WO2006079209A1 - Pulmonary compositions comprising a vitamin a compound and a surfactant and uses thereof - Google Patents

Abstract

The current invention is directed towards a pulmonary composition comprising a vitamin A compound in combination with a pulmonary surfactant, wherein said vitamin A compound is miscible or soluble with said surfactant. The invention also provides a method of treating a pulmonary disorder by administering the pulmonary composition to the lungs of a subject (e.g., intratracheally).

Description

PULMONARY COMPOSITIONS AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. provisional application number

60/646,653 filed January 26, 2005, which is hereby incorporated by reference.

FIELD OF THE INVENTION

The invention is, in general, in the field of drug delivery. More specifically, the invention provides methods and compositions for pulmonary delivery of a combination of a Vitamin A compound and a surfactant, for the treatment of pulmonary disorders.

BACKGROUND OF THE INVENTION

Vitamin A is a generic name for a group of fat soluble compounds which have the biological activity of the primary alcohol, retinol, and are present in three natural forms: retinol, retinaldehyde and retinoic acid. Retinol is a dietary component present in food sources of animal origin and is also formed from its precursor β-carotene, which is present in food sources of plant origin (Mactier and Weaver, 2005). At the molecular level, Vitamin A compounds and derivatives interact with cytoplasmic retinoid receptors that act as transcription factors, and thus regulate gene expression. Vitamin A also has antioxidant properties thought to be due in part to the hydrophobic chain of polyene units that quench singlet oxygen (Palace et al., 1999). The antioxidant properties of Vitamin A are supported by indications that Vitamin A deficiency produces an increase in markers of oxidant stress. In vitro, and in physiologic animal models, Vitamin A has been implicated in the regulation and promotion of growth and differentiation of many cells, promotion of alveolar repair, protection against hyperoxia mediated cell-cycle arrest, stimulation of surfactant synthesis, and improvement of pulmonary vascularization (Ozer et al, 2005; Maden and Hind, 2004; Snyder et al., 2005). In lungs, the functional consequences of vitamin A deficiency include decreasing septation, surfactant production and loss of ciliated cells. This results in impaired mucociliary clearance and loss of the protective antioxidant-rich layer of the lungs, thereby rendering the airway to injury and infection (Thomas and Hazinski, 2000). Vitamin A deficiency in laboratory animals produces histopathological changes in the respiratory system such as necrosis and metaplasia that can be reversed by adequate Vitamin A administration.

Bronchopulmonary dysplasia or BPD (also referred to as chronic lung disease or CLD) is a pulmonary disorder that is clinically diagnosed based upon oxygen dependence for a specific period of time after birth. Pathologically, BPD is characterized by a decreased alveolarization of the lung, a localized inflammatory response, pulmonary infections and impaired pulmonary vascular growth (D'Angio and Maniscalco, 2004; Stenmark and Abman, 2005). Risk factors for BPD include premature birth, respiratory failure, oxygen supplementation and mechanical ventilation (Northway, 2001). Premature infants are prone to Vitamin A deficiency because they fail to accumulate Vitamin A in the third trimester of gestation. As a result, premature infants have low concentrations of vitamin A and low plasma retinol/RBP (retinol binding protein) molar ratios (Hustead et al., 1984). In addition, even if normal vitamin A (retinol) levels are present in pre-term infants, hepatic and lung storages of retinyl esters are often reduced (Tammela et al., 1999). This is further exacerbated by the fact that premature infants start feeding late, the absorption of Vitamin A is low because of low bile secretion and lipase production in the immature gut and the metabolic needs for Vitamin A are high (Shenai, 1999; Greene et al., 1987; Hustead et al., 1984; Darlow et al., 2002; Haas et al., 2002). Vitamin A supplementation is thought to protect the lungs of preterm infants from oxidative damage that occurs as a result of exogenous oxygen administration.

Vitamin A administration has proved problematic or ineffective in many instances. Vitamin A is currently administered to infants via multiple intramuscular injections (Wardle et al., 2001), which invasive and time consuming, while oral supplementation with Vitamin A may not be efficacious (Ambalavanan N et al., 2003). Intravenous administration of Vitamin A has also proved problematic because Vitamin A degrades in the light and there is significant adsorption to the tubing (Haas et al., 2002). Furthermore, Vitamin A may have toxicity effects or drug interactions at high doses when administered systemically. For example, infants receiving large doses of vitamin A have exhibited increased intracranial pressure and vomiting. In addition, in many cases liver toxicity is a major concern with large doses of vitamin A.

SUMMARY OF THE INVENTION The invention provides, in part, a pulmonary composition that combines a surfactant with a vitamin A compound that may be miscible or soluble in the surfactant. The pulmonary composition may be administered to the lungs of a subject (e.g., intratracheally) for the treatment of a pulmonary disorder.

In one aspect, the invention provides a method of treating a pulmonary disorder in a subject by administering to the subject an effective amount of a pulmonary composition including a vitamin A compound in combination with a surfactant, where the vitamin A compound may be miscible or soluble with the surfactant.

In an alternative aspect, the invention provides a pulmonary composition including a vitamin A compound in combination with a surfactant, where the Vitamin A compound may be miscible or soluble with the surfactant. In an alternative aspect, the invention provides the use of an effective amount of a pulmonary composition including a vitamin A compound in combination with a surfactant, where the vitamin A compound may be miscible or soluble with the surfactant, for the preparation of a medicament for treating a pulmonary disorder in a subject.

In an alternative aspect, the invention provides a kit for treating a pulmonary disorder, the kit including a Vitamin A compound and a surfactant, where the vitamin A compound may be miscible or soluble with the surfactant, together with instructions for preparation of a pulmonary composition.

In an alternative aspect, the invention provides a method of selecting a Vitamin A compound suitable for pulmonary administration, by combining the Vitamin A compound with a surfactant and determining whether the Vitamin A compound may be soluble or miscible in the surfactant, where a Vitamin A compound that may be soluble or miscible in the surfactant may be suitable for pulmonary administration.

In alternative embodiments of the various aspects, the pulmonary composition further includes an effective amount of an alcohol, such as ethanol. In alternative embodiments of the various aspects, the pulmonary composition may be an intratracheal composition.

In alternative embodiments of the various aspects, the pulmonary disorder may be neonatal respiratory distress syndrome, bronchopulmonary dysplasia, meconium aspiration syndrome, persistent pulmonary hypertension, or adult respiratory distress syndrome. The subject may be a neonate, such as a premature neonate.

In alternative embodiments of the various aspects, the administering may be performed intratracheally.

In alternative embodiments of the various aspects, the surfactant may be derived from a natural source, such as bovine lungs. The surfactant may be BLES®. In alternative embodiments of the various aspects, the vitamin A compound may be a vitamin A ester, e.g., retinyl palmitate, retinyl formate, retinyl acetate, retinyl propionate, retinyl butyrate, retinyl valerate, retinyl isovalerate, retinyl hexanoate, retinyl heptanoate, retinyl octanoate, retinyl nonanoate, retinyl decanoate, retinyl undecandate, retinyl laurate, retinyl tridecanoate, retinyl myristate, retinyl pentadecanoate, retinyl heptadeconoate, retinyl stearate, retinyl isostearate, retinyl nonadecanoate, retinyl arachidonate, retinyl behenate, retinyl linoleate, retinyl oleate, retinyl ethyl ester, or retinyl capryl ester.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a line graph showing the minimum (min) and maximum (max) ranges of surface activity for 5000 IU/mL retinyl acetate + ethanol + the surfactant BLES ® over 52 consecutive pulsations in 2.5 minutes in 3 separate runs as measured with the pulsating bubble surfactometer. Figure 2 is a line graph showing the minimum (min) and maximum (max) ranges of surface activity for the surfactant BLES ® over 52 consecutive pulsations in 2.5 minutes in 3 separate runs as measured with the pulsating bubble surfactometer.

Figures 3A-D are bar graphs indicating hemodynamic, pulmonary function, blood gas and hepatic Vitamin A levels derived from three experimental groups in which a surfactant deficiency was created: Group I: Intermittent positive pressure ventilation

(IPPV) with 100% O2 (denoted as C in the graphs); Group II: BPPV with 100% O2 + the surfactant BLES® (5nύ7kg) (denoted as BLES in the graphs); Group III: IPPV with 100% O2+ Vitamin A (25,000 IU/kg) and the surfactant BLES® (5ml/kg) (denoted as BLES + vit A in the graphs), pre- and post-treatment.

DETAILED DESCRIPTION OF THE INVENTION The invention provides, in part, a pulmonary composition that combines a surfactant with a vitamin A compound that is miscible or soluble in the surfactant. A "pulmonary composition" as used herein is a pharmaceutical composition that is suitable for delivery to the lungs of a subject in need thereof. The pulmonary composition may be administered (e.g., intratracheally) to the lungs of a subject, for example, for the treatment of a pulmonary disorder. In some embodiments, the pulmonary composition of the invention provides a rapid, non-invasive and/or cost effective route of administration. In some embodiments, the pulmonary composition of the invention avoids problems associated with absorption of a vitamin A compound from the gastrointestinal tract and controls hepatic release and cellular uptake. In some embodiments, the pulmonary composition of the invention decreases toxicity or systemic or local side effects associated with vitamin A administration. In some embodiments, the pulmonary composition of the invention provides rapid delivery of the active agent to the lungs. In some embodiments, the pulmonary composition of the invention enables delivery of effective doses of the active agent to the lungs. In some embodiments, the pulmonary composition of the invention is particularly suited for intratracheal administration. In some embodiments, the pulmonary composition of the invention is suited for administration to the lungs of a neonate, such as a preterm infant. In alternative embodiments, a pulmonary composition including retinyl acetate and BLES® is suitable, as both retinyl acetate and BLES® are well tolerated clinically.

By "pulmonary disorder" is meant a lung disorder. In some embodiments, a pulmonary disorder according to the invention is a disorder relating to endogenous pulmonary surfactant deficiency or dysfunction, hi some embodiments, a pulmonary disorder according to the invention is a disorder that benefits from improved oxygenation. In some embodiments, a pulmonary disorder according to the invention is a disorder that may be treated by the administration of a surfactant, or a disorder in which a surfactant may be administered as an aspect of treatment. Pulmonary disorders according to the invention include, without limitation, the following disorders.

Neonatal respiratory distress syndrome (RDS) is one of the most common lung disorders in premature infants and causes increasing difficulty in breathing. The disease is caused by a lack of lung surfactant, which normally appears in mature lungs. A long term complication of RDS is BPD (Karcher et al., 2005); Bronchopulmonary dysplasia (BPD) develops most often in premature babies who are born with underdeveloped lungs. It is characterized by inflammation and scarring in the lungs associated with delayed lung growth. (Jobe, 1999). BPD (also referred to as chronic lung disease or CLD) is clinically diagnosed based upon oxygen dependence for a specific period of time after birth. Although the time frame for oxygen dependence has evolved, the current clinical diagnosis is based upon oxygen requirement at 36 weeks post-conceptual age. Infants with BPD may have low birth weights, e.g., 500-150Og.

Meconium aspiration syndrome: Meconium is the first intestinal discharge from newborns. Meconium-stained amniotic fluid may be aspirated during labor and delivery, causing neonatal respiratory distress. Because meconium is rarely found in the amniotic fluid prior to 34 weeks' gestation, meconium aspiration chiefly affects infants at term and postterm. (Clark DA., 1987).

PPHN: Persistent pulmonary hypertension (PPHN) of the newborn is a cardiopulmonary disorder causing neonatal respiratory distress. It is characterized by systemic arterial hypoxemia secondary to elevated pulmonary vascular resistance with resultant shunting of pulmonary blood flow to the systemic circulation. (Dworetz et al., 1889).

Adult respiratory distress syndrome (ARDS): Acute respiratory distress syndrome (ARDS) is a life-threatening condition in which inflammation of the lungs and accumulation of fluid in the air sacs leads to low blood oxygen levels. ARDS is a serious condition with respiratory failure seen in the pediatric population as well. ARDS usually requires hospitalization and intensive care. It can be brought on by various problems with the lungs or numerous other medical conditions. (Martin M et al., 2005). A "surfactant" is in general a material that is capable of reducing the surface tension of a liquid in which it is dissolved. A surfactant may be a protein/lipid composition that is produced endogenously in the lungs and is critical for oxygen uptake. A surfactant may be a natural surfactant (e.g., derived from animal lungs, e.g., bovine lungs or porcine lungs) or may be synthetic (e.g., Exosurf®). Surfactants include naturally occurring surfactants such as L-α-phosphatidylcholine dipalmitoyl (DPPC). Other surfactants include, but are not limited to, diphosphatidyl glycerol (DPPG), hexadecanol, fatty alcohols such as polyethylene glycol (PEG), polyoxyethylene-9-lauryl ether, a surface active fatty acid (pamitic acid), sorbitan trioleate, glycholate and bovine surfactant extract (BLES®). Any surfactant that is suitable for administration to the lungs of a subject may be used in the compositions and methods of the invention.

Vitamin A compounds

Vitamin A is a generic name for a group of fat soluble compounds which have similar biological activity to the primary alcohol, retinol. In general, any Vitamin A compound that is suitable for pharmaceutical use and is soluble or miscible with a surfactant, for example, a surfactant that is suitable for administration to the lungs of a subject, may be used in the compositions and methods of the invention. Vitamin A compounds include, but are not limited to, derivatives of retinol, such as an ester thereof, or retinoic acid or an ester thereof. In some embodiments, a suitable Vitamin A compound may be a retinyl ester, such as (without limitation): retinyl palmitate, retinyl formate, retinyl acetate, retinyl propionate, retinyl butyrate, retinyl valerate, retinyl isovalerate, retinyl hexanoate, retinyl heptanoate, retinyl octanoate, retinyl nonanoate, retinyl decanoate, retinyl undecandate, retinyl laurate, retinyl tridecanoate, retinyl myristate, retinyl pentadecanoate, retinyl heptadeconoate, retinyl stearate, retinyl isostearate, retinyl nonadecanoate, retinyl arachidonate, retinyl behenate, retinyl linoleate, retinyl oleate, retinyl ethyl ester, retinyl capryl ester, etc. Other Vitamin A compounds include but are not limited to retinal, retinoic acid (such as 13-cis retinoic acid and all-trans retinoic acid), retinaldehyde, etc.

A vitamin A compound is "miscible or soluble" in a surfactant when examined macroscopically or microscopically. Macroscopic evaluation (Eggert et al., 1982; Wong et al., in press) may include vigorous agitation of a composition including a vitamin A compound and a surfactant prior to visual inspection to determine the presence of opalescence, separation of solutions, gross precipitation or crystallization using a strong light and a dark background. Preparations not showing the above physical signs may be considered macroscopically miscible. Microscopic evaluation may be performed using a light microscope (Carl Zeiss West Germany) at standard magnification (10 to 10Ox) and determining evidence of precipitation and crystallization. Separation of solutions and/or denaturation of BLES was observed using fluorescent microscope.

Briefly, macroscopic and microscopic analysis may be performed as follows. Samples are examined under at least three different conditions by a single observer: immediately after preparation (t = 0 h), 24 hours after preparation at room temperature (t = 24 h), and 3 hours after incubation in a 37°C water bath (t = 27 h). Under each condition, macroscopic and microscopic analyses are performed. The macroscopic analysis involves vigorous agitation of each mini-bag prior to inspection for the presence of haze or gross precipitation using a strong light and a dark background. The microscopic analysis involves examination of two drops of each solution using a light microscope (Carl Zeiss, West Germany) under powers of 10x, 4Ox, and 10Ox to detect the presence of crystals. Precipitation is deemed to occur with the presence of a single crystal. Three determinations are performed for each sample. This technique is validated by verifying that the limit of precipitation detected microscopically does not vary over multiple samples of the same Vitamin A solution. While the macroscopic and microscopic evaluation method may be used to assess the solubility of a Vitamin A compound, any method known in the art to assess miscibility or solubility of a compound may be used.

Animal Models

A suitable Vitamin A compound may be combined with a surfactant as described herein or known to a person of skill in the art, and administered to an animal model of a pulmonary disorder. For example, a primate model of BPD, as described by Coalson et al., 2000), or a rat, baboon, or sheep model of BPD, may be employed. As an example of animal model of a pulmonary disorder such as BPD, rat pups are exposed to high environmental O₂ concentrations during the period of alveolar development. This model recapitulates the histological pattern (anomalies in lung structure) seen in lungs of newborns with BPD: i.e. large and few alveoli (air sacs), with decreased septation ("fine tissue strings" that subdivide the air sacs to form true alveoli) and decreased lung capillaries (fine vessels that run below the alveoli to exchange oxygen, O₂ and carbon dioxide, CO₂). Because the newborn rat is incompletely-alveolarized at birth, this is a useful model to study postnatal influences on alveolarization.

Rat pups and their dams are placed in sealed Plexiglas chambers (BioSpherix, Redfield, NY) on day 4 of life (P4). Rat pups are exposed to hyperoxia (95% O₂, BPD- group) or normoxia (21%, control group) beginning on P4. Environmental O₂ is measured continuously with an analyzer (BioSpherix, Redfield, NY). Dams that provide equal nutrition to each litter are switched every 48 hours between the hyperoxic and normoxic chambers to prevent damage to their lungs (adult animals tolerate hyperoxia less well than newborn animals). Litter size is adjusted to 12 pups to control for effects of litter size on nutrition and growth. The chamber is dehumidified and temperature controlled and is opened 10 minutes every 48 hours to change bedding, food and water, exchange dams and check conditions of pups. At P 15, rat pups are euthanized with intraperitoneal pentobarbital and lungs are processed for lung histology.

Drug delivery (VitA in 0.9% NaCl, BLES, BLES + vit A) into the airways of newbon rats is performed through intratracheal puncture at P4. After halothane anesthesia, the trachea i exposed through a neck-incision. The drug (25μl) is then delivered through a tracheal punctur with a short, 30-gauge needle (Becton-Dickinson, Oakville, ON). The incision is then suturei (6.0 Prolene, Ethicon, Germany) and rat pups are allowed to recover. Lungs are prepared for light microscopy by tracheal infusion, using a 4% glutaraldehyde solution at a pressure of 20 CmH₂O. The trachea are then be ligated and the lungs immersed in fixative overnight at 4⁰C. The lung are sampled systematically, processed and embedded in paraffin. Transverse sections are cut 5μm thick, stained with hematoxylin and eosin, and examined under light microscopy. Lung morphometry is assessed using lung volume and the mean linear intercept method.

Pulmonary Compositions, Dosages, And Administration

Pulmonary compositions according to the invention may be used to treat or prevent a pulmonary disorder in a subject in need thereof. As used herein, "treatment" includes prophylaxis or prevention of a pulmonary disorder. Pulmonary compositions according to the invention include a combination of a Vitamin A compound with a surfactant. In some embodiments, the pulmonary composition includes an alcohol (e.g., ethanol, methanol, propyl alcohol, propanol, isopropyl alcohol, butanol, hexanol, heptanol, octanol, ethyl glycerol, glycerol, etc.). In some embodiments, ethanol is a suitable alcohol for use in humans. In general, any alcohol that is suitable as a solvent for a Vitamin A compound can be used, if it is also suitable for administration to a subject, such as human subject (e.g., a term or preterm infant).

The amount of alcohol to be included in the pulmonary composition may be any value from about 0.1% to about 25% v/v, or from about 0.5% to about 20% v/v, or from about 1% to about 15% v/v, or from about 5% to about 10% v/v, of the total volume of the pulmonary composition, hi some embodiments, the amount of alcohol to be included in the pulmonary composition may be about 0.95% to about 1.5% v/v, e.g., about 0.99% v/v, of the total volume of the pulmonary composition. In some embodiments, the amount of alcohol to be included in the pulmonary composition may be about 0.25 to about 1 mL out of a total volume of 5mL. In some embodiments, the volume of alcohol may be minimized with respect to the volume of surfactant and vitamin A compound, to minimize any potential toxicity associated with the alcohol.

The amount of vitamin A compound to be included in the pulmonary composition may range from any value between about 1 to about 150,000 IU, or about 100 to about

100,000 IU, or about 1000 to about 50,000 IU, or about 5000 to about 10,000 IU. In some embodiments, the amount of vitamin A compound may be about 5000 IU per mL.

If desired, treatment with a pulmonary composition according to the invention may be combined with more traditional and existing therapies for pulmonary disorders, such as oxygen administration, intubation and ventilation, continuous positive airway pressure (CPAP), inhaled NO (nitric oxide), parenteral vasoactive drugs, liquid ventilation, aerosols, parenteral vasodilatators, etc. Treatment with a pulmonary composition according to the invention may also be combined with therapies such as agents that promote vascular development, such as IL- 13 or vascular endothelial growth factor (VEGF), or anti-inflammatory agents, such as antagonists of metalloproteinases, or superoxide dismutase.

Pulmonary compositions of the invention can be provided in any pharmaceutically acceptable carrier, in a form suitable for administration to mammals, for example, humans, cattle, sheep, etc. In some embodiments, the pulmonary composition is provided in a form suitable for administration to neonates.

Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the compounds to subjects suffering from or presymptomatic for a pulmonary disorder, hi general, the pulmonary composition is formulated such that a Vitamin A compound is solubilized in a suitable surfactant and administered to a subject. In some embodiments, the Vitamin A compound is incorporated directly into the surfactant. Such directly compositions have the advantage of reducing the number of steps or manipulations to be performed by medical personnel perform prior to administering the composition. In some embodiments, the Vitamin A compound (e.g., retinyl acetate) is mixed with the surfactant (e.g., BLES®) at the time of administration. Any appropriate route of pulmonary administration may be employed, for example, intratracheal or aerosol administration. In some embodiments, the pulmonary composition is not an aerosol or inhalable composition. The pulmonary formulations may be in the form of liquid solutions or suspensions. Methods well known in the art for making formulations are found in, for example, "Remington's Pharmaceutical Sciences" (19^th edition), ed. A. Gennaro, 1995, Mack Publishing Company, Easton, Pa.

Pulmonary compositions according to the invention may be administered chronically or intermittently. "Chronic" administration refers to administration of the pulmonary composition(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. "Intermittent" administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature. For therapeutic or prophylactic compositions, the pulmonary compositions are administered to an individual in an amount sufficient to enhance lung compliance, or to enhance oxygenation, depending on the pulmonary disorder. The pulmonary composition may be administered as a single "bolus" dose or in multiple doses.

An "effective amount" of a pulmonary composition according to the invention includes a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as improved lung compliance or oxygenation, as well as decreased time on ventilatory support and oxygen supplementation, decreased length of hospitalisation, reduced risk of bronchopulmonary dysplasia, which is associated with decreased oxygen requirement at 36 weeks corrected gestational age, decreased use of anti-inflammatory medications, decreased risk of developmental delay, and/or decreased number of rehospitalisations for respiratory problems. A therapeutically effective amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the compound to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as improved lung compliance or oxygenation. Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount. A suitable range for therapeutically or prophylactically effective amounts of a vitamin A compound may range from any value between about 1 to about 150,000 IU, or about 100 to about 100,000 IU, or about 1000 to about 50,000 IU, or about 5000 to about 10,000 IU in a single dose. In some embodiments, the amount of vitamin A compound may be about 5000 IU per mL of the pulmonary composition.

It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners. The amount of active compound(s) in the composition may vary according to factors such as the disease state, age, sex, and weight of the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. In general, compounds of the invention should be used without causing substantial toxicity. Toxicity of the compounds of the invention can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LD50 (the dose lethal to 50% of the population) and the LDlOO (the dose lethal to 100% of the population). In some circumstances however, such as in severe disease conditions, it may be necessary to administer substantial excesses of the compositions.

As used herein, a "subject" may be a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc. The subject may be a neonate clinical patient, a clinical trial volunteer, an experimental animal, etc. The subject may be a premature neonate, i.e., born before 37 weeks of gestation. The subject may be suspected of having or at risk for having a pulmonary disorder, be diagnosed with a pulmonary disorder, be an experimental subject induced to have a pulmonary disorder, or be a control subject that is confirmed to not have a pulmonary disorder. Diagnostic methods for various pulmonary disorders and the clinical delineation of pulmonary disorder diagnoses are known to those of ordinary skill in the art.

Various alternative embodiments and examples of the invention are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention.

EXAMPLE I: Miscibility and/or solubility of Vitamin A Compounds with Surfactant

The miscibility and surface activity were tested in solutions of surfactant supplemented with vitamin A compounds. More specifically, miscibility/solubility and surface activity of bovine surfactant (BLES Biochemicals Inc., London, ON, Canada) ± alcohol (brand name: alcohol (dehydrated) injection USP 100% v/v; generic name: ethyl alcohol injection USP 100% v/v from Sandoz Canada Inc., Boucherville, QC, Canada) ± emulsifiers (tween 20 Sigma-Aldrich, Canada; tylaxapol: 4-(l,l,3,3-tetramethylbutyl) phenolpolymer with formaldehyde and όxirane from Sigma-Aldrich, Canada) supplemented with 5000 or 50000 IU/mL were tested using the following vitamin A compounds: retinyl acetate, retinyl palmitate, 13-cis retinoic acid, and all-trans retinoic acid. Miscibility relates to two solutions (BLES + vit A in ethanol). Solubility relates to a solution and solids (BLES + vit A).

For the BLES + vitamin A preparation, 5000 and 50000 IU vitamin A were dissolved directly and vortexed into 1 mL BLES. For the BLES + vitamin A + alcohol preparation, a stock solution of 50000 IU/mL vitamin A dissolved in alcohol was vortexed with 1 mL BLES. The 5000 IU/mL vitamin A solution was obtained by dilution of the 50000 IU/mL before mixing with 1 mL BLES. For the "BLES + vitamin A + emulsifiers" preparation, same as above using tylaxopol (25 mg) or tween 20 instead of alcohol. Briefly, the vitamin A compound (retinyl acetate) was dissolved directly into the dehydrated alcohol 100% v/v before further mixing it with BLES. 0.089g of retinyl acetate powder (2.83M IU/g) was measured into a microcentrifuge tube, and 50OuL of 100% v/v dehydrated alcohol was added, to give a 500,000 IU/mL retinyl acetate preparation, and the resulting suspension was mixed thoroughly using a vortex until dissolved. 5OuL of the retinyl acetate preparation was added to 5mL BLES® and the resulting 25,000 IU retinyl acetate was administered to a lkg animal. The amount of alcohol in the final preparation is 5OuL, giving a total volume of 505OuL that was administered.

Miscibility/solubility was determined at room temperature and at 37⁰C immediately upon mixing the preparation and 24h thereafter by testing visually for macroscopic appearance and microscopically using light and fluorescence for precipitation. Macroscopic evaluation (Eggert, 1982) was performed in a plastic test tube after vigorous agitation prior to visual inspection to determine the presence of opalescence, separation of solutions, gross precipitation or crystallization using a strong light and a dark background. Preparations not showing the above physical signs were considered macroscopically miscible. Using light microscope (Carl Zeiss West Germany) at standard magnification (10 to 10Ox) evidence of precipitation and crystallization was sought in all preparations, while separation of solutions and/or denaturation of BLES was observed using fluorescent microscope. Surface activity was measured for the vitamin A derivatives showing miscibility in surfactant. Immediately after mixing the "BLES + vitamin A + ethanol" preparation, surface activity was measured using a pulsating bubble surfactometer (PBS) as described by Enhorning (Enhorning G, 1977). The mixture of 138 μL of the preparation diluted in 1362 μL of diluent (240 μL absolute ethanol in 260 μL purified water) was kept at 37⁰C for 90 minutes prior to determination of surface tension. The preparation was then loaded and allowed to equilibrate for three minutes in the small volume chamber of the PBS which communicates with ambient air through a chimney. The motion of a piston of the PBS produces a negative pressure in the chamber until a bubble is formed at the air-liquid interface in the chimney. As the bubble is subjected to 20 pulsations per minutes for 2¹A minutes, surface tension is recorded. Under optimal conditions a surfactant will induce after a few pulsations the surface tension to diminish to zero during expiration. During each pulsation the bubble formed at the surface of the air/surfactant interface will oscillate between a maximal surface tension when the bubble has the smallest size at baseline and a minimal surface tension with the larger bubble size (Enhorning G, 2001). Three runs were performed per sample. A BLES reference sample was run as control.

As indicated in Table I, retinol acetate (5000 IU/mL) exhibited good miscibility (macro and microscopically) with surfactant after premixing vitamin A with ethanol. Furthermore, retinol palmitate exhibited miscibility (macro and microscopically) with surfactant alone after preheating at 37⁰C. Figure 1 shows that after 20 pulsations BLES + retinyl acetate + ethanol induces a significant effect on surface activity, as surface tension drops similarly to the drop observed with BLES®.

EXAMPLE II: In vivo oxygenation status in animals treated with surfactant with or without vitamin A

To determine the effects of the administration of surfactant- vitamin A preparation on pulmonary mechanics, gas exchange and hemodynamics in vivo, twelve piglets (1-3 days age, 1.02-2.275 kg) were anesthetized, mechanically ventilated and instrumented. More specifically, mixed strain newborn piglets weighing 1.2 to 2.0 kg were obtained on the first or second day of life. The animals were placed in the supine position under a radiant heat lamp to maintain their body temperature, and were maintained in the same position for the duration of the study. Rectal temperature probe was positioned. A fluid-filled catheter was positioned in the distal oesophagus. Initial anaesthetic induction was by inhaled halothane 4%, later reduced to 2%. An incision in the midline of the neck allows the insertion of a catheter through the external jugular vein into the right atrium, through which maintenance IV fluid of 0.9% saline at 3-5 ml/kg/hour was infused. A catheter was placed in the common carotid artery, a tracheostomy was performed, and 0.2 mg/kg of acepromazine then given as well as a bolus of 10 mg/kg of fentanyl, followed by a continuous infusion of fentanyl of 5 mg/kg/h. Halothane was discontinued, assisted ventilation started and paralysis obtained using pancuronium bromide 0.1 mg/kg every 45 minutes. Ventilator settings: 30 breaths per minute, 20 cmH₂O peak inspiratory pressure (PIP), 4cm H₂O positive end expiratory pressure (PEEP) and an inspiratory time (Ti) of 0.4 seconds. Pressure was not changed throughout the study, only the ventilator rate was adjusted at baseline to give normal PaCO₂ and not exceeding an I:E ratio of 1:1. Through the right internal jugular vein, a 5 Fr sheath was inserted.

Lung injury was induced by multiple lung lavages with saline to induce hypoxemic respiratory failure (paO2 <80 mmHg in 100% O₂), utilizing a modification of the method described by Lachman¹⁰. More specifically, 35 ml/kg of normal saline, warmed to 37° C was administering through the endotracheal tube from a height 60 cm above the piglet's head. Recovery of saline was accomplished by passive drainage by lowering the connecting tube 60 cm below the piglet's head. This drainage was increased by gentle chest compressions. The average duration of the lavage procedure was 100 second. Between lavages (6 min apart), VT was maintained as baseline values by adjusting the PIP. Arterial blood gases will be monitored every two -four lavages. Lavages will be continued until the PaO₂ remains below 80 mm Hg for 20 min. The number of saline lavages usually is around 13. Then the piglet was ventilated for additional 90 min keeping VT and PEEP constant. Following this, a second series of saline lung lavages was performed (mean duration 96 sec) to induce further lung injury Soos et al., 1996). This series was identical to the first series, including the same end point. The number of saline lavages required to meet criteria for this series is 4. About 30 minutes after the last lavage, surfactant was administered.

The surfactant used was bovine surfactant extract (BLES; BLES Biochemicals Inc., London, Ontario, Canada).

Post saline-lavage, the acidotic and hypoxic piglets were randomly assigned to one of the following three groups (4 piglets in each):

• Group I: Intermittent positive pressure ventilation (IPPV) with 100% 02

• Group II: IPPV with 100% 02 + BLES (5ml/kg) • Group III: IPPV with 100% 02+ vitamin A (retinyl acetate) (25,000

IU/kg) and BLES (5ml/kg)

Hemodynamics, lung compliance, and blood gases were measured and recorded pre- (following saline lavage) and post-treatment (hourly till 4h following randomization). Lung compliance during gas ventilation was measured by a single breath expiratory occlusion technique. Data acquisition was done at baseline (pre and post meconium) and then every 30 minutes for the duration of the study (360 minutes). At the end of the ^' experiment (360 minutes), the animals were euthanized by intravenous overdose of phenobarbital (30 mg/kg IV). The analogue outputs of the pressures (ventilator,), heart rate and saturation were digitized (Data Translation DT 2801A) and acquired at 24 Hz (Asyst 4.0, Keithly Instruments Inc., Taunton, MA). The digitized signals were stored on the hard disk of a 486 IBM compatible computer and backed up onto floppies. Later offline analysis was utilized. For purposes of analysis, the acquired data were analyzed at baseline (pre and post meconium) and then every 30 minutes for the duration of the study (360 minutes).

Liver (main site of vitamin A storage) was sampled at 4 hours for retinol determination by HPLC. The data are presented as the mean +/- the standard deviation (n=4) and were analyzed by ANOVA with a p<0.05 as statistically significant.

Vitamin A determination:

Vitamin A was measured in liver by HPLC as reported (Chessex et al., 2005). Briefly, Vitamin A was measured as follows.

Chromatography was performed using Beckman Coulter System Gold chromatographic equipment (Mississauga, ON, Canada) consisting of a 125 pump, a manual injector with a 50 ml sample loop, a UV/VIS 166 detector. The data were collected and analyzed with the Karat 32 software. A Synergi 4μ Hydro-RP 80 A (250 x 44.6 mm) from Phenomenex was used.

Vitamin E (α-tocopherol), used as a control, vitamin A (all-trø/M-retinol), and retinyl acetate were purchased from Sigma- Aldrich Canada Ltd., Oakville, ON. The standard stock solution of the vitamins were prepared in ethanol at concentration 1, 2, 4, 8, 10 μg/ml and stored at -2O⁰C. HPLC-grade methanol, hexanes, and ethanol were Fisher Scientific Company, Nepean ON, Canada.

The mobile phase consisted of 100%MeOH filtered through a PTFE 0.45 μm filter (Millipore C, Bedford, MA) and degassed before each HPLC session.

Stock standard solutions of vitamin A (100 mg/1), vitamin E (100 mg/1), retinyl acetate (20 mg/1) were prepared in ethanol. A combination of five different concentrations of from 1 to 10 μg/ml of these standards were prepared and used to obtain calibration curves. All solutions were stored at -2O⁰C. The liver was homogenized and vitamin A was extracted with solvent. To 0.50 ml of each sample, 50 μl of retinyl acetate (internal standard, 20 mg/ml) and 2 ml of ethanol- water (1:1) mixture were added. Samples were vortex mixed for 1 min followed by addition of 2 ml of hexanes and vortex mixed again for 1 min. The samples were then centrifuged at 24000 rpm for 10 min, the supernatant (hexanes phases) containing the vitamins were removed and the aqueous phases extracted once more time with 2 ml hexanes. The hexanes fraction were combined and dried under a stream nitrogen gas at 3O⁰C. The dried samples were reconstituted with 500 μl of ethanol, filtered on 0.45 μm filter (Millipore C, Bedford, MA) and a portion of 50 μl was injected into the HPLC system. Each sample was run in triplicate.

Table II

Retinol levels were significantly higher (p<0.001 in animals receiving retinyl acetate.

As seen in Table II and Figures 3 A-D, post treatment, oxygenation and lung compliance were higher (p<0.001) in groups II & III compared to controls, indicating that intratracheal administration of surfactant with and without vitamin A significantly improved gas exchange and compliance compared to controls. As demonstrated in Figures 3A-C, BLES plus Vitamin A significantly improved oxygenation, but did not alter CO₂ levels or lung compliance relative to BLES alone. Furthermore, intratracheal administration of vitamin A in combination with a bovine surfactant (BLES®) did not alter the efficacy of the surfactant in vivo. As demonstrated in Figure 3D, hepatic Vitamin A levels significantly increased after administration of Vitamin A plus BLES, indicating a favorable bioavailable profile. Hepatic levels of retinol were higher (p<0.001) in animal receiving vitamin A compared to groups I and II. Furthermore, Vitamin A was detectable in the liver in vivo, indicating that intratracheal delivery makes Vitamin A bioavailable and that, using this route of administration, there was rapid uptake of retinyl acetate by the liver. Without being bound to any particular theory, the synergistic effect of vitamin A + surfactant on oxygenation may indicate an effect of vitamin A on the induction of vascular endothelial growth factor (VEGF), as Vitamin A and VEGF are implicated in regulating lung maturation (Maeno et al, 2002; Morrisey and Savani, 2003).

EXAMPLE III: Administration of a Vitamin A/surfactant Combination to an Infant A suitable surfactant is defrosted/warmed to room temperature. For a 1 kg infant,

25000 IU of a Vitamin A compound that is miscible or soluble in the surfactant (e.g., retinyl acetate) is optionally dissolved in 0.05 mL of an alcohol (e.g., ethanol), and combined with 5 mL of a surfactant (e.g., BLES®) yielding a total volume of 5.05 mL. The resulting pulmonary compound is administered to the patient through an intratracheal tube as follows. The endotracheal tube is appropriately positioned, and suction is applied, if necessary to clear the airway. The pulmonary composition is instilled intratracheally through a small catheter placed in the infant's endotracheal tube and aliquots are administered per manufacturer's instructions. The adequacy of ventilation is assessed by observing chest expansion/abdominal excursion and monitoring tidal volume/transcutaneous PCO₂. Ventilation pressure/tidal volume is adjusted as required during and after administration. Satisfactory SpO₂ZPaO₂ is maintained by adjusting FiO₂ as required. If oxygenation improves rapidly after administration, then FiO₂ is reduced according to oximeter/blood gas results. The infant is positioned baby during and after administration as per manufacturer's instructions.

REFERENCES

The following publications are incorporated by reference:

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Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. In the specification, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to", and the word "comprises" has a corresponding meaning. Citation of references herein shall not be construed as an admission that such references are prior art to the present invention. All publications are incorporated herein by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein and as though fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a pulmonary disorder in a subject, the method comprising administering to said subject an effective amount of a pulmonary composition comprising a vitamin A compound in combination with a surfactant, wherein said vitamin A compound is miscible or soluble with said surfactant.

2. The method of claim 1 wherein said pulmonary composition further comprises an effective amount of an alcohol.

3. The method of claim 1 or 2 wherein said alcohol is ethanol.

4. The method of any one of claims 1 to 3 wherein said pulmonary disorder is neonatal respiratory distress syndrome, bronchopulmonary dysplasia, meconium aspiration syndrome, persistent pulmonary hypertension, or adult respiratory distress syndrome.

5. The method of claim 4 wherein said pulmonary disorder is bronchopulmonary dysplasia.

6. The method of any one of claims 1 to 5 wherein said subject is a neonate.

7. The method of claim 6 wherein said neonate is a premature neonate.

8. The method of any one of claims 1 to 7 wherein said pulmonary composition is an intratracheal composition.

9. The method of any one of claims 1 to 8 wherein said administering is performed intratracheally.

10. The method of any one of claims 1 to 9 wherein said surfactant is derived from a natural source.

11. The method of claim 10 wherein said natural source is bovine lungs.

12. The method of any one of claims 1 to 9 wherein said surfactant is BLES®.

13. The method of any one of claims 1 to 12 wherein said vitamin A compound is a vitamin A ester.

14. The method of claim 13 wherein said vitamin A ester is selected from the group consisting of one or more of retinyl palmitate, retinyl formate, retinyl acetate, retinyl propionate, retinyl butyrate, retinyl valerate, retinyl isovalerate, retinyl hexanoate, retinyl heptanoate, retinyl octanoate, retinyl nonanoate, retinyl decanoate, retinyl undecandate, retinyl laurate, retinyl tridecanoate, retinyl myristate, retinyl pentadecanoate, retinyl heptadeconoate, retinyl stearate, retinyl isostearate, retinyl nonadecanoate, retinyl arachidonate, retinyl behenate, retinyl linoleate, retinyl oleate, retinyl ethyl ester, and retinyl capryl ester.

15. The method of any one of claims 1 to 12 wherein said vitamin A compound is retinyl acetate..

16. A pulmonary composition comprising a vitamin A compound in combination with a surfactant, wherein said Vitamin A compound is miscible with said surfactant.

17. The pulmonary composition of claim 16 further comprising an effective amount of an alcohol.

18. The pulmonary composition of claim 17 wherein said alcohol is ethanol.

19. The pulmonary composition of any one of claims 16 to 18 wherein said pulmonary composition is for intratracheal administration.

20. The pulmonary composition of any one of claims 16 to 19 wherein said surfactant is derived from a natural source.

21. The pulmonary composition of claim 20 wherein said natural source is bovine lungs.

22. The pulmonary composition of any one of claims 16 to 19 wherein said surfactant is BLES®.

23. The pulmonary composition of any one of claims 16 to 22 wherein said vitamin A compound is a vitamin A ester.

24. The pulmonary composition of claim 23 wherein said vitamin A ester is selected from the group consisting of one or more of retinyl palmitate, retinyl formate, retinyl acetate, retinyl propionate, retinyl butyrate, retinyl valerate, retinyl isovalerate, retinyl hexanoate, retinyl heptanoate, retinyl octanoate, retinyl nonanoate, retinyl decanoate, retinyl undecandate, retinyl laurate, retinyl tridecanoate, retinyl myristate, retinyl pentadecanoate, retinyl heptadeconoate, retinyl stearate, retinyl isostearate, retinyl nonadecanoate, retinyl arachidonate, retinyl behenate, retinyl linoleate, retinyl oleate, retinyl ethyl ester, and retinyl capryl ester.

25. The pulmonary composition of any one of claims 16 to 22 wherein said vitamin A compound is retinyl acetate.

26. Use of an effective amount of a pulmonary composition comprising a vitamin A compound in combination with a surfactant, wherein said vitamin A compound is miscible with said surfactant, for the preparation of a medicament for treating a pulmonary disorder in a subject.

27. The use of claim 26 wherein said pulmonary composition further comprises an effective amount of an alcohol.

28. The use of claim 26 or 27 wherein said alcohol is ethanol.

29. The use of any one of claims 26 to 28 wherein said pulmonary disorder is neonatal respiratory distress syndrome, bronchopulmonary dysplasia, meconium aspiration syndrome, persistent pulmonary hypertension, or adult respiratory distress syndrome.

30. The use of claim 29 wherein said pulmonary disorder is bronchopulmonary dysplasia.

31. The use of any one of claims 26 to 30 wherein said subject is a neonate.

32. The use of claim 31 wherein said neonate is a premature neonate.

33. The use of any one of claims 26 to 32 wherein said pulmonary composition is an intratracheal composition.

34. The use of any one of claims 26 to 33 wherein said administering is performed intratracheally.

35. The use of any one of claims 26 to 34 wherein said surfactant is derived from a natural source.

36. The use of claim 35 wherein said natural source is bovine lungs.

37. The use of any one of claims 26 to 36 wherein said surfactant is BLES®.

38. The use of any one of claims 26 to 37 wherein said vitamin A compound is a vitamin A ester.

39. The use of claim 38 wherein said vitamin A ester is selected from the group consisting of one or more of retinyl palmitate, retinyl formate, retinyl acetate, retinyl propionate, retinyl butyrate, retinyl valerate, retinyl isovalerate, retinyl hexanoate, retinyl heptanoate, retinyl octanoate, retinyl nonanoate, retinyl decanoate, retinyl undecandate, retinyl laurate, retinyl tridecanoate, retinyl myristate, retinyl pentadecanoate, retinyl heptadeconoate, retinyl stearate, retinyl isostearate, retinyl nonadecanoate, retinyl arachidonate, retinyl behenate, retinyl linoleate, retinyl oleate, retinyl ethyl ester, and retinyl capryl ester.

40. The use of any one of claims 26 to 40 wherein said vitamin A compound is retinyl acetate.

41. A kit for treating a pulmonary disorder, the kit comprising a Vitamin A compound and a surfactant, together with instructions for preparation of the pulmonary composition of any one of claims 1 to 22.

42. A method of selecting a Vitamin A compound suitable for pulmonary administration, the method comprising combining the Vitamin A compound with a surfactant and determining whether the Vitamin A compound is soluble or miscible in the surfactant, wherein a Vitamin A compound that is soluble or miscible in the surfactant is suitable for pulmonary administration.