Introduction

The endocannabinoid system (ECS) is a sophisticated and intricate signalling network involved in different biological processes, within both neural and non-neural tissues. It has been described as maintaining homeostatic equilibrium in response to environmental factors and metabolic stress conditions (Lutz 2020).

The ECS can be conceptually defined as a network comprising cannabinoid receptors such as cannabinoid receptor type 1 (CB1), cannabinoid receptor type 2 (CB2), G protein-coupled receptor 55 and 119 (GPR55, GPR119), and related receptors such as transient receptor potential vanilloid (TRPV) and peroxisome proliferator-activated receptor (PPAR). It also includes endocannabinoids (eCBs) such as anandamide (AEA) and 2-Arachidonoylglycerol (2-AG), endocannabinoid-like signaling molecules, proteins involved in their transport, enzymes responsible for endocannabinoid synthesis and catabolism, and the genes encoding these proteins (Kibret et al. 2023; Migliaro et al. 2023). Nevertheless, offering a detailed explanation of the ECS remains difficult due to the ongoing discovery and development of its potential components, receptors, and pathways.

Coined as the “endocannabinoidome” (Di Marzo and Wang 2015; Fraguas-Sánchez et al. 2018), this system plays a vital role in maintaining homeostatic functions, exhibiting antioxidant, hypotensive, immunosuppressive, anti-inflammatory, and pain-relieving effects. The distribution of cannabinoid receptors in the brain hints at physiological involvement in movement control, perception, sleep, appetite regulation, inhibition of learning and memory, emotional state regulation, and neuroprotection. The ECS affects vasomotor function, fertility, and even tumor cell proliferation (Fraguas‐Sánchez et al. 2018).

Knowledge of this intrinsic system prompted exploration into how active ingredients, particularly phytocannabinoids (pCBs) contained in Cannabis sativa, interact with it, leading to both therapeutic and psychotropic effects. In veterinary medicine, consideration of Cannabis derivatives for therapeutic purposes emerged a few years ago. Potential applications include pain management, neurological conditions, well-being, gastrointestinal health, dermatologic diseases, and oncology (Di Salvo et al. 2023). With growing awareness of the therapeutic potential of Cannabis derivatives in veterinary medicine and the legalization of cannabinoids in some states, veterinarians and pet owners are increasingly exploring cannabinoid products for their companion animals (Kogan et al. 2019a, b). Indeed, according to the analysis carried out by Zion Market Research, the CBD pet market was estimated to be about 257.6 Million US dollars in 2023, and it is expected to increase up to 2,967.40 Million US dollars at the end of 2032 (https://www.zionmarketresearch.com/report/cbd-pet-market; accessed 15 July 2024).

This paper aims to review the molecular mechanisms by which pCBs carry out their physiological and pharmacological actions and to summarize data published so far about the expression of the ECS and the pharmacokinetics, efficacy, and tolerability of Cannabis derivatives in the main species of veterinary interest.

Mode of action of phytocannabinoids

Based on their sources, cannabinoids can be classified as eCBs that are specifically synthesised in animal cells, pCBs mainly found in cannabis plant, and synthetic cannabinoids (sCBs), designed to mimic the effects of natural cannabinoids. Endocannabinoids and pCBs have both hydrophobic proprieties but different chemical structures. The eCBs are endogenous lipid compounds composed of a long-chain polyunsaturated fatty acid tail and a polar head containing functional groups like amide, ester, ether, or hydroxy groups, while pCBs are terpenophenolic substances containing tricyclic, bicyclic, and monocyclic structures that can exists in different isomers (Maccarrone et al. 2023). Although eCBs and pCBs differ in their chemical structures, they exhibit overlapping effects, although with some variability, through target receptors, signaling pathways, and enzymes involved in their metabolism. Indeed, the ECS was originally identified by studying the mechanism of action of the psychotropic cannabis substance, Δ9 -tetrahydrocannabinol (THC) (Silver 2019) which is the major components of the Cannabis plant extract. Meanwhile, cannabidiol (CBD) represent the most abundant non-psychoactive compound of Cannabis plants. Further, other active compounds including cannabigerol (CBG), Cannabigerovarin (CBGV), cannabichromene (CBC), cannabielsoin (CBE), cannabicyclol (CBL), cannabinol (CBN), (CBC), cannabinodiol (CBND), cannabitriol (CBT), cannabidivarin (CBDV), tetrahydrocannabivarin (THCV), as well as their analogues (such as Δ8-THC and Δ10-THC, among others), their acid derivatives (THCA, CBDA, CBGA, CBGVA, CBCA, CBDVA, etc.) and other minor cannabinoids, terpenes, and flavonoids have been extracted from different Cannabis species (Maccarrone et al. 2023; Mechoulam 2023). This plethora of bioactive molecules makes it difficult to define the specific effects of Cannabis extracts but provides “lead” molecules for therapeutic and medical applications. Thus, the understanding of pharmacological properties and potential effects of pCBs is still superficial and often inferred from findings obtained in studies primarily focused on the pathways activated by the interaction between eCBs and CB receptors (Maccarrone et al. 2023; Mechoulam 2023). Nevertheless, it is accepted that pCBs act by binding specific receptors of eCBs in animal cells and by imitating their effects. Most of the findings reviewed in this section have been extrapolated from mouse and human studies.

Cannabinoids may engage with various receptors including: G protein-coupled receptors (GPCRs) such as CB1, CB2, GPR55, GPR119, and GPR18, that are transmembrane proteins with an extracellular binding; Transient Receptor Potential Vanilloid (TRPV) 1, 2, 3, 4 also located on the plasma membrane but having intracellular binding sites; nuclear PPARs receptors by which cannabinoids may regulate gene expression (Gomez-Canas et al. 2023) (Fig. 1). Further, CB1 has been described in the mitochondrial membrane. The ability to interact with various receptors with different downstream pathways is considered the basis for the different, sometimes opposite effects of various pCBs (Maccarrone et al. 2023).

Fig. 1
figure 1

Schematic representation of the downstream pathways of cannabinoid receptors described in the text. (Created with BioRender.com)

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G protein-coupled receptors (GPCRs)

Cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2) (numbered in the order of their discovery) represent the most extensively investigated cannabinoid receptors. CB1 and CB2 differ in tissue distribution, affinity and intrinsic activity to the various pCBs. In particular, THC has a high affinity for CB1 compared to CBD, which interacts mainly with CB2; CBD may also act as a negative allosteric modulator of CB1. CBG and CBC have a relatively low affinity for CB1 and CB2 receptors, while CBN has been considered a partial agonist at CB1 receptors. Surprisingly, even if THCV has a structure like THC, it everts a higher affinity for CB2 than CB1 (McPartland et al. 2015), The ability to recognize different receptors with different affinities results into different effects of pCBs, that also reflect the specific cellular and tissue localization of these receptors (Blebea et al. 2024). Overall, the CB1 receptor is widely expressed in the brain, primarily localized at the presynaptic axon terminals (both excitatory glutamatergic and inhibitory γ-amino-butyric acid (GABA)-ergic) of neurons, as well as in astrocytes and microglia; CB2 receptors are predominantly found in immune system cell components (as monocyte-derived cells and lymphocytes) and in peripheral tissues including endothelial cells, lungs, tonsils, enteric neurons, synovial membrane, and skin keratinocyte (Bie et al. 2018; Zou 2018). Moreover, CB2 receptors have been observed within the central nervous system (CNS) such as the amygdala, hippocampus, cerebellum, cerebral cortex, striatum, etc. Similarly, CB1 has been detected in peripheral tissues including the liver, pancreas, small intestine, and skeletal muscle (Lim et al. 2023; Miranda-Cortés et al. 2023). Species specific localization of receptors is described in the next paragraph. The activation of both CB1 and CB2, coupled with G proteins (mainly Gi/o-types), leads to inhibition of the adenylyl cyclase activity with reduction of cellular cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) activity (Ye et al. 2019; Maccarrone et al. 2023). Moreover, CB1 receptors are also coupled, via G proteins, to potassium channels, and their activation is followed by the hyperpolarization of postsynaptic neurons (Irving et al. 2017; Maccarrone et al. 2023). CB1 and CB2 stimulation can also lead to the activation of mitogen-activated protein kinase (MAPK) signaling pathways, extracellular kinase-1 and − 2 (ERK1/2), p38 and p42/p44 MAPKs, and c-Jun N-terminal kinase (JNK), involved in the regulation of cell proliferation and death.

The activation of MAP/ERK/JNK pathways by pCBs can also be mediated via GPR55, GPR119, and GPR18 (Irving et al. 2017). Even if GPR55 endogenous ligand is mainly the phospholipid lysophosphatidylinositol (LPI), CBD has been reported as its potent antagonist (Ryberg et al. 2007; Whyte et al. 2009). GPR55 has been evidenced in the brain, in the peripheral system and in various tissues co-localized with CB1 and CB2. GPR55 was found associated with different G-proteins (Gαq/11, Gα12, Gα13, or Gα12/13) in the stimulation of intracellular Ca2+ release through Rho signalling pathway (Whyte et al. 2009; Morales and Reggio 2017). However, the activation of GPR55 can have also effects on the antioxidant response mediated by nuclear factor erythroid 2-related factor 2 (Nrf2) and ERK pathways (Irving et al. 2017). Cannabidiol has also been proposed as an antagonist of GPR18, widespread in almost all tissues (Morales et al. 2020; Senn et al. 2020).). The activation of GPR18 by its endogenous ligand N-arachidonoyl glycine (NAGly) can induce intracellular Ca2+ mobilization (Console-Bram et al. 2014). Similar effects can be exerted by THC via GPR18, in a dose dependent manner (Senn et al. 2020). Otherwise, CBD can block/reduce the effects of both NAGly and THC on GPR18 (Irving et al. 2017). These receptors seem to be also involved in the effects of pCBs and metabolic pathways via insulin signalling and cAMP (Soga et al. 2005).

Transient receptor potential vanilloid (TRPV)

The anti-hyperalgesic effects of CBD, and in some cases its anti-inflammatory properties, appear to be mediated by TRPV1 (Etemad et al. 2022). Phitocannabinoids such as CBD, THC and CBN, CBG, CBC and Δ9-THCV show different TRPV type 1–4 agonistic activities (Etemad et al. 2022). Overall, TRPVs are non-selective cation channels allowing calcium influx into various types of cells (Huang et al. 2024). TRPV1 may be activated by CBD and minor cannabinoids but not by Δ9-THC (Starkus et al. 2019), which activates TRPV2. Cannabidiol has high potential to bind TRPV1 and with slight differences TRPV2 as well as TRPV3, TRPV4, and transient receptor potential ankyrin subtype 1 (TRPA1) (Etemad et al. 2022). Working as a TRPV1 potent agonists, CBD induces a rapid desensitization of TRPV1 channels, leading to a decrease in calcium influx (Etemad et al. 2022). Indeed, CBD-induced anti-hyperalgesia was nullified by the TRPV1 antagonist capsazepine but not by CB1 or CB2 antagonists (Costa et al. 2004). Similarly, the protective effects against lipopolysaccharides (LPS)-stimulated macrophages induced by CBD occurs via TRPV1, but not CB1 or CB2 receptors, by modulating enhanced Nrf2, toll-like receptor 4 (TLR4) activity, and nuclear factor kappa B (NF-κB) activity (Rajan et al. 2016). Cannabidiol also reduces the increase in inflammatory cytokines and chemokines induced by various inflammatory stimuli via TRPV1 (Etemad et al. 2022; Peyravian et al. 2020) through downstream signalling pathways following the TRPV1 activation including phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) and the extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinases/mitogen-activated protein kinase (JNK/MAPK) and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) (Peyravian et al. 2020).

The anticancer effects of CBD can be mediate by both CB2 and TRPV1 receptors. Indeed, it has been reported that CBD can induce apoptosis, modulate cell adhesion, reduce cells viability and promote endoplasmic reticulum (ER) stress as well as oxidative stress (Etemad et al. 2022). Depending on cell types, these effects seem to be mediate by different pathways involving p42/44 MAPK, p38 MAPK, poly ADP ribose polymerase (PARP), transcription factor 4 (ATF4); most of them are related to increase of Ca2+ and TRPV1 activity. Moreover, through TRPV1, CBD can modulate autophagy via ERK1/2 activation and Akt but not dependent on the mammalian target of rapamycin complex 1 (mTORC1) (Vrechi et al. 2021).

Cannabidiol has been proposed as the most active pCB for the TRPV2 channel (Etemad et al. 2022). Moreover, the understanding of how CBD affects TRPV2 is hindered by the absence of a specific ligand, and CBD is still employed as an agonist to elucidate the function of TRPV2. In addition to its ability in desensitise the TRPV2 channel, CBD stimulate the synthesis of TRPV2 and its translocation to the plasma membrane. Some antitumoral effects of CBD have been described through TRPV2 and not via other CB receptors (i.e. CB1, CB2, or TRPV1) (Etemad et al. 2022). Cannabidiol resulted able to regulate PI3K/Akt pathway and mTORC1 for promoting cell differentiation and autophagy (Nabissi et al. 2015).

Even if CBD has affinity for TRPV3 and TRPV4, its efficacy was limited at both channels (Etemad et al. 2022).

Nuclear receptors and gene expression

Phytocannabinoids like eCBs, could act by regulating the gene expression trough some above described signaling pathway, as well as by interacting directly with nuclear receptor/transcription factors including some members of the PPAR family (PPARα, PPARβ/δ, and PPARγ) (Khosropoor et al. 2023). In particular, CBD is an agonist of the PPARγ: it can promote the formation of PPARγ-retinoid X receptor (RXR) heterodimers inducing the transcription of specific genes involved in metabolism (of glucose and lipids), immune response and inflammation. Moreover, CBD increases the PPARγ expression (Hegde et al. 2015; O’Sullivan 2016). Cannabidiol can activate the Nrf2 and inhibits NF-kB signaling also through PPARγ (Khosropoor et al. 2023).

Overall, cannabinoids can act as redox modulators, exerting antioxidant and pro-oxidant effects depending on the dose, time of exposure, and specific cell types (Rybarczyk et al. 2023). Moreover, it has been demonstrated that the antioxidant effects of CBD can occur via TRPV (as reported above) or PPARγ, both of which can promote the nuclear translocation and activation of Nrf2. This transcription factor, by binding to the antioxidant response element (ARE), activates the expression of phase II antioxidant enzymes, including glutathione S-transferase (GST), catalase (CAT), heme oxygenase-1 (HO-1), and manganese-dependent superoxide dismutase (Mn-SOD), thus modulating the cellular antioxidant response (Rybarczyk et al. 2023). Notably, pCBs and specifically CBD and 9Δ-THC exhibited antioxidants proprieties linked to their phenolic groups. They can act as chain-breaking antioxidants like vitamin E (Dawidowicz et al. 2021).

Further, as NF-kB activation depends upon the regulation of cellular redox status, CBD is also considered involved in the crosstalk between NF-kB and Nrf2 (Atalay Ekiner et al. 2022). Cannabinoids are known as downregulators of NF-kB signalling, which has a crucial role in the activation of various pro-inflammatory gene expressions encoding e.g. IL-1β, IL-6, TNF-α, and COX-2 (Atalay Ekiner et al. 2022). The nuclear translocation of active forms (p50, p52, p65, RelB or c-Rel) can occur only after degradation of IkB inhibitors that could be reduced by CBD also via PPARγ (Khosropoor et al. 2023). Cannabidiol seems to prevent NLR family pyrin domain containing 3 (NLRP3)-inflammasome pathway activation by suppressing the expression of key genes like NLRP3 and caspase 1 (Martinez Naya et al. 2023).

Toll-like receptors

The anti-inflammatory effects of pCBs can be mediated by Toll-like receptors (TLRs) activated by pathogen-associated molecules, acting within the innate immune system (Cui Sun et al. 2024). Indeed, CBD inhibits TLR1-induced IL-1β secretion and TLR2-induced Interferon Gamma-induced Protein 10 (IP-10), IL-1β, IL-6, and TGF-β1 secretion in human monocytes (Sermet et al. 2021). Both THC and CBD also reduce TLR3-induced IP-10 release and IFN-β protein expression, and CBD inhibits TLR4-induced TNFα production in macrophages (Carlisle et al. 2002). Similarly, CBD can impact TLR 5–8 signaling, whereas cannabinoids can also exacerbate TLR-induced inflammatory signaling in a dose-dependent manner and depending on the type of TLR agonism (Cui Sun et al. 2024).

Opioids and GABA receptors

Finally, pCBs have been proposed as alternative to opioids for pain management (Ang et al. 2023). Indeed, THC and CBD can both also work as allosteric modulators of the opioid receptor µ and δ (Kathmann et al. 2006). In particular, CBD can serve in the control of glutamatergic signalling via N-methyl-D-aspartate (NMDA) receptor-mediated seizures in vivo (Rodríguez-Muñoz et al. 2018). Moreover, CBD can act on GABAergic neurotransmission affecting CNS excitability, probably modulating the composition and arrangement of different subunits that characterise GABA receptors (Ruffolo et al. 2022). On the contrary, Δ(9)-THC does not have any effects on GABA receptors (Lile et al. 2014).

Localization of cannabinoid receptors

The first studies to define the presence of the ECS were carried out in animal species and it has been found in both vertebrate (mammals, birds, reptiles, and fish) and invertebrates (such as sea urchins, leeches, mussels, nematodes, crustaceans, protozoa, onychophorans) (Della Rocca and Di Salvo 2020; Silver 2019). In non-mammalian species, cannabinoid receptors have been identified in birds (i.e. parrots, zebrafinch, poultry) (Alonso-Ferrero et al. 2006; Stincic and Hyson 2008; Divín et al. 2022), reptiles and fish (i.e. goldfish, zebrafish) (Silver 2019). Notably, it has been demonstrated that the ECS is highly conserved between Danio rerio (zebrafish) and mammals, exhibiting high similarity to that in rodents and human with a parallel CB receptors expression pattern (Lachowicz et al. 2023). Recently, several studies focused on the identification and localization of cannabinoid receptors in various animal species (dogs, cats, horses cattles and pigs) to suggest a therapeutic potential role of Cannabis derivatives in veterinary medicine. Table 1 synthetizes the localization of cannabinoid receptors in the different tissues and cells of dogs, cats and horses.

Table 1 Localization of cannabinoid receptors in dogs, cats and horses
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Localization of CB receptors in dogs

Dogs express a large number of CB1 receptors in the brain, specifically in clastrum and neocortex, cerebellar cortex, midbrain, medulla oblongata, gray matter of spinal cord, trigeminus, basal ganglia, cochlear nucleus, olfactory bulb and hippocampus (Pirone et al. 2016; Freundt-Revilla et al. 2017; Silver 2019). In the hippocampus, a different expression of CB1 receptors was observed in dogs affected by idiopathic or structural epilepsy when compared with healthy subjects. Specifically, a significant decrease of CB1 receptors was observed in dogs with idiopathic epilepsy, while an increase of the same receptors was reported in dogs with structural epilepsy (Kostic et al. 2023). At the dorsal root ganglia level, besides CB1 receptors, also TRPV1, GPR55 and CB2 receptors were identified. In satellite glial cells, a strong presence of CB2 receptors but also of PPARα and TPVR1 was observed in old dogs. A clear presence of CB2 receptors was observed in Schwann cells and cells surrounding blood capillaries and, although at a lesser extent, in smooth muscular blood vessels (Chiocchetti et al. 2019). The distribution and density of cannabinoid receptors show interspecies differences. In dogs, CB1 receptors are significantly more expressed in hind brain than in humans (Silver 2019).

At the gastrointestinal tract level, a large presence of cannabinoid receptors, both CB1 and CB2 and also GPR55 and PPARα was observed. Specifically, immunoreactivity for cannabinoid receptors was found in the lamina propria (CB1, CB2 and GPR55), epithelial cells (CB1), mast cells (CB2), immunocytes (CB2), blood vessels (CB2 and PPARα), smooth muscle cells (CB2, GPR55 and PPARα), macrophages (GPR55), neurons and glial cells of the submucosal plexus (CB2) and glial cells of the myenteric plexus (PPARα) (Galiazzo et al. 2018).

Cannabinoid receptors CB1 and CB2 are also present in skin of healthy dogs and dogs with atopic dermatitis, with a stronger immunoreactivity in diseased subjects (Campora et al. 2012). More recently, also GPR55, TPVR1, PPARα and ankyrin 1 (TRPA1), besides CB1 and CB2 receptors, were found in keratinocytes of healthy and atopic dogs, and an overexpression of CB2 receptor and TPRA1 was observed in those with atopic dermatitis (Chiocchetti et al. 2022a). Receptors such CB2, GPR55, TPVR1, PPARα and TRPA1 were also found in inflammatory infiltrate of the skin of atopic dogs (Chiocchetti et al. 2022b).

Other location of canine CB1 receptor include salivary glands as parotid and mandibular glands (Dall’Aglio et al. 2010), hair follicles (Mercati et al. 2012), and embryo (Pirone et al. 2015).

Both CB1 and CB2 receptors were found in canine mast cell tumors (Rinaldi et al. 2022), immune cells (Brown et al. 2023) and synovial cells of canine hip and stifle (Zamith Cunha et al. 2023a). Synoviocytes express immunoreactivity also for GPR55 (Zamith Cunha et al. 2023a).

Localization of CB receptors in cats

Similarly to dogs, also in cats PPARα, CB1 and CB2 receptors were overexpressed in the skin of subjects with dermatitis due to hypersensitivity compared to healthy ones (Miragliotta et al. 2018).

A role of ECS was also hypothesized in cats with feline chronic gingivostomatitis: CB1, CB2, GPR55 and TRPA1 were markedly upregulated in the oral mucosa of affected cats when compared to healthy ones (Polidoro et al. 2021). As in dogs, cannabinoid (CB1 and 2) and cannabinoid-related receptors (GPR55, PPARα and TRPA1) are widely expressed in the gastrointestinal tract of cats (Stanzani et al. 2020).

Lastly, CB1 receptors are also expressed in feline arterial smooth muscle cells (Gebremedhin et al. 1999), ovary and oviduct (Pirone et al. 2017) and in synovial membrane of cats with synovitis (Ruel et al. 2022).

Localization of CB receptors in horses

In horses, the most recent scientific literature has focused on the identification of cannabinoid receptors in the synovial membrane of metacarpophalangeal joint. CB1 and CB2 receptors, as well as TRPV1, GPR55 and PPARα, were found in synovia of healthy horses (Miagkoff et al. 2023; Zamith Cunha et al. 2023c). Miagkoff et al. (2023) found a relationship between the expression of CB1 and CB2 receptors in the synovia and the grade of synovitis or osteoarthritis in equine metacarpophalangeal joints. They observed that CB1 and CB2 were overexpressed with increased degree of synovitis, while CB1 receptors significantly decreased with the increase of osteoarthritis scores (Miagkoff et al. 2023).

Cannabinoid receptors were also widely distributed in ileum (CB1 and CB2 receptors, PPARα and TRPA1), skin (CB1 and CB2 receptors), in the trigeminal ganglion (CB1, CB2, TRPV1, GPR55 and PARRγ) and dorsal root ganglia (CB1 and CB2, PPARα and γ, TRPV1, TRPA1, GPR3 and GPR55) (Chiocchetti et al. 2021; Galiazzo et al. 2021, 2022; Kupczyk et al. 2022; Zamith Cunha et al. 2023b).

Localization of CB receptors in other animal species

The information available regarding other animal species is very scarce. CB2 receptors were found in bovine fetal pancreas (Dall’Aglio et al. 2017), while CB1 receptor was identified in pig clastrum (Pirone et al. 2020) and myenteric plexus of pig ileum together to TRPV1 and TRPA1 (Toschi et al. 2021). Moreover, the expression of genes encoding CB1 receptor in spermatozoa of bull was correlated to animal fertility: a major expression of this gene was found in sample from high fertile bulls compared to low fertile ones (Kumar et al. 2018).

Pharmacokinetics of phytocannabinoids in animal species

The understanding of the pharmacokinetics (PK) of a drug is crucial for defining an accurate dosage regimen and avoiding its empirical use. Consequently, the increasing interest in the therapeutic application of pCBs in veterinary clinical practice has led to a growing number of publications on their pharmacokinetics in various animal species in recent years. Studies aimed at defining the kinetic behavior of cannabinoids primarily focus on CBD, especially following oral administration in dogs (Bartner et al. 2018; Gamble et al. 2018; Deabold et al. 2019; Chicoine et al. 2020; Fernández-Trapero et al. 2020; Wakshlag et al. 2020; Kulpa et al. 2021; Vaughn et al. 2021; Doran et al. 2022; Polidoro et al. 2022; Tittle et al. 2022; Corsato Alvarenga et al. 2023; Della Rocca et al. 2023; Limsuwan et al. 2024). More recently, similar studies have been conducted in horses (Ryan et al. 2021; Turner et al. 2022; Williams et al. 2022; Yocom et al. 2022; Eichler et al. 2023a, 2023b; Sánchez De Medina et al. 2023; Thomson et al. 2024) and cats (Deabold et al. 2019; Kulpa et al. 2021; Wang et al. 2022; Jukier et al. 2023; Rozental et al. 2023a; Lyons et al. 2024). However, as of now, the pharmacokinetic profile of CBD has not been fully elucidated in any of these species.

Absorption and bioavailability

According to the authors’ knowledge, only a few experimental studies, one in dogs and two in horses, have administered CBD both orally and intravenously, allowing for the estimation of its oral bioavailability. The results indicated a bioavailability of less than 19% in dogs (Samara et al. 1988) and 14% in horses (Sánchez De Medina et al. 2023; Turner et al. 2022). A poor bioavailability may correspond to a lack of therapeutic efficacy, and from a pharmacokinetic perspective, a significant interindividual variability is more likely to be observed (Millar et al. 2020).

A considerable variability in CBD plasma concentrations following oral administration was observed in the mentioned studies in dogs, horses, cats, but also in rabbits (Rooney et al. 2022), calves (Meyer et al. 2022), and parrots (Sosa-Higareda et al. 2023). The reduced oral bioavailability of CBD has been attributed to both poor intestinal absorption and high hepatic metabolism (Perucca and Bialer 2020). Various formulations, such as microencapsulated CBD oil beads, CBD-infused oil (Bartner et al. 2018), nanoemulsion, water-soluble and semisolid forms (Limsuwan et al. 2024), CBD-rich hemp extract in soft gel capsules and sesame oil (Tittle et al. 2022), soft chews containing a CBD/CBDA predominant extract, and the same extract diluted in different oils (Wakshlag et al. 2020), were tested. Different routes of administration that can avoid first-pass metabolism, as sublingual (Fernández-Trapero et al. 2020), transdermal (Bartner et al. 2018), intranasal, intrarectal (Polidoro et al. 2022), and oral transmucosal (Della Rocca et al. 2023), were evaluated, but unfortunately, conclusive results were not achieved. It is important to note that the diverse experimental conditions used in various studies, such as sampling times, the number of samples, the number of animals enrolled, age, sex, and breed, make comparisons among them very difficult.

After multiple oral escalation doses of CBD (up to a maximum dose of 30.5 mg/kg) in cats, the observed maximum concentration (Cmax) was about half that obtained with co-administration of a THC/CBD oil orally administered in escalating doses up to a maximum of 10.6 mg/kg of CBD (Kulpa et al. 2021). This result led to the hypothesis that THC may improve CBD absorption (Rozental et al. 2023a). More recently, an increase in the oral bioavailability of CBD in rats was observed following oral administration of CBD as a full spectrum compared to an equal dose of CBD alone. In the same study, the authors also demonstrated, through in vitro assays, that THC can increase the intestinal permeability of CBD and decrease its efflux (Berthold et al. 2023).

Previously, a greater Cmax and Area Under Curve (AUC) have already been observed for CBDA following oral administration of a full spectrum extract (CBDA in co-presence of other cannabinoids) when compared with the administration of pure CBDA in mice (Anderson et al. 2021). Regarding this issue, the different types of cannabinoids (full spectrum, purified, or synthetic) used in various pharmacokinetic studies introduce another variable in elucidating their kinetic behavior.

In humans, it was observed that a high-fat meal enhances the absorption of CBD, allowing for Cmax and AUC values about four times higher than those resulting from a fasted state (Taylor et al. 2018). Similar results have been reported in cats, where a significantly higher AUC was observed for orally administered CBD 30 min after a meal compared to its administration in the fasting state (Jukier et al. 2023). In dogs, the data obtained are not entirely conclusive; even if the Cmax value of CBD was significantly higher under fed conditions compared with a fasted state, no significant difference was observed for AUC values (Doran et al. 2022). It is essential to underline that, in this last study, only three subjects per group were enrolled, and for one dog, the greater values of Cmax and AUC were obtained in the fasted condition. On the contrary, following oral administration of an oil containing CBD and CBDA in rabbits, the AUC values of both cannabinoids were significantly greater in the fasted state than after a slurry meal (Rooney et al. 2022). Also, for THC, CBG, and CBGA, a greater extent of oral absorption, with higher values for both Cmax and AUC, was observed in fasted dogs compared to fed dogs (Łebkowska-Wieruszewska et al. 2019; Amstutz et al. 2022).

Contrarily to single administration, after oral treatment with a CBG and CBGA-rich hemp extract twice a day for two weeks in fed and fasted dogs, no significant differences in blood concentrations of cannabinoids were observed between the two different food states (Amstutz et al. 2022). Changes in the extent of absorption can be due to species-specific differences and/or depend on the nature and amount of ingested food (Deng et al. 2017). The variables linked to food can sometimes also influence the rate of absorption, as observed following the administration of THC in fed dogs where time to maximum concentration (Tmax) resulted in 5 h compared to 1.25 h observed in fasted subjects (Łebkowska-Wieruszewska et al. 2019).

Additionally, the acid forms are generally more absorbed than the corresponding non-acid products. This phenomenon was observed after single oral co-administration of CBD and CBDA in similar concentrations in dogs (Wakshlag et al. 2020; Tittle et al. 2022), cats (Wang et al. 2022), rabbits (Rooney et al. 2022), horses (Thomson et al. 2024) and parrots (Sosa-Higareda et al. 2023), as well as following transdermal application, twice a day for two weeks, in dogs (Hannon et al. 2020). In all cases, both Cmax and AUC values of CBDA were at least double that of CBD. This was also observed for other acid derivatives, such as THCA vs. THC in dogs, cats and horses (Wakshlag et al. 2020; Tittle et al. 2022; Wang et al. 2022; Thomson et al. 2024) and CBGA vs. CBG in cats and dogs (Amstutz et al. 2022; Wang et al. 2022). It should be highlighted that, after multiple oral administrations, the differences in absorption of CBD and CBDA, observed after a single administration, were attenuated and similar in both dogs and parrots (Wakshlag et al. 2020; Sosa-Higareda et al. 2023).

Following oral administration of industrial hemp (flower material in gelatine capsules) in cattle, the average Cmax of THCA-A, CBCA, and CBDVA was 12.1, 12.3, and 13.1 ng/mL, respectively. Considering that cattle were exposed to very different doses of these cannabinoids (117.4 mg of THCA-A, 101.3 mg of CBCA, and 3.5 mg of CBDVA), it is possible to suppose that CBDVA has the highest oral bioavailability from cattle rumen compared to the other acid cannabinoids (Kleinhenz et al. 2020).

Distribution

Following intravenous administration of THC in swine and CBD in horses, a large apparent volume of distribution was computed, indicating a deep distribution of these cannabinoids in the organism (Schaefer et al. 2016; Sánchez De Medina et al. 2023). In swine, intravenously administered THC was primarily distributed in bile fluid, lung, and perirenal and abdominal adipose tissue. However, it was also detected in the brain, heart, liver, kidney, spleen, and muscle (Schaefer et al. 2017). In sheep, after being fed for 56 days with diets containing different percentages of hemp stubble, with THC content less than or equal to 0.001% on a dry matter basis, the cannabinoid was detected in kidney fat, subcutaneous fat, and loin meat, although not in all animals (Krebs et al. 2021). In sheep, when fed for 22 days with a pellet diet containing 42% green hemp (partly in full flower), THC was detected in the subcutaneous fat of 4 out of 6 animals at the end of the treatment, and 35 days later it was found in all animals, albeit at lower concentrations. These results led the authors of the study to hypothesize the existence of a redistribution of THC from other body compartments into fat (Stevens et al. 2022). Further, cannabinoid can accumulate in milk; following seven days of feeding with hemp silage, which had low cannabinoid concentrations, cow’s milk showed detectable levels of Δ9-THC, Δ9-THCA, Δ9-THCV, CBD, CBN, and CBDV. After an additional six days with a higher cannabinoid content in the diet, the milk concentrations of Δ9-THC, Δ9-THCV, and CBD were higher than plasma concentrations by over 6, 3, and 11 times, respectively (Wagner et al. 2022).

Finally, CBD was detected in the synovial fluid of 5 out of 6 horses following a single oral administration of 3 mg/kg of CBD in sunflower lecithin oil. Furthermore, its synovial concentrations consistently increased at five weeks after oral administration of the same formulation at 1.5 mg/kg twice daily, indicating a probable cumulative effect in this compartment (Yocom et al. 2022).

Although higher blood concentrations were observed for acidic cannabinoid derivatives than their non-acidic counterparts following the administration of equal doses, they do not appear to have the same ability to cross the blood-brain barrier. Indeed, the brain/plasma AUC ratios of CBD and CBDA following intraperitoneal administration in mice resulted in 0.51 (Deiana et al. 2012) and 0.04 (Anderson et al. 2019), respectively. Tiny brain/plasma AUC ratios, between 0.02 and 0.04, were also observed for other acid derivatives (CBDVA, CBGVA, and CBGA), while for CBCA and THCA, their presence at the central nervous system level was not detectable at all (Anderson et al. 2019).

Metabolism

In terms of cannabinoid metabolism, as lipophilic molecules, pCBs undergo extensive biotransformation reactions. In humans, CBD is primarily converted into the active metabolite 7-hydroxy cannabidiol (7-OH-CBD) and is further metabolized into 7-carboxy cannabidiol (7-COOH-CBD), which is the most abundant circulating metabolite; 6α- and 6β-hydroxy cannabidiol (6-OH-CBD) are also formed but in smaller amounts (Mechoulam and Hanuš 2002; Pérez-Acevedo et al. 2020; Sitovs et al. 2024). Regarding hydroxylated metabolites, 6-OH-CBD was the major metabolite in dogs following the oral administration of a cannabis herbal extract. 7-OH-CBD was only intermittently observed (Chicoine et al. 2020). Similarly, in cats treated with the same formulation at the same doses, only 6-OH-CBD was detected, albeit with blood concentrations 2.5 fold smaller than in dogs (Lyons et al. 2024), indicating species differences in biotransformation. In humans, 7-OH-CBD is predominantly formed via CYP2C19 and CYP2C9 isozymes, while in a canine in vitro study, the main enzyme of the CYP2C subfamily (CYP2C21) is of secondary importance in CBD metabolism compared to CYP1A2. In humans, CYP1A2 appears to be less involved in CBD metabolism and does not form the active metabolite 7-OH-CBD (Court et al. 2024).

Similar to what is observed in humans, a greater presence of 7-COOH-CBD compared to the hydroxylated metabolites was reported in the serum of dogs (Chicoine et al. 2020; Wakshlag et al. 2020; Vaughn et al. 2021), cats (Wang et al. 2022), and horses (Ryan et al. 2021; Turner et al. 2022; Eichler et al. 2023a, 2023b). In cats, after oral administration of 11 escalating doses of both CBD and CBD/THC combined oil, the concentration of the metabolite 7-COOH-CBD at 24 h post-dosing was significantly higher following treatment with combined oil, even if the total dose of CBD administered was smaller (Kulpa et al. 2021). Conversely, in parrots, following a single oral administration of 30/32 mg/kg of CBD/CBDA-rich hemp extract, this metabolite was not quantifiable in serum (Sosa-Higareda et al. 2023). In this last study, the predominant metabolite observed was 11-OH-THC, due to a concentration of THC and THCA in the formulation such that the administered dose was 1.23 and 0.39 mg/kg, respectively. These results indicate another species-specific difference in cannabinoid biotransformation (Sosa-Higareda et al. 2023).

THC is another cannabinoid that undergoes extensive metabolism, and the main biotransformation products observed in humans are 11- hydroxy tetrahydrocannabinol (11-OH-THC) and 11- carboxy tetrahydrocannabinol (11-COOH-THC) (Lucas et al. 2018). In dogs, following single sublingual administration of Sativex (dose equal to 8.1 mg of THC and 7.5 mg of CBD), the hydroxylated metabolite, 11-OH-THC, was observed, albeit at the limit of detection; its Cmax and AUC increased following two weeks of treatment. 11-COOH-THC was not tested for its presence in serum (Fernández-Trapero et al. 2020). Sporadically, 11-OH-THC was observed in canine serum after oral administration of CBD/CBDA-rich hemp extract in different oils (Wakshlag et al. 2020) and Cannabis herbal extract (Chicoine et al. 2020); the latter study is the only one to report, albeit only for some samples, the detectable presence of 11-COOH-THC in dogs. The formation of 11-OH-THC was also observed in cats following oral administration of 11 escalating doses of both THC alone and CBD/THC combined oils. Surprisingly, 4 h after the end of administrations, a significantly higher peak concentration was observed following CBD/THC combined oil, even if the maximum THC dose administered with this formulation was 8.4 mg/kg vs. 41.5 mg/kg of the THC-alone oil (Kulpa et al. 2021). In a more recent study neither 11-OH-THC and 11-COOH-THC were detected in blood of cats orally treated with 1:20 THC: CBD cannabis herbal extract at the dose of 0.25 mg/kg in THC (Lyons et al. 2024).

Elimination

Cannabidiol and its metabolites are excreted in feces and urine (Bradley et al. 2022; Ryan et al. 2021; Samara et al. 1990). The presence of different metabolites in dogs’ urine confirms the existence of its extensive metabolism in this species (Samara et al. 1990). Both 7-OH-CBD and 7-COOH-CBD were detected in equine urine up to 72 h after oral administration of CBD. However, it is necessary to underline that urine samples were exposed to the β-glucuronidase enzyme, so the two detected metabolites also account for any conjugate derivatives (Ryan et al. 2021). Cannabidiol, as such, was also found in canine urine, although in lower amounts than observed in feces (Bradley et al. 2022). In the opinion of the authors of this review, however, it cannot be ruled out that the higher concentrations of CBD in feces are related to its unabsorbed portion rather than its biliary elimination.

Efficacy and tolerability of phytocannabinoids in dogs, cats, horses and cattle

Dogs

Most studies aimed at evaluating the efficacy and tolerability of Cannabis derivatives in veterinary medicine are mainly focused on CBD in the canine species. Pathologies considered include pain (especially from osteoarthritis), epilepsy, behavioral and skin disorders.

The efficacy and safety of cannabis derivatives in treating chronic pain have been tested in ten studies, while only two studies dealt with the use of CBD in acute painful conditions.

From the randomized, placebo-controlled, double-blind, crossover clinical trials conducted by Gamble et al. (2018), Brioschi et al. (2020), and Verrico et al. (2020) to evaluate the therapeutic effects of different Cannabis formulation containing mainly CBD in dogs with chronic osteoarthritic (OA) pain, CBD seemed able to significantly reduce pain and increase locomotor activity, thus improving dogs’ quality of life (Gamble et al. 2018; Brioschi et al. 2020; Verrico et al. 2020). However, these results are not in line with those obtained by Mejia et al. (2021) in their double-blind, randomized, placebo-controlled, crossover clinical study conducted on OA patients, where no differences were noted between groups for any of the recorded outcome measures (Mejia et al. 2021). In addition to the above studies, two pilot studies (Martello et al. 2019; Shilo-Benjamini et al. 2023), a non-blinded observational study (Kogan et al., 2020) and two case reports (Coelho et al. 2021; Shilo-Benjamini et al. 2022), all conducted in OA dogs, have also reported the positive therapeutic effects of CBD administration on pain and locomotion. Very recently, a double-blind, placebo-controlled study was also performed in owned dogs with chronic pain (the origin of which was not specified). The study was a 16-week evaluation of dogs in either placebo or hemp oil phase, each lasting 8 weeks. Results showed a decrease in pain scores as well as in plasma levels of proinflammatory cytokines. However, the lack of washout between the phases was a procedural limitation of this study. More detailed information on these cited studies is reported in Table 2.

Table 2 Summary of studies on the efficacy of Cannabis derivatives in dogs
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In the only published randomized, placebo-controlled, blinded clinical trial aimed at determining the impact of capsules containing a CBD/CBDA-rich hemp oil (2–2.5 mg/kg orally twice daily for four weeks) on acute post-operative pain in dogs following tibial plateau levelling osteotomy (TPLO), pain scores (obtained by CBPI), degree of lameness, degree of weight-bearing, or radiographic healing of the osteotomy did not differed significantly between placebo and CBD/CBDA groups at any point, but an increase in alkaline phosphatase (ALP) activity was observed (Klatzkow et al. 2023). However, a previous study where a CBD/CBDA-rich hemp oil was given at 5 mg/kg in postsurgical intervertebral disc disease suggested lower postsurgical pain scores compared to placebo based on blinded veterinary assessment (Wright 2022).

Very recently, the anesthetic sparing effect of a single oral transmucosal full spectrum CBD-rich extract (6 mg/kg of total pCBs) has been demonstrated in 9 dogs. Indeed, a reduction of 23% on propofol dose necessary for induction was obtained in CBD treated dogs with respect to a placebo group, indicating that pCBs could be an adjunct option in anesthesia (Hasckel Gewehr et al. 2024).

The efficacy and tolerability of CBD in treating epilepsy in dogs have been investigated in three randomized, placebo-controlled, double-blinded clinical trials, and in one case series. The three randomized controlled clinical trials (Garcia et al. 2022; McGrath et al. 2019; Rozental et al. 2023b) produced promising results, as all studies showed a reduction in seizures frequency (total seizures or seizure days) in the CBD treated dogs compared to placebo. However, the case series by Mogi and Fukuyama (2019) reported different and sometimes contradictory results in the three evaluated dogs (Mogi and Fukuyama 2019) (see Table 2 for a detailed description of these studies).

It is noteworthy that in the study by Rozental et al. (2023b) the interactions between antiseizure drugs (ASD), such as phenobarbital, potassium bromide, zonisamide and levetiracetam, and CBD treatment did not affect the percentage change of total seizures or seizure days. Additionally, there was no evidence of differences between CBD administration and placebo regarding the percentage change from baseline in ASD concentrations for any of the measured ASDs (Rozental et al. 2023b). A previous study by Doran et al. (2022), which explored the drug-drug interactions between CBD and phenobarbital in healthy dogs, did not find any significant pharmacokinetic interactions between the two drugs. This indicates that, at least in healthy dogs, CBD and phenobarbital can be co-administered without notable alterations in their respective pharmacokinetics (Doran et al. 2022).

Five scientific studies on the clinical efficacy of CBD in treating behavioral disorders have been published so far: a replicated 4 × 4 Latin square design experiment (Morris et al. 2020), a placebo-controlled study (Corsetti et al. 2021), two blinded, placebo-controlled, parallel-design study (Hunt et al. 2023; Flint et al. 2024) and a pilot study (Masataka 2024). The efficacy of CBD treatment was evaluated in different stressful conditions, such as fear-inducing stimuli, shelter dogs, separation from caregivers ad car travels. In all studies but the one by Morris et al. (2020) (which results did not provide strong support for the anxiolytic effect of CBD in dogs), the efficacy of CBD in reducing stress in treated dogs was evidenced (see Table 2 for details).

Recently, four studies investigating the efficacy of CBD and CBD/CBDA as a treatment for canine skin diseases have also been published. They account for a randomised, double-blinded and placebo-controlled trial (Loewinger et al. 2022), a double-blinded and placebo-controlled trial (Mariga et al. 2023), a retrospective case series (Mogi et al. 2022) and a case report (Da Silva et al. 2024). The first three studies were aimed to evaluate the effect of CBD on lesion’s severity and pruritus in dogs affected by atopic dermatitis. While the severity of the lesions was not improved by CBD treatment in any of these three studies, an improvement in itching was observed in studies by Loewinger et al. (2022) and Mogi et al. (2022). It is noteworthy that in these studies the CBD-based solution was administered orally; meanwhile, humans’ studies have demonstrated a potential therapeutic value of CBD applied topically for skin diseases (Martins et al., 2022). The case report was instead intended to test the efficacy of a CBD treatment in a dog suffering with discoid lupus erythematosus (DLE) and resistant to conventional treatments. Cannabidiol was able to significantly improve skin lesions within a few weeks of treatment, and after 1 year the dog remained clinically stable on a low dose of CBD (Da Silva et al. 2024). Table 2 reports more detailed information on the studies cited above.

A fifth study (a randomized complete block design, placebo-controlled study) intended to determine the influence of CBD (1.25 mg/kg or 2.5 mg/kg orally twice daily for three weeks) on the dogs’ daily activity, showed that CBD did not alter the total daily activity points or activity duration, but reduced total daily scratching compared with the control, albeit not statistically (Morris et al. 2021).

From this summary of studies, it is possible to conclude that some evidence exists supporting the beneficial role of CBD for adverse conditions in dogs, including OA pain, seizures, behavioral and skin problems. However, it is worth reporting the conclusions of two studies aimed to summarize the evidence of efficacy and safety of the use of cannabis for the treatment of animal disease and to assess the risk of bias regarding the obtained results. The first study (a systematic review of randomized clinical trials) included six trials meeting the inclusion criteria (i.e., Gamble et al. 2018; McGrath et al. 2019; Brioschi et al. 2020; Verrico et al. 2020; Corsetti et al. 2021; Mejia et al. 2021) and found that all of them presented a certain risk of bias (classified as low risk, some concern or high risk) accounting for one or more of the following items: randomization process, deviation from intended interventions, missing outcome data, measurement of the outcome, and selection of the reported results (Lima et al. 2022). The second study (a systematic review and meta-analysis of animal intervention studies) examined the results obtained by Gamble et al. 2018; Brioschi et al. 2020; Kogan et al. 2020; Verrico et al. 2020; and Mejia et al. 2021; addressing six types of bias: selection bias, performance bias, detection bias, attrition bias, reporting bias, and other, across 10 domains (with each domain categorized as high, unclear, or low risk of bias). Each of the identified study presented a certain level of bias, and the Authors concluded that the evidence is very uncertain to confirm the clinical efficacy of CBD treatment (Patikorn et al. 2023). Overall, both systematic reviews indicate that the results of published studies, despite being randomized, double-blinded, and placebo-controlled, should be interpreted with caution. Greater attention to study design and the definition and measurement of outcomes is needed in future research to strengthen the evidence regarding the therapeutic benefits of CBD for dogs.

In the matter of tolerability, in addition to the above studies (see Table 2 for tolerability results), other research has evaluated the safety of Cannabis derivatives, often together with their pharmacokinetics.

In the study by McGrath et al. (2018), diarrhea, vomiting, erythematous pinnae, transient isosthenuria, hyposthenuria or proteinuria, and increased ALP activity, nasal discharge, salivary staining, lameness, prolapsed nictitan and hyperthermia were observed after the administration of CBD oil, CBD microencapsulated and CBD cream (10 or 20 mg/kg) twice daily orally or transdermal for six weeks in 30 dogs. Loose stool and vomiting (food or bile products) were also reported by Deabold et al. (2019) after treatment with an oil (embedded in soft chews) containing 1 mg/kg of CBD and 1 mg/kg of CBDA, orally twice daily for 12 weeks in 8 dogs. Mild and self-limiting gastrointestinal signs (mainly hypersalivation) and transient increase of ALP were reported when a CBD hemp oil (1, 2, 4, or 12 mg/kg) was given orally once daily for four weeks in 20 dogs (Vaughn et al. 2021).

Increased ALP activity was also observed by Bradley et al. (2022), who administered CBD hemp oil at 4 mg/kg orally daily for 26 weeks in 40 subjects. Tittle et al. (2022) reported vomiting, loose stools and occasional episodes of licking, grimacing and chomping after oral treatment with 2 mg/kg CBD/CBDA-rich soft gel and hemp oil twice daily for four weeks in 8 dogs, while Doran et al. (2022) described the occurrence of vomiting, hyporexia, anorexia and an increase in serum ALP activity after the administration of 5, 10 or 20 mg/kg of pure CBD in MCT oil twice daily for two weeks in 9 dogs (3 x dosage).

In a study conducted by Chicoine and colleagues, neurological signs (head bobbing, hyperesthesia, ataxia or swaying, among others) were observed in six dogs treated with a 1:20 THC: CBD Cannabis herbal extract with a dosage of 10 mg/kg of CBD and 0,5 mg/kg of THC (Chicoine et al. 2020).

Additionally, a case report published by Simpson et al. (2020) described widespread cutaneous erythema and ulceration associated with anorexia and diarrhea in a dog. These symptoms occurred five days after the dog received an oral hemp oil formulation for anxiety, with a dosage of 0.3 mg/kg of CBD administered once daily (Simpson et al. 2020).

Very recently, the tolerability of oils containing different cannabinoids (broad spectrum CBD, broad spectrum CBD with CBG, or broad spectrum CBD with CBDA at 5 mg total cannabinoids/kg body weight/day) given orally was evaluated in a randomized, non-blinded, negative controlled, parallel design 90-day repeat dose study in 8 dogs/group. Clinical examinations, body weights, food consumption, serum hematology and biochemistry, coagulation parameters, and urinalysis were conducted at prefixed experimental times during the treatment period and up to 2 weeks after dosing. No somnolence, adverse effetcts (AEs) or serious AEs were reported during the study. The most common abnormal observation was diarrhea. Some hematology and clinical chemistry parameters showed statistically significant within-treatment group changes compared to baseline value. However, most of these changes were either transient or were within reference ranges. No significant abnormalities were recorded for any urinalysis parameters evaluated (Bookout et al. 2024).

The long-term tolerability of an industrial hemp broad spectrum extract with 94.5% CBD, 4.6% CBG, 0.3% CBDV, and 0.6% CBCH, administered orally to healthy dogs for 36 weeks at dosages of 5 and 10 mg/kg/day was also assessed in a randomized placebo-controlled study. Adverse events were recorded daily. Cell blood count and blood biochemistry profiles were monitored every 4 weeks, as well as physical examination (temperature, heart rate, respiratory rate, weight, and body fat index, observations of skin, eyes, nose, mouth and teeth, heart and lungs, the abdomen, lymph nodes, mentation and personality, activity level, and vocalization). The most prevalent AEs were gastrointestinal (soft feces or diarrhea with tenesmus and straining), and all observed AEs were mild. Dogs in the 10 mg/kg group had a higher frequency of soft feces than the 5 mg/kg group and placebo. All dogs dosed with both CBD oil doses had overall higher ALP activity than those given placebo, and monthly variations with a significant time effect were observed (Corsato Alvarenga et al. 2024).

Besides clinical trials, a preclinical/preregistration study was conducted in healthy male and females Beagle dogs to study the toxicology of a purified CBD extract (Epidiolex™). Dogs were treated over 39 weeks either with 0 mg/kg/day (control group C), 10, 50 and 100 mg/kg/day. Soft/liquid/mucoid feces, reduced body weight, marked increases in ALP (up to 8-fold compared to C), and liver changes, such as hepatocyte hypertrophy associated with increased liver weight and macroscopic enlargement, were observed at all doses, as well as consistent decreases in heart rate in males at the higher dose (Food and Drug Administration Application 2018).

From the above findings, an oral administration of CBD 2 mg/kg once daily and up to 20 mg/kg/ twice daily appeared well-tolerated with mild side effects, observed in both healthy and diseased animals. This favorable safety profile aligns with the findings of a study conducted by Vaughn et al. (2020), which utilized increasing oral doses of CBD, reaching up to 62 mg/kg (Vaughn et al. 2020).

To the best of the authors’ knowledge, only one paper has been published so far on the physiological effect of CBG and its acid derivative (CBGA) in dogs. Following an oral twice-daily administration of an equal mix of CBG and CBGA at a dosage of 2 mg/kg for two weeks in 6 fasted and fed dogs, physical examination, comprehensive blood analyses, and serum chemistry assessments conducted throughout the two weeks indicated the absence of any adverse events during this short-term dosing trial. Indeed, notwithstanding some changes between baseline and week 2, in some cases statistically significant, no values for any parameters were outside of the normal reference range established by the Cornell Veterinary Diagnostic Laboratory Clinical Pathology Services, and physical examinations performed throughout the treatment period found no observable abnormalities regarding activity, neurological deficits, or behavior at any stage of the study. Interestingly, rises in liver enzymes were not observed in this study. ALP, in particular, decreased in both the fasted and fed states (Amstutz et al. 2022).

Cats

Compared to dogs, there is a significantly smaller number of scientific studies concerning the use of Cannabis derivatives in cats. However, some information can be found even for this animal species, with Cannabis derivatives tested in a placebo-controlled trial evaluating the effectiveness and safety of CBD for pain management in feline chronic gingivostomatitis (FCGS) (Coelho et al. 2023), and in a case report on a cat with chronic osteoarthritic pain (Gutierre et al. 2023).

In the first study, CBD was administered orally within a multimodal treatment (including butorphanol and meloxicam) in cats with FCGS undergoing partial or total dental extractions. The intervention was positively associated with improved Stomatitis Disease Activity Index (SDAI) score and a marked relief of oral pain after the surgery using the Composite Oral Pain Scale Canine/Feline (COPS-C/F) (Coelho et al. 2023).

In the case report on a cat with osteoarthritis, pain scored with the Feline Musculoskeletal Pain Index (FMPI) decreased of 8 points, from 13 on day 0 (before treatment) to 5 points on day 30 (Gutierre et al. 2023).

More details on the two cited studies are reported in Table 3).

Table 3 Summary of studies on the efficacy of Cannabis derivatives in cats
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When tolerability was assessed within a pharmacokinetic study conducted in 8 healthy cats dosed orally with a CBD-infused fish oil (50/50% mix of CBD and CBDA) at 2 mg/kg twice daily for 84 days, no changes in physical examination and few changes in the mean cell counts (slight decrease in eosinophil counts) and serum chemistry parameters (a single cat with elevated alanine aminotransferase (ALT) level during treatment, a decrease in Blood Urea Nitrogen, triglycerides and creatine kinase activity over time) were observed, suggesting the relative safety of the oral supplementation over 12 weeks. Cats commonly displayed excessive licking and head shaking with oil administration (Deabold et al. 2019).

Two years later, Kulpa et al. (2021) evaluated the safety and tolerability of up to 11 escalating doses of orally delivered cannabis oils predominant in CBD, THC, or both CBD and THC in 20 healthy cats in a placebo-controlled, blinded study. Clinical observations, complete blood counts (CBCs) and clinical chemistry were evaluated as outcomes. All cats safely reached the highest doses of 30.5 mg/kg CBD, 41.5 mg/kg THC, or 13.0:8.4 mg/kg CBD: THC. Any observed AEs were mild, temporary, and resolved without medical intervention. Constitutional symptoms like lethargy and hypothermia, as well as neurological symptoms such as ataxia and ocular symptoms like protrusion membrana nictitans, were more prevalent with oils containing THC (both CBD/THC and THC oils). There were no significant changes in hematology or clinical chemistry observed among the different treatment groups (Kulpa et al. 2021).

Recently, Coltherd et al. (2024) published the results of a randomized, blinded, and placebo-controlled study with CBD or placebo administered orally at 4 mg/kg twice daily for 26 weeks in 10 cats/group. All biochemistry and hematology data showed no clinically significant differences between supplement group but ALT, for which a statistical equivalence (at 2-fold limits) was found.

Horses

To the best of the authors’ knowledge, only three clinical studies on the efficacy of CBD in horses are available so far, with information detailed in Table 4.

Table 4 Summary of studies on the efficacy of Cannabis derivatives in horses
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Ellis and Contino (2021) published a case study concerning a 4-year-old Quarter Horse mare with a 5-week history of strong sensitivity to touch near the withers/shoulder region, probably due to an insect bite. Conventional therapeutic approaches did not improve the clinical signs. After treatment with CBD, the condition significantly improved after 36 h. An attempt to reduce the dose by half was made, resulting in a recurrence of the clinical signs after one day. The dose was, therefore, adjusted to the initial level, and gradually decreased during two months without repeated presence of increased sensitivity and the owner describing a 90% improvement (Ellis and Contino 2021).

Another case report was published by (Cunha et al. 2023), who described a 22-year-old mare suffering from chronic crib-biting and wind-sucking that started at the age of 7 years old and got progressively worse over the years, and the successful outcome of four weeks-therapy with CBD (Cunha et al. 2023).

Recently, the efficacy of CBD oil in relieving pain in 12 horses with osteoarthritis was also evaluated in a randomized study (Interlandi et al. 2024). The CBD treatment was included in a classical pharmacological regimen, with a control group treated with the traditional analgesic (phenylbutazone) only. A significant reduction in the Horse Chronic Pain Scale (HCPS) scores was seen in both groups. However, lower HCPS scores were recorded in the CBD group (Interlandi et al. 2024).

Moreover, in 2020, a patent application (US 10,624,936 B2) concerning the use of CBD in stress and anxiety disorders in horses described the calming effects of CBD in 7 horses of different ages and sexes, where a single administration of a water-based CBD formulation at doses of 50 or 100 mg/horse (depending on the size of the animal) dramatically improved the behavior associated with stress or anxiety (Denapoli and Denapoli 2020).

Besides the described clinical studies, a preliminary experimental study was also conducted (McIver et al. 2020). The study was aimed to evaluate the effect of a 1% CBD extract in 2 mL of manuka honey applied topically daily for 42 days, compared to the sole manuka honey, on second intention wound healing in distal limb wounds of 6 horses. The study failed to demonstrate any difference in wound healing variables (wound area, daily healing rate, days to complete healing) between the two treatment groups (McIver et al. 2020).

Finally, it’s worth mentioning the study by Turner et al. (2023), who evaluated the effects of a broad spectrum hemp extract (98% CBD) on immune function (by measuring inflammatory cytokines and antibody responses to vaccination) and health parameters in senior horses. Horses were vaccinated with an equine influenza vaccine and orally-dosed with CBD (2 mg/kg: 13 horses) or control (soy oil: 14 horses) daily for 90 days. A significant decrease was determined for whole blood inflammatory cytokine expression of IFN-γ at day 60, and for IL6 at day 60 and 90 in CBD-treated horses when compared to control horses. CBD did not significantly affect any other immune factors, hemagglutination inhibition titers, or health parameters (Turner et al. 2023).

In the matter of safety, CBD has been referred to as a generally well-tolerated substance in horses (Draeger et al. 2021a; St. Blanc et al. 2022). Indeed, the blinded, placebo-controlled study conducted by St. Blanc et al. (2022), where daily oral supplementation of CBD (150 mg) was given for 56 days in 10 horses, did not reveal any difference from placebo regarding blood count, biochemical panel, as well as sedation and ataxia. Similarly, in horses (n. 30) randomly treated orally with CBD (0.75 or 150 mg/kg) or placebo twice daily for 28 days, body weight, body condition score, and blood chemical parameters were not adversely affected following supplementation (Leise et al. 2023).

Mild hypocalcemia was seen in all horses (n. 12), and elevated liver enzymes were observed in 8/12 horses after administration of CBD (0.5–1.5 mg/kg orally twice daily for six weeks). However, these changes improved or normalized within ten days after the final CBD dose (Yocom et al. 2022).

More recently, Eichler and co-workers performed two randomized, blinded, placebo-controlled studies where CBD was provided as a single oral administration at escalating doses (0.2, 1 and 3 mg/kg in 3, 3 and 5 horses, respectively) (study 1), and as multiple oral administration (3 mg/kg twice daily for 15 days in 6 horses) (study 2). In the first study, behavioral parameters, as measured using the FaceSed (facial sedation scale for horses) and the Horse Grimace Scale, as well as heart rate (HR) and heart rate variability (HRV), were evaluated at 0, 1, 2, 4 and 12 h after CBD or placebo administration. In the second study, blood and saliva cortisol were also measured throughout the study period besides behavioral and heart parameters. Both the single and multiple CBD administration trials revealed no statistically significant effect in horses’ behavioral observations, HR and HRV, and cortisol levels (Eichler et al. 2023a, 2024). Draeger and co-workers previously obtained similar findings on HR after administering 100 mg of CBD or placebo once daily for six weeks. However, treated horses exhibited less reactivity after six weeks of supplementation, suggesting that CBD supplementation may lower reactivity in horses (Draeger et al. 2021b).

Cattle

Only one study is available so far where a cannabis derivative has been evaluated in the bovine species. In this study, 8 cattle were fed with industrial hemp (IH) once a day for 14 days, receiving a target daily dose of 5.5 mg/kg of CBDA, and behavior, serum cortisol, serum haptoglobin, liver enzymes, serum amyloid A, and prostaglandin E2 concentrations were evaluated compared with a control (CNTL) group. The IH group showed increased lying behavior compared to the CNTL group. Cortisol, and prostaglandin E2 concentrations were lower in the IH than the CNTL group, and no differences for haptoglobin or serum amyloid A were observed. Authors thus suggested that feeding IH with a high CBDA content for 14 days increases lying behavior and decreases biomarkers of stress and inflammation in cattle (Kleinhenz et al. 2022).

Conclusion

The discovery of the ECS and the recognition of its involvement in multiple physiological processes opened the scenario of a possible modulation of its functions using exogenous cannabinoids as therapeutic intervention.

For instance, the involvement of the dorsal root ganglia in chronic pain and the expression of CB receptors at this level, allow to hypothesize a role of modulation of their activity in the management of animal pain (Chiocchetti et al. 2019, 2021; Galiazzo et al. 2022). The different expression disease-correlated of CB1 receptors in dogs with idiopathic and structural epilepsy may be noteworthy in the development of specific therapeutic protocols for dogs (Kostic et al. 2023). Similarly, an intervention on ECS could be hypothesized to manage feline chronic gingivostomatitis or dermatitis of dogs and cats with hypersensitivity, by virtue of the overexpression of cannabinoids receptors in these subjects compared to healthy ones (Campora et al. 2012; Miragliotta et al. 2018; Polidoro et al. 2021; Chiocchetti et al. 2022a, b).

However, even if there are some evidences of a beneficial role of pCBs for adverse conditions in animals, the results obtained so far are still not always consistent because of some limitations (e.g. small sample size, concomitant administration of other drugs, subjective evaluations by owners and veterinarians as well as the large variability of the pCBs content in the used formulations, etc.) (Di Salvo et al. 2023).

It is therefore clear that further investigations need to better understand and identify how possible interventions at the ECS level can be exploited in veterinary therapy.