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The use of in-vitro liver models to predict the in-vivo pharmacokinetics of paracetamol in the horse

Abstract

Introduction

Pain management in Horses

Pain management in horses has been widely discussed within the equine veterinary industry (reference). There is now particular focus on the management of pain for the wellbeing and welfare in horses (Mama and Hector, 2019). The improved attitude to pain management and use of analgesic medication is thought to have changed due to the enhancement of pain assessment as well as our shifting thought process that animals feel pain (Flecknell, 2008). The use of analgesia perioperatively, post trauma and for treatment of musculoskeletal pain is commonly used by veterinarians in both small animal, equine practice and large animal practice. However, the challenges of pain management include potential side effects of medication, cost and efficacy (Sanchez, 2014). Vets must also take into consideration the use of pain management drugs in performance horses where there are many restrictions of usage of various medications, including those used in pain management (Mama and Hector, 2019). Currently Non-steroidal anti-inflammatory drugs (NSAIDs) are the most frequently used for the management of pain, inflammation and pyrexia in both small animals and horses (surgeons, 2019) (Matthews, 2007). While NSAID’s provide adequate analgesia, they are known to have many adverse side effects (Knych, 2017)

Paracetamol

Paracetamol (Acetaminophen) is a small and moderately lipid soluble weak organic acid (Neirinckx et al., 2010). It has long been used in human medicine in the treatment of pain and fever. Like NSAIDs Paracetamol is used for analgesia, however, studies into the pharmacodynamic activity of paracetamol have shown that unlike NSAIDs, paracetamol has little anti-inflammatory effects (Ghanem et al., 2016). This is likely to be because paracetamol does not inhibit cyclooxygenase (cox) enzymes within peripheral tissues and tends to target more centrally (Wishart et al., 2008)

The exact mechanism of action is still not fully understood but it is believed that paracetamol has some inhibition of cox 3 enzymes and serotonergic (5-HT₃) receptors (Hanson and Maddison, 2008), there is also some inhibition of cox 1 and 2 enzymes although to a lesser degree than NSAIDs (Anderson, 2008). It’s analgesic actions may also involve 5-HT₃, opioid, nitric oxide and cannabinoid pathways (Sharma and Mehta, 2013), and studies have also shown the antipyretic actions are likely attributed to a direct action on the heat regulating centres within the hypothalamus (Wishart et al., 2008). It has been suggested that a combination of these pathways is involved (Sharma and Mehta, 2013).

Administration of paracetamol is commonly given orally but can be given via intravenous injection (BSAVA, 2011). Absorption occurs primarily within the small intestine by passive diffusion if given orally (Raffa et al., 2014), bioavailability varies across species (Neirinckx et al., 2010), and metabolism occurs within the liver by first order kinetics (Wishart et al., 2008). The major metabolites formed via phase II metabolic pathways are pharmacologically inactive sulphate and glucuronide conjugates (Yoon et al., 2016; Mazaleuskaya et al., 2015). A small fraction of paracetamol is converted via phase I oxidation to the reactive metabolite N-acetyl-p-benzoquinone (NAPQI) which is then inactivated via the binding of glutathione (Hinson et al., 2010). In the healthy animal the majority of these metabolites are renally excreted (Forrest et al., 1982). Toxicity occurs following overdose. There is formation of NAPQI in excessive quantities due to saturation of other pathways which depletes cellular glutathione (Yoon et al., 2016). This leads to damage of cellular protein and subsequent liver necrosis (Ramachandran and Jaeschke, 2017) add in structure and metabolic pathway picture (there is one on google that is a picture of the liver.. add as a figure and say underneath what it is etc

Paracetamol use in pain management of horses

Paracetamol is currently being used in horses as either part of multimodal analgesia (Matthews, 2007), or as an alternative to NSAIDs which have been shown to have many detrimental side effects such as gastrointestinal ulceration and renal damage (Mercer, 2018). Studies have shown increased paracetamol efficacy in the treatment of a laminitic pony (West et al., 2011), and in combination with an NSAID during a study into horses with induced foot pain (Foreman JH, 2015). Paracetamol has been used as a marker for gastric emptying in multiple species. A study of gastric emptying in ponies proved useful to show the rate in which the stomach empties as paracetamol passes through the stomach unchanged and is absorbed passively from the small intestine (Doherty et al., 1998). As well as being absorbed in a similar way to other species, paracetamol also tends to follow the same pathways of metabolism as humans, dogs and pigs (Ian wilson reference, paracetamol). Previous studies in horses have shown paracetamol is rapidly absorbed and has a high bioavailability (91%) compared to the dog (45%) (Neirinckx et al., 2010). It was shown  that horses displayed a lower hepatic intrinsic clearance capability (Neirinckx et al., 2010) and a longer half-life (Mercer et al., 2019) than other species, this was thought to be due to higher plasma protein binding and therefore less exposure of drug to hepatic enzymes (Neirinckx et al., 2010). Put all Neirinckx reference info together, then put mercer info on half life at the end… to support what Neirinckx has stated Safety margins have been confirmed in humans, dogs and cats however, little research has been carried out into  the toxic doses  in horses. A study investigating paracetamol toxicity in horses showed a dose 25 % higher than the recommended 20 mg/kg dose did not cause hepatic, renal or red blood cell injury (Foreman, 2018). Currently paracetamol use in horses is off label as there is yet to be a specific equine formulation of the drug.

Control of drug administration in horse racing

Drug doping and the inappropriate use of medications are a threat to the integrity and reputation of the racing industry and may give unfair advantage to competitors and threaten the welfare of the horse (British_Horseracing_Authority, 2019). Anti-doping policies are characterised by a set of values which ensure competitions provide a level playing field for all individuals taking part (Cunningham et al., 2010). Most racing authorities operate under medication rules set by the International Federation of Horseracing Authorities (IFHA). The European Horserace Scientific Liaison Committee (EHSLC) and the Fédération Equestre Internationale (FEI) have gone further and established policies that aim to distinguish between illicit substances where the objective is to detect any trace of drug exposure (parent or metabolite) and therapeutic substances which may still be detected long after administration. Highly sensitive analytical mass spectroscopy and chromatography techniques are used (Wong et al., 2015), along with Pharmacokinetic/pharmacodynamic principles which involve the estimation of both irrelevant plasma and urine concentrations (Cunningham et al., 2010). Irrelevant plasma and urine concentrations are required to establish harmonised screening limits (HSL’s). HSL’s are determined by the EHSLC and are used to formulate a detection time (DT). A DT is the time in which the urinary or plasma concentration of a drug in all horses involved in a clinical trial are below that of the HSL. DT’s provide information to veterinarians and enables them to determine a suitable withdrawal time (WT) for medications being used. The addition to the rules set out by the IFHA allow for horses to be properly treated by veterinarians and ensures the horses welfare is upheld (Cunningham et al., 2010).

Paracetamol uses in horseracing

According to the Association of Racing Commissioners International (ARCI), drugs are classified based on their pharmacological effects, drug use patterns and the appreciation of the drug use. Paracetamol comes under class four of this classification. Drugs within this class four category are mild analgesics or muscle relaxants without prominent central nervous system effects and are used primarily as therapeutic medication which may have an impact on performance. Currently there is no literature on withdrawal times or serum levels of paracetamol for racehorses

I am not sure this is true but have done extensive online research and cannot find anything on any of the official racing websites

Hepatic drug metabolism

The liver is predominantly by hepatocytes (Kmiec, 2001). Drugs and other chemicals are usually metabolised by enzymes situated within the hepatocytes (Schaffner, 1975). The process involves the conversion of substances which are lipophilic (non-polar) into substances which are hydrophilic (polar) and therefore more easily excreted from the body (Mittal et al., 2015). For many drugs, metabolism occurs within the smooth endoplasmic reticulum of liver hepatocytes (Gibson, 2001), and happens in two phases. A phase one reaction is the addition of a functional group which increases the polarity of the compound. The most important enzyme group of phase one reactions are the cytochrome P450s (CYP450) (Susa, June, 2019). Phase one processes occur via oxidation, reduction or hydrolysis (Royal_Society_of_Chemistry, 2000-2009). Phase two reactions involve the conjugation of the compound with an endogenous substance, the most common reactions are glucuronidation, methylation, conjugation with amino acids such as glutamine or glycine or sulfoconjugation (Gibson, 2001). This further increases polarity and allows for excretion of the compound from the body.

In-vivo and In-vitro pharmacokinetics (general principles)

In-vivo research provides the most accurate model to assess hepatic drug metabolism as live animals provide precise representation of drug clearance pathways and enzymatic systems used (Tingle and Helsby, 2006). Clearance , clinically measured through hepatic, renal, biliary parameters, clearance required for dosing, key parameter for steady state concentration. SS concentration equation However, in-vivo studies come with vast costs and ethical considerations (Moran et al., 2016, Scarth et al., 2010). In-vitro techniques have been developed to provide specific and convenient methods in predicting the in-vivo drug clearance within an individual, thus reducing the number of animals used within in-vivo studies (Shibany et al., 2016). While in-vitro methods are common in some species including humans and rats (Lavé et al., 1999) (Agius, 1987), in-vitro methods for the predictions of drug clearance in horses is limited. The use of hepatocytes, microsomes, precision cut liver slices, perfused liver models and recombinant enzymes are techniques used for human in-vitro studies (Groneberg et al., 2002). Equine hepatocytes have been successfully obtained and have been used in pharmacological studies. Freshly isolated hepatocytes and more recently cryopreserved hepatocytes have shown to possess similar enzymatic capabilities and therefore equally useful in drug screening studies (Shibany et al., 2016). Although fresh hepatocytes are superior in accurately predicting in-vivo metabolism, their viability gradually decreases over six hours (Blanchard et al., 2004). Therefore, cryopreserved hepatocytes may be a superior option for long term hepatocyte storage and continued availability (Shibany et al., 2016). The use of human liver microsomes have provided a convenient way to study cytochrome-mediated drug metabolism (Jia and Liu, 2007). Advantages of microsomal use are cost and ease of use. However, some phase two enzymes are not found within microsomes and the addition of specific co-factors are required for drug metabolism (Brandon et al., 2003). For this reason, microsomes do not represent the complete internal physiological environment of the animal and are inferior to the use of hepatocytes (Jia and Liu, 2007).

In-vitro In-vivo  extrapolation (IVIVE)

IVIVE describes the relationship of hepatic drug clearance between in-vivo and in-vitro models (Gibson, 2001). Experiments using an in-vitro model rey on the determination of the intrinsic hepatic clearance (Cl_int). Cl_int is measured as the livers ability to remove a drug in the absence of hepatic blood flow and protein binding (Birkett, 2010). Cl_int is calculated using V_max (the maximum reaction rate) and K_m (concentration of substrate at half the V_max value) (Berg et al., 2002). Use of scaling factors (SFs) and the weight of the liver are then necessary in order to calculate the entire liver Cl_int. In-vitro data generated by microsomes requires knowledge of microsomal protein per gram of liver (MPPGL) whereas with hepatocytes, the hepatocellularity per gram of liver (HPGL) is required (Wilson et al., 2003). Standard SF’s using methods involving DNA, CYP and protein content are used to calculate the whole liver Cl_int (Carlile et al., 1997). There is variability of parameters however, allows to predict range of values within a population (reference- intrinsic clearance variability)

Hypothesis

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