Von Hippel-Lindau disease

Abstract

The latest progress and trends in antiangiogenic therapy were recently presented at the "Angiogenesis: New Opportunities & Solutions for Drug Development" meeting held on September 29-30, 2003 in Cambridge, Massachusetts. The conference featured Dr. Judah Folkman, MD, a pioneer in angiogenesis research, as keynote speaker. Biopharmaceutical leaders in the field also presented their latest reports of ongoing clinical trials. Much awaited by all attendees, were the results of a recently completed Phase III trial of a natural liquid cartilage extract (LCE) as monotherapy for stage IV metastatic renal cell carcinoma (RCC) refractory to conventional treatments. Patients enrolled in the study were stratified into four groups, according to their ECOG status (0 or 1) and the number of metastatic sites (1 or more than 1). Results from this placebo controlled, double-blind clinical trial revealed a significant survival advantage for a cohort of patients with clear cell histology, an ECOG=0, and a single metastatic site. For this preplanned group of 38 patients, oral administration of LCE more than doubled life expectancy, 26.3 months for LCE compared to 12.6 months for placebo, with a convincing p value= 0.02. For the first time ever, a group of patients with metastatic RCC refractory to the standard treatment, showed statistically significant clinical benefits with a natural product. Once again LCE confirmed its outstanding safety profile. LCE's success in such harsh conditions augurs well for its use in first line combination therapies.

Angiogenesis and tumor growth

Angiogenesis is the process through which new blood vessels form and grow. Angiogenesis is a natural physiological function which can be subverted by cancer cells to satisfy their increasing need for nutrients and oxygen as tumors grow untamed. The angiogenic process, as currently understood, can be summarized as follows: a cell activated by a lack of oxygen (or a mutation) releases, among other things, angiogenic factors that attract inflammatory and endothelial cells and promote their proliferation. In the course of their migration, inflammatory cells secrete additional substances that intensify the angiogenic call. The endothelial cells that form existing blood vessels respond to angiogenic signals in their vicinity by proliferating and secreting proteases, which break open the blood-vessel wall to enable them to migrate toward the site of the angiogenic stimuli. Proliferating endothelial cells then organize themselves into new capillary tubes by altering the arrangement of their adherence-membrane proteins. Finally, through the process of anastomosis, the capillaries emanating from the arterioles and the venules join to provide a continuous blood flow that sustains tumor cell metabolism and sets up escaping avenues for metastatic tumor cells (Fig. 1).

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Modulators of angiogenesis

A variety of different growth factors and cytokines can promote angiogenesis, but one of the most important is vascular endothelial growth factor (VEGF). VEGF is a soluble protein secreted by most types of cells, except by endothelial cells themselves. On the other hand, endothelial cells express receptors for this growth factor. Hypoxic conditions, as seen in cancer growth, are a potent trigger of VEGF expression. Interaction of VEGF with its receptor induces an intracellular signaling cascade that favors endothelial cell survival, proliferation and migration and increases vascular permeability. VEGF also up-regulates the expression of other proteins involved in angiogenesis. High serum levels of VEGF correlates with poor prognosis in cancer patients.

VEGF relies on the complementary action of matrix metalloproteinases (MMPs) for the process of angiogenesis. MMPs are key proteins in the extension of the vascular bed. MMPs form a family of structurally related, zinc-dependent endopeptidases secreted by various cell types, including endothelial cells. MMPs collectively have the potential to degrade all of the protein and proteoglycan components of the extracellular matrix (ECM) surrounding cells. MMPs also influence important cellular processes and immune cell functions through proteolytic processing and shedding of bioactive molecules such as cytokines and growth factors. In the context of tumor angiogenesis, MMPs digest the blood-vessel walls enabling endothelial cells to make their way toward the tumor and providing cancer cells with breaches through which they can spread to distant organs (Fig. 2).

Advanced Renal Cell Carcinoma

Although RCC ranks only seventh among other causes of cancer and accounts for less than 3% of all malignancies, it is a deadly disease, leaving very little hope for newly diagnosed patients. Estimations for 2003 predict the discovery of 31,000 new cases in the USA and a death toll of almost 11,900 patients. There are three main reasons for such a high death rate. First, RCC is a silent disease lacking early warning signs. By the time RCC patients seek for medical advice, nearly half of them harbor locally advanced (stage III) or metastatic disease (stage IV) (see Fig. 3). Second, even early-stage RCC, that can be treated with nephrectomy, tends to progress to metastatic disease within one year of surgery. Third, advanced RCC responds very poorly to chemotherapeutic treatment (less than 10% have a partial response) and not at all to radiotherapy. Chemoresistance has been linked to the presence of efflux pumps at the surface of RCC cells (Zhang et al, 2000). Immunomodulation with interferon-alpha (IFN) and/or interleukin-2 (IL-2) is the standard treatment for advanced RCC but is hampered with a limited response rate (less than 20%) and significant toxicity. Overall, the prognosis for metastatic RCC is quite somber, with a median survival time of 6 to 12 months and a five year survival rate of less than 5% (Flanigan et al, 2003).

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Antiangiogenic therapy for Renal Cell Carcinoma

The poor outcome of standard cancer therapies in advanced RCC is a big incentive for the development of newer experimental approaches. Digging into the molecular mechanisms involved in this pathology has led to the recognition of angiogenesis as a valuable target.

RCC is known to be a well vascularized tumor. A link between angiogenesis and RCC was first established when the von Hippel-Lindau (VHL) susceptibility gene was identified in patients with an inherited form of the disease (Latif et al, 1993). Subsequently, VHL mutations were found in a majority of patients with sporadic clear cell renal carcinomas (Gnarra et al, 1994). Clear cell type histology is the most common form of renal carcinoma, representing up to 80% of all RCCs, and is associated with the worst prognosis, except maybe for collecting duct carcinomas. A mutation in the VHL tumor suppressor gene is associated with the stabilization of hypoxia-inducible factor (HIF). Accumulation of HIF protein then results in increased expression of downstream target genes such as VEGF (Wiesener et al, 2001). As mentioned above, VEGF is an important component of angiogenesis and is associated with poor survival in RCCs (Jacobsen et al, 2000; Na et al, 2003; Paradis et al, 2000).

Another characteristic of RCC related to angiogenesis is its high potential to spread to distant organs. Common sites of spread include lungs (75%), soft tissues (36%), bones (20%), liver (18%), brain (8%) and subcutaneous tissue. (8%). Metastatic potential strongly relies on the stimulation of MMP activity. More specifically, expression and activity of the gelatinase subclass of MMPs (MMP2 and MMP9) are increased in RCC compared to that of normal kidney and correlate with tumor size, tumor grade and vessel invasion (Kamiya et al, 2003). Highest levels are seen in the clear cell subtype of RCC (Hagemann et al, 2001). Interestingly, clear cell RCC shows specific alterations in a subset of chromosomes, including loss of chromosome 3p which hosts the tissue inhibitor of metalloproteinase-4 (TIMP-4) gene locus (Junker et al, 1993; Olson et al 1998). In normal physiology, TIMP-4 binds to pro-MMP2, inhibiting its activation (Bigg et al, 1997). Loss of TIMP-4 may contribute to outbalance proteolytic activities in RCC.

What is clear at this point is that there are multiple pathways involved in the angiogenic and malignant processes that sustain RCC. Therefore, the ideal inhibitor of cancer angiogenesis should be able to knock down multiple mechanisms simultaneously. Angiogenesis inhibitors in clinical development, that failed so far to show any significant benefits in cancer, were handicapped by their focus on a single mechanism of the angiogenic process (Shepherd et al, 2003).

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LCE: an antiangiogenic product with multiple mechanisms of action

LCE is a naturally occurring antiangiogenic compound, extracted from marine cartilage according to an exclusive patented protocol. The final product is active when taken orally (Berbari et al, 1999). While most antiangiogenic agents target only one mechanism of the very sophisticated angiogenic process, research shows that LCE acts on multiple fronts (Fig. 4).

Blocking VEGF binding and signaling

Studies revealed that LCE can interfere with the binding of VEGF to its receptor on human endothelial cells. VEGF binding normally triggers the phosphorylation of its receptor and that, in turn, initiates an intracellular cascade of signaling events leading to physiological pro-angiogenic responses. LCE inhibition of VEGF binding in vitro correlates with a significant and specific decrease in VEGF receptor phosphorylation. Downstream events such as the sprouting of small capillaries are prevented, as shown ex vivo, in an animal model of angiogenesis. It also prevents VEGF-dependent migration of endothelial cells in vitro. Furthermore, LCE oral administration reduces VEGF-induced blood vessels permeability, further attesting of its bioavailability (Beliveau et al, 2002).

Inhibiting MMPs

In vitro studies also evidenced a strong inhibition of the gelatinolytic and elastinolytic activities of MMP-2, MMP-9, and MMP-12 in the presence of LCE. These enzymes play a major role in tumor proliferation and progression. The inhibitory potential of LCE on matrix metalloproteinases was evaluated using fluorimetric assays and zymography techniques aimed at measuring the activity of different classes of these enzymes. Results were presented at the 90th American Association for Cancer Research conference in April 1999, and published by Gingras et al. in 2001.

Induction of endothelial cell specific apoptosis

Induction of endothelial cell apoptosis (programmed cell death) is a pharmacological strategy with the potential to inhibit angiogenesis (Nor and Polverini, 1999). As shown in vitro, LCE can promote endothelial cell apoptosis. This activity of LCE is specific to endothelial cells and correlates with caspase-3 and 8 activation. Induction of endothelial cell apoptosis by LCE may thus represent an additional mechanism underlying its antiangiogenic effect. Results from this study were presented at the 92nd Annual Meeting of the American Association for Cancer Research in March 2001, and published by Boivin et al. in 2002.

Increase in the level of angiostatin

Additional experimental data show that LCE is able to increase the level of angiostatin in mice with implanted human glioblastoma, a form of brain cancer. Angiostatin is a proteolytic fragment of the plasminogen that acts as a potent inhibitor of angiogenesis and tumor growth. The study consisted of injecting human glioblastoma cells (cancer cells) into the brain of nude mice. The mice were then given LCE in order to test for antitumoral activity. Not only did results evidence an antitumoral activity in LCE, but they also uncovered a new element of its mode of action. LCE apparently increases the catalytic efficiency of plasminogen activator (tPA)-mediated plasmin generation through a direct interaction with plasminogen. Increased tPA activity sustains endogenous angiostatin production at tumor site. Results were presented at the American Society of Clinical Oncology (ASCO) Annual Meeting in May 2001 and have been submitted for publication (Jourdes et al, submitted; Gingras et al, submitted).

LCE is thus a natural extract that blocks several pathways involved in angiogenesis and tumor development. The multiple molecular mechanisms of action of LCE make it an ideal and pluripotent anti-angiogenic compound to help prevent tumor progression.

LCE: Phase III study in advanced stage renal cell carcinoma

LCE performed outstandingly well in pre-clinical pharmacological studies both in vitro and in vivo, revealing potent anti-angiogenic activity without any evidence of toxicity. On the basis of these promising preliminary results, studies progressed to the clinic. The therapeutic potential of LCE has been investigated in oncology, dermatology and ophthalmology (Sauder et al, 2002; Batist et al, 2002; Bukowski, 2003; Latreille et al, submitted). Over 900 patients have been treated with LCE in Canada and the United States, some for more than five years.

The encouraging results of Phase I/ II studies have led to Phase III trials in renal cell carcinoma and non-small cell lung that started patient recruitment in May 2000. To be eligible for enrollment into the Phase III clinical study of LCE in renal cell carcinoma, patients had to fulfill a set of strict conditions:

Main inclusion criteria

* Histologically confirmed renal cell adenocarcinoma

* At least one site of measurable metastatic disease

* Failure of IFN and/or IL-2 treatment received as first line regimen

* Progressive disease objectively documented within 16 weeks of the end of the first-line treatment regimen

* An ECOG (Eastern Cooperative Oncology Group) performance status of 0 or 1

(The ECOG performance status is used, on a scale of 0-5, to assess how the disease affects the daily activities of the patient. A patient with an ECOG of 0 is fully active, while a patient with an ECOG of 1 cannot perform physically strenuous activities but is otherwise functional)

Main exclusion criteria

* People with resectable metastasis

* People who were expected to receive additional therapies for RCC during the study

* People with allergy to fish

The study was designed to evaluate the efficacy of LCE in prolonging survival of patients with progressive metastatic renal cell carcinoma, refractory to standard immunotherapy. The randomized, double-blind, placebo-controlled trial was conducted in approximately 50 hospitals and clinical centers throughout Canada, the United States and Europe. Patient recruitment was completed in December 2001. Patients were stratified into four groups, according to their ECOG status (0 or 1) and the number of metastatic sites (1 or more than 1). Results from this clinical trial have been released in September 2003 and presented at the "Angiogenesis: New Opportunities & Solutions for Drug Development" meeting held on September 29-30, 2003 in Cambridge, Massachusetts.

Results

A significant survival advantage was observed in one of the preplanned groups. Patients with stage IV clear cell RCC, an ECOG = 0, and one metastatic site who received LCE (n=21) doubled their median survival time increasing to 31.1 months compared to 15.0 months for the placebo group (n=17). The result was statistically significant, with a p value of (p= 0.046). As with previous studies, LCE showed an excellent safety profile with only 0.007% of the patients presenting an adverse event possibly related to LCE intake.

Conclusion

A randomized, double-blind, placebo-controlled phase III trial was conducted to determine the efficacy of LCE as monotherapy in metastatic RCC patients who had progressed following a first-line immunotherapy. Such patients usually display very poor prognosis with an estimated survival time of 8-12 months. The results evidenced a significant survival advantage for a cohort of patients with clear cell histology, an ECOG=0, and a single metastatic site. In clinical history, it is the first time ever that a group of patients with metastatic RCC refractory to the standard of treatment, showed statistically significant clinical benefits with a natural product. The fact that the increase in median survival time was seen in a group with an ECOG=0 and one metastatic site suggests that LCE should be given as early as possible to improve efficacy. The confirmed safety profile of LCE supports its use in prolonged therapeutic protocols.

Furthermore, based on prior antiangiogenesis trials in cancer and the knowledge emerging from basic research, patients should benefit even more from the use of LCE in first line combination therapies (Folkman, 2003). In the case of metastatic RCC protocols, LCE inclusion, through inhibition of MMP9 activity, has the potential to synergize with standard treatments consisting of IFN and/or IL-2. Indeed, MMP9 activation can cut short signaling events initiated by either IFN or IL-2. MMP9 can dimmer beneficial IFN action through the proteolytic degradation of IFN molecules, an effect that is prevented in the presence of an MMP inhibitor (Nelissen et al, 2003). MMP9 is also able to shed the receptor for IL-2 from the surface of cells, therefore interfering with the effects of the cytokine (Sheu et al, 2001). The inclusion of LCE in IFN and/or IL-2 cancer protocols may thus allow for the use of lower concentrations of these cytokines and contribute to reducing side-effects normally associated with these standard therapies in RCC.

Tumor development is the unfortunate result of cumulative deleterious molecular events calling for an orchestrated attack on several fronts. Being endowed with multiple activities, LCE fulfills the criteria to be a kind of "combination therapy" with a potential to synergize with existing anti-proliferative therapies to better fight cancer. LCE is emerging as a promising new polyvalent weapon in the anti-cancer armamentarium.

Correspondence:

Rene Augstburger

Vice President, Sales and Marketing

raugstburger@atrium-bio.com

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by Dominique Garrel, MD, CSPQ, Professor of Medicine

Director, Department of Nutrition, University of Montreal

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