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.

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.

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.

Patients' perspective on awake craniotomy for brain tumors-single center experience in Brazil.

BACKGROUND: Awake craniotomy with brain mapping is the gold standard for eloquent tissue localization. Patients' tolerability and satisfaction have been shown to be high; however, it is a matter of debate whether these findings could be generalized, since patients across the globe have their own cultural backgrounds and may perceive and accept this procedure differently. METHODS: We conducted a prospective qualitative study about the perception and tolerability of awake craniotomy in a population of consecutive brain tumor patients in Brazil between January 2013 and April 2015. Seventeen patients were interviewed using a semi-structured model with open-ended questions. RESULTS: Patients' thoughts were grouped into five categories: (1) overall perception: no patient considered awake craniotomy a bad experience, and most understood the rationale behind it. They were positively surprised with the surgery; (2) memory: varied from nothing to the entire surgery; (3) negative sensations: in general, it was painless and comfortable. Remarks concerning discomfort on the operating table were made; (4) postoperative recovery: perception of the postoperative period was positive; (5) previous surgical experiences versus awake craniotomy: patients often preferred awake surgery over other surgery under general anesthesia, including craniotomies. CONCLUSIONS: Awake craniotomy for brain tumors was well tolerated and yielded high levels of satisfaction in a population of patients in Brazil. This technique should not be avoided under the pretext of compromising patients' well-being.

Survival rate and perioperative data of patients who have undergone hemipelvectomy: a retrospective case series.

BACKGROUND: Hemipelvectomy is a major orthopedic surgical procedure indicated in specific situations. Although many studies discuss surgical techniques for hemipelvectomy, few studies have presented survival data, especially in underdeveloped countries. Additionally, there is limited information on anesthesia for orthopedic oncologic surgeries. The primary aim of this study was to determine the survival rate after hemipelvectomy, and the secondary aims were to evaluate anesthesia and perioperative care associated with hemipelvectomy and determine the influence of the surgical technique (external hemipelvectomy [amputation] or internal hemipelvectomy [limb sparing surgery]) on anesthesia and perioperative care in Brazil. METHODS: This retrospective case series collected data from 35 adult patients who underwent hemipelvectomy between 2000 and 2013. Survival rates after surgery were determined, and group comparisons were performed using the Kaplan-Meier method and the log-rank test. Mantel-Cox test and multiple linear regression analysis with stepwise forward selection were performed for univariate and multivariate analyses, respectively. RESULTS: Mean survival time was 32.8 ± 4.6 months and 5-year survival rate was 27 %. Of the 35 patients, 23 patients (65.7 %) underwent external hemipelvectomy and 12 patients (34.3 %) underwent internal hemipelvectomy. The survival rate was significantly higher in patients with bone tumors than in those with soft tissue sarcomas (P = 0.024). The 5-year cumulative probability of survival was significantly lower in patients who underwent external hemipelvectomy than in those who underwent internal hemipelvectomy (P = 0.043). In the univariate and multivariate analyses, only advanced disease stage (3 and 4) was identified as a significant independent predictor of reduced survival (P = 0.0003). Balanced general anesthesia combined with epidural block was the most frequent anesthesia technique. Median intraoperative crystalloid volume and red blood cell transfusions were 3500 mL and 2 units, respectively. CONCLUSIONS: Overall mean survival time after hemipelvectomy was 32.8 months. Advanced disease stage might be independently associated with reduced survival. Smaller amounts of fluids and transfusions were administered and time to discharge was shorter. Acute and chronic pain as well as wound complications are still important challenges in hemipelvectomy.

A Time-Based and Intratumoral Proteomic Assessment of a Recurrent Glioblastoma Multiforme.

Tumors consist of cells in different stages of transformation with molecular and cellular heterogeneity. By far, heterogeneity is the hallmark of glioblastoma multiforme (GBM), the most malignant and aggressive type of glioma. Most proteomic studies aim in comparing tumors from different patients, but here we dive into exploring the intratumoral proteome diversity of a single GBM. For this, we profiled tumor fragments from the profound region of the same patient's GBM but obtained from two surgeries a year's time apart. Our analysis also included GBM's fragments from different anatomical regions. Our quantitative proteomic strategy employed 4-plex iTRAQ peptide labeling followed by a four-step strong cation chromatographic separation; each fraction was then analyzed by reversed-phase nano-chromatography coupled on-line with an Orbitrap-Velos mass spectrometer. Unsupervised clustering grouped the proteomic profiles into four major distinct groups and showed that most changes were related to the tumor's anatomical region. Nevertheless, we report differentially abundant proteins from GBM's fragments of the same region but obtained 1 year apart. We discuss several key proteins (e.g., S100A9) and enriched pathways linked with GBM such as the Ras pathway, RHO GTPases activate PKNs, and those related to apoptosis, to name a few. As far as we know, this is the only report that compares GBM fragments proteomic profiles from the same patient. Ultimately, our results fuel the forefront of scientific discussion on the importance in exploring the richness of subproteomes within a single tissue sample for a better understanding of the disease, as each tumor is unique.

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.

Morphometric analysis of the medial opticocarotid recess and its anatomical relations relevant to the transsphenoidal endoscopic endonasal approaches.

BACKGROUND: The medial opticocarotid recess (MOCR) is located in the posterior wall of the sphenoid sinus, medial to the junction of the optic canal (OC) and the carotid prominence (CP). There is controversy in the literature in relation to the presence of the MOCR and its constancy, which is relevant when approaching the skull base through an endoscopic route. METHODS: The morphometric relations of the MOCR with the surrounding structures were studied in 18 cadaveric specimens after endoscopic endonasal approach (EEA). RESULTS: The distance between both MOCR was 11.06 ± 1.14 mm; the distance between the MOCR and the lateral opticocarotid (LOCR) recess was 5.56 ± 0.85 mm; the distance between the MOCR and the suprasellar recess was 3.72 ± 0.49 mm; the angle between the MOCR plane and the OC 13.32 ± 2.30°; the angle between the MOCR plane and the CP 13.50 ± 2.68° and; the angle between the OC and the CP 26.81 ± 4.26°. All measurements showed low variability, with low standard deviation and interquartile range. No relations were found between any of the measurements. CONCLUSIONS: The MOCR may be used as a reference point for precise location of structures during EEA. Objective measurements may be especially useful in cases with distorted sphenoid bone anatomy.

Intranasal administration of perillyl alcohol activates peripheral and bronchus-associated immune system in vivo.

Perillyl alcohol (POH) presents antitumoral activity but clinical application is hampered by adverse effects following oral administration. This work aimed to verify the cytotoxic effect of intranasal POH administration in the histology of lung, liver, brain; the cellularity and function of peripheral and bronchoalveolar-associated immune system. C57 adult mice received 1-min inhalation with POH, vehicle 70 % ethanol or saline buffer, once (84 µg/day) or twice (164 µg/day) during five consecutive days, and were killed 72 h after treatment. Spleen, cervical and mesenteric lymph nodes were removed for (3)H-thymidine proliferation assay, leukocyte cellularity and flow cytometry analysis. Peripheral blood and bronchoalveolar lavage cells were collected to assess cellularity and immunoglobulin (IgA, IgM) levels. Intranasal POH did not alter body weight or liver, brain and lung morphology, but increased splenocyte and cervical lymph node cell proliferation, and IgM production without altering peripheral lymphocyte subsets. Treatment also increased the percentage of alveolar macrophages (83 %) and IgA-producing lymphocytes (15 %), a pattern characteristic of activated bronchoalveolar innate immune system. Intranasal administration of POH activated peripheral immune system and innate immunity of bronchus-associated lymphoid tissue, thus suggesting a possible role for POH as a chemotherapeutic drug also in pathological processes affecting the lung.

Analysis of EGF+61A>G polymorphism and EGF serum levels in Brazilian glioma patients treated with perillyl alcohol-based therapy.

PURPOSE: Malignant gliomas are associated with alteration in EGF/EGFR signaling. Functional EGF+61A>G polymorphism is implicated with risk, recurrence, and progression of glioma. This study aimed to establish a putative association of EGF+61A>G with risk of glioma development, production of angiogenic growth factor EGF, and the response to perillyl alcohol administered by intranasal route. METHODS: The study included 83 patients with recurrent glioma enrolled in Phase I/II trial for intranasal perillyl alcohol therapy and subjects without cancer (n = 196) as control group. DNA was extracted from blood samples, EGF genotype performed with PCR-RFLP assay, and EGF circulating levels by enzyme immunoassay. Adequate statistical tests were performed to verify associations between polymorphism and glioma risk, and genotype correlation with EGF circulating levels. The log-rank test was also used to evaluate differences on patient survival. RESULTS: Patients with primary glioblastoma had high frequency of AA genotype (p = 0.037) and A allele (p = 0.037). Increased EGF circulating levels were observed in glioma patients with AA (p = 0.042), AG (p = 0.006), and AA + AG (p = 0.008) genotypes compared with GG. Patients with GG genotype showed increased but not significant (p > 0.05) survival rate, and EGF levels lower than 250 pg/mL was consistently (p = 0.0374) associated with increased survival. CONCLUSION: Presence of EGF+61A>G polymorphism in Brazilian subjects was associated with glioma risk and increased circulating EGF levels. Better response to perillyl alcohol-based therapy was observed in a group of adult Brazilian subjects with lower EGF levels.

Chemo-resistant protein expression pattern of glioblastoma cells (A172) to perillyl alcohol.

Glioblastoma multiform (GBM) is by far the most malignant glioma. We have introduced a new treatment for GBMs that comprises the inhalation of a naturally occurring terpene with chemotherapeutic properties known as perillyl alcohol (POH). Clinical trial results on recurrent GBM patients showed that POH extends the average life by more than eight months, temporarily slows tumor growth, and in some cases even decreases tumor size. After approximately seven months, the tumor continues to grow and leads to a dismal prognosis. To investigate how these tumors become resistant to POH, we generated an A172 human glioblastoma cell culture tolerant to 0.06 mM of POH (A172r). We used Multidimensional Protein Identification Technology (MudPIT) to compare the protein expression profile of A172r cells to the established glioblastoma A172 cell line. Our results include a list of identified proteins unique to either the resistant or the nonresistant cell line. These proteins are related to cellular growth, negative apoptosis regulation, Ras pathway, and other key cellular functions that could be connected to the underlying mechanisms of resistance.

Ras pathway activation in gliomas: a strategic target for intranasal administration of perillyl alcohol.

INTRODUCTION: Targeted therapy directed at specific molecular alterations is already creating a shift in the treatment of cancer patients. Malignant gliomas commonly overexpress the oncogenes EGFR and PDGFR and contain mutations and deletions of the tumor suppressor genes PTEN and TP53. Some of these alterations lead to activation of the P13K/Akt and Ras/MAPK pathways, which provide targets for therapy. Perillyl alcohol (POH), the isoprenoid of greatest clinical interest, was initially considered to inhibit farnesyl protein transferase. Follow-up studies revealed that POH suppresses the synthesis of small G proteins, including Ras. Intranasal delivery allows drugs that do not cross the blood-brain barrier to enter the central nervous system. Moreover, it eliminates the need for systemic delivery, thereby reducing unwanted systemic side effects. MATERIALS AND METHODS: Applying this method, a phase I/II clinical trial of POH was performed in patients with relapsed malignant gliomas after standard treatment: surgery, radiotherapy, and chemotherapy. POH was administrated in a concentration of 0.3% volume/volume (55 mg) four times daily in an interrupted administration schedule. The objective was to evaluate toxicity and progression-free survival (PFS) after six months of treatment. The cohort consisted of 37 patients, including 29 with glioblastoma multiforme (GBM), 5 with grade III astrocytoma (AA), and 3 with anaplastic oligodendroglioma (AO). Neurological examination and suitable image analysis (computed tomography (CT), magnetic resonance imaging (MRI)) established disease progression. Complete response was defined as neurological stability or improvement of conditions, disappearance of CT/MRI tumor image, and corticosteroid withdraw; partial response (PR) as > or =50 reduction of CT/MRI tumor image, neurological stability, or improvement of conditions and corticosteroid requirement; progressive course (PC) as > or =25 increase in CT/MRI tumor image or the appearance of a new lesion; and stable disease as a lack of any changes in the CT/MR tumor image or neurological status. RESULTS: After six months of treatment, PR was observed in 3.4% (n=1) of the patients with GBM and 33.3% (n=1) with AO; stable disease in 44.8% (n=13) with GBM, 60% (n=3) with AA, and 33.3% (n=1) with AO; and PC in 51.7% (n=15) with GBM, 40% (n=2), with AA and 33.3% (n=1) AO. PFS (sum of PRs and stable disease) was 48.2% for GBM, 60% for AA, and 66.6% for AO patients. CONCLUSIONS: The preliminary results indicate that intranasal administration of the signal transduction inhibitor POH is a safe, noninvasive, and low-cost method. There were no toxicity events and the regression of tumor size in some patients is suggestive of antitumor activity.

Preliminary results from a phase I/II study of perillyl alcohol intranasal administration in adults with recurrent malignant gliomas.

BACKGROUND: Activation of the p21-ras signaling pathway from aberrantly expressed receptors promotes the growth of malignant human astrocytomas. Perillyl alcohol has shown to have both chemopreventive and chemotherapeutic activities in preclinical studies. The underlying action mechanism(s) of POH has yet to be delineated but may involve effects on the TGF-beta and/or the Ras signaling pathways. The intranasal delivery allows drugs that do not cross the BBB to enter the CNS; moreover, it eliminates the need for systemic delivery, thereby reducing unwanted systemic side effects. METHODS: We are conducting a phase I/II study to evaluate the antitumoral activity of POH intranasal delivery in a 4x daily schedule in patients with recurrent MG. The objective was to determine PFS at 6 months and the safety for POH in adult patients who failed conventional treatment. Assessments were performed every 27 days. Thirty-seven patients with progressive disease after prior surgery, radiotherapy, and at least temozolomide-based chemotherapy were enrolled, 29 of whom had GBM, 5 who had anaplastic astrocytoma, and 3 had AO. RESULTS: One patient (3.4%) with GBM and 1 patient (33.3%) with AO achieved partial response; 13 patients (44.8%) with GBM, 3 patients (60%) with AA, and 1 (33.3%) with AO achieved stable disease; 15 (51.7%) patients with GBM, 2 (40%) patients with AA, and 1 (33.3%) with AO showed progressive disease. Progression-free survival (partial response and stable disease) was 48.2% for patients with GBM, 60% for patients with AA, and 66.6% for patients with AO. CONCLUSIONS: There were no toxicity events. Perillyl alcohol is well tolerated and regression of tumor size in some patients is suggestive of antitumor activity. This work discusses POH intranasal delivery as a potential adjuvant therapeutic strategy for patients with malignant gliomas.

Anti-thrombin as a prognostic biomarker candidate for patients with recurrent glioblastoma multiform under treatment with perillyl alcohol.

Perillyl alcohol (POH) is a naturally occurring monoterpene with antiangiogenic and anti-tumoral properties. This chemotherapeutic agent has proven effectiveness in several clinical trials, including an ongoing phase I, comprising patients with recurrent glioblastoma multiform (GBM) under treatment with POH by intranasal administration. Proteomics offers tools to distinguish states of biological systems according to protein expression differences and therefore, can be used to gain pathological insights and to search for disease follow-up biomarkers. In this work, a differential gel electrophoresis (DIGE) proteomic approach was used to search for plasma proteins that correlated with the disease progression in 10 of these patients. Our results pointed antithrombin (down) and fibrinogen (up) regulated after a four months treatment deserving to be further verified as prognostic markers for this treatment. Possible links between tumor progression and anti-thrombin expression level are also discussed.

Recent advances in the molecular genetics of malignant gliomas disclose targets for antitumor agent perillyl alcohol.

Tumors of glial origin such as glioblastoma multiforme (GBM) comprise the majority of human brain tumors. Despite advances in surgery, radiation, and chemotherapy, the prognosis for patients with malignant glioma has not improved, emphasizing the need for a search for new chemotherapeutic drugs. Deregulated p21-Ras function, as a result of mutation, overexpression, or growth factor-induced overactivation, contributes to the growth of GBM. The monoterpene perillyl alcohol (POH) has preventive and therapeutic effects in a wide variety of preclinical tumor models and is currently under phase I and phase II clinical trials. As inhibition of posttranslational isoprenylation of Ras, a family of proteins that are involved in signal transduction is among the drug-related activities observed in this compound; POH may be a potential chemotherapeutic agent for GBM. Intranasal delivery is a practical and noninvasive approach that allows therapeutic agents that do not cross the blood-brain barrier to enter the central nervous system, reducing unwanted systemic side effects. This article describes the effect of intranasal delivery of POH in a patient with relapsed GBM.

Perillyl alcohol induces apoptosis in human glioblastoma multiforme cells.

Standard treatment of glioblastoma multiforme consisting of surgical resection, radiation and/or chemotherapy is rarely curative, emphasizing the need for new chemotherapeutic drugs. The monoterpene perillyl alcohol (POH) has preventive and therapeutic effects in a wide variety of pre-clinical tumor models and is currently under phase I and II clinical trials. In the present study, we analyzed its effect on human glioblastoma cell lines (U87 and A172) and a primary cell culture derived from a human glioblastoma tumor specimen (GBM-1). Using MTT, we showed that POH inhibits the viability of glioblastomas in a concentration-dependent way. Glioblastoma cell lines treated with POH showed morphological alterations characteristic of apoptosis. Analysis of cell cycle and quantification of DNA fragmentation, in cells stained with propidium iodide (PI), confirmed the apoptotic effect of POH on glioblastomas. These data support the potential usefulness of perillyl alcohol as an effective chemotherapeutic agent for patients with recurrent glioblastoma multiforme.