RBx 7796: A novel inhibitor of 5-lipoxygenaseR. K. Shirumalla1, K. S. Naruganahalli1, S. G. Dastidar1, V. Sattigeri2, G. Kaur1, C. Deb1, J. B Gupta1, M. Salman 2, A. Ray1
1 Department of Pharmacology, New Drug Discovery Research, Ranbaxy Research Laboratories, Plot No-20, Sector-18, Udyog Vihar, Gurgaon, Haryana 122015, India, Fax: ++91-124-2343544, e-mail: [email protected]
2 Department of Medicinal Chemistry, New Drug Discovery Research, Ranbaxy Research Laboratories, Gurgaon, Haryana, India
Received 9 March 2006; returned for revision 17 May 2006; accepted by I. Ahnfelt-Rønne 19 May 2006
Published Online First 13 October 2006
Abstract. Objective: To evaluate the pharmacological pro-fi le of RBx 7796, a novel 5-lipoxygenase inhibitor.Materials and methods: RBx 7796 was evaluated for 5-lipoxygenase inhibitory potential using human recombinant enzyme and profi led for selectivity against 12 and 15 lipox-ygenase. RBx 7796 was evaluated in cell based assay for inhibition of A23187 induced LTB4 release from isolated neutrophils. Ex vivo activity was evaluated for inhibition of A23187 induced LTB4 release in blood from treated rats. In vivo effi cacy of RBx 7796 was profi led in LPS induced neu-trophilia model in rats and also in ovalbumin induced bron-choconstriction and airway infl ammation models in guinea pigs. Results: RBx 7796, a novel chemotype, showed competitive inhibition of 5-lipoxygenase enzyme with an IC50 of 3.5 ± 1.1 µM. RBx 7796 offered >100 fold selectivity against other related enzymes – 12 and 15 lipoxygenase. RBx 7796 inhib-ited release of LTB4 from human and rat neutrophils in vitro. Upon administration to rats, RBx 7796 inhibited A23187 induced LTB4 release from rat neutrophils. Upon repeated administration, dosed once daily, RBx 7796 inhibited LPS induced neutrophil infl ux in rat airway. RBx 7796 also in-hibited allergen induced bronchoconstriction and eosinophil infl ux in guinea pig airway in a dose dependent manner. Conclusion: The results suggest that RBx 7796, a novel chemotype, is an orally effi cacious inhibitor of 5-lipoxyge-nase enzyme that is effective against both neutrophilic and eosinophilic airway infl ammation and shows potent inhibi-tion with once daily administration.
Incidence of infl ammatory airway diseases like bronchial asthma and chronic obstructive pulmonary disease is on
the rise world over [1–3]. The molecular and cellular basis of both diseases have been reviewed extensively [4–7]. It has now become clear that products of arachidonic acid breakdown, cysteinyl leukotrienes and leukotriene B4, play important roles in the initiation and perpetuation of infl ammatory processes in asthma and COPD, respec-tively [8–15]. 5-Lipoxygenase is the enzyme that catalyses fi rst step in the conversion of arachidonic acid to cysteinyl leukotrienes and leukotriene B4 [16]. This enzyme is ideally suited to be target for drug development because of its ability to control genera-tion of cysteinyl leukotrienes and leukotriene B4. Synthetic inhibitors of 5-lipoxygenase enzyme have shown protective effect in experimental animal models of airway infl ammation and in human bronchial asthma [17–22]. Although, consid-erable effort has been made to design an effective inhibitor of 5-lipoxygenase, at the present time the most successful chemotype belongs to N-hydroxyurea category [16, 23–26]. This chemotype reportedly suffers from poor bioavailabil-ity; inhibitor specifi city and safety issues like hepatic and renal abnormality, myelosuppression, mild gastrointestinal abnormality etc [26-27]. Zileuton, the FDA approved 5-lipoxygenase inhibitor also suffers from poor potency, poor pharmacokinetic properties, cytochrome P450 liabilities [28] and hepatotoxicity due to formation of a metabolite 2-acetyl benzothiophene [29]. Efforts to move away from the N-hydroxyurea chemotype have not really been successful. Several molecules have shown need for reducing environ-ment for inhibiting 5-lipoxygenase enzyme. Some of these molecules were effective in acute infl ammation models but failed to offer protection in chronic models of infl ammation in experimental animals and in clinic [16, 26, 30, 31]. Re-ports exist to suggest that long chain fatty acids [32, 33] and molecules like α-Tocopherol [34] with long carbon chain can inhibit 5-lipoxygenase, albeit, with weak II-potency. Through extensive medicinal chemistry efforts, we have dis-covered RBx 7796 (Clafrinast) – a distinct chemical entity from N-hydroxyurea, that inhibits 5-lipoxygenase enzyme in a competitive manner and exhibits effi cacy in several experi-mental animal models of airway infl ammation. Correspondence to: Abhijit Ray
518 R. K. Shirumalla et al. Infl amm. res.
Materials and methods
Animals
Male Wistar rats (270–300 g) and male Dunken–Hartly Guinea pigs (350–500 g) were housed in temperature and humidity controlled envi-ronment, on a 12 h light/dark cycle with free access to food and water. All experiments were conducted as per the guidelines of the Institutional Animal Ethics Committee and those of Government of India, and all efforts were made to minimise the animals suffering and the number of animals used.
Materials
RBx 7796 and Montelukast were obtained from Ranbaxy Laboratories Ltd. (New Delhi, India). Zileuton was obtained from Beijing Medik-ing Pharmaceutical Co. Ltd., (Beijing, China). LTB4 ELISA kit was obtained from Assay designs (Ann Arbor, MI, USA). Human recom-binent 5-lipoxygenase, porcine leukocyte 12-lipoxygenase and soybean 15-lipoxygenase were sourced from Cayman Chemicals Co. (Ann Ar-bor, MI, USA). LPS, Ovalbumin, Ca2+-ionophore (A23187), Arachi-donic acid and dithiothreitol were obtained from Sigma-Aldrich Chemie Gmbh (Steinheim, Germany). 14C Arachidonic acid was from Amersham Biosciences (Piscataway, NJ, USA).
5-Lipoxygenase Assay Assay was carried out in 96 well UV plate containing 100 µL reaction mixture (DTT, 200 µM; ATP, 100 µM; and calcium chloride, 100 µM; in phosphate buffered saline) in the absence and presence of different con-centrations of RBx 7796 or Zileuton (100 nM–30 µM) and 12 U (3 U/µl) of human recombinant 5-lipoxygenase (Cayman Chemicals Co., USA). Reaction mixture was incubated at 37 °C for 5 min, and reaction initiated by adding 1 µl of 1 mM freshly prepared arachidonic acid. Increase in absorbance was monitored at 234 nm for 10 min [35]. A plot of absorb-ance vs. time curve was prepared and area under curve (AUC) was com-puted for each well. Percent inhibition of AUC for different treatments was calculated with respect to the difference between the Arachidonic acid stimulated and negative control values, to compute IC50 values. The same assay was repeated in the absence of DTT to mimic non-reducing conditions and IC50 values were computed.
In another series of experiments, neutrophil lysate was also used as enzyme source. In this assay blood was collected from healthy human volunteers in sodium heparin containing vacutainers (Becton Dickinson, USA) and neutrophils were isolated following standard technique [36]. Whole human blood (10 ml) was layered on top of 2.5 ml dextran (5 % w/v) and allowed to sediment for 1 h. Supernatant was collected and layered on 5 ml fi coll (Histopaque®-density 1.083, Sigma-Aldrich Co, USA.) and centrifuged at 3,000 rpm for 30 min at 20 °C. Erythrocytes in the cell pellet were lysed by hypotonic shock (2 ml of ice cold dis-tilled water followed by 1 ml of 3 × NaCl) and centrifuged at 3,000 rpm for 10 min at 20 °C. Cell pellet was resuspended in phosphate buffered saline (PBS), until use. Viability of the neutrophils was assessed by try-pan blue exclusion and was found to be greater than 95 %. The isolated neutrophils at a concentration of 1–5 × 108 cells/ml were homogenized on ice for 3 × 15 s. pulse in a homogenizer (Bransonic ultrasonic cleaner, Branson Ultrasonics Corp, USA.). The cells were centrifuged at 10,000 × g for 30 min at 4 °C, the supernatant collected and used immediately for assay.
Reaction mixture (100 µL) containing (DTT, 1 mM; ATP, 1 mM; cal-cium chloride, 2 mM; and indomethacin 10 µM; in phosphate buffered saline) was incubated in the absence and presence of RBx 7796 or Zi-leuton (100 nM–30 µM) and 85 µl of cell lysate (0.2 mg protein/well) at 37 °C for 5 min. Reaction was initiated by adding freshly prepared 14C arachidonic acid (from a stock solution of 50 µCi/ml radioactive arachi-donic acid, 0.01 µCi was added per well and made upto fi nal concentra-tion of 27 µM with cold arachidonic acid) and incubated in a shaking water bath for a further 20 min. The reaction was terminated with 16 µl of 0.5N hydrochloric acid. 75 µl of ice-cold methanol was added to the
mixture and was stored at –20 °C till assayed by HPLC. Samples were subjected to separation by HPLC (Waters Corporation, USA) using sol-vent containing acetonitrile: methanol: water: acetic acid in the ratio of 70: 15: 14: 1, with pH 5.6. HPLC was run at a fl ow rate of 1 ml/min and 30 min/sample. 5-LO product (5-HETE) and the substrate (arachi-donic acid) were monitored by a radioactive fl ow scintillation analyzer (PerkinElmer, USA) connected to the HPLC. The retention time for arachidonic acid varied between 9.5–11 min and that of 5-HETE varied between 3.5–4.5 min. Results were expressed as percent control.
Nature of InhibitionThe nature of inhibition by RBx 7796 in the 5-lipoxygenase inhibition assay was studied, in the presence of different concentrations of arachi-donic acid (15, 10, 5, 2, 1 & 0.5 µM). Effect of RBx 7796 at 5 µM was studied and arachidonic acid (AA) levels per min/ml/mg were calcu-lated. The double-reciprocal Lineweaver-Burke plot of 1/V vs. 1/S was plotted to determine the nature of inhibition.
15-Lipoxygenase AssayReaction mixture (100 µL) (DTT, 1mM; ATP, 1 mM; calcium chlo-ride, 2 mM; in phosphate buffered saline) containing 30U of soybean 15 lipoxygenase (Cayman Chemicals Co., USA) was incubated in the absence and presence of RBx 7796 or Zileuton (100 nM–600 µM) for 5 min at 37 °C. 5 min later freshly prepared 14C-arachidonic acid (from a stock solution of 50 µCi/ml radioactive arachidonic acid, 0.01 µCi was added/well and made upto fi nal concentration 27 µM with cold arachi-donic acid) was added to the reaction mixture and incubated in a shak-ing water bath for a further 20 min. The reaction was terminated with 16 µL of 0.5N hydrochloric acid. 75 µL of ice-cold methanol was added to the mixture and was stored at –20 °C till assayed by HPLC. For HPLC analysis, a solvent containing acetonitrile: methanol: water: acetic acid in the ratio of 70: 15: 14: 1, with pH 5.6 was used. HPLC was run at a fl ow rate of 1 ml/min and 30 min per sample. 15-LO product (15-HETE) and the substrate (arachidonic acid) were monitored by a radioactive fl ow scintillation analyzer, connected to the HPLC. The retention time for arachidonic acid varied between 9.5–11 min and that of 15-HETE varied between 3.5–4.5 min. Results are expressed as percent control and the IC50 values of test compounds are reported.
12-Lipoxygenase Assay100 µL reaction mixture (DTT, 1 mM; ATP, 1 mM; calcium chloride, 2 mM; and indomethacin 10 µM; in phosphate buffered saline) was in-cubated in the absence and presence of RBx 7796 or Zileuton (10 µM) and 5.3U of porcine 12 lipoxygenase (Cayman Chemicals Co., USA), for 5 min at 37 °C. Reaction was initiated by adding freshly prepared 14C arachidonic acid (from a stock solution of 50 µCi/ml radioactive arachi-donic acid, 0.01 µCi was added/well and made upto fi nal concentration 27 µM with cold arachidonic acid). Reaction mixture was incubated in a shaking water bath for a further 20 min. The reaction was terminated with 16 µl of 0.5N hydrochloric acid. 75 µl of ice-cold methanol was added to the mixture and it was stored at –20 °C till assayed by HPLC. For HPLC analysis, samples were subjected to separation by HPLC us-ing solvent containing acetonitrile: methanol: water: acetic acid in the ratio of 70: 15: 14: 1, with pH 5.56. HPLC was run at a fl ow rate of 1 ml/min and 30 min per sample. 12-LO product (12-HETE) and the substrate (arachidonic acid) were monitored by a radioactive fl ow scintillation analyzer, connected to the HPLC. The retention time for arachidonic acid varied between 9.5–11 min and that of 12-HETE varied between 3.5–4.5 min. Results were expressed as percent control.
Cell based assaysPreparation of neutrophils Washed neutrophils were prepared following standard technique [36] and whole blood from healthy human volunteers or anesthetized wistar rats, was added to dextran (5 %) in the ratio of 1 (dextran): 4 (whole blood) and kept undisturbed for about 1 h. Supernatant containing leu-kocytes was centrifuged at 3,000 rpm for 10 min, pellet was resuspended in 2 ml of phosphate-buffered saline pH 7.4(PBS) plus 1 mg/ml glucose (PG buffer). Suspension was layered over 5 ml of fi coll (Histopaque®-
Vol. 55, 2006 Pharmacological profi le of RBx 7796 519
and 20 guinea pigs were exposed to 0.1 % w/v ovalbumin or PBS for 10 min, and with 1 % ovalbumin for 30 min on day 21. Guinea pigs were treated with RBx 7796 (0.1, 1 and 10 mg/kg) or Montelukast (0.1, 1 and 10 mg/kg) or vehicle once daily from day 18 and continued for 5 days. Zileuton was tested at 0.5, 5 and 15 mg/kg, administered twice daily for 4 days. Ovalbumin / PBS challenge was performed 2 h after dif-ferent drug treatment. Guinea pigs were fasted for 12 h before each treatment.
Ovalbumin induced early phase bronchoconstrictionOn day 21, 2 h after drug treatment or vehicle administration, basal res-piratory parameters were recorded using Whole body Plethysmograph (Biosystem XA software, Buxco Electronics, USA) followed by chal-lenge with 1 % ovalbumin/PBS for 10 min duration. For recording basal respiratory parameters, 10 consecutive 1 min readings were averaged. Each 1 min reading represents an average of each breadth taken in that 60 s duration. Following PBS/Ovalbumin challenge data was recorded for 120 min, which represented hundred and twenty recordings one min apart. Each 1 min recording was an average of all the breath in 1 min. PenH, at any chosen time point post challenge was, expressed as percent of basal response. These values were plotted against time using Graph-pad prism software (GraphPad Software Inc, USA) and Area Under the Curve (AUC) was computed. The percent inhibition was computed us-ing the following formula.
AUCOVA - AUCTEST
% Inhibition = × 100 AUCOVA - AUCCON
Where,AUCOVA = AUC in vehicle treated group challenged with ovalbumin AUCTEST = AUC in group treated with a given dose of test compoundAUCCON = AUC in vehicle treated group challenged with PBS
Ovalbumin induced airway infl ammationTwentyfour hours after the fi nal ovalbumin challenge BAL was per-formed using Hank’s balanced salt solution (HBSS). Collected lavage fl uid was centrifuged at 3,000 rpm for 5 min, at 4 °C. Pellet was collected and resuspended in 1-ml HBSS. Total leukocyte count was performed in the resuspended sample. A portion of suspension was cytocentrifuged and stained with Leishmann’s stain for differential leukocyte count. To-tal leukocyte and eosinophil counts were expressed as cell count (mil-lions cells/ml of BAL). Eosinophil was also expressed as percent of total leukocyte count. The percent Inhibition was computed using the follow-ing formula.
EosOVA - EosTEST
% Inhibition = × 100 EosOVA – EosCON
Where,EosOVA = eosinophil count in vehicle treated group challenged with ovalbumin.EosTEST = eosinophil count in group treated with a given dose of test compoundEosCON = eosinophil count in vehicle treated group challenged with PBS
Statistical analysis
The program ‘Graph Pad Prism 4.0’ was used for statistical compar-isons. Percentage of inhibition was computed by comparing individual values in treatment group with mean values of control group. Statisti-cal signifi cance of each parameter in different treatment groups was determined by comparing with respective vehicle control group using one-way analysis of variance followed by Dunnett’s ‘t’ test for multiple comparison. A p level of ≤ 0.05 was considered to be statistically signifi -cant. Student’s t-test was used for comparison of two unpaired observa-tions. Nonlinear regression was used to estimate IC50 values.
density 1.083, Sigma-Aldrich Co, USA) and centrifuged at 3,000 rpm for 30 min. PMNL (in the pellet) were collected, depleted of erythro-cytes by osmotic shock, washed and resuspended at 0.2 × 106 cells/ml, in PG buffer until use. Viability of the neutrophils was assessed by trypan blue exclusion test and was found to be >95 %.
A23187 induced LTB4 releaseNeutrophil suspension (0.2 × 106 cells/ml) was incubated in polysty-rene microtitre plates with RBx 7796 or Zileuton for 1 h at 37 °C and 0.25 mM Ca++/Mg++ was added in the fi nal 3 min of incubation period. Reaction was initiated by adding 0.3 µg/ml A23187 (Sigma-Aldrich Co, USA) and continued for 10 min at 37 °C. Reaction was stopped by adding 80 µL of cold methanol [37]. LTB4 released was assayed using ELISA kits. The amount of LTB4 released was quantifi ed and percent inhibition of LTB4 release was calculated with respect to the difference between the A23187 stimulated and negative control cells, to compute IC50 values. In some experiments, arachidonic acid was added to the reaction mixture and inhibitory effect of RBx 7796 on A23187 induced LTB4 release was monitored.
In some experiments, at the end of an hour of incubation with test compound, cells were washed with 10 volume of buffer and centrifuged (800 g; 10 min). Cells were resuspended in PBG, incubated for 15 min in the absence or presence of RBx 7796/Zileuton with 0.25 mM Ca++/Mg++
being added for the fi nal 3 min. LTB4 released after A23187 challenge was assayed as described above.
Ex vivo LTB4 Release in ratsOvernight fasted male Wistar rats (180–220 gm) were treated with RBx 7796 (0.1, 1, 3 and 10 mg/kg) (dissolved in 3 % tween 80) or vehicle p.o. once daily for 5 days. Zileuton at a dose of 10 mg/kg served as standard. One-hour post dosing on day one and on day 5, blood was collected under light ether anaesthesia from retro-orbital plexus. Whole blood (180 µl) was mixed with PBG (20 µl) and incubated at 37 °C for 15 min. LTB4 synthesis was initiated with A23187 (10 µg/ml; 30 min). LTB4 was assayed using ELISA kit as described above. The amount of LTB4 was quantitated and fold-shift in LTB4 release w.r.t. negative control was calculated.
LPS induced neutrophilia in ratsMale Wistar rats were dosed orally once daily for fi ve days with vehi-cle, RBx 7796 (0.01, 0.1, 1 and 10 mg/kg) or Montelukast (0.1, 1 and 10 mg/kg). Zileuton was dosed at 0.05, 0.5 and 5 mg/kg, twice daily (i. e., 2 h before and then 6 h after LPS challenge) for one day. On the day of experiment, animals were exposed to LPS (100 µg/ml) dissolved in phosphate buffered saline (PBS) for 40 min by inhalation route. One group of normal saline treated rats were exposed to phosphate buffered saline for 40 min and served as negative control. Twenty-four hours after LPS challenge, bronchoalveolar lavage was performed, using Hank’s balanced salt solution (HBSS). Lavage fl uid was centrifuged at 3,000 rpm for 5 min, at 4 °C. Pellet was collected and resuspended in 1-ml HBSS. Total leukocyte count was performed in the resuspended sample. A portion of suspension was cytocentrifuged and stained with Leishmann’s stain for differential leukocyte count. Neutrophil count was expressed as cell count (millions cells/ml of BAL). Data was ex-pressed as % inhibition, which was computed using the following for-mula NeuLPS - NeuTEST
% Inhibition = × 100 NeuLPS - NeuCON
Where,NeuLPS = neutrophil count in vehicle treated group challenged with LPS NeuTEST = neutrophil count in group treated with a given dose of test compoundNeuCON = neutrophil count in group not challenged with PBS
Allergen induced bronchoconstriction and airway eosinophilia in guinea pigsGuinea pigs were sensitised on days 0, 7 and 14 with 50-µg ovalbumin and 10 mg aluminium hydroxide injected intraperitoneally. On days 19
520 R. K. Shirumalla et al. Infl amm. res.
Results
Inhibition of 5- Lipoxygenase enzyme activity
RBx 7796 was assayed for 5-LO inhibition using human recombinant 5-lipoxygenase under reducing (in the presence of DTT) or in non reducing condition (absence of DTT). Un-der reducing condition, RBx7796 inhibited human recom-binant 5-lipoxygenase activity in a concentration dependent manner with an IC50 of 3.5 ± 1.1 µM (Fig. 2a). Zileuton also inhibited the enzyme activity with an IC50 of 3.8 ± 2.0 µM (Fig. 2a). Both RBx 7796 and Zileuton inhibited 5-lipoxy-genase activity in human neutrophil lysate with IC50 of 5 ± 0.7 µM and 1.1 ± 0.1 µM, respectively (Table 1). RBx 7796 inhibited enzyme activity with an IC50 of 2.4 ± 0.5 µM (Table 1) in the absence of DTT.
RBx 7796 inhibited release of LTB4 from isolated neu-trophils obtained from rat and human blood with an IC50 of 2.9 ± 0.8 µM and 5 ± 0.8 µM, respectively (Table 1 and Fig. 2b). Zileuton inhibited LTB4 release from rat (2.2 ± 0.5 µM) and human (1.3 ± 0.2 µM) neutrophils.
Washing human neutrophils, 1h post-incubation with Zileuton, attenuated inhibitory potential of Zileuton (IC50 = 44.6±15.0 µM) as compared to unwashed samples (IC50 = 2.4 ± 0.9 µM), showing a 20 fold increase in IC50 values (Table 2). On re-incubating washed cells with Zileuton, the activ-
ity recovered with an IC50 value of 1.7 ± 0.1 µM, indicating washing of neutrophils did not change the integrity or vi-ability of neutrophils. However, unlike Zileuton, in washed neutrophils treated with RBx 7796 not much change in IC50 was observed as shown in table 2, indicating slow dissocia-tion rate of RBx 7796 from target and could be responsible for longer duration of action seen with RBx 7796. A similar trend was also observed in washed rat neutrophils treated with RBx 7796 where the IC50 value in unwashed prepara-tion was 1.3 ± 0.1 µM and on washing it was 1.5 ± 0.2 µM, showing no change in IC50 values between unwashed and
O CH3CH3
CH3 CH3 CH3
CH3CH3
OHCH3
O O
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Fig. 1. Chemical structure of RBx 7796 (Sodium salt of 1-O-dodecyl 2,3-O-isopropylidene-5,6-dideoxy-5-N-[4-(2-hydroxy-2-oxoethyl)phenylaminocarbonyl] amino-b-L-gulofuranoside), Zileuton and a-Tocopherol.
Fig. 2a. 5-lipoxygenase inhibitory activity of RBx 7796Recombinant human 5-lipoxygenase was incubated in the presence of increasing concentrations of RBx 7796 or Zileuton (100 nM–30 µM) in a reaction mixture containing DTT, ATP and calcium chloride (Reac-tion was monitored at 234 nm for 10 min. Area under curve (AUC) was computed from the plot of absorbance vs. time and percent inhibition of AUC for different treatments was calculated to compute IC50 values). Each data point represents mean ±SEM of 4 independent experiments done in duplicate.
Fig. 2b. In-vitro effect of RBx 7796 on A23187 induced LTB4 release from human neutrophil.Washed human neutrophils were incubated with increasing concentra-tions of RBx 7796 (empty circle)/Zileuton (inverted triangle). A23187 induced LTB4 release was estimated by ELISA kits and expressed as % inhibition as compared to vehicle treatment. Each point represents mean ±S.E.M. of 9–23 experiments.
Vol. 55, 2006 Pharmacological profi le of RBx 7796 521
washed neutrophils (Table 2) again indicating that RBx 7796 may be dissociating slowly from its target.
Nature of inhibition
In order to establish nature of interaction of RBx 7796 with 5-lipoxygenase enzyme, we evaluated the effect of increas-ing concentration of arachidonic acid on enzyme activity in the presence and absence of RBx 7796 (5 µM). As shown in Figure 3, Km of 5-lipoxygenase in the absence of RBx 7796 was 5.1 µM. RBx 7796 (5 µM) increased Km to 7.5 µM. Maximum velocity of enzyme action (Vmax) was not altered, indicating competitive nature of inhibition.
In a different series of experiment, we investigated the effect of exogenous arachidonic acid on the ability of RBx 7796 to inhibit A23187 induced LTB4 release. As shown in Figure 4, in the absence of exogenous arachidonic acid RBx 7796 dose dependently inhibited A23187 induced LTB4 release with an IC50 of 4.9 µM. Supplementation with 3 µM arachidonic acid increased the IC50 of RBx 7796 to 15.1 µM. Further increasing the arachidonic acid concentra-
tion to 30 µM resulted in increase of RBx 7796 IC50 value to 59.3 µM.
Selectivity study
The selectivity of RBx 7796 was assessed against related en-zymes. Activity of RBx 7796 against 15-Lipoxygenase was screened using soybean 15-lipoxygenase. RBx 7796 did not show any signifi cant inhibition against soybean 15-lipoxy-genase up to a concentration of 600 µM (Table 1), indicating a >100 fold selectivity. In this assay Zileuton has also not shown any signifi cant inhibition upto 600 µM, indicating a similar selectivity profi le for soybean 15 lipoxygenase as RBx 7796. RBx 7796 was also screened for 12-lipoxyge-nase inhibitory activity, using porcine leukocyte 12-lipox-ygenase. RBx 7796 was screened upto a concentration of 600 µM and no appreciable inhibition was observed, again demonstrating an IC50 values of >600 µM with >100-fold selectivity. Zileuton in this assay showed an IC50 values of 56 µM with selectivity of less than 50 fold as shown in the Table 1.
Table 1. In vitro potency and selectivity of RBx 7796 vs. Zileuton, a known selective 5-lipoxygenase inhibitors.5-lipoxygenase inhibitory potential of RBx 7796 was evaluated using human recombinant enzyme or human neutrophil lysate in reducing (in the presence of DTT) as well as non-reducing milieu. Selectivity for 12 and 15 lipoxygenase was evaluated using porcine leukocyte 12 lipoxygenase and soybean 15-lipoxygenase.Cell based inhibitory potential was evaluated in isolated neutrophils from human and rat blood. Neutrophils were incubated with RBx 7796 or Zileuton and the effect on A23187 induced LTB4 release was measured.
Table 2. Effect of RBx 7796 and Zileuton on A23187 induced LTB4 release in wash and unwashed neutrophils.Heparinized blood was collected from human volunteers or rat, neutrophils separated and incubated with RBx 7796 or Zileuton or vehicle. The equili-brated cells were either challenged with A23187 in presence of 0.25 mM calcium or washed with 10 times volume of buffer and resuspended cells were then challenged with A23187. LTB4 release was estimated by ELISA kits and expressed as % inhibition as compared to vehicle treatment and IC50 were computed.
A23187 induced LTB4 release (IC50 values)
Treatment Isolated Human neutrophils Isolated Rat neutrophils
A23187 induced LTB4 release from whole blood of rats treated with vehicle of RBx 7796 was 6466 ± 685 pg/ml. Pretreatment with Zileuton (10 mg/kg) before A23187 chal-lenge, blunted the ability of A23187 to release LTB4 (Fig. 5). LTB4 release in Zileuton treated rats was 2093 ± 302 pg/ml, an inhibition of 68 ± 5 %.
Rats treated with RBx 7796 at a dose of 0.1, 1 and 10 mg/kg, one hour before A23187 challenge, showed an LTB4 release of 4463 ± 412, 4793 ± 657 and 4943 ± 546 pg/ml respectively. This effect on LTB4 release was neither dose dependent nor statistically signifi cant (Fig. 5).
In another set of experiment, rats were treated with RBx 7796 or its vehicle once daily, for fi ve days and on the 5th
day 1 h post treatment whole blood was collected and chal-lenged with A23187. In vehicle treated animals a mean LTB4 release was 8934 ± 688 pg/ml which was not different from the vehicle treated group after single oral administration. Pretreatment with RBx 7796 – 0.1, 1 and 10 mg/kg once dai-ly for 5 days, showed a dose dependent inhibition of LTB4 release. LTB4 released in rats treated with RBx 7796 – 0.1, 1 and 10 mg/kg once daily for 5 days were 6362 ± 1042, 5397 ± 935 and 4263 ± 762 pg/ml respectively in response to A23187 challenge. This inhibitory effect was statistically signifi cant at a dose of 1 and 10 mg/kg, which represented 40 and 52 % inhibition, respectively (Fig. 5).
LPS induced airway neutrophiliaTotal leukocyte count and total neutrophil count in bron-choalveolar lavage fl uid in rats challenged with phosphate buffered saline (PBS) was 13.8 ± 2 and 2.6 ± 0.46 million cells (n = 11) respectively. Neutrophils constituted 21.8 ± 3.8 % of total bronchoalveolar lavage cell population. Eosi-nophils were 1.2 ± 0.9 % with mononucleated cells constitut-ing remaining of the 77 % of total BAL cell population. LPS challenge increased total leukocyte count in the lavage fl uid to 29.8 ± 6.3 million cells and total neutrophil count to 18.6 ± 4.3 million cells (Fig. 6a). Neutrophils constituted 63.2 ±
-0.5 0.0 0.5 1.0 1.5 2.0
0.1
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0.7RBx 77965-Lipoxygenase
Km=5.1µM Vmax=26.9r2=0.96 Ki= 7.5µM
1/[AA]
1/v
Fig. 3. Competitive interaction of RBx 7796 with 5-LipoxygenaseKinetic analysis of human recombinant 5-lipoxygenase inhibition by 5 µM RBx 7796 presented as Lineweaver-Burke plot. The enzyme assay was performed with of RBx 7796 at 5 µM, in the presence of different concentrations of Arachidonic acid (0.5 µM, 1 µM, 2 µM, 5 µM, 8 µM, 10 µM, 15 µM) by recording increase in absorbance at 234 nm. The dou-ble-reciprocal Lineweaver-Burke plot of 1/V vs. 1/S was plotted.
Fig. 4. In-vitro effect of arachidonic acid on the activity of RBx 7796 on A-23187 induced LTB4 release from human neutrophils.A-23187 induced LTB4 release was monitored. Human neutrophils in-cubated with increasing concentrations of RBx 7796 in the absence or presence of arachidonic acid. Each data point represents mean ±S.E.M.
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Fig. 5. Ex vivo LTB4 release after single and multiple (5 day) oral admin-istration of RBx 7796 in rats.Heparinized blood was collected from rats treated with either RBx 7796-(0.1, 1 and 10 mg/kg-empty bars) or vehicle (0.25 % Tween 80-solid bar) or Zileuton-10 mg/kg (grey bar) after (A) single oral administration and (B) after dosing once daily for 5 days. LTB4 released from whole blood was monitored after A23187 challenge and expressed as pg/ml of whole blood. Each bar represents LTB4 release expressed as mean ±S.E.M. of 6–12 animals. (*) Represents signifi cantly different from respective vehicle treated group (one way ANOVA) and p ≤ 0.05 was considered signifi cant.
Vol. 55, 2006 Pharmacological profi le of RBx 7796 523
5 % of total cells in the lavage fl uid following LPS challenge with mononucleated cells being reduced to 36.7 ± 4.9 %. Eosinophils were only 0.1 %.
In preliminary experiments, we observed that single ad-ministration of RBx 7796 did not have any effect on cellular profi le in rats following LPS challenge. Consequently, we resorted to multiple dosing and settled on a fi ve day dosing regimen. Upon repeated daily administration for fi ve days, RBx 7796 reduced total neutrophil count in a dose depend-ent manner (Fig. 6) to 13.7 ± 2.4, 9.9 ± 4.7, 5.8 ± 1.85 and 5.8 ± 1.9 million cells/ml of BAL fl uid at the doses of 0.01, 0.1, 1 and 10 mg/kg, respectively. These neutrophil counts amounted to an inhibition of 31 %, 55 %, 80 % and 87 % re-spectively, in comparison to LPS control group animals and was statistically signifi cant at 1 and 10 mg/kg RBx 7796.
Figure 7, shows the effect of RBx 7796 on LPS induced LTB4 release in BAL fl uid. LTB4 release in the BAL fl uid of PBS challenged animals was 360 ± 114 pg/ml. LPS chal-lenge increased the BAL LTB4 levels to 1155 ± 387 pg/ml. Single administration of RBx 7796 did not show a signifi cant inhibition of LTB4 release in BAL fl uid. Repeated admin-istration of RBx 7796 for 5 days completely inhibited LPS induced LTB4 release in BAL fl uid (Fig. 7).
We also tested the effect of Zileuton and Montelukast on airway neutrophilia in rats. Zileuton (0.1, 1 and 10 mg/kg, in 2 divided doses) reduced the number of neutrophils in the lavage fl uid in a dose dependent manner. A peak inhibition of 83 % was observed at a dose of 10 mg/kg (dosed 2 h before and 6 h post LPS challenge). However, Montelukast treated for 5 days did not lower number of neutrophils in lavage fl uid up to a dose of 10 mg/kg, the highest dose tested in the study.
Ovalbumin induced bronchoconstriction
Bronchoconstriction in guinea pigs following ovalbumin challenge was recorded for 2 h. Area under the curve for PenH vs. time response plot was computed. Basal PenH values in different treatment groups were 0.53 to 0.6 units. In guinea pigs challenged with phosphate buffered saline
an AUC of 7180 ± 1553 units was observed. As shown in Figure 8a, the AUC value in ovalbumin challenged guinea pigs increased by nearly 7 fold as compared with phosphate buffered saline challenged animals and was 53758 ± 7757 units. Pretreatment of guinea pigs with RBx 7796 reduced ovalbumin induced increase in AUC in a dose dependent manner to 53320 ± 7614, 29046 ± 5634 and 16325 ± 4095 units at the doses of 0.1, 1 and 10 mg/kg, respectively. This translated into 0.9, 53 and 80 % protection respectively. A statistically signifi cant inhibition was observed with 10 mg/kg. RBx 7796.
In the same model Zileuton and Montelukast were also tested in a separate study. Effect of Zileuton and Montelu-kast was expressed as percent inhibition using the formula described under the methods section of ovalbumin induced bronchoconstriction. Montelukast was also tested at doses of 0.1, 1 and 10 mg/kg treated once daily for 5 days. Montelu-kast treatment showed a dose dependent inhibition of oval-bumin induced bronchoconstriction (Fig. 8b) and the effect was statistically signifi cant at dose of 10 mg/kg with a 60 % inhibition, which was comparable to RBx 7796. Treatment with Zileuton (0.5, 5 and 15 mg/kg, twice daily) showed a dose dependent inhibition of ovalbumin induced bronchoc-onstriction (Fig. 8b). A statistically signifi cant effect was produced at a dose of 5 mg/kg, bid.
Ovalbumin induced airway infl ammation
Twentyfour hours after last ovalbumin challenge, animals were sacrifi ced and bronchoalveolar lavage was performed. Total cell count in bronchoalevolar lavage fl uid of guinea pig challenged with PBS was 66.3 ± 18.2 million cells of which 17.4 ± 4.9 million were eosinophils (26.2 ± 2.4 %). Mononu-cleated cells were 62.0 ± 2.1 % with 11.8 ± 1.9 % neutrophils. Ovalbumin challenge signifi cantly increased total cell count (150 ± 38.1 million cells) and eosinophil count (94.3 ± 22.1 million cells) as also the % eosinophilia (64.8 ± 3.2 %) in bronchoalveolar lavage fl uid. Consequently, mononeucleat-ed cell as well as neutrophil percent in BAL fl uid decrease to
*
0
4
8
12
16
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24
LPS-Vehicle PBS-Vehicle
Neu
trop
hil c
ount
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ion
cells
/ml)
**
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24
RBx 7796-0.1mg/kg
RBx 7796-1mg/kg
RBx 7796-10mg/kg
RBx 7796-30mg/kg
Fig. 6. Effect of RBx 7796 on LPS induced airway infl ammation in male wistar rats.Rats were treated with RBx 7796 once daily for 5 days and challenged with LPS (for details see the text). Twenty-four hours later, broncho-alveolar lavage was performed and neutrophil counts were estimated in the BAL. Each bar represents mean ±S.E.M. of 6–11 animals. (*) Represents signifi cant difference of the neutrophil count in the treatment groups from respective LPS control group using one way ANOVA and p ≤ 0.05 was considered signifi cant.
***
0
400
800
1200
1600
Vehicle-LPS RBx 7796 -1mg/kg (1 day)
RBx 7796 -1mg/kg (5 day)
Zileuton -10mg/kg (1 day)
Vehicle-PBS
LTB
4 in
BA
L flu
id (p
g/m
l)
Fig. 7. Effect of RBx 7796 on LPS induced airway LTB4 release in male wistar rats.Rats were treated once with Zileuton or with RBx 7796-1 mg/kg or RBx 7796-1 mg/kg once daily for fi ve consecutive days. On the day of ex-periment, 2 h after vehicle/drug administration, animals were exposed to LPS (100 µg/ml) or PBS for 40 min. Twenty-four hours after LPS/PBS challenge, bronchoalveolar lavage was performed and LTB4 released was estimated. Each bar represents mean ±S.E.M. of 6–11 animals. (*) Represents signifi cantly different in the treatment groups from LPS control group using one-way ANOVA and p ≤ 0.05 was considered signifi cant.
524 R. K. Shirumalla et al. Infl amm. res.
30.7 ± 3.3 % and 4.6 ± 1.3 % respectively, although the total mononucleated cell population or neutrophil count remained unchanged as compared to PBS challenged animals.
RBx 7796 produced in a dose dependent manner, de-crease in eosinophil infi ltration as indicated by decrease in the percent eosinophil in BAL fl uid. The peak inhibitory effect was seen with 30 mg/kg of RBx 7796. As shown in Figure 9a, RBx 7796 lowered total eosinophil count in bron-
choalveolar lavage fl uid in a dose dependent manner with 74.6 ± 18.2, 46.6 ± 12.2 and 30.4 ± 12.9 million cells/ml of BAL fl uid which were 26, 62 and 83 % inhibition at the doses of 0.1, 1 and 10 mg/kg respectively. This effect was statistically signifi cant at doses of 10 mg/kg.
We also tested the effect of Zileuton and Montelukast on ovalbumin induced airway infl ammation in guinea pigs. Effect of Zileuton and Montelukast were expressed as percent inhibi-tion using the formula described under the methods section
*0
10203040506070
LPS-Control PBS-Control
AU
C(%
initi
al P
enh.
min
) x10
3
*
010203040506070
RBx 7796-0.1mg/kg
RBx 7796 -1mg/kg
RBx 7796-10mg/kg
Fig. 8a. Effect of Ovalbumin challenge on bronchoconstriction ex-pressed as AUC (Penh Vs Time) in RBx 7796 treated guinea pigs.AUC was obtained by plotting % Initial-PenH (Index of airway resist-ance) Vs time (0–120 min), of guinea pigs challenged with either oval-bumin or PBS. Guinea pigs were treated with RBx 7796 for 5 days and challenged with ovalbumin (For details see text). Each point represents mean ±S.E.M. of 5–12 animals. (*) Represents signifi cant difference of the AUC in the treatment groups from ovalbumin control group using one-way ANOVA and p ≤ 0.05 was considered signifi cant.
Montelukast
*
-40
-20
0
20
40
60
80
100
120
0.1 1 10
Dose (mg/kg)
% in
hibi
tion
Zileuton
**
0
20
40
60
80
100
120
1 10 30
Dose (mg/kg)
% in
hibi
tion
(A)
(B)
Fig. 8b. Effect of Ovalbumin challenge on bronchoconstriction ex-pressed as AUC (Penh Vs Time) in Montelukast or Zileuton treated guinea pigs.AUC was obtained by plotting % Initial-PenH (Index of airway resist-ance) Vs time (0–120 min), of guinea pigs challenged with ovalbumin. Guinea pigs were treated with (A) Montelukast or (B) Zileuton for fi ve days, as described in the text and challenged with ovalbumin. Each point represents mean ±S.E.M. of 5–12 animals. (*) Represents signifi cant difference of the AUC in the treatment groups from respective ovalbu-min control group using one way ANOVA and p ≤ 0.05 was considered signifi cant. Percent inhibition was computed as described in the text.
*
0
20
40
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Ovalbumin Control PBS Control
Eosi
noph
il co
unt
(mill
ion
cells
/ml)
*
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40
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120
RBx7796-0.1mg/kg RBx7796-1mg/kg RBx7796-10mg/kg
Fig. 9a. Effect of RBx 7796, on airway infl ammation in ovalbumin chal-lenged guinea pigs. Eosinophil counts were obtained in bronchoalveolar lavage fl uid of ovalbumin sensitised and challenged guinea pigs. Guinea pigs were treated with RBx 7796 for 5 days, as described in the text and challenged with ovalbumin. Each point represents mean ±S.E.M. of 5–12 animals. (*) Represents signifi cant difference of the eosinophil count in the treat-ment groups from ovalbumin control group using one-way ANOVA and p ≤ 0.05 was considered signifi cant.
Montelukast
0
20
40
60
80
100
120
0.1 1 10
Dose (mg/kg)
% in
hibi
tion
Zileuton
*
0
20
40
60
80
100
120
1 10 30
Dose (mg/kg)
% in
hibi
tion
(A)
(B)
Fig. 9b. Effect of Montelukast and Zileuton on airway infl ammation in ovalbumin challenged guinea pigs. Eosinophil counts were obtained in bronchoalveolar lavage fl uid of ovalbumin sensitised and challenged guinea pigs. Guinea pigs were treated with (A) Montelukast or (B) Zileuton for fi ve days, as described in the text and challenged with ovalbumin. Each point represents mean ±S.E.M. of 5–12 animals. (*) Represents signifi cant difference of the eosinophil count in the treatment groups from respective ovalbumin control group using one-way ANOVA and p ≤ 0.05 was considered sig-nifi cant. Percent inhibition was computed as described in the text.
Vol. 55, 2006 Pharmacological profi le of RBx 7796 525
of ovalbumin induced airway infl ammation. Treatment with Zileuton reduced total airway eosinophil count. A statistically signifi cant inhibitory effect was seen at a dose of 5 mg/kg, bid (Fig. 9b). Treatment with 15 mg/kg, bid dose did not increase inhibitory effect any further. Montelukast dosed 0.1, 1 and 10 mg/kg decreased total eosinophil count in the BAL fl uid of guinea pigs challenged with ovalbumin (Fig. 9b). However, this effect did not attain statistical signifi cance.
Discussion
In this article we have shown 5-lipoxygenase inhibitory activity of RBx 7796 using a recombinant human 5-lipoxy-genase enzyme and human neutrophil lysate. We have also demonstrated that RBx 7796 inhibits arachidonic acid induced LTB4 release from neutrophils of rat and human volunteers, a 5-lipoxygenase dependent process. In addition, RBx 7796 inhibited A23187 induced LTB4 release ex vivo in rats. We have shown that RBx 7796 inhibits 5-lipoxygenase in a competitive manner, and exhibits reasonable degree of selectivity against related enzymes like 12 and 15-lipoxyge-nase. Finally, we have demonstrated that RBx 7796 exhibits anti-infl ammatory effect in rat model of neutrophilic airway infl ammation and guinea pig model of allergen induced bronchoconstriction and eosinophilic airway infl ammation.
A considerable need exists to broaden the therapeutic options in bronchial asthma and COPD. 5-Lipoxygenase inhibitors can fi ll the void because of the vantage position 5-lipoxygenase enzyme occupies in the arachidonic acid catalysis cascade. This enzyme can regulate generation of both cysteinyl leukotriene and leukotriene B4 [16]. Thus an effective inhibitor of the enzyme can very likely be useful in the management of eosinophilic airway infl ammation of bronchial asthma and neutrophilic infl ammation of COPD. A main stumbling block in fi nding an effective inhibitor is the availability of appropriate and distinct non N-hyrdroxy urea chemotype. In this regard we have discovered RBx 7796, which is a chemically distinct entity from Zileuton and re-lated N-hyroxy urea class of inhibitors of 5-lipoxygenase. Like Zileuton [17, 18], RBx 7796 inhibited 5-lipoxygenase enzyme and calcium ionophore induced LTB4 release from neutrophils, a process related to inhibition of 5-lipoxygenase enzyme activity, with very similar potency.
The nature of interaction of RBx 7796 with 5-lipoxyge-nase enzyme appeared to be competitive based on classical enzyme kinetic experiments as well as shift on concentration response curve of RBx 7796 in the presence of arachidonic acid [38]. Potency of many competitive inhibitors, e. g. ZD 2138, ZM 230487, BWA4C, CJ-13610 [16, 38, 39] is related to oxidative status of its environment. As a result competitive inhibitors exhibit greater potency of enzyme inhibition in the reducing environment in the presence of DTT as well as in preparations where cell membrane is intact. The fact that RBx 7796 exhibited very similar potency in cell free and cell based assays as well as in the presence or absence of DTT, indicated that this molecule is insensitive to redox status of its milieu. We compared inhibitory effect of RBx 7796 on other enzymes that use arachidonic acid as substrate. No ap-preciable inhibition of 15-Lipoxygenase or 12 Lipoxygenase was apparent up to 100 µM.
Neutrophils play an important role in the pathophysiol-ogy of COPD [7, 10, 40]. Neutrophils release chemokine like LTB4 that bring in more and activate more neutrophils to release their cellular and granular content including pro-teolytic enzymes like neutrophil elastase. Elevated levels of neutrophils and LTB4 can be detected in sputum of COPD patients. Inhibition of neutrophil migration to airway is con-sidered to be a worthwhile intervention in COPD [7, 40]. 5-Lipoxygenase inhibitors have been reported to inhibit A23187 induced LTB4 release, ex vivo [17, 18]. RBx 7796 inhibited ex vivo LTB4 release from neutrophils following repeated administration. The lack of signifi cant inhibitory effect of RBx 7796 upon single administration is not clear. It may be possible that RBx 7796 has a delayed onset of action and therefore did not show signifi cant inhibition after single administration. Upon multiple dosing, due to its slow disso-ciation from target in neutrophils prolonged inhibitory effect is observed. However, no evidence of drug accumulation has been found in pharmacokinetic studies.
In experimental animals, airway neutrophilia can be in-duced using LPS [41, 42]. It is believed that LPS challenge leads to activation of 5-lipoxygenase enzyme [43] and in-hibitors of 5-lipoxygenase, inhibit LPS induced plasma pro-tein leakage [44]. In this study also, both Zileuton and RBx 7796 inhibited LPS induced total cell infl ux and neutrophil infl ux in a dose dependent manner. RBx 7796 also reduced LPS induced increase in LTB4 level and protein level in the bronchoalveolar lavage fl uid. However, cysteinyl leukot-riene receptor antagonist, Montelukast, did not exhibit any inhibitory effect on LPS induced airway neutrophil infl ux. This suggests that inhibition of 5-lipoxygenase enzyme may be a better option to address the infl ammation of COPD compared to cysteinyl leukotriene receptor antagonism.
Human bronchial asthma is usually characterized by an early acute bronchoconstriction, followed by late phase bron-choconstriction response, airway hyperresponsiveness and eosinophilic airway infl ammation [45]. The allergic guinea pig has been used extensively to evaluate compounds for anti-asthma activity. This model exhibits allergen-induced bronchoconstriction and eosinophilic airway infl ammation characteristic of allergic bronchial infl ammation [46]. Evi-dence suggest that allergen induced bronchoconstriction in guinea pig is mediated through leukotriene generation [11, 19, 47–49]. RBx 7796 when administered once daily 2 h be-fore allergen challenge, inhibited ovalbumin induced bron-choconstriction in guinea pigs. In this model effi cacy of RBx 7796 was comparable to that of Montelukast, in agreement with dominant role of cysteinyl leukotrienes in bronchocon-striction.
Late phase bronchoconstriction and airway hyperrespon-siveness refl ect underlying airway infl ammation. Cysteinyl leukotrienes have been recognized to be potent chemotactic factors for eosinophils [8]. In agreement with involvement of leukotrienes in early bronchoconstriction we observed eosinophil infl ux into the airway of allergic guinea pigs. RBx 7796 inhibited, in a dose dependent manner, ovalbu-min induced increase in eosinophil count in the lavage fl uid. However, Montelukast was much less effective in lowering eosinophil count. Although, the reason for this is not known clearly, it has been suggested that in guinea pigs, LTB4 is a powerful chemoattractant for eosinophils [9]. Thus, greater
526 R. K. Shirumalla et al. Infl amm. res.
effi cacy of RBx 7796 may be attributed to its ability to re-move both cysteinyl leukotrienes and LTB4, while Monte-lukast was restricted to inhibiting only the cysteinyl leukot-riene mediated response. This may also suggest that in real life bronchial asthma where neutrophils may contribute to-wards airway infl ammation, 5-lipoxygenase inhibition may appear to be a better strategy to counter airway infl ammation than blocking cysteinyl leukotriene receptors.
In summary, in our attempt to discover a 5-lipoxygenase inhibitor we have succeeded in moving away from the N-hy-droxy urea chemotype. RBx 7796 is a competitive inhibitor that, unlike other reported competitive inhibitors, does not lose potency in a non-reducing environment. RBx 7796 ex-hibited effi cacy both in acute and chronic models of airway reactivity and infl ammation. The fact that the molecule was effi cacious by oral route, indicates RBx 7796 may not suffer from bioavailability issues and its slow dissociation from the target may only add to this advantage. A case in point being Zileuton, which was dosed twice daily for fi ve days in al-lergen induced bronchoconstriction models while RBx 7796 was dosed only once daily. It is our observation that follow-ing oral administration in rat and dog, RBx 7796 exhibits bioavailability to the extent of 30–50 % (data not shown). Also RBx 7796 has shown reasonably potent in vivo activity in experimental animal models of airway reactivity and air-way infl ammation, which is comparable to Zileuton. We do not know the reason for potent in vivo activity of RBx 7796. It is commonly known that 5-lipoxygenase inhibitors exhibit comparable in vivo effi cacy compared to their more potent receptor antagonist counterparts. A case in point is Zileuton, which is nearly 1,000 fold less potent than Montelukast in in vitro studies, but in a clinical situation it is as effi cacious as Montelukast [3, 20–22]. This may indicate that inhibitors of 5-lipoxygenase may address disease pathophysiology more effi ciently than cysteinyl leukotriene receptor antagonists.
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