Antitumor Efficacy of Liposome-Encapsulated NVP-BEZ235 Combined with Irreversible Electroporation for Head and Neck Cancer.

Irreversible electroporation (IRE) kills tumor cells by the delivery of short pulses of strong electric fields. However, the field strength decreases with distance from the treatment center. When IRE cannot eradicate the entire tumor mass, the surviving tumor cells can regrow. NVP-BEZ235 is a dual PI3K/mTOR inhibitor that has been administered orally in clinical trials. However, its hydrophobicity and poor water solubility make NVP-BEZ235 difficult to deliver to target areas. To improve its pharmacokinetics and therapeutic efficacy, we have encapsulated NVP-BEZ235 in a liposome (termed as L-BEZ). Our current study focuses on the long-term antitumor efficacy of IRE and intratumoral injection of L-BEZ in HN5 head and neck cancer xenografts in nude mice. We compared in vitro efficacy, as well as the effect on tumor size and growth rate in vivo, between IRE alone, IRE + oral BEZ, and IRE + L-BEZ over the course of two months. All animals in the control group were sacrificed by day 36, due to excess tumor burden. Tumors treated with IRE alone grew faster and larger than those in the control group. IRE + oral BEZ suppressed tumor growth, but the growth rate increased to that of the controls toward the end of 21 days. Only IRE + L-BEZ eradicated the tumor masses, with no palpable or extractable tumor mass observed after two months. The combination of IRE and L-BEZ could effectively eradicate tumors and prevent recurrence.

Delayed Onset of Symptoms After a Rattlesnake Bite in a Renal Transplant Patient: A Case Report.

Introduction: The United States is home to two major families of venomous snakes, Crotalids and Elapids. The Crotalid family, also known as pit vipers, is well known for being among the most frequent causes of snakebites reported. Crotalid envenomation can present with local findings, hematologic toxicity, and systemic toxicity. Identification of envenomated patients is key to determining who needs antivenom. Most sources recommend an observation period of six to eight hours after the snakebite to determine whether the bite was "dry" or the patient was exposed to venom. Case Report: We present the case of a 33-year-old patient with a history of renal transplantation who had delayed onset of symptoms of envenomation 18 hours after an initial emergency department observation. The patient did well after a course of antivenom and was discharged on hospital day three. Conclusion: The patient's immunosuppressive regimen may have delayed the onset of clinical symptoms, thus delaying treatment. To the best of our knowledge, this is the first case reported of a patient presenting with a delayed onset of initial snakebite envenomation symptoms.

Long-Term Biocatalyst Performance: Mechanistic Prediction and Continuous Non-Isothermal Testing.

The assessment of the operational stability of biocatalysts by conventional direct determination of the total turnover number (TTN), a useful indicator of lifetime biocatalyst productivity, via continuous isothermal experiments tends to be time-consuming, material-intensive, and prone to disturbances, especially in case of rather stable catalysts. In the present work, we present and validate two alternative methods for estimating the TTN of a biocatalyst for any desired operating temperature. The first method is a mechanistic approach, built upon mathematical derivation of enzyme deactivation models derived from first principles, in which TTN can be calculated from two straightforward isothermal biochemical batch measurements. The second method relies on a few non-isothermal, continuous-mode experiments in conjunction with mathematical modeling to determine the intrinsic deactivation parameters of the biocatalyst. We verify both methods on the test case of TEM-1 ß-lactamase-catalyzed penicillin G (Pen G) hydrolysis. Both alternative methods provide estimates of TTN which are typically within a factor of two to five or less of the values measured directly via lengthy, costly, and error-prone conventional isothermal aging tests. Therefore, both the mechanistic approach and the non-isothermal continuous approach are extremely valuable tools to enable calculation of catalyst cost contribution in continuous processing and to eliminate underperforming candidates in search of the most stable biocatalyst.

Utilizing Simple Biochemical Measurements to Predict Lifetime Output of Biocatalysts in Continuous Isothermal Processes.

The expected product yield of a biocatalyst during its useful lifetime is an important consideration when designing a continuous biocatalytic process. One important indicator of lifetime biocatalyst productivity is the dimensionless total turnover number (TTN). Here, a method is proposed for estimating the TTN of a given biocatalyst from readily-measured biochemical quantities, namely the specific activity and the deactivation half-life, measured under identical conditions. We demonstrate that this method may be applied to any enzyme whose thermal deactivation follows first-order kinetics, regardless of the number of unfolding intermediates, and that the TTN method circumvents the potential problems associated with measuring specific catalyst output when a portion of the enzyme is already unfolded. The TTN estimation was applied to several representative biocatalysts to demonstrate its applicability in identifying the most cost-effective catalyst from a pool of engineered mutants with similar activity and thermal stability.

Antitumor efficacy of liposome-encapsulated NVP-BEZ 235 in combination with irreversible electroporation.

Irreversible electroporation (IRE) is an emerging minimally invasive tumor ablation technique that delivers short pulses of strong electric fields and kills cancer cells by disrupting their cell membranes with the electric pulses. However, clinical studies report that more than 10% of local tumor recurrences occur at the original ablated site. NVP BEZ-235 (BEZ) is a dual PI3K/mTOR inhibitor that has substantial anticancer effects. However, the clinical trials of BEZ was not satisfactory because of its low bioavailability and high toxicity, which stemmed from the use of oral administration of high doses over a long period of time. In this research, we prepared a liposomal formulation of BEZ (L-BEZ) for intratumoral injection and studied its antitumor efficacy alone and in combination with IRE. We hypothesized that IRE could release BEZ from the liposomes and that the combination could decrease tumor viability. Our results show that IRE released BEZ from its liposomal encapsulation. The combination of L-BEZ and IRE killed more Hep3B tumor cells in vitro than did L-BEZ or IRE alone and also inhibited cancer cell proliferation in nude mice bearing Hep3B xenografts. Combination of chemotherapeutic agent loaded nanoparticles could enhance the antitumor efficacy of IRE.

Deactivation of TEM-1 ß-Lactamase Investigated by Isothermal Batch and Non-Isothermal Continuous Enzyme Membrane Reactor Methods.

The thermal deactivation of TEM-1 ß-lactamase was examined using two experimental techniques: a series of isothermal batch assays and a single, continuous, non-isothermal assay in an enzyme membrane reactor (EMR). The isothermal batch-mode technique was coupled with the three-state "Equilibrium Model" of enzyme deactivation, while the results of the EMR experiment were fitted to a four-state "molten globule model". The two methods both led to the conclusions that the thermal deactivation of TEM-1 ß-lactamase does not follow the Lumry-Eyring model and that the T(eq) of the enzyme (the point at which active and inactive states are present in equal amounts due to thermodynamic equilibrium) is at least 10 °C from the T(m) (melting temperature), contrary to the idea that the true temperature optimum of a biocatalyst is necessarily close to the melting temperature.