PTI-125 Reduces Biomarkers of Alzheimer's Disease in Patients.

BACKGROUND: The most common dementia worldwide, Alzheimer's disease is often diagnosed via biomarkers in cerebrospinal fluid, including reduced levels of Aß1-42, and increases in total tau and phosphorylated tau-181. Here we describe results of a Phase 2a study of a promising new drug candidate that significantly reversed all measured biomarkers of Alzheimer's disease, neurodegeneration and neuroinflammation. PTI-125 is an oral small molecule drug candidate that binds and reverses an altered conformation of the scaffolding protein filamin A found in Alzheimer's disease brain. Altered filamin A links to the α7-nicotinic acetylcholine receptor to allow Aß42's toxic signaling through this receptor to hyperphosphorylate tau. Altered filamin A also links to toll-like receptor 4 to enable Aß-induced persistent activation of this receptor and inflammatory cytokine release. Restoring the native shape of filamin A prevents or reverses filamin A's linkages to the α7-nicotinic acetylcholine receptor and toll-like receptor 4, thereby blocking Aß42's activation of these receptors. The result is reduced tau hyperphosphorylation and neuroinflammation, with multiple functional improvements demonstrated in transgenic mice and postmortem Alzheimer's disease brain. OBJECTIVES: Safety, pharmacokinetics, and cerebrospinal fluid and plasma biomarkers were assessed following treatment with PTI-125 for 28 days. Target engagement and mechanism of action were assessed in patient lymphocytes by measuring 1) the reversal of filamin A's altered conformation, 2) linkages of filamin A with α7-nicotinic acetylcholine receptor or toll-like receptor 4, and 3) levels of Aß42 bound to α7-nicotinic acetylcholine receptor or CD14, the co-receptor for toll-like receptor 4. DESIGN: This was a first-in-patient, open-label Phase 2a safety, pharmacokinetics and biomarker study. SETTING: Five clinical trial sites in the U.S. under an Investigational New Drug application. PARTICIPANTS: This study included 13 mild-to-moderate Alzheimer's disease patients, age 50-85, Mini Mental State Exam ≥16 and ≤24 with a cerebrospinal fluid total tau/Aß42 ratio ≥0.30. INTERVENTION: PTI-125 oral tablets (100 mg) were administered twice daily for 28 consecutive days. MEASUREMENTS: Safety was assessed by electrocardiograms, clinical laboratory analyses and adverse event monitoring. Plasma levels of PTI-125 were measured in blood samples taken over 12 h after the first and last doses; cerebrospinal fluid levels were measured after the last dose. Commercial enzyme linked immunosorbent assays assessed levels of biomarkers of Alzheimer's disease in cerebrospinal fluid and plasma before and after treatment with PTI-125. The study measured biomarkers of pathology (pT181 tau, total tau and Aß42), neurodegeneration (neurofilament light chain and neurogranin) and neuroinflammation (YKL-40, interleukin-6, interleukin-1ß and tumor necrosis factor α). Plasma levels of phosphorylated and nitrated tau were assessed by immunoprecipitation of tau followed by immunoblotting of three different phospho-epitopes elevated in AD (pT181-tau, pS202-tau and pT231-tau) and nY29-tau. Changes in conformation of filamin A in lymphocytes were measured by isoelectric focusing point. Filamin A linkages to α7-nicotinic acetylcholine receptor and toll-like receptor 4 were assessed by immunoblot detection of α7-nicotinic acetylcholine receptor and toll-like receptor 4 in anti-filamin A immunoprecipitates from lymphocytes. Aß42 complexed with α7-nicotinic acetylcholine receptor or CD14 in lymphocytes was also measured by co-immunoprecipitation. The trial did not measure cognition. RESULTS: Consistent with the drug's mechanism of action and preclinical data, PTI-125 reduced cerebrospinal fluid biomarkers of Alzheimer's disease pathology, neurodegeneration and neuroinflammation from baseline to Day 28. All patients showed a biomarker response to PTI-125. Total tau, neurogranin, and neurofilament light chain decreased by 20%, 32% and 22%, respectively. Phospho-tau (pT181) decreased 34%, evidence that PTI-125 suppresses tau hyperphosphorylation induced by Aß42's signaling through α7-nicotinic acetylcholine receptor. Cerebrospinal fluid biomarkers of neuroinflammation (YKL-40 and inflammatory cytokines) decreased by 5-14%. Biomarker effects were similar in plasma. Aß42 increased slightly - a desirable result because low Aß42 indicates Alzheimer's disease. This increase is consistent with PTI-125's 1,000-fold reduction of Aß42's femtomolar binding affinity to α7-nicotinic acetylcholine receptor. Biomarker reductions were at least p ≤ 0.001 by paired t test. Target engagement was shown in lymphocytes by a shift in filamin A's conformation from aberrant to native: 93% was aberrant on Day 1 vs. 40% on Day 28. As a result, filamin A linkages with α7-nicotinic acetylcholine receptor and toll-like receptor 4, and Aß42 complexes with α7-nicotinic acetylcholine receptor and CD14, were all significantly reduced by PTI-125. PTI-125 was safe and well-tolerated in all patients. Plasma half-life was 4.5 h and approximately 30% drug accumulation was observed on Day 28 vs. Day 1. CONCLUSIONS: PTI-125 significantly reduced biomarkers of Alzheimer's disease pathology, neurodegeneration, and neuroinflammation in both cerebrospinal fluid and plasma. All patients responded to treatment. The magnitude and consistency of reductions in established, objective biomarkers imply that PTI-125 treatment counteracted disease processes and reduced the rate of neurodegeneration. Based on encouraging biomarker data and safety profile, approximately 60 patients with mild-to-moderate AD are currently being enrolled in a Phase 2b randomized, placebo-controlled confirmatory study to assess the safety, tolerability and efficacy of PTI-125.

Phenylalanine and tyrosine synthesis under primitive earth conditions.

Phenylacetylene can be synthesized in substantial yields from various hydrocarbons by high temperatures, electric discharges, and ultraviolet light. Phenylacetylene is hydrated to phenylacetaldehyde by way of both nucleophilic and radical additions of H(2)S followed by hydrolysis of the thtioaldehyde. The addition of NH(3) and HCN to phenylacetaldehyde yields phenylalanine nitrile which is hydrolyzed to phenylalanine. A small yield of tyrosine is obtained from the radical addition of H(2)S to phenylacetylene. This sequence of reactions is a possible mechanism for the synthesis of these amino acids on the primitive earth.

Cyclic adenosine and guaosine monophosphates and gucagon: effect on liver membrane potentials.

Cyclic adenosine monophosphate, cyclic guanosine monophosphate, glucagon, and isoproterenol each hyperpolarized perfused rat liver cells. The hyperpolarization followed a time course similar to the stimulated increase in potassium efflux and was preceded by the increase in calcium efflux. The hyperpolarization induced by cyclic adenosine monophosphate was blocked by tetracaine. The similarity of the action of the cyclic nucleotides to that of glucagon supports the hypothesis that cyclic adenosine monophosphate is the secondary messenger mediating the action of glucagon.

Primitive earth synthesis of nicotinic acid derivatives.

Nicotinonitrile, 2-cyanopyridine, and 4-cyanopyridine can be synthesized under primitive earth conditions by the action of electric discharges on ethylene and ammonia. The electric discharge first synthesizes pyridine and hydrogen cyanide, which react in the discharge to form the cyanopyridines. Nicotinonitrile would have hydrolyzed in the primitive ocean to nicotinamide and nicotinic acid.

Activation of protein kinase(s) by glucagon and cyclic AMP in the rat liver. Relationship to metabolic effects.

Although it is known that protein kinases are activated by cyclic AMP, the role of the activated kinase in the gluconeogenic response to cyclic AMP is not known. Therefore, we examined whether the inhibition of the gluconeogenic response in the liver is due to an interference with the activation of protein kinase in the following situations: (1) adrenalectomy, (2) Na+-free perfusate, (3) administration of local anesthetic. We measured protein kinase activity indirectly by measuring incorporation of 32P into proteins of the perfused liver, and directly by measuring the enzyme activity. We found no significant inhibition of activation of protein kinase in the above experimental conditions. It seems that in the intact liver, activation of protein kinase by itself is not sufficient to evoke metabolic responses. In order to clarify whether the requirement for ion redistribution is specific for the gluconeogenic response or not, the lipolytic and antilipogenic effects of glucagon and cyclic AMP were examined. Na+-free perfusate, local anesthetic or high K+ did interfere with the lipolytic and antilipogenic responses to these agents just as it interfered with the gluconeogenic response. It is likely that ion redistribution evoked by glucagon and cyclic AMP is essential to the expression of most, if not all, metabolic effects.

Antagonistic effect of insulin on glucagon-evoked hyperpolarization. A correlation between changes in membrane potential and gluconeogenesis.

In the perfused rat liver, administration of glucagon causes a hyperpolarization of the liver cell membrane and increases gluconeogenesis. Insulin, a hormone which is known to antagonize the effect of glucagon on gluconeogenesis also blocks the hyperpolarizing effect of glucagon. Because of this inhibitory effect of insulin of the glucagon-evoked hyperpolarization, a systematic study of possible correlation between changes in membrane potential and gluconeogenesis was undertaken. The membrane potential was changed by valinomycin, tetracaine, or by varying the ionic composition of the perfusate. A highly significant correlation between changes in membrane potential and the rate of gluconeogenesis was noticed. The possibility was raised that changes in membrane potential might exert an influence on metabolic process by a yet unknown mechanism.

Characterization of the hormone-sensitive Ca2+ uptake activity of the hepatic endoplasmic reticulum.

The characteristics and kinetics of calcium uptake activity were studied in isolated hepatic microsomes. The sustained accumulation of calcium was ATP- and oxalate-dependent. Glucagon increased microsomal Ca2+ uptake upon either in vivo injection, or in vitro perfusion of the hormone in the liver. In contrast, the effect of insulin depended on the route of administration. Calcium accumulation by subsequently isolated hepatic microsomes increased when insulin was injected intraperitoneally whereas it decreased when the hormone was perfused directly into the liver. These effects of glucagon and insulin were dose dependent. When insulin was added to the perfusate prior to the addition of glucagon, insulin blocked the glucagon-stimulated increase in microsomal Ca2+ uptake. Cyclic AMP mimicked the effect of glucagon on microsomal Ca2+ accumulation when the cyclic nucleotide was perfused into the liver. The effects of glucagon and insulin on the kinetics of hepatic microsomal Ca2+ uptake were investigated. In microsomes isolated from perfused rat livers treated with glucagon the V of the uptake was significantly increased over the control values (12.2 vs. 8.6 nmol Ca2+ per min per mg protein, P less than 0.02). In contrast, the addition of insulin to the perfusate significantly decreased the V of Ca2+ uptake by subsequently isolated microsomes (6.8 vs. 8.3 nmol Ca2+ per min per mg protein, P less than 0.05). However, neither hormone had an effect on the apparent Km for Ca2+ (4.1 +/- 0.5 microM) of the reaction. The effect of these hormones on the activity of Ca2+-stimulated ATPase was also studied. No significant changes in either V or Km for Ca2+ of the enzymatic reaction were detected.

Liposome encapsulated tetracaine lowers blood glucose.

Tetracaine, a local anesthetic, was previously shown to block hormonal stimulation of gluconeogenesis and glycogenolysis ( Friedmann , N. and Rasmussen, H. (1970) Biochim. Biophys. Acta 222, 41-52). In the present studies tetracaine incorporated into liposomes (phospholipid vesicles) was injected into intact rats and epinephrine was administered an hour later. Liposomal tetracaine blocked 50% of the hyperglycemic response. When tetracaine, incorporated into liposomes, was injected into diabetic rats it reduced transiently, but significantly, blood glucose levels. Equivalent doses of free tetracaine were toxic. These studies indicate that liposomal drug administration might be developed into a tool to influence hepatic metabolism and, consequently, blood glucose levels.

A comparison between 45Ca2+ and atomic absorption in calcium flux determinations in perfused rat liver.

Calcium efflux from perfused rat liver following the administration of Ca2+ releasing agents was measured with two methods, 45Ca2+ labeling and atomic absorption. The values obtained with atomic absorption were usually higher than the values obtained with 45Ca2+. These indicate that the intracellular Ca2+ did not equilibrate with the perfusate ca2+ during the 90-minute labeling period. A similar conclusion was reached by measuring the liver 45Ca2+ and 40Ca2+ content. In addition, the types of albumin added to the perfusate influenced the amounts of Ca2+ released.

Calcium sequestration in the liver.

Hepatic parenchymal cells maintain intracellular total and cytosolic free Ca2+ levels by: entry of Ca2+ through channels, extrusion of Ca2+ by an outwardly directed Ca2+ pump, and controlled sequestration into intracellular pools. The mechanism of Ca2+ inflow is poorly characterized. The plasma membrane Ca2+ channels seem to share some of the characteristics of Ca2+ channels in excitable cells, but also differ from them. The outwardly directed plasma membrane Ca2(+)-ATPase is a calmodulin independent, P-type enzyme. Ca2+ uptake into the endoplasmic reticulum is due to the activity of a different Ca2(+)-ATPase, which is similar in molecular weight and shares antigenic determinants with the sarcoplasmic reticulum enzyme. In addition, mitochondria and nuclei also take up calcium. The exact mechanism by which Ca2+ is released from intracellular organelles is not well known. Several mechanisms for Ca2+ release from the endoplasmic reticulum were reported, including IP3 and GTP-induced. The most effective identified way of eliciting Ca2+ release from microsomal fraction is by the oxidation of critical -SH groups. This mechanism is likely to be involved in the rise of cytosolic Ca2+ observed in many situations of hepatocellular injury. In addition to being sequestered into subcellular organelles, some of the intracellular Ca2+ is bound to specific Ca2+ binding proteins. Both calmodulin and members of the annexin family were identified in the liver. Stimulation of the liver with gluconeogenic hormones results in increased Ca2+ entry into the cell, the release of Ca2+ from intracellular pools, and an oscillatory increase in free cytosolic Ca2+ levels. Extensive research is still needed for the elucidation of the exact mechanisms by which these events occur.

Cyclic nucleotide-gated channels in non-sensory organs.

Cyclic nucleotide-gated channels represent a class of ion channels activated directly by the binding of either cyclic-GMP or cyclic-AMP. They carry both mono and divalent cations, but select calcium over sodium. In the majority of the cases studied, binding of cyclic nucleotides to the channel results in the opening of the channel and the influx of calcium. As a consequence, cytosolic free calcium levels increase leading to the modifications of calcium-dependent processes. This represents and important link in the chain of events leading to the physiological response. Cyclic nucleotide-gated channels were discovered in sensory cell types, in the retina, and in olfactory cells, and were extensively studied in those cells. However, it is becoming increasingly evident that such channels are present not only in sensory systems, but in most, if not all, cell types where cyclic nucleotides play a role in signal transduction. A hypothesis is presented here which attributes physiological importance to these channels in non-sensory organs. Four examples of such channels in non-sensory cells are discussed in detail: those in the liver, in the heart, in the brain, and in the testis with the emphasis on the possible physiological roles that these channels might have in these organs.

The effects of Mg2+ on rat liver microsomal Ca2+ sequestration.

The effects of Mg2+ on the hepatic microsomal Ca2(+)-sequestering system was tested. Ca2(+)-ATPase activity and Ca2+ uptake were both dependent on the concentration of free Mg2+, reaching maximum levels at 2 mM. The effects of Mg-ATP were also influenced by the concentration of free Mg2+, being maximally effective at a ratio of 1:1. The results suggest that Mg2+ influences Ca2+ sequestration at various steps, namely in addition to forming the substrate of the Ca2(+)-ATPase reaction, Mg-ATP, Mg2+ stimulates the reaction at an additional step, as indicated by its stimulatory effect on the Ca2(+)-ATPase reaction and on Ca2+ uptake, even at optimal Mg-ATP levels. The stimulatory effect of Mg2+ was evident at various pH levels tested, and it was nucleotide specific. The stimulatory effect of Mg2+ might be exerted at the dephosphorylation step of the enzymatic reaction or at an other, yet undefined, site. The results demonstrate a plural effect of Mg2+ on the hepatic microsomal sequestration system. This indicates that, depending on its magnitude, changes in Mg2+ distribution might influence cytosolic Ca2+ levels.

45Ca2+ uptake and phospholipid methylation in isolated rat liver microsomes.

The effects of glucagon, epinephrine and insulin on hepatic phospholipid methylation were studied. Glucagon, either injected into rats or added to perfused livers, stimulated methylation in subsequently isolated microsomes. Epinephrine also increased phospholipid methylation. Insulin by itself did not influence the rate of the reaction, but, when administered prior to glucagon, it blocked the effect of the latter. The possibility that the observed stimulation of phospholipid methylation might be causally linked to the reported stimulation by glucagon of 45Ca2+ uptake in subsequently isolated liver microsomes was examined. Both the substrate and the competitive inhibitor of the methylation reaction, S-adenosylmethionine and S-adenosylhomocysteine, had profound effect on the rate of phospholipid methylation, without having comparable effects on Ca2+ uptake. S-adenosylmethionine in increasing concentration stimulated methylation four-fold, while no significant changes in 45Ca2+ uptake were seen. S-adenosylhomocysteine did not inhibit 45Ca2+ uptake even at levels causing more than 95% decrease in methylation. In conclusion, while both phospholipid methylation and 45Ca2+ uptake seem to be hormonally controlled, the correlation between these two processes was not sufficient to support the notion that the changes in 45Ca2+ uptake are caused by the changes in phospholipid methylation.

Different localization of inositol 1,4,5-trisphosphate and ryanodine binding sites in rat liver.

The distribution of inositol 1,4,5-trisphosphate and ryanodine binding sites between plasma membrane, microsomal, and mitochondrial fractions of rat liver were compared. IP3 bound mostly to the plasma membrane fraction (Kd = 6 nM; Bmax = 802 fmol/mg protein). Some IP3 binding sites were also present in the microsomal and mitochondrial fractions (Kd = 2.5 and 2.9 nM; Bmax = 35 and 23 fmol/mg protein respectively). The possibility that these binding sites are due to contamination of the fractions with plasma membrane cannot be excluded. Binding of IP3 to the plasma membrane was inhibited by heparin but not by either caffeine or tetracaine. High-affinity ryanodine binding sites were present mostly in the microsomal fraction (Kd = 13 nM; Bmax = 301 fmol/mg protein). Lower affinity binding sites were also found to be present in the mitochondrial and plasma membrane fractions. Binding of ryanodine to the microsomal fraction was inhibited by both caffeine and tetracaine but not by heparin. These data demonstrate that IP3 and ryanodine binding sites are present in different cellular compartments in the liver. These differences in the localization of the binding sites might be indicative of their functional differences.

A fast in vitro effect of glucocorticoids on hepatic lipolysis.

A fast and direct effect of dexamethasone on liver metabolism is reported. In the perfused rat liver, addition of dexamethasone directly to the perfusate is followed by an increase in the level of free fatty acids (FFA) and glycerol. These effects of dexamethasone are evident within 30 minutes of hormone administration. The response to dexamethasone in livers from normal and adrenalectomized rats is similar. While dexamethasone and glucagon both had a lipolytic effect, these effects are not additive.

Glucagon-stimulated respiration and intracellular Ca2+.

The effects of extra- and intracellular Ca2+ on glucagon-stimulated respiration were examined in perfused rat liver. Glucagon increased the uptake of O2 to a significantly greater extent in Ca2+-containing perfusate than in Ca2+-free perfusate. If, however, the livers were perfused first with Ca2+-containing perfusate for 60 min in order to load the hormone-sensitive Ca2+ pool(s) and subsequently with Ca2+-free perfusate, glucagon was able to stimulate O2 uptake to the same extent in Ca2+-free, as in Ca2+-containing perfusate. These experiments support previous observations of a connection between Ca2+ and the hormonal stimulation of respiration, but indicate a role for intracellular, rather than extracellular, Ca2+ in the process.

Expression of photoreceptor cyclic nucleotide-gated cation channel alpha subunit (CNGCalpha) in the liver and skeletal muscle.

Glucagon and beta-adrenergic agents increase cAMP levels and stimulate Ca2+ influx in liver cells. There is no consensus as to the mechanism by which these hormones stimulate the influx of Ca2+. Using mouse retinal rod CNGCalpha cDNA probes, we cloned rat liver and skeletal muscle, and human hepatic CNGCalpha subunit sequences showing 97-100% identity with the human rod channel. In order to assess channel activity, the effect of cyclic nucleotides on free intracellular Ca2+ levels of isolated hepatocytes was measured. Dibutyryl-cAMP was more effective in increasing free Ca2+ levels than dibutyryl-cGMP. These data indicate that the CNGCalpha subunit is expressed in both the liver and skeletal muscle possibly mediating hormonal effects on ion fluxes.

Reduction of ryanodine binding and cytosolic Ca2+ levels in liver by the immunosuppressant FK506.

The mechanism of action of the immunosuppressant FK506 in the liver was studied. The hypothesis was tested that FK506 exerts its effect in the liver by interacting with the ryanodine-binding Ca2+ release channel. Two types of experiments were carried out: (1) [3H]-ryanodine binding studies with isolated microsomal fractions, and (2) cytosolic-free Ca2+ ([Ca2+]i) measurements with the intracellular Ca(2+)-indicator fura-2. The inclusion of FK506 in the incubation medium significantly decreased the binding of [3H]-ryanodine to liver microsomes. The Bmax of binding in control experiments was 405 fmol/mg protein; the presence of FK506 decreased the Bmax to 157 fmol/mg protein. Measurements of [Ca2+]i in the presence and absence of FK506 showed a decrease in [Ca2+]i in the presence of FK506. The data support the notion that FK506 interacts with the ryanodine binding Ca2+ channel in the liver and suggest a critical role for the ryanodine-binding Ca2+ channel in the hepatic responses to FK506. The interaction between FKBP-12 (FK506 binding protein) and the ryanodine-binding Ca2+ channel may be an essential link in the chain of events by which FK506 alters Ca(2+)-dependent cellular processes.

Demonstration of ryanodine-induced metabolic effects in rat liver.

The effects of ryanodine, a plant alkaloid which alters Ca2+ sequestration in the liver, on O2 uptake and gluconeogenesis were measured. Ryanodine administration to perfused rat liver resulted in the stimulation of O2 uptake and of gluconeogenesis. Because ryanodine does not affect directly mitochondrial respiration, its stimulatory effect on O2 uptake in the whole cell is likely to be secondary to the increased cytosolic free Ca2+ levels.

Effects of ryanodine on calcium sequestration in the rat liver.

Ryanodine, a highly toxic alkaloid known to react specifically with the Ca2+ release channels in sarcoplasmic reticulum (SR), was employed to study Ca2+ sequestration in the liver. Ryanodine at a 200 microM concentration increased cytosolic free Ca2+ levels and phosphorylase a activity in isolated hepatocytes. These effects may involve microsomal Ca2+ sequestration, because ryanodine, in the presence of inhibitors of mitochondrial Ca2+ uptake, at concentrations of 1 nM, 1 microM, 50 microM and 100 microM decreased 45Ca2+ retention in permeabilized hepatocytes. This inhibition of Ca2+ retention by ryanodine was not due to inhibition of the microsomal Ca(2+)-ATPase. Dantrolene, a compound shown previously to inhibit ryanodine binding in the liver, also decreased 45Ca2+ retention in permeabilized hepatocytes, and activated phosphorylase a. These results show that ryanodine administration alters calcium sequestration in liver. The possibility of the existence of a ryanodine-sensitive Ca(2+)-release channel in liver is discussed.