Identification and Optimization of a Series of 8-Hydroxy Naphthyridines with Potent In Vitro Antileishmanial Activity: Initial SAR and Assessment of In Vivo Activity

Visceral leishmaniasis (VL) is a parasitic infection that results in approximately 26 000–65 000 deaths annually. The available treatments are hampered by issues such as toxicity, variable efficacy, and unsuitable dosing options. The need for new treatments is urgent and led to a collaboration between the Drugs for Neglected Diseases initiative (DNDi), GlaxoSmithKline (GSK), and the University of Dundee. An 8-hydroxynaphthyridine was identified as a start point, and an early compound demonstrated weak efficacy in a mouse model of VL but was hampered by glucuronidation. Efforts to address this led to the development of compounds with improved in vitro profiles, but these were poorly tolerated in vivo. Investigation of the mode of action (MoA) demonstrated that activity was driven by sequestration of divalent metal cations, a mechanism which was likely to drive the poor tolerability. This highlights the importance of investigating MoA and pharmacokinetics at an early stage for phenotypically active series.


■ INTRODUCTION
According to the World Health Organization (WHO), neglected tropical diseases (NTDs) affect in excess of 1 billion people, the majority being in the most impoverished areas of the world. 1 Of these NTDs, visceral leishmaniasis (VL) remains one of the most challenging to treat. VL is caused by infection with the protozoan parasites Leishmania donovani and Leishmania infantum, which are transmitted through the bite of female phlebotomine sand flies. 2 Following infection, parasites reside predominantly in the liver, spleen, and bone marrow, and if left untreated, the disease is invariably fatal. Although it is challenging to precisely determine the number of people affected by VL, WHO estimates suggest that 50 000−90 000 new infections result in an annual death toll of between 26 000 and 65 000, with the vast majority of cases occurring in India, Bangladesh, Sudan, Ethiopia, and Brazil. 3 Although there are a number of available treatments, all suffer from major issues that limit their use. Miltefosine, the only orally available treatment, is teratogenic so can only be prescribed to women of childbearing age alongside contraception, and also shows other side effects, as well as variable efficacy. 4 Liposomal amphotericin B (Ambisome) is widely effective in Asia, but its use is limited by the need for intravenous (iv) administration which requires hospitalization, and it also requires a cold-chain for storage. An alternative aminoglycoside antibiotic paromomycin is much less expensive but demonstrates variable efficacy and requires a long course of painful intramuscular injections. 5 Finally, pentavalent antimonials, such as sodium stibogluconate, have been widely used since the 1940s, but they are cardiotoxic, require parenteral administration, and are largely ineffective in large areas of India due to high levels of drug resistance. Although new compounds have recently entered the development pipeline, 6 including two preclinical candidates developed within this collaboration (DDD853651/GSK3186899 and DDD1305143/GSK3494245) as well as the Novartis compound GNF6702, which have been disclosed in recent publications, 7 there is still an urgent need for new treatments with improved safety profiles, more straightforward administration, lower costs, and also with alternate modes of action.
One of the major challenges for drug discovery for VL is the lack of robustly validated drug targets in Leishmania spp. For this reason, to identify suitable start points for drug discovery, compound libraries are screened directly against parasites in vitro, leading to phenotypically active compounds with unknown mechanisms of action. An additional confounding factor in attempts to identify chemical start points is the fact that in the human host, parasites are found within macrophages, where they reside within a parasitophorous vacuole. Therefore, relevant high-content screens, suitable for high-throughput screening (HTS), require culture of parasites in macrophages, typically differentiated THP-1 cells. 8 For any compounds to be identified as hits in these screens, they would be required to have suitable physicochemical properties to cross a number of cell membranes, across various pH gradients, as well as having antiparasitic activity. As a result, hit rates are extremely low in these assays, typically below 0.1%. 9 Nevertheless, compound series that are active in this assay and can be developed to give suitable properties for in vivo dosing have a high likelihood of success in rodent models of VL.
Using this intramacrophage assay, an HTS screen of a GlaxoSmithKline (GSK) collection of 1.8 M compounds was performed against L. donovani. 10 The hits identified were screened in secondary antiparasitic assays, assessed for nonspecific cytotoxicity in HepG2 cells, clustered, and filtered based on favorable physicochemical properties, resulting in the identification of 33 chemical series and 75 singletons. One of the identified series was exemplified by 1 (Table 1), an 8hydroxynaphthyridine, which was shown to have potency against the parasite (pEC 50 = 6.5) with ∼100-fold selectivity over the human THP-1 cell line (pEC 50 = 4.5). This compared favorably to the current treatments, amphotericin (pEC 50 = 6.7) and miltefosine (pEC 50 = 5.4). Upon further profiling, 1 was shown to have reasonable aqueous (aq) solubility (219 μM) but low stability in mouse liver microsomes (Cl i = 18 mL/min/g). On the basis of this, 1 was selected as a suitable start point for a hit-to-lead program.
The principal aim of this work was to identify analogues of 1 with a suitable profile for dosing in a mouse efficacy model of VL as rapidly as possible, to demonstrate that the series had the potential to progress into lead-optimization. Therefore, the initial chemistry program focused on understanding the structure−activity relationship (SAR) of the series, with an aim of identifying compounds with improved solubility and metabolic stability, as well as suitable potency for in vivo studies. Our targets were to achieve pEC 50 > 5.8, aqueous solubility >200 μM, and mouse liver microsomal clearance of <5.0 mL/min/g, as these criteria had been used previously to identify chemical series likely to have in vivo efficacy. 12

■ RESULTS AND DISCUSSION
Lack of knowledge regarding the molecular target of compound 1 made optimization challenging, with no guide as to the potential pharmacophore, or which vectors were most likely to positively influence activity. We therefore focused on utilizing tractable chemistry that would facilitate a rapid exploration of SAR. Also, to maintain good solubility and hopefully improve metabolic stability, we aimed to reduce, or at least maintain, the Log D of the initial analogues. This led us to focus on the triazole substituent, as well as the 5-position of the naphthyridine as initial points for exploration. SAR of 7-Triazolyl Analogues. Variations to the benzyl substituent of the triazole (Table 2), including substitutions on the phenyl position (exemplified by 2), or on the methylene (exemplified by 3), led to a ∼10-fold loss in potency compared to 1, although 3 did show an improvement in metabolic stability. A truncated analogue 4 was inactive, but we were encouraged by its improved solubility and metabolic stability. We thus replaced the 4-chlorophenyl group of 1 with more polar substituents, with the aim of regaining in vitro potency while maintaining a favorable absorption, distribution, metabolism, and excretion (ADME) profile. Morpholine-substituted 5 and pyrrolidinonesubstituted 6 were synthesized and indeed proved to be both soluble and metabolically stable, although both compounds were essentially inactive. Switching to an amide as an isosteric replacement for the triazole was also investigated, and the matched pairs (7 vs 1) showed similar levels of potency, although the amide did not appear to show any clear advantage over the triazole as its mouse liver microsomal clearance was still not below the targeted 5 mL/min/g. Table 1 a Table 2 Journal of Medicinal Chemistry pubs.acs.org/jmc Article We next switched attention to the naphthyridyl 5-position. Initially, nitrogen-linked analogues were investigated. While both methylamine 8 and dimethylamine 9 were essentially inactive, larger amines such as p-chlorobenzylamine 10 and morpholine 11 both had pEC 50 values above 5, with good solubility and low clearance. Cyclic amides, such as 12, proved to be inactive, as did sultam 13, presumably due to the reduced electron density in the aromatic ring. Compound 13 was of particular interest as 5-sultam-substituted naphthyridines had been previously reported in a series of integrase inhibitors and were shown to impart very good pharmacokinetics (PK) properties. 13 Indeed, the lead compound from this series progressed as far as phase II clinical trials (compound 30; Table 5). Further exploration of N-linked analogues failed to deliver compounds with the necessary potency for progression to in vivo studies, so we switched our focus to carbon-linked analogues. Interestingly, the unsubstituted phenyl analogue 14 showed reasonable potency (pEC 50 = 5.9), and further analogues showed ortho-substitution to be beneficial, with the o-trifluoromethyl analogue 15 giving a significant increase in antiparasitic activity (pEC 50 = 7.0), possibly driven by the increased lipophilicity, and the o-methoxy analogue 16 showing a good balance of potency, stability, and solubility (pEC 50 = 6.2, Cl i = 1.4 mL/min/g and aqueous solubility >250 μM). Replacement of phenyl by aromatic heterocycles (such as pyridyl or pyrazolyl) was also explored, as was substitution on the other side of the naphthyridine (2-, 3-, and 4-positions), but these changes led to only weakly active compounds (data not shown).
Profiling of Compound 16. Compounds 15 and 16 both showed a promising balance of potency, solubility, and metabolic stability. Because of having higher metabolic stability (in mouse liver microsomes) and lower Chrom Log D, compound 16 was progressed into a VL in vivo efficacy study, carried out in our previously described VL mouse model. 7 Mice were dosed orally with the standard antileishmanial drug miltefosine, or with 16 dosed intraperitoneal (ip) two times daily for 5 days post infection (although 16 had a suitable profile for oral dosing, we elected to dose ip to maximize exposure and increase our chances of demonstrating in vivo proof of concept for the series). Parasite load was determined in the livers of animals 3 days after cessation of treatment, and parasite burden was expressed in Leishman Donovan units (LDUs, the mean number of amastigotes per liver cell × mg weight of liver). The blood exposure of compound 16 was also determined in dosed animals on days 1 and 5 to better understand the PK/ pharmacodynamics (PD) relationship of the series. According to our project criteria, a compound needs to reduce parasite burden by >70% before being considered suitable for progression to lead-optimization, while a reduction of >95% would be considered suitable for a preclinical development candidate. 12 In the study, miltefosine behaved as expected, reducing parasite levels by >99% at 30 mg/kg qd. After twice daily ip dosing at 50 mg/kg, compound 16 reduced parasite burden in mouse liver by 46%. This provided an early proof of concept for this series but fell short of our target of >70% parasite reduction.
Upon examining the blood samples taken on days 1 and 5, it was clear that 16 was rapidly cleared from blood, with unbound concentrations of compound exceeding EC 99 only during the first hour post-dose (Table 3). Further examination of the samples revealed the presence of glucuronidated adducts of 16, suggesting secondary metabolism as the key driver of the low exposure.
Glucuronidation is a means of increasing water solubility of small molecules, facilitating their elimination from the body in urine. It involves transfer of the glucuronic acid component of uridine diphosphate glucuronic acid to a suitable substrate, catalyzed by UDP-glucuronosyltransferase (UGT), and occurs mainly in the liver. 14 Glucuronidation occurs at nucleophilic sites such as R−OH, R−NH 2 , or R−COOH, which can be present in the small molecule, or generated via phase I metabolism. Due to the low rates of microsomal clearance of 16, alongside our in vivo data, we surmised that glucuronidation was occurring on the parent compound, most likely at the phenolic OH or the triazole N−H. Also, the observed reduction of parasite load, despite the high in vivo clearance, suggested that reducing glucuronidation to increase the duration of exposure above EC 99 would be a key strategy to progress the series toward lead-optimization.
As a means of measuring glucuronidation in vitro, 16 was assessed in a mouse liver hepatocyte assay. Unfortunately, 16 showed similar stability to that seen in mouse liver microsomes (0.9 mL/min/g in microsomes vs 1.4 mL/min/g in hepatocytes) with negligible amounts of the glucuronide adduct being observed. This suggested that there was little involvement of the hepatic UDP-glucuronosyltransferase in the in vivo phase II metabolism. This result made series progression challenging, as there was no way to determine the potential impact of glucuronidation without running an in vivo study, limiting our understanding of the SAR surrounding the observed secondary metabolism.
One possible strategy to identify compounds with improved metabolic stability would be to reduce lipophilicity. This had been shown previously to be a potential strategy for reducing glucuronidation; 15 however, as shown in Figure 1, this was very challenging within this compound series. Looking at measured log D (Chrom Log D 7.4 ), 11 analogues with Chrom Log D 7.4 values below 3 were generally only weakly active, with pEC 50 's above 5.8 only being achieved where Chrom Log D 7.4 was greater than 3. In our experience, this is a common problem when trying to optimize series phenotypically, where increasing potency without increasing lipophilicity is very challenging; this highlights a key advantage of running structure-enabled programs. Because of this, alternative approaches to improving metabolic stability were required. Ld InMac is the intramacrophage assay carried out in THP-1 cells with L. donovani amastigotes. Data are the result of at least three independent replicates, and standard deviations are ≤0. 4. b Cl i is mouse liver microsomal intrinsic clearance. c Aq solubility is kinetic aqueous solubility. d ND means not determined. SAR of the Naphthyridine Core. Our initial approach to reduce potential phase II metabolism within the series was therefore to investigate the SAR around the phenolic hydroxyl group and the triazole. Understanding which of these features was important for antiparasitic activity might enable us to synthesize potent analogues with lower potential for glucuronidation. As shown in Table 4, removal or methylation of the naphthyridine 8-hydroxyl group led to a loss of antiparasitic activity compared to 16 (17 and 18, respectively), although 18 was close to the targeted pEC 50 of 5.8. Also, replacement of triazole with oxadiazole to remove the nucleophilic N−H led to a loss of activity (19). Synthesis of other analogues to explore the naphthyridine SAR proved extremely challenging within the triazole subseries, so to more rapidly address this, we switched attention to the bioisosteric replacement of triazole with amide; this change had previously been seen to have limited effect on potency (e.g., 1 vs 7) and allowed much more straightforward synthesis of the analogues of interest. Thus, deletion of the naphthyridine N-6 of 7 led to a compound that was toxic to the host THP-1 cells, and deletion of naphthyridine N-1 led to loss of antiparasitic activity (20 and 21, respectively). Moving to a scaffold that trapped the phenolic OH as a carbonyl removed all antiparasitic activity despite the parent amides having pEC 50 values >6.0 (comparing 22 to 28 and 23 to 27). Finally, we examined Raltegravir (24), an inhibitor of human immunodeficiency virus (HIV) integrase marketed as a treatment for HIV. 16 As shown in Figure 2, 24 contains the key acceptor− donor−acceptor binding motif identified within the naphthyridine series. Although 24 was inactive in our in vitro Leishmania assays, we surmised that transferring the known SAR of the naphthyridine core onto the Raltegravir scaffold could be a viable strategy to regain activity. Unfortunately, as exemplified by 25, none of the analogues based on this scaffold were active. From this round of synthesis, we concluded that we were unlikely to identify active compounds without the acceptor− donor−acceptor binding motif of the 7-substituted-1,6-naphthyridin-8-ol core.
SAR of 7-Carboxamide Analogues. Previous SAR demonstrated that replacing the triazole with an amide led to compounds such as 7 that retained antiparasitic activity. Since 7 itself was metabolically unstable (Cl i = 10 mg/mL/g), we became interested in transferring the SAR from the triazole subseries (e.g., 16) to investigate its impact on metabolic stability, with a particular focus on glucuronidation. To this end, we synthesized 26 (Table 5) as a direct analogue of triazole 16. Although it did not show sufficient potency for progression into efficacy studies, it had reasonable aqueous solubility (219 μM) and good metabolic stability (Cl i = 0.9 mL/min/g) and was therefore progressed into a mouse PK study to assess the extent of in vivo glucuronidation. After dosing (50 mg/kg ip) and analyzing the metabolites generated ( Figure 3), there was little evidence of glucuronidation, with the major metabolism observed being hydroxylation. As significant quantities of parent were still present 8 h post-dose, this supported a strategy of switching to the amide series to reduce phase II metabolism and improve in vivo exposure.
With this in mind, we further explored the SAR of the amide subseries, as shown in Table 5. We synthesized a set of benzyl amides, where 4-methoxy analogue 27 and α-methyl-4-fluoro analogue 28 both gave very encouraging profiles, meeting progression criteria in terms of potency, metabolic stability, and clearance. Alternatively, nonaromatic amides were explored, and although none were identified with suitable profiles for progression, cyclopropylmethyl analogue 29 did demonstrate reasonable potency. Due to its impressive in vitro potency, we selected 28 for progression into an in vivo PK study. However, upon dosing (50 mg/kg ip), the compound proved to be toxic, rapidly giving symptoms (within 3.5 h) requiring termination of the experiment.
We noted that a related compound from Merck, L-870,810 (30), 17 was reported as a clinical candidate targeting HIV integrase, which progressed as far as phase II clinical trials. Compound 30 was inactive in our in vitro efficacy assays, but the report, alongside the in vivo data for 26, suggested that compounds with substitutions in the naphthyridine 5-position could have suitable profiles for in vivo studies. Also, introducing substituents into the 5-naphthyridyl position had been a successful strategy for improving metabolic stability and potency in the triazole subseries. We therefore investigated a range of 5substituted naphthyridyl analogues (Table 5). THP-amine 31 and morpholine 32 lost potency compared to parent compound 7, so we switched attention back to 5-phenylnaphthyridines. Previously identified groups were combined (the 4-methoxyphenyl of 16 and the cyclopropylmethyl amide of 29), leading to 33, which unfortunately did not deliver the expected increase in potency. Further combinations of nonaromatic amides with differently substituted 5-phenylnaphthyridines were synthesized, and while changing the phenyl substituent gave flat SAR and no advantage over previous compounds, exploration of the amide led to 34 with a trans-4-methoxycyclopropylamide.
Compound 34 gave a good balance of potency, solubility, and microsomal stability and was therefore selected for a mouse PK study. Disappointingly, when dosed at 50 mg/kg ip, the mice again displayed the similar symptoms as with compound 28, and the study was terminated after 60 min.
Profiling of 16, 28, and 34. Due to the encouraging results within the series (16 showing low-level efficacy and 26 giving a good PK profile), we were keen to understand the origins of the observed toxicity of 28 and 34. As previously mentioned, compound 30 progressed as far as phase II clinical trials as an HIV integrase inhibitor, and although it was inactive against VL, we were keen to assess whether there was scope to progress our related series further. To this end, two studies were conducted in parallel; a screen against receptors with known links to toxicity and investigation of mode of action (MoA).
First, the compounds which were poorly tolerated in mice, 28 and 34, were screened against a panel of >30 receptors with Journal of Medicinal Chemistry pubs.acs.org/jmc Article known links to in vivo toxicity (GSK-enhanced cross-screen panel (eXP)). 18 Compound 28 gave a pIC 50 value of 5.4 against monoamine oxidase A, highlighting a slight risk of drug−drug interactions and possible side effects, and a pIC 50 value of 4.9 in a phenotypic cell health assay, suggesting possible effects on mitochondrial integrity that could lead to an increased risk of Table 4 a Ld InMac is the intramacrophage assay carried out in THP-1 cells with L. donovani amastigotes. Data are the result of at least three independent replicates, and standard deviations are ≤0. 4. b Cl i is mouse liver microsomal intrinsic clearance. c Aq solubility is kinetic aqueous solubility. d ND means not determined.
Journal of Medicinal Chemistry pubs.acs.org/jmc Article hepatotoxicity. Compound 34 also showed potency in the cell health assay, alongside activity in a bile salt export pump (BSEP) assay (pIC 50 value of 4.8, hepatotoxicity risk) and a phospholipidosis assay. From this, it was not clear whether the effects seen in the receptor screen were related to the observed in vivo toxicity. Alongside these screens, mode-of-action studies were initiated focusing on 10, 16, and 28, as representatives of both the triazole and amide subseries. We were particularly interested in confirming that the compounds inhibited a shared target, identifying off-target effects, and understanding the source of the observed toxicity. The results of these studies have been reported previously and demonstrated that the compounds act as nonspecific chelators of divalent metal cations, in particular Zn 2+ , Fe 2+ , and Cu 2+ , and that this property is likely responsible for their antiparasitic activity. 19 Indeed, the propensity of these compounds to nonspecifically chelate divalent cations may well explain the observed in vivo toxicity associated with this series.  Table 5 a Ld InMac is the intramacrophage assay carried out in THP-1 cells with L. donovani amastigotes. Data are the result of at least three independent replicates, and standard deviations are ≤0. 4. b Cl i is mouse liver microsomal intrinsic clearance. c Aq solubility is kinetic aqueous solubility. d ND means not determined.
Journal of Medicinal Chemistry pubs.acs.org/jmc Article Chemistry. To access the required analogues, a number of different approaches were needed, as illustrated in scheme 1−3. Compounds 1 and 2 were previously reported, 20 and synthesis of the remaining 7-triazolyl-8-hydroxy naphthyridines started from 7-cyanonaphthyridine 35 20 (Scheme 1), where cyclization with substituted hydrazides under acidic conditions led to compounds 3−6. To access 5-substituted analogues, 35 could be brominated with N-bromosuccinimide (NBS) to give 36 and subsequently cyclized to give 37, which was treated with a suitable amine to give 8−11. For 12 and 13, protection of the phenol proved to be necessary. Hence, 36 could be tosylprotected to give 38, followed by either Buchwald−Hartwig coupling to introduce the pryrrolidinone (12) or copper coupling with 1,2-thiazinane 1,1-dioxide to introduce the sultam (13). Alternatively, 36 could be directly coupled with phenylboronic acid to give 14, or benzyl-protected to give 39, which could be coupled to give 15 and 16.
To fully explore the SAR around the naphthyridine ring, and also to introduce alternative heterocycles to replace the triazole, a number of analogues required bespoke synthesis. Analogues 17, 18, 20, and 21 were synthesized according to established procedures and are described in the Supporting Information, with the synthesis of 19,22,23, and 25 highlighted in Scheme 2. Thus, 5-bromo-8-methoxy-1,6-naphthyridine-7-carboxylic acid 40 was cyclized with acetylhydrazide to give 41, followed by Suzuki coupling and deprotection of the 8-methoxy group to give oxadiazole 19. Compound 22 was synthesized from 3chloropicolinic acid via conversion to the acid chloride, condensation with ethyl 3-(dimethylamino)acrylate, cyclization with methylamine, and ester hydrolysis to give 42. This was then coupled with 4-fluoro-α-(R)-methylbenzylamine to give 22. To access 23, methyl 3-fluoropicolinate was condensed with tertbutyl acetate to give 43, which was treated with 4acetamidobenzenesulfonyl azide (ABSA), PBu 3 , then 1,8diazabicyclo [5.4.0]undec-7-ene (DBU)/iodomethane to give cyclized 44, which was subsequently hydrolyzed and coupled with 4-methoxybenzylamine to give 23. Finally, 25 was synthesized according to a previously published route such that 2-cyanoanisole was treated with N-methylhydroxylamine and cyclized to 45. Thermal rearrangement gave 46, which was treated with 4-fluoro-α-(R)-methylbenzylamine to give 25. 21 7-Carboxamide analogues were synthesized from the corresponding ester 47 according to Scheme 3. Amides 7 and 27−29 were synthesized directly from the ester by treating with the relevant amine at high temperature. To access the desired 6functionalized analogues, 47 was brominated with NBS to give 48, tosyl-protected to give 49, and converted to sultam 30. 22 Alternatively, 48 could be treated with 4-chlorobenzylamine to give 51a, then coupled with another amine to give 31 and 32.
Compound 49 could also be coupled with 2-methoxyphenylboronic acid and hydrolyzed to give 50, which was coupled with trans-4-methoxycyclohexylamine to give 34. Alternatively, 48 could be treated directly with a relevant amine to give 51b and 51c, with subsequent Suzuki coupling with 2-methoxyphenylboronic acid giving 26 and 33.

■ CONCLUSIONS
To identify new compound series with the potential to be developed as new therapeutics for VL, a collection of 1.8 M compounds from the GSK corporate collection was screened against L. donovani in vitro. One hit series identified from this, exemplified by 1, was selected for a hit-to-lead program. In vivo studies of an early compound, 16, demonstrated that the series had the potential to reduce parasite burden, but that glucuronidation was a potential barrier to series progression. Scaffold hopping from the core triazole to an amide was a key strategy for progressing the series, leading to 28 with a very good in vitro profile. Dosing of 28 identified an issue with toxicity for the series and further chemistry failed to identify compounds that did not carry this liability. MoA studies suggested that the antiparasitic activity, and the toxicity, was likely driven by chelation of divalent metal cations. Based on these findings, we concluded that attempting to develop compounds within this series that would separate antiparasitic activity from inherent toxicity would be extremely challenging and unlikely to succeed. With this in mind, work on the series was halted. This demonstrates the importance of understanding the mode of action from a very early stage in the drug discovery process, when working to progress phenotypically active hit compounds.
Kinetic Aqueous Solubility Assessment and Intrinsic Clearance Experiments. These assays were conducted as previously described. 23 Chrom Log D pH7. 4 . This assay was conducted as previously described. 11 In vivo Mouse Efficacy Studies. In vivo studies were carried out as previously described. 12 Ethical Statements. Mouse and Rat Pharmacokinetics. All animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK/Dundee University Policy on the Care, Welfare, and Treatment of Animals.
In Vivo Efficacy. All regulated procedures, at the University of Dundee, on living animals were carried out under the authority of a project license issued by the Home Office under the Animals (Scientific Procedures) Act 1986, as amended in 2012 (and in compliance with EU Directive EU/2010/63). License applications will have been approved by the University's Ethical Review Committee (ERC) before submission to the Home Office. The ERC has a general remit to develop and oversee policy on all aspects of the use of animals on University premises and is a subcommittee of the University Court, its highest governing body.