BAM-15

Research Overview

BAM-15 represents a significant advancement in mitochondrial uncoupler research compounds, developed to overcome the safety limitations of classical uncouplers like DNP while maintaining potent metabolic enhancement effects. This novel small molecule functions as a selective mitochondrial protonophore, facilitating proton transport across the inner mitochondrial membrane independently of ATP synthase. By uncoupling oxidative phosphorylation, BAM-15 converts energy that would normally generate ATP into heat production, effectively increasing metabolic rate and energy expenditure without proportional ATP depletion.

The compound’s development emerged from research into safer mitochondrial uncouplers with therapeutic potential for metabolic diseases, obesity, and related conditions. Unlike DNP, which has a narrow therapeutic window and can dangerously elevate body temperature, BAM-15 demonstrates self-limiting properties in preclinical models due to its mild acidic nature and selective mitochondrial targeting. The compound accumulates specifically in mitochondria driven by membrane potential, providing built-in safety mechanisms absent in earlier uncouplers.

Research applications of BAM-15 span multiple areas of metabolic investigation including obesity research, type 2 diabetes models, non-alcoholic fatty liver disease (NAFLD) studies, insulin resistance mechanisms, mitochondrial dysfunction investigation, thermogenesis pathway analysis, and energy expenditure modulation. Laboratory protocols examine BAM-15’s effects in cell culture systems, isolated mitochondria preparations, and preclinical animal models to characterize its metabolic and physiological effects.

Molecular Characteristics

Complete Specifications:

• CAS Registry Number: 1803860-99-0
• Molecular Weight: 500.6 Da
• Molecular Formula: C28H27ClFN3O3
• IUPAC Name: [Complex substituted phenylhydrazone derivative]
• Chemical Classification: Mitochondrial uncoupler, protonophore
• Appearance: Yellow to orange solid powder
• Solubility: DMSO, ethanol, limited aqueous solubility (vehicle required for biological studies)

The molecular structure of BAM-15 incorporates specific design features that confer its selective mitochondrial targeting and improved safety profile. The compound contains lipophilic regions enabling membrane permeation, along with ionizable groups that facilitate proton shuttling across lipid bilayers. Unlike DNP, which readily crosses all cellular membranes and disrupts pH gradients throughout the cell, BAM-15’s structural characteristics result in preferential accumulation in mitochondria and selective uncoupling activity at the inner mitochondrial membrane.

The compound’s mechanism involves facilitated proton transport through the lipid bilayer of the inner mitochondrial membrane. In its protonated form, BAM-15 crosses the membrane, releases the proton in the mitochondrial matrix (high pH side), and returns in deprotonated form to pick up another proton from the intermembrane space (low pH side). This cycling dissipates the proton gradient that normally drives ATP synthesis, converting the energy of fuel oxidation into heat rather than chemical energy storage.

Mechanism of Action

Mitochondrial Uncoupling Process:

BAM-15 functions by disrupting the normal coupling between the electron transport chain and ATP synthesis. Under normal physiological conditions, the electron transport chain pumps protons from the mitochondrial matrix to the intermembrane space, creating an electrochemical gradient. This proton-motive force drives ATP synthase to produce ATP as protons flow back into the matrix. Mitochondrial uncouplers short-circuit this process by providing an alternative pathway for proton return that bypasses ATP synthase.

Key Mechanistic Features:

1. Selective Mitochondrial Targeting: BAM-15 accumulates specifically in mitochondria driven by the organelle’s membrane potential. This selectivity reduces effects on other cellular compartments and contributes to the compound’s improved safety profile compared to non-selective uncouplers.

2. Protonophoric Activity: As a weak lipophilic acid, BAM-15 shuttles protons across the inner mitochondrial membrane, dissipating the proton gradient without generating ATP. This increases oxygen consumption and fuel oxidation while reducing ATP production efficiency.

3. Dose-Dependent Effects: BAM-15 demonstrates dose-dependent uncoupling activity in research models. At lower concentrations, partial uncoupling increases metabolic rate without depleting cellular ATP to dangerous levels. At higher concentrations, more complete uncoupling occurs with proportionally greater metabolic effects.

4. Self-Limiting Properties: The compound exhibits self-limiting characteristics in preclinical studies. As uncoupling increases and membrane potential decreases, the driving force for BAM-15 accumulation in mitochondria diminishes, providing an intrinsic safety mechanism against excessive uncoupling.

5. Preserved Mitochondrial Function: Unlike compounds that damage mitochondria, BAM-15 maintains mitochondrial structure and function in research models. The organelles remain capable of oxidative phosphorylation, simply with reduced coupling efficiency between respiration and ATP synthesis.

Pharmacokinetic Profile in Research Models

Research characterizing BAM-15 pharmacokinetics in preclinical models reveals properties important for experimental design:

Absorption and Distribution:

• Oral bioavailability demonstrated in rodent models
• Requires appropriate vehicle formulation for aqueous delivery
• Distributes to metabolically active tissues with high mitochondrial content
• Tissue accumulation correlates with mitochondrial density

Metabolism and Elimination:

• Subject to hepatic metabolism via phase I and phase II pathways
• Multiple metabolites identified in preclinical pharmacokinetic studies
• Elimination primarily through hepatobiliary route
• Plasma half-life: Several hours in rodent models (species-dependent)

Duration of Action:

• Metabolic effects persist beyond detectable plasma concentrations
• Suggests tissue/mitochondrial retention or sustained biological effects
• Duration of action influences dosing frequency in research protocols
• Cumulative effects observed with repeated administration

These pharmacokinetic characteristics inform research protocol design regarding dosing regimens, timing of measurements, and experimental duration. Understanding BAM-15’s pharmacokinetic profile is essential for interpreting research results and designing experiments that capture relevant metabolic effects.

Research Applications

Obesity and Weight Management Research

BAM-15 serves as a valuable research tool for investigating mechanisms of energy expenditure and obesity:

• Energy Expenditure Studies: Investigation of metabolic rate enhancement, oxygen consumption increases, and heat production (thermogenesis) in research models
• Weight Loss Mechanism Research: Examination of body weight reduction mechanisms independent of caloric restriction or exercise
• Adipose Tissue Research: Studies on white adipose tissue reduction, fat oxidation enhancement, and body composition changes
• Metabolic Efficiency Investigation: Analysis of coupling efficiency modulation and its effects on energy balance
• Dietary Intervention Studies: Research combining BAM-15 with various dietary protocols (high-fat diet, caloric restriction, etc.)

Research protocols utilize diet-induced obesity models, genetic obesity models (ob/ob, db/db mice), and body composition analysis techniques to characterize BAM-15’s effects on weight management parameters.

Metabolic Disease Research

Laboratory studies investigate BAM-15 in metabolic dysfunction models:

• Type 2 Diabetes Research: Investigation of glucose homeostasis improvement, insulin sensitivity enhancement, and glycemic control mechanisms
• Insulin Resistance Studies: Examination of insulin signaling pathway restoration and glucose uptake enhancement in insulin-resistant models
• Non-Alcoholic Fatty Liver Disease (NAFLD): Research on hepatic steatosis reduction, liver fat content decrease, and hepatic insulin sensitivity
• Metabolic Syndrome Models: Investigation of multi-parameter metabolic improvements (glucose, lipids, body weight, blood pressure)
• Lipid Metabolism Research: Studies on triglyceride reduction, fatty acid oxidation enhancement, and lipid partitioning

Experimental approaches include glucose tolerance tests, insulin tolerance tests, hyperinsulinemic-euglycemic clamps, and metabolic cage studies to comprehensively assess metabolic parameters.

Mitochondrial Function Research

BAM-15 serves as a research tool for investigating mitochondrial biology:

• Mitochondrial Respiration Studies: Analysis of oxygen consumption rates, respiratory chain activity, and coupling efficiency measurements
• ROS Production Research: Investigation of reactive oxygen species reduction through mild uncoupling effects
• Mitochondrial Biogenesis: Studies examining compensatory mitochondrial proliferation and mitochondrial content increases
• Oxidative Stress Research: Examination of oxidative damage reduction through ROS mitigation mechanisms
• Mitochondrial Quality Control: Investigation of mitophagy, mitochondrial dynamics, and quality control pathway activation

Research protocols employ isolated mitochondria preparations, permeabilized cell systems, and intact cell respiration measurements using Seahorse analyzers or Clark-type oxygen electrodes.

Thermogenesis and BAT Research

Laboratory investigations examine BAM-15 effects on thermogenic tissues:

• Brown Adipose Tissue (BAT) Studies: Investigation of BAT activation, thermogenic gene expression, and brown fat function
• UCP1-Independent Thermogenesis: Research on thermogenic mechanisms independent of uncoupling protein 1 (UCP1)
• Beige Adipocyte Research: Examination of white adipose tissue browning and beige adipocyte formation
• Thermoregulation Studies: Analysis of body temperature regulation and cold tolerance in research models
• Thermogenic Capacity Research: Investigation of maximal thermogenic capacity and limiting factors

Experimental approaches include cold exposure studies, thermogenic gene expression analysis (UCP1, PGC-1α, Dio2), and temperature monitoring in research models.

Aging and Longevity Research

Emerging research areas investigate BAM-15 in aging biology:

• Metabolic Health Span Research: Investigation of age-related metabolic decline and healthspan extension mechanisms
• Caloric Restriction Mimetics: Studies examining whether mild uncoupling mimics beneficial effects of caloric restriction
• Age-Related Disease Models: Research on metabolic dysfunction, neurodegeneration, and cardiovascular aging
• Cellular Senescence: Investigation of senescent cell accumulation and mitochondrial dysfunction in aging
• Longevity Pathway Research: Examination of AMPK activation, SIRT1 modulation, and other longevity-associated pathways

Research protocols utilize aged animal models, accelerated aging models, and longevity outcome measurements to investigate BAM-15’s effects on aging-related parameters.

Cardiovascular and Metabolic Research

Research applications extend to cardiovascular system investigation:

• Cardiac Metabolism Studies: Examination of cardiac mitochondrial function, myocardial energy metabolism, and cardiac efficiency
• Atherosclerosis Research: Investigation of metabolic factors in atherosclerotic plaque development and cardiovascular disease models
• Blood Pressure Research: Studies examining effects on hypertension models and blood pressure regulation
• Endothelial Function: Research on vascular health, endothelial function, and nitric oxide pathway modulation
• Heart Failure Models: Investigation of cardiac energetics and metabolic approaches to heart failure research

Laboratory protocols include isolated heart perfusion studies, cardiac function assessment via echocardiography, and cardiovascular biomarker analysis.

Neurological and Neuroprotection Research

Emerging applications in neurological research include:

• Neuroprotection Studies: Investigation of mitochondrial uncoupling effects on neuronal survival and neuroprotection
• Neuroinflammation Research: Examination of inflammatory pathway modulation in neurological models
• Neurodegenerative Disease Models: Studies on Parkinson’s disease, Alzheimer’s disease, and other neurodegenerative conditions
• Brain Metabolism Research: Investigation of brain energy metabolism, neuronal mitochondrial function, and cerebral metabolic rate
• Cognitive Function Studies: Examination of metabolic effects on learning, memory, and cognitive performance

Research protocols employ neurological disease models, behavioral testing, and brain tissue analysis to characterize neurological effects.

Comparison to Other Mitochondrial Uncouplers

BAM-15 vs. DNP (2,4-Dinitrophenol):

DNP represents the classical mitochondrial uncoupler with a long history in research and unfortunate history of human use. Key differences between BAM-15 and DNP in research models:

• Safety Profile: BAM-15 demonstrates superior safety margins in preclinical studies compared to DNP’s narrow therapeutic window
• Selectivity: BAM-15 shows selective mitochondrial targeting, while DNP uncouples all cellular membranes
• Self-Limiting Properties: BAM-15 exhibits intrinsic dose-limiting effects; DNP does not, leading to runaway thermogenesis risk
• Temperature Effects: BAM-15 shows reduced hyperthermia risk compared to DNP in research models
• Cellular pH Effects: BAM-15 preferentially affects mitochondrial pH gradients; DNP disrupts pH throughout the cell

These differences make BAM-15 a valuable research tool for investigating mitochondrial uncoupling with improved experimental safety.

BAM-15 vs. Natural Uncouplers:

Comparison to endogenous uncoupling mechanisms:

• UCP1 (Uncoupling Protein 1): BAM-15 provides UCP1-independent uncoupling, valuable for research in UCP1-knockout models or tissues lacking brown adipose tissue
• Fatty Acids: Long-chain fatty acids provide mild uncoupling activity; BAM-15 offers more potent and consistent uncoupling for research applications
• Synthetic vs. Physiological: BAM-15 as a synthetic uncoupler allows investigation of uncoupling independent of endogenous regulatory mechanisms

Laboratory Handling and Storage Protocols

Storage of Solid Compound:

• Store at -20°C to -80°C in original container
• Protect from light exposure, moisture, and oxygen
• Desiccated storage environment strongly recommended
• Inert atmosphere (nitrogen or argon) recommended for long-term storage
• Stability data available for 12+ months at -20°C under proper conditions

Solubilization and Vehicle Preparation:

BAM-15 has limited aqueous solubility requiring appropriate vehicles for biological studies:

• Stock Solution Preparation: Dissolve in DMSO at 10-50 mM concentration for stock solution
• Working Solution Preparation: Dilute stock into appropriate vehicle for cell culture or in vivo administration
• Common Vehicles for In Vivo Research:
• 5-10% DMSO in PBS or saline
• PEG-400 based formulations
• Cremophor/ethanol/saline mixtures
• Cyclodextrin complexation for improved aqueous delivery

• Cell Culture Applications: Final DMSO concentration should not exceed 0.1-0.5% to avoid vehicle effects on cells

Stock Solution Storage:

• DMSO stock solutions: Store at -20°C in aliquots to avoid freeze-thaw cycles
• Protect from light and moisture
• Single-use aliquots recommended to maintain compound integrity
• Document preparation date and concentration for laboratory records

Handling Precautions:

• Minimize exposure to air, light, and moisture during weighing and solubilization
• Use amber vials or aluminum foil protection for light-sensitive storage
• Allow vial to warm to room temperature before opening to prevent condensation
• Work in well-ventilated area or fume hood when preparing solutions

Quality Assurance and Analytical Testing

Each BAM-15 batch undergoes comprehensive analytical characterization:

Purity Analysis:

• High-Performance Liquid Chromatography (HPLC): ≥98% purity
• Analytical method: Reversed-phase HPLC with UV detection
• Multiple wavelength detection to identify potential impurities
• Peak integration following USP guidelines for accurate purity determination

Structural Verification:

• Mass Spectrometry (LC-MS or ESI-MS): Confirms molecular weight 500.6 Da
• Nuclear Magnetic Resonance (NMR): Structural characterization via ¹H-NMR and ¹³C-NMR
• Infrared Spectroscopy (IR): Functional group verification
• Melting point determination: Confirms chemical identity

Contaminant Testing:

• Residual solvents: Gas chromatography to quantify synthesis solvents
• Heavy metals: ICP-MS analysis per USP standards
• Water content: Karl Fischer titration
• Bacterial endotoxin: LAL test if applicable for biological studies

Stability Testing:

• Accelerated stability studies under various conditions
• Forced degradation studies to identify potential degradation products
• Long-term stability data under recommended storage conditions
• Photostability testing to confirm light sensitivity

Documentation:

• Certificate of Analysis (COA) provided with each batch
• Spectroscopic data (NMR, MS, IR) available upon request
• Batch-specific quality control results traceable by lot number
• Third-party analytical verification available for independent confirmation

Research Considerations

Experimental Design Factors:

Researchers should carefully consider multiple factors when designing BAM-15 experiments:

1. Dose Selection and Optimization:

• Dose-response studies essential to identify appropriate concentrations
• In vitro: Typical range 0.1-10 μM depending on cell type and experimental duration
• In vivo: Preclinical studies report oral doses ranging from 5-50 mg/kg depending on species, model, and objectives
• Start with lower doses and escalate to identify optimal experimental concentrations

2. Vehicle Considerations:

• Vehicle selection impacts bioavailability and experimental outcomes
• Include appropriate vehicle control groups in all experiments
• Test vehicle effects independently, particularly with DMSO-containing formulations
• Document exact vehicle composition for reproducibility

3. Temporal Factors:

• Acute vs. chronic administration produces different metabolic adaptations
• Time course studies help identify optimal measurement timepoints
• Consider pharmacokinetic parameters when timing measurements
• Metabolic effects may include both immediate uncoupling and adaptive responses

4. Metabolic Assessment Methods:

• Indirect calorimetry for energy expenditure and respiratory exchange ratio
• Body composition analysis (MRI, DEXA, EchoMRI)
• Glucose and insulin measurements (GTT, ITT, clamps)
• Mitochondrial function assessment (respirometry, Seahorse analysis)
• Molecular analysis (gene expression, protein levels, phosphorylation states)

5. Control Considerations:

• Vehicle controls matched to experimental groups
• Pair-feeding controls to separate effects of reduced food intake from direct metabolic effects
• Positive controls (other uncouplers, metabolic modulators) where appropriate
• Temperature-controlled housing when studying thermogenic effects

Safety Considerations in Research:

Even in controlled laboratory settings, researchers should be aware of BAM-15’s biological activity:

• Monitor core body temperature in animal studies, particularly at higher doses
• Assess general health parameters (activity, appearance, body weight)
• Include safety endpoints in research protocols (body weight loss limits, temperature thresholds)
• Observe for signs of excessive metabolic stress or adverse effects
• Use appropriate dose escalation approaches when establishing new protocols

Mechanism Investigation:

BAM-15’s effects involve multiple interconnected pathways requiring careful dissection:

• Primary Effect: Mitochondrial uncoupling and increased oxygen consumption
• Secondary Effects: ATP/ADP ratio changes, AMPK activation, metabolic adaptations
• Compensatory Responses: Mitochondrial biogenesis, increased food intake, altered fuel utilization
• Tissue-Specific Effects: Different tissues show varying sensitivity to uncoupling

Comprehensive mechanism investigation requires multi-level analysis from molecular pathways to whole-organism physiology.

Integration with Other Research Approaches

Combination with Other Compounds:

Research investigating combination approaches may examine:

• Metabolic Compounds: Combined with metformin, GLP-1 analogs, or other metabolic modulators
• Exercise Mimetics: Combined with AMPK activators or other exercise-mimicking compounds
• Dietary Interventions: Combined with caloric restriction, ketogenic diet, or time-restricted feeding
• Thermogenic Compounds: Combined with beta-3 agonists or other thermogenic activators

Combination studies require careful controls to separate individual effects from synergistic interactions.

Genetic Model Research:

BAM-15 research in genetic models provides mechanistic insights:

• Obesity Models: ob/ob, db/db mice, Zucker rats
• Diabetes Models: Various genetic diabetes models
• UCP1 Knockout Models: Investigating UCP1-independent thermogenesis
• Metabolic Gene Knockouts: Examining specific pathway requirements for BAM-15 effects

Genetic models help identify necessary pathways and mechanisms underlying BAM-15’s metabolic effects.

Comparative Analysis with Related Research Compounds

SLU-PP-332:

Another research mitochondrial uncoupler with distinct properties:

• Similar mechanism but different chemical structure
• Comparative research helps identify structure-activity relationships
• May show tissue-specific or potency differences

Niclosamide:

FDA-approved anthelmintic with mitochondrial uncoupling properties:

• Repurposed for metabolic research applications
• Provides comparison point for uncoupling effects
• Additional biological activities beyond uncoupling complicate interpretation

BAM-15 Advantages for Research:

• Developed specifically as mitochondrial uncoupler with improved safety profile
• Extensive characterization in metabolic research models
• Selectivity for mitochondria reduces off-target effects
• Growing body of published research literature for protocol reference

Compliance and Safety Information

Regulatory Status:

BAM-15 is provided as a research chemical for in-vitro laboratory studies and preclinical research only. This compound has not been approved by the FDA or any regulatory agency for human therapeutic use, dietary supplementation, or medical applications. DNP’s history of misuse and dangerous outcomes in humans underscores the importance of maintaining BAM-15 strictly within research contexts.

Intended Use:

• In-vitro cell culture and mitochondrial studies
• In-vivo preclinical research in approved animal models
• Laboratory investigation of mitochondrial uncoupling mechanisms
• Academic and institutional research applications
• Metabolic research and mechanism investigation

NOT Intended For:

• Human consumption or administration
• Therapeutic treatment or diagnosis
• Dietary supplementation or weight loss applications
• Veterinary therapeutic applications
• Athletic enhancement or performance
• Any use outside controlled research settings

Safety Protocols:

Researchers must follow appropriate laboratory safety practices:

• Use appropriate personal protective equipment (lab coat, gloves, safety glasses)
• Handle in well-ventilated areas or chemical fume hood
• Avoid skin contact and inhalation of powder or solutions
• Follow institutional chemical safety guidelines
• Be aware of the compound’s biological activity affecting mitochondrial function
• Dispose of waste according to local regulations for chemical/biological waste
• Consult Safety Data Sheet (SDS) for detailed safety information
• Maintain proper documentation of compound handling and usage

Institutional Requirements:

• Appropriate institutional review board (IRB) or institutional animal care and use committee (IACUC) approvals required for research involving animals
• Chemical hygiene plan compliance
• Proper training in handling bioactive compounds
• Emergency procedures in place for accidental exposure

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