Delivery of Dihydroergotamine Mesylate to the Upper Nasal Space for the Acute Treatment of Migraine: Technology in Action.

Oral tablets account for the majority of medications used to acutely treat migraine, but relief can be limited by their rates of dissolution and absorption. The nose is an attractive alternative route of drug delivery since it provides patient convenience of at-home use, gastrointestinal (GI) avoidance, and rapid absorption of drugs into systemic circulation because of its large surface area. However, the site of drug deposition within the nasal cavity should be considered since it can influence drug absorption. Traditional nasal devices have been shown to target drug delivery to the lower nasal space where epithelium is not best-suited for drug absorption and where there is an increased likelihood of drug clearance due to nasal drip, swallowing, or mucociliary clearance, potentially resulting in variable absorption and suboptimal efficacy. Alternatively, the upper nasal space (UNS) offers a permeable, richly vascularized epithelium with a decreased likelihood of drug loss or clearance due to the anatomy of this area. Traditional nasal pumps deposit <5% of active drug into the UNS because of the nasal cavity's complex architecture. A new technology, Precision Olfactory Delivery (POD®), is a handheld, manually actuated, propellant-powered, administration device that delivers drug specifically to the UNS. A dihydroergotamine (DHE) mesylate product, INP104, utilizes POD technology to deliver drug to the UNS for the acute treatment of migraine. Results from clinical studies of INP104 demonstrate a favorable pharmacokinetic profile, consistent and predictable dosing, rapid systemic levels known to be effective (similar to other DHE mesylate clinical programs), safety and tolerability on the upper nasal mucosa, and high patient acceptance. POD technology may have the potential to overcome the limitations of traditional nasal delivery systems, while utilizing the nasal delivery benefits of GI tract avoidance, rapid onset, patient convenience, and ease of use.

Nasal Delivery of Acute Medications for Migraine: The Upper Versus Lower Nasal Space.

The acute treatment of migraine requires effective drugs that are well tolerated and provide rapid and consistent pain relief. Oral tablets are the most commonly used acute treatment for migraine; however, their effectiveness is limited by the rate of gastrointestinal (GI) tract absorption and first-pass hepatic metabolism, and they may not be ideal for patients experiencing GI motility issues. Nasal delivery is an attractive alternative route as it may circumvent GI tract absorption, avoid first-pass metabolism in the liver, and potentially reduce the frequency of GI adverse events. The large surface area and high vascularity within the nose may permit rapid absorption of therapeutics into the systemic circulation, allowing for rapid onset of action. However, the site of drug deposition (upper versus lower nasal cavity) may influence drug pharmacokinetics. Most approved nasal migraine therapies target the lower nasal space where the epithelium is less permeable, and they may be quickly cleared away due to increased ciliary function or dripping from the nose or swallowing, resulting in variable absorption and limited bioavailability. Together with its abundant vascularization, relative mucosal thickness stability, and low clearance rates, the upper nasal space harnesses the benefits of nasal delivery to potentially maximize drug efficacy.

The SNAP 101 Double-Blind, Placebo/Active-Controlled, Safety, Pharmacokinetic, and Pharmacodynamic Study of INP105 (Nasal Olanzapine) in Healthy Adults.

OBJECTIVE: INP105 is a drug-device combination of olanzapine and technology that delivers a powder formulation of olanzapine to the vascular-rich upper nasal space. This study evaluated the pharmacokinetics, pharmacodynamics, safety, and tolerability of single ascending doses of INP105, olanzapine intramuscular (OLZ IM), and olanzapine oral disintegrating tablet (OLZ ODT). METHODS: This was a phase 1, active and double-blind placebo comparator-controlled, ascending-dose, 2-period, incomplete-block, 1-way crossover study in 40 healthy subjects, randomized to single doses of OLZ IM (5 or 10 mg) or OLZ ODT (10 mg) in Period 1 and then 1 of 3 doses (5 mg, 10 mg, or 15 mg) of INP105 or placebo in Period 2 between July and October 2018. Sedation and attention were evaluated by visual analog scale (VAS), the Agitation/Calmness Evaluation Scale (ACES), and the Digit Symbol Substitution Test (DSST). RESULTS: At equivalent doses, INP105 provided similar area under the drug concentration-time curve (AUC) from time 0 to the last measurable concentration, AUC from time 0 to infinity, and maximum observed concentration (Cmax) as OLZ IM and greater Cmax than but similar AUCs to OLZ ODT. Median time to maximum concentration was less for INP105 (15, 10, and 9.5 min for 5 mg, 10 mg, and 15 mg, respectively) than for OLZ IM (20 and 15 min for 5 mg and 10 mg, respectively) or OLZ ODT (120 min). Effects as measured with the VAS, ACES, and DSST with INP105 5 mg were comparable to those with OLZ IM 5 mg, with earlier onset for INP105 10 mg and 15 mg and greater effects than placebo and OLZ ODT. The incidence of treatment-emergent adverse events with INP105 5 mg, 10 mg, and 15 mg was 80%, 66.7%, and 75%, respectively, compared to 90% and 100% for OLZ IM 5 mg and 10 mg, respectively, and 83.3% for OLZ ODT; most common were dizziness, hypotension, and orthostatic symptoms. CONCLUSIONS: INP105 has rapid absorption and pharmacodynamic effects and may represent an effective, convenient, noninvasive, and well-tolerated alternative for treating acutely agitated patients by self- or caregiver administration in the home, community, or hospital environments. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03624322.

Dihydroergotamine (DHE) - Then and Now: A Narrative Review.

OBJECTIVE: To provide a narrative review of clinical development programs for non-oral, non-injectable formulations of dihydroergotamine (DHE) for the treatment of migraine. BACKGROUND: Dihydroergotamine was one of the first "synthetic drugs" developed in the 20th century for treating migraine. It is effective and recommended for acute migraine treatment. Since oral DHE is extensively metabolized, it must be given by a non-oral route. Intravenous DHE requires healthcare personnel to administer, subcutaneous/intramuscular injection is challenging to self-administer, and the approved nasal spray formulation exhibits low bioavailability and high variability that limits its efficacy. Currently there are several attempts underway to develop non-oral, non-injected formulations of DHE. METHOD: A systematic search of MEDLINE/PubMed and ClinicalTrials.gov databases, then narrative review of identified reports, focusing on those published in the last 10 years. RESULTS: Of 1881 references to DHE from a MEDLINE/PubMed search, 164 were from the last 10 years and were the focus of this review. Further cross reference was made to ClinicalTrials.gov for 19 clinical studies, of which some results have not yet been published, or are studies that are currently underway. Three nasal DHE products are in clinical development, reawakening interest in this route of delivery for migraine. Other routes of DHE administration have been, or are being, explored. CONCLUSION: There is renewed appreciation for DHE and the need for non-oral, non-injected delivery is now being addressed.

STOP 101: A Phase 1, Randomized, Open-Label, Comparative Bioavailability Study of INP104, Dihydroergotamine Mesylate (DHE) Administered Intranasally by a I123 Precision Olfactory Delivery (POD® ) Device, in Healthy Adult Subjects.

OBJECTIVE: Investigate the safety and pharmacokinetics (PK) of INP104, intranasal dihydroergotamine mesylate (DHE) administered via a Precision Olfactory Delivery (POD® ) device, (Impel NeuroPharma, Seattle, WA) vs intravenous (IV) DHE and DHE nasal spray (Migranal® ) in healthy adult subjects. METHODS: This was a Phase 1, open-label, randomized, single-dose, 3-period, 3-way crossover study. Subjects received a single dose of A) INP104 1.45 mg (a drug-device combination product composed of DHE and the I123 POD device); B) DHE 45® Injection (IV) 1.0 mg; and C) DHE by Migranal® Nasal Spray 2.0 mg. Plasma levels of DHE and the major bioactive metabolite, 8'OH-DHE, were measured, and PK parameters were determined for both. Comparative bioavailability (BA) was assessed by calculating the ratio of the geometric means between treatments for Cmax and AUC0-inf on the ln-transformed data. Safety was assessed from adverse events, vital signs, electrocardiograms, and clinical laboratory values. RESULTS: Thirty-eight subjects were enrolled, 36 were dosed with at least 1 IP and 27 were included in the evaluation of PK and comparative BA. DHE plasma levels following INP104 1.45 mg administration reached 93% of Cmax by 20 minutes and were comparable to IV DHE 1.0 mg by 30 minutes (1219 ng/mL for INP104 vs 1224 ng/mL for IV DHE), which was the Tmax for INP104. From 30 minutes onward, DHE levels for INP104 closely matched those of IV DHE to 48 hours, the last time point measured. In comparison, the Cmax for Migranal was 299.6 pg/mL (approximately 4-fold less than INP104) and occurred at 47 minutes, 17 minutes later than INP104. Plasma DHE AUC0-inf were 6275, 7490, and 2199 h*pg/mL for INP104, IV DHE, and Migranal, respectively. Variability (coefficient of variation [CV%]) for Cmax and AUC0-inf for INP104 compared to Migranal indicated more consistent delivery with INP104. In the BA comparison using the PK population (subjects who had received all 3 treatments), the ratios of geometric means (percent) for Cmax and AUC0-inf were 7.9% and 74.2%, respectively, for INP104: IV DHE, and 445% and 308% for INP104: Migranal. Mean plasma concentration profiles for 8'-OH-DHE were proportionately lower and followed a similar profile to the parent compound, regardless of route of administration (IN vs IV) or delivery system (Migranal vs INP104). Treatment emergent AEs (TEAEs), of mostly mild intensity, were reported by 15/31 (48.4%), 21/32 (65.6%), and 14/34 (41.2%) subjects after INP104, IV DHE, and Migranal, respectively. Treatment-related TEAEs occurred in 6/31 (19.4%), 16/32 (50.0%), and 4/34 (11.8%) subjects after INP104, IV DHE, and Migranal, respectively. CONCLUSION: INP104 met the predefined statistical criteria for comparative bioavailability with IV DHE and Migranal. The shorter time to reach Cmax and at 4 times the plasma concentration of DHE in comparison to Migranal combined with a favorable tolerability profile support further investigation of INP104 as an effective, well tolerated, and non-invasive treatment for acute episodic migraine.

Positron Emission Tomography Assessment of the Intranasal Delivery Route for Orexin A.

Intranasal drug delivery is a noninvasive drug delivery route that can enhance systemic delivery of therapeutics with poor oral bioavailability by exploiting the rich microvasculature within the nasal cavity. The intranasal delivery route has also been targeted as a method for improved brain uptake of neurotherapeutics, with a goal of harnessing putative, direct nose-to-brain pathways. Studies in rodents, nonhuman primates, and humans have pointed to the efficacy of intranasally delivered neurotherapeutics, while radiolabeling studies have analyzed brain uptake following intranasal administration. In the present study, we employed carbon-11 radioactive methylation to assess the pharmacokinetic mechanism of intranasal delivery of Orexin A, a native neuropeptide and prospective antinarcoleptic drug that binds the orexin receptor 1. Using physicochemical and pharmacological analysis, we identified the methylation sites and confirmed the structure and function of methylated Orexin A (CH3-Orexin A) prior to monitoring its brain uptake following intranasal administration in rodent and nonhuman primate. Through positron emission tomography (PET) imaging of [11C]CH3-Orexin A, we determined that the brain exposure to Orexin A is poor after intranasal administration. Additional ex vivo analysis of brain uptake using [125I]Orexin A indicated intranasal administration of Orexin A affords similar brain uptake when compared to intravenous administration across most brain regions, with possible increased brain uptake localized to the olfactory bulbs.

Aerosol-stable peptide-coated liposome nanoparticles: a proof-of-concept study with opioid fentanyl in enhancing analgesic effects and reducing plasma drug exposure.

Previously, we reported a novel pressurized olfactory drug (POD) delivery device that deposits aerosolized drug preferentially to upper nasal cavity. This POD device provided sustained central nervous system (CNS) levels of soluble morphine analgesic effects. However, analgesic onset of less soluble fentanyl was more rapid but brief, likely because of hydrophobic fentanyl redistribution readily back to blood. To determine whether fentanyl incorporated into an aerosol-stable liposome that binds to nasal epithelial cells will enhance CNS drug exposure and analgesic effects and reduce plasma exposure, we constructed Arg-Gly-Asp (RGD) liposomes anchored with acylated integrin-binding peptides (palmitoyl-Gly-Arg-Gly-Asp-Ser). The RGD liposomes, which assume gel phase membrane structure at 25 °C, were stable under the stress of aerosolization as only 2.2 ± 0.5% calcein leakage was detected. The RGD-mediated integrin binding of liposome is also verified to be unaffected by aerosolization. Rats treated with fentanyl in RGD liposome and POD device exhibited greater analgesic effect, as compared with the free drug counterpart (AUC(effect) = 1387.1% vs. 760.1% MPE*min), whereas approximately 20% reduced plasma drug exposure was noted (AUC(0-120) = 208.2 vs. 284.8 ng min/mL). Collectively, fentanyl incorporated in RGD liposomes is physically and biologically stable under aerosolization, enhanced the overall analgesic effects, and reduced plasma drug exposure for the first 2 h.

Intranasal delivery of growth differentiation factor 5 to the central nervous system.

CONTEXT: Growth differentiation factor 5 (GDF5), in addition to its role in bone and joint development, protects dopaminergic (DA) neurons from degeneration, and is a potential therapeutic agent for Parkinson's disease. Its large size and insolubility at physiologic pH are obstacles for drug administration to the central nervous system (CNS) in humans. OBJECTIVE: In this study, formulations to deliver GDF5 to the brain using intranasal (IN) administration were developed. MATERIALS AND METHODS: IN administration of GDF5 in acidic buffer, 20 mM sodium acetate (NaAc) at pH 4.25, was performed in rats. Also, a lipid microemulsion (LME) comprised of olive oil and phosphatidylserine (PS) was used to formulate GDF5 at neutral pH for IN administration. Tissue concentrations of GDF5 were determined by both gamma counting and enzyme-linked immunosorbent assay (ELISA). RESULTS: IN administration of GDF5 in acidic buffers bypassed the blood-brain barrier (BBB), resulting in delivery to the brain with limited systemic exposure. IN administration of GDF5-LME increased drug targeting to the midbrain eightfold when compared to IN administration of GDF5 in acidic buffer. DISCUSSION AND CONCLUSION: This study is the first to show that GDF5 can be formulated at neutral pH and can be directly delivered to the CNS via IN administration, with biologically relevant concentrations in the midbrain where it may be used to treat Parkinson's disease.

Enhanced analgesic responses after preferential delivery of morphine and fentanyl to the olfactory epithelium in rats.

BACKGROUND: Centrally acting opioid analgesics such as morphine and fentanyl are effective, but their efficacy is often limited by a delayed response or side effects resulting from systemic first pass before reaching the brain and the central nervous system (CNS). It is generally accepted that drugs applied to the nasal cavity can directly access the brain and the CNS, which could provide therapeutic advantages such as rapid onset and lower systemic exposure. The olfactory region of the nasal cavity has been implicated in facilitating this direct nose-to-CNS transfer. If the fraction of opioid administered to the olfactory region could be improved, there could be a larger fraction of drug directly delivered to the CNS, mediating greater therapeutic benefit. METHODS: We have developed a pressurized olfactory delivery (POD) device to consistently and noninvasively deposit a majority of drug on the olfactory region of the nasal cavity in Sprague-Dawley rats. Using the tail-flick latency test and analysis of plasma and CNS tissue drug exposure, we compared distribution and efficacy of the opioids morphine and fentanyl administered to the nasal olfactory region with the POD device or the nasal respiratory region with nose drops or systemically via intraperitoneal injection. RESULTS: Compared with nose drop administration, POD administration of morphine resulted in a significantly higher overall therapeutic effect (area under the curve [over the time course] [AUC](effect)) without a significant increase in plasma drug exposure (AUC(plasma)). POD of morphine resulted in a nose-to-CNS direct transport percentage of 38% to 55%. POD of fentanyl led to a faster (5 vs 10 minutes) and more intense analgesic effect compared with nasal respiratory administration. Unlike intraperitoneal injection or nose drop administration, both morphine and fentanyl given by the POD device to olfactory nasal epithelium exhibited clockwise (plasma) versus effect hysteresis after nasal POD administration, consistent with a direct nose-to-CNS drug transport mechanism. CONCLUSIONS: Deposition of opioids to the olfactory region within the nasal cavity could have a significant impact on drug distribution and pharmacodynamic effect, and thus should be considered in future nasally administered opioid studies.

Effects of localized hydrophilic mannitol and hydrophobic nelfinavir administration targeted to olfactory epithelium on brain distribution.

Many nasally applied compounds gain access to the brain and the central nervous system (CNS) with varying degree. Direct nose-to-brain access is believed to be achieved through nervous connections which travel from the CNS across the cribriform plate into the olfactory region of the nasal cavity. However, current delivery strategies are not targeted to preferentially deposit drugs to the olfactory at cribriform. Therefore, we have developed a pressurized olfactory delivery (POD) device which consistently and non-invasively deposited a majority of drug to the olfactory region of the nasal cavity in rats. Using both a hydrophobic drug, mannitol (log P = -3.1), and a hydrophobic drug, nelfinavir (log P = 6.0), and POD device, we compared brain and blood levels after nasal deposition primarily on the olfactory region with POD or nose drops which deposited primarily on the respiratory region in rats. POD administration of mannitol in rats provided a 3.6-fold (p < 0.05) increase in cortex-to-blood ratio, compared to respiratory epithelium deposition with nose drop. Administration of nelfinavir provided a 13.6-fold (p < 0.05) advantage in cortex-to-blood ratio with POD administration, compared to nose drops. These results suggest that increasing the fraction of drug deposited on the olfactory region of the nasal cavity will result in increased direct nose-to-brain transport.

Novel Gd nanoparticles enhance vascular contrast for high-resolution magnetic resonance imaging.

BACKGROUND: Gadolinium (Gd), with its 7 unpaired electrons in 4f orbitals that provide a very large magnetic moment, is proven to be among the best agents for contrast enhanced MRI. Unfortunately, the most potent MR contrast agent based on Gd requires relatively high doses of Gd. The Gd-chelated to diethylene-triamine-penta-acetic acid (DTPA), or other derivatives (at 0.1 mmole/kg recommended dose), distribute broadly into tissues and clear through the kidney. These contrast agents carry the risk of Nephrogenic Systemic Fibrosis (NSF), particularly in kidney impaired subjects. Thus, Gd contrast agents that produce higher resolution images using a much lower Gd dose could address both imaging sensitivity and Gd safety. METHODOLOGY/PRINCIPAL FINDINGS: To determine whether a biocompatible lipid nanoparticle with surface bound Gd can improve MRI contrast sensitivity, we constructed Gd-lipid nanoparticles (Gd-LNP) containing lipid bound DTPA and Gd. The Gd-LNP were intravenously administered to rats and MR images collected. We found that Gd in Gd-LNP produced a greater than 33-fold higher longitudinal (T(1)) relaxivity, r(1), constant than the current FDA approved Gd-chelated contrast agents. Intravenous administration of these Gd-LNP at only 3% of the recommended clinical Gd dose produced MRI signal-to-noise ratios of greater than 300 in all vasculatures. Unlike current Gd contrast agents, these Gd-LNP stably retained Gd in normal vasculature, and are eliminated predominately through the biliary, instead of the renal system. Gd-LNP did not appear to accumulate in the liver or kidney, and was eliminated completely within 24 hrs. CONCLUSIONS/SIGNIFICANCE: The novel Gd-nanoparticles provide high quality contrast enhanced vascular MRI at 97% reduced dose of Gd and do not rely on renal clearance. This new agent is likely to be suitable for patients exhibiting varying degrees of renal impairment. The simple and adaptive nanoparticle design could accommodate ligand or receptor coating for drug delivery optimization and in vivo drug-target definition in system biology profiling, increasing the margin of safety in treatment of cancers and other diseases.

Intranasal deferoxamine provides increased brain exposure and significant protection in rat ischemic stroke.

Deferoxamine (DFO) is a high-affinity iron chelator approved by the Food and Drug Administration for treating iron overload. Preclinical research suggests that systemically administered DFO prevents and treats ischemic stroke damage and intracerebral hemorrhage. However, translation into human trials has been limited, probably because of difficulties with DFO administration. A noninvasive method of intranasal administration has emerged recently as a rapid way to bypass the blood-brain barrier and target therapeutic agents to the central nervous system. We report here that intranasal administration targets DFO to the brain and reduces systemic exposure, and that intranasal DFO prevents and treats stroke damage after middle cerebral artery occlusion (MCAO) in rats. A 6-mg dose of DFO resulted in significantly higher DFO concentrations in the brain (0.9-18.5 microM) at 30 min after intranasal administration than after intravenous administration (0.1-0.5 microM, p < 0.05). Relative to blood concentration, intranasal delivery increased targeting of DFO to the cortex approximately 200-fold compared with intravenous delivery. Intranasal administration of three 6-mg doses of DFO did not result in clinically significant changes in blood pressure or heart rate. Pretreatment with intranasal DFO (three 6-mg doses) 48 h before MCAO significantly decreased infarct volume by 55% versus control (p < 0.05). In addition, post-treatment with intranasal administration of DFO (six 6-mg doses) immediately after reperfusion significantly decreased infarct volume by 55% (p < 0.05). These experiments suggest that intranasally administered DFO may be a useful treatment for stroke, and a prophylactic for patients at high risk for stroke.

Intranasal tat alters gene expression in the mouse brain.

Intranasal (IN) delivery of HIV-1 Tat in aging mice was investigated as a possible model for HIV-1 infection in the brain. After IN administration, the distribution of [(125)I]-labeled Tat in the brains of Swiss Webster mice was evaluated by autoradiography and gamma counting. [(125)I]-labeled Tat was detected at the highest concentrations in the olfactory bulb, cervical nodes, and trigeminal nerve tract. In another experiment, APPSw transgenic mice were used to model chronic Tat exposure. The mice were treated intranasally with 6 mug Tat (n = 4) or vehicle (n = 4) three times per week for 4 weeks. Total RNA was isolated from the frontal cortex, and differential gene expression analysis was performed using gene microarrays. Gene ontology profiles indicated innate immunity, inflammatory and apoptotic responses. Five genes of interest in the Tat-treated mice that were significantly elevated in the microarrays were validated by RT-PCR. One gene, the Toll-like receptor 9 (Tlr9), has previously been shown to activate signaling cascades leading to innate immunity and enhanced HIV-1 gene expression. A second gene, Fas, plays a key role in neuroinflammation. Two cysteine-rich cytokines associated with chemotaxis were elevated: MCP-1 (Ccl2), which is chemotactic for monocytes, and Ccl17 (TARC), which is chemotactic for lymphocytes. Finally, the gene sestrin was significantly elevated and has been associated with oxidative stress, in particular amyloid beta-induced oxidative stress. This IN Tat model of neuroinflammation may be useful to study HIV-1-induced neurodegeneration.

Molecular modeling of the calmodulin binding region of calcineurin.

The Homology module within Insight-II was used to model residues 374-420, sequences missing in the coordinates of resolved structure of the catalytic subunit of calcineurin. The modeling was done in two segments. The calmodulin binding region from residues 389 to 420 was modeled based on the structure of two other proteins having calmodulin binding domains with the same 1-8-14 structural motif as calcineurin. The link region (residues 374-389) between the calmodulin binding region and the solved core sequence was generated as a random loop and two residues at the C-terminal end of the sequence were added to the model using the EndRepair function within Homology. The model was refined using the Discover module of Insight-II with energy minimization. The Builder module was used to merge the modeled regions with the solved structure of calcineurin (residues 14-373). A final refinement step was done for the joined calcineurin model. From the model, it was predicted that the calmodulin and cyclophilin binding regions seem to be proximal. Biochemical experiments provided evidence that cyclosporin-A influenced calmodulin binding and activation of calcineurin consistent with overlapping binding regions.

Identification of mouse brain proteins associated with isoform 3 of metallothionein.

Using immunological approaches and mass spectrometry, five proteins associated with metallothionein-3 in mouse brains have been identified. Metallothionein-3 and associated proteins were isolated using immunoaffinity chromatography over immobilized anti-mouse brain MT3 antibody. Proteins in the recovered pool were separated by SDS-polyacrylamide gel electrophoresis, and distinct bands were excised and the proteins digested using trypsin. Peptides were extracted and analyzed using electrospray ionization mass spectrometry. Initial identification was done comparing the identified peptide mass:charge ratios to the MASCOT database. Confirmation of proteins was accomplished by sequencing of selected peptides using tandem mass spectrometry and comparison to the MASCOT database. The proteins were heat-shock protein 84 (mouse variant of heat-shock protein 90), heat-shock protein 70, dihydropyrimidinase-like protein 2, creatine kinase, and beta actin. Independently using antibodies against metallothionein-3, creatine kinase, and heat-shock protein 84 showed that all three proteins were coimmunoprecipitated from whole mouse brain homogenates with each of the three antibodies. Mixing purified samples of metallothionein and human brain creatine kinase also generated a complex that could be immunoprecipitated either by anti-metallothionein-3 or anticreatine kinase antibody. These data are consistent with metallothionein-3 being present in the mouse brain as part of a multiprotein complex providing new functional information for understanding the role of metallothionein-3 in neuronal physiology.