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.

Developmental characterization of the gene for laminin alpha-chain in sea urchin embryos.

We describe the isolation and characterization of a cDNA clone encoding a region of the carboxy terminal globular domain (G domain) of the alpha-1 chain of laminin from the sea urchin, Strongylocentrotus purpuratus. Sequence analysis indicates that the 1.3 kb cDNA (spLAM-alpha) encodes the complete G2 and G3 subdomains of sea urchin a-laminin. The 11 kb spLAM-alpha mRNA is present in the egg and declines slightly in abundance during development to the pluteus larva. The spLAM-alpha gene is also expressed in a variety of adult tissues. Whole mount in situ hybridization of gastrula stage embryos indicates that ectodermal and endodermal epithelia and mesenchyme cells contain the spLAM-alpha mRNA. Immunoprecipitation experiments using an antibody made to a recombinant fusion protein indicates spLAM-alpha protein is synthesized continuously from fertilization as a 420 kDa protein which accumulates from low levels in the egg to elevated levels in the pluteus larva. Light and electron microscopy identify spLAM-alpha as a component of the basal lamina. Blastocoelic microinjection of an antibody to recombinant spLAM-alpha perturbs gastrulation and skeleton formation by primary mesenchyme cells suggesting an important role for laminin in endodermal and mesodermal morphogenesis.

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.

Matrix metalloproteinase inhibitors disrupt spicule formation by primary mesenchyme cells in the sea urchin embryo.

The primary mesenchyme cells of the sea urchin embryo construct an elaborate calcareous endoskeletal spicule beginning at gastrulation. This process begins by ingression of prospective primary mesenchyme cells into the blastocoel, after which they migrate and then fuse to form a syncytium. Skeleton deposition occurs in spaces enclosed by the cytoplasmic cables between the cell bodies. Experiments are described which probe the role of proteases in these early events of spicule formation and their role in the continued elaboration of the spicule during later stages of embryogenesis. We find that several inhibitors of metalloproteinases inhibit the continuation of spiculogenesis, an effect first reported by Roe et al. (Exp. Cell Res. 181, 542-550, 1989). A detailed study of one of these inhibitors, BB-94, shows that fusion of primary mesenchyme cells still occurs in the presence of the inhibitor and the formation of the first calcite granule is not impeded. Continued elaboration of the spicule, however, is completely stopped; addition of the inhibitor during the active elongation of the spicule stops further elongation immediately. Removal of the inhibitor allows resumption of spicule growth. The inhibition is accompanied by almost complete cessation of massive Ca ion transport via the primary mesenchyme cells to the spicule. The inhibitor does not prevent the continued synthesis of several spicule matrix proteins. Electron microscopic examination of inhibited primary mesenchyme cells shows an accumulation of characteristic vesicles in the cytoplasm. Gel zymography demonstrates that although most proteases in homogenates of primary mesenchyme cells are not sensitive to the inhibitor in vitro, a protease of low abundance detectable in the medium of cultured primary mesenchyme cells is inhibited by BB-94. We propose that the inhibitor is interfering with the delivery of precipitated calcium carbonate and matrix proteins to the site(s) of spicule growth.

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.

An N-linked carbohydrate-containing extracellular matrix determinant plays a key role in sea urchin gastrulation.

During the process of gastrulation in the sea urchin embryo, the vegetal plate invaginates to form the archenteron, a long narrow tube that extends across the blastocoel. Rearrangement of cells within the archenteron is thought to be a key component of the process of archenteron elongation. While these cell rearrangements have been well described, the mechanism of the rearrangements and the coordination of the movements of individual cells that results in the elongation of the archenteron are not well understood. We have identified a monoclonal antibody, called ECM 1, that recognizes an N-linked carbohydrate-containing epitope on several high-molecular-weight basal lamina glycoproteins. In an attempt to block the function of this determinant in development, the ECM 1 antibody was injected into the blastocoel of living embryos and the effects on morphogenesis were determined. Injection of intact ECM 1 IgG or monovalent Fab fragments blocks cell rearrangements during secondary invagination and cell movements during segmentation of the gut. Glycopeptides derived from the glycoproteins recognized by ECM 1 also inhibit cell rearrangements during secondary invagination when injected into the blastocoel. The inhibitory activity of these peptides is eliminated by digestion with N-glycosidase F or pronase, enzymes that also disrupt the ECM 1 determinant. ECM 1 recognizes a nonuniformly distributed determinant in the basal lamina and blastocoel matrix. This determinant is stored in cytoplasmic granules in the unfertilized egg and is deposited into the basal lamina by the blastula stage. The determinant becomes concentrated in the basal lamina in the vegetal region of the embryo early in gastrulation. At the prism stage, the determinant accumulates in the basal lamina and blastocoel matrix in the ventral region of the embryo. These data indicate that the vegetally localized, N-linked carbohydrate-containing determinant recognized by ECM 1 plays an important role in cell movements during archenteron morphogenesis in the sea urchin embryo.

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.

Anesthesia influences the outcome from experimental spinal cord injury.

The effect of anesthesia upon the functional outcome after experimental spinal cord injury (SCI) was studied in 221 rats subjected to graded weight drop contusion in the thoracic cord. Neurologic function was assessed in a blinded fashion for one week after injury using a modification of the method of Tarlov. The post-mortem concentrations of serotonin and its metabolite were measured in injured and surrounding spinal tissues in a subset of animals in order to estimate the survival of descending long-tract axons. In initial studies using non-ventilated animals where body temperature was not controlled (n = 130), halothane anesthesia was associated with significantly better neurologic scores at all levels of injury (50, 100 and 250 g.cm) in comparison to pentobarbital. In a second experiment under these conditions (n = 53) the effect of halothane was observed after a 50 g.cm injury in comparison to both pentobarbital and nitrous oxide. Improved neurologic recovery was accompanied by the preservation of normal serotonin and metabolite concentrations in spinal tissue caudal to the site of injury. These values did not differ from those measured in sham-operated animals. Separate experiments (n = 12) revealed halothane's preservation of somatosensory-evoked responses during the early postinjury period in animals showing improved neurologic recovery. Subsequent experiments (n = 12) were performed to assess the effect of oxygen supplementation and the control of rectal temperature and a separate series of acute experiments (n = 14) examined arterial blood pressure responses to injury in halothane- and pentobarbital-anesthetized animals.(ABSTRACT TRUNCATED AT 250 WORDS)

The somatosensory evoked potential predicts neurologic deficits and serotonergic pathochemistry after spinal distraction injury in experimental scoliosis.

The validity of the somatosensory evoked potential as an intraoperative spinal cord monitor was evaluated in an experimental model of scoliosis in the rat and a Harrington distraction model of injury. Under these conditions, it was found that any change in latency or amplitude of the major negative wave above a certain level was a significant predictor of an adverse neurologic outcome. Changes in latency of 4% or greater and changes in amplitude of 50% or greater were unequivocal indicators of spinal cord injury. Postmortem analyses of the spinal neurotransmitter serotonin revealed that apparent false-positive results of the SEP were, in fact, true-positive results.