Background
==========

Vitamin A and its biologically active metabolites, the retinoids, are essential for the maintenance of normal epithelial differentiation \[[@B1]\]. Retinoid-deficient epithelial tissues acquire a premalignant phenotype, which is characterized by enhanced mitotic activity and loss of differentiation \[[@B2]\]. Retinoids have been used as chemopreventive and chemotherapeutic agents for various human cancers \[[@B3]\].

The antiproliferative and differentiative effects of retinoids are mediated through two classes of nuclear receptors, the RARs and RXRs. Both classes consist of three members, α, β and γ \[[@B4]\]. All these receptors are ligand-activated transcription factors. They activate transcription by binding to retinoic acid response elements (RAREs) located in the promoter regions of the target genes. One of the targets of retinoid receptors is the RAR-β gene itself. The RAR-β gene encodes for four transcripts and is expressed in normal epithelial tissue, where its expression is up-regulated by treatment with retinoic acid (RA) \[[@B5],[@B6]\]. Recent studies have demonstrated that in many malignant cell lines derived from various carcinomas the level of RAR-β2 mRNA (one of four transcripts of this gene) is decreased or undetectable \[[@B7]-[@B9]\]. A decrease of RAR-β2 mRNA level (compared to normal tissues) was also observed in carcinomas of cervix \[[@B10]\], and in carcinomas of head and neck \[[@B11]\]. These findings indicate that the specific loss of RAR-β2 mRNA expression may be an important event in tumorogenesis. This hypothesis has been supported by an observation that RAR-β2 can function as a tumor suppressor gene in two different carcinoma cell lines \[[@B12],[@B13]\]. Thus, a defect in RAR-β2 expression may be one of the events that lead the tumor cells to the escape from growth regulation.

The etiology of cancer of the uterine cervix has been associated with several types of human papillomaviruses (\"high-risk\" HPVs) \[[@B14]\]. In addition to growth regulation, the product of RAR-β gene can inhibit transcription of the viral oncogenes (E6 and E7 genes) in high-risk HPV-immortalized cells \[[@B15]\]. The decrease of the expression of negative regulator of viral oncogene may be an additional important step on the way towards malignant progression of HPV-positive cells in this case.

It has been shown that the retinoic acid can induce an increase of RAR-β2 expression in carcinoma cells \[[@B16]\] and in lesions of the oropharynx \[[@B17]\]. RA treatment has inhibited transformation of human keratinocytes by HPV-16 \[[@B18]\] and has enhanced regression of cervical moderate dysplasia \[[@B19]\]. Beneficial effects of RA treatment of advanced cervical squamous cell carcinomas (in combination with interferon α) have been demonstrated in clinical trials \[[@B20]\]. On the other hand, the existence of retinoic acid-insensitive cancer cell lines, including cervical carcinoma cell lines, and the lack of clinical effects of retinoids treatments in some cases are well established \[[@B21],[@B22]\].

The mechanism of the down-regulation and the loss of RA-inducible expression of RAR-β2 in cervical carcinomas and cell lines derived from them is not well understood yet. Recent findings have shown that hypermethylation of the promoter region is implicated in the transcriptional silencing of many tumor suppressor genes and several other genes functionally important in neoplastic process \[[@B23]\]. The promoter regions of many genes, including RAR-β2, contain CpG islands, that are protected from methylation in normal cells \[[@B24]\]. Aberrant methylation of CpG islands in tumors serves as an alternative pathway (in addition to intragenic mutations and loss of chromosomal loci) for complete inactivation of tumor suppressor genes. Hypermethylation of normally unmethylated CpG islands in the 5\' region of the RAR-β2 gene has been observed in breast and colon cancer cell lines, that do not express RAR-β2 mRNA \[[@B25]-[@B29]\].

In this study we examined whether the methylation could be responsible for the deficit of RAR-β2 gene expression in cervical squamous cell carcinomas.

Materials and Methods
=====================

Tissue specimens
----------------

Specimens of cervical tumor were collected in the Department of Radiosurgery of Russian Cancer Research Center: 20 specimens of invasive squamous cell carcinoma with low and moderate levels of differentiation and I, II and III FIGO stages and 20 specimens of morphologically normal tissues of cervix from the same patients. All tumors were HPV16- or HPV18-positive by PCR analysis.

Cell lines
----------

The human cervical carcinoma cell HeLa, SiHa and CaSki (American Type Culture Collection, Rockville, MD) were maintained in DMEM, supplemented with 10% FCS and 2 mM glutamine.

DNA and RNA preparations
------------------------

DNA and RNA were isolated from frozen tissues homogenized in the 4.5 M solution of guanidine thiocyanate by centrifugation through cesium chloride cushion, as described previously \[[@B30]\].

Northern blot analysis
----------------------

10 μg of total RNA was fractionated in 0.8% formaldehyde agarose gel and transferred to \"Hybond C extra\" nitrocellulose membrane (Amersham, UK) according to Sambrook et al. \[[@B31]\]. The membranes were hybridized with ^32^P-labelled 650 bp and 610 bp EcoRI fragments of RAR-β2-specific DNA. In order to control the relative amounts of RNA the membranes were hybridized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA probe. Hybridization was performed in 5 × SSC at 42°C in the presence of 50% formamide. The membranes were washed in 2 × SSC, 1% SDS at 65°C and were exposed to X-ray film at -70°C.

Southern blot analysis
----------------------

10 μg of DNA was digested overnight with MspI or HpaII restriction enzymes (10 units/μg of DNA, SibEnzyme, Novosibirsk, Russia). The restriction fragments were separated in 1% agarose gel, transferred to nylon membrane (Zeta-Probe, Bio-Rad, USA) and hybridized with ^32^P-labelled PCR DNA probe. PCR DNA probe was prepared by PCR amplification of a 680 bp fragment of RAR-β2 promoter and exon_3 with primers: forward, 5\'-ATT TGA AGG TTA GCA GCC CG and reverse 5\'-CAT TCG GTT TGG GTC AAT CC, in the following conditions: 94°C for 3 min, 30 amplification cycles (94°C, 30 sec; 56°C, 30 sec; 72°C, 1 min) and 72°C, 5 min . Hybridization and washing conditions were as described for RNA.

Methylation-specific PCR (MSP) assay \[[@B32]\]
-----------------------------------------------

Bisulfite conversion reaction was carried out according to S.J. clark et al. \[[@B33]\] with minor modifications. DNA (2 μg) was digested with RsaI restriction enzyme (SibEnzyme, Novosibirsk, Russia), denaturated by addition of NaOH to a final concentration 0.3 M, incubated for 15 minutes at 37°C and 3 minutes at 95°C, followed by cooling on ice. Sodium bisulfite and hydroquinone were added to the denatured DNA to final concentration 3.6 M and 0.5 mM, respectively. The sample was overlaid with mineral oil and incubated at 65°C for 16 hours. The DNA was purified using QIAEX II kit (Qiagen), desulfonated by addition of NaOH to a final concentration 0.3 M and incubation at 37° for 15 min, neutralized with 3 M ammonium acetate (pH 7.0), precipitated with 3 volumes of ethanol, resuspended in H~2~O and stored at -20°C.

Primers that were specific for the upper strand of bisulfite-modified DNA and did not contain CpG dinucleotides in the original sequences were used in the first round of PCR: forward 5\'-AAG TGA GTT GTT TAG AGG TAG GAG GG and reverse 5\'-CCT ATA ATT AAT CCA AAT AAT CAT TTA CC \[[@B28]\]. 5-20 ng of DNA was amplified with these primers in the following conditions: 94°C, 5 min; 35 amplification cycles (94°C, 15 sec; 53°C, 15 sec and 72°C, 30 sec) and 72°C, 5 min. The obtained DNA fragments were amplified in the second round of PCR with two set of primers specific for the upper strand of bisulfite-modified DNA, which contains CpG dinucleotides: 1) forward 5\'-TTG AGA ATG TGA GTG ATT TGA and reverse 5\'-AAC CAA TCC AAC CAA AAC AA for the unmethylated sequence (U), 2) forward 5\'-TCG AGA ACG CGA GCG ATT CG and reverse 5\'-GAC CAA TCC AAC CGA AAC GA for the methylated sequence (M). The PCR was performed under the following conditions: 94°C, 2 min; 40 cycles (94°C, 30 s; 50°C, 30 s and 72 °C, 30 s for U-primers) and 35 cycles (94°C, 30 s; 58°C, 30 s and 72°C, 30 s for M-primers) and 72°C, 5 min. The PCR products were fractionated in the 3% agarose gel and photographed.

Results
=======

RAR-β2 mRNA expression
----------------------

The level of the expression of RAR-β2 mRNA was evaluated in 20 samples of invasive squamous cell carcinomas of uterine cervix at low and moderate levels of differentiation and in the morphologically normal tumor-adjacent tissues of cervix in order to select a group of tumors with deficient expression of RAR-β2 (Figure [1B](#F1){ref-type="fig"}, Table [1](#T1){ref-type="table"}). The level of RAR-β2 mRNA expression was significantly decreased in 8 out of 20 tumors constituting 5 -- 48% of the expression level of the corresponding morphologically normal tissue. An extremely low (undetectable) level of mRNA expression was observed in one tumor, as well as in the adjacent morphologically normal tissue (Fig. [1B](#F1){ref-type="fig"}, lanes 5,6; Table [1](#T1){ref-type="table"}, case 7). Thus, in 40% of cervical SCC the expression of the RAR-β2 mRNA was significantly decreased. An extremely low level of mRNA expression, wich was observed earlier in three cell lines HeLA, SiHa and CaSki \[[@B22]\], was confirmed in our experiment (data are not presented).

![**A. Restriction map of promoter-exon region of RARβ2 gene for methylation-sensitive Hpa II enzyme.** The bolt line shows the 680 bp DNA probe generated by PCR of the promoter and exon 3. The expected fragment sizes which are recognized by this probe are shown in the bottom part of the map. The transcription start site is indcated by broken arrow. The positions of the primers for MSP assay are indicated by arrows. The 146 bp fragment is the region analyzed for CpG methylation by MSP assay. The triangles show the positions of two RAREs within the promoter region. H -- HpaII sites, the positions of the Hpa II sites in relation to the trascription start site are indicated by fugures in bp. **B. Northern blot analysis of total RNA isolated from cervical carcinomas (T) and adjacent cervical normal tissues (N).** The blots were hybridized with ^32^P labeled DNA probes for RARβ2 and GAPDH. Lanes1, 2 -- 4N, 4T, lanes 3, 4 -- 5N, 5T, lanes 5,6 -- 7N, 7T respectively. Case numbers correspond to that indicated in table [1](#T1){ref-type="table"}. The arrows show the positions of 28S and 18S RNA. **C. Southern blot analysis of DNA isolated from cervical carcinomas (T) and adjacent cervical normal tissues (N).** The blots were hybridized with 680 bp ^32^P labeled DNA probe for RARβ2. The bold arrow shows the positions of a minimal 1.1 kbp fragment, recognized by this probe. Lanes 1, 3, 5, 7, 9, 11, 13, 15 -- DNA digested by MspI; lanes 2, 4, 6, 8, 10, 12, 14, 16 -- DNA digested by HpaII; lanes 1, 2 -- 9N; lanes 3, 4 -- 9T; lanes 5,6 -- 4N; lanes 7, 8 -- 4T; lanes 9, 10 -- 7N; lanes 11,12 -- 7T; lanes 13,14 -- SiHa; lanes 15, 16 -- HeLa. Case numbers correspond to that indicated in table [1](#T1){ref-type="table"}. **D. MSP assay of DNA isolated from cervical carcinomas (T), adjacent cervical normal tissues (N) and leucocytes (L).** All DNA samples were treated with sodium bisulfite before amplification of a 146 bp fragment with methylation-specific primers (M) and unmethylation-specific primers (U). Sample numbers on the top of the gel correspond to that indicated in table [1](#T1){ref-type="table"}; mr -- molecular weight marker.](1471-2407-2-4-1){#F1}

::: {#T1 .table-wrap}
::: {.caption}
######

Methylation Status of RAR β2 5\' region in cervical SCC and cervical cancer cell lines
:::

  Case number or type of cells   RNA expression %^\*^   Southern blot analysis   MSP assay
  ------------------------------ ---------------------- ------------------------ -----------
  1                              5                      nd                       M
  2                              19                     M                        Nd
  3                              48                     M                        M
  4                              22                     M                        M
  5                              26,5                   M                        M
  6                              18                     M                        M
  7                              ^\*\*^                 M                        M
  8                              19                     nd                       M
  9-20                           95-103                 U                        U
  HeLa                           ^\*\*^                 M                        M
  SiHa                           ^\*\*^                 M                        M
  CaSki                          ^\*\*^                 nd                       M

^\*^ The expression levels of RAR β2 mRNA in carcinomas relative to morphologically normal tissues (%), determinations by Northern blot analysis; the RAR β2 mRNA levels have been normalized to GAPDH. ^\*\*^ Expression of RAR β2 mRNA was extremely low in tumor and morphologically normal tissue or cell lines. nd -- not done
:::

Methylation status of the RAR-β2 5\' region
-------------------------------------------

The methylation status of the 5\' region of the gene was studied in order to determine if this DNA modification, which inhibits transcription, contributes to the inhibition of the expression of the RAR-β2 mRNA. The study was carried out by two independent methods: by Southern blot analysis and by methylation-specific PCR assay. The localization of the recognition sites for the methylation-sensitive restriction enzyme Hpa II within the promoter region and the first transcribed exon of the RAR-β2 transcript (exon 3) is presented on the fig. [1A](#F1){ref-type="fig"}. The sizes of the fragments, which are produced when the restriction sites are methylated and can not be digested by Hpa II, are indicated in the lower part of the fig. [1A](#F1){ref-type="fig"}. The control digestion of DNA by Msp I (cuts at the identical to Hpa II site even if the cytosine residue is methylated) produced only one major band 1.1 kbp (Fig. [1C](#F1){ref-type="fig"}, lanes 1, 3, 5, 7, 9, 11, 13, 15). The absence of the 1.1 kbp band, as well as that of 1.15 -- 1.6 kbp, after the digestion of the DNA by Hpa II indicates that the H1-H5 sites are methylated (Fig. [1C](#F1){ref-type="fig"}, lanes 8, 10, 12, 14, 16). The presence of 1.1 kbp band and the absence of any additional bands indicates that these sites are unmethylated (Fig [1C](#F1){ref-type="fig"}, lanes 2, 4, 6). As demonstrated by Southern blot analysis, all analyzed sites were methylated in 6 out of 8 carcinomas with the deficit of the RAR-β2 mRNA (Table [1](#T1){ref-type="table"}). It was impossible to perform this analysis in two carcinomas due to lack of adequate amounts of DNA. Methylation of this region was found in the morphologically normal tissue in the case 7 (Fig. [1C](#F1){ref-type="fig"}, lane 10). This result correlated with the absence the mRNA expression in this samples (Fig. [1B](#F1){ref-type="fig"}, lane 5). Two cervical cancer cell lines, showed methylation of this region also (Fig [1C](#F1){ref-type="fig"}, lanes 14, 16).

The same samples were subjected to the MSP assay. The MSP assay allows detection of methylated, as well as of unmethylated alleles after the modification of DNA by sodium bisulfite. The presence of nonmalignant cells (fibroblasts, blood cells and some normal epithelial cells) in tumor specimens does not interfere with the ability to detect methylation. The sensitivity of this assay to detect methylated alleles in the background of unmethylated alleles is \> 10^-4^\[[@B32]\]. The result of a typical experiment with a set of primers, that amplify a 146 bp product in the region of H4 and H5 sites of Hpa II (5\' untranslated region, 5\' UTR), is presented on the fig. [1D](#F1){ref-type="fig"}. The presence of an amplification product obtained in the reaction with methyl-specific primers (M) indicates for the methylation (Fig. [1D](#F1){ref-type="fig"}, samples IT, 3T, 7T). The absence of this product indicates for the absence of the methylated alleles (Fig. [1D](#F1){ref-type="fig"}, samples 13T, 9T, 13L and all N samples). Amplification with primers, which are specific for the unmethylated sequence (U), demonstrated that this region is mostly demethylated. It should be noted, that in all tumor samples, that bear the methylated alleles, we detected at least traces of the products of amplification with U primers. Apparently, the nonmalignant cells, that are present in tumor specimens serve as a source of this product. Both primer pairs (M and U) do not react with DNA, which was not treated with bisulfite (data not shown). Thus, the presence of an amplification product in the reaction with the U primers serves as a control of a correct DNA modification by bisulfite in the case of a negative reaction with the M primers. The results of the MSP assay confirmed the methylated status of DNA in the 5\' UTR region of RAR-β2 gene in tumors with decreased level of mRNA expression and in all cervical cancer cell lines examined. (Table [1](#T1){ref-type="table"}). In addition to one case, which was detected by Southern blot analysis, two patients were found to produce a poor amplification with the M primers in morphologically normal tissues (data not shown).

Thus, methylation of CpG site in the promoter and the 5\' UTR region of RAR-β2 gene was detected in all 8 tumor samples with deficient expression of the RAR-β2 mRNA and all cervical cancer cell lines. Methylation of this region was not found in the tumors with normal expression levels of the RAR-β2 mRNA.

Discussion
==========

RAR-β2 is a putative tumor suppressor. Complete or partial inhibition of the expression of the gene was observed in many tumor cell lines and in primary human tumors \[[@B7]-[@B11],[@B22]\]. It was demonstrated in this study, that in 8 out of 20 SCC of cervix a deficient expression of the RAR-β2 mRNA was observed in comparison to the matched morphologically normal tissue of cervix.

The mechanism of the inhibition of RAR-β2 expression remains obscure. It was demonstrated in the tumor cells lines derived from breast and colon carcinomas, that the expression of RAR-β2 mRNA is silenced by methylation of the promoter and the first exon, that is expressed in the RAR-β2 transcript. Treatment of DNA by demethylating agent 5-aza-2\'-deoxycytidine restored the inducibility of the gene by all-*trans*-retinoic acid (ATRA) in many, but not all tumor cell lines. For breast carcinomas a relationship between methylation status and decreased RAR-β2 expression was found only in invasive grade II tumors. In grade III tumors the RAR-β2 expression was never observed, regardless the methylation status \[[@B28]\]. In all 8 invasive SCC of cervix and in three cell lines, analyzed in our study, the deficit of the expression of the RAR-β2 mRNA was associated with methylation of the promoter and the region of the first exon, including the H3 site close to the RARE (Figure [1A](#F1){ref-type="fig"}). The methylation of this site plays an important role in the repression of the gene transcription in the breast cancer cells \[[@B29]\]. However, recently 5 cervical cancer cell lines were revealed in that the inhibition of the RAR-β2 mRNA expression was not associated with the methylation of the same region of the gene \[[@B34]\]. Apparently, some other events together with methylation of the promoter region are involved in gene inactivation in certain cancer cell lines and invasive carcinomas. In particular, overexpression of Nur 77 gene has been found to be associated with a loss of RAR-β2 inducibility and RA resistance in lung cancer cells \[[@B35]\].

In our study the methylated alleles of RAR-β2 were absent in the majority of morphologically normal tissues of cervix. However, methylated alleles of the gene were found in three samples of the normal tissue, which were adjacent to the tumors with methylated status of RAR-β2. It can be suggested, that methylation of the normal tissues of cervix is an early preneoplastic event, that favours the development of malignancy. Our data provide only indirect evidence that methylation and the loss of RAR-β2 expression are early steps of cervical carcinogenesis. Recently this suggestion was supported by the study, where the methylated alleles of the gene were found in 11% and 29% of low grade and of high grade cervical intraepithelial neoplasias respectively \[[@B34]\]. This authors found the methylation of RAR-β2 gene in 5 out of 17 invasive carcinomas also. Taken together these data indicate that methylation of RAR-β2 gene is frequent and early event during cervical cancerogenesis.

Conclusions
===========

These findings suggest that methylation of the 5\' region of RAR-β2 gene may contribute to gene silencing and that methylation of this region may be an important and early event in cervical carcinogenesis. Cervical carcinomas may serve as a potential target for the DNA methylation inhibitors due to a close correlation between the deficit of RAR-β2 mRNA expression and methylation of this gene. These inhibitors in combination with retinoids might be clinically useful in future as chemopreventive and chemotherapeutic agents for patients with cervical dysplasias and carcinomas.

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

<>

Acknowledgements
================

We thank Dr. O.V. Sacharova for providing the clinical information and Dr. A.F. Kisseleva for technical assistance. This work was supported by grants 97-04-50140 and 01-04-49225 from the Russian Foundation of Basic Science.


Background
==========

Interest in the peer review process and research aimed at determining the method of obtaining the best quality reviews has grown in recent years. \[[@B1]\] Checklists have been developed that aim to guide reviewers when assessing the quality of papers, but little evidence exists concerning the extent of agreement between two referees when evaluating the same paper. In addition, little is known about which dimensions of a checklist are likely to result in greater agreement between referees.

There were two aims of this study: (1) to examine inter-rater agreement of the quality of abstracts submitted to a primary care research conference (Annual Meeting of the South West Association of University Departments of General Practice, Exeter 2000, UK), and (2) to compare the scores of abstracts accepted and rejected for the meeting.

Materials and Methods
=====================

Abstracts were anonymised and scored using a structured assessment comprising seven categories: (1) importance of the topic (2) originality (3) overall quality of the study design (4) appropriateness of the design used (5) achievement of aim (6) contribution to academic primary care (7) likelihood of provoking discussion. For comparison purposes, we have classified the assessment of categories 1, 2, 6 and 7 as more \'subjective\' in nature, and categories 3, 4 and 5 as more \'objective\'. Between one (poor) and four (excellent) marks were awarded for each category, giving a maximum possible score of 28 marks. Every abstract was assessed independently by two referees (AM and AG).

Agreement between referees was assessed using intraclass correlation coefficients (ICC), a chance corrected measure of agreement. \[[@B2]\] The ICC indicates perfect agreement only if the two assessments are numerically equal and is preferable to the more usual (Pearson) correlation coefficient. The crude ICC is lowered by any systematic differences between referees\' scores. In terms of a plot of the two referees\' scores, a line with a non-zero intercept will further lower the ICC irrespective of any disagreement, represented by deviation of the slope of the line away from unity and scatter around the line. In a further analysis, this effect was investigated by subtracting the mean difference for each component from the higher of the two referees\' scores. The ICCs were then recalculated, giving estimates of agreement corrected for both systematic differences and chance. There are no universally applicable standard values for the ICC that represent adequate agreement, but the following convention is used here to aid interpretation: ICC \<0.20 \'slight agreement\'; 0.21--0.40 \'fair agreement\'; 0.41--0.60 \'moderate agreement\'; 0.61--0.80 \'substantial agreement\'; \>0.80 \'almost perfect agreement\'.

Scores from referees from three different institutions were summed to give each abstract an overall score. Abstracts were ranked by this overall score and the top 45 were accepted for oral presentation at the meeting. Of the 52 abstracts refereed by AM and AG, mean total scores of those accepted and rejected for the meeting were compared using an unpaired t test.

Results
=======

Chance corrected agreement between the two referees\' scores measured using crude ICCs was greater for the three components relating to design and execution of the study (Table [1](#T1){ref-type="table"}: items 3 to 5) compared to those relating to more subjective elements of the abstract (Table [1](#T1){ref-type="table"}: items 1, 2, 6, 7). After adjustment for systematic differences in referees\' scores, ICCs for items 3 to 5 remained highest, demonstrating fair to moderate agreement.

::: {#T1 .table-wrap}
::: {.caption}
######

Inter rater agreement between two referees for 52 abstracts submitted for a primary care research conference
:::

  **Component**                               **Mean difference (AG minus AM)**   **Crude ICC**   **Adjusted ICC**
  ------------------------------------------- ----------------------------------- --------------- ------------------
  1\. importance of the topic                 0.71                                0               0.24
  2\. originality                             0.27                                0               0.01
  3\. overall quality of the study design     0.40                                0.30            0.40
  4\. appropriateness of the design used      0.17                                0.40            0.41
  5\. achievement of aim                      -0.12                               0.44            0.45
  6\. contribution to academic primary care   0.25                                0.20            0.25
  7\. likelihood of provoking discussion      0.15                                0.22            0.24
  Overall score                               1.85                                0.31            0.41

ICC = intraclass cluster coefficient
:::

A total of 76 abstracts were submitted for the meeting. Of 52 received by the authors for assessment, 26 were accepted for oral presentation (Table [2](#T2){ref-type="table"}). Abstracts accepted for the meeting had a significantly higher mean score than those that were rejected (95% CI for difference 1.3 to 4.1, p = 0.0003) (Table [2](#T2){ref-type="table"}).

::: {#T2 .table-wrap}
::: {.caption}
######

Summary statistics of abstracts accepted and rejected for oral presentation at a primary care research conference
:::

             **N**   **Mean score**   **SD**   **Range (possible 7 to 28)**
  ---------- ------- ---------------- -------- ------------------------------
  Accepted   26      17.4             2.7      11 to 21.5
  Rejected   26      14.6             2.3      11 to 19.5
:::

Discussion
==========

This study has shown that when using a structured assessment form, two independent reviewers were more likely to agree on design or methodological components of a checklist than on subjective components of abstracts submitted for an annual research meeting. Abstracts accepted for the meeting had significantly higher total scores, but overlapped considerably with rejected abstracts. This was due to acceptance for the meeting being determined by an overall aggregate of scores awarded by referees from three institutions.

While the subject of inter-reviewer agreement on different components of a checklist is relatively under-researched, some previous studies offer support for our finding that agreement is better when reviewers can be more objective in their assessments. Among a group of reviewers asked to rate a series of review articles, agreement on scientific quality of the papers was very high (60% of ICCs \> 0.7) both within and between groups with varying levels of research training and expertise. \[[@B3]\] All 10 dimensions of the checklist that reviewers rated could be regarded as objective. Divergent reviewers have been identified in a study comparing an overall rating score that indicated a recommendation to publish rather than individual dimensions of a review checklist. \[[@B4]\]

This study does have limitations. Importantly, we assessed agreement between only two reviewers on a relatively small number of abstracts. This could be addressed by having more abstracts assessed by a greater number of reviewers. However the study was conducted pragmatically within the time and administrative constraints of a small annual scientific meeting rather than submissions to a journal over an extended period. Another limitation is that the reviewer checklist was constructed prior to conceiving the study. If future meetings are to be used to investigate the content of structured reviewer assessments, such checklists should be constructed with specific hypotheses in mind.

Characteristics associated with good peer review are age under 40 years and training in epidemiology or statistics, \[[@B5]\] characteristics that applied to both reviewers in the present study. Structured assessment forms that ask the reviewer for their opinion of a paper\'s interest, originality or likelihood of provoking discussion may be more likely to result in scores that reflect the reviewer\'s own research interests. This is not necessarily a criticism -- it is perhaps only natural that individuals will differ in their opinions of how interesting they find, and think others will find, a particular paper. It is interesting that the two components with the lowest agreement, importance of the topic and originality of the study, both require more knowledge about a specific subject area than either of the other two subjective questions. Journal editors and meeting organisers should be aware that including subjective components in review checklists may result in greater disagreement between reviews.

Conclusions
===========

This study provides some evidence that inclusion of subjective components in a review checklist may result in greater disagreement between reviewers. An interesting area for further research would be to investigate the effects of attaching different weights to subjective and objective components of a checklist, or to exclude subjective components altogether from overall quality scores and simply use them a guide to acceptance or rejection.

Competing interests
===================

None declared.

![Difference between referees\' scores versus mean score](1472-6963-2-8-1){#F1}

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

<>

Acknowledgements
================

We would like to thank Mrs Sylvia Smith for collation of all referees\' responses. During the period in which this work was conducted, AM was supported by a UK Medical Research Council Training Fellowship in Health Services Research. TF is supported by a UK NHS R&D Primary Care Career Scientist Award.


Background
==========

During the first days after stroke, between one and two fifths of the patients develop fever or subfebrile temperatures. \[[@B1]-[@B4]\] Increased temperatures have been associated with relatively large infarct volumes, high case fatality, and poor functional outcome, even after adjustment for initial stroke severity. \[[@B2]-[@B6]\] The period in which hyperthermia is associated with poor outcome is probably limited to the first 12 or 24 hours from stroke onset. \[[@B6],[@B7]\]

The harmful effects of an early rise in body temperature have been attributed to increased cerebral metabolic demands, \[[@B8]\] changes in the blood-brain barrier permeability, acidosis, and an increased release of excitatory amino acids. \[[@B9]\] In animal models of temporary focal cerebral ischemia, mild intra-ischaemic hyperthermia increased infarct volume, \[[@B10]\] whereas mild hypothermia reduced infarct size. \[[@B11]\]

The above suggests that a pharmacological reduction of temperature in patients with acute ischaemic stroke may improve their functional outcome. Previously, we studied the effect on body temperature of high (6 g daily) and medium dose (3 g daily) paracetamol (acetaminophen) in a randomised placebo controlled trial of 3 × 25 patients (PAPAS: paracetamol (acetaminophen) in patients with acute stroke). \[[@B12]\] In the high dose paracetamol group, mean body temperature at 12 and 24 hours after start of treatment was 0.4°C (95% CI:0 -- 0.8°) lower than in the placebo group. Koennecke conducted a comparable study of 40 patients with acute stroke. He showed that treatment with paracetamol in a daily dose of 4000 mg resulted in a substantial reduction of the proportion of patients with body temperatures over 37.5°C, but he did not estimate the size of the temperature reduction. \[[@B13]\]

Kasner et al. conducted a partly blinded trial of paracetamol against placebo in 39 patients with haemorrhagic or ischemic stroke, and oberved a difference of 0.2°C in body temperature. The patients who received paracetamol tended to be more often hypothermic (\<36.5°C) and less often hyperthermic (\>37.5°C), but these results were not statistically significant. \[[@B14]\] In this study, a lower dose (3.9 g daily) was used than in our study (6 g daily).

Several studies in adults and children with fever suggest a larger antipyretic effect from ibuprofen than from paracetamol. \[[@B15]-[@B17]\] The effect of ibuprofen on body-core temperature in normothermic and subfebrile patients with stroke, who may have a disturbance of thermoregulation,\[[@B18]\] has not been studied.

Pharmacological properties and toxicity of ibuprofen
----------------------------------------------------

Ibuprofen is a prostaglandin-synthetase inhibitor with analgesic, anti-inflammatory, and antipyretic features. It belongs to the group of propionic acid derivatives. It is usually well tolerated, has almost no side effects or drug interactions at therapeutic doses, and it affects the platelet aggregation in doses of more than 1 g by reversible inhibition of cyclo-oxygenase.

After oral administration, ibuprofen is rapidly absorbed; the maximum concentration is reached after 1 to 2 hours. Rectal administration leads to a slightly slower absorption. Ibuprofen is bound for 90% to 99% to plasma proteins, but at usual concentrations only a small part of the binding sites is occupied.

Ibuprofen is mainly distributed in the plasma compartment; the distribution volume is approximately 0.15 I/kg body weight. Elimination through the urine is almost complete within 24 hours. The metabolites do not have any detectable activity. More than 90% of a dose is found back as metabolites and their conjugates in the urine. Less than 1% is excreted as unchanged ibuprofen. The plasma half-life is 1.5 to 2.5 hours. Gastrointestinal side effects, including epigastric pain, nausea, heartburn, and sensations of fullness, are experienced by 5 to 15% of patients taking ibuprofen. The frequency of these side effects is lower than that of indomethacin or aspirin. A meta-analysis even showed that there is no significant difference in adverse effects in patients treated with ibuprofen in daily doses up to 1200 mg compared with patients given placebo. \[[@B19]\] Estimates of the incidence of gastro-intestinal haemorrhage in patients with acute stroke range from 0.1% to 3%. \[[@B20],[@B21]\] Whether treatment with non-steroidal anti-inflammatory drugs (NSAID) increases the risk of this complication in these patients is likely, but unknown.

Other side effects have been reported incidentally. These include toxic amblyopia, fluid retention, edema, skin rashes, and thrombocytopenia. Several cases of aseptic meningitis, probably caused by ibuprofen, have been reported. Allopecia has been described in negroid women who also used relaxing hair lotions and steroids simultaneously. Teratogenic effects have not been reported. \[[@B22]\]

Pharmacological properties and toxicity of paracetamol
------------------------------------------------------

Paracetamol (N-acetyl-p-aminophenol) is a para-aminophenol derivative and the major metabolite of phenacetin. It has a weak inhibitory effect on peripheral biosynthesis (which accounts for its weak anti-inflammatory activity), but it is a potent inhibitor of prostaglandin production within the central nervous system. This presumably accounts for its analgesic and antipyretic properties. It is rapidly absorbed through the gastrointestinal tract and uniformly distributed through most body fluids. After oral administration peak plasma levels are reached after 30 min to 1 hour. \[[@B23]\] Rectal administration leads to a slightly slower absorption, but effective plasma levels are reached after approximately one hour. \[[@B24],[@B25]\] Acetaminophen is mainly conjugated in the liver, and then excreted in the urine. Plasma half-life is 1.5 to 3 hours. Following a dose of 140 mg/kg bodyweight or more, the glucuronide pathway may become saturated, and (hepato-) toxicity may result. In patients with chronic liver failure, the threshold for toxicity is lower. \[[@B23]\] These patients will therefore be excluded from the present study.

Methods and design
==================

This study is a three-armed, randomised, double-blind, placebo-controlled trial, performed in the university hospitals of Rotterdam and Utrecht, and in the regional hospital \"Eemland\" in Amersfoort, The Netherlands.

Aim of the study
----------------

To study the effect of high-dose ibuprofen and to confirm the previously observed reducing effect of high-dose paracetamol on body temperature, and to study their safety in normothermic and subfebrile patients with acute ischaemic stroke.

Study medication
----------------

Patients will be treated for 5 days with paracetamol or ibuprofen in daily doses of 6 × 1000 mg or 6 × 400 mg, respectively, or with placebo. The study drug will be administered through identical capsules. The first gift of medication may be given as a suppository. The study medication will be provided in white paper boxes, numbered consecutively with a medication number. Each box contains 70 identical capsules containing paracetamol 500 mg, ibuprofen 200 mg, or placebo, and one suppository, containing paracetamol 1000 mg, ibuprofen 400 mg, or placebo. Every gift of medication consists of 2 capsules except the first gift, which is one suppository. The study-population will be stratified according to the time since onset of stroke (0 to 12 hours vs 12 to 24 hours).

Randomisation and treatment schedule
------------------------------------

Treatment allocation will be random. The randomisation will be blocked in lots of six. A computer-generated list containing the medication number and information on the nature of the treatment (placebo, paracetamol, or ibuprofen) will reside with the independent safety committee and with the study pharmacist. Treatment allocation of any patient who is in the study will be disclosed before termination of the study if the safety committee deems this necessary. Local clinical investigators may apply for disclosure of the treatment allocation of an individual patient by the study pharmacist. This will be reported to the safety committee and to the steering committee by the study pharmacist.

Pain should not be treated with NSAID. We advise codeine, 4 to 6 times 10 mg daily, or tramadol, 4 times 25 to 50 mg daily. When the treating physician judges that fever should be treated in a patient, the study medication should be stopped. The clinical investigators are advised to prescribe low-dose aspirin in all patients.

Outcomes
--------

The primary outcome will be body temperature at 24 hours from start of treatment. Secondary outcomes will be change in baseline temperature at 1 and 5 days from start of treatment, and time with elevated body temperature (\>37.0°C) (area under the curve) during the first 24 hours and the first five days. The tertiary outcome is functional outcome at 1 month, as determined by the scores on the modified Rankin Scale (mRS) and Barthel Index (BI).

Setting
-------

Ziekenhuis Eemland Amersfoort is a 600 bed regional hospital. Annually, 150 patients with acute stroke are admitted. The University Medical Centre Utrecht has 1043 beds, and admits a similar number of patients with acute ischaemic stroke. The University Hospital Rotterdam has 1229 beds. Annually, 250 patients with acute stroke are admitted. All three hospitals have an acute stroke unit and a dedicated stroke team.

Study organization
------------------

The study personnel consists of an executive committee (RJM, EJB, DWJD, HMAvG, HBvdW), an advisory committee (PJK, UK), and an independent data-monitoring and safety committee.

The co-ordinating centre for data management is located in Rotterdam. The investigators are responsible for patient selection and inclusion, and data acquisition. The investigators will notify the data management centre of newly included patients and of serious adverse events within 72 hours.

The data-management centre is responsible for prompt data entry. Every week, a progress report entailing all serious adverse events (infection, gastro-intestinal haemorrhage, liver function disturbance, neurological deterioration, death, or other) will be provided by the executive committee. These data will be combined with the randomisation scheme by the secretary of the data-monitoring and safety committee, and summarised in a simple table. This table is forwarded to the other members of the data-monitoring and safety committee. The members will acknowledge receipt of the summary table. Upon receipt of the safety data, each member of the safety committee will notify the advisory committee of his approval of continuation of the study. If one or more members of the safety committee express a concern with regard to the safety of the study treatment, the chairman of the safety committee will summon the members of the safety committee and try to reach a consensus or majority advise. In light of the small number of patients in the study, no formal statistical stopping rule is given.

Time path
---------

In the previous study carried out in the same centres with similar inclusion criteria, it took 8 months to include 75 patients, i.e. 2 patients per week. It is therefore not unrealistic to take eight months for patient inclusion, one month to complete the follow-up and to close the database, and one month to report the study. The study has started in December 2000, and is expected to be completed at the end of the year 2001.

Inclusion criteria
------------------

Patients will be included if they have an acute ischaemic anterior circulation stroke, a body temperature \>36.0°C or \<39.0°C, a CT scan that is compatible with acute ischaemic stroke, a focal deficit without rapid improvement, and a possibility to start treatment within 24 h after stroke onset.

Exclusion criteria
------------------

Criteria for exclusion from this study are: 1) severe aphasia, defined as an aphasia score of 2 or 3 on the National Institutes of Health Stroke Scale (NIHSS); 2) treatment with an NSAID deemed necessary; 3) hypersensitivity to ibuprofen or paracetamol, (chronic) liver failure or cirrhosis; 4) (chronic) renal failure; 5) history of alcohol abuse; 6) active gastric ulcer disease or a history of peptic ulceration or gastro-intestinal hemorrhage in the preceding year; 7) colitis ulcerosa; 8) pregnancy; 9) use of corticosteroids; 10) a severe concomitant medical condition that could affect the assessment of the effect of the study medication on temperature; 11) residual neurological impairment resulting from a previous stroke that may hamper the assessment of functional outcome; 12) death appearing imminent, and 13) no informed consent given.

Study activities
----------------

In the first 24 hours, tympanic temperature will be measured in both ears every 2 hours. During the remaining 6 days, temperature will be measured twice a day. On the first day, rectal temperature will be measured at start of treatment (t = 0) and at t = 24 hours.

Day 0 is defined as the time period between onset of stroke and inclusion in the study. All baseline investigations are therefore carried out on day 0. Day 1 commences directly after the start of treatment with the study medication. All time-periods in the study are measured relative to the time of start of treatment.

At baseline, the medical history, including previous strokes or transient ischaemic attacks, will be assessed and a general and neurological examination will be carried out. The NIHSS is used to assess stroke severity at onset. The mRS is used to determine the patients\' pre-stroke functional status. Laboratory investigations will include a full blood count, glucose, electrolytes, creatinin, AST, ALT, AF, LDH, γGT, total bilirubin, APTT and PTT, and CRP. A brain CT will be done before inclusion in the study.

Body-temperature will be measured with a tympanic thermometer\[[@B26],[@B27]\] at 2-hourly intervals during the first 24 hrs after start of treatment, and thereafter every 12 hours until day 7. Temperatures will be taken from both ears, and averaged. The baseline temperature (at the start of treatment) and temperature at 24 hours after start of treatment will be determined by both rectal and tympanic thermometry. Compliance will be measured by counting leftover pills, and by a registry of the medication that was actually taken. Safety laboratory assessments will be carried out on day 6, including hemoglobin, AST, ALT, AF, LDH, γGT, total bilirubin and CRP.

At the follow-up visit on day 28, mRS is used to assess functional outcome. \[[@B28]\] The score on the BI will also be assessed. Based on the neurological examination and the results of CT, duplex, and other studies, etiological stroke type will be determined according to the (modified) Trial of Orgaran in Acute Stroke (TOAST) classification. \[[@B29]\]

Safety concerns and adverse events
----------------------------------

One should be careful not to overlook infections in patients who are treated according to the study protocol. Therefore, treating physicians are advised to lower their thresholds for clinical suspicion of infection to 38°C, to monitor the heart rate carefully, and start diagnostic studies and antibiotic treatment earlier than usual. The decision to start antipyretic mediciation in feverish patients is left to the discretion of the treating physician. On such an occasion, the study medication should be stopped.

Serious adverse events are defined as any deterioration in health status that is potentially life threatening, and death, within the study-monitoring period (day 0 to day 7). This includes any life-threatening infection, such as pneumonia, sepsis, or urinary tract infection that needs antibiotic treatment according to the judgement of the treating physician, any liver function disturbance (AST, ALT, AF, or total bilirubin levels exceeding twice the local upper limit of normal), gastro-intestinal haemorrhage, and neurological deterioration (i.e. decrease in consciousness level of more than one point on the Glasgow coma scale, or a decrease of two or more points on the NIHSS). All serious adverse events have to be reported within 72 hours to the independent data-monitoring and safety committee.

Ethical considerations
----------------------

Each patient will be given written information about the study. Informed consent will be obtained verbally and in writing before inclusion. If the patient is unable to write, written consent will be obtained from an appropriate source (spouse, child, or parent). The medical ethics committees of the three hospitals have approved this protocol.

Analysis
--------

The study results will be analysed on intent-to-treat basis, but an \"on-treatment\" analysis of primary and secondary endpoints will be carried out. There will be no formal interim analysis. The main results of the study will be presented as the mean difference in temperature between each treatment group and the placebo group. The precision of these estimates will be expressed with 95% confidence intervals, based on the *t* distribution. No adjustments for multiple comparisons will be made. Multiple linear regression will be used to adjust for possible confounding factors, such as age, stroke type, and stroke severity. The main analysis will be carried out according to the intent to treat principle, but an on treatment analysis will be provided. The analysis will be carried out with the STATA 7.0 statistical package. \[[@B30]\]

Sample size
-----------

Seventy-five (3 × 25) patients will be included in the study. In a recent cohort study of 162 patients admitted within 48 hours from onset, conducted at the University Hospital Rotterdam, mean rectal temperature was 36.6°C, with a standard deviation of 0.6°C. In order to detect a difference in core body temperature at 24 hours from onset of 0.5°C, with a significance level of α = 0.05, and power 1--β = 0.80, we need at least 23 patients in each treatment group; this is rounded to 25. The primary outcome occurs 24 hours after start of treatment. We expect more than 90% compliance in this period. We therefore need only 2 extra patients per treatment for lost patients in the on-treatment analysis.

Publication of study results
----------------------------

All publications will be authored by the members of the executive committee and the advisory committee. Co-authors are supposed to have contributed to the design, the analysis and actual reporting of the study; their centres are expected to include patients roughly proportional to the number of acute stroke patients that are admitted annually.

Discussion
==========

We report the protocol of a phase II trial that aims to assess the feasibility of early temperature reduction in normothermic or subfebrile patients with acute stroke. This study intends to confirm the previously observed temperature-reducing effect of paracetamol (6 g daily) and to assess the effect of ibuprofen 2.4 g daily.

The study is powered to find a difference of 0.5°C between active treatment and placebo. Half a degree Celsius does not seem much by itself, but body temperature has been shown to be a very strong prognostic factor in patients with acute stroke. For every degree Celsius of body temperature the risk of poor outcome increases by 2.2. The earlier the increase in temperature occurs, the stronger the relationship between brain damage and high temperature seems to be. \[[@B6],[@B7]\]

Because of the consistently observed relation between hyperthermia and poor outcome after stroke, it is commonly advised to treat all stroke patients with a raised temperature with antipyretic drugs. \[[@B31],[@B32]\] However, a causal relationship between hyperthermia and poor outcome has never been demonstrated, and subfebrile temperatures and fever may just be epiphenomena of severe brain damage. In addition, there is no evidence that a pharmacological reduction of increased temperatures will lead to an improved outcome. Preventive administration of antipyretics may even mask infections and lead to delayed treatment with antibiotics. For these reasons, we think that the efficacy of antipyretic drugs to improve outcome after stroke has to be proven in a randomised controlled trial before treatment with these agents is accepted as a standard procedure. For the same reasons, we consider it ethical to withhold antipyretics from stroke patients with subfebrile temperatures in the placebo arm of such a trial.

When the effect of paracetamol or ibuprofen on body temperature in acute stroke has been established, a large, pragmatic phase III study will be undertaken to assess the effect of these cheap and well known drugs on functional outcome after stroke.

The PISA investigators are
==========================

Executive committee: EJ van Breda, DWJ Dippel, RJ Meijer, HB van de Worp, HMA van Gemert. Data-management: EJ van Breda, H Hilkemeijer, L Kasijo. Statistical analysis: DWJ Dippel. Data monitoring and safety committee: A Algra, (chairman), J van Gijn, PA van Doom, A Koudstaal-Overduin (secretary). Advisory committee: PJ Koudstaal, LJ Kappelle. Writing committee: EJ van Breda, HB van der Worp, HMA van Gemert, RJ Meijer, LJ Kappelle, PJ Koudstaal, and DWJ Dippel. Participating centers: Ziekenhuis Eemland Amersfoort, The Netherlands: HMA van Gemert, A Hovestadt, JBS Boringa. University Hospital Rotterdam: DWJ Dippel, MPJ van Goor, RJ Meijer, D Siepman, E Maasland, SLM Bakker, F van Kooten, A Wijnhoud. University Medical Centre Utrecht: LJ Kappelle, HB van der Worp.

Competing interests
===================

None declared.

Financial support
=================

The study is funded by the Stichting Neurovasculair Onderzoek Rotterdam.

List of abbreviations
=====================

NSAID -- non-steroidal anti-inflammatory drug

mRS -- modified Rankin Scale

BI -- Barthel Index

NIHSS -- National Institutes of Health Stroke Scale

AST -- aspartate aminotransferase

ALT -- alanine aminotransferase

AF -- alkaline phosphatase

LDH -- lactate dehydrogenase

γGT -- gamma glutamyl transpeptidase

APTT -- activated partial thromboplastin time

PTT -- prothrombin time

CRP -- C-reactive protein

CT -- computed tomography

TOAST -- Trial of Orgaran in Acute Stroke

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

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Acknowledgements
================

Special thanks to DR Paul Janssen, pharmacologist at the University Hospital Rotterdam, who prepared the study medication and gave some useful advice on the study design, and to Dr Paul Mulder (Dept of Epidemiology and Biostatistics), who provided the randomisation codes.


Background
==========

The weak opioid codeine is widely used in the management of pain. Various studies have demonstrated a synergistic analgesic effect of an opioid-NSAID combination \[[@B1]-[@B4]\], particularly if repeated doses are given. \[[@B5]-[@B7]\] The synergistic effect is thought to be caused by the known different pharmacodynamic mechanisms, opioids acting via opioid receptors in the central nervous system, NSAIDs affecting the synthesis of prostaglandins due to inhibition of the enzyme cyclooxygenase. NSAIDs have also been postulated to display additional antinociceptive effects in the central nervous system. \[[@B8]\] Moreover, a synergistic effect is also possible via a pharmacokinetic interaction between the two classes of drugs: NSAIDs may decrease the renal excretion of the pharmacologically active metabolite of morphine M-6-G (morphine-6-glucuronide).\[[@B9]\] We have previously demonstrated a marked inhibition of codeine glucuronidation in human liver tissue homogenate by diclofenac (K~i~ of 7.9 μM).\[[@B10]\] Others have shown \[[@B11]\] a more than 50% inhibition of the glucuronidation of the codeine structural analogue, dihydrocodeine, by 50 μM diclofenac in vitro. Codeine is predominantly metabolised by glucuronidation to C-6-G (codeine-6-glucuronide) (Figure [1](#F1){ref-type="fig"}). Minor metabolic pathways include N-demethylation to norcodeine and O-demethylation to morphine.\[[@B12],[@B13]\] The latter is catalysed by the polymorphically expressed CYP2D6. \[[@B14]-[@B16]\] There is increasing evidence, that the analgesic effect of codeine is mediated by its O-demethylated metabolite morphine \[[@B17],[@B18]\] and that the glucuronidated metabolite M-6-G possesses even greater analgesic potency than morphine itself.\[[@B19]\] In humans, the analgesic activity of C-6-G has not been reported; however, antinociceptive responses after intracerebroventricular administration have been reported in rats. \[[@B20]\] Since in vitro findings may not necessarily be of clinical relevance, we aimed to investigate whether diclofenac inhibits codeine glucuronidation *in vivo* in healthy volunteers in terms of pharmacokinetics, analgesic efficacy and side effects.

![The chemical structures of codeine and its major metabolites in humans.](1472-6904-2-2-1){#F1}

Methods
=======

Subjects
--------

Twelve healthy male volunteers, median age 31 years (range 26--42 years), median weight 77 kg (range 63--95 kg), median BMI 23.9 kg/m^2^ (range 19.9--28.4 kg/m^2^) participated in the study. One subject had to be replaced because of additional drug intake during the study.

The study was approved by the ethics committee of the local medical board (Landesärztekammer Baden Württemberg) according to the Declaration of Helsinki (1996 Sommerset). All volunteers gave their written informed consent prior to study inclusion.

The volunteers were healthy according to history, physical examination and laboratory tests, had no history of drug abuse and did not take any regular medication. The volunteers had previously been genotyped for CYP2D6 by allele-specific PCR \[[@B21],[@B22]\] and all were predicted to be extensive metabolisers (EM). In addition, the subjects were phenotyped with respect to sparteine oxidation polymorphism with a single dose of 100 mg sparteine sulphate and were classified as EMs according to the metabolic ratio (MR) of sparteine and its 2- and 5-dehydrometabolite.\[[@B23]\] In order to limit the effect of the variability of CYP2D6 on metabolic capacity, volunteers with a MR\<1 were generally included in the study, with one subject having a MR of 2.8 (intermediate metaboliser).

Study design
------------

The study was designed as a randomised, placebo-controlled, double-blind, cross-over trial. Randomisation was performed using the computer program Sampsize 2.0 (Blackwell Science Ltd., Machin, Campbell, Fayers, Pinol). Each volunteer received on the study days 1 and 8 in random order either 100 mg codeine phosphate + 50 mg diclofenac sodium **or** 100 mg codeine phosphate + placebo. Codeine phosphate was provided as tablets (Codeinum Phosphoricum forte Compretten^®^, Glaxo Welcome GmbH/Cascan GmbH, Hamburg, Germany), diclofenac sodium and placebo were provided as capsules of identical appearance (manufactured by Contract Pharma GmbH & Co KG, Murr, Germany).

Blood and urine sampling
------------------------

On study days 1 and 8 the following identical procedures were carried out: Blood samples were taken before drug administration as well as 0.25, 0.5, 0.45, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 24, 35 and 36 hours after drug administration. Blood samples were centrifuged after 30 min. and serum was stored at -20°C until analysed. Urine was collected before drug administration and from 0--12, 12--24 and 24--36 hours after drug administration. The volume was measured and an aliquot of each collection was stored at -20°C until analysed.

Determination of analgesic effect
---------------------------------

The analgesic effects of codeine with and without simultaneous administration of diclofenac or placebo were assessed using the cold pressor test as previously described.\[[@B17]\] Briefly, the cold pressor test apparatus consisted of temperature-controlled water baths of 35 ± 0.5°C and 1.0 ± 0.5°C (ice-water bath). The nondominant forearm was placed into the warm-water bath for exactly 2 minutes. Fifteen seconds before transferring the forearm into the cold-water bath, a blood pressure cuff was inflated to 20 mmHg below the diastolic blood pressure and the eyes were covered with eye-patches. Subjects placed their forearm in a fixed position with the fingers wide apart into the cold-water bath (for a maximum time period of 2 min). They were instructed to clearly indicate the time of the first pain sensation as well as the time of intolerable pain, at which the forearm was removed from the cold-water bath. The time from the immersion of the forearm into the cold-water bath to the first pain sensation was measured, and is defined as the pain threshold. The pain tolerance is defined as the time from immersion of the forearm into the cold-water bath until the time of intolerable pain. A training session was carried out before the study to screen out volunteers with a pain tolerance of less than 15 sec or more than 120 sec and to familiarise the volunteers with the study procedure. The pain experiments were conducted on study days before drug administration (10--15 min before drug administration) and 1, 1.5, 2, 2.5, 3, 3.5, 4 and 6 hours after drug administration. Subsequently, the changes from baseline were determined and for each study day the area under the pain threshold change versus time curve and the area under the pain tolerance change versus time curve were calculated.

Determination of adverse events
-------------------------------

Adverse events were evaluated by the following method: The volunteers were required to list each symptom (fatigue, headache, dizziness, blurred vision, nausea, itching, exanthema and any other symptom) on a visual analogue scale (VAS) rated from 0 (not present) to 10 (most severe) at zero h (baseline, before drug intake), 2, 4 and 6 hours after drug intake. The values were corrected for the baseline measurement at t = 0 h.

Analytical measurement of codeine and its metabolites
-----------------------------------------------------

Codeine and its metabolites were determined using HPLC-electrospray mass spectrometry analogous to a previously published method\[[@B24]\] with minor modifications. Samples (1.0 ml of plasma, or 100 μl of urine diluted with 900 μl of water) were spiked with the deuterated internal standards (10 μl of standard mix with 50 pmol/μl codeine-G-glucuronide-d~3~, 5 pmol/μl codeine, 10 pmol/μl morphine-3-glucuronide-d~3~, 5 pmol/μl morphine-6-glucuronide-d~3~ and 1 pmol/μl morphine-d~3~) and extracted automatically using end-capped C~2~ solid-phase extraction columns. For the determination of the conjugates of norcodeine and normorphine, urine was hydrolysed\[[@B25]\] prior to extraction. The mobile phases used for HPLC were: (A) 1 % acetic acid in water and (B) 1 % acetic acid in acetonitrile. HPLC separation was achieved on a LiChrospher 100 RP-18 (Merck, Darmstadt, Germany) end-capped analytical column (125 × 3 mm I.D., 5 μm particle size) at a flow rate of 0.5 ml/min using a linear gradient from 8 % B to 40 % B in 8 min. The mass spectrometer (HP 1100 MSD, Hewlett-Packard, Waldbronn, Germany) was operated in the selected ion monitoring mode using the respective MH^+^ ions, *m/z* 476 for codeine-6-glucuronide, *m/z* 300 for codeine, *m/z* 303 for codeine-d~3~, *m/z* 286 for norcodeine, *m/z* 462 for morphine-3-glucuronide and morphine-6-glucuronide and *m/z* 465 for the deuterated morphine glucuronides, *m/z* 286 for morphine, *m/z* 289 for morphine-d~3~ and *m/z* 272 for normorphine. The limits of quantification achieved with this method were 0.5 pmol/ml for the morphine-glucuronides and morphine, 2 pmol/ml for codeine, normorphine and norcodeine and 5 pmol/ml for codeine-6-glucuronide with coefficients of variation less than 12 %.

Analytical measurements of diclofenac
-------------------------------------

Following the addition of 100 μl of a 10 μg/ml solution of 4\'-methoxydiclofenac in water for internal standardisation and 2 ml 10 mM phosphate buffer pH 6.5 to 1 ml of serum, samples were extracted by an automated solid phase extraction procedure using an ASPEC XL (Gilson, Villiers Ie Bel, France). Samples were applied on Bakerbond SPE™ C~18~ extraction columns (3 ml, 500 mg) which were equilibrated with 3 ml methanol and 3 ml of phosphate buffer. The columns were washed with 2 ml of phosphate buffer and 2 ml of phosphate buffer: methanol 6:4 (v/v) and eluted with phosphate buffer: methanol 2:8 (v/v). The solvents were evaporated and the residue was resuspended in 150 μl of the mobile phase. A 100 μl aliquot was injected on a HPLC-system consisting of an autosampler SIL 9A, a solvent delivery system LC 9A and an UV-detector (Shimadzu, Duisburg, Germany). Separation was performed on a Waters Spherisorb ODS1 5 μm 125 × 4.6 mm column equipped with a guard column filled with the same material (Bischoff, Leonberg, Germany) using 8 mM tetrabutylammonium bromide in 10 mM phosphate buffer pH 6.5: 65:30:5 (v/v/v) at a flow rate of 1 ml/min. Diclofenac and the internal standard 4\'-methoxydiclofenac were detected at 282 nm at a retention time of 11.0 and 12.4 min, respectively. Recovery was between 85 and 100.5%. Calibration curves were linear over a concentration range from 10 to 2000 ng/ml (r^2^ ranging from 0.9993 to 1). Inter-assay variability (n = 9) was 14.2, 8.3, and 7.8% for 30, 300, and 2000 ng/ml, respectively. Accuracy was within 10% at these concentrations.

Pharmacokinetic evaluation
--------------------------

Standard noncompartmental analysis for calculations using serum concentration-time data was performed using TOPFIT 2.0 (Gustav Fischer Verlag 1993).

The following pharmacokinetic parameters were determined from serum concentration-time data and urine concentration data for codeine (cod), codeine-6-glucuronide (C-6-G), norcodeine (NC), morphine (morph), morphine-3-glucuronide (M-3-G), morphine-6-glucuronide (M-6-G) and normorphine (NM):

C~max~- peak serum concentration \[pmol ml^-1^\], obtained from the visual inspection of the serum concentration-time curves

t~max~- time to reach peak serum concentrations \[h\], obtained from the visual inspection of the serum concentration-time curves

t~1/2~- terminal phase half-life \[h\] calculated according to ln(2)/λ~z~, the terminal elimination rate constant λ~z~ was determined from the slope of the regression line of In(concentration) vs time

AUC~0-∞~- area under the serum concentration curve \[pmol ml^-1\*^h\], calculated by the trapezoidal rule, the segment to infinity was calculated from the last concentration measured by dividing the last concentration measured by the elimination rate constant (λ~z~)

ratio AUC\' s- AUC~0-∞~ codeine/AUC~0-∞~ morphine

Ae- cumulative amount of codeine, C-6-G, norcodeine, norcodeine-glucuronide, morphine, M-3-G, M-6-G, normorphine and normorphine-glucuronide \[% of dose\] excreted in urine

CLo- apparent oral clearance of codeine calculated using dose/AUC~0-∞~ \[ml min^-1^\]

CL~cod→c-6-G~- metabolic clearance of codeine to C-6-G calculated as Ae C-6-G/AUC~0-∞~ codeine \[ml min^-1^\]

CL~cod→NC~- metabolic clearance of codeine to norcodeine calculated as (Ae norcodeine + Ae norcodeine-glucuronide)/AUC~0-∞~ codeine \[ml min^-1^\]

CL~cod→morph~- metabolic clearance of codeine to morphine calculated as (Ae morphine + Ae M-3-G + Ae M-6-G + Ae normorphine + Ae normorphine-glucuronide)/AUC~0-∞~ codeine \[ml min^-1^\]

CL~morph→M-3-G~- metabolic clearance of morphine to M-3-G calculated as Ae M-3-G/AUC~0-∞~ morphine \[ml min^-1^\]

CL~morph→M-6-G~- metabolic clearance of morphine to M-6-G calculated as Ae M-6-G/AUC~0-∞~ morphine \[ml min^-1^\]

CL~morph→NM~ metabolic clearance of morphine to normorphine calculated as (Ae normorphine + Ae normorphine-glucuronide)/AUC~0-∞~morphine \[ml min^-1^\]

CL~ren\ cod~- renal clearance of codeine calculated as Ae codeine/AUC~0-∞~ codeine \[ml min^-1^\]

CL~ren\ morph~- renal clearance of morphine calculated as Ae morphine/AUC~0-∞~ morphine \[ml min^-1^\]

CL~ren\ C-6-G~- renal clearance of C-6-G calculated as Ae C-6-G/AUC~0-∞~ C-6-G \[ml min^-1^\]

CL~ren\ NC~- renal clearance of norcodeine calculated as Ae norcodeine/AUC~0-∞~ norcodeine \[ml min^-1^\]

CL~ren\ M-3-G~- renal clearance of M-3-G calculated as Ae M-3-G/AUC~0-∞~ M-3-G \[ml min^-1^\]

CL~ren\ M-6-G~- renal clearance of M-6-G calculated as Ae M-6-G/AUC~0-∞~ M-6-G \[ml min^-1^\]

CL~ren\ NM~- renal clearance of normorphine calculated as Ae normorphine/AUC~0-∞~ normorphine \[ml min^-1^\]

Statistical analysis
--------------------

In this cross over study, 12 subjects were required to ensure a 80% power of detecting a potentially clinically relevant difference in pharmacokinetic parameters of 30% with a variability of 25% at the 5% significance level for α. Pharmacokinetic data are presented as mean ± 95% CI. Pharmacokinetic data of codeine and its metabolites after administration of codeine + placebo vs. codeine + diclofenac are compared using the Wilcoxon matched pairs test. In addition, AUC and C~max~ of codeine and C-6-G were investigated in terms of bioequivalence: Point estimates (geometric means) and 90% CI were given for the ratios test/reference (test: pharmacokinetic parameters under consideration after codeine + diclofenac, reference: pharmacokinetic parameters under consideration after codeine + placebo). Test was considered bioequivalent to reference if 90% CI of log transformed AUC ratios were within 0.80--1.25 and C~max~ ratios were within 0.75--1.34.

Adverse events are presented in two different manners: 1.) The number of subjects reporting *any* side effect after administration of codeine + placebo vs. codeine + diclofenac were compared using the chi square test. 2.) At different time points (2, 4 and 6 h after drug administration) VAS-rated mean sum scores of all reported side effects after administration of codeine + placebo vs. codeine + diclofenac were calculated; the mean sum scores are compared using the paired Wilcoxon matched pairs test at the different time points. The area under the pain threshold versus time curve and the area under the pain tolerance versus time curve (corrected to baseline at t = 0 h) are presented as mean ± SD and are compared using Wilcoxon matched pairs test. A p value of less than 0.05 was considered significant.

Results
=======

Pharmacokinetic data
--------------------

Similar serum concentrations of codeine and its metabolite C-6-G were observed after treatment with codeine + placebo vs. codeine + diclofenac (Figure [2](#F2){ref-type="fig"}). Peak serum concentrations (C~max~) of codeine (764; 602--924 pmol ml^-1^ vs. 821; 663--980 pmol ml^-1^) and C-6-G (5263; 4738--5788 pmol ml^-1^ vs. 5621; 5167--6074 pmol ml^-1^), did not differ significantly between the two different treatments, nor did the time to attain peak serum concentrations (t~max~) and the terminal half-life t~1/2~ (Table [1](#T1){ref-type="table"}). The concentrations of codeine and its metabolites in the one intermediate metaboliser (MR = 2.8) were within the range of the concentrations of the other volunteers with MR\<1, however as expected the metabolites formed via O-demethylation (morphine, morphine glucuronides and normorphine) were the lowest or second lowest observed.

![Serum concentration time profile of codeine and codeine-6-glucuronide after administration of codeine + placebo vs. codeine + diclofenac (Mean, SD, n= 12) □ codeine + placebo ♦ codeine + diclofenac](1472-6904-2-2-2){#F2}

::: {#T1 .table-wrap}
::: {.caption}
######

Time to attain peak serum concentrations (t~max~) and terminal half-life of codeine and its metabolites (n = 12).
:::

                                codeine + placebo mean (95% CI)   codeine + diclofenac mean (95% CI)   p level
  ----------------------------- --------------------------------- ------------------------------------ ---------
  t~max~ codeine \[h\]          0.69 (0.51--0.87)                 0.71 (0.51--0.91)                    n.s.
  C~max~ codeine \[pmol/ml\]    764 (602--924)                    821 (663--980)                       n.s.
  t~1/2~ codeine \[h\]          3.37 (3.16--3.59)                 3.43 (3.24--3.62)                    n.s.
  t~max~ C-6-G \[h\]            1.13 (0.89--1.35)                 1.10 (0.87--1.34)                    n.s.
  C~max~ C-6-G \[pmol/ml\]      5263 (4738--5788)                 5621 (5167--6074)                    n.s.
  t~1/2~ C-6-G \[h\]            3.54 (3.39--3.68)                 3.54 (3.36--3.73)                    n.s.
  t~max~ morphine \[h\]         0.63 (0.45--0.80)                 0.67 (0.53--0.81)                    n.s.
  C~max~ morphine \[pmol/ml\]   22.4 (12.3--32.5)                 24.3 (13.7--35.0)                    n.s.
  t ~1/2~ morphine \[h\]        8.78 (5.97--11.59)                8.54 (6.74--10.61)                   n.s.
  t~max~ M-6-G \[h\]            1.5 (1.37--1.64)                  1.35 (1.06--1.65)                    n.s.
  C~max~ M-6-G \[pmol/ml\]      63.4 (38.7--88.1)                 68.8 (41.2--96.5)                    n.s.
  t~1/2~M-6-G \[h\]             7.77 (7.40--8.13)                 8.10 (7.57--8.63)                    n.s.
:::

The AUCs of codeine did not differ between the two treatments (Table [2](#T2){ref-type="table"}). In addition, the two treatment, based on AUCs and C~max~ of codeine and C-6-G, could be considered bioequivalent: Point estimates of the test/reference ratios and 90% CI were 1.007 (0.998--1.017) for AUC codeine, 1.004 (1.001--1.008) for AUC of C-6-G, 1.015 (0.990--1.042) for C~max~ of codeine and 1.008 (1.001--1.016) for C~max~ of C-6-G. However, a small but significant increase in the AUC of C-6-G (+ 5.5%) was observed in subjects after codeine + diclofenac (Table [2](#T2){ref-type="table"}). In terms of morphine formation, the ratios of AUC codeine/AUC morphine did not reveal a significant difference (53.4; 10.5--96.4 95% CI vs. 45.2; 16.4--74.0 95% CI) between the two treatments.

::: {#T2 .table-wrap}
::: {.caption}
######

Area under the serum concentration time curve (AUC~0-∞~) of codeine and its metabolites (n = 12).
:::

  \[pmol ml^-1\*^h\]   codeine + placebo mean (95% CI)   codeine + diclofenac mean (95% CI)   p level
  -------------------- --------------------------------- ------------------------------------ ---------
  AUC codeine          2416 (2025--2807)                 2515 (2176--2855)                    n.s.
  AUC C-6-G            29550 (26190--32910)              31120 (28150--34090)                 \< 0.05
  AUC norcodeine       342 (257--428)                    338 (228--448)                       n.s.
  AUC morphine         94.5 (52.9--136)                  92.1 (53.8--131)                     n.s.
  AUC M-3-G            2137 (1298--2976)                 2259 (1427--3091)                    n.s.
  AUC M-6-G            425 (254--596)                    471 (298--644)                       \< 0.05
  AUC normorphine      540 (396--684)                    549 (387--710)                       n.s.
:::

Peak serum concentrations (C~max~) of morphine (22.4 pmol ml^-1^; 12.3--32.5 95% CI vs. 24.3 pmol ml^-1^; 13.7--35.0 95% CI) and M-6-G (63.4 pmol ml^-1^; 38.7--88.1 95% CI vs. 68.8 pmol ml^-1^; 41.2--96.5 95% CI) did not differ significantly between the two different treatments, as well as the time to attain peak serum concentrations (t~max~) and the terminal half-life t~1/2~ (Table [1](#T1){ref-type="table"}). AUC of morphine did not differ between the two treatments (Table [2](#T2){ref-type="table"}). A small but significant increase of the AUC of M-6-G (+10.8%) was observed after codeine + diclofenac (Table [2](#T2){ref-type="table"}). Serum concentration-time curves of morphine, M-3-G and M-6-G are displayed in Figure [3](#F3){ref-type="fig"}.

![Serum concentration time profile of morphine, morphine-3- and morphine-6-glucuronide after administration of codeine + placebo vs. codeine + diclofenac (Mean, SD, n= 12) □ codeine + placebo ♦ codeine + diclofenac](1472-6904-2-2-3){#F3}

No significant differences were observed in either the apparent oral clearance of codeine (CLo) (1805 ml min^-1^; 1498--2112 95% CI vs. 1700 ml min^-1^; 1470--1929 95% CI), metabolic clearance of codeine to C-6-G, norcodeine and morphine, of morphine to M-3-G, M-6-G and normorphine or renal clearance of codeine and its metabolites between the two treatments (Tables [3](#T3){ref-type="table"} and [4](#T4){ref-type="table"}). The total amount excreted of codeine and its metabolites excreted was 81.3% of dose (76.3--86.3 95% CI) vs. 84.9% of dose (81.8--88.0 95% CI) and did not differ between the two treatment groups. The urinary recoveries of codeine and its metabolites are listed in Table [5](#T5){ref-type="table"}.

::: {#T3 .table-wrap}
::: {.caption}
######

Partial metabolic clearances of codeine and morphine after administration of 100 mg codeine phosphate (n = 12).
:::

  Cl~met~ \[ml min^-1^\]   codeine + placebo mean (95% CI)   codeine + diclofenac mean (95% CI)   p level
  ------------------------ --------------------------------- ------------------------------------ ---------
  codeine → C-6-G          1134 (882--1385)                  1104 (928--1279)                     n.s.
  codeine → morphine       174 (118--230)                    176 (126--226)                       n.s.
  codeine → norcodeine     102 (69.1--136)                   97.1 (76.2--118)                     n.s.
  morphine → M-3-G         2631 (1227--4036)                 2550 (1527--3573)                    n.s.
  morphine → M-6-G         702 (362--1042)                   692 (446--938)                       n.s.
  morphine → normorphine   3090 (431--5749)                  2600 (1456--3744)                    n.s.
:::

::: {#T4 .table-wrap}
::: {.caption}
######

Renal clearances of codeine and metabilites after administration of 100 mg codeine phosphate (n = 12).
:::

  Cl ~ren~ \[ml min^-1^\]   codeine + placebo mean (95% CI)   codeine + diclofenac mean (95% CI)   p level
  ------------------------- --------------------------------- ------------------------------------ ---------
  codeine                   66.5 (54.6--78.3)                 64.2 (53.6--74.9)                    n.s.
  C-6-G                     88.6 (76.4--101)                  86.6 (78.6--94.5)                    n.s.
  norcodeine                220 (187--253)                    233 (208--259)                       n.s.
  morphine                  117 (72.4--162)                   114 (83.6--144)                      n.s.
  M-3-G                     88.2 (76.1--100)                  86.2 (77.1--95.4)                    n.s.
  M-6-G                     122 (108--136)                    114 (101--127)                       n.s.
  normorphine               123 (106--141)                    133 (115--152)                       n.s.
:::

::: {#T5 .table-wrap}
::: {.caption}
######

Urinary excretion of codeine and metabolites after administration of 100 mg codeine phosphate (n = 12).
:::

  Ae \[% of dose\]          codeine + placebo mean (95% CI)   codeine + diclofenac mean (95% CI)   p level
  ------------------------- --------------------------------- ------------------------------------ ---------
  codeine                   3.74 (3.33--4.15)                 3.89 (3.10--4.68)                    n.s.
  C-6-G                     62.2 (57.0--67.5)                 64.7 (60.7--68.7)                    n.s.
  norcodeine                1.76 (1.35--2.18)                 1.86 (1.36--2.36)                    n.s.
  norcodeine-glucuronide    3.87 (3.01--4.73)                 4.01 (3.02--5.00)                    n.s.
  morphine                  0.22 (0.13--0.32)                 0.23 (0.14--0.33)                    n.s.
  M-3-G                     4.40 (2.81--5.99)                 4.66 (3.10--6.22)                    n.s.
  M-6-G                     1.21 (0.77--1.64)                 1.31 (0.83--1.78)                    n.s.
  normorphine               1.56 (1.91--1.93)                 1.73 (1.34--2.11)                    n.s.
  normorphine-glucuronide   2.30 (1.80--2.79)                 2.50 (1.99--3.01)                    n.s.
:::

Diclofenac was detected in the serum of all subjects after codeine + diclofenac with mean peak serum concentration of 4.4 nmol/ml (3.77--5.06 95% CI) after 1.73 h (1.21--2.26 95% CI) (see Table [6](#T6){ref-type="table"}).

::: {#T6 .table-wrap}
::: {.caption}
######

Pharmacokinetic parameters of diclofenac after oral administration of 50 mg diclofenac sodium together with 100 mg codeine phosphate (n = 12).
:::

                       mean (95% CI)
  -------------------- -------------------
  C~max~ \[nmol/ml\]   4.42 (3.77--5.06)
  t~max~ \[h\]         1.73 (1.21--2.26)
  AUC \[h nmol/ml\]    5.63 (4.77--6.50)
  t~1/2~ \[h\]         0.97 (0.68--1.26)
:::

Pharmacodynamic data and adverse events
---------------------------------------

The area under the pain threshold-time as well as the area under the pain tolerance-time curves did not differ significantly after administration of codeine + placebo vs. codeine + diclofenac (8.68 ± 12.7 vs. 4.87 ± 9.82 s\*h and 23.3 ± 57.5 vs. 13.9 ± 35.6 s\*h, respectively). The time courses of pain threshold and pain tolerance are displayed in Figure [4](#F4){ref-type="fig"}.

![Pain threshold after administration of codeine + placebo vs. codeine + diclofenac, corrected to baseline at t = 0 (Mean, SD, n= 12) ▪ codeine + placebo • codeine + diclofenac. **B** Pain tolerance after administration of codeine + placebo vs. codeine + diclofenac, corrected to baseline at t = 0 (Mean, SD, n= 12) ▪ codeine + placebo • codeine + diclofenac](1472-6904-2-2-4){#F4}

Both codeine + placebo and codeine + diclofenac were well tolerated by the volunteers. Only minor side effects occurred and no rating higher than 5 was reported on a VAS scale. The number of subjects reporting any adverse event from VAS rated from 0 (not present) to 10 (most severe) at 2, 4 and 6 hours after drug intake is displayed in table [7](#T7){ref-type="table"} and did not differ significantly between the two treatments. Also, no significant differences could be observed concerning the mean sum scores of all reported side effects at the given time points (Table [8](#T8){ref-type="table"}).

::: {#T7 .table-wrap}
::: {.caption}
######

Number of subjects reporting any adverse event
:::

                   codeine + placebo   codeine + diclofenac
  ---------------- ------------------- ----------------------
  fatigue          10                  8
  headache         4                   1
  dizziness        6                   5
  blurred vision   3                   3
  nausea           2                   2
  itching          0                   0
  exanthema        0                   0
  others           0                   0
:::

::: {#T8 .table-wrap}
::: {.caption}
######

Mean of sum scores of all adverse events
:::

  time after drug administration   codeine + placebo mean ± SD   codeine + diclofenac mean ± SD
  -------------------------------- ----------------------------- --------------------------------
  2 h                              4.3 ± 4.4                     2.3 ± 2.1
  4 h                              1.6 ± 1.5                     1.3 ± 1.7
  6 h                              2.9 ± 2.8                     1.7 ± 1.7
:::

Discussion
==========

The possible pharmacokinetic interaction between codeine and diclofenac could have clinical implications: One could have speculated that by inhibiting codeine glucuronidation other metabolic pathways of codeine, especially O-demethylation to morphine may then be favoured in extensive metabolizers of CYP2D6, resulting in elevated morphine serum levels and a greater analgesic effect, and, possibly also increased adverse effects. A direct influence of diclofenac on O-demethylation of codeine to morphine via interaction with CYP2D6 has been excluded.\[[@B26]\]

Considering our previous in vitro data\[[@B10]\], it was important to verify whether codeine glucuronidation was inhibited by diclofenac in vivo after administration of a commonly used dose.

However, after single dose administration of codeine + diclofenac in healthy volunteers we did not observe major changes in the pharmacokinetics of codeine and its metabolites compared to codeine + placebo. In this study, codeine and diclofenac were given in a dose commonly used for clinical treatment of pain. In addition, no significant differences in the ratios AUC codeine/ AUC morphine were detected after the two different treatments, indicating that the formation of morphine from codeine remained unaffected. A slight increase in the AUCs of C-6-G (+5.5%) and M-6-G (+10.8%) after the diclofenac containing regimen was observed, reaching statistical significance (p \< 0.05). This might be a result of inhibition of renal excretion of both glucuronides by diclofenac or its glucuronide. In our opinion, these minor changes are not of clinical relevance after single dose administration. Nevertheless, the observed non significant decrease in renal clearance especially of M-6-G in the diclofenac containing regimen, might play a role during chronic treatment with opioids.

In this study, we did not investigate a possible influence of codeine on the pharmacokinetics of diclofenac because it has been previously demonstrated that codeine does not influence the relative bioavailability of diclofenac in vivo.\[[@B27]\] The pharmacokinetic results obtained from this study do not confirm our previous in vitro data with human liver tissue homogenates, which revealed a marked non-competitive inhibition of codeine-6-glucuronidation caused by diclofenac (K~i~ of 7.9 μM in a concentration range of 100--10000 μM codeine and 0.5--100 μM diclofenac).\[[@B10]\] Although UGT (UDP-glucuronosyltransferase) 2B7, the UGT catalysing codeine-6-glucuronidation, morphine-6-glucuronidation and diclofenac glucuronidation \[[@B28]\], is known to be expressed in the intestine,\[[@B29]\] we did not favour a potential interaction of codeine-6-glucuronidation at the level of the small intestine because of its very low intrinsic clearance compared to liver tissue in vitro.\[[@B10]\]

The pharmacokinetic data for codeine and its metabolites obtained in our study are supported by the data obtained by other investigators.\[[@B12],[@B13],[@B30]\] The codeine peak serum concentrations observed in our study ranged from 517 to 1481 pmol ml^-1^ and therefore did not reach the concentrations used in our previous in vitro investigation.

Kirkwood et al. could demonstrate a more than 50% inhibition of the glucuronidation of the codeine structural analogue dihydrocodeine by 50 μM diclofenac. \[[@B11]\] In our study, mean peak serum concentrations of diclofenac were 4.4 nmol/ml (range 3.01--6.21 nmol/ml) and were in the same range as reported by Davies et al.\[[@B31]\] Mean t~max~ of diclofenac was 1.73 h, so peak serum concentrations of diclofenac were later than the t~max~ of C-6-G and M-6-G (table [1](#T1){ref-type="table"}). In addition, diclofenac is known to be highly bound to serum protein (\>99.7%).\[[@B31]\] Therefore it is likely that diclofenac, at least after administration of a common single dose, does not achieve serum levels fast enough and high enough to inhibit codeine glucuronidation in vivo. Furthermore, it is unlikely that a multiple dose regimen of diclofenac (e.g. 50 mg tid) would lead to the cumulation of diclofenac serum levels due to the short elimination half-life of diclofenac (mean t~1/2~ 0.97 h in our study). Therefore, in our opinion, steady-state administration of diclofenac will also not alter codeine glucuronidation in vivo. However, the data obtained in this study cannot exclude a pharmacokinetic / pharmacodynamic interaction at steady-state. Concerning codeine glucuronidation, our pharmacokinetic results demonstrate that in vitro conditions studied did not necessarily reflect the in vivo conditions at therapeutic doses. In terms of side effects, both codeine + placebo as well as codeine + diclofenac were well tolerated by the volunteers in our study. Only minor side effects occurred and no rating higher than 5 was reported on a VAS scale. There are several studies demonstrating the benefit of the combination of NSAIDs plus opioids in comparison to opioids alone in the treatment of postsurgical pain, pain induced by arthrosis and chronic pain in cancer patients. \[[@B1]-[@B7]\] In one study, after a single dose of 100 mg diclofenac rectally given to postsurgical patients receiving morphine PCA, a significant decrease in hourly morphine consumption and plasma morphine and morphine-6-glucuronide were observed after administration of diclofenac, but with a lag time of more than 5 hours.\[[@B32]\]

In our study, we used the cold pressor test for analysing the analgesic effects of opioids because it has been proven to be sensitive to the effects of codeine and morphine in several studies.\[[@B17],[@B18]\] In contrast, this test is not appropriate to investigate the efficacy of NSAIDs alone.\[[@B33]\] In our study, the area under the pain threshold versus time curve and the area under the pain tolerance versus time curve did not differ significantly after the two treatments over the time intervals studied. In case of a pharmacokinetic interaction between codeine and diclofenac leading to higher serum levels of morphine and M-6-G, an increase in pain threshold and pain tolerance would have been postulated. A placebo part of the study was omitted because the primary objective was the assessment of a pharmacokinetic interaction.

Because we could exclude a pharmacokinetic interaction between codeine and diclofenac, the synergistic analgesic effects in the above mentioned studies are likely to result solely from the different pharmacodynamic mode of actions. Most postsurgical patients and many cancer patients have an inflammatory component to their pain, which responds to cyclooxygenase inhibition. Furthermore, NSAIDs have been reported to have a specific effect in malignant bone pain. \[[@B34]\]

Conclusions
===========

A single dose of diclofenac does not alter the glucuronidation of codeine in healthy volunteers, which is in contrast to recent in vitro data. The formation of morphine from codeine was not affected. The combination of codeine and diclofenac was well tolerated.

List of abbreviations used
--------------------------

CI: confidence interval

MR: metabolic ratio

NSAIDs: non steroidal anti-inflammatory drugs

C-6-G: codeine-6-glucuronide

M-6-G: morphine-6-glucuronide

UGT: UDP-glucuronosyltransferase

Competing interests
===================

None declared

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

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Acknowledgements
================

We are grateful for the excellent technical assistance of Mrs. Anja Riebe, Mrs. Erika Schneider, Mrs. Sonja Seefried and Mrs. Monika Seiler.

Funding: The study was fully supported by the Robert Bosch-Foundation, Stuttgart, Germany