Poor outcomes in patients with sepsis undergoing emergency laparotomy and laparoscopy are attenuated by faster time to care measures.

AIM: Emergency laparotomy and laparoscopy (EmLap) are amongst the commonest surgical procedures, with high prevalence of sepsis and hence poorer outcomes. However, whether time taken to receive care influences outcomes in patients requiring antibiotics for suspected infection remains largely unexplored. The aim of this work was to determine whether (1) time to care contributes to outcome differences between patients with and without suspected infection and (2) its impact on outcomes only amongst those with suspected infection. METHOD: Clinical information was retrospectively obtained from the 2017-2018 Emergency Laparotomy and Laparoscopic Scottish Audit (ELLSA). Time to care referred to six temporal variables describing radiological investigation, anaesthetic triage and surgical management. Outcome measures [mortality, readmission, hospital death, postoperative destination and length of stay (LoS)] were compared using adjusted and unadjusted regression analyses to determine whether the outcome differences could be explained by faster or slower time to care. RESULTS: Amongst 2243 EmLap patients [median age 65 years (interquartile range 51-75 years), 51.1% female], 892 (39.77%) received antibiotics for suspected infection. Although patients with suspected infection had faster time to care (all p ≤ 0.001) and worse outcomes compared with those who did not, outcome differences were not statistically significant when accounted for time (all p > 0.050). Amongst those who received antibiotics, faster time to care was also associated with decreased risk of postoperative intensive care unit (ICU) stay and shorter LoS (all p < 0.050). CONCLUSION: Worse outcomes associated with infection in EmLap patients were attenuated by faster time to care, which additionally reduced the LoS and ICU stay risk amongst those with suspected infection.

Current Status and Future Perspectives on MRNA Drug Manufacturing.

The coronavirus disease of 2019 (COVID-19) pandemic launched an unprecedented global effort to rapidly develop vaccines to stem the spread of the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2). Messenger ribonucleic acid (mRNA) vaccines were developed quickly by companies that were actively developing mRNA therapeutics and vaccines for other indications, leading to two mRNA vaccines being not only the first SARS-CoV-2 vaccines to be approved for emergency use but also the first mRNA drugs to gain emergency use authorization and to eventually gain full approval. This was possible partly because mRNA sequences can be altered to encode nearly any protein without significantly altering its chemical properties, allowing the drug substance to be a modular component of the drug product. Lipid nanoparticle (LNP) technology required to protect the ribonucleic acid (RNA) and mediate delivery into the cytoplasm of cells is likewise modular, as are technologies and infrastructure required to encapsulate the RNA into the LNP. This enabled the rapid adaptation of the technology to a new target. Upon the coattails of the clinical success of mRNA vaccines, this modularity will pave the way for future RNA medicines for cancer, gene therapy, and RNA engineered cell therapies. In this review, trends in the publication records and clinical trial registrations are tallied to show the sharp intensification in preclinical and clinical research for RNA medicines. Demand for the manufacturing of both the RNA drug substance (DS) and the LNP drug product (DP) has already been strained, causing shortages of the vaccine, and the rise in development and translation of other mRNA drugs in the coming years will exacerbate this strain. To estimate demand for DP manufacturing, the dosing requirements for the preclinical and clinical studies of the two approved mRNA vaccines were examined. To understand the current state of mRNA-LNP production, current methods and technologies are reviewed, as are current and announced global capacities for commercial manufacturing. Finally, a vision is rationalized for how emerging technologies such as self-amplifying mRNA, microfluidic production, and trends toward integrated and distributed manufacturing will shape the future of RNA manufacturing and unlock the potential for an RNA medicine revolution.

Prognostic value of estimated glomerular filtration rate in hospitalised older patients (over 65) with COVID-19: a multicentre, European, observational cohort study.

BACKGROUND: The reduced renal function has prognostic significance in COVID-19 and it has been linked to mortality in the general population. Reduced renal function is prevalent in older age and thus we set out to better understand its effect on mortality. METHODS: Patient clinical and demographic data was taken from the COVID-19 in Older People (COPE) study during two periods (February-June 2020 and October 2020-March 2021, respectively). Kidney function on admission was measured using estimated glomerular filtration rate (eGFR). The primary outcomes were time to mortality and 28-day mortality. Secondary outcome was length of hospital stay. Data were analysed with multilevel Cox proportional hazards regression, and multilevel logistic regression and adjusted for individual patient clinical and demographic characteristics. RESULTS: One thousand eight hundred two patients (55.0% male; median [IQR] 80 [73-86] years) were included in the study. 28-day mortality was 42.3% (n = 742). 48% (n = 801) had evidence of renal impairment on admission. Using a time-to-event analysis, reduced renal function was associated with increased in-hospital mortality (compared to eGFR ≥ 60 [Stage 1&2]): eGFR 45-59 [Stage 3a] aHR = 1.26 (95%CI 1.02-1.55); eGFR 30-44 [Stage 3b] aHR = 1.41 (95%CI 1.14-1.73); eGFR 1-29 [Stage 4&5] aHR = 1.42 (95%CI 1.13-1.80). In the co-primary outcome of 28-day mortality, mortality was associated with: Stage 3a adjusted odds ratio (aOR) = 1.18 (95%CI 0.88-1.58), Stage 3b aOR = 1.40 (95%CI 1.03-1.89); and Stage 4&5 aOR = 1.65 (95%CI 1.16-2.35). CONCLUSION: eGFR on admission is a good independent predictor of mortality in hospitalised older patients with COVID-19 population. We found evidence of a dose-response between reduced renal function and increased mortality.

Lipid nanoparticle-mediated siRNA delivery for safe targeting of human CML in vivo.

Efficient and safe delivery of siRNA in vivo is the biggest roadblock to clinical translation of RNA interference (RNAi)-based therapeutics. To date, lipid nanoparticles (LNPs) have shown efficient delivery of siRNA to the liver; however, delivery to other organs, especially hematopoietic tissues still remains a challenge. We developed DLin-MC3-DMA lipid-based LNP-siRNA formulations for systemic delivery against a driver oncogene to target human chronic myeloid leukemia (CML) cells in vivo. A microfluidic mixing technology was used to obtain reproducible ionizable cationic LNPs loaded with siRNA molecules targeting the BCR-ABL fusion oncogene found in CML. We show a highly efficient and non-toxic delivery of siRNA in vitro and in vivo with nearly 100% uptake of LNP-siRNA formulations in bone marrow of a leukemic model. By targeting the BCR-ABL fusion oncogene, we show a reduction of leukemic burden in our myeloid leukemia mouse model and demonstrate reduced disease burden in mice treated with LNP-BCR-ABL siRNA as compared with LNP-CTRL siRNA. Our study provides proof-of-principle that fusion oncogene specific RNAi therapeutics can be exploited against leukemic cells and promise novel treatment options for leukemia patients.

The role of the transition metal copper and the ionophore A23187 in the development of Irinophore C™.

PURPOSE: A liposomal irinotecan formulation referred to as Irinophore C relies on the ability of copper to complex irinotecan within the liposome. It is currently being evaluated for critical drug-loading parameters. Studies presented here were designed to determine the optimum copper concentration required for the effective encapsulation and retention of irinotecan into liposomes. METHODS: Distearoylphosphatidylcholine/cholesterol liposomes were formulated using buffers containing various copper or manganese concentrations, and irinotecan loading was determined in the presence and absence of divalent metal ionophore A23187. The rate and extent of irinotecan encapsulation and the rate of irinotecan release from the liposomes were assessed. The amount of copper retained inside liposomes following irinotecan loading and the effect of copper on membrane permeability were determined. RESULTS: Efficient (>98%) irinotecan loading was achieved using encapsulated copper concentrations of 50 mM. However, irinotecan release was copper concentration dependent, with a minimum 300 mM concentration required for optimal drug retention. The presence of copper increased liposomal membrane permeability. CONCLUSION: Results explain why irinotecan loading rates are enhanced in the presence of formulations prepared with copper, and we speculate that the Irinophore C formulation exhibits improved drug retention, due to generation of a complex between copper and irinotecan.

Rh2 or its aglycone aPPD in combination with docetaxel for treatment of prostate cancer.

BACKGROUND: Docetaxel is one of the few chemotherapeutic drugs that are considered highly effective when used to treat prostate cancer patients that have relapsed and/or metastatic disease, it is therefore reasonable to expect further improvements in treatment outcomes when it is combined with other therapeutic agents active in prostate cancer. This study assesses the combination of well tolerated and orally bioavailable formulations of ginsenoside Rh2 or its aglycone aPPD with docetaxel. METHODS: The in vitro activity of Rh2, aPPD, and docetaxel was determined in four prostate cancer cell lines: PC-3, LNCaP, DU145, and C4-2. Combinations of Rh2 or aPPD with docetaxel were assessed using the constant ratio combination design. Combination Indices (CI) and Dose Reduction Indices (DRI) were subsequently estimated using Calcusyn. In vivo efficacy studies and Immunohistochemical analyses (PC-3 model) were also evaluated. RESULTS: In PC-3, DU145 and C4-2 prostate cancer cells combinations of Rh2 or aPPD with docetaxel were predominantly additive or synergistic. Combinations of Rh2 + docetaxel and aPPD + docetaxel caused established PC-3 tumors to regress from their initial size by 15% and 27%, respectively. Tumor cell proliferation rate (measured by Ki-67 positive cells) was significantly lower for combinations of Rh2 + docetaxel and aPPD + docetaxel, compared to animals treated with docetaxel alone. CONCLUSIONS: Rh2 and aPPD can be combined with docetaxel to yield additive or synergistic activity in vitro and in vivo. Pending further assessment of toxicity and pharmacodynamic behavior, this study supports testing of combinations of ginsenoside Rh2 or its aglycone aPPD with docetaxel in a clinical setting.

Targeted Inhibition of the NUP98-NSD1 Fusion Oncogene in Acute Myeloid Leukemia.

NUP98-NSD1-positive acute myeloid leukemia (AML) is a poor prognostic subgroup that is frequently diagnosed in pediatric cytogenetically normal AML. NUP98-NSD1-positive AML often carries additional mutations in genes including FLT3, NRAS, WT1, and MYC. The purpose of our study was to characterize the cooperative potential of the fusion and its associated Neuroblastoma rat sarcoma (NRAS) mutation. By constitutively expressing NUP98-NSD1 and NRASG12D in a syngeneic mouse model and using a patient-derived xenograft (PDX) model from a NUP98-NSD1-positive AML patient, we evaluated the functional role of these genes and tested a novel siRNA formulation that inhibits the oncogenic driver NUP98-NSD1. NUP98-NSD1 transformed murine bone marrow (BM) cells in vitro and induced AML in vivo. While NRASG12D expression was insufficient to transform cells alone, co-expression of NUP98-NSD1 and NRASG12D enhanced the leukemogenicity of NUP98-NSD1. We developed a NUP98-NSD1-targeting siRNA/lipid nanoparticle formulation that significantly prolonged the survival of the PDX mice. Our study demonstrates that mutated NRAS cooperates with NUP98-NSD1 and shows that direct targeting of the fusion can be exploited as a novel treatment strategy in NUP98-NSD1-positive AML patients.

Irinophore C: a liposome formulation of irinotecan with substantially improved therapeutic efficacy against a panel of human xenograft tumors.

PURPOSE: To assess the pharmacokinetics, tumor drug accumulation, and therapeutic activity of Irinophore C, a novel liposomal formulation of irinotecan (CPT-11). EXPERIMENTAL DESIGN: The plasma lactone/carboxy levels of CPT-11 and SN-38 were determined in mice after a single i.v. dose of irinotecan (Camptosar), or Irinophore C, and the plasma t(1/2), plasma area under the curve, plasma C(max), and plasma clearance were calculated. Further, plasma and tumor drug levels were also measured in tumor-bearing mice following Irinophore C treatment. The efficacy of Irinophore C was compared with that of Camptosar in five s.c. human tumor xenografts using single-dose treatment (LS 180), a total of three doses administered at 4-day intervals (H460), or a total of three doses administered at 7-day intervals (Capan-1, PC-3, and HT-29). RESULTS: Compared with Camptosar, Irinophore C mediated an 8-fold increase in t(1/2), a 100-fold increase in C(max), a 1,000-fold increase in area under the curve, and a 1,000-fold decrease in clearance for the active lactone form of CPT-11. Further, the plasma and tumor SN-38 lactone levels were consistent for at least 48 h post-Irinophore C injection. Camptosar treatment (40 mg/kg) mediated a delay in the time required for tumors to increase to four times their pretreatment size compared with controls (T-C). T-Cs ranged from 2 days (LS 180 model) to 18 days (PC-3 model). Irinophore C (40 mg/kg) engendered T-Cs ranging from 14 days (LS 180 model) to 87 days (Capan-1 model). CONCLUSION: Irinophore C improved CPT-11/SN-38 pharmacokinetics, promoted tumor drug accumulation, and increased therapeutic efficacy in a panel of five distinct human tumor xenografts.

Irinophore C, a novel nanoformulation of irinotecan, alters tumor vascular function and enhances the distribution of 5-fluorouracil and doxorubicin.

PURPOSE: To examine the antitumor effects of Irinophore C, a nanopharmaceutical formulation of irinotecan, on the tissue morphology and function of tumor vasculature in HT-29 human colorectal tumors. EXPERIMENTAL DESIGN: Fluorescence microscopy was used to map and quantify changes in tissue density, tumor vasculature, hypoxia, and the distribution of Hoechst 33342, a perfusion marker, and the anticancer drug, doxorubicin. Noninvasive magnetic resonance imaging was used to quantify Ktrans, the volume transfer constant of a solute between the blood vessels and extracellular tissue compartment of the tumor, as a measure of vascular function. Following treatment with Irinophore C, 19F magnetic resonance spectroscopy was used to monitor the delivery of 5-fluorouracil (5-FU) to the tumor tissue, whereas scintigraphy was used to quantify the presence of bound [14C]5-FU. RESULTS: Irinophore C decreased cell density (P = 8.42 x 10(-5)), the overall number of endothelial cells in the entire section (P = 0.014), tumor hypoxia (P = 5.32 x 10(-9)), and K(trans) (P = 0.050). However, treatment increased the ratio of endothelial cells to cell density (P = 0.00024) and the accumulation of Hoechst 33342 (P = 0.022), doxorubicin (P = 0.243 x 10(-5)), and 5-FU (P = 0.0002) in the tumor. Vascular endothelial growth factor and interleukin-8, two proangiogenic factors, were down-regulated, whereas the antiangiogenic factor TIMP-1 was up-regulated in Irinophore C-treated tumors. CONCLUSIONS: Irinophore C treatment improves the vascular function of the tumor, thereby reducing tumor hypoxia and increasing the delivery and accumulation of a second drug. Reducing hypoxia would enhance radiotherapy, whereas improving delivery of a second drug to the tumor should result in higher cell kill.

A novel liposomal irinotecan formulation with significant anti-tumour activity: use of the divalent cation ionophore A23187 and copper-containing liposomes to improve drug retention.

We determined whether the method used to encapsulate irinotecan into 1,2-distearoyl-sn-glycero-phosphocholine/cholesterol (DSPC/Chol; 55:45 mol%) liposomes influenced: (i) irinotecan release rate and (ii) therapeutic efficacy. DSPC/Chol (55:45 mol%) liposomes were prepared with: (i) unbuffered CuSO4; (ii) buffered (pH 7.5) CuSO4; (iii) unbuffered MnSO4 and the ionophore A23187 (exchanges internal metal2+ with external 2H+ to establish and maintain a transmembrane pH gradient); and (iv) unbuffered CuSO4 and ionophore A23187. All formulations exhibited >98% irinotecan encapsulation (0.2 drug-to-lipid molar ratio; 10 min incubation at 50 degrees C). Following a single intravenous injection (100mg/kg irinotecan) into Balb/c mice, the unbuffered CuSO4 plus A23187 formulation mediated a half-life of irinotecan release of 44.4h; a >or=4-fold increase compared to the other liposome formulations. This surprising observation demonstrated that the CuSO4 plus A23187 formulation enhanced irinotecan retention compared to the MnSO4 plus A23187 formulation, indicating the importance of the divalent metal. A single dose of the CuSO4 plus A23187 formulation (50mg/kg irinotecan) mediated an 18-fold increase in median T-C (the difference in days for treated and control subcutaneous human LS 180 adenocarcinoma xenografts to increase their initial volume by 400%) when compared to a comparable dose of Camptosar. Improved irinotecan retention was associated with increased therapeutic activity.

Microfluidic Production and Application of Lipid Nanoparticles for Nucleic Acid Transfection.

Lipid nanoparticles (LNPs) are established in the biopharmaceutical industry for efficient encapsulation and cytosolic delivery of nucleic acids for potential therapeutics, with several formulations in clinical trials. The advantages of LNPs can also be applied in basic research and discovery with a microfluidic method of preparation now commercially available that allows preparations to be scaled down to quantities appropriate for cell culture. These preparations conserve expensive nucleic acids while maintaining the particle characteristics that have made LNPs successful in later stages of genetic medicine development. Additionally, this method and the resulting LNPs are seamlessly scalable to quantities appropriate for in vivo models and development of nucleic acid therapeutics.The present work describes the methodology for preparing LNPs loaded with siRNA, mRNA or plasmids using a commercially available microfluidic instrument and an accompanying transfection kit. Guidelines for application to cultured cells in a well-plate format are also provided.

Irinotecan-cisplatin interactions assessed in cell-based screening assays: cytotoxicity, drug accumulation and DNA adduct formation in an NSCLC cell line.

PURPOSE: The use of in vitro drug cytotoxicity assays for the assessment of drug-drug interactions that lead to synergy may not take into account the many cellular determinants responsible for combination effects. Administration of the anticancer drug CPT-11, for example, is associated with rapid conversion of drug from its active lactone form to the inactive carboxylate form. Thus it is difficult to model, in vitro, the behavior of this drug when used as a single agent and when used in a combination setting, this factor may contribute to the interactions measured. Therefore, the objective of this study was to examine the influence of CPT-11 lactone ratio on the cellular accumulation of CPT-11 when used as a single agent and under conditions where it is used in combination with cisplatin. METHODS: A fixed ratio experimental design was used and drug ratios of CPT-11 and cisplatin were judged to be antagonistic, additive, or synergistic to the non-small cell lung cancer cell line, H460, on the basis of the median effect analysis methodology of Chou and Talalay. The influence of extracellular pH on CPT-11 accumulation was evaluated at pH 7.4 and pH 6.6 when the drug was added immediately to the cells or first pre-equilibrated at the indicated pH. These studies were completed in the presence and absence of cisplatin. RESULTS: When CPT-11 was added as a single agent to cells in pH = 7.4 media, the drug underwent hydrolysis to the carboxylate form; however, there was a rapid accumulation of the CPT-11 lactone form which peaked at 3,800 pmol/mg protein by 30 min and drops to 570 pmol/mg protein by 24 h. In pH = 6.6 media, accumulation of CPT-11 lactone was substantially lower over a 60 min timecourse; however, the cellular uptake measured at 24 h was comparable to that observed when the drug was added into pH 7.4 media. When evaluating CPT-11 lactone accumulation in a combination setting with cisplatin no significant difference in either CPT-11 lactone accumulation or cisplatin accumulation was observed, suggesting that drug interactions that led to synergy were mechanistically based. Results are presented which suggest that when cisplatin and CPT-11 are used in combination, there was a significant prolongation of platinum association with DNA compared to results obtained when cisplatin was used alone. CONCLUSION: These results suggest that the CPT-11 lactone to carboxylate ratio does not influence the accumulation of the active CPT-11 lactone form in H460 cells and that CPT-11 does not influence cisplatin uptake when used in combination. It is argued, therefore, that the improved cytotoxicity between CPT-11 and cisplatin, as determined using cell-based assay, has the potential to be preserved in vivo assuming the optimal drug-drug ratio and concentration can be effectively delivered to the tumor.

Ratiometric dosing of anticancer drug combinations: controlling drug ratios after systemic administration regulates therapeutic activity in tumor-bearing mice.

Anticancer drug combinations can act synergistically or antagonistically against tumor cells in vitro depending on the ratios of the individual agents comprising the combination. The importance of drug ratios in vivo, however, has heretofore not been investigated, and combination chemotherapy treatment regimens continue to be developed based on the maximum tolerated dose of the individual agents. We systematically examined three different drug combinations representing a range of anticancer drug classes with distinct molecular mechanisms (irinotecan/floxuridine, cytarabine/daunorubicin, and cisplatin/daunorubicin) for drug ratio-dependent synergy. In each case, synergistic interactions were observed in vitro at certain drug/drug molar ratio ranges (1:1, 5:1, and 10:1, respectively), whereas other ratios were additive or antagonistic. We were able to maintain fixed drug ratios in plasma of mice for 24 hours after i.v. injection for all three combinations by controlling and overcoming the inherent dissimilar pharmacokinetics of individual drugs through encapsulation in liposomal carrier systems. The liposomes not only maintained drug ratios in the plasma after injection, but also delivered the formulated drug ratio directly to tumor tissue. In vivo maintenance of drug ratios shown to be synergistic in vitro provided increased efficacy in preclinical tumor models, whereas attenuated antitumor activity was observed when antagonistic drug ratios were maintained. Fixing synergistic drug ratios in pharmaceutical carriers provides an avenue by which anticancer drug combinations can be optimized prospectively for maximum therapeutic activity during preclinical development and differs from current practice in which dosing regimens are developed empirically in late-stage clinical trials based on tolerability.

Gene silencing in the development of personalized cancer treatment: the targets, the agents and the delivery systems.

The advent of sophisticated experimental tools that can probe the molecular pathology of cancer has revealed a number of genes and gene families that could prove attractive targets for cancer therapy. Thus, gene silencing strategies have been envisioned to treat cancer by targeting the cancer cell's capacity to: (I) resist conventional treatment methods (chemotherapy and radiotherapy), (II) promote angiogenesis, and (III) metastasize and/or to survive microenvironments that normally would promote cell apoptosis/necrosis. The realization of such strategies is limited by the lack of pharmaceutically-viable technologies that enable the safe and effective delivery of gene-targeting agents to neoplastic cells following systemic administration. There are many reasons for this, including an incomplete understanding of how cancer cells respond when genes are silenced. Further the pharmacokinetic and pharmacodynamic attributes of gene therapy products are not well understood. This review will discuss gene therapy strategies that have been developed based on gene inhibition by the use of antisense oligonucleotides, ribozymes and RNA interference (RNAi). In this context, several particularly promising targets will be described, with a focus on strategies that have progressed to the stage where clinical trials have been initiated. The review highlights product development strategies that emphasize non-viral systemic formulations and the potential for delivery systems to become an enabling technology for development of effective gene therapy products.

Copper-topotecan complexation mediates drug accumulation into liposomes.

These studies describe the role of transition metal ions in the liposomal encapsulation of topotecan. Liposomes (1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (CH) (55:45, mole ratio)) were prepared with manganese (Mn), copper (Cu), zinc (Zn) or cobalt (Co) ion gradients (metal inside). Subsequently, topotecan was added to the liposome exterior (final drug-to-lipid ratio (mol/mol) of 0.2) and drug encapsulation was measured as a function of time and temperature. No drug loading was achieved with liposomes containing Co or Zn. Topotecan could be encapsulated into Mn-containing liposomes only in the presence of the ionophore, A23187 suggesting that a transmembrane pH gradient was necessary. However, Cu-containing liposomes, in the presence or absence of an imposed pH gradient, efficiently encapsulated topotecan. It has been reported that Cu(II) can form transition metal complexes with camptothecin; therefore, the Cu-topotecan interaction was characterized in solution as a function of pH. These investigations demonstrated that topotecan inhibited formation of an insoluble Cu hydroxide precipitate. Cryo-TEM analysis of the topotecan-loaded Cu liposomes showed electron-dense intravesicular precipitates. Further studies demonstrated that only the active lactone form of the drug was encapsulated and this form predominated in Cu-containing liposomes. Copper complexation reactions define a viable methodology to prepare liposomal camptothecin formulations.

Microfluidics: a transformational tool for nanomedicine development and production.

Microfluidic devices are mircoscale fluidic circuits used to manipulate liquids at the nanoliter scale. The ability to control the mixing of fluids and the continuous nature of the process make it apt for solvent/antisolvent precipitation of drug-delivery nanoparticles. This review describes the use of numerous microfluidic designs for the formulation and production of lipid nanoparticles, liposomes and polymer nanoparticles to encapsulate and deliver small molecule or genetic payloads. The advantages of microfluidics are illustrated through examples from literature comparing conventional processes such as beaker and T-tube mixing to microfluidic approaches. Particular emphasis is placed on examples of microfluidic nanoparticle formulations that have been tested in vitro and in vivo. Fine control of process parameters afforded by microfluidics, allows unprecedented optimization of nanoparticle quality and encapsulation efficiency. Automation improves the reproducibility and optimization of formulations. Furthermore, the continuous nature of the microfluidic process is inherently scalable, allowing optimization at low volumes, which is advantageous with scarce or costly materials, as well as scale-up through process parallelization. Given these advantages, microfluidics is poised to become the new paradigm for nanomedicine formulation and production.

Liposomal irinotecan: formulation development and therapeutic assessment in murine xenograft models of colorectal cancer.

PURPOSE: The purpose is to demonstrate whether an appropriately designed liposomal formulation of irinotecan is effective in treating mice with liver-localized colorectal carcinomas. EXPERIMENTAL DESIGN: Irinotecan was encapsulated in 1,2-distearoyl-sn-glycero-3-phosphocholine/cholesterol (55:45 molar ratio) liposomes using an ionophore (A23187)-generated transmembrane proton gradient. This formulation was evaluated in vivo by measuring plasma elimination of liposomal lipid and drug after i.v. administration. Therapeutic activity was determined in SCID/Rag-2M mice bearing s.c. LS180 tumors or orthotopic LS174T colorectal metastases. RESULTS: Drug elimination from the plasma was significantly reduced when irinotecan was administered in the liposomal formulation. At 1 hour after i.v. administration, circulating levels of the liposomal drug were 100-fold greater than that of irinotecan given at the same dose. High-performance liquid chromatographic analysis of plasma samples indicated that liposomal irinotecan was protected from inactivating hydrolysis to the carboxylate form. This formulation exhibited substantially improved therapeutic effects. For the LS180 solid tumor model, it was shown that after a single injection of liposomal irinotecan at 50 mg/kg, the time to progress to a 400-mg tumor was 34 days (as compared with 22 days for animals treated with free drug at an equivalent dose). In the model of colorectal liver metastases (LS174T), a median survival time of 79 days was observed after treatment with liposomal irinotecan (50 mg/kg, given every 4 days for a total of three doses). Saline and free drug treated mice survived for 34 and 53 days, respectively. CONCLUSIONS: These results illustrate that liposomal encapsulation can substantially enhance the therapeutic activity of irinotecan and emphasize the potential for using liposomal irinotecan to treat liver metastases.

The formulation of lipid-based nanotechnologies for the delivery of fixed dose anticancer drug combinations.

The introduction of combination chemotherapeutic regimens for the treatment of childhood leukaemia in the 1960s provided the proof-of-principle that cytotoxic drugs were capable of curing cancer. However, in the four decades since this discovery, the majority of cancers still cannot be cured by chemotherapy. Clinical evidence supports the hypothesis of Goldie and Coldman that treating cancers with all the available effective agents simultaneously provides the greatest chance of eliciting a cure. Unfortunately, for traditional cytotoxic agents with narrow therapeutic indices, life-threatening toxicity precludes combination chemotherapy regimens employing multiple agents. This review discusses the concept of fixed dose combination chemotherapy with emphasis on capturing therapeutic efficacy described as synergistic as a basis for improving the effectiveness of combination chemotherapy. The use of lipid-based nanotechnologies, focusing on liposomes, as an enabling technology to facilitate the delivery of cytotoxic agents to the tumour site at concentrations and/or drug ratios judged to be synergistic will be discussed. It is envisaged that the development of this model system will be supported by cell-based screening technologies, pharmacokinetic and pharmacodynamic parameters and mathematical models describing therapeutic drug:drug interactions (the Median Effect Principle of Chou and Talalay). Experiments using preclinical models are presented to support the benefits of drug delivery systems as a foundation for fixed dose anticancer drug combinations. The ultimate goal of this research is to prepare a 'single vial' fixed dose combination product that encompasses both traditional cytotoxic agents and new molecularly targeted modalities with optimum therapeutic effects and acceptable toxicity.

Microfluidic-based manufacture of siRNA-lipid nanoparticles for therapeutic applications.

A simple, efficient, and scalable manufacturing technique is required for developing siRNA-lipid nanoparticles (siRNA-LNP) for therapeutic applications. In this chapter we describe a novel microfluidic-based manufacturing process for the rapid manufacture of siRNA-LNP, together with protocols for characterizing the size, polydispersity, RNA encapsulation efficiency, RNA concentration, and total lipid concentration of the resultant nanoparticles.