WO2017182044A1

WO2017182044A1 - New modified synthetic peptides derived from honey proteins in cancer control

Info

Publication number: WO2017182044A1
Application number: PCT/EG2017/000008
Authority: WO
Inventors: Khaled Mohy Eldin ELAWADAN
Original assignee: Elawadan Khaled Mohy Eldin
Priority date: 2017-03-05
Filing date: 2017-03-05
Publication date: 2017-10-26
Also published as: WO2017182044A4

Abstract

Many trials were done to get more potent and stable anti-cancer peptides. Synthetic peptides derived from honey were modified to obtain promising peptides in cancer control. We modified the first two amino acids of the N-terminus and the last three amino acids of the C-terminus to be in the D-form instead of the L-form plus we added a NH2-group to the N-terminus and a COOH-group to the C-terminus. We found that the following peptides with the sequences from N-terminus to C-terminus have more potent anti-cancer effects plus more stability in human serum than the non-modified peptides of patent W0/2014/040605. Seven peptides similar to peptide F of patent W0/2014/040605 were postulated of more or less similar actions for further assessment experiments and maybe modification.

Description

New modified synthetic peptides derived from honey proteins in cancer control

Technical field:

Drug discovery, anti-cancer peptides. BACKGROUND ART:

We have four main lines in management of malignancy.

Surgery: Surgery can be used to prevent, treat, stage (determine how advanced the cancer is), and diagnose cancer. In relation to cancer treatment, surgery is done to remove tumors or as much of the cancerous tissue as possible. It is often performed in conjunction with chemotherapy or radiation therapy.

For those whose cancer is not treatable, palliative surgery may be an option to relieve pain that may be caused by the cancer. Palliative surgery is not intended to treat or cure the cancer, or even to prolong life, but more to lessen discomfort.

Chemotherapy: Chemotherapy is a type of cancer treatment that uses of drugs to eliminate cancer cells. Unlike surgery, chemotherapy affects the entire body, not just a specific part. It works by targeting rapidly multiplying cancer cells. Unfortunately, other types of cells in our bodies also multiply at high rates, like hair follicle cells and the cells that line our stomachs. This is why chemo can cause side effects like hair loss and an upset stomach.

Chemotherapy is most commonly given by pill or intravenously (IV), but can be given in other ways. A single type of chemotherapy, or a combination of drugs, may be prescribed for a specific length of time. Like surgery, chemotherapy can be prescribed alone, in conjunction with radiation therapy or biologic therapy.

Radiation Therapy: Radiation therapy uses certain types of energy to shrink tumors or eliminate cancer cells. It works by damaging a cancer cell's DNA, making it unable to multiply. Cancer cells are highly sensitive to radiation and typically die when treated. Nearby healthy cells can be damaged as well, but are resilient and are able to fully recover. Radiation therapy may be given alone, along with chemotherapy, and/or with surgery. The decision to combine radiation therapy with other types of treatment depends on the stage of cancer and other factors

Biologic or Targeted Therapy: Biologic therapy is a term for drugs that target characteristics of cancerous tumors. Some types of targeted therapies work by bl ocking the biological processes of tumors that allow tumors to thrive and grow. Other types of therapies cut off the blood supply to the tumor, causing it to basically starve and die because of a lack of blood.

Targeted therapy is used in select types of cancer and is not available for everyone. It is given in conjunction with other cancer treatments.

Problem and Defect:

Although of many methods in management of malignancy like surgery, chemotherapy, radiotherapy, immunotherapy . . . etc.

Many types of cancer still have very poor prognosis.

It is enough for anybody to have a look at the main causes of death in any country to know that cancer is one of them and to discover how the challenge and defect in its treatment.

Many researches aims were just to improve of the already present methods.

Anyhow, I think we must introduce something new rather than just thinking of improvement of traditional methods to overcome this challenge.

DISCLOSURE OF INVENTION

Many trials were done to get more potent and stable anti-cancer peptides from our previous published patent peptides (WO/2014/040605).

We changed the first two amino acids of N terminus to D-form as well as we did for the last tree amino acids in C terminus.

Also N terminus was modified by adding NH2 group and C terminus was also modified by adding COOH group.

As we recognize peptide F from our previous patent as a lead peptide, peptide Fland F2 were modified and peptide F3 was added to our group as a promising peptide in cancer control. We found that those peptides with the sequences from N Terminus to C terminus have more potent anti cancer effects plus more stability in human serum than our postulated peptides of patent WO/2014/040605.

Al- NH2-(d-Met)(d- Ala) ILTYVYVF AVLFIAK(d-Lys)(d-Lys)(d-Lys)- COOH

El - NH2- (d-Met)(d-Gln) LRVLFFFL FVATISKK(d-Lys)(d-Lys)(d-Lys)- COOH

Fl - NH2- (d-Met)(d-Thr)KWLLL VVCLGIACQ K(d-Lys)(d-Lys)(d-Lys)- COOH

F2- NH2-(d-Met)(d-Thr) KWLLLWC LGIACQDVTSA(d-Lys)(d-Lys)(d- Lys)-COOH

F3- NH2- (d-Met)(d-Thr)RLFMLVCL GIVCQGTT(d-Gly)(d-Asn)(d-Ile)- COOH

Seven peptides similar to peptide Fl were postulated of more or less similar actions for further assessment experiments and may be modification .

1- NH2-(d-Met)(d-Thr) RWLFMVACLGIACQGAIVR(d-Lys)(d-Lys)(d-Lys)-COOH

2- NH2-(d-Met)(d-Thr) KWLLLMVCLGIACQNIRGG(d-Lys)(d-Lys)(d-Lys)-COOH

3- NH2-(d-Met)(d-Thr) TWLLLWCLGIACQGITSV(d-Lys)(d-Lys)(d-Lys)-COOH

4- NH2-(d-Met)(d-Thr) NWLLLIVCLSIACQDVTSA(d-Lys)(d-Lys)(d-Lys)-COOH

5- NH2-(d-Met)(d-Thr) RWLFMVACLGIACQGAILR(d-Lys)(d-Lys)(d-Lys)-COOH

6- NH2-(d-Met)(d-Ile) RWLLLMYLGIAVRCYRHSF(d-Lys)(d-Lys)(d-Lys)-COOH

7- NH2-(d-Met)(d-Ser) FNIWWLILYFSIVCQAKAH(d-Lys)(d-Lys)(d-Lys)-COOH

Using 3 letters symbols for amino acid:

Al - NH2- (d-Met)(d- Ala) lie Leu Thr Tyr Val Tyr Val Phe Ala Val Leu Phe Ile Ala Lys(d-Lys)(d-Lys)(d-Lys)-COOH

El - NH2- (d-Met)(d-Gln) Leu Arg Val Leu Phe Phe Phe Leu Phe Val Ala Thr Ile Ser Lys Lys(d-Lys)(d-Lys)(d-Lys)-COOH

Fl - NH2- (d-Met)(d-Thr) Lys Trp Leu Leu Leu Val Val Cys Leu Gly Ile Ala Cys Gin Lys Lys (d-Lys)(d-Lys)(d-Lys)-COOH F2- NH2- (d-Met)(d-Thr) Lys Trp Leu Leu Leu Val Val Cys Leu Gly Ile Ala Cys Gin AspVal Thr Ser Ala(d-Lys)(d-Lys)(d-Lys)-COOH

F3- NH2- (d-Met)(d-Thr) Arg Leu Phe Met Leu Val Cys Leu Gly Ile Val Cys Gin Gly Thr Thr(d-Gly)(d-Asn)(d-Ile)-COOH

1- NH2-(d-Met)(d-Thr) Arg Trp Leu Phe Met Val Ala Cys Leu Gly Ile Ala Cys Gin Gly Ala Ile Val Arg(d-Lys)(d-Lys)(d-Lys)-COOH

2- NH2-(d-Met)(d-Thr) Lys Trp Leu Leu Leu Met Val Cys Leu Gly Ile Ala Cys Gin Asn Ile Arg Gly Gly(d-Lys)(d-Lys)(d-Lys)-COOH

3- NH2-(d-Met)(d-Thr) Thr Trp Leu Leu Leu Val Val Cys Leu Gly Ile Ala Cys Gin Gly Ile Thr Ser Val(d-Lys)(d-Lys)(d-Lys)-COOH

4- NH2-(d-Met)(d-Thr) AsnTrp Leu Leu Leu Ile Val Cys Leu Ser Ile Ala Cys Gly Asp Val Thr Ser Ala(d-Lys)(d-Lys)(d-Lys)-COOH

5- NH2-(d-Met)(d-Thr) Arg Trp Leu Phe Met Val Ala Cys Leu Gly Ile Ala Cys Gin Gly Ala He Leu Arg(d-Lys)(d-Lys)(d-Lys)-COOH

6- NH2-(d-Met)(d-Ile) Arg Trp Leu Leu Leu Met Tyr Leu Gly Ile Ala Val Arg Cys Tyr Arg His Ser Phe(d-Lys)(d-Lys)(d-Lys)-COOH

7- NH2-(d-Met)(d-Ser) Phe Asn Ile Trp Trp Leu He Leu Tyr Phe Ser Ile Val Cys Gin Ala Lys Ala His(d-Lys)(d-Lys)(d-Lys)-COOH

So, we ask protection of those peptides sequences as it can be used as a drug in cancer control.

Experimental Data:

In vitro:

Study no l:Effects of peptides A, E, F, Al, El & Fl on cell proliferation in 3 different cell lines.

This project was aimed to evaluate the peptides A, E & F related to Al, El & Fl effects on C6 glioma cell line, U251 human glioma cell line, UW3 cell line proliferations.

10 mg per each samples of A, E, F, Al, El & Fl was dissolved in 1 mL DPBS to make the stock solution which will be added into the wells directly. 0.1% TFA solution was applied as the control. Stability of these peptides in human serum at room temperature were found to be around 70-80% after 12h opposite the case of our data for peptide F before modification that was around 10-16% after only one hour. a) Plate the cells into the 48 well plate in DMEM media + 10% fetal calf serum + 1 % penicillin & streptomycin + 1% fungizon

b) Add 20 μL stock solutions of the compounds into the wells.

c) 20 μL0.1 % TFA solution was used as the control.

d) Incubate the plate at 37°C &5%co2 condition.

e) After around 24 hour,48 hours and 72 hours Cells were harvested of 0.05%

trypsin and 0.02% EDTA,

f) Manual method was used in cell counting through heamocytometer.

Results:

Better inhibitory effects for peptide Al, El &F1 upon C6, U251 & UW3 proliferation compared to peptides A, E and F.

Study no 2:Effects of peptides Al, El & Fl on cell proliferation in 3 different cell lines.

This project was aimed to evaluate the effects of peptides Al, El, Flon

C6 glioma cell line, U251 human glioma cell line, UW3 cell line proliferations.

10 mg per each samples of (Al, El, Fl) was dissolved in 1 mL DPBS to make the stock solution which will be added into the wells directly. 0.1% TFA solution was applied as the control. a) Plate the cells into the 24 well plate in DMEM media + 10% fetal calf serum + 1 % penicillin & streptomycin + 1% fungizon

b) Add 40 μL stock solutions of the compounds into the wells.

c) 40 μL0.1 % TFA solution was used as the control.

d) Incubate the plate at 37°C &5%co2 condition.

e) After around 24 hour,48 hours and 72 hours Cells were harvested of 0.05%

trypsin and 0.02% EDTA, Manual method was used in cell counting through heamocytometer.

Results:

As shown in figure.1,2,3 very good inhibitory result for peptide Fl upon C6, U251 & UW3 proliferation compared to peptide Al and El.

Study no 3: Effects of peptides Fl, F2 & F3 on U251 cell proliferation

This project was aimed to evaluate the effects of peptides F1,F2 & F3 on

C6 glioma cell line proliferation.

10 mg per each samples of (F1,F2 &F3) was dissolved in 1 mL DPBS to make the stock solution which will be added into the wells directly. 0.1% TFA solution was applied as the control.

A. Plate the cells into the 24 well plate in DMEM media +10% fetal calf serum +1% penicillin & streptomycin + 1% fungizon.

B. Add 40 μL stock solutions of the compounds into the wells.

C. 40 μL0.1% TFA solution was used as the control.

D. Incubate the plate at 37°C &5%co2 condition.

E. After around 24 hour,48 hours and 72 hours Cells were harvested of 0.05% trypsin and 0.02% EDTA.

F. Manual method was used in cell counting through heamocytometer.

Results: As shown in fig.4 excellent inhibitory result for peptide F3 upon U251 proliferation compared to peptide Fl & F2.

Previous experimental data: Materials & Methods:

In Vitro study 1 :

Objective:

This project was aimed to evaluate the peptides A, E, F and AEF effect on 15 tumor cell lines MG63, U87MG, SH-4, RD, KP1 , 5637, 2774, ML-1 , Cal-27, Colo-205, 769P.EOL-1 , HLE, Calu-3 and MDA-MB-436 with cell viability assay.

Test and control article:

6 samples of peptides (1 A, 2B, 3C, 4D, 5E, 6F) in powder were stored at -20°C. 10 mg samples of (1 A, 5E, 6F) was dissolved in 1 ml DPBS to make the stock solution which will be added into the wells directly. 0.1% TFA solution was applied as the control.

Test System

Reagents and materials

a) MEM Alpha Medium, Cat. # 32561-037, Lot. # 1245518

Gibco b) RPMI 1640 Medium, Cat. # SH30809.01 B, Lot . # NYA0779 Hyclone c) DMEM Medium, Cat. # 11960-044, Lot. # 1267442 Gibco d) FBS, Cat. # 04-001-1 A , Lot. # 615303 Hyclone e) Penicillin-Streptomycin, Cat. # SV30010 , Lot. # J130071

Thermo

f) 96 well assay plate, Cat. # 3610 , Lot. # 27012035

Corning g) CellTiter, Cat. # G755B , Lot. # 0000047328 Promega Assay Procedures

g) Plate the cells into the 96 well assay plate in a density of 5000 and 10000 cells/well for adherent and suspension cell lines respectively with 50 μL complete cell culture medium.

h) Add 10 μL stock solutions of the compounds into the wells.

i) 10 μL0.1 % TFA solution was used as the control for data analyzing. j) Add 40 μL of complete medium into the wells to reach a total volume of 100pL

k) Incubate the plate at 37°C for 72 hours . I) Add 50 LCelltiterGlo assay mix solution to each well and mix gently at room temperature for 10 minutes, then read the luminescence with PherastarPlus (Molecular Devices).

Results

The peptides (A,E,F and their combination AEF) were prepared and tested according to the protocols described above. The cell growth inhibition results were shown in the figures of supplementary final report data.

As shown, very good response for peptide F upon 14 cell lines out of 15 cell line then peptide A and lastly peptide E; while we got excellent response when we added peptide F,E&A in one group.

EOL-1 cells viability assay results were not presented in figure 1 , EOL-1 cells were very sensitive to 0.1 %TFA control. Only about 13% cell viability was detected with 1%TFA treated EOL-1 cells as compared with medium without TFA treated EOL-1 cells. So the data was presented as either normalized by 0.1 %TFA control or medium control for EOL-1 cells.

In Vitro Study 2:

2 Objectives

This project was aimed to evaluate peptides A+F effect (of five

concentrations) on 4 tumor cell lines U87MG, MDA-MB-468, K562 &A375 - as being proposed for further in vivo studies- with cell viability assay and apoptosis assay.

3 Test and Control Articles

Growth inhibitory assay was performed with 10ug, 25ug, 50ug, 75ug and 100ug of each peptide.

(A+F) per well. Peptide A and peptide F were stored at -20°C. 5mg samples of peptide A and peptide F were dissolved in 500ul DPBS to make the stock solution which will be added into the wells directly. 0.1 % TFA solution was applied as the control.

4 Test System

4.1 Reagents and materials Name Cat. No. Lot. No. Vendor

MEM Alpha 11095-080 1179276 Gibco

MDM 12440-046 841374 Hyclone

DMEM 10566-016 1300085 Gibco

FBS 10099-141 1227693 Gibco

96 well assay plate 3610 27012035 Corning

CellTiterGloKit G755B 0000047328 Promega

Caspase3/7 G811A 0000014788 Promega

4.2 Assay Procedures:

a) Plate the cells into the 96 well assay plate in a density of 5000 and 10000 cells/well for adherent and suspension cell lines respectively with 50 μΙ_ complete cell culture medium.

b) Add 10 μL stock solutions of the compounds into the wells.

c) 10 μL 1% TFA solution was used as the control for data analyzing.

d) Add 40 μL of complete medium into the wells to reach a total volume of 100 μL.

e) Incubate the plate at 37°C for 72 hours .

f) Add 50 μL CelltiterGlo assay mix solution to each well and mix gently at room temperature for 10 minutes, then read the luminescence with

PherastarPlus (Molecular Devices).

5 Results

5.1 Growth inhibition assay:

The peptides (combination A+F) were prepared and tested according to the protocols described above. The cell growth inhibition results were shown in the figures 5 & 6 below.

Apparent tumor cell inhibition for all tested lines increases with increased peptides doses.

5.2 Apoptosis assay:

Apoptosis Effect of Compound A+F

on Tumour Cell(TFA as control).

As shown in figure7 high apoptosis results of peptides A+F in U87MG & MDA-MB-468, moderate apoptosis in K562 and mild apoptosis in A375 cell line.

In Vivo study 1

Dosage and acute toxicity:

Before the experiment, we did pilot study to mimic the treatment and studied mouse tolerance to i.v. injection of the peptides. We tested i.v. injection of mixed peptide A+F in nude mice at different doses: (1 ) 50 ug/g per peptide, which is a total of 100 ug peptide /g; (2) 25 ug/g per peptide and (3) 12.5 ug/g per peptide. In group (1), 2 out of 2 mice died immediately after injection; in group (2), 3 of 3 mice tolerated the does but showed weak and reluctant to move after the injection but recovered in a few hours; in groups (3), 3 of 3 mice tolerated well without much stress. The combination of Peptide A+F in i.v route showed strong acute toxicity at a relatively high dosage while in s.c route can tolerate more than 100 ug/g per peptide, total of 200 ug peptides/g. This is consistent with the observation in rats with i.v. injection of Peptide F for the PK study.

Efficacy study of peptide A&F in U67 s.c xenograft tumor model:

1. Purpose To evaluate the anti-tumor efficacy of peptide A and F combination in human glioblastoma U87 subcutaneous (s.c.) xenograft tumor model.

2. Material

2.1 Cell line U87, the cells were cultured in DME +10% FBS+1% P/S antibiotics.

2.2 Animal 2.2.1Animal species and strain: 20 BALB/c nude mice

2.2.2 Sex and age: Female, 6-8 weeks old, (20±2)g

2.2.3 Breeder/supplier: SLAC Laboratory Animal Co. Ltd.

2.2.4 Test Facility: In Vivo Pharmacology, GenScriptlnc USA.

2.2.5 Acclimation: 7 days

2.2.6Room: SPF

2.2.7 Room temperature : (22-25)□

2.2.8 Room relative humidity: (40-70)%

2.2.9Room pressure and air change: (10-20) Pa; (10-15) times/h

2.2.10 Light cycle: 12-hour light cycle (8:00-20:00) withi 2-hour dark cycle

2.2.11 Animal housing: 5 mice / cage by treatment group

2.2.12Food: Free access to food, autoclaved and irradiated, SLAC Laboratory Animal Co. Ltd.

2.2.13 Water: Free access to water from local supply (filtered by Mol animal ultrapure water system and autoclaved)

The study protocol and all the animal use was approved by the IACUC of GenScript.

2.3 Reagents

2.3.1 DMEM medium: Invitrogen, Cat No: 11875093

2.3.2 FBS: Invitrogen, Cat No: 100999-141

2.3.3 P/S antibiotics: Invitrogen, Cat No: 15070-063

2.3.4 Trypsin-EDTA: Invitrogen, Cat No: 25200-072

2.3.5 P/S antibiotics: Invitrogen, Cat No: 15070- 63

2.3.6 Matrigel: BD, Cat No: 354234Order Number: 335369

2.3.7.1 Test article (nasal) name: Peptide A Supplier: Client

Physical description: White powder Storage condition: -20°C

2.3.7.2 Test article (nasal) name: Peptide F Supplier: Client

Physical description: White powder Storage condition: -20°C

2.3.8 Drug formulation: Use normal saline as solvent. 2.3.9 Solution storage condition: The peptide solution should be kept at 4°C and used within 48 hours.

3. Procedure

3.1 Cell amplification and implantation U87 cells were cultured and s.c.

injected into nude mice (right flank). A total number of 2.0x106 tumor cells per mouse were suspended in 0.1 ml_ of PBS/ matrigel mixture (1 :1 ) and injected (12 tumor-bearing mice are required).

3.2 Groups and dosage When tumor size reached a volume of 100-200mm3 , U87 tumor-bearing nude mice were randomly assigned into 3 groups (4 mice/group) and started dosing immediately:

Group 1 was administered with vehicle and served as control group.

Group 2 was administrated with peptide A+F (10 g/g of mouse weight with each peptide, i.v., biw, 3 injections; then 20 μg/g intratumorals.c injection, every other day, 2 injections; therefore 5 injection in total ).

Group 3 was administrated with peptide A+F (10 μg/g of mouse weight with each peptide, i.v., only a single dose at first week; then 20 μg/g

intratumorals.c injection, every other day, 2 injections; therefore 3 injections in total).

The administration period lasted 12 days.

• For the peptide injection: Mix the peptide A solution and peptide F solution before each injection and inject them together.

3.3 Physical examination If abnormal appearance and behavior or signs of morbidity and/or mortality were seen through the cage side observation, the veterinarian would be notified and proper physical examination and/or necropsy would be performed. „

Frequency: Daily just after the cell inoculation and twice a day thereafter starting on first dosing.

3.4 Measurement The tumor volumes, body weight of tumor-bearing mice were measured twice a week and mice morbidity was recorded immediately before measuring the tumor volume and body weight throughout the whole experiment. 3.5 Terminal procedures and necropsy

3.5.1 Early death/unscheduled necropsy If a mouse died on study, the time of death would be estimated as closely as possible and recorded, and the mouse would be necropsied as soon as possible. If the necropsy could not be performed immediately, the mouse would be refrigerated (not frozen) to minimize tissue autolysis and would be necropsied no later than 12 hours following death. If a mouse appeared in poor condition or in morbidity, the mouse was euthanized after discussing with the client. It might be euthanized (as described below) per the Testing Facility's policies on humane care of animals. If the tumor burden was larger than 2000mm3 or the body weight loss was greater than 20% baseline body weight, the mice would be euthanized. If the weight dropped significantly, the data would be viewed with a caveat. All unscheduled-necropsy mice would be necropsied immediately, or, if this could not be performed, the mouse would be refrigerated to minimize autolysis and necropsy no later than 12 hours after death.

3.5.2 Scheduled necropsy At the end of the experiment, tumor bearing mice were euthanized with C02 by the end of the study. The tumor samples were harvested, weighed and photographed.

4. Endpoints

4.1 Tumor volume Tumor volume was measured twice a week in two dimensions using a caliper, and the volume was expressed in mm3 using the formula: V = 1/2*axb2 where a and b were the long and short diameters of the tumor, respectively.

4.2 Body weight The body weight of tumor-bearing mice was measured and recorded twice a week after administration. If the body weight loss was greater than 20% baseline body weight, stop dosing or decreased dosage would be carried out.

4.3 Tumor weight The tumor masses were weighed at the end of the experiment after being harvested. Three tumors, one from each group, were fixed in 10% formalin for paraffin block preparation.

4.4 Inhibition Rate Inhibition rate (%)=(average tumor weight of control group-average tumor weight of test group)/ average tumor weight of control group* 100%

5. Results

5.1 Tumor growth curve Figure 8. Tumor growth curve The data show that the tumor volumes of two treatment groups were inhibited compared to Saline group from Day 9 to Day 12 post treatment, but not significantly.

5.2 Body weight change curve . Body weight change curve The body weights of the mice who received peptice A+F were not significantly reduced after dosing.

5.3 Photo of tumor mass and tumor weight The tumor masses were harvested and weighedTumor weight Tumor weights of two treatment groups were decreased compared to Saline group without statistically significant difference. 5.4 Inhibition Rate:The data demonstrate that combination of peptide A and F moderately inhibited U87 tumors.c.xenograft growth, but not significantly Figure 9.

6. Conclusion Human glioblastoma U87 xenograft grew well in saline group (the control group), demonstrating that the U87 xenograft model was successfully established in nude mice in this study. The combination of Peptide A and peptide F moderately inhibited the growth of U87 human glioblastoma s.c.xenograft. The treatment in Group 2 with 5 injections demonstrated better efficacy than that in Group 3 with 3 injections in cancer suppression. However, the inhibition was not statistically significant. Body weight loss was not observed in either treatment group.

IN VIVO STUDY 2: PHARMACOKINETIC STUDY :

1. Study purpose To evaluate the pharmacokinetic parameters of peptide F in Wistar rats following intravenous (i.v.) administration.

2. Material

2.1. Animal 2.1.1. Animal species and strain: Wistar rat, 3 rats

2.1.2. Gender and age: Female, 200-220 g 2.1.3. Breeder/supplier: Shanghai SLAC Experimental Animal Center

2.1.4. Test Facility:ln Vivo Pharmacology Dept., GenScript Inc. Ltd

2.1.5. Adaptation: 7 days

2.1.6. Room: SPF level

2.1.7. Room temperature: (22-25) °C

2.1.8. Room relative humidity: 40-70%

2.1.9. Light cycle: 12-hour light cycle (8:00-20:00) withl 2-hour dark cycle

2.1.10. Animal hosting: 3 rats / cage

2.1.11. Food: Free access to food, autoclaved and irradiated, Shanghai SLAC Laboratory Animal Co. Ltd.

2.1.12. Water: Free access to water from local supply (filtered by

MolanimalUltrapure Water Systemand autoclaved) The study protocol and all the animal use have been approved by the IACUC of GenScript.

2.2. Reagents and Equipment

2.2.1. Test Article Test article (nasal) name: Peptide F Supplier.Client

Physical description: White powder Storage condition: -20°C Purity: 95.91 Molecular weight:2432.19 Formulation: Use saline as solvent.

3. Procedure

3.1. Peptide F was administrated to the rats via i.v. route at a single dose of 10pg /gm.

3.2. Plasma samples were collected at 0, 5, 15, 30 minutes; and at 1 , 2, 4, 6, 8, 24 hours (totally 10 time points) after drug administration for each rat (3 rats in all). All the samples will be kept in clean tubes and stored frozen at -80°C for further analysis.

3.3. The compound concentration in plasma samples were analyzed by a validated LC-MS/MS method.

4. LC-MS/MS Analysis

4.1 Instrument

An Agilent Technologies 1260 infinity liquid chromatographic system was used. Detection was performed on an API4000 mass spectrometer equipped with TurbolonSpray (ESI) Interface (Applied Biosystems, Concord, Ontario, Canada).Analyst 1.5.2 software packages (Applied Biosystems) were used to control the LC-MS/MS system, as well as for data acquisition and processing.

4.2 Sample Preparation

An 80 uL aliquot of plasma samples were spiked into a 96-well plate, and 160 uL of acetonitrile containing internal standard were added for protein precipitation. The vortexes plate ,and then centrifuged at 4000 rpm for 10 min. Supernatant was transferred into a new 96-well plate and mixed with 1 :1 volume of H2O and the final solution was injected for LC-MS/MS analysis.The whole procedure was operated on ice.

4.3 Chromatography Condition

Chromatographic separation was achieved on the Phenomenex Luna 3u NH2 100A (100x2.0mm) column. The column temperature was maintained at ambient temperature (25°C). The flow rate was maintained at 0.2 mL/min and the following mobile phases were used:

A : Water/Acetonitrile (90:10), 2% Formic acid

B : Acetonitrile

5. Data Analysis

Peak areas of analyte and internal standard were calculated using Analyst™ 1.5.2 software (Applied Biosystems). Regression analysis was performed in this software and the standardize on linear curve fit will 1/x or 1/x2 in the first instance. Acceptance criteria should be ±30% of target value for standards and QC's.

A minimum of five calibration points must be used in the quantification. At least three out of the four QC standards must be within the ±30% target value. If the sample concentrations are significantly above the lowest QC point (100ng/ml), then this QC maybe disregarded and the run accepted.

At least one of the high QC's must be within the target acceptance. Plasma versus time data was analyzed by non-compartmental approaches using the WinNonlin version 5.2 software programs.

6. Pharmacokinetic Parameters As shown in table 1 harmacokinetic Parameters of Peptide F after i.v.

administration (10 ug/g) showed very short half life.

Table 1. Pharmacokinetic parameters of peptide F after i.v. administration (lOug/g)

Stability in Plasma and Stability after Sample Preparation

Table 2. The Stability in Plasma at room temperature

Peptide F showed not stable in plasma at room temperature,the standard samples preparation steps has to be on the ice.

Table 3. Stability after sample preparation (4°C)

~20hours Peptides sequences list sheet:

Using one letter symbol for amino acid( except for D-form):

Al- NH2-(d-Met)(d- Ala) ILTYVYVF AVLFIAK(d-Lys)(d-Lys)(d-Lys)- COOH

El - NH2-(d-Met)(d-Gln) LRVLFFFL FVATISKK(d-Lys)(d-Lys)(d-Lys COOH

F 1 - NH2-(d-Met)(d-Thr)KWLLLVVCLGIACQKK(d-Lys)(d-Lys)(d-Lys)- COOH

F2- NH2-(d-Met)(d-Thr) KWLLLWC LGIACQDVTSA(d-Lys)(d-Lys)(d- Lys)-COOH

F3- NH2-(d-Met)(d-Thr)RLFMLVCL GIVCQGTT(d-Gly)(d-Asn)(d-Ile)- COOH

Seven peptides similar to peptide Fl were postulated to be of more or less similar actions for further assessment experiments and may be modification .

1- NH2-(d-Met)(d-Thr)RWLFMVACLGIACQGAIVR(d-Lys)(d-Lys)(d-Lys)-COOH

2- NH2-(d-Met)(d-Thr)KWLLLMVCLGIACQNIRGG(d-Lys)(d-Lys)(d-Lys)-COOH

3- NH2-(d-Met)(d-Thr)TWLLLWCLGIACQGITSV(d-Lys)(d-Lys)(d-Lys)-COOH

4- NH2-(d-Met)(d-Thr)NWLLLIVCLSIACQDVTSA(d-Lys)(d-Lys)(d-Lys)-COOH

5- NH2-(d-Met)(d-Thr)RWLFMVACLGIACQGAILR(d-Lys)(d-Lys)(d-Lys)-COOH

6- NH2-(d-Met)(d-Ile)RWLLLMYLGIAVRCYRHSF(d-Lys)(d-Lys)(d-Lys)-COOH

7- NH2-(d-Met)(d-Ser)FNIWWLILYFSIVCQAKAH(d-Lys)(d-Lys)(d-Lys)-COOH

Using 3 letters symbols for amino acid:

Al - NH2- (d-Met)(d- Ala) Ile Leu Thr Tyr Val Tyr Val Phe Ala Val Leu Phe Ile Ala Lys(d-Lys)(d-Lys)(d-Lys)-COOH

El - NH2- (d-Met)(d-Gln) Leu Arg Val Leu Phe Phe Phe Leu Phe Val Ala Thr He Ser Lys Lys(d-Lys)(d-Lys)(d-Lys)-COOH

Fl - NH2- (d-Met)(d-Thr) Lys Trp Leu Leu Leu Val Val Cys Leu Gly Ile Ala Cys Gin Lys Lys (d-Lys)(d-Lys)(d-Lys)-COOH

F2- NH2- (d-Met)(d-Thr) Lys Trp Leu Leu Leu Val Val Cys Leu Gly Ile Ala Cys Gin AspVal Thr Ser Ala(d-Lys)(d-Lys)(d-Lys)-COOH

2- NH2-(d-Met)(d-Thr) Lys Trp Leu Leu Leu Met Val Cys Leu Gly Ile Ala Cys Gin Asn He Arg Gly Gly(d-Lys)(d-Lys)(d-Lys)-COOH

3- NH2-(d-Met)(d-Thr) Thr Trp Leu Leu Leu Val Val Cys Leu Gly Ile Ala Cys Gin Gly He Thr Ser Val(d-Lys)(d-Lys)(d-Lys)-COOH

Claims

Claims:

Those peptides with these sequences from N Terminus to C terminus have more potent anti cancer effects plus more stability in human serum than our postulated peptides of patent WO/2014/040605 as we changed the first two amino acids of N terminus to D-form and as well as we did for the last tree amino acids in C terminus..

Also N terminus was modified by adding NH2 group and C terminus was modified by adding COOH group.

As we recognize peptide F from our previous patent as a lead peptide, peptide Fland F2 were modified and peptide F3 was added to our group as a promising peptide in cancer control.

Al- NH2-(d-Met)(d- Ala) ILTYVYVF AVLFIAK(d-Lys)(d-Lys)(d-Lys)- COOH

El - NH2- (d-Met)(d-Gln) LRVLFFFL FVATISKK(d-Lys)(d-Lys)(d-Lys)- COOH

Fl - NH2- (d-Met)(d-Thr)KWLLLVVCLGlACQKK(d-Lys)(d-Lys)(d-Lys) - COOH

F2- NH2-(d-Met)(d-Thr) KWLLLVVC LGIACQDVTSA(d-Lys)(d-Lys)(d- Lys)-COOH

F3- NH2- (d-Met)(d-Thr)RLFMLVCL GIVCQGTT(d-Gly)(d-Asn)(d-Ile)- COOH

Seven peptides similar to peptide F were postulated to be of more or less similar actions for further assessment experiments and may be modification .

1- NH2-(d-Met)(d-Thr) RWLFMVACLGIACQGAIVR(d-Lys)(d-Lys)(d-Lys)-COOH

2- NH2-(d-Met)(d-Thr) KWLLLMVCLGIACQNIRGG(d-Lys)(d-Lys)(d-Lys)-COOH

3- NH2-(d-Met)(d-Thr) TWLLLVVCLGIACQGITSV(d-Lys)(d-Lys)(d-Lys)-COOH

4- NH2-(d-Met)(d-Thr) NWLLLIVCLSIACQDVTSA(d-Lys)(d-Lys)(d-Lys)-COOH

5- NH2-(d-Met)(d-Thr) RWLFMVACLGIACQGAILR(d-Lys)(d-Lys)(d-Lys)-COOH

6- NH2-(d-Met)(d-Ile) RWLLLMYLGIAVRCYRHSF(d-Lys)(d-Lys)(d-Lys)-COOH

7- NH2-(d-Met)(d-Ser) FNIWWLILYFSIVCQAKAH(d-Lys)(d-Lys)(d-Lys)-COOH So, we ask protection of those peptides squences as it can be used as a drug in cancer control.