2. Universidad Cardenal Herrera-CEU

The incidence of fungal pulmonary infections is known to be on the increase, and yet there is an alarming gap in terms of marketed antifungal therapies that are available for pulmonary administration. Amphotericin B (AmB) is a highly efficient broad-spectrum antifungal only marketed as an intravenous formulation. Based on the lack of effective antifungal and antiparasitic pulmonary treatments, the aim of this study was to develop a carbohydrate-based AmB dry powder inhaler (DPI) formulation, prepared by spray drying. Amorphous AmB microparticles were developed by combining 39.7 % AmB with 39.7 % γ-cyclodextrin, 8.1 % mannose and 12.5 % leucine. An increase in the mannose concentration from 8.1 to 29.8 %, led to partial drug crystallisation. Both formulations showed good in vitro lung deposition characteristics (80 % FPF < 5 µm and MMAD < 3 µm) at different air flow rates (60 and 30 L/min) when used with a DPI, but also during nebulisation upon reconstitution in water.

The protozoans Giardia duodenalis and Cryptosporidium spp. are common causes of gastrointestinal disease in humans and animals. While both are commonly documented in domestic animals, few studies have analysed their presence in wildlife. To assess the prevalence of both parasites in wild boar (Sus scrofa) in the Valencian Community (eastern Spain), 498 wild boar faecal samples were collected from 2018 to 2022. Cryptosporidium spp. was detected by performing a nested PCR targeting a 578 bp sequence of the small subunit ribosomal RNA gene (SSU rRNA), followed by sequencing and phylogenetic analysis. For G. duodenalis, a qPCR amplifying a fragment of 62 bp from the SSU rRNA was employed. Positive samples were genotyped for glutamate dehydrogenase and β-giardin genes. Different epidemiological factors were considered potential modulating variables in the transmission of both parasites. G. duodenalis prevalence was 1.20%, while Cryptosporidium spp. prevalence reached 21.7%. Coinfection was observed in 0.2%. Genotyping of G. duodenalis isolates only detected genotype E. Two species of Cryptosporidium spp. were identified: Cryptosporidium scrofarum and Cryptosporidium suis. The results of this study demonstrate that the exposure to Cryptosporidium spp. in wild boars is high, particularly among young individuals belonging to the Typical Mediterranean climate. Moreover, the probability of infection is dependent on both the season and the density of wild boars. On the other side, exposure to G. duodenalis seems scarce and is influenced, in turn, by the climate. Both Cryptosporidium species detected in the present study have been reported in humans. Due to wild boar increasing in number and their colonisation of urban and peri-urban areas, this could represent an inherent health risk for the human population.

Metal complexes have gained a huge interest in the biomedical research in the last decade because of the access to unexplored chemical space with regards to organic molecules and to present additional functionalities to act simultaneously as diagnostic and therapeutic agents. Herein, we evaluated the interaction of two polytopic polyaza ligands and their zinc complexes with DNA and RNA by UV thermal denaturation, fluorescence and circular dichroism spectroscopic assays. The zinc coordination was investigated by X-ray diffraction and afforded the structure of the binuclear zinc complex of PYPOD. Thermal denaturation of DNA and RNA and fluorimetry analysis revealed preferential binding of the zinc-PHENPOD complexes towards GC-containing DNA in contrast to the free ligands. On the other hand, PYPOD metal complexes, compared to the free ligand, stabilized AT-based DNA (B-form) better than AU-RNA (A-form). With regards to single stranded RNA, the binuclear complex of PHENPOD and the free ligand can efficiently identify polyadenylic acid (poly A) among other RNA sequences by circular dichroism spectroscopy. The antimicrobial activity in S. aureus and E. coli bacteria showed the highest activity for the free ligands and their trinuclear zinc complexes. This work can provide valuable insights into the impact of the nuclearity of polytopic polyaza ligands in the binding to DNA/RNA and the antimicrobial effect.

Microsporidia are widely spread obligate intracellular fungal pathogens from vertebrate and invertebrate organisms, mainly transmitted by contaminated food and water. This study aims to detect the presence of major human-pathogenic microsporidia, i.e., Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, and Encephalitozoon cuniculi, in the gastrointestinal tract of commercially harvested marine fish from Mediterranean coast of the Comunidad Valenciana, Eastern Spain. A total of 251 fish, 138 farmed fish and 113 wild fish from commercial fishing were tested by SYBR Green real-time PCR, enabling the simultaneous detection of the four targeted species. E. intestinalis/hellem was found in 1.45% of farmed fish and 7.96% of wild fish, while Enterocytozoonidae was detected in 2.90% and 18.58% of farmed and wild fish, respectively. E. cuniculi was not detected in any of the analyzed specimens. To the authors’ knowledge, this is the first report of E. intestinalis/hellem in fish, particularly in marine fish. Although the role of fish in these species’ epidemiology remains unknown, this finding points out a potential public health risk linked to fish consumption. Further studies are necessary to characterize these microsporidia in fish hosts better and to elucidate their epidemiological role.

Fish not only harbor host-specific species/genotypes of Cryptosporidium, but also species like zoonotic C. parvum or anthroponotic C. hominis, which can pose a risk for fish consumers. This study aims to investigate fish cryptosporidiosis in an important aquaculture and fishery area of the Western Mediterranean (Comunidad Valenciana, Spain). We analyzed 404 specimens belonging to the following three groups: cultivated fish (N = 147), wild synanthropic fish (N = 147) and wild fish from extractive fisheries (N = 110). Nested PCR targeting the 18S rRNA gene, followed by sequencing and phylogenetic analysis, were performed. Positive isolates were also amplified at the actin gene locus. An overall prevalence of 4.2% was detected, with the highest prevalence in the synanthropic group (6.1%). C. molnari was identified in thirteen specimens from seven different host species. Zoonotic C. ubiquitum was detected in two European sea bass (Dicentrarchus labrax). One isolate similar to C. scophthalmi was detected in a cultivated meagre (Argyrosomus regius), and one isolate, highly divergent from all the Cryptosporidium species/genotypes described, was identified from a synanthropic round sardinella (Sardinella aurita). This study contributes to increasing the molecular data on fish cryptosporidiosis, expanding the range of known hosts for C. molnari and identifying, for the first time, zoonotic C. ubiquitum in edible marine fishes, pointing out a potential health risk.

Chagas disease (CD) is a parasitic zoonosis endemic in Central and South America affecting nearly 10 million people, with 100 million people at high risk of contracting the disease. Treatment is only effective when received at the early stages of the disease and it involved two drugs (nifurtimox (NFX) and benznidazole (BNZ)). Both treatments require multiple daily administrations of high doses, suffer from variable efficacy and insufficient efficacy in chronic CD, many side effects, and a very long duration of treatment that results in poor compliance, while combined available therapies that lead to reduced duration of treatment are not available and polypharmacy reduces compliance and increases the cost further. Here we present self-nanoemulsified drug delivery systems (SNEDDS) able to produce easily scalable combined formulations of NFX and BNZ that can allow for tailoring of the dose and can be easily converted to oral solid dosage form by impregnation on mesoporous silica particles. SNEDDS demonstrated an enhanced solubilisation capacity for both drugs as demonstrated by flow-through studies and in vitro lipolysis studies. High loading of SNEDDS to Syloid 244 and 3050 silicas (2:1 w/w) allowed clinically translatable amounts of both NFX and BNZ to be loaded. Tablets prepared from NFX-BNZ combined SNEDDS loaded on Syloid 3050 silicas demonstration near complete dissolution in the flow through cell apparatus compared to NFX and BNZ commercial tablets respectively (Lampit® and Rochagan®). NFX-BNZ-SNEDDS demonstrated nanomolar efficacy in epimastigotes and amastigotes of T. cruzi with acceptable selectivity indexes and demonstrated enhanced survival and reduced parasitaemia in acute murine experimental models of CD. Thus, the results presented here illustrate the ability for an easily scalable and personalised combination oral therapy prepared from GRAS excipients, enabling treatment access worldwide for the treatment of CD.

Diarrhea diseases caused by the intestinal protozoan parasite Giardia intestinalis are a major global health burden. Moreover, there is an ongoing need for novel anti-Giardia drugs due to drawbacks with currently available treatments. This paper reports on the isolation and structural elucidation of six new flavonoids (1–6), along with twenty-three known ones (7–29) from the Piper species. Their structures were established by spectroscopic and spectrometric techniques. Flavonoids were tested for in vitro antiprotozoal activity against Giardia intestinalis trophozoites. In addition, structure-activity relationship (SAR) and in silico ADME studies were performed to understand the pharmacophore and pharmacokinetic properties of these natural compounds. Eight flavonoids from this series exhibited remarkable activity in the micromolar range. Moreover, compound 4 was identified as having a 40-fold greater antiparasitic effect (IC50 61.0 nM) than the clinical reference drug, metronidazole (IC50 2.5 M). This antiprotozoal potency was coupled with an excellent selectivity index (SI 233) on murine macrophages and in silico drug-likeness. SAR studies revealed that the substitution patterns, type of functional group, and flavonoid skeleton played an essential role in the activity. These findings highlight flavonoid 4 as a promising candidate to develop new drugs for the treatment of Giardia infections.

Giardiasis is a parasitism produced by the protozoa Giardia intestinalis that lives as trophozoite in the small intestine (mainly in the duodenum) attached to the intestinal villus by means of billed discs. The first line treatment is metronidazole, a drug with high bioavailability, which is why to obtain therapeutic concentrations in duodenum, it is necessary to administer high doses of drug to patients with the consequent occurrence of side effects. It is necessary to developed new therapeutical approaches to achieve a local delivery of the drug. In this sense, we have developed gated mesoporous silica microparticles loaded with metronidazole and with a molecular gate pH dependent. In vitro assays demonstrated that the metronidazole release is practically insignificant at acidic pHs, but in duodenum conditions, the metronidazole delivery from the microparticles is effective enough to produce an important parasite destruction. In vivo assays indicate that this microparticulate system allows to increase the concentration of the drug in duodenum and reduce the concentration in plasma avoiding systemic effects. This system could be useful for other intestinal local treatments in order to reduce doses and increase drug availability in target tissues.

The development of new molecules for the treatment of leishmaniasis is, a neglected parasitic disease, is urgent as current anti-leishmanial therapeutics are hampered by drug toxicity and resistance. The pyrrolo[ 1,2-b]isoquinoline core was selected as starting point, and palladium-catalyzed Heck-initiated cascade reactions were developed for the synthesis of a series of C-10 substituted derivatives. Their in vitro leishmanicidal activity against visceral (L. donovani) and cutaneous (L. amazonensis) leishmaniasis was evaluated. The best activity was found, in general, for the 10-arylmethyl substituted pyrroloisoquinolines. In particular, 2ad (IC50 ¼ 3.30 mM, SI > 77.01) and 2bb (IC50 ¼ 3.93 mM, SI > 58.77) were approximately 10-fold more potent and selective than the drug of reference (miltefosine), against L. amazonensis on in vitro promastigote assays, while 2ae was the more active compound in the in vitro amastigote assays (IC50 ¼ 33.59 mM, SI > 8.93). Notably, almost all compounds showed low cytotoxicity, CC50 > 100 mg/mL in J774 cells, highest tested dose. In addition, we have developed the first Perturbation Theory Machine Learning (PTML) algorithm able to predict simultaneously multiple biological activity parameters (IC50, Ki, etc.) vs. any Leishmania species and target protein, with high values of specificity (>98%) and sensitivity (>90%) in both training and validation series. Therefore, this model may be useful to reduce time and assay costs (material and human resources) in the drug discovery process.

The abietane-type diterpenoids are significant bioactive compounds exhibiting a varied range of pharmacological properties. In this study, the first synthesis and biological investigation of the new abietane-diterpenoid (+)-4-epi-liquiditerpenoid acid (8a) together with several of its analogs are reported. The compounds were generated from the readily available methyl callitrisate (7), which was obtained from callitrisic acid present in Moroccan Sandarac resin. A biological evaluation was conducted to determine the effects of the different functional groups present in these molecules, providing basic structure–activity relationship (SAR) elements. In particular, the ferruginol and sugiol analogs compounds 10–16 were characterized by the presence of a phenol moiety, higher oxidization states at C-7 (ketone), and the hydroxyl, methyl ester or free carboxylic acid at C19. The biological profiling of these compounds was investigated against a panel of six human solid tumor cell lines (HBL-100, A549, HeLa, T-47D, SW1573 and WiDr), four parasitic Leishmania species (L. donovani, L. infantum, L. guyanensis and L. amazonensis) and two malaria strains (3D7 and K1). Furthermore, the capacity of the compounds to modulate gamma-aminobutyric acid type A (GABAA) receptors ( 1 2 2s) is also described. A comparison of the biological results with those previously reported of the corresponding C18-functionalized analogs was conducted.