Cannabis Extracts Research Articles

Role of Cannabinoids and Terpenes in Cannabis-Mediated Analgesia in Rats.

Introduction: Cannabis sativa has been used for centuries in treating pain. However, the analgesic role of many of its constituents including terpenes is unknown. This research examined the contributions of terpenes (volatile oil) and cannabinoids in cannabis-mediated analgesia in rats. Methods: Animals received intraperitoneal administration of either vehicle, 10.0 or 18.0 mg/kg morphine, or various doses of the extract without terpenes, isolated terpenes, Δ9-tetrahydrocannabinol (THC), or the full extract. Thirty minutes later animals were tested on hotplate and tail-flick tests of thermal nociception. One week later, rats received a second administration of test articles and were tested 30 min later in the abdominal writhing test of inflammatory nociception. Results: In the thermal assays, hotplate and tail-flick latencies for morphine-treated rats were dose dependent and significantly higher than vehicle-treated animals. All the cannabinoid compounds except for the isolated terpenes produced dose-dependent increases in hotplate and tail-flick latencies. In the inflammatory nociceptive assay, animals treated with vehicle and isolated terpenes demonstrated increased abdominal writhing, whereas all the cannabinoid compounds significantly decreased abdominal writhing responses. Conclusions: Overall, THC alone produced robust analgesia equivalent to the full cannabis extract, whereas terpenes alone did not produce analgesia. These data suggest the analgesic activity of cannabis is largely mediated by THC, whereas terpenes alone do not cause alterations in cannabis-mediated analgesia.

Tandem Mass Spectrometric Quantification of 93 Terpenoids in Cannabis Using Static Headspace Injections.

The therapeutic effect of Cannabis largely depends on the content of its pharmacologically active secondary metabolites, mainly phytocannabinoids, flavonoids, and terpenoids. Recent studies suggest there are therapeutic effects of specific terpenoids as well as synergistic effects with other active compounds in the plant. Although Cannabis contains an overwhelming milieu of terpenoids, only a limited number are currently reported and used for metabolic analysis of Cannabis chemovars. In this study, we report the development and validation of a method for simultaneous quantification of 93 terpenoids in Cannabis air-dried inflorescences and extracts. This method employs the full evaporation technique via a static headspace sampler, followed by gas chromatography-mass spectrometry (SHS-GC/MS/MS). In the validation process, spiked terpenoids were quantified with acceptable repeatability, reproducibility, sensitivity, and accuracy. Three medical Cannabis chemovars were used to study the effect of sample preparation and extraction methods on terpenoid profiles. This method was further applied for studying the terpenoid profiles of 16 different chemovars acquired at different dates. Our results demonstrate that sample preparation methods may significantly impact the chemical fingerprint compared to the nontreated Cannabis. This emphasizes the importance of performing SHS extraction in order to study the natural terpenoid contents of chemovars. We also concluded that most inflorescences expressed relatively unique terpenoid profiles for the most pronounced terpenoids, even when sampled at different dates, although absolute concentrations may vary due to aging. The suggested method offers an ideal tool for terpenoid profiling of Cannabis and sets the scene for more comprehensive works in the future.

Bioefficacy of different insecticides on cowpea aphid (Aphis craccivora Koch)

Aphid (Aphis craccivora Koch) is one of the serious pests of cowpea. Bioefficacy of four insecticides with different concentrations namely Jholmol (125 ml/L), Neemix (2 ml/L), Cannabis extract (100 g/L), Chlorpyrifos 50% EC and Cypermethrin 5% EC (2 ml/L) along with control were evaluated against aphid at Research Station of Agriculture and Forestry University, Rampur, Chitwan, Nepal in 2018. Five treatments; four different insecticides and control were laid out in a randomized complete block design with four replications. The results indicated that all the insecticides were significant for aphid reduction. The effects were higher in Neemix followed by Jholmol and cannabis extract. The yield of cowpea was significantly highest (11.10 t/ha) with Neemix application, while lowest in control (7.58 t/ha). The highest net profit (NRs.61670) was obtained in Neemix followed by treatments of Jholmol (NRs.46175) and Cannabis extract (NRs.35815). The lowest incremental Cost/Benefit ratio was obtained in Neemix (1.341) and followed by treatments of, jholmol (1/1.12) and Cannabis extract (1/0.85), respectively. Thus, application of Neemix @ 2 ml/L could be used in cowpea to control aphid with high yield.

Cannabis sativa L. extract and cannabidiol inhibit in vitro mediators of skin inflammation and wound injury.

Skin inflammatory diseases result from complex events that include dysregulation and abnormal expression of inflammatory mediators or their receptors in skin cells. The present study investigates the potential effect of a Cannabis sativa L. ethanolic extract standardized in cannabidiol as antiinflammatory agent in the skin, unraveling the molecular mechanisms in human keratinocytes and fibroblasts. The extract inhibited the release of mediators of inflammation involved in wound healing and inflammatory processes occurring in the skin. The mode of action involved the impairment of the nuclear factor-kappa B (NF-κB) pathway since the extract counteracted the tumor necrosis factor-alpha-induced NF-κB-driven transcription in both skin cell lines. Cannabis extract and cannabidiol showed different effects on the release of interleukin-8 and vascular endothelial growth factor, which are both mediators whose genes are dependent on NF-κB. The effect of cannabidiol on the NF-κB pathway and metalloproteinase-9 (MMP-9) release paralleled the effect of the extract thus making cannabidiol the major contributor to the effect observed. Down-regulation of genes involved in wound healing and skin inflammation was at least in part due to the presence of cannabidiol. Our findings provide new insights into the potential effect of Cannabis extracts against inflammation-based skin diseases.

Cannabis for medical purposes : Impact of the law of 6March 2017 on physicians

The law on the amendment of the narcotics law and other regulations, which was accepted by consensus by the German Parliament, significantly expanded the options for the use of cannabis-based medicinal products. In individual cases, already approved cannabis-based medicinal products can also be prescribed outside the approved indication at the expense of the statutory health insurance funds (GKV). The cost of treatment with cannabis flowers and extracts as well as dronabinol, which are not approved under the drug law, will also be covered by the GKV upon application. Physicians must advise their patients on the options of treatment with cannabis-based medicinal products and support them in applying for reimbursement. The prescription of unauthorized medical cannabis products poses particular challenges, as there is no summary of product characteristics that is mandatory for authorized finished products. In addition, physicians are obliged to participate in an accompanying survey. To this end, they must send data to the Federal Institute for Drugs and Medical Devices providing information about treatment with cannabis-based medicinal products. When prescribing medical cannabis products as defined in the Act of 6March 2017 amending the law on narcotic drugs and other regulations, physicians assume aspecial responsibility that goes far beyond the responsibility for the use of authorized finished medicinal products.

The heterogeneity and complexity of Cannabis extracts as antitumor agents.

The Cannabis plant contains over 100 phytocannabinoids and hundreds of other components. The biological effects and interplay of these Cannabis compounds are not fully understood and yet influence the plant’s therapeutic effects. Here we assessed the antitumor effects of whole Cannabis extracts, which contained significant amounts of differing phytocannabinoids, on different cancer lines from various tumor origins. We first utilized our novel electrospray ionization liquid chromatography mass spectrometry method to analyze the phytocannabinoid contents of 124 Cannabis extracts. We then monitored the effects of 12 chosen different Cannabis extracts on 12 cancer cell lines. Our results show that specific Cannabis extracts impaired the survival and proliferation of cancer cell lines as well as induced apoptosis. Our findings showed that pure (-)-Δ9-trans-tetrahydrocannabinol (Δ9-THC) did not produce the same effects on these cell lines as the whole Cannabis extracts. Furthermore, Cannabis extracts with similar amounts of Δ9-THC produced significantly different effects on the survival of specific cancer cells. In addition, we demonstrated that specific Cannabis extracts may selectively and differentially affect cancer cells and differing cancer cell lines from the same organ origin. We also found that cannabimimetic receptors were differentially expressed among various cancer cell lines and suggest that this receptor diversity may contribute to the heterogeneous effects produced by the differing Cannabis extracts on each cell line. Our overall findings indicate that the effect of a Cannabis extract on a specific cancer cell line relies on the extract’s composition as well as on certain characteristics of the targeted cells.

Paradoxical Patterns of Sinusoidal Obstruction Syndrome-Like Liver Injury in Aged Female CD-1 Mice Triggered by Cannabidiol-Rich Cannabis Extract and Acetaminophen Co-Administration.

The goal of this study was to investigate the potential for a cannabidiol-rich cannabis extract (CRCE) to interact with the most common over-the-counter drug and the major known cause of drug-induced liver injury–acetaminophen (APAP)–in aged female CD-1 mice. Gavaging mice with 116 mg/kg of cannabidiol (CBD) [mouse equivalent dose (MED) of 10 mg/kg of CBD] in CRCE delivered with sesame oil for three consecutive days followed by intraperitoneally (i.p.) acetaminophen (APAP) administration (400 mg/kg) on day 4 resulted in overt toxicity with 37.5% mortality. No mortality was observed in mice treated with 290 mg/kg of CBD+APAP (MED of 25 mg/kg of CBD) or APAP alone. Following CRCE/APAP co-administration, microscopic examination revealed a sinusoidal obstruction syndrome-like liver injury–the severity of which correlated with the degree of alterations in physiological and clinical biochemistry end points. Mechanistically, glutathione depletion and oxidative stress were observed between the APAP-only and co-administration groups, but co-administration resulted in much greater activation of c-Jun N-terminal kinase (JNK). Strikingly, these effects were not observed in mice gavaged with 290 mg/kg CBD in CRCE followed by APAP administration. These findings highlight the potential for CBD/drug interactions, and reveal an interesting paradoxical effect of CBD/APAP-induced hepatotoxicity.

Cannabis for medicinal use: development of pharmacopoeia monographs as aquality standard

Herbal drugs and extracts, like all pharmaceutical starting materials used in the manufacture of medicinal products, must have an appropriate pharmaceutical quality. Corresponding quality standards are described in the individual monographs of the pharmacopoeia according to §55 of the German Drug Law. This includes information on ingredients and active substance content, among other things. This article describes the development of the Cannabis Flower Monograph for the German Pharmacopoeia (DAB) and the quality requirements and storage conditions contained therein. The state of development of monographs for the European Pharmacopoeia is also presented.After it was announced that there would be new legal regulations for the medical use of cannabis flowers and cannabis extracts, the first work on the cannabis flower monograph for the DAB began as early as 2015. First, amonograph on cannabis flowers was published in May 2016 in the German Drug Codex (DAC). The monograph was replaced in May 2017 by the publication of the DAB monograph. Arevised version of the DAB monograph has been in force since April 2018 as anational quality standard.Aharmonised cannabis flower monograph for the European Pharmacopoeia is currently being prepared to replace national quality standards. In addition, the German Pharmacopoeia and subsequently the European Pharmacopoeia develops monographs for preparations from cannabis flowers. In future, harmonised monographs in the European Pharmacopoeia will make it possible to avoid multiple testing according to the respective national standards and to facilitate analyses in laboratories and pharmacies.

Disposition of oral delta-9 tetrahydrocannabinol (THC) in children receiving cannabis extracts for epilepsy

Introduction: Although over half of US states have legalized marijuana for medical indications, there is limited research in use in the pediatric population. The objective was to evaluate the disposition of oral tetrahydrocannabinol (THC) in children receiving cannabis extracts for pediatric epilepsy.Methods: Prospective, observational study, evaluating the disposition of oral THC in children receiving cannabis extracts. Subjects were less than 18 years of age, receiving oral cannabis for pediatric epilepsy. Subjects included in the study had at least 2 detectable THC and related metabolite plasma concentrations during serial blood draw over a 10–12 h study period.Results: Nine subjects with a median age of 11 years (IQR 4.75) were included in the study, with oral doses ranging from 0.02 mg/kg to 1.59 mg/kg. Peak plasma concentrations (0.8 to 3.6 ng/ml) in most patients were achieved within 2 hours, while acute phase elimination half-life ranged from 1 to 5 hours. THC-COOH and glucuronide remained elevated through the study period. There was significant variation between the dose ingested and peak concentrations (R2 = 0.05).Conclusion: In pediatric patients receiving oral THC cannabis extracts, mean time to peak plasma concentrations was 2–7 hours, while mean acute phase elimination half-life was 4.0 hours. THC-COOH and THC-COOH glucuronide metabolites persisted throughout the 10–12 hour study period. Large variation and no correlation was noted between dose of THC by weight and peak concentrations, suggesting variation of bioavailability amongst pediatric population or inaccurate reporting of THC contents.

The relationship between cannabis/hemp use in foods and processing methodology

A diversity of commercial food products can be made from cannabis and hemp extracts, using both crude or winterized ‘oils’, distillates, and isolates, as documented in the literature. The manufacturing of these food-related end products involves processes such as extraction, phase separation, and distillation under vacuum. There is preference for consumer-friendly solvents that are generally regarded as safe (GRAS) such carbon dioxide, ethanol, water, and propane or butane; and triglyceride-based oils. Frequently CO2 is preferred as a solvent because it is environmentally benign, consumer-friendly, and selective as an extraction solvent. Here, we invoke solubility parameter theory (SPT) to explain the solubility/miscibility behavior of the above solvents toward cannabinoid, terpene, and non-polar contaminating solutes that must be removed using the above processes. This approach can also be used in formulating various food products, in which cannabis extracts and distillated are infused into a food matrix. Note is made of the fact that the infused cannabinoids are converted under the influence of heat and over time to other cannabinoids. Considering the difficulty of retaining terpenes during the above processing steps, back-addition of terpenes is frequently practiced. Numerous examples are provided of foods that are infused with defined cannabis/hemp extracts and distillates, and an example of an appropriate analysis and requisite labeling associated with a cannabinoid-infused product provided.

Comparison of different methods for the extraction of cannabinoids from cannabis

Cannabis oils, namely concentrated cannabis extracts, are getting plenty of attention because of their therapeutic potential for treatment of patients with cancer, HIV, multiple sclerosis and several other pathologies. Here we propose the use of ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE) as alternative methods to the current protocols followed by pharmacists, the only authorized to manipulate standardized Cannabis. A third method, consisting of the use of Tween 20 as surfactant, was considered. Our best extraction methodology for commercial hemp extraction was applied to medicinal cannabis. Here we report the results obtained for ‘Eletta campana’, ‘Carmagnola selezionata’, Bediol®, FM2® and Bedrocan®.

Uso do canabidiol (CBD) no tratamento da doença de Parkinson e suas comorbidades

Fundamento: A doença de Parkinson (DP) é um transtorno neurodegenerativo crônico caracterizado por sintomas motores (bradicinesia, tremores, rigidez) e não motores (transtornos psicóticos, do humor e do sono). Os tratamentos farmacológicos disponíveis não são eficazes para uma parcela significativa dos pacientes. Pesquisas recentes sugerem que o fitocanabinóide canabidiol (CBD) poderia ser eficaz no tratamento de alguns sintomas da DP. Objetivos: Revisar os estudos pré-clínicos e clínicos dos efeitos do CBD nos sintomas da DP. Métodos: Revisão narrativa dos principais estudos básicos e clínicos sobre os efeitos do CBD nos sintomas da DP. Devido ao número pequeno de estudos clínicos, pesquisas com outros canabinóides também foram incluídas. Resultados: No total, 15 artigos foram incluídos na revisão: 5 estudos pré-clínicos envolvendo a administração de CBD e 10 estudos com pacientes envolvendo a administração de diferentes canabinóides (maconha, nabilona, CBD). A maioria dos estudos básicos mostraram um efeito positivo do CBD em comportamentos ou alterações bioquímicas relacionadas à DP. Estudos observacionais e clínicos com uso de maconha fumada, extrato oral de maconha, nabilona (canabinóide sintético) e CBD sugerem que estes canabinóides podem reduzir sintomas motores (bradicinesia, tremores, rigidez) e não motores (transtornos psicóticos, do humor e do sono, qualidade de vida) da DP. Além disso, são substâncias bem toleradas e com poucos efeitos adversos significativos. Conclusão: Embora o CBD tenha demonstrado resultados favoráveis tanto em estudos pré-clínicos como em estudos clínicos, estas evidências ainda não são suficientes para indicar o uso deste canabinóide em pacientes com distúrbios do DP. Novos estudos controlados devem ser realizados com diferentes dosagens de CBD para replicar estes dados.

Development of a validated method for the qualitative and quantitative analysis of cannabinoids in plant biomass and medicinal cannabis resin extracts obtained by super-critical fluid extraction

The social push for the therapeutic use of cannabis extracts has increased significantly over recent years. Cannabis is being used for treatment for conditions such as epilepsy, cancer and pain management. There are a range of medicinal cannabis products available, but the use of cannabis resin obtained by super critical fluid extraction, often diluted in oil, is becoming increasingly more prominent. Much of the research on cannabis has focused on plant biomass or the final therapeutic product with a concerning lack of information on the intermediate resin. This study aims to bridge the gap between current methods of analysis for biomass and the final therapeutic product by describing a fully developed and validated ultra-high-performance-liquid-chromatography method with diode array detection (UHPLC-DAD) for the qualification and quantification of the cannabinoids CBDA, CBD, CBN, THC, CBC and THCA, in medicinal cannabis biomass and resin obtained by super-critical fluid extraction (SFE). The method was validated for specificity, linearity, limit of detection (LOD), limit of quantitation (LOQ), precision, accuracy, robustness, spike recovery and stability in accordance with the Validation of Analytical Procedures: Text and Methodology Q2 to meet the requirements of the International Council for Harmonisation (ICH), Therapeutic Goods Authority (TGA) and the Food and Drug Administration (FDA) test method validation regulations.

Analytical quality by design: Development and control strategy for a LC method to evaluate the cannabinoids content in cannabis olive oil extracts

Cannabidiol (CBD) and Δ9-tetrahydrocannabinol (Δ9-THC) are considered as the most interesting cannabinoids in Cannabis sativa L. for the clinical practice. Since 2013, the Italian law allows pharmacists to prepare and dispense cannabis extracts to patients under medical prescription, and requires the evaluation of CBD and Δ9-THC content in cannabis extracts before sale. Cannabis olive oil extracts are prepared from dried female cannabis inflorescences, but a standard protocol is still missing. In this study, a fast RP-HPLC/UV method has been developed to quantify CBD and Δ9-THC in cannabis olive oil extracts. The analytical quality by design strategy has been applied to the method development, setting critical resolution and total analysis time as critical method attributes (CMAs), and selecting column temperature, buffer pH and flow rate as critical method parameters. Information from Doehlert Design in response surface methodology combined to Monte-Carlo simulations led to draw the risk of failure maps and to identify the method operable design region. The method was validated according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines and then implemented in routine analysis. A control strategy based on system control charts was planned to monitor the developed method performances. Evaluation data were recorded over a period of one year of routine use, and both the CMAs showed values within the specifications in every analysis performed. Hence, a new risk evaluation for the future performances of the method was achieved by using a Bayesian approach based on the routine use data, computing the future distribution of the two CMAs. Finally, a study focusing on the monitoring of CBD and Δ9-THC concentrations in cannabis olive oil extracts was carried out. The developed method was applied to 459 extracts. The statistical analysis of the obtained results highlighted a wide variability in terms of concentrations among different samples from the same starting typology of cannabis, underlining the compelling need of a standardised procedure to harmonise the preparation of the extracts.

Corrigendum: Potential Clinical Benefits of CBD-Rich Cannabis Extracts Over Purified CBD in Treatment-Resistant Epilepsy: Observational Data Meta-analysis.

[This corrects the article DOI: 10.3389/fneur.2018.00759.].

Análise crítica sobre edulcorantes e flavorizantes empregados na correção sensorial de extratos de Cannabis para uso pediátrico

O tratamento terapêutico com extratos de cannabis representa, por vezes, a única alternativa no controle da epilepsia refratária infantil. Cannabis sativa é uma planta proibida no Brasil, mas a importação de extratos de cannabis foi autorizada para uso médico em 2015. Entretanto, estas formulações não são específicas para uso infantil comprometendo a adesão ao tratamento devido à palatabilidade. Este estudo objetivou realizar uma análise crítica dos edulcorantes e flavorizantes usados na formulações de extratos de cannabis registrados como suplementos alimentares e empregados no Brasil no controle de epilepsia refratária na população pediátrica. Foram estudadas as composições dos extratos disponíveis no mercado e realizadas buscas em bases de dados e sites de agências reguladoras. Dentre as alternativas disponíveis no mercado, apenas uma estava adequada, porque, incorporava um flavorizante de elevada aceitação, além de glicerina, que mascara o sabor desagradável do óleo de cânhamo. Foram sugeridas formulações sem restrições ao tipo de óleo incorporado, ausentes de edulcorantes e com pelo menos uma estratégia de mascaramento de sabor, como a incorporação de glicerina ou a incorporação de flavorizantes. Estas propostas podem guiar a elaboração de extratos que aumentem a adesão infantil a terapia.

Manejo del dolor crónico con extracto de Cannabis, en un grupo de pacientes de la ciudad de Manizales

El dolor crónico es un problema de salud pública. En la última década se han introducido diversos fármacos para el manejo de diversos tipos de dolor, y entre ellos se cuenta con los cannabinoides. Materiales y métodos: en el presente estudio se evaluó el manejo del dolor crónico de un grupo de pacientes de la ciudad de Manizales, mediante cinco mezclas diferentes de Δ9-Tetrahidro cannabinol (Δ9-THC) y cannabidiol (CBD). Resultados: En promedio, los valores de la escala numérica del dolor (1-10), antes de la exposición a las cinco concentraciones de extracto de cannabis fue de: 7,33; 7,75; 7,33; 7,94 y 7,33 respectivamente; posteriormente los valores fueron de 3,50; 4,40; 4,00; 4,72 y 3,17 respectivamente. Conclusión: Puede concluirse que se observó una disminución en el nivel de dolor mediante el uso de extractos a base de cannabis, observándose un efecto similar entre los diferentes extractos utilizados.

Occupational cannabis exposure and allergy risks

ObjectivesCannabis allergy has mainly been described following recreational use but some cases also point to cannabis sensitisation as a result of occupational exposure. As a consequence, little is known on...

Co-administration of nicotine ameliorates cannabis-induced behavioral deficits in normal rats: role of oxidative stress and inflammation

Nicotine (Nic) and cannabis are considered to be the most abused drugs worldwide that are progressively taken concomitantly. The present study aimed to investigate the modulatory effect of Nic on cannabis extract–induced neuro-inflammation, oxidative status, and the associated behavioral/biochemical alterations. Nic (0.25 mg/kg) and/or cannabis extract expressed as ∆9-tetrahydrocannabinol (THC10/20; 10 and 20 mg/kg) were given intraperitoneally for 30 days to Wistar rats. Nic shortened the floating time in forced swimming test, increased locomotion in the open field test, and decreased escape latency in the Morris water maze when co-administered with THC. These effects were associated with the inhibition of THC-mediated elevations in brain interleukin-1 beta, lipid peroxidation, superoxide dismutase, and ascorbic acid. Additionally, Nic increased serum butyrylcholinesterase (BChE) when combined with THC without affecting the serum acetylcholinesterase enzyme. The combinations spiked the brain glucose content above normal. In conclusion, the co-administration of Nic reduced THC-induced depressive-like behavior and memory impairment as well as hypo-locomotion associated with THC20. Such effects could be linked to Nic-mediated inhibition of brain oxidative stress, inflammation, and decreased serum BChE deactivity.

Treatment of infantile spasms.

SummaryThe treatment of infantile spasms is challenging, especially in the context of the following: (1) a severe phenotype with high morbidity and mortality; (2) the urgency of diagnosis and successful early response to therapy; and (3) the paucity of effective, safe, and well‐tolerated therapies. Even after initially successful treatment, relapse risk is substantial and the most effective therapies pose considerable risk with long‐term administration. In evaluating any treatment for infantile spasms, the key short‐term outcome measure is freedom from both epileptic spasms and hypsarrhythmia. In contrast, the most important long‐term outcomes are enduring seizure‐freedom and measures of intellectual performance in later childhood and adulthood. First‐line treatment options—namely hormonal therapy and vigabatrin—display moderate to high efficacy but also exhibit substantial side‐effect burdens. Data on efficacy and safety of each class of therapy, as well as the combination of these therapies, are reviewed in detail. Specific hormonal therapies (adrenocorticotropic hormone and various corticosteroids) are contrasted. Those etiologies that prompt specific therapies are reviewed briefly, as are an array of second‐line therapies supported by less‐compelling data. The ketogenic diet is discussed in greater detail, with a focus on the limitations of numerous available studies that generally suggest that it is efficacious. Special discussion is allocated to cannabidiol—the investigational therapy that has received the most attention, and which is already in use in the form of various artisanal cannabis extracts. Finally, a treatment algorithm reflecting the concepts and controversies discussed in this review is presented.