The dinitrobenzamide mustard prodrugs, PR-104A and SN27686, for use in a novel MNDEPT cancer prodrug therapy approach.

Directed enzyme prodrug therapy is a highly promising anti-cancer strategy. However, the current technology is limited by inefficient prodrug activation and the dose-limiting toxicity associated with the prodrugs being tested; to overcome these limitations, the dinitrobenzamide mustard prodrugs, PR-104A and SN27686, have been developed. The present study will assess both of these prodrugs for their potential uses in a novel magnetic-nanoparticle directed enzyme prodrug therapy strategy by determining their kinetic parameters, assessing the products formed during enzymatic reduction using HPLC and finally their ability to cause cell death in the ovarian cancer cell line, SK-OV-3. It was shown for the first time that the dinitrobenzamide mustard prodrugs are able to be reduced by the genetically modified nitroreductases, NfnB-cys and YfkO-cys, and that these enzyme/prodrug combinations can induce a significant cell death in the SK-OV-3 cell line, highlighting the potential for both enzyme/prodrug combinations for use in magnetic-nanoparticle directed enzyme prodrug therapy.

Optimized method for analysis of ethanolamines, hydrolysis products of nitrogen mustards, from urine samples using LC-MS/MS.

Highly polar ethanolamines (EAs), excreted in urine, are hydrolysis products of nitrogen mustards (NMs), which are prohibited by the Chemical Weapons Convention (CWC). The methods established for biological matrices are essential for verification analysis of the CWC related chemicals. This paper describes a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method developed for qualitative and quantitative analysis of EAs, N-ethyldiethanolamine (EDEA), N-methyldiethanolamine (MDEA) and triethanolamine (TEAOH) from urine samples. After optimization of sample preparation and chromatographic conditions, the method was fully validated. Silica solid-phase extraction (SPE) cartridges and a porous graphite carbon (PGC) column were selected for validation studies. The method is linear from 5 to 500, 0.5 to 250, and 0.5 to 500 ng/mL for TEAOH, EDEA, and MDEA, respectively. It is also precise and accurate. A minimum sample amount of 0.5 mL urine was used. The limit of quantification using this approach was 0.4, 5.5, and 6.3 ng/mL for MDEA, EDEA and TEAOH, respectively. The combination of the PGC column and high pH eluents in analysis retained and separated the studied EAs. Retention times were 2.11, 2.56 and 2.98 min for MDEA, EDEA and TEAOH, respectively. The method is applicable for verification analysis of the CWC.

Amustaline-glutathione pathogen-reduced red blood cell concentrates for transfusion-dependent thalassaemia.

Transfusion-dependent thalassaemia (TDT) requires red blood cell concentrates (RBCC) to prevent complications of anaemia, but carries risk of infection. Pathogen reduction of RBCC offers potential to reduce infectious risk. We evaluated the efficacy and safety of pathogen-reduced (PR) Amustaline-Glutathione (A-GSH) RBCC for TDT. Patients were randomized to a blinded 2-period crossover treatment sequence for six transfusions over 8-10 months with Control and A-GSH-RBCC. The efficacy outcome utilized non-inferiority analysis with 90% power to detect a 15% difference in transfused haemoglobin (Hb), and the safety outcome was the incidence of antibodies to A-GSH-PR-RBCC. By intent to treat (80 patients), 12·5 ± 1·9 RBCC were transfused in each period. Storage durations of A-GSH and C-RBCC were similar (8·9 days). Mean A-GSH-RBCC transfused Hb (g/kg/day) was not inferior to Control (0·113 ± 0·04 vs. 0·111 ± 0·04, P = 0·373, paired t-test). The upper bound of the one-sided 95% confidence interval for the treatment difference from the mixed effects model was 0·005 g/kg/day, within a non-inferiority margin of 0·017 g/kg/day. A-GSH-RBCC mean pre-transfusion Hb levels declined by 6·0 g/l. No antibodies to A-GSH-RBCC were detected, and there were no differences in adverse events. A-GSH-RBCCs offer potential to reduce infectious risk in TDT with a tolerable safety profile.

Striatal spreading depolarization: Possible implication in levodopa-induced dyskinetic-like behavior.

OBJECTIVE: Spreading depolarization (SD) is a transient self-propagating wave of neuronal and glial depolarization coupled with large membrane ionic changes and a subsequent depression of neuronal activity. Spreading depolarization in the cortex is implicated in migraine, stroke, and epilepsy. Conversely, spreading depolarization in the striatum, a brain structure deeply involved in motor control and in Parkinson's disease (PD) pathophysiology, has been poorly investigated. METHODS: We characterized the participation of glutamatergic and dopaminergic transmission in the induction of striatal spreading depolarization by using a novel approach combining optical imaging, measurements of endogenous DA levels, and pharmacological and molecular analyses. RESULTS: We found that striatal spreading depolarization requires the concomitant activation of D1-like DA and N-methyl-d-aspartate receptors, and it is reduced in experimental PD. Chronic l-dopa treatment, inducing dyskinesia in the parkinsonian condition, increases the occurrence and speed of propagation of striatal spreading depolarization, which has a direct impact on one of the signaling pathways downstream from the activation of D1 receptors. CONCLUSION: Striatal spreading depolarization might contribute to abnormal basal ganglia activity in the dyskinetic condition and represents a possible therapeutic target. © 2019 International Parkinson and Movement Disorder Society.

Discovery and Optimization of Novel Hydrogen Peroxide Activated Aromatic Nitrogen Mustard Derivatives as Highly Potent Anticancer Agents.

We describe several new aromatic nitrogen mustards with various aromatic substituents and boronic esters that can be activated with H2O2 to efficiently cross-link DNA. In vitro studies demonstrated the anticancer potential of these compounds at lower concentrations than those of other clinically used chemotherapeutics, such as melphalan and chlorambucil. In particular, compound 10, bearing an amino acid ester chain, is selectively cytotoxic toward breast cancer and leukemia cells that have inherently high levels of reactive oxygen species. Importantly, 10 was 10-14-fold more efficacious than melphalan and chlorambucil for triple-negative breast-cancer (TNBC) cells. Similarly, 10 is more toxic toward primary chronic-lymphocytic-leukemia cells than either chlorambucil or the lead compound, 9. The introduction of an amino acid side chain improved the solubility and permeability of 10. Furthermore, 10 inhibited the growth of TNBC tumors in xenografted mice without obvious signs of general toxicity, making this compound an ideal drug candidate for clinical development.

Drug-DNA adducts as biomarkers for metabolic activation of the nitro-aromatic nitrogen mustard prodrug PR-104A.

PR-104A is a clinical-stage nitrogen mustard prodrug that is activated for DNA alkylation by reduction of a nitro group to the corresponding hydroxylamine (PR-104H) or amine (PR-104M). Metabolic reduction is catalysed by flavoreductases such as cytochrome P450 oxidoreductase (POR) under hypoxia, or by aldo-ketoreductase 1C3 (AKR1C3) independently of hypoxia. The unstable reduced metabolites are challenging to measure in biological samples, and biomarkers of the metabolic activation of PR-104A have not been used in the clinical evaluation of PR-104 to date. Here, we employ a selected reaction monitoring mass spectrometry assay for DNA crosslinks to assess the capacity of human cancer cells to bioactivate PR-104A. We also test whether the more abundant DNA monoadducts could be used for the same purpose. DNA monoadducts and crosslinks from PR-104A itself, and from its reduced metabolites, accumulated over 4 h in AKR1C3-expressing TF1 erythroleukaemia cells under hypoxia, whereas intracellular concentrations of unstable PR-104H and PR-104M reached steady state within 1 h. We then varied rates of PR-104A reduction by manipulating hypoxia or reductase expression in a panel of cell lines, in which AKR1C3 and POR were quantified by targeted proteomics. Hypoxia or reductase overexpression induced large increases in PR-104A sensitivity (inhibition of proliferation), DNA damage response (γH2AX formation), steady-state concentrations of PR-104H/M and formation of reduced drug-DNA adducts but not DNA adducts retaining the dinitro groups of PR-104A. The fold-change in the sum of PR-104H and PR-104M correlated with the fold-change in reduced crosslinks or monoadducts (R2 = 0.87 for both), demonstrating their potential for assessing the capacity of cancer cells to bioactivate PR-104A.

Does C60 fullerene act as a transporter of small aromatic molecules?

C60 fullerene is reported to directly interact with biomolecules, such as aromatic mutagens or anticancer drugs. Therefore, it is extensively studied for its potential application in the fields of drug delivery and chemoprevention. Understanding the nature of fullerene-drugs interactions might contribute to optimization and modification of the existing chemotherapy systems. Possible interactions between ICR-191, a model acridine mutagen, with well-established biophysical properties and mutagenic activity, and C60 fullerene aqueous solution were investigated by broad range of biophysical methods, such as Dynamic Light Scattering, Isothermal Titration Calorimetry, and Atomic Force Microscopy. Additionally, to determine biological activity of ICR-191-C60 fullerene mixtures, Ames mutagenicity test was employed. It was demonstrated that C60 fullerene interacts non-covalently with ICR-191 and has strong affinity to bacterial membranes. The obtained results provide practical insight into C60 fullerene interactions with aromatic compounds.

Engineering a Multifunctional Nitroreductase for Improved Activation of Prodrugs and PET Probes for Cancer Gene Therapy.

Gene-directed enzyme-prodrug therapy (GDEPT) is a promising anti-cancer strategy. However, inadequate prodrugs, inefficient prodrug activation, and a lack of non-invasive imaging capabilities have hindered clinical progression. To address these issues, we used a high-throughput Escherichia coli platform to evolve the multifunctional nitroreductase E. coli NfsA for improved activation of a promising next-generation prodrug, PR-104A, as well as clinically relevant nitro-masked positron emission tomography-imaging probes EF5 and HX4, thereby addressing a critical and unmet need for non-invasive bioimaging in nitroreductase GDEPT. The evolved variant performed better in E. coli than in human cells, suggesting optimal usefulness in bacterial rather than viral GDEPT vectors, and highlighting the influence of intracellular environs on enzyme function and the shaping of promiscuous enzyme activities within the "black box" of in vivo evolution. We provide evidence that the dominant contribution to improved PR-104A activity was enhanced affinity for the prodrug over-competing intracellular substrates.

Phase I/II study of the hypoxia-activated prodrug PR104 in refractory/relapsed acute myeloid leukemia and acute lymphoblastic leukemia.

We previously demonstrated vast expansion of hypoxic areas in the leukemic microenvironment and provided a rationale for using hypoxia-activated prodrugs. PR104 is a phosphate ester that is rapidly hydrolyzed in vivo to the corresponding alcohol PR-104A and further reduced to the amine and hydroxyl-amine nitrogen mustards that induce DNA cross-linking in hypoxic cells under low oxygen concentrations. In this phase I/II study, patients with relapsed/refractory acute myeloid leukemia (n=40) after 1 or 2 prior treatments or acute lymphoblastic leukemia (n=10) after any number of prior treatments received PR104; dose ranged from 1.1 to 4 g/m(2). The most common treatment-related grade 3/4 adverse events were myelosuppression (anemia 62%, neutropenia 50%, thrombocytopenia 46%), febrile neutropenia (40%), infection (24%), and enterocolitis (14%). Ten of 31 patients with acute myeloid leukemia (32%) and 2 of 10 patients with acute lymphoblastic leukemia (20%) who received 3 g/m(2) or 4 g/m(2) had a response (complete response, n=1; complete response without platelet recovery, n=5; morphological leukemia-free state, n=6). The extent of hypoxia was evaluated by the hypoxia tracer pimonidazole administered prior to a bone marrow biopsy and by immunohistochemical assessments of hypoxia-inducible factor alpha and carbonic anhydrase IX. A high fraction of leukemic cells expressed these markers, and PR104 administration resulted in measurable decrease of the proportions of hypoxic cells. These findings indicate that hypoxia is a prevalent feature of the leukemic microenvironment and that targeting hypoxia with hypoxia-activated prodrugs warrants further evaluation in acute leukemia. The trial is registered at clinicaltrials.gov identifier: 01037556.

Renal human organic anion transporter 3 increases the susceptibility of lymphoma cells to bendamustine uptake.

Chronic lymphatic leukemia (CLL) is often associated with nephritic syndrome. Effective treatment of CLL by chlorambucil and bendamustine leads to the restoration of renal function. In this contribution, we sought to elucidate the impact of organic anion transporters (OATs) on the uptake of bendamustine and chlorambucil as a probable reason for the superior efficacy of bendamustine over chlorambucil in the treatment of CLL. We examined the effects of structural analogs of p-aminohippurate (PAH), melphalan, chlorambucil, and bendamustine, on OAT1-mediated [(3)H]PAH uptake and OAT3- and OAT4-mediated [(3)H]estrone sulfate (ES) uptake in stably transfected human embryonic kidney-293 cells. Melphalan had no significant inhibitory effect on any OAT, whereas chlorambucil reduced OAT1-, OAT3-, and OAT4-mediated uptake of PAH or ES down to 14.6%, 16.3%, and 66.0% of control, respectively. Bendamustine inhibited only OAT3-mediated ES uptake, which was reduced down to 14.3% of control cells, suggesting that it interacts exclusively with OAT3. The IC50 value for OAT3 was calculated to be 0.8 µM. Real-time PCR experiments demonstrated a high expression of OAT3 in lymphoma cell lines as well as primary CLL cells. OAT3-mediated accumulation of bendamustine was associated with reduced cell proliferation and an increased rate of apoptosis. We conclude that the high efficacy of bendamustine in treating CLL might be partly contributed to the expression of OAT3 in lymphoma cells and the high affinity of bendamustine for this transporter.

Endothelial Akt1 mediates angiogenesis by phosphorylating multiple angiogenic substrates.

The PI3K/Akt pathway is necessary for several key endothelial cell (EC) functions, including cell growth, migration, survival, and vascular tone. However, existing literature supports the idea that Akt can be either pro- or antiangiogenic, possibly due to compensation by multiple isoforms in the EC when a single isoform is deleted. Thus, biochemical, genetic, and proteomic studies were conducted to examine isoform-substrate specificity for Akt1 vs. Akt2. In vitro, Akt1 preferentially phosphorylates endothelial nitric oxide synthase (eNOS) and promotes NO release, whereas nonphysiological overexpression of Akt2 can bypass the loss of Akt1. Conditional deletion of Akt1 in the EC, in the absence or presence of Akt2, retards retinal angiogenesis, implying that Akt1 exerts a nonredundant function during physiological angiogenesis. Finally, proteomic analysis of Akt substrates isolated from Akt1- or Akt2-deficient ECs documents that phosphorylation of multiple Akt substrates regulating angiogenic signaling is reduced in Akt1-deficient, but not Akt2-deficient, ECs, including eNOS and Forkhead box proteins. Therefore, Akt1 promotes angiogenesis largely due to phosphorylation and regulation of important downstream effectors that promote aspects of angiogenic signaling.

Reactive oxygen species (ROS) inducible DNA cross-linking agents and their effect on cancer cells and normal lymphocytes.

Reducing host toxicity is one of the main challenges of cancer chemotherapy. Many tumor cells contain high levels of ROS that make them distinctively different from normal cells. We report a series of ROS-activated aromatic nitrogen mustards that selectively kill chronic lymphocytic leukemia (CLL) over normal lymphocytes. These agents showed powerful DNA cross-linking abilities when coupled with H2O2, one of the most common ROS in cancer cells, whereas little DNA cross-linking was detected without H2O2. Consistent with chemistry observation, in vitro cytotoxicity assay demonstrated that these agents induced 40-80% apoptosis in primary leukemic lymphocytes isolated from CLL patients but less than 25% cell death to normal lymphocytes from healthy donors. The IC50 for the most potent compound (2) was ~5 µM in CLL cells, while the IC50 was not achieved in normal lymphocytes. Collectively, these data provide utility and selectivity of these agents that will inspire further and effective applications.

Competitive interactions of anti-carcinogens with serum albumin: a spectroscopic study of bendamustine and dexamethasone with the aid of chemometrics.

Interactions between the anti-carcinogens, bendamustine (BDM) and dexamethasone (DXM), with bovine serum albumin (BSA) were investigated with the use of fluorescence and UV-vis spectroscopies under pseudo-physiological conditions (Tris-HCl buffer, pH 7.4). The static mechanism was responsible for the fluorescence quenching during the interactions; the binding formation constant of the BSA-BDM complex and the binding number were 5.14×10(5)Lmol(-1) and 1.0, respectively. Spectroscopic studies for the formation of BDM-BSA complex were interpreted with the use of multivariate curve resolution - alternating least squares (MCR-ALS), which supported the complex formation. The BSA samples treated with site markers (warfarin - site I and ibuprofen - site II) were reacted separately with BDM and DXM; while both anti-carcinogens bound to site I, the binding constants suggested that DXM formed a more stable complex. Relative concentration profiles and the fluorescence spectra associated with BDM, DXM and BSA, were recovered simultaneously from the full fluorescence excitation-emission data with the use of the parallel factor analysis (PARAFAC) method. The results confirmed that on addition of DXM to the BDM-BSA complex, the BDM was replaced and the DXM-BSA complex formed; free BDM was released. This finding may have consequences for the transport of these drugs during any anti-cancer treatment.

A novel fluorometric assay for aldo-keto reductase 1C3 predicts metabolic activation of the nitrogen mustard prodrug PR-104A in human leukaemia cells.

Aldo-keto reductase 1C3 (AKR1C3, EC 1.1.1.188) metabolises steroid hormones, prostaglandins and xenobiotics, and activates the dinitrobenzamide mustard prodrug PR-104A by reducing it to hydroxylamine PR-104H. Here, we describe a functional assay for AKR1C3 in cells using the fluorogenic probe coumberone (a substrate for all AKR1C isoforms) in conjunction with a specific inhibitor of AKR1C3, the morpholylurea SN34037. We use this assay to evaluate AKR1C3 activity and PR-104A sensitivity in human leukaemia cells. SN34037-sensitive reduction of coumberone to fluorescent coumberol correlated with AKR1C3 protein expression by immunoblotting in a panel of seven diverse human leukaemia cell lines, and with SN34037-sensitive reduction of PR-104A to PR-104H. SN34037 inhibited aerobic cytotoxicity of PR-104A in high-AKR1C3 TF1 erythroleukaemia cells, but not in low-AKR1C3 Nalm6 pre-B cell acute lymphocytic leukaemia (B-ALL) cells, although variation in PR-104H sensitivity confounded the relationship between AKR1C3 activity and PR-104A sensitivity across the cell line panel. AKR1C3 mRNA expression showed wide variation between leukaemia patients, with consistently higher levels in T-ALL than B-ALL. In short term cultures from patient-derived paediatric ALL xenografts, PR-104A was more potent in T-ALL than B-ALL lines, and PR-104A cytotoxicity was significantly inhibited by SN34037 in T-ALL but not B-ALL. Overall, the results demonstrate that SN34037-sensitive coumberone reduction provides a rapid and specific assay for AKR1C3 activity in cells, with potential utility for identifying PR-104A-responsive leukaemias. However, variations in PR-104H sensitivity indicate the need for additional biomarkers for patient stratification.

Creation and screening of a multi-family bacterial oxidoreductase library to discover novel nitroreductases that efficiently activate the bioreductive prodrugs CB1954 and PR-104A.

Two potentially complementary approaches to improve the anti-cancer strategy gene-directed enzyme prodrug therapy (GDEPT) are discovery of more efficient prodrug-activating enzymes, and development of more effective prodrugs. Here we demonstrate the utility of a flexible screening system based on the Escherichia coli SOS response to evaluate novel nitroreductase enzymes and prodrugs in concert. To achieve this, a library of 47 candidate genes representing 11 different oxidoreductase families was created and screened to identify the most efficient activators of two different nitroaromatic prodrugs, CB1954 and PR-104A. The most catalytically efficient nitroreductases were found in the NfsA and NfsB enzyme families, with NfsA homologues generally more active than NfsB. Some members of the AzoR, NemA and MdaB families also exhibited low-level activity with one or both prodrugs. The results of SOS screening in our optimised E. coli reporter strain SOS-R2 were generally predictive of the ability of nitroreductase candidates to sensitise E. coli to CB1954, and of the kcat/Km for each prodrug substrate at a purified protein level. However, we also found that not all nitroreductases express stably in human (HCT-116 colon carcinoma) cells, and that activity at a purified protein level did not necessarily predict activity in stably transfected HCT-116. These results highlight a need for all enzyme-prodrug partners for GDEPT to be assessed in the specific context of the vector and cell line that they are intended to target. Nonetheless, our oxidoreductase library and optimised screens provide valuable tools to identify preferred nitroreductase-prodrug combinations to advance to preclinical evaluation.

Diflavin oxidoreductases activate the bioreductive prodrug PR-104A under hypoxia.

The clinical agent PR-104 is converted systemically to PR-104A, a nitrogen mustard prodrug designed to target tumor hypoxia. Reductive activation of PR-104A is initiated by one-electron oxidoreductases in a process reversed by oxygen. The identity of these oxidoreductases is unknown, with the exception of cytochrome P450 reductase (POR). To identify other hypoxia-selective PR-104A reductases, nine candidate oxidoreductases were expressed in HCT116 cells. Increased PR-104A-cytotoxicity was observed in cells expressing methionine synthase reductase (MTRR), novel diflavin oxidoreductase 1 (NDOR1), and inducible nitric-oxide synthase (NOS2A), in addition to POR. Plasmid-based expression of these diflavin oxidoreductases also enhanced bioreductive metabolism of PR-104A in an anoxia-specific manner. Diflavin oxidoreductase-dependent PR-104A metabolism was suppressed >90% by pan-flavoenzyme inhibition with diphenyliodonium chloride. Antibodies were used to quantify endogenous POR, MTRR, NDOR1, and NOS2A expression in 23 human tumor cell lines; however, only POR protein was detectable and its expression correlated with anoxic PR-104A reduction (r(2) = 0.712). An anti-POR monoclonal antibody was used to probe expression using human tissue microarrays; 13 of 19 cancer types expressed detectable POR with 21% of cores (185 of 874) staining positive; this heterogeneity suggests that POR is a useful biomarker for PR-104A activation. Immunostaining for carbonic anhydrase 9 (CAIX), reportedly an endogenous marker of hypoxia, revealed only moderate coexpression (9.6%) of both CAIX and POR across a subset of five cancer types.

Initial testing of the hypoxia-activated prodrug PR-104 by the pediatric preclinical testing program.

BACKGROUND: PR-104 is rapidly hydrolyzed to PR-104A in vivo, which is activated by reduction to the corresponding 5-hydroxylamine (PR-104H) and amine (PR-104M) to produce DNA interstrand cross-links. PR-104 activation can occur via hypoxia-dependent reductases and also independently of hypoxia by aldo-keto reductase (AKR) 1C3. PROCEDURES: PR-104A was tested against the PPTP in vitro panel (10 nM to 100 µM), and PR-104 in vivo using a weekly × 6 schedule at its maximum tolerated dose (MTD) of 550 mg/kg. Subsequently PR-104 was tested at 270 and 110 mg/kg. Pharmacokinetics for PR-104 and its metabolites were determined, as were levels of AKR1C3 RNA and protein in xenografts. RESULTS: In vitro, the leukemia models were most sensitive to PR-104A. In vivo, PR-104 induced objective responses at its MTD in 21/34 solid tumor models and maintained complete responses against 7/7 acute lymphoblastic leukemia (ALL) models. At 270 mg/kg and lower dose levels, PR-104 did not induce solid tumor regressions, suggesting a steep dose-response relationship. Pharmacokinetic analysis suggests higher systemic exposures to PR-104A and its metabolites in mice compared to those achievable in patients. Levels of AKR1C3 protein did not correlate with tumor responsiveness. CONCLUSIONS: As monotherapy, PR-104 demonstrated a high level of activity against both solid tumor and ALL models at its MTD, but the activity was almost completely lost at half the MTD dose for solid tumors. Pharmacokinetic data at the PR-104 MTD from human trials suggest that PR-104 metabolites may not reach the plasma exposures in children that were associated with high-level preclinical activity.

Glucuronidation of anticancer prodrug PR-104A: species differences, identification of human UDP-glucuronosyltransferases, and implications for therapy.

PR-104, the phosphate ester of a dinitrobenzamide mustard [PR-104A; 2-((2-bromoethyl)-2-{[(2-hydroxyethyl) amino] carbonyl}-4,6-dinitroanilino)ethyl methanesulfonate], is currently in clinical trial as a hypoxia- and aldo-keto reductase 1C3 (AKR1C3)-activated prodrug for cancer therapy. Here, we investigate species (human, dog, rat, mouse) differences in metabolism to the corresponding O-glucuronide, PR-104G, and identify the human UDP-glucuronosyltransferase (UGT) isoforms responsible. After intravenous PR-104, plasma area under the concentration-time curve ratios (PR-104G/PR-104A) decreased in the order of dog (2.3) > human (1.3) > mouse (0.03) > rat (0.005). The kinetics of uridine 5'-diphosphoglucuronic acid-dependent glucuronidation by liver microsomes in vitro fitted the single-enzyme Michaelis-Menten equation with similar K(m) (∼150 µM) but differing V(max) (472, 88, 37, and 14 nmol/h/mg for dog, human, rat, and mouse, respectively), suggesting that facile glucuronidation is responsible for the anomalously rapid clearance of PR-104A in dogs. In vitro-in vivo extrapolation of PR-104A glucuronidation kinetics is consistent with this also being a major clearance pathway in humans. Recombinant UGT screening identified UGT2B7 as the only commercially available human isoform able to conjugate PR-104A, and UGT2B7 protein concentrations were highly correlated (r = 0.93) with PR-104A glucuronidation by liver microsomes from 24 individuals. The active hydroxylamine metabolite of PR-104A, PR-104H, was also glucuronidated by UGT2B7, although with slightly lower specificity and much lower rates. UGT2B7 mRNA expression was highly variable in human tumor databases. Glucuronidation of PR-104A greatly suppressed nitroreduction by AKR1C3 and NADPH-supplemented anoxic human liver S9 (9000g postmitochondrial supernatant). In conclusion, PR-104A is glucuronidated by UGT2B7 with high specificity and seems to make a major contribution to clearance of PR-104A in humans, but it also has the potential to confer resistance in some human tumors.

Bendamustine: new perspective for an old drug in lymphoproliferative disorders.

Bendamustine is an old bifunctional alkylating agent that also has potential antimetabolite properties, and only partial cross-resistance with other alkylators. Although it was synthesized in 1963 only few validated study results exist from this early period. More recently, its peculiar mechanism of action has reawakened interest in this drug that has been extensively studied in indolent non-Hodgkin lymphoma (NHL) and in chronic lymphocytic leukemia (CLL). Experience has also been reported in high-grade NHL and Hodgkin disease. Based on its unique structure, various strategies can be used for mechanism-based combination chemotherapeutic regimens with bendamustine. Moreover, data indicates that bendamustine when combined with rituximab is a valid therapeutic choice for patients with CLL or low-grade NHL demonstrating refractoriness to standard chemotherapy regimens. Furthermore, its documented favorable toxicity profile makes it a particularly useful treatment option for elderly patients.

Optimization of the N-lost drugs melphalan and bendamustine: synthesis and cytotoxicity of a new set of dendrimer-drug conjugates as tumor therapeutic agents.

Bendamustine and melphalan are very promising alkylating drugs with applicability in the treatment of various tumoral diseases, e.g., chronic lymphocytic leukemia (CLL) or breast cancer. However, numerous adverse effects limited their use. Therefore, 1,3,5-tris(3-aminopropyl)benzene (G0) and its G1 analogue 3,5-bis(3-aminopropyl)-N-(3-{3,5-bis[3-{3,5-bis(3-aminopropyl)benzoylamino}propyl]phenyl}propyl)benzamide were selected to design cytostatic drug-dendrimer conjugates to achieve tumor cell accumulation by endocytosis as already demonstrated for platinum complexes. The dendrimers act as carriers and an N-(2-hydroxyethyl)maleimide spacer between drug and carrier should guarantee a selective release of the cytostatics in the tumor cells. The resulting cytotoxicity was determined in vitro using the human MCF-7 and MDA-MB-231 breast cancer cell lines. It was demonstrated that melphalan caused cytotoxic effects depending on its free amino group (Boc protection strongly decreased the activity) but independent of a derivation of the carboxylic group (dendrimers and spacer binding). Esterification of bendamustine with the N-(2-hydroxyethyl)maleimide spacer strongly increased the hydrolytic stability of the N-lost moiety, so antiproliferative effects were yet observed in vitro.