Background Art

Three traditional lines were used for about a century or even more in malignancy treatment; surgery, radiotherapy and chemotherapy with a very poor prognosis still found in many types of malignancy and finally inevitable death.

Biological, immunological and targeted therapy were introduced few decades agoas a fourth line in cancer management to overcome this problem, but still in need of a lot of researches and improvements to achieve the goal and triumph over the tragedy of cancer in this battle.

Problem and Defect:

Many types of cancer still have a poor or very poor prognosis, despite using all four available lines of cancer control.

DISCLOSURE OF INVENTION*

J1d)

NH2-(d-Pro)(d-Phe)KISI(d-His)(d-Leu)-COOH

Peptide J1d with the above sequence is a modified Jellein l(found in MRJPl) peptide where we changed the first two amino acids of N terminus to D-form and as well the case we did for the last two amino acids in C terminus.

N terminus and C terminus of this peptide werealso modified by adding NH2 group and COOH group consecutively,

Ap3d)

NH2-(d-Leu)I(d-Asp)AN(d-Val)(d-Phe)(d-Ala)-COOH

Peptide Ap3d with the above sequence is a modified last 8 amino acids peptide of Apisimin(found in MRJPl )wherethe first & third amino acids of N terminus of this peptide were changed to D-form and as well as we did for the last three amino acids in C terminus.

N terminus and C terminus were also modified by adding NH2 group and COOH group consecutively. Neither peptide J1d nor peptide Ap3d alone have any inhibitory effects upon malignant cells while you get a statistically significant effect on cancer cells either in vitro or/and in vivo when using both peptides simultaneously.

Peptide K2 was introduced as a new peptide consisted of both peptide Ap3d and peptide J1d to form a new one peptide named K2 with its sequence:

NH2-(d-Leu) I (d- Asp) AN VF APFKISI(d-Hi s)(d-Leu)-COOH

Fortunately, we got the same statistically significant inhibitory effects for peptide K2 upon malignant cells in vitro and in vivo as that we got when using both peptides Ap3d & J1d simultaneously.

Needless to say, that the changes of amino acids for ail peptides from L form to D form in N terminus and C terminus, were to increase stability and potency of these peptides.

These peptides showed statistically significant effects in vivo and were confirmed by Pharmalegacy company, Shanghai, China;while we could not get those cancer effects before for our postulated peptides of patents WO/2014/040605&W02017182044.

Experimental Data:

In vitro:

Peptides J1, J1d, J2 & J2d were sent to Chales River Lab, Germany to be tested versus 3 different tumor cell lines and no effect for these peptides was recorded for our in vitro studies; J1 (Jellein 1), J1d (modified Jellein 1), J2 (Jellein 2)& J2d(modified Jellein 2).

Peptides J3d, J4d, Ap2d & Ap3d also showed no effects for cancer cells where they were tested in the same lab ( Charles River).

Even after asking to add peptide Ap3d to J3d and to J4d, no cancer cell effect was there. J3d (modified Jellein 3), J4d (modified Jellein 4), Ap2d (modified peptide froml6 amino acids C terminus of Apisimin) & Ap3d (modified peptide from 8 amino acids C terminus of Apisimin).

In vivo:

Efficacy study of peptide Jld, Ap3d &K2 in U87 s.c xenograft tumor model:

This study was done for us in Pharmalegacy company labs, Shanghai, China. 1. Purpose to evaluate the anti-tumor efficacy of peptide J1d plus peptide Ap3d in one group and peptide K2 in another group 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 2.2.1 Animal 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, Pharmalegacy, Shanghai, China.

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) and 12-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 Pharmalegacy.

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 J1d & Ap3d Supplier: Client Physical description: White powder Storage condition: -20°C

2.3.7.2 Test article (nasal) name: Peptide K2 Supplier: Client

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

2.3.8 Drug formulation: Use water as solvent for peptide J1d and K2 and PBS for peptide Ap3d. 2.3.9 Solution storage condition: The peptide solution should be prepared and used freshly with every injection.

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 (32 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 (8 mice/group) and started dosing immediately:

Group 1 was administered with vehicle(saline) and served as a control group.

Group 2 was administrated with peptide K2 (20 mg/kg of mouse weight with peptide, S.C., every other day, 4 injections; then 20 mg/kg intra tumor injection, every other day, till end of the study.

Group 3 was administrated with peptide J1d+Ap3d (10 mg/kg of mouse weight with each peptide, i.v. every other day, 4 injections; then 10 mg/kg intra tumor injection, every other day, till end of the study.

The administration period lasted 3- 4 weeks.

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 of the tumor volumes, body weight of tumor-bearing mice were taken 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 was measured twice a week in two dimensions using a caliper, and the volume was expressed in mm3 using the formula: V = l/2*axb2 where a and b were the long and short diameters of the tumor, respectively. Relative tumor volume RTV is calculated as follows: RTV=Vt/V0, where Vt is the volume on each day, and V0 is the volume at the beginning of treatment.

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&Relative tumor growth curve:

Figure 1& 2 .

The data show that the tumor volumes and relative tumor volume of two treatment groups were inhibited compared to Saline group from Day 11 post treatment, especially with change to intra-tumor injections; being a statistically significant to a very significant. 5.2 Body weight change curve: Figure 3.

The body weights of the mice who received peptide J1d+Ap3d and peptide K2 were not significantly reduced after dosing.

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 J1d and peptide Ap3d in one group and peptide K2 in another group showed a statistically significant inhibition of the growth of U87 human glioblastoma s.c.xenograft. Body weight loss was not statistically important in either treatment group.

Peptides sequences list sheet:

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

Ap3d)

NH2-(d-Leu)I(d-Asp)AN(d-Val)(d-Phe)(d-Ala)-COOH J Id)

NH2-(d-Pro)(d-Phe)KISI(d-His)(d-Leu)-COOH

K2)

NH2-(d-Leu) I (d-Asp)ANVFAPFKISI(d-His)(d-Leu)-COOH Using 3 letters symbols for amino acid:

Ap3d)

NH2-(d-Leu) lie (d-Asp)Ala Asn (d-Val)(d-Phe)(d-Ala)-COOH J Id)

NH2-(d-Pro)(d-Phe) Lys lie Ser Ile (d-His)(d-Leu)-COOH K2)

NH2-(d-Leu) Ile (d-Asp)Ala Asn Val Phe Ala Pro phe Lys Ile Ser Ile(d-His)(d-Leu)-COOH