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Primate testing in Europe

Critique of studies observed at HLS

Studies observed at HLS included an HIV vaccine; a diabetes drug; an incontinence drug; an anti-inflammatory (painkiller); and a blood clotting drug. The nature of the procedures and the suffering of the monkeys has been described earlier. Here, we examine the flaws in the scientific rationale behind these tests.

Study of vaccine for HIV positive individuals

72 monkeys were used to investigate the toxicity and bio-distribution of a therapeutic (not preventative) vaccine based on a chimpanzee virus vector [carrier][172]. Although “…. most AIDS vaccine researchers are highly sceptical that it will be feasible to manipulate the immune response in infected individuals to significantly improve their health status”[173].

The rationale for testing in primates at HLS was that the vaccine involves adenoviral vectors, and in the past adenoviruses have triggered Disseminated Intravascular Coagulation (DIC) in humans[174]. However, using animal data to determine the safety profiles of Adenoviral (Ad) vectors has proved ineffectual and dangerous. An NIH report on the safety trials of an Ad vector, which resulted in the death of one participant, stated “Critically, researchers need to examine more thoroughly the validity of animal model systems used for determining pharmacodynamic and toxicity profiles of vectors”[174].

The legacy of animal research in the field of HIV vaccines with rhesus macaques is one of failure, with researchers noting that despite tests with simian and simian-human immunodeficiency virus (SIV/SHIV) infected primates, only the outcome of long-term human efficacy trials will demonstrate the effects of a prophylactic vaccine on HIV-1 infection[175]. Animal models failed in the Merck STEP Ad5 vaccine trial, with the conclusion “…vaccine protection studies that used challenge with a chimeric simian-human immunodeficiency virus (SHIV89.6P) in macaques did not predict the human trial results…. [Ad5] did cause a substantial reduction in viral load and a preservation of CD4T cell counts after infection, findings that were not reproduced in the human trials”[176].

The monkeys in the HLS study were not infected with a virus, but were simply being used to investigate toxicity and bio-distribution of the compound – something that could clearly have been assessed using a raft of alternative human based methods (see replacements, p.30) and researching the already available data.

Study of diabetes drug

Four monkeys were used to investigate the pharmacokinetics (activity, speed, metabolization) of this peptide with possible anti-diabetic properties. Over a period of three months, each monkey was taken from its home cage and bled 84 times. At the end of the study they were simply killed and disposed of without necropsies being performed[145].

The effects and actions of the hormones being studied vary between species “Receptors for glucagon and physiologic incretins (e.g.,GIP) are present on adipocytes in animals, but it is not clear if they are also present on human adipocytes...”[177]. Others have found a clear species difference in the activity of the drug in humans and non-human primates “In the baboon, blockade of the effect of GLP-1 could only be achieved at…., five times the concentration that we have found to effectively block GLP-1 in humans”[178].

It is also significant that the drug being tested was just another version of existing diabetes therapies, which have been shown to be successful in human use[179]. Adding to a saturated market with a variant on existing therapies underscores the lack of any scientific justification for tests on primates and highlights the failure to properly assess the product using existing data. This would seem a clear candidate for examination by advanced non-animal techniques rather than yet more animal tests. Interestingly, even in fundamental research elsewhere, human pancreas cells are being used to examine the role of GLP-1 – highlighting that there are no shortage of alternatives in this field[180].

Study of incontinence drug

49 monkeys were force-fed (by oral gavage) daily for a year in order to assess the toxicity of an incontinence drug[146]. We have discussed earlier, the misleading results from monkey tests, and this incontinence study provides some stark examples:

In 2002 researchers ascertained which neuro-transmitters and receptors in the body are activated to effect bladder relaxation and thus urine storage. They concluded the “monkey is the only instance of detrusor relaxation being mediated by the β3- adrenoceptor alone”, although they admit there may be a tiny contribution from another subtype of adrenoceptor[181]. A paper discussing the human bladder reports “stimulation of both β2- and β3- adrenoceptors can relax the bladder”[182]. So in the monkey, one type of adrenoceptor is almost solely responsible, in humans two types of receptors are responsible.

Whilst the purpose of the HLS test was to establish toxicity rather than the action of the drug, when there is such a difference between the species, the model must be questioned.

This supports the need for closer scrutiny of commercial regulatory testing.

A 2004 review of the incontinence drug market found “there are at least 13 other incontinence drugs currently in phase II development or higher in the US, Europe and Japan. Even if only a handful of these agents are eventually approved, the market is set to become extremely competitive”[183].

This appears to be another example of a drug candidate simply progressing through standard tests on animals without that process being informed by existing data.
The use of QSARs (Quantitive Structure Activity Relationships) for existing data, for example, and other techniques outlined later, would have provided an approach more relevant to humans.

Humab anti-inflammatory (painkiller) study

36 monkeys were used to investigate the toxicity/action of the test substance “fully human monoclonal antibodies” known as BTT” which has an anti-inflammatory action which works by a reducing the extravasation of leucocytes by inhibition of vascular adhesion protein 1 (VAP1)[184].

Researchers elsewhere (including some working for the HLS client that commissioned this test) are using human tissue rather than animal models, which avoids the problem of species differences. “We have earlier shown that an anti-VAP-1 mAb blocks extravasation of PMNs [polymorphonuclear leukocytes or granulocytes] into inflamed peritoneum in rabbits and in vitro binding of human PMNs to myocardial vessels in frozen sections of human hearts with a reperfusion injury”[185].

Furthermore, BTT has already undergone some human clinical trials. The first-in-human study with “Biotie fully human VAP-1 monoclonal antibody BTT-1023” was recently reported. Two participants, on the highest dose, reported facial flushing, one of these had facial oedema. These “are not uncommon events in association with intravenous administration of therapeutic protein drugs”[186].

An indication of how even with human based test data at the fingertips of the companies, products continue to march relentlessly through standard animal tests.
The use of primates in the development of the new generation of ‘biologicals’ in the drug industry is on the rise[187]. It is of some concern that testing biologicals on primates is simply becoming the latest convention rather than something that has been assessed in a rational way. A discussion on the practicality of replacing non-human primates in the preclinical safety testing of monoclonal antibodies concluded that “It is clear that from a scientific perspective there are some cases in which the use of Old World primates is not appropriate and other approaches should be considered routinely”[188].

Blood clotting drug study

56 monkeys were used to study “a recombinant form of Von Willebrand factor [developed by the customer]…derived from a culture”. The animals were dosed and bled and killed and necropsied after either a day or two weeks[156].

Von Willebrand disease (vWD) is an inherited blood disorder, characterised by a deficiency or defect in the blood component called von Willebrand factor (vWF)[189]. The symptoms of vWD include: frequent nose bleeds, bruising easily, heavy menstrual flow and excessive bleeding following surgery, dental work or childbirth[190].

Recombinant vWF (rec vWF) is produced from mammalian cells[191], and it has been known since 1999 that “rec vWF exhibited activities comparable with plasma-derived vWF, such as platelet binding, platelet aggregation, collagen binding and coagulation factor VIII (FVIII) binding”[191].

The justification given for the use of non-human primates in this study was that the compound being investigated contained Tween 80, which can cause sensitivity in dogs. It would appear that this is a concession to problems with the dog as a test subject, rather than a testament to the monkey being a good model. Tween 80 is known to cause adverse effects in some humans[192]. In fact, vWD is so rare in primates that a 2002 paper which described type 3 vWD in a rhesus macaque, stated that “this is the first report of vWD in a non-human primate”[193].

It was reported that the aim of this study was development of a recombinant form of von Willebrand Factor. The HLS client says “The majority of therapeutic clotting factors continue to be processed using blood-derived components. By applying Baxter’s proven proprietary blood-free processing technology, we are working to develop a therapeutic option that will eliminate the potential risk of blood-borne pathogen transmission for people with von Willebrand disease”[194]. However, the contention that the new drug is needed due to problems with existing plasma derived vWF is debatable. Wilate®, is a new generation, double virus inactivated vWF / Factor VIII concentrate, which undergoes processes “aggressive enough to inactivate viruses efficiently, but yet gentle enough to maintain the structural integrity and function of the VWF and FVIII molecules”[195].

Almost a decade before these tests on monkeys, vWD experiments on pigs, dogs and mice, had led to the conclusion that “some differences cannot be avoided” and that “…one should be cautious about extrapolating the results from animals to humans”[196].

It is clear that the use of available human data is preferable to animal studies for such a disease. As far back as 2001, a study was conducted to examine the efficacy of a high-purity factor VIII/von Willebrand concentrate for treatment and prevention of bleeding. The study used 81 patients representing all 3 types of vWD and concluded “the concentrate effectively stopped active bleeding and provided adequate haemostasis for surgical or invasive procedures[197].

Advanced scientific techniques to replace the use of primates in regulatory and commercial testing

Developments in science and technology have provided new techniques to replace animals, which provide data relevant to humans.

An intelligent, cross-disciplinary approach is needed, which draws upon the very best in technology.
There is a clear and urgent imperative: by the time primates and dogs have been selected for testing, other animals have already suffered and died for the same products. Replacement of primates is an achievable goal.

Microdosing and Acceleratory Mass Spectrometry (AMS): A ‘microdose’ is defined as less than one hundredth of the proposed pharmacological dose but never exceeding 100µg[199]. Drug levels from microdosing can be measured in any biological sample such as plasma or urine to determine ADME (absorption, distribution, metabolism and secretion) and pharmacokinetic characteristics of a drug. Analysis uses an Accelerator Mass Spectrometer (AMS)[200], which can count individual atoms and has the ability to detect a liquid compound even after just one litre of it has been diluted in the ocean[201]. A recent EU study over a period of 31 months demonstrated the value of microdosing in drug development[202], comparing microdosing data to animal tests. For example, rat data for the compound phenobarbital over-predicted the clearance of the drug in humans, and under-predicted the compound’s half-life (measure of drug metabolism). The microdosing data proved more accurate and was 80% predictive of ADME in people[203].

This demonstrates that microdosing and AMS is significantly more accurate than primate, dog and rodent models. Microdosing could accelerate drug development. Preclinical studies can take 18 months and cost $3-5 million. Microdosing can reduce the time to 4 to 6 months and the cost to $0.35m per new molecule[204]. Other options for replacement through microdosing strategies include PET (positron emission tomography)[205] and human volunteers. It has been concluded that microdosing, could improve selection of new compounds and reduce failures[206].

QSARs (Quantitative Structure Activity Relationships) computer modelling; correlates a compounds’ structure and properties with its activity. QSAR is used in drug design and environmental risk assessment[207], it can play a significant role in assessing toxicity and pharmacokinetics and can be used to determine target organ or system doses[208].

Derek for windows (DfW) is an expert knowledge base system (a computer program that applies rules), which predicts a chemical’s toxicity from its molecular structure[209] by applying QSARs and other knowledge rules [209].

Human cell lines: An EU project, ‘vitrocellomics’ aimed to provide “…new, efficient in vitro prevalidation models, which will significantly reduce the use of animal experimentation for prediction of human drug metabolism by 60-80%”[210,211]. Specialised cell cultures, such as hepatocytes (liver cells), allow researchers to conduct multiple studies on multiple days using hepatocytes from a single donor to assess intra-assay variability and to study multiple endpoints (i.e. transport and metabolism)[212]. A variety of in vitro systems have been under development, derived mainly from the liver, kidney and brain [208].

Human tissue use: Pharmaceutical companies use liver tissue to provide biological data and safety test compounds; “animal drug metabolism is very different to human and it may be more appropriate to use human liver tissue early in a new drugs life to establish metabolites that may be toxic to humans”[213].

Scaffolds and 3-Dimensional (3D) cultures can be formed in different tissue, and used as models for pharmaceutical and drug discovery. The scaffold can be made from synthetic or natural materials, with differing scientific advantages[214]. Tissue can be constructed to recreate whole body systems, such as the human artificial immune system, which assesses a substance’s interaction with the immune system[215].

High throughput screening: This technique, involving robotics and sophisticated control software, rapidly analyses compounds for drug discovery, often to generate starting points for drug development[216].

Biochips: These show the effect on different cells in the body and how toxicity is altered when the compound is broken down (metabolized) in the human body. They provide “comprehensive toxicity data very quickly and cheaply” and can provide data on the toxicity on different human organs[217].

Toxicogenomics: This seeks to translate data about genetic variation and gene expression into an understanding of the biological systems in organisms, including humans, and the effects of changes in the systems on the organism’s health. Toxicogenomics may improve understanding in processes such as reproductive toxicity and nongenotoxic carcinogenesis, usually carried out in long-term animal studies, and “help treat people at the greatest risk of diseases caused by environmental pollutants or toxicants”[218]. Antidote Europe has developed a novel approach to toxicogenomics by using miniaturized DNA chips, in combination with sequential exposure of two different cell types, approximating what would occur in a whole body and call the approach ‘Scientific Toxicology Program’ (STP)[219].

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