DE3216026A1 - TECHNETIUM (99M) MARKED ISONICOTINIC HYDRAZIDE, METHOD FOR THE PRODUCTION THEREOF AND A PHARMACEUTICAL AGENT CONTAINING THIS COMPOUND - Google Patents

Description

PROF. DR. DR. J. REITSTÖTTER DR. WERNER KINZEBACH

DR. ING. WOLFRAM BUNTE (, _Boa -, _e7 e)

REITSTÖTTER. KINZEBACH Bc PARTNER POSTBOX 7BO. D-BOOO MUNICH 43

PATENT LAWYERS APPROVED REPRESENTATIVES AT THE EUROPEAN PATENT OFFICE EUROPEAN PATENT ATTORNEYS

REFERENCE: RE

TELEPHONE: (OBS) 2 71 05 83 TELEX: OB21S2O8 ISAR D BAUERSTRASSE 22. D-BOOO MUNICH AO

VNR 104 523

April 29, 1982

OUR FILES: OUR REF:

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IKEDA MOHANDO CO., Ltd.

No. 24, Yokohoonji, Kamiichi-cho,

Nakaniikawa-gun,

Toyama, Japan

Technetium (99m) -labeled isonicotonic acid hydrazide, Process for the preparation thereof and a pharmaceutical composition containing this compound

POSTAL ADDRESS; D-8OOO MUNICH 43. POST BOX 7BO

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The invention relates to technetium (99m) -labeled isonicotinic acid hydrazide (hereinafter referred to as " ^m Tc isoniazid"), a process for the preparation of this compound and a pharmaceutical agent for tumor diagnosis which contains "Vcc-isoniazid" as the main component.

So far, a radioactive gallium salt of citric acid has been used (hereinafter referred to as "Ga-Citrate") for tumor diagnosis. This Ga-Citrate is due to its great affinity for various carcinomas as Means useful for scanning, but because there is a high affinity not only for the tumor, but also for it has other inflammatory tissues one has great difficulty in distinguishing between them. Another disadvantage of Ga-Citrate is that that, because of the dangers arising from the action of Ga on the body, it is only minor Amounts can be administered so that satisfactory scintigraphic images are not obtained.

In addition, the production costs are relatively high. 30th

In view of the problems mentioned above, Ga citrate is considered to be un>
Routine checkups.

ft 7

one found Ga-Citrate unsuitable for use at

In recent years technetium (99m) has been dedicated (hereinafter referred to as "Tc") as a very useful radioactive agent for use in tumor diagnosis

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increasing attention because

1. The time during which the organs are exposed to radiation can be reduced considerably,

99m
since Tc has a short half-life of 6 hours, and

2. Tc. Due to the high energy of the ^ - rays (140 KeV) has sufficient permeation in the tissue and, in addition , emits U- rays without emission of ß-rays, so that in vivo examinations can be carried out without patients to expose to a dangerous dose of radiation.

At present, nuclear medicine diagnosis using Tc in the form of agents with various ligands and ³¹¹ Tc is widely used, taking advantage of the beneficial properties of these ligands for determining the functions and shapes of most of the organs of the human body such as the brain, spinal cord, thyroid gland , Lungs, heart, liver, hepatobiliary duct, spleen, kidney, bones, placenta, salivary glands and the like. ° The utility of ^ Tc in the field of tumor diagnosis is limited despite its excellent physical and chemical properties. In particular, it is used practically only for the following investigations: In the form of a free pertechnetate solution

for the diagnosis of brain tumor based on the distribution of Tc in the brain when the blood-cerebrospinal fluid barrier is damaged based on the brain tumor, or to diagnose bone tumor or metastatic bone damage with the help of bone scanning using an agent with Tc and a ligand that accumulates in the bone, such as pyrophosphate or methylene

M / 23 104

diphosphonate, or to generate a negative image of the space-filling damage from liver cancer or liver metastases with the aid of a tc-marked colloid, such as a phytate or sheep colloid, and liver scanning, the function of the reticuloendothelial phagocytes being used. This means that at the present time a tumor-affinity based tumor imaging agent containing a ¹¹¹ Tc labeled ligand has not been practically used.

It is therefore desirable to use compounds for in vivo studies which are produced by labeling with the ^m Tc which is considered to be ideal for such studies. ^{However, m} Tc-labeled compounds which give satisfactory results in this area are practically unknown. US Pat. No. 4,017,596 describes radio-pharmaceutical chelates (including those of technetium 99ni).

The object of the present invention was to create ^m Tc-labeled isonicotinic acid hydrazide which does not have the above-mentioned disadvantages, a process for the production of ^ c-labeled isonicotinic acid hydrazide and a pharmaceutical agent for tumor-

^ O diagnose, which contains ^m Tc-labeled isonicotinic acid hydrazide as the main ingredient.

This task is solved by creating: 35

M / 23 104 ~ ^ ~

1) of Tc-labeled isonicotinic acid hydrazide,

2) a process for the preparation of Tc-labeled isonicotinic acid hydrazide, which is characterized is that one contains a pertechnetate (99m) with a reducing agent in an isonicotinic acid hydrazide Solution under acidic, alkaline or neutral conditions and reduced

3) a pharmaceutical agent for tumor diagnosis containing Tc-labeled isonicotinic hydrazide as a main ingredient.
15th

The inventive Tc-isoniazid is due to the

99m described excellent properties of Tc a very effective diagnostic tool. It is particularly useful as a scanning agent for tumor examination, since the tumor / tissue ratio is extremely high, as will be described later in the examples, and more so too the accumulation in a transferred tumor easily

can be distinguished.
25th

When using Tc-isoniazid as an agent for tumor scanning, it occurs in an amount of 1 to 100 mC, preferably 5 to 25 mC based on the unit weight of a living body for use. The usage of

¹¹¹ Tc isoniazid in such amounts leads to sufficient permeation in tissue during in vivo scanning that the amount of radiation to which a patient is exposed can be considerably reduced, and to

satisfactory scintigraphic images. 35

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With regard to the toxicity of ^m Tc-isoniazid, it should be noted that it is impossible to administer Tc in such amounts that a toxic effect is noticeable. It has been confirmed that using ^m Tc-isoniazid within the above does not cause a toxicity problem.

of Tc-isoniazid within the range given above

The ^m Tc isoniazid according to the invention can easily be prepared by reducing a pertechnetate (99m), for example sodium pertechnetate, with a reducing agent in an aqueous solution which contains isonicotinic hydrazide, which is frequently used as an anti-tuberculosis agent.

An aqueous, ^m Tc-pertechnetate solution can be, for example, a sodium Tc-pertechnetate solution, which can be prepared by a known method, namely by "milking" a commercially available generator for radioactive molybdenum (Mo) - -technetium (Ültra-Technekow, Diichi RI Kenkyojo, Tokyo, Japan). Generators of this type are described, for example, in US Patents 3 833 509, 3 830-746, 3 468 808, 3 382 152. The concentration of ¹¹¹ Tc that can be produced by "milking" the Mo- ^m Tc generator is generally around 1 mC / ml to 100 mC / ml.

Reducing agents which can be used for the process according to the invention are for example iron-II-sulfate, iron-II-chloride, Tin (II) chloride, hydrogen sulfite, sodium borohydride and sodium thiosulfate.

3215026

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When carrying out the reaction under acidic conditions, inorganic acids are used, for example Sulfuric acid, hydrochloric acid and nitric acid, or organic acids, for example ascorbic acid, in Combination with the above-mentioned reducing agent. Iron (II) sulfate is preferably used in combination with sulfuric acid or tin-II-chloride in combination with hydrochloric acid.

Conditions when carrying out the reaction under alkalischen- loading ■ for example by using sodium hydroxide, potassium hydroxide, sodium bicarbonate or the like. 15th

According to the invention, the isoniazid is used in one

-7 _3

Amount of about 10 to about 10 moles, preferably

-6 -4
wise 10 to 10 moles, per 1 mC of a pertechnetate. The reducing agent can also be used in an amount of

-7 -4 -6 "

about 10 to about 10 moles, preferably 10 to 10 ~ moles, are used per 1 mC of a pertechnetate will.

The reaction temperature is not critical for the process according to the invention. Although the reaction can be carried out, for example, within a range from 5 to 100 ^° C., it is conveniently and economically advantageously carried out at room temperature,

ie 10 to 30 ⁰ C, through.
30th

The pH of the reaction solution is most preferably within a range of 1.0 to 7.0, whereas the pH under neutral or alkaline conditions within a range of 7 to 11, preferably 7 to 9, is set.

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^{The 111} Tc isoniazid aqueous solution thus prepared can be administered to a living body either as such or after adjusting the pH to about 7.0 by neutralization with alkali or acids.

^{If the m} Tc-isoniazid solution according to the invention is used as such for an investigation and still unreacted ^ TcO. and free Tc are present, the solution can be filtered with the aid of a suitable filter and purified by column chromatography using, for example, an ion exchange resin, a pure Tc-isoniazid solution being obtained.

The agent for tumor diagnosis according to the invention can, if desired, according to the method described above and is used within a period of 5 to 60 minutes after its manufacture. The length of time within which the agent must be used is influenced by various factors, such as the half-life of Tc (6 hours), and the time

99m the for the enrichment of the Tc in an infested Body part is required.

99m
The one used to enrich the Tc in an infested one

Body part time required varies depending on the mode of administration of the invention Means, the disease to be examined, etc. This time is generally 3 to 48 hours, one will however, prefer that type of administration that leads to an accumulation within a period of 8 to 24 hours leads.

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In view of the above, it is most preferred to administer the agent according to the invention by intravenous To give injection. Of course, others within the scope of the invention can also be used Modes of administration such as oral and intraperitoneal administration can be used.

When administering the agent according to the invention by injection, it is most preferred as a dispersant to use physiological saline solution. This allows damage to occur of the living body which are expected to occur when other dispersants are used.

Since the agent of the present invention is soluble in water, it therefore becomes an ideal agent for in vivo scanning considered.

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- AU

example 1

4 ml of a 24.4% H ₂ SO. Solution are added to 60 mg FeSO. · 7Η ₂ Ο, which is dissolved by stirring for 5 minutes at room temperature. 1 ml (approx. 10 mC) of a sodium pertechnetate solution (Na ^m Tc0.), Obtained by milking a commercially available

Mo- ^ Tc generator (Ultratechnekow, Daiichi RI Kenkyujo) and stirs the mixture another 5 minutes. One takes from the received

I ^ solution of a 1 ml sample and added 80 mg of isoniazid while stirring for 10 minutes at room temperature, a solution being obtained. After completion of the reaction, 9N sodium hydroxide is added to the reaction mixture to adjust the pH to 7.0. Then 1 ml of a phosphate buffer (50 ml of a 0.2 M KH ₀ PO. And 35 ml of a 0.2 M NaOH; pH 7.2) is added and the resulting solution is stirred for 10 minutes. This solution is filtered using a membrane filter (pore size: 0.22 µm) using an ^m Tc-isoniazid solution

^ ° a marking ratio of about 80% is obtained. A paper chromatographic analysis of the solution obtained shows that the compound obtained is pure Form is present and no impurities, such as TcO. ,

contains.
30th

Example 2

Dissolve 500 ml of isoniazid in 40 ml of distilled water

and 4 ml of 1N hydrochloric acid are added to the solution. 1 ml of a 1N hydrochloric acid solution which contains _{200 mg of SnCl 0} '2H _{0 O per 10 ml is added to the reaction mixture.} Afterward

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the mixture is mixed with 5 ml of a 0.2 N aqueous sodium bicarbonate solution and filled with distilled Water to 100 ml. The solution thus obtained is filtered using a membrane filter (pore size: 0.22 μm). 2 ml of this solution are mixed with 2 ml (about 20 mC) of the sodium pertechnetate solution (Na TcO.) described in Example 1 and stir the Mix for a minute, using a ^ Tc-isoniazid solution with a labeling ratio of about 80%. A paper chromatographic analysis of the obtained solution shows that the so produced

bond is present in pure form and does not contain impurities such as ^99m Tc0 ₄ ~.

Application example 1

Yoshida - Sarcomatose cells are transplanted subcutaneously in 10 Donryu rats in the area of the thigh. 6 days after transplant, each rat with an intramuscular mastocytoma is intravenously in the tail 1.3 ml (0.5 mC) of a solution of the ^ Tc-isoniazid combination prepared according to Example 1 (diluted with physiological saline solution). Eighteen hours after the injection, the rats are urethane anesthetized and killed by drawing blood from the main artery. Tumor, chest muscles, liver, Spleen, kidney, lungs, stomach, pancreas, parietal, brain, thymus, heart, thyroid and sex glands separated out. The weight and radioactivity of each organ are determined. From the ratio of the measured Radioactivity value to the total value of radioactivity administered becomes the degree of radioactivity accumulation determined (% dose / g). The results obtained are summarized in Table I below:

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Tabel

Degree of R & dioactivity accumulation (% dose / g)

tumor 0.23 + _ 0.01 blood 0.12 H ₁ 0.00 Pectoral muscle 0.01 + _ 0.00 liver 0.28 + _ 0.02 spleen 0.35 J ₁ 0.03 kidney 1.68 _ + 0.12 lung 0.23 + _ 0.04 stomach 0.06 + _ 0.00 pancreas 0.13 _ + 0.01 thyroid 0.57 _ + 0.04 Parietal bone 0.02 + _ 0.00 heart 0.12 + _ 0.00 brain 0.00 i 0.00 Thyimus 0.02 J ₁ 0.01 Testicles 0.03 + 0.01

The image of the abdomen of a rat taken with an FR camera, which was treated according to the method described in the application example, is shown in the figure. The photograph shows the accumulation of radioactivity in the Yoshida sarcomatosis cells that were transplanted into the lower left thigh area and metastatic damage in the lower part of the body. The accumulation in the middle of the body occurs mainly in the kidney.

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Application example 2

Ehrlich ascites tumor cells are transplanted subcutaneously into male ddY mice in the area of the thigh in an amount of 1 × 10 ⁶ cells / 0.1 ml per mouse.

Ten days after the transplant, the mice (10 mice each) that have a tubercle with a.

Have a diameter of about 1 cm, intravenously in the tail 0.2 ml (40 μθ) of the prepared according to Example 1

99m
provided solution that contains the Tc-isoniazid combination and that is diluted with physiological saline solution, or as a control 0.2 ml (5 \ iC) one

Ga citrate solution injected. 18 hours after the injection the mice were anesthetized with urethane and by removing the blood from the axillary artery killed. Tumor, pectoral muscle, liver, spleen, kidney, lung, stomach, pancreas, cranium, brain, Thymus, heart, thyroid, and sex glands were separated out. The weight and radioactivity of each organ were determined. From the ratio of the measured radioactivity value to the total value of the administered radioactivity was the degree of Radioactivity accumulation determined (% dose / g). The results obtained are shown in Table II.

Table II

Organ Degree of radioactivity accumulation (% dose / g)

^99m Tc-Isoniazid ⁶⁷ Ga-Citrate

Tumor 1.57 + 0.15 2.04 + _ 0.08

Blood 0.88 + 0.13 1.40 + 0.08

Chest muscle 0.50 + 0.06 0.61 + 0.05

.. .- "* -" + 0 - - 4th , 55 3216026 -YES- ± ° 2 , 23 M / 23 104 2.17 ± ο 3 , 31 1 0.16 liver 0.75 t ο , 34 1 , 58 i 0.17 spleen 5.42 ± ° , 12 2 , 63 ± 0/10 kidney 2.08 i ° , 75 1 , 24 + 0 ₇ 11 lung 0.92 i ° , 32 1 , 78 J ₁ 0.28 stomach 0.64 i ο , 21 10 , 02 ± 0.27 pancreas 0.92 i ο , 10 0 , 83 1 0.13 thyroid 0.36 + 0 , 14 0 , 09 i 0.58 Os parietal 1, 06 ± ° , 06 2 , 20 +. 0.08 heart 0.02 + 0 , 16 0 , 99 ± 0 '° 1 brain 0.40 , 01 I 0.25 Thymus 0.56 , 05 + 0.49 Testicles , 08

Application example 3

Yoshida sarcomatosis cells are transplanted subcutaneously into the thigh region in 10 Donryu rats. Six days after the transplant, each rat that has an intramuscular mastocytoma will be intravenous in the tail 0.9 ml (0.5 mC) of a solution containing the according to example 2 (diluted with physiological cooking

99m
saline solution) contains the Tc-isoniazid combination. Eighteen hours after the injection, the rats are anesthetized with urethane and killed by taking blood from the main artery. Tumor, chest muscles, liver, spleen, kidney, lungs, stomach, pancreas, parietal bone, brain, thymus, heart, thyroid, sex glands are removed. The weight and radioactivity of each organ are measured. The degree of radioactivity accumulation is determined from the ratio of the measured radioactivity value to the total value of the administered radioactivity (% dose / g). The results obtained are shown in Table III.

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Table III

Organ degree of radioactivity

accumulation (% dose / g)

Tumor 0.31 + _ 0.01

Blood 0.05 + _ 0.01

Chest muscles 0.01 + _ 0.00

Liver 0.16 + 0.05

Spleen 0.15 + _ 0.08

Kidney 1.35 + _ 0.53

Lungs 0, 1 0 + _ 0.07

Stomach 0.10 + 0.06

Pancreas 0.03 + _ 0.01

Thyroid 0.08 _ + 0.02

Os parietale 0.01 _ + 0.01

Heart 0.02 + _ 0.01

Brain O ₇ OO + _ O ₇ OO

Thymus 0.01 + _ 0.01

Testicles 0.01 _ + 0.00

From the results compiled in Tables I to III it can be seen that when using the Tc-isoniazids according to the invention the tumor / tissue ratio is high and that, moreover, the accumulation in the transferred tumor was clearly differentiated can be. The u? C-isoniazid of the present invention is therefore useful as an agent for tumor scanning

useful.

It is known that the acute toxicity (LDj.-) determined by oral, subcutaneous and intravenous administration of isoniazid in mice is 142 mg / kg, 160 mg / kg and 153 mg / kg (Am. Rev. Tuberc. 65, 376, 1952).

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_{The LD 5Q} value determined in rats is known to be 650 mg / kg for oral administration (Merck Index, £, 680, 76) and for subcutaneous administration to 329 mg / kg (Journal of Pharmacology and Experimental Therapeutics, 119, 444, 57 ). The administration of

99m

Tc-labeled isoniazid on the human body

does not cause any toxicity problems at the dose that can be used for tumor scanning.

259 / Hch

Claims (1)

M / 23 104 claims 10 1. Technetium (99m) -labeled isonicotinic acid hydrazide. 2. Process for the production of technetium (99m) -labelled Isonicotinic hydrazide according to Claim 1, characterized in that a pertechnetate is used (99m) with a reducing agent in an isonicotinic acid hydrazide-containing Solution reduced under., Acidic, alkaline or neutral conditions. 3. The method of claim 2, wherein the pertechnetate Is sodium pertechnetate. 4. The method of claim 2, wherein the reducing agent a combination of ferrous sulfate and Sulfuric acid is.
30th 5. The method of claim 2, wherein the reducing agent Tin (II) chloride. 6. The method of claim 2, wherein the reaction under alkaline conditions using a Compound selected from sodium hydroxide, M / 23 104
1 -2- Potassium hydroxide and sodium bicarbonate will. 7. The method of claim 6, wherein the reduction at a temperature of about 5 is performed to about 100 ⁰ C. 8. The method according to claim 7, wherein the reduction is carried out at a temperature between 10 and 30 ^0C . 9. The method of claim 2, wherein the reaction solution has a pH of 1.0 to 7.0. 10. The method of claim 6, wherein the pH is on 7.0 to 11.0 is set. - | 1 . The method of claim 10 wherein the pH is 7.0 to 9.0 is set. 12. Pharmaceutical agent containing technetium (99m) -markii
part. labeled isonxcotinic acid hydrazide as the main component