**4. Antitumor effects of ivermectin-animal data**

In a wide-range of pre-clinical studies, rodent models of human xenografts of glioblastoma, leukemia, breast and colon carcinomas, as well as a variety of murine cell lines in syngeneic models have consistently shown ivermectin to possess robust antitumor effect at a median dose of 5 mg/Kg [12, 13, 15, 17, 18]. We present a review of some results of anticancer studies of ivermectin in animal below.

#### **4.1 Glioblastoma**

Two independent glioblastoma xenograft SCID mice models were established by subcutaneous injection of U87 or T98G cells, and the rodents were subsequently treated with intraperitoneal ivermectin at 40 mg/Kg. The treated mice had demonstrated significant tumor growth inhibition but maintained normal behavior and retained their weight [12]. A separate study using 3 mg/Kg of ivermectin showed a 50% decrease in tumor size and there was near complete regression of tumors at 10 mg/Kg. Ki67 staining also confirmed that glioma cell proliferation was decreased in ivermectin-treated animals compared to controls [17].

### **4.2 Colon and lung cancers**

Melotti et al. used H358 human metastatic lung bronchioalveolar carcinoma cells and DLD1 colorectal adenocarcinoma cells to test the antitumor effects of ivermectin. The animals received intraperitoneal injections of cyclodextrin-conjugated ivermectin daily at 10 mg/kg after tumor establishment. Subsequently, it was found that tumors responded to ivermectin with a near 50% reduction of growth and a repressed lung cancer WNT-TCF signature and enhanced p21 levels [13].

#### **4.3 Breast cancer**

Ivermectin was evaluated in an orthotopic breast cancer model with human MDA-MB-231 cells subcutaneously injected in the mammary fat pad of NOD-SCID mice. Xenografts treated with ivermectin grew at a slower rate than those of the control group, and the size and weight of control tumors were macroscopically larger than that of ivermectin-treated tumors [15]. Another study tested JC murine breast cancer cells in Balb/c mice treated with a dose of 3 mg/Kg of ivermectin. Treatment reduced tumor size more than 60% with no changes in weight or behavior of the study animals when compared with controls [22]. Recently it was demonstrated the ivermectin at a dose of 5 mg/Kg induces immunogenic cell death hallmarks with large numbers of intratumoral CDA4+ and CD8+ T cells in a 4 T1 murine tumor model. Thus, ivermectin turns cold tumors into hot ones which allows for marked synergy with check point inhibitor nivolumab, leading to major antitumor effects and most importantly, protective immunity [26].

### **4.4 Leukemia**

Human leukemia (OCI-AML2 and K562) and murine leukemia (MDAY-D2) cells were injected subcutaneously into NOD/SCID mice which were subsequently treated with 3 mg/Kg (human equivalent dose of 0.240 mg/Kg) of ivermectin or control in water via oral gavage. Upon follow-up, the treated mice had up to 70% decrease in their tumor burden without any gross sign of organ toxicity, and treatment led to increased apoptosis in OCI-AML2 xenografts [18]. It must be remarked that most of the in vivo studies to evaluate the antitumor effects of ivermectin dose ranging from 3 to 10 mg/Kg. These mice doses translate into human to 0.240 to 0.810 mg/Kg which are clinically attainable [27].
