Rabies: New Methods of Treatment

Site of entry of the Rabies virus from the nose and
oral cavity; and new methods of treatment of Rabies
using olfactory mucosa and by breaking BBB.
To be presented at
RABIES IN ASIA CONFERECNE (RIACON)
HEALD IN HANOI 0N SEPTEMBER 10TH2009
T.R. Shantha, MD, PhD, F.A.C.A,
115 Bayberry Hills,
McDonough, GA 30253.
Email: shantha35@aol.com
Phone/Fax: 7 70-507-6564, Cell: 678-640-7705
www.3E-Technologies.com
Previously: Associate Prof. Emory University School of medicine,
Yerkes Regional Primate Center of Atlanta. Hon Prof. Medical College
of Georgia. Director: Southeastern Pain Inst. Associate Chairman:
Medical Center Anesthesia Associates. Director: Integrated Medical
Specialists. Visiting Prof. JJM Medical College
Baer GM, ShanthaveerappaTR and Bourne GH: Studies on the
pathogenesis of fixed rabies virus in rats. Bull World HlthOrg, 33:783-
794 (1965).
Baer G, Shantha TR and Bourne GH: The pathogenesis of street rabies
virus in rats. Bull World HlthOrg, 38(1):119-125 (1968).
What did the above studies show
•Sciatic Nerve section before rabies virus injection prevents itsspread
•Cutting nerve 10 hours after injection does not prevent the spread, before it does prevent the spread
•Crushing the nerve, Wallerian degeneration does not prevent the viral spread indicating intact axons are not needed for viral spread
•Injection of rabies antiserum around and underneath the perineural epithelium did not prevent the spread of the virus.
•Removal of the perineural epithelium slows down the viral spreadbut does not spread
•"These findings once again point to the Schwànncells, the endoneurium, or associated tissue spaces as the principal progression routes forrabies virus"
•It is interesting to note the complete lack of inflammationin any of the infected sciatic nerves even though viral titresin some nerves was high as we observe in most rabies infected brains.
•Based on our studies over 4 decades, we want to explore how the rabies virus gets into nerve fasciculus, by inhalation, oral routes.

Medial (lateral) walls of the nose with nerves and blood vessels as the route of Rabies virus and bacterial entry into the brain.






Medial wall of the nose with olfactory and other nerves as the route of Rabies virus (bacterial) entry into the brain, and SAS (Fig.modifiedafter Grays Anatomy)










Figures I.-4: Olfactory mucosa of Rhesus monkey showing Succinic dehydrogenase, Lactic dehydrogenase, Aldolase, and Glucose -6-phosphate dehydrogenase preparation showing very strong positiveactivity in the dendritic process of receptor cells Z. Zellforsch. 103. 291—319 (1970). The mucus coat is very lightly positive and Rabies virus CAN sticks to this mucus lining












Olfactory Mucosa and nerve fasciculi of the Rhesus Monkey
based on our studies to show how the olfactory axons are
surrounded by perineural epithelium as they travel to the olfactory
bulb through the cribriform plate of the Ethmoid bone
(Shantha & Nakajima Z. Zellforsch. 103. 291—319,1970)










Olfactory nerves surrounded by Perineural Epithelial cells and route taken by the rabies virus from the olfactory mucosa to enter the olfactory bulb through the axons and sub perineural epithelial space to SAS












Olfactory nerve fasciculi surrounded by multiple layers of Perineural Epithelial cells which acts as a route taken by the rabies virus from the olfactory mucosa to enter the olfactory bulb and subarachnoid space CSF












Olfactory nerves surrounded by Perineural Epithelial cells layer which acts as a route taken by the rabies virus from the olfactory mucosa to enter the olfactory bulb











Olfactory Mucosa with rabies virus or other infecting agents on the surface sticking to the mucus coating and their passage to the brain through axons and sub perineural spaces and BV (diagrammatic)









Perineural Epithelial cells covering olfactory nerves continuing with pia-arachnoid mater covering and SAS of the olfactory bulb which can acts as passage for the transfer of rabies virus from the olfactory mucosa









Complex Glomeruli of the olfactory bulb like a massive distribution stop though which rabies virus reaches the olfactory tract and brain










Glomeruli are made up of1.Axons from olfactory neurons2.Mitral cell dendrites3.Tufted cells4.Granule cells5.Periglomerular cellsCentrifugal fibers from1.Locus Coeruleus2.Ralphie nuclei3.N. Diagonal band of Broca
Routes of Rabies virus spread to central Nervous System From the Olfactory bulb connections and SAS -CSF (modified from Guyton)










Rabies viral Spread from Subarachnoid space after entry into olfactory system (after Shantha)










The vasculature of the hypothalamic median eminence,
infundibulum, and the rest of the hypophysis cerebri
through which rabies virus can spread if there is
viremia (from Gray’s anatomy and Netter Ciba
symposium).









Hematogenousspread of rabies virus through Circumvetricularorgan (Citation: PreussMR, et al. (2009) PL0S Pathog5(6): e1000485. doi:1O.1371/journal.ppat.1000485)
CircumventricularOrgans
which can leak rabies virus to the
surrounding neuropile and CSF; and their
role in spread of Rabies Virus
There are several areas of the brain known as "circumventricularorgans". where the BBB is weak which allows substances to cross into the brain somewhat freely.The circumventricularorgans include:
•Pineal gland: Secretes melatonin and neuroactivepeptides. Associated with circadian rhythms.
•Neurohypophysis(posterior pituitary):Releases neurohormoneslike oxytocin and vasopressin into the blood.
•Area postrema: "Vomiting center": when a toxic substance enters the bloodstream it will get to the area postremaand may cause the animal to throw up. In this way, the animal protects itself by eliminating the toxic substance from its stomach before more harm can be done.
•Subfornical organ: Important for the regulation of body fluids.
•Vascular organ of the lamina terminalis: A chemosensory area that detects peptides and other molecules.
•Median eminence: Regulates anterior pituitary through release of neurohormones
•To these I would add Ependymal liningof the ventricles and central canal, choroid plexus, arachnoid villi, pia materof the brain and spinal cord and emerging nerve rootsof the CNS and Spinal cord .

Taste Bud in the Tongue through which rabies virus can enter axons and pass on to brain (after Guyton)TS cilia










Taste bud with pore, cilia projections, mucus coating at the pore to which rabies virus can gets attached. The taste nerve fibers then convey them to brain (Grays Anatomy)










This color-enhanced SE image depicts a taste bud on the tongue. The human tongue has about 10,000 tastebuds that are involved with detecting salty, sour, bitter, sweet and savourytaste perceptions. The rabies virus can enters the taste buds through these pores and then passes on to the taste conveying nerve fibers to the brain stem











•Image: David Gregory & Debbie Marshall, WellcomeImages


Taste bud, pore, mucus coating, nerve supply and its perineural epithelium coverings based on our studies which can acts as a route taken by rabies virus to enter the CNS









Shantha and Bourne, vgusActaAnat52:95-100 (1963).
Electron micrograph of Taste bud base showing the PE cells covering of the nerve fasciculi as they as they emerge from the taste buds










Central Nervous System connections from the taste buds from which rabies virus can spread to enter brain stem nucleus tractus solitarius through V,VII, IX and X cranial nerves ( Diagram From Guyton)















Nerve fasciculi and axonal coverings based our studies spanning 15 years. Perineural epithelium plays a role in spread of rabies virus-It delays virus entry into nerve fasciculi (Baer et al, Shantha and Bourne)









Perineural Epithelium with blood vessel supply to nerve fasciculi which can carry rabies virus to the axons











BV as it enters nerve fasciculi and the Virchow-Robin space around the BV which can also transmit rabies virus to nerve fasciculi interior from outside the nerve fasciculi from the interstitial tissue









Virchow-Robin Space in Peripheral nervous system which can conduct the rabies viruses to nerve fasciculi interior (Shantha T.R. perivascular Virchow-Robin space in peripheral nerves. V 17, #15, Jan-Feb-Supple, Regional anesthesia, 1992)










EM of Cross section of rat Sciatic nerve and sympathetic chain NF showing various types of NF and node of Ranvier where rabies virus can easily enter the axons (SFNT1968)










A three-dimensional reconstruction of the architecture
of intestinal villi and subjacent wall
through which rabies virus can enter the Auerbachs
and Meissner plexus (Modified From Gray’s anatomy).










Transmission electron micrograph of’the columnar epithelium lining the murinesmall intestine,
showing a mucus-secreting goblet cell between two absorptive enterocytes cells which bear
microvilli. The cells rest on a delicate basal lamina deep to which is the vascular lamina propria.
(Magnification 4,800.) (Prepared and photographed by Mr. DerrickJ. Lovell, Department of
Anatomy, Guy’s Hospital Medical School, Note the rabies virus can easily enter deeper layers of
intestines without coming in contact with brush border of enterocytes.
Rabies virus can get attached to the surface of the goblet cellsand
enter the deeper layers of the intestines nerve plexus & BV.










Rabies virus entry into myenteric nerve plexus in the intestines











Diagram showing the entire autonomic nervous system
is covered by Perineural epithelial cells under which
rabies viruses can easily enter the spinal cord and CNS













(From. Shantha and Bournezeitschritcfur Zellforschung61:742-753 (1641).











Treatment Outlines:
1.Post Exposure Prophylaxis (PEP);
2. Treatment of full blown Rabies
The following CDC reports available on Internet describes prophylaxis and treatment
1.Manning SE, Rupprecht CE, FishbeinD, Ct al.: Human rabies prevention—United States, 2008: recommendations of the Advisory Committee on Immunization Practices. CDC MMWR MorbMortal Wkly Rep 2008, 57(No. RR-3):1—28.This up-to-date document gives detailed recommendations concerning the prevention of human rabies after exposure.
2. Rabies Treatment Protocol; Wisconsin Treatment of Rabies cases (after Willoughby et al 2004): Checklist & schema for treatment of rabies version 3..All materials ©2009 The Medical College of Wisconsin, Inc. Last updated June 2009.
Rabies Cases survival and Ketamine treatment: success and failures

•Five cases of rabies have survived so far. They all had Post exposure Prophylaxis (PEP) and intensive care.
•One patient of bat rabies in US survived (Willougbyet al 2004). This patient did not receive PEP.She was treated with Ketamine and other antiviral therapies with critical care. There are reports of 3 other caseswhich survived in a similar treatment (CDC personal communication)
•10th case of rabies recovery: MjnisteriodaSaudein Brazil: Rabies, human survival, bat—Brazil (Pernambuco). ProMED-mail Web site; 20081114.3599. Available at http://www.promedmail.org. Accessed February 2009. This patients received PEP
•Latest Cases of MILWAUKEE TREATMENT PROTOCOLFailure: 13
patients were treated using Milwaukee method and antiviral agent
Ketamine did not survive the rabies virus attack.
(Wilde H, Hemachudha,T, Jackson AC: Viewpoint: management of human
rabies. ‘Trans R Soc TropMed Hyg2008, 102:979—982. A.C. Jackson,
Update on Rabies Diagnosis and Treatment. Current Infectious Disease
Reports 2009, 11:296—301)

What was the denominator in those who survived from rabies?
Survival of Rabies patients impinges on the early appearance of rabies virus neutralizing antibodies in serumand CSF(Watson et al., 2007 immune responses after rabies infection. Arch. Neurol. 64,1355—1 356).
To survive, rabies virus neutralizing antibodies presence in the CSF is a must
Where do we go from here?

•I do believe that one day we will have a cure for human rabies. "SEEK YOU SHALL FIND"(MATT 7:7)still holds good in all phases of our research and life; we need to keep seeking answer to this dreaded deadly disease.
•I do believe that there will be development of effective (with least untoward reaction), one or two shot prophylactic vaccine to be used in PEP.
•I want to introduce today the method of treatment which I do believe can be effective and save some if not all rabies patients..

The principle methods I propose to treat Full Blown Rabies Cases are (patented)

1.Intranasal administration of antirabies therapies including Monoclonal antirabies and anti TNF MAB (Etanarcept) to reduce the brain inflammation using insulin as enhancer of uptake form olfactory mucosa and carry them to the brain.
2.Antirabies therapy through subarachnoid space with insulin
3.Intravenous or intra-arterial antirabies therapy after BREAKING THE BLOOD BRAIN BARRIER to get it into the substance of the brain with insulin pretreatment to enhance the NEUROPILE uptake.
4.Intravetricualr delivery of antirabies therapies with insulin.
5.Enhance the immune system by orally administering immune stimulating component of the rabies virus and / or other such immune enhancer through NG tube or Gstro-jeunostomytube (or IM with insulin) to stimulate the plasma cells(180,000 /cubic mm) in the lamina propria of the intestines, to produce neutralizing rabies antibodies.
6.Lower the metabolism of the brain by lowering the blood sugar level, what I call INTERMEIDATE HYPOGLYCEMIA every two to 6 hours once to enhance the uptake of neutralizing antirabies antibodies and therapeutic agents.
7.Supportive critical care therapy with Critical care monitoring.












TREATMENT OF RABIES CASES
THERAPEUTIC PLAN-PART ONE

•Intranasal delivery of antirabies antibodies (human MAB), Nerve growth factors, erythropoietin, insulin, Insulin growth factor-I, and other antiviral therapies using insulin as uptake, therapeutic activity enhancer and helper in dissipations therapeutic agents to the CNS to attack the rabies virus
•This is a method of going "through" and / or "behind" the BBB.
Intranasal olfactory mucosal delivery of
Antirabies therapies
Advantages and disadvantages
Advantages
•Painless
•Ease of use
•No shot needed
•Avoids first pass metabolism –improving bioavailability over oral and rectal doses
•Nose-brain pathway allows direct delivery to the cerebral spinal fluid
•Compliance not an issue -easy and fast to deliver to any patient

Disadvantages
•Limited medications that can be delivered in this fashion
•Many medications are not adequately concentrated to achieve ideal dosing volumes
•Mucosal health impacts absorption
How administered
Ideally, drug doses will be divided in half, and each nostril received half the dose, which doubles the absorptive surface area.
Intranasal olfactory mucosal delivery of
Antirabies therapies

1.Rapid appearance of 1 ‘25-labeled nerve growth factor in the olfactory bulbs, cerebrum, and brain stem is more consistent with entry oflabel through intercellular clefts in the olfactory epithelium and extracellular transport along the olfactory neural pathway to reach the cerebrospinal fluid and brain than with uptake by olfactory neuronsand subsequent intracellular axonal transport. (Drug Delivery, Volume 4, Issue April 1997 , pages 87 –92)
2.Olfactory and trigeminal-associated extracellular pathways to rapidly elicit biological effects at multiple sites within the brain and spinal cord(Neuroscience 2004, 127:481-496)).
3.Kinetic studies showed that intranasallyadministered plasmid DNA reached the brain with a 2,595-fold higherefficiency than intravenously administered plasmid DNA (J Mol Med. 2007 Jan;85(1):75-83. Epub2006)

Intranasal –olfactory delivery catheter balloon device (Shantha-patent pending)
TREATMENT OF RABIES CASES
THERAPEUTIC PLAN-PARTTWO

1.Going through the BBB by Breaking the Blood Brain Barrier and deliver therapeutic agents against rabies virus.(By use of endogenous transport systems, and carrier-mediated transporters such as glucose and amino acid carriers; receptor-mediated transcytosisfor insulinor transferrin; and blocking of active efflux transporters such as p-glycoprotein. USE OF INSULINAND INDUCTION OF INTERMEDIATE HYPOGLYCMIA DESCRIBED IN OUR PATENTED METHOD INCORPORATES MANY OF THESE PRINCIPLES).
2.After breaking the BBB, the antirabies viral therapy can be administered IV and / or Intra arterial method by directly infusing internal carotid arteries on both sides to deliver into the brain.
3.By linking transferrin with rod-shaped semiconductor nanocrystals (quantum rods) and Nanoparticles-based platform which 'tricks' the BBB into allowing the entry ofthe nanoparticle into the brain, using an approach that draws parallel to the 'TROJAN HORSE'concept. They are not yet available.

The BBB can be broken down by:

•Hyperosmolitity:a high concentration of a substance in the blood can open the BBB.
•Vasoactive substancessuch as Brandykinins opens BBB
•Insulinenhances the transfer of therapeutic agents from theBBB, and membranes covering the nervous system to neuropile
•Hypertension: high blood pressure opens the BBB.
•Developmental: the BBB is not fully formed at birth. Microwaves: exposure can open the BBB.
•Radiation: exposure can open the BBB.
•Infectious agents: exposure can open the BBB.
•Trauma, Ischemia, Inflammation, Pressure: can open the BBB.
•HIFU-High intensity focused ultrasound opens BBB
400 mile longCapillary system with astrocytes feet surrounding their wall contributing to BBBwhich prevent effective anti rabies viral therapeutic agents reaching neuropile










in the early 1980s, Edward A. Neuwelt, M.D., an OHSU neurosurgeon, pioneered a unique method of outwitting the brain's protective blood-brain barrier. By temporarily opening this barrier, chemotherapy and Viral -tumor specific antibodiescan pass into the brain and reach the tumor. Dr. Neuwelt continues to devote his efforts to research

















TREATMENT OF RABIES CASES
THERAPEUTIC PLAN-PART THREE

•Subarachnoid space (SAS) and CSF delivery of antirabies therapeutic agents including but not limited to human antirabies MAB though continuous SAS catheter or injection into Cisterna Magna or close to the SAS at Cervical spine levels using insulin as uptake and therapeutic agent activity enhancer and dissipater of the therapeutic agents.

Subarachnoid –CSF delivery of antirabies
therapeutic agents including antirabies MAB
and ketamine with insulin
(continuous or intermittent delivery)










Virchow-Robin space entry of antirabies therapeutic agents with use of Insulin deposited in the SAS-CSF(such a space is also found in peripheral nerve fasciculi Shantha 1992 ASRA)
Subarachnoid and CSF delivery of immune substance and antiviral agents

•Therapeutic agents have hard time entering the neuropile from SAS because the astroglial cells feet attach to the pia mater and which acts as barrier:PIALBRAIN BARRIER.
•From CSF and Pia, the drugs can penetrate up to 6mmsurface of the brainunder normal circumstances.
•Some of the therapeutic agents can enter through Virchow-Robin space, but it is minimal and will not be able attack the virus in deeper depth of the brain.
•Our method of use of insulin with therapeutic agentswill allow penetration into deeper depths of the brain to eradicated the offending agent at least from the surface of the brain, spinal cord and proximal part of emerging cranial and spinal nerves.

TREATMENT OF RABIES CASES
THERAPEUTIC PLAN –PART FOUR

•Intravetricualr injectionof therapeutic agents against rabies virus using insulin to enhance their uptake, activity and facilitate distribution through the Ependymal lining using Ommaya or other delivery system
•This is a method of going "through" and / or "behind" the BBB.
•***Strategies for drug delivery by intracerebral implantationand convection-enhanced distribution be avoided.

Antirabies therapeutic agents (MAB and other therapeutic agents) administered with Insulin to the ventricles of the brain though a Ommaya reservoir and infusion port using Huber needleOmmaya reservoiris an intraventricularcathetersystem used for the delivery of drugs (e.g. chemotherapy, antiviral and antibacterial therapy) into the cerebrospinal fluid. It was originally described in 1963 by AyubK. Ommaya, a neurosurgeon
Therapeutic agent uptake of Antirabies virus therapies: from Ependyma to Pia mater after Intravetricualr delivery with insulin. Neuralgia are shown in green (from Gray’s anatomy)











TREATMENT OF RABIES CASES
THERAPEUTIC PLAN –PART FIVE

•Enhance the immune system by orally administering immune stimulating component of the rabies virusand / or other such immune enhancer through NG tube or Naso-Gstro-jejunostomytube to stimulate the plasma cells (180,000 cells /cubic mm)in the lamina propria of the intestines, to produce neutralizing rabies antibodies.
•Can inject the antibody stimulating rabies virus component IM, IV, or SQ using insulinto enhance its uptake, distribution resulting in stimulation of viral antibodies by the immune system.
•Start with low doses and increase the dose every day.

TREATMENT OF RABIES CASES
THERAPEUTIC PLAN –PART SIX

•Lower the metabolism of the brain by lowering the blood sugar level, what I call INTERMEIDATE HYPOGLYCEMIA(40-60 mg%)every6 to 12 hours once to enhance the uptake of neutralizing antirabies antibodies and other therapeutic agents into neuropile and to enhance the uptake, enhance the activity and dissipation of therapeutic agents.

TREATMENT OF RABIES CASES
THERAPEUTIC PLAN –PART SEVEN

•Critical care monitoring as needed and as available –see the publication on the internet about Milwaukee protocol and follow the guide lines they describe.
•Monitor fluid intake and out put. Avoid over hydration and maintain the BP enough to perfuse the brain. Use all supportive therapies to maintain hear, lung, and kidney functions.
•Maintain Electrolyte balance
•Patient on respirator should receive enough to maintain 100% oxygen saturation to avoid lung damage.
•Sedation with ketamine and midazolam
•Keep paralyzed if the patient is fighting and having muscle spasms by using non depolarizing muscle relaxants.

Antirabies Viral therapy-Part I

1.***No medications are given intrathecally (MP)-Iam opposed to it. Give small antirabies human monoclonal antibodies (MAB) with other antiviral agents including Ketamine with 3-5 units of insulin pretreatment.
2.***Avoid rabies immunization of the patient after onset of clinical symptoms (MP)-I am for giving small doses rabies antigen; increasing the dose every day through the Naso-Gastro-Jejunostomy tubes. Intramuscular antirabies immunization can be administered mixed with 1-2 units of insulinfor rapid absorption and dissipation to immune system stimulation.
3.Avoid administration of rabies-specific antiserum (MP)-I am for giving intranasal, SAS, and Intracisternal delivery of MAB and other anti rabies viral agents with insulin.

Antirabies Viral therapy-Part II

1.Avoid administration of IFNa-I am for administering MAB against TNFand cytokines to lower the brain inflammation such as Etanarcept with insulin.
2.Ribavirinis no longer recommended(MP)–I will administer small doses intranasal and other routes because it is a prodrug, which when metabolized resembles purineRNAnucleotides. In this form it interferes with RNA metabolism required for viral replication. Why not add qhininesulfate, it is antiviral?
3.Amantadineis administered orally or intranasal & other routes here in described. It does Passes through BBBand has both anti Parkinson's and antiviral effect. It may block reproduction of the VIRUSand decrease the ability of the virus to get into the cells.Believed to release brain dopamines from nerve endings in Parkinson's.

Antirabies Viral therapy-Part III

•Ketamineis much debated and it is opposed by some rabies researchers dueto failure of save rabies patients.
•I would use it intranasal (IN), SAS, Intracisternal, IV or intraarterial routes. Studies show that it acts at the level of rabies virus genome transcription and prevents it multiplication. It is also non a competitive blockerof the NMDA receptors on the nerve cell. It sedates at the same time blocks the virus multiplication. I have used this drug on patients since 1968. It is one of the safest drug. I will use it on rabies patients though the lateststudies are disappointing.
1.IN-Erythropoietin:rhEPOi.n. reduced infarct volume, brain swelling and cell damage in the ischemic hemispheres, and improved behavioral dysfunction 24 h after cerebral ischemia (NeurosciLett. 2005 Oct 14;387(1):5-10)
•IN-IGF-I and Insulin: reduced apoptosis, infartsizee, imporvedsensory and motorofunction in stroke (J Stroke CV Dis. 2004 Jan-Feb;13(1):16-23).
•Any latest antirabies antiviral therapywith insulin uptake, activity and dissipation enhancer.
•Magnesium can be used to relax synaptic discharge.
•Provide antipyretic therapy if the body temperature raises.

Antirabies Viral therapy-Part IV
Use of human antirabies MAB

1.Use recombinant fully human anti-rabies monoclonal antibodythat specifically binds a broad variety of rabies virus isolates and inhibits the ability of the virus to infect cells and cause the disease or prevent their further spread and pathology by neutralizing them.
2.Advantages:
•They are likely to be less adverse immunogenic in humans than non-human antibodies.
•Rabies virus inactivation can occur as soon as the antibody reaches sites of infection.
3.Combining MAB with insulin as enhancer of uptake and therapeuticaction, the rabies virus can be effectively neutralized providing a cure for this incurable disease and act as effective PEP therapy
4.Combining other therapeutic antiviral agents can augment the effectiveness of the antirabies therapy.
5.IMPORTANT: These MAB and rabies antigens should be used with insulin (patented method) as part of pre and post exposure prophylaxis.
6.Use Human monoclonal antirabies antibodies(MAB) intranasal, IV, SAS, Intracisternal methods with insulin as uptake, activity anddistribution enhancer.