Alterations in the Gut Microbiota of Rats Chronically Exposed to Volatilized Cocaine and Its Active Adulterants Caffeine and Phenacetin.

A role of the gut microbiota in influencing brain function and emotional disorders has been suggested. However, only a few studies have investigated the gut microbiota in the context of drug addiction.Cocaine can be smoked (i.e., crack or coca paste) and its consumption is associated with a very high abuse liability and toxicity. We have recently reported that cocaine base seized samples contained caffeine and phenacetin as main active adulterants, which may potentiate its motivational, reinforcing, and toxic effects. However, the effect of volatilized cocaine and adulterants on the gut microbiota remained unknown. In the present study, we evaluated the effect of volatilized cocaine and two adulterants on the structure, diversity, and functionality of the gut microbiota in rats. Animals were chronically exposed to the fume of cocaine, caffeine, and phenacetin during 14 days. At the end of the treatment, feces were collected and the structure, composition, and functional predictions of the gut microbiota were analyzed. Cocaine significantly decreased the community richness and diversity of the gut microbiota while both cocaine and phenacetin drastically changed its composition. Phenacetin significantly increased the Firmicutes-Bacteroidetes ratio compared to the control group. When the predicted metagenome functional content of the bacterial communities was analyzed, all the treatments induced a dramatic decrease of the aromatic amino acid decarboxylase gene. Our findings suggest that repeated exposure to volatilized cocaine, as well as to the adulterants caffeine and phenacetin, leads to changes in the gut microbiota. Future studies are needed to understand the mechanisms underlying these changes and how this information may support the development of novel treatments in drug addiction.

Turpentine oil induced inflammation decreases absorption and increases distribution of phenacetin without altering its elimination process in rats.

Plasma concentrations and pharmacokinetics of phenacetin, a CYP1A2 substrate were determined in normal and experimentally induced inflamed rats by turpentine oil to know the role of inflammation on the pharmacokinetics of phenacetin and formation of its active metabolite (paracetamol) by CYP1A2 in wistar albino rats, weighing about 200-250 g that were randomly divided into two groups consisting six in each group. Rats in group I (control) received phenacetin (150 mg kg(-1), PO) where as group II received phenacetin 12 h after induction of inflammation by turpentine oil (0.4 mL, i.m). Blood samples were collected from retro orbital plexus at pre-determined time intervals prior to and at 0.166, 0.33, 0.67, 1.5, 2, 4, 8 and 12 h post-administration of phenacetin. Plasma was separated and analyzed for phenacetin and its metabolite paracetamol by HPLC assay. Based on plasma concentrations of phenacetin and its metabolite paracetamol, the pharmacokinetic parameters were determined by compartmental methods. C(max) of phenacetin was significantly (p < 0.01) decreased to 19.50 ± 2.74 µg mL(-1) in inflamed conditions compared to 38.13 ± 2.20 µg mL(-1) obtained in normal rats. Except, for significant (p < 0.001) increase in volume of distribution at steady state (V(dss)) from 2.87 ± 0.37 to 8.03 ± 1.26 L kg(-1) and increased the rate of absorption with shorter absorption half-life (t(1/2ka)) for phenacetin in inflammation. None of the pharmacokinetic parameters of either phenacetin or its metabolite paracetamol were affected. It can be concluded that turpentine oil induced inflammation has no role on the activity of CYP1A2 in rats, as the plasma concentrations and pharmacokinetic parameters of paracetamol were found unaltered.

Phenacetin pharmacokinetics in CYP1A2-deficient beagle dogs.

Phenacetin is widely used as an in vitro probe to measure CYP1A2 activity across species. To investigate whether phenacetin can be used as an in vivo probe substrate to phenotype CYP1A2 activity in dogs, beagle dogs previously genotyped for a single nucleotide polymorphism that yields an inactive CYP1A2 protein were selected and placed into one of three groups: CC (wild-type), CT (heterozygous), or TT (homozygous mutants). The dogs were dosed with phenacetin orally at 5 and 15 mg/kg and intravenously at 15 mg/kg. Plasma samples were analyzed by liquid chromatography-tandem mass spectrometry, and phenacetin and its primary metabolite, acetaminophen, were monitored. After intravenous dosing, all groups showed similar exposure of phenacetin irrespective of genotype. After oral dosing at 15 mg/kg, the exposure of phenacetin in CC and CT dogs was similar, but phenacetin exposure was 2-fold greater in TT dogs. Exposure of the metabolite, acetaminophen, was similar in all groups; however, the mean acetaminophen/phenacetin ratio in TT dogs was 1.7 times less than that observed in CC dogs. Similar trends between the groups of dogs with respect to phenacetin exposure were also observed after a lower 5 mg/kg p.o. dose of phenacetin; however, a proportionally greater amount of acetaminophen was generated. Although oral exposure of phenacetin was 2-fold higher and acetaminophen exposure was 2-fold lower in CYP1A2-deficient (TT) dogs, these results were considered modest and suggest that phenacetin is not a selective or robust in vivo probe to measure CYP1A2 enzyme activity in the dog.

Use of prediction methods to estimate true density of active pharmaceutical ingredients.

True density is a fundamental and important property of active pharmaceutical ingredients (APIs). Using prediction methods to estimate the API true density can be very beneficial in pharmaceutical research and development, especially when experimental measurements cannot be made due to lack of material or sample handling restrictions. In this paper, two empirical prediction methods developed by Girolami and Immirzi and Perini were used to estimate the true density of APIs, and the estimation results were compared with experimentally measured values by helium pycnometry. The Girolami method is simple and can be used for both liquids and solids. For the tested APIs, the Girolami method had a maximum error of -12.7% and an average percent error of -3.0% with a 95% CI of (-3.8, -2.3%). The Immirzi and Perini method is more involved and is mainly used for solid crystals. In general, it gives better predictions than the Girolami method. For the tested APIs, the Immirzi and Perini method had a maximum error of 9.6% and an average percent error of 0.9% with a 95% CI of (0.3, 1.6%).

Effect of acetaminophen, a cyclooxygenase inhibitor, on Morris water maze task performance in mice.

Although the mechanism of action of acetaminophen (AAP) is not fully understood, some studies suggest that AAP and phenacetin (PHE) are selective cyclooxygenase (COX)-3 inhibitors. To examine the participation of COX-3 in memory formation, water maze performance was studied in mice treated with AAP, PHE or other COX inhibitors. Mice received intraperitoneal injections of drugs immediately after each training session. Administration of high-dose AAP [302.3 mg/kg (IC50 for COX-2)] or PHE [179.2 mg/kg (IC50 for COX-2)] and of non-specific (indomethacin: 20 mg/kg) or specific COX-2 (NS-398: 10 mg/kg) inhibitor impaired the performance in hidden platform (HP) not visible platform (VP) tasks, whereas low-dose (15.1 mg/kg) AAP facilitated performance in HP and VP tasks. The facilitation of performance by low-dose AAP was reversed by co-administration with a 5-HT(1/2) receptor antagonist (methysergide: 0.47 mg/kg). The middle-dose [69.5 mg/kg (IC50 for COX-3)] of AAP, the PHE [17.9 mg/kg (IC50 for COX-3)] and a specific COX-1 inhibitor (piroxicam: 10-20 mg/kg) did not influence performance in either task. These results suggest that the memory impairment by high-dose AAP and PHE and facilitation of performance by low-dose AAP could involve endogenous COX-2 and serotonergic neuronal activity, but not COX-3, respectively.

Bladder tumor associated with phenacetin abuse: a case report and a review of the literature.

We herein report the case of a bladder tumor in an 85-year-old man who had been engaged in phenacetin abuse. He had been taking phenacetin owing to migraine headaches since he was 45 year of age. His total intake of phenacetin was approximately 7.3 to 11.5 kg over a period of years. He visited the Department of Urology in our hospital due to gross hematuria and pain on urination. IVP and a pelvic CT scan revealed a tumor mass on the right lateral wall of the urinary bladder. TUR-BT was performed. A histopathological examination of the resected specimen was diagnosed as urotherial carcinoma, grade 2∼3, pT2N0M0. To our acknowledge, only 24 cases of urotherial tumors owing to phenacetin abuse have been previously reported in the Japanese literature, making this the 25 th such case to be reported in Japan.

Identification of phenacetin metabolites in human urine after administration of phenacetin-C2H3: measurement of futile metabolic deacetylation via HPLC/MS-SPE-NMR and HPLC-ToF MS.

The metabolism of acetyl-labelled phenacetin-C2H3 was investigated in man following a single (150 mg) oral dose. Urine samples were collected at predose, 0-2 h and >2-4 h post-dose, and samples from each time-point were then analysed directly using 1H-nuclear magnetic resonance (NMR) spectroscopy. The phenacetin metabolites acetaminophen (paracetamol) glucuronide, sulphate and the N-acetyl-L-cysteinyl conjugate were identified by this method, and all showed clear evidence of the loss of the original 2H3-acetyl label and its replacement with 1H3 (futile deacetylation). The observed percentage futile deacetylation by 1H-NMR spectroscopy was measured as approximately 20% in each metabolite (about 2% of the recovered dose). After sample preparation by solid-phase extraction on a C18 solid-phase extraction (SPE) cartridge, further profiling was performed using high-performance liquid chromatography/mass spectrometry-solid-phase extraction-nuclear magnetic resonance (HPLC/MS-SPE-NMR) confirming futile deacetylation had taken place as indicated by NMR spectroscopy on both the isolated acetaminophen glucuronide and L-cysteinyl-metabolites. Additional analysis by high-performance liquid chromatography-time-of-flight mass spectrometry (HPLC-ToF MS) identified further phenacetin metabolites, and from these data the mean percentage of futile deacetylation was measured as 31% +/- 2% for the acetylated phenacetin metabolites. A number of non-acetylated metabolites were also detected in the sample via HPLC-ToF MS. The results showed that phenacetin underwent a transient formation via a number of toxic intermediates to a much greater extent than had been observed in similar studies on acetaminophen. These results may contribute to the understanding of the analgesic nephropathy reported following chronic phenacetin consumption.

Evaluation of the Xpa-deficient transgenic mouse model for short-term carcinogenicity testing: 9-month studies with haloperidol, reserpine, phenacetin, and D-mannitol.

As part of the international evaluation program coordinated by ILSI/HESI, the potential of DNA repair deficient Xpa-/- mice and the double knockout Xpa-/-.p53+/- mice for short term carcinogenicity assays was evaluated. For comparison also wild-type C57BL/6 mice (WT) were included in these studies. Four test compounds were administered to groups of 15 male and 15 female Xpa-/- mice, Xpa-/-.p53+/- mice and WT mice for 39 weeks. The model compounds investigated were haloperidol, reserpine (nongenotoxic rodent carcinogens, putative human noncarcinogens), phenacetin (genotoxic rodent carcinogen, suspected human carcinogen), and D-mannitol (noncarcinogen in rodents and humans). The test compounds were administered as admixture to rodent diet at levels up to 25 mg/kg diet for haloperidol, 7.5 mg/kg diet for reserpine, 0.75% for phenacetin, and 10% for D-mannitol. These levels included the maximum tolerable dose (MTD). Survival was not affected with any of the test compounds. Haloperidol, reserpine and D-mannitol were negative in the carcinogenicity assay with Xpa-/- and Xpa-/-.p53+/- mice, showing low and comparable tumor incidences in controls and high-dose animals. The results obtained with phenacetin may be designated equivocal in Xpa-/-.p53+/- mice, based on the occurrence of a single rare tumor in the target organ (kidney) accompanied by a low incidence of hyperplastic renal lesions and a high incidence of karyomegaly. These results are in agreement with the currently known carcinogenic potential of the 4 test compounds in humans.

Caffeine as a promoter of analgesic-associated nephropathy--where is the evidence?

Individual groups of nephrologists - in their responsibility for their patients - initiated a most controversial discussion whether or not caffeine - coformulated to analgesics - might initiate or sustain analgesic overdosing. The original sources (data) of such suspicion have got lost during the debate of the last two decades. Therefore, it seemed to be appropriate to investigate the original data background and the reasons why nephrologists started to suspect caffeine as a stimulant of analgesic overdosing by employing a systematic and exhaustive review of primary nephrological publications. Their selection followed a precise selection plan, including all epidemiological studies on analgesic-associated nephropathy, the original papers of all groups having been involved in those studies, further originals from the mainly involved countries (academically, politically), and any literature thereof cited as a proof. The following results emerged from the investigation: (i) The epidemiological studies warranted no conclusion about a role of caffeine in prompting excessive analgesic use. (ii) The identified groups of nephrologists provided not substantial data to advocate the said suspicion, except for the observation of a preferential choice of phenacetin-containing combinations, especially powder preparations. (iii) Only two cited original data sources revealed drug-seeking behaviour with phenacetin-containing preparations which subsided, after phenacetin was banned from the respective markets. Conclusively, it appears that there is no substantial data to support a pivotal role of caffeine in initiating or sustaining analgesic overdosing. However, there is strong data that phenacetin, by its psychotropic properties, may have caused drug-seeking behaviour and thus led to analgesic overdosing. This conclusion is convincingly supported by thorough pharmacokinetic investigations. Note: All caffeine-related statements within the reviewed literature have been collected in tables (referred to as Table SX) which are provided in full text for check on the following website: http://www.blackwellpublishing.com/products/journals/suppmat/FCP/FCP174/FCP174sm.htm

Controlled release from solid dispersion composed of poly(ethylene oxide)-Carbopol interpolymer complex with various cross-linking degrees of Carbopol.

Solid dispersion composed of the poly(ethylene oxide) (PEO)-Carbopol((R)) (CP) interpolymer complex containing phenacetin (PHE) was prepared by using six grades of CP having various cross-linking degrees. We attempted to control the medicine release from the PEO-CP solid dispersion by varying the CP grade. The powder X-ray diffraction pattern and differential scanning calorimetry curves suggested that PHE existed in the amorphous state, and PEO in the crystalline state disappeared in the solid dispersions. The release profile of PHE varied depending on the CP grade. A small release rate was observed at CP910 and CP971P that are cross-linked at low and middle degrees, respectively. The Fourier transform-infrared (FT-IR) spectra showed that the amount of the PEO-CP complex formed by hydrogen bonding changed depending on the CP grade. With the cross-linked CP, a good correlation was observed between the hydrogen bonding percent and the percent released of the PHE after 60 min (D(60 min)), indicating that PHE release was controlled by the amount of PEO-CP complex formation in the solid dispersion. These results show that it is feasible to control the medicine release from PEO-CP solid dispersion by varying the CP grade.

Role of CYP1A2 in the toxicity of long-term phenacetin feeding in mice.

The mechanisms underlying phenacetin-induced toxicity and carcinogenicity are not clear. In particular, it is not known whether these effects are mediated by metabolic activation of the drug. CYP1A2 is known to metabolize phenacetin in vitro. To determine the role of this enzyme in vivo, the toxicity and carcinogenicity of phenacetin was examined in Cyp1a2-null mice (that lack CYP1A2). Six- to 8-week-old wild type (+/+) or null (-/-) mice were fed either a control diet, or one containing 1.25% phenacetin, ad libitum for up to 67 weeks. Representative groups of mice were examined for phenacetin-induced toxicity and carcinogenicity after 36, 48, 58, or 67 weeks of feeding. Consistent with the known role of CYP1A2 in phenacetin metabolism, plasma levels of phenacetin were higher and acetaminophen levels lower in the (-/-) mice fed phenacetin compared to phenacetin-fed (+/+) controls. Weight gain was significantly depressed in both groups of phenacetin-fed mice after 4 weeks of feeding, and continued to be lower for the remainder of the experiment, compared to controls. Hepatomegaly and splenomegaly were more severe in (-/-) mice but present in both genotypes fed phenacetin at all time points assessed. Histological analysis of liver, kidney, spleen, and urogenital tract also revealed a differential response in the (-/-) mice fed phenacetin compared to (+/+) mice fed the same diet. Further, mortality was the most severe in the (-/-) mice fed phenacetin than in all other groups. Despite significant toxicity in (-/-) mice fed phenacetin, only one renal carcinoma was found among them. Results from this work demonstrate that, in the absence of CYP1A2, phenacetin is more toxic than in controls. This provides evidence that metabolism of phenacetin by CYP1A2 alters toxicity in vivo, and suggests that alternate CYP1A2-independent metabolic pathways contribute to its toxicity.

Analgesic nephropathy in rodents.

While it is clear that humans suffer from "classic" analgesic nephropathy, the causative agents and mechanisms are still not known. A review of the literature revealed that chronic acetaminophen exposure does not produce renal papillary necrosis in rodents or humans. In contrast, while chronic aspirin exposure to rodents results in renal papillary necrosis with renal morphological and functional changes similar to that described in humans, epidemiological studies do not implicate aspirin alone in human analgesic nephropathy. The difference in the effects of aspirin in humans and rats may be due to the inability of epidemiological studies to detect aspirin-induced analgesic nephropathy or more likely to the fact that species differences exist, with the rat being more sensitive than humans. With respect to combinations of aspirin and acetaminophen, with or without caffeine, there are minimal tightly controlled studies. In addition, there is little evidence of enhanced renal papillary necrosis in rodents treated with aspirin and acetaminophen combinations. In summary, it remains to be determined what chemical entities cause "classic" analgesic nephropathy in humans and the mechanisms of this toxicity such that preventative measures can be instituted. Elucidation of the mechanisms of analgesic nephropathy has been hampered due to the lack of animal models that closely mimic the human disease. Rodents do not appear to be an appropriate model.