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Understanding the Role of a Mycobacterium Phlei Immunostimulant in Veterinary Medicine

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Understanding the Role of a Mycobacterium Phlei Immunostimulant in Veterinary Medicine Understanding the Role of a Mycobacterium phlei Immunostimulant in Veterinary Medicine Maira Medellin-Peña, MD, MSc, PhD This information is for presentation within a scientific forum or in response to technical service inquiries. Contains non-label approved information. Not for mass distribution. P a g e | 1 CONTENTS About the Mycobacterium Cell Wall Fraction Technology...................................................................... 2 Immune System Response ........................................................................................................................ 3 Amplimune................................................................................................................................................ 5 K99 E. coli scours ................................................................................................................................. 5 Effect on bovine blood leukocyte populations ...................................................................................... 6 Effect on health and performance parameters ....................................................................................... 6 Mycoplasma bovis in dairy cattle .......................................................................................................... 7 Equimune .................................................................................................................................................. 8 Equine Respiratory Disease Complex (ERDC) .................................................................................... 8 Vaccine Adjunct .................................................................................................................................... 9 Immunocidin ........................................................................................................................................... 10 Mixed mammary tumor & mammary adenocarcinoma .................................................................. 10 Osteosarcoma .................................................................................................................................. 11 Transmissible Venereal Tumors ..................................................................................................... 12 Appendicular Osteosarcoma ........................................................................................................... 13 Transitional Cell Carcinoma ........................................................................................................... 13 Salivary Gland Carcinoma .............................................................................................................. 13 Lymphoma ...................................................................................................................................... 13 Immunocidin Equine ............................................................................................................................... 14 Equine Sarcoids............................................................................................................................... 14 Settle........................................................................................................................................................ 16 Future Research....................................................................................................................................... 20 Concluding Remarks ............................................................................................................................... 21 References ............................................................................................................................................... 22 Understanding the Role of a Mycobacterium phlei Immunostimulant in Veterinary Medicine P a g e | 2 NovaVive Inc. (NovaVive) acquired a mycobacterium cell wall fraction (MCWF) technology in December 2014. This technology was developed by Bioniche Life Sciences Inc. (Bioniche) for a range of veterinary products; the treatment of cancer in animals and as an immunotherapeutic to treat viral and bacterial diseases. Bioniche, now Telesta Therapeutics Inc., has further developed this technology for the treatment of bladder cancer (MCNA) in humans. NovaVive acquired the veterinary rights to the technology platform after Bioniche sold its Animal Health division to Vétoquinol S A of France in April 2014. About the Mycobacterium Cell Wall Fraction Technology Mycobacterium species have been shown to be profound stimulants of the immune system. Many researchers have shown mycobacteria to be capable of fighting infections and malignancies (Zbar, 1979; Yarkoni and Rapp, 1980; Yarkoni et al., 1982; Mallick et al., 1985; Chin et al., 1996; Cleveland et al., 1996; Filion et al., 1999; Reader et al., 2001; Young et al., 2004). In addition, studies with other microorganisms have shown an ability to stimulate the host immune system, leading to inhibition of cancer progression. Examples include: • the BCG vaccine strain Mycobacterium bovis, used in the treatment of superficial bladder cancer (Alexandroff et al., 1999), or • attenuated Salmonella Typhimurium (Pawelek et al., 1997; Tome et al., 2013) used as a targeted anti- cancer vector, and • anaerobic bacteria of the Clostridium genus (Dang et al., 2001; Nair et al., 2014) proven to enhance tumor regression in mice, and • coryneform organisms such as Rhodococcus equi (Alkemade et al., 1998), Propionebacterium acnes (Ko et al., 1981), and Corynebacterium parvum (Halpern et al., 1966; Mathé et al., 1973). One drawback is that most of the organisms demonstrating this ability are pathogenic. It has also been identified that specific microbial infections or the use of vaccines or therapies that mimic or, to a certain extent, repeat the immune response evoked by the intrusion of a pathogen, can elicit activation of macrophages and lymphocytes. This activation leads to the production of cytokines; especially interleukin 1 beta (IL-1β), IL-12, interferon gamma (IFNγ) and tumor necrosis factor alpha (TNF-α). These cytokines are cytotoxic agents that possess anticancer and antibacterial activity (Johnson, 1991; Neville & Pezzella, 1994; Gee et al., 2009; Engel & Neurath, 2010; Patyar et al., 2010). The selection and enhancement of a nonpathogenic saprophytic mycobacterium (Mycobacterium phlei), together with proprietary growth and extraction techniques used by NovaVive, have produced a line of products (Amplimune®, Equimune®, Immunocidin®, Settle®) that have successfully avoided many of the side effects associated with other mycobacterial derived products [Freund's complete adjuvant (Goubau et al., 1989), Bacillus Understanding the Role of a Mycobacterium phlei Immunostimulant in Veterinary Medicine P a g e | 3 Calmette-Guerin (BCG) vaccine (Lamm et al., 1992; Curtis and Soloway, 1998; Barza et al., 1998; Kim et al., 2000)]. One aspect of the MCWF formulations, now marketed by NovaVive, is the excellent safety profile in all species and by all routes of administration. Immune System Response Application of NovaVive's MCWF immunotherapeutic technology has been shown to trigger a rapid and broad response to infection by stimulation of the innate and adaptive immunity. MCWF contains purified fragments that consist of high concentrations of muramyl dipeptides (MDP), trehalose dimycolate (TDM); lipid mycolic acid (MA); and glycolipid lipoarabinomannan (LAM), among others. These compounds have been found to display antitumoral and immunostimulating activities (Chedid et al., 1972; Bernstein et al., 1991; Filion et al., 1999; Filion et al., 2000; Morales et al., 2001). It has been demonstrated that Mycobacterium phlei DNA (in the form of short oligonucleotides) when associated with the bacterial cell wall, inhibited the proliferation of bladder cancer cells by inducing apoptosis (Morales et al., 1995; Filion and Phillips, 1997; Filion et al., 1999). • Lipid mycolic acid (MA) has been found to stimulate transitory neutrophils, as well as provoke the production of IL-12, IFNγ, IL-6, and myeloperoxidase while suppressing IL-10 production and enhancing MCWF has been TNFα production (Korf et al., 2005). IL-12 (sometimes referred to, as the anticancer cytokine) has been related to the antiangiogenic activity demonstrated to interact associated with MCWF (Voest et al., 1995). MCWF was able to with common signalling stimulate levels of IL-12 equivalent to lipopolysaccharide (Filion pathways used by et al., 2000). different cell types of the • Trehalose dimycolate (TDM), or cord factor, is a known inducer of the immune system. pro-inflammatory cytokines IL-1β, IL-6 and TNFα (Indrigo et al., 2003). • Muramyl dipeptides (MDP), the smallest peptidoglycan component of MCWF, up-regulates the production of IL-2, IL-4, IL-10, IL-12 and IFNγ (Filion et al., 1999; Filion et al., 2000). • Glycolipid lipoarabinomannan (LAM) has been identified as a trigger for the production of TNF-α, GM- CSF, TGFβ, IL-1α, IL-β, IL-6, IL-8, IL-10, and IL-12, as well as being a chemoattractant for monocytes and neutrophils (Strohmeier and Fenton, 1999). Mycobacterium phlei DNA associated with the bacterial cell wall (cell wall complex), as well as purified M. phlei DNA, was shown to be a potent inducer of IL-6, IL-8, IL-12, IL-18 and TNF-α synthesis by monocytes and macrophages in vitro and in vivo (Filion et al., 1999; Filion et al., 2000), and was capable of inhibiting tumor cell division and inducing apoptosis in cancer cells (Filion
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