Pentazocine

The S1R model protein was taken from PDB entry 6DJZ, S1R with haloperidol. The protein is a trimeric membrane protein containing a cupin fold [50 (link)] with each protomer bound to haloperidol and an N-terminal alpha helix that is inserted into the membrane. First, the protein was prepared for docking using the protein preparation tools of Sybyl (Tripos Corp). Missing side-chains were added in structurally reasonable positions. None of these were in the active site. The haloperidol was removed and hydrogens were added. The two carboxylates in the active site, Aps126 and Glu172, were in the deprotonated state. Minor side-chain clashes were repaired visually. Gatseiger–Huckel charges were added and the structure energy-minimized in steps: hydrogens alone, then side-chains and backbone, followed by the whole molecule. The Tripos force field was used with Simplex initial optimization, followed by Powell and terminated when the gradient reached 0.05 Kcal/mole. The molecule was then removed and used for docking experiments.
Dockings were performed with Autodock4 [106 (link)] utilizing AutodockTools. A box of approximate dimensions 30 × 30 × 30 angstroms was centered in the cupin pocket and included the majority of the pocket as well as the alpha 4 and alpha 5 helices that are juxtaposed on the membrane surface. This is including His154, Asp126, and Glu172. Both (+) pentazocine, an agonist, and haloperidol, an antagonist, were used as controls to validate that the docking procedure was correct. Haloperidol, with a proton on the nitrogen and, thus, positively charged, was found to dock in a similar way as found in the crystal structure 6DJZ with the nitrogen hydrogen-bonded to Glu172. Autodock produced the top 30 lowest energies and clustered them into groups. The docking reproduced the crystal structure in the top 10 best (lowest energy) examined. Initially, (+)-pentazocine would not dock into 6DJZ, because it interfered with Ala185 in helix 4. When helix 4 was moved slightly and energy-minimized, (+)-pentazocine docked as found in the crystal structure 6DK1, with the top 10 best dockings reproducing the interactions of the positively charged ring nitrogen interacting with Glu172. In both control compounds, Asp126 was deprotonated. The Autodock scoring is an addition of 4 intermolecular-contributing Kcal/mol energy terms as the sum of Intermolecular Energy (VdW + Hbond + desolvation + electrostatics), plus total internal energy, plus torsional energy, minus the unbound system energy. The comparison docking of (+)-pentazocine and SPH is shown in Figure 6. The positively charged nitrogen hydrogen bonded to the Glu172 as expected. In some of the less energetic dockings, the Tyr120 hydroxyl proton is involved in interactions, but Tyr103 hydroxyl is donated to the carbonyl in the interior of the binding site, stabilizing the orientation of the carboxylate on Glu172. The docking of SPH and DMS was performed under the same. The two endogenous sphingolipids, SPH and DMS, docked similarly to the control (+)-pentazocine in 6DJZ S1R. In all dockings, the protein was static and the ligand was randomly torsioned and then docked into the protein in the defined box.

Knowledge was measured by adapting the Palliative Care Knowledge Test (PCKT) questionnaire [10 (link)]. The questionnaire has twenty items and is divided into five domains: philosophy (items 1 and 2), pain (items 3 to 8), dyspnoea (items 9 to 12), psychiatric problems (items 13 to 16), and gastrointestinal problems (items 17 to 20). Participants answer every question as either “true”, “false”, or “unsure”. One point is given for a correct answer and zero points for incorrect and “unsure” answers. The total score ranges from 0 to 20 and is converted into a mean and standard deviation (SD). A higher score indicates higher knowledge. The translated English version was used in this study and a few modifications were made to suit the local setting. Three items regarding specific drug use (pentazocine and buprenorphine hydrochloride) were substituted since these drugs are not widely used in Malaysia. Item number four, pentazocine, was changed to oxycodone as the latter is more commonly used for pain control in Malaysian settings. Item number six, which is “The effect of opioids should decrease when pentazocine or buprenorphine hydrochloride is used together after opioids are used”, was changed to “Even if breakthrough pain occurs when opioids are taken on a regular basis, the next dose should not be given earlier than scheduled.” Item number 12, “Anticholinergic drugs or scopolamine hydrobromide are effective for alleviating bronchial secretions of dying patients” was changed to “Evaluation of dyspnoea should be based on the subjective report of patients”. Preliminary testing of the questionnaire was done in a pilot study yielding a Cronbach alpha of 0.7.
Attitudes were measured using Frommelt Attitude Toward Care of the Dying-form B (FATCOD Form B) [11 ]. This form has been used and evaluated for validity and reliability, and it has demonstrated an interrater agreement of 1.0 and a Pearson’s Coefficient of 0.93 for test-retest reliability. FATCOD Form B consists of 30 Likert-type items scored on a five-point scale from 1 (Strongly Disagree), 2 (Disagree), 3 (Uncertain), 4 (Agree), to 5 (Strongly Agree). The instrument is made up of an equal number of positively and negatively worded items. Items 1, 2, 4, 16, 18, 20, 21, 22, 23, 24, 25, 27, and 30 are positively-worded statements. All others were negatively worded. The total score, ranging from 30 to 150, was converted into a mean and standard deviation with higher scores indicating more positive attitudes.