NZ718717B2 - Rapamycin for the treatment of lymphangioleiomyomatosis - Google Patents

Abstract

The present invention relates to methods and compositions for treating lymphangioleiomyomatosis in a human subject in need of such treatment. The methods comprise administering to the subject via inhalation an aerosol composition comprising microparticles of rapamycin.

Description

RAPAMYCIN FOR THE TREATMENT OF LYMPHANGIOLEIOMYOMATOSIS FIELD OF THE INVENTION The present invention relates to methods and pharmaceutical compositions comprising rapamycin for pulmonary delivery, preferably by inhalation, for the prophylaxis and treatment of lymphangioleiomyomatosis.

BACKGROUND OF THE INVENTION Lymphangioleiomyomatosis (LAM) is a multisystem disease affecting 30-40% n with tuberous sis x (TSC), an often-fatal disease which is characterized by the read proliferation of abnormal smooth -like cells that grow aberrantly in the lung.

The proliferation of these cells red to as LAM cells) leads to the ion of cysts in the lungs and fluid-filled cystic structures in the axial lymphatics (referred to as lymphangioleiomyomas). The result is progressive cystic destruction of the lung parenchyma, obstruction of lymphatics, airways, and progressive respiratory failure. In addition, LAM cells can form tumors. These are generally slow growing hamartomas referred to as angiomyolipomas. Renal angiomyolipomas can lead to renal failure in LAM patients. The abnormal proliferation ofLAM cells is caused at least in part by an inactivating mutation in one ofthe tuberous sis complex tumor suppressor genes, TSCl or TSC2. The TSC genes are negative regulators of the mammalian target ofrapamycin (mTOR). The mTOR pathway is an important l point for cell growth, metabolism, and cell survival. As a consequence of the inactivation ofTSC genes, LAM cells show constitutive activation ofmTOR and many other kinases in the mTOR pathway including Akt, and 86K.

LAM generally occurs in women of child-bearing age although it may also occur in men.

While it is most prevalent in women having TSC, it can also occur in persons who do not have clinical manifestations of TSC, as well as those who do not have germline mutations in the TSCl or TSC2 tumor suppressor genes. These cases are referred to as sporadic LAM. Thus, LAM can occur as a sporadic, non-heritable form as well as in association with us sclerosis complex.

Although LAM can progress slowly, it ultimately leads to respiratory failure and death.

Ten years after the onset of ms 55% of patients are breathless, 20% are on oxygen and % are deceased. See e. g., Johnson et al. 2004 Thorax. Survival and e progression in UK patients with lymphangioleiomyomatosis. There is no currently approved drug for the treatment or prophylaxis ofLAM. The primary treatment options include the off-label use of oral rapamycin (sirolimus, which is FDA approved for the prophylaxis of organ rejection and renal transplantation, see below), or off— label use of oral everolimus.

Rapamycin is a macrocyclic triene antibiotic produced by Streptomyces copicus.

See e. g., US. Pat. No. 3,929,992. Rapamycin is an inhibitor of mTOR. The immunosuppressive and anti-inflammatory properties ofrapamycin initially indicated its use in the transplantation field and in the treatment of mune diseases. For example, it was shown to prevent the ion of l (IgE-like) antibodies in response to an albumin ic nge, to t murine T-cell activation, and to prolong survival time of organ grafts in histoincompatable rodents. In rodent models of autoimmune disease, it sses immune- mediated events associated with systemic lupus erythematosus, collagen-induced arthritis, autoimmune type I diabetes, autoimmune myocarditis, experimental allergic encephalomyelitis, graft-versus-host disease, and autoimmune uveoretinitis.

Rapamycin is also referred to by its generic drug name, sirolimus (see for example, ANDA #201578, by Dr. Reddys Labs Ltd., approved May 28, 2013). Sirolimus is FDA approved and marketed in the United States for the prophylaxis of organ rejection and renal lantation under the trade name RAPAMUNE by Wyeth (Pfizer). It is in the form of an oral solution (1 mg/ml) or tablet (multiple ths). Wyeth (Pfizer) also markets a derivative by the tradename TORISEL (temsirolimus) for the ent of advanced renal cell carcinoma, which is administered intravenously. Temsirolimus is a water-soluble prodrug of sirolimus. Cordis, a division of Johnson & n, markets a sirolimus-eluting coronary stent under the tradename CYPHER. In this context, the antiproliferative effects of sirolimus prevent restenosis in ry arteries following balloon angioplasty. US 2010/0305150 to Berg et al. (Novartis) describes rapamycin tives for treating and ting neurocutaneous disorders, such as those mediated by TSC including tuberous sclerosis, as well as those mediated by neurofibromatosis type 1 (NF-1). Rapamycin and its derivatives are further described in Nishimura, T. et a]. (2001) Am. J. Respir. Crit. Care Med. 163:498-502 and in US. Pat. Nos. 6,384,046 and US 823.

Rapamycin use in its clinically approved context has several known adverse effects including lung ty (the RAPAMUNE label warns that it is not indicated for lung transplant patients), increased cancer risk, and diabetes-like symptoms. Rapamycin is associated with the occurrence ofpulmonary toxicity, usually in the form of interstitial pneumonitis, but pulmonary alveolar proteinosis has also been documented. See for example, Nocera et al., Sirolimus Therapy in Liver Transplant ts: An Initial Experience at a Single Center, Transplantation Proceedings (2008), 40(6), 1950-1952; Perez et al., titial Pneumonitis Associated With Sirolimus in Liver Transplantation: A Case Report, lantation Proceedings (2007), 39(10), 3498-3499; Hashemi—Sadraei et al., Sirolimus-associated diffiJse alveolar hemorrhage in a renal lant ent on long-term agulation, Clinical Nephrology (2007), 68(4), 238-244; Pedroso et al., Pulmonary alveolar proteinosis - a rare pulmonary toxicity of Sirolimus, Transplant International (2007), 20(3), 291-296. The cause ofrapamycin-induced pulmonary toxicity is not known.

Severe respiratory adverse events have also been associated with mus use as an anti- cancer therapy under chronic stration resulting in circulating blood concentrations greater than 1 nanogram/mL range. For example, the lung toxicity of the Sirolimus prodrug, temsirolimus, was nted in a 2009 report noting that “interstitial lung disease is a rare side effect of temsirolimus treatment in renal cancer patients”. Aparicio et al., Clinical & Translational Oncology (2009), 11(8), 499-510; Vahid et al., Pulmonary complications of novel antineoplastic agents for solid tumors, Chest (2008) 133:528-538. In addition, a 2012 meta- analysis concluded that 10% of cancer patients administered temsirolimus or everolimus may experience mild grade ty with a worsening of quality of life and, in some case, interruption oftherapy. See Iacovelli et al., Incidence and risk ofpulmonary toxicity in patients treated with mTOR tors for malignancy. A meta—analysis ished trials, Acta oncologica (2012), 51(7), 873-879. Furthermore, safety pharmacology studies performed in rats with temsirolimus showed reductions in respiratory rate as well as alveolar macrophage infiltration and inflammation in the lungs (see Pharmacology Review for temsirolimus NDA 22088 available from the US FDA website). These adverse effects were observed under ions of relatively high concentrations of the drug in the circulating blood volume as a result of systemic stration.

Despite its potential for toxicity to the lung, orally administered rapamycin has shown inary promise as a potential LAM therapy. See New Eng. J. ne 364: 1595-1606 (2011) and review by Hammes and Krymskaya, Harm. Cancer 4(2):70-7 (2013); see also Ando et al. Respir Investig. 51(3): 175-8 (2013) “The efficacy and safety of low-dose Sirolimus for treatment of lymphangioleiomyomatosis”. But the clinical evidence also indicates the limitations ofrapamycin in this context and the need for improved therapies and therapeutic regimens for the treatment of LAM. The primary limitations mycin are the need to use the drug chronically, and most importantly, that rapamycin is associated with other adverse events (in addition to potential lung toxicities). For example, in a 24 month non-randomized open label trial ted in 20 patients, sirolimus administered orally was tested for its ability to reduce angiomyolipomas, which are slow growing hamartomas that can lead to renal failure in patients with TSC or sporadic LAM. Bissler et al. (2008) Sirolimus for angiomyolipoma in tuberous sclerosis complex or lymphangioleiomyomatosis. N Engl JMed 358(2): 140—151. In that study, angiomyolipomas regressed “somewhat” during the treatment period but tended to increase after therapy stopped. Serious adverse events ated with sirolimus ed diarrhea, pneumonia, pyelonephritis, cellulitis (from an animal bite), itis, and hemorrhage of a renal angiomyolipoma. Dosing was based on the serum target levels that would t rejection in renal transplant ts and ranged from 1 to 15 ng/ml (blood mus . In another similar study (phase 2, non-randomized open label trial) 16 patients with TSC or sporadic LAM were treated with oral sirolimus for up to 2 years. Davies et al (2011) Sirolimus therapy for angiomyolipoma in tuberous sclerosis and sporadic lymphangioleiomyomatosis: a phase 2 trial.

Clin Cancer Res 17(12):4071—4081. In that study, steady state blood levels of sirolimus were 3- ng/ml with more than half of the patients maintained on maintenance levels of 3-6 ng/ml.

Sirolimus treatment showed sustained regression ofrenal angiomyo lipomas. However, while tumor response was maintained with continuation of therapy, little further shrinkage occurred during the second year of treatment. Adverse events associated with sirolimus included oral mucositis, respiratory infections, and proteinuria. In another study of 10 LAM patients with documented progression, sirolimus was discontinued in 3 patients because of s recurrent lower atory tract infection or sirolimus- induced nitis. Neurohr et al., Is sirolimus a therapeutic option for patients with progressive pulmonary lymphangioleiomyomatosis? Respiratory Research (2011), 12:66. That study concluded that “sirolimus might be considered as a eutic option in rapidly declining LAM patients” but noted that its “administration may be associated with severe respiratory adverse events requiring ent cessation in some patients” and that ntinuation of mus is mandatory prior to lung transplantation.” Finally, a 12 month randomized, double-blind 89 patient clinical trial was conducted with 46 patients having LAM, followed by a 12 month observation period. McCormack et al (2011) Efficacy and safety of sirolimus in lymphangioleiomyomatosis. NEngl JMed 364: 1595—1606.

Patients were maintained at sirolimus blood levels of 5-15 ng/ml. In this study, sirolimus treatment ized lung function, reduced serum VEGF-D levels, and was associated with a reduction in symptoms and improved quality of life. But stabilization of lung on required continuous ent. Importantly, all of these clinical studies utilized oral formulations of mus. This is because an aerosol formulation of rapamycin for delivery directly to the lungs was considered highly unlikely to succeed in view ofrapamycin’s well-known lung toxicity, as exemplified by the articles cited above.

A US. patent application by Lehrer published in 2013 reflects the view that “[r]apamycin (sirolimus) cannot be safely inhaled because of its well-documented lung toxicity, interstitial pneumonitis”. See US 20130004436, citing Chhajed et a]. (2006) -374. The Lehrer patent application is directed to compositions and s for treating and preventing lung cancer and lymphangioleiomyomatosis. Although some earlier publications, such as US. Patent No. 5,080,899 to Sturm et al. (filed February 1991) and US Patent No. 5,635,161 (filed June 1995), contain some generic description ofrapamycin formulated for delivery by inhalation, such generic ptions were orted by any evidence and came before the many reported nces ofrapamycin-induced lung toxicity that appeared following its more widespread adoption as an immunosuppressant in the transplantation context and as an inhibitor of cellular proliferation in the ancer context, as ced by the reports discussed above.

W0 201 1/163600 describes an aerosol formulation of tacrolimus, which like rapamycin is a macrolide lactone. But tacrolimus is a distinct chemical entity from sirolimus and the molecular target of tacrolimus is calcineurin, not mTOR, and unlike rapamycin, tacrolimus did not show lung toxicity and in fact is ted for preventing rejection ing lung transplantations.

In view of the wide-spread ition of the ial for rapamycin-induced lung toxicity, a pharmaceutical composition comprising rapamycin for pulmonary delivery in the treatment ofLAM was not considered to be a viable therapeutic option in humans.

Delivery of drugs to the lung by way of inhalation is an important means of treating a variety of conditions, including such common local conditions as cystic fibrosis, pneumonia, bronchial asthma and chronic obstructive pulmonary disease, some systemic conditions, ing hormone replacement, pain management, immune deficiency, erythropoiesis, diabetes, lung cancer, etc. See review by Yi et al. J. Aerosol Med. Palm. Drug Deliv. 23: 181-7 (2010).

Agents indicated for treatment of lung cancer by inhalation include cisplatin, carboplatin, taxanes, and anthracyclines. See e.g., US. Pat. Nos. 6,419,900; 6,419,901; 6,451,784; 6,793,912; and US. Patent Application Publication Nos. US 2003/0059375 and US 2004/0039047. In on, bicin and temozolomide administered by inhalation have been ted for treating lung metastases. See e.g., US. Pat. No. 7,288,243 and US. Patent Application Publication No. 2008/0008662.

BRIEF DESCRIPTION OF THE FIGURES flgLel: LC-MS/MS Chromatogram of 10.6 ng/mL Rapamycin (top) and Internal Standard (bottom) in Mouse Blood.

F_igLe2: Representative Chromatograms of 10.6 ng/mL Rapamycin (top) and Internal Standard m) in Mouse Lung Homogenate.

F_igLe3: Calibration Curve for Rapamycin in Mouse Blood.

F_igLe4: Calibration Curve for Rapamycin in Mouse Lung Homogenate.

F_igLe5: Representative Chromatograms mycin (top) and Internal Standard (bottom) in Blood from Mouse 2-07 Administered Rapamycin by OPA.

F_igLe6: entative Chromatograms ofRapamycin (top) and Internal Standard (bottom) in Lung Homogenate from Mouse 2-07 Administered Rapamycin by OPA. figm: S6 Phosphorylation in Mouse Lung Following CPA and oral administration of rapamycin.

F_igLe8: Predicted rapamycin blood concentrations for ary administration repeated once daily.

SUMMARY OF THE INVENTION The present invention is based, in part, upon the discovery of surprising pharmacokinetics ofrapamycin when it was delivered directly to the lungs in animal studies. Unexpectedly, direct administration to the lungs resulted in a higher concentration of rapamycin in the lung tissue compared to blood. This is in contrast to what would be expected as highly lipophilic small molecule drugs such as rapamycin lly diffuse rapidly from the lung into the circulatory system and then ribute uniformly within the volume of distribution. In addition, the drug persisted in the lung at concentrations sufficiently high to have a therapeutic . Finally, direct administration to the lung also failed to produce any acute or c toxicity in the respiratory tract, another unexpected outcome in view of the association ofrapamycin with pulmonary toxicity, particularly in the form of interstitial pneumonitis.

As bed herein, there is a need for pharmaceutical formulations ofrapamycin, its prodrugs, derivatives, and analogues, delivered directly to the lungs, preferably by inhalation, in order to provide an effective local treatment and laxis of diseases and disorders affected by the TOR signaling pathway. Such localized treatment reduces or eliminates the toxicities and adverse events, including those associated with elevated concentrations ofrapamycin in the blood resulting from systemic delivery of the drug. The present invention addresses this need.

The present invention provides compositions ofrapamycin for delivery directly to the lungs which provide an amount of rapamycin effective to inhibit mTOR signaling in the lung with low or no toxicity to lung , and resulting in blood levels mycin of less than about 1 ng/ml. The compositions of the invention are expected to present an improved safety profile, especially with respect to their chronic or prolonged use, compared to current dosage forms ofrapamycin which result in persistent blood trations in the range of 1 ng/ml to 15 ng/ml. This is expected due to the low pulmonary toxicity of the present compositions combined with the probability of no or substantially fewer adverse events due to systemic exposure to rapamycin in view of the very low blood levels ofrapamycin ed. ingly, the compositions of the invention are expected to demonstrate a greater therapeutic index compared to existing dosage forms delivered via the gastrointestinal tract or intravenously.

The present invention is directed to itions and methods for the treatment and prophylaxis ofLAM using a pharmaceutical composition comprising rapamycin designed for pulmonary delivery, preferably by inhalation. In one embodiment, the invention provides a method for the treatment and laxis ofLAM in a human subject in need of such treatment, the method comprising administering to the subject an aerosolizable composition sing rapamycin (also referred to as sirolimus), or a prodrug or derivative thereof The compositions ofthe invention may be used alone, or in ation with one or more additional therapies or therapeutic regimens for the treatment ofLAM. In addition, the compositions provided herein may comprise rapamycin, or a prodrug or derivative thereof, as the sole therapeutic agent in the composition, or the cin may be ated with one or more additional therapeutic agents in a single dosage form.

Accordingly, the present invention provides a ceutical dry powder composition for pulmonary ry comprising an amount of microparticles of a drug, particles of a carrier, and one or more optional excipients, for use in treating lymphangioleiomyomatosis in a human subject, the drug being selected from rapamycin, or a g or tive thereof.

In one embodiment, the amount of drug in the composition is from 50 to 500 micrograms, from 50 to 250 micrograms, or from 50 to 150 micrograms.

In one embodiment, the drug is rapamycin. In one embodiment, the drug is selected from the group consisting of everolimus, temsirolimus, ridaforolimus, umirolimus, and zotarolimus.

In one embodiment, the microparticles consist of particles of drug having mean diameters from about 0.1 to 10 s or from about 1 to 5 microns. In one embodiment, the particles have a mean diameter of about 1.5 to 4 s, about 1.5 to 3.5 microns, or about 2 to 3 The carrier may be selected from the group consisting of arabinose, glucose, fructose, ribose, mannose, sucrose, trehalose, lactose, maltose, starches, dextran, mannitol, lysine, e, isoleucine, dipalmitylphosphatidylcholine, lecithin, polylactic acid, poly (lactic-co-glutamic) acid, and xylitol, and mixtures of any ofthe foregoing. In one embodiment, the carrier comprises or consists of a blend oftwo different carriers. The particles of carrier may have diameters g from to 200 microns, from 30 to 100 microns, or less than 10 s. Where the carrier consists of a blend oftwo different carriers, each carrier consists of les of a different size range, ed as average particle diameter. In one embodiment, the carrier consists of a blend oftwo different carriers, a first carrier and a second carrier. The first carrier consists of particles having diameters ranging from about 30-100 s and the second carrier consists of particles having diameters of less than 10 microns. The ratio of the two different carriers is in the range of from 3:97 to 97:3. In one embodiment, the carrier consists of a blend oftwo different e carriers.

The drug to carrier ratio in the powder may be from 0.5 % to 2 % (w/w). In one embodiment, the drug to carrier ratio in the powder is l % (w/w).

The amount of drug in the composition may be from about 0.5 % to 20 % (w/w) based upon total weight of the composition. In one embodiment, the amount of drug is from about 1 % to 2 % (w/w).

In one embodiment, the one or more optional excipients is present in the composition and is selected from a phospholipid and a metal salt of a fatty acid, and mixtures of the foregoing. In one embodiment, the phospholipid is selected from dipalmitylphosphatidylcholine and lecithin.

In one embodiment, the metal salt of a fatty acid is magnesium stearate. In one embodiment, the excipient or excipients is coated on the carrier particles in a weight ratio of excipient to large carrier particle ranging from 0.01 to 0.5%.

In one embodiment, the amount of drug in the compositions is an amount effective to inhibit the biological activity of mTORCl. In one embodiment, the amount of drug is an amount effective to t the phosphorylation of the 86K protein. In one embodiment, the amount of drug is an amount effective to achieve a respirable dose of from 5 to 400 micrograms delivered to the lung. In one embodiment, the respirable dose is about 10, about 50, about 100 or about 250 micrograms. In one embodiment, the amount of drug is an amount effective to produce a blood trough level in the subject of less than 5 ng/ml, less than 2 ng/ml, less than 1 ng/mL less than 0.5 ng/ml, or less than 0.25 ng/ml. In one embodiment, the blood trough level is less than 1 ng/ml, less than 0.5 ng/ml or less than 0.25 ng/ml. In one embodiment, the amount of drug is an amount ive to produce a tration of drug in the lung tissue of from 1 ng/g to 1 microgram (ug)/g. In one ment, the concentration of drug in the lung tissue is about 10 ng/g, about ng/g, about 50 ng/g, about 100 ng/g, or about 200 ng/g. In one ment, the drug persists in lung at therapeutic levels of about 1 ng/g, about 10 ng/g, about 25 ng/g, about 50 ng/g, or about 100 ng/g for a period oftime after administration, preferably to a human t, the period oftime selected from about 6 to 10 hours, about 6 to 14 hours, about 6 to 24 hours, and about 6 to 72 hours. In one embodiment, the period oftime is selected from about 10 hours, about 14 hours, about 24 hours, and about 72 hours.

In one embodiment, the composition has a fine particle fraction (FPF) greater than 20% with a corresponding fine particle dose (FPD) ranging from 10 micrograms to 2 milligrams, preferably less than 0.5 milligrams, following 1 to 12 months or 1 to 36 months of storage. In one embodiment the dose delivered to patient , the delivered dose (DD) or emitted dose (ED), ranges from 25 micrograms to 2.5 milligrams, preferably less than 0.5 milligrams.

In one embodiment, the composition further ses one or more additional therapeutic agents. The one or more additional therapeutic agents may be selected from an estrogen nist (e.g., letrozole, tamoxifen), a statin (e.g., simvastatin), a src inhibitor (e.g., saracatinib), and a VEGF receptor inhibitor (e.g., pazopanib). In one embodiment, the one or more additional therapeutic agents is selected from letrozole, fen, simvastatin, saracatinib, pazopanib, and imatinib.

In one embodiment, the composition delivers an amount of drug effective to improve the subject’s pulmonary on as measured by forced vital capacity (FVC) and forced expiratory volume . In one embodiment, the composition delivers an amount of drug ive to reduce the size or amount of pleural effusion detectable by radiologic examination.

In one embodiment, the composition is adapted for once daily administration.

In one embodiment, the composition is produced by a wet ing process comprising the steps of preparing an aqueous suspension of drug, subjecting the drug suspension to microfluidization, and spray-drying the resulting particles to form a dry powder.

In one embodiment, the drug is rapamycin, the carrier consists of a blend of two different lactose carriers, the first carrier consists of particles having e diameters ranging fiom about -100 microns and the second carrier consists of particles having average diameters of less than microns, the ratio of the two different carriers is about 9?:3 to 3:97, and the amount of rapamycin is from 25 to 1400 micrograms.

The invention also provides a unit dosage form for treating lymphangioleiomyomatosis sing the composition of any of claims 1 to 31, wherein the amount of drug is from about to 2500 micrograms, from 25 to 250 micrograms, or from 50 to 150 micrograms. In one embodiment, the amount of drug is from about 50 to 250 micrograms. In one ment, the dosage form is a e le for use in a dry powder inhaler . In one embodiment, the capsule contains from 1 mg to 100 mg ofthe powder or from 10 mg or 40 mg of the powder. The capsule may be a gelatin, plastic, or cellulosic e, or in the form of a foil/foil or foil/plastic r le for use in a DPI device.

The invention also provides a pharmaceutical package or kit comprising a composition or unit dosage form described herein, and instructions for use.

The invention also provides a dry powder ry device comprising a reservoir containing a composition or unit dosage form described herein. The reservoir may be an integral chamber within the device, a capsule, or a blister. In one embodiment, the device is selected from Plastiape® RSOl Model 7, Plastiape® RSOO Model 8, XCaps®, Handihaler®, Flowcaps® TwinCaps®, and Aerolizer®. [3 6] The invention also provides a method for treating lymphangioleiomyomatosis in a human subject in need of such treatment, the method comprising administering to the subject via inhalation a composition or unit dosage form described herein. _1()_ DETAILED DESCRIPTION OF THE INVENTION [3 7] The present invention provides s and compositions for the treatment and prophylaxis ofLAM in a human subject in need of such treatment. The human subject in need of such treatment is one who has been diagnosed with LAM. In one embodiment, the human subject is a woman. In one embodiment, the human subject is a man. In one embodiment, the human subject has been diagnosed with tuberous sclerosis complex. In one ment, the human subject has been sed with sporadic LAM. In one embodiment, the methods comprise administering to the subject via inhalation a composition comprising rapamycin in a suitable carrier, and optionally one or more additives. The term “rapamycin” is used generically throughout this disclosure to refer to cin itself, also referred to as mus, as well as to its prodrugs (such as temsirolimus) and derivatives. Derivatives of rapamycin e compounds that are structurally similar to rapamycin, are in the same chemical class, are rapamycin analogs, or are pharmaceutically acceptable salts ofrapamycin or its derivatives.

Further description and es of rapamycin, its prodrugs, and derivatives are provided in the following section.

The compositions described herein are aerosolizable compositions le for ing respirable particles or droplets containing rapamycin, or a prodrug or derivative f, (collectively referred to as ). In one embodiment, the drug is selected from sirolimus, everolimus, and temsirolimus. In one embodiment, the drug is sirolimus. The compositions may comprise the drug, a carrier, and optionally one or more additives. The compositions may be in the form of an aqueous solution, a dry powder, or a mixture of one or more pharmaceutically acceptable propellants and a carrier, as described in detail in the section below entitled “Compositions for Inhalation”.

The present invention also provides s for the treatment and prophylaxis ofLAM in a human subject in need of such treatment, the methods comprising the step ofpulmonary administration of a composition of the invention to the subject. In one embodiment, the administered dose ofrapamycin is sufficient to achieve a blood trough level of drug in the subject from 0.01 to 0.15 ng/ml, from 0.075 to 0.350 ng/ml, from 0.150 to 0.750 ng/ml, from 0.750 to 1.5 ng/ml, or from 1.5 to 5 ng/ml. In one embodiment, the administered dose of rapamycin is sufficient to achieve a blood trough level of drug in the subject of less than 5 ng/ml, less than 2 ng/ml, less than 1 ng/ml, or less than 0.5 ng/ml. In one embodiment, the administered dose ofrapamycin is ient to e a concentration ofrapamycin in lung tissue in the _11_ range of from 1 ng/g to 1 ug/g. Preferably, the aforementioned therapeutic levels ofrapamycin are achieved by administering a composition bed herein once a day and ably the total daily dose ofrapamycin administered to the subject is less than 2 mg or less than 1 mg per day.

Further aspects of pulmonary delivery and dosing, including combination ies, are described in the section below entitled “Pulmonary Administration and Dosing”.

The methods and compositions of the invention are effective to treat LAM in a subject in need of such treatment, preferably a human subject. The amount of drug effective to treat LAM (the “effective amount” or “therapeutically effective amount”) refers to the amount of drug (e. g., rapamycin) which is sufficient to reduce or ameliorate the progression, severity, and/or duration ofLAM or one or more symptoms of LAM, to prevent the advancement ofLAM, cause the regression ofLAM, or prevent the pment or onset of one or more symptoms associated with LAM, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., a prophylactic or therapeutic agent) with respect to the severity or onset of one or more symptoms ofLAM, or with respect to the pment or progression ofLAM. In specific embodiments, with respect to the treatment ofLAM, a therapeutically effective amount refers to the amount of a therapy (e.g., therapeutic agent) that inhibits or reduces the proliferation ofLAM cells, inhibits or reduces the spread of LAM cells (metastasis), or s the size of a tumor or improves PVC or FEVl or reduces the amount ofpleural on detectable by radiologic ation. In a preferred embodiment, a therapeutically effective amount of a therapy (e.g., a therapeutic agent) reduces the proliferation ofLAM cells or the size of a tumor by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% relative to a control (e.g., phosphate buffered saline (“PBS”)). Thus, in the t of the methods of the invention, the terms “treat”, “treatment”, and “treating” refer to the reduction of the severity, duration, or progression ofLAM or one or more symptoms associated with LAM. In specific embodiments, these terms may refer to the inhibition of eration or reduction in proliferation ofLAM cells, the tion or reduction in the spread (metastasis) ofLAM cells, or the development or ssion of a LAM-associated cancer, or the reduction in the size of a LAM-associated tumor, or the reduction or the involvement of axial lymphatics.

In one embodiment ofthe methods of the invention, the rapamycin is administered in a dose ive to improve the subject’s pulmonary function as measured by forced vital capacity _12_ (PVC) and forced tory volume (FEVl). In another embodiment, the rapamycin is administered in a dose effective to reduce the size or amount of pleural effiasion in the t that is detectable by radio logic examination. In one embodiment, the rapamycin is administered in a dose effective to improve one or more of the following: functional residual capacity, serum VEGF-D, quality of life and functional performance, 6 minute walk ce, and diffusing capacity of the lung for carbon de. In one embodiment, rapamycin delivered via a pulmonary route achieves blood levels ofrapamycin effective to limit the growth ofLAM- related tumors in the lungs and at sites distant from the lung. In one embodiment, the efficacy of the administered dose ofrapamycin is measured by any one or more of the foregoing.

In certain embodiments, the methods of the invention are effective to manage LAM in a subject having LAM. In this context, the terms “manage”, “managing”, and “management” refer to the beneficial effects that a subject derives from a y which does not result in a cure. In one embodiment, LAM is d in the subject if its progression is slowed or stopped during treatment with cin according to the methods of the invention. In another embodiment, LAM is managed in the subject if one or more symptoms ated with LAM is ameliorated or stabilized (i.e., the symptom does not worsen during the course of treatment).

In one embodiment, the methods ofthe invention are directed to subjects who are “non- responsive” or “refractory” to a currently available y for LAM. In this t, the terms “non-responsive” and “refractory” refer to the subject’s response to therapy as not clinically adequate to relieve one or more symptoms associated with LAM. The terms “subject” and “patient” are used interchangeably in this invention disclosure. The terms refer to an animal, ably a mammal including a non-primate (e.g., a cow, pig, horse, cat, dog, rat, and mouse) and a e (e.g., a chimpanzee, a monkey such as a cynomolgous monkey and a human), and more preferably a human. In a preferred ment, the subject is a human.

The terms “prevent”, “preventing” and “prevention” refer to the prevention of the recurrence, development, progression or onset of one or more symptoms ofLAM resulting from the administration of one or more compounds identified in accordance the methods of the invention or the administration of a combination of such a compound and a known therapy for a e or disorder.

In the context of the pharmaceutical itions of the invention, a “carrier” refers to, for example, a liquid or solid material such as a solvent, a diluent, stabilizer, adjuvant, excipient, auxiliary agent, propellant, or vehicle with which rapamycin is formulated for delivery. _13_ Examples of pharmaceutically acceptable carriers for use in the compositions of the invention include, without limitation, dry powder carriers such as lactose, mannose, amino acids, extrin, dipalmitylphosphatidylcholine, hydrocarbon and arbon propellants, compressed gases, sterile liquids, water, buffered , ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), oils, detergents, suspending agents, carbohydrates (e.g., glucose, lactose, sucrose or dextran), antioxidants (e. g., ascorbic acid or glutathione), chelating agents, low molecular weight proteins, or suitable mixtures thereof.

Preferably, in the context of the dry powder aerosol formulations ofrapamycin, the carrier, if present, is selected from the group consisting of a saccharide and a sugar alcohol. In one ment, the carrier, if t, is lactose.

The term “pharmaceutically acceptable” indicates approval by a regulatory agency of the Federal or a state government or listed in the US. Pharmacopeia or other generally recognized pharmacopeia such as the European Pharmacopeia, for use in animals, and more particularly in humans. One method for solubilizing poorly water soluble or water insoluble drugs is to form a salt of the drug or to prepare a prodrug that is more soluble itself or that can be used to form a water e salt of the prodrug. Methods for forming salts and pharmaceutically able salt forms are known in the art and include, t limitation, salts of acidic or basic groups that may be present in the drug or prodrug of interest. Compounds that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that can be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including but not limited to sulfuric, citric, maleic, acetic, oxalic, hydrochloride, romide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid e, tartrate, oleate, tannate, pantothenate, rate, ascorbate, succinate, maleate, gentisinate, fiamarate, gluconate, glucaronate, rate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, esulfonate, enesulfonate and pamoate (i.e., 1,1’-methylene-bis-(2-hydroxy oate)) salts. Compounds that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts and, ularly, calcium, magnesium, sodium m, zinc, potassium, and iron salts. _14_ In one embodiment, the methods and compositions of the invention utilize a water soluble prodrug or derivative ofrapamycin, preferably temsirolimus or related compound. In one embodiment, the methods and compositions of the invention utilize rapamycin (sirolimus).

Rapamycin Rapamycin is a yclic lactone produced by Streptomyces hygroscopicus Its chemical ) name is (3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)- 2,13, -14,21,22,23,24,25,26,27,32,33,34,34a-hexadecahydro-9,27-dihydroxy[(1R) [(18,3R,4R)hydroxymethoxycyclohexyl]methylethyl]-10,21-dimethox-y— 6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclohentriacontine- 1,5,11,28,29(4H,6H,31H)—pentone.

Its molecular formula is C51H79N013 and its molecular weight is 914.172 g/mol. Its structure is shown below. i 3 -\. '» ‘. V .\ WV» é...\. {:3 V.»- v 4 \ :l' ask-V, § 9 .-«~‘" \x- \v” N3" \~\ k z § KNNMN‘WQ £2} (.-"§r:\\\g§i{}§‘§ ' A" \ “ ‘z "i; ‘. '- .‘ 3w?" "2% 3:35“- fi\ ‘3. .‘ 3* - s; «R. ..-\~.\ fix -‘*§;\- 3-“) -\:\‘\,.,~“ [\m‘” x???» - 1\;:$“" Nags» «\f -.~c ..

Rapamycin is a white to off-white powder and is considered insoluble in water, having a very low solubility of only 2.6 ug/ml. It is freely soluble in benzyl alcohol, chloroform, e, and acetonitrile. The water insolubility ofrapamycin presents special technical problems to its formulation. In the context of its formulation as an oral dosage form, it has been prepared as an oral solution in the form of a solid dispersion (WO 97/03654) and a tablet containing nanosized (less than 400 nm) particles (US 591). But these procedures suffer from substantial variation in the dissolution of the active and therefore its bioavailability. Another method of formulation utilizes the crystalline powder. According to art-recognized methods, the transformation of the crystalline form of a low solubility drug to its ous form can significantly increase its solubility. While this is also true for rapamycin, the amorphous form is ely chemically unstable. Pharmaceutical dosage forms comprising amorphous rapamycin _15_ (sirolimus) are described in WO 06/03923? and WO 06/094507 (modified release formulation comprising sirolimus and glyceryl monostearate at a concentration of 49.25%). An improved stable oral dosage form ofrapamycin is described in US 8,053,444. The dosage form employs a fatty acid ester and a polymer (e.g., polyvinylpyrrolidone (PVP), hydroxypropylcellulose (HPC) or ypropylmethylcellulo se (HPMC)) in the composition to increase the stability of sirolimus without adversely affecting its release rate. According to US 8,053,444, a fatty acid ester concentration exceeding 10% w/w suppresses the e rate of sirolimus from the ation and so should be avoided because it can lead to insufficient absorption fiom the gastrointestinal tract. The preferred concentration of fatty acid ester rol ester) is 1% to 5% or 5% to 9%. In one ment, the aerosol rapamycin compositions of the present invention do not n a fatty acid ester in combination with a r. In one embodiment, the aerosol rapamycin compositions ofthe invention contain a fatty acid ester at a concentration exceeding % or exceeding 12% by weight ofthe composition.

Rapamycin and its tives (including analogs) and prodrugs suitable for use in the compositions and methods ofthe invention include rapamycin (sirolimus) and prodrugs or derivatives thereofwhich are inhibitors ofthe mTOR ar signaling pathway, and preferably inhibitors of mTOR itself. In one embodiment, a rapamycin derivative or prodrug is an mTOR inhibitor selected from the group consisting of everolimus tor, RAD001), temsirolimus (CCI-779), ridaforolimus ously known as deforolimus; AP23573), umirolimus (Biolimus A9), zotarolimus (ABT-578), novolimus, myolimus, AP23841, KU-0063794, INK-128, EX2044, EX3855, EX7518, AZD08055 and 081027. Further derivatives are known to the skilled person and include, for example, an O-substituted derivative in which the hydroxyl group on the cyclohexyl ring of sirolimus is replaced by —OR1, in which R1 is optionally a substituted alkyl, inoalkyl, or aminoalkyl.

In one embodiment, the compound for use in the aerosol formulations and s of the invention is a rapamycin derivative selected from the group consisting of everolimus, temsirolimus, ridaforolimus, umirolimus, and zotarolimus. The chemical structures of everolimus, olimus, ridaforolimus, umirolimus, and zotarolimus are shown below. fifiimcfimmwe“N In one embodiment, the compound for use in the aerosol formulations and methods of the ion is an mTOR inhibitor selected from the group ting of KU-0063794, AZD8055, INKl28, and 081-027. The chemical structures of the mTOR inhibitors KU-0063794, AZD8055, INKl28, and 081-027 are shown below.

Kfifi‘fixfififi -l8- Particularly preferred for use in the methods and compositions of the invention are sirolimus, temsirolimus, and everolimus. In one embodiment, the compound for use in the aerosol formulations and methods of the invention is selected from the group ting of sirolimus, temsirolimus, and everolimus. In one ment, the compound is sirolimus or everolimus. itions for tion The invention provides pharmaceutical compositions adapted for administration by inhalation comprising rapamycin, or a prodrug or derivative thereof, in the form of an aqueous solution, a dry powder, or a mixture of one or more pharmaceutically acceptable propellants and a carrier. In one embodiment, the rapamycin is encapsulated in a pharmaceutically acceptable compound, material, or matrix. In one embodiment, the rapamycin is encapsulated in a liposomal formulation or a non-liposomal formulation.

The compositions ofthe invention are aerosolizable formulations ofrapamycin le for pulmonary drug delivery in a human subject by inhalation. The term “aerosol” is used in this context to mean a dal system in which the dispersed phase is composed of either solid or liquid particles and in which the dispersal medium is a gas. In one embodiment, the gas is air and the formulation is a solution formulation suitable for administration via a nebulizer or a dry powder formulation suitable for stration via dry powder inhaler device. Generally, respirable particles or droplets will have a mean diameter in the range of 0.10 to 10 microns.

The size of the particles or droplets is selected to ze targeted ry either to the lungs themselves (z'.e., where the lung is the target tissue) or systemically (where the lungs are utilized _19_ as an alternative route for systemic administration). The size will preferably be in the range of about 0.5 to 5 microns where the lung itself is the therapeutic target, or less than 3 microns for systemic delivery via the lung. Size is measured according to methods known in the art and described, for example, in the US. Pharmacopeia at Chapters 905 and 601. For example, it is measured as Mass Median Aerodynamic Diameter (MMAD). In one embodiment, the average or mean er of the particles comprising the compositions described herein is measured as MMAD.

In one ment, the dispersed phase of the aerosol is composed of liquid particles or droplets. In this t, the terms d les” and ets” are used hangeably. In this embodiment, the formulation of the invention is a solution formulation. In one ment, the dispersed phase of the aerosol is composed of solid particles. In this embodiment, the formulation of the invention is a dry powder formulation. Micronized particles of this size can be produced by methods known in the art, for example by mechanical grinding (milling), precipitation from subcritical or supercritical solutions, spray-drying, freeze-drying, or lyophilization.

Generally, inhaled particles are subject to deposition by one oftwo mechanisms: impaction, which y predominates for larger particles, and sedimentation, which is ent for smaller particles. Impaction occurs when the momentum of an inhaled particle is large enough that the particle does not follow the air stream and encounters a physiological surface. In contrast, sedimentation occurs primarily in the deep lung when very small particles which have traveled with the inhaled air stream encounter physiological surfaces as a result ofrandom diffusion within the air stream. The aerosol formulations of the invention are preferably adapted to maximize their deposition either by impaction (in the upper airways) or by sedimentation (in the alveoli), in order to achieve the desired therapeutic efficacy.

The amount of drug delivered to the t from a delivery device, such as a nebulizer, pMDI or DPI device, is referred to as the delivered dose. It can be ted in vitro by determining the amount of drug emitted from the delivery device in a ted inhalation maneuver. This is termed emitted dose (ED) as measured according to methods known in the art, for examples those set out in the US. and European Pharmacopeias, e. g., at Chapter 601 and Chapter 905 of the USP. Accordingly, “emitted dose” is considered equivalent to the delivered dose. _20_ The amount of drug delivered from the delivery device to the lungs of the patient is termed the respirable dose. It can be estimated in vitro by determining the fine particle dose (FPD) as measured using cascade impactors, such as a Next Generation Impactor (NGI) according to methods known in the art, for examples those set out in the US. and European Pharmacopeias, e. g., at Chapters 601 and 905 of the USP.

The amount of drug released in fine, inhalable particles from a delivery device is referred to as the fine particle on (FPF) of the formulation. FPF is the fraction of drug in the red dose that is potentially respirable. Thus, FPF is the ratio ofFPD to ED (emitted, or delivered dose). These characteristics of the formulation are measured according to methods known in the art, for examples those set out in the US. and European Pharmacopeias, e.g., at Chapter 601 of the USP and monograph 2.9.18 of the Pharm Europa.

In one ment, the aerosolizable rapamycin formulations of the present ion have an FPF greater than 20% with a ponding FPD ranging from 10 micrograms to 2 milligrams, preferably less than 0.5 milligrams, even after prolonged storage, e.g., after 1 to 12 months or after 1 to 36 months of storage. In one embodiment the dose delivered to the patient, the delivered dose (DD) or d dose (ED), ranges from 25 micrograms to 2.5 milligrams, preferably less than 0.5 milligrams.

In certain embodiments the rapamycin is encapsulated in a pharmaceutically acceptable compound, material, or matrix. In one ment, the rapamycin is encapsulated in a liposomal formulation or non-liposomal formulation. s on itions In one embodiment, the aerosolizable composition of the invention is an aqueous solution ation ofrapamycin adapted for pulmonary delivery via a nebulizer, including jet, vibrating mesh, and static mesh or orifice zers. Thus, the solution formulation is adapted to enable aerosol droplet formation in the resp irable range of from about 0.1 to 10 micron diameter, as described above. In one ment, the composition is a nebulizable aqueous solution ation consisting of rapamycin (sirolimus) or a prodrug or derivative thereof, dissolved in water, ethanol, and a low molecular weight polyo], and optionally including a surface active agent. In one embodiment, the aqueous solution formulation has a Viscosity below 20 mPa-s, below 10 mPa-s, or below 5 mPa—s, and a surface tension of at least 45 dynes/cm, preferably greater than 60 dynes/cm. Preferably, the formulation has a viscosity below 5 mPa-s, and a _21_ surface n above 45 dynes/cm. In one embodiment, the composition has a viscosity below mPa-s, a viscosity below 10 mPa-s, or a viscosity below 5 mPa-s and a surface tension of at least 45 dynes/cm, preferably greater than 60 dynes/cm.

In one ment, the aqueous solution formulation consists of rapamycin, water, ethanol, and a low molecular weight polyol selected from glycerol and propylene glycol. In one embodiment, the aqueous on formulation consists of rapamycin, water, and a low molecular weight polyol selected from glycerol and propylene glycol, with the ethanol being optional. The formulation may also optionally contain a non- ionic surfactant, preferably PEG 100, or a polysorbate, preferably Polysorbate 80 (“P880”), a phospholipid, preferably a natural phospholipid such as lecithin, and ably hydrogenated soya lecithin, and an antioxidant or stabilizer, preferably disodium EDTA. In one embodiment, the non-ionic surfactant is ed from the group consisting ofpolyethylene glycol (PEG) PEG 100, PEG 1000, and Polysorbate 80 (also referred to as TweenTM 80, sorbitan monooleate, or polyoxyethylene sorbitan ), and mixtures thereof.

The amount mycin in the aqueous solution is from about 0.001% to 0.01 % weight t (% wt or % w/w) based on the total weight of the solution. In one embodiment, rapamycin is present in solution at a concentration of about 0.01 mg/ml to about 0.1 mg/ml. In one ment, the amount ofrapamycin is from 0.001 % to 0.01% w/w based upon total weight of the solution.

In one embodiment, the concentration ofrapamycin in solution is from about 0.01 to .1 mg/ml, the amount of the low molecular weight polyol is from 5 to 35 % w/w, the amount of ethanol is present in the amount of 5-20 % w/w, and the amount of the non-ionic surfactant is from 1 to 200 parts per million (ppm) w/w. Preferably, the amount of nic surfactant is less than 100 ppm (w/w). The amounts ofthe optional antioxidant/stabilizer from zero to less than 0.01% w/w.

In one embodiment, the aqueous solution formulation of the invention does n_ot n one or more additives or excipients selected from the group consisting of polyethylene glycol, in, EDTA, a block copolymer, and a cyclodextrin.

The aqueous solution formulation is a single phase aqueous solution in which the rapamycin is completely ved. The main co-solvents in the formulation are ethanol and a low molecular weight polyol selected from glycerol and propylene glycol. The rapamycin is not in suspension or emulsion, nor can the solution be described as a colloidal on or dispersion. _22_ The aqueous solution formulation of the ion lacks colloidal structures such as micelles or liposomes. The amount ofphospholipid, ifpresent, is too small to form liposomes or to itate the rapamycin. And the combined amount ofphospho lipid and non- ionic surfactant is too small to modify surface tension. Consequently, neither the phospholipid nor the non- ionic surfactant is present in amounts sufficient to act as a surfactant in the traditional sense. In this context, the term surfactant refers to an agent that acts to lower the e tension of the solution or the interfacial tension between the liquid and any solid drug particles in solution such that the surfactant acts as a detergent, wetting agent, emulsifier, or dispersing agent. Instead, the non- ionic surfactant in the solution formulation of the invention serves to block adsorption of the drug to the hylene container in which the final product is packaged, y preventing loss of drug potency via tion to the container.

Um Awmflmgyinmmemmmmmmflmammmmsdmbnfianmbnfiasmgemmm aqueous solution in which the rapamycin is completely dissolved, the solution lacks micelles or liposomes, and the solution is not an emulsion, dispersion, or suspension.

In one embodiment, the solution formulation is sterile. In one embodiment, the solution formulation is sterile ed through a 0.2 micron filter. In one embodiment, the solution formulation is not ized by heat, such as by autoclaving, or by radiation.

In one ment, the invention provides a package containing one or more containers or vials (these terms are used interchangeably) filled with the sterile aqueous solution formulation. Preferably, the containers are unit dose containers. In one embodiment, the containers are polymer vials, preferably polyethylene vials. In one embodiment, the container or vial filled with the sterile aqueous solution formulation of the ion is produced by a process comprising the steps of forming the vial by blow molding and immediately thereafier filling the vial with the sterile-filtered formulation of the invention under aseptic conditions, followed by goflmvmhmmwmmwmkfifismbd In one embodiment, the aqueous aerosol formulation of the invention comprises or consists of the ing rapamycin (or a prodrug or derivative thereof) from about 0.001% to 0.01% w/w, propylene glycol from about 5% to 35% w/w, ethanol from about 5% to 20% w/w, Polysorbate 80 from about 1 to 200 ppm w/w, lecithin from about 1 to 100 ppm w/w, and _23_ water, where the amount ofwater is sufficient to achieve a concentration of the cin between and 0.01 to 0.1 milligrams/milliliter. Optionally, a stability enhancer could be added such as disodium EDTA at levels below 0.01% wt/wt.

For s and other non-pressurized liquid systems, a variety of nebulizers (including small volume nebulizers) are available to aerosolize the formulations. Compressor-driven nebulizers incorporate jet technology and use compressed air to te the liquid aerosol. Such devices are commercially available from, for example, Healthdyne Technologies, Inc.; Invacare, Inc.; Mountain Medical Equipment, Inc.; Pari atory, Inc.; Mada Medical, Inc; Puritan- Bennet; Schuco, Inc., DeVilbiss Health Care, Inc.; and Hospitak, Inc. Ultrasonic nebulizers rely on mechanical energy in the form of vibration of a piezoelectric l to generate able liquid droplets and are commercially available from, for example, Omron Healthcare, Inc. and DeVilbiss Health Care, Inc.

In one embodiment, the aqueous aerosol formulation of the invention is delivered via a vibrating nebulizer available from Aerogen, Pari, Philips, or Omron. In one embodiment, the aqueous aerosol ation of the invention is packaged in a container suitable for use with a vibrating mesh nebulizer, for example, the Aeroneb® Go (Aerogen, distributed by Philips Respironics), I-Neb® ps) or E-Flow® (Pari), or similar nebulizer. In one embodiment the aqueous aerosol formulation of the invention is delivered via an orifice nebulizer such as the Respimat® from Boeringher—Ingelheim.

Thus, in one ment the invention es a pharmaceutical composition in the form of a zable aqueous solution suitable for administration by inhalation to a human subject, the aqueous solution consisting ofrapamycin or a g or derivative thereof, preferably selected from sirolimus, everolimus, and olimus, water, ethanol, and a low molecular weight polyol. In one embodiment, the low lar weight polyol is glycerol or propylene glycol, or a mixture thereof. In one embodiment, the composition further comprises a nonionic surfactant selected from the group consisting ofPEG 100, PEG 1000, and polysorbate 80, and mixtures thereof. In one ment, the amount of nonionic surfactant in the formulation is from 1 to 200 ppm w/w, preferably less than 100 ppm w/w, based upon the weight ofthe formulation. In one embodiment, the composition fiarther comprises a phospholipid, an antioxidant or chemical stabilizer. In one embodiment, the amount of antioxidant or chemical stabilizer in the formulation is less than 0.01 % w/w based upon the weight of the formulation. In _24_ one embodiment, the idant or chemical stabilizer is EDTA. In one embodiment, the amount ofrapamycin in the ation is from 0.001 to 0.01 % w/w based upon the weight of the formulation.

In one embodiment, the composition does not contain one or more additives or excipients selected from the group consisting of polyethylene glycol, lecithin, EDTA, a block copolymer, and a cyclodextrin.

In one embodiment, the ition lacks colloidal structures ed from es and liposomes.

In one embodiment, the composition is suitable for administration Via any one of a jet nebulizer, a ing mesh nebulizer, a static mesh nebulizer, and an orifice nebulizer.

In one embodiment, the composition has a viscosity below 20 mPa-s, preferably below mPa-s, most preferably below 5 mPa—s, and a surface tension of at least 45 dynes/cm, ably at least 50 dynes/cm.

The invention also provides a method ofmanufacturing a ceutical composition of the invention in the form of a nebulizable aqueous solution, the method comprising sterile filtering the solution through a filter with pore size of 0.2 microns or less and collecting the sterile filtrate in tion vessel under aseptic conditions. In one embodiment, the method of manufacturing filrther comprises transferring the sterile filtrate into a container e under aseptic conditions. In one embodiment, the container closure is a unit-dose polyethylene vial. In one embodiment, the vial is produced by blowmolding immediately before the e filtrate is transferred to the Vial. In one embodiment, the method further comprises the step ofthermally sealing the Vial immediately after the sterile filtrate is transferred to the Vial.

Dry Powder Compositions In one embodiment, the aerosolizable composition of the invention is a dry powder comprising micronized particles mycin, or a prodrug or derivative thereof, as the therapeutic agent (also referred to as “drug”), the les having diameters fiom 0.1 to 10 microns and a mean diameter of between about 0.5 to 4.5 microns, about 1 to 4 microns, about 1 to 3.5 microns, about 1.5 to 3.5 microns, or about 2 to 3 microns. The dry powder formulation is suitable for use in either a dry powder inhaler device (DPI) or a pressurized metered dose inhaler (pMDI). The amount ofrapamycin in the dry powder is from about 0.5 to 20% (w/w) based on _25_ total weight of the powder. In one embodiment, the amount ofrapamycin is about 1% or 2% (w/w).

In one embodiment, ized rapamycin is produced by wet polishing or jet milling as bed below to generate diameters in the range of about 0.5 to 4.5 microns, about 1 to 4 s, or about 2 to 3 microns, and the rapamycin les are d onto lactose carrier particles in a drug/carrier ratio ranging from 0.5-2% w/w with a preferred ratio of 1%.

In one embodiment, the drug particles are lightly compacted into a frangible matrix which is contained within the delivery device (a dry powder inhaler). Upon actuation the delivery device abrades a portion of the drug particles from the matrix, and disperses them in the inspiratory breath delivering the drug particles to the respiratory tract. Alternatively the drug particles may be a free flowing powder contained within a reservoir in the delivery device (a dry powder inhaler). The reservoir can be an integral chamber within the device, or a e, blister or similar preformed reservoir that is inserted into the device prior to actuation. Upon actuation the device dispersed a n of the drug particles from the reservoir and disperses them in the inhalation breath delivering the drug particles to the respiratory tract.

In one embodiment, the dry powder composition consists of drug particles and a r selected from the group ting of arabinose, glucose, se, ribose, mannose, sucrose, trehalose, lactose, maltose, starches, dextran, mannitol, leucine, lysine, isoleucine, dipalmitylphosphatidylcholine, lecithin, ctic acid, poly (lactic-co-glutamic) acid, and xylitol, or mixtures of any ofthe foregoing. In one embodiment, the carrier is lactose, particularly in the form of the monohydrate. In one embodiment, the dry powder composition comprises a blend oftwo or more carriers.

In one ment the dry powder composition comprises drug and a blend of at least two different carriers. In one embodiment, the drug to carrier ratio is in the range of from about 0.5 to 20% (w/w). In one embodiment, the drug particles have diameters g from 0.1 to 10 microns with a mean er of about 1 to 4, 1 to 3.5, or 1.5 to 3.5, or 2 to 3 microns. The carrier particles may have diameters ranging from 2 to 200 microns.

In one ment, the composition is contained in a blister pack or a reservoir of a DPI device. In one embodiment, the dry powder composition is preloaded into a gelatin, starch, cellulosic, or polymeric capsule, or a foil/foil or foil/plastic blister suitable for use in a DPI device. Each capsule or blister may contain from 1 to 100 milligrams of the dry powder composition. The capsules or blisters may be inserted into a dry powder inhaler (DPI) device such as Aerolizer®, ape® RS01 Model 7, and Plastiape® RS00 Model 8, XCaps®, FlowCaps®, Arcus®, Diskhaler® or Microdose®. Upon actuating the DPI device, the capsules or blisters are ruptured and the powder is dispersed in the inspiratory breath, delivering the drug to the atory tract.

In one embodiment, the dry powder composition is contained in a dry powder inhaler (DPI) device selected from Accuhaler®, ConixTM, Rotahaler®, TwinCaps®, XCaps®, FlowCaps®, Turbuhaler®, NextHaler®, CycloHaler®, Revolizer TM , Diskhaler®, Diskus®, Spinhaler, Handihaler®, Microdose Inhaler, GyroHaler®, OmniHaler®, Clickhaler®, Duohaler® (Vectura), and ARCUS® inhaler (Civitas Therapeutics). In one embodiment, the invention provides a DPI device containing a dry powder composition described herein. In one embodiment the device is selected from the group consisting of XCaps, FlowCaps, Handihaler, TwinCaps, Aerolizer®, Plastiape® RSOl Model 7, and Plastiape® RSOO Model 8. In one embodiment, the device containing the composition is selected from the group consisting of a ler®, an OmniHaler®, a aler®, a Duohaler®, and an ARCUS® inhaler.

The r particles are preferably of larger size (greater than 5 microns) so as to avoid deposition of the r material in the deep lung. In one embodiment, the carrier particles have diameters g from 1 to 200 microns, from 30 to 100 microns, or less than 10 microns. In one embodiment the carrier particles are a blend oftwo carriers, one with particles of about 30- 100 s and the other with particles less than 10 s. The ratio of the two different carriers is in the range of from 3:97 to 97:3. In one embodiment, the dry powder composition consists of 0.5 -20% (w/w) drug to carrier ratio, the drug particles having diameters from 0.1 to 10 microns with a mean diameter less than 3.5 microns. In one embodiment, the carrier material is a crystalline carrier material. Preferably, the crystalline carrier material is one which is at least 90%, ably greater than 95% crystalline and in which no or substantially no water is absorbed by the carrier under conditions of 80% or lower relative humidity at room ature.

Examples of such crystalline rs are lactose monohydrate and glucose monohydrate. The amount of carrier is from 1 to 99.0 % or more of the formulation by dry weight of the , preferably 5 to 99%, 10 to 99%, 20 to 99%, 30 to 99%, 40 to 99%, or 50 to 99%.

In one embodiment, the dry powder composition is contained within a reservoir in the delivery device (a dry powder inhaler). The reservoir can be an integral chamber within the device, or a capsule, blister or similar preformed oir that is inserted into the device prior to _27_ actuation. Upon actuation the device dispersed a n of the drug particles from the reservoir and disperses them in the inspiratory breath delivering the drug particles to the respiratory tract.

In one embodiment, drug is present as a fine powder with a pharmaceutically acceptable carrier. In the context, the term “fine” refers to a particle size in the inhalable range, as discussed above. Preferably, the drug is micronized such that the particles have a mean diameter in the range of 10 microns or less. In one embodiment, the mean diameter (MMAD or Dv50) ofthe particles mycin (or a prodrug or derivative thereof) in dry powder ition described herein is from 0.5 to 10 microns, from 0.5 to 6 microns, from 1 to 5 microns, from 1 to 4 microns, from 1 to 3 microns, or from 2 to 3 microns. The MMAD or Dv50 value is the particle size below which 50% of the volume of the tion occurs.

In one embodiment, the dry powder formulation of rapamycin further comprises one or more additives selected from the additives described below. In one ment, the one or more ves comprises or consists ofmagnesium stearate. In one aspect of this embodiment, the ium stearate is present in amounts of 0.001 to 10% by dry weight of the powder, preferably in amounts of from 0.01 to 5% or 0.01 to 2%. In another embodiment, the additive comprises or consists of a phospholipid, such as lecithin (which is a mixture of phosphatidylcho lines) in an amount of 0.1% to 1% by dry weight of the powder, preferably 0.2% to 0.6%. In one aspect of this ment, the additive is coated onto the carrier material prior to or simultaneously with a step of ng the carrier with the particles of rapamycin. This can be accomplished, for example, by utilizing a high energy mixing step to coat the carrier with the additive, or a long duration of low energy mixing, or a combination of low and high energy mixing to achieve the desired level of coated carrier material. Low energy devices for mixing dry powders to form blends are known in the art and include, for example, V-blenders, double cone blenders, slant cone rs, cube blenders, bin blenders, horizontal or vertical drum blenders, static continuous blenders, and dynamic continuous rs. Other, higher energy devices e high shear mixers known to those skilled in the art.

In certain embodiments, the dry powder is contained in a capsule. In one embodiment the capsule is a gelatin capsule, a c capsule, or a cellulosic capsule, or is in the form of a foil/foil or foil/plastic blisters. In each instance, the capsule or blister is suitable for use in a DPI device, preferably in dosage units together with the r in amounts to bring the total weight of powder in each capsule to from 1 mg to 100 mg. Alternatively, the dry powder may be contained in a reservoir of a multi-dose DPI device.

The particle size of the rapamycin can be reduced to the desired microparticulate level by conventional methods, for example by grinding in an air-jet mill, ball mill or vibrator mill, by wet polishing, microprecipitation, spray drying, lyophilization or recrystallization from subcritical or supercritical solutions. Jet milling or ng in this context refers to micronization of dry drug particles by mechanical means. Micronization techniques do not require making a solution, slurry, or suspension of the drug. Instead, the drug particles are mechanically d in size. Due to the relatively high energy that is employed by micronization, in certain embodiments it is desirable to include a carrier material in a co- micronized e with the rapamycin. In this context, the carrier material absorbs some of the energy of micronization which otherwise could adversely affect the ure of the rapamycin.

In one ment, rapamycin particles in a size range of from 1 to 4 or from 2 to 3 microns are produced by a jet milling method.

Wet polishing as bed in USZOl3/020371? involves using high shear to reduce the particle size of the drug particles in a suspension or slurry. Wet polishing can include just the drug particles or additional particulates termed milling media. In one ment, the particle size of the rapamycin can be reduced to the desired level using a wet polishing s, which comprises wet milling, specifically by cavitation at ed re, where rapamycin is suspended in water or other solvent where it is insoluble, and then is followed by spray drying of the suspension to obtain rapamycin as a dry powder. In one embodiment, rapamycin particles in a size range of from 1 to 4 or from 2 to 3 microns are produced by a wet polishing method that comprises ing a suspension ofrapamycin, subjecting the suspension to microfluidization, and spray-drying the resulting particles to form a dry powder. The rapamycin may be suspended in an anti-solvent selected from the group consisting of propyl or butyl alcohol, water, and ethyl e. In one embodiment, the suspension is an aqueous suspension.

Spray drying generally involves making a solution, , or suspension of the drug, atomizing the solution, slurry, or suspension, to form particles and then evaporating the solution, sMwmmmmMmmwmmbmeWWMUMWMMmflmymwwmmmwflm formed under subcritical or ritical conditions. The evaporation step can be lished by elevating the temperature of the atmosphere into which the atomization , or by decreasing the pressure, or a combination of both. In one embodiment, the powder formulation comprising rapamycin is made by spray drying an aqueous dispersion of rapamycin to form a dry powder consisting of aggregated particles ofrapamycin having a size suitable for pulmonary _29_ delivery, as described above. The aggregate particle size can be adjusted (increased or decreased) to target either the deep lung or upper respiratory sites, such as the upper bronchial region or nasal mucosa. This can be accomplished, for example, by increasing the concentration of rapamycin in the spray-dried dispersion or by increasing the droplet size generated by the spray dryer.

Alternatively, the dry powder can be made by -drying (lyophilization) the aqueous drug solution, dispersion, or emulsion, or by a combination of spray-drying and freeze-drying.

In one embodiment, the dry powder ation is made by freeze-drying an aqueous dispersion ofrapamycin, and one or more optional additives. In one embodiment, the powders contain aggregates of rapamycin and an additive, if present, wherein the ates are within a respirable size range as described above.

In one embodiment, the aqueous dispersion ofrapamycin and the one or more optional additives further comprises a dissolved diluent such as lactose or mannitol such that when the dispersion is freeze-dried, able diluent particles, each containing at least one embedded drug particle and additive particle, if present, are formed.

In one ment, the dry powder comprises cin loaded liposomes. Drug- loaded liposomes can be produced by methods known in the art, for example using the technique described for tacrolimus in M. Chougale, et al. Int. J. dicine 2:625-688 (2007). Briefly, rwmmflmhyhgmmwpmmmmMflwmmefiBHDJmmmmfimdmemmdwdma mixture of ol and chloroform and then subjected to dry thin film formation, e.g., in Rotaevaporator. The liposomes are hydrated and the liposomal dispersion is passed through a high-pressure nizer for size ion. The resultant pellets are characterized for vesicle size and percent drug entrapment and pellets equivalent to the desired amount ofrapamycin are then dispersed in a suitable medium and subjected to spray-drying to obtain particles of the d size for inhalation. The spray dried powder can be filled into capsules, canisters, or blister packs for stration.

In one embodiment the dry powder particles can be produced by precipitation from a ritical or subcritical solution.

The dry powder compositions may be contained in a suitable dry powder inhaler device, or in a capsule or blister for use in such a device. Examples of such devices are provided above and include Accuhaler®, Aerolizer®, the ape® RSOl Model 7, the Plastiape® RSOO Model 8, M, Rotahaler®, TwinCaps®, XCaps®, FlowCaps®, Turbuhaler®, NextHaler®, _30_ CycloHaler®, Revolizer TM Microdose , ler®, Diskus®, Spinhaler, aler®, Inhaler, GyroHaler®, OmniHaler®, Clickhaler®, or Duohaler® (Vectura), or a breath-actuated ARCUS® inhaler as Therapeutics). In one embodiment, the invention provides a DPI device containing a dry powder composition described herein. In one embodiment the device is selected from the group consisting of XCaps, FlowCaps, Handihaler, TwinCaps, Aerolizer®, the Plastiape® RSOl Model 7, and the Plastiape® RSOO Model 8.

Propellant-Based Formulations In another embodiment of the invention, the rapamycin is ated in a propellant- based formulation which may also be referred to cally herein as “a pMDI formulation”. A pMDI formulation is suitable for delivery by a device such as a pressurized d dose inhaler (pMDI). In one embodiment, the composition comprises rapamycin, a propellant, and a vegetable oil or pharmaceutically able derivative of a vegetable oil. The propellant is preferably ed from l,l,l,2—tetrafluoroethane (HFAl34a) and l,l,l,2,3,3,3- heptafluoropropane (HFA227), or mixtures thereof. In one embodiment, the vegetable oil is selected from olive oil, safflower oil, and n oil. The rapamycin may be in solution or in suspension in the propellant. In this t, “in suspension” refers to where the rapamycin is present in particulate form sed in the propellant. In one ment, the cin is micronized and is t in suspension in the propellent. In one embodiment, the formulation fithher comprises a wetting agent or co-solvent such as ethanol. In one embodiment, the formulation fiarther comprises a polyhydroxy alcohol such as propylene glycol.

Suitable propellants are known in the art and include, for example, halogen-substituted hydrocarbons, for example fluorine-substituted methanes, ethanes, propanes, butanes, cyclopropanes or cyclobutanes, particularly l,l,l,2-tetrafluoroethane (HFAl34a) and 2,3,3,3-heptafluoropropane (HFAZZ?), or es thereof.

In one embodiment, the formulation comprises micronized rapamycin, ethanol, a suitable propellant such as HFA 134a, HFA 227, or a mixture of suitable propellants, and optionally one or more surfactants. In one embodiment, the formulation fithher comprises a lubricant.

In one embodiment, the formulation comprises rapamycin, a propellant, and a vegetable oil. In one , the formulation does not comprise an additive or surfactant. For example, the formulation does not comprise ethanol, a polyhydroxy alcohol (e.g., propylene glycol), or a surfactant (e. g., sorbitan trioleate, sorbitan monooleate, or oleic acid). _31_ In one embodiment, the propellant-based formulation comprises compressed air, carbon dioxide, nitrogen or a liquefied propellant ed from the group ting of n-propane, n- butane, isobutane or mixtures thereof, or l,l,l,2-tetrafluoroethane (HFAl34a) and l,l,l,2,3,3,3- uoropropane (HFA227), or mixtures f, with or without a polar co-solvent such as an alcohol. The composition can be a solution or a suspension. For suspensions the drug particles have diameters from 0.1 to 10 microns with a mean diameter less than 3.5 microns.

The propellant-based formulation is prepared by methods known in the art, for example by wet milling the coarse rapamycin, and optional additive, in liquid propellant, either at ambient pressure or under high pressure conditions. In certain embodiments, the additive is a surfactant which serves to prevent aggregation (caking or crystallization), to facilitate uniform dosing, and (or alternatively) to provide a favorable fine particle fraction (FPF). In one , the surfactant is selected from sorbitan trioleate, an eate, or oleic acid. Alternatively, dry powders containing drug particles are prepared by spray-drying or freeze-drying aqueous dispersions of the drug particles as discussed above and the resultant powders dispersed into le propellants for use in conventional pressurized metered dose inhalers (pMDIs). In one embodiment, the inhalation device is a RespimatTM.

In one embodiment, the propellant-based aerosol rapamycin formulations of the invention are stable against particle size grth or change in the crystal morphology of the rapamycin over prolonged periods of time.

Process for Manufacturing Sterile Unit Dose Forms In one embodiment, the compositions of the invention are e compositions. In one embodiment, the sterile compositions are sterile unit dose forms. In one embodiment, the sterile unit dosage form is a capsule suitable for use in a nebulizer device.

In one embodiment, the finished composition is ized in its container-closure by heat, e. g., aving, or by ion. In one embodiment, the component parts of the composition are first sterilized by a suitable process ing sterile ion for liquid components and radiation or autoclaving for solids or liquids, the s r comprising maintaining the sterility of the sterile components by packaging in hermetic containers, combining the components in a mixing vessel in the appropriate proportions, and filling the resulting product into a container closure, all performed in an aseptic suite. This process has the disadvantage of being expensive and requiring difficult aseptic handling techniques. Accordingly, it is used _32_ primarily to process particulate suspensions or colloidal dispersions, liposomal formulations, or emulsions, which cannot be passed through a submicron filter for ization. Finally, in one embodiment, the finished composition is sterile filtered through a submicron filter, preferably a 0.2 micron filter. In one embodiment, the compositions of the invention are single-phase aqueous solutions sterilized via a filtration sterilization process. In contrast, emulsions and liposomal formulations are typically not sufficiently stable under the high shear conditions of a filtration sterilization process and so are not preferred for this process.

In one ment, the compositions of the invention are single-phase s solutions which are filled into a container-closure, e.g., a vial, formed of a polymer, preferably polyethylene, or alternatively a glass vial. Autoclaving and ion are not suitable where the vial is a polymer vial because of the high likelihood of creating chemical instability in the drug and/or formulation excipients, as well as in the ner, and due to the generation of undesirable impurities. In one embodiment, the compositions of the invention are sterilized by a process that does not include heat laving) or radiation, and instead includes a filtration sterilization process. Preferably, in accordance with this embodiment, the single-phase aqueous solutions ofrapamycin are sterilized by filtration through a filter having a pore size less than or equal to 0.2 microns. In one embodiment, the sterile filtrate is collected in a collection vessel located in an aseptic suite. In one embodiment, the sterile filtrate is transferred from the collection vessel into a container closure in an aseptic suite. Preferably the container closure is a r vial, preferably a unit dose vial, and most preferably a polyethylene unit dose vial. In one embodiment, the polymer vial is formed by blowmolding immediately before it is filled and then thermally sealed ately after filling. This que may be also referred to as “form- fill-seal” or a “blow-fill”. This technique is particularly advantageous in the context of the compositions of the invention which are single-phase aqueous solutions of cin because this process does not require heat or radiation, both ofwhich may e either the drug itself, the formulation excipients, or the container e. ary Administration and Dosing The present invention provides compositions and methods for the treatment and prophylaxis ofLAM by administering rapamycin to the respiratory tract, preferably to the lungs, by inhalation. ary delivery is preferably accomplished by inhalation of the aerosol h the mouth and throat into the lungs, but may also be accomplished by inhalation of the _33_ aerosol through the nose. Thus, in one embodiment the aerosol is delivered intranasally. In another embodiment, the aerosol is delivered perorally.

The compositions and methods ofthe invention advantageously provide for the targeted delivery of a therapeutically effective amount ofrapamycin to the lungs while simultaneously reducing to very low or undetectable levels the amount ofrapamycin in the blood and available ically. In one embodiment, the amount of cin in a single dose of a dry powder composition described herein is from about 5 to 500 micrograms or from about 100 to 300 micrograms, or from about 50 to 250 micrograms. The targeted delivery of low dose rapamycin directly to the lungs while minimizing ic exposure provides for an improved therapeutic index compared to oral dosage forms.

In one ment, administration of rapamycin by inhalation according to the methods ofthe invention ses the therapeutic index of rapamycin. In this t, as applied to human subjects, the therapeutic index is a ratio that compares the dose that es a therapeutic effect (ED50) to the dose that produces a toxicity (TD50) in 50% of the population.

The ratio is ented as TDso/EDSO. In one embodiment, stration ofrapamycin by inhalation ing to the methods of the invention reduces one or more toxicities associated with orally administered rapamycin, thereby increasing the therapeutic index ofrapamycin.

The invention includes aerosolizable formulations in the form of solutions and powders.

Accordingly, the rapamycin may be administered according to the methods of the ion in the form of an aqueous aerosol, a dry powder aerosol, or a propellant-based aerosol.

In one embodiment, the administered dose ofrapamycin is sufficient to achieve a blood trough level in the subject of from of from 0.01 to 0.15 ng/ml, from 0.075 to 0.350 ng/ml, from 0.150 to 0.750 ng/ml, from 0.750 to 1.5 ng/ml or from 1.5 to 5 ng/ml. In one embodiment, the administered dose ofrapamycin is sufficient to achieve a blood trough level in the subject of less than 5 ng/ml, less than 2 ng/ml, less than 1 ng/ml, or less than 0.5 ng/ml.

In one embodiment, the administered dose ofrapamycin is sufficient to produce a concentration ofrapamycin in lung tissue in the range of from 1 ng/g to 1 ug/g.

In one embodiment, the stered dose ofrapamycin is from 10 to 100 micrograms, from 50 to 250 rams, from 100 to 500 micrograms (0.1 to 0.5 milligrams), fiom 500 to 1000 micrograms (0.5 to 1 milligrams) or from 1000 to 2000 micrograms (1 to 2 milligrams). In one embodiment, the amount ofrapamycin administered is less than 1 milligrams, less than 0.75 _34_ milligram, less than 0.5 milligrams or less than 0.25 milligrams. Preferably, the amount of rapamycin administered is less than 0.5 milligrams.

In one embodiment, the rapamycin is administered once daily.

In one embodiment, the total daily dose mycin is in the range of from 10 to 100 micrograms, from 50 to 250 micrograms, from 100 to 500 micrograms (0.1 to 0.5 milligrams), from 500 to 1000 micrograms (0.5 to l milligrams) or from 1000 to 2000 micrograms (1 to 2 milligrams). In one embodiment, the total daily dose ofrapamycin is less than 1 milligram, less than 100 micrograms, less than 50 micrograms, less than 10 micrograms, or less than 1 microgram. In one embodiment, the total daily dose ofrapamycin is less than 500 nanograms, less than 250 nanograms, less than 100 nanograms, less than 50 ams, or less than 10 nanograms. In one embodiment, the total daily dose ofrapamycin administered to the t is less than 2 mg or less than 1 mg per day.

In one embodiment, a composition of the invention is administered once per day to the subject. In one ment, a composition of the invention is administered twice or three times a day. Preferably, the composition is administered once or twice daily, or less than once daily.

In one embodiment, the methods ofthe invention comprise administering rapamycin via a pulmonary route in combination with one or more onal therapeutic agents selected from the group consisting of a statin, progesterone, tamoxifen, gonadotropin-releasing hormone (GnRH) agonists, cline, a src inhibitor, an autophagy inhibitor (e. g. hydroxychloroquine), a VEGF-C or -D inhibitor, and a VEGF receptor inhibitor. In one embodiment, the one or more onal therapeutic agents is selected from a statin, terone, tamoxifen, and gonadotropin—releasing hormone (GnRH) agonists. In one embodiment, the one or more additional therapeutic agents is selected from an estrogen nist, a statin, a src inhibitor, and a VEGF-R tor. In one embodiment, the one or more onal therapeutic agents is selected from the group consisting of letrozole, tamoxifen, simvastatin, saracatinib, pazopanib, imatinib, and combinations thereof. The one or more additional agents may be stered by the same or a ent route of administration as the rapamycin. For example, the agent may be administered by inhalation, intranasally, orally or intravenously.

In one embodiment, the methods of the invention se administering rapamycin via a pulmonary route in combination with one or more additional therapies. In one embodiment, the one or more additional therapies is selected from anti—estrogen therapy, hormonal therapy, anti-cancer chemotherapy, and radiation therapy. In one embodiment, the methods of the _35_ ion comprise administering rapamycin via a pulmonary route in ation with anti- estrogen therapy or hormone therapy.

In certain embodiments, the methods include pulmonary administration of a composition ofthe invention as the primary therapy. In other embodiments, the administration of a composition of the invention is an adjuvant therapy. In either case, the methods of the invention contemplate the administration of a composition of the invention in combination with one or more additional therapies for the treatment of a disease or disorder. The terms “therapy” and “therapies” refer to any , protocol and/or agent that can be used in the tion, treatment, management or amelioration of a disease or disorder, or one or more ms thereof. In certain embodiments, a therapy is ed from chemotherapy, radiation therapy, hormonal therapy, and strogen therapy.

Preferably, the administration of a pharmaceutical composition comprising rapamycin or a prodrug or derivative thereof according to the methods of the invention in combination with one or more additional therapies provides a synergistic response in the subject having LAM. In this context, the term “synergistic” refers to the efficacy of the combination being more effective than the additive effects of either single therapy alone. In one embodiment, the synergistic effect of combination cin therapy according to the invention permits the use of lower s and/or less frequent administration of at least one therapy in the combination compared to its dose and/or frequency outside of the combination. In another embodiment, the synergistic effect is manifested in the avoidance or reduction of adverse or unwanted side effects associated with the use of either therapy in the combination alone.

Nebulizer Delivery In one ment, the rapamycin is ated as an s solution suitable for nebulization and delivered via a zer. For aqueous and other non-pressurized liquid systems, a variety of nebulizers (including small volume nebulizers) are available to aerosolize the formulations. Compressor-driven nebulizers incorporate jet logy and use compressed air to generate the liquid aerosol. Such devices are commercially available fiom, for example, Healthdyne Technologies, Inc.; Invacare, Inc.; Mountain Medical ent, Inc; Pari Respiratory, Inc.; Mada Medical, Inc.; Puritan-Bennet; Schuco, Inc., DeVilbiss Health Care, Inc.; and Hospitak, Inc. Ultrasonic zers rely on mechanical energy in the form of vibration of a piezoelectric crystal to generate respirable liquid droplets and are cially available from, for example, Omron Healthcare, Inc. and DeVilbiss Health Care, Inc. The nebulizer may be, for example, a conventional pneumatic nebulizer such as an airjet nebulizer, or an ultrasonic nebulizer, which may n, for example, from 1 to 50 ml, commonly l to 10 ml, of the on formulation.

In one embodiment, the aqueous solution formulation of the invention is adapted for administration with a nebulizer comprising a vibrating or fixed mesh. For example, devices such as an AERx® (Aradigm), RESPIMAT® (Boehringer eim), I-Neb® (Philips), or MicroAire® (Omron) in which drug solution is pushed with a piston or pneumatic pressure, or with a piezoelectric crystal through an orifice or mesh. Alternatively, the solution can be pumped through a vibrating mesh nebulizer such as the E-Flow® (Pari) or Aeroneb® Go (Aerogen). These devices allow much smaller nebulized volumes, e.g., 10 to 100 111, and higher delivery efficiencies than tional nebulizers.

Dry Powder Delivery In one embodiment, the dry powder itions of the invention are delivered by a non- propellant based dry powder inhaler (DPI) device. In one embodiment, the powder is contained in capsules of gelatin or plastic, or in rs, suitable for use in a DPI device. In one embodiment, the powder is supplied in unit dosage form and in dosage units of from 5 mg to 100 mg ofpowder per capsule. In another embodiment, the dry powder is contained in a reservoir of a multi-dose dry powder inhalation device. In one embodiment, the inhaler device comprises an aerosol vial provided with a valve d to deliver a metered dose, such as 10 to 100 ul, 6. g. 25 to 50 ul, of the composition, i.e. a device known as a metered dose inhaler.

In one embodiment, the DPI device is a blister based device such as the GyroHaler® or the OmniHaler® (both from a), a reservoir based device such as the Clickhaler® or Duohaler® (Vectura), and the ARCUS® inhaler (Civitas Therapeutics). In one embodiment, the DPI device is ed from PulmatrixTM, and Hovione ps and XCapsTM. In one embodiment the device is selected from the group ting of XCaps, ape® RSOI Model 7, and ape® RSOO Model 8.

In one embodiment, the DPI device is selected from the group consisting of Accuhaler®, Aerolizer®, the Plastiape® RSOl Model 7, the Plastiape® RSOO Model 8, ConixTM, Rotahaler®, TwinCaps®, XCaps®, FlowCaps®, Turbuhaler®, NextHaler®, CycloHaler®, Revolizer TM Microdose Inhaler, GyroHaler®, , ler®, Diskus®, Spinhaler, Handihaler®, _37_ OmniHaler®, Clickhaler®, or Duohaler® (Vectura), or a breath-actuated ARCUS® inhaler (Civitas Therapeutics).

In one embodiment, the DPI device is selected from the group consisting of ArcusTM, AspirairTM, AxahalerTM, BreezhalerTM, alerTM, Conix DryTM, CricketTM, DreamboatTM, GenuairTM, GeminiTM, InspiromaticTM, iSPERSETM, MicroDoseTM, Next DPITM, ProhalerTM, PulmojetTM, PulvinalTM, SolisTM, M, Taper DryTM, TrivaiTM, NovolizerTM, PodhalerTM, SkyehalerTM, SpiromaxTM, Twincaps/FlowcapsTM, and TurbuhalerTM. In one ment, the DPI device is adapted to deliver the dry powder from a capsule or blister containing a dosage unit of the dry powder or a multi-dose dry powder inhalation device adapted to deliver, for example, 5-25 mg of dry powder per actuation. pMDI Delivery In another embodiment, the rapamycin is red in the form of aerosolized particles from a pressurized container or dispenser that contains a le propellant as described above in connection with propellant-based formulations. In one embodiment, the r is a propellant driven inhaler, such as a pMDI device, which releases a metered dose of rapamycin upon each actuation. A typical pMDI device comprises a canister containing drug, a drug metering valve, and a mouthpiece. In one aspect of this embodiment, the rapamycin is formulated as a suspension in the propellant. In the context ofthis embodiment, the rapamycin is made into a fine powder which is ded in the liquefied propellant or propellant blend. The suspension is then stored in a sealed canister under ent pressure to maintain the lant in liquid form. In another embodiment, the rapamycin is formulated as a solution. In the context of this ment, the rapamycin is solubilized in the liquefied lant or propellant blend. In one embodiment, the formulation further comprises a stabilizer in an amount suitable to stabilize the formulation against settling, creaming or flocculation for a time sufficient to allow reproducible dosing of the rapamycin after agitation of the formulation. The stabilizer may be present in excess in an amount of about 10 part by weight to about 5000 parts by weight based on one million parts by total weight of the aerosol formulation. In one embodiment, the fluid carrier is 1,1,1,2-tetrafluoroethane, 2,3,3,3-heptafluoropropane or a mixture f. In another embodiment, the fluid carrier is a arbon (e.g., n—butane, propane, isopentane, or a mixture thereof). The composition may further comprise a co-solvent (e.g., ethanol or other suitable cosolvent In one embodiment ofthe methods ofthe ion, the aerosol formulation comprising rapamycin fithher comprises an additional drug. In one aspect of this embodiment, the additional drug is ed from the group consisting of corticosteroids, estrogen receptor antagonists, anticholinergics, beta-agonists, non-steroidal anti-inflammatory drugs, macrolide antibiotics, bronchodilators, leukotriene receptor inhibitors, muscarinic antagonists, cromolyn sulfate, and combinations thereof.

Additives The aerosol compositions of the invention may contain one or more additives in addition to any r or diluent (such as lactose or mannitol) that is present in the formulation. In one embodiment, the one or more ves comprises or consists of one or more surfactants.

Surfactants lly have one or more long tic chains such as fatty acids which enables them to insert directly into the lipid structures of cells to enhance drug penetration and absorption. An empirical parameter commonly used to characterize the relative hydrophilicity and hydrophobicity of surfactants is the hydrophilic-lipophilic balance (“HLB” value).

Surfactants with lower HLB values are more hydrophobic, and have greater solubility in oils, while surfactants with higher HLB values are more hydrophilic, and have r lity in aqueous solutions. Thus, hilic surfactants are generally considered to be those compounds haVing an HLB value greater than about 10, and hydrophobic surfactants are generally those having an HLB value less than about 10. However, these HLB values are merely a guide since for many surfactants, the HLB values can differ by as much as about 8 HLB units, depending upon the empirical method chosen to determine the HLB value.

Among the surfactants for use in the aerosol compositions of the invention are polyethylene glycol (PEG)-fatty acids and PEG-fatty acid mono and diesters, PEG glycerol esters, alcohol-oil transesterification ts, polyglyceryl fatty acids, propylene glycol fatty acid esters, sterol and sterol derivatives, polyethylene glycol sorbitan fatty acid , polyethylene glycol alkyl ethers, sugar and its derivatives, polyethylene glycol alkyl phenols, polyoxyethylene-polyoxypropylene (POE-POP) block copolymers, an fatty acid esters, ionic surfactants, fat-soluble vitamins and their salts, water-soluble vitamins and their amphiphilic derivatives, amino acids and their salts, and organic acids and their esters and anhydrides. Each ofthese is described in more detail below. _39_ PEG Fatty Acid Esters Although polyethylene glycol (PEG) itself does not function as a surfactant, a variety of tty acid esters have useful surfactant properties. Among the PEG-fatty acid monoesters, esters of lauric acid, oleic acid, and stearic acid are most useful in embodiments of the present ion. Preferred hilic surfactants include PEG-8 e, PEG-8 oleate, PEG-8 stearate, PEG-9 oleate, PEG-10 laurate, PEG-10 oleate, PEG-12 laurate, PEG-12 oleate, PEG-15 oleate, PEG-20 e and PEG-20 oleate. The HLB values are in the range of 4-20.

Polyethylene glycol fatty acid diesters are also suitable for use as surfactants in the compositions of embodiments of the present invention. Most preferred hydrophilic surfactants include PEG-20 dilaurate, PEG-20 dioleate, PEG-20 distearate, PEG-32 dilaurate and PEG-32 dioleate. The HLB values are in the range of 5-15.

In general, mixtures of surfactants are also useful in embodiments of the t invention, including mixtures oftwo or more commercial surfactants as well as mixtures of surfactants with another additive or additives. Several PEG—fatty acid esters are marketed commercially as mixtures or mono- and diesters.

Polyethylene Glycol Glycerol Fatty Acid Esters Preferred hydrophilic surfactants are PEG-20 glyceryl laurate, PEG-30 yl laurate, PEG-40 glyceryl laurate, PEG-20 glyceryl oleate, and PEG-30 glyceryl oleate.

Alcohol-Oil Transesterification Products A large number of surfactants of different degrees of hydrophobicity or hydrophilicity can be prepared by reaction of ls or polyalcohol with a variety of natural and/or hydrogenated oils. Most commonly, the oils used are castor oil or enated castor oil, or an edible vegetable oil such as corn oil, olive oil, peanut oil, pahn kernel oil, apricot kernel oil, or almond oil. red ls include glycerol, propylene , ethylene glycol, polyethylene glycol, sorbitol, and pentaerythritol. Among these alcohol-oil transesterifled surfactants, preferred hydrophilic surfactants are PEG-35 castor oil (Incrocas-35), PEG-40 hydrogenated castor oil (Cremophor RH 40), PEG-25 trioleate (TAGAT.RTM. TO), PEG-60 corn glycerides (Crovol M70), PEG—60 almond oil (Crovol A70), PEG-40 palm kernel oil (Crovol PK70), PEG- 50 castor oil (Emalex C—50), PEG-50 hydrogenated castor oil (Emalex HC—SO), PEG-8 caprylic/capric glycerides (Labrasol), and PEG-6 ic/capric glycerides (Softigen 767).

Preferred hobic surfactants in this class include PEG-5 hydrogenated castor oil, PEG-7 _40_ hydrogenated castor oil, PEG-9 hydrogenated castor oil, PEG-6 corn oil (Labrafil.RTM. M 2125 CS), PEG-6 almond oil (Labrafil.RTM. M 1966 CS), PEG-6 apricot kernel oil (LabrafilRTM. M 1944 CS), PEG-6 olive oil (Labrafil.RTM. M 1980 CS), PEG-6 peanut oil fil.RTM. M 1969 CS), PEG-6 hydrogenated palm kernel oil (Labrafil.RTM. M 2130 BS), PEG-6 pahn kernel oil (Labrafil.RTM. M 2130 CS), PEG-6 triolein (Labrafil.RTM.b M 2735 CS), PEG-8 corn oil (Labrafil.RTM. WL 2609 BS), PEG-20 corn glycerides (Crovol M40), and PEG-20 almond glycerides l A40).

Polyglyceryl Fatty Acids Polyglycerol esters of fatty acids are also suitable surfactants for use in embodiments of the present invention. Among the polyglyceryl fatty acid esters, preferred hydrophobic surfactants include polyglyceryl oleate (Plurol e), polyglyceryl-2 dioleate (Nikkol DGDO), polyglyceryl-10 trio leate, polyglyceryl te, polyglyceryl laurate, yceryl ate, polyglyceryl palmitate, and polyglyceryl linoleate. Preferred hydrophilic surfactants include yceryl-10 laurate (Nikkol Decaglyn l—L), polyglyceryl-10 oleate (Nikkol Decaglyn 1-0), and polyglyceryl-10 mono, dioleate (Caprol.RTM. PEG 860), polyglyceryl-10 stearate, polyglyceryl-10 laurate, polyglyceryl- 10 myristate, polyglyceryl— 10 palmitate, polyglyceryl-10 linoleate, yceryl-6 stearate, polyglyceryl-6 laurate, polyglyceryl-6 myristate, polyglyceryl- 6 palmitate, and polyglyceryl-6 linoleate. Polyglyceryl polyricinoleates (Polymuls) are also preferred surfactants.

Propylene Glycol Fatty Acid Esters Esters ofpropylene glycol and fatty acids are suitable surfactants for use in embodiments ofthe present invention. In this surfactant class, preferred hydrophobic tants include propylene glycol monolaurate (Lauroglycol FCC), propylene glycol leate (Propymuls), ene glycol monooleate (Myverol P-06), propylene glycol dicaprylate/dicaprate (Captex.RTM. 200), and propylene glycol noate x.RTM. 800).

Sterol and Sterol Derivatives Sterols and derivatives of sterols are suitable surfactants for use in embodiments of the present invention. Preferred derivatives include the polyethylene glycol derivatives. A preferred surfactant in this class is PEG-24 cholesterol ether (Solulan C-24). _41_ Polyethylene Glycol Sorbitan Fatty Acid Esters A variety of PEG-sorbitan fatty acid esters are available and are suitable for use as surfactants in embodiments of the present invention. Among the PEG-sorbitan fatty acid , preferred surfactants include PEG-20 an monolaurate (Tween-20), PEG-20 sorbitan monopalmitate (Tween-40), PEG-20 sorbitan monostearate (Tween-60), and PEG-20 sorbitan monooleate (Tween-80). hylene Glycol Alkyl Ethers Ethers ofpolyethylene glycol and alkyl alcohols are le surfactants for use in embodiments of the present invention. Preferred ethers include PEG-3 oleyl ether (Volpo 3) and PEG-4 lauryl ether (Brij 30).

Sugar and its Derivatives Sugar derivatives are suitable surfactants for use in embodiments of the present invention. Preferred tants in this class include sucrose monopalmitate, sucrose monolaurate, decanoyl-N-methylglucamide, n—decyl—B—D—glucopyranoside, n-decyl—B-D- maltopyranoside, n-dodecyl-B-D-glucopyranoside, n—dodecyl—B-D-maltoside, heptanoyl-N— methylglucamide, n-heptyl- B-D-glucopyranoside, n-heptyl-B-D-thioglucoside, n-hexyl-B-D- yranoside, nonanoyl-N-methylglucamide, n-nonyl-B-D-glucopyranoside, octanoyl-N- methylglucamide, n-octyl-B-D-glucopyranoside, and octyl-B-D-thioglucopyranoside.

Polyethylene Glycol Alkyl s Several PEG-alkyl phenol surfactants are available, such as -lOO nonyl phenol and PEG-lS-lOO octyl phenol ether, Tyloxapol, octoxynol, nonoxynol, and are suitable for use in embodiments of the present invention.

Polyoxyethylene-Polyoxypropylene (POE-POP) Block Copolymers The POE-POP block copolymers are a unique class of polymeric surfactants. The unique ure of the surfactants, with hydrophilic POE and hydrophobic POP es in well-defined ratios and positions, provides a wide variety of surfactants suitable for use in ments of the present invention. These surfactants are available under various trade names, ing Synperonic PE series (ICI); Pluronic.RTM. series (BASF), Emkalyx, Lutrol (BASF), Supronic, Monolan, Pluracare, and Plurodac. The generic term for these polymers is “poloxamer” (CAS _42_ 90036). These polymers have the formula: HO(C2H40)a(C3H60)b(C2H40)aH where “a” and “b” denote the number ofpolyoxyethylene and polyoxypropylene units, respectively.

Preferred hydrophilic surfactants of this class include Poloxamers 108, 188, 217, 238, 288, 338, and 407. Preferred hydrophobic surfactants in this class include Poloxamers 124, 182, 183, 212, 331, and 335.

Sorbitan Fatty Acid Esters Sorbitan esters of fatty acids are suitable surfactants for use in embodiments of the present invention. Among these esters, preferred hydrophobic surfactants e sorbitan monolaurate (Arlacel 20), sorbitan monopalmitate (Span-40), sorbitan monooleate (Span-80), sorbitan monostearate.

The sorbitan monopalmitate, an amphiphilic derivative of Vitamin C (which has Vitamin C ty), can serve two important ons in solubilization systems. First, it possesses effective polar groups that can modulate the microenvironment. These polar groups are the same groups that make vitamin C itself bic acid) one of the most water-soluble organic solid compounds available: ascorbic acid is soluble to about 30 wt/wt % in water (very close to the solubility of sodium chloride, for example). And second, when the pH increases so as to convert a fraction of the ascorbyl palmitate to a more e salt, such as sodium ascorbyl palmitate.

Ionic Surfactants Ionic surfactants, including cationic, c and zwitterionic tants, are suitable hydrophilic tants for use in embodiments of the present invention. Preferred ionic surfactants include quaternary ammonium salts, fatty acid salts and bile salts. Specifically, preferred ionic tants include benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, docecyl trimethyl ammonium bromide, sodium docecylsulfates, dialkyl methylbenzyl ammonium chloride, onium de, domiphen bromide, dialkylesters of sodium sulfonsuccinic acid, sodium dioctyl sulfosuccinate, sodium cholate, and sodium taurocholate. These quaternary ammonium salts are preferred ves. They can be dissolved in both organic solvents (such as ethanol, e, and e) and water. This is especially useful for medical device coatings because it simplifies the preparation and coating process and has good adhesive properties. Water insoluble drugs are commonly dissolved in organic solvents. _43_ Fat-Soluble ns and Salts Thereof Vitamins A, D, E and K in many oftheir various forms and provitamin forms are considered as fat-soluble Vitamins and in addition to these a number of other vitamins and n sources or close relatives are also fat-soluble and have polar groups, and relatively high octanol-water partition coefficients. Clearly, the general class of such nds has a history of safe use and high benefit to risk ratio, making them usefiJl as additives in embodiments of the present invention.

The following examples of fat-soluble n derivatives and/or sources are also usefiJl as additives: Alpha-tocopherol, beta-tocopherol, gamma-tocopherol, tocopherol, tocopherol acetate, erol, 1-alpha-hydroxycholecal-ciferol, Vitamin D2, vitamin D3, alpha-carotene, beta-carotene, gamma-carotene, vitamin A, fursultiamine, methylolriboflavin, octotiamine, prosultiamine, vine, vintiamol, dihydrovitamin K1, menadiol diacetate, menadiol dibutyrate, menadiol disulfate, menadiol, Vitamin K1, vitamin K1 oxide, vitamins K2, and vitamin K—S(II). Folic acid is also of this type, and although it is water-soluble at physiological pH, it can be formulated in the free acid form. Other derivatives of fat-soluble vitamins usefill in embodiments of the t invention may easily be ed via well-known chemical reactions with hilic les.

Water-Soluble Vitamins and their Amphiphilic Derivatives Vitamins B, C, U, pantothenic acid, folic acid, and some of the menadione-related vitamins/provitamins in many of their various forms are considered water-soluble vitamins.

These may also be conjugated or complexed with hydrophobic moieties or multivalent ions into amphiphilic forms having relatively high octanol-water partition coefficients and polar groups.

Again, such compounds can be of low toxicity and high benefit to risk ratio, making them useful as additives in embodiments of the present invention. Salts of these can also be usefiJl as ves in the present invention. Examples of water-soluble vitamins and derivatives include, without limitation, acetiamine, benfotiamine, pantothenic acid, amine, cyclothiamine, dexpanthenol, niacinamide, nicotinic acid, pyridoxal S-phosphate, nicotinamide ascorbate, riboflavin, riboflavin phosphate, ne, folic acid, menadiol diphosphate, menadione sodium ite, menadoxime, vitamin B12, vitamin K5, vitamin K6, vitamin K6, and Vitamin U. Also, as mentioned above, folic acid is, over a wide pH range including physiological pH, water- soluble, as a salt. _44_ Compounds in which an amino or other basic group is present can easily be modified by simple acid-base reaction with a hydrophobic containing acid such as a fatty acid (especially , oleic, myristic, tic, stearic, or 2-ethylhexanoic acid), low-solubility amino acid, benzoic acid, salicylic acid, or an acidic fat-soluble vitamin (such as vin).

Other compounds might be obtained by reacting such an acid with another group on the vitamin such as a hydroxyl group to form a linkage such as an ester linkage, etc. Derivatives of a water- soluble vitamin containing an acidic group can be generated in ons with a hobic group-containing reactant such as stearylamine or riboflavine, for example, to create a compound that is useful in embodiments of the present invention. The linkage of a palmitate chain to vitamin C yields ascorbyl palmitate.

Amino Acids and Their Salts Alanine, arginine, asparagines, aspartic acid, cysteine, e, glutamic acid, glutamine, glycine, ine, proline, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, valine, and their derivatives are other useful additives in embodiments of the invention.

Certain amino acids, in their zwitterionic form and/or in a salt form with a monovalent or multivalent ion, have polar groups, relatively high octanol—water partition coefficients, and are usefiJl in embodiments of the present invention. In the context of the present sure we take “low-solubility amino acid” to mean an amino acid which has solubility in unbuffered water of less than about 4% (40 mg/ml). These include cystine, tyrosine, tryptophan, leucine, isoleucine, phenylalanine, asparagine, ic acid, glutamic acid, and methionine. c Acids and Their Esters and ides Examples are acetic acid and anhydride, benzoic acid and ide, acetylsalicylic acid, diflunisal, 2-hydroxyethyl salicylate, diethylenetriaminepentaacetic acid dianhydride, ethylenediaminetetraacetic dianhydride, maleic acid and anhydride, succinic acid and anhydride, diglycolic anhydride, glutaric anhydride, ascorbic acid, citric acid, tartaric acid, lactic acid, oxalic acid aspartic acid, nicotinic acid, 2-pyrrolidonecarboxylic acid, and 2-pyrrolidone.

These esters and ides are soluble in organic solvents such as ethanol, acetone, methyl ethyl ketone, ethyl acetate. The water insoluble drugs can be dissolved in organic solvent with these esters and anhydrides, then coated easily on to the medical device, then hydrolyzed _45_ under high pH conditions. The hydrolyzed anhydrides or esters are acids or alcohols, which are water soluble and can effectively carry the drugs off the device into the vessel walls.

EXAMPLES The ion is further described in the following examples, which do not limit the scope of the invention bed in the claims.

Example 1: Aqueous aerosol formulation An exemplary aqueous formulation ofrapamycin was prepared using the following components.

Comonent Mass Fraction w/w raoam cin 0.01 % ethanol 250 25 % prop lene 1 col 25 % pol sorbate 80 0.002 % water 50 % Total ng Procedure: in a 1000 ml amber volumetric flask, blend 250 propylene glycol with 250 ethanol until uniform. Then sequentially dissolve first 100 mg rapamycin then mg polysorbate 80 in the propylene glycol and ethanol solution. Add water to bring the volumetric to 1000 ml and stir or sonicate until uniform and all the rapamycin is dissolved. Store at controlled temperature away from light.

Example 2: Dry Powder Formulation Batch 06RP68.HQ00008 and 06RP68.HQ00009. These two ations are each a blend of micronized drug (rapamycin) particles dispersed onto the surface of lactose carrier particles. The final composition of each batch comprises 1% (w/w) drug particles having a mean er of about 2.60 microns and 3.00 microns, respectively. Drug particles having a suitable size range are made by wet polishing (06RP68.HQ00008) or jet milling (06RP68.HQ00009), as described below. While this example used 1% (w/w) rapamycin, a range 0.5 to 20% is practicable. The carrier particles consist of a blend oftwo carriers, Respitose® SV003, present at 95.5% (w/w) and having le sizes of about 30 to 100 microns (equivalent spherical er), and Respitose® LH300 hale 300) present at 5.5% (w/w) and having particle sizes less than 10 microns alent spherical diameter). After blending, the blends were assayed to confirmed homogeneity and drug content of 1%.

To reduce drug particle agglomeration and aid in the aerosolization of drug les several other excipients are optionally included. al excipients include olipids, such as dipalmitylphosphatidylcholine (DPPC) and lecithin, and metal fatty acid salts, such as magnesium stearate. These can be coated on the carrier particles in weight ratio of excipient to large carrier particle ranging from 0.01 to 0.5%.

Capsule Filling: 20 milligrams of the powder blends from Batch 06RP68.HQ00008 and Batch 06RP68.HQ00009 were loaded into size #3 HPMC capsules to produce drug product. For these blends it was feasible to load from 5 to 35 milligrams of drug into #3 size capsules and empty greater than 95% of the loaded blend from the e upon actuation in Plastiape® RS01 Model 7or Plastiape® RSOO Model 8 s at flow rates ranging from 60 to 100 liters per minute.

Example 3: Determination ofrapamycin in lung and blood following administration by oropharyngeal aspiration (CPA) and oral gavage to C57BL6 mice This study was ted to evaluate the concentration ofrapamycin in male C57BL/6 mice after administration ofrapamycin at a very high target dose of 1 mg/kg by gavage and oropharyngeal aspiration (OPA). A method for the analysis ofrapamycin in mouse blood and lung homogenate was developed using liquid chromatography with tandem mass spectrometry detection (LC-MS/MS). Calibration curves ofrapamycin using triplicate concentrations were ed between 1 ng/mL and 2000 ng/mL in mouse blood, and between 2 ng/mL and 20,000 ng/mL in mouse lung homogenate. Accuracy, precision and linearity were within expected In pilot studies, the efficiency of vehicle delivery to the lungs via oropharyngeal aspiration with a volume of 50 [LL per mouse was evaluated by administration of Evans Blue dye. The presence of blue dye only in lungs was verified Visually, and the absence of blue dye in the stomach demonstrated that delivery to the stomach was avoided in the procedure used.

Rapamycin was administered to male C57BL/6 mice (N=6) by gavage at a dose of 1.0 mg/kg either orally or via OPA. The oral dose was formulated using pharmaceutical oral liquid formulation Rapamune Oral® ). Rapamycin for OPA was prepared by dissolving the test article in an appropriate volume of ethanol, and then addition of an riate volume ofwater _47_ to prepare a 10% ethanol solution at a concentration of 1 mg rapamycin/mL. Rapamycin was administered to 2 groups of 6 male C57BL/6 mice by OPA under ane anesthesia. An additional group of 6 mice received vehicle only (10% ethanol in water). At 1 h after stration a group of 6 mice receiving oral and OPA rapamycin were euthanized, and blood was obtained by cardiac puncture, and the lungs removed. The remaining mice in each group administered rapamycin or vehicle by OPA were observed for an additional 3 days. At the 72-h necropsy, blood was obtained by cardiac puncture and the lungs removed. No adverse effects were observed in rapamycin- or vehicle-treated mice in the 72 h period following dosing.

The concentration ofrapamycin was determined in the collected blood and in lung homogenate by LC-MS/MS. At 1 h following OPA ofrapamycin, the concentration of rapamycin was ~6 fold higher in lung tissue (3794 i 1259 ng/g tissue) than in blood (641 i 220 ng/ml). Following oral stration of a r dose ofrapamycin, the 1-h lung and blood trations ofrapamycin were 71 i 43 ng/g and 23 i 16 ng/mL, respectively. Lung homogenate concentrations following OPA were 53-fo 1d higher than those measured following oral administration of the same high dose (1 mg/kg) ofrapamycin. The data suggests that ry of lower doses ofrapamycin to lung (dose levels that do not saturate system) will result in rapamycin levels in the lung that can be ed by oral dosing but with significantly less cin in the blood than occurs with oral dosing.

Materials and Methods Test Substance: Sirolimus (Rapamune, cin) MW 914.172, C51N79N012, CAS NUMBER: 531239. Source (for oral gavage): ne Oral® (Pfizer) for oral stration, Lot No.: MWGT, Expiration: 07/ 16. Source (for OPA): Rapamycin (Sirolimus) solid, LC Laboratories, Woburn MA, Lot No.: ASW-127, Expiration: 12/2023.

Animals: Male C57BL/6 mice, approximately 8 weeks of age, from Charles River Laboratories, Inc, Raleigh, NC. Animals were fed Certified Purina Rodent Chow #5002 and were ed tap water ad libitam. The analysis of each feed batch for nutrient levels and possible contaminants was performed by the supplier, examined by the Study Director, and maintained in the study records. The feed was stored at approximately 60-70 °F, and the period ofuse did not exceed six months from the milling date. Mice were housed (one per cage) in polycarbonate cages with stainless steel bar lids accommodating a water bottle. Cage sizes are approximately 11.5" x 7.5" x 5" high (70 sq. in. floor space) for mice. Contact bedding was Sani- Chips hardwood chips (P. J. Murphy Forest ts Co.; lle, NJ). Mice were quarantined for a period of 5 days before use on a study. A veterinarian or qualified designee examined the animals prior to their release from quarantine. Temperature and relative humidity in RTI animal rooms were continuously monitored, controlled, and recorded using an automated system /Barber-Colman k 8000 System with Revision 4.4.1 for ® software [Siebe Environmental Controls (SEC)/Barber-Colman Company; Loves Park, IL]). The target environmental ranges were 64—79 0F (18 OC - 26 0C) for temperature and 30-70% relative humidity, with a 12-h light cycle per day. At the end of the in-life phase, the mice were euthanized by overexposure to carbon e.

Test Chemical Preparation: Evans Blue was prepared at 0.5% w/V in sterile distilled water. Rapamune Oral® was administered as supplied for oral dosing. Rapamycin (solid) was dissolved in ethanol and diluted with sterile distilled water to provide a final concentration of 0.5 mg/mL in10% ethanol.

Dosing: Each animal was weighed prior to dosing to determine the amount of dose to be stered. A single gavage dose was administered using a lOO-uL glass syringe ton, Reno, NV) fitted with a ball-tipped 20-G stainless steel gavage dosing needle (Popper & Sons Inc., New Hyde Park, NY). The dose administered to each animal was determined from the weight of the filll syringe minus that of the empty syringe. The dosing time was recorded. Dosing of animals was spaced apart to allow blood collection at the appropriate times. The dose formulations stered to each group are shown below.

For oropharyngeal aspiration group animals, a single dose ofrapamycin (50 uL) was administered to each mouse under isoflurane anesthesia, using a 100 uL glass syringe (Hamilton, Reno, NV) fitted with a ball-tipped 24-G stainless steel gavage dosing needle (Popper & Sons Inc., New Hyde Park, NY). The mouse was weighed prior to dosing, and the dose ofrapamycin administered was recorded by weight. Each mouse was anesthetized with isoflurane, and restrained with the mouth open. The tongue was held to one side of the mouth with forceps, and the dose was slowly injected into the distal part of the oral cavity. The nostrils were covered with a finger for two breaths to ensure aspiration (Rao et al., 2003).

Table 1: Study Design Summary _49_ ————n._<50—<m1——Evans Blue Rapamycin Oral lun- 4-OA--1--Vehicle .0 blood, lung lun_ Collection of Blood and Lung Samples: At study termination (l or 72 h after dosing), mice were anesthetized by exposure to CO2, and blood was collected by cardiac puncture with dipotassium EDTA as anticoagulant. Lung tissue was d and divided into the right and left lung. The left lung was used for analysis, and the right lung flash frozen in liquid nitrogen and stored at -70 0C for fithher analysis.

Analysis of Samples for Rapamycin by LC-MS/MS: An LC—MS/MS method for analysis mycin in lung and blood was prepared based on the published method of Wu et al. (2012).

The volumes ofblood and lung homogenate were reduced substantially from the published method. Triamcinolone was used as internal standard.

Lung homogenate was prepared by homogenization ofweighed lung s with 2.8- mm ball hearings in a homogenizer with tissue + zed water (1:3 w/v) in a SPEX SamplePrep 2010 Geno/Grinder.

The concentrations of standards were arranged so that each standard came from an alternate stock rd. A six-point calibration curve, each made in triplicate, was employed for analyte quantitation. A simple linear regression model with or without ing was employed for curve fitting. The concentration range determined was from 1-2000 ng/mL in blood and 2— 2000 ng/mL in lung homogenate.

The following method performance parameters were considered acceptable; the coefficient of ination, r2, of 20.98 for concentration-response relationship; an cy of S i 15% (for concentrations above LOQ) or S i 20% (for concentration at LOQ) of the nominal value. r2 was greater than 0.999 in all analysis.

Thirty (30) uL of matrix, 30 uL of spiking solution (methanol for blanks and samples), 10 uL Internal standard solution (in MeOH) and 90 uL of MeOH were pipetted into microcentrifiage tubes, vortexed briefly, then centrifuged for 6 min at 10,000 RPM at ~4 °C. ts (90 uL) of supernatant were transferred to LC vial inserts, and then analyzed by LC-MS/MS (Table 2).

Table 2: LC-MS/MS Method Column Waters Acquity UPLC HSS T3 1.8 um, 2.1 x 50 mm with VanGuard 2.1 x 5 mm HSS T3 1.8 Mobile Phase A 10 mM Ammonium e in water, 0.1% acetic acid MobilephaseB Gradient 70% A for 1 min, a linear gradient to 5% A from 1—3 min, held for 1 min, a linear gradient to 70% A from 4-5.1 min, and held at 70% until 6 min 931.70%6470 Triamcinolone (IS MRM) 395.30—>357.20 Data tion and Reporting: Study data was collected and reported in the DebraTM system version .72 (Lablogic Systems Ltd, Sheffield, England). This includes data for animal body weights, dose administered, dose time, and sample collection times. Calculations of dose administered and sample collection times were reported with the DebraTM .

Results Rapamycin Analysis: The analysis ofrapamycin was set up of sample volumes of 30 uL ofblood and lung homogenate. Example chromatograms are shown for rapamycin and internal standard in blood and lung (flgm l and 2). Prior to the generation of study samples, triplicate calibration curves were generated for lung and blood, to verify method performance. The calibration range was from 1.0 —2000 ng/mL for blood and 1 — 20,000 ng/mL for lung homogenate. Lung homogenate was prepared with 1 g of lung tissue nized in 3 volumes ofwater, to yield a 1:4 homogenate. ation curves are shown in flgm 3 and 4 for blood, lung nate, and solvent. _51_ Oropharyngeal tion: Prior to the stration ofrapamyc in by oropharyngeal aspiration, administration of Evans Blue was used to verify that the CPA delivered the dose to the lungs. Mice were anaesthetized with isoflurane and administered Evans Blue by OPA, using a syringe ed with a blunt needle. Immediately following OPA, the mice were euthanized and the lungs and stomach ed visually to ensure that the Evans Blue dye was delivered to the lungs, and was not delivered to the stomach. Four mice were successfully stered Evans Blue with all of the dye appearing to be d in the lungs and none in the stomach.

Rapamycin Administration: The weight of dose solution administered was determined by weighing the charged e with dose solution prior to dosing, and weighing following dosing.

The weight of dose solution administered was used to calculate the amount ofrapamycin administered. The time of dosing was recorded as 0. Animals in groups 2 and 3 were euthanized at l h afier dosing. Animals in groups 4 and 5 were ed for 72 h after dosing. No significant clinical signs were observed in any of the groups.

Rapamycin Analysis in Blood and Lung: Rapamycin was analyzed in mouse blood and left lung homogenate in all of the samples collected (flgm 6 and 7). Samples of the right lung from each animal were saved for potential further analysis. Summary data for the samples are provided in Table 3.

Table 3: Concentration of Rapamycin in Blood and Lung Following Oral and Oropharyngeal (OPA) Administration of Rapamycin to Mice (1 mg/kg) Animal No Route ofAdmin Time post--dose Lung (ng/g Blood (ng/ml) (h) tissue) 3-13 Gavage 1 109.8 49.85 3-14 Gavage 1 24.66 11.3 _52_ 3-15 Gavage 1 122.8 28.7 3-16 Gavage 1 54 28.35 3- 17 Gavage 1 <LOQ 2.845 3-18 Gavage l 43 19.35 Mean 71 23 For all sample sets, a triplicate calibration curve was analyzed with the sequence of standard set, sample replicate 1, standard set, sample replicate sample 2, standard set. At 1 h following GPA of rapamyc in, the concentration ofrapamycin was ~6 fold higher in lung tissue (3794 :: 1259 ng/g tissue) than in blood (641 i 220 ng/ml). Following oral administration of a similar dose ofrapamycin, the 1-h lung and blood concentrations ofrapamycin were 71 :: 43 ng/g and 23 :: 16 ng/mL, respectively. Lung homogenate concentrations following OPA were 53-fold higher than those ed ing oral administration of same high dose (1 mg/kg) of cin. sion This study investigated the concentration ofrapamycin in blood and lung tissue following administration ofrapamycin by gavage in a commercial oral formulation, and by oropharyngeal administration (OPA) as a suspension prepared in 10% aqueous ethanol. No adverse effects were observed in rapamycin- or vehicle-treated mice up to 72 h following dosing Via OPA. Prior to administration ofrapamycin, an ical method was developed, and the administration of a dye into the lung by OPA was verified. The concentrations of cin in lung following OPA _53_ were 6-fold higher than in blood. At 72 h afier OPA, rapamycin was below the limit of quantitation in blood, but was detectable in lung. This study indicated that rapamycin is available systemically following pulmonary administration, and that lung tissue concentrations greatly exceed that of blood at early and late time points following delivery to the lung.

These results further demonstrate that rapamycin delivered directly to the lung achieves an unexpectedly high local concentration of drug in lung tissue compared to the blood. This result was ly unexpected from what is known about the pharmacology mycin, which predicts an imately equal concentration of the drug in lung tissue and the blood because rapamycin is known to distribute evenly throughout bodily tissues and should be cleared rapidly from the lung due to its high lipophilicity. Accordingly, these s indicate that direct administration ofrapamycin to the lungs should be able to achieve a high enough delivered dose for therapeutic efficacy while at the same time achieving almost undetectable systemic availability, thereby eliminating the toxicities associated with oral administration that are due to systemic exposure to the drug. While toxicity to the lung itself is also of concern in view of earlier studies, the s here further unexpectedly indicate that relatively high amounts of rapamycin were not acutely toxic to lung .

References Crowe, A., Bruelisauer, A. & Duerr, L. (1999). Absorption and intestinal lism of SDZ- RAD and rapamycin in rats. Drug Metabolism and Disposition, 27, 627—632.

Rao, G. V. S., Tinkle, S., an, D. N., Antonini, J. M., Kashon, M. L., Salmen, R., Hubbs, A. F. (2003). Efficacy of a technique for exposing the mouse lung to particles aspirated from the pharynx. Journal of logy and Environmental . Part A, 66(15), 1441—52. doi:10.1080/15287390306417.

Wu, K., Cohen, E. E. W., House, L. K., z, J ., Zhang, W., Ratain, M. J., & Bies, R. R. (2012). Nonlinear population pharmacokinetics of sirolimus in patients with advanced cancer.

CPT: Pharmacometrics & Systems Pharmacology, 1(October), e17. doi:10.1038/psp.2012.18.

Example 4: S6 Phosphorylat ion in Mouse Lung Following Oral and CPA Administration of Rapamycin As discussed above, our experiments showing the tissue distribution ofrapamycin in lung and blood following oral administration and CPA demonstrated that direct administration _54_ ofrapamycin to the lungs should be able to achieve a high enough delivered dose for therapeutic efficacy while at the same time achieving very low ic exposure to the drug, thereby simultaneously improving therapeutic efficacy and eliminating many of the toxicities associated with oral administration mycin. To validate this approach, we used the presence of phosphorylated S6 protein in murine lung tissue as a biomarker for mTOR activity. In the mouse strain used (C57b1/6), the mouse airway and alveolar epithelial cells have constitutively active (phosphorylated, “p”) S6 protein. The S6 protein is typically phosphorylated by S6K which is downstream ofmTORCl and is activated, for example, ream of growth factors such as epidermal growth factor (EGF), AKT, ERK, and RSK. mTORCl promotes cell growth and proliferation by stimulating ic processes such as biosynthesis of , ns, and organelles, and suppressing catabolic processes such as autophagy. The mTORCl pathway senses and integrates intracellular and extracellular signals, including growth factors, oxygen, amino acids, and energy status, in order to regulate a wide range of processes, such as protein and lipid synthesis and autophagy. mTORCl is acutely sensitive to rapamycin.

In the present study, lung tissue was taken from the C57b1/6 mice treated as discussed above, either with vehicle (n=6), or 1 mg/kg cin administered via OPA (n=6) or via oral gavage (n=6) at two time points post dosing, 1 hr and 72 hours. As discussed above, following OPA at 1 hr, rapamycin was detected at 641 ng/ml in the blood and 3794 ng/g tissue in the lung, and at 72 hrs was still able in the lung at 12.5 ng/g while being undetectable in the blood at that time point. Conversely, following oral (gavage) administration, at 1 hr, rapamycin was detected at 23 ng/ml in the blood and 71 ng/g tissue in the lung, and at 72 hrs was undetectable in either the lung or blood. As shown by the data in Figures 1 and 2, the level of phosphorylated S6 (pS6) was reduced substantially by both CPA and orally administered cin at 1 hr and remained suppressed at 72 hr for OPA. pS6 was t in the vehicle control e these mice have constitutively active mTOR signaling. These data show that a delivered dose mycin sufficient to achieve about 70 ng/g drug in the lung substantially abrogates mTOR signaling in the lung tissue as measured by pS6 protein and that mTOR signaling remains suppressed at levels as low as 12.5 ng/g. These results validate our approach to utilize inhaled cin for the treatment of diseases and disorders such as LAM, which is characterized by aberrantly high mTOR pathway activity by demonstrating that inhaled rapamycin can be red at much lower doses than orally administered rapamycin to simultaneously achieve high therapeutic efficacy and very low toxicity. _55_ e 5: Size Reduction of Rapamycin for Inhalable Compositions Particle size ofrapamycin was reduced to a target range of 2.0um < DV50 < 3.0um using either a wet polishing or jet milling s. For jet milling, a lab scale MCOne unit from Jetpharma was used with the following ing conditions: venturi pressure 2-4 bar, milling pressure 3-5 bar, feed rate 90 g/h. For wet polishing, feed suspensions were prepared using purified water. A microfluidics high pressure nizer was used for the size ion step and the resulting suspension was spray-dried. Details of the wet polishing s are set forth below.

The high pressure homogenizer used for the size reduction step of the wet polishing process was a pilot-scale Microfluidics High Pressure Homogenizer equipped with an auxiliary processing module (200 micron) and a 100 micron interaction r was used. The unit was operated at ~455 bar (~30 bar in the intensifier module hydraulic pressure). After microfluidization the fluid was d by spray drying to generate a dry powder. A laboratory scale spray dryer, SD45 (BUCHI, model B-290 Advanced) was equipped with a two fluid nozzle (cap and diameter were 1.4 and 0.7 mm, respectively). Two cyclones in series were used (being the first the standard Buchi cyclone and the second the erformance Buchi cyclone) to collect the dried product. The spray drying unit was operated with nitrogen and in single pass mode, i.e. without recirculation of the drying nitrogen. The aspirator, blowing nitrogen, was set at 100% of its capacity (flow rate at maximum capacity is approximately 40 kg/h). The flow rate ofthe atomization nitrogen was adjusted to a value in the rotameter of 40 :: 5 mm. Before feeding the product suspension, the spray dryer was stabilized with purified water, during which the flow rate was adjusted to 6 mein (20% in the peristaltic pump). The inlet temperature was adjusted to e the target outlet temperature (45°C). Afier stabilization of the temperatures, the feed of the spray dryer was commuted from purified water to the t suspension (keeping the same flow rate used during stabilization) and the inlet temperature once again adjusted in order to achieve the target outlet temperature. At the end of the stock suspension, the feed was once more commuted to purified water in order to rinse the feed line and perform a controlled shut down. The dry product in the collection flasks under both cyclones was weighed and the yield calculated as the mass percentage of the dry product in relation to the total solids in the suspension fed to the high re homogenizer.

Particle size distribution was ed by laser diffraction. Solid state characterization (for polymorphic form and purity) was performed by high pressure liquid chromatography (HPLC), X-ray powder diffraction (XRPD), and differential scanning calorimetry . Water content was determined by the Karl Fischer method.

Jet milling produced crystalline rapamycin powder with a monodisperse particle size distribution having DVlO of 1.5 s, a DV50 of 2.? microns and a DV 90 of 4.9 microns, as shown in the table below.

Wet polishing produced crystalline rapamycin powder with a monodisperse particle size distribution having a Dle of 1.0 microns, a DV50 of 2.4 microns and a DV 90 of .0 microns.

Both methods produced particles of rapamycin within the target range and r process showed an impact on polymorphic form or purity of the rapamycin. The tables below show in—process control data for the jet milling and wet ing processes. The data indicate that both processes were able to produce API particle sizes within the target range without impacting API purity or polymorphic form.

Table 4: Jet Milling Data XRPD Similarto API s(iro|imus) ctogram Assavmw/w) Table 5: Wet Polishing Data XRPD Similarto API s(iro|imus) diffractogram Wow/WI —a_ Example 6: Aerosol Performance Testing ofDry Powder Compositions The capsules produced in the example above were placed into the device indicated in the tables below and actuated. The aerosol performance red from the devices/capsules containing blends from Batch 06RP68.HQ00008 and Batch 06RP68.HQ00009 were characterized using a next generation impactor (NGI) according to the methods described in Chapters 905 and 601 of the USP. The aerosols were tested at flow rates of 60 and 100 liters per minute (LPM). The fine particle dose (FPD) and fine le fraction (FPF) are shown in the tables below. Mass median aerodynamic diameters (MMAD) and geometric standard deviations (GSD) are also shown.

Table 6: .HQ00008 (Wet Polished) + Plasitape RSOl Model 7 60 LPM 100 LPM Table 7: 06RP68.HQ00008 (Wet Polished) + Plastiape RSOO Model 8 60 LPM 100 LPM Table 8: 06RP68.HQ00009 (Jet ) + Plastiape RSOl Model 7 60 LPM 100 LPM Table 9: 06RP68.HQ00009 (Jet Milled)+ Plastiape RSOO Model 8 60 LPM 100 LPM Based on these aerosol performance data, the wet polished drug particles are preferred.

They resulted in a higher fine le dose, higher fine particle fraction, a particle size distribution that would exhibit ation into both the central and peripheral lung regions, and would have less oral deposition. e 7: Pharmacokinetic ng of rapamyc in Based on the aerosol performance 06RP68.HQ00008 (Wet Polished) + Plasitape RSOl Model as shown above, and the results of animal ments in Example 3, it can be expected that delivery of inhaled rapamycin directly to the lung in humans will similarly result in tent lung concentrations that are sufficiently high to be therapeutically effective, but with low ic exposure (low blood concentrations) thereby ively minimizing side effects due to systemic exposure. A two compartment, pharmacokinetic model was developed to predict the concentrations in the blood and lungs in humans after repeat QD dosing using the formulation and DPI inhaler in Table 7. For the pharmacokinetic model, human PK parameters from the Rapamune® (NDA 21-110, and NDA 21—083) y basis of approval were used: the volume of distribution was assumed to be 780 liters, clearance was 0.0003/minute, and elimination half life was 42.3 hours (assuming equivalency to rapamycin IV dosing).

Absorption half life ofrapamycin from the lung was estimated to be approximately 0.5 hours, similar to other highly lipophilic compounds, such as fluticasone proprionate for which lung absorption data is available. Bioavailability ofrapamycin depositing in the lung was assumed to be approximately 100%. Bioavailability mycin absorbed by the GI route through oropharyngeal deposition or removal from the upper airways by mucociliary clearance was assumed to be 14% as ed in the Rapamune® summary basis for approval. For a typical human inspiratory er at a flow rate of 60 liters per minute, as shown in Table 7, the fine particle dose was 57 micrograms, and the fine particle fraction was 40%.

The model predicts achieving an average steady state concentration after 11 days as shown in Figure 8. From the figure it can be seen that once daily repeat dosing of 57 rams delivered to the lungs results in trough blood concentrations of approximately 0.150 nanograms/ml, substantially below the concentrations of 5-15 ng/ml reported in McCormack et al. (2011), “Efficacy and safety of sirolimus in lymphangioleiomyomatosis”, N Engl JMed 364: 1595—1606. Assuming a lung tissue mass of 850 grams, no metabolism in the lung and a lung absorption half life or 30 minutes, 57‘ micrograms rapamycin delivered to the lungs would result in eutic levels in the lung tissue, with local lung concentrations of rapamycin as high as imately 60 ng/gram.

EQUIVALENTS Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the ion described herein. Such equivalents are intended to be encompassed by the following claims.

All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was ically and dually indicated to be incorporated by reference in its entirety for all purposes.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications ofthe invention in addition to those described herein will become apparent to those skilled in the art from the foregoing ption and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Claims (29)

What is claimed is:

1. A pharmaceutical dry powder composition for pulmonary delivery comprising an amount of microparticles of rapamycin, particles of a carrier, and ally one or more excipients, wherein the microparticles of rapamycin have a mass median aerodynamic diameter (MMAD) of from 0.5 to 6 s.

2. The composition of claim 1, wherein the microparticles have an MMAD of from 1 to 3 microns or from 2 to 3 microns.

3. The ition of claim 1 or 2, wherein the carrier is selected from the group consisting of arabinose, glucose, se, ribose, mannose, sucrose, trehalose, lactose, maltose, starches, dextran, mannitol, lysine, leucine, isoleucine, dipalmitylphosphatidylcholine, in, polylactic acid, poly c-co-glutamic) acid, and xylitol, or mixtures of any of the foregoing.

4. The composition of any one of claims 1 to 3, n the les of carrier have diameters ranging from 1 to 200 microns, from 30 to 100 microns, or less than 10 microns.

5. The composition of any one of claims 1 to 4, wherein the carrier comprises or consists of a blend of two different carriers, a first carrier and a second carrier.

6. The composition of claim 5, wherein the carrier consists of a blend of two different lactose carriers.

7. The composition of claim 5 or 6, wherein the first r consists of particles having diameters ranging from about 30-100 microns and the second carrier consists of particles having diameters of less than 10 microns.

8. The composition of claim 7, wherein the ratio of the two different carriers is in the range of from 3:97 to 97:3.

9. The composition of any one of claims 1 to 8, wherein the drug to carrier ratio in the powder is from 0.5 % to 2 % (w/w).

10. The composition of claim 9, n the drug to carrier ratio in the powder is 1 % (w/w).

11. The ition of any one of claims 1 to 10, wherein the amount of drug is from 0.5 to 20% (w/w) based upon total weight of the composition.

12. The composition of claim 11, wherein the amount of drug is about 1 % to 2 % (w/w).

13. The ition of any one of claims 1 to 12, wherein the one or more excipients is present and is selected from a phospholipid and a metal salt of a fatty acid.

14. The composition of claim 13, wherein the phospholipid is selected from dipalmitylphosphatidylcholine and lecithin.

15. The composition of claim 13, wherein the metal salt of a fatty acid is magnesium stearate.

16. The composition of any one of claims 13 to 15, wherein the one or more excipients is coated on the carrier particles.

17. The composition of any one of claims 1 to 16, wherein the amount of rapamycin is an amount effective to inhibit the ical activity of mTORC1.

18. The composition of any one of claims 1 to 17, wherein the amount of rapamycin in the composition is from 50 to 500 ug, from 50 to 250 ug, or from 50 to 150 ug.

19. The composition of any one of claims 1 to 18, wherein the composition has a fine particle fraction (FPF) greater than 20% with a corresponding fine particle dose (FPD) ranging from 10 micrograms to 2 milligrams, or less than 0.5 milligrams, ing 1 to 12 months or 1 to 36 months of storage.

20. The composition of any one of claims 1 to 19, further comprising one or more additional therapeutic agents.

21. The composition of claim 20, wherein the one or more additional therapeutic agents is selected from an estrogen nist, a statin, a src inhibitor, and a VEGF-R inhibitor.

22. The composition of claim 20, n the one or more additional therapeutic agents is selected from the group consisting of letrozole, tamoxifen, simvastatin, saracatinib, pazopanib, imatinib, and combinations thereof.

23. The composition of any one of claims 1 to 22, where the composition is adapted for once daily administration.

24. A method for making the composition of any one of claims 1 to 23, the method comprising a wet polishing process comprising the steps of preparing an aqueous sion of drug, subjecting the drug suspension to micronization, and drying the resulting particles to form a dry powder.

25. A pharmaceutical e or kit comprising the composition of any one of claims 1 to 23, and instructions for use.

26. A dry powder delivery device comprising a reservoir containing the composition of any one of claims 1 to 23.

27. The dry powder delivery device of claim 26, wherein the reservoir is an integral chamber within the device, a capsule, or a blister.

28. A method for making the composition of any one of claims 1 to 23, the method comprising mechanically reducing the particle size of the rapamycin by jet milling.

29. Use of the composition of any one of claims 1 to 23 in the cture of a medicament for treating lymphangioleiomyomatosis.