Essay 2: paragraph flashcards

2. Based on cortisol’s physiological mechanisms of action, design pre-clinical tests for a new corticoid steroid, with particular attention on how you would monitor efficacy and side effect profiles.

INTRODUCTION: PHYSIOLOGICAL ANCHOR - How do cortisol’s physiological mechanisms of action guide the design of pre-clinical tests for a new corticoid steroid?

• Cortisol exerts its anti-inflammatory effects by:

  • binding intracellular glucocorticoid receptors (GRα)

  • which translocate to nucleus and regulate gene expression.

• Beneficial efficacy:

  • through trans-activation of anti-inflammatory genes via glucocorticoid response elements (GRE)

  • adverse effects result from inappropriate activation of metabolic pathways.

• Pre-clinical development:

  • must be grounded in these mechanisms.

• Test design should:

  • assess anti-inflammatory efficacy

  • alongside key metabolic and systemic side-effect liabilities

  • to establish an improved therapeutic index.

1st PARAGRAPH: EFFICACY ARM 1: GRE REPORTER ASSAY - How can GRE-mediated trans-activation be measured to assess glucocorticoid efficacy?

• Start with a whole-cell GRE reporter assay:

  • to establish whether the steroid retains the desired trans-activation pathway

• A549 lung epithelial cells:

  • engineered to stably express a luciferase gene

  • under control of four tandem GRE consensus sequences.

• When exposed to the steroid:

  • activation of receptor should drive luciferase expression,

  • producing measurable luminescent signal.

• Cells treated with range of concs:

  • picomolar to micromolar levels

  • prednisolone = reference standard.

• Potency expressed as pEC50 value

  • maximal efficacy compared to prednisolone.

• Compound considered promising:

  • if = nanomolar potency (pEC50 ≥ 8.5) & at least 90% of prednisolone’s max effect.

• To confirm signal is receptor-mediated:

  • parallel experiments with GRα knock-down performed

  • loss of signal rules out off-target toxicity.

2nd PARAGRAPH: EFFICACY ARM 2: TNF-α WHOLE BLOOD ASSAY - Why is TNF-α suppression in human whole blood used to assess glucocorticoid efficacy and safety?

• Immunosuppression is the therapeutic aim of corticosteroids

  • next step = test steroid in more integrated human system.

• Heparinised whole blood from healthy volunteers:

  • incubated with compound

  • then challenged with lipopolysaccharide (LPS), a potent stimulator of cytokine release.

• Key read-out:

  • suppression of tumour necrosis factor-alpha (TNF‑α), measured by ELISA.

• LPS stimulation:

  • induces TNF-α release

  • models inflammatory challenge

• Clinically useful inhaled/topical steroid:

  • should achieve an IC50 of 30 nM or lower.

• Reversibility (equally important) after a wash-out period:

  • ≥ half of cytokine production should recover within 24 hours.

• Importance of reversibility:

  • limits prolonged immunosuppression

  • of the hypothalamic-pituitary-adrenal (HPA) axis.

• Ex-vivo IC50 correlates closely with effective dose in:

  • Brown-Norway rat asthma model

  • translational confidence.

3rd PARAGRAPH: SIDE-EFFECT ARM 1: HEPATIC GLUCOSE OUTPUT - How can gluconeogenic liability of a new corticoid steroid be assessed pre-clinically?

• Classic side effects of cortisol = stimulation of hepatic gluconeogenesis:

  • mediated by increased transcription of PEPCK.

• To model this liability:

  • HepG2 hepatocyte cultures r exposed to the steroid

  • glucose output measured following provision of lactate and pyruvate substrates.

• Increased glucose output indicates:

  • metabolic side-effect liability

  • risk of hyperglycaemia and diabetes

• Prednisolone produces 3-fold increase in glucose release at 100nM.

• Candidate compound must remain below 1.5-fold at same conc

  • retaining ≥ 80% of its anti-TNF activity.

• Ensures a therapeutic index of 5≤.

• Compounds that fail:

  • re-engineered with structural mods,

  • such as 9α‑fluoro and 16α‑methyl substitutions,

  • known to reduce hepatic activation w/o compromising pulmonary efficacy.

4th PARAGRAPH: SIDE-EFFECT ARM 2: 3-D HUMAN SKIN MODEL - Why are 3-D human skin models used to assess corticosteroid-induced epidermal atrophy?

• Dermal thinning:

  • another adverse effect of potent steroids.

• To asses:

  • employ three-dimensional human skin cultures at an air-liquid interface

  • mimic in vivo epidermal structure

  • are more predictive than monolayer cultures

• Cultures are treated daily:

  • with topical cream containing steroid

  • for 1 week.

• On day 8:

  • tissues are fixed, stained and measured for epidermal thickness.

• 15% reduction as a threshold of concern.

• Compound must produce ≤5% reduction:

  • still suppressing interleukin-6 release by at least 60%.

• Dual read-out provides early indication:

  • whether compound can deliver anti-inflammatory efficacy w/o compromising skin integrity.

5th PARAGRAPH: SIDE-EFFECT ARM 3: HPA-AXIS SUPPRESSION- How is HPA-axis suppression assessed and why is reversibility critical in corticosteroid development?

• Systemic steroid exposure can suppress:

  • ACTH release

  • endogenous corticosterone production

• Animal dosing allows assessment of:

  • basal stress-axis function

  • Sprague-Dawley rats are dosed subcutaneously for 7 days.

• Key biomarkers:

  • plasma corticosterone and ACTH levels

  • measured promptly after sacrifice to avoid handling artefacts.

• 50% reduction in basal corticosterone = regulatory red flag.

• Candidate must preserve:

  • at least 80% of endogenous corticosterone

  • and still demonstrate immunosuppressive activity in splenocyte proliferation assays.

• Significant reduction indicates:

  • central HPA-axis suppression

• Wash-out phase:

  • assesses reversibility of suppression

  • w ACTH levels required to return to baseline within 48 hours.

• Reflects the latest EMA draft guidance on topical glucocorticoids.

CONCLUSION: INTEGRATED RISK–BENEFIT SCORE - Why is an integrated risk–benefit score essential in the pre-clinical development of a new corticoid steroid?

• Data from all efficacy & safety arms:

  • integrated into weighted composite score.

• All contribute equally to the index:

  • GRE-mediated trans-activation (efficacy)

  • TNF-α suppression (functional immunosuppression)

  • hepatic glucose output (metabolic liability)

  • epidermal atrophy (local toxicity)

• Combining endpoints into a composite score:

  • enables objective comparison to prednisolone

  • supports go/no-go decision-making

• A score of 3≤, relative to prednisolone set at one:

  • qualifies compound for longer-term studies.

• Early medicinal chemistry iterations:

  • demonstrated that structural mods can raise index from 2.1 to 3.4 w/o loss of potency.

• If margins are maintained:

  • programme will deliver an inhaled corticosteroid w anti-inflammatory strength of fluticasone

  • but w reduced systemic exposure.

• Such advance would align with NHS initiative to reduce steroid-induced diabetes in patient with severe asthma.