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Background: Fostroxacitabine bralpamide (fostrox) is an orally administered liver-targeted troxacitabine-based nucleotide prodrug currently undergoing phase 1/2a clinical trial in advanced hepatocellular carcinoma (HCC), in combination with pembrolizumab or lenvatinib (NCT03781934). In phase 1 monotherapy fostrox has demonstrated proof-of-concept in advanced HCC, intrahepatic cholangiocarcinoma and liver metastasis from gastrointestinal solid tumors. Liver-selective fostrox-induced DNA-damage and tumor cell killing has the potential to enhance the anti-tumor activity in combination with checkpoint blockade, and inhibition of angiogenesis leads to increased levels of the active metabolite of fostrox. We therefore investigated a triple combination of fostrox with anti-PD1 and lenvatinib in nonclinical tumor models in vivo. Methods: Combinations of fostrox with anti-PD1 and lenvatinib treatment were evaluated in the subcutaneous syngeneic mouse CT26 model1. Groups of 8 mice were randomized using the Matched Distribution method based on tumor size day 1. Treatment was with fostrox (BID days 1-5, p.o.), anti-PD1 (BIW for 3 weeks, i.p.), and lenvatinib (QD for 21 days, p.o.). Tumours were measured three times weekly during the dosing phase, and statistical differences between the treatment groups was analyzed by two-way ANOVA. Pharmacodynamic response to fostrox, induction of DNA-damage (phospo-ser139-H2AX), was assessed by immunohistochemistry (IHC). Tumor infiltrating lymphocytes (TILs) were assessed by IHC evaluating the expression level of CD8, CD4, LAG-3, and PD-L1. Results: Treatment with the triple combination of fostrox (30mg/kg) plus anti-PD1 (3mg/kg) and lenvatinib (5mg/kg) showed a significantly (p Conclusions: The triple combination of fostrox with anti-PD1 and lenvatinib showed enhanced efficacy in a nonclinical tumor model, and changes in TILs were consistent with increased immune-mediated anti-tumor activity. The results indicate a potential for increased anti-tumor efficacy using a triple combination of fostrox plus checkpoint inhibition and anti-angiogenic therapy. 1The study was approved by the Institutional Animal Care and Use Committee (IACUC) of CrownBio UK, and conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) Citation Format: Fredrik G. Oberg, Sujata Bhoi, Malene Jensen, Karin Tunblad, Hans Wallberg. A triple combination of fostrox (MIV-818) with immune checkpoint and kinase inhibition shows increased anti-tumor efficacy in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2691.

309 Background: MIV-818 is an orally administered troxacitabine (TRX)-based nucleotide prodrug. It is highly metabolized by human hepatocytes, directing high levels of the chain-terminating nucleotide tri-phosphate to the liver, while minimizing exposure to other organs. The tri-phosphate is incorporated into DNA during replication, which leads to DNA double strand breaks, DNA damage responses, and cytotoxicity. Methods: Patients (pts), ≥18 years, ECOG performance status < 1, adequate organ function, with treatment-refractory hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (iCCA) or liver metastases (LM) from solid tumors were enrolled. In phase Ia, MIV-818 monotherapy was administered in an intra-patient dose-escalation design with doses of 3-70mg for 3-5 days in cycles of 21 days. Phase Ib is an inter-patient dose escalation in a 3+3 cohort design and is currently ongoing. Treatment is given until disease progression or unacceptable toxicity. The primary objective is to assess safety and tolerability and, for phase Ia, also to establish the starting dose for phase Ib. Key secondary objective was to evaluate the overall response rate based on RECIST v1.1. The pharmacokinetics of MIV-818 and its metabolites were evaluated, and on-treatment liver biopsies were collected to assess the pharmacokinetics and the pharmacodynamic effects of MIV-818. Results: Nine patients (7M; 2F), ECOG performance status 0 (n=3) or 1 (n=6), median age = 57 years (range: 50-84) with HCC (2 pts), iCCA (1) or LM (6) from solid tumors (mainly GI tract), previously treated with median 2 (1-5) lines of therapy, were included in phase Ia. Patients were dosed up to 60 mg 5 days/week. The most common treatment related AEs were those in the hematological system: neutropenia grade (gr) 1-4, neutropenic sepsis gr 4; thrombocytopenia gr 1-2, anemia gr 1-2. Elevated bilirubin gr 1-3 and liver enzymes gr 3 were also reported. Most of the AEs were reversible. Five pts discontinued due to AE and 4 pts discontinued due to progressive disease outside the liver after 2-4 cycles. Starting dose of phase Ib was determined to be 40mg 5 days/week. Tumor biopsies showed evidence of selective DNA damage in tumor tissue, including in hypoxic regions, with minimal or no impact of MIV-818 observed in healthy liver tissue. Conclusions: MIV-818 had an acceptable safety and tolerability profile. Biomarker data of liver biopsies demonstrated a selective effect of MIV-818 on cancer cells. Updated safety and efficacy data from phase Ib will be presented at the meeting. (NCT03781934). Clinical trial information: 2018-000995-14.

Cathepsin S inhibitors attenuate mechanical allodynia in preclinical neuropathic pain models. The current study evaluated the effects when combining the selective cathepsin S inhibitor MIV-247 with gabapentin or pregabalin in a mouse model of neuropathic pain. Mice were rendered neuropathic by partial sciatic nerve ligation. MIV-247, gabapentin, or pregabalin were administered alone or in combination via oral gavage. Mechanical allodynia was assessed using von Frey hairs. Neurobehavioral side effects were evaluated by assessing beam walking. MIV-247, gabapentin, and pregabalin concentrations in various tissues were measured. Oral administration of MIV-247 (100-200 µmol/kg) dose-dependently attenuated mechanical allodynia by up to approximately 50% reversal when given as a single dose or when given twice daily for 5 days. No behavioral deficits were observed at any dose of MIV-247 tested. Gabapentin (58-350 µmol/kg) and pregabalin (63-377 µmol/kg) also inhibited mechanical allodynia with virtually complete reversal at the highest doses tested. The minimum effective dose of MIV-247 (100 µmol/kg) in combination with the minimum effective dose of pregabalin (75 µmol/kg) or gabapentin (146 µmol/kg) resulted in enhanced antiallodynic efficacy without augmenting side effects. A subeffective dose of MIV-247 (50 µmol/kg) in combination with a subeffective dose of pregabalin (38 µmol/kg) or gabapentin (73 µmol/kg) also resulted in substantial efficacy. Plasma levels of MIV-247, gabapentin, and pregabalin were similar when given in combination as to when given alone. Cathepsin S inhibition with MIV-247 exerts significant antiallodynic efficacy alone, and also enhances the effect of gabapentin and pregabalin without increasing side effects or inducing pharmacokinetic interactions.

Purpose: Investigation of immune-modulating activity of MIV-818. Background: MIV-818, a phosphoramidate prodrug of troxacitabine was designed to target the active metabolite troxacitabine-triphosphate to the liver while minimizing systemic exposure. MIV-818 is being developed for the treatment of cancer in the liver, and is currently being evaluated in a phase 1/2 study. Preclinical studies have demonstrated that some DNA damaging agents may further enhance antitumor immune responses in addition to direct cytotoxic effects. We therefore investigated whether MIV-818 induces similar immunomodulating effects as single agent or in combination with pembrolizumab. Methods: Immune-mediated tumor cell killing was determined by co-culturing labelled SKOV3 cancer cells with peripheral blood mononuclear cells (PBMC) and quantified over time using the IncuCyte ZOOM system. Effects on inflamed host tumor microenvironments (TMEs) were investigated by cytokine profiling co-cultures of activated PBMCs, primary fibroblasts or endothelial cells and HT29 colon adenocarcinoma cells. Effects on Staphylococcal enterotoxin B (SEB)-stimulated PBMC were determined by quantifying thymidine incorporation, and cytokine expression in supernatants. Results: Co-incubation of SKOV3 and PBM cells with MIV-818 induced a substantially higher level of apoptosis in the cancer cells (74±25%) compared to co-incubation in the absence of MIV-818 (37±23%). MIV-818 inhibited the proliferation of SKOV-3 cells in the absence PBMCs, but with no significant induction of apoptosis (IC50 7.5nM; ≤5% apoptosis). In a more complex model of the immune TME, MIV-818 treatment impacted inflammatory and angiogenesis-related responses in both the stromal and vascular co-culture systems, increasing IFNγ, IL-10, IL-17A, and TNFα in a manner comparable to that of pembrolizumab alone while also inhibiting IL-6. Combining MIV-818 with pembrolizumab further enhanced an immune-stimulatory effect by increasing IL-10, IL-17A and IFNy, while also modulating immune-related (GranB, IL-2, IL-6), and inflammation-related cytokines (VCAM-1, IP-10, TNFα). MIV-818 demonstrated dose-dependent inhibitory effect on proliferation of SEB-stimulated PBMCs. Despite this growth inhibitory effect, it led to a strong, dose-dependent, increase in IL-2 levels as a single agent which was further enhanced in combination with pembrolizumab. Conclusions: MIV-818 exerts favorable immunomodulating effects in vitro by increasing immune cytokine levels and the PBMC-mediated killing of cancer cells. These effects are further enhanced in combination with pembrolizumab and support future combination of MIV-818 with PD-(L)1 inhibitors in patients with HCC and other liver cancers. The effects of MIV-818 on tumor immune activation markers are being assessed the ongoing phase 1/2 clinical trial. Citation Format: Fredrik G Oberg, Karin Tunblad, Ana Slipicevic, Richard Bethell, Mark R Albertella. MIV-818 stimulates an antitumor immune response in vitro and enhances the effects of pembrolizumab [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C056. doi:10.1158/1535-7163.TARG-19-C056

Background MIV-711 is a highly potent and selective cathepsin K inhibitor. The current article summarizes the therapeutic effects of MIV-711 on joint pathology in rabbits subjected to anterior cruciate ligament transection (ACLT), and the prophylactic effects on joint pathology in dogs subjected to partial medial meniscectomy, two surgical models of osteoarthritis (OA). Methods Starting 1 week after surgery, rabbits were dosed daily via oral gavage with either MIV-711 or vehicle (n = 7/group) for 7 weeks. The four treatment groups were: (1) sham + vehicle; (2) ACLT + vehicle; (3) ACLT + MIV-711, 30 µmol/kg and (4) ACLT + MIV-711, 100 µmol/kg. Subchondral bone and articular cartilage structures were assessed by µCT, histomorphometry, and scoring. Dogs subjected to partial medial meniscectomy received either MIV-711 (30 µmol/kg) or vehicle (n = 15/group) via oral gavage once daily, starting 1 day before meniscectomy, for 28 days. Cartilage degradation was assessed at the macroscopic and microscopic levels. The exposures of MIV-711 were assessed in both studies and biomarkers reflecting bone resorption (HP-1 in rabbits, CTX-I in dogs) and cartilage degradation (CTX-II) were measured. Results In ACLT rabbits, MIV-711 decreased HP-1 levels by up to 72% (p

A major hallmark of Alzheimer's disease (AD) is the deposition of amyloid-β (Aβ) peptides in amyloid plaques. Aβ peptides are produced by sequential cleavage of the amyloid precursor protein by the β amyloid cleaving enzyme (BACE) and the γ-secretase (γ-sec) complex. Pharmacological treatments that decrease brain levels of in particular the toxic Aβ42 peptide are thought to be promising approaches for AD disease modification. Potent and selective BACE1 inhibitors as well as γ-sec modulators (GSMs) have been designed. Pharmacological intervention of secretase function is not without risks of either on- or off-target adverse effects. One way of improving the therapeutic window could be to combine treatment on multiple targets, using smaller individual doses and thereby minimizing adverse effect liability. We show that combined treatment of primary cortical neurons with a BACE1 inhibitor and a GSM gives an additive effect on Aβ42 level change compared with the individual treatments. We extend this finding to C57BL/6 mice, where the combined treatment results in reduction of brain Aβ42 levels reflecting the sum of the individual treatment efficacies. These results show that pharmacological targeting of two amyloid precursor protein processing steps is feasible without negatively interfering with the mechanism of action on individual targets. We conclude that targeting Aβ production by combining a BACE inhibitor and a GSM could be a viable approach for therapeutic intervention in AD modification.

To characterise the pharmacokinetics of dofetilide in patients and to identify clinically relevant parameter-covariate relationships. To investigate three different modelling strategies in covariate model building using dofetilide as an example: (1) using statistical criteria only or in combination with clinical irrelevance criteria for covariate selection, (2) applying covariate effects on total clearance or separately on non-renal and renal clearances and (3) using separate data sets for covariate selection and parameter estimation. Pooled concentration-time data (1,445 patients, 10,133 observations) from phase III clinical trials was used. A population pharmacokinetic model was developed using NONMEM. Stepwise covariate model building was applied to identify important covariates using the strategies described above. Inclusion and exclusion of covariates using clinical irrelevance was based on reduction in interindividual variability and changes in parameters at the extremes of the covariate distribution. Parametric separation of the elimination pathways was accomplished using creatinine clearance as an indicator of renal function. The pooled data was split in three parts which were used for covariate selection, parameter estimation and evaluation of predictive performance. Parameter estimations were done using the first-order (FO) and the first-order conditional estimation (FOCE) methods. A one-compartment model with first order absorption adequately described the data. Using clinical irrelevance criteria resulted in models containing less parameter-covariate relationships with a minor loss in predictive power. A larger number of covariates were found significant when the elimination was divided into a renal part and a non-renal part, but no gain in predictive power could be seen with this data set. The FO and FOCE estimation methods gave almost identical final covariate model structures with similar predictive performance. Clinical irrelevance criteria may be valuable for practical reasons since stricter inclusion/exclusion criteria shortens the run times of the covariate model building procedure and because only the covariates important for the predictive performance are included in the model.

The objective was to evaluate the influence of probenecid on the blood-brain barrier (BBB) transport of morphine-6-glucuronide (M6G). Microdialysis probes were placed in the striatum and into the jugular vein of Sprague-Dawley rats. Each probe was calibrated in vivo using retrodialysis by drug. M6G was administered as a 4-h exponential i.v. infusion, and the experiment was repeated the following day with the addition of probenecid. The data were analysed using NONMEM. An integrated model including the total arterial concentrations, the dialysate concentrations in brain and blood, and the recovery measurements, was developed. The extent of BBB transport, expressed as the ratio between clearance into the brain and clearance out of the brain (CL(in)/CL(out)), was estimated as 0.29 on both days, indicating that efflux transporters act on M6G at the BBB. However, the probenecid-sensitive transporters are not involved in the brain efflux, as the ratio was unaltered although probenecid was co-administered. In contrast, the systemic elimination of M6G decreased by 22% (p

Results The area under the concentration-time curve (AUC) ratio of unbound morphine in brain tissue to plasma was 0.64 (95% confidence interval 0.40, 0.87) in 'better' brain tissue ( P < 0.05 vs. the subcutaneous fat/plasma ratio), 0.78 (0.49, 1.07) in 'worse' brain tissue and 1.00 (0.86, 1.13) in subcutaneous fat. The terminal half-life and T max were longer in the brain vs. plasma and fat, respectively. The relative recovery for morphine was higher in 'better' than in 'worse' brain tissue. The T max value tended to be shorter in 'worse' brain tissue.

Purpose. The objective of this study was to investigate the possible influence of probenecid on morphine transport across the blood-brain barrier (BBB) in rats.

Background: Many systemic chemotherapeutics have failed to show efficacy in hepatocellular carcinoma (HCC), often because systemic toxicity prevents efficacious liver levels of the drug from being reached. MIV-818, a nucleotide prodrug of troxacitabine-monophosphate (TRX-MP) has been designed as a novel approach to deliver high levels of the chain-terminating nucleotide troxacitabine-triphosphate (TRX-TP) to the liver after oral dosing while minimizing systemic exposure. We investigated MIV-818 and troxacitabine using in vivo models in order to identify therapeutic levels of TRX-TP required in the tumors. Methods: MIV-818 or troxacitabine were administered to nude mice with subcutaneous Hep3B or Huh7 xenografts. LC-MS/MS was used to assess MIV-818 and its metabolites. Effects on tumor growth, plasma AFP, inhibition of proliferation and induction of DNA damage were examined and correlated with exposures to TRX-TP in the tumor. Quantitative immuno-fluorescent histology was used to assess DNA damage (pH2AX) proliferation (BrdU), and hypoxia (pimonidazole). Results: Compared to the parent nucleoside troxacitabine, MIV-818 has increased potency of inhibition of HCC cell line growth, increased conversion to its active metabolite TRX-TP and in vitro properties optimized for oral bioavailability and liver targeting, including permeability and intestinal stability. MIV-818 also shows strong synergistic anti-proliferative activity with sorafenib in a number of HCC cell lines in vitro. Pronounced tumor growth inhibition of 70-100% and extensive tumor growth delays of up to 26 days were observed in the Hep3B xenograft model following a five day period of dosing. 12-32-fold induction of DNA damage was seen throughout the tumor sections, consistent with the expected mechanism of action, and with associated inhibition of proliferation. Clear PD responses were apparent even in hypoxic regions of the tumor, indicating effective distribution of TRX-TP even far from blood vessels. DNA damage persisted for up to 7 days after the final dose, demonstrating long-lasting effects and indicating that intermittent dosing is likely to be effective. Similar dosing regimens given to Huh7 xenograft models resulted in significant tumor growth inhibition and induction of DNA damage. Intratumoral TRX-TP exposures across both models were correlated with anti-tumor effects including DNA damage induction, proliferation inhibition and tumor growth inhibition. Conclusions: We have identified TRX-TP exposures required for pronounced anti-tumor effects to give a comprehensive understanding of PK-PD-efficacy relationships for the active metabolite of MIV-818. These data could be used to guide dosing and dose selection in clinical studies. MIV-818 is currently in preclinical development in preparation for the initiation of clinical trials in patients with advanced HCC and other liver cancers. Citation Format: Mark Albertella, Biljana Rizoska, Alastair Kyle, Andrew Minchinton, Annelie Linqvist, Sanja Juric, Susanne Sedig, Karin Tunblad, Fredrik Öberg, Björn Classon, Anders Eneroth, John Öhd, Richard Bethell. Defining exposure-PD and efficacy relationships with the novel liver-targeting nucleotide prodrug MIV-818 for the treatment of liver cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5101. doi:10.1158/1538-7445.AM2017-5101

Corrected QT interval (QTc) prolongation in humans is usually predictable based on results from preclinical findings. This study confirms the signal from preclinical cardiac repolarization models (human ether-a-go-go-related gene, guinea pig monophasic action potential, and dog telemetry) on the clinical effects on the QTc interval. A thorough QT/QTc study is generally required for bioavailable pharmaceutical compounds to determine whether or not a drug shows a QTc effect above a threshold of regulatory interest. However, as demonstrated in this AZD3839 [(S)-1-(2-(difluoromethyl)pyridin-4-yl)-4-fluoro-1-(3-(pyrimidin-5-yl)phenyl)-1H-isoindol-3-amine hemifumarate] single-ascending-dose (SAD) study, high-resolution digital electrocardiogram data, in combination with adequate efficacy biomarker and pharmacokinetic data and nonlinear mixed effects modeling, can provide the basis to safely explore the margins to allow for robust modeling of clinical effect versus the electrophysiological risk marker. We also conclude that a carefully conducted SAD study may provide reliable data for effective early strategic decision making ahead of the thorough QT/QTc study.

Blood-brain barrier transport and brain distribution ofmorphine-6-glucuronide in relation to the antinociceptive effect inrats--pharmacokinetic/pharmacodynamic modelling.

The aims were to quantify the in vivo time-course between the oral dose, the plasma and brain exposure and the inhibitory effect on Amyloid β (Aβ) in brain and cerebrospinal fluid, and to establish the correlation between in vitro and in vivo potency of novel β-secretase (BACE1) inhibitors. BACE1-mediated inhibition of Aβ was quantified in in vivo dose- and/or time-response studies and in vitro in SH-SY5Y cells, N2A cells, and primary cortical neurons (PCN). An indirect response model with inhibition on Aβ production rate was used to estimate unbound in vivo IC 50 in a population pharmacokinetic-pharmacodynamic modeling approach. Estimated in vivo inhibitory potencies varied between 1 and 1,000 nM. The turnover half-life of Aβ40 in brain was predicted to be 0.5 h in mouse and 1 h in guinea pig. An excellent correlation between PCN and in vivo potency was observed. Moreover, a strong correlation in potency was found between human SH-SY5Y cells and mouse PCN, being 4.5-fold larger in SH-SY5Y cells. The strong in vivo-in vitro correlation increased the confidence in using human cell lines for screening and optimization of BACE1 inhibitors. This can optimize the design and reduce the number of preclinical in vivo effect studies.

To develop an integrated model for microdialysis data that incorporates all data including the recovery measurements in one model, and to compare this model to a previous model and the results from a noncompartmental analysis.The models were developed in NONMEM. The modes of analysis were compared with respect to parameter estimates, model structures, gained mechanistic insight, and practical aspects.Both modeling approaches resulted in similar model structures. The parameter estimates in blood and brain from the models and the results from the noncompartmental analysis were comparable. Using the integrated model all data, that is, the total arterial concentrations, the venous and brain dialysate concentrations, and the recovery measurements, were analyzed simultaneously.The theoretical benefits of the integrated model are related to the inclusion of the recovery in the model and the use of all collected data as it was observed. Thus, all data are described in a single model, corrections for the recovery and the protein binding are done within the model, and the dialysate observations are described by the integral over each collection interval. Thereby, the variability and the uncertainty in the model parameters are handled correctly to give more reliable parameter estimates.

During pathologic conditions such as meningitis and traumatic brain injury the function of the blood-brain barrier (BBB) is disturbed. In the present study we examined the cerebral pharmacokinetic pattern of morphine in the intact brain and during experimentally induced meningitis using a pig model. Secondly, the use of intracerebral microdialysis as a potential tool for monitoring damage in the BBB by studying the pharmacokinetics of morphine is addressed.Six pigs were studied under general anaesthesia. One occipital and two frontal microdialysis probes and one pressure transducer were inserted into the brain tissue. Another probe was placed into the jugularis interna. Morphine 1 mg kg(-1) was administered as a 10-min infusion, and morphine concentrations were then measured for 3 h. Meningitis was subsequently induced by injecting lipopoly-saccharide into the cisterna magna. When meningitis was established, the morphine experiment was repeated.The unbound area under the concentration-time curve (AUCu) ratio of morphine in brain to blood was 0.47 (0.19) during the control period, and 0.95 (0.20) (P0.001) during meningitis. The increase in the brain/blood AUCu ratio during meningitis implies decreased active efflux and increased passive diffusion of morphine over the BBB. The half-life of morphine in brain was longer than in blood during both periods, and was unaffected by meningitis.This study demonstrates that the morphine exposure to the brain is significantly increased during meningitis as compared with the control situation.

The objective of this study was to investigate the possible influence of probenecid on morphine transport across the blood-brain barrier (BBB) in rats.Microdialysis probes, calibrated using retrodialysis by drug, were placed into the striatum and jugular vein of seven Sprague-Dawley rats. Morphine was administered as a 4-h exponential infusion. The experiment was repeated the next day with the addition of probenecid, administered as a bolus dose (20 mg/kg) followed by a constant infusion (20 mg/kg/h). Models for BBB transport were built using the computer program NONMEM.The steady-state ratio of 0.29 +/- 0.07 of unbound morphine concentration in brain to that in blood indicates that morphine is actively effluxed at the BBB. Probenecid co-administration increased the ratio to 0.39 +/- 0.04 (p0.05). Models in which probenecid influenced the brain efflux clearance rather than the influx clearance, well described the data. The half-life in brain increased from 58 +/- 9 min to 115 +/- 25 min when probenecid was co-administered. Systemic clearance of morphine also decreased upon probenecid co-administration, and M3G formation was decreased.This study indicates that morphine is a substrate for the probenecid-sensitive transporters at the BBB. Co-administration of probenecid decreased the brain efflux clearance of morphine.