Field of the Invention:
The present invention is in the field of Huntington’s disease. The invention provides a drug screening kit and the pharmaceutical composition for Huntington’s disease.
The invention also provides the drug screening kit for the screening of potential hydrophobic drug molecules and specifically in finding suitable protein aggregation inhibitors having therapeutic value.
In a preferred embodiment, the present invention has been described with reference to Huntington’s disease. However, the scope of the invention shall not be considered limiting to Huntington’s disease, rather, the described embodiments including pharmaceutical composition, screening kit & method and other embodiments in the present application shall be applicable for other neurodegenerative diseases as well.
Background of the Invention:
Neurodegeneration is the umbrella term for the progressive loss of structure or function of neurons, including death of neurons. Many neurodegenerative diseases including Amyotrophic lateral sclerosis, Parkinson's, Alzheimer's, and Huntington's occur as a result of neurodegenerative processes.
Molecular mechanisms underlying neurodegenerative diseases are poorly understood. A common feature, however, is a slow accumulation of protein aggregates in specific regions and selected neuron populations in the brain. In the Huntington’s disease (HD), aggregates of N-terminal huntingtin (htt) fragments with a pathogenic number of glutamine residues (> 37) are found. Studies with different types of HD models showed that in neurons, both normal and mutant htt proteins localize to several different subcellular compartments, such as endosomes, presynaptic, clathrin-coated vesicles and dendritic plasma membrane (Harjes and Wanker 2003). In contrast, htt inclusion bodies developed in cell lines expressing large N-terminal htt fragments incorporate multi-vesicular membranes, autophagosomes and mitochondria into their surfaces (Kegel et al. 2000; Qin et al. 2004). Both the broad subcellular localization and the presence of membranous structures in the aggregates strongly suggest a direct interaction of htt with lipid bilayers.
The htt exon 1 (HDex1) protein with a pathogenic polyglutamine (polyQ) stretch aggregates in a time- and concentration-dependent manner in vitro and in cell culture models (Scherzinger et al. 1997, 1999; Poirier et al. 2002). In addition, the presence of HDex1 with extended polyQ is sufficient to induce HD-resembling neuropathological changes in transgenic (tg) R6/2 mice (Mangiarini et al. 1996; Davies et al. 1997 ; Carter et al. 1999).
For discovering of novel therapeutic lead molecules, diverse chemical molecules are screened against a validated drug target. To start a long journey of drug discovery, molecules are initially screened using in vitro assays. Many hydrophobic molecules present in chemical libraries are slightly soluble in water and require dimethyl sulfoxide (DMSO) for complete solubilization. These molecules are then added to the aqueous solution containing target molecule to understand a response of inhibition or modulation of the target property. These assays are performed in tubes or microplates made up of polystyrene or polypropylene material. Often, addition of DMSO soluble molecules in such tubes result in nonspecific surface adsorption and the molecules are lost from the solution. Thereby, it represents as if a given molecule hasn’t worked against a particular target. Hence, it becomes very necessary to find out efficient drug screening assay.
Huntington’s disease is one amongst many neurodegenerative and systemic origin, where aggregation is the major driver of the disease pathology. In order to address this problem many groups are trying to test different molecules in order to either prevent aggregate formation or to halt its progression. Molecules that worked on other protein aggregation diseases are also tried considering that the final aggregates formed are similar in characteristics. One such molecule is Calmidazolium chloride (CLC) which was found to enhance the rate of formation of Aß (1-40) protofibrils and arrest of mature fiber formation. This has implication in Alzheimer’s disease and also provided a way to study intermediates species which are otherwise difficult to capture. Same molecule however was not found effective on Polyglutamine in a microtiter plate format. Moreover, CLC has been shown as a Ca+2 calmodulin (CaM) antagonist and was in use as an investigational drug for calcium channel blockers for more than two decades. It was shown to inhibit CaM dependent protein phosphorylation with IC50 of 10-20 µM and to work as pain killer by blocking TRPV1 channel and shown to dock to the pore loop domain of TRPV1. It was also reported that addition of CLC to protein resulted in cloudiness due to its inherent insolubility in water. Moreover, CaM was shown to be involved in Huntington’s pathogenesis. It regulates Transglutaminase 2 (TG2) which is involved in cross linking of huntingtin (htt). Also, CaM binds to htt with expanded polyglutamine with high affinity and become part of cross-linked aggregates. Thus, finding a molecule which can show bispecific property i.e. target protein aggregation as well as block CaM association with Htt and TG2 will have high therapeutic value.
It is observed that the hydrophobic drug molecules with little solubility in water needs special composition design for their use. For example, molecules like paclitaxel (taxol) are prone to surface adsorption and lost from solution in due course of time. Different methods are employed for making stable composition with use of excipients ranging from small molecules to macromolecules. But prevention of such undesired adsorption in experimental assay for testing novel drug molecules is largely ignored till date. Mostly these molecules fail to move ahead in screening assays not due to their inefficiency of action but, due to their susceptibility to vial surface adsorption. To accelerate the drug discovery process, high throughput methods are used with final outcome as readout of an assay. This put more emphasis on the end result, rather that intermediate changes due to which numerous lead molecules can be missed. Interestingly, no one check the status of a drug molecule in an assay, but only look at the outcome of the assay. Use of high throughput screens further aggravate the problem, where hundreds of molecules tested together, leaving no chance to examine individual molecules. Also, it is important to look at reasons for failure of any drug in a particular assay so as to develop more scientific knowhow for better assay design.
US 20060079447 discloses a method for producing a stabilized Aß protofibrillar aggregate comprising contacting a Aß peptide with a stabilizing compound like calmidazolium chloride (CLC).
US 20040063612 discloses use of calmidazolium chloride for inhibiting nitric oxide synthase III (NOS III) in a mammal. US ‘612 and US ‘447 discloses about the use of calmidazolium chloride.
US 20050009847 discloses methods of promoting neurogenesis by contacting neuronal tissue with intracellular cAMP elevating agents and intracellular Ca2+ elevating agents. US ‘847 discloses use of calmidazolium chloride as inhibitor of cAMP-specific phosphodiesterase.
WO 2004045592 discloses methods of promoting neurogenesis by contacting neuronal tissue with intracellular cAMP elevating agents and intracellular Ca2+ elevating agents. WO ‘592 discloses calmidazolium chloride as inhibitor of cAMP-specific phosphodiesterase.
WO 1999040435 discloses a method for identifying a protein folding inhibitor. The method comprises steps of contacting a protein biosynthetic system under protein synthesis conditions with at least a compound to be tested and determining whether said test compound increases the ratio of unfolded protein to folded protein.
In view of the existing art, there is needed a pharmaceutical composition having the preventive and therapeutic action for the treatment of Huntington disease.
Also, there is needed a kit useful for screening the potential drug for Huntington disease or other neurodegenerative diseases.

Object(s) of the Invention:
A primary object of the present invention is to overcome the drawbacks of the prior art.

Yet another object of the present invention is to provide a pharmaceutical composition for neurodegenerative disease, preferably Huntington’s disease, which has dual function of treatment and preventive action i.e. treating the disease and simultaneously preventing the disease to occur.
Yet another object of the present invention is to provide a pharmaceutical composition which can target protein aggregation as well as block CaM association with Htt and TG2.
Yet another object of the present invention is to provide a pharmaceutical composition based on 1-[Bis(4-chlorophenyl)methyl]-3-[2,4-dichloro-ß-(2,4-dichlorobenzyloxy)phenethyl]imidazolium chloride) useful for the treatment of Huntington’s disease.
Yet another object of the present invention is to provide a process for the preparation of the above mentioned pharmaceutical composition.
Yet another object of the present invention is to provide a highly efficient drug screening kit for finding potential hydrophobic drug molecule useful for the treatment of neurodegenerative disorder.
Yet another object of the present invention is to provide a highly efficient drug screening kit for finding potential hydrophobic drug molecule useful for the treatment of Huntington’s disease
Yet another object of the present invention is to provide a drug screening kit for testing a novel drug molecule.
Yet another object of the present invention is to provide a kit based on the reagents which reduces the adsorption susceptibility of a test drug molecule to vial’s surface or container’s surface and thus increases the possibility of successful action of the drug to occur and simultaneously eliminates the possibility of any false results.
Yet another object of the present invention is to provide an in-vitro drug screening kit for screening a surface susceptible hydrophobic drug for a neurodegenerative disease.
Yet another object of the present invention is to provide an in-vitro kit for screening protein aggregation inhibitors.
Yet another object of the present invention is to provide a method of testing a drug molecule using the above kit.
Summary of the Invention:

In one aspect of the Invention, there is provided a preventive and therapeutic composition for a neurodegenerative disease comprising:

a) drug present in an amount ranging from 1 µM to 100 µM;
b) serum albumin present in an amount ranging from 1 µM to 100 µM;
c) an aqueous phase present in an amount ranging from 10 to 99.9% v/v;
d) organic phase/solvent present in an amount ranging from 0.1 to 20% v/v;
e) Physiological buffer present in an amount ranging from 10 mM to 50 mM while salt is present in an amount ranging from 100 to150 mM.
f) pH of buffer is in range from pH 3-8.

In another aspect of the Invention, there is provided a preventive and therapeutic composition for Huntington's disease, wherein the composition comprises 1-[Bis(4-chlorophenyl)methyl]-3-[2,4-dichloro-ß-(2,4-dichlorobenzyloxy)phenethyl]imidazolium chloride as the active drug which is present in an amount ranging from 1 µM to 100 µM; bovine serum albumin present in an amount ranging from 1 µM to 100 µM; an aqueous phase present in an amount ranging from 10 to 99.9% v/v; DMSO as the organic phase/solvent present in an amount ranging from 0.1 to 20% v/v and phosphate ions based physiological buffer present in an amount ranging 10 mM to 50 mM.
In another aspect of the Invention, there is provided an in-vitro drug screening kit for testing a surface susceptible hydrophobic drug molecule for its therapeutic action towards a neurodegenerative disease. The kit comprises:

a) Reaction mixture (A) comprising aqueous based solution of reagents comprising a physiological buffer, an antimicrobial agent and a serum albumin, wherein said reaction mixture (A) is formulated by preparing an aqueous based solution by dissolving said physiological buffer followed by an antimicrobial agent and a serum albumin in water characterized in that the serum albumin, being already present in the dissolved state in water, is added pursuant to the addition of said antimicrobial agent to the water;

b) Reaction mixture (B) comprising a test drug molecule being present in the dissolved state in an organic solvent/phase and an aggregating peptide being present in the dissolved state in water with a determined pH, wherein said reaction mixture (B) is added to said reaction mixture (A), to cause reaction, followed by incubation at 37 degree celsius,
and wherein the absence of self assembly of the peptide during said reaction of reaction mixture (A) and reaction mixture (B) indicates the therapeutic action of said drug towards the neurodegenerative disease

Brief description of drawing(s):
Figure 1: illustrates hydrophobic drug Calmidazolium Chloride (100 µM) adsorption to surface of polypropylene vial with time
Figure 2: illustrates soluble fraction of hydrophobic drug at different temperatures in presence of phosphate buffered saline with 10% DMSO (v/v), determined by RP-HPLC
Figure 3: illustrates the soluble fraction of hydrophobic drug after 24 hours of incubation at different temperatures in presence of 100 % DMSO (v/v) and in presence of Phosphate buffered saline with 10% DMSO (v/v). There is also shown the recovery of adsorbed drug from surface of polypropylene vial after incubation for 24 hours
Figure 4: illustrates soluble fraction of hydrophobic drug at different buffer compositions after 24 h incubated at 37 oC
Figure 5: illustrates particle size distribution of CLC (Intensity %) (left scale & empty bars) and count rate (right scale & stripped bars) at different buffer compositions after 24 h incubated at 37 oC.
Figure 6: illustrates particle size distribution (Number %) of hydrophobic drug at different concentrations in phosphate buffered saline.
Figure 7: illustrates soluble fraction of hydrophobic drug in presence of different polypropylene surface and in presence of different macromolecule, after 24h incubated at 37 oC.
Figure 8 (a): illustrates particle size distribution (Volume %) of hydrophobic drug alone, macromolecule alone in phosphate buffered saline and drug in presence of BSA; (b) illustrates diffusion coefficient of hydrophobic drug alone, macromolecule alone in phosphate buffered saline and drug in presence of macromolecule.
Figure 9 (a): illustrates soluble fraction of aggregation prone HDex1 peptide with time incubated at 37 oC. HDex1 20 µM, CLC 100 µM and BSA 5 µM; (b) shows soluble fraction of aggregation prone HDex1 peptide after 48 hours of incubation at 37 oC. Drug 100 µM and BSA 5 µM.
Figure 10: illustrates change in diffusion coefficient of aggregation prone HDex1 peptide with time incubated at 37 oC, monitored by dynamic light scattering. HDex1 20 µM, Drug 100 µM and BSA 5 µM.
Figure 11: illustrates (a) albumin anti-adsorption action at 50 µM; (b) BSA anti-adsorption action at low pH (PBS pH3)
Figure 12: illustrates soluble fraction of aggregation prone Q35P10 peptide after 1 day and 6 days of incubation at 37 oC. Q35P10 15 µM, CLC 100 µM and Fatty acid free BSA 50 µM.
Figure 13: illustrates BSA anti-adsorption action on IWP-2, an effective therapeutic option for cancer and neurodegenerative diseases
Figure 14: illustrates schematic representation of steps involved in testing assay of the screening kit.
Figure 15: illustrates schematic representation of the steps involved in the preparation of the pharmaceutical composition for a neurodegenerative disease as described in the present invention.
Figure 16: illustrates soluble fraction of hydrophobic drug (CLC) in presence 50 ?M BSA, after 6 days and 30 days incubated at 4 oC and 25 oC.
Figure 17: illustrates soluble fraction of hydrophobic drug (CLC) in presence different concentration of macromolecule BSA, after 24 h and 96 h incubated at 37 oC.
Detailed description of the Invention:
The present invention in accordance with the above stated objectives provides a highly effective pharmaceutical composition acting as a protein aggregation inhibitor useful for the treatment of Huntington’s disease.
The pharmaceutical composition targets the aggregation process of Huntington’s gene encoded Huntington’s exon1 (HDex1) peptide fragment, which is validated in animal and cell models as a target for therapeutic intervention in Huntington’s disease.
The composition shows the bispecific property i.e. target protein aggregation as well as block CaM association with Htt and TG2, which are believed to be involved in cross linking of Huntington’s and causing cross-linked aggregates respectively. Thus, the composition has the preventive and therapeutic action for the Huntington’s disease.
The pharmaceutical composition of the present invention is though specific for Huntington’s disease, however the scope of invention shall not be considered as restrictive and the composition may be equally found efficient in the treatment of other neurodegenerative genetic disorder related diseases as well.
In an embodiment the composition comprises a drug present in an amount ranging from 1 µM to 100 µM, serum albumin present in an amount ranging from 1 µM to 100 µM, an aqueous phase present in an amount ranging from 10 to 99.9% v/v, organic phase/solvent present in an amount ranging from 0.1 to 20% v/v, physiological buffer present in an amount ranging from 10 mM to 50 mM.
In an embodiment, the drug molecule is present in the dispersed state. Approximately 90 to 100% of the drug molecule particles present are smaller than 1 ?m or 2 ?m, according to the intensity distribution and volume distribution as determined by dynamic light scattering.
In an embodiment, the aqueous solution comprises water.
In an embodiment, the aqueous solution contains organic solvent other than water.
In an embodiment, the organic solvent/phase is aprotic polar solvent like DMSO.
In an embodiment, the composition comprises Calmidazolium chloride (CLC) i.e. 1-[Bis(4-chlorophenyl)methyl]-3-[2,4-dichloro-ß-(2,4-dichlorobenzyloxy)phenethyl]imidazolium chloride as the drug molecule and Bovine serum albumin as amongst the active ingredients. The composition been tested through in vitro suspension based assay for aggregation inhibition of HDex1 fragment.
Calmidazolium chloride (CLC) is an amphipathic weak base with almost no solubility in water. It carries positive charge with imidazolium ring at the center and large hydrophobic region at periphery.
The composition based on CLC and Bovine serum albumin (which actually forms complex with each other) inhibits the HDex1 aggregation by twenty-five folds as compared to four fold inhibition obtained with only CLC as illustrated in Example 1 and corresponding figure 9. The difference in the monomer concentration of HDex1 after 48 hrs of incubation is evident from Figure 9. The last bar when compared to the first bar, demonstrates the difference in monomer concentration of HDex1 after 48 hrs of incubation.
In a preferred embodiment of the invention, the pharmaceutical composition comprises hydrophobic drug and BSA.
The Composition comprises hydrophobic drug and serum albumin in a molar ration from 1:1 to about 20:1, wherein the drug molecule has aqueous solubility of at least 100 ?M or 200 ?M in Composition.
The serum albumin may be of Human origin or Bovine origin or recombinant. The serum albumin can be fatted or defatted- globulin containing or free. The anti adsorption action of albumin with respect to with/without fatty acid is illustrated in Figure 11(a). Thus, from figure 11(a), it is evident that fat free, with fat and human serum albumin works to arrest adsorption of CLC.
In a preferred embodiment, the present invention has been described with reference to Bovine Serum Albumin (BSA), however the scope of the invention shall not be considered as restrictive to BSA only, rather the invention covers use of other albumins like HSA in order to achieve the presently described various aspects of the invention. The invention may also work with recombinant human albumin.
The molar ratio of hydrophobic drug to bovine serum albumin is about 20:1 or lesser, however preferably more than 5:1 or 1:1. The bovine serum albumin is present in concentration ranging from 1 to 200 ?M while the hydrophobic drug is present in concentration from 0.001 to 300 ?M.
In an embodiment of the invention, the pharmaceutical composition comprises a physiological buffer.
In an embodiment, the phosphate buffer saline comprises 10 mM to 50 mM Phosphate buffer, 100 mM to 200 mM NaCl.
In another embodiment, the physiological buffer is preferably a phosphate buffered saline.
In another embodiment, the physiological buffer comprises phosphate ions and has a pH in the range of 3 to 8.
The phosphate buffer saline comprises 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4 with pH adjusted with HCl or NaOH.
In another embodiment, there is provided a process of preparing the above defined preventive and therapeutic composition.

The process comprises the following steps:

a) preparing an aqueous based solution of a physiological buffer and serum albumin by dissolving in water. The serum albumin being already present in the dissolved state in water is added pursuant to the addition of said buffer to the water;

b) adding the drug molecule, being already present in the dissolved state in an organic phase/solvent, to the solution resulting from step (a) followed by incubation at 37 degree celsius to obtain the preventive and therapeutic composition of the present invention.
In the other important embodiment of the invention, there is provided a drug screening kit for finding the potential hydrophobic drug molecules, particularly protein aggregation inhibitors, effective in the treatment of Huntington’s disease or other neurodegenerative disease.
In another embodiment of the invention, there is provided an in-vitro drug screening kit for testing a surface susceptible hydrophobic drug molecule for its therapeutic action towards a neurodegenerative disease. Thus, the kit is very useful for screening the hydrophobic molecules susceptible to vial surface adsorption like Tamoxifen, Lapachone, Indomethacin, Digoxin, Midazolam, Propranolol, Procaine, Verapamil etc.

The component of the kit comprises following reagents:

i) a reaction mixture (A) comprising aqueous based solution of reagents comprising a physiological buffer, an antimicrobial agent and a serum albumin. The reaction mixture A is formulated by preparing an aqueous based solution by dissolving said physiological buffer followed by an antimicrobial agent and a serum albumin in water characterized in that the serum albumin, being already present in the dissolved state in water, is added pursuant to the addition of said antimicrobial agent to the water;

ii) a reaction mixture (B) comprising a test drug molecule being present in the dissolved state in an organic solvent/phase and an aggregating peptide being present in the dissolved state in water with a determined pH. The reaction mixture B is added to the reaction mixture A, to cause reaction, followed by incubation at 37 degree celsius,

following the reaction between the reaction mixture (A) and (B), the absence of self assembly of the peptide during the reaction of reaction mixture A and reaction mixture B indicates the therapeutic action of the drug towards a neurodegenerative disease.

In an embodiment, the antimicrobial agent comprises sodium azide and alike reagents with similar chemical properties.

In another embodiment, the azide is a sodium azide present in an amount of 0.05%.

In another embodiment, the hydrophobic drug molecule comprises a protein aggregation inhibitor.

In another embodiment, the aggregation peptide is selected from a group comprising of an aggregation protein peptide, insulin, mutant Huntingtin (mhtt), synuclein, medin, IAPP, crystalline, serum amyloid A (SAA).

In another embodiment, the hydrophobic drug is available in an amount upto 70% for reaction in the screening assay.

In another embodiment, the drug screening kit is based on the reagents which reduces the adsorption susceptibility of said subject drug molecule to vial’s surface or container’s surface and thus increases the possibility of successful reaction of the drug to occur and simultaneously eliminates the possibility of any false results.
In another aspect of the invention, there is provided a method for testing a drug, using the above described testing kit, wherein the method comprises treating the prospective hydrophobic drug with the aggregating peptide, wherein disentangling of said aggregating peptide indicates said drug to be effective for a neurodegenerative disease.

The method is useful for the successful testing of hydrophobic molecule susceptible to vial surface adsorption.

The aggregation peptide is selected from a group comprising of an aggregation protein peptide, insulin, mhtt, synuclein, medin, IAPP, crystalline, serum amyloid A (SAA).
In another embodiment, the drug screening assay of the present invention has been described with reference to screening of drugs for Huntington’s disease, however the scope of invention shall not be considered as restrictive and covers use of the screening assay for identification of drug, particularly hydrophobic drug, having potential in the treatment of other neurodegenerative genetic disorder related diseases as well. Further, the stable complex formed can be tested for developing liquid composition for the CLC drug.
In an embodiment, the screening assay comprises composition based on serum albumin, a hydrophobic drug and a physiological buffer. The physiological buffer comprises phosphate ions and has a pH in range of 6 to 8. The physiological buffer is preferably phosphate buffered saline. The phosphate buffered saline consists of 100-200 mM Sodium chloride.

In an embodiment, the screening assay comprises composition based on aggregating peptide, which is present in the concentration ranging from 0.01-200 ?M. The aggregating peptide may be any aggregation protein peptide, insulin, mhtt, synuclein, medin, IAPP, crystalline, serum amyloid A (SAA). In a preferred embodiment, the aggregating peptide is mhtt and HDex1 peptide of mhtt.

In the screening assay of a preferred embodiment of the invention, the hydrophobic drug prone to adsorption is available between 50 to 100%, preferably 80%, during aggregation assay by HPLC-sedimentation at 100 ?M of BSA.

In the screening assay of yet another preferred embodiment, the hydrophobic drug prone to adsorption is available above 70%, during aggregation assay by HPLC-sedimentation at 5?M of BSA. The hydrophobic drug prone to adsorption is dissolved in DMSO and may be present in complex with BSA in aqueous solution. The hydrophobic drug prone to adsorption dissolved in DMSO is added to aqueous solution containing BSA.

In an embodiment, the aggregation prone peptide is added to aqueous solution containing hydrophobic drug and BSA.

In an aspect of the invention, the screening assay comprises an antimicrobial agent, present in the concentration ranging from 0.01-0.09% v/v. The antimicrobial agent is preferably sodium azide.

In another embodiment of the invention, the composition of hydrophobic drug and BSA (or other albumin) can be used for screening of other hydrophobic molecules prone to surface adsorption in anti-aggregation studies in aqueous solution.

The HDex1 monomer concentration in the presence of drug and BSA is above 50% to 65% after 48h while its concentration in the presence of drug (only) ranges less than 25% after 48 h.
Experiments:
To establish the importance of preventing hydrophobic drug adsorption to the container surface, peptide aggregation assay was setup in the presence of drug (CLC) and monitored by high pressure liquid chromatography (HPLC) assay. Interestingly, drug showed aggregation inhibition effect for initial 30% of reaction and smaller inhibitory effect thereafter. It was anomalous behavior, that after initial retardation of aggregation, reaction finished almost similar to that of control without drug. Notable, it was found that with time CLC was lost from the reaction mixture however the inventors were able to unmask it by the use of RP-HPLC technique, which not only quantify target protein but also resolve other solution constituents, simultaneously. Use of commonly used fluorescence or light scattering techniques might have missed this important change. Therefore, characterisation was done for determining the behaviour of CLC alone in aqueous solution, before establishing its action on HDex1 aggregation. The experiments show about the inhibition tendency of the aggregation of HDex1 fragment with CLC in a suspension assay, which was found to be marginal. This was mainly due to the non-availability of CLC in the solution and its adsorption to the testing tube. Therefore, it is very crucial for finding a composition which makes CLC available for the reaction and prevent its adsorption to the vials. Thus, the present invention provides a composition based on CLC and bovine serum albumin (BSA). This composition based on the presence of BSA composition did not allow CLC molecule to adsorb to the vials.
Since, the serum albumin and CLC binding is also seen at low pH, thus the present screening kit and the assay also work for those proteins which tend to aggregate at low pH. In other words, the complex of CLC and Serum albumin work as inhibitor of aggregation at low pH, as illustrated in Figure11(b).

Characterisation of the behaviour of CLC alone in aqueous solution:
An experiment was performed for the characterization of the behaviour of CLC alone in aqueous solution, before establishing its action on HDex1 aggregation. To elucidate the adsorption phenomenon, individual investigation was performed for all the steps of the assay that could potentially accelerate the adsorption. First, it was tested whether CLC was lost during centrifugation step used to pellet down aggregates. There was a retrieval of only about 10% of CLC from the pellet after centrifugation at centrifugal force ranging from 600 ? g to 119500 ? g (RCF). Pellet was analyzed by solubilizing it in DMSO but 90% of the lost product was not retrieved. This hints towards loss of product even before centrifugation step, precluding centrifugal force induced effect. Thus, adsorption to polypropylene vial surface was performed for probing CLC loss. To ascertain this, DMSO washing of vial was performed in which CLC was incubated. DMSO was chosen as solvent due to high solubility of CLC. Interestingly, the experiment retrieved almost the entire lost compound from first wash itself. This proved that indeed CLC was adsorbed to the vial surface on incubation at 37oC. Further, the kinetics of CLC adsorption was investigated. When amount of CLC adsorbed normalized to available surface area was plotted against time, it was observed that there was a rapid adsorption initially (Fig. 1). It was followed by sudden decline and then to a stationary phase, where minimal adsorption took place. Further, experiments were performed for finding the contribution of incubation temperature. It was found that the adsorption happened at all temperatures and slight increase in adsorption was observed at lower temperature (Fig. 2 & 3).

Effect of buffer on CLC adsorption:
Finally, the effect of buffers on CLC adsorption, after ruling out other plausible factors was examined. When CLC was incubated in 100% DMSO, compound remained in solution with no loss to surface due to adsorption (Fig. 3). The effect of different buffer and ionic strengths was tested. Surprisingly, it was found that pH is not contributing alone towards adsorption. It was further observed that there was no adsorption in water pH 5.5, Phosphate buffer pH 7 (without salt) and 20% formic acid pH 2 (used to arrest HDex1 aggregation reaction) (Fig. 4). These results prompted to testify ionic strength as driving force to adsorption, as CLC in phosphate buffer without salt did not show any adsorption. To experimentally examine this, different sodium chloride concentrations ranging from 0 mM to 150 mM dissolved in water were used. Throughout this experiment pH was ranging from 5.5 to 6. It was observed that the increase in adsorption with increasing salt concentration, with adsorption at 150 mM NaCl almost equivalent to what was found in PBS (Fig. 5). 150 mM NaCl was tried as it is more relevant to physiological condition and most commonly used concentration in PBS buffer. Also, increase in particle size and scattered light intensity (kcps) in presence of NaCl (Fig. 5) was observed. Interestingly, CLC formed sub-micron sized assemblies in PBS and did not show comparable change in size with increasing concentration from 5 µM to 100 µM (used in aggregation assay) (Fig. 6). Only change in scattered intensity (kcps) was observed which indicated reduction in number of particles with decrease in concentration. Large sized particles observed were in accordance with the size observed for few other hydrophobic molecules (as described in Ilevbare G and Taylor L. 2013. Liquid-Liquid Phase Separation in Highly Supersaturated Aqueous Solutions of Poorly Water-Soluble Drugs: Implications for Solubility Enhancing Composition s, Cryst Growth Des, 13, 1497 and Wang T., Joshi S. B., Kumru O. S., Telikepalli S., Middaugh C. R., Volkin D. B. (2013b). Case studies applying biophysical techniques to better characterize protein aggregates and particulates of varying size, in Biophysics for Therapeutic Protein Development, edNarhi L., editor. (New York, NY: Springer Publishing; 205–243). The larger particle size in aqueous solution indicated self-assembly of CLC perhaps due to its low water solubility. The formation of these aggregates might be responsible for surface adsorption, as when present in monomeric state in DMSO, there was no adsorption. Although the aggregation phenomenon of small molecule is not completely understood, but it was observed in case of few dyes like congored and many other lipophilic drugs. Micelles like structures were also observed in case of presence of both hydrophobic and hydrophilic groups on same molecule (as described in “Case studies applying biophysical techniques to better characterize protein aggregates and particulates of varying size, in Biophysics for Therapeutic Protein Development” by Wang et al.) Recently liquid-liquid phase separation was associated with this kind of aggregation behaviour (as described in “Liquid-Liquid Phase Separation in Highly Supersaturated Aqueous Solutions of Poorly Water-Soluble Drugs: Implications for Solubility Enhancing Composition s” by Ilevbare et al).
Further, having established the factors contributing to CLC adsorption, experiments were conducted to prevent adsorption with the aim of applying it to aggregation assay. For most of the solution based peptide aggregation assays, physiological relevant buffers like phosphate buffered saline (PBS) are used. Thereafter, widely used technique of coating tube surface with BSA was tried as it acts as blocking agent. However, the surprising effect was that, there was not significant improvement in preventing adsorption of CLC, though it prevents other proteins adsorption. Thereafter, experiment was performed using BSA in solution rather than adsorbing it on tube wall. Interestingly, there was a significant decrease in CLC adsorption at 100 µM concentration of BSA (Fig. 7). Even at lower concentration of 5 µM, BSA in soluble form was able to alleviate CLC surface adsorption. This was interesting advancement, as use of other techniques failed to prevent adsorption phenomenon. On the contrary, the low protein binding Eppendorf LoBind tube was also tested, but no decrease in CLC adsorption was found by its use (Fig. 7). However, the specific adsorption phenomenon of the present invention was specific to BSA, as low molecular weight protein lysozyme failed to show such effect. Another set of experiments were performed to probe into the series of events behind such interesting result. DLS of samples containing BSA and CLC was performed and it was found that there was a complex formation between BSA and CLC in solution (Fig. 8A). New peak in DLS (Volume %) in the presence of CLC and BSA was found and significant change in diffusion coefficient of assemblies formed were also observed (Fig. 8B).The formation of this complex was found to be responsible for keeping drug in soluble form which remains in solution state even after centrifugation at 119500 ? g (RCF).
To test the functionality of CLC present as BSA-CLC complex, HDex1 peptide aggregation assay was carried out. It was observed that there was 25 fold decrease in HDex1 aggregation in presence of complex and 4 fold decrease in CLC alone (Fig. 9A & 9B). Further, significant difference in diffusion coefficients of the assemblies formed by HDex1, HDex1 CLC and HDex1 BSA-CLC was found (Fig. 10). In case of HDex1 alone and with CLC, there was rapid decline in diffusion coefficient, indicating formation of larger assemblies. Abrupt change for HDex1 CLC was surprising, but can be attributed to formation of few higher ordered assemblies which scatter more light. Contrary to this, there was no rapid fall in diffusion coefficient of assemblies formed in case of HDex1 present along with BSA-CLC complex (Fig. 10). Also, there was no change for BSA-CLC complex, indicating stability of complex formed. By considering the data obtained it was a surprising effect in terms of wherein self-associating and adsorbate CLC molecule acted as a better aggregation inhibitor in presence of BSA.
The invention is further described with the help of non-limiting example:
Example 1:
Calmidazolium Chloride (CLC, 1- [Bis(4-chlorophenyl)methyl]-3-[2,4-dichloro-ß-(2,4-dichlorobenzyloxy)phenethyl]imidazolium chloride) was purchased from sigma.
HDex1 peptide having following sequence MATLEKLMKAFESLKSFQQQQQQQQ QQQQQQQQQQQQQQQQQQQQQQQQQQQPPPPPPPPPPKK was purchased from Keck biotechnology resource laboratory, Yale University, New Haven, CT, USA. Trifluoroaceticacid (TFA), 1,1,1,3,3,3-Hexafluoro-2-Propanol (HFIP), sodium azide and formic acid were purchased from sigma. Bovine serum albumin (97% purity) was purchased from SD fine-chem. HPLC grade water and acetonitrile were purchased from Merck.
The sample was prepared by using CLC stock. 1mM CLC stock was prepared in DMSO. HDex1 peptide was purified, lyophilized and disaggregated as per published protocols. 0.1 mg of HDex1 peptide was dissolved in 1 mL of TFA: HFIP (1:1) and kept at room temperature for 12 h in dark. Solvent was evaporated under purge of nitrogen and kept in desiccator for 2 h. Disaggregated peptide was dissolved in dissolved in aqueous TFA solution (pH 3). Peptide was centrifuged at 80,000 rpm (303,824 ? g using Thermo S80-AT2 fixed angle rotor) for 4 h at 4 oC. Aggregation reaction was carried out in phosphate buffered saline (PBS) pH 7 with 0.05% sodium azide as anti-microbial agent and incubated at 37 oC in polypropylene tube. Reaction was set up with 20 µM of peptide and 100 µM CLC. Only CLC and peptide were used as controls and blank as negative control. Final DMSO amount in reaction mixture is 10% (v/v). Reaction was monitored by taking small aliquots of sample intermittently at different time points and centrifuged at 119500 ? g for 30 minutes. 70% of supernatant was aspirated and 20% formic acid was added to stop ongoing reaction. Sample was analyzed using reverse phase high performance liquid chromatography (RPHPLC) at 215 nm wavelength on Agilent HPLC 1260 infinity system. ZORBAX Eclipse Plus C18 Rapid Resolution column (4.6 × 100 mm, 3.5 µm) was used with column thermostat kept at 25 oC. Water and acetonitrile each containing 0.05% TFA were used as mobile phase. Adsorption of CLC was tested after 24 h of incubation unless stated otherwise. The comparison between two groups was performed using two-tailed unpaired student’s t-test and difference was considered statistically significant when P < 0.05.
For complex formation, constituents were added in following order: first water (pH 3) was added followed by PBS, sodium azide, BSA and, CLC. No physical perturbation was provided to sample for complex formation. In case of test sample HDex1 peptide was added at end, after all other components.
While, for Dynamic Light Scattering (DLS), analysis was performed on Malvern Zetasizer Nano ZS90 equipped with He–Ne laser operating at awavelength of 633 nm and an angle of 90°. Reaction components were filtered through 0.22 µm PVDF syringe filter before starting reaction, to avoid dust particles. All the DLS measurements were performed at 25 ºC. The experimental results clearly show that Bovine serum albumin (BSA) when present in aqueous solution of aggregation assay prevents surface adsorption of a hydrophobic molecule (CLC), thus makes it available to act on HDex1 aggregating peptide during suspension assay.
Particularly, the use of BSA in aqueous solution of aggregation assay to make complex of BSA-CLC to prevent surface adsorption of hydrophobic molecule and the surprising increase in the aggregation inhibition activity of HDex1 by BSA-CLC complex contributes towards the technical advancement of the invention. The Drug:BSA ration used in this assay has been 20:1.

Example 2:
Q35P10 peptide having following sequence (KKQQQQQQQQQQQQQQQQQQQQQQQ QQQQQQQQQQQQPPPPPPPPPPKK was purchased from Keck biotechnology resource laboratory, Yale University, New Haven, CT, USA. All other reagents including drug were similar to example 1.
Q35P10 peptide was purified, lyophilized and disaggregated as per published protocols. 0.5 mg of Q35P10 peptide was dissolved in 1 mL of TFA: HFIP (1:1) and kept at room temperature for 12 h in dark. Similar procedure was followed as mentioned in example 1.
For complex formation, constituents were added in following order: first water (pH 3) was added followed by PBS, sodium azide, 50 µM BSA and, CLC. No physical perturbation was provided to sample for complex formation. In case of test sample Q35P10 peptide was added at end, after all other components.
Drug:BSA ratio used in this assay is 2:1.
The results (as illustrated in figure 12) show that BSA-CLC complex at 6th day of assay prevents aggregation of Q35P10 peptide by more than 20% compared to control. Q35P10 peptide is part of HDex1 peptide implicated in Huntington’s disease.
Example 3:
IWP-2 is a potent WNT-3A inhibitor. IWP-2 can also downregulate the transcriptional activity of the Wnt/ß-catenin signaling pathway. It is a cell-permeable benzothiazolyl-acetamide compound that inhibits the cellular Wnt processing and secretion via selective blockage of MBOAT (membrane-bound O-acyltranferase) family member Porcn- (Porcupine) mediated Wnt palmitoylation. The compound was tested for being a therapeutic option for cancer and neurodegenerative diseases.
The sample was prepared by using IWP2 stock. 200 µM IWP2 stock was prepared in DMSO. For complex formation, constituents were added in following order: first water was added followed by PBS, sodium azide, 50 µM BSA and IWP2. No physical perturbation was provided to sample for complex formation. Sample was incubated at 37 oC in polypropylene tube.
The Drug:BSA ratio used in this assay is 3:4.
The results (as illustrated in figure 13) shows that BSA prevented vial surface adsorption of IWP-2, thus enhancing availability of drug for potential therapeutic application in cancer and neurodegenerative diseases.
Example 4
50 ?M CLC was incubated with 20 ?M BSA and kept at 4 oC and 25 oC to check stability of complex formed. CLC concentration in solution was monitored at 6th day and 30th day.
The results (as illustrated in figure 16) shows negligible CLC surface adsorption at 25 oC while there was loss of 20% CLC at 4 oC consistent with previous observation of enhances surface adsorption at low temperature. The composition remains stable till 30th day as well.

Example 5
To demonstrate the effective BSA concentration sufficient to prevent vial surface adsorption, different concentration of BSA were used keeping CLC at 50 ?M concentration. BSA: CLC ratio was kept as follows 1:50, 1:10, 1:5, 2:5, 1:1 and 2:1.
The results (as illustrated in figure 17) shows inhibition of vial surface adsorption for BSA: CLC ratio of =1:10. Only for ratio 1:50, where BSA was 1 ?M and CLC was 50 ?M, 40% loss of CLC was observed.

Example 6:
The invention also provides a kit. The kit comprises reaction mixture (A) and reaction mixture (B). The reaction mixture (A) is prepared by dissolving the phosphate buffer saline followed by sodium azide in water. Thereafter, in the resulting solution, serum albumin which is already present in the dissolved state in water is added which makes up the final reaction mixture. This is followed by addition of the test molecule to be tested, which is present in the dissolved state in an organic solvent/phase.
While, the reaction mixture (B) contains a aggregation prone peptide/protein. The aggregating peptide to be tested is present in the dissolved state in water.

While testing a drug molecule for its therapeutic action towards the neurodegenerative disease, the reaction mixture B is added to the reaction mixture A, to cause reaction, followed by incubation of the solution at 37 degree Celsius where the absence of any self assembly of the aggregation prone peptide during the reaction of reaction mixture A and reaction mixture B indicates the therapeutic action of the drug towards the disease.
,CLAIMS:We Claim:

1. A preventive and therapeutic composition for a neurodegenerative disease comprising:
a) drug present in an amount ranging from 1 µM to 100 µM;
b) serum albumin present in an amount ranging from 1 µM to 100 µM;
c) an aqueous phase present in an amount ranging from 10 to 99.9% v/v;
d) organic phase/solvent present in an amount ranging from 0.1 to 20% v/v;
e) Physiological buffer present in an amount ranging 10 mM to 50 mM

2. The composition as claimed in claim 1, wherein said drug comprises 1-[Bis(4-chlorophenyl)methyl]-3-[2,4-dichloro-ß-(2,4-dichlorobenzyloxy)phenethyl]imidazolium chloride.

3. The composition as claimed in claim 1, wherein approximately 90 to 100% of said drug molecule particles are smaller than 1µm or 2µm.

4. The composition as claimed in claim 1, wherein said organic phase/solvent comprises DMSO.

5. The composition as claimed in claim 1, wherein said hydrophobic drug and serum albumin is present in a molar ratio ranging from 1: 1 to 20:1, preferably in a ratio ranging from 5:1 or 1:1.

6. The composition as claimed in claim 1, wherein said serum albumin is sourced from a Human or Bovine or may be Recombinant.

7. The composition as claimed in claim 1, wherein said physiological buffer having pH of 7.4 comprises phosphate ions.

8. The composition as claimed in claim 7, wherein said physiological buffer is a phosphate buffer saline comprising 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4 with pH adjusted with HCl or NaOH.

9. The composition as claimed in claim 1, wherein said neurodegenerative disease comprises Huntington's disease, Amyotrophic lateral sclerosis, Parkinson's disease. The composition as claimed in claim 1, wherein said composition prevents protein aggregation.

10. A preventive and therapeutic composition for Huntington's disease as claimed in claim 1, wherein said composition comprises 1-[Bis(4-chlorophenyl)methyl]-3-[2,4-dichloro-ß-(2,4-dichlorobenzyloxy)phenethyl]imidazolium chloride as the drug present in an amount ranging from 1 µM to 100 µM; bovine serum albumin present in an amount ranging from 1 µM to 100 µM; an aqueous phase present in an amount ranging from 10 to 99.9% v/v; DMSO as the organic phase/solvent present in an amount ranging from 0.1 to 20% v/v; and phosphate ions based physiological buffer present in an amount ranging 10 mM to 50 mM.

11. The composition as claimed in claim 10, wherein said composition prevents and treats mhtt and peptide fragment thereof.

12. The composition as claimed in claim 10, wherein said composition inhibits Huntington’s exon 1 peptide fragment by 25 fold as compared to the four fold inhibition obtained by CLC.

13. The process of preparing the preventive and therapeutic composition of the preceding claim(s), said process comprising:

a) preparing an aqueous based solution of said physiological buffer and said serum albumin by dissolving in water wherein said serum albumin being already present in the dissolved state in water is added pursuant to the addition of said buffer to the water;
b) adding said drug molecule, being already present in the dissolved state in an organic phase/solvent, to the solution resulting from step (a) followed by incubation at 37 degree celsius to obtain the said preventive and therapeutic composition of claim 1.

14. An in-vitro drug screening kit for testing a surface susceptible hydrophobic drug molecule for its therapeutic action towards a neurodegenerative disease, said kit comprises:
a) Reaction mixture A comprising aqueous based solution of reagents comprising a physiological buffer, an antimicrobial agent and a serum albumin, wherein said reaction mixture A is formulated by preparing an aqueous based solution by dissolving said physiological buffer followed by an antimicrobial agent and a serum albumin in water characterized in that the serum albumin, being already present in the dissolved state in water, is added pursuant to the addition of said antimicrobial agent to the water;
b) Reaction mixture B comprising said test drug molecule being present in the dissolved state in an organic solvent/phase and an aggregating peptide being present in the dissolved state in water with a determined pH, wherein said reaction mixture B is added to said reaction mixture A, to cause reaction, followed by incubation at 37 degree celsius,
and wherein the absence of self assembly of said test drug molecule during said reaction of reaction mixture A and reaction mixture B indicates the therapeutic action of said drug towards the neurodegenerative disease
15. The kit as claimed in claim 14, wherein said antimicrobial agent comprises sodium azide.
16. The kit as claimed in claim 14, wherein said hydrophobic drug molecule comprises protein aggregation inhibitor.
17. The kit as claimed in claim 16, wherein said aggregation peptide is selected from a group comprising of an aggregation protein peptide, insulin, mhtt, synuclein, medin, IAPP.
18. The kit as claimed in claim 16, wherein said hydrophobic drug is available in an amount upto 70% for reaction in the screening assay.