Sahar Salah

El Din

El Souda

Azza Abdelmageed Matloub

Françoise Nepveu

Alexis Valentin

Christine Roques

Phenolic composition and prospective anti-infectious properties of Atriplex lindleyi

Keywords:

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Malaria is one of the most dangerous tropical parasitic diseases. About 198 million cases of malaria infection and an estimated 584 million deaths were reported in 2013, mostly among children living in Africa where almost one child dies every minute [1]. It also poses a risk to travellers and immigrants, with imported cases increasing in non-endemic areas [START_REF] Pereira | Transfusion-transmitted protozoal infections: what is the risk in non-endemic countries?[END_REF]. Therefore, endemic countries started to adopt artemisinin-based combination therapies for uncomplicated malaria [START_REF]Guidelines for the treatment of malaria[END_REF]. However, in the Greater Mekong Subregion, Plasmodium falciparum (P. falciparum) has become resistant to almost all available antimalarial medicines. In addition, there is a real risk that multidrug resistance will emerge soon in other parts of the subregion as well [START_REF]Status report on artemisinin resistance[END_REF]. Therefore, the current research aimed at the development of new antimalarial agents.

The genus Atriplex comprises of 200 species and belongs to the subfamily Chenopodiaceae. Although this genus is considered as common weeds, it has always been used as folk remedy. In the Arab world, Atriplex halimus is used as a laxative, stomach pain killer, against intestinal worms, to regulate gall bladder excretions and to treat chest ailments. The Bedouin tribes in the Negev use the plant for treating muscular pain and intestinal diseases in animals, to feed sheep and goats, and to flavor cooking. The leaves are edible but too salty to be eaten alone [START_REF] Said | Ethnopharmacological survey of medicinal herbs in Israel, the Golan Heights and the West Bank region[END_REF]. The chemical composition of Atriplex species is very complicated as it contains flavonol as the major components [START_REF] Benzarti | Environmental and economical opportunities for valorisation of the genus Atriplex: new insight[END_REF][START_REF] Awaad | Novel flavonoids with antioxidant activity from a Chenopodiaceous plant[END_REF], triterpene saponins [START_REF] Kamal | Anticholinesterse and antioxidant investigations of crude extracts, subsequent fractions, saponins and flavonoids of Atriplex laciniata L.: potential effectiveness in Alzheimer's and other neurological disorders[END_REF][START_REF] Jabrane | Glaucasides A-C, three saikosaponins from Atriplex glauca L. var. ifiniensis (Caball) Maire[END_REF], alkaloids and amino acids [START_REF] Emam | Bioactive constituents of Atriplex halimus plant[END_REF], coumarins and ecdysterols [START_REF] Ael | Secondary metabolites and antifertility potential of Atriplex farinosa Forssk[END_REF]. Therefore, Atriplex species have been reported as cytotoxic [START_REF] Jabrane | Glaucasides A-C, three saikosaponins from Atriplex glauca L. var. ifiniensis (Caball) Maire[END_REF][START_REF] Capua | Cytotoxicity of Atriplex confertifolia[END_REF], antimicrobial [START_REF] Salazar | Activity of medicinal plants, used by native populations from Sonora, Mexico, against enteropathogenic bacteria[END_REF], antioxidant [START_REF] Benhammou | Antioxidant activity of methanolic extracts and some bioactive compounds of Atriplex halimus[END_REF], antidiabetic [START_REF] Chikhi | Antidiabetic activity of aqueous leaf extract of Atriplex halimus L. (Chenopodiaceae) in streptozotocin-induced diabetic rats[END_REF], and to reduce male fertility in rats [START_REF] Ael | Secondary metabolites and antifertility potential of Atriplex farinosa Forssk[END_REF].

Of the Atriplex species, Atriplex lindleyi Moq. (A. lindleyi) synonym: Atriplex halimoides, known as Lindley's saltbush, is widely spread in Egypt, especially in dry areas. The hepatorenal protective potential of Lindley's saltbush was evaluated [START_REF] Matloub | Chemo-protective effect on hepato-renal toxcicity and cytotoxic activity of lipoidal matter of Atriplex lindleyi Moq[END_REF], and its antidiabetic, antihepatotoxic, antimicrobial and anti-inflammatory activities were reported [START_REF] L -S H A Fa E A M , E L -S H A Z L Y A M , H E M D A N | antiinflammatory, antimicrobial activities and chemical constituents of Atriplex inflata[END_REF][START_REF] Mohammed | Biological investigation of some wild Aizoaceae and Chenopediaceae species growing in Egypt[END_REF]. In addition, several compounds were isolated such as flavonoids, quercetin, quercetrin, isoquercetin and isorhamnetin-3-O-glucopyranoside besides lupeol, ecdesterol, 3,23-dihydroxybetulin, 20-hydroxyecdysone,olean-12-en-3,11dione, β-amyrenone, erythrodiol and cholesterol [START_REF] Matloub | Chemo-protective effect on hepato-renal toxcicity and cytotoxic activity of lipoidal matter of Atriplex lindleyi Moq[END_REF][START_REF] L -S H A Fa E A M , E L -S H A Z L Y A M , H E M D A N | antiinflammatory, antimicrobial activities and chemical constituents of Atriplex inflata[END_REF][START_REF] Mohammed | Biological investigation of some wild Aizoaceae and Chenopediaceae species growing in Egypt[END_REF]. The present work deals with the identification of phenolic constituents and the investigation of the antiplasmodial, antimicrobial and radical scavenging effect of A. lindleyi extracts.

Materials and methods

General experiment

Ultraviolet spectra were recorded by using UV-vis double beam UWD-3500 spectrophotometer (Labomed, Inc. USA). Electrospray ionization mass (ESIMS) was obtained from an LCQ Advantage Thermo/Finnigan spectrometer (Thermo Finnigan, USA). Nuclear magnetic resonance (NMR) spectra were measured on JEOL EX- 500 spectrometer (Tokyo, Japan) with 500 MHz for 1 H-NMR and 125 MHz for 13 C-NMR by using methyl sulfoxide-d 6 as solvent and as internal reference. Analytical thin layer chromatography (TLC) was done on TLC cellulose and silica gel 60 F 254 (200 mm layer thickness) (Merck; Darmstadt, Germany). Chromatograms were visualized under UV light at 366 nm before and after sprays of aluminium chloride reagent.

The phenolic acids were analyzed by using high performance liquid chromatography (HPLC), HPLC Agilent 1100 series (Waldbornn, Germany), equipped with quaternary pump (G1311A), degasser (G1322A); the column used was Zorbax 300SB C18 (Agilent Technologies, USA). Injection was carried out at wave length of 280 nm for separation of phenolic compounds. Reference compounds were purchased from Sigma-Aldrich (Steinheim, Germany), and column chromatography for fractionation was employed by using Polyamide 6S (Riedel-de-Haen AG, Seelze Haen AG, D-30926 Seelze Hanver, Germany). The purification was performed on Sephadex LH-20 (Pharmacia, Sweden) column. All chemicals of analytical grade were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Solvents were purchased from Merck (Darmstadt, Germany).

Plant material

A. lindleyi Moq. aerial parts were collected in March from Road of Suez, Egypt. A voucher specimen (No. 954) was kept in the Herbarium of National Research Centre.

Extraction and isolation

Dried powdered aerial parts of A. lindleyi (350 g) were successively extracted with petroleum ether (40-60 °C), chloroform and 70% aqueous ethanol (23.0, 12.5 and 12.0 g), respectively. The aqueous ethanol extract was subjected to reversed phase liquid chromatography on polyamide eluted with water gradient and decreased polarity with methanol. Fractions of 100 mL were collected and monitored on TLC silica plates by using ethyl acetate: formic acid: acetic acid: water (100:11:11:27 v/v/v/v) as developing solvent. To obtain three substantial fractions, major compounds were isolated and purified on Sephadex LH20 subcolumn. Purity of isolated compounds was inspected through two-dimensional TLC cellulose plates eluted with solvent systems: (1) 15% HOAc (water: acetic acid 85:15 v/v), (2) BAW (butanol: acetic acid: water 4:1:5 v/ v/v, upper layer).

Acid hydrolysis

Acid hydrolysis was performed by using 6% aqueous hydrochloric acid at 100 °C for 2 h. For aglycone detection, the final mixture was extracted with ethyl acetate, then the aqueous layer was neutralized for determination of the released sugar moiety by using silica gel plates with the mixture of n-propanol: ethyl acetate: water (7:2:1 v/v/ v); aniline phthalate was employed as a spray for color detection of the sugars [START_REF] El-Kassem | Digalacturonide flavones from Egyptian Lantana camara flowers with in vitro antioxidant and in vivo hepatoprotective activities[END_REF]. Characterizations of compound 3 were as follows: ESIMS (-ve) m/z: 609 [M-H] -, 447 [M-H-glucose] -, 463 [M-H-rhamnose] -, 301 [M-H-glucose-rhamnose] -; 1 H-NMRδ: 12.64 (1H, s, 5-OH), 7.62 (1H, d, J = 2.5 Hz, H-2'), 7.60 (1H, dd, J = 8.5, 2.5 Hz, H-6'), 6.85 (1H, d, J = 8.5 Hz, H-5'), 6.80 (1H, d, J = 2.5 Hz, H-8), 6.43 (1H, d, J = 2.5 Hz, H-6), 5.49 (1H, d, J = 6.9 Hz, H-1'), 5.55 (1H, s, H-1''), 1.12 (3H, d, J = 6.0 Hz, -CH3); 13

Characterizations of compounds

HPLC of phenolic acids

To prepare the fraction containing phenolic compounds, one gram of the aqueous ethanol extract was re-extracted with normal butanol in separating funnel, and the butanol fraction was evaporated. The residue was re-dissolved in methanol, then shaken with ether, allowed to stand. Finally, the supernatant was filtered, dried and subjected to HPLC as described above, by using mobile phase and acetonitrile: phosphate buffer (86:14 v/v) at flow rate of 1 mL/min.

Antiplasmodial assay

Antiplasmodial activity was tested according to the protocol previously reported [START_REF] Souda | Powerful antioxidant and pro-oxidant properties of Cassia roxburghii DC. leaves cultivated in Egypt in relation to their anti-infectious activities[END_REF]. Roswell Park Memorial Institute 1640 medium [BioWhittaker, Cambrex (catalogue No. BE12-702F), Belgium] containing L-glutamine (BioWhittaker, catalogue No. BE17-605E), 25 mmol/L 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (BioWhittaker, catalogue No. 17-737F), and 10% human serum (Etablissement Français du Sang, Toulouse, France), was used to cultivate P. falciparum. Human red blood cells (RBCs) (group O ±) were obtained from Etablissement Français du Sang. They were extensively washed with Roswell Park Memorial Institute medium to remove plasma and leucocytes. Leucocytefree erythrocytes were stored at 50% hematocrit for a maximum period of 21 days. P. falciparum asexual blood-stage parasites were propagated by incubation at 37 °C in P. falciparum culture media at 3%-5% hematocrit in a controlled atmosphere (5% CO 2 , 100% relative humidity).

Parasitized RBCs were maintained in 25 cm 2 culture flasks (TPP, Switzerland, reference No. 90025). Reference drugs, chloroquine and artesunate were obtained from Sigma (reference No. C6628) and Cambrex, respectively. Chloroquine was dissolved in culture medium and artesunate in ethanol (stock solutions of 10 mg/mL) and stored at -20 °C before tested. For extract assays, serial extract dilutions were made in P. falciparum culture media and added to 96-well (TPP) culture plates. Plasmodium-infected RBCs were distributed at 2% parasitaemia (2% hematocrit) in 96-well microtiter plates with different drug concentrations and incubated for 48 h at 37 °C and 5% CO 2 .

[3H]-Hypoxanthine (Perkin-Elmer) was added 24 h after the beginning of the incubation. At the end of incubation (48 h), microtiter plates were frozen and thawed, and each well was harvested onto a glass-fiber filter paper. The quantity of incorporated [3H]-hypoxanthine was determined with a β-counter (1450-Microbeta Trilux, Wallac-PerkinElmer). Growth-inhibition percentages were plotted as a semilogarithmic function of drug concentration. The IC 50 values were determined by linear regression analysis on the linear segments of the curves. Assays were repeated three times for each concentration. Controls were carried out to assess the background (negative control) and parasite growth (positive control).

Antimicrobial assays

Antimicrobial activity was tested according to the method previously reported [START_REF] Souda | Powerful antioxidant and pro-oxidant properties of Cassia roxburghii DC. leaves cultivated in Egypt in relation to their anti-infectious activities[END_REF]. The extracts were dissolved in water/ dimethylsulfoxide (DMSO) to obtain an initial concentration of 1 000 µg/mL. The resulting solutions were then diluted in microtiter plates in trypcase soja medium (Biomérieux, Craponne, France) for bacteria and Sabouraud (Biomérieux, Craponne, France) for fungi and yeast. Gram-positive strains, Staphylococcus aureus (S. aureus) CIP 4.83 and Enterococcus hirae (E. hirae) CIP 5855, and Gram-negative strains, Pseudomonas aeruginosa (P. aeruginosa) CIP 82118 and Escherichia coli CIP 53126, were used for the antimicrobial assays. For the antifungal assay, cultures of the yeast C. albicans IP 48.72 and mould Aspergillus niger (A. niger) IP 1431.83 were employed. Strains were obtained from the collection of the Pasteur Institute (Paris, France). Microbial suspensions were prepared in sterile distilled water to obtain final inoculums of 10 6 cells/mL and 10 5 spores/mL for bacteria and fungi, respectively.

Minimal inhibitory concentrations (MICs) and minimal germicidal concentrations [(minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC)] were determined after incubation of the bacterial strains at 37 °C and the fungal strains at 22.5 or 30.0 °C for 24 h (36 h for A. niger) in the presence of serial dilutions of the test compounds. The MIC was defined as the concentration of compound at which no macroscopic sign of cellular growth was detected in comparison to the control without antimicrobial compound. The MBC/MFC was determined by subcultivation on corresponding agar plates after incubation of the bacterial strains at 37 °C for 24 h and fungal strains at 22.5 or 30.0 °C for 48 h.

MBC/MFC was defined as the concentration of compound at which no macroscopic sign of cellular growth was detected in comparison to the control without antimicrobial compound on subculturing. All the experiments were carried out in duplicate at each concentration. In order to ensure that DMSO per se did not interfere with the antimicrobial activity evaluation of the products under assay, a control test was also performed containing inoculated broth supplemented with only DMSO at the same dilutions used in our experiments.

2,2-diphenyl-1-picrylhydrazyl assay

The antioxidant activity was evaluated by using the method previously reported [START_REF] Souda | Powerful antioxidant and pro-oxidant properties of Cassia roxburghii DC. leaves cultivated in Egypt in relation to their anti-infectious activities[END_REF]. The stable 2,2-diphenyl-2-picrylhydrazyl nitrogen-centered free radical (DPPH • ). DPPH in ethanol (300 µmol/ L, 100 µL) was added to 200 µL of the test compounds at different concentrations in the appropriate solvents (MeOH, EtOH, MeOH/ H 2 O 2:1, v/v). Each mixture was then mixed thoroughly and the absorbance was recorded every 5 min for 30 min, by using a microplate reader (UV Max, Molecular Devices). The decrease in DPPH • absorbance (at λ 530 nm) was monitored. DPPH • solution (100 µL) in the respective solvent (200 µL) served as the blank.

All tests were performed in triplicate. The radical scavenging activity of the samples (antioxidant activity) was expressed in terms of IC 50 (concentration in mg/L required for a 50% decrease in DPPH • absorbance). A plot of absorbance vs. concentration was made to establish the standard curve and to calculate the IC 50 . Ascorbic acid and Trolox (water-soluble form of vitamin E) were used as positive controls.

Results

Column chromatography

Column chromatography of aqueous ethanol extract afforded eight flavonoids, namely, quercetin-7-O-arabinoside-3-Oneohesperidosides (1), quercetin-3-O-arabinopyranosyl(1→6)glucopyranoside (2), quercetin-3-O-glucopyranoside-7-Orhamnopyranoside (3), quercetin-3-O-glucoside,7-O-arabinose (4), schaftoside (5), quercimeritrin (6), isorhamnetin-3-O-glucoside [START_REF] Awaad | Novel flavonoids with antioxidant activity from a Chenopodiaceous plant[END_REF] and quercetin [START_REF] Kamal | Anticholinesterse and antioxidant investigations of crude extracts, subsequent fractions, saponins and flavonoids of Atriplex laciniata L.: potential effectiveness in Alzheimer's and other neurological disorders[END_REF]. Their structural elucidation was established by comparing the UV, ESIMS and/or NMR spectral data with reference data from available literature [START_REF] Andersen | Flavonoids: chemistry, biochemistry and applications[END_REF][START_REF] Agrawal | Carbone-13 NMR of flavonoids[END_REF][START_REF] Azimova | Kaempferol-3-O-β-D-(6"-O-pcoumaroyl) glucopyranoside[END_REF]. While the absolute configuration of the sugar was elucidated through chemical degradation and comparison with an authentic standard. To the best of our knowledge, compounds 1-6 were isolated from A. lindleyi aerial parts for the first time.

In the case of compound 1, the negative mode ESI of compound 1 showed molecular ion at m/z 741 [M-H] -. The MS data showed fragment ion at m/z 609 [M-H-132] -, 489 [M-H-132-120] -, and 301 [M-H-132-308] -, indicating successive elimination of pentose, rhamnose and hexose, respectively. In addition, fragment ion at m/ z 308 was indicative of compound having the disaccharide structure, rutinose or neohesperidose linked through an -O-glycosidic bond. The fragmentation patterns of flavonoid O-neohesperidosides were more complicated in comparison with their rutinoside analogues. A major difference was found in the [M-H-120] -ion in the MS2 spectrum, which was a common feature of all the flavonoid O-neohesperidosides [START_REF] Liu | Advances of modern chromatographic and electrophoretic methods in separation and analysis of flavonoids[END_REF]. The low intensity of fragment ion m/ z 609 due to loss of pentose indicates its position at C7 [START_REF] Andersen | Flavonoids: chemistry, biochemistry and applications[END_REF][START_REF] Ablajan | Structural characterization of flavonol 3,7-di-O-glycosides and determination of the glycosylation position by using negative ion electrospray ionization tandem mass spectrometry[END_REF]. The chromatographic behavior and UV supported a 3,7-glycosylated flavonolstructure. Additional confirmation was obtained by acid hydrolysis and co-chromatography with authentic sugars. These emphasized that this compound was identified as quercetin-7-Oarabinoside-3-O-neohesperidosides.

Compound 2 showed purple spot when examined under UV at λ 365 nm, suggesting substituted 3 hydroxyl group, which was confirmed by UV-shift reagents. The 1 H-NMRδ showed the expected signal in the aromatic region for -3-O-substituted quercetin, signals of two anomeric protons of sugar doublet at δH 5.2 (J = 7.65 Hz) and 5.3 (J = 6.85 Hz); their coupling constants matched the values of β-D-glucopyranoside and α-L-arabinopyranoside, respectively [21-23]. This was confirmed by the observed two anomeric carbon of sugar at 101.3 and 103.2 ppm in 13 C-NMRδ and the appearance of molecular ions in ESI/MS at m/z 595 [M-H] -and fragment ion at m/z 301 [M-H-294] -due to loss of hexose and pentose units. Acid hydrolysis indicated the presence of glucose and arabinose. The 13 C-NMR spectra showed significant down field shift of 6 ppm for the C6 signal of glucose due to interglycosidic linkage indicating that the glucopyranoside unit directly attached to the aglycone and arabinopyranoside was a terminal sugar. Altogether, compound 2 was identified as quercetin 3-O-α-L-arabinopyranosyl(1'''→6'')-β-Dglucopyranoside [START_REF] Krenn | Flavonoids from Achillea nobilis L[END_REF].

For compound 3, the MS, 1 H-NMR and 13 C-NMR data were in agreement with those reported for quercetin-3-O-glucopyranoside-7-O-rhamnopyranoside [START_REF] Song | Several flavonoids from Capsella bursa-pastoris (L.) Medic[END_REF].

In the situation of compound 4, the UV absorbance of compound with shift reagents indicated a flavonol moiety substituted at C7 and C3. ESI/MS of compound 4 showed a mass molecular ion at m/ z 595 [M-H] -. The MS2 fragment ion at m/z 463 [M-H-132] -was corresponding to the loss pentose moiety. Further, MS3 fragment ion was observed at m/z 301 [M-H-132-162] -indicating the loss of a hexose unit. The sugars were identified by acid hydrolysis and cochromatography with authentic sugars, arabinose and glucose. The intensity of the fragment resulting from loss of hexose was higher than that obtained from loss of pentose, indicating glucose attached to C3 meanwhile arabinose was at C7. The compound was identified as quercetin-3-O-glucoside,7-O-arabinoside [START_REF] Ablajan | Structural characterization of flavonol 3,7-di-O-glycosides and determination of the glycosylation position by using negative ion electrospray ionization tandem mass spectrometry[END_REF].

For compound 5, all data were in accordance with those of apigenin-6-C-glucopyranoside-8-C-arabinopyranoside (schaftoside) [START_REF] De Oliveira | Flavonoids from leaves of Mauritia flexuosa[END_REF].

For compound 6, chemical shifts values at δ H 6.44 for H-6, δ H 6.81 for H-8, δc 147.9 (C-2) and δc 160.1 for (C-7) suggested 7-O-glycosylated flavonoids, while anomeric proton at δ 5.10 with J = 7.7 Hz indicated the β configuration. Results of UV, MS, 1 H-NMR and 13 C-NMR were in agreement with those previously reported in the literature of quercetin-7-O-glucopyranoside (quercimeritrin) [START_REF] Andersen | Flavonoids: chemistry, biochemistry and applications[END_REF][START_REF] Agrawal | Carbone-13 NMR of flavonoids[END_REF][START_REF] Azimova | Kaempferol-3-O-β-D-(6"-O-pcoumaroyl) glucopyranoside[END_REF].

For compound 7, the UV, ESIMS, 1 H-NMR results were corresponding to those of isorhamnetin-3-O-glucoside [START_REF] Andersen | Flavonoids: chemistry, biochemistry and applications[END_REF][START_REF] Agrawal | Carbone-13 NMR of flavonoids[END_REF][START_REF] Azimova | Kaempferol-3-O-β-D-(6"-O-pcoumaroyl) glucopyranoside[END_REF].

For compound 8, ESIMS and 1 H-NMR results matched those reported to quercetin [START_REF] Andersen | Flavonoids: chemistry, biochemistry and applications[END_REF][START_REF] Agrawal | Carbone-13 NMR of flavonoids[END_REF][START_REF] Azimova | Kaempferol-3-O-β-D-(6"-O-pcoumaroyl) glucopyranoside[END_REF].

HPLC of phenolic acid fraction

HPLC analysis of phenolic acid fraction led to identification of five compounds which were compiled in Table 1. Ellagic acid (26.4%) and cathechin (31.3%) were predominant ones.

Antiplasmodial and cytotoxic activities

The antiplasmodial effect of tested extracts and compounds against P. falciparum was expressed as concentration that killed 50% of parasites (IC 50 ) in Table 2. All extracts possessed moderate lethal effect IC 50 ranging from 10 to 50 μg/mL. Among the tested flavonoids, compound 8 (IC 50 = 9 μg/mL) was the most potent followed by compound 6 then compound 2 and finally compound 3. Extracts exhibited cytotoxicity with 50% cytotoxicity concentration (CC 50 ) values ranging from 27-33 µg/mL on the mammalian MCF-7 cell line.

Antimicrobial activity

The antimicrobial activities were summarized in Table 3. The results showed that extracts demonstrated antibacterial activity with MIC and MBC ranging from 62-125 µg/mL and 125-500 µg/mL, respectively, exhibiting similar inhibitory effect on E. hirae and other three germs. Petroleum ether and chloroform extracts were more active on P. aeruginosa than reference drugs (nalidixic acid, bifonazole, clotrimazole).

Meanwhile, petroleum ether and aqueous ethanol extracts exhibited antimicrobial activity against S. aureus similar to reference drugs. On the other hand, all the extracts showed the same antifungal effect on the tested fungi. The yeast C. albicans was more susceptible to the extracts with MIC values of 62.5 µg/mL.

Radical scavenging ability

The IC 50 of extracts in DPPH absorbance represented in Table 4 revealed that the petroleum ether extract had no radical scavenging ability at the tested concentration.

Discussion

In spite of extensive studies, the structure-activity relationships for the antiplasmodial action remain unclear. Here we found that the hydrophilic extract which comprises predominantly flavonoids and phenolic acids showed radical scavenging ability and antiplasmodial activity. Our detailed investigation of the isolated compounds used as antiplasmodial showed that the aglycone with free hydroxyl groups was the most active compound and its activity decreased upon the substitution of hydroxyl groups. We also observed that the 7-O-subtituted flavonol was relatively more active than 3-Osubtituted and that the disubstitution at 7-O and 3-O was unfavorable to the antiplasmodial activity. From our data, it can be concluded that the free OH considerably increased the antiplasmodial activity, which is reduced when the free OH is occupied by sugar moiety. These results are supported by an earlier study [START_REF] Tasdemir | Inhibition of Plasmodium falciparum fatty acid biosynthesis: evaluation of FabG, FabZ, and FabI as drug targets for flavonoids[END_REF], which focused on the efficacy of different flavonoids and gallic acid esters of catechins toward three important enzymes, i.e. β-ketoacyl-ACP-reductase, β-hydroxacyl-ACP-dehydratase and enoyl-ACP-reductase involved in the fatty acid biosynthesis of P. falciparum strain. The authors found that the activity of flavonoids was attributed to the combination of two (catechol) or three hydroxyl group in ring B (regardless of an additional hydroxyl group at C-3). They also reported that the presence of the double bond between C-2 and C-3 was essential to keep the planarity structure of flavonoids with respect to activity. Another study found that a bulky substituent such as sugar moiety at C-3 of flavonol forced the ring B out of the plane of the ACring [START_REF] Moalin | A planar conformation and the hydroxyl groups in the B and C rings play a pivotal role in the antioxidant capacity of quercetin and quercetin derivatives[END_REF]. These studies explained why compound 6 was relatively active compared to compound 2 while compound 3 was inactive. These results are in agreement with those deduced by Moalin et al. regarding the antioxidant capacity of quercetin derivatives [START_REF] Moalin | A planar conformation and the hydroxyl groups in the B and C rings play a pivotal role in the antioxidant capacity of quercetin and quercetin derivatives[END_REF]. Another parameter is the oxidative stress which is considered beneficial for killing parasites and for weakening the adherence between infected RBCs and endothelial cells or monocytes. It was reported that compounds with high oxidation potential may be used as relatively efficient antiplasmodial agents with low mammalian cell cytotoxicity [START_REF] Grellier | Role of singleelectron oxidation potential and lipophilicity in the antiplasmodial in vitro activity of polyphenols: comparison to mammalian cells[END_REF]. In our study, both chloroform and ethanol extracts displayed higher radical scavenging ability, antiplasmodial activity and lower cytotoxicity than petroleum ether extract. In contrast, the petroleum ether extract showed no radical scavenging ability, relatively lower antiplasmodial activity and higher cytotoxicity than the ethanol extract. The constituents of petroleum ether extract have been recently studied for their hepatorenal protection and cytotoxic effects [START_REF] Matloub | Chemo-protective effect on hepato-renal toxcicity and cytotoxic activity of lipoidal matter of Atriplex lindleyi Moq[END_REF]. Despite of the limited antimicrobial activity of extracts, it is noteworthy that P. aeruginosa and C. albicans were the most susceptible microorganisms. Particularly, both organisms are frequently coexisting opportunistic pathogens, forming microbial biofilms that can lead to severe illness and serious threat to human health [START_REF] Kart | Activity of disinfectants against multispecies biofilms formed by Staphylococcus aureus, Candida albicans and Pseudomonas aeruginosa[END_REF].

To the best of our knowledge, this is the first study to investigate the possible antimalarial effect of A. lindleyi extracts. Evaluating the extracts and compounds revealed promising antiplasmodial activity of ethanol extract and some components. Moreover, this plant can be useful as prophylactic against C. albicans and P. aeruginosa. The identified compounds were reported for the first time from this species excluding compounds 7 and 8.

  13 C-NMRδ: 157.9 (C-2), 133.7 (C-3), 177.8 (C-4), 161.7 (C-5), 99.2 (C-6), 164.9 (C-7), 94.1 (C-8), 157.2 (C-9), 104.3 (C-10), 122.1 (C-1'), 115.8 (C-2'), 145.3 (C-3'), 149.1 (C-4'), 115.6 (C-5'), 121.5 (C-6'), 103.2 (C-1''), 74.4 (C-2''), 76.8 (C-3''), 70.5 (C-4''), 77.3 (C-5''), 67.8 (C-6''), 101 (C-1'''), 70.9 (C-2'''), 72.9 (C-3'''), 70.9 (C-4'''), 65.4 (C-5'''). ESIMS (-ve) m/z: 595 [M-H] -.

  C-NMRδ: 156.8 (C-2), 133.7 (C-3), 177.7 (C-4), 160.9 (C-5), 99.5 (C-6), 161.7 (C-7), 94.4 (C-8), 156.8 (C-9), 105.7 (C-10), 121.8 (C-1'), 115.3 (C-2'), 145.0 (C-3'), 148.9 (C-4'), 116.4 (C-5'), 121.1 (C-6'), 100.8 (C-1'), 74.2 (C-2'), 76.6 (C-3'), 70.3 (C-4'), 77.8 (C-5'), 61.1 (C-6'), 99.5 (C-1''), 70.0 (C-2''), 70.2 (C-3''), 71.7 (C-4''), 69.9 (C-5''), 18.0 (C-6''). Characterizations of compound 4 were UV (λ max ) nm: (NaOMe): 269, 400; (AlCl 3 ): 275, 330sh, 433; (AlCl 3 + HCl): 269, 300sh, 360sh, 400; (NaOAc): 262, 408 and (NaOAc + H 3 BO 3 ): 260, 378; ESIMS (-ve) m/z: 595 [M-H] -, 463 [M-H-arabinose] -, 301 [M-Hglucose-arabinose] -. Characterizations of compound 5 included UV (λ max ) nm: (MeOH): 271, 338; 1 H-NMRδ: 8.02 (2H, d, J =8.5 Hz, H-2', 6'), 6.91 (2H, d, J = 8.65 Hz, H-3', 5'), 6.81 (1H, s, H-3); 6-C-β-Glc: 4.73 (1H, d, J = 9.8 Hz, H-1ʹʹ); 8-C-α-Ara: 4.83 (1H, d, J = 9.7 Hz, H-1ʹʹʹ); 13 C-NMRδ: 182.3 (C-4), 163.6 (C-2), 161.2 (C-7), 161.2 (C-5), 160.8 (C-4ʹ), 153.4 (C-9), 128.8 (C-2ʹ, 6ʹ), 121.3 (C-1ʹ), 116.0 (3ʹ, 5ʹ), 109.3 (C-6), 103.7 (C-8), 103.0 (C-10), 102.5 (C-3); 6-C-β-Glc: 74.3 (C-1), 68.4 (C-2), 79.4 (C-3), 70.3 (C-4), 79.0 (C-5), 60.8 (C-6); 8-C-α-Ara: 74.9 (C-1), 69.8 (C-2), 73.7 (C-3), 70.0 (C-4), 70.3 (C-5). ESIMS (m/ z): 563 [M-H] -, 473 [M-90] -, 443 [M-120] -, 383 [M-180] -, 353 and 297. Compound 6 was observed in yellow fluorescence under UV at λ 360 nm. Its characterizations were as follows: UV (λ max ) nm: (MeOH): 259, 267sh, 378; (+NaOMe): 291, 241sh, 367, 456; (+AlCl 3 ): 275, 259sh, 332sh, 439; (+AlCl 3 /HCl): 271, 300sh, 363, 426; (+NaOAc): 286, 375, 426; (+NaOAc/H 3 BO 3 ): 261, 289, 384; ESIMS (-ve) m/z: 463 [M-H] -, m/z 301 [M-H-162] -; 1 H-NMRδ: 12.60 (1H, s, 5-OH), 7.29 (1H, d, J = 1.5 Hz, H-2ʹ), 7.24 (1H, d, J = 8.4 Hz, H-6ʹ), 6.85 (1H, d, J = 8.4 Hz, H-5ʹ), 6.36 (1H, br s, H-8), 6.19 (1H, brs, H-6), 5.10 (1H, d, J =7.7 Hz, H-1ʹʹ); 13 C-NMRδ: 147.9 (C-2), 134.6 (C-3), 181.2 (C-4), 161.8 (C-5), 99.3 (C-6), 160.1 (C-7), 94.1 (C-8), 157.7 (C-9), 104.4 (C-10), 121.6 (C-1ʹ), 115.9 (C-2ʹ), 145.7 (C-3ʹ), 149.0 (C-4ʹ), 116.1 (C-5ʹ), 121.2 (C-6ʹ), 100.2 (C-1ʹʹ), 74.5 (C-2ʹʹ), 77.6 (C-3ʹʹ), 71.3 (C-4ʹʹ), 75.4 (C-5ʹʹ), 65.

Table 1

 1 HPLC analysis of phenolic acids.

	Compounds	Retention time (min)	Relative (%)
	Gallic acid	3.00	5.3
	Caffeic acid	3.50	15.4
	Ellagic acid	4.00	26.4
	Cathechin	6.90	31.3
	Coumaric acid	11.17	21.1

Table 2

 2 Antiplasmodial activity and cytotoxicity of A. lindleyi extracts and some isolated compounds.

	Sample	IC 50 a ± SD (μg/mL)	CC 50 ± SD (μg/mL) b	Selectivity index c
		FcB1 strain	MCF-7	MCF-7/FcB1
	Pet ether extract	33	27.00	0.82
	Chloroform extract	28.500 0 ± 0.500 0	32.00	1.02
	Ethanol extract	33.000 0 ± 1.000 0	30.00	0.91
	Compound 2	63	Not tested	-
	Compound 3	> 100	Not tested	-
	Compound 6	33	Not tested	-
	Compound 8	9	Not tested	-
	Chloroquine	0.072 0 ± 0.015 0	9.70	134.00
	(n = 5)
	Sodium artesunate	0.002 4 ± 0.001 2 c	3.97 d	1 654.00
	Values were expressed as mean percentage of three replication ± SD; a :
	Drug concentration that killed 50% of FcB1 strain; b : Drug concentration
	(CC 50 ) needed to cause 50% decrease of the cellular viability of MCF-7; c :
	Cytotoxicity/antiplasmodial activity ratio;

d : Nepveu et al., 2010[29].

Table 4

 4 The DPPH radical scavenging activity (IC 50 ) of the extracts compared with vitamin C and Trolox as standard antioxidants.

	Sample	IC 50 a (mg/L) ± SD
	Pet ether extract	> 667
	Chloroform extract	332.46 ± 16.21
	Ethanol extract	345.70 ± 2.77
	Trolox	8.36 ± 0.05
	Ascorbic acid	3.80 ± 0.08
	Each value was expresssed as mean percentage of three replication ± SD; a :
	Concentration that inhibited DPPH absorbance by 50%.

Table 3

 3 Antibacterial and antifungal activity of A. lindleyi extracts. µg/mL. Thanks for French Centre for Culture and for Cooperation, French Embassy in Cairo, Egypt, for awarding a fellowship to Sahar Salah El Din El Souda. The authors thank the taxonomist Dr. Mona Marzouk for authenticating the plant material as well as Eliane Pelissou and Severine Maurel for technical assistance.

	Sample	S. aureus	P. aeruginosa	E. hirae	Escherichia coli	A. niger	C. albicans
		MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MFC	MIC	MFC
	Pet ether extract	125	500	62.5	250	125.00 > 500.00	125.00	500.00	125.00	-	62.50	125.00
	Chloroform extract > 500	> 500	62.5	125	125.00 > 500.00	125.00	500.00	125.00	-	62.50	125.00
	Ethanol extract	125	500	125.0	250	125.00 > 500.00	125.00	500.00	125.00	-	62.50	125.00
	Nalidixic acid	125	125	> 125.0 > 125	6.25	6.25	3.12	3.12	-	-	-	-
	Bifonazole	> 125	> 125	> 125.0 > 125	> 125.00 > 125.00	> 125.00 > 125.00	3.12	-	125.00 > 125.00
	Clotrimazole	> 125	> 125	> 125.0 > 125	> 125.00 > 125.00	> 125.00 > 125.00	1.56	-	3.12	6.25

Acknowledgments

This work was supported by the European Commission (FP6-LSH-20044-2.3.0-7, STREP No. 018602, Redox Antimalarial Drug Discovery, READ-UP). We thank J. P. Nallet for his scientific

Conflict of interest statement

We declare that we have no conflict of interest.


Michael Kreuter
email: kreuter@uni-heidelberg.de

M Toby

Maher

J Tamera

Maria Corte

Molina-Molina

Axmann Frank Judit

Gilberg

Kirchgaessler Vincent Klaus-Uwe

Cottin

T M Maher

T J Corte

M Molina-Molina

J Axmann

Á F Gilberg

K.-U Kirchgaessler

V Cottin

Pirfenidone in Unclassifiable Interstitial Lung Disease: A Subgroup Analysis by Concomitant Mycophenolate Mofetil and/or Previous Corticosteroid Use

Introduction: There are currently no approved treatments solely for unclassifiable interstitial lung disease (uILD); however, a recent trial showed this population can benefit from pirfenidone. We report a subgroup analysis of this trial to assess the effects of immunomodulators (concomitant mycophenolate mofetil [MMF] and/or previous corticosteroids) with pirfenidone in patients with uILD. Methods: This was a multicenter, international, double-blind, randomized, placebo-controlled phase II trial of patients with progressive fibrosing uILD (NCT03099187). Patients were randomized (1:1) to receive pirfenidone

2403 mg/day or placebo. This analysis assessed forced vital capacity (FVC) change from baseline measured using site spirometry (key secondary endpoint) and safety over 24 weeks by concomitant MMF use at randomization (prespecified analysis) and/or previous corticosteroid use (post hoc analysis). Results: Overall, 253 patients were randomized, including 45 (17.8%) patients (pirfenidone, n = 23; placebo, n = 22) receiving concomitant MMF with/without previous corticosteroids (MMF subgroup); 79 (31.2%) patients (pirfenidone, n = 44; placebo, n = 35) receiving previous corticosteroids without MMF (corticosteroids/no-MMF subgroup); and 129 (51.0%) patients (pirfenidone, n = 60; placebo, n = 69) not receiving concomitant MMF or previous corticosteroids (no-corticosteroids/no-MMF subgroup). At 24 weeks, difference in mean (95% confidence interval) FVC change from baseline between pirfenidone and placebo was -55.4 mL (-206.7, 96.0; P = 0.4645) in the MMF subgroup; 128.4 mL (-6.4, 263.3; P = 0.0617) in the corticosteroids/no-MMF subgroup; and 115.5 mL (35. [START_REF] Ryerson | Update on the diagnosis and classification of ILD[END_REF]195.9; P = 0.0052) in the no-corticosteroids/no-MMF subgroup. All subgroups generally exhibited a similar pattern of treatment-emergent adverse events. Conclusion: Although limited by design and small sample sizes, this analysis suggests pirfenidone may be less effective in patients with uILD receiving concomitant MMF, whereas a beneficial treatment effect was observed in patients not receiving concomitant MMF regardless of previous corticosteroid use. Pir Diagnosis of a specific interstitial lung disease (ILD) is important for identifying the most appropriate management strategy and informing disease prognosis; however, despite thorough investigation by a multidisciplinary team, some patients are diagnosed with unclassifiable ILD (uILD).

There are currently no approved treatments solely indicated for uILD; however, a recent 24-week phase II clinical trial that evaluated the efficacy and safety of pirfenidone versus placebo in patients with progressive fibrotic uILD suggested that this population of patients can benefit from pirfenidone treatment.

The subgroup analysis of this phase II clinical trial aimed to assess the effects of immunomodulators (concomitant mycophenolate mofetil [MMF] and/or previous corticosteroids) with pirfenidone in patients with uILD.

What was learned from the study?

Despite the limitation of a small sample size, this subgroup analysis suggests that pirfenidone may be less effective in patients with uILD receiving MMF at randomization, whereas a beneficial treatment effect for pirfenidone on forced vital capacity change was observed in patients not receiving MMF at randomization regardless of previous corticosteroid use; pirfenidone was well tolerated regardless of MMF and/or corticosteroid use.

Further research is needed to explore these findings in a larger group of patients in a study appropriately designed to determine the effects of concomitant immunomodulators with pirfenidone in patients with progressive uILD.

INTRODUCTION

Interstitial lung diseases (ILDs) are a large, heterogeneous group of diseases characterized by abnormalities of pulmonary interstitium or alveoli, including fibrosis [START_REF] Ryerson | Update on the diagnosis and classification of ILD[END_REF]. Although some ILDs have a progressive fibrosing phenotype similar to idiopathic pulmonary fibrosis (IPF), which is the most common form of idiopathic interstitial pneumonia [START_REF] Cottin | Presentation, diagnosis and clinical course of the spectrum of progressive-fibrosing interstitial lung diseases[END_REF], the clinical course of other ILDs is variable [START_REF] Ryerson | Update on the diagnosis and classification of ILD[END_REF][START_REF] Guler | Heterogeneity in unclassifiable interstitial lung disease. A systematic review and meta-analysis[END_REF][START_REF] Skolnik | Unclassifiable interstitial lung disease: a review[END_REF].

Diagnosis of a specific ILD is important for identifying the most appropriate management strategy and informing disease prognosis [START_REF] Ryerson | Update on the diagnosis and classification of ILD[END_REF][START_REF] Guler | Heterogeneity in unclassifiable interstitial lung disease. A systematic review and meta-analysis[END_REF][START_REF] Skolnik | Unclassifiable interstitial lung disease: a review[END_REF]. However, despite thorough investigation by a multidisciplinary team, some patients are diagnosed with unclassifiable ILD (uILD) due to nonspecific or conflicting clinical, radiological, or histopathological findings or where invasive diagnostic procedures are inappropriate or not possible [START_REF] Ryerson | Update on the diagnosis and classification of ILD[END_REF][START_REF] Guler | Heterogeneity in unclassifiable interstitial lung disease. A systematic review and meta-analysis[END_REF][START_REF] Skolnik | Unclassifiable interstitial lung disease: a review[END_REF]. Adding to the heterogeneity of the uILD population is a subgroup of patients that meet criteria for the research classification scheme of interstitial pneumonia with autoimmune features (IPAF), who often fall within the category of uILD [START_REF] Guler | Heterogeneity in unclassifiable interstitial lung disease. A systematic review and meta-analysis[END_REF][START_REF] Fischer | An official European Respiratory Society/American Thoracic Society research statement: interstitial pneumonia with autoimmune features[END_REF].

Small retrospective cohort studies and a case study have shown potential benefits of immunomodulators including mycophenolate mofetil (MMF) and corticosteroids in some patients with IPAF [START_REF] Chartrand | Clinical features and natural history of interstitial pneumonia with autoimmune features: a single center experience[END_REF][START_REF] Mccoy | Mycophenolate therapy in interstitial pneumonia with autoimmune features: a cohort study[END_REF][START_REF] Wiertz | Cyclophosphamide in steroid refractory unclassifiable idiopathic interstitial pneumonia and interstitial pneumonia with autoimmune features (IPAF)[END_REF][START_REF] Sasano | Long-term clinical course of anti-glycyl tRNA synthetase (anti-EJ) antibody-related interstitial lung disease pathologically proven by surgical lung biopsy[END_REF] or other uILDs [START_REF] Leung | Unclassifiable interstitial lung disease: an unresolved diagnostic dilemma[END_REF]. Moreover, immunomodulators are frequently used as concomitant medications in patients with fibrosing ILDs in clinical studies or in clinical practice [START_REF] Distler | Nintedanib for systemic sclerosis-associated interstitial lung disease[END_REF][START_REF] Cottin | Nintedanib and immunomodulatory therapies in progressive fibrosing interstitial lung diseases[END_REF][START_REF] Wijsenbeek | Progressive fibrosing interstitial lung diseases: current practice in diagnosis and management[END_REF].

There are currently no approved treatments solely indicated for uILD, but nintedanib is now approved for progressive fibrosing ILDs, including uILD, on the basis of its efficacy and safety demonstrated in the INBUILD study [START_REF] Cottin | Nintedanib and immunomodulatory therapies in progressive fibrosing interstitial lung diseases[END_REF][START_REF] Flaherty | Nintedanib in progressive fibrosing interstitial lung diseases[END_REF]. Moreover, a recent 24-week phase II clinical trial that evaluated the efficacy and safety of pirfenidone versus placebo in patients with progressive fibrotic uILD suggested that this population of patients can benefit from antifibrotic treatment with pirfenidone [START_REF] Maher | Pirfenidone in patients with unclassifiable progressive fibrosing interstitial lung disease: a double-blind, randomised, placebo-controlled, phase 2 trial[END_REF]. It should be noted that the primary endpoint (forced vital capacity [FVC] measured by home spirometry) could not be analyzed in this trial because of technical and analytical issues with home spirometry. Therefore, the beneficial effect of pirfenidone in patients with uILD was based on the analysis of the key secondary endpoints, including change in FVC measured by site spirometry, an outcome which has been utilized as a primary endpoint in clinical trials for IPF [START_REF] Richeldi | Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis[END_REF][START_REF] King Te | A phase 3 trial of pirfenidone in patients with idiopathic pulmonary fibrosis[END_REF] and other progressive fibrosing ILDs [START_REF] Flaherty | Nintedanib in progressive fibrosing interstitial lung diseases[END_REF][START_REF] Behr | Pirfenidone in patients with progressive fibrotic interstitial lung diseases other than idiopathic pulmonary fibrosis (RELIEF): a double-blind, randomised, placebocontrolled, phase 2b trial[END_REF]. Moreover, pirfenidone treatment was efficacious and well tolerated in patients with other non-IPF progressive fibrosing ILDs, including connective tissue disease-associated ILD, fibrotic nonspecific interstitial pneumonia, chronic hypersensitivity pneumonitis, and asbestos-induced lung fibrosis, in the RELIEF trial [START_REF] Behr | Pirfenidone in patients with progressive fibrotic interstitial lung diseases other than idiopathic pulmonary fibrosis (RELIEF): a double-blind, randomised, placebocontrolled, phase 2b trial[END_REF].

Here, we report a subgroup analysis of the phase II trial of pirfenidone in patients with uILD to assess the effects of immunomodulators (concomitant MMF and/or previous corticosteroids) with pirfenidone in these patients.

METHODS

This was a multicenter, international, doubleblind, randomized, placebo-controlled phase II trial of patients with progressive fibrosing uILD (ClinicalTrials.gov: NCT03099187), the methods of which have been previously described [START_REF] Maher | Pirfenidone in patients with unclassifiable progressive fibrosing interstitial lung disease: a double-blind, randomised, placebo-controlled, phase 2 trial[END_REF][START_REF] Maher | Pirfenidone in patients with unclassifiable progressive fibrosing interstitial lung disease: design of a double-blind, randomised, placebo-controlled phase II trial[END_REF]. The trial was done in accordance with the ethical principles of the Good Clinical Practice guidelines and the Declaration of Helsinki, and local laws for countries in which the research was done. Informed consent was obtained from each participant by the study investigator before any study-specific screening procedures were done.

Patients were randomized in a 1:1 ratio to receive pirfenidone 2403 mg/day or placebo. Randomization was stratified by concomitant MMF use. Patients were permitted to receive a maximum MMF dose of 1.5 g twice daily; otherwise, the dosage was at the discretion of the investigator. Patients who were not taking MMF at the time of enrollment were not permitted to start MMF during the trial. Patients were not permitted to receive any other immunomodulators during the trial or to receive treatment with high-dose systemic corticosteroids (i.e., more than 15 mg/day of prednisolone or equivalent) for more than 4 weeks during the trial.

The primary endpoint was predicted mean change in FVC (in milliliters) measured using daily home spirometry over 24 weeks. As previously reported, the primary endpoint could not be analyzed because of technical issues with the home spirometers and the inclusion of patients with a small number of readings collected within a short time window [START_REF] Maher | Pirfenidone in patients with unclassifiable progressive fibrosing interstitial lung disease: a double-blind, randomised, placebo-controlled, phase 2 trial[END_REF]. Examples of FVC readings recorded at home included daily values of less than 0.5 L or greater than 6 L, and predicted increases of 33 L at 24 weeks, which are clearly impossible. As the statistical assumptions for applying a Student's t test could not be fulfilled, the pre-specified model could not be applied to the home spirometry data [START_REF] Maher | Pirfenidone in patients with unclassifiable progressive fibrosing interstitial lung disease: a double-blind, randomised, placebo-controlled, phase 2 trial[END_REF]. Secondary endpoints included change in FVC from baseline measured by spirometry during clinic visits, change in percentage predicted carbon monoxide diffusing capacity (DLco) from baseline, and change in 6-min walk distance (6MWD) from baseline.

Primary and secondary efficacy endpoints were assessed in the intention-to-treat (ITT) population, which included all randomly assigned patients. Safety was assessed in the safety analysis set (SAS), which included all randomly assigned patients who received at least one dose of study drug. The incidence and severity of treatment-emergent adverse events (TEAEs) and withdrawals from study treatment or study discontinuations were recorded.

In this subgroup analysis, we investigated baseline characteristics, changes in FVC (in milliliters and percentage predicted) from baseline measured using site spirometry, change in percentage predicted DLco from baseline, change in 6MWD from baseline, and safety by concomitant MMF use at randomization (prespecified analysis), and/or previous corticosteroid use (all patients who started corticosteroids prior to or at baseline; post hoc analysis), all over 24 weeks. It should be noted that patients who received corticosteroids after baseline were not included in the analyses because those patients started treatment at different times and for different durations, which may have confounded the results of the analyses.

The change in FVC (percentage predicted and in milliliters) between baseline and week 24 measured by site spirometry was estimated from a linear regression model. The mean change in FVC was compared between treatment groups using a Student's t test with a two-sided significance level of 5%. Changes in percentage predicted FVC, percentage predicted DLco, and 6MWD were compared between treatment groups using a rank analysis of variance model, with change from baseline used as an outcome variable and standardized rank baseline value used as a covariate. Categorical changes in percentage predicted FVC (greater than 5% and greater than 10%) were compared between treatment groups using a Cochran-Mantel-Haenszel test. Categorical changes in percentage predicted DLco (greater than 15%) were compared between treatment groups using logistic regression or Fisher's exact test, where appropriate. Categorical changes in 6MWD (greater than 50 m) were compared between treatment groups using logistic regression.

RESULTS

Overall, 253 patients were randomized and included in the ITT analysis set (pirfenidone, n = 127; placebo, n = 126). The SAS included 251 patients (pirfenidone, n = 127; placebo, n = 124). The present analysis included 45 (17.8%) patients (pirfenidone, n = 23; placebo, n = 22) who received concomitant MMF treatment with or without previous corticosteroid treatment (MMF subgroup), 79 (31.2%) patients (pirfenidone, n = 44; placebo, n = 35) who received previous corticosteroid treatment without MMF (corticosteroids/no-MMF subgroup), and 129 (51.0%) patients (pirfenidone, n = 60; placebo, n = 69) who received no concomitant MMF treatment or previous corticosteroid treatment (no-corticosteroids/no-MMF subgroup). In total, 21 (91.3%) and 17 (77.3%) patients receiving concomitant MMF treatment also received concomitant corticosteroid treatment during the double-blind period in the pirfenidone and placebo groups, respectively. At 24 weeks, the mean (95% confidence interval [CI]) predicted FVC change from baseline was -132.8 mL (-253.2, -12.4) in the pirfenidone group and -77.5 mL (-176.6, 21.6) in the placebo group for the MMF subgroup (difference -55.4 mL [-206.7, 96.0]; P = 0.4645); -16.5 mL (-100.7, 67.7) in the pirfenidone group and -144.9 mL (-256.7, -33.2) in the placebo group for the corticosteroids/no-MMF subgroup (difference 128.4 mL [-6.4, 263.3]; P = 0.0617); and 12.8 mL (-54.1, 79.7) in the pirfenidone group and -102.7 mL (-151.5, -53.8) in the placebo group for the no-corticosteroids/no-MMF subgroup (difference 115.5 mL [35. [START_REF] Ryerson | Update on the diagnosis and classification of ILD[END_REF]195.9]; P = 0.0052; Fig. 1). Categorical change in FVC of greater than 5% or greater than 10%, change in percentage predicted DLco, and change in 6MWD by concomitant MMF and previous corticosteroid use are shown in Table 2.

Generally, a similar pattern of TEAEs was observed across all subgroups (Table 3). The incidence of treatment-related TEAEs was slightly higher in the MMF subgroup (82.6%) than the other subgroups (68.2-68.3%) for pirfenidone-treated patients. The incidence of treatment-related TEAEs was lower in the corticosteroids/no-MMF subgroup (37.1%) than in the other subgroups (49. [START_REF] Guler | Heterogeneity in unclassifiable interstitial lung disease. A systematic review and meta-analysis[END_REF]

DISCUSSION

Although the overall results of the uILD study suggested that patients with progressive fibrosing uILD can benefit from pirfenidone over 24 weeks, this subgroup analysis suggests that pirfenidone may be less effective in patients receiving concomitant MMF at randomization versus those not receiving MMF. A numerically beneficial treatment effect for pirfenidone versus placebo on FVC change was observed regardless of previous corticosteroid use in patients not receiving MMF, although the beneficial effect in the corticosteroids/no-MMF subgroup was not statistically significant. This was most likely as a result of small samples sizes and because the study was not designed nor powered to show differences between corticosteroid subgroups. It is important to note that although the patient demographics and clinical characteristics were generally similar irrespective of concomitant MMF and/or previous corticosteroid use, mean percentage predicted FVC at baseline was higher in the no-corticosteroids/ no-MMF subgroup compared with the MMF and corticosteroids/no-MMF subgroups. This difference in FVC at baseline may have contributed to the lack of statistically significant effect of pirfenidone on FVC decline in the MMF and corticosteroids/no-MMF subgroups versus the no-corticosteroids/no-MMF subgroup. In general, FVC measurements can be highly variable between patients, and small sample sizes can lead to large CIs, which were also observed in these analyses. Variable FVC measurements suggest that efficacy may differ depending on patients' genotype and/or phenotype, and as PTs nodular rash, rash, rash erythematous, rash generalized, rash macular, rash maculopapular, rash papular, rash pruritic, rash follicular, exfoliative rash, solar dermatitis, solar urticarial, sunburn, erythema, dry skin such, more research and accurate disease genotyping and phenotyping are required.

The SENSCIS study of nintedanib in systemic sclerosis-associated (SSc)-ILD suggested that MMF may have an additive effect in reducing FVC decline [START_REF] Distler | Nintedanib for systemic sclerosis-associated interstitial lung disease[END_REF]; however, the present study indicated that pirfenidone may be less effective with concomitant MMF use. This may be because SENSCIS was in a patient population with SSc-ILD, since immunomodulators including MMF have been shown to benefit this population as a result of the autoimmune nature of the disease [START_REF] Tashkin | Cyclophosphamide versus placebo in scleroderma lung disease[END_REF][START_REF] Tashkin | Mycophenolate mofetil versus oral cyclophosphamide in scleroderma-related interstitial lung disease (SLS II): a randomised controlled, doubleblind, parallel group trial[END_REF]. However, uILD populations are more heterogeneous than those with a specific ILD diagnosis, such as SSc-ILD [START_REF] Skolnik | Unclassifiable interstitial lung disease: a review[END_REF], so immunomodulators may not be consistently beneficial in this population. For example, it has been previously suggested that treatment with the immunomodulator cyclophosphamide may be beneficial for patients with IPAF but may be less effective in patients with non-IPAF uILD [START_REF] Wiertz | Cyclophosphamide in steroid refractory unclassifiable idiopathic interstitial pneumonia and interstitial pneumonia with autoimmune features (IPAF)[END_REF]. Moreover, the PANTHER-IPF trial showed that treatment with immunomodulators (a combination of prednisone, azathioprine, and N-acetylcysteine) was associated with detrimental outcomes including increased rate of death and hospitalization versus placebo in patients with IPF [START_REF] Raghu | Prednisone, azathioprine, and N-acetylcysteine for pulmonary fibrosis[END_REF]. Considering that some patients with uILD have a similar disease course to IPF again suggests that immunomodulators may not benefit all patients with uILD. It is important to note that a greater proportion of patients (48.4%) in SENSCIS were receiving MMF compared with the present study (17.8%) and that previous corticosteroid use was not investigated in SEN-SCIS [START_REF] Distler | Nintedanib for systemic sclerosis-associated interstitial lung disease[END_REF].

Unlike the present study, the INBUILD study reported that the introduction of immunomodulatory therapies, including MMF, during the study did not affect the benefit of nintedanib in patients with progressive fibrosing non-IPF ILD on reducing FVC decline versus placebo, although fewer patients were receiving MMF in INBUILD than in the present study (2.7% vs 17.8% [at randomization]) since this was a restricted therapy in INBUILD [START_REF] Cottin | Nintedanib and immunomodulatory therapies in progressive fibrosing interstitial lung diseases[END_REF]. Moreover, INBUILD reported that the use of glucocorticoids at baseline (54.4% of patients) did not affect the benefit of nintedanib on reducing FVC decline versus placebo, although it should be noted that high-dose (greater than 20 mg/day) glucocorticoids were restricted [START_REF] Cottin | Nintedanib and immunomodulatory therapies in progressive fibrosing interstitial lung diseases[END_REF]; similarly, the present study showed a beneficial treatment effect for pirfenidone on FVC change in patients not taking MMF regardless of previous corticosteroid use (31.2% of total patients). Likewise, the RELIEF trial showed that pirfenidone treatment was efficacious in patients with other non-IPF progressive fibrosing ILDs, the majority of whom were receiving immunomodulators at baseline (73% and 89% in the pirfenidone and placebo groups, respectively) [START_REF] Behr | Pirfenidone in patients with progressive fibrotic interstitial lung diseases other than idiopathic pulmonary fibrosis (RELIEF): a double-blind, randomised, placebocontrolled, phase 2b trial[END_REF].

The safety profile of pirfenidone versus placebo was generally similar between all the subgroups. The incidence of treatment-related TEAEs was higher in the MMF subgroup than the no-corticosteroids/no-MMF subgroup in pirfenidone-treated patients; this increase in treatment-related TEAEs appears to be driven by an increase in mild-to-moderate GI disorders in the MMF subgroup. The LOTUSS trial of pirfenidone in SSc-ILD also found that the safety profile of pirfenidone was comparable in the patients who did or did not take concomitant MMF; however, there was a lower proportion of patients reporting severe TEAEs or TEAEs leading to discontinuation in the MMF versus no-MMF subgroup [START_REF] Khanna | An open-label, phase II study of the safety and tolerability of pirfenidone in patients with scleroderma-associated interstitial lung disease: the LOTUSS trial[END_REF], which was not the case in the current study.

Limitations of the present analyses include the low patient numbers, meaning that these data should be interpreted with caution and firm conclusions cannot be drawn. In addition, the analysis by corticosteroid use was a post hoc analysis (leading to subgroups of different sizes as a result of corticosteroid use not being a stratification factor, unlike the analysis by MMF use). The analysis by MMF use was restricted to use of MMF treatment at randomization, whereas the analysis by corticosteroid use included all patients who started corticosteroids prior to or at baseline. Therefore, it is unclear whether patients in the MMF or corticosteroid subgroups received these treatments throughout the study. Furthermore, no clinical information relating to the reasons for treating patients with MMF was available, but it is possible that the clinical features of patients who received MMF were different to those who did not. Additionally, patients with uILD are a heterogeneous population [START_REF] Guler | Heterogeneity in unclassifiable interstitial lung disease. A systematic review and meta-analysis[END_REF]; some have a predominantly fibrotic phenotype like IPF whereas others have a predominantly inflammatory phenotype (including patients with IPAF) or a combination of both fibrotic and inflammatory phenotypes, which may mean that the effect of pirfenidone treatment varies on a case-by-case basis. It should be noted that the study included patients who did not have a biopsy, which is reflective of clinical reality where many patients will not or cannot have a biopsy. It is therefore possible that some of these patients may have had an underlying pathological pattern of usual interstitial pneumonia, which could have led to a diagnosis of IPF if they had a biopsy. Although, it is important to note that even patients with a low-confidence IPF diagnosis were excluded from the study. However, the exclusion of patients with a low-confidence IPF diagnosis, as well as patients who received corticosteroids after baseline, may have contributed to the small sample size, and the patients who were included in the current analyses may not be reflective of patients with uILD treated during real-world clinical practice. A further limitation of these analyses is that patients were not permitted to receive any other immunomodulators during the trial. Although MMF and corticosteroids are the common agents of choice, other immunomodulators may also be used to treat patients with uILD (e.g., cyclophosphamide); as such, further evaluation including a broader range of immunomodulators is required.

CONCLUSION

Although limited by design and small sample sizes, this subgroup analysis suggests that pirfenidone may be less effective in patients with uILD receiving MMF at randomization, whereas a beneficial treatment effect for pirfenidone on FVC change was observed in patients not receiving MMF at randomization regardless of previous corticosteroid use. Pirfenidone was well tolerated in patients with uILD regardless of MMF and/or corticosteroid use. Further research is needed to explore these findings in a larger group of patients in a study appropriately designed to determine the effects of concomitant immunomodulators with pirfenidone in patients with progressive fibrosing uILD.

Funding. This study is sponsored by F. Hoffmann-La Roche, Ltd., Basel, Switzerland. This trial was initially developed as part of an NIHR-funded Clinician Scientist Fellowship awarded to Toby M. Maher (NIHR reference CS-2013-13-017) and subsequently adapted following discussions with F. Hoffmann-La Roche, Ltd. The Advances in Therapy Rapid Service Fees and Open Access Fees are funded by F. Hoffmann-La Roche, Ltd. Prior Presentation. Some of the results of the study were presented as a poster at the virtual American Thoracic Society Congress 2020 (August 5-10, 2020).

Authorship. All named authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship of this article, take responsibility for the integrity of the work as a whole, and have given their approval for this final version to be published.

Author Contributions. All authors were involved in the conception and/or design of the work and interpretation of study results, contributed to the brief report from the outset, and read and approved the final draft. Data analysis was performed by FG. All authors vouch for the

Fig. 1

 1 Fig. 1 Mean (95% CI) change from baseline to week 24 in FVC (measured by site spirometry) for pirfenidone versus placebo by concomitant MMF use at randomization and/or previous corticosteroid use. FVC (mL) decline at week 24 measured by site spirometry and estimated from linear regression model. Two-sided 95% CI for mean value is based on percentiles of the t distribution. CI confidence

  Photosensitivity c

  Medical Writing, Editorial, and Other Assistance. Medical writing support was provided by Adele Edwards, PhD, of CMC AFFI-NITY, McCann Health Medical Communications, funded by F. Hoffmann-La Roche, Ltd.

Table 1

 1 Demographic and baseline characteristics of the intention-to-treat population (n = 253)

	Pirfenidone

  Overall, 33 patients in the study had an IPAF diagnosis (pirfenidone, n = 15 and placebo, n = 18); 12 of these patients were in the MMF subgroup (pirfenidone, n = 6; placebo, n = 6).Demographics and baseline characteristics of the ITT population are shown in Table1.

	0 (0.0) Demographics were similar between all con-29.3 (5.3) 79.0 (20.8) 51.8 (13.8) (n = 64) 82.0 (21.3)
	comitant MMF and previous corticosteroid
	subgroups (mean age 63.4-70.4 years; male
	1 (2.9) 50.7-60.9%; white race 91.3-100.0%). Mean 28.9 (5.3) 66.7 (17.0) (n = 34) 48.0 (13.8) (n = 32) (n = 34) percentage predicted FVC at baseline measured by site spirometry was similar between the MMF 71.2 (20.9) subgroup (68.7-72.6%) and corticosteroids/no-
	MMF subgroup (66.7-68.2%) but was slightly
	higher in the no-corticosteroids/no-MMF sub-
	29.8 (5.7) 30.2 (5.6) group (79.0-80.6%). Mean percentage predicted 80.6 (18.0) (n = 59) 72.6 (12.4) DLco (43.9-51.8%) and mean 6MWD (373.5-412.6 m) at baseline were similar 49.0 (11.2) (n = 57) 46.4 (11.2) (n = 21) between all concomitant MMF and previous 83.6 (18.5) (n = 58) 78.0 (14.6) corticosteroid subgroups.
	30.1 (6.7)		68.2 (17.5)	(n = 43)		45.3 (13.2)	(n = 40)		70.5 (17.5)	(n = 43)
	30.8 (6.0)		68.7 (18.1)			43.9 (9.5) (n = 22)			72.4 (16.8)
	Body mass index,	kg/m 2	Percentage	predicted FVC,	%	Percentage	predicted DLco,	%	Percentage	1 , predicted FEV	%

Table 3

 3 TEAEs in the safety analysis set (n = 251)

	Pirfenidone

  drug-induced liver injury that include an elevated ALT or AST in combination with either an elevated bilirubin or clinical jaundice, as defined by Hy's law (ALT or AST [

	) 12 (17.9)	PT Medical Dictionary for Regulatory Activities
	Data are n (%)	ALT alanine aminotransferase, AST aspartate aminotransferase, GI gastrointestinal, MMF mycophenolate mofetil,	Preferred Term, SOC system organ class, ULN upper limit of normal TEAE treatment-emergent adverse event,	3 9 ULN ? total bilirubin [ 2 9 ULN)

a Cases of potential b SOC GI disorders c PTs photodermatosis, photosensitivity reaction, pruritus, pruritus allergic, pruritus generalized d

ACKNOWLEDGEMENTS

We would like to thank the patients, their family members, and participating staff at all of the study centers.

accuracy of the content included in the final brief report.

Disclosures. Michael Kreuter has received fees for speaking and/or organizing education from AstraZeneca, Bayer, Boehringer Ingelheim, Chiesi, ERS, F. Hoffmann-La Roche, Ltd., GlaxoSmithKline, and Novartis; has received fees for consulting from Boehringer Ingelheim, F. Hoffmann-La Roche, Ltd., Galapagos, GlaxoSmithKline, and InterMune; and has received research funding, including institutional funding, from BMBF, Boehringer Ingelheim, DFG, the German Center for Lung Research (DZL), F. Hoffmann-La Roche, Ltd., Hopp Stiftung, InterMune, Lilly, Lungenfibrose E.V., Medac, Olympus, UKGM, and WATL. Toby M. Maher is supported by an NIHR Clinician Scientist Fellowship (NIHR reference CS-2013-13-017) and a British Lung Foundation Chair in Respiratory Research (C17-3); has received industry-academic research funding from GlaxoSmithKline R&D and UCB; has served on a clinical trial advisory board for GlaxoSmithKline R&D; has received stock options from Apellis; and has received consultancy or speaker fees from AstraZeneca, Bayer, Biogen Idec, Boehringer Ingelheim, Cipla, F. Hoffmann-La Roche, Ltd., GlaxoSmithKline R&D, ProMetic, Samumed, and UCB. Tamera J. Corte has received unrestricted educational grants, travel assistance, and speaker fees from, and has served on advisory boards for, Boehringer Ingelheim; has received unrestricted educational grants and speaker fees and has served on advisory boards for F. Hoffmann-La Roche, Ltd.; has received unrestricted educational grants from Actelion, Bayer, Galapagos, and Sanofi; and has served on advisory boards for AstraZeneca, Bristol Myers Squibb, and Promedior (now a fully owned subsidiary of F. Hoffmann-La Roche, Ltd.). Maria Molina-Molina, or her institution, has received grants from AstraZeneca, Boehringer Ingelheim, BRN, Chiesi, F. Hoffmann-La Roche, Ltd., GlaxoSmithKline, and InterMune. Judit Axmann and Klaus-Uwe Kirchgaessler are employees and shareholders of F. Hoffmann-La Roche, Ltd. Frank Gilberg is an employee of F. Hoffmann-La Roche, Ltd. Vincent Cottin reports personal fees and non-financial support from Actelion, F. Hoffmann-La Roche, Ltd., and Promedior (now a fully owned subsidiary of F. Hoffmann-La Roche, Ltd.); grants, personal fees, and non-financial support from Boehringer Ingelheim; personal fees from AstraZeneca, Bayer/MSD, Celgene/ BMS, FibroGen, Galapagos, Galecto, Novartis, PureTech, RedX, Sanofi, and Shionogi, outside the submitted work.

Compliance with Ethics Guidelines. The trial was done in accordance with the ethical principles of the Good Clinical Practice guidelines and the Declaration of Helsinki, and local laws for countries in which the research was done. Informed consent was obtained from each participant by the study investigator before any study-specific screening procedures were done.

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