Europ. J. Cancer Vol. 12, pp. 865-870. Pergamon Press 1976. Printed in Great Britain

Fate and Distribution of [14C] Succinyl Neocarzinostatin in Rats* HIROSHI MAEDA, NAOKI YAMAMOTO and AKIRA YAMASHITA'f Department of Microbiology and Department of Anatomy~ Kumamoto University Medical School, Kumamoto, Japan, 860.

Abstract--In vivo distribution of the [14C] succinyl derivative of the antitumor antibiotic, neocarzinostatin (NCS), was studied in rats. The nature of the derivative is almost identical to ttu,parental NCS. The radioactivity of the derivative within 20 rain after i.v. injection was found to have accumulated in large amounts in such organs as the kidney, bladder, ovary-uterus, bone marrow, tongue, intestine, lung, skin, stomach and muscle. The levels in the plasma and bladder, although partly due to decomposed components, were high even after 1 hr. Thymus, spleen, and eye werefound to contain only a small amount of this derivative. No appreciable amount of radioactivity wasfound in the brain. The accumulation of radioactivity in the kidney was greater than 6.9 ~ of input. Within 20 rain, 45.5 ~ of the drug was found in urine, indicating a very rapid turnover. The desuccinylation of the derivative was much milder than anticipated while degradation by proteolysis of NCS was the main mechanism of inactivation which was followed by excretion. The nature of accumulated molecules in the kidney and bladder was largely high molecular weight components (mean weight > 6000). NCS seems to be excretedinto the urine both in the degradedforms and in almost intactforms.

NCS orally into mice [8]. No attention was paid for the proteolytic breakdown of the drug during these experiments, which was found to be the major inactivation pathway [9]. Based on the present results, we have applied NCS for the treatment of human bladder cancer, which was found to be one of the main objectives in this study, and have obtained excellent results (S. Sakamoto, J. Ogata and H. Maeda).* We prepared radioactive NCS by succinylation [11-13] based on its structure [14-16] as previously reported. The chemically modified NCS (bis succinyl NCS) retained most of its original properties, such as molecular size, acidic nature, amino acid sequence and composition, conformation and biological activities [12]. The succinyl derivative was almost as active as the original compound at 0.25/~g/ml against hepatoma derived cell line (He) and a lymphoblastoid cell line (P3HR-1) but slightly weak activity against human embryonic lung fibroblasts (Flow 2000). A strong in vivo activity was also confirmed. The succinyl derivative was found to penetrate into cytosol

INTRODUCTION NEOCARZINOSTATIN(NCS) is the first antibiotic of protein nature (mean weight 10,700) of known chemical structure now at the preliminary clinical stage in cancer chemotherapy [1-4]. Previously, the potential usefulness of the drug was shown experimentally in mice [5, 6] and in human [1]. Until recently NCS was known to be useful for acute myelogenous leukemia [2,3] and stomach cancer [4]. However, the specific organs or tumors in vivo have been rather unclear due to the difficulties in the preparation of a radiolabelled derivative of the antibiotic NCS. Fujita et al. reported in a study which was based on the distribution of residual antibacterial activity of intact NCS after i.v. administration in mice and rabbits [7] where extremely rapid inactivation made detailed analysis impossible. Recently, Toriyama et al. reported on a similar work giving

Accepted 26 M a y 1976. *A part of this investigation was supported by the Cancer Grant (II) for 1974 from the Ministry of Education, Science and Culture, Japan. Reprint requests should be addressed to H.M. JfPresent address: Department of Anatomy, Hamamatsu University of Medicine, Hamamatsu, Japan.

*Seven out of eight patients with bladder carcinoma, treated with NCS, have been cured completely without recurrence over six months [10].

865

866

Hiroshi Maeda, Naoki Yamamoto and Akira Yamashita

a n d onto/into cell-nucleus as indicated b y a u t o r a d i o g r a p h y [13]. Its desuccinylation did not occur b u t proteolysis did proceed in vitro with serum [9, 13]. T h e present investigation is the first systematic study o f the catabolic b r e a k d o w n o f protein antibiotics in vivo. T h e results will p r i m a r i l y shed light on which specific organs N C S can effect as a cancer c h e m o t h e r a p y agent in humans.

MATERIAL AND METHODS Material NCS and the succinylafion p r o c e d u r e with [14C] succinic a n h y d r i d e were the same as previously r e p o r t e d [11, 13]. T h e succinyl N C S was more acidic ( a b o u t 0.5 p H unit in pI) t h a n NCS, b u t otherwise very similar to the original NCS. I t has the same antigenicity as NCS. T h e specific radioactivity o f the material used was 2.38 x 10 6 dis/rain per m g protein (about 24 m C i / m m o l e ) .

Animals, treatments and preparations of specimens Female and male rats o f D A strain weighing 95-140 g were used for the experiments. O n e m g of the drug, dissolved in 0.5 ml of 0.01 M p h o s p h a t e buffered saline (0-15 M NaC1) (PBS, p H 7-0), was injected intravenously t h r o u g h a tail vein while the rats were u n d e r ether anesthesia. Since the original N C S has an in vivo half-life of less t h a n 10 rain [7], only the distribution o f the d r u g at 20 and 60 min was studied. T w e n t y and sixty min after injections, each rat was anesthetized again and all of the blood was i m m e d i a t e l y replaced b y 10 ml o f PBS infused t h r o u g h the inferior v e n a cava and recovered from the cross sected a b d o m i n a l aorta. T h e infusion process was repeated three times (30 ml in total) until complete replacem e n t was ascertained b y the disappearance o f the color o f blood in the organs such as the lung a n d liver. E a c h o r g a n was r e m o v e d i m m e d i a t e l y and weighed b y a torsion balance after r e m o v a l o f excess liquid with soft tissue paper. T h e D P M counts o f urine in T a b l e 1 were o b t a i n e d b y placing the weighed b l a d d e r in vials with 10 ml o f PBS and rinsing for a b o u t 40 rain. Bone m a r r o w specimens were o b t a i n e d b y applying a hydrostatic pressure to one side o f femurs using a 23 gauge needle a t t a c h e d to a syringe filled with 1 ml of cold PBS. T h e c o n t e n t o f the bone cavity (marrow) was dispersed b y repeating the flushing processes into a scintillation vial. T h e e m p t y bones thus obtained were used as bone specimens. T h e specimens o f skins were obtained from

Table 1. Distribution of radioactivity of [14C] succinyl neocarzinostatin in organs or tissues of rats After 20 rain

After 60 rain

Specimens Mean dis/min Kidney Bladder Ovary-uterus Bone marrow Blood plasma Lung Skin Tongue Stomach Muscle Large intestine Lymph nodes Small intestine Testicles Adrenal gland Pancreas Liver Submaxillarysalivary gland Bone Heart Thymus Eye Spleen Brain Cellular fraction in blood* Hypophysis~" Urine+* (out of I g bladder)

S.E.

151,570+ 15,661 108,759 + 28,756 14,502+ 5878 12,291_+ 673 10,246 + 639 8261 + 2664 7243 _+ 767 7076 _+ 611 5558 _+ 706 4289_+ 1302 4041 _+ 3884 _+

938 499

Mean dis/mAn

S.E.

99,514_+ 6333 133,423 _+29,556 3973§ 5394_+ 1002 5541 _+ 198 1785_+ 390 4278 _+ 353 5158 + 907 2693 _+ 471 1482_+ 206 1487 _+ 1604 _+

266 762

3336_+ 1033 --3305 _+ 445 2339_+ 417 2290 _+ 466

939+ 77 1350 [1_+ 62 6684_+ 2469 1223_+ 113 1575 _+ 114

2279 _+ 106 2251 _+ 1115 2179 _+ 246 2072 _+ 505 1880 -+ 317 1209 _+ 126 96 _+ 28

1375 _+ 1026-+ 1032 _+ 927 -+ 599 -+ 827 _+ 108 _+

100 79 206 139 194 71 8

951 _+ 4050 _+

1001 _+ 3822 -t-

195 689

129 680

5 4 6 9 - + 1254

6110_+ 1230

Data are the mean of four rats at 20 min and 60 rain respectively and DPM counts were expressed as per gr (wet wt.) organ. The body weight of each rat was adjusted to 100 g by calculation. *Cellular components were washed with PBS two times by centrifugation (1500 rpm x 5 mln). tThe weight of hypophysls was about 5 rag, and thus the errors of 20% was unavoidable. +*Diffusible counts from 1 g bladder is assigned as urine arbitrary. See Table 2. §Sample from one rat. ]ISamples from three rats. back-hide and those o f muscles were from rectus abdominis, free o f connective tissue. Contents o f stomach and small and large intestines were r e m o v e d b y gentle rinsing with PBS.

Measurement of radioactivity A whole or a p a r t o f the organs or tissues was oxidized by combustion with S a m p l e O x i d i z e r M o d e l 306 o f P a c k a r d Inc., and [a4C]O2 gas

Distribution o f Succinyl Neocarzinostatin, An Antitumor Protein

was trapped in Carbosorb (Packard Inst. Co. Inc. Downers Grove, Ill). Oxidation of samples was carried with the aid of oxygen gas and cellulose powder, and with Combustaid (Packard) when necessary. The scintillation liquid contained both Carbosorb and Permafluor V (12: 13) (Packard). Bone marrow cells, blood plasma and cellular fraction of the blood were solubilized with Soluene solubilizers and with aliquots of 30% H2Oz as described in the procedure of Packard Manual [17], and the radioactivity was counted. Analysis o f molecular sizes o f degradation products

of

:vcs The validity of the [14C] derivative, particularly the lack of desuccinylation of the succinyl NCS is shown on Fig. 1. As soon as the blood sample was taken, it was mixed with

-A "~ o

200

160

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I I I

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l

120

8 0 --

:~'

4c-

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I0

each fraction. Calibrations of the molecular weight were carried out with myoglobin (mean weight 17,000), actinomycin D (mean weight, 1,255), phenol red (mean weight 354) and acetic acid (mean weight 60). The molecular sizes of [14C] containing fragments obtained from homogenates of various specimens were investigated with a column (1.5 x60 cm) of Sephadex G-25 (Fine) and eluted by PBS (2 ml per fraction). The calibrations of the column with molecular references were the same as that for G-15. The samples of each organ (about 1 g) were prepared by homogenization with a Sorval homogenizer at 4°C with 2 ml PBS containing DFP and HgCI2 to give a final concentration of 0.1%, and then centrifuged at 5500rpm for 15 min. Urine specimens were collected on filter papers and the urinal spots were cut out and extracted with about 4 ml of PBS.

RESULTS

15. d

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20

30

40

Tube No.

Fig. 1. Breakdown of [z4C] succin~l neocarzinostatin in vivo (Rat) as revealed by Sephadex G-15. The open circle-solid line and the solid circle-dotted line indicate plasma samples obtained 20 min and 60 min after injection of the drug respectively. Note the decrease of the first peak near the void volume after 60 rain and the increase in amount and the size shift to the low molecular wt. region of the second peak thus indicating an extensive degradation of the protein.

heparin, HgC12 and di-isopropyl fluorophosphate (DFP), tihe latter two to minimize proteolysis. The final concentrations used were 10 units/ml for heparin and 0-1% each for the latter. Then the plasma was separated from cellular components by centrifugation. Subsequently, 0-1-0.:3 ml of plasma were applied to a Sephadex G.-15 column (1.5 x46 cm) and eluted with distilled water. Elution with the latter produced a better separation of the peaks than PBS. Radioactivity was monitored on

The molecular sieve chromatography with Sephadex G-15 indicated that there was no desuccinylation of the derivative during 20 or 60 min in vivo treatment, however, a peptide bond cleavage of NCS which resulted in a peak of smaller molecular size was noticeable after 60 min (Fig. 1). The results summarized on Table 1 show the distribution. The radioactivity was found to be distributed throughout the whole body within 20 min except the brain. The brain shows the least amount in the uptake even after 60 min. At the initial 20 min, the highest radioactive counts per gram of specimen was found in the kidney followed by the bladder, ovary-uterus, bone marrow, lung, tongue, stomach, muscle and skin. The high activity found in the bladder after 60 rain was partly attributed to urine origin (back diffusion) since the smaller peptide fragments account for more than 60% in plasma (Fig. 1) and high counts were found in the diffusible fraction and in the urine (Tables 1 and 2). The recovery of the drug in various parts of the body is shown in Table 2. The very rapid excretion of the drug is noteworthy. In 20 min, 45.5% of the radioactivity was found in the urine (Table 2). This radioactivity is due to large molecules and partly to small molecules (Fig. 2). The molecular sizes of the drug in various organs after 20 min were of larger molecular weight. For instance, the first peak (mol. weight > 6000) in such organs as the kidney, lung, muscle (not shown), and liver was composed of 78, 90, > 90 and 550/0 of the

868

Hiroshi Maeda, Naoki Yamamoto and Akira Yamashita

Table 2. Recovery of [x4C] radioactivity of succinyl neocarzinostatin in various organs and specimens 20 min after intravenous injection

(b)

O) ...:

2 d

Organs or specimens

Total Wet weight dis/min of organs per organ (g)* ( x 10-3)

i

I

I

E

I

f

I

I

I

%of recovery

7

7

O

0

Kidney Blood plasma Liver Small intestine Lung Stomach Large intestine Bladder Tongue Pancreas Skin Muscle Bone Bone marrow Urine Blood cell fraction

1.1 4.0 t 5-7 3.5 0.9 1.1 1.3 0.045 0.2 0.33 13.0J" 30t 101" 2.3:~ in 20 min§ 3.2~"

166.0 41.1 13"1 11.7 7.36 6.15 5.26 4.89 1.41 0.77 93.7 128.8 22.5 67-0 1083.4 3-05

6.97 1.73 0"55 0.49 0.31 0"26 0"22 0.21 0.058 0-032 3.94 5.4 0.95 2.81 45-5 0.128

x

x

z 7

i 20

30

40

Tube

No.

total cpm respectively. The total recovery of radioactivity in the supernatant from the homogenates of kidney, lung, liver, and urine were 24.1, 69.2, 80.3 and 74.6% respectively after one process of homogenization and extraction (Fig. 2A-D). Only in the liver was the second peak (mol. weight ~ 1000) found to be at significantly higher indicating the degradation of NCS in this organ in addition to the blood. The [14C] derivatives in urine showed that NCS was excreted very rapidly, mainly as a large molecular size compound. Although smaller molecular size fragments are eluted, majority of the components are of intermediate size component when compared to the other patterns from other organs (Fig. 2B). The liver extract showed, on the contrary, more c/min in the smaller fragments (44-8%) than any of the other organs tested indicating that the degradation of this drug in the liver was one of the main pathways (Fig. 2D). In the lung, about 9% of c/min was found in the smaller fragments. Less than 10% of the total c/min in muscle was also smaller fragments (not shown). When the radioactivities at 20-min and 60-mi~ intervals were compared (Table 1),

2o

50

30 Tube

40

50

No.

(d)

(c)

u

--I-~

*The wet weights of organs represented here are the average values of two rats about 100 g body weight. "~Estimated. ~.Estimated from G. Hudson's data in J. Anatomy 92~ 150 (1958). §Excreted in the initial 20 rain on filter papers followed by extraction.

u

i

i

I

u

,

I

--r-~

i

?

~

I

~

t

,

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0 x

x

¢

¢

c; o

~0.,

>,

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30

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No.

50

20

30 Tube

40

50

No.

Fig. 2. Analysis of molecular size distribution in various organs of [14C] succinyl neocarzinostatin 20 min after intravenous injections by Sephadex G-25 column. The preparations and fractionations were carried out as described under Material and Methods. (A), (B), (C) and (D) indicate the samples prepared from kidney, urine, lung and liver. A back ground cpm of 20 has been subtracted from each fraction. Each point represents an average of triplicate runs. In (B), two curves show the same data, but the solid circles were in expanded scale (ordinate at right).

the pattern of excretion and retention of c/min can easily be seen. A pronounced fall in the radioactivity was observed in the ovary-uterus, lung, intestine, muscle, and eyes, O n the other hand, relatively good retention or accumulation, nearly 50% or more, was observed, in the kidney, urine, bone marrow, tongue, liver, adrenal gland, blood cells and skin. The hypophysis, which is outside the blood brain barrier, had high values in contrast to the brain. The relatively high counts in the skin and muscle indicate that the absolute amount of the drug in these parts should be quite large in a whole body as shown in Table 2.

Distribution of Succinyl Neocarzinostatin, An Antitumor Protein

DISCUSSION Since the prime requisite for drug action is its accessibility to particular tissues or organs, we have designed our present experiments with the use of the radioactive derivative. The drug concentration w]hether intact or degraded forms varied from one organ to the other 60 min after drug injection. The degradation studies clarified that more than 60% of the original NCS was distributed as molecular size of about 1000 (Fig. 1) in plasma within 60 min. The highest values found in the bladder after 60 min (Table 1) could be due to the excretion and the accumulation in urine. A back-diffusion to the bladder tissue was anticipated (details to be reported elsewhere). The radioactivities in the lung and ovary-uterus fell to about 21-8% and 27"4% to the values found at 20 min respectively. In other organs a similar or a less radioactivity than that of 20 min was found indicating relatively good retension of the drug. The low DPM counts found in the liver or spleen, both of which have well developed networks of blood vessels, could have been resulted by washing out in the flushing process partly, but nevertheless the same procedure did yield high values in lung and kidney. These findings suggest that main areas of accumulation of this antibiotic may include the tumors in the kidney and bladder, followed by bone marrow, ovary-uterus, lung, tongue, stomach, muscle, large and small intestine, lymph nodes and skin. Despite comparatively low activity in the: liver, it still may be a good area due to its vascularity. Brain and thymus seem to be poor areas perhaps due to their anatomical architectures of blood vessels, such as blood brain barrier and blood thymus barrier. The high counts in the hypophysis may be related to its lack of such a barrier. Recent publication on toxicological studies of NCS in dogs showed renal lesions in survivors which can be rationalised by the high accumulation of the drug in these organs [18]. More

than 6.9% of the drug is accumulated in the kidney in 20 min as shown in Table 2. Previous studies of the distribution of NCS in vivo [7, 8] had drawbacks. These experiments were based on the residual antibacterial activity of NCS and were done without the use of inhibitors of proteolysis. The bacterial assay requires an incubation period of 1-2 hr for drug diffusion as well as an overnight for bacterial growth. Since the drug has a half life of about 10 min in vivo, the bioassay leads to much lower activity and makes it impossible to calculate the recovery rate. In addition, there are known inhibitory or unfavorable substances to bacterial growth in vivo. On the other hand, the present investigation reveals the target organs of NCS much more directly and with higher degree of sensitivity than did the bioassay method. The study of breakdown of succinyl NCS, observed previously in vitro [13] with serum and cell homogenate and in vivo in the present study, showed, however, that desuccinylation of the derivative is not marked. Therefore, the distribution of [14C] radioactivity, obtained here, is not measuring [14C] succinic acid, but the fragment of succinylated polypeptide molecules of NCS within the experimental time. Thus the present results can be extended the distribution of the parental NCS molecule, in which an overall close similarity exists. Due to the well known development of penetrated capillaries in tumors in vivo, the accessibility of this drug to the tumors itself should be more favorable than to the normal organs.

Acknowledgements--We thank Prof. Y. Hinuma of the Department of Microbiology for his interest and encouragements, Dr. H. Ouchi of the Department of Pharmacology for discussions, Dr. Y. Koyama of Kayaku Antibiotic Res. Laboratory for his supply of NCS, Dr. Dave T. Osuga of University of California, and Dr. James Shaw of University of California Medical School, Davis, Calif., for their careful readings and suggestions during preparation of the manuscript and Mrs. M. Fujii for the typing.

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M. TAKAHASHI, K. TORIYAMA, H. MAEDA, M. KIKUCHI, K. KUMAGAIand N, ISHIDA, Clinical trials of a new antitumor polypeptide: neocarzinostatin (NCS). Tohoku d. exp. Med. 98~ 273 (1969). K HIRAm, O. KAMIMURA, I. TAKAHASHI, T. NAOAO, K. KITAJIMA and S. IRINO,Neocarzinostatin, une approche nouvelle dans la chimioth6rapie des leucdmies aigu~s. Nouv. Rev.frarff. Hdmat. 13, 445 (1973). K. HIRAm, K. KITAJIMA,T. NAOAO,I. TAKAHASHI,H. KINOSHITA,O. KAMIMURA, H. HAYASHI, Y. MORIWAKI, H. CHIN and H. SANADA,Treatment of acute leukemia with neocarzinostatin. Igaku no Ayumi (Progress in Medicine) 87~ 18 (1973).

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