Dapansutrile

Dapansutrile (OLT1177) is an inhibitor of the NLRP3 (nucleotide-binding domain leucine-rich repeat and pyrin domain containing receptor 3) inflammasome.[1]

Figure 1. NLRP3 protein domains and multiple NLRP3 forms the NLRP3 Inflammasome. The NLRP3 protein is composed of three domains: NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), the apoptosis speck-like protein( ASC), and Caspase-1.[2] Image created in biorender.com.
Dapansutrile
Names
IUPAC name
3-methylsulfonylpropanenitrile
Identifiers
3D model (JSmol)
ChEMBL
ChemSpider
ECHA InfoCard 100.255.888
EC Number
  • 821-352-9
KEGG
UNII
Properties
C4H7NO2S
Molar mass 133.17 g·mol−1
Hazards
GHS pictograms
GHS Signal word Warning
H302, H315, H319, H335
P261, P264, P270, P271, P280, P301+312, P302+352, P304+340, P305+351+338, P312, P321, P330, P332+313, P337+313, P362, P403+233, P405, P501
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
Infobox references

An inflammasome can be defined as an immune system receptor that induces inflammation through the activation of caspase 1 and caspase 11 when it is triggered by damaged cells, microbial pathogens, and stress.[3] NLRP3 is a canonical inflammasome.[3] The NLRP3 inflammsome comprises of NLRP3, the apoptosis spec-like protein (ASC) and the caspase-1[2] (Figure 1). The NLRP3 inflammasome forms by binding to pattern recognition receptors (PRRs) and damage associated molecular patterns (DAMPS) that activate caspase 1 which then signals for the secretion of pro-inflammatory cytokines IL-1β and IL-18 resulting in pryoptosis[3],.[4][5] Constant activation of the NLRP3 inflammasome is believed to play a direct or indirect role in acute arthritis, atherosclerosis and various neurodegenerative diseases such as multiple sclerosis (MS), Alzheimer’s disease (AD), and Parkinson’s disease (PD)[6],[2],.[7] This drug was developed by Olactec Theraputics with the purpose of decreasing IL-1ß peripheral inflammation by binding to the NLRP3 protein and inhibiting the formation of the NLRP3 inflammasome. Interestingly, dapansutrile has also been found to reduce levels of pro inflammatory cytokines IL-18 without interfering with TNF𝛂 levels.[1]  Stressed cells in the system can ignite the NLRP3 inflammasome which in turn produces the secretion inflammatory cytokines such as IL-1ß and IL-18 (see § inflammasome ). Dapansutrile has tested in clinical trials and has been proposed as a beneficial compound for the remedy of osteoarthritis, and gouty arthritis.[1] Nevertheless, other preclinical research has proposed dapansutrile to be potentially beneficial for heart failure and multiple sclerosis.[1]

Molecular Structure and Properties

Dapansutrile is a ẞ-sufinyl nitrile compound with four carbons, seven hydrogens one nitrile, 2 oxygens and one sulfur (Figure 2).[8][9] The molecular formula of this compound is  C4H7NO2S, and it carries a molecular weight of 133. 7g/mol. Compound acknowledged by the names of dapansutrile, OLT1177 and  3-(methylsulfonyl)propanenitrile, and Propanenitrile, 3-(methylsulfonyl)[8].

Figure 1. Molecular Structure of Dapansutrile[10]
Figure 2. Molecular Structure of Dapansutrile.
Dapansutrile (OLT1177)
Molecular Formula[8] C4H7NO2S
Molecular Weight[10] 133.7g/mol
IUPAC Name[8] 3-(methylsulfonyl)propanenitrile
ACD/Index Name[8] Propanenitrile, 3-(methylsulfonyl)
Olatec Therapeutics Name[8] OLT1177
International Non-Proprietary Name[8] Dapansutrile
Storage[11] Store at -20℃
CAS Number [11] 54863-37-5
PubChem ID[11] 12714644
Smiles[11] C[S](=O)(=O)CCC#N
Experimental Solubility[12] Soluble to:

100mM in DMSO

20nM in ethanol

Formal Charge[12] 0
Hydrogen Bond Donor Count[12] 0
Hydrogen Bond Acceptor Count[12] 3
Rotatable Bond Count[12] 2
Topological Polar Surface Area[12] 66.3Å2
Heavy Atom Count[12] 8
Isotope Atom Count[12] 3

Synthesis

OLT1177 is synthesized by alkylation of sodium methanesulfinate with 3- boromopropionitrile. This reaction produces the crude substance: methylsulfonylpropionitrile. Methylsulfonylpropionitrile, is then biotransformed into dapansutrile by purification through dissolution into acetone, filtration of the sodium bi-product, solvent exchange and recrystallization from ethanol respectively.[9]

Dapansutrile's Mechanism of Action
Figure 3. Dapansutrile's Mechanism of Action. A. Depicts the activation and oligomerization of the NLRP3 inflammsome caused by PAMPS and DAMPS. B. When Dapansutrile is adminstered it binds to the NLRP3 protein and if DAMPS and PAMPS signals are present, Dapansutrile will inhibit NLRP3 inflammasome activation. This leads to an inhibition of the NLRP3 secretion of cytokines (IL-1ẞ and IL-18) and caspase-1 mediated pryopoptosis.[2][5][9] Image created in biorender.com.

Dapansutrile's Reaction Pathways

Dapansutrile denoted as a ẞ-sufonyl nitrile molecule. Its mechanism of action induces a Pinner reaction which is initiated by reacting with thiols, alcohols and amines. Thus, leading to the formation of thiomidates, imidates and amidines respectively.[13] The nitrile group of OLT1177 is still not denoted as a covalent, noncovalent, irreversible or non-reversible inhibitor as there are currently no studies about its reactivity.[9][13] Nevertheless, some researchers believe that dapansutrile promotes inhibition of NLRP3 through covalent bonds (Figure 4).[13]

Dapansutrile Mechanism of Action. A. Reaction pathway with sulphur. B.Reaction pathway with oxygen. C. Reaction pathway with nitrogen.[13]
Figure 4. Reaction Pathways of Dapansutrile (OLT1177)[13] A. Reaction pathway of Dapansutrile with sulphur. B.Reaction pathway of Dapansutrile with oxygen. C. Reaction Pathway of Dapansutrile with nitrogen.

Summary Biological Effects of Dapansutrile[13][9]
Compound ( Reversible or Non-Reversible) Not Known
Primary Mechanism of Action Inhibits:

- ATPase of NLRP3

- NLRP3-ASC

- NLRP3 capase 1 interactions

- NLRP3 assembly

Selectivity Selective to NLRP3 inflammasome
IL-ẞ inhibition %(uM) 60% (0.0001–10 μM)
NLRP3 ATPase inhibition %(uM) 60% (1 μM)
Pryoptosis Inhibition %(uM) 25–40% (0.001–10 μM)
Other Targets Src; Fyn; HcK; STAT3; NF-kB
Cytotoxicity Tc50 ± SE (uM) Non toxic in healthy volunteers (up to 1 gram a day for 8 days in oral form)

Pharmacokinetics

Selectivity

Dapansutrile targets the inhibition of the NLRP3 ATPase and thus blocks the activation of the inflammasome’s ASC, and capase-1 interaction; thereby preventing the assembly of the inflammasome and inflammatory signals such as IL-1ẞ and IL-18.  This drug also inhibits pyroptosis. Nevertheless, the drug does not impact the mRNA levels of the nlrp3, asc, capase 1, il1ẞ and il18 genes.[9]

Research of dapansutrile presents that the compound solely inhibits the NLRP3 inflammasome. Murine macrophages were used and stimulated with lipopolysaccharides and either flagellin or the dsDNA analog Poly(dA:dT) to activate inflammasomes such as NLRC4 and AIM 2 respectively. When Dapansutrile was added they found no difference in the release of TNFα and Il-1ẞ cytokines to when these inflammasomes were activated.[9]

Other Drug Targets

Other known targets of dapansutrile include several phosphorylated kinases such as Src; Fyn; HcK; STAT3.[13] Human monocyte derived macrophages (HMDM) cells were stimulated with lipopolysaccharide, and nigericin. Dapansutrile was then added and they found a 26%, 35%, 43% and 33% reduction of these phosphorylated kinases respectively.[9]

Dosages

In vitro: Human derived macrophages were cultured to study the effect of dapansutrile cytokine production. It was found that dapansutrile at a 1uM dose inhibited secretion of IL-1ẞ by 60%, and IL-18 levels by 70%. The drug was also found to selectively inhibit pyroptosis at 10uM.[9]

In vivo: A phase one clinical trial of 35 subjects was conducted to establish the safety of dapansutrile. The daily mean plasma concentration maximum ( Cmax) for a single oral dose of dapansutrile was 2,700 ng/mL for the 100-mg dose, 9,800 ng/mL for the 300 mg dose and 32,00 ng/mL for the 1,000 mg dose (Figure 5).[9] Dapansutrile was also studied if given repeatedly once per day for 8 days with a dose of either 100 mg, and 300 mg, or 1,000 mg. The subjects mean plasma concentration on day 8 were 4,800 ng/mL for the 100 mg dose, 15,800 ng/mL for the 300 mg dose and 41,400 ng/mL for the 1,000 mg dose.[9]

Figure 5. Plasma Concentration after a Single Oral Dose of OLT1177 versus Time. Plasma concentration for a single dose of OLT1177: 100mg of OLT1177 (red), 300mg (blue), 1,000mg (green). Reposted with permission.[9]
Dapansutrile (OLT1177) Pharmacokinetics (PK) after Single Oral Dose[9]
Mean PK Characteristic (units) 100 mg

N=5

 300 mg

N=5

 1000 mg

N=5

AUC0-t (h*ng/ml) 76457.07 324650.71 918963.5
AUC0-24 (h*ng/ml) 40121.19 157378.19 461809.56
Residual Area % 0.71 0.74 1.12
Cmax (ng/ml) 2,700 9,800 32,000
T1/2 el(h) 23.01 22.8 24.15
Kel (h) 0.0309 0.0317 0.0307
Cl/F (L/h) i.e oral clearance 1.32 0.97 1.11
Vd/F (L) 43.95 31.86 37.63
Dapansutrile (OLT1177) Pharmacokinetics (PK) after Multiple Oral Dose (8 days)[9]
Mean PK Characteristic (units) N 100 mg

N=5

 N 300 mg N 1000 mg
AUC0-τ (h*ng/ml) 5 70288.75 3 260409.24 5 7231127.44
Cmax (ng/ml) 5 4,800 4 15,800 5 41,400
Cmin(ng/ml) 5 1,710 4 6,820 5 18,700
Cave(ng/ml) 5 2,930 3 10,900 5 30,100
T1/2 el(h) 5 16.01 3 21.34 5 24.72
Clss/F(L/h) 5 1.47 3 1.17 5 1.49
Vd/F (L) 5 35.22 3 36.19 5 48.56

Pharmacodynamics

Safety and Efficacy of Dapansutrile

In the Marchetti et al study, seven of the 35 subjects reported adverse events. Five of the cases were in the single dose study while two cases were in the multi-regimen group. However, the adverse effects were considered unrelated to dapansutrile.[9] Subjects presented no changes in blood pressure (systolic and diastolic), urinalysis, heart rate, liver function enzymes or acute phase proteins in the cohort after an 8-day trial with up to daily 1,000 mg dosing of dapansutrile.[9]

Adverse Events[9]
Single Dose Administration of OLT1177 Multidose Administration of OLT1177
Dose Adverse Event Dose Adverse Event
Placebo Headache 100 mg Diarrhea
100 mg Eczema 100 mg Headache
1000 mg Migraine
1000 mg Back pain

Potential Therapeutic Applications

Neurodegenerative diseases

Multiple Sclerosis:

Multiple sclerosis (MS) is a neurodegenerative disease characterized by the immune system deteriorating myelin. Myelin damage leads to the disruption of neuronal signaling, and dysregulrated inflammatory levels.[14] Dapansutrile was used in the Experimental Autoimmune Encephalomyelitis (EAE) mouse model to understand its possible underlying effects for MS. It was found that mice fed with the dapansutrile diet protected the mice from demyelination in the spinal cord as well as decreased their levels of interleukins IL-1ẞ and IL-18.[15] Currently, it is unclear whether the drug could inhibit microglial reactivity, but currently it has no known benefits to aid in the prevention of dementia and cognitive function.[1]

Inflammation

Gouty Arthritis:

Gouty arthritis is an inflammatory joint disorder, partly induced to the activation of the NLRP3 inflammasome and excess IL-1ẞ activation which leads to gout attacks.[16] This is mildly due to the excess uric acid in the blood which them promotes the formation of uric acid crystals which are then found in the joints.Uric acid is a known danger signal and induce the cleavage signal of the capase-1 NLRP3 inflammasome.[17]

Animal Model Experiments: In 2018, two murine models were used to validate Dapansutrile as a drug beneficial for acute joint inflammation. They injected zymosan or monosodium urate crystals in order to induce gouty arthritis in mice. OLT1177 was then either injected intraperitoneally or through their diet. In essence, the drug reduced joint inflammation, as well as interleukin levels[6] demonstrating its therapeutic benefit for this disease.

Clinical Trials: As for clinical trials, Olatec Therapeutics also conducted a phase 2.a trials where Dapansutrile was given to 29 subjects with gouty arthritis.[1]

Cardiovascular Diseases

Acute Myocardial Infraction/ Heart Attack:

One of the major downstream effects of having coronary artery disease is possibility of having an acute myocardial infraction or heart attack.[2] In mouse models, Dapansutrile was found to decrease infarct size a dose-dependent manner.[2]

Heart Failure:

Olatec Therapeutics conducted a phase 1 clinical trial for Dapansutrile as a potential therapeutic for systolic heart failure.[18] They have carried out a Phase 1 double blinded study with a total of 30 subjects to assess the drug's safety and pharmacodynamics. The drug was given in capsule form an the subjects were divided in 3 cohorts. Each cohort had 8 subjects taking an oral capsule with 100 mg Dapansutrile while 2 subjects were given the placebo capsule. Although the completion date of this clinical trial was in November 21, 2019 the results of the study have not yet been published.[18]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) / COVID-19:

Dapansutrile has been proposed by a number of scientists as a measure to reduce cardiovascular outcomes that seem to be brought on by COVID-19.[19] The rationale behind using Dapansutrile is to inhibit the NLRP3 inflammasome and reduce the chances of a cytokine storm which is seems to cause multi-organ failure in COVID-19 patients.[19] Subjects with Covid-19 have shown to have an increased concentration of pro-inflammatory cytokines resulting in the cytokine storm, and thus producing an exhaustion of lymphocytes.[20] NLRP3 not only activates cytokines but other key players that can inflict myocardial damages, and Acute Respiratory Distress Syndrome (ARDS), and its inhibition has been found to deter these outcomes.[19][21] Thus, dapansutrile is proposed as a possible mediator to relief and prevent COVID-19 symptoms and effects.

References
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