JPS625919A - Neocarzinostatin linked to transferrin - Google Patents

Abstract

NEW MATERIAL:Tf-NCS [Melting point; 230 deg.C (decomposition); Elementary analysis; C, 44-50%; H, 6-7%; N, 13-16%; S, 0.3-0.8%; Molecular weight; 90,000-130,000 (measured by the gel filtration method); Solubility; Soluble in water and insoluble in methanol, etc. Color reaction; Positive to Rydon-Smith reagent, biuret reaction, etc.]. USE:A selective carcinostatic agent. PREPARATION:Transferrin Tf-NH2 is reacted with SPDP expressed by formula I to give a compound expressed by formula II. Neocarzinostatin NCS-NH2 is then reacted with the SPDP expressed by formula I to afford a compound expressed by formula III, which is reacted with a compound expressed by formula IV to give a compound expressed by formula V. The above-mentioned compound expressed by formula II is reacted with the compound expressed by formula V to afford the aimed Tf-NCS expressed by formula VI which is an excellent carcinostatic agent surrounded by cancer cells having Tf receptors to cause DNA damage and exhibit cellulicidal effect of 2-10 times based on the NCS.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides transferrin (hereinafter referred to as "Tf")
Transferrin-conjugated neocarzinostatin (hereinafter referred to as [Tf-NC8j) is a novel selective anticancer substance that has high affinity for cancer cells to which neocarzinostatin (hereinafter referred to as "NC8") is chemically bound. ) regarding.

[Conventional technology and its problems]

NC8 is composed of 113 amino acids and has a molecular weight of approximately 1ioo.
It is a compound in which a chromophore with a molecular weight of 700 is bound to the acidic protein of O, and it has a strong anticancer effect, and is currently widely used in clinical practice.It has particularly high therapeutic effects on gastric cancer, pancreatic cancer, acute whitening disease, etc. ing.

However, like other anticancer drugs, NC8 may also partially act on normal cells in addition to cancer cells, resulting in side effects.

Therefore, in recent years, much research has been carried out to improve the disadvantages of such side effects. An attempt has been made to bind to cancer cells to have selectivity only for cancer cells (Japanese Patent Application Laid-open No. 199635/1983). However, these monoclonal antibodies have the disadvantage that they are effective only against cancer cells that specifically bind to the antibodies, and that human-derived monoclonal antibodies are difficult to obtain.

On the other hand, it is known that normal human liver and kidney cells have a small amount of Tf receptor, and cancer cells that actively divide and proliferate have a large amount of Tf receptor.
cologia) vol 5. l Q 9-12
p. 0 (1983)).

[Failure to solve the problem]

In view of these circumstances, the present inventor conducted intensive research and found that when NC8 is chemically bonded to Tf, NC8 reaches and accumulates in cancer cells using Tf as a carrier, and is specific to cancer cells. The present invention was completed based on the discovery that a selective anticancer substance with specific properties can be obtained.

That is, the present invention provides Tf-NCS in which Tf and NC8 are combined at an average molar ratio of 1:4.

T f -NCS of the present invention is produced, for example, according to the following reaction formula and through the following four steps.

(1) N-succinimidyl-5- in Tf (I)
(2-pyridyldithio)propionate (r 5PDP
(referred to as j) (■) to obtain Tf-pyridyldithiopropionate (I).

(2) NCS (IV) is reacted with N-succinimidyl-5-(2-pyridylthio)propionate (old) to form NC8-pyridyldithiopropionate (V).
get.

(3) NC8-pyridyldithiopropionate (V) is reacted with dithiothreitol (Vl) to obtain NC8-mercaptopropionate (■).

(4) Tf-pyridyldithiopropione) (
I) is reacted with NC8-mercaptopropionate (■) to obtain Tf-NC8 (■).

1st step = (I) (old hat) 2nd step: (■) (old (V) 3rd step: 10) L, SH-H8-CH2CH2-C-NH-1%
Lower (■) Fourth step: (I) (Vlf) (■) (In the formula, NC3-NH2 represents neocarzinostatin, anal-NI (2 represents transferrin) Tf used as a raw material in this method is produced by precipitating human plasma saturated with ferric iron with a reduced molecular weight and separating and purifying it with a cellulose ion exchange column after dialysis (Biophysical Chemistry, vol. 27. A2 +
19-24, 1983).

This method is carried out, for example, as follows.

Conversion of Tf to pyridyldithiopropionate (first step)
To do this, add Tf to a phosphate buffer solution containing 0.1M sodium chloride (pH 7 to 8) so that the concentration is 0.5 to 5 mg/ml.
5PDP dissolved in ethanol and a small amount of ethanol in this
f. Add and react. It is preferable to use 5PDP in a molar amount 2 to 20 times that of Tf, and to carry out the reaction at a temperature of 20 to 30°C for 30 minutes to 1 hour. The degree of pyridyldithiopropionation can be adjusted by the amount of 5PDP used and the pH of the reaction solution. ) (I) can be obtained. After the reaction, unreacted 5PDP and by-produced N-hydroxysuccinimide may be separated by gel filtration using a column such as Sephadex G-25.

NC3 (■l pyridyldithiopropionation (second
Step) is the same as the first step, with 5PD at pH 6-7.
The reaction is carried out by reacting 2 to 10 times the mole of P. Since NC8 is unstable to alkali, after the reaction, Sephadex G-25 equilibrated with 0.1M acetate buffer (pH 4 to 5) was used.
The unreacted S PDP was removed by gel filtration using a column such as
By separating the by-produced N-hydroxysuccinimide, NC8-pyridyldithioprobionate (V) in which one or two amine groups of NC8 are converted to pyridyldithiopropionate is obtained.

To thiolate (third step) NC8-pyridyldithioprobionate (Vl), add dithiothreitol (Vl) to (Vl) for 15 minutes at room temperature in an acetate buffer at pH 4-5.
This is done by reacting for ~30 minutes. After the reaction,
If unreacted dithiothreitol and by-product pyridine-2-thione are separated using a column such as Sephadex G-25, NC3-mercaptoprobionate (■) can be obtained.

To obtain Tf-NC8 (fourth step), NC8-mercaptopropionate (■) and Tf-pyridyldithiopropione-) (I [) to react. The reaction was carried out using 10 to 20 times the molar amount of (■) to (I) at 20 to 30°C.
It is preferable to carry out the reaction at a temperature of 5 to 48 hours.

After the reaction, unreacted (I) and (■) are separated using a gel filtration column such as Sephadex G-100 or G-150 to obtain a Tf-NC3 conjugate (■).

In the above reaction steps, the decrease in NC8 activity is extremely small, and the target product is thus obtained at a recovery rate of 75 to 85%.The Tf-NC8 of the present invention has the following physical properties.

■Melting point 230°C (decomposition) or higher ■Elemental analysis value 044~50chi, ■6~7chi, N13~
16%, 80.3-0.8% ■Molecular weight 90,000-130,000 (gel filtration method) ■Solubility Soluble in water, insoluble in methanol, ethanol ■Color reaction Lydon-Smith reagent, Buret reaction,
Phenol sulfuric acid reaction and potassium thiocyanate reaction are positive ■ Ultraviolet absorption spectrum Figure 1 ■ Infrared absorption spectrum Figure 2 ■ Bonded molar ratio Tf content determined by Bio-Rad protein assay method, NC8 content determined from Micrococcus luteus antibacterial activity When calculated, Tf580pP/ml (7,7n
NC85oou/ml (27,8nM
It was l. As a result, the average binding molar ratio of both substances is Tf:
NC3=1:4.

(2) Isoelectric focusing Acrylamide desk electrophoresis with a pH gradient of 2.5 to 6.5 shows bands different from Tf and NC8 (Figure 3).

The seeds were completely fused in the agar (Figure 4).

0 immunoelectrophoresis As a result of performing electrophoresis at 32 mA for 1 hour using 0.06M barbital buffer (pH 8,6), Tf moved to the cathode side and NC8 moved to the anode side, and Tf-NC8 moved slightly more than NC8. It moved to the cathode side (Fig. 5).

[Effect]

The biological properties of Tf-NC8 of the present invention are as follows.
62 cells (2,5X10'/ml)' with 0.1% BSA
The cells were washed five times with RPMI 1640 medium containing 5 times to remove as much Tf as possible in the medium. In Ependle 7 tube, K562 cells (200p13), H”I-Tf
50μ13 (1.25nM, radioactivity 3.OX11
03cp/n54), Tf-NC850μk at various concentrations
The cells were mixed at 4° C. for 45 minutes, washed twice with phosphate buffer, and the radioactivity of +26I-Tf bound to the cells was measured. The results are shown in FIG.

As a result, the binding of 12111-'l'f to 562 cells was clearly inhibited by Tf-NCS, and since Tf-NCS and 1211-'ff competed for the Tf receptor, Tf- NCS is Tf
It is clear that it has an affinity for receptors.

+2) Intracellular metabolism of Tf-NC8 After Tf is taken into the cell, it liberates Fe, which is then released outside the cell and recycled. T f -NCS in experiment ■
Although it has been revealed that Tf-NCS also binds to Tf receptors, we investigated how Tf-NCS bound to cells is metabolized.

K562 cells in RPMI 1640 with 0.1% BSA added
Wash 5 times with the medium of
Adjust and distribute 3X107 to 70, and add enough 125I-Tf or 12” I-T f-N to each.
CS was added and incubated for 45 minutes at 4°C to saturate Tf receptors on the cell surface.

Next, the cells were washed twice with ice-cold RPMI 1640 medium to remove Tf or Tf-NC8.
°C, 5% Co20.1% BSA added RPMI 1640
K562 cells were cultured for 120 minutes in a
The radioactivity released at 00μk and the radioactivity precipitated with lQ%TcA were measured. The results are shown in FIG.

l2BI-Tf has 90% of the activity released into the supernatant at 10
4TCA precipitable polypeptide, whereas “5
I -T f -NCS was only 65% 10-tidlychloroacetic acid (TCA) precipitable material. That is, Tf-NCS is injected into the cell in larger quantities than Tf, metabolized, and releases NC8 into the cell, and this recycling occurs repeatedly, thereby exerting an antitumor effect.

(3) 562 cells were treated with 1% FCS and RPMI 1640 to inhibit DNA synthesis.
100 fi adjusted to 1 x 106/rnl in culture medium.
Dispense T f -NCS at various concentrations into 13 tubes.
Or add 100μp of NC3Q, 37℃, 5μco,
Incubated for 0 minutes. Then RPMI 1640
Cells were incubated with The radioactivity of 3H-thymidine incorporated into the cells was measured using a scintillation counter. The results are shown in FIG.

This is obvious (Tf-NC8 is in the low concentration region (0,
2-5 u/tnl) at NC8 and 2-1
It showed 0-fold inhibition of DNA synthesis.

(4) To inhibit cell growth, incubate 562 cells with 10 lXl0 in FC8RPMI.
'/rILl, 100 μp was dispensed into each well, and 50 μp of Tf-NC8 or NC8 at each concentration was added.

The cells were cultured at 37° C. and 5% CO2, sampled every day, and stained with trypan blue to count the number of living cells in each well. The results are shown in FIG. As is clear from this, T f-NC8 has the same unit (lu/Id
The growth of 562 cells is significantly more inhibited than that of NC8.

As is clear from the above tests, Tf-NC8 is introduced into cancer cells that have Tf receptors, causes DNA damage, and exhibits a cell-killing effect, and exhibits a cell-killing effect 92 to 10 times that of NC8. It is an excellent anticancer drug.

〔Example〕

Next, an example will be given and explained.

Example 1 500 ml of collected plasma was saturated with ferric chloride and then salted out with ammonium 35 sulfate, and the supernatant was further diluted with 65
Salt out with ammonium sulfate and collect the precipitate. The collected precipitate was 0.05 M)! JS hydrochloric acid pH 7,4
After dialysis overnight against the same buffer, adsorption onto a DEAE cellulose column equilibrated with the same buffer was performed.
05M to 0.35M) was eluted with a concentration gradient using lithium-hydrochloric acid (pH 7.4), and a pink peak was collected.

After concentrating with Amicon Mempuran membrane (VM-101), it was adsorbed on a preparative high performance liquid chromatography column (Toyo Soda Co., Ltd., HLC-803Al, and eluted with phosphate buffer to obtain a peak with a molecular weight of 5oooo. Yield 300
■.

Example 2 Tf50m9 obtained in Example 1 was dissolved in 10 Tne of phosphate buffer (pH 7.5) containing 0.1 M-salt, and N-succinimidyl-5-(2-pyridyldithio)propionate (hereinafter referred to as 5PDP) Add 1.8 mg (abbreviated as ) in 100 μp of ethanol and heat at 25°C.
Stir for 30 minutes. After the reaction, the Tf-
Byridyl dithiopropionate 4 Q ml f, H obtained. The Tf content was 1, Q mg/mlT:.

Example 3 NC8200m9 was dissolved in 25rnl of 0.1M phosphate buffer (pH 7,0) containing 0.1M sodium chloride, and SP DP 28.4 mg x ta/#3 was added to this.
ml and stirred at 25°C for 30 minutes in the dark. After stirring, the reaction solution was diluted with 0.1M salt containing 0.1M salt.
Pass through a Sephadex G-25 250mlO column equilibrated with acetic acid buffer (pH 4,51) and elute with the same buffer to remove low molecular weight portions to obtain 55ml of NC8-pyridyldithiopropionate solution.IQOmM was added to this solution. After adding 5 ml of dithiothreidol aqueous solution and stirring under spectroscopic light at room temperature for 30 minutes, the mixture was passed through a Sephadex G-25 column equilibrated with a pH 4.5 buffer to obtain 70 ml of NC3-mercaptopropione. .

The NC8 concentration of this solution was 2.2 mg/ml.

Example 4 Tf-pyridyldithiopropione obtained in Example 2) 1
15 ml of NC8-mercaptopropionate obtained in Example 4 was added to 5 mJ, and the pH of IN-NaOH was adjusted to 7.
After adjusting the temperature to 2, the mixture was stirred at 25°C for 20 hours in the dark. After stirring, the reaction solution was filtered by ultrafiltration (molecular weight: 10,000) through a 7 m
Concentrate to 0.1 in advance. Sephadex G-100 column 2 equilibrated with OIM phosphate buffered saline
00rILl and eluted with the same buffer, T f
-20 ml of NCS fraction was obtained.

The elution pattern from the column is shown in FIG.

[Brief explanation of drawings]

Figure 1 is the ultraviolet absorption spectrum of T f -NCS, Figure 2 is the infrared absorption spectrum of the same substance, Figure 3 is a diagram of the same substance, Figure 5 is the immunoelectrophoresis diagram of the same substance, and Figure 6 is the same substance. Fig. 7 shows the intracellular metabolism of the substance, and Fig. 8 shows the DN of the substance.
Figure 9 shows the inhibition of 562 cell growth by the same substance. Figure 10 shows the reaction solution of Tf-pyridyldithiopropionate and NC8-mercaptopropionate in Sephadex G. It is a figure showing an elution pattern when purified with a -100 column. Above Figure 6. 5th cause Tf-NC8 concentration (nM) Figure 7 1'. ■-Tf ”'l-Tf-
NC5 Deception Procedures Amendment (Method) October 21, 1985

Claims (1)

[Claims] 1. The following physical properties of transferrin and neocarzinostatin combined at an average molar ratio of 1:4 (1) Melting point 230°C (decomposition) or higher (2) Elemental analysis value C44-50%, H6 ~7%, N1
3-16%, S0.3-0.8% (3) Molecular weight 90,000-130,000 (gel filtration method) (4) Solubility Soluble in water, insoluble in methanol and ethanol (5) Color reaction Lydon Smith reagent , Biuret reaction, phenol sulfuric acid reaction and potassium thiocyanate reaction are positive (6) Ultraviolet absorption spectrum (7) Infrared absorption spectrum (7) Infrared absorption spectrum (Figure 2) Transferrin-bound neocarzinostatin.