Anaemia as a risk stratification tool for symptomatic patients referred via the two-week wait pathway for colorectal cancer.

Introduction Anaemia is associated with cancer. In 2014 a new form was introduced in our department requesting a haemoglobin (Hb) result on every two-week wait referral for suspected colorectal cancer (CRC). The aim of this study was to review the impact of this intervention. In particular, the significance of any evidence of anaemia (without additional indices) was investigated. Methods A review was conducted of 1,500 consecutive suspected CRC referrals recorded prospectively over a 10-month period. Data on demographics, referral Hb, referral criteria and outcomes were analysed. Anaemia was defined according to World Health Organization criteria (Hb <120g/l for women, Hb <130g/l for men). Results Overall, 1,015 patients were eligible for inclusion in the study. Over a third (38.2%) were documented as anaemic on referral. These patients were three times more likely to be diagnosed with CRC than non-anaemic patients (odds ratio [OR]: 3.22, 95% confidence interval [CI]: 1.87-5.57). Using a more stringent threshold (Hb <100g/l for women and <110g/l for men), they were four times more likely to have CRC (OR: 4.27, 95% CI: 2.35-7.75). Almost a quarter (23.7%) were actually anaemic at the time of referral but not referred with anaemia. In this subgroup, there was a 2.8-fold increase in risk of CRC diagnosis compared with non-anaemic patients (adjusted OR: 2.77, 95% CI: 1.55-4.95). Conclusions Nearly a quarter of patients not referred with iron deficiency anaemia had evidence of anaemia and this was still associated with a higher rate of CRC detection. A full blood count alone might help to risk stratify symptoms such as change in bowel habit in patients on urgent pathways and identify those cases most likely to benefit from invasive investigation.

Methylene-linked bis-phenylbenzimidazoles - a new scaffold to target telomeric DNA/RNA hybrid duplex.

We report a series of novel methylene-linked bis-phenylbenzimidazoles intercalators that stabilize telomeric DNA/RNA hybrid (tDRH) structures by up to 7.2 °C at a 1 µM ligand concentration while having negligible affinity for DNA/DNA duplexes, although with a low affinity for quadruplex DNA. We have used molecular modelling studies to rationalize this selectivity, concluding that the methylene spacer between the terminal benzimidazole and phenylene moieties plays a key role in facilitating the bis-intercalating process. This scaffold may be used to develop chemical tools or new therapeutics to selectively target the telomeric DNA/RNA duplex without affecting normal genomic DNA.

Non-small cell lung cancer with distal cutaneous metastases in a patient with a previously treated colorectal carcinoma.

Cutaneous manifestations of visceral carcinomas are scarce, occurring in around 0.7-12% of internal malignancies. Lung cancer is one of the most common sources of skin metastasis, particularly in male patients. We present a case of cutaneous metastasis in a man with concurrent lung lesions and a previously treated colorectal carcinoma. Immunohistochemistry markers for both skin and lung lesions were strongly positive for carcinoembryonic antigen and cytokeratin 20, suggesting an intestinal primary tumour. However, colonoscopy excluded new and metastatic bowel lesions. After multidisciplinary team meetings, which reviewed the clinical, radiological and immunohistochemistry findings, it was concluded to be a non-small cell lung cancer with skin metastasis. This case presented an interesting diagnostic challenge, and highlighted the importance of cross-specialty liaison and investigation to reach the correct diagnosis.

ABCB1 genetic polymorphism influences the pharmacology of the new pyrrolobenzodiazepine derivative SJG-136.

ATP-binding cassette transporter P-glycoprotein (ABCB1) is responsible for the multidrug resistance (MDR1) phenotype observed in cancer cells. SJG-136, a new pyrrolobenzodiazepine dimer, is a sequence-dependent DNA crosslinking agent and substrate of ABCB1. We previously showed that colon cancer cell lines expressing high levels of ABCB1 showed a lower sensitivity to SJG-136. Here, we show that in 3T3 isogenic fibroblasts, ABCB1 genetic polymorphism differentially affects ABCB1 gene expression and transport function. However, this genotype-phenotype relationship was not observed in immortalized lymphocytes, which expressed 10- to 1000-fold less ABCB1 than colon cancer cell lines. Consistent with this, the cytotoxicity of SJG-136 in 3T3 fibroblasts was affected by ABCB1 genetic polymorphism but not in immortalized lymphocytes. ABCB1 genetic polymorphism is therefore likely to affect drug sensitivity in tissues expressing high levels of the transporter and in which significant variability is observed.

Fludarabine-mediated suppression of the excision repair enzyme ERCC1 contributes to the cytotoxic synergy with the DNA minor groove crosslinking agent SJG-136 (NSC 694501) in chronic lymphocytic leukaemia cells.

In this study, we set out to establish whether fludarabine could enhance the DNA interstrand crosslinking capacity of SJG-136 in primary human chronic lymphocytic leukaemia (CLL) cells and thereby offer a rationale for its clinical use in combination with SJG-136. SJG-136 rapidly induced DNA crosslinking in primary CLL cells which was concentration-dependent. Further, the level of crosslinking correlated with sensitivity to SJG-136-induced apoptosis (P=0.001) and higher levels of crosslinking were induced by the combination of SJG-136 and fludarabine (P=0.002). All of the samples tested (n=40) demonstrated synergy between SJG-136 and fludarabine (mean combination index (CI)=0.54+/-0.2) and this was even retained in samples derived from patients with fludarabine resistance (mean CI=0.62+/-0.3). Transcription of the excision repair enzyme, ERCC1, was consistently increased (20/20) in response to SJG-136 (P<0.0001). In contrast, fludarabine suppressed ERCC1 transcription (P=0.04) and inhibited SJG-136-induced ERCC1 transcription when used in combination (P=0.001). Importantly, the ability of fludarabine to suppress ERCC1 transcription correlated with the degree of synergy observed between SJG-136 and fludarabine (r(2)=0.28; P=0.017) offering a mechanistic rationale for the synergistic interaction. The data presented here provides a clear indication that this combination of drugs may have clinical utility as salvage therapy in drug-resistant CLL.

Preclinical pharmacology of the pyrrolobenzodiazepine (PBD) monomer DRH-417 (NSC 709119).

The pyrrolobenzodiazepine monomer DRH-417 is a member of the anthramycin group of anti-tumor antibiotics that bind covalently to the N2 of guanine within the minor groove of DNA. DRH-417 emerged from the EORTC-Drug Discovery Committee and NCI 60 cell line in vitro screening programs as a potent antiproliferative agent with differential sensitivity towards certain cancer types such as melanoma, breast and renal cell carcinoma (mean IC(50) = 3 nM). DRH-417 was therefore tested for in vivo activity. The maximum tolerated dose (MTD) was established as 0.5 mg/kg given i.p. Marked anti-tumor activity was seen in two human renal cell cancers, one breast cancer and a murine colon tumor model (p<0.01). A selective HPLC (LC/MS) analytical method was developed and plasma pharmacokinetics determined. At a dose of 0.5 mg kg(-1), the plasma AUC was 540 nM h (197.1 ng h ml(-1)) and the peak plasma concentration (171 nM [62.4 ng ml(-1)]) occurred at 30 min., reaching doses levels well above those needed for in vitro antiproliferative activity. Genomic profiling of in vivo sensitive tumors revealed that the latter have an activated insulin-like growth factor signaling pathway.

Influence of P-glycoprotein expression on in vitro cytotoxicity and in vivo antitumour activity of the novel pyrrolobenzodiazepine dimer SJG-136.

SJG-136 is a novel pyrrolobenzodiazepine dimer analogue that acts as a minor-groove interstrand DNA cross-linking agent. The present study investigated the impact of ABCB1 (mdr-1) expression on the activity of SJG-136 using both in vitro and in vivo systems. SJG-136 was highly potent in the colon cancer cell lines HCT-116, HT-29 and SW620 (IC50 0.1-0.3 nM). However, HCT-8 and HCT-15 cells expressing significant levels of mdr-1 were less sensitive (IC50 2.3 and 3.7 nM, respectively) using a SRB assay. The cytotoxicity was increased in HCT-15 and A2780(AD) in presence of 5 microg/ml verapamil. Mdr-1 mRNA expression was determined by qRT-PCR and correlated to SJG-136 IC50s (r2=0.86, P=0.0001). Isogenic 3T3 cells expressing mdr-1 cDNA (3T3 pHamdr-1) were less sensitive to SJG-136 than the parental 3T3 cells (IC50 208 and 6.3 nM, respectively). Finally, SJG-136 (120 microg/kg/d dx5) was highly active against A2780 xenografts (SGD=275) but not A2780(AD) xenografts (SGD=67).

Synthesis of the first example of a C2-C3/C2'-C3'-endo unsaturated pyrrolo[2,1-c][1,4]benzodiazepine dimer.

We report the first example of a C2-C3/C2'-C3'-endo unsaturated pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer 16 synthesised through a new and efficient route, thus establishing that C2-C3-endo unsaturation enhances both cytotoxicity and DNA-binding affinity in A-Ring-linked PBD dimers but to a lesser extent than C2/C2'-exo-unsaturation. This new route has allowed the preparation of multi-gram quantities of the related clinical candidate 1 and should lead to more structurally diverse PBD dimer analogues.

Design, synthesis, and evaluation of a novel pyrrolobenzodiazepine DNA-interactive agent with highly efficient cross-linking ability and potent cytotoxicity.

A novel sequence-selective pyrrolobenzodiazepine (PBD) dimer 5 (SJG-136) has been developed that comprises two C2-exo-methylene-substituted DC-81 (3) subunits tethered through their C8 positions via an inert propanedioxy linker. This symmetric molecule is a highly efficient minor groove interstrand DNA cross-linking agent (XL(50) = 0.045 microM) that is 440-fold more potent than melphalan. Thermal denaturation studies show that, after 18 h incubation with calf thymus DNA at a 5:1 DNA/ligand ratio, it increases the T(m) value by 33.6 degrees C, the highest value so far recorded in this assay. The analogous dimer 4 (DSB-120) that lacks substitution/unsaturation at the C2 position elevates melting by only 15.1 degrees C under the same conditions, illustrating the effect of introducing C2-exo-unsaturation which serves to flatten the C-rings and achieve a superior isohelical fit within the DNA minor groove. This behavior is supported by molecular modeling studies which indicate that (i) the PBD units are covalently bonded to guanines on opposite strands to form a cross-link, (ii) 5 has a greater binding energy compared to 4, and (iii) 4 and 5 have equivalent binding sites that span six base pairs. Dimer 5 is significantly more cytotoxic than 4 in a number of human ovarian cancer cell lines (e.g., IC(50) values of 0.0225 nM vs 7.2 nM, respectively, in A2780 cells). Furthermore, it retains full potency in the cisplatin-resistant cell line A2780cisR (0.024 nM), whereas 4 loses activity (0.21 microM) with a resistance factor of 29.2. This may be due to a lower level of inactivation of 5 by intracellular thiol-containing molecules. A dilactam analogue (21) of 5 that lacks the electrophilic N10-C11/N10'-C11' imine moieties has also been synthesized and evaluated. Although unable to interact covalently with DNA, 21 still stabilizes the helix (Delta T(m) = 0.78 degrees C) and has significant cytotoxicity in some cell lines (i.e., IC(50) = 0.57 microM in CH1 cells), presumably exerting its effect through noncovalent interaction with DNA.

Design and synthesis of novel pyrrolobenzodiazepine (PBD) prodrugs for ADEPT and GDEPT.

Three N10-(4-nitrobenzyl)carbamate-protected PBD prodrugs (9a, 9b and 15) have been synthesized and evaluated for potential use in nitroreductase-based ADEPT and GDEPT therapies. An approximately 100-fold activation was observed for the DC-81 prodrug 9a.

Effect of C2-exo unsaturation on the cytotoxicity and DNA-binding reactivity of pyrrolo[2,1-c][1,4]benzodiazepines.

A series of novel C2-exo unsaturated pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) has been synthesised via a versatile pro-C2 ketone precursor. C2-exo-unsaturation enhances both DNA-binding reactivity and in vitro cytotoxic potency.

Effect of C2/C3-endo unsaturation on the cytotoxicity and DNA-binding reactivity of pyrrolo[2,1-c][1,4]benzodiazepines.

A series of novel C2,C3-endo unsaturated pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) has been synthesised via cleavage of the N10-Alloc protecting group from appropriate precursors. Biophysical and biological evaluations show that the presence of C2/C3-endo unsaturation in the PBD C-ring enhances both DNA-binding reactivity and in vitro cytotoxic potency.

The gain-of-function G389R variant of the beta1-adrenoceptor does not influence blood pressure or heart rate response to beta-blockade in hypertensive subjects.

Mutation scanning of the beta1-adrenoceptor gene has identified a polymorphism, G389R, that markedly affects G-protein coupling of the receptor and resulting cAMP production. We have investigated the effect of this functionally active polymorphism on clinical response to beta-adrenoceptor blockade. Two cohorts of untreated hypertensive patients randomly assigned to a beta1-selective beta-blocker at the start of antihypertensive therapy were studied retrospectively to see if the G389R polymorphism influenced the response in terms of blood pressure and heart rate. The blood pressure and heart rate responses to treatment were assessed 4 weeks later and compared with the G389R genotype, ascertained by PCR/restriction fragment length polymorphism. The falls in blood pressure and heart rate for the first group (n = 92) by genotype were: GG, 20.1 +/- 3.5/13.9 +/- 2.7 mmHg (systolic/diastolic blood pressure), 18.4 +/- 2.2 beats/min; GR, 20.0 +/- 2.2/15.0 +/- 1.3 mmHg, 16.5 +/- 1.5 beats/min; RR, 20.8 +/- 2.3/13.4 +/- 1.1 mmHg, 16.0 +/- 1.4 beats/min. For the second group (n = 55) the corresponding falls were: GG, 17.0 +/- 4.3/11.2 +/- 3.4 mmHg, 12.0 +/- 3.5 beats/min; GR, 16.6 +/- 1.8/14.4 +/- 1.1 mmHg, 13.1 +/- 2.1 beats/min; RR, 18.0 +/- 1.6/13.0 +/- 1.4 mmHg, 14.4 +/- 1.4 beats/min. The G389R genotype also failed to have a significant effect on pretreatment blood pressure or heart rate in either group. These data suggest that, despite clear functional differences between the G389R receptor variants expressed in vitro, the polymorphism does not affect the haemodynamic response of hypertensive subjects to chronic beta1-adrenoceptor blockade.

Synthesis, in vitro antiproliferative activity, and DNA-binding properties of hybrid molecules containing pyrrolo[2,1-c][1, 4]benzodiazepine and minor-groove-binding oligopyrrole carriers.

The synthesis, biological activity, and DNA-binding properties of a series of four hybrids prepared by combining polypyrrole minor groove binders and pyrrolo[2,1-c][1,4]benzodiazepine (PBD) 13, related to the naturally occurring anthramycin (3) and DC-81 (4), have been described, and structure-activity relationships have been discussed. These hybrids 22-25 contain from one to four pyrrole units, respectively. To investigate sequence selectivity and stability of drug/DNA complexes, DNase I footprinting and arrested polymerase chain reaction (PCR) were performed on human c-myc oncogene, estrogen receptor gene, and human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR) gene sequences. The antiproliferative activity of the hybrids has been tested in vitro on human myeloid leukemia K562 and T-lymphoid Jurkat cell lines and compared to antiproliferative effects of the natural product distamycin A 1, its tetrapyrrole homologue 17, DC 81 (4), and the PBD methyl ester 12. The results obtained demonstrate that the hybrids 22-25 exhibit different DNA-binding activity with respect to both distamycin A 1 and PBD 12. In addition, a direct relationship was found between number of pyrrole rings present in the hybrids 22-25 and stability of drug/DNA complexes. With respect to antiproliferative effects, it was found that the increase in the length of the polypyrrole backbone leads to an increase of in vitro antiproliferative effects, i.e., the hybrid 25 containing the four pyrroles is more active than 22, 23, and 24 both against K562 and Jurkat cell lines.

Design, synthesis, and evaluation of a novel sequence-selective epoxide-containing DNA cross-linking agent based on the pyrrolo[2, 1-c][1,4]benzodiazepine system.

Synthetic routes have been investigated to prepare a novel C8-epoxide-functionalized pyrrolo[2,1-c][1,4]benzodiazepine 6 as a potential sequence-selective DNA cross-linking agent (Wilson et al. Tetrahedron Lett. 1995, 36, 6333-6336). A successful synthesis was accomplished via a 10-step route involving a pro-N10-Fmoc cleavage method that should have general applicability to other pyrrolobenzodiazepine (PBD) molecules containing acid- or nucleophile-sensitive groups. During the course of this work, a one-pot reductive cyclization procedure for the synthesis of PBD N10-C11 imines from nitro dimethyl acetals was also discovered, although this method results in C11a racemization which can reduce DNA binding affinity and cytotoxicity. The target epoxide 6 was shown by thermal denaturation studies to have a significantly higher DNA-binding affinity than the parent DC-81 (3) or the C8-propenoxy-PBD (15), which is structurally similar but lacks the epoxide moiety. The time course of effects upon thermal denaturation indicated a rapid initial binding phase followed by a slower phase consistent with the stepwise cross-linking of DNA observed for a difunctional agent. This was confirmed by an electrophoretic assay which demonstrated efficient induction of interstrand cross-links in plasmid DNA at concentrations >1 microM. Higher levels of interstrand cross-linking were observed at 24 h compared to 6 h incubation. A Taq polymerase stop assay indicated a preference for binding to guanine-rich sequences as predicted for bis-alkylation in the minor groove of DNA by epoxide and imine moieties. The pattern of stop sites could be partly rationalized by molecular modeling studies which suggested low-energy models to account for the observed binding behavior. The epoxide PBD 6 was shown to have significant cytotoxicity (45-60 nM) in the A2780, CH1, and CH1cis(R) human ovarian carcinoma cell lines and an IC(50) of 0.2 microM in A2780cis(R). The significant activity of 6 in the cisplatin-resistant CH1cis(R) cell line (IC(50) = 47 nM) gave a resistance factor of 0.8 compared to the parent cell line, demonstrating no cross-resistance with the major groove cross-linking agent cisplatin.

Effect of A-ring modifications on the DNA-binding behavior and cytotoxicity of pyrrolo[2,1-c][1,4]benzodiazepines.

Several A-ring-modified analogues of the DNA-binding antitumor agent DC-81 (5) have been synthesized in order to study structure-reactivity/cytotoxicity relationships. For two molecules (23 and 30) the modifications required the addition of a fourth ring to give the novel dioxolo[4,5-h]- and dioxano[5,6-h]pyrrolo[2,1-c][1, 4]benzodiazepin-11-one (PBD) ring systems, respectively. Another three analogues (34, 38, and 48) have the native benzenoid A-ring replaced with pyridine, diazine, or pyrimidine rings to give the novel pyrrolo[2,1-c][1,4]pyridodiazepine, pyrrolo[2,1-c][1, 4]diazinodiazepine, and pyrrolo[2,1-c][1,4]pyrimidinodiazepine systems, respectively. The other new analogues (16a,b) have extended chains at the C8-position of the DC-81 structure. During the synthesis of these compounds, a novel tin-mediated regiospecific cleavage reaction of the dioxole intermediate 18 was discovered, leading to the previously unknown iso-DC-81 (20). In addition, an unusual simultaneous nitration-oxidation reaction of 4-(3-hydroxypropoxy)-3-methoxybenzoic acid (8) was found to produce 3-(4-carboxy-2-methoxy-5-nitrophenoxy)propanoic acid (9), a key intermediate, in high yield. In general, the results of cytotoxicity and DNA-binding studies indicated that none of the changes made to the A-ring of the PBD system significantly improved either binding affinity or cytotoxicity in comparison to DC-81. This result suggests that the superior potency of natural products such as anthramycin (1), tomaymycin (2), and sibiromycin (3) is due entirely to differences in C-ring structure, and in particular exo or endo unsaturation at the C2-position and C2-substituents containing unsaturation. This study also provided information regarding the influence of A-ring substitution pattern on the relative stability of the interconvertible N10-C11 carbinolamine, carbinolamine methyl ether, and imine forms of PBDs.

Elevated maternal serum relaxin concentrations throughout pregnancy in singleton gestations after superovulation.

OBJECTIVE: To test the hypothesis that superovulation results in elevated maternal circulating relaxin concentrations throughout the second and third trimesters of pregnancy, independent of the pattern of hCG secretion. METHODS: Two groups of women with singleton gestations were studied: a group of nine women who achieved pregnancy after stimulation with human menopausal gonadotropin and a group of six women who achieved pregnancy without prior stimulation. Peripheral blood samples were drawn approximately every 5 weeks throughout the second and third trimesters. Serum relaxin concentrations were measured using a human relaxin-specific enzyme-linked immunosorbent assay; hCG was measured by an immunofluorometric assay. RESULTS: The stimulated group had significantly higher relaxin levels throughout pregnancy (P=.007, multivariate analysis of variance) than did nonstimulated controls. The mean relaxin level in stimulated patients was 1.78 ng/mL (95% confidence interval [CI] 1.5, 2.17) and in nonstimulated subjects the level was 0.73 ng/mL (95% CI 0.59, 1.25). Spline fits demonstrated that stimulated patients had higher relaxin levels throughout the second and third trimesters. There was no significant difference in hCG concentrations between the two groups (P=.61). CONCLUSION: In singleton gestations after superovulation, maternal serum relaxin concentrations are significantly higher throughout the second and third trimesters of pregnancy. These differences are independent of the pattern of hCG secretion. It appears that luteal relaxin secretion is controlled by factors in addition to hCG.