Title:  Use of intracellular calcium chelators to increase surfactant secretion in the lungs

United States Patent:  6,797,728

Issued:  September 28, 2004

Inventors:  Strayer; David S. (Newtown Square, PA)

Assignee:  Thomas Jefferson University (Philadelphia, PA)

Appl. No.:  045904

Filed:  November 7, 2001

Abstract

Pulmonary surfactant is required in order to reduce surface tension in the lungs so that less effort is needed to reinflate the lungs after exhalation. A number of diseases and conditions exist that disrupt the normal flow of surfactant secretion, resulting in respiratory distress or failure. The present invention provides a method of treating a patient in respiratory distress syndrome wherein a surfactant deficiency has occurred, thereby restoring a normal respiratory function.

SUMMARY OF THE INVENTION

An object of the invention is to present a method of treating or preventing a respiratory distress syndrome in a mammal by administering a therapeutically effect amount of an agent that activates surfactant secretion in the mammal.

It is a further object of the present invention that the therapeutic agent used in the method of treatment or prevention of a respiratory distress syndrome uses at least one intracellular calcium chelator. In one embodiment of the invention the intracellular calcium chelator is BAPTA-AM. The BAPTA-AM is between 25 and 100 .mu.M.

It is another object of the present invention that the therapeutic agent used in the method of treatment or prevention of a respiratory distress syndrome enhances secretion of surfactant from type II pneumocytes. In one embodiment the therapeutic agent acts by altering the endoplasmic reticulum free calcium concentration ([Ca⁺² ]_l ] in the type II pneumocytes.

It is also an object of the present invention that the therapeutic agent is administered by aerosol, nebulization or liquid instillation.

DESCRIPTION OF THE INVENTION

Methods

Animals

Specified-pathogen free, female Sprague-Dawley rats (180-200 g) were purchased from Charles River Laboratories. The animals were used as sources of alveolar type II cells within 1 week of receipt.

Chemicals and Reagents

SP-A from bovine lung was purified and analyzed as described. (Dobbs, et al, AM. Rev. Respir. Dis. 134, 141-145, 1986). BAPTA AM and TPEN were purchased from Molecular Probes, Inc. (Corvallis, Oreg.). After initial dose-response studies, 25 .mu.M BAPTA-AM was used to load type II cells.

Type II Cell Cultures

Freshly isolated type II cells were prepared according to standard techniques, originally described by Dobbs (Dobbs, et al, AM. Rev. Respir. Dis. 134, 141-145, 1986), and later modified by Sen and Chander (Sen and Chander, Biochem. J. 298: 681-687, 1994). The use of these techniques has been previously reported. (Strayer, et al, Ex. Cell Res. 226: 90-97, 1996; Strayer, et al, Rec. Signal Transd. 7: 111-120, 1997). Briefly, lungs of anesthetized rats were cleared of blood and treated with elastase endotracheally (Worthington Biochemical, Freehold, N.J.) to obtain free cells. The cells were separated from lung debris and plated on bacteriological plates coated with normal rat IgG (Sigma Chemical Co., St. Louis, Mo.). After 1 hr, free cells were collected by panning, pelleted, and resuspended (0.8x10⁶ cells/ml) in minimum essential medium (MEM)+10% fetal bovine serum (FCS, Life Technologies, Gaithersburg, Md.).

Secretion Studies

Secretion studies were performed as described by Sen and Chander. (Sen and Chander, Biochem. J. 298: 681-687, 1994). Briefly, 0.8x10⁶ alveolar type II cells were cultured for 18 h in MEM with 10% FBS+0.5 .mu.Ci of [methyl-³ H]-choline (Amersham, Arlington Heights, Ill.) to label cellular phospholipids (PL) metabolically. Cells were washed to remove non-adherent cells and unincorporated radioactivity. After replacing this medium with fresh MEM, plates were incubated for 15 min. SP-A (100 ng/ml) was added to selected plates, followed by incubation for an additional 15 min.; secretagogues were then added at the appropriate concentrations [control (no addition), ATP (500 .mu.M), TG (10 .mu.M), ionomycin (1 .mu.M), PMA (50 ng/ml)]. At this point (t=0) in each experiment, some samples were removed for analysis of secreted labeled lipids to establish a baseline. Incubation proceeded for an additional 2 h for all other plates. Following this incubation, media were removed and centrifuged (15 min., 300xg) to pellet cells that detached during incubation. These cells were later pooled with those recovered from the plates. Lipids from both media and plates were extracted (Sen and Chander, Biochem. J. 298: 681-687, 1994) after adding [methyl-¹⁴ C]-DPPC (Amersham, Arlington Heights, Ill.) as a recovery standard, and egg phosphatidylcholine (Sigma Chemical Co., St. Louis, Mo.) as a carrier lipid. Recovered lipids were dissolved in Scintiverse II fluid (Fisher Scientific, Pittsburgh, Pa.) and the radioactivity of these lipid extracts measured with a scintillation counter (Beckman Instruments, Fullerton, Calif.). ³ H-labeled phospholipid recovery was normalized to recovery of [methyl-¹⁴ C]-DPPC. Phospholipid secretion was determined as percent secretion=(cpm in lipids recovered from mediax100)/(cpm in lipids recovered from media and plates).

In some studies involving the effect of calcium depletion on phospholipid synthesis, synthesis of glycerol-based lipids was also measured by quantitating the incorporation of ³ H-glycerol into surfactant lipids. Cell culture and lipid extraction were performed similarly to those described above except that [1,2,3-³ H]-glycerol (NEN Life Science Products, Inc., Boston, Mass.) was used instead of [methyl-³ H]choline.

Type II Cell Viability Assays

Viability of type II cells was tested both by trypan blue exclusion, and by tetrazolium/formazan assay (Promega Corp., Madison, Wis.), which measures mitochondrial dehydrogenases. These enzymes are only present in active form in living cells and are absent in nonviable cells. (Cory, et al, Cancer Comm. 3: 207-212, 1991). For such studies, an assay solution containing the tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfonyl)-2H -tetrazolium, inner salt; MTS) and an electron-coupling reagent (phenazine methosulfate; PMS) in MEM is added to the cells. MTS is bioreduced by dehydrogenase enzymes found in metabolically active cells into a formazan compound that is soluble in tissue culture medium and directly measurable from its absorbance at 490 nm. Correction was made for the small effect of BAPTA itself on this reaction by running a parallel assay on plates with no cells.

Intracellular Calcium Measurements

10⁶ freshly isolated type II cells were added to 35 mm tissue culture plates, each containing a polylysine (Sigma Chemical Co.) coated 25 mm diameter coverslip and incubated (37^oC., 5% CO₂) for at least 2 hr. Twenty min before use, Fura2/AM (Molecular Probes) was added (final concentration, 3 .mu.M) to each coverslip. After Fura2 loading, the coverslips were washed with cell incubation buffer (121 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂ PO₄, 1.2 mM MgSO₄, 2 mM CaCl₂, 10 mM glucose, 5 mM NaHCO₃, 0.2% BSA, and 10 mM HEPES/NaOH, pH 7.4) and inserted into a thermostatically regulated chamber (37^oC.). Cell incubation buffer was added and the whole assembly placed on the stage of a Nikon Diaphot inverted microscope. At appropriate times (see infra), secretagogues in the appropriate concentration (see supra) were added. Fluorescence images of the cells were collected by exciting the cells alternately at 340 and 380 nm and measuring emission at 500 nm using a charge-coupled device camera (Photometrics, Inc.) as described previously. (Rooney, et al, Biol. Chem 264: 17131-17141, 1989). Image acquisition and analysis were done with Macintosh computers running customized image-processing software. The ratio (340/380) of Fura2 excitation was calibrated to [Ca²⁺ ]_i as described. (Rooney, et al, Biol. Chem 264: 17131-17141, 1989; Grynkiewicz, et al, J. Biol. Chem. 260: 3440-3450, 1985).

ER Calcium Concentration

Type II cells were prepared as described and plated on glass coverslips that had been pretreated with poly-L-lysine (Sigma, 0.01%, 20^oC., 15 mins). After incubation overnight at 37^oC. in MEM-10% FCS, the cells were washed three times with serum-free MEM and once with loading buffer (121 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂ P0₄, 1.2 mM MgSO₄, 2% BSA, 10 mM glucose, 20 mM HEPES, 2 mM CaCl₂). The cells were loaded in loading buffer containing 10 .mu.M Fura-2FF(AM) (Teflabs, Austin, Tex.) and 0.06% Pluronic Acid (Molecular Probes, Inc.) for two hours with shaking at 37^oC. The coverslip was washed in calcium-free imaging buffer (121 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂ PO₄, 1.2 mM MgSO₄, 10 mM glucose, 20 mM HEPES), inserted into a thermostatically regulated chamber (37^oC.), and mounted on an inverted fluorescence microscope in 1 ml of imaging buffer. Fluorescence imaging following excitation at 340 nm and 380 nm was performed (supra).

During imaging MnCl₂ was added at time=0 to a final concentration of 500 .mu.M followed by either SP-A, ionomycin, or TG (final concentrations 100 ng/ml, 1 .mu.M and 4 .mu.M respectively).

Therapeutic Methods and Compositions

Therapeutic agent as used herein refers to an agent which is a biologically-active synthetic or natural substance, other than alveolar surfactant proteins themselves, that is useful for treating a medical or veterinary disorder or trauma, preventing a medical or veterinary disorder, or regulating the physiology of a human being or animal. Preferred pharmaceutical agents are those which are useful in treating disorders localized in or near the lungs or respiratory tract, including, but not limited to, infant or adult respiratory distress syndrome, oxygen toxicity associated with respirator therapy, pneumonia, bronchitis, asthma, emphysema, tuberculosis, chronic obstructive pulmonary disorders, and lung cancer.

"Respiratory distress syndrome" includes many conditions characterized by respiratory distress or failure. The following conditions represent some examples of respiratory distress or failure, but as this list is exemplary it is not intended to limit the use of the present invention: traumatic shock; Gram negative septic shock; sock due to other infectious organisms besides Gram negative bacteria; toxic shock; fluid loss or blood volume depletion other than traumatic shock; allergic reactions; allergic reactions to inhaled allergens; allergic reactions to ingested allergens; allergic reactions to administered allergens (either iatrogenically or otherwise); pneumonitis or pneumonia due to infectious agents; pneumonitis or pneumonia due to infectious agents where the infectious agent is bacterial; pneumonitis or pneumonia due to infectious agents where the infectious agent is fungal; pneumonitis or pneumonia due to infectious agent is a protozoan; pneumonitis or pneumonia due to infectious agents where the infectious agent is a multicellular organism; pneumonitis or pneumonia due to infectious agents where the infectious agent is mycoplasma; pneumonitis or pneumonia due to infecious agents where the infectious agent is Pneumocystis carinii; toxic pneumonitis; toxic pneumonitis where the toxin is inhaled; toxic pneumonitis where the toxin is ingested; toxic pneumonitis where the toxin is injected; toxic pneumonitis where the toxin is applied topically; primary organ failure in an organ other than the lungs; primary organ failure in an organ other than the lungs where that organ is the heart; primary organ failure in an organ other than the lungs where that organ is the liver; primary organ failure in an organ other than the lungs where that organ is the kidneys; primary organ failure in an organ other than the lungs where that organ is the digestive system; primary organ failure in an organ other than the lungs where that organ is the endocrine system (e.g., thyroid, adrenal, parathyroid); a reaction to an administered pharmacologic agent, including administered blood or blood products, pharmaceuticals and anesthetics; a tumor, benign or malignant, of the lungs or other organs; a developmental abnormality, including immaturity of the lungs; a developmental abnormality, including immaturity of the cardiovascular system; a developmental abnormality of the chest or chest wall; a developmental abnormality of the diaphragm; a developmental abnormality, including immaturity, of the digestive system; ionizing radiation.

The invention provides methods of treatment and prophylaxis by administration to a subject of an effective amount of a therapeutic, i.e., an intracellular calcium chelator. In a preferred aspect, the therapeutic is substantially purified. The subject is preferably an animal, including, but not limited to, animals such as cows, pigs, chickens, etc., and is preferably a mammal, and most preferably a human.

Various delivery systems are known and can be used to administer a therapeutic of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, expression by recombinant cells, receptor-mediated endocytosis (Wu and Wu, J. Biol. Chem. 262:4429-4432, 1987), construction of a therapeutic nucleic acid as part of a retroviral or other vector, etc.

The active compounds disclosed herein are administered to the lung(s) of a subject by any suitable means. Active compounds are preferably administered by administering an aerosol suspension of respirable particles comprised of the active compound or active compounds, which the subject inhales. The active compound can be aerosolized in a variety of forms, such as, but not limited to, dry powder inhalants, metered dose inhalants, or liquid/liquid suspensions. The respirable particles may be liquid or solid. The particles may optionally contain other therapeutic ingredients such as amiloride, benzamil or phenamil, with the selected compound included in an amount effective to inhibit the reabsorption of water from airway mucous secretions, as described in U.S. Pat. No. 4,501,729.

The particulate pharmaceutical composition may optionally be combined with a carrier to aid in dispersion or transport. A suitable carrier such as a sugar (i.e., lactose, sucrose, trehalose, mannitol) may be blended with the active compound or compounds in any suitable ratio (e.g., a 1 to 1 ratio by weight).

Particles comprised of the active compound for practicing the present invention should include particles of respirable size, that is, particles of a size sufficiently small to pass through the mouth or nose and larynx upon inhalation and into the bronchi and alveoli of the lungs. In general, particles ranging from about 1 to 10 microns in size (more particularly, less than about 5 microns in size) are respirable. Particles of non-respirable size which are included in the aerosol tend to deposit in the throat and be swallowed, and the quantity of non-respirable particles in the aerosol is preferably minimized. For nasal administration, a particle size in the range of 10-500 .mu.m is preferred to ensure retention in the nasal cavity.

Liquid pharmaceutical compositions of active compound for producing an aerosol may be prepared by combining the active compound with a suitable vehicle, such as sterile pyrogen free water. The hypertonic saline solutions used to carry out the present invention are preferably sterile, pyrogen-free solutions, comprising from one to fifteen percent (by weight) of the physiologically acceptable salt, and more preferably from three to seven percent by weight of the physiologically acceptable salt.

Aerosols of liquid particles comprising the active compound may be produced by any suitable means, such as with a pressure-driven jet nebulizer or an ultrasonic nebulizer. See, e.g., U.S. Pat. No. 4,501,729. Nebulizers are commercially available devices which transform solutions or suspensions of the active ingredient into a therapeutic aerosol mist either by means of acceleration of compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation.

Suitable formulations for use in nebulizers consist of the active ingredient in a liquid carrier, the active ingredient comprising up to 40% w/w of the formulation, but preferably less than 20% w/w. The carrier is typically water (and most preferably sterile, pyrogen-free water) or a dilute aqueous alcoholic solution, preferably made isotonic, but may be hypertonic with body fluids by the addition of, for example, sodium chloride. Optional additives include preservatives if the formulation is not made sterile, for example, methyl hydroxybenzoate, antioxidants, flavoring agents, volatile oils, buffering agents and surfactants.

Aerosols of solid particles comprising the active compound may likewise be produced with any solid particulate therapeutic aerosol generator. Aerosol generators for administering solid particulate therapeutics to a subject produce particles which are respirable and generate a volume of aerosol containing a predetermined metered dose of a therapeutic at a rate suitable for human administration. One illustrative type of solid particulate aerosol generator is an insufflator. Suitable formulations for administration by insufflation include finely comminuted powders which may be delivered by means of an insufflator or taken into the nasal cavity in the manner of a snuff. In the insufflator, the powder (e.g., a metered dose thereof effective to carry out the treatments described herein) is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in situ and the powder delivered by air drawn through the device upon inhalation or by means of a manually-operated pump. The powder employed in the insufflator consists either solely of the active ingredient or of a powder blend comprising the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant. The active ingredient typically comprises from 0.1 to 100 w/w of the formulation.

A second type of illustrative aerosol generator comprises a metered dose inhaler. Metered dose inhalers are pressurized aerosol dispensers, typically containing a suspension or solution formulation of the active ingredient in a liquified propellant. During use these devices discharge the formulation through a valve adapted to deliver a metered volume, typically from 10 to 200 .mu.l, to produce a fine particle spray containing the active ingredient. Suitable propellants include certain chlorofluorocarbon compounds, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof. The formulation may additionally contain one or more co-solvents, for example, ethanol, surfactants, such as oleic acid or sorbitan trioleate, antioxidant and suitable flavoring agents.

The present invention considers the route of administration of the therapeutic agent, the amount of therapeutic agent delivered to the injured region of the lung and the length of time which the therapeutic agent is delivered to the injured region of the lung to be determined by the physician in a particular situation. In addition, the periodicity of the treatment and the number of treatments administered during each period is dependent on the condition of the patient and the response of that patient to the treatment.

Results

Phorbol Ester Treatment Does Not Alter Cytosol Calcium

The effect of the phorbol ester secretagogue, phorbol myristate acetate (PMA), on [Ca²⁺ ]_i was examined. The lack of alteration in [Ca²⁺ ]_i on PMA treatment of type II cells has been reported. (Sano, et al, Am. J. Physiol. 253: C679-C686, 1987; Pian, et al, Biochim. Biophys. Acta 960: 43-53, 1988). The effect of PMA on type II cell [Ca²⁺ ]_i was tested by digital imaging fluorescence microscopy, which confirmed that PMA did not alter [Ca²⁺ ]_i in type II cells.

Calcium Depletion Reduces Surfactant Secretion Induced by Io and TG, but Not by PMA

ATP, terbutaline, Io and TG all elicit biphasic increases in [Ca²⁺ ]_i, reflecting both calcium release and calcium influx. (Strayer, et al, Rec. Signal Transd. 7: 111-120, 1997). SP-A, acting through its type II cell membrane receptor, inhibits both the surfactant secretion and Ca²⁺ transients stimulated by ATP, Io and TG (Strayer, et al, Rec. Signal Transd. 7: 111-120, 1997). PMA, in contrast, stimulates surfactant secretion without altering [Ca²⁺ ]_i. To test whether secretion induced by these secretagogues required calcium signaling, secretagogue activities in Ca²⁺ -depleted type II cells was studied.

Type II cells were incubated overnight and loaded with a ³ H-phospholipid (PL) precursor in Ca²⁺ -free medium. They were then treated with ATP, Io, TG or PMA, still in calcium-free medium, and PL secretion was measured 2 h later. Calcium depletion in this manner prevented PL secretion stimulated by ATP and Io, and partially inhibited that stimulated by TG. It did not alter PMA-stimulated surfactant secretion.

An altered PL labeling due to the incubation of cells without Ca²⁺ might account for the observed changes. Surfactanc secretion is expressed, relative to internal standards, as a percentage of total PL secreted in 2 h. Thus, altered labeling efficiency should not cause observed decreases in secretion in Ca²⁺ -depleted cells. To assess the effects of depleting cells of Ca²⁺ on PL synthesis, labeled PL (cpm ³ H-choline or ³ H-glycerol incorporated into PL/0.8x10⁶ cells) in type II cells that were cultured with and without Ca²⁺ overnight were compared. Overnight culture without calcium did not affect PL synthesis (Table 1).

Secretagogue Activity is Restored by Adding Calcium

The importance of calcium in the activity of these secretagogues was studied in Ca²⁺ -deprived cells by replenishing calcium, incubating the cells for 15 min., and then adding secretagogues. Adding Ca²⁺ back in this way restored PL secretion in response to Io and TG. Re-adding Ca²⁺ also increased PMA-induced secretion, but this effect was not statistically significant.

Before adding secretagogues, ³ H-choline-containing medium is removed and the cells are washed. An enhanced PL synthesis from the small pool of unincorporated ³ H-choline within the cells might account for the apparent increased secretion during the 2 h between re-adding Ca²⁺ and the assay. Therefore, the total ³ H-PL after overnight incubation was compared to that after 2 additional hours of culture, both without secretagogue. The additional 2 h during the assay did not increase PL synthesis significantly (207,175+78,555 cpm overnight, vs. 203,554+73,101 cpm over-night+2 h; P=0.640 by Wilcoxson signed rank test).

Chelation of Intracellular Calcium Stimulates Surfactant Secretion

Ca²⁺ depletion inhibits PL secretion that is induced by Ca²⁺ -releasing secretagogues (supra), and secretagogue activity is restored by re-adding Ca²⁺. Secretagogue-induced changes in [Ca²⁺ ]_i is central to surfactant secretion, i.e., increased [Ca²⁺ ]_i due to release from ER stores could signal type II cell activation to secrete PL. Therefore, the effects of chelating calcium on surfactant secretion was tested. BAPTA-AM, an esterified EGTA analog, does not bind Ca²⁺ and crosses cell membranes freely. It is de-esterified in the cytosol, whereupon it binds Ca²⁺. If increases in [Ca²⁺ ]_i elicited by Ca²⁺ -releasing secretagogues is important in PL secretion, BAPTA should decrease surfactant secretion. However, the results reveal that BAPTA is a powerful secretagogue at concentrations between 25-100 .mu.M.

BAPTA chelates calcium and other cations. TPEN is a chelator of heavy metal cations. It crosses cell membranes but binds calcium much more weakly than BAPTA. To test whether chelation of other cations explains BAPTA secretagogue activity, TPEN was tested along with BAPTA. TPEN showed no secretagogue activity, even at 150 .mu.M.

BAPTA-AM is not known to be toxic to experimental animals or cells. In these studies, cellular viability was determined in two ways. Viable cells enumerated by counting trypan blue-excluding cells. Neither BAPTA-AM nor TPEN at these concentrations had any effect on cell viability, compared to each other or to cultures of untreated type II cells. In addition, cellular viability in BAPTA-AM-treated cultures was measured using the tetrazolium/formazan assay for mitochondrial dehydrogenase. (Cory, et al, Cancer Comm. 3: 207-212, 1991). These data also indicate that the viability of BAPTA-AM-treated type II cells does not differ from that of control cells.

Thus, BAPTA-AM, an inhibitor of cellular calcium and commercially available from chemical companies, does not inhibit surfactant secretion. This inhibitor causes a huge increase in surfactant secretion. This increased secretory activity is very large and highly reproducible. Within 4 hours of treatment, type II cells typically release about 2/3 of their stored surfactant, while control cells release about 7% in this time period.

BAPTA-induced PL Secretion is Inhibited by SP-A

SP-A inhibits secretagogue activities of ATP, Io and TG, all of which release Ca²⁺ from ER stores (Strayer, et al, Exp. Cell Res. 226: 90-97, 1996; Strayer, et al, Rec. Signal Transd. 7: 111-120, 1997), as well as that of PMA, which does not alter [Ca²⁺ ]_i (Dorn, C C, et al., Br. J. Pharmacol., 97:163-170, 1989; Kuroki, Y., et al., Biochem. J., 115: 115-119, 1991). BAPTA is a powerful secretagogue that binds cytosol calcium, buffering [Ca²⁺ ]_i. Analysis of whether SP-A altered BAPTA-induced surfactant secretion was determined. Type II cells were pretreated with SP-A, then exposed to BAPTA-AM (loading concentration, 25 .mu.M). Following SP-A treatment, BAPTA induced significantly less surfactant secretion than in control cells that were not exposed to SP-A (Table 2).

BAPTA Elicits Surfactant Secretion by Chelating Calcium

To ascertain that chelation of Ca²⁺, and not other cations, was responsible for BAPTA secretagogue activity, type II cells were maintained in Ca²⁺ -free medium overnight, and then during and after treatment with BAPTA-AM (25 .mu.M). Depleting cell Ca²⁺ prevented BAPTA secretagogue activity. Secretagogue activity was restored by re-adding calcium after overnight depletion. Thus, like secretagogues that release Ca²⁺ from stores, BAPTA-induced surfactant secretion requires Ca²⁺.

Calcium Influx is not Necessary for Secretagogue Activity

Calcium signaling in many cell types involves both release from stores and transmembrane influx. To test whether Ca²⁺ influx is important in Ca²⁺ signaling of surfactant secretion, type II cells were cultured in standard MEM containing 1.5 mM Ca²⁺ overnight during PL labeling. This medium was exchanged for Ca²⁺ -free medium just before the addition of the secretagogues. Despite some variability, most likely due to assaying secretion in non-equilibrium conditions, when Ca²⁺ influx is prevented, i.e., by removing extracellular Ca²⁺ during the assay, the surfactant secretion elicited by BAPTA or Io is not significantly altered. Statistically significant but small decreases in TG- and PMA-induced secretion were noted.

Secretagogue Activities of Calcium-releasing Secretagogues Are Not Additive, Except to that of PMA

If the several secretagogues stimulate surfactant secretion via the same pathway, their activities should overlap and their secretagogue activities should not be additive. If, however, two secretagogues stimulate surfactant secretion via different pathways, surfactant secretion following treatment with both simultaneously may exceed that elicited by either alone. PMA induces surfactant secretion without increasing [Ca²⁺ ]_i, even in calcium depleted cells. Its secretagogue activity could thus reflect a different signaling pathway or, less likely, action at a different point in the same pathway. Combinations of calcium-releasing secretagogues and PMA were tested for secretagogue activity. Adding two calcium-releasing secretagogues together did not increase surfactant secretion beyond that seen with either individually. However, when PMA was added to a Ca²⁺ -releasing secretagogue, the observed secretagogue activities were additive.

Cell viability was measured in all studies. Some combinations of secretagogues-PMA+BAPTA and BAPTA+TG--were toxic to type II cells, thus the additivity of these secretagogue activities could not be assessed.

Effect of BAPTA on Cytosol Calcium Concentration

BAPTA, a calcium chelator, while requiring calcium to activate surfactant secretion, should not alter [Ca²⁺ ]_i. To ascertain this, [Ca²⁺ ]_i was followed in type II cells loaded with fura2, and then exposed to BAPTA-AM. As measured by a decrease in the ratio of fura2 excitation at 340 nm compared to 380 nm, BAPTA-AM caused [Ca²⁺ ]_i to decrease somewhat, compared to mock-treated cells. Thus, BAPTA-AM is a calcium dependent secretagogue that does not increase [Ca²⁺ ]_i.

Alterations in Lumenal Calcium Concentration in Response to Calcium-releasing Secretagogues, PMA, BAPTA, and SP-A

Decreases in [Ca²⁺ ]_l, rather than increases in [Ca²⁺ ]_i, were hypothesized to be the critical regulator of surfactant secretion by all secretagogues, with the exception of PMA. This theory predicts that BAPTA-AM, and other calcium-dependent secretagogues, would decrease [Ca²⁺ ]_l, but that PMA would not. Thus, type II cells were loaded with fura2ff. This fluorophore has a much lower affinity for calcium in the .mu.M range than fura2 does, and is therefore used to assess changes in [Ca²⁺ ]_l. Type II cells were also treated with Mn²⁺, which binds cytoplasmic fura2ff and prevents its binding to Ca²⁺. (Dorn, et al, Br. J. Pharmacol. 97: 163-170, 1989; Kuroki, et al, Biochem. J. 115: 115-119, 1991; Miyata, et al, Am. J. Physiol. 261: H1123-H1134, 1991). Any changes in the lumenal free calcium concentration were measured as changes in the ratio of fura2ff excitation at 340 nm to that at 380 nm. As predicted, BAPTA decreased [Ca²⁺ ]_l, as did Io. PMA had no effect.

Effect of SP-A on Lumenal Calcium Concentration

To explain SP-A inhibition of both secretagogue-induced Ca²⁺ release and secretagogue-induced surfactant secretion, SP-A was hypothesized to further increase the stores' avidity for calcium, increasing Ca²⁺ uptake, and rendering lumenal Ca²⁺ less susceptible to release. SP-A alone should then increase [Ca²⁺ ]_l. This was tested as in type II cells loaded with fura2ff and treated with Mn2+(supra). SP-A caused increased [Ca²⁺ ]_l in Type II cells.

Discussion

BAPTA Does Not Change [Ca²⁺ ]_i

The essential nature of calcium signaling in surfactant secretion implies an important role for [Ca²⁺ ]_l in that signaling. The ability of BAPTA to stimulate surfactant secretion is key to understanding both signaling by Ca²⁺ -releasing secretagogues, and by Ca²⁺ inhibition by SP-A.

The secretagogue activities of ATP, Io, and TG are all inhibited by depleting cytosol calcium, and are restored by replenishing calcium. PMA-stimulates secretion, which appears to circumvent calcium signaling in type II cells, and is not affected by depleting cytosol calcium.

TG-induced secretion is less completely inhibited by depleting cytosol Ca²⁺ than is secretion induced by other calcium-releasing secretagogues. The reasons for this are not known. TG may, for example, access a store of calcium (such as high affinity binding sites) not easily depleted by overnight culture without Ca²⁺, and not accessible to other secretagogues.

Ca²⁺ influx is not a major participant in signaling surfactant secretion. When Ca²⁺ is removed from the medium at assay time BAPTA- or Io-induced secretion are not affected. Small decreases in TG- and PMA-induced secretion seen in these studies are of borderline statistical significance (P=0.03).

Calcium starvation does not make surfactant totally unavailable for secretion, as PMA-induced secretion is largely unaffected. PL synthesis is unaffected, incorporation of choline and glycerol precursors into PL is similar in calcium-starved and control cells.

In this regard the ability of a cation chelator, BAPTA, to stimulate type II cells to secrete surfactant is illuminating. The cation involved in BAPTA-induced secretion is calcium. TPEN, which chelates transition metals like iron, zinc, etc. well, and Ca²⁺ poorly (Aballay, et al, Biochem. J. 312: 919-23, 1995; Richardson, et al, J. Biol. Chem. 268: 11528-11533, 1993), does not stimulate surfactant secretion. More importantly, BAPTA secretagogue activity is eliminated by depleting Ca²⁺ and restored by replacing it. Another group reported using BAPTA in type II cell cultures, but at 5 .mu.M. (Benito, et al, Mol. Cell. Biochem. 189: 169-176, 1998). In the present invention, the dose-response studies in the concentration range of 5 .mu.M are below the threshold for BAPTA to stimulate surfactant secretion. Stimulation of surfactant secretion with BAPTA occurs between 25-100 .mu.M.

The ability of BAPTA to stimulate secretion does not involve depleting the total cellular pool of calcium, as overnight Ca²⁺ deprivation itself does not elicit large-scale surfactant secretion. There are several possible explanations for these findings. As in other systems (Lillie, et al Phil. Trans. Royal Soc. Lond. Ser. B. 336: 25-34, 1992; Lillie, et al, Biochem. J. 288: 181-187, 1992), Ca²⁺ is needed further downstream for surfactant secretion, independently of signaling. BAPTA buffering cellular Ca²⁺ does not block such a calcium-requiring step, while depleting cellular calcium does. Calcium also helps to maintain the structure of ER stores, and its depletion will distort these stores and render them less effective in transducing secretion-activating signals. (Blatter and Weir, Biophys. J. 58: 1491-1499, 1990).

From the perspective of Type II cell surfactant secretion, BAPTA-AM acts like a Ca²⁺ -releasing secretagogue. It causes surfactant secretion in a dose-dependent manner; is inhibited by depleting calcium; does not require calcium influx; and is inhibited by SP-A. However, unlike calcium-releasing secretagogues, BAPTA does not raise [Ca²⁺ ]_i. Thus, calcium signaling in type II cell PL secretion is unlikely to be related to increased [Ca²⁺ ]_i.

[Ca⁺² ]_l is the Critical Determinant for Surfactant Secretion

Calcium in ER stores is in equilibrium between free and protein-bound states. If the free calcium in stores is chelated or released, the equilibrium shifts, decreasing the amount of bound calcium. BAPTA in its esterified form will easily access ER stores (Miyata, et al, Am. J. Physiol. 261: H1123-H1134, 1991; Blatter and Wier, Biophys. J., 58: 1491-1499, 1990) where it is de-esterified. The high affinity of BAPTA for calcium (160 nM) competes effectively with the calcium-binding proteins there, and decreases bound calcium. Thus, calcium signaling activates surfactant secretion by altering [Ca²⁺ ]_l, not by increasing [Ca²⁺ ]_i.

ER calcium ([Ca2+]₁) was measured directly, applying a combination of fura2ff and Mn²⁺ to quench cytosolic fura2ff fluorescence. This approach has been used previously as a means to measure [Ca²⁺ ]_l. (Renard-Rooney, et al, J. Biol. Chem. 268: 23601-23610, 1993; Dorn, et al, Br .J Pharmacol., 97: 163-170, 1989; Kuroki, et al, Biochem. J. 115: 115-119, 1991; Miyata, et al, Am. J. Physiol. 261: H1123-H1134, 1991). This analysis highlighted the fact that BAPTA and calcium-releasing secretagogues all decreased [Ca²⁺ ]_l. PMA, which does not release calcium from stores, had no effect on [Ca²⁺ ]_l. These findings are best explained by postulating that surfactant secretion, as elicited by calcium-active secretagogues, is triggered by a decrease in the free calcium ([Ca²⁺ ]_l) and/or bound calcium in the ER. Alternatively, the process of calcium release, rather then the consequent decrease in [Ca²⁺ ]_l, causes secretion.

ER calcium stores regulate, directly or indirectly, lamellar body movement to the cell surface for extrusion. For example, Ca²⁺ release may alter the conformation of a Ca²⁺ -binding protein that spans the ER membrane, such as IP3R. (Mikoshiba, K, Curr. Opin. Neurobiol, 7: 339-345. 1997). In changing configuration, IP3R may initiate a signal to move lamellar bodies to the cell surface.

The observations using SP-A both support the importance of [Ca²⁺ ]_l in signaling surfactant secretion and suggest a means by which SP-A inhibits secretagogue-induced surfactant secretion. SP-A binds a type II cell membrane receptor (SPAR), inhibiting surfactant secretion. (Strayer, et al, Exp. Cell Res. 226: 90-97, 1996; Rice, et al, J. Appl. Physiol. 63: 692-698, 1987; Kuroki, et al, Biochem. J. 263: 17596-17602, 1988; Kuroki, et al, Proc. Natl. Acad. Sci. USA 85: 5566-5570, 1988). It also modulates the secretagogue-induced Ca²⁺ transients. (Sano, et al, Am. J. Physiol. 253: C679-C686, 1987; Strayer, et al, Rec. Signal Transd. 7: 111-120, 1997; Pian, et al, Biochim. Biophys. Acta 960: 43-53, 1988; Dorn, et al, Br. J. Pharmacol, 97: 163-170, 1989). It is noteworthy that Io- and TG-induced increases in [Ca²⁺ ]_i were also partially inhibited by SP-A (and restored if SP-A-SPAR interaction is blocked with anti-SPAR antibody, (Strayer, et al, Exp. Cell Res. 226: 90-97, 1996), since Io and TG act on ER stores directly. The ability of SP-A to decrease secretagogue-induced increases in [Ca²⁺ ]_i implies that SP-A alters the availability of calcium for release.

The fact that SP-A increased [Ca²⁺ ]_l in a high percentage of type II cells, then, both helps substantiate the proposed mechanism for the activity of calcium-releasing secretagogues and implies that the activity of SP-A in inhibiting their action is by increasing [Ca²⁺ ]_l. The mechanism(s) by which SP-A does this is unclear. SP-A might, for example, increase the avidity with which stored calcium is bound, or, alternatively, increase the activity of the calcium pump across the ER store membrane.

The studies with PMA emphasize the suggested relationship between the ability of a secretagogue to elicit calcium transients and its dependence on calcium for signaling secretion. PMA secretagogue activity circumvents calcium signaling. (Strayer, et al, Rec. Signal Transd. 7: 111-120, 1997). PMA also does not alter [Ca²⁺ ]_l. SP-A inhibition of PMA-induced secretion probably involves mechanisms other than SP-A-induced increases in [Ca²⁺ ]_l.

Combining calcium-releasing secretagogues does not increase secretion, only PMA augments secretion that is elicited by individual calcium-releasing secretagogues. Combinations including BAPTA+TG and BAPTA+PMA killed the cells. Apoptosis associated with TG has been previously reported. (Bian, et al, Am. J. Physiol. 272: C1241-1249, 1997; He, et al, J. Cell Biol. 138: 1219-1228, 1997). Still, these data imply that PMA activates secretion either via a different signaling mechanism or, less likely, distal to calcium release in the pathway activated by calcium-releasing secretagogues.

The extent to which the current data apply to other systems of Ca²⁺ -related secretion is not clear. In some experimental systems, BAPTA has no clear effect on secretory activity (Rotondo, et al, Thromb. Haemost. 78: 919-925, 1997), while in many experimental systems BAPTA chelation of Ca²⁺ inhibits cellular secretion. (Ko, et al, Br. J. Pharmacol, 121: 150-156, 1997; Vainio, et al, J. Cell Physiol., 169: 538-543, 1996). In cell types other than type II cells, BAPTA inhibited secretagogue activities of the same compounds whose secretagogue activities it mimicked here. (Suchard, et al, J. Innumol, 152: 290-300, 1994; Xu, et al J. Cell Sci. 109: 1605-1613, 1996). Several studies describe BAPTA and, for example, Io having parallel effects in cellular secretion. (Xu, et al J. Cell Sci. 109: 1605-1613, 1996). However, BAPTA did not activate secretion in any of these systems.

The present invention focuses attention on calcium in stores and away from [Ca²⁺ ]_i, as a key effector in signaling type II pneumocyte surfactant secretion. The extent to which similar mechanisms may operate in other calcium-dependent cell activation pathways in other cell types is not known. These findings have clinical ramifications. An intracellular chelator of divalent cations, for example BAPTA-AM, is used to treat conditions wherein an increase in surfactant secretion is desired. These conditions include (see also supra), but are not limited to, infection; respiratory distress syndrome; pulmonary injury such as trauma, inhalation or toxic exposure, drowning, external irradiation, administration of a compound that causes pulmonary injury, or a pulmonary complication of a systemic disease.

Claim 1 of 6 Claims

What is claimed is:

1. A method of treating a respiratory distress syndrome in a mammal, comprising administering a therapeutically effective amount of an agent comprising an intracellular calcium chelator that activates pulmonary surfactant secretion without increasing the cytosolic free calcium concentration ([Ca⁺² ]i) in said mammal, wherein said intracellular calcium chelator comprises BAPTA-AM.

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