Description

Drugs exert their effects via specific interactions with target proteins and these depend on the concentration of drugs, which is maintained by the balance of dose rate versus clearance. The clearance rate is proportional to half-life of a drug and is dictated by specific enzymes.

Pharmacokinetics. A brief description of various components of pharmacokinetics is provided here.

Absorption and distribution of drugs. All drugs, regardless of the method of administration, must cross membranes to be absorbed into the circulation to reach the desired target organ. Membranes are composed of a lipid bilayer and are hydrophobic. Drugs with high lipid solubility and low molecular weight can cross the membranes easily, but large molecular weight drugs, such as proteins, have difficulty doing so and require active membrane transport carriers to cross the membranes.

Bioavailability of the drug is the amount and rate of drug entering the systemic circulation. It is determined by comparing the area under the plasma concentration curve and the area under the time curve after intravenous administration. Bioavailability is affected by several factors including the "first pass effect," ie, extensive metabolism of the drug in the portal circulation after oral ingestion prior to reaching the systemic circulation.

Most drugs are transported across membranes by passive diffusion, a mechanism that depends on the concentration gradient across the membrane. The amount of drug delivered to a tissue after absorption into the general circulation is determined by the blood flow rate and by the plasma concentration of the drug. A drug is delivered to the brain before it can affect the brain.

Drug metabolism and elimination. The term “elimination” refers to the removal of the drug from the body without the drug undergoing any change. This occurs mainly by renal clearance into urine. This is the major method of elimination for most of the drugs. Metabolism refers to the disappearance of a drug by chemical transformation into another compound called a metabolite. The metabolite may be inactive, or in some cases it is the active form of the drug administered as a prodrug.

Drug metabolism involves enzyme action. Several enzyme systems are involved, but the most common is the family of cytochrome P(pigment)-450 enzymes. The liver is the major site of drug metabolism. Several drugs can elevate the level of hepatic drug metabolizing enzymes, a phenomenon referred to as “enzyme induction,” and may result in an increase in the metabolism of a variety of substrates. The level of cytochrome Ps in brain, approximately 0.5% to 2% of that in the liver, is too low to significantly influence the overall pharmacokinetics of drugs and hormones in the body, but it has specific functions in brain, such as regulation of the levels of endogenous GABA(A) receptor agonists, maintenance of brain cholesterol homeostasis, and elimination of retinoids.

Desorption electrospray ionization mass spectrometry, a proteomic technique, can be used for imaging drugs and metabolites in tissues as a part of study of pharmacokinetics. For example, clozapine can be detected and quantified directly from histological sections of brain and other organs without chemical treatment.

Microdialysis, a technique for monitoring extracellular changes in living tissue, can be applied in neuropharmacology to substances present in the extracellular space, such as neurotransmitters and metabolites transported between cells and capillaries in the extracellular fluid.

Pharmacokinetic parameters of CNS drugs govern their pharmacodynamic responses. The cerebral blood flow represents the upper limit of the rate of brain penetration as it exposes the brain to the maximum amount of drug delivered and, hence, limits the entry of the drug into the brain in vivo. The presence of membrane transporters in the blood-brain barrier is another pathway to modulate the bioavailability of neuroactive drugs in the brain. Therefore, the early study of metabolism of a newly discovered drug can enable determination of efficacy, toxicity, reduction of side effects, and withdrawal of the drug from the market if indicated (07).

Pharmacodynamics. The interaction between the drug and the receptor results in a pharmacological response. The basic molecular mechanisms are being uncovered. If the molecular mechanism is unknown, the drug action is considered at the next highest biochemical or physiological level. Some of the fundamentals of drug action on the central nervous system will be considered here briefly.

Receptors. These are found in the postsynaptic side of the synapse. Each receptor consists of a protein molecule embedded in the membrane of the cell that contains active sites that bind to a specific chemical or neurotransmitter. When a receptor is stimulated, changes take place, either at the receptor through chemically gated ion channels or within the cell using transmembrane transduction; this activates second messengers to carry out secondary effects. Receptors in CNS disorders have accessory proteins expressed in specific neuronal pathways, which provide an opportunity for discovery of drugs with improved drug efficacy and reduced side effects; eg, targeting a TARP auxiliary subunit of AMPA receptors selectively modulates neuronal excitability in specific forebrain pathways relevant to epilepsy (31). Other receptors such as ion channels, gated by glutamate, γ-aminobutyric acid, and acetylcholine, also have associated proteins, which may be druggable.

Principles of synaptic transmission. Almost all drugs acting on the central nervous system produce their effects by modifying chemical synaptic transmission. The basic sequence of events is:

Impulse conduction >> transmitter release >>

signal transduction >> pharmacologic response

Neurotransmitters. Nearly all central nervous system neurons communicate by means of chemical transmitters or neurotransmitters that can be classified as:

Neurons store and release neurotransmitters and neuropeptides. Both are stored in secretary vesicles that release their contents by exostosis when cytoplasmic levels of calcium are raised.

Neurotransmitters play an important role in functions of the central nervous system. Studies investigating mechanisms of attention in sensory areas indicate that glutamatergic and cholinergic systems could contribute to increased processing abilities at the cellular and network level during states of top-down attention (09).

One of the most important functions of norepinephrine is its role as the neurotransmitter released from the sympathetic neurons affecting the heart. An increase in norepinephrine from the sympathetic nervous system increases the rate of contractions of the heart. As a stress hormone, norepinephrine affects parts of the brain, such as the amygdala, where attention and responses are controlled. Along with epinephrine, norepinephrine also underlies the fight-or-flight response, directly increasing heart rate, triggering the release of glucose from energy stores, and increasing blood flow to skeletal muscle. It increases oxygen supply to the brain. Norepinephrine can also suppress neuroinflammation when released diffusely in the brain from the locus coeruleus.

Synaptic transmission. Synaptic transmission has presynaptic and postsynaptic components. On the presynaptic side, neurotransmitters are synthesized and stored in synaptic vesicles in the neuron in a manner that will result in appropriate release into the synaptic cleft on stimulation. They interact with the appropriate receptor in the synaptic cleft that is coupled to second messenger systems with specific postsynaptic functions. The neurotransmitter may be degraded by enzymes or taken up by neurotransmitter transporters back into the neuron and into the vesicle. The reuptake process is mostly active and involves reuptake pumps generally applicable to serotonin, norepinephrine, and dopamine. Other neurotransmitters, such as acetylcholine, are degraded by enzymes in the synaptic vesicle. The breakdown products (choline) are subsequently reused for further transmitter resynthesis.

When released, neurotransmitters are effective only if they interact with receptors on their target cells. The specificity of neuronal interaction is determined by the type of transmitter released and by the type of receptors.

Cell signaling pathways. About one fifth of all human genes encode proteins involved in signal transduction. Neuronal viability is maintained through a complex interacting network of signaling pathways that can be disturbed in response to a multitude of cellular stresses and pathogenic factors. The current aim is intelligent design of therapeutic agents that can specifically correct disease-specific signaling alterations by targeting individual proteins. Protein kinases are the most important among substrates in cell signaling pathways relevant to neurologic disorders.

Sites of drug action in the central nervous system. Drugs acting on the central nervous system can be divided into the following categories according to the target molecules:

Receptors as sites of drug action. Receptors are involved in the action of most drugs, except drugs such as osmotic diuretics and general anesthetics that may not involve receptors as they are currently defined. First the drugs (ligands) must come close to the receptors and should be recognized before they bind to the receptor. This is possible because receptors possess molecular recognition capabilities. The receptors are embedded in a cellular or subcellular membrane and facilitate communication between the 2 sides of the membrane.

Receptor identification and classification is based on ligand (usually radiolabeled) binding specificity. Drugs may be agonists or antagonists of receptors. An agonist is a drug or a ligand that binds to the same site as the endogenous ligand and produces the same signal. An antagonist is a drug that binds to the site used by the endogenous ligand and acts competitively to diminish or block the signal produced by the endogenous signal.

Receptors that control ion channels. These can be ligand-gated, voltage-gated, or G protein-coupled.

(1) The voltage-gated potassium, sodium, and calcium channels control nerve impulse conduction and frequency. Because of this, they are common targets for anesthetics.

(2) Ligand-gated ion channels are pharmacologic receptors with specific drug binding sites and are regulated by chronic drug action. The binding sites are usually multiple. Ligand or transmitter-gated ion channels form a class of multisubunit membrane-spanning receptors that are essential for rapid signal transduction. In this class, the transmitter molecule itself operates the opening or closing of the channel by binding to a site on the receptor. In the nervous system, voltage-gated ion channels mediate action potentials and trigger transmitter release, whereas ligand-gated channels are responsible for chemical signaling mediated by classical fast-acting neurotransmitters.

(3) Neurotransmitters also trigger slower transmitter responses that are mediated by G protein-coupled receptors.

Gating refers to a conformational change in ion channels that is triggered by ligand binding. Ligand-activated ion channels are preferably termed transmitter-gated ion channels and divided into 2 categories: extracellularly activated, and intracellularly activated. The former includes nACh (nicotinic or neuronalACh), GABA(A), glycine, serotonin, and glutamate receptors, which are more relevant to the central nervous system drugs. Metabotropic glutamate receptor 5 (mGluR5) is a biomarker as well as a drug target for neuropsychiatric disorders and can be studied with mGluR5-specific PET radiotracers to understand the pathophysiology of relevant diseases and to speed up drug development for these (35).

The gating behavior of channels has many parallels with the allosteric behavior of enzymes, with the binding of ligand providing the free energy necessary to maintain the channel in the open conformation. For fast-acting neurotransmitters, the concentration of neurotransmitter in the synaptic cleft is high for a brief period and the receptors quickly reach the fully ligated but closed state. The rising and falling phases of synaptic response largely reflect the unbinding rate and the channel opening and closing rates. Various mechanisms, desensitization, channel block, and allosteric modulation affect the gating. Desensitization refers to the loss of response (conductance) during the continued presence of agonist. Rapid desensitization may be responsible for the termination of the neuronal response to neurotransmitters at some synapses.

Receptors coupled to guanine nucleotide-binding-proteins. Guanine nucleotide-binding proteins play an essential role in signal transduction, a complex communication mechanism whereby extracellular stimuli known as ligands activate an intracellular "messenger service." Guanine nucleotide-binding proteins couple extracellular signals, such as hormones and neurotransmitters, to membrane enzymes. Biochemical, electrophysiological, and cloning studies have shown that guanine nucleotide-binding proteins are also involved in the coupling process of membrane receptors to ion channels. Guanine nucleotide-binding protein can regulate ion channels directly or via second messengers such as cAMP (cyclic adenosine monophosphate), formed by the interaction of activated guanine nucleotide-binding protein with membrane-associated enzymes. Messages transmitted by the guanine nucleotide-binding protein signaling pathway can switch on or off a set of genes or control the passage of ions across the cell membrane. About 50% to 60% of all clinically relevant drugs exert their actions via guanine nucleotide-binding protein-coupled receptors. These drugs include serotonin modulators and opioid analgesics.

Most of the drugs are receptor modulators and produce their effect by enhancing or blocking signal transmission at the receptor. Some receptors for drugs acting on the central nervous system are as follows:

Acetylcholine receptor channels. Neuronal nicotinic receptors modulate fast synaptic transmission mediated by the glutamate and GABA. They are involved in the pathogenesis of several neurologic disorders, including Alzheimer and Parkinson disease. The activity of the different subtypes of neuronal nicotinic receptors is also subject to modulation by drugs such as galantamine. Some of these compounds can be used for the treatment of conditions in which neuronal nicotinic receptor function or expression is known to be altered.

Many toxins, chemicals, and therapeutic drugs are known to block neuronal nicotinic or acetylcholine receptor channels. These include the following drugs:

Cholinergic therapies are widely used because cholinergic dysfunction is involved in several neurologic and psychiatric disorders and focus on increasing the availability of acetylcholine (ACh). However, dysfunction of muscarinic subtypes of ACh receptors are involved in many of these diseases. Therefore, cholinergic therapeutics targeted to the M1 and M4 muscarinic receptors is also required for treating several disorders associated with cognitive dysfunction and behavioral symptoms (11).

Dopamine receptors. In the central nervous system, dopamine is involved in the control of locomotion and cognition. The actions of dopamine are mediated by 5 different receptor subtypes, D1 to D5, that are members of a large G protein coupled-receptor family. D1 receptors are mainly postsynaptic and D2 receptors are found in both pre- and postsynaptic sides of the synapse. D3 and D4 are expressed in areas of the brain serving cognition and affect and have an increased affinity for neuroleptics. These may serve as targets for neuroleptics devoid of side effects.

GABA-A receptors. GABA was the first neurochemical to satisfy all neurotransmitter criteria. Synaptic inhibition in the central nervous system is mediated by channels gated by GABA. Each isoform consists of 5 homologous or identical subunits surrounding a central chloride ion-selective channel gated by GABA (39). GABA(A) belongs to a family of ligand-gated ion channels.

Interaction of various drugs with GABA(A) receptors has been studied in detail. Some the important drugs that act on GABA(A) receptors are:

Several subtypes of the GABA(A) receptor have been identified, and this information is used for improving the safety profile of some of the drugs acting on these receptors. For example, anxiolytics that are partial agonists of GABA(A) receptors have less adverse effects compared to conventional benzodiazepine anxiolytics.

GABA(B) receptor is a distinct class of GABA receptors found in the central nervous system for which baclofen is an agonist. Major pharmacological effects of gamma-hydroxybutyric acid, used for the treatment of narcolepsy and alcoholism as well as abused as the recreational drug, are usually considered to be mediated by GABA(B) receptors. However, with the availability of molecular tools, certain GABA(A) receptor subtypes have been discovered as high-affinity binding sites and potential mediators of gamma-hydroxybutyric acid effects (05).

Glutamate receptor channels. Glutamate is the principal excitatory neurotransmitter in the brain. When glutamate is released from nerve terminals and interacts with postsynaptic glutamate receptors, it leads to the opening of cationic channels. Glutamate receptors, located on virtually all neurons in the central nervous system, function as ligand-gated channels, and are essential components of cell-cell communication in the nervous system. Although glutamate has been known to be a neuroexcitant since the 1950s, the role of glutamate-gated ion channels in central synaptic transmission was widely accepted during this decade. Glutamate receptors mediate a substantial portion of fast excitatory transmission in the brain and consist of 2 types: (1) N-methyl-D-aspartate and (2) non-N-methyl-D-aspartate (amino-methyl propionic acid/kainate).

Because glutamate plays an important role in several central nervous system disorders, there is a considerable interest in the development of competitive N-methyl-D-aspartate antagonists as drugs. Therapeutic targets for N-methyl-D-aspartate antagonists are as follows.

Noncompetitive N-methyl-D-aspartate antagonists. The earlier drugs of this category were compounds that acted at the level of the ion channel (noncompetitive N-methyl-D-aspartate antagonists) and included potent antagonists such as dizocilpine (MK-801). They penetrated readily into the central nervous system but possessed undesirable side effects such as sedation and behavioral effects. Some drugs of this category are under investigation as neuroprotectives in stroke.

Serotonin receptors. The action of serotonin receptors is mostly believed to be mediated by members of 5-HT1-5. The application of molecular cloning techniques has enabled the discovery of novel serotonin receptors such as 5-HT6 and 5-HT7 for which there was no prior functional or pharmacologic data. A classification of serotonin receptors is shown in Table 2 along with the agonists and antagonists when known. There are 14 serotonin receptors with different properties. This far exceeds the number of receptors known for any other neurotransmitter.

Signal transduction mechanisms of serotonin receptors in the brain are shown in Table 2. Among the characterized serotonin receptors of the central nervous system, the type 3 receptor subtype (5-HT3R) is the only one known to be a ligand-gated ion channel. Its early pharmacologic characterization and mapping by radioligand and binding autoradiography suggested that this receptor may, among other actions, regulate dopamine release in the nigrostriatal pathway. Following the cloning of this receptor, direct cellular localization was made possible by in situ hybridization and immunohistochemical analysis and it was shown that 5-HT3R-expressing neurons are mainly GABA-containing cells.

The question arises as to why there are so many serotonin receptors in the central nervous system. It is believed that multiple neurotransmitter receptors provide the nervous system with a capacity for coding and decoding serotonin-mediated neuronal transmission that could not take place with a single neurotransmitter receptor.

Table 2. Signal Transduction Mechanism of Serotonin Receptors in the Brain

The 5-HT6 receptors appear to regulate cholinergic neurotransmission in the brain. 5-HT6 receptor antagonists might have a role in the treatment of learning and memory disorders. 5-HT7 has been implicated in the pathophysiology of several disorders including those involving smooth muscle within the vascular system. Widespread distribution of 5-HT7 within the brain suggests multiple central roles that are being investigated. These include a neuroendocrine role as well as links to circadian rhythms and depression.

Central adenosine A-2A receptors. Adenosine and its various receptor subtypes play multiple functions in the modulation of several central nervous system functions. A-2A receptors play an important role in the modulation and release of several neurotransmitters and neuromodulators. Physiological effects and therapeutic potential of A-2A receptors are as follows.

2A receptor agonists decrease locomotor activity and pentylenetetrazol-induced seizures but increase slow-wave and paradoxical sleep. These compounds are being developed for epilepsy, sleep disorders, and psychoses.

2A receptor antagonists decrease excitatory amino acid release and have a neuroprotective effect. One example is decrease of infarct size in experimental cerebral ischemia (see Neuroprotection for central nervous system disorders).

G-protein coupled receptors. These are an important class of drug targets that exist as proteins on the surface membranes of all cells. They are also referred to as "serpentine" or "7-TM" receptors, as they cross the membrane 7 times. The G-protein coupled receptors are a superfamily of proteins accounting for approximately 1% of the human genome and are associated with a wide range of therapeutic categories including CNS diseases. Purified multiple G-protein coupled receptors in a functional form can be used for the identification of tight-binding ligands. There are estimated to be about 2000 of them within the human body, but to date only approximately 100 G-protein coupled receptors are well-characterized with known ligands, of which only about half are currently targets of commercial drugs. Approximately 60% of all currently available prescription drugs interact with these receptors (22). 5-HT receptors are associated with protein networks that are important for their coupling to the signaling machinery. Techniques are available to insert fluorescent labels into receptors in intact cells, making it possible to sense receptor activation via changes in their fluorescence (01).

An example of a drug interaction with G-protein coupled receptors is the antimigraine drug sumatriptan, which acts at the 5-HT(1B/1D) receptor. Many drugs of abuse signal through receptors that couple to G-protein coupled receptors, so the factors that control their signaling are likely to be important to the understanding of drug abuse. Drugs that could inhibit their activity would be useful in preventing the development of, or in treating, drug dependence.

Sigma-1 receptors. These receptors are involved in the modulation of various neurotransmitter systems and play important roles in the pathophysiology of neuropsychiatric diseases. They have a high affinity to psychotropic drugs. Sigma-1 receptors are assumed to serve as a regulator of adenosine triphosphate production and bioenergetics within the cell (16).

Role of astrocytes in neuropharmacology. Astrocytes are specialized glial cells with complex functions. They release several transmitters that affect neuronal activity and synaptic transmission (15). They respond to various insults by reactive gliosis, which plays a role in the pathomechanism of several diseases. Therefore, astrocytes may be valuable targets for the development of novel therapies for neurologic disorders.

Microglia are associated with neurons and astrocytes, thereby acting as regulators of neurotransmission. On activation, microglia cells rapidly release small amounts of adenosine triphosphate, and astrocytes amplify this release (34). Because of its role in neurologic disorders, pathological activation of microglia is also a target for therapeutic intervention. Pharmacological and brain imaging studies show that microglia play a role in regulating neurotransmitter synthesis and immune cell activation, indicating their involvement in the pathophysiology of bipolar disorder that may translate into improved therapies (40).

Role of myelin in neuropharmacology. A myelin-centered model of human brain function has been proposed to explain why psychotropic treatments ranging from antipsychotics to serotonin reuptake inhibitors and acetylcholinesterase inhibitors have been shown to be effective for a wide spectrum of disorders ranging from autism, schizophrenia, and depression to Alzheimer disease (04). Many of these treatments for neuropsychiatric disorders share complex signaling pathways such as Akt and glycogen synthase kinase-3 that affect plasticity and repair of myelination. These pathways respond to neurotransmitters, neurotrophins, and hormones and may contribute to the mechanisms of action of current therapeutics by promoting myelination.

Role of gene expression in neuropharmacology. Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product, which is usually a protein. In chronic treatment with a drug acting on the CNS, drug-induced protein expression leads to accumulation of alterations that eventually manifest as therapeutic effects. The profile of drug-induced gene expression in the brain is determined by activity of different neurotransmitter systems and response of various types of cells. Comparison of gene expression differences between various drugs as determined by microarrays has provided a new way to classify the different antidepressants and to predict their cellular targets (28).

Systems neuropharmacology. Because most neuropsychic disorders are polygenic with involvement of multiple mechanisms, their response to single-target drugs is unsatisfactory. An approach based on systems biology, termed “systems pharmacology,” is more appropriate because it considers that a disease expresses a disturbance of the network of interactions underlying organismic functions, rather than alteration of single molecular components (32). Systems pharmacology aims to restore a disturbed network via multitargeted drugs. The term used for CNS drugs is “systems neuropharmacology.” A similar approach, synergistic network pharmacology, has been applied to drug discovery for stroke by using both cellular in vitro and mouse in vivo models. In one study, a primary target, NADPH oxidase type 4, was used to identify a related co-target, nitric oxide synthase, and inhibition of both were found to be highly synergistic in achieving significant reduction of infarct volume, direct neuroprotection, and blood-brain-barrier stabilization (06). CNS quantitative systems pharmacology is gradually evolving into a mature technology and could have the capability to move beyond the prediction of effect on biomarkers toward functional clinical responses of pharmaceutical interventions in CNS diseases (13).

Cheminformatics. Analysis of data from new compounds targeting multiple complex networks (drug-target interactome network) in the nervous system is challenging. Cheminformatics (application of informatics in chemistry) tools are being used in translational research from medicinal chemistry to neuropharmacology (37). Artificial neural network algorithms may be helpful for predicting the interactions of drugs and targets in CNS interactome.

Clinical applications

Drug delivery to the central nervous system. Drugs need to be delivered to the nervous system to be effective in neurologic disorders.

Gene therapy of neurologic disorders. Gene therapy can be broadly defined as the transfer of defined genetic material to specific target cells for the ultimate purpose of preventing or altering a specific disease state.

Cell therapy for neurologic disorders. Cell therapy for neurologic disorders means the use of cells of neural or nonneural origin to replace, repair, or enhance the function of the disturbed or damaged nervous system. Action of stem cells may be enhanced by certain drugs.

Nanobiotechnology and neurotherapeutics. Nanobiotechnology is contributing to improving our understanding of the functions of the nervous system and diagnosis of neurologic disorders (25). Nanofibers provide scaffolds for regeneration of the central nervous system. Nanobiotechnology is contributing to neurotherapeutics in 3 ways:

Limitation of translation of animal experimental studies to human neurotherapeutics. A major challenge for neuropharmacologists developing new drugs for neurologic disorders is that successes in animal disease models are not necessarily reproducible in human patients, ie, failure of translation. Mice are popular test subjects for human neurotherapeutics because their brain is made up of similar types of brain cells as humans. However, brain cells in mice express genes that are different from the ones in human brain cells. For example, a mouse's serotonin receptors are not found on the same cells that they are found in humans, and a drug that increases serotonin levels in the brain, such as a SSRI used to treat depression, might deliver it to vastly different cells in mice than in humans (18). These species-specific features indicate the importance of directly studying the drug action on the human brain.

Suggestions for improving discovery of disease-modifying therapies. Most of the conventional drugs for neurologic disorders provide only symptomatic relief. Possible reasons for the shortage of disease modifying therapies for neurologic disorders include complexity and variability of human brain diseases, poor understanding of brain pharmacokinetics and pharmacodynamics, species differences, flaws in preclinical studies, and failure of poorly designed clinical trials. To address some of these problems, a platform for CNS drug discovery has been developed comprising: (1) drug screening of primary adult human brain cells, (2) human brain tissue microarray analysis of drug targets, and (3) high-content phenotypic screening methods (10).

An additional approach to improve clinical translation is the use of a human brain-based drug discovery platform to validate brain drug targets as well as to directly test drugs for efficacy on human brain cells developed from patient-derived induced pluripotent stem cell (iPSC) reprograming. However, cells generated by this method do not quite represent the cells of the brain although they help to identify genetic abnormalities associated with the disease and test drugs against these. Organoid 3-dimensional brain structures are an improvement for testing drugs, but they also fail to capture the connectivity and environment of the human brain. Further improvements include human brain tissue microarrays and human brain cell culture.

Human brain tissue microarrays. Advantages of tissue microarrays over conventional histological methods include unbiased and standardized assessments as well as efficient utilization of human brain samples. This information is used to guide the development of valid animal models of brain disease that incorporate complexity of most brain disorders. There is also a pressing need to incorporate more variations in human diseases into animal models. Animal models should closely match symptoms in humans. Humanized mice expressing human brain validated targets would likely improve the translatability of model systems. A study by demonstrated that a phosphodiesterase-4D allosteric inhibitor protects against memory loss and neuronal atrophy induced by oligomeric Abeta 1-42 and provides useful insight into the potential role of compensatory mechanisms in Alzheimer disease in a model of oligomeric Abeta 1-42 neurotoxicity (08).

In vivo study of drug action in the central nervous system in human patients. Several methods have been used for studying drug action on the brains of experimental animals, but few are available for use in living patients. Some of these are the following:

Biomarkers. A biomarker is a characteristic that can be objectively measured and evaluated as an indicator of a physiological as well as pathological process or pharmacological response to a therapeutic intervention. Any specific molecular alteration of a cell on DNA, RNA, metabolite, or protein level can be referred to as a molecular biomarker (23). Among the current applications of biomarkers, those for drug discovery and development are of the most important. Biomarkers are used in new approaches to remedy the shortage of new drugs and the current lengthy and expensive process of drug development. Biomarkers can be used to predict and confirm target binding, to determine mechanism of action of a drug, pharmacokinetics, and toxicity, to monitor disease status, to stratify patients, and to determine treatment efficacy in clinical trials.

The ideal biomarker for CNS drug development should recognize the mechanism of action of a potential new therapy (mechanism-based biomarker), and the relation between biomarker endpoint and intervention should have a biologically plausible explanation. Biomarker endpoints need to be investigated in both animals and humans, as the extrapolation of animal models of disease to human pathology is often uncertain. The validation process is required for a better definition of the biomarker sensitivity, specificity, positive and negative predictive value, accuracy, likelihood ratio of positive and negative tests, discriminant validity, and sensitivity to change and to treatment difference.

Neuroimaging plays an important role in neuropharmacology and provides key biomarkers as bridges from the laboratory to the clinic in central nervous system drug development where quantitative biomarkers as surrogate measures of efficacy are often lacking and clinical trial endpoints can be confounded by high placebo response. Imaging can be used preclinically to select candidate drug molecules and clinically to facilitate proof of concept testing and optimization of resources through prioritization of decision making during the development of new therapeutics.

Electroencephalography. Quantitative pharmaco-EEG uses the computer-analyzed EEG, and has been used in determining the pharmacodynamic profiles of psychotropic drugs at the central nervous system level. Therapeutic effects of the antidepressant trazodone on its target symptoms (depressed mood, anxiety, and insomnia) were identified by early human pharmaco-EEG and subsequent sleep laboratory studies. It is an inexpensive and efficient screening tool to generate data regarding a drug’s central nervous system potential and dose response in humans during phase 1 and 2 studies. In healthy volunteers, such a study should provide the following information about a compound:

(1) If a compound exerts a measurable effect on the human central nervous system that is distinguishable from placebo, and that is the minimum central nervous system-effective dose.

(2) Whether any demonstrable central nervous system effect is dose- or time-related.

(3) For correlating the compound’s pharmacodynamic activity with pharmacokinetic measures.

(4) The potential clinical indication of a compound based on comparison of the compound’s profile(s) against those contained in a database.

Quantitative EEG changes induced by the acute administration of standard neuroleptics or novel antipsychotics in healthy subjects as well as in patients with schizophrenia may be used to predict response to antipsychotic therapy. This technique has been used mostly during drug development but applications of pharmaco-EEG, together with pharmacokinetic-pharmacodynamic modeling in everyday clinical practice of neurology can also be considered. One caveat is that electrophysiological effects of neuroactive drugs are somewhat nonspecific and require correlation with other parameters.

Magnetoencephalography. A recording of the magnetic fields of the brain, magnetoencephalography is recognized as a complement to EEG in neurophysiology and is under investigation as a neurodiagnostic tool. Although EEG records the electrical potentials on the scalp, magnetoencephalography records the magnetic fields produced by the same electrical activity. Magnetoencephalography is being used to explore the effect of various pharmacological agents on the brain. With a resolution of milliseconds, magnetoencephalography enables exploration of focal cortical effects of psychopharmacological agents. It can be combined with functional MRI.

Brain imaging. PET and MRI are powerful tools for studying the action of drugs used in the treatment of neurologic disorders. Technological advances in imaging have made it possible to noninvasively extract information from the human brain regarding a drug's mechanism and site of action. Distribution, density, and activity of receptors in the brain can be visualized by radioligands labeled for PET, and the receptor binding can be quantified by appropriate tracer kinetic models. PET has been used for determining indirect drug-induced metabolic changes and for characterization of functional interactions of neurotransmitter systems by assaying drug-induced displacement of specific receptor ligands. With PET, compounds labeled with appropriate isotopes can be used to determine the amount of a drug that reaches the target in the brain, the minimum effective dose, and the duration of action required to elicit the desired therapeutic response. PET techniques have been used for over a decade to examine the functions of histaminergic neurons in the living human brain. Progression of Parkinson disease can be assessed by (18)F-dopa PET.

Fluctuations and dysregulation in brain networks can occur in neurologic disorders and drug addiction. Resting state MRI is used to assess continuous spontaneous activity of the brain to identify distinct patterns of large-scale brain networks and can also be used to study effect of drugs on these fluctuations (30).

PET and fMRI can be used simultaneously to correlate changes in receptor occupancy with hemodynamic changes in the brain. An experimental study in nonhuman primates has demonstrated that administration of D2/D3 dopamine receptor antagonist [(11)C]raclopride to nonhuman primates causes an increase in cerebral blood volume and a reduction in binding potential that are localized to the dopamine-rich striatum (38). This method has potential clinical applications in diagnosis and monitoring of therapy in clinical studies. Apart from the use of MRI in monitoring the effect of treatment on brain lesions, functional MRI has been used to study gene expression in the brain in vivo. fMRI provides the means to image molecular processes and screen active drugs in vivo. Pharmacologic MRI (phMRI) is the use of drugs as measured stimuli to study their effects on the brain and can provide information beyond that obtained by conventional fMRI (27). It is possible to measure parameters reflecting neurotransmitter release and binding associated with the pharmacokinetics or the pharmacodynamics of drugs. Brain imaging has contributed to determining not only the molecular targets and pharmacokinetics of methylphenidate, but also the connectivity and brain networks activated by treatment (41). Hybrid PET-MRI techniques enable multimodal tracking of the effect of methylphenidate on neurotransmission.

MRI can also be used to study the pathomechanism of drug-induced neurologic disorders. Use of diffusion-weighted imaging has shown that reversible metronidazole-induced encephalopathy might be caused by cytotoxic edema.

Although fMRI is proving useful in pharmacological studies, there are some limitations. Drugs might interfere with the mechanisms that give rise to the pharmacological fMRI signal. Brain imaging findings, combined with clinical correlations, can be used as surrogate biomarkers of response even for the later stages of drug development. However, a pitfall, which needs to be avoided, is failure to interpret the imaging data in an appropriate disease- and drug-specific manner. A series of experimental measures has been suggested to improve the interpretability of blood oxygen level-dependent fMRI studies to investigate the effect of diseases and pharmacological agents on brain activity (20). These include simultaneous EEG-fMRI, cerebral blood volume, and rate of metabolic oxygen consumption measurements.

Effect of drugs on the developing brain. Psychotropic drugs are being increasingly used in children. Drug exposure during sensitive periods of brain development may have beneficial long-term effects, but also harmful delayed consequences (03). Study of the effects of stimulants, antidepressants, and antipsychotics on brain maturation should be taken into consideration in the treatment of adults as well.

Chronopharmacology as applied to the central nervous system. The term "chronopharmacology" is applied to variations in the effect of drugs according to the time of their administration during the day. Mammalian biological functions are organized according to circadian rhythms (lasting about 24 hours). They are coordinated by a biological clock situated in the suprachiasmatic nuclei of the hypothalamus. These rhythms persist under constant environmental conditions, demonstrating their endogenous nature. Some rhythms can be altered by disease. Chronotherapy is modulation of treatment over a 24-hour period based on rhythms of disease and chronopharmacology.

“Chronopharmacokinetics” is defined as the predictable changes observed in the plasma levels of drugs and in the parameters used to characterize the pharmacokinetics of a drug. Half-life of a drug can vary as a function of the hour of administration. Central nervous system drugs with documented time-dependent changes in pharmacokinetics are:

Knowledge of chronopharmacology is important in designing a suitable method of drug delivery for 2 reasons:

(1) Knowledge of the circadian pattern of disease, eg, strokes in early hours of morning, may enable the optimal drug delivery at the critical time.

(2) Blood levels of a drug such as an antiepileptic can be maintained at a constant level to compensate for the variation.

Shift work can interfere with mechanisms regulating drug kinetics in peripheral compartments and action at selective brain sites, either directly or through effects on the gastrointestinal or hormonal cycles. Night-shift workers also have alterations in their circadian rhythms. Caution should be exercised in drug administration in inappropriate phases of the circadian cycles. Risk of inadequate drug dosing or unexpected effects of drugs should be considered in such subjects who are under long-term treatment.

Therapeutic drug monitoring. Therapeutic drug monitoring (TDM) is a means of monitoring drug levels in the blood and is defined by the International Association of Therapeutic Drug Monitoring & Clinical Toxicology as follows:

“Therapeutic drug monitoring is a multi-disciplinary clinical specialty aimed at improving patient care by individually adjusting the dose of drugs for which clinical experience or clinical trials have shown that it improved outcome in the general or special populations.”

Therapeutic range of a drug is usually depicted as the upper and lower levels, and the drug is usually effective within this range. Higher levels may lead to toxicity, and lower levels to loss of efficacy. The lower limit of therapeutic range, or the trough, is set to provide approximately 50% of the maximum therapeutic effect, whereas the upper limit (peak) is defined by toxicity, not therapeutic effect. Some patients may achieve therapeutic effects at levels below the established range, whereas others may experience toxicity while still in the established range. These may be due to individual genetic variations.

Many drugs are used without monitoring of blood levels, as their dosage can generally be varied according to the clinical response of the patient to that drug. However, this is impossible with some drugs, as insufficient levels can result in undertreatment or development of drug resistance, and excessive levels can lead to drug toxicity. Indications for therapeutic drug monitoring include the following:

Drugs that require monitoring include antiepileptics, analgesics, antidepressants, and antipsychotics. These are important for the management of epilepsy, psychiatric disorders, and pain. Most clinical laboratories provide these services.

Approved drugs for neurologic disorders. Selected approved drugs are listed according to therapeutic categories in Table 3.

Table 3. Selected Approved Drugs for Neurologic Disorders

Future of neuropharmacology. Considerable progress has been made in the availability of new drugs for neurologic disorders but a need remains for more drugs. Most of the new drugs will be biological, ie, cell and gene therapies, antisense oligonucleotides, RNA inhibitors, microRNAs, monoclonal antibodies, and vaccines. The focus will be on targeted delivery to the CNS and nanobiotechnologies will play an important role. Examples of disease-modifying therapies include natalizumab for multiple sclerosis, antisense therapies eteplirsen and golodirsen for Duchenne muscular dystrophy, and nusinersen for spinal muscular atrophy.

Natural history of a disease and the “neuroecological” milieu of the molecular target of drug action in the disease state may provide better information for future discovery efforts (14). Models of CNS diseases and drugs that best suit the individual patient will be important factors in drug discovery.

Application of advances in technologies for mapping the spatial and temporal patterns of neural activity, based on an improved understanding of brain function in health as well as disease, is the discovery of next-generation neurotherapeutics for neuropsychiatric disorders. High-throughput technology to generate large-scale brain activity maps with machine learning for predictive analysis enables screening of drug clusters that are associated with known therapeutic categories by systems neuropharmacology approach, eg, antiseizure-like drug leads and validation of their therapeutic effects in the pentylenetetrazol zebrafish seizure model (29).