Aplicaci´ on a datos reales

Due to its association with the degree of disease inflammation and clinical disease activity (Rho et al., 2009), NAMPT has been identified as a possible therapeutic target for the development of new RA drugs (Brentano et al., 2007). Preliminary studies have demonstrated that suppressing NAMPT with a highly specific small molecule inhibitor named APO866 limits the effects of RA in a murine collagen-induced arthritis model. This effect was seen when treatment was administered before the appearance of arthritis, and also in established disease (Busso et al., 2008). In fact, in vivo studies indicated that APO866 had comparable efficacy to anti-TNF treatment (Busso et al., 2008) and Nowell et al; unpublished). Therefore the last part of this review will provide a historical perspective on APO866, including its interactions with NAMPT and the findings of work involving this molecule.

1.4.1 Discovery of APO866

Angiogenesis is the process involving the growth of new blood vessels from pre- existing vessels. In normal tissues this is a vital process in growth, development and wound healing. However, excessive angiogenesis is associated with tumour formation. In 2003 researchers working for Fujisawa Deutschland GMBH developed a number of different compounds with the aim of inhibiting/reducing angiogenesis. One such compound was (E)-N-[4-(1-benzoylpiperidin-4-yl) butyl]-3-(pyridin-3-yl) acrylamide, which was designated FK866 (Beidermann et al., 2003). Since its development this compound has been in the possession of a number of different pharmaceutical companies. In 2005 the Fujisawa Pharmaceutical Co., Ltd merged with Yamanouchi Pharmaceutical Co., Ltd to form Astellas Pharma GmbH. In October 2005 Swiss biopharmaceutical company Apoxis SA acquired exclusive worldwide development and marketing rights to the drug and renamed it APO866, although it is still often referred to in some literature by its former names FK866 and WK175 (Apoxis, 2005). Finally, in 2007 Apoxis was acquired by TopoTarget, which has continued to initiate clinical studies of APO866 in order to understand its biologic effects.

1.4.2 Assigning in vitro functionality of APO866

Wosikowski et al (2002) published the first study of APO866 in vitro. In a screen of novel anti-tumour agents, they discovered that APO866 had distinct characteristics to conventional chemotherapies: it induced apoptosis in cells, with no DNA-damaging effects which can lead to genomic instability and generation of resistant tumour phenotypes (Wosikowski et al., 2002). They stimulated a human monocytic cell line (THP-1) with APO866 for four days, and found that this agent caused a time and dose- dependent decrease in cell metabolic activity, as determined by a cell proliferation assay.

They also observed a concurrent increase in the proportion of apoptotic cells. They discovered that it exerted these effects by interfering with NAD+ _{biosynthesis, resulting} in decreased cellular NAD+ _{content (Wosikowski et al., 2002). Prolonged incubation of} cells with APO866 altered mitochondrial membrane potential. This disruption of mitochondrial function led to activation of caspase 3, which is a crucial event in the final degradation phase of apoptosis. These data suggest that depletion of intracellular NAD + _{is a trigger that initiates the apoptotic cascade, resulting in an anti-tumour effect} (Wosikowski et al., 2002).

In 2003 Hasmann and Schemainda explored the pro-apoptotic effects of APO866 further in human liver carcinoma (HepG2) cells. Like Wosikowski et al (2002), they observed that APO866 decreased metabolic activity and cell number, whilst increasing the proportion of non-viable cells. These effects were observed along with a concurrent decrease in intracellular NAD+_{. They sought to determine which NAD}+ _biosynthesis pathway APO866 was involved in, using radiolabelled NAD+ _{precursors Nam and NA.} APO866 caused rapid inhibition of Nam-mediated NAD+ _{production, with no discernible} effect on NA-mediated NAD+ _{levels. Additionally, no accumulation of radiolabelled NMN} was observed; suggesting that NAMPT and not the downstream enzyme NMNAT was the target for APO866 (Hasmann and Schemainda, 2003).

1.4.3 Molecular basis of APO866 inhibition of NAMPT activity

In 2006 groups working on the crystal structure of NAMPT co-crystallised it with APO866, as well as its reaction product NMN, in order to determine the molecular basis of NAMPT enzymatic activity and inhibition (Kim et al., 2006, Khan et al., 2006). NAMPT is a dimer in solution, and is only active in this dimeric form. APO666 is tightly bound in a tunnel at the interface of this dimer. It competes directly with Nam, and therefore acts as a competitive inhibitor of NAMPT activity (figure 1.10). This inhibition is highly specific to NAMPT, as the structurally similar NAPRT does not contain a tunnel at its dimer surface, which is the molecular basis for APO866 inhibition. Mutations of the NAMPT binding site abolished inhibition by APO866 (Khan et al., 2006).

Figure 1.10 The molecular basis of NAMPT inhibition by APO866

A) Ribbon diagram of the NAMPT dimer (in cyan and gold) co-crystallised with NMN (shown in green and red). B) Interaction of NAMPT dimer with APO866 (shown in black). Images adapted from (Hasmann and Schemainda, 2003).

1.4.4 Assessing impact of APO866 in vivo

Once the anti-tumour effects of APO866 were established, Drevs and colleagues (2003) set out to investigate the anti-tumour, anti-metastatic and anti-angiogenic potency of this agent in a murine renal cell carcinoma model (RENCA) (Drevs et al., 2003). This was achieved by inoculating mice with RENCA tumour cells to initiate tumour growth, before treating orally with various doses of APO866. Mice were sacrificed on day 21 and analysed for primary tumour weight and volume, lung metastases and tumour vessel density. It was found that doses of ≥10 mg/kg showed significant anti-tumour and non- significant anti-metastatic activity on RENCA primary tumours, with all doses resulting in anti-angiogenic activity (Drevs et al., 2003). In a subsequent study the metabolic effects of APO866 were investigated in a mouse mammary carcinoma model in vivo. Groups of mice were given two different dosages of the drug, administered by intra-peritoneal (i.p.) injection. The lower dosage treatment resulted in retarded tumour growth compared to controls, whilst the higher dose also caused reduced mammary carcinoma tumour radiation sensitivity. Histological annexin staining of APO866-treated tumours also showed evidence of apoptosis compared with controls (Muruganandham et al., 2005).

Although most interest in APO866 is focused on its potential as an anti-cancer agent, it is now emerging that APO866 may also be useful in inflammatory and autoimmune diseases. Busso et al found that APO866 treatment reduced intracellular

NAD+ _{levels and pro-inflammatory cytokine secretion by inflammatory cells in vitro. In}

vivo, animals with collagen-induced arthritis (CIA) were treated with APO866 by i.p.

Injection, resulting in reduced disease severity, comparable to anti-TNFα drug etanercept. They observed that APO866 caused a reduction in circulating TNFα levels during endotoxemia in mice (Busso et al., 2008). In light of these findings, the Nowell research group have further investigated the in vivo efficacy of APO866 in CIA animals with the use of osmotic minipumps. Early findings suggest that APO866 ameliorates disease in treated animals in a time and dose-dependent manner.

1.4.5 Clinical studies involving APO866

Preclinical studies in rats and monkeys showed that lymphocytes are most sensitive to APO866, resulting in lymphocytopenia and reticulocytopenia (Holen et al., 2002). Following on from these findings Holen et al (2008) carried out an open-label Phase I clinical trial (Holen et al., 2008). Patients with solid tumour malignancies were given escalating doses of APO866 as continuous 96 hour infusions. The group aimed to determine dose-limiting toxicities of APO866, in addition to its pharmacokinetics, efficacy and effect on VEGF levels. VEGF is an important signalling protein in vasculogenesis and angiogenesis, which can contribute to disease when over expressed by providing a blood supply to solid tumours. APO866 was generally well tolerated, although some patients suffered from a drop in platelet levels, but recovered quickly at the end of the study. Plasma VEGF levels appeared to drop, but due to the small sample size this effect was not significant. None-the-less, a few patients showed improvement. Although APO866 has potential as a single agent, it may work best if administered in combination with other anti-tumour agents (Holen et al., 2008). TopoTarget subsequently initiated a Phase II study to determine the efficacy and safety of 3 cycles of APO866 for the treatment of advanced cutaneous melanoma (Topotarget, 2009b), as well as a Phase I/II study for the treatment of B-Chronic Lymphocytic Leukaemia (B-CLL) (Topotarget, 2009a). These studies have recently been completed, and a third Phase II study on APO866 in cutaneous T cell lymphoma (CTCL) is currently underway (Topotarget, 2011).