DE2001002A1 - Radioactive material and process for its manufacture - Google Patents

Description

United Kingdom Atomic Energy Authority

11, Charles II Street, London S. ¥. 1, England

For this application, the priority will be taken from the UK Patent application No. 2009/69 dated January 13, 1969 claimed

Radioactive material and process for its manufacture

Baokground of the invention

The present invention relates to radioactive materials and, more particularly, to radioactive materials and Compositions which can be used for diagnostic purposes.

The compound inulin is a polyecharide with a Molecular weight approximately equivalent to 5000 and consists of about 30-35 fructose units, which are end-to-end are connected in a straight chain. The main use of this compound in medicine was in the measurement of the glorae-

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rular degree of filtration (G.F.R.), which is used as the index of Kidney function can be used. It should be noted that the inulin is injected into the bloodstream and, since it is physiologically inert, it is freely filtered through the urine into the urine unchanged. The inulin in the urine is usually by a colorimetric method which involves the hydrolysis of the inulin to fructose, has been determined. It should be easy to see that this determination is tedious and also by the Complicating the need to determine blank values to account for the presence of glucose in the Urine may be present.

In order to avoid some of the difficulties mentioned above, a radioactive material has been proposed based on inulin to manufacture and use the "marked" / labeled / inulin. It is known that, provided the radioactivity is tightly bound, the application of a radioactive material for diagnostic purposes is an uncomplicated and safe procedure, and the determination of the material is relatively easy because it is by means of a conventional detector for radioactivity can be counted and it does not need to be any perform a chemical separation.

The problem of blind determinations is also minimized. A common radioactive label is carbon-H.

The labeled inulin previously proposed and manufactured was made through reacting the inulin with hydrogen oyanide which contained carbon-Η, to obtain a nitrile. This nitrile then became hydrolyzed to give a carboxylic acid group with

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Carbon-14. This special compound is named differently "Inulin carboxyl-CI4", ^u Carboxy-Inulin-C14 "or" Inulin (carboxylic acid-014) "", and this latter term is to be used below Inulin (carboxylic acid-CH) has been found to be extremely satisfactory. However, some doubts have been expressed that it might not behave in exactly the same way as the unmodified inulin due to the presence of the carboxyl group. that there must be no difference in the physiological behavior of the labeled inulin in comparison with the unlabeled material, because this would render the previously collected knowledge useless.

Objects of the invention

One subject of the invention is a new or improved labeled inulin.

Another subject matter of the invention is the creation of a diagnostic composition which has such a marked Includes inulin.

Another, alternative subject matter of the invention is to create a. Process for the production of such a labeled inulin.

Description of the invention

According to one side of the present invention, a labeled inulin is provided, which consists of inulin with a Hydroxymethyl group, the carbon atom of which is a carbon-14 atom. In the following this compound "Inulin" (hydroxymethyl-CH) ".

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According to a further aspect of the present invention, a diagnostic composition is provided which consists of a solution of inulin (hydroxymethyl-C14) in physiological inert saline.

It should be noted that the new labeled inulin does not contain carboxylic acid groups and the chemical Group introduced by the labeling process is a hydroxymethyl group which is already in fructose units is present so that there should be no physiological difference between the newly marked Inulin and the inactive or unlabeled inulin. It must also be noted that the determination of the new labeled inulin is essentially the same as the determination of inulin (carboxylio acid-CH).

It should also be stated that inulin is physiologically inert, and that inulin (hydroxymethyl-Ci4) is equally inert, the small amount of radioactivity, thanks to the 0-14 emitting only soft ß-rays, of no significance whatsoever for those in it existing doses and concentrations. The preferred method of manufacturing the material is such that it is of high, for example better than 98%, radio-chemical purity and a suitable concentration of the diagnostic composition for injection into the bloodstream between 1.6 mg / ml and 5.0 mg / ml or more specifically 2 mg / ml. Ee is estimated that 20 mg corresponds to up to 50 Ny 01. The amount to be injected or "Doeie ^H is not very dependent rom Kr ^ Jpergewioht, but only upon the generation of sufficient Aktlvitätskoncentration in Planne ,. In the case of laboratory animals such as laninohen the dose appropriately

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Way 1 My Oi / kg, while this could be 2 My Oi / kg for a human. These doses should be compared with the conventional inulin dosage of 200 mg / kg or 500 mg / kg.

Now, according to another aspect of the invention, there is a process for the production of inulin (hydroxymethyl-CH) in starting from inulin (carboxylic acid-014-), to methylate this to the methyl ester and then to reduce the ester to the alcohol.

In short, the preferred method of preparation is to mix the acid with diazomethane to methylate methanol and ether and then reduce the methyl ester to alcohol with sodium borohydride. That Product can then be fractionated to give a narrow molecular weight range around 5000.

Preferably method of manufacture

In order to make the present invention easier to understand, the detailed preparation of inulin (hydroxymethyl-CH) to be discribed.

This production uses inulin (carboxylic) as a starting point acld-CH), which is from the radiochemical Oentre, Amersham and the preparation of this material from unlabelled inulin is not described. 0.43 grams of inulin (carboxylic acid-014) with a specific Activity of 1.4 microours per milligram was powdered and mixed with 1.5 ml of methanol. Two milliliters one 0.14 molar solution of diazomethane in ether was added

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and the mixture was stirred in a flask at room temperature for 20 minutes. The volatiles in the vial were then removed by pumping under high vacuum. The non-volatile residue was dissolved at 4O ⁰ G in 15 milliliters ammoniumhydroydhaltigem water (1 ml ammonia with specific gravity of 0.880 per 150 milliliters of water). A solution of 60 milligrams of sodium borohydride in 3 milliliters v / water was added, and the mixture was stirred at room temperature for 45 minutes and then evaporated to the dry state (to dryness) ^{in a water bath with a temperature of not higher than 35 ° C.} The remaining gel was dissolved in 3 milliliters of water and 10 milliliters of methanol was added to cause the product to precipitate as a fine powder, which was filtered off and dried. The treatments with diazomethane and sodium borohydride were repeated on the residue.

The dry powdery product of these two preparations was mixed, dissolved in 5 milliliter of water and applied to a 200 milliliter column of "Sephadex G-25" and eluted with water in 5 milliliter fractions. The numbered fractions 12-20 of the eluate contained the largest proportion, which was of the same molecular weight range as compared to the starting material inulin (caboxylic acid-G14), and these fractions were combined and concentrated to dryness on a water bath at a temperature not higher 35 ⁰ G. This process gives 0 ', 36 grams of a solid material, which is a 04 / o yield.

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Comparison with the previous technique

Experiments using infrared spectroscopy have shown that there is no carboxyl group in the new labeled inulin is present. In order to simulate the glomerular filtration, the newly labeled inulin was mixed with unlabeled Inulin is mixed, and efforts have been made to identify the "two induvidues by electrophoresis and gel filtration." to separate, but these attempts were unsuccessful. It must be noted that it is possible to use unlabeled inulin from inulin (carboxylic acid-G14) by electrophoresis to separate. W.

Brief description of the drawings

For a better understanding of the experiments, reference should be made to the accompanying drawings, in which:

1A and 1B are graphs showing which show the total elimination versus time, and

Figures 2A and 2B are graphs showing the specific activity of excreted inulin versus time. * k

In the following tests, the following diagnostic compositions were used:

Table I.

Short name Active component dose mg In unlabeled inulin 200 mg
fiGi. InCO ₂ H Inulin
(carboxylic acid-GH) U mg InGH ₂ OH Inulin
(hydroxymethyl-GH) (10

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Rabbits were given intravenously through an ear vein with a solution of natural inulin and one of the labeled ones Derivatives injected. Urine samples were taken at intervals after the injection, and the inulin content and determined the radioactivity. The specific activity of the inulin in the urine could match the specific activity of the starting material can be compared. This technique would eliminate any difference between the marked and non- ' reveal labeled inulin inside or outside the kidneys.

Urine samples were collected (every 10 minutes for the first hour, then approximately every 20 minutes during the next two hours) by washing the bladder with two volumes of saline (20 ml each). The time for each sample was determined upon removal of the second wash from the bladder.

Testing for radioactivity was by liquid scintillation counting using a Nuclear Chicago Mark I liquid scintillation counter. The urine samples were diluted to 100 ml and a duplicate sample (1 ml) added to 10 ml of Triton X-100 scintillation solution. All swatches were chilled to 4- ⁰ G and kept in the dark for 30 minutes before counting. No chemiluminescent agent was observed. Appropriately predetermined base counts were subtracted from each specimen, and the specimens counted until a net specimen count showed a standard deviation of less than 1 jt. The correct functioning of the counting process was determined by the channel ratio method and was routinely 60-60 ft.

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The colorimetric determination of the inulin was carried out by acid hydrolysis of the inulin to fructose and the latter was estimated by Nakamura's modification of the method of Roe and Epstein (see Agr.Biol.Chem. (Tokyo), 32 , 696 (1968). This modification gives a fructose: glucose color result of 100: 1, thus trivializing the glucose base value in the samples. Replicated samples were generally within $ 2.5 of the average. Standard fructoae samples were determined simultaneously with the test samples. This technique allowed the Expressing the inulin content of the samples directly in ag The fructose standard values, which covered the range of the experimental values, followed the Lambert-Beer law.

The cumulative total values of inactive inulin and the labeled inulin derivative excreted in the urine at each sampling time were taken into account. Table II shows these totals one hour, two hours and three hours after the start of injection of the inactive and active materials. It can be seen that a large proportion (about 50-60 ° β) of the inulin is boiled off within the first hour of the experiment, and that about 80 $ of it was eliminated within three hours of beginning.

Table II

Total cumulative excretion for inulin, inulin (carboxylic acid C 14) and inulin (hydroxymethyl-G14) _t expressed as the percentage of the injected material excreted in the urine at the respective times.

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- ίο -

animal Injected
materials Tent in min.
after
injection Percentage
on rejected
denem active
material Percentage
on rejected
denem inactive
ven material R3 InCO ₂ H 55 55.2 58.2 + In 120 71.9 75.8 180 81.7 7.3 R4 InCO ₀ H 53 51.0 54.4 + In 123 69.1 75.6 178 74.5 83.7 R5 InCO ^ H 55 68.4 78.7 + In 125 80.5 94.0 170 81.7 96.7 R6 InCO ₂ H 65 35.0 37.2 + In 135 48.3 52.0 180 63.0 68.9 R7 InCH ₂ OH 60 50.6 49.2 + In 130 68.4 67.2 175 72.7 71.6 R8 InCHpOH 6 5 37.1 31.9 + In 130 42.3 39.4 170 45.0 43.7 R9 InCH ₂ OH 60 61.3 61.4 + In 120 75.4 75.1 180 81.9 82.0 E1O InCH ₀ OH 6b 59.7 60.0 + In 120 76.1 76.7 170 81.6 82.6 R11 InCH ₀ OH 30th 49.5 49.5 + In 60 64.8 65.1

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'- 11 -

It can be observed that there is an obvious and the continuously increasing difference between inulin (carboxylic aoid-C14) and unlabelled Inulin is present. The excretion of inulin (carboxylic acid-C14) falls markedly below that of unlabelled Inulin, as can be seen from Fig. 1 '(A), which these shows growing difference between active and inactive material for a typical experiment. The excretion of inulin (carboxylic acid-C14) decreases, so that there there appears to be a non-reversible retention of active material within the body.

The other indicator shows inulin (hydroxymethyl-C14) a good reference ratio between the total values of active and inactive material, which was eliminated at any time (see Table II and Figure 1 (B)).

The ratio of excreted labeled material: excreted inactive material was determined for each experiment with both markings in front of the calculated from the table for the cumulative eliminations (table II). The average value this ratio and its standard deviation (S.D.) are for the experimental sets with have been computed for each derivative and have resulted in the following:

Derivative R, ratio mean of

marked

Derivative to specially natural 1.

Inulin before the total end values S.D.

Inulin '

(carboxylic

acid-OH) 0.899 0.017

Inulin

(hydroxymethyl-

C14) 1.006 0.007

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The application of the Student _t test (η = 4) shows that the deviation of inulin (carboxylic acid-C14) from the behavior of natural inulin (R = 1000) is highly significant is (p <0.01). On the other hand, the inulin (hydroxymethyl-G14) a high degree of consistency with natural inulin.

13 The specific activity of inulin in the urine

Read the criterion of cumulative total eliminations is useful in determining the existence of differences between inulin and labeled inulin because the retention of the marker in the body can easily be demonstrated. The cumulative total eliminations may not have been precisely balanced by a slight inaccuracy in the estimation of the samples which earlier with were taken in the experiment and contained large amounts of markers and inulin. Hence there is a more sensitive one Evaluation criterion which shows the differences between the labeled compound and the natural inulin is, in the specific activity of the inulin in each resulting urine subset.

This was calculated from the radioactivity and the inulin content of each fraction and expressed as the specific activity of this fraction at the center of each excretion period. The specific activity was compared with that of the injected material. These specific activities naturally show a scatter around the trend line, and the slope "b ^{M of} the latter has been computed for the least-sqaree line (law of least squares), (y χ a + bx), for the regression of the specific activity The norm deviation dtr slope and the value for the section ( ⁿ a ⁿ ) were also computed.

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FIG. 2A shows a typical trend line for inulin (carboxylic acid C14) while FIG. 2B shows the same trend line for inulin (hydroxymethyl-G14) shows. The data for Each run is listed in Table III, which is the slope of the return line, expressed as a percentage Reflects change in the initial specific activity, as well as the nominal deviation of the slope, as well expressed as a percentage, the computed portion of the Line expressed as a percentage of the initial specific activity and the level of importance of the deviation the slope of zero.

Table III

The change in specific activity (S.A.) in urine over time

The section "a" and the slope "b" of the least-sqares Regression line, y = a + bx are expressed in percentages, or percentage changes per hour in the initial Activity.

animal Injected Change in Norm-Afr- Initial

R3 InCO ₂ H R4 R5 R6 InCH ₀ OH 2 R7) R8) R9) Rio} R11

Material of the S.A. 'b' which specific

in for each StcFe SDv ^ V activity 'a ^f "\

fe / stde? E p ^x;

-6.0 2.2 98.0 <0.05

-11.7 1.1 99.3 <0.001

-12.7 2.7 95.6 <0.001

- 4.4 1.3 99.4 <0.01

- 0.7 1.3 102 _f 9 K.3. -4.2 2.4 108.4 "

+ 2.2 2.0 100.1 "

- 2.1 1.3 101.3 "

- 0.3 5.3 98.0 »

* 'ρ rtpresents the true unity that N.H. that

b - 0 correct 1st. It was obtained using the • £ test · and the student £ test · (n * 10 - 12)

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The results of the specific activity for inulin (carboxylic acid-C14) show that it cannot be regarded as a good indicator for natural inulin in this system. In this there is a continuous decrease in the specific activity of the urine produced throughout the entire experiment. This drop shows animal-to-animal variations and is very highly significant in two experiments (p <0.001), highly significant in one (p <0.01), and significant in the fourth (p <0.05). The mean value of the slope 'b' for the four experiments was - 8.7 1 ° ( d = 1.7 $; ρ <0.001), values which are highly significant in their deviation from zero.

Inulin (hydroxymethyl-CH), on the other hand, can be seen as a good identifier for unlabelled inulin. No value of the slope 'b ¹ shows any significant deviation from zero. Therefore, it shows no significant change in the specific activity over the duration of the experiment even at the end when the concentration level of the inulina in the plasma is only about 6 mg per hundred. In addition, the standard deviation of the slope is small, which means that the 95 ° β> confidence limits for the magnitude of the slope covers a narrow range (about 4.5 $ on both sides of the mean) (see FIG. 4)

The mean value of 'b ¹ for inulin (hydroxymethyl-C14) is -1.0 $> ( d = 2.9 fi), a value which in turn indicates that this labeled inulin derivative is not separated from the inulin in this system.

Any little differences in physiology Application between the labeled inulin derivative and the unlabelled inulin would be in one

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reflect a relatively large change in the specific activity of the inulin in the urine, particularly towards the end of the experiment when the plasma concentration levels of each are low. If, for example, the labeled derivative is excreted at about 5 pounds per hour, is comparatively slower, whether it is excreted by an interrenal process or secreted from the circulation by a process outside the kidneys, the specific activity of the inulin in the urine is after three hours at 75 ^{ΰ ρ} have fallen to its original value. even a 2 ^£ ^ strength difference would still _ of 88 $ of the original in ™ give a specific activity in the urine after three hours.

The invention also relates to modifications of the enclosed Claim 1 outlined embodiment and relates above all to all features of the invention, which are disclosed individually - or in combination - throughout the description and drawing.

Claims

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Claims (5)

- 16 69 178 Dr. Bü / A January 9, 1970 Claims 1 / ΰ-e denoted B inulin derivative, "consisting of Inulin with a hydroxymethyl group whose carbon atom is a carbon-14 atom. 2. Diagnostic composition, consisting of a solution of inulin (hydroxymethyl-C14) in physiological, inert saline solution. 3. Diagnostic composition according to claim 2, in which the concentration of inulin (hydroxymethyl-C14) is between 1.6 mg / m1 and 5 mg / ml. 4. Process for the production of inulin (hydroxymethyl-C14), which dyrin consists of taking inulin (carboxylic acid-C14), and then methylating this to methyl ester to reduce the ester to alcohol. 5. The method according to claim 4, in which the Me thylier; -; - " is carried out using diazomethane. ■ ·, " ^: learn: '" according to claim > \ oaer 5, -i -i which: die ) \ (i (' ^: ' ί : r.;, Ur'"^; . '." Use of Katri unborhydi'i.dd ^ ror.R- 0 Ü S 8 c Blank page