Is the Fate of Clinical Candidate Arry-520 Already Sealed? Predicting Resistance in Eg5–Inhibitor Complexes

Arry-520 is an advanced drug candidate from the Eg5 inhibitor class undergoing clinical evaluation in patients with relapsed or refractory multiple myeloma. Here we show by structural analysis that Arry-520 binds stoichiometrically to the motor domain of Eg5 in the conventional allosteric loop L5 pocket in a complex that suggests the same structural mechanism as other Eg5 inhibitors. We have previously shown that acquired resistance through mutations in the allosteric binding site located at loop L5 in the Eg5 structure appears to be independent of the inhibitors' scaffold, which suggests that Arry-520 will ultimately have the same fate. When Arry-520 was assessed in two cell lines selected for the expression of either Eg5(D130A) or Eg5(L214A) STLC-resistant alleles, mutations previously shown to convey resistance to this class of inhibitors, it was inactive in both. Surprisingly, when the cells were challenged with ispinesib, another Eg5 inhibitor, the Eg5(D130A) cells were resistant, but those expressing Eg5(L214A) were strikingly sensitive. Molecular dynamics simulations suggest that subtle differences in ligand binding and flexibility in both compound and protein may alter allosteric transmission from the loop L5 site that do not necessarily result in reduced inhibitory activity in mutated Eg5 structures. Whilst we predict that cells challenged with Arry-520 in the clinical setting are likely to acquire resistance through point mutations in the Eg5 binding site, the data for ispinesib suggests that this resistance mechanism is not scaffold independent as previously thought, and new inhibitors can be designed that retain inhibitory activity in these resistant cells

Assessment of the stability of exogenous gamma hydroxybutyric acid (GHB) in stored blood and urine specimens

OBJECTIVE: The aim of this work is to test the stability of exogenous GHB in whole blood and urine samples collected from living and deceased GHB free-users, spiked with known concentrations of GHB and stored at different temperatures (–20°C, 4°C and 20°C) up to 4 weeks. MATERIALS AND METHODS: GHB was added to GHB-free ante-mortem blood and urine samples at the concentration of 5 and 10 mg/L, respectively whereas in post-mortem blood and urine specimens at 50 and 10 mg/L respectively. All samples were stored at three different temperatures: –20°C, 4°C and 20°C and extracted and analyzed at three days, 1 week, 2 weeks, 3 and 4 weeks in duplicate. No preservatives were added. GHB was quantified by GC-MS after LLE according to a previously published method. RESULTS: Post-mortem blood specimens showed a reduction of GHB levels higher than 10% only after a period of 4 weeks of storage for samples kept at +4°C and +20°C, whereas samples stored at –20°C showed a mean reduction of 8.7%. In post-mortem urine samples, there was a mean reduction of GHB levels higher than 20% at all storage temperatures, after 4 weeks of storage. Antemortem blood samples showed a reduction of GHB levels lower than 10% only after 3 days of storage at –20°C and at +4°C (samples stored at +20°C showed a mean reduction of 10.4%). After 4 weeks of storage, there was a mean reduction of GHB concentrations higher than 20% at all storage temperatures. Ante-mortem urine samples showed a reduction of GHB levels higher than 10% after just 3 days of storage for samples kept at all tested temperatures. After 4 weeks of storage, there was a mean reduction of GHB concentrations higher than 25% at all storage temperatures. CONCLUSIONS: According to our findings, it would be useful to perform GHB analysis both in blood and urine specimens within 3 days of sampling and the specimens should be stored at –20°C or 4°C in order to avoid instability issues

A fatal iatrogenic right vertebral injury after transoral odontoidectomy and posterior cervical stabilization for a type II odontoid fracture

ingleseThe authors present a singular case of an iatrogenic right vertebral artery injury, involving a 67 year-old man, who reported a type II odontoid fracture (Anderson and D'Alonzo Classification) and posterior atlantoaxial dislocation following a road traffic accident. A small injury involving the right vertebral artery occurred as a consequence of transoral odontoidectomy and posterior cervical stabilization. It was caused by bone spicules of spinal origin and their presence was confirmed by the histological section of the right vertebral artery at the level of C1-C2. The case confirms how iatrogenic vertebral artery injuries during cervical spine surgery may be potentially lethal, especially where complications arise some days after surgery

A fatal accidental subarachnoid injection of lidocaine and levobupivacaine during a lumbar paravertebral block

Paravertebral block (PVB) is the technique of injecting a local anesthetic solution alongside the vertebral column, close to where the spinal nerves emerge, resulting in unilateral somatic and sympathetic nerve blockade. Here is reported a fatal case involving a 60-year-old woman with spondylitis arthropathy, who developed cardiac and respiratory arrest 40 min after receiving an accidental subarachnoid injection (L5-S1 bilaterally) of depomedrol lidocaine and levobupivacaine. A complete autopsy including histological and toxicological analyses was performed in order to establish the cause of death. Liquid/ liquid extraction (LLE) and GC–MS analysis were performed according to a previously published method. Lidocaine and bupivacaine were detected both in blood, at concentrations of 14.8 mg/L and 13.3 mg/L respectively, and in cerebrospinal fluid (CSF) at concentrations of 287.1 mg/L and 464.2 mg/L respectively. Both lidocaine and bupivacaine were also detected in the urine. The toxicological findings along with the autopsy allowed us to establish that the accidental subarachnoid injection of lidocaine and levobupivacaine had led to a progressive hypotension and normovolaemic shock caused by a severe ganglionic block, determining the patient’s deat

Hypertrophic cardiomyopathy associated with polyarteritis nodosa: A case of sudden cardiac death

This case concerns a rare sudden cardiac death characterized by macroscopic and microscopic postmortem findings of hypertrophic cardiomyopathy and polyarteritis nodosa. A complete autopsy was carried out, and histological and histochemical methods were employed. The cause of death was acute multifocal ischemic myocitolitic damage caused by both myocardial structural alteration attributable to hypertrophic cardiomyopathy (widespread interstitial fibrosis and multifocal myocyte disarray) and coronary arteritis attributable to polyarteritis nodosa. This is the first case in which the cause of death was attributed to both diseases

Structure–Activity Relationship ofS-Trityl-l-Cysteine Analogues as Inhibitors of the Human Mitotic Kinesin Eg5

The human kinesin Eg5 is a potential drug target for cancer chemotherapy. Eg5 specific inhibitors cause cells to block in mitosis with a characteristic monoastral spindle phenotype. Prolonged metaphase block eventually leads to apoptotic cell death. S-trityl-L-cysteine (STLC) is a tight-binding inhibitor of Eg5 that prevents mitotic progression. It has proven antitumor activity as shown in the NCI 60 tumor cell line screen. It is of considerable interest to define the minimum chemical structure that is essential for Eg5 inhibition and to develop more potent STLC analogues. An initial structure-activity relationship study on a series of STLC analogues reveals the minimal skeleton necessary for Eg5 inhibition as well as indications of how to obtain more potent analogues. The most effective compounds investigated with substitutions at the paraposition of one phenyl ring have an estimated K-i(app) of 100 nM in vitro and induce mitotic arrest with an EC50 of 200 nM