RASP (reactive aldehyde species) sequestering agent
- Aldehydes (e.g., malondialdehyde, 4-hydroxynonenal): ADX-102 covalently binds to these aldehyde species. [1]
Reproxalap sequesters reactive aldehyde species that are elevated under inflammatory conditions . RASP enhance NF-κB, inflammasome upstream pro-inflammatory signaling cascades by binding to amino and thiol groups on receptors and kinases, and can also bind to phosphatidylethanolamine, a key component for maintaining ocular surface moisture . By reducing pro-inflammatory RASP levels, reproxalap reduces PKCα activity and blocks caspase 3/7 activation .
Reproxalap targets reactive aldehyde species (RASP), which are pro-inflammatory and cytotoxic lipid oxidation products. It covalently binds to aldehydes, including malondialdehyde and 4-hydroxynonenal, thereby sequestering them and preventing their pathological effects. By reducing the activity of protein kinase C alpha (PKCα) and blocking caspase 3/7 activation, it mitigates aldehyde-induced cellular damage and inflammation.

ADX-102 is a novel small molecule that covalently binds aldehydes, including malondialdehyde and 4-hydroxynonenal, which have been shown to mediate inflammatory pain.[1]

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Reproxalap protects cells from the cytotoxicity of C18:0-al. In vitro, the compound reduces PKCα activity and blocks caspase 3/7 activation, thereby exerting anti-inflammatory and pain-relieving effects . Reproxalap has demonstrated the ability to trap free aldehydes, diminish inflammation, and protect key cellular constituents from aldehyde damage .
In vitro, Reproxalap demonstrates protective effects against aldehyde-induced cytotoxicity. It protects FAA-K1A cells from the cytotoxicity of octadecanal (C18:0-al) at concentrations of 100-300 μM. In BEAS-2B and 16HBE cells, it dose-dependently reverses the slowing of cell migration induced by cigarette smoke extract. The compound shows no significant change in cell viability at low concentrations (0.1-10 μM), but decreases viability at high millimolar concentrations. It also inhibits the metabolism of C18:0-al to fatty alcohol (octadecanol).

- In a carrageenan-induced acute inflammatory pain mouse model, ADX-102 mediated dose-dependent reductions in nociceptive behavior. [1]
- In a Complete Freund's Adjuvant (CFA)-induced acute inflammatory pain mouse model, ADX-102 mediated dose-dependent reductions in nociceptive behavior. The data imply that ADX-102 may differentially affect thermal and mechanical pain pathways. [1]
- In a Phase 2b clinical trial for dry eye disease (January – July 2018), reproxalap ophthalmic solutions demonstrated statistically significant improvements versus vehicle across multiple symptom and sign measures. Improvements in symptoms and signs were observed as early as two weeks. [2]
- In the Phase 2b trial, reproxalap (0.25%) improved the ocular dryness score (OD & 4-Symptom Questionnaire, scale 0-5) compared to vehicle in the total population (baseline average score = 3.1) and in the above-median baseline population (baseline average score = 3.6). [2]
- In the Phase 2b trial, reproxalap (0.25%) improved the overall ocular discomfort score (0-5) compared to vehicle in the total population (baseline average = 2.9) and above-median baseline population. [2]
- In the Phase 2b trial, reproxalap (0.25%) improved the ocular stinging score (0-5) compared to vehicle in the total population (baseline average = 1.4) and above-median baseline population. [2]
- In the Phase 2b trial, reproxalap (0.25%) improved the fluorescein staining score (nasal, 0-4) compared to vehicle in the total population (baseline average = 1.9) and above-median baseline population (baseline average = 2.3). Responder analyses demonstrated statistical superiority of reproxalap over vehicle for staining improvement and for combined improvement of both ocular dryness and staining. [2]

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In the CFA-induced pain model, treatment with 100 mg/kg QD or 100 mg/kg BID ADX-102 resulted in statistically significant reductions in thermal hypersensitivity as measured by the Hargreave’s plantar test (Figure 1). Only the 100 mg/kg BID dose of ADX-102 showed an effect on mechanical sensitivity, as assessed by the Von Frey force test (Figure 2). Paw thickness, a measure of swelling, showed a modest effect at the 30 and 100 mg/kg BID doses (Figure 3).[1]

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In the carrageenan-induced pain model, ADX-102 treatment resulted in statistically significant reductions in thermal hypersensitivity at ADX-102 doses of 30 mg/kg BID and 100 mg/kg BID (Figure 4), but did not affect mechanical hypersensitivity (Figure 5). Paw thickness, a measure of swelling, showed a modest effect at the 100 mg/kg QD dose (Figure 6). There were no changes in body weight as a result of treatment in any group.[1]

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In animal models, by blocking RASP, reproxalap has been shown to inhibit Th1- and Th2-mediated inflammation . In Phase III clinical trials for dry eye disease, patients receiving reproxalap showed significant reduction in ocular redness just 10 minutes after entering a dry eye chamber compared to vehicle control, with effects sustained through the last measured time point (90 minutes) . Among 132 participants, reproxalap was statistically superior to vehicle for relieving ocular discomfort (P=0.004), with 70% of patients showing response to reproxalap . Reproxalap also significantly reduced ocular redness and increased Schirmer scores (a measure of tear production) in patients .
In vivo, Reproxalap exhibits anti-inflammatory and pain-relieving effects. In animal models, it shows a dose-dependent reduction in pain-related behaviors at doses of 30-100 mg/kg, administered once or twice daily. It has demonstrated clinical efficacy in reducing eye inflammation in conditions such as dry eye disease, allergic conjunctivitis, and noninfectious anterior uveitis.

The in vitro enzyme/receptor binding assay for Reproxalap involves measuring its ability to covalently bind to reactive aldehydes. The compound is incubated with aldehydes such as malondialdehyde or 4-hydroxynonenal, and the formation of the adduct is quantified using mass spectrometry or HPLC. Its effect on PKCα activity is assessed using a kinase activity assay with a specific peptide substrate, measuring the incorporation of radioactive phosphate or using a fluorescence-based detection system.

In vitro cell-based assays utilize BEAS-2B and 16HBE cell lines to evaluate cytoprotective effects. Cells are treated with Reproxalap at concentrations ranging from 0.1 μM to 10 mM for 24 hours. Cell viability is assessed using standard assays such as MTT or CellTiter-Glo. To study protection against aldehyde toxicity, FAA-K1A cells are treated with C18:0-al in the presence or absence of the compound, and apoptosis is measured by quantifying caspase 3/7 activation.

The effect of ADX-102 on acute inflammatory pain was tested in the carrageenaninduced and Complete Freund’s Adjuvant (CFA)-induced models in mice [1].
In vivo animal experiments for Reproxalap are conducted in models of ocular inflammation and pain. The compound is administered topically or via oral gavage at doses ranging from 30 to 100 mg/kg. Efficacy is evaluated by measuring pain-related behaviors, scoring ocular surface staining, and quantifying inflammatory cytokine levels in tear fluid or corneal tissue. The reduction in these parameters is used to assess the compound's anti-inflammatory and analgesic effects.

Reproxalap has a molecular weight of 236.07, XLogP of 2.27, topological polar surface area of 59.14 Ų, 3 hydrogen bond acceptors, 2 hydrogen bond donors, 1 rotatable bond, and breaks 0 Lipinski's rules . In clinical studies, reproxalap is administered topically as a 0.25% ophthalmic solution, with rapid onset of action observed as early as 10 minutes after administration . Cross-over and parallel clinical trials evaluated efficacy data ranging from minutes to up to 12 weeks post-administration . Storage conditions: Powder is stable for 3 years at -20°C and 2 years at 4°C; solutions are stable for 6 months at -80°C and 1 month at -20°C .
Reproxalap is a small molecule with a molecular weight of 236.70 g/mol. It is soluble in DMSO (100 mg/mL) and ethanol (47 mg/mL), but is insoluble in water. For in vivo studies, it can be formulated as a homogeneous suspension in CMC-Na at ≥5 mg/mL or as a clear solution using 5% DMSO, 40% PEG300, 5% Tween 80, and 50% ddH2O. Powder formulations are stable for up to 3 years at -20°C and 2 years at 4°C.

- In the Phase 2b dry eye disease clinical trial (0.1% and 0.25% reproxalap ophthalmic solutions), no observed safety concerns were reported, and the drug was generally well tolerated. Predominantly mild instillation site irritation was reported. Discontinuation rates: 3/100 (3%) for 0.1% reproxalap, 12/100 (12%) for 0.25% reproxalap, and 1/100 (1%) for vehicle. These rates are consistent with recent Phase 2 dry eye disease clinical trials. [2]
Toxicological data for Reproxalap indicate that it is generally well-tolerated in preclinical and clinical studies. In vitro, it shows no significant change in cell viability at concentrations up to 100 μM in 16HBE cells and up to 10 μM in BEAS-2B cells. At high concentrations (1-10 mM), it significantly decreases cell viability. As a topical ocular agent, systemic exposure is expected to be minimal, reducing the risk of systemic toxicity.

[1]. Susan Macdonald, et al. ADX-102, a novel aldehyde trap, reduces nociceptive behavior in mouse models of carrageenan and CFA induced pain.https://ir.aldeyra.com/static-files/527c5795-0792-4322-9f47-69c79879415a
[2]. Reproxalap Phase 2b Dry Eye Disease Results.https://ir.aldeyra.com/static-files/c642ef41-fe14-47d1-ba0e-d2b6a90f6a57

- ADX-102 is a novel aldehyde trap that covalently binds aldehydes such as malondialdehyde and 4-hydroxynonenal, which mediate inflammatory pain. Aldehyde signaling represents a novel therapeutic target for pain treatment. [1]
- In the Phase 2b dry eye disease trial, the primary objective was achieved: endpoint selection and sample size powering were confirmed for Phase 3 clinical trials. The pathway to registration trials was confirmed with ocular dryness symptom score, ocular staining score, and 0.25% reproxalap dose. Aldeyra plans to discuss results with regulatory authorities and expects to initiate Phase 3 clinical trials in 2019. [2]
- Reproxalap has a novel mechanism of action with the potential to address the two major forms of dry eye disease. [2]
- Current prescription options for dry eye disease may take up to six weeks or longer to have an effect, whereas reproxalap showed early and consistent symptom improvements in Phase 2b trial. [2]
- Reproxalap is in late-stage development for dry eye disease and allergic conjunctivitis – two medical conditions with significant overlap. Anticipated milestones: dry eye disease Phase 3 program initiation in 2019; allergic conjunctivitis ALLEVIATE Phase 3 trial results in late 2018/early 2019. [2]
- Pipeline: Reproxalap ocular is in Phase 2/3 for dry eye disease, allergic conjunctivitis, and noninfectious anterior uveitis. Reproxalap dermal is in Phase 3 for Sjögren-Larsson Syndrome. [2]

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Reproxalap (formerly known as ADX 102 or NS-2) is a small molecule inhibitor developed by Aldeyra Therapeutics. It is currently being investigated for its efficacy in treating dry eye, allergic conjunctivitis, non-infectious anterior uveitis, and Sjögren-Larsson syndrome. NS-2 was granted orphan drug designation for its research in the treatment of Sjögren-Larsson syndrome.

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Mechanism of Action
Reproxalap inhibits reactive aldehydes (RASPs) and is currently being investigated for its efficacy in treating dry eye, allergic conjunctivitis, non-infectious anterior uveitis, and Sjögren-Larsson syndrome.
Reproxalap is an investigational drug that has not yet received regulatory approval. It has been evaluated in clinical trials for the treatment of dry eye disease, allergic conjunctivitis, and noninfectious anterior uveitis. The compound is also being investigated for Sjögren-Larsson syndrome (SLS), a rare genetic disorder characterized by ichthyosis and neurological symptoms. Its mechanism of action as a RASP inhibitor represents a novel approach to treating ocular surface inflammation.

C12H13CLN2O

236.697421789169

236.071

C, 60.89; H, 5.54; Cl, 14.98; N, 11.84; O, 6.76

916056-79-6

16088030

Typically exists as Light yellow to yellow solid at room temperature

2.4

2

3

1

16

259

0

CC(C)(C1=C(C=C2C=C(C=CC2=N1)Cl)N)O

GUHFUVLKYSQIOQ-UHFFFAOYSA-N

InChI=1S/C12H13ClN2O/c1-12(2,16)11-9(14)6-7-5-8(13)3-4-10(7)15-11/h3-6,16H,14H2,1-2H3

2-(3-amino-6-chloroquinolin-2-yl)propan-2-ol

NS-2; NS2 NS 2; ADX-102; Reproxalap; 916056-79-6; 2-(3-amino-6-chloroquinolin-2-yl)propan-2-ol; ADX-102; Reproxalap [USAN]; NS-2; ALD-102; F0GIZ22IJH; ADX102; ADX 102,

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~422.48 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.56 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (10.56 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)