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.

Properties of several protein kinases that copurify with rat spinal cord neurofilaments.

Several protein kinases that copurify with neurofilaments (NF) were identified and each kinase was assessed for its ability to phosphorylate NF proteins. NFs were isolated using an axonal flotation procedure and the kinases were extracted from NFs with 0.8 M KCl. NF kinases were incubated with peptide substrates for selected protein kinases, [32P]ATP and protein kinase cofactors and inhibitors to characterize the kinases. Using peptide substrates, three types of kinase were identified, and a fourth was identified using NF protein as substrate. The first three kinases were the catalytic subunit of cAMP-dependent protein kinase, calcium-calmodulin dependent protein kinase II and a cofactor-independent kinase that phosphorylated prepro VIP sequence 156-170 and was inhibited by heparin. Using NF proteins as substrate, a fourth kinase was identified which was cofactor-independent and was not inhibited by heparin. Neither cofactor-independent kinase was casein kinase II. NF proteins were phosphorylated in vitro on serine and threonine, primarily by the two cofactor-independent kinases. Using [alpha-32P]8-N3ATP for affinity labeling, one kinase of 43,800 Da was identified. Thus, in addition to cAMP-dependent protein kinase and calcium-calmodulin dependent protein kinase II, two kinases have been found which are primarily responsible for NF phosphorylation in vitro and are cofactor-independent.

Monoclonal antibodies as probes of structure, function and isoenzyme forms of the type II regulatory subunit of cyclic AMP-dependent protein kinase.

We have attempted to review studies which have utilized monoclonal antibodies in the analysis of various aspects of type II cAMP-dependent protein kinase. It is readily apparent that the monoclonal antibodies directed against RII can be used in a number of ways to assess the structure and function of this protein. Monoclonal antibodies have been used to identify specific structural aspects of the protein, down to the level of amino acid sequence. These have included identification of relationships between several functional domains and the antigenic sites recognized by different antibodies. Monoclonal antibodies also have been used to specifically identify distinct isoenzyme forms of RII. The antibodies were shown to have virtually complete specificity for heart-type RII despite relatively few amino acid substitutions in neural-type RII. This discrimination was utilized to show that purified brain RII is composed of a small fraction of protein similar to the heart isozyme while the bulk of the protein is a distinct isozyme form. It is anticipated that future studies using antibodies specific for individual forms will be a valuable approach to analyzing the distribution and functions of different forms. Monoclonal antibodies have also been used as probes of RII in tissue extracts to examine phosphorylation of this subunit in intact tissue. Monoclonal antibodies should continue to provide powerful probes of the structure and function of this and other protein kinases.

Autophosphorylation of rat liver type II cAMP-dependent protein kinase.

A monoclonal antibody was prepared against the regulatory subunit (RII) of rat liver type II cAMP-dependent protein kinase. Autophosphorylated and nonphosphorylated RII in extracts from rat liver or hepatocytes were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and quantified by immunoblot analysis with this antibody. Under basal conditions, 90% of hepatocyte RII was in the phosphorylated form. Incubating hepatocytes with 8-bromo-cAMP and a phosphodiesterase inhibitor resulted in activation of cAMP-dependent protein kinase and glycogenolysis but did not affect phospho RII levels. RII phosphorylation was also unaffected by the inclusion of sufficient insulin to cause a decrease in cAMP-dependent protein kinase activity and glycogenolysis. The results indicate that unlike other cell types, dissociation of rat hepatocyte type II cAMP-dependent protein kinase does not result in dephosphorylation of RII. The biochemical basis for the apparent lack of RII dephosphorylation in intact hepatocytes was examined by comparison with smooth muscle where RII is rapidly dephosphorylated. Rat liver extract contained 4-fold less RII and had an 80-fold lower rate of dephosphorylation of endogenous RII compared to bovine smooth muscle extract. The differences in the rates of RII dephosphorylation in tissue extracts were not observed using purified RII from either tissue. These data suggested that the slow rate of RII dephosphorylation in rat hepatocytes is due to a difference in the susceptibility of endogenous rat liver RII to dephosphorylation rather than a difference in phosphatase activity.

Immunological characterization of the region of tau protein that is bound to Alzheimer paired helical filaments.

Tau protein is known to be present in the paired helical filaments (PHFs) of Alzheimer brains. This study investigated the fragments of tau protein that remain bound to pronase-treated PHFs and conditions that lead to the release of these tau fragments from the core structure of the PHF. Antibody 423 reacted with PHFs and with fetal rat tau but not with adult rat tau, pig tau, or recombinant human tau. Three other antibodies that react with the tubulin binding region of tau only reacted with PHFs after they were disrupted with formic acid or guanidine. Other antibodies that recognize tau sequences C terminal to the tubulin binding region also recognized pronase-treated PHFs. Antibodies SMI34 and T3P that recognize phosphorylated epitopes were reactive with pronase-treated PHFs. Tau fragments from the PHF were solubilized by acid or guanidine treatment. These findings suggest that the fragments of tau that are bound to PHFs and protected from pronase digestion include sequences from the tubulin binding region to the C terminus of tau. In addition, some of these sequences appear to be conformationally or post-translationally modified.

Harmful effect of megadoses of vitamins: electroencephalogram abnormalities and seizures induced by intravenous folate in drug-treated epileptics.

Folic acid was administered intravenously during constant EEG monitoring to eight epileptic subjects who had received diphenylhydantoin therapy for more than 1 year. Six of the subjects displayed low folate levels by Lactobacillus casei assay in plasma and/or whole blood. Six patients received 75 mg of folate intravenously over a 30-min period without clinical effect of EEG change. One patient exhibited an increase in spike discharges on the EEG in comparison to the baseline pattern, but no clinical change or seizure while receiveing 150 mg of folate in a 30-min period. One patient displayed a 2-sec burst of spike and slow wave activity on the EEG after receiveing 7.2 mg of folate in a 3-min period. A tonic-clonic seizure occured after the infusion of 14.4 mg of folate, and again after infusion of 19.2 mg of folate. There appear to be wide variation in the ability of drug-treated epileptic subjects to tolerate infusions of folic acid. These variations help explain conflicting reports in the literature concerning the effect of folic acid administration on seizure control. Megadoses of folic acid should be employed with great caution in all subjects, but particularly among epileptics. Information is lacking as to whether folate might induce seizures in certain ostensibly normal individuals. It is postulated that folic acid exerts a direct effect among sensitive subjects on existing pathways concerned with the metabolism of folate, histidine, or other important brain metabolites. The mechanism of the anticonvulsant action of diphenylhdantoin remains unknown, but there is considerable evidence to suggest that it interferes with the deamination of histidine.

Two apparent glucose-6-phosphate dehydrogenase variants in normal XY males: G6PD Alabama.

A six-year-old black boy who had transient hemolysis after a viral infection was found to have mildly decreased red cell glucose-6-phosphate dehydrogenase (G6PD) activity (1.25 IU/g hemoglobin). Two G6PD bands, both slightly faster than normal G6PD B, were seen on electrophoresis in both the propositus as well as in his maternal grandfather. This is an unexpected finding, since the G6PD gene is located on the long arm of the X chromosome that is subject to X-chromosome inactivation, and available evidence indicates that it is present as a single functional copy in the human genome. The obvious possibility of duplication of the X chromosome was eliminated by cytogenetic analysis with G-banding. G6PD duplication is unlikely, since peripheral blood granulocytes, platelets, and lymphocytes; cultured skin and bone marrow fibroblasts; and Epstein-Barr virus-stimulated lymphocytes yielded only a single electrophoretic band with mobility identical to the slower band seen in crude red blood cell hemolysate. Study of partially purified red blood cell hemolysate G6PD also yielded a single band with identical mobility. Kinetic studies of the enzyme in the propositus and in three generations of his family identified a unique, previously unpublished G6PD mutant that is herein designated G6PD Alabama. Red blood cells were separated by density gradient into a reticulocyte-enriched, an intermediate, and a dense, older portion. Two distinct enzyme bands were identified on electrophoresis of hemolysate from the reticulocyte-enriched portion, but not from the other two portions. It is postulated that two transcriptional products of the mutant G6PD gene exist; one with a short half-life and detectable only in young red blood cells, and another with a longer half-life present in all cells. The existence of two distinct mutant genes in the genome or a unique post-translational form of the mutant G6PD detected only in reticulocytes cannot be excluded.

Phosphorylation of tau protein in tau-transfected 3T3 cells.

The tau protein of Alzheimer paired helical filaments (PHFs) is aberrantly phosphorylated, as evidenced by its reactivity with several phosphate-dependent antibodies. We sought to identify whether this unusual phosphorylation state exists in tau expressed by transfected NIH 3T3 fibroblasts. Immunoblot analysis of cell clones transfected with constructs for either the 3-repeat or 4-repeat isoforms of tau revealed two tau bands, with the lower band migrating with unmodified tau in each case. Antibodies T3P and tau-1 were used to probe these bands, as they also react with PHF-tau in a phosphate-dependent manner. The epitopes for both antibodies were phosphorylated in both tau isoforms. Only the upper band was phosphorylated at the T3P site whereas phosphorylation at the tau-1 site was not always associated with a shift of tau mobility on gels. Tau in both bands was soluble, in contrast to PHF-tau, and was competent to bind to exogenously added bovine microtubules. Colchicine treatment of the cells resulted in an inhibition of phosphorylation at both sites, through an unknown mechanism. In conclusion human tau expressed in 3T3 cells was phosphorylated at the T3P and tau-1 sites as is PHF-tau, although no PHFs formed and the phosphorylated tau was competent to bind to microtubules.

Human tau isoforms confer distinct morphological and functional properties to stably transfected fibroblasts.

Tau protein is a neuronal microtubule-associated protein that promotes the assembly and stability of microtubules. To evaluate the biological significance of tau isoform diversity, NIH-3T3 cells were stably transfected with cDNAs encoding each of the six isoforms present in human brain. Cells expressing different isoforms developed distinct morphologies. Cell lines expressing 3-repeat tau isoforms developed large flat cell bodies while cells expressing 4-repeat isoforms had small, round cell bodies. All transfected cell lines, except those expressing the shortest tau isoform, displayed very long thin neurite-like processes. Tau colocalized with microtubules in both the cell body and the long processes in all of the tau-transfected cells. Tau also displayed a diffuse amorphous staining pattern that was concentrated around the cell nucleus. Microtubule bundling was not enhanced in any of the transfected cells as compared to untransfected controls. The transfected cells showed increased resistance to colchicine treatment. Thus, different tau isoforms can confer unique cellular morphologies to 3T3 cells and can alter the susceptibility of these cells to a microtubule depolymerizing agent.

Aggregation of tau protein by aluminum.

Aluminum has been detected in Alzheimer neurofibrillary tangles, but the significance of its presence is unknown. The principal component of tangles is the paired helical filament (PHF), comprised of tau protein. We investigated whether aluminum could induce tau protein to form filaments or aggregate. When 10 microM bovine tau or non-phosphorylated recombinant human tau was combined with 400 microM or more aluminum, tau protein appeared to aggregate, observed as a dose-dependent decrease in electrophoretic mobility on SDS-PAGE. Tau appeared as a smear above the region of the expected tau bands and, at higher aluminum doses, failed to enter the gel. A tau fragment encompassing the microtubule binding domains did not show decreased mobility in the presence of aluminum, but did form aggregates that failed to electrophorese. However no fibrillar structures were observed in the aluminum-treated tau samples when observed by electron microscopy. The effect of aluminum on tau mobility was reversed by incubating with 1 mM deferoxamine. In contrast, the morphology of PHF fibrils was unaffected by deferoxamine treatment and the characteristic abnormal mobility of PHF-tau was not reduced by deferoxamine. This suggests that aluminum is not, by itself, a significant factor in maintaining the assembly of PHF-tau as fibrils or in its abnormal mobility on SDS gels. Aluminum treatment of 3T3 fibroblasts transfected with human tau resulted in toxicity, but did not change tau expression levels or induce tau aggregation. In conclusion, aluminum appears to induce isolated tau protein to aggregate in a phosphate-independent way, without the formation of fibrils.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation of tau by proline-directed protein kinase (p34cdc2/p58cyclin A) decreases tau-induced microtubule assembly and antibody SMI33 reactivity.

Tau protein was evaluated as a substrate for a proline-directed protein kinase (p34cdc2/p58cyclin A) which recognizes the phosphorylation site motif X-Ser/Thr-Pro-X. The shortest human tau isoform, expressed as a recombinant protein, was phosphorylated to a stoichiometry of 2 mol phosphate/mol tau. Phosphoamino acid analysis revealed phosphorylation of both serine and threonine residues. Phosphorylation of recombinant tau resulted in a decreased ability to induce microtubule assembly but had no effect on the final extent of microtubule formation or on the rate of cold-induced microtubule disassembly. Phosphorylation of tau by the proline-directed protein kinase completely blocked immunoreactivity with antibody SMI33. Phosphorylation did not create the epitopes for the phosphate-dependent antibodies SMI31 or SMI34. Antibody SMI33 recognizes neurofibrillary tangles after treatment with alkaline phosphatase, suggesting that the proline-directed protein kinase may phosphorylate tau at sites that are phosphorylated in Alzheimer's disease.

Phosphorylation, calpain proteolysis and tubulin binding of recombinant human tau isoforms.

In this study, the phosphorylation, calpain hydrolysis and tubulin binding of three recombinant human tau isoforms were examined. The three isoforms used in these studies were tau with three (T3) or four (T4) tandemly repeated tubulin binding domains located in the carboxy-terminal half of the molecule; and tau with four-tandem repeats and a 58-amino acid insert in the amino terminus (T4L). Both cAMP-dependent protein kinase (cAMP-PK) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) readily phosphorylated the three human tau isoforms, although cAMP-PK phosphorylated them to a significantly greater extent than CaMKII. Phosphorylation of T3, T4 and T4L by cAMP-PK or CaMKII resulted in the slowed migration of the protein bands on sodium dodecyl sulfate-polyacrylamide gels and a shift of the isoelectric variants to more acidic positions on two-dimensional non-equilibrium pH gradient electrophoresis gels compared with controls. However, the phosphorylation-induced changes in the electrophoretic migration of the tau isoforms were unique for each kinase. Two-dimensional phosphopeptide maps and sequential phosphorylation experiments indicate that cAMP-PK phosphorylates sites in the human tau isoforms that are phosphorylated by CaMKII, as well as unique sites that are not phosphorylated by CaMKII. T3, T4 and T4L were hydrolyzed similarly by calpain; however, the calpain proteolysis of the recombinant tau isoforms was significantly faster than the proteolysis of human or bovine tau. Phosphorylation of the isoforms by either cAMP-PK or CaMKII did not alter the rate or extent of calpain proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of tau in the polymerization of peptides from beta-amyloid precursor protein.

The composition of paired helical filaments (PHFs), the intracellular amyloid fibrils that accumulate in the brains of Alzheimer patients, is not completely known. We investigated whether synthetic peptides from beta-amyloid precursor protein (APP) can form PHF-like fibrils. Two peptides formed fibrils morphologically similar to PHFs. The presence of tau protein, a known PHF component, greatly enhanced the numbers of fibrils formed from one peptide, from the C-terminus of APP, and became associated with the fibrils. A tau fragment corresponding to the tubulin-binding region was sufficient to induce fibril formation. Tau did not alter fibril formation by the other peptide, which was from the beta/A4 region of APP. These results raise the possibility that a C-terminal fragment of APP, along with tau, may be involved in PHF formation. Thus the proteolytic processing of APP may generate fragments that contribute to both amyloids and both histopathologic lesions of Alzheimer's disease.

Academic performance of reapplicants to a medical school.

This study examined the academic performance of 399 reapplicants who had entered the University of Alabama School of Medicine between 1978 and 1984. Compared with the first-time applicants, the reapplicants had lower preadmission measures and a higher rate of academic difficulty during medical school. However, applicant status (that is, first-time or reapplicant) added nothing to the prediction of academic difficulty over what could be predicted by preadmission measures alone: the students with lower preadmission measures were more likely to experience academic difficulty, regardless of their applicant status. Based on these results, reapplicants should not be viewed negatively simply because they are reapplicants.

Case report: alpha G-Philadelphia, beta O-Arab, and beta C globins present in a single patient.

The case of a 7-month-old Nigerian child who presented with anemia and microcytosis is described. Hemoglobin electrophoresis studies revealed a band with pronounced cathodic mobility. This represented a heterohybrid hemoglobin tetramer composed of an alpha-globin mutant, G-Philadelphia (alpha GPhil), and two variant beta-globin chains, beta C and beta O-Arab. The absolute amounts of alpha GPhil found in the propositus were less than expected for an alpha 2-globin gene product. It has not been established whether alpha G-Philadelphia interacting with beta O-Arab and beta C globin chains is the cause of the microcytosis.

Acupuncture: does it have a place in military general practice?

Acupuncture can be learnt by doctors in a short space of time. Its mode of action is becoming increasingly understood and attempts are being made for statistical evaluation to allow for Western medical acceptance. After attending a basic course in acupuncture, the author describes his first one hundred cases. The preponderance of military patients, chronicity of the presenting complaints and the promising results obtained, illustrates the potential use of such a simple technique in military general practice.