Phase I clinical trial and pharmacodynamic evaluation of combination hydroxychloroquine and doxorubicin treatment in pet dogs treated for spontaneously occurring lymphoma

Abstract

Autophagy is a lysosomal degradation process that may act as a mechanism of survival in a variety of cancers. While pharmacologic inhibition of autophagy with hydroxychloroquine (HCQ) is currently being explored in human clinical trials, it has never been evaluated in canine cancers. Non-Hodgkin lymphoma (NHL) is one of the most prevalent tumor types in dogs and has similar pathogenesis and response to treatment as human NHL. Clinical trials in canine patients are conducted in the same way as in human patients, thus, to determine a maximum dose of HCQ that can be combined with a standard chemotherapy, a Phase I, single arm, dose escalation trial was conducted in dogs with spontaneous NHL presenting as patients to an academic, tertiary-care veterinary teaching hospital. HCQ was administered daily by mouth throughout the trial, beginning 72 h prior to doxorubicin (DOX), which was given intravenously on a 21-d cycle. Peripheral blood mononuclear cells and biopsies were collected before and 3 d after HCQ treatment and assessed for autophagy inhibition and HCQ concentration. A total of 30 patients were enrolled in the trial. HCQ alone was well tolerated with only mild lethargy and gastrointestinal-related adverse events. The overall response rate (ORR) for dogs with lymphoma was 93.3%, with median progression-free interval (PFI) of 5 mo. Pharmacokinetic analysis revealed a 100-fold increase in HCQ in tumors compared with plasma. There was a trend that supported therapy-induced increase in LC3-II (the cleaved and lipidated form of microtubule-associated protein 1 light chain 3/LC3, which serves as a maker for autophagosomes) and SQSTM1/p62 (sequestosome 1) after treatment. The superior ORR and comparable PFI to single-agent DOX provide strong support for further evaluation via randomized, placebo-controlled trials in canine and human NHL.

Keywords: autophagy; canine model; doxorubicin; hydroxychloroquine; lymphoma.

Figure 1. Progression-free interval. Progression-free interval was determined for the 27 lymphoma patients receiving the HCQ and DOX combination. Median time to progression was 4.9 mo.

Figure 2. Assessment of pharmacodyanmic response. Concentrations of hydroxychloroquine (HCQ) and the metabolite N-desethylhydroxychloroquin (DHCQ) in dogs administered 12.5 mg/kg HCQ daily were significantly higher in tumor tissues compared with plasma [HCQ (P < 0.0001) and DHCQ (P = 0.0003)] (A). There was no significant correlation between plasma and tumor HCQ or DHCQ concentrations (Pearson correlation = 0.143 and 0.238, P = 0.695 and 0.537) (B). PBMCs were isolated from whole blood before and 3 d post HCQ administration. The representative plot shows gating of cells and histograms demonstrate the increase of LC3 positive cells after HCQ administration (C). Overall there was a significant increase in the mean fluorescence intensity post HCQ. Representative EM images of PBMCs taken pre and post HCQ administration. There is an increase in autophagic vesicles after HCQ treatment, indicated by arrows (D). Tumor biopsies were taken before and 3 d post HCQ administration. Western blot analysis was performed on biopsies to determine expression of LC3-II. Though not significant, both increased overall after treatment (E).