JP2972334B2 - Purified protein and biologically active therapeutics - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to drugs, particularly drugs used to treat human tumors.

Currently, tumors are treated with chemotherapy, radiation therapy or surgical procedures. Each of these treatments has disadvantages.

It would be advantageous to avoid the disadvantages of chemotherapy, radiation therapy and surgical procedures.

One object of the present invention is to provide a pharmaceutical treatment for human tumors.

Another object of the present invention is to provide a treatment with fewer side effects than known treatments.

Yet another object of the present invention is to provide a therapy for use with more than one type of tumor.

Yet another object of the present invention is to improve known therapies for the treatment of human tumors.

According to the present invention, there is provided a medicament for treating a human tumor. The drug is a pure protein with a molecular weight of about 12,000 daltons by mass spectrometry (about 14,500 daltons by electrophoresis), a characteristic isoelectric point by isoelectric focusing, and a characteristic amino acid composition. It is advantageous, but not necessary, that the drug be derived from the frog egg that is subjected to fertilization. That is, in a preferred embodiment, the drug is rana pipiens
Derived from frog embryos and eggs. Embryo development is conveniently stopped prior to gastrulation and preferably at or before the blastocyst (128 cell) stage, and the embryo is homogenized in the presence of a weakly acidic buffer and then centrifuged. Obtain the supernatant. This is then subjected to ion exchange chromatography and size-exclusion chromatography.

When this drug is used in vitro in combination therapy with other drugs, in some cases, the results of this combination treatment are much more bioactive than expected.

One of the other drugs is marketed under the trademark TAMOXIFEN and the other drug is marketed under the trademark STELAZINE.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention may be better understood with reference to the following non-limiting drawings, which are set forth in the following description.

FIG. 1 is a flowchart of the method in a preferred embodiment of the present invention, and FIG. 2 shows the amino acid sequence of the drug.

FIG. 3 shows how the combination treatment of this drug and STELAZINE is more active against human lung A-549 cancer than the drug or STELAZINE separately.

FIG. 4 shows that treatment with this drug and TAMOXIFEN in combination with human pancreatic ASPC-
It shows how large the activity is against one adenocarcinoma.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A. Preparation of an Embryo / Egg Mixture In a preferred embodiment, eggs of rana pipiens are produced by induced ovulation (to develop in a highly controlled manner) and externally of female frogs. Fertilize under controlled conditions. Ovulation is induced between September and March. In other months, induced ovulation is not possible. The reason is ranapipi
ens spontaneously ovulates in April, and its breeding season lasts from May to August.

Large and healthy energetic pregnant female rana pipiens are selected for induced ovulation. These were isolated from male rana pipiens and placed in an aquarium filled with 1 inch of tap water for 6 days for 6 days.
Keep at ℃. While this temperature is preferred, other temperatures can be used if other conditions are appropriately adjusted during the fertilization process. That is, the rate of egg development depends on temperature.

For each scalpel selected, each petri dish is preferably filled with 10 cubic centimeters of tested spring water. Tested spring water is fresh spring water that has been tested to maintain the life of rana pipiens and its embryos.

Advantageously, ovulation is induced by introducing five newly isolated female rana pipiens pituitaries into the body of each selected female. A male pituitary gland may be used in place of a female pituitary gland, but one female pituitary is equal to two male pituitaries and the amount used must be adjusted appropriately.

Place the appropriate number of pituitaries in the corresponding liquid-containing Petri dishes. Bring each selected female to room temperature (22 ° C). For each dish, the pituitary is introduced into a syringe and into the lower right quadrant of the corresponding selected female abdomen with an 18 gauge needle.

Next, the selected female is placed in a water tank containing 1 inch of spring water. Place a flat rock at the bottom of each aquarium so that the female rests on it and can be above water level.
(This is advantageous because pregnant females may be lethargic and may drown if not kept above water level.) Cover the aquarium with warehouse cloth (this allows frogs to To prevent it from jumping out of the aquarium), and conveniently kept at room temperature (22 ° C.) for 48 hours. The eggs produced by the pregnant females are then fertilized according to a preferred embodiment outside the female frog, conveniently in petri dishes, and preferably using four petri dishes for each pregnant female.

To achieve fertilization, female rana pipiens are sacrificed (ether over-anesthesia) and their testes are removed to remove connective tissue and fat. Sufficient males are sacrificed to provide at least four testes in each Petri dish of eggs to be fertilized (ie, 16 testes per pregnant female). This amount gives the optimal amount of sperm suspension when considering the size of the Petri dish. (Conveniently, the pituitary gland of the male rana pipiens is also removed and later used to induce ovulation in other females.) Four testes are then placed in each Petri dish and the testes disintegrated (by dissection) To form a milky sperm suspension.
Testes must be disrupted in such a way that sperm are not destroyed. The eggs are then removed by squeezing the abdomen of each pregnant female towards the back. The spawn of each female is evenly distributed on four suspension-filled Petri dishes. This prevents over concentration of eggs in the dish.

The eggs are left in the suspension for 3 to 4.5 hours at room temperature. During the first hour, the sperm suspension and the eggs in the dish are intermittently agitated so that the eggs are always covered with the sperm suspension. After 3-4.5 hours, the dishes are examined under a dissecting microscope and observed for signs of division. When 80% division of the rana pipiens embryo is observed, the corresponding dish is collected. At that point, the embryo is at least at the 4-cell stage of development. The 4-cell stage is used as an indicator because it establishes cleavage and indicates that the observation of fertilization is not a misjudgment.

The collected dishes contain both normal embryos (fertilized eggs) and unfertilized eggs, since 100% division of the embryos is not usually achieved in 3 to 4.5 hours. This mixture is often referred to as a fertilized egg mixture and refers to the presence of both eggs and embryos. In a preferred embodiment, 80% fission is used as an indicator,
The ratio of eggs to embryos in the mixture will be approximately 1: 4.

The whole eggs that have been subjected to fertilization are then scraped into a container and stored frozen at -15 to -20C. This preservation is not essential to the practice of the present invention, which is preferred only when it is convenient to carry out the subsequent processing in batch.

B. Mechanical processing of fertilized eggs If the mixture is frozen, thaw it in any way that will not cause overheating. The thawed or unfrozen mixture is homogenized in the presence of a weakly acidic buffer, preferably under laminar airflow to prevent contamination.

In a preferred embodiment, the mixture of fertilized eggs is treated at room temperature with 0.15 M sodium acetate (pH 4.8-4.0) using 2 volumes of buffer per volume of the mixture of fertilized eggs.
Mix with 9). The buffer need not be sodium acetate, but need to be weakly acidic, in a preferred embodiment, S-Sepharose chromatography is performed and sodium acetate has a good buffer in the pH range of 4-5.8. Sodium acetate is used because it is a liquid (S-Sepharose exchange column is effective in this area). Homogenization is performed with Waring Blender until all eggs are visually destroyed,
Any sanitary method that does not require the Waring Blender to achieve complete homogenization can be used. Homogenization ends when the suspension is homogeneous and the undamaged egg is no longer visible. The homogenate is then stirred for 2 hours at room temperature and stored frozen until further processing is performed.

In a preferred embodiment, the two stages of the homogenized and agitated fertilized egg mixture (the mixture is stored frozen for at least two weeks and then thawed) are centrifuged (4 ° C to 8 ° C). In the first step, the stirred homogenate is centrifuged at an average acceleration of 34,000 × G and the resulting supernatant is secured. The time required for this step is typically 60 minutes, but it is necessary to obtain a clear, gel-free supernatant, the time being increased as necessary to achieve this. In the second stage, the precipitate obtained in the first stage of the centrifugation is rehomogenized as described above. The homogenized precipitate is again centrifuged as described above, and the supernatant obtained is combined with the supernatant obtained in the first step.

The time, speed and other conditions of the centrifugation step described above are not necessary for the practice of the present invention, but have been optimized for the preferred embodiment of the present invention.

Filter each batch of supernatant through a layer of DEAE-Sepharose equilibrated in extraction buffer (0.15 M sodium acetate, pH 4.8-4.9) while decanting. This removes the residue that clogs the column in the processing steps described below, but DEAE-Sepharose is not particularly necessary and may be replaced by another suitable filter.

In a preferred embodiment, the filtered extract is assayed for biological activity on a given cell line. This is not required, but is advantageous for two main reasons.
The first reason is that in a process scaled for commercial production, the next processing step is performed after the combined batches of filtered extracts. Examining and discarding the batch of inert filtered extract prevents the undesirable incorporation of the non-bioactive batch into the bioactive batch. Another reason is that although the next processing step is expensive, the identification and elimination of non-biologically active substances can save substantial costs that are lost in the processing. However, although the assay is currently preferred, the assayed batch of the filtered extract has never been found to be inert, and thus the use of the assay must be considered entirely optional.

Assays of practice used in the preferred embodiment include human submandibular epidermoid carcinoma A-235 cells and a tetrazolium compound sold under the MTT trademark (Chemicon International, Id.
nc., 100 Lomita Street, E1 Segundo, California). However, this is not necessary and other bioassays may be used. Alternatively, the bioassay is unnecessary if there are alternative non-bioassays that have good correlation with the bioassay.

The filtered extract is conveniently and immediately subjected to a further purification step.

C. Ion exchange chromatography The filtered extract is subjected to ion exchange chromatography. In a preferred embodiment, the ion exchange chromatography is performed in two steps with slightly different conditions. First
In this step, the active protein is first isolated and essentially free of endotoxin. In the second step, the protein is purified from other proteins that have been co-purified with the active protein in the first step, as well as from any remaining endotoxins.

In a preferred embodiment, the first ion exchange chromatography step is performed using a column 11 cm in diameter and 20 cm in length. The conditions described below have been optimized for columns of this size. However, if columns of different dimensions are used, the conditions may vary.

In a preferred embodiment, the purpose of the two successive ion exchange chromatography steps is to separate proteins with an isoelectric point pI of 9.5 to 10.5 (as determined by isoelectric focusing) before separating the proteins by size. Release. In the first ion exchange chromatography step, the filtered extract is adjusted to pH 5.2 with ammonium hydroxide and applied to a column packed with S-Sepharose. The column is equilibrated with 0.15 M sodium acetate buffer (pH 5.2) and the column is developed with a continuous linear gradient (0-0.5 M) of sodium chloride in the equilibration buffer. These conditions are not essential for the practice of the present invention, but are advantageous and are believed to produce bioactive proteins in high yields at this time.

In a preferred embodiment, the eluted protein is diluted two-fold with pyrogen-free water and subjected to a second ion exchange chromatography step performed under different conditions. This second ion exchange chromatography step is performed on a second column packed with S-Sepharose 11 cm in diameter and 15 cm in length. The column is equilibrated with 0.15 M sodium acetate buffer (pH 5.2) and developed with a continuous linear concentration gradient (0-0.3 M) of sodium chloride in the equilibration buffer.

Advantageously, both chromatography steps are 18-20
C. (air conditioning room), but this is not critical. Column chromatography is known to be more efficient above 4 ° C. (refrigerator) and the process is performed at the highest temperature commensurate with the stability of the purified drug.

In a preferred embodiment, the eluate from the second ion exchange step is washed with an aqueous solution of ammonium hydroxide at pH
Make alkaline to 6-7 and ultrafiltrate using a 5000 dalton molecular weight cut off membrane and concentrate by discarding the permeate. Suitable membranes are Spectora-Por (S
pectrum Medical Industries) and Amicon YM5 (Am
icon), but other membranes and other enrichment methods may be substituted.

D. Size Exclusion Chromatography Next, the concentrated material was bio-gel P-
Apply 30 gel to a column packed and equilibrated with 0.075 ammonium bicarbonate. Isolate the major protein peak.

These particular conditions are not particularly required for the practice of the present invention, and other sizes, gels and other size exclusion methods may be substituted. However, it is recommended that size exclusion chromatography be used next to ion exchange chromatography. The reason for this is that performing the chromatography in this order makes it possible to use a reasonably sized column for size exclusion chromatography.

E. Final treatment The eluate from the size exclusion column is sterile filtered through a 0.22 micron filter and then lyophilized. These processing steps are standard in the pharmaceutical industry and are not part of the present invention. The resulting formulation does not contain live bacteria.

Biological activity of the drug According to the results of in vitro and in vivo confirmed animal tests, the drug was tested on human submandibular epidermal carcinoma A-253 cells and human ovarian adenocarcinoma N
It turns out that it has activity with respect to IH-OVCAR-3 cells. The drug also showed activity against human leukemia HL-60 cells, human COLO320DM cells originally isolated from colon adenocarcinoma, human LOX melanoma, and human lung squamous cell carcinoma HT-520 cells.

In vitro data show that a combination of a drug sold under the trademark TAMOXIFEN and this drug could
It shows that the activity is much greater for human pancreatic ASPC-1 adenocarcinoma than would be expected given the separate activity of XIFEN. In vitro data also show that a combination of the drug and the drug sold under the trademark STELAZINE could be more effective against human lung A-549 cancer than would be expected given the separate activities of the drug and STELAZINE. Shows much higher activity.

A preferred embodiment of the invention was tested using a cell culture assay. In this assay, cell lines of known growth for a given period of time are treated with a test substance and the growth of the treated cells is compared to the growth normally expected from untreated cells.

The drug was dissolved in phosphate buffered saline (PBS) to give a 1 mg / ml stock solution.

TAMOXIFEN is produced by Sigma Chemical Co., St. Louis MO and is a trademark for (Z-1-p-dimethylaminoethoxyphenyl-1,2-diphenyl-1-butene) citrate. In the experiments described here, TAMOXIFEN was dissolved in absolute ethanol and diluted in RPMI 1640 medium (produced by Hazleton Research Products, Lenexa, KS) to make a 1 mM stock solution (final concentration of ethanol 11%). Obtained.

STELAZINE is manufactured by SK & F Co., Carolina, PR, a subsidiary of SmithKline Beckman CO., (10
-[3- (4-methylpiperazin-1-yl) -propyl] -2-trifluoromethylphenothiazine). In the experiments described here, STEL
AZINE was diluted with RPMI 1640 medium to obtain a 1 mM stock solution.

The assay system was ASPC-1 human pancreatic adenocarcinoma cell line and A
A -549 human lung cancer cell line was used. American Ty for both strains
obtained from pe Culture Collection (Accession number is ASP
ATCC CRL 1682 for C-1 and A for A-549
TCC CCL 185). Both cell lines RPMI 1640 medium (Hazl
eton Research Products), 20% (ASPC-1)
Or 10% (A-549) heat-inactivated fetal bovine serum (Gibco Life
Technologies, Grand Island NY), and 10,
Supplemented with an antibiotic-antimycotic solution consisting of 000 units of penicillin, 10 mg of streptomycin per ml and 25 μg of fungizone per ml (complete growth medium). A-
2000 live cells (50 μl per well) for the 549 cell line
) And for the ASPC-1 cell line at a density of 4000 viable cells (50μ) in 96-well tissue culture plates manufactured by Falcon, Oxnard, CA. Cell numbers were based on growth curve characteristics previously determined for 7 days of culture. The cells are left for 24 hours, then the drug and / or TAMOXIFEN or STELAZINE per well
50μ of the solution was added. The following final concentrations were used.

a) drug, 20 ng to 10 μg / ml, b) TAMOXIFEN, 10 μM for ASPC-1, and c) STELAZINE, 5 μM.

Plates were incubated for an additional 6 days at 37 ° C. and 5% carbon dioxide atmosphere. As a result, the total assay time was 7 days (1 day for cells to stand and 6 days for incubation). The viable cell rate was then determined using the MIT colorimetric assay using a Bio-Rad EIA microtiter plate reader.

The number of cells was determined by Reichert ScientificIns, Buffalo, NY.
AO-Spencer “Brightline” manufactured by truments
It was determined by direct counting using a Neubauer line in a hemocytometer.
All solutions used for this purpose were from Hazleton Research
Manufactured by Products. Wash the adhered cells three times with Hanks' Balanced Salt Solution,
Treated with 2 ml of a solution of 0.25% trypsin-0.02% EDTA in buffered saline for about 30 seconds. Remove the solution and allow the cells to
Left for 0 min, then suspended in 10 ml of complete growth medium.
0.25 ml of this cell suspension was diluted with 0.75 ml of complete growth medium, then 1 ml of 0.5% trypan blue solution was added, and the viable cells were counted.

Figures 3 and 4 show the results of the above experiment. These tables are expressed as equilibrium growth inhibition rates. That is, they show efficacy in preventing the tested treatment from growing the test cancer cells over a one week assay period. That is, the higher the number, the higher the biological activity on the cell line used during the experiment.

These results indicate that, where indicated, the drug and ASP combined with STELAZINE in the case of A-549 human lung cancer
It demonstrates that in the case of C-1 human pancreatic adenocarcinoma, the biological activity of the drug combined with TAMOXIFEN is much greater than would be expected from the biological activity of the individual drug alone. This can be seen from the ED ₅₀ numbers on the far right of the table. These numbers represent the calculated equally effective dose. The numbers shown are the amounts required to halve the viability of the cells undergoing the assay. That is, the dose required to achieve the same biological activity as the number of ED ₅₀ is small is small.

Chemical Analysis and Drug Composition The drugs described above are chemically well characterized. The drug is a protein isolated from rana pipiens,
As long as the final product has the following chemical characteristics and structure:
It is believed that it may be produced by genetic engineering.

The drug is a pure protein (ie, it is clear that it is homogeneous by routine tests used to assay protein homogeneity). Electrophoretic molecular weight of drug is approximately 14,5.
00 Dalton. Calculation of the molecular weight from the amino acid sequences listed below indicates that the molecular weight should be 11,819 daltons. However, despite all efforts to remove metal ions, they were determined by mass spectrometry because they may be bound to proteins and may contain various isotopes. The molecular weight of the drug is 12,430 daltons. Considering this discrepancy, the molecular weight of the drug determined by mass spectrometry is believed to be about 12,000 daltons. The drug has an isoelectric point pI 9.5 to 10.5 as determined by isoelectric focusing. The drug has a blocked amino end group and is substantially free of carbohydrates (as determined by the anthrone and orcinol methods).

 The drug has the following amino acid composition:

Drugs were sequenced. As shown below, the total length of the sequence is believed to be 104 residues. The N-terminus of this protein is pyroglutamic acid (<Glu). This is a cyclized derivative of glutamic acid that lacks the free amino acids needed for direct sequencing and thus "blocks" the N-terminus of the protein.

When the short fragment described in U.S. Pat. No. 4,882,421 was cleaved using pyroglutamate aminopeptidase, pyroglutamic acid was removed from this short fragment and sequencing could begin at the second residue. This cleavage strongly indicates that the N-terminus is pyroglutamic acid, since only pyroglutamate aminopeptidase cleaves pyroglutamic acid. The presence of pyroglutamic acid was further confirmed by mass spectral analysis of the referenced short fragment. The molecular weight of this short fragment, determined by mass spectrometry, agreed well with the weight calculated assuming the presence of pyroglutamic acid and did not match the weight calculated assuming the presence of glutamic acid.

 This drug has the following amino acid sequence:

The C-terminus at position 104 was identified as cysteine.

While the preferred embodiments have been described above, the scope of the invention is limited only by the following claims.

────────────────────────────────────────────────── ─── Continued on the front page (56) References WO 89/9606 (WO, A1) (58) Fields investigated (Int. Cl. ⁶ , DB name) C07K 14/435 WPI (DIALOG) GaveSeq CA (STN ) REG (STN)

Claims (2)

(57) [Claims] 1. A protein having the following amino acid sequence: 2. The protein according to claim 1, and 10- [3
-(4-Methylpiperazin-1-yl) -propyl]-
An agent for treating a tumor comprising 2-trifluoromethylphenothiazine.