Prophylaxis and Treatment of Inflammation with Pentacyclic Chemotype of Uncaria tomentosa.

Context: Anti-inflammatory drugs and biologics can effectively treat inflammatory conditions and diseases but can also cause burdensome side effects. Many patients prefer to use time-tested, traditional medicinal products and dietary supplements, but such products seldom receive the rigorous testing required for regulatory approvals of drugs and biologics. Thus, it is advantageous to demonstrate by experimentation the pharmacological and toxicological properties of unapproved natural products and compared to benchmark approved drugs or biologics. Objective: The studies intended to evaluate Samento®, a commercial hydro-alcoholic extract of the pentacyclic chemotype of Uncaria Tomentosa (Willd.) DC, for the prophylactic and treatment effects of irritant-induced inflammation and antigen-induced arthritic inflammation in two rat models. The studies were also intended to create a clinical bridge rationale for the use of Samento® in the treatment of inflammation in humans, with a suggested allometrically scaled starting dose. Design: The research team performed two in vivo animal model studies of induced inflammation in rats. Setting: The studies took place at the Universidad Peruana Cayetano Heredia in Lima, Peru. Prophylaxis Model of Irritant-Induced Inflammation: Holtzman rats in five groups (five each per group) were administered 14 daily doses of Samento® at 250, 500, and 1000 mg/kg of wet weight, or 40 mg/kg of naproxen sodium as a positive control, or nothing as a negative control. At 14 days the right legs of the rats were challenged by injection into the connective tissue by the chemical irritant carrageenan. Two hours thereafter the weight of the irritant-injected right leg was compared to the non-injected left leg of each rat, to determine the level of irritant-induced inflammation. Treatment Model of Antigen-Induced Arthritic Inflammation: Arthritic inflammation was induced in five groups of Lewis rats (five each per group) by intradermal injection of the tail with non-allogeneic, bovine type II collagen in incomplete Freund's adjuvant. A sixth group was not injected with collagen antigen as a non-induced control. Ten days after the induction of arthritic inflammation in the five injected groups, individual groups were treated with Samento® at 250, 500, and 1000 mg/kg of wet weight daily for 21 days, or 0.2 mg/kg of methotrexate twice per week as a positive control, or nothing as a negative control. At 21 days the animals were assessed for antigen-induced arthritic inflammation and assigned an analog score ranging from 0 to 4. Results: The research team confirmed the presence of pentacyclic oxindoles and the absence of tetracyclic oxindoles within Samento®. In the prophylactic model, 14 days of pretreatment with oral Samento® produced dose-dependent, statistically significant reductions in inflammation in the rats' leg weights between the carrageenan induction and postintervention, for the 250, 500, and 1000 mg/kg Samento® groups (P < .05 for all groups). The 1000 mg/kg group had a 74% anti-inflammatory effect versus 97% with the 40 mg/kg of naproxen sodium in the positive control group. In the treatment model, 21 days of oral administration of Samento® produced dose-dependent, statistically significant reductions in arthritic inflammation postintervention, for the 250, 500, and 1000 mg/kg Samento® groups (P < .05 for all groups). Treatment with the highest tested dose of the extract (1000 mg/kg) yielded an 85% anti-inflammatory effect versus 90% with 0.2 mg/kg of methotrexate in the positive control group. Conclusions: The two rat models revealed that the Uncaria phytotherapy Samento® was an effective prophylactic as well as a treatment for induced inflammation in rats. The highest dose of the pentacyclic chemotype of Uncaria approached the anti-inflammatory activities of the established benchmark pharmaceutical positive controls. The results in the two rat models suggest an allometrically scaled starting dose of Samento® of 4 g daily in humans.

GlucoMedix®, an extract of Stevia rebaudiana and Uncaria tomentosa, reduces hyperglycemia, hyperlipidemia, and hypertension in rat models without toxicity: a treatment for metabolic syndrome.

BACKGROUND: The objective of this in vivo study is to evaluate in five rat models the pharmacologic effects and toxicity of a commercial hydro-alcoholic extract, GlucoMedix®, derived from Stevia rebaudiana and the pentacyclic chemotype of Uncaria Tomentosa (Willd.) DC, for use as a treatment for metabolic syndrome. The extract contains phytochemicals of Stevia (e.g., steviol glycosides) and Uncaria (e.g., pentacyclic oxindole alkaloids, but lacks tetracyclic oxindole alkaloids). METHODS: The pharmacologic assessments in three rat models include reductions in chemically induced hyperglycemia, hyperlipidemia (cholesterol and triglycerides), and hypertension, all of which are comorbidities of metabolic syndrome. Acute toxicity and 28-day subacute toxicity were assessed in rat models at doses higher than those used in the efficacy models. RESULTS: The acute oral toxicity was evaluated in Holtzman rats and the extract did not produce acute toxic effects or lethality, with the LD50 > 5000 mg/kg (extract wet weight). Furthermore, subacute oral toxicity was evaluated in rats for 28 days at daily doses as high as 2000 mg/kg without toxicity or abnormal clinical chemistry or hematological effects. Daily oral doses of 250 - 1000 mg/kg were used to evaluate the treatment effects in hyperglycemic (alloxan-induced and glibenclamide-controlled), hyperlipidemic (cholesterol-induced and atorvastatin-controlled), and hypertensive (L-NAME-induced and enalapril-controlled) rat models. Alloxan-induced hyperglycemia was reduced in a dose-dependent manner within 28 days or less. Cholesterol-induced hyperlipidemic rats exhibited dose-dependent reductions in cholesterol and triglycerides at 21 days. Furthermore, GlucoMedix® produced a dose-dependent decrease in systolic and diastolic arterial blood pressure in L-NAME-induced hypertensive rats at 28 days. CONCLUSIONS: The five in vivo rat models revealed that the all-natural phytotherapy GlucoMedix® is a safe and effective treatment for hyperglycemia, hyperlipidemia, and hypertension. This extract is expected to affect multiple comorbidities of metabolic syndrome, without any acute or subacute oral toxicity in humans. Although multiple prescription drugs are well known for the treatment of individual comorbidities of metabolic syndrome, no drug monotherapy concurrently treats all three comorbidities.

Dual-histamine receptor blockade with cetirizine - famotidine reduces pulmonary symptoms in COVID-19 patients.

BACKGROUND: The COVID-19 pandemic due to SARS-CoV-2 infection can produce Acute Respiratory Distress Syndrome as a result of a pulmonary cytokine storm. Antihistamines are safe and effective treatments for reducing inflammation and cytokine release. Combinations of Histamine-1 and Histamine-2 receptor antagonists have been effective in urticaria, and might reduce the histamine-mediated pulmonary cytokine storm in COVID-19. Can a combination of Histamine-1 and Histamine-2 receptor blockers improve COVID-19 inpatient outcomes? METHODS: A physician-sponsored cohort study of cetirizine and famotidine was performed in hospitalized patients with severe to critical pulmonary symptoms. Pulmonologists led the inpatient care in a single medical center of 110 high-acuity patients that were treated with cetirizine 10 mg b.i.d. and famotidine 20 mg b.i.d. plus standard-of-care. RESULTS: Of all patients, including those with Do Not Resuscitate directives, receiving the dual-histamine receptor blockade for at least 48 h, the combination drug treatment resulted in a 16.4% rate of intubation, a 7.3% rate of intubation after a minimum of 48 h of treatment, a 15.5% rate of inpatient mortality, and 11.0 days duration of hospitalization. The drug combination exhibited beneficial reductions in inpatient mortality and symptom progression when compared to published reports of COVID-19 inpatients. Concomitant medications were assessed and hydroxychloroquine was correlated with worse outcomes. CONCLUSIONS: This physician-sponsored cohort study of cetirizine and famotidine provides proof-of-concept of a safe and effective method to reduce the progression in symptom severity, presumably by minimizing the histamine-mediated cytokine storm. Further clinical studies in COVID-19 are warranted of the repurposed off-label combination of two historically-safe histamine receptor blockers.

Dense chitosan surgical membranes produced by a coincident compression-dehydration process.

High density chitosan membranes were produced via a novel manufacturing process and used as implantable resorbable surgical membranes. The innovative method utilizes the following three sequential steps: (1) casting an acidic chitosan solution within a silicon mold, followed by freezing; (2) neutralizing the frozen acidic chitosan solution in alkaline solution to facilitate polymerization; and (3) applying coincident compression-dehydration under a vacuum. Resulting membranes of 0.2-0.5 mm thickness have densities as high as 1.6 g/cm(3). Inclusion of glycerol prior to the compression-dehydration step provides additional physical and clinical handling benefits. The biomaterials exhibit tensile strength with a maximum load as high as 10.9 N at ~2.5 mm width and clinically relevant resistance to suture pull-out with a maximum load as high as 2.2 N. These physical properties were superior to those of a commercial reconstituted collagen membrane. The dense chitosan membranes have excellent clinical handling characteristics, such as pliability and 'memory' when wet. They are semipermeable to small molecules, biodegradable in vitro in lysozyme solution, and the rates of degradation are inversely correlated to the degree of deacetylation. Furthermore, the dense chitosan membranes are biocompatible and resorbable in vivo as demonstrated in a rat oral wound healing model. The unique combination of physical, in vitro, in vivo, and clinical handling properties demonstrate the high utility of dense chitosan membranes produced by this new method. The materials may be useful as surgical barrier membranes, scaffolds for tissue engineering, wound dressings, and as delivery devices for active ingredients.

Methods for gene expression profiling in dermatology research using DermArray nylon filter DNA microarrays.

Here we present methods of gene expression profiling using nylon filter deoxyribonucleic acid (DNA) microarrays and radiolabeled and nonradiolabeled hybridization probes. DermArray(R) nylon filter DNA microarrays were designed specifically for use in dermatology research. A patent-pending method was used to select approx 4400 highly informative, sequence-verified human cDNA clones for this DNA micro array. Using DermArray(R) filters, biomarkers have been discovered for normal and pathologic cells from skin, and for responses to dermatologic drugs. As an example, gene expression profiling was performed with hydroquinone-treated SKMel-28 cells, a melanoma cell line. Also included are the methods for bioinformatic analysis using Pathwaystrade mark software.

Regulation of gene expression in inflammatory bowel disease and correlation with IBD drugs: screening by DNA microarrays.

Potential biomarkers for Crohn's disease (CD) and ulcerative colitis (UC) were identified from two sets of full thickness pathologic samples utilizing DermArray and PharmArray DNA microarrays relative to uninvolved (Un) colon or normal colon. Seven of the over-expressed genes were verified using quantitative RT-PCR (i.e., TMPT, FABP1, IFI27, LCN2, COL11A2, HXB, and metallothionein). By correlating gene expression profiles between inflammatory bowel disease (IBD) tissue samples and IBD drug-treated cell cultures it might be possible to identify new candidate molecular target genes for IBD therapy and drug discovery. Potential biomarkers for CaCo2 cell cultures, which are routinely used as a GI tract surrogate model for in vitro pharmacokinetic studies, treated with azathioprine, 5-aminosalicylic acid, metronidazole, and prednisone were also identified from another experiment. Metallothionein mRNA expression was found to be down-regulated in azathioprine-treated CaCo2 cells, and was coincidentally up-regulated in the CD sample, thus resulting in an anti-correlation. These results suggest that this new screening methodology is feasible, that metallothioneins might be biomarkers for azathioprine therapy in vivo in CD, and that azathioprine might mechanistically down-regulate metallothionein gene expression. Correlations were also observed between IBD samples and either metronidazole- or 5-aminosalicylic acid-treated CaCo2 cells. Similar comparisons of disease tissue samples in vivo vs drug-treated cell cultures in vitro might reveal new mechanistic insights concerning established or experimental drug therapies. This affordable in vitro methodology is promising for expanded studies of IBD and other diseases.

Cross-species application of cDNA microarrays to profile gene expression using UV-induced melanoma in Monodelphis domestica as the model system.

This study established the utility of cross-species application of the cDNA microarray technique for investigating differential gene expression. Using both total RNA and mRNA samples recovered from two opossum cell lines derived from UVB-induced melanoma, we analyzed expression of ca. 4400 genes on the human DermArray DNA microarrays. The signals generated on the DermArrays were clear, strong, and reproducible. A cDNA dot blot consisting of differentially expressed genes representative of different functional clusters was used to validate the DermArray results. We also cloned a Monodelphis gene, keratin 18 (KRT18), and characterized its expression patterns in tumor samples of different progression stages. Up-regulated expression was observed for the KRT18 gene in advanced melanomas, a finding consistent with the DermArray analysis. These results provide evidence that cross-species application of cDNA microarrays is a useful strategy for investigating gene expression patterns in animal models for which species-specific cDNA microarrays are not available.

Biomarkers of human cutaneous squamous cell carcinoma from tissues and cell lines identified by DNA microarrays and qRT-PCR.

Squamous cell carcinoma (SCC) is the second most common form of skin cancer in Caucasians. Here we report on the identification of biomarkers of human cutaneous SCC cell lines in vitro and tissue samples in vivo using DermArray and PharmArray DNA microarrays, consisting of ca. 7400 unique human cDNAs. Differentially expressed genes were identified in two facial skin SCC cell lines (SCC 12 and SCC 13) compared to normal keratinocytes, and three cutaneous SCC tissue samples compared to normal skin. Quantitative validations of up- and down-regulated biomarkers were performed by qRT-PCR on 23 biomarker genes for the cell lines and 20 biomarker genes for the tumor tissues. In addition, three oral SCC cell lines were also included in the qRT-PCR validations for comparison, and the biomarker profiles were highly similar between the cutaneous and the oral SCC cell lines for all 23 biomarkers examined. The expression profiles for a variety of non-cutaneous SCC types, such as head-and-neck, oral, and lung, have been previously published. This report is the first to describe biomarkers for cutaneous SCC in two contexts, in vitro and in vivo. Although there was minimal overlap between the two different contexts using DNA microarrays, five genes were found common to both the cell lines and tissues, namely fibronectin 1, annexin A5, glyceraldehyde 3-phosphate dehydrogenase, zinc-finger protein 254, and huntingtin-associated protein interacting protein. Some of our previously published biomarkers of normal keratinocytes were down-regulated in SCC, suggestive of the dedifferentiated status of the transformed cells. While recent reports have identified some of the same genes as SCC biomarkers, for instance in head-and-neck cancer, thereby validating our approach, we have identified some novel biomarkers for cutaneous disease. These biomarker lists may be useful in molecular diagnostics of non-melanoma skin cancer, and a subset of the biomarkers might serve as suitable targets for drug discovery efforts of therapies for SCC.

DNA microarrays and likelihood ratio bioinformatic methods: discovery of human melanocyte biomarkers.

In this article, some of the advantages and limitations of DNA microarray technologies for gene expression profiling are summarized. As a model experiment, DermArray DNA microarrays were utilized to identify potential biomarkers of cultured normal human melanocytes in two different experimental comparisons. In the first case, melanocyte RNA was compared with vastly dissimilar non-melanocytic RNA samples of normal skin keratinocytes and fibroblasts. In the second case, melanocyte RNA was compared with a primary cutaneous melanoma line (MS7) and a metastatic melanoma cell line (SKMel-28). The alternative approaches provide dramatically different lists of 'normal melanocyte' biomarkers. The most robust biomarkers were identified using principal component analysis bioinformatic methods related to likelihood ratios. Only three of 25 robust biomarkers in the melanocyte-proximal study (i.e. melanocytes vs. melanoma cells) were coincidentally identified in the melanocyte-distal study (i.e. melanocytes vs. non-melanocytic cells). Selected up-regulated biomarkers of melanocytes (i.e. TRP-1, melan-A/MART-1, silver/Pmel17, and nidogen-2) were validated by qRT-PCR. Some of the melanocytic biomarkers identified here may be useful in molecular diagnostics, as potential molecular targets for drug discovery, and for understanding the biochemistry of melanocytic cells.

A method to improve selection of molecular targets by circumventing the ADME pharmacokinetic system utilizing PharmArray DNA microarrays.

DNA microarrays may be used to identify potential molecular targets for drug discovery. Yet, DNA microarray experiments provide massive amounts of data. To limit the choice of potential molecular targets, it may be desirable to eliminate genes coincidentally up-regulated in tissues implicated in absorption, distribution, metabolism, and excretion (ADME) pharmacokinetics. DNA microarray experiments were performed to demonstrate a gene-exclusion approach using as an example RNA samples of neural origin, i.e., a human neuroblastoma cell line (SK-N-SH) and brain tissue, as the intended hypothetical site(s) of drug action. Biomarkers were identified using PharmArray DNA microarrays. The lists of neuroblastoma and neural biomarkers were constrained by limiting selection to the subset of genes that were not highly expressed in three transformed cell lines from liver, colon, and kidney (HepG2, Caco-2, and 786-O, respectively) that are routinely used as representatives of the ADME system during in vitro pharmacology and toxicology experiments. Principal component analysis methods with likelihood ratio-related bioinformatic tools were utilized to identify robust potential biomarker genes for the three ADME-related cell lines, neuroblastoma, and normal brain. Biomarkers of each sample were identified and selected genes were validated by qRT-PCR. Hundreds of biomarkers of the three ADME-related cell types, representing hepatocytes, kidney epithelium, and gastrointestinal tract, may now be used as a valuable database to restrict selection of biomarkers as potential molecular targets from the intended samples (e.g., neuroblastoma in this work). In addition to biomarker discovery per se, this demonstration suggests that our model method may be viable to help restrict gene lists during selection of potential molecular targets for subsequent drug discovery.

Biomarkers of human skin cells identified using DermArray DNA arrays and new bioinformatics methods.

Biomarker genes of human skin-derived cells were identified by new simple bioinformatic methods and DNA microarray analysis utilizing in vitro cultures of normal neonatal human epidermal keratinocytes, melanocytes, and dermal fibroblasts. A survey of 4405 human cDNAs was performed using DermArray DNA microarrays. Biomarkers were rank ordered by "likelihood ratio" algorithms and stringent selection criteria that have general applicability for analyzing a minimum of three RNA samples. Signature biomarker genes (up-regulated in one cell type) and anti-signature biomarker genes (down-regulated in one cell type) were determined for the three major skin cell types. Many of the signature genes are known biomarkers for these cell types. In addition, 17 signature genes were identified as ESTs, and 22 anti-signature biomarkers were discovered. Quantitative RT-PCR was used to verify nine signature biomarker genes. A total of 158 biomarkers of normal human skin cells were identified, many of which may be valuable in diagnostic applications and as molecular targets for drug discovery and therapeutic intervention.