Browsing by Subject "farmakologia"
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(Helsingin yliopisto, 2011)Neurons can be divided into various classes according to their location, morphology, neurochemical identity and electrical properties. They form complex interconnected networks with precise roles for each cell type. GABAergic neurons expressing the calcium-binding protein parvalbumin (Pv) are mainly interneurons, which serve a coordinating function. Pv-cells modulate the activity of principal cells with high temporal precision. Abnormalities of Pv-interneuron activity in cortical areas have been linked to neuropsychiatric illnesses such as schizophrenia. Cerebellar Purkinje cells are known to be central to motor learning. They are the sole output from the layered cerebellar cortex to deep cerebellar nuclei. There are still many open questions about the precise role of Pv-neurons and Purkinje cells, many of which could be answered if one could achieve rapid, reversible cell-type specific modulation of the activity of these neurons and observe the subsequent changes at the whole-animal level. The aim of these studies was to develop a novel method for the modulation of Pv-neurons and Purkinje cells in vivo and to use this method to investigate the significance of inhibition in these neuronal types with a variety of behavioral experiments in addition to tissue autoradiography, electrophysiology and immunohistochemistry. The GABA(A) receptor γ2 subunit was ablated from Pv-neurons and Purkinje cells in four separate mouse lines. Pv-Δγ2 mice had wide-ranging behavioral alterations and increased GABA-insensitive binding indicative of an altered GABA(A) receptor composition, particularly in midbrain areas. PC-Δγ2 mice experienced little or no motor impairment despite the lack of inhibition in Purkinje cells. In Pv-Δγ2-partial rescue mice, a reversal of motor and cognitive deficits was observed in addition to restoration of the wild-type γ2F77 subunit to the reticular nucleus of thalamus and the cerebellar molecular layer. In PC-Δγ2-swap mice, zolpidem sensitivity was restored to Purkinje cells and the administration of systemic zolpidem evoked a transient motor impairment. On the basis of these results, it is concluded that this new method of cell-type specific modulation is a feasible way to modulate the activity of selected neuronal types. The importance of Purkinje cells to motor control supports previous studies, and the crucial involvement of Pv-neurons in a range of behavioral modalities is confirmed.
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(Helsingin yliopisto, 2016)Major depressive disorder is one of the most significant causes of disability worldwide. Currently, the main treatment options for depression are psychotherapy and antidepressant drugs that pharmacologically target the monoamine systems such as serotonin transporter (SERT) blocker fluoxetine. It has been hypothesized that impairments in synaptic function and plasticity, caused for example by stress, could underlie the manifestation of depression. Moreover, in rodent models chronic treatment with antidepressant drugs has been shown to enhance plasticity of the adult brain via brain-derived neurotrophic factor (BDNF). The effects of BDNF mediated via its receptor tropomyosin receptor kinase B (TrkB) promote synapse function and thus could facilitate recovery from depression. Interestingly, antidepressant drugs with different main pharmacological targets seem to share the ability to activate the TrkB receptor, however, the mechanisms how antidepressant drugs activate TrkB are not known. The delayed onset of action and limited therapeutic efficacy of antidepressant drugs has promoted interest toward finding more rapid-acting and effective treatment options for depression. Electroconvulsive therapy has been the treatment of choice for treatment resistant depressed patients, however, side effects and the disrepute among general public has limited its use. Recently, subanesthetic doses of dissociative anesthetic ketamine have been shown to rapidly alleviate depression symptoms in depressed patients who do not respond to conventional antidepressant drugs. The effects of ketamine on mood appear already couple of hours after single intravenous infusion and last for about one week. Ketamine has been shown to induce mammalian target of rapamycin (mTOR) via BDNF-TrkB signaling, rapidly promote synaptogenesis and alter neural network function. Furthermore, in small human studies another anesthetic isoflurane has rapidly alleviated symptoms of depressed patients. Yet, the potential of isoflurane in the treatment of depression has not been studied in large clinical trials. Since TrkB receptor is involved in regulation of synaptic plasticity, drugs that act as agonists or positive allosteric modulators of TrkB could be potentially beneficial in the treatment of CNS disorders characterized by impaired plasticity. The first aim of our studies was to develop a platform suitable for high-throughput screening of compounds regulating TrkB activity. We developed an in situ ELISA (enzyme-linked immunosorbent assay) method that detects phosphorylated TrkB receptors from cultured cells. The main advantage of the in situ ELISA compared to conventional ELISA is that the cells are cultivated directly on the ELISA plate making the additional transfer step of the cellular material from the cell culture plate to the ELISA plate unnecessary. To further validate the in situ ELISA method, we conducted a proof-of-concept screening of a small chemical library and found several compounds that dose-dependently activated TrkB receptor or inhibited BDNF-induced TrkB activation. The second aim was to examine the mechanism how the antidepressant drugs activate TrkB. Interestingly, we found that antidepressant drugs activate TrkB independently of BDNF. Moreover, SERT, the main pharmacological target of fluoxetine, was not required for the fluoxetine-induced TrkB activation. Furthermore, the antidepressant-induced TrkB activation was developmentally regulated. The ability of antidepressants to activate TrkB appeared around postnatal day 12. Interestingly, at this same developmental timepoint (P12) the ability of BDNF to activate TrkB decreased dramatically. Finally, we aimed to characterize the neurobiological basis for the possible antidepressant effects of isoflurane. We found that brief isoflurane anesthesia rapidly and transiently activated the TrkB-mTOR signaling and produced antidepressant-like behavioral response in the forced swim test in a TrkB-dependent manner. Single isoflurane treatment also produced an antidepressant-like phenotype in behavioral paradigms that normally require chronic treatment with conventional antidepressant drugs, suggesting that isoflurane may have rapid antidepressant effects similar to ketamine. Moreover, isoflurane facilitated hippocampal long-term potentiation when measured 24 hours after the treatment and affected the general neural network function by increasing activity of the parvalbumin-positive inhibitory interneurons in the hippocampus. In conclusion, our results improve the understanding of the mechanism of action of conventional antidepressant drugs and provide plausible neurobiological basis for the antidepressant effects of isoflurane. Our findings also support examining further the potential of anesthetics in the treatment of depressed patients who do not respond to the current treatment options.
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AMPA-type glutamate receptors and behavioural neuroplasticity : Bidirectional role of GluA1 subunits (Helsingin yliopisto, 2012)Neuroplasticity is defined as an ability of the nervous system to change when it responds to modified environmental or internal conditions. Functional neuroplasticity enables a change in behaviour and ultimately provides a major mechanism for enhanced survival in a given environment. There are two main types of plasticity, positive and negative plasticity. Positive plasticity describes abilities and brain processes that benefit an animal. Negative plasticity is related to processes that tend to be harmful. In this study, positive neuroplasticity was investigated in relation to aggressive behaviour and processing of novel signals; processes that are both required for successful survival in a changing habitat. Negative plasticity was studied in several stages of addiction, a psychiatric disease in which normal plasticity is interfered by drugs of abuse eventually leading to compulsive drug use. The neurotransmitter glutamate has a well-established role in chemical signalling between neurons, and an emerging role as a major component regulating neuroplasticity. Glutamate receptors of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type are tetramers composed of subunits GluA1-4 and they mediate a majority of the fast synaptic neurotransmission, that is also implicated in neuroplastic processes. Here, the AMPA receptor subunit GluA1-deficient mouse line (GluA1-KO mouse line) was studied using behavioural and neurochemical approaches. GluA1-KO mouse line provides an alternative approach to study the importance of AMPA receptors in neuroplasticity, as there are no subunit-selective pharmacological ligands available. GluA1-KO mice displayed less propensity to change their agonistic behaviour when exposed to an altered social environment in comparison with wild-type (WT) control mice, increased responsiveness towards spatial novelty followed by deficient habituation, decreased capability to develop tolerance to benzodiazepine flurazepam, and defective morphine-induced state-dependency. Taken together, these results propose a bidirectional role for GluA1 subunit in positive and negative neuroplastic processes. In summary, these results strengthen the role of GluA1 subunit in neuroplasticity, and may assist future drug development in disorders in which neuroplasticity plays a role.
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(Helsingin yliopisto, 2013)Major depressive disorder (MDD) affects millions of people every year and produces significant human suffering and economic burden to society. The symptomatology of MDD is heterogeneous and multidimensional, and only two core symptoms, depressed mood and anhedonia, are frequently shared by patients. Consequently, modeling of MDD is challenging, and only depression-related phenomena, not depressed mood itself, can be examined in animals. MDD is commonly treated with antidepressant drugs (or antidepressants, ADs). However, monoamine-based ADs act in a delayed-onset manner and often exhibit only moderate clinical efficacy. Electroconvulsive therapy (ECT) remains the treatment of choice for treatment-resistant depression (TRD) and for cases for which a rapid clinical response is required. Given the practical and ethical limitations of ECT, the development of fast-acting ADs is needed. Importantly, the NMDA receptor antagonist ketamine has been shown to produce rapid and long-lasting AD effects in TRD patients. Changes in the levels and signaling of neurotrophin brain-derived neurotrophic factor (BDNF) have been associated with the etiology of MDD. However, studies of genetically modified mice expressing altered levels of BDNF have not provided a solid link between BDNF deficiency and depression-related behavior. By contrast, emerging evidence indicates that the effects of ADs are mediated by BDNF and its tropomyosin-related kinase B receptor, TrkB. ADs enhance BDNF-TrkB signaling and thereby facilitate neuronal plasticity in the brain. Recent evidence indicates that these changes in plasticity lead to the restoration of juvenile-type plasticity in the adult rodent cortex, which allows environment-driven reorganization of brain networks. Based on these data, the network theory of AD action was formulated. However, it is unclear if this concept can be generalized to diverse neuronal networks. The main aims of this thesis were to investigate the importance of TrkB signaling in the anxiety- and depression-like behavioral phenotype in mice, to examine the role of BDNF-TrkB signaling in the antidepressant-like effects of glutamatergic drugs in mice, to study the network theory of ADs in a mouse fear extinction paradigm and to investigate the behavioral effects of adult fluoxetine treatment in mice exposed to fluoxetine early in life. When examining TrkB signaling-deficient mice (TrkB.T1), we observed that young and aged TrkB.T1 mice exhibited alterations in their exploration and emotional behavior and increased behavioral despair. These findings suggest that altered TrkB signaling leads to depression-like behavior, and thus, TrkB.T1 mice may be used as a genetic model of depression. We next studied selected glutamatergic drugs in behavioral despair models and determined that, similar to their effects in humans, ketamine and the AMPA receptor potentiator LY 451646 produce an antidepressant-like effect in mice. In contrast to classical ADs, these drugs were also effective in BDNF heterozygote knock-out mice. Furthermore, neither of these drugs influenced BDNF protein or Trk-phosphorylation levels in wild-type or BDNF-deficient mice. These data suggest that the antidepressant-like effects of ketamine may be independent of BDNF-TrkB signaling. Disturbances in the serotonergic system during early development may cause permanent behavioral effect in adult animals. In our study, early life exposure to fluoxetine, an AD that enhances serotonergic transmission, led to specific and persistent behavioral changes in adult animals. Intriguingly, adult fluoxetine treatment normalized some of these changes. We therefore examined whether fluoxetine can enable plastic changes in fear circuits in mice in conjunction with an environmental stimulus. We observed that the combination of fear exposure and fluoxetine treatment produced permanent fear extinction in the classical fear conditioning paradigm in mice. Importantly, neither fluoxetine nor extinction alone produced permanent fear erasure. This finding supports the network theory of AD action and clinical observations demonstrating the superiority of the combination of drug administration and psychotherapy for the treatment of post-traumatic stress disorder and depression. In conclusion, these data strengthen the connection between BDNF-TrkB signaling and the antidepressant-like effects of classical ADs and support the network hypothesis of AD action. In addition, these results also suggest that there may be fast-acting AD treatments with a mechanism of action that is independent of BDNF-TrkB signaling.
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(Helsingin yliopisto, 2013)Acute pain is an important warning signal, however, neuropathic pain and often chronic pain,lack a physiological function. Pain is a major clinical challenge and especially chronic and neuropathic pain are difficult to treat. On individual level, pain causes occupational and functional disability, suffering, and impairs quality of life. On a macro level pain and its direct and indirect consequences cause multi-billion expenses. Genetic factors and mechanisms underlying susceptibility to chronic pain have recently raised significant scientific interest. COMT-gene, which codes for catechol-O-methyltransferase (COMT), is subject for genetic polymorphic variation and COMT polymorphisms modulate pain and opioid analgesia in humans. The effects of COMT on pain and opioid responses were studied in rodents and humans. In mice, COMT deficiency was associated with altered stress- and morphine-induced analgesia reflecting weakened capacity of endogenous pain modulation and changes in opioidergic transmission. In normal mice, COMT inhibitors reduced thresholds of mechanical nociception and shortened thermal nociceptive latencies and thus increased nociceptive sensitivity in models of acute and inflammatory pain. Pronociceptive effects were COMT-dependent. In the spinal nerve ligation model of neuropathic pain in rats nitecapone decreased nociceptive symptoms - cold and mechanical hyperalgesia and allodynia. In humans, genetic variation of COMT gene was associated with pain phenotypes. The associations were strongest for the experimental pain phenotypes but also clinical pain phenotypes, such as acute postoperative pain, showed associations (uncorrected p=0.006-0.007)with three single nucleotide polymorphisms (SNPs). The strongest effect was observed in the SNP located in the 3´UTR-region of COMT, rs887200, pointing to importance of this region in regulation of nociceptive phenotypes and confirming the results in rodents. Together, these results confirm the role of COMT in pain and opioid responses. The antiallodynic effects of COMT inhibitors should be further studied in neuropathic pain since the safety and efficacy of current therapies are not satisfactory. In humans, mutations in COMT gene affect pain. The predictive value of individual SNPs, however, is limited and several SNPs of COMT as well as other genetic factors should be included in the same analysis or treatment algorithms possibly utilizing a haplotypic approach. Finally, the effects of most SNPs associated with pain phenotypes on COMT expression and activity are not known and should be explored in further studies.
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(Helsingin yliopisto, 2010)Parkinson s disease (PD) is a neurodegenerative disorder associated with a progressive loss of dopaminergic neurons of the substantia nigra (SN). Current therapies of PD do not stop the progression of the disease and the efficacy of these treatments wanes over time. Neurotrophic factors are naturally occurring proteins promoting the survival and differentiation of neurons and the maintenance of neuronal contacts. Neurotrophic factors are attractive candidates for neuroprotective or even neurorestorative treatment of PD. Thus, searching for and characterizing trophic factors are highly important approaches to degenerative diseases. CDNF (cerebral dopamine neurotrophic factor) and MANF (mesencephalic astrocyte-derived neurotrophic factor) are secreted proteins that constitute a novel, evolutionarily conserved neurotrophic factor family expressed in vertebrates and invertebrates. The present study investigated the neuroprotective and restorative effects of human CDNF and MANF in rats with unilateral partial lesion of dopamine neurons by 6-hydroxydopamine (6-OHDA) using both behavioral (amphetamine-induced rotation) and immunohistochemical analyses. We also investigated the distribution and transportation profiles of intrastriatally injected CDNF and MANF in rats. Intrastriatal CDNF and MANF protected nigrostriatal dopaminergic neurons when administered six hours before or four weeks after the neurotoxin 6-OHDA. More importantly, the function of the lesioned nigrostriatal dopaminergic system was partially restored even when the neurotrophic factors were administered four weeks after 6-OHDA. A 14-day continuous infusion of CDNF but not of MANF restored the function of the midbrain neural circuits controlling movement when initiated two weeks after unilateral injection of 6-OHDA. Continuous infusion of CDNF also protected dopaminergic TH-positive cell bodies from toxin-induced degeneration in the substantia nigra pars compacta (SNpc) and fibers in the striatum. When injected into the striatum, CDNF and GDNF had similar transportation profiles from the striatum to the SNpc; thus CDNF may act via the same nerve tracts as GDNF. Intrastriatal MANF was transported to cortical areas which may reflect a mechanism of neurorestorative action that is different from that of CDNF and GDNF. CDNF and MANF were also shown to distribute more readily than GDNF. In conclusion, CDNF and MANF are potential therapeutic proteins for the treatment of PD.
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CDNF in an experimental model of Parkinson's disease : studies on gene therapy and protein infusion (Helsingin yliopisto, 2014)Neurodegenerative diseases are characterized by progressive loss of distinct neuronal populations. In Parkinson s disease (PD) the most prominent cell loss is seen in the dopamine (DA) neuron population in the substantia nigra pars compacta (SNpc). The resulting decrease in striatal DA levels causes dysregulation of neuronal circuits controlling movement and leads to motor symptoms typical to the disease. As for other neurodegenerative diseases, there are no available treatments that would interfere with the degenerative process in PD. The purpose of this work was therefore to test the therapeutic potential of long-term delivery of the neurotrophic factor (NTF) cerebral dopamine neurotrophic factor (CDNF) in the rat partial 6-hydroxydopamine (6-OHDA) lesion model of PD. When injected unilaterally in the striatum, 6-OHDA causes progressive dose-dependent loss of DA neurons in the SNpc accompanied by asymmetrical motor impairment. The 6-OHDA model used in our NTF studies (2x10 µg 6-OHDA) showed a stable lesion progression with a cell loss at two weeks post-lesion corresponding to that seen in PD at symptom onset. In the 6-OHDA model, the DAergic system is traditionally evaluated using immunodetection methods or measurements of tissue neurotransmitter levels. Imaging methods, such as single-photon emission computed tomography (SPECT), allows in vivo detection of neuronal circuits, and together with the DA transporter (DAT) radioligand 2β-carbomethoxy-3β-(4-[123I]iodophenyl)tropane ([123I]β-CIT), SPECT/CT provided reliable estimations of the DA cell degeneration showing high correlation to immunohistochemical findings. The method is sensitive and selective and provides substantial benefits in pre-clinical research allowing longitudinal studies in living animals. The neuroprotective effect of CDNF was studied by applying the NTF intrastriatally as two-week protein infusion with osmotic pumps, or as gene therapy with a recombinant adeno-associated viral vector in 6-OHDA-lesioned rats. Both CDNF delivery methods normalized the amphetamine-induced rotational asymmetry and provided partial protection of the tyrosine hydroxylase (TH) reactive DAergic cells in the SNpc and DA fibers in the striatum. As for GDNF, there were indications of retrograde transport of CDNF, but contrary to what has been reported for GDNF, CDNF did not affect the intact rat DAergic system. In addition, there were differences between the treatments in the capacity to induce sprouting of TH-reactive fibers. Our results confirm that CDNF can be considered as a potential therapy in PD, and that the neuroprotective mechanism of CDNF differs from that of GDNF.
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(Helsingin yliopisto, 2019)Ethanol use disorders affect a vast number of people worldwide. In some individuals, controlled ethanol intake can gradually progress via ethanol abuse into addiction, characterized by escalated, uncontrolled and compulsive ethanol seeking and intake despite its negative consequences. A negative emotional state is common when ethanol is not available. The relapsing nature of this chronic disease also makes it difficult to treat. As the clinical efficiency of the currently available pharmacotherapies is relatively low, new treatment strategies are needed. The µ- and κ-opioidergic systems interacting with the brain’s reward pathway have been suggested to be central in controlling ethanol intake. The µ-opioidergic system is attributed to the rewarding and positive reinforcing effects of ethanol while the κ-opioidergic system is attributed to its negative reinforcing effects. It has been suggested that the µ-opioidergic system is more important in controlling ethanol intake while intake is still under control, while the role of the κ-opioidergic system increases as ethanol intake becomes more chronic, compulsive and relapsing. The main aim of this thesis was to clarify the role of µ- and κ-opioidergic mechanisms in the nucleus accumbens shell, a main brain area of the reward pathway, in controlling intermittent and relapse-like ethanol intake in rats. The used paradigms can roughly be considered to model aspects of ethanol intake before and after addiction has developed. Selective µ- or κ-opioid receptor agonists and antagonists were administered locally into the nucleus accumbens shell and systemic injections were used to elucidate this brain area’s overall role in controlling ethanol intake. These studies were undertaken as there is a gap in the knowledge on how the µ- and κ-opioidergic systems interacting with the nucleus accumbens shell affect ethanol intake and addiction-related behaviors per se. A high innate µ-opioidergic tone in the nucleus accumbens shell of alcohol-preferring Alko Alcohol (AA) rats has been proposed to account for their high ethanol preference but pharmacological studies are lacking. As local infusions of a selective µ-opioid receptor antagonist increased and agonist tended to decrease intermittent ethanol intake, the results support the notion that nucleus accumbens shell µ-opioidergic mechanisms participate in controlling ethanol intake and reward in AA rats. The role of nucleus accumbens shell κ-opioidergic mechanisms in controlling intermittent ethanol intake has not been extensively studied. Intra-accumbens shell administration of a selective κ-opioid receptor agonist had no effect but JDTic, a selective κ-opioid receptor antagonist, showed a weak long-term ethanol intake decreasing effect in AA rats. When these results are combined with the long-term decreasing effects shown after systemic JDTic administration, the results suggest that κ-opioid receptors are indeed able to control intermittent ethanol intake and the nucleus accumbens shell is one site participating in mediating these effects. The effects of JDTic on relapse-like ethanol intake in Long-Evans rats was examined because of the positive results from the previous study, earlier reports suggesting an increased tone of the accumbal κ-opioidergic system as ethanol addiction evolves and the lack of knowledge on what role the nucleus accumbens shell κ-opioid receptors have in relapse to ethanol intake. Both intra-accumbens shell and systemic JDTic attenuated relapse-like ethanol intake. These results suggest that the κ-opioidergic system interacting at least with the nucleus accumbens shell participates in controlling relapse-like ethanol intake. As the reference drug naltrexone, a non-selective antagonist, administered systemically also inhibited relapse-like ethanol intake, µ- and possibly also δ-opioidergic systems seem to have a role in mediating relapse. Taken together, these findings suggest that µ- and κ-opioidergic mechanisms are important in controlling intermittent ethanol intake and relapse to ethanol intake and the nucleus accumbens shell is one anatomical site mediating these effects. The results also suggest that selective κ-opioid receptor antagonism could be a feasible treatment strategy for ethanol use disorders.
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(Helsingin yliopisto, 2021)Most neurodegenerative diseases (NDD) are characterized by accumulation of toxic protein species leading to the loss of movement and cognitive disorders. In the two most common NDD Parkinson´s disease (PD) and Alzheimer´s disease (AD), there are almost 60 million patients worldwide. In the U.S, there were one million PD patients and an estimated economic burden of 51.9 billion dollars in 2017. As there is no disease-modifying therapy available, NDD lead to loss of life quality and eventually to untimely death. Current drug therapies for PD can only relieve symptoms of the disease by increasing dopaminergic activity in the brain but they do not address the underlying mechanisms of the disease such as clearance of detrimental protein aggregates. In previous studies, prolyl oligopeptidase (PREP) has been shown to negatively regulate autophagy and enhance the aggregation of alpha-synuclein (αSyn). Thus, PREP inhibitors have been shown to reduce aggregation and increase clearance of αSyn aggregates via enhanced autophagy. These mechanisms are mediated by protein-protein -interactions (PPI) of PREP with other proteins. Several small molecular inhibitors of PREP have been developed to target memory disorders, cancer, and NDD. However, none of these PREP inhibitors are in therapeutical use. The aim of this study was to investigate the effect of these inhibitors in two neurodegeneration-related functions of PREP: autophagy and αSyn dimerization. A further aim was to synthesize novel PREP ligands targeted to modulate these PPI-mediated functions of PREP. In the first part of this study, a structurally diverse set of 12 potent PREP inhibitors were tested in in vitro assays of autophagy and αSyn dimerization. The study revealed that the least potent inhibitor with IC50-value of 1010 nM had the most prominent effect on reduction of αSyn dimers and enhancing autophagy, while some of the highly potent inhibitors with IC50-value ranging from 0.32 to 1.2 nM did not have an effect on autophagy or αSyn dimerization. In the second part of the study, novel 4-phenylbutanoyl-α-aminoacyl-2(S)-tetrazolyl-pyrrolidines were designed and synthesized to target αSyn dimerization. Synthesized compounds had IC50-values ranging from 12 nM to 200 000 nM, but they all reduced αSyn dimerization in a cellular assay. Furthermore, molecular docking studies showed that these novel compounds have another putative binding mode to PREP compared to typical PREP inhibitors. These findings combined with the findings from the first part of the study suggest that the PREP mediated reducing effect on αSyn dimerization and enhancing effect on autophagy is not dependent on the magnitude of PREP inhibition but more likely due to conformational stabilization in PREP caused by ligand binding. Another speculative explanation is that PREP has another binding site, which modulates the conformation of PREP and thereby the PPI mediated functions of PREP. In the third part of the study, a human αSyn transgenic mouse, carrying A30P and A53T point mutations in αSyn was characterized and evaluated as a model for early onset PD. The model displayed behavioral alternations at an early age with differences in dopaminergic neurotransmission in the nigrostriatal pathway and accumulation of oligomeric αSyn leading to decreased tyrosine hydroxylase in the striatum and substantia nigra. Furthermore, we evaluated the effect of seven days treatment by the novel PREP inhibitor, HUP-55, in this model. Results in this study showed that HUP-55 was able to decrease the level of oligomeric αSyn in the striatum and substantia nigra. In summary, this study revealed and highlighted the disconnected structure-activity relationships between inhibition of the proteolytic activity and the PPI mediated functions of PREP. Moreover, this finding led to the development of a novel PREP ligand that was successfully tested in a PD mouse model.
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(Helsingin yliopisto, 2011)Type 2 diabetes is a risk factor for the development of cardiovascular disease. Recently, the term diabetic cardiomyopathy has been proposed to describe the changes in the heart that occur in response to chronic hyperglycemia and insulin resistance. Ventricular remodelling in diabetic cardiomyopathy includes left ventricular hypertrophy, increased interstitial fibrosis, apoptosis and diastolic dysfunction. Mechanisms behind these changes are increased oxidative stress and renin-angiotensin system activation. The diabetic Goto-Kakizaki rat is a non-obese model of type 2 diabetes that exhibits defective insulin signalling. Recently two interconnected stress response pathways have been discovered that link insulin signalling, longevity, apoptosis and cardiomyocyte hypertrophy. The insulin-receptor PI3K/Ak pathway inhibits proapoptotic FOXO3a in response to insulin signalling and the nuclear Sirt1 deacetylase inhibits proapoptotic p53 and modulates FOXO3a in favour of survival and growth. --- Levosimendan is a calcium sensitizing agent used for the management of acute decompensated heart failure. Levosimendan acts as a positive inotrope by sensitizing cardiac troponin C to calcium and exerts vasodilation by opening mitochondrial and sarcolemmal ATP-sensitive potassium channels. Levosimendan has been described to have beneficial effects in ventricular remodelling after myocardial infarction. The aims of the study were to characterize whether diabetic cardiomyopathy associates with cardiac dysfunction, cardiomyocyte apoptosis, hypertrophy and fibrosis in spontaneously diabetic Goto-Kakizaki (GK) rats, which were used to model type 2 diabetes. Protein expression and activation of the Akt FOXO3a and Sirt1 p53 pathways were examined in the development of ventricular remodelling in GK rats with and without myocardial infarction (MI). The third and fourth studies examined the effects of levosimendan on ventricular remodelling and gene expression in post-MI GK rats. The results demonstrated that diabetic GK rats develop both modest hypertension and features similar to diabetic cardiomyopathy including cardiac dysfunction, LV hypertrophy and fibrosis and increased apoptotic signalling. MI induced a sustained increase in cardiomyocyte apoptosis in GK rats together with aggravated LV hypertrophy and fibrosis. The GK rat myocardium exhibited decreased Akt- FOXO3a phosphorylation and increased nuclear translocation of FOXO3a and overproduction of the Sirt1 protein. Treatment with levosimendan decreased cardiomyocyte apoptosis, senescence and LV hypertrophy and altered the gene expression profile in GK rat myocardium. The findings indicate that impaired cardioprotection via Akt FOXO3a and p38 MAPK is associated with increased apoptosis, whereas Sirt1 functions in counteracting apoptosis and the development of LV hypertrophy in the GK rat myocardium. Overall, levosimendan treatment protects against post-MI ventricular remodelling and alters the gene expression profile in the GK rat myocardium.
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(Helsingin yliopisto, 2018)Acute myocardial infarction is a life-threatening condition that occurs as a result of reduced blood flow in the cardiac muscle, eventually leading to tissue damage. In infarcted areas, cardiomyocytes have insufficient ability to proliferate and replace the injured cells, which is associated with a deficient pumping capacity. A strictly regulated combinatorial interplay of transcription factors, e.g., GATA4, NKX2-5, TBX5, and MEF2C, orchestrates cardiac type gene expression during the cardiomyocyte differentiation and maturation processes. The aim of the present study was to (i) characterize the protein-protein interaction of the cardiac transcription factors GATA4-NKX2-5, (ii) evaluate the chemical agents that modify the synergy of GATA4-NKX2-5 in vitro, (iii) examine the capacity of the lead compound to promote myocardial repair in vivo after myocardial infarction and other cardiac injuries and (iv) study the structural features of the compound important for metabolism and cytotoxicity. Integration of the experimental mutagenic data with computational modeling suggests that the structural architecture of the GATA4-NKX2-5 interaction resembles the protein structure of the conserved DNA binding domain of nuclear receptors. Fragment-based screening, reporter gene-based optimization and pharmacophore searching were utilized to identify the most potent lead compound targeting the GATA4-NKX2-5 interaction: N-[4- (diethylamino)phenyl]-5-methyl-3-phenylisoxazole-4-carboxamide. This compound presented anti-hypertrophic effects in vitro and cardioprotective effects in vivo. In addition, structural analysis of the lead compound revealed the signature molecular features for metabolism and cytotoxicity. Current drug treatments are able to delay, but not prevent the progress of the heart failure; therefore, modulators of protein-protein interactions of key transcription factors may represent a novel class of pharmaceuticals for cardiac remodeling and repair.
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(typis Frenckellianis, 1845)
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Distribution, regulation and physiological roles of catechol-O-methyltransferase (COMT) in rodents (Helsingin yliopisto, 2012)Catechol-O-methyltransferase (COMT) metabolises catecholamines such as dopamine, noradrenaline and adrenaline, which are well-characterised neurotransmitters that play important roles in the regulation of physiological processes. The COMT enzyme exists in membrane-bound (MB-COMT) and soluble (S-COMT) forms that are both encoded by the same gene. Presently, the necessity and physiological importance of the existence of two COMT isoenzymes remains partially unknown. The aim of this thesis was to provide new insights into the specific distribution patterns of MB-COMT and S-COMT, to verify the proposed hormonal regulation of COMT and to investigate its possible physiological role in feeding behaviour. We used two different study designs to address the distribution of COMT in the brain. First, we utilised dopaminergic and noradrenergic toxins to assess the presence of COMT in presynaptic neurons originating from selected brain areas. Successful lesioning of the neurons was confirmed by measuring dopamine and noradrenaline levels in their respective projection areas. However, despite successful lesioning, no changes in COMT protein expression or activity could be noted, indicating that COMT is not present in presynaptic dopaminergic and noradrenergic neurons. The specific distribution patterns of MB-COMT and S-COMT were studied in an S-COMT-deficient mouse line (lacking the S-COMT form of COMT). By comparing these mice to wild type mice, we found that the general distribution patterns of both COMT isoforms were similar between the genotypes. Notably, S-COMT-deficient mice did not exhibit nuclear COMT staining, indicating that MB-COMT is not present in cell nuclei. Furthermore, S-COMT-deficient mice allowed us to examine the association of MB-COMT with the plasma membrane. Contrary to previous assumptions, MB-COMT is not associated with the plasma membrane, but instead may be attached to intracellular cytosolic membranes. The proposed regulatory function of oestrogens on COMT activity was studied by subjecting male and female Wistar rats to different hormonal treatments over a two-week period. Antagonising/modulating the effects of oestrogen via the administration of tamoxifen increased COMT activity and expression. Unexpectedly, increasing oestradiol levels in male rats also led to the upregulation of COMT expression in several tissues, underscoring the importance of COMT in the clearance of biologically active oestrogen metabolites. Finally, we used female rodents (rats and mice) to address the possible role of COMT in feeding behaviour. Our studies revealed that acute COMT inhibition affects the feeding pattern of rodents; COMT-inhibited animals have significantly more ''long'' meals (lasting 300 sec or more) than vehicle-treated animals. Notably, a relatively short starvation period of 16 h induced a slight, albeit not significant, decrease in prefrontal and striatal COMT activity in mice. This finding is interesting, especially as COMT activity is thought to be rather robust, and may suggest the increased importance of COMT during altered physiological status. In conclusion, although the specific roles of MB-COMT and S-COMT remain partly obscure, their absence from presynaptic dopaminergic and noradrenergic neuron terminals suggests a secondary (and perhaps more modulatory) role of COMT in neurotransmitter metabolism in the brain. Furthermore, the observed decrease in COMT activity after a relatively short starvation period suggests that COMT has an increased importance during certain physiological states. Finally, the observed regulatory function of oestrogens on COMT activity and protein expression in vivo may be of clinical importance as COMT inhibitors are used as adjuncts in the treatment of PD.
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(Helsingin yliopisto, 2009)Tobacco smoking is a worldwide health problem. Nicotine is generally accepted as the addictive substance in tobacco smoke. In addition to causing cancer, cardiac, vascular and pulmonary diseases, tobacco smoking has been suggested to act as a gateway drug to other drugs of abuse. The purpose of this study was to find out, whether chronic nicotine administration and its cessation potentiate the effects of morphine, and the mechanisms behind this. The study was performed in mice that received nicotine chronically via drinking water. This method of administration mimics human smoking in that the mice receive nicotine intermittently during their active time and that their plasma nicotine concentrations resemble those found in human smokers. First, we found that after chronic oral nicotine treatment mice are sensitized to the locomotor activity increasing effects of morphine. However, cocaine s stimulating effect was not enhanced after chronic nicotine treatment, suggesting that the enhanced stimulation is due to changes in the regulation of dopaminergic system. Second, we noticed that mice pretreated chronically with nicotine required smaller doses of morphine to display place conditioning than control mice, suggesting that chronic nicotine treatment enhances morphine s reinforcing effects. However, we found no changes in the number, affinity or functional activity of the µ-opioid receptors, which mediate morphine s effects, suggesting a minor role for the alterations of this receptor in the enhanced effects of morphine. Third, we found that nicotine pretreatment enhances the morphine-induced increase in nigrostriatal and mesolimbic extracellular dopamine levels, which are likely involved in the behavioural changes observed after chronic nicotine treatment. Furthermore, morphine s effect on GABAergic transmission in the ventral tegmental area/substantia nigra after chronic nicotine treatment was opposite to that in controls; morphine increased extracellular GABA in nicotine-pretreated mice. No changes in GABAB-receptor function were found after nicotine treatment. The changes in GABA transmission are possibly involved in the alterations in the morphine-induced dopamine transmission after chronic nicotine treatment. Last, chronic nicotine treatment altered cerebral levels of brain-derived neurotrophic factor (BDNF) and phosphorylated cAMP response element binding protein (pCREB), which are markers of neuronal plasticity. Chronic nicotine decreased BDNF and pCREB levels in the ventral tegmental area and nucleus accumbens, respectively. BDNF levels increased during nicotine abstinence, suggesting that mechanisms of neuronal plasticity are activated after the cessation of chronic nicotine treatment, which could be related to the long-lasting neuronal changes behind addiction. We also found nicotine-morphine interactions in pCREB levels in the soma areas of dopaminergic neurons, showing that nicotine and morphine interact also at the molecular level. These alterations may be involved in the behavioural and neurochemical nicotine-morphine interactions. In conclusion, these experiments suggest that chronic nicotine treatment profoundly enhances morphine s effects on striatal dopaminergic pathways and thereby enhances its effects on locomotion and reinforcement. The alterations in neuronal plasticity markers found in dopaminergic brain areas most probably are involved in these changes. Also, alterations in midbrain GABAergic systems may contribute. These experiments indicate that chronic nicotine exposure leads to cerebral neurochemical and molecular changes that facilitate the addictive properties of morphine. Thus, tobacco smoking may alter the addictive properties of other drugs of abuse.
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(Helsingin yliopisto, 2008)Diet high in dairy products is inversely associated with body mass index, risk of metabolic syndrome and prevalence of type 2 diabetes in several populations. Also a number of intervention studies support the role of increased dairy intake in the prevention and treatment of obesity. Dairy calcium has been suggested to account for the effect of dairy on body weight, but it has been repeatedly shown that the effect of dairy is superior to the effect of supplemental calcium. Dairy proteins are postulated to either enhance the effect of calcium or have an independent effect on body weight, but studies in the area are scarce. The aim of this study was to evaluate the potential of dairy proteins and calcium in the prevention and treatment of diet-induced obesity in C57Bl/6J mice. The effect of dairy proteins and calcium on the liver and adipose tissue was also investigated in order to characterise the potential mechanisms explaining the reduction of risk for metabolic syndrome and type 2 diabetes. A high-calcium diet (1.8%) in combination with dietary whey protein inhibited body weight and fat gain and accelerated body weight and fat loss in high-fat-fed C57Bl/6J mice during long-term studies of 14 to 21 weeks. α-lactalbumin, one of the major whey proteins, was the most effective whey protein fraction showing significantly accelerated weight and fat loss during energy restriction and reduced the amount of visceral fat gain during ad libitum feeding after weight loss. The microarray data suggest sensitisation of insulin signalling in the adipose tissue as a result of a calcium-rich whey protein diet. Lipidomic analysis revealed that weight loss on whey protein-based high-calcium diet was characterised by significant decreases in diabetogenic diacylglycerols and lipotoxic ceramide species. The calcium supplementation led to a small, but statistically significant decrease in fat absorption independent of the protein source of the diet. This augments, but does not fully explain the effects of the studied diets on body weight. A whey protein-containing high-calcium diet had a protective effect against a high-fat diet-induced decline of β3 adrenergic receptor expression in adipose tissue. In addition, a high-calcium diet with whey protein increased the adipose tissue leptin expression which is decreased in this obesity-prone mouse strain. These changes are likely to contribute to the inhibition of weight gain. The potential sensitisation of insulin signalling in adipose tissue together with the less lipotoxic and diabetogenic hepatic lipid profile suggest a novel mechanistic link to explain why increased dairy intake is associated with a lower prevalence of metabolic syndrome and type 2 diabetes in epidemiological studies. Taken together, the intake of a high-calcium diet with dairy proteins has a body weight lowering effect in high-fat-fed C57Bl/6J mice. High-calcium diets containing whey protein prevent weight gain and enhance weight loss, α-lactalbumin being the most effective whey protein fraction. Whey proteins and calcium have also beneficial effects on hepatic lipid profile and adipose tissue gene expression, which suggest a novel mechanistic link to explain the epidemiological findings on dairy intake and metabolic syndrome. The clinical relevance of these findings and the precise mechanisms of action remain an intriguing field of future research.
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(Helsingin yliopisto, 2017)Tobacco use is the leading cause of preventable death worldwide. Nicotine is the primary addictive component of tobacco, and repeated nicotine exposure often leads to dependence in humans. Nicotine is one of the most commonly co-used substances among polysubstance abuse patients and combined use of nicotine and other drugs of abuse, such as opioids, increases the use of one or both substances. The health consequences associated with polysubstance abuse exceed those of either drug alone. The current pharmacotherapeutic options are ineffective among opioid-substituted patients and the levels of successful smoking cessation are low. At the cellular level, nicotine and opioids have their own molecular mechanisms of action, yet both drugs increase the activity of the reward pathway by increasing dopamine transmission. The purpose of these studies was to investigate the possible effects of different opioid ligands on human neuronal nicotinic acetylcholine receptors (nAChRs) expressed in cell cultures. Our results showed that morphine has a partial agonist effect at α4β2 nAChRs, a very weak antagonist effect at α3* nAChRs and a positive synergistic effect with nicotine on α7 nAChR function. We found that methadone acts as a non-competitive antagonist (NCA) at α4β2 and α3* nAChRs. We also confirmed that methadone is a human α7 nAChR agonist. In the prolonged studies with methadone and morphine, we found that human α3*, α4β2 and α7 nAChRs are differentially regulated by prolonged exposure to methadone and morphine. Buprenorphine was shown to be a weak antagonist at α4β2, α3*, and α7 nAChRs, and codeine had a positive modulatory effect on α4β2 nAChRs and a weak NCA effect on α3* nAChRs. Oxycodone seemed to have a mixed competitive/non-competitive effect on α4β2 nAChRs and a weak NCA effect on α3* nAChRs. Tramadol was shown to be a NCA of α3* nAChRs and a weak NCA of α4β2 nAChRs. Naloxone and naltrexone were mixed competitive/non-competitive antagonists of α4β2 nAChRs, weak NCAs of α3* nAChRs and weak antagonists of α7 nAChRs. Taken together, these studies showed that many opioid ligands have effects on nAChRs that are independent of their agonist or antagonist properties at opioid receptors. These findings suggest that some effects of the nicotine opioid interaction seen in humans can be partially mediated through the receptor-level interplay of these substances. These results, together with earlier findings, highlight the complexity of different nAChRs and the multiplicity of responses to opioid ligands.
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(Helsingin yliopisto, 2010)Oral calcium sensitizer levosimendan and its active metabolite OR-1896 improve cardiac functions and prevent harmful cardiac remodeling in an experimental heart failure Calcium sensitizers is a novel class of inotropic agents that may possess potential advantages for the treatment of acute decompensated heart failure and other low output heart failure situations as compared with conventional inotropic drugs. Levosimendan, the only calcium sensitizer in clinical use at the moment, mediates its cardiac effect by the calcium sensitization of the contractile protein troponin C. Besides increasing the strength of cardiac contractions, levosimendan also exerts vasodilatory effects through opening of the sarcolemmal and mitochondrial ATP-sensitive potassium channels. The major elimination route of levosimendan is conjugation with glutathione to cysteine and cysteinylglycine conjugates. A minor metabolism route is reduction of levosimendan in intestine by bacteria into metabolite OR-1855, which is further acetylated to OR-1896, a long-lasting metabolite sharing the pharmacological properties of the parent compound. Currently levosimendan is used only as 24-h systemic infusion. The present study aimed at exploring the cardiovascular effects of oral calcium sensitizers in two different animal models of heart failure, namely in hypertensive and salt-sensitive Dahl/Rapp rats on high salt diet (a model of hypertensive heart failure with preserved systolic function), and in spontaneously diabetic Goto-Kakizaki rat with experimental myocardial infarction induced by coronary artery ligation (a model of post-infarct heart failure with impaired systolic functions). We were able to demonstrate that, in Dahl/Rapp rats, both levosimendan and OR-1896 prevented mortality, produced a transient decrease in blood pressure, prevented cardiac hypertrophy and improved cardiac contractility. These beneficial effects were associated with decreased cardiac ANP mRNA expression, a marker of pressure/volume overload, attenuation of oxidative stress, inflammatory response and cellular senescence. In Goto-Kakizaki rats, levosimendan and OR-1896 prevented MI-induced systolic heart failure, pronounced cardiac hypertrophy in the remote area, and sustained cardiomyocyte apoptosis. The beneficial effects of calcium sensitizers were associated with decreased myocardial ANP, inflammatory IL-6, and fibrogenic CTGF mRNA expressions, and corrections of MI-induced disturbances in calcium-handling proteins (SERCA2, Na+-Ca2+-exhanger). In conclusion, findings from the present study suggest a therapeutic role for oral calcium sensitizers in the treatment of hypertension-induced heart failure with preserved systolic function as well as in the prevention of post-infarct heart failure with decreased systolic function.
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(Helsingin yliopisto, 2016)Midbrain dopamine neurons exert a powerful influence on behavior and their dysfunction is associated with many neurological and neuropsychiatric diseases, including Parkinson s disease (PD). Dopamine neurons are large, complex and sensitive cells. Hence, their survival and correct function requires coordinated action of various transcription and regulatory factors both during development and aging. Potentially, one such factor is glial cell line-derived neurotrophic factor (GDNF). Ectopically applied GDNF is best known for its potent ability to protect and restore damaged dopaminergic neurons both in vitro and in vivo. GDNF-based therapies have been tested in clinical trials with PD patients with variable success. However, the function of endogenous GDNF in brain dopamine system development, aging and disease is poorly understood. Improvement in GDNF-based therapies requires better understanding of the physiological functions of GDNF in the brain. The current knowledge of endogenous GDNF function remains obscure, mainly due to the lack of proper animal models. The present study investigated the regulatory role of endogenous GDNF in the development, maintenance and function of midbrain dopamine neurons utilizing novel mouse models: GDNF conditional knock-out (cKO) mice and GDNF hypermorphic (GDNFh) mice over-expressing GDNF from the endogenous locus. GDNF cKO mice enable GDNF deletion solely from the central nervous system during embryonic development or later in adulthood, preserving its vital role in kidney development. Midbrain dopamine systems of these new mouse strains were studied with immunohistochemical, neurochemical, pharmacological, behavioral and molecular biology methods. We found more substantia nigra dopaminergic cells and elevated striatal dopamine levels in immature and adult GDNFh mice. In cKO mice, dopamine levels and cell numbers were unaltered, even upon aging, and regardless of the timing of GDNF deletion. Both mouse strains exhibited enhanced dopamine uptake, while responses to amphetamine were augmented in GDNFh mice and reduced in cKO mice. GDNFh mice also released more dopamine and GDNF elevation protected them in a lactacystin-based model of PD. Overall, dopamine neurons were more sensitive to moderate elevation than complete absence of endogenous GDNF, which suggests that they can adaptively compensate for GDNF loss. This highlights the limitation of broadly utilized gene deletion approaches in analyzing gene function. Our results indicate a clear role for endogenous GDNF in midbrain dopamine neuron development and function, but also demonstrate that GDNF is not required for their maintenance during aging. Furthermore, the ability of endogenous GDNF to protect animals in a PD model without the side effects associated with ectopic GDNF application suggests that elevation in endogenous GDNF levels may be an important future route for PD therapy.
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(Helsingin yliopisto, 2021)Parkinson’s disease (PD) is a chronic neurodegenerative disorder characterized by progressive loss of nigrostriatal dopamine neurons and propagating Lewy body pathology. Dopamine depletion in the striatum gives rise to the cardinal motor symptoms of PD. Current PD medications are based on replenishing striatal dopamine and provide symptomatic relief to the motor deficits. However, troublesome adverse effects and diminished efficacy complicate their long-term use. There is a great unmet medical need for a therapy that could slow or halt the progression of the disease. Neurotrophic factors (NTFs) are secreted proteins that promote neuronal growth, differentiation and survival. They are able to prevent the progression of neurodegeneration and restore aberrant neuronal function in a variety of preclinical models of PD. Nonetheless, outcomes from clinical trials have been disappointing. The purpose of this work was to characterize the effects of cerebral dopamine neurotrophic factor (CDNF), mesencephalic astrocyte-derived neurotrophic factor (MANF) and novel small molecule receptor tyrosine kinase RET agonists (BT13 and BT44) on nigrostriatal dopamine system and support their preclinical development as potential neurotrophic therapies of PD. To further clarify the functional effects of glial cell line-derived neurotrophic factor (GDNF), CDNF and MANF in the normal rat brain, microdialysis measurements were performed after a bolus injection of NTFs into the striatum. We saw augmented stimulus-evoked dopamine release and elevated dopamine turnover in the striatum of MANF-injected rats. GDNF injection increased in vivo tyrosine hydroxylase (TH) and catechol-O-methyltransferase activity and decreased monoamine oxidase A activity. These data are relevant when considering exogenously administered NTFs as a potential therapeutic approach for PD, since they have to be compatible with the existing dopaminergic medications of the patients. We also investigated the distribution properties of 125I-labeled and unlabeled CDNF after a nigral injection to intact rats. CDNF readily diffused into the brain areas surrounding the injection site and colocalized with TH-immunoreactive neurons in the substantia nigra. We did not detect active transportation of CDNF to distal brain areas. This characterization provides valuable insights into the selection of optimal delivery site and protocol for CDNF therapy. Our in vitro assays showed that RET agonists BT13 and BT44 were able to induce RET phosphorylation and activate downstream pro-survival signaling cascades Akt and ERK. They also supported the survival of cultured midbrain dopamine neurons from wild-type, but not from RET knockout, mice. The functional effects of BT13 and BT44 were evaluated in a unilateral 6-hydroxydopamine rat model of PD, where both compounds alleviated amphetamine-induced turning behavior. BT44 also showed potential to restore striatal TH-immunoreactive fibers. As blood-brain barrier penetrating compounds, BT13 and BT44 serve as promising leads that can be further developed into a disease-modifying therapy for PD.
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(Helsingin yliopisto, 2009)Glaucoma is a group of progressive optic neuropathies causing irreversible blindness if not diagnosed and treated in the early state of progression. Disease is often, but not always, associated with increased intraocular pressure (IOP), which is also the most important risk factor for glaucoma. Ophthlamic timolol preparations have been used for decades to lower increased intraocular pressure (IOP). Timolol is locally well tolerated but may cause e.g. cardiovascular and pulmonary adverse effects due to systemic absorption. It has been reported that approximately 80% of a topically administered eye drop is systemically absorbed. However, only limited information is available on timolol metabolism in the liver or especially in the human eye. The aim of this work was to investigate metabolism of timolol in human liver and human ocular tissues. The expression of drug metabolizing cytochrome P450 (CYP) enzymes in the human ciliary epithelial cells was studied. The metabolism of timolol and the interaction potential of timolol with other commercially available medicines were investigated in vitro using different liver preparations. The absorption of timolol to the aqueous humor from two commercially available products: 0.1% eye gel and 0.5% eye drops and the presence of timolol metabolites in the aqueous humor were investigated in a clinical trial. Timolol was confirmed to be metabolized mainly by CYP2D6 as previously suggested. Potent CYP2D6 inhibitors especially fluoxetine, paroxetine and quinidine inhibited the metabolism of timolol. The inhibition may be of clinical significance in patients using ophthalmic timolol products. CYP1A1 and CYP1B1 mRNAs were expressed in the human ciliary epithelial cells. CYP1B1 was also expressed at protein level and the expression was strongly induced by a known potent CYP1B1 inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The CYP1B1 induction is suggested to be mediated by aryl hydrocarbon receptor (AHR). Low levels of CYP2D6 mRNA splice variants were expressed in the human ciliary epithelial cells and very low levels of timolol metabolites were detected in the human aqueous humor. It seems that negligible amount of CYP2D6 protein is expressed in the human ocular tissues. Timolol 0.1% eye gel leads to aqueous humor concentration high enough to achieve therapeutic effect. Inter-individual variation in concentrations is low and intraocular as well as systemic safety can be increased when using this product with lower timolol concentration instead of timolol 0.5% eye drops.
