Preparation and evaluation of chitosan based thermoreversible gels for intraperitoneal delivery of 5-fluorouracil (5-FU)

Sterile thermoreversibly gelling systems based on chitosan-glycerol phosphate were developed for intraperitoneal delivery of the antineoplastic agent 5-FU. The formulation was evaluated for gelling characteristics and in vitro drug release. Drug free gels were evaluated for in vitro cytotoxicity in L-929 mouse fibroblast cells. Drug loaded gels were subjected to acute toxicity studies in Swiss albino mice via intraperitoneal route and efficacy studies via intratumoral injections in subcutaneous colon carcinoma bearing BALB/c mice. The formulations gelled reversibly in 8 min at 37 oC and provided prolonged release of the drug. Drug free systems showed dose dependent cytotoxicity in fibroblast cells, while in vivo studies revealed a 2.8 fold increase in LD50 of 5-FU administered intraperitonealy as the developed system. Tumor volume measurements showed comparable efficacy of 5-FU administered as gel and commercial injection with greatly improved safety profile of the former as adjudged from mortality and body weight measurements

The evolution of lung cancer and impact of subclonal selection in TRACERx

Lung cancer is the leading cause of cancer-associated mortality worldwide. Here we analysed 1,644 tumour regions sampled at surgery or during follow-up from the first 421 patients with non-small cell lung cancer prospectively enrolled into the TRACERx study. This project aims to decipher lung cancer evolution and address the primary study endpoint: determining the relationship between intratumour heterogeneity and clinical outcome. In lung adenocarcinoma, mutations in 22 out of 40 common cancer genes were under significant subclonal selection, including classical tumour initiators such as TP53 and KRAS. We defined evolutionary dependencies between drivers, mutational processes and whole genome doubling (WGD) events. Despite patients having a history of smoking, 8% of lung adenocarcinomas lacked evidence of tobacco-induced mutagenesis. These tumours also had similar detection rates for EGFR mutations and for RET, ROS1, ALK and MET oncogenic isoforms compared with tumours in never-smokers, which suggests that they have a similar aetiology and pathogenesis. Large subclonal expansions were associated with positive subclonal selection. Patients with tumours harbouring recent subclonal expansions, on the terminus of a phylogenetic branch, had significantly shorter disease-free survival. Subclonal WGD was detected in 19% of tumours, and 10% of tumours harboured multiple subclonal WGDs in parallel. Subclonal, but not truncal, WGD was associated with shorter disease-free survival. Copy number heterogeneity was associated with extrathoracic relapse within 1 year after surgery. These data demonstrate the importance of clonal expansion, WGD and copy number instability in determining the timing and patterns of relapse in non-small cell lung cancer and provide a comprehensive clinical cancer evolutionary data resource

The evolution of non-small cell lung cancer metastases in TRACERx

Metastatic disease is responsible for the majority of cancer-related deaths. We report the longitudinal evolutionary analysis of 126 non-small cell lung cancer (NSCLC) tumours from 421 prospectively recruited patients in TRACERx who developed metastatic disease, compared with a control cohort of 144 non-metastatic tumours. In 25% of cases, metastases diverged early, before the last clonal sweep in the primary tumour, and early divergence was enriched for patients who were smokers at the time of initial diagnosis. Simulations suggested that early metastatic divergence more frequently occurred at smaller tumour diameters (less than 8 mm). Single-region primary tumour sampling resulted in 83% of late divergence cases being misclassified as early, highlighting the importance of extensive primary tumour sampling. Polyclonal dissemination, which was associated with extrathoracic disease recurrence, was found in 32% of cases. Primary lymph node disease contributed to metastatic relapse in less than 20% of cases, representing a hallmark of metastatic potential rather than a route to subsequent recurrences/disease progression. Metastasis-seeding subclones exhibited subclonal expansions within primary tumours, probably reflecting positive selection. Our findings highlight the importance of selection in metastatic clone evolution within untreated primary tumours, the distinction between monoclonal versus polyclonal seeding in dictating site of recurrence, the limitations of current radiological screening approaches for early diverging tumours and the need to develop strategies to target metastasis-seeding subclones before relapse

Genomic–transcriptomic evolution in lung cancer and metastasis

Intratumour heterogeneity (ITH) fuels lung cancer evolution, which leads to immune evasion and resistance to therapy. Here, using paired whole-exome and RNA sequencing data, we investigate intratumour transcriptomic diversity in 354 non-small cell lung cancer tumours from 347 out of the first 421 patients prospectively recruited into the TRACERx study. Analyses of 947 tumour regions, representing both primary and metastatic disease, alongside 96 tumour-adjacent normal tissue samples implicate the transcriptome as a major source of phenotypic variation. Gene expression levels and ITH relate to patterns of positive and negative selection during tumour evolution. We observe frequent copy number-independent allele-specific expression that is linked to epigenomic dysfunction. Allele-specific expression can also result in genomic–transcriptomic parallel evolution, which converges on cancer gene disruption. We extract signatures of RNA single-base substitutions and link their aetiology to the activity of the RNA-editing enzymes ADAR and APOBEC3A, thereby revealing otherwise undetected ongoing APOBEC activity in tumours. Characterizing the transcriptomes of primary–metastatic tumour pairs, we combine multiple machine-learning approaches that leverage genomic and transcriptomic variables to link metastasis-seeding potential to the evolutionary context of mutations and increased proliferation within primary tumour regions. These results highlight the interplay between the genome and transcriptome in influencing ITH, lung cancer evolution and metastasis

Antibodies against endogenous retroviruses promote lung cancer immunotherapy

B cells are frequently found in the margins of solid tumours as organized follicles in ectopic lymphoid organs called tertiary lymphoid structures (TLS). Although TLS have been found to correlate with improved patient survival and response to immune checkpoint blockade (ICB), the underlying mechanisms of this association remain elusive. Here we investigate lung-resident B cell responses in patients from the TRACERx 421 (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy) and other lung cancer cohorts, and in a recently established immunogenic mouse model for lung adenocarcinoma. We find that both human and mouse lung adenocarcinomas elicit local germinal centre responses and tumour-binding antibodies, and further identify endogenous retrovirus (ERV) envelope glycoproteins as a dominant anti-tumour antibody target. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted inhibition of KRAS(G12C) in the mouse model. ERV-reactive antibodies exert anti-tumour activity that extends survival in the mouse model, and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Finally, we find that effective immunotherapy in the mouse model requires CXCL13-dependent TLS formation. Conversely, therapeutic CXCL13 treatment potentiates anti-tumour immunity and synergizes with ICB. Our findings provide a possible mechanistic basis for the association of TLS with immunotherapy response

DEVELOPMENT AND EVALUATION OF A POLOXAMER- AND CHITOSAN-BASED IN SITU GEL-FORMING INJECTABLE DEPOT

Objective: The present study is intended to investigate the applicability of poloxamer- and chitosan-based temperature induced in situ injectable gelling depot for once a week therapy as an intramuscular injection employing olanzapine as a model drug. Methods: The thermosetting gel was prepared by admixture of a solution of poloxamer P127 and a solution of olanzapine and chitosan in aqueous acetic acid. The resultant formulation was characterized for gelation temperature, gelation time, viscosity, syringeability, pH, drug content, and in vitro drug release. The in vitro release of olanzapine from the gelled depot was followed using USP paddle type II apparatus in conjunction with a dialysis bag. The gel was injected ex vivo into chicken muscle and observed by subsequent dissection. Results: The formulation was designed to have a phase transition temperature of 34°C and gelled in <10 s at 37°C. Addition of chitosan imparted favorable rheological properties to the poloxamer gel and resulted in a pseudoplastic mixture with low viscosity in the sol state and higher viscosity post gelation. The preparation had a pH of 5.4, appropriate drug content and readily passed through a 20 gauge needle. The release of olanzapine was unhindered by the dialysis bag. Following an initial bust, a sustained, zero-order release of the remainder of drug was observed up to 9 days. The injectable was found to form a compact depot when evaluated ex vivo. Conclusion: The developed system showed several features which make it a suitable vehicle for sustained intramuscular delivery of drugs

DEVELOPMENT OF ORODISPERSIBLE TABLETS OF LORATADINE CONTAINING AN AMORPHOUS SOLID DISPERSION OF THE DRUG IN SOLUPLUS® USING DESIGN OF EXPERIMENTS

Objective: The objective of the present work was to develop an orodispersible tablet of loratadine, an orally active, non-sedating anti-histaminic, belonging to BCS Class II. The drug was prepared as a solid dispersion using Soluplus® as carrier and formulated into an optimal tablet using Design of Experiments. Methods: Solid dispersions of loratadine with varying ratios of Soluplus® were prepared by solvent evaporation and subjected to solubility study in simulated salivary fluid. Selected composition was characterized by differential scanning calorimetry and X-ray diffraction and formulated into an orodispersible tablet by direct compression after addition of suitable excipients. DOE based on a full factorial design was used to optimize the product using a trial version of JMP software, so as to obtain a tablet with low friability, rapid disintegration and maximal drug dissolution within 5 min. The optimized tablet was prepared and evaluated for several attributes, including in vivo disintegration and palatability. Results: A solid dispersion prepared with a 1: 4 ratio of loratadine: Soluplus® was found to show a 130-fold increase in drug solubility in the simulated salivary fluid. X-ray diffraction revealed loratadine in amorphous form. The exercise using DOE for optimization of the orodispersible tablet formula served to balance the proportion of crospovidone as super disintegrant and PVP as dry binder and yielded a formulation with good mechanical strength, rapid in vitro disintegration (39 sec) and dissolution of 93.78% of the drug within 5 min. When evaluated in vivo, the tablets were found to disintegrate in about 60 secs and were reported to be palatable. Conclusion: A patient-friendly dosage form containing a highly soluble form of loratadine was prepared and could be of potential benefit in offering quick relief from allergic conditions

Synthesis of Thiolated Alginate and Evaluation of Mucoadhesiveness, Cytotoxicity and Release Retardant Properties

Modification of polymers by covalent attachment of thiol bearing pendant groups is reported to impart many beneficial properties to them. Hence in the present study, sodium alginate–cysteine conjugate was synthesized by carbodiimide mediated coupling under varying reaction conditions and the derivatives characterized for thiol content. The thiolated alginate species synthesized had bound thiol content ranging from 247.8±11.03–324.54±10.107 ΅mol/g of polymer depending on the reaction conditions. Matrix tablets based on sodium alginate-cysteine conjugate and native sodium alginate containing tramadol hydrochloride as a model drug were prepared and mucoadhesive strength and in vitro drug release from the tablets were compared. Tablets containing 75 mg sodium alginate-cysteine conjugate could sustain release of 10 mg of model drug for 3 h, whereas 90% of the drug was released within 1 h from corresponding tablets prepared using native sodium alginate. An approximately 2-fold increase in the minimal detachment force of the tablets from an artificial mucin film was observed for sodium alginate–cysteine conjugate as compared to native sodium alginate. In vitro cytotoxicity studies in L-929 mouse fibroblast cells studied using an MTT assay revealed that at low concentrations of polymer, sodium alginate–cysteine conjugate was less toxic to L-929 mouse fibroblast cell line when compared to native sodium alginate. Hence, thiolation is found to be a simple route to improving polymer performance. The combination of improved controlled drug release and mucoadhesive properties coupled with the low toxicity of these new excipients builds up immense scope for the use of thiolated polymers in mucoadhesive drug delivery systems