FORM 2
THE PATENT ACT 1970
(39 of 1970)
AND
The Patents Rules, 2003
PROVISIONAL SPECIFICATION
(See section 10 and rulel3) 1. TITLE OF THE INVENTION:
"NOVEL DRUG DELIVERY SYSTEM " 2. APPLICANT(S):
(a) NAME: Nano Therapeutics Pvt. Ltd.
(b) NATIONALITY: Indian Company incorporated under the Indian
Companies ACT, 1956
(c) ADDRESS: R-6/8, Jayashreedhan Soc, Bangur Nagar, Goregaon (West),
Mumbai-400090, Maharashtra, India.
3. PREAMBLE TO THE DESCRIPTION:
The following specification describes the invention

FIELD OF THE INVENTION:
The present invention relates to the field of targeted drug delivery, more particularly, relates to the field of vascular medicine. More particularly, the invention relates to novel drug delivery system comprising a therapeutic formulation which creates a local therapeutic effect at diseased site in mammalian vessels providing long-term retention of a highly concentrated drug in particular macrocyclic lactone within the target tissue.
BACKGROUND OF THE INVENTION:
Targeted drug delivery is a useful approach for the treatment of many localized pathologies as the concentration of systemically administered drugs is often limited due to circulation thus resulting in side effects of the drug as well as failure to achieve the desired therapeutic doses at the diseased sites. On the other hand, a much lower amount of drug is required, if the drug can be targeted to the site of pathology. Furthermore, a much higher local concentration of drug can be achieved as compared to systemic administration. The efficiency of targeted drug delivery is often compromised by the fact that the local concentration of the drug may decrease rather soon.
Often, subsequent to an intravascular procedure neointima proliferation and vascular injury remodeling occurs in the blood vessel of humans, more specifically in the heart, as well as in vulnerable peripheral blood vessels like the carotid artery, iliac artery, femoral and popliteal arteries. This results in a narrowing of the vessel lumen, causing restricted flow and pre-disposing to an ischemic event.
Although, some recently published clinical studies have suggested that selected patients may benefit from the administration of a single sirolimus compound over a period of time systemically (oral) to help control cellular neointimal proliferation caused by stenting, such oral use of single sirolimus compounds have not yet demonstrated adequate or consistent reduction in neointimal proliferation, even with daily use, relative to that achieved with a drug eluting stent.

Medical devices such as coronary stents coated with various forms of drug eiuting coatings containing sirolimus drugs have shown promise at controlling vascular wall proliferation following vascular injury and/or vascular reperfusion procedures such as balloon angioplasty and/or mechanical stent deployment.
In many patients, a vascular injury location develops into a narrowed or stenotic region, restricting flow and predisposing the vessel to a major thrombotic event, most commonly known as a heart attack (clot occlusion) or blood flow occlusion in the arm, leg , kidneys and brain commonly referred to as peripheral occlusion. Once an occluded blood vessel has been "opened up" and/or mechanically cleared of the occluding thrombus which has formed in this narrowed region of the diseased vessel, this narrowed area must be mechanically altered to increase the cross section flow diameter. Hence, it is now widely accepted clinically, that mechanically opening the constricted flow area of the vessel, together with the use of a single compound of sirolimus, delivered locally from a stent, provides the best outcome for minimizing re-stenosis. Today, a preferred drug eiuting format includes application, e.g.. coating, of a single sirolimus compound on the surface of a radially expandable metal tube. This is generally called a drug eiuting stent. Drugs such as Sirolimus (rapamycin) and its analogs, and paclitaxel have been shown to reduce cellular neointimal proliferation following restenosis. No known mechanical suppression means has been found to prevent or suppress cellular proliferation from occurring, which left untreated can cause re-stenosis within the vessel lumen within weeks of a vascular injury. Local delivery of a single sirolimus or its analogs or taxol compound has been shown to be the most effective means to minimize uncontrolled cellular proliferation after vascular injury.
In spite of the clinical benefits of using a sirolimus compound locally on a drug eiuting stent, experimental studies conducted so far have shown that such sirolimus like agents do not fully suppress inflammation and delay healing in and around the localized tissue area of the medical device and its drug eiuting location. Lack of endothelial cell coverage during delayed healing induced by rapamycin exhibits a high potential for luminal thrombosis. As an example, when two separate rapamycin drug eiuting stents are placed into an overlapping condition within a rabbit's iliac vessel, the amount of inflammation

induced by the overlapping drug coating increases nearly two fold, as determined by the lack of smooth muscle cell proliferation, and massive amounts of fibrin found deposited by the blood at such locations. Sirolimus like compounds in particular, inhibit growth factor driven proliferation of smooth muscle cells following vascular injury. This suggests a potential for therapeutically treating vascular injury vessel disease locally and minimizing restenosis following percutaneous transluminal angioplasty (PTCA). For example, vascular injury events have been shown to cause uncontrolled proliferation of smooth muscle cells in human. Vascular injury also results from endothelial cell disruption and vascular wall injury induced by mechanical means, such as during balloon angioplasty to radially expand the vessel and from stent deployment. Injured blood vessels may self-perpetuate a "chronic" repair process which includes a series of biological events whereby growth factors stimulate proliferation of smooth muscle cells, resulting in internal vessel thickening and excessive vessel narrowing. This may be countered with a sirolimus eluting stent. However, this technique often requires that patients must be kept on powerful anti-platelet Clopidogrel medications and ASA (aspirin) to prevent "in stent" thrombus due to the lack of endothelial cell coverage at these locations as a result of the deployment of a drug eluting stent.
The use of micro bubbles to target drug delivery by Jeane M Tsutsui, Feng Xie, and Richard Thomas Porter Cardiovasc Ultrasound. 2004; 2: 23. (Published online 2004 November 16. doi: 10.1186/1476-7120-2-23) discloses 'ultrasound-mediated micro bubbles destruction' as an innovative method for noninvasive delivering of drugs and genes to different tissues. Microbubbles are used to carry a drug until a specific area of interest is reached, and then ultrasound is used to burst the microbubbles, causing site-specific delivery of the bioactive materials. Furthermore, the ability of albumin-coated microbubbles to adhere to vascular regions with glycocalix damage or endothelial dysfunction is another possible mechanism to deliver drugs even in the absence of ultrasound. However, it has been reported that the application of ultrasound to contrast agents creates extravasation points in skeletal muscle capillaries and this phenomenon is dependent on the applied ultrasound power. High intensity ultrasound can rupture capillary vessels, resulting in deposit of protein and genetic material into the tissues.

These effects were directly related to the mechanical index. These studies indicate that although high-energy ultrasound seems to be necessary to mduce tissue permeability facilitating local drug delivery, it may also have significant bioeffects in the myocardium. Therefore, the optimal ultrasound parameters to enhance drug delivery with microbubbles needs to be further investigated.
Further improvements in localized drug delivery system, particularly drug eluting stents are exemplified in US7056339 and US7041130.
US7056339 discloses a biological agent of interest which is entrapped within a matrix comprising biodegradable polymers, loaded into channels on the surface of a stent. The matrix allows for release, usually sustained release, of the entrapped agent. The stent and matrix is sheathed with a covalently bound gel containing non-biodegradable polymer.
US7041130 describes devices for the controlled release of one or more drugs into a patient. The said devices include an implantable stent, at least two reservoirs in the stent, and a release system contained in each of the at least two reservoirs, wherein the release system may have one or more drugs for release at a controlled rate. Reservoir caps optionally are provided to control the time at which release of the one or more drugs is initiated. The release system comprises one or more drugs in a biodegradable or water-soluble matrix which comprises one or more synthetic polymers-
In an article by Mehili J, Kastrati A, Wessely R, et al; (Circulation. 2006;113:273-279) 'Randomized Trial of a Nonpolymer-Based Rapamycin-Eluting Stent Versus a Polymer-Based Paclitaxel-EIuting Stent for the Reduction of Late Lumen Loss', the investigators found that the polymer-free, on-site coated, rapamycin-eluting stent was not inferior to the polymer-based, paclitaxel-eluting stent in reducing neointimal proliferation as the incidences of angiographic and clinical restenosis were virtually the same in both study groups.
Although the drug-carrying polymer was a key component to the development of drug-eluting stents, it has become a source of concern and controversy due to possible thrombogenic and long-term pro-inflammatory effects. The use of a polymer-free porous

stent may circumvent such concerns. However, on-site stent coating may be somewhat cumbersome and time-consuming.
The major problem of the intravascular drug delivery is that after being delivered to the site of pathology, the drug is rapidly (in a few minutes) washed out from the site of delivery by the bloodstream. Restenosis is a long process (initial phase takes a few weeks and final changes develop after a few months), and it would be desirable to keep a drug for a long period of time at the site of potential restenosis after local delivery. Incorporation of the drug into polymers or micro particles or hydrogels with (controlled) drug release is suggested as an approach for prolongation of drug retention (local drug delivery with controlled drug release, LDD-CDR).
However, the vessel wall appears to be an anatomic barrier to drug delivery using polymers or micro particles; they penetrate poorly into the vascular wall and are found mostly in vasa vasorum and dissections within the intima after angioplasty. Drug-containing hydrogel can be incorporated in stents, so that the drug is slowly released from the hydrogel next to the vessel wall. In this technique, the storage of the drug is located next to the target tissue, but not therein; thus, the drug, after being released from the reservoir compartment, has to penetrate the neighbouring vascular tissue.
Besides LDD, another alternative to systemic drug treatment is cell-targeted drug delivery (CTDD). In this approach, the drug, endowed with an affinity to a pathology-specific epitope exposed on the cells in the locus of pathology, is supposed to find the site of pathology and concentrate therein due to binding to these epitopes. Within this approach, most of the efforts are concentrated on the anticancer therapy, since a number of cancer-specific epitopes are identified on various malignant cells. The approach is most attractive for the cases where the location of pathology is not precisely known and the local delivery of drug is not possible. Normally, a highly specific antibody should be generated to provide the specific delivery of the drug to the targeted cell population in the presence of a great excess of the irrelevant tissue epitopes.

A variant of this approach is to use a ligand to a cellular receptor as a vehicle for targeting a drug to the cell. In any case, with cell-directed targeting, even if local drug delivery is used, only a small portion of the overall administered drug will bind to the target cells, because cell-associated epitopes (especially pathology-specific ones) represent only a minor fraction of all epitopes present in the tissue. In the blood vessel wall, cells occupy only a small fraction of the volume of the tissue. So, in the case of intravascular local delivery, the major part of the drug will be washed out by the bloodstream. In terms of retention of the drug, which has bound to the target cells, this approach is not much different from the conventional systemic or local drug delivery.
Once bound to the cell surface, the drug may be either internalized or shedded from the cellular membrane within hours or, at longest, days.
Conventional Medical devices such as coronary stents coated with various forms of drug eluting coatings like polymers containing sirolimus drugs have shown promise at controlling vascular wall proliferation following vascular injury and/or vascular reperfiision procedures such as balloon angioplasty and/or mechanical stent deployment. The drug from the stent is delivered to the vessel wall in a programmed fashion over a period ranging from days to several weeks. The drug concentration in the arterial tissue thus achieved using this technique ranges from 1 ng/mg to 20 ng/mg of tissue for a dose of 140 micrograms/cm2 of stent surface area. The drug concentration in the blood ranges from 1 to 5 ng/ml of blood. Typically a stent of diameter 3.0 mm and length of 19 mm has a total drug loading of nearly 175 micrograms of drug on it.
A cursory review of prior art suggests that there is still need in art to develop a more efficient mode of drug delivery particularly in the field of vascular medicine or cancer medicine, where the local drug delivery is desired to achieve the desired therapeutic effect. In view of the above, the present inventors have developed a novel and more user friendly targeted drug delivery system using non-polymeric micelles as drug carriers to achieve localized high drug concentration at target tissues. The novel method of instant target drug delivery system is neither programmed to release the drug over a period of time nor utilizes any polymers, instead utilizes non-polymeric particles to deliver the drug

to target site and also easy to administer so as to achieve the desired localized drug delivery.
DETAILED DESCRIPTION OF THE INVENTION:
In accordance with the above, the present invention discloses a novel pharmaceutical formulation for targeted local drug delivery. The invention further discloses pharmaceutical compositions and drugs suitable for this method of targeted drug delivery.
In a preferred embodiment, the method is designed to provide a long-term retention of a highly concentrated drug in the site of pathology. A principal feature of the approach of the subject invention, which distinguishes it from othef drug targeting and local drug delivery systems, is that in particular, the drug is pre bounded to the long chain fatty acids used as a drug carrier which when interacts with water present in the blood, forms micelles thereby it prevents the drug to be washed away in the blood stream. These micelles then enter the site of pathology and support for accumulation of a high concentration of the drug for several weeks, providing a rather uniform distribution of the bound drug throughout the tissue.
Broadly, this invention can be useful for treatment of a variety of pathologies, which involve a local abnormality in any soft tissue, to which a drug can be delivered locally to the wall of any hollow "vessel" in the human body, either via an intra-tissue injection or via a catheter or stent.
Besides treatment of vascular pathologies, the invention can also be used for local intervention in cancer with vascular access, and drug delivery in any other tubular structures within the human organism, such as urethra, prostate, fallopian tubes etc. Furthermore, the invention can be useful in the cases where the drug can be locally injected with a needle into a site of a local pathology in a soft tissue. In the field of vascular medicine, the invention can be applied for the treatment or prevention of atherosclerosis, restenosis and thrombosis.

Accordingly, in a preferred embodiment, the present invention provides a method of providing therapeutic formulation at diseased sites in mammalian vessels for creating a local therapeutic effect. This method provides long-term retention of a highly concentrated drug in particular, macrocyclic lactone (such as rapamycin or its analogs) within the target tissue. The invention utilizes in particular, specific non polymeric carriers pie bounded by macrocyclic lactone for penetrating the "leaky" endothelium & delivering the macrocyclic lactone at high tissue concentration even at very low doses using parenteral or local delivery routes using catheters or stents.
Accordingly, the novel formulation of the present invention which is to be delivered to the pathologic target, comprises a macrocyclic lactone effector moiety coupled to a non polymeric vehicle moiety, where the non polymeric vehicle is capable of not being washed away by blood stream and penetrating the 'leaky endothelium' thus entering the vascular wall providing long-term retention of the therapeutic effector therein. An important application of the present method of drug delivery is in treatment of vascular disease, particularly artherosclerosis, restenosis & thrombosis.
According to another preferred embodiment, the present invention discloses pharmaceutical compositions and drugs suitable for this novel method of drug delivery system. The novel method according to the invention is designed to provide a long-term retention of a highly concentrated drug in the site of pathology. The drug suitable for this method of drug delivery system is selected from the group consisting of sirolimus, tacrolimus Everolimus, bioiimus, paclitaxel,etc.
Other examples of drugs include antiproliferative substances such as actinomycin D, or derivatives and analogs thereof. The drugs can also fall under the genus of antineoplastic, antiinflammatory, antiplatelet, anticoagulant, antifibrin, antithrombin, antimitotic, antibiotic, antiallergic and antioxidant substances. Examples of such antineoplastics and/or antimitotics include paclitaxel, docetaxel, methotrexate, azathioprine, vincristine, vinblastine, fluorouracil, doxorubicin hydrochloride, and mitomycin. Examples of such antiplatelets, anticoagulants, antifibrin, and antithrombins include sodium heparin, low molecular weight heparins, heparinoids, hirudin, argatroban, forskolin, vapiprost,

prostacyclin and prostacyclin analogues, dextran, D-phe-pro-arg-chloromethylketone (synthetic antithrombin), dipyridamole, glycoprotein IIb/IIIa platelet membrane receptor antagonist antibody, recombinant hirudin, and thrombin inhibitors such as Angiomax™ (Biogen, Inc., Cambridge, Mass.) Examples of such cytostatic or antiproliferative agents include angiopeptin, angiotensin converting enzyme inhibitors such as captopril, cilazapril or lisinopril calcium channel blockers such as nifedipine, colchicine, fibroblast growth factor (FGF) antagonists, fish oil (omega 3-fatty acid), histamine antagonists, lovastatin monoclonal antibodies (such as those specific for Platelet-Derived Growth Factor (PDGF) receptors), nitroprusside, phosphodiesterase inhibitors, prostaglandin inhibitors, suramin, serotonin blockers, steroids. thioprotease inhibitors, triazolopyrimidine (a PDGF antagonist), and nitric oxide. An example of an antiallergic agent is permirolast potassium. Other therapeutic substances or agents which may be appropriate include alpha-interferon, genetically engineered epithelial cells, tacrolimus, dexamethasone, and rapamycin and structural derivatives or functional analogs thereof, such as 40-O-(2-hydroxy)ethyl-rapamycin, 40-O-(3-hydroxy)propyl-rapamycin, 40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, and 40-O-tetrazole-rapamycin.
A principal feature of the approach of the subject invention, which distinguishes it from other drug targeting and local drug delivery systems is that, in particular, the drug is pre bounded to the long chain fatty acids used as a drug carrier which when interacts with water present in the blood forms micelles there by it prevents the drug to be washed away in the blood stream. These micelles then enter the site of pathology and support for accumulation of a high concentration of the drug for several weeks, providing a rather uniform distribution of the bound drug throughout the tissue.
Further, the current invention do not utilize any polymers and also not programmed to release the drug over a period of time instead utilizing pre bounded drug with long chain fatty acids, which form micelles when interacts with blood. These micelles enter the tissue immediately and make the drug localized there for a time period ranging from days to weeks. Generally, the dosage or concentration of the drug required to produce a favorable therapeutic effect should be less than the level at which the drug produces toxic effects and greater than the level at which non-therapeutic results are obtained.

The inventive mode of local drug delivery as described above can be delivered through stents/orthopedic device/dental device/ENT device or by directly injecting the formulation at the preferred site to achieve the desired the therapeutic effect.
In one preferred embodiment, the delivery of the drug can be accomplished by coating the inventive formulation on the stent using various coating techniques like spraying, dipping etc. Alternatively, the formulation can be delivered using catheter or other instrumental means. The formulation can also be delivered using parenteral route or may be by oral route. The drug concentration in tissue thus achieved using this method ranges from I ng/mg to 300 ng/mg of tissue and the drug concentration in the blood ranges from 0 to 2 ng/ml of blood for a total drug dose of 55 micrograms in the formulation.
Regular systemic delivery of Sirolimus to prevent restenosis using Rapamune tablets of 1 or 2 mg used for renal transplant patients. The local drug concentration thus achieved at the site of restenosis ranges from 1 ng/mg to 5 ng/mg of tissue with high drug concentration in the blood. All the symptoms of side effects for use of sirolimus seen in the patients and the dose achieved in the tissue were not effective to prevent restenosis effectively.
On the other hand, the novel formulation of the current invention when used systemically, the tissue concentration thus achieved using this ranges from 1 ng/mg to 300 ng/mg of tissue and the drug concentration in the blood ranges from 0 to 2 ng/ml of blood for a total drug dose of 55 micrograms in the formulation. Therefore, the high drug concentration in the tissue as achieved using a very low systemic dose (55 micrograms) administered just once is sufficient to prevent restenosis.
Moreover, the disadvantages associated with the drug eluting stents is the presence of drug & polymer for extended period of time results in incomplete healing and partial endothelialization resulting in acute/subacute and late thrombosis. For these reasons patients are put on long term antiplatelet therapy.
However, the current invention successfully overcomes the above disadvantage using the novel formulations as well as novel method of drug delivery. Since the formulation do

not use polymer and therefore, the drug is immediately transferred to the tissue and hence there is no residual drug remains on the stent. Therefore, there will be complete healing of endothelial coverage on the stent and hence there is no need to keep the patient on long term anti platelet therapy.
As compared to polymer based drug delivery from stent, the current technology requires almost 1/41 the drug dose to achieve the required efficacy.
The conventional systemic administration of drug in the form of oral dosage tablets, the local tissue concentration thus achieved is not effective in preventing the proliferation. Moreover, the higher blood concentration resulting from this dose results in drug induced side effects. However, the current invention further ameliorate the drug induced side effects associated with systemic administration by using the inventive technology as the inventive technology provides higher drug concentration at diseased site even at lower dose thereby minimizing the drug concentration in blood stream and hence devoid of any side effects.
Thus the present invention is able to achieve higher drug concentration at the diseased site even at low dose to improve efficacy with out any side effects.
Industrial applicability:
Broadly, this invention can be useful for treatment of a variety of pathologies, which involve a local abnormality in any soft tissue, to which a drug can be delivered locally, to the wall of any hollow "vessel" in the human body, either via an intra-tissue injection or via a catheter or stent. Besides treatment of vascular pathologies, the invention can also be used for local intervention in cancer with vascular access, and drug delivery in any other tubular structures within the human organism, such as urethra, prostate, fallopian tubes etc. Furthermore, the invention can be useful in the cases where the drug can be locally injected with a needle into a site of a local pathology in a soft tissue. In the field of vascular medicine, the invention can be applied for the treatment or prevention of atherosclerosis, restenosis and thrombosis.

The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.
Examples:
Example 1
Drug coating solution preparation:
A solution containing 1.10 gm of Castor oil and 10 gm of pure Ethanol (Solution A) was
prepared.
310 mg of Sirolimus was added and mixed with 1990 mg of solution (A) and added 6.0
gm of Ethanol (Solution B) and then added 1.5 mg of Butylated Hydroxytoluene (BHT)
into solution (B) and mixed the solution until all solute dissolves properly. The solution
thus prepared can be used to coat multiple stents.
Example 2
Drug Coating Process description:
Drug coating process was performed using Sono-tek micromist medicoat spray coating machine (SONO-TEK Corporation, USA).
Drug coatings have been applied to the surface of a medical device by spray-coating with a coating formulation as prepared above. The spray-coating method has been frequently used because of its excellent features, fully controlled by sono-tek program, for e.g., good efficiency, high accuracy and control over the amount or thickness of coating and drug distribution through out the stent length as well.
The Spray coating Sono tek machine containing one micro mist nozzle which carries the nitrogen gas, vertically and two side nozzles horizontally, one is for drug spraying formulation and another is for pre wetting, spraying of ethanol solvent to remove the

excess drug/castor oil from stent. The process sequence is first purging of nitrogen gas to remove the foreign particles, if any, second is drug formulation, then pre wetting, further spraying of drug formulation and finally pre wetting again. This whole process was controlled by machine spray fully automatic program.
To achieve the good coating process objectives, invented method which is efficient and highly controlled to realize a very uniform coating on even a medical device having intricate surfaces. Specifically, a coating formulation, which comprises castor oil. BHT and a solvent, is applied to the surface using a single feed ultrasonic nozzle. This apparatus comprise 'a medicoat bench top glove box type enclosure' and drug solution and pre wetting (ethanol) solution syringe fitted in pump beside of the coating chamber. To apply the coating formulation, the formulation is placed into syringe and fitted in to the pump. Afterwards, droplets of the electrically charged coating formulation were created and dispensed through the opening of nozzle and deposited onto the grounded surface to form a coating on the surface of the medical device.
The coatings produced by the method of the present invention are very uniform. In particular, when a coating formulation is applied to a stent having a tube-like sidewall and openings therein, the coating on both the inside surface of the stent's sidewall and that on the outside surface of the stent's sidewall are uniform. Additionally, the method of the present invention provides a much more efficient means for applying a coating formulation to the surface of a medical device. More specifically, in the present method approximately up to 70% of the coating formulation that is sprayed is deposited on the surface.
Another advantage of the method of the present invention is that, because the atomizing of gas is conducted solely by electrostatic forces, each droplet has very little kinetic energy or moves at very slow velocity. Accordingly, a spray mist of such droplets is less likely to miss the target surface.

Weight measurements were taken before and after coating. Substantial, weight grain is considered a measure of coating deposition. After weighing, the stents coating morphology was observed under microscope for quality inspection.
Further, all the drug coated stents were kept in the vacuum oven at 29" Hg for minimum 12 hrs to maximum 24 hr for vacuum drying to remove the residuals of ethanol. After drying, final quality inspection was carried out to check the coating smoothness, surface morphology, and coating coverage.
Dated this 8th day of October, 2008