- Department of General and Specialized Surgery, Antonio Pedro University Hospital, Fluminense Federal University, Rio de Janeiro, Brazil
- Memphys-Center for Biomembrane Physics, University of Southern Denmark, Odense, Denmark
- Department of Experimental Therapeutics, Beckman Research Institute of City of Hope, California, USA
- Department of Microbiology and Immunology, Keck School of Medicine, University of Southern California, California, USA
- Retired Professor from the Rio de Janeiro Federal University, Rio de Janeiro, Brazil
- Department of Cellular and Molecular Biology, Institute of Biology, Fluminense Federal University, Rio de Janeiro, Brazil
Clovis Orlando da Fonseca
Department of Cellular and Molecular Biology, Institute of Biology, Fluminense Federal University, Rio de Janeiro, Brazil
DOI:10.4103/2152-7806.173301Copyright: © 2016 Surgical Neurology International This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.
How to cite this article: da Fonseca CO, Khandelia H, Marcela D’Alincourt Salazar, Axel H. Schönthal, Osório C. Meireles, Quirico-Santos T. Perillyl alcohol: Dynamic interactions with the lipid bilayer and implications for long-term inhalational chemotherapy for gliomas. Surg Neurol Int 05-Jan-2016;7:1
How to cite this URL: da Fonseca CO, Khandelia H, Marcela D’Alincourt Salazar, Axel H. Schönthal, Osório C. Meireles, Quirico-Santos T. Perillyl alcohol: Dynamic interactions with the lipid bilayer and implications for long-term inhalational chemotherapy for gliomas. Surg Neurol Int 05-Jan-2016;7:1. Available from: http://surgicalneurologyint.com/surgicalint_articles/perillyl-alcohol-dynamic-interactions-with-the-lipid-bilayer-and-implications-for-long%e2%80%91term-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.
Keywords: Chemotherapy for gliomas, intranasal administration, long-term survival, membrane potentials, molecular dynamics simulation, perillyl alcohol
The development of resistance to chemotherapeutic agents is commonly observed during the course of cancer therapy and represents a major clinical challenge.[
Accumulating evidence shows that proliferation, as well progression of human malignant astrocytoma involves activation of the Ras oncoprotein.[
Literature provides numerous examples where synthetic drugs or naturally occurring substances exert biological effects via their interactions with the plasma membrane. Such interactions may involve disrupting the integrity and biophysical properties of the lipid bilayer, increasing its permeability, disturbing the conformation of membrane proteins, or directly binding to membrane lipids.[
Based on the above-outlined membrane interactions of Ras proteins, in combination with considerations of the amphipathic character of POH, it is conceivable that POH might play an important role in the inhibition of Ras, thereby affecting a key signaling pathway leading to pleiotropic outcomes.[
Despite some progress, there remains an urgent medical need for cancer treatments that are more effective and avoid the development of the MDR phenotype. At the same time, it is desirable that such treatments are safe and easily tolerated, which usually mandates a lack of toxic impact on normal cells. We, therefore, investigated the interactions between the Ras inhibitor POH and cellular membranes, using molecular dynamic (MD) simulations to provide a biophysical description as to how lipid bilayer-mediated pharmacological effects might be involved in generating therapeutic outcomes of long-term POH inhalation treatment. Prior MD simulations of POH and related terpenes showed that the terpenes all partitioned into the lipid bilayer, thereby altering membrane properties. In this study, we measured the dipole potential of the lipid bilayer and found that it was significantly modified by POH.
Molecular dynamics simulations
The simulations were set up in a manner similar to that previously described.[
The potential was symmetrized around the bilayer center to correct for overall bilayer translation during the simulation.
This clinical study was approved by the Rio de Janeiro Federal University Hospital Ethics Committee and the Brazilian Ministry of Health (CONEP 9681 Number 124 25000.009267/2004-25). It was carried out at the Hospital Medical School of the Fluminense Federal University. Before inclusion in the protocol, patient signed a written informed consent to enroll the Phase I/II clinical trial. POH was formulated for inhalation delivery and the preparation supplied by the Multidisciplinary Laboratory of Pharmaceutical Sciences at Rio de Janeiro Federal University. POH (55 mg; 0.3% v/v), was administered by inhalation 4 times daily totaling 266.8 mg/daily.
This prospective study was carried out from July 2006 to December 2014 with patients attending the outpatient Neurosurgical Unit in the Antonio Pedro University Hospital and included in the Phase I/II clinical trial to assess the efficacy of intranasal administration of the POH. Eligibility criteria included patients older than 18 years with recurrent malignant glioma with at least two relapses, measurable contrast-enhancing tumor image on magnetic resonance, Karnofsky index ≥70%, adequate hematological clinic laboratory-based measures, stable heart rhythm, and no clinical evidence of congestive heart failure or unstable angina. Exclusion criteria included pregnancy, hematological malignancy, occurrence of seizures, concomitant infectious or inflammatory processes, and acute cerebrovascular or hemorrhagic events.
Among 198 patients in our study, we observed that long-term treatment (up to 5 years) with daily intranasal POH administration improved and stabilized the condition of 19 LGG patients. In this study presented here, the cohort included, after appropriate informed consent, 19 adult patients with LGG. Diagnosis and histological classification came from biopsy and were formally designated the LGG by the World Health Organization (Grade II). Adverse events were graded according to Common Terminology Criteria for Adverse Events-Version 4.0 (CTCAE). These 19 patients did not undergo radiotherapy because the clinical management was observation and imaging control.
Drug administration and dose escalation
POH was formulated for intranasal delivery and the preparation supplied by the University Pharmacy was according to the following patents BR Application Number 0107262-5 December 17, 2001. POH (Sigma Chem. Co., St Louis, MI, USA) 0.3% v/v POH (55 mg) was administered by inhalation 4 times daily. All patients received POH 4 times daily by intranasal (inhalation) delivery from initial dose (66.7/dose; totaling 266.8 mg/daily).
Magnetic resonance imaging
Magnetic resonance imaging (MRI) was used to correlate the initial symptoms with clinical presentation. Tumor size was determined by performing axial contrast-enhanced scans, where the largest perpendicular diameter of the enhanced lesion was used as the scale in order to keep the comparison constant with the same type of axial imaging (MRI).
Interaction of perillyl alcohol with plasma membranes
As hypothesized, all POH molecules partition into the lipid-water interface with the hydrophobic part of the molecule interacts with the lipid acyl tails, and the −OH group interacts with the lipid head groups. The membrane dipole potential is significantly modified by POH [
(a) A snapshot from a molecular dynamic simulation of an H-Ras lipid-anchored in a dimyristoylphosphatidylcholine bilayer. The dimyristoylphosphatidylcholine lipids are shown in silver stick models with the noncarbon heavy atoms at the glycerol and head group regions colored in yellow. The H-Ras anchor is shown in a space-filling model with carbon in light green, oxygen in red, nitrogen in blue, and sulfur in orange. Lipid molecules that were on top of the anchor and prevented visualization were removed for clarity, as were water molecules and hydrogen atoms. (b) The dipole potential across a dimyristoylphosphatidylcholine bilayer in the presence and absence of perillyl alcohol. The potential was symmetrized around the bilayer center to correct fluctuations from bulk translation of the center of mass of the lipid bilayer during the simulation
Clinical activity of intranasal perillyl alcohol in low-grade glioma patients
Our previous studies[
According to CTCAE, 2 patients had nasal aching after prolonged use with 133.4 mg/dose (533.6 mg/daily) POH. After topic treatment, POH dose was reduced (266.8 mg/daily) and patients improved clinical condition without any further complaint. Prolonged POH inhalation chemotherapy at 266.8 mg/daily did not cause cumulative toxicity, and neither altered clinical chemistry (hepatic, renal, and lung) nor hematological parameters. At the start of treatment, patients frequently reported an intense headache (33%); however, there were no further complaints of seizures, nausea, mental confusion, lack of memory or language deficits, visual dizziness, behavioral alterations, or sleepiness.
A factor that greatly influenced the overall survival and response of malignant glioma patients to long-term intranasal POH treatment was the presence of peritumoral edema that contributed to clinical symptoms (intense headache, dizziness, focal neurological deficits, and seizures) and morbidity, and eventually favored glioma cell invasion to other brain structures. In this subgroup studied here, there was no presence of peritumoral edema in the MRI taken before patient inclusion in POH treatment.
Noteworthy, among 198 patients in our study, we observed that long-term treatment (up to 5 years) with daily intranasal POH administration as a single chemotherapeutic drug improved and stabilized the condition of 19 LGG patients [
Representative MRIs of LGG patients under intranasal POH treatment. One patient presented reduction of tumor size after 3 years (b) and 6 years (c) of POH treatment in comparison with first image obtained before treatment (a) Conversely, compared with initial MRI (d) a second patient maintained stable disease after 3 years (e) and 5 years (f) of treatment with POH
This study demonstrated that long-term POH intranasal administration was well tolerated and effective in halting progression in LGG patients. As gliomas are highly multidrug resistant, and changes in physiological parameters may be linked to their high degree of malignancy, this study pari passu, quantified the interaction of POH with a DMPC lipid bilayer. Thus, we aimed to explain the mechanism by which POH is effective primarily in tumor cells and not in normal cells. Our results demonstrated that POH modifies the dipole potential, and can thus influence the properties of proteins residing in the lipid bilayer such as Ras, which has been implicated in malignant gliomas.[
Thus, POH as an amphipathic drug can modulate membrane lipids of drug resistant cells, leading to alterations in the bilayer's biophysical properties. As a consequence, this may facilitate drug transport and delivery, thereby overcoming drug resistance. As well, the dipole potential, with its much stronger electric field, may impact membrane proteins and result in alterations of their activity. This idea is supported by studies performed on the Na/K-ATPase ion pump,[
It is well established that an acid-base complex equilibrium, involving surrounding medium and the lipid bilayer, determines the electrical properties of cellular membranes.[
Geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP) are derived from mevalonate, whose production is catalyzed by 3-hydroxy-3-methylglutaryl-CoA reductase. Prenylation by FPP, as well as GGPP, is required for membrane insertion and oncogenic function of Ras- and Rho-proteins triggering the stimulation of the Ras-Raf-MEK-ERK pathway.[
Data in literature indicates that LGG may dedifferentiate into a more malignant astrocytoma or glioblastoma multiforme, a process that appears to take place within approximately 5 years after the diagnosis.[
In summary, our results demonstrate that POH effectively enters into the cell membrane and might exert its multivariate clinical effects via modulation of the lipid bilayer components of the cellular membrane. This reduces the formation of tubular networks through inhibition of posttranslational isoprenylation of Ras. Consequently, intranasal administration of POH could be an effective novel strategy for future therapeutic efforts regarding prevention of dedifferentiation in LGGs and further make an important contribution to overcome induction of acquired drug resistance. We propose that in the near future the synthesis of biologically active hybrid molecules, containing POH as a carrier conjugated to drugs specifically targeting critical regulators of cell proliferation, may become a potentially effective addition to therapeutic regimens for brain tumors.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
HK is funded by Lundbeck Foundation. The computations were carried out on the Gardar supercomputer in Iceland.
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