Claims:Claims
We Claim:
1. A pharmaceutical composition of biocompatible nanoparticle or nanoparticle aggregate comprising a) a core comprising ferromagnetic material; b) a double layer of biocompatible shell surrounding the core; and c) a targeting moiety.
2. The pharmaceutical composition of claim 1 wherein the nanoparticle or nanoparticle aggregate is coated with positively charged amino acid and wherein the amino acid is selected from the group consisting of lysine, arginine and histidine .
3. The pharmaceutical composition of claim 1 wherein the biocompatible shell of the nanoparticle has an outer diameter of 10 - 65nm, preferably 14 - 50nm and more preferably 20nm.
4. The pharmaceutical composition of claim 1 wherein the biocompatible shell of the nanoparticle is made of a material selected from the group consisting of PEG, poly(ethylene oxide) oligomer or polymer, polyecaprolactone, polylactide, polyglycolide, and copolymers thereof, polyoxypropylene, polyoxyethylene-polyoxypropylene diblock, polyoxyethylene-polyoxypropylene-polyoxyethylene triblock, and any mixture thereof, arranged into two layers.
5. The pharmaceutical composition of claim 1 wherein the ferromagnetic core material is selected from the group consisting of iron, nickel, cobalt, gadolinium, samarium, neodymium, boron, aluminium or a mixture thereof.
6. The pharmaceutical composition of claim 1 wherein the ferromagnetic core material is an oxide, an hydroxide or a metal.
7. The pharmaceutical composition of claim 1 wherein the targeting moiety of nanoparticle comprises a material selected from the group consisting of negatively charged amino acid, such as aspartic acid, or glutamic acid, oligomers, polymers, and combination thereof.
8. The pharmaceutical composition of claims 1 to 7, further comprising a targeting agent attached via non-covalent or covalent interactions to the polymeric material of the nanoparticle.
9. A pharmaceutical composition comprising a biocompatible nanoparticle or nanoparticle aggregate, and targeting agent in the presence of external magnetic field source wherein the nanoparticle is retained in the lungs upon use of an external magnet.
10. The pharmaceutical composition of claim 9 wherein the external magnetic field source is uniform and unidirectional and is an electromagnet or Magnetic Resonance Imaging (MRI) equipment.
11. A delivery vehicle to target drug to lungs comprising a biocompatible nanoparticle or nanoparticle aggregate comprising a) a core comprising ferromagnetic material; b) a double layer of biocompatible shell surrounding the core; and c) a targeting moiety.
12. A method to target drug to lungs comprising a biocompatible nanoparticle or nanoparticle aggregate comprising a) a core comprising ferromagnetic material; b) a double layer of biocompatible shell surrounding the core; and c) a targeting moiety.
13. A method of use of a biocompatible nanoparticle or nanoparticle aggregate comprising a) a core comprising ferromagnetic material; b) a double layer of biocompatible shell surrounding the core; and c) a targeting moiety for delivering drug to lungs in lung-related conditions
14. The method of use of a biocompatible nanoparticle or nanoparticle aggregate as claimed in claim 13, wherein the lung-related condition is selected from the group consisting of lung cancer, lung infection, lung edema, asthma, COPD, pneumonia, cystic fibrosis and tuberculosis.
15. The method of use of a biocompatible nanoparticle or nanoparticle aggregate as claimed in claim 13, wherein the drug is selected from the group consisting of cytotoxic drugs, antibodies, protein drugs, nucleic acid based drugs, anti-infectictives, steroids and antibiotics.
16. The method of use of a biocompatible nanoparticle or nanoparticle aggregate as claimed in claim 13, wherein the drug is selected from the group consisting of irinotecan, cisplatin, methotrexate, montelukast, theophylline, aclidinium, roflumilast, isoniazid, rifampicin, amoxicillin and azithromycin.
, Description:Magnetic nanoparticle formulations for targeted delivery of drugs to lungs for treatment of pulmonary diseases
Related Application
This application is being filed as a complete application with the Indian Patent Office.

Field of Invention
This application provides a novel nanoparticle that can selectively deliver therapeutic compounds to the lungs. The particle also contains magnetic moieties so that the drug can be retained longer in the lungs by use of an external magnet. This nanoparticle can be used for treatment of lung cancer and other diseases such as asthma, COPD, pneumonia, cystic fibrosis and TB that require delivery of therapeutics directly to lung.

Background of invention
Direct delivery of therapeutics to lungs has been a challenge. In different disease conditions involving lungs, different problems have been faced by discovery scientists for developing therapeutics that can specifically target lungs.

The major drawback of cytotoxic drugs is that since they do not differentiate between cancer cells and normal healthy cells thus killing all cells that come in contact with them. As a result of which there is collateral damage to several tissues along with the tumour resulting in adverse side effects. Typical side effects with Irinotecan, for example, are diarrhoea and myelosuppression.

Cytotoxic drugs such as Irinotecan are first line therapy for most cancers since they are effective across a variety of cancers. If they are ineffective then second line or additive therapy is more personalized treatment with mutation specific monoclonal antibodies. The other benefit of cytotoxic drugs is that they are cost-effective relative to the tailored monoclonal antibodies by a significant factor of tenfold or so.

One way to make these drugs more effective and reduce their side effects is to target them largely to the organ that is afflicted with cancer while sparing other organs and tissues. There have been attempts to do this using ligands attached to drugs that could bind to counter receptors on the tissues of interest [See Bioconjugate Chem. 2010, 21(5), 979–987 ]. Another method adopted is to link drugs to monoclonal antibodies specific for antigens expressed on tumour cells [British Journal of Cancer, 2016, 114, 362–367].

Lung is the most vascularised organ in the body with capillaries extensively formed for the exchange of oxygen. Almost 70 to 80 of the blood vessels in the body are present in the lung. Because exchange of gases is the major function of the lung, the endothelial lining of the lung capillaries is the thinnest of all organs to allow for crossing over of the gasses.
The endothelium is lined richly with negatively charged heparin sulfate proteoglycans. One of the functions they serve binding of positively charged growth factors, matrix protein and certain enzymes. Once a molecule is bound to to the surface heparin sulfate proteoglycan, it usually undergoes transcytosis and is transferred across the endothelium as an intact molecule. Keeping this in view, Applicants have devised a new delivery vehicle that targets drug delivery to the lung.

As an example, in the present invention, Applicants have exploited the densely populated capillary endothelium in the lung to deliver Irinotecan containing nanoparticles whose surface is decorated with positive charge.

Brief Description of Figures
Figure 1. Transmission electron micrograph of a) Iron Oxide (IO) magnetic nanoparticles; b) PEGylated Irinotecan loaded IO nanoparticles; c) double PEGylated Irinotecan loaded nanoparticles and their corresponding size distribution (d,e,f). According to this data, the size of the nanoparticles even after incorporation of the second layer of PEG increased only by 2-3 nm in average, and diameter was in the range of 10-16 nm.
Figure 2. Magnetic properties of NPs: a) using external magnetic field; b) EPR data confirms magnetic properties of VT-28.
Figure 3. FTIR spectra of F3O4 nanoparticles - confirms structure of F3O4
Figure 4. FTIR spectra of PEG-coated F3O4 nanoparticles – confirms PEGylation
Figure 5. Figure 10. Solutions of VT-287 in PBS buffer: A-3.125 mg in 0.5 ml of lysine solution injected to 1 ml PBS buffer - 0.25 mg/ml; B-3.125 mg in 0.5 ml of lysine solution injected to 2.5 ml PBS buffer; C- 6.25mg in 0.5 ml of lysine solution injected to 1 ml PBS buffer - 0.5 mg/ml; D- 6.25 mg in 0.5 ml of lysine solution injected to 2.5 ml PBS buffer.
Figure 6. Flocculation of VT-287 in presence of Heparin
Figure 7. Stability study of VT-287 and Irinotecan hydrochloride in PBS buffer
Figure 8. Stability study of VT-287 and Irinotecan hydrochloride in Human plasma
Figure 9. Permeability of Irinotecan from VT-287 in HULEC-5a cell line. Intracellular concentration of Irinotecan, µM was measured (n=3 for each condition and time point) and the mean is plotted. Error bars are S.D.
Figure 10. a) Tissue distribution of Irinotecan after a single dose administration, in Balb/c mice, Irinotecan concentration in plasma and tissue samples are reported; b) Comparative lung concentration of Irinotecan from VT-287 vs. free Irinotecan after a single dose administration in Balb/c mice.
Figure 11. Concentration of SN-38 in lung tissue in a single dose PK/TD study carried out in Balb/c mice (n=3). A comparison of the data from administration of naked Irinotecan and VT-287 (2 studies) at 5mg/kg b.w. equivalent is presented.
Figure 12. Concentration of SN-38 in lung tissue in New Zealand rabbits. A comparison of the SN-38 concentration in lung tissue measured at two time points, upon administration of a single dose of Irinotecan or VT-287 (both at 0.5mg/kg b.w. equivalent of Irinotecan) is plotted (n=2). Error bars represent S.D.
Figure 13. Lung concentrations (Irinotecan) in male New Zealand Rabbits, ng/g.
Figure 14. Lung concentrations (SN-38) in male New Zealand Rabbits, ng/g.
Figure 15. Effect of VT-287 and Irinotecan on cell proliferation in lung cancer cell line A549 (NSCLC).
Figure 16. Effect of VT-287 and Irinotecan on cell proliferation in lung cancer cell line NCI-H1299 (NSCLC).
Figure 17. Effect of VT-287 and Irinotecan on cell proliferation in NCI-H187cell line.
Figure 18. Effect of VT-287 and Irinotecan on cell proliferation in NCI-H69cell line.

Detailed Description
The present invention delivers positive charge coated nanoparticles containing active pharmaceutical ingredient, Irinotecan as an example, to the densely populated capillary endothelium in the lung.
The present invention provides a nanoparticle or nanoparticle aggregate comprising a) a core comprising ferromagnetic material; b) a double layer of biocompatible shell surrounding the core; and c) a targeting moiety.
In one embodiment, the surface of the nanoparticle is coated with positively charged amino acids such as lysine bound to the surface by ionic bonds. The positive charge on surface allows the nanoparticle to engage with the heparin sulfate moiety on the endothelium.
In another embodiment, the invention provides momentary interaction allowing transcytosis of the nanoparticle across the endothelium into the lung space/cells. Since lungs have the most amount of capillaries this mechanism allows the nanoparticle to be delivered predominantly to the lungs and not to other organs.
In another embodiment, the nanoparicle is biocompatible.
In another embodiment, the nanoparticle can be used singly or as an aggregate of nanoparticles. The nanoparticle aggregate can be activated and used similar to that of single nanoparticle.
The nanoparticle is essentially spherical, circular or round in shape and the outer diameter of the nanoparticle is in the range of 6 nm to 50nm.
The biocompatible shell of the nanoparticle has an outer diameter of 10 - 65nm, preferably 14 - 50nm and more preferably 20nm.
The biocompatible shell is made of a material selected from the group consisting of PEG, poly(ethylene oxide) oligomer or polymer, polycaprolactone, polylactide, polyglycolide, and copolymers thereof, polyoxypropylene, polyoxyethylene-polyoxypropylene diblock, polyoxyethylene-polyoxypropylene-polyoxyethylene triblock, and any mixture thereof, arranged into two layers.

The ferromagnetic core material is selected from the group consisting of iron, nickel, cobalt, gadolinium, samarium, neodymium, boron, aluminium or a mixture thereof.
The ferromagnetic core material is an oxide, an hydroxide or a metal.
In a preferred embodiment the nanoparticle comprises a targeting moiety attached via non-covalent or covalent interactions to the polymeric material of the biocompatible shell of the nanoparticle.

The targeting moiety of nanoparticle comprises a material selected from the group consisting of negatively charged amino acid, such as aspartic acid or glutamic acid, oligomers, or polymers and combination thereof.

The nanoparticle of the invention is used for delivery of any therapeutic that needs to target the lung. Thus the nanoparticle of the invention intends to deliver therapeutic to lungs in conditions including but not limited to cancer, specially lung cancer, lung infection, cystic fibrosis, lung edema, asthma, pneumonia and COPD. The therapeutic or drug or the compound of interest, for example a cancer chemotherapeutic is incorporated into the nanoparticle via covalent or non-covalent interaction.
Since the nanoparticle serves as a delivery platform, it can be loaded with other drugs that are required to act primarily in the lung. Thus the same nanoparticle formulation can be used to deliver any other cancer therapeutic including but not limited to cytotoxic drugs, antibodies, protein and nucleic acid based treatments. Additionally the nanoparticle of the invention is used to treat other lung diseases such as but not limited to asthma, COPD, pneumonia, cystic fibrosis and tuberculosis, more effectively.

VT-287, the nanoparticle presented as an example in the current specification, is composed of a self-contained nanoparticle core, loaded with the drug Irinotecan. This allows for the nanoparticle core of VT-287 to serve as a delivery platform to deliver drugs other than Irinotecan to the lung. It can be loaded with cytotoxic drugs (such as Cisplatin, Methotrexate etc) to treat lung cancer; drugs for asthma and COPD (Montelukast, Theophylline, Aclidinium, Roflumilast); or anti-infectives to treat lung infections such as TB and pneumonia (Isoniazid, Rifampicin, Amoxicillin, Azithromycin). Drug delivery using the lung-targeted VT287 platform will ensure increased efficacy at the site of the disease, as we have demonstrated in the case of Irinotecan. In addition, the impact of long-term treatment with drugs such as steroids and antibiotics that have a broad systemic side-effect profile will be significantly reduced.
Magnetic field source is generally used for triggering or generating therapeutic activity. In this context, the nanoparticle of the invention along with the targeting agent is used in combination with an external magnetic field in order to be able to retain the said nanoparticle in the lungs upon use of an external magnet. The magnetic field source is selected from electromagnet or magnetic resonance imaging (MRI) equipment.
The invention also encompasses a process of drug loading, involving attachment of the selected drug to the biocompatible polymeric shell of the nanoparticle via non-covalent or covalent interactions and includes double PEGylation/ double drug loading process.

The Examples provided herein are illustrative and are for better understanding of the invention and should not be considered as a limitation in any way.
Example 1.
Synthesis and Drug loading of PEG-double layer nanoparticles VT-287 (Schematic 1)
1. Synthesis of magnetic nanoparticles Fe3O4: Magnetic nanoparticles (NPs) were prepared using modified co-precipitation method according to published procedures [See British Journal of Cancer, 2016, 114, 362–367; Journal of Science and Health at The University of Alabama, 2010, 7, 16-18].
0.1M Ferric chloride hexahydrate (FeCl3 .6H2O) (250 mg in 9.2 ml of water) and 0.05M Ferrous chloride tetrahydrate (FeCl2 .4H2O) (99.4 mg in 9.2 ml of water) were dissolved in deoxygenated nano-pure water at 50 ?C under nitrogen, using magnetic stirrer (the reaction performed in 2-neck 250 ml RB, equipped with magnetic bar, nitrogen pipe-line with needle placed inside solution, and nitrogen balloon on the top). After all salts are dissolved, sodium hydroxide solution (NaOH, 2.5 M, prepared as 1.5 g dissolved in 15ml) was added drop-wise into the reaction mixture with vigorous stirring, until the pH value reached 9 (initially added 1.5 ml, checked pH, then added required amount, usually approx. another 0.5 ml, checking pH often). The solution was stirred for 30 min at 50 ?C under nitrogen, the magnetic nanoparticles were collected by magnetic field separation (using small magnet placed close to an RB followed by decanting the solution), washed one time with diluted HCl solution (one drop of HCl in 5 ml of water) to reach pH 7, then washed 4-5 times with 20 ml of deionized water. Wet NPs were subjected directly to the next step of PEGylation without drying.The process yields ~160 mg of dry NPs.

Schematic 1. Pictographic representation of nanoparticles composition
The nanoparticles prepared thus were investigated by the Transmission Electron Microscopy (Tecnai G2FEI F12 transmission electron microscope (TEM) at an accelerating voltage of 120 kV), the TEM data showed that nanoparticles are mono-dispersed with size in range of 8?2 nm (Figure 1a, d.)

2. PEGylation of Iron Oxide nanoparticles (Schematics 2, 3):
Fe3O4 NPs (160 mg) were dissolved in PEG-2000 solution (850 mg dissolved in 50 ml of deionized water) under N2 using sonication (10 min) at 40° C. The solution of PEG with Fe3O4 NPs in one-neck 250 ml RB was de-gassed using nitrogen (pass nitrogen flow through needle for 10 min), the RB then placed in ultra-sound bath pre-heated to 40?C, and sonicated for 10 min. The RB was shifted to an oil-bath, equipped with condenser (e.g. Dimroth Condenser), and nitrogen balloon. Temperature was increased to 160° C in oil bath, aiming 90 ° C in reaction mixture. The reaction runs strictly under inert atmosphere till black color solution turned brown (approx. 2.5-3 h). Finally, reaction mixture was allowed to cool down to RT, shifted portion-wise into centrifuge tubes (20 ml), and NPs were collected by centrifugation for at 4000 rpm 10-15 minutes, followed by removal of water by decantation with a magnet placed outside of the tube. The NPs were transferred back into 250 ml RB, 80 ml of pure water was added, and stirred at RT for 10 min under nitrogen atmosphere. The water in the reaction mixture was replaced with pure water and this step was repeated two more times, so as to remove excess free PEG completely from the NPs.

Schematic 2. Synthesis and drug loading of nanoparticles (IO- Iron (II,III) oxide , Fe3O4; PEG- Polyethylene glycol; Ir- Irinotecan hydrochloride; K- lysine)

Schematic 3. The technology chat flow

The magnetic bar was removed from the reaction mixture, water was decanted using external magnet, the slightly wet NPs were shifted to a Petri dish and dried at 40°C under nitrogen for 24 h or lyophilized (at -80°C, overnight).
Magnetic properties of NPs were checked at every step using external magnet (see Figure 2a, b).
TEM study of PEGylated NPs confirmed the size in range of 8-10 nm (Figure 1b, e.). FT-IR (Figures 3, 4) confirmed composition of the nanoparticles and successful PEGylation. The IR spectra of iron oxide Fe3O4 (Figure 3) exhibited string bands in the low frequency region (1000 – 400 cm-1) attributed to the Fe-O bond vibration of Fe3O4. The peak at ~3400 cm-1 is attributed to the stretching vibrations of –OH (corresponds to OH- absorbed by iron oxide NPs). The Figure 4 shows stretch band at 1099 cm-1 and the vibrational band at 1344 cm-1 corresponding to C-O-C ether bonds after PEGylation of iron oxide nanoparticles. The bands around 2900 and 955 cm-1 correspond to –CH2- stretching vibrations and –CH out of plane bending vibrations, respectively (Figure 4). These peaks are strong evidence that PEG covered the nanoparticles surface. Additionally all main peaks in PEG-coated NPs' spectra are shifted in comparison with spectra of NPs before coating, indicating change in environment of the particle after polymer coating.

Example 2.
1. Drug loading in PEGylated Iron Oxide NPs (Schematics 2, 3):
A. Primary drug loading: 6 mg of Irinotecan?HCl trihydrate was dissolved in 3ml of nanopure water; then 3 mg of PEGylated NPs were dispersed in the same solution with magnetic stirring at RT. The solution was stirred for 120h (5 days; measured absorbance using UV spectrophotometer at 0 and 5 days or checking drug loading by LC-MS-MS). After 120h drug loaded NPs were washed 2 times with cold (T= 1-5?C) lysine solution (1mg/ml, 2ml each, 4 times shake sidewise), and a solvent was removed with syringe using magnet to keep NPs aside. Solution of Glycerol (10 ?L of solution: 0.005 mg in 1ml of water, 5% wt.) was added to the reaction mixture and the drug loaded NPs were snap frozen in liquid nitrogen, lyophilized and dried at -60?C for 5 hours (NPs can be storedafter snap freezing at -80?C until ready for lyophilisation).
B. Secondary layer and drug loading (VT-287): 3.5 ml of Irinotecan?HCltrihydratewas dissolved in 3.5 ml of water, and 3 mg of lysine were added to the above solution with magnetic stirring at RT. Then, 10 mg of single layer NPs (protocol A) were added to the above mixture, followed by addition of 900mg of PEG -2000 portion-wise, the mixture was sonicated for 3 minutes (using ultrasound bath, RT), stirred at RT for 20 minutes. Magnetic bar was removed from reaction mixture, NPs were isolated the using external magnet, washed 2 times with cold (T= 1-5?C) lysine solution (1mg/ml, 2ml each, 4 times shake sidewise), solvent was removed with syringe using magnet to keep NPs aside. Further, NPs were snap frozen in liquid nitrogen and lyophilized for 5 hours at -60?C (NPs can be stored after snap freezing at -80?C until ready for lyophilisation).
Magnetic properties of VT-287 were confirmed by applying external magnetic field (Figure 2a) and by Electron Paramagnetic Resonance (EPR) spectroscopy (Figure 2b). The presence of signal in EPR spectra firmly supports paramagnetic properties of sample VT-287.
TEM study of VT-287 shows size in range of 10-16 nm (Figure 1c, f.). According to this data, the size of the nanoparticles after incorporation of the second layer of PEG increased only by 2-3 nm in average. Drug loading percentage was1-4%, measured using LC-MS-MS. Zeta potential was found to be -18.5Mv. Table 1 further provides the characterization data.
Table 1. Characterization of VT-287 formulation
IONPs, diameter (only metal core, TEM) 6 - 8 nm
VT-287 NPs - double layer, diameter (TEM) 10-16 nm
pH of NPs after drug loading 1 ÷ 2
pH of NPs after water wash 5.4
pH of NPs after lysine wash 9.2
pH of lysine solution (0.5mg/0.5 ml) 10.0
Conformation of PEGylation
(-CH2-O-CH2-) 664 cm-1
(FTIR spectroscopy) PEG C-O-C
1352 and 1102 cm-1,
Fe–O 569 cm-1
Fe3O4 NPs -OH 1457.94cm-1 and 1616.08cm-1
PEG-OH 3409.58cm-1
Iron core to PEG ratio (from TEM data) 3 : 1 (mass) 2.6 : 1 (mol)
Iron core to Irinotecan ratio 105 : 1 (mass) 450 : 1 (mol)
PEG-2000 to Irinotecan ratio 33 : 1 (mass) 165 : 1 (mol)
Drug loading (LC-MS/MS) 1-4 %
labs (UV-Spectrometry; auto zero - in water) 277
Zeta potential -18.5
Colour Light brown(when dry),
Dark brown(when dispersed in water)
EPR signal Yes

2. Method to prevent aggregation of nanoparticles VT-287 and keep it in solution
In order to check stability of VT-287 with and without lysine wash, nanoparticles VT-287 were dispersed in water with or without lysine (1 mg/ml) and dried using speedvac for 15 h, then dry nanoparticles VT-287 were re-dispersed in water and pictures were taken at 0 min, 5 min and 10 min after dispersion. Introduction of positively charged amino acid lysine (physical interaction) prior to drying step helps to prevent aggregation (solutions B). At the same time chemical conjugation of lysine molecules to VT-287 nanoparticles did not help to prevent aggregation after drying step. Nanoparticles in solution of lysine-congugate-VT-287 re-dispersed in water after conjugation to lysine, precipitated after 10 min, similarly to nanoparticles without any lysine treatment. The reason for this is that when lysine is covalently conjugated with the nanoparticle, the amino groups in lysine are engaged in the conjugation and as such the nanoparticle is no longer positively charged. Solutions of nanoparticles VT-287 treated with lysine were stable in PBS buffer for more than 2hrs (at different concentrations) and complete precipitation was observed at 6 hrs at room temperature (Figure 5).

3. Heparin induced flocculation of nanoparticles VT-287
This experiment was carried out to show that the lysine coated charged nanoparticles binds to heparin sulfate moiety on the lung blood vessel endothelial surface. This was demonstrated in vitro by incubating VT-287 with heparin solution. To demonstrate this, VT-287 with varying concentrations of heparin and flocculation was incubated and monitored. 3.125 mg of VT-287 (equivalent to Irinotecan 0.25 mg/ml, drug loading 4%) were re-suspended in 0.5 ml of lysine solution (1 mg/ml). This suspension was injected at 37°C inPBS buffer solutions (pre-incubated at 37°C for 30 min before addition of VT-287,10 ml) with heparin at 4X, 3X, 2X, and 1X(to lysine by mol, with average molecular weight 3,500g/mol) and PBS buffer alone as a control. Photographs were taken at different time points upto 2 hrs. The difference in solutions was observed starting from 15 minutes, and interaction of VT-287 with heparin in concentration-dependant manner becomes obvious by 75 minutes (Figure 11). In control solution without heparin VT-287 precipitated completely by 75 min. At the same time heparin-containing solutions were more stable at this time, and even when flocculation occurs they were visually smaller, which indicates interaction of heparin molecules with lysine molecules on the surface of nanoparticles VT-287.

Example 3.
1. Stability study of VT-287 and Irinotecan hydrochloride in PBS buffer and human plasma.
a) In PBS Buffer: Working solutions of test/ reference item(s) in phosphate buffer (pH 7.0) saline were prepared at concentration of 1 µM equivalent of Irinotecan by using 1 mM DMSO stocks for Irinotecan hydrochloride trihydrate (0.677 mg of Irinotecan hydrochloride trihydrate in 1 ml of DMSO) and in lysine buffer for VT-287 at concentrations of 3.33 mM lysine buffer stock (2.26 mg of VT-287 in 1 ml of 0.05% w/v lysine buffer). For Irinotecan hydrochloride trihydrate, 1 µL of 1 mM DMSO stock was spiked to 1 ml of PBS such that the final concentration of DMSO is 0.1%. For VT-287, 10 µL of lysine buffer stock was spiked to 1 ml of PBS. The samples were incubated at 37ºC for 120 minutes, with shaking at 400 rpm. At 0.00, 0.25, 0.5, 1.00, 2.00 and 24 hr, an aliquot of 100 µl sample was removed. To 100 µl of removed sample aliquot in pre-labeled centrifuge tubes 200 µl of acetonitrile containing internal standard (haloperidol) was added, vortexed for 30 seconds, centrifuged for 10min at the speed of 10000 rpm at 10ºC. After centrifugation, ~200 µl sample was transferred into labeled auto sampler vials for LC-MS-MS analysis. The Irinotecan from formulation VT-287 was as stable as free Irinotecan in phosphate buffer upto 24 hrs (Figure 7).
b) In Human plasma: Working solutions of test/ reference item(s) in Human plasma (pH-7.40) were prepared at concentration of 1 µM equivalent ofIrinotecan by using 1 mM DMSO stocks for Irinotecan hydrochloride trihydrate (0.677 mg of Irinotecan hydrochloride trihydrate in 1 ml of DMSO) and in Lysine buffer for VT-287 at concentrations of 3.33 mM lysine buffer stock (2.26 mg of VT-287 in 1 ml of 0.05% w/v lysine buffer). For Irinotecan hydrochloride trihydrate, 1 µl of 1 mM DMSO stock was spiked into 1 ml of human plasma such that the final concentration of DMSO is 0.1%. For VT-287 (active ingredient of Irinotecan in VT-287 was found to be 3 %), 10 µl of lysine buffer stock was spiked into 1 ml of human plasma. The samples were incubated at 37ºC for 24hr, with shaking at 400 rpm. At 0.00, 0.25, 0.5, 1.00, 2.00 and 24 hr, an aliquot of 100 µl sample were removed. To 100 µl of removed sample aliquot in pre-labeled centrifuge tubes added 200 µl of acetonitrile containing internal standard (haloperidol), vortexes for 30 seconds, and then centrifuged for 10min at the speed of 10000 rpm at 10ºC. After centrifugation, ~200 µL sample was transferred into pre-labeled auto sampler vials for LC-MS/MS analysis. The Irinotecan from formulation VT-287 was as stable as free Irinotecan in human plasma up to 24 hrs (Figure 8).

Example 4
In vitro permeability study for VT-287 using HuLEC-5a cell line.
Dosing solution of test item VT-287 at 5 µM (equivalent concentration of Irinotecan) was prepared by spiking 80 µl of 0.5 mM lysine buffer stock (Irinotecan equivalent concentration) to 7920 µL of MCDB 131 media (with serum). Dosing solution of test item Irinotecanhydrochloride trihydrate at 5 µM was prepared by spiking 8 µL of 5 mM DMSO stock to 7992 µl of MCDB 131 media (with serum). Dose formulation analysis was performed on the batch of VT-287 used for the study and Irinotecan loading was determined to be 0.97% and this data was used to calculate Irinotecan-equivalent concentration of VT-287 for the experiments.The dosing solutions of test items were added to HuLEC-5a cell monolayers and incubated for specified incubation period (15 min and 2 hour individually, with or without a magnet under the cell culture plate) at 37 ± 1 ºC with 5 ± 1 % CO2 using a CO2 Incubator. In this permeability study, VT-287 was tested at 5 µM test concentration (equivalent concentration of Irinotecan). After specified incubation period, the spent media was removed from all the wells and monolayers were washed with ice cold PBS (pH-7.40). The cell monolayers were lysed and samples was subjected for extraction. The extracted samples were submitted for LC-MS-MS analysis to measure Irinotecan concentration. A significant concentration of Irinotecan was detected inside the cells when incubated with VT-287 (Figure 9), at 15 minutes as well as after 2 hours. These results indicate that VT-287 is able to permeate the endothelial cells, and deliver physiologically relevant concentration of Irinotecan to the cells. In the presence of a magnet, two-fold more Irinotecan was detected inside the cells at both time points, suggesting an increased penetration of the iron containing formulation in the presence of magnet.

Example 5
Targeted delivery of Irinotecan the lung by VT-287
Adult healthy male BALB/c mice aged 7-10 weeks were used for experimentation after a minimum 3 days of acclimatization. Fed animals were administered with test item a) Irinotecan hydrochloride trihydrate in a recommended vehicle (Sterile water for Injection) by intravenous route with a dose of 5 mg/kg b.w and at dose volume of 10 ml/kg b.w or b) VT-287 in a recommended vehicle (lysine buffer) by intravenous route with a dose of 150 mg/kg body weight at dose volume of 10 ml/kg b.w. Under mild isoflurane anesthesia, blood specimens were collected by retro-orbital puncture method using capillary tubes into pre-labeled tubes containing anticoagulant (K2EDTA; 2 mg/ml blood) during the next 4 hours of post-dose. After blood collection animals were euthanized and the organs under study were collected, blotted, weighed and transferred into pre-labelled containers. The tissue samples were added to 1 ml deionized water and homogenized by using T10 basic homogenizer (ULTRA-TURRAX®) on ice. After homogenization samples were stored at -80 ± 5 ºC until analysis. Concentrations of the analyte Irinotecan and active metabolite (SN-38) in tissue samples were determined by using API 3200 Q-trap LC-MS-MS system, after homogenization.

In the first detailed pharmacokinetics and tissue distribution study carried out with VT287 in Balb/c mice, Irinotecan was found to be highly enriched in lung tissue (among others) in mice administered VT-287 (Figure 15a). In a second follow up PK study carried out in Balb/c mice, Irinotecan levels in the lung and select organs were measured in a head-head comparison between VT-287 and Irinotecan of the same dose. These results show that Irinotecan levels in the lung were consistently several fold higher than in the case of Irinotecan alone (Figure 15b). The concentration of the active metabolite SN-38 was also found to be enhanced in the lung tissue, especially in comparison with naked Irinotecan (Figure 11).

Example 6
Conversion of Irinotecan to the active metabolite SN38 in vivo
Irinotecan gets metabolized into SN38 in vivo and this conversion is essential for the potent cytotoxicity of Irinotecan against cancer cells. The mouse studies were carried out as described in the previous section. Concentrations of the analyte Irinotecan and active metabolite (SN-38) in tissue samples were determined by using API 3200 Q-trap LC-MS-MS system, after homogenization.
Male New Zealand White Rabbits were administered the test items by intravenous bolus route via marginal ear vein. The first group animals received the VT-287 test item in a solution form containing 0.05 % (w/v) of lysine buffer (0.5 mg equivalent to Irinotecan Hydrochloride); the second group animals received VT-287 test item in a solution form containing 0.05 % (w/v) of lysine buffer (0.5 mg equivalent to Irinotecan Hydrochloride) under influence of magnet and the third group animals received plain Irinotecan in a solution form containing 100 % (w/v) of Sterile Water for Injection. Rabbit were restrained and blood samples were collected by auricular artery puncture method using 21 – 22 gauge needle with a syringe into pre-labeled tubes containing anticoagulant (K2EDTA; 2 mg/ml blood) during the next 0.5 hours of post-dose. After blood collection animals were euthanized by over dose of Sodium thiopental; and lung and brain samples were collected, blotted, weighed and transferred into pre-labeled containers.Concentrations of the analyte Irinotecan and active metabolite (SN-38) in tissue samples were determined by using API 3200 Q-trap LC-MS-MS system, after homogenization.
Both in the case of Balb/c mice, as well as New Zealand rabbits, the concentration of SN-38 found in the lung tissues upon administration of VT-287 was found to be significantly higher, than upon administration of an equivalent concentration of Irinotecan alone. This is illustrated in Figures 11 and 12. Figure 11 shows a comparison of the AUC of SN-38 from 2 independent mouse studies conducted with VT-287 with a similar study conducted with Irinotecan. There is 5-7 fold more SN-38 in the lung in the case of VT-287 as compared to Irinotecan administration.
Figure 12 shows the lung concentration of SN-38 at two different time points in a rabbit study (n=2). Here, SN-38 is undetectable in the lung tissue upon administration of 0.5mg/kg of nakedIrinotecan. However, with the administration of the same Irinotecan equivalent of VT-287, a significant concentration of SN-38 is detected in the lung, suggesting an improved, specific delivery to this tissue by the formulation.

Example 7
Magnet mediated increase in Irinotecan and SN-38 concentration in the lung
Comparative (magnet vs. non-magnet) single dose intravenous pharmacokinetics study of VT-287 was conducted in Male New Zealand White Rabbits (methods are described in the previous sections), to evaluate the following: a) Organ (Brain/Lung) Targeting of Irinotecan when compared to Plasma; and b) Retention in Brain/Lungs under the influence of Magnet (Figures 13 and 14). With VT-287, 10-fold selective targeting of Irinotecan to the lung compared to plasma was observed (Figure 13). Moreover, a statistical difference in retention of Irinotecan from 10 min. to 30 min. in lungs under the influence of magnet was also noted.We believe that the improved retention in lungs resulted in over 2-3 fold increase in the levels of active metabolite SN-38 (the actual anti-cancer agent) under the influence of magnet (Figure 19).

Example 8
Effect of VT-287 and Irinotecan on cell proliferation in lung cancer cell lines.
Cells were seeded in a 96-well plate at 1x104 cells per well and incubated overnight at 37°C, 5% CO2. Test compound VT-287 and Irinotecan hydrochloride trihydrate were added to the cells at: 0.781, 1.562, 3.125, 6.25, 12.5, 25.0, 50.0 and 100 µM conc. along with controls (0.1% DMSO and 0.05 mg/ml of lysine) in triplicates/concentrations and incubated at 37°C, 5% CO2 for 96 hours. Cell viability assay was performed using Alamar blue (resazurin). 20µl of resazurin reagent (0.15mg/ml) was added to each well and incubated for 3 hour at 37°C, 5% CO2. Fluorescence was read at 530/590 nm in a multi well-plate reader. Data is presented as the % inhibition cells vs drug concentration compared to the vehicle control.
A549 (NSCLC): In A549 cell line (Figure 15), VT-287 showed a dose dependent inhibition of cell proliferation and results were comparable to Irinotecan after 96 hours of incubation.
NCI-H1299 (NSCLC): In NCI-H1299 cell line (Figure 16), Irinotecan showed a dose dependent inhibition of cell proliferation, while VT-287 did show comparable (not dose dependent) inhibition of cell proliferation after 96 hours of incubation.
Effect of VT-287 and Irinotecan on NCI-H187 cell proliferation after 96 hours: In NCI-H187 cell line, Irinotecan showed a dose dependent inhibition of cell proliferation and at 100µM conc. about 80% cell death was observed, while VT-287 did show dose dependent inhibition of cell proliferation after 96 hours of incubation, at 100µM Conc., percent inhibition was 50% (Figure 17).

Effect of VT-287 and Irinotecan on NCI-H69 cell proliferation after 96 hours: In NCI-H69 cell line, both Irinotecan and VT-287showed a dose dependent inhibition of cell proliferation and results were comparable after 108 hours of incubation (Figure 18).

To summarize, inall the four lung cancer cell lines (SCLC and NSCLC cell lines) tested, the cytotoxic effect of VT-287 was observed to be dose dependant over the range of concentrations employed in most cell lines and comparable to Irinotecan of equivalent concentration. Taken together with the in vitro and in vivo results described above, this suggests that when delivered to the lung, VT-287 will be efficacious at killing lung cancer cells as well as the well-characterized cytotoxic drug Irinotecan.

The results presented herein indicate that VT-287 is a stable nano-formulation that is able to transport and deliver Irinotecan in its active form. The physical properties of VT-287, we believe, prevent aggregation in solution, and the novel idea of coating the nanoparticle with positively charged lysine facilitates interaction with specific cell-surface receptors thus leading to targeted delivery and permeability in lungs .We have shown that VT-287 is able to penetrate the endothelial cells and deliver physiologically relevant concentration of Irinotecan to the intracellular compartment. This delivery and intracellular retention is enhanced in the presence of a magnet due to the presence of iron in VT-287. The in vivo studies that were carried out in two species (rodent and non-rodent) demonstrate that VT-287 is able to selectively target Irinotecan delivery to the lung, and result in a significantly higher concentration of the active metabolite SN-38 in the lung in both species. Further, we have also shown that under the influence of a magnet, there is improved retention of VT-287 in the lung, resulting in further enhancementof SN-38. Finally, we have demonstrated that the Irinotecan delivered via VT-287 is just as potent as Irinotecan in killing lung cancer cells, using cytotoxicity studies carried out with 4 different lung cancer cell lines. Taken together, our results strongly suggest that VT287 will be a potent, targeted, efficacious and safe therapy for lung cancer.
Data presented herein is representative of how the nanoparticle of the invention can deliver therapeutic compounds to lungs. It is stated that the cytotoxic drug irinotecan is merely a representative example to establish the efficacy of the nanoparticle as delivery platform and it can be extended to other therapeutics and disease conditions as described in this specification The data is merely representative and cannot be construed to be limiting the invention in any way.