KROKODIL: THE RESURFACE OF AN OLD DRUG

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Street-like synthesis, product chemical analysis and

toxicological evaluation

Emanuele Amorim Alves

TESE DE DOUTORAMENTO APRESENTADA À FACULDADE DE MEDICINA DA UNIVERSIDADE DO PORTO EM CIÊNCIAS FORENSES

Porto, 2017

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Dissertação de candidatura ao grau de Doutor em Ciências Forenses apresentada

à Faculdade de Medicina da Universidade do Porto

Dissertation thesis for the degree of Doctor of Philosophy in Forensic Sciences submitted to the Faculty of Medicine of Porto University

Orientador: Professor Doutor Ricardo Jorge Dinis Oliveira (Professor Auxiliar com Agregação da Faculdade de Medicina da Universidade do Porto);

Co-orientador: Professor Doutor Félix Dias Carvalho (Professor Catedrático da Faculdade de Farmácia da Universidade do Porto);

Co-orientador: Professor Doutor Annibal Duarte Pereira Netto (Professor Titular do Departamento de Química Analítica do Instituto de Química da Universidade Federal Fluminense).

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Aos meus Avós, Germano e Sebastiana (in memorian).

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AUTHOR’S DECLARATION

Under the terms of the Decree-Law nº 74/2006, of March 24th, it is hereby declared that the following original articles were prepared in the scope of this dissertation.

PUBLICATIONS

Articles in international peer-reviewed journals

Theoretical Background

I. Alves EA, Grund JPC, Afonso CM, Netto ADP, Carvalho F, Dinis-Oliveira RJ. The Harmful Chemistry Behind Krokodil (desomorphine) synthesis and mechanism of toxicity. Forensic Science International. 249: 207-213, 2015.

Original Research

II. Alves EA, Brandão P, Magalhães T, Carvalho F, Dinis-Oliveira RJ. Fatal Intoxications in the North of Portugal: 12 years of Retrospective Analysis. Current Drug Safety. 12:39-45, 2017.

III. Alves EA, Soares JX, Afonso CM, Grund JPC, Agonia AS, Cravo SM, Netto ADP, Carvalho F, Dinis-Oliveira RJ. The Harmful Chemistry Behind Krokodil: Street-like synthesis ans Product Analysis. Forensic Science International. 257: 76-82, 2015. IV. Alves EA, Agonia AS, Cravo SM, Afonso CM, Netto ADP, Bastos ML, Carvalho F,

Dinis-Oliveira RJ. GC-MS Method for the Analysis of Thirteen Opioids, Cocaine and Cocaethylene in Whole Blood Based on Modified QuEChERS Extraction. Current

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V. Neves JF, Alves EA, Soares JX, Cravo SM, Silva AMS, Netto ADP, Carvalho F, Dinis-Oliveira RJ, Afonso CM. Data Analysis of “Krokodil” Samples Obtained by Street-like Synthesis. Data in Brief. 6:83-88, 2016.

VI. Alves EA, Brandão P, Neves JF, Cravo SM, Soares JX, Duarte JA, Grund JPC, Afonso CM, Netto ADP, Carvalho F, Dinis-Oliveira RJ. Repeated subcutaneous administrations of Krokodil causes skin necrosis and internal organs toxicity in Wistar rats. Human Psychopharmacology: Clinical and Experimental. In press, 2017. DOI: 10.1002/hup.2572.

V. Soares JX, Alves EA, Silva AMN, Figueiredo NG, Cravo SM, Rangel M, Netto ADP, Carvalho F, Dinis-Oliveira RJ, Afonso CM. The street-like synthesis of krokodil results in the formation of an enlarged cluster of known and new morphinans. Submitted, 2017.

Abstracts in international peer-reviewed journals

Original Research

I. Alves EA, Agonia AS, Cravo SM, Afonso CM, Netto ADP, Bastos ML, Carvalho F, Dinis-Oliveira RJ. Validation of a Modified QuEChERS Extraction/GC-MS Methodology for Quantification of Drugs of Abuse in Human Samples.

Toxicology Letters. 238 (Suppl. 2): S376, 2015.

II. Alves EA, Brandão P, Neves JF, Cravo SM, Soares JX, Duarte JA, Grund JPC, Otiashvilli D, Afonso CM, Netto ADP, Carvalho F, Dinis-Oliveira RJ. Short-term Toxicodynamics of Krokodil in Wistar Rats Following Repeated Subcutaneous Administration. Toxicology Letters. 258 (Suppl. 2): S130, 2016.

III. Alves EA, Soares JX, Silva AM, Neves JF, Cravo SM, Silva AM, Rangel M, Afonso CM, Netto ADP, Carvalho F, Dinis-Oliveira RJ. Identification of a Complex Mixture of Opioids on Krokodil Street-like Samples. Toxicology

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ix Under the terms of the referred Decree-Law, the author declares that she afforded a major contribution to the conceptual design and technical execution of the work, interpretation of the results and manuscript preparation of the published articles included in this thesis.

The candidate performed the experimental work with a doctoral fellowship (245844/2012-0) supported by the “Conselho Nacional de Pesquisa e Tecnologia - CNPq”, which also participate with grants to attend in international meetings and for the graphical execution of this thesis. The Faculty of Pharmacy of the University of Porto (Portugal) provided the facilities and logistic support.

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ACKNOWLEDGMENTS

A PhD is not a lonely journey, but one full of pain, joy, surprises and new discoveries. During my working time, lots of people helped me, part of them became part of my life, other helped me to be a better human being and a better scientist. I would like to show my gratitude to all of them. I may have forgotten names but they know how important they are to me. THANK YOU!!

At first, I would like to thank to my Supervisor, Prof. Ricardo Dinis for his orientation, guidance and big smile. Thanks for believe in this crazy and energic girl who appeared in the middle of a Master presentation to ask him if he would like to be her supervisor. I can say all the words in the world to express how I enjoyed being his student but it will be not enough. With him I learn how to be a better student, better researcher and better person.

I have no words to express how big is my gratitude to Prof. Annibal Duarte, my co-supervisor. Without his guidance, I would probably not be able to achieve such accomplishment. He knows me since I was an undergraduate student and he was by my side in the worst moment of my life, helping me to not give up. His intelligence, knowledge, professionalism always coupled to a big heart, which I was privileged to know. He always had a word of strength during all the moments I thought I was not able to finish this thesis. Thanks for being professor, friend and almost like a father. I hope to continue working with him during my next scientific journey.

I would like to extend my gratitude to Prof. Félix Carvalho, my co-supervisor, for helping and for his amazing ideas and contribution to this work. All the opportunities, the ideas and the support during my thesis process will never be forgotten.

I would like to thank to Prof. Carlos Afonso, my “almost co-supervisor”, for the support, ideas and amazing moments of discussion about our beautiful organic chemistry. His giant knowledge is only not bigger than his heart. He introduced me to his team, he gave me a place to work under his guidance, and he was always available to discuss

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xi results, chemistry, life, philosophy and “beattles”. He embraced my work as his own and gave me wings to fly. I will never forget him and I hope I will have the chance to keep working with him in the future.

I am very grateful to Dr. Jean Paul Grund for his contribution. Without him I think it would have been impossible to comprehend the nature of the street synthesis of krokodil. I liked his approach and kindness since we met at the airport. Thanks for believing in me and in my work. It was such a joy to find a truly friend in him. I hope to continue our partnership for a long time. Thanks for all the support and friendly words in moments that I really lost my faith.

There is no doubt that the synthesis process of krokodil is at the core of this dissertation. The Folk Chemistry drug synthesis described in this thesis was primarily informed by and based on a meticulous ethnographic video observation of the production of krokodil in Tbilisi, Georgia. Produced by Dr. David Otiashivilli and his colleagues from ACESO and Alternative Georgia (Tbilisi, Georgia) in collaboration with local people who use drugs, this video was core to accomplishing my main research objectives, as it allowed for the accurate replication of the drug's synthesis in our laboratory. I am therefore truly and deeply grateful to Dr. Otiashivilli and his team and to the people featuring in the video, who generously allowed to film them during these intimate activities and agreed to use of the video for scientific and public health purposes. Without their crucial contribution, this thesis would not have seen the light of day.

I would like to thank to Dr. Teresa Magalhães, coordinator of the PhD program in Forensic Sciences from FMUP, for believe in me and give me the opportunity to be her student. All the kind and encouraging words will be forever in my heart.

I am thankful to Ms. Amélia Castro and Ms. Maria João Alves for all the attention, kidness and consideration that both gave to me during my work.

I am grateful to Professor José Alberto Duarte from CIAFEL. Without his help and guidance through the histologic world I could not be able to finish this work. I also would like to thank to Ms. Celeste Resende’s contribution.

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I’m gratified to Professor Artur Silva for his collaboration with the Nuclear Magnetic Resonance (NMR) analyzes.

I acknowledge “Conselho Nacional de Pesquisa (CNPq)” for my doctoral fellowship (245844/201-0) and for the financial support of this dissertation.

I acknowledge “REQUIMTE”, associated laboratory, for the financial support of the laboratory work.

To Sara Cravo who without her help, friendship and partnership this thesis would not be possible. Thanks for all the discussion with a hot tea, all the ideas and support, especially concerned about the GC-MS. She is not just a colleague but a true friend that I am thankful to entered into my life.

To Gisela Adriano, the amazing lab worker. She inspired me to be a better person and a better scientist. In her I found someone to trust and admire.

To Pedro Brandão, my partner in “crime”. Without him my lab work would be miserable and sad. He brought the happiness to my PhD. His help and partnership put me up in so many times I was sad that I can’t even count. Thanks for believe in me, be my friend and help me when nobody wanted to. I am so blessed to have friends like him.

To José Soares for his contribution in the beginning of the work and with the Orbitrap analysis.

To Ana Sofia Agonia, my first Master student, my friend and partner. Thanks for all the help in the process of validation of QuEChERS methodology. Her dedication was inspiring. It was a great honor to be her Master’s co-supervisor.

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xiii To Cátia Faria for her prompt support whenever I need it. For all the cookies, the supporting words and smiles. I can’t thank her enough.

To Ana Margarida, my in vivo assays partner. She gave me the guidance and helped me to be more confident with my laboratory animals. Without her, the experiments would be impossible. Thanks for being my friend and to teach me how to deal with the animals and to take care of them.

To my friends from the Toxicology Department, Maria João Valente, Renata Silva and Catarina Chaves. Thanks for the friendship, lunch times and smiles. Of course, I have to thank them for all the help! They are amazing!

To my friend and partner Juliana Garcia for helping me and teaching me so much about the in vivo assays and toxicological experiments. Also, she is a friend to keep, an amazing person and the sweetest doctor that I ever meet. I am very proud to be her friend.

To my second Master student, Mariana Soares, for all the help with the toxicity assays, especially at moments when I had a lot of plaques to analyze. Thanks for her supporting words and for trusting me to be her Master’s co-supervisor.

To André Silva for all the help and knowledge he shared about LC-MS and Orbitrap.

To my Portuguese friends who became my family during this amazing journey, João Duarte, Frederico Mattos and Albino Gomes. They make me feel so warm and loved. In special to Fátima Quadros who was my safety harbor, my sister and my confident. Love her!

To my CESPU friends Joana Barbosa and Juliana Faria for all the kidness and friendship. I love to work with them!

To my friends in Brazil that never forgot me even with an ocean between us, Simone Mendes, Cristina Stork, Erika Guimarães, Beatriz Benjamin, Nel Gonçalves and Adriana Honorato. I love them.

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To my boss, friend and confident, Etelcia Molinaro. Only her to believe in me, to allow me to think about a PhD. Thanks for all the words, all the love and all the support that I needed to leave my life, my family and friends and jump into my dream. She is much more than a boss, she is the best boss I ever had, the best travel partner, the best friend.

To my fiancé, Daniel Lopes, for all the unconditional, unmeasurable and unbelievieble love. He is a light in my life, my hope for an amazing future. He is my romantic movie with the most beautiful end. I love him so much!

To my brothers, Carlos Germano and Luiz Carlos, who always encouraged me and loved me truly.

To my sisters-in-law, Rose Balbino and Amanda Indaia, for all the love and support. They are sisters from another mother.

To my Mom, Carmen Andrade, who believed me, encouraged me and gave me the strength to never give up. I am so proud to be her daughter. I love her deeply.

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ABSTRACT

Opioids have a major role in fatal intoxications in Europe. Despite the rehabilitation programs for opioids abuse and the policy of user’s decriminalization adopted by the Portuguese government, the use of opioids has been increasing, especially methadone. After a study by our group where the fatal intoxications profile in Portugal over 12 years were analyzed, we realized that fatal intoxications are a topic of great relevance in today’s society and the opioids still have to be considered. In that work, we analyzed postmortem forensic medical reports with positive results for the presence of xenobiotics. Cases of fatal intoxications involving opioids come on fifth place, namely due to accidental overdoses.

“Krokodil” is the street name for the homemade injectable mixture that has been used as a cheap substitute for heroin. Its use begun in Russia and Ukraine and actually is being spread over several other countries. Desomorphine is the semi-synthetic opioid claimed to be the main component of krokodil and considered to be responsible for its addictive and psychoactive characteristics. Due the fast spreading of krokodil trough Europe, it is highly likely that it is just a matter of time until this drug becomes a widespread problem. Thinking about this possibility, the initial part of this thesis is based on the study of krokodil and the development and validation of methodologies to detect this drug as a crude sample or in biological samples antemortem and postmortem. For this purpose, our group developed a street-like synthesis to obtain krokodil samples that most resemble to the street samples in Russia, Ukraine and Georgia.

Krokodil is a homemade drug usually prepared by people who inject drugs (PWID). The starting materials for krokodil synthesis are codeine tablets, alkali solutions, organic solvent, acidified water, iodine and red phosphorus, all of which are easily available in retail outlets, such as supermarkets and drugstores. The resulting product is a light brown liquid that is called krokodil. In the initial part of the thesis, we aimed to understand the chemistry behind “krokodil” synthesis by mimicking the steps in its street synthesis followed by PWID. The successful synthesis was confirmed by the presence of desomorphine and two additional morphinans. Subsequentely, we developed and validated an analytical gas chromatography-electron impact/mass spectrometry (GC-EI/MS) methodology for the quantification of desomorphine and codeine in the krokodil concoction. This approach yielded samples of krokodil representative of those observed

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xvii by field research and furthermore resulted in the development of an effective analytical methodology for the quantification of desomorphine, the major morphinan found in krokodil samples.

With reliable samples and a validated detection method available, we developed a methodology for the forensic analysis of biological samples. Forensic labs usually have low budgets and very short deadlines to work with. With this in mind, we modified an extraction method that is known to be cheaper and faster than the standard extraction methods, in order to apply it in this forensic context. QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) is a methodology previously developed to extract pesticides from vegetables and fruits and has been successfuly applied for different analytical approaches. In this part of the thesis, a rapid and less laborious modified QuEChERS extraction method for the quantification of 13 opioids [codeine, morphine, heroin, 6-acetylmorphine (6-AM), desomorphine, ethylmorphine, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP), papaverine, tramadol, O-desmetyltramadol (M1) and, tapentadol], cocaine and cocaethylene in whole blood, followed by gas chromatography-mass spectrometry, was developed and validated. The validated method was applied to whole blood samples collected from volunteers undergoing opioid rehabilitation treatment, and rats exposed to desomorphine, tramadol and tapentadol. The developed method could provide a rapid, effective and “greener” process for the determination of a wide range of opioid drugs in whole blood samples and can be applied at clinical and forensic antemortem and

postmortem analysis.

When people who inject krokodil (PWIK) arrive at the emergence services often present a great variety of serious morbidity signs and symptoms, including thrombophlebitis, ulcerations, gangrene, necrosis and internal organ damage. It has been suggested that these effects result from the toxic components used in or produced in the drug’s rudimentary synthesis in makeshift laboratories, in people’s kitchen or basements. Pharmaceutically produced desomorphine has not been associated with these injuries observed in people who inject krokodil, but information on the mechanisms of toxicity of krokodil is lacking. To understand these mechanisms, we conducted an in vivo assay using Wistar male rats as experimental model. Animals were divided into seven groups and exposed subcutaneously to NaCl 0.9% (control group), krokodil mixture free of psychotropic content (blank krokodil group), pharmaceutical-grade desomorphine 1 mg/Kg and four different concentrations of krokodil synthetized following a “domestic”

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protocol (1, 0.5, 0.25 and 0.12 mg/Kg). The injections were performed one per day during five consecutive days. Animals were monitored daily and euthanized 24 hours after the last administration. Biochemical and histological analysis were carried out. The obtained results showed that the continuous use of krokodil may cause injury at the injection area, with formation of necrotic zones. The biochemical results evidenced alterations on cardiac and renal biomarkers of toxicity, namely CK, CK-MB and uric acid. Significant alteration in kidney and heart levels of reduced and oxidized glutathione suggested that oxidative stress may be involved in krokodil mediated toxicity. Although urinary biomarkers such as N-acetyl-β-glucosaminidase evidenced slight alterations, histological analysis revealed only mild alterations. Cardiac congestion was the most relevant finding of continuous krokodil administration. High phosphorous concentration was responsible for the presence of a Kidney Chronic Disease like Syndrome (CKD-like) which result in a descompensation of the calcium levels, which affected the heart. The present findings, contribute notably to comprehension of the local and systemic toxicological impact of this complex drug mixture on major organs, and will hopefully be useful for the development of appropriate treatment strategies towards the toxicological effects of krokodil.

Due to its homemade character, krokodil is composed of a large and complex mixture of different substances, namely other opioids, which may synergistically enhance the effects of desomorphine. In addition, we confirmed the important role in krokodil’s toxicity of phosphorous, one of the reactants used by PWIK. In order to better understand the chemical complexity, krokodil was analyzed by HPLC-DAD-ESI-MS/MS with LTQ Orbitrap XL analyzer. By making use of exact mass, at least 34 morphinan derivatives were found, including morphine, not previously described. Moreover, liquid and solid state 31P-NMR were also employed in order to study the inorganic and organic natures of the mixture. Phosphorous species such as phosphoric acid and phosphates were identified as important components of the mechanism of synthesis as previous described. Comprehension of krokodil’s chemical composition is an important step towards effective prophylactic and therapeutic measures.

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xix In conclusion, the results of this thesis suggest that the toxicity of krokodil is the consequence of both the very high acidity and the high concentration of phosphorous, which is injected intravenously. Ulcerations and gangrenous limbs are directly connected to the degradation of the tissue around the injection place. The high phosphorus content is probably responsible for the main internal organs toxicity such jaw osteonecrosis and renal and cardiac damage. Different species of organophosphate and phosphorous species present in the krokodil mixture may may be responsible for the cognitive symptoms observed in PWIK, as this group of substances is known as an inhibitor of the enzyme acetylcholinesterase.

We hope that our results will support clinicians and harm reduction workers to better treat and care for people who inject krokodil and other homemade drugs.

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RESUMO

Os opioides têm um papel importante nos casos de intoxicações fatais na Europa. Apesar da existência de programas de reabilitação para usuários de opioides e uma política de descriminalização adotada pelo governo Português, o uso de opioides tem vindo a crescer, especialmente a metadona. No decurso de um estudo do nosso grupo onde foi analisado o perfil das intoxicações fatais em Portugal durante 12 anos, percebemos que as intoxicações fatais são um tema de grande relevância na sociedade de hoje, nas quais os opioides ocupam um lugar de destaque. Neste estudo foram analisados os relatórios médicos forenses de necrópsias com resultados positivos para a presença de xenobióticos. Casos de intoxicações fatais envolvendo os opioides aparecem em quinto lugar, normalmente devido a sobredosagens acidentais.

"Krokodil" é o nome de rua para a mistura injectável caseira que tem sido utilizada como um substituto mais barato para a heroína. O seu uso começou na Rússia e na Ucrânia, e de fato vem-se espalhando por vários outros países. A desomorfina é o opiáceo semi-sintético conhecido por ser o principal componente do krokodil e considerado responsável por suas características viciantes e psicoativas. Devido à rápida propagação de krokodil na Europa, é uma questão de tempo até que torne num problema generalizado. Pensando sobre esta possibilidade, a parte inicial desta tese baseou-se no estudo do krokodil objetivando o desenvolvimento e a validação de metodologias para sua detecção, tanto como amostra bruta como em amostras biológicas antemortem e postmortem. Com esse fim, nosso grupo procedeu a uma síntese de rua para a obtenção de amostras de krokodil que fossem o mais representativas quanto possível de amostras produzidas por usuários na Rússia, Ucrânia e Geórgia.

O krokodil é uma droga caseira geralmente preparada pelos próprios usuários. Os materiais de partida para a sua síntese são comprimidos de codeína, soluções alcalinas, solvente orgânico, água acidificada, iodo e fósforo, todos facilmente disponíveis em supermercados e drogarias. O produto resultante é um líquido castanho claro que é chamado de krokodil. Nesta parte inicial da tese, o objetivo era compreender a química envolvida na síntese do krokodil imitando os passos seguidos pelos próprios usuários. O sucesso da síntese realizada pelo grupo foi confirmada pela presença de desomorfina e outros dois morfinanos. Subsequentemente, desenvolvemos e validamos uma metodologia baseada em Cromatografia Gasosa acoplada à Espectrometria de Massas

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(CG-EM) para a determinação e quantificação de desomorfina e codeína na mistura do krokodil. Esta metodologia validou a veracidade da amostra sintetizada sendo esta representativa de amostras de krokodil apreendidas na rua e, além disso, resultou no desenvolvimento de uma metodologia efetiva para a quantificação da desomorfina, o principal morfinano encontrado nas amostras de krokodil.

Com amostras representativas e um método validado, desenvolvemos e validamos uma metodologia para a análise forense de amostras biológicas. Os laboratórios forenses geralmente possuem baixos orçamentos e prazos muito limitados para a realização das análises. Com esta informação em mente, adaptou-se um método de extração conhecido por ser mais barato e rápido do que os métodos padrão para extração de amostras, com o objetivo de ser aplicado no contexto forense. QuEChERS (Rápido, Fácil, Barato, Eficaz, Robusto e Seguro) é uma metodologia previamente desenvolvida para extrair pesticidas de legumes e frutas e tem sido aplicada com sucesso em diferentes abordagens analíticas. Nesta parte da tese, o método de QuEChERS modificado foi desenvolvido para a quantificação de 13 opióides [codeína, morfina, heroína, 6-acetilmorfina (6-AM), desomorfina, etilmorfina, metadona, 2-etilideno-1, 5-dimetil-3,3-difenillpirrolidina (EDDP), 2-etil-5-metil-3,3-difenil-1-pirrolina (EMDP), papaverina, tramadol, O-desmetiltramadol (M1) e o tapentadol], cocaína e cocaetileno, em sangue total para posterior detecção por Cromatografia Gasosa acoplada à Espectrometria de Massas. O método validado foi aplicado a amostras de sangue total recolhido de voluntários submetidos a tratamento de reabilitação de opiáceos, e em ratos expostos a desomorfina, tramadol e tapentadol. O método desenvolvido mostrou ser um processo rápido, eficaz e mais "verde" para a análise de uma grande variedade de drogas e opioides em amostras de sangue total, podendo vir a ser aplicada a casos clínicos e forenses.

Pessoas que injetam krokodil, quando atendidas em urgências hospitalare, apresentam uma grande variedade de graves sinais e sintomas, incluindo tromboflebite, ulcerações, gangrena, necrose e danos em órgãos internos. Tem sido sugerido que estes efeitos resultam da produção rudimentar do krokodil em laboratórios caseiros, cozinhas e caves. A desomorfina farmaceuticamente produzida sozinha não está associada às lesões observadas nas pessoas que injetam krokodil, mas informação sobre os mecanismos de toxicidade do krokodil ainda é escassa. Para entender estes mecanismos foi realizado um ensaio in vivo utilizando ratos machos Wistar como modelo experimental. Os animais foram divididos em sete grupos e expostos por via subcutânea a NaCl 0,9% (grupo controle), a uma mistura krokodil livre de desomorfina (grupo

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xxiii krokodil branco), de desomorfina pura 1 mg / Kg e quatro concentrações diferentes de krokodil sintetizados seguindo o processo de rua previamente estudado, com concentrações de 1, 0,5, 0,25 e 0,12 mg/kg. As injecções foram realizadas diariamente durante cinco dias consecutivos. Os animais foram monitorados e sacrificados 24 horas após a última administração. Foram realizadas análises bioquímicas e histológicas. Os resultados obtidos mostraram que o uso contínuo de krokodil pode causar lesões na área de injecção, com a formação de zonas necróticas. Os resultados bioquímicos evidenciaram alterações em biomarcadores cardíacos e renais de toxicidade, tais como CK, CK-MB e ácido úrico. As alterações significativas nos níveis de glutationa reduzida e oxidada no rim e no coração sugerem que o stresse oxidativo pode estar envolvido na toxicidade do krokodil. Embora biomarcadores urinários, como N-acetil-β-glicosaminidase tenham indicado efeitos nefrotóxicos, a análise histológica revelou apenas alterações discretas. A congestão cardíaca foi o achado mais relevante da administração contínua do krokodil. Altos níveis de fósforo foram responsáveis por efeitos semelhantes ao da síndrome renal crónica (SRC) com descompensação dos níveis de cálcio sanguíneo, o que possivelmente afetou o coração. Estes resultados contribuirão significativamente para a compreensão do impacto toxicológico local e sistémico desta droga em órgãos importantes, e será útil para o estabelecimento de estratégias de tratamento adequadas tendo em vista os efeitos toxicológicos do kroodil.

Devido ao seu caráter caseiro, o krokodil é composto por uma mistura complexa de substâncias diferentes, incluindo outros opióides que podem sinergicamente ampliar os efeitos de desomorfina. Além disso, confirmamos o importante papel do fósforo, um dos reagentes usados na síntese, na toxicidade do krokodil. A fim de melhor compreender a complexidade química do krokodil, amostras sintetizadas por esta metodologia foram analisadas por HPLC-DAD-ESI-MS/MS com analisador LTQ Orbitrap XL. Fazendo uso da massa exata, pelo menos 34 derivados morfinanos foram encontrados, incluindo morfina, não descrita previamente. Além disso, a Ressonância Nuclear Magnética do Fósforo (31P-NMR) em estado líquido e em estado sólido foram também empregues para estudar as espécies orgânicas e inorgânicas contendo fósforo na mistura. Espécies de fósforo tais como ácido fosfórico e fosfatos foram identificadas como importantes componentes do mecanismo de síntese descrito anteriormente. Conhecer a composição química da krokodil foi é passo importante no avanço da implementação de medidas profiláticas e terapêuticas.

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Em conclusão, os resultados desta tese sugerem que a toxicidade do krokodil é uma consequência de tanto sua alta acidez como da elevada concentração de fósforo, injetados intravenosamente. Ulcerações e amputação de membros estão diretamente conectados à degradação dos tecidos ao redor do local de injeção. A alta concentração de fósforo no krokodil poderá ser responsável pela toxicidade sistêmica como a osteonecrose da mandíbula e os danos cardíacos e renais. Diferentes espécies de organofosforados e espécies de fósforo estão presentes dentro da mistura krokodil e podem estar relacionados aos sintomas cognitivos observados nas pessoas que injetam krokodil, já que este grupo de substâncias são conhecidas como inibidoras da enzima acetilcolinesterase.

Esperamos que os resultados deste trabalho possam auxiliar os médicos e equipes de resgaste e emergência, assim como equipes responsáveis no diminuição de riscos no uso de drogas, no maior entendimento e compreensão para o tratamento das pessoas que injetam krokodil e outras drogas com as mesmas características.

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Part I - General Introduction__________________________________________________

2

ABBREVIATIONS LIST

1_{H NMR, proton nuclear magnetic resonance} 31_{P-NMR, phosphorous nuclear magnetic resonance} 6-AM, 6-acetylmorphine

CK, creatinine kinase

CKD, chronic kidney disease CK-MB, creatinine kinase - MB DAD, diode-array

EDDP, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine EI, electron impact mode

EMA, European Medicines Agency

EMDP, 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline ER, emergency room

ESI, electrospray ionization

FTIR, Fourier transform infrared spectroscopy HI, hydriodic acid

HPLC, high performance liquid chromatography M1, O-desmethyltramadol

MS, mass spectrometry NaCl, sodium chloride

PWID, people who inject drugs PWIK, people who inject krokodil

QuEChERS, quick, easy, cheap, effective, rugged and safe TLC, thin layer chromatography

UV, ultraviolet Vis, visible

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OUTLINE OF THE THESIS

The present thesis is structured in four main parts:

PART I

1. GENERAL INTRODUCTION

In Part I, the present section, a general overview about krokodil is presented as a review paper.

2. GENERAL AND SPECIFIC OBJECTIVES OF THE THESIS

The general and specific objectives of the thesis are provided. PART II – ORIGINAL RESEARCH

The Part II is divided in six chapters, corresponding to the original articles published during the thesis development, and describes the experimental work in order to answer the questions that derived from the general and specific objectives of the thesis.

PART III

This section it is divided in three major points:

1. INTEGRATED OVERVIEW OF THE PERFORMED STUDIES – the studies undertaken are integrated in a harmonized form;

2. CONCLUSIONS - the conclusions that can be taken from this thesis are summarized;

3. DIRECTIONS FOR FUTURE RESEARCH – future studies are projected.

PART IV

The references used in the PART III are listed.

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AUTHOR’S DECLARATION ... VII PUBLICATIONS ... VII Articles in international peer-reviewed journals ... vii Abstracts in international peer-reviewed journals ... viii ACKNOWLEDGMENTS ... X ABSTRACT ... XVI RESUMO ... XXV ABBREVIATIONS LIST ... XXVI OUTLINE OF THE THESIS ... XXVIII TABLE OF CONTENTS ... XXX PART I - 1. GENERAL INTRODUCTION ... 1 REVIEW ARTICLE ... 3 PART I - 2. GENERAL AND SPECIFIC OBJECTIVES OF THE THESIS ... 13 PART II - 1. ORIGINAL RESEARCH ... 13 CHAPTER I ... 21 CHAPTER II ... 31 CHAPTER III ... 41 CHAPTER IV ... 53 CHAPTER V ... 61 CHAPTER VI ... 76 PART III - 1. INTEGRATED OVERVIEW OF THE PERFORMED STUDIES . 117 PART III – 2. CONCLUSIONS ... 127 PART III - 3. DIRECTIONS FOR FUTURE WORK ... 132 PART IV - 1. REFERENCES ... 136

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PART I

1. GENERAL INTRODUCTION

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__________________________________________________Part I - General Introduction

6

REVIEW ARTICLE

The harmful chemistry behind krokodil (desomorphine) synthesis and mechanisms of toxicity

Reprinted from Forensic Science International 249: 207–213

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_______________________________Part I – General and Specific Objectives of the Dissertation

2. GENERAL AND SPECIFIC OBJECTIVES OF THE DISSERTATION

PART I

2. GENERAL AND SPECIFIC OBJECTIVES

OF THE DISSERTATION

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The global aim of this thesis was to study the chemical composition of krokodil and its toxicity, to understand the role of their components on the toxicity presented by people who inject krokodil (PWIK).

It is expected that an increase of the knowledge in this field, resulting from this thesis, will provide data street-like synthesis, product chemical analysis and toxicological evaluation of krokodil. It is also expected that the evaluation of damaged organs and the extension of the effects by krokodil use can support the physicians working at Emergence Rooms (ER) of what is expect and which measures have to be taken, immediately or not, when a PWIK is admitted in a ER.

The hypothesis derived from these general objectives supported the specific goals of the original research corresponding to the six chapters of this thesis, as described bellow:

CHAPTER I

In this chapter, general aspects of opioids toxicity and its importance in Portugal were evaluated considering two aspects:

(i) Description of the prevalence of opioids in fatal intoxications in Portugal during 12 years (2001 to 2013);

(ii) Evaluation of the data of opioid addiction in Portugal.

CHAPTER II

Due the lack of cases of krokodil seizures in Portugal the need of krokodil samples to performe further studies was addressed following two objectives:

(i) The development of a successful methodology of street-like synthesis to prepare representative krokodil samples in the laboratory using the same procedures adopted by PWIK;

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(ii) The evaluation of the composition of these samples. With this purpose, an analytical methodology using GC-MS to detect and quantify desomorphine (the main active substance in krokodil) in krokodil street samples, was developed and validated in house.

CHAPTER III

In order to further proceed and improve the knowledge about the toxicological effects of krokodil and its main active compound (desomorphine), further analytical developments were necessary. Furthermore, other compounds were included in the analytical method development due the concomitant use of several drugs by addict persons (polydrug use). These methodologies involved the development of:

(i) A new modified QuEChERS extraction methodology to extract 13 opioids, including desomorphine, and also, cocaethylene and cocaine, from blood. The methodology was then adapted to be used in a variety of matrices;

(ii) The validation of a GC-MS methodology to analyze biological samples using the modified QuEChERS method as extraction process.

CHAPTER IV

The composition of krokodil samples was not previous described and part of its toxicity is believed to be related to by-products and reactants, especially phosphorous. Also, additional information concerning the physico-chemical characteristics of krokodil such as pH, color, odor and UV data was not available in literature. In order to fullfil this lack of information, the following study was implemented to evaluate:

(i) the physico-chemical characteristics of krokodil and additional profile studies such as 31P-MNR.

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CHAPTER V

This chapter dealt with the most important aspect regarding krokodil use and addicition. The study of its toxicological effects upon major organs, using Wistar male rats as an experimental model. This part of this study involved the study of:

(i) the short-term toxicity of krokodil in Wistar male rats;

(ii) oxidative stress, biochemical alterations and histological alterations following the administration of krokodil during five consecutive days.

CHAPTER VI

The investigation of the presence of other chemical compounds besides desomorphine in the complex mixture that krokodil represents was also studied using:

(i) a LC-MS/MS with Exact Mass characterization. This technique allowed the identification of other morphinans that occur in the krokodil mixture.

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____________________________________________________Part II – Original research

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PART II

1. ORIGINAL RESEARCH

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141 CHAPTER I

Fatal Intoxications in the North of Portugal: 12 Years of Retrospective Analysis

Reprinted from Current Drug Safety 12: 39-45

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GRAPHICAL ABSTRACT

Graphical abstract of Fatal Intoxications in the North of Portugal: 12 Years of Retrospective Analysis

22

Part I – General and Specific Objectives of the Dissertation_______________________________

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Outflow cannula inserted in the left ventricle. 2. Clamp to fix the outflow cannula. 3. Ligature placed around pulmonary and aortic arteries. 4. Inflow cannula placed just before the bifurcation of the pulmonar artery.

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CHAPTER II

The Harmful Chemistry Behind Krokodil: Street-like Synthesis and Product Analysis

Reprinted from Forensic Science International 257: 76-82

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Part II – Original research ____________________________________________________

Visual aspect of the homemade krokodil

Part II – Original research____________________________________________________

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CHAPTER III

GC-MS Method for the Analysis of Thirteen Opioids, Cocaine and Cocaethylene in Whole Blood Based Modified QuEChERS Extraction

Reprinted from Current Pharmaceutical Analysis 13: 215-223.

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Scheme of the experimental steps for QuEChERS extraction of samples

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CHAPTER IV

Data Analysis of Krokodil Samples Obtained by Street-like Synthesis

Reprinted from Data in Brief 6: 83-88

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CHAPTER V

Repeated Subcutaneous Administrations of Krokodil Causes Skin Necrosis and Internal Organs Toxicity in Wistar Rats: Putative Implications for Human Users

Reprinted from Human Psychopharmacology: Clinical and Experimental, In press DOI: 10.1002/hup.2572

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Part II – Original research ____________________________________________________ Part II – Original research____________________________________________________

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Part II – Part II – Original researchOriginal research ____________________________________________________ ____________________________________________________

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CHAPTER VI

The street-like synthesis of krokodil results in the formation of an enlarged cluster of known and new morphinans

Article Submitted Elsewhere

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Full Title: THE STREET-LIKE SYNTHESIS OF KROKODIL RESULTS IN THE FORMATION OF AN ENLARGED CLUSTER OF KNOWN AND NEW MORPHINANS

Running Head: “krokodil” synthesis and analysis

Authors’ names and institutional addresses:

José Xavier Soares5*†, Emanuele Amorim Alves1,2,3,4*†, André M. N. Silva6, Natália Guimarães de Figueiredo7, Sara Manuela Cravo8, Maria Rangel9, Annibal Duarte Pereira Netto10, Félix Carvalho1, Ricardo Jorge Dinis-Oliveira*1,2,4Ψ, Carlos Manuel Afonso*8,11Ψ

†_{The first two authors contributed equally to this work.} Ψ_{Co-principal investigators of this study.}

1_{LAQV, REQUIMTE, Department of Chemical Sciences, Laboratory of Applied} Chemistry, Faculty of Pharmacy, University of Porto.

2_{UCIBIO, REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences,} Faculty of Pharmacy, University of Porto, Porto, Portugal.

3_{Department of Legal Medicine and Forensic Sciences, Faculty of Medicine, University} of Porto, Porto, Portugal.

4_{EPSJV – Polytechnic School of Health Joaquim Venâncio, Oswaldo Cruz Foundation,} Rio de Janeiro, Brazil.

5_{IINFACTS - Institute of Research and Advanced Training in Health Sciences and} Technologies, Department of Sciences, University Institute of Health Sciences (IUCS), CESPU, CRL, Gandra, Portugal.

6_{REQUIMTE, UCIBIO, Department of Chemistry and Biochemistry, Faculty of} Sciences, University of Porto, Porto, Portugal.

7_{Laboratory of Tobacco and Derivatives, Analytical Chemistry Division, National} Institute of Technology, Rio de Janeiro, Brazil.

8_{Department of Chemical Sciences, Laboratory of Organic and Pharmaceutical} Chemistry, Faculty of Pharmacy, University of Porto, Porto, Portugal.

9_{LAQV, REQUIMTE, Institute of Science Abel Salazar, University of Porto, Porto,} Portugal.

10_{Department of Analytical Chemistry, Chemistry Institute, Fluminense Federal} University, Niterói, Brazil.

11_{Interdisciplinary} _Center _of _Marine _and _{Environmental} _{Investigation} (CIIMAR/CIMAR), Porto, Portugal.

*Corresponding authors:

____________________________________________________ Part II – Original research

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José Xavier Soares Email: jfxsoares@ff.up.pt

Emanuele Alves

Email: manuhpa@hotmail.com

Ricardo Jorge Dinis-Oliveira Email: ricardinis@med.up.pt

Carlos Afonso

Email: cafonso@ff.up.pt

Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto,

Porto, Portugal

Rua José Viterbo Ferreira nº 228 4050-313 Porto, Portugal

Fax: 00351 226093390 Phone: +351 220428597

ABSTRACT

“Krokodil” is the street name for a homemade injectable drug that has been used as a cheap substitute for heroin. Codeine is the opioid starting material for krokodil synthesis and desomorphine is the opioid claimed to be the main component of krokodil and the main responsible for its addictive and psychoactive characteristics. However, due to its peculiar manufacture, using cheap raw materials, krokodil is composed by a large and complex mixture of different substances. In order to shed some light upon the chemical complexity of krokodil, its profiling was conducted by reverse phase high pressure liquid chromatography coupled to photodiode array detector (RP-HPLC-DAD) and by liquid chromatography coupled to high resolution mass spectrometry (LC-ESI-IT-Orbitrap-MS). Besides desomorphine, codeine and morphine, profiting from the high resolution mass spectrometry data, an endeavor to study the morphinans content in krokodil was set for the first time. Considering codeine as the only morphinan precursor and the possible chemical transformations that can occur during krokodil synthesis, the morphinan chemical space was designed and 95 compounds were defined. By making use of the morphinan chemical space in krokodil, the exact masses featured by high resolution mass spectrometry, and the morphinan mass fragmentations patterns, a targeted identification approach was designed and implemented. The proposed 95 morphinans were searched using the full scan chromatogram of krokodil and findings were validated by mass fragmentation of the correspondent precursor ions (MS2 spectra). Following this effort,

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a total of other 42 morphinans were detected, highlighting the fact that these other morphinans may contribute to the psychotropic effects of krokodil.

Keywords: krokodil; opioids; morphinans; LC-ESI-IT-Orbitrap-MS; analgesia.

INTRODUCTION

“Krokodil” is a complex psychotropic drug mixture resulting from a homemade synthesis process using easy access starting materials and used as a cheap substitute for heroin (Alves et al., 2015a; Alves et al., 2015c; Baquero Escribano et al., 2016; Dinis-Oliveira et al., 2012; Florez et al., 2017; Grund et al., 2013; Hakobyan and Poghosyan, 2017; Katselou et al., 2014; Poghosyan et al., 2014). Krokodil firstly appeared around 2002/3 in Siberia and its use spread throughout other Russian areas and former Soviet Republic countries and more recently across Western Europe (Grund et al., 2013; Jolley et al., 2012; Piralishvili et al., 2014). Desomorphine is the semi-synthetic opioid claimed to be the main component of krokodil and considered to be responsible for its addictive and psychoactive characteristics. However, due to its nature, the synthesis produces not only desomorphine but also other side-products as previously described in krokodil samples and in syringes and biological fluids of PWIK (Alves et al., 2015a; Savchuk et al., 2008). Several toxic effects have been reported and claimed to be related to impurities, namely jaw osteonecrosis presenting as alveolar process exposure in the oral cavity, infections by HIV and hepatitis A, B, and C, skin and venous damage, including ulcers, scaly and rough phlebitis, like a crocodile skin around the injection sites, gangrene and limb amputations (Alves et al., 2015a; Alves et al., 2015c; Baquero Escribano et al., 2016; Dinis-Oliveira et al., 2012; Florez et al., 2017; Grund et al., 2013; Hakobyan and Poghosyan, 2017; Katselou et al., 2014; Poghosyan et al., 2014).

Clinical cases related to PWIK and also animals exposed to krokodil indicate high

tolerance to the pain induced by this corrosive and necrotizing formulation, having at the time pleasure with administration (Alves et al., 2017a). We therefore hypothesized that,

besides desomorphine, krokodil may uncover new morphinan derivatives capable of

relieving moderate and severe pain. For the purpose of identifying such derivatives, a suitable analytical methodology, capable of identifying complex mixtures is required.

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A great variety of separation techniques coupled to mass spectrometry (MS) have been used for the identification of different compounds in clinical and forensic toxicology due to its capacity to generate information both on the molecular mass and the structure of molecules (Alves et al., 2017b; Carey et al., 2013; Dunn and Allison, 2007; Gallidabino et al., 2011; Helfer et al., 2015; Nicdaeid et al., 2013; Thevis and Volmer, 2012; Williams et al., 2009). With the advent of high resolution mass spectrometry (HRMS), exact masses could be determined and new perspectives were opened for LC-HRMS allowing the study of complex mixtures. Since its first description (Makarov, 2000) and its commercial introduction in 2005, the Orbitrap mass analyzer has demonstrated great sensitivity and high resolving power (Makarov et al., 2006; Perry et al., 2008). This high-resolution mass analyzer is particularly attractive due to its great sensitivity and scan rate, which is ideal for online coupling with liquid chromatography separation. Indeed, coupling of liquid chromatography (LC) to Orbitrap mass spectrometers equipped with an electrospray ionization source (ESI) has been providing an analytical instrument of great utility in different areas of analytical and forensic chemistry (Ojanpera et al., 2012; Thevis and Volmer, 2012). Therefore, the use of LC-Orbitrap-MS data analysis with the aid of informatics tools, such as Mass Frontier® and Compound Discoverer®, may be considered of great value for the identification of substances in krokodil, namely unknown morphinan derivatives as a representative skeleton of a large class of opioids (Hellerbach et al., 1966). Indeed, clinical cases related to PWIK and also animals exposed to krokodil well the pain a corrosive and necrotizing formulation having at the time pleasure with administration (Alves et al., 2017a). We hypothesized that krokodil may uncover new therapeutic molecules to relief of moderate and severe pain. Therefore, considering the

above rationale, the goals of this work were to contribute a better knowledge of krokodil

providing new insights into the chemical profile and detecting the different morphinans present in krokodil. Morphinans were identified by comparison with reference standards (desomorphine, codeine and morphine) and by making use of exact masses and mass fragmentations provided by LC-ESI-IT-Orbitrap-MS.

MATERIAL AND METHODS

Reagents and standards

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For krokodil synthesis, gasoline, alkali solutions for cleaning pipes and matchboxes were purchased from local retail stores in Porto, Portugal. Hydrochloric acid 37% was purchased from VWR Prolabo®_{. Codeine-containing capsules, iodine tinctures, hydrogen} peroxide and commercial ethanol 96% were purchased from local pharmacies in Porto, Portugal. For High-Performance Liquid Chromatography (HPLC) analysis, Chromasolv acetonitrile and LC-MS grade formic acid were obtained from Sigma-Aldrich®. Ultrapure (MilliQ) water was used throughout the study.

Krokodil samples

All the krokodil samples were produced by street-like synthesis as previous described (Alves et al., 2015b). The synthesis procedure was repeated ten times, and a representative pool of the obtained products were used for further analysis.

Reversed phase high performance liquid chromatography with photodiode array detection

(RP-HPLC-DAD) conditions

For DAD analysis, a Dionex Ultimate 3000 HPLC Basic system (Thermo Scientific), equipped with an auto-sampler and a DAD detector was used. For chromatographic separation, a C18 Hypersil GOLD with 1.9 µm particle size, 50 mm L and 2.1 mm ID (Thermo Scientific) equipped with a C18 BEH VanGuard (5 mm L × 2.1 mmID) (Waters) guard column was used. Column temperature was set to 40ºC. Eluent A was aqueous formic acid (1%) and eluent B was formic acid (1%) in acetonitrile. Samples (10 µL) were injected directly into the column and washed over for 5 minutes with an isocratic flux of 98% eluent A and 2% eluent B at a flow rate of 300 µl/min. Subsequently to the initial 5 min wash, a 30 min linear gradient from 2% to 40% buffer B was applied. The eluent composition was then raised to 100% buffer B over 15 min and thus maintained for 10 additional min in order to allow column cleaning. The column was re-equilibrated in 98% buffer A/2% buffer B before any further analysis. DAD spectra were acquired for the full length of the chromatographic run between 200 and 600 nm. Chromeleon 7.1 SR2 software Thermo Fisher Scientific managed chromatographic data. Prior to use, mobile phase solvents were degassed in an ultrasonic bath for 15 min. The identification of desomorphine, codeine and morphine was established based on the comparison with ____________________________________________________ Part II – Original research