Introduction

Autism affects 1–2% of children in the United States.1, 2 Dozens of single genes and chromosomal copy number variants (CNVs)3 increase the relative risk of autism spectrum disorder (ASD) nearly 5–50 times over the current background risk. Yet no single gene or CNV causes ASD in 100% of children who carry the mutation,4 and no single DNA mutation accounts for more than 1–2% of all ASD.5 Specific environmental factors have also been shown to increase the risk of ASD.6, 7 However, no single child has all of the known genetic risk factors for ASD, or is exposed to all the same environmental risks. Although the noncore symptoms of ASD are highly heterogeneous from child to child, making each child unique, the same core features used for diagnosis – abnormalities in social communication, restricted interests, repetitive behaviors, adherence to routine, and/or atypical sensory behaviors – are by definition expressed in every child. One approach to addressing the challenge of many etiologies of ASD is to define a common pathophysiology that can contribute to the core diagnostic symptoms, regardless of the initiating genetic and environmental triggers. We hypothesized that there is a conserved cellular response to metabolic perturbation or danger that is shared by all children with ASD. This is called the cell danger hypothesis.8 Aspects of the cell danger response (CDR) are also referred to as the integrated stress response.9-11 Preclinical studies showed that the cell danger response in mice produced a treatable metabolic syndrome that was maintained by purinergic signaling. Antipurinergic therapy with suramin corrected both the behavioral and metabolic features of these genetic and environmental mouse models of ASD.12-14

The formulation of the cell danger hypothesis was based on the recognition that similar metabolic pathways were coordinately regulated as an adaptive response to cellular threat regardless of whether the perturbation was caused by a virus,15 a bacterium,16 genetic forms of mitochondrial disease,10 or neurodevelopmental disorders with complex gene–environment pathogenic mechanisms like autism.17 These metabolic pathways traced to mitochondria. Mitochondria are responsible for initiating and coordinating innate immunity18 and produce stereotyped changes in oxidative metabolism under stress19 that lead to the regulated release of purine and pyrimidine nucleotides like ATP and UTP through cell membrane channels.20 Inside the cell, ATP is an energy carrier. Outside the cell, extracellular ATP (eATP) is a multifunctional signaling molecule, a potent immune modulator,21 and a damage‐associated molecular pattern (DAMP) that can activate microglia, and trigger IL‐1β production and inflammasome assembly.22 Extracellular purines like ATP, ADP, and adenosine, and pyrimidines like UTP are ligands for 19 different purinergic (P2X, P2Y, and P1) receptors.23 The intracellular concentration of ATP (iATP) in mammalian cells is typically 1–5 mmol/L,24 but drops when ATP is released through membrane channels under stress. Typical concentrations of extracellular adenine nucleotides in the unstirred water layer at the cell surface where receptors and ligands meet are about 1–10 μmol/L, near the effective concentration for most purinergic receptors,25 but can increase when ATP is released during cell stress. Concentrations of eATP in the blood are another 500 times lower (10–20 nmol/L).26 Purinergic effectors like ATP are also coreleased with canonical neurotransmitters like glutamate, dopamine, and serotonin during depolarization at every synapse in which they have been studied23 and play key roles in activity‐dependent synaptic remodeling.27 These and other features28-30 led us to test the hypothesis that the CDR8 was maintained by purinergic signaling.12-14

Suramin has many actions. One of its best‐studied actions is as an inhibitor of purinergic signaling. It is the oldest member of a growing class of antipurinergic drugs (APDs) in development.31 Suramin was first synthesized in 1916,32 making it one of the oldest manmade drugs still in medical use. It is used to treat African sleeping sickness (trypanosomiasis), and remains on the World Health Organization list of essential medications. Concerns about the toxicity of high‐dose suramin arose when the cumulative antitrypanosomal dose was increased 5 times or more over several months to treat AIDS or kill cancer cells during chemotherapy. When blood levels were maintained over 150 μmol/L for 3–6 months at a time to treat cancer, a number of dose‐limiting side effects were described.32 These included adrenal insufficiency, anemia, and peripheral neuropathy. In contrast, mouse studies suggested that high‐dose suramin was not necessary to treat autism‐like symptoms. These studies showed that low‐dose suramin that produced blood levels of about 5–10 μM was effective in treating ASD‐like symptoms and did not produce toxicity even when used for at least 4 months.12, 14

Here, we report the findings of the Suramin Autism Treatment‐1 (SAT‐1) trial, the first direct test of suramin, the cell danger hypothesis, and the relevance of abnormal purinergic signaling in children with ASD. These data help form the foundation for future studies that will test the safety and efficacy of suramin, provide fresh directions for the development of new antipurinergic drugs, and add support to the hypothesis that a potentially treatable metabolic syndrome may contribute to the pathogenesis of autism.