Animal drug research


Animal value and the propriety of using them in drug research has been a subject of public debate for several decades in many parts of the world due to lack of consensus on its ethical implications. The understanding of diseases and their treatment largely cannot be done without the use of animals because isolated enzymes cannot serve as models for what can happen in a single real cell, and no culture of cells can recapitulate what goes on in a real organism. Also, most disease pathology is not manifest or at least not recognised at the cellular level, but at tissue, organ or whole organism level. Animal research is considered a last resort to be used only when there is no alternative method. In the UK, strict regulations and a licensing system mean that animals must be looked after properly and may not be used if there is any other way of doing a piece of research (Jensen et al, 2008).The Home Office (UK) has legislated that experimentation is only permitted when there is no alternative research technique and the expected benefits of the research outweigh any possible adverse effects that might be caused to the animals used (Home Office UK, 2009). Careful selection of the type of animal to be used for research is very important as animals usually do not emulate exactly what goes on in the human body and some animals are better models for different kinds of research, so animals are only used to test if the newly researched compounds have the expected effects and are observed for any general unwanted effects or toxicity. Many candidate drugs fail this stage and thus do not proceed to the clinical phase I trials and therefore never make it to the registration and marketing stage. A lot of pressure is put on pharmaceutical companies by law governing bodies because by law, all human medicines must first be tested for safety in animals before they are tested for safety and efficacy in humans and these laws are tightly monitored by strict standards to ensure and monitor how this work is performed. The public especially the animal right activists criticise the use of animals for research.

The focus of this review will be on essential hypertension and antihypertensive agents and how animals have contributed positively to our understanding of the diseases and the development of suitable treatment in different kinds of patients. The ability to develop preventive and effective treatments for hypertension, a polygenic disease will depend on animal models that mimic the human subject metabolically and pathophysiologically and will develop lesions comparable to those in humans. The rat is the most useful, economic, and valid model for studying hypertension and exploring effective therapeutic approaches (Zadelaar et al., 2007), but more recently several candidate genes for hypertension have been used to produce several strains of transgenic mice for gain of function, gene targeted or knockout mice for loss of function (Sugiyama et al, 2001) and wild type (C57) mice (Lemmer, 2006). These genetically engineered mouse strains have provided an insight into the development of antihypertensive drugs and the mechanism of blood pressure (BP) regulation.

Hypertension can be studied using a genotype-driven approach and a phenotype driven approach (Sugiyama et al, 2001). The genetic form of hypertension and the mechanism of the diseases have been well studied and established but the genes that cause the common form of the disease are still not known, so developing specific drugs that target specific cells and tissues and lack of specificity of the agents for the targets have been a major therapeutic challenge in the pharmaceutical industries and health-care systems. Hypertension and the methods of treatment are still a widely researched area and a major area of interest by pharmaceutical companies because it affects almost one billion people worldwide and its prevalence is expected to increase from 26.4% in 2000 to 29.2% in 2025 and this prevalence increases with age (Kearney et al., 2005). It has been shown that for every 20mmHg increase in systolic blood pressure or for every 10mmHg increase in diastolic blood pressure, there's a doubling in the risk of other types of cardiovascular diseases (20), this is why there is so much interest in this area of research and animal experimentation is therefore inevitable. These hypertensive models are enriched with blood pressure raising alleles of genes and are employed to localise the quantitative trait locus (QTL) for blood pressure (Deng, 1998). Animal models with genetic manipulation have thus helped generate a wealth of valuable information and opened a new era for hypertension and antihypertensive research.


Animal experimentation is a very large and important aspect of the discovery and development process as over 100 million vertebrates are used worldwide for drug testing and about 10-11 million in the European Union (Home Office UK, 2009). Drug research is a very long, complex, costly and highly risky process and involves several stages starting from identification of a medical need to administration of the new drug to the patients ( 1). This process can take between 12 – 24 years for a single medicine. From past researches, only approximately 1 out of 15–25 drug candidates survives the detailed safety and efficacy testing (in animals and humans) required for it to become a marketed product and used in the treatment of diseases(Lombardino & Lowe, 2004).

Generally, the creation of a new drug begins with the testing of several selected chemical compounds in applicable biological tests. Major subsequent steps then follow and these include detecting relevant biological activity, a 'hit' for a structurally novel compound in vitro, then finding a related compound with in vivo activity in an appropriate animal model, followed by maximizing this activity through the preparation of analogous structures, and finally selecting one compound as the drug development candidate (Lombardino & Lowe, 2004). In vivo studies which involve the use of animals determine the effects of test compounds in integrated systems, selectivity in the whole body, long term effects, pharmacokinetics, assess safety, toxicology and set the clinical range. Non-animal procedures are used for the majority of biomedical research, so animal studies are used alongside these other types of research. Such procedures include the study of cells and tissues grown in the laboratory, computer-modelled systems, and human patients, volunteers or populations (Jensen et al, 2008).


Animals are used in the late stages of drug research process to test potential drug candidates for affinity, potency and selectivity at the targets sites, measuring metabolism, suggesting toxicity and measuring systemic effects of the drugs. The choice of animal model for drug discovery process depends on therapeutic effect required by the research process (Rang, 2006) because naturally occurring disease produce various biochemical abnormalities which are characterised differently in humans and in animal models.

Animals used in meaningful experimental procedures must be held long enough in approved facilities to insure that they are not incubating infectious diseases. They should be verified free of both internal and external parasites. The physical examination of each animal should include the rectal temperature and an evaluation of the mucous membranes. An electrocardiogram (ECG) should be carried out to rule out congenital or acquired arrhythmias that might render the animal unfit for protocols involving the cardiovascular system (Gross, 2009). Animals must be well hydrated especially those that are to undergo long surgical procedures except in cases where hydration would cause harm.

The Animals (Scientific Procedures) Act 1986 put in place an unrelenting system of controls on scientific work on living animals, including the need for a project, a personal licence and certification for the agency carrying out the research; stringent safeguards on animal pain and suffering; and general requirements to ensure the care and welfare of animals (Home Office UK, 2008).The introduction of the “three Rs” also acts as a guide for the use of animals in most countries:

Reduction refers to methods that enable researchers to obtain comparable levels of information from fewer animals, or to obtain more information from the same number of animals.

Replacement refers to the preferred use of non-animal methods over animal methods whenever it is possible to achieve the same scientific aim and valuable data.

Refinement refers to methods that alleviate or minimize potential pain, suffering or physiological distress and enhance welfare for the animals used. This involves using pain relievers and analgesics where significant pain is likely to occur.

Rodents especially rats and mice are the closest accessible mammalian transgenic animal models for the genetic analysis of human diseases for the following reasons; 1). their relatively rapid regeneration time 2). their genomic sequence is closely related to the human genome 3). they have very similar gene order and relative positioning of large chromosomal regions 4). they show defined symptoms closely related to that of human and hence are widely used for drug testing. However, the problem with using this model is the difficulty in determining the genes that control specific diseases compared with the human disease. Other organisms less closely related to human but are used for gene manipulation and gene-phenotypic relation of diseases, include the fruit fly, Drosophila melanogaster, the flatworm, Caenorhabditis elegans and Saccharomyces cerevisiae, a yeast. These are more often used than vertebrates but are not strictly regulated by law. They enable the generation of potential animal models for drug testing and mechanical studies at the cellular level. The use of these organisms has an advantage over higher organisms because their genes are easily manipulated (Rang, 2006). Animals are selected on the basis of their size, regeneration time, understanding of their genetic and genomic sequence and how suitable they are for the disease models to be researched.


Hypertension is characterised by an increase in systolic blood pressure i.e more than 140mm Hg and diastolic blood pressure greater than 90mm Hg. It is caused by an increase in calcium levels in smooth muscle cells of the blood vessels, lymphocytes, platelets and other cells (Jennings and Zanstra, 2009). In hypertension, there are major changes in structure of the blood vessels, increased vascular resistance leading to vascular remodelling. Vascular resistance and rarefaction (changes to structure of small blood vessels) contribute to end organ damage. For example, two-kidney, one-clip Goldblatt hypertensive rats develop increased aortic impedance, decreased arterial compliance and thickening of these vessels owing to smooth muscle cell hypertrophy (Rosendorff, 1996).

Blood pressure is regulated by many pathways, the most important are the renin-angiotensin system, the sympathetic nervous-catecholamine system, the natriuretic peptide system; the nitric oxide system; and vasopressin, endothelin, prostaglandin, and kallikrein-kinin (Sugiyama et al, 2001). Clinically and experimentally it is well established that hypertension leads to the development of atherosclerosis (Zhou et al, 2008), particularly when associated with diabetes, smoking or dyslipidemia and targets the endothelium, where the upregulation of molecules such as monocyte chemoattractant protein (MCP)-1 and lectin-like oxidised low density lipoprotein receptor (LOX)-1, associated with an increase in reactive oxygen species (ROS) and a decrease in nitric oxide (NO) bioactivity play a critical role in linking hypertension and atherogenesis (Schulman et al, 2006). It contributes to a wide range of cardiovascular events including stroke, myocardial infarction, heart failure, renal failure and sudden death (Chobian et al, 2003) which are as a consequence of progressive structural and functional changes in the vasculature.

The variability of high blood pressure in human can be affected by environmental or genetic factors or both. There are two main types of hypertension; the primary or essential hypertension and the secondary hypertension. Other rare types include; malignant hypertension, isolated systolic hypertension, white coat hypertension and resistant hypertension.

Essential hypertension is a multi-factorial and complex disease involving several genes that have, thus far, defied complete characterization. This type of hypertension which affects about 95% of hypertensive patients (Sugiyama et al, 2001) is characterised by an elevated blood pressure for which a single underlying cause is not known. This review will focus on treatment of this type of hypertension as this is the major area of concentration of the development of antihypertensive agent. Studies in the SHR have suggested that the rennin-angiotensin-aldosterone system might play an important role in the pathogenesis of this disease. These studies include determining whether the inheritance of a restriction fragment length polymorphism (RFLP) marking the renin gene of the SHR was associated with greater blood pressure than the inheritance of a RFLP marking the renin gene of a normotensive control rat, the Lewis rat (Churchill et al, 1997). This disease is not inherited in a classical Mendelian fashion but the genes responsible are usually mapped by QTL analysis and identified by positional cloning because rodent blood pressure QTL often correspond with regions of the human genome containing genes affecting blood pressure (Sugiyama et al, 2001. Feng et al, 2009). This QTL is defined as a locus genetically determining a difference in blood pressure between two contrasting rat strains usually a hypertensive and a normotensive strain (Deng, 1998).

Animal models of this disease suggest a change in vasculature which increases cardiac output and overperfusion of tissues followed by vasoconstructive influences and remodelling of the vasculature, increasing the area of the vascular wall (Jennings & Zanstra, 2009). Pathophysiologic factors of this type of hypertension include increased sympathetic nervous system activity, overproduction of sodium-retaining hormones and vasoconstrictors, inappropriate renin secretion, deficiencies of vasodilators, such as prostacyclin, nitric oxide (NO), and the natriuretic peptides, alterations in expression of the kallikrein–kinin system and renal salt handling, diabetes mellitus, obesity, increased activity of vascular growth factors, alterations in adrenergic receptors, ionotropic properties of the heart and altered cellular ion transport (Oparil, Zaman, & Calhoun, 2003). The afflicted person risks disability and death from myocardial infarction and stroke (Lake-Bruise and Sigmund, 2000).

The other forms of hypertension arise as a consequence of external factors or diseases and are often curable once these factors are removed.


Individuals risk of developing high BP differ, but the general risk factors apart from the genetic causes include age, ethnicity, gender, family history, smoking, activity level, diet, medical problems (including kidney diseases, diabetes, sleep apnea) and stress


Two two major systems affected by hypertension are the brain and kidneys. Studies in transgenic rat models and now supplemented by behavioural and brain imaging studies in humans have improved the understanding of the brain and its initiation of high BP. The brain is essential in hypertension. It initiates the disease at the early stage and is functionally impaired during the maintenance of the disease failing to regulate blood pressure and characterising functional deficit in information processing (Jennings and Zanstra, 2009). It also has a target for the late phase of the disease due to a high risk factor of stroke seen in hypertensive patients. Untreated hypertension progress to levels that delay integrity of cerebral vessels in the late phase.

Blood pressure is modulated by functional circuitry linking the hypothalamus nucleus tractus solitarius and the rostral ventrolateral medulla (Guyenet, 2006). Hypertensive patients often show reduced neural responses to cognitive tasks, altered neuropsychological performance, and altered brain networks in response to psychological challenge (Jennings and Zanstra, 2009). Several studies are still being carried out to prove the association of hypertension with increased aging of the brain and this was seen in the reduction of the brain grey matter, white matter hyperintensities as well as increased sulcal and ventral size due to brain atrophy (Raz, 2005)

The kidneys play a primary role in the pathogenesis and maintenance of increased blood pressure. They have an effect on blood pressure by regulating blood volume via salt and water balance. This regulation is tightly controlled by a neuroendocrine regulatory system, renin-angiotensin (RAS) system, the most active component is a vasoconstrictor called the angiotensin II. The activation of this vasoconstrictor promotes pro inflammatory factors leading to vascular injury and vascular remodelling (Guyton, 1991). The hypothalamus and sympathetic innervation control the kidney function and the effects of angiotensin in the brain via sympathetic efferents to regulate blood volume. Hypertension also hastens the ageing of the circulatory system (Lakatta, 1989, 1990).


There are two (2) approaches to hypertension management:

1. Non-pharmacological approach: behavioural and lifestyle modifications have been seen as an alternative for the management of blood pressure. These behavioural changes include: reduction in alcohol intake, reduction in cigarette smoking, relaxation, physical activity, obesity control, reduction in dietary sodium (Tudor, 1993), consumption of less saturated fat.

2. Pharmacological approach: various classes of antihypertensive drugs are prescribed according to the patients profile; race, age, family history and prevalence of the disease and presence of other diseases for example, patients with diabetes and hypertension are first treated with ACE inhibitors and then a diuretic if further blood pressure reduction is required (Nash, 2007).

Hypertensive patients respond differently to the different classes of antihypertensive drugs and this is as a result of the nature of the disease especially in patients with essential hypertension, the heterogeneity causes variable pathogenesis leading to detectable variability in individual responses to different agents. The main classes of antihypertensive drugs are: ACE inhibitor and AT1 receptor blockers, beta-adrenoceptor blockers, diuretics, angiotensin receptor antagonists and calcium-channel blockers which differ in the nature of their therapeutic action and reduction in blood pressure(Dickerson, et al., 1999). Treatment using these drugs can usually be divided into first line, second line, third line and last resorts (Tudor, 1993). But this order of treatment and the drugs used differs in different patients according to individual's profile. Antihypertensive agents are suitable for the initiation and maintenance of treatment either as monotherapy or in some combinations. (Giuseppe et al.). The main classes of treatment drugs are analysed below.

Calcium Channel Blockers: The effects of calcium antagonists are mediated by blocking the calcium current by acting on the L-type (Cav1) calcium channel in plasma membrane and therefore reducing calcium current, causing vasodilation even at low concentrations (Lemmer, 2006) while the action of other antihypertensive agents are associated with the modification of membrane receptor function, stimulation of the formation of the cyclic 3', 5'-adenosine monophosphate (cAMP) (Menshikov et al., 1988). Examples are verapamil, nifedipine, nicardipine, nimodipine, diltiazem. These drugs except verapamil are used in combination with other antihypertensive drugs such as beta-blockers.

Thiazide Diuretics: diuretics are often used in combination with other antihypertensive agents are used in most patients as first line antihypertensives to achieve the goal of therapy. These include hydrochlorothiazide (HCTZ), sulfonamide derivatives chlorthalidone, bendroflumethiazide, metolazone, indapamine. HCTZ has never been shown to reduce cardiovascular morbidity or mortality, although it increases antihypertensive efficacy of whatever drug with which it is combined (Kaplan, 2009). On the other hand chlorthalidone has been shown repeatedly to reduce cardiovascular mortality and morbidity in random trials. Potent diuretics even in low doses often result in lower serum potassium, enough to cause cardiac arrest (Kaplan, 2009), a combination of this class of drugs with other antihypertensive agents such as aldosterone blocker spironolactone and eplerenone can reduce side effects and provide maximal antihypertensive efficacy.

Thiazide diuretics inhibit reabsorption by sodium-chloride symporter ( 3) located in the distal convoluted tubules of the kidney. This transport system moves both sodium and chloride into the cell using the free energy produced by the sodium, potassium ATPase. Sodium is pumped out of the epithelial cell via this transport system in the basolateral membrane while Cl- exits the cell via a chloride channel. This increase in sodium delivery increases potassium loss (potentially causing hypokalemia) and stimulates the aldosterone-sensitive sodium pump to increase sodium reabsorption in exchange for potassium and hydrogen ion, which are lost to the urine. The increased hydrogen ion loss can lead to metabolic alkalosis. Part of the loss of potassium and hydrogen ion by these diuretic results from activation of the renin-angiotensin-aldosterone system that occurs as a result of reduced blood volume and arterial pressure. Increased aldosterone stimulates sodium excretion (natriuresis) and increases potassium and hydrogen ion excretion into the urine and this has a direct effect on arteriolar vasodilation, volume depletion and reduction of peripheral vascular resistance (Nash, 2007), therefore lowering blood pressure. The efficacy of these drugs is derived from their ability to reduce blood volume, cardiac output, and with long-term therapy, systemic vascular resistance

Ace-Inhibitors And AT1 Receptor Blockers. Hypertension can occur at elevated or at normal plasma angiotensin (Ang) II levels. Ang II, which is generated from angiotensin I by the angiotensin-converting enzyme (ACE), is the principal effector of the renin-angiotensin system (RAS) (Zadelaar, et al., 2007) binds to Ang II subtype 1 receptors (AT1 receptors) ( 4), narrows blood vessel and increases BP (Jensen et al, 2008).

ACE inhibitors are able to reduce blood pressure in patients and in animal models such as the normotensive controls, STNx model or chronic renal hypertensive rats, including the 2-kidney, 1-clip (2K–1C) and the 1-kidney, 1-clip (1K–1C) hypertensive rat (Miyazaki et al, 2006. Rosendorff, 1996). But this is still a source of debate as there is conflicting evidence regarding whether vascular ACE is increased in experimental hypertension when compared with human hypertension. Examples of ACE inhibitors include captopril, enalapril, fosinopril, perinopril, ramipril, temocapril, quinapril, zofenopril. AT1 receptors blockers include candesartan, irbesartan, losartan, olmesartan, telmisartan and valsartan.

Ang II acts through 2 receptor subtypes, the AT1 and AT2 receptors. The long-term effects of angiotensin II are mediated by the complex signaling pathways involving G-proteins, receptor and non-receptor kinases, MAPKs, and others and these have been studied in TGR models. Classical effects mediated by the AT1 receptor include vasoconstriction, aldosterone, and vasopressin release, sodium and water retention, and sympathetic facilitation with increased release of catecholamines and inhibition of the neuronal uptake of norepinephrine (Lemmer, 2006).

These inhibitors not only reduce blood pressure, they also increase plasma renin activity.

Beta-Blockers: β-Adrenoceptor antagonists can be divided into 4 main groups: non-selective (e.g. propranolol, oxprenolol), β1-selective (e.g. bisoprolol, metoprolol, atenolol), compounds with intrinsic sympathomimetic/agonist activity (ISA, e.g. pindolol, carteolol) and β-adrenoceptor antagonists with additional activities (e.g. α-adrenoceptor blockade [carvedilol] or NO release [nebivolol]) (Lemmer, 2006). These antagonists bind to the contracting myocytes and prevent normal ligand (epinephrine or norepinephrine) activity at the β-adrenoreceptor site and therefore decrease heart rate and decrease BP. Several studies in normotensive and hypertensive rats and mice have shown beta-blockers to reduce ischaemic events from occurring in hypertensive patients.

Combination Therapy In Hypertension Management: Studies in normotensive and hypertensive rats have shown that hypertensive patients often require 2 or more antihypertensive drugs to reach the desired goal of a diastolic blood pressure of less than 90mmHg and a systolic blood pressure of less than 140mmHg because the pathophysiology of hypertension is multifactorial. Well formulated drug combination reduces blood pressure via complementary mechanism that increases the efficacy of the drug and reduces the adverse effects compared with high-dose single antihypertensive drug administration. These combinations include angiotensin-converting enzyme (ACE) inhibitors with calcium channel blocker (CCB) or diuretic; beta-blockers with diuretic; or angiotensin receptor blocker (ARB) with diuretic. High risk and uncomplicated hypertension are most efficiently treated with low-dose thiazide diuretic with an ARB ( 5) because ARB shows more blood pressure lowering efficacy than ACE-inhibitors and other classes of antihypertensive agents (Nash, 2007). ARBs increase risk of other cardiovascular events in high risk patient including those with heart failure, myocardial infarction and left ventricular dysfunction (Pfeffer et al, 2003).

Diuretics are useful for achieving blood pressure control in combination therapy but other classes of antihypertensives such as the renin-angiotensin system (RAS) blockers are important for treating high risk patients for example diabetic patients (McMurray at al, 2003)..


Animal models of hypertension are the most used in cardiovascular research and this has led to effective treatment of the disease in human. These models include the congenic models such as the spontaneously hypertensive rats (SHR) and the Wistar-Kyoto (WKY), the model with subtotal nephrectomy (STNx), normotensive control rats (Sprague-Dawley [SPD], [SPD] Dahl salt-sensitive (DSS) rat, DOCA (deoxycorticosterone acetate) -salt, two-kidney one-clip dog model, and the transgenic TGR(mRen2)27 (TGR) model (Gross, 2009) for human secondary hypertension (Lemmer, 2006). The condenic models are a well-known experimental model to study primary hypertension (Amaya & Marta, 2009), they are genetically identical and thus aid the search for genes that cause this disease. However, this model reflects a rare subtype of human hypertension, primary hypertension inherited in a Mendelian fashion, they show hypertriacylglycerolaemia, abdominal obesity and hypertension (Reaven et al, 1989) but nevertheless are similar in alterations in the rennin-angiotensin-aldosterone characterised in patients with the heritable form of essential hypertension. Stroke-prone SHR (SHRSP) is a rat model that develops severe hypertension. SHRSP rats develop hypertension-related disorders, such as nephropathy, cardiac hypertrophy and atherosclerosis, similar to human essential hypertension and 100% of them die to stroke(Yamori et al, 1978). The transgenic rats provide a powerful tool to study the influence of genes on the pathogenesis of hypertension. The TGR model bears the murine Ren-2 gene cloned from the DAB[2] mouse strain and provides a monogenic model of hypertension in which the genetic basis is known (Langheinrich et al, 1996).

Responsiveness to antihypertensive drugs varies in different rat models, for example all rat models exhibited cardiac hypertrophy and impaired endothelium-dependent relaxation but severe forms such as heart failure, stroke and kidney failure were model dependent.


The use of animals is considered an unavoidable requirement but despite their relevance in the drug research industry, animal experiments have several limitations; they are usually expensive and are subject to ethical and legal constraints. They are therefore kept to a bare minimum and experimental variability is generally often a problem (Rang, 2006). Therefore using animals for experiments must be carefully planned such that information would be derived in an efficient manner and such information will reliable and effective for the stated purpose. Benefits to man must outweigh the suffering of the animals and the 3R's (Reduction, replacement and Refinement) must be applied to all experimental and research procedures using animals.

Essential hypertension, a polygenic disease remains a widely researched area, hence the need of animal models for research, particularly ones with hereditary transmitted hypertension to study the genetics of the disease. Analysis of candidate genes for hypertension in SHRs may give insights into the causes of high BP or into the hypertensive process in subsets of humans with primary hypertension. Molecular design of new animal models will answer more questions and open new possibilities for studying the mechanisms involved in hypertension. Series of research and clinical trials have proven combination therapy to be the most effective treatment for essential hypertension as individual antihypertensive agents have not been so effective

It is generally accepted that multiple endogenous systems contribute to the regulation of BP, however, the regulation of the rhythmicity of BP and its disease-induced disturbances are not fully understood. Furthermore, there is some debate on whether and to what degree human rhythms in BP and HR are endogenous in nature. Therefore, animal models of primary and secondary hypertension can contribute to a better understanding of the mechanisms involved. Different strains of normotensive, hypertensives and transgenic rats as well as wildtype and knock-out mice are used to address this issue (Lemmer, 2006). The aim of this review was to emphasise the importance of animals in drug research and to increase the understanding of hypertension as a disease and how they have helped in the development of suitable treatment.

This project will be based on the development of a CAL package for second year RSM students of the Faculty of Life Sciences, University of Manchester and will focus on the treatment of hypertension using agonists and antagonists at different receptor sites in animal models such as dogs and cat. Experimental data will be used to plot graphs using the Cardiolab package.


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