WO2018150362A1

WO2018150362A1 - Magnetic nanoparticle formulations for targeted delivery of drugs to lungs for treatment of pulmonary diseases

Info

Publication number: WO2018150362A1
Application number: PCT/IB2018/050952
Authority: WO
Inventors: Marina RAJADURAI; Pushkar KULKARNI; Aarti SEVILIMEDU; Uday Saxena
Original assignee: Vegrandis Therapeutics Pvt. Ltd.
Priority date: 2017-02-16
Filing date: 2018-02-16
Publication date: 2018-08-23

Abstract

Cytotoxic drugs such as irinotecan are used as first line therapy for a variety of cancers. Cytotoxic drugs are very effective at killing the tumour cells but have an undesirable side effect because they also destroy normal healthy cells. Secondly, even if the drugs enter a tissue they may be transported out by transport proteins in the cells thereby reducing their concentration overtime, resulting in decreased efficacy. One way to increase their efficacy and reduce the side effects is to selectively target them to the cancer containing organ/tissue. We have designed a novel nanoparticle that can selectively deliver irinotecan to the lungs. It also contains iron molecules 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 lung diseases.

Description

Magnetic nanoparticie formulations for targeted delivery of drugs to lungs for treatment of u monary diseases

Related Application

This appl cation is being fi ed as a c mplete application with the Indian Patent Office..

Field of invention:

This application provides a nove 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 t e lungs by use of arc external magnet. This nanoparticle can be used for treatment of lung cancer and other diseases such as as hma, 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 cancel cells and normal healthy cells thus killing ail ceils 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 rnyeiosuppression.

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 tine 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 dru s 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 Btocanjugate Chem, 2010., 21(5}_t 979—987 ] . Another method adopted: is to Sink drugs to monoclonal antibodies specific for antigens expressed on tumour cells [British Journal of Cancer, 2016, .114, 362— 3S7|. 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 th body are present in the lung. Because exchange of gases is the major function of the lung, the endothelial lining of the Sung 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 king.

As art example, in the present invention, Applicants have exploited the densely populated capillar endothelium in the lung to deliver irinotecan containing nanoparticles whose surface is decorated: with positive charge. Brief Description of Figures

Figure I. Transmission electron micrograph of a iron Oxide flO} magnetic anopartlcles; b) PEGyiated Irinotecan loaded 10 nanopartlcles; c) double PEGyiated irinoteean loaded rtanoparticles and their corresponding size distribution (d,e,f). According to this data, the size of the nartoparticles even after incorporation of the second layer of PEG increased only by 2-3 ran in average, and diameter was in the range of 10-16 nm.

Figure 2. Magnetic properties of MPs: a) using external magnetic field; b) EP data confirms ma neti properties of VT-2S.

Figure 3. FTIft spectra of F3O4 nanoparti tes - confirms structure of F3Q4

Figure 4, FTI spectre of PEG -coated F3O4 rsanooarticies— confirms FESylation

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 mi PBS buffer - 0.25 mg ml; 8-3.125 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 ral; D- 6.25 mg in 0.5 ml of lysine solution injected to 2.5 ml PSS buffer.

Figure ¾. Flocculstson 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 ami irinotecan hydrochloride in Human plasma

Figure 3. Permeability of Irinotecan from VT-287 in HULEC-5a cell line, intracellular concentration of Irinotecan, uM was measured n=3 for each condition and time point) and the mean s platted. Error bars are S.D.

Figure 10. a) Tissue distribution of irinotecan after a single dose administration, in Satb/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 Baib/c mice.

Figure 11. Concentration of SN-38 in lung tissue in a single dose PK TD study carried out irt 3aib/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 o 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 O.Smg/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 king cancer ceil line A549 (NSCLC).

Figure IS. Effect of VT-287 and irinotecan on ceil proliferation in lung cancer celt line MO- H129S i SCLCi.

Figure 1.7, Effect of VT-287 and irinotecan on cell proliferation in NCi-H187cell line.

Figure IS. Effect of VT-287 and irinotecan on ceil proliferation in NCJ-H69ceil line.

Detailed Description

The present invention delivers positive charge coated nanoparti ies 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 com rising a) s core comprising ferromagnetic material; b) a double layer of biocompatible shell

surrounding the core; and c} a targeting moiety .

i one embodiment, the surface of the rjanopartlcie 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, i 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 orga ns...

in another embodiment, the nanopa lcie is biocompatible.

in another embodiment, the nanopartfcSe can be used singly or as an aggregate of nanopartlcles. 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 rtanoparticle Is In the range of δ nra to SGnm.

The biocompatible shell of the nanoparticle has an outer diameter of 10- 65nm, preferably

14 - SQnm and more preferably 20nm,

The biocompatible shell is made of a material selected from the group consisting of PEG, polyethylene oxide) oligomer or polymer, po!yeaprolactone, pofylactide, potygiycoiide, and copolymers thereof, polyoxypropyiene, poiyoxyethylene-polyoxypropyiene dibiock, polyoxyethyierte-polyoxypropylene-poiyoxyethyEene triblock, and any mixture thereof, arranged into two layers. Th 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 meta!.

in a preferred embodiment the nanoparticle comprises a targeting moiety attached via noi covalent or covalent interactions to the polymeric material of the biocompatible shell of the nanoparticle. The targeting moiety of nan apart! die comprises a materia! selected: from the group consisting of negatively charged amino acid., such as aspart c acid or glutamic add, oligomers, or polymers and combination) thereof, The nanopartide of the Invention is used for delivery of any therapeutic that needs to target the lung. Thus the nanopartide of the invention intends to deliver therapeutic to lungs, in conditions including but not. limited to cancer, specially Jung cancer, lung infection,, cystic fibrosis, lung edema,, asthma, pneumonia and COPO. The therapeutic or drug or the compound of interest, for example a cancer dierootherapeutic is incorporated into the nanopartide via covsient or non-covalent interaction.

Since the nanopartide serves as a delivery platform, i can be loaded with other drugs that are required to act primarily in the lung. Thus the same nanopartide formulation cars be used to deliver any other cancer therapeutic induding but not limited to cytotoxic drugs, antibodies, protein and nucleic acid based treatments. Additionally the nanop rtide 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 nanopartide presented as a example in the current specification, is composed of a self-contained nanopartide core, loaded with the drug Irinotecan. This allows for the nanopartide care of VT-287 to serve as a deiivery platform to deliver drugs other than

Irtnoteean to the lung. It can be loaded with cytotoxic: drugs {such as Gsplatin, Methotrexate etc) to treat lung cancer; drugs for asthma and COPD (Montelukast, Theophylline,

Aclidinlum, Rofiumliast); or anti-lnfectives to treat lung infections such as TB and pneumonia (feoniazid, t arrspictn, Amoxicillin, Azithromycin). Drug deiivery using the iung- targeted VT287 platform will ensure Increased efficacy at the site of the disease, as we have demonstrated in the case of ftlnoiecan. 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 nanopartide 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 nanoparttde in the lungs upon use of an external magnet. The magnetic field source is selected from electromagnet or magnetic resonance imaging (MR!) equipment.

The invention: also encompasses a: process of drug loading, involving attachment of the selected drug to the biocompatible polymeric shelf of the nanoparticie via non-covalent or covalent Interactions and includes double PEGylatksn/ 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 leading of PES- ou his. layer nanoparti les VT-287 (Sshematiic 1)

1. Synthesis of magnetic nanopartkles FejQ*: Magnetic nanoparttcles (MPs) we e prepared using modifsed co -precipitation method according to published procedures fSee British Journal of Cancer, 2016, 114, 362—367; Journal of Science and Heakk at The University of Alabama, 2010, 7,. 16-18] .

O-I Ferric chloride hexahydrate (FeCI₃ ,6¾0) {250 mg in 9.2 mi of water) and 0.O5

Ferrous chloride tetrahydrate {FeCla .4HaO) (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-necfe 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 nanoparti les were collected by magnetic field separation (using small magnet placed close to an R8 followed by decanting the solution), washed one time with diluted HCI solution {one drop of HCI in 5 ml of water) to reach pH 7, then washed 4-5 times wit h 20 ml of deionezedi water. Wet NPs were subjected directly to the next step of PEGylation without drying.Tbe process yi lds '""160 mg of dry NPs.

The nanoparttdes prepared thus we ; investigated by the ^'Tra mtesim. El&cXmn Microscopy (Tecnai GZFEi F12 transmission electron microscope ( E^?) at a ^' accelerating voltage of 120 kV-j, the TEM data. showed that nanoparti es -are mono-dispersed with size In range of 8+2 nm (Figure ia, d,)

2, PESyistiort of irttn Oxide naRoparticlesiSebematics 2, 3J:

Fs₃&> MPs |1BQ mg) were _:dissdysd PEG-2QQ solution {SSQ. g- dissolved_. in SO ml o deionszed w-ater) under !% using sonscation [10 mm} at 40^*£. The solution of f^G.with Feg i

for 10-_.min _> the SB:ihen .placed In ultra-sound bath pre-heated t 40*C, and sonicated f r 10 ■mm. The !RB as shifted to an oil-bath quipped with .condenser (e.g. Q rriroth Condenser and nitrogen -baOoon. Temperature was increased to S£ C m &^' sl hath, aiming 90 ^* C ts reaction mixture- The reaction runs strict y under inert atmosphere till black, color solution ^'turned brown fap.prox, 2-5-3 h^^■■Finally, reaction mixture was allowed to cool down ¾o RT, shifted portion-wise Into centrifuge tbb.es (2δ rnl _f and MPs were collected by cehtt"l¾gation for at 000 rpffl 10^:-15: i¾ptes_f followed by removal of water by- cle-cantstlon .with a magnet placed outside of the tybe. Th P were transferred back into 250 ml R8, SB mi of pyre water was added, arscl stirred at_. RT for^' -10 mm ^'-under mtra-gen 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 P E¾3 completely from the $Fs.

sO^

Schematic 3. The technology chat fiow

The magnetic bar was removed from: the reaction mixture, wate was decanted using external magnet,, the slightly wet MPs wer shifted to a Petri dish and dried at 40^*C under nitrogen for 24 h or lyophifized fat -SO'C, overnight!.

Magnetic properties of NPs were cheeked at every step using external magnet (see Figure 2a, bj.

JEM study of PEGyiated Ps confirmed the siz in range of 8-10 nrn (Figure lb, e.}. FT-i (Figures 3, 4} confirmed composition of the nanaparttdes and successful PEGylation. The IR spectra of iron oxide Fej&i {Figure 3} exhibited string hands in the low frequency region

{ m - 400 cm^'1} attributed to the Fe-Q bond vibration of Fe₃0₄. The peak a t "-^'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 1093 cm^"" and the vibrations! band at 1344 cm^"" corresponding to C-O-C ether bonds after PEGylatfen of iron oxide nanopartacles. The bands around 2900 and 955 m^'* correspond to— CH_?- stretching vibrations and -CH out of plane bending vibrations,, respectively {Figure 4). These peaks are strong evidence that PEG covered the nanoparticies surface. Additionally alt main peaks in PEG-coated Ps¹ spectra are shifted in comparison with spectra of Ps before coating,. Indicating change in environment of the particle after polymer coating.

Example 2.

1. Drug loading in PEGyiated: Iron Oxide NPs f Schematics 2, 3):

A. Primary drug loading: 6 rng of irinotecan-HCl tri ydrate was dissolved in 3ml of nanopu e water; then 3 mg of PEGyiated NPs were dispersed n the same solution with magnetic stirring at R.T. The solution was stirred far 120h {5 days; measured absorfoance 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 {i gfml, 2ml each_., 4 times shake sidewise), and a solvent was removed with syringe using magnet to keep MiPs aside. Solution of Glycerol (10 jiiL of solution: 0.005 mg in 1ml of water, 5% vet.) was added to the reaction mixture and the drug loaded NPs were snap frozen in liquid nitrogen, yophllized and dried at -60°C for 5 hours {NPs can be storedafter snap freezing at -S0^cC until: ready for lyophsiisation). B. Secondary layer and drug; loading {VT-2S7): 3.5 ml of Irinotecan -HCltrlhydrate as dissolved in 3.5 mt of water, and 3 mg of lysine were added to the above solution with ma netic stirring at RT. Then, 10 mg of single layer MPs (protocol A) were added to the above mixture, followed by addition; of 900 mg of PEG -2000 portion -wise, the mixture was sonicated for 3 minutes {using ultrasound bath, RT), stirred at ST for 20 minutes. Magnetic bar was removed from reaction mixture, MPs were isolated the using external; magnet, washed 2 times with cold fj= 1-S^CC) lysine solution {Irng mi, 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 lyoph^'siiied for 5 hours at -60°C {HPs cars be stored after snap freezing at -S0^¾C until ready for fyophilisation}.

Magnetic properties of VT-287 were confirmed by applying external magnetic, fi d Figure 2a) and by Electron Paramagnetic Resonance (EPS) spectroscopy {Figure 2b}. The presence of signal in EPR spectra firmly supports paramagnetic properties of sample VT-287.

TEM tudy of VT-287 shows size in range of 10-16 nm {Figure lc, f.J. .According to this da a, the size of the nanoparticles after incorporation of the second layer of PEG Increased only by 2-3 rsm in average. Drug loading percentage wasl-4%, measured using LC-MS-MS, Zeta potential was found to be -I8.5Mv. Table 1 further provides the characterization data. Table 1, Characterization of FT-287 ^' fonnutati&n

2, thod to prevent aggregation of nanoparti ies VT-287 and keep it In solution

i order to check stability of VT-287 with and without lysine wash, nanopartides VT-287 were dispersed in water with or without lysine (1 rog mi) a d dried using speedvac for 15 h, then dry nan op articles VT-287 were re-dispersed: In water and pictures were taken: at 0 min, 5 mm and 10 mm after dispersion, i troduction of positively charged amino acid lysine f physical interaction) prior to drying step helps to prevent aggregation (solutions B), At the same time chemical conjugation of lysine molecules to VT-287 nanopartides did not help to prevent aggregation after drying step. Nanopartides in solution of Iysine-congugate-VT-287 re-dispersed in water after conjugation to lysine, precipitated after 10 mm, similarly to nanopartides without any lysine treatment. The reason for this is that when lysine is cova!entiy conjugated with the nanopartie!e, the amino groups in: lysine are engaged in the conjugation and as such the nanopartide is no longer positively charged. Solutions of nanopartides 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 indused floecyfatfon of nanopsitieies VT-2S This experiment was carried out to show that the lysine coated charged nanoparti es b^'mds 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 rrig ^'mi, drug loading 4%) were re- suspended Sn G.5 ml of lysine solution (1 rog/rnl). This suspension was injected at 3J^"'C inPBS buffer solutions (pre-incubated at 37^cCfor 30 mm before addition of VT-287,10 mi) with heparin at X, 3X, 2X, and lX to lysine by mol, with average molecular weight 3,£ 30g/mol) and PBS buffer alone as a control. Photographs were taken at different time points opto 2 hrs. The difference in solutions was observed starting from 15 minutes, and interaction of VT-287 wsth 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 fioccutation occurs they were visually smaller, which indicates interaction of heparin molecules with lysine molecules OR the surface of oanopartkles VT-287.

Example 3.

1, Stability stud of VT-2S7 and trinotecan hydrochloride in PBS buffer and human lasms.

a) In PBS Buffer: Working, solutions of test/ reference sterols} in phosphate buffer (pH ?.0| saline were prepared at concentration: of 1 μΜ equivalent of trinotecan by using 1 mM DMSO stocks for trinotecan hydrochloride tdhydrate f 0.677 mg of irinotecan hydrochloride trshydrate in 1 ml of DiMSO) and in lysine buffer for VT-287 at concentrations of 3.33 mM lysine buffer stock {2.26 mg of VT-287 in 1 nil of 0.05% w/V lysine buffer). For trinotecan hydrochloride trt ydrate, .1 μ of 1 mM DMSO stock was spiked; to 1 mi of P8S such that the final concentration of DMSO is 0,1%. For VT-287, 10 pL of lysine buffer stock was spiked to 1 mi of PBS. The samples were incubated at 37⁹Cfor 120 minutes, with shakin at 400 rpm. At 0.00, 0.25, 0.5, 1,00, 2.00 and 24 hr, an aliquot of 100 μ| sampl was removed. To 100 pj of removed sample aliquot: in pre-bbeled centrifuge tubes 200 ui of acetonitrlie containing internal standard {haioperidol} was added, vortexed for 30 seconds, centri uged for 10m Irs at the speed of 10000 rpm at 10-C. After centrifugatfon, '"^'200 μί sample was transferred into labeled auto sampler vials for LC-iMS- S analysis- The Irinotecan from formulation VT- 287 was as stable as free irinotecan in phosphate buffer opto 24 hrs (Figure 7),

h\ in Human plasma: Working solutions of test/ reference item{s) In Human plasma (pH- 7,40| were prepared at concentration of 1 Μ 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 μί of 1 mM DMSO stock was spiked into 1 ml of human plasma such that the final concentration of DMSO h 0.1%. For VT-287 {active ingredient of

Irinotecan in VT-287 was found to be 3 %}, 10 μ! of lysine buffer stock was spiked Into 1 ml of human plasma. The samples were incubated at 37-C for 24hr, with shaking at 400 rpm. At Q.0G, 0.25,. 0.5, 1.00, 2.00 and 24 hr, an aliquot of 100 pi sample were removed. To 100 pi of removed sample aliquot In pre-iafoeied centrifuge tubes added 200 μ\ of acetonitrile containing Internal standard (haioperidol), vortexes for 30 seconds, and then eentrtfuged for lOmin at the speed of 10000 rpm at 10⁵ After centrifugation, ~200 μΐ sample was transferred Into pre-laheled: 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-Sa cell line.

Dosing solution of test Item VT-287 at 5 μίΥΙ (equivalent concentration of Irinotecan) was prepared by spiking 80 μί of 0.5 mM lysine buffer stock (Irinotecan equivalent

concentration} to 7920 μί of MCD8 131 media (with serum). Dosing solution of test item Irinotecan hydrochloride trihydrate at 5 μ was prepared by spiking 8 ΐ of 5 mM DMSO stock to 7992 μί of MCD& 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-equivaient concentration of VT-287 for the experiments.The dosing solutions of test items were added to HuLEC-5a cell monolayers end incubated for specified incubation period {15 min and 2 hour individually, with or without a magnet under the cell culture plate) at 37 ± 1 ^SC with 5 ± 1 % &¾ using a G¾ incubator. In this permeability study, VT-287 was tested at 5 μΜ test concentration {equivalent concentration of Irinotecan). After specified incubation period, the spent media was removed from all the wells and monolayers were washed w th ice cold PBS (pH-7.40). The el monolayers were iysed and samples was subjected for extraction. The extracted samples were submitted for LC-MS- S 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 weft 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 celts.. 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 S

Targeted: delivery of irinotecafi the lung by VT-2S7

Adult healthy male BALB/e 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 tri hydrate 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 bj VT-287 in a recommended vehicle (lysine buffer} by intravenous route with a dose of ISO mg/kg body weight at dose volume of 10 ml/kg b.w. Under mild Isoftursne anesthesia, blood specimens were collected by retro-orbital puncture method using capillary tubes into pre- labeled tubes containing anticoagulant K2E0TA; 2 mg mi blood J 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-ia elted containers. The tissue samples were added to 1 ml deio ized water and homogenized by using T10 basic homogenizer ULTRA-TURRAX*) on ice, After homogenization samples were stored at -80 ± 5 -C until analysis. Concentrations of the analyte irinotecan and active metabolite (SM-3S) in tissue samples were determined by using API 3200 Q-trap LC-MS-MS system, after

homagenization. in the first detailed pharmacokinetics and tissue distribution study carried out with VT287 In 3aib/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 P . study carried out In Ba h c mice, irinotecan levels in the lung and select organs were measured In a head-head comparison between VT-2S7 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-.3Swas also found to be enhanced in the lung tissue, especially in comparison with naked Irinotecan {Figure 11).

Example 8

Conversion of irinotecatu to the active metabolite SN3S in vivo

irinotecan gets metabolized Into 5 38 in viva 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 anaiyte Irinotecan and active metabolite (SN-38) in tissue samples were determined by usin 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-2S7 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 l sin 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/ ) 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 ( ₂EDTA; 2 mg/rnl 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 t ransferred into pre-iabeled contalners.Concentrations of the anaiyte Irinotecan and active metabolite (SM-38) in tissue samples were determined by using API 3200 Q-trap LC-MS-MS system., after homogenization.

Soth in the case of Ba!b/c mice, as well as ew 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 show 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:.

igure 12 shows the lung concentration of SN-38 at two different time points m a rabbit study (rs=2). Here, SN-38 is undetectable in the lung tissue upon administration of Ct-Srrtg/kg of rtakedirinotecan. However, with the administration of the same irinotecan equivalent of VT-287, a significant concentration of S -38 is detected in the lung, suggesting an improved, specific delve y to this tissue by the formulation.

Example 7

Magnet meditated increase in irinotecan and concentration m the lung

Comparative (magnet vs. non-magnet) single dose Intravenous pharmacokinetics study of VT-287 was conducted m Male Hew Zealand White Rabbits (methods are described in the previous sections}, to evaluate the fallowing: 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 tangs resulted in aver 2-3 fold increase in the levels of active metabolite SR-38 (the actual anti-cancer agent) under the influence of magnet (Figure 19).

Example S

Eff ect of VT-287 and Irinotecan on cell proliferation in lung cancer ceil lines.

Cells were seeded fn a 96- welt plate at 1x104 cells per well and incubated overnight at 37"C₄ 5% 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 μΜ cone, along with controls (0.1% DMiSO and 0.05 mg/rrsi of lysine) in triplicates/concentrations and incubated at 37^eC, 5% CQ2 for 96 hours. Cell viability assay was performed using Afamar blue (resaiurin). 20 μί of resazurin reagent (O.lSmg ml) was added to each well and incubated for 3 hour at 37'C, 5% COj. Fluorescence was read at: 530/590 nrn in a mufti well-plate reader. Data is presented as the % inhibition cells vs drug concentration compared to the vehicle control. A54S {NSCIC}: In A5 9 cell line {Figure 15}_... VT-287 showed a dose dependent Inhibition of ceil proliferation and results were comparable to Irinotecan after 96 hours of Incubation, NCI-H1293 f SCLC|; Irt NG-H1299 cell fine {Figure 16% lirinotecan showed a dose dependent inhibition of ceti proliferation, while VT-287 did show comparable (not dose dependent) inhibition of cell proliferation after 36 hours of incubation.

Effect of VT-2&7 and !rinoteean on &Q-H187 cell proliferation after SS hours:: in NCI -H 187 ceil line, irinotecan showed a dose dependent inhibition of cell proliferation and at IQQp cone, about 80% ceil death was observed, while VT-287 did show dose dependent inhibition of cell proliferation after 96 hours of Incubation, at ΙΟΟμΜ Cone, percent inhibition was 50% (Figure 17),

Effect of VT-287 and !rirtotecan on CI-H69 cell proliferation after 36 hours: in NC1-H69 cell fine, 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, insii the four lung cancer cell lines |S€LC and NSCLC ceil lines) tested, the cytotoxic effect of ^'VT-287 was observed to be dose dependant over the range of concentrations employed in most cell tines 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 narto-formulation that Is able to transport and deliver Irinotecan So its active form. The physical properties of VT-287, we believe, prevent aggregation in solution, and the novel idea of coating the nanc-parti e with: positively charged lysine facilitates interaction with specific ceii-surf ce 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-3S Irs the lung in both species.. Further, we have also shown that under the Influence of a mag et, there is smpraved retention of VT- 2S7 in the lung, resulting In further enhancernentaf S -38... Finally., we have demonstrated that the Irinotecan delivered via VT-28? is just as potent as Irinotecan In killing lung cancer celts, using cytotoxicity studies carried out with 4 different lung cancer cell lines. Taken together, our results strongly suggest that VT287 will fee a potent., ta geted, efficacious and safe therapy for kmg cancer.

Data presented herein is representative of how the nanopartscle 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 nanopartide 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 anno be construed to be limiting the i ven io In any way.

Claims

Claims:

1, A pharmaceutical com osition of biocompatible nanopartide or nanoparticie aggregate comprising 3} a core comprising ferromagnetic material is) a double layer of biocompatible shell surrounding the core.: and c) a targeting moiety.

2, Tihe pharmaceutical com sition of claim 1 wherei n the nanoparticie or nanoparticie aggregate is coated with positively charged ami o add and wherein the amino acid is seiected from the grou consisting of lysine, arginme and hsstidine ,

3, Tihe pharmaceutical composition of claim 1 wherein the biocompatible shell of the nanoparttde has an outer diameter of 10 - 65nm, preferably 14 - 50nro and more preferably iOnni.

4, The pharmaceutical composition of claim 1 wherein the biocompatible shell of the nanoparticie is mad of a material seiected from the group consisting of PEG, poiyfethylene oxide} oligomer or polymer, poiyscaprolaetone, poiylactlde, poUyglyoolade, and copolymers thereof, polyoxyprapylene, polyoxyethylene-poiyoxypropyiene dSb ck, poiyoxyethyiene- poiyoxypropyiene-poiyoxyethylene triblock, and any mixture thereof, arranged into two layers...

5, Tihe pharmaceutical com position of claim 1 wherein the fe rromagnetic core material is seiected from the group consisting of iron, nickel, cobalt,, gadolinium., samarium, neodymiurn, boron, aluminium or a: mixture thereof,

6, Tihe pharmaceutical composition of claim 1 wherein the f rromagnetic core material Is an oxide, an hydroxide or a metal.

7, The pharmaceutical composition of claim 1 wherein the ta rgeting moiety of nanoparticie comprises a material seiected from the group consisting of negatively charged amino acid, such as aspartic acid, or glutamic acid, oligomers, polymers,, and combination thereof,.

8, Tihe pharmaceutical composition of claims 1 to 7, further comprisin 3 targeting agent attached via non-covaient or covalent interactions to the polymeric material of the nanoparticie.

9, A pharmaceutical composition comprising a biocompatible nanoparticie or nanoparticie aggregate, and targeting agent In the presence of external magnetic field source wherein tihe nanoparticie is retained in the lungs upon use of an external magnet.

10, The pharmaceutical composition of claim 9 wherein the externa! 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 nanoparticie or nanoparticie 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 nanoparticie or rtanopartEcie 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 nanoparticie or nanoparticie aggregate comprising a) a core comprising ferromagnetic material; b) a double layer of biocompatible shell surrounding the core; and c) s targeting moiety for delivering drug to lungs irt lung-related conditions

14. The method of use of a biocompatible nanoparticie or nanoparticie aggregate as claimed in claim 13, wherein the lung-related condition is selected from the group consisting of lung cancer, lung infection, Sung edema, asthma, COPD, pneumonia, cystic fibrosis and tuberculosis..

15. The method of use of a biocompatible nanoparticie or nanoparticie aggregate as ciatmed in claim 13, wherein the drug is selected from the group consisting of cytotoxic drugs, antibodies, protein drugs, nucleic add based drugs, anti-infectictives, steroids and antibiotics,

16. The method of use of a biocompatible nanoparticie or nanoparticie aggregate as claimed in claim 13, wherein the drug is selected from the group consisting of irSnotecan, cisplatin, methotrexate, montelukast, theophylline, adtdintum, raflumilast, isoniazid, rifampi in, amoxicillin and azithromycin.