PDE4B (IC50 = 7.2 nM)

Nerandomilast, with IC50 values of 35 nM, suppresses the release of IL-2 caused by phytohemagglutinin P and TNF-α induced by lipopolysaccharides in human peripheral blood monolayer cells. and 9 nM [2]. With an IC50 value of 91 nM, nerandomilast suppresses TNF-α release in rat whole blood[2].

---

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.

---

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).

---

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).

---

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).

In rats, nerandomilast (Example 2) at 1.0 mg/kg reduced intestinal transit without significantly altering body weight (0, 0.3, 1.0, and 3.0 mg/kg; oral; single dose)[1]. Nerandomilast has an ED50 value of 0.1 mg/kg and can reduce inflammation in rat lung tissue[1]. In a dose-dependent way, nerandomilast (0.01, 0.1, and 1.0 mg/kg; oral; single dose) decreases TNF-α release in mouse plasma produced by lipopolysaccharides [2]. Male Suncus Murinus and Wistar rats' bronchoalveolar lavage fluid is not affected by lipopolysaccharide-induced neutrophil entrance when nerandomilast (0.1, 0.3, and 1.0 mg/kg; oral; single dosage) is administered[2]. Nerandomilast (orally administered twice daily for six days at doses of 2.5 mg/kg and 12.5 mg/kg) significantly reduces bleomycin-induced injury in mice[2].

---

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).

---

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).

---

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).

---

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.

---

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.

---

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.

---

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: Oral gavage; single dose.
Experimental Results: Had minimal toxic and side effects on the intestines and stomach of rats, demonstrating biosafety.

---

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: Oral gavage; single dose or twice (two times) daily for 6 days.
Experimental Results: Effectively improved inflammation in lung tissue and decreased the pro-inflammatory factor TNF-α release.

---

Lipopolysaccharide-Induced Neutrophil Influx Into the Bronchoalveolar Lavage Fluid of Male Suncus Murinus and Wistar Rats [2]
Suncus 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.

---

Bleomycin Mouse Model [2]
Adult, 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).

---

Silica-Induced Lung Inflammation and Fibrosis in the Mouse [2]
BI 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.

The efficacy and potency of BI 1015550 in the disease models will depend on other pathophysiologic aspects, and on the pharmacokinetics and availability of the compound during 24 h, which will be below the maximal plasma concentration due to the rather short half-life of the compound. [2]

[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 abilities of oral selective phosphodiesterase 4 (PDE4) inhibitors enabled the approval of roflumilast and apremilast for use in chronic obstructive pulmonary disease 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 showing a preferential enzymatic inhibition of PDE4B. In vitro, 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 rat whole blood. In vivo, oral BI 1015550 shows potent anti-inflammatory activity in mice by inhibiting LPS-induced TNF-α synthesis ex vivo and in Suncus murinus by inhibiting neutrophil influx into bronchoalveolar lavage fluid stimulated by nebulized LPS. In Suncus murinus, PDE4 inhibitors induce emesis, a well-known gastrointestinal side effect limiting the use of PDE4 inhibitors in humans, and the therapeutic ratio of BI 1015550 appeared to be substantially improved compared with roflumilast. Oral BI 1015550 was also tested in two well-known mouse models of lung fibrosis (induced by either bleomycin or silica) under therapeutic conditions, and appeared to be effective by modulating various model-specific parameters. To better understand the antifibrotic potential of BI 1015550 in vivo, its direct effect on human fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) was investigated in vitro. BI 1015550 inhibited transforming growth factor-β-stimulated myofibroblast transformation and the mRNA expression of various extracellular matrix proteins, as well as basic fibroblast growth factor plus interleukin-1β-induced cell proliferation. Nintedanib overall was unremarkable in these assays, but interestingly, the inhibition of proliferation was synergistic when it was combined with BI 1015550, leading to a roughly 10-fold shift of the concentration-response curve to the left. In summary, the unique preferential inhibition of PDE4B by BI 1015550 and its anticipated improved tolerability in humans, plus its anti-inflammatory and antifibrotic potential, suggest BI 1015550 to be a promising oral clinical candidate for the treatment of IPF and other fibro-proliferative diseases. [2]

---

The present paper describes the preclinical pharmacology of Nerandomilast/BI 1015550, a novel oral PDE4 inhibitor that preferentially inhibits PDE4B.
Among the large superfamily of PDE isoenzymes, Nerandomilast/BI 1015550 selectively inhibits PDE4, and so far, has not shown any other off-target effects in screening against a panel of enzymes and receptors (Cerep high-throughput profile and Cerep non-kinase enzyme profile, Euriofins Cerep, Celle L’Escevault, France). Compared with roflumilast, the only oral PDE4 inhibitor on the market for a lung indication, BI 1015550 shows a unique profile in inhibiting PDE4 subtypes by a preferential inhibition of PDE4B (Table 1). The use of earlier PDE4 inhibitors, including roflumilast and apremilast, has been hampered by gastrointestinal side effects such as nausea, emesis, and/or diarrhea in humans. Although not proven, there is some evidence that these side effects are linked to inhibition of the PDE4D subtype (Giembycz, 2002). In this respect, the roughly 10-fold selectivity of BI 1015550 for inhibition of PDE4B versus PDE4D may be of clinical importance. It may also explain why in Suncus murinus the therapeutic ratio (inhibition of LPS-induced neutrophil influx into the lung versus induction of emesis) of BI 1015550 is superior to roflumilast (Figure 4; Table 2). More importantly, ongoing phase I clinical trials with BI 1015550 support the excellent gastrointestinal tolerability and safety of this compound in humans (data not shown). Another aspect of the use of BI 1015550 in humans relates to toxicity evident in animal studies. The major toxicity of PDE4 inhibitors, especially in the rat, is the induction of a vasculopathy (vasculitis, arteritis) in different tissues, which in some ways is paradoxical given that PDE4 inhibition is strongly linked to anti-inflammatory efficacy (see below). Although induction of vasculitis has never been reported in patients for the two oral PDE4 inhibitors roflumilast and apremilast, which have been on the market for many years, there is still some concern on this potentially serious side effect for the introduction of new PDE4 inhibitors. Dietsch et al. were the first to report stimulation of IL-6 in the rat by the PDE4 inhibitor IC542 (Dietsch et al., 2006). Since IL-6 is a possible candidate for induction of vasculitis in the rat, the lack of stimulatory effect of BI 1015550 on IL-6 in the human setting (Figure 2B) may indicate that, in contrast to rats, the risk for vasculitis induction by BI 1015550 in humans may be quite low. In addition, it is interesting to note that apremilast was reported to be clinically active in Behcet’s disease, a form of vasculitis characterized by inflammation of blood vessels, by resolution of oral ulcers, one of the most common symptoms (Hatemi et al., 2015).

Despite the alleviated inhibition of the PDE4D subtype, the well-known anti-inflammatory and immune-modulating properties of PDE4 inhibitors are still evident for Nerandomilast/BI 1015550. Qualitatively similar to roflumilast (Hatzelmann and Schudt, 2001), BI 1015550 appeared to be a potent inhibitor of TNF-α and IL-2 in human PBMCs (Figure 1), which, most likely, reflects the impact of BI 1015550 on monocytes and T lymphocytes, respectively. The efficient inhibition of TNF-α was also evident in whole blood (Figure 2), which is of importance because this assay format (ex vivo) can be used as a pharmacodynamic biomarker during clinical studies, as shown first by Timmer et al. for roflumilast (Timmer et al., 2002). Although the higher IC50 value of BI 1015550 for TNF-α inhibition in human whole blood (670 nmol/L) compared with human PBMCs (35 nmol/L) may be explained partly by the moderate plasma protein binding of the compound (77%), the loss of potency is higher than expected. In an effort to mimic the clinical situation, the potent and efficient inhibition of LPS-stimulated TNF-α in whole blood ex vivo was demonstrated for the mouse model in this study (Figure 3). It is clear that the ED50 of oral BI 1015550 (0.04 mg/kg) in this mouse model was much lower than in the mouse lung fibrosis disease models (bleomycin, silica) discussed below. However, in the former ex vivo model, blood is taken close to the maximal plasma concentration of the compound, and, although TNF-α as the pharmacologic read-out may contribute to lung fibrosis pathology, it is not the only relevant mediator. The efficacy and potency of BI 1015550 in the disease models will depend on other pathophysiologic aspects, and on the pharmacokinetics and availability of the compound during 24 h, which will be below the maximal plasma concentration due to the rather short half-life of the compound.

The aberrant wound-healing process in lung fibrosis passes through an inflammatory phase, with the involvement of inflammatory cells (in particular macrophages, monocytes, neutrophils, and T lymphocytes) and increased levels of cytokines (e.g. TNF-α and IL-1ß) and growth factors (e.g. TGF-ß and CTGF), creating a biochemical environment that supports chronic tissue remodeling and fibrosis. Based on this working hypothesis of the pathophysiology of lung fibrosis, the anti-inflammatory (inhibition of TNF-α, inhibition of monocytes, inhibition of neutrophil influx into the lung) and immune-modulatory (inhibition of IL-2, inhibition of T cells) characteristics of BI 1015550/Nerandomilast can be expected to contribute to improvement of fibrosis, at least to some extent. Therefore, we were interested to see whether BI 1015550 exerts antifibrotic effects in distinct animal models, and further, if the compound has direct effects on fibroblasts and myofibroblasts.

Bleomycin has been widely used in rodents to model pulmonary fibrosis, to understand mechanisms involved in fibrogenesis, and to evaluate potential new therapies. However, it should be noted that although bleomycin-induced pulmonary fibrosis mimics many features of human disease, fibrosis resolves in rodents whereas in humans it is generally irreversible. Furthermore, whilst human pulmonary fibrosis predominantly impacts the peripheral airways, in rodents it is often found more prominently around the central airways. Despite these restrictions, the bleomycin model is still a good tool to assess the efficacy of potential compounds in general as proof of principle (Moeller et al., 2008; Jenkins et al., 2017). The therapeutic treatment regimen of Nerandomilast/BI 1015550 attenuated some important aspects of disease pathology, including FVC and Cstat decline (Figure 5). These therapeutic effects were statistically significant at the highest BI 1015550 dose. In addition, lung tissue density was improved to the same extent, although this effect did not reach statistical significance (Table 3). With regards to the Ashcroft score, BI 1015550 only showed a non-significant trend towards inhibition. For other experimental parameters investigated (BALF protein and monocytes, airway compliance), no effects of BI 1015550 were detected. The lack of effect of BI 1015550 on inflammatory mediators in BALF as well as the minimal effects on histologically assessed lung fibrosis in the present study are in contrast to bleomycin mouse studies reported by others (Cortijo et al., 2009; Udalov et al., 2010). We can only speculate that fibrotic changes in the mouse bleomycin model performed under our conditions are not captured by our scoring system, originally designed to assess human pulmonary fibrosis. It might also be possible that the changes in tissue volume do not only reflect fibrotic changes, but are related to other remodeling aspects like neovascularization in our model, as shown by Ackermann et al. (Ackermann et al., 2017). Differences versus previous studies may also be due to differing treatment durations and timings of data collection. Regardless of the nature of such changes, improvement in functional parameters, such as lung function, suggests a positive impact of BI 1015550 in pulmonary tissue remodeling. This assumption is supported by the positive findings for BI 1015550 in the silica-induced lung fibrosis model in the mouse. In contrast to bleomycin, which induces reversible lung fibrosis, silica particles induce a progressive type of lung fibrosis, which resembles the human counterpart of a progressive fibrosing ILD. To the best of our knowledge, a PDE4 inhibitor that preferentially inhibits PDE4B has not been evaluated in this animal model before. As shown in Table 4, BI 1015550 in a therapeutic regimen achieved dose-dependent improvements in semi-quantitative scores of granuloma formation, inflammation, and fibrosis, although these effects did not reach statistical significance. In BALF, macrophages and neutrophils were significantly inhibited at the highest dose, while the inhibition of MPO and KC did not reach statistical significance.

Overall, the beneficial effects of Nerandomilast/BI 1015550 in two lung fibrosis models are in agreement with preclinical data in the literature that selective PDE4 inhibitors could be clinically effective not only in lung fibrosis but, based on animal data, probably also in fibrosis of other organs (e.g. liver, kidney, colon, and/or skin). This hypothesis is supported by several studies showing that selective PDE4 inhibitors are able to directly target fibroblasts in addition to inflammatory and immune-competent cells. By using relevant cells, namely human lung fibroblasts from patients with IPF, we were able to support this by showing that BI 1015550 inhibits TGF-ß-stimulated transformation into myofibroblasts (Figure 6A). This indicates a differential mode of action for BI 1015550 compared with nintedanib, which was inactive under these conditions and did not enhance the efficacy of BI 1015550. Furthermore, BI 1015550 attenuated TGF-β-induced Col1, Col3, and FN gene expression (Figures 6B–D), which are important constituents of fibrotic extracellular matrix, and the combination with nintedanib at 100 nmol/L showed additive effects in inhibiting Col3 expression. In addition, BI 1015550 inhibited bFGF plus IL-1β-induced cell proliferation. Nintedanib by itself, at a relevant concentration (100 nmol/L), inhibited the proliferation by only 15%, but, most strikingly, the combination of BI 1015550 plus nintedanib resulted in synergistic inhibitory effects and shifted the concentration–response curve to the left by about 10-fold (Figure 6E). Inhibition of human lung fibroblast proliferation and myofibroblast transformation in vitro might contribute to potential clinical efficacy of BI 1015550 in IPF. Furthermore, the combination of BI 1015550 with nintedanib might yield synergistic therapeutic effects in patients with IPF and other fibrotic lung diseases. This kind of synergism between a PDE4 inhibitor that preferentially inhibits PDE4B and nintedanib has not been reported before.

In summary, BI 1015550/Nerandomilast is an oral preferential inhibitor of PDE4B with suggested improved tolerability in humans compared with the selective oral PDE4 inhibitors on the market; the preclinical profile suggests that this compound is a promising oral clinical drug candidate for the treatment of IPF and other ILDs. A phase II trial of BI 1015550 versus placebo in patients with IPF is currently ongoing (NCT04419506). Future clinical studies will show whether BI 1015550 will be able to provide substantial efficacy alone, or whether the combination with IPF standard therapies such as nintedanib may be preferred in order to fully exploit the therapeutic potential of BI 1015550. [2]

C20H25CLN6O2S

448.969501256943

448.144

C, 53.50; H, 5.61; Cl, 7.90; N, 18.72; O, 7.13; S, 7.14

1423719-30-5

Nerandomilast dihydrate

166177189

Off-white to light yellow solid powder

1.3

2

9

5

30

624

1

ClC1C=NC(C2CCN(C3N=C4CC[S@](C4=C(N=3)NC3(CO)CCC3)=O)CC2)=NC=1

UHYCLWAANUGUMN-SSEXGKCCSA-N

InChI=1S/C20H25ClN6O2S/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)/t30-/m1/s1

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

nerandomilast; 1423719-30-5; BI 1015550; BI-1015550; nerandomilast [INN]; Nerandomilast (JAN/INN); I5DGT51IB8; NERANDOMILAST [USAN];

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: 10 mg/mL (22.27 mM)

Solubility in Formulation 1: 5 mg/mL (11.14 mM) 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; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 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.

---

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