PDE4B (IC50 = 7.2 nM)

In human PBMCs, nerandomilast (dihydrate) suppresses the release of IL-2 caused by phytohemagglutinin P (Phytohemagglutinin P) and TNF-α induced by lipopolysaccharides, with IC50 values of 9 nM and 35 nM, respectively [2]. Nerandomilast (dihydrate) has an IC50 value of 91 nM and suppresses TNF-α release in rat whole blood[2].

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Nerandomilast/BI 1015550 Preferentially Inhibits Phosphodiesterase 4B [2]
Nerandomilast/BI 1015550 preferentially inhibited hydrolysis of cAMP by PDE4B with an IC50 of 10 nmol/L, compared with 248 nmol/L for PDE4A, 8,700 nmol/L for PDE4C, and 91 nmol/L for PDE4D (Table 1). Under similar assay conditions, human PDE7A and human PDE3A were only weakly affected, with calculated IC50 values of 14 μmol/L and 120 μmol/L, respectively. Likewise, human PDE1C was only inhibited with high IC50 values (46 μmol/L and 85 μmol/L, respectively, using cAMP and cGMP as substrate), and the IC50 value for human PDE9A (substrate cGMP) was >100 μmol/L. BI 1015550 did not inhibit human PDE2A and PDE5, nor bovine PDE6.

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BI 1015550/Nerandomilast Inhibits Tumor Necrosis Factor-α and Interleukin-2 Release of Purified Human Peripheral Blood Mononuclear Cells [2]
In human PBMCs, BI 1015550 inhibited LPS-induced TNF- α release with an IC50 of 35 nmol/L (Figure 1A), and inhibited phytohemagglutinin P-induced IL-2 release with an IC50 of 9 nmol/L (Figure 1B).

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BI 1015550/Nerandomilast Inhibits Lipopolysaccharide-Induced Tumor Necrosis Factor-α Release in Human and Rat Whole Blood In Vitro, and Stimulates Interleukin-6 in Rat, but Not in Human, Whole Blood [2]
In rat whole blood, BI 1015550 completely inhibited TNF-α release with an IC50 value of 91 nmol/L. In human whole blood, BI 1015550 inhibited TNF-α release up to 70–80% with an IC50 value of 670 nmol/L (Figure 2A). Under identical experimental conditions, high concentrations of BI 1015550 resulted in a fourfold concentration-dependent increase of IL-6 compared with LPS-treated samples without added PDE4 inhibitor in rat whole blood (Figure 2B). In contrast, in human whole blood, high concentrations of BI 1015550 did not result in an IL-6 increase, but rather resulted in a concentration-dependent reduction of IL-6 release with a maximum inhibition of about 30–40% (Figure 2B).

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BI 1015550/Nerandomilast Shows Complementary Activity on Human Myofibroblast Transformation Compared with Nintedanib and a Synergistic Effect in Combination with Nintedanib on Fibroblast Proliferation [2]
BI 1015550 inhibited α-SMA protein expression of TGF-β-stimulated IPF-LF with an IC50 of 210 nmol/L. Combination with nintedanib 10–100 nmol/L did not result in additional inhibitory efficacy (Figure 6A). Nintedanib alone up to the highest concentration of 100 nmol/L had no inhibitory effect on α-SMA protein expression. BI 1015550 attenuated TGF-β-induced Col1, Col3, and FN mRNA expression, with IC50 values of 269, 213, and 246 nmol/L, respectively (Figures 6B–D). The combination with nintedanib at 100 nmol/L showed additive effects in inhibiting Col3 mRNA expression (Figure 6C). BI 1015550 inhibited bFGF plus IL-1β-induced cell proliferation with an IC50 of 255 nmol/L. Nintedanib (100 nmol/L) alone inhibited proliferation by 15%. The combination of BI 1015550 plus 100 nmol/L nintedanib resulted in synergistic inhibitory effects and shifted the concentration–response curve to the left towards an IC50 of 23 nmol/L (Figure 6E). The combination of BI 1015550 with pirfenidone (100 µmol/L) did not yield any additional inhibitory effects in the two described fibroblast in vitro assays used (data not shown).

Nerandomilast (dihydrate) (Example 2) at 1.0 mg/kg reduced intestinal transit in rats but had no discernible effect on body weight (0, 0.3, 1.0 and 3.0 mg/kg; oral; single dosage) [1]. With an ED50 value of 0.1 mg/kg, nerandomilast (dihydrate) can reduce inflammation in rat lung tissue[1]. In a dose-dependent way, lipopolysaccharides-induced TNF-α release in mouse plasma is decreased by nerandomilast (dihydrate) (0.01, 0.1, and 1.0 mg/kg; oral; single dose)[2]. Male Suncus Murinus and Wistar rats' bronchoalveolar lavage fluid is prevented from being invaded by lipopolysaccharide-induced neutrophil entrance by nerandomilast (dihydrate) (0.1, 0.3, and 1.0 mg/kg; oral; single dosage) [2]. Nerandomilast (dihydrate) (oral; twice daily for six days; 2.5 mg/kg and 12.5 mg/kg) significantly enhances bleomycin damage in mice [2].

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BI 1015550/Nerandomilast Inhibits Lipopolysaccharide-Induced Tumor Necrosis Factor-α Release in Whole Blood Ex Vivo in Mice [2]
In an initial experiment, BI 1015550 at 3 mg/kg inhibited TNF-α by 93% (data not shown). In a subsequent experiment, a dose–response relationship was shown when mice were treated with BI 1015550 at doses of 0.01, 0.1, and 1 mg/kg. The dose of BI 1015550 resulting in half-maximal inhibition (ED50) of LPS-induced TNF-α release was determined to be 0.04 mg/kg (Figure 3).

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BI 1015550/Nerandomilast Inhibits Lipopolysaccharide-Induced Neutrophil Influx Into the Bronchoalveolar Lavage Fluid of Male Suncus Murinus and Wistar Rats [2]
To directly compare efficacy and tolerability, the in vivo activity of BI 1015550 was assessed in the BALF of Suncus murinus exposed to nebulized LPS.
Untreated control animals not exposed to LPS exhibited only a very low number of neutrophils in the BALF. Exposure to LPS led to a strong influx of neutrophils into the BALF. Treatment of the animals with Nerandomilast/BI 1015550 at doses of 0.1, 0.3, and 1 mg/kg led to a dose-dependent inhibition of the LPS-induced neutrophil influx into the BALF. The ED50 calculated was 0.6 mg/kg (Figure 4). Roflumilast (0.3, 1, and 3 mg/kg) was used as a reference (ED50 = 1 mg/kg).
BI 1015550/Nerandomilast (doses 0.01, 0.1, and 1 mg/kg) and roflumilast (doses 0.3, 1, and 3 mg/kg) inhibited LPS-induced lung neutrophil influx in male Wistar rats with an ED50 of 0.1 mg/kg and 1 mg/kg, respectively (data not shown).

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BI 1015550/Nerandomilast Has a Low Emetic Potential in Male Suncus Murinus [2]
After administration of Nerandomilast/BI 1015550 at a dose of 0.5 mg/kg (close to the ED50 determined in the neutrophil influx model), 3 of 24 animals tested showed emesis, with 0.1 mean emetic events per animal (data not shown). With 6 mg/kg BI 1015550 (∼10 times the ED50 determined in the neutrophil influx assay), 5 out of 24 (21%) animals showed emesis, with 0.3 mean emetic events per animal (Table 2). With roflumilast at 10 mg/kg (10x ED50), emesis was induced in 10/24 animals (42%, with a mean of 0.7 events per animal). The emetic potential of BI 1015550 was comparable to untreated animals (2 out of 24 tested animals showed emesis, with 0.1 mean emetic events per animal) and animals treated with vehicle (0.5% Natrosol) (3 out of 24 tested animals showed emesis, with 0.1 mean emetic events per animal).

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BI 1015550/Nerandomilast is Active in the Therapeutic Bleomycin Model in Mice [2]
Typically in this model, animals lose body weight approximately 3 days after bleomycin administration, but then gain weight at a normal rate from day 8 onwards. There was no significant effect of Nerandomilast/BI 1015550 treatment on weight gain (data not shown).
Following bleomycin challenge, there was a decrease in FVC. The lower dose of Nerandomilast/BI 1015550 (2.5 mg/kg) induced a small numerical, but non-significant improvement. However, the higher dose of BI 1015550 (12.5 mg/kg b.i.d.) was associated with a statistically significant improvement in FVC of 41% (p < 0.05) (Figure 5A; Table 3).
Following bleomycin challenge, there was an impairment in the pulmonary pressure-volume (PV) loops. The lower dose of Nerandomilast/BI 1015550 induced a small numerical, but non-significant improvement in PV loops. However, again the higher BI 1015550 dose was associated with a statistically significant improvement in PV loops of 40% (p < 0.05) (Figure 5B). The calculated Cstat at a pressure of 30 cm/H2O was similarly improved (Figure 5C; Table 3).
Administration of bleomycin significantly increased lung tissue density assessed by µCT, whereas treatment with BI 1015550 at the higher dose numerically reduced the ratio of dense fibrotic tissue to total lung volume by 39% (Table 3), although this was not statistically significant compared with untreated animals.

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BI 1015550/Nerandomilast is Active in a Therapeutic Murine Model of Progressive Lung Fibrosis Induced by Silica Particles [2]
A single intranasal administration of silica resulted in robust lung inflammation, with a marked increase in total cells, macrophages, neutrophils, and lymphocytes in the BALF, as well as several pro-inflammatory mediators like MPO or KC. Nerandomilast/BI 1015550 was administered orally in a therapeutic regimen (day 10–30) at doses of 0.25, 0.75, and 2.5 mg/kg, and the results are summarized in Table 4. BI 1015550 dose-dependently improved microscopic scores for granuloma formation, fibrosis, and inflammation, although these parameters did not reach statistical significance. In BALF, the highest dose of BI 1015550 (2.5 mg/kg) inhibited macrophages and neutrophils (p < 0.5) among the cells investigated. Other BALF parameters (MPO and KC) were also inhibited substantially at the higher doses (0.75 and 2.5 mg/kg). However, these effects again did not reach statistical significance. Lung weight was reduced by BI 1015550, although this effect was not dose dependent.

Recombinant Phosphodiesterase Activity In Vitro with Scintillation Proximity Assay [2]
The plasmids were transformed into DH10Bac bacteria, SF9 insect cells were transfected with the bacmid DNA, and the resulting baculoviruses were stored and used for further rounds of infections. For protein production, SF9 cells were infected until a cytopathic effect was visible (after about 72 h), the SF9 cells were harvested and broken up by 3 freeze/thaw cycles, and by shearing 10 times through a 0.1 mm cannula using a syringe. The cytoplasmic cell extract was separated by centrifugation (10 min, 14.000 × g, 4°C). The protein content was measured and an equal volume of 87% (v/v) glycerol was added prior to freezing at −20°C. The Phosphodiesterase Scintillation Proximity Assays (TRKQ7090 for [3H]cAMP, and TRKQ7100 for [3H]cGMP) were performed essentially according to the instruction manual. Briefly, serial dilutions of Nerandomilast/BI 1015550 were added to the assays [final dimethyl sulfoxide (DMSO) concentration of 1.1% (v/v)]. Assay solutions containing recombinant baculoviral-expressed PDE (to give an activity of 5.000–20.000 cpm corresponding to 5–20% of total activity added), 50 mmol/L Tris/HCl pH 7.5, 8.3 mmol/L MgCl2, 1.7 mmol/L EGTA, and 10 µL radiolabeled substrate (0.05 µCi in H2O) were incubated for 1 h at 30°C (final volume 100 μL). The reaction was stopped by addition of 50 µL of yttrium silicate scintillation proximity assay beads (17.8 mg/mL, 18 mM zinc sulfate in H2O) and the amount of produced [3H]AMP/[3H]GMP associated was determined using a Wallac Microbeta scintillation counter.

Tumor Necrosis Factor-α and Interleukin-2 Release in Human Peripheral Blood Mononuclear Cells [2]
For preparation of peripheral blood mononuclear cells (PBMCs), 200 mL freshly drawn human blood from healthy donors was mixed with 50 mL acid-citrate-dextrose solution (38 mmol/L citric acid, 75 mmol/L tri-sodium citrate and 121 mmol/L glucose) and 50 mL Hanks buffered saline solution, overlaid on Ficoll and centrifuged for 30 min at 300 x g. PBMCs were diluted in RPMI-1640 medium containing 6% (v/v) autologous plasma to a concentration of 5 × 106 cells/mL. PBMCs were incubated at 37°C, 5% CO2, and 95% humidity in the presence or absence of Nerandomilast/BI 1015550 and stimulated either with 100 ng/mL LPS (serotype 055:B5) for 4 h (TNF-α assay), or with 10 µg/mL phytohemagglutinin P for 20 h (IL-2 assay). Supernatants were taken and cytokines measured by enzyme-linked immunosorbent assay.

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Tumor Necrosis Factor-α and Interleukin-6 Release in Human and Rat Whole Blood [2]
Heparinized whole blood was collected from the Aorta abdominalis of WI (Han) male ratsand from healthy male human donors, and treated with 1 µL (rat) or 2.5 µL (human) of Nerandomilast/BI 1015550 or vehicle [final DMSO concentration 0.5% (v/v)]. Final assay volumes were 200 μL (rat) and 500 μL (human). After incubation for 30 min at 37°C, 95% humidity, and 5% CO2, cultures were treated with LPS [final concentration 10 µg/mL (rat), 0.1 µg/mL (human)] or saline. After 7 h, plates were centrifuged at 3.200 × g at 4°C for 10 min and plasma was used for cytokine measurement. Meso Scale Discovery (MSD) rat and human pro-inflammatory panels were used for detection of TNF-α and IL-6 in rat plasma [diluted 1:2 (v/v)]) and human plasma [negative control undiluted, other samples diluted 1:200 (v/v)] according to the manufacturer’s instructions.

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Human Fibroblast Functions [2]
Fibroblasts from donors with IPF (IPF-LF) were grown in fibroblast basal medium supplemented with FGM-2 SingleQuot Kit Supplements and Growth Factors. Cells were grown in a humidified incubator at 37°C and 5% CO2. All assays were performed at passage 7 or 8. For assay set-up, cells were seeded in fibroblast growth medium plus supplements in assay-relevant densities. For TGF-β-stimulated assays, cells were seeded at 4,500 cells per well in 96-well plates. Proliferation assays were performed in 96-well plates at an initial seeding cell density on day 0 of 2,000 cells per well. After 24-h, the cell culture medium was changed to starvation medium (fibroblast basal medium without supplements). After a 24-h starvation period, cells were pre-incubated for 30 min with different concentrations of Nerandomilast/BI 1015550 plus/minus different concentrations of nintedanib and/or 1 nmol/L PGE2 and stimulated with the assay-relevant stimulus for the indicated time in the presence of the compound.

Animal/Disease Models: Rats[1].
Doses: 0, 0.3, 1.0 and 3.0 mg/kg.
Route of Administration: po (oral gavage); single dose.
Experimental Results: Had minimal toxic and side effects on the intestines and stomach of rats, demonstrating biosafety.

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Animal/Disease Models: Male Suncus Murinus and Wistar rats; mice[2].
Doses: 0.01, 0.1, 0.3, 1.0, 2.5 or 12.5 mg/kg.
Route of Administration: po (oral gavage); single dose or twice (two times) daily for 6 d.
Experimental Results: Effectively improved inflammation in lung tissue and decreased the pro-inflammatory factor TNF-α release. \n

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\n\nLipopolysaccharide-Induced Neutrophil Influx Into the Bronchoalveolar Lavage Fluid of Male Suncus Murinus and Wistar Rats [2]
\nSuncus murinus is phylogenetically closer to primates than rodents and has been shown to be quite sensitive to emesis induction by archetypal PDE4 inhibitors like rolipram and denbufylline (Sawanishi et al., 1997). Compared with other animal species used to study emesis induction by PDE4 inhibitors, like ferrets, dogs, minipigs, or monkeys, Suncus murinus allows the testing of a sufficient number of animals to provide a high statistical power. Nerandomilast/BI 1015550 or roflumilast was suspended in 0.5% Natrosol (hydroxyethylcellulose). Male Suncus murinus (weight 50–55 g) or male Wistar rats (200–250 g), eight animals per group, were used. Animals were pre-treated with oral Nerandomilast/BI 1015550 (doses of 0.1, 0.3, and 1.0 mg/kg) or roflumilast (doses 0.3, 1, and 3 mg/kg) 30 min before LPS exposure. For this purpose, animals in the positive control and treatment groups were put into a circular Plexiglas chamber with 16 pie-like compartments for one animal each, and were consecutively exposed to nebulized LPS for 30 min. The LPS solution used for nebulization contained 1 mg/mL LPS in phosphate-buffered saline (PBS). Nebulization was performed with the commercially available PARI Master® nebulizer with a PARI Master® LL adapter. 4 h after LPS exposure, the animals were anesthetized and euthanized by cervical dislocation. After sacrifice, the trachea was cannulated and bronchoalveolar lavage was performed by instilling and re-aspirating two-times 1 mL or 5 mL (rat) lavage buffer (PBS +2% bovine serum albumin). BALF volumes were recorded manually. Determination of neutrophil numbers in BALF was performed using a blood hemacytometer.\n

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\n\nBleomycin Mouse Model [2]
\nAdult, test-naïve, male C57BL6/6J mice, aged 10–12 weeks from Charles River were used (weight 25–27 g). The model was performed essentially as described by Ackermann et al. (2017). Briefly, bleomycin 1 mg/kg was administered intratracheally in an application volume of 2 mL/kg body weight. Animals were weighed daily. A body weight loss of 20% or more automatically resulted in euthanization. Nerandomilast/BI 1015550 (at final doses of 2.5 and 12.5 mg/kg) was applied by oral gavage using a dose volume of 10 mL/kg twice daily (b.i.d.) from day 8 until day 13. On day 12, animals were anesthetized using 3–4% isoflurane, and lung density was assessed by micro-computed tomography (µCT) analysis with a Quantum FX µCT system with cardiac gating (without respiratory gating). Images were analyzed using MicroView 2.0 software. The Hounsfield unit (HU) corresponding to the peak of the HU-histogram for the segmented pixels was used as a measure of fibrosis. After µCT analysis, animals were allowed to awaken from anesthesia. On day 14, lung function [pressure-volume loops, FVC, and static lung compliance (Cstat)] was measured with a FlexiVent system. After the lung function measurement, animals were sacrificed by an overdose of Narcorene and lungs were lavaged with two-times 0.8 mL of PBS. BALF was used to determine differential cell counts and the number of monocytes. Left lung lobes were fixed with 4% paraformaldehyde and inflated under 20 cm water pressure for 20 min. Samples were embedded in paraffin, sectioned (3 μm thickness), and stained for hematoxylin and eosin (HE) and Masson’s trichrome to assess general morphology and fibrotic change following standard histopathology operating protocols. Images were taken with an AxioCam MRm microscope camera using AxioVision software. Lung sections stained with Masson’s trichrome were assessed for severity of pulmonary fibrosis using the Ashcroft score. The protocol used is therapeutic, i.e. treatment commenced at a time where lung fibrosis should have developed, in line with the recommendations of the American Thoracic Society panel on IPF models (Jenkins et al., 2017).\n

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\n\nSilica-Induced Lung Inflammation and Fibrosis in the Mouse [2]
\nBI 1015550/Nerandomilast was given at doses of 0.25, 0.75, and 2.5 mg/kg b.i.d. in Natrosol 0.5% (10 mL/kg) starting from day 10 after silica instillation until day 30 (therapeutic regimen). 8-week-old BL6 mice were used. Details of the experimental methods are described elsewhere (Lo Re et al., 2010). Briefly, mice received silica particles at 2.5 mg/mouse by intranasal instillation. Control mice received the respective saline solution by intranasal instillation. Mice were killed 30 days after silica administration and macroscopic changes were recorded at post-mortem analysis. Lung weight was measured, BALF for total cell and differential cell counts as well as myeloperoxidase (MPO) activity and keratinocyte-derived cytokine (KC, the counterpart of human GRO protein) determination was generated, and lung histology (staining with HE and chromotrope-aniline blue with semi-quantitative analysis for the estimation of inflammation, granuloma, and fibrosis scores was done.\n

The efficacy and potency of BI 1015550 in disease models will depend on other pathophysiological aspects, as well as the pharmacokinetics and availability of the compound over 24 hours, as plasma concentrations over 24 hours will be lower than maximum plasma concentrations due to the compound's short half-life. [2]

The most common side effects of Jascayd (≥5%) included diarrhea, COVID-19, upper respiratory tract infection, depression, weight loss, decreased appetite, nausea, fatigue, headache, vomiting, back pain, and dizziness. Diarrhea was the most common adverse reaction leading to treatment discontinuation, occurring most frequently in patients receiving 18 mg nalandoxalate (4%) or 9 mg nalandoxalate (3%) in combination with antifibrotic therapy, and less frequently (1%) in patients receiving placebo in combination with antifibrotic therapy. Notably, 1% of patients receiving 18 mg nalandoxalate who were not concurrently receiving nintedanib discontinued treatment due to diarrhea, while no such discontinuation occurred in patients receiving 9 mg nalandoxalate or placebo.

[1]. Piperidino-dihydrothienopyrimidine sulfoxides and their use for treating COPD and asthma. United States. US9150586.

[2]. BI 1015550 is a PDE4B Inhibitor and a Clinical Drug Candidate for the Oral Treatment of Idiopathic Pulmonary Fibrosis. Front Pharmacol. 2022 Apr 20:13:838449.

The anti-inflammatory and immunomodulatory effects of orally administered selective phosphodiesterase 4 (PDE4) inhibitors have led to their approval for the treatment of chronic obstructive pulmonary disease (COPD) and psoriasis/psoriatic arthritis, respectively. However, the antifibrotic potential of PDE4 inhibitors has not yet been explored clinically. Nerandomilast/BI 1015550 is a novel PDE4 inhibitor with preferential enzyme inhibition of PDE4B. In vitro studies have shown that BI 1015550 inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α) and phytohemagglutinin-induced interleukin-2 synthesis in human peripheral blood mononuclear cells, as well as LPS-induced TNF-α synthesis in human and mouse whole blood. In vivo studies have shown that orally administered BI 1015550 exhibits potent anti-inflammatory activity by inhibiting LPS-induced in vitro TNF-α synthesis in mice and by inhibiting the influx of neutrophils stimulated by nebulized LPS into the bronchoalveolar lavage fluid of rays. In stingrays, PDE4 inhibitors cause vomiting, a common gastrointestinal side effect limiting the human application of PDE4 inhibitors, while the therapeutic index of BI 1015550 appears to be significantly higher than that of roflumilast. Furthermore, oral BI 1015550 also demonstrated efficacy under treatment conditions in two commonly used mouse models of pulmonary fibrosis (induced by bleomycin or silica, respectively), with its mechanism of action being the modulation of multiple model-specific parameters. To better understand the in vivo antifibrotic potential of BI 1015550, we investigated its direct effects on human fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) in vitro. BI 1015550 inhibited transforming growth factor-β-stimulated myofibroblast transformation and mRNA expression of various extracellular matrix proteins, as well as basic fibroblast growth factor and interleukin-1β-induced cell proliferation. In these experiments, nintedanib performed poorly overall, but interestingly, when used in combination with BI 1015550, it showed a synergistic effect on cell proliferation inhibition, resulting in a shift of about 10-fold to the left of the concentration-response curve. In summary, the unique preferential inhibition of PDE4B by BI 1015550 and its expected good tolerability in humans, as well as its anti-inflammatory and anti-fibrotic potential, suggest that BI 1015550 is a promising oral clinical candidate for the treatment of IPF and other fibroproliferative disorders. [2] This article describes the preclinical pharmacological properties of a novel oral PDE4 inhibitor, Nerandomilast/BI 1015550, which preferentially inhibits PDE4B. Within the vast PDE isoenzyme superfamily, Nerandomilast/BI 1015550 selectively inhibits PDE4 and has, to date, shown no other off-target effects in screening against a range of enzymes and receptors (Cerep high-throughput screening and Cerep non-kinase screening, Euriofins Cerep, Celle L'Escevault, France). Compared to roflumilast, currently the only commercially available oral PDE4 inhibitor for treating lung diseases, BI 1015550 exhibits unique PDE4 subtype inhibitory properties by preferentially inhibiting PDE4B (Table 1). Early PDE4 inhibitors, including roflumilast and apremilast, were limited by gastrointestinal side effects such as nausea, vomiting, and/or diarrhea in humans. Although not yet confirmed, there is evidence that these side effects are related to the inhibition of the PDE4D subtype (Giembycz, 2002). In this regard, the approximately 10-fold higher selectivity of BI 1015550 for PDE4B compared to PDE4D may have clinical significance. This may also explain why, in stingrays (Suncus murinus), BI 1015550 showed a better treatment ratio (the ratio of LPS-induced neutrophil infiltration in the lungs to induced vomiting) than roflumilast (Figure 4; Table 2). More importantly, ongoing Phase I clinical trials of BI 1015550 support good gastrointestinal tolerability and safety in humans (data not shown). Another aspect of the human application of BI 1015550 relates to the toxicity observed in animal studies. The main toxicity of PDE4 inhibitors, particularly in rats, is the induction of vascular lesions (vasculitis, arteritis) in various tissues, which is somewhat contradictory given the strong association between PDE4 inhibition and anti-inflammatory efficacy (see below). Although the two oral PDE4 inhibitors, roflumilast and apremilast, have been marketed for many years, no patients have reported inducing vasculitis, but this potentially serious side effect remains a concern with the introduction of novel PDE4 inhibitors. (Dietsch et al.) Dietsch et al. (2006) first reported that the PDE4 inhibitor IC542 could stimulate IL-6 expression in rats. Since IL-6 is a potential candidate factor for inducing vasculitis in rats, and BI 1015550 did not show any stimulatory effect on IL-6 in humans (Figure 2B), this may suggest that the risk of BI 1015550 inducing vasculitis in humans is likely low compared to rats. Furthermore, it is noteworthy that apremilast has been reported to have clinical activity against Behcet's disease (a type of vasculitis characterized by vascular inflammation), alleviating one of its most common symptoms—oral ulcers (Hatemi et al., 2015). Despite a reduced inhibitory effect on the PDE4D subtype, the well-known anti-inflammatory and immunomodulatory properties of PDE4 inhibitors remain evident in Nerandomilast/BI 1015550. Similar in nature to roflumilast (Hatzelmann and Schudt, 2001), BI 1015550 appears to be a potent inhibitor of TNF-α and IL-2 in human peripheral blood mononuclear cells (PBMCs) (Figure 1), which likely reflects the effects of BI 1015550 on monocytes and T lymphocytes, respectively. Effective inhibition of TNF-α was also observed in whole blood (Figure 2), which is significant because this assay (in vitro) can serve as a biomarker of pharmacodynamics in clinical studies, as first demonstrated by Timmer et al. with roflumilast (Timmer et al., 2002). Although the IC50 value of BI 1015550 against TNF-α in human whole blood (670 nmol/L) was higher than that in human peripheral blood mononuclear cells (PBMCs) (35 nmol/L), which may be partly attributed to the compound's moderate plasma protein binding (77%), its potency loss was still greater than expected. To simulate clinical conditions, this study demonstrated in a mouse model that the compound exhibits potent inhibitory activity against LPS-stimulated TNF-α in isolated whole blood (Figure 3). Clearly, in this mouse model, the ED50 value of orally administered BI 1015550 (0.04 mg/kg) was significantly lower than that in the mouse pulmonary fibrosis model discussed below (bleomycin, silica). However, in the aforementioned in vitro models, blood samples were collected near the peak plasma concentration of the compound. While TNF-α, as a pharmacological marker, may be associated with pulmonary fibrosis pathology, it is not the only relevant mediator. The efficacy and potency of BI 1015550 in the disease model will depend on other pathophysiological factors, as well as the compound's pharmacokinetics and bioavailability over 24 hours. Due to the compound's short half-life, its 24-hour plasma concentration will be lower than the maximum plasma concentration. The abnormal wound healing process in pulmonary fibrosis involves an inflammatory phase, in which inflammatory cells (especially macrophages, monocytes, neutrophils, and T lymphocytes) are involved, and the levels of cytokines (e.g., TNF-α and IL-1β) and growth factors (e.g., TGF-β and CTGF) are elevated, creating a biochemical environment conducive to chronic tissue remodeling and fibrosis. Based on the working hypothesis of pulmonary fibrosis pathophysiology, the anti-inflammatory (inhibition of TNF-α, inhibition of monocytes, inhibition of neutrophil infiltration in the lungs) and immunomodulatory (inhibition of IL-2, inhibition of T cells) properties of BI 1015550/Nerandomilast hold promise for improving fibrosis to some extent. Therefore, we are interested in whether BI 1015550 exerts anti-fibrotic effects in different animal models and whether this compound has direct effects on fibroblasts and myofibroblasts. Bleomycin has been widely used in rodent models of pulmonary fibrosis to understand the mechanisms of fibrosis and evaluate potential new therapies. However, it is noteworthy that while bleomycin-induced pulmonary fibrosis mimics many features of the human disease, fibrosis in rodents can regress, whereas in humans it is generally irreversible. Furthermore, human pulmonary fibrosis primarily affects the peripheral airways, while rodent fibrosis is more commonly found around the central airways. Despite these limitations, bleomycin models remain a good tool for evaluating the efficacy of potential compounds and for validating their mechanisms of action (Moeller et al., 2008; Jenkins et al., 2017). The Nerandomilast/BI 1015550 treatment regimen alleviated some important aspects of the disease pathology, including a decrease in FVC and Cstat (Figure 5). These therapeutic effects were statistically significant at the highest BI 1015550 dose. Additionally, lung tissue density was also improved to a similar degree, although this improvement did not reach statistical significance (Table 3). Regarding the Ashcroft score, BI 1015550 showed only a non-significant trend toward inhibition. For experimental parameters in other studies (bronchial lavage fluid proteins and monocytes, airway compliance), no effect of BI 1015550 was detected. In this study, BI 1015550 had no effect on inflammatory mediators in bronchoalveolar lavage fluid and had minimal effect on histologically assessed pulmonary fibrosis, contrary to the results reported in other bleomycin mouse studies (Cortijo et al., 2009; Udalov et al., 2010). We can only speculate that the fibrotic changes observed in the bleomycin mouse model established under our experimental conditions were not captured by the scoring system originally designed to assess human pulmonary fibrosis. Changes in tissue volume may reflect not only fibrotic alterations but may also be related to other aspects of remodeling in the model (e.g., angiogenesis), as shown by Ackermann et al. (Ackermann et al., 2017). Differences from previous studies may also stem from variations in treatment duration and data collection time. Regardless of the nature of these changes, the improvement in functional parameters such as lung function suggests that BI 1015550 has a positive effect on lung tissue remodeling. This hypothesis is supported by the positive results of BI 1015550 in a mouse silica-induced pulmonary fibrosis model. Unlike bleomycin, which induces reversible pulmonary fibrosis, silica particles induce a progressive pulmonary fibrosis, similar to progressive fibrotic interstitial lung disease (ILD) in humans. To our knowledge, PDE4 inhibitors that preferentially inhibit PDE4B have not been evaluated in this animal model before. As shown in Table 4, BI 1015550 achieved dose-dependent improvements in the semi-quantitative scores of granuloma formation, inflammation, and fibrosis in the treatment regimen, although these effects did not reach statistical significance. In bronchoalveolar lavage fluid (BALF), macrophages and neutrophils were significantly suppressed in the highest dose group, while the inhibition of myeloperoxidase (MPO) and Kupffer cells (KC) did not reach statistical significance. Overall, the beneficial effects of Nerandomilast/BI 1015550 in two pulmonary fibrosis models are consistent with preclinical data in the literature, suggesting that selective PDE4 inhibitors may not only have clinical efficacy in pulmonary fibrosis but, based on animal data, may also be effective for fibrosis in other organs (e.g., liver, kidney, colon, and/or skin). Multiple studies support this hypothesis, demonstrating that selective PDE4 inhibitors can directly target fibroblasts in addition to inflammatory and immune-active cells. Using relevant cells, namely human lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF), we demonstrated that BI 1015550 can inhibit the conversion of TGF-β-stimulated fibroblasts into myofibroblasts (Figure 6A). This indicates that the mechanism of action of BI 1015550 differs from that of nintedanib, which is ineffective under these conditions and does not enhance the efficacy of BI 1015550. Furthermore, BI 1015550 attenuated TGF-β-induced expression of Col1, Col3, and FN genes (Figures 6B-D), molecules that are important components of the extracellular matrix in fibrotic cells. The inhibitory effect on Col3 expression was additive when BI 1015550 was used in combination with 100 nmol/L nintedanib. In addition, BI 1015550 also inhibited bFGF and IL-1β-induced cell proliferation. Nintedanib alone, at the relevant concentration (100 nmol/L), only inhibited 15% of cell proliferation. Most significantly, however, the combination of BI 1015550 and nintedanib produced a synergistic inhibitory effect, shifting the concentration-response curve approximately 10-fold to the left (Figure 6E). In vitro inhibition of human lung fibroblast proliferation and myofibroblast transformation may help explain the potential clinical efficacy of BI 1015550 in idiopathic pulmonary fibrosis (IPF). In addition, the combination of BI 1015550 and nintedanib may have a synergistic therapeutic effect on patients with IPF and other fibrotic lung diseases. The synergistic effect between this PDE4 inhibitor that preferentially inhibits PDE4B and nintedanib has not been reported before. In summary, BI 1015550/Nerandomilast is an oral PDE4B inhibitor that preferentially inhibits PDE4B and appears to be better tolerated in humans compared to commercially available selective oral PDE4 inhibitors; preclinical results suggest that this compound is a promising oral candidate for the treatment of idiopathic pulmonary fibrosis (IPF) and other interstitial lung diseases (ILD). A phase II clinical trial (NCT04419506) of BI 1015550 versus placebo in IPF patients is currently underway. Future clinical studies will reveal whether BI 1015550 can exert significant efficacy on its own, or whether it needs to be used in combination with standard IPF therapies such as nintedanib to fully realize the therapeutic potential of BI 1015550. [2]

C20H29CLN6O4S

485.00

484.16595

Nerandomilast;1423719-30-5

170902251

Typically exists as solid at room temperature

4

11

5

32

624

0

C1CC(C1)(CO)NC2=NC(=NC3=C2S(=O)CC3)N4CCC(CC4)C5=NC=C(C=N5)Cl.O.O

HNNFNSBDBHISSY-UHFFFAOYSA-N

InChI=1S/C20H25ClN6O2S.2H2O/c21-14-10-22-17(23-11-14)13-2-7-27(8-3-13)19-24-15-4-9-30(29)16(15)18(25-19)26-20(12-28)5-1-6-20;;/h10-11,13,28H,1-9,12H2,(H,24,25,26);2*1H2

[1-[[2-[4-(5-chloropyrimidin-2-yl)piperidin-1-yl]-5-oxo-6,7-dihydrothieno[3,2-d]pyrimidin-4-yl]amino]cyclobutyl]methanol;dihydrate

Nerandomilast dihydrate; BI1015550; BI-1015550

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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