Effects of a Combination of Polynucleotide and Hyaluronic Acid for Treating Osteoarthritis.

Knee osteoarthritis (OA), an age-related degenerative disease characterized by severe pain and disability, is treated using polynucleotides (PNs) and hyaluronic acid (HA). The intra-articular (IA) injection of HA has been studied extensively in both animal models and in humans; however, the efficacy and mechanisms of action remain unclear. In addition, there has been a paucity of research regarding the use of PN alone or in combination with HA in OA. To investigate the effect of the combined injection of PN and HA in vivo, pathological and behavioral changes were assessed in an OA model. Anterior cruciate ligament transection and medial meniscectomy were performed in Sprague-Dawley rats to create the OA animal model. The locomotor activity improved following PNHA injection, while the OARSI grade improved in the medial tibia and femur. In mild OA, TNFα levels decreased histologically in the PN, HA, and PNHA groups but only the PNHA group showed behavioral improvement in terms of distance. In conclusion, PNHA exhibited anti-inflammatory effects during OA progression and improved locomotor activity regardless of the OARSI grade.

Polynucleotides Suppress Inflammation and Stimulate Matrix Synthesis in an In Vitro Cell-Based Osteoarthritis Model.

Osteoarthritis (OA) is characterized by degeneration of the joint cartilage, inflammation, and a change in the chondrocyte phenotype. Inflammation also promotes cell hypertrophy in human articular chondrocytes (HC-a) by activating the NF-κB pathway. Chondrocyte hypertrophy and inflammation promote extracellular matrix degradation (ECM). Chondrocytes depend on Smad signaling to control and regulate cell hypertrophy as well as to maintain the ECM. The involvement of these two pathways is crucial for preserving the homeostasis of articular cartilage. In recent years, Polynucleotides Highly Purified Technology (PN-HPT) has emerged as a promising area of research for the treatment of OA. PN-HPT involves the use of polynucleotide-based agents with controlled natural origins and high purification levels. In this study, we focused on evaluating the efficacy of a specific polynucleotide sodium agent, known as CONJURAN, which is derived from fish sperm. Polynucleotides (PN), which are physiologically present in the matrix and function as water-soluble nucleic acids with a gel-like property, have been used to treat patients with OA. However, the specific mechanisms underlying the effect remain unclear. Therefore, we investigated the effect of PN in an OA cell model in which HC-a cells were stimulated with interleukin-1ß (IL-1ß) with or without PN treatment. The CCK-8 assay was used to assess the cytotoxic effects of PN. Furthermore, the enzyme-linked immunosorbent assay was utilized to detect MMP13 levels, and the nitric oxide assay was utilized to determine the effect of PN on inflammation. The anti-inflammatory effects of PN and related mechanisms were investigated using quantitative PCR, Western blot analysis, and immunofluorescence to examine and analyze relative markers. PN inhibited IL-1ß induced destruction of genes and proteins by downregulating the expression of MMP3, MMP13, iNOS, and COX-2 while increasing the expression of aggrecan (ACAN) and collagen II (COL2A1). This study demonstrates, for the first time, that PN exerted anti-inflammatory effects by partially inhibiting the NF-κB pathway and increasing the Smad2/3 pathway. Based on our findings, PN can potentially serve as a treatment for OA.

Efficacy of intradermal hyaluronic acid plus polynucleotides in vulvovaginal atrophy: a pilot study.

OBJECTIVE: This study aimed to evaluate the safety and efficacy of vulvovaginal intradermal injections of polynucleotides (PN) combined with hyaluronic acid (HA) in postmenopausal women affected by vulvovaginal atrophy (VVA). MATERIALS AND METHODS: Postmenopausal women affected by VVA were treated with vulvar and vaginal intradermal injections of one prefilled syringe of 2 ml PN/HA every 2 weeks for four sessions. Patients were evaluated at T0 (baseline), T1 (after session 4) and T2 (1 month after session 4). Evaluation of the treatment was assessed by three international validated questionnaires: Vaginal Health Index (VHI), Vulvar Health Index (VuHI) and Female Sexual Function Index (FSFI). The Wilcoxon matched-paired signed-rank test was used to compare the differences in VHI, VuHI, FSFI and FSFI domains within the groups. Statistical significance was set at p < 0.05. RESULTS: Fifty patients were included in the study (mean age 59.9 ± 7.6 years). Overall, the VHI, VuHI and FSFI reported statistically significant differences between baseline and T1 (p < 0.001) and between baseline and T2 (p < 0.001). All FSFI domains registered a statistically significant increase between baseline and T2 (p < 0.001). No complications or side effects were observed. CONCLUSIONS: Vulvovaginal intradermal injection of PN/HA is a safe, effective treatment, is not expensive and is a reproducible procedure in postmenopausal women with VVA.

Efficacy of Intra-Articular Polynucleotides Associated With Hyaluronic Acid Versus Hyaluronic Acid Alone in the Treatment of Knee Osteoarthritis: A Randomized, Double-Blind, Controlled Clinical Trial.

OBJECTIVE: Pain and range of motion loss are the main clinical features of osteoarthritis (OA). Hyaluronic acid (HA) is one of the infiltrative therapies for OA treatment; however, its effectiveness is a matter of an ongoing debate in clinical practice. Polynucleotides (PNs), a DNA-derived macromolecule with natural origin and trophic activity, were found to favor cell growth and collagen production, in preclinical and clinical studies regarding cartilage regeneration. This study aimed at evaluating whether injection of PNs, in combination with HA [PNs associated with HA (PNHA)], can ameliorate pain and function of knees affected by OA, more than HA alone. DESIGN: A randomized, double-blind, controlled clinical trial. PATIENTS: The study enrolled 100 patients, then randomized to receive PNHA or HA alone (3 weekly knee I.A. injections). INTERVENTIONS AND MAIN OUTCOME MEASURES: Pain reduction, decrease of proinflammatory synovial fluid (SF) factors, and improvement in knee function were evaluated by Knee Society Score and WOMAC scores, after 2, 6, and 12 months and by biochemical and immunoenzymatic analyses of SF at the end of the treatment. RESULTS: Knee Society Score total score and pain item significantly ameliorated in both groups, showing better results in PNHA- than in the HA-treated group. A significant reduction in the WOMAC score was observed over time for both groups. No significant adverse events were reported in either group. CONCLUSIONS: These findings suggest that I.A. injection of PNs, in combination with HA, is more effective in improving knee function and pain, in a joint affected by OA, compared with HA alone.

Polydeoxyribonucleotide injection in the treatment of patients with carpal tunnel syndrome: Retrospective preliminary study.

BACKGROUND: Corticosteroid injection is beneficial in treating carpal tunnel syndrome (CTS) due to its anti-inflammatory effects. However, its side effects limit widespread usage. Recently, several studies have found that polydeoxyribonucleotide offers anti-inflammatory capabilities with fewer side effects, making it an ideal alternative. Nevertheless, there has been no study on its effectiveness in patients with CTS. Therefore, we evaluate the effectiveness of polydeoxyribonucleotide in patients with CTS. Based on the criteria, 30 patients with CTS who received two-consecutive polydeoxyribonucleotide injections (with a week interval) were initially included. METHOD: Patients with CTS were investigated retrospectively. To evaluate the effectiveness of polydeoxyribonucleotide in patients with CTS, numeric rating scale (NRS), cross-sectional area (CSA) of the median nerve, and severity and functional status scores of CTS based on the Boston Carpal Tunnel Syndrome Questionnaire (BCTQ) were assessed. RESULTS: There was a significant improvement in the NRS, CSA, and functional and severity scores of BCTQ after two-consecutive polydeoxyribonucleotide injections (P < .05). CONCLUSION: In conclusion, although more research is needed to evaluate the effectiveness of polydeoxyribonucleotide in patients with CTS, the findings here suggest that polydeoxyribonucleotide may be a viable alternative to corticosteroids in patients with CTS.

Anti-inflammatory Effect of DNA Polymeric Molecules in a Cell Model of Osteoarthritis.

The DNA polymeric molecules polydeoxynucleotide (PDRN) and polynucleotide (PN) can be used as new alternative treatment for osteoarthritis (OA); however, the underlying mechanisms are not fully understood. In this study, we investigated the effect of PDRN and PN on gene-expression profiles in a cell model of OA using transcriptome analysis. Under hypoxic conditions, human chondrosarcoma cells were stressed for 24 h in the presence of interleukin (IL)-1ß and subsequently treated with PDRN, PN, or hyaluronic acid (HA) for another 24 h, followed by transcriptome analysis. The results of the transcriptome study comprising differentially expressed genes were analyzed using the Database of Annotation Visualization and Integrated Discovery program, which yielded Kyoto Encyclopedia of Genes and Genomes pathways. Toll-like receptor (TLR)- and nucleotide-binding oligomerization domain-like receptor (NLR)-signaling pathways were related between the IL-1ß group and the group treated with DNA polymeric molecules. The genes involved in the TLR- and NLR-signaling pathways were validated using real-time quantitative polymerase chain reaction and western blot. Among these genes, IL-6, IL-1ß, IL-8, and chemokine (C-C motif) ligand 3 were dramatically upregulated in the IL-1ß group, but significantly downregulated in the group treated with DNA polymeric molecules. Specifically, PN treatment resulted in a greater decrease in the expression of these genes as compared with PDRN treatment. Both PDRN and PN treatments were involved in the anti-inflammatory response associated with OA progression, with PN treatment exhibiting additional anti-inflammatory properties relative to PDRN treatment. These results provide insight into potential therapeutic approaches involving PDRN and PN treatment of OA.

Effect of multiple intra-articular injections of polynucleotides on treatment of intractable knee osteoarthritis: A case report.

RATIONALE: Knee osteoarthritis (KOA) is a chronic joint degenerative disease. Intra-articular injection (IAI) of hyaluronic acid (HA) is widely used to treat KOA. However, some HA injections have no effect at all. Polynucleotides (PN) are recently noted as a valid substitute for HA. PATIENT CONCERNS: A 61-year-old female was admitted to the pain center with symptoms of pain over the knee and warmth feeling with stiffness in the left knee. The patient reported chronic severe pain in the left knee area despite 6 times IAI of HA. She had past medical history of breast cancer and thyroid cancer. DIAGNOSES: She was diagnosed as having KOA. INTERVENTIONS: Ultrasound-guided IAI of PN was carried out 3 times in 3 weeks. OUTCOMES: She was followed-up for more than 5 months with good improvement in intractable knee pain without any adverse event. LESSONS: IAI of PN is an efficient therapeutic option for KOA treatment if HA injection is unsuccessful.

A phase III, randomized, double-blind, matched-pairs, active-controlled clinical trial and preclinical animal study to compare the durability, efficacy and safety between polynucleotide filler and hyaluronic acid filler in the correction of crow's feet: a new concept of regenerative filler.

The Rejuran® is a new filler product made from purified polynucleotides. Here we present data from an animal study and a clinical trial to examine the durability, efficacy and safety of the Rejuran® on crow's feet. For the animal study, 25 mice were divided into three groups: Group 1 received phosphate buffered saline (PBS); Group 2 were treated with Yvoire®; and Group 3 were treated with Rejuran®. The durability and efficacy of each treatment were assessed by microscopy and staining. In the clinical trial, 72 patients were randomized to receive Rejuran® treatment for crow's feet on one side and Yvoire-Hydro® on the contralateral side, at a ratio of 1:1. Repeated treatments were performed every two weeks for a total of three times, over a total of 12 weeks' observation. All injections and observations of efficacy and safety were performed by the same two investigators. In the animal study, the Rejuran® group showed similar durability and inflammatory response to the Yvoire® group. Upon efficacy assessment, the Rejuran® group showed the greatest elasticity and collagen composition, and a significant difference in skin surface roughness and wrinkle depth. In the clinical trial, the primary and secondary objective efficacy outcome measure showed no statistical significance between the two groups, and in safety outcomes there were no unexpected adverse effects. Our data suggest that the Rejuran®, as a new regenerative filler, can be useful to reduce wrinkles, by showing evidence for its efficacy and safety.

A randomized double-blind clinical trial on the treatment of knee osteoarthritis: the efficacy of polynucleotides compared to standard hyaluronian viscosupplementation.

BACKGROUND: This randomized, double-blind, parallel-group clinical trial aims to assess the equivalence of intra-articular polynucleotides compared to standard hyaluronic acid (HA) viscosupplementation in the treatment of knee osteoarthritis (OA). METHODS: 75 patients affected by knee OA were assessed for eligibility and 72 were enrolled and randomized to receive either intra-articular polynucleotides (Condrotide-36 patients) or hyaluronic acid (Hyalubrix-36 patients) at the Orthopedic Institute "Gaetano Pini" (Milan). All patients underwent three intra-articular injections of Condrotide or Hyalubrix with an interval of 1week. Participants, care givers, and investigators responsible for outcome assessment were all blinded to group assignment. Primary outcome measurements (KOOS and pain level (1) at rest, (2) at weight-bearing and (3) during physical activity) were evaluated at baseline (T0) and after one (T1), two (T2), six (T6), ten (T10), and 26 (T26)weeks. Secondary measurements included the determination of COMP serum levels at T0, T6 and T26. RESULTS: The reduction of pain and the increase of KOOS values from baseline were statistically significant for both treatments; nevertheless, for parameter KOOS "symptoms" the treatment with Condrotide showed significant results already after twoweeks (at T2 p=0.003) while the results obtained with Hyalubrix became significant only after 18 weeks (at T18 p=0.01). No significant adverse events were reported. CONCLUSIONS: Condrotide is as effective as Hyalubrix in reducing knee OA symptoms but showed an earlier response on pain reduction and can therefore be considered a valid alternative to the use of HA in the treatment of OA, avoiding the adverse events of NSAIDs and of intra-articular corticosteroids.

Carbon nanotubes enhance CpG uptake and potentiate antiglioma immunity.

PURPOSE: Stimulation of toll-like receptor-9 (TLR9) by CpG oligodeoxynucleotides (CpG) has been shown to counteract the immunosuppressive microenvironment and to inhibit tumor growth in glioma models. Because TLR9 is located intracellularly, we hypothesized that methods that enhance its internalization may also potentiate its immunostimulatory response. The goal of this study was to evaluate carbon nanotubes (CNT) as a CpG delivery vehicle in brain tumor models. EXPERIMENTAL DESIGN: Functionalized single-walled CNTs were conjugated with CpG (CNT-CpG) and evaluated in vitro and in mice bearing intracranial GL261 gliomas. Flow cytometry was used to assess CNT-CpG uptake and antiglioma immune response. Tumor growth was measured by bioluminescent imaging, histology, and animal survival. RESULTS: CNT-CpG was nontoxic and enhanced CpG uptake both in vitro and intracranial gliomas. CNT-mediated CpG delivery also potentiated proinflammatory cytokine production by primary monocytes. Interestingly, a single intracranial injection of low-dose CNT-CpG (but not free CpG or blank CNT) eradicated intracranial GL261 gliomas in half of tumor-bearing mice. Moreover, surviving animals exhibited durable tumor-free remission (>3 months), and were protected from intracranial tumor rechallenge, demonstrating induction of long-term antitumor immunity. CONCLUSIONS: These findings suggest that CNTs can potentiate CpG immunopotency by enhancing its delivery into tumor-associated inflammatory cells.

Amphipathic DNA polymers exhibit antiviral activity against systemic murine Cytomegalovirus infection.

BACKGROUND: Phosphorothioated oligonucleotides (PS-ONs) have a sequence-independent, broad spectrum antiviral activity as amphipathic polymers (APs) and exhibit potent in vitro antiviral activity against a broad spectrum of herpesviruses: HSV-1, HSV-2, HCMV, VZV, EBV, and HHV-6A/B, and in vivo activity in a murine microbiocide model of genital HSV-2 infection. The activity of these agents against animal cytomegalovirus (CMV) infections in vitro and in vivo was therefore investigated. RESULTS: In vitro, a 40 mer degenerate AP (REP 9) inhibited both murine CMV (MCMV) and guinea pig CMV (GPCMV) with an IC50 of 0.045 microM and 0.16 microM, respectively, and a 40 mer poly C AP (REP 9C) inhibited MCMV with an IC50 of 0.05 microM. Addition of REP 9 to plaque assays during the first two hours of infection inhibited 78% of plaque formation whereas addition of REP 9 after 10 hours of infection did not significantly reduce the number of plaques, indicating that REP 9 antiviral activity against MCMV occurs at early times after infection. In a murine model of CMV infection, systemic treatment for 5 days significantly reduced virus replication in the spleens and livers of infected mice compared to saline-treated control mice. REP 9 and REP 9C were administered intraperitoneally for 5 consecutive days at 10 mg/kg, starting 2 days prior to MCMV infection. Splenomegaly was observed in infected mice treated with REP 9 but not in control mice or in REP 9 treated, uninfected mice, consistent with mild CpG-like activity. When REP 9C (which lacks CpG motifs) was compared to REP 9, it exhibited comparable antiviral activity as REP 9 but was not associated with splenomegaly. This suggests that the direct antiviral activity of APs is the predominant therapeutic mechanism in vivo. Moreover, REP 9C, which is acid stable, was effective when administered orally in combination with known permeation enhancers. CONCLUSION: These studies indicate that APs exhibit potent, well tolerated antiviral activity against CMV infection in vivo and represent a new class of broad spectrum anti-herpetic agents.

Immunotherapy for colorectal cancer.

There have been significant improvements in the diagnosis and treatment of colorectal cancer over the past 15 years. However, some 30% of patients with colorectal cancer have disseminated disease at presentation, and furthermore, 50% of patients initially believed to be cured by surgery subsequently relapse and die of the disease. Novel treatment concepts based on understanding the molecular signatures that separate tumor from normal epithelium, such as immunotherapy, are aimed at abolishing microscopic residual disease post standard treatment. The authors provide an overview of progress in the development of specific and nonspecific immunotherapies and explain why definition of end-points and early translation of immunotherapy into the adjuvant field are key to effective use of such agents in the clinical setting.

Comparative pharmacology of site directed antithrombin agents. Implication in drug development.

Beside the direct inhibition of thrombin and its regulatory functions, many of the newer antithrombin agents produce several additional effects, unrelated to their anticoagulant actions. Synthetic peptide inhibitors are capable of producing fibrinolytic compromise by virtue of their actions on fibrinolytic enzymes such as t-PA, plasmin, urokinase and protein Ca. In addition, the low molecular weight arginine-containing peptides are also known to produce hemodynamic and hemostatic deficits. The designs of the ongoing clinical trials are largely empirical because of the non-availability of valid pharmacologic and toxicologic data on thrombin inhibitors. In contrast to heparin, none of the thrombin inhibitors produce endogenous release of tissue factor pathway inhibitor (TFPI) in the experimental and clinical settings. These observations suggest that beside the direct inhibition of thrombin, these agents also produce multiple additional effects that can significantly contribute to their pharmacologic and toxicologic profile.

[The prevention and treatment of experimental radiation injuries to the skin by using the Nucliderm-gel preparation]. / Profilaktika i lehcenie éksperimental;'nykh luchevykh povrezhdenii kozhi s pomoshch'iu preparata nukliderm-gel'.

Radiopreventive and radiotherapeutic efficacy of nuclyderm-gel was studied on tails of mice exposed to fractionated gamma-radiation of 90 Gy (15 + 30 + 45 Gy) and 75 Gy (45 + 30 Gy). Daily seven-day treatment with nuclyderm-gel prior to irradiation with increasing doses (15 + 30 + 45 Gy) and the subsequent continuous therapy were shown to delay and reduce skin reactions. Nuclyderm-gel was particularly effective when used therapeutically. The treatment of exposed areas, with the three-fold dose fractionation, was the optimum therapy scheme.