Volume 9, Issue 1, Pages 1-18 (February 2006)

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Tyrosine kinase inhibitors and gemcitabine: New treatment options in pancreatic cancer?

Received 22 September 2005; received in revised form 5 February 2006; accepted 14 February 2006. published online 17 April 2006.

Abstract

Pancreatic cancer (PCa) is one of the most lethal malignancies in humans. Gemcitabine is the current standard chemotherapy of advanced PCa but it is still far from optimal and novel therapeutic strategies are urgently needed. For the near future, tyrosine kinase inhibitors (TKIs) hold great promise as a therapeutic strategy. Tyrosine kinases (TKs) play a pivotal role in intercellular signal transduction and regulate crucial processes of tumor cells such as proliferation, migration, survival and angiogenesis. Several TKs – such as EGFR, VEGFR, PDGFR and Src – are known to be overexpressed or constitutively activated in PCa. Hence, blocking receptor tyrosine kinases (RTKs) and non-receptor, cytoplasmic tyrosine kinases (CTKs) represents a rational approach to treat PCa. In particular, cetuximab and erlotinib, the monoclonal antibodies against EGFR-1 (ErbB-1) showed promising activity in Phase II and Phase III trials and their combination with gemcitabine resulted in synergistic antitumor activity. In addition, small antiangiogenic molecules such as VEGFR-2 inhibitors, PDGFR inhibitors and multiple receptor targeting agents are under active investigation. Association of chemoresistance with the activity of certain tyrosine kinases (e.g. ErbB-1 and Src) has been described for pancreatic cancer and makes a strong case for combining gemcitabine with TKIs. Combinations of different TKIs might also be used to target the cancer cell micro-environment. Detailed molecular characterization of tumor cells and combinations of appropriate TKIs with cytotoxic agents such as gemcitabine are expected to lead to improved therapy of pancreatic cancer.

Keywords: Molecular targeting, Targeted therapy, Signal transduction, VEGFR, EGFR, PDGFR, SRC, RTK, TKI, Receptor tyrosine kinase, Tyrosine kinase inhibitor, Gemcitabine

Article Outline

• Abstract

• 1. Introduction

• 2. Standard chemotherapy of pancreatic cancer

• 2.1. 5-Fluorouracil

• 2.2. Gemcitabine and combinations

• 3. Tyrosine kinases and cancer

• 4. EGF- receptor targeting in pancreatic cancer

• 4.1. Antibody type inhibitors (EGF-receptor MABs)

• 4.1.1. Cetuximab (Erbitux, IMC-C225)

• 4.1.2. Panitumumab (ABX-EGF)

• 4.1.3. Matuzumab (EMD72000)

• 4.1.4. Trastuzumab (Herceptin)

• 4.2. Small-molecule inhibitors (EGF-receptor TKIs)

• 4.2.1. Gefitinib (Iressa, ZD1839)

• 4.2.2. Erlotinib (Tarceva, OSI 774)

• 4.2.3. EKB-569

• 4.2.4. PKI-166 (CGP75144)

• 4.2.5. Lapatinib (GW572016)

• 5. Targeting VEGF- receptors in pancreatic cancer

• 5.1. Antibody type inhibitors (VEGF-receptor MABs)

• 5.1.1. DC101, IMC-1C11, IMC-2C6 and IMC-1121

• 5.2. Small-molecule inhibitors (VEGF-receptor TKIs)

• 5.2.1. Vatalanib (PTK787/ZK222584)

• 5.2.2. Zactima (ZD6474)

• 5.2.3. Semaxanib (SU5416), SU6668 and Sutent (SU11248)

• 5.2.4. Other VEGFR - TKIs (ZD4190, AZD2171, CEP4214, CEP7055, AG0137336)

• 6. Targeting PDGFR and multiple pathways

• 6.1. Imatinib (Gleevec, STI-571)

• 6.2. Other multi-targeting PDGFR-TKIs: Sorafenib (BAY43-9006), Leflunomide (SU101)

• 7. Src kinase inhibition in pancreatic cancer

• 7.1. Pyrazolopyrimidines: PP1 and PP2

• 7.2. AZM475271

• 7.3. Other Src inhibitors: siRNA, AP23846, SKI-606, AZD05230

• 8. Chemoresistance and TKIs in pancreatic cancer

• 9. Future perspectives

• 10. Conclusion

• References

• Copyright

1. Introduction

Pancreatic cancer (PCa) is one of the most lethal malignancies in humans and continues to be a major medical challenge in the western world. The incidence rate of about 10 per 100,000 equals the mortality rate, underscoring the devastating nature of this disease (Van Cutsem et al., 2004). The median overall survival is 3–6 months with a 12-month survival rate of 10% and a 5-year survival rate of less than 3% (Bramhall et al., 1995, Jemal et al., 2002). The very poor prognosis is due to late symptoms and the inability to detect this disease at early stages. Even with current diagnostic modalities, the inaccessible location of the pancreas often makes the diagnosis of pancreatic cancer a real challenge. Pancreatic cancer spreads early and most of the patients show liver or lymph node metastases at the time of diagnosis. Even though painless jaundice occurs in about 50% of patients with resectable lesions of the pancreatic head, in general early symptoms are non-specific, and jaundice may also occur as a late symptom of large tumors of the body of the gland. Pain is the most frequent symptom and is present in 80% of patients with locally advanced tumors and metastatic disease (Van Cutsem et al., 2004). Nearly 90% of pancreatic neoplasms are adenocarcinomas, arising from the exocrine ductal system. They are most often multicentric and 75% are located in the head of the pancreas. The etiology of pancreatic cancer is not well understood but risk factors such as smoking, chronic pancreatitis and positive family history are implicated (Ahlgren, 1996b, Hruban et al., 1998, Li et al., 2004a). Only a detailed understanding of the molecular biology of this highly aggressive disease will lead to effective diagnosis, prevention and treatment.

To date, surgical resection is the only potentially curative treatment, but the majority of patients are not surgical candidates due to advanced disease or significant co-morbidity (Brand, 2001, Rosewicz and Wiedenmann, 1997). For over 80% of pancreatic carcinoma patients, palliative treatment protocols represent the only reasonable therapeutic option. Pancreatic cancer is relatively resistant to both chemotherapy and radiation therapy and hence patient survival rates have been only modestly improved by standard therapeutic regimens (Brand and Tempero, 1998). Consequently, there is a great demand for the development of new effective drugs and alternative therapeutic approaches.

2. Standard chemotherapy of pancreatic cancer

2.1. 5-Fluorouracil

Until recently, the most commonly administered treatment for patients with advanced or metastatic pancreatic cancer has been palliative 5-fluorouracil (5-FU)-based chemotherapy. However, response rates have rarely exceeded 10%, with no consistent effect on disease-related symptoms, quality of life or survival (Ahlgren, 1996a, Diaz-Rubio, 2004, Oettle and Riess, 2002). Studies comparing the efficacy of 5-FU monotherapy with either 5-FU combination regimens (5-FU plus cisplatin, doxorubicin or doxorubicin/mitomycin) (Cullinan et al., 1985; Ducreux et al., 2002) or 5-FU-based radio-chemotherapy (Kalser and Ellenberg, 1985; Klinkenbijl et al., 1999) have shown better response rates for some regimens, but also increased toxicity and no significant overall survival benefit, suggesting little or no clinical advantage for these combinations.

2.2. Gemcitabine and combinations

To date, gemcitabine has emerged as the cornerstone of chemotherapy for advanced and metastatic pancreatic cancer. Gemcitabine is a pyrimidine analog (Fig. 1) with a wide spectrum of antitumor activity (Abbruzzese, 1996). It is metabolized intracellulary by nucleoside kinases to the active species gemcitabine-diphosphate (dFdCDP) and gemcitabine-triphosphate (dFdCTP). Incorporation of dFdCTP into DNA is responsible for the cytotoxic effects of gemcitabine, via inhibition of DNA synthesis, DNA repair and ultimately via induction of apoptosis. Moreover, gemcitabine is a radio-sensitizing agent which acts specifically in the S and G1/S Phase of the cell cycle. Based on the results from different randomized trials, first-line and second-line gemcitabine treatment in patients with advanced pancreatic cancer reveal significant advantages compared to 5-FU treatment regarding disease stabilization, overall survival and clinical benefit response (CBR) measured as a decrease in pain, functional impairment and weight loss (Burris et al., 1997, Carmichael et al., 1996; Casper et al., 1994, Rothenberg et al., 1996). Moreover, preliminary results from the first adjuvant trial of gemcitabine in pancreatic cancer patients indicate that patients with earlier stages of disease may also benefit (Neuhaus et al., 2005). Initial analysis of the data from 243 patients was completed in March 2005, showing almost twice as long disease-free survival after surgery for treated patients; 14.2 months for the gemcitabine group compared with 7.5 months for the observation group (p<0.001). However, the results obtained with gemcitabine monotherapy in advanced disease have been modest (reported median survival, 4–6 months) necessitating a search for combination partners to improve therapeutic outcome.

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Fig. 1. Gemcitabine (2′,2′-difluoro-2′-deoxycytidine), a pyrimidine analog with a broad-spectrum of antitumor activity.

A growing number of Phases I and II studies compare gemcitabine single-agent therapy for advanced disease with gemcitabine plus 5-FU combination treatment with and without folinic acid, using various treatment schedules. Most of these studies report low toxicity and partial or complete responses (reviewed by Oettle and Riess, 2002). Higher rates of stable disease in the combination arm were found in most of the trials, and some showed significant longer overall survival compared to treatment with either agent alone. However, a recently published Phase III study could not confirm this issue (Berlin et al., 2002). Therefore, data from ongoing Phase III studies must be reviewed before a definitive statement can be made regarding the value of this cytotoxic combination.

Many other gemcitabine-based combination regimens are currently under clinical investigation (Kulke, 2005). One potential group of partners are platinum agents (Philip, 2002), which have been used successfully with gemcitabine in other solid malignancies such as lung cancer. Several studies have focused on gemcitabine plus cisplatin. The two agents have different mechanisms of action and non-overlapping dose-limiting toxicities. There is evidence that gemcitabine is not cross-resistant with cisplatin in other solid tumors (Lund and Neijt, 1996, Philip, 2002). A gemcitabine–cisplatin combination treatment of advanced pancreatic cancer improved objective response rates, progression-free survival and overall survival (up to 11 months) in different Phase II trials (Brodowicz et al., 2000, Colucci et al., 1999, Heinemann et al., 2000, Philip et al., 2001). However, a very recent meta-analysis of larger Phase II and Phase III trials did not show a significant advantage for gemcitabine–cisplatin combinations over gemcitabine monotherapy, regarding 6-month survival rate, clinical benefit and toxicity (Xie et al., 2006). Studies comparing gemcitabine monotherapy with gemcitabine–oxaliplatin combinations showed better response rates, clinical improvement and disease-free survival in the combination arm, but failed to show improvement in overall survival (Alberts et al., 2003, Louvet et al., 2005). Similarly, combination of gemcitabine and irinotecan resulted in superior response rates but not in improvement of progression-free or overall survival (Rocha Lima et al., 2002, Rocha Lima et al., 2004, Stathopoulos et al., 2003). Other combinations with gemcitabine, such as the addition of docetaxel, pemetrexed and exatecan mesylate, have proved disappointing in larger trials (Jacobs, 2002, Kindler, 2002, O’Reilly et al., 2004, Richards et al., 2004, Stathopoulos et al., 2001). Regarding three- and four-drug regimens, the combination of gemcitabine, cisplatin, epirubicin and infusional fluorouracil (PEFG), developed by Reni et al. (2005), has been most promising. However, the survival benefit remained small.

Taken together, gemcitabine represents the current standard drug for cytotoxic therapy of advanced pancreatic cancer, and surgical patients might benefit from adjuvant gemcitabine therapy. Combination regimens of gemcitabine with other cytotoxic drugs have shown promising activity in Phase II studies, but treatment with these combinations in a randomized setting has usually not translated into survival benefit. New conventional cytotoxic agents and other gemcitabine combinations might improve survival for patients with pancreatic cancer, but the improvement is likely to be small. Therefore, novel therapeutic strategies are urgently needed. Among these strategies, targeting cancer-specific molecular defects holds the greatest promise for the near future.

3. Tyrosine kinases and cancer

Tyrosine kinases (TKs) represent a major subclass of protein kinases, which play a pivotal role for intracellular signal transduction. Crucial cellular processes are regulated by TK signaling, such as adhesion, proliferation, migration, invasion, differentiation, metabolism, angiogenesis, survival and apoptotic cell death. TKs generally activate downstream target proteins through phosphorylation or provide binding sites for protein–protein interactions. TKs are further divided into receptor TKs (RTKs) and non-receptor, cytoplasmic TKs (CTKs) (Levitzki, 1999, Levitzki and Gazit, 1995).

RTKs possess an extracellular ligand-binding domain, a transmembrane domain, and an intracellular catalytic domain (Fig. 2). RTKs are specifically activated by growth factors, such as EGF, VEGF, FGF, PDGF and many others (Fry et al., 1994, Kovalenko et al., 1994, Levitzki and Gazit, 1995, Mohammadi et al., 1997). The intracellular kinase domains of RTKs can be further divided into those containing a stretch of amino acids separating the kinase domain (e.g. VEGFR and PDGFR) (Bender et al., 2004, Board and Jayson, 2005) and those in which the kinase domain is continuous (e.g. EGFR and HER2/neu) (Fig. 2).

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Fig. 2. Two methods of blocking tyrosine kinase-dependent growth factor signaling are shown. (1) Inhibition of receptor–ligand interaction by receptor specific monoclonal antibodies (MABs); (2) inhibition of intracellular tyrosine kinase activity by small-molecule tyrosine kinase inhibitors (TKIs). RTK: receptor tyrosine kinase (growth factor receptor); CTK: cytoplasmic tyrosine kinase (intracellular signaling protein).

Cellular tyrosine kinases (CTKs) are located in the cytoplasm, nucleus or are anchored in the inner leaflet of the plasma membrane (Levitzki, 1999) (Fig. 2). They are grouped into eight families – SRC, JAK, ABL, FAK, FPS, CSK, SYK and BTK – each family consisting of several members (Levitzki, 1999). Of those CTKs whose functions are known, many – such as SRC – are involved in cell growth (Osherov and Levitzki, 1994).

In pancreatic cancer many TKs were found to be activated, such as EGFR (ErbB-1), HER-2/neu (ErbB-2), VEGFR-2, PDGFR-α, c-KIT, FGFR-1, CSF1R, SRC and others. Activation of the oncogenes coding for these enzymes can lead to the production of kinases that are constitutively active in the absence of a stimulus. Indeed, more than 70% of the known oncogenes and proto-oncogenes in cancer code for TKs. Blocking tyrosine kinase activity therefore represents a rational approach to cancer therapy.

Therapeutic strategies for targeting RTKs include blocking extracellular receptor domains on tumor cells (antibody type inhibitors), blocking intracellular kinase–substrate interaction or inhibiting the enzyme's ATP binding site (small-molecule type inhibitors) (Fig. 2). Advances in the understanding of the oncogenic activation of RTKs have been matched by the identification of new structural classes of tyrosine kinase inhibitors (TKIs) with improved potency, specificity and efficacy (Garcia-Echeverria and Fabbro, 2004). Indeed, half of the 65 kinase inhibitors currently in clinical trials are targeting RTKs. Interference with downstream signal transduction has also become possible since TKIs were developed which specifically target intracellular cascades by blocking CTKs such as Src and others.

4. EGF- receptor targeting in pancreatic cancer

The epidermal growth factor receptor family consists of four tyrosine kinase receptors including ErbB-1 (EGFR) and ErbB-2 receptor (HER-2/neu), ErbB-3 (HER-3) and ErbB-4 (HER-4) (Arteaga, 2001). ErbB-1 and ErbB-2 receptor expression and over-expression has been observed in various solid malignancies and has been most extensively studied in human breast cancer (Arteaga, 2002). Many of the features of the malignant phenotype, such as increased proliferation, survival and angiogenesis are associated with the signaling networks that involve EGF-receptors (EGFRs). Dysregulation of their pathways contributes to the development and progression of malignancies (Fig. 3). ErbB-1 and ErbB-2 receptor activation leads to elevated MAP-kinase (MAPK) and PI3-kinase (PI3K) recruitment. The PI3K-pathway suppresses apoptosis by activating the AKT anti-apoptotic protein kinase, while MAPK-pathway activation is associated with tumor cell proliferation (Craven et al., 2003) (Fig. 3). In addition, EGFRs as well as their ligands EGF and TGFα are involved in VEGF-dependent tumor angiogenesis (Arteaga, 2002). ErbB-1 receptor expression may be detected in up to 90% of pancreatic tumors and ErbB-2 expression in up to 21% of pancreatic tumors (Lemoine et al., 1992, Saad and Hoff, 2004, Safran et al., 2001, Xiong and Abbruzzese, 2002). Moreover, co-expression of ErbB-1 and at least one of its ligands by tumor cells has been associated with poor prognosis in pancreatic cancer (Dong et al., 1998, Lemoine et al., 1992, Yamanaka et al., 1993). This coexpression might function as an autocrine loop to constantly stimulate tumor cell proliferation and therefore blockade of ErbB-1 activity should result in suppression of tumor growth. Two classes of EGF-receptor inhibitors are available: monoclonal antibodies (MABs) that inhibit ligand binding to EGFRs and small-molecule tyrosine kinase inhibitors (TKIs) that inhibit the tyrosine kinase activity of EGFRs by interfering with ATP binding (Fig. 2). Since a large proportion of patients with pancreatic cancer overexpress ErbB-1, various ErbB1-MABs and ErbB1-TKIs are under active investigation (Table 1, Table 2).

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Fig. 3. EGF-receptor binding leads to tyrosine kinase phosphorylation and downstream activation of different signal processing pathways including PI3-kinase and MAP-kinase pathways. Subsequent effects on gene transcription result in transformation of the tumor cell, promoting survival, proliferation, migration and other tumor-specific actions.

Table 1.

Clinical trials on pancreatic cancer treatment using TKIs and MABs with and without gemcitabine, published until February 2006


Reference	Journal/Abstract	Drug	Drug-ID	Class	Type of trial			Additional Therapy		Disease	Pat. (n)	Age (y)	Molecular target		Toxicity	PR (%)	SD (%)	TTP (mo.)	Surv. (mo.)	1 year (%)
					Phase	rand.	mc.	CTX	RTX				Expr. (%)	Entry Crit.
Xiong et al. (2004)	J. Clin. Oncol.	Cetuximab	IMC-C225	ErbB1-MAB	II	−	+	Gemc.	−	Advanced, recurrent	41	61	95	+	Mild	12.2	63.4	3.8	7.1	31.7
Graeven et al. (2004)	J. Clin. Oncol.	Matuzumab	EMD-72000	ErbB1-MAB	I	−	−	Gemc.	−	Advanced	17		94	−	Moderate+1 severe		65
Safran et al. (2004)	Cancer Invest.	Trastuzumab		ErbB2-MAB	I	−	−	Gemc.	−	Metastatic	34		16	+	Mild-moderate	6			7.0	19
Moore et al. (2005)	ASCO–GI-Symposium	Erlotinib	OSI-774	ErbB1-TKI	III	+	+	Gemc.	+	Advanced	569			−	Mild-moderate	9	49			24
Iannitti et al. (2005)	Am. J. Clin. Oncol.	Erlotinib	OSI-774	ErbB1-TKI	I	−	−	Gemc.+Paclitaxel	+	Locally -advanced	13	62		−	Mild	46			14
Morgan et al. (2003)	Proc. Am. Soc. Clin. Oncol.	EKB-568	EKB-569	ErbB1-TKI	I	−	−	Gemc.	−	Advanced	29	60		−	Mild-moderate
Safran et al. (2006)	ASCO–GI-Symposium	Lapatinib	GW572016	ErbB-1/-2-TKI	I	−	−	Gemc.+Oxaliplatin	−	Advanced	11	62		−	Mild-moderate	27
Kuo et al. (2006)	ASCO–GI-Symposium	Vatalanib	PTK787/ZK222584	pan-VEGFR-TKI	I	−	−	Gemc.	−	Advanced	8	59		−	Mild	25	75
Chen et al. (2005)	Cancer Lett.	Imatinib	STI-571	PDGFR-TKI, (multi-targeting)	II	+	−	[Gemc.]	−	Advanced	26	59 [63]	100	−	Mild-moderate	0	0	1.0	2.0

rand.: randomized; mc.: multicenter trial; CTX: chemotherapy; RTX: radiation; Expr.: expression; Entry Crit.: entry criterion; PR: partial response; SD: stable disease; TTP: time to progression; Surv.: survival; mo.: month; year: 1 year survival rate; advanced: locally advanced+metastatic; ErbB1: EGF-receptor 1; ErbB2: EGF-receptor 2 (HER-2/neu); pan-VEGFR: vascular endothelial growth factor receptors 1-3; PDGFR: platelet derived growth factor receptor; MAB: monoclonal antibody; TKI: tyrosine kinase inhibitor; Gemc.: gemcitabine; []: control group.

Table 2.

Ongoing clinical trials on TKIs and MABs (Plus gemcitabine) for pancreatic cancer treatment, listed by the National Institute of Health (USA), February 2006


Class	Drug	Phase	Combined drugs	Stage	Therapy	Patients (n)	Type of trial	Sponsors	NIH-protocol	Study ID	Status
EGF-receptor MAB	Cetuximab	II	Irinotecan, Docetaxel	Metastatic	Palliative	92	Multicenter, randomized	Eastern Cooperative Oncology Group, NCI	NCT00042939	ECOG-E8200	Recruiting
	Cetuximab+Erlotinib	II	Gemcitabine, Bevacizumab	Unresectable, locally advanced, not amendable to radiation	Palliative	54–126	Multicenter, randomized	University of Chicago, NCI	NCT00091026	NCI-6580, UCCRC-13200A	Recruiting
	Cetuximab	III	Gemcitabine	Unresectable, locally advanced or metastatic	Palliative	704	Multicenter, randomized, open-label	Southwest Oncology Group, NCI	NCT00075686	SWOG-S0205	Recruiting
	Trastuzumab	II	Gemcitabine and radiation	Regionally confined	Adjuvant–palliative	50		NCI	NCT00005926	00-C0161	Completed

EGF-receptor TKI	Gefitinib	II	Docetaxel	Unresectable, advanced	Palliative		Non-randomized, open-label, active control	University of Pittsburgh, AstraZeneca	NCT00177242	04-027	Recruiting
	Erlotinib	I	Gemcitabine	Locally advanced or metastatic	Palliative	30	Multicenter, dose-escalation	OSI Pharmaceuticals	NCT00033241	NCI-V02-1694, OSI-774-155	Completed
	Erlotinib	I	Gemcitabine and radiation	Potentially resectable	Neo-adjuvant	8	Non-randomized, open-label, uncontrolled	Indiana University School of Medicine, Genentech	NCT00216307	IUCRO-0130	Recruiting
	Erlotinib	I	Gemcitabine, radiation	Unresectable, locally advanced	Palliative	19–28	Non-randomized, open-label, dose-escalation	Memorial Sloan-Kettering Cancer Center, NCI	NCT00063947	NCI-5441, MSKCC-03031	Recruiting
	Erlotinib	I/II	Gemcitabine, Capecitabine, Bevacizumab	Advanced disease			Non-randomized, open-label, uncontrolled	Royal Marsden NHS Foundation Trust, Hoffmann-La Roche Pharmaceuticals	NCT00260364	TARGET 1	Recruiting

VEGF-receptor TKI	Vatalanib	II	−/−	Advanced or metastatic, where Gemcitabine first line therapy failed	Palliative	65	Non-randomized, open-label, uncontrolled	Pancreatic Research Team, university of Arizona Cancer Center	NCT00226005	PCRT04-001	Recruiting
	Vatalanib	I/II	Gemcitabine	Unresectable, locally advanced or metastatic	Palliative	24	Non-randomized, open-label, active control	Stanford University, Novartis	NCT00185588	CPTK787AUS08	Recruiting

PDGF-receptor TKI	Imatinib	II	Gemcitabine	Unresectable, locally advanced or metastatic	Palliative	28	Non-randomized, open-label, uncontrolled	University of Medicine New Jersey, Novartis	NCT00161213	CINJ #070501	Recruiting
Src TKI	AZD0530	I/II	Gemcitabine	Unresectable, locally advanced or metastatic	Palliative	60	Multicenter, open-label, dose-escalation	National Cancer Institute of Canada	NCT00265876	CAN-NCIC-IND173	Recruiting

EGF: epidermal growth factor; VEGF: vascular endothelial growth factor; PDGF: platelet derived growth factor; Src: cytoplasmic tyrosine kinase; TKI: tyrosine kinase inhibitor; NCI: National Cancer Institute (USA); NIH: National Institute of Health (USA).

4.1. Antibody type inhibitors (EGF-receptor MABs)

4.1.1. Cetuximab (Erbitux, IMC-C225)

Cetuximab (Erbitux, IMC-C225; ImClone/Merck AG) is a chimeric (human-mouse) monoclonal antibody (MAB) directed against ErbB-1 that received FDA approval in May 2004 for colorectal cancer treatment. The anti-tumor effect of cetuximab has been studied in different preclinical models of human pancreatic carcinoma, both as a single agent and in combination with gemcitabine (Arnoletti et al., 2004, Bruns et al., 2000a, Buchsbaum et al., 2002, Huang et al., 2003; Overholser et al., 2000, Sclabas et al., 2003). Using the human pancreatic cancer cell line L3.6pl and orthotopic tumor models, it has been shown that cetuximab effectively blocks ErbB-1 autophosphorylation in vitro and in vivo (Bruns et al., 2000a). In this study, cytostatic effects in vitro were modest since cetuximab alone inhibited cell growth by only 20%, but additive cytotoxic effects were observed when gemcitabine was added. In vivo application of cetuximab resulted in profound inhibition of orthotopic tumor growth and metastases, due to induction of apoptosis and suppression of tumor cell proliferation. The in vivo effect was further enhanced when gemcitabine was added to the regiment. Moreover, apoptosis of endothelial cells and subsequent lower microvessel density was found after cetuximab treatment. This probably was due to down-regulation of VEGF and interleukin-8 (IL-8) expression in tumor cells. The data suggested that, in addition to the antiproliferative activity of cetuximab in experimental pancreatic cancer, some antiangiogenic activity contributes to the antitumor effects. Cetuximab also affected heterotopic tumor growth of s.c. implanted BxPC-3 pancreatic tumor xenografts (Overholser et al., 2000). Using the combination of cetuximab, gemcitabine and radiation as treatment for different pancreatic cancer cell lines, Buchsbaum et al. could induce the highest levels of apoptosis in vitro and complete regression of established MiaPaCa-2 tumors in vivo. (Arnoletti et al., 2004, Buchsbaum et al., 2002). The combination therapy was more effective than any single or double modality treatment, suggesting chemosensitizing and radiosensitizing properties of cetuximab. However, partial resistance to this combination therapy was found in BxPC-3 pancreatic tumors, which possibly was caused by variable downstream signaling (Arnoletti et al., 2004, Huang et al., 2003). Recently, Xiong et al. (2004) have reported the results of their Phase II study combining gemcitabine and cetuximab in patients with advanced and recurrent pancreatic cancer (Table 1). These patients had received no previous chemotherapy and were screened for intratumoral ErbB-1 expression, which was present in 95% of the patients. Forty-one patients were enrolled in the trial and received gemcitabine 1000mg/m2 once weekly for 7 weeks in addition to cetuximab 400mg/m2 once weekly (first week) followed by 250mg/m2 once weekly for 6 weeks. Toxicities were typically mild, and 12.2% of the patients achieved a partial response, while 63.4% had stable disease. The median time to progression was 3.8 month, the median over all survival was 7.1 month and the 1-year survival rate was 31.7% (Table 1). Therefore, the combination therapy of cetuximab and gemcitabine has promising activity against advanced pancreatic cancer when compared to gemcitabine monotherapy. Further Phase II and Phase III trials are currently underway to further assess this combination regimen in unresectable and metastatic pancreatic cancer (Table 2).

4.1.2. Panitumumab (ABX-EGF)

Panitumumab (ABX-EGF; Abgenix) was the first fully humanized IgG2 monoclonal antibody specific for ErbB-1 receptor. Panitumumab was generated using XenoMouse technology and seems to offer immunological and pharmacokinetic advantages over human-mouse chimeras like cetuximab. Panitumumab inhibits ErbB-1 mediated signal transduction and cell proliferation. Animal studies have shown Panitumumab to be active against a variety of human tumor xenografts that express ErbB-1, including pancreatic carcinoma cells (BxPC-3) (Yang et al., 2001). Moreover, Yang et al. reported that administration of Panitumumab alone resulted in complete eradication of established tumor xenografts without additional chemotherapy (Yang et al., 1999). The first results of clinical trials in different solid tumors appear promising (Foon et al., 2004), but specific data for patients with pancreatic cancer are not yet available.

4.1.3. Matuzumab (EMD72000)

Matuzumab (EMD72000; Merck AG) is another fully humanized MAB directed against ErbB-1 which binds to ErbB-1 with high affinity and specificity (Bangard et al., 2005). A longer half-life of this agent makes it unique among other ErbB-1 antibodies. Therefore, one administration of matuzumab every 2–3 weeks may be sufficient (Tabernero et al., 2003). A recent animal study (Bangard et al. 2005) assessed the antitumor and antiangiogenic effect of matuzumab in orthotopic pancreatic tumor models (Bangard et al. 2005). Matuzumab-treated rats showed significantly less tumor progression and reduced tumor angiogenesis. Furthermore, 17 patients with advanced pancreatic cancer were recently enrolled in a Phase I trial of matuzumab plus gemcitabine (Graeven et al., 2004) (Table 1). Escalating doses of matuzumab, 400mg/week (1), 800mg/2 week (2), or 800mg/week (3) followed by a weekly dose of 1000mg/m2 gemcitabine were applied, and combination therapy was overall well-tolerated. However, one possibly treatment-related Guillain–Barree syndrome occurred (3) and one non-treatment-related death (1). Stable disease occurred in 3/5 (1), 3/4 (2) and 5/8 patients (3), respectively (65%), with continuous treatment for up to 50 weeks, suggesting biologically effective doses in all three treatment groups. Further clinical trials are planned.

4.1.4. Trastuzumab (Herceptin)

Trastuzumab (Herceptin; Genentech) is a recombinant humanized MAB directed against ErbB-2 (HER-2/neu), which exhibits cytostatic activity on breast and prostate cancer cells that overexpress the HER-2 oncogene. Trastuzumab was the first MAB, which received FDA approval in September 1998 for breast cancer treatment. Following the reported success of trastuzumab in breast cancer patients, investigators preceeded to explore the efficacy of this agent in other solid tumors. ErbB-2 expression in pancreatic cancer is less frequent than in breast cancer and overexpression was detected by immunohistochemistry in 16, 17, 19.6 and 21% of pancreatic cancer patients (Novotny et al., 2001, Safran et al., 2001, Safran et al., 2004, Saxby et al., 2005). Trastuzumab induced tumor growth inhibition in vitro and in vivo in different pancreatic cell lines with high ErbB-2 expression levels (Büchler et al., 2001). Cell lines with low levels of this protein did not respond significantly to the antibody. In another animal study, the same authors could even increase anti-tumor and anti-metastatic activity when docetaxel was added to gemcitabine–trastuzumab combination therapy (Büchler et al., 2005). This might be due to the fact, that pancreatic tumor cell resistance to taxanes (e.g. docetaxel) is at least in part mediated by ErbB-2. In the first clinical trial combining gemcitabine and trastuzumab in ErbB-2-overexpressing patients with advanced pancreatic carcinoma (Safran et al., 2004), thirty-four patients were enrolled and received gemcitabine 1000mg/m2 per week for 7 of 8 weeks, followed by 3 of every 4 weeks, and trastuzumab 4mg/kg loading dose, followed by 2mg/kg/week. Toxicity was similar to gemcitabine alone and 13 patients (41%) had either a partial response or a >50% reduction in tumor marker CA 19-9. The median survival was 7 months, and 1-year survival was 19% (Table 1). Therefore, the response rate for trastuzumab and gemcitabine was similar to gemcitabine alone, but the median survival of 7-month in metastatic pancreatic cancer suggests a modest benefit for some patients. Another adjuvant trial of trastuzumab and gemcitabine chemoradiation therapy for regionally confined pancreatic cancer was completed and results are awaited (Table 2). However, infrequent ErbB-2 overexpression may limit the future role of trastuzumab in pancreatic cancer therapy.

4.2. Small-molecule inhibitors (EGF-receptor TKIs)

4.2.1. Gefitinib (Iressa, ZD1839)

Gefitinib (Iressa, ZD1839; AstraZeneca), is an orally bioavailable, small-molecule TKI which specifically targets ErbB-1 signaling. Gefitinib was the first TKI approved by FDA for advanced lung cancer treatment (NSCLC) in May 2003 (Cella et al., 2005, Thatcher et al., 2005) and is currently evaluated alone or in combination in numerous clinical trials for several other tumor types (Cools et al., 2005). Gefitinib inhibited the EGF-induced phosphorylation of ErbB-1 and MAPK and completely blocked EGF-induced cell proliferation in different pancreatic cancer cell lines in vitro (Li et al., 2004b). Moreover, colony formation and invasiveness were significantly inhibited in this study, using low concentrations of gefitinib. Phase II results for gefitinib in combination with docetaxel are awaited for PCa (Table 2).

4.2.2. Erlotinib (Tarceva, OSI 774)

Erlotinib (Tarceva, OSI-774; Genentech) is another specific and reversible small-molecule ErbB-1 TKI that has received FDA approval for advanced NSCLC in November 2004. Evaluation of erlotinib in pancreatic cancer cell lines in vitro revealed significant growth inhibition in five of six cell lines which expressed ErbB-1 (Durkin et al., 2003). In orthotopic tumor xenografts, it was demonstrated that erlotinib significantly inhibited the phosphorylation of ErbB-1 and other downstream kinases and significantly promoted gemcitabine-induced apoptosis in vivo (Ng et al., 2002). Recently, erlotinib has been shown to induce prolonged survival in pancreatic cancer patients when added to gemcitabine in a clinical Phase III trial (Moore, 2005; Moore et al., 2005) (Table 1). In this study, 569 patients with advanced pancreatic cancer were randomized to receive gemcitabine 1000mg/m2/week for 7 of 8 weeks and then for 3 of every 4 weeks plus either erlotinib 100mg p.o. per day or a placebo in a double-blinded fashion. Patients had not received prior systemic chemotherapy, and ErbB-1 status was not an entry criterion. The addition of erlotinib to gemcitabine was associated with a small but significant improvement in performance status and 1-year overall survival (24% versus 17%) when compared with gemcitabine alone. Quality-of-life analysis revealed similar results for both groups. Another Phase I study analyzed erlotinib therapy in combination with chemoradiation followed by maintenance erlotinib for locally advanced pancreatic cancer (Iannitti et al., 2005). The maximum tolerated dose of erlotinib was 50mg/d when given in combination with weekly gemcitabine (75mg/m2), paclitaxel (40mg/m2) and radiation (50.4Gy), and maintenance erlotinib of 150mg/d was well-tolerated. Other palliative trials and one neoadjuvant trial on erlotinib–gemcitabine combinations with and without radiation therapy are currently recruiting PCa patients (Table 2). Regulatory approval of erlotinib in advanced pancreatic cancer is being sought.

4.2.3. EKB-569

EKB-569 (Wyeth) is a selective irreversible ErbB-1 specific TKI that inhibits the growth of cancer cells that overexpress ErbB-1 in vitro and in vivo, including pancreatic cancer cells (Wissner et al., 2002). An initial Phase I trial evaluated a combination therapy of gemcitabine and EKB-569 in 29 patients with advanced pancreatic cancer (Morgan et al., 2003) (Table 1). In this open-label, dose-escalation study EKB-569 was administered orally daily and gemcitabine was administered i.v. on days 1, 8 and 15 in a 28-day cycle. Dose-limiting toxicities were grade three diarrhea and elevated transaminases at doses of 100mg/day. An additional 18 patients were enrolled at the maximum tolerated dose (MTD) and treated with 25mg EKB-569 orally each day, and gemcitabine 750mg/m2. This combination therapy has generally been well-tolerated in patients with advanced pancreatic cancer and was recommended for further study. Additional efficacy data have not been reported yet.

4.2.4. PKI-166 (CGP75144)

PKI-166 (CGP75144; Novartis), a pyrrolo-pyrimidine derivative, is a small-molecule TKI with dual specificity for ErbB-1 and ErbB-2 (EGFR, HER-2/neu). Its mechanisms of action on pancreatic tumor cells as well as on tumor associated endothelial cells have been studied using different in vitro and in vivo models (Baker et al., 2002, Bruns et al., 2000b, Solorzano et al., 2001b). PKI-166 could block ErbB-1 autophosphorylation effectively in vitro and in vivo, and cytotoxic effects of gemcitabine could be enhanced in vitro by the addition of non-cytostatic concentrations of PKI-166 (Bruns et al., 2000b). In vivo experiments showed that PKI-166 application resulted in profound inhibition of growth and metastatic spread of orthotopically implanted pancreatic tumors, showing maximal efficacy in combination with gemcitabine (Bruns et al., 2000b), and minimal toxicity when administered three times a week (Solorzano et al., 2001b). PKI-166/gemcitabine combination therapy induced apoptosis and suppressed proliferation of tumor cells and tumor associated endothelial cells (Baker et al., 2002, Bruns et al., 2000b). This resulted in lower microvessel density, which might be the result of an indirect antiangiogenic effect of PKI-166 due to reduced VEGF and IL-8 expression of tumor cells (Bruns et al., 2000b). Moreover, a direct antiangiogenic effect of PKI-166 resulted from ErbB-1 inhibition on tumor associated endothelial cells (Baker et al., 2002). This dual antineoplastic and antiangiogenic effect of PKI-166 in experimental pancreatic cancer has to be confirmed in clinical settings.

4.2.5. Lapatinib (GW572016)

Lapatinib ditosylate (GW572016, GlaxoSmithKline) is another reversible dual-specific TKI against ErbB-1 and ErbB-2 which was developed for the potential treatment of advanced solid tumors (Burris et al., 2005, Spector et al., 2005), and exhibited relevant antitumor activity against breast and lung cancer (Burris, 2004, Chu et al., 2005). Originally, GSK had hoped to file for regulatory approval of lapatinib in 2004 but this has been extended to 2006 to allow more time for completion of clinical trials. The first results of preclinical studies on pancreatic cancer were presented recently (Baerman et al., 2005). Lapatinib inhibited EGFR dependent proliferation and colony formation in different pancreatic cancer cell lines through inhibition of MAPK and AKT pathways (Fig. 3). Very recently this year, preliminary results from a Phase I trial were published, where lapatinib was used with either gemcitabine or a combination of gemcitabine and oxaliplatin (GEMOX) for the treatment of advanced pancreatico-biliary cancer (Safran et al., 2006) (Table 1). Lapatinib could be administered with full dose gemcitabine in this study, toxicities were moderate and some dramatic responses were demonstrated in PCa patients with diffuse liver and peritoneal metastases. Therefore, further evaluation of gemcitabine–lapatinib combinations for the treatment of pancreatic cancer is indicated.

5. Targeting VEGF- receptors in pancreatic cancer

Vascular endothelial growth factor (VEGF) and its receptors are over-expressed in pancreatic cancer (Baker et al., 2001, Büchler et al., 2002). Since the VEGF growth factor family is one of the most important groups of pro-angiogenic molecules, which facilitate tumor growth and spread (Dvorak, 2002), blockage of their angiogenic effects might be a promising tool to stop tumor progress. Several studies were undertaken to show efficacy of anti-VEGF treatment. Most of these studies tested neutralizing antibodies against mature VEGF protein or one of its isoforms. Based on those preclinical and clinical trials, bevacizumab (Avastin; Genentech), a humanized MAB against VEGF, is a new candidate of targeted therapy for colorectal and other cancers (Motl, 2005). Another strategy for inhibition of pro-angiogenic signalling is blocking the VEGF-receptors by receptor antibodies or TKIs. Three VEGF-receptors are high affinity transmembrane receptors which together form the flt-subfamily of receptor tyrosine kinases (VEGFR-1, VEGFR-2 and VEGFR-3). VEGFR-1 and -2 are expressed in the vasculature of VEGF-expressing tumors as well as on tumor cells, forming an autocrine loop of growth stimulation. Binding of VEGF to VEGFR-1 and VEGFR-2 on vascular endothelial cells initiates a downstream activation of signaling proteins including, PI3K, GAP, MAPK and others, and results in neo-angiogenesis (Dvorak, 2002). VEGFR-3 expression becomes restricted mainly to the lymphatic endothelium of adult tissues. VEGFR-3 activation may induce lymphangiogenesis, but has very little effect on blood capillaries (Kaipainen et al., 1995). The following paragraphs will review recent results of VEGFR-MABs and -TKIs in experimental and clinical human PCa treatment.

5.1. Antibody type inhibitors (VEGF-receptor MABs)

5.1.1. DC101, IMC-1C11, IMC-2C6 and IMC-1121

DC101 (ImClone) is a specific rat anti-murine VEGFR-2 MAB, which inhibits VEGF binding to its murine receptor VEGFR-2 (flk-1). IMC-2C6 and IMC-1121 (both ImClone) are fully human MABs (IgG1) directed against the corresponding human VEGFR-2 species (KDR) (Zhu et al., 2003). IMC-2C6 and IMC-1121 have replaced IMC-1C11 (ImClone), a human-mouse chimeric antibody against VEGFR-2, which was found to induce anti-chimeric antibodies in up to 50% of colorectal cancer patients (Posey et al., 2003). By binding specifically to VEGFR-2, IMC-2C6 and IMC1121 are able to block VEGF-induced receptor activation and subsequently migration and proliferation of human endothelial cells. In pancreatic cancer models, DC101 treatment resulted in decreased microvessel density, tumor hypoxia (suggestive of an antiangiogenic effect), decreased tumor cell proliferation, increased tumor cell apoptosis and extensive tumor necrosis of established human BxPC-3 xenografts (Prewett et al., 1999). In the orthotopic pancreatic tumor model L3.6p1, DC101 resulted in a decrease of tumor vessel count, lymphatic metastasis and tumor growth (Bruns et al., 2002). The DC101 effects were enhanced by gemcitabine administration resulting in reduced tumor cell proliferation and increased apoptotic death of endothelial cells. Clinical results with VEGFR-MABs are pending.

5.2. Small-molecule inhibitors (VEGF-receptor TKIs)

5.2.1. Vatalanib (PTK787/ZK222584)

Vatalanib (PTK787/ZK222584, PTK/ZK or CGP-79787; Novartis/Schering) is a selective small-molecule pan-VEGFR-TKI, which targets VEGFR-1, -2 and -3. Vatalanib was co-developed by Schering and Novartis to block angiogenic and lymphangiogenic signaling of VEGF-receptors in cancer. In orthotopic pancreatic cancer models, vatalanib was shown to reduce tumor growth and microvessel density of human tumor xenografts (Solorzano et al., 2001a). Combination therapy with gemcitabine produced the largest growth inhibition of pancreatic tumors (81%), a significant decrease of lymph node and liver metastasis and significant increase in animal survival. Moreover, a triple combination of vatalanib, gemcitabine and the EGFR inhibitor PKI-166 reduced pancreatic tumor growth by 97% (Baker et al., 2002) (see also Section 4.2.4). Phases I and II trials on vatalanib therapy for metastatic or unresectable PCa with or without gemcitabine are currently recruiting patients (Table 2). Preliminary results of eight patients treated with gemcitabine (escalated from 700 to 850mg/m2 and 1000mg/m2/week, i.v.) and vatalanib (1250mg p.o. daily) were presented very recently (Kuo et al., 2006). In this Phase I study, the combination treatment of vatalanib and gemcitabine was well-tolerated, and 25% partial responses and 75% stable diseases were seen during this combination regimen (Table 1). Novartis reaffirmed its plans to submit vatalanib for regulatory approval in 2007.

5.2.2. Zactima (ZD6474)

Zactima (ZD6474; AstraZeneca) is a reversible small-molecule VEGFR-TKI which shows potent activity against VEGFR-2 (KDR) and some additional effects on VEGFR-3 (flt-4), ErbB-1 and RET kinase. Consistent with its inhibition of tumor angiogenesis, once-daily oral dosing of Zactima had antitumor effect in a panel of histologically diverse human xenografts including pancreatic cancer (reviewed by Ryan and Wedge, 2005). The combined inhibition of VEGFRs and ErbB-1 by Zactima may produce synergism, and resistance to ErbB-1 inhibition may be overcome (Taguchi et al., 2004). In a preclinical model of advanced, metastatic pancreatic cancer, Zactima showed significant antiangiogenic activity, inhibited primary tumor growth and reduced the number of metastases (Bruns et al., 2003). This effect was most evident in combination with gemcitabine. However, no clinical results for pancreatic cancer treatment are available so far.

5.2.3. Semaxanib (SU5416), SU6668 and Sutent (SU11248)

Semaxanib (SU5416; Sugen/Pfizer) was one of the first VEGFR-targeting drugs and the first small-molecule VEGFR-TKI to be evaluated in large-scale clinical trials (Eskens, 2004). Semaxanib displays 20-fold selectivity to VEGFR-2 compared to PDGFR and shows almost no activity against EGFR or FGFR (Fong et al., 1999), which is in contrast to SU6668 (Sugen/Pfizer), a second-generation synthetic derivate of Semaxanib, which additionally inhibits FGFR, PDGFR and c-KIT (Laird et al., 2002). Both compounds were tested as single agents and in combination with antineoplastic agents (e.g. gemcitabine) in various pancreatic tumor models, in which they attenuated tumor growth and spread via anti-angiogenic activity (Bergers et al., 2003, Bocci et al., 2004, Davis et al., 2005, Griffin et al., 2002). However, in clinical trials neither Semaxanib nor SU6668 displayed marked biological activity in primary human tumors (Davis et al., 2005), but exhibited severe toxicities including thromboembolic events, prompting Pfizer to discontinue the development of Semaxanib and SU6668. Pfizer is now testing the successor compound Sutent (SU11248), a multi-targeting TKI which primarily targets VEGFRs but also other kinases such as c-KIT, PDGFR and Flt-3 (O’Farrell et al., 2003). Antineoplastic activity was reported in Phase I studies, with partial responses in renal cell carcinoma. Trials for pancreatic cancer have to be conducted.

5.2.4. Other VEGFR - TKIs (ZD4190, AZD2171, CEP4214, CEP7055, AG0137336)

ZD4190 (AstraZeneca) is a VEGFR-1 and VEGFR-2 TKI, which revealed anti-proliferative activity on endothelial cells in vitro and antitumor activity in different human tumor xenograft models (Wedge et al., 2000). AZD2171, also developed by AstraZeneca, is a new, highly potent, ATP-competitive TKI of VEGFR-2, which was shown to reduce VEGF-induced angiogenesis in vitro and experimental tumor growth in vivo (Wedge et al., 2005). AZD2171 is undergoing Phase I trials for solid tumors, and once daily oral treatment at doses <45mg was generally well-tolerated in patients with advanced cancers (Drevs et al., 2005). CEP-7055 (Cephalon/Sanofi-Aventis) is the orally active dimethylglycine ester of CEP-5214, a reversible TKI with pan-VEGFR activity (Gingrich et al., 2003), and has been shown to cause inhibition of angiogenesis and tumor growth. The antitumor efficacy of long-term CEP-7055 administration in a pancreatic carcinoma model (ASPC-1) was independent of initial tumor volume and reversible on drug withdrawal (Ruggeri et al., 2003). In a Phase I trial, a favorable toxicity profile was shown for a twice-daily schedule (Pili et al., 2003). Another new oral pan-VEGFR-TKI is AG013736 (Pfizer), a molecule that also inhibits PDGFR and c-KIT. AG013736 was shown to cause early morphological changes in endothelial cells, pericytes and basement membranes of vessels in spontaneous pancreatic islet cell tumors of RIP-Tag2 transgenic mice (Inai et al., 2004). Within 24h, endothelial fenestrations in RIP-Tag2 tumors disappeared, vascular sprouting was suppressed, and blood flow ceased in some vessels. By 1 week, vascular density decreased more than 70% and VEGFR-2 and VEGFR-3 expression was reduced in surviving endothelial cells. The first clinical Phase I study in patients with advanced solid tumors identified a dose of 5mg AG013736 twice daily for further clinical testing (Rugo et al., 2005).

6. Targeting PDGFR and multiple pathways

Platelet derived growth factor (PDGF) was one of the first polypeptide growth factors identified that signals through tyrosine kinase receptors on the cell surface (PDGFRs). Since then, several related genes have been identified constituting a family of ligands (primarily PDGF-A and -B) which bind to their receptors with different binding affinities (Heldin and Westermark, 1999). Two distinct PDGF receptor types have been identified: PDGFR-α and PDGFR-β. When PDGF isoforms bind to the extracellular portion of their receptors, the receptors undergo homo- or hetero-dimerization (composed by one α- and one β-chain), followed by autophosphorylation and tyrosine kinase activation. Once the PDGF receptors are thus activated, a variety of events can occur, including stimulation of cell growth, proliferation, differentiation, migration and inhibition of apoptosis (Chen et al., 2005, Heldin and Westermark, 1999, Li et al., 2003). Dysregulation of these cellular events may result in tumorigenesis (Hwang et al., 2003). PDGFR signaling plays an important role in the autocrine stimulation of cancer cells, and in more subtle paracrine interactions involving stimulation of adjacent stroma cells, vascular pericytes and angiogenesis. PDGFRs and PDGFs are overexpressed in various solid tumors, including glioblastoma, prostate cancer, some gastro-intestinal stromal tumors (GIST), and pancreatic cancer (Ebert et al., 1995, George, 2001, Li et al., 2003, Pietras and Hanahan, 2005). The first evidence of PDGF expression in pancreatic cancer was provided by Ebert et al. (1995). The authors identified PDGF and PDGFR-β in pancreatic cancer cell lines PANC-1 and HPAF. Moreover, expression of PDGF ligands and PDGFRs was demonstrated in human pancreatic cancer specimens and surrounding stromal cells. Since then, a couple of studies have examined the role of PDGF expression and PDGF receptors in pancreatic cancer (Chen et al., 2005, Hwang et al., 2003, Li et al., 2003, Yamamoto et al., 1996).

6.1. Imatinib (Gleevec, STI-571)

The most important PDGFR inhibitor is imatinib (Gleevec, STI571; Novartis), a small-molecule TKI which also targets c-KIT and BCR-Abl. Imatinib was primarily developed for the treatment of chronic myelogenous leukaemia (Abl), and it is also used for the treatment of c-KIT positive GIST (approved by FDA in May 2001). As PDGFRs are expressed in the stroma of the majority of solid tumors, imatinib was tested in different animal models that express PDGFRs. However, when supplied as a single agent, imatinib had generally no effect on tumor growth. A significant response was obtained only when imatinib was used in combination with chemotherapeutic agents. This might be due to increased chemotherapeutic drug uptake by tumor cells as a result of the reduction of PDGFR-dependent interstitial fluid pressure (Pietras et al., 2001). Accordingly, Bergers et al. (2003) documented that PDGFRs are functionally important for the maintenance of vascular pericytes, presenting imatinib as a resonably selective pericyte-targeting drug (Bergers et al., 2003). In another preclinical model, Pietras and Hanahan (2005) showed that otherwise intractable end-stage pancreatic islet cell tumors could be controlled by using a combination regimen of imatinib, VEGFR-targeting and metronomic chemotherapy. Imatinib therapy resulted in pericyte detraction and consequently in a re-sensitization of the mature vasculature to concomitant antiangiogenic therapy and chemotherapy (Pietras and Hanahan, 2005). For pancreatic adenocarcinoma variable results were reported. Farivar et al. (2003) did not find significant anti-proliferative activity in vitro, when imatinib was tested in different human pancreatic cancer cell lines (BxPC-3, MiaPaCa-2, PANC-1). In contrast, Li et al. (2003) reported 50% growth inhibition (GI50) in vitro at concentrations of 21, 26 and 31.5μM for BxPC-3, MiaPaCa-2 and AsPC-1 cells, respectively. However, these effects were apparently not mediated through a blockade of PDGFR tyrosine kinases, since PDGF did not stimulate pancreatic cell growth and did not lead to PDGFR- or MAP-kinase phosphorylation in this study. As indicated by its relatively high GI50 concentrations, the anti-proliferative activity of imatinib appeared to be of cytotoxic origin. Consequently, the authors of this article questioned the clinical relevance of imatinib in pancreatic cancer. Hwang et al. (2003) demonstrated that phosphorylated (activated) PDGFRs are expressed in both tumor cells and endothelial cells of human pancreatic cancer specimen as well as of experimental L3.6pl tumors, when implanted in nude mice. Combination therapy of imatinib and gemcitabine resulted in significant growth inhibition of pancreatic tumor xenografts and liver metastases while imatinib monotherapy failed to inhibit tumor progression. Very recently, a clinical trial was published by Chen et al. (2005) who randomly treated 26 patients with unresectable pancreatic cancer using either gemcitabine (1000mg/m2/wk, i.v.) or imatinib (2×400mg/d, p.o.). Generally, imatinib was well-tolerated in this study and QOL was similar in both treatment groups. Nevertheless, in this small series, imatinib did not exhibit a significant effect on pancreatic cancer growth and progression. No prolonged survival was observed when compared with gemcitabine standard therapy, and treatment response was independent of c-KIT and PDGFR-β expression in imatinib-treated patients (Table 1). However, combination treatment of gemcitabine and imatinib was not studied in this trial. In conclusion, the clinical relevance of imatinib monotherapy in pancreatic cancer seems to be rather doubtful, since it has failed to inhibit pancreatic cancer growth in vitro, in animal models and in a clinical trial, when applied as a single agent. On the other hand, combination therapy with gemcitabine was effective in experimental tumor models and should be studied clinically. A clinical Phase II trial currently recruits patients with advanced pancreatic cancer for further evaluation of an imatinib–gemcitabine combination therapy (Table 2).

6.2. Other multi-targeting PDGFR-TKIs: Sorafenib (BAY43-9006), Leflunomide (SU101)

In addition to the above-mentioned broad-spectrum RTK inhibitors SU6668, Sutent (SU11248) and AG013736, Vatalanib (PTK787/ZK222584) also inhibits PDGFRs at higher concentrations (see also Sections 5.2.1, 5.2.3, 5.2.4). Recently, other small-molecule PDGFR inhibitors have been developed with different specificities. Sorafenib (BAY43-9006; Bayer) is a novel RAF kinase inhibitor that inhibits cell proliferation and angiogenesis (Wilhelm et al., 2004). Subsequent characterization revealed it to be a multitargeted inhibitor with activity against PDGFR-β, VEGFR-2, VEGFR-3 and c-KIT among others. BAY43-9006 inhibits the MAPK pathway in several tumor cell lines and corresponding tumor xenograft models, including pancreas, colon and breast (Wilhelm et al., 2004). Phases I and II studies were conducted in advanced solid tumors and showed promising results. Phase III trials are in progress (Ahmad and Eisen, 2004, Awada et al., 2005, Richly et al., 2004).

Leflunomide (Arava, SU101; Aventis) is an immunomodulatory drug, which was originally developed for the treatment of rheumatoid arthritis and is currently being investigated for immunosuppression in organ transplantation. Leflunomide is a small-molecule PDGFR-TKI which recently has demonstrated broad-spectrum anti-tumor activity in preclinical studies, and antitumor activity in advanced prostate cancer (Ko et al., 2001). Neither of those two drugs has been tested specifically in pancreatic cancer patients, but BAY43-9006 in particular might be a possible candidate due to its broad activity spectrum.

7. Src kinase inhibition in pancreatic cancer

Src kinases are cytoplasmic, non-receptor tyrosine kinases (CTKs), which mediate signaling from many types of receptors including receptor tyrosine kinases (RTKs), integrins, and G-protein-coupled receptors (Fig. 2). They are critically involved in a variety of intracellular signaling pathways which promote cell survival, proliferation, motility, adhesion, transformation and angiogenesis (Summy and Gallick, 2003). RTKs that signal through Src kinases include EGFRs, PDGFRs and FGFRs. The Src family consists of eight members: three ubiquitously expressed (c-Src, c-Yes and Fyn), whereas the other five mainly expressed in haematopoietic cells (Lck, Hck, Fgr, Lyn and Blk). c-Src represents the 60-kDa protein product of the c-src proto-oncogene, overexpression of which has been associated with tumorigenesis, metastasis and invasion. Src family kinases – most notably c-Src – are frequently overexpressed or aberrantly activated in a variety of epithelial cancers, particularly in colon and breast cancer, but also in pancreatic cancer (Lutz et al., 1998, Summy and Gallick, 2003). Further, the extent of increased c-Src activity often correlates with malignant potential and patient survival. Lutz et al. (1998) found c-Src overexpression in 13 of 13 human pancreatic cancer specimens in comparison to normal pancreatic tissue, and elevated c-Src kinase activity in 14 of 17 pancreatic cancer cell lines (Lutz et al., 1998). It has been shown that activated c-Src expression in pancreatic carcinoma cells resulted in upregulation of IGF-1 receptor expression (Flossmann-Kast et al., 1998), reduced expression of E-cadherin (Menke et al., 2001) and increased production of IL-8 and VEGF (Trevino et al., 2005a, Trevino et al., 2005b), leading to increased cell proliferation, decreased cell–cell adhesion, enhanced migration and increased angiogenesis, respectively. Moreover, there is some evidence that Src kinase expression plays a role in the development of chemoresistance, and Src inhibition would enhance chemosensitivity of tumors (Grant and Dent, 2004). Given the fact that Src kinases participate in so many aspects of tumor progression and metastasis, strategies to target Src kinase activity alone and in combination with cytotoxic drugs might prove effective in anti-cancer therapy.

7.1. Pyrazolopyrimidines: PP1 and PP2

Pyrazolopyrimidines are experimental TKIs of the Src kinase family, which have been tested in different tumor models. The synthetic pyrazolopyrimidine PP1 (Calbiochem/Merck AG) is a novel, potent and cell permeable inhibitor of Src family kinases, that is >800 times more selective for mutant Src compared to wild-type Src and also blocks Fyn. PP2 (AG1879; Calbiochem/Merck AG) acts similarly to PP1 but also inhibits Lck, Fyn and Hck. Ito et al. (2003) proposed that Src kinase activity might be crucial for the invasive potential of pancreatic cancer since a substantial fraction of c-Src is localized in focal adhesions, regulates integrin-dependent signaling, and is critically involved in cell–matrix interactions. To test their hypothesis, they applied PP1 on pancreatic cancer cell lines in vitro (BxPC-3, MiaPaCa-2 and PANC-1). PP1 treatment completely suppressed Src phosphorylation, significantly inhibited activity of matrix metalloproteinases (MMP2, 40%; MMP9 35%), resulted in up to 50% growth inhibition, and induced more than 90% suppression of cellular invasion of all cell lines (Ito et al., 2003). Regarding chemoresistance of pancreatic cancer, Duxbury et al. (2004a) found higher levels of total Src expression, Src phosphorylation and Src activity in the gemcitabine-resistant pancreatic cancer cell line PANC-1GemRes, relative to the PANC-1 mother cell line. RRM2, a putative chemoresistance enzyme, was overexpressed in PANC-1GemRes. In vitro exposure to PP2 enhanced gemcitabine chemosensitivity, attenuated gemcitabine resistance and suppressed RRM2 expression in PANC-1GemRes cells, whereas constitutively activated Src increased gemcitabine resistance. In vivo, combination therapy of PP2 and gemcitabine substantially decreased tumor growth and inhibited metastatic spread in orthotopic PANC-1GemRes xenografts (Duxbury et al., 2004a). In summary, pyrazolopyrimidines appear to possess anti-invasive, anti-proliferative and chemosensitizing activity in experimental pancreatic cancer.

7.2. AZM475271

AZM475271 (AstraZeneca) is a novel orally bioavailable anilinoquinazoline inhibitor of c-Src, which was recently tested for pancreatic cancer treatment in nude mice. Yezhelyev et al. (2004) showed antitumor and antimetastatic activity of AZM475271 in orthotopic implanted human pancreatic cancer. AZM475271 reduced tumor volume by 40%, inhibited tumor cell proliferation, increased apoptosis and inhibited tumor angiogenesis. Moreover, combination treatment with AZM475271 and gemcitabine reduced tumor volume by 90% and completely suppressed metastatic tumor spread. The authors suggested a chemosensitizing effect of AZM475271 to the cytotoxic effect of gemcitabine (Yezhelyev et al., 2004).

7.3. Other Src inhibitors: siRNA, AP23846, SKI-606, AZD05230

Using a c-Src-specific, small interfering RNA (siRNA), Duxbury et al. (2004b) found that Src kinase expression and activity are directly correlated with gemcitabine chemoresistance in pancreatic cancer cell lines. In that study c-Src specific siRNA increased gemcitabine-induced, caspase-mediated apoptosis and decreased AKT-kinase activity (Duxbury et al., 2004b). AP23846, another novel ATP-based c-Src kinase inhibitor was recently shown to be approximately 10-fold more potent in solid tumor cell lines than PP2 (Summy et al., 2005). AP23846 reduced cellular migration, VEGF expression and IL-8 expression in L3.6pl pancreatic cancer cells in vitro. SKI-606, a dual tyrosine kinase inhibitor of Src and Abl was also evaluated recently for its antineoplastic activity. SKI-606 inhibited Src autophosphorylation and tyrosine phosphorylation of FAK, a substrate of Src in colon cancer cell lines. Moreover, SKI-606 inhibited colony formation, proliferation and tumor growth of experimental colon cancer and CML (Golas et al., 2003, Golas et al., 2005). However, no data on pancreatic cancer have been published. New insights into downstream signaling mechanisms, including activation of STAT3, PDK1 and AKT, further corroborate the importance of Src family kinases in pancreatic tumorigenesis and chemoresistance. Despite our rather clear understanding of Src family kinases as pro-oncogenes, only one Src family kinase inhibitor has entered clinical trials, so far: AZD0530 (Table 2). AZD0530 is a novel selective, orally active Src kinase inhibitor, that has low antiproliferative activity, but inhibits tumor cell adhesion, migration and invasion (Green et al., 2004). A clinical Phase I/II multicenter trial is currently evaluating AZD0530 in combination with gemcitabine for the treatment of patients with unresectable or metastatic pancreatic cancer (Table 2).

8. Chemoresistance and TKIs in pancreatic cancer

In addition to promoting the malignant phenotype of tumor cells by activation of multiple transformation pathways, several tyrosine kinases have been implicated in the pre-existence and development of cell resistance to chemotherapeutic drugs. Links between chemoresistance and the activity of various receptor tyrosine kinases (e.g. ErbB-1) and non-receptor tyrosine kinases (e.g. Src) have been described (Grant and Dent, 2004). For these reasons, TKIs may not only act as antitumor agents in their own right, but also as agents that modulate tumor cell resistance to cytotoxic drugs. The Src kinase inhibitor PP2 effectively promoted gemcitabine lethality in gemcitabine resistant pancreatic cancer cell lines (see also Section 7.1) (Duxbury et al., 2004b). However, no synergism for PP2 and other chemotherapeutic agents was shown in the same study. This finding suggests that synergism between TKIs and cytotoxic agents, when it occurs, may be highly cell-type and drug-specific. Chemoresistance is a highly complex, pleiotropic phenomenon and more mechanistic studies are needed to predict whether a TKI will overcome resistance to an individual cytotoxic agent. Chemoresistance may stem from enhanced activation of a drug efflux mechanism (e.g. multidrug resistance protein) (Aszalos and Ross, 1998), specific biochemical disturbances that antagonize the conversion of a cytotoxic drug to its active form (e.g. elevated ribonucleotide reductase) (Goan et al., 1999), or increased expression of anti-apoptotic proteins conferring resistance to a broad range of cytotoxic agents. Src inhibition in Duxbury's study was associated with several events, including reduction in ribonucleotide reductase expression, inactivation of AKT kinase, and diminished activity of E2F-1. Which of these downstream events was most directly responsible for enhanced gemcitabine sensitivity remains to be determined. However, all this makes a strong argument for pursuing the strategy of combining gemcitabine with TKIs in pancreatic cancer.

9. Future perspectives

During the past decade, an enormous amount of knowledge has been collected for the rational design of molecular therapies for cancer patients. In particular, tyrosine kinase inhibitors represent a major breakthrough in the development of molecular targeted therapy. Preclinical evidence for a broad activity of TKIs in pancreatic cancer is encouraging and the first large-scale clinical trials revealed promising results (e.g. cetuximab and erlotinib) (Moore et al., 2005, Trevino et al., 2005b, Xiong et al., 2004). However, other clinical trials in pancreatic cancer patients have been disappointing (e.g. trastuzumab) (Safran et al., 2004) (Table 1). Tumor heterogeneity and activity of alternative signaling pathways may partly explain the therapeutic failures seen in those trials. Basically, if we want to use TKIs in pancreatic cancer, the molecular targets must be expressed by the tumor. Moreover, the efficacy of therapy depends on the differential level of target expression by tumor cells versus normal cells. This means that the targeted pathway must be activated in the tumor cells. The success of trastuzumab and bevacizumab in breast cancer and colorectal cancer, respectively, has shown that selection of appropriate patients is critical to demonstrate efficacy of a given drug. Conversely, poor patient selection strategies might dilute the clinical effect of a potentially effective drug. Therefore, we will have to focus not only on the pursuit of new therapeutic agents, but also on tools that allow us to prospectively identify patients who are more likely to benefit from a certain therapy (Broxterman and Georgopapadakou, 2005). The relative therapeutic merits of receptor antibodies versus small-molecule inhibitors, highly selective TKIs versus multi-targeting agents, and reversible versus irreversible TKIs are not well defined at this juncture; clinical benchmarking studies are required to address these issues. Some agents might be more effective when used in early stages of pancreatic cancer, and multi-targeting compounds might be required to overcome redundancies in deregulated signaling cascades (e.g. BAY43-9006). For example, irreversible ErbB-1 inhibitors (e.g. EKB569), dual ErbB-1/ErbB-2 (e.g. PKI-166) and pan-ErbB receptor inhibitors may have better antitumor activity in pancreatic cancer. Future studies will have to investigate combinations of different TKIs and combinations with other targeted agents as well as with cytotoxic drugs to inhibit multiple mechanisms of tumor progression. This should counteract the problem of complex kinase profiles and chemoresistance in pancreatic cancer, and possibly result in greater antitumor activity. In particular, combined inhibition of the EGFR and VEGFR pathways may produce synergistic results, as shown preclinically for PKI-166 and Vatalanib. Clinical trials that combine TKIs with chemotherapy are underway, and the first results from ongoing and completed Phase III trials of cetuximab and erlotinib are very promising.

10. Conclusion

The clinical development of tyrosine kinase inhibitors is far from simple and we need to better understand biological and clinical criteria for patient selection and how to best use the different available agents in pancreatic cancer. Targeting not only the tumor cells but also the tumor cell environment, accurate molecular characterization of the tumor and combination of TKI therapy with conventional chemotherapy (e.g. gemcitabine), will help optimize therapeutic outcome in pancreatic cancer. This may ultimatively improve the presently grim prognosis of pancreatic cancer patients.

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Department of Surgery, Klinikum Grosshadern, Ludwig-Maximilian-University Munich, Marchioninistrasse 15, 81377 Munich, Germany

Corresponding author. Tel.: +49 89 7095 0; fax: +49 89 7095 6574.

PII: S1368-7646(06)00004-5

doi:10.1016/j.drup.2006.02.002

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