Cruel experiments on marmosets in misguided attempt to help humans

Animal Aid has investigated cruel experiments on marmoset monkeys, carried out to test combinations of drugs to treat Parkinson’s Disease, by one of the UK’s leading universities.

The experiments, published in 2017 and carried out at King’s College London, were jointly authored with a Belgian biotech company. They involved marmosets being chemically paralysed to suffer Parkinson’s Disease-like symptoms, to try to mimic the disease in patients, before being dosed with L-DOPA, a drug used to treat Parkinson’s for decades and known to cause significant side effects and distress, including uncontrollable body tremors. Following this, the marmosets were force-fed a combination of two test drugs (Radiprodil and Tozadenant) to investigate how the drugs alleviated Parkinson’s disease symptoms, but without the complications of L-DOPA, which causes increasing side effects with long-term use.

The study was carried out to confirm previous experiments by some of the same authors in brain-damaged rats, with the aim of evaluating the test drug combination   ‘in a model which more closely resembles the clinical situation‘. Despite this, the uncertainty of using marmosets is highlighted by the conclusion that ”Although encouraging, these preclinical data need to be confirmed in the clinic’.

The use of monkeys in scientific research is not only scientifically flawed and immensely cruel, but also very expensive. As a result, they are reused wherever possible. The marmosets used in this study were intended for use again, in further experiments, within a couple of weeks. In the UK during 2016 alone, 2,440 non-human primates (including marmosets, macaques and tamarins) were used in a total of 3,569 procedures, meaning that over 1100 procedures involved the use of monkeys who had already been used once.

Title: Antiparkinsonian effects of the “Radiprodil and Tozadenant” combination in MPTP-treated marmosets

Published: PLOS One, 2017

Jointly authored by King’s College London and UCB BioPharma, UK/Belgium

Twelve monkeys were first injected daily for five days with MPTP (1-methyl-4-phenyl-1, 2, 3, 6,-tetrahydropyridine), a well-known agent used for decades in experimental primate research. MPTP causes severely debilitating, distressing effects and Parkinson-like symptoms, including paralysis and total loss (or extreme slowness) of movement, rigidity, loss of vocalisation, lack of muscle movement including eye rigidity, lack of coordination, uncontrollable body tremors and hunched posture. These were described as ‘stable motor deficits‘ in the study.

The cruel and inhumane effects of MPTP are well known, after its repeated use for many years in countless procedures to cause seriously debilitating effects in primates. Indeed, as noted by the authors of this study, the level of harm caused was such that half-size cages ‘were used for severely disabled animals immediately after MPTP’ dosing.

The next stage of the experiment (within a few months of MPTP treatment) involved the marmosets being dosed with the Parkinson’s drug L-DOPA (by daily force feeding) for up to 28 days to induce symptoms of mild to severe dyskinesia (uncontrollable body spasms, writhing and twisted body posture). These effects are well known and expected as shown by many previous, similar experiments in marmosets.

Immediately prior to the start of the next stage of the experiment below, they were force-fed another ‘pre-dose’ of L-DOPA and their behaviour recorded for five hours, before a second dose was given (along with a novel object (e.g. tennis ball) added to each cage to assess movement activity) before being observed for a further five hours. This ‘Pre L-DOPA test’ was carried out to ensure that the marmosets were still ‘primed’ to express moderate to severe dyskinesia and so that the effects of L-DOPA could be compared to those observed in the combination drug tests. This resulted in twelve marmosets (from a total of fourteen) who were considered suitably ‘primed’, and so were selected for use in the main study.

The next (and main) protocol of the study was aimed at determining whether a combination of two drugs could alleviate the Parkinson-like symptoms of MPTP, without the adverse effects of L-DOPA (dyskinesia), which acts on dopaminergic receptors in the brain to exert its effects. The test drugs chosen for this study – Radiprodil and Toxadenant – were selected because of their action on other types of non-dopaminergic brain receptors, therefore with the aim of avoiding the motor-disabling side effects of L-DOPA.

To do this, the marmosets were isolated in behavioural cages and dosed – again by force feeding – with one of four treatments; i) both drugs; ii) Radiprodil only; iii) Toxedenant only or iv) a control solution. The doses selected were based on previous studies in rats. Five hours later, they received a repeat dose and at the same time, new objects were put into their cages, in the same way as in the previous L-DOPA tests.

During ten-hour observation periods, the monkeys endured repeated blood samples being taken every 30 minutes from their thigh (femoral) veins and recordings were made of their movement and levels of disability. They were allowed access to water, but no food until the observation period was complete. The ten-hour experiments were performed twice weekly, with a two-day drug ‘washout’ period in between. The study continued until all twelve animals had received all four treatments.

The study report states that ‘Several methods were used to minimize any suffering for the primates.’ These include:

• ‘Specialised post MPTP treatment after care was provided and included hand-feeding of fluids and liquidised high protein/energy food, bathing and cleaning and use of padded/insulated recovery units equipped with heated blankets.’
• ‘Extensive use of wooden ladders/cage furniture to assist mobility of bradykinetic animals’ [NB – bradykinetic means having a slowed ability to start and continue with movement and a reduced ability to adjust your body position].

Whilst the provision of these adaptations may appear to be a good thing, for the monkeys, it is important to highlight that as MPTP injection causes severe immobilisation, hand feeding would be essential for severely disabled animals who cannot feed themselves. This means the animals only need these adaptions because of what has been done to them. The terrible state these monkeys would be left in, by being given the chemical MPTP, is chilling. For an animal to be so disabled by a chemical that they are unable to eat or drink by themselves and need to be bathed is truly horrific.

These animals are intelligent and are usually capable of running, jumping and grooming themselves and others – the inability to do these things shows how terrible MPTP is for both the physical and mental well-being of these animals. The padded ‘units’ also suggest that the animals could be falling about, so require padding in order not to be injured. Insulation and heated blankets also suggest how terribly poorly the monkeys are likely to feel. Half-size cages are also mentioned as being used for ‘severely disabled animals immediately after MPTP dosing‘. Additionally, features such as puzzle feeders are likely to be used by the marmosets only when their condition had improved, following the effects of MPTP and L-DOPA dosing.

The study concluded that when the test drugs were given together, the Parkinsonian-induced marmosets showed a significant increase in motor activity, when compared to each of the drugs alone. However, compared to L-DOPA, the combination drugs took longer to elicit an effect and once reached, the effect was longer-lasting but did not achieve the same level of improved movement as L-DOPA. After the combination treatment, the ‘Radiprodil alone’ treatment showed the next greatest increase in motor activity with the ‘Tozadenant alone’ treatment showing the least effect, being closest in comparison to the control group. However, it is also important to note that a significant increase in activity was observed at the five-hour ‘halfway’ stage, when objects were placed in the cages to stimulate movement and allow behavioural scoring. The authors state that this was done to demonstrate the pharmacological effect of the drug combination and account for the marmosets becoming habituated to their environment, as well as eliminating the possibility that they were hyperstimulated by the drugs and instead demonstrating natural exploratory behaviour. This was considered to be consistent with previous studies in brain-damaged rats.

The study also noted a lack of side effects (dyskinesia) in comparison to L-DOPA, noting that ‘only three subjects demonstrated very minor dyskinesia’.

Nevertheless, the study concluded that the behaviour observed with the drug combination in the monkeys remains hypothetical, given a lack of evidence which would only be obtained from clinical trials in patients.

This experiment, like so many before it, has caused extensive distress and debilitating effects to non-human primates in both the injection of MPTP as well as force feeding of L-DOPA and test drugs, causing deliberately severe side effects in attempts to mimic Parkinson’s in humans. This presents grave ethical problems in the causing of deliberate suffering. Scientifically, the research is problematic as, it lacks human relevance. There are several inherent problems with the use of marmosets and other NHPs in Parkinson’s disease research, as highlighted by the landmark Weatherall report published in 2006 [1] which outlined a number of  limitations cited by researchers when trying to model Parkinson’s in non-human primates, including:

• Primate use has confounded human disease research by conflicting with human data and failed to deliver treatments for Parkinson’s and other major diseases.
• There are many differences in type, persistence and onset of Parkinson’s symptoms between humans and monkeys.
• Monkeys suffer acute and severe symptoms of deliberately induced Parkinson’s, whereas Parkinson’s progression suffered by patients is a slow and chronic deterioration.
• Genetic, age-related, psychological and environmental factors linked to Parkinson’s in humans cannot be studied in animal models.

Furthermore, and perhaps most critically, despite being fully supportive of the continuation of NHP research, the Weatherall report also states that ‘through lack of appropriate data it has not been possible to provide an overall picture of the outcomes of research of this kind over recent years’.

A subsequent review in 2011 by an expert panel retrospectively analysed ten years of research using non-human primates, of which two thirds involved neuroscience studies, including Parkinson’s disease research. One assessment ‘that the review panel found most difficult to establish with confidence was the impact of a specific piece of research on developments in medicine’.[2]

This lack of evidence of medical benefit was also reiterated in a 2013 report by the (then) Animal Procedures Committee (APC, now the Animals in Science Committee (ASC)) on the severity of suffering experienced by NHPs used in neuroscience research[3].

This study seeks to show how effects seen in many preclinical animal tests, for the types of drugs used in this study, fail to be reproduced in patients, leading to investigation of drug combinations which might be more effective than each drug alone.  While this is a reasonable argument for potential improvement of clinical efficacy in patients, a fundamental problem is that marmosets, as well as other NHPs (e.g. macaques) fail to accurately represent human Parkinson’s.

The uncertainty of using marmosets is further highlighted by the authors’ statement that ‘Although encouraging, these preclinical data need to be confirmed in the clinic’.

The two drugs given to the marmosets have already been tested in some clinical trials in patients. The first, Tozadenant, has undergone phase IIb clinical trials and is entering phase III trials in patients ¹. Furthermore, this drug has been used in several clinical trials, both alone and in combination with other drugs (including L-DOPA) for Parkinson’s and other conditions^[4].The second drug, Radiprodil was originally developed as a pain treatment. However, it failed to be effective during a phase II patient trial which was stopped as a result [5] [6].There is also a wealth of combination therapy trial data to treat Parkinson’s disease [7].  Directly human-relevant data from all of these studies provides more reliable evidence than non-clinical data from marmosets and other species, both for Parkinson’s and other conditions.

While the authors concluded that the combination of Radiprodil and Tozadenant was more effective than either drug alone, it is important to note key differences found in each of these drugs when tested in rats, marmosets and humans previously. For example, Tozadenant was considered generally non-effective and not significantly different from the control treatment during this study, based on treatment and observation in marmosets. However, phase II clinical trials in patients concluded a successful effect with Tozadenant and that phase III trials were warranted[8].

It is also feasible that a number of human-based approaches could and should have been investigated to test the drug combination used. Given that the study appears to have proceeded directly to the use of marmosets, and given the extensive history of using marmosets and other animals, it is unclear whether any intelligent, human-relevant strategies were considered first.

Furthermore, regulatory non-clinical (animal) testing guidelines published by the European Medicines Agency (EMA) on combination drug therapy[9] intended for use in patients provide guidance on ‘the non-clinical strategies to be considered when developing a fixed combination based on the different data available in order to support the safe human use as well as avoid unnecessary repetition of animal studies’ . The guidelines also state that depending on the test data already available on the individual drugs and their classification as approved or new active substances, the relevant guidance should be sought as to which animal studies need to be carried out.

Given that both of the test drugs used are not entirely new substances and they have already been tested in (or are currently undergoing) patient trials with some considerable human data available, Animal Aid questions whether adequate investigation of all existing data was carried out, to determine if further marmoset experiments were fully justified from a regulatory perspective. This is not clear from the study report, which only states the approval, use and procedures performed on marmosets, with no explanation of whether any data review, weight of evidence or alternative strategies were considered. This is a particularly critical point, given a history of highly controversial and purely experimental procedures on marmosets at KCL, by some of the same research authors, for example to investigate the effects of ecstasy (MDMA) on Parkinson’s symptoms, despite the fact that ecstasy would never be approved as a treatment for the disease^[10].

As explained above, the analysis of this study raises not only ethical but scientific problems with the repeated use of marmosets in severely invasive procedures, that have been repeatedly used, over decades, to deliberately induce Parkinson’s-like symptoms. Animal Aid questions whether any review of existing data (in both animals and patients) was adequately performed, as well as whether there was any consideration of human-relevant approaches and intelligent testing strategies to avoid the use of further primates.

[1] The use of non-human primates in research (2006). A working group report chaired by Sir David Weatherall FRS FMedSci

[2 ] Review of Research Using Non-Human Primates (2011). Report of a panel chaired by Professor Sir Patrick Bateson FRS

[3] Review of the assessment of cumulative severity and lifetime experience in non-human primates used in neuroscience research.(2013) Animal Procedures Committee (APC).

[4] US National Library of Medicine. List of clinical trials for Tozadenant. Accessed 11^th April 2018.

[5] US National Library of Medicine. Study of the Safety and Efficacy of RGH-896 in Patients With Diabetic Peripheral Neuropathic Pain Accessed 11^th April 2018.

[6] Radiprodil Trial fails to show reductions in daily pain scores. (2010). Pharmaceutical Business Review.

[7] Selection of clinical trial listings for combination therapy in Parkinson’s Disease. Clincaltrials.gov [https://clinicaltrials.gov/ct2/results?cond=Parkinson&term=combination+&cntry=&state=&city=&dist=]

[8] Hauser, R. et al. (2014) Tozadenant (SYN115) in patients with Parkinson’s disease who have motor fluctuations on levodopa: a phase 2b, double-blind, randomised trial. Lancet Neurol. 13(8):767-76.

[9] European Medicines Agency (EMA) Guideline on the non- clinical development of fixed combinations of medicinal products

[10] Iravani, M.,et al. (2003) 3,4-Methylenedioxymethamphetamine (Ecstasy) Inhibits Dyskinesia Expression and Normalizes Motor Activity in 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Treated Primates. Journal of Neuroscience. 23 (27) 9107-9115