Accepted Manuscript Title: Nanomedicine in veterinary oncology Author: Tzu-yin Lin, Carlos O. Rodriguez Jr, Yuanpei Li PII: DOI: Reference:

S1090-0233(15)00076-3 http://dx.doi.org/doi:10.1016/j.tvjl.2015.02.015 YTVJL 4430

To appear in:

The Veterinary Journal

Accepted date:

11-2-2015

Please cite this article as: Tzu-yin Lin, Carlos O. Rodriguez Jr, Yuanpei Li, Nanomedicine in veterinary oncology, The Veterinary Journal (2015), http://dx.doi.org/doi:10.1016/j.tvjl.2015.02.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Review

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

Nanomedicine in veterinary oncology Tzu-yin Lin a,*, Carlos O. Rodriguez Jr b, Yuanpei Li c a

Department of Internal Medicine, School of Medicine, University of California-Davis, Sacramento, CA 95817, USA b Department of Veterinary Surgical and Radiological Sciences, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616, USA c Department of Biochemistry and Molecular Biology, School of medicine, University of California-Davis, Sacramento, CA 95817, USA

* Corresponding author. Tel.: +1 916 7035081. E-mail address: [email protected] (T.-Y. Lin). Highlights

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chemistry, biology and material sciences. 

24 25

Nanoparticle-based agents are currently under intensive investigation for cancer treatment in humans and animals.



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Nanomedicine is an interdisciplinary field that combines medicine, engineering,

Nanoparticle-based agents can overcome several limitations associated with conventional oncology protocols.



Nanoparticles can be applied for cancer diagnosis and imaging.

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Abstract

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Nanomedicine is an interdisciplinary field that combines medicine, engineering,

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chemistry, biology and material sciences to improve disease management and can be especially

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valuable in oncology. Nanoparticle-based agents that possess functions such as tumor targeting,

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imaging and therapy are currently under intensive investigation. This review introduces the basic

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concept of nanomedicine and the classification of nanoparticles. Because of their favorable

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pharmacokinetics, tumor targeting properties, and resulting superior efficacy and toxicity profiles,

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nanoparticle-based agents can overcome several limitations associated with conventional

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diagnostic and therapeutic protocols in veterinary oncology.

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The two most important tumor targeting mechanisms (passive and active tumor targeting)

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and their dominating factors (i.e. shape, charge, size and nanoparticle surface display) are

41

discussed. The review summarizes published clinical and preclinical studies that utilize different

42

nanoformulations in veterinary oncology, as well as the application of nanoparticles for cancer

43

diagnosis and imaging. The toxicology of various nanoformulations is also considered. Given the

44

benefits of nanoformulations demonstrated in human medicine, nanoformulated drugs are likely

45

to gain more traction in veterinary oncology.

46 47

Keywords: Nanomedicine; Nanoparticles; Chemotherapy; Drug delivery; Oncology; Veterinary

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Introduction Nanotechnology is an emerging field that has shown great promise in the development of

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novel diagnostic, imaging and therapeutic agents for a variety of diseases, including cancer

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(Davis et al., 2008). It exploits the improved and often novel physical, chemical and biological

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properties of materials on a nanometer scale. Nanomedicine is defined as the application of

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nanotechnology to medical diagnosis, therapy and prevention.

55 56

Nanoformulations seek to overcome several limitations of conventional drugs, including

57

toxicity, poor water solubility, instability (e.g. small interfering RNA, or siRNA) and

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pharmacokinetic (PK) properties, and may also contribute to the advancement of personalized

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medicine and to the customization of healthcare (Eifler and Thaxton, 2011). Taking advantage of

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versatile payloads, favorable PKs, unique tumor targeting properties with both passive and active

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mechanisms, and an overall superior efficacy and toxicity profile, these nanoscale ‘theranostic’

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(therapeutic-diagnostic, i.e. combining therapeutic and diagnostic purposes) formulations represent

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potential breakthroughs for cancer therapy and have created a new field known as ‘cancer

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nanomedicine’ (Chow and Ho, 2013).

65 66

Companion animals, such as cats and dogs, spontaneously develop various types of

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cancers, such as oral squamous cell carcinoma (SCC), mammary carcinoma, osteosarcoma (OSA)

68

and transitional cell carcinomas, which closely resemble cancers in humans (Rowell et al., 2011).

69

Consequently, spontaneous cancers in cats and dogs have been proposed as the best animal

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models for human cancers and have been used in preclinical studies for novel drug development,

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including nanoformulated drugs or imaging probes (De Vico et al., 2005; Withrow and Wilkins,

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2010; Rowell et al., 2011).

73 74 75

This review focuses on nanoformulations that have been reported in preclinical studies using companion animals or in early phase clinical trials in veterinary medicine.

76 77

Nanoparticle classification and tumor targeting properties

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Composition of nanoparticles

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Nanoparticles can be categorized as inorganic or ‘solid’ (gold, iron oxide, quantum dots

80

and carbon nanotubes), or as organic or ‘soft’ (liposomes, dendrimers, polymeric micelles, and

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protein aggregates (Yu et al., 2012; Bao et al., 2013; Cheng et al., 2014). Each nanoparticle

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category has distinct advantages and limitations; for instance, quantum dots and iron oxide

83

particles have well known fluorescence imaging capability and magnetic resonance imaging

84

(MRI) contrast properties, respectively, but are limited as drug delivery vehicles (Lovell et al.,

85

2011; Li et al., 2014). Conversely, liposomes and polymeric micelles are used clinically for drug

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delivery, but offer limited applications as imaging agents.

87 88

Novel nanomedicine platforms can be developed that synthesize hybrid nanoparticles.

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Specifically, these synthetic theranostic nanoparticles merge the therapeutic potential of the

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polymeric soft nanoparticle domains with the diagnostic properties of inorganic solid

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nanoparticles. Most recently, a few novel organic theranostic nanoplatforms have been touted for

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their potential applications in optical imaging, MRI, positron emission tomography (PET),

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chemotherapy, photodynamic therapy (PDT) and photothermal therapy (Lovell et al., 2011; Li et

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al., 2014).

95 96 97

Factors associated with passive tumor targeting The tumor vasculature and lymphatic vessels are known to be leaky to macromolecules.

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Thus, nanoparticles can preferentially accumulate in tumors via enhanced permeability and

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retention (EPR) effects (Matsumura and Maeda, 1986) (Fig. 1). Size, surface charge and shape

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dictate the interaction of nanoparticles in living subjects. These nanoparticle-specific properties

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will affect their PK, biodistribution and diffusivity, and consequently determine their in vivo

102

efficacy and toxicity profiles.

103 104

Nanoparticle size affects the rate of nanoparticle intratumoral deposition through the EPR

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effect and therapeutic efficacy. The optimal nanoparticle size for passive tumor targeting is ~10-

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100 nm (Davis et al., 2008). Hydrophilic components, such as polyethylene glycol (PEG), have

107

been used to coat the surface of nanoparticles to minimize their interaction with blood proteins

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and reduce their subsequent sequestration by macrophages (Gref et al., 1994; Zahr et al., 2006;

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Schipper et al., 2009). Unfortunately, the generation of anti-PEG IgM results in accelerated

110

blood clearance (ABC) and decreased liposomal drug circulation time (Suzuki et al., 2012; Abu

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Lila et al., 2013) (Table 1, liposomal topotecan in dogs) with repeated PEGylated liposome

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administration.

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The surface charge (i.e. positive, neutral or negative) and density also need to be optimized to prolong the blood circulation time, minimize non-specific clearance and prevent

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loss to undesired locations. In addition, the shape of nanoparticles affects their blood circulation,

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ability to marginate and binding affinity, and therefore the rate of tumor deposition and

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therapeutic efficacy; for example, rods and hollow cubes enter tumors more readily than discs or

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spheres (Wang et al., 2013b).

120 121

Active cancer targeting strategy

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Active targeting is more attractive than passive EPR because of improved efficiency,

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specific delivery of more therapeutic drugs/probes to target sites and the potential for

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individualized treatment. As shown in Fig. 1, taking advantage of specific cancer cell surface

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receptors (e.g. folate, transferrin, asialoglycoprotein, integrins, epidermal growth factor

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receptors, CD44) and unique tumor microenvironment signaling molecules (e.g. vascular

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endothelial growth factor, matrix metalloproteinases, αvβ3 integrin), a broad variety of ligands

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(e.g. antibodies, single-chain Fv fragments, peptides, small molecules, aptamers) can be bound to

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the surface of nanoparticles for cancer-targeting therapy (Zhang et al., 2007; Dhar et al., 2008;

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McCarron et al., 2008; Lu et al., 2009). Compared to the EPR effect alone, these ‘active’

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targeting methods enhance delivery, allow for deeper tumor penetration, and prolong drug

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retention within both the blood and the tumor, resulting in superior anti-cancer efficacy,

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specificity and biodistribution.

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Our laboratory has identified a urinary bladder cancer specific peptide, PLZ4, which

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specifically recognizes dog and human neoplastic, but not normal or inflamed urothelial cells,

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fibroblasts or white blood cells. When PLZ4 is displayed on the surface of micelles by the self-

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assembly of telodendrimers, these ‘active bladder cancer targeting micelles’ are considered to be

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theranostic because they can deliver chemotherapeutic drugs (doxorubicin or paclitaxel) and

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fluorescence dyes to bladder cancer cells (Fig. 2). These micelles enhance the anti-cancer

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efficacy in a bladder xenograft mouse model.

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Nanoparticle-based drug delivery systems for veterinary oncology Nanoparticles have been used to deliver chemotherapeutic drugs, small molecule

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inhibitors and cytokines to tumor sites. Different formulations may enhance the therapeutic index,

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increase the maximally tolerated dose (MTD), exhibit preferable PKs or improve the toxicity

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profile of existing drugs. The nanoparticle approach has also offered a second chance for drugs

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that are effective but toxic, poorly water soluble drugs, or unstable molecules (e.g. siRNA) for

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applications in veterinary clinical studies. Nanocarriers and their payloads, delivery routes, and

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neoplastic targets evaluated in companion animals are summarized in Table 1.

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Paclitaxel nanoformulations

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The first US Food and Drug Administration (FDA) approved nanoformulation in

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veterinary oncology is Paccal Vet, which consists of paclitaxel (PTX)-loaded micelles. It

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received conditional approval in 2014 for the treatment of canine mammary carcinoma and

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squamous cell carcinoma. Although paclitaxel is effective against a broad range of human

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cancers, its use in veterinary medicine has been hampered by hypersensitivity reactions to the

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PTX carrier, cremaphor. Paccal Vet also improved the overall response rate and biological

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observed response rate of canine malignant mast cell tumors (MCTs) when compared to

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lomustine (Vail et al., 2012). In a subsequent study, 59% complete or partial response rates were

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documented in 29 grade II-III canine MCTs (Rivera et al., 2013).

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Doxorubicin nanoformulations Liposomes have been used to deliver chemotherapeutic drugs (doxorubicin; DOX),

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cytokines (interleukin 2, IL-2), small molecule inhibitors and immune active reagents (muramyl

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tripeptide phosphatidyl ethanolamine, MTP) in veterinary oncology (MacEwen et al., 1989;

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Khanna et al., 1997; Poirier et al., 2002; Hauck et al., 2006). DOX-encapsulated (PEGylated or

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hybrid) liposomes have been investigated in dogs and cats for their clinical efficacy against

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sarcomas (Poirier et al., 2002; Sorenmo et al., 2007; Kleiter et al., 2010), carcinomas (Hauck et

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al., 2006) and round cell tumors (Kisseberth et al., 1995; Vail et al., 1998; Stettner et al., 2005).

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Doxil is the first FDA-approved liposomal DOX in human medicine and has also been studied in

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veterinary oncology (Barenholz, 2012). In the clinical trial of Doxil, 51 tumor-bearing dogs were

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enrolled and a 25.5% overall response rate was observed (Vail et al., 1998). Interestingly,

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intraperitoneal administration of Doxil failed to demonstrate improvement in overall survival or

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prevention of peritoneal recurrence when compared to conventionally formulated and

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administered free DOX given post-operatively to dogs with splenic hemangiosarcoma (HSA)

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(Sorenmo et al., 2007).

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Doxil (1 mg/kg) has been used in cats with vaccine-associated sarcoma (VAS), with a

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39% overall response rate and a median progression time of 84 days. This was similar to cats

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receiving free DOX (Poirier et al., 2002). Overall, Doxil was well-tolerated and had less

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cardiotoxicity, despite similar efficacy. However, despite this favorable toxicity profile, cost has

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prevented its widespread use in veterinary medicine.

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Immunomodulator nanoformulations Liposomes have been used to deliver the immunomodulator MTP for various cancers in

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dogs (OSA, HSA, malignant melanoma and mammary carcinoma) and cats (mammary

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carcinoma) (MacEwen et al., 1989, 1999; Fox et al., 1995; Kurzman et al., 1995; Vail et al.,

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1995). Dogs with OSA and splenic HSA receiving liposomal MTP had significantly prolonged

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disease-free survival, overall survival and/or prolonged time to relapse (Kleinerman et al., 1995;

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Kurzman et al., 1995; Vail et al., 1995). However, liposomal MTP failed to prolong post-surgical

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survival rates in dogs with oral malignant melanoma or mammary carcinoma (MacEwen et al.,

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1999; Teske et al., 1998). There is still a strong interest in the use of liposomal MTP in the

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treatment of human OSA and the formulation is currently in early clinical trials in humans in

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Europe. The drug has not received FDA approval in the United States and hence its availability

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is restricted.

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Nanoparticle-mediated photodynamic and photothermal therapy Nanoparticle-mediated photodynamic therapy (PDT) and photothermal therapy (PTT) are

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interesting alternative approaches for cancer treatment. First and second generation

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photosensitizers have poor water solubility, equivocal cancer selectivity and often accumulate in

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the skin for prolonged periods of time after therapy, which leads to photosensitivity and sunburns.

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Nanoformulated photosensitizers are currently considered as third generation photosensitizers

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and presumably have better tumor targeting property.

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A preliminary trial in feline cutaneous SCC using a liposomal formulation of the

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lipophilic photosensitizer meta-(tetrahydroxyphenyl) chlorin (m-THPC)-mediated PDT resulted

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in a 100% complete response rate, a 75% overall 1-year control rate and a 20% recurrence rate.

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No obvious systemic toxicity was noted but, importantly, untoward local skin reactions occurred

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in only 15% of the patients (Buchholz et al., 2007).

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An orthotopic canine transmissible venereal tumor (cTVT) brain tumor model has been

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tested for gold nanoshell-assisted PTT. After infusion, the nanoshells specifically accumulated in

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the intracranial cTVT through the disrupted blood-brain barrier. Intratumoral laser illumination

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thermally ablated the cTVTs (average temperature 65.8 ± 4.1 °C). Importantly, the temperature

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achieved in the vehicle-treated control dog (53.1 °C) resulted in minimal normal tissue damage

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due the lack of nanoshell accumulation (Schwartz et al., 2009). Currently, The Ohio State

218

University has an open enrolment for dogs with solid tumors for a phase I study on gold

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nanorods-mediated PTT (Table 2).

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Alternative administrating route for nanoparticles As well as intravenous (IV) administration, nanoparticles have also been given via

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intrathecal (Kitamura et al., 1996), intratumoral (Kitamura et al., 1996), inhalational (Khanna et

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al., 1997) and intravesicular (Lu et al., 2011) routes. Canine transitional cell carcinoma of the

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urinary bladder has been used to evaluate intravesicular administration of PTX-loaded gelatin-

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based nanoparticles with minimal systemic absorption and toxicity (Lu et al., 2011). Comparing

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neoplastic to normal urothelial cells using PTX-gelatin nanoparticle, a differential uptake as high

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as 360-fold at the same tissue depth was observed (Lu et al., 2011).

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A carrier that can penetrate the blood-brain barrier is usually required for targeting brain

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tumor cells via systemic administration; however, Dickinson et al. (2010) bypassed this

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requirement by directly injecting liposomal CP-11 (a topoisomerase inhibitor)/gadolinium (Gd)

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into canine spontaneous gliomas. Drug distribution and tumor response was monitored by MRI;

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5/9 dogs exhibited decreased tumor volume (40-88%) (Dickinson et al., 2010). To date, no

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liposomal formulations administered IV to dogs with brain tumors have been studied in a similar

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manner. It is tempting to speculate that nanoparticles could be created to penetrate the blood-

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brain barrier and deliver their theranostic payload to brain tumors, thus leading to similar or

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better results without the necessity for technically demanding intrathecal injections. However,

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further experiments are necessary to demonstrate feasibility.

240 241 242

Nanoparticles for cancer diagnosis and imaging applications Rapid advances in the nanoprobe field have created a novel targeted non-invasive method

243

to evaluate both the tumor and its microenvironment. These advancements further enable

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scientists to detect, control biodistribution and monitor treatment in real time (Shin et al., 2013).

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However, there are very few limited studies on the application of nanoparticles for imaging of

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cancers in companion animals.

247 248

Several inorganic nanoparticles have been tested in veterinary medicine. In client-owned

249

dogs with spontaneous thyroid carcinoma and OSA, gum arabic-stabilized gold nanocrystals

250

have been injected intratumorally as contrast agents. Computed tomography (CT) images

251

showed effective accumulation at the tumor sites with a contrast enhancement of 12 δ-HU

252

(Chanda et al., 2014). Similarly, Technetium-99m-labelled liposomes provided scintigraphic

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imaging with a four-fold image enhancement of feline sarcomas undergoing tumor hyperthermia

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(Matteucci et al., 2000). These studies support the potential use of nanoformulations to detect

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tumors, monitor drug delivery, control drug release and assess treatment outcomes.

256 257

Canine spontaneous glioma has been used as a model for drug delivery, imaging, and

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therapy due to its striking similarities to human glioma. It is a challenge to treat glioma

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systemically due to the blood-brain barrier. Convection-enhanced delivery (CED) of therapeutic

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agents has circumvented the blood-brain barrier by allowing direct infusion of test articles

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directly into tumors via one to multiple catheters. Nanotechnology offers a multifunctional

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platform allowing real-time imaging and monitoring of drug delivery, while sequentially

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accurately evaluating therapeutic efficacy. As mentioned above, through CED, Dickinson et al.

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(2010) co-infused CPT-11/Gd-encapsulated liposomes into dogs with spontaneous grade III

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astrocytoma and used concurrent real-time MRI visualization. The distribution, location and

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leakage of the infusate could be observed on T1-weighted images; coupling treatment with

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imaging (theranostic) helped to optimize infusion parameters and the interpretation of outcomes

268

(Dickinson et al., 2010).

269 270

Our group has studied the biodistribution of indodicarbocyanines (DiD, a near infrared,

271

NIR, fluorescent dye)/PTX co-loaded PLZ4-micelles (PNMs) via optical imaging in a canine

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urinary bladder cancer orthotopic mouse model. PNMs exhibited significantly higher

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accumulation in tumors than non-targeted micelles as assessed by NIR fluorescent signals (Lin et

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al., 2012). This selectivity toward cancer suggested that our PNMs could carry and deliver

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chemotherapeutic drugs to tumor sites and could be used for intravesicular tumor detection in

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dogs. This possibility is currently being assessed in a clinical trial in dogs with urinary bladders.

277 278

Several other nanomaterials have been injected into dogs to test their potential for

279

diagnosing tumors or for mapping tumor draining lymph nodes. For example, cetuximab-

280

conjugated magnetic iron-oxide nanoparticles and liposome-encapsulated Gd were infused in

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dog brains to evaluate toxicity and their potential application in glioma diagnosis and treatment

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(Dickinson et al., 2008; Platt et al., 2012). Lymph node metastases are the hallmark for various

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malignant cancers. Lymphadenectomy, along with primary tumor resection, may achieve better

284

long-term cancer control and prevent local recurrences. Iodized oil emulsion has been injected

285

into the gastric submucosa so that draining lymph nodes could be identified by CT and

286

lymphangiography (Lim et al., 2012). Furthermore, lymphotropic iodinated nanoparticles have

287

been injected subcutaneously near oral malignant melanomas to detect cancerous lymph nodes

288

(Wisner et al., 1996).

289 290 291

Toxicology Nanoparticle formulation not only improves drug delivery but also largely changes the

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PKs of chemotherapeutic drugs, resulting in reduced toxicity and side effects (De Jong and Borm,

293

2008). Some effective but poor water soluble chemotherapeutic drugs, such as PTX, can now be

294

used in veterinary oncology. Because of the emerging recognition of companion animals with

295

spontaneous cancers as animal models with great potential for human oncology research, many

296

anti-cancer nanoformulations have been tested in dogs, rabbits and cats to predict safety issues

297

and to determine the recommended starting dose in human clinical trials. Although most

Page 13 of 32

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molecules are not aimed at veterinary species, those preclinical studies offer a wealth of

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information regarding potential toxicity in animals.

300 301

Toxicity associated with doxorubicin nanoformulations

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DOX-containing liposomes were studied for their toxicity in dogs and rabbits in the late

303

1990s. DOX is an effective chemotherapeutic drug, but cardiotoxicity limits its use in dogs and

304

humans. Dogs treated with Doxil showed no evidence of cardiotoxicity clinically or

305

pathologically, while dogs treated with free DOX exhibited marked cardiotoxicity. Similarly,

306

rabbits receiving Doxil had lower evidence of cardiotoxicity (16%) compared with free DOX

307

treated rabbits (67%) (Working et al., 1999). Findings in humans reflect these preclinical

308

findings; Doxil effectively minimizes cardiomyopathy even when patients receive high

309

cumulative doses (Gabizon et al., 2004; Uyar et al., 2004). Although the Doxil formulation

310

prolonged DOX circulating time in dogs and changed its biodistribution, resulting in decreased

311

cardiotoxicity, there was a higher incidence of skin toxicity, such as palmar-plantar

312

erythrodysesthesias (PPE), the dose-limiting event in dogs and humans. Pyridoxine (vitamin B6)

313

delays and decreases the development and severity of PPE in lymphoma-bearing dogs receiving

314

Doxil (Vail et al., 1998). In comparison, myelosuppresion with free DOX treatment is usually the

315

dose-limiting toxicity (Vail et al., 1997).

316 317

Doxil has also been evaluated in cats with vaccine-associated sarcoma. Delayed

318

nephrotoxicity and cutaneous toxicity occurred in 23% and 22% of treated cats, respectively

319

(Poirier et al., 2002). Overall, Doxil has been considered well-tolerated in cancer veterinary

320

patients and has demonstrated decreased cardiotoxicity with manageable cutaneous toxicity.

Page 14 of 32

321 322

Another albumin-binding DOX prodrug, the 6-maleimidocaproyl hydrazone derivative of

323

doxorubicin (DOXO-EMCH), was tested in dogs to determine the recommended starting dose in

324

humans and to evaluate potential toxicities. From the time of injection up to 3 h, dogs showed

325

dose-dependent allergic reactions, along with clinical signs of extensive skin redness, swelling,

326

and salivation. At the high dose, some animals also developed post-injection motor hypoactivity,

327

hair loss and dermatitis (Kratz et al., 2007). Of note, the free DOX MTD is approximately 2.25-

328

2.5 mg/kg in dogs, while DOXO-EMCH can double the dose of the free DOX MTD (Kratz et al.,

329

2007).

330 331

Toxicity associated with plaxitel nanoformulations

332

PTX, a mitotic inhibitor, is a potent anti-cancer drug in humans; however, despite

333

aggressive premedication in dogs and cats, cremophor EL-induced side effects largely limits its

334

veterinary use (Poirier et al., 2004; Kim et al., 2014). Nanotechnology has enabled the

335

production of a canine-tolerated PTX (Paccal Vet; PTX-loaded micelles; MTD 150 mg/m2).

336 337

In the first randomized trial of Paccal Vet in dogs with high grade MCTs, the most

338

common adverse event was grade 3-4 neutropenia, followed by the gastrointestinal toxicity

339

(emesis, anorexia and diarrhea) (Vail et al., 2012). Increased liver enzymes were noted, but to a

340

less extent than in the control dogs that received lomustine, a standard chemotherapeutic drug.

341

While 33% of dogs receiving lomustine were withdrawn due to clinically relevant hepatotoxicity,

342

only 2% of dogs receiving Paccal Vet did so (Vail et al., 2012). These findings were confirmed

Page 15 of 32

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in later trials using dogs with high grade solid tumors and MCTs (Rivera et al., 2013; von Euler

344

et al., 2013).

345 346

Nanoparticulate PTX in saline (Crititax, CTI52010) has an MTD of 120 mg/m2 in dogs

347

and also causes grade 4 neutropenia and grade 1-2 gastrointestinal toxicity at higher doses

348

(Axiak et al., 2011). Intraparenchymal microsphere-PTX (Paclimer) delivery to the brain is not

349

associated with any systemic toxicity or myelosuppression. Although no neurotoxicity was found,

350

local wound infection and brain abscess were present on occasions (Pradilla et al., 2006). These

351

results were as expected; only local exposure occurred without systemic absorption.

352 353

Since dogs are more sensitive to the development of anaphylaxis in response to treatment

354

with various PTX formulations, they have also been used to evaluate allergic reactions after

355

systemic administration of PTX microemulsions (Wang et al., 2011). Compared to dogs

356

receiving PTX (in cremophor EL/ethanol), dogs receiving PTX microemulsion exhibited

357

significantly less severe allergic reactions. Premedication with prednisone, diphenhydramine,

358

cimetidine and dexamethasone significantly inhibited allergic reactions induced by PTX and

359

PTX microemulsion in dogs. The group of dogs receiving PTX microemulsion with

360

premedication exhibited the least toxicity (Wang et al., 2011).

361 362

Shi et al. (2013) studied the toxicity and PKs of docetaxel-loaded arginine-stabilized

363

mPEG-PDLLA polymeric micelles in dogs and showed that this micelle formulation also

364

improved the acute toxicity that occurred with free docetaxel injection. Taken together, PTX

365

nanoformulations have superior toxicity profiles when to conventional, poorly water soluble

Page 16 of 32

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PTX. Most adverse effects to the nanoformulations were transient, clinically silent, and

367

manageable.

368 369 370

Toxicity of other chemotherapeutic drug nanoformulation Liposomal curcumin causes reversible hematuria, while higher doses (40 mg/kg) caused

371

irreversible acute hemolysis accompanied by hematuria. Hemolysis was more likely related to

372

oxidative damage from the drug (Helson et al., 2012). CED CPT-11 liposome induces mild

373

transient proprioceptive deficits, local hemorrhage and perivascular inflammation following

374

direct intraparenchymal delivery (Dickinson et al., 2008).

375 376

Interestingly, the toxicity profiles of the same nanoformulation may vary across species.

377

For instance, in mice, liposome encapsulated vincristine (an organic nanoparticle) caused

378

significant less toxicity when compared to free vincristine following intravascular injection,

379

while dogs exhibited comparable toxicity between those two molecules (gastrointestinal toxicity

380

was the dose-limiting factor when given as a single high-dose) (Kanter et al., 1994). Toxicology

381

studies in veterinary medicine are lacking, especially for those inorganic nanomaterials.

382 383

Conclusions

384

The field of nanotechnology is growing rapidly. The technology has enabled the clinical

385

use of several chemotherapeutic drugs that were originally too toxic, too unstable, or difficult to

386

formulate. Veterinary medicine is poised to take advantage of these advances. In humans,

387

nanoparticle drug formulation has shown clinically superior tumor targeting ability, favorable

388

patient PK/biodistribution, and an overall improved toxicity profile. There are an increasing

Page 17 of 32

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number of nanoformulations receiving approval from regulatory agencies in human medicine,

390

while the first paclitaxel nanoformulation was approved by the FDA for companion animal use

391

in 2014. Despite these advances, most formulations are still in their very early development.

392

More and different formulations are expected to be tested and approved for clinical use and for

393

development as theranostic nanoformulations, which will allow for convenient detection,

394

treatment and imaging follow-up of tumors in a single platform. Most importantly, advances in

395

targeting of tumors or tumor environments will further contribute to the development of

396

individualized medicine. All of these steps will eventually influence cancer treatment in human

397

and veterinary medicine.

398 399 400 401

Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.

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786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804

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805 806

Fig. 1. Passive and active tumor targeting mechanisms of nanoparticles. Blood vessels in tumors

807

are relatively ‘leaky’ and thus allow nanoparticles to escape the blood circulation and enter

808

tumor tissues. Through surface modification with tumor targeting molecules, nanoparticles are

809

able to further specifically recognize tumor cells after extravasation. Then, through recognition

810

of the unique cancer marker, receptor-mediated endocytosis occurs. In general, active cancer

811

targeting strategy delivers greater amounts of drug and has deeper tumor penetration than

812

conventionally administered chemotherapy or uncoated nanoparticles.

813 814

Fig. 2. Active targeting of nanomicelles of canine urinary bladder cancers. The canine urinary

815

bladder cancer cell line, K9TCC-PU-Axc (kindly provided by Dr. D. Knapp from Purdue

816

University) was cultured on an eight-well chamber slide. Cells were then treated with 0.1 mg/mL

Page 27 of 32

817

PLZ4 (bladder cancer targeting peptide) coated nanomicelles (NM) loaded with DID (red

818

fluorescence dye) (PLZ4-NM-DID) or with NM-DID (non-targeting) for 20 min. Hoechst 33342

819

was used for nuclear staining (blue). Imaging was acquired using DeltaVision (GE healthcare

820

Life Science). Bar = 15m.

821 822

Page 28 of 32

823 824 825

Table 1 Nanoformulation for drug delivery, imaging, and toxicity in veterinary oncology.

Animal

Formulation/drugs

Route

Dog

Peg liposome/Doxorubicin (Doxil)

Liposome/Doxorubicin Liposome/Doxorubicin Liposome/Doxorubicin

Liposome/Muramyl tripeptide

Toxicity

References

IP

Tumor type (special application) Splenic HSA

PPES and anaphylactic reaction

IV

Non-Hodgkin’s lymphoma

Less cardiotoxicity than free doxorubicin

IV IV IV Urinary bladder submucosal injection IV

cTVT Sarcomas and carcinomas Myeloma

Grade 4 neutropenia, liver failure and renal damage

Teske et al., 2011 Sorenmo et al., 2007 Amantea et al., 1999 Working et al., 1999 Vail et al., 1998 Stettner et al., 2005 Hauck et al., 2006 Kisseberth et al., 1995 Kiyokawa et al., 1999

OSA with pulmonary metastasis Malignant melanoma Splenic HSA Appendicular OSA Mammary carcinoma Pulmonary carcinoma with metastases

Liposome/Interleukin-2 Liposome/SN-38 Liposome/Cisplatin

Inhalation IV IV

Liposome/Paclitaxel (PTX)

IV

Liposome/Vinblastine

IV

Hybrid liposome/BCNU Liposome/CPT-11 and gadoteridol

Intra-thecal Intra-tumoral

Meningeal gliomatosis Glioma (MRI)

Liposome/Cisplatin (CPI77) Liposome/Clodronate Liposome/Curcumin

IV

OSA

IV IV

Malignant histiocytosis

Peg liposome/Topotecan

IV

Liposome/SL052 Liposome-DNA complex (e.g. endostatin DNA)

IV and IA IV

Prostate cancer (PDT) HSA

Liposome/Interleukin-2 plasmid Lipid nanoemulsion/Vincristine/Pr ednisone Iodized oil emulsion

IV

Soft tissue sarcoma (Vaccine) OSA with lung metastases

IV

lymphoma

Cancerous lymph node (LN) mapping (CT lymphography)

Micelle/PTX (Paccal Vet)

Gastric submucosal injection IV

PLZ4-Micelle/PTX

IV

Nanoparticle/PTX (Crititax or CTI52010) Microsphere/PTX (Paclimer) Arginine-stabilized mPEGPDLLA polymeric micelles/Docetaxel Polymer/Docetaxel (Nanoxel-OM) Microemulsion/PTX Gelatin nanoparticles/PTX Hyaluronan conjugated cisplatin DOX-albumin

IV

Gold nanoshell

IV Intra-prostate

Mild fever post injection, and otherwise no significant toxic effects

Minimal toxicity; increased cell counts Injection site swelling and hematology changes Acute anaphylaxis-like reactions, nephrotoxicosis and substantial myelosuppression Lung nanoparticle accumulation, but no hemolysis Anorexia, weight loss, pyrexia, myelosuppression and gastrointestinal toxicity Mild lymphocytic pleocytosis in CSF, mild transient proprioceptive deficits, focal hemorrhage and focal mild perivascular non-suppurative encephalitis Acute anaphylaxis-like signs after infusion

Brief single episode of reversible hematuria; 40 mg/kg caused acute hemolysis with hematuria (oxidant effect) Accelerated blood clearance phenomenon

Grade 2/3 mast cell tumors(MCTs)

Acute urinary retention

MacEwen et al., 1989 Kleinerman et al., 1995 MacEwen et al., 1999 Vail et al., 1995 Kurzman et al., 1995 Teske et al., 1998 Khanna et al., 1997 Pal et al., 2005 Marr et al., 2004 Zhao et al., 2011 Wang et al., 2013a Kanter et al., 1994 Zhong et al., 2014 Kitamura et al., 1996 Dickinson et al., 2008, 2010 Vail et al., 2002 Hafeman et al., 2010 Helson et al., 2012 Li et al., 2012 Zhao et al., 2012 Xiao et al., 2010 U’Ren et al., 2007 Kamstock et al., 2006 Dow et al., 2005

Leucopenia

Lucas et al., 2013

Lim et al., 2012

Transient grade 3/4 neutropenia and grade 1/2 leukopenia; transient myelosuppression

High grade solid tumors Bladder cancer (mouse model)

Vail et al., 2012 Rivera et al., 2013 von Euler et al., 2013 Lin et al., 2012 Axiak et al., 2011

Intraparenchymal IV

Grade 4 neutropenia; grades 1 and 2 gastrointestinal toxicity (at high dose) Wound infection; no systemic toxicity, myelosuppression or neurotoxicity Less acute toxicity than free docetaxel

IV

Neutropenia; mild weight loss; no hypersensitivity reaction

Lee et al., 2011

Improved hypersensitivity reaction compared to free PTX

Wang et al., 2011 Lu et al., 2011 Venable et al., 2012

Systemic histamine-like reaction after injection at high dose; no toxicity at low dose

Kratz et al., 2007

IV Intra-vesical Intra-tumoral

Bladder cancer Soft tissue sarcomas

IV Orthotopic cTVT in brain Normal prostate (NIR imaging and PTT)

Pradilla et al., 2006 Shi et al., 2013

Schwartz et al., 2009 Schwartz et al., 2011

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Cat

Rabbit Pig

826 827 828 829 830

Gold nanoparticle

Intra-tumoral

Cetuximab conjugated iron oxide nanoparticles Doxil

Intra-cranial

Thyroid carcinoma and OSA (CT scan) MRI

Chanda et al., 2014

IV

Vaccine-associated sarcoma

Liposome/Doxorubicin + radiotherapy Liposome/MTP

IV

Soft tissue sarcomas

Kleiter et al., 2010

IV

Mammary adenocarcinoma

Fox et al., 1995

Liposome/Cisplatin Liposome/Cis-bisneodecanoato-trans-R,R-1,2diaminocyclohexane platinum (II) Liposome/Meta(tetrahydroxyphenyl)chlorin e Magnetic nanoparticles/feline granulocyte macrophagecolony stimulating factor plasmid or interleukin-2 or interferon-γ Liposome/Technetium-99m Doxil Lymphotropic iodinated nanoparticle

IV IV

None

Platt et al., 2012

Delayed nephrotoxicosis; localized hyperpigmentation and alopecia (chin); mild transient gastrointestinal signs; sudden explained death

Vail et al., 1997 Poirier et al., 2002

Increased cholesterol levels 2 days after injection; pyrexia Transient pyrexia; lethargy; vomiting; inappetence; acute infusion reaction; thrombocytopenia; cumulative myelosuppression; liver/kidney toxicity

Thamm and Vail, 1998 Fox et al., 1999

IV

Cutaneous squamous cell carcinoma (PDT)

Mild local toxicity, such as erythema and edema

Buchholz et al., 2007

Intra-tumoral

Fibrosarcoma

None

Jahnke et al., 2007 Huttinger et al., 2008

IV IV Peri-tumoral

Sarcoma (planar scintigraphy/PET) Progressive cardiomyopathy Cutaneous malignant melanoma (cancerous lymph node mapping)

Matteucci et al., 2000 Working et al., 1999 Wisner et al., 1996

IV, intravenous injection; IP, intraperitoneal injection; IA, intra-arterial injection; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; HSA, hemangiosarcoma; cTVT, canine transmissible venereal tumor; OSA, osteosarcoma; BAL, bronchoalveolar lavage; CSF, cerebrospinal fluid; PPES, palmar-plantar erythrodysesthesia; PDT, Photodynamic therapy; PTT, Photothermal therapy; MRI, Magnetic resonance imaging; CT, computed tomography; NIR, Near infrared red.

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831 832 833

Table 2 Selective clinical trials on nanoformulations in veterinary medicine.

Animal Dog/cat

Nanoformulation Cisplatin hyaluronan nanoparticles

Dog

Gold nanorod mediated photothermal therapy Radioactive gold nanoparticles Temozolomide loaded polycaprolactone magnetite nanoparticles Doxorubicin loaded nanoparticle Liposomal curcumin Liposomal clodronate Paccal Vet (micellar paclitaxel)

Route Intra-tumoral and peri-tumoral IV

Tumor type Oral squamous cell carcinoma and malignant melanoma Solid tumor

Organization University of Missouri

IV IV

Primary prostate tumor Glioma

IV IV IV IV

Appendicular osteosarcoma Lung cancer Soft-tissue sarcoma Histiocytic sarcoma (phase II)

University of Missouri University of Illinois and University of Chicago University of Illinois University of California-Davis Colorado State University University of Tennessee at Knoxville

Ohio State University

834 835

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836

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