Core Function – Oxidative Phosphorylation (OXPHOS)

The canonical role of the mitochondrion is the synthesis of adenosine triphosphate (ATP), the universal energy currency of the cell, through the process of oxidative phosphorylation (OXPHOS).¹ This highly efficient metabolic pathway is the culmination of cellular respiration, converting the chemical energy stored in nutrients into a usable form. The process begins upstream with catabolic pathways such as glycolysis and fatty acid beta-oxidation, which break down glucose and fats to produce key intermediates. These intermediates feed into the citric acid cycle (or Krebs cycle) within the mitochondrial matrix, generating high-energy electron carriers, primarily nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2).²

These electron carriers are the essential fuel for the electron transport chain (ETC), a series of five large protein-lipid enzyme complexes (designated Complexes I-V) embedded within the highly folded inner mitochondrial membrane (IMM).² NADH and FADH2 donate their electrons to the ETC at Complex I and Complex II, respectively. The electrons are then passed sequentially through the chain—from Complex I/II to coenzyme Q10, then to Complex III, cytochrome c, and finally to Complex IV—in a series of precisely controlled oxidation-reduction reactions.³ The ultimate electron acceptor at the end of this chain is molecular oxygen, which is reduced to form water.³

The transfer of electrons is a highly exergonic process, and the energy released at Complexes I, III, and IV is harnessed to actively pump protons () from the mitochondrial matrix across the IMM into the intermembrane space.³ This action establishes a powerful electrochemical gradient, comprising both a pH differential (the matrix becomes alkaline, around pH 8) and a voltage differential (the matrix becomes negative, approximately -140 mV).⁴ This stored potential energy is termed the ‘proton motive force’.³ The final stage of OXPHOS is catalyzed by Complex V, more commonly known as ATP synthase. This remarkable molecular machine acts as a rotary motor; as protons flow back down their electrochemical gradient into the matrix through a channel in the  subunit of ATP synthase, they drive the rotation of the  subunit, which in turn catalyzes the phosphorylation of adenosine diphosphate (ADP) to ATP.⁴ The efficiency of this system is staggering: the complete oxidation of a single glucose molecule via OXPHOS yields approximately 30 to 32 molecules of ATP, a dramatic increase over the mere two ATP molecules produced by glycolysis alone.³ This immense energy output is what fuels virtually all essential biological functions.

Integral Hub for Cellular Homeostasis

Beyond its bioenergetic role, the mitochondrion is a critical nexus for numerous other cellular processes. It is the gatekeeper of the intrinsic pathway of apoptosis, or programmed cell death.⁵ This process is fundamental for embryonic development (e.g., sculpting digits), tissue homeostasis (balancing cell division with cell death), and the elimination of damaged or potentially cancerous cells.⁵ The release of mitochondrial intermembrane space proteins, most notably cytochrome c, into the cytosol is a key initiating event that triggers a cascade of caspase proteases, leading to the orderly dismantling and disposal of the cell.⁵

Mitochondria are also central to maintaining cellular calcium homeostasis, sequestering and releasing calcium ions to modulate a wide range of signaling pathways. Furthermore, as a natural consequence of electron transport, mitochondria are the primary intracellular source of reactive oxygen species (ROS)—chemically reactive molecules like superoxide radicals that are formed when electrons prematurely leak from the ETC and react with oxygen.⁸ While ROS play roles in cell signaling at low levels, their overproduction leads to oxidative stress, a damaging state where cellular components like lipids, proteins, and DNA are oxidized, contributing to cellular dysfunction and aging.⁸ The mitochondrion is thus a finely balanced organelle, where efficient energy production is inextricably linked to the control of cell death and redox signaling.

1.2 Mitochondrial Dysfunction as a Unifying Pathogenic Hub

Given the organelle’s centrality, it is logical that its failure, termed “mitochondrial dysfunction,” is a profoundly pathogenic state. This term encompasses a spectrum of defects, including impaired ATP production, leading to a cellular energy crisis; excessive generation of ROS, causing widespread oxidative damage; dysregulated calcium buffering, which disrupts cellular signaling; and the inappropriate activation of apoptotic pathways, leading to unwarranted cell death.

This pathological state has been identified as a core, unifying component in a vast and diverse array of human diseases. These range from rare, monogenic mitochondrial diseases, such as Barth syndrome and primary mitochondrial myopathies, to highly prevalent chronic conditions.⁹ The consequences of mitochondrial failure are most pronounced in tissues and organs with high metabolic demands and a heavy reliance on OXPHOS for their function. The heart, which produces and consumes approximately 6 kg of ATP daily, is exquisitely vulnerable, making mitochondrial dysfunction a key driver of cardiomyopathy and heart failure.¹¹ The brain and central nervous system are similarly susceptible, with mitochondrial impairment implicated in the pathogenesis of neurodegenerative diseases like Parkinson’s and Alzheimer’s disease. Skeletal muscle, which requires immense energy for contraction, is the primary site of pathology in mitochondrial myopathies, characterized by weakness and exercise intolerance.¹⁰ The photoreceptors of the retina, which have one of the highest metabolic rates in the body, are also highly vulnerable, linking mitochondrial dysfunction to the progression of diseases like dry age-related macular degeneration (AMD).¹⁴ This growing understanding of mitochondrial dysfunction as a common pathogenic denominator across numerous debilitating conditions has firmly established the mitochondrion as one of the most attractive and promising targets for novel therapeutic intervention.

Restoration of Cristae Architecture and ETC Supercomplex Stability

By associating with cardiolipin, SS-31 is theorized to shield this critical phospholipid from the damaging effects of oxidative stress.¹² This protective interaction helps to preserve the structural and functional integrity of the IMM. A foundational effect is the restoration of proper cristae architecture, which is often disorganized and fragmented in diseased states.²⁰ Re-establishing the correct membrane curvature and folding is a prerequisite for efficient energy production. Concurrently, the SS-31-cardiolipin interaction promotes the stability and re-assembly of the ETC supercomplexes.²² In states of mitochondrial dysfunction where oxidative damage to cardiolipin has caused these supercomplexes to dissociate, SS-31 helps to restore their crucial organization. This structural stabilization is the first and most fundamental step in its restorative action.

Enhancement of ATP Synthesis

The direct and most important consequence of a more organized membrane and a more stably coupled ETC is a significant improvement in mitochondrial respiration and ATP synthesis.¹⁹ By promoting the proper function of the ETC supercomplexes, SS-31 ensures a more fluid and efficient transfer of electrons along the respiratory chain, which in turn enhances the process of oxidative phosphorylation.²⁰ This leads to a restoration of cellular ATP levels, providing energy-starved cells with the fuel required for normal function, maintenance, and repair.¹⁹ Further research has elucidated an even more direct role in this process. SS-31 has been shown to bind directly to key components of the ATP transport and production machinery, including the Adenine Nucleotide Translocator (ANT) and ATP Synthase (Complex V) itself.²⁴ This interaction appears to improve the mitochondrion’s sensitivity to ADP, the primary substrate for ATP synthesis, thereby increasing the overall rate and efficiency of ATP production.²⁴

Dual-Mechanism Mitigation of Oxidative Stress

SS-31 mitigates the damaging effects of oxidative stress through two distinct and complementary mechanisms. Initial hypotheses regarding its function centered on its direct antioxidant properties. However, a more comprehensive understanding has emerged, repositioning its primary action further upstream, at the very source of pathological ROS production.

1. Upstream Prevention (Primary Mechanism): The principal source of pathological ROS in the cell is a “leaky” and inefficient electron transport chain. When supercomplexes are disorganized, electron transfer is impaired, allowing electrons to escape the chain prematurely and react with molecular oxygen to form superoxide radicals.¹⁸ By stabilizing cardiolipin and improving the coupling and efficiency of the ETC, SS-31 fundamentally reduces this electron leakage.¹⁹ This action prevents the formation of ROS at its source and is now considered the principal mechanism by which SS-31 exerts its profound antioxidant effects. This upstream prevention is a far more powerful and fundamental therapeutic action than mere downstream scavenging.
2. Downstream Scavenging (Secondary Mechanism): The inclusion of a dimethyltyrosine (Dmt) residue in SS-31’s structure endows it with the ability to act as a direct free radical scavenger.¹⁹ This modified amino acid can directly interact with and neutralize existing oxygen radicals, such as hydroxyl and peroxynitrite radicals, thereby inhibiting downstream damage like lipid peroxidation. This direct scavenging capacity serves as a secondary, supportive mechanism to the primary effect of preventing ROS formation.

This evolution in understanding—from viewing SS-31 as a simple ROS scavenger to recognizing it as a fundamental bioenergetic stabilizer—is critical. It explains why the compound has such profound effects on restoring ATP production and why it holds broad therapeutic potential across diseases characterized by a primary energy failure, a pathology that is more fundamental than oxidative stress alone.

Inhibition of Apoptosis

SS-31 also exerts potent anti-apoptotic effects by stabilizing the IMM. In healthy mitochondria, cardiolipin anchors cytochrome c to the inner membrane. During cellular stress, oxidative damage can cause cardiolipin to translocate to the outer mitochondrial membrane, facilitating the release of cytochrome c into the cytosol—a critical, committing step in the intrinsic apoptotic cascade.⁵ By binding to and stabilizing cardiolipin, SS-31 helps to prevent this release, thereby inhibiting the initiation of programmed cell death.¹² It has also been shown to inhibit the opening of the mitochondrial permeability transition pore (mPTP), a large, non-selective channel whose prolonged opening leads to mitochondrial swelling, rupture, and subsequent cell death.

A key theme that emerges from this mechanistic analysis is the compound’s remarkable selectivity for pathological states. Numerous studies, including ex vivo experiments on cardiac tissue from explanted failing human hearts, have demonstrated that SS-31 significantly improves function in diseased, energy-depleted mitochondria but has little to no effect on the function of healthy mitochondria.¹⁹ This suggests that SS-31 is not an indiscriminate metabolic “booster” but rather a targeted “stabilizer.” In a healthy mitochondrion, where cardiolipin is intact and the ETC machinery is already optimally configured into supercomplexes, there is no disorganized pathological substrate for the drug to act upon. Its effect is conditional on the presence of dysfunction. This selective action likely underpins its highly favorable safety profile and suggests a high therapeutic index, as it leaves normal cellular processes unperturbed.

Pathophysiology

Barth syndrome (BTHS) represents a prototypical disease of cardiolipin deficiency, making it an ideal indication to test the therapeutic hypothesis of SS-31. BTHS is an ultra-rare, X-linked genetic disorder caused by mutations in the TAFAZZIN gene.¹¹ This gene encodes the enzyme tafazzin, which is responsible for the final remodeling step in cardiolipin biosynthesis.²² A defect in this enzyme leads to a profound deficiency of mature, functional cardiolipin and a concomitant accumulation of an abnormal precursor, monolysocardiolipin (MLCL).¹¹ This altered lipid profile severely compromises mitochondrial structure and function, leading to the characteristic clinical triad of cardiomyopathy, skeletal myopathy with debilitating fatigue and exercise intolerance, and chronic or intermittent neutropenia.¹¹

The TAZPOWER Trial

The pivotal clinical study for SS-31 in this population was the Phase 2/3 TAZPOWER trial. The initial part of the study was a 28-week, randomized, double-blind, placebo-controlled crossover trial.²⁶ This rigorous design, however, failed to demonstrate a statistically significant benefit for SS-31 on the primary endpoints, which included the 6-Minute Walk Test (6MWT).²⁵ This outcome was initially a significant setback.

However, the subsequent open-label extension (OLE) phase of the trial, where all participants received daily subcutaneous SS-31, yielded dramatically different and highly promising results. Over long-term treatment, patients demonstrated clinically meaningful and statistically significant improvements across multiple domains. By week 168 of the OLE, patients showed a cumulative improvement in the 6MWT distance of 96.1 meters from their OLE baseline.³⁶ They also exhibited significant increases in muscle strength as measured by handheld dynamometry and improved scores on patient-reported outcome measures for fatigue.²¹ Perhaps most notably, long-term treatment was associated with significant improvements in cardiac function. Echocardiographic analysis revealed a 27% increase in average cardiac stroke volume and favorable trends in left ventricular end-diastolic and end-systolic volumes, suggesting positive cardiac remodeling was occurring.¹² These functional gains were correlated with favorable changes in the MLCL:CL biomarker ratio, providing a physiologic basis for the observed clinical benefits.³⁵

The discrepancy between the short-term crossover results and the long-term OLE results points to a critical mismatch between the trial design and the biological reality of the disease. BTHS is a chronic condition characterized by long-standing structural and functional deficits in high-energy tissues. The mechanism of SS-31 involves stabilizing membranes and improving bioenergetics, which would theoretically enable a slow process of cellular repair and tissue remodeling. The TAZPOWER results strongly suggest that this restorative process takes significantly longer than the 12 weeks allotted in the crossover phase to manifest in measurable functional gains. A short-term trial design may be fundamentally unsuited to capture this type of therapeutic benefit.

The Regulatory Saga

The regulatory journey for SS-31 in BTHS has been a protracted and complex saga, reflecting the broader challenges of drug development for ultra-rare diseases. Following the mixed results of TAZPOWER, Stealth BioTherapeutics’ New Drug Application (NDA) initially received a “refusal to file” letter from the U.S. Food and Drug Administration (FDA) in 2021, which cited the lack of a single, adequate, well-controlled trial.³⁷

Despite this, persistent and organized advocacy from the patient community, their families, and clinical experts ultimately led the FDA to convene a Cardiovascular and Renal Drugs Advisory Committee meeting in October 2024.³⁸ This meeting resulted in a 10-6 vote concluding that the drug was effective, a non-binding but influential recommendation in favor of approval.³⁹ Following further review and delays, the FDA issued a complete response letter in May 2025, declining to approve the application in its current form but, for the first time, proposing a viable path forward for resubmission for accelerated approval.³⁷ This path was based on using the improvement in knee extensor muscle strength—which had increased by over 45% in the TAZPOWER trial—as an intermediate clinical endpoint that is reasonably likely to predict clinical benefit.³⁹

Following a rapid resubmission by the company in August 2025, the FDA granted Accelerated Approval to FORZINITY™ (SS-31) on September 19, 2025, for the treatment of Barth syndrome in patients weighing at least 30 kg.²⁷ This landmark decision represents the first-ever approved therapy for BTHS and the first approved mitochondria-targeted therapeutic. The approval journey is a powerful case study in modern drug regulation, demonstrating the FDA’s increasing willingness to exercise regulatory flexibility for diseases with high unmet need. It highlights the acceptance of evidence from OLEs, comparisons to natural history controls, and the use of novel intermediate endpoints when traditional, large placebo-controlled trials are unfeasible. It also underscores the transformative power of patient advocacy in shaping regulatory outcomes.

Pathophysiology

Primary Mitochondrial Myopathies (PMM) are a clinically and genetically heterogeneous group of disorders characterized by impaired mitochondrial oxidative phosphorylation that predominantly affects skeletal muscle.¹⁰ The genetic basis can lie in either the mitochondrial DNA (mtDNA), which encodes 13 essential protein subunits of the ETC, or the thousands of genes in the nuclear DNA (nDNA) that are required for mitochondrial structure, function, and maintenance.¹⁰ Patients typically suffer from progressive muscle weakness, severe fatigue, and exercise intolerance.⁴⁴

The MMPOWER-3 Trial

The MMPOWER-3 trial was a large, pivotal Phase 3 study designed to evaluate the efficacy of a 40 mg daily subcutaneous dose of SS-31 in 218 adults with genetically confirmed PMM.²⁸ It was conceived as a “basket” study, enrolling a broad population of patients with a wide variety of underlying mtDNA and nDNA mutations. The trial ultimately failed to meet its co-primary endpoints: over 24 weeks, there was no statistically significant difference between the SS-31 and placebo groups in the change from baseline on either the 6MWT or the Primary Mitochondrial Myopathy Symptom Assessment (PMMSA) total fatigue score.²⁸

The Crucial Subgroup Analysis

While the overall result was negative, the most significant finding from MMPOWER-3 emerged from a pre-specified subgroup analysis. This analysis revealed a striking divergence in treatment response based on the location of the pathogenic mutation. The subgroup of patients with nDNA mutations (n=59), particularly those with defects in genes required for mtDNA maintenance and replication (the “mtDNA replisome,” such as POLG and TWNK), demonstrated a statistically significant and clinically meaningful improvement in the 6MWT, walking on average 25.2 meters further than their placebo counterparts.²³ Conversely, the larger subgroup of patients with primary mtDNA mutations (n=162) showed no improvement compared to placebo.²³

This differential outcome provides a compelling, mechanistically plausible rationale for the trial’s overall result and represents a critical scientific learning. SS-31’s mechanism is to stabilize cardiolipin and organize structurally normal ETC proteins into functional supercomplexes. In patients with primary mtDNA mutations, the ETC proteins themselves are often structurally flawed due to the mutation. While SS-31 can stabilize the membrane environment, it cannot repair a fundamentally broken protein component. In contrast, the myopathy in patients with nDNA replisome defects often arises from secondary mtDNA instability (e.g., multiple deletions), but the ETC protein subunits encoded by the remaining mtDNA may be structurally normal. In this context, SS-31’s ability to organize these structurally sound proteins into more efficient supercomplexes could be highly effective. Therefore, the “failure” of MMPOWER-3 was not necessarily a failure of the drug, but rather a failure of a “one-size-fits-all” trial design that did not account for this critical genetic heterogeneity.

The NuPOWER Trial

This crucial learning has directly informed the path forward. Stealth BioTherapeutics has since initiated the NuPOWER trial, a Phase 3 study specifically designed to evaluate SS-31 in the nDNA-PMM patient population that demonstrated a positive response in MMPOWER-3.²³ This trial, which is now fully enrolled, embodies the principles of precision medicine and represents a critical test of the refined therapeutic hypothesis generated from the previous study.²⁹

Pathophysiology

Dry age-related macular degeneration (AMD) is a progressive retinal disease and a leading cause of irreversible blindness in older adults.¹⁴ Its pathogenesis is strongly linked to chronic oxidative stress and mitochondrial dysfunction within the high-energy-demand photoreceptors and the supportive retinal pigment epithelium (RPE).¹⁵ This bioenergetic failure leads to the progressive damage and death of these critical retinal cells, culminating in the loss of central vision, which is essential for tasks like reading and recognizing faces.¹⁴

The ReCLAIM-2 Trial

SS-31’s potential in this area was evaluated in the Phase 2 ReCLAIM-2 clinical trial, which randomized 176 patients with dry AMD and geographic atrophy (GA), an advanced form of the disease, to receive either SS-31 or placebo for 12 months.³⁰ The trial did not meet its co-primary efficacy endpoints, which were based on functional measures of low-luminance best-corrected visual acuity (LL-BCVA) and the rate of GA lesion growth as measured by fundus autofluorescence.³⁰

The Landmark Anatomical Finding

Despite the failure to meet the functional primary endpoints, the trial yielded a highly significant and promising result on a key anatomical endpoint measured by advanced retinal imaging (spectral-domain optical coherence tomography, or SD-OCT). Treatment with SS-31 resulted in a 43% reduction in the rate of photoreceptor loss compared to placebo, as quantified by the progression of Ellipsoid Zone (EZ) attenuation (nominal ).³⁰ The EZ is a specific hyperreflective band visible on OCT imaging that corresponds to the mitochondria-rich layer of the photoreceptor inner segments.⁴⁸ The integrity of the EZ is a direct structural surrogate for photoreceptor health, and its attenuation or loss precedes and predicts the loss of visual function.⁴⁸

The ReCLAIM-2 results suggest that preserving the cellular machinery of vision (the photoreceptors) may be a more sensitive and reliable early measure of a neuroprotective drug’s effect than subjective and often variable functional tests. Vision loss in AMD is the direct result of photoreceptor death. The logical conclusion is that preserving the structure of these cells today is reasonably likely to predict the preservation of their function tomorrow.

The ReNEW and ReGAIN Program

Following these results, and in a landmark decision for the field, the FDA confirmed that EZ attenuation is an approvable primary clinical trial endpoint in dry AMD.⁵² This has paved the way for a large, global Phase 3 program, consisting of two identical pivotal trials named ReNEW and ReGAIN.¹⁵ These trials, which are currently enrolling patients, will use the rate of change in the macular area of photoreceptor loss (EZ attenuation) as their primary endpoint.²³ The success of this program could validate a new paradigm for developing neuroprotective therapies in ophthalmology, where preserving retinal structure is accepted as a primary measure of therapeutic benefit.