The following is an article I am posting in various AD forums and blogs.
William A. DeGroodt, President/COO of Kurve Technology has expressed interest in conducting clinical trials, and has contacted the licensing department at Amgen.
Disclosure: I have no interests in Kurve Technology or Amgen. I am an Alzheimer's advocate on behalf of my wife, Linda, who has AD at 51.
___________________________________________
Enbrel Treatment to Reverse Symptoms in Alzheimer's:
Perispinal or Intranasal?
Robert Lee; AD Advocate rpl20080225.2 tumates@gmail.com

Publicity following a paper published in the Journal of Neuroinflammation has raised worldwide interest.
Rapid cognitive improvement in Alzheimer's disease following perispinal etanercept administration
Edward L Tobinick, Hyman Gross
Journal of Neuroinflammation 2008, 5:2 (9 January 2008)

Enbrel (etanercept), a biologic medication taken by more than 470,000 people worldwide, is a type of protein called a tumor necrosis factor (TNF) blocker. It blocks the action of a substance your body's immune system makes called TNF. It was approved for human use in 1998.
The paper's Abstract states: "Substantial basic science and clinical evidence suggests that excess tumor necrosis factor-alpha (TNF-alpha) is centrally involved in the pathogenesis of Alzheimer's disease." Finding a method allowing etanercept to bypass the blood brain barrier is the basis for both the technique described in the paper and the results shown from the treatment.
Enbrel is distributed by Amgen and Wyeth. http://www.enbrel.com/ .
The perispinal etanercept technique is patented by the Institute of Neurological Research, http://www.nrimed.com . The single patient discussed in the paper was a follow up to a 6 month 15 person trial in 2006. The perispinal etanercept treatment has also been offered to AD patients on a private pay basis for three years at the Institute of Neurological Research clinic. There are currently no other clinics known to offer the off-label perispinal etanercept AD treatment or formal clinical trials known to be underway.

The online Journal of Neuroinflammation publication of the Tobinick & Gross paper was on 1/9/08, with over 42,000 online reads in 6 weeks.
Various Alzheimer's online community forums have responded to caregiver peer requests for AD patients to share experiences with INR's perispinal etanercept treatment.
Forum postings have offered week by week anecdotal confirmation that for many patients perispinal injections with Enbrel partially reverse Alzheimer's symptoms and improve patient quality of life.
Enbrel, according to prescribing information, dissipates in 4 to 8 days. Long term therapy requires a maintenance program with weekly injections to sustain results. Even if the perispinal etanercept injections were available in locations other than INR's Los Angeles clinic, it is a burden for AD patients as injections require the clinical setting described in the paper:
"Twenty-five mg of etanercept in 1 cc of sterile water was administered by posterior cervical interspinous injection in the midline with a 27 gauge needle at the C6–7 interspace followed by Trendelenburg positioning with the head dependent for five minutes, as previously described, to effect entry of etanercept into the cerebrospinal venous system."
Results demonstrated by this method are reportedly due to etanercept molecules bypassing the blood brain barrier.

Contrast perispinal with intranasal.

Many companies are developing technology aimed at nasal drug delivery to the CNS.
Intranasal etanercept Alzheimer's treatment should now be explored as it has the potential of non-invasive, self dosing ease for use for the patient. If intranasal etanercept treatment duplicates the reported results of perispinal injections in the INR clinical studies, the impact for Alzheimer's sufferers could be profound.
An intranasal drug/device's popularity could exceed the success of the Enbrel SureClic autoinjector. A collaberation with Amgen providing premeasured dosing packs for a device like Kurve Technology's ViaNase electronic Atomizer could provide a non-invasive, simple, safer transport method to the same end goal as a perispinal injection: bypassing the blood brain barrier with the drug. http://www.kurvetech.com/TechnologyNosetobrain.asp
For Alzheimer's patients, the opportunity for symptom reversal, home treatment, travel, and freedom from weekly clinic visits for required injections will be compelling.
Enbrel has been available for 10 years, with 470,000 current users and known risks.
Most moderate to severe Alzheimer's patients eventually find available treatment ineffective in halting or reversing the disease that is inevitably fatal.
A case may be made for an Expedited IRB Review Procedure for intranasal Enbrel delivery as an AD treatment, especially in the advanced stages.

Hi Bob,

This is VERY INTERESTING! I'm still planning on taking my Mom in for the treatments as soon as they call me, but I will be watching for this much safer and less costly treatment in the future.

Thank you for sharing this!

Felicia

where can i sign my mother up?

Look through www.clinicaltrials.gov for studies recruiting near you if you want to help with the research.
This one in Kansas is testing intranasal insulin:
http://clinicaltrials.gov/ct2/show/NCT00581867?cond=%22Alzheimer+Disease%22&rank=160

Anyone hear any more about this?

Believe that Dr. T included this concept in his procedural patents covering the use of Enbrel as a tool in fighting Alzheimer's. Think that the idea died. If someone is going to risk possible litigation from procedural patent infringement, why should they bother to go through the effort of 'inventing'?

Also one more question..is the shot given at C3-4 or C6-7?

Thank you.

Momota---I'm not sure of the answer to your question. Think that you might be able to find it by looking back over the last 3 or 4 pages of the Enbrel for medication thread. If you don't have the time to go wading through, you might e-mail Bob Lee. Perhaps he will be kind enough to tell you where he believes Linda's doctor administered her injections.

FYI - I came across this today:

TNF-alpha Blockers Pose Higher Risk for Fungal Infections

The prescribing information for tumor necrosis factor (TNF)-alpha blockers must carry stronger warnings about the potential for opportunistic fungal infections, the FDA mandated on Thursday.

Despite the existing warnings, "health care professionals are not consistently recognizing cases of histoplasmosis and other invasive fungal infections, leading to delays in treatment," the agency says.


The FDA has examined 240 reports of histoplasmosis among patients receiving Humira (adalimumab), Enbrel (etanercept), and Remicade (infliximab). Treatment was delayed in at least 21 of these patients because the infection was not immediately recognized; 12 died. The agency has also reviewed one report of the condition in a patient using Cimzia (certolizumab).

Other fungal infections noted in patients on TNF-alpha blockers include coccidioidomycosis and blastomycosis; some of these patients have died.

FDA news release
Related Journal Watch link(s):
Journal Watch Dermatology coverage of recent study showing link between TNF blockers and fungal infections (Subscription required)

Does anyone know of a practitioner who is administrating perispinal enbrel in the NC or NY area?

IMC - I believe the doctor in NYC is Dr. Gayatri Devi (212) 517-6881. From what I've heard, tho, she's VERY pricey.

Bob Lee, Where did you obtain the procudure for perispinal enbrel (landmarks and dosing)?

Thank you DebZ

Has Kurve Technology progressed in its idea of a clinical trial of Enbrel via nasal spray?

ST---I doubt if Kurve Technology has made any progress. 2 of the reasons for this lack of progress may be:

Kurve cannot do a clinical trial without the permission of Amgen

Unless they have been amended...Dr. T's patents include intranasal delivery.

You can do research without infringing a patent. It's only when you try selling a product based on the patent that infringement comes in to play. And even then, the person who holds the patent has quite a bit of time before he has to file a lawsuit against the infringement before he loses his rights, and it may be to that person's advantage to wait -- to get more of an idea how well the product may sell. (And, of course, it might turn out that claims associated with intranasal administration are indefensible ... or maybe even Tobinick's entire patent is indefensible.)

(It doesn't sound like Tobinick had Amgen's permission to use their injectable product ... are there special circumstances that would require Kurve to obtain permission to evaluate an intranasal form?)

quote:

(And, of course, it might turn out that claims associated with intranasal administration are indefensible ... or maybe even Tobinick's entire patent is indefensible.)

I'm neither a doctor nor a lawyer...but... is an idea that I have and still believe might prove credible.

I'm not talking about whether intranasal enbrel might be effective. You can get a patent but have some of its claims, or even the entire patent, turn out to be invalid -- "indefensible" -- when you try to enforce it.

Plenty of people do research on concepts that appear to be covered by one or more patents, to see how valuable the concept might actually be. Once you decide it has value and you want to commercialize it, then you decide whether you want to license the patent, or just go right ahead and infringe it, and see what happens after that. The patent holder may not have the stomach (or resources) for a court battle, and even if he does, the courts may decide against him. Patent law is not exactly what you'd call "clear cut."

There are all sorts of things that can invalidate a patent that purportedly covers a perfectly viable technical or scientific concept.

A new article, in the Journal of Neuroimmunology, has published about a future potential anti-TNF agent for treatment of AD via the intranasal route.

The abstract and link to the full article is available at: http://www.ncbi.nlm.nih.gov/pubmed/19733918

The article is entitled:
Intranasal delivery of ESBA105, a TNF-alpha-inhibitory scFv antibody fragment to the brain.
The article cites two of Dr. Tobinick's articles.

The current problem regarding intranasal delivery of anti-TNF agents is that sinusitis is one of the most common forms of infection complicating use of anti-TNF therapeutics, so intranasal delivery of anti-TNF agents is not recommended.

I have pasted the entire PDF File into this post since you can't get the full article on the internet without subscribing. The tables/diagrams are not included. If you're interested in seeing the file, please e-mail me and I will send it to you.

Felicia
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Please cite this article as: Furrer, E., et al., Intranasal delivery of ESBA105, a TNF-alpha-inhibitory scFv antibody fragment to the brain, J. Neuroimmunol. (2009), doi:10.1016/j.jneuroim.2009.08.005
1. Introduction
Antibody-based therapies have proven efficacious in a variety of
diseases and due to their high target specificity are generally
considered to be associated with improved safety profiles as
compared to small chemical molecules. Current technologies allow
for the generation of antibodies characterized by specific binding to
virtually any protein target in the human body. Thus, antibodies
represent a pharmacologic class that could be used for the treatment
of a majority of human diseases including disorders of the central
nervous system (CNS). However, the high molecular weight of conventional
monoclonal antibodies of about 150 kDa prevents them
from efficient penetration into tissues and through tissue barriers
following systemic administration (Banks, 2008; Ottiger et al., 2009;
Reilly et al., 1995). Only few full-length antibodies were described
to penetrate the blood–brain-barrier (BBB) following systemic administration.
Such antibodies most probably enter the CNS via extracellular
pathways (Banks et al., 2002) and the concentrations that
reached the CNS usually correspond to at most a few percent of
plasma concentrations only. Similar limitations regarding the penetration
of biological barriers such as epithelia apply also for smaller
size Fab fragments (∼50 kDa).
In contrast, single-chain Fv antibody fragments (scFv) (26.3 kDa)
may efficiently penetrate certain tissue barriers, due to their more
efficient extravasation formblood vessels and improved diffusion in the
extracellular matrix (Thurber and Wittrup, 2008). We have recently
shown that ESBA105, a TNF-alpha inhibitory scFv penetrates through
ocular epithelial barriers and reaches therapeutic concentrations in
different ocular compartments including the vitreous humor and retina
(Furrer et al., 2009; Ottiger et al., 2009). ESBA105most probably reaches
the inner compartments of the eye by passive diffusion through tight
junctions within the extracellular space. Thus, the scFv format qualifies
for topical applications and it is hypothesized that its excellent tissue
penetration properties may also facilitate delivery into the CNS
following either intranasal or systemic administration.
A number of high molecular weight proteins have been successfully
delivered to the CNS by intranasal administration in rodents and
monkeys (Dhanda et al., 2005; Frey, 2002; Thorne et al., 2008). It was
previously shown that intranasally administered proteins can migrate
along the olfactory and trigeminal nerves to reach the CNS (Ma et al.,
2007; Thorne and Frey, 2001; Thorne et al., 2008, 2004). In humans
peptide hormones were successfully delivered to the cerebrospinal
fluid by intranasal administration (Born et al., 2002). Thus, proteins
might bypass the blood–brain-barrier via direct migration from the
nasal cavity to the brain involving two mechanisms, a slow intraneuronal
and/or a fast extraneuronal pathway. The latter includes
absorption of the drug through the nasal olfactory epithelium
followed by transport possibly within perineural or lymphatic
channels or through perivascular spaces directly into the brain
parenchyma and/or the cerebrospinal fluid (Frey, 2002; Illum, 2000;
Thorne and Frey, 2001; Thorne et al., 2004). Efficacy of intranasally
administered proteins has been demonstrated in a variety of rodent
disease models (Capsoni et al., 2002; De Rosa et al., 2005; Xiao et al.,1998). In addition, in humans intranasal administration of insulin
improved mood and memory and further had a positive effect on
memory in patients suffering from mild cognitive impairment or even
Alzheimer's disease (Benedict et al., 2004). Interestingly, intranasal
insulin did not increase circulating insulin concentrations and had no
effect on plasma glucose levels (Benedict et al., 2004). Therefore and
in contrast to systemic injection, intranasal administration may
represent an application method supporting efficient delivery of
high molecular weight drugs directly to the CNS while avoiding high
exposure in the blood circulation, thus lowering the probability of
systemic side effects. Although CNS concentrations of agonistic
proteins following intranasal delivery are sufficiently high to exert a
biological effect, the question remains whether this holds true as well
for drugs that exert their effect by inhibition of protein–protein
interaction, as such inhibitory compounds usually require a significant
local excess over the target proteins.
Thus, the aim of this study was a) to investigate the pharmacokinetics
of intranasal application of an scFv antibody fragment in the
mouse, and b) to evaluate the feasibility of the intranasal delivery
route for the TNF-alpha inhibitory antibody fragment ESBA105 as a
potential therapy for neurodegenerative disorders such as Alzheimer's
disease, Parkinson's disease and multiple sclerosis.
2. Materials and methods
2.1. Materials
All chemicals were purchased from Sigma Aldrich, Buchs, Switzerland,
if not mentioned differently.
2.2. Recombinant expression and purification of ESBA105
ESBA105, an anti-TNF-alpha single-chain antibody fragment with a
molecular weight of 26.3 kDa, was expressed in and purified from
Escherichia coli as described earlier (Furrer et al., 2009; Ottiger et al.,
2009). Briefly, ESBA105 was produced by recombinant expression in E.
coli BL21(DE3), refolding from inclusion bodies and subsequent size exclusion
chromatography. For animal studies ESBA105was formulated
at 10 mg/ml (for intranasal administration) or 0.5 mg/ml (for intravenous
injection) in 50mM sodium phosphate, 150 mM NaCl, pH 6.5.
The endotoxin content as determined in the LAL clotting assay was
below 0.1 EU in all formulations used for in vivo experiments.
2.3. Animals
Eight to ten week old male Balb/c mice (Charles River, Sulzfeld,
Germany) were housed in groups of five under a 12-hour light/dark
cycle. Food and water were provided ad libitum. All performed animal
experiments were approved by the local Swiss Veterinary Authority in
accordance with Swiss Animal Welfare Laws.
2.4. Establishment of intranasal administration
For efficient and specific drug delivery into the CNS the applied
substance should remain in the nasal cavity. However, in a variety of
studies the intranasally applied substance was found not only in the
nasal cavity but also in the respiratory system and the gastrointestinal
tract due to breathing and ingestion (Eyles et al., 1999; Klavinskis
et al., 1999; Lundholm et al., 1999; Trolle et al., 2000). From the
respiratory as well as from the gastrointestinal tract, the substance
may be absorbed into circulation. Consequently, direct transport from
the nasal cavity to the brain can hardly be distinguished from systemic
absorption through respiratory and gastrointestinal tract and subsequent
transport across the BBB. Aspects, such as anaesthesia, animal
position during and post substance administration as well as volume
and frequency of administration may influence the residence time of
the compound in the nasal cavity. In order to a) optimize residence
time in the nasal cavity and b) minimize distribution into lungs and
stomach, we evaluated different administration techniques using
Evans blue as a dye. Balb/c mice were intranasally dosed with 0.3%
Evans blue in 0.9% NaCl. Administration was performed as described
in Table 1. At predefined time points animals were sacrificed by CO2
inhalation. Lungs and stomach were harvested and visually inspected
for the presence of Evans blue. First we examined the distribution of a
single dose to either anaesthetized or alert mice that were held in a
supine position during the administration. The dye was found in the
lungs as well as in the stomach regardless of whether the animals
were conscious or not. Also keeping the anaesthetized animals for 30–
50 min in the supine position instead of only 3 min, or splitting the
total volume to 10 μl doses that were administered in 5 min intervals
alternating between the two nares, did not reduce Evans blue delivery
to the lungs and stomach. In order to minimize flowing of the dye
out of the nasal cavity, volumes as low as 2 μl were applied to
anaesthetized animals. Optimal results with only minimal traces of
Evans blue in the lungs and total absence of dye in the stomach were
obtained by keeping the animals under isoflurane (Provet, Lyssach,
Switzerland) anaesthesia in a supine position and treating each nare
with 2 μl Evans blue at 5 min intervals until a total of 40 μl was
reached (45 min) (Table 1). Therefore, this technique was applied for
intranasal administration of ESBA105 in all experiments.
2.5. Intranasal and intravenous administrations of ESBA105
Prebleeds of all animals were collected ten days before the intranasal
or intravenous dosing with ESBA105. Intranasal administration
of ESBA105 was carried out under isoflurane (Provet, Lyssach,
Switzerland) anaesthesia. Mice were placed in a supine position and
a total of 40 μl (400 μg) ESBA105 was administered by pipette in 2 μl
drops, treating each nare every 5 min over a total of 45 min. For the
intranasal PK study, four animals were sacrificed at 1, 2, 4, 6, 8, 10, 12,
and 24 h after the first intranasal instillation. In some experiments
3 mM Pz-peptide (4-Phenylazobenzoxycarbonyl-Pro-Leu-Gly-Pro-DArg;
Bachem, Bubendorf, Switzerland), a penetration enhancer that
facilitates the transport of paracellular markers by triggering opening
of tight junctions in a transient, reversible manner (Yen and Lee,
1994), was added to the ESBA105 formulation. The addition of the Pzpeptide
to the formulation was well tolerated by the animals and no
difference in behavior was observed compared to mice that were
treated with ESBA105 without Pz-peptide. Four animals were
sacrificed at 1, 2, and 4 h after the first administration. For intravenous
injection, mice were placed in a restrainer and 40 μg (80 μl) ESBA105
was administered in a bolus injection into the tail vein. The intravenous
dose was chosen to best approximate the systemic exposure
according to the area under the blood concentration-time curve
(AUC) observed over a 4 h period with intranasal administration of
400 μg ESBA105. Two animals were sacrificed at each time point (1, 2,
and 4 h). At the time of sacrifice mice were deeply anaesthetized with
a mixture of ketamine (Ketasol100, 65 mg/kg; Pharmacy, Schlieren,
Switzerland), xylazine (Rompun, 13 mg/kg; Provet, Lyssach, Switzerland)
and acepromazine (Prequillan, 2 mg/kg; Arovet, Zollikon,
Switzerland). A blood sample was collected by heart puncture before
perfusing the mice with 20 ml PBS. The brains were carefully
harvested and dissected into olfactory bulb, cerebrum including
thalamus and hypothalamus, cerebellum and brainstem. The tissues
were weighed, frozen on dry ice and stored at −80 °C until analysis.
2.6. Tissue preparation
100 μl lysis buffer (10 mM Tris, pH 7.4, 0.1% SDS, with proteinase
inhibitor cocktail (Roche Diagnostics, Rotkreuz, Switzerland)) was
added per 15 mgof brain tissue. Tissueswere sonicated for 5 s (8 cycles,
100% intensity) (Sonoplus, Bandelin, Berlin, Germany), centrifuged and the supernatants were subjected to ELISA based determination of
ESBA105 concentrations.
2.7. Quantification of ESBA105 in serum and brain tissue
ESBA105 concentrations were determined by triplicate measurements
of each sample in a direct ELISA. 96-well plates (NUNCMaxiSorp;
Omnilab, Mettmenstetten, Switzerland) were coated with 0.5 μg/ml
human TNF-alpha (Peprotech, London, UK) in PBS overnight at 4 °C.
Between each of the following steps plates were washed three times
with TBS-T (0.005% Tween20; Axon Lab, Baden-Dättwyl, Switzerland)
using a microplatewasher (ASYS Atlantis, Salzburg, Austria). Unspecific
binding sites were saturated by 1.5 h incubation in PBS/1% BSA/0.2%
Tween20. Predilutions of each sample were prepared in dilution buffer
(PBS, 0.1% BSA, 0.2% Tween20) containing 10% of the respective matrix
(olfactory bulb, cerebrum, cerebellum, brainstem or serum). Standard
reference dilution series (50–0.5 ng/ml) of ESBA105 was prepared in
dilution buffer/10% respective matrix. Prediluted samples and standard
reference dilutions were then added to the wells and plates were
incubated for 1.5 h at roomtemperature. Bound ESBA105 was detected
with a biotinylated affinity purified polyclonal rabbit anti-ESBA105
antibody (AK3A, ESBATech, Schlieren, Switzerland) that was diluted
1:20,000 in dilution buffer (1.5 h, room temperature). AK3A, in turn,
was detected with poly-horseradish peroxidase streptavidin (Stereospecific
Detection Technologies, Baesweiler, Germany) at a concentration
of 0.2 ng/ml dilution buffer. POD (Roche Diagnostics, Rotkreuz,
Switzerland) was used as peroxidase substrate and the color reaction
was stopped after 2 to 20 min (depending on color intensity) by the
addition of 1 M HCl. Absorbance was measured at 450 nm in a plate
reader (Sunrise; Tecan, Maennedorf, Switzerland) and ESBA105 concentrations
in samples were calculated from a standard curve using
polynomial second-order regression as best fit for the standard curves
(r2>0.98) (GraphPad Prism 4.03; GraphPad Software, Inc., San Diego,
CA). The minimum quantifiable concentration (LOQ) of ESBA105 was
5 ng/ml in the serum and 33 ng/ml in the brain tissue, respectively.
Undiluted samples that resulted in signals below the lower limit of
quantitationwere set to LOQ for mathematical evaluation and graphical
display.
3. Results
3.1. Efficient delivery of ESBA105 to the CNS following intranasal
application
Following intranasal administration to mice, ESBA105 reached
significant concentrations in all analyzed brain regions. High
concentrations were measured already at the first sampling time
point, i.e. 1 h after the first administration. Maximum ESBA105
concentrations (Cmax) in the cerebellum and brainstem were reached
within 1h after the first instillation, whereas concentrations in the
olfactory bulb and cerebrum peaked slightly later at 2 h. ESBA105
levels then declined in all brain regions and a clear second, however,
lower peak was observed in the olfactory bulb, the cerebellum and the
brainstem after 6 to 12 h (Fig. 1) indicating that two different
migration routes are likely to exist. Highest concentrations were
measured in the olfactory bulb and the brainstem. In the olfactory
bulb which is connected with the nasal cavity through the olfactory
system (N. olfactorius), concentrations culminated at 9455 ng/ml and
were even higher in the brainstem (11067 ng/ml) which is connected
with the nasal passages through the peripheral trigeminal system (N.
trigeminus) (Table 2). Cmax in the cerebrum (975 ng/ml) was slightly
delayed (2 h) and about seven to ten times lower than in the
cerebellum or the olfactory bulb, respectively. This finding can be
explained by the hypothesis that ESBA105 first reaches the olfactory
bulb and the brainstem and from there distributes to the cerebrum
and cerebellum. Similar to the brainstem and cerebellum, Cmax in
serum was reached at 1 h after the first administration of ESBA105
and peaked a second time between 5 and 10 h. Interestingly, ESBA105
levels remained almost constant during the last 12 h (Fig. 1).
3.2. Direct delivery of ESBA105 into CNS after intranasal administration
To determine whether ESBA105 migrates to the CNS directly from
the nasal cavity or possibly, via systemic absorption and subsequent
trans-BBB delivery to the brain, we compared intranasal administration
side by side with intravenous injection. The intravenous dose of
ESBA105 (40 μg) was chosen such that a similar systemic exposure
was expected for both routes (the intranasal dose was 400 μg). In case
delivery to the CNS would mainly occur from the circulation through
the BBB, both routes should result in similar ESBA105 concentrations
in the CNS. Indeed, following intravenous injection, ESBA105 was
detected in all analyzed regions, except the cerebrum where
concentrations were below the lower limit of quantitation. However,
considerably higher drug concentrations were measured in all brain
regions following intranasal administration (Fig. 2). Maximum
ESBA105 levels in the cerebellum and brainstem upon intranasal
dosing of 400 μg were about 10- to 18-fold higher when compared to
intravenous injection of 40 μg and Cmax in the olfactory bulb was even
more than 60-fold higher for intranasal versus intravenous administration
(Table 3). The two different doses following intranasal and
intravenous administrations were intended to produce similar
systemic exposures, thus, allowing discrimination between the two
migration routes, namely direct transport from the nasal cavity to the
CNS or delivery across the BBB. Nevertheless, serum concentrations
were clearly lower after intranasal administration (Fig. 2) reaching
6006 ng/ml while Cmax following intravenous injection was more
than 10-fold higher (63709 ng/ml) (Table 3). No detectable concentrations
were observed in the cerebrum following intravenous
injection of 40 μg. In contrast, application of identical total doses for
both routes (400 μg) resulted in 12.9-fold higher ESBA105 concentrations
in the cerebrum for the intranasal route (Table 3, bottom row).
Following intravenous injection,maximal concentrations (Cmax) and
exposures (AUC) in olfactory bulb, cerebellum and brainstem reached
similar values with 202,257, and 174 ng/ml for Cmax and 448,567, and
416 ng-h/ml for AUC, respectively. Cmax in the brain tissues following
intravenous injection was at 2 h and no ESBA105 could be detected at
Table 1
Administration scheme and presence of dye following intranasal delivery of 0.3% Evans blue dye.
Application Anaesthesia Sacrificed
(after first
administration)
Evans blue
Volume Interval Nare Isoflurane Duration Lungs Stomach
1×40 μl – Both No – 50 min + ++
1×40 μl – Both Yes 3 min 50 min + (+)
1×50 μl – Both Yes 3 min 3 min + +
1×50 μl – Both Yes 3 min 30 min ++ ++
1×50 μl – Both Yes 3 min 50 min ++ ++
10×10 μl 5 min Alternating Yes 45 min 55 min +++ +++
10×(2+2 μl) 5 min 2 μl per nare Yes 45 min 50 min − −
E. Furrer et al. / Journal of Neuroimmunology xxx (2009) xxx-xxx 3
ARTICLE IN PRESS

4 h. In contrast, following intranasal administration clearly higher
concentrationsweremeasured in all brain regions. Highest valueswere
obtained for the olfactory bulb (Cmax: 12586 ng/ml; AUC: 23130 ng-h/
ml) followed by the brainstem (Cmax: 3169 ng/ml; AUC: 7942 ng-h/ml),
cerebellum (Cmax: 2819 ng/ml; AUC: 5908 ng-h/ml) and cerebrum
(Cmax: 1831 ng/ml; AUC: 2951 ng-h/ml). In contrast to intravenous
injection, there were still detectable concentrations of ESBA105 in all
brain regions 4 h after intranasal administration.
Importantly, dose adjusted exposures (AUC/mg) were higher for
the intranasal route in all brain regions. This difference is most pronounced
for the cerebrum where relative bioavailability is almost 8-
fold higher with intranasal versus intravenous application (Table 3).
This is in sharp contrast to the situation in serum, where the dose
adjusted exposure is about 33-fold lower following intranasal
administration than after intravenous injection. These results demonstrate
that ESBA105 is able to penetrate from the blood across the
BBB into the CNS. However, delivery to the CNS is much more efficient
following intranasal administration (Table 3).
3.3. Improved delivery of ESBA105 to olfactory bulb in the presence of a
penetration enhancing peptide
Certain excipients were shown to enhance penetration of large
proteins, such as ESBA105 across ocular epithelia (Ottiger et al., 2009).
For example Pz-peptide was demonstrated to have penetration
enhancing effects in vitro and ex vivo (Chung et al., 1998; Yen and Lee,
1994, 1995). Therefore, we investigated whether the addition of 3 mM
Pz-peptide enhances the delivery of ESBA105 to the brain. Indeed, in the
presence of Pz-peptide, Cmax in the olfactory bulb, cerebrum and cerebellum was reached earlier (1 instead of 2 h after first dosing)
(Table 4). Furthermore, the addition of Pz-peptide resulted in a 2- to 3-
fold increase in Cmax in the olfactory bulb and cerebrum (7309 to
15786 ng/ml and 1133 to 3417 ng/ml, respectively) while Cmax in the
brainstem remained unchanged. Tissue-to-blood ratios for Cmax were
clearly higher in the olfactory bulb and cerebrum with the coadministration
of ESBA105 and Pz-peptide than with ESBA105 alone
(Fig. 3A). The effect on the delivery to cerebellum, brainstemand serum was, however, less pronounced. In summary, Pz-peptide can enhance
the delivery of largemolecularweight proteins to the olfactory bulb and
the cerebrum without increasing systemic exposure (Fig. 3). Therefore,
for therapeutic applications, Pz-peptide bears the potential to enhance
drug delivery without adding to the risk for systemic side effects
(Table 4).
4. Discussion
Intranasal administration has proven to be an efficient noninvasive
method to deliver agonistic proteins of limited molecular
weight directly to the CNS. In fact, agonists such as interferon-beta
(INF-beta), vascular endothelial growth factor (VEGF), nerve growth
factor (NGF), transforming growth factor-beta I (TGF-beta), insulinlike
growth factor I, and insulin were successfully delivered from the
nasal cavity to the brain most likely directly via perineural migration
(De Rosa et al., 2005; Francis et al., 2009; Ma et al., 2008; Thorne et al.,
2008, 2004; Yang et al., 2009). Importantly, intranasal administration
of NGF, TGF-beta and insulin led to effective concentrations in the
CNS.
In this study we present for the first time a pharmacokinetic
analysis of an antagonistic protein with a high molecular weight.
ESBA105, a TNF-alpha inhibitory single-chain Fv antibody fragment,
was administered intranasally (400 μg) as well as systemically by
intravenous injection (40 μg). Two different doses were chosen for
intranasal and intravenous administrations as it was intended to
obtain similar systemic exposures, thus, being able to discriminate
between the two migration routes, direct transport from the nasal
cavity to the brain or delivery across the BBB. Efficiency of delivery to
the brain for both routes was compared. Following intravenous
injection, Cmax in the olfactory bulb, cerebellum and brainstem was in
the same range, whereas no detectable concentrations were found in
the cerebrum at this dose. In contrast, following intranasal administration,
much higher levels of ESBA105 were found in all brain regions
and concentrations in the olfactory bulb were considerably higher
than in all other brain regions. Thus, following local delivery, ESBA105
distributed in a region-dependent manner with a preference for the
olfactory bulb and brainstem, the two regions that are directly
connected with nasal passages via the olfactory and the peripheral
trigeminal system.
Direct transport from the nasal cavity to the CNS is thought to
involve two mechanisms (Fig. 4). The first pathway, an extracellular
and rapid transport route, leads i) along the olfactory nerve to the
olfactory bulb after migrating through intercellular clefts in the
olfactory epithelium as it was demonstrated for HRP (Balin et al.,
1986) and ii) along the trigeminal nerve, which connects the nasal
passages with the brainstem (Thorne et al., 2004). The second
pathway, which was shown to be much slower (Balin et al., 1986),
involves endocytosis of proteins into olfactory sensory neurons and
intracellular transport along axons into the olfactory bulb and
subsequent distribution in CNS. Our data support the theory of direct
migration from the nasal cavity along N.olfactorius and N.trigeminus to
the olfactory bulb and brainstem, respectively. Significant ESBA105
levels were detected already one hour after the first administration,
which represents the earliest time point in this study. Peak
concentrations were measured between 1 and 2 h and a second
peak was observed after 8 (cerebellum), 10 (brainstem) and 12 h
(olfactory bulb). It is hypothesized that the early peaks result from
extracellular, perineural transport along N. olfactorius and N. trigeminus.
It is also hypothesized that later peaks may result from
intracellular transport within the axons, although alternate explanations
are possible, and further investigation will be necessary. It is
rather unlikely that these late peaks result from blood-to-brain
transport across the BBB, because ESBA105 concentrations in the
circulation after 8 to 12 h were at just about the same levels as they
were at this time in the brain. Therefore direct blood-to-brain
transport could only account for the late peaks if ESBA105 would
freely pass the BBB to reach a bioavailability in the CNS of
approximately 1 (∼100%). This is certainly not the case as tissue-toblood
ratios (based on AUC) following intravenous injection did never
exceed a value of 0.01 for all brain regions (extracted from Table 3).
Interestingly, the addition of Pz-peptide enhanced the uptake of
ESBA105 into the CNS following intranasal administration without
increasing systemic exposure. Maximal concentrations increased 2-
fold in the olfactory bulb and 3-fold in the cerebrum. Tissue-to-blood
ratios (based on AUC or Cmax) increased mainly for the olfactory bulb
and the cerebrum in the presence of Pz-peptide. Specific enhancement
of local but not systemic absorption following topical application
of Pz-peptide containing beta-adrenergic antagonists to the eye
was reported before by Chung et al. (1998). Pz-peptide stimulates calcium flux across colonic segments at the level of amiloride sensitive
Na+ channel, thereby triggering intracellular biochemical changes
that ultimately result in tight-junctional opening and enhance
paracellular solute transport. This mechanism, however, does not
seem to be involved in Pz-peptide improved paracellular permeability
in other tissues, such as the cornea and the conjunctiva, since blockage
of Na+ and Na+/H+ exchangers did not affect Pz-peptide penetration
across these tissues.
In this study, local bioavailability and systemic exposure following
repeated intranasal application was compared with bolus intravenous
injection. Consequently, relative comparisons of the two routes are
rather qualitative than quantitative. Nevertheless, CNS concentrations
of ESBA105 were considerably higher following repeated intranasal
administration while systemic exposure was significantly lower when
compared to a bolus intravenous injection. Thus, intranasal administration
of ESBA105 might be an attractive and safe approach for the
treatment of neurological disorders in which TNF-alpha plays a crucial
role. Elevated TNF-alpha levels in CNS have been demonstrated for
example in Alzheimer's disease (AD) (Katsuse et al., 2003), Parkinson's
disease (PD) (Mogi et al., 1994; Nagatsu et al., 2000), and multiple
sclerosis(MS) (Hofman et al., 1989; Rieckmann et al., 1995; Selmaj et al.,
1991; Sharief and Hentges, 1991). TNF-alpha concentrations in the brain
tissue of PD patients were elevated by 366% and concentrations of
68.9±23.0 pg permg of proteinwere reported—levels that could readily
be blocked with intranasal ESBA105 as a 16,000-fold excess (w/w) was
reached in the cerebrumand even amore than 100,000-fold excess in all
other analyzed brain regions in the mouse. However, the volume of a
human brain is more than 3000-fold higher than the volume of amouse
brain, and, despite a 7.5-fold larger surface area of the olfactory region in
humans (Gross et al., 1982; Illum, 2000), it is likely that the amount of
scFv delivered to a human brain will be significantly lower. Further, not
only the relative distribution of the scFv in the respective brain regions
may vary between rodents and primates but also local elimination
kinetics may change if the scFv is able to bind to its endogenous target.
Nonetheless, concentrations of an intranasally applied TNF-alphainhibitory
scFv may well be in the therapeutic range even in the
human situation, since as little as a 16-fold excess of ESBA105 over TNFalphawas
sufficient to block inflammation and cartilage degeneration in
rats (Urech et al., 2009). Furthermore, repeated applicationwould result
in higher steady-state concentrations. Clues that modulation of TNFalpha
signaling may have a positive effect on cognitive performance in
AD patients were obtained from a recent clinical study where patients
suffering frommild to severe ADwere treated with perispinal infusions
of etanercept. Patients who received etanercept improved in cognitive
performance compared to patientswho were treatedwith placebo over
a period of 6 month (Tobinick et al., 2006; Tobinick and Gross, 2008). In
MS not only elevated TNF-alpha levels were reported in the cerebrospinalfluid
(CSF) and serumof patients butwere also detected at the site
of active MS lesions (Hofman et al., 1989). TNF-alpha levels correlated
with severity of the lesions (Beck et al., 1988; Maimone et al., 1991;
Sharief and Hentges, 1991). Neutralization of TNF-alpha in an
experimental autoimmune encephalomyelitis transgenic mouse
model abrogated the autoimmune demyelination (Ruddle et al.,
1990; Selmaj et al., 1991). However, a phase II randomized, placebocontrolled
clinical trial with intravenous lenercept in MS was stopped
due to dose-dependent increase in attack frequency (MS Study Group,
1999). Rare events of demyelination have also been reported during
anti-TNF-alpha therapy in other indications (Fromont et al., 2009;
Sicotte and Voskuhl, 2001). Therefore, demyelination may be of
concern for therapies with TNF-alpha inhibitors, specifically for the
treatment of MS. However, demyelination under anti-TNF-alpha
therapy occurred in very low frequency and may possibly represent
less of a problem for disorders of the CNS in which demyelination is
not directly related to disease symptoms, such as AD or PD. Further,
upper respiratory tract infections and sinusitis were reported to be
related to systemic anti-TNF-alpha therapies (Keating and Perry,
2002). Thus, there is a certain probability that such events may be
more frequent following intranasal application of a TNF-alpha
inhibitory molecule.
ESBA105 is a potent TNF-alpha inhibitory scFv that is highly
species selective and binds exclusively to TNF-alpha from human and
non-human primates. The ability of ESBA105 to efficiently penetrate
through certain epithelial barriers, such as the cornea epithelium
following topical application to the eye has been demonstrated (Furrer
et al., 2009; Ottiger et al., 2009). This finding has triggered clinical
trials in ophthalmology (clinicaltrials.gov Identifiers: NCT00820014;
NCT00823173). Further, the impact of its ability to penetrate into the
cartilage is currently evaluated in a first clinical trial in osteoarthritis
of the knee (Clinicaltrials.gov identifier: NCT00819572). Due to its
species selectivity, efficacy could not be assessed in rodent models.
However, results of the pharmacokinetic study presented here, suggest
that intranasal administration of ESBA105 may represent a promising
therapeutic approach for the therapy of degenerative diseases in the
central nervous system.
--------------------------------------------
Acknowledgements
The authors thank Anja Wittig for excellent technical assistance
and Dr. Peter Lichtlen for critical review of the manuscript.
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Please cite this article as: Furrer, E., et al., Intranasal delivery of ESBA105, a TNF-alpha-inhibitory scFv antibody fragment to the brain, J. Neuroimmunol. (2009), doi:10.1016/j.jneuroim.2009.08.005

THank you Felicia,

It seems like TNF inhibitor is becoming an idea some folks are becoming more interested in. My family is still overwhelmed by the cost of trying the treatment. And it is breaking my heart and spirit thinking that it may help. Tonight my Mum could not tell my Dad why she wanted to keep her clothes on in bed and the frustration ended in slight aggression and tears. Breaks my heart to see their relationship changing in this way. (and mine with them I guess, I stroked my mum's hair as I tucked her in...and reassured her just like I did with my wee kids upstairs an hour earlier. I am quite determined to find a way to try this with my mum but cannot help but feel time is running out and I may be kidding myself that it is possible with the financial reality of it. Anyway thanks for this forum.

ST

ST - have you considered giving Axona a try?? $70 a month using the online coupon. Most important thing is to start out slowly with the amount of powder you use - maybe 1/4 packet for a few days and then slowly increase. One common side effect is diarrhea. Be patient with it - they say sometimes it takes 45 days or so to start working. Also, it does work best for those not carrying the APOE4 gene.

Deb is right, and Axona should be available through a neurologist. Until the time comes that you can try the Enbrel, Axona may stabilize her for a while, I've heard about some good results. But yes, it takes a while before you'll know anything.

Keep the faith,

Felicia