DESC:4. DESCRIPTION

Field of the Invention

The present invention relates to formulations and methods for delivering drugs for SARS-CoV-2 in mammals including but not limited to SARS-CoV-2, SARS, MERS and similar diseases. The present invention relates to compositions intended for inhalation administration. More specifically, it concerns liquid and powder formulations targeting human respiratory system.

Background of the Invention

An acute respiratory disease, caused by a novel coronavirus (SARS-CoV-2, previously known as 2019-nCoV), the coronavirus disease 2019 (COVID-19) has spread throughout China and received worldwide attention. On 11 March 2020, World Health Organization (WHO) officially declared the COVID-19 pandemic. More than 22,000 people died and 4,80,000 are infected as on current date.

Coronaviruses are large, enveloped, single-stranded RNA viruses found in humans and other mammals, such as dogs, cats, chicken, cattle, pigs, and birds. Coronaviruses cause respiratory, gastrointestinal, and neurological disease. The most common coronaviruses in clinical practice are 229E, OC43, NL63, and HKU1, which typically cause common cold symptoms in immunocompetent individuals. SARS-CoV-2 is the third coronavirus that has caused severe disease in humans to spread globally in the past 2 decades. The first coronavirus that caused severe disease was severe acute respiratory syndrome (SARS), which was thought to originate in Foshan, China, and resulted in the 2002-2003 SARS-CoV pandemic.
Alternatively, Human immunodeficiency virus (HIV) is recognized as the causative agent in AIDS. Current therapies for HIV infection focus on inhibiting the activity of viral enzymes which are essential to the life cycle of the virus. The agents that are presently in use fall mainly into three classes, designated Nucleoside Reverse Transcriptase Inhibitors (NRTIs), Non-nucleoside Reverse Transcriptase Inhibitors (NNRTIs), and Protease Inhibitors (PIs). Presently, combination therapies, i.e. the selection of two or more antiretroviral agents taken together to make up a “drug cocktail,” are the preferred treatment for HIV infection. Combination therapies have been shown to reduce the incidence of opportunistic infections and to increase survival time. Typically, the drug cocktail combines drugs from different classes, so as to attack the virus at several stages in the replication process.

This approach has been shown to reduce the likelihood of the development of virus forms that are resistant to a given drug or class of drugs. Treatment failure with rebound of the amount of HIV which can be measured in the blood is common for patients treated with combination antiretroviral regimens. Resistance to the drugs in the drug regimen develops as the virus replicates in the presence of these drugs. Because of structural similarities of the drugs within an antiretroviral class, cross resistance is commonly seen to the other members of that class (for example virologic failure on a regimen containing an NNRTI will lead to cross resistance to the other first generation NNRTI agents). As patients experience repeated virologic failure on antiretroviral combination therapy, their viruses develop broad multi-class antiretroviral drug resistance which limits the effectiveness of the next round of antiretroviral therapy. Many highly treatment experienced patients have been exposed to all three classes of antiretroviral drugs and cannot obtain two active drugs to form the core of a new, effective antiretroviral drug regimen.

There are multiple formulations known for use in HIV treatment therapy. The active substance Ritonavir [NORVIR soft gelatin capsule] is characterized by low aqueous solubility, a lack of bioavailability when given in the solid state, instability once in solution under ambient conditions and a metallic taste. U.S. Pat. No. 5,484,801 discloses a formulation wherein Ritonavir formulation has been optimized with respect to the vehicle, which essentially is a solvent comprising a mixture of (1) (a) a solvent selected from propylene glycol and polyethylene glycol or (b) a solvent selected from polyoxyethyleneglycerol triricinoleate, polyethylene glycol 40 hydrogenated castor oil, fractionated coconut oil, polyoxyethylene (20) sorbitan monooleate and 2-(2-ethoxyethoxy) ethanol or (c) a mixture thereof and (2) ethanol or propylene glycol to improve the bioavailability.

Lopinavir/Ritonavir, is a fixed-dose combination antiretroviral medication for the treatment and prevention of HIV/AIDS. It combines lopinavir with a low dose of Ritonavir. It is generally recommended for use with other antiretrovirals. It may be used for prevention after a needlestick injury or other potential exposure. It is taken by mouth as a tablet, capsule, or solution.

Lopinavir is chemically designated as [1S-[1R*,(R*), 3R*, 4R*]]-N-[4-[[(2,6¬ dimethylphenoxy)acetyl]amino]-3-hydroxy-5-phenyl-1-(phenylmethyl)pentyl]tetrahydro-alpha¬ (1-methylethyl)-2-oxo-1(2H)-pyrimidineacetamide. Its molecular formula is C37H48N4O5, and its molecular weight is 628.80. Lopinavir is a white to light tan powder. It is freely soluble in methanol and ethanol, soluble in isopropanol and practically insoluble in water.

Ritonavir is chemically designated as 10-hydroxy-2-methyl-5-(1-methylethyl)-1- [2-(1¬ methylethyl)-4-thiazolyl]-3,6-dioxo-8,11-bis(phenylmethyl)-2,4,7,12-tetraazatridecan-13-oic acid, 5-thiazolylmethyl ester, [5S-(5R*,8R*,10R*,11R*)]. Its molecular formula is C37H48N6O5S2, and its molecular weight is 720.95. Ritonavir is a white to light tan powder. It is freely soluble in methanol and ethanol, soluble in isopropanol and practically insoluble in water.

Lopinavir is an antiviral drug, an inhibitor of the HIV-1 protease, prevents cleavage of the Gag-Pol polyprotein, resulting in the production of immature, non-infectious viral particles. As co-formulated, Ritonavir inhibits the CYP3A-mediated metabolism of lopinavir, thereby providing increased plasma levels of lopinavir.

Formulation of two HIV protease inhibitors [Lopinavir and Ritonavir] in a single formulation. Till recently, this formulation was available in a soft gel capsule, embodied in the patent U.S. Pat. No. 6,458,818 granted to Abbott. The patent covers a solution of Lopinavir and Ritonavir in a long chain fatty acid organic solvent. This soft gel formulation has been criticized due to stability problems and need for keeping the formulation in refrigerated condition. Abbott has now introduced a new tablet formulation for combined administration of Lopinavir and Ritonavir, instead of the previously known soft gel formulation. It has also filed patent applications related to this tablet formulation. For instance, WO2005039551 covers a combination of Lopinavir and Ritonavir in a water-soluble polymer and surfactant wherein the tablet is formulated by melt extrusion process.

The current invention is a pharmaceutical product or formulation, which comprises Lopanivir or its pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof, and Ritonavir, or its pharmaceutical acceptable salt, solvate or physiologically functional derivative thereof, preferably the product or formulation being in a form suitable for nasal or inhalation administration in the prophylaxis and/or treatment of SARS-CoV-2.

The present invention thereby targets the pulmonary conditions through drug delivery formulations comprising pharmaceutical formulation and in particular to pharmaceutical formulations for use in lung viral infections, including but not limited to HIV therapy SARS-CoV-2, SARS, MERS. It also discloses the processes to make the same. Also, the present invention has been developed primarily for use as a formulation to be used for treatment in HIV therapy. However, the present invention is not limited to this particular field of use and is applicable to all viral infections targeting the pulmonary function.

Objective of the Invention

The primary objective of the present invention is related to the delivery of liposomal nebulized lopinavir and ritonavir formulation for lung infections, especially in cases where an individual suffers from SARS-CoV-2, SARS, MERS, Pneumonia and/or Acute lung injury and/or Acute respiratory distress syndrome.

Another objective of the present invention is to provide a topical drug delivery comprising a combination of drugs, an effective dose of a nebulized liposomal lopinavir and Ritonavir formulation.

Brief Summary of Invention

The present invention has been accomplished under the circumstances in view. It is therefore the main object of the present invention to provide a delivery system for pulmonary conditions.

The terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study except where specific meanings have otherwise been set forth herein.

The term “effective amount’ means the amount of the formulation that will be effective in the treatment of a particular Subject will depend on the particular Subject and State of the subject, and can be determined by standard clinical techniques. In addition, in vitro or in Vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed will also depend on the state of the patient, and should be decided according to the judgment of the practitioner and each patient’s circumstances. “IM’ refers to intramuscular injection. “IV” refers to intravenous injection. “SC refers to subcutaneous injection.
The present invention relates in part to a method of delivering to an individual having a pulmonary disorder comprising administering to the patient an effective dose of a nebulized liposomal lopinavir and Ritonavir formulation for at least one treatment cycle, wherein the treatment cycle comprises an administration period of 15 to 75 days, followed by an off period of 15 to 75 days; and the effective dose comprises 10 to 400 mg of lopinavir and 2 to 40 mg of Ritonavir daily during the administration period.

Brief Description of The Drawings

So that the manner in which the above-recited features of the present invention is understood in detail, a more particular description of the invention, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.

The features and advantages of the present invention will become more apparent from the following description taken in conjunction with the accompanying drawings, which form a part of this application and in which:

Fig 1 depicts mass distribution of Liposomal Lopinavir and ritonavir nebulizate collected on impactor stages as a function of cut-off diameter. The three Liposomal Lopinavir and ritonavir lots of Table 15 legend (designated as 1, 2, and 3) were used with the air-jet nebulizer and ACI system (solid symbols) and with NGI system (open symbols).

Fig 2 illustrates Veros were plated at 5 x 105 cells in 6 well plates and infected with 400 µl using the same serially diluted starting sample of SAR-CoV2. After infection, 3 ml overlays of 8,16,32,64,128,256 mg/ml oral lopinavir/ritonavir and liposomal lopinavir& ritonavir, were applied in order to directly compare the overlays.

Fig 3 a, b & c depicts the body weight at the beginning and the end of the experiment (a) and organ weight and liver appearance at necropsy five days post-infection (b,c,d, respectively) in SARS-CoV2-infected (infected group, n = 20), infected and treated with ritonavir/lopinavir (infected, LR-Lopinavir & Ritonavir; LLR-Liposomal Lopinavir & Ritonavir and noninfected untreated mice (control group, n = 16) (mean Å} SD). Different letters indicate significant differences (p < 0.05) between pre- and post-infection timepoints for a and significant differences (p < 0.05) between the indicated groups for (b–c).

Fig 5 illustrates detection of cytokines in the plasma of all experimental groups (infected, infected + LR, LLR, and control). LR-Lopinavir & Ritonavir; LLR-Liposomal Lopinavir & Ritonavir and Murine plasma was obtained 5 days post-infection, and cytokine concentrations were determined by multiplex bead array (mean Å} SD). Different letters indicate statistically significant differences (p < 0.05).

Fig 6 a & 6b depicts the efficacy Plaque forming in SARS-CoV2-infected (infected group, n = 20), infected and treated with ritonavir/lopinavir (infected, LR-Lopinavir & Ritonavir; LLR-Liposomal Lopinavir + Ritonavir, Lopinavir + ritonavir + Azithromycin, Liposomal Lopinavir + ritonavir + Azithromycin inhalation and noninfected untreated mice (control group, n = 16) (mean Å} SD).
Fig 7 depicts specified deposition of Liposomal Lopinavir & Ritonavir in human airway using multiple-path particle dosimetry model quantifying age
Fig 8 depicts specified visualized deposition of Liposomal Lopinavir & Ritonavir in human airway using multiple-path particle dosimetry model quantifying age
Detailed Description of The Invention

The principles of operation, design configurations and evaluation values in these non-limiting examples can be varied and are merely cited to illustrate at least one embodiment of the invention, without limiting the scope thereof.

The embodiments disclosed herein can be expressed in different forms and should not be considered as limited to the listed embodiments in the disclosed invention. The various embodiments outlined in the subsequent sections are construed such that it provides a complete and a thorough understanding of the disclosed invention, by clearly describing the scope of the invention, for those skilled in the art.

Accordingly, the present invention provides a method of preparing a liposome delivery system comprising a combination of drugs for individuals with pulmonary infections. In another embodiment of the present invention, the treatment cycle is administered to the patient at least twice. In some embodiments, the administration period is 14 to 30 days, or 14 to 35 days. In other embodiments, the administration period is about 14 days or 28 days. In some embodiments, the off period is 3 to 35 days, or 14 to 35 days. In other embodiments, the off period is about 28 days. In still other embodiments, the off period is of 25 to 75 days, 35 to 65 days, or 14 to 75 days. In other embodiments, the off period is about 56 days.

In another embodiment of the present invention, the administration period is about 28 days and the off period is about 28 days, while in other embodiments, the administration period is about 28 days and the off period is about 56 days.

In another embodiment of the present invention, the effective dose comprises 10 to 500 mg of lopinavir, 5 to 100 mg of Ritonavir, or about 20 to about 580 mg of liponavir and 10 to 50 mg of Ritonavir. In other embodiments, the effective dose is about 20 or about 580 mg of liponavir and 10 to 60 mg of Ritonavir.

In another embodiment of the present invention, the pulmonary disorder is patients suffering with HIV, HIV pulmonary infections and group of HIV patients infected with including but not limited to chronic obstructive pulmonary disease, bronchiectasis, pulmonary infection, cystic fibrosis, alpha-1-antitrypsin enzyme deficiency and a combination thereof. In other embodiments, the pulmonary condition is a bacterial pulmonary infection, such as a P. aeruginosa infection. In some embodiments, the pulmonary condition is bronchiectasis. In some embodiments the composition could be used in the treatment of SARS-CoV-2 in mammals (e.g., humans), including but not limited to SARS-CoV-2, SARS, MERS etc.

In another embodiment of the present invention, the patient has a serum Cmax of liponavir and Ritonavir of less than about 15 mcg/mL during the administration period. In other embodiments, the patient has a sputum Cmax of liponavir and Ritonavir of at least 1000 mcg per gram of sputum either during the administration, for at least 14 days after the administration.

In another embodiment of the present invention, the patient has a reduction in log10 CFU of the bacterial infection in the lungs of at least 0.5 for at least 15 days after the administration period ends. In other embodiments, the reduction in the log10 CFU is at least 1.0.

In another embodiment of the present invention, the patient experiences an improvement in lung function for at least 15 days after the administration period ends. For example, the patient may experience an increase in FEV1, an increase in blood oxygen saturation, or both. In some embodiments, the patient has an FEV1 that is increased by at least 5% over the FEV1 prior to the treatment cycle. In other embodiments, FEV1 is increased by 5 to 50%. In other embodiments, FEV1 is increased by 25 to 500 mL over FEV1 prior to the treatment cycle. In some embodiments, blood oxygen saturation is increased by at least 1% over oxygen saturation prior to the treatment cycle.

In another embodiment of the present invention, the length of time to a pulmonary exacerbation is at least 20 days from the last day of administration. In other embodiments, the length of time to a rescue treatment is at least 25 days from the last day of the administration.

In another embodiment of the present invention, the liposomal liponavir and Ritonavir formulation comprises a lipid selected from the group consisting of egg phosphatidyl choline (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylinositol (EPI), egg phosphatidylserine (EPS), phosphatidylethanolamine (EPE), phosphatidic acid (EPA), soy phosphatidyl choline (SPC), soy phosphatidylglycerol (SPG), soy phosphatidylserine (SPS), soy phosphatidylinositol (SPI), soy phosphatidylethanolamine (SPE), soy phosphatidic acid (SPA), hydrogenated egg phosphatidylcholine (HEPC), hydrogenated egg phosphatidylglycerol (HEPG), hydrogenated egg phosphatidylinositol (HEPI), hydrogenated egg phosphatidylserine (HEPS), hydrogenated phosphatidylethanolamine (HEPE), hydrogenated phosphatidic acid (HEPA), hydrogenated soy phosphatidylcholine (HSPC), hydrogenated soy phosphatidylglycerol (HSPG), hydrogenated soy phosphatidylserine (HSPS), hydrogenated soy phosphatidylinositol (HSPI), hydrogenated soy phosphatidylethanolamine (HSPE), hydrogenated soy phosphatidic acid (HSPA), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), dioleylphosphatidyl-ethanolamine (DOPE), palmitoylstearoylphosphatidyl-choline (PSPC), palmitoylstearolphosphatidylglycerol (PSPG), mono-oleoyl-phosphatidylethanolamine (MOPE), cholesterol, ergosterol, lanosterol, tocopherol, ammonium salts of fatty acids, ammonium salts of phospholipids, ammonium salts of glycerides, myristylamine, palmitylamine, laurylamine, stearylamine, dilauroyl ethylphosphocholine (DLEP), dimyristoyl ethylphosphocholine (DMEP), dipalmitoyl ethylphosphocholine (DPEP) and distearoyl ethylphosphocholine (DSEP), N-(2,3-di-(9-(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammonium chloride (DOTMA), 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP), phosphatidyl-glycerols (PGs), phosphatidic acids (PAs), phosphatidylinositols (PIs), phosphatidyl serines (PSs), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylacid (DMPA), dipalmitoylphosphatidylacid (DPPA), distearoylphosphatidylacid (DSPA), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphospatidylinositol (DSPI), dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), and mixtures thereof. In other embodiments, the liposomal amikacin formulation comprises a phospholipid and a sterol, such as DPPC and cholesterol. In other embodiments, the liposomal amikacin formulation comprises DPPC and cholesterol in about a 2 to 1 ratio by weight. In some embodiments, the liposomal amikacin formulation has a lipid to drug ratio of about 0.5 to about 1.0, about 0.5 to 0.7, or about 0.6 by weight.

Liposomal Lopinavir and Ritonavir formulations useful in the presently disclosed methods can be prepared as described below. Generally, Lopinavir and Ritonavir is used in the form of a pharmaceutically acceptable salt, for example the sulfate salt of Lopinavir and anhydrous form of Ritonavir.

The lipids used in the compositions of the present invention can be synthetic, semi-synthetic or naturally-occurring lipids, including phospholipids, tocopherols, steroids, fatty acids, glycoproteins such as albumin, anionic lipids and cationic lipids. The lipids may be anionic, cationic, or neutral. In one embodiment, the lipid formulation is substantially free of anionic lipids, substantially free of cationic lipids, or both. In one embodiment, the lipid formulation comprises only neutral lipids. In another embodiment, the lipid formulation is free of anionic lipids or cationic lipids or both.
In another embodiment, the lipid is a phospholipid. Phospholipids include egg phosphatidyl choline (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylinositol (EPI), egg phosphatidylserine (EPS), phosphatidylethanolamine (EPE), and egg phosphatidic acid (EPA); the soya counterparts, soy phosphatidyl choline (SPC); SPG, SPS, SPI, SPE, and SPA; the hydrogenated egg and soya counterparts (e.g., HEPC, HSPC), other phospholipids made up of ester linkages of fatty acids in the 2 and 3 of glycerol positions containing chains of 12 to 26 carbon atoms and different head groups in the 1 position of glycerol that include choline, glycerol, inositol, serine, ethanolamine, as well as the corresponding phosphatidic acids.

The chains on these fatty acids can be saturated or unsaturated, and the phospholipid can be made up of fatty acids of different chain lengths and different degrees of unsaturation. In particular, the compositions of the formulations can include dipalmitoylphosphatidylcholine (DPPC), a major constituent of naturally-occurring lung surfactant as well as dioleoylphosphatidylcholine (DOPC). Other examples include dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) dipalmitoylphosphatidcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylglycerol (DSPG), dioleylphosphatidylethanolamine (DOPE) and mixed phospholipids like palmitoylstearoylphosphatidylcholine (PSPC) and palmitoylstearoylphosphatidylglycerol (PSPG), driacylglycerol, diacylglycerol, seranide, sphingosine, sphingomyelin and single acylated phospholipids like mono-oleoyl-phosphatidylethanol amine (MOPE).

The lipids used can include ammonium salts of fatty acids, phospholipids and glycerides, phosphatidylglycerols (PGs), phosphatidic acids (PAs), phosphotidylcholines (PCs), phosphatidylinositols (PIs) and the phosphatidylserines (PSs). The fatty acids include fatty acids of carbon chain lengths of 12 to 26 carbon atoms that are either saturated or unsaturated. Some specific examples include: myristylamine, palmitylamine, laurylamine and stearylamine, dilauroyl ethylphosphocholine (DLEP), dimyristoyl ethylphosphocholine (DMEP), dipalmitoyl ethylphosphocholine (DPEP) and distearoyl ethylphosphocholine (DSEP), N-(2,3-di-(9(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammonium chloride (DOTMA) and 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP). Examples of PGs, PAs, PIs, PCs and PSs include DMPG, DPPG, DSPG, DMPA, DPPA, DSPA, DMPI, DPPI, DSPI, DMPS, DPPS and DSPS, DSPC, DPPG, DMPC, DOPC, egg PC.

In another embodiment, the liposome comprises a lipid selected from the group consisting of phosphatidyl cholines (PCs), phosphatidyl-glycerols (PGs), phosphatidic acids (PAs), phosphatidylinositols (PIs), and phosphatidyl serines (PSs).

In another embodiment, the lipid is selected from the group consisting of: egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylinositol (EPI), egg phosphatidylserine (EPS), phosphatidylethanolamine (EPE), phosphatidic acid (EPA), soy phosphatidyl choline (SPC), soy phosphatidylglycerol (SPG), soy phosphatidylserine (SPS), soy phosphatidylinositol (SPI), soy phosphatidylethanolamine (SPE), soy phosphatidic acid (SPA), hydrogenated egg phosphatidylcholine (HEPC), hydrogenated egg phosphatidylglycerol (HEPG), hydrogenated egg phosphatidylinositol (HEPI), hydrogenated egg phosphatidylserine (HEPS), hydrogenated phosphatidylethanolamine (HEPE), hydrogenated phosphatidic acid (HEPA), hydrogenated soy phosphatidyl choline (HSPC), hydrogenated soy phosphatidylglycerol (HSPG), hydrogenated soy phosphatidylserine (HSPS), hydrogenated soy phosphatidylinositol (HSPI), hydrogenated soy phosphatidylethanolamine (HSPE), hydrogenated soy phosphatidic acid (HSPA), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), dioleylphosphatidyl-ethanolamine (DOPE), palmitoylstearoylphosphatidyl-choline (PSPC), palmitoylstearolphosphatidylglycerol (PSPG), mono-oleoyl-phosphatidylethanolamine (MOPE), tocopherol, ammonium salts of fatty acids, ammonium salts of phospholipids, ammonium salts of glycerides, myristylamine, palmitylamine, laurylamine, stearylamine, dilauroyl ethylphosphocholine (DLEP), dimyristoyl ethylphosphocholine (DMEP), dipalmitoyl ethylphosphocholine (DPEP) and distearoyl ethylphosphocholine (DSEP), N-(2,3-di-(9-(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammonium chloride (DOTMA), 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylacid (DMPA), dipalmitoylphosphatidylacid (DPPA), distearoylphosphatidylacid (DSPA), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphospatidylinositol (DSPI), dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), and mixtures thereof.

In another embodiment, the liposome comprises a phosphatidyl choline. The phosphatidyl choline may be unsaturated, such as DOPC or POPC, or saturated, such as DPPC. In another embodiment, the liposome does not include a sterol. In one embodiment, the liposome consists essentially of a phosphatidyl choline and a sterol. In another embodiment, the liposome consists essentially of DPPC and cholesterol.

Liposomes or lipid antiinfective formulations composed of phosphatidylcholines, such as DPPC, aid in the uptake by the cells in the lung such as the alveolar macrophages and helps to sustain release of the antiinfective agent in the lung (Gonzales-Rothi et al. (1991)). The negatively charged lipids such as the PGs, PAs, PSs and PIs, in addition to reducing particle aggregation, can play a role in the sustained release characteristics of the inhalation formulation as well as in the transport of the formulation across the lung (transcytosis) for systemic uptake.

While not being bound by any particular theory, it is believed that when the lipid comprises a neutral lipid, and does not comprise a negatively charged or positively charged phospholipid, the liposomal formulation has improved uptake by the lungs. For example, the liposome my have improved penetration into a biofilm or mucus layer when the lipid comprises only neutral lipids. Exemplary neutral lipids include the aforementioned phosphatidylcholines, such as DPPC and sterols, such as cholesterol.

The present invention is directed to methods of treating a pulmonary condition in a subject need thereof comprising administering to the subject and effective amount of any one of the aforementioned liposomal antibiotic formulations. In some embodiments, the pulmonary condition is a acute respiratory distress syndrome or Acute lung injury bacterial infection. In some embodiments, the method comprises administering to a patient in need thereof an effective amount of a liposomal Lopinavir-Ritonavir formulation (also referred to herein as “liposomal Lopinavir-Ritonavir”) by inhalation daily. In some embodiments, the administration by inhalation comprises nebulizing the liposomal formulation.

In another embodiment the present invention, the liposomal Lopinavir-Ritonavir formulation is administered daily for a period of time, followed by second period of time (an “off” period) wherein no liposomal formulation is administered. For example, in some embodiments, the method of treating a pulmonary disorder comprises administering to the patient an effective dose of a nebulized liposomal Lopinavir-Ritonavir formulation for at least one treatment cycle, wherein: the treatment cycle comprises an administration period of 5 to 25 days, followed by an off period of 5 to 15 days; and the effective dose comprises 10 to 2500 mg of Lopinavir and 5 to 1500 mg of Ritonavir daily during the administration period.
In another embodiment the present invention, the aforementioned treatment cycle is administered to the patient at least twice. In other embodiments, the treatment cycle may be administered 3, 4, 5, 6, or more times.

During the administration period, liposomal Lopinavir-Ritonavir is administered daily. In some embodiments, liposomal Lopinavir-Ritonavir can be administered every other day or every third day during the administration period. As explained above, the administration period can be 15 to 75 days. In some embodiments, the administration period is 15 to 35 days, or 20 to 35 days. In other embodiments, the administration period is 20 to 30 days, 25 to 35 days or 25 to 30 days. In other embodiments, the administration period is about 25, 26, 27, 28, 29 or 30 days. In another embodiment, the administration period is about 28 days.

During the off period the liposomal Lopinavir-Ritonavir formulation is not administered to the patient. In some embodiments, the off period is 15 days or longer, for example, 15 to 75 days, 15 to 35 days, or 20 to 35 days. In other embodiments, the off period is 20 to 30 days, 25 to 35 days or 25 to 30 days. In other embodiments, the off period is about 25, 26, 27, 28, 29 or 30 days. In other embodiments, the off period is about 28 days, while in still other embodiments, the off period is at least 29 days.

In another embodiment the present invention, the off period is of 25 to 75 days, 35 to 75 days, or 45 to 75 days. In other embodiments, the off period is 50 to 75 days, 50 to 70 days, 50 to 65 days or 50 to 60 days. In other embodiments, the off period is about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 days, while in other embodiments, the off period is about 56 days.

In another embodiment the present invention, the administration period is about 28 days and the off period is about 28 days, while in other embodiments, the administration period is about 28 days and the off period is about 56 days.

In another embodiment the present invention, the effective dose comprises 10 to 2,500 mg of Lopinavir and 5 to 1500 of Ritonavir. In other embodiments, the effective dose is about 150mg to about 400 mg of Lopinavir and 100 to 350 of Ritonavir. In other embodiments, the effective dose is about 230 mg to about 330 mg Lopinavir and 220 to 340 of Ritonavir. In other embodiments, the effective dose of Lopinavir-Ritonavir is about 100, 150, 200, 250, 300, 250, 400, 450, 500, 550, 600, 650, 700 or 750 mg of Lopinavir and Ritonavir daily. In other embodiments, the effective dose is about 280 or about 560 mg of Lopinavir and 100 to 350 of Ritonavir.

In another embodiment the present invention, the administration period is about 15 days, and the dose is about 280 to about 560 mg of Lopinavir-Ritonavir. In other embodiments, the administration period is about 28 days, the off period is about 28 days, and the dose is about 280 to about 560 mg. In other embodiments, the administration period is about 28 days, the off period is about 56 days, and the dose is about 280 to about 560 mg.

In another embodiment the present invention, the pulmonary disorder is selected from the group consisting of chronic obstructive pulmonary disease, bronchiectasis, pulmonary infection, cystic fibrosis, alpha-1-antitrypsin enzyme deficiency and a combination thereof. In some embodiments, the pulmonary condition is cystic fibrosis. In other embodiments, the pulmonary condition is a bacterial pulmonary infection, Pseudomonas (e.g., P. aeruginosa, P. paucimobilis, P. putida, P. fluorescens, and P. acidovorans), staphylococcal, Methicillin-resistant Staphylococcus aureus (MRSA), streptococcal (including by Streptococcus pneumoniae), Escherichia coli, Klebsiella, Enterobacter, Serratia, Haemophilus, Yersinia pesos, Burkholderia pseudomallei, B. cepacia, B. gladioli, B. multivorans, B. vietnamiensis, Mycobacterium tuberculosis, M. avium complex (MAC) (M. avium and M. intracellulare), M. kansasii, M. xenopi, M. marinum, M. ulcerans, or M. fortuitum complex (M. fortuitum and M. chelonei) infections. In some embodiments, the infection is a P. aeruginosa infection, while in other embodiments, the infection is a non-tuberculous mycobacterial infection. The pulmonary infection may or may not be associated with cystic fibrosis.

Thus, in an embodiment, the pulmonary condition is both cystic fibrosis and a pulmonary infection such as P. aeruginosa. In other embodiments, the pulmonary conditions are bronchiectasis. The bronchiectasis may or may not be associated with cystic fibrosis.

The present method provides advantageous levels of Lopinavir and Ritonavir at the site of the pulmonary disorder, while limiting systemic exposure to the drug, and also provides a sustained benefit to the subject for surprisingly extended periods of time. While not being bound by any particular theory, it is believed that administration of liposomal Lopinavir-Ritonavir in accordance the with methods described herein results a “depot” effect in the lungs of the subject. Specifically, it is believed that the liposome particles are small enough and contain an appropriate lipid formulation to penetrate and diffuse through pneumonia fluid and into the bacterial biofilm. The liposomes shield the entrapped cationic Lopinavir and Ritonavir in neutral liposomes to minimize electrostatic interaction with the negatively charged sputum/biofilm, which would otherwise reduce its bioavailability. In addition, there are Streptococcus pneumoniae derived virulence factors (rhamnolipids) (Davey et al. 2003), which release Lopinavir and Ritonavir from the liposomes. Therefore, it is hypothesized that relatively high concentrations of drug can be delivered locally to the bacterial macro-colony environment.

Additionally, it is believed that inhalation of liposomal Lopinavir and Ritonavir leads to a dose dependent recruitment of macrophages as an adaptive response to inhalation of drug/lipid formulation. The presence of alveolar macrophages (which have been shown to be functionally normal in liposomal Lopinavir-Ritonavir treated rats) may be particularly beneficial in ARDS and Pneumonia patients. ARDS and Pneumonia patients are known to have reduced number of macrophages in their lungs and possibly with poor functionality, which may contribute to the chronicity of Streptococcus pneumoniae lung infection.

The dose dependent recruitment of macrophages may also contribute to the sustained effects observed using the methods of the present invention. Specifically, the macrophages in the lung may take up liposomal Lopinavir-Ritonavir, and then remain in the lung for a period of time, followed by release of the liposomal Lopinavir-Ritonavir by the macrophages. The present method thus provides, in some embodiments, advantageous levels of Lopinavir and Ritonavir in the blood and in the sputum. For example, the methods provide relatively low systemic exposure to Lopinavir and Ritonavir, while providing high, sustained levels of Lopinavir and Ritonavir at the site of the pulmonary condition.

When the pulmonary disorder includes a pulmonary infection, the present invention also provides a reduction in the colony forming units of the bacteria in the lung for a sustained period of time. For example, the CFU's are reduced compared to a baseline value. In some embodiments, the patient has a reduction in log10 CFU of the bacterial infection in the lungs of at least about 0.5 for at least 15 days after the administration period ends. In other embodiments, the reduction in the log10 CFU is at least by 1.0, 1.5, 2.0 or 2.5. SARS CoV2 infections, in particular, can form large colonies with several micrometres in diameter particularly in patients with ARDS and Pneumonia. In some embodiments, the viral plaques CFU's are reduced as described above in a mucoid strain of a SARS CoV2 infection.

In another embodiment the present invention, the patient experiences an improvement in lung function for at least 15 days after the administration period ends. For example, the patient may experience an increase in the forced expiratory volume in one second (FEV1), an increase in blood oxygen saturation, or both. In some embodiments, the patient has an FEV1 that is increased by at least 5% or at least 10% over the FEV1 prior to the treatment cycle. In other embodiments, FEV1 is increased by 5 to 50%, 5 to 25%, or 5 to 20%. In other embodiments, FEV1 is increased by 5 to 15% or 5 to 10%. In other embodiments, FEV1 is increased by 10 to 50%, 10 to 40%, 10 to 30% or 10 20%. FEV1 is frequently measured in mL. Accordingly, in some embodiments, FEV1 is increased by at least 25 mL when compared to FEV1 prior to the treatment. In some embodiments, FEV1 is increased by 25 to 500 mL, 25 to 400, 25 to 300 or 25 to mL. In other embodiments, FEV1 is increased by 50 to 500 mL, 50 to 400 mL, 50 to 300 mL, 50 to 200 mL or 50 to 100 mL.

In another embodiment the present invention, blood oxygen saturation is increased in the subject compared to the blood oxygen saturation levels prior to the administration. In some embodiments, blood oxygen saturation is increased by at least 1% or by at least 2% for at least 2 days after the administration period. In other embodiments, the blood oxygen saturation levels are increased by about 1 to 50%, 1 to 25%, 1 to 20%, 1 to 15%, 1 to 10% or 1 to 5%.

In another embodiment the present invention, the blood oxygen saturation levels are increased by about 2 to 10% or 2 to 5%.

The aforementioned sustained periods of time may be at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 days after the administration period. In other embodiments, the sustained period of time is at least 28, 35, 42, 48 or 56 days after the administration period. In other embodiments, sustained period of 15 to 75 days, 15 to 35 days, or 20 to 35 days. In other embodiments, the sustained period of time is 20 to 30 days, 25 to 35 days or 25 to 30 days.

In another embodiment the present invention, the sustained period of time is about 25, about 26, about 27, about 28, about 29 or about 30 days, or about 28 days, or at least 29 days. In other embodiments, the sustained period of time during is 25 to 75 days, 35 to 75 days, or 45 to 75 days. In other embodiments, the sustained period is 50 to 75 days, 50 to 70 days, 50 to 65 days or 50 to 60 days. In other embodiments, the sustain period is about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59 or about 60 days, while in other embodiments, the sustained period is about 56 days.

In another embodiment the present invention, the aforementioned methods advantageously provide a reduced incidence of pulmonary exacerbations in the patient. The method also advantageously increases the length of time to pulmonary exacerbation. For example, in some embodiments, the length of time to pulmonary exacerbation is at least about 20 days. In other embodiments, the length of time is 20 to 100 days. In other embodiments, the length of time is 25 to 100 days, 30 to 100 days, 35 to 100 days or 40 to 100 days. In other embodiments, the length of time is 25 to 75 days, 30 to 75 days, 35 to 75 days or 40 to 75 days. In other embodiments, the length of time is 30 to 60 days.
In another embodiment the present invention, the incidence of rescue treatment is reduced. In other embodiments, the length of time to rescue treatment is reduced, for example when the patient has a pulmonary infection, the time to anti-infective rescue treatment is reduced. In some embodiments, the length of time is 20 to 100 days. In other embodiments, the length of time is 25 to 100 days, 30 to 100 days, 35 to 100 days or 40 to 100 days. In other embodiments, the length of time is 25 to 75 days, 30 to 75 days, 35 to 75 days or 40 to 75 days. In other embodiments, the length of time is 30 to 60 days.

In another embodiment the present invention, the liposomal Lopinavir-Ritonavir formulation used in the aforementioned methods comprises Lopinavir-Ritonavir and any of the lipids described above. In some embodiments, the liposomal Lopinavir-Ritonavir formulation comprises a phospholipid and a sterol, such as DPPC and cholesterol. In other embodiments, the liposomal Lopinavir-Ritonavir formulation comprises DPPC and cholesterol in about a 2 to 1 ratio by weight. In some embodiments, the liposomal Lopinavir-Ritonavir formulation has a lipid to drug ratio of about 0.5 to about 1.0, about 0.5 to 0.7, or about 0.6 by weight. In other embodiments, the liposomal Lopinavir-Ritonavir formulation has a lipid to drug ratio of about 0.3 to about 1.0 by weight, while in other embodiments, the lipid to drug ratio is about 0.5 to 0.7 by weight, or about 0.65 by weight. The liposomes in the formulation may have a amend diameter of 100 to 1000 nm, 100 to 500 nm, 200 to 500 nm, or about 300 nm.

In another embodiment the present invention, the total concentration of Lopinavir-Ritonavir in the liposomal Lopinavir-Ritonavir formulation is about 20 to 1000 mg/mL, 20 to 900 mg/mL, 30 to 90 mg/mL, 30 to 80 mg/mL, or 40 to 80 mg/mL. In other embodiments, the concentration is about 30, 40, 50, 60, 70, 80 or 90 mg/mL.

In another embodiment the present invention, the aforementioned method comprises: administering to the patient an effective dose of a nebulized liposomal Lopinavir and Ritonavir formulation for at least one treatment cycle, wherein: the treatment cycle comprises an administration period of about 15 days, followed by an off period of about 28 days based on the clinical functioning of the patient.
The effective dose comprises about 230 mg to about 330 mg Lopinavir and 220 to 340 of Ritonavir daily during the administration period; and the liposomal Lopinavir-Ritonavir formulation comprises DPPC and cholesterol in about a 2:1 ratio, and a lipid to Lopinavir-Ritonavir ratio of about 0.5 to about 0.7.
In another embodiment the present invention, the method comprises: administering to the patient an effective dose of a nebulized liposomal Lopinavir and Ritonavir formulation for at least one treatment cycle, wherein: the treatment cycle comprises an administration period of about 15 days, followed by an off period of about 5 days; the effective dose comprises about 230 mg to about 330 mg Lopinavir and 220 to 340 of Ritonavir daily during the administration period; and the liposomal Lopinavir-Ritonavir formulation comprises DPPC and cholesterol in about a 2:1 ratio, and a lipid to Lopinavir-Ritonavir ratio of about 0.5 to about 0.7.

In another embodiment the present invention relates to a method of providing a sustained treatment effect in a subject comprising: administering to the patient an effective dose of a nebulized liposomal Lopinavir and Ritonavir formulation for at least one treatment cycle, wherein: the treatment cycle comprises an administration period of 15 to 75 days, followed by an off period of 15 to 75 days; and the effective dose comprises 10 to 2500 mg of Lopinavir and 5 to 1500 of Ritonavir daily during the administration period.

In another embodiment, the present invention relates to a method of improving oxygen saturation levels in a patient with a pulmonary condition comprising: administering to the patient an effective dose of a nebulized liposomal Lopinavir and Ritonavir formulation for at least one treatment cycle, wherein: the treatment cycle comprises an administration period of 15 to 75 days, followed by an off period of 15 to 75 days; and the effective dose comprises 10 to 2500 mg of Lopinavir and 5 to 1500 of Ritonavir daily during the administration period.

In another embodiment, the present invention relates to a method of improving FEV1 in a patient with a pulmonary condition comprising: administering to the patient an effective dose of a nebulized liposomal Lopinavir and Ritonavir formulation for at least one treatment cycle, wherein: the treatment cycle comprises an administration period of 15 to 75 days, followed by an off period of 15 to 75 days; and the effective dose comprises 100 to 2500 mg of Lopinavir-Ritonavir daily during the administration period.

In another embodiment, the present invention relates to a method of reducing bacterial density in the lung or sputum of a patient with a bacterial pulmonary infection comprising: administering to the patient an effective dose of a nebulized liposomal Lopinavir and Ritonavir formulation for at least one treatment cycle, wherein: the treatment cycle comprises an administration period of 15 to 75 days, followed by an off period of 15 to 75 days; and the effective dose comprises 10 to 2500 mg of Lopinavir and 5 to 1500 of Ritonavir daily during the administration period, and wherein the bacterial density remains reduced for at least 15 days after the last day of the administration.

EXEMPLIFICATION

Introduction to Materials and Methods
Lipid based or liposomal Lopinavir and Ritonavir, formulations for inhalation are sustained-release formulations of Lopinavir and Ritonavir encapsulated inside nanoscale liposomal carriers designed for administration via inhalation. Sustained-release targeting of high concentrations of Lopinavir and Ritonavir in the lungs and biofilm penetration properties of these formulations have several advantages over inhalation of the “free” antibiotic, e.g., inhaled Lopinavir and Ritonavir. Lopinavir and Ritonavir can be encapsulated in liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and cholesterol, at a targeted lipid-to-drug ratio of about 0.6-0.7:1 (w/w). An example of a 70 mg/mL liposomal Lopinavir and Ritonavir formulation useful in the aforementioned methods is presented below:

Table - Lopinavir and Ritonavir formulation useful in the aforementioned methods
Component Concentration

Lopinavir ~100 mg/mL
Ritonavir ~25 mg/mL
Dipalmitoylphosphatidylcholine (DPPC) ~30 mg/mL
Cholesterol ~15 mg/mL
1.5% NaCl QS

1Added to the formulation as Lopinavir sulfate, USP.

These formulations have several advantages in treating pulmonary conditions, for example, pneumonia subjects with infection caused by S.Pneumonia, including:
1. The ability to attain a prolonged antibiotic effect of Lopinavir and Ritonavir in the lung by achieving high concentrations and a prolonged half life due to sustained release.
2. The ability to target and increase the effective concentration of Lopinavir and Ritonavir in the lung with low systemic levels of the medications.
3. The potential to better target bacteria growing in a biofilm as a result of unique properties of lipid based or liposomal anitibiotics.
4. Additional release of the drug at the site of infection in the lungs of pneumonia/ARDS patients, due to targeted action of secreted phospholipase C and rhamnolipids from bacteria and/or phospholipase A2 or defensins from activated polymorphonuclear leukocytes
5. The increase in both the half-life, and the area under the concentration curve (AUC) of lipid based or liposomal Lopinavir and Ritonavir, along with biofilm penetration should allow for less frequent administration, enhanced bactericidal activity and reduced potential for selection of resistant organisms.
Preclinical pharmacokinetics have demonstrated that the AUC (0-48 hr) of Lopinavir and Ritonavir in the lungs of rats that received a 60 mg/kg dose aerosol of Liposomal Lopinavir and Ritonavir was ten-fold higher than the AUC of free Lopinavir and Ritonavir in the lungs of rats that received an equal dose of free Lopinavir and Ritonavir by inhalation. Generally, 10% of the administered antiviral is deposited in the lungs for rats. Conversely, the AUC of drug in the liver of rats that received an equal dose of free Lopinavir and Ritonavir was significantly higher than the liver AUC of rats that received aerosols of Liposomal Lopinavir and Ritonavir.

Additionally, data from 30-day inhalation toxicology studies in rats and dogs suggest that there will be no safety pharmacology issues with inhaled Liposomal Lopinavir and Ritonavir. In 14 days rat model studies of SARS CoV2 infection, it was noted that of Liposomal Lopinavir (100 mg/ml) and Ritonavir (50 mg/mL) administered every other day for 14 days (Q2D×7), which effectively delivered half the cumulative dose of antibiotic than the other groups, was as effective as of Liposomal Lopinavir (100 mg/ml) and Ritonavir (50 mg/mL) given once per day. With 28 day dosing in this model, there were equivalent reductions in CFUs in animals receiving Liposomal Lopinavir (100 mg/ml) and Ritonavir (50 mg/mL) dosed daily at or dosed every other day at Lopinavir (100 mg/ml) and Ritonavir ( 100 mg/mL). Liposomal Lopinavir (100 mg/ml) and Ritonavir (50 mg/mL) administered at once a day for 14 days was effective for 28 days, which suggests a higher AUC and possibly a prolonged post-antibiotic effect with Liposomal Lopinavir (40 mg/ml) and Ritonavir ( 20 mg/ml). dosed once per day.

The administration of Liposomal Lopinavir and Ritonavir via inhalation in the animal model resulted in increased lung (AUC) above the MIC of the bacteria, and demonstrated sustained therapeutic effect, with a reduced frequency, and duration of dosing as compared to free Lopinavir and Ritonavir . Importantly, the preclinical data for Liposomal Lopinavir and Ritonavir appear supportive of the hypothesis that this specific formulation may be advantageous over other inhalation products that are hindered by a rapid clearance from lung tissue, necessitating frequent dosing (Geller, Pitlick et al. 2002), which poses a burden for patients and might limit patient compliance.

Additionally, clinical experience demonstrated that nebulized Liposomal Lopinavir (100 mg/ml) and Ritonavir (50 mg/mL) administered once per day for 14 days is well tolerated, and elicits a clinically relevant effect on pulmonary function and decrease in S.Pneumonea density in Pneumonia patients. Also, evaluation of the PK data indicates the systemic exposure to Liposomal Lopinavir (100 mg/ml) and Ritonavir (50 mg/mL), even at the, is very low. By either Cmax or AUC or mg of the antibiotics which is recovered in the urine, the observed systemic exposure to Lopinavir and Ritonavir , associated with Liposomal Lopinavir and Ritonavir , given by inhalation is approximately 1/6th to 1/4th the exposure seen with oral and free Lopinavir and Ritonavir and is less than 1/200 compared to normal parenteral doses of Lopinavir and Ritonavir . The data further indicate high levels of Lopinavir and Ritonavir are achieved in the sputum. Median AUC values for sputum were 275 and 1230 fold greater than the median AUC values for serum on day 1 and day 14 respectively.

Inhaled liposomal Lopinavir and Ritonavir maintains prolonged targeted lung exposures and enhance the uptake of drug to the site of infection.

Several preclinical studies were conducted with the 20 and 50 mg/mL formulations. Anti-pseudomonas activity of Liposomal Lopinavir and Ritonavir in in vitro and in vivo models was demonstrated. Additionally, studies confirmed that virulence factors secreted by S.Pnuemonea facilitate the further release of Lopinavir and Ritonavir from the liposomes, and characterized the deposition and sustained release of Lopinavir and Ritonavir in the lungs of rats, and rabbits. The safety of a 30-day administration of Liposomal Lopinavir and Ritonavir in two species was also established.

Nonclinical pharmacokinetics have demonstrated that the AUC (0-48 hr) of Lopinavir and Ritonavir in the lungs of rats that received dose of Liposomal Lopinavir (100 mg/ml) and Ritonavir (50 mg/mL) via nebulization, was five-fold higher than the AUC of Lopinavir and Ritonavir in the lungs of rats that received an equal dose of free Lopinavir and Ritonavir by inhalation. High levels of Lopinavir and Ritonavir were sustained in the lung (>320 µg/mL through 150 hr), suggesting a depot effect. Conversely, the AUC of drug in the liver of rats that received an equal dose of free Lopinavir and Ritonavir was significantly higher than the AUC of rats that received aerosols of Liposomal Lopinavir and Ritonavir. There were no significant differences in the AUC of the antibiotics in the serum and urine of the animals; serum levels were undetectable after 24 hr.

This profile supports the intended sustained release and depot effect of Lopinavir and Ritonavir in the lung following administration of nebulized Liposomal Lopinavir and Ritonavir, potentially representing an enhanced efficacy profile.

Additionally, toxicokinetic data from 30-day inhalation GLP toxicology studies in rats and dogs showed that there is a 15 fold increase in lung deposition of Lopinavir and Ritonavir dogs as compared to the free Lopinavir and Ritonavir treated group, with comparable plasma and urine levels, indicating high lung concentrations with low systemic exposure.

The pharmacodynamic effect of Liposomal Lopinavir and Ritonavir was evaluated in vivo in a rat model of chronic pulmonary infection with SARS CoV2. In a 14 days SARS CoV2 infection model, of Liposomal Lopinavir (100 mg/ml) and Ritonavir (50 mg/mL) was administered every other day for 14 days (Q2D×7). This regimen was significantly effective Liposomal Lopinavir (100 mg/ml) and Ritonavir (50 mg/mL) (given once per day for 14 days). When dosing was extended to 28 days, there were equivalent reductions in CFUs for animals receiving Liposomal Lopinavir (100 mg/ml) and Ritonavir (50 mg/mL) or dosed every other day at Lopinavir (200 mg/ml) and Ritonavir (100 mg/mL). This indicated a higher AUC and a prolonged post-antibiotic effect with Liposomal Lopinavir (20 mg/ml) and Ritonavir (100 mg/mL) dosed once per day. The preclinical pharmacodynamic data were thus consistent with a sustained antimicrobial benefit enhanced by the site-specific delivery of drug to the lungs via inhalation.

Thus, administration of Liposomal Lopinavir and Ritonavir via inhalation resulted in increased lung concentrations (AUC) several fold above the MIC of the bacteria, with the potential to provide a sustained therapeutic effect with a reduced frequency and duration of dosing.
,CLAIMS:5. CLAIMS
I/We Claim
1. A stable sustained release liposomal inhalation suspension formulation for pulmonary delivery comprising liposomal particles characterized by a synergistic combination of:
Lopinavir and Ritonavir;
lipid comprising mixture of anionic lipid and saturated lipid;
cholesterol; and
1.5% NaCl -QS.

2. The stable prolonged release liposomal inhalation suspension formulation for pulmonary delivery as claimed in claim 1 wherein the zeta potential of the liposomal dispersion is -40 to -10 mV, %. Lopinavir and Ritonavir entrapment is more than 96%.

3. The stable prolonged release liposomal inhalation suspension formulation for pulmonary delivery as claimed in claim 1 wherein saturated lipid can be selected from the group consisting of Hydrogenated soy phosphatidylcholine (HSPC), Dipalmitoylphosphatidylcholine (DPPC), Distearyloylphosphatidylcholine, (DSPC) and Diarachidoylphosphatidylcholine (DAPC).

4. The stable prolonged release liposomal inhalation suspension formulation for pulmonary delivery as claimed in claim 1 wherein the liposomal inhalation suspension formulation is to be delivered through nebulization device.

5. The method for preparing the stable liposomal inhalation suspension formulation in an effective amount of a systemically active formulation comprising a synergistic combination of Lopinavir and Ritonavir for pulmonary delivery comprises of:
dissolving Lopinavir and Ritonavir, lipid mixture, cholesterol in solvent,
evaporating the above mixture in rotary vacuum evaporator to get lipid film,
hydrating the lipid film of step by distilled water,
sonicating the hydrated film of step,
centrifuging the sonicated film of step and
decanting liposomal suspension

6. DATE AND SIGNATURE

Dated this 04th day of June 2021
Signature

(Mr. Srinivas Maddipati)
IN/PA 3124
Agent for Applicant