Synthetic eicosapeptide inhibitor of postsynaptic density protein 95 (PSD-95) with neuroprotective activity mediated by disruption of NMDA receptor excitotoxic signaling
A cell-permeant Tat-conjugated peptide designed to uncouple NMDA receptor activation from downstream neurotoxic signaling by inhibiting the scaffolding protein PSD-95, advanced through Phase 3 clinical trials for acute ischemic stroke neuroprotection.
Abstract
NA-1, designated nerinetide in clinical development and also known by its research name Tat-NR2B9c, is a synthetic, cell-permeant eicosapeptide composed of the 11-residue cell-membrane transduction domain of the human immunodeficiency virus type 1 (HIV-1) Tat protein fused to the nine C-terminal residues of the GluN2B (NR2B) subunit of the N-methyl-D-aspartate (NMDA) glutamate receptor. The compound was designed to inhibit the protein-protein interaction between postsynaptic density protein 95 (PSD-95) and the GluN2B subunit, thereby uncoupling NMDA receptor activation from the downstream excitotoxic signaling cascade that drives neuronal injury in ischemic stroke and related conditions, without blocking normal NMDA receptor ion channel function or calcium influx. The foundational discovery was reported by Aarts et al. in Science in 2002, demonstrating that perturbation of the NMDA receptor-PSD-95 interaction reduced excitotoxic neuronal death in vitro and focal ischemic brain damage in rats. Subsequent preclinical work extended the neuroprotective efficacy to nonhuman primates, where Cook et al. (2012) demonstrated greater than 50 percent reduction in stroke volume in cynomolgus macaques subjected to transient middle cerebral artery occlusion and treated with Tat-NR2B9c at 2.6 mg/kg intravenous. The compound was advanced into clinical development by NoNO Inc., a Toronto-based biotechnology company founded by the originating investigator Michael Tymianski. The Phase 2 ENACT trial (Hill et al. 2012, Lancet Neurology) in 185 patients undergoing endovascular aneurysm repair demonstrated safety and a significant reduction in the number of procedure-related ischemic infarcts on diffusion-weighted magnetic resonance imaging. The Phase 3 ESCAPE-NA1 trial (Hill et al. 2020, Lancet) in 1105 patients with acute ischemic stroke undergoing endovascular thrombectomy did not meet its primary endpoint of improved 90-day modified Rankin Scale outcomes in the overall population, but a pre-specified subgroup analysis revealed a clinically meaningful 9.5 percent absolute benefit in patients who did not receive concurrent alteplase, with subsequent mechanistic work demonstrating that plasmin generated by alteplase degrades the nerinetide peptide in vivo. The Phase 3 ESCAPE-NEXT trial (2025, Lancet) in 850 patients undergoing thrombectomy without prior thrombolysis did not confirm the subgroup benefit. The Phase 2 FRONTIER trial (2025, Lancet) in 532 patients with suspected acute cerebral ischemia treated by paramedics in the prehospital setting demonstrated safety and feasibility but did not meet primary efficacy endpoints. Pharmacokinetically, nerinetide exhibits a plasma half-life of approximately 5 to 10 minutes following intravenous bolus administration, consistent with rapid peptide clearance by endogenous proteases and tissue distribution. The clinical dose is 2.6 mg/kg intravenous (maximum 270 mg) administered as a single infusion. The safety profile across all clinical trials has been favorable, with adverse event rates comparable to placebo; the only notable signal is a small excess of serious hypotension events immediately following infusion. This monograph reviews the chemistry, structure, and synthesis of nerinetide; the PSD-95 inhibitory mechanism in molecular detail; the pharmacokinetic profile; the preclinical neuroprotection evidence across species; the complete clinical trial record; sourcing and quality considerations; reconstitution and handling; stack-interaction considerations; the adverse event and safety record; and a comparative assessment of five PSD-95 pathway neuroprotective candidates against nerinetide on five competency standards.
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Synthetic aliphatic sympathomimetic amine with indirect adrenergic and dopaminergic activity
A branched-chain aliphatic amine originally developed by Eli Lilly as the inhaled nasal decongestant Forthane, subsequently repurposed as an ergogenic dietary supplement ingredient, and characterized pharmacologically as an indirect sympathomimetic acting principally through norepinephrine and dopamine release with substrate-like dopamine transporter regulation.
Abstract
1,3-Dimethylamylamine (1,3-DMAA; 4-methylhexan-2-amine; methylhexanamine) is a synthetic aliphatic sympathomimetic amine first introduced by Eli Lilly and Company in 1944 under the trade name Forthane as an inhaled nasal decongestant and voluntarily withdrawn from the market in 1983. The compound re-entered commercial circulation in 2006 when it was marketed as an ergogenic dietary supplement ingredient under the name Geranamine, following the regulatory removal of ephedrine alkaloids from the United States supplement market in 2004. The pharmacology of 1,3-DMAA is that of an indirect sympathomimetic: the compound acts as a norepinephrine and dopamine releasing agent, inhibits the norepinephrine transporter (NET) at low micromolar concentrations, and exhibits substrate-like regulation of the dopamine transporter (DAT) including competitive inhibition of dopamine uptake, binding at the S1 substrate site, induction of outward-facing-open to outward-facing-closed conformational transitions, and stimulation of DAT endocytosis through cocaine- and protein kinase A-sensitive mechanisms. These monoaminergic actions produce dose-dependent cardiovascular stimulation (vasoconstriction, elevation of systolic and diastolic blood pressure, tachycardia), central nervous system arousal, and thermogenic effects consistent with the broader sympathomimetic amine class that includes ephedrine, amphetamine, and phenylpropanolamine.
The sole formal human pharmacokinetic study (Schilling et al., 2013; n = 7 healthy men; single oral dose of 25 mg) reported a peak plasma concentration of approximately 70 ng/mL, time to peak of 3.57 hours, terminal elimination half-life of 8.45 hours, oral clearance of 20.02 L/hr, and volume of distribution of 236 L, indicating extensive tissue partitioning. Physiological monitoring in that study documented modest increases in heart rate and diastolic blood pressure that remained within normal clinical ranges at the 25 mg dose. However, case reports and postmarketing surveillance have associated substantially higher doses of 1,3-DMAA (often in combination with caffeine and other stimulants in multi-ingredient pre-workout and weight-loss supplements) with serious cardiovascular events including cerebral hemorrhage, myocardial infarction, cardiac arrest, hepatotoxicity, and death. The United States Food and Drug Administration has received at least 86 reports of illness and death associated with DMAA-containing supplements and has determined that 1,3-DMAA is not a dietary ingredient; its inclusion in products marketed as dietary supplements is unlawful under United States federal law. Preclinical abuse liability assessment (Dolan and Gatch, 2015) demonstrated that 1,3-DMAA fully substituted for cocaine and partially substituted for methamphetamine in drug discrimination assays, produced conditioned place preference in mice at intermediate doses, and generated dose-dependent locomotor depression, indicating psychostimulant-like abuse potential.
The compound is banned or restricted in the United States, Canada, Australia, New Zealand, the United Kingdom, Brazil, Sweden, Finland, and Switzerland, and is listed as a prohibited substance by the World Anti-Doping Agency. It is not approved as a medicine in any jurisdiction as of the date of this monograph. This monograph reviews the chemistry, synthesis, and structural classification of 1,3-DMAA; the historical development and regulatory trajectory; the molecular pharmacology at monoamine transporters and adrenergic receptors; the limited human pharmacokinetic record; preclinical pharmacology including abuse liability; the clinical and adverse-event evidence base; sourcing and analytical verification considerations; reconstitution and handling; stack interaction and combination risks; the comprehensive adverse-event and safety signal; and a comparative assessment of five sympathomimetic amine stimulants against 1,3-DMAA on five competency standards (novelty, effect size, promising potential, side-effect profile, and overall validation).
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Type I transmembrane protein and circulating endocrine factor of the glycosyl hydrolase family 1 superfamily with obligate FGF23 co-receptor and pleiotropic anti-aging functions
A kidney-derived transmembrane glycoprotein and its shed soluble ectodomain, identified through insertional mutagenesis in 1997 as a suppressor of aging phenotypes in mice, functioning both as an obligate co-receptor for fibroblast growth factor 23 in mineral metabolism and as a circulating endocrine factor that inhibits insulin/IGF-1, Wnt, TGF-beta, and NF-kappaB signaling pathways with demonstrated neuroprotective, cardioprotective, and renoprotective activity in preclinical models.
Abstract
Alpha-Klotho (alpha-KL) is a 130 kDa type I single-pass transmembrane protein encoded by the KL gene on human chromosome 13q13.1, first identified in 1997 by Kuro-o et al. through characterization of an insertional mutation in mice that produced a syndrome of accelerated aging encompassing soft tissue calcification, arteriosclerosis, skin atrophy, osteoporosis, emphysema, gonadal dysplasia, and dramatically shortened lifespan [1]. The protein comprises a short intracellular domain, a single transmembrane helix, and a large extracellular region containing two tandem glycosyl hydrolase family 1 (GH1) domains, designated KL1 and KL2, which share sequence homology with family 1 beta-glucosidases but lack catalytic activity against conventional substrates due to substitutions in the active-site residues. The extracellular domain undergoes proteolytic shedding by ADAM10, ADAM17, and BACE1, generating a soluble ectodomain (sKL) comprising KL1 and KL2 that circulates as an endocrine factor detectable in plasma, cerebrospinal fluid, and urine. A shorter secreted isoform containing only KL1 is produced by alternative mRNA splicing.
In its membrane-bound form, alpha-Klotho functions as an obligate co-receptor for fibroblast growth factor 23 (FGF23), forming a ternary complex with FGF receptor 1c (FGFR1c) that is essential for phosphaturic signaling in the renal proximal tubule and for suppression of 1,25-dihydroxyvitamin D3 (calcitriol) synthesis. The crystal structure of the alpha-Klotho/FGFR1c/FGF23 ternary complex, resolved by Chen et al. (2018) at 3.0 angstrom resolution, demonstrated that the KL2 domain of alpha-Klotho cradles FGF23 with a receptor-binding arm extending from the KL1-KL2 interdomain cleft, creating a composite binding surface for FGF23 engagement [2]. Loss of this co-receptor function produces the hyperphosphatemia, hypervitaminosis D, and ectopic calcification that characterize the kl/kl mouse phenotype and that are recapitulated in FGF23 knockout mice.
Independent of the FGF23 co-receptor function, soluble alpha-Klotho acts as a circulating endocrine factor with pleiotropic anti-aging activity. Characterized signaling activities include inhibition of the insulin/insulin-like growth factor 1 (IGF-1) pathway through suppression of receptor autophosphorylation; suppression of Wnt/beta-catenin signaling through direct binding to Wnt ligands; inhibition of transforming growth factor beta (TGF-beta) type II receptor signaling and downstream Smad phosphorylation; suppression of NF-kappaB-driven inflammatory transcription; and regulation of ion channel and transporter activity in the renal tubule, including TRPV5, TRPV6, ROMK1, and the Na+/K+-ATPase [3, 4, 5]. These FGF23-independent activities are the molecular basis for the broader anti-aging, neuroprotective, cardioprotective, and anti-fibrotic effects observed in gain-of-function and supplementation studies.
Circulating soluble alpha-Klotho levels decline with age in humans, beginning approximately in the fourth decade of life, and are markedly reduced in chronic kidney disease, where loss of renal alpha-Klotho expression precedes and contributes to the mineral and bone disorder, cardiovascular calcification, and accelerated aging phenotype of uremia [6]. Epidemiological studies have identified inverse associations between circulating soluble alpha-Klotho concentrations and all-cause mortality, cardiovascular events, and cognitive decline in community-dwelling older adults [7]. The KL-VS haplotype (defined by the F352V and C370S variants, rs9536314 and rs9527025) has been associated in some cohorts with altered klotho secretion, cortical brain volume, and cognitive resilience in aging, though replication across large cohorts remains inconsistent [8, 9].
Preclinical studies have demonstrated that recombinant alpha-Klotho protein administration, adeno-associated virus-mediated KL gene transfer, and transgenic KL overexpression produce renoprotection in ischemia-reperfusion injury and unilateral ureteral obstruction models; cardioprotection with attenuation of left ventricular hypertrophy and fibrosis; suppression of vascular calcification; and cognitive enhancement in aged, young, and alpha-synuclein transgenic mice through NMDA receptor-dependent glutamatergic mechanisms [10, 11, 12, 13]. A 2023 study in aged nonhuman primates demonstrated that a single subcutaneous injection of a klotho protein fragment enhanced spatial and working memory, representing the first primate cognitive enhancement data for the compound [14].
As of 2026, alpha-Klotho is in early clinical development. Klothea Bio launched a Phase 1b randomized, double-blind, placebo-controlled trial of AKL003, an alpha-Klotho mRNA therapeutic administered intravenously, in healthy adult volunteers in February 2026. Klotho Neurosciences is advancing KLTO-202, a KL gene therapy, toward first-in-human studies for amyotrophic lateral sclerosis. No alpha-Klotho protein or gene therapy product has received regulatory approval in any jurisdiction. Recombinant human alpha-Klotho protein is available from multiple research suppliers (R&D Systems, Abcam, Sino Biological, Thermo Fisher) for in vitro and preclinical applications. This monograph reviews the molecular identification, structural biology, receptor pharmacology, preclinical pharmacology across organ systems, the emerging clinical evidence base, sourcing and handling considerations, stack interactions, safety signal, and a comparative assessment of five anti-aging intervention candidates against alpha-Klotho on five competency standards.
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Polypeptide bioregulator complex derived from bovine retinal tissue with retinoprotective and neurotrophic activity
A tissue-derived polypeptide fraction developed at the St. Petersburg Institute of Bioregulation and Gerontology as a retinoprotective agent, distinguished from conventional neuroprotective ophthalmologics by its multi-peptide composition, tissue-specific gene expression regulation, and clinical application across glaucoma, diabetic retinopathy, and hereditary retinal dystrophies.
Abstract
Retinalamin is a complex of water-soluble polypeptide fractions with molecular weight not exceeding 10,000 daltons, isolated from bovine retinal tissue and developed as a retinoprotective peptide bioregulator by Vladimir Khavinson and colleagues at the St. Petersburg Institute of Bioregulation and Gerontology of the Russian Academy of Medical Sciences. The compound is registered in the Russian Federation and several Commonwealth of Independent States jurisdictions as a lyophilized preparation for parabulbar and intramuscular injection, manufactured by Geropharm LLC under the ATC code S01XA (other preparations for eye disease treatment). Retinalamin represents a distinct pharmacological class within ophthalmic neuroprotection: rather than a single molecular entity with a defined receptor target, it consists of a heterogeneous mixture of short-chain polypeptides (predominantly 2 to 7 amino acid residues in length) that act through tissue-specific modulation of gene expression, stimulation of intracellular protein synthesis, regulation of lipid peroxidation, and normalization of cellular membrane function in retinal photoreceptors, retinal pigment epithelium, and Mueller glial cells.
The mechanistic basis of Retinalamin activity has been characterized through in vitro and in vivo models at the St. Petersburg Institute. In Xenopus laevis early gastrula ectoderm assays, Retinalamin demonstrated concentration-dependent induction of neuronal differentiation including brain, retinal, and pigment epithelium lineages, establishing the compound as a morphogenetic peptide regulator with tissue-specific inductive capacity. In cell culture, Retinalamin and the related synthetic tetrapeptide Epithalon stimulated proliferation of retinal and pigmented epithelial cells in a tissue-specific and concentration-dependent manner. The proposed molecular mechanism involves sequential binding of constituent short peptides to promoter regions of genes involved in retinal cell differentiation, survival, and metabolic homeostasis, thereby modulating transcriptional activity and downstream protein expression. Pharmacodynamic effects observed in preclinical and clinical settings include stimulation of photoreceptor and retinal cellular element function, improvement of functional interactions between retinal pigment epithelium and photoreceptor outer segments, enhancement of Mueller cell activity and glutamate inactivation, normalization of vascular permeability, and reduction of oxidative stress through regulation of lipid peroxide metabolism.
Clinical evidence for Retinalamin spans multiple retinal pathologies. In compensated primary open-angle glaucoma, a 180-patient randomized controlled trial (Egorov et al. 2019) demonstrated that intramuscular Retinalamin produced significant retinoprotective effects, with improvement in mean deviation index from negative 5.52 to negative 4.82 decibels, stabilization of ganglion cell complex thickness (versus progressive thinning in controls), and preservation of pattern electroretinography amplitudes over the study period. A subsequent 147-patient randomized trial (Strakhov et al. 2020) demonstrated that biannual Retinalamin courses over 24 months arrested development of glaucomatous optic neuropathy, with retinal nerve fiber layer thickness remaining stable in treated patients versus declining from 83.5 to 76.7 micrometers in controls. In diabetic retinopathy, a 56-patient comparative study (Malakhova et al. 2024) provided objective structural and functional evidence of positive retinal changes with intramuscular Retinalamin in early-stage disease. In hereditary retinal dystrophies, long-term observational data (Razumovskiy et al.) demonstrated that a first course of Retinalamin improved visual acuity in 58.1 percent and visual fields in 64.5 percent of retinal degeneration patients, with repeated courses over 23 to 25 years preserving residual vision in 55.6 percent of patients and preserving object vision in 11.1 percent. In retinal abiotrophy, residual vision was preserved in 100 percent of treated cases. A 498-patient glaucoma study (Erichev et al. 2020) comparing intramuscular, retrobulbar, and combined administration routes demonstrated comparable efficacy across delivery methods, with total threshold retinal sensitivity increasing by 122 to 274 decibels across glaucoma stages.
The safety profile of Retinalamin is characterized by low adverse event rates. The principal reported events are local injection site reactions (pain, redness, swelling) and rare hypersensitivity reactions including anaphylactic shock and angioneurotic laryngeal edema. The compound is contraindicated during pregnancy (absence of clinical safety data) and in pediatric populations under 18 years for most indications. Pharmacokinetic characterization in the conventional sense is not feasible owing to the multi-component polypeptide composition, which does not permit standard absorption, distribution, metabolism, and elimination analysis of individual constituents. The standard clinical regimen consists of 5 to 10 milligrams administered once daily by parabulbar or intramuscular injection for 5 to 10 days, with courses repeated every 3 to 6 months. Retinalamin is not approved by the United States Food and Drug Administration, the European Medicines Agency, or other major Western regulatory authorities. The clinical evidence base is derived predominantly from Russian-language literature published in Vestnik Oftalmologii and related journals. Investigators outside the Russian Federation should approach the compound as a research-grade peptide preparation requiring independent analytical verification and should interpret the clinical literature with attention to the methodological standards and reporting conventions of the source publications.
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Synthetic 17-alpha-alkylated anabolic-androgenic steroid derived from 5-alpha-dihydrotestosterone
A potent orally active 2-alpha,17-alpha-dimethylated dihydrotestosterone derivative with a high anabolic-to-androgenic dissociation ratio, originally synthesized by Syntex in 1956, never marketed pharmaceutically, and classified as a Schedule III controlled substance following its illicit distribution as an over-the-counter designer steroid supplement beginning in 2005.
Abstract
Superdrol (methasterone; 2-alpha,17-alpha-dimethyl-5-alpha-androstan-17-beta-ol-3-one) is a synthetic, orally active anabolic-androgenic steroid (AAS) of the 5-alpha-dihydrotestosterone (DHT) structural class. The compound was first synthesized by researchers at Syntex Corporation in 1956 and characterized in a 1959 publication as a potent orally active anabolic agent exhibiting only weak androgenic activity. Despite this favorable preclinical dissociation profile, methasterone was never advanced to clinical development or marketed as a prescription pharmaceutical. The compound resurfaced in 2005 when it was introduced to the United States consumer market under the trade name Superdrol, sold as an over-the-counter dietary supplement and marketed deceptively as a prohormone to circumvent the Anabolic Steroid Control Act of 1990. In preclinical rat bioassays using methyltestosterone as the reference standard, methasterone demonstrated approximately 400 percent anabolic potency and 20 percent androgenic potency, yielding a Q-ratio (anabolic-to-androgenic dissociation index) of 20, among the highest reported for any oral AAS. The compound’s oral bioavailability (approximately 50 percent) is conferred by the 17-alpha-methyl group, which protects the steroid nucleus from hepatic first-pass metabolism but simultaneously renders the compound hepatotoxic through a mechanism common to all C17-alpha-alkylated androgens. Methasterone is non-aromatizable owing to its 5-alpha-reduced A-ring saturation, and therefore does not produce estrogenic effects such as gynecomastia or water retention. Hepatotoxicity is the principal and most serious adverse effect: a distinctive pattern of bland cholestatic liver injury, characterized by severe hyperbilirubinemia with only modest aminotransferase elevation, has been documented in multiple case series and case reports. A comprehensive literature review of 52 reported cases identified a median presentation bilirubin of 314 micromol/L, peak bilirubin of 705 micromol/L occurring approximately 28 days after cessation, and resolution over a median of 90 days with supportive care alone; no deaths or liver transplantations were reported in the published literature. Acute kidney injury occurred in 43 percent of cases, with peak creatinine correlating with peak bilirubin. The World Anti-Doping Agency placed methasterone on its prohibited list in 2006. The United States Drug Enforcement Administration classified methasterone as a Schedule III controlled substance in January 2012 under the Controlled Substances Act, and the Designer Anabolic Steroid Control Act of 2014 further expanded regulatory authority over designer steroids of this class. Methasterone has no approved medical indication in any jurisdiction. This monograph documents the complete chemistry, synthesis, pharmacology, pharmacokinetics, hepatotoxicity profile, clinical case evidence, sourcing considerations, handling, combination interactions, adverse event signal, and a comparative assessment against five alternative oral anabolic-androgenic steroids (oxandrolone, oxymetholone, stanozolol, methyltestosterone, and epistane) on five competency standards.
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A synthetic structural analog of tadalafil in which the piperazinedione N-methyl group is replaced by a primary amine, yielding an unapproved phosphodiesterase type 5 inhibitor principally encountered as an undeclared adulterant in dietary supplements marketed for erectile dysfunction.
Abstract
Aminotadalafil, formally (6R,12aR)-2-amino-6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione (CAS 385769-84-6), is a synthetic structural analog of tadalafil, the selective cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5) inhibitor marketed as Cialis for the treatment of erectile dysfunction, pulmonary arterial hypertension, and benign prostatic hyperplasia. The single structural modification that distinguishes aminotadalafil from the parent drug is the replacement of the N-methyl substituent on the 2-position of the piperazinedione (diketopiperazine) ring with a primary amino group (NH2), a change that alters hydrogen-bonding capacity, basicity, and physicochemical properties while preserving the core beta-carboline-fused diketopiperazine scaffold responsible for PDE5 active-site recognition. Aminotadalafil retains inhibitory activity at PDE5 and exhibits greater than 100 percent cross-reactivity with anti-tadalafil polyclonal antibodies, confirming close structural and immunochemical homology with the parent compound [1]. The compound has not undergone formal preclinical toxicology, human pharmacokinetic characterization, or clinical efficacy evaluation in any regulatory jurisdiction, and it is not approved for human use by any national medicines authority. Its principal significance in the biomedical literature arises from its repeated identification as an undeclared adulterant in dietary supplements, herbal products, and electronic cigarette liquids marketed for sexual enhancement, where it poses uncharacterized risks to consumers who are unaware of its presence and who may be concurrently taking nitrate-containing medications or other agents that interact with the cGMP-nitric oxide signaling pathway [2, 3, 4, 5].
The analytical chemistry literature on aminotadalafil is substantial. The compound has been identified and structurally characterized by proton and carbon-13 nuclear magnetic resonance spectroscopy, electrospray ionization mass spectrometry, Fourier transform infrared spectroscopy, and ultraviolet spectrophotometry in dietary supplement matrices across multiple continents, including reports from Latin America, Asia, Europe, and North America [2, 6, 7]. Validated high-performance liquid chromatography with diode array detection (HPLC-DAD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods permit simultaneous identification and quantification of aminotadalafil alongside other PDE5 inhibitor analogs (hydroxythiohomosildenafil, thiosildenafil, dimethylsildenafil, thiodimethylsildenafil) in complex supplement matrices [3, 8]. A dimeric interaction product of aminotadalafil has also been isolated from adulterated health food products, suggesting that the compound undergoes degradation or condensation reactions under storage conditions encountered in supplement manufacture [9].
The pharmacological characterization of aminotadalafil is limited. No peer-reviewed study has reported a direct IC50 determination for aminotadalafil against recombinant human PDE5, and no selectivity panel across the eleven mammalian phosphodiesterase families has been published. The compound’s activity is inferred from its structural homology to tadalafil (IC50 approximately 1.8 to 5.0 nanomolar against PDE5) and from the immunochemical cross-reactivity data. Structure-activity relationship studies of the broader tadalafil analog series indicate that modifications at the 2-position of the diketopiperazine ring modulate PDE5 affinity, and that the (6R,12aR) stereochemistry derived from L-tryptophan is essential for potent inhibition [10, 11]. Aminotadalafil preserves this stereochemistry. No human pharmacokinetic, dose-response, or safety data exist; toxicological risk assessment must therefore rely on extrapolation from tadalafil and on the general pharmacology of the PDE5 inhibitor class. This monograph reviews the chemistry, structural pharmacology, inferred pharmacokinetics, regulatory history, detection methodology, sourcing considerations, and comparative assessment of aminotadalafil against five alternative PDE5 inhibitor analogs encountered in the adulterated supplement landscape.
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NSI-189 is a small molecule developed at Neuralstem to stimulate hippocampal neurogenesis (the birth of new neurons in the dentate gyrus). Preclinical rodent studies showed proliferation of neural progenitor cells, hippocampal volume increase, and antidepressant-like behavior in chronic stress models, generating considerable hope that this could be a fundamentally new class of antidepressant. Phase 1 trials were uneventful. Phase 2 trials in major depression in 2014 and 2017 failed to beat placebo on the primary endpoints, and Neuralstem ended development. It briefly became popular in nootropic communities sourced as a research chemical, on the strength of secondary cognitive endpoints in the failed trials. The clinical case is essentially closed. Not stocked by Kodiac. This monograph is provided for research and educational reference.
Intrigue 0–100 blends mechanism novelty, evidence strength, and translational potential. Kodiac editorial, not peer-reviewed.
Benzylpiperazine-aminopyridine neurogenic compound with indirect brain-derived neurotrophic factor modulation and hippocampal neurogenesis stimulation
A first-in-class small molecule neurogenic agent discovered through phenotypic screening of human hippocampal neural stem cells, developed for major depressive disorder and under investigation for cognitive impairment, diabetic neuropathy, and post-traumatic stress disorder.
Abstract
NSI-189 (amdiglurax; ALTO-100) is a benzylpiperazine-aminopyridine small molecule identified through a phenotypic screen of approximately 10,000 compounds against human hippocampal neural stem cells and advanced as a first-in-class hippocampal neurogenesis stimulator for the treatment of major depressive disorder (MDD). The compound was discovered by Karl Johe and colleagues at Neuralstem, Inc. (Germantown, Maryland) and is now under development by Alto Neuroscience (Mountain View, California) under the designation ALTO-100. NSI-189 is mechanistically distinct from all marketed antidepressants: it has no detectable activity at serotonin, norepinephrine, or dopamine transporters, no binding at 52 standard neurotransmitter receptor and ion channel targets, and no activity across a panel of 900 kinases. Instead, the compound stimulates proliferation and neurogenic differentiation of hippocampal neural stem cells in vitro with low-micromolar potency and, on oral administration to rodents at 10 to 30 mg/kg/day, produces dose-dependent increases in hippocampal volume (up to 66 percent at 30 mg/kg in mice), upregulation of brain-derived neurotrophic factor (BDNF), stem cell factor (SCF), glial cell line-derived neurotrophic factor (GDNF), and vascular endothelial growth factor (VEGF), and activation of the TrkB/Akt signaling pathway. The morphological effects are confined to the dentate gyrus of the hippocampus and the subventricular zone; no structural changes have been observed elsewhere in the brain. A bell-shaped dose-response relationship is observed in preclinical hippocampal volume endpoints, with 100 mg/kg producing less effect than 30 mg/kg, suggesting an optimal range for neurogenic stimulation.
Clinical development has proceeded through Phase 1 (41 healthy volunteers, 2011), Phase 1b (24 MDD patients, Fava et al. 2016, published in Molecular Psychiatry), and Phase 2 (220 MDD outpatients, Papakostas et al. 2020, published in Molecular Psychiatry). The Phase 1b trial demonstrated safety and tolerability at 40, 80, and 120 mg daily for 28 days, with medium-to-large effect sizes on the Symptoms of Depression Questionnaire (SDQ) and the Cognitive and Physical Functioning Questionnaire (CPFQ). The Phase 2 trial, conducted using a sequential-parallel comparison design across 12 weeks, did not meet its primary endpoint (change from baseline on the Montgomery-Asberg Depression Rating Scale, MADRS) at either 40 mg or 80 mg daily. However, 40 mg daily produced statistically significant improvements on the SDQ (pooled mean difference -8.2; Cohen’s d = -0.64 in Stage 2; p = 0.04), the CPFQ (pooled mean difference -1.9; p = 0.03), and several objective cognitive measures on the CogScreen battery (Cohen’s d ranging from 0.12 to 1.12 for significant measures). Hippocampal volume was not significantly changed in MDD patients at the studied doses and duration, despite the robust preclinical volumetric signal.
Alto Neuroscience acquired the NSI-189 program in October 2021 and redesignated the compound ALTO-100. A Phase 2b trial (301 adults with MDD, 34 U.S. sites, 6 weeks, biomarker-enriched design using a cognitive memory test) reported topline results in 2024: ALTO-100 did not demonstrate statistically significant improvement in MADRS versus placebo in the biomarker-defined population and did not meet secondary endpoints. The compound was well tolerated, with headache, nausea, and abnormal dreams as the most common adverse events at rates similar to placebo. A Phase 2b trial in bipolar depression is expected to report in 2026, and the compound remains under investigation for post-traumatic stress disorder.
Preclinical pharmacology extends beyond depression. NSI-189 reverses cognitive and motor deficits in a rat model of ischemic stroke (30 mg/kg oral), ameliorates central and peripheral neuropathy in mouse models of type 1 and type 2 diabetes (10 to 30 mg/kg oral), enhances synaptic plasticity and reverses motor and cognitive impairments in a mouse model of Angelman syndrome through TrkB/Akt pathway activation, and enhances long-term potentiation in hippocampal slice preparations in vitro. The compound has linear pharmacokinetics across the 40 to 120 mg/day clinical dose range, an oral Tmax of 1 to 2 hours, a plasma elimination half-life of 17.4 to 20.5 hours supporting once-daily dosing, and achieves steady state within 4 to 5 days.
This monograph reviews the chemical identity and synthesis of NSI-189; the discovery through phenotypic screening; the molecular pharmacology and neurotrophic factor cascade; the comprehensive pharmacokinetic profile; the preclinical evidence base across depression, stroke, neuropathy, and Angelman syndrome models; the clinical evidence base from Phase 1 through Phase 2b; sourcing, reconstitution, and handling considerations; stack interactions; the adverse-event and safety signal; and a structured comparative assessment of five neurogenesis-associated compounds (fluoxetine, ketamine, agomelatine, psilocybin, and 7,8-dihydroxyflavone) against NSI-189 on five competency standards. The compound is not approved by any regulatory authority for any indication. It is sold as a research-grade preparation; investigators should obtain analytical confirmation of identity and purity on every lot.
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17-alpha-alkylated anabolic-androgenic steroid; synthetic dihydrotestosterone derivative with potent erythropoietic and anabolic activity
A 17-alpha-alkylated oral anabolic-androgenic steroid derived from dihydrotestosterone, developed at Syntex in the late 1950s for the treatment of anemias characterized by deficient red blood cell production, distinguished by potent erythropoietic stimulation, marked nitrogen retention, and significant hepatotoxic liability.
Abstract
Oxymetholone, marketed as Anadrol-50 (Syntex, later Unimed Pharmaceuticals, now Alaven Pharmaceutical) and Anapolon (Imperial Chemical Industries and generics), is a synthetic 17-alpha-alkylated derivative of dihydrotestosterone first described by Ringold et al. at Syntex in 1959 and introduced into clinical medicine in the early 1960s for the treatment of anemias, osteoporosis, and catabolic wasting states. The compound is the 2-hydroxymethylene analog of 17-alpha-methyl-dihydrotestosterone (mestanolone), bearing an unusual hydroxymethylene substituent at the C2 position that confers oral bioavailability through resistance to first-pass hepatic inactivation and that can be metabolically cleaved to yield mestanolone as an active metabolite. Oxymetholone is one of the most potent oral anabolic-androgenic steroids in clinical use, exhibiting a high ratio of anabolic to androgenic activity in classical levator ani and ventral prostate bioassays while paradoxically demonstrating low direct binding affinity for the androgen receptor in competitive displacement studies. The principal pharmacodynamic effects are stimulation of erythropoiesis through increased renal erythropoietin production (with urinary erythropoietin levels elevated up to fivefold at therapeutic doses), promotion of positive nitrogen balance and skeletal muscle protein synthesis, and a poorly characterized but clinically significant estrogenic activity that occurs despite the compound’s structural inability to undergo aromatization, possibly through direct activation of estrogen receptor alpha. The compound received approval from the United States Food and Drug Administration for the treatment of anemias caused by deficient red blood cell production, including acquired aplastic anemia, congenital aplastic anemia, myelofibrosis, and hypoplastic anemias due to the administration of myelotoxic drugs. In the mid-1970s, the FDA restricted the approved indication exclusively to anemias characterized by deficient erythropoiesis, withdrawing prior approvals for osteoporosis and general catabolic states. Clinical investigation has subsequently extended to HIV/AIDS-associated wasting (Hengge et al. 2003 Phase III trial demonstrating 3.0 to 3.5 kg weight gain over 16 weeks at 100 to 150 mg daily), antithrombin III deficiency, and pediatric growth failure, with varying degrees of success. The hepatotoxic liability of the 17-alpha-alkylated structure is the principal safety concern: cholestatic jaundice occurs in approximately 1 percent of treated patients, typically within 1 to 4 months of initiation; peliosis hepatis (blood-filled sinusoidal cysts) develops with prolonged administration and has resulted in fatal hepatic rupture and hemorrhage in case reports; and hepatocellular carcinoma and hepatic adenoma have been reported after 2 to 15 years of continuous use, predominantly in patients with Fanconi anemia and aplastic anemia receiving chronic androgen therapy. The compound is classified as a Schedule III controlled substance in the United States under the Controlled Substances Act and is subject to equivalent regulatory controls in most jurisdictions. This monograph reviews the chemistry, synthesis, and structure-activity relationships of oxymetholone; the androgen receptor, erythropoietic, and estrogenic pharmacology; the limited but clinically relevant pharmacokinetic record; the preclinical pharmacology in animal models of anemia and wasting; the clinical evidence base across anemia, HIV wasting, and ancillary indications; sourcing and quality verification for research applications; reconstitution and handling; stack interactions and combinations with other anabolic and therapeutic agents; the adverse-event and safety signal including detailed hepatotoxicity characterization; and a comparative assessment of five alternative anabolic-androgenic agents (oxandrolone, nandrolone decanoate, stanozolol, testosterone enanthate, danazol) against oxymetholone on five competency standards.
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A benzhydrylsulfinylmethylthiazole developed at the University of Vienna as a next-generation modafinil analogue, distinguished from the parent compound by approximately 30-fold selectivity for the dopamine transporter over the norepinephrine transporter, five-fold superior blood-brain barrier penetration, and a preclinical cognitive enhancement profile spanning memory acquisition, cognitive flexibility, motivation, and social cognition in rodent models of aging, early-life stress, and fetal alcohol spectrum disorders.
Abstract
CE-123 (5-((benzhydrylsulfinyl)methyl)thiazole; CAS 1879038-73-9) is a synthetic modafinil analogue and atypical dopamine transporter (DAT) inhibitor developed at the Department of Pharmaceutical Chemistry, University of Vienna, under the direction of Gert Lubec as part of a structure-activity exploration of heterocyclic diphenylmethylsulfinyl derivatives designed to achieve higher selectivity and affinity at the dopamine transporter than the parent compound modafinil. The active enantiomer, (S)-CE-123, inhibits DAT-mediated dopamine reuptake with an EC50 of approximately 2.76 micromolar in HEK293 cells stably expressing human DAT, with approximately 30-fold selectivity over the norepinephrine transporter (NET) and greater than 400-fold selectivity over the serotonin transporter (SERT). The compound interacts with the outward-facing conformation of DAT to block substrate access without triggering reverse transport or vesicular release, a mechanism that defines the atypical DAT inhibitor class and is associated with lower abuse liability than substrate-type releasers such as amphetamine. Pharmacokinetic characterization in Sprague-Dawley rats demonstrates that (S)-CE-123 achieves an unbound brain-to-plasma concentration ratio (Kp,uu,brain) of 0.5, compared to 0.1 for R-modafinil, indicating approximately five-fold superior blood-brain barrier penetration. Hepatic metabolism proceeds via CYP2C19, CYP3A, and CYP2B6, with a 9.3-fold faster hepatic clearance rate compared to modafinil. The principal metabolite (M1) is formed by hydroxylation of one of the aromatic rings.
The preclinical pharmacology of CE-123 spans multiple cognitive domains tested in several rodent models. In the spatial hole-board paradigm in male Sprague-Dawley rats, daily oral CE-123 at doses of 1 and 10 mg/kg improved both memory acquisition and memory retrieval, with significantly increased reference memory indices and shortened latency to find baited holes. In the attentional set-shifting task, CE-123 at 0.3 and 1.0 mg/kg increased cognitive flexibility (reduced extra-dimensional shift errors) without increasing impulsivity. In aged (26-month) Lister Hooded rats, (S)-CE-123 markedly enhanced motivation and performance in a new-to-learn operant discrimination task and in a cooperation assay of social cognition, with post-treatment proteomic analysis of prefrontal cortex synaptosomes revealing modulation of pathways involved in synaptic vesicle recycling, receptor-mediated endocytosis, and alpha-synuclein membrane localization. In a maternal separation model of early-life stress, CE-123 restored spatial memory deficits in adolescent rats with sex-dependent effects favoring females, and normalized maternal-separation-induced upregulation of DAT and dopamine D1 receptor expression in the prefrontal cortex and hippocampus. In a neonatal ethanol exposure model of fetal alcohol spectrum disorders, CE-123 at 3 and 10 mg/kg attenuated locomotor hyperactivity and ameliorated reversal learning impairment. Neurochemical microdialysis studies in freely moving rats demonstrated that (S)-CE-123 at 10 mg/kg intraperitoneally increased extracellular dopamine in the infralimbic/prelimbic cortex with a pharmacodynamic profile distinct from R-modafinil, and produced only a low and transitory dopamine increase in the nucleus accumbens shell, consistent with reduced reinforcing potential.
No human clinical trials of CE-123 have been published or registered as of the date of this monograph. The compound is not approved by any regulatory authority for human use. It is supplied as a research-grade preparation by multiple chemical vendors at greater than 98 percent purity. A process development and scale-up synthesis has been published (ACS Omega, 2023), establishing a scalable route to the (S)-enantiomer. This monograph reviews the chemistry, synthesis, and stereochemistry of CE-123; the DAT inhibitor pharmacology in molecular and functional detail; the pharmacokinetic characterization including blood-brain barrier penetration; the preclinical cognitive pharmacology across multiple behavioral paradigms and disease models; sourcing and quality verification; reconstitution and handling; stack-interaction considerations; adverse-event signal from preclinical studies; and a comparative assessment of five alternative cognitive-enhancing DAT-active compounds against CE-123 on five competency standards. The compound is strictly a research tool at this stage of development; investigators should obtain analytical confirmation of identity and purity on every lot and should not extrapolate preclinical findings to human dose-response without appropriate regulatory and ethical authorization.
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Endogenous immunostimulatory tetrapeptide derived from the CH2 domain of immunoglobulin G
A spleen-dependent, IgG-derived tetrapeptide that activates phagocytic cells through neuropilin-1 receptor binding, stimulating macrophage phagocytosis, chemotaxis, and tumoricidal activity, and serving as the structural parent of the anxiolytic heptapeptide Selank.
Abstract
Tuftsin (L-threonyl-L-lysyl-L-prolyl-L-arginine) is an endogenous tetrapeptide corresponding to residues 289 through 292 of the CH2 domain of the immunoglobulin G (IgG) heavy chain. First identified in 1970 by Victor A. Najjar and Keisuke Nishioka at the Tufts University School of Medicine during investigations of phagocytosis by polymorphonuclear granulocytes, the peptide derives its name from its institutional origin and has since become the prototype endogenous immunostimulatory peptide. Tuftsin is not synthesized de novo but is liberated from a larger IgG-associated polypeptide termed leukokinin through the sequential action of two proteases: tuftsin-endocarboxypeptidase, a splenic enzyme that cleaves the C-terminal bond of the tetrapeptide within the intact immunoglobulin, and leukokinase, a serine protease on the outer membrane of phagocytic cells that releases the active N-terminal tetrapeptide from the partially processed fragment. This two-step enzymatic liberation places the spleen as an obligate organ in tuftsin physiology and accounts for the well-documented tuftsin deficiency, impaired phagocytic function, and increased susceptibility to overwhelming bacterial infection observed following splenectomy.
The molecular pharmacology of tuftsin centers on its binding to neuropilin-1 (NRP1), a single-pass transmembrane glycoprotein that also serves as a co-receptor for vascular endothelial growth factor (VEGF) and transforming growth factor beta (TGF-beta). Tuftsin binds the b1 domain of neuropilin-1 through a motif similar to the C-terminal sequence encoded by exon 8 of VEGF165, and signals through TGF-beta receptor 1 (a co-receptor of NRP1) via the canonical Smad3 phosphorylation pathway, with concurrent reduction in Akt phosphorylation. At the cellular level, tuftsin binding stimulates phagocytosis, pinocytosis, chemotaxis, respiratory burst (superoxide anion and hydrogen peroxide generation), antigen presentation, and tumoricidal activity in monocytes, macrophages, neutrophils, microglia, and Kupffer cells. Intracellular calcium serves as a critical second messenger in tuftsin-mediated phagocyte activation. In neuroinflammatory models, tuftsin promotes an anti-inflammatory M2 microglial phenotype shift, suppresses pro-inflammatory Th1 responses, upregulates Th2 responses, and expands regulatory T cell populations.
Pharmacokinetically, tuftsin is an extremely labile peptide in vivo, with a plasma half-life of approximately 16 minutes owing to rapid degradation by serum aminopeptidases and carboxypeptidases. Oral bioavailability is negligible because of gastric peptidase destruction. The rapid enzymatic clearance has been the principal barrier to clinical development. Initial clinical studies conducted at the Weizmann Institute of Science demonstrated that tuftsin is nontoxic in humans when administered intravenously at doses up to 5 mg per injection in patients with advanced malignancy; a Phase II study in 25 patients with various advanced cancers reported leucocytosis and increased natural killer activity with two partial responses among 16 evaluable patients, confirming biological activity but insufficient monotherapy efficacy.
The most significant translational legacy of tuftsin is the development of Selank (Thr-Lys-Pro-Arg-Pro-Gly-Pro), a synthetic heptapeptide analog in which the tuftsin sequence is extended at the C-terminus with a Pro-Gly-Pro tripeptide motif that confers resistance to enzymatic degradation and facilitates blood-brain barrier penetration. Selank was developed at the Institute of Molecular Genetics of the Russian Academy of Sciences and is approved in Russia as a nasal spray for generalized anxiety disorder and neurasthenia. Selank exhibits pronounced anxiolytic, nootropic, and immunomodulatory activity through mechanisms including allosteric modulation of GABA-A receptors and modulation of monoamine neurotransmitter metabolism, and does so without the sedation, tolerance, or dependence associated with benzodiazepines.
Beyond Selank, tuftsin has been developed as a targeting ligand for liposomal and nanoparticle drug delivery systems. Palmitoyl tuftsin grafted onto liposome surfaces enables selective binding to phagocytic cells and has demonstrated augmented antitumor efficacy of encapsulated cytotoxic agents (etoposide, doxorubicin, curcumin) against fibrosarcoma and Ehrlich ascites carcinoma in murine models. Tuftsin-bearing liposomes have also been used to deliver antimicrobial agents to macrophage-resident intracellular pathogens including Leishmania and Plasmodium species.
This monograph reviews the chemistry, enzymatic biogenesis, and structural biology of tuftsin; the neuropilin-1 receptor pharmacology and downstream signaling; the pharmacokinetic constraints and stabilization strategies; the preclinical pharmacology across immunostimulatory, anti-inflammatory, antitumor, and anti-infective applications; the clinical evidence base; sourcing and quality verification; reconstitution and handling; stack interactions; adverse events and safety; and a comparative assessment of five immunostimulatory peptide candidates (Selank, thymosin alpha-1, muramyl dipeptide, GM-CSF, and thymopentin) against tuftsin on five competency standards.
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