Overcoming PARPi resistance: Preclinical and clinical evidence in ovarian cancer.

Ovarian cancer is the fifth cause of cancer-related deaths in women with high grade serous carcinoma (HGSOC) representing the most common histological subtype. Approximately 50 % of HGSOC are characterized by deficiency in homologous recombination (HR), one of the main cellular pathways to repair DNA double strand breaks and one of the well-described mechanisms is the loss of function of the BRCA1 or BRCA2 genes. Inhibition of the poly-ADP-ribose polymerase (PARP) is synthetic lethal with HR deficiency and the use of PARP inhibitors (PARPi) has significantly improved the outcome of patients with HGSOC with a greater benefit in patients with BRCA1/2 deficient tumors. However, intrinsic or acquired resistance to PARPi inevitably occurs in most HGSOC patients. Distinct heterogeneous mechanisms underlying the resistance to PARPi have been described, including a decrease in intracellular drug levels due to upregulation of multidrug efflux pumps, loss of expression/inactivating mutations in the PARP1 protein, restoration of HR and the protection of the replicative fork. Deciphering the molecular mechanisms of resistance to PARPi is of paramount importance towards the development of new treatment strategies and/or novel pharmacological agents to overcome this chemoresistance and optimize the treatment regimen for individual HGSOC patients. The current review summarizes the mechanisms underlying the resistance to PARPi, the available preclinical and clinical data on new combination treatment strategies (with chemotherapy, anti-angiogenic agents and immune checkpoint inhibitors) as well as agents under investigation which target the DNA damage response.

Effects of short-to-long term enzyme replacement therapy (ERT) on skeletal muscle tissue in late onset Pompe disease (LOPD).

AIMS: Pompe disease is an autosomal recessive lysosomal storage disorder resulting from deficiency of acid α-glucosidase (GAA) enzyme. Histopathological hallmarks in skeletal muscle tissue are fibre vacuolization and autophagy. Since 2006, enzyme replacement therapy (ERT) is the only approved treatment with human recombinant GAA alglucosidase alfa. We designed a study to examine ERT-related skeletal muscle changes in 18 modestly to moderately affected late onset Pompe disease (LOPD) patients along with the relationship between morphological/biochemical changes and clinical outcomes. Treatment duration was short-to-long term. METHODS: We examined muscle biopsies from 18 LOPD patients at both histopathological and biochemical level. All patients underwent two muscle biopsies, before and after ERT administration respectively. The study is partially retrospective because the first biopsies were taken before the study was designed, whereas the second biopsy was always performed after at least 6 months of ERT administration. RESULTS: After ERT, 15 out of 18 patients showed improved 6-min walking test (6MWT; P = 0.0007) and most of them achieved respiratory stabilization. Pretreatment muscle biopsies disclosed marked histopathological variability, ranging from an almost normal pattern to a severe vacuolar myopathy. After treatment, we detected morphological improvement in 15 patients and worsening in three patients. Post-ERT GAA enzymatic activity was mildly increased compared with pretreatment levels in all patients. Protein levels of the mature enzyme increased in 14 of the 18 patients (mean increase = +35%; P < 0.05). Additional studies demonstrated an improved autophagic flux after ERT in some patients. CONCLUSIONS: ERT positively modified skeletal muscle pathology as well as motor and respiratory outcomes in the majority of LOPD patients.

Changes in Lipid Composition During Manganese-Induced Apoptosis in PC12 Cells.

Lipid composition of membranes is fundamental to modulate signaling pathways relying on lipid metabolites and/or membrane proteins, thus resulting in the regulation of important cell processes such as apoptosis. In this case, membrane remodeling is an early event important for the activation of signaling leading to cell death and removal of apoptotic cells. In the present study, we analyzed phospholipid, cholesterol and fatty acid content during apoptosis induced by manganese in PC12 cells. Lipid analysis of whole cells and detergent-resistant membranes was carried out by HPLC/GC. Results showed that apoptosis is associated with changes in lipid composition detectable in whole cell extracts, namely cholesterol, phosphatidylserine and phosphatidylethanolamine decreases. Noteworthy, phosphatidylserine level reduction was detectable before to the detection of apoptosis, in correlation with our previous study carried out by radioactive labelling. By contrast, phosphatidylserine and phosphatidylethanolamine changes were not detected in detergent resistant membranes, which instead showed an altered composition in phosphatidylinositol, phosphatidylcholine and sphingomyelin in apoptotic cells.

SAKK57/16 Nab-paclitaxel and Gemcitabine in Soft Tissue Sarcoma (NAPAGE): a phase I/II trial.

BACKGROUND: To determine whether the combination of nab-paclitaxel with gemcitabine has activity in patients with pretreated soft tissue sarcoma (STS). PATIENTS AND METHODS: NAPAGE is a phase Ib/II clinical trial investigating the combination of nab-paclitaxel (nab-pc) with gemcitabine employing two cohorts. One of a dose-de-escalation phase and one of expansion. In phase I, nab-pc was given at 150 mg/m2 in combination with gemcitabine 1000 mg/m2 every two weeks, until disease progression or unacceptable toxicity. This dose was recommended for phase II (RP2D), as there was no dose limiting toxicity (DLT) or discontinuations due to adverse events (AEs). The primary endpoint of the phase II was progression-free rate (PFR) at 3 months (H0: 20%, H1:40%). The secondary endpoints included progression free survival (PFS), overall survival (OS), AEs, objective response and patient-reported outcomes (PRO). Efficacy analysis was by intention to treat. RESULTS: The 3-month PFR was 56.4% (95% confidence interval CI: 39.6-72.2%). The 3-month and 6-month PFS were 58.4% (95% CI: 41.3-72.1%) and 44.6% (95% CI: 28.4-59.5%), respectively. Median PFS was 5.3 months (95% CI: 1.4-8.2) and median OS was 12.8 months (95% CI: 10.5-39.2). The most common treatment-related grade ≥ 3 AE were neutropenia (18%), followed by anemia (2.6%), hypertension (2.6%) and alanine aminotransferase increase (2.6%). Grade 1 and grade 2 peripheral sensory neuropathy (PNP) occurred in 15.4% and 20.5%, respectively. No grade 3-4 PNP was reported. CONCLUSIONS: Combining nab-pc and gemcitabine is safe. Promising activity is observed in pretreated STS patients with manageable toxicity. This regimen should be considered for further exploration.

A systematic review of phase II trials exploring anti-PD-1/PD-L1 combinations in patients with solid tumors.

BACKGROUND: A high number of combinations of PD-1/PD-L1 inhibitors with other anti-cancer therapies are in clinical development. The usefulness of phase II trials in evaluating their efficacy and safety is unclear. MATERIALS AND METHODS: We performed a systematic search on PubMed and Cochrane Library for phase II trials of PD-1/PD-L1 inhibitors in combination with other anti-cancer therapies (systemic therapy and/or radiotherapy) published between January 1st 2018 and December 31st 2020. Study design, primary endpoint and main outcomes were registered for each paper. RESULTS: 119 articles reporting on 65 regimens were included in our analysis. Backbone agents were more frequently PD-1 inhibitors (pembrolizumab = 47, nivolumab = 41, camrelizumab = 3) followed by anti-PD-L1 (durvalumab = 19, atezolizumab = 6, avelumab = 3). Therapeutic partners were other immunotherapeutic agents (n = 46), targeted therapies (n = 40), chemotherapy (n = 22) or radiotherapy (n = 11). The majority of articles reported on single-arm trials (n = 87, 73%) and response rate was the most frequent primary endpoint (n = 69, 58%). Objective responses, registered in 109 (92%) articles, ranged between 0% and 91%. The incidence of grade 3 or higher treatment-related adverse events, clearly reported in 97 (82%) articles, spanned from 0 to 100%. Five combinations received regulatory approval by Food and Drug Administration or European Medicine Agency for 9 different indications, based on the results of a phase II trial (n = 3) or on a confirmatory phase III trial (n = 6). CONCLUSIONS: The landscape of phase II trials evaluating PD-1/PD-L1 inhibitors with other anticancer therapies is heterogeneous. Combinations of two immunotherapeutic agents have been the most investigated. Only a minority of indications (8%) granted regulatory approval.

cDNA cloning and Escherichia coli expression of UK114 tumor antigen.

Experimental evidence indicates that the antineoplastic effects of UK101, a goat liver perchloric acid extract, is likely due to one of its constituent proteins: the 14 kDa protein named UK114. The cDNA encoding UK114, obtained by PCR methodologies, contains an open reading frame coding for a protein of 137 amino acids with a theoretical molecular mass of 14298 Da. It shows high sequence homology with a 14 kDa protein identified in human, rat and Mus musculus tissues which is likely involved in the inhibition of cell-free protein synthesis. Northern blot analysis indicated that the transcript is present in variable amounts in a wide range of human tissues. Genomic Southern blots revealed that the UK114 mRNA in goat as well as in human is encoded by a single gene, as is the case in rat. The expression system for UK114 was constructed under the control of the PL promoter from bacteriophage lambda and the cDNA coding region has been highly expressed in Escherichia coli as a thioredoxin fusion protein. The recombinant UK114, purified to homogeneity, is immunoreactive to rabbit antisera prepared against UK101 or native UK114, as well as to sera of UK101-treated cancer patients. It inhibits cell-free protein synthesis at 8 microM concentration.

Modifications of glycosphingolipid profile and synthesis in normal rat fibroblasts and in syngeneic neoplastic cells at different subculture stages.

Glycosphingolipids are plasma membrane macromolecules involved in diversified recognition functions on the cell surface resulting in modulation of cell adhesion and differentiation. As the in vitro cellular system of the neoplastic cell line SGS/4A and syngeneic normal fibroblasts (FG) represents a useful tool for studies on molecular mechanisms regulating cell adhesion, neoplastic transformation and cellular ageing, we studied the changes of glycosphingolipid and of the enzymes involved in their metabolism in both cultured cells at different subculture stages. The FG subculture progression induces a drastic decrease of total glycosphingolipid content with consistent alterations in the molecular composition. In particular, a significant decrease of GM(3), a slight increase of GD(1a), the disappearance of 'b'-series gangliosides and the drastic reduction of triosylceramides were observed. On the contrary, the increasing number of SGS/4A subcultures, characterized by a specific and different glycosphingolipid composition as compared with FG cells, does not cause modifications. Although glycosyltransferase activity levels quite well parallel the glycosphingolipid patterns and can account for the noted variations, the mRNA expression analysis of two glycosyltransferases suggests that the in vitro cell ageing of normal rat fibroblasts causes drastic changes in the glycosphingolipid profile through the regulation, at either the transcriptional or post-translational level, of some biosynthetic enzymes.

Thermal transformations and stability of organometallic materials with electrical and optical properties: the case of polycrystalline cis-[Ir(CO)2Cl(C5H5N)].

In this paper the solid-state transformations under heating of cis-[Ir(CO)2Cl(C5H5N)] are discussed. The complexity of the transformations was revealed by integrating infrared spectroscopy, conventional and bidimensional X-ray diffraction, thermal analysis, and hot stage optical microscopy. During heating anisotropic expansion of the lattice along the Ir-Ir stacking takes place. Then cis-[Ir(CO)2Cl(C5H5N)] undergoes an irreversible solid-solid phase transition to a lattice of higher symmetry followed by a reversible transition into the amorphous phase. Under proper cooling a partial recrystallization takes place. Experiments in the presence of oxygen must be carried out in short time periods to avoid oxidation from Ir(I) to Ir(III).

Adult polyglucosan body disease: clinical and histological heterogeneity of a large Italian family.

Adult Polyglucosan Body Disease (APBD) is a rare inherited leukodystrophy associated with axonal polyneuropathy, mainly reported in persons of Ashkenazi-Jewish descent. We describe three Italian siblings at disease onset, presenting in their fifties with a combination of pyramidal and ataxic signs, mild demyelinating neuropathy on neurophysiological investigation (1/3 cases) and transient symptoms (1/3). A leucoencephalopathy with infratentorial lesions without enhancement and medullary/spine atrophy was demonstrated on brain/spine MRI (3/3). Muscle biopsy was normal in 2/3; both muscle and nerve biopsy showed polyglucosan bodies in the sibling with polyneuropathy. This indicated a need for GBE1 sequencing, which revealed a novel missense mutation (c.1064G>A; p.Arg355His) and one previously described (c.1604A>G; p.Tyr535Cys) in all siblings. We highlight that peripheral neuropathy, deemed as disease hallmark, may be missing and that transient symptoms are confirmed as early disease manifestations. The pattern of damage at neuro-imaging described recurs irrespective of clinical presentation, constituting a unifying diagnostic clue.

Purification, characterization and partial amino acid sequences of carnitine palmitoyl-transferase from human liver.

Carnitine palmitoyl-transferase has been extracted with 0.5% Tween-20 from human liver homogenate and purified to homogeneity. The purified enzyme has a native Mr of 274 kDa. The subunit Mr is of 66 kDa, as shown by SDS-PAGE and immunoblots obtained with antibodies raised against human CPT. Purified CPT shows high affinity for palmitoyl-CoA and palmitoyl-carnitine and is not inhibited by malonyl-CoA. Seven tryptic peptides and the N-terminal of purified human CPT have been sequenced, and found homologous to rat CPT sequence. Both antibodies and peptide sequences are important tools for the investigation of the molecular basis of CPT deficiency in man.

The primary structure of UK114 tumor antigen.

UK114 is a tumor antigen expressed by various malignant neoplasms. The complete amino acid sequence of UK114 purified from goat liver has been determined by automated Edman degradation of CNBr and endoproteinase Lys-C peptides. The protein contains 137 amino acid residues. which corresponds to a molecular mass of 14,229 Da. MALDI-TOF analysis resulted in a molecular weight of 14,290, suggesting that the N-terminal Met residue is acetylated. Sequence comparison shows that UK114 from goat liver (1) has 77% identity with a previously described 23 kDa protein from rat liver (Levy-Favatier et al. (1993) Eur. J. Biochem. 212, 665-673), (2) shares a very high degree of similarity with a family of prokaryotic and eukaryotic hypothetic proteins whose function have not yet been characterized, and (3) exhibits a significant similarity to a group of tumor-associated antigens which belongs to a superfamily of heat shock proteins, acting as possible targets for the host's antitumor immunity.

Investigation of surface properties of some polymers by a thermodynamic and mechanical approach: possibility of predicting mucoadhesion and biocompatibility.

Mucoadhesive properties of several polymers, such as sodium alginate, hydroxypropylmethyl cellulose, scleroglucan, xanthan gum, polyacrylic acid (Carbopol), and poly co-(methyl vinyl ether-maleic anhydride) (Gantrez), have been investigated by comparing a thermodynamical and a mechanical approach. Surface properties of polymers in the dry state have been studied by contact angle measurements and thermodynamical parameters derived by using different equations. This tensile adhesive strength of polymers in hydration conditions was measured by a modified DuNoy tensiometer. Comparison of the two different approaches has allowed us to conclude that thermodynamical consideration on surface energy can be used to evaluate mucoadhesive properties of materials. Data obtained with the two methods yielded a good linear correlation. Calculation of surface free energy of the considered materials also allowed a prediction of the water-polymer interface free energy: biocompatibility, defined according to the minimal interfacial free energy concept, could consequently be evaluated.

Mathematical modelling of drug permeation through a swollen membrane.

This work proposes two different mathematical models (linear and numerical) able to simulate the drug permeation through a swollen membrane sandwiched by two external layers (trilaminate system). Moreover, a solid drug dissolution phenomenon in the donor compartment may be accounted for. Indeed, this is a situation that may often occur in permeation experiments. An insufficient stirring of the donor and of the receiver volume may give rise to two sandwiching layers and the target of a constant drug concentration in the donor compartment may be accomplished by putting a solid drug amount in the saturated donor solution. The linear model shows the advantage of having an analytical expression which extremely simplifies the calculation of the drug diffusion coefficient D inside the membrane. Its main drawback lies in the fact that it works only for thin trilaminate systems. The numerical model is more general than the linear one, as it works for all kind of trilaminate thickness and it may account for a solid powder dissolution in the donor compartment. Of course, it does not have an analytical solution and, thus, the D determination is less easy to perform as the numerical model is more time consuming than the linear one. These two models are then compared with the classical approach developed by Flynn and Barrie in order to better define its validity limits.

Experimental determination of the theophylline diffusion coefficient in swollen sodium-alginate membranes.

In this paper attention is focused on the determination of the drug diffusion coefficient in a swollen polymeric membrane referring to a recent mathematical model (linear model). The main advantage deriving from its use is that, despite its analytical nature and its ability to account for the most important aspects characterising a permeation experiment, it can also be applied in the case of thick membranes. To check the model reliability, a comparison is made with a more complex numerical model and with a largely employed model in terms of data fitting quality. To this purpose, particular care is devoted to the experimental and theoretical tools employed to calculate the auxiliary parameters required by the three models, and with the aim of getting a drug diffusion coefficient value as accurate as possible. Theophylline was chosen as model drug owing to its wide employment in the pharmaceutical field. Membranes were prepared with sodium alginates hydrogels at three different polymer concentrations. The present analysis demonstrates the reliability of the linear model and reveals that the theophylline diffusion coefficient is not significantly affected by the polymer concentration. Indeed, such a parameter is reflected in different membrane thicknesses rather than in different mesh sizes of the polymeric network.

Dietary sphingolipids in colorectal cancer prevention.

Sphingolipids are widespread membrane components that are found in all eukaryotic cells. They consist of a long chain sphingoid-base, usually sphingosine, which is acylated at the 2-amino position, forming a ceramide. All together, sphingolipids may represent the most structurally diverse category of lipids in nature. There is no known nutritional requirement for sphingolipids. Nonetheless, studies with experimental animals have shown that consumption of sphingolipids inhibits colon carcinogenesis, reduces serum low-density lipoprotein cholesterol and elevates high-density lipoproteins, which suggest that they are 'functional' components of food. In animal models (CF1 mice) sphingomyelin supplementation reduces the number of aberrant colonic crypt foci by approximately 70% and, with longer feeding, reduces the number of colonic adenocarcinomas. A possible mechanism of action of sphingolipids in suppressing colon carcinogenesis is that exogenously supplied sphingolipids bypass a sphingolipid signalling defect that is important in cancer (for example, a loss of cellular sphingomyelin turnover to produce ceramide and sphingosine). Indirect evidence suggests that sphingolipids can inhibit colon cancer in humans: sphingosine and ceramide induce apoptosis in a human adenocarcinoma cell line and feeding sphingolipids to Min mice reduces the number of colon tumours.

Acyclovir permeation through rat skin: mathematical modelling and in vitro experiments.

The aim of this work is to characterise the skin permeation properties of a male rat by means of a purely diffusive mathematical model based on Fick's second law. Additionally, in the attempt of proposing a reliable tool allowing the skin permeability (or resistance) determination on the basis of experimental data, the model automatically accounts also for two typical experimental conditions. In particular, drug dissolution in the donor environment and receiver sampling technique (part of the receiver volume is withdrawn and immediately replaced by fresh solvent) are considered. The results of this characterisation are then compared with those coming from a common simplified approach. Acyclovir is chosen as model drug and a thermostatic (37 degrees C) Franz cell apparatus is used to perform permeation experiments. This study suggests that Acyclovir permeation through the rat skin can be well described by the proposed model and that some differences arise in the evaluation of the full-skin resistance performed by means of our model or the usual simpler approach.

The effect of compression on the capillary microstructure of tablets.

The effect of compaction pressure on the capillary characteristics of compacts of a crystalline drug and of a pure polymer was studied by means of penetration and porosimetry measurements. Not only the constant but also the exponent m of the general penetration volume-time relationship V = Kmtm proved to be strongly dependent on the pressure: as the compaction pressure is increased, the value of m tends to decrease. The capillary penetration data also showed the development of a marked heterogeneity in the structure of the polymer compacts at the lower compaction pressures: at these pressures the surface layers showed a much lower penetrability than the inner core of the compacts, whereas at the higher pressures the structure of the compacts was homogeneous. Furthermore, on the basis of a generally used equation describing the powder consolidation process, it was possible to relate the most relevant variation of the kinetics of capillary penetration into the drug compacts to the deformation of the particles. It is concluded that the capillary penetration measurements can give useful information in the understanding of the behaviour of pharmaceutical powders under compression.

[Controlled clinical trial of parsalmide in patients with subacute and chronic arthro-neuro-mypathies]. / Sperimentazione clinica controllata di parsalmide in pazienti affetti da artro-neuro-miopatie subacute e croniche

Controlled experiments were carried out to compare the anti-inflammatory, analgesic, muscle-relaxing and anxiolytic-tranquillizing activities of a new compound, parsalmide, and indomethacin. The study was conducted on 40 patients of both sexes suffering from syndromes involving neuromotor deficiency of various origin and the double blind between patient, technique was used. Overall evaluation of the activities of the two drugs did not reveal statistically significant differences although parsalmide was more effective with "pain", "tumefaction" and "anxiety" symptoms. This latter finding is particularly interesting owing to the frequent association of neuroanxiety disturbances in such patients. General and local tolerance was good with both drugs.

Glycosphingolipid expression in solid tumours and transformed cell lines.

Glycosphingolipids are assumed to play a crucial role in cell-cell and cell-substrate interactions, including cell adhesion, proliferation, differentiation and apoptosis. Furthermore, cell surface glycolipid profile changes in the so called "social disorders", such as malignant transformation. To better investigate these modifications, the ganglioside composition in different solid tumours and in two transformed cell lines was analyzed. In some of these models we also tried to correlate the pattern of gangliosides to the key enzymes involved in their metabolism. The results we obtained can be summarized as follows:(1), meningiomas with or without chromosome 22 deletion: predominance of ganglioside GD3 in the former and of ganglioside GM3 in the latter. Correlation between GM3/GD3 ratio and SAT-2 activity; (2), mammary carcinomas developed in MMTV/c-neu transgenic mice: accumulation of GM3-derived species. The different ganglioside distribution seems to correlate with the tumour size; (3), Sarcoma Galliera-strain cells SGS/3A and normal syngenic murine fibroblasts FG: transformed cells exhibit a lower activity of sialyltransferases (SAT-1, SAT-2, SAT-4) compared to normal fibroblasts, suggesting a possible correlation with the ganglioside pattern. The neuraminidase activity seems to correlate to the glycoprotein sialic acid content; (4), 3T3 normal murine fibroblasts and SVT2 transformed cells: GM3 is absent in 3T3, while it accounts for the main ganglioside species in SVT2. On the contrary, GM2 present in a large amount in normal fibroblasts, is practically absent in transformed cells. No correlation has been observed between ganglioside profile and glycosyltransferase activities so far examined.