The safe disposal of unused opioid drugs is an area of regulatory concern. While toilet flushing is recommended for some drugs to prevent accidental exposure, there is a need for data that can support a more consistent disposal policy based on an assessment of relative risk. For drugs acting at the Mu-opioid receptor (MOR), published measurements of binding affinity (K(i)) are incomplete and inconsistent due to differences in methodology and assay system, leading to a wide range of values for the same drug thus precluding a simple and meaningful relative ranking of drug potency. Experiments were conducted to obtain K(i)'s for 19 approved opioid drugs using a single binding assay in a cell membrane preparation expressing recombinant human MOR. The K(i) values obtained ranged from 0.1380 nM (sufentanil) to 12.486 μM (tramadol). The drugs were separated into three categories based upon their K(i) values: K(i)> 100 nM (tramadol, codeine, meperidine, propoxyphene and pentazocine), K(i)=1-100 nM (hydrocodone, oxycodone, diphenoxylate, alfentanil, methadone, nalbuphine, fentanyl and morphine) and K(i) < 1 nM (butorphanol, levorphanol, oxymorphone, hydromorphone, buprenorphine and sufentanil). These data add to the understanding of the pharmacology of opioid drugs and support the development of a more consistent labeling policies regarding safe disposal.

Aging of organophosphorus (OP)-compound-inhibited neuropathy target esterase (NTE) is the critical event that initiates OP-compound-induced delayed neurotoxicity (OPIDN). Aging has classically been considered to involve side-group loss from phosphylated NTE, rendering the enzyme refractory to reactivation. N,N'-Diisopropylphosphorodiamidofluoridate (mipafox, MIP)-inhibited NTE has been thought to age quickly; however, it can be reactivated under acidic conditions. The present study was undertaken to determine whether MIP-inhibited human recombinant NTE esterase domain (NEST) ages classically by isopropylamine loss. Diisopropylphosphorofluoridate (DFP), the oxygen analogue of MIP, was used for comparison. Kinetic values for DFP against NEST were as follows: k(i) = 17 200 +/- 180 M(-1) min(-1); reactivation t(1/2) approximately 90 min at pH 8.0 and approximately 60 min at pH 5.2; k(4) = 0.108 +/- 0.041 min(-1) at pH 8.0 and 0.181 +/- 0.034 min(-1) at pH 5.2. Kinetic values for MIP against NEST were as follows: k(i) = 1880 +/- 61 M(-1) min(-1); reactivation t(1/2) = 0 min at pH 8.0 and approximately 60 min at pH 5.2; aging was complete at all time points tested at pH 8.0, but no aging occurred at pH 5.2. Mass spectrometry revealed a mass shift of 123.0 +/- 0.6 Da for the active site peptide peak of aged DFP-inhibited NEST, corresponding to a monoisopropyl phosphate adduct. In contrast, the analogous mass shift for aged MIP-inhibited NEST was 162.8 +/- 0.6 Da, corresponding to the intact N,N'-diisopropylphosphorodiamido adduct. Thus, MIP-inhibited NEST does not age by isopropylamine loss. However, because kinetically aged MIP-inhibited NEST yields an intact adduct capable of reversible deprotonation, aging could occur by proton loss. Indeed, MIP-inhibited NEST does not age at pH 5.2 but ages immediately and completely at pH 8.0. Therefore, we conclude that the MIP-NEST conjugate ages by deprotonation rather than classical side-group loss.

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Aging of phosphylated serine esterases, e.g., acetylcholinesterase (AChE) and neuropathy target esterase (NTE), renders the inhibited enzymes refractory to reactivation. This process has been considered to require postinhibitory side group loss from the organophosphorus moiety. Recently, however, it has been shown that the catalytic domain of human NTE inhibited by N,N'-diisopropylphosphorodiamidofluoridate (mipafox, MIP) ages by deprotonation. For mechanistic understanding and biomarker development, it would be important to know the identity of the MIP adduct on target esterases after inhibition and aging occurred. Accordingly, the present study was performed to determine if MIP-inhibited human AChE ages by side group loss or an alternate method, e.g., deprotonation. Diisopropylphosphorofluoridate (DFP), the oxygen analogue of MIP, was used for comparison, because DFP-inhibited AChE is known to age by net loss of an isopropyl group. Kinetics experiments were done with DFP and MIP against AChE to follow the time course of inhibition, reactivation, and aging for each inhibitor. MS studies of tryptic digests from kinetically aged DFP-inhibited AChE revealed a mass shift of 122.8 +/- 0.7 Da for the active site peptide (ASP) peak, corresponding to the expected monoisopropylphosphoryl adduct. In contrast, the analogous mass shift for kinetically aged MIP-inhibited AChE was 80.7 +/- 0.9 Da, corresponding to a phosphate adduct. Because this finding was unexpected, the identity of the phosphoserine-containing ASP was confirmed by immunoprecipitation followed by MS. The results indicate that aging of MIP-inhibited AChE proceeds by displacement of both isopropylamine groups. Further research will be required to elucidate the detailed mechanism of formation of a phosphate conjugate from MIP-inhibited AChE; however, knowledge of the identity of this adduct will be useful in biomarker studies.

Elucidating mechanisms of aging of esterases inhibited by organophosphorus (OP) compounds is important for understanding toxicity and developing biomarkers of exposure to these agents. Aging has classically been thought to involve net loss of a single side group from the OP moiety of phosphylated esterases, rendering the enzyme refractory to reactivation. However, recent evidence has shown that acetylcholinesterase (AChE) and the catalytic domain of human neuropathy target esterase (NEST) undergo aging by alternative mechanisms following their inhibition with N,N'-diisopropylphosphorodiamidofluoridate (mipafox, MIP). This study was performed to determine whether MIP-inhibited butyrylcholinesterase (BChE) ages conventionally, by net loss of a single side group, or by an alternate route, e.g., reversible deprotonation or displacement of both isopropylamine groups, as recently observed for MIP-inhibited NEST and AChE, respectively. Diisopropylphosphorofluoridate (DFP), the phosphate analogue of the phosphoroamidate MIP, was used for comparison. Kinetic values for MIP against BChE were as follows: ki = (1.28 +/- 0.053) x 10(6) M-1 min-1; k3 = 0.004,15 +/- 0.000,27 min-1; k4 = 0.008,49 +/- 0.000,99 min-1. Kinetic values for DFP against BChE were as follows: ki = (1.83 +/- 0.18) x 10(6) M-1 min-1; k3 = 0.004,88 +/- 0.000,24 min-1; k4 = 0.0121 +/- 0.0028 min-1. Mass spectrometric studies revealed a mass shift of 123.4 +/- 0.7 Da for the active-site peptide peak of aged DFP-inhibited BChE, corresponding to a monoisopropylphosphate adduct. Similarly, the analogous mass shift for aged MIP-inhibited BChE was 122.4 +/- 0.7 Da, corresponding to a monoisopropylphosphoroamido adduct. Therefore, we conclude that the MIP-BChE conjugate ages by loss of a single isopropylamine group, in contrast to MIP-inhibited AChE or NEST.

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FDA and PhUSE cohosted a Computational Science Symposium (CSS) in 2012 that brought stakeholders from the pharmaceutical industry and government to work collaboratively to solve common needs and challenges. A nonclinical informatics workgroup was formed, dedicated to improving nonclinical assessments and regulatory science by identifying, collecting, and prioritizing key needs and challenges in the field and then establishing an innovative framework for addressing them in a collaborative manner. This paper discusses the process and outcomes of the nonclinical informatics workgroup during the CSS and describes an approach which crossed organizational barriers to optimize computational science for nonclinical assessment.