Foslevodopa/Foscarbidopa: A New Subcutaneous Treatment for Parkinson's Disease.

OBJECTIVE: The aim was to demonstrate that continuous s.c. infusion of a soluble levodopa (LD)/carbidopa (CD) phosphate prodrug combination effectively delivers stable LD exposure via a minimally invasive and convenient mode and has the potential to treat Parkinson's disease (PD) patients who are not well controlled on oral medication. METHODS: Foslevodopa and foscarbidopa were prepared and the equilibrium solubility and chemical stability examined in aqueous media with different values of pH. Solutions of foslevodopa/foscarbidopa (ratios ranging from 4:1 to 20:1) were prepared by dissolving pH-adjusted lyophilized materials in water and infused s.c. in healthy volunteers for ≤72 hours. Frequent blood samples were collected to measure LD and CD exposure, and safety was monitored throughout the study. RESULTS: Foslevodopa/foscarbidopa (ABBV-951) demonstrates high water solubility and excellent chemical stability near physiological pH, enabling continuous s.c. infusion therapy. After s.c. infusion, a stable LD pharmacokinetic (PK) profile was maintained for ≤72 hours, and the infusion was well tolerated. INTERPRETATION: Preparation of foslevodopa and foscarbidopa enables preclinical and clinical PK, safety, and tolerability studies in support of their advancement for the treatment of PD. In phase 1 clinical trials, foslevodopa/foscarbidopa demonstrates consistent and stable LD plasma exposure, supporting further studies of this treatment as a potentially transformational option for those suffering from PD. ANN NEUROL 2021;90:52-61.

Potential mechanisms of target-independent uptake and toxicity of antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are a promising therapeutic modality for oncology indications. The concept of an ADC platform is to increase the therapeutic index (TI) of chemotherapeutics through more selective delivery of cytotoxic agents to tumor cells while limiting exposure to healthy normal cells. Despite the use of antibodies targeting antigens abundantly and/or exclusively expressed on cancer cells (i.e., target cells), dose limiting toxicities (DLTs) in normal cells/tissues are frequently reported even at suboptimal therapeutic doses. Although advancement of ADC technology has helped to optimize all three key components (i.e., mAb, linker, and payload), DLTs remain a key challenge for ADC development. Mechanisms of ADC toxicity in normal cells/tissues are not clearly understood, but the majority of DLTs are considered to be target-independent. In addition to linker-drug instability contributing to the premature release of cytotoxic drug (payload) in circulation, uptake/trafficking of intact ADCs by both receptor-dependent (FcγRs, FcRn and C-type lectin receptors), and-independent (non-specific endocytosis) mechanisms may contribute to off-target toxicity in normal cells. In this article, we review potential mechanisms of target-independent ADC uptake and toxicity in normal cells, as well as discuss components of ADCs which may influence these mechanisms. This information will provide a deeper understanding of the underlying mechanisms of ADC off-target toxicity and prove helpful toward improving the overall TI of the next generation of ADCs.

Fishing for teratogens: a consortium effort for a harmonized zebrafish developmental toxicology assay.

A consortium of biopharmaceutical companies previously developed an optimized Zebrafish developmental toxicity assay (ZEDTA) where chorionated embryos were exposed to non-proprietary test compounds from 5 to 6 h post fertilization and assessed for morphological integrity at 5 days post fertilization. With the original 20 test compounds, this achieved an overall predictive value for teratogenicity of 88% of mammalian in vivo outcome [Gustafson, A. L., Stedman, D. B., Ball, J., Hillegass, J. M., Flood, A., Zhang, C. X., Panzica-Kelly, J., Cao, J., Coburn, A., Enright, B. P., et al. (2012). Interlaboratory assessment of a harmonized Zebrafish developmental toxicology assay-Progress report on phase I. Reprod. Toxicol. 33, 155-164]. In the second phase of this project, 38 proprietary pharmaceutical compounds from four consortium members were evaluated in two laboratories using the optimized method using either pond-derived or cultivated-strain wild-type Zebrafish embryos at concentrations up to 100µM. Embryo uptake of all compounds was assessed using liquid chromatography-tandem mass spectrometry. Twenty eight of 38 compounds had a confirmed embryo uptake of >5%, and with these compounds the ZEDTA achieved an overall predictive value of 82% and 65% at the two respective laboratories. When low-uptake compounds (≤ 5%) were retested with logarithmic concentrations up to 1000µM, the overall predictivity across all 38 compounds was 79% and 62% respectively, with the first laboratory achieving 74% sensitivity (teratogen detection) and 82% specificity (non-teratogen detection) and the second laboratory achieving 63% sensitivity (teratogen detection) and 62% specificity (non-teratogen detection). Subsequent data analyses showed that technical differences rather than strain differences were the primary contributor to interlaboratory differences in predictivity. Based on these results, the ZEDTA harmonized methodology is currently being used for compound assessment at lead optimization stage of development by 4/5 of the consortium companies.

Summary of the HESI consortium studies exploring circulating inhibin B as a potential biomarker of testis damage in the rat.

The Developmental and Reproductive Toxicity Technical Committee of the Health and Environmental Sciences Institute hosted a working consortium of companies to evaluate a new commercially available analytic assay for Inhibin B in rat serum or plasma. After demonstrating that the kit was stable and robust, the group performed a series of independent pathogenesis studies (23 different compound/investigator combinations) designed to examine the correlation between the appearance of lesions in the testis and changes in circulating levels of Inhibin B. These studies were reported individually in the previous articles in this series (this issue), and are discussed in this paper. For roughly half of these exposures, lesions appeared well before Inhibin B changed. A few of the studies showed a good correlation between seminiferous tubule damage and reduced circulating Inhibin B levels, while for seven exposures, circulating Inhibin B was reduced with no detectable alteration in testis histology. Whether this indicates a prodromal response or a false-positive signal will require further investigation. These exceptions could plausibly suggest some value of circulating Inhibin B as a useful biomarker in some circumstances. However, for roughly half of these exposures, Inhibin B appeared to be a lagging biomarker, requiring significant damage to the seminiferous tubules before a consistent and credible reduction in circulating levels of Inhibin B was observed.

The inhibin B response to testicular toxicants ethylene glycol monomethyl ether or dibromoacetic acid in male rats.

BACKGROUND: This study was conducted as part of an ILSI-HESIconsortium effort to assess the utility of circulating inhibin B as an early biomarker of testicular toxicity in rats. METHODS: Two known testicular toxicants were selected for use in this study: ethylene glycol monomethyl ether (EGME) and dibromoacetic acid (DBAA). EGME (200 mg/kg/day), DBAA (250 mg/kg/day), or vehicle control (0.2% hydroxypropyl methylcellulose [HPMC]) was administered orally to male rats for 3, 6, or 14 consecutive days. On study days 4, 7, and 15, serum was collected for evaluation of inhibin B levels from all surviving animals and a subset of animals was necropsied from each of the control, EGME, and DBAAgroups. RESULTS: Administration of EGMEresulted in spermatocyte degeneration in late stage tubules and spermatocyte depletion to stage III on day 4, progressing to loss of spermatocytes and round spermatids to stage VI by day 7 and continued germ cell loss and degeneration of elongating spermatids by day 15. Inhibin B levels among EGME-treated animals progressively decreased relative to their respective controls at all time points. Administration of DBAA was associated with spermatid retention at all three time points and abnormal residual bodies at days 7 and 15. Inhibin B levels among DBAA-treated animals decreased progressively relative to their respective controls on days 7 and 15. CONCLUSIONS: Serum inhibin B levels in rats provided a signal of testicular toxicity for each of these known testicular toxicants administered at high levels; however, histopathology provided the earliest evidence of toxic effects.

Pre- and postnatal development in the cynomolgus monkey following administration of ABT-874, a human anti-IL-12/23p40 monoclonal antibody.

BACKGROUND: ABT-874 is an anti-IL-12/23 monoclonal antibody that binds to the p40 subunit of human IL-12 and IL-23. As part of its preclinical safety assessment, studies were conducted to assess its potential effects on pre- and postnatal development in cynomolgus monkeys. METHODS: In the embryo-fetal development studies, ABT-874 was administered once weekly subcutaneously to adult female cynomolgus monkeys at doses of 0, 5, 25, or 100 mg/kg during gestation days (GD) 20 to 48. Fetuses were examined for external, visceral, and skeletal development on GD 100 or 150. In the pre- and postnatal study, ABT-874 was administered once weekly subcutaneously to adult female cynomolgus monkeys at doses of 10, 50, or 200 mg/kg from GD 20 through postpartum day 182. Infants were examined from birth up to 9 months of age for morphological and functional development. Potential effects on the infant immune system were evaluated by immunophenotyping of peripheral blood lymphocytes and by T-dependent antibody response to KLH. RESULTS: There was no ABT-874-related maternal toxicity or adverse effects on fetuses or infants. ABT-874 was present in maternal and fetal serum at GD 100 and 150, and in infant serum through day 63 postbirth. ABT-874 was also present at low levels in breast milk through postpartum day 175. CONCLUSIONS: Exposure of cynomolgus monkey fetuses and infants to ABT-874 had no adverse effects on embryo-fetal or postnatal development.

Male and female fertility assessment in the cynomolgus monkey following administration of ABT-874, a human Anti-IL-12/23p40 monoclonal antibody.

BACKGROUND: ABT-874 is an anti-IL-12/23 monoclonal antibody that binds to the p40 subunit of human IL-12 and IL-23. As part of its preclinical safety assessment, studies were conducted to asses its potential effects on the reproductive system in male and female cynomolgus monkeys. METHODS: Sexually mature male cynomolgus monkeys (n = 6/group) were administered once weekly subcutaneous doses of 0, 5, 25, or 100 mg/kg ABT-874 for 13 weeks. Four monkeys/group were necropsied at the end of the 13-week dosing period and two monkeys/group were necropsied following an 8-week recovery period. Endpoints assessed in these males included sperm parameters such as sperm count and morphology, male reproductive hormones, and testes histopathology. Sexually mature female cynomolgus monkeys (n = 6/group) were administered subcutaneous doses of 0, 5, 25, or 100 mg/kg/week ABT-874 for two menstrual cycles, and recovery was subsequently assessed in each of these animals over two menstrual cycles. Endpoints assessed in these females included menses and reproductive hormone levels. RESULTS: In both the male and female fertility studies, there were no unscheduled deaths and there was no evidence of toxicity. In male monkeys, there were no ABT-874-related effects on sperm count or motility, histopathology of the testes or effects on testosterone and inhibin B levels. In addition, menstrual cycle length, progesterone, 17ß-estradiol, and luteinizing hormone levels in female monkeys were comparable among control and ABT-874-treated groups. CONCLUSIONS: These results demonstrate that ABT-874 had no adverse effects on reproductive hormones or fertility parameters in male or female cynomolgus monkeys.

Effects of SCH 486757, a nociceptin-1 receptor agonist, on fertility and reproductive hormone levels in female CRL:CD®[SD] rats.

BACKGROUND: SCH 486757 is a nociceptin-1 receptor agonist that was in development as an antitussive. Studies were conducted to characterize its effects on female fertility and to examine its potential modes of action. METHODS: Female rats were administered up to 20 mg/kg SCH 486757 before/during mating through gestation day (GD) 7; female fertility and embryonic development were assessed on GD 14. In a subsequent study, pregnant rats were dosed up to 50 mg/kg SCH 486757 from GD 0 to 7. Reproductive hormones were assessed on GD 1, 3, 5, and 7, and embryonic development was assessed on GD 14. A subset of dosed dams were allowed to deliver, were subsequently re-mated, and reproductive hormones and fertility were assessed on GD 7 and 14, respectively. To determine the effects of SCH 486757 on nonpregnant rats, doses of up to 50 mg/kg SCH 486757 were administered for 4 days beginning on the day of estrus; reproductive hormones were assessed after the final dose. RESULTS: Female rats administered ≥20 mg/kg SCH 486757 exhibited abnormal estrous cycles; decreased fertility, number of corpora lutea, and implantation sites; and increased pre- and postimplantation loss. In general, administration of SCH486757 was associated with lower luteinizing hormone (LH) progesterone (P4), and estradiol (E2) levels in pregnant rats. These effects on fertility/embryonic development and reproductive hormones exhibited reversibility post dosing. Nonpregnant rats in the 50-mg/kg group exhibited apparent decreases in P4 and E2 levels, with no apparent effects on LH values. CONCLUSIONS: The SCH 486757-related effects on fertility and embryonic development were likely the result of decreases in P4, E2, and/or LH, rather than being due to decreased prolactin levels.

Developmental and reproductive toxicology studies in IL-12p40 knockout mice.

BACKGROUND: Interleukin (IL)-12 is a cytokine that can exert regulatory effects on T and NK cells. This study was designed to identify potential developmental and reproductive hazards associated with IL-12p40 knockout in mice. METHODS: In the combined fertility and teratology study, female F0 C57/BL6 wild-type control mice and female F0 C57/BL6 IL-12p40 homozgyous knockout mice were assessed for estrous cyclicity, sperm, and mating parameters. Pregnant females were euthanized on gestation day (GD) 18 and their fetuses were assessed for external, visceral, and skeletal development. In the peri and postnatal development study, the F1 wild-type control and IL-12p40 knockout mice were assessed for developmental landmarks, sexual development, passive avoidance, motor activity, and morris water maze. RESULTS: The IL-12p40 knockout male mice exhibited decreased testis weights when compared to the wild-type control group; however, this finding was not considered adverse, as it had no apparent functional effects on mating, fertility, and pregnancy rates or sperm motility. The IL-12p40 knockout group exhibited effects on estrous cycle length, passive avoidance, morris water maze, and motor activity when compared to the wild-type control group. However, since these findings were small in magnitude, transient and/or had no apparent effects on subsequent growth and development, they were not considered adverse. CONCLUSIONS: These results demonstrate that although IL-12p40 homozygous knockout in mice exhibited effects on developmental and reproductive parameters, these effects were relatively minor and were not considered adverse.

Assessment of hydroxypropyl methylcellulose, propylene glycol, polysorbate 80, and hydroxypropyl-ß-cyclodextrin for use in developmental and reproductive toxicology studies.

BACKGROUND: A series of studies were conducted to assess Polysorbate 80 (PS80), Propylene Glycol (PG), and Hydroxypropyl-ß-Cyclodextrin (HPßCD), when compared with Hydroxypropyl Methylcellulose (MC) in developmental and reproductive toxicology (DART) studies. METHODS: In the rat fertility study, 20 mg/kg MC, 10 mg/kg PS80, 1,000 mg/kg PG, 500 mg/kg HPßCD or 1,000 mg/kg HPßCD were administered orally before/during mating, and on gestation Day (GD) 0-7, followed by an assessment of embryonic development on GD 14. In the rat and rabbit teratology studies, the doses of MC, PS80, PG, and HPßCD were the same as those in the fertility study. In these teratology studies, pregnant females were dosed during the period of organogenesis, followed by an assessment of fetal external, visceral, and skeletal development. RESULTS: In the rat fertility and rat teratology studies, PS80, PG, and HPßCD did not exhibit toxicity, when compared with MC. Similarly, in the rabbit teratology study, there was no PS80 or PG-related toxicity, when compared with MC. However, individual rabbits in the 500 and 1,000 mg/kg HPßCD groups exhibited maternal toxicity, which included stool findings, decreased food consumption, and body weight gain. Furthermore, one rabbit each in the 500 and 1,000 mg/kg HPßCD groups exhibited evidence of abortion, which was considered secondary to maternal toxicity. CONCLUSIONS: Although HPßCD was not well tolerated in rabbits at doses of 500 and 1,000 mg/kg, PS80 and PG were comparable to MC and should be considered for use in developmental and reproductive toxicology studies.

Effects of the histamine H1 antagonist chlorcyclizine on rat fetal palate development.

BACKGROUND: The effects of histamine H1 antagonist chlorcyclizine on rat palate development were characterized following in utero exposure. METHODS: To identify the optimum dose for inducing cleft palate, pregnant rats were administered 30, 60, or 90 mg/kg chlorcyclizine on Gestation Days 11 to 14. Fetal palate gene expression was also assessed after 90 mg/kg chlorcyclizine at 8, 15 and 30 hours post-dose on Gestation Day 14 using microarray and qRT-PCR. RESULTS: Rats in the 60- and 90-mg/kg groups exhibited adverse clinical signs and body weight loss. Rats in the 90-mg/kg group also demonstrated increases in late resorptions and decreases in fetal weight. Effects in the low-dose group were limited to decreases in body weight gain. Fetal assessment on Gestation Day 21 revealed that findings were limited to the 60- and 90-mg/kg groups, and included cleft palate (80% of litters for both groups), high arched palate, small nose, micrognathia, high domed head, digits shortened/absent and small limb. The fetal incidence of cleft palate was higher at 90 mg/kg, thus this dose was selected to assess palate gene expression. The altered genes associated with chlorcyclizine-induced cleft palate included Wnt5a, Bmp2, Bmp4, Fgf10, Fgfr2, Msx1, and Insig1 but the magnitude of the change was relatively small (1.5- to 2-fold). CONCLUSIONS: Expression of several genes involved in palate, limb and digit development was altered in the fetal palate following in utero exposure to chlorcyclizine. The subtle perturbation and interplay of these genes may have profound effects on the dynamics of fetal palate development.

Effects of an antagonist of neurokinin receptors 1, 2 and 3 on reproductive hormones in male beagle dogs.

BACKGROUND: SCH 206272, a neurokinin 1, 2, and 3 receptor antagonist, administered to beagle dogs results in testicular toxicity. Therefore, a series of experiments were conducted to determine whether this observed toxicity was associated with changes in reproductive hormones and hypothalamic gonadotrophin releasing hormone (GnRH) levels. METHODS: Male beagle dogs were administered 30 mg/kg SCH 206272 for up to 7 days. Blood samples were collected at the end of the dosing period for reproductive hormone analysis. Male reproductive organs were stained with hematoxylin and eosin and the hypothalamus was stained for GnRH. RESULTS: Intact male dogs exhibited SCH 206272-related decreases in pulsatility and magnitude of luteinizing hormone (LH) and testosterone, which were associated with seminiferous tubule degeneration, oligospermia, and epithelial atrophy in the prostate gland. Neutered dogs also exhibited SCH 206272-related decreases in LH and FSH. In a subsequent reversibility study, intact male dogs exhibited decreased LH, testosterone, and FSH, which exhibited recovery by 2 weeks post-dosing; however, seminiferous tubule degeneration and oligospermia did not exhibit recovery by 2 weeks post-dosing. Dogs administered SCH 206272 also exhibited an increase in mean number of GnRH-containing neurons in the hypothalamus and an increase in GnRH mRNA/neuron, which exhibited recovery by 2 weeks post-dosing. CONCLUSIONS: SCH 206272-dosed dogs exhibited rapid decreases in reproductive hormones and subsequent testicular pathology. Collectively, these changes in hormone levels suggest that the observed SCH 206272-related reproductive tract findings are the result of alterations in hypothalamic-pituitary-gonadal function. However, a direct effect on the testes cannot be definitively ruled out.

Comparative effects of interferon alpha-2b and pegylated interferon alpha-2b on menstrual cycles and ovarian hormones in cynomolgus monkeys.

BACKGROUND: The covalent modification of interferon (IFN) alpha2b with monomethyoxy polyethylene glycol (PEG) reduces its clearance rate and increases its half-life. High doses of interferon (IFN) alpha2b have previously been shown to affect maintenance of pregnancy in rhesus monkeys. Given the role of ovarian hormones in reproductive function and pregnancy, this study was conducted to assess the effects of PEG-IFNalpha2b or IFNalpha2b (comparative control) on ovarian hormones and menstrual cyclicity in cynomolgus monkeys. In addition, the potential for reversibility of PEG-IFNalpha2b or IFNalpha2b-related observations was assessed. METHODS: Monkeys were administered 3,105 microg/m(2) human recombinant (hr) IFNalpha2b or 52, 262, or 4,239 microg/m(2) PEG-hr-IFNalpha2b every other day for one menstrual cycle, followed by a post-dose period of up to two menstrual cycles. RESULTS: Monkeys administered 3,105 microg/m(2) hr-IFNalpha2b or 52, 262, or 4,239 microg/m(2) PEG-hr-IFNalpha2b exhibited transient decreases in food consumption, leukocyte and erythrocyte parameters. Monkeys administered 3,105 microg/m(2) hr-IFNalpha2b exhibited lengthened menstrual cycles that were associated with a delay in reaching peak ovarian hormone levels and lower respective peak concentrations. Similarly, monkeys administered 4,239 microg/m(2) PEG-hr-IFNalpha2b exhibited lengthened menstrual cycles and a delay in reaching peak ovarian hormone levels and slightly lower respective peak concentrations. Post-dosing menstrual cycle length, estradiol and progesterone profiles exhibited evidence of recovery in both the hr-IFNalpha2b and the high-dose PEG-hr-IFNalpha2b groups. CONCLUSIONS: Administration of hr-IFNalpha2b or PEG-hr-IFNalpha2b at high doses to cynomolgus monkeys resulted in similar effects on menstrual cycles, estradiol and progesterone profiles, and exhibited evidence of reversibility upon cessation of dosing. These results suggest that the previously observed high-dose IFNalpha-related effects on the maintenance of pregnancy in monkeys are likely the result of altered ovarian function.

Diagnosing complicated grief: a closer look.

Over the past decade, a number of researchers have proposed a separate DSM category for complicated grief. Recently, there have been attempts to determine empirically the number and nature of variables comprising the complicated-grief syndrome. The present research addresses one such procedure for defining these variables. Combining a past methodology that demonstrated the relative utility of one classification of complicated grief (Worden, 1991) with dimensional concepts derived from other classifications, the present research concludes that a relatively small number of variables account for the concept. These findings are discussed not only in terms of previous research on Worden's categorization and more recent classifications, but, more importantly, in terms of the more global theoretical and methodological issues surrounding the definition(s) of complicated grief.

Age-related changes of the somatotropic axis in cloned Holstein calves.

To determine if the development of the somatotropic axis in somatic clones (clones) is similar to that in heifers produced by artificial insemination (controls), serum samples were collected every 30 min for 6 h, once per month, for 7 mo from 4 clones generated from a 13-yr-old cow and from 4 age-matched controls. Average concentrations of growth hormone (GH) were not different between clones and controls, and GH concentrations declined over time in controls. Average concentrations of insulin-like growth factor I (IGF-I) were less in clones than controls, and IGF-I concentrations increased over time in both groups. Concentrations of IGF-binding protein 3 (IGFBP-3) were greater in controls than in clones and did not change over time. Average IGFBP-2 concentrations did not change over time and were not different between clones and controls. Clones and controls were challenged with GH-releasing hormone (GHRH) (3 microg/100 kg body weight) and somatostatin (somatotropin release-inhibiting factor [SRIF]) (1.87 and 5 microg/100 kg body weight) at 14 mo of age. GHRH-induced GH secretion was greater and SRIF inhibition of GHRH-induced GH was less in clones than in controls. We speculate that some of the differences between clones and controls in concentrations of GH, IGF-I, and IGFBP-3 may be related to the genetic merit of the animals. Although there were differences in concentrations of components of the somatotropic axis between these clones and their age-matched controls, the values recorded were all within the range reported for calves of similar ages.