Endeavours made by trade associations, pharmaceutical companies and regulators in the replacement, reduction and refinement of animal experimentation in safety testing of pharmaceuticals.

Following the European Commission decision to develop a roadmap to phase out animal testing and the signing of the US Modernisation Act, there is additional pressure on regulators and the pharmaceutical industry to abandon animal experimentation in safety testing. Often, endeavours already made by governments, regulators, trade associations, and industry to replace, reduce and refine animal experimentation (3Rs) are unnoticed. Herein, we review such endeavours to promote wider application and acceptance of 3Rs. ICH guidelines have stated 3Rs objectives and have enjoyed many successes driven by global consensus. Initiatives driven by US and European regulators such as the removal of the Abnormal Toxicity Test are neutralised by reticent regional regulators. Stream-lined testing requirements have been proposed for new modalities, oncology, impurity management and animal pharmacokinetics/metabolism. Use of virtual controls, value of the second toxicity species, information sharing and expectations for life-threatening diseases, human specific or well-characterised targets are currently being scrutinised. Despite much effort, progress falls short of the ambitious intent of decisionmakers. From a clinical safety and litigation perspective pharmaceutical companies and regulators are reluctant to step away from current paradigms unless replacement approaches are validated and globally accepted. Such consensus has historically been best achieved through ICH initiatives.

Industry experiences with immune-mediated findings in biotherapeutic nonclinical toxicology studies.

With the growth of monoclonal antibodies and other proteins as major modalities in the pharmaceutical industry, there has been an increase in pharmacology and toxicity testing of biotherapeutics in animals. Animals frequently mount an immune response to human therapeutic proteins. This can result in asymptomatic anti-drug antibody formation, immune complexes that affect drug disposition and/or organ function such as kidney, cytokine release responses, fatal hypersensitivity, or a range of reactions in between. In addition, an increasing number of oncology therapeutics are being developed that enhance or directly stimulate immune responses by a variety of mechanisms, which could increase the risk of autoreactivity and an autoimmune-like syndrome in animals and humans. When evaluating the risk of biotherapeutics prior to entering the clinic, the nonclinical safety data may include any of these responses and it is critical to understand whether they represent a safety liability for humans. The DruSafe Leadership group of the IQ Consortium conducted a survey of industry to understand sponsors' experiences with these immune reactions in nonclinical studies related to both immunogenicity and pharmacologically-mediated immune perturbations. The survey covered what pathways were affected, how the immune responses were presented, how the company and health authorities interpreted the data and whether the immune responses were observed in the clinic. Additionally, the survey gathered information on association of these findings with anti-drug antibodies as well as sponsor's use of immunogenicity predictive tools. The data suggests that the ability of a biotherapeutic to activate the immune system, intended or not, plays a significant role on characteristics of the response and whether theys are translatable.

Role of P-glycoprotein in the brain disposition of seletalisib: Evaluation of the potential for drug-drug interactions.

Seletalisib is an orally bioavailable selective inhibitor of phosphoinositide 3-kinase delta (PI3Kδ) in clinical development for the treatment of immune-mediated inflammatory diseases. The present study investigated the role of P-gp in seletalisib disposition, especially brain distribution, and the associated risks of interactions. Seletalisib was found to be actively transported by rodent and human P-gp in vitro (transfected LLC-PK1 cells; Km of ca. 20 µM), with minimal or no affinity for the other tested transporters. A distribution study in knockout rats (single oral dosing at 750 mg kg-1) showed that P-gp restricts the brain disposition of seletalisib while having minimal effect on its intestinal absorption. Restricted brain penetration was also observed in cynomolgus monkeys (single oral dosing at 30 mg kg-1) using brain microdialysis and cerebrospinal fluid sampling (Kp,uu of 0.09 and 0.24, respectively). These findings opened the question of potential pharmacokinetic interaction between seletalisib and P-gp inhibitors. In vitro, CsA inhibited the active transport of seletalisib with an IC50 of 0.13 µM. In rats, co-administration of high doses of CsA (bolus iv followed by continuous infusion) increased the brain distribution of seletalisib (single oral dosing at 5 mg kg-1). The observed data were found aligned with those predicted by in vitro-in vivo extrapolation. Based on the same extrapolation method combined with literature data, only very few P-gp inhibitors (i.e. CsA, quinine, quinidine) were predicted to increase the brain disposition of seletalisib in the clinical setting (maximal 3-fold changes).

Isolation of a Microsporum canis gene family encoding three subtilisin-like proteases expressed in vivo.

Microsporum canis is the main agent of dermatophytosis in dogs and cats and is responsible for frequent zoonosis. The pathogenesis of the disease remains largely unknown, however. Among potential fungal virulence factors are secreted keratinolytic proteases, whose molecular characterization would be an important step towards the understanding of dermatophytic infection pathogenesis. M. canis secretes a 31.5 kDa keratinolytic subtilisin-like protease as the major component in a culture medium containing cat keratin as the sole nitrogen source. Using a probe corresponding to a gene's internal fragment, which was obtained by polymerase chain reaction, the entire gene encoding this protease named SUB3 was cloned from a M. canislambdaEMBL3 genomic library. Two closely related genes, termed SUB1 and SUB2, were also cloned from the library using as a probe the gene coding for Aspergillus fumigatus 33 kDa alkaline protease (ALP). Deduced amino acid sequence analysis revealed that SUB1, SUB2, and SUB3 are secreted proteases and show large regions of identity between themselves and with subtilisin-like proteases of other filamentous fungi. Interest ingly, mRNA of SUB1, SUB2, and SUB3 were detected by reverse transcriptase nested-polymerase chain reaction from hair of experimentally infected guinea pigs. These results show that SUB1, SUB2, and SUB3 encode a family of subtilisin-like proteases and strongly suggest that these proteases are produced by M. canis during the invasion of keratinized structures. This is the first report describing the isolation of a gene family encoding potential virulence-related factors in dermatophytes.

Evaluation of immunogenicity and protective efficacy of a Microsporum canis metalloprotease subunit vaccine in guinea pigs.

In order to identify protective immunogens against Microsporum canis infection, a purified recombinant keratinolytic metalloprotease (r-MEP3) was tested as a subunit vaccine in experimentally infected guinea pigs. Both humoral and cellular specific immune responses developing towards r-MEP3 were evaluated, by enzyme-linked immunosorbent assay and by in vitro lymphocyte transformation tests respectively. Vaccination induced a strong antibody response, and a significant but transient lymphoproliferative response against the protein. However, the protocol failed to prevent fungal invasion or development of dermatophytic lesions. These results show that under the present experimental conditions, r-MEP3 specific antibodies are not protective against a challenge exposure. They also suggest that in the same model, the induction of cell-mediated immunity towards r-MEP3 is not sufficient, indicating the need for further research in the field of specific immune mechanisms involved in M. canis dermatophytosis.

Recombinant expression and antigenic properties of a 31.5-kDa keratinolytic subtilisin-like serine protease from Microsporum canis.

A secreted 31.5-kDa keratinolytic subtilase (SUB3; AJ431180) is thought to be a Microsporum canis virulence factor and represents a candidate for vaccination trials. In this study, the recombinant keratinase (r-SUB3) was produced by the Pichia pastoris expression system and purified to homogeneity. Recombinant SUB3 displayed identical biochemical properties with the native protease. Experimentally cutaneously infected guinea pigs showed specific lymphoproliferative response towards r-SUB3, while no specific humoral immune response was induced except for one animal. The heterologous expression of SUB3 provides a valuable tool for addressing further investigations on the role of this keratinase in the specific cellular immune response and on its use in vaccination trials in the cat.

Secreted metalloprotease gene family of Microsporum canis.

Keratinolytic proteases secreted by dermatophytes are likely to be virulence-related factors. Microsporum canis, the main agent of dermatophytosis in dogs and cats, causes a zoonosis that is frequently reported. Using Aspergillus fumigatus metalloprotease genomic sequence (MEP) as a probe, three genes (MEP1, MEP2, and MEP3) were isolated from an M. canis genomic library. They presented a quite-high percentage of identity with both A. fumigatus MEP and Aspergillus oryzae neutral protease I genes. At the amino acid level, they all contained an HEXXH consensus sequence, confirming that these M. canis genes (MEP genes) encode a zinc-containing metalloprotease gene family. Furthermore, MEP3 was found to be the gene encoding a previously isolated M. canis 43.5-kDa keratinolytic metalloprotease, and was successfully expressed as an active recombinant enzyme in Pichia pastoris. Reverse transcriptase nested PCR performed on total RNA extracted from the hair of M. canis-infected guinea pigs showed that at least MEP2 and MEP3 are produced during the infection process. This is the first report describing the isolation of a gene family encoding potential virulence-related factors in dermatophytes.

Humoral and cellular immune response to a Microsporum canis recombinant keratinolytic metalloprotease (r-MEP3) in experimentally infected guinea pigs.

In order to better understand the host-fungus relationship in Microsporum canis dermatophytosis and to identify major fungal antigens, the immune response to a crude exoantigen preparation and to a purified recombinant keratinolytic metalloprotease (r-MEP3) was evaluated in guinea pigs experimentally infected with M. canis. Humoral and cellular immune responses were assessed from day 0 to day 57 post-infection (PI), the former by enzyme-linked immunosorbent assay (ELISA) and the latter via a lymphocyte proliferation assay. Infected guinea pigs developed humoral and cellular responses to both M. canis exoantigen and r-MEP3, while no specific immune response to these antigens was observed in control animals. This is the first report on the development of both humoral and cell-mediated immune responses to a purified keratinase in M. canis dermatophytosis.

A recombinant 31.5 kDa keratinase and a crude exo-antigen from Microsporum canis fail to protect against a homologous experimental infection in guinea pigs.

A Microsporum canis recombinant 31.5 kDa keratinase and a M. canis crude exo-antigen were tested as vaccines in an experimental infection model in guinea pigs. Animals were vaccinated subcutaneously three times at two-week intervals with either the keratinase, the exo-antigen or the adjuvant alone. Cutaneous challenge was performed blindly. Both humoral and cellular-specific immune responses to M. canis antigens were evaluated every 14 days, while a blind evaluation of clinical lesion development and fungal persistency in skin were monitored weekly. Vaccination induced very high and significant (P < 0.01) antibody responses towards both antigens. High cell-mediated immune responses to both immunogens were also induced by vaccination. After challenge, however, scores reflecting the severity of dermatophytic lesions did not differ significantly between vaccinated and control groups at any time after challenge. These results suggest that, in the guinea pig, the induction of specific immune responses against the M. canis-secreted antigens used in this study are not protective against challenge exposure.