Antide

What is Antide and what is its complete chemical structure?

Antide is a synthetic decapeptide LHRH antagonist with the chemical structure N-Ac-D-Nal(2),D-Phe(pCl),D-Pal(3),Ser,Lys(Nic),D-Lys(Nic),Leu,Lys(iPr),Pro,D-Ala-NH2. The full nomenclature breaks down as follows:

• Nal(2) represents 3-(2-naphthyl)alanine

• Phe(p-Cl) represents 3-(4-chlorophenyl)alanine

• Pal(3) represents 3-(3-pyridyl)alanine

• Lys(Nic) represents N epsilon-nicotinoyllysine

• Lys(iPr) represents N epsilon-isopropyllysine

This peptide was designed with specific D-amino acid substitutions and side chain modifications to optimize receptor binding and antagonist properties while minimizing histamine release, a common side effect of earlier LHRH antagonists.

How does Antide's mechanism of action differ from traditional reproductive hormone modulators?

Unlike conventional reproductive hormone modulators such as GnRH agonists that initially stimulate hormone release before downregulation, Antide functions as a competitive antagonist that directly blocks LHRH (GnRH) receptors without initial stimulation. This mechanism provides immediate suppression of luteinizing hormone and follicle-stimulating hormone, avoiding the "flare effect" seen with agonists.

The direct antagonism mechanism makes Antide particularly valuable in research contexts where immediate hormonal suppression is required without the initial hormone surge, such as studying time-sensitive reproductive processes or in models where even temporary hormonal stimulation could confound results.

What experimental parameters should be considered when comparing Antide to other LHRH antagonists?

When designing comparative studies between Antide and other LHRH antagonists, researchers should consider:

• Receptor binding affinity and selectivity

• Antiovulatory potency (ED50 values)

• Duration of action (Antide shows significant activity when injected 44 hours before challenge with agonists)

• Histamine-releasing potential (Antide releases negligible histamine)

• Oral bioavailability (Antide shows 73% activity at 600 μg and 100% at 1200 μg orally)

• Formulation influences (Antide shows similar effectiveness in water and corn oil)

How can researchers optimize solid-phase synthesis protocols for Antide and its analogs?

Solid-phase peptide synthesis (SPPS) of Antide requires specific optimizations:

• Protection strategy: Utilize orthogonal protection schemes for the multiple modified lysine residues (positions 5, 6, and 8)

• Coupling conditions: For sterically hindered amino acids (especially D-amino acids at positions 1, 2, 3, 6, and 10), extended coupling times or stronger coupling reagents may be necessary

• Monitoring: Implement quantitative ninhydrin tests between coupling steps to ensure complete reactions

• Cleavage conditions: Optimize to preserve the sensitive side chain modifications while ensuring complete removal from the resin

• Purification parameters: Develop specific HPLC gradients that can separate closely related deletion peptides

Researchers should conduct small-scale pilot syntheses to optimize these parameters before proceeding to larger-scale production for biological testing. Analytical characterization should include mass spectrometry, amino acid analysis, and circular dichroism to confirm both sequence and conformational properties.

What structural modifications of Antide yield enhanced pharmacological profiles?

Structure-activity relationship studies have identified several key modifications that enhance Antide's pharmacological profile:

The most potent analog identified replaced Lys(Nic) with Lys(Pic) at position 5 and D-Lys(Nic) with cis-D-Ala(PzAC) at position 6, yielding N-Ac-D-Nal(2),D-Phe(pCl),D-Pal(3),Ser,Lys(Pic),cis-D-Ala(PzAC),Leu,Lys(iPr),Pro,D-Ala-NH2, where:

• Lys(Pic) represents N epsilon-picoloyllysine

• Ala(PzAC) represents 3-(4-pyrazinylcarbonylaminocyclohexyl)alanine

This analog demonstrated significantly enhanced potency with 73% antiovulatory activity at just 0.25 μg and 100% at 0.5 μg, compared to Antide's 36% at 0.5 μg and 100% at 1.0 μg.

These findings suggest positions 5-6 are particularly important for optimizing receptor interactions and biological activity, providing direction for further rational design efforts.

What pharmacokinetic models best describe Antide's unusual duration of action?

Antide exhibits an unusually prolonged duration of action for a peptide antagonist, remaining active 44+ hours after administration. Several pharmacokinetic models may explain this phenomenon:

• Receptor binding kinetics model: Extremely slow dissociation rate (koff) from the LHRH receptor

• Depot formation model: Formation of a subcutaneous or tissue depot with slow release into circulation

• Metabolic stability model: Resistance to proteolytic degradation due to D-amino acid incorporation

• Enterohepatic recirculation model: For oral administration, potential reabsorption after biliary excretion

Researchers investigating Antide's pharmacokinetics should design studies that can distinguish between these mechanisms by:

• Measuring plasma concentrations over extended timeframes (>72 hours)

• Conducting tissue distribution studies with radiolabeled compound

• Performing receptor occupancy studies at various timepoints

• Identifying and quantifying metabolites in biological fluids

Understanding the mechanism behind Antide's extended duration would provide valuable insights for designing next-generation LHRH antagonists with optimized pharmacokinetic properties.

How can Antide be utilized in neuroendocrine research beyond reproductive applications?

Antide's properties make it valuable for broader neuroendocrine research:

• Hypothalamic-pituitary-gonadal axis mapping: As a specific LHRH receptor antagonist, Antide can be used to selectively block one component of complex neuroendocrine cascades

• Neuropeptide receptor distribution studies: When radiolabeled, Antide can help identify and quantify LHRH receptor populations in various tissues

• Sexual dimorphism research: Investigating sex-specific differences in LHRH signaling pathways

• Developmental neuroendocrinology: Studying the establishment and maturation of reproductive neuroendocrine systems

When designing such studies, researchers should consider:

• Appropriate dosing based on the specific sensitivity of their experimental model

• Timing relative to developmental or physiological stages of interest

• Potential for species differences in receptor binding affinities

• Complementary approaches using genetic or antibody-based methods for validation

What methodological considerations are critical when evaluating Antide's oral bioavailability?

Antide's unusual oral bioavailability for a peptide requires specialized methodology for accurate assessment:

• Formulation variables:

  • Although initial studies showed similar results between water and corn oil formulations , researchers should evaluate:

  • pH effects on stability and absorption

  • Potential for enhancers (e.g., penetration enhancers or enzyme inhibitors)

  • Protection strategies against gastric degradation

• Sampling protocol design:

  • Multiple plasma timepoints to capture absorption and elimination phases

  • Collection of both portal and systemic blood samples to assess first-pass effects

  • Fecal analysis to determine unabsorbed fraction

  • Bile collection to evaluate enterohepatic cycling

• Analytical methods:

  • Development of sensitive LC-MS/MS methods for plasma quantification

  • Immunoassay techniques for metabolite identification

  • In vitro intestinal permeability models for mechanistic studies

• Pharmacokinetic analysis:

  • Non-compartmental analysis for basic parameters

  • Population pharmacokinetic modeling for inter-subject variability

  • Physiologically-based pharmacokinetic (PBPK) modeling for mechanistic insights

This comprehensive approach allows researchers to determine not just bioavailability values but also the mechanisms underlying Antide's exceptional oral absorption properties.

How should researchers design dose-response studies to accurately characterize Antide's antiovulatory effects?

Rigorous dose-response characterization requires careful experimental design:

• Dose selection criteria:

  • Begin with broad range covering sub-effective to fully effective doses (e.g., 0.1-2.0 μg)

  • Include sufficient intermediate doses to accurately calculate ED50

  • Consider logarithmic dose spacing for greatest statistical power

• Control groups:

  • Vehicle control (negative)

  • Reference LHRH antagonist (positive control)

  • Agonist challenge group ([D-Qal(3)6]LHRH)

• Evaluation endpoints:

  • Primary: Direct ovulation measurement through laparoscopy/necropsy

  • Secondary: Hormonal measurements (LH, FSH, estradiol, progesterone)

  • Tertiary: Histological examination of reproductive tissues

• Statistical analysis approach:

  • Probit or logit transformation for quantal responses

  • Four-parameter logistic model for continuous variables

  • Calculation of relative potency compared to reference antagonist

This design ensures both accurate characterization of the dose-response relationship and sufficient statistical power to detect meaningful differences between Antide and its analogs or other LHRH antagonists.

What are the primary analytical challenges in detecting and quantifying Antide in biological matrices?

Researchers face several analytical challenges when working with Antide:

• Extraction efficiency issues:

  • Peptide adsorption to labware

  • Protein binding in plasma samples

  • Matrix effects in complex biological samples

• Sensitivity limitations:

  • Low circulating concentrations after physiologically active doses

  • Need for sub-nanogram/mL detection limits

  • Signal suppression in mass spectrometry

• Specificity challenges:

  • Discrimination between parent compound and metabolites

  • Potential for immunoassay cross-reactivity with endogenous peptides

  • Separation of closely related analogs in structure-activity studies

• Method development strategies:

  • Optimized solid-phase extraction protocols with carefully selected sorbents

  • Immunoaffinity purification prior to instrumental analysis

  • Derivatization approaches to enhance MS detection

  • Use of surrogate peptides after enzymatic digestion

Researchers should validate their analytical methods across multiple matrices (plasma, urine, tissue homogenates) with careful attention to recovery, matrix effects, and stability under storage and processing conditions.

How can computational approaches enhance structure-based design of next-generation Antide analogs?

Modern computational methods offer powerful tools for Antide optimization:

• Molecular dynamics simulations:

  • Explore conformational space of Antide and analogs

  • Evaluate stability of receptor-ligand complexes

  • Identify key binding interactions and residence times

• Homology modeling and docking:

  • Create refined LHRH receptor models based on related GPCRs

  • Predict binding modes of Antide and proposed analogs

  • Calculate binding free energies to prioritize synthesis candidates

• Quantitative structure-activity relationship (QSAR) modeling:

  • Develop predictive models based on existing analogs

  • Incorporate 3D descriptors and pharmacophore features

  • Validate with external test sets of known compounds

• Machine learning approaches:

  • Train neural networks on available structure-activity data

  • Implement generative models for novel peptide design

  • Utilize transfer learning from related peptide classes

Researchers should implement ensemble approaches combining multiple computational methods, with experimental validation cycles to refine models. This integrated approach can significantly reduce the number of compounds requiring synthesis while increasing the probability of identifying improved analogs.

What methodological advances could improve translation of Antide research from preclinical to clinical applications?

Bridging the preclinical-clinical gap requires methodological innovations:

• Improved model systems:

  • Humanized animal models with human LHRH receptors

  • Patient-derived organoids for personalized response testing

  • Physiologically-based pharmacokinetic (PBPK) models calibrated across species

• Translational biomarkers:

  • Development of non-invasive hormone monitoring techniques

  • Identification of early response markers predictive of long-term outcomes

  • Standardization of assays across preclinical and clinical settings

• Formulation and delivery innovations:

  • Controlled-release systems for sustained activity

  • Targeted delivery approaches for tissue-specific effects

  • Stabilization techniques to enable practical clinical formulations

• Clinical trial design considerations:

  • Adaptive designs using early biomarker data

  • Patient stratification based on receptor polymorphisms

  • Crossover designs for comparative efficacy assessment

These methodological advances would accelerate the translation of fundamental Antide research into clinically meaningful applications, potentially expanding its utility beyond reproductive medicine into broader therapeutic areas.