Analogues of DNA minor groove cross-linking agents incorporating aminoCBI, an amino derivative of the duocarmycins: Synthesis, cytotoxicity, and potential as payloads for antibody-drug conjugates.

A Pd-catalysed amination method is used to convert seco-CBI, a synthetic analogue of the alkylating subunit of the duocarmycin natural products, from the phenol to amino form. This allows efficient enantioselective access to the more potent S enantiomer of aminoCBI and its incorporation into analogues of DNA minor groove cross-linking agents. Evaluation in a panel of nine human tumour cell lines shows that the bifunctional agents containing aminoCBI are generally less cytotoxic than their phenolCBI analogues and more susceptible to P-glycoprotein-mediated resistance. However, all bifunctional agents are potent cytotoxins, some in the sub-pM IC50 range, with in vitro properties that compare favourably with established microtubule-targeted ADC payloads.

Spatially-resolved pharmacokinetic/pharmacodynamic modelling of bystander effects of a nitrochloromethylbenzindoline hypoxia-activated prodrug.

PURPOSE: Hypoxia-activated prodrugs (HAPs) have the potential for eliminating chemo- and radiation-resistant hypoxic tumour cells, but their activity is often compromised by limited penetration into hypoxic zones. Nitrochloromethylbenzindoline (nitroCBI) HAPs are reduced in hypoxic cells to highly cytotoxic DNA minor groove alkylating aminoCBI metabolites. In this study, we investigate whether a lead nitroCBI, SN30548, generates a significant bystander effect through the diffusion of its aminoCBI metabolite and whether this compensates for any diffusion limitations of the prodrug in tumour tissue. METHODS: Metabolism and uptake of the nitroCBI in oxic and anoxic cells, and diffusion through multicellular layer cultures, was characterised by LC-MS/MS. To quantify bystander effects, clonogenic cell killing of HCT116 cells was assessed in multicellular spheroid co-cultures comprising cells transfected with cytochrome P450 oxidoreductase (POR) or E. coli nitroreductase NfsA. Spatially-resolved pharmacokinetic/pharmacodynamic (PK/PD) models, parameterised by the above measurements, were developed for spheroids and tumours using agent-based and Green's function modelling, respectively. RESULTS: NitroCBI was reduced to aminoCBI by POR under anoxia and by NfsA under oxia, and was the only significant cytotoxic metabolite in both cases. In spheroid co-cultures comprising 30% NfsA-expressing cells, non-metabolising cells were as sensitive as the NfsA cells, demonstrating a marked bystander effect. Agent-based PK/PD models provided good prediction of cytotoxicity in spheroids, while use of the same parameters in a Green's function model for a tumour microregion demonstrated that local diffusion of aminoCBI overcomes the penetration limitation of the prodrug. CONCLUSIONS: The nitroCBI HAP SN30548 generates a highly efficient bystander effect through local diffusion of its active metabolite in tumour tissue.

Factors affecting recovery after arthroscopic labral debridement of the hip.

PURPOSE: The purpose of this study was to develop and validate a model predicting whether patients would have shorter-than-typical or longer-than-typical recoveries after hip arthroscopy for labral tears. METHODS: We retrospectively reviewed 268 cases of hip arthroscopy implemented between 2000 and 2007 by 2 orthopaedic surgeons at our institution. The development cohort consisted of patients with magnetic resonance angiography-identified labral tears and a history and physical examination consistent with either labral pathology or loose bodies. Univariate analysis targeted preoperative patient characteristics correlated with the risk of longer-than-typical recoveries. Multivariate logistic regression was applied to generate an algorithm predicting risk of longer-than-typical recovery based on baseline characteristics. The algorithm was tested in the validation sample of 52 patients who were treated in 2007 and was found to be valid. RESULTS: Five predictors for longer-than-typical recovery were identified: Workers' Compensation status, female gender, use of pain medications, presence of a limp, and presence of a lateral labral tear. The multivariate algorithm was developed and successfully validated. CONCLUSIONS: This study identifies many new predictors of recovery, and it also corroborates those that have already been identified. The 5 predictors for longer-than-typical recovery identified by our validated multivariate algorithm were Workers' Compensation status, female gender, use of pain medications, presence of a limp, and presence of a lateral labral tear. LEVEL OF EVIDENCE: Level IV, therapeutic case series.

Predictors of bone loss in revision total knee arthroplasty.

Revision total knee arthroplasty (RTKA) requires preoperative planning to enable the reconstruction of bony deficiencies. The objective of this project was to identify predictors of bone loss management at RTKA based on the preoperative failure mode and patient demographics known preoperatively. We retrospectively reviewed 245 consecutive RTKA procedures in which the same revision knee system was utilized. Patient demographic and treatment data were recorded, and locations of bone loss were identified based on the reconstructive management. We identified significant predictors for use of femoral augments at all four positions. Several predictors significantly predisposed to use of a thick (>19 mm) polyethylene; however, no predictors of tibial augments were significant. Although the reconstruction of bone loss is primarily based on the intraoperative assessment, these findings may provide additional information to help the surgeon prepare for difficult revision procedures.

Synthesis and structure-activity relationships of soluble 8-substituted 4-(2-chlorophenyl)-9-hydroxypyrrolo[3,4-c]carbazole-1,3(2H,6H)-diones as inhibitors of the Wee1 and Chk1 checkpoint kinases.

Pyrrolo[3,4-c]carbazoles bearing solubilising basic side chains at the 8-position retain potent Wee1 and Chk1 inhibitory properties in isolated enzyme assays, and evidence of G2/M checkpoint abrogation in several cellular assays. Co-crystal structure studies confirm that the primary binding to the Wee1 enzyme is as described previously, with the C-8 side chains residing in an area of bulk tolerance.

Synthesis and structure-activity relationships of N-6 substituted analogues of 9-hydroxy-4-phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H)-diones as inhibitors of Wee1 and Chk1 checkpoint kinases.

A series of N-6 substituted 9-hydroxy-4-phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H)-diones were prepared from N-substituted (5-methoxyphenyl)ethenylindoles. The target compounds were tested for their ability to inhibit the G2/M cell cycle checkpoint kinases, Wee1 and Chk1. Analogues with neutral or cationic N-6 side chains were potent dual inhibitors. Acidic side chains provided potent (average IC(50) 0.057 microM) and selective (average ratio 223-fold) Wee1 inhibition. Co-crystal structures of inhibitors bound to Wee1 show that the pyrrolo[3,4-c]carbazole scaffold binds in the ATP-binding site, with N-6 substituents involved in H-bonding to conserved water molecules. HT-29 cells treated with doxorubicin and then target compounds demonstrate an active Cdc2/cyclin B complex, inhibition of the doxorubicin-induced phosphorylation of tyrosine 15 of Cdc2 and abrogation of the G2 checkpoint.

Antitubercular Nitroimidazoles Revisited: Synthesis and Activity of the Authentic 3-Nitro Isomer of Pretomanid.

A published study of structural features associated with the aerobic and anaerobic activities of 4- and 5-nitroimidazoles had found that the 3-nitro isomer of pretomanid, 8, displayed interesting potencies, including against nitroreductase mutant Mycobacterium tuberculosis. However, recent nuclear magnetic resonance analyses of two trace byproducts, isolated from early process optimization studies toward a large-scale synthesis of pretomanid, raised structural assignment queries, particularly for 8, stimulating further investigation. Following our discovery that the reported compound was a 6-nitroimidazooxazole derivative, we developed a de novo synthesis of authentic 8 via nitration of the chiral des-nitro imidazooxazine alcohol 26 in trifluoroacetic or acetic anhydride, and verified its identity through an X-ray crystal structure. Unfortunately, 8 displayed no antitubercular activity (MICs > 128 µM), whereas the second byproduct (3'-methyl pretomanid) was eight-fold more potent than pretomanid in the aerobic assay. These findings further clarify target specificities for bicyclic nitroimidazoles, which may become important in the event of any future clinical resistance.

Influence of a Basic Side Chain on the Properties of Hypoxia-Selective Nitro Analogues of the Duocarmycins: Demonstration of Substantial Anticancer Activity in Combination with Irradiation or Chemotherapy.

A new series of nitro analogues of the duocarmycins was prepared and evaluated for hypoxia-selective anticancer activity. The compounds incorporate 13 different amine-containing side chains designed to bind in the minor groove of DNA while spanning a wide range of base strength from pKa 9.64 to 5.24. The most favorable in vitro properties were associated with strongly basic side chains, but the greatest in vivo antitumor activity was found for compounds containing a weakly basic morpholine. This applies to single-agent activity and for activity in combination with irradiation or chemotherapy (gemcitabine or docetaxel). In combination with a single dose of γ irradiation 50 at 42 µmol/kg eliminated detectable clonogens in some SiHa cervical carcinoma xenografts, and in combination with gemcitabine using a well-tolerated multidose schedule, the same compound caused regression of all treated A2780 ovarian tumor xenografts. In the latter experiment, three of seven animals receiving the combination treatment were completely tumor free at day 100.

4-Phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione inhibitors of the checkpoint kinase Wee1. Structure-activity relationships for chromophore modification and phenyl ring substitution.

High-throughput screening has identified a novel class of inhibitors of the checkpoint kinase Wee1, which have potential for use in cancer chemotherapy. These inhibitors are based on a 4-phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione template and have been shown by X-ray crystallography to bind at the ATP site of the enzyme. An extensive study of the effects of substitution around this template has been carried out, which has identified substituents which lead to improvements in potency and selectivity for Wee1. While retention of the maleimide ring and pendant 4-phenyl group is necessary for potency, replacement of the carbazole nitrogen by oxygen is well tolerated and results in improved Wee1 selectivity against the related checkpoint kinase Chk1. Wee1 potency and selectivity are also enhanced by the incorporation of lipophilic functionality at the 2'-position of the 4-phenyl ring, and Wee1 selectivity against Chk1 is favored by C3-C5 alkyl substitution of the carbazole nitrogen. These studies provide a basis for the design of active analogues of the pyrrolocarbazole lead with improved physical properties.

Robotic testing of proximal tibio-fibular joint kinematics for measuring instability following total knee arthroplasty.

Pain secondary to instability in total knee arthroplasty (TKA) has been shown to be major cause of early failure. In this study, we focused on the effect of instability in TKA on the proximal tibio-fibular joint (PTFJ). We used a robotics model to compare the biomechanics of the PTFJ in the native knee, an appropriately balanced TKA, and an unbalanced TKA. The tibia (n = 5) was mounted to a six-degree-of-freedom force/torque sensor and the femur was moved by a robotic manipulator. Motion at the PTFJ was recorded with a high-resolution digital camera system. After establishing a neutral position, loading conditions were applied at varying flexion angles (0°, 30°, and 60°). These included: internal/external rotation (0 Nm, ±5 Nm), varus/valgus (0 Nm, ±10 Nm), compression (100 N, 700 N), and posterior drawer (0 N, 100 N). With respect to anterior displacement, external rotation had the largest effect (coefficient = 0.650; p < 0.0001). Polyethylene size as well as the interaction between polyethylene size and flexion consistently showed substantial anterior motion. Flexion and mid-flexion instability in TKA have been difficult to quantify. While tibio-femoral kinematics is the main aspect of TKA performance, the effects on adjacent tissues should not be overlooked. Our data show that PTFJ kinematics are affected by the balancing of the TKA.

Prospective randomized evaluation of the need for blood transfusion during primary total hip arthroplasty with use of a bipolar sealer.

BACKGROUND: Blood loss during total hip arthroplasty can be substantial and may lead to adverse patient outcomes and increased health-care costs. Many blood-management options are available for these procedures. The purpose of the present study was to test the hemostatic efficacy of a bipolar sealer used during total hip arthroplasty in order to determine whether its use results in significantly lower transfusion requirements and/or improved clinical, functional, and health-related quality-of-life outcomes in healthy patients. METHODS: This prospective, single-center, randomized, double-blinded study was designed to enroll a total of 140 patients. Patients with a low preoperative hemoglobin level or a history of bleeding abnormalities and other medical conditions were excluded. Patients were randomized to either the treatment arm (radiofrequency energy with use of the Aquamantys 6.0 bipolar sealer) or control arm (standard Bovie electrocautery). The primary outcome measure was the transfusion requirement, and the secondary outcome measures were intraoperative estimated blood loss, postoperative hemoglobin levels, perioperative narcotic usage, length of hospital stay, postoperative pain scores, and postoperative function as measured with the Harris hip score and the Short Form-12 quality-of-life score. RESULTS: Seventy-one patients were assigned to the treatment arm, and sixty-nine were assigned to the control arm. The mean number of units of blood transfused for all patients in the study and control arms were 0.38 and 0.44, respectively (p = 0.72). The transfusion requirements were similar in the two groups, with fifteen of seventy-one patients in the treatment arm and fourteen of sixty-nine patients in the control arm requiring a transfusion (p = 0.9). No significant differences were detected between the groups in terms of estimated blood loss, postoperative hemoglobin levels, perioperative narcotic usage, length of hospital stay, postoperative pain scores, Harris hip scores, or Short Form-12 scores. CONCLUSIONS: In this patient population, there were no significant differences between the treatment and control groups in terms of the need for blood transfusions or overall blood loss. Given these findings, we have discontinued the use of this bipolar sealing device in uncomplicated primary total hip arthroplasty patients at our institution. LEVEL OF EVIDENCE: Therapeutic Level I. See Instructions to Authors for a complete description of levels of evidence.

Preparation and antitumour properties of the enantiomers of a hypoxia-selective nitro analogue of the duocarmycins.

Racemic 2-{[1-(chloromethyl)-5-nitro-3-{5-[2-(dimethylamino)ethoxy]indol-2-carbonyl}-1,2-dihydro-3H-benzo[e]indol-7-yl]sulfonyl}aminoethyl dihydrogen phosphate, a synthetic nitro derivative of the duocarmycins, is a hypoxia-selective prodrug active against radiation-resistant tumour cells at nontoxic doses in mice. An intermediate in the synthesis of this prodrug was resolved by chiral HPLC and the absolute configuration assigned by X-ray crystallography. The intermediate was used to prepare the prodrug's enantiomers, and also the enantiomers of the active nitro and amino metabolites. In vitro analysis in the human cervical carcinoma cell line SiHa showed that both nitro enantiomers are hypoxia-selective cytotoxins, but the "natural" S enantiomer is at least 20-fold more potent. Examination of extracellular amino metabolite concentrations demonstrated no enantioselectivity in the hypoxia-selective reduction of nitro to amino. Low levels of amino derivative were also found in aerobic cell suspensions, sufficient to account for the observed oxic toxicity of the nitro form. At an equimolar dose in SiHa-tumour bearing animals, the (-)-R enantiomer of the prodrug was inactive, while the (+)-S enantiomer caused significantly more hypoxic tumour cell kill than the racemate. At this dose, the combination of (+)-S-prodrug and radiation eliminated detectable colony-forming cells in four out of five treated tumour-bearing animals.

The use of navigation in total knee arthroplasty for patients with extra-articular deformity.

Computer-assisted navigation for total knee arthroplasty provides high technology instrumentation that may improve the technique for restoring the normal lower limb mechanical axis. This study evaluated the use of computer-assisted navigation in 7 patients (9 total knee arthroplasties) with a radiographic femoral extra-articular deformity. Postoperatively, the mechanical axis deviated medially by a mean of 1.3 degrees +/- 0.9 degrees (range, -0.2 degrees to 2.5 degrees ). Early patient outcomes showed an increase in the average preoperative to postoperative Knee Society Scores (from 62 to 92, P < .05), function scores (from 52 to 83, P < .05), and range of motion (from 4 degrees -74 degrees to 0.6 degrees -98 degrees , P < .05). These results support the use of computer-assisted navigation as effective high technology instrumentation in recreating an acceptable mechanical axis in patients with distorted anatomical landmarks.

Tricyclic [1,2,4]triazine 1,4-dioxides as hypoxia selective cytotoxins.

A series of novel tricyclic triazine-di- N-oxides (TTOs) related to tirapazamine have been designed and prepared. A wide range of structural arrangements with cycloalkyl, oxygen-, and nitrogen-containing saturated rings fused to the triazine core, coupled with various side chains linked to either hemisphere, resulted in TTO analogues that displayed hypoxia-selective cytotoxicity in vitro. Optimal rates of hypoxic metabolism and tissue diffusion coefficients were achieved with fused cycloalkyl rings in combination with both the 3-aminoalkyl or 3-alkyl substituents linked to weakly basic soluble amines. The selection was further refined using pharmacokinetic/pharmacodynamic model predictions of the in vivo hypoxic potency (AUC req) and selectivity (HCD) with 12 TTO analogues predicted to be active in vivo, subject to the achievement of adequate plasma pharmacokinetics.