Mast-cell stabilizer

Lodoxamide suppresses compound 48/80-induced histamine release as well as ionophore-induced 45Ca influx and related histamine release in purified rat peritoneal mast cells[1]. Lodoxamide significantly and dose-dependently inhibits eosinophils' chemotactic response to fMLP and IL-5. In addition, following IgA-dependent activation, lopidosamide can potently block the release of eosinophil peroxidase. It can also somewhat block the release of eosinophil cationic protein and eosinophil-derived neurotoxin[2].

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Recent reports describe the beneficial use of Lodoxamide, an anti-allergic compound, for the treatment of asthma and allergic conjunctivitis. Lodoxamide is known as a mast cell stabilizer, however, the association of a significant clinical improvement with a specific decrease in eosinophil infiltrate suggested possible direct effects of lodoxamide on eosinophils. The chemotactic response of eosinophils to fMLP as well as to IL-5, in vitro, was very significantly and dose-dependently inhibited by Lodoxamide. Lodoxamide was also able to strongly inhibit the release of eosinophil peroxidase after IgA-dependent activation and, to a lesser extent, the release of eosinophil cationic protein and eosinophil-derived neurotoxin. Moreover, the release of cytotoxic mediators evaluated in an antibody-dependent cytotoxicity assay against parasitic targets was also significantly reduced, not only in the case of human eosinophils but also in a rat eosinophil-mast cell model of cytotoxicity. Taken together, these results indicate that lodoxamide can exert potent inhibitory effects on eosinophil activation in vitro combined with a strong inhibition of eosinophil attraction, leading therefore to a reduction in their pathological potential in vivo. [2]

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Eosinophil Chemotaxis [2]
The eosinophil chemotactic response to fMLP (formyl- methionine-leucine-phenylalanine) has been first evaluated, since fMLP is a potent chemoattractant for eosinophils and also since such small peptides are naturally found in subjects, deriving from ubiquitous bacterial flora. Six patients with eosinophilia have been included in the study. The mean absolute values of the chemotaxis assay for the 6 patients are presented in figure 1. Four concentrations of Lodoxamide ranging between 0.01 ng/ml and 10 µg/ml have been used in the presence of one optimal concentration of fMLP (10–6 mol/l) determined by preliminary dose-response studies.

The results presented in figure 1 clearly indicate the potent and dose-dependent inhibitory effects of Lodoxamide on eosinophil chemotactic response to fMLP, reaching more than 60% inhibition at 100 ng/ml and 80% inhibition for 10 µg/ml lodoxamide. The results were highly significant with p < 0.001 for each concentration (Student’s t test for paired experiments).

In a second series of experiments, the role of Lodoxamide was evaluated on the chemotactic response of eosinophils to IL-5, a chemotactic agent specific for eosinophils. The mean results obtained with eosinophils purified from 4 different patients are presented in figure 2. A statistically significant inhibition was obtained for the two highest concentrations, reaching 48% inhibition with 10 µg/ml and more than 80% inhibition with 50 µg/ml lodoxamide.

To determine whether inhibition of eosinophil chemotaxis by Lodoxamide could be due to a cytotoxic effect, the release of LDH, a marker of cell lysis, was measured in the supernatants of eosinophils incubated with lodoxamide for 2–18 h. No significant LDH release was observed, even with the highest concentration of lodoxamide (data not shown).

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Effects of Lodoxamide on the Release of Granule Proteins [3]
Previous studies have shown that eosinophils could release their granule content after activation with immobilized [11] or cross-linked IgA [5]. The effects of lodoxamide were investigated on the release of EPO, ECP and EDN by eosinophils after activation with IgA-anti-IgA complexes. Lodoxamide was used at 10 µg/ml, a concentration known to induce optimal chemotaxis inhibition. Results were presented as absolute values for each patient, since individual mediator release upon stimulation strongly differed from patient to patient. The results presented in figure 3 showed that lodoxamide induces a very potent inhibition of EPO release by IgA-activated eosinophils in the 6 patients tested (mean inhibition = 93%). The effects of lodoxamide on ECP release by eosinophils activated with IgA-anti-IgA immune complexes were investigated. Results presented in figure 3 showed that lodoxamide (10 µg/ml) induced a decrease in ECP release by eosinophils in 3 of 7 patients (mean inhibition = 35%). The addition of Lodoxamide to eosinophils from 6 different hypereosinophilic patients significantly inhibited the release of EDN in 4 of 6 patients, with a mean inhibition of 51%.

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Effects of Lodoxamide on Eosinophil-Mediated Cytotoxicity [3]
Antibody-dependent eosinophil-mediated cytotoxicity involves the release of several cationic proteins contained in eosinophil granules, namely EPO, ECP, MBP and to a lesser extent EDN [1]. This experimental procedure thus represents a sensitive and functional assay to evaluate the modulatory effects of drugs able to inhibit eosinophil activation. Addition of increasing concentrations of lodoxamide to human eosinophils led to a partial but statistically significant inhibition of cytotoxicity (fig. 4A, n = 4 experiments). Since lodoxamide is able to inhibit the release of rat mast cell mediators in vitro and in vivo [15, 16], and since rat mast cell-eosinophil interactions are required to induce antibody-dependent cell-mediated cytotoxicity [13], we have also investigated the role of Lodoxamide in rat eosinophil-mediated antibody-dependent cell-mediated cytotoxicity. The results presented in figure 4B clearly indicate a dose-dependent inhibition of cytotoxicity, reaching more than 90% inhibition for the highest concentration of lodoxamide (p < 0.0001). Moreover, this microscopical procedure revealed that lodoxamide had no effect, neither on eosinophil nor on target viability.

Lodoxamide has been shown to exhibit cromolyn-like activity in rat peritoneal mast cell assay (PCA) model3 and Ascaris antigen-sensitized rhesus monkeys. In Ascaris-sensitized anesthetized rhesus monkeys, lodoxamide administered intravenously, orally, or intrabronchially by aerosol dramatically reduces the increased respiratory frequency and decreased tidal volume induced by antigen challenge[1]. As evidenced by increased oxygenation, decreased microvascular permeability, and increased compliance, adding Lodoxamide tromethamine to the University of Wisconsin or Euro-Collins solution significantly reduces lung reperfusion injury[3]. Sleep, coughing, sputum production, and daytime breathing difficulties all improve in patients receiving lodoxamide tromethamine treatment[4].

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Lodoxamide tromethamine (U-42,585E) is a new drug intended for prophylaxis of mast cell-mediated allergic disease. It is a water-soluble, cromolyn-like agent with demonstrated activity in rat peritoneal mast cell assay, rat percutaneous anaphylaxis (rat PCA) and sensitized rhesus monkey airway system. Ten allergen-sensitive asthmatics were pretreated with Lodoxamide (0.01, 0.1, or 1.0 mg) or placebo, then challenged with serial dilutions of allergen extract. Analysis of allergen dose-response curve parameters shows that pretreatment with lodoxamide offers significant protection against experimental allergen-induced bronchoconstriction. At 0.01 mg, Lodoxamide was effective in over half the subjects tested. Administration of lodoxamide by inhalation at doses of 0.1 and 1.0 mg uniformly allowed subjects to tolerate significantly larger doses of inhaled allergen. Side effects observed at these doses were minimal[1].

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After 6 hours of ischemic storage: Lodoxamide tromethamine-enhanced Euro-Collins solution decreased alveolar-arterial oxygen difference from 539 to 457 (p = 0.004), increased oxygen tension from 119 to 205 mm Hg (p = 0.006), and decreased capillary filtration coefficient from 3.9 to 2.0 (p < 0.001); Lodoxamide tromethamine-enhanced University of Wisconsin solution decreased alveolar-arterial oxygen difference from 546 to 317 (p < 0.001), increased oxygen tension from 166 to 335 mm Hg (p < 0.001), and decreased capillary filtration coefficient from 3.0 to 1.7 (p < 0.001). After 12 hours of ischemic storage, lodoxamide tromethamine-enhanced Euro-Collins solution decreased alveolar-arterial oxygen difference from 588 to 485 (p < 0.001), increased oxygen tension from 100 to 161 mm Hg (p = 0.012), decreased capillary filtration coefficient from 6.2 to 2.6 (p < 0.001), and increased compliance from 0.12 to 0.21 (p < 0.001); lodoxamide tromethamine-enhanced University of Wisconsin solution decreased alveolar-arterial oxygen difference from 478 to 322 (p < 0.001), increased oxygen tension from 214 to 335 mm Hg (p < 0.001), decreased capillary filtration constant from 4.2 to 2.0 (p < 0.001), and increased compliance from 0.20 to 0.25 (p < 0.001).
Conclusions: Addition of Lodoxamide tromethamine to Euro-Collins or University of Wisconsin solution results in a marked decrease in lung reperfusion injury as demonstrated by increased oxygenation, decreased microvascular permeability, and increased compliance. These results are relevant as Euro-Collins and University of Wisconsin solutions are the most common clinically used lung preservation solutions. This study also highlights the deleterious role of resident mast cells in preservation injury [3].

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The efficacy of Lodoxamide tromethamine in the treatment of asthma was studied in a 16-week double-blind, placebo-controlled study of 68 perennial allergic subjects with asthma. Patients received either lodoxamide tromethamine, 0.25 mg four times daily, or placebo, administered by metered-dose inhaler. Response to treatment was assessed by analyzing changes in asthma symptoms, inhaled bronchodilator requirements, and pulmonary function when compared to a 2-week baseline period. Patients treated with lodoxamide tromethamine demonstrated an improvement in daytime breathing difficulty, cough, sputum production, and sleep (p less than 0.01 to 0.05), but improvement was not significantly different from that demonstrated by placebo-treated patients. Patients from both treatment groups were able to reduce their inhaled bronchodilators (p less than 0.01), but again no significant difference was apparent between lodoxamide tromethamine and placebo treatment, nor were there any differences in peak expiratory flow rate or FEV1 between the two groups. Seven patients who received lodoxamide tromethamine withdrew because of a sensation of heat and gastrointestinal symptoms. Thus, although lodoxamide tromethamine possesses potent mast cell-stabilizing activity in vitro, we have failed to demonstrate any useful long-term effect in the treatment of mild allergic asthma.

Chemotaxis Assay. [2]
The measurement of leukocyte chemotaxis was assessed by a modification of the Boyden micropore filter technique. Briefly, the assay was carried out in a 48-well microchemotaxis assembly. Cell suspensions of eosinophils in HBSS adjusted to 1 × 106 cells/ml were placed with or without Lodoxamide (at different concentrations), in the upper chamber separated from the leukoattractant solution by a 5-µm-pore-size polycarbonate membrane filter. The stimulus compartment received either control buffer (HBSS) for spontaneous migration measurement, or the attractant reagent: fMLP (10–6 mol/l) or human IL-5 (10 ng/ml); optimal concentrations had been determined in preliminary dose-dependent experiments. Migration was carried out at 37 °C in humidified air with 5% CO2 for 2 h. The filters were then fixed and stained with Giemsa. The numbers of eosinophils that had migrated were enumerated microscopically in four random high-power fields in quadruplicate wells. The percentage of inhibition obtained with Lodoxamide was calculated by comparison to the positive control (migration of untreated cells in response to the respective chemoattractant reagent), according to the formula:

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Lactate Dehydrogenase (LDH) Assay. [2]
Eosinophil lysis was followed by the measurement of one cytoplasmic marker, LDH, in supernatants of eosinophils incubated for 2–18 h with medium, with Triton as positive control or with different concentrations of Lodoxamide. LDH was evaluated by a colorimetric assay.

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Eosinophil Activation and Mediator Release. [2]
Highly purified eosinophils from patients were incubated with secretory IgA and anti-IgA mAb at respective final concentrations of 15 and 20 µg/ml in RPMI medium. After 2–18 h at 37 °C, the cells were centrifuged, the supernatants were collected and stored at –20 °C until the measurements of granule proteins could be performed. The determination of EPO was carried out by a chemiluminescence (CL) assay as previously described. Briefly, 50 µl of each supernatant were transferred into plastic tubes containing 50 µl of D-luciferin (160 µmol/l in Tris HCl buffer, 0.01 mol/l, pH 6), 50 µl of luminol (200 µg/ml in Tris HCl buffer, pH 6) and 50 µl of H2O2 (1.35 mmol/l in Tris HCl buffer, pH 6). The enzymatic reaction was started by adding the cell supernatant, and light emission was monitored by a photometer. Only cell populations with at least 90% eosinophils were submitted to the EPO release assay. Results were expressed in CL units. ECP release was measured in the 2-hour supernatants by double antibody radioimmunoassay. The radioactivity in the pellet was then measured and is inversely proportional to the quantity of ECP in the sample. Results were expressed in nanograms per milliliter on the basis of a standard curve obtained with purified ECP. Eosinophil-derived neurotoxin (EDN) was measured in the 18-hour supernatants by a commercially available radioimmunoassay and the results were expressed in nanograms per milliliter. The results of the three release assays (EPO, ECP and EDN) are presented as absolute values for each individual patient, from which the spontaneous release values, in the absence of IgA anti-IgA, have been subtracted.

Experimental protocol [3]
Isolated lungs were flushed with either modified EC solution (modified with 2.5 ml 50% magnesium sulfate and 50 ml 50% dextrose per liter) or UW solution, with 40 U insulin and 16 mg dexamethasone per liter) and stored in the same solution for 6 or 12 hours. Control lungs were flushed and stored using standard solution, and experimental lungs were flushed and stored using solution with 10 μmol/L of LT. At each storage time there were two experimental and two control groups consisting of six lungs each: standard EC, standard UW, EC+LT (EC+L), and UW+Lodoxamide/LT (UW+L).

Absorption, Distribution and Excretion
In a study of twelve healthy adult volunteers, topical administration of lodoxamide tromethamine ophthalmic solution 0.1%, one drop in each eye four times per day for ten days, did not result in any measurable lodoxamide plasma levels at a detection limit of 2.5 ng/mL.
Urinary excretion is the major route of elimination.

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Biological Half-Life
Elimination half-life was 8.5 hours in urine.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Although no published data exist on the use of lodoxamide during lactation, maternal milk levels are likely to be very low after the use eye drops. To substantially diminish the amount of drug that reaches the breastmilk after using eye drops, place pressure over the tear duct by the corner of the eye for 1 minute or more, then remove the excess solution with an absorbent tissue.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

[1]. Protective effect opf lodoxamide tromethamine on allergen inhalation challenge. J Allergy Clin Immunol. 1980 Oct;66(4):286-94.

[2]. Inhibitory effects of lodoxamide on eosinophil activation. Int Arch Allergy Immunol. 1998 Jun;116(2):140-6.

[3]. Addition of a mast cell stabilizing compound to organ preservation solutions decreases lung reperfusion injury. J Thorac Cardiovasc Surg. 1998 Mar;115(3):631-6; discussion 636-7.

[4]. Inhaled lodoxamide tromethamine in the treatment of perennial asthma: a double-blind placebo-controlled study. J Allergy Clin Immunol. 1985 Jul;76(1):83-90.

Lodoxamide Tromethamine is the tromethamide salt form of lodoxamide, a synthetic mast cell stabilizing compound with anti-inflammatory activity. Upon administration to the eye, lodoxamide tromethamide inhibits type I immediate hypersensitivity reaction by preventing the antigen-stimulated calcium influx into mast cells, thereby inhibiting release of histamine. Lodoxamide tromethamide also prevents the release of slow-reacting substances of anaphylaxis (SRS-A) and inhibits eosinophil chemotaxis.
See also: Lodoxamide (has active moiety).

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Lodoxamide is an organooxygen compound and an organonitrogen compound. It is functionally related to an alpha-amino acid.
Lodoxamide is a mast-cell stabilizer for topical administration into the eye. Mast-cell stabilizers, first one approved being cromolyn sodium, are used in treatment of ocular hypersensitivity reactions such as vernal conjunctivitis. These conditions often require treatment with anti-inflammatory medications such as ophthalmic NSAIDs or topical steroids which may cause systemic or toxic effects long-term. Although less effective than topical steroids at decreasing inflammation, mast-cell stabilizers offer another treatment option and exhibit minimal adverse effects. Lodoxamide is marketed under the brand name Alomide by Alcon.
Lodoxamide is a Mast Cell Stabilizer. The physiologic effect of lodoxamide is by means of Decreased Histamine Release.
Lodoxamide is a synthetic mast cell stabilizing compound with anti-inflammatory activity. Lodoxamide appears to inhibit the antigen-stimulated calcium transport across the mast cell membrane, thereby inhibiting mast cell degranulation and the release of histamine, leukotrienes, and other substances that cause hypersensitivity reactions. Lodoxamide also inhibits eosinophil chemotaxis. When applied topically to the eye, this drug prevents the symptoms associated with keratitis or conjunctivitis.
See also: Lodoxamide Tromethamine (has salt form).

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Drug Indication
Indicated in the treatment of the ocular disorders referred to by the terms vernal keratoconjunctivitis, vernal conjunctivitis, and vernal keratitis.
FDA Label

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Mechanism of Action
Although lodoxamide's precise mechanism of action is unknown, it is postulated that it prevents calcium influx into mast cells upon antigen stimulation and therefore stabilizes the membrane. By stabilizing the mast cell membrane from degranulation, lodoxamide consequently inhibits the release of intracellular histamine and other chemoattractant factors that primarily cause ocular symptoms. Lodoxamide's mechanism of action may be similar to cromolyn sodium, as both exhibit cross-tachyphylaxis.

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Pharmacodynamics
Lodoxamide is a mast cell stabilizer that inhibits the in vivo Type 1 immediate hypersensitivity reaction. Lodoxamide therapy inhibits the increases in cutaneous vascular permeability that are associated with reagin or IgE and antigen-mediated reactions.

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In conclusion, Lodoxamide seems to represent a very efficient drug, which is able to inhibit eosinophil chemotactic response to fMLP and IL-5 and to decrease the release of cytolytic granule proteins. The functional relevance of these results is confirmed by the potent inhibitory effects of Lodoxamide on eosinophil-mediated cytotoxicity. It is interesting to note that lodoxamide at 10 µg/ml concentration could induce partial but significant inhibition of each parameter of eosinophil activation (chemotactic response, mediator release and antibody-dependent cytotoxicity). These different effects could be synergistic in vivo, needing therefore lower concentrations. A combination of the effects of lodoxamide on mast cell activation (previously described) as well as on eosinophil chemotaxis and degranulation suggests that lodoxamide could dramatically reduce the pathological potential of eosinophils in tissues and represents a very efficient drug for the treatment of eosinophilic diseases, including allergic conjunctivitis. [2]

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This study also indicates that EC and UW, the two most commonly used clinical lung preservation solutions, are not optimal because they were both significantly improved by adding Lodoxamide. In the development of new lung preservation solutions, we believe there may be a benefit to considering the role of the mast cell in reperfusion injury and incorporating mast cell stabilizing compounds into the solutions. [3]

C15H17CLN4O9

553.90

553.142

C, 41.20; H, 5.10; Cl, 6.40; N, 12.64; O, 34.66

63610-09-3

Lodoxamide; 53882-12-5

11192129

White to light yellow solid powder

827.5ºC at 760 mmHg

454.3ºC

12

15

10

37

543

0

ClC1C(=C([H])C(C#N)=C([H])C=1N([H])C(C(=O)O[H])=O)N([H])C(C(=O)O[H])=O.O([H])C([H])([H])C(C([H])([H])O[H])(C([H])([H])O[H])N([H])[H].O([H])C([H])([H])C(C([H])([H])O[H])(C([H])([H])O[H])N([H])[H]

JJOFNSLZHKIJEV-UHFFFAOYSA-N

InChI=1S/C11H6ClN3O6.2C4H11NO3/c12-7-5(14-8(16)10(18)19)1-4(3-13)2-6(7)15-9(17)11(20)21;2*5-4(1-6,2-7)3-8/h1-2H,(H,14,16)(H,15,17)(H,18,19)(H,20,21);2*6-8H,1-3,5H2

2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-chloro-5-cyano-3-(oxaloamino)anilino]-2-oxoacetic acid

Lodoxamide Tromethamide; LODOXAMIDE TROMETHAMINE; 63610-09-3; Alomide; Lodoxamide Trometamol; Lodoxamide tromethamine salt; UNII-50LV9A548L; U-42,585E; 50LV9A548L;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

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

DMSO: ~20 mg/mL (~36.1 mM)
Water: ~19 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.76 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.76 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.76 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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