Download
1 / 66
660 likes | 806 Views
ADMEAssorbimentoDistribuzioneMetabolismoEscrezione. ADME : definizioni. . ASSORBIMENTOVelocit: dipende da diversi fattori; per un dato farmaco proporzionale alla sua concentrazione a livello del sito di assorbimentoEntit: definita dalla competizione tra velocit di assorbimento e veloc
E N D
4. Figure 1. The multiple mechanisms of transport through the
intestinal epithelium. After oral administration, a drug can be
absorbed into the systemic circulation via passive diffusion
through the cells (transcellular transport), between cells via
cell–cell junctions (paracellular transport) or via a transporter
(active transport). P-glycoprotein (P-gp) is a transporter found
associated with the plasma membrane of intestinal mucosal
epithelium that actively pumps some drugs back into the
intestinal lumen after they are absorbed into the cells.
Figure 1. The multiple mechanisms of transport through the
intestinal epithelium. After oral administration, a drug can be
absorbed into the systemic circulation via passive diffusion
through the cells (transcellular transport), between cells via
cell–cell junctions (paracellular transport) or via a transporter
(active transport). P-glycoprotein (P-gp) is a transporter found
associated with the plasma membrane of intestinal mucosal
epithelium that actively pumps some drugs back into the
intestinal lumen after they are absorbed into the cells.
12. Figure 2. The Caco-2 permeability assay. Caco-2 cells are
cultured as confluent monolayers on the porous membrane of
an inner well situated within an outer well. Transport studies are
performed by placing the compounds to be studied in the inner
well (apical side) and monitoring the amount of the test
compound in the outer well (basolateral side). For this assay to
be useful, the Caco-2 cell barrier needs to have tight cell–cell
junctions. This is usually achieved by culturing the cells for
>21 days. However, it has since been demonstrated that tight
cell–cell junctions and the expression of P-glycoprotein can be
achieved after only three days in culture
Figure 2. The Caco-2 permeability assay. Caco-2 cells are
cultured as confluent monolayers on the porous membrane of
an inner well situated within an outer well. Transport studies are
performed by placing the compounds to be studied in the inner
well (apical side) and monitoring the amount of the test
compound in the outer well (basolateral side). For this assay to
be useful, the Caco-2 cell barrier needs to have tight cell–cell
junctions. This is usually achieved by culturing the cells for
>21 days. However, it has since been demonstrated that tight
cell–cell junctions and the expression of P-glycoprotein can be
achieved after only three days in culture
17. Fig 1. — Mechanism of action of P-glycoprotein (P-gp) inhibitors, showing normal P-gp function in the plasma membrane of a cancer cell during chemotherapy. Activation of the efflux pump by the hydrolysis of a bound ATP molecule drives the cytotoxic drug molecules out of the cell.
Fig 2. — Competitive inhibition of the P-glycoprotein transporter. First and second-generation modulators compete as a substrate with the cytotoxic
agent for transport by the pump. This limits the efflux of the cytotoxic agent, increasing its intracellular concentration.
Fig 3. — Noncompetitive inhibition of the P-glycoprotein transporter. Third-generation inhibitors of P-gp, such as tariquidar, bind with high affinity
to the pump but are not themselves substrates. This induces a conformational change in the protein, thereby preventing ATP hydrolysis and
transport of the cytotoxic agent out of the cell, resulting in an increased intracellular concentration.
Fig 1. — Mechanism of action of P-glycoprotein (P-gp) inhibitors, showing normal P-gp function in the plasma membrane of a cancer cell during chemotherapy. Activation of the efflux pump by the hydrolysis of a bound ATP molecule drives the cytotoxic drug molecules out of the cell.
Fig 2. — Competitive inhibition of the P-glycoprotein transporter. First and second-generation modulators compete as a substrate with the cytotoxic
agent for transport by the pump. This limits the efflux of the cytotoxic agent, increasing its intracellular concentration.
Fig 3. — Noncompetitive inhibition of the P-glycoprotein transporter. Third-generation inhibitors of P-gp, such as tariquidar, bind with high affinity
to the pump but are not themselves substrates. This induces a conformational change in the protein, thereby preventing ATP hydrolysis and
transport of the cytotoxic agent out of the cell, resulting in an increased intracellular concentration.
21. Figure 1 | The blood–brain barrier. a | The blood–brain barrier (BBB) is formed by endothelial cells at the level of the cerebral capillaries. These endothelial cells interact with perivascular elements such as basal lamina and closely associated astrocytic end-feet processes, perivascular neurons (represented by an interneuron here) and pericytes to form a functional BBB. b | Cerebral endothelial cells are unique in that they form complex tight junctions (TJ) produced by the interaction of several transmembrane proteins that effectively seal the paracellular pathway. These complex molecular junctions make the brain practically inaccessible for polar molecules, unless they are transferred by transport pathways of the BBB that regulate
the microenvironment of the brain. There are also adherens junctions (AJ), which stabilize cell–cell interactions in the junctional zone. In addition, the presence of intracellular and extracellular enzymes such as monoamine oxidase (MAO), ?-glutamyl transpeptidase (?-GT), alkaline phosphatase, peptidases, nucleotidases and several cytochrome P450 enzymes endow this dynamic interface with metabolicactivity. Large molecules such as antibodies, lipoproteins, proteins and peptides can also be transferred to the central compartment by receptor-mediated transcytosis or
non-specific adsorptive-mediated transcytosis. The receptors for insulin, low-density lipoprotein (LDL), iron transferrin (Tf) and leptin are all involved in transcytosis.
P-gp, P-glycoprotein; MRP, multidrug resistance-associated protein family.Figure 1 | The blood–brain barrier. a | The blood–brain barrier (BBB) is formed by endothelial cells at the level of the cerebral capillaries. These endothelial cells interact with perivascular elements such as basal lamina and closely associated astrocytic end-feet processes, perivascular neurons (represented by an interneuron here) and pericytes to form a functional BBB. b | Cerebral endothelial cells are unique in that they form complex tight junctions (TJ) produced by the interaction of several transmembrane proteins that effectively seal the paracellular pathway. These complex molecular junctions make the brain practically inaccessible for polar molecules, unless they are transferred by transport pathways of the BBB that regulate
the microenvironment of the brain. There are also adherens junctions (AJ), which stabilize cell–cell interactions in the junctional zone. In addition, the presence of intracellular and extracellular enzymes such as monoamine oxidase (MAO), ?-glutamyl transpeptidase (?-GT), alkaline phosphatase, peptidases, nucleotidases and several cytochrome P450 enzymes endow this dynamic interface with metabolicactivity. Large molecules such as antibodies, lipoproteins, proteins and peptides can also be transferred to the central compartment by receptor-mediated transcytosis or
non-specific adsorptive-mediated transcytosis. The receptors for insulin, low-density lipoprotein (LDL), iron transferrin (Tf) and leptin are all involved in transcytosis.
P-gp, P-glycoprotein; MRP, multidrug resistance-associated protein family.
22. Fig. 4: Phase contrast microscopic images of conditionally immortalized cells forming the blood-brain barrier. Conditionally immortalized rat brain capillary endothelial cell line, TR-BBB (a); astrocyte cell line, TR-AST (b); and pericyte cell line, TR-PCT (c). Magnification X100. Fig. 4: Phase contrast microscopic images of conditionally immortalized cells forming the blood-brain barrier. Conditionally immortalized rat brain capillary endothelial cell line, TR-BBB (a); astrocyte cell line, TR-AST (b); and pericyte cell line, TR-PCT (c). Magnification X100. Fig. 4: Phase contrast microscopic images of conditionally immortalized cells forming the blood-brain barrier. Conditionally immortalized rat brain capillary endothelial cell line, TR-BBB (a); astrocyte cell line, TR-AST (b); and pericyte cell line, TR-PCT (c). Magnification X100.Fig. 4: Phase contrast microscopic images of conditionally immortalized cells forming the blood-brain barrier. Conditionally immortalized rat brain capillary endothelial cell line, TR-BBB (a); astrocyte cell line, TR-AST (b); and pericyte cell line, TR-PCT (c). Magnification X100.
23. Fig. 1. Schematic diagrams of the blood–brain barrier [BBB, (a)] and blood–cerebrospinal fluid barrier [BCSFB (b)]. The tight junction is a specific feature of brain capillary endothelial cells and choroid epithelial cells. It minimizes the penetration of drugs from circulating blood into the brain parenchyma and cerebrospinal fluid via the paracellular route. Therefore, drugs have to penetrate the CNS transcellularly. These barriers restrict the penetration of drugs that have low intrinsic permeability across the lipid bilayer because of their high MW and/or high hydrophilicity. This leads to poor drug distribution in the brain. Both brain capillary endothelial cells and choroid epithelial cells provide a barrier function to protect the CNS and are referred to as the blood–brain barrier and the blood–cerebrospinal fluid barrier, respectively. Transporters play an important role in restricting the penetration of drugs into the CNS by removing them into the circulating blood. (c) shows the mechanisms of transport that occur in the blood capillaries of other tissues where cell–cell junctions are not as tight as in the BBB and BCSFB. Abbreviation: CSF, cerebrospinal fluid. Fig. 1. Schematic diagrams of the blood–brain barrier [BBB, (a)] and blood–cerebrospinal fluid barrier [BCSFB (b)]. The tight junction is a specific feature of brain capillary endothelial cells and choroid epithelial cells. It minimizes the penetration of drugs from circulating blood into the brain parenchyma and cerebrospinal fluid via the paracellular route. Therefore, drugs have to penetrate the CNS transcellularly. These barriers restrict the penetration of drugs that have low intrinsic permeability across the lipid bilayer because of their high MW and/or high hydrophilicity. This leads to poor drug distribution in the brain. Both brain capillary endothelial cells and choroid epithelial cells provide a barrier function to protect the CNS and are referred to as the blood–brain barrier and the blood–cerebrospinal fluid barrier, respectively. Transporters play an important role in restricting the penetration of drugs into the CNS by removing them into the circulating blood. (c) shows the mechanisms of transport that occur in the blood capillaries of other tissues where cell–cell junctions are not as tight as in the BBB and BCSFB. Abbreviation: CSF, cerebrospinal fluid.
24. Fig. 1: Establishing conditionally immortalized cell lines from transgenic animals harboring temperature-sensitive SV40 large T-antigen gene (tsA58 T-antigen gene). The strategy for establishing conditionally immortalized cell lines from transgenic animals is shown here. The tsA58 T-antigen gene product is stably expressed in all tissues in transgenic animals and is inactive at 37°C. When isolated tissue cells are cultured at 33°C, the gene product turns into the activated form and some cells acquire immortalized characteristics. The cloned cell lines are obtained by colony isolation for the rapidly growing cells. Fig. 1: Establishing conditionally immortalized cell lines from transgenic animals harboring temperature-sensitive SV40 large T-antigen gene (tsA58 T-antigen gene). The strategy for establishing conditionally immortalized cell lines from transgenic animals is shown here. The tsA58 T-antigen gene product is stably expressed in all tissues in transgenic animals and is inactive at 37°C. When isolated tissue cells are cultured at 33°C, the gene product turns into the activated form and some cells acquire immortalized characteristics. The cloned cell lines are obtained by colony isolation for the rapidly growing cells.
25. a | Brain endothelial cells are grown on filter inserts together with glial cells at the bottom of 6-, 12- or 24-well culture plates. b | Glial soluble factors secreted in the culture medium induce the blood-brain barrier (BBB) phenotype in the capillary endothelium. This experimental design can be used for compound screening in the drug discovery process in the pharmaceutical industry but is also well suited for studying mechanistic aspects of BBB transport as well as other biological and pathological processes. c | Illustration of a typical experimental design which allows a co-culture of brain endothelial cells and glial cells74. Vimentin immunostaining shows a confluent brain endothelial cell monolayer with non-overlapping morphology and typical spindle shaped cells (top right panel). The continuous marginal localization of the tight junction protein occludin reflects the tightness of the barrier and the cerebral origin of the capillary endothelial cells (middle panel). In the bottom right panel, staining for glial fibrillary acidic protein (GFAP) (red) shows astrocytes within the glial cell population and ED-1 staining (green) highlights the presence of microglia. Scale bar represents 25 m.
a | Brain endothelial cells are grown on filter inserts together with glial cells at the bottom of 6-, 12- or 24-well culture plates. b | Glial soluble factors secreted in the culture medium induce the blood-brain barrier (BBB) phenotype in the capillary endothelium. This experimental design can be used for compound screening in the drug discovery process in the pharmaceutical industry but is also well suited for studying mechanistic aspects of BBB transport as well as other biological and pathological processes. c | Illustration of a typical experimental design which allows a co-culture of brain endothelial cells and glial cells74. Vimentin immunostaining shows a confluent brain endothelial cell monolayer with non-overlapping morphology and typical spindle shaped cells (top right panel). The continuous marginal localization of the tight junction protein occludin reflects the tightness of the barrier and the cerebral origin of the capillary endothelial cells (middle panel). In the bottom right panel, staining for glial fibrillary acidic protein (GFAP) (red) shows astrocytes within the glial cell population and ED-1 staining (green) highlights the presence of microglia. Scale bar represents 25 m.
26. Fig. 5: In vitro blood-brain barrier models constructed with conditionally immortalized cell lines. (a) Non-contact co-culture model: endothelial cells are cultured with astrocytes and pericytes using cell culture inserts without contact. Endothelial cells are cultured on the upper side of the inserts. Astrocytes and pericytes are cultured on plates. The proteins secreted from astrocytes or pericytes interacts with endothelial cells through the pores on the inserts. (b) Culture with conditioned medium model: conditioned medium is prepared from a single culture of either astrocytes or pericytes. The conditioned medium contains proteins secreted from astrocytes or pericytes. Then, endothelial cells are cultured with conditioned medium of astrocytes or pericytes. (c) Contact co-culture model: endothelial cells are cultured with astrocytes and pericytes using a cell culture insert with contact. Endothelial cells are cultured on the upper side of the insert. Astrocytes and pericytes are cultured on the opposite side of the insert. In addition to the soluble factor effect, this system produces a partial contact effect. (d) Mixed culture model: endothelial cells, astrocytes and pericytes are cultured in the same culture plate. Right side panel, TR-BBB cells are isolated from a mixture of TR-BBB and TR-AST cells by means of a cell sorter with anti-Tie2 (an endothelial cell-specific marker) antibody labelling. Red, yellow and blue cells indicate endothelial cells, astrocytes and pericytes, respectively. Fig. 5: In vitro blood-brain barrier models constructed with conditionally immortalized cell lines. (a) Non-contact co-culture model: endothelial cells are cultured with astrocytes and pericytes using cell culture inserts without contact. Endothelial cells are cultured on the upper side of the inserts. Astrocytes and pericytes are cultured on plates. The proteins secreted from astrocytes or pericytes interacts with endothelial cells through the pores on the inserts. (b) Culture with conditioned medium model: conditioned medium is prepared from a single culture of either astrocytes or pericytes. The conditioned medium contains proteins secreted from astrocytes or pericytes. Then, endothelial cells are cultured with conditioned medium of astrocytes or pericytes. (c) Contact co-culture model: endothelial cells are cultured with astrocytes and pericytes using a cell culture insert with contact. Endothelial cells are cultured on the upper side of the insert. Astrocytes and pericytes are cultured on the opposite side of the insert. In addition to the soluble factor effect, this system produces a partial contact effect. (d) Mixed culture model: endothelial cells, astrocytes and pericytes are cultured in the same culture plate. Right side panel, TR-BBB cells are isolated from a mixture of TR-BBB and TR-AST cells by means of a cell sorter with anti-Tie2 (an endothelial cell-specific marker) antibody labelling. Red, yellow and blue cells indicate endothelial cells, astrocytes and pericytes, respectively.
27. Fig. 1: Establishing conditionally immortalized cell lines from transgenic animals harboring temperature-sensitive SV40 large T-antigen gene (tsA58 T-antigen gene). The strategy for establishing conditionally immortalized cell lines from transgenic animals is shown here. The tsA58 T-antigen gene product is stably expressed in all tissues in transgenic animals and is inactive at 37°C. When isolated tissue cells are cultured at 33°C, the gene product turns into the activated form and some cells acquire immortalized characteristics. The cloned cell lines are obtained by colony isolation for the rapidly growing cells. Fig. 1: Establishing conditionally immortalized cell lines from transgenic animals harboring temperature-sensitive SV40 large T-antigen gene (tsA58 T-antigen gene). The strategy for establishing conditionally immortalized cell lines from transgenic animals is shown here. The tsA58 T-antigen gene product is stably expressed in all tissues in transgenic animals and is inactive at 37°C. When isolated tissue cells are cultured at 33°C, the gene product turns into the activated form and some cells acquire immortalized characteristics. The cloned cell lines are obtained by colony isolation for the rapidly growing cells.
28. Fig. 2: Conditionally immortalized cell lines of rat blood-organ barriers. Conditionally immortalized cell lines were established from the cells forming the barrier system, including blood-brain barrier (BBB) (a), blood-cerebrospinal fluid barrier (BCSFB) (b), inner blood-retinal barrier (iBRB) (c) and blood-placental barrier (BPB) (d). Brain capillary endothelial cells, choroid plexus epithelial cells, retinal capillary endothelial cells and syncytiotrophoblast cells form the barrier in the BBB, BCSFB, iBRB and BPB, respectively. The cells surrounding capillary endothelial cells, such as astrocytes, pericytes and Müller cells, regulate the barrier functions of endothelial cells. The name of each cell line is indicated by red text. Fig. 2: Conditionally immortalized cell lines of rat blood-organ barriers. Conditionally immortalized cell lines were established from the cells forming the barrier system, including blood-brain barrier (BBB) (a), blood-cerebrospinal fluid barrier (BCSFB) (b), inner blood-retinal barrier (iBRB) (c) and blood-placental barrier (BPB) (d). Brain capillary endothelial cells, choroid plexus epithelial cells, retinal capillary endothelial cells and syncytiotrophoblast cells form the barrier in the BBB, BCSFB, iBRB and BPB, respectively. The cells surrounding capillary endothelial cells, such as astrocytes, pericytes and Müller cells, regulate the barrier functions of endothelial cells. The name of each cell line is indicated by red text.
29. Fig. 2: Conditionally immortalized cell lines of rat blood-organ barriers. Conditionally immortalized cell lines were established from the cells forming the barrier system, including blood-brain barrier (BBB) (a), blood-cerebrospinal fluid barrier (BCSFB) (b), inner blood-retinal barrier (iBRB) (c) and blood-placental barrier (BPB) (d). Brain capillary endothelial cells, choroid plexus epithelial cells, retinal capillary endothelial cells and syncytiotrophoblast cells form the barrier in the BBB, BCSFB, iBRB and BPB, respectively. The cells surrounding capillary endothelial cells, such as astrocytes, pericytes and Müller cells, regulate the barrier functions of endothelial cells. The name of each cell line is indicated by red text. Fig. 2: Conditionally immortalized cell lines of rat blood-organ barriers. Conditionally immortalized cell lines were established from the cells forming the barrier system, including blood-brain barrier (BBB) (a), blood-cerebrospinal fluid barrier (BCSFB) (b), inner blood-retinal barrier (iBRB) (c) and blood-placental barrier (BPB) (d). Brain capillary endothelial cells, choroid plexus epithelial cells, retinal capillary endothelial cells and syncytiotrophoblast cells form the barrier in the BBB, BCSFB, iBRB and BPB, respectively. The cells surrounding capillary endothelial cells, such as astrocytes, pericytes and Müller cells, regulate the barrier functions of endothelial cells. The name of each cell line is indicated by red text.
36. Figure 1 | Routes of elimination of the top 200 most prescribed drugs in 2002. Metabolism represents the listed clearance mechanism for 73% of the top 200 drugs. Of the drugs cleared via metabolism, about three-quarters are metabolized by members of the cytochrome P450 (CYP) superfamily. For the CYP-mediated clearance mechanisms, the majority of drug oxidations (46%) were carried out by members of the CYP3A family; followed by 16% by CYP2C9; 12% for both CYP2C19 and CYP2D6; 9% for members of the CYP1A family; and 2% for both CYP2B6 and CYP2E1 (Ref. 9). UGT, uridine diphosphate glucuronyl transferase. Figure 1 | Routes of elimination of the top 200 most prescribed drugs in 2002. Metabolism represents the listed clearance mechanism for 73% of the top 200 drugs. Of the drugs cleared via metabolism, about three-quarters are metabolized by members of the cytochrome P450 (CYP) superfamily. For the CYP-mediated clearance mechanisms, the majority of drug oxidations (46%) were carried out by members of the CYP3A family; followed by 16% by CYP2C9; 12% for both CYP2C19 and CYP2D6; 9% for members of the CYP1A family; and 2% for both CYP2B6 and CYP2E1 (Ref. 9). UGT, uridine diphosphate glucuronyl transferase.
38. Figure 3. Preparation of liver microsomes and hepatocytes.
Liver microsomes can be prepared from previously frozen
tissues, whereas hepatocytes can only be prepared from a
freshly isolated liver. Hepatocytes can either be plated onto
collagen-coated plates as primary cultures or used in suspension
for studies of metabolism, cytotoxicity and drug–drug
interaction. However, cultured hepatocytes are required for use
in enzyme induction studies. Hepatocytes can be cryopreserved
and stored in liquid nitrogen for later use, which greatly
enhances the utility of this experimental system, particularly
where human hepatocytes are concerned.
Figure 3. Preparation of liver microsomes and hepatocytes.
Liver microsomes can be prepared from previously frozen
tissues, whereas hepatocytes can only be prepared from a
freshly isolated liver. Hepatocytes can either be plated onto
collagen-coated plates as primary cultures or used in suspension
for studies of metabolism, cytotoxicity and drug–drug
interaction. However, cultured hepatocytes are required for use
in enzyme induction studies. Hepatocytes can be cryopreserved
and stored in liquid nitrogen for later use, which greatly
enhances the utility of this experimental system, particularly
where human hepatocytes are concerned.
41. Figure 4. Metabolic stability screening assay. This assay can be
performed using 96-well plates with a porous membrane at the
bottom. Test compounds are mixed with either hepatocytes or
liver microsomes and incubated for a period of time. After
incubation, an organic solvent, such as acetonitrile, is added to
stop the reaction and to extract any non-covalently bound
compounds from macromolecules. The plates are then
centrifuged so that the liquid fraction can be filtered into a
second 96-well recipient plate, and the concentration of test
compound is determined using, for example, LC–MS.
Figure 4. Metabolic stability screening assay. This assay can be
performed using 96-well plates with a porous membrane at the
bottom. Test compounds are mixed with either hepatocytes or
liver microsomes and incubated for a period of time. After
incubation, an organic solvent, such as acetonitrile, is added to
stop the reaction and to extract any non-covalently bound
compounds from macromolecules. The plates are then
centrifuged so that the liquid fraction can be filtered into a
second 96-well recipient plate, and the concentration of test
compound is determined using, for example, LC–MS.
44. Figure 5. (a) P450 inhibition assay. Test compounds are preincubated with either liver microsomes or hepatocytes followed by the addition of P450 substrates. After further incubation, the rate of metabolism is determined by quantification of specific metabolites. P450 inhibition is indicated by a dose-dependent decrease in activity. For screening assays, a single concentration of the test compound is used, but for more extensive studies of inhibitory potential, multiple concentrations of test compounds can be used for the determination of IC50 or Ki values.
(b) P450 induction assay. This assay uses cultured hepatocytes that are treated with the test compounds for >2 days.
Isoform-specific P450 substrates are then added to the cells to quantify the activities of specific P450 isoforms. Induction is indicated by an increase in activity in treated cells compared with untreated control cells. This is a robust assay: to date, all known inducers of human P450 isoforms induce the relevant enzymes in cultured human hepatocytes.
Figure 5. (a) P450 inhibition assay. Test compounds are preincubated with either liver microsomes or hepatocytes followed by the addition of P450 substrates. After further incubation, the rate of metabolism is determined by quantification of specific metabolites. P450 inhibition is indicated by a dose-dependent decrease in activity. For screening assays, a single concentration of the test compound is used, but for more extensive studies of inhibitory potential, multiple concentrations of test compounds can be used for the determination of IC50 or Ki values.
(b) P450 induction assay. This assay uses cultured hepatocytes that are treated with the test compounds for >2 days.
Isoform-specific P450 substrates are then added to the cells to quantify the activities of specific P450 isoforms. Induction is indicated by an increase in activity in treated cells compared with untreated control cells. This is a robust assay: to date, all known inducers of human P450 isoforms induce the relevant enzymes in cultured human hepatocytes.
46. Of the 38 drugs withdrawn from the market between 1994 and 2006, the majority was toxic to liver or heart. To date, mitochondrial impairment has been implicated in the observed toxicities by cerivastatin, troglitazone, and tolcapone. Of the 38 drugs withdrawn from the market between 1994 and 2006, the majority was toxic to liver or heart. To date, mitochondrial impairment has been implicated in the observed toxicities by cerivastatin, troglitazone, and tolcapone.
48. The surface electrocardiogram (ECG), which provides information on the electrical events occurring within the heart, is obtained by placing electrodes on the surface of the body. Typically, the P wave reflects atrial depolarization, the QRS complex reflects ventricular depolarization and the T wave is indicative of ventricular repolarization. The QRS complex is produced by the upstroke (phase 0) of the action potential. The isoelectric S–T segment corresponds to the plateau (phase 2), whereas the T wave is indicative of ventricular repolarization (phase 3). The resting membrane potential corresponds to phase 4. The duration of the QT interval on the ECG is defined as the duration between the beginning of the QRS complex and the end of the T wave. It is a reflection of ventricular action potential duration, and represents the time during which the ventricles depolarize, and repolarize. Numerous overlapping ionic currents contribute to determining the morphology and duration of the ventricular action potential duration (see text).The surface electrocardiogram (ECG), which provides information on the electrical events occurring within the heart, is obtained by placing electrodes on the surface of the body. Typically, the P wave reflects atrial depolarization, the QRS complex reflects ventricular depolarization and the T wave is indicative of ventricular repolarization. The QRS complex is produced by the upstroke (phase 0) of the action potential. The isoelectric S–T segment corresponds to the plateau (phase 2), whereas the T wave is indicative of ventricular repolarization (phase 3). The resting membrane potential corresponds to phase 4. The duration of the QT interval on the ECG is defined as the duration between the beginning of the QRS complex and the end of the T wave. It is a reflection of ventricular action potential duration, and represents the time during which the ventricles depolarize, and repolarize. Numerous overlapping ionic currents contribute to determining the morphology and duration of the ventricular action potential duration (see text).
49. Torsades de Pointes is characterized by an abnormally prolonged QT interval, the presence of morphological changes on the T wave, and the occurrence of rapid polymorphic ventricular tachyarrhythmias with a distinctive twisting morphology of the QRS complex around the isoelectric baseline. The initiation of the arrhythmia is characterized by a short-long-short sequence, followed by a pause and a normal beat with a prolonged QT interval Torsades de Pointes is characterized by an abnormally prolonged QT interval, the presence of morphological changes on the T wave, and the occurrence of rapid polymorphic ventricular tachyarrhythmias with a distinctive twisting morphology of the QRS complex around the isoelectric baseline. The initiation of the arrhythmia is characterized by a short-long-short sequence, followed by a pause and a normal beat with a prolonged QT interval
51. (a) Model of voltage-sensitive oxonol redistribution mechanism. (b) Fluorescence micrograph of CHO-K1 cells exciting the oxonol dye, bis-(1,2-dibutylbarbituric acid) trimethine oxonol [DisBAC4(3)] directly with 535 nm light,
(a) Model of voltage-sensitive oxonol redistribution mechanism. (b) Fluorescence micrograph of CHO-K1 cells exciting the oxonol dye, bis-(1,2-dibutylbarbituric acid) trimethine oxonol [DisBAC4(3)] directly with 535 nm light,
52. (c) Model of fluorescence resonance energy transfer (FRET) mechanism. The distance, and hence FRET, between the stationary donor and the mobile accptor at the plasma membrane is dependent on voltage.
(d) Fluorescence micrograph of the same cells as in (b) loaded with the FRET-based voltage sensor dyes. The image shows the localized fluorescence emission obtained when exciting the coumarin-lipid donor,
(c) Model of fluorescence resonance energy transfer (FRET) mechanism. The distance, and hence FRET, between the stationary donor and the mobile accptor at the plasma membrane is dependent on voltage.
(d) Fluorescence micrograph of the same cells as in (b) loaded with the FRET-based voltage sensor dyes. The image shows the localized fluorescence emission obtained when exciting the coumarin-lipid donor,
54. Figure 6. Screening assay for hepatotoxicity. Hepatocytes are treated with the test compounds and cell viability is determined using various endpoints. The endpoints commonly used include: quantification of ATP content, release of cytoplasmic enzymes, mitochondrial functions, dye uptake, macromolecular synthesis and cellular glutathione content. For screening assays, a single concentration of test compound is usually used; however, cytotoxicity can be further studied using multiple concentrations, enabling the determination of dose–response curves and EC50 values.
Figure 6. Screening assay for hepatotoxicity. Hepatocytes are treated with the test compounds and cell viability is determined using various endpoints. The endpoints commonly used include: quantification of ATP content, release of cytoplasmic enzymes, mitochondrial functions, dye uptake, macromolecular synthesis and cellular glutathione content. For screening assays, a single concentration of test compound is usually used; however, cytotoxicity can be further studied using multiple concentrations, enabling the determination of dose–response curves and EC50 values.
55. Model of mitochondrial toxicity screening. Early identification of mitochondrial liabilities during compound series selection and lead selection using in vitro screens allows for structure–activity relationship (SAR) studies to circumvent it. Cell-based screening assays are available for initial identification of general cytotoxicity. If a mitochondrial etiology is suspected, assessment of isolated mitochondrial function can also be performed in screening mode. If positive, additional in vitro mechanistic studies can examine effects on OXPHOS respiration, permeability transition, reactive oxygen and nitrogen centered (ROS), membrane potential (??m), and mitochondrial DNA (mtDNA) status, among others. Such data enable structure–activity relationship (SAR) studies necessary to circumvent mitochondrial toxicity. Mitochondrial assessment should be completed before a compound moves into further development and is elevated to drug candidate level but should certainly also be triggered if a compound has a negative response in an animal model. Classical assays such as electron microscopy and enzyme-linked assays of the individual respiratory complexes are available for assessing mitochondrial status in tissues. In addition, newer histochemical and immunohistochemical techniques are being developed assays that will help illuminate mechanism of mitochondrial impairment. Fostered by the organelle and cell studies, animal models are also being developed that better reveal mitochondrial toxicity, and hence better predict clinical outcome. One such model is a manganese superoxide dismutase (MnSOD) knockdown mouse that more sensitively detects drug-induced mitochondrial toxicity [71]. Model of mitochondrial toxicity screening. Early identification of mitochondrial liabilities during compound series selection and lead selection using in vitro screens allows for structure–activity relationship (SAR) studies to circumvent it. Cell-based screening assays are available for initial identification of general cytotoxicity. If a mitochondrial etiology is suspected, assessment of isolated mitochondrial function can also be performed in screening mode. If positive, additional in vitro mechanistic studies can examine effects on OXPHOS respiration, permeability transition, reactive oxygen and nitrogen centered (ROS), membrane potential (??m), and mitochondrial DNA (mtDNA) status, among others. Such data enable structure–activity relationship (SAR) studies necessary to circumvent mitochondrial toxicity. Mitochondrial assessment should be completed before a compound moves into further development and is elevated to drug candidate level but should certainly also be triggered if a compound has a negative response in an animal model. Classical assays such as electron microscopy and enzyme-linked assays of the individual respiratory complexes are available for assessing mitochondrial status in tissues. In addition, newer histochemical and immunohistochemical techniques are being developed assays that will help illuminate mechanism of mitochondrial impairment. Fostered by the organelle and cell studies, animal models are also being developed that better reveal mitochondrial toxicity, and hence better predict clinical outcome. One such model is a manganese superoxide dismutase (MnSOD) knockdown mouse that more sensitively detects drug-induced mitochondrial toxicity [71].
56. Mitochondrial function can be inhibited in many ways in addition to inhibition of electron transport and uncoupling of it from the membrane potential. For example, impairment of exchange of requisite substrates via membrane transporters, inhibition of metabolic pathways that fuel respiration, and direct effects of drugs on cardiolipin can acutely undermine mitochondrial function. Such acute effects frequently accelerate autoxidation of electron transport components that yields oxygen-centered and nitrogen-centered free radicals. By contrast, drugs that impair DNA replication or protein synthesis will diminish mitochondrial and hence bioenergetic capacity over a longer time period. In addition, many drugs can precipitate irreversible mitochondrial collapse via formation of the permeability transition pore leading to release of pro-apoptotic factors such as cytochrome c. Drugs that alter the normal equilibrium between pro-apoptotic and anti-apoptotic proteins, such as Bak/Bax and Bcl-2, among many others, can also induce mitochondrial failure. Mitochondrial function can be inhibited in many ways in addition to inhibition of electron transport and uncoupling of it from the membrane potential. For example, impairment of exchange of requisite substrates via membrane transporters, inhibition of metabolic pathways that fuel respiration, and direct effects of drugs on cardiolipin can acutely undermine mitochondrial function. Such acute effects frequently accelerate autoxidation of electron transport components that yields oxygen-centered and nitrogen-centered free radicals. By contrast, drugs that impair DNA replication or protein synthesis will diminish mitochondrial and hence bioenergetic capacity over a longer time period. In addition, many drugs can precipitate irreversible mitochondrial collapse via formation of the permeability transition pore leading to release of pro-apoptotic factors such as cytochrome c. Drugs that alter the normal equilibrium between pro-apoptotic and anti-apoptotic proteins, such as Bak/Bax and Bcl-2, among many others, can also induce mitochondrial failure.
59. DL50
66. Stages in the drug discovery process. The drug discovery process begins with the identification of a medical need, including a judgement on the adequacy of existing therapies (if there are any). From this analysis, together with an appraisal of the current knowledge about the target disease, will come hypotheses on how to possibly improve therapy — that is, what efficacy, safety or mechanistically novel improvements will advance the method of drug treatment for patients with the target disease? On the basis of these hypotheses, specific objectives will be set for the project. Then, testing selected chemicals in appropriate biological tests can begin. Key subsequent steps in the process include detecting relevant biological activity (a 'hit') for a structurally novel compound in vitro, then finding a related compound with in vivo activity in an appropriate animal model, followed by maximizing this activity through the preparation of analogous structures, and finally selecting one compound as the drug development candidate. This drug candidate then undergoes toxicological testing in animals, as required by law. If the compound passes all these tests, all the accumulated research data are assembled and submitted as an Investigational New Drug Application (IND) to the Food and Drug Administration (FDA) in the United States (or comparable agency in other countries) before clinical trials are initiated. In the clinic, there is sequential evaluation in normal human volunteers of toleration (Phase I), efficacy and dose range in patients (Phase II), followed by widespread trials in thousands of appropriate patients to develop a broad database of efficacy and safety. For the few (4–7%) drug candidates that survive this series of development trials, a New Drug Application (NDA) that contains all the accumulated research data is filed for thorough review by the experts at the FDA. Only with their approval can the new drug be offered to doctors and their patients to treat the disease for which it was designed.
Stages in the drug discovery process. The drug discovery process begins with the identification of a medical need, including a judgement on the adequacy of existing therapies (if there are any). From this analysis, together with an appraisal of the current knowledge about the target disease, will come hypotheses on how to possibly improve therapy — that is, what efficacy, safety or mechanistically novel improvements will advance the method of drug treatment for patients with the target disease? On the basis of these hypotheses, specific objectives will be set for the project. Then, testing selected chemicals in appropriate biological tests can begin. Key subsequent steps in the process include detecting relevant biological activity (a 'hit') for a structurally novel compound in vitro, then finding a related compound with in vivo activity in an appropriate animal model, followed by maximizing this activity through the preparation of analogous structures, and finally selecting one compound as the drug development candidate. This drug candidate then undergoes toxicological testing in animals, as required by law. If the compound passes all these tests, all the accumulated research data are assembled and submitted as an Investigational New Drug Application (IND) to the Food and Drug Administration (FDA) in the United States (or comparable agency in other countries) before clinical trials are initiated. In the clinic, there is sequential evaluation in normal human volunteers of toleration (Phase I), efficacy and dose range in patients (Phase II), followed by widespread trials in thousands of appropriate patients to develop a broad database of efficacy and safety. For the few (4–7%) drug candidates that survive this series of development trials, a New Drug Application (NDA) that contains all the accumulated research data is filed for thorough review by the experts at the FDA. Only with their approval can the new drug be offered to doctors and their patients to treat the disease for which it was designed.
