Phase II study of the oxygen saturation curve left shifting agent BW12C in combination with the hypoxia activated drug mitomycin C in advanced colorectal cancer

BW12C (5-[2-formyl-3-hydroxypenoxyl] pentanoic acid) stabilizes oxyhaemoglobin, causing a reversible left-shift of the oxygen saturation curve (OSC) and tissue hypoxia. The activity of mitomycin C (MMC) is enhanced by hypoxia. In this phase II study, 17 patients with metastatic colorectal cancer resistant to 5-fluorouracil (5-FU) received BW12C and MMC. BW12C was given as a bolus loading dose of 45 mg kg−1over 1 h, followed by a maintenance infusion of 4 mg kg−1h−1for 5 h. MMC 6 mg m−2was administered over 15 min immediately after the BW12C bolus. The 15 evaluable patients had progressive disease after a median of 2 (range 1–4) cycles of chemotherapy. Haemoglobin electrophoresis 3 and 5 h after the BW12C bolus dose showed a fast moving band consistent with the BW12C-oxyhaemoglobin complex, accounting for approximately 50% of total haemoglobin. The predominant toxicities – nausea/vomiting and vein pain – were mild and did not exceed CTC grade 2. Liver31P magnetic resonance spectroscopy of patients with hepatic metastases showed no changes consistent with tissue hypoxia. The principle of combining a hypoxically activated drug with an agent that increases tissue hypoxia is clinically feasible, producing an effect equivalent to reducing tumour oxygen delivery by at least 50%. However, BW12C in combination with MMC for 5-FU-resistant colorectal cancer is not an effective regimen. This could be related to drug resistance rather than a failure to enhance cytotoxicity. © 2000 Cancer Research Campaig

Crystallographic investigation of the cooperative interaction between trimethoprim, reduced cofactor and dihydrofolate reductase.

The structure of the complex between E. coli (RT500) form I dihydrofolate reductase, the antibacterial trimethoprim and NADPH has been determined by X-ray crystallography. The inhibitor and cofactor are in mutual contact. A flexible chain segment which includes Met 20 is in contact with the inhibitor in the presence of NADPH, but more distant in its absence. By contrast, the inhibitor conformation is little changed with NADPH present. We discuss these observations with regard to the mutually cooperative binding of these ligands to the protein, and to the associated enhancement of inhibitory selectivity shown by trimethoprim for bacterial as opposed to vertebrate enzyme

Pyrrolo[2,3-d]pyrimidines and pyrido[2,3-d]pyrimidines as conformationally restricted analogues of the antibacterial agent trimethoprim.

Conformationally restricted analogues of the antibacterial agent trimethoprim (TMP) were designed to mimic the conformation of drug observed in its complex with bacterial dihydrofolate reductase (DHFR). This conformation of TMP was achieved by linking the 4-amino function to the methylene group by one- and two-carbon bridges. A pyrrolo[2,3-d]pyrimidine, a dihydro analogue, and a tetrahydropyrido[2,3-d]pyrimidine were synthesized and tested as inhibitors of DHFR. One analogue showed activity equivalent to that of TMP against DHFR from three species of bacteria. An X-ray crystal structure of this inhibitor bound to Escherichia coli DHFR was determined to evaluate the structural consequences of the conformational restriction

Refined crystal structures of Escherichia coli and chicken liver dihydrofolate reductase containing bound trimethoprim.

Refined crystal structures are reported for complexes of Escherichia coli and chicken dihydrofolate reductase containing the antibiotic trimethoprim (TMP). Structural comparison of these two complexes reveals major geometrical differences in TMP binding that may be important in understanding the stereo-chemical basis of this inhibitor's selectivity for bacterial dihydrofolate reductases. For TMP bound to chicken dihydrofolate reductase we observe an altered binding geometry in which the 2,4-diaminopyrimidine occupies a position in closer proximity (by approximately 1 A) to helix alpha B compared to the pyrimidine position for TMP or methotrexate bound to E. coli dihydrofolate reductase. One important consequence of this deeper insertion of the pyrimidine into the active site of chicken dihydrofolate reductase is the loss of a potential hydrogen bond that would otherwise form between the carbonyl oxygen of Val-115 and the inhibitor's 4-amino group. In addition, for TMP bound to E. coli dihydrofolate reductase, the inhibitor's benzyl side chain is positioned low in the active-site pocket pointing down toward the nicotinamide-binding site, whereas, in chicken dihydrofolate reductase, the benzyl group is accommodated in a side channel running upward and away from the cofactor. As a result, the torsion angles about the C5-C7 and C7-C1' bonds for TMP bound to the bacterial reductase (177 degrees, 76 degrees) differ significantly from the corresponding angles for TMP bound to chicken dihydrofolate reductase (-85 degrees, 102 degrees). Finally, when TMP binds to the chicken holoenzyme, the Tyr-31 side chain undergoes a large conformational change (average movement is 5.4 A for all atoms beyond C beta), rotating down into a new position where it hydrogen bonds via an intervening water molecule to the backbone carbonyl oxygen of Trp-24

Receptor-based design of dihydrofolate reductase inhibitors: comparison of crystallographically determined enzyme binding with enzyme affinity in a series of carboxy-substituted trimethoprim analogues.

By the use of molecular models of Escherichia coli dihydrofolate reductase (DHFR), analogues of trimethoprim (TMP) were designed which incorporated various 3'-carboxyalkoxy moieties in order to acquire ionic interactions with positively charged active-site residues. Certain of these compounds have shown exceptionally high affinity for this enzyme. For example, the 3'-(carboxypentyl)oxy analogue was found to be 55-fold more inhibitory than TMP toward E. coli DHFR (Ki = 0.024 nM vs. 1.32 nM for TMP). X-ray crystallographic studies of E. coli DHFR in binary complexes with TMP and two members of this acid-containing series of compounds defined the binding of these inhibitors and showed the carboxyl group of the latter two inhibitors to be ionically bound to Arg-57. These observations were in agreement with postulated binding modes that were based on receptor modeling

Contribution of conformational stability of hen lysozyme to induction of type 2 T-helper immune responses

It is important to identify characteristics that confer on proteins the potential to induce allergenic sensitization and allergenic disease. Protein allergens carry T-cell epitopes that are capable of inducing a type 2 T helper (Th2) cell response. There is limited information regarding factors that govern the allergenicity of proteins. We previously reported that a decrease in the conformational stability of hen-egg lysozyme (HEL) enhanced its capacity to activate HEL-specific T cells owing to the increased susceptibility to intracellular antigen processing. To determine whether the conformational stability of HEL makes for a critical contribution to allergenic sensitization in vivo, we immunized BALB/c mice with HEL derivatives of different conformational stability, but which retained a similar three-dimensional structure. The magnitude of in vivo T-cell responses, evaluated by ex vivo proliferative responses of lymph node T cells from mice primed with various HEL derivatives, was inversely correlated with conformational stability, as was interferon-γ (IFN-γ) and interleukin-4 (IL-4) production by splenic T cells in response to HEL. Immunization of the least stable derivative led to a potent IL-4 response and to immunoglobulin E (IgE) antibody production. We propose that the intrinsic allergenicity of proteins can be determined by the degree of conformational stability