Evaluation of antidepressant drugs

Evaluation of antidepressant drugs

Need for new drugs:

1. High suicide rates in severe depression even if pharmacotherapy is available.
   1. 1/3^rd patients are resistant.
   1. Some patients show low compliance. All presently available class of drugs are a/w numerous s/e.

In-vitro assay.

Receptor binding assay:

• Isolate the appropriate cells with receptors of interest.
• Add radioactive ligand in the presence and absence of the test drug.
• Count the receptor ligand binding by liquid scintillography.
• The test drug displaces the known ligand from the receptor.
• Alpha adrenoceptor assay: rat cortex, 3H-Yohimbine
• Muscarnic receptor assay: Rat cerebellum, 3H-Quinuclidinyl benzilate (QNB)
• Monoamine oxidase inhibition: Inhibition of type A and type B monoamine oxidase activities in rat brain synaptosomes

Measurement of beta-adrenoceptor stimulated adenylate cyclase:

• Take male SD rats and treat them with desipramine or vehicle for 14 days.
• Isolate the brains and take slices.
• Add 3H-adenine and incubate with buffer and NA.
• Take the supernatant and count for radioactivity.
• The activity of adenylate cyclase is calculated as the conversion of [3H]-adenine to [3H]-cyclic AMP.
• After treatment with antidepressants, this conversion rate is not altered without stimulation by noradrenaline, but significantly reduced in slices treated with the maximally stimulating concentration of 100 µM noradrenaline.

Assays for determining decreased uptake of the amines: (Norepinephrine/Dopamine/Serotonin)

• Isolate the hypothalamus or corpora striata and homogenize it with sucrose solution.
• Centrifuge and take the supernatant.
• Add 3H-NE or 3H-DA or 3H-5HT to the supernatant, add a buffer and centrifuge again after adding test drug or control.
• Aspirate the supernatant and measure the monoamine by liquid scintillography.
• Compare IC50 b/w test and control.

• Additional assays: Binding to monoamine transporters. Monoamine oxidase inhibition assays
• Information that we get after in-vitro assays: Which receptors do the drug attaches to. Class of the drug. Possible s/e to look for in animal in-vivo experiments.

In Vivo

1. Gross behavior test.

2. Test based on inhibition of amine uptake.

3. Test Based on anticholinergic activity.

4. Test based on depletion of biogenic amine.

5. Hypermotility in olfactory-bulbectomized rats

• Gross behavioral models.

Forced swimming test.

• Animals: Wistar rats of  either sex weighing  200-225 gms.
• Plexiglas cylinder (h= 40 c.m., d=18 c.m. containing 15 c.m. of water)
• Duration of treatment
• Acute: 1 days
• Chronic: 13 days

Final Observation à Immobility time during 6 mins (360 seconds)

Adv: Relative simplicity

• Sensitive to a large number of atypical antidepressants otherwise inactive in the more classical tests.

Limitation

• Stimulants like amphetamine and caffeine, also reduce duration of immobility.
• Differentiation is done by measurement of locomotor Activity by open field test.

Tail suspension test in mice

• The immobility displayed by rodents when subjected to an unavoidable and inescapable stress reflect behavioral despair.
• mice ( 20–25 g) àTest drug is given i.p. 30 minutes prior to testing.
• The mice are suspended on the edge of a shelf above a table top by adhesive tape placed approximately 1 cm from the tip of the tail. The duration of immobility is recorded for a period of 5 min.
• EVALUATION
• The percentage of animals showing the passive behavior is counted and compared with vehicle treated controls.
• Using various doses, ED50 values can be calculated.

Disadv: Several mouse strains are inherently resistant to TST.

Learned helplessness model.

• Animals exposed to inescapable and unavoidable electric shocks in one situation later fail to escape shock in a different situation when escape is possible.
• Male SD rats (300 g)
• Apparatus – Box with a grid floor having a platform which can be inserted through one side wall to allow a jump-up escape response.
• Training – Exposure to electric shock (0.7 mA) for 1 h on a schedule of 10 s of shock/min.
• The platform is not available during training.
• This training resulted in 80% acquiring learned helplessness behavior.
• Shock is given without escape chance for 1 hour.
• Then platform is provided as a route of escape.
• Antidepressants increase the escaping behavior as compared to controls.
• EVALUATION
• A drug is considered to be effective, if the learned helplessness is reduced and the number of failures to escape is decreased.

Muricide behavior in rats

• Male Sprague-Dawley rats 
• Only rats consistently killing mice within 5 min after presentation are used for the test.
• Drugs are injected i.p. to the rats before the test. Mice are presented 30, 60 and 120 min after drug administration.
• EVALUATION
• Failure to kill a mouse within 5 min is considered inhibition of muricidal behavior.
• ED50 is calculated, the ED50 is defined as the dose which inhibits mouse killing in 50% of the rats.
• The mouse-killing behavior is also inhibited d-amphetamine some antihistamines  and some cholinergic drugs.
• Other models: catalepsy antagonism in chicken

Tests based on inhibition of amine reuptake

Potentiation of NE toxicity in mice:

• sublethal doses are given to mice of 3 mg/kg noradrenaline.
• This is followed by administration of the test drug.
• The mortality rate is assessed 48 h post-dosing. ED50 is calculated.
• Test drugs which increase mortality can be thought to decrease NE reuptake

Potentiation of 5HTP (5 hydroxy tryptophan) in mice:

• Test drug is given at 0 mins.
• This is followed 30 min later by Pargyline which inhibits MAO.
• At 120 mins 5HTP is given.
• 5HTP is converted to serotonin.
• If test drug decreases serotonin reuptake then mice show characteristic head twitches.
• If the test drug inhibits MAO then the head twitches are seen even without administration of pargyline.

Potentiation of 5HTP (5 hydroxy tryptophan) in rats.

• In contrast to mice exhibiting head twitches, rats show continuous forelimb clonus

• Test based on anticholinergic activity

Compulsive gnawing in mice.

•  Treatment of rodents with apomorphine causes compulsive gnawing instead of vomiting due to dopaminergic stimulation.
• Anticholinergics shift the balance between Ach & dopamine resulting in an enhancement of apomorphine effect.
• Ach à increase gnawing
• Mice are injected s.c. with 10 mg/kg apomorphine + test drug/vehicle at the same time.
• Immediately, mice are placed in a cage with corrugated paper the corrugation facing upwards for 1 hour.
• The mice start to bite into the paper causing fine holes or tear the paper.
• The template having 10 rectangle windows divided into 10 areas of the same size are placed over the corrugated paper.
• In a total of 100 areas number of bites is checked.
• Percentage of damaged paper in calculated.
• This behavior is enhanced by antidepressants.
• Not only antidepressants, but also centrally acting anticholinergics and antihistaminics are active in this test.

Tests based on depletion of monoamines

• Tetrabenazine antagonism in mice
• Tetrabenazine (TBZ) induces a depletion of biogenic amines (e.g. noradrenaline, dopamine, serotonin) without affecting their de novo synthesis.
• Catalepsy and ptosis are used as criteria.
• Antidepressants antagonize  the effect of TBZ

• Reserpine induced hypothermia in mice.
  • Depletion of biogenic amines (noradrenaline, 5-hydroxytryptamine, dopamine) in the brain also induces hypothermia in rodents.
  • The decrease of body temperature induced by reserpine is antagonized by antidepressants.
  • Rectal temperature is recorded every hour.
  • The difference in temperature from vehicle controls is calculated for each time and the maximal difference is scored.
  • The differences are then statistically compared using the t-test.
  • The reversal of hypothermia is not specific for antidepressants. The fall in body temperature can also be antagonized by amphetamines, and some antipsychotic agents (chlorpromazine).

• Hypermotility in olfactory bulbectomized rats:
  • Bilateral olfactory bulbectomy in the rat is associated with changes in exploratory behavior that are reversed by chronic, but not acute treatment with antidepressant drugs.
  • The animals are allowed to recover for 14 days after surgery.
  • the animals are treated s.c. with the test drug / standard / vehicle once daily for 14 days.
  • The behavior of the animals is tested from the 12th day onwards.
  • The rats are placed singly in the center of an open field apparatus.
  • Ambulation (no. of squares crossed), rearing (forepaws raised from the floor), grooming and defecation (no. of fecal boli) scores are recorded for a 3 min period of observation.

Other models

• Apomorphine- induced hypothermia in mice.
• Yohimbine toxicity enhancement.
• Tryptamine seizure potentiation in rats.
• Serotonin syndrome in rats.
• Sexual behavior in male rats.

• Clinical evaluation.
• Various scales available for evaluation of depression are: HAM-D (Hamilton depression rating scale), MADRS (Montgomery ASberg Depression rating scale), CGI (Clinical global impression), Becks Depression inventory (BDI).

• Phase 1.
• Objectives: Safety, tolerability, PK and PD.
• Groups: Young or elderly. Elderly volunteers are more appropriate.
• Dosing: Single ascending dose study in young. Multiple ascending dose study in elderly.
• Endpoints: AE, Vital signs, Lab parameters, Physical and neurological examination.
• PK: Polymorphic hydroxylation
• PD: Amine pressor test (assessment of peripheral effects of antidepresants), Psychomotor performance (DSST, CFFT), Quantitative EEG.

• Phase 2 and 3: Conducted usually for a period of 12 weeks.
• Objectives: Primary: Compare the efficacy and safety in treatment arm a/c/t placebo arm in decreasing symptoms of MDD. Secondary: onset of antidepressant action, duration of antidepressant action and peak of antidepressant action.
• Inclusion: DSM IV TR. Specific level of MADRS or HAMD. Becks anxiety inventory to exclude comorbid anxiety. No other mental, neurological and medical comorbidities.
• Endpoints:
• Primary: change from baseline in MADRS score. Change from Baseline in HAMD score. Snaith Hamilton Pleasure Scale (SHAPS) in which a score of <2 indicates anhedonia. Addiction research center inventory (ARCI ) which measures the reinforcing or dysphoric effects of the test drug. Profile of mood states (POMS) measures drug induced changes in mood.
• Secondary: Time to onset of a consistent decrease in depressed mood as measured by VAS. Number of patients achieving HAM-D <7. Adverse evens in both the groups.
• Design: double blind, randomized, placebo-controlled, parallel-group study