JPS60155125A - Agent for phenylketonuria - Google Patents

Abstract

PURPOSE:To use a fibroin inclusion compound of phenylalanine ammonia-lyase as an oral agent for phenylketonuria having improved shelf stability and improved activity. CONSTITUTION:An orgal agent for phenylketonuria comprising a fibroin inclusion compound of phenylalanine ammonia-lyase (PAL for short). The fibroin inclusion compound of PAL is prepared by subjecting a mixed aqueous solution of PAL and fibroin to salting-out with a salt (e.g., ammonium sulfate, sodium citrate, etc.). In the preparation, a blending ratio of PAL and fibroin is preferably 400-1,500 units/g. A dosage form of administration is preferably an enteric agent (e.g., enteric tablet, enteric capsule). The fibroin inclusion compound of PAL contributes to stabilization of phenylalanine decomposition activity of PAL and manifestation of effect.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides phenylnaranine encapsulated in fibroin.
-Relates to an oral phenylketonuria drug consisting of ammonia lyase (hereinafter referred to as PAL).

□ Raninylketonuria is a disease based on a congenital disorder of metabolism of fu;nylalanine. Newborn infants with this disorder are congenitally deficient in phenylalanine hydroxyl-transforming enzyme, which is an enzyme that metabolizes phenylalanine.
When children are raised on a diet with a diet of 90%, the concentration of nylalanine in their blood increases abnormally by 1, resulting in serious and irreversible disorders in their mental development. Therefore, newborns are now tested for phenylnalanine hydroxylase deficiency, and those with hair loss are fed a diet that does not contain phenylalanine (phenylalaten has been removed). The technique used is unavoidable. Moreover, this dietary restriction needs to be continued for 5 to lθ years. Such treatment imposes not only an unbearable mental and physical burden on the patients themselves, but also their guardians, as well as an economic burden.

□ Therefore, □ a means of giving PAL together with normal food (from which phenylalanine has not been removed) has been proposed.

In other words, by giving PAL with food, phenylnaranine in the food is absorbed from the intestinal tract before EPA'L.
This is a method for decomposing lualanine into cinnamic acid. However, the disadvantage of this method is that P
It has been pointed out that AL is rapidly inactivated by bu'0+nase (specially donated lipsin) in duodenal secretions (Biochem).

J, (IHi) pages 715-723].

In addition, attempts have been made to use PAL by filling it into enteric-coated capsules, but the effect of lowering blood phenylalanine concentration is not sufficient, and there is a need for further pharmaceutical improvements (The Lancet, Feb. -rury
23, pp. 392-394 (1980)].

] In view of the circumstances mentioned above, the present inventors conducted various studies with the aim of providing a better and more practical PAL preparation.
We found that the fibroin inclusion complex of AL plays a major role in stabilizing the phenylalanine degrading activity of PAL and exerting its effect, and based on this knowledge, we completed the present invention.

First, the production of the PAL fibroin inclusion body of the present invention (hereinafter referred to as PAL-fibroin inclusion body) will be described below.
I will explain.

PALi fibroin inclusions are produced by salting out a mixed Taniki solution of PAL and fibroin.

The mixed aqueous solution of PAL and fibroin has a concentration of lθ~1
00 units/- of 5 layers of PAL with potassium phosphate buffer (pH 3.5) 4;
III/v) Prepared by mixing nofifuroin aqueous solution.

The appropriate mixing ratio of PAL and fibroin in the following is 200 to 3000 u of PAL per 1 g of fibroin.
nits, and when a mixed aqueous solution in this range is salted out as described below, the PAL activity per Ig is 50 to 200.
units of PAL-fibroin inclusions are obtained.

If the amount of PAL used with respect to fibroin is below the range specified above, the PAL activity per Ig of the resulting PAL-fibroin inclusion complex will be small; yield of activity) decreases.

In addition, a particularly preferable mixing ratio of PAL and fibroin is 400 to 1500 units/g, and in this case, P
The AL-fibroin entrapping activity is high, and the degree of entrapping of PAL (yield of PAL activity) is also good.

Next, the above-mentioned mixed aqueous solution of PAL and fibroin is added to an aqueous salt solution for salting out to precipitate a PAL-fibroin inclusion.

Various inorganic and organic salts can be used as the salts, but ammonium sulfate and sodium citrate are preferred.

PAL-fibroin inclusions are precipitated by salting out, so collect them by filtration and add 1ml of 5mM potassium phosphate solution (p) 1
8.0 to 7.5).

Note that the preparation, salting out, and washing of the above-mentioned mixed aqueous solution are preferably carried out at 10° C. or lower.

Finally, the above PAL-fibroin inclusion complex was added to 5mM
The PAL-fibroin inclusion complex of the present invention is obtained as a white dry powder by suspending it in a potassium phosphate buffer (PH 8.0 to 7.5) and drying it under reduced pressure at low temperature, preferably freeze-drying.

The PAL-fibroin inclusion complex of the present invention has excellent properties as shown in the results of Test Examples E to I, and is useful as a drug for phenylketonuria.

In order to use the PAL-fibroin inclusion complex of the present invention as a drug for phenylketonuria, a small particle size is preferable, and according to a conventional method, the particle size is 800 μm or less, usually 50 to 200 μm.
used as

In order to actually use the PAL-fibroin complex of the present invention as a drug for treating phenylketonuria, it is appropriate to use it as an enteric coating agent.

Typical examples of enteric-coated agents include enteric-coated tablets and enteric-coated capsules, which are manufactured according to conventional methods.

The amount of the PAL-fibroin complex of the present invention used as a drug for phenylketonuria depends on the amount of phenylalanine contained in the diet. - PA per gram of phenylalanine in the diet as expected
The PAL-fibroin composite of the present invention having an L activity of 50 to 200 units is used.

Note that the PAL-fibroin complex of the present invention is taken immediately before or after a meal, but the latter is rather preferable.

The present invention will be explained below with reference to Test Examples and Examples.

The method for measuring PAL activity in Test Examples and Examples is as follows.

D, S,'Hodgin's c7) Method (J,
Biol, Chew, Vol. 246, 297? Page (18
71)], o, i + n Lis-HCl buffer containing 0.833 mM phenylalanine (pH 8, 5, 30
℃) was added to PAL test solution 20, and the amount of cinnamic acid produced was quantified by measuring ultraviolet absorption (211 Onm) over time.

The activity of PAL that produced 14 mol of cinnamic acid per minute under the above conditions was defined as 1 unit.

[Test Example I] Storage stability (1) Sample A... PAL-fibroin inclusion body obtained in (3) of Example 1 (110 units/g, product of the present invention) B.
...Purified PAL solution obtained in (1) of Example 1 (313
, 8 units/d) in 0.31Q vials (
White solid obtained by lyophilization (11.8 units/vial, control) (2) Test method Specimen A Ioomg (general, 11 units) was dispensed into a vial (inner volume 5aQ). ), sealed tightly, and stored together with sample B in a desiccator kept at 4°C and 40°C.

Samples were taken over time, suspended or dissolved in 50mM) Lis-HCl buffer (PH8, 5), and used as a PAL test solution to measure PAL activity.

(3) Results As shown in Table 1, PAL-fibro of the present invention [Test Example ■] Stability to Chymotrypsin (1) Specimen □ Test-1 Same specimens (A, B) as on the left were used.

(2) - Test method □ Integral A and B) 1 5 hM each) Liss-HCl buffer □ solution (p
The PAL activity was adjusted to about 1.1 units/- by suspending it in H8. 20 g of IN chymotrypsin hydrochloride ('''!/'Guma Inc., type 1-5) solution was added and incubated at 30°C.

The PAL concentration of each PAL test solution sampled over time was kept constant. ' (3) □ Results □ Q 2 Table #As shown, the PAL-fibroin complex of the present invention exhibits strong resistance to chymotrypsin.
This means that when the PAL-fibroin complex of the present invention is used as a drug for phenylketonuria, it is less likely to become inactive in the duodenum.

[Test Example III] PAL activity in vivo Phenylalanine is decomposed by PAL in the intestinal tract to produce cinnamic acid, which is absorbed and transferred into the blood. Therefore, by quantifying cinnamic acid in the blood, the degree of decomposition of phenylalanine in the intestinal tract (and thus PAL activity) can be measured.

(1) Sample The same specimens (A, B) as in the test example were used.

(2) Test method Samples A and B were each mixed with 50mM potassium phosphate buffer (
4. Suspended or dissolved in pH 7.2); Own it
It was set as s/-.

Next, a group of 4 rats (SD strain, male, 6 weeks old, 9 rats, 17 rats)
The abdomen was opened under ether anesthesia, and each of the above solutions was administered into the duodenum at a rate of 20 units/g of resting body. Next, phenylalanine (30mg/+N
100 m of 50+nM potassium phosphate slow draw solution of Q
The drug was administered into the duodenum at a dosage of 1.5 g/kg body weight, and the abdominal incision was quickly sutured.

Immediately thereafter, heart blood was collected over time using a syringe (heparinized).

Next, 50 J of plasma obtained by centrifuging each of these direct solutions.
Cold methanol 250JLJ1 was added to L to remove protein.

50 g of each of the obtained methanol fractions was subjected to reverse phase high performance liquid chromatography to quantify cinnamic acid (Cl1n
, Chew, Vol. 28, p. 2282 (1882)). Elution was with acetonitrile 35:5% acetic acid 65 (V/
V) (7) The mixture was used at a flow rate of 2-/min.

(3) Results As shown in Table 3, the PAL-fibroin complex of the present invention showed strong PAL activity when administered into the duodenum of rats, and its action time was also long [Test Example ■] Acute toxicity (L
D5G) The PAL-buiproin complex (A) obtained in Example 1 (3) was suspended in physiological saline at a concentration of 100 mg/aQ.

Next, this suspension was orally administered to a group of 10 rats (ddY strain, male, 5 years of age, 9 weight, 28-30 g) & 2.5 g/kg body weight, and observed for 1 week, and no abnormalities were observed. .

Therefore, the LD5 of the PAL-buiproin complex of the present invention
0 is 2.5 g/kg body weight or more.

Example I Production of PAL-fibroin inclusion complex (1)
Production of PAL Fr1tz et al. medium (J, Bial, Chew, 5
Volume 251, page 484B (197B) elf,) L
400 volumes of improved medium supplemented with 1% polypeptone were inoculated with the yeast ``Wang Frog Shande'' IFo 05513, and 3
The cells were cultured for 14 hours under the conditions of 0°C, 9 aeration, 300 m/sin, and 250 rpm stirring to obtain bacterial cells weighing 7.2 kg in wet weight.

1- The bacterial cells were added to twice the amount of 50mM Tris-HCl buffer (pH
11,5) and suspended in Dyno-Mill (Illill
y A, manufactured by Bach-oben, 111) IK-15
! l;! The crushed solution was centrifuged and the crude PAL solution (PAL total activity; approximately 5 (IQQQuni
ts) was obtained.

Next, 3 volumes of the above crude PAL solution (total PAL activity; approximately 1500
Quants) was heated to 55°C for 10 minutes, then cooled to 50mM.
) It was diluted 2.5 times with Lis-HCl buffer (PUA, 5), and after centrifugation, the supernatant was concentrated to 900-fold using a hollow fiber concentrator (manufactured by Amicon, Model DC-10).

Ammonium sulfate was added to this concentrated solution to make a 15% saturated ammonium sulfate solution, which was then subjected to column chromatography using Phenyl Sepharose CL-48 (manufactured by Pharmacia). (eluted by the gent method).

PAL high activity fractions were collected, concentrated, and then dialyzed against 5+s% potassium phosphate buffer (pH 8,5) to obtain approximately 2% of the purified PAL solution.
80 d (PAL total activity; approximately 1 l 1000 units, 3
9. Elunits/aQ)" was obtained.

In addition, the specific activity of ice crystals (t, the method of Owry et al. (J.

Biol, Chew, Vol. 193, p. 2B5 (Sumi 851)), the PAL activity relative to the amount of protein (mg) was 2.3 units/mg.

(2) After solubilizing the fibroin production machine Kiki according to the method described in Example 1 of JP-A-58-15887, the 5χ (W/V) of fibroin was dialyzed using distilled water. A solution was obtained.

(3) Production of PAL-fibroin inclusion complex (1) above
The obtained purified PAL solution 25-(PALila activity; 10.00 units+) and the fibroin solution 40d obtained in (2) above (fibroin content 2.0 g)
were mixed.

This PAL-fibroin mixed solution 1'43% saturated ammonium sulfate (50q% potassium phosphate buffer (P) 113.5)
IJL4: 2B 3g of ammonium sulfate was dissolved and the pH was adjusted to 8.5 with potassium acetate. 1g of the solution was added dropwise with vigorous stirring for about 3 minutes, and left in 4A for 30 minutes. . .

), the precipitated PAL-buiproin complex was injected three times.
TE5mMIJ 78 Cali,') Mu1a'al solution (p
H8,5) Washed well with 400- to obtain PAL-fibroin inclusions.

, the above PAL-fibroin inclusion body is 30-5s+
Resuspended in M potassium phosphate buffer (pH 8.5) and lyophilized overnight using a conventional method, the mixture was passed through a $100 mesh sieve to obtain 2.46 g of the desired PAL--iB0i' complex (product PAL activity per gram was 110 units).

Example 2 Production of PAL-fibroin inclusions 5% (W
/V) Specific activity 2.1 obtained in the same manner as in Example 1 (1) in 20 d of fibroin aqueous solution (containing 1 g of fibroin)
Purified PAL solution of units/tag (3B, 4un
250 units and 5
00units+, 1250units, 2500u
Add a considerable amount to the nits.

PAL-
Fibroin inclusions were obtained.

As shown in Table 4, when the amount of PAL used for fibroin is 250 units, the PAL activity of the target product is small;
Also, at 2500 units, the utilization rate of PAL used (
PAL activity yield) is poor.

Example 311! PAL-buiproin complex obtained according to Molten Tablet Example 1 (1
61 units/g) , , , , , , 50.0 g
Corn starch 25.0 g
Lactose 23.5 g Magnesium stearate 1.5 g
The above ingredients were mixed uniformly and uncoated tablets having a diameter of 8+ am and a weight of 15011 g were prepared according to the tablet manufacturing method specified in the Japanese Pharmacopoeia General Rules for Preparations.

Next, the uncoated tablets were coated in a conventional manner using a mixed solution consisting of 10 parts of hydroxypropyl methyl cellulose phthalate, 3 parts of triacetin, 50 parts of ethanol, and 50 parts of methylene chloride (W/W).

The total PAL activity in one enteric-coated tablet is approximately 12 units.

Example 4 Enteric-coated capsule PAL-fibroin inclusion complex obtained in Example 3 (181u
nits/3) 5011 g was filled into enteric coated hard gelatin capsules.

The total PAL activity of one capsule is approximately 4 units.

Procedural amendment (voluntary) January 1, 1985 Manabu Shiga, Commissioner of the Patent Office 2, Name of the invention: Agent for phenylketonuria 3, Relationship with the person making the amendment Patent applicant address: Sumida Go, Sumida-ku, Tokyo No. 17-4, 5-80, Tomobuchi-cho 1-chome, Bejima-ku, Osaka City, Osaka 534, Kanebo Co., Ltd. Patent Department Telephone: (08) 921-1251 4. Date of amendment order (voluntary) 6. Specification subject to amendment Column 7 of "Detailed Description of the Invention", Contents of Amendment (1) ru nuitg J on page 1, line 2 of the specification
Correct to nits J.

(2) In Table 2, page 11 of the specification, "residual activity rate" is corrected to "r activity remaining rate".

(3) Insert r (pH7,2)J next to "phosphorus potassium buffer" on the 3rd to 2nd lines from the bottom of page 12 of the specification.

(4) In the ^UC column of Table 3 on page 14 of the specification, “ρ~12
0vin(pH) J (5) Specify page 11, 2nd "rat" as mouse
Correct to.

(8) rWil on page 12, bottom line to page 1B, line 1 of the specification
lyA, manufactured by Bachoben, rWilly A,
Corrected to 1 made by Bachofen.

(7) “Fraction” on page 18 of the specification, line 5 from the bottom, “fraction”
Correct.

(8) On page 11 of the specification, 2, ``Obtained in Example 3'' is corrected to ``1: Used in Example 3''.

(8) On page 11 of the specification, 2, "about 4 units" is corrected to "about 8 unitsJ."

-H

Claims (1)

[Scope of Claims] 1) An intravenous drug for phenylketonuria consisting of a fibroin-encompassed complex of phenylalanine ammonia-lyase 12) The drug package is enteric-coated tablets or enteric-coated capsules.
The preparation according to claim 1, which