Design, synthesis and antitumor evaluation of phenyl N-mustard-quinazoline conjugates

A series of N-mustard-quinazoline conjugates was synthesized and subjected to antitumor studies. The N-mustard pharmacophore was attached at the C-6 of the 4-anilinoquinazolines via a urea linker. To study the structure-activity relationships of these conjugates, various substituents were introduced to the C-4 anilino moiety. The preliminary antitumor studies revealed that these agents exhibited significant antitumor activity in inhibiting various human tumor cell growths in vitro. Compounds 21b, 21g, and 21h were selected for further antitumor activity evaluation against human breast carcinoma MX-1 and prostate PC-3 xenograft in animal model. These agents showed 54-75% tumor suppression with low toxicity (5-7% body-weight changes). We also demonstrate that the newly synthesized compounds are able to induce DNA cross-linking through alkaline agarose gel shift assay and inhibited cell cycle arrest at G2/M phase.     

Human T cells targeted with anti-T3 cross-linked to antitumor antibody prevent tumor growth in nude mice

Human peripheral blood T cells were tested for the ability to prevent tumor growth in nude mice when targeted with anti-T3 cross-linked to antitumor antibodies. LS174T human colon adenocarcinoma cells were mixed with human PBL coated either with anti-T3 (Fab) cross-linked to 315F6 (Fab) (an antitumor monoclonal antibody) or with no antibody, and were injected subcutaneously into nude mice. Tumor growth was totally inhibited at effector to target (E:T) ratios of 7.0:1 and 2.1:1, and was partially inhibited at 0.7:1 with antibody-coated PBL, but was not inhibited by uncoated PBL. T cell-mediated protection against tumor growth occurred when an antitumor was physically cross-linked to anti-T3. Neither a mixture of unlinked anti-T3 and antitumor antibodies nor anti-human MHC class I cross-linked to antitumor antibody prevented tumor growth. Whereas in vitro cytotoxicity was mediated exclusively by T8+ cells and was augmented by brief exposure of effector cells to IL 2, tumor neutralization in vivo was mediated by both T4+ and T8+ cells and was not significantly stimulated by prior exposure of the cells to IL 2. We conclude that human T cells, when targeted with appropriate antibody heteroaggregates, can specifically inhibit tumor growth at low E:T ratios, and that cells mediating tumor neutralization in vivo may differ from those mediating cytotoxicity in vitro.     

Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates

The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.     

Stability and localization of biotin-labeled murine monoclonal antibody to human gastric cancer in normal mice and nude mice-human tumor models

We examined the tissue localization of biotin-labeled murine monoclonal antibody (MAb) S202 directed against the human scirrhous gastric carcinoma cell line MK-01 in normal and tumor-bearing mice after intravenous (IV) administration. The biotin-labeled MAb proved to be stable in vivo under normal conditions, antibody titer being 1:256 at 4 hr after IV injection. At 24 hr after injection, the tumor was stained by the avidin-biotin-peroxidase complex (ABC) method. Biotin-labeled MAb was found to be suitable for detection of the xenografted tumor of nude mice. This study provides new information concerning the dynamics of the distribution of biotin-labeled MAb in vivo.     

New potential anticancer agent of carborane derivatives: selective cellular interaction and activity of ferrocene-substituted dithio-o-carborane conjugates

The large diversity of structures and unique bonding modes of organometallic complexes make them possible to act as promising candidate therapeutic agents. In this study, the new type of ferrocene-substituted dithio-o-carborane conjugates (FcSB1, FcSB2, and FcSBCO) has been synthesized, and their in vitro antineoplastic activities have been explored by means of the electrochemical study, the real time cell electronic sensing (RT-CES) system, and biological assays. The conjugate-cell interactions were first monitored by electrochemistry, and the results show different cell uptake efficiency for FcSB1, FcSB2, and FcSBCO toward target cells. Both the highly hydrophobic ferrocenyl and carboranyl groups render the conjugates able to rapidly enter cells and exert acute cytotoxicity after 4 h incubation in serum-free media. However, FcSB1, FcSB2, and FcSBCO display different inhibition efficiencies toward SMMC-7721 and HepG2 cancer cells via the G(0)/G(1) arrest mechanism in a physiological environment. The anticancer activity is in the order FcSB2 > FcSB1 > FcSBCO, which is parallel to the order of the redox potentials of the ferrocenyl groups in the three complexes. In particular, FcSB1 and FcSB2 display a potent selective inhibition effect on the proliferation of the cancer cell lines SMMC-7721 and HepG2, but almost no effect on the normal cell line, the human embryonic lung fibroblast (HELF) cells. Thus, these results may provide some clues for use of the ferrocene-carborane conjugates in developing anticancer drugs.     

In vitro antitumor activity of 2'-deoxy-5-fluorouridine-monoclonal antibody conjugates

5-Fluorouracil (5-FU) is an anticancer drug used in patients for the treatment of gastric and breast cancer and used either alone or in combination with methotrexate is one of the few drugs with some effect on colon cancer. 2'-Deoxy-5-fluorouridine (5-FUdr) (1) is an analogue based on 5-FU and can be covalently linked to a murine anti-Ly-2.1 monoclonal antibody (mAb) with the active ester derivative of 2'-deoxy-5-fluoro-3'-O-(carboxypropanoyl)uridine (5-FUdr-succ) (4). Such immunoconjugates can contain up to 42 residues of drug, although the most antibody activity was retained when substitution ratios were between 10 and 25 molecules of drug to mAb. In a cytotoxicity assay, 50% inhibition of [3H]deoxyuridine incorporation (IC50) with a murine Ly-2.1+ve thymoma cell line was 6 nM for 5-FUdr-anti-Ly-2.1, which is 12-fold more than that for free 5-FUdr (IC50 = 0.51 nM) but similar to that of 5-FUdr-succ (IC50 = 5.2 nM). The 5-FUdr-monoclonal antibody conjugates (5-FUdr-mAb) were 100-fold more active on the Ly-2.1+ve E3 cell line than on the Ly-2.1-ve BW5147 OU- cell line. The high in vitro activity and specificity of 5-FUdr-MoAb conjugates indicates that potent in vivo activity of these conjugates should be expected.     

鼠抗人hMIC-l单克隆抗体抑制裸鼠移植人胰腺癌体内研究

目的：初步研究鼠抗人hMIC-l单克隆抗体对胰腺癌体内给药的抗肿瘤活性,为hMIC-l抗体应用于肿瘤治疗提供实验依据。方法2种人胰腺癌细胞株PANC-1和SW1990各腋窝皮下接种24只Balb/c裸鼠,共计48只皮下接种荷瘤裸鼠分别随机共分为8组,每组6只荷瘤鼠。模型对照组荷瘤裸鼠腹腔注射0.9％氯化钠注射液（10 mL/kg,NS,Biw,ip ×8）,阳性对照组荷瘤裸鼠腹腔注射键择（50 mg/kg,qw,ip ×4）,鼠抗人hMIC-l单克隆抗体组分别荷瘤鼠尾静脉内注射鼠抗人hMIC-l单克隆抗体（10 mg/kg,Biw,ip ×8）共4周或（2 mg/kg,Biw,ip ×8）共4周。观察荷瘤裸鼠日常表现、肿瘤生长、实验后肿瘤组织切片HE染色后光镜下的组织形态学改变。结果荷瘤裸鼠尾静脉内注射鼠抗人hMIC-l单克隆抗体组裸鼠（10 mg/kg,Biw,iv ×8）肿瘤生长缓慢,瘤体明显小于模型对照组,并呈现量效关系。镜下观察鼠抗人hMIC-l单克隆抗体组实验后肿瘤组织切片胰腺结构破坏,有大量淋巴细胞浸润,肿瘤细胞明显坏死,细胞溶解。结论尾静脉内注射鼠抗人hMIC-l单克隆抗体（10 mg/kg,Biw,iv ×8）能有效抑制裸鼠移植人胰腺癌PANC-1肿瘤生长,使瘤组织坏死、结构破坏。     

Immune responses to hapten--lipopolysaccharide conjugates. 1. Modulation of in vivo antibody responses to the polysaccharide

The immune responses of mice to various lipopolysaccharides (LPS) and hapten-LPS conjugates were compared. We found that some strains of mice ($\text{A}\text{J}$ and BALB/c) produced equivalent amounts of anti-LPS antibody after the injection of either LPS or hapten-LPS conjugates. In contrast, however, other strains of mice (C57BL/6J, C3H/St, DBA/1J, DBA/2J, and Swiss) produced fewer anti-LPS-antibody-secreting cells after stimulation with hapten-LPS conjugates than did mice injected with unsubstituted LPS. The covalent coupling of hapten to LPS changed neither the mitogenic capacity nor the antigenicity of the LPS. The differences in the magnitude of antibody responses to hapten-LPS and LPS in these latter strains of mice occurred in the absence of mature T lymphocytes and was restricted to the primary immune response. Furthermore, these differential responder mice (C57BL/6J) did produce anti-LPS antibody when primed with LPS before challenge with the hapten-LPS conjugate. These data are discussed with respect to both the modulatory capacity of the hapten-LPS in the regulation of the primary immune response to LPS and the biochemical and structural requirements of the hapten-LPS conjugate for immunogenicity.     

Conjugation of metallothionein to a murine monoclonal antibody

A method of conjugation of the metal-binding protein, metallothionein, to an anticarcinoma murine monoclonal antibody, B72.3, and its F(ab')2 fragment has been developed utilizing the heterobifunctional crosslinking reagent, succinimidyl 4-(N-maleimidomethyl)-cyclohexane 1-carboxylate. This crosslinking reagent is first reacted with the free amines on the immunoglobulin. After removal of unreacted crosslinker, conjugation is affected through a sulfhydryl group on metallothionein. Under the conditions employed all immunoglobulin aggregates contained metallothionein. The degree of undesired aggregation is directly proportional to the number of metallothioneins attached to the immunoglobulin. This aggregation can be controlled by the amount of crosslinker and metallothionein presented to the immunoglobulin. The immunoglobulin conjugate retains full immunoreactivity and can be readily purified from the unreacted metallothionein and high molecular weight aggregates. The metallothionein-B72.3 conjugate functions as an efficient and stable chelator of radiometals. Thus metallothionein-monoclonal antibody conjugates have potential utility in cancer diagnosis and therapy.     

In vivo antitumor activity of hydrosoluble derivatives of 7-hydroxycholesterols

Sodium bis-hemisuccinates of 7 beta- and 7 alpha-hydroxycholesterols are moderately water-soluble. They have been tested intraperitoneally against the murine Krebs-II carcinoma, grown as an ascitic tumour, and their action has been compared with that of usual chemotherapeutic drugs, cyclophosphamide, 5-fluoro-uracil, and methotrexate. The hydroxycholesterol derivatives show a faster and stronger activity (life prolongation), and lead to the complete disappearance of the tumour in about 1/3 of the cases, even with one single injection. Similar results have been obtained (on fewer cases) with two other experimental ascitic tumours, the S-180 sarcoma and the ZHC hepatoma. The mechanism of action is not known; it appears to be very different from that of the usual anti-cancer chemotherapeutic agents.     

Antitumor agents. 256. Conjugation of paclitaxel with other antitumor agents: evaluation of novel conjugates as cytotoxic agents

Sixteen different taxoid conjugates were prepared by linking various anticancer compounds, including camptothecin (CPT), epipodophyllotoxin (EP), colchicine (COL), and glycyrrhetinic acid (GA), at the 2'- or 7-position on paclitaxel (TXL, 1) through an ester, imine, amine, or amide bond. Newly synthesized conjugates were evaluated for cytotoxic activity against replication of several human tumor cell lines. Among them, TXL-CPT conjugates, 8-10, were more potent than TXL itself against the human prostate carcinoma cell line PC-3 (ED(50)=14.8, 3.1, 19.4nM compared with 55.5nM), and conjugate 10 was also 8-fold more active than TXL against the LN-CAP prostate cancer cell line. These compounds also possessed anti-angiogenesis ability as well as lower inhibitory effects against a normal cell line (MRC-5). Thus, conjugates 8-10 are possible antitumor drug candidates, particularly for prostate cancer.     

Multivalent poly(ethylene glycol)-containing conjugates for in vivo antibody suppression

Poly(ethylene glycol) (PEG) was incorporated into multivalent conjugates of the N-terminal domain of beta(2)GPI (domain 1). PEG was incorporated to reduce the rate of elimination of the conjugates from plasma and to putatively improve their efficacy as toleragens for the suppression of anti-beta(2)GPI antibodies and the treatment of antiphospholipid syndrome (APS). Three structurally distinct types of multivalent platforms were constructed by incorporating PEG into the platform structures in different ways. The amount of PEG incorporated ranged from about 5000 g per mole to about 30000 g per mole. The platforms were functionalized with either four or eight aminooxy groups. The conjugates were prepared by forming oxime linkages between the aminooxy groups and N-terminally glyoxylated domain 1 polypeptide. The plasma half-life of each conjugate, labeled with (125)I, was measured in both mice and rats. The half-lives of the conjugates ranged from less than 10 min to about 1 h in mice, and from less than 3 h to about 19 h in rats. The ability of five tetravalent conjugates to suppress anti-domain 1 antibodies in immunized rats was also measured. Incorporation of PEG in the conjugates significantly reduced the doses required for suppression, and the amount of reduction correlated with the amount of PEG incorporated.     

Conjugation of DM1 to anti-CD30 antibody has potential antitumor activity in CD30-positive hematological malignancies with lower systemic toxicity

ABSTRACT

An anti-CD30 antibody-drug conjugate incorporating the antimitotic agent DM1 and a stable SMCC linker, anti-CD30-MCC-DM1, was generated as a new antitumor drug candidate for CD30-positive hematological malignancies. Here, the in vitro and in vivo pharmacologic activities of anti-CD30-MCC-DM1 (also known as F0002-ADC) were evaluated and compared with ADCETRIS (brentuximab vedotin). Pharmacokinetics (PK) and the safety profiles in cynomolgus monkeys were assessed. Anti-CD30-MCC-DM1 was effective in in vitro cell death assays using CD30-positive lymphoma cell lines. We studied the properties of anti-CD30-MCC-DM1, including binding, internalization, drug release and actions. Unlike ADCETRIS, anti-CD30-MCC-DM1 did not cause a bystander effect in this study. In vivo, anti-CD30-MCC-DM1 was found to be capable of inducing tumor regression in subcutaneous inoculation of Karpas 299 (anaplastic large cell lymphoma), HH (cutaneous T-cell lymphoma) and L428 (Hodgkin’s disease) cell models. The half-lives of 4 mg/kg and 12 mg/kg anti-CD30-MCC-DM1 were about 5 days in cynomolgus monkeys, and the tolerated dose was 30 mg/kg in non-human primates, supporting the tolerance of anti-CD30-MCC-DM1 in humans. These results suggest that anti-CD30-MCC-DM1 presents efficacy, safety and PK profiles that support its use as a valuable treatment for CD30-positive hematological malignancies.     

尼罗红示踪纳米制剂在荷瘤裸鼠体内活体成像的应用

目的拟将尼罗红包裹入纳米制剂中,利用其荧光特性来考察纳米制剂在荷瘤鼠体内的肿瘤靶向性与组织分布情况。方法采用CCK-8法检测尼罗红纳米乳[NRNE(O)]对H1688的细胞毒性。建立BALB/c裸鼠肺癌模型,灌胃给予尼罗红混悬液(NRS)及NRNE(O),通过小动物活体成像系统观察其在荷瘤裸鼠中荧光强度的动态分布情况。结果 NRNE(O)具有较低的细胞毒性和良好的生物相容性,并且在体内的吸收量及肿瘤部位的蓄积量均高于NRS;NR在纳米制剂中能快速、稳定、清晰地反映纳米制剂在小鼠体内的动态分布情况。结论本研究中制备的NRNE(O)的稳定性好,为尼罗红作为示踪剂应用于纳米制剂在动物体内的研究提供了重要的研究手段。     

Selective in vivo antitumor effects of monoclonal anti-I-A antibody on B cell lymphoma

In a study of the biologic consequences of using monoclonal antibodies (mAb) with specificity for I-A for the elimination of an I-A-bearing B cell lymphoma, it was found that, despite the presence of I-A on a number of normal cell types and the propensity of anti-I-A to induce modulation of I-A and I-E on normal cells in vivo, a substantial effect on lymphoma growth could be measured in mAb-treated hosts. Unlike I-A on normal cells, tumor I-A failed to modulate in vivo, and 50% of animals could be cured of lymphoma by multiple doses of anti-I-A mAb. With a sensitive spleen tumor colonization assay, it was shown that neither T lymphocytes nor natural killer cells were involved in tumor elimination by anti-I-A mAb. In addition, C3 depletion only minimally affected the ability of anti-I-A to inhibit tumor growth, suggesting that complement-dependent lysis of tumor cells was not a major mechanism. Spleen cells from long term survivors of tumor challenge and mAb treatment functioned normally as antigen-presenting cells and in the recognition of alloantigens, and serum Ig levels were somewhat higher than in untreated mice; thus, such therapy can be carried out without compromising the immune reactivity of long term survivors.     

Antibacterial and mutagenic activities of new isothiocyanate derivatives

Nine newly synthetized isothiocyanate derivatives were demonstrated to posses antibacterial and genotoxic activitiesin vitro. 4-Hydroxybutyl isothiocyanate exhibited a broad antibacterial effect, with MIC values of 762 μmol/L forStaphylococcus aureus andEscherichia coli. Ethyl 4-methylsulfoxidobutanoate had the highest antibacterial activity in Gram-positive bacteria, the MIC value being 425 μmol/L forS. aureus. The highest tested concentrations of ethyl 4-isothiocyanatobutanoate and 4-hydroxybutyl isothiocyanate produced a bacteriocidal effect in Gram-positive bacteria. The compounds showed no mutagenic effects onSalmonella typhimurium tester strains TA 98 and TA 100, either in the absence or in the presence of a metabolically active microsomal S9 fraction from rat liver using standard Ames test.     

Synthesis and antitumor activity of peptide-paclitaxel conjugates.

Paclitaxel (Pac) is the most important anticancer drug used mainly in treatment of breast, lung, and ovarian cancer and is being investigated for use as a single agent for treatment of lung cancer, advanced head and neck cancers, and adenocarcinomas of the upper gastrointestinal tract. In this work, we present the synthesis of five 2'-paclitaxel-substituted analogs in which paclitaxel was covalently bound to peptides or as multiple copies to synthetic carriers. Ac-Cys(CH(2)CO-2'-Pac)-Arg-Gly-Asp-Arg-NH(2), Folyl-Cys(CH(2)CO-2'-Pac)-Arg-Gly-Asp-Ser-NH(2), Ac-[Lys-Aib-Cys(CH(2)CO-2'-Pac)](2)-NH(2), Ac-[Lys-Aib-Cys(CH(2)CO-2'-Pac)](3)-NH(2) and Ac-[Lys-Aib-Cys(CH(2)CO-2'-Pac)](4)-NH(2) were synthesized using 2'-halogeno-acetylated paclitaxel derivatives. Paclitaxel conjugates showed greater solubility in water than paclitaxel and inhibited the proliferation of human breast, prostate, and cervical cancer cell lines. Although all synthesized compounds had an antiproliferative activity, the Ac-[Lys-Aib-Cys(CH(2)CO-2'-Pac)](4)-NH(2) derivative showed improved biological activity in comparison with paclitaxel in cervical and prostate human cancer cells.     

In vivo andin vitro antitumor activity of mitomycin C conjugates at 7-N position through a linker containing thiocarbamate bond with CD10 monoclonal antibody

Through a linker containing thiocarbomate bound to the 7-N position of mitomycin C (MMC), conjugates with a monoclonal antibody to CD10 (NL-1) were prepared, and their antitumor activities were examined. All five conjugates, except one, showedin vitro cytotoxity to two CD10⁺ lymphoid cell lines superior to MMC. The conjugate displaying the highest cytotoxicity was selected and further tested against three CD10⁺ and two CD10 lymphoid cell linesin vitro. The conjugate with NL-1 antibody demonstrated higher cytotoxic activity against CD10+ tumor cells than the control conjugate with normal immunoglobulin, while there was no significant difference, when tested against CD10^– tumors. The cytotoxic activity of the NL-1 conjugate to CD10+ tumors was significantly blocked by NL-1 antibody. In vivo antitumor activity of the NL-1 conjugate was then tested against a CD10⁺ tumor transplanted to nude mice, and side effects were recorded. The NL-1 conjugate (4 mg/kg) showed anin vivo antitumor effect similar to MMC (2 mg/kg), which is at nearly maximal tolerable dose; the latter induced decreases in numbers of leukocytes and platelets, while the former did not, suggesting less side effect by the NL-1 conjugate. Since MMC demonstrates a broad spectrum of antitumor activity, the conjugate, as such, may be applicable for the treatment of cancer patients.     

Titanium salan complexes displays strong antitumor properties in vitro and in vivo in mice

The anticancer activity of titanium complexes has been known since the groundbreaking studies of Köpf and Köpf-Maier on titanocen dichloride. Unfortunately, possibly due to their fast hydrolysis, derivatives of titanocen dichloride failed in clinical studies. Recently, the new family of titanium salan complexes containing tetradentate ONNO ligands with anti-cancer properties has been discovered. These salan complexes are much more stabile in aqueous media. In this study we describe the biological activity of two titanium salan complexes in a mouse model of cervical cancer. High efficiency of this promising complex family was demonstrated for the first time in vivo. From these data we conclude that titanium salan complexes display very strong antitumor properties exhibiting only minor side effects. Our results may influence the chemotherapy with metallo therapeutics in the future.