Isozyme-specific histone deacetylase 1/2 inhibitor K560 attenuates oxidative stress-induced retinal cell death.

Oxidative stress-induced damage is an underlying mechanism in the pathogenesis of age-related retinal diseases. Here, we examined the effects of K560, a potential candidate drug for the treatment of these diseases, on oxidative stress-induced retinal cell death. K560 is a novel isozyme-specific inhibitor of histone deacetylase 1 and 2 (HDAC1/2). Histone acetylation in retinal lysates and dissociated retinal cells was detected with a western blot analysis and cell-based enzyme-linked immunosorbent assay (ELISA), respectively. The viability of mouse retinal cells was measured with an alamarBlue assay. We used immunohistochemistry for RNA binding protein with multiple splicing (RBPMS) to visualize the retinal ganglion cells (RGCs) of mice. An ELISA analysis indicated that histone acetylation was enhanced in dissociated mouse retinal cells treated with K560. The cell viability assay indicated that K560 attenuated both exogenous hydrogen peroxide-induced and endogenous oxidative stress-induced cell death in dissociated retinal cells. Western blot analysis indicated that intravitreal K560 administration enhanced the acetylation of histones H3 and H4 in mouse retinal lysates. To examine the effect of K560 on oxidative stress-induced RGC death, we performed whole-mount immunohistochemistry for RBPMS on retinas dissected from eyes treated with K560 or vehicle on day one, and K560 or vehicle and NMDA on day two. Quantification of RBPMS-immunopositive cells indicated that K560 attenuated NMDA-induced RGC death. Taken together, our findings suggest that administration of a HDAC1/2-specific inhibitor K560 may be effective in the treatment of oxidative stress-mediated retinal degeneration and have less cytotoxicity than other known HDAC inhibitors, which are known to target a wide range of HDAC family members.

MDMX elevation by a novel Mdmx-p53 interaction inhibitor mitigates neuronal damage after ischemic stroke.

Mdmx and Mdm2 are two major suppressor factors for the tumor suppressor gene p53. In central nervous system, Mdmx suppresses the transcriptional activity of p53 and enhances the binding of Mdm2 to p53 for degradation. But Mdmx dynamics in cerebral infarction remained obscure. Here we investigated the role of Mdmx under ischemic conditions and evaluated the effects of our developed small-molecule Protein-Protein Interaction (PPI) inhibitors, K-181, on Mdmx-p53 interactions in vivo and in vitro. We found ischemic stroke decreased Mdmx expression with increased phosphorylation of Mdmx Serine 367, while Mdmx overexpression by AAV-Mdmx showed a neuroprotective effect on neurons. The PPI inhibitor, K-181 attenuated the neurological deficits by increasing Mdmx expression in post-stroke mice brain. Additionally, K-181 selectively inhibited HDAC6 activity and enhanced tubulin acetylation. Our findings clarified the dynamics of Mdmx in cerebral ischemia and provide a clue for the future pharmaceutic development of ischemic stroke.

[Isoform Selectivity of HDAC Inhibitors Has a Significant Effect on PD-L1 Expression in the Triple-negative Cancer Cell Line MDA-MB-231].

Various reports have been published in recent years on the effects of histone deacetylase (HDAC) inhibitors on programmed death ligand 1 (PD-L1) expression in cancer cells. The combination therapy of immune checkpoint inhibitors and HDAC inhibitors utilizing these effects has attracted attention as a new clinical treatment of triple-negative breast cancers. We investigated how the expression level of PD-L1 changes depending on the type of HDAC inhibitor exposed to triple-negative breast cancer cell line MDA-MB-231. We found that the mRNA expression level of PD-L1 was significantly decreased by Vorinostat and K-32 (pan-HDAC inhibitors) at high concentrations exhibiting low cell viability, while it was increased by high concentrations of K-560 (HDAC1,2 inhibitor) and Entinostat (HDAC1,3 inhibitor). On the other hand, the mRNA level of PD-L1 was increased by all of these HDAC inhibitors at low concentrations showing high cell viability. Of particular note, K-32 induced more PD-L1 mRNA than all the other HDAC inhibitors at the lowest concentration of 0.5 µM. This finding might suggest the usefulness of pan-HDAC inhibitors in clinical treatment in combination with immune checkpoint inhibitors.

New 5-Aryl-Substituted 2-Aminobenzamide-Type HDAC Inhibitors with a Diketopiperazine Group and Their Ameliorating Effects on Ischemia-Induced Neuronal Cell Death.

We previously synthesized new 5-thienyl-substituted 2-aminobenzamide-type HDAC1, 2 inhibitors with the (4-ethyl-2,3-dioxopiperazine-1-carboxamido) methyl group. K-560 (1a) protected against neuronal cell death in a Parkinson's disease model by up-regulating the expression of XIAP. This finding prompted us to design new K-560-related compounds. We examined the structure activity relationship (SAR) for the neuronal protective effects of newly synthesized and known K-560 derivatives after cerebral ischemia. Among them, K-856 (8), containing the (4-methyl-2,5-dioxopiperazin-1-yl) methyl group, exhibited a promising neuronal survival activity. The SAR study strongly suggested that the attachment of a monocyclic 2,3- or 2,5-diketopiperazine group to the 2-amino-5-aryl (but not 2-nitro-5-aryl) scaffold is necessary for K-560-related compounds to exert a potent neuroprotective effect.

Potential Application of 5-Aryl-Substituted 2-Aminobenzamide Type of HDAC1/2- Selective Inhibitors to Pharmaceuticals.

Diverse histone deacetylase (HDAC) inhibitors have been developed to date. They control not only histone modification but also gene expression of diverse proteins and thus are expected to provide useful therapeutic effects on various diseases, including cancers, psychiatric and cognitive disorders and neurodegenerative diseases, as well as cardiovascular and diabetic diseases. Some isoform-nonselective HDAC inhibitors have been successfully used for clinical treatments of the haematological malignancies, including advanced forms of cutaneous T-cell lymphoma, refractory peripheral T-cell lymphoma and multiple myeloma. However, the nonselective HDAC inhibitors threaten to cause adverse effects, such as thrombocytopenia, nausea, fatigue and cardiotoxicity, and their influence on the health care of patients is of concern. It is therefore anticipated that the development of isoform-selective HDAC inhibitors may offer more efficacy and less toxicity. Recently, a number of 5- aryl-substituted 2-aminobenzamide series of HDAC1/2-selective inhibitors have been synthesized, and their useful biological activities have been reported. In this review, we introduce the recent development of synthetic and biological studies on 5-aryl-substituted 2-aminobenzamide-type HDAC1/2 inhibitors, together with the latest research findings on biology of broad-spectrum HDAC inhibitors. In addition, we refer to the possibility of their application in clinical therapies.

Discovery of new low-molecular-weight p53-Mdmx disruptors and their anti-cancer activities.

Although several p53-Mdm2-binding disruptors have been identified to date, few studies have been published on p53-Mdmx-interaction inhibitors. In the present study, we demonstrated that o-aminothiophenol derivatives with molecular weights of 200-300 selectively inhibited the p53-Mdmx interaction. S-2-Isobutyramidophenyl 2-methylpropanethioate (K-178) (1c) activated p53, up-regulated the expression of its downstream genes such as p21 and Mdm2, and preferentially inhibited the growth of cancer cells with wild-type p53 over those with mutant p53. Furthermore, we found that the S-isobutyryl-deprotected forms 1b and 3b of 1c and S-2-benzamidophenyl 2-methylpropanethioate (K-181) (3c) preferentially inhibited the p53-Mdmx interaction over the p53-Mdm2 interaction, respectively, by using a Flag-p53 and glutathione S-transferase (GST)-fused protein complex (Mdm2, Mdmx, DAPK1, or PPID). In addition, the interaction of p53 with Mdmx was lost by replacing a sulfur atom with an oxygen atom in 1b and 1c. These results suggest that sulfides such as 1b, 3b, 4b, and 5b interfere with the binding of p53-Mdmx, resulting in the dissociation of the two proteins. Furthermore, the results of oral administration experiments using xenografts in nude mice indicated that 1c reduced the volume of tumor masses to 49.0% and 36.6% that of the control at 100 mg/kg and 150 mg/kg, respectively, in 40 days.

A novel histone deacetylase 1 and 2 isoform-specific inhibitor alleviates experimental Parkinson's disease.

With increased histone deacetylase (HDAC) activity and histone hypoacetylation being implicated in neurodegeneration, HDAC inhibitors have been reported to have considerable therapeutic potential. Yet, existing inhibitors lack specificity and may show substantial adverse effect. In this study, we identified a novel HDAC1/2 isoform-specific inhibitor, K560, with protective effects against 1-methyl-4-phenylpyridinium (MPP(+))- and/or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neuronal death in both in vitro and in vivo Parkinson's disease model. K560 attenuated cell death induced by MPP(+) in differentiated SH-SY5Y cells through the sustained expression of an antiapoptotic protein, X-linked inhibitor of apoptosis (XIAP). Inhibition of XIAP expression by locked nucleic acid antisense oligonucleotides abolished the protective effect of K560. Inactivation of mitogen-activated protein kinase cascades, reduced p53 phosphorylation, and down-regulation of p53-upregulated modulator of apoptosis on K560 treatment were also observed. Furthermore, pre- and post-oral administration of K560 to mice prevented MPTP-induced loss of dopaminergic neurons in substantia nigra, suggesting that selective inhibition of HDAC1 and HDAC2 by K560 may pave the way to new strategies for Parkinson's disease treatment.

Influence of the carbamate fungicide benomyl on the gene expression and activity of aromatase in the human breast carcinoma cell line MCF-7.

The carbamate fungicide benomyl reportedly inhibited the growth of the human breast cancer cell line MCF-7 by inducing apoptosis. However, influence of benomyl on the expression and activity of aromatase of MCF-7 cells remains to be examined, since benomyl was identified as an endocrine disruptor. We here confirmed through cell cycle analysis and immunofluorescence staining that benomyl damaged microtubules and caused apoptosis. We also found that benomyl inhibited histone deacetylase (HDAC) 1 and accumulated acetylated histone H3 in MCF-7 cells. Additionally, benomyl enhanced the levels of aromatase protein and mRNA, albeit at high concentrations. It is thus likely that benomyl enhanced the promoter activity of the aromatase gene via acetylation of histone H3 as does the HDAC inhibitor Vorinostat. In conclusion, benomyl remains to be a risk factor as an endocrine disruptor for breast cancer.

Synergistic antitumor effect of a combination of paclitaxel and carboplatin with nobiletin from Citrus depressa on non-small-cell lung cancer cell lines.

Non-small-cell lung carcinomas do not sufficiently respond to cancer chemotherapeutic drugs. Combination effects of cancer chemotherapy drugs (paclitaxel and carboplatin) with nobiletin or powdered Shiikuwasha extract from Citrus depressa were examined by isobologram and combination index analyses. It was demonstrated that the combination generated a synergistic inhibitory effect against the proliferation of the human non-small-cell lung carcinoma cell lines A549 and H460 and that of the two chemotherapy drugs, paclitaxel was responsible for this synergistic effect. Furthermore, the percentage of apoptotic cells was decreased with increasing rates of nobiletin to paclitaxel and carboplatin. These findings were considered to be attributed to the ability of nobiletin to regulate cells in the G1 phase, which escaped cell death initiated by paclitaxel and carboplatin. An antitumor activity assay showed that this combination significantly suppressed the growth of subcutaneous A549 tumor xenografts in nude mice.

Blockade of the ERK pathway enhances the therapeutic efficacy of the histone deacetylase inhibitor MS-275 in human tumor xenograft models.

The ERK pathway is up-regulated in various human cancers and represents a prime target for mechanism-based approaches to cancer treatment. Specific blockade of the ERK pathway alone induces mostly cytostatic rather than pro-apoptotic effects, however, resulting in a limited therapeutic efficacy of the ERK kinase (MEK) inhibitors. We previously showed that MEK inhibitors markedly enhance the ability of histone deacetylase (HDAC) inhibitors to induce apoptosis in tumor cells with constitutive ERK pathway activation in vitro. To evaluate the therapeutic efficacy of such drug combinations, we administered the MEK inhibitor PD184352 or AZD6244 together with the HDAC inhibitor MS-275 in nude mice harboring HT-29 or H1650 xenografts. Co-administration of the MEK inhibitor markedly sensitized the human xenografts to MS-275 cytotoxicity. A dose of MS-275 that alone showed only moderate cytotoxicity thus suppressed the growth of tumor xenografts almost completely as well as induced a marked reduction in tumor cellularity when administered with PD184352 or AZD6244. The combination of the two types of inhibitor also induced marked oxidative stress, which appeared to result in DNA damage and massive cell death, specifically in the tumor xenografts. The enhanced therapeutic efficacy of the drug combination was achieved by a relatively transient blockade of the ERK pathway. Administration of both MEK and HDAC inhibitors represents a promising chemotherapeutic strategy with improved safety for cancer patients.

Induction of melanogenesis by 4'-O-methylated flavonoids in B16F10 melanoma cells.

Agents to control melanogenesis are in demand for the development of cosmetics to improve pigmentation disorders of skin and hair. In this study, we examined and evaluated the effects of flavonoids on melanogenesis in the melanogenic cells model, murine B16F10 melanoma cells. In the course of this study, we found that incubation of the cells in a medium containing 10 µM of the 4'-O-methylated flavonoids, diosmetin (4'-O-methylluteolin), acacetin (4'-O-methylapigenin) or kaempferide (4'-O-methylkaempferol), increased the melanin contents of the cells 3- to 7-fold higher than the control cells. The concentration-dependence test revealed that 20 µM acacetin showed the highest effect, up to 33-fold higher than the vehicle. On the other hand, the corresponding 4'-OH-type flavonoids, luteolin, apigenin and kaempferol, had a significantly smaller effect. Furthermore, by evaluating the melanogenic proteins, we found that the cells treated with 4'-O-methylated flavonoids showed higher tyrosinase activity, as well as upregulation of tyrosinase expression, preceded by activation of cAMP response element binding protein (CREB) and extracellular signal-regulated kinases types 1 and 2 (ERK1/2). These results indicate that the 4'-O-methyl group of flavonoids plays an important role in the induction of melanogenesis by activating its major signal transduction pathway through the upregulation of phospho-CREB in murine B16F10 melanoma cells.

Anti-tumor activity of new orally bioavailable 2-amino-5-(thiophen-2-yl)benzamide-series histone deacetylase inhibitors, possessing an aqueous soluble functional group as a surface recognition domain.

New orally bioavailable 5-(thiophen-2-yl)-substituted 2-aminobenzamide-series histone deacetylase inhibitors were synthesized. These compounds possess a morpholine or piperadine-derived moiety as an aqueous soluble functional group. Among them, 8b, having a 4-ethyl-2,3-dioxopiperazine-1-carboxamide group as a surface recognition domain, showed promising inhibitory activities against HCT116 cell growth and HDAC1/2. Notably, unlike MS-275, this compound did not induce apoptosis in the cell cycle tests. We therefore conducted antitumor tests of 8b and MS-275 against HCT116 cell xenografts in nude mice. Compound 8b reduced the volume of tumor mass to T/C: 60% and 47% at 45 and 80mg/kg over 16days, respectively. These values were comparable to the rate (T/C: 51% at 45mg/kg) for MS-275. Furthermore, 8b, at neither 45 nor 80mg/kg, induced the weight loss which was observed in the mice given MS-275 at 45mg/kg.

Antitumor effect of liposomal histone deacetylase inhibitor-lipid conjugates in vitro.

Histone deacetylase inhibitor (HDACI), suberoylanilide hydroxamic acid (SAHA), approved by the Food and Drug Administration (FDA) for the treatment of cutaneous T cell lymphoma, is a promising new treatment strategy for various cancers. In this study, we hypothesized that a liposomal formulation of HDACI might efficiently deliver HDACI into tumors. To incorporate HDACI efficiently into the liposomal membrane, we synthesized six HDACI-lipid conjugates, in which polyethylene glycol(2000) (PEG(2000))-lipid or cholesterol (Chol) was linked with a potent hydroxamic acid, HDACI, SAHA or K-182, by cleavable linkers, such as ester, carbamide and disulfide bonds. Liposomal HDACI-lipid conjugates were prepared with distearoylphosphatidylcholine (DSPC) and HDACI-Chol conjugate or with DSPC, Chol and HDACI-PEG-lipid conjugates, and their cytotoxicities were evaluated for human cervix tumor HeLa and mouse colon tumor Colon 26 cells. Among the liposomes, liposomal oleyl-PEG(2000)-SAHA conjugated with SAHA and oleyl-PEG(2000) via a carbamate linker showed higher cytotoxicity via hyperacetylation of histone H3 and induction of caspase 3/7 activity. These results suggested that liposomal HDACI-lipid conjugates may be a potential tool for cancer therapy.

A potent inhibitor of SIK2, 3, 3', 7-trihydroxy-4'-methoxyflavon (4'-O-methylfisetin), promotes melanogenesis in B16F10 melanoma cells.

Flavonoids, which are plant polyphenols, are now widely used in supplements and cosmetics. Here, we report that 4'-methylflavonoids are potent inducers of melanogenesis in B16F10 melanoma cells and in mice. We recently identified salt inducible kinase 2 (SIK2) as an inhibitor of melanogenesis via the suppression of the cAMP-response element binding protein (CREB)-specific coactivator 1 (TORC1). Using an in vitro kinase assay targeting SIK2, we identified fisetin as a candidate inhibitor, possibly being capable of promoting melanogenesis. However, fisetin neither inhibited the CREB-inhibitory activity of SIK2 nor promoted melanogenesis in B16F10 melanoma cells. Conversely, mono-methyl-flavonoids, such as diosmetin (4'-O-metlylluteolin), efficiently inhibited SIK2 and promoted melanogenesis in this cell line. The cAMP-CREB system is impaired in A(y)/a mice and these mice have yellow hair as a result of pheomelanogenesis, while Sik2(+/-); A(y)/a mice also have yellow hair, but activate eumelanogenesis when they are exposed to CREB stimulators. Feeding Sik2(+/-); A(y)/a mice with diets supplemented with fisetin resulted in their hair color changing to brown, and metabolite analysis suggested the presence of mono-methylfisetin in their feces. Thus, we decided to synthesize 4'-O-methylfisetin (4'MF) and found that 4'MF strongly induced melanogenesis in B16F10 melanoma cells, which was accompanied by the nuclear translocation of TORC1, and the 4'-O-methylfisetin-induced melanogenic programs were inhibited by the overexpression of dominant negative TORC1. In conclusion, compounds that modulate SIK2 cascades are helpful to regulate melanogenesis via TORC1 without affecting cAMP levels, and the combined analysis of Sik2(+/-) mice and metabolites from these mice is an effective strategy to identify beneficial compounds to regulate CREB activity in vivo.

New microtubule polymerization inhibitors comprising a nitrooxymethylphenyl group.

We have designed cancer antiproliferative compounds, starting from aniline or phenol derivative, which comprise one or two nitrooxymethylphenyl groups as do the hybrid drugs NCX4040 and NCX530. Compound 2a with p-nitrooxymethylbenzoyl-oxy and -amino groups as well as 8a with a p-nitrooxymethylbenzoylamino group showed more promising effects than NCX4040 against human colon and breast cancer cells. Since 2a and 8a, but not NCX4040, arrested human colon carcinoma HCT116 cells in the M phase, the former two compounds may inhibit cell growth differently from NCX4040. Merged images of immunofluorescence-stained α-tubulin and Hoechst-stained nuclei in human fibrosarcoma HT1080 cells showed that 2a and 8a disrupted microtubule formation just as did vincristine, the tubulin polymerization inhibitor. In experiments in vivo, the intraperitoneal administration of 8a at 80 mg/kg/day reduced the growth of HCT116 xenografts in nude mice to T/C 55%.

Therapeutic effect of arctiin and arctigenin in immunocompetent and immunocompromised mice infected with influenza A virus.

Arctiin and its aglucone, arctigenin from the fruits of Arctium lappa L. showed potent in vitro antiviral activities against influenza A virus (A/NWS/33, H1N1) (IFV). Based on the data from time-of-addition experiments and on release tests of progeny viruses, arctigenin was assumed to interfere with early event(s) of viral replication after viral penetration into cells, and to suppress the release of progeny viruses from the host cells. Arctiin was orally effective against either IFV-inoculated normal or 5-fluorouracil (5-FU)-treated mice, being less effective as compared with oseltamivir. Noticeably, arctiin produced a larger amount of virus-specific antibody than those of control and oseltamivir in sera collected from 5-FU-treated mice. Furthermore, oral treatment of 5-FU-treated mice with arctiin did not induce any resistant viruses, although the same treatment with oseltamivir induced resistant viruses at a 50% frequency. When the combination of arctiin and oseltamivir was administered to normal mice infected with IFV, the virus yields in both bronchoalveolar lavage fluids and lungs were significantly reduced relative to those in the mice treated with arctiin or oseltamivir alone. Thus, monotherapy of arctiin or combined therapy of arctiin with oseltamivir would be another treatment option for influenza.

New orally bioavailable 2-aminobenzamide-type histone deacetylase inhibitor possessing a (2-hydroxyethyl)(4-(thiophen-2-yl)benzyl)amino group.

New 2-aminobenzamide-type histone deacetylase (HDAC) inhibitors were synthesized. They feature a sulfur-containing bicyclic arylmethyl moiety-a surface recognition domain introduced to increase in cellular uptake-and a substituted tert-amino group which affects physicochemical properties such as aqueous solubility. Compound 22 with a (2-hydroxyethyl)(4-(thiophen-2-yl)benzyl)amino group reduced the volume of human colon cancer HCT116 xenografts in nude mice to T/C 67% by oral administration at 45mg/kg, which was comparable to the rate (T/C 62%) for a positive control, MS-275. Western blot analyses as well as cell cycle and TUNEL assays by flow cytometry suggested that the two compounds inhibited the growth of cancer cells via similar mechanisms.

Histone deacetylase inhibitor prodrugs in nanoparticle vector enhanced gene expression in human cancer cells.

We developed histone deacetylase inhibitor (HDACI) prodrugs to enhance the expression of the external genes transfected into human cells with cationic nanoparticles (NPs). We synthesized five kinds of lipid-linked HDACI prodrugs in which n-dodecanoic acid or cholesterol is linked with a potent HDACI, K-182, by an ester bond or a disulfide carbonate linker. The prodrugs were able to admix as a component of NPs, although the intact K-182 was not incorporated into NPs. Namely, NPs composed of cholesteryl-3beta-carboxyamidoethylene-N-hydroxyethylamine and Tween 80 with the 10 mol% K-182 prodrug were prepared as a DNA vector to transfect plasmid DNAs into human prostate cancer cells, PC-3, or human breast cancer cells, Sk-Br-3. The NPs containing K-182 prodrugs with n-dodecanoic acid exhibited two to four times higher the gene expression than the original NPs. The enhancement of the gene expression will be due to the hyperacetylation of core histones caused by intact K-182 degraded from the prodrug in the vector incorporated into the cells.

Inhibitory effect of (-)-epigallocatechin and (-)-epigallocatechin gallate against heregulin beta1-induced migration/invasion of the MCF-7 breast carcinoma cell line.

Migration/invasion is involved in the multiple steps of metastasis, resulting in a poor prognosis of breast cancer. (-)-Epigallocatechin-3-gallate (EGCG) in green tea inhibits the metastasis of some cancer cell lines. Cell migration/invasion assays using Boyden chambers demonstrated that (-)-epigallocatechin (EGC), another green tea catechin, inhibited heregulin-beta1 (HRG)-induced migration/invasion of MCF-7 human breast carcinoma cells to approximately the same extent as EGCG. Assays of cytoskeletal reorganization, Western blotting and immunoprecipitation suggested that EGCG inhibited this migration/invasion by suppressing the HRG-stimulated activation of epidermal growth factor receptor-related protein B2 (ErbB2)/ErbB3/protein kinase B (Akt), whereas EGC did so through pathways including the disruption of the HRG-stimulated activation of ErbB2/ErbB3 but not Akt. EGC, as well as EGCG, could play an important role against the promotion of metastasis of breast cancer cells.

Attempt to detect natural anticancer compounds--protein binding through precursor ion scan and MS/MS measurements.

A 2'-succinyltaxol-bovine serum albumin (BSA) conjugate was prepared as an antigen to produce an anti-taxol monoclonal antibody by immunizing mice. Formation of a linkage between hapten and protein is usually confirmed by the UV or fluorescamine method. However, it was difficult to confirm the binding of 2'-succinyltaxol to BSA by these methods owing to the similar UV absorption maxima of 2'-succinyltaxol (273 nm) and BSA (280 nm). In the present study, we therefore conducted a mass spectrometric analysis using the precursor ion scan and MS/MS techniques to confirm the formulation of the 2'-succinyltaxol-BSA conjugate in the following way: The conjugate was subjected to thermal denaturalization, dithiothreitol (DTT)-reduction, iodoacetamide-alkylation and trypsin-digestion, affording a peptide fragment mixture. This was then analyzed by electrospray ionization (ESI)-MS in the positive mode by scanning the peaks containing a mass of 854 corresponding to taxol. The detected peaks were in turn subjected to MS/MS measurements. Among them, a peak at m/z 1247.4 was found to be a peptide fragment containing Lys (epsilon-2'-succinyltaxol), demonstrating the formulation of the 2'-succinyltaxol-BSA conjugate. In order to confirm the feasibility of this analytical method, the deacetylvinblastine (deacetylVLB)-BSA antigen which produced the anti-VLB monoclonal antibody (MAb-10-A9), was subjected to the same analytical treatment as above, giving a peak at m/z 851.3 originating from a Lys (epsilon-deacetylVLB). Thus, this new method could serve as an additional tool for confirmation of the formation of hapten-protein conjugates which are difficult to detect by the above spectrophotometric methods.