Perillyl Alcohol Promotes Relaxation in Human Umbilical Artery.

BACKGROUND: Perillyl alcohol (POH) is a monoterpenoid found in plant essential oils and has been shown to relax murine vessels, but its effect on human vessels remains poorly studied. OBJECTIVE: The study aimed to characterize the effect of POH on human umbilical arteries (HUA). METHODS: Rings of HUA were obtained from uncomplicated patients and suspended in an organ bath for isometric recording. The vasorelaxant effect of POH in HUA was evaluated on basal tone and electromechanical or pharmacomechanical contractions, and possible mechanisms of action were also investigated. RESULTS: POH (1-1000 µM) altered the basal tone of HUA and completely relaxed HUA rings precontracted with KCl (60 mM) or 5-HT (10 µM), obtaining greater potency in the pharmacomechanical pathway (EC50 110.1 µM), suggesting a complex interference in the mobilization of extra- and intracellular Ca2+. POH (1000 µM) inhibited contractions induced by BaCl2 (0.1-30 mM) in a similar way to nifedipine (10 µM), indicating a possible blockade of L-type VOCC. In the presence of potassium channel blockers, tetraethylammonium (1 mM), 4-aminopyridine (1 mM), or glibenclamide (10 µM), an increase in the EC50 value of the POH was observed, suggesting a modulation of the activity of BKCa, KV, and KATP channels. CONCLUSION: The data from this study suggest that POH modulates Ca2+ and K+ ion channels to induce a relaxant response in HUA.

Perillyl alcohol for pediatric TP53- and RAS-mutated SHH-medulloblastoma: an in vitro and in vivo translational pre-clinical study.

PURPOSE: Inhalation of perillyl alcohol (POH) recently emerged as an investigational promising antiglioma strategy. However, little attention has been paid to its therapeutic potential for other brain tumors, especially in the pediatric setting. METHODS: The effects of POH were explored in medulloblastoma cell models belonging to the SHH variant with activation of RAS (ONS-76) or with TP53 mutations (DAOY and UW402), by means of proliferation and invasion assays. Interactions with methotrexate, thiotepa, or ionizing radiation were also assessed. Mice bearing subcutaneous tumors were treated with intraperitoneal injections. Alternatively, animals with intracranial tumors were exposed to intranasal POH alone or combined with radiation. Tumor growth was measured by bioluminescence. Analyses of cytotoxicity to the nasal cavity were also performed, and the presence of POH in the brain, lungs, and plasma was surveyed through chromatography/mass spectrometry. RESULTS: POH decreased cell proliferation and colony formation, with conspicuous death, though the invasive capacity was only affected in the NRAS-mutated cell line. Median-drug effect analysis displayed synergistic combinations with methotrexate. Otherwise, POH showed to be a reasonable radiosensitizer. In vivo, intraperitoneal injection significantly decreased tumor volume. However, its inhalation did not affect orthotopic tumors, neither alone or followed by cranial irradiation. Nasal cavity epithelium showed unimportant alterations, though, no traces of POH or its metabolites were detected in tissue samples. CONCLUSION: POH presents robust in vitro antimedulloblastoma effects and sensitizes cell lines to other conventional therapeutics, reducing tumor volume when administered intraperitoneally. Nevertheless, further improvement of delivery devices and/or drug formulations are needed to better characterize its effectiveness through inhalation.

Limonene and Perillyl Alcohol Derivatives: Synthesis and Anticancer Activity.

Limonene and perillyl alcohol are natural monoterpenes that have attracted the attention of medicinal chemists due to their promising anticancer activities. Considering this, both compounds were explored as scaffolds to obtain various derivatives with anticancer activity. In this review, the data are organized for the first time, with a focus on the synthetic methods and strategies to obtain the derivatives throughout the period from 2000 to 2020. A brief discussion regarding the structure and activity relationships of the most active derivatives, stereoisomers, and their mechanisms of action is presented. Among the active compounds, a series of limonenes with thiosemicarbazone groups and perillyl alcohol hybrids with glycosides or drugs are illustrated. Taking all of this into account, this review may help researchers develop new promising anticancer candidates based on the structures of limonene and perillyl alcohol.

Anti-Tumor Efficiency of Perillylalcohol/ß-Cyclodextrin Inclusion Complexes in a Sarcoma S180-Induced Mice Model.

The low solubility and high volatility of perillyl alcohol (POH) compromise its bioavailability and potential use as chemotherapeutic drug. In this work, we have evaluated the anticancer activity of POH complexed with ß-cyclodextrin (ß-CD) using three complexation approaches. Molecular docking suggests the hydrogen-bond between POH and ß-cyclodextrin in molar proportion was 1:1. Thermal analysis and Fourier-transform infrared spectroscopy (FTIR) confirmed that the POH was enclosed in the ß-CD cavity. Also, there was a significant reduction of particle size thereof, indicating a modification of the ß-cyclodextrin crystals. The complexes were tested against human L929 fibroblasts after 24 h of incubation showing no signs of cytotoxicity. Concerning the histopathological results, the treatment with POH/ß-CD at a dose of 50 mg/kg promoted approximately 60% inhibition of tumor growth in a sarcoma S180-induced mice model and the reduction of nuclear immunoexpression of the Ki67 antigen compared to the control group. Obtained data suggest a significant reduction of cycling cells and tumor proliferation. Our results confirm that complexation of POH/ß-CD not only solves the problem related to the volatility of the monoterpene but also increases its efficiency as an antitumor agent.

Intranasal administration of perillyl alcohol-loaded nanoemulsion and pharmacokinetic study of its metabolite perillic acid in plasma and brain of rats using ultra-performance liquid chromatography/tandem mass spectrometry.

Perillyl alcohol (POH) is a monocyclic terpene that has strong antitumor activity. Brain tumors are particularly difficult to treat with therapeutic agents, and clinical trials have shown their low tolerance through oral administration. We proposed the entrapment of POH into an oil-in-water chitosan nanoemulsion aiming its intranasal administration for brain targeting. An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantitation of total metabolite perillic acid (PA) in plasma and brain of rats. The rat samples containing the metabolite were treated by liquid-liquid extraction with acetonitrile. The mobile phase was 0.1% formic acid in water (solvent A) and 0.1% formic acid in methanol (solvent B), at a flow rate of 0.3 mL min-1 in gradient elution. The chromatography was run for 10 min, and analytical curves were built in acetonitrile, plasma, and brain. The PA was detected in positive ion mode with multiple reaction monitoring. The method has shown high selectivity, sensitivity, and throughput. The low quantification limits of 162, 178, and 121 ng mL-1 for acetonitrile, brain, and plasma, respectively, indicate a good detectability of the method. The repeatability and precision observed were within the limits recommended in the literature. The accuracy of the method was verified through high recovery rates (106-118%). The validated method was successfully applied to the pharmacokinetic study of the metabolite PA after the intranasal administration of free or POH-loaded nanoemulsion in rats. The results showed that chitosan nanoemulsion improved the plasma and brain bioavailability of POH, representing a promising alternative to free POH treatment.

Adjuvant effect of low-carbohydrate diet on outcomes of patients with recurrent glioblastoma under intranasal perillyl alcohol therapy.

BACKGROUND: Standard of care for glioblastoma (GB), consisting of cytotoxic chemotherapy, steroids, and high-dose radiation, induces changes in the tumor microenvironment through its effects on glucose availability, which is a determinant for tumor progression (TP). Low-carbohydrate diet (LCD) reduces the glucose levels needed to drive the Warburg effect. METHODS: To investigate LCD's effect on GB therapy, we have begun a clinical trial using LCD as an addition to intranasal perillyl alcohol (POH) for recurrent GB (rGB) patients. This study involved 29 individuals and evaluated, over a period of 1 year, the adjuvant effect of LCD associated with POH therapy in terms of toxicity, extent of peritumoral edema, reduced corticosteroid use, seizure frequency, and overall survival. POH group (n = 14) received solely intranasal POH without specific diet regimen, whereas POH/LCD group (n = 15) received intranasal POH in combination with nutritional intervention. Patients' assessment was based on clinical reviews and magnetic resonance data. RESULTS: In the 1-year follow-up, the POH/LCD group showed a 4.4-fold decrease in the proportion of patients who needed treatment with corticosteroids, as well as a reduction in tumor size and peritumoral edema, as compared to the POH group. While 75% of patients undergoing POH treatment experienced seizures, this fraction was reduced to 56% in the POH/LCD group. A 2.07-fold increase in the proportion of patients with stable disease, along with a 2.8-fold decrease in the proportion of patients with TP, was seen in the POH/LCD group. CONCLUSION: The results presented in this study show that the LCD associated with intranasal POH therapy may represent a viable option as adjunctive therapy for rGB to improve survival without compromising patients' quality of life. Prospective cohort studies are needed to confirm these findings and validate the efficacy of this novel therapeutic strategy.

Limonene, a food additive, and its active metabolite perillyl alcohol improve regeneration and attenuate neuropathic pain after peripheral nerve injury: Evidence for IL-1ß, TNF-α, GAP, NGF and ERK involvement.

BACKGROUND: Limonene (LIM) and its main metabolite perillyl alcohol (POH) are ingredients found in food with promising chemical entities due to their pharmacological profile. In this study, we hypothesized that LIM and POH are two molecules capable of accelerating the regenerative process and alleviating neuropathic pain. METHODS: Animals were treated daily (LIM, POH and saline) for 28 days and during this period evaluated for mechanical hyperalgesia, astrocyte participation by immunofluorescence for GFAP, and ELISA was used to quantify IL-1ß and TNF-α in the spinal cord. Western blot analysis of the following proteins was also performed: GFAP, GAP-43, NGF and ERK. For motor deficit analysis, tests were performed to assess hind paw muscle strength and footprints through gait (SFI). RESULTS: Both POH and LIM accelerated the regenerative process and improved motor deficits comparing to positive control; however, POH was more effective, particularly between the 2nd and 3rd week after the nerve injury, increasing GAP-43, NGF and the phosphorylated ERK immunocontent. Moreover, POH and LIM were able to reduce hyperalgesia and astrocytosis. CONCLUSIONS: Both substances, LIM and POH, improved the regeneration process and sensory and motor function recovery in the PNI model in mice by mitigating the inflammatory reactions and up-regulating the neurotrophic process.

Intranasal perillyl alcohol therapy improves survival of patients with recurrent glioblastoma harboring mutant variant for MTHFR rs1801133 polymorphism.

BACKGROUND: Polymorphisms in MTHFR gene influence risk and overall survival of patients with brain tumor. Global genomic DNA (gDNA) methylation profile from tumor tissues is replicated in peripheral leukocytes. This study aimed to draw a correlation between rs1801133 MTHFR variants, gDNA methylation and overall survival of patients with recurrent glioblastoma (rGBM) under perillyl alcohol (POH) treatment. METHODS: gDNA from whole blood was extracted using a commercially available kit (Axygen) and quantified by spectrophotometry. Global gDNA methylation was determined by ELISA and rs1801133 polymorphism by PCR-RFLP. Statistical analysis of gDNA methylation profile and rs1801133 variants included Mann-Whitney, Kruskal-Wallis, Spearman point-biserial correlation tests (SPSS and Graphpad Prism packages; significant results for effect size higher than 0.4). Prognostic value of gDNA methylation and rs1801133 variants considered survival profiles at 25 weeks of POH treatment, having the date of protocol adhesion as starting count and death as the final event. RESULTS: Most rGBM patients showed global gDNA hypomethylation (median = 31.7%) and a significant, moderate and negative correlation between TT genotype and gDNA hypomethylation (median = 13.35%; rho = - 0.520; p = 0.003) compared to CC variant (median = 32.10%), which was not observed for CT variant (median = 33.34%; rho = - 0.289; p = 0.06). gDNA hypermethylated phenotype (median = 131.90%) exhibited significant, moderate and negative correlations between TT genotype (median = 112.02%) and gDNA hypermethylation levels when compared to CC (median = 132.45%; rho = - 0,450; p = 0.04) or CT (median = 137.80%; rho = - 0.518; p = 0.023) variants. TT variant of rs1801133 significantly decreased gDNA methylation levels for both patient groups, when compared to CC (d values: hypomethylated = 1.189; hypermethylated = 0.979) or CT (d values: hypomethylated = 0.597; hypermethylated = 1.167) variants. Positive prognostic for rGBM patients may be assigned to gDNA hypermethylation for survivors above 25 weeks of treatment (median = 88 weeks); and TT variant of rs1801133 regardless POH treatment length. CONCLUSION: rGBM patients under POH-based therapy harboring hypermethylated phenotype and TT variant for rs1801133 had longer survival. Intranasal POH therapy mitigates detrimental effects of gDNA hypomethylation and improved survival of patients with rGBM harboring TT mutant variant for MTHFR rs1801133 polymorphism. TRIAL REGISTRATION: CONEP -9681- 25,000.009267 / 2004. Registered 12th July, 2004.

Simultaneous measurement of perillyl alcohol and its metabolite perillic acid in plasma and lung after inhalational administration in Wistar rats.

The inhalational administration of drugs is a practical and non-invasive approach with the potential to reduce side effects and with a quick onset of therapeutic activity. Perillyl alcohol (POH) is a monoterpene with antitumor activity that currently is undergoing clinical evaluation as an inhalational anticancer agent. A detection method was developed that will be applicable to pharmacokinetic studies of not only POH, but also its longer-lived main metabolite, perillic acid (PA), in lung tissue and plasma after inhalational delivery. The anticancer activity of POH was investigated in vitro with the use of various lung cancer cell lines. Toxicity was established by a standard MTT assay, and apoptosis markers were analyzed by Western blot. For the detection of POH and PA in lungs and plasma, albino Wistar rats were used that were exposed to POH inhalation. Tissues were subjected to chromatographic separation on an Agilent Zorbax Eclipse XDB C18 column, followed by detection of absorption in the ultraviolet (UV) range. In vitro, POH exerted cytotoxic activity against six different lung tumor cell lines, and apoptotic cell death was indicated by induction of active caspase 3 and cleavage of poly (ADP-ribose) polymerase 1 (PARP1). These results demonstrate that inhalational delivery of POH results in effective biodistribution and metabolism of POH in the systemic circulation. In addition, our study introduces a simple, rapid HPLC-UV method with high accuracy for simultaneous detection of POH and its metabolite PA in plasma, and for sensitive detection of PA in lung tissue, which should prove useful for applications in clinical studies.

Synthesis and Antiproliferative Activity of New Cyclodiprenyl Phenols against Select Cancer Cell Lines.

Six new cyclodiprenyl phenols were synthesized by direct coupling of perillyl alcohol and the appropriate phenol. Their structures were established by IR, HRMS and mainly NMR. Three human cancer cell lines-breast (MCF-7), prostate (PC-3) and colon (HT-29)-were used in antiproliferative assays, with daunorubicin and dunnione as positive controls. Results described in the article suggest that dihydroxylated compounds 2⁻4 and monohydroxylated compound 5 display selectivity against cancer cell lines, cytotoxicity, apoptosis induction, and mitochondrial membrane impairment capacity. Compound 2 was identified as the most effective of the series by displaying against all cancer cell lines a cytotoxicity close to dunnione antineoplastic agent, suggesting that the cyclodiprenyl phenols from perillyl alcohol deserve more extensive investigation of their potential medicinal applications.

Hydroxyl Group and Vasorelaxant Effects of Perillyl Alcohol, Carveol, Limonene on Aorta Smooth Muscle of Rats.

The present study used isometric tension recording to investigate the vasorelaxant effect of limonene (LM), carveol (CV), and perillyl alcohol (POH) on contractility parameters of the rat aorta, focusing in particular on the structure-activity relationship. LM, CV, and POH showed a reversible inhibitory effect on the contraction induced by electromechanical and pharmacomechanical coupling. In the case of LM, but not CV and POH, this effect was influenced by preservation of the endothelium. POH and CV but not LM exhibited greater pharmacological potency on BayK-8644-induced contraction and on electromechanical coupling than on pharmacomechanical coupling. In endothelium-denuded preparations, the order of pharmacological potency on electrochemical coupling was LM < CV < POH. These compounds inhibited also, with grossly similar pharmacological potency, the contraction induced by phorbol ester dibutyrate. The present results suggest that LM, CV and POH induced relaxant effect on vascular smooth muscle by means of different mechanisms likely to include inhibition of PKC and IP3 pathway. For CV and POH, hydroxylated compounds, it was in electromechanical coupling that the greater pharmacological potency was observed, thus suggesting a relative specificity for a mechanism likely to be important in electromechanical coupling, for example, blockade of voltage-dependent calcium channel.

Circulating Cell-Free DNA as a Prognostic and Molecular Marker for Patients with Brain Tumors under Perillyl Alcohol-Based Therapy.

Tumor infiltration into brain tissue usually remains undetected even by high-resolution imaging. Molecular markers are used to increase diagnostic accuracy, but with limited continuous monitoring application. We evaluated the potential of circulating cell-free DNA (cfDNA) as a molecular indicator of the response to therapy by the intranasal administration (ITN) of perillyl alcohol (POH) in brain tumors. The cohort included 130 healthy subjects arranged as control-paired groups and patients at terminal stages with glioblastoma (GBM, n = 122) or brain metastasis (BM, n = 55) from stage IV adenocarcinomas. Serum cfDNA was isolated and quantified by fluorimetry. Compared with the controls (40 ng/mL), patients with brain tumors before ITN-POH treatment had increased (p < 0.0001) cfDNA median levels: GBM (286 ng/mL) and BM (588 ng/mL). ITN-POH treatment was significantly correlated (rho = -0.225; p = 0.024) with survival of >6 months at a concentration of 599 ± 221 ng/mL and of.

Efficacy of a ketogenic diet with concomitant intranasal perillyl alcohol as a novel strategy for the therapy of recurrent glioblastoma.

It has been hypothesized that persistent ketotic hypoglycemia represents a potential therapeutic strategy against high-grade gliomas. Perillyl alcohol (POH) is a non-toxic, naturally-occurring, hydroxylated monoterpene that exhibits cytotoxicity against temozolomide-resistant glioma cells, regardless of O6-methylguanine-methyltransferase promoter methylation status. The present study aimed to evaluate the toxicity and therapeutic efficacy of intranasal POH when administered in combination with a ketogenic diet (KD) program for the treatment of patients with recurrent glioblastoma. The 32 enrolled patients were divided into two groups, KD or standard diet, with intranasal POH treatment (n=17 and n=15, respectively). The nutritional status and anthropometric parameters of the patients were measured. Patients that adhered to the KD maintained a strict dietary regimen, in addition to receiving 55 mg POH four times daily, in an uninterrupted administration schedule for three months. Neurological examination and magnetic resonance imaging analysis were used to monitor disease progression. A total of 9/17 patients in the KD group survived and maintained compliance with the KD. After three months of well-tolerated treatment, a partial response (PR) was observed for 77.8% (7/9) of the patients, stable disease (SD) in 11.1% (1/9) and 11.1% (1/9) presented with progressive disease (PD). Among the patients assigned to the standard diet group, the PR rate was 25% (2/8 patients), SD 25% (2/8) and PD 50% (4/8 patients). The patients assigned to the KD group presented with reduced serum lipid levels and decreased low-density lipoprotein cholesterol levels. These results are encouraging and suggest that KD associated with intranasal POH may represent a viable option as an adjunct therapy for recurrent GBM.

Patient with Recurrent Glioblastoma Responding Favorably to Ketogenic Diet Combined with Intranasal Delivery of Perillyl Alcohol: A Case Report and Literature Review

Introduction Monoterpene perillyl alcohol (POH) is cytotoxic to temozolomideresistant glioma cells, regardless of its O6-methylguanine-methyltransferase (MGMT) promoter methylation status. Moreover, adherence to a ketogenic diet (KD) produced successful outcomes in preclinical and clinical studies in the glioma setting. Case Presentation A 54-year-old Caucasian man had a confirmed diagnosis of refractory glioblastoma multiforme (GBM). The immunohistochemical evaluation was negative for methylation, and failed to detect mutations in the isocitrate dehydrogenase (IDH) 1 and 2 genes. In January 2016, the patient was enrolled in a clinical trial combining daily intranasal delivery of POH in combination with a KD. The KD was administered concomitantly with inhalation of POH (55 mg, 4 times a day) in an uninterrupted administration schedule for 3 months. Results The combination treatment was well-tolerated. The nutritional status and anthropometric measurements of the patient were measured. Adherence to the KD was confirmed by measuring the levels of ketone bodies in the urine. Throughout the treatment, a reduced frequency of seizures was observed. After three months of adherence to the treatment, the patient presented with weight loss, reduced body fat, increased water retention, and a slight increase in bone and muscle mass. A follow-up magnetic resonance imaging (MRI) scan after 3 months of treatment revealed marked reduction of the enhancing lesion. Conclusion Intranasal delivery of POH combined with concomitant adherence to a KD appeared to have a beneficial therapeutic effect in a patient with recurrent GBM. Further studies are needed to evaluate the efficacy of this therapeutic strategy in a larger cohort of treatment-refractory GBM patients.

Introdução O monoterpeno álcool perílico (AP) é citotóxico para linhagens celulares de glioblastoma, independentemente do status do promotor de metilação O6-metilguaninametiltransferase (MGMT). Além disso, a adesão à dieta cetogênica (DC) produziu resultados bem sucedidos em desenho de estudos pré-clínicos e clínicos de glioma. Relato de Caso Homem, 54 anos, caucasiano, com diagnóstico de glioblastoma multiforme (GBM) recidivo. A avaliação imuno-histoquímica foi negativa para metilação e não detectou mutações do gene da isocitrato desidrogenase 1 e 2 (IDH1 IDH2). Em janeiro de 2016, o paciente foi inscrito em um ensaio clínico da administração intranasal diária do AP combinada a DC. A DC foi administrada concomitantemente com inalação de AP (55 mg, 4 vezes ao dia) em um cronograma de administração ininterrupto durante 3 meses. Resultados O tratamento combinado foi bem tolerado. O estado nutricional e as medidas antropométricas do paciente foram avaliadas. Aderência a DC foi confirmada pela presença de corpos cetônicos na urina. Ao longo do tratamento, observou-se redução da frequência de convulsões. Após três meses de adesão ao tratamento, o paciente apresentou perda de peso, redução da gordura corporal, melhor hidratação e um aumento discreto da massa óssea e muscular. O acompanhamento da ressonância magnética após 3 meses de tratamento revelou redução acentuada do volume da lesão. Conclusão A administração intranasal do AP combinada a DC sugere ter um efeito terapêutico benéfico em pacientes com GBM recorrente. São necessários mais estudos para avaliar a eficácia desta estratégia terapêutica em uma coorte maior de pacientes com GBM refratários.

Perillyl alcohol, a pleiotropic natural compound suitable for brain tumor therapy, targets free radicals.

Monoterpenes such as limonene and perillyl alcohol (POH) are promising natural compounds with pro-oxidant properties partly due to endoplasmic reticulum (ER) stress-induced cytotoxicity, and antioxidant activity owing to their activity as free radical scavengers, inhibition of coenzyme Q synthesis, activation of antioxidant-responsive elements (inducing detoxification enzymes) and induction of apoptosis. Activation of ER-stress responses generates reactive oxygen species (ROS), which are highly reactive free radicals mainly produced during mitochondrial electron transfer for adenosine triphosphate (ATP) synthesis. When cells are subjected to oxidative stress conditions, there is an accumulation of ROS that can lead to irreversible cell injury caused primarily by lipid peroxidation, protein aggregation and/or DNA damage. Malignant tumors, such as glioblastoma multiforme, display elevated rates of oxygen consumption, necrosis and abnormal structural microvasculature. Alterations in the tumor microenvironment are tightly linked to tumor progression and occur as a result of activation of complex signaling networks involving inter-clonal cooperation, cell-matrix interactions and an ongoing inflammatory response leading to genetic and epigenetic alterations. This review will focus on the pro- and anti-oxidant activities of POH, which are greatly dependent on the respective ROS levels within the tumor microenvironment and involve the ER stress response system. As well, some critical aspects of tumor-associated metabolic changes and the consequences of endogenous ROS production for tumor progression will be discussed.

Orofacial antinociceptive activity of (S)-(-)-perillyl alcohol in mice: a randomized, controlled and triple-blind study.

This study investigated the antinociceptive effects of (S)-(-)-perillyl alcohol (PA) on orofacial nociception in Swiss male mice using formalin-, capsaicin-, and glutamate-induced pain tests. For each test, eight animals per group were pre-treated intraperitoneally by a blinded investigator with PA (50 or 75mg/kg), morphine, or vehicle (saline+0.2% Tween 80). The treatment was performed before the induction of orofacial nociception by injecting formalin, capsaicin, or glutamate solution into the right area of the upper lip. The orofacial nociceptive behaviour was timed in all tests by an investigator who was blinded to the treatments. The statistical analysis was performed using confidence intervals (CI), the effect size, and power. PA blocked the orofacial nociceptive behaviour at both doses tested (P<0.05) similarly to morphine (P>0.05), in all tests. The effect size was high in the phase 1 formalin test for 50mg/kg PA (95% CI 0.48-2.31, power 84.6%) and 75mg/kg PA (95% CI 0.82-2.76, power 96.2%), in phase 2 for 75mg/kg PA (95% CI 0.44-2.26, power 82.3%), and in the glutamate test for 75mg/kg PA (95% CI 1.11-3.16, power 99.2%). These findings show strong evidence for the antinociceptive properties of PA in the orofacial region.

Perillyl alcohol: Dynamic interactions with the lipid bilayer and implications for long-term inhalational chemotherapy for gliomas.

BACKGROUND: Gliomas display a high degree of intratumor heterogeneity, including changes in physiological parameters and lipid composition of the plasma membrane, which may contribute to the development of drug resistance. Biophysical interactions between therapeutic agents and the lipid components at the outer plasma membrane interface are critical for effective drug uptake. Amphipathic molecules such as perillyl alcohol (POH) have a high partition coefficient and generally lead to altered lipid acyl tail dynamics near the lipid-water interface, impacting the lipid bilayer structure and transport dynamics. We therefore hypothesized that glioma cells may display enhanced sensitivity to POH-induced apoptosis due to plasma membrane alterations, while in non-transformed cells, POH may be expelled through thermal agitation. METHODS: Interactions between POH and the plasma membrane was studied using molecular dynamics simulations. In this phase I/II trial, we set up to evaluate the clinical effectiveness of long-term (up to 5 years) daily intranasal administration of POH in a cohort of 19 patients with low-grade glioma (LGG). Importantly, in a series of clinical studies previously published by our group, we have successfully established that intranasal delivery of POH to patients with malignant gliomas is a viable and effective therapeutic strategy. RESULTS: POH altered the plasma membrane potential of the lipid bilayer of gliomas and prolonged intranasal administration of POH in a cohort of patients with LGG halted disease progression with virtually no toxicity. CONCLUSION: Altogether, the results suggest that POH-induced alterations of the plasma membrane might be contributing to its therapeutic efficacy in preventing LGG progression.

In Vivo Anti-Tumor Activity and Toxicological Evaluations of Perillaldehyde 8,9-Epoxide, a Derivative of Perillyl Alcohol.

Recent studies have revealed the high cytotoxicity of p-menthane derivatives against human tumor cells. In this study, the substance perillaldehyde 8,9-epoxide, a p-menthane class derivative obtained from (S)-(-)-perillyl alcohol, was selected in order to assess antitumor activity against experimental sarcoma 180 tumors. Toxicological effects related to the liver, spleen, kidneys and hematology were evaluated in mice submitted to treatment. The tumor growth inhibition rate was 38.4%, 58.7%, 35.3%, 45.4% and 68.1% at doses of 100 and 200 mg/kg/day for perillaldehyde 8,9-epoxide, perillyl alcohol and 25 mg/kg/day for 5-FU intraperitoneal treatments, respectively. No toxicologically significant effect was found in liver and kidney parameters analyzed in Sarcoma 180-inoculated mice treated with perillaldehyde 8,9-epoxide. Histopathological analyses of the liver, spleen, and kidneys were free from any morphological changes in the organs of the animals treated with perillaldehyde 8,9-epoxide. In conclusion, the data suggest that perillaldehyde 8,9-epoxide possesses significant antitumor activity without systemic toxicity for the tested parameters. By comparison, there was no statistical difference for the antitumor activity between perillaldehyde 8,9-epoxide and perillyl alcohol.

Evaluation of the cytotoxicity of structurally correlated p-menthane derivatives.

Compounds isolated from essential oils play an important role in the prevention and treatment of cancer. Monoterpenes are natural products, and the principal constituents of many essential oils. The aim of this study was to investigate the cytotoxic potential of p-menthane derivatives. Additionally, analogues of perillyl alcohol, a monoterpene with known anticancer activity, were evaluated to identify the molecular characteristics which contribute to their cytotoxicity, which was tested against OVCAR-8, HCT-116, and SF-295 human tumor cell lines, using the MTT assay. The results of this study showed that (-)-perillaldehyde 8,9-epoxide exhibited the highest percentage inhibition of cell proliferation (GI = 96.32%-99.89%). Perillyl alcohol exhibited high cytotoxic activity (90.92%-95.82%), while (+)-limonene 1,2-epoxide (GI = 58.48%-93.10%), (-)-perillaldehyde (GI = 59.28%-83.03%), and (-)-8-hydroxycarvotanacetone (GI = 61.59%-94.01%) showed intermediate activity. All of the compounds tested were less cytotoxic than perillyl alcohol, except (-)-perillaldehyde 8,9-epoxide (IC50 = 1.75-1.03 µL/mg). In general, replacement of C-C double bonds by epoxide groups in addition to the aldehyde group increases cytotoxicity. Furthermore, stereochemistry seems to play an important role in cytotoxicity. We have demonstrated the cytotoxic influence of chemical substituents on the p-menthane structure, and analogues of perillyl alcohol.

Long-term outcome in patients with recurrent malignant glioma treated with Perillyl alcohol inhalation.

AIM: This retrospective study aimed to evaluate the long-term response and toxicity of recurrent malignant glioma patients to inhalation chemotherapy with perillyl alcohol (POH). PATIENTS AND METHODS: The cohort included 117 men and 81 women with primary glioblastoma multiforme (GBM; n=154), grade III astrocytoma (AA; n=26) and anaplastic oligodendroglioma (AO; n=5). POH inhalation schedule 4-times daily started with 66.7 mg/dose; 266 mg/day and escalated up to 133.4 mg/dose; 533.6 mg/day. Clinical toxicity and overall survival following treatment were compared with tumor size, topography, extent of peritumoral edema and histological classification. RESULTS: Adhesion to the protocol was high (>95%), POH (533.6 mg/daily) occasionally caused nose soreness but rarely nosebleed. Tumor size, peritumoral edema and the oligodendroglial component influenced response to treatment. CONCLUSION: After 4 years under exclusive POH treatment, 19% of patients still remain in clinical remission. Long-term POH inhalation chemotherapy is a safe and non-invasive strategy efficient for recurrent malignant glioma.