Exam 3

Drug approval steps

1. preclinical testing

2. Investigational New Drug Application (NED)

3. Phase 1

4. Phase 2

5. Phase 3

Phase 1

In Phase 1, the drug is tested in a small group of healthy volunteers to evaluate its safety, determine a safe dosage range, and identify side effects. (20-80 people)

Pharmacokinetics = 2nd goal

only 5% of drugs move end up being approved

Phase 2

In Phase 2, the drug is tested in a larger group of people (tens to hundreds of people) who have the condition it aims to treat. This phase focuses on assessing the drug's effectiveness and further evaluating its safety.

phase 3

In Phase 3, the drug is tested in a large group of patients (hundreds to thousands) to confirm its effectiveness, monitor side effects, and compare it to commonly used treatments. This phase provides a more comprehensive understanding of the drug's benefits and risks.

Note that participants are healthier than in real life.

Drug Generics

Produced/marketed once a drug’s patent has ended.

How do drug companies attempt to prevent generics

1. “pay to delay”

2. patent infringement suits

3. Product hopping

Product Hopping

A strategy used by brand-name drug manufacturers to change or reformulate a drug shortly before its patent expires, (includes altering dosage, formulation, routes of administration, or dosing schedule) incentivizing consumers to switch to the new version, thereby delaying generic competition.

Components of stress

1. stressful expirenceexperience

2. processing

3. adaptation

Components of anxiety

1. fear or worry of future

   1. increase muscle tension

   2. restfulness

   3. impaired concentration

   4. increased irritability

   5. activation of autonomic nervous system aka “fight-or-flight”

steroids: cycling pattern

A method used by bodybuilders or athletes where they alternate between periods of steroid use ("cycles") and periods of abstaining to minimize side effects/tolerance and maintain gains.

typically 6-12 weeks long between cycles.

cycling + pyramiding

is a technique used in steroid use where athletes gradually increase the dose (pyramiding) during a cycle before tapering off to prevent side effects.

aids in reducing possible withdrawal.

Metabolic tolerance

is the body's reduced response to a drug or substance after repeated use, often resulting in the need for higher doses to achieve the same effect.

There is an increase in p450 liver microsomal enzymes that metabolize alcohol

Cross-tolerance

is a phenomenon where tolerance to one substance leads to a reduced response to another pharmacologically similar substance.

ie> due to upregulation of p450 in the livers of smokers —> they can break caffeine down faster.

Cross-tolerance can be @ the receptor level: GABAA

Pharmacodynamic tolerance

neurons adapt to the continued presence of drugs by making compensatory changes in cell function

All addictive drugs BLANK dopamine levels in the BLANK

increase, nucleus accumbens

What type of information do glutamatergic neurons carry, and where do they originate? (addiction lecture)

sensory motor information from cerebral cortex

What type of information do dopaminergic neurons carry, and where do they originate? (addiction lecture)

reward related information, from ventral tegmental area

What is the role of the Nucleus Accumbens (NAc) in this process?

The Nucleus Accumbens plays a crucial role in the reward circuitry of the brain, integrating signals related to pleasure and reinforcement from various areas and facilitating motivated behavior.

What is the significance of the co-incidence of glutamate and dopamine signals?

It strengthens the synaptic connection between the glutamatergic input (cue or action) and the NAc neuron, which is a cellular mechanism of associative learning.

Dopamine release serves as a prediction error signal

1. burst of dopamine signifies a reward that hasn’t been predicted

2. When monkey learns cues that predict a reward, DA release begins with the cue in expectation of reward and ceases once reward is presented

3. if reward fails to occur, DA neurons cease firing, generating a decrease in DA levels that correlates with the time when the expected reward did not come.

4. Once environmental cues for rewards are learned, DA-ergic neurons fire back to baseline level.

What makes addictive drugs differ in reward pathways/DA

They also cause a DA release, to the brain interprets them as “better than expected” which maximizes drug seeking.

3 key factors of relapse

1. delivering a small dose

2. subjecting subject to stress

3. environmental cues previously paired with the drug

Pharmacokinetics of Methamphetamine

Liver metabolism:

• chief metabolite: amphetamine

  • both are active

  • peak plasma concentrations at 3-6hrs (oral)

  • half life is approx. 12 hours

• When taken via IV or smoked —> an initial rush followed by euphoria

Mechanisms of action of amphetamines

1. causes several transporters for NTs to reverse (different than blocking as it causes actual release into synaptic cleft)

2. DA, 5-HT, and NE spill into synapse

3. Also blocks MAO, so more DA available for packing into vesicles

GHB PD/PK

• usually sold as a solution in water

  • is rapidly absorbed from the GI tract

  • readily crosses the BBB

• Low doses produce an alcohol-like experience

  • mild euphoria

  • relaxation

  • social disinhibition

Higher doses of GHB? Overdosing

• lethargy

• ataxia

• slurred speech

• dizziness

• nausea

• vomiting

ODing

• respiratory depression

• potential for seizures

Receptor target for GHB

• likely some action through GABAb

  • metabotropic GABA receptor (gpcr)

  • tendency to inhibit signaling in neurons

  • action is often presynaptic- inhibits NT release

• GABAb knockoutmich have high affinity GHB binding sites but don’t respond to GHB treatment —> most effects of GHB are blocked by GABAb antagonists

Inhalants: Behavioral and neural effects:

• CNS depressant

  • is best explained by the effects on ionotropic receptors —> enhance function of inhibitory GABAa receptors

• Inhibit the activity of excitatory NMDA glutamate and nicotinic cholinergic receptors

• Inhalants reduce CNS excitability and cause behavioral impairment in much the same way as alcohol.

Binge eating disorder and Lisdexamfetamine

• vyvanse

• increases monoamine NTs in the synaptic cleft

  • via inhibiting reuptake, MAO, and disrupting vesicular release

ghrelin: production site and effect (relevant for feeding)

• stomach and neurons in the hypothalamus

• appetite

Anadamide: production site and effect (relevant for feeding)

• small intestine

• appetite

Insulin: production site and effect (relevant for feeding)

• pancreas

• satity & glycogen/lipid storage

leptin: production site and effect (relevant for feeding)

• adipose tissue (long term) & Stomach (short term)

• satiety

• Regulates the amount of fat stored in the body

• reduced in sleep deprivation

CCK: production site and effect (relevant for feeding)

• produced in the small intestine

• stimulates digestion ( release of digestive enzymes from exocrine pancreas, bile from gallbladder, and H+ from pariertal cells in stomach) and promotes satiety

PYY: production site and effect (relevant for feeding)

• produced in the intestine (ileum and colon)

• promotes satiety

Vagal mechanism of satiety

• afferent signals in the vagus conveyed to the brain function to limit meal size

  • information from stretch receptors in the stomach wall

  • sensors in the portal blood vessels for

    • cck

    • glucose

    • osmolality

    • pH

Bilateral lesions placed in the region of the ventro____ hypothalamus INCREASE appetite

medial

Bilateral lesions placed in the region of the ventro____ hypothalamus DECREASE appetite

lateral

Leptin infused in the brain ventricles of insulin-deficient diabetic animals dose WHAT to blood glucose

normalizes blood glucose

Glucose transporters and its location

GLUT-1: Erythrocytes and BBB

GLUT-2: beta cells, renal tubular cells, liver, and intestinal epithelial cells

GLUT-3: Neurons and placenta

GLUT-4: striated muscle and adipose tissue, aka INSULIN REGULATED GLUCOSE

Types of herbal remedies

Infusions: Hot water extracts of herbs

Tinctures: 100% alcoholic extracts of herbs

elixirs: alcoholic extracts of herbs (12-38%)

syrups: extracts of herbs with syrup or honey

Concentrations of “active ingredients” in herbs very depending on

1. geographic location: soil, climate, UV exposure, and plant comp.

2. time of harvest: ripeness

3. enhancer use: pesticides, herbicides, and fertilizers (organic vs. natural vs conventional)

4. harvesting method

5. storage/shipping conditions: shelf life

Cocaine metabolism:

Metabolized by butyrylcholinesterase through hydrolysis, liver cholinestrases, and liver cytochrome p450 34A

using cocaine with alcohol can lead up to a 30% increase in cociain levels in the blood —> half life is 1 h in the plasma

liver cytochrome p450 34A

• Inhibited by grapefruit juice

• Substrates include acetaminophen, codeine, cyclosporin, diazepam, erythromycin, and chloroquine

6 common adverse effects of cocaine in pregnancy use

Common Adverse Effect of Cocaine use in Pregnancy

1- Restricts blood flow to the uterus, cause fetal hypoxia.

2- Uterine contractions, CNS infarction, Heart defects.

3- Persistent neonatal arterial hypertension.

4- Decreased neonatal weight and size (¯ head circumference).

5- Sudden infant death syndrome, Intellectual disability.

6- Babies may be irritable at birth and exhibit symptoms such as: tremor, hypertension, abnormal reflexes, tachypnea, autonomic instability, vomiting, diarrhea, seizures and poor feeding.

True or false: Neuroinflammation is ALWAYS a negative thing

FALSE: example: synaptic pruning

synaptic pruning

is a natural process in the brain that removes excess neurons and synapses, allowing for more efficient neural connections.

TBI mouse models: what reduces inflammation and improves functional recovery

Anti-inflammatory treatments minocycline can help reduce inflammation and promote recovery in TBI mouse models.

Borrelia Burgdorferi aka Lyme disease

• bacterial infection

• red rash, fever, headaches, tiredness

• Later can lead to —> loss of ability to move one or both sides of the face, joint pains, severe headaches with neck stiffness, or heart palpitations stiffness

• Treatments: antibiotics- first choice, usually doxycycline

Treatment for brain eating amoebas

• anti fungal drug: amphotericin b

  • binds to cell membrane’s sterols of the pathogen

  • cell disruption and death

  • fatality is still greater than 95% even with treatment.

Multiple sclerosis

• autoimmune disease

• presence of T cells that recognize oligodendrocyte myeline specific antigens

• activated t-cells cross the bbb and secrete pro-inflamatory cytokines, leading to demyelination and neurodegeneration in the central nervous system.

Anoctamin 2 and MS

protein in oligodendrocytes that is involved in myelin regulation and may contribute to nerve repair processes in multiple sclerosis.

is the target in MSand is implicated in disrupting normal myelin function.

Disease modifying treatments of MS focus on…

Reducing frequency and severity of relapses and accumulation of lesions

Interferon beta (1a and 1b) in MS

• Increases suppressor T cell function

• these t cells maintain tolerance to self antigens and decrease the self attack of MS

Drug treatment of MS: Copaxone (glatiramer acetate)

• synthetic protein

  • random combination of four amino acids that are found in myelin

• May work as acting as a decoy target for the immune system

Drug treatment of MS: Novantrone (mitoxantrone)

• chemotherapy

• Suppresses components of the immune system like T & b cells and macrophages

Drug treatment of MS: Tysabri (natalizumab)

• monoclonal antibody (lab-produced protien designed to mimic the function of natural antibodies) against cell adhesion molecule alpha4-intergrin

• hinders movement of t cells across the bbb

• the side effects makes this reserved as a second line drug

Drug treatment of MS: Gilenya (fingolimod)

sphingosine 1-phosphate receptor modulator

• GPCR

• causes retention of lymphocytes ( a kind of white blood cell) in lymph nodes

• prevents their entry to the CNS

SMA: common signs and symptoms

• movement difficulties (walking, sitting up, carrying objects)

• Problems with breathing

• Problems with swallowing

• Flaccidity

• atrophy

• hypotonia

• fasciculations

• hyporeflexia

4 types of SMA:

Type 1: onset before 6 months, no sitting

Type 2: onset 6-18 months, sitting but no walking

Type 3: onset after 12 months, walking

Type 4: onset after 30 years, normal

What causes SMA?

Mutation in the survivor motor neuron gene 1.

SMN2 plays a secondary role in symptom severity.

Purpose of SMN1 gene

creates a protein that supports the motor neurons.

SMN genes in healthy individuals vs people with SMA

SMA: both copies of SMN1 are either missing or mutated

healthy: have 2 copies of SMN1 and about 1-2 copies of SMN2 in each cell

SMN genes and severity of SMA

most severe types of SMA have only one copy of SMN2 while less severe cases have 4+ copies of SMN2 in each cell.

More copies of SMN2 compensate for the loss of SMN1’s protein making power.

Spinraza

• antisense oligonucleotides

• only for 5q SMA

• targets the SMN2 gene and enables it to produce more functional, full length SMN proteins.

• Cannot cross BBB so it is directly injected into the CSF via lumbar puncture

Risdiplam

• small molecule drug that targets the “back up” SMN2 to produce more SMN protein

• only used to treat 5q SMA —> can be taken orally but oral solution can only be stored at room temp for more than a total of 120 hours

• is incompatible with reproductive function

Zolgensma

• gene therapy that delivers a working copy of the SMN1 gene to motor neurons

• made from parts of AAV9 (cannot make copies of its self so it does not spread from person to person)

Different Variants of ALS

SOD1, C9ORF72, TARDBP, and FUS

SOD1 gene:

• implicated in approx 20% of familial ALS

• SOD1 serves as an antioxidant

• may protect against superoxide produced by mitochondria

• 110 SOD1 mutations linked to ALS

C9ORF72 gene:

• present in 40% of familial and 10% of sporadic ALS

• abnormal expansion of a GGGGCC hexanucleotide repeat is common genetic cause of ALS —> loss of function of this gene

TARDBP and FUS

DNA binding/repair/regulation

Mechanisms that contribute to cell death:

• Excitotoxicity from excessive glutamate signaling

• Aggregation of proteins

• Breakdown of axonal transport w/ loss of neurofilament structure

• Reduced production of ATP

• Neuroinflammation

• Triggering of cell death pathways

Primary hits of ALS

• inherited or sporadic mutations in related genes

• negatively affect the initial anti-inflammation phase

Secondary hits of ALS

• aging

• toxicity

• traumatic injuries

• CNS inflammation

Sets off the rapidly progressive stage marked with uncontrollable inflammation

FDA approved drug for ALS

• only one!

• Riluzole: presynaptic inhibitor of glutamate release —> blocks glutamate-mediated excitotoxicity

  • mechanism of action: blocks Na channels, reducing AP

• may reduce symptoms severity

• doesnt effect muscle strength

• may delay the need for intubation and ventilatory support.

Edaravone

• used for stroke & ALS

• an antioxidant —> VERY expensive in the US (145k)

• 2024 study: drug was associated with SLIGHTLY slower progression, mortality rates decreased SLIGHTLY

• side effects: confusion, gait disturbance, headache, eczema , and contacy dermatitis

AT-1501 (now ‘tegoprubart’)

• early stage clinical trial for ALS

• phase 2a trails as of 2022

• Antibody against the ligand of CD40

• CD40: receptor that is present on many cell types —> target seems to be macrophages

  • Secondary activation pathway for macrophages

  • Macrophages play an important role in cleaning up dead cells

  • But they are sources of microbicidal reactive oxygen species

• Binding to ligand to cut down on immune cell activation

Tuberous Sclerosis Complex (TSC)

• rare multisystem autosomal dominant genetic disease that causes noncancerous tumors to grow

  • brain and eyes

  • kidneys and heart

  • Liver and Lungs

  • Skin

• Combination of symptoms: seizures, intellectual disability, developmental delay, skin abnormalities, lung disease, and kidney disease.

Treatments for TSC

1. mTOR inhibitors: slows down tumor growth and prevent complications

   i.e., Everolimus: selective for mTORC1 protein complex —> mRNA translation is hindered so tumor growth is inhibited

2. Anti seizure medications: control seizures

3. medications: treat skin lesions

4. surgeries: to remove tumors

HD symptoms:

• develops between 30-50 years but they can appear as early as age 2 and as late as 80

• Hallmark: uncontrollable movement of the arms, legs, head, face and upper body “chorea” aka dance

• caused decline in thinking and reasoning skills

• causes brain changes that lead to alterations in mood

• OCD is another common symptom

HD degeneration of the brain

Striatum: key player in motor control

“dying back” pattern: Neuronal degeneration in HD

Affected neurons begin to exhibit signs of synaptic and axonal alterations early in the disease process:

abnormalities in the phosphorylation of axonal proteins

abnormal accumulation of membrane-bounded organelles (blue circles) in axons

Axonal degeneration steadily advances in a retrograde fashion, and nuclear/neuritic Htt aggregates (red stars) become evident

First line treatments for HD

• They try to REDUCE DA-ergic signaling

Tetrabenazine:

• blocks VMAT2

• Prevents the uptake of catecholamines into vesicles

• Monoamine levels are reduced because they are not packaged into protective vesicles

Potential side effects since depletion of monoamines can lead to increased psychological symptoms

DA antagonists may also be used to supresse unwanted movements

Anticonvulsants, anxiolytics and antidepressants are also prescribed to deal with psychological aspects of the disease

Early stages HD: the aggregates delay axonal transport resulting in…

failed delivery of GABA(A) —> hyperkinesia

In the late stage, trafficking of NMDA receptors in ___ is affected, what doe this cause?

striatal neurons —→ bradykinesia

AMT-130

Used for HD

• reduce the production of normal and mutated huntingtin protein by targeting the HTT gene’s mRNA

• uses AAV5 to deliver microRNA

• is administered directly into the caudate nucleus and the putamen

Two-year trial update:

• 12 people

• was shown to significantly slow the progression of the disease by a mean of 80%

Rabies

transmitted via bites —> or “inhalation” of the virus

• symptoms occur within 1 week- 1 year of contact

• RETROGRADE TRAVEL FROM SITE TO BRAIN

• universally fatal

• Post-exposure prophylaxis with vaccine is very effective when administered with first 24h post-exposure

• one dose of human rabies immunoglobulin (HRIG) and four doses of rabies vaccine over a 14-day period

• very $$$ (600$ after insurance in 2005 in IU health center/ or several thousands in hospital)

Peru and rabies

In 8 people in peru antibodies are found with no explanation…

We think that these people were lightly bitten during the night, but were not exposed to enough of the virus to develop a full infection

Signs and symptoms of rabies

1. light or partial paralysis

2. Hydrophobia (larynx)

3. anxiety

4. insomnia

5. Confusion

6. agitation

7. abnormal behavior

8. paranoia

9. terror

10. hallucinations

11. delirium

12. Coma

13. Death

Two types of HSV and what HSV is as a whole

• HSV: double stranded DNA virus

• HSV-1: oral infections

• HSV-2: genital infection

HSV and associations

• Postive status: associated with cognitive defects, bipolar, and AD

• APOE-epsilon4 allele carriers are predisposed to AD once infected

HSV infection

• virus enters the nerves at the site of primary infection, migrates to the cell body of the neuron and becomes inactive in the ganglion

• the body produces antibodies to that particular type of HSV as a tool to prevent a 2nd infection of that type at a different site.

• HSV-1: seroconversion after an oral infection prevents additional HSV-1 infections such as whitlow, genital herpes, and herpes of the eye —> seems to reduce symptoms if the person has HSV-2 later (can still be contracted)

Stages of PD

Identifiers of PD

• less movement of facial muscles —> reduced facial expression = “mask like”

• bradykinesia —> also results in small handwriting, decreased speaking volume and monotone speech

• rigidity in the joints is another common symptoms —> shuffling gait

Key component in PD:

• protien aggregation

• lewy bodies consist of α-synuclein and other proteins

• healthy cells: a-synuclein mediates vesicle movement at axon temrinals

• lewy bodies are formed by protien misfolding and proteasomal dysfunction

• they interrupt cell function and trigger apoptotic cell death

6-OHDA

• neurotoxin in PD models

• doesn’t cross BBB —> selective for DA neurons —> stereotaxic injections

• Causes rapid apoptosis —> difficult to study disease progression of PD

• lots of neuro inflammation

Abscisic acid:

• phytohormone (plant hormones) found in various brain regions of several mammals

• exhibits neuroprotective properties

MPTP

dopamine neurotoxin used to produce animal models of PD

• formed by MAO-B

• in astrocytes and 5-HT ergic neurons

• transported by dopamine transporter into cells

MitoPark mouse model

mitochondrial transcription factor was inactivated in mice models

• depletion in mitochondrial DNA

• loss of gene transcripts

• deficiency in respiratory chain

• ends with cell death

Symptoms were reversed by L-dopa treatment —> doesn’t prevent cell death but replaces DA that dead neurons are no longer producing

Levodopa (L-DOPA)

• metabolite of tyrosine and is the immediate precursor of DA

• pathway continues to form NA and EPI

• can cross BBB but not dopamine

• advantage: it leaves neurons in the driver seat -→ DAergic neurons fire as before contrast to agonist