Effectiveness and safety of REBACIN as a non-invasive intervention for persistent high-risk human papillomavirus infection: A real-world prospective multicenter cohort study.

BACKGROUND: We previously reported that REBACIN effectively eliminates persistent high-risk human papillomavirus (hrHPV) infection. Here, we conducted a prospective multicenter cohort study to evaluate the safety and effectiveness of REBACIN, taking into account factors such as specific hrHPV subtype and patient's age. METHODS: According to inclusion/exclusion criteria and participant willingness, 3252 patients were divided into REBACIN group while 249 patients into control group. Patients in REBACIN group received one course treatment of intravaginal administration of REBACIN while no treatment in control group. After drug withdrawal, participants in both groups were followed up. RESULTS: The clearance rate of persistent hrHPV infection in REBACIN group was 60.64%, compared to 20.08% in control group. Specifically, the clearance rates for single-type infection of HPV16 or HPV18 were 70.62% and 69.23%, respectively, which was higher than that of HPV52 (59.04%) or HPV58 (62.64%). In addition, the single, double, and triple/triple+ infections had a clearance rate of 65.70%, 53.31%, and 38.30%, respectively. Moreover, 1635 patients under 40 years old had a clearance rate of 65.14%, while it was 55.08% for 1447 patients over 40 years old. No serious adverse effects were found. CONCLUSION: This study confirmed that REBACIN can effectively and safely eliminate persistent hrHPV infection, which the clearance rate of HPV16/18 is higher than that of HPV52/58, the clearance rate of single-type infection is higher than that of multiple-type infections, and the clearance rate in young patients is higher than that in elder patients, providing a guidance for REBACIN application in clearing hrHPV persistent infection in real-world settings. CLINICAL TRIAL REGISTRATION: Chinese Clinical Trial Registry Registration Number: ChiCTR1800015617 http://www.chictr.org.cn/showproj.aspx?proj=26529 Date of Registration: 2018-04-11.

REBACIN® inhibits E6/E7 oncogenes in clearance of human papillomavirus infection.

Previous studies have demonstrated that REBACIN® intervention eliminates persistent high-risk human papillomavirus (hrHPV) infection. The initial establishment and subsequent progression of cervical cancer mainly depends on two major oncogenes, E6/E7, and previous studies have proposed E6/E7 oncogenes as a target for therapeutic drug development. The aim of this study was to investigate in vitro and in vivo whether REBACIN® inhibits E6/E7 oncogenes for elucidating the mechanism of REBACIN® in the clearance of persistent hrHPV infection. In vitro, after REBACIN® treatment, the growth of both Ca Ski and HeLa cervical cancer cells containing the E6/E7 oncogenes was prevented. In line with this finding is that E6/E7 expression was inhibited, which can be counteracted by the co-application of anti-REBACIN® antibody. These studies demonstrated that REBACIN® can effectively inhibit the growth of cervical cancer cells via targeting HPV E6/E7 expression. To further verify this finding in clinic, 108 volunteer patients with persistent hrHPV infections were randomly divided into REBACIN®, recombinant human interferon alpha-2b (Immunological drug control), or no-treatment blank control groups, received intravaginal administration of REBACIN®, interferon or no-treatment every other day for three months, and then followed up for E6/E7 mRNA assay. In REBACIN® group, 68.57% of patients showed complete clearance of HPV E6/E7 mRNA, which was significantly higher compared to 25.00% in the interferon immunological drug control group and 20.00% in blank control group, confirming that REBACIN® is potently efficacious on clearing persistent hrHPV infections via inhibition of HPV E6/E7 oncogenes. Clinical trial registration: http://www.chictr.org.cn/historyversionpuben.aspx?regno=ChiCTR2100045911, identifier ChiCTR2100045911.

Case Report: Noninvasive Clinical Intervention of REBACIN® on Histologic Regression of High Grade Cervical Intraepithelial Neoplasia.

High-risk human papillomavirus (hrHPV) persistent infection is the major cause of cervical cancer. Clinical intervention of hrHPV-associated high-grade squamous intraepithelial lesion (HSIL) is critical to prevent cervical cancer, and current treatment is surgery (an invasive therapy). However, some patients refuse to do so for an afraid of potential adverse effects on future fertility or other concerns which creates a critical need for development of non-invasive therapeutic strategies. Here, we report for the first time the cases of non-invasive intervention with REBACIN®, a proprietary antiviral biologics, in clinical treatment of HSIL. From 12,958 visiting patients assessed for eligibility, 18 HSIL-patients with cervical intraepithelial neoplasia-grade 2, positive of both diffused overexpression of p16 and high-risk HPV were enrolled in this non-invasive clinical intervention mainly due to concerns of future fertility. REBACIN® was administered intravaginally every other day for 3 months (one-course) except during menstrual period, and were followed up for 6-36 months for the examination of high-risk HPV DNA, cervical cytology, and histopathology. After one to three course treatments, most cases (16/18) displayed both the regression from HSIL (CIN2) to normal cervical cytology and clearance of high-risk HPV infection. Further studies demonstrated REBACIN® significantly suppressed HPV16 E7 oncoprotein expression in a human cervical cancer cell line, which is consistent with previous finding that REBACIN® inhibits the growth of tumors induced by expression of E6/E7 oncogenes of either HPV16 or HPV18. This report indicates REBACIN® as a novel effective non-invasive clinical intervention for HSIL-patients as well for high-risk HPV persistent infection, providing a new clinical option for the non-invasive treatment of hrHPV-associated high-grade squamous intraepithelial lesion, which is worthy of further research on clinical validation and application.

REBACIN® as a noninvasive clinical intervention for high-risk human papillomavirus persistent infection.

The development of highly sensitive HPV-genotyping tests has opened the possibility of treating HPV-infected women before high-grade lesions appear. The lack of efficient intervention for persistent high-risk HPV infection necessitates the need for development of novel therapeutic strategy. Here we demonstrate that REBACIN®, a proprietary antiviral biologics, has shown potent efficacy in the clearance of persistent HPV infections. Two independent parallel clinical studies were investigated, which a total of 199 patients were enrolled and randomly divided into a REBACIN®-test group and a control group without treatment. The viral clearance rates for the REBACIN® groups were 61.5% (24/39) and 62.5% (35/56), respectively, for the two independent parallel studies. In contrast, the nontreatment groups showed self-clearance rates at 20.0% (8/40) and 12.5% (8/64). We further found that REBACIN® was able to significantly repress the expression of HPV E6 and E7 oncogenes in TC-1 and Hela cells. The two viral genes are well known for the development of high-grade premalignancy lesion and cervical cancer. In a mouse model, REBACIN® was indicated to notably suppress E6/E7-induced tumor growth, suggesting E6 and E7 oncogenes as a potential target of REBACIN®. Taken together, our studies shed light into the development of a novel noninvasive therapeutic intervention for clearance of persistent HPV infection with significant efficacy.

Extraction of high-quality RNA from rubber tree leaves.

A specific technique capable of producing high-quality RNA for rapid amplification of cDNA ends (RACE) was established for challenging tissues: leaves of the rubber tree. Total RNA was extracted by cetyltrimethylammonium bromide (CTAB)-LiCl combined with TRIzol reagent. The isolated RNA was highly intact. With RNA as template, full-length cDNA was obtained (NCBI, AY461413) by RACE.

Chromogranin A deficiency in transgenic mice leads to aberrant chromaffin granule biogenesis.

The biogenesis of dense-core secretory granules (DCGs), organelles responsible for the storage and secretion of neurotransmitters and neuropeptides in chromaffin cells, is poorly understood. Chromogranin A (CgA), which binds catecholamines for storage in the lumen of chromaffin granules, has been shown to be involved in DCG biogenesis in neuroendocrine PC12 cells. Here, we report that downregulation of CgA expression in vivo by expressing antisense RNA against CgA in transgenic mice led to a significant reduction in DCG formation in adrenal chromaffin cells. The number of DCGs formed in CgA antisense transgenic mice was directly correlated with the amount of CgA present in adrenal medulla. In addition, DCGs showed an increase in size, with enlargement in the volume around the dense core, a phenomenon that occurs to maintain constant "free" catecholamine concentration in the lumen of these granules. The extent of DCG swelling was inversely correlated with the number of DCGs formed, as well as the amount of CgA present in the adrenal glands of CgA antisense transgenic mice. These data indicate an essential role of CgA in regulating chromaffin DCG biogenesis and catecholamine storage in vivo.

Sorting of carboxypeptidase E to the regulated secretory pathway requires interaction of its transmembrane domain with lipid rafts.

Carboxypeptidase E (CPE) functions as a regulated secretory pathway sorting receptor for several prohormones, including pro-opiomelanocortin (POMC), proenkephalin and proinsulin. The association of CPE with lipid rafts in the trans -Golgi network and secretory granule membranes is necessary for its sorting receptor function. We now provide evidence that a domain within the C-terminal 25 residues of CPE functions as a signal for both raft association and the sorting of CPE to the regulated secretory pathway. A fusion protein containing the extracellular domain of the human interleukin-2 receptor Tac (N-Tac) and the C-terminal 25 amino acids of CPE was transfected into Neuro2A cells. This fusion protein floated in sucrose density gradients, indicating raft association, and co-localized with chromogranin A (CGA), a secretory granule marker. To define further a minimum sequence required for raft association and sorting, deletion mutants of CPE that lacked the C-terminal four or 15 residues (CPE-Delta4 and CPE-Delta15 respectively) were transfected into a clone of CPE-deficient Neuro2A cells. In contrast with full-length CPE, neither CPE-Delta4 nor CPE-Delta15 floated in sucrose density gradients. The sorting of both CPE-Delta4 and CPE-Delta15 to the regulated secretory pathway was impaired, as indicated by significantly increased basal secretion and a lack of response to stimulation. Additionally, there was a significant decrease in the co-localization of mutant CPE immunofluorescence with CGA when compared with full-length CPE. Finally, the sorting of the prohormone POMC to the regulated pathway was impaired in cells transfected with either CPE-Delta4 or CPE-Delta15. We conclude that the sorting of CPE to the regulated secretory pathway in endocrine cells is mediated by lipid rafts, and that the C-terminal four residues of CPE, i.e. Thr(431)-Leu-Asn-Phe(434), are required for raft association and sorting.