Assessment of cerebral drug occupancy in humans using a single PET-scan: A [11C]UCB-J PET study.

PURPOSE: Here, we evaluate a PET displacement model with a Single-step and Numerical solution in healthy individuals using the synaptic vesicle glycoprotein (SV2A) PET-tracer [11C]UCB-J and the anti-seizure medication levetiracetam (LEV). We aimed to (1) validate the displacement model by comparing the brain LEV-SV2A occupancy from a single PET scan with the occupancy derived from two PET scans and the Lassen plot and (2) determine the plasma LEV concentration-SV2A occupancy curve in healthy individuals. METHODS: Eleven healthy individuals (five females, mean age 35.5 [range: 25-47] years) underwent two 120-min [11C]UCB-J PET scans where an LEV dose (5-30 mg/kg) was administered intravenously halfway through the first PET scan to partially displace radioligand binding to SV2A. Five individuals were scanned twice on the same day; the remaining six were scanned once on two separate days, receiving two identical LEV doses. Arterial blood samples were acquired to determine the arterial input function and plasma LEV concentrations. Using the displacement model, the SV2A-LEV target engagement was calculated and compared with the Lassen plot method. The resulting data were fitted with a single-site binding model. RESULTS: SV2A occupancies and VND estimates derived from the displacement model were not significantly different from the Lassen plot (p = 0.55 and 0.13, respectively). The coefficient of variation was 14.6% vs. 17.3% for the Numerical and the Single-step solution in Bland-Altman comparisons with the Lassen plot. The average half maximal inhibitory concentration (IC50), as estimated from the area under the curve of the plasma LEV concentration, was 12.5 µg/mL (95% CI: 5-25) for the Single-Step solution, 11.8 µg/mL (95% CI: 4-25) for the Numerical solution, and 6.3 µg/mL (95% CI: 0.08-21) for the Lassen plot. Constraining Emax to 100% did not significantly improve model fits. CONCLUSION: Plasma LEV concentration vs. SV2A occupancy can be determined in humans using a single PET scan displacement model. The average concentration of the three computed IC50 values ranges between 6.3 and 12.5 µg/mL. The next step is to use the displacement model to evaluate LEV occupancy and corresponding plasma concentrations in relation to treatment efficacy. CLINICAL TRIAL REGISTRATION: NCT05450822. Retrospectively registered 5 July 2022 https://clinicaltrials.gov/ct2/results? term=NCT05450822&Search=Search.

Effects of escitalopram on synaptic density in the healthy human brain: a randomized controlled trial.

Selective serotonin reuptake inhibitors (SSRIs) are widely used for treating neuropsychiatric disorders. However, the exact mechanism of action and why effects can take several weeks to manifest is not clear. The hypothesis of neuroplasticity is supported by preclinical studies, but the evidence in humans is limited. Here, we investigate the effects of the SSRI escitalopram on presynaptic density as a proxy for synaptic plasticity. In a double-blind placebo-controlled study (NCT04239339), 32 healthy participants with no history of psychiatric or cognitive disorders were randomized to receive daily oral dosing of either 20 mg escitalopram (n = 17) or a placebo (n = 15). After an intervention period of 3-5 weeks, participants underwent a [11C]UCB-J PET scan (29 with full arterial input function) to quantify synaptic vesicle glycoprotein 2A (SV2A) density in the hippocampus and the neocortex. Whereas we find no statistically significant group difference in SV2A binding after an average of 29 (range: 24-38) days of intervention, our secondary analyses show a time-dependent effect of escitalopram on cerebral SV2A binding with positive associations between [11C]UCB-J binding and duration of escitalopram intervention. Our findings suggest that brain synaptic plasticity evolves over 3-5 weeks in healthy humans following daily intake of escitalopram. This is the first in vivo evidence to support the hypothesis of neuroplasticity as a mechanism of action for SSRIs in humans and it offers a plausible biological explanation for the delayed treatment response commonly observed in patients treated with SSRIs. While replication is warranted, these results have important implications for the design of future clinical studies investigating the neurobiological effects of SSRIs.

Stress Hormone Dynamics Are Coupled to Brain Serotonin 4 Receptor Availability in Unmedicated Patients With Major Depressive Disorder: A NeuroPharm Study.

BACKGROUND: A prominent finding in major depressive disorder (MDD) is distorted stress hormone dynamics, which is regulated by serotonergic brain signaling. An interesting feature of the cerebral serotonin system is the serotonin 4 receptor (5-HT4R), which is lower in depressed relative to healthy individuals and also has been highlighted as a promising novel antidepressant target. Here, we test the novel hypothesis that brain 5-HT4R availability in untreated patients with MDD is correlated with cortisol dynamics, indexed by the cortisol awakening response (CAR). Further, we evaluate if CAR changes with antidepressant treatment, including a selective serotonin reuptake inhibitor, and if pretreatment CAR can predict treatment outcome. METHODS: Sixty-six patients (76% women) with a moderate to severe depressive episode underwent positron emission tomography imaging with [11C]SB207145 for quantification of brain 5-HT4R binding using BPND as outcome. Serial home sampling of saliva in the first hour from awakening was performed to assess CAR before and after 8 weeks of antidepressant treatment. Treatment outcome was measured by change in Hamilton Depression Rating Scale 6 items. RESULTS: In the unmedicated depressed state, prefrontal and anterior cingulate cortices 5-HT4R binding was positively associated with CAR. CAR remained unaltered after 8 weeks of antidepressant treatment, and pretreatment CAR did not significantly predict treatment outcome. CONCLUSIONS: Our findings highlight a link between serotonergic disturbances in MDD and cortisol dynamics, which likely is involved in disease and treatment mechanisms. Further, our data support 5-HT4R agonism as a promising precision target in patients with MDD and disturbed stress hormone dynamics.

Common HTR2A variants and 5-HTTLPR are not associated with human in vivo serotonin 2A receptor levels.

The serotonin 2A receptor (5-HT2AR) is implicated in the pathophysiology and treatment of various psychiatric disorders. [18 F]altanserin and [11 C]Cimbi-36 positron emission tomography (PET) allow for high-resolution imaging of 5-HT2AR in the living human brain. Cerebral 5-HT2AR binding is strongly genetically determined, though the impact of specific variants is poorly understood. Candidate gene studies suggest that HTR2A single nucleotide polymorphisms including rs6311/rs6313, rs6314, and rs7997012 may influence risk for psychiatric disorders and mediate treatment response. Although known to impact in vitro expression of 5-HT2AR or other serotonin (5-HT) proteins, their effect on human in vivo brain 5-HT2AR binding has as of yet been insufficiently studied. We thus assessed the extent to which these variants and the commonly studied 5-HTTLPR predict neocortex in vivo 5-HT2AR binding in healthy adult humans. We used linear regression analyses and likelihood ratio tests in 197 subjects scanned with [18 F]altanserin or [11 C]Cimbi-36 PET. Although we observed genotype group differences in 5-HT2AR binding of up to ~10%, no genetic variants were statistically significantly predictive of 5-HT2AR binding in what is the largest human in vivo 5-HT2AR imaging genetics study to date. Thus, in vitro and post mortem results suggesting effects on 5-HT2AR expression did not carry over to the in vivo setting. To any extent these variants might affect clinical risk, our findings do not support that 5-HT2AR binding mediates such effects. Our observations indicate that these individual variants do not significantly contribute to genetic load on human in vivo 5-HT2AR binding.

Impaired behavioural pain responses in hph-1 mice with inherited deficiency in GTP cyclohydrolase 1 in models of inflammatory pain.

BACKGROUND: GTP cyclohydrolase 1 (GTP-CH1), the rate-limiting enzyme in the synthesis of tetrahydrobiopterin (BH4), encoded by the GCH1 gene, has been implicated in the development and maintenance of inflammatory pain in rats. In humans, homozygous carriers of a "pain-protective" (PP) haplotype of the GCH1 gene have been identified exhibiting lower pain sensitivity, but only following pain sensitisation. Ex vivo, the PP GCH1 haplotype is associated with decreased induction of GCH1 after stimulation, whereas the baseline BH4 production is not affected. Contrary, loss of function mutations in the GCH1 gene results in decreased basal GCH1 expression, and is associated with DOPA-responsive dystonia (DRD). So far it is unknown if such mutations affect acute and inflammatory pain. RESULTS: In the current study, we examined the involvement of the GCH1 gene in pain models using the hyperphenylalaninemia 1 (hph-1) mouse, a genetic model for DRD, with only 10% basal GTP-CH1 activity compared to wild type mice. The study included assays for determination of acute nociception as well as models for pain after sensitisation. Pain behavioural analysis of the hph-1 mice showed reduced pain-like responses following intraplantar injection of CFA, formalin and capsaicin; whereas decreased basal level of GTP-CH1 activity had no influence in naïve hph-1 mice on acute mechanical and heat pain thresholds. Moreover, the hph-1 mice showed no signs of motor impairment or dystonia-like symptoms. CONCLUSIONS: In this study, we demonstrate novel evidence that genetic mutations in the GCH1 gene modulate pain-like hypersensitivity. Together, the present data suggest that BH4 is not important for basal heat and mechanical pain, but they support the hypothesis that BH4 plays a role in inflammation-induced hypersensitivity. Our studies suggest that the BH4 pathway could be a therapeutic target for the treatment of inflammatory pain conditions. Moreover, the hph-1 mice provide a valid model to study the consequence of congenital deficiency of GCH1 in painful conditions.

Reliability of three versus five saliva sampling times for assessing the cortisol awakening response.

The cortisol awakening response (CAR) describes the sharp increase in cortisol secretion within 60 min after awakening. A summary of the CAR, the area under the cortisol curve above the awakening cortisol value (AUCi) is a widely used biomarker in health research. Estimation of the AUCi rely on a number of collected salivary samples at fixed time intervals (i.e., 5 samples in 15 min intervals) starting from awakening. Little empirical work has been executed to investigate the impact of reducing sampling times on AUCi estimation, which could potentially improve participant compliance and reduce operational costs. This study aimed to assess the reliability and validity of using 3-sample AUCi versus 5-sample AUCi, i.e., systematic and random fluctuations based on a large dataset from healthy and case individuals (total n = 537). We showed that the ideal timing of 3-sampling times was 0-30-60 min with a median difference in AUCi of - 8 nmol*h/L and interquartile range of 65 nmol*h/L among healthy individuals, and - 12 nmol*h/L and 78 nmol*h/L among case individuals. We subsequently validated the 3-sample AUCi by re-analyzing three published association studies. Overall, we obtained similar p-values with 3-sample AUCi when compared to 5-sample AUCi, while smaller effect sizes and standard errors were observed. In conclusion, despite a less precise estimation of the AUCi itself, our data support that the AUC measure of the CAR, based on three samples collected at 0-30-60 min from awakening, provides reliable results in association studies.

Kinetic models for estimating occupancy from single-scan PET displacement studies.

The traditional design of PET target engagement studies is based on a baseline scan and one or more scans after drug administration. We here evaluate an alternative design in which the drug is administered during an on-going scan (i.e., a displacement study). This approach results both in lower radiation exposure and lower costs. Existing kinetic models assume steady state. This condition is not present during a drug displacement and consequently, our aim here was to develop kinetic models for analysing PET displacement data. We modified existing compartment models to accommodate a time-variant increase in occupancy following the pharmacological in-scan intervention. Since this implies the use of differential equations that cannot be solved analytically, we developed instead one approximate and one numerical solution. Through simulations, we show that if the occupancy is relatively high, it can be estimated without bias and with good accuracy. The models were applied to PET data from six pigs where [11C]UCB-J was displaced by intravenous brivaracetam. The dose-occupancy relationship estimated from these scans showed good agreement with occupancies calculated with Lassen plot applied to baseline-block scans of two pigs. In summary, the proposed models provide a framework to determine target occupancy from a single displacement scan.

Genetic contributions to brain serotonin transporter levels in healthy adults.

The serotonin transporter (5-HTT) critically shapes serotonin neurotransmission by regulating extracellular brain serotonin levels; it remains unclear to what extent 5-HTT levels in the human brain are genetically determined. Here we applied [11C]DASB positron emission tomography to image brain 5-HTT levels and evaluated associations with five common serotonin-related genetic variants that might indirectly regulate 5-HTT levels (BDNF rs6265, SLC6A4 5-HTTLPR, HTR1A rs6295, HTR2A rs7333412, and MAOA rs1137070) in 140 healthy volunteers. In addition, we explored whether these variants could predict in vivo 5-HTT levels using a five-fold cross-validation random forest framework. MAOA rs1137070 T-carriers showed significantly higher brain 5-HTT levels compared to C-homozygotes (2-11% across caudate, putamen, midbrain, thalamus, hippocampus, amygdala and neocortex). We did not observe significant associations for the HTR1A rs6295 and HTR2A rs7333412 genotypes. Our previously observed lower subcortical 5-HTT availability for rs6265 met-carriers remained in the presence of these additional variants. Despite this significant association, our prediction models showed that genotype moderately improved prediction of 5-HTT in caudate, but effects were not statistically significant after adjustment for multiple comparisons. Our observations provide additional evidence that serotonin-related genetic variants modulate adult human brain serotonin neurotransmission.

An in vivo Pig Model for Testing Novel Positron Emission Tomography Radioligands Targeting Cerebral Protein Aggregates.

Positron emission tomography (PET) has become an essential clinical tool for diagnosing neurodegenerative diseases with abnormal accumulation of proteins like amyloid-ß or tau. Despite many attempts, it has not been possible to develop an appropriate radioligand for imaging aggregated α-synuclein in the brain for diagnosing, e.g., Parkinson's Disease. Access to a large animal model with α-synuclein pathology would critically enable a more translationally appropriate evaluation of novel radioligands. We here establish a pig model with cerebral injections of α-synuclein preformed fibrils or brain homogenate from postmortem human brain tissue from individuals with Alzheimer's disease (AD) or dementia with Lewy body (DLB) into the pig's brain, using minimally invasive surgery and validated against saline injections. In the absence of a suitable α-synuclein radioligand, we validated the model with the unselective amyloid-ß tracer [11C]PIB, which has a high affinity for ß-sheet structures in aggregates. Gadolinium-enhanced MRI confirmed that the blood-brain barrier was intact. A few hours post-injection, pigs were PET scanned with [11C]PIB. Quantification was done with Logan invasive graphical analysis and simplified reference tissue model 2 using the occipital cortex as a reference region. After the scan, we retrieved the brains to confirm successful injection using autoradiography and immunohistochemistry. We found four times higher [11C]PIB uptake in AD-homogenate-injected regions and two times higher uptake in regions injected with α-synuclein-preformed-fibrils compared to saline. The [11C]PIB uptake was the same in non-injected (occipital cortex, cerebellum) and injected (DLB-homogenate, saline) regions. With its large brain and ability to undergo repeated PET scans as well as neurosurgical procedures, the pig provides a robust, cost-effective, and good translational model for assessment of novel radioligands including, but not limited to, proteinopathies.

Evaluation of the α-synuclein PET radiotracer (d3)-[11C]MODAG-001 in pigs.

BACKGROUND: A positron emission tomography (PET) radiotracer to neuroimage α-synuclein aggregates would be a crucial addition for early diagnosis and treatment development in disorders such as Parkinson's disease, where elevated aggregate levels are a histopathological hallmark. The radiotracer (d3)-[11C]MODAG-001 has recently shown promise for visualization of α-synuclein pre-formed fibrils (α-PFF) in rodents. We here test the radiotracer in a pig model where proteins are intracerebrally injected immediately before scanning. Four pigs were injected in one hemisphere with 150 µg α-PFF, and in the other hemisphere, either 75 µg α-PFF or human brain homogenate from either dementia with Lewy bodies (DLB) or Alzheimer's disease (AD) was injected. All pigs underwent one or two (d3)-[11C]MODAG-001 PET scans, quantified with the non-invasive Logan graphical analysis using the occipital cortex as a reference region. RESULTS: The α-PFF and AD homogenate injected brain regions had high uptake of (d3)-[11C]MODAG-001 compared to the occipital cortex or cerebellum. BPND values in 150 µg α-PFF injected regions was 0.78, and in the AD homogenate injected regions was 0.73. By contrast, the DLB homogenate injected region did not differ in uptake and clearance compared to the reference regions. The time-activity curves and BPND values in the 150 µg and 75 µg injected regions of α-PFFs show a dose-dependent effect, and the PET signal could be blocked by pretreatment with unlabeled MODAG-001. CONCLUSION: We find that both α-PFF and AD brain homogenates give rise to increased binding of (d3)-[11C]MODAG-001 when injected into the pig brain. Despite its limited specificity for cerebral α-synuclein pathology, (d3)-[11C]MODAG-001 shows promise as a lead tracer for future radiotracer development.

Stress-Hormone Dynamics and Working Memory in Healthy Women Who Use Oral Contraceptives Versus Non-Users.

Background: Women who use oral contraceptives (OCs) may have a higher risk of developing a depression, which is associated with both vulnerability to stress and cognitive dysfunction. OCs disrupt the hypothalamic-pituitary-gonadal (HPG) axis by suppressing endogenous sex steroid production including estradiol. The HPG axis and the hypothalamic-pituitary-adrenal (HPA) axis are known to interact, possibly through modulations driven by estradiol. OCs may affect HPA regulation capacity, i.e., disturb cortisol dynamics such as the cortisol awakening response (CAR), and influence cognition such as working memory (WM). We hypothesize that OC use is associated with blunted cortisol dynamics and impaired WM performance relative to non-users. Methods: Data from 78 healthy women in the reproductive age were available from the CIMBI database. We evaluated if CAR and WM differed between OC users (n=25) and non-users (n=53) and if the level of estradiol modulated the OC use effect on CAR or WM in generalized least square models. Results: We found that OC users had a blunted CAR (p= 0.006) corresponding to a 61% reduction relative to non-users; however, no estradiol-BY-OC use interaction effect was observed on CAR. Also, OC users had higher cortisol levels at awakening compared to non-users (p = 0.03). We observed no effect of OC use or an estradiol-BY-OC use interaction effect on WM. Also, within the OC user group, neither CAR nor WM was associated with suppressed estradiol. CAR was not associated with WM. Conclusion: Healthy women who use OCs have blunted cortisol dynamics relative to non-users. However, we could not detect OC use effects on working memory in our sample size. We speculate that disrupted cortisol dynamics may be important for the emergence of depressive symptoms in OC users.

Associations between the cortisol awakening response and patient-evaluated stress and mood instability in patients with bipolar disorder: an exploratory study.

OBJECTIVE: The Cortisol Awakening Response (CAR) measured as the transient increase in cortisol levels following morning awakening appears to be a distinct feature of the HPA axis. Patients with bipolar disorder (BD) experience daily stress, mood instability (MI) and studies have shown disrupted HPA-axis dynamics. AIMS: to evaluate (1) patient-evaluated stress against the CAR, (2) associations between the CAR and mood symptoms, and (3) the effect of smartphone-based treatment on the CAR. METHODS: Patients with BD (n = 67) were randomized to the use of daily smartphone-based monitoring (the intervention group) or to the control group for six months. Clinically rated symptoms according to the Hamilton Depression Rating Scale 17-items (HDRS), the Young Mania Rating Scale (YMRS), patient-evaluated perceived stress using Cohen's Perceived Stress Scale (PSS) and salivary awakening cortisol samples used for measuring the CAR were collected at baseline, after three and six months. In the intervention group, smartphone-based data on stress and MI were rated daily during the entire study period. RESULTS: Smartphone-based patient-evaluated stress (B: 134.14, 95% CI: 1.35; 266.92, p = 0.048) and MI (B: 430.23, 95% CI: 52.41; 808.04, p = 0.026) mapped onto increased CAR. No statistically significant associations between the CAR and patient-evaluated PSS or the HDRS and the YMRS, respectively were found. There was no statistically significant effect of smartphone-based treatment on the CAR. CONCLUSION: Our data, of preliminary character, found smartphone-based patient-evaluations of stress and mood instability as read outs that reflect CAR dynamics. Smartphone-supported clinical care did not in itself appear to disturb CAR dynamics.

Blocking of efflux transporters in rats improves translational validation of brain radioligands.

BACKGROUND: Positron emission tomography (PET) is a molecular imaging technique that can be used to investigate the in vivo pharmacology of drugs. Initial preclinical evaluation of PET tracers is often conducted in rodents due to the accessibility of disease models as well as economic considerations. Compared to larger species, rodents display a higher expression and/or activity of efflux transporters such as the P-glycoprotein (P-gp). Low brain uptake could, therefore, be species-specific and uptake in rodents not be predictive for that in humans. We hypothesized that a better prediction from rodent data could be achieved when a tracer is evaluated under P-gp inhibition. Consequently, we compared the performance of eight neuroreceptor tracers in rats with and without P-gp inhibition including a specific binding blockade. This data set was then used to predict the binding of these eight tracers in pigs. METHODS: PET tracers targeting serotonin 5-HT2A receptors ([18F]MH.MZ, [18F]Altanserin, [11C]Cimbi-36, [11C]Pimavanserin), serotonin 5-HT7 receptors ([11C]Cimbi-701, [11C]Cimbi-717 and [11C]BA-10) and dopamine D2/3 receptors ([18F]Fallypride) were used in the study. The brain uptake and target-specific binding of these PET radiotracers were evaluated in rats with and without inhibition of P-gp. Rat data were subsequently compared to the results obtained in pigs. RESULTS: Without P-gp inhibition, the amount of target-specific binding in the rat brain was sufficient to justify further translation for three out of eight evaluated tracers. With P-gp inhibition, results for five out of eight tracers justified further translation. The performance in pigs could correctly be predicted for six out of eight tracers when rat data obtained under P-gp inhibition were used, compared to four out of eight tracers without P-gp inhibition. CONCLUSIONS: P-gp strongly affects the uptake of PET tracers in rodents, but false prediction outcomes can be reduced by evaluating a tracer under P-gp inhibition.

Differential effects of repeated low dose treatment with the cannabinoid agonist WIN 55,212-2 in experimental models of bone cancer pain and neuropathic pain.

Pain due to bone malignancies is one of the most difficult types of cancer pain to fully control and may further decrease the patients' quality of life. Animal models of chronic pain conditions resulting from peripheral inflammatory reactions or nerve injuries are responsive to treatment with cannabinoid agonists. However, the use of cannabinoid agonists in humans may be hampered by CNS related side effects and development of tolerance. In the present study, we investigated the effect of repeated low dose administration of the synthetic cannabinoid agonist WIN 55,212-2 on bone cancer pain and neuropathic pain in mice. In addition, we investigated the development of CNS related side effects and tolerance. We found that 0.5 mg/kg/day for 18 days reduced pain related behavior and expression of spinal glial fibrillary acidic protein in the bone cancer pain model but not in the neuropathic pain model. Furthermore, this treatment strategy was not found to induce measurable CNS related side effects or tolerance. Cancer cell viability assays and bone volume fraction assessed by micro computed tomography (microCT) demonstrated that these effects were not due to changes in cancer progression. The difference in WIN 55,212-2 efficacy between the bone cancer and neuropathic pain models may reflect the different pain generating mechanisms, which may be utilized in designing new therapeutic drugs.

Heterozygous mutations in GTP-cyclohydrolase-1 reduce BH4 biosynthesis but not pain sensitivity.

Human studies have demonstrated a correlation between noncoding polymorphisms of "the pain protective" haplotype in the GCH1 gene that encodes for GTP cyclohydrolase I (GTPCH1)-which leads to reduced tetrahydrobiopterin (BH4) production in cell systems-and a diminished perception of experimental and clinical pain. Here, we investigate whether heterozygous mutations in the GCH1 gene which lead to a profound BH4 reduction in patients with dopa-responsive dystonia (DRD) have any effect on pain sensitivity. The study includes an investigation of GCH1-associated biomarkers and pain sensitivity in a cohort of 22 patients with DRD and 36 controls. The patients with DRD had, when compared with controls, significantly reduced levels of BH4, neopterin, biopterin, and GTPCH1 in their urine, blood, or cytokine-stimulated fibroblasts, but their pain response with respect to non-painful stimulation, (acute) stimulus-evoked pain, or pain response after capsaicin-induced sensitization was not significantly different. A family-specific cohort of 11 patients with DRD and 11 controls were included in this study. The patients with DRD were heterozygous for the pain protective haplotype in cis with the GCH1 disease-causing mutation, c.899T>C. No effect on pain perception was observed for this combined haplotype. In conclusion, a reduced concentration of BH4 is not sufficient to alter ongoing pain sensitivity or evoked pain responses.

Effects of the dimeric PSD-95 inhibitor UCCB01-144 in mouse models of pain, cognition and motor function.

NMDAR antagonism shows analgesic action in humans and animal pain models, but disrupts cognitive and motor functions. NMDAR-dependent NO production requires tethering of the NMDAR to neuronal NO synthase (nNOS) by the postsynaptic density protein-95 (PSD-95). Perturbing the NMDAR/PSD-95/nNOS interaction has therefore been proposed as an alternative analgesic mechanism. We recently reported that UCCB01-125, a dimeric PSD-95 inhibitor with limited blood-brain-barrier permeability, reduced mechanical hypersensitivity in the complete Freund's adjuvant (CFA) inflammatory pain model, without disrupting cognitive or motor functions. Here, we investigated the analgesic efficacy in the CFA model of UCCB01-144, a PSD-95 inhibitor with improved blood-brain-barrier permeability. To extend the comparison of UCCB01-125 and UCCB01-144, we also tested both compounds in the spared nerve injury (SNI) model of neuropathic pain. Potential cognitive effects of UCCB01-144 were examined using the social transmission of food preference (STFP) test and the V-maze test, and motor coordination was assessed with the rotarod test. UCCB01-144 (10mg/kg) reversed CFA-induced mechanical hypersensitivity after 1h, and completely normalised sensitivity after 24h. In the SNI model, UCCB01-144 (30mg/kg) partially reversed hypersensitivity after 1h, but no effect was observed after 24h. UCCB01-125 did not affect SNI-induced hypersensitivity. Rotarod performance was unaffected by UCCB01-144, but 30mg/kg UCCB01-144 impaired performance in the STFP test. Collectively, UCCB01-144 reversed both CFA and SNI-induced hypersensitivity, but the efficacy in the SNI model was only transient. This suggests that enhanced BBB permeability of PSD-95 inhibitors improves the analgesic action in neuropathic pain states.

Intraplantar injection of tetrahydrobiopterin induces nociception in mice.

Tetrahydrobiopterin (BH4) is implicated in the development and maintenance of chronic pain. After injury/inflammation, the biosynthesis of BH4 is markedly increased in sensory neurons, and the pharmacological and genetic inhibition of BH4 shows analgesic effects in pre-clinical animal pain models. Intrathecal injections of BH4 have been shown to induce and enhance pain-like behaviours in rats, suggesting that under chronic pain conditions BH4 may act by facilitating central sensitisation. So far it is unknown whether BH4 acts on peripheral sites of the somatosensory system or whether BH4 per se provokes nociceptive pain behaviours. The purpose of this study was therefore to investigate the acute nociceptive effects of intraplantar injection of BH4. BH4 was found to induce dose-dependent licking/biting of the paw lasting 5 min, which was not observed following an injection of biopterin (inactive BH4 metabolite). Paw swelling, measured as paw thickness and weight, was not observed after BH4 injection. To explore possible mechanisms of action of BH4, the effect of local pre-treatment with indomethacin, Nω-nitro-L-arginine methyl ester, Nω-nitro-L-arginine, capsazepine and ruthenium red was tested. Morphine served as a positive control. Intraplantar pre-injection of morphine dose-dependently inhibited BH4-induced nociception, while none of the other compounds showed any statistical significant antinociception. These results suggest that BH4 exhibits nociceptive properties at peripheral sites of the somatosensory system, proposing an as yet unexplored involvement of BH4 in peripheral nociceptive processes. However, this appears not to be mediated through nitric oxide and prostaglandin release or by activation of the transient receptor potential vanilloid 1.

GCH1 variants, tetrahydrobiopterin and their effects on pain sensitivity.

Background A great proportion of the variation in pain experience and chronicity is caused by heritable factors. Within the last decades several candidate genes have been discovered either increasing or decreasing pain sensitivity or the risk of chronic pain in humans. One of the most studied genes is the GCH1 gene coding for the enzyme GTP cyclohydrolase 1 (GCH1). GCH1 catalyses the initial and rate-limiting step in the biosynthesis of tetrahydrobiopterin (BH4). The main function of BH4 is regulation of monoamine and nitric oxide biosynthesis, all involved in nociceptive signalling. Methods In this topical review we focus on the implication of the GCH1 gene and BH4 in painful conditions. We discuss experimental evidence from our group in relation to relevant research publications evaluating the BH4 pathway in pain. Studies assessing the role of GCH1 and BH4 in pain consist of human and animal studies, including DOPA-responsive dystonia (DRD) patients and hph-1 mice (a genetic mouse model of DRD) having mutations in the GCH1 gene as well as preclinical studies with the GCH1 inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP). The hypothesis is that genetic and pharmacological reduction of GCH1 would result in lower pain sensitivity. Results Previous studies have demonstrated that a particular "pain protective" GCH1 haplotype, found in 15% of the general human population, is linked to decreased pain sensitivity. We further support these findings in DRD patients, showing normal thresholds to mechanical and thermal stimuli, whereas a trend towards lower pain sensitivity is seen following chemical pain sensitisation. Consistent with these observations, non-injured hph-1 mice displayed normal mechano- and thermosensation compared to wild-type mice. After peripheral inflammation with Complete Freund' Adjuvant or sensitisation with capsaicin the mutant mice exhibited lower sensitivity to mechanical and heat stimuli. Moreover, hph-1 mice showed decreased nociception in the first phase of the formalin test. Several studies report analgesic effects of GCH1 inhibition with 90-270 mg/kg DAHP in rat models of inflammatory and neuropathic pain. However, we could not completely replicate these findings in mice. Fairly higher doses of DAHP (≥270 mg/kg) were needed to reduce inflammatory pain in mice, but the window between antinociception and toxic effects was small, since 400 mg/kg DAHP affected motor performance and general appearance. Also, the analgesic effects were marginal in mice compared to that observed in rats. Conclusions Variations in the GCH1 gene in both humans and mice appear to regulate pain sensitivity and pain behaviours, particularly after pain sensitisation, whereas pain sensitivity to phasic mechanical and thermal stimuli is normal. Moreover, pharmacological inhibition of GCH1 shows antinociceptive effects in preclinical pain studies, though our studies imply that GCH1 inhibition may have a small therapeutic index. Implications The implication of the GCH1 gene in pain may increase our understanding of the risk factors of chronic pain development and improve current pain therapy by personalised medicine. In addition, inhibition of GCH1 provides a potential target for analgesic drug development, though GCH1 inhibitors should possess local or partial effects to avoid serious side-effects to the central nervous system and cardiovascular system.

Anxiety- and depression-like phenotype of hph-1 mice deficient in tetrahydrobiopterin.

Decreased tetrahydrobiopterin (BH4) biosynthesis has been implicated in the pathophysiology of anxiety and depression. The aim of this study was therefore to characterise the phenotype of homozygous hph-1 (hph) mice, a model of BH4 deficiency, in behavioural tests of anxiety and depression as well as determine hippocampal monoamine and plasma nitric oxide levels. In the elevated zero maze test, hph mice displayed increased anxiety-like responses compared to wild-type mice, while the marble burying test revealed decreased anxiety-like behaviour. This was particularly observed in male mice. In the tail suspension test, hph mice of both sexes displayed increased depression-like behaviours compared to wild-type counterparts, whereas the forced swim test showed a trend towards increased depression-like behaviours in male hph mice, but significant decrease in depression-like behaviours in female mice. This study provides the first evidence that congenital BH4 deficiency regulates anxiety- and depression-like behaviours. The altered responses observed possibly reflect decreased hippocampal serotonin and dopamine found in hph mice compared to wild-type mice, but also reduced nitric oxide formation. We propose that the hph-1 mouse may be a novel tool to investigate the role of BH4 deficiency in anxiety and depression.

UCCB01-125, a dimeric inhibitor of PSD-95, reduces inflammatory pain without disrupting cognitive or motor performance: comparison with the NMDA receptor antagonist MK-801.

Excessive N-Methyl-d-aspartate receptor (NMDAR)-dependent production of nitric oxide (NO) is involved in the development and maintenance of chronic pain states, and is mediated by postsynaptic density protein-95 (PSD-95). By binding to both the NMDAR and neuronal NO synthase (nNOS), PSD-95 mediates a specific coupling between NMDAR activation and NO production. NMDAR antagonism shows anti-nociceptive action in humans and animal models of chronic pain but is associated with severe disturbances of cognitive and motor functions. An alternative approach to modulate the NMDAR-related activity is to perturb the NMDAR/PSD-95/nNOS complex by targeting PSD-95, thereby decreasing NO production without interfering with the NMDAR ion channel function. Here, we compared the effects of a dimeric PSD-95 inhibitor, UCCB01-125, and the NMDAR antagonist, MK-801, on mechanical hypersensitivity in the complete Freund's adjuvant (CFA) model of inflammatory pain. To examine side-effect profiles we also compared the effects of UCCB01-125 and MK-801 in tests of attention, long-term memory, and motor performance. When administered concurrently with CFA, both MK-801 and UCCB01-125 prevented the development of CFA-induced mechanical hypersensitivity 1 and 24 h after treatment. Moreover, UCCB01-125 was found to reverse CFA-induced hypersensitivity when administered 24 h after CFA treatment, an effect lasting for at least 3 days. At the dose reducing hypersensitivity, MK-801 disrupted attention, long-term memory, and motor performance. By contrast, even high doses of UCCB01-125 were devoid of side-effects in these tests. The data suggest that PSD-95 inhibition is a feasible strategy to prevent both development and maintenance of chronic inflammatory pain, while avoiding NMDAR antagonism-related side-effects.