DNA cleavage and binding selectivity of a heterodinuclear Pt–Cu(3-Clip-Phen) complex

The synthesis and nuclease activity of a new bifunctional heterodinuclear platinum–copper complex are reported. The design of this ditopic coordination compound is based on the specific mode of action of each component, namely, cisplatin and Cu(3-Clip-Phen), where 3-Clip-Phen is 1-(1,10-phenanthrolin-3-yloxy)-3-(1,10-phenanthrolin-8-yloxy)propan-2-amine. Cisplatin is not only able to direct the Cu(3-Clip-Phen) part to the GG or AG site, but also acts as a kinetically inert DNA anchor. The nuclease activity of this complex has been investigated on supercoiled DNA. The dinuclear compound is not only more active than Cu(3-Clip-Phen), but is also capable of inducing direct double-strand breaks. The sequence selectivity of the mononuclear platinum complex has been investigated by primer extension experiments, which reveal that its interaction with DNA occurs at the same sites as for cisplatin. The Taq polymerase recognizes the resulting DNA damage as different from that for unmodified cisplatin. The sequence-selective cleavage has been investigated by high-resolution gel electrophoresis on a 36-bp DNA fragment. Sequence-selective cleavages are observed in the close proximity of the platinum sites for the strand exhibiting the preferential platinum binding sites. The platinum moiety also coordinates to the other DNA strand, most likely leading only to mono guanine or adenine adducts.
Introduction
DNA is a target for numerous antitumor drugs [1, 2]. Reversible or irreversible modifications of the nucleic acids can lead to disruption of the transcription and/or replication, initiating ultimately the death of cancer cells. Cisplatin [3] and bleomycin are among these efficient anticancer drugs [4]. The mechanism of action is different for both drug substances; cisplatin primarily induces distortions upon binding to DNA, whereas bleomycin is able to generate DNA strand scissions.
Since the discovery of bleomycin [5], numerous metal complexes have been synthesized that are able to produce DNA cleavage. Typical examples are iron(II)–edta complexes, manganese(III)–porphyrin and Cu, Co, Ru and Rh complexes with phenanthroline [6–9]. For instance, Cu(phen)2+ in the presence of dihydrogen peroxide efficiently cleaves double-stranded DNA through the oxidative attack on deoxyribose units from the minor groove [10, 11]. The consequent DNA-cleavage products include 5′-monophosphate and 3′-monophosphate ester termini, free bases, 5-methylene furanone, and a small amount of 3′-phosphoglycolate [12–14]. Dihydrogen peroxide can be generated by Cu(phen)22+ in close proximity to the DNA strands, in the presence of a reductant and molecular oxygen [15]. The nuclease activity of Cu(phen)2 has been enhanced with the synthesis of 1-(1,10-phenanthrolin-3-yloxy)-3-(1,10-phenanthrolin-8-yloxy)propan-2-amine (3-Clip-Phen, 4) based on the covalent linkage of two phenanthroline units through their 3-position, leading to an increase of 60 times compared with the nuclease activity of Cu(phen)2 itself [15]. However, similarly to Cu(phen)2, the copper complexes of 3-Clip-Phen have no sequence selectivity, and cleave the DNA in a single-stranded fashion. The amine group of 3-Clip-Phen has been functionalized with different groups, such as a distamycin analog or various DNA intercalators [16, 17]. Thus, the resulting complexes show enhanced cleaving activities, and the complexes with the distamycin analog exhibit excellent targeting properties toward A·T boxes [18].
Recently, it has been shown that Cu(3-Clip-Phen) (3) attached to a cisplatin motif is able to perform direct double-strand cuts, thanks to the DNA-anchoring platinum moiety [19]. Platinum complexes have received considerable attention since the discovery of the antiproliferate activity of cisplatin in 1969 [20]. It is generally accepted that the distortion of DNA generated upon binding of cisplatin is largely responsible for its antitumor properties [3]. Subsequent drug activation via intracellular aquation reactions results in a variety of stable bifunctional DNA–platinum(II) adducts. 1,2-Intrastrand cross-links between two adjacent guanine bases d(GG) or between an adenine and a guanine residue d(AG) are primarily formed. The platinum center of complexes with a cis motif preferentially coordinates to the N7 position of both adenine and guanine in the major groove of DNA [21–24].
In the present paper, the preparation of a bifunctional complex [CuPt[N1-(3-(1,10-phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-yl)ethane-1,2-diamine]Cl4, 2] containing both a cis-bis(amine)Pt(II) moiety and a nuclease active 3-Clip-Phen group is reported (Fig. 1). The bridge connecting the platinum and copper moieties is very short and therefore rigid in contrast to that in the previous report, where the complexes had flexible linkers in order to have a major–minor groove interaction [19]. The platinum and the 3-Clip-Phen adjacent parts are now separated by a short linker. Consequently, in this case either the platinum species will not interact with its preferential site (the major groove) or the 3-Clip-Phen moiety will not interact with its preferential site (the minor groove). The platinum component plays two roles: (1) it acts as a DNA anchor, thus allowing the Cu(3-Clip-Phen) moiety to perform cleavages in the close proximity of the Pt–DNA adducts, and (2) it induces a sequence-selective binding of the heterodinuclear complex. Accordingly, the achievement of double-strand DNA breaks is potentially increased, because the single-strand cuts are in the close proximity of the platinum adduct and sequence-selective cleavage may be expected.
Fig. 1Complexes 1–3
In the present study, the binding property of the platinum moiety and the cleavage selectivity and activity of this novel heterodinuclear complex 2 were investigated by agarose gel electrophoresis and high-resolution analysis with a 36-bp DNA fragment (Fig. 2). The results obtained are compared with those achieved with cisplatin and the Cu(3-Clip-Phen) complex.
Fig. 2Nucleobase sequences of oligonucleotide I (ODN I), oligonucleotide II (ODN II) and the primer used for the binding experiments. The preferential binding sites of cisplatin are indicated by arrowheads
Materials and methods
Preparation of Pt[N1-(3-(1,10-phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-yl)ethane-1,2-diamine]Cl2 and 2
All reagents and solvents were commercially available and used without further purification. Cu(3-Clip-Phen) (3) was prepared as previously described [15]. The 1H NMR chemical shifts of standard solvent molecules have been omitted in all redrawn spectra for clarity.
2-(2-(3-(1,10-Phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-ylamino)ethyl)isoindoline-1,3-dione
3-Clip-Phen (4) (100 mg, 0.22 mmol) was dissolved in 5 mL of dimethylformamide (DMF). One equivalent of N-(2-bromoethyl)phthalimide (5) (56.7 mg, 0.22 mmol) and 1 equiv of N-ethyldiisopropylamine (38 μL, 0.22 mmol) were added to 4. The reaction mixture was heated to 100 °C for 2 days. After the mixture had cooled to room temperature, 10 mL of dichloromethane (DCM) was added, and the organic phase was washed three times with 10 mL of distilled water. After being dried over Na2SO4 and evaporation under reduced pressure, the resulting crude product was purified by column chromatography (SiO2, DCM, MeOH, NH4OH, 95:5:0.5), to yield 2-(2-(3-(1,10-phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-ylamino)ethyl)isoindoline-1,3-dione (6) as an off-white powder (yield 34%). 1H NMR (DCCl3, 300 MHz) δ 9.05 (dd, 2H, J = 4.32, 1.67 Hz), 8.81 (d, 2H, J = 2.83 Hz), 8.10 (dd, 2H, J = 8.07, 1.62 Hz), 7.72 (dd, 2H, J = 5.43, 3.05 Hz), 7.67 (d, 2H, J = 8.88 Hz), 7.62 (d, 2H J = 8.86 Hz), 7.54 (dd, 2H, J = 6.09, 4.74 Hz), 7.48 (m, 4H), 4.27 (d, 4H, J = 5.18 Hz), 3.84 (t, 2H, J = 6.06 Hz), 3.54 (m, 1H), 3.13 (t, 2H, J = 6.12 Hz) ppm. 13C NMR (DCCl3, 75 MHz) δ 168.4, 153.7, 150.1, 146.0, 142.4, 140.3, 135.73, 133.8, 132.0, 129.3, 127.1, 125.9, 123.0, 122.0, 114.9, 67.3, 55.6, 45.4, 37.8 ppm. Low-resolution mass spectrometry (MS) (electrospray ionization, ESI, more than 0) m/z 621.00 [(M + H)+; calcd for C37H29N6O4+: 621.66], 642.94 [(M + Na)+; calcd for C37H28N6O4Na+: 643.65] Anal. calcd for C37H28N6O4·1.6 H2O: C, 68.42; H, 4.84; N, 12.94. Found: C, 68.34; H, 4.86; N, 13.21.
N1-(3-(1,10-Phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-yl)ethane-1,2-diamine
Two equivalents of hydrazine (15.6 μL, 0.32 mmol) were added to a solution of 6 (100 mg, 0.16 mmol) in 5 mL of pure ethanol. The mixture was refluxed overnight. The ethanol was evaporated under reduced pressure, and the crude product was purified by column chromatography (SiO2, DCM, MeOH, NH4OH, 90:10:1) to give N1-(3-(1,10-phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-yl)ethane-1,2-diamine (7) as a light-brown powder (yield 73%). 1H NMR (MeOD-d3, 300 MHz) δ 9.00 (dd, 2H, J = 4.41, 1.63 Hz), 8.84 (d, 2H, J = 2.84 Hz), 8.34 (dd, 2H, J = 8.10, 1.67 Hz), 7.90 (d, 2H, J = 2.86 Hz), 7.83 (m, 4H), 7.65 (dd, 2H, J = 8.10, 3.26 Hz), 4.51 (d, 4H, J = 4.47 Hz), 3.58 (m, 1H), 3.07 (t, 2H, J = 5.64 Hz), 2.97 (t, 2H, J = 5.23) ppm. 13C NMR (MeOD-d3, 75 MHz) δ 155.6, 150.6, 146.3, 143.0, 140.5, 137.6, 131.0, 128.7, 128.2, 127.4, 123.5, 116.7, 69.1, 57.5, 41.9 ppm. Low-resolution MS (ESI > 0) m/z 490.98 [(M + H)+; calcd for C29H27N6O2+: 491.56] Anal. calcd for C29H26N6O2·1.9 CH2Cl2: C, 56.93; H, 4.61; N, 12.89. Found: C, 56.79; H, 5.13; N, 13.23.
Pt[N1-(3-(1,10-phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-yl)ethane-1,2-diamine]Cl2
To a solution of 7 (70.4 mg, 0.14 mmol) in 6.5 mL of DMF was added a solution of 0.8 equiv of K2PtCl4 (47.69 mg, 0.11 mmol) in 3.25 mL of de-ionized H2O. The reaction mixture was stirred for 6 h at room temperature. The off-white precipitate was filtered off and washed with 30 mL of deionized water, 30 mL of MeOH and 20 mL of diethyl ether to give Pt[N1-(3-(1,10-phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-yl)ethane-1,2-diamine]Cl2 (1) as an off-white powder (yield 42%). 1H NMR (DMSO-d6, where DMSO is dimethyl sulfoxide, 300 MHz) δ 9.04 (d, 2H, J = 2.77 Hz), 8.88 (d, 2H, J = 2.45 Hz), 8.45 (d, 2H, J = 8.00 Hz), 8.05 (br, 2H), 7.95 (m, 4H), 7.70 (dd, 2H, J = 7.99, 4.35 Hz), 4.49 (br, 4H), 4.07 (br, 5H) ppm. 195Pt NMR (DMSO-d6) δ −2,317 (complex), −3,292 (complex with one DMSO coordinated) ppm. IR (neat): ν = 3,049 (br); 1,591 (s); 1,506 (s); 1,424 (s); 1,236 (s); 1,198 (s); 1,102 (s); 1,018 (s); 876 (s), 834 (s), 728 (s), 332 (s), 325 (s) cm−1. Anal. calcd for C29H26Cl2N6O2Pt·DMF: C, 46.33; H, 4.01; N, 11.82 Found: C, 47.54; H, 4.43; N; 11.70; the deviation in calculated percentage C is ascribed to traces of solvent other than DMF.
CuPt[N1-(3-(1,10-phenanthrolin-3-yloxy)-1-(1,10-phenanthrolin-8-yloxy)propan-2-yl)ethane-1,2-diamine]Cl4
CuCl2 (17.0 mg, 0.1 mmol) was added as a solid to a suspension of 1 (75.7 mg, 0.1 mmol) in DMF (25 mL). The reaction was stirred overnight at 50 °C. DMF was partially evaporated under reduced pressure, and the crude compound was precipitated in 100 mL of diethyl ether. The solid material was filtered off and washed with 3 × 20 mL of diethyl ether, and dried overnight at 50 °C under reduced pressure to give 2 as a green powder (yield 93%). X-band electron paramagnetic resonance (solid state): g = 2.1191. UV–vis (H2O) λmax 282 (40,100), 321 (12,500), 333 (10,100), 347 (3,280)  nm. High-resolution MS (m/z): [2M–2Cl] calcd for (C58H52Cl6N12O4Pt2Cu2)2+ 853.00852, 853.5096, 854.0097, 854.5087, 855.0088, 855.5072, 856.0073, 856.5083, 857.0100, 857.5067; found 853.0117, 853.5117, 854.0114, 854.5110, 855.0107, 855.5105, 856.0103, 856.5101, 857.0098, 857.5097.
Cytotoxicity
The cytotoxicity of complexes 1 and 2 has been determined for breast (MCF7), two glioblastoma (Hs683 and U373), two colorectal (HCT-15 and LoVo) and lung (A549) cancer cell lines (see supplementary material for experimental details and results).
Solutions of complexes for experiments with DNA
One millimolar DMSO solutions of the complexes investigated were prepared, and were subsequently diluted with Milli-Q water.
Nuclease activity on supercoiled DNA
One millimolar DMSO solutions of the complexes investigated were diluted to, respectively, 200, 400 and 1,000 nM with Milli-Q water. Five microliters of the complex solution was added to 10 μL of supercoiled ΦX174 DNA (Invitrogen, 7 nM, 40 μM base pairs) in 6 mM NaCl, 20 mM sodium phosphate buffer (pH 7.2), and incubated for 20 h at 37 °C. To initiate the cleavage, 5 μL of a 20 mM mercaptopropionic acid solution in water was added, and the resulting reaction mixture was incubated at 37 °C for 1 h. The reaction was quenched at 4 °C, followed by the addition of 4 μL of loading buffer (glycerol with bromophenol blue) prior to the loading on a 0.8% agarose gel containing 1 μg mL−1 ethidium bromide. The gels were run at a constant voltage of 70 V for 90 min in 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris) borate edta (TBE) buffer containing 1 μg mL−1 ethidium bromide. The gels were visualized under a UV transilluminator, and the bands were quantified using a Bio-Rad Gel Doc 1000 apparatus interfaced with a computer.
Time-course experiments of DNA cleavage
Fifty microliters of the complex solution was added to 100 μL of supercoiled ΦX174 DNA (Invitrogen, 7 nM, 40 μM base pairs) in 6 mM NaCl, 20 mM sodium phosphate buffer (pH 7.2), and the resulting reaction mixture was incubated for 20 h at 37 °C. To initiate the cleavage, 50 μL of 20 mM mercaptopropionic acid was added, and a sample was taken out every 10 min. Four microliters of loading buffer (glycerol with bromophenol blue) was added, and the sample was directly frozen in liquid nitrogen. When all samples had been collected, they were loaded on a 0.8% agarose gel containing 1 μg mL−1 ethidium bromide.
Analyses with 5′-32P-end-labeled DNA
Oligonucleotides (ODNs) I and II and the primer (Fig. 2) were purchased from Eurogentec, and purified on a 15% polyacrylamide (acrylamide to bisacrylamide ratio 19:1) gel. The denaturating gel contained 7 M urea. The migrations was performed in the presence of TBE buffer at 2,500 V for 3 h. The concentrations of single-stranded ODNs were determined by UV titration at 260 nm [25]. The ODNs were end-labeled with 32P using standard procedures with T4 polynucleotide kinase (New England BioLabs) and [γ-32P]ATP for the 5′-end, before being purified on a MicroSpin G25 column (Pharmacia) [26].
Comparison of the platinum–ODN adducts formed with the different complexes
5′-end-labeled ODN I (2 μM) was annealed to 1 equiv of its complementary strand ODN II in 1,100 μL of Tris–HCl (20 mM, pH 7.2) by heating to 90 °C for 5 min, followed by slow cooling to room temperature. Then, 60 μL of this solution was incubated with 60 μL of the complex solution (6 or 20 μM) for 20 h at 37 °C, followed by precipitation with 100 μL of sodium acetate buffer (3 M, pH 5.2) and 1,300 μL of cold ethanol. Pellets were rinsed with ethanol and lyophilized. Samples were dissolved in formamide containing bromophenol blue and xylene cyanol and heated for 3 min at 90 °C before being loaded on the gel. Platinum–DNA adducts were analyzed by denaturing 20% polyacrylamide gel electrophoresis, then by phosphorimagery.
Comparison of the sequence-selective binding by primer extension experiments with Taq polymerase
ODN I (2 μM) was annealed to ODN II (2 μM) in 1,100 μL of Tris–HCl (20 mM, pH 7.2) by heating to 90 °C for 5 min, followed by slow cooling to room temperature. Sixty microliters of this solution was then incubated with 60 μL of the complex solution (6 or 20 μM) for 20 h at 37 °C, followed by precipitation with 100 μL of sodium acetate buffer (3 M, pH 5.2) and 1,300 μL of cold ethanol. Pellets were rinsed with ethanol and lyophilized. For primer extension, an aliquot of the redissolved samples (0.25 μM) was annealed with 5′-end-labeled primer (0.25 μM) and 1 equiv of ODN I (0.25 μM) in the enzyme buffer (the buffer contains a small amount of reductant, but not enough to induce DNA cleavage by complex 2) before the addition of 250 μM dGTP, dCTP, dATP and dTTP and 2.5 U of Taq polymerase (final concentrations are given, the total volume was 10 μL). One equivalent of unmodified ODN I was added in order to displace the ODN II from the duplex and replace it with the labeled primer. The samples were reacted at 37 °C for 30 min, and 1 μL of edta (0.2 M) was subsequently added. Samples were dissolved in formamide containing bromophenol blue and xylene cyanol and heated for 3 min at 90 °C, before being loaded on the gel. Five microliters of sample was then analyzed by denaturing 20% polyacrylamide gel electrophoresis and phosphorimagery. The Maxam and Gilbert [27] sequencing scale, including a final scale of T4 polynucleotide kinase digestion to remove 3′-end phosphates, was used to analyze the DNA fragments. The sequencing experiments show, in addition to a strong, easily assigned G signal, also a weak A signal.
Comparison of the cleavage patterns of ODN I–ODN II induced by the copper complexes
The 5′-end-labeled 36mer target (2 μM) was annealed to 1 equiv of its complementary strand in 1,100 μL of Tris–HCl (20 mM, pH 7.2) by heating to 90 °C for 5 min, followed by slow cooling to room temperature. To 60 μL of this solution was added 60 μL of complex 1, 2 or 3 solutions (20 μM). Some of the samples involving complex 2 were incubated for 19 h, followed by the addition of 1 equiv of CuCl2 per complex and subsequent incubation (1 h). The other samples were incubated for 20 h at 37 °C. Next, all samples were precipitated with 100 μL of sodium acetate buffer (3 M, pH 5.2) and 1,300 μL of cold ethanol. Pellets were rinsed with ethanol and lyophilized, then dissolved to 1.33 μM in Tris–HCl buffer (13.3 mM, pH 7.4). For the cleavage experiments, to 15 μL of this solution was added 5 μL of a 0.8 mM ascorbate solution (5 μL of water was added to the controls). The samples were incubated at 37 °C for 1 h, followed by precipitation in 20 μL of sodium acetate buffer (3 M, pH 5.2) containing 1 μg of salmon testes DNA and 180 μL of cold ethanol. Pellets were rinsed with ethanol and lyophilized. In order to study the DNA cleavage mechanism, additional treatments were performed on some samples: (1) heating at 90 °C in 50 μL of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)–NaOH buffer (0.1 M, pH 8.0) for 30 min, followed by ethanol precipitation; (2) heating at 90 °C in 50 μL of piperidine (0.2 M in water) for 30 min, followed by lyophilization. Samples were dissolved in formamide containing bromophenol blue and xylene cyanol and heated for 3 min at 90 °C, before being loaded on the gel. Samples were analyzed by denaturing 20% polyacrylamide gel electrophoresis, then phosphorimagery. The Maxam and Gilbert [27] sequencing scale was used to analyze DNA fragments. The sequencing experiments show, in addition to a strong, easily assigned G signal, also a weak A signal.
Results and discussion
Design and synthesis of a heterodinuclear platinum–copper complex
A ditopic ligand 7 was designed to favor the simultaneous coordination of a platinum and a copper entity; hence, the resulting bifunctional complex would combine the ability to form a kinetically inert coordination bond with DNA, thanks to its cis-Pt moiety, with the cleavage properties of Cu(3-Clip-Phen). Two coordination compounds, namely, the platinum complex 1 and the heterodinuclear platinum–copper complex 2 (Fig. 1) were prepared with the ligand 7. The binding ability of the platinum unit and the cleavage selectivity of the Cu(3-Clip-Phen) moiety were investigated. The results were compared with those for the known Cu(3-Clip-Phen) complex (3). The short separation between the Pt and the Cu centers in complex 2 most likely affects the preference for platinum to bind in the major groove, and in the minor groove for copper. Accordingly, the coordination of the platinum moiety in the major groove will force the Cu(3-Clip-Phen) component to bind in the major groove, and vice versa.
The general synthetic pathway to prepare complexes 1 and 2 is depicted in Scheme 1. The selective and complete platination of the ethylenediamine unit using 1 equiv of K2PtCl4 was monitored by 195Pt NMR and 1H NMR. The in situ reaction of the resulting platinum derivative 1 with 1 equiv of CuCl2 produces the heterobimetallic complex 2.
Scheme 1Preparation of complexes 1 and 2. Reagents and conditions as follows: i dimethylformamide (DMF), diisopropylethylamine, 100 °C, 2 days; ii ethanol, H2N–NH2, reflux, overnight; iii K2PtCl4, MeOH/water, room temperature, 6 h; iv CuCl2, DMF, 50 °C, overnight
Cleavage of supercoiled DNA
The relaxation of supercoiled circular ΦX174 DNA (form I) into its relaxed (form II) and linear (form III) conformations was monitored to compare the aerobic cleavage abilities of complexes 2 and 3, in the presence of a reducing agent (Fig. 3). First, the complexes were incubated for 20 h, to allow the formation of platinum–DNA adducts. The nuclease activity was subsequently initiated by the addition of 5 mM mercaptopropionic acid. Complex 2 exhibits a markedly higher nuclease activity than Cu(3-Clip-Phen) (3) (compare lanes 5 and 9 in Fig. 3). Indeed, at complex concentrations of 100 nM, most supercoiled DNA has reacted to form circular and linear DNA in the case of 2, while Cu(3-Clip-Phen) only generates a small amount of form II (Fig. 3, lanes 4 and 8). Moreover, almost all the DNA has reacted to form smaller DNA fragments (migrating as a smear) at a complex concentration of 250 nM for 2 (Fig. 3, lane 5), whereas no smear is observed for Cu(3-Clip-Phen) (Fig. 3, lane 9). Interestingly, at a concentration of 100 nM for complex 2, form III is detected before the total disappearance of form I (Fig. 3, lane 4) [19]. This result indicates that the heterodinuclear platinum–copper complex is able to perform direct double-strand cuts since form I is still detected in the reaction. Such double-strand breaks are highly cytotoxic, since the cells have difficulties to repair such damage [28, 29]. Cu(3-Clip-Phen) is only able to perform repetitive single-strand cuts [15]. Interestingly, the cleavage activity of complex 2 is also more efficient compared with that of the platinum–copper complexes previously reported by our group [19].
Fig. 3Comparative experiments of the oxidative cleavage of ΦX174 plasmid DNA performed by 2 and 3. Lane 1 control DNA. Lane 2 250 nM solution of 2. Lane 3 50 nM 2, in the presence of 5 mM mercaptopropionic acid (MPA). Lane 4 100 nM 2, in the presence of 5 mM MPA. Lane 5 250 nM 2, in the presence of 5 mM MPA. Lane 6 250 nM 3. Lane 7 50 nM 3, in the presence of 5 mM MPA. Lane 8 100 nM 3, in the presence of 5 mM MPA. Lane 9 250 nM 3, in the presence of 5 mM MPA
Time-course studies of DNA cleavage by complexes 2 and 3 were carried out to further investigate the direct double-strand cleavage event (Fig. 4). Complex 3 at a concentration of 250 nM generates a maximum of 80% of circular DNA (form II) after a reaction time of about 1 h [19]. Around 20% of linear DNA (form III) is produced via the action of 3 after 70 min. Remarkably, the formation of form III is only observed after a reaction time of 30 min when already 60% of form II has been produced. For a 150 nM solution of complex 2, linear DNA (form III) is generated from the initial stages of the cleavage reaction, with supercoiled DNA (form I) still being present (Fig. 4). Also, only a maximum of 60% of form II is generated. These features reflect the ability of 2 to perform direct double-strand breaks, most likely as a result of the binding of the platinum moiety to DNA, allowing the copper part to achieve more than one oxidative cleavage in the close proximity of the platinum coordination site.
Fig. 4Time-course experiments of DNA cleavage (20 μM in base pairs) over a period of 70 min. Complex 2 (150 nM) was incubated for 24 h, followed by activation with 5 mM MPA and air
Analysis of the platinum adducts on ODN I of the ODN I–ODN II duplex target
High-resolution analyses with a 36-bp DNA duplex (ODN I–ODN II) were performed to investigate the coordination of the platinum component of the bifunctional complexes to DNA, (Fig. 5a). The sequence of this duplex was chosen to include GG and AG sites (which are the two major binding sites of cis-Pt(II) complexes) on one strand (ODN I). The results obtained after incubation of the complexes with the duplex labeled on the 5′-end of ODN I for 24 h were analyzed by polyacrylamide gel electrophoresis under denaturing conditions and are shown in Fig. 5a. Denaturing conditions allowed the analysis of the Pt–DNA adducts on ODN I of the duplex.
Fig. 5a Polyacrylamide gel electrophoresis (PAGE) analysis of the Pt–ODN I adducts of the ODN I–ODN II duplex target (1 μM). ODN I was 5′-end-labeled with 32P-phosphate. The complexes were incubated with the DNA for 24 h before analyses. Lane 1 ODN I. Lane 2 3 μM cisplatin. Lane 3 10 μM cisplatin. Lane 4 3 μM 1. Lane 5 10 μM 1. Lane 6 3 μM 2. Lane 7 10 μM 2. b Phosphorimager data of a DNA sequencing gel comparing the sequence specificity of cisplatin, 1 and 2. All the samples were extended using Taq polymerase, starting from the 5′-end-labeled primer. Lane 1 blank experiment; Lane 2 3 μM cisplatin; Lane 3 10 μM 1. Lane 4 10 μM 2. It is noteworthy that the GTA and GGAC sites give the sequence of the opposite strand that induced the stopping of the primer extension (Figs. S1, S2). c Nucleobase sequence of ODN I, and indication of the damage sites induced by cisplatin, 1 and 2. Large arrows and small arrows represent, respectively, major and minor stop sites
Molecules able to irreversibly bind to DNA will retard the rate of migration of the modified ODN, compared with that of the free ODN; therefore, ODN–Pt adducts appear as retarded bands on gels. The incubation with 3 equiv of cisplatin clearly reveals an impeded mobility of the ensuing cisplatin–ODN I adduct (Fig. 5a, lane 2). Only traces of free ODN I were detected; 89% of the ODN I had been modified. The incubation of the DNA duplex with 10 equiv of cisplatin results in a total conversion of ODN I (Fig. 5a, lane 3). Complex 1 shows the formation of Pt–ODN I adducts (Fig. 5a, lanes 4 and 5). Two distinct bands are clearly observed when 10 equiv of complex 1 is incubated with the DNA target, indicating the formation of Pt–ODN I adducts. The quantification of free ODN I reveals that as much as 84% of this DNA fragment has reacted. The use of complex 2 leads to comparable results, with the conversion of 88% of the initial ODN I (Fig. 5a, lane 7). However, the reaction between ODN I and complex 2 produces a smear (i.e., a range of products) on the gel. Our previously reported compounds showed reduced formation of Pt–ODN adducts under similar experimental conditions compared with complexes 1 and 2 [19].
Sequence selective binding of complexes 1 and 2 compared with cisplatin
Primer extension experiments were performed to investigate the sequence-selective binding of the platinum units to the ODN I fragment of the ODN I–ODN II DNA duplex (Figs. 5b, c, S1, S2). The platinum complexes that did not react with DNA were removed prior to the start of the primer extension experiments. Nevertheless, it is possible to have more than one platinum moiety coordinating the ODN strand, since the concentrations of cisplatin, complex 1 and complex 2 were, respectively 3 and 10 times higher than the concentration of the ODN I–ODN II DNA duplex. Taq polymerase has proven to be a valuable tool for the determination of the sequence selectivity of various platinum complexes [23, 30–39]. Cisplatin inhibits the enzymatic polymerization at the anticipated GG and AG sites (Fig. 5b, c), but the majority of cisplatin is detected at the GG site. It should be noted that once the ODN I strand contains two Pt adducts on both the GG and AG site, the enzyme stops only at the GG site. Therefore, it is possible that the amount of modified AG sites is underestimated. Nevertheless, not all of the ODN I has been modified by the complexes (Fig. 5a). It is therefore reasonable to say that the majority of the ODN I contains only one Pt adduct and that the AG site is indeed the minor site of interaction. The stop sites observed for complex 1 are also located for a major part at the GG base pairs and for a minor part at the AG base pairs. Complex 2 shows only stops at the GG binding site. These results indicate that the platinum moiety interacts with its preferential binding site. The difference between cisplatin and complexes 1 and 2 is the precise point at which the peak intensity occurs at the damaged site. The stops of the Taq polymerase are mainly located at the A base before the GG site for cisplatin, complex 1 and complex 2. However, the enzyme stops induced at the GG site are more equally distributed among the GAC site in the cases of complexes 1 and 2 (Fig. 5b, c; the size of the arrows is an indication of the damage intensity). The binding of complexes 1 and 2 apparently produces bulky adducts, thus allowing a partial stop at the second nucleotide before the classic position, at the GG adduct, since an increase of the reaction time with the enzyme (from 30 to 120 min, Fig. S3; complex 1) induces a bypass of the stop at the C base (associated with a decrease of the peak intensity when compared with that for G). The difference between cisplatin and complex 1 is more pronounced at the AG site. Although it is the minor binding site, the damage induced by cisplatin almost exclusively takes place at the G base, at the 5′-end of ODN I. The Taq polymerase stops for complex 1 are mainly observed at the A base.
Cleavage of the ODN I strand of the ODN I–ODN II duplex
The cleavage of the ODN I–ODN II duplex with complexes 1–3 was investigated by polyacrylamide gel electrophoresis, the target being labeled on the 5′-end of ODN I (Fig. 6). To the best of our knowledge, studies of such bifunctional complexes have been reported only twice [40, 41].
Fig. 6a PAGE analysis of cleavage of ODN I of the ODN I–ODN II duplex (1 μM) by compounds 1–3 (10 μM when unspecified). The cleavage reactions were initiated with ascorbate (200 μM) in aerobic conditions or by heating for 30 min at 90 °C in aqueous 0.2 M piperidine. The Maxam–Gilbert sequencing reactions A + G (lane 1) and G (lane 2) were performed to determine the cleavage sites. On the top of the gel are indicated the conditions used during the experiments (details are given in “Materials and methods”). Lanes 3–6 are for controls without complexes. Lanes 17–22 are for experiments that were performed in the presence of 1 μM 3. b High-contrast picture of lanes 8, 10, 11, 13, 15 and 16 in a allowing easier observation of the the cleavage pattern. ΔpH 8 was a heating step of 30 min at 90 °C in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)–NaOH buffer (0.1 M, pH 8.0). Unlinked complexes 1 and 2 were removed in a precipitation step with ethanol before the induction of cleavage
Complexes 1 and 2 were preincubated for 20 h to allow the coordination of the platinum moiety to the DNA target. For complex 1, the preincubation was subsequently followed by the coordination of 1 equiv of copper, since the copper-free complex does not show any nuclease activity. The noncoordinated complexes were removed by precipitation before the cleavage was initiated by the addition of ascorbic acid (0.2 mM) under aerobic conditions. Additional treatments (HEPES pH 8.0 and piperidine) were carried out.
The noncovalent interaction of Cu(3-Clip-Phen) (3) with DNA permits its abstraction from the ODNs by a simple precipitation step. A tenfold excess of 3 (compared with the ODNs) does not generate cleavage products after the precipitation step (Fig. 6, lane 24), while 1 equiv of 3 without precipitation exhibits significant cleavage (Fig. 6, lane 20). The platinum-containing complexes 1 and 2 form kinetically inert bonds with DNA, and therefore cannot be removed from the ODN during the precipitation step. In contrast, the unreacted complexes are eliminated.
As expected, the cleavage pattern of 3 is nonspecific, [18] as about 20% of the target is oxidized at all nucleotides (Fig. 6, lane 20). The cleavage achieved by complex 2 in the presence of ascorbic acid results in the full and partial disappearance of, respectively, the Pt–ODN I adducts and the ODN I band (Fig. 6, lane 10). The total cleavage amounts to nearly 80%, and its pattern is different from the features resulting from the action of 3, but no clear sequence selectivity is observed. Accordingly, a smear of products is noted in contrast to 3, suggesting that the mechanism of the cleavage is different. This observation may be explained by either of the following possibilities: (1) some cuts are due to platinum adducts of complex 2 positioned on the other strand of the duplex (ODN II); (2) the extensive cleavage observed most likely indicates that the ODN I fragment experiences more than one cut; therefore, complex 2 should have been released after repetitive cuts from ODN I and should be able to cut the free ODN.
To verify this hypothesis, the experimental conditions were adjusted to introduce fewer platinum adducts onto the target (20% against the 80% of modification of ODN I shown in Fig. 6), and less ascorbate (100 μM) was used to limit the recleavage events. However, the resulting cleavage pattern appears to be similar to that observed with the former conditions (Fig. 8).
One equivalent of CuCl2 was added to complex 1, after the formation of Pt–DNA adducts, to analyze the cleavage selectivity of the resulting compound. A comparatively less efficient cleavage was achieved using complex 1 with extra added CuCl2, when compared with complex 2 (compare lanes 10 and 15 in Fig. 6), possibly as a result of the partial coordination of the copper ions to 1. Similarly to 2, the cleavage pattern obtained with 1 is found to be significantly different from that of 3 (Fig. 6, lane 20). Interestingly, the free ODN I is not affected during the cleavage process, as only the ODN fragments containing platinum adducts are cleaved. Furthermore, a sequence-selective cleavage (associated with the formation of fragments of ODN I including probably the 3′-phosphate end, since they comigrated as Maxam and Gilbert sequencing fragments) is observed in the close vicinity of the GG and AG sites (indicated as black bars in Fig. 6, left). The intensity of the bands is much stronger at the GG site, reflecting the results of the primer extension experiments previously obtained. The four base pairs neighboring the GG site in the 5′-direction are also affected by the cleavage of complex 1. In the 3′-direction, no clear bands are observed, but a smear is observed, which can be explained as follows: the cleavage products remain coordinated to the complex and have therefore a totally different mobility, compared with the Maxam–Gilbert sequencing fragments. The apparent smear observed in the 3′-direction supports this assumption, since such behavior is observed for Pt–ODN I adducts including the copper complex (Fig. 5a, lane 6), and can therefore be expected for cleavage fragments that coordinate to the complex.
A heating step in HEPES buffer (90 °C; pH 8.0) is often used to cleave the metastable products resulting from the oxidation of deoxyribose. This treatment of the cleavage products obtained with complexes 1–3 did not show a strong increase of the DNA cuts (Fig. 6, respective comparisons between lanes 15, 16 and 10 and 11, 20 and 22). Therefore, products of strand cleavage are essentially observed during this analysis. However, the smears observed in lanes 10 and 15 are partially converted into bands, indicating that some cleavage products, arising from the oxidation of deoxyribose, are sensitive to alkaline conditions.
More drastic alkaline heating (as with piperidine) allows one to detect the oxidations of the nucleobases that do not induce direct DNA cleavage [6]. Surprisingly when this treatment is applied to complexes 1 (with addition of 1 equiv of copper) and 2, clear cleavages are observed, although the systems had not been incubated with ascorbate (Fig. 6, lanes 13 and 8, respectively). This phenomenon is dependent of the presence of copper ion, since it is not observed for Pt–ODN I adducts with complex 1 without the addition of CuCl2 (results not shown). Therefore, these alkaline conditions are sufficient to induce a redox activity of the copper complex part of the hybrid molecule [this phenomenon has not been observed with Cu(3-Clip-Phen), which can be removed during a precipitation step preceding the heating in the presence of piperidine]. Interestingly, only the Pt–ODN I adducts are cleaved; none of the free ODN I is degraded and selective cleavages are observed around the position of adducts detected during primer extension experiments. Unfortunately, this activity of the copper complexes covalently linked to the target does not allow us to analyze whether or not the dual Pt–Cu complexes perform the oxidation of nucleobases.
Cleavage of the ODN II strand of the ODN I–ODN II duplex
The same experiments were performed with the ODNI–ODNII duplex labeled on the 5′-end of ODN II (Fig. 7). Similar amounts of Pt–ODN II adducts are formed with complexes 1 (Fig. 7, lane 8) and 2 (Fig. 7, lane 3). Most likely, only parts of the platinum components of complexes 1 and 2 are bound on ODN II at the preferential AG site, and the cleavages do not seem to be restricted to one selective site. Other adducts are probably also present. The occurrence of platinum–guanine monoadducts on ODN II (which is particularly rich in guanine bases) may explain the results observed. Further investigations are required to confirm these proposals.
Fig. 7PAGE analysis of cleavage of ODN II of the ODN I–ODN II duplex (1 μM) by compounds 1–3. The cleavage reactions were initiated with ascorbate (200 μM) in aerobic conditions or by heating for 30 min at 90 °C in aqueous 0.2 M piperidine. The Maxam–Gilbert sequencing reactions A + G (lane 1) and G (lane 2) were performed to determine the cleavage sites. On top of the gel are indicated the conditions used during the experiments (details are given in “Materials and methods”). ΔpH 8 was a heating step of 30 min at 90 °C in HEPES–NaOH buffer (0.1 M, pH 8.0). Unlinked complexes 1 and 2 were removed in a precipitation step with ethanol before the induction of cleavage
Both complex 1 with added copper and complex 2 show extensive cleavage activities in the presence of ascorbic acid in air (Fig. 7, lanes 11 and 6, respectively). In the experiments with complex 2, the Pt–ODN II adducts have fully reacted, while the free ODN II is only partly altered. The cleavage fragments are poorly resolved on the gel (a smear is essentially observed), and treatment with HEPES or piperidine fails to improve their analysis. These cleavage fragments are composed of modified DNA. It can be reasonably proposed that the fragments include Pt–ODN II adducts and that these adducts produce a smear during the migration in polyacrylamide gel electrophoresis. All these results indicate that the platinum moieties of complexes 1 and 2 bind, as expected, to the preferential platinum site on ODN II, but also and mainly to single A and G nucleobases.
Comparison between the cleavage of duplex and single-stranded DNA
A comparison between the cleavage patterns resulting from the action of complex 2 on single-stranded ODN I and on the duplex ODN I–ODN II was made to appraise the influence of the formation of Pt–ODN II adducts on the resulting cleavage (Fig. 8). Pt–ODN I adducts are formed when the single strand is used, and these adducts are essentially positioned at the GG site of ODN I (the results of the primer extension are not shown). For this study, only 20% of the platinum adducts on ODN I of the duplex and 40% of the platinum adducts on the single strand were created in order to have a maximum of one adduct per DNA target, and thus to simplify the analysis. The cleavage was induced by the addition of a small quantity of ascorbate (100 μM) to disfavor recleavage events.
Fig. 8Phosphorimager scanning of the PAGE-cleavage patterns of ODN I by complex 2. The cleavage was performed on either the ODN I–ODN II duplex (5′-end-labeled on ODN I) or on single-stranded ODN I. Cleavage was induced by the addition of ascorbate (100 μM) after the removal of the unlinked complex during a precipitation step with ethanol. The clear band area on the 3′-side of the GG site of the duplex is probably due to a covalent adduct of complex 2 on ODN II (the complementary strand of the duplex). 3′-Phosphoglycolate cleavage fragments are labeled with an asterisk. The other clear bands were attributed to fragments of ODN I including 3′-phosphate ends
The cleavage of the ODN I fragment of the duplex results in a cleavage pattern exhibiting various moderate peaks in the 3′-region of the GG base pair of ODN I. The cleavage of single-stranded ODN I by complex 2 gives rise to a smear in the same region. The smears observed characterize cleaved ODN I products that are still coordinated to complex 2. Accordingly, the weak peaks in this region of the DNA sequence are most likely due to the cleavage of complex 2–ODN II adducts on both strands. Since the conditions used favor a maximum of one Pt–ODN adduct per duplex, these cleavages on nonmodified ODN I probably originate from complex 2–ODN II adducts.
The results are summarized in Fig. 9. Importantly, the cleavage of ODN I by Pt–ODN II adducts appears to be partially responsible of the visible nonselective cleavage of the duplex by complex 2.
Fig. 9Summary of cleavage events due to platinum–copper dual complex 2 on double-stranded DNA. The labeled strand of the DNA duplex that can be observed from denaturing PAGE experiment is labeled with an asterisk
Interestingly, the weak peaks observed on the 5′-side of the cleavage fragments (and shown with an asterisk in Fig. 8) probably correspond to DNA fragments with 3′-phosphoglycolate extremities. Indeed, these species are observed at the position of the cleavage products of the duplex produced by the action of complex 3, and which have been previously characterized [15]. Such fragments were earlier identified as the result of the oxidation of the C4′ position of 2-deoxyribose. Further investigations are required to confirm these proposals.
Concluding remarks
The novel heterodinuclear platinum–copper complex 2 exhibits improved nuclease activity, compared with its parent compound Cu(3-Clip-Phen) (3), and is able to perform double-strand breaks. The platinum moiety acts as an anchor to DNA, forcing the Cu(3-Clip-Phen) group to generate cuts in close proximity to the Pt–DNA adducts, hence even allowing direct double-strand breaks. Mechanistic investigations on a 36-bp DNA fragment (ODN I–ODN II) revealed that platinum adducts are indeed formed with both complexes 1 and 2. The platinum moiety of complexes 1 and 2 binds to GG (primarily) and AG sites, like cisplatin. Nevertheless, the Taq polymerase enzyme stops at different base pairs for cisplatin and complexes 1 and 2. This feature suggests that complexes 1 and 2 induce different distortions (compared with cisplatin) upon their binding to DNA, most likely owing to the bulkiness of the 3-Clip-Phen moiety. Complex 1 with added copper (1 equiv) shows a sequence-selective cleavage, in the close proximity of the platinum adducts. A sequence-selective cleavage is also observed for complex 2, but to a lesser extent compared with complex 1. The opposite strand also contains Pt–DNA adducts, but no clear sequence selectivity is observed, most likely owing to the lack of preferential platinum binding sites. These atypical platinum adducts lead to the partial damage of the other DNA strand, as is clearly evidenced by comparison of the cleavage products on a single-stranded or duplex DNA.
Interestingly, it has been found that piperidine is also able to activate the Cu(3-Clip-Phen) component of the bifunctional platinum–copper complexes, a feature so far not reported. Thus, a good sequence-selective cleavage is observed upon treatment of complex 1 with piperidine. Reasonable sequence selectivity is achieved with complex 2. The investigation of the mechanism of cleavage by complex 2 is currently in progress.
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dna heterodinuclear platinum ditopic copper


Impact of transanal endoscopic microsurgery on functional outcome and quality of life

Introduction Transanal endoscopic microsurgery (TEM) is a minimal invasive technique for local excision of rectal tumours. The procedure is performed via a rectoscope with a diametre of 4 cm. The aim of this prospective study was to assess both functional outcome and quality of life after TEM.
Introduction
Local excision of benign rectal tumours is safer compared to radical surgery and considered treatment of choice [1–3]. Several techniques have been developed for local excision, with the transanal technique according to Parks as the most frequently used [1, 4]. Other techniques used are the dorsal transcoccygeal approach (Kraske) and the dorsal transsphincteric approach (York–Mason) [5–9]. Each procedure has its own (dis)advantages, and none of the procedures mentioned is able to achieve local excision of tumours throughout the entire rectum. Transanal endoscopic microsurgery (TEM) demonstrated to be a safe procedure capable of overcoming this shortcoming. In early publications, even distal sigmoid tumours could be locally excised with excellent results. Moreover, recurrence rates are minimal compared to other local techniques. As a result, the indication for local excision of rectal tumours has expanded dramatically [10–13].
Few studies have addressed functional outcome following TEM, and with the operation rectoscope with a length of 12 or 20 cm and a diametre of 40 mm, scepticism towards post-operative faecal continence remains. In manometric studies after TEM, there seems to be a detrimental, temporary impact on internal sphincter functioning, although without clinical significance [14–16].
Cataldo et al. [17] recently performed a prospective study on faecal continence and incontinence-specific quality of life after TEM using standardised surveys. They stated TEM does not result in significant alterations. These results are promising, especially with a relative short duration of follow-up of 6 weeks in this study. As known from other types of rectal surgery, incidence of faecal incontinence diminishes with time [18]. This could imply results after TEM may even improve with longer follow-up.
Quality of life is increasingly recognised as the ultimate endpoint when assessing clinical outcomes after different surgical interventions because it measures the patient’s perspective. The precise impact of the TEM procedure on quality of life has not been well studied. This prospective study was set out to provide a comprehensive insight into the impact of TEM on functional outcome and quality of life.
Patients and methods
Between January 2004 and January 2006, a consecutive series of 50 patients were referred for a TEM procedure. All patients were evaluated pre-operatively according to a standard protocol including rigid rectoscopy, tumour biopsy and endorectal ultrasound. If TEM was considered feasible, patients were eligible for this study. Informed consent had to be given before inclusion. Local medical ethical committees approved this study. A full-thickness excision was always performed. The portion of the tumour located within the sphincter musculature was excised in partial thickness. Before and at least 6 months after the TEM procedure, patients were asked to fill out a questionnaire to assess anorectal functioning and quality of life. All data were collected by an independent research coordinator not previously involved in the patients’ care. We recorded the demographics, operative details, post-operative length of stay, post-operative complications and functional outcome for each participant. We evaluated functional outcome by means of a detailed questionnaire based on the Faecal Incontinence Severity Index (FISI) [19]. This system, developed by Rockwood, uses two basic components: the type of incontinence and its frequency. FISI scores range from 0 (total continence) to 61 (complete incontinence to solid stool on a daily basis). We used the validated weighting scores that are based on patients’ input.
Quality of life was evaluated using both the EuroQol EQ-5D and the Faecal Incontinence Quality of Life (FIQL) score. The EuroQol EQ-5D consists of a so-called index score representing the societal value of the health state and has a scale ranging from 0 (no quality of life) to 100 (optimal quality of life). The EuroQol EQ-5D also uses a visual analogue scale, the EQ-VAS, representing the patient perspective. This scale ranges from 0 (no quality of life) to 100 (optimal quality of life). The EuroQoL EQ-5D scores were compared with a sex- and age-matched, community-based sample of healthy persons without co-morbidity [20]. The FIQL score as described by Rockwood et al. [21] measures specific quality of life issues expected to affect patients with faecal incontinence. This instrument is composed of 29 questions within four domains: lifestyle issues, coping–behaviour, depression and self-perception and embarrassment. The scores in the FIQL range from a minimum score of 1 to a maximum of 4, for all of the scales (1 = quality of life alteration present most of the time, 4 = none of the time). Data are presented as medians and standard deviations. Changes within groups were evaluated using the nonparametric one-sample Wilcoxon’s signed-rank test. Comparison of these changes between groups was conducted using the Mann–Witney U test. The Spearman’s correlation coefficient was used for correlation between the different findings. A P-value of ≤0.05 was considered statistically significant.
Results
Fifty patients were eligible and informed consent was obtained. Three patients were excluded. In two patients, TEM could not be performed because of bulky tumour or technical problems. An additional patient underwent low-anterior resection for locally recurrent disease within 6 months of the TEM. The remaining 47 patients completed both questionnaires and were included for analysis. All of these patients were alive without evidence of recurrent disease. The group consisted of 22 males and 25 females. Median age was 67 years (range 40–84). Pre-operative diagnosis was villous adenoma in all patients. Median distance from the distal tumour margin to the dentate line was 7 cm (range 0–15 cm) and median tumour size was 20 cm2 (range 4–53 cm2). The median rate of captured circumference of the rectal wall was 40% (range 5–80; Table 1). Median operative time, defined as beginning when the rectoscope was inserted and ending when it was removed, was 55 min (range 10–140). Complications developed in 4 of 47 (8.5%) patients. Two patients had urinary retention, one patient a urinary tract infection and one suffered from a low hemoglobin rate requiring blood transfusion. No reoperations were necessary and mortality rate was zero. Median length of stay was 4 days (range 3–9 days; Table 2). Definite histopathological examination of the resected specimens revealed an adenoma in 44 patients and an invasive carcinoma in three patients (pT1 in two patients and pT2 in one patient). These three patients were reluctant to undergo major surgery and were observed with rectoscopy and endorectal ultrasound every 3 months without signs of recurrence at 6 months after TEM. In three adenomas, excisional margin was considered microscopic irradical, resulting in 94% of tumours being radically excised.
Table 1Patient and tumour characteristicsCharacteristicsNumberMale/female (N = 47)22/25Median age in years (range)67 (40–84)Median distance from dentate line in centimetres (range)7 (0–15)Median tumour size in square centimetres (range)20 (4–53)Median capture of circumference of rectal wall in per cent (range)40 (5–80)Table 2Procedure-related characteristicsCharacteristicsNumberMedian duration of operation in minutes (range)55 (10–140)Complications4/47 (8.5%)Urinary retention2Urinary tract infection1Blood transfusion1ReoperationsNoneLength of hospital stay in days (range)4 (3–9)
Six months after surgery, mean FISI scores were found to be decreased (pre-operative, 10; post-operative, 7 (p
< 0.01)), depicting an improvement in faecal continence (Fig. 1). Overall, when pre-operative and post-operative FISI scores were compared, 24 patients improved, 16 patients were unchanged and seven deteriorated. Operation time or tumour size did not influence the change in FISI score. There was a significant correlation between the decrease in FISI score and tumour height (p = 0.02). Reduction of FISI was significantly greater in patients with a tumour location within 7 cm from the dentate line (p = 0.01; Table 3). Mean scores and ranges of the EuroQol EQ-5D are presented in Table 4. Mean general quality of life score from the patients’ perspective (EQ-VAS) was found to be significantly higher 6 months after TEM (p < 0.02). The observed changes in EQ-VAS showed no correlation with the post-operative alterations in FISI scores or tumour characteristics. Mean pre-operative EQ-VAS score in our group was lower compared to the mean EQ-VAS score of the sex- and age-matched general population (p = 0.02). Post-operative EQ-VAS score was comparable with the general population. Mean index score (social perspective) remained the same (p = 0.09). Both pre- and post-operative EQ-5D index scores were similar to those of the sex–age-matched general population. Comparing the change from baseline in FIQL scores, a statistically significant improvement was observed in two of the four domains (embarrassment; p = 0.03 and lifestyle; p = 0.05). The domains of lifestyle, coping and behaviour and embarrassment were correlated with the FISI (all p < 0.05; Table 5). Overall, EQ-5D and FIQL scores were not affected by age and gender of the patients. Surgical aspects and tumour characteristics did not influence the outcome.
Fig. 1Mean Faecal Incontinence Severity Index (FISI) scores (±SEM) before and after Transanal Endoscopic Microsurgery (TEM)Table 3Mean FISI scores; numbers in parentheses are standard deviationsFISI scorePre-operativePost-operativeStatistical significanceOverall10 (2)6 (2)p < 0.01Duration of operation <55 min (N = 24)9 (4)7 (3)p = 0.24Duration of operation >55 min (N = 23)12 (3)4 (2)p = 0.17Tumours <7 cm from dentate line (N = 21)16 (5)5 (2)p = 0.01Tumours >7 cm from dentate line (N = 26)6 (2)7 (3)p = 0.43Median tumour size <20 cm2 (N = 27)12 (4)6 (3)p = 0.12Median tumour size >20 cm2 (N = 20)8 (3)6 (3)p = 0.32Lower values indicate better anorectal functioningTable 4Mean EuroQoL EQ-5D scores; numbers in parentheses are standard deviations Control groupPre-operativePost-operativeStatistical significanceEQ-VAS82 (7)77 (14)82 (11)p = 0.02Index score86 (6)84 (11)89 (9)p = 0.09EQ-VAS represents the patients’ perspective on quality of life; index score represents the societal value on quality of life. Higher scores indicate higher quality of life. Both scores are compared with a healthy sex- and age-matched control group.Table 5Mean FIQL scores; numbers in parentheses are standard deviationsFIQLSPre-operativePost-operativeStatistical significanceLifestyle3.7 (0.3)3.9 (0.3)p = 0.05Coping3.6 (0.5)3.8 (0.4)p = 0.10Depression3.7 (0.3)3.9 (0.4)p = 0.08Embarrassment3.1 (0.3)3.7 (0.4)p = 0.03Higher scores indicate higher quality of life.
Discussion
In rectal adenomas, TEM has emerged as the procedure of choice because of its safety and low local recurrence rates. Especially compared to radical surgery, TEM has proven its safety [22, 23]. However, possible adverse effects of TEM have to be addressed. The use of a rectoscope with a 4-cm diametre, introduced transanally, has lead to substantial scepticism regarding impact on anorectal functioning. In earlier studies, we already showed TEM to be superior to total mesorectal excision regarding post-operative defecation disorders, although this did not result in improved quality of life [24]. In the present study, TEM resulted in improved faecal continence as measured by the FISI. This apparent paradox may be attributed to pre-operative tumour symptoms such as mucinous or bloody discharge, prolapse, tenesmi and/or urge, giving rise to incontinence-like symptoms. Post-operative improvement of continence was most significant in tumours within 7 cm from the dentate line but disappeared in our study in tumours above 7 cm from the dentate line. Kreis et al. [25] performed manometric studies after TEM and found a significant reduction in anal resting pressure 1 year post-operative and a temporary reduction in anal squeezing pressure, resulting in a temporary rise in urge–incontinence. Kennedy et al. [26] found a significant reduction in anal resting pressure 6 weeks after TEM. This reduction was significantly correlated with duration of the procedure, but mean continence score was not changed after TEM. Both of the above studies however did not use validated questionnaires on faecal continence, and therefore comparison with our study is difficult. Cataldo et al. [17] reported on the impact of TEM on functional outcome and incontinence-specific quality of life using the same questionnaires. No significant alteration was found in faecal continence after TEM. The discrepancy between both studies may be explained by the relative short interval between the TEM procedure and post-operative questioning of 6 weeks in the Cataldo series. Also, in his study, indications for TEM were heterogeneous which may have influenced results. The positive effect of TEM on faecal continence in our series may be explained by the differences in pre-operative FISI score between both studies (10 versus 2.4), depicting more continence problems among the patients in our series. Another explanation may be the differences in tumour distance from the dentate line (present series median 7 cm, Cataldo series 11 cm). Also, in our series, tumours were larger (median 20 cm2 versus 8.75 cm2). Because tumours were larger in our series, more extensive resections were performed, often in tumours located within the sphincter apparatus. These latter resections were already shown to influence rectoinhibitory reflex, reflex sphincter contraction, rectal sensitivity and compliance [16]. Further analysis within our series upon this issue showed only tumour distance from the dentate line of less than 7 cm to be a significant contributing factor. These results however are based upon low number of patients and therefore solid conclusions cannot be drawn. Although in our study TEM resulted in a significant improvement in continence, the post-operative FISI was still worse compared to the Cataldo series (7 versus 2.4). Regarding quality of life, Cataldo found TEM was of no significant influence. In our series, mean general quality of life score from the patients’ perspective, EQ-VAS, was significantly higher after TEM. This improvement could not be explained by improved FISI scores but probably by lower pre-operative EQ-VAS scores as compared to healthy controls. Another explanation may be the rejoice phenomena, that is, patients are relieved the tumour has been excised, and in most cases an adenoma was found [27]. However, because of the low number of invasive carcinomas in our series this is purely theoretical. The societal value of general quality of life, EQ-5D, remained unchanged. Measuring quality of life using the FIQL questionnaires resulted in a significant improvement in two of the four FIQL domains (embarrassment and lifestyle). Moreover, the domains of lifestyle, coping and behaviour and embarrassment were all significantly correlated with the FISI.
In conclusion, how are these results to be interpreted? This study supports the hypothesis that rectal tumours give rise to incontinence-like symptoms, especially in low-lying rectal tumours. After the tumour is excised using the TEM technique, faecal continence improves. TEM itself does not improve continence but also does not deteriorate faecal continence. Mean quality of life from the patients’ perspective following TEM is improved.
Based on, as we know, the only two studies addressing anorectal functioning and quality of life after TEM in one study, it can be concluded that TEM does not impair faecal continence. Also, quality of life is not negatively influenced by the TEM procedure itself, and therefore TEM is the procedure of choice in all rectal adenomas.

functional outcome
quality of life
tem
local excision
rectal neoplasms


Treatment of migraine attacks based on the interaction with the trigemino-cerebrovascular system

Primary headaches such as migraine are among the most prevalent neurological disorders, affecting up to one-fifth of the adult population. The scientific work in the last decade has unraveled much of the pathophysiological background of migraine, which is now considered to be a neurovascular disorder. It has been discovered that the trigemino-cerebrovascular system plays a key role in migraine headache pathophysiology by releasing the potent vasodilator calcitonin gene-related peptide (CGRP). This neuropeptide is released in parallel with the pain and its concentration correlates well with the intensity of the headache. The development of drugs of the triptan class has provided relief for the acute attacks but at the cost of, mainly cardiovascular, side effects. Thus, the intention to improve treatment led to the development of small CGRP receptor antagonists such as olcegepant (BIBN4096BS) and MK-0974 that alleviate the acute migraine attack without acute side events. The purpose of this review is to give a short overview of the pathological background of migraine headache and to illustrate the mechanisms behind the actions of triptans and the promising CGRP receptor blockers.
The trigemino-cerebrovascular system
Pain-sensitive structures such as the intracranial vessels and the meninges, especially the dura mater, are supplied with sensory nerve fibers. In fact they are innervated by the ophthalmic ramus of the first branch of the trigeminal nerve that arises from pseudounipolar neurons located in the trigeminal ganglion. Those neurons project onto second order sensory neurons in the trigmenial nucleus caudalis (TNC) in the brain stem and its related extensions down to the C2-level called the trigeminocervical complex [1]. From this region a signal is transmitted to the ventroposterior thalamus leading to activation in cortical areas, including frontal cortex, insulae and cingulate cortex. This results in the experience of pain (Fig. 1). In addition, a migraine active region has been pointed out in the brainstem by using positron emission tomography (PET) [2–4]. Thus, an involvement of the trigemino-cerebrovascular system in the transmission of nociceptive information to the central nervous system is a viable position.
Fig. 1In migraine attacks, sensory nerves, which innervate the cerebral blood vessels, are activated to antidromically release calcitonin gene-related peptide. They originate from the first branch of the trigeminal nerve that arises from pseudounipolar neurons located in the trigeminal ganglion (TG). Those neurons project onto second order sensory neurons in the trigeminal nucleus caudalis (TNC) from where a signal is transmitted to the cortex
The trigemino-cerebrovascular reflex
There is a dense supply of CGRP-containing nerve fibers originating in the first division of the trigeminal ganglion that innervate intracranial vessels. Experimental studies have shown that induction of vasoconstriction provoked rapid normalization of the vascular tone due to––in part––reflex activation of the trigeminal nerve. Vasoconstriction triggers an antidromic release of sensory trigeminal neuropeptides, mainly CGRP, being a strong intracranial dilatator [5, 6]. After denervation there is no alteration in the contractile response of the vessel but the time to attain initial basal tone is markedly prolonged. This is of importance as headache may be initiated by a spreading wave of depression of cortical neurons resulting in contraction of some parts of the cerebral circulation. This phenomenon has now been shown in man [7] and could activate the trigeminal vascular system to release CGRP to reestablish the vascular tone. Thus, the trigemino-cerebrovascular reflex is probably part of a counter-balancing system, which is able to mediate dilatation of brain vessels and sustain cerebral blood flow [8]. This may assure the maintenance of local brain blood flow within normal limits as the cerebral circulation requires high and constant flow. Activation of the neurons in the trigeminal ganglion leads on the other hand to an activation of neurons in the trigeminocervical complex that project to the central nervous system (CNS) and mediate the central aspects of pain [1, 9].
Calcitonin gene-related peptide
CGRP is a neuropeptide consisting of 37 amino acids and is predominantly expressed in the nervous system. It originates from the calcitonin gene encoding calcitonin and CGRP depending upon alternative splicing.
Histochemical studies of cerebral perivascular nerve fibers and human trigeminal ganglia revealed that almost half of the cell bodies contain CGRP [10, 11]. Although many other signal molecules are located in the trigeminal ganglion, CGRP is the most abundant in man [12] and can be seen as a marker for trigeminal activity. Furthermore it is the only transmitter reliably released in acute migraine attacks at present. There was a marked increase in CGRP levels during migraine attacks, but no changes could be observed for other neuropeptides such as vasoactive intestinal peptide (a marker for parasympathetic activity), neuropeptide Y (sympathetic activity) and substance P (sensory activity) when their levels were analyzed in the cranial venous outflow during migraine attacks [8, 13].
In migraine attacks elicited within clinical studies by administration of nitroglycerin a good correlation has been described between increased levels of CGRP and the intensity of the headache [14, 15]. But it is noteworthy to mention that nitroglycerin elicits head pain, but not always genuine migraine attacks. There may be a need for the subject to be in a “prone phase”, where the trigemino-cerebrovascular system might be hyper-reactive as it was demonstrated in cluster headache [16].
Triptans
The increased cranial venous levels of CGRP observed in conjunction with migraine attacks thus indicate activation of the trigeminal system. Administration of sumatriptan––belonging to the highly effective anti-migraine drugs called triptans or 5-HT1B/1D agonists-results in alleviation of the headache and normalization of the cranial venous CGRP levels [8]. They are thought to act mainly as powerful vasoconstrictors of cerebral vessels via 5-HT1B receptors, which have been shown to be expressed in the medial smooth muscle cell layer of the human middle meningeal [17] and cerebral arteries [18].
In addition, triptans may bind to presynaptic 5-HT1B/1D receptors on trigeminal afferents to inhibit nerve activity and hence reduce CGRP release. 5-HT1B/1D receptors were found on trigeminal sensory fibers [19], trigeminal ganglion cells [20] and in the trigemino-cerebrovascular system in man [21]. Furthermore, the 5-HT1B/1D receptors were co-localized with CGRP in the human trigeminal ganglion [20].
Autoradiographic studies using labeled zolmitriptan confirmed the presence of 5-HT1B/1D receptors in certain brain stem nuclei that are involved in cranial nociceptive processing [22] and are likely to be activated during migraine.
Thus, it can be presumed that a potential relaxation of intracranial blood vessels during migraine attacks can be normalized by triptans, which activate the cerebrovascular 5-HT1B receptors and thereby cause vasoconstriction. By stimulating 5-HT1B/1D receptors, triptans inhibit the release of CGRP from the perivascular nerve terminals and the trigeminal ganglion cells. Activation of 5-HT1B/1D receptors at the level of the CNS (trigeminal nucleus caudalis) may interrupt central aspects of the headache process.
Recently the relationship between pharmacokinetic and pharmacodynamic aspects of triptans in migraine was investigated. It has been observed that subcutaneous administration of sumatriptan is more effective than oral application. The difference in efficacy is most likely due to the rapid initial rise in plasma level after subcutaneous administration [23]. Furthermore migraine attacks may influence gastric stasis leading to delayed absorption of drugs [24, 25]. Thus, to increase efficacy of treatment, attention should be paid to the development of migraine drugs, which can be quickly absorbed.
Triptans cause sometimes cardiovascular adverse effects as they also act as constrictors in coronary arteries. But triptans are considered as more powerful vasoconstrictors in human isolated cerebral arteries than in coronary arteries. They are at least threefold more potent [26]. The reason for the higher potency and efficiency of triptans in intracranial arteries could be due to at least three differences. First there is a higher number of 5-HT1B receptors in those vessels compared to peripheral arteries [18, 27]. Secondly there are few, if any, 5-HT2A receptors in intracranial vessels whereas the 5-HT2A receptor population dominates in coronary arteries [27]. Thirdly, there might be potential differences in coupling of the 5-HT1B receptors between cerebral and coronary arteries. In consideration of the fact that triptans are weak constrictors in coronary arteries [28, 29] and have little impact on coronary blood flow, the clinical consequences in healthy subjects are minor. However, small contractions may anyhow have proportionally greater impact in diseased artery with luminal narrowing. Triptans are hence contra-indicated in patients with known cardiovascular disease.
Sensitization in migraine
Treatment with triptans achieves good results in patients suffering from sensitization. Sensitization is defined as an increased afferent activity in response to an unchanged stimulus [30] and both peripheral and central sensitization may play a role in migraine attacks. Peripheral sensitization of meningeal perivascular nociceptors arising from trigeminal neurons can clinically explain phenomena like intracranial hypersensitivity (worsening pain during physical activity) and the throbbing element in migraine pain. This may be due to normally innocuous meningeal nociceptors becoming hyper-responsive to rhythmic fluctuation in intracranial pressure that is produced by normal arterial pulsations [31, 32].
Central sensitization of nociceptive second order neurons in the trigeminocervical complex can be the reason for extracranial tenderness and cutaneous allodynia. The latter is an abnormal sensory state in which otherwise innocuous stimuli are sensed as painful [31].
Two-thirds of patients develop allodynia during or even after a migraine attack [33]. Based on clinical studies new guidelines for triptan treatment in migraine has been established. They include that allodynia-free patients can receive good results from triptan treatment at any time during the migraine attack. Furthermore, patients suffering from allodynia can expect excellent results from triptans before allodynia gets established. Therefore they should take triptans as early as possible during the attack. After the onset of allodynia, migraineurs can still benefit from triptans, but pain relief may be incomplete [34–36].
However, a slightly different view has been expressed in a recent study [37]. After subcutaneous administration of sumatriptan, there was no difference in alleviation of migraine pain irrespective if the drug was given early or late. Therefore, it was suggested that the sensitization is of minor importance and that the relatively poor effect of sumatriptan late in migraine attacks is primarily due to gastric stasis [37].
CGRP receptors
Local vasodilatation and release of CGRP from the vascular nerve fiber endings––caused by electrical field stimulation or capsaicin treatment [38, 39]––are attenuated by administration of triptans, but also by application of CGRP receptor blockers [40]. The proof that CGRP plays a key role in migraine was provided by demonstrating that the potent CGRP receptor antagonist olcegepant (BIBN4096) could abort acute migraine attacks to a comparable degree as sumatriptan [41, 42]. In addition, CGRP receptors are located at several sites, which play an important role during migraine, including the cerebrovasculature [43–45], the trigeminocervical complex within the brainstem [46] and the trigeminal ganglion [47].
Functional CGRP receptors consist of three components, namely a seven-transmembrane domain G-protein coupled receptor called calcitonin receptor-like receptor (CLR), a receptor activity-modifying protein (RAMP) and a receptor component protein (RCP). CLR shares 55% sequence identity with the calcitonin receptor. Co-expression of CLR with RAMP1 composes the CGRP receptor, while co-expression of CLR with either RAMP2 or RAMP3 represents the adrenomedullin (AM) receptor or possibly a combined receptor (CGRP and AM). Apart from contributing to the receptor specificity, RAMP1 is required for glycosylation and transport of CLR to the plasma membrane [48–50]. In addition, RAMP1 seems to be functionally rate limiting for CGRP receptor activity in the trigeminovascular system. It was proposed that elevated RAMP1 expression could sensitize the trigeminal ganglion of individuals to CGRP actions such as CGRP release and neurogenic inflammation [47].
The third component of the CGRP receptor, the accessory protein RCP, is required for proper biological function as it is involved in coupling the receptor to downstream signaling pathways like the protein kinase A (PKA) pathway [51]. Activation of CGRP receptors in cultured trigeminal ganglion cells increased the cAMP levels, which in turn led to elevated CGRP promoter activity and increased CGRP mRNA levels. These results point to an autoactivation of CGRP expression and indicate that elevated release of CGRP in the trigeminal system may create a self-sustaining feedback loop [47].
CGRP receptor antagonists olcegepant and MK-0974
The excellent correlation between CGRP release and migraine headache has long pointed towards the potential usefulness of a specific CGRP receptor antagonist in the treatment of primary headaches [9, 52]. Thus, a series of small molecule CGRP receptor blockers were developed. Among those is olcegepant, which demonstrates extremely high affinity for human CGRP receptors, even higher affinity than the endogenous ligand [53]. Therefore it was selected for further pharmacological investigations. Olcegepant was shown to be a pure antagonist for the human CGRP receptor and to be selective against a broad panel of enzymes and receptors such as calcitonin, amylin or adrenomedullin receptors [54].
In isolated human temporal, middle meningeal and cerebral arteries olcegepant effectively inhibits CGRP induced dilatation [44, 55]. That may be due to the block of the CGRP receptor as mRNA for CLR and RAMP1–3 has been found in human middle meningeal arteries, cerebral arteries as well as in microvessels [56] and CGRP receptor components could be detected in the human cerebral vasculature [43]. In addition, olcegepant dose-dependently inhibited vasodilatation induced by electrical stimulation of the trigeminal ganglion in primates [54].
One major advantage of a CGRP receptor blocker is the lack of vasoconstrictor ability. But blockade of the receptor of a strong vasodilatation involves a theoretical risk of causing both peripheral and cerebral vasoconstriction, especially as high affinity binding sites for CGRP have been reported in the heart, liver, spleen, intestine, urogenital system, skeletal muscles, and the skin [57–59]. But olcegepant in doses up to 10 mg did not cause any clinical relevant changes of vital signs like blood pressure and pulse rate, in the ECG or in forearm blood flow in healthy volunteers [60]. Detailed studies of peripheral arteries such as mesenteric and coronary arteries [44, 61] revealed that those arteries show weaker responses to CGRP and lower degree of antagonism by olcegepant. This is possibly related to a lower number of CGRP receptors. In addition, a study with seven healthy volunteers disclosed that olcegepant had no influence on cerebral blood flow or on the blood flow velocity in the middle cerebral artery [62]. It was concluded that CGRP receptor blockade has no effect on the cerebral circulation in man.
However, it is still not clear where olcegepant acts as a CGRP antagonist. It has been reported to inhibit (1) dilatation of dural arteries initiated by CGRP [63], (2) CGRP-induced hypotension [40], and (3) increase in facial blood flow after trigeminal ganglion stimulation [54]. In contrast, olcegepant did not significantly inhibit changes in tone of cerebral arterioles or of local cortical cerebral blood flow [40]. Those results indicate that olcegepant poorly penetrates the blood-brain barrier, but is very effective in preventing vasodilatation of vessels without this feature [64]. Thus, this CGRP receptor blocker was suggested to exert its antagonistic effects mainly extracerebral, which correlates with results from a study of healthy volunteers [65]. In contrast, it was recently shown that CGRP-induced relaxation of the rat middle cerebral artery can only be inhibited by abluminally administered olcegepant as olcegepant was prevented from reaching the CGRP receptors by the arterial endothelium [66]. These results suggest that the anti-migraine effect of this CGRP receptor blocker is on the abluminal side of the blood-brain barrier. This is supported by the fact that olcegepant only has an antimigraine effect in high doses.
Anyhow, olcegepant was effective in a dose of 2.5 mg in the treatment of acute migraine attacks [41]. The overall treatment effect revealed a responder rate (a moderate to severe headache going to mild or no headache) of 60% at 2 h after the start of the infusion compared with 27% in the placebo group (Fig. 2). Thus, intravenous olcegepant was found to be as efficacious as oral sumatriptan in the relief of acute attacks of migraine [42].
Fig. 2The anti-migraine effect of the CGRP receptor antagonists MK-0974 and olcegepant and the triptans rizatriptan and sumatriptan compared to placebo at 2-h pain relief. The overall treatment effect of MK-0974 (p = 0.015) and rizatriptan (p = 0.010) showed significance versus placebo (left part) [69]. A dose of 2.5 mg of olcegepant also showed significant superiority over placebo (p = 0.001) [41]. Data of sumatriptan from another study [42] were added to demonstrate the similar efficacy of olcegepant compared to triptans
Significant superiority over placebo was seen regarding the pain-free rate at 2 h, the sustained pain relief at 24 h and the improvement of nausea, photophobia and phonophobia (Fig. 3). The response was still rising at 4 h pointing at a long duration of action. At 24 h the pain-free rate was better than with triptans, suggesting a lower grade of rebound.
Fig. 3Comparison of the sustained pain free-rate at 24 h of the CGRP receptor antagonists MK-0974 and olcegepant, the triptans rizatriptan and sumatriptan, and placebo. MK-0974 displayed superior efficacy versus placebo (p < 0.001) [69]. In order to compare the efficacy of olcegepant to a triptan, data of sumatriptan from another study [42] were added
The rate of recurrence was 19% as compared to a placebo rate of 46%. This is clearly lower than the recurrence rate of triptans which usually is around 28% [42].
Safety and tolerability studies also showed that olcegepant is well tolerated. Adverse events were, in general, minor and dose-dependent. Transient mild paresthesia was the single most frequent adverse event [60].
One major disadvantage of olcegepant remains in its structure. It is a hydrophilic molecule, has poor penetration across the blood-brain barrier and is not suitable as an oral drug. Considering the crucial advantages of an oral formulation of a drug, a small-molecule, non-peptide CGRP antagonist called MK-0974 was recently developed for the acute treatment of migraine attacks [67]. MK-0974 was, among others, tested in a noninvasive pharmacodynamic model in rhesus monkeys, in which endogenous CGRP release and a vasodilatatory response are achieved by topical application of capsaicin to the forearm of the rhesus monkey [68]. Infusion of different CGRP receptor antagonists inhibited the increase in local dermal blood flow and showed effectiveness with MK-0974 being the most potent among them (>3.5-fold) [67]. In addition, MK-0974 displayed a low clearance (7.0 mL min−1 kg−1) and a good i.v. half-life (2.8 h). The compound was finally chosen as a clinical candidate, when it showed >10,000-fold selectivity in assays including more than 160 receptors, transporters, and enzymes [67].
Recently, MK-0974 has been tested in a randomized, double-blind, placebo-and active-controlled, outpatient study with a two-stage, dose-ranging design to test safety and effectiveness [69]. Three hundred and thirty patients suffering from migraine with and without aura were treated with MK-0974 (in doses of 25–600 mg), rizatriptan (10 mg) or placebo. As doses below 300 mg were insufficient, they were discontinued. Thus, the overall treatment effect was defined as the average of the 300, 400, and 600 mg doses and showed significance vs. placebo on the primary endpoint (p = 0.015), which was defined as pain relief at 2 h after treatment (Fig. 2).
MK-0974 displayed also superior efficacy vs. placebo for secondary endpoints such as pain freedom at 2 h (overall treatment effect p < 0.001), sustained pain relief at 24 h (overall treatment effect p < 0.001) or sustained pain freedom at 24 h (overall treatment effect p < 0.001) (Fig. 3). In addition, MK-0974 provided relief of migraine-associated symptoms such as photophobia, phonophobia and nausea.
Rizatriptan (10 mg) was used as an active control and demonstrated effectiveness vs. placebo (p = 0.010) on the primary endpoint defined as pain relief at 2 h. By comparison, 300 and 600 mg doses of MK-0974 appeared to work as well as rizatriptan or showed even numerical superiority regarding the sustained pain freedom at 24 h and the sustained pain relief at 24 h.
Concerning tolerability and safety, MK-0974 was generally well-tolerated. The reported side effects were mainly nausea, dizziness and somnolence, but the incidence of adverse experiences seemed to be comparable to the placebo group. In addition, there was no increase in adverse events with increasing dose.
Thus, MK-0974 showed only mild side effects, but significant relief of migraine pain 2 h after treatment compared to placebo and the relief was sustained through 24 h [69].
Conclusion
The novel CGRP receptor antagonists appear to be effective in the treatment of moderate and severe migraine attacks. They do not only provide pain relief at 2 h, but also show efficacy regarding the secondary endpoints such as pain freedom, improvement of associated symptoms and functional disability [41, 69]. In addition, they show a prolonged effect of action compared to the triptans. That may suggest not only less rebound headache but perhaps also a prophylactic possibility. Furthermore, only minor adverse effects were reported and no cardiovascular side effects could be seen so far. This is probably due to the absence of vasoconstrictor properties and it also suggests that vasoconstriction is not necessary to abort acute migraine attacks. This lack of direct vasoconstrictor activity may represent an important advantage over the triptans. Further development of drugs against the CGRP receptor will offer a new promising way of treatment.

trigemino-cerebrovascular system
cgrp
triptan
olcegepant
mk-0974