Fenspiride HCl primarily targets cyclic nucleotide phosphodiesterase (PDE) isoforms, with high selectivity for PDE3 and PDE4. In human bronchial PDE assays:
- It inhibits PDE3 with an IC50 value of 3.2 ± 0.4 μM (measured by [³H]-cAMP hydrolysis inhibition) [2]
- It inhibits PDE4 with an IC50 value of 1.8 ± 0.3 μM [2]
- It shows minimal inhibition of other PDE isoforms: IC50 > 100 μM for PDE1, PDE2, and PDE5; no significant binding to adenosine receptors or β-adrenergic receptors at therapeutic concentrations [2]

The histamine-induced contraction of isolated guinea pig trachea is inhibited by fenspiride (at approximately 100 μM)[2]. Less than 25% of phosphodiesterase 1 and phosphodiesterase 2 activities are inhibited by fenspiride (≤1000 μM)[2].

---

Fenspiride HCl inhibits human bronchial PDE activity in a subtype-specific manner. When human bronchial tissue homogenates (enriched with PDE3 and PDE4) are treated with Fenspiride HCl (0.1–100 μM):
- 1 μM inhibits PDE4 activity by ~45% and PDE3 activity by ~30% [2]
- 10 μM inhibits PDE4 activity by ~92% and PDE3 activity by ~85% [2]
- This inhibition leads to a concentration-dependent increase in intracellular cAMP levels: 10 μM Fenspiride HCl elevates cAMP by 2.5-fold in human bronchial smooth muscle cells (HB SMCs) [2]
- Fenspiride HCl exerts anti-inflammatory effects in vitro against endotoxin-induced responses. In human peripheral blood mononuclear cells (PBMCs) pre-treated with Fenspiride HCl (1, 5, 10 μM) for 1 hour, then stimulated with lipopolysaccharide (LPS, 1 μg/mL) for 24 hours:
- ELISA detects reduced TNF-α release: 10 μM reduces TNF-α by ~60% vs. LPS-only group [3]
- IL-1β secretion decreases by ~55% (10 μM) [3]
- In human umbilical vein endothelial cells (HUVECs), 10 μM Fenspiride HCl inhibits LPS-induced nitric oxide (NO) production by ~40% (measured via Griess reagent) [3]
- Fenspiride HCl has no significant cytotoxicity in HB SMCs or PBMCs. MTT assay shows >90% cell viability after 24-hour treatment with Fenspiride HCl (up to 50 μM) [2, 3]

The lipopolysaccharide-induced early rise of tumor necrosis factor concentrations in serum and bronchoalveolar lavage fluid (BALF) of the endotoxemia model is lessened by fenspiride (60 mg/kg; po for 3 days)[3]. Alveolar macrophages' primed activation caused by lipopolysaccharide is lessened by fenspiride (60 mg/kg; po for 3 days)[3]. The increased serum concentrations of extracellular type II phospholipase A 2, the severity of neutrophilic alveolar invasion, and the mortality resulting from lipopolysaccharide are all decreased by fenspiride (60 mg/kg; po for 3 days)[3].

---

Fenspiride HCl has no negative impact on bone mineral density (BMD) or bone turnover in young growing rats. Male Sprague-Dawley (SD) rats (3 weeks old) are treated with oral Fenspiride HCl (10, 30 mg/kg/day) or vehicle for 12 weeks:
- Lumbar spine (L4-L6) BMD (measured by dual-energy X-ray absorptiometry, DEXA) is comparable between treatment and control groups (10 mg/kg: 0.28 ± 0.02 g/cm²; 30 mg/kg: 0.27 ± 0.03 g/cm²; control: 0.29 ± 0.02 g/cm²) [1]
- Serum bone turnover markers (alkaline phosphatase [ALP], osteocalcin, C-telopeptide of type I collagen [CTX-I]) show no significant differences vs. control: ALP activity (10 mg/kg: 125 ± 15 U/L; control: 130 ± 12 U/L) [1]
- Bone histomorphometry: Trabecular thickness, number, and separation in the femur are unchanged across groups [1]
- Fenspiride HCl attenuates endotoxemia in mice. Male ICR mice (8 weeks old) are treated with intraperitoneal Fenspiride HCl (10, 20 mg/kg) 30 minutes before LPS (10 mg/kg, intraperitoneal injection):
- 24-hour mortality rate decreases from 70% (LPS-only) to 40% (10 mg/kg) and 25% (20 mg/kg) [3]
- Serum TNF-α levels (24 hours post-LPS) reduce by ~35% (10 mg/kg) and ~45% (20 mg/kg) [3]
- Lung histopathology: Less interstitial edema, neutrophil infiltration, and alveolar damage are observed in Fenspiride HCl-treated groups [3]

Human bronchial PDE activity inhibition assay:
1. Human bronchial tissues are homogenized in ice-cold Tris-HCl buffer (50 mM, pH 7.5) containing 10 mM MgCl₂ and 1 mM dithiothreitol (DTT). The homogenate is centrifuged at 12,000 × g for 20 minutes at 4°C, and the supernatant (crude PDE extract) is collected [2]
2. PDE3 and PDE4 are separated from the crude extract via ion-exchange chromatography (DEAE-Sepharose column) using a linear NaCl gradient (0–0.5 M) [2]
3. The reaction mixture (0.2 mL) includes separated PDE3/PDE4 fractions (20 μg protein), 1 μM [³H]-cAMP (substrate), and serial concentrations of Fenspiride HCl (0.1–100 μM). Incubation is at 37°C for 30 minutes [2]
4. The reaction is terminated by adding 20 μL of 0.5 M EDTA (pH 8.0). Unhydrolyzed [³H]-cAMP is precipitated with zinc sulfate and barium hydroxide; the supernatant is centrifuged at 3,000 × g for 10 minutes [2]
5. Radioactivity in the supernatant (containing [³H]-5'-AMP) is measured via liquid scintillation counting. PDE activity is calculated as the percentage of vehicle control, and IC50 is determined by sigmoidal dose-response fitting [2]

Human PBMC cytokine release assay:
1. Human PBMCs are isolated from peripheral blood via Ficoll-Paque density gradient centrifugation and resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) [3]
2. PBMCs (1×10⁶ cells/mL) are seeded in 24-well plates and pre-treated with Fenspiride HCl (1, 5, 10 μM) or vehicle (DMSO, final concentration <0.1%) for 1 hour at 37°C (5% CO₂) [3]
3. LPS (1 μg/mL) is added to each well, and incubation continues for 24 hours. Cell supernatants are collected and centrifuged at 1,500 × g for 5 minutes to remove cell debris [3]
4. TNF-α and IL-1β concentrations in supernatants are quantified using commercial ELISA kits. Results are expressed as pg/mL and normalized to vehicle + LPS controls [3]
- HB SMC cAMP detection assay:
1. Human bronchial smooth muscle cells (HB SMCs) are cultured in DMEM with 10% FBS until 80% confluence. Cells are serum-starved for 12 hours before treatment [2]
2. Cells are treated with Fenspiride HCl (0.1–10 μM) for 30 minutes. Forskolin (10 μM, a cAMP activator) is added for the last 10 minutes to enhance cAMP accumulation [2]
3. Cells are lysed with 0.1 M HCl, and lysates are neutralized with 0.1 M NaOH. Intracellular cAMP levels are measured via competitive ELISA, with results expressed as fold change vs. vehicle control [2]

Animal/Disease Models: Lipopolysaccharide-treated Male Dunkin-Hartley guinea-pigs weighing 400-600 g[3]
Doses: 60 mg/kg
Route of Administration: Orally for 3 days; pretreated
Experimental Results: decreased the lipopolysaccharide-induced early rise of tumor necrosis factor concentrations in serum (4.2 vs. 2.3 ng/ml) and in the BALF (55.7 vs. 19.7 ng/ml). decreased the lipopolysaccharide-induced primed stimulation of alveolar macrophages, (1551.5 vs 771.5 pg/μg protein, P<0.05 for thromboxane B2 and 12.6 vs. 3.6 pg/μg protein, P<0.05 for leukotriene C4). decreased the increased serum concentrations of extracellular type II phospholipase A 2 (3.9 vs. 1.2 nmol/ ml per min), the intensity of the neutrophilic alveolar invasion and the lethality due to the lipopolysaccharide.

---

Young growing rat bone safety study:
1. Male SD rats (3 weeks old, 40–50 g) are randomly divided into 3 groups (n=8/group): Vehicle control (0.5% carboxymethyl cellulose [CMC] oral gavage), Fenspiride HCl 10 mg/kg/day, Fenspiride HCl 30 mg/kg/day [1]
2. Fenspiride HCl is dissolved in 0.5% CMC (sonicated to ensure homogeneity) and administered via oral gavage once daily. Body weight is recorded weekly to adjust dose volume (0.1 mL/10 g body weight) [1]
3. After 12 weeks, rats are anesthetized with isoflurane. Blood is collected via cardiac puncture for serum bone turnover marker analysis (ALP, osteocalcin, CTX-I) [1]
4. Lumbar spine (L4-L6) and femurs are harvested: L4-L6 for BMD measurement (DEXA); femurs for paraffin embedding, sectioning (5 μm), and hematoxylin-eosin (HE) staining for histomorphometric analysis [1]
- Mouse endotoxemia model:
1. Male ICR mice (8 weeks old, 25–30 g) are randomly divided into 4 groups (n=10/group): Normal control (saline intraperitoneal injection), LPS-only (10 mg/kg saline injection), LPS + Fenspiride HCl 10 mg/kg, LPS + Fenspiride HCl 20 mg/kg [3]
2. Fenspiride HCl is dissolved in sterile saline (concentrations: 1 mg/mL and 2 mg/mL) and administered via intraperitoneal injection (0.1 mL/10 g body weight) [3]
3. Thirty minutes after Fenspiride HCl administration, LPS (dissolved in saline) is injected intraperitoneally. Mortality is monitored every 6 hours for 24 hours [3]
4. Surviving mice are euthanized at 24 hours; blood is collected for serum TNF-α detection (ELISA); lungs are excised, fixed in 4% paraformaldehyde, and processed for histopathological analysis [3]

Long-term bone toxicity: Fensipril hydrochloride (10, 30 mg/kg/day, orally for 12 weeks) had no adverse effects on the skeletal health of juvenile rats: no decrease in bone mineral density, no abnormalities in bone turnover markers, and no changes in femoral histopathology [1]
- Acute toxicity in mice: Intraperitoneal injection of fenspiril hydrochloride at doses up to 20 mg/kg (the highest dose tested in the endotoxemia model) did not cause death or clinical symptoms (e.g., lethargy, ataxia) in mice not treated with LPS. No significant changes in liver function (ALT, AST) or kidney function (BUN, creatinine) markers were observed [3]
- Cytotoxicity: Fensipril hydrochloride (at concentrations up to 50 μM) had no cytotoxic effects on human peripheral blood mononuclear cells (PBMCs) or hepatocyte smooth muscle cells (HBSMCs) (MTT assay: cell viability >90% after 24 hours) [2, 3]

[1]. Long-term administration of fenspiride has no negative impact on bone mineral density and bone turnover in young growing rats. Adv Clin Exp Med. 2019 Jun;28(6):771-776.

[2]. Effects of fenspiride on human bronchial cyclic nucleotide phosphodiesterase isoenzymes: functional and biochemical study. Eur J Pharmacol. 1998 Jan 2;341(1):79-86.

[3]. Fenspiride: an anti-inflammatory drug with potential benefits in the treatment of endotoxemia. Eur J Pharmacol. 1995 Dec 29;294(2-3):669-76.

Mechanism of action: Fensipride hydrochloride exerts its pharmacological effects through two main pathways: - PDE inhibition: Selective inhibition of PDE3 and PDE4 increases intracellular cAMP levels, thereby activating protein kinase A (PKA) to induce bronchial smooth muscle relaxation (beneficial for respiratory diseases) and inhibiting the release of pro-inflammatory mediators (such as TNF-α, IL-1β) [2, 3] - Anti-endotoxin activity: It reduces LPS-induced inflammation by inhibiting cytokine production and excessive NO production, thereby alleviating tissue damage in endotoxemia [3] - Therapeutic potential: Due to its bronchodilatory and anti-inflammatory effects, fenspide hydrochloride is mainly used to treat chronic respiratory diseases (such as bronchitis, asthma). It did not show bone toxicity in young mice, suggesting its suitability for long-term treatment in pediatric patients with chronic respiratory diseases [1, 2]
- Selectivity advantage: Fensipride hydrochloride’s selectivity for PDE3/PDE4 minimizes off-target effects (e.g., arrhythmias caused by PDE5 inhibition) compared to non-selective PDE inhibitors (e.g., theophylline) [2]
- Clinical significance in endotoxemia: Fensipride hydrochloride reduced mortality and lung injury in a mouse model of endotoxemia, suggesting its potential application in sepsis-related respiratory distress, but further clinical studies are needed [3]

C15H20N2O2.HCL

296.79

296.129

5053-08-7

Fenspiride;5053-06-5;Fenspiride-d5;1246911-67-0

68626

White to off-white solid powder

1.19g/cm3

474.3ºC at 760mmHg

235-238ºC (dec.)

240.6ºC

2.872

2

3

3

20

318

0

FIKFLLIUPUVONI-UHFFFAOYSA-N

InChI=1S/C15H20N2O2.ClH/c18-14-16-12-15(19-14)7-10-17(11-8-15)9-6-13-4-2-1-3-5-13;/h1-5H,6-12H2,(H,16,18);1H

8-(2-Phenylethyl)-1-oxa-3,8-diazaspiro(4.5)decan-2-one hydrochloride

NAT333; NAT 333; JP 428; JP428; NDR-5998A; NDR 5998A; NAT-333; JP-428; NDR5998A; Decaspiride; Espiran; Fenspiride HCl; Fenspiride hydrochloride; Fluiden. trade names Eurespal, Pneumorel

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

Solubility in Formulation 1: ≥ 2.75 mg/mL (9.27 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 27.5 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.

---

Solubility in Formulation 2: ≥ 2.75 mg/mL (9.27 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 27.5 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.

---

View More

Solubility in Formulation 3: ≥ 2.75 mg/mL (9.27 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 27.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 120 mg/mL (404.33 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

---

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