PHA3032 NotesTheme 2: Pharmacology of Mood and Behaviour

Lecture 5 – Schizophrenia and Antipsychotic Drugs

BackgroundSchizophrenia is a relatively common disorder affecting about 1% of the population at a flat rate. The onset of the disease commonly occurs between the ages of 15 – 30. It is likely to have some component that allows for a genetic transmission of the disorder, however environmental factors contribute greatly to the extent and severity of the disease.

There are no definitive physiological or biochemical diagnostic tests, and the disease is identified by symptomology: flat affect, bizarre delusions, prominent hallucinations, functioning in work, social relations and self-care are diminished. The symptoms are divided into three main modalities: positive, negative and cognitive symptoms. Positive symptoms include delusions and auditory hallucinations. Negative symptoms include social withdrawal, a lack of drive and a blunting of emotions. Cognitive symptoms include lack of attention, deficits in memory and problems with executive function.

Neurochemical DeficitIt is known that psychotic symptoms arise from cocaine and amphetamine use (suggesting an elevation in dopamine). There are psychotic symptoms that arise from NMDA channel blocker overuse as seen with PCP and ketamine. Finally hallucinations can arise from LSD, the activation of 5-HT2A receptors. As such, schizophrenia probably involves a range of receptor classes.

However the most evidence for a neurochemical deficit in schizophrenia is that there is an over activation of the dopaminergic system. It has been shown that during a psychotic episode there is an increase in D2 receptor occupancy, and post mortem studies show an increase in number of D2 receptors in the schizophrenic brain.

The dopaminergic pathway that is most relevant in schizophrenia is the mesocortical/mesolimbic pathway. The pathway arises in the ventral tegmental area (VTA) where all the cell bodies are, and the axons leave this area to innervate the nucleus accumbens and the prefrontal cortex. This pathway is largely concerned with behaviour (agitation, psychosis, cognitive

functions, language and affect).

Drug TargetsHistorically patients were sedating using antihistamines. Chlorpromazine is a prototypical agent still used in schizophrenia today, traditionally used as a strong sedating agent with mild antihistamine action. Chlorpromazine turned out to be a potent antagonist at dopamine receptors. All potent antipsychotic/neuroleptic agents are potent antagonists of dopamine receptors, and clinical potency of most of these agents correlates with the blockade of D2 dopamine receptor sites.

The inhibition of actions of dopamine acting through the mesolimbic pathway include psychomotor slowing, emotional quietening, affective indifference and inhibition of aggression. These are effect that we want to see in a dopamine antagonist to combat the symptoms of schizophrenia. However, the other two dopaminergic pathways (nigrostriatal and tuberohypophyseal) will also have their actions blocked, which will give us unwanted effects. As such, most of the major unwanted side effects are due to the extension of the pharmacological effects following the blockade of D2 receptors at these other, unwanted sites.

Inhibition of the action of dopamine acting through the nigrostriatal pathway result in “extrapyramidal” symptoms (EPS) or motor disturbances. Drug induced Parkinsonism or rigidity/bradykinesia can be observed.

Inhibition of actions of dopamine mediated through the tuberohypophyseal dopaminergic pathway lead to an increase in prolactin release (hyperprolactinaemia). This can cause gynacomastia and amenorrhea (in females).

Antipsychotics/neuroleptics fall into several classes: classical antipsychotics (e.g. chlorpromazide, haloperidol) and atypical/2nd generation antipsychotics (e.g. clozapine). These are the three main drugs to remember.

Classical Antipsychotics30% of schizophrenic patients respond completely to antipsychotic/neuroleptic therapy. Many respond only partially. Classical antipsychotics work really well for the positive symptoms of schizophrenia, but the negative and cognitive symptoms tend to be resistant to these classical antipsychotics. While they’re not curative, they certainly help reduce hospitalisation.

The current hypothesis for the mechanism of action of antipsychotics is that there is an over activation of dopamine receptors in the nucleus accumbens. The regulation of the mesolimbic

pathway is important in many emotional behaviours and contributes strongly to the positive symptoms of psychosis, and by blocking D2 receptors you treat these positive symptoms.

If you look at the classical antipsychotics more closely, they’re not particularly ‘tidy’ drugs. Chlorpromazine blocks D2 receptors, as well as H1, M, 5-HT2, and α receptors. Most of the classical antipsychotics resemble chlorpromazine.

Interestingly haloperidol only blocks D2 receptors (selective), and does not cause as many side effect. Why is this?

This is because by only blocking D2 receptors, haloperidol does not cause sedation, postural hypotension, dry mouth/blurred vision/constipation or weight gain. These side effects are due to the blockade of various other receptors (left) which are only caused by chlorpromazine.

If chlorpromazine produces more side-effects due to its non-selective blocking mechanism, why is it preferred? It is because it produced less of the Parkinsonism effects (EPS). It does this because dopamine neurons usually exert a tonic inhibitory influence on cholinergic neurons within the striatum. A D2 blockade removes this inhibitory influence and intensifies the activity of the cholinergic system (leading to EPS). The fact that chlorpromazine is also an M receptor antagonist helps curb this side effect.

Atypical AntipsychoticsThough chlorpromazine is still used, nowadays the atypical antipsychotics are more widely prescribed. This is because they treat a wider range of symptoms – atypical antipsychotics treat the negative AND positive symptoms well (variable response with cognitive symptoms) c.f. classical antipsychotics which only treat the positive symptoms.

Why is clozapine (an atypical antipsychotic) so good? It shares a similar pharmacological profile to chlorpromazine with one exception: it is a potent antagonist of 5-HT2C receptors. It is due to this particular blockade of 5-HT2C

receptors that it is able to treat a larger range of symptoms.

Where it is hypothesised that the over-activation of the mesolimbic pathway, in the nucleus accumbens, is responsible for the positive symptoms of psychosis, there is also a hypothesis that there is a hypo-activation of the mesocortical pathway, in the pre-frontal cortex, which is responsible for the negative and cognitive symptoms of psychosis.

So how do we increase the dopamine activity of the mesocortical pathway?

In the prefrontal cortex there are no D2 receptors. So adding a D2 agonist will not help. The receptors here are D1 – dopaminergic neurons will release

dopamine and it will act on D1 receptors in the prefrontal cortex. However, dopaminergic neurons are negative regulated by 5-HT – that is, the activation of 5-HT2A receptors cause a decrease in the amount of dopamine released. A potent atypical antipsychotic that is strong at blocking 5-HT2A

receptors is able to remove this level of regulation and increase dopamine release, changing the hypo-activation of the mesocortical pathway into a more normal level of activation.

Drugs

Chlorpromazine. Typical antipsychotic. Blocks D2 receptors, as well as H1, M, 5-HT2, and α receptors. Used to treat schizophrenia. Because of its anti-muscarinic properties, there are less EPS observed. Most sedating, most likely to cause hypotension. Only treats positive symptoms of schizophrenia.

Haloperidol. Typical antipsychotic. Only blocks D2 receptors, so less side effects, but most likely to cause EPS. Only treats positive symptoms of schizophrenia.

Clozapine. Atypical antipsychotic. Similar profile to chlorpromazine, but it can also block 5-HT2C receptors so can treat a larger range of symptoms. Treats negative and positive symptoms of schizophrenia.

Lecture 6 – Anxiety and Anxiolytics, Sedatives and OCD

BackgroundAnxiety is a normal emotion experienced at some time by virtually all people. It is characterised by an unsettling feeling of uncertainty and apprehension. It can involve restlessness, agitation, tachycardia, sweating, GI disturbances, weeping and sleep disturbances. This ensures survival, but in excess can cripple an individual. Anxiety and panic disorders are often chronic illnesses in some patients, this is characterised by remissions and exacerbations – recurrence of the illness can be due to situational stress.

The area of the CNS which controls the fear response is the amygdala. It recruits different brain regions to experience feelings of fear/avoidance, endocrine output of fear, autonomic output of fear and the re-experiencing of fear. As such, it is important to look at the neurotransmitters that are present in the amygdala to determine what’s causing anxiety. Some of the transmitter systems that drive amygdala activity include noradrenaline, GABA and 5-HT.

hSert is the human 5-HT transporter. The expression and activity of this transported is regulated by a promoter called 5HTTLPR. This 5HTTLPR is only present in humans and primates, and alleles are either identified as being “short” (S) or “long” (L). It has been shown that the S variant results in lower SERT expression and activity. Humans with the 5HTTLPR SS genotype have a greater predisposition towards anxiety and depression related traits.

BenzodiazepinesBenzodiazepines have been the mainstay of treatment for anxiety, but antidepressant drugs such as TCAs, MAOIs, RIMAs, SSRIs have been shown to be effective in treating some forms of anxiety. Benzodiazepines act by enhancing the actions of GABA at the GABAA site.

GABA is the major inhibitory neurotransmitter in the CNS. GABAA receptors are ligand gated receptors (ionotropic), and are widely distributed among the CNS. The activation of GABAA receptors results in increases in Cl- ion permeability and entry of this ion into the cell, resulting in hyperpolarisation (and inhibition) of the neuron. Benzodiazepines do not do anything on their own, they only augment the effects of the endogenous neurotransmitter GABA.

The GABAA receptor is formed from a combination of 5 subunits forming the ion channel receptor. You can get different GABAA receptors formed depending on which subunits you combine. There is a family of GABAA receptors that arise from the combinations of different subunits. Benzodiazepines will only respond to GABAA receptors that are comprised of α1, α2, α3 and α5 subunits.

So it is the α subunit that is essential for the modulation of the GABAA receptor by BDZs (and contains the BDZ binding site). It is the α subunit which determines the affinity and efficacy of BDZ binding. As mentioned above, only GABAA receptors that are α1, α2, α3 or α5 containing are sensitive to BDZs – α4 or α6 containing GABAA receptors DO NOT interact with BDZs.

Benzodiazepines that work on the α2 and α3 containing subunits are usually responsible for the anxiety-relieving effects. If your benzodiazepine causes sedation, then it most likely targets α1-containing GABAA receptors. The α5 subunits in the hippocampus are associated with the memory impairing effects of BDZs.

Choice of BDZAll BDZs are relatively non-selective. They all reduce anxiety and aggression, produce sedation and induce sleep, reduce muscle tone and co-ordination, and have an anti-convulsant effect. Clinical efficacy for BDZs are the same i.e. they are all targeting the same receptor, their pharmacodynamics

don’t change. However, their pharmacokinetic characteristics vary considerably. The speed of onset is determined by lipid solubility (access to CNS), duration of action is dependent on metabolism, and longer durations BDZs usually form active metabolites.

So diazepam is quite long lasting as it is metabolised into a range of active metabolites before it is rendered inactive. Chlordiazepoxide is also long lasting. Something like lorazepam is much shorter acting however, because there are not secondary active metabolites, they are immediately metabolised and excreted.

Commonly Used BDZsFlurazepam – used for insomnia – 1-2 hours ½ life

Nitrazepam – used for insomnia – 20-30 hours ½ life

Diazepam – used for anxiety, panic attacks – 20-90 hours ½ life

Using BDZs to Treat AnxietyTreatment usually involved brief interrupted courses of 2-4 weeks duration with a tapered withdrawal of drug. In panic disorder, continuous treatment for as long as 6 months to a year may be necessary. Periodic breaks from treatment (and/or dose reduction) are useful in determining whether continued drug use in necessary. Withdrawal is similar to alcohol withdrawal.

BDZs are relatively safe in terms of overdosage compared with alcohol, and have predictable side effects. These side effects include excessive sedation (fatigue, poor concentration, confusion) as well as ataxia/muscle weakness. Some tolerance develops to both these side effects, with little tolerance developing to their anti-anxiety effects. There is also a decrease in REM sleep with a rebound following discontinuing treatment. Additional side effects include disinhibition (feelings of anger or irritability) as well as anterograde amnesia – BDZs obliterate memory of events experienced while under their influence. There is some psychological dependence, and strong physical dependence.

BDZs in InsomniaShort-term insomnia is best treated by a DBZ for 2 days to 1 week (if necessary). Long-term insomnia usually requires that BDZs be used for a prolonged period. These should be re-appraised within a month, and short-to-median ½ life BDZs are used e.g. temazepam, nitrazepam.

Zolpidem is a new selective BDZ for insomnia. It is technically not a benzodiazepine, but it does target the same targets as BDZs (α1 containing receptors). It shows good sedative action, with little anticonvulsant or muscle relaxing activity. It also has a rapid onset, and is short acting (3-6 hours).

5-HT and AnxietyThere are a multitude of 5-HT types, and they all serve various roles. For example, 5-HT1C and 5-HT1D are associated with migraine, whereas 5-HT2A receptors are important in schizophrenia. Interestingly,

all of the receptor subtypes are also strongly linked with anxiety. So new therapies for anxiety disorders don’t have to just focus on GABAA receptors, but 5-HT receptors are also a good candidate. This is why some antidepressants can be effective in anxiety, as they also target 5-HT receptors.

BuspironeBuspirone is a partial agonist at 5-HT1A receptors. Paradoxically, through activation of 5-HT1A receptors, it inhibits 5-HT transmission. Importantly, it has no action at BDZ receptors. The benefits of using buspirone over BDZs is that there is less potential for sedation and psychomotor impairment, as well as less potential for abuse and dependence. They have similar efficacy to BDZs in generalised anxiety, but are less effective than BDZs in acute panic

attacks. Buspirone also has a slower onset of action than BDZs (weeks vs. days) – improvement noted within 1-2 weeks, with further improvement noted over 4-6 weeks. Some of the side effects though include dizziness, nausea, headache, some drowsiness/insomnia, nervousness and fatigue.

Obsessive Compulsive Disorder (OCD)OCD involves recurring obsessions and/or compulsions. They are excessive and unreasonable, which cause marked distress and consume time, interfering with normal life. The prevalence of OCD is approximately 1-5% - symptoms include cleaning, washing, checking, arranging, counting, repeating and collecting. SSRIs (antidepressants) and behavioural therapy is useful for moderate to severe OCD.

Drugs

Flurazepam. GABAA agonist at the BDZ site. Used for insomnia. 1-2 hours ½ life

Nitrazepam. GABAA agonist at the BDZ site Used for insomnia. 20-30 hours ½ life

Diazepam. GABAA agonist at the BDZ site Used for anxiety, panic attacks. 20-90 hours ½ life, as it has a number of active metabolites.

Zolpidem. GABAA agonist at the BDZ site, though not structurally a BDZ. Little anti-convulsant or muscle relaxing side effects. Used for insomnia. 3-6 hour ½ half-life.

Buspirone is a partial agonist at 5-HT1A receptors but inhibits 5-HT transmission. Used for generalised anxiety disorder. Less potential for sedation and psychomotor impairment, as well as less potential for abuse and dependence. Slower onset of action vs. BDZ (weeks)

Lecture 7 – Anti-depressant and Anti-manic Drugs

BackgroundThe incidence of depression in the total population is about 5%. Only about 30% of depressed individuals are in treatment, and up to 15% of severely depressed individuals will commit suicide. It can be characterised by a depressed mood, apathy, weight/appetite changes, sleep disturbances, psychomotor agitation/retardation, and fatigue, feelings of guilt/worthlessness, executive dysfunction and suicidal ideation. Depression is twice as likely in women, peak onset is 20-40 years.

Biological Basis for DepressionThe main hypothesis for the biological basis of depression is the monoamine hypothesis – that there is something wrong with transmitters like noradrenaline or 5-HT. This hypothesis was extended to suggest that it was a lack of these transmitters. Evidence in support of this hypothesis was that be depleting these transmitters, depressive effects were seen. Also, by augmenting the levels of these neurotransmitters in individuals who were depressed seemed to help.

Most of the drugs for depression work in a very simple way. They either target the monoamine uptaker by blocking it, or by targeting the MAO and preventing it from breaking down the neurotransmitter.

If this was the only factor that caused depression, then you would expect to see an immediate increase in mood after using these drugs once the plasma levels are recorded and the biochemical effect is accounted for. Instead, it actually takes a long period of time before an improvement in mood is observed. This suggest there is not just blocking of the uptake transporter or of MAO, but some long-term process that is being targeted.

As such there is a modified hypothesis that takes into account monoamine receptors. The evidence to support that it is not just neurotransmitter levels that cause depression, is the observation that depression is associated with an abnormal upregulation of monoamine receptors – elevated 5-HT2A receptors observed in frontal cortex of depression suicides. This new modified hypothesis suggests that antidepressant drugs act by elevating monoamine levels, which then results in a down-regulation (normalisation) of monoamine neurotransmitter receptors. Particularly implicated at 5-HT2, as well as α and β adrenoceptor subtypes.

This is not the full story however, as there is some evidence to suggest that depression is associated with neuronal loss in the hippocampus and frontal cortex. It is also thought that depression is associated with a hyperactive hypo-pituitary axis – increased cortisol levels cause cortisol-dependent

stress related atrophy of neurons within these regions, associated increased in glutamate levels and potential for excitotoxic damage.

TCAsTricyclic antidepressants (TCAs) are the original class of antidepressants. Desipramine is the most well known in this class. TCAs block the re-uptake of NA and 5-HT to varying degrees, though most usually favour blockade of NA re-uptake.

All TCAs have at least 2 other actions: blockade of muscarinic receptors (anti-SLUD), blockade of H1 receptors (sedation) or blockade of α1-adrenoceptors (orthostatic hypotension).

TCAs are toxic. Overdose results in atrial/ventricular extrasystoles, sudden death – ventricular fibrillation. This is due to anti-muscarinic effect and blockade of NA reuptake.

SSRIsSSRIs are very selective and specifically block the re-uptake of 5-HT. A classic example of an SSRI is fluoxetine. Increased 5-HT release in combination with increased synaptic transmission ultimately results in a down-regulation of postsynaptic 5-HT2A receptors.

SSRI side effects are generally acute and are attenuated over time. The side effects are attributed to the intial increase in 5-HT stimulating a whole range of receptors (5-HT2A, 5-HT2C, 5-HT3 and 5-HT4) before there is a down-regulation of 5-HT reuptake transporters.

In terms of long-term side effects, the is little sedation/insomnia, no orthostatic hypotension, no anticholinergic effects and no cardiac toxicity (c.f. TCAs)

However, SSRIs should not be administered in conjunction with other MAOIs, SSRIs, TCAs, St John’s Wort, Sumatriptan or ecstasy. Can cause serotonin syndrome (excessive increase in synaptic 5-HT

levels) characterised by: agitation, confusion, sweating, shivering, tremor, diarrhoea, fever, in-coordination.

Irreversible MAOIsThese include agents like tranylcypromine. These prefer to target (but don’t always) MAOA which breaks down NA and 5-HT. By inhibiting MAO, you prevent a breakdown of NA and 5-HT, so synaptic levels of these both increase. These irreversible MAOIs tend to be non-selective and block MAOB as well, and they bind to MAO and completely destroy its function. Enzyme activity returns with the resynthesis of new enzyme (2-3 weeks). Ultimately they increase cytoplasmic and released levels of NA and 5-HT.

Side effect include insomnia, postural hypotension, atropine-like side effects (though less than TCAs) and sexual dysfunction (loss of libido/impotence). There is also an issue with the cheese reaction and irreversible MAOIs: they can inhibit MAO present in gut and liver, which allows unmetabolised dietary monoamines to have free access to body, which can overload NA and 5-HT stores in the nerves leading to hypertensive crisis.

RIMAsReversible monoamine oxidase inhibitors (RIMAs) include moclobamide. RIMAs bind reversibly to MAOA, they are selective for it, and will NOT lead to cheese reaction. Can cause insomnia and nausea, but does not cause postural hypotension or atropine like side-effects.

SARIsSerotonin antagonist and reuptake inhibitors (SARIs) include nefazadone. SARIs are a selective 5-HT reuptake inhibitor (less potent than either TCAs or SSRIs. Predominantly, they are a selective antagonist for 5-HT2A receptors – reducing the adverse effects associated with initial 5-HT receptor stimulation, so effective in limiting anxiety that SSRIs tend to cause initially, also have a particular benefit in sleep disturbances and less effect on sexual function.

Bipolar Disorder“Manic depression” – depression alternates with mania. Lithium stabilises mood though the mechanism of action is largely unclear. It is possible that it permeates the voltage depended Na+ channels and is not pumped out. This could allow it to accumulate in excitable tissues, blocking the release of monamines, enhancing their reuptake? Seriously, who knows? It works.

Drugs

Desipramine. Most well known TCA.

Theme 3: Hormones and Endocrine Mechanisms

Lecture 9 – Hypothalamic-Pituitary Hormones

BackgroundThe endocrine system regulates many body activities and consists of a variety of organs that secrete substances directly into the blood which in turn affect the function of target tissues. Why might we want to target the endocrine system? As a replacement in hormone deficient states (e.g. thyroid hormone for hypothyroidism), to modify malfunction of endocrine systems (e.g. carbimazole for hyperthyroidism), to alter normal function where this is an inconvenience (e.g. contraceptive pill), and to analyse the functional integrity of the endocrine system (e.g. somatotrophin for GH release).

How can drug agents influence endocrine systems? They can mimic or block the actions of endogenous hormones. Consider a partial agonist, it may act as an agonist in the absence of endogenous hormones or an antagonist in the presence of endogenous hormones by competing for the same sites.

Hypothalamic-pituitary axisThis is known as the co-ordination centre of the endocrine system and controls or influences the activity of other endocrine organs. The pituitary gland is about the size of a pea in a human brain and is responsible for the synthesis and release of “trophic” hormones (trophic means to stimulate the activity of another endocrine gland). Because it regulates the secretion of numerous hormones, it is known as the master gland. In addition to releasing trophic hormones, it can also release hormones which act directly on target tissues.

The pituitary gland has a connection with the hypothalamus, and is split into two lobes. The anterior pituitary, or adenohypophysis, can release trophic hormones which can act on the thyroid (to produce TSH), can act on the adrenal cortex to release ACTH, as well as acting on the ovaries and testes to release FSH and LH. In addition to releasing these trophic hormones, it can also release primary hormones such as prolactin which can act on mammary glands, and growth hormone (GH) for bone growth.

The posterior pituitary, or neurohypophysis, only releases primary hormones that act directly on the tissue, and include oxytocin (can act on muscles of the uterus) and antidiuretic hormone (ADH, acts on kidney tubules).

The Posterior PituitaryThe posterior pituitary consists largely of terminals of nerve cells which lie in hypothalamic nuclei. So the hormones are synthesised in the hypothalamus, get transported down the hypophyseal tract, where they reside in the posterior pituitary i.e. peptides get synthesised in the hypothalamus, pass down along axons into the posterior pituitary, then into the blood. The nerve endings are in close association with the capillaries in the posterior pituitary, which is important as the hormones are synthesised and then released into the blood vessels where they can circulate around the body.

Vasopressin and oxytocin are structurally similar, so it is no surprise that you can occasionally have them bind to each other’s receptors. There are three types of vasopressin receptors: V1A, V1B and V2. There is one oxytocin receptor (OT).

OxytocinOxytocin contracts the smooth muscle of the uterus – used to stimulate labour and to treat postpartum haemorrhage. Oxytocin receptor antagonists can be used as well to prevent the onset of labour (agents include atosiban and barusiban), which although are just as good as B2 agonists, they are far less effective than nifedipine. In addition, oxytocin can be used to cause milk release during lactation (it contracts the myoepithelial cells surrounding mammary gland alveoli). Oxytocin has also been linked to positive social interactions – mother-infant bonding, love and pair bonding, sexual arousal and behaviour. It has been shown to enhance social contact and promote social cohesion – increases trust and reduces fear, is calming.

Antidiuretic Hormone (ADH)Arginine vasopressin (AVP) is involved in the control of body water, and is released in response to an increase in blood osmolality (or decrease in blood volume). Vasopressin activates various cell surface receptors such as V1A on blood vessels to cause vasoconstriction, V1B in the anterior pituitary to cause ACTH release, and V2 in renal collecting tubules to cause an increase expression of water channels and increased water reabsorption.

Diabetes Insipidus – ADH deficiencyPatients have increased thirst; polydipsia, polyuria. This is due to either a decrease in vasopressin being released (neurogenic DI) or a decreased response to vasopressin (nephrogenic DI). Normally vasopressin will be released from the posterior pituitary and act on V2 receptors on collecting ducts, causing increased water channel expression and thus increased reuptake of water. In neurogenic DI, there is a reduced amount of ADH being secreted from the posterior pituitary, less receptor activation, less expression of water channels, less water reabsorption. However in nephrogenic DI, the right amount of ADH is being released from the posterior pituitary, but the V2 receptors are missing or unresponsive, so less water channel expression and less reuptake.

Pituitary DI – treat with vasopressin analogues. Vasopressin has a very short half-life, so treat with desmopressin (V2 selective, so treats problem in collecting duct without causing hypertension or other issues if it was V non-selective).

SIADH – ADH excessSyndrome of Inappropriate ADH Secretion (SIADH) is caused by a small cell carcinoma in the lung, causing ectopic vasopressin secretion, resulting in hypertension and fluid retention. Treated with V receptor antagonists such as demeclocycline.

The Anterior PituitaryThe anterior pituitary is composed of a heterogeneous collection of numerous cell types. It is connected with the hypothalamus via the hypothalamic-pituitary portal vascular system.

Hypothalamic-releasing factors (except DA) are peptides, i.e. somatostatin, TRH, GnRH, VIP.

Anterior pituitary hormones are peptides/proteins, i.e. ACTH, PRL, GH, TSH, FSH/LH

Posterior pituitary hormones are peptide, i.e. oxytocin, vasopressin.

As such, none of them are orally active.

Hypothalamic Control of Anterior Pituitary and RegulationHormones get released from the hypothalamus, travel down the capillary bed, to affect the cells of the anterior pituitary. These ‘releasing factors’ act of specific GPCRs (to alter cAMP, IP3 or Ca+). Most of these hypothalamic-releasing factors are secreted in a cyclical or pulsatile manner. Their therapeutic effects can vary depending on frequency and pattern of administration.

When the hypothalamus releases a hormone to act on the pituitary, it then releases a trophic hormone which acts on an endocrine gland to release a primary hormone. This primary hormone then negatively feeds back on the hypothalamus to prevent it from continuing the cycle.

An example of this is with gonadotrophic releasing hormone (GnRH) which is released from the hypothalamus to act on the anterior pituitary, to release FSH or LH. These hormones then act on the ovaries to regulate the amount of oestrogen or progesterone being released, and these hormones in a negative feedback fashion modulate both the hypothalamus and the anterior pituitary to stop their production.

But what can go wrong? Sometime there can be too much or too little of the primary hormone. This could be due to a number of abnormalities: dysfunction of the primary endocrine organ (e.g. gonads, thyroid), dysfunction of

regulatory organs (e.g. hypothalamus, pituitary), or maybe the end (target) tissue is unresponsive to the hormone.

Disorders of the PituitaryHyperpituitarism is generally caused by pituitary tumours and is characterised by increased release of two or more pituitary hormones. Treatment involves surgery or radiation therapy. In contrast, hypopituitarism may affect one or more endocrine systems, and treatment involves the replacement of these primary hormones.

ProlactinProlactin receptors are tyrosine kinase-linked receptors and are important for the development and maintenance of lactation. Levels are increased during pregnancy and breast feeding, at night and during stress. Dopamine exerts a tonic inhibitory control over the release of prolactin (this occurs normally) although when prolactin is released it is usually episodic. TRH, VIP and oxytocin can enhance the release of prolactin.

Though there are no disorders caused by a lack of prolactin release, too much can result in infertility disorders. Bromocriptine can be used as a dopamine agonist to treat prolactin excess. Adverse effects include nausea and vomiting, and a tolerance develops to these side effects but not to the therapeutic effects. Though cabergoline is better tolerated and has better efficacy, bromocriptine is older and we have more safety data for it.

Growth Hormone (Somatotrophin)GH receptors are tyrosine kinase-kined. They stimulate linear body growth, regulate cellular metabolism and have anabolic effects (also simulate lipolysis, production of free fatty acids, increase blood glucose). GH is released episodically and is stimulated by GHRH (somatorelin) while it is inhibited by GHIH (somatostatin). GH can also be released by factors such as exercise, stress and hypoglycaemia.

Lecture 10 & 11 – Gonadal Hormones

BackgroundSteroids that are produced by the gonads mediate the endocrine functions of the gonads. In the testes, the steroid there are androgens which make you feel like superman. For ladies, the ovaries produce oestrogen and progesterone. The main functions of the gonadal steroids are involved in sex differentiation and differential expression of secondary sex characteristics, and control of fertility.

Gonadal steroids can be split into androgens, progestins and oestrogens. When we think of male sex hormones we typically think of testosterone, and while it certainly plays a key role in the effects we see, dihydrotestosterone is also important – bot are androgens. Progesterone is a progestin and is required for the maintenance of pregnancy. The group of oestrogens are largely responsible for feminising characteristics. All 3 steroid types are synthesised from cholesterol.

Control of Sex Hormone SynthesisThe hypothalamus releases GnRH which stimulates the anterior pituitary to release FSH and LH to then produce testosterone. There are two different types of cells which can produce testosterone. In Leydig cells, LH increases testosterone synthesis. In Sertoli cells, the FSH can lead to an increase in ABP (androgen binding protein) which ultimately leads to an increase in testosterone.

Likewise in ovarian hormone synthesis, the hypothalamus releases GnRH to act on the anterior pituitary, causing it to release FSH and LH, which then cause the production of oestrogen and progesterone in the ovary. Oestrogen plays a key role in the thickening of the endometrium, where LH is involved in the ovulation or release of the egg. Sequence goes oestrogen increase first, then progesterone increase. LH increases androgen synthesis in Thecal cells (increasing oestrogen) and FSH increases aromatase in Granulosa cells which break down androgen into oestrogen.

Gonadal Steroid PharmacokineticsAll of these steroids are lipid soluble. This is important as they have to be able to pass through the cell membrane to bind to intracellular receptors. Most are subjected to extensive hepatic metabolic inactivation, so they cannot be given orally and we must synthesis compounds that can avoid this process. In the blood these hormones can bind to plasma proteins such as albumin, but they can also bind to SHBG (sex hormone binding globulin), so if we change the amount of SHBG levels in the blood we can change the amount of free steroid available.

There are separate receptors for each steroid hormone i.e. progesterone receptors, androgen receptors, oestrogen receptors. There are some cross reactivity observed, for example prolonged progestin levels can have androgenic effect. There are some subtypes, mostly our knowledge is limited to oestrogen receptor subtypes though. These nuclear receptors regulate gene expression.

Target cell activation can depend of blood levels of free hormone, the relative numbers of the receptor for that hormone in the target cell, the affinity of the bond between hormone and receptor, as well as the number of cofactors recruited and their availability.

When we think about various drugs we typically think about them having an agonist or an antagonist effect. However, there are selective steroid receptor modulators that can have different effects at different tissues (ligand-specific actions) due to the recruitment of different co-factors. For example SERMs (selective oestrogen receptor modulators) which include tamoxifen and raloxifen may be an antagonist for typical nuclear hormone receptors, they are an agonist at certain GPCRs. There are also androgen-based modulators (SARMs) as well as progesterone-based modulators (SPRMs)

Physiological ActionsTestosterone is involved in the development of the secondary sex characteristics, increased muscle

and fat distribution. DHT can also have a number of effects such as increased facial hair, prostate etc.

Oestrogen has a number of different effects. It plays an important role in the growth and differentiation of the uterus (to do with the endometrium), it’s also involved in the growth of breast tissue. It also has other effects not related to the sex organs: it has neuroprotective effects in various CNS disorders, plays an important role in vasodilation, etc.

The actions of progesterones include metabolic changes (CHO and fat metabolism), increase in body temperature, have a depressant/hypnotic effect on the brain, mammary glandular development, and modulates action of oestrogen on uterus. Is known as the “hormone of pregnancy”.

Replacing Hormones in Deficient StatesSymptoms vary depending on age of onset, could be caused by hypogonadism following ovariectomy/castration, menopause or andropause. In these cases the aim is to replace the effects on peripheral tissue by perhaps adding hormones.

Reducing Innapropriate Growth of Hormone-dependent TissuesTreatment aim is to inhibit the effect of oestrogen/dihydrotestosterone on these tissues. We can do this by inhibiting the synthesis or activation of the receptor. Here are three examples:

1. Some breast cancer cells express large amounts of oestrogen receptors, which stimulate tumour growth. By using an oestrogen antagonist or an agent that can decrease the rate of oestrogen synthesis, we can slow tumour growth.

2. Likewise in BPH (benign prostate hyperplasia) and prostate cancer, in the prostate the presence of testosterone can lead to prostate tissue growth so we can use synthesis inhibitors and androgen receptor antagonists to inhibit this growth.

3. Finally in endometriosis there is a benign ectopic growth of endometrial tissue which grows and regresses with menstrual cycle. Because it is oestrogen dependent, we can decrease oestrogen synthesis (by decreasing GnRH).

Altering Normal Function Where This is InconvenientI.e. contraception. The treatment aim in this case is to upset hormonal control of ovulation/spermatogenesis. By adding exogenous steroid hormone, you negatively feedback on the anterior pituitary, which no longer produces FSH and LH, so no spermatogenesis/ovulation occurs.

Restoring Dysfunction of the Hypothalamic-Pituitary-Reproductive Axis

1. Polycystic Ovarian Syndrome (PCOS)This occurs as a result of an increase in serum androgen. Results in abnormal ovulation, masculinisation, excess hair growth etc. Mechanism is unclear, but the LH hypothesis suggests that and increase in LH causes an increase in androstenedione. Insulin hypothesis: increased insulin = decreased SHBG = Increased free testosterone. Ovarian hypothesis = dysregulation of sex steroid synthesis in thecal cells leads to androgen synthesis. Treatment involves oestrogen and/or antiandrogen.

Treatment StrategiesIn constructing treatment strategies you can either affect the synthesis of certain steroid hormones by stimulating or inhibiting these processes, you can block their actions by using antagonists, or you can replace the hormones.

We can either increase FHS/LH leading to increased steroidogenesis, perhaps in order to stimulate ovulation; or we can decrease FSH/LH (decreased steroidogeneis) and directly inhibit steroid hormone synthesis perhaps for use in endometriosis, prostate cancer or breast cancer.

Modulation of the Gonadotropin Axis

We can use GnRH agonists (e.g. goserelin, nafarelin) which can be released in a pulsatile manner (mimic endogenous release to stimulate the anterior pituitary) or in a continuous manner (which inhibits the anterior pituitary).

An example in PCOS, you can use clomiphene (an antioestrogen compound) which works by targeting oestrogen receptors that are located within the anterior pituitary. By blocking these receptors, you block the negative feedback mechanism and thus increase the amount of FSH and LH.

In contrast danazol targets the negative feedback loop, and by targeting this it can drive down FSH and LH and thus decrease testosterone and oestrogen.

GonadotrophinsFollitropin (recombinant FSH)Lutropin (recombinant LH)Human chorionic gonadotrophin (HCG) - acts as LH

Aromatase InhibitorsUsed in the treatment of metastatic breast cancer (oestrogen responsive). Aromatase is the key enzyme involved in the conversion of testosterone to oestrogen. Examples include aminogluethimide, anastrozole, exemestane.

Hormone Receptor Antagonists

“Anti-androgens” can be used to preococious puberty, benign prostatic tumours, hirsuitism and acne.

o Flutamide, cyproterone, spirolactone all act to block the androgen-receptor complex. Good to use in females as it still allows testosterone to be produced and thus broken down into oestrogen.

SERMs (selective oestrogen receptor modulators) can be used for breast cancer, anovulatory infertility, osteoporosis and menopausal treatment.

o Have agonist activity in bone, lipids, endometrium and coagulation. Has antagonist activity in breast and pituitary.

TamoxifenAntioestrogenic –mammary tissue. Oestrogenic in plasma lipids, bone, endometrium. Used for treatment of breast cancer.

ClomipheneAntagonist at pituitary oestrogen receptors. Pituitary gonadotrophins induce ovulation. Partial agonist in ovaries. Used for anovulatory infertility.

RaloxifeneAntioestrogenic in breast and endometrial tissue. Oestregenic in bone. Used for treatment of osteoporosis.

TiboloneAntioestrogenic in endometrial tissue. Oestrogenic in bone, breast tissue. Used for treatment of osteoporosis and menopausal symptoms.

AntiprogesteronesProgesterone is the key hormone in pregnancy and plays an important role in getting the endometrial tissue ready for the fertilised egg. Menstruation occurs with the withdrawal of progesterone. So if we antagonise progesterone we get uterine bleeding. Pregnancy is also dependent on progesterone, and if we antagonise progesterone you get an abortion.

MifeprisoneProgesterone receptor antagonist. Used in pregnancy termination (RU486). Emergency contraception in lower doses

DanazolWeak progestational and androgenic activity. Inhibition of pituitary LH/FSH secretion. Atrophic changes in endometrium. Use = endometriosis.

Therapeutic Uses of Oestrogens and ProgestinsCan be used for ovulation suppression/menstrual disorders including endometriosis, contraception, dysmenorrhoea, pre-menstrual tension. They can also be used as replacement therapy in hypo-ovarian conditions e.g. post-menopausal hormone replacement.

Hormonal contraceptives can either be oestrogen only (just targeting the negative feedback of the anterior pituitary) or oestrogen and progesterone (targeting both negative feedback of anterior pituitary and the hypothalamus). They act to inhibit ovulation, thicken cervical mucous to make it harder for sperm to get through the cervix, and to reduce the receptivity of the endometrium.

Oestrogen also has adverse, systemic effects. It can increase the production of liver proteins (bad), cause the growth and proliferation of breast tissue (risk factor for cancer) as well as increase growth and differentiation of primary sex organs (risk factor for endometrial cancer). These can be counter-balanced by adding progesterone to counteract these effects.

Long-term use of oestrogen containing contraceptives confer a risk of deep vein thrombosis and pulmonary embolism, which is related to dose of oestrogen and type of progesterone (increased risk if >35 and smoker). Potential increased risk of breast cancer. Increased risk of gallbladder disease. ALTHOUGH there is a decreased risk of ovarian cancer, potentially due to the decreased amount of circulating gonadotrophins.

MenopauseDecreased ovarian function. Signs include loss of periods, genital atrophy, bladder overactivity, hot flashes, decreased libido, lack of energy, loss or memory, mood swings and irritability. Long-term consequences of menopause include increased risk of disease: increased bone loss (decreased density, osteoporosis), increased risk of coronary heart disease (myocardial infarction/fatal strokes) and potentially increased risk of Alzheimer’s disease.

Whilst there are negative effects of oestrogen, there are also positive effects which include neuroprotective effects, maintenance of bone density and cardioprotection. This make hormone replacement therapy a good option to use in menopause.

Theme 4: Neuropharmacology of Neurodegenerative Diseases and Epilepsy

Lecture 12 – Excitotoxicity and Neurodegeneration

BackgroundIt is important to know that in general, neurons of the CNS cannot divide (with a few exceptions). Neurodegenerative diseases such as Parkinson’s or Alzheimer’s disease are progressive, and current therapy does not treat the underlying cause, only the symptoms. It is important for us to understand the pathological process to allow for the development of targets that can stop or reverse neurodegeneration (neuroprotective agents/targets) or targets that are applicable to other processes in the brain that lead to brain damage.

Neurodegeneration is the dysfunction and/or loss of neurons/synapses and there are a number of pathological processes that are common including: excitotoxicity, ageing/oxidative stress, neuroinflammatory processes, mitochondrial dysfunction, changes to protein folding and aggregation.

ExcitotoxicityExcitotoxicity is the phenomenon that occurs when there is over-activation of the excitatory glutamate system. Activation of NMDA receptors allows for the entry of Na+ and Ca2+ ions, causing an increase in intracellular Ca2+ and the activation of Ca2+ dependent enzymes (kinases, lipases, proteases, NOS) ultimately causing cell death.

Stroke is a good example of excitoxicity. It is associated with the loss of cellular homeostasis (loss of transporter function). GLUT levels are tightly regulated by EAATs, which work by sucking up extracellular glutamate into the cell to maintain levels, and so when we get a loss of energy (due to ischaemic or haemorrhagic stroke) there is a loss of energy, a loss of transporter regulation and reversal of EAATs. This causes glutamine to be pumped into the ECF,

causing excessive ECF glutamate concentration and thus excessive NMDA receptor activation.

Neurodegeneration involves a continuum of effects. The breakdown of cell membrane integrity causes cell death through the process of necrosis. This involves the depletion of glucose/ATP, failure of Na+, K+, ATPase and other ion pumps, cellular swelling, run down of membrane potentials, further glutamate release and stimulation of NMDA receptors, as well as the activation of Ca2+ dependent enzymes.

Once these Ca2+ dependent enzymes are activated they can cause the activation of proteases, the activation of lipases, activation of NOS, the activation of endocucleases, the activation of oxygen-derived free radicals and the activation of neuroinflammatory processes.

There is also an apoptosis component, a late stage response (hours to days). The mitochondria are critical in the regulation of apoptosis and it involves alterations in mitochondrial permeability, release of cytochrome C, activation of mitochondrial pathway and the activation of caspases, all to cause apoptosis.

Oxidative Stress and Formation of ROSSuperoxide (O2 ) is probably the most common of the free radicals, it is generated enzymatically by ∙NOX (NADPH oxidase found in macrophages/reactive microglia). Other free radicals include OH- , ∙H2O2 and ONOO-. ROS attack key molecules (such as enzymes, membrane lipids, DNA and mitochondria) hydroxyl free radicals and peroxynitrite are the most destructive.

Defensive mechanisms against ROS include several antioxidant enzymes and enzyme systems, including: SOD (superoxide dismutase), catalase and glutathione peroxidase.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease causing death of neurons in the spinal cord, brainstem and motor cortex. Prognosis = progressive paralysis followed by death in 3-5 years. ALS may be sporadic or familiar – 20% of all ALS cases are heritable, and of these, 20-30% are caused by gain-of-toxic function mutations in Cu, Zn-SOD (SOD1)

Oxidative Stress in Parkinson’s DiseaseTo treat PD, often dopamine is given. However there is a consequence of this. The metabolism of DA by MAOB can lead to the production of oxygen free radicals. Because the DA-ergic terminals are sensitive to the elevated oxidative stress, you get further cellular damage and tissue necrosis.

ApoptosisApoptosis is cell suicide, or programmed cell death. The cells are dismantled and remains are removed by macrophages. It is a process that is essential in embryogenesis and tissue homeostasis (i.e. shedding of intestinal lining, regression of glands, pathophysiology of cancer, autoimmune diseases).

There are two main pathways by which apoptosis occurs: the death receptor pathway (intrinsic pathway) and the mitochondrial pathway. The death receptor pathway occurs by activation of tumour necrosis factor receptor family (TNFR), which activate caspase-8, which can in turn activate caspase-3 (the key executioner protein) for apoptosis. The mitochondrial pathway is activated by DNA damage and withdrawal of cell survival factors. This DNA damage activates the p53 pathway and the activation of pro-apoptotic factors. These pro-apoptotic factors, such as Bax, promote the release of cytochrome C from the mitochondria which activates caspase-9.

NeuroinflammationNeuroinflammation is the first line of defence against injury and infection. Excessive inflammatory response is a source of additional injury. Brain inflammation occurs as a result of activation and recruitment to the areas affects of glia (microglia)/astrocytes. These activated microglia/reactive astrocytes occur in Alzheimer’s, Parkinson’s, Multiple Sclerosis, AIDS, dementia, trauma and stroke.

Activated microglia can generate reactive oxygen species causing oxidative damage. Reactive astrocytes generate other reactive oxygen species via myeloperoxidase (MPO). Activated microglia contribute to neuronal damage (neurodegenerative diseases) via the release or production of cytokines such as TNFα (activation of apoptosis), stimulating the expression of other enzymes (e.g.

NOS and the generation of toxic levels of NO) as well as the recruitment of other inflammatory cells to the area affected.

Drugs in NeuroprotectionOxidative Stress – use of free radical scavengers i.e. vitamin E

Neuroinflammation – COX-2 inhibitors/anti-inflammatory steroids, ibuprofen, inhibition of cytokines

Apoptosis – caspase inhibitors

Lecture 13 – Drugs for Parkinson’s Disease and Huntington’s Disease

Parkinson’s disease was first described in 187 by James Parkinson – “as essay of the shaking palsy”. Common disorder up to 1% (aged population over 60). PD has a long latency/is progressive, and this makes early diagnosis impossible. In the clinical phase there is difficulty walking, fatigue, limb discomfort, clumsiness. 70% show a resting tremor that is worsened by stress and decreases with action. There is rigidity and bradykinesia, as well as impaired postural reflexes. Furth signs include micrographia, difficulty swallowing, cognitive problems and the presence of Lewy Bodies post-mortem.

The aetiology of Parkinson’s disease is questionable. There are some evidence that ageing/oxidative stress is involved, potentially environmental toxins, and there is a minor genetic susceptibility. We are left with an idiopathic disease, without one underlying cause. The striatum (caudate + putamen) brain region in those who have Parkinson’s shows a loss of dopamine. This dopamine is lost because there is a loss of cell bodies which project from the substantia nigra into the striatum.

It is the nigrostriatal dopaminergic system which collects and integrates the signals from the cortex as well as put a sequence to the movements – to ensure that the right muscles are moving at the right times. In this way, it prepares the motor system for the next movement in a given sequence.

Why is there a latency to the disease? The reason you do not observe symptoms until the individual is in their 60s, 70s or 80s is because there is quite a lot of adaptive capacity within the nigrostriatal dopaminergic system. It is possible to lose up to 80% of dopaminergic neurons without observing any symptoms, only after further loss will you observe mild symptoms getting increasingly worse. So by the time the disease has been characterised, there is

already a potential loss of 80-90% of dopaminergic neurons. The reason why this adaptive capacity exists is because DAergic neurons mediate their activity through D2 receptors, and symptoms don’t occur until ~80% of neurons are lost because the D2 receptors proliferate and increase in number to initially compensate for the loss of dopamine.

Rational Therapy for PDBecause the pathology is so well understood, there are clear therapies to treat the symptoms of PD. These include increasing synaptic concentrations of dopamine (1), direct activation of dopamine receptors with dopamine agonists (2), prevention of dopamine metabolism (3) and altering the efficacy of interacting neurotransmitters (4).

1. Increasing Synaptic Dopamine

Increasing synaptic dopamine is key, and probably the most common therapy. The conversion of DOPA to dopamine occurs via the enzyme DOPA decarboxylase. We can’t give individuals dopamine as it does not cross the blood-brain barrier, so DOPA is used as replacement therapy (levodopa). After adding this precursor, DOPA decarboxylase (DDC) converts DOPA to dopamine in the remaining neurons. 80% of patients show improvement in function, and 20% are restored to normal function with this therapy.

There are some issues with levodopa though, it doesn’t last very long (plasma ½ life of 2hrs). Another issue is that while DDC is present in the CNS, it is also present in the periphery – more than 90% of levodopa is converted to DA in the periphery by peripheral DDC – so less than 1% enters the brain for conversion to DA in the CNS.

As such, to optimise levodopa therapy it is usually combined with inhibitors of DDC that are unable to cross the BBB. This allows for a greater proportion of levodopa to reach the CNS, and allows concentrations in the plasma to be higher for longer. Some of the DDC inhibitors that are supplied in combination with levodopa include carbidopa and benserazide.

One of the main side effects of levodopa therapy include dyskinesia (abnormal involuntary movements) caused by too much dopamine and dopamine receptor activation, potentially due to fluctuations in plasma levels owing to the difficulty of giving the proper dose.

2. Direct Activation of Dopamine Receptors

Forget about dopamine, let’s use a D2 receptor agonist. Using a DA agonist means that you do not require the CNS for metabolism and storage, there is increased reliability in dose by dose effects, and many DA agonists have a longer ½ life than levodopa. Potentially less likely to result in dyskinesias for some reason. All of these agents that promote a clear cut improvement in PD symptomology are D2 agonists, and include apomorphine, bromocriptine and pergolide.

Side effects from these agents are due to peripheral and central DA receptor stimulation. Nausea, vomiting and hypotension are observed (peripheral effects) – these occur when DA activates the chemoreceptor trigger zone (CTZ) and can be treated with domperidone which is a useful peripheral DA antagonist.

There are also some psychological side effects such as delusions, mania and anxiety hallucinations due to excessive DA stimulation of the mesocortical pathways (see schizophrenia).

3. Prevention of Dopamine Metabolism

When dopamine is released it is taken back up by the nerves, so there is a degree of recycling. MAO is the enzyme that breaks down dopamine. There are two types of this enzyme, and the one we want to target is MAOB which is the type that is most common in the brain.

One compound that is a very selective MAOB inhibitor is selegiline. It can be used alone or in combination with other agents for PD. Can be given without restriction (i.e. no cheese reaction). Minor side effects include insomnia/headache.

What happens when you inhibit MAOB and/or DDC is that other enzyme systems start to become more relevant. An inhibition of DDC is associated with an increase in other pathways for levodopa metabolism. COMT metabolism results in production of 3-methyl-dopa. This competes with levodopa for transport across the BBB and is associated with a poor response to levodopa. COMT inhibitors include tolcapone and entacapone.

4. Altering the Efficacy of Interacting Neurotransmitters

Dopamine neurons usually exert a tonic inhibitory influence on cholinergic neurons within the striatum. PD removes this inhibitory influence and intensifies the activity of the cholinergic system, so one strategy is to diminish this level of activity.

Muscarinic antagonists provide mild relief in the early stage of PD – 25% of patients are adequately controlled with muscarinic antagonists. Benzotropine is such an agent. All of these agents are centrally active, uncharged, and able to cross the BBB. As they are anti-muscarinic agents their side effects are predictable: dry mouth, constipation, urinary retention, mental confusion.

Parkinson’s Disease PolypharmacyWhen treating PD, you can use a whole range of drugs. For example selegeline can be combined with levodopa, or combined with DA agonists or muscarinic antagonists.

Huntington’s DiseaseHD is an inherited neurodegenerative disorder which is autosomal dominant and largely displays 100% penetrance. The age of onset is 35-45 years, and affects approximately 4-10 individuals per 100,000 (relatively rare). Death occurs 10-15 years after symptoms are observed, so by this stage the gene is potentially passed on to offspring.

The symptoms of HD are quite the opposite to what is observed in PD. It is characterised by chorea (irregular, unpredictable, purposeless, rapid movements), impaired voluntary movement, jerks, cognitive deficits and severe rigidity in late stages.

HD actually affects GABA, completely different transmitter. In this disease there is a selective loss of GABA neurons within the striatum. GABAergic neurons exert an inhibitory influence on DA within the striatum. This loss, leads to DA hyperactivity, which accounts for the motor hyperactivity. Most drugs are poorly effective or only effective in the short term. It is possible to use DA antagonists (such as chlorpromazine), GABA agonists (such as baclofen) or tetrabenazine (depletes dopamine within the brain).

Lecture 13 – Epilepsy and Anti-Epileptic Drugs

BackgroundEpilepsy and seizures are two different things. A seizure is a clinical manifestation of

abnormal/excessive synchronous excitation of a population of cortical neurons. Epilepsy is defined as 2 or more recurrent, unprovoked seizures. Epilepsy affects 0.5-1% of the population, and drugs are affective in 70% of cases (though 20% do not respond at all to drugs).

Partial seizures begin locally and remain localised, with symptoms depending on brain regions involved. Simple partial seizures show motor signs, sensory symptoms and autonomic symptoms. Complex partial seizures show impaired consciousness and various clinical manifestations that vary with origin and spread of seizure – temporal lobe epilepsy most common.

Generalised seizures involve the whole brain and are associated with a loss of consciousness. Toni-clonic seizures show strong contraction of musculature (rigid extensor spasm)/involuntary vocalisation. Absence/petit mal seizures are common in children and are characterised by stopping current action and vacant stares with little or no motor disturbances.

Epilepsy can be acquired due to physical injury to the brain from things such as tumours, strokes, CNS infections etc. 50% of patients with head trauma develop a seizure disorder. An initial seizure can lead to pathological events which can lead to future vulnerability. Genetic epilepsy involves mutations in structural elements of the brain such as receptors or ion channels.

The main mechanism of action of all anti-epileptic drugs is to target the neuronal excitability. It is still unclear whether there is too much neuronal excitation occurring, or whether there is too little neuronal inhibition. To model epilepsy, a number of different methods can be used. Convulsants such as bicuculline and picrotoxin inhibit GABAA receptor activation. To model complex partial seizures, kainic acid can be used as an agonist at glutamate receptors which results in seizure activity with systemic/IV administration. Also, repeated stimulation to a group of neurons can result in a sustained enhanced excitability of the neurons which can initiate seizure activity weeks after the stimulus.

GABAergic Transmission as a TargetMost of the anti-epileptic agents in use seek to enhance GABA function, not so much block glutamate activity. GABA is metabolised by the enzyme GABA-T (GABA transaminase). The inhibition of this enzyme leads to substantial increases in neuronal and extra-neuronal levels of GABA enhanced levels of neuronal inhibition/quiescence. Alternatively, as GABA is removed from its site of

action by reuptake through a specific transporter, blocking this transporter leads to increases in extra-neuronal levels of GABA and enhanced neuronal inhibition. So here are potentially to mechanisms to target. Valproate and vigabatrin target GABA-T, whereas tiagabine targets GABA reuptake transporter.

Another key target in epilepsy is the GABA receptor itself. Benzodiazepines and barbiturates tend to be used which target the allosteric site and potentiate the effects of GABA. Diazepam and phenobarbitone are used in epilepsy.

Na+ Channels as TargetsIf we want to stop electrical activity (the action potential) we must affect Na+ ions and their movement. Action potentials are an all-or-none depolarisation of the cell membrane, once propagated it spreads to all parts of the cell. During seizures there are neurons firing at a high frequency. To effectively target Na+ channels in epilepsy, we need to utilise a use-dependent blockade of Na+ channels, that is preferentially block neurons that are firing at high frequency without interfering with the firing of neurons which are in a “normal” state.

Ca2+ Channels as TargetsAbsence seizures are exclusively associated with T-type Ca2+ channel activation. The T-type Ca2+ channels within the thalamus provide a “pacemaker” current that produces bursting intrinsic firing, leading to absence seizures. Ethosuximide specifically blocks these T-type Ca2+ (low voltage activated) channels.

Gabapentin also works through Ca2+ channels, but a different type, known as the high voltage activated (HVA) Ca2+ channel. Some partial seizures are associated with HVA Ca2+ channel activation.

Comparative Unwanted Effects of Major Anti-Epileptic Drugs and Drug InteractionsSedation and confusion are common, as are skin rashes. Phenytoin also produces gum hyperplasia, acne and hiruitism ,and it shown zero order kinetics (narrow safety margin).

The cytochrome P450 enzyme system in the liver is responsible for the metabolism of many drugs. These enzymes have broad specificity and several enzymes can metabolise a drug. The enzyme activity within this system can be induced or inhibited. An example of an inducer of CYP450 is phenobartbitol and phenytoin, whereas valproate is an inhibitor of CYP450.

Some anti-epileptic drugs can cause miscarriage and foetal abnormalities. Phenytoin affects palate/heart, and valproate causes spinal bifida.

Lecture 15 – Alzheimer’s Disease and Dementia

BackgroundDementia is a significant loss of intellectual abilities severe enough to interfere with social or occupational functioning. Mild cognitive impairment is significant memory loss without the loss of other cognitive functions. Forgetfulness is a common symptom associated with ageing. Many things can cause or lead to dementia, with neurodegenerative disorders being the most common. Currently 1 in 4 people over the age of 85 are affected by dementia, and it is the third leading cause of death in Australia after heart disease and stroke. The most common condition resulting in dementia is Alzheimer’s disease.

Neuropathy of Alzheimer’s DiseaseAD is the most common neurodegenerative disease, affecting more than 20million people worldwide. Familial AD <10% of cases, much more prevalent is late onset AD (>90%, age related). AD is characterised by a shrinkage of the temporal cortex/frontal lobe by as much as 20%. The two hallmarks which allow us to identify AD are the presence of amyloid plaques and neurofibrillary tangles.

Amyloid plaques are formed outside of neurons and are composed of β-amyloid (Aβ). When Aβ is in its soluble form it is toxic to neurons when it is taken up by the neurons, and when it is in its insoluble form they clump together outside neurons to form plaques which release oxygen-derived free radicals, disrupt potassium and calcium channels, and cause vasoconstriction and blood vessel injury.

Neurofibrillary tangles form inside neurons and occur as a result of production of abnormal form of tau. Tau normally forms cross bridges between microtubules and keeps them in stable configurations. Microtubules collapse, and tau proteins clump together to form neurofibrillary tangles.

Diagnosis of Alzheimer’s DiseaseDiagnosing is problematic, and involves post-mortem confirmation. Cognitive tests such as a Mini Mental State Examination (MMSE) can be performed, but they are subjective. Biomarkers in CSF, looking for Aβ and tau are invasive and unreliable. Neuroimaging is expensive, but MRI can look for hippocampal atrophy, and a PET tracer compound can bind to Aβ plaques. Cognitive symptoms of AD include impairment in memory/concentration, disorientation, alterations in personality, aphasia, apraxia, agnosia.

Cholinergic HypothesisIt has been identified that there is a selective loss of cholinergic neurons arising from the basal forebrain in AD. Typically there is a 70% loss of choline acetyl transferase (ChAT), causing decreased

ACh release. Treatment for this is largely focused on replacing ACh: increasing ACh synthesis, adding choline as a precursor, augmenting the release, stimulating post-synaptic receptors, and reducing the degradation of ACh by inhibiting AChE or BuChE.

Drug Treatments

Synthetic precursors – choline is not effective at driving ACh production

Cholinesterase inhibitors – AChE inhibitors do have beneficial effects on the cognitive and behavioural symptoms of AD

Direct activation of cholinergic receptors – agonists poorly effective

Tacrine is a reversible cholinesterase inhibitor, but is not used very often due to high liver toxicity (25%). The most widely prescribed reversible cholinesterase inhibitor is donepezil which is highly selective for AChE and has a long ½ life.

Mematine was approved by the FDA in 2008. It is an antagonist for NMDA receptors and shows moderate affinity. The rationale for its use in AD is that the overactivation of NMDA receptors has been implicated in many neurodegenerative states. However, it only treats symptoms and it not thought to affect the pathology.

Other targets for AD include cognitive enhancers that treat the symptoms, disease modifying therapies – such as Aβ therapies, kinase inhibtors to block tau hyperphosphorylation and anti-aggregation therapies.

Aβ Drug Therapies

Rosiglitazone supresses β-secretase activity. Phase III clinical trial, but the FDA has warned of cardiac risk as an adverse event.

Semegacestat inhibits Aβ production by inhibiting γ-secretase at the active site. Phase III clinical trial halted as it failed to slow disease progression and increased risk of skin cancer.

Tramiprosate is a glucosaminoglucan mimetic which prevents Aβ fibril formation and aggregation, currently in Phase III clinical trials

Aβ Immunization TherapiesActive ImmunizationAN-1972 – Anti- Aβ vaccine. Patients developed significant Aβ antibody titres. Stopped because aseptic meningo encephalitis in some patients, attributed to cytotoxic T cells/immune reactions.

Passive ImmunizationBapineuzumab is a humanised anti-Aβ monoclonal antibody – 18 month treatment no significant effect on cognitive measures

Solanezumab is a monoclonal antibody that binds specifically to soluble Aβ, promotes Aβ clearance from brain through the blood

Tau-based TherapiesThere are two main therapeutic approaches:

1. Modulation of tau-phosphorylating kinases- Protein phosphatase 2A inibitors

2. Inhibition of tau aggregation and/or promote aggregate disassembly- Methylthioninium chloride – widely used histology dye, tau anti-aggregant

Lecture 16 –The Immune Response as a Drug Target in Stroke

BackgroundStroke is the second largest cause of death in Australia (after heart attack) and the major cause of disability. One in five people having a first-ever stroke die within one month, one in three die within a year. There are two types of strokes: ischaemic (85-90%) and haemorrhagic (10-15%). Neurons have very high energy demands but they cannot store ATP – it is the boring lecture. So I will come back to it.

Theme 5: Hormones and Endocrine Mechanisms

Lecture 17 – Agents That Affect Bone Mineral Homeostasis

BackgroundBone provides structure and support, a space for haemopoiesis, as well as a storage site for calcium

(and phosphate). There is approximately 1000g of calcium stored in the body, 99% of that is within the bone in crystalline form. The bone is the site of calcium deposition and reabsorption – it is a source of calcium when plasma levels fall, and is a storage site for calcium when plasma levels are too high.

Bone is made up of two major types: cortical bone which is the typical long, hard and dense bone you think of, and trabecular bone which is spongy and has a honeycomb-like structure to absorb force. The proportion of these two types of bone varies in different skeletal sites.

Bone HomeostasisIn order for bone to maintain its strength, it must undergo a continuous process of reformation. Bone is constantly being broken down in areas where it may be damaged, absorbed into the circulation as free calcium, and new bone is laid down in its place. Bone homeostasis and remodelling occurs particularly in response to mechanical stress. It is known that trabecular bone has a faster turnover rate than cortical bone. This homeostasis is a balance between the activity of osteoblasts (which cause bone deposition) and osteoclasts (which cause bone resorption).

Osteoclasts are in a precursor form that is inactive, and they get activated by osteoblasts. When there is stimulation and differentiation of osteoclasts, they turn into a multinucleated cell that sits down on top of the bone which release enzymes to break down the bone. As they start to break down the bone you get the release of free calcium and phosphate, as well as some cytokines, and when they’re released they stimulate the activity of osteoblasts to lay down new bone matter.

The way that osteoblasts activate osteoblasts is via the RANK/RANK-L system. Osteoclast progenitors express RANK on their cell surface. Osteoblasts express the RANK ligand, so when they come into contact with osteoclasts they activate them into multinucleated osteoclast which resorbs bone. Osteoblasts also produce osteoprotegerin (OPG) which is like a soluble form of the RANK. This allows them to bind to RANKL and prevent binding to osteoclasts, preventing excessive osteoclast activation and bone resorption.

Parathyroid hormone (PTH) – released in response to low calcium levels, tries to increase again, does this by recruiting and activating osteoclasts. At high concentrations it can inhibit osteoblast activity. All of this promotes the calcium going back into the plasma.

Vitamin D – vitamin D levels go up in response to low calcium levels. Promotes the maturation of osteoclasts & indirectly stimulates their activity. Promotes the calcium going back into the plasma.

Calcitonin – Opposite. Inhibits osteoclast activity.

Calcitriol – increases expression of the RANKL more osteoclast activity.

Glucocorticoids – at physiological levels stimulate osteoblast activity. At increased (pharmacological) levels stimulate osteoclast activity.

Oestrogens – decrease osteoclast activity (increases OPG), opposes PTH. Works to slow bone turnover and bone loss

OsteoporosisPeak bone mass occurs around age 30, and is determined by genetic factors, endogenous factors (hormones) and exogenous factors (diet, drugs, physical activity). Bone loss occurs with age of 0.5-1.0% per year. Osteoporosis is a reduced bone mass/density due to greater resorption happening than deposition. As you would imagine some factors that can contribute to osteoporosis include decreased oestrogen, TSH, calcium/VitD, OPG, as well as increased PTH and RANKL.

As trabecular bone has a faster turnover rate than cortical bone and is generally responsible for weight bearing, it’s not surprising that when this imbalance occurs the bones that are most affected are those that have high trabecular content i.e. hip, spine, wrist.

Pharmacological treatments for osteoporosis are considered when people with presence of history of osteoporotic fracture, or when their bone density is >2.5 SD below young mean value. Drug

treatments aim to decrease bone resorption in order to maintain calcium levels and prevent fractures. They can be classified as either antiresorptive agents that inhibit the bone resorption, or as bone anabolic agents which stimulate bone formation.

Calcium and/or Vitamin DUsed as a preventative treatment. Meta-analysis shows a reduction in fracture rates. May reduce bone loss if there is low dietary calcium intake. Treatment though is less effective than other treatments when used as a sole therapy, so recommended to take with other anti-resorptive agents.

Colecalciferol/ergocalciferolVitamin D precursor, evidence to suggest it can increase bone mineral density and reduce falls in older people.

HRT e.g. oestrone and/or progesteroneOnly really appropriate if high risk of osteoporosis but with no other risk factors, due to the increased risk of endometrial cancer, breast cancer, cardiovascular disease. Can decrease loss in bone density

SERMS e.g. raloxifeneOestrogenic activity in bone. Can increase bone density and decrease risk of vertebral fractures in postmenopausal women. Antioestrogenic activity in breast, so will not promote breast cancer. Still retains adverse cardiovascular effects.

CalcitoninReduced bone resorption by inhibiting osteoclast activity. Not really more effective than raloxifene

BisphosphonatesAnalogues of pyrophosphate, are concentrated in bone and incorporated into the mineralised matrix. They work to decrease the resorption of bone by inhibiting the formation and activity of osteoclasts. Leads to increased BMD and strength, and a decrease in fractures. Serves as a 1st line management for postmenopausal osteoporosis. As they are embedded in the bone matrix, when osteoclasts attach to and degrade the bone they release these bisphosphonates which are toxic to the osteoclasts, decreasing their activity. Issue with patient compliance however. Adverse events include atypical fractures (rare) and osteonecrosis of jaw (very rare)

DenosumabA new monoclonal Ab against RANKL. It causes the number and function of osteoclasts to be reduced. Mops up the RANKL before it can activate and differentiate osteoclasts. Increased BMD, decreased resorption. Approved for the treatment of osteoporosis in postmenopausal women.

PTH/PTH fragments e.g. teriapartideParadoxically anabolic effect if given intermittently at low doses. Causes bone anabolism due to the stimulation of osteoblast development and activity. Increased BMD and decreased risk of vertebral fractures in postmenopausal women with established osteoporosis. Treatment restricted to 18 months as animal studies show increased incidence of osteosarcoma.

Strontium ranelate2 molecules of strontium combined with organic ranelic acid. Inhibits bone resorption and stimulates bone formation. Decreases RANKL and increased OPG.

Lecture 18 – Pancreatic Hormones and Antidiabetic Drugs

BackgroundDiabetes mellitus is the term given when a patient has chronic hyperglycaemia. It is a metabolic disorder affective 1.7 million Australians. Signs and symptoms include excessive thirst, excessive urination, glucose in urine, loss of weight (in children) and tiredness. These symptoms occur because the kidneys have a certain threshold for the reabsorption of glucose. If that is exceeded, then glucose spills into the urine. This causes the kidney to produce more urine to fix this gradient (osmotic diuresis), and the frequent urination leads to thirst and dehydration.

Type 1 DiabetesAlso known as “juvenile-onset” or “insulin-dependent” diabetes. In this disease, the β cells in the pancreas that produce insulin are destroyed by autoimmune response. This can occur following infection (virus) where the β cells are attacked, antibodies are produces and an autoimmune response develops. Sometimes the symptoms may not appear for a few years as 90% of β cells must be wiped out before symptoms are seen. Happens in the young population and treatment involves insulin injections and diet modification.

Type 2 DiabetesAlso known as “mature onset” or “non-insulin dependent” diabetes. In this case, the β cells are usually functioning pretty well and producing insulin, but what happens is that there is resistance to the effects of the insulin. This happens slowly over time, usually developing in middle/late life. Many who have type 2 diabetes are overweight. Majority have this form (85-90%). Treatment involves change in diet/exercise, oral hypoglycaemics and insulin. There is a strong genetic component, involving a resistance to circulating insulin, a reduction in receptor numbers and/or an altered release mechanism.

InsulinIn the pancreas there are α, β and δ cells. It is the β cells which generate and secrete insulin. How is insulin released? Ingesting food and nutrients causes the release of glucose from the liver into the blood, which causes the β cells to release insulin. Parasympathetic nerves stimulate β cells to produce insulin. There is a continual basal release on insulin, but a surge when feeding. Out of all the insulin that is generated, about 50% that reaches the liver via the portal vein is destroyed and never reaches general circulation. When insulin binds to its receptor, the receptor gets translocated internally within the cell. In type 2 diabetes, after having such high levels of circulating insulin

chronically, the receptors mostly get internalised so there are no longer enough receptors on the cell surface to respond to circulating insulin. Insulin stimulates glucose uptake in skeletal muscle, amino acid uptake and facilitates protein synthesis. It preserves fat stores in adipose tissue, and inhibits glycogenolysis and gluconeogenesis.

GlucagonThe hormone of the ‘fasting state’. Released by the α cells of the pancreas. Provides ‘fuel’ between meals. Secretion is stimulated by amino acids, stress and hypoglycaemia. Glucagon acts predominantly on the liver to increase blood glucose to provide energy. As such, promotes the breakdown of glycogen stores into glucose, as well as gluconeogenesis.

Despite effective treatment, diabetic complications can include: large blood vessel disease (atherosclerosis) kidney disease (nephropathy), nerve damage (neuropathy) and eye damage/blindness (retinopathy).

A more sensitive measure to test for diabetes is glycosylated haemoglobin, rather than simply testing blood glucose levels. Glycosyated haemoglobin means sugar molecules attached to haemoglobin, and though blood glucose levels are a current ‘snapshot’, this is a more long-term accurate measurement.

TreatmentFor Type 1 diabetes, insulin is injected as well as dietary modifications. End of story.

For type 2 diabetes, after dietary modifications, insulin sensitizers can be give (such as metformin and glitazones) or drugs that increase insulin secretion (such as sulfonylureas and glitinides).

Metformin is the first line choice against type 2 diabetes, because it combats the insulin resistance and makes the target tissues more sensitive to insulin and will facilitate glucose uptake. Not sure how it works. Does NOT stimulate insulin release. Has a very low risk of causing hypoglycaemia, and

can be used in combination with sulphonylureas and insulin. Side effects include weight loss, nausea and diarrhoea.

Glitazones increase tissue sensitivity to insulin. Promote increased glucose transporters? Inhibit hepatic gluconeogenesis. Bind to nuclear receptor PPARγ. Less efficacious than metformin/sulphonylureas. Slow onset (weeks?). Low risk hypoglycaemia. Can be used in combination. Side effects include weight gain.

Sulphonylureas stimulate insulin release from the pancreas. Require functioning β cells. Block ATP-sensitive K+ channels (depolarisation). Useful in early stages of diabetes. High protein binding (90-95%), can cause hypoglycaemia and weight gain.

Stil 2 lectures in this theme to come back to. But boring so move on.

Theme 6: Pain and Addiction

Lecture 21 – Pain Systems and Analgesic Agents

BackgroundPain is a subjective experience which is usually a direct response to things such as tissue damage, inflammation or cancer etc. There is acute pain and chronic pain. Acute pain you would describe as a really excessive, noxious stimuli. Chronic pain is usually just a change in the physiological perception of pain – so you may detect pain for a relatively mild stimuli. Pain perception is via nociceptive afferent neurons. Their nerve terminals at are various sites in the periphery, which project back to the spinal cord.

So if you have an injury, you’ll have these nociceptive afferent neurons being stimulated. These nerve terminals in the periphery are stimulated by endogenous mediators that are released in response to tissue damage: bradykinin, 5-HT, prostaglandin, ATP, H+, etc.

How do the neurons transmit their signal? By depolarisation of Na+ channels. If you look at prostaglandins for example, when they are released to local tissue in response to injury they act on

receptors which can lead to the opening of voltage-gated Na+ channels, causing an influx of Na+ ions into the cell, depolarisation and excitation and transmission of signal. Some patients can have a mutation in this sodium channel meaning they do not feel pain. That’s why blocking these channels pharmacologically is an effective way of blocking transmission and inhibiting the pain response.

Prostaglandins are synthesised by arachidonic acid by cyclooxygenase (COX) and are released during inflammation. They do not directly cause pain but can enhance the effects of pain producing events. They work by facilitating the opening of voltage gated Na+ channels. PGE2 is the one that increases sensitivity to pain. When we use NSAIDs, we inhibit COX and so stop the production of PGE2, causing analgesic and anti-inflammatory effects for mild-moderate pain.

Excitation of these pain neurons is not just by excitation of sodium channels, there is also a type of channel present in these neurons called TRP channels. Activation of TRP channels leads to a large influx of Na+ and Ca2+. These channels can be activated by a number of different ways: by bradykinin phosphorylating them, and directly by capsaicin (chili), high temperatures. Caspaicin results in a large influx of Ca2+ ions by targeting the VR1 receptor. Alternatively, can also release substance P and cause propagation of pain transmission. When capsaicin activates the neuron it causes a really large increase in Ca2+ initially (and sever pain) which is enough to cause the nerve terminals to degenerate (weeks to recover). Potential future target for pain relief?

When the afferent neurons transmit their signal, which neurotransmitters are released at the level of the spinal cord? This could be an important target in analgesia. Substance P is abundant in nociceptive primary afferent neurons (produced and released), looks like it is a pain transmitter in the periphery. Its actions are mediated via NK1 receptors. Glutamine is also an important co-transmitter.

Opioids and Opioid ReceptorsOpioids are endogenous or synthetic compounds that produce ‘morphine-like’ effects. They produce a mixture of euphoric/dysphoric and depressant effects. Opioids are agonists at their receptor systems. Opioids have actions in the periphery, the spinal cord, and in the CNS, and they will have an analgesic effect at each of these sites.

Morphine mimics the effects of endogenous opioids such as endorphins and enkephalins. Endogenous opioids are small peptides (a neuropeptide transmitter) that are released from nearby neurons. In the CNS and periphery they are involved in the modulation of pain. There are three different types of opioid receptors: µ-receptors, δ-receptors and κ-receptors. It is the µ-receptors which mediate most of the analgesic effects of opioid (as well as some of the adverse events).

Opioid receptors are G-protein coupled receptors which when activated target a G inhibitory subunit, causing a decrease in cAMP. In addition what this receptor activation does is it opens K+ channels causing hyperpolarisation, decreasing neuronal excitability. It also inhibits Ca2+ ion entry. In total causes decreased neuronal excitability and decreased neurotransmitter release.

Opioids, Pain and Analgesia“Fast” pain – better treated with NSAID, “slow/dull” pain good for opioids.