Genetic Pharmacotherapy: A New Approach to Drug Development for Schizophrenia Celia Gellman 1,2 , Susana Mingote 1,2 , Yvonne Wang 1,2 , Inna Gaisler-Salomon 1,3 , Stephen Rayport 1,2 1 Department of Psychiatry, Columbia University; 2 Department of Molecular Therapeutics, New York State Psychiatric Institute; and 3 Department of Psychology, Haifa University •Cost to bring drug to market has increased from $802 million in 2002 to $1.8 billion (Paul et al., 2010) •Despite this – number of new innovative medicines is decreasing. Over the past 5 years, there have been 50% fewer new molecular entities (NME) approved compared with the 5 years previous (Paul et al., 2010) •The major factor is high rates of late-stage drug failure. 66% of drug failure due to lack of therapeutic efficacy (Arrowsmith, 2011) •Target selection may be one of the most important determinants of attrition and thus overall R&D productivity (Paul et al., 2010) •We propose‘genetic pharmacotherapy’as a proof-of-concept tool to cut down on late-stage losses by expediting target validation prior to drug development Figure 1. Cre-lox recombination is a method of removing targeted DNA sequences from the genome via Cre recombinase (from the P1 Bacteriophage), which excises DNA between the Cre recognition sites (dubbed loxP) and recombines the flanking strands. A mouse transgenically modified with loxP sites flanking a portion of the gene of interest (a floxed gene) is bred with a mouse that carries the Cre transgene; the resulting F 1 progeny will have an excision/recombination event at all floxed alleles in tissue where Cre is present. Sources: www.scq.ubc.ca/targeting-your-dna-with-the-crelox-system cre.jax.org/introduction.html Figure 2. The DNA recombination event can be controlled temporally by a chimeric version of Cre that is attached to a modified estrogen receptor (ERT2). Though usually sequestered to cytoplasm, endogenous estrogen receptors move to the nucleus to act as a transcription factor when bound by estrogen. The modified ERT2 is likewise sequestered to the cytoplasm (a) but has virtually no affinity for endogenous estrogen; instead it enters the nucleus upon binding the high-affinity, high-specificity ligand tamoxifen (b), only then allowing Cre access to irreversibly recombine genomic DNA (c and d). How genetic pharmacotherapy could have accelerated the validation of D3 receptor antagonists for SCZ treatment Figure 3. Tetracycline transactivator (tTA)-controlled gene expression. In “Tet-off” gene regulation, the effector protein tTA binds to the TetO promoter, which activates transcription of the target gene. Upon the administration of doxycycline, tTA changes conformation and ceases to activate the target gene’s transcription. In the “Tet-on” system, a modified “reverse” tTA (rtTA) binds TetO only in the presence of doxycycline. Source: Lewandowski, 2001. Why develop a D3 receptor antagonist? •Antipsychotic therapy with classical neuroleptics induces extrapyramidal side effects (EPS; movement disorders) that contribute to patient non-adherence •Pharmacologic goal (1980s): Block mesolimbic dopaminergic transmission selectively while leaving unaffected the nigrostriatal connections implicated in motor control (Fig 4) Tet-off Tet-on • In classical drug development, therapeutic efficacy of drug targeting cannot be tested until selective ligands are available • D3 receptor ligand development efforts have yet to produce selective ligand • Therefore, it remains to be seen if selectively targeting D3 is therapeutic Figure 7. Complete excision of glutaminase exon 1 at DNA level following tamoxifen-induced Cre-mediated recombination and reduction in GLS1 expression. (a) Transgene map is shown with primer locations. (b) In the first two lanes of the gel, primers A and C were used to amplify DNA from Gls1 lox/+ (#1) and CagCreERT2::Gls1 lox/+ (#2) animals. Animal #1 (lane 1) shows two bands – one for the wt allele and the other containing the loxP site. In the Cre animal (lane 2) the loxP flanked sequence has been excised eliminating the primer A site. Primers X and C were used to demonstrate the recombination. The span between the primer sites in the unexcised DNA from the control (no Cre) animal is too long for amplification, so there is no band (lane 3), while the Cre-animal (lane 4) shows just the recombined band. (c) GLS1 mRNA levels are about 50% of control (no Cre) in hippocampus (Hipp), prefrontal cortex (PFC), in the left (L) and right (R) hemispheres of the Cre animals. Figure 4. Differential effects of an atypical neuroleptic and propsychotic drugs on mesolimbic versus nigrostrial dopaminergic transmission. (a) Prolonged administration (21 days) of classical neuroleptics L-sulpiride, Haloperidol and chlorpromazine decreases the number of spontaneously firing cells in rat VTA and SNc cells, whereas this effect is restricted to only VTA neurons with the atypical neuroleptic clozapine. (Adapted from Chiodo and Bunney, 1983.) (b) Acute amphetamine (1 mg/kg) and cocaine (5 mg/kg) administration increases synaptic dopamine concentrations preferentially in the nucleus accumbens (the post-synaptic target of the VTA), compared with the caudate nucleus (the post-synaptic target of the SNc). Source: Di Chiara and Imperato, 1988. a b Figure 5. Distribution of d2 and d3 receptors in coronal sections of the rat brain. (a) [ 125 I] iodosulpride binding corresponds to D2 receptor distribution. The distribution of D2 and D3 mRNA (b and c, respectively) is shown by selective 32 P labeled probes. CPu, caudate putamen; Acb, nucleus accumbens. Source: Sokoloff et al., 1990. a b c a b c d Genetic pharmacotherapy could accelerate targets’ proof-of-concept earlier in the drug development process and halt pursuit of targets doomed to fail …thereby reducing costs associated with each successful drug launch •Technique enables investigators to test targets with perfect specificity in advance of developing a specific ligand •In transgenic mice, target protein is up- or down- regulated at DNA level after the addition of an exogenous inducer Two well-established strategies : •Tamoxifen-inducible ubiquitous CreERT2 mice (Figures 1 and 2) •Tetracycline-regulatable ubiquitous tTA mice (Figure 3) •Induce gene knock-down in adulthood – avoid developmental confound of traditional knockouts (Gingrich and Hen, 2000) Concept of genetic pharmacotherapy Rising drug development costs – how to enhance productivity? • In 1990, D3 receptor was cloned • its expression is restricted to mesolimbic striatum (Fig 5) • means of achieving pharmacologic goal? • 20 years later, despite many efforts, such a drug has not been developed; whether D3 antagonists would be therapeutic remains to be determined • Inducing D3 knockdown via inducible-Cre technology could show whether D3 receptor antagonists are unequivocally worth the investment How genetic pharmacotherapy is advancing Gls1 inhibition validation Prioritizing target validation in drug development to increase R&D efficiency • Schizophrenia appears to be a disorder of excessive glutamate release, possibly due to deficits in interneurons (Belforte, 2010). • Hippocampal hyperactivity seen in SCZ (Schobel et al., 2009) may be related to this phenomenon through loss of interneuron-mediated inhibition. • In lieu of attempting to rescue lost neurons, one therapeutic strategy could be to use drug targeting that mitigates hyperactivity. • The contribution of the enzyme glutaminase to glutamate stores is use-dependent -- glutaminase becomes more important with increased glutamatergic activity (Masson et al., 2006; Tani et al., 2010). • Our laboratory has shown that mice haploinsufficient for glutaminase (Gls1 hets) exhibit a lower cerebral blood volume profile (an indicator of metabolic activity) in the hippocampus compared to controls—the reverse of what is seen in schizophrenic patients (Gaisler-Salomon, 2009). • Gls1 hets exhibit a‘schizophrenia resilience’phenotype with attenuated responsiveness to amphetamine (Fig. 6a), diminished dopamine release (Fig. 6b), and enhanced latent inhibition (Fig. 6c). Figure 6. Attenuated behavioral and neurochemical response to amphetamine in GLS1 hets. (a) Ambulatory distance was measured before and after i.p. injection of amphetamine (2 mg/kg) or saline (arrow). Amphetamine-treated Gls1 hets respond as saline-treated animals of either genotype. (b and c) Blunted amphetamine-induced dopamine release in Gls1 hets. (d) Gls1 hets exhibit potentiated latent inhibition, one of the hallmarks of antipsychotic treatment. Source: Gaisler-Salomon et al., 2009. Can Gls1 het‘schizophrenia resilience’phenotype be induced in adulthood, when disease has onset? •To eliminate the developmental confound of the Gls1 het phenotype, we can mimic administration of a Gls1 inhibitor by inducing the mutation in adulthood. •iCagCre::Gls lox/+ mice show complete DNA recombination following tamoxifen injections, and about a 50% reduction in glutaminase levels with qPCR (Fig. 7). •We are currently doing behavioral assays for resilience •The next step will be to apply this strategy in mouse models of schizophrenia to see whether we can‘treat’the mice. • Missteps in drug development may be due to a disconnect between a thorough understanding of disease mechanism and its effective application to ligand development • Genetic pharmacotherapy – crossing an inducible global deletor mouse with a conditional mouse – enables systematic target validation prior to developing new molecular entities: Conclusions References Arrowsmith J (2011) Trial watch: Phase III and submission failures: 2007-2010. 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Nature 347:146-151. Tani H, Dulla CG, Huguenard JR, Reimer RJ (2010) Glutamine is required for persistent epileptiform activity in the disinhibited neocortical brain slice. J Neurosci 30:1288-1300. a b Primers A,C Primers X,C Lane 1 2 3 4 c 0 0.2 0.4 0.6 0.8 1 1.2 Hipp PFC control expression L R 341 385 390 Copyright protected 1000 Posters. Copyright protected. F1000 Poste t protected. F1000 Posters. Copyright protected. F1000 Posters. Copyright ers. Copyright protected. F1000 Posters. Copyright protected. F1000 Posters. Copyright protected. F1 yright protected. F1000 Posters. Copyright protected. F1000 Posters. Copyright protected. F1000 Posters. ed. F1000 Posters. Copyright protected. F1000 Posters. Copyright protected. F1000 Posters ters. Copyright protected. F1000 Posters. Copyright protected. F ht protected. F1000 Posters. Copyrigh F1000 Pos