DESCRIPTION

FIELD OF INVENTION

The present invention relates to the denaturing of amyloids resulting from protein which is associated to disorders like type II diabetes, Creutzfeldt Jacob (CJD) and Alzheimer's. An agent that has a potential to degrade these misfolded protein aggregates is critically needed. This invention addresses the denaturation of the aggregates caused by these misfolded proteins by the enzyme lumbrokinase.

BACKGROUND OF INVENTION

Amyloid related diseases are the current problem all over the world leading a large population
towards the deadly diseases like type II diabetes, CJD and Alzheimer's disease. So far only a few
remedies have been found that can dissociate the senile plaques or subcutaneous amyloid deposits in
the patients affected with these diseases. Several natural products have been suggested to dissociate
these prpteinaceous deposits (1-2), but the effectiveness of such agents is still questionable.
Lumbrokinase (LK) and Nattokinase (NK) are serine proteases and NK has been patented for its
amyloid degradation properties (3), but LK has not been reported so far as an agent for degrading
amyloids. Here we are describing the action of LK on amyloid, dissociation in comparison to thej
standard amyloid degrading enzyme NK. LK is highly stable in gastrointestinal tract and thus highly suitable for an oral administration. LK is present in earthworm, Lumbricus rubellus, and is a collection of six proteolytic isozymes (EC 3.4.21). The molecular weight of LK is in the range of 25 kDa to 32 kDa and its isoelectric pH ranges from 3 to 5.

OBJECT OF INVENTION

The present process of degradation of misfolded protein aggregates by lumbrokinase gives an effective avenue for development of potent agents for protein misfolding disease.

This is a novel enzyme that can be used to degrade insulin amyloids as well as amyloids related to
neurodegenerativedisorders.

This novel enzyme is capable of degrading the protein aggregates both in vitro and in vivo.

i i
This novel enzyme can also be used for sterilization purposes where the place or instruments are affected with infectious protein aggregates.

STATEMENT OF INVENTION

Lumbrokinase is a novel enzyme that can be used for the dissociation or degradation of misfolded protein aggregates that are detrimental to human beings and found in conditions like diabetes and different neurodegenerative disorders.
SUMMARY OF THE INVENTION

The invention relates to the application of lumbrokinase.

In one aspect, the invention relates methods of degrading insulin amyloid using lumbrokinase enzyme.

Lumbrokinase can be used to digest amyloids present in the human body because it is stable in the gastrointestinal tract and also stable at temperature up to 65 °C.

In one aspect, the invention relates to the denaturing of pre-formed amyloid aggregates and inhibiting the amyloid formation, deposition or accumulation in amyloidosis.

The therapeutically effective amount of lumbrokinase may be administered through supplement, aerosol spray or directly through infusion or localized injection.

In another aspect the invention relates to the denaturing or inhibiting amyloids that are formed through in vitro environment. The method comprises of the effect of lumbrokinase by adding to these amyloid fibrils and its potential to denature the amyloids.

In another aspect the invention relates to the denaturing of amyloids that are formed through in vivo environment. The method comprises the effect of intramuscular injection of amyloid and amyloid with LK and subsequent observation of amyloid tagged fluorescent images under in vivo imaging system for a designated time.

BRIEF DESCRIPTION OF DRAWINGS

FIGURE 1 shows a photograph of SDS-PAGE gel showing purified Lumbrokinase (indicated by an arrow).
FIGURE 2 shows the fibrinolytic activity of LK compared with a fibrinolytic enzyme NK. It depicts the clot degradation after treatment with different amounts of LK and NK. The fold increase of degradation is found to be 1.25 fold higher in LK compared to NK at the concentration used. So we have normalized the enzyme amount for further experiments based on this result and NK is taken as a standard enzyme for comparison.

FIGURE 3 shows the turbidity of insulin amyloids at 600 nm as a marker amount of protein aggregation respectively. For insulin amyloid treated with LK it is seen that the turbidity is decreased upon treatment with the enzymes respectively. The growth of insulin is exponential up to 9 h and reaches a plateau after 12 h of incubation (Figure 3 A). ThT fluorescene intensity at 479 nm of insulin solution showing the growth of amyloid fibrils (Figure 3B).

FIGURE 4 shows the fluorescence intensity of a dye ThT of insulin amyloids only and insulin amyloids treated with LK with comparison NK respectively. ThT specifically binds to amyloids and yields enhanced fluorescence. Thus, the intensity of fluorescence is directly proportional to the amount of amyloids present in the sample.

FIGURE 5 shows the size of insulin amyloid fibrils using DLS at 24 h of incubation at 65 °C and its subsequent size after treating with enzymes. Insulin can grow to yield matured fibrils upon incubation at 65 °C.

Table I shows the size of insulin amyloid fibrils after NK and LK treatment for 1 h post fibrillation.

FIGURE 6 shows the visualization of the insulin amyloid fibrils after 9 h of fibrillation using atomic force microscopy (AFM) before and after treatment with LK.

FIGURE 7 (A) CD spectra of insulin amyloid at 12 h (black) and that of insulin amyloid after
F te&tmentQ\ffi;lftfi^,(fi) ConteTit^of secobdaiyStEuaurelinlonlyiiisulin amyloid (IA)
and amyloid which is degraded by LK (IA+LK).

FIGURE 8 The toxicity of insulin amyloid (IA) on PC 12 cell at various concentration of insulin amyloids which are treated with LK (IA+LK).

FIGURE 9 (A) The in vivo imaging images using only insulin, and insulin with LK at different time intervals. (B) ROI values of \n vivo images, measured by using IVIS LUMIA System. The fluorescence intensity at different time post injection is shown for only insulin amyloids and insulin amyloid treated with LK.
DETAILED DESCRIPTION OF THE INVENTION

Amyloids are proteinaceous aggregates having a predominant p-sheet structure, and the formation of amyloid plaque is the key pathological feature of various amyloid diseases. The transition of native soluble proteins into insoluble fibrils are the common symptoms of Alzheimer's disease, Parkinson disease, prion-associated encephalopathy's and type II diabetes (4-7). The aggregated state of this protein could be permeable to cell membranes after attachment and could induce cellular dysfunction and apoptosis (5). Thus, the denaturing of this amyloid aggregates could lead to a therapeutic approach for the treatment of amyloid diseases. Insulin hormone had been routinely administered into the subcutaneous tissue for an antidiabetic medication. Repeated insulin injection could result in localized amyloidosis. The amyloid cytotoxicity was found to be an early mechanism resulting in the death of insulin-producing islet P cells (8). Due to the structural similarity of insulin amyloid to the other category of amyloidogenic proteins such as Alzheimer's p and prion protein, insulin can be considered as a model amyloid protein for studying the denaturing effect on amyloid aggregates (9).

The aggregated state of this protein could be permeable to cell membranes after attachment can induce the cellular dysfunction and apoptosis. Thus, the denaturing of this amyloid aggregates could lead to a therapeutic approach for the treatment of amyloid diseases. Insulin hormone is routinely administered into the subcutaneous tissue for an antidiabetic medication. Repeated insulin injection resulted in localized amyloidosis. Several quinones have also been reported to degrade insulin fibrils, but the effect of LK on insulin amyloid degradation has not been explored (10). Although NK has been patented for reducing and degrading Alzheimer's p amyloid (3), no such report exists studying the effect of LK on amyloid degradation. Thus, the study to isolate a stable enzyme for amyloid degradation is warranted. Experiments were conducted to see the potential of Lumbrokinase (LK) towards degradation of pre-formed insulin amyloid.

EXAMPLE 1

Materials

Huminsulin 30/70 40 IU/ml, manufactured by Eli Lilly and Company (India) Limited was procured from local pharmacy. NK plus Astaxanthin & CoqlO from Hawaiian Herbals, USA; Thioflavin T (ThT) and Sephadex G-75 from Sigma Aldrich and other chemicals were purchased locally. Earthworm extract was procured from Xi'an Great and Excellent Natural Product Co., Ltd. China.

Extraction and Purification of LK

LK was extracted from earthworm extract following the protocol of Li et. al (2015) (11) with little
modification. Commercially available earthworm extract suspended in 100 ml saline and

HiK>Wol5en@e9 5txo§nandltherl filtered with guaze.
After centrifugation at 3,000 rpm for 30 min at 4 °C, the supernatant was aspirated. The saturation of ammonium sulfate was prepared according to the table, and certain amount of ammonium sulfate was added to make saturation 20S, and then allowed to stand overnight at 4 °C. The protein peilets thus obtained by ammonium sulfate saturation was resolubilized with phosphate buffer (PBS, 10.mM, pH=7.4) and dialyzed overnight at 4 °C. The dialyzed solution was kept in the refrigerator and purified next day by sephadex G-75 gel filtration chromatography followed by DEAE cellulose-52 ion exchange chromatography. The purity of LK was checked by SDS PAGE (Figure 1) and eluent was stored at -20°C for further use. The final yield of the pure enzyme was 37 % and the protein concentration was 0.4025 mg/ml. This solution was used for further amyloid degradation studies and taken as the source of LK.

In vitro fibrinolytic activity

To normalize the enzyme activity of LK, in vitro fibrinolytic activity was assayed according to Prasad et.al (2006) (12). The standardization of the earthworm extracted LK with NK was done by fibrinolytic assay using blood clot degradation (Figure 2A and B). The percentage of clot degraded was taken as a measure for its fibrinolytic activity. The clot degradation ability of NK and LK is shown in Figure 1. Figures 1 A and 1 B depict the clot degradation after treatment with different amounts of NK and LK. Figure 1 C compares the clot degrading efficiency of these enzymes. The results showed that the fibrinolytic activity of LK was 1.15 fold, 1.2 fold and 1.4 fold of NK at enzyme amounts of 100 ul, 150 ul and 200 ul respectively for clot lysis. Thus the average activity of LK obtained from the earthworm is 1.25 fold higher than NK. Based on these results 12.5 ul of NK was used for every 10 ul of LK for further experiments.

EXAMPLE 2

Insulin amyloid formation and degradation

Insulin amyloid formation was induced in a similar way as done by Lee et.al (2007) (13). Huminsulin 30/70 40 1U /ml was incubated at 65 °C to form insulin aggregates and the amount of fibrillation at designated time (2 h, 4 h, 6 h, 9 h, 12 h, and 24 h) was monitored. Amyloid degradation study was done according to Hsu et. al (2009) (3). The fibrinolytic activity of LK was found to be 1.25 fold higher than NK. Thus, for every 10 ul of LK, 12.5 \i\ of NK was utilized for further experiments with equal activity. Briefly, at each designated time, 12.5 ul NK or 10 ul LK was added to 179 ul of pregrown insulin amyloid samples respectively and incubated at 40 °C for 1 h to allow enzymatic degradation reaction. After incubation, the fibril degradation status was compared with insulin fibrils formed till that time, by turbidity assay, ThT fluorescence and DLS. For enzymatic degradation study we withdraw sample at each respective time from 65 °C to confirm its amyloid formation by ThT assay and then incubated with enzyme LK and NK at 40 °C for 1 h to study degradation status by Turbidity assay, ThT assay, DLS and CD and AFM.

EXAMPLE 3

Turbidity assay of insulin amyloid

The absorbance at 600 nm is extensively used to quantify insoluble protein aggregates (fibrils) (14). The insulin amyloid aggregation and its degradation using NK and LK.at different time intervals were measured by turbidity assay using Shimadzu UV-1800 UV-VIS double beam a s>e€tTopHotBnfeterC E CBENNA-I 87/10/2016 1.1 ■- 1 8

Our results on turbidity assay of insulin fibril growth also corroborates with the findings of ThT fluorescence study. The turbidity at 600 nm for insulin fibrils at different time intervals (2 h, 4 h, 6 h, .. ■ 9 h, 12 h, 24 h and 36 h) is shown in Figure 3 (A). The turbidity values indicating the dissociation of fibrils by the enzymes NIC and LK is also depicted in Figure 3 (A). The insulin fibrils were allowed to grow at 65 °C and at different time points were further incubated with the enzymes for 1 h. The decrease in the amount of aggregated proteins was obtained by measuring the absorbance at 600 nm for different enzyme treated samples.

EXAMPLE 4

ThT fluorescence assays of insulin amyloid

ThT gives fluorescence on binding with amyloids and is the key molecule to monitor amyloid formation (15). For each sample (control and enzyme treated) 200 ul ThT (20 uM) was added to 30 u.1 insulin amyloid. 20 ul of this solution was diluted with 3 ml of distilled water to measure the fluorescence monitoring emission from 440-600 nm after exciting at 412 nm using FP-8300 Jasco Spectrofluorimeter.

The growth of insulin fibrils as obtained from ThT fluorescence study at different time intervals (2 h, 4 h, 6 h, 9 h, 12 h, 24 h, 32 h and 36 h) is shown in Figure 3 (B). ThT gives specific fluorescence for amyloid fibril morphology and it was found that the fibril growth has a lag phase upto 4 h, an exponential phase upto 10 h and then a stationary phase was found upto 36 h.

The insulin fibril degradation kinetics was also monitored using ThT fluorescence. The ThT fluorescence intensity at different time interval (2 h, 4 h, 6 h, 9 h, 12 h, 24 h) for 1 h incubation of the enzymes shows a significant decrease in case of both LK and NK treated samples compared to control insulin fibrils at that time (Figure 4). At 2 h, 4 h, 6 h and 9 h, it was observed that LK could degrade higher amount of insulin fibrils compared to NK. The percentage of insulin fibril dissociation is shown in Figure 3 B. For the insulin sample at 12 h the degradation with both the enzymes was (for LK-76 % and NK- 62 %). After 24 h, insulin forms fully gown fibrils and the enzymatic degradation for 1 h dissociates fibrils up to 78% by LK.

EXAMPLE 6

Dynamic light scattering (DLS) analysis of insulin amyloid

DLS was used to measure the size of insulin fibrils in control and after treatment with NK and LK at different time intervals using a Malvern nano ZS90 zeta sizer (10). In 1 ml PBS, 10 ul of sample after proper mixing was added and allowed to equilibrate for 1 min. The hydrodynamic diameter obtained from the DLS data was taken as a measurement for fibril size.

The particle size distribution was investigated using DLS for insulin fibrils and enzyme treated insulin fibrils at different time intervals (Figure 5). At 2 h the size of insulin aggregates were 600 nm which decreased to 448 nm and 488 nm upon NK and LK treatment respectively. With increase in the incubation time the size of insulin fibrils increased and at 9 h the size was observed to be 4198 nm. NK treatment reduced the size to 259 nm & 3106 nm where as treatment with LK reduced the fibril size to 299 nm & 999 nm (Table I). The presence of two different sizes indicated a heterogeneous aggregation of fibrils after degradation with the enzymes; although the size has reduced upon NK and LK treatment. This can be justified by the fact the 1 h incubation with the enzyme was not sufficient for degrading the amount of insulin fibrils formed due to 9 h incubation. Thus, a longer time of incubation with the enzyme may decrease the size into a smaller and a monodispersed one. After 12 h and 24 h the size of insulin fibrils decreased due to inaccessibility to the longer fibrils for the analysis as the sample increased its viscosity. The size distribution of the insulin fibrils as revealed by DLS studies is shown in Table I. '
EXAMPLE 7

Atomic Force Microscopy (AFM) Analysis of insulin amyloid

The dissociation of insulin amyloids using LK was visualized using an AFM equipped with a Nanoscope HID controller (Digital Instruments Inc., Santa Barbara, CA) in the tapping mode according to Girigoswami et.al (2008) (15). Glass coverslips were cleaned with aqua regia and dried in a dirt free atmosphere. A drop of sample was taken in the coverslip and was dried overnight in a dust free atmosphere and taken for AFM analysis. Representative images in each case were obtained by scanning different samples and at least five spots over the entire surface.

Mature amyloid fibrils were observed at 9 h incubation of insulin. LK treated insulin fibrils degraded
into smaller fibrils was observed. The mature fibrils at 12 h sample (data not shown) are efficiently !:
degraded by LK. These results suggest that LK extracted from the earthworm has a capacity to degrade insulin amyloid fibrils.
EXAMPLE 8

Circular Dichroism (CD) study

Circular dichroism (CD) was measured for the samples of insulin amyloids and LK treated insulin amyloids. Briefly, the sample was placed in a 1-mm quartz cell and the CD spectrum between 200 and 250 nm recorded on a J-715 CD spectrometer (JASCO, Japan). The bandwidth was set to 2 nm and the step resolution was 0.1 nm.

To see structural changes, we recorded circular dichroism in which the pre-formed insulin amyloid is digested by enzyme LK has shown negative ellipticity at 218 nm in the CD spectra, which indicates the loss of p sheet in pre-formed insulin amyloid (Figure 7A). Insulin amyloid sample shows high P sheet content compare to LK treated insulin amyloid (Figure 7B).

EXAMPLE 9

In vitro toxicity assay

To study the effect of toxicity of the amyloid fragments after degradation with LK, we have carried
out MTT assay using PC 12 cells as described earlier (14). Undifferentiated PC 12 cells were
maintained in DMEM supplemented with 10 % FBS and 1 % antibiotic solution at 37 °C, 5 % C02,
humidified atmosphere. The cells were sub-cultured two times a week to maintain them in the
exponential phase. Cells at density of 104cells/well were seeded in 24 well plates and allowed to
adhere for 24 h. After 24 h, the cells were treated with different concentrations of insulin amyloid
and LK treated insulin amyloid: The plates were incubated overnight and after 24 h^ 100 ul of MTT
at 5 mg/ml was added to each well at dark. The plates were further incubated at 37 °C for 4 h to
develop the color of formazan crystals. After 4 h the purple formazan crystals were dissolved using
DMSO and the absorbance at 570 nm was recorded. The percent cell viability was then calculated
with respect to the untreated control.
The degradation of insoluble aggregate may form soluble aggregates, which can be more toxic than the matured insoluble fibrils. So it will be very much interesting to see the toxicity of disintegrated particle (product of Amyloid digestion by LK) on PC 12 cells. At 18 uM of Insulin amyloid cell viability was observed upto 85.18 % where as insulin amyloid treated with LK at 18 uM showed cell viability upto 96.54 % (Figure 8). MTT assay suggest there is no toxicity of disintegrated particle compared to control (cells treated with only insulin amyloid) Treating of amyloid with LK may attenuate the cytotoxicity which has been observed in PC 12 cell.

EXAMPLE 10

In vivo live animal imaging

Adult zebrafish (Danio rerio) was used for studying the in vivo effect of LK on insulin amyloid. Zebrafishes were maintained in rectangular glass tanks in distilled water with dry worms and live worms for feeding and maintained at an average temperature of 27± 4°C, and 14 h in light and 10 h dark condition (16). Air pump was attached for proper oxygen circulation inside water. Insulin amyloid was formed according to Lee et.al (13) and confirmed by ThT fluroscence assay. For subcutaneous injection in zebrafish the sample was prepared as follows. 10 ul LK from stock solution was added to 179 ul of pregrown insulin amyloid samples respectively. 10 ul of ThT (26 uM) was added in 100 ul sample of the above solution. Finally 12.5 ul of the solution was injected subcutaneously in the zebrafish using a microsyringe. For control without the enzymes only insulin was injected with ThT at the same concentrations mentioned above. Fluorescence images were acquired by using IVIS Kinetic from Caliper Life Sciences according to Chen et. al., (2013) (17) with excitation at 440 nm (slitwidth 5 nm) and emission using specific filter up to 2 h post injection. Just after the injection the fishes were placed on the stage of the instrument and the image is acquired. They were replaced immediately after image capture into water tank. The process was repeated at an interval of 30 min for 2 h. The fishes retained normal activity during the study. The fluorescent images were captured and analyzed. After acquiring images in IVIS Kinetic Lumia imaging system, we traced the fluorescence intensity region of interest (ROI) by using ROI Tool in IVIS system following user manual and compared the ROI intensity among control fish and the fish those are treated with enzyme.

The in vivo degradation potential of the enzymes LK on insulin fibrils is visualized by IVIS Kinetic Lumia imaging system. Figure 9A shows the fluorescent images of zebrafish at different time intervals post injection with insulin and insulin LK respectively. The fluorescence intensity with respect to the time was plotted in the figure 9B. There was a regular increment in the fluorescence intensity for insulin amyloid confirms the growth in amyloid. 81% signal intensity was reduced for there is 98% reduction was observed for LK treated fish (Figure 9B). Therefore, it clearly shows remarkable change in the emission intensity after treatment with enzymes but LK. The in vivo study confirms that the LK has a better capacityfor the reduction of amyloid fibrils.

In conclusion, this study suggests the potential of the enzyme LK extracted from earthworm for the degradation of human insulin amyloids in vitro and in vivo. All the observations revealed that LK is a better option for the degradation and reduction of amyloid fibrils and it can be used as a potent amyloid degrading agent for insulin induced amyloidosis. The amyloid denaturing capacity of LK for other amyloids like Alzheimer's A|3 and Prion amyloid is to be explored further.

Further Aspects and Utilization of the invention Therapeutic Application

Prior to administration of Lumbrokinase it can be formulated with suitable carrier. One who is suffering for type II diabetes, Prion disease or Alzheimer's disease can take Lumbrokinase orally.

Lumbrokinase can be modulated with appropriate suitable carrier, diluents or recipients like dextrose, sucrose, water syrup, talc etc. The Lumbrokinase optimum compositions can be formulated in a unit dosage form, each dosage containing from about 1 to about 10,000 mg of Lumbrokinase, more usually about 250 or 500 to about 1,000 or 2,000 mg. sterile injection can be prepared with Lumbrokinase or can be prepared with freeze dried powder of Lumbrokinase. The Lumbrokinase for neurodegenerative disease can be optimized by to cross the blood brain barrier. Lumbrokinase can be administered directly through intraventricular, subcutaneous, intraperitoneal, and intradermal injection.

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CLAIMS

1. We claim a method of dissolving or degradation and/or disintegration of pre-formed insulin amyloid fibrils in an in vitro environment, the method comprising: adding to the in vitro environment a composition comprising an effective amount of LK to degrade insulin amyloids.
2.
3. We claim a method of dissolving or degradation and/or disintegration of pre-formed insulin amyloid fibrils in an in vivo environment, the method comprising: adding to the in vivo environment a composition comprising an effective amount of LK to degrade insulin amyloids.
4.
5. We claim a method of subcutaneous insulin amyloid clearance which is associated with type II diabetes.
6.
7. The method of claim 1, wherein the amyloid associated with type 2 diabetes and can be a model protein for Alzheimer's and/or a Prion disease.
8.
9. The method of claim 1, wherein the pre-formed amyloid comprises the object like surgical instruments, a pharmaceutical product, and animal feed and LK can be used to sterilize them.
10.
11. The method of claim 2, wherein the in vivo clearance of amyloid by LK comprises the tissue/organ which has been affected with amyloids example kidney, heart and brain.