Tramadol and Codeine Stacking/Boosting Dose Exposure Induced Neurotoxic Behaviors, Oxidative Stress, Mitochondrial Dysfunction, and Neurotoxic Genes in Adolescent Mice.

In spite of the increasing epidemic of pharmaceutical opioids (codeine and tramadol) misuse and abuse among the adolescents, little is known about the neurotoxic consequences of the widespread practice of tramadol and codeine abuse involving increasing multiple doses across days, referred to as stacking and boosting. Hence, in this study, we replicated stacking and boosting doses of tramadol, codeine alone, or in combination on spontaneous motor activity and cognitive function in adolescent mice and adduced a plausible mechanism of possible neurotoxicity. Ninety-six adolescent mice were randomly distributed into 4 groups (n = 24 per group) and treated thrice daily for 9 days with vehicle, tramadol (20, 40, or 80 mg/kg), codeine (40, 80, or 160 mg/kg), or their combinations. Exposure of mice to tramadol induced hyperactivity and stereotypic behavior while codeine exposure caused hypoactivity and nootropic effect but tramadol-codeine cocktail led to marked reduction in spontaneous motor activity and cognitive function. In addition, tramadol, codeine, and their cocktail caused marked induction of nitroso-oxidative stress and inhibition of mitochondrial complex I activity in the prefrontal cortex (PFC) and midbrain (MB). Real-time PCR expression profiling of genes encoding neurotoxicity (RT) showed that tramadol exposure upregulate 57 and downregulate 16 neurotoxic genes, codeine upregulate 45 and downregulate 25 neurotoxic genes while tramadol-codeine cocktail upregulate 52 and downregulate 20 neurotoxic genes in the PFC. Findings from this study demonstrate that the exposure of adolescents mice to multiple and increasing doses of tramadol, codeine, or their cocktail lead to spontaneous motor coordination deficits indicative of neurotoxicity through induction of oxidative stress, inhibition of mitochondrial complex I activity and upregulation of neurotoxicity encoding genes in mice.

Codeine alters female reproductive function by targeting ovarian steroidogenesis and folliculogenesis via the induction of oxidative stress, inflammation, and apoptosis.

The rise in the abuse of codeine raises concerns about its impact on the health of users, and little has appeared on its effect on the female reproductive function. Therefore, this study evaluated the impact of codeine on female reproductive function. We administered codeine at low (2 mg/kg) and high (5 mg/kg) doses to female animals prior to mating for 8 weeks. In comparison with a vehicle-treated group, we then assessed the impact of codeine on body weight gain and ovarian weight, female sexual behaviour, ovarian steroidogenesis, and folliculogenesis. The role of oxidative stress, inflammation, and apoptosis were also evaluated. Codeine at either dose elicited a profound deficit in the absolute and relative ovarian weight, indicative of ovarian toxicity. Also, codeine induced female sexual dysfunction, and suppressed ovarian steroidogenesis and folliculogenesis, with degeneration of the ovarian cytoarchitecture and follicles. The effects of codeine were associated with a rise in ovarian hydroxyl radical generation and oxidative stress, evident by an increase in ovarian malondialdehyde, a reduction in reduced glutathione, and a decline in the activities of ovarian enzymatic antioxidants. In addition, codeine triggered an increase in the ovarian concentration of inflammatory cytokines, TNF-α and IL-1ß, and myeloperoxidase activity. Furthermore, codeine caused an increase in 8-hydroxydeoxyguanosine (8OHdG), ovarian DNA fragmentation, and caspase-3 activity, suggestive of genotoxicity and apoptosis respectively. The current study provides some of the first evidence for the adverse effects of prolong codeine use on female sexual function, ovarian steroidogenesis, and folliculogenesis. It also emphasizes the reproductive health consequences of drug abuse.

Zebrafish (Danio rerio) larvae show behavioral and embryonic development defects when exposed to opioids at embryo stage.

Opioid abuse continues to plague society, and in recent years, there has been an epidemic, leading to increased addiction and death. It is poorly understood how prenatal opioid use affects the lives of children. The aim of this work was to evaluate the effect of early embryonic codeine or morphine exposure in zebrafish (Danio rerio), examining gastrulation progression (epiboly), teratogenic effects, mortality and locomotor behavior response to light/dark cycles. Zebrafish embryos were exposed to codeine or morphine (designated C or M) at 1, 5 or 10 mg/L (designated 01, 05 or 10, respectively) from 3 to 24 h postfertilization (hpf) or from 3 to 48 hpf (designated -24 or - 48 for 1 or 2 days of exposure, respectively). The C10-24, C01-48, C05-48 and C10-48 groups showed significantly smaller eyes than control larvae at 7 days postfertilization (dpf). Locomotor behavior of control larvae in light/dark cycles showed greater swimming time and distance in dark cycles. Two-day codeine exposure produced strong effects, showing no significant response due to light/dark cycles in distance moved. Morphine exposed groups showed similar effects as observed in 2-day codeine exposed groups, showing less large movement activity and also no significant difference between inactive duration in response to light/dark cycles. In conclusion, we observed low teratogenic effects and mortality effects. Animals exposed to high levels and higher exposure times of opioids were hypoactive, relative to controls, in the dark period. Future studies will be needed to understand the neural defects producing behavior changes.

Codeine exerts cardiorenal injury via upregulation of adenine deaminase/xanthine oxidase and caspase 3 signaling.

AIMS: Codeine treatment has been shown to be associated with glucolipid deregulation, though data reporting this are inconsistent and the mechanisms are not well understood. Perturbation of glutathione-dependent antioxidant defense and adenosine deaminase (ADA)/xanthine oxidase (XO) signaling has been implicated in the pathogenesis of cardiometabolic disorders. We thus, hypothesized that depletion of glutathione contents and upregulation of ADA/XO are involved in codeine-induced glucolipid deregulation. The present study also investigated whether or not codeine administration would induce genotoxicity and apoptosis in cardiac and renal tissues. MATERIALS AND METHODS: Male New Zealand rabbits received per os distilled water or codeine, either in low dose (4 mg/kg) or high dose (10 mg/kg) for 6 weeks. KEY FINDINGS: Codeine treatment led to reduced absolute and relative cardiac and renal mass independent of body weight change, increased blood glucose, total cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL-C), as well as increased atherogenic indices and triglyceride-glucose index (TyG). Codeine administration significantly increased markers of cardiac and renal injury, as well as impaired cardiorenal functions. Codeine treatment also resulted in increased cardiac and renal malondialdehyde, Advanced Glycation Endproducts (AGE) and 8-hydroxydeoxyguanosine (8-OH-dG), and myeloperoxidase (MPO), ADA, XO, and caspase 3 activities. These observations were accompanied by impaired activities of cardiac and renal proton pumps. SIGNIFICANCE: Findings of this study demonstrate that upregulation of ADA/XO and caspase 3 signaling are, at least partly, contributory to the glucolipid deregulation and cardiorenal injury induced by codeine.

Codeine-induced sperm DNA damage is mediated predominantly by oxidative stress rather than apoptosis.

ABSTRACTBackground: Opioids have been implicated to induce infertility. Although codeine remains the most used opioid for recreational purpose, no study has documented its effect on sperm quality. Elucidating the effect of codeine on sperm cells and the associated mechanisms may provide an insight into preventing drug-induced sperm damage. Twenty-one New Zealand white rabbits were randomized into three groups; control and codeine-treated. The codeine-treated groups received either 4 or 10mg/kg b.w of codeine for six weeks.Results: Codeine treatment led to significant decrease in sperm count, motility, viability, normal morphology, and sperm membrane integrity. This was associated with significant rise in sperm DNA fragmentation, oxidative damage, and caspase 3 activity. The percentage of sperm DNA fragmentation correlates positively with 8-hydroxy-2'-deoxyguanosine, a biomarker of oxidative DNA damage, and caspase 3 activity, a biomarker of apoptosis. The observed correlation was stronger between sperm DNA fragmentation and oxidative DNA damage than sperm DNA fragmentation and caspase 3 activity.Conclusion: This study revealed that chronic codeine exposure causes sperm DNA fragmentation and poor sperm quality primarily via oxidative stress rather than activation of caspase 3-dependent apoptosis. Findings of the present study may explain drug-induced male factor infertility, particularly, those associated with opioid use.

Testicular toxicity following chronic codeine administration is via oxidative DNA damage and up-regulation of NO/TNF-α and caspase 3 activities.

BACKGROUND: Codeine, a 3-methylmorphine, and other related opioids have been implicated in androgen suppression, although the associated mechanisms remain unclear. AIM: Therefore, the objective of the current study was to elucidate the in vivo molecular mechanisms underlying codeine-induced androgen suppression. METHODS: This study made use of twenty-one healthy male rabbits, distributed into three groups randomly, control and codeine-treated groups. The control had 1ml of normal saline daily p.o. The codeine-treated groups received either 4mg/kg b.w of codeine or 10mg/kg b.w of codeine p.o. for six weeks. Reproductive hormonal profile, testicular weight, testicular enzymes, oxidative and inflammatory parameters, testicular DNA fragmentation, histological examination and apoptosis marker were evaluated to examine the effects of codeine use. KEY FINDINGS: Oral administration of codeine resulted in testicular atrophy and alterations in testicular histomorphology, elevated testicular enzymes, and suppression of circulatory and intra-testicular testosterone. These changes were associated with a marked rise in oxidative markers and decline in the activities of testicular enzymatic antioxidants, as well as oxidative DNA damage, inflammatory response, testicular DNA fragmentation, and caspase-dependent apoptosis (p<0.05). SIGNIFICANCE: In conclusion, chronic codeine use resulted in testicular degeneration and testosterone suppression, which is attributable to TNF-α/nitric oxide-/oxidative stress-mediated caspase-dependent apoptotic testicular cell death and loss of testicular function.

Assessment of sexual behaviour and fertility indices in male rabbits following chronic codeine use.

BACKGROUND: Codeine is the latest trend of drug abuse, particularly in Nigeria and regarded as the gateway to the abuse of other substances. OBJECTIVES: The present study examined the effects of graded doses of codeine on sexual behaviour and fertility profile. MATERIALS AND METHODS: Rabbits were either administered normal saline (0.2 mL), 4mg/kg b.w of codeine (low dose), or 10mg/kg b.w of codeine (high dose) p.o for 6 weeks. RESULTS AND DISCUSSION: Findings of the study showed that codeine administration significantly increased libido as witnessed by significantly short mount latency (ML), intromission latency (IL), post-ejaculatory interval (PEI) and significantly increased mount frequency (MF), intromission frequency (IF) and ejaculation latency (EL). Furthermore, codeine caused a marked rise in penile reflexes evident by a significant increase in erections, quick flips, long flips and total penile reflexes. However, copulatory efficiency and fertility index were significantly lower in codeine-treated groups when compared with the control. Serum levels of testosterone were also significantly lower in the treated groups. CONCLUSIONS: The present study demonstrates that codeine-induced enhancement of sexual performance is via a testosterone-independent mechanism. It also reveals that although codeine enhances copulatory locomotor activity, it is a potential risk factor for infertility.

Aquatic Phytotoxicity to Lemna minor of Three Commonly Used Drugs of Addiction in Australia.

The manufacturing and consumption of drugs of addiction has increased globally and their widespread occurrence in the environment is an emerging concern. This study evaluated the phytotoxicity of three compounds: methamphetamine, codeine and morphine; commonly reported in Australian urban water, to the aquatic plant Lemna minor under controlled conditions. L. minor was sensitive to lower drug concentrations when administered in multi-compound mixtures (100-500 µg L-1) than when applied individually (range 600-2500 µg L-1), while no adverse effects were observed at environmentally-relevant concentrations (1-5 µg L-1) detected in wastewater effluent. In conclusion, the results show that the concentrations of these compounds discharged into the environment are unlikely to pose adverse phytotoxic effects. These three compounds are known to be the most stable of their group under such conditions indicating that with this respect it is safe to use recycled water for existing regulated reclaimed purposes including agricultural or parklands irrigation or replenishing surface and groundwater. However, more research on the analysis of methamphetamines and opiates in municipal effluents is needed to reassure the likely environmental hazard of these neuroactive drug classes to aquatic organisms. Given the ever-growing production and aquatic disposal of discharge wastewater globally, this study provides timely and valuable insights into the likely drug-related impacts of effluent disposal on aquatic plants in receiving environments.

Metatox - Web application for generation of metabolic pathways and toxicity estimation.

Xenobiotics biotransformation in humans is a process of the chemical modifications, which may lead to the formation of toxic metabolites. The prediction of such metabolites is very important for drug development and ecotoxicology studies. We created the web-application MetaTox ( http://way2drug.com/mg ) for the generation of xenobiotics metabolic pathways in the human organism. For each generated metabolite, the estimations of the acute toxicity (based on GUSAR software prediction), organ-specific carcinogenicity and adverse effects (based on PASS software prediction) are performed. Generation of metabolites by MetaTox is based on the fragments datasets, which describe transformations of substrates structures to a metabolites structure. We added three new classes of biotransformation reactions: Dehydrogenation, Glutathionation, and Hydrolysis, and now metabolite generation for 15 most frequent classes of xenobiotic's biotransformation reactions are available. MetaTox calculates the probability of formation of generated metabolite - it is the integrated assessment of the biotransformation reactions probabilities and their sites using the algorithm of PASS ( http://way2drug.com/passonline ). The prediction accuracy estimated by the leave-one-out cross-validation (LOO-CV) procedure calculated separately for the probabilities of biotransformation reactions and their sites is about 0.9 on the average for all reactions.

Potential of the strain Raoultella sp. KDF8 for removal of analgesics.

The bacterial strain KDF8 capable of growth in the presence of diclofenac and codeine analgesics was obtained after chemical mutagenesis of nature isolates from polluted soils. The strain KDF8 was identified as Raoultella sp. based on its morphology, biochemical properties, and 16S rRNA gene sequence. It was deposited in the Czech Collection of Microorganisms under the number CCM 8678. A growing culture efficiently removed diclofenac (92% removal) and partially also codeine (about 30% degradation) from culture supernatants within 72 h at 28 °C. The degradation of six analgesics by the whole cell catalyst was investigated in detail. The maximum degradation of diclofenac (91%) by the catalyst was achieved at pHINI of 7 (1 g/L diclofenac). The specific removal rate at high concentrations of diclofenac and codeine increased up to 16.5 mg/gCDW per h and 5.1 mg/gCDW per h, respectively. HPLC analysis identified 4'-hydroxydiclofenac as a major metabolite of diclofenac transformation and 14-hydroxycodeinone as codeine transformation product. The analgesics ibuprofen and ketoprofen were also removed, albeit to a lower extent of 3.2 and 2.0 mg/gCDW per h, respectively. Naproxen and mefenamic acid were not degraded.

Repeated subcutaneous administrations of krokodil causes skin necrosis and internal organs toxicity in Wistar rats: putative human implications.

OBJECTIVE: "Krokodil" is the street name for an impure homemade drug mixture used as a cheap substitute for heroin, containing desomorphine as the main opioid. Abscesses, gangrene, thrombophlebitis, limb ulceration and amputations, jaw osteonecrosis, skin discoloration, ulcers, skin infections, and bleeding are some of the typical reported signs in humans. This study aimed to understand the toxicity of krokodil using Wistar male rats as experimental model. METHODS: Animals were divided into seven groups and exposed subcutaneously to NaCl 0.9% (control), krokodil mixture free of psychotropic substances (blank krokodil), pharmaceutical grade desomorphine 1 mg/kg, and four different concentrations of krokodil (containing 0.125, 0.25, 0.5, and 1 mg/kg of desomorphine) synthesized accordingly to a "domestic" protocol followed by people who inject krokodil (PWIK). Daily injections for five consecutive days were performed, and animals were sacrificed 24 hr after the last administration. Biochemical and histological analysis were carried out. RESULTS: It was shown 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, creatine kinase, creatine kinase-MB, and uric acid. Significant alteration in levels of reduced and oxidized glutathione on kidney and heart suggested that oxidative stress may be involved in krokodil-mediated toxicity. Cardiac congestion was the most relevant finding of continuous krokodil administration. CONCLUSIONS: These 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 human toxicological effects of krokodil.

Fundamentals of Clinical Pharmacology With Application for Pregnant Women.

Medication use is common in pregnancy, yet for most medications the optimal formulation and dosage have not been described specifically for pregnant women. Often, adverse effects are only discovered anecdotally or after extensive off-label use occurs. Since pharmacologic research that includes pregnant women is sparse and animal studies are often not applicable to the human fetus, providers must use knowledge of drug behavior and normal physiologic changes of pregnancy to personalize treatment for pregnant women. In this review, we present an overview of the basic concepts of clinical pharmacology: pharmacokinetics, pharmacodynamics, and pharmacogenomics. The normal physiologic changes of pregnancy are presented as a framework to understand alterations in drug behavior. A clinical vignette that addresses 4 pregnancy scenarios involving medications-preterm birth, vaccination, herpes simplex virus infection, and codeine toxicity-is provided to illustrate application of core clinical pharmacologic concepts. Discussion of relevant literature illustrates the challenges of offering individualized pharmacologic therapy in pregnancy.

Spontaneous bone formation after mandible segmental resection in "krokodil" drug-related jaw osteonecrosis patient: case report.

We report a case of a 48-year-old male patient with "krokodil" drug-related osteonecrosis of both jaws. Patient history included 1.5 years of "krokodil" use, with 8-month drug withdrawal prior to surgery. The patient was HCV positive. On the maxilla, sequestrectomy was performed. On the mandible, sequestrectomy was combined with bone resection. From ramus to ramus, segmental defect was formed, which was not reconstructed with any method. Post-operative follow-up period was 3 years and no disease recurrence was noted. On 3-year post-operative orthopantomogram, newly formed mandibular bone was found. This phenomenon shows that spontaneous bone formation is possible after mandible segmental resection in osteonecrosis patients.

Desomorphine (Krokodil): An overview of its chemistry, pharmacology, metabolism, toxicology and analysis.

BACKGROUND: "Krokodil" or "Crocodile" is an illegal homemade desomorphine drug obtained from chemical reactions of commercial codeine drugs with several other powerful and highly toxic chemical agents increasing its addiction and hallucinogenic effects when compared with other morphine analogues. METHODS: This paper summarizes a complete review about an old drug called desomorphine (Krokodil), presenting its chemistry, pharmacology, metabolism, toxicology and analysis. RESULTS: It is of particular interest and concern because this cheaper injectable semisynthetic opioid drug has been largely used in recent years for recreational purposes in several Eastern European as well as North and South American countries, despite known damage to health that continuous use might induce. These injuries are much stronger and more aggressive than morphine's, infecting and rotting skin and soft tissue to the bone of addicts at the point of injection in less than three years, which, in most cases, evolves to death. On this basis, it is imperative that literature reviews focus on the chemistry, pharmacology, toxicology and analysis of dangerous Krokodil to find strategies for rapid and effective determination to mitigate its adverse effects on addicts and prevent consumption. CONCLUSIONS: It is crucial to know the symptoms and consequences of the use of Krokodil, as well as METHODS: for identification and quantification of desomorphine, contaminants and metabolites, which can help the forensic work of diagnosis and propose actions to control and eradicate this great danger to public health around the world.

[Multislice computed tomography in the diagnosis of toxic phosphorus necrosis of the jaw].

Objective: To estimate the possibilities of using and systematizing computed tomographic findings in patients with toxic phosphorus necrosis of the jaw. Material and Methods: The investigation enrolled 87 patients diagnosed as having toxic phosphorus osteonecrosis. Radiation examination consisted of two stages: primary and repeated radiologic examinations in the postoperative period (final examination before hospital discharge). All the patients underwent skull X-ray and multislice computed tomography (MSCT). Results: Clinical and radiation examination revealed toxic phosphorus osteonecrosis of the maxilla and mandible in 29 (33%) cases. Osteonecrosis affected only the mandible in 40 (46%) cases and only the maxilla in 18 (21%) cases. In all the patients, computed tomography showed main trends in the X-ray semiotics of toxic phosphorus necrosis of the facial skeleton, such as periostitis; osteosclerosis; development a lesion having a "soap-bubble" appearance; nonspecific and inflammatory bone destruction. The bone, being destroyed, was replaced by pus; inflammatory granulations were absent; osteonecrosis occurred. These processes were characterized by the absence of an obvious demarcation zone along the edges of the process. Sequestration commonly occurred to form sinus tracts. The process involved the adjacent bones; there were reactive changes in the accessory sinuses. Conclusion: MSCT data are of highly informative value in evaluating the status of bone tissue and teeth and in detecting a concomitant abnormality in patients with osteonecrosis of the facial skeleton and may be used to plan surgical treatment for this category of patients.

[New insights into the role of pholcodine in the treatment of cough in 2013?]. / Quel rôle pour la pholcodine dans le traitement de la toux, en 2013 ?

Pholcodine is an opioid that has been widely used worldwide since 1950 for the treatment of non-productive cough in children and adults. The results of early preclinical studies but also those of recent clinical trials have shown the antitussive efficacy of pholcodine to be superior to that of codeine, of longer duration, and with an equivalent or safer toxicity profile. Also, there is no risk of addiction. Concern had been raised over a possible cross-sensitisation with neuromuscular blocking agents. While a recent assessment of the available data by the European Medicines Agency (EMA) has confirmed the favourable risk-benefit ratio of pholcodine, further studies are needed to clear this point.

Is there a role for therapeutic drug monitoring with codeine?

Codeine is an old and commonly used analgesic agent for mild to moderate pain. It is the prototypical "prodrug" in that its analgesic effect is almost wholly dependent on its biotransformation to morphine, a process that is mediated by the polymorphic cytochrome P450 2D6 enzyme. As such, interindividual variability in codeine metabolism and response is a clinical reality, and there has been much progress in characterizing the genetic causes of this variability in diverse populations. Yet despite the potential for both life-threatening adverse reactions and lack of therapeutic effect, codeine is not commonly indicated for therapeutic drug monitoring. This review will discuss the relative role of pharmacogenetics and therapeutic drug monitoring in predicting and/or maintaining adequate and safe analgesia with codeine. The review will end on a discussion of how the marriage of these 2 fields may provide new insights into the mechanisms of codeine-induced toxicity and analgesia.

More codeine fatalities after tonsillectomy in North American children.

In 2009 we reported the fatal case of a toddler who had received codeine after adenotonsillectomy for obstructive sleep apnea syndrome. The child was an ultra-rapid metabolizer of cytochrome P4502D6 (CYP2D6). We now report 3 additional fatal or life-threatening cases from North America. In the 2 fatal cases, functional gene duplications encoding for CYP2D6 caused a significantly greater production of potent morphine from its parent drug, codeine. A severe case of respiratory depression in an extensive metabolizer is also noted. These cases demonstrate that analgesia with codeine or other opioids that use the CYP2D6 pathway after adenotonsillectomy may not be safe in young children with obstructive sleep apnea syndrome.

[Pharmacogenomics in routine medical care]. / Pharmakogenomik in der Praxis.

Pharmacogenomics investigates inherited differences in drug responses including beneficial and adverse reactions. While a considerable amount of evidence for genetic influences on drug responses has been accumulated within the last decade, predominantly in small studies, its value in routine therapy is still a matter of debate. The aim of this review is to discuss well established examples where pharmacogenomic techniques can improve routine treatment. Examples include genotyping of CYP2D6 in the context of antidepressant therapy, analysis of TPMT variants for the prediction of mercaptopurine-induced bone marrow depression, VKORC1 and CYP2C9 analyses for a better control of anticoagulant administration and the SLCO1B1 variant in the context of statin-induced myopathies.