**7. HA and receptor antagonists in clinical trials**

Since it is evident that HA and its receptors play an important role in BCA and other tumours, it is unsurprising that reagents blocking HA metabolism are being assessed as therapeutic agents in certain types of cancer. In pre-clinical models, Kultti *et al. (*2009) demonstrated that the HAS inhibitor 4-MU (4-Methylumbelliferone) specifically depletes intracellular levels of UDP-Glc-UA (Kakizaki *et al*., 2004) by serving as a glucoronidation substrate in A2058 melanoma cells, MCF-7, MDA-MB-361 BCA cells, SKOV-3 ovarian, and UT-SCC118 squamous carcinoma cells. Additionally, Lokeswar *et al.* (2010) used 4-MU to block growth of human prostate cancer cell line xenografts in immunocompromised mice. 4-MU induces apoptosis in these tumours and also strongly inhibits cell proliferation, motility and invasion. These effects can be reversed by addition of HA, which demonstrates that, although 4-MU does not specifically block HAS and has other off target effects, its effects on tumour cell growth result from inhibition of HAS (Ekici *et al*., 2004).

HA has also proven to be a good adjunct therapeutic option *in vivo* in human cancers since it promotes targeting of active anti-cancer compounds. For example, when patients with Calmette-Guérin refractory bladder cancer were included in a Phase I clinical trial using Paclitaxel-HA (ONCOFID-P-BTM) for treatment of their cancers, 60% of the patients treated exhibited a clinical response with minimal toxicity reported (Bassi *et al*., 2010). HA has been successfully used to carry/target other chemotherapeutics, thus reducing cytotoxic side effects of the active drug. Hyung *et al.* (2008) demonstrated the efficacy of HA-coated drug carriers by delivering doxorubicin to MDA-MB-231 and ZR-75-1 human BCA cell lines (Hyung *et al*., 2008). Similarly, after coating nanoparticles containing paclitaxel with HA, cytotoxicity is reduced while cellular uptake of the drug by S-180 sarcoma cell line is enhanced 9.5 fold *in vitro* and in a mouse model (He *et al*., 2009).

In light of fairly recent evidence for the display of CD44 on BCA tumour initiator cells, interest in developing CD44 targeted therapies has increased. Riechelmann *et al.* (2008) exploited the potential of CD44 in a Phase I clinical trial using an antimicrotubule agent (mertansine) and a monoclonal antibody to CD44v6 (bivatuzumab), (BIWI 1), to treat patients with recurrent or metastatic head and neck squamous cell carcinoma (Riechelmann *et al*., 2008). The response to the treatment was unexpectedly variable and the trials using these agents were stopped after one patient died of toxic epidermal necrolysis (Tijink *et al*., 2006). Targeting the HA binding ability of activated CD44 may result in decreased toxicity.

RHAMM peptide vaccination (e.g. R3, which is HLA-A2-restricted) has recently been assessed in PhaseI/II clinical trials for treatment of MM, AML, and CLL (Giannopoulos *et al*., 2010, Greiner *et al*., 2008, 2010, Schmitt *et al*., 2008). Additionally, vaccination with DC pre-stimulated against the same peptide has also undergone Phase I and II clinical trials for treatment of CLL (Hus *et al*., 2008). Vaccination with RHAMM peptide has the attractive advantage of very low toxicity because it is not expressed in healthy bone marrow tissue. RHAMM vaccination resulted in leukemic blast lysis, blast reduction in the bone marrow and avoided the need for blood transfusions for one patient. Furthermore, an immunological response, marked by an increase in T cell frequency, was observed in 70% of AML, MM, and MDS patients in an initial study (Schmitt *et al*., 2008). Subsequently, RHAMM peptide was shown to be non-toxic at high dosage (1000 µg/vaccination), however, there was no dose-dependent effect, indicating that RHAMM is an effective therapeutic target even at low levels (Greiner *et al*., 2010). A similar response was seen in CLL patients vaccinated with RHAMM peptide, as well as RHAMM peptide-stimulated DC.

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Clinical response was correlated with an increase in CD8+ T cell proliferation and in some cases a decrease in Treg population. Interestingly, in B-CLL patients with clinical response to vaccination with stimulated DC cells, the CD8+ cytotoxic T cell and IL-12 anti-tumour response was increased, whereas the Treg cell population was decreased (Hus *et al*., 2008). In a Phase I study of CLL patients vaccinated with RHAMM peptide, there was no correlation between clinical response and Treg population dynamics (Giannopoulos *et al*., 2010). This strategy has not yet been used for BCA, although, as RHAMM is a prognostic marker for BCA. and overexpressed in many cases which currently do not have a specific targeted therapeutic option (e.g. basal subtype) and also given the magnitude of the response, along with such low toxicity, it is an approach which merits further consideration.
