Gastroprotective effects of D-002 (beeswax alcohols) and Lyprinol® on experimentally-induced gastric ulceration in rats

Introduction: D-002, a mixture of beeswax alcohols, has been effective in osteoarthritis models and for reducing osteoarthritis symptoms. Unlike the classic anti-inflammatory drugs, D-002 elicits gastroprotective rather than gastrotoxic effects. Lyprinol, used for ameliorating inflammation and arthritic symptoms, improves gastrointestinal dysfunction symptoms in osteoarthritis subjects. Both D-002 and Lyprinol inhibit cyclooxygenase and 5?lipoxygenase activities, and have been similarly effective for reducing inflammation experimentally. Objective: to compare the effects of D-002 and Lyprinol on gastric mucosa of normal and experimentally-induced ulcer rats. Methods: ulcer indexes were measured in normal rats and in rats with ethanol or pylorus ligation-induced ulcers, in which gastric volume and mucus secretion were also measured. Normal rats were randomized into a vehicle control, one acetic salicylic acid (150 mg/kg), three D-002, three Lyprinol groups; rats with ethanol-ulcers into a vehicle control, three D-002 and three Lyprinol-treated groups; and the experiment on pylorus ligation included a negative control and eight pylorus-ligated groups: one vehicle control, three D-002, three Lyprinol, one omeprazole 10 mg/kg. In all cases, D-002 and Lyprinol (50, 200 and 400 mg/kg) were given orally. Results: unlike D-002 and Lyprinol (50-400 mg/kg), acetic salicylic acid increased ulcer indexes and the incidence of ulcers versus the vehicle control. Single oral doses of D-002 (50-400 mg/kg) or Lyprinol (200 and 400 mg/kg) decreased significantly (p<0.01) and in a similar way ulcer indexes versus the ethanol-positive control. D-002 and Lyprinol (50-400 mg/kg) lowered significantly (p<0.01) and comparably ulcer indexes in rats with pylorus ligation versus the positive controls. D-002 (200 and 400 mg/kg) decreased gastric volume and increased gastric mucus secretion versus the positive control whereas only Lyprinol 400 mg/kg increased the gastric mucus secretion but without modifying the gastric volume. Omeprazole significantly reduced ulcer index (p<0.05) and gastric volume (p< 0.01), with no change in mucus secretion. Conclusion: D-002 and Lyprinol did not show gastrotoxic effects and similar efficacy in protecting against ethanol and pylorus ligation-induced gastric ulceration in rats(AU)

Introducción: el D‒002, una mezcla de alcoholes de la cera de abejas, efectivo en modelos de osteoartritis y para reducir los síntomas de la misma. A diferencia de los medicamentos antiinflamatorios clásicos el D‒002 produce efectos gastroprotectores más que efectos gastrotóxicos. El Lyprinol, usado para disminuir la inflamación y los síntomas artríticos, mejora los síntomas de disfunción gastrointestinal en sujetos con dicha enfermedad. D‒002 y Lyprinol inhiben las actividades de cyclooxigenasa y 5‒lipooxigenasa, y son similarmente efectivos para reducir la inflamación en modelos experimentales. Objetivo: comparar los efectos del D‒002 y el Lyprinol sobre la mucosa gástrica de ratas normales y de ratas con úlcera gástrica inducida experimentalmente. Métodos: se determinó el índice de úlcera en ratas normales y en ratas con úlceras gástricas inducidas por etanol e inducidas por ligadura de píloro, en las cuales se midió el volumen gástrico y la secreción de mucus. Las ratas normales se distribuyeron en un grupo control (vehículo), uno con ácido acetil salicílico (150 mg/kg), tres con D‒002 y tres con Lyprinol; las ratas con úlcera inducida por etanol en un grupo control (vehículo), tres con D‒002 y tres con Lyprinol; y el experimento con ligadura de píloro en un grupo control (vehículo), tres D‒002, tres Lyprinol y uno con omeprazol (10 mg/kg). En todos los casos, el D‒002 y el Lyprinol (50, 200 y 400 mg/kg) se administraron por vía oral. Resultados: el ácido acetil salicílico, no el D‒002 ni el Lyprinol (50‒400 mg/kg), incrementó el índice de úlceras y la incidencia de úlceras comparadas con el grupo control. Dosis orales únicas de D‒002 (50‒400 mg/kg) o Lyprinol (200 y 400 mg/kg) redujeron significativa (p<0,01) y similarmente el índice de úlceras comparado con el grupo control positivo con úlceras por etanol. El D‒002 y el Lyprinol (50‒400 mg/kg) redujeron significativamente (p<0,01) y comparablemente el índice de úlceras en ratas con ligadura de píloro comparado con el grupo control positivo. El D‒002 (200 y 400 mg/kg) redujo el volumen gástrico e incrementó la secreción de mucus gástrico respecto al grupo control positivo; mientras solo el Lyprinol 400 mg/kg aumentó la secreción de mucus gástrico pero sin modificar el volumen gástrico. El omeprazol redujo significativamente el índice de úlcera (p<0,05) y el volumen gástrico (p<0,01), sin modificar la secreción de mucus. Conclusiones: el D‒002 y el Lyprinol no presentaron efectos gastrotóxicos, y protegieron con eficacia similar de las úlceras gástricas inducidas por etanol y por ligadura del píloro en ratas(AU)

Effects of octacosanol and triacontanol alcohols on ciclooxygenase and 5-lipoxygenase enzyme activities in vitro / Efecto sobre la actividad in vitro de las enzimas cicloxigenasa y 5-lipoxigenasa de los alcoholes octacosanol y el triacontanol

INTRODUCTION: policosanol, a mixture of eight primary aliphatic alcohols purified from sugar cane wax, contains octacosanol as major component. D-002, a mixture of six primary aliphatic alcohols purified from beeswax, presents triacontanol as the main component. Although both substances are high molecular weight alcohol mixtures, they have different compositions and pharmacological effects such as their distinct effects on arachidonic acid metabolism enzymes; whereas policosanol inhibits cyclooxygenase (COX)-1, D-002 inhibits COX and 5-lipoxygenase (5-LOX) activities. OBJECTIVE: to study the effects of octacosanol and triacontanol, which are main components of policosanol and D-002, respectively on the COX and the 5-LOX enzyme in vitro activities. METHODS: triacontanol and octacosanol were suspended in a Tween-20/H2O (2 %) (0.6-5000 g/mL) vehicle. The effects of adding these alcohols on COX-1, COX-2 and 5-LOX enzymes activities were assessed in rat platelet microsomes, rat seminal vesicle microsomes and rat polymorphonuclear (PMN) preparations, respectively. Indomethacin (0.4µg/mL) was used as reference inhibitor of COX-1 and COX-2, and Lyprinol as 5-LOX inhibitor. RESULTS: octacosanol showed significant, marked (70% with highest concentration) (IC50=143.54 g/mL) and dose-dependent (r=0.991, p <0.001) inhibitory action on COX-1 activity. However, Triacontanol did not affect COX-1, but inhibited significantly, depending on dose (r=0.985, p <0.001) the COX-2 activity to 50 % with 1250 g/mL. In contrast, octacosanol did not change COX-2 activity. Indomethacin inhibited both COX-1 and COX-2 by 83 %. Octacosanol addition was ineffective whereas triacontanol had significant, dose-dependent (r=0.978, p<0.001) and marked effect (79 %) on the 5-LOX activity (IC50=58.74 g/mL). Lyprinol inhibited 5-LOX by 89 %. The inhibitions induced by octacosanol and triacontanol were competitive. CONCLUSIONS: in vitro addition of octacosanol and triacontanol caused differential effects on COX-1, COX-2 and 5-LOX enzyme activities. Whereas octacosanol markedly inhibited COX-1 activity and did not change those of COX-2 and 5-LO, triacontanol markedly inhibited 5-LOX activity, but had moderate effect on COX-2 and did not change COX-1 activity.

INTRODUCCIÓN: el policosanol, mezcla de ocho alcoholes purificados de la cera de la caña de azúcar, contiene octacosanol como componente mayoritario. El D-002, mezcla de seis alcoholes alifáticos primarios purificada de la cera de abejas, presenta triacontanol como el componente mayoritario. Aunque ambas sustancias son mezclas de alcoholes de alto peso molecular, exhiben diferente composición y perfil farmacológico como son sus efectos sobre las enzimas del metabolismo del ácido araquidónico: mientras el policosanol inhibe la actividad de ciclooxigenasa (COX)-1, el D-002 inhibe las actividades de la COX y la 5-lipooxigenasa (5-LOX). OBJETIVO: investigar los efectos del octacosanol y el triacontanol, principales componentes del policosanol y el D-002, respectivamente, sobre las actividades de las enzimas COX y 5-LOX in vitro. MÉTODOS: el policosanol y el triacontanol se suspendieron en vehículo Tween-20/H2O (2 %) (0.6-5000g/mL). Los efectos de la adición de estos alcoholes sobre las actividades de las enzimas COX-1, COX-2 y 5-LOX se evaluaron en microsomas de plaquetas de ratas, microsomas de vesículas seminales de ratas y en preparaciones de polimorfonucleares (PMN) de ratas, respectivamente. Se utilizó indometacina (0.4 µg/mL) como inhibidor de referencia de COX-1 and COX-2 y Lyprinol como inhibidor de 5-LOX. RESULTADOS: la adición de octacosanol inhibió la actividad de COX-1 de modo significativo, marcado (70 % con la concentración mayor) (CI50=143.54 g/mL) y dependiente de la dosis (r=0.991, p <0.001). La adición de triacontanol, sin embargo, no afectó COX-1, pero inhibió de modo significativo y dependiente de la dosis (r=0.985, p <0.001) la actividad de la COX-2 hasta 50 % con 1250 g/mL. En contraste, el octacosanol no modificó la actividad de la COX-2. La indometacina inhibió COX-1 y COX-2 en un 83 %. Mientras la adición del octacosanol no fue efectiva, el triacontanol inhibió de modo significativo, dependiente de la dosis r=0.978, p <0.001) y marcadamente (79 %) la actividad de la 5-LOX (CI50=58.74 g/mL). El Lyprinol inhibió la 5-LOX en un 89 %. Las inhibiciones inducidas por el octacosanol y el triacontanol fueron competitivas. CONCLUSIONES: la adición in vitro de octacosanol y triacontanol produjo efectos diferenciales sobre las actividades enzimáticas de COX-1, COX-2 y 5-LOX. Mientras el octacosanol inhibió marcadamente la actividad de COX-1, sin afectar COX-2 y 5-LOX; el triacontanol inhibió marcadamente 5-LOX, pero moderadamente COX-2, y no cambió la actividad de COX-1.

Evaluation of the effect of D-002, a mixture of beeswax alcohols, on osteoarthritis symptoms

BACKGROUND/AIMS: Nonsteroidal anti-inflammatory drugs relieve osteoarthritis (OA) symptoms but cause adverse effects. D-002, a mixture of beeswax alcohols, is effective against experimental OA. A pilot study found that D-002 (50 mg/day) for 8 weeks improves OA symptoms. The aim of this study was to investigate the effects of D-002 (50 to 100 mg/day) administered for 6 weeks on OA symptoms. METHODS: Patients with OA symptoms were double-blindly randomized to D-002 (50 mg) or placebo for 6 weeks. Symptoms were assessed by the Western Ontario and McMaster Individual Osteoarthritis Index (WOMAC) and the visual analog scale (VAS) scores. Patients without symptom improvement at week 3 were titrated to two daily tablets. The primary outcome was the total WOMAC score. WOMAC pain, joint stiffness and physical function scores, VAS score, and use of rescue medications were secondary outcomes. RESULTS: All randomized patients (n = 60) completed the study, and 23 experienced dose titration (two in the D-002 and 21 in the placebo groups). At study completion, D-002 reduced total WOMAC (65.4%), pain (54.9%), joint stiffness (76.8%), and physical function (66.9%) WOMAC scores, and the VAS score (46.8%) versus placebo. These reductions were significant beginning in the second week, and became enhanced during the trial. The use of rescue medication by the D-002 (6/30) group was lower than that in the placebo (17/30) group. The treatment was well tolerated. Seven patients (two in the D-002 and five in the placebo group) reported adverse events. CONCLUSIONS: These results indicate that D-002 (50 to 100 mg/day) for 6 weeks ameliorated arthritic symptoms and was well tolerated.

Effects of D-002, a mixture of high molecular weight beeswax alcohols, on patients with nonalcoholic fatty liver disease

BACKGROUND/AIMS: Nonalcoholic fatty liver disease (NAFLD) is intimately related to insulin resistance and ranges from a benign course to liver fibrosis and cirrhosis. NAFLD management mainly involves dietary modification and weight loss. Although no fully successful pharmacological intervention is available, alternative therapies to treat NAFLD have shown promising results. Experimental studies have shown that D-002, a mixture of beeswax alcohols with antioxidant effects, is hepatoprotective. The aim of this study was to investigate the efficacy and safety of D-002 in patients with NALFD. METHODS: Fifty patients with NAFLD were randomized to receive a placebo or D-002 (100 mg/day) for 24 weeks. The primary endpoint was a significant ultrasonography-detected reduction of liver fat infiltration versus a placebo. Secondary endpoints were decreases in the homeostatic model assessment (HOMA) index, insulin levels, serum liver enzymes, increases in plasma total antioxidant status (TAS) and improved clinical symptoms versus the placebo recipients. RESULTS: At randomization, all indicators were comparable in both groups. At study completion, seven (28.0%) D-002-patients, but none of the placebo recipients, exhibited a normal liver echo pattern on ultrasonography (p < 0.01). Also, D-002 significantly reduced (p < 0.01 vs. baseline and placebo) the HOMA index and insulin levels and increased the TAS, but did not affect other parameters. The proportion of D-002-patients (12/25, 48.0%) showing symptom improvement was higher (p < 0.001) than that of the placebo group (1/25, 4.0%). The treatment was safe and well tolerated. Three patients in each group withdrew from the study. CONCLUSIONS: D-002 (100 mg/day) improved ultrasonographic findings, indicators of insulin resistance, plasma TAS and clinical evolution on NAFLD patients. Further studies, however, are needed to confirm these results.

Effects of D-002 on aspirin-induced ulcers and neutrophil infiltration on the gastric mucosa / Efecto del D-002 sobre la úlcera inducida por aspirina asociada al infiltrado de neutrófilos en la mucosa gástrica

Introduction: the gastric mucosa is susceptible to the effects of aggressive factors, which are counterbalanced by mucosal defensive factors. Acid peptic diseases result from the imbalance between these aggressive and defensive factors. Aspirin-induced ulcer is a model of NSAIDs-induced damage in which neutrophil infiltration plays a key role. Objective: this paper investigates the protective effect of D-002 against aspirin-induced ulcers and associated neutrophil infiltration in the gastric mucosa. Methods: rats were randomized into six groups of 8 rats each. A negative vehicle control, and five aspirin (300 mg/kg)-treated groups: a positive control, orally treated with the vehicle, three with D-002 (25, 50 and 100 mg/kg, respectively) and other with 10 mg/kg Omeprazole. Five hours after induced damage the rats were sacrificed. The stomachs were removed and opened, and lesions examined macroscopically and microscopically. Ulcer indexes and neutrophil infiltration per ulcer areas were measured. Results: all positive, none negative, controls exhibited aspirin-induced ulcers. Oral treatment with D-002 (25-100 mg/kg) dose-dependently and significantly reduced aspirin-induced gastric lesions (37 to 75 ), the mean number of microscopic ulcers (40 to 72 por ciento) and neutrophil infiltration (41.7 to 83.1 por ciento) in the rat gastric mucosa. Conclusion: Oral treatment with D-002 (25-100 mg/kg) effectively protects against aspirin-induced ulcers and decreases the neutrophil infiltration in the gastric mucosa induced by aspirin ulceration

Introducción: la integridad de la mucosa gástrica depende del balance entre los factores agresivos y defensivos. La úlcera inducida por aspirina es un modelo de daño por antiinflamatorios no esteroidales en el cual el infiltrado de neutrófilos desempeña una función fundamental. Objetivo: evaluar el efecto protector del D-002 sobre la úlcera inducida por aspirina asociada al infiltrado de neutrófilos en la mucosa gástrica. Métodos: las ratas fueron aleatorizadas en seis grupos de ocho animales cada uno. Un control negativo con vehículo y cinco grupos tratados con aspirina (300 mg/kg): un control positivo, tratado por vía oral con vehículo, tres grupos con D-002 (25, 50 and 100 mg/kg respectivamente) y otro con omeprazol 10 mg/kg. Cinco horas después de inducido el daño las ratas fueron sacrificadas y se extrajeron sus estómagos para su análisis morfológico. Se determinó el índice de úlcera, el número de úlceras microscópicas y el número de neutrófilos por área ulcerada. Resultados: todos los controles positivos y ninguno negativo mostraron lesiones en la mucosa. El tratamiento por vía oral con D-002 (25-100 mg/kg) redujo de modo significativo y dependiente de la dosis el índice de úlceras gástricas (37-75 percent), el promedio de úlceras microscópicas (40- 72 percent) y la infiltración de neutrófilos (41,7-83,1 percent) en la mucosa de las ratas. Conclusiones: el tratamiento por vía oral con D-002 (25-100 mg/kg) protege la mucosa gástrica de las ratas del daño inducido por aspirina, lo que disminuye el índice de úlcera y el infiltrado de neutrófilos

Efecto secuestrador del D-002 sobre radicales hidroxilo en mucosa gástrica / Scavenger effect of D-002 on hydroxyl radicals in the gastric mucosa

Introducción: el agente causal de la ulceración gástrica está asociado al desequilibrio entre factores agresivos y defensivos que actúan sobre la mucosa gástrica. El D-002, mezcla de seis alcoholes alifáticos primarios superiores purificada de la cera de abejas, produce efectos gastroprotectores mediados por múltiples mecanismos y reducción de la peroxidación lipídica en la mucosa gástrica. Objetivo: determinar si el D-002 es capaz de capturar el radical hidroxilo añadido in vitro o generado in vivo en ratas con úlcera gástrica inducida por indometacina. Métodos: En la experiencia in vitro el D-002 se añadió a concentraciones entre 0,9 y 1 000 mg/mL. En la experiencia in vivo las ratas se distribuyeron en seis grupos: un control negativo y cinco que recibieron indometacina: un control positivo tratado con el vehículo, tres con D-002 (5, 25, y 100 mg/kg, respectivamente, p.o.) y otro con omeprazol (20 mg/kg i.p.). Los tratamientos se administraron una hora (vehículo y D-002) o 30 min (omeprazol), respectivamente, antes de inducir las úlceras. En ambas experiencias se tomaron alícuotas de mucosa gástrica, y se determinó el daño a la 2-desoxirribosa por el radical hidroxilo. Resultados: la administración oral del D-002, no in vitro, protegió a la 2-desoxirribosa del daño oxidativo de modo marcado, significativo y dependiente de la dosis con respecto al control positivo. Conclusiones: los resultados indican que la capacidad del D-002 (25 y 100 mg/kg) administrado por vía oral para secuestrar el radical hidroxilo, generado en la mucosa gástrica por la indometacina, pudiera contribuir a sus efectos antioxidantes y gastroprotectores sobre el daño que los antiinflamatorios no esteroideos producen sobre la mucosa gástrica.

Introduction: the etiology of the gastric ulceration is associated to the imbalance between aggressive and defensive factors acting upon the gastric mucosa. D-002, a mixture of 6 higher primary alcohols purified from the beeswax, cause some multiple mechanism-mediated gastroprotective effects and decrease of lipid peroxidation in the gastric mucosa. Objective: to determine whether D-002 can scavenge the in vivo added or in vivo generated hydroxyl radical in rats with indometacin-induced gastric ulcer or not. Methods: For the in vitro experiment, D-002 was added at concentrations 0.9 and 1 000 mg/mL For the in vivo experiment, the rats were randomized into 6 groups: one negative control, and five indometacin-treated groups as follows a positive excipient-treated control, three under D-002 treatment (5, 25 or 100 mg/kg, respectively, p.o.), and another group treated with Omeprazole (20 mg/kg i.p.). These lines of treatment were given 1 hour (excipient and D-002) or 30 min (Omeprazole) prior to inducing the ulcers. In both experiments, aliquots from the gastric mucosa were taken and the damage infringed to 2-deoxiribose by the hydroxyl radical was determined. Results: oral administration of D-002, rather than in vitro addition, significantly protected 2-desoxiribose from the oxidative damage depending on the dosage as compared to the positive control. Conclusions: these results indicate that the ability of the orally administered D-002 (25 and 100 mg/kg) to scavenge the hydroxyl radical endogenously generated on the gastric mucosa by indometacin could contribute to its antioxidant and gastroprotective effects against the damage that the non-steroidal anti-inflammatory drugs carry to the gastric mucosa.