A liposome-based formulation containing equol, dihomo-γ-linolenic acid and propionyl-l-carnitine to prevent and treat hair loss: A prospective investigation.

Hair loss is a common aesthetic disorder that can be triggered by genetic, inflammatory, hormonal, and environmental factors acting on hair follicles and their life cycle. There are several types of hair loss that differ in causes, symptoms, and spatial and temporal progression. Androgenic alopecia, a common form of hair loss, is the consequence of a decreased microcirculation of the scalp as well as the toxic action of elevated dihydrotestosterone levels on the hair bulbs. In the present study, the lotions TRINOV Lozione Anticaduta Uomo and TRINOV Lozione Anticaduta Donna, containing dihomo-γ-linolenic acid (DGLA), S-equol, and propionyl-l-carnitine, were tested on 30 men and 30 women (mean age of men was 46.6 ± 6.4 years; mean age of women was 49.5 ± 9.0) with signs of androgenic alopecia, respectively. DGLA is a precursor of the prostaglandin PGE1, which acts by improving microcirculation; S-equol inhibits 5α-reductases, thus preventing the transformation of testosterone into dihydrotestosterone; and propionyl-l-carnitine promotes lipid metabolism, stimulating energy production. These three molecules are loaded into liposomes for their effective transdermal delivery. Daily topical applications of the lotions resulted in a hair count that significantly increased for women and marginally increased for men after 6 months of treatment. Furthermore, significant increase in anagen hair and a significant decrease in telogen hair were observed starting from 3 months in male and 1 month in female patients. Thus, the formulations under investigation were effective in attenuating androgenic alopecia-related hair loss in men and women.

[Hydrating cleansing: efficacy and tolerability of new formulations with hyaluronic acid 0.2% for intimate hygiene in the woman in fertile age and menopause].

Dehydration of genital areas, known as vaginal dryness, frequently affects woman's life, from fertile to postmenopausal period, and must be treated with specific products, including proper intimate cleansing. Based on that, two new cleanser formulations, characterized by Hyaluronic Acid 0.2% as main ingredient, have been developed (Hyalo Gyn® Intimo Active and Hyalo Gyn® Intimo Advance), with the aim to provide an effective hydration of the vaginal, vulvar, perianal and anal areas beyond guaranteeing daily intimate hygiene. The two intimate cleansing mousses have been formulated in mousse and differentiated, in terms of ingredients, accordingly to the different physiological conditions and female needs of fertile age and menopause respectively. Clinical trials conducted on women of childbearing age and menopause showed that daily cleansing with these intimate washes promotes hydration of vaginal tissues thanks to the presence of hyaluronic acid. In particular, the cleansing mousse for fertile women gives also a soothing effect, offers antimicrobial protection and a long lasting feeling of freshness. The cleansing mousse for menopausal women provides a freshness sensation, improves elasticity and tonicity and exerts an anti-smell and anti-oxidative effect.

A site-selective hyaluronan-interferonα2a conjugate for the treatment of ovarian cancer.

While interferon alpha (IFNα) is used in several viral and cancer contexts, its efficacy against ovarian cancer (OC) is far from being incontrovertibly demonstrated and, more importantly, is hindered by heavy systemic side effects. To overcome these issues, here we propose a strategy that allows a targeted delivery of the cytokine, by conjugating IFNα2a with an aldehyde-modified form of hyaluronic acid (HA). The resulting HA-IFNα2a bioconjugate was biochemically and biologically characterized. The conjugation with HA did not substantially modified both the antiviral function and the anti-proliferative activity of the cytokine. Moreover, the induction of STAT1 phosphorylation and of a specific gene expression signature in different targets was retained. In vivo optical imaging biodistribution showed that the i.p.-injected HA-IFNα2a persisted into the peritoneal cavity longer than IFNα2a without being toxic for intraperitoneal organs, thus potentially enhancing the loco-regional therapeutic effect. Indeed, in OC xenograft mouse models bioconjugate significantly improved survival as compared to the free cytokine. Overall, HA-IFNα2a bioconjugate disclosed an improved anticancer efficacy, and can be envisaged as a promising loco-regional treatment for OC.

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.

Hyaluronic Acid as a Protein Polymeric Carrier: An Overview and a Report on Human Growth Hormone.

Hyaluronic acid (HA) is a natural polysaccharide primarily present in the vitreous humor and in cartilages where it plays a key structural role in organizing the cartilage extracellular matrix. HA is used in a wide range of applications including treatment of arthritis (as a viscosupplementation agent for joints) and in a variety of cosmetic injectable products. Its safety profile is thus well established. Thanks to its high biocompatibility and targeting properties, HA has also been investigated for use as a carrier of anticancer drugs and, recently, also of proteins. Its role in the last case is a particularly challenging one as dedicated coupling chemistries are required to preserve the protein's conformation and activity. This study focuses on the state of the art on protein HAylation. New data from our laboratory on the local delivery of specific biologics to joints will also be outlined.

Peritoneal tumor carcinomatosis: pharmacological targeting with hyaluronan-based bioconjugates overcomes therapeutic indications of current drugs.

Peritoneal carcinomatosis still lacks reliable therapeutic options. We aimed at testing a drug delivery strategy allowing a controlled release of cytotoxic molecules and selective targeting of tumor cells. We comparatively assessed the efficacy of a loco-regional intraperitoneal treatment in immunocompromised mice with bioconjugates formed by chemical linking of paclitaxel or SN-38 to hyaluronan, against three models of peritoneal carcinomatosis derived from human colorectal, gastric and esophageal tumor cell xenografts. In vitro, bioconjugates were selectively internalized through mechanisms largely dependent on interaction with the CD44 receptor and caveolin-mediated endocytosis, which led to accumulation of compounds into lysosomes of tumor cells. Moreover, they inhibited tumor growth comparably to free drugs. In vivo, efficacy of bioconjugates or free drugs against luciferase-transduced tumor cells was assessed by bioluminescence optical imaging, and by recording mice survival. The intraperitoneal administration of bioconjugates in tumor-bearing mice exerted overlapping or improved therapeutic efficacy compared with unconjugated drugs. Overall, drug conjugation to hyaluronan significantly improved the profiles of in vivo tolerability and widened the field of application of existing drugs, over their formal approval or current use. Therefore, this approach can be envisaged as a promising therapeutic strategy for loco-regional treatment of peritoneal carcinomatosis.

A hyaluronic acid-salmon calcitonin conjugate for the local treatment of osteoarthritis: chondro-protective effect in a rabbit model of early OA.

Osteoarthritis (OA) is characterized by chronic degeneration of joints, involving mainly the articular cartilage and the underlying bone, and severely impairing the quality of life of the patient. Although with limited efficacy, currently available pharmacological treatments for OA aim to control pain and to retard disease progression. Salmon calcitonin (sCT) is a drug which has been shown to have therapeutic effects in experimental arthritis by inhibiting both bone turnover and cartilage degradation and reducing the activities of matrix metalloproteinases (MMP). High molecular weight hyaluronic acid (HA) is used as a lubricant in OA therapy, and, interestingly, HA polymers may normalize the levels of MMP-1, -3 and -13. We demonstrated that sCT rapidly clears from the knee joint of rat animal model, after intra-articular (i.a.) administration, and it induces systemic effects. Here, sCT was conjugated to HA (200kDa) with the aim of prolonging the residence time of the polypeptide in the joint space by reducing its clearance. An aldehyde derivative of HA was used for N-terminal site-selective coupling of sCT. The activity of sCT was preserved, both in vitro and in vivo, after its conjugation and the i.a. injection of HA-sCT did not trigger any systemic effects in rats. The efficacy of HA-sCT treatment was tested in a rabbit OA model and clear chondro-protective effect was proven by macro- and microscopic assessments and histological findings. Our results indicate that HAylation of sCT increases the size of the polypeptide in a stable covalent manner and delays its passage into the blood stream. We conclude that HA conjugation prolongs the anti-catabolic effects of sCT in joint tissues, including the synovial membrane and cartilage.

The role of the conformational profile of polysaccharides on skin penetration: the case of hyaluronan and its sulfates.

The literature data suggest the capacity of biomacromolecules to permeate the human skin, even though such a transdermal permeation appears to be governed by physicochemical parameters which are significantly different compared to those ruling the skin permeation of small molecules. On these grounds, the present study was undertaken to investigate the in vitro diffusion properties through the human epidermis of hyaluronic acid and their sulfates. Low- and medium-molecular-weight hyaluronic acids and the corresponding derivatives at two degrees of sulfation were then tested. In vitro studies evidenced that the sulfated polymers permeate better than the corresponding hyaluronic acid, despite their vastly greater polarity, while the observed permeation markedly decreases when increasing the polymer's molecular weight regardless of the sulfation degree. Using a fluorescent-labeled polysaccharide, it was also evidenced that hyaluronans have a great affinity for corneocytes and likely cross the stratum corneum mainly through a transcellular route. The molecular-dynamics study revealed how the observed permeations for the investigated polysaccharides can be rationalized by monitoring their conformational profiles, since the permeation was found to be directly related to their capacity to assume extended and flexible conformations.

Conjugation of hyaluronan to proteins.

Polymer conjugation has been widely exploited to prolong half-life and reduce immunogenicity of therapeutic proteins. Here, the potentials of hyaluronic acid (HA) have been investigated by studying the conjugates with two model enzymes, trypsin and RNase A, and with insulin. As the direct coupling of proteins to the HA's carboxylic groups can cause cross-linking problems, a hyaluronan-aldehyde derivative has been synthesized for N-terminal site-selective conjugation. HA conjugation, termed HAylation, preserved the activities of enzymes and their thermal stabilities. Insulin HAylation was studied by preparing two conjugates with different peptide loadings (32% and 17%, w/w). Noticeably, the conjugate with the lower loading showed the greater effect on blood glucose level. The 17% HA-insulin conjugate showed a lowering effect on blood glucose level for up to 6h, while free insulin exhausted its action after 1h. This study highlights the potentials of hyaluronan-aldehyde for protein delivery.

Paclitaxel-hyaluronan hydrosoluble bioconjugate: mechanism of action in human bladder cancer cell lines.

OBJECTIVES: A previously described hydrosoluble paclitaxel-hyaluronan bioconjugate appears particularly well suited for treatment of superficial bladder cancer because of its in vitro cytotoxic profile against urothelial carcinoma (UC) cell lines and in vivo biocompatibility. The aim of this work was to assess the mechanism of action of the bioconjugate in UC cells. MATERIALS AND METHODS: Expression of CD44 and RHAMM hyaluronan-binding receptors in RT-4 and RT-112/84 UC cell lines, interaction of fluorochrome-labeled bioconjugate with tumor cells, CD44 modulation upon incubation with the compound or free hyaluronan, and caspase activation were assessed by flow cytometry. Cytotoxicity was studied by the MTT assay. Analysis of bioconjugate intracellular localization and effects on ß-tubulin organization was carried out by confocal microscopy. RESULTS: The paclitaxel-hyaluronan bioconjugate bound to UC tumor cells entered intracellular compartments through a saturable and energy-dependent mechanism that involved CD44, as assessed by blocking with specific antibody. Upon internalization, the bioconjugate accumulated into lysosomes where the esteric bond between paclitaxel and the hyaluronan moiety was cleaved, leading to cytoplasmic diffusion of the free drug, caspase activation, and disruption of the ß-tubulin microtubular mesh with subsequent cell death. CONCLUSIONS: Conjugation of paclitaxel to hyaluronan results in a new chemical entity, characterized by selective targeting to polymer receptors on plasma membrane and cell entry through receptor-mediated endocytosis, followed by lysosomal accumulation. Ultimately, the active molecule is released, fully preserving the cytotoxic potential and profile of clinically used free paclitaxel.

Pharmacokinetic profile of Oncofid-S after intraperitoneal and intravenous administration in the rat.

OBJECTIVES: Oncofid-S is a bio-conjugate molecule obtained from the binding of campthotecin, SN-38, to hyaluronic acid. In view of a possible clinical development for loco-regional treatment of peritoneal carcinomatosis, this study aimed to establish the pharmacokinetic profile of Oncofid-S after single intraperitoneal or intravenous administration in the rat. METHODS: Single-dose intraperitoneal or intravenous administrations of Oncofid-S were performed. Groups of six rats were sacrificed at various times (up to 24 and 72 h in i.p. and i.v. study, respectively) after drug injection. Trunk blood, livers and spleens were collected for subsequent analysis. Total SN-38 was assayed by HPLC. KEY FINDINGS: We found that Oncofid-S was poorly absorbed after intraperitoneal injection, the estimated AUC0­72 being less than 2%. The drug was distributed in liver,but not spleen, and was eliminated with a terminal half-life of 16 h. After intravenous dosing, Oncofid-S was found in liver as well as in spleen. CONCLUSIONS: Here we have demonstrated that Oncofid-S administered intraperitoneally in the rat was poorly absorbed into the systemic circulation, even after the administration of an extremely high dose. This finding reinforces the rationale for developing Oncofid-S in the loco-regional intraperitoneal treatment of peritoneal carcinomatosis in man.

CD44-targeting for antitumor drug delivery: a new SN-38-hyaluronan bioconjugate for locoregional treatment of peritoneal carcinomatosis.

An innovative approach for cancer therapy implies the use of drugs covalently conjugated to macromolecular carriers that specifically target molecules over-expressed on tumor cells. This drug delivery strategy may allow a controlled release of the drug and a high targeting selectivity on tumor cells, increasing drug cytotoxicity and decreasing its undesirable side effects. We provide in vitro and in vivo preclinical data on the antitumor efficacy of ONCOFID™-S, a new bioconjugate of hyaluronic acid (HA) with SN-38 (the CPT11 active metabolite), that support the validity of the drug delivery strategy implying the use of HA as macromolecular carrier of antineoplastic drugs, an approach based on the over-expression of its target CD44 (the receptor for HA-mediated motility) in a wide variety of cancers. We show that ONCOFID™-S exerts a strong in vitro anti-proliferative activity on CD44 over-expressing rat DHD/K12/trb colon adenocarcinoma cells, as well as on gastric, breast, oesophageal, ovarian and lung human cancer cells, higher than that exerted by unconjugated SN-38. We also demonstrated the in vivo anti-tumor efficacy of locoregional treatment with ONCOFID™-S on two pre-clinical models of colorectal cancer (CRC) in BDIX rats: a) syngeneic model of subcutaneous tumor; b) syngeneic model of metastatic tumor induced by injection of cells into the peritoneal cavity, mimicking the clinical situation of peritoneal carcinomatosis. Specifically, in the latter model ONCOFID™-S is able to dramatically reduce all parameters indicative of a poor prognosis in peritoneal metastatization of CRC without any myelotoxicity or mesothelial inflammation. We propose this CD44-targeted therapeutic strategy for locoregional treatment of peritoneal carcinomatosis from CRC, against which systemic chemotherapy results almost inefficient.

Synthesis, characterization and in vitro cytotoxicity studies of a macromolecular conjugate of paclitaxel bearing oxytocin as targeting moiety.

The present study describes the experimental synthetic procedure and the characterization of a new polyaspartamide macromolecular prodrug of paclitaxel, bearing oxytocin residues as targeting moieties. In vitro stability studies of bioconjugate, performed in media mimicking biological fluids (buffer solutions at pH 7.4 and 5.5) and in human plasma, evidenced the high stability of the targeting portion (oxytocin)-polymer linkage and the ability of this conjugate to release linked paclitaxel in a prolonged way in plasma. Moreover, preliminary in vitro antiproliferative studies, carried out on MCF-7 cells, that are oxytocin receptor positive cells, showed that the polymeric conjugate has the same cell growing inhibition ability of free drug.

Reversibly stable thiopolyplexes for intracellular delivery of genes.

Novel polyaspartamide non-viral carriers for gene therapy were synthesized by introducing, on the same polymer backbone, positively charged groups, for electrostatic interactions with DNA, and thiol groups for the formation of disulfide bridges between polymer chains. The introduction of thiols was aimed to have a vector with low redox potential sensitivity: disulfide crosslinking in fact, being stable in extracellular environment, allowed either to have stable complexes in plasma, that can protect DNA from metabolism, or to be reduced inside the cell, where the excess of glutathion in reduced form maintains a low redox potential. The consequent destabilization of the complex after disulfide cleavage can release DNA selectively inside the cells. Alpha,beta-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) was used as starting polymer being a highly water-soluble synthetic polymer, already proposed with success as therapeutic carrier by our group. In this study, PHEA was firstly functionalised with ethylendiamine, obtaining a well defined copolymer with pendant primary amine groups (PHEA-EDA), to which N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and 3-(carboxypropyl)trimethyl-ammonium chloride (CPTA) were linked in two subsequent steps, allowing the introduction of thiol and cationic groups respectively. Finally DTT treatment lead to the final PHEA-EDA-SH-CPTA thiopolycation, named PESC. The present work describes the synthesis and characterization of the thiopolycation PESC. 1H NMR spectroscopy detected the derivatization molar degrees in SPDP and CPTA; the formation of DNA complexes (thiopolyplexes), their stability in the presence of polyanions and the ability to release DNA under reductive conditions were studied by agarose gel electrophoresis. DNase II degradation study was carried out to detect the ability of thiopolyplex to stabilize DNA towards enzymatic metabolism. Thiopolyplexes were then characterized by Dynamic Light Scattering (DLS) and Zeta Potential analysis. Finally, in vitro toxicity profile (MTT) and gene transfer efficiency (Luciferase assay) were carried out to evaluate thiopolyplex biocompatibility, safety and efficacy to be used as gene delivery system.

Synthesis and characterization of polyaminoacidic polycations for gene delivery.

The properties as non viral gene vector of a protein-like polymer, the alpha,beta-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) were exploited after its derivatization with 3-(carboxypropyl)trimethyl-ammonium chloride (CPTA) as molecule bearing a cationic group, in order to obtain stable polycations able to condense DNA. PHEA was firstly functionalized with aminic pendant groups by reaction with ethylenediamine (EDA) obtaining the alpha,beta-poly(N-2-hydroxyethyl)(2-aminoethylcarbamate)-d,l-aspartamide (PHEA-EDA) copolymer. We demonstrated that polymer functionalization degree is easily modulable by varying reaction conditions, so allowing to produce two PHEA-EDA derivatives at different molar percentage of amine groups. Subsequently, the condensation reaction of PHEA-EDA copolymers with CPTA yielded alpha,beta-poly(N-2-hydroxyethyl)(2-[3-(trimethylammonium chloride)propylamide]-amidoethylcarbamate)-d,l-aspartamide (PHEA-EDA-CPTA) polycation derivatives. In vitro studies were carried out to evaluate polycations ability to complex DNA and to protect it from nuclease degradation. Obtained results demonstrated the good ability of our new PHEA polycationic derivatives, PHEA-EDA-CPTA, to complex and condense genomic material, neutralizing its anionic charge even at very low polycation/DNA weight ratio. Finally, PHEA-EDA-CPTA polycations were characterized by in vitro cytotoxicity studies to evaluate their effects on the viability of HuH-6 human hepatocellular carcinoma cells by MTS assay. No cytotoxicity was evidenced by both polycationic derivatives after 48h of incubation at all tested concentrations.