTD/RP2D was 75-mg daily for 5 days. Preliminary pharmacokinetic data indicate that the oral agent undergoes moderately rapid absorption, that exposure is related to dose, and that rapamycin is a major metabolite. Phase II trials in patients with RCC, 163 breast carcinoma, 164, 165, 166 and mantle cell lymphoma (MCL)167 indicated that temsirolimus may induce objective responses and/or prolong progression-free survival (PFS) compared to historical data.

 

In the RCC phase II study, 111 patients were randomly assigned to receive 25, 75, or 250 mg of temsirolimus weekly as a 30-minute IV infusion. The reported objective response rate was 7%, and minor responses were noted in 26% of patients. Median time to tumor progression was 5.8 months, and median survival was 15 months. Neither toxicity nor efficacy was significantly influenced by dose. Eight of the nine patients treated with a single dose of 25, 75, or 250 mg had evidence of S6K activity inhibition in peripheral blood mononuclear cells (PBMCs) that was independent of the administered dose.168

A phase II trial of 109 advanced breast cancer patients, the majority having previously been treated with chemotherapy, found no significant difference in efficacy among patients who received 75- or 250-mg weekly doses.164, 165, 166 However, toxicity was higher among patients receiving 250 mg weekly. Among the 98 evaluable patients, the objective response rate was 10% (10/98), and the median response duration was 5.4 months. No patients with HER-2 negative tumors had any significant response. Four patient tumors were negative for PTEN, and three of the patients with these tumors had objective tumor responses. Three specimens had HER-2 gene overexpression as shown by Herceptin test or FISH analysis, and two of the patients with these tumors had objective responses to the drug. These findings suggest that PTEN mutation and/or HER-2 overexpression in breast cancer may predict response to mTOR inhibitors.

The most promising activity has been reported in an abstract from a phase II study in which a 250-mg dose of temsirolimus is administered intravenously once weekly to previously treated patients with MCL. An overall objective response rate of 44% was seen among 18 eligible patients.167 The 250-mg dose was not well tolerated; therefore, the trial has been modified to evaluate a lower dose (25 mg).

Two studies evaluating temsirolimus with standard agents suggest enhanced toxicity at relatively modest doses of temsirolimus. Temsirolimus given intravenously once weekly over a dose range of 5 to 25 mg with interferon-α given subcutaneously 3 times 6 million units (MU) weekly169 necessitated dose reductions or delays in 7 of 20 patients because of hyperlipidemia, leukopenia, and hyperglycemia. In the second study, patients were treated with escalating doses of temsirolimus administered intravenously followed by leucovorin (LV), 200 mg/m2 administered intravenously over 1 hour, and 5-fluorouracil (5-FU), 2, 000 to 2, 600 mg/m2 via 24-hour IV infusion.170 Stomatitis was the dose-limiting toxicity for the 75-mg/m2 dose, and 2 of 15 patients who received 45 mg/m2temsirolimus with 5-FU and LV died from mucositis with bowel perforation. No pharmacokinetic interaction between temsirolimus and 5-FU was observed. Thus it appears that enhanced toxicity may be seen with combinations of temsirolimus and some standard anticancer agents.

Everolimus (RAD001)

Everolimus is the 42-O-(2-hydroxyethyl) derivative of rapamycin (Fig. 25.6). Preclinical studies have shown dose-dependent inhibition of tumor growth and reduced tumor vascularity.171, 172, 173 Everolimus also potentiated the anticancer activity of a number of agents, including paclitaxel, gemcitabine, and gefitinib.171, 172, 173

Recently, a phase I study of everolimus administered orally 1 day a week was preliminarily reported.171 Among patients receiving doses of 5 to 30 mg once weekly, everolimus was well tolerated, with only mild to moderate anorexia, fatigue, rash, mucositis, headache, hyperlipidemia, and gastrointestinal toxicities. Pharmacokinetic results showed that exposure (AUC) increased in proportion to dose. A plateau in peak plasma concentration occurred at doses greater than or equal to 20 mg, and the terminal half-life of the agent was 26 to 38 hours. Although the MTD was not defined, at doses of 20 mg or greater, seven of eight patients exhibited inhibition of S6K1 activity in PBMCs for at least 7 days. The data from the nonclinical and clinical studies suggest that weekly administration of 20 mg everolimus in patients gives plasma concentrations and sustained S6K1 inhibition equivalent to the pharmacokinetic and pharmacodynamic changes that correlate with antitumor effects in rodents treated at an equivalent dose and schedule, 171, 172, 174 and 20 mg once weekly was identified as the initial dosage for subsequent clinical investigation.172

A phase I study evaluating everolimus combined with gemcitabine found that 600 mg/m2 gemcitabine plus 20 mg everolimus per week was not tolerated in a majority of patients due to myelosuppression. No apparent pharmacokinetic interaction was observed. The results of this trial are consistent with the experience with temsirolimus in combination with standard anticancer agents and suggest that modifications of dose and possibly schedule of administration of rapamycins may be required to optimize combination regimens.

Figure 25.6 Chemical structure of everolimus. (Reproduced with permission from Huang S, Bjornsti MA, Houghton PJ. Rapamycins: mechanism of action and cellular resistance. Cancer Biol Ther 2003;2:222–232.)

AP23573

AP23573 was identified among a series of semisynthesized phosphorus-containing, C43-modified rapamycin analogs.175 AP23573 is not a prodrug for rapamycin.175 AP23573 potently inhibits human tumor cell line proliferation in vitro and induces partial tumor regressions in several xenograft models.156Tumor mTOR signaling, measured by levels of phosphorylated ribosomal S6 protein, was completely abolished for 2 to 3 days after a single administration of 1 mg/kg AP23573. As in the case of other rapamycins, in vitro studies showed that the antitumor activity of AP23573 adds to that of cytotoxic agents such as camptothecin, cisplatin, and 5-FU (ref. 156) as well as docetaxel, doxorubicin, topotecan, and trastuzumab.176

Two phase I trials, evaluating single daily IV doses of drug for 5 consecutive days every 14 days154, 155 and single weekly doses, 177, 178 are underway. When AP23573 was administered as a 30-minute IV weekly infusion, its side effects were generally mild to moderate and reversible. The DLT was oral mucositis at 100 mg. Other grade 1 to 2 toxicities included anorexia, diarrhea, fatigue, mucositis/stomatitis, rash, and anemia. In the second study, AP23573 was administered as a 30-minute IV infusion daily for 5 days every 2 weeks in 4-week cycles.154, 155 As with the weekly schedule, dose-limiting mucositis (grade 3) occurred in two of four patients at 28 mg (140 mg total dose). Other reported toxicities included grade 3 neutropenia, thrombocytopenia, and rash and grade 1 to 2 fatigue, mucositis/ stomatitis, anemia, and leukopenia. The RP2D is 18.75 mg daily for 5 days.

Pharmacokinetic analyses in these studies showed modest interindividual variability and nonproportional increase in AUC and peak concentration with dose. Unlike temsirolimus, no appreciable rapamycin was measurable. Clearance increased with dose, consistent with the saturation of distribution sites. On the weekly schedule, the drug concentration generally remained above in vitro antiproliferation IC50 levels until the next weekly dose. The terminal half-life was 50.5 to 65.7 hours for the daily-for-5-days schedule and 39.2 to 52 hours for the weekly schedule.

Pharmacodynamic assessment of target inhibition in PBMCs was determined by measuring phospho-4E-BP1 inhibition. Phospho-4E-BP1 levels were reduced by at least 90% within 1 hour after infusion of AP23573, 179 they remained reduced by more than 70% 48 hours after dosing, and this level of inhibition persisted in some patients for 7 to 10 days. AP23573 levels greater than 10 ng/mL generally correlated with greater than 70% inhibition of phospho-4E-BP1.

Conclusions

mTOR inhibitors appear to be well tolerated, and there has been some evidence of antitumor activity in cancer patients. The most common toxicities seen are skin reactions, stomatitis, and myelosuppression. Hyperlipidemia and hyperglycemia have also been reported. These adverse effects are transient, reversible, and generally mild to moderate in severity. To date, there has been no evidence of clinically significant immunosuppression with intermittent schedules.

For temsirolimus and everolimus, the RP2Ds are below the agents' MTDs; conversely, the RP2Ds of AP23573 are the MTDs. Pharmacodynamic assays assessing inhibition of either S6K1 or 4E-BP1 phosphorylation in PBMC samples from patients treated with temsirolimus, everolimus, or AP23573 demonstrate that inhibition of mTOR kinase targets tracks with blood levels of the agents. Whether the degree and duration of inhibition in human tumors are similar to those observed in PBMCs is currently unknown. The RP2Ds of the agents appear to be within the range that can be expected to induce target inhibition based on preclinical models.

Antitumor activity has been reported among patients with a variety of malignancies with all agents. With the exception of MCL, the response rates have been low. It seems likely that only a subset of patients will have tumors sensitive to single-agent mTOR inhibitors. Limited clinical results describing correlations between objective responses seen in breast cancer patients with tumors that have HER-2 amplification or PTEN mutations suggest that the nonclinical results demonstrating correlations between enhanced signaling through the PI3K-Akt pathway may correlate with activity. Limited experience with temsirolimus or everolimus combined with cytotoxic agents suggests that doses well below the MTDs of mTOR inhibitors may be sufficient to accentuate the toxicity of standard agents. Whether antitumor activity will also be enhanced remains an unanswered question.

17-ALLYLAMINOGELDANAMYCIN (17-AAG) AND 17-(DIMETHYLAMINOETHYLAMINO)- 17-DEMETHOXYGELDANAMYCIN (17-DMAG)

Introduction

The benzoquinoid ansamycin antibiotics, first isolated from the actinomycete Streptomyces hygroscopicus var. geldanus var. nova180 include geldanamycin and its semisynthetic derivatives 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) (see Fig. 25.7 for structure of geldanamycin and its derivatives). These small molecules inhibit the chaperone function of the heat shock protein Hsp90181 and are currently being evaluated in phase I and II clinical trials. The parent compound, geldanamycin, is broadly cytotoxic in the NCI 60-cell line screen182; its poor solubility and unacceptable liver toxicity in dogs precluded testing in humans.

Figure 25.7 Structure of geldanamycin and its derivatives.

Because 17-AAG is less toxic than geldanamycin in rats183 and caused growth inhibition in breast, 184 melanoma, 185 and ovarian mouse xenograft models, the NCI initiated phase I trials in 1999. Development of 17-DMAG followed soon afterwards.

Background

In the mid-1980s, while searching for compounds to inhibit the activity of malfunctioning oncogenes, 186, 187 Uehara and his associates discovered the benzoquinone ansamycin antibiotics. These compounds were able to convert transformed Rous sarcoma virus-infected rat kidney cells to normal morphology. Although initially ascribed to inhibition of protein tyrosine kinase activity, the activity was subsequently shown to be related to binding to the 90-kd heat shock protein Hsp90, resulting in inhibition of src-Hsp90 heteroprotein complex formation.188

Heat shock proteins (Hsps, or molecular chaperones) are among the most abundant proteins found in mammalian cells.189 Hsps are highly conserved proteins necessary for the conformational stability and functional activity of a variety of client proteins that mediate various signal transduction pathways and in some cases possess oncoprotein activity. Hsp90 also activates steroid hormone receptors (e.g., estrogen and androgen). In association with several different co-chaperones, including Hsp70 and p23, Hsp90 forms heteroprotein complexes.190 The N-terminal domain of Hsp90 contains the binding site for ATP/ADP, is highly conserved, and binds geldanamycin.191, 192 Inhibition of Hsp90 leads to accumulation of misfolded client proteins that are then targeted for polyubiquitination and degradation by the proteasome.193

The geldanamycins' affinity for Hsp90′s ATP-binding pocket makes them appealing therapeutic agents for treating a variety of oncologic conditions that are driven by or express clients of Hsp90.194, 195 Figure 25.8A, Figure 25.8B(a and b) depicts this process.

To date, more than 100 client proteins of Hsp90 have been identified.196 Oncologists are particularly interested in the Hsp90 client proteins listed below.181,184, 188, 197, 198, 199, 200

·     Metastable signaling proteins (e.g., soluble kinases Raf-1, AKT, IKK)

·     Mutated signaling proteins (e.g., p53, KIT, FLT3, B-Raf)

·     Chimeric signaling proteins (e.g., NPM-ALK, Bcr-Abl)

·     Steroid receptors and bHLH/Pas domain transcription factors (e.g., androgen/estrogen/progesterone receptors, HIF-1α)

·     Transmembrane tyrosine kinases—immature versus mature (e.g., HER2, EGFR, MET, KIT, IGFR)

Mechanism of Action

The geldanamycins appear to simultaneously inhibit overlapping signaling pathways and tumor cell receptors that depend on these pathways for proliferation and survival signaling. The NCI is exploring single-agent studies in diseases known to express clients of Hsp90: melanoma, anaplastic large cell lymphoma, mastocytosis, medullary thyroid carcinoma, renal cell carcinoma, Her-2-positive breast cancer, and hormone-refractory prostate cancer.

Figure 25.8 A Hsp90 Chaperone Complex Cycling and Conformational Shuttle. A wide variety of client/receptor proteins, including oncoproteins, require chaperoned folding to achieve an active conformation for signaling and receptor/ligand interactions. 1. The client/receptor protein initially binds to Hsp40. 2. Following the initial binding of Hsp40, additional co-chaperones, such as Hsp7O, are recruited for the formation of early complexes. 3. Hsp40 is released and the binding of Hip/Hop defines the formation of the intermediate Hsp90 chaperone complex. 4. The mature Hsp90 complex is formed following the release of Hsp40 and binding of Hip. This event leads to the entry of the complex into a shuttle that is ATP dependent and maintains the client/receptor in an actively-folded conformation. The active conformation then proceeds to signaling or leads to regulated degradation through E3 ligase-mediated ubiquitination. 5. The conformational shuttle is driven by ATP exchange and hydrolysis, and occurs in response to binding of other co-chaperones (Neckers and Neckers, Expert Opin Emerging Drugs, 2002;7:277–288.)

In addition, phase I combination studies were undertaken early in development to evaluate 17-AAG's biomodulatory and molecular-targeted effects with current chemotherapy for acute myelogenous leukemia (AML) and chronic lymphocytic leukemia (CLL) and with gemcitabine/cisplatin, imatinib mesylate, bortezomib (PS-341), irinotecan, Raf-1 kinase inhibitor, rituximab, and the taxanes.

Effects of Geldanamycin Derivatives on p53, p185erbB2, and Raf-1

In vitro experiments with geldanamycin derivatives demonstrated that doses four to five times greater than the cytotoxic IC50 values (IC50 = 6–600 nM) were required to achieve maximal effects on mutant p53, p185erbB2, and Raf-1.201

Burger et al. studied melanoma xenografts that were sensitive (MEXF 276 [T/C = 6%]) or resistant (MEXF 514 [T/C = 60%]) to 17-AAG in addition to other derivative cell lines in order to better define the inhibition of Hsp90 chaperone function.185 Hsp90 was abundantly expressed in 17-AAG–responsive MEXF 276 tumors, but expressed at lower levels in 17-AAG–resistant MEXF 514 tumors and in normal tissues. Hsp90 expression diminished markedly in the sensitive MEXF 276 tissue but not in the resistant MEXF 462 tumors treated with 80 mg/kg 17-AAG.185 Apoptosis occurred concurrently with diminished Hsp90 expression in MEXF 276 tissues: the apoptotic index rose from 9 to 45% during drug treatment. When cell lines were exposed to concentrations of 17-AAG that cause total growth inhibition, a rapid decline in Hsp90 in the sensitive MEXF 276 cells was accompanied by translocation of Hsp90 from the

P.563


cytoplasm and nucleus to cell membranes. In the resistant MEXF 514, 17-AAG did not alter Hsp90 levels. After 8 hours of exposure to 17-AAG in MEXF 276 cells, Hsp90 depletion and down-regulation of Raf-1 and p185erbB2 were observed.

Figure 25.8 B The Hsp90 Chaperone/Client/Receptor Complex and Proteasome-Mediated Degradation The Hsp90 chaperone supercomplex cycles from early to intermediate to mature complex formation after client/receptor binding. Drive by ATP hydrolysis during the binding of co-chaperones including CyP40, p50cdc37, p23, immunophilins, FKBP51, 52 and Aha, the chaperone supercomplex will release the client in normoxic conditions. VHL binding proceeds E3 ligase binding, by which clients are polyubiquitinated and thereby targeted for proteasomal degradation.

Lower single-doses of 17-AAG in the 500 nM range were adequate to produce a reduction in Raf-1 protein levels detected by Western blotting at 24 hours in both HCT116 and HT29 colon adenocarcinoma cell lines.202 In addition to Raf-1 inhibition, 17-AAG inhibited constitutive MAP kinase phosphorylation detected in HCT116 cells. Raf-1 expression was evaluated in a variety of colon adenocarcinoma cells treated with iso-effective doses of 17-AAG. The minimum effective concentration of 17-AAG was defined as that sufficient to deplete Raf-1 protein at 24 hours. In addition, total and phosphorylated Erk1/2 and c-Akt were also depleted, proliferation was inhibited in a dose-dependent manner, and an increase in floating apoptotic cells was seen.203 This is the first report of 17-AAG–induced apoptosis mediated by the phosphoinositol-3-kinase/Akt pathway. With one exception, the expression of Bad, Bax, and Bag-1 was similar for all cell lines, and phosphorylation of Bad at serine-136 but not at serine-112 was inhibited by 17-AAG.

Effects of Geldanamycin Derivatives on Other Oncoproteins and Receptors

The Bcr-Abl TK is a client protein that is chaperoned by Hsp90.204, 205, 206, 207 Bhalla et al. studied the apoptotic effects of 17-AAG on Bcr-Abl levels in vitro. Intracellular expression of Bcr-Abl and c-Raf protein levels were decreased following exposure to 17-AAG, as was Akt kinase activity. The binding of Bcr-Abl shifted from Hsp90 to Hsp70 after exposure to 17-AAG and induced the proteasomal degradation of Bcr-Abl, as was previously reported by An et al.205Treatment with the proteasome inhibitor bortezomib (PS-341) and 17-AAG resulted in down-regulation of Bcr-Abl levels and inhibition of apoptosis of both the p185- and p210-expressing cell lines. Shiotsu et al. reported that an oxime derivative of radicicol (another Hsp90 inhibitor distinct from 17-AAG), when used as a single agent, induced erythroid differentiation in K562 leukemia cells through destabilization of Bcr-Abl association with Hsp90.206 Further, Blagoskonny et al. demonstrated that Hsp90 inhibition with quite low doses of geldanamycin (90 nM), while not apoptotic on its own, selectively sensitized Bcr-Abl–expressing leukemia cells to cytotoxic chemotherapeutic agents such as doxorubicin and paclitaxel.207 Imatinib mesylate–sensitive and imatinib mesylate–resistant Bcr-Abl–positive acute and chronic myelogenous leukemia cells both exhibit a reduction in Bcr-Abl expression when treated with 17-AAG.208, 209

Growth inhibition has been observed after treatment with 17-AAG in several breast cancer cell lines with differing levels of oncogene p185erbB2 expression. Cells with wild-type and mutated Rb appear to induce apoptosis differentially after exposure to 17-AAG.210, 211, 212 Thus, the Rb context of the breast cancer cellular environment seems to dictate response. The breast cell lines with p185erbB2amplification were 10- to 100-fold more sensitive to treatment with 17-AAG. Apoptosis occurred earlier and at lower concentrations in cells overexpressing p185erbB2. The p185erbB2 expression was down-regulated to almost undetectable levels 24 hours following treatment with 17-AAG. Down-regulation was associated with a number of cell cycle effects:

·     arrest of cells predominantly in G1

·     accumulation of hypophosphorylated Rb

·     down-regulation of cyclin D expression

·     transient differentiation characterized by flattening and enlarging of the cytoplasm, intracellular lipid accumulation, and induction of milk fat globule proteins with apoptosis210, 211, 212

17-AAG–mediated inhibition of the Akt kinase–dependent pathway resulted in a posttranscriptional decrease in cyclin D.210 17-AAG does not affect PI3 kinase. Although phosphorylated AKT and Akt kinase activities were lost within 30 minutes of treatment with 17-AAG, this preceded the change in Akt protein in cells with high p185erbB2 Akt protein expression. However, activated Akt decreased gradually over 24 hours in breast cancer cells with low p185erbB2 expression. Differentiation was not seen in Rb-negative cells, although these cells did undergo apoptosis following drug treatment.210, 211 Transfection of Rb into cells that are not replete with Rb led to G1 block, differentiation, and apoptosis. These studies suggest that the geldanamycins will be most effective in specific tumors and cellular environments in combination with other drugs.

The 17-AAG (200 nM)–mediated G1 arrest observed in wild-type Rb-positive non–small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cell lines resulted in the loss of CDK4, CDK6, and cyclin E expression following a 6-hour exposure, but no changes in the levels of D-type cyclins were observed.213 Rb-negative lung cancer cell lines did not undergo G1 arrest when exposed to 17-AAG at concentrations from 200 nM to 5 µM.

Using MCF-7 and T47D human breast cancer cells, Bagatell et al. demonstrated that steroid receptors were destabilized and depleted by 17-AAG, geldanamycin, and KF58333.214 Estrogen-supplemented, T47D-bearing SCID mice exhibited a marked reduction of progesterone receptor levels in the uterus and tumor following intraperitoneal (IP) administration of 17-AAG (75-mg/kg IP dose per day for 2 days). Three-week growth delay, after treatment with a 50-mg/kg IP dose per day for 5 days, was observed in established hormone-responsive MCF-7 and T47D xenografts. The role of 17-AAG–mediated alterations in steroid hormone receptor levels and clinical activity of 17-AAG will need to be established in future studies.

Nonclinical Rationale for Disease-Specific and Combination Studies

An extensive body of nonclinical work provides the rationale for clinical combination studies of the geldanamycins. The concepts supporting a combination of 17-AAG with imatinib mesylate were reviewed above. Combining the geldanamycins with other agents documented to be active in a specific disease seem rational under the following circumstances:

·     the specific disease expresses client proteins.

·     the specific disease relies on a client protein in a specific pathway.

·     the disease is known to have a molecularly targeted critical pathway.

Preliminary results from colony-forming assays used to investigate the effects of 17-AAG and cytotoxic agents in two ovarian cell lines, A2780 and SKOV3, show that drug interactions with 17-AAG are both drug-specific and cell line–specific.215 17-AAG alone caused a G1 block in A2780 cells and inhibited cell cycle progression in G1-synchronized SKOV3 cells but did not produce a G1 block in SKOV3 cells growing asynchronously. In both cell lines, 17-AAG and gemcitabine were generally antagonistic, while doxorubicin and docetaxel were generally additive with 17-AAG. 17-AAG and cisplatin were additive in A2780 cells and mildly synergistic in SKOV3 cells, whereas 17-AAG and etoposide were antagonistic in A2780 cells and moderately synergistic in SKOV3 cells.

17-AAG and cisplatin were additive in the colon adenocarcinoma cell line HCT116 but antagonistic in the colon cell line HT29.216 17-AAG and cisplatin were also antagonistic in HCTp5.2 cells, which express dominant negative p53. Induction of c-Jun by cisplatin was unaffected by 17-AAG in HCT116 cells and greatly diminished in HT29 cells. These preliminary results suggest that the combination of 17-AAG and cisplatin is additive in colon cancer cells with normal p53 but antagonistic in p53-deficient lines. Interference with cisplatin-induced apoptosis is a possible reason for 17-AAG's antagonistic action in HT29 cells.

The ability of 17-AAG to sensitize NSCLC cells expressing high levels of p185erbB2 to paclitaxel-mediated growth arrest and apoptosis was sequence-dependent.217 Exposure of NSCLC cell lines with low (H460 and H1299) or high (H358, H322, H661, H522) constitutive levels of p185erbB2 to 17-AAG (20 or 80 nM) for 24 hours resulted in a significant dose-dependent reduction of p185erbB2 levels in all lines. Exposure of the NSCLC cells to 17-AAG for 96 hours resulted in a dose-dependent inhibition of NSCLC cell proliferation, with estimated IC50 values of 60 to 180 nM. With concurrent exposure to 17-AAG and paclitaxel, the paclitaxel IC50 was significantly reduced (P ≤ 0.001) in cell lines that had high constitutive levels of p185erbB2. The cytotoxic effects were synergistic in cells expressing high levels of p185erbB2 but only additive in cells expressing low levels of p185erbB2. However, when cells were pretreated with 17-AAG for 24 hours before concurrent exposure to both agents, the paclitaxel IC50 values were increased. Flow-cytometric analysis revealed that 17-AAG induced a G1 phase arrest in NSCLC cells, which may have rendered these cells refractory to the cytotoxic effects of subsequent paclitaxel treatment, since cells are resistant to microtubule damage during the G1 phase.218

The tumoricidal and antiangiogenic effects of the paclitaxel–17-AAG combination were evaluated in H358 tumor xenografts (a lung cancer line expressing high levels of p185erbB2) in nude mice.219, 220 VEGF and p185erbB2 levels were reduced 2-fold and 3-fold, respectively, in H358 cells exposed to 17-AAG, and sensitivity to paclitaxel was enhanced 3- to 25-fold. Apoptosis induction was significantly increased in cells treated with the combination compared with cells treated with paclitaxel alone (32.6% vs. 9.5%, P = 0.02). Combinations of 17-AAG (10 or 25 mg/kg) and paclitaxel (1 mg/kg) were administered intraperitoneally per week for 4 weeks to nude mice bearing H358 tumor xenografts. The survival of animals treated with the combination was significantly longer (P < 0.01) than the survival of animals treated with 17-AAG or paclitaxel alone. VEGF expression and a strong reduction of capillary density occurred in tumors treated with either 17-AAG or 17-AAG–paclitaxel; however, a substantial degree of apoptosis was noted only in tumors treated with the combination. The antiproliferative and apoptotic effects induced by paclitaxel or doxorubicin in breast cancer cells with high expression of p185erbB2 were enhanced at concentrations of 17-AAG that down-regulated Akt kinase.210 In cells with intact Rb, pretreatment with 17-AAG decreased paclitaxel-induced apoptosis; however, apoptosis was enhanced when 17-AAG was given after paclitaxel. The schedule dependency was not observed in Rb-negative cells or with doxorubicin.

The Chk1 pathway has emerged as a critical moderator of cellular responses activated by replication stress and various types of DNA damage.221, 222 Chk1, an Hsp90 client, is lost when cells are treated with 17-AAG. When U937 monocytic leukemia cells are treated concurrently with minimally toxic concentrations of 17-AAG (400 nM) and UCN-01 (a Chk1 inhibitor; 75 nM), apoptosis as a result of mitochondrial injury occurred.223 Arlander et al. showed that the combination of gemcitabine and 17-AAG was markedly more effective at killing ML-1 cells than either drug alone.224

Recently, Schrump et al. reported that acetylation is a critical regulator of Hsp90 function.225 Although histone deacetylase inhibitors (HDACIs) are so named because they induce hyperacetylation of the amino-terminal lysine residues of core nucleosomal histones, causing chromatin remodeling and altered gene expression, 226 HDACIs also promote hyperacetylation of various nonhistone proteins, including Hsp90. For example, Schrump et al. showed that, in NSCLC cells, the depsipeptide HDACI FR901228 induced Hsp90 hyperacetylation, antagonized the binding of ATP to Hsp90, remodeled Hsp90 co-chaperone complexes, and promoted ubiquitin-dependent and proteasome-dependent degradation of several Hsp90 client proteins, including mutated p53, ErbB1, ErbB2, and c-Raf.225 Bhalla et al. have confirmed and extended these initial observations, demonstrating that, similar to FR901228, the HDACI LAQ824 induced Hsp90 acetylation, inhibited its binding to ATP, and stimulated enhanced degradation of the Hsp90 clients ErbB2, Akt, and c-Raf-1227 as well as Bcr-Abl.204, 228 17-AAG has been reported to act synergistically with HDACIs such as suberoylanilide hydroxamic acid (SAHA) to induce mitochondrial damage, caspase activation, and apoptosis in HL-60 human promyelocytic and Jurkat lymphoblastic leukemia cells.229, 230

Oxygen homeostasis is critical in the cancer cell environment and is tightly regulated in both tumor and normal tissues. Regulation occurs through hypoxia-inducible factor 1 (HIF-1) and the highly homologous HIF-2 proteins. HIF-1 is an Hsp90 client protein.231 HIF-1 proteins function as nuclear transcription factors and transactivate numerous target genes, many of which are implicated in the promotion of angiogenesis, such as VEGF, and in the adaptation to hypoxia.232, 233 These labile proteins are expressed in low concentrations in normoxic cells; their stability and activation increase severalfold in hypoxic conditions. Von Hipple-Lindau factor (VHL)–mediated protein binding in normoxia accounts for the instability. HIF is not degraded in hypoxic conditions because VHL function is compromised.

Both the geldanamycins and radicicol, another Hsp90 inhibitor, diminish the transcription of HIF-induced downstream effectors like VEGF231.234, 235, 236Proteasomal degradation of HIF is critical to the modulation of its downstream effectors. Geldanamycin decreases HIF expression by promoting the protein's VHL-independent proteasomal degradation.235 Geldanamycin has been shown to induce proteosomal degradation of HIF-1α in prostate cancer cells237 and to block HIF-1 induction, leading to the diminished cellular migration of U78MG, LN229, and U251MG glioma cells.238

The c-Met receptor TK and its ligand, hepatocyte growth factor (HGF), play a pivotal role in angiogenesis, cellular motility, differentiation, growth, and invasion in a variety of solid tumors, 239 including colorectal cancer (CRC)240, 241, 242, 243 and SCLC.244 The geldanamycin analogs have been shown to disrupt the c-Met/HGF axis.245 Treatment of VoLo human colon cancer cell lines with c-Met antisense oligonucleotides decreased c-Met protein levels, leading to programmed cell death246; however, the effectiveness of CRC treatment via inhibition of Hsp90-c-Met interactions by the geldanamycins has not been studied yet. No direct association was seen between Hsp90 and c-Met itself.

Oncogene-producing, dominant, gain-of-function mutations of receptor protein tyrosine kinases (RTKs) can confer uncontrolled proliferation, disordered differentiation, or uncontrolled survival through activation of multiple downstream signaling cascades.247 These mutated oncogenic RTKs depend on Hsp90 for appropriate folding and activity. One such gain of function mutation involves the RET receptor TK and is associated with human cancer and several human neuroendocrine diseases. Point mutations of RET are responsible for multiple endocrine neoplasia type 2 (MEN2A, MEN2B) and familial medullary thyroid carcinoma (FMTC). Somatic gene rearrangements juxtaposing the TK domain of RET to heterologous gene partners are found in papillary carcinomas of the thyroid (PTC).247, 248, 249

To study inhibition of RET TK activity using growth inhibition assays in MTC cells, the activities of two TK inhibitors and 17-AAG were tested in the TT MTC cell line.250 Following treatment with 200 µM genistein, a soy-based inhibitor in the Ras/PI3K pathway, or 6 µM 17-AAG for 48 hours, RET TK activity was inhibited by 87% and 72%, respectively. At the highest drug concentrations tested (100 µM imatinib mesylate, 139 µM genistein, and 3.41 µM 17-AAG), TT cell proliferation at 48 hours was inhibited by 89%, 90%, and 94%, respectively. Thus, 17-AAG, as well as other Hsp90 inhibitors, is an attractive pharmacologic agent for use in systemic therapy in patients with recurrent metastatic MTC for which nonsurgical therapy has been ineffective.

Metabolism

In human or murine hepatic microsome assays, 17-aminogeldanamycin (17-AG), a diol, and an epoxide are the three major metabolites of 17-AAG.251 The 17-AAG diol was the major metabolite in human hepatic microsomes, followed by 17-AG; in contrast, 17-AG was the most abundant metabolite in murine microsomes. Acrolein, a nephrotoxin, is a potential by-product of the 17-AG metabolite. Finally, the epoxide is probably formed by addition of oxygen across the double bond of the allylamino side chain. CYP3A4 enzymatic metabolism is responsible for 17-AG and epoxide formation. Microsomal epoxide hydrolase catalyzes the conversion of the diol to 17-AG, which does not undergo further microsomal metabolism. 17-AAG metabolites are active and may have clinical significance. The biologically active epoxides and acrolein may induce toxic effects in humans.251 Pharmacodynamic studies show that the 17-AG metabolite is as active as 17-AAG in decreasing cellular p185erbB2 in human breast cancer SKBr3 cells in culture.252 17-AG causes growth inhibition in six human colon cancer lines and three ovarian cancer cell lines.253

The quinone-metabolizing enzyme DT-diaphorase may alter 17-AAG's antitumor activity and toxicologic properties.253 17-AAG growth inhibitory activity was increased 32-fold by transfection of the active DT-diaphorase gene NQO1 into the DT-diaphorase–deficient BE human colon carcinoma cell line, and concomitant depletion of Raf-1 and mutant p53 protein confirmed the Hsp90 inhibition mechanism of action. Increased growth inhibition was not observed with the parent compound, geldanamycin. The increased sensitivity to 17-AAG in cell lines transfected with NQO1 was also seen in xenograft models.

In contrast to 17-AAG, 17-DMAG appears to be only minimally metabolized by CYP3A4.254 Therefore, intestinal CYP3A4 should not impede 17-DMAG's oral activity. 17-AG does not appear to be a metabolite of 17-DMAG based on the lack of conversion at the 17 position of the compound. The marked metabolic differences between 17-AAG and 17-DMAG suggest that they may have distinct toxicity profiles and therapeutic indices.

In vitro Antitumor Studies

17-AAG exhibited a clear differential pattern of activity in the NCI in vitro cancer screen, with a mean concentration causing 50% growth inhibition (GI50) of 0.19 µM and a mean concentration resulting in total growth inhibition value of 18.7 µM. Overall, the melanoma cell line panel was the most sensitive to 17-AAG, but greater than average sensitivity was demonstrated by individual cell lines in the leukemia, NSCLC, colon, ovarian, breast, prostate, and renal cancer panels.

Banerji et al. describe studies designed to provide the basis for selection of molecular pharmacodynamic markers for a clinical trial of 17-AAG.255 The CML cell line K562 and normal human peripheral blood lymphocytes/ mononuclear cells (PBMCs) were treated with 17-AAG in vitro, and client proteins were measured by Western blotting. Raf-1 and the src family kinase member Lck were depleted following 24 hours of exposure to ≥500 nM 17-AAG. Hsp70 was also induced in response to 17-AAG. Hsp70, Hsp28, and Hsf1 were induced in K562 cells by geldanamycin, suggesting that the functional inactivation of Hsp90 may stimulate the expression of heat shock proteins in this cell line through activation of Hsf1.256 When nude mice bearing human ovarian cancer xenografts were treated with a single IP dose of 17-AAG (80 mg/kg), client protein changes were observed at 24 hours in A2780 but not in CH1 tumors.255

Four cell lines (H460, H358, H322, and H661) that express varying levels of ErbB1 (EGFR) and ErbB2 (HER2/neu) oncogenes were assayed for expression of these oncogenes, the cell adhesion molecule E-cadherin, secretion of the matrix metalloproteinase 9 (MMP-9), VEGF, and their ability to invade Matrigel after 48-hour exposure to 17-AAG.257 17-AAG significantly depleted erbB1 or erbB2 levels in NSCLC cells expressing high levels of these proteins and effectively inhibited their growth, with IC50 values ranging from 50 to 90 nM. Drug treatment also enhanced E-cadherin expression in H322 and H358 cells and inhibited secretion of MMP-9 and VEGF secretion by tumor cells. 17-AAG diminished hypoxia-induced up-regulation of VEGF expression as well as growth factor–mediated augmentation of MMP-9 secretion. It profoundly inhibited the ability of H322 and H358 cells to migrate through Matrigel in response to chemoattractant. Thus, 17-AAG's ability to inhibit the metastatic phenotype of lung cancer cells may make it a novel pharmacologic agent for specific molecular intervention in lung cancer.

Recently, Mimnaugh et al. showed that combining a low dose of 17-AAG (50 nM) with a low and clinically achievable dose of the proteasome inhibitor Velcade (PS-341, bortezomib) resulted in a markedly enhanced cytotoxicity that was correlated with a dramatic accumulation of insoluble polyubiquitinated proteins.258 These investigators also demonstrated that transformed cells were much more sensitive to this drug regimen than were nontransformed cells, suggesting that the combination of an Hsp90 inhibitor and a proteasome inhibitor may prove particularly toxic to cancer cells.

In Vivo Antitumor Studies

Antitumor activity of 17-AAG has been demonstrated in melanoma, 185 breast, 259 ovarian, 255 and colon253 xenograft models.

Mice with established BT-474 (high-level p185erbB2 expression) xenografts were treated with 17-AAG (20, 50, or 75 mg/kg) by daily IP injection for 5 days every 3 weeks.260, 261 Dose-dependent growth inhibition occurred without excess toxicity in the 17-AAG–treated animals. Maximum tumor regression (58%) at the 75 mg/kg–dose level was noted on day 25. 17-AAG cleared rapidly, with no drug detectable in serum by 10 hours. Expression of p185erbB2 and phosphorylated AKT decreased 1 hour after a single IP dose of 17-AAG 50 mg/kg, and the loss persisted 24 hours posttreatment. Total AKT protein was not changed.

Mice with established CWR22 prostate cancer xenografts using both the androgen-dependent parental line and androgen-independent sublines have been treated with 5-day cycles of 17-AAG (25 or 50 mg/kg) or vehicle alone.262 Dose-dependent growth inhibition was noted without excess toxicity in the treated groups using both intermittent and continuous dosing schedules. A 74% loss of p185erbB2 and a 58% loss of AR expression were noted 2 hours posttreatment.

The growth of C6 glioma xenografts in nude mice263 was greatly inhibited when 17-AAG (80 mg/kg) was injected intraperitoneally for 9 days (4 days the first week and 5 days the second week), starting 13 days after C6 cell implantation. The differences in tumor volumes between the 17-AAG group and a vehicle control group were significant (P = 0.017) at all time points from day 18 to day 35. The mean tumor volume of the 17-AAG group on day 35 (the last day tumor volume was measured) was less than one third that of the tumor volume of the vehicle control group.

Combination with Radiation

Exposure of human prostate tumor cells (LNCaP), grown as spheroids, to 17-AAG (100 or 1, 000 nM) for 96 hours before or after LET (low linear energy transfer), high dose rate irradiation (2 or 6 Gy) demonstrated supra-additive sequence-dependent responses.264 Although incubation with 17-AAG or irradiation alone resulted in growth delays, all spheroids, except 6 of 23 exposed to 1, 000 nM 17-AAG, regrew to control volumes after the delay. After the sequence consisting of 6 Gy of radiation followed by 100 nM 17-AAG, 5 of 12 spheroids failed to regrow, and after the reverse sequence (100 nM 17-AAG followed by 6 Gy of radiation), 10 of 12 failed to regrow. After the 1, 000 nM 17-AAG and 6 Gy sequences, all spheroids failed to regrow. Similarly, Bisht et al. reported that 17-AAG and 17-DMAG are both potent radiosensitizers in vitro and in vivo.265 These investigators showed that treatment of two human cervical carcinoma cell lines with these Hsp90 inhibitors resulted in an enhanced response to radiation within 6 to 48 hours after drug exposure. In addition, clinically achievable amounts of 17-AAG dramatically sensitized tumor xenografts to single and fractionated courses of irradiation and enhanced programmed and nonprogrammed cell death in the tumors.

Toxicity

Single dose range–finding, multiple dose range–finding, 5-day daily dose, and multiple-dose/DMSA formulation toxicity studies have been conducted in rats. Additionally, single dose range–finding/microdispersed formulation, multiple dose range–finding reconstituted lyophilized formulation, and 5-day daily dose with microdispersed and DMSA-formulated 17-AAG have been conducted. In those studies, the following trends were noted:

·     Doses of geldanamycin exceeding 5 mg/kg in rats were generally toxic, leading to death.

·     A single dose of microdispersed 17-AAG could be given in doses up to 25 mg/kg in both rats and dogs; the MTD when given daily for 5 days was 25 mg/kg per day for rats and 7.5 mg/kg per day for dogs.

·     Lyophylized 17-AAG was tolerated in rats at doses up to 30 mg/kg when given daily or twice daily and in dogs at doses of 10 mg/kg per day.

·     Hepatotoxicity, renal failure, and gastrointestinal toxicities (mainly emesis and diarrhea) were the DLTs in both species. Dogs also experienced gallbladder toxicities.

For 17-DMAG, similar studies were conducted, with the following results:

·     When given intravenously or intraperitoneally, the maximum daily dose was 12 to 15 mg/m2 per day in rats and 8 mg/m2 per day in dogs.

·     The main DLTs in both species were renal, gastrointestinal, hepatobiliary, and bone marrow effects.

Nonclinical Pharmacokinetics

Plasma pharmacokinetics (PK) of 17-AAG were measured by HPLC following IV administration of 27, 40, or 60 mg/kg to CD2F1 mice.266 By noncompartmental analysis, AUCs (402, 625, and 1, 739 µg/mL.min, respectively) increased proportionally for the lower doses, but a greater than linear increase was observed at the highest dose. Analysis with the trapezoidal function gave more linear AUCs: 375, 624, and 1, 373 µg/mL.min, respectively, for the doses used. Total body clearance varied from 34.5 to 66.3 mg/kg per minute. The plasma data were best approximated by a two-compartment, open, linear model. Terminal half-lives (t½)were 73, 87, and 361 minutes following doses of 27, 40, and 60 mg/kg, respectively. In dogs given 1-hour IV infusions of 2 to 10 mg/kg per day for 5 days, the mean t½ was dose-independent and ranged from 46 to 73 minutes.267

In a preliminary report of studies performed in normal mice and SCID mice bearing MDA-MB-453 xenografts, both 17-AAG and 17-AG levels were below detection in normal tissues 7 hours after a single injection of 40 mg/kg 17-AAG.259 However, 17-AAG and 17-AG levels were 0.5 to 1 µg/g in tumor tissue for more than 48 hours.

The pharmacokinetic-pharmacodynamic relationships for 17-AAG were investigated in nude mice bearing human ovarian cancer xenografts CH1 and A2780.268,269 Following a single IP dose of 80 mg/kg, the half-lives in plasma, liver, and tumor were 0.88, 0.86, and 7.5 hours in the A2780-bearing mice and 0.89, 1.73, and 3.86 hours in the CH1-bearing mice, respectively, confirming other reports of differential drug accumulation in tumor.268 There was no tumor response on the single-dose regimen, and Western blotting showed a minimal induction of Hsp70 at 16 to 24 hours in A2780 xenografts but not in CH1 xenografts. Tumor response was obtained with multiple dosing. The growth delay was greater in A2780 tumors (6.8 days) than in H1 tumors (2 days), and tumor growth resumed 2 to 4 days after dosing ceased. On day 4, expression of Raf-1, Lck, and CDK4 was reduced and expression of Hsp70 was increased in the mouse peripheral blood leukocytes (PBLs).269 With the exception of Lck, which is not expressed in A2780 tumors, these changes were mirrored in the tumor tissue. These preliminary reports suggest that the use of PBLs to measure pharmacodynamic endpoints may be possible in clinical trials. The same markers are being used to guide the phase I study of 17-AAG at the investigator's institution.

Clinical Pharmacology

Plasma PKs were described in patients entered in a phase I trial of 17-AAG given daily for 5 days every 3 weeks.270, 271, 272

One patient each was treated at dose levels of 10, 14, 20, and 28 mg/m2, eight patients at 40 mg/m2, and seven patients at 56 mg/m2. A two-compartment, open model best fit the PK data.271 Mean values for terminal t½, clearance, and Vdss were 2.5 ± 0.5 hours, 41.0 ± 13.5 L/hour, and 86.6 ± 34.6 L/m2, respectively. Peak plasma concentrations reached 3, 170 ± 1, 310 nM at 56 mg/m2.271 Using noncompartmental analysis of data from patients treated with 56 mg/m2, the average values for 17-AAG and 17-AG, respectively, were as follows: Cmax = 2, 080 and 770 nM; AUC = 6, 708 and 5, 558 nM.hour; terminal t½ = 3.8 and 8.6 hours.272 Clearances of 17-AAG and 17-AG were 19.9 and 30.8 L/m2 per hour, and the Vdss values were 92 and 203 L/m2, respectively. Over all dose levels, the total amount of drug recovered in urine was 10.6% for 17-AAG and 7.8% for 17-AG. There were no significant differences between day 1 and day 5 PK values. The MTD was 40 mg/m2, a dose at which Hsp90 inhibition would be expected. Another phase I trial that used the same daily 3 5 schedule provided PK data for the 80 mg/m2 dose.273 The t½ was 1.5 hours, and the peak plasma level was 2, 700 nM at 30 minutes. Plasma levels at 1, 6, 24, 72, and 96 hours were 1, 930, 190, 36, 63, and 57 nM, respectively. For the active metabolite, 17-AG, the t½ was 1.75 hours, and the peak plasma level was 607 nM at 1 hour. 17-AG plasma levels at 0.5, 6, 24, 72, and 96 hours were 262, 138, 46, 101, and 39 nM, respectively. Thus, concentrations exceeded in vitro and xenograft concentrations of 10 to 500 nM for cell kill.

TABLE 25.3 PHARMACOKINETIC DATA FOR 17-AAG

Schedule

d×5

 

 

d×5

qwk×3 q4wk

qwk

 

Parameter (units)

10–56 mg/m2

40 mg/m2

56 mg/m2

80 mg/m2

15–112 mg/m2

10–450 mg/m2

450 mg/m2

17-AAG

Sample size

n = 15

n = 5

n = 7

n = 1

n = 9

n = 22

NA

Cmax (nM)

530–3170

1860 ± 660

2080

2700 (30 min)

16710

AUC(INF)(nM.h)

6708

t½ (hr)

2.5 ± 0.5

3.8

1.5

2.8

CL

41.0 ± 13.5 L/hr

19.9 L/hr/m2

26.6 (12.5–293.1)L/hr/m2

47.3 L/hr

Vdss (L/m2)

86.6 ± 34.6

92

186 L

72-hr urinary recovery

10.6%

Reference

(271, 272)

(271)

(272)

(273)

(274, 275)

(276)

(276)

17-AG

Sample size

n= 1

Cmax (nM)

770

607 (60 min)

AUC(INF) (nM.hr)

5558

t½ (hr)

8.6

1.75

4.6

CL L/hr/m2

30.8

Vdss(L/m2)

203

72-hr urinary recovery

7.8%

Reference

(272)

(272)

(273)

NA, not available.

TABLE 25.4 DCTD, NCI–SPONSORED 17-AAG AND COMBINATION PHASE I CLINICAL TRIALS

No. of Patients Planned/ Disease Type

Agent(s)

Dose/Schedule

Toxicities

70/Solid tumor

17-AAG

Dose escalation from 150 mg/m2 (level 1) to 480 mg/m2 (level 5) IV twice/wk for 2 wks followed by a 1 wk rest for patients with solid tumors; in adult leukemia patients, the rest is omitted

 

36/Solid tumor

17-AAG

Dose escalation from 150 mg/m2 (level 1) to 480 mg/m2 (level 5) IV twice/wk for 2 wks followed by a 1 wk rest for patients with solid tumors; in adult leukemia patients, the rest is omitted

38/Solid tumor

17-AAG

Dose escalations from 40 mg/m2/day (level 1) to 301 mg/m2/d (level 7) given on days 1, 4, 15, and 18 of a 4-wk cycle

Gr 2 hepatitis, gr 3 nausea, dypsnea

96/Solid tumor and refractory hematological malignancies

17-AAG

150 mg/m2 twice/wk for 12 wks and escalated by 40% with each cohort; an MTD is defined independently for each population

Nausea, vomiting secondary to pancreatitis, and gr 3 fatigue

24/Solid tumor

17-AAG

220 mg/m2/wk for 12 wks, escalating to 700 mg/m2/wk

Gr 3 reversible hepatitis

130/Solid tumor

17-AAG

Cohort 1: from 10 mg/m2/dose to 603 mg/m2/dose on days 1, 8, and 15 in a 28-day cycle
Cohort 2: from 10 mg/m2/dose to 603 mg/m2/dose on days 1, 4, 8, and 11 in a 21-day cycle

Gr 4 elevated SGOT, dypsnea, hypoxia

66/Solid tumor

17-AAG, gemcitabine and cisplatin

Cohort A: 17-AAG 154 mg/m2 IV over 1 hr on days 1 and 8, every 21 days; gemcitabine 500 mg/m2 IV over 30 min on days 1 and 8, every 21 days; cisplatin 30 mg/m2 IV over 2 hrs on days 1 and 8, every 21 days
B, C, D: An MTD of 17-AAG 154 mg/m2 IV over 1 hr on days 1 and 8, every 21 days; gemcitabine 750 mg/m2 IV over 30 min on days 1 and 8, every 21 days; cisplatin 40 mg/m2 IV over 2 hrs on days 1 and 8, every 21 days

30/Solid tumor

17-AAG and docetaxel

Schedule 1: Docetaxel 55 to 75 mg/m2 IV over 1 hr on day 1, every 21 days; 17-AAG 80 to 650 mg/m2 IV over 1 hr on day 1, every 21 days
Schedule 2: Docetaxel 35 mg/m2 IV over 1 hr on days 1, 8, and 15 every 28 days; 17-AAG 160 to 450 mg/m2 IV over 1 hr on days 1, 8, and 15 every 28 days

35/Solid tumor

17-AAG and paclitaxel

17-AAG 100 to 225 mg/m2 IV over 1 hr on days 1, 4, 8, 11, 15, and 18 every 28 days
Paclitaxel 80 mg/m2 IV over 1 hr on days 1, 8, and 15 every 28 days

18/CML

17-AAG and imatinib

Imatinib 600 mg/day PO once a day started 4–5 days prior to the first 17-AAG treatment; 17-AAG 20 to 60 mg/m2 days 1 and 4 of wks 1 and 2, each 3 wks

Just opening

36/ALL

17-AAG and cytarabine

Cytarabine 400 mg/m2/day CIV days 1–5; 17-AAG 100 mg/m2 to 400 mg/m2 IV over 60 min on days 3 and 6; repeat 30 ± 5 days after marrow recovery or hospital discharge

30/CLL

17-AAG, fludarabine and rituximab

17-AAG 100 to 360 mg/m2 IV over 60 min on days 1, 4, 8, 11, 15 and 18 of a 28-day cycle; fludarabine 25 mg/m2 IVPB will be administered on days 1–5; rituximab day 1, cycle 1, 100 mg IVPB over 4 hrs; day 3, cycle 1, 375 mg/m2 using standard escalation; day 5, cycle 1, 375 mg/m2 using standard escalation

74/Hematologic unspecified

17-AGG and bortezomib

17-AAG 100 mg/m2 to 250 mg/m2 administered over 1 hr immediately prior to PS-341 0.7 to 1.3 mg/m2 on days 1, 4, 8, and 11 of each cycle

42/Solid tumor

17-AAG and bortezomib

17-AAG 100 mg/m2 to 250 mg/m2 administered over 1 hr immediately prior to PS-341 0.7 to 1.3 mg/m2 on days 1, 4, 8, and 11 of each cycle

27/Solid tumor

17-AAG and BAY 43-9006

BAY 43-9006 400 mg BID starting 2 wks prior to 17-AAG 100 mg/m2to 250 mg/m2 on days 1, 8, and 15 every 28 days

46/Solid tumor

17-AAG and irinotecan

Irinotecan 85 mg/m2 to 125 mg/m2 followed by 17-AAG 220 mg/m2to 450 mg/m2 once/wk for 2 wks in a 21-day cycle

 

TABLE 25.5 DCTD, NCI–SPONSORED 17-AAG PHASE II CLINICAL TRIALS

No. of Patients Planned/Disease Type

Dose/Schedule

40/Ovarian epithelial cancer stage IV

17-AAG 220 mg/m2 IV over 1 hr on days 1, 4, 8, and 11, every 21 days

26/Clear cell carcinoma of the kidney

17 AAG 300 mg/m2 IV over 1–2 hrs on days 1, 8, 15, every 28 days

36/Malignant mast cell neoplasm

17-AAG 220 mg/m2 IV over 1 hr on days 1, 4, 8, and 11, every 3 wks

58/Renal cell carcinoma stage IV

17-AAG 220 mg/m2 IV over 60–90 min twice/wk for 2 wks; cycle = 21 days

50/Malignant melanoma stage IV

17-AAG 450 mg/m2 IV over 1–2 hr every wk × 6 wks, every 8 wks

72/Medullary thyroid cancer

17-AAG 220 mg/m2 IV over 1 hr on days 1, 4, 8, and 11, every 21 days

25/Malignant melanoma stage IV

17-AAG 450 mg/m2 IV over 1 hr once every 7 days, for 12 wks

41/Breast cancer stage IV

17-AAG 220 mg/m2 IV over 1 hr on days 1, 4, 8, and 11, every 21 days

28/Prostate cancer stage IV

17 AAG 300 mg/m2 IV over 1–2 hrs on days 1, 8, 15, every 28 days

70/Mantle cell lymphoma

17-AAG 220 mg/m2 IV over 1 hr on days 1, 4, 8, and 11, every 21 days

TABLE 25.6 DCTD, NCI–SPONSORED 17-DMAG PHASE I CLINICAL TRIALS

No. of Patients Planned/Disease Type

Dose/Schedule

30/Solid tumor

17-DMAG 2.5 mg/m2 to 40 mg/m2 IV wkly × 3

40/Solid tumor

17-DMAG 1mg/m2 to 40 mg/m2 IV over 1 hr on days 1 and 4 each wk; cycle = 4 wks

30/Solid tumor

17-DMAG 1.25 mg/m2 to 10 mg/m2 IV over 1 hr each wk; cycle = 4 wks

60/Solid tumor

17-DMAG 1.5 mg/m2 to 19 mg/m2 IV over 1 hr daily × 5, every 21 days

Preliminary PK and pharmacodynamic data have also been reported from a phase I trial of 17-AAG given once weekly for 3 out of every 4 weeks.274, 275 The median clearance of 17-AAG from plasma samples (n = 9) drawn on day 1 was 412 mL/m2 per minute (range 208 to 4, 885). The Cmax increased linearly with dose, and the t½ was 166 ± 115 minutes.275 The t½ for 17-AG was 277 minutes (4.6 hours). 17-AAG was a substrate for both the CYP3A4 and CYP3A5 enzyme systems.275

Pharmacokinetic data for 17-AAG from NCI-sponsored trials are summarized in Table 25.3.

Clinical Trial Data

A phase I Institute of Cancer Research (UK) trial of 17-AAG in solid tumors used a once weekly administration schedule. The starting dose was 10 mg/m2 per week administered intravenously once weekly in a cohort of three patients. Doses were doubled in each succeeding cohort.277 Adverse events included grade 1 to 2 nausea and grade 1 to 2 fatigue in 3 and 9 of the first 15 patients, respectively. One patient experienced grade 3 vomiting at the 80 mg/m2 per week dose. Grade 3 nausea and vomiting occurred in two of six patients treated at the 320 mg/m2 per week dose, following which the dose was escalated by 40% to 450 mg/m2 per week.276 The DLT at 450 mg/m2 per week was a grade 3 to 4 elevation of AST/ALT in one of six patients.278 A total of 28 patients have been treated on this trial. Among the six patients treated in the 320 to 450 mg/m2 per week dose range, two patients showed stable disease for 27 and 91 weeks, respectively.

PD marker analysis of tumor biopsies done before and 24 hours after treatment in nine patients showed depletion of c-Raf in four of seven samples (where the marker was expressed) and CDK4 depletion and Hsp70 induction in eight of the nine samples.278 At the highest dose level, PK analysis indicated a t½ of 5.8 ± 1.9 hours, a Vdss of 274 ± 108 L, clearance of 35.5 ± 16.6 L/h, and a Cmax of 16.2 ± 6.3 µM (ref. 278), which is above the levels of 375 nM to10 µM reported to inhibit Hsp90 in vitro.279 Although an MTD was not established in this trial, the RP2D is likely to be 450 mg/m2 per week, as there was evidence of tumor target inhibition at that dose level.278 Analysis of duration of target inhibition is ongoing.

The NCI, has sponsored 17 phase I studies (7 single agent and 10 combination) to evaluate 17-AAG. An overview of these trials is presented in Table 25.4 and Table 25.5. Table 25.6 shows the four trials currently being planned or conducted with 17-DMAG. Of note, dosing was adjusted based on results from early phase I work. In one study, patients with advanced solid tumors were treated with a 60-minute IV infusion for 5 consecutive days every 3 weeks. An MTD of 40 mg/m2 per dose was established.271 In a second study, patients with advanced solid tumors who received daily doses for 5 days every 3 weeks reached an MTD of 80 mg/m2 per dose.273 Increasing the dosing interval to days 1, 8, and 15 of a 3-week cycle resulted in an MTD of 308 mg/m2 per dose, 274 and this protocol was amended to alter the dosing to days 1, 4, 8, and 11 based on PK endpoints. Additionally, when patients were dosed once weekly for 4 weeks, dosing could be escalated to 450 mg/m2 per dose.276, 277 Thus, protocols have been amended to reflect the best dosing schedule.

The Hsp90 inhibitors are a class of agents that affect a diverse group of client proteins involved in oncogenesis. Many of these clients are expressed in a disease-specific fashion. The development of these inhibitors as biomodulators is complex and not necessarily governed by standard approaches. The clinical approach taken with the Hsp90 inhibitors was to proceed simultaneously with single-agent phase II studies as well as disease-specific combinations that would be used to evaluate the biomodulatory effects of the geldanamycins. The ongoing clinical trials outlined in the tables will be used to assess activity of the agents in a disease-specific fashion and to provide a response comparison for the phase I combinations in order to proceed into disease-specific phase II investigations. As these studies mature and reach completion, the role of Hsp90 inhibitors in the treatment of cancer should be better defined with regard to their activity and molecular targeted effects.

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