DESC:FIELD OF INVENTION
The present invention relates to the field of pharmaceutical drug delivery systems and demonstrated development and characterization of the bleomycin sulfate loaded phospholipid lipid nanoparticle for enhanced tumor delivery of bleomycin sulfate in brain tumor. The phospholipid nanoparticle enhanced the permeation of bleomycin sulfate through blood brain barrier, Sustained release pattern, low biodistribution, enhanced circulation half-life, less lung toxicity and superior antitumor efficacy as compared to commercial formulation (Bleocip).

BACKGROUND OF INVENTION
Brain tumor accounted for death of 250,000 people a year globally, making up less than 2% of cancers. In children younger than 15, brain tumors are second only to acute lymphoblastic leukemia as the most common form of cancer. Brain tumors mainly originate from glial cells and are classified as gliomas. Malignant gliomas represent an incurable disease; indeed, after surgery and chemotherapy, recurrence appears within a few months, and mortality has remained high in the last decades. This is mainly due to the heterogeneity of malignant gliomas, indicating that current available treatment option is not effective for all patients. In this regard, the advent of nanomedicine represents a strategic tool for the treatment of malignant brain tumors.
Phospholipid based nanoparticle (aptly called as phytosomes) has been an attractive carrier due to multiple reasons like biocompatible, non-toxic, able to improve the pharmacokinetics of the encapsulated drug and accumulation in a solid tumor via the enhanced permeability and retention (EPR) effect. Currently Phospholipid based nanoparticle has been used as a carrier in various filed including they have now been extensively researched as novel delivery systems with great potentiality in various medicinal fields including cardiovascular, hepatoprotective, anti-cancer applications. Recently it has widely investigated as a drug delivery carrier for targeting brain. Shihong el al demonstrated the delivery of phospholipid nanoparticle (R780-phospholipid micelles) in brain tumor by using Near infrared fluorescent imaging. The result of study concluded that IR780- phospholipid micelles showed enhanced intra-tumoral accumulation and preferred intracranial tumor accumulation and potent NIRF signal intensity in glioma orthotopic models at a real-time, non-invasive manner.
Mancini et al compared the brain delivery of phenolic compounds through liposome and phospholipid nanoparticles (Aptly named phytosomes). The phospholipid nanoparticles demonstrated enhanced encapsulation/binding efficiency, enhanced delivery of antioxidants into the brain, in comparison to brain.
Bleomycin (BLM), a kind of cytostatic antibiotic complex produced by a strain of Streptomyces verticillus, and was found to be highly effective against human squamous cell carcinoma. Various studies had conducted to demonstrate the efficacy of bleomycin sulfate in brain tumor. Hayakawa et al conducted clinical study to demonstrated uptake of bleomycin in human brain tumor. The results of the study demonstrated that a relatively large amount of bleomycin may be taken up selectively in glioma after intravenous administration, and about two-thirds of the compounds distributed in glioma may remain in the so-called 'active form. Tribhawan et al revealed the survival rate of rat was enhanced after treatment with bleomycin.
Bleomycin sulphate (BLM) is a non-ribosomal peptide which consists of a mixture of BLM A2 (N1-[3-(dimethylsulfonio) propyl]-bleomycinamide) and B2 (N1-[4-(amino imino methyl) amino] butyl]-bleomycinamide). Instrumentally, BLM inhibits the proliferation of cancer cells by breaking the double helical structure of DNA. Bleomycin sulphate is reported to have applicability in glioblastoma. The parenteral bioavailability of BLM is reported to be 70% and 45% following intramuscular and intraperitoneal administrations. However, despite significant systemic bioavailability’s, BLM is labeled as a class III drug in armamentarium of Biopharmaceutical Classification System (BCS) due to the traits of high solubility and low permeability which do not allow bleomycin sulphate to cross blood brain barrier. After intravenous injection of therapeutic doses of bleomycin only small amounts could be measured in glioma tissue obtained at operation in patients with malignant gliomas. Moreover, Bleomycin is inactivated by a cytosolic cysteine proteinase enzyme, bleomycin hydrolase. The enzyme is widely distributed in normal tissues with the exception of the skin and lungs, both targets of bleomycin toxicity include lung toxicity and also responsible for the rapid clearance of bleomycin sulfate from the body with short half-life (T1/2 = 2 hrs). Therefore, there is an urgent need to develop a formulation through which bleomycin sulfate can easily permeate through blood brain barrier with minimum lung toxicity.
The current invention provides novel phospholipid nanoparticles formulation for delivery of anticancer agents such as bleomycin sulfate (BLM). The developed formulation showed significantly higher antitumor efficacy, circulation half-life and safety profile as compared to the current clinical formulation Bleocip. Additionally, the present invention delivered very less amount to lungs, therefor it is deprived of lung toxicity as observed in case of clinical formulation. Apart from this tailored formulation of bleomycin sulphate possess high encapsulation efficiency, high drug loading capacity and low particle size with reduced pulmonary toxic effect. The method of preparation inculcated in present invention is simple and easy to scale up.

Novelty of the invention
The present invention significantly improves the biopharmaceutics of difficult-to-deliver drugs such as BLM through blood brain barrier. In addition, about 2-3- and 4-5-fold increase in the total area under curve for bleomycin A2 and bleomycin B2 was observed in case of bleomycin sulfate loaded phospholipid nanoparticles as compared to bleocip in brain. A significantly smaller elimination rate constant (KE), volume of distribution (Vd) and clearance rate (CLTotal) were observed for bleomycin sulfate lipid nanoparticle (PB3) as compared to bleocip in brain. In addition, in lungs value of Cmax and area under curve for bleomycin A2 and Bleomycin B2 was found to be lower for bleomycin sulfate loaded lipid nanoparticle over market preparation (bleocip)which corresponds accumulation of lower amount of bleomycin sulfate lipid nanoparticles in lungs. Despite, in plasma about 2-3- and 5-6-fold increase in the half life for bleomycin A2 and bleomycin B2 was observed in case of bleomycin sulfate loaded phospholipid nanoparticles as compared to bleocip. In nutshell, the developed formulation is novel among its field and has potential to improve the deliverability of various anticancer drugs such as bleomycin sulfate.
Inventive steps
The phospholipid nanoparticles in the present invention were prepared initially by complex formation between bleomycin sulfate and phospholipid followed by formation of nano size range particles. Exhaustive optimization of the process variables was carried out to maximize the encapsulation efficiency and drug loading and low particle size within the formulation. Further the careful selection of appropriate method led to a surprising increase in an effectiveness of the final formulation in improving the deliverability of bleomycin sulfate.
Industrial applications
Globally Chemotherapeutic drugs for brain cancer treatment market are witnessing significant growth. Bleomycin sulphate has largest market share in the anticancer drug market for brain cancer. Our tailored formulation has industrial application because use, it is Easy scale up, Machinery used in preparation of nanostructure particles is simple and easy to handle. Excipients like phospholipid used in preparation of Nanostructured lipid particles are cheap in cost. Nanostructure phospholipid particles can easily prepare at lab level, small scale level and large-scale level. Excellent properties of Nanostructured lipid particle make them attractive drug carrier system even for pharmaceutical companies. Several companies like Pharma Sol DDS Transo Plex, AlphaRx Vancomycin and Gentamicin products with VansolinTM and ZysolinTM trade names currently developing several nanostructure lipid particles for treatment of various life-threatening disease like pneumonia.
OBJECTIVE OF INVENTION
The main objective of the present invention is to develop phospholipid nanoparticles capable of delivering various difficult-to deliver drugs such as bleomycin sulfate efficiently to brain with less amount deliver in lung.
Another objective of the present invention is to improve a circulation half-life of the drug in physiological conditions and thereby therapeutic efficacy of the formulation without compromising safety profile as compared to the clinical formulation and free drug.
The development of pharmaceutical stable and efficacious formulation with high drug loading, low particle size, which is of high industrial adaptability and applicability is yet another objective of the present invention.

SUMMARY OF INVENTION
The present invention provides a novel phospholipid nanoparticles formulation of various difficult-to-deliver drugs such as bleomycin sulfate with enhanced circulation half-life, enhanced brain delivery, superior therapeutic efficacy and higher safety profile as compared to currently marketed clinical formulation bleocip.
In on embodiment HPLC method of analysis of bleomycin sulfate in bulk form, market preparation and nanoparticle was developed and validated as per ICH guideline. In all parameters %RSD was observed to be less than 2%.

In one embodiment identification of Bleomycin sulfate and other excipient including Phospholipid, cryoportectant was executed by using several spectroscopy techniques i.e., FTIR, DSC, XRD and NMR.

The present invention also provides a process of formation of phospholipid nanoparticle formulation comprising the steps of:
Formation of drug phospholipid complex involves the solubilizing drug and egg phospholipid in suitable organic solvents and reflux for a certain period of time.
Formation of thin film layer of phospholipids and drug.
Hydration of the film formed in step (b) using market saline under suitable conditions
Probe sonication for a suitable time in ice bath solution at 60 amplitudes to yield phospholipid nanoparticles of desired vesicle size and PDI.

In one embodiment, the solvent includes but is not limited to, for example, chloroform, methanol
or mixtures thereof.

In one embodiment, complex formation between drug and phospholipid was carried out by the reflux of drug phospholipid organic solution at 50°C for 4 hours.

In one embodiment of the present invention, the hydration of the film is carried out by using the
suitable market saline followed by vortex for 5 min.

In another embodiment of the present invention, the suitable time for the probe sonication can be
5 min (4 sec on time and 5sec off time).

The phospholipid nanoparticles of the present invention were prepared by thin film hydration technique and systemically optimized for various critical process variables such as Liposolubility of drug phospholipid complex, particle size, poly dispersity index (PDI), drug loading.

In one embodiment of the present invention, Liposolubility of drug phospholipid complex in n-dichloromethane was significantly increased (~10 times) as compared with pure drug

In one embodiment of the present invention, drug loading capacity of phosholipid nanoparticle was ranges from 15-18% and 5-8% for bleomycin A2 and bleomycin B2 of drug phospholipid complex in n-dichloromethane was significantly increased (~10 times) as compared with pure drug

In one embodiment of the present invention, the particle size of nanoparticle ranges from 120-170nm.

In yet another embodiment of the present invention, the phospholipid nanoparticles posed sustained drug release upto 24 h.

In one embodiment of the present invention, the phospholipid nanoparticles formulation of the present invention shows significantly higher in-vitro cytotoxicity as compared to free drug and bleocip.

In a further embodiment, the phospholipid nanoparticle of the present invention shows 2-3- and 4-5-fold increase in the total area under curve for bleomycin A2 and bleomycin B2 was observed in case of bleomycin sulfate loaded phospholipid nanoparticles as compared to bleocip in brain. In lungs 2-3-fold less amount for bleomycin A2 and bleomycin B2 was observed from phospholipid nanoparticle as compared to bleocip. In plasma 1-2-fold less amount for bleomycin A2 and bleomycin B2 was observed from phospholipid nanoparticle as compared to bleocip.

In one embodiment, the phospholipid nanoparticles of the present invention show 2-3 -fold and
5-6 fold enhancement in circulation half-life for bleomycin A2 and bleomycin B2 as compared to bleocip. In brain, phospholipid nanoparticles of the present invention show 1-2 -fold and 4-5 fold enhancement in circulation half-life for bleomycin A2 and bleomycin B2 as compared to bleocip.

The phospholipid nanoparticles formulation was found to stable up to 12 months when tested as per ICH guidelines and posed significant level of industrial adaptability and applicability.

BRIEF DESCRIPTION OF THE FIGURES
Figure 1 illustrate HPLC chromatogram of bleomycin sulfate in bulk form
Figure 2 illustrates images of FTIR spectrum of bleomycin sulfate and bleomycin sulfate phospholipid complex a) FT-IR spectrum of pure drug bleomycin sulfate b) FT-IR spectrum of phospholipid lipoid E 80 c) FT-IR spectrum of 1:3 millimolar ratio drug phospholipid complex
Figure 3 illustrates images of 31P NMR spectra of bleomycin sulfate and bleomycin sulfate phospholipid complex a) P31 NMR spectrum of phospholipid lipoid E 80 b) 31P NMR spectrum of bleomycin sulfate-phospholipid lipoid E 80 complex 1:3 millimolar
Figure 4 illustrates images of DSC thermogram of bleomycin sulfate and bleomycin sulfate phospholipid complex
Figure 5 (a, b) A binding mode of bleomycin A2 into the binding sites of phospholipid E 80; (c) Bleomycin sulfate with interpolated charge surface; (d) Surface representations of phospholipid E 80 with bleomycin A2 (Hydrogen bonding).
Figure 6 illustrates images of particle size of bleomycin sulfate loaded phospholipid nanoparticles (PB3)
Figure 7 illustrates TEM images of bleomycin sulfate loaded phospholipid nanoparticles.
Figure 8 illustrates Release profiles of bleomycin sulfate phospholipid nanoparticle and bleocip in market saline for 24 h.
Figure 9 illustrate XRD Diffractogram of formulation bleomycin sulfate loaded phospholipid nanoparticles (PB3) at different time interval a) XRD Graph of bleomycin sulfate loaded lipid nanoparticle (PB3) at Zero Month b): XRD Graph of bleomycin sulfate loaded lipid nanoparticle (PB3) at first month c) XRD Graph of bleomycin sulfate loaded lipid nanoparticle (PB3) at Third month d) XRD Graph of bleomycin sulfate loaded lipid nanoparticle (PB3) at Six month.
Figure 10 illustrate DSC thermogram of formulation bleomycin sulfate loaded phospholipid nanoparticles (PB3) at different time interval a) DSC Graph of bleomycin sulfate loaded lipid nanoparticle (PB3) at Zero Month (PB3) at Zero Month b) DSC Graph of bleomycin sulfate loaded lipid nanoparticle (PB3) at first month (PB3) at first month c) DSC Graph of bleomycin sulfate loaded lipid nanoparticle (PB3) at Third month (PB3) at Third month d) DSC Graph of bleomycin sulfate loaded lipid nanoparticle (PB3) at Six month
Figure 11 illustrate TEM image of formulation bleomycin sulfate loaded phospholipid nanoparticles (PB3) at different time interval a) TEM image of bleomycin sulfate loaded lipid nanoparticle (PB3) at Zero-month b) TEM image of bleomycin sulfate loaded lipid nanoparticle (PB3) at Six month
Figure 12 illustrate Graph between concentration and percentage cell viability of different formulation of bleomycin sulfate
Figure 13 illustrate Qualitative cell Uptake study. Where, (I) Image with 10X magnification (II) Image with 40X magnification. In both images I and II, (A) NPs Formulation (B) Free FITC. In all the images (a) Images under the FITC channel; Panel (b) Corresponding differential interface contrast images of cells (c) Superimposition of panel (a) and panel (b).
Figure 14 illustrate A) LCMS chromatogram of bleomycin A2 B) LCMS chromatogram of bleomycin B2
Figure 15 illustrates drug plasma/tissue concentration of bleomycin sulfate phospholipid nanoparticle and bleocip in brain, lungs and plasma.
Figure 16 illustrates Histopathological Examination of Control; H& E stain A) Mild focal Astrocyte like cells (Yellow arrow), Mild focal Microvascular proliferation (red arrow), Pleomorphic cell (Black arrow), Minimal necrosis (Green arrow) B) Mild focal Astrocyte like cells (Yellow arrow), Mild focal Infiltration of inflammatory cells (Black arrow).
Figure 17 illustrates Histopathological Examination of Reference (Market Preparation); H& E stain A) Image for reference: glioblastoma: necrosis, cells with astrocyte-like processes and marked nuclear pleomorphism, mitoses and microvascular proliferation (Red arrow) B) Animal No 07: Mild presence of astrocyte like cells (Yellow arrow), Minimal necrosis (Black arrow) area of Hemocyanin (arrow head) C) presence of inflammatory cells, Vascular proliferation (Black arrow) 40X D) presence of hemocyanin areas, Pleomorphic cells (Black arrow) 40X
Figure 18 illustrates Histopathological Examination of Test (Bleomycin sulfated loaded nanoformulation); H& E stain A) (a) Minimal focal astrocyte like cells (Yellow arrow), 40X B) Minimal focal pleomorphic cells (Yellow arrow), 40X C) Minimal infiltration of inflammatory cells, 10X D) Minimal focal pleomorphic cells (Yellow arrow), Minimal focal infiltration of inflammatory cells (Black arrow), 40X

DETAILED DESCRIPTION OF THE INVENTION
Accordingly, the present invention provides bleomycin sulfate loaded phospholipid nanoparticle for enhanced brain tumor delivery of anticancer agents comprising:
a) an anticancer agent as an active ingredient; and
b) Phospholipid nanoparticle composed of complexation between drug and phospholipid wherein, the anticancer agent is physically complex and entrapped within the liposomes.
According to one aspect of the present invention, the anticancer agent is selected from a group comprising bleomycin sulfate, docetaxel, paclitaxel, doxorubicin, methotrexate, hydroxyurea, mercaptopurine, cyclophosphamide, tamoxifen, imatinib, gefitinib, trimetrexate and their salts or polymorphs thereof. According to another aspect of present invention, the anticancer agent is bleomycin sulfate.

In one embodiment of the present invention preferentially egg phospholipid was selected. But selection is not limited to egg phospholipid, soya phospholipid, phosphatidylcholine, Glycerophospholipid, Hydrogenated egg phospholipid, Hydrogenated soya phospholipid, phosphatidylethanolamine, phosphatidylserine

In one embodiment of present invention HPLC method of quantitative determination of bleomycin sulfate in bilk form, market preparation and in nanoparticle preparation was developed and validated as per ICH guidelines.

In one embodiment identification of bleomycin sulfate in bulk form, excipients including phospholipid and existence of any physical and chemical interaction between bleomycin sulfate and excipients in physical mixture were executed by several spectroscopy techniques including FTIR, DSC XRD, and Proton NMR. Observed characteristic peaks in different spectroscopic techniques was concordant with functional groups present in structure of respective phospholipid lipoid E 80 and bleomycin sulfate, which confirmed the purity of component and also confirmed absence of any physical and chemical interaction between bleomycin sulfate and excipients in physical mixture

In one embodiment of the present invention, the drug phospholipid complex was prepared by using reflux process and further nanoparticle was prepared by using thin film hydration technique and subjected to exhaustive optimization. The optimization parameters included Drug: phospholipid: molar ratio, drug loading, particle size, sonication time.

In another embodiment, the drug phospholipid complex of the present invention was characterized for Liposolubility of complex, NMR, DSC, FTIR for confirmation of complex formation
In another embodiment, phospholipid nanoparticles containing the drug phospholipid complex followed of the present invention was characterized for particle size and distribution, ? potential, encapsulation efficiency, percentage drug loading, in-vitro drug release and storage stability.

One of the embodiments of the present invention provides a freeze drying of the phospholipid nanoparticle using freeze drying with mannitol as cryoprotectant.

In another embodiment of the present invention, morphology of the phospholipid formulation was confirmed to spherical using TEM analysis. The particle size was in well correlation with that observed using zeta size.
In another embodiment of the present invention, the phospholipid nanoparticle was subjected to in vitro cell cytotoxicity studies against U87 glioma cancer cell lines. The results revealed significantly higher cytotoxicity of developed formulation as compared to clinical formulation bleocip. The bleomycin sulfate nanoformulation exhibited enhanced efficacy against glioma cell line with an IC50 value of 2.2µM, which was significantly lower than IC50 value of market preparation of bleomycin sulfate (14.8 µM). Blank NLPs did not induce any cytotoxicity to cervical cancer cells.
In one embodiment, permeation of bleomycin sulfate from market preparation and from optimized phospholipid nanoparticle formulation was compared through Bovine brain micro vessel endothelial cell monolayers, the result revealed the amount of bleomycin sulfate from phospholipid nanoparticle (PB3) was found to be higher than market preparation.

In one embodiment bioanalytical method was developed using in plasma as well as tissue and validated as per ICH guideline by using LCMS.

In another embodiment of the present invention, in-vivo pharmacokinetic studies of the phospholipid nanoparticles revealed significantly higher circulation half-life and higher amount of bleomycin sulfate in brain of Swiss albino mice as compared to the clinical formulation bleocip.

Another embodiment of the present invention provides pharmaceutical stability of for 12 months to the phospholipid nanoparticle when evaluated as per ICH guidelines.

The present invention provides an enhanced brain tumor delivery of anti-cancer drugs such as Bleomycin sulfate without compromising the safety profile of the formulation and has great potential in improving the patient compliance by virtue of reduction in dosing and fewer side effects.

In one embodiment, in vitro characterization parameters of market preparation and phospholipid nanoparticle was summarized, which demonstrated that our tailored formulation having advantages over the market preparation.

EXAMPLES

The invention is illustrated by the following examples which are only meant to illustrate the invention and not to act as limitations. All embodiments apparent to a process there in the art are
deemed to fall within the scope of the present invention.

Example 1: Preparation of drug phospholipid complex
Drug Phospholipid complex was prepared by the solvent evaporation method with slight modifications. Drug and Phospholipid E 80 were co solubilized in different ratio in a 20 ml methanol. The mixture was refluxed at 50°C for 4 hr. Further the organic solvent was rotary evaporated under vacuum at 40°C until dried film was obtained. The dried residues were gathered and were stored at -20°C until further use.

Example 2: Preparation of drug phospholipid complex containing phospholipid nanoparticles
Nano-structured lipid particle was prepared by hydrating drug phospholipid complex with 10ml of saline followed by vortexing for 5 min. and followed probe sonication for 5 min in an ice bath to obtain uniform dispersion. The dispersion of Bleomycin sulfate nanoparticles so obtained was finally subjected to lyophilisation (Vertis, Gardiner, New York) using D-mannitol as a cryoprotectant to obtain the product in dry powder form. Blank Nano-structured lipid particle was formed using same above process.
Optimization of Drug phospholipid complex
Sr. No. Optimization parameters Variables Remarks
1 Drug: Phospholipid Ratio (millimolar) 1:1
1:2
1:3
2:1
3:1
1:5 Liposolubility: 2.017±0.333 bleomycin A2 and 1.294±0.208 bleomycin B2 in dichloromethane in mg (3ml)
Reflux: 50°C for 4 hr.
2 Liposolubility in dichloromethane (mg/3ml) Amount of bleomycin A2 Amount of bleomycin B2 Drug: Phospholipid Ratio (millimolar): 1:2 and 1:3
Reflux: 50°C for 4 hr.
0.299±0.068 0.195±0.038
0.324±0.000 0.236±0.019
2.017±0.333 1.294±0.208
0.560±0.032 0.323±0.028
0.181±0.001 0.120±0.008
0.2231±0.000 0.1182±0.001
Optimization of bleomycin sulfate loaded phospholipid nanoparticles
Sr. No. Optimization parameters Variables Remarks
1 Drug: Phospholipid Ratio (millimolar) 1:1 Percentage drug loading : 16.998±0.645 bleomycin A2 and 6.662± 0.227 bleomycin B2 for 1:3 and 19.794± 0.094 bleomycin A2 and 7.881± 0.314 bleomycin B2 for 1:2
Vortexing : 5min
Probe sonication: Amplitude 60, Sonication time: 5mim, 4 sec on time and 5sec off time.
1:2
1:3
2 Percentage drug loading (w/w) Bleomycin A2 Bleomycin B2 Drug: Phospholipid Ratio (millimolar): 1:2 and 1:3
Vortexing : 5min
Probe sonication: Amplitude 60, Sonication time: 5mim, 4 sec on time and 5sec off time.
11.131±0.092 2.581± 0.717
19.794± 0.094 7.881± 0.314
16.998± 0.645 6.662± 0.227
2 Particle size (nm) 251 Drug: Phospholipid Ratio (millimolar): 1:2 and 1:3
Vortexing: 5min
Probe sonication: Amplitude 60, Sonication time: 5mim, 4 sec on time and 5sec off time.
167.9
160.2±18.73

Based on the optimization studies carried out, a set of parameters were found to be yielding phospholipid nanoparticles with vesicle size in a range of <160nm, entrapment efficiency <75%, and PDI <0.250. These optimized parameters are as mentioned in Table1.

Table 1: Optimized parameters for preparation of bleomycin sulfate loaded phospholipid nanoparticles
Sr. No. Optimization parameters Variables
1 Drug: Phospholipid Ratio (millimolar) 1:3 (Bleomycin sulfate: 30.32%w/w
(Egg phospholipid: 49.59%w/w)
2 D-mannitol 20.08%w/w
3 Percentage drug loading (w/w) Bleomycin A2
16.998± 0.645 Bleomycin B2
6.662± 0.227
4 Particle size (nm) 160.2±18.73
5 Probe Sonication Amplitude 60, Sonication time: 5mim, (4 sec on time and 5sec off time).

Example 3 Analytical method development and validation using HPLC
Chromatographic Conditions
Instrumentation: The HPLC (Shimadzu, Kyoto, Japan) instrument was equipped with two LC-10 ATVP pumps, SPD-10AVP UV-Visible detector, and Rheodyne injector with a 50 ??L loop. The results were acquired and processed using Shimadzu LC-solution version 6.42 software for data acquisition and processing.
Mobile Phase: Mobile phase was prepared by transferring 0.96 gm of anhydrous 1-pentane sulphonic acid sodium salt and 1.86 gm of EDTA disodium salt to a 1000 ml volumetric flask. The pH was adjusted to 4.3 using glacial acetic acid (GAA) or ammonia solution. The mobile phase was filtered through 0.45 µm filter (Millipore) and degassed before use. In parallel, a gradient of 10% to 40 % methanol was also used as a part of mobile phase.
Flow rate: 1.5 ml/min.
Absorption maxima: 254 nm
Injection Volume: 10µl
Column: Nucleodur C18 (250mm × 4.6mm I.D., 5 ??m)

Validation and Forced degradation study developed HPLC method of bleomycin sulfate
The concentration ranges were selected for linearity parameter in between 1250-3750µg/ml. HPLC chromatogram of bleomycin A2 and bleomycin B2 was demonstrated in figure 1. In each parameter % RDS was found to be less than 2%. In forced degradation study, bleomycin sulfate having maximum degradation in acidic media, basic media, oxidative media and observed less degradation in UV media.
Table 2: Validation parameters of HPLC analysis of bleomycin sulfate.
S.no. Parameter Bleomycin A2 Bleomycin B2
1 Concentration range (µg/ml) 1250-3750 1250-3750
2 Retention time 38.84min. 47.46min.
3 Media Triple distilled water
4 Regression Equation Y=2361X-73107 Y=1061X-37124
5 Slope 2361 1061
6 Intercept 73107 37124
7 Correlation coefficient 0.998 0.998
8 Accuracy % Recovery
8.1 80% 63.662 28.718
8.2 100% 63.89133 28.641
8.3 120% 63.402 29.20467
9 Precision (%RSD) Less than 2%
10 Robustness (%RSD) Less than 2%
Table 3: Degradation of bleomycin sulfate in different media.
Conditions Conc. (µg/ml) Time period % Degradation of A2 % Degradation of B2
Acid Degradation 2500 1 hr 99.356 82.0194
2500 2 hr 99.408 83.0215
2500 4 hr 99.876 88.772
Basic Degradation 2500 1 hr 99.351 78.289
2500 2 hr 99.409 79.887
2500 4 hr 99.876 87.273
Oxidative Degradation 2500 1 hr 98.231
99.423
2500 2 hr 98.101 99.184
2500 4 hr 99.538 99.679
Thermal Degradation 2500 1 hr 1.1247 99.42
2500 2 hr 12.330 99.18
2500 4 hr 12.196 99.67
UV Degradation 2500 1 hr 6.385 4.988
2500 2 hr 5.603 -0.675
2500 4 hr 8.307 4.685

Example 4: Physicochemical characterization of the prepared formulations
The prepared bleomycin sulfate loaded phospholipid nanoparticles were characterized as follows:
Liposolubility or Equilibrium solubility study of bleomycin sulfate phospholipid complex
The equilibrium solubility studies of the pure drug and complex was carried out in saline and dichloromethane by shake flask method. Accurately weighed quantities of the pure drug, and complex were taken separately in culture tube. Subsequently, 3ml dichloromethane was added to each culture tube and vortex for 15 min to obtain uniform dispersion. Furthermore 3 ml of saline was added into dispersion and kept on the water bath shaker for 24 hr at 25°C and 100 rpm. After 24 hr dispersion was transferred into separating funnel with continuous shaking for 5min. followed separating funnel was allowed to stand for 15 min. Both the phases were separated and collected indifferent vials each phase was centrifuged 15000rpm for 10min. to separate insolubilized drug. Dichloromethane fraction was subjected for extraction of the drug using methanol as the extracting solvent to determine solubility of drug into organic phase. The amount of drug solubilizes in aqueous and organic phase was estimated by RP-HPLC

Table 4: Equilibrium solubility millimolar ratio drug phospholipid complex in dichloromethane
Formulation code Amount of bleomycin A2 in dichloromethane in mg (3ml) Amount of bleomycin B2 in dichloromethane in mg (3ml)
1:3 2.017±0.333 1.294±0.208

Liposolubility of phospholipid complex in n-dichloromethane was significantly increased (~10 times) as compared with pure drug. However, it was not significantly enhanced by mere mixing of drug and phospholipid. This indicated the enhancement of Liposolubility of hydrophilic drug, following complexation with phospholipid.

Confirmation of bleomycin sulfate- phosholipid complex by spectroscopies techniques
FT-IR (Fourier transform infrared spectroscopy)
The spectrum was recorded drug phospholipid complex 1:1, 1:3, 1:5 millimolar ratios by using infrared spectrophotometer Perkin Elmer - Spectrum RX-IFTIR. Samples were prepared in KBr disk (2 mg sample in 200 mg KBr) with a hydrostatic press at a force of 40 psi for 4 min. The scanning range employed was 400-4000 cm-1 at a resolution of 4 cm-1. Figure 2
The FTIR spectrum of the drug phospholipid complex displayed the changes in wavenumber as compare to FTIR spectrum of pure drug and phospholipid.

Phosphorous NMR (31P NMR)
Process: 31P NMR spectra of samples were recorded on a MHz JEOL JNM ECS400 in a 5-mm broad band probe at 25°C as previously reported. Samples (Lipoid E 80 and bleomycin sulfate-lipoid E 80 complex (1:1, 1:3 millimolar ratio) were dissolved in CD3OD. Figure 3
In 31P NMR spectra, chemical shift d (ppm) of phosphorous in phospholipid showed value of 0.7274, which was shifted to 0.7543 in drug phospholipid complex, confirmed existence of interactions between phospholipid and bleomycin sulfate and formation of drug phospholipid complex

DSC (Differential scanning calorimeter)
Process: DSC thermogram of drug phospholipid complex was recorded in a Differential Scanning Calorimeter (Shimadzu, Model no: DSC-60). Samples were sealed in aluminum pan and scanned between 25°C and 300°C with heating rate of 10 °C per minute under an atmosphere of dry nitrogen. Figure 4
DSC thermogram of bleomycin sulfate-phospholipid E 80 complex showed complete disappearance of the endothermic peaks of the individual component and exhibited a broad new peak at about 98.76 °C (DHf = 108.62 J/g) was supported the interaction between bleomycin sulfate molecule and phospholipid E 80 lipid.

Molecular docking
The structure of phospholipid and bleomycin sulfate was draw by using Chem Bio Draw Ultra 12.0, minimized using MMFF94 and used for docking. The docking free energy was -1.753 Kcal/mol. Figure 5
Figure 4 (a, b) showed binding mode of bleomycin sulfate with phospholipid E 80, the hydrogen of -NH- between bithiazole and threonine fragments making interaction with oxygen of phospholipid E 80 with distance of 1.81 Å, hydrogen of a-D-mannose amine with oxygen of -C=O, with 1.97 Å. The two hydrophobic arms of phospholipid hold carbohydrate (a-D-mannose, a-L-glucose) and pyramidinyl propionamide fragments. Figure 53 (c, d), surface representation of phospholipid E 80 with bleomycin sulfate.

Size, size distribution and zeta potential
The particle size of tailored lipid nanoparticle was measured by Particle size analyzer, UK. Briefly 10 mg sample of each lyophilized nanoformulation was suspended separately in distilled water and sonicated to remove air bubbles. The zeta potential of each nanoformulation was measured at similar concentration using zeta sizer. Figure 6

Encapsulation efficiency and Percentage drug loading
10 mg of lyophilized bleomycin sulfate loaded lipid nano-particle was dispersed into 5 ml of saline to obtain dispersion. Dispersion was centrifuged at 15,000 rpm for 15 min at 4°C. The supernatant liquid was then filtered through 0.22 µm membrane filter and assayed by HPLC
Percentage encapsulation efficiency and drug loading were calculated as follows
Percentage drug encapsulation efficiency =(Initial Amount of drug-Final amount of drug)/(Initial amount of drug)×100
Percentage drug loading =(Weight of drug in phospholipid nanoparticle)/(Weight of phospholipid nanoparticle)×100
Table 5 represents various quality attributes of bleomycin sulfate loaded phospholipid nanoparticle formulations.

Table 5: Critical quality attributes of bleomycin sulfate loaded phospholipid nanoparticles
Formulation Size(nm) PDI ZP(mV) %EE of Bleomycin A2 % EE of Bleomycin B2 % drug loading of Bleomycin A2 % drug loading of Bleomycin B2
BLM phospholipid nanoparticles (PB3) 160.2±18.73 0.237±0.070 -21.02±2.50 72.953±2.769 64.043± 2.188 16.998± 0.645 6.662± 0.227
Values are expressed in mean ± SD (n=6);
BLM: Bleomycin sulfate

Shape and morphology of prepared formulations
The surface morphology of lyophilized lipid nanoparticle was examined using transmission electron microscope (FTI Tecnai F20). An aqueous suspension of nanoparticle was drop cast onto a carbon coated grid which was then air dried at room temperature before loading into microscope, maintained at a voltage of 80 KV. Figure 7
TEM images revealed that the surface topography of PB3 formulation indicated that nanoparticle was smooth, spherical in shape and small in size.

In-vitro drug release
Freeze dried bleomycin sulphate loaded lipid nanoparticle powder equivalent to 9.8 mg of bleomycin sulphate drug or 15 units of bleomycin drug was added into a 10ml media (saline). Saline was used as a dissolution medium that was maintained at 37°C and 100 rpm at predetermined time intervals 0, 0.083, 0.17, 0.5, 1, 2, 4, 6, 12, 24, 1 ml dissolution medium was withdrawn and replaced with fresh medium to maintain the sink condition. The entire samples were filtered through 0.22µm membrane filter. Figure 8
Percentage release of drug from three formulations (Marketed formulation of Bleomycin sulfate, Bleomycin sulfate lipid nanoparticles) was calculated and present in figure 7. Bleomycin sulfate loaded nanostructured phospholipid particles demonstrated a sustained release pattern 54.4015±1.63% within 24hrs while bleomycin sulfate lipid particles and marketed formulation of bleomycin sulfate.

Example 5: Storage stability studies
Percentage entrapment and drug loading
Percentage entrapment and drug loading bleomycin sulfate lipid nanoparticle at 4°C, 25°C/60%RH and 40°C/75%RH over different time interval is as given in below table.
Table 6: Percentage entrapment and drug loading of bleomycin sulfate loaded lipid nanoparticle (PB3) at different time interval over 4°C
S.no. Month Percentage entrapment of bleomycin A2 Percentage drug entrapment of bleomycin B2 Percentage drug loading of bleomycin A2 Percentage drug loading of bleomycin B2
1 Zero month 69.182±1.9613 57.739± 2.867 14.64± 0.90 5.49± 0.71
2 First Month 65.117±2.9138 55.306± 2.729 13.83± 1.82 5.25± 0.47
3 Third month 65.50±5.2168 55.192± 3.382 13.95± 2.34 5.25± 0.74
4 Six month 64.884±3.8459 54.616± 8.094 13.74± 1.26 5.22± 1.17
5 Twelve month 63.016±2.58 53.256±1.471 13.71± 1.96 5.006± 0.39

Table 7: Percentage entrapment and drug loading of bleomycin sulfate loaded lipid nanoparticle (PB3) at different time interval over 25°C/60%RH
S.no. Month Percentage entrapment of bleomycin A2 Percentage drug entrapment of bleomycin B2 Percentage drug loading of bleomycin A2 Percentage drug loading of bleomycin B2
1 Zero month 69.182±1.9613 57.739± 2.8671 14.64± 0.90 5.49± 0.71
2 First Month 65.17±1.10 56.77± 3.83 13.80±1.05 5.41± 0.81
3 Third month 64.98±2.17 53.39± 4.49 13.75±0.71 5.095± 0.86
4 Six month 64.48±1.14 52.31± 0.29 13.66±1.16 4.97± 0.42
5 Twelve month 63.62±1.60 52.21± 4.73 13.83±1.76 4.92± 0.70

Table 8: Percentage entrapment and drug loading of bleomycin sulfate loaded lipid nanoparticle (PB3) at different time interval over 40°C/75%RH
S.no. Month Percentage entrapment of bleomycin A2 Percentage drug entrapment of bleomycin B2 Percentage drug loading of bleomycin A2 Percentage drug loading of bleomycin B2
1 Zero month 69.182±1.9613 57.739± 2.8671 14.64± 0.90 5.49± 0.71
2 First Month 64.71±1.45 54.03± 3.26 13.70±0.91 5.14± 0.73
3 Third month 63.65±2.08 50.83± 3.73 13.47±0.70 4.84± 0.76
4 Six month 61.74±0.84 50.37± 1.41 13.08±1.03 4.78± 0.30
5 Twelve month 60.78±3.88 50.75± 4.17 13.24±2.19 4.77± 0.63

Result: The nanoformulation did not showed the major changes in percentage drug entrapment and percentage drug loading at all three storage condition.

Particle size and Zeta Potential
Table 9: Particle size, PDI and Zeta Potential of bleomycin sulfate loaded lipid nanoparticle (PB3) at different time interval over 4°C
S.no. Month Particle size (nm) PDI Zeta Potential (mv)
1 Zero month 160.2±18.73 0.237±0.070 -21.02±2.500
2 First Month 215.96±17.044 0.122± 0.018 -
3 Third month 236.63±11.55 0.219± 0.0070 -19.3
4 Six month 284.53±26.34 0.279± 0.079 -18.5
5 Twelve Month 263.34±77.85 0.297± 0.080 -12.05

Table 10: Particle size, PDI and Zeta Potential of bleomycin sulfate loaded lipid nanoparticle (PB3) at different time interval over 25°C/60%RH
S.No. Month Particle size (nm) PDI
1 Zero Month 160.2±18.73 0.237±0.070
2 First Month 264.6±13.16 0.233±0.01
3 Third month 275.9±35.21 0.256±0.02
4 Six month 271.98±34.83 0.233±0.03
5 Twelve month 291±5.47 0.20±0.06

Table 11: Particle size, PDI and Zeta Potential of bleomycin sulfate loaded lipid nanoparticle (PB3) at different time interval over 40°C/75%RH
S.No. Month Particle size PDI
1 Zero Month 160.2±18.73 0.237±0.070
2 First Month 306.36±32.91 0.270±0.24
3 Third month 404.03±13.16 0.30±0.30
4 Six month 324.33±3.00 0.16±0.01
5 Twelve month 475.9±142.92 0.32±0.26

Result: The nanoformulation did not showed the major changes in Particle size and PDI at all three storage conditions except the storage condition 40°C/75%RH displayed the slightly higher size of particles.

TEM analysis
Process: The surface morphology of lyophilized lipid nanoparticle was examined using transmission electron microscope (FTI Tecnai F20). An aqueous suspension of nanoparticle was drop cast onto a carbon coated grid which was then air dried at room temperature before loading into microscope, maintained at a voltage of 80 KV.

Transmission electron microscopy image of bleomycin sulfate loaded lipid nanoparticle (PB3) at different time interval over 4°C, 25°C/60%RH and 40°C/75%RH
The particle shape is also a key parameter in therapeutic efficacy analysis of a nanoformulation. TEM was used to visualize the morphology of lipid nanoparticles at different time interval over storage period. The surface topography of PB3 formulation indicated that nanoparticle was smooth respectively.
Example 6: In vitro cell line study using U87 glioma cell line

MTT assay
Cell viability will be determined by standard colorimetry based, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assay using 96 wells microtiter plate. 5×103 U87 glioblastoma cells will be plate in 200 µL of the serum DMEM medium and would replace with serum free-DMEM after 24 hr of incubation period. U87 cells then expose continuously with market preparation of bleomycin sulfate, bleomycin sulfate loaded lipid nanoparticles, and respective blank nanoparticles sample at a concentration ranging from 0.5 µM to 500 µM (0.5, 1, 5, 20, 40, 100, 250, 500) for three different time intervals 24 hr, 48 hr and 72 hr. At the end of treatment, medium will be removed and control and treated cells will be incubated with MTT (0.5 mg/mL) for 4 h at 37 °C. The cells will lyse and the formazan crystals will be dissolved using 100 µl of dimethyl sulfoxide. The absorbance will be measure at 570 nm using 630 nm as reference wavelength by ELISA (Enzyme linked immunosorbent assay) reader. The cytotoxicity of Market preparation of bleomycin sulfate and bleomycin sulfate loaded nanoparticle (PB3) was measured against U87 (Glioma cell line) and expressed in terms of IC50 (concentration of drug required to kill 50% of the cells) value. The bleomycin sulfate nanoformulation exhibited enhanced efficacy against glioma cell line with an IC50 value of 2.2µM, which was significantly lower than IC50 value of market preparation of bleomycin sulfate (14.8 µM). Blank NLPs did not induce any cytotoxicity to cervical cancer cells. Figure 12

In vitro bovine brain micro vessel endothelial cell assay
Bovine brain micro vessel endothelial cell monolayers packed on a polycarbonate membrane were placed in a Side-By-Side Franz diffusion cell containing 10 ml of continuously stirred physiological assay buffer (122 mM NaCl, 3.0 mM KCl, 1.2 mM MgSO4, 25 mM NaHCO3, 0.4 mM K2HPO4, 1.4 mM CaCl2, 10 mM D-glucose, and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) on each side at 37°C. At time 0, bleomycin sulfate pure drug and bleomycin sulfate loaded lipid nanoparticle equivalent to 9.8 mg of bleomycin sulfate was added to the donor chamber, and 0.1ml aliquots were removed from the receptor chamber at various time points (15, 30, 60, 90, and 120 min) and stored for high-performance liquid chromatography (HPLC) analysis. An equal volume of assay buffer was added to replace the aliquots removed.

Table 12: Amount of bleomycin sulfate permeated form market preparation and phospholipid nanoparticle (PB3) in mg
Time (min.) Amount of bleomycin sulfate permeated form market preparation in mg Amount of bleomycin sulfate permeated form phospholipid nanoparticle (PB3) in mg
15 0.065±1.67 0.061±2.01
30 0.098±1.37 1.38±2.34
60 0.14±1.19 4.39±1.89
90 0.64±2.01 7.15±2
120 0.98±2.39 7.48±1.11
180 1.01±2.2 7.46±1.74

To examine the ability of bleomycin sulfate from market preparation and phospholipid nanoparticle to cross the blood-brain barrier, we used a well-characterized in vitro assay. The amount of bleomycin sulfate from market preparation can cross a layer of cultured brain microvascular endothelial cells separating a donor and receiver chamber was determined and compared with amount of bleomycin sulfate permeated from phospholipid nanoparticle (PB3). Bleomycin sulfate from phospholipid nanoparticle was detected in the receiver chamber at higher amount 7.48±1.11mg as compare to market preparation of 0.98±2.39mg at 120 min.

Cellular uptake
Briefly, U87 MG cells were grown in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin (PAA, Austria) under controlled conditions (5% CO2 atmosphere and 37 °C). Once 90% confluence of cell culture medium was attained, cells were washed thrice with Hank’s Buffered Salt Solution (HBSS) Solution (PAA, Austria) and harvested by 0.25% trypsin- EDTA solution (Sigma) followed by culturing the cells at a density of 50,000 cells/well in 96 well plate (Costars, Corning Incorporated) for subsequent qualitative and quantitative uptake studies. Qualitative uptake study was carried out to study the uptake bleomycin sulfate NPs, where FITC encapsulated NPs were prepared same method as method used for preparation of bleomycin sulfate NPs. The cell was incubated with free FITC and FITC loaded NPs (equivalent to 1 µg/ml of free FITC) for 4 h and extracellular particles were removed by washing with HBSS. The cells were observed under confocal laser microscope (CLSM) (Olympus FV1000).
CLSM image as shown in figure 13 cellular uptake of intensity of dye from phospholipid nanoparticle was found to be higher than free dye has been showing the higher cellular uptake of phospholipid nanoparticle.

Example 7: Bio-analytical Method development and validation in plasma, tissue by using LCMS
LCMS method development
Instrumentation
The LCMS/MS (DS Sciex)
Mobile Phase: Mobile phase A: Acetonitrile in 0.1% TFA- 12%
Mobile phase B: 50Mm Ammonium formate in 0.1% formic acid- 88%
Flow rate: 0.4 ml/min.
Auto samples temperature: 5°C
Column over temperature: 40°C
Injection Volume: 10µl
Column: Waters Symmetry, 100*4.6mm, 5µ
Run time: 6.0min.
Retention time: Bleomycin A2: 2.75±0.5 min.
Bleomycin B2: 4.75±0.5 min.
Kanamycin (ISD: 1.16 ±0.5 min.
MS/MS condition
Mode: Positive
Collision energy:
Bleomycin A2: 45
Bleomycin B2: 39
Kanamycin (ISD: 12
Ion spray voltage: 2500
Heater temperature: 400
Run time: 6.0min.
LCMS chromatogram of bleomycin A2 and bleomycin B2 was demonstrated in figure 14. In each parameter % RDS was found to be less than 2%.

Preparation of Plasma, Brain and Lung samples for LCMS/MS analysis
Aliquots 200µl plasma samples in prelabeled polypropylene tubes were mixed with 50 µl CuSo4, Vortex few seconds and keep it for 25min. Add 50 µl of internal standard to plasma samples and vortex for 30min. Add 100 µl of 0.5% formic acid in water, vortex few seconds. Further add 3.0 ml of methanol, vortex for 10min at 1800rpm and centrifuge at 5000 rpm for 5min. at 50°C. Transfer about 2.8ml of supernatant organic layer into p prelabeled polypropylene tubes and evaporate in dry nitrogen. Reconstitute the dry residues with 150 µl of reconstitution solution and vortex for 2 min. Transfer the samples in autosampler and analyze using LCMS. In case of brain lung samples, crus h the samples and process the method as used in preparation of plasma samples.

In vivo pharmacokinetic studies
The study was performed to assess the ‘Pharmacokinetic and Bioavailability study of Bleomycin sulfate preparations Nano formulation (1.65mg of Bleomycin/ml) and Market preparation (2mg of Bleomycin /ml) in plasma and determination of drug concentration in different organs (Brain, Lungs) of Swiss albino mice’. The study design consists 66 Male animals divided into two groups; G1 (AID: 1 to 33) & G2 (AID 33-66). Both test and reference item were administered via intravenous at dose level of 5 units (15mg of formulation is equivalent to 5units of bleomycin sulfate). After dosing, blood samples were collected from G1 (AID: 1 to 33) & G2 (AID 33-66) group of animals from 1:00 minute to 24:00 hours post dosing at the time points of 1, 5, 15 and 30 minutes, 1hr, 2hr, 4hr, 6hr, 8hr, 12hr and 24hr. Approximately (~300µl) blood was collected through retro orbital plexus using capillary tube into pre-labeled K3EDTA vacutainers (containing K3EDTA as anticoagulant) and was kept cool on ice pack prior to centrifugation. Centrifugation was done at 3000 rpm, 4°C for 10 minutes. At each time point, blood sample was collected from three animals of each group (G1 and G2) and were euthanized (within 15minutes) with CO2 and the lungs and brain were collected. Both plasma and organ sample were stored at -70°C until the analysis. Plasma and organ samples were assayed by LCMS/MS method developed at bio-analytical facility, corporate office, Sipra labs Limited. The method was specific for the determination of bleomycin-A2 and bleomycin-B2 of the bleomycin injection. Pharmacokinetic parameters such as kel, t ½(hrs), Vd, CLtotal, Cmax, AUC (0-t), AUC (0-8), and tmax for plasma, organ (Brain and Lungs) concentration profile was estimated. Figure 15
The phospholipid nanoparticle of the present invention shows 2-3- and 4-5-fold increase in the total area under curve for bleomycin A2 and bleomycin B2 was observed in case of bleomycin sulfate loaded phospholipid nanoparticles as compared to bleocip in brain. In lungs 2-3-fold less amount for bleomycin A2 and bleomycin B2 was observed from phospholipid nanoparticle as compared to bleocip. In plasma 1-2 fold less amount for bleomycin A2 and bleomycin B2 was observed from phospholipid nanoparticle as compared to bleocip. The phospholipid nanoparticles show 2-3 -fold and 5-6 fold enhancement in circulation half-life for bleomycin A2 and bleomycin B2 as compared to bleocip. In brain, phospholipid nanoparticles of the present invention show 1-2 -fold and 4-5 fold enhancement in circulation half-life for bleomycin A2 and bleomycin B2 as compared to bleocip.

Table 13: Pharmacokinetic parameters of various bleomycin sulfate loaded formulations in brain
Form Kel T ½(hrs) Vd, CL Totoal Tmax
hrs Cmax
ng/ml AUC (0-t) ng/ml AUC(0-8)
hrs.ng/ml
Bleomycin A2 Conc. Brain
Reference
(Market preparation) 0.0709 9.7719 0.0411 0.0029 0.0166 60.7660 255.8721 342.9029
Test
(Bleomycin sulfate lipid nanoparticles formulation code PB3) 0.0692 10.0238 0.0167 0.0012 1.0000 60.2366 663.9724 867.4855
Bleomycin B2 Conc. Brain
Reference
(Market preparation) 0.1558 4.4492 0.7869 0.1226 0.0166 8.8433 6.7319 8.1568
Test
(Bleomycin sulfate lipid nanoparticles formulation code PB3) 0.0345 20.0864 0.8253 0.0285 0.5000 4.8200 14.5364 35.1112

Table 14: Pharmacokinetic parameters of various bleomycin sulfate loaded formulations in lungs
Form Kel T ½(hrs) Vd, CL Totoal Tmax
hrs Cmax
ng/ml AUC (0-t) ng/ml AUC(0-8)
hrs.ng/ml
Bleomycin A2 Conc. Lung
Reference
(Market preparation) 0.2916 2.3767 0.0888 0.0259 0.0166 51.0400 34.3885 38.5943
Test
(Bleomycin sulfate lipid nanoparticles formulation code PB3) 0.0860 8.0574 0.7162 0.0616 0.0833 10.2933 11.8141 16.2314
Bleomycin B2 Conc. Lung
Reference
(Market preparation) 0.1953 3.5497 0.0490 0.0096 0.0166 85.5700 98.6545 104.5950
Test
(Bleomycin sulfate lipid nanoparticles formulation code PB3) 0.1019 6.8015 0.1890 0.0193 0.0833 32.6566 42.4107 51.9289

Table 15: Pharmacokinetic parameters of various bleomycin sulfate loaded formulations in plasma
Form Kel T ½(hrs) Vd, CL Totoal Tmax
hrs Cmax
ng/ml AUC (0-t) ng/ml AUC(0-8)
hrs.ng/ml
Bleomycin A2 Conc. Plasma
Reference 0.2944 2.3544 0.0004 0.0001 0.0833 13690.813 9376.0767 9393.7277
Test 0.1531 4.5266 0.0022 0.0003 0.2500 4248.9466 2975.0883 2994.6577
Bleomycin B 2 Conc. Plasma
Reference 0.2550
2.7182 0.0002 0.0001 0.0833 22517.4766 19173.5399 19283.2096
Test 0.0496 13.9656 0.0020 0.0001 0.2500 12985.4800 9372.3748 9840.6843
All values are expressed as mean ± SEM (n=3)

ACUTE INTRAVENOUS TOXICITY STUDY OF BLEOMYCIN SULPHATE NANOFORMULATION IN RODENTS
Study design
• Toxicity guideline: OECD Guideline 423
• Protocol No: NIP/07/2020/PC/393
• Animal: Female rats
• Number of animals: 06
• Dose: 210 mg/kg
• Dose volume: 2.5 ml/kg gm body weight
• Duration: 14 Day

Observation
All the animals were observed for death or mortality, Lacrimation, Salivation, Bizarre Behavior, Body weight, Food consumption during the observation period of 14 days and Gross Necropsy at end of the study.

Result
•Death or mortality- No mortality; Lacrimation- No lacrimation; Salivation- No salivation.
• Bizarre Behavior - Not observed.
• Gross Necropsy: All the major organs were found to be normal in size and appearance
• Body weight: Total body weight gain was found to normal in 14-day duration
• Food consumption: Food consumption by each animal was found to be normal for 14 days.

Conclusion
Base on the data obtained for Acute Intravenous toxicity study of Bleomycin Sulphate Nanoformulation in Rodents at the dose of 210 mg/kg, it can state that the Maximum Tolerable Dose (MTD) was found to be 210 mg in experimental animals (rat).

REPEATED DOSE 28-DAYS INTRAVENOUS TOXICITY STUDY OF BLEOMYCIN SULPHATE NANOFORMULATION IN RODENTS
Study design
• Toxicity guidelines: OECD Guidelines 407
• Animal Ethical Protocol Approval No: NIP/07/2020/PC/393
• Animal: Male and Female rats
• Number of animals: 72
• Duration: 42 Days (28 Days +14 Days Reversal)
Table 16: Details of Group
Group No. Group details Dose No. of Animals
G1 Normal control Phosphate saline buffer (PBS) 10 ml/kg i.v. (6M + 6F)
G2 Low Dose (1/10 of LD 50) 21 mg/kg i.v. (6M + 6F)
G3 Mid Dose (1/5 of LD 50) 42 mg/kg i.v. (6M + 6F)
G4 High Dose (1/3 of LD 50) 70 mg/kg i.v.. (6M + 6F)
G5 Reversal Control (RC) Phosphate saline buffer (PBS) 10 ml/kg i.v. (6M + 6F)
G6 Reversal Control of High Dose 70 mg/kg i.v.. (6M + 6F)

Reversal control and Reversal control of 5 x Therapeutic dose will be kept for additional 14 days after treatment.

Preparation of The Test Item
The test item Bleomycin Sulphate Nanoformulation was dissolved in water and administered through intravenous (i.v.) route. The details of the doses and the concentration of the dosing solutions are given in test method section.
Table 17: Test Method
Animal model and Rout of administration Group No. Dose Frequency of dose No. of Animals
Rat and Intravenous G1 Normal control Once a week 12 (6M+6F)
Once a week
G2 Low dose : 21 mg/kg i.v. Once a week 12 (6M+6F)
Once a week
G3 Mild dose: 42 mg/kg i.v. Once a week 12 (6M+6F)
Once a week
G4 High dose: 70 mg/kg i.v. Once a week 12 (6M+6F)
Once a week
G5 Reversal control Once a week 12 (6M+6F)
Once a week
G6 High dose:70 mg/kg i.v.. Once a week 12 (6M+6F)
Once a week

Test Procedure
Animals of different treatment group and doses were administered intravenous at dose volume of 2.5 ml/kg gm body weight. Animals were assessed for their behavior changes after each exposure. Moreover, after the exposure period on 28 day hematological and biochemical parameters were investigated. However, animal studies are subject to variation owing to age, sex, body weight, species, and environmental factors.

Result
Body Weight Recording
The progress in the body weights of each animal recorded prior to the test item administration (Day 0) and on Day 7, 14, 21, 28, 35 and 42 of the experiment.
Clinical Observation
Clinical signs observed so far in all the animals of different treatment groups are presented as follows:
• Lacrimation: Not observed.
• Salivation: Not observed.
• Bizarre Behavior: Not observed.
• Grooming: Normal
• Rearing: Normal
• Fur Quality: No change
• Posture: No abnormality in posture detected.
Clinical Biochemistry
Biochemical parameters are presented in table 57 and 58 (Male and Female) respectively.
Hematological parameters are presented in table 59 and 60 for rats (Male and Female) respectively. Organ Weight
At the end of experiment at 28 day and 42 days, all the animals were sacrificed and their visceral organs were collected and weighed.
Histo-Pathology Examination
Detailed histopathological observations of the animals of various groups and doses were found to be normal in size, color and appearance.
Conclusion
Based on the data obtained, it can be stated that, Bleomycin Sulphate nanoformulation at low dose (1/10 of MTD), mid dose (1/5 of MTD) was found to be safe. The No Observed Adverse Effect level (NOAEL) was found to be 21 mg/kg and Low Observed Adverse Effect Level (LOAEL) was found to be 70 mg/kg.

IN-VIVO ANTITUMOR EFFECTS OF BLEOMYCIN SULFATE NANOFORMULATION (PB3)
Objective: The objectives of the study was to standardize the method for development of xenograft glioblastoma brain tumor model in Swiss albino mice and evaluate the antitumor effects of Bleomycin sulfate nanoformulation.

10.2 Summary: Summary of the study is as follows:
Table 18: Summary of In vivo brain tumor model
Study No. PRADO/B-2106
Reference item Market Preparation (Bleochem, Manufacturer: Zydus celexa (equivalent to 8.25 mg /15 units of bleomycin) solubilized into 5ml of water for injection, Batch No: BYW1162, BYW1162, BYW1162)
Test Item Bleomycin Sulfate Nanoformulation (35 mg of bleomycin sulfate test formulation in one vails (equivalent to 8.25mg/15 units of bleomycin) was solubilized into 5ml of water for injection.)
Study Title Evaluation of Efficacy of ‘Bleomycin Sulfate and Bleomycin Sulfate Nanoformulation’ in Xenograft Glioblastoma Model in Swiss Albino Mice
Route Intravenous
Dose Vehicle (0mg/kg) and standard Market Preparation (5 mg/kg) for G2, Bleomycin sulfate nanoformulation (5 mg/kg) for G3
No. of Groups 3 (4 Females/ Group)
The objective of this study was to standardize the method for development of xenograft glioblastoma brain tumor model in Swiss albino mice and evaluate the antitumor effects of Bleomycin sulfate nanoformulation in comparison with the standard Market Preparation.
The study design comprised of 3 groups, each containing 4 female mice per group. All the mice were anesthetized with Xylazine + Ketamine anaesthesia (Dose 16 + 120 mg/kg) and were injected with 1 x 106 cells per animal intracranially using the 24G needle. For confirmation of development of glioblastoma one animal from control group was sacrificed on day 21. Bleomycin sulfate nanoformulation (5 mg/kg) was administered to each animal from G3 group and standard Market Preparation was administered to each animal from G2 group. Animals from control group (G1) received vehicle.
Parameters evaluated include mortality, clinical signs, gross and histopathological examination. One animal sacrificed on day 21 and all remaining survived animals sacrificed on day 36, did not show any adverse gross pathological lesions in any organ when compared with control. Brain slices observed grossly using magnifying glass showed small whitish spot at the site of injection.
Histopathology observations revealed that in disease control animal tumour cells with typical morphology of astrocyte like cells, pleomorphism were present, cell edema and microvascular proliferation found around the tumor cells. When observed with high power, nuclear pleomorphism, mitoses and microvascular proliferation were seen. These typical characteristics indicated successful development of tumor model.
The treatment of reference and treatment groups showed reduced number of tumor cells, reduction in the nuclear pleomorphism, mitoses and microvascular proliferation. Small areas of hemocyanin were noted, usually reflects subsided haemorrhage. This might be due to treatment of reference and test item induced death of some rapidly proliferating endothelial cells in tumor vasculature, leading to vessel leakage. Based on present study conditions, it can be concluded that there was successful development of glioblastoma model in non- immunocompromised mice model, evaluation of potency of glioma cells and standardization of method of intracranial inoculation. Incidence and severity of histopathological observations was maximum in disease control followed by reference group and test group. Although, these observations are based only on histopathological observations they are indicating that the bleomycin sulfate nanoformulation have better efficacy compared with reference group. The results are indicating that the bleomycin sulfate nanoformulation have better efficacy compared with reference group. However, based on these observations it is recommended to develop this model using immunocompromised mice and to increase the observation period which will help to get more clear results for interpretation However, it is recommended to develop this model using immunocompromised mice and to increase the observation period for longer duration.

Development of xenograft Glioblastoma model
Process: The glioma cell spheroids were received from Institute of Bioinformatics and Biotechnology, Savitribai Phule Pune University Pune India. All the mice were anesthetized with Xylazine + Ketamine anaesthesia (Dose xx mg/kg) and were injected with 1 x 106 cells per animal intracranially using the 24G needle. For confirmation of development of glioblastoma one animal from control group was sacrificed on day 21. Brain tissues were collected for histopathological examination.
Experimental Design
The rats were allocated to different treatment groups as follows:
Table 19: Details of Treatment Groups
Group No. Group Treatment Dose
(mg/kg) Animal Numbers
Females
G1 Control Tumour+ Vehicle 0 01-04
G2 Reference Tumour+ Bleomycin sulfate Market Preparation 5 05-08
G3 Test Tumour+ Bleomycin sulfate nanoformulation (PB3) 5 09-12

Dose Administration
Process: After confirmation of tumor development on day 21, bleomycin sulphate-nanoformulation and bleomycin sulfate market preparation at the dose 5 mg/kg of body weight were administered intravenously weekly for two weeks. Control group (G1) animals were administered with vehicle alone.

Observations
Following observations were recorded from all the animals.

Mortality and Clinical Signs Observations
All the animals were observed once daily throughout study period for clinical signs, and twice daily for morbidity and mortality except on the day of necropsy.

Necropsy and Gross Pathology
One animal from control group was sacrificed on day 21 to confirm proper administration of cells and development of tumor cells. On day 36, all the animals were sacrificed using CO2 asphyxiation and subjected for detailed gross pathology observations which included careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The brains collected were fixed in 10% neutral buffer formalin, after fixation, brains were sliced into coronal sections (parallel with the cell plantation road) and observed grossly using magnifying glass specifically for development of tumour at the injection site.
Histopathology
For histopathological examination of brain slices, were processed routinely, embedded in paraffin wax in such a way that the injection site was exposed, various serial sections were taken at 5 µ thick, stained with H&E stain and observed under microscope (Figure 16-18).
Results
Development of Brain tumor model
The histopathological observations of brain tissues, showed presence of tumour cells at the site of injection, indicated that it is possible to develop glioblastoma brain tumour model in non-immunocompromised Swiss albino mice, however need to be given more time.
Mortality and Clinical Signs Observations
All the animals were observed once daily throughout study period for clinical signs, and twice daily for morbidity and mortality except on the day of necropsy. All animals survived throughout the entire duration of the study. (Table 61-62)
Necropsy and Gross Pathology
One animal sacrificed on day 21 and all remaining survived animals sacrificed on day 36, did not show any adverse gross pathological lesions in any organ when compared with control. Brain slices observed grossly using magnifying glass showed small whitish spot at the site of injection. (Table 63-64).

Histopathology
Histopathology observations revealed that in disease control animals’ tumour cells with typical morphology of astrocyte like cells, pleomorphism were present, cell edema and microvascular proliferation found around the tumor cells. When observed with high power, nuclear pleomorphism, mitoses and microvascular proliferation were seen. These typical characteristics indicated successful development of tumor model.
The treatment of reference and treatment groups showed reduced number of tumor cells, reduction in the nuclear pleomorphism, mitoses and microvascular proliferation. Small areas of hemocyanin were noted, usually reflects subsided haemorrhage. This might be due to treatment of reference and test item induced death of some rapidly proliferating endothelial cells in tumor vasculature, leading to vessel leakage. Incidence and severity of histopathological observations was maximum in disease control followed by reference group and test group. Although, these observations are based only on histopathological observations they are indicating that the bleomycin sulfate nanoformulation have better efficacy compared with reference group. However, based on these observations it is recommended to develop this model using immunocompromised mice and to increase the observation period which will help to get more clear results for interpretation (Table 15-16).
Table 20: Histopathology Findings- Females
Tissue/ Findings/Sex Females
Dose mg/kg/day Control Reference Test
Group G1 G2 G3
Number Examined 4 4 4
Brain
Astrocyte like cells
Minimal Focal 2 1 2
Mild Focal 3 3 1
Infiltration of inflammatory cells
Minimal Focal 0 1 2
Mild Focal 4 3 1
Microvascular proliferation
Minimal Focal 2 3 1
Mild Focal 2 1 0
Necrosis
Minimal Focal 1 1 0
Note: 1 = Minimal, 3 = Mild, 4 = Focal

Table 21: Individual Histopathology Observations- Females
Animal Number Observations
Astrocyte like cells Infiltration of Inflammatory cells Microvascular proliferation Necrosis
G1- Control
01 Mild Mild Mild No abnormality detected
02 Mild Mild Minimal No abnormality detected
03 Minimal Mild Minimal Minimal
04 Mild Mild Mild No abnormality detected
G2- Reference
05 Mild Mild Minimal No abnormality detected
06 Minimal Mild Minimal No abnormality detected
07 Mild Minimal Mild Minimal
08 Mild Mild Minimal No abnormality detected
G2- Test
09 Minimal Minimal No abnormality detected No abnormality detected
10 Mild Mild No abnormality detected No abnormality detected
11 Minimal Minimal Minimal No abnormality detected
12 No abnormality detected Minimal No abnormality detected No abnormality detected

ADVANTAGES OF THE PRESENT INVENTION
1. The present invention poses significant advantages over the current clinical therapy of bleomycin sulfate.
2. The present invention provides a novel formulation and drug delivery strategy to simplify
the complications associated with various difficult-to-deliver drugs such as bleomycin sulfate.
3. The present invention provides a novel phospholipid nanoparticle formulation wherein the formulation is exhibited higher drug loading, low particles size, enhanced accumulation of drug in brain, less amount in lungs and higher circulation half-life as compared to bleocip.
4. The present invention provides a method of preparation wherein the method has higher level of industrial scalability and adaptability.

WE CLAIMS:

1.A composition of phospholipid nanoparticles for enhanced brain tumor delivery of anticancer drugs, wherein said phospholipid nanoparticles comprise of:
i) bleomycin sulfate as anticancer drug;
ii) a phospholipid selected from group comprising of egg phospholipid, soya phospholipid, phosphatidylcholine, glycerophospholipid, hydrogenated egg phospholipid, hydrogenated soya phospholipid, phosphatidylethanolamine, phosphatidylserine;
iii) a cryoprotectant selected from group comprising Mannitol, Trehalose, Mannitol, Trehalose, Lactose, Sucrose, Acetamide, Pyridine-N-Oxide, Albumin, Propylene glycol, Ammonium acetate, Ribose, Choline magnesium, chloride sodium, bromide, Serine, Diethyl glycol, Sodium chloride, Dimethylacetamide, Sodium bromide, Dimethyl sulfoxide, Sodium iodide, Ethanol, Sodium sulfate, Erythritol, Sorbitol, EG, Sucrose, Glycerol, Triethylene glycol, Glucose, Trimethylamine, Formamide, Acetate, Glycerophosphate, Xylose, Proline, Valine.
wherein the said phospholipid nanoparticles are formed by complexation between said anticancer drug and said phospholipid in a ratio of 1:1 to 1:5.
wherein particle size of said phospholipid nanoparticles ranges from 50- 170nm.
2.The composition as claimed in claim 1, wherein said phospholipid is egg phospholipid.
3.The composition as claimed in claim 1, wherein said cryoprotectant is mannitol.
4.The composition as claimed in claim 1, wherein drug loading capacity of said phospholipid nanoparticles are found in a range of 5-20% for bleomycin A2 and 2-8% for bleomycin B2.
5.The Composition as claimed in claim 1, wherein encapsulation efficacy of said phospholipid nanoparticles are found in a range of 25-75% for bleomycin A2 and 15-65% for bleomycin B2.
6.The composition as claimed in claim 1, wherein brain tumor delivery of said anticancer agent is increased by 2-5 fold from said phospholipid nanoparticles.
7.A method of preparing a composition as claimed in claim 1 for enhanced brain tumor delivery of anticancer drugs, said method comprising the steps of:
i. solubilizing anticancer drug and egg phospholipid in suitable organic solvents and reflux for a certain period of time for the complexation.
ii. Forming of thin film layer of phospholipids and anticancer drug.
iii. Hydration of the film formed in step (b) using market saline under suitable conditions.
iv. Performing probe sonication for a suitable time in ice bath solution at 60 amplitude to yield phospholipid nanoparticles of desired vesicle size and PDI.
8.The method of preparation of composition as claimed in claim 6,wherein said phospholipid nanoparticles processed as pharmaceutically acceptable dosage forms.