Detection and metabolic investigations of a novel designer steroid: 3‐chloro‐17α‐methyl‐5α‐androstan‐17β‐ol

In 2012, seized capsules containing white powder were analyzed to show the presence of unknown steroid‐related compounds. Subsequent gas chromatography–mass spectrometry (GC‐MS) and nuclear magnetic resonance (NMR) investigations identified a mixture of 3α‐ and 3β‐ isomers of the novel compound; 3‐chloro‐17α‐methyl‐5α‐androstan‐17β‐ol. Synthesis of authentic reference materials followed by comparison of NMR, GC‐MS and gas chromatography‐tandem mass spectrometry (GC‐MS/MS) data confirmed the finding of a new ‘designer’ steroid. Furthermore, in vitro androgen bioassays showed potent activity highlighting the potential for doping using this steroid. Due to the potential toxicity of the halogenated steroid, in vitro metabolic investigations of 3α‐chloro‐17α‐methyl‐5α‐androstan‐17β‐ol using equine and human S9 liver fractions were performed. For equine, GC‐MS/MS analysis identified the diagnostic 3α‐chloro‐17α‐methyl‐5α‐androstane‐16α,17β‐diol metabolite. For human, the 17α‐methyl‐5α‐androstane‐3α,17β‐diol metabolite was found. Results from these studies were used to verify the ability of GC‐MS/MS precursor‐ion scanning techniques to support untargeted detection strategies for designer steroids in anti‐doping analyses. Copyright © 2015 John Wiley & Sons, Ltd.     

The Anti‐tumor Effects of Androstene Steroids Exhibit a Strict Structure–Activity Relationship Dependent upon the Orientation of the Hydroxyl Group on Carbon‐17

Androstene steroids are metabolites of dehydroepiandrosterone and exist as androstene‐diols or ‐triols in α‐ and β‐epimeric forms based upon the placement of the hydroxyl groups relative to the plane of the Δ⁵cycloperhydrophenanthrene ring. 5‐Androstene‐3β,17β‐diol (3β,17β‐AED) functions to upregulate immunity and the addition of a third hydroxyl group at C‐7 in the α‐ or β‐orientation (3β,7α,17β‐AET and 3β,7β,17β‐AET, respectively) enhances the immunological activity of the molecule. In contrast, 5‐androstene‐3β,17α‐diol (3β,17α‐AED) possesses potent anti‐tumor activity. We synthesized a new androstene by adding a third hydroxyl group at C‐7 to make 5‐androstene‐3β,7α,17α‐triol (3β,7α,17α‐AET) and compared the anti‐tumor activity of this steroid to the four existing androstenes. The results showed that this modification reduced the activity of 3β,17α‐AED. The ranking of the anti‐tumor activities of these steroids and their IC50 on human glioblastoma and lymphoma cells was: 3β,17α‐AED (～10 μm ) > 3β,7α,17α‐AET (～30 μm ) >> 3β,7α,17β‐AET (～150 μm )> 3β,7β,17β‐AET (not achievable) ≥ 3β,17β‐AED (not achievable). 3β,17α‐AED and 3β,7α,17α‐AET induced autophagy in T98G glioblastoma cells and apoptosis in U937 lymphoma cells. These results indicate that the position of the hydroxyl group on C‐17 dictates the anti‐tumor activity of the androstenes and must be in the α‐configuration, demonstrating a strict structure–activity relationship.     

Discovery of Novel 2‐(piperidin‐4‐yl)‐1H‐benzo[d]imidazole Derivatives as Potential Anti‐Inflammatory Agents

A novel 2‐(piperidin‐4‐yl)‐1H‐benzo[d]imidazole derivative 5 with good anti‐inflammatory activity was identified from our in‐house library. Based on hit compound 5 , two series of 2‐(piperidin‐4‐yl)‐1H‐benzo[d]imidazole derivative 6a – g and 7a – h were designed and synthesized as novel anti‐inflammatory agents. Most of synthesized compounds exhibited good inhibitory activity on NO and TNF‐α production in LPS‐stimulated RAW 264.7 macrophages, in which the compound 6e showed most potent inhibitory activity on NO (IC₅₀ = 0.86 μm ) and TNF‐α (IC₅₀ = 1.87 μm ) production. Further evaluation revealed that compound 6e displayed more potent in vivo anti‐inflammatory activity than ibuprofen did on xylene‐induced ear oedema in mice. Additionally, Western blot analysis revealed that compound 6e could restore phosphorylation level of IκBα and protein expression of p65 NF‐κB in LPS‐stimulated RAW 264.7 macrophages.     

Effect of bile acids on the binding of drugs and dyes to human albumin

Chenodeoxycholic, cholic, deoxycholic and taurodeoxycholic acids were found to inhibit the binding of 2-(4'-hydroxybenzeneazo)benzoic acid, methyl orange, sulphadimethoxine and warfarin to human albumin. In addition, glycodeoxycholic acid inhibited the binding of sulphadimethoxine and warfarin. In contrast, these bile acids did not inhibit the binding of phenylbutazone. The extent of displacement was in the rank order of: dihydroxy acids (chenodeoxycholic and deoxycholic) greater than trihydroxy acid (cholic) greater than conjugates (glycodeoxycholic and taurodeoxycholic). Thus the introduction of polar groups into the steroid nucleus of bile acids reduces their effectiveness as binding inhibitors. Displacement was usually accompanied by a decrease in the apparent association constant which suggests that the mechanism of displacement may be competitive.     

New oxymesterone metabolites in human by gas chromatography‐tandem mass spectrometry and their application for doping control

Oxymesterone (17α‐methyl‐4, 17β‐dihydroxy‐androst‐4‐ene‐3‐one) is one of the anabolic androgenic steroids (AAS) banned by the World Anti‐Doping Agency (WADA). The biotransformation of oxymesterone is performed in vitro by human heptocytes and human urinary metabolic profiles are investigated after single dose of 20 mg to two adult males as well. Cell cultures and urine samples were hydrolyzed by β‐glucuronidase, extracted, and reacted with N‐Methyl‐N‐trimethylsilyltrifluoroacetamide (MSTFA), ammonium iodide (NH₄I), and dithioerythritol. After derivatization, a gas chromatography triple quadruple tandem mass spectrometry (GC‐MS/MS) using full scan and MS/MS modes was applied. The total ion chromatographs of the blank and the positive samples are compared, and 7 new metabolites were found. In addition to the well‐known 17‐epioxymesterone, oxymesterone is metabolized by 4‐ene‐reduction, 3‐keto‐reduction, 11β‐hydroxylation, and 16ξ‐hydroxylation. Based on the behavior of the MS/MS results of product ion and precursor ion modes, a GC‐MS/MS method has been developed monitoring these metabolites. The structures of metabolite 2 and 4 are tentatively identified as 17α‐methyl‐3β, 17β‐dihydroxy‐5α‐androstane‐4‐one and 17α‐methyl‐3α, 4ξ, 17β‐trihydroxy‐5α‐androstane, respectively. Detection of oxymesterone using new metabolites M2 and M4 can extend the detection window up to 4 days since the parent steroid was not detectable. Copyright © 2015 John Wiley & Sons, Ltd.     

In vivo metabolism of the designer anabolic steroid hemapolin in the thoroughbred horse

Hemapolin (2α,3α‐epithio‐17α‐methyl‐5α‐androstan‐17β‐ol) is a designer steroid that is an ingredient in several “dietary” and “nutritional” supplements available online. As an unusual chemical modification to the steroid A‐ring could allow this compound to pass through antidoping screens undetected, the metabolism of hemapolin was investigated by an in vivo equine drug administration study coupled with GC‐MS analysis. Following administration of synthetically prepared hemapolin to a thoroughbred horse, madol (17α‐methyl‐5α‐androst‐2‐en‐17β‐ol), reduced and dihydroxylated madol (17α‐methyl‐5α‐androstane‐2β,3α,17β‐triol), and the isomeric enone metabolites 17β‐hydroxy‐17α‐methyl‐5α‐androst‐3‐en‐2‐one and 17β‐hydroxy‐17α‐methyl‐5α‐androst‐2‐en‐4‐one, were detected and confirmed in equine urine extracts by comparison with a library of synthetically derived reference materials. A number of additional madol derivatives derived from hydroxylation, dihydroxylation, and trihydroxylation were also detected but not fully identified by this approach. A yeast cell‐based androgen receptor bioassay of available reference materials showed that hemapolin and many of the metabolites identified by this study were potent activators of the equine androgen receptor. This study reveals the metabolites resulting from the equine administration of the androgen hemapolin that can be incorporated into routine GC‐MS antidoping screening and confirmation protocols to detect the illicit use of this agent in equine sports.     

Metabolic studies of 1‐testosterone in horses

1‐Testosterone (17β‐hydroxy‐5α‐androst‐1‐en‐3‐one), a synthetic anabolic steroid, has been described as one of the most effective muscle‐building supplements currently on the market. It has an anabolic potency of 200 as compared to 26 for testosterone. Apart from its abuse in human sports, it can also be a doping agent in racehorses. Metabolic studies on 1‐testosterone have only been reported for human in the early seventies, whereas little is known about its metabolic fate in horses. This paper describes the studies of in vitro and in vivo metabolism of 1‐testosterone in horses, with the aim of identifying the most appropriate target metabolites to be monitored for controlling the misuse or abuse of 1‐testosterone in racehorses. Six in vitro metabolites, namely 5α‐androst‐1‐ene‐3α,17β‐diol (T1a), 5α‐androstane‐3β,17β‐diol (T2), epiandrosterone (T3), 16,17‐dihydroxy‐5α‐androst‐1‐ene‐3‐one (T4 & T5), and 5α‐androst‐1‐ene‐3,17‐dione (T6), were identified. For the in vivo studies, two thoroughbred geldings were each administered orally with 800 mg of 1‐testosterone by stomach tubing. The results revealed that the parent drug and eight metabolites were detected in urine. Besides the four in vitro metabolites (T1a, T2, T3, and T5), four other urinary metabolites, namely 5α‐androst‐1‐ene‐3β,17α‐diol (T1b), 5α‐androst‐1‐ene‐3β,17β‐diol (T1c), 5α‐androstane‐3α,17α‐diol (T7) and 5α‐androstane‐3β,17α‐diol (T8) were identified. This study shows that the detection of 1‐testosterone administration is best achieved by monitoring the parent drug, which could be detected for up to 30 h post‐administration. Copyright © 2012 John Wiley & Sons, Ltd.     

3‐Acetyl‐11‐keto‐β‐boswellic acid loaded‐polymeric nanomicelles for topical anti‐inflammatory and anti‐arthritic activity

Objectives The aim of this study was to develop 3‐acetyl‐11‐keto‐β‐boswellic acid (AKBA)‐loaded polymeric nanomicelles for topical anti‐inflammatory and anti‐arthritic activity. Methods Polymeric nanomicelles of AKBA were developed by a radical polymerization method using N‐isopropylacrylamide, vinylpyrrolidone and acrylic acid. The polymeric nanomicelles obtained were characterized by Fourier transform infrared (FTIR), transmission electron microscopy (TEM) and dynamic light scattering (DLS). In‐vitro and in‐vivo evaluations of AKBA polymeric nanomicelles gel were carried out for enhanced skin permeability and anti‐inflammatory and anti‐arthritic activity. Key findings TEM and DLS results demonstrated that polymeric nanomicelles were spherical with a mean diameter approximately 45 nm. FTIR data indicated a weak interaction between polymer and AKBA in the encapsulated system. The release of drug in aqueous buffer (pH 7.4) from the polymeric nanomicelles was 23 and 55% after 2 and 8 h, respectively, indicating sustained release. In‐vitro skin permeation studies through excised abdominal skin indicated a threefold increase in skin permeability compared with AKBA gel containing the same amount of AKBA as the AKBA polymeric nanomicelles gel. The AKBA polymeric nanomicelle gel showed significantly enhanced anti‐inflammatory and anti‐arthritic activity compared with the AKBA gel. Conclusions This study suggested that AKBA polymeric nanomicelle gel significantly enhanced skin permeability, and anti‐inflammatory and anti‐arthritic activity.     

Reduced camptothecin sensitivity of estrogen receptor‐positive human breast cancer cells following exposure to di(2‐ethylhexyl)phthalate (DEHP) is associated with DNA methylation changes

Di(2‐ethylhexyl)phthalate (DEHP) has been considered as an estrogen receptor alpha (ERα) agonist due to its ability to interact with ERα and promote the cell proliferation of ERα‐positive breast cancer cells. The impact of DEHP on the chemical therapy in breast cancer is little known. Two breast cancer cell lines, MCF‐7 (ERα‐dependent) and MDA‐MB‐231 (ERα‐independent) were examined. We found that DEHP impaired the effectiveness of camptothecin (CPT) and alleviated the CPT‐induced formation of reactive oxygen species in ERα‐positive MCF‐7 cells, but not in ERα‐negative MDA‐MB‐231 cells. DEHP also significantly protected MCF‐7 cells against the genotoxicity of CPT. Genome‐wide DNA methylation profiling revealed that after 48 hours of exposure to 100 μM DEHP, MCF‐7 cells exhibited a significant change in their DNA methylation pattern, including hypermethylation of 700 genes and hypomethylation of 221 genes. The impaired therapeutic response to CPT in DEHP‐exposed MCF‐7 cells is probably mediated by epigenetic changes, especially through Wnt/β‐catenin signaling. A zebrafish xenograft model confirmed the disruptive effect of DEHP on CPT‐induced anti‐growth of MCF‐7 cells. In summary, DEHP exposure induces acquired CPT‐resistance in breast cancer cells and epigenetic changes associated with Wnt/β‐catenin signaling activation are probably depending on an ER‐positive status.     

Unambiguous identification and characterization of a long‐term human metabolite of dehydrochloromethyltestosterone

In doping control analysis, the characterization of urinary steroid metabolites is of high interest for a targeted and long‐term detection of prohibited anabolic androgenic steroids (AAS). In this work, the structure of a long‐term metabolite of dehydrochloromethyltestosterone (DHCMT) was elucidated. Altogether, 8 possible metabolites with a 17α‐methyl‐17β‐hydroxymethyl – structures were synthesized and compared to a major DHCMT long‐term metabolite detected in reference urine excretion samples. The confirmed structure of the metabolite was 4α‐chloro‐18‐nor‐17β‐hydroxymethyl‐17α‐methyl‐5α‐androst‐13‐en‐3α‐ol.     

Solidagenone from Solidago chilensis Meyen inhibits skin inflammation in experimental models

Solidagenone (SOL) is a labdane‐type diterpenoid found in Solidago chilensis, a plant traditionally used to treat skin diseases, kidney pain and ovarian inflammation. In this study, the topical anti‐inflammatory activity of SOL was evaluated using in vivo and in silico assays. Croton oil‐, arachidonic acid (AA)‐ and phenol‐induced ear oedema mouse models were applied in the in vivo studies. Myeloperoxidase (MPO) and N‐acetyl‐β‐D‐glucosaminidase (NAG) activities and tumour necrosis factor alpha (TNF‐α), interleukin‐6 (IL‐6) and nitric oxide (NO) levels were determined, as well as histopathological analyses were conducted. Interaction profiles between SOL and cyclooxygenase‐1 (COX‐1), cyclooxygenase‐2 (COX‐2), glucocorticoid receptor, estradiol‐17‐β‐dehydrogenase and prostaglandin‐E(2)‐9‐reductase were established using molecular docking. SOL significantly inhibited croton oil‐, AA‐ and phenol‐induced ear oedema (P < .001) at doses of 0.1, 0.5 and 1.0 mg/ear. The MPO and NAG activities and TNF‐α, IL‐6 and NO levels were decreased (P < .001). The histopathological data revealed that inflammatory parameters (oedema thickness, leucocyte infiltration and vasodilatation) were reduced by treatment with SOL at doses of 0.1, 0.5 and 1.0 mg/ear. The docking study showed that SOL interacts with COX‐1 and prostaglandin‐E(2)‐9‐reductase through hydrogen bonding, inhibiting these enzymes. These results indicate that SOL may be a promising compound for the treatment of cutaneous inflammatory disorders and has potential as a topical anti‐inflammatory agent.     

Investigation of anti‐inflammatory,nitric oxide donating,vasorelaxation and ulcerogenic activities of 1, 3‐diphenylprop‐2‐en‐1‐one derivatives in animal models

The main aim of this work is to find out novel chemical moieties with potent anti‐inflammatory and vasorelaxant activities with reduced gastric toxicities. For fulfilling the above aim, here we investigated novel chalcones (1, 3‐diphenylprop‐2‐en‐1‐one derivatives) with nitric oxide (NO) and hydrogen sulphide (H₂S) donating potency for anti‐inflammatory activity by carrageenan‐induced rat paw oedema. These molecules then further evaluated for in‐vitro NO‐releasing potency and vasorelaxation effect on isolated adult goat aortic tissue. The promising molecules were further screened for ulcerogenic activity in the rat model. The tested compounds produced % inhibition in paw oedema ranging from 29.16% to 79.69% and standard drug Diclofenac sodium produced 85.30% reduction in paw oedema after 5 hours. Out of this dataset, compounds AI1, AI7, Ca1, B2, B10, D2, and E8 showed 73.01%, 79.69%, 75.02%, 75.46%, 74.35%, 73.9% and 74.35% reduction in paw oedema respectively, which is approximately 80%–90% to that of standard Diclofenac sodium. The compound Ca1 was found to release 0.870 ± 0.025 mol/mol of NO and standard Glyceryl trinitrate (GTN) was found to release 0.983 ± 0.063 mol/mol of NO. The compound Ca1 produced 950.2 μmol/L of EC₅₀ whereas standard GTN produced 975.8 μmol/L of EC₅₀ for aortic smooth relaxation. The compounds Ca1 produced 0.1117 of ulcer index which is far less than that of standard Diclofenac sodium (1.148). The potent lead molecules were further evaluated to understand the mechanism of vasorelaxation by using specific antagonists or blockers of NO and H₂S.     

Isopropoxy‐Carvacrol,a Derivative Obtained from Carvacrol,Reduces Acute Inflammation and Nociception in Rodents

Monoterpenes, compounds mainly presented in essential oils, have important pharmacological actions. Isopropoxy‐carvacrol (IPC) is a derivative of the monoterpene carvacrol, and its pharmacological properties have not yet been investigated. The aim of this study was to analyse the acute anti‐inflammatory and antinociceptive properties of IPC. Mice (25–30 g) and rats (150–230 g) were pre‐treated (i.p.) with IPC at the doses of 10, 30 or 100 mg/kg or vehicle (Tween 80, 0.5%), 30 min. before injection of the phlogistic agents. Both the first and the second phases of formalin‐induced nociception were significantly reduced by IPC (100 mg/kg). Injection of carrageenan in mice paw reduced the threshold of stimulus intensity, applied with an analgesymeter, necessary to cause paw withdrawal, which was significantly reduced by 100 mg/kg of IPC. The area under curve (0–4 hr) of rat paw oedema induced by injection of carrageenan was also significantly diminished by the administration of IPC (100 mg/kg). Administration of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) markedly increased mice ear oedema and myeloperidase (MPO) activity. Topical co‐administration of IPC (0.3–3 mg/ear) during the induction did not affect TPA‐induced ear oedema, but significantly decreased MPO activity in the ears, when compared with the vehicle. In in vitro experiments, IPC reduced lipoperoxidation induced by different stimuli, showed nitric oxide scavenger activity and did not interfere with murine macrophage viability in concentrations up to 100 μg/mL. These results demonstrate that IPC exerts acute anti‐inflammatory and antinociceptive activities, suggesting that it may represent an alternative in the development of new future therapeutic strategies.     

Radiosynthesis of [123I]N‐(3‐iodoprop‐(2E)‐enyl)‐2α‐(imino‐methyl)‐3β‐(3′,4′‐dichlorophenyl) nortropane as a potential SPET tracer for dopamine transporter

The radiosynthesis of a novel tropane derivative [¹²³I]KUC‐25019, [[¹²³I];N‐(3‐iodoprop‐(2E)‐enyl)‐2α‐(imino‐methyl)‐3β‐(3′,4′‐dichlorophenyl)nortropane], a potential inhibitor of the dopamine transporter is reported. The synthetic routes include the preparation of standard reference, the stannyl precursor and the ¹²³I‐labeling synthesis. The no‐carrier‐added ¹²³I‐labeling has about 20% yield, the specific activity of [¹²³I]KUC‐25019 is > 107 GBq/µmol and the radiochemical purity of [¹²³I] KUC‐25019 is >95%. Copyright © 2002 John Wiley & Sons, Ltd.     

α‐Lipoic acid prolongs survival and attenuates acute kidney injury in a rat model of sepsis

Acute kidney injury is a frequent and serious complication in patients with severe sepsis. α‐Lipoic acid (ALA), a naturally occurring dithiol compound, has been shown to possess anti‐inflammatory and anti‐oxidative properties. In the present study we investigated whether ALA could attenuate acute kidney injury and improve survival in a rat model of sepsis. Rats were subjected to caecal ligation and puncture (CLP) to induce sepsis. α‐Lipoic acid (200 mg/kg) was administered by oral gavage either immediately (early treatment) or 12 h after the surgical procedure (delayed treatment). Both early and delayed ALA treatment effectively prolonged survival, improved pathological damage in kidney tissues and reduced serum blood urea nitrogen and creatinine levels in CLP‐induced septic rats. Furthermore, early treatment with ALA markedly inhibited the release of tumour necrosis factor‐α, interleukin (IL)‐6 and IL‐1β into the serum and reduced mRNA and protein expression of inducible nitric oxide synthase and high mobility group box 1 in kidney tissues from CLP‐induced rats. Finally, CLP‐induced nuclear factor‐κB activation in kidney tissues was significantly suppressed by early ALA treatment. Together, the results indicate that ALA is able to reduce mortality and attenuate acute kidney injury associated with sepsis, possibly by anti‐inflammatory actions. α‐Lipoic acid may be a promising novel agent for the treatment of conditions associated with septic shock.     

Design and Synthesis of 2‐Phenoxynicotinic Acid Hydrazides as Anti‐inflammatory and Analgesic Agents

A series of 2‐phenoxynicotinic acid hydrazides were synthesized and evaluated for their analgesic and anti‐inflammatory activities. Several compounds having an unsubstituted phenyl/4‐pyridyl or C‐4 methoxy substituent on the terminal phenyl ring showed moderate to high analgesic or anti‐inflammatory activity in comparison to mefenamic acid as the reference drug. The compounds with highest anti‐inflammatory activity were subjected to in vitro COX‐1/COX‐2 inhibition assays and showed moderate to good COX‐1 and weak COX‐2 inhibition activities.     

Synthesis of New N‐Substituted 5‐Arylidene‐2,4‐thiazolidinediones as Anti‐Inflammatory and Antimicrobial Agents

A novel series of 5‐arylidene ‐ 2,4‐thiazolidinediones (TZDs) 2a – p was synthesized from the condensation of 3‐((2‐phenylthiazol‐4‐yl)methyl)thiazolidine‐2,4‐dione with different benzaldehyde derivatives. All the structures were confirmed by their spectral (IR, ¹H NMR, ¹³C NMR and mass) and elemental analytical data. The new molecules were evaluated in vivo as anti‐inflammatory agents in an acute experimental inflammation, evaluating the acute phase bone marrow response and phagocyte activity. All compounds, excepting one, reduced the absolute leukocytes count due to the lower neutrophil percentage. Phagocytary index was decreased by the same molecules, while only half of them reduced the phagocytary activity. The effect was superior to meloxicam, the reference anti‐inflammatory drug, for the majority of the TZD derivatives. The new molecules were also investigated for their antimicrobial properties on Gram‐positive and Gram‐negative bacteria and one fungal strain. Two compounds ( 2e and 2n ) manifested growth inhibition capacity on all the tested strains.     

Enhanced synergistic anti‐Lewis lung carcinoma effect of a DNA vaccine harboring a MUC1‐VEGFR2 fusion gene used with GM‐CSF as an adjuvant

In order to achieve a synergistic effect on anti‐tumour and anti‐angiogenesis activity, we designed and constructed a DNA vaccine that expresses MUC1and VEGFR2 in the same reading frame. The aim of this study was to investigate the anti‐tumour activity of this DNA vaccine. Furthermore, we also investigated the enhanced synergistic anti‐Lewis lung carcinoma effect of this DNA vaccine by using GM‐CSF as an adjuvant. A series of DNA plasmids encoding MUC1, VEGFR2, GM‐CSF, and their conjugates were constructed and injected into mice intramuscularly (i.m.) followed by an electric pulse. The humoral and cellular immune responses after immunization were detected by enzyme‐linked immunosorbent assay (ELISA) and enzyme‐linked immunospot (ELISPOT), respectively. To evaluate the anti‐tumour efficacy of these plasmids, murine models with MUC1‐expressing tumours were generated. After injection into the tumour‐bearing mouse model, the plasmid carrying the fusion gene of MUC1 and VEGFR2 showed stronger inhibition of tumour growth than the plasmid expressing MUC1 or VEGFR2 alone, which indicated that MUC1 and VEGFR2 could exert a synergistic anti‐tumour effect. Furthermore, mice vaccinated with the combination of the GM‐CSF expressing plasmid and the plasmid carrying the fusion gene of MUC1 and VEGFR2 showed an increased inhibition in the growth of MUC1‐expressing tumours and prolonged mouse survival. These observations emphasize the potential of the synergistic anti‐tumour and anti‐angiogenesis strategy used in DNA vaccines, and the potential of the GM‐CSF gene as an adjuvant for DNA vaccines, which could represent a promising approach for tumour immunotherapy.