Int. J. lmrnunopharmac., Vol. 15, No. 2, pp. 175-183, 1993. Printed in Great Britain.

0192-0561/93 $6.00 + .00 Pergamon Press Ltd.

©1993 International Society for lmmunopharrnacology.

IMMUNOMODULATING ACTIVITIES OF ORALLY ADMINISTERED SMANCS, A POLYMER-CONJUGATED DERIVATIVE OF THE

PROTEINACEOUS ANTIBIOTIC NEOCARZINOSTATIN, IN AN OILY FORMULATION

FuJIo SUZUKI, *t KAORI MATSUMOTO,* DAVID A. SCHMITT,* RICHARD B. POLLARD* and HIROSHI MAEDA ~

*Department of Internal Medicine, University of Texas Medical Branch; tShriners Burns Institute, Galveston, TX 77555, U.S.A.; and *Department of Microbiology, Kumamoto University Medical School,

Kumamoto 862, Japan

(Received 19 June 1992 and in final form 2 October 1992)

Abstract - - Immunomodulatory effects of oily formulated SMANCS, a polymer conjugated derivative of the proteinaceous antibiotic neocarzinostatin, after oral administrations to mice was investigated. The oral administrations of SMANCS, dissolved in medium-chain triglyceride containing phosphatidylcholine and polyglycerine dioleate (oily SMANCS), resulted in: (1) augmentation of NK cell activity in naive mice, (2) activation of macrophage cytostasis in naive mice, (3) enhancement of the generation of cytotoxic T-lymphocytes in mice immunized with allogeneic lymphocytes, (4) production of circulating interferon in naive mice, and (5) increase of delayed-type hypersensitivity response in mice immunized with sheep red blood cells. The degree of immunomodulation orally stimulated with oily SMANCS was similar to that of the immunomodulation induced by i.v. or i.p. administrations of aqueous formulated SMANCS (aqueous SMANCS). Although SMANCS is a protein drug that may be digestable by various enzymes present in the stomach, in the present study immunomodulating activities of SMANCS were clearly demonstrated when an oily formulation of the compound was administered to mice orally. Since aqueous SMANCS administered parenterally and oily SMANCS administered orally exhibit the same immunomodulatory activities and the former has demonstrated antitumor activity in man and animals, the latter may possess this same antitumor activity.

Neocarzinostatin (NCS), an acidic single-chain polypeptide with 113 amino acid residues (Mr 12,000), was the first protein antibiotic to be utilized as a chemotherapeutic agent in human malignancy (Maeda, 1981). Recently, Maeda and co-workers synthesized a lipophilic high-molecular-mass derivative of NCS, which they called SMANCS (Mr 16,000), by chemical conjugation of poly(styrene- maleic acid) (Maeda, Takeshita & Kanamaru, 1979; Maeda, Matsumoto, Konno, Iwai & Ueda, 1984; Maeda, Ueda, Morinaga & Matsumoto, 1985). Because lipophilic SMANCS exhibits water and lipid solubility (Maeda et al., 1979, 1985), SMANCS dissolved in lipiodol has been administered to more than 300 patients with liver cancer, often by catheterizing the respective feeding arteries under

X-ray monitoring (Konno et al., 1983, 1984; Maeda, 1986). By this method, SMANCS can be delivered and deposited in tumor tissue over a long period of time without major systemic side effects, and obvious clinical activity in patients with unresectable liver cancer has been demonstrated. Recently, oily formulations of SMANCS (oily SMANCS) have been prepared using medium-chain triglyceride and their effective intestinal absorption observed after oral administration (Oka et al., 1990). Although protein drugs are often ineffective when they are administered orally because of their susceptibility to the enzymes of the digestive tract, oily SMANCS could be effectively absorbed gastrointestinally after oral administration. Since parenteral administrations of aqueous SMANCS has already been shown to

tAuthor to whom correspondence should be addressed: Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX 77555-0882, U.S.A.

175

176 F. SUZUKI et aL

stimulate various immune parameters in healthy or tumorous animals and in patients with malignancy (Oda, Morinaga & Maeda, 1986; Suzuki, Munakata & Maeda, 1988; Suzuki, Pollard & Maeda, 1989; Suzuki, Pollard, Uchimura, Munakata & Maeda, 1990), in the present study the immunomodulating activities of oily SMANCS administered orally were examined in mice. It has been reported (Suzuki et al., 1990) that antitumor effector cells, such as cytotoxic macrophages generated by the stimulation of parenterally administered aqueous SMANCS, play an important role in the inhibition of tumor growth by the compound in solid tumors implanted into mice.

EXPERIMENTAL PROCEDURES

Reagents

SMANCS was synthesized by Kuraray Co., Ltd (Kurashiki, Japan), as described previously (Maeda et al., 1979, 1984, 1985). Medium-chain triglyceride (MCT), phosphatidylcholine and polyglycer- ine(6)dioleate were gifts from Nippon Oil and Fat Co., Ltd (Tokyo, Japan). MCT is a synthetic material containing about 75070 caprylic acid and 20070 capric acid. Murine IFN-y was supplied from the Shionogi Pharmaceutical Co., Ltd (Ohsaka, Japan). Anti-mouse IFN-y Antiserum (anti-IFN-y) was kindly supplied by the Department of Microbiology, the University of Texas Medical Branch, Galveston, TX. Murine IFN-a /p and anti- mouse IFN-a/fl polyclonal IgG (anti-IFN-a/fl) was purchased from the Lee Biomolecular Research Laboratories Inc. (San Diego, CA).

Animals, cells and viruses Eight-week-old inbred BALB/c, C57BL/6, and

C3H/HeJ mice of both sexes were obtained from the Jackson Laboratory (Bar Harbor, ME). The tumor ceils used as target ceils for the cytotoxic tests were YAC-1 (NK cells), RLc~I (NK cells and macro- phages), and EL-4 (cytotoxic T-lymphocytes) cells. These tumor cells were maintained in RPMI-1640 medium supplemented with 10070 heat-inactivated fetal bovine serum (FBS), penicillin (100 IU/ml), and streptomycin (10/ag/ml) (complete medium). Monolayer cultures of murine L cells (L-Galveston cells) were grown in Eagle's modified minimum essential medium (EMEM) supplemented with 10070 FBS and antibiotics. The Indiana strain of vesicular stomatitis virus (VSV) was grown in L-Galveston cells and stored frozen ( - 70°C) until use. Sheep red

blood cells (SRBC) were separated from fresh sheep blood in our laboratory.

Preparation of aqueous and oily formulations o f SMANCS

SMANCS was dissolved in saline to form the aqueous formulation just prior to its use in the experiments. An oily formulation of SMANCS was prepared as described previously (Oka et al., 1990). Briefly, 100mg of SMANCS and 200mg of phosphatidylcholine were dissolved in 70 ml of 0.01070 (NH4)2SO4 solution and lyophilized. The lyophilized materials were added to MCT containing 5070 polyglycerine(6)dioleate and suspended using a vortex mixer. This procedure facilitated dissolution of the drug in oil. The final concentration of oily SMANCS was 4 mg/ml and it was diluted with the MCT solution to the appropriate concentrations for use in the animal experiments. As a control, the same procedure was repeated without SMANCS and oily solution obtained (MCT solution) was administered to mice in the same doses and schedules with that of oily SMANCS.

Assay of NK cell-mediated cytotoxicity Mononuclear cells (SMNC) from spleens of

BALB/c mice were prepared by F i c o l l - Hypaque sedimentation, as described previously (Suzuki & Pollard, 1982). In some experiments splenic effector cells were treated with a 1 : 100 dilution of anti- asialo GMj antiserum (Wako Chemicals U.S.A., Inc., Dallas, TX) or a 1 : 500 dilution of anti-Thy 1.2 monoclonal antibody (mAb) (Accurate Chemical and Scientific Corp., Westbury, NY) followed by treatment with a 1 : 15 dilution of complement (C) (Low-tox-M Rabbit C, Cedarlane Laboratories, Hornby, ON) as described previously (Kasai, Yoneda, Habu, Maruyama, Okumura & Tokunaga, 1981; Suzuki, Brutkiewicz & Pollard, 1986). The cells were washed with complete medium and assayed for NK cell activity. Cytotoxicity mediated by NK cell activity against YAC-1 target cells at an effector-to-target cell ratio of 50 : 1 was determined by a standard 4-h S~Cr-release assay, as described previously (Aso, Suzuki, Yamaguchi, Hayashi, Ebina & Ishida, 1985). All assays were performed in triplicate.

Assay of macrophage-mediated cytostasis Peritoneal exudate cells (PEC) were used as

effectors for the activated macrophage (A-Mdp) cytostatic assay. As the occasion demanded, these cells were fractionated into adherent and

Orally Administered SMANCS

nonadherent cells, as described previously (Suzuki & Pollard, 1982). Removal of phagocytic cells from PEC was performed by treatment with carbonyl-iron (Suzuki & Pollard, 1982), where more than 99070 of phagocytic cells were removed from the PEC by this technique. To eliminate T-cell or NK cell popu- lations, 1,8 × 107 cells/ml of PEC were mixed with anti-Thy 1.2 mAb or anti-as]alp GM~ antiserum, as mentioned above (Asp et al., 1985). Cells were then washed once with RPMI medium, resuspended in a 1 : 15 dilution of C, and incubated at 37°C for 60 min. The surviving cells were washed twice with complete medium and adjusted to the desired concentration for the cytostatic assay. As described previously (Suzuki et al., 1990), cytostasis of A-M+ preparations was detected by a [3H]-thymidine incorporation inhibition technique against RLcrl leukemia cells (Suzuki et al., 1990). Percentage of A-M+-cytostasis was calculated by the following formula

°70 cytostasis = ](counts/min of control-counts/rain of experimental)/counts/min of control] x 100

where counts/rain of control equals target cells alone and counts/min of experimental equals target ceils and A-M+/ preparations. Spontaneous cytostasis was calculated as above, where counts/rain of control equals target cells alone and counts/rain of experimental equals target cells and SMNC obtained from normal mice. All assays were performed in triplicate and results shown in the table and figure were presented as the mean of these three assays.

Assay o f allospecific cytotoxic T-iymphocytes As described previously (Munakata, Suzuki &

Maeda, 1990), C3H/HeJ mice (H-2*) received a subcutaneous (s.c.) immunization of 2 × 10 7 EL-4 cells (H-2 ~) grown in vitro. One day before immunization, mice were treated once with aqueous SMANCS (i.v., 1.6 mg/kg; oral, I0 mg/kg) or oily SMANCS (oral, 10 mg/kg). Ten days postimmuni- zation, mice were killed by cervical dislocation and the spleens were removed. SMNC prepared from these immune spleen cells were subjected to the assay of cytotoxic T-lymphocytes (CTL). For the characterization of effector cells, SMNC from immunized mice were treated with anti-Thy 1.2 mAb followed by C, then assayed for their cytotoxic activities. Allospecific CTL activities were deter- mined by a standard 5~Cr-release assay against EL-4

177

target cells (Munakata et al., 1990). The following formula was used to calculate the percentage of specific release

% specific release = ](test counts/rain - spontaneous counts/rain)/ (maximal counts/rain- spontaneous counts/min)l × I00.

Spontaneous release by target cells was approximately 8°70 of the maximum release. All samples were assayed in triplicate and each experiment was repeated at least two times with similar effects. For simplification, the values were presented as the mean since the standard deviation in triplicate was always less than 1007o.

Induction and titration o f interferon For detection of circulating interferon (IFN),

serum specimens were obtained from mice treated with aqueous SMANCS (i.v., 1.6 mg/kg) or oily SMANCS (p.o., 10 mg/kg) at appropriate intervals after the treatments, as described previously (Suzuki, Munakata & Maeda, 1988). The ant]viral activity was determined by means of a plaque reduction assay with murine L-Galveston cells and vesicular stomatitis virus (VSV) as described previously (Suzuki et al., 1988). To determine trypsin, heat, and acid stabilities of the ant]viral activity, serum specimens were treated with 200 ~g/ml of trypsin at 37°C for 2 h, incubated at 56°C for 30 min, or dialyzed against 0.2 M g lyc ine- HCL buffer (pH 2.0) at 4°C for 24 h (Asp et al., 1985; Suzuki et aL, 1988). These specimens were then assayed for their ant]viral activities. To determine species specificity of the ant]viral activity, serum specimens were assayed with human embryonic lung fibroblasts or rabbit RK-13 cells and compared with titers obtained against murine L-Galveston cells (Suzuki et al., 1988). Serum specimens were also treated with anti-IFN-a/fJ, anti-IFN-), antibody, or their mixture before subjection to the IFN assay, as described (Suzuki, Pollard & Maeda, 1989).

Induction and titration o f delayed-type hypersensi- tivity (DTH)

C57BL/6 mice were immunized i.v. with 0.2 ml of 0.01070 sheep red blood cells (SRBC). A 10 mg/kg oral dose of oily and aqueous SMANCS or a 1.6 mg/kg i.v. dose of aqueous SMANCS was administered to these mice one day before, just after, and one day after the immunization. Four days after the immunization, one foot pad per mouse was measured for thickness with a microcaliper and was

178

240 , \ 180

120

2; 60

0 4 8 12 16 20 24 28 32 36 40

Hours after administration

Fig. 1. Interferon induction in mice treated orally with oily SMANCS. Mice were stimulated with either an aqueous (1.6 mg/kg, i.v.) or an oily (10 mg/kg, oral) form of SMANCS. At various intervals after the stimulation, serum specimens were obtained and were assayed for their antiviral activities in L-Galveston cells infected with VSV. Symbols: - - O - - , mice treated with aqueous SMANCS; - - O - - , mice treated with oily SMANCS; - - - A - - , mice

treated with MCT solution.

then challenged with an injection of 0.03 ml of 20% SRBC in sterile saline. Twenty-four hours after the challenge, the original foot pad was remeasured and the thickness was compared to the size obtained prior to the challenge. The percent increase in thickness was then computed by comparing the two measure- ments (Askenase, Hayden & Gershon, 1975).

RESULTS

1FN induction by oily SMANCS

Figure 1 shows the IFN response induced by oily (10mg/kg, oral) and aqueous (1.6 mg/kg, i.v.) SMANCS in naive mice. Blood samples from 5 mice per group were taken at 4-h intervals after the drug administration. In the group treated with oily SMANCS, the antiviral activity was detected at 12 h and peak levels of 240 U/ml were obtained in serum by 24 h. The antiviral titer decreased gradually during the next 16 h. The antiviral titer was also detected in the circulation of mice treated with aqueous SMANCS. The peak titer of the antiviral activity was demonstrated in serum of mice 20 h after the treatment. These antiviral activities induced by both forms of SMANCS showed species specificity, inability to inactivate the challenge virus

F. SuzuKi et al.

directly, and sensitivity to trypsin, lower pH (pH 2.0), and temperature (56°C, 30 min) (data not shown). These results indicate that the antiviral activities induced by both forms of SMANCS in the circulation of mice were characterized as an IFN. In addition, treatment of serum IFN with anti IFN-y antiserum neutralized 48% of IFN activity induced by oily SMANCS, while 55% of it was neutralized by treatment with anti-IFN-g/3 antiserum. These results indicate that the circulating IFN induced by oily SMANCS in mice contained both IFN-y and IFN-~/3 equally (Table I). However, antiviral activity was not demonstrated when serum specimens from mice various hours after the treatment with MCT solution were assayed for their IFN activities.

Cytostasis o f A-Mdp from mice treated with oily SMANCS

The ability of peritoneal macrophages from mice treated with aqueous SMANCS (i.v., 1.6 mg/kg; oral, 10 mg/kg), oily SMANCS (oral, 10 mg/kg), MCT solution (0.2 ml/mouse, oral), or saline to inhibit the growth of RLcrl target cells was examined. As shown in Fig. 2, PEC from mice treated with aqueous SMANCS i.v. or oily SMANCS orally inhibited the growth of target cells when they were co-cultured. However, PEC from mice orally treated with aqueous SMANCS or MCT solution did not inhibit the growth of target cells significantly. PEC from mice treated with aqueous (i.v.) or oily (oral) SMANCS 3 - 5 days after the treatment showed peak levels of cytostasis against target cells.

To characterize the cellular activity induced by oily SMANCS, plastic-adherent cells (PAC prepared from PEC) were (1) treated with anti-Thy 1.2 mAb plus C to eliminate T-cells, (2)treated with anti- asialo GM~ antiserum plus C to abolish NK cells, and (3) incubated with carbonyl-iron to eliminate phagocytic cells. These effector cells were then assayed for their cytostatic activities against RLc,1 target cells at an effector-to-target cell ratio of 10 : 1. As shown in Table 2, the cytostatic activity of PEC remained in the PAC population and it was clearly reduced by elimination of phagocytic cells. The activity of PAC was unchanged after depletion of asialo GM~ + cells and Thy 1 + cells. These results indicate that peritoneal effector cells generated in mice after oral administrations of oily SMANCS possess the characteristics of M~.

Augmentation o f NK cell activity in spleens o f mice treated with oily SMANCS orally

The effect of oily SMANCS, administered orally to naive mice, on splenic NK cell cytotoxicity

Orally Administered SMANCS

Table 1. Neutralization of various IFNs by treatment with anti-IFN-e//3, anti-lFN-), antisera, or their mixtures antibodies

179

IFN titer (U/ml) (070 loss of activity)

IFN Anti-lFN-a//J Anti-IFN-y A mixture of antibodies

IFN-a//3, 100 U/ml <8 (>92) 99 (1) <8 (>92) IFN-y, 100 U/ml 98 (2) <8 (>92) <8 (>92) Serum IFN, 120 U/ml 54 (55) 63 (48) <8 (>93)

Serum IFN was prepared from mice 24 h after oily SMANCS administration (10 mg/kg, oral). Murine IFN-a/p (0.5 ml, 200 U/ml), murine IFN-y (0.5 ml, 200 U/ml), and serum IFN (0.5 ml, 240 U/ml) were mixed with equal volumes of anti-IFN-a//3, anti-IFN-y and their mixture (200-IFN-neutralizing U/ml), respectively, and then, their antiviral activities were assayed in L-Galveston cells infected with VSV.

50

4 0

20

10

L

_1 [ I I L I I 1 I I I

Untreated 1 2 3 4 5 6 7 8 9 10

Days after administration

Fig. 2. Cytostasis of peritoneal macrophages derived from oily SMANCS-treated mice. Peritoneal exudate cells were prepared from mice treated with an aqueous (i.v., 1.6 mg/kg; oral, 10 mg/kg) or an oily (p.o., 10 mg/kg) form of SMANCS. Macrophage cytostasis against RLcyl target cells was assayed by a [~Hl-thymidine incorporation inhibition test at an effector to target cell ratio of I0 : 1. Symbols: - - © - - , mice treated with a 1.6 mg/kg i.v. dose of aqueous SMANCS; - - O - - , mice treated with a 10 mg/kg oral dose of oily SMANCS; - - • - - , mice treated with a 10 mg/kg oral dose of aqueous SMANCS;

_ _ A _ _ , mice treated with MCT solution.

was examined. SMNC from mice treated with a 10 m g / k g oral dose of aqueous S M A N C S or 0.2 ml oral dose o f M C T solution did not show detectable levels of NK cell activity against YAC-1 target cells. Administrat ion of a 10 m g / k g oral dose of oily SMANCS, as well as a 1.6 m g / k g i.v. dose of aqueous S M A N C S to naive mice resulted in augmentat ion of NK cell activity (Fig. 3). Since it has been reported (Kasai et al., 1981) that murine NK cell activity could be eliminated by t reatment of effector cells with anti-asialo GM1 antiserum and C, but not eliminated by the t reatment of anti-Thy 1.2 mAb

and C, splenic effector cells were treated with both blockers and C before being subjected to the NK cell assay. As shown in Table 3, t reatment of effector cells with anti-asialo GMj antiserum and C completely abolished their killing activities, while no decrease of cytotoxicity was found following treatments with normal rabbit serum or anti-Thy 1.2 mAb and C. These results indicate that this cellular cytotoxicity, which is augmented by oral adminstrations of oily SMANCS, is mediated by NK cells.

Generation o f allospecific CTL in spleens o f immune mice treated with oily SMANCS

Various effector cells, which included SMNC from naive or immunized mice that were treated with oily (oral) or aqueous (oral, i.v.) SMANCS, were tested for their cytotoxicities by the standard 5~Cr-release assay against EL-4 target cells. Allospecific CTL activity in the immunized control group treated with M C T solution or saline was shown to be 15.9 or 14.8°70 lysis, respectively, while 31.9°70 lysis (215070 augmentation) or 33.4070 lysis (227070 augmentation) was demonstrated in killing activity of SMNC from mice treated with a 1.6 m g / k g i.v. dose o f aqueous SMANCS or a 10 m g / k g oral dose of oily SMANCS, respectively. However , oral t reatment of immunized mice with a 10 m g / k g dose of aqueous SMANCS did not increase the killing activity of their splenic ceils (Table 4). Addit ional experiments were performed to confirm the nature and specificity of CTL generated by the stimulation of allogeneic lymphocytes com- bined with oily SMANCS. As described previously (Kobayashi, Aso, Ishida, Maeda, Pollard & Suzuki, 1990), SMNC from immunized mice treated with oily S M A N C S were treated with anti-Thy 1.2 mAb and C to deplete T-lymphocytes before subjection to the CTL assay. As shown in Table 5, t reatment of effector cells with mAb followed by C abrogated

180 F. SUZUKI et al.

Table 2. Characterizations of cytostatic activities of peritoneal exudate cells from mice treated with oily SMANCS

Effector cells Lysis (070) Reduction (070)

Peritoneal exudate cells (PEC) Plastic adherent cells (PAC) from PEC Non-PAC from PEC PEC treated with carbonyl-iron

33.0 ± 8.6 0 39.3 ± 9.8 0 9.2 ± 3.2* 72 6.1 ± 4.4* 82

PAC + C 32.5 ± 9.1 0 PAC + anti-asialo GM~ + C 30.2 ± 11.5 9 PAC + anti-Thy 1.2 mAb + C 31.8 + 10.6 4

PEC were harvested from mice 5 days after treatment with a 10 mg/kg oral dose of oily SMANCS. Cytostasis of effector cells was assayed by [3H]-thymidine incorporation inhibition technique at an effector to target (RLc,1) cell ratio of 10 : 1.

*Student's t-test, P<0.001.

20 - -

._~ lO

I I I I I I 0 1 2 3 4 5 6

Days after administration

Fig. 3. Augmentation of NK cell activities in mice treated orally with oily SMANCS. Splenic mononuclear cells were prepared from mice treated with aqueous (i.v., 1.6 mg/kg; oral, 10 mg/kg) or oily (oral, 10 mg/kg) SMANCS. The NK cell activity of these cells against YAC-I target cells was assayed by a standard ~Cr-release assay at an effector to target cell ratio of 50 : 1. Symbols: - - ( 2 ) - - , mice treated with a 1.6 mg/kg i.v. dose of aqueous SMANCS; - - @ - - , mice treated with a 10 mg/kg oral dose of oily SMANCS; - - A - - , mice treated with a 10 mg/kg oral dose of aqueous SMANCS; - - A - - , mice treated with

MCT solution.

Effector cells induced by the H-2 ~ stimulation were effective against EL-4 lymphoma cells and the same preparat ion of CTL induced by the H-2 ~ stimulation inhibited the growth of RLcel leukemia cells. However , the growth of EL-4 was not inhibited by the H-2 d stimulated effector cells. Similarly, the growth of RLo ' I cells was demonstrated when the assay was performed with the 1-1-2 b stimulated effector cells (data not shown). These data seem to indicate the aUospecificity of effector functions of CTL generated in this study.

Augmentat ion o f D T H response in mice by oily S M A N C S treatment

Effect o f oily SMANCS administered orally on D T H response in mice 4 days after the immunizat ion with SRBC was examined. Mice, immunized with SRBC, were treated with oily SMANCS one day before, just after, and one day after the immuni- zation. Four days after the sensitization, these mice were challenged with SRBC and footpad swelling was measured 24 h after the challenge with a microcaliper. As shown in Table 6, 67% of the D T H response was augmented in immunized mice by the treatment with oral oily SMANCS, while 53°7o of the response was enhanced in mice by the treatment with aqueous S M A N C S (i.v., 1.6 mg/kg) .

their killing activity. To examine specificity of effector cells, we also generated CTL from C57BL/6 mice (H-2 ~) immunized with RLc¢I cells (H-2 a) and treated orally with oily SMANCS, by the method described above. The CTL activity was measured by the 5~Cr-release assay against RLcrl target cells (H-2~), as well as EL-4 thymoma cells (H-2O).

D I S C U S S I O N

These studies presented herein have shown that an oily formulat ion o f SMANCS, a conjugat ion o f poly (styrene-maleic acid) and protein antibiotic N C S ' ,

Orally Administered SMANCS

Table 3. Cytotoxic activities of splenic mononuclear cells (SMNC) treated with anti-asialo GM~ antiserum or anti-Thy 1.2 monoclonal antibody (mAb) followed by complement (C)

181

Effector cells treatment Lysis (%) Reduction (%)

Medium 18.9 _+ 2.7 0 C alone 18.0 +__ 3.2 5 Normal rabbit serum + C 19.1 +_ 3.0 0 Anti-asialo GM~ antiserum + C 2.4 +_ 1.7" 87 Anti-Thy 1.2 antibody + C 17.7 +__ 3.6 6

SMNC (1.0 × 107 cells/ml) harvested from mice 4 days after oral treatment of oily SMANCS were treated with C (1 : 15 dilution), anti-asialo GM~ antiserum (1 : 100 dilution) plus C, normal rabbit serum (1 : 16 dilution) plus C or anti-Thy 1.2 mAb (1 : 500 dilution) plus C, before being tested for their NK cell activities. Cytotoxicity was determined by the 5~Cr-release assay at an effector to target cell ratio of 50 : 1

*Student's t-test, P<0.001.

Table 4. Effect of oral administration of oily SMANCS on the generation of allospecific CTL in C3H/HeN mice immunized with EL-4 tumor cells

Source of Specific effector cells Treatments lysis (%) Augmentation (%)

Normal mice Saline (i.v., 0.1 ml/mouse) 5.7 0 Normal mice MCT solution (p.o., 0.2 ml/mouse) 5.8 ~ 102 Normal mice Aqueous SMANCS (i.v., 1.6 mg/kg) 7.1 ~ 125 Normal mice Aqueous SMANCS (p.o., 10 mg/kg) 5.9 ~ 104 Normal mice Oily SMANCS (p.o., 10 mg/kg) 7.0 ~ 123

Immunized mice Saline (i.v., 0.1 ml/mouse) 14.8 0 Immunized mice MCT solution (p.o., 0.2 ml/mouse) 15.9 ~ 107 Immunized mice Aqueous SMANCS (i.v., 1.6 mg/kg) 31.9" 215 Immunized mice Aqueous SMANCS (p.o., 10 mg/kg) 14.1 0 Immunized mice Oily SMANCS (p.o., 10 mg/kg) 33.4* 227

C3H/HeN mice (H-2 k) were immunized i.p. with 5 × 106 cells/mouse of EL-4 lymphoma cells (H-2b). SMNC prepared from these mice 10 days after the immunization were measured for their CTL activities against EL-4 target cells at an effector to target cell ratio of 100 : 1. One day before immunization, mice were treated with MCT solution, aqueous, or oily SMANCS.

Student's t-test, *P<0.001, +not significant.

expressed var ious i m m u n o m o d u l a t i n g activities in mice when it was adminis te red orally. W h e n a 10 m g / k g dose of oily S M A N C S was admin is te red to mice orally, IFN activities were demons t r a t ed in the circulat ion. The level of the peak activity was f o u n d in sera of mice 24 h af ter the s t imula t ion and was a lmost equal to the IFN activity induced in mice 20 h af ter i.v. s t imula t ion wi th aqueous SMA N CS. These ant ivi ra l activities in sera induced by oily or aqueous S M A N C S were conf i rmed to be the result o f IFN activity by proper t ies such as species specificity, acid and heat stabil i ty, t ryps in sensitivity, and sensitivities to polyclonal ant i - IFN-a/ /3 an t i serum, ant i - IFN-y ant ibodies , and their mixtures. Serum IFN induced

by oral admin i s t r a t ion o f oily S M A N C S con ta ined abou t 55°7o IFN-a/ /3 and 4807o IFN-y activities. The activity of NK cells in SMNC f rom mice t rea ted orally wi th oily S M A N C S was also significantly augmented . However , augmen ta t i on of NK cell activity was no t demons t r a t ed in S M N C f rom mice t reated wi th aqueous S M A N C S orally. The NK cell activity augmen ted by oily S M A N C S was depleted by in vitro t r ea tmen t with ant i -asialo GM~ an t i se rum fol lowed by C, but no t an t i -Thy 1.2 m A b plus C. These results suggested tha t splenic effector cells demons t r a t ed in oily SMANCS- t r ea t ed mice were NK cells. A-M+ were also demons t r a t ed in P E C of mice orally t rea ted with oily SMANCS. The

182 F. SUZUKI et al.

Table 5. Effect of anti-Thy 1.2 monoclonal antibody (mAb) treatment on the activity of killer cells from spleens of mice l 1 days after oral treatment of oily SMANCS

Source of Treatment Lysis Reduction effector cells of effector cells (%) (%)

Normal mice C alone 4.2 + 1.1 0 Normal mice mAb + C 3.9 _+ 0.9* 7

Immunized mice C alone 19.6 _+ 2.6 0 Immunized mice mAb + C 3.6 _+ 0.5* 82

Immunized mice treated with oily SMANCS C alone 28.3 _+ 3.3 0 Immunized mice treated with oily SMANCS mAb + C 3.1 _+ 0.8* 89

Splenic effector cells from mice immunized with EL-4 cells were treated in vitro with mAb and/or complement (C), and their cytotoxic activities were determined by the 5tCr-release assay against EL-4 target cells at an effector to target cell ratio of 100 : 1. Oily SMANCS (10 mg/kg, p.o.) was administered to immunized mice one day before immunization.

Student's t-test, *P<0.001, *not significant.

Table 6. Effect of oily SMANCS on delayed type hypersensitivity induced in mice by SRBC immunization

Footpad swelling (mm) Enlargement Significance Treatment (mean _+ S.D.) (o7o) (P)

Saline, i.v., 0.l ml/mouse

MCT solution, p.o., 0.2 ml/mouse

Aqueous SMANCS, i.v., 1.6 mg/kg

Aqueous SMANCS, p.o., 10 mg/kg

Oily SMANCS, p.o., 10 mg/kg

0.913 __. 0.011 0

0.929 _+ 0.019 102 N.S.

1.401 _+ 0.015 153 <0.00l

0.998 _+ 0.012 109 N.S.

1.523 + 0.018 167 <0.001

C57BL/6 mice were immunized i.v. with 0.2 ml of a SRBC solution (0.01o7o). Oily and aqueous SMANCS was administered orally (both oily and aqueous formulations) or i.v. (aqueous SMANCS) to these mice one day before, just after, and one day after the immunization. As a control, immunized mice were treated with MCT solution (0.2 ml/mouse, orally) by the same schedules to that of oily SMANCS. Four days after sensitization, these mice were challenged in the footpad with a 0.03 ml dose of 20°7o SRBC solution. Footpad swelling was measured 24 h after the challenge with a microcaliper.

cytostat ic activity o f P E C f rom mice t reated with oily S M A N C S was ab roga ted when phagocyt ic cells or P A C were specifically e l iminated f rom P E C popula t ions . However , cytostat ic activity of P E C was unchanged when they were t reated with ant i - asialo GMz an t i se rum or an t i -Thy 1.2 m A b plus C. These results indicated tha t A-Md? were generated f rom rest ing macrophages by the s t imula t ion of oily S M A N C S adminis te red orally. The allospecific C T L was genera ted in spleens o f immunized mice af ter admin i s t r a t ion of aqueous (i.v., 1.6 m g / k g ) or oily (oral, 10 m g / k g ) SMA N C S. The CTL activity in S M N C was augmen ted by 215°70 (aqueous, i .v.) and

227% (oily, oral) af ter the t r ea tment of mice with SMANCS. W h e n S M N C f rom immunized mice orally adminis te red with oily S M A N C S were t reated in vitro with an t i -Thy 1.2 m A b plus C, 89°70 of the CTL activity o f effector cells were abrogated . The activity o f CTL induced in immunized mice t reated with oily S M A N C S was allospecific. Fur ther , D T H induced in mice by SRBC immuniza t ion was enhanced signif icantly by the oral admin i s t ra t ion of oily SMANCS.

Taken all together , orally adminis te red S M A N C S in oily fo rmula t ion expressed equally i m m u n o m o d u - lat ing activities in mice compared with tha t of i.v.

Orally Administered SMANCS 183

admin i s t r a t ion of aqueous S M A N C S , suggesting t ha t the pa t ien t can stay in thei r h o m e and be able to equally a n t i t u m o r efficacy between o r a l - oily and take this medicine and the toxicity of the c o m p o u n d a q u e o u s - i . v , admin i s t ra t ions of S M A N C S . The is reduced as compared to i.v. admin is t ra t ion . advantages o f the oral admin i s t r a t ion o f S M A N C S is

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