Reversible and Species-Specific Depigmentation Effects of AZD3293, a BACE Inhibitor for the Treatment of Alzheimer's Disease, Are Related to BACE2 Inhibition and Confined to Epidermis and Hair.

BACKGROUND: AZD3293 (also known as LY3314814) is a novel, potent, non-selective BACE1/BACE2 inhibitor currently in Phase 3 clinical development for the treatment of Alzheimer's disease. OBJECTIVES: The purpose of these studies was to characterize the effects, putative mechanism, and reversibility of hypopigmentation following treatment with AZD3293 in pigmented Long-Evans rats, Beagle dogs, human cell cultures, and humans. DESIGN: Nonclinical studies were conducted in Long-Evans pigmented rats, and both young and older Beagle dogs using a variety of oral dose levels of AZD3293 or AZD3839 (BACE inhibition reference compound; used in older dogs only) for dosing durations of 13 to 26 weeks. In vitro studies of normal human epidermal melanocytes and reconstituted human epidermis were also conducted. Skin biopsy data from a multiple-dose Phase 1 clinical study of AZD3293 (NCT01795339) are also reported. SETTING: Nonclinical in vivo and in vitro studies were conducted in laboratory settings in the US, Canada, and France; the multiple dose clinical study was conducted in a specialized inpatient setting in the US. PARTICIPANTS: Beagle dogs: 13-week study N=36 young (8-10 mo) animals; 39-week study N=64 young animals; and a second 13-week study N=32 older (30-32 mo) animals. Long-Evans rats: N=68 animals. Multiple-dose clinical study: only data for subjects enrolled in Part 2 of this study are included in this report (N=16). INTERVENTIONS: AZD3293 was the primary intervention used in these studies. AZD3839, a relatively BACE1-selective reference inhibitor compound was used in one group in the 13 week study in older Beagle dogs and one in vitro assessment. Finally, AZ1340, another relatively BACE1-selective reference inhibitor compound was used only in one in vitro assessment. MEASUREMENTS: Measurements for the nonclinical studies in dogs and rats included macroscopic observation and assessment of skin biopsies via histopathology, immunochemistry, and electron microscopy. Measurements for the in vitro studies included melanocyte premelanosome protein (PMEL) processing, cytotoxicity, melanin synthesis, Pmel17 labeling, and melanocyte dendricity. Measurements in the clinical study included scoring of melanin content in skin biopsies taken before and after dosing with AZD3293 over 14 days at dose levels up to 150 mg. RESULTS: Depigmentation in rats and dogs was limited to skin, hair, and mucosa with no effects on other pigmented tissues. At a cellular level depigmentation was observed within a week of treatment, whereas the appearance of depigmentation in skin and hair did not become apparent until, at earliest, 4 weeks of treatment. The depigmentation effects were reversible, not associated with degenerative or inflammatory changes, and were dose- and species-dependent in severity. Full recovery of melanization was observed at the microscopic (cellular) level and at least partial recovery was seen in the macroscopic appearance of animals by the end of the 12-week recovery period in both rats and dogs. Interestingly, no changes in melanin production or melanocyte morphology were seen in human primary melanocytes or reconstituted human epidermis in vitro. Finally, there were no changes in melanization level in skin biopsies following 12 days of daily AZD3293 treatment at doses of AZD3293 up to 150 mg/day in human subjects. CONCLUSIONS: AZD3293, a novel, potent, non-selective BACE1/BACE2 inhibitor is in development as a potentially disease-modifying treatment for Alzheimer's disease. Chronic nonclinical studies in Beagle dogs and pigmented rats showed macroscopic and microscopic hypopigmentation effects of AZD3293 that were limited to skin, hair, and mucosa. These effects were shown to be reversible in both species. Analysis of data from nonclinical and in vitro studies suggests that hypopigmentation is caused by BACE2 inhibition resulting in accumulation of a premelanosome protein fragment, which interrupts the normal production of melanin. No macroscopic or microscopic reports of hypopigmentation were observed in a Phase 1 clinical study following 13 doses of AZD3293 over 14 days at dose levels up to 150 mg/day. These data suggest that hypopigmentation is species-specific and humans appear to be least sensitive to the depigmentation effect caused by BACE2 inhibition.

Sleep/wake states of preterm infants: stability, developmental change, diurnal variation, and relation with caregiving activity.

Using time-lapse video, we recorded the sleep/wake states of 95 preterm infants, born under 1500 g and cared for in three hospitals, for three 24-hr periods at 33 and 35 weeks conceptional age. The videotapes were scored in 5-min epochs for quiet sleep (QS); active sleep (AS); wakefulness (Wa); bout lengths of QS (QSBL), AS (ASBL), and waking (WaBL); nursing/caregiving periods (Crgv); bout lengths of Crgv (CrgvBL); and time out of the crib (OOC). The infants' sleep showed significant individual differences at each age, over age, and from day to nighttime. They showed developmental changes in QS, AS, Wa, and QSBL. QS and QSBL were positively related to caregiving time; ASBL was related to maternal age; and QS was related to gestational age and birth weight. Thus, very low birth weight preterm infants show marked stability and developmental change in the organization of the sleep/wake states from a very early age, and their states are related to demographic variables as well as temporal measures of caregiving.

The breathing bear: effects on respiration in premature infants.

The sleep states and the regularity of quiet sleep (QS) respiration were investigated in premature infants who were provided a "breathing" teddy bear. The bear (BrBr) is a source of optional rhythmic stimulation that reflects the breathing rate of the individual infant it is with. At 33 weeks CA, 19 premature infants were given a BrBr and 17 were given a nonbreathing bear (N-BrBr). At 35 weeks CA, and again at 45 weeks CA, a 1-2-h interfeed motility recording was obtained. These analog signals were scored for active sleep, QS, and wakefulness; and each 10-s epoch of QS was judged for regularity of respiration using a four-point rating scale. At 35 weeks, the BrBr babies showed slower and more regular respiration during QS. At 45 weeks, the BrBr babies showed more QS and less active sleep. At both ages, only the BrBr babies showed a correlation between respiratory regulatory and the amount of QS. The findings suggest facilitation of neurobehavioral development as well as entrainment from optional stimulation, which reflects one of the infant's own biological rhythms.

Premature infants seek rhythmic stimulation, and the experience facilitates neurobehavioral development.

This was a clinical trials study of self-regulation of rhythmic stimulation in preterm infants. Infants were enrolled in three regional hospitals and followed in four outlying hospitals. Forty-five premature infants, 22 males and 23 females, enrolled at 29-33 weeks conceptional age (CA) received in the isolette either a "breathing" teddy bear (set to breathe at one-half the infant's quiet sleep respiration rate) or a nonbreathing bear. Using time-lapse videorecording at a 60:1 ratio, subjects were recorded for 3 days at the beginning of the Intervention period and again for 3 days, 2 weeks later. After discharge from the hospital, the sleep of the subjects was monitored in the home for a 24-hour period on weeks 1, 2, 3, 4, and 5 after expected date of birth (postterm). Infants with a breathing bear spent more time in contact with the bear, and increased their contact over the two weeks. Postterm, the "breathing bear babies" showed more quiet sleep and a greater increase in quiet sleep over weeks. The results indicate that premature infants ("prematures") are capable of organizing their motility to express a preference for rhythmic stimulation, and that the experience facilitates neurobehavioral development.