A Non-Covalent Antibody Complex for the Delivery of anti-cancer drugs

Antibody drug conjugates (ADCs), which are obtained by coupling a potent cytotoxic agent to a monoclonal antibody (mAb), are traditionally bound in a random way to lysine or cysteine residues, with the final product's heterogeneity having an important impact on their activity, characterization, and manufacturing. A new antibody drug delivery system (ADS) based on a non-covalent linkage between a Fc-binding protein, in this case Protein A or Protein G, and a mAb was investigated in the effort to achieve greater homogeneity and to create a versatile and adaptable drug delivery system. Recombinant staphylococcal Protein A and streptococcal Protein G were chemically PEGylated at the N-terminus with a 5 kDa and a 20 kDa PEG, respectively, yielding two monoconjugates with a mass of 48 50 and 48 45 kDa. Circular dichroism studies showed that both conjugates preserved secondary structures of the protein, and isothermal titration calorimetry experiments demonstrated that their affinity for mAb was approximately 107 M-1. Upon complexation with a mAb (Trastuzumab or Rituximab), in vitro flow-cytometry analysis of the new ADSs showed high selectivity for the specific antigen expressing cells. In addition, the ADS complex based on Trastuzumab and Protein G, conjugated with a heterobifunctional 20 kDa PEG carrying the toxin Tubulysin A, had a marked cytotoxic effect on the cancer cell line overexpressing the HER2/neu receptor, thus supporting its application in cancer therapy

Cisplatin liposome and 6-amino nicotinamide combination to overcome drug resistance in ovarian cancer cells

Ovarian cancer is an aggressive and lethal cancer usually treated by cytoreductive surgery followed by chemotherapy. Unfortunately, after an initial response, many patients relapse owing mainly to the development of resistance against the standard chemotherapy regime, carboplatin/paclitaxel, which is also affected by heavy side effects. In view to addressing such issues here, an association of liposomal cisplatin with 6-amino nicotinamide is investigated. It is known that resistant cells increase their demand for glucose, which is partially redirected toward the pentose phosphate pathway (PPP). Interestingly, we have found that also a cisplatin-resistant subclone of the ovarian cancer cells IGROV1 switch their metabolism toward the glycolytic pathway and rely on PPP to elude cisplatin cytotoxicity. The drug 6-amino nicotinamide, an inhibitor of the enzyme glucose-6-phosphate dehydrogenase (the rate-limiting step of the PPP) can restore the sensitivity of resistant cells to cisplatin. Then, to reduce the toxicity of cisplatin and prolong its action, a lyophilized stealth liposomal formulation of cisplatin was developed. The combination treatment of liposomal cisplatin and 6-amino nicotinamide showed promising cytotoxic activities in drug-resistant cells and a prolonged pharmacokinetics in rats, thus opening the way for a new therapeutic option against ovarian cancer

Drug conjugation to hyaluronan widens therapeutic indications for ovarian cancer

Management of ovarian cancer still requires improvements in therapeutic options. A drug delivery strategy was tested that allows specific targeting of tumor cells in combination with a controlled release of a cytotoxic molecule. To this aim, the efficacy of a loco-regional intraperitoneal treatment with a bioconjugate (ONCOFID-S) derived by chemical linking of SN-38, the active metabolite of irinotecan (CPT-11), to hyaluronan was assessed in a mouse model of ovarian carcinomatosis. In vitro, the bioconjugate selectively interacted with ovarian cancer cells through the CD44 receptor, disclosed a dose-dependent tumor growth inhibition efficacy comparable to that of free SN-38 drug, and inhibited Topoisomerase I function leading to apoptosis by a mechanism involving caspase-3 and -7 activation and PARP cleavage. In vivo, the intraperitoneal administration of ONCOFID-S in tumor-bearing mice did not induce inflammation, and evidenced an improved therapeutic efficacy compared with CPT-11. In conclusion, SN-38 conjugation to hyaluronan significantly improved the profile of in vivo tolerability and widened the field of application of irinotecan. Therefore, this approach can be envisaged as a promising therapeutic strategy for loco-regional treatment of ovarian cancer

Using ductwork to improve supply plenum temperature distribution in underfloor air distribution (UFAD) system

Cool supply air flowing through the underfloor plenum is exposed to heat gain from both the concrete slab (conducted from the warm return air on the adjacent floor below the slab) and the raised floor panels (conducted from the warmer room above). The magnitude of this heat gain can be quite high, resulting in undesirable loss of control of the supply air temperature from the plenum into the occupied space (sometimes referred to as thermal decay). These warmer supply air temperatures can make it more difficult to maintain comfort in the occupied space (without increasing airflow rates), particularly in perimeter zones where cooling loads reach their highest levels. How to predict plenum thermal performance is one of the key design issues facing practicing engineers – evidence from completed projects indicates that excessive thermal decay can be a problem. Current research at the Center for the Built Environment (CBE) at the University of California, Berkeley indicates that unless slab insulation or other means is used to reduce plenum heat gain, one of the strategies that should be considered consists in providing the coolest supply air into the perimeter plenum zones, allowing warmer plenum temperature in interior plenums. The three approaches that have been used to accomplish this include: • Use plenum inlets with higher inlet velocities directed at critical perimeter locations. • Use ductwork (or DuctSox) to deliver cool air to/towards the perimeter. This is the subject of this research work. • Instead of the typical interior plenum inlet locations, consider designing the plenum with perimeter inlets (shafts), if possible. As described above, one of the recommended strategies for addressing thermal decay problems in UFAD systems is the use of ductwork (flexible or rigid) within the underfloor plenum to deliver cool air preferentially to perimeter zones or other critical areas of high cooling demand. Several tests, in an underfloor plenum facility, were done to investigate the use of ductwork to improve supply plenum temperature distribution. Experimental tests have shown that using ductwork, especially vented duct with solid end cap, can cause significant pressure rising in the HVAC system. The test results were used to calibrate and validate a CFD model. This model was compared with a previous CFD model developed and validated at the Center for the Built Environment, used by the researcher to investigate the temperature distributions in an underfloor plenum for different ways (number of inlets and inlet momentum) of supplying air into the plenum. The two models are representative of the first two strategies proposed before to reduce perimeter air temperatures in the underfloor plenum. Analyzing the simulation results comparing the models under the same operative conditions it is possible to conclude that: • Delivering air into the plenum with a high momentum to reach the perimeter loses its capability to reduce perimeter air temperature under partial load conditions. More than this, under part load conditions (25%) there is a reversed result, i.e., the perimeter air is warmer than interior air.; • Under the same load condition, using ductwork produces always lower perimeter temperatures; • Delivering fresh air into the perimeter plenum with high momentum could reduce the perimeter temperatures but not the heat flux, while using a well designed ductwork reduces the heat flux and, as a consequence, the perimeter temperatures.I sistemi per la distribuzione dell’aria sottopavimento (UFAD), utilizzati nella climatizzazione degli ambienti, espongono l’aria fresca che circola nel plenum allo scambio termico con il solaio (riscaldato dall’aria di ritorno nell’ambiente sottostante) e con il pavimento rialzato ( riscaldato dall’ambiente sovrastante). L’entità di questo scambio termico può essere rilevante, poiché ne consegue una di controllo sulla temperatura dell’aria di mandata (denominato Thermal Decay). L’aumento non desiderato della temperatura di mandata può creare difficoltà nel mantenimento del comfort termico nell’ambiente, in particolare nelle zone perimetrali dove i carichi termici raggiungono il loro picco. Un lavoro condotto presso il Center for the Built Environment (CBE) ha evidenziato che, in assenza di un adeguato isolamento del solaio, dovrebbero essere prese in considerazione strategie per la riduzione della temperatura nelle zone perimetrali del plenum, anche a discapito di un aumento della stessa nelle parti interne. Le tre principali strategie utilizzate a questo scopo sono: • immissione di aria nel plenum ad alta velocità, al fine di raggiungere le zone perimetrali dell’underfloor; • utilizzo di condutture aerauliche(sia in metallo che in tessuto) per portare l’aria direttamente nelle zone critiche del plenum; • considerare la possibilità di immettere l’aria nelle zone perimetrali del plenum anziché nella parte centrale. Lo sviluppo e l’analisi del secondo punto qui esposto è l’oggetto di questo lavoro. Utilizzando il modello di UFAD in scala 1:1, presso l’Università di Berkeley, in California, si sono potuti investigare i profili di temperatura in pavimenti rialzati dotati di tale sistema. I test hanno evidenziato fin da subito il problema dell’aumento di pressione nel sistema HVAC legato alle perdite di carico nei condotti. Il problema è ancora più marcato quando viene utilizzata una particolare tipologia di condotto che possiamo definire ventilato. Tale elemento, in tessuto nella versione analizzata, presenta lungo due lati opposti delle file di fori che permettono di distribuire l’aria in modo più efficiente. I risultati sperimentali sono stati utilizzati per validare un modello CFD che simuli la soluzione impiantistica oggetto di analisi. Utilizzando il modello CFD appena descritto ed un secondo, testato e validato presso il CBE, che simula la prima delle strategie illustrate in precedenza, è stato possibile confrontare le due soluzioni in situazioni reali di funzionamento. Per rendere le simulazioni il quanto più possibile coerenti con un caso reale, le condizioni al contorno sono state ottenute con il programma di simulazione energetica Energy Plus. La simulazione energetica è stata eseguita per il giorno di progetto estivo, per un pavimento rialzato situato al secondo di tre piani di uno stabile uso ufficio. Come zona climatica è stata scelta la località di Sacramento, California. Si è giunti così alle seguenti conclusioni: • mandare l’aria nel plenum ad alta velocità non garantisce la riduzione di temperatura nelle zone perimetrali in condizioni di carico parziale. La distribuzione di temperatura nel plenum, inoltre varia notevolmente al variare del carico di raffrescamento richiesto, rendendo così difficile predire le performance termiche dell’underfloor; • dal confronto delle due tipologie di distribuzione dell’aria sotto il pavimento emerge che nel caso si utilizzino condutture aerauliche le temperature perimetrali sono, per ogni condizioni di carico, inferiori a quelle registrate nel caso di mandate ad alta velocità; • la distribuzione delle temperature in plenum che utilizzano i condotti d’aria rimane pressoché invariata per ogni condizione di carico, facilitando così il controllo delle temperature e del comfort termico nell’ambiente condizionato; • l’utilizzo di condutture, unito ad un’attenta analisi delle strategie di distribuzione dell’aria attraverso le stesse, consente una riduzione del flusso termico tra plenum e ambienti circostanti fino al 15% del totale

Culturing muscle fibres in hanging drop: A novel approach to solve an old problem

Background Information. The satellite cells (SCs) associated with muscle fibres play a key role in postnatal growth and regeneration of skeletal muscle. Commonly used methods of isolation and in vitro culture of SCs lead to the mixture of their subpopulations that exist within muscle. To solve this problem, we used the well established technique, the hanging drop system, to culture SCs in a three-dimensional environment and thus, to monitor them in their original niche. Results. Using hanging drop technique, we were able to culture SCs associated with the fibre at least for 9 days with one transfer of fibres to the fresh drops. In comparison, in the classical method of myofibres culture, that is, on the dishes coated with Matrigel, SCs leave the fibres within 3 days after the isolation. Cells cultured in both systems differed in expression of Pax7 and MyoD. While almost all cells cultured in adhesion system expressed MyoD before the fifth day of the culture, the majority of SCs cultured in hanging drop still maintained expression of Pax7 and were not characterised by the presence of MyoD. Among the cells cultured with single myofibre for up to 9 days, we identified two different subclones of SCs: low proliferative clone and high proliferative clone, which differed in proliferation rate and membrane potential. Conclusions. The hanging drop enables the myofibres to be kept in suspension for at least 9 days, and thus, allows SCs and their niche to interact each other for prolonged time. In a consequence, SCs cultured in hanging drop maintain expression of Pax7 while those cultured in a traditional adhesion culture, that is, devoid of signals from the original niche, activate and preferentially undergo differentiation as manifested by expression of MyoD. Thus, the innovative method of SCs culturing in the hanging drop system may serve as a useful tool to study the fate of different subpopulations of these cells in their anatomical location and to determine reciprocal interactions between them and their niche

A novel PEG–haloperidol conjugate with a non-degradable linker shows the feasibility of using polymer–drug conjugates in a non-prodrug fashion

A PEG–haloperidol conjugate containing a non-biodegradable linker was synthesised. Incubation with rat plasma demonstrated excellent linker stability, and competition radioligand binding assays demonstrated retained binding to the D2-receptor. In silico studies predicted that the conjugate will not cross the blood–brain barrier (BBB), thus potentially restricting haloperidol action to one side of the BBB