C.49A. 9° Conférence Européenne sur le Sida

(Varsovie 25-29 octobre 2003).

ANNEXE

Textes soumis au congrès en collaboration avec l'association POSITIFS.

1. Accepté sous forme Poster :

THE SCORPION VENOM MODEL OF AIDS : ALPHA-DEFENSIN IS HOMOLOGOUS TO HIV 1 NEF IN THREE DIMENSIONS (3D). TACRINE WITH SILYMARIN AS A RESCUE FOR FAILURE AND HIV-1 CHEMORESISTANCE.

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2. Abstracts non retenus.

a) Immune Reconstitution by DHEA - A significant Clinical Case.

b) MOLECULAR MIMICRY BETWEEN HIV 1 NEF AND HUMAN MDM2 (MOUSE DOUBLE MINUTE 2), A LIGAND OF P53, THE MAJOR TUMOR SUPPRESSOR PROTEIN IN ONCOLOGY.

1 THE SCORPION VENOM MODEL OF AIDS : 
ALPHA-DEFENSIN IS HOMOLOGOUS TO HIV 1 NEF
IN THREE DIMENSIONS (3D). TACRINE WITH SILYMARIN
AS A RESCUE FOR FAILURE AND HIV-1 CHEMORESISTANCE.

TRAN Guy Mong Ky ^{2, 5}, CAPRANI Adrien ^{2, 3}, NGUYEN Dat Xuong ¹, KIRKIACHARIAN Serge ¹, MAURISSON Gilbert ^{2, 4}, FREDJ Gilles^{* 5}.

¹ University Paris-Sud XI, Service of Therapeutic Chemistry, Faculty of Pharmacy, rue Jean-Baptiste Clément 92290 Châtenay-Malabry, University Paris-Sud XI.
² Association Positifs, 75 Paris, France,
³ CNRS UMR 7057,
⁴ Centre Medical Europe, Paris (E-mails : positifs@positifs.org, mkg_tran@yahoo.fr, sergekirkiacharian@cep.u-psud.fr,caprani@ccr.jussieu.fr,maurisson@club-internet.fr
⁵ University Paris-Sud XI, 31 Avenue du Bois 92290 Chatenay Malabry, France.

*^{, 5} In Memoriam. ⁵ Pharmacy, Hospital Paul Brousse, Villejuif, France.

Communication acceptée sous forme de Poster.

INTRODUCTION:

HIV-1 remains a fantastic mutator, with millions of quasispecies, and this drug escape capacity on the long term, inherent to the absence of reverse trancriptase proofreading, remains an unsolved problem for great pharmaceutical companies implicated in antivirals and vaccines.

Despite some encouraging advances [enfuvirtide (T-20) ; tipranavir and atazanavir, both with ritonavir boosting], management of heavily treatment-experienced AIDS patients represents an ongoing challenge. Katlama's GIGHAART with 9 drug salvage therapy (2003) did not solve the problem. As the number of affected patients continues to increase, research in this area should be an urgent priority (Montaner J.S.G., 2003). We present here 2 breakthroughs :

The first, a biological one, is the discovery of a-defensin as the major natural immunity in long term non progressors (Zhang, 2003), and as a-defensin is a member of the scorpion venom toxin family (Bontems F., 1991), the direct consequence is a renewal of the scorpion venom concept of AIDS we defend since 1989 (Tran M.K.G.) ;
The second, is a logical transposition of fundamental research in clinical settings, namely utilisation of tacrine, tetrahydroaminoacridine (Fredj G.), a modifier of the scorpion receptor (the voltage-dependent-Na+ channel) (Schauf C.L., 1987) in chemoresistant patients. The new data for tacrine is the better tolerance and higher dosage by addition of silymarin (Legalonâ), a hepatoprotector.

As Nef will be presented further as an homologous protein of a-defensin, we first comment on Nef (see : Nef reviews).

 

THE MAIN FUNCTIONS OF NEF.

HIV 1 Nef is a complex protein, a kind of multicolor patchwork endowed with numerous apparently unrelated functions ; Nef is a crucial protein, despite its classification as an " accessory " protein, because quantitatively Nef is by far the most abundant protein in infected T lymphocytes and macrophages : It represents 80% of the total intra-cellular messenger RNA, compared with only 2% for Tat and 18% for Rev (Robert-Guroff M., 1990). There is an abundant Nef expression in brain astrocytes of dementia patients (Ranki A., 1995). The most important Nef biological functions, among others, were :

- HIV infectivity,
- Bone marrow CD34+ and CD133+ hematopoietic progenitor cell ligand (Calenda V., 1994 ; Tran M.K.G., 2002),
- scorpion venom toxin (Werner T., 1991 ; Tran M.K.G., Eur. Conf. HIV, Hamburg, 1997),
- dementia (Ranki A., 1995) (Tran M.K.G., 2001)
- superantigen (Torres B.A., 1994), like another retrovirus LTR 3' orf (open reading frame) [the mouse mammary tumor virus (MMTV) superantigen (Wang Y., 1995 ; Liu B., 2001)]
- CD4 & HLA-A, -B & -C down-regulation,
- p53 ligand mdm2, (Tran M.K.G., 2003, unpublished).

In summary : Greenway A.L. (2002) found that Nef NH2 terminus (residues 1-57) has mdm2 properties, i.e. binds to and inhibits p53, a key protein in cancerology, the major tumor suppressor protein, involved in 50% of all cancers and also a " stemness " marker abundantly overexpressed in embryonic stem cells (Ramalho-Santos M ., 2002). Mdm2 (Sigalas I., 1997) denomination stems for mouse double minute extrachromosomal nuclear bodies [evolving in the homogenous staining region (H.S.R.) of the oncogenic amplified DNA region in cancer]. We found a molecular mimicry spanning an hexapeptide GAVTSS (Gly Ala Val Thr Ser Ser) common to many HIV-1 Nefs and mdm2 ; the homology extended downstream to this hexapeptide (VSQDLD is read in reverse sense):

This is strengthened by an homology in the same region between mdm2 (28-DKEESVE-34) and human polyoma T antigen (6-NREESME-12), a known p53 ligand. Phosphorylation of the hydroxylated residues threonine (T) and serines (S) of the sequence GAVTSS may explain the functionality of these amino acids and their conservation in the molecular mimicry. Nef is a new member of the large viral p53 inhibitors family (human papillomavirus E6, polyomavirus T antigen, adenovirus E1B, hepatitis B protein X). Nef role could be important in AIDS oncogenesis : Kaposi sarcoma, lymphoma, papillomavirus uterine cervical cancer and ano-rectal cancer. Deciphering of the p53 binding sequence may be an opportunity to synthetize short chiral peptidomimetic antagonists or retroinverso peptides designed as anticancer drugs.

Nef deletion (in the scorpion venom region and LTR U3) has been observed in long term survivor patients (Rhodes D.L., 2000 ; Deacon N.J., 1995 ; Soriano V., 1995). On the other hand, Zhang L. (2002) found that the protective effect of CD8 cytotoxic T lymphocytes in long survivors originated from the secretion of high levels of a-defensin 1, 2 & 3. [members of the scorpion venom family (Bontems F., 1991)].

OBJECTIVES:

1) Our initial objective was to elucidate the mechanism of protection against HIV-1 in long term non progressors, in order to find a-defensin-like drugs inhibiting HIV-1. As the first result was an homology between a-defensin and Nef (see below), and highlighted the scorpion venom concept of AIDS, we applied the results of this biological " molecular mimicry " to medical field and consequently drug utilisation : Tacrine, a modifier of Na+ channel (Schauf C.L., 1987) (the receptor of scorpion venoms) is an exemple of such drugs, and

2) we had then as a second objective : To reassess the clinical value of tacrine in chemoresistent patients. For this purpose, we associated tacrine to silymarin to decrease its hepatotoxicity, as it is known in Alzheimer's disease (Allain H., 1999).

METHODS:

We compared the amino acid sequences [Los Alamos database (Kuiken C. et al., 2002)] and the three dimensional (3D) structures of a-defensins (Hill C.P., 1991), HIV-1 Nef (Lee C.H., 1996) and scorpion venoms (about 100 sequences of our personnal data), in the Nef scorpion venom-like region we determined in 1997 (Tran M.K.G., 1997).

RESULTS:

The cysteine bridged scorpion structure is found in a-defensin (Bontems F., 1991) .

Nef and a-defensin 3D structure superimposition are shown :

 

HAART SWITCH TO TACRINE FOR ALLEVIATING CHEMORESISTANCE.

Nef and a-defensin superimposed on each other in 3D, suggesting that the a -defensin protective action may be a competitive inhibition with Nef. We and others (Brack-Werner R., 1991; Werner T., 1991 ; Tran M.K.G., 1993) have shown that Nef contains scorpion venom sequences, and on the other part a-defensins belong to the scorpion venom family (Bontems F., 1991).

Thus the scorpion venom model of AIDS we proposed earlier (Tran M.K.G., 1989) seems to be confirmed here, and this new data strongly boosted the clinical use of tacrine. Enfuvirtide (T-20) which blocked cell fusion, virus entry and reduced the viral load (Lazzarin A., 2003 ; Lalezari J.P., 2003) also confirmed the scorpion model, because 3D structure analysis (Tran M.K.G., 2002) revealed an homology with the active site tryptophan (W38) containing loop of the North African scorpion venom Androctonus Australis Hector (AaH II), the most powerful scorpion in the world. Every AA could be superimposed on the AaH II 3D structure.

(QWASL is the chimpanzee SIV gp41)

(in bold, active site).

Scorpion Bot IX

K

V

R

D

G

Y

-

I

V

45

15

44

37

38

39

40

41

Scorpion AaH II

V

R

D

G

Y

-

I

V

A

F

N

Q

W

A

S

P

HIV1 gp41

K

V

R

Q

G

Y

S

P

L

E

L

D

K

W

A

S

L

HIV1 and SIV gp41 variants

A,

F,

N,

Q

In electrophysiological voltage-clamp studies on axons, tacrine has been demonstrated to be a modifier of voltage-dependent Na+ channels (Schauf, 199). Interestingly, tacrine also increased the total number of polymorphonuclear neutrophils (PMN) (i.e. precisely the source of defensin production) in clinical trial of patients : This specificity of PMNs increase seems remarquable enough to be noted, as it does not seem to exist with other known anti-HIV-1 drugs (Fredj G., personnal communication).

 

ASSOCIATION OF TACRINE WITH SYLIMARINE TO REDUCE HEPATOTOXICITY.

We decided to reassess the prior clinical success of tacrine used alone, compared to AZT, in 1989 (Dr Dietlin François, Newpharm documentation, France. The name of this society is now Newmed, France). In all earlier clinical trials (Fredj G., 1992, 1989), the hepatotoxic (nausea, vomiting) limitation of tacrine was obvious, because doses given could not generally exceed 150 mg/day (3 times 50 mg/day), and as tacrine efficiency was strongly dose-dependent, the benefit with this upper limit dose was not as spectacular as with anti-protease (although it was certain and superior to the standard drug AZT) in about 75% patients. However, despite its lower strength in terms of efficiency (if compared to anti-protease), tacrine had the enormous advantage not to create any chemoresistance, even in the very long term (the greatest follow up is more than 13 years tacrine treatment) because its target was a cellular one, the voltage-dependent Na+ channels. Contrarily to all other anti-HIV-1, whose targets are all viral (reverse transcriptase, antiprotease, envelope,É), no chemoresistence develop with tacrine, because -of course- a cellular protein does not mutate as did HIV-1 so tremendously : So even with the most hypermutating strength, the virus can never escape to tacrine, even if it successfully finally become chemoresistant to with all the other drugs. This very interesting data stems from the fundamental concept of a cellular, immuable, non mutating target. However tacrine Achilles' heel remains its slight hepatotoxicity, that was sufficient to obligate to stop the drug if transaminases rose too high. Although this hepatotoxicity has never been accompanied with death on the many thousands (about 6000) of treated Alzheimer's patients (Parke-Davis Laboratory Cognexâ), it is sufficient to limit the upper limit of tacrine dose to 150 mg/day, which is clearly unsufficient to obseve a frank effect on HIV-1. In summary, tacrine is not a 100 meters sprinter or a 1500 meters runner, like anti-proteases , but is rather a winner on a marathon distance : Its efficacy remains intact, alone or associated with other drugs (if not hepatotoxic) after 13 years follow-up (namely, in clinical translation, for life). To our knowledge, it is the only anti-HIV-1 drug with such a persistence of very very long term efficiency. It can be compared to penicillin prevention of rheumatic fever. So we asked if it could be possible to ameliorate the hepatotolerance by adjunction to tacrine of a hepatoprotective drug, such as silymarin (Legalonâ) from the thistle Mary Silybum Marianum. We chose silymarin for 2 reasons : First, in Alzheimer's disease, a 33% reduction of hepatotoxicity was observed by Allain H .(1999) when tacrine was used in association with silymarin. Second, our work and others on silymarin use in hepatitis C virus (HCV) infection revealed its utility in alleviating HCV severity (the randomised, double blind clinical study conducted during 5 years by Pr Poynard T., hospital Pitié-Salpêtrière, Paris, France, in about 100 patients versus placebo will be published soon) but other patients were also successfully treated by one of us M.G. (3 patients) and Meulan Pascal of the association " S.O.S. hépatite C " in France (10 patients), in the latter case in association with pentoxyfilline. Generally speaking, silymarin is a non specific hepatoprotective drug, with a very large spectrum of liver diseases, including hepatotoxicity induced by CCl4, D-galactosamine, drugs, alcoholism, hepatitis frog virus 3 and Amanita Phalloides intoxication (Legalonâ : Madaus Laboratory documentation). We have demonstrated (Tran M.K.G., 2002) that some of these causal agents (and also hepatitis B virus pre-S1) have a common base : The molecular mimicry to hepatotrophic Fas ligand. This the case for phallotoxins from the mushroom Amanita Phalloides and hepatitis C virus core, hence the clinical study conducted in HCV infections with silymarin (precisely silibinine, a more biodisponible compound from silymarin).

 

CASE REPORT.

A case of multitherapy HAART arrest (he experienced all available drugs) , a 50 years old male, seropositive since 20 years, infected with cytomegalovirus (CMV) [a well known hepatotoxic virus, which induced a lethal hepatitis in a child treated with didanosine (D.D.I.) by Blanche S., in Paris (Lacaille F., 1995)] and suffering from CMV retinitis & colitis, was switched to tacrine associated with silymarin (this couple decreased hepatotoxicity by 33%), with T4 increase in one month from 80 to 120 and a fall of viral load from 150,000 to 40,000; the hepatoprotection conferred by silymarin permitted to avoid any severe liver damage ; tacrine efficiency realized at least a therapeutic window with possible HAART resensibilisation. This patient presented nausea and vomiting when tacrine doses were increased further to 150 mg/day (50mg x 3), so tacrine was stopped permitting the transaminases decrease to normal level; for the moment, at the time of this Conference, he takes enfurvitide (T-20) and saquinavir, indinavir and 3TC( T4 = 250 ; V.L. = 5,000), but in the future tacrine is programmed to be taken again progressively and slowly without any complication. In Alzheimer's disease, it is well known that tacrine can be stopped and taken again, without, curiously, any reapparition of the first transaminase elevation. This is a very enigmatic phenomenon, unique to tacrine, as it is well known that a hepatotoxic drug must never be taken a second times. Susceptibility to tacrine hepatoxicity is increased by a genetic polymorphism [glutathione-S-transferase (GST) alleles M1 & T1 deficiencies (Simon T., 2000)], but to date we did not look for this genetic marker. Pentoxyfilline (Trental â), an anti-TNF-a, may also be an interesting hepatoprotector (Akriviadis E, 2000 ; Vasil'ev V.S., 1990) but a study noted an in vitro enhancement of cytomegalovirus (Staak K. 1997), so we did not use pentoxyfilline in this CMV infected patient.

But he received vitamin E and selenium, as vitamin E is hepatoprotector against tacrine in hepatocyte cell culture (Dogterom).

Pyridoxine (vitamin B6) deficiency must be searched and corrected, because pyridoxine is an efficient treatment of pregnancy nausea and vomiting (Magee L.A., 2002).

In summary, Tacrine with silymarin is able to rescue the 10-15% chemoresistant patients not responding to multitherapy, by at least a first mechanism of resensitization to ineffective (by escape mutation) or toxic (metabolic, mitochondrial, cardio-vascular) drugs. The complete window permitted by tacrine allows HIV-1 to change its population by a replacement of a HAART-resistent to a -sensitive variant. In the same time, tacrine inhibit any HIV-1 velleity and assumed all the security to the patient during the window. Secondly, after the tacrine alone window, ideally, it is possible to reassociate the new drugs (if not hepatotoxic) to tacrine without any difficulty.

Other patients are planned on this protocol of switch to tacrine + sylimarin (or more biodisponible silibinin), after HAART stop for inefficiency or toxicity. The population concerned in primary intention is the 10% HAART chemoresistent one. But this is not limitative, as any HIV-1 is concerned, as far as Tacrine can be associated with any anti-HIV-1 drug if not hepatotoxic. Interestingly, tacrine is not expensive and very easy to synthetise chemically.

CONCLUSION:

Nef and a-defensin superimposed on each other in 3D, suggesting that the a-defensin protective action is a competitive inhibition with Nef.

Thus the previous scorpion venom model of AIDS (Tran M.K.G., 1989) is confirmed and this new data strongly boosted the clinical use of tacrine. Interestingly, tacrine increased the total number of polymorphonuclear neutrophils (precisely the a-defensin producing cells) in clinical trial (Fredj G.). A case of multitherapy HAART arrest with cytomegalovirus infection was switched to tacrine associated with silymarin (decreasing hepatotoxicity by 33%), with T4 increase in one month from 80 to 120 and a fall of viral load from 150.000 to 40.000 ; the hepatoprotection conferred by sylimarin permits the avoidance of any severe liver damage & the tacrine efficiency permitted at least a therapeutic window with possible resensibilisation. Tacrine with silymarin is able to rescue the 10% chemoresistent patients not responding to multitherapy.

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 2.a. Immune Reconstitution by DHEA - A significant Clinical Case.

Caprani A.¹, Richert A.², Maurisson G.³

(1,2) Association Positifs, Paris , Université Paris 7;
(3) Centre Médical Europe, Paris; Association Positifs, Paris

Background : DHEA prevents TH1 and TH2 imbalance, known to contribute to the progression of HIV. Moreover, HIV+ patients show generally a decline of DHEA blood level

Method : Blood DHEA sulphate(DHEAS), cortisol, CD4 and CD8 counts were measured for an HIV+ patient presenting initially a low DHEAS/cortisol level, during supplementation by 7-keto DHEA for 9 months

Results : A DHEA supplementation of a 60 year old osteoporotic patient HIV+ since 20 years, with a stable biological and clinical state ( CD4 =325±20/mm3 ; viral load< 50 copies/ml ; DHEAS ª 700ng/ml; no opportunistic affections during the preceding 2 years), was instituted in order to increase mass bone after failure of orthodoxic therapies. Supplementation was done with 7-keto DHEA (no hormon-dependent cancer risks) ; 50mg per os daily the first 3 months and 75mg daily the next 6 months.
After 3 months, blood DHEAS level increases to 1433ng/ml and CD4 increases to 407 (+25%). At 6 and 9 months, DHEAS level increases to 2700ng/ml and CD4 to 501 (+54%)
In addition, at 6 months, mass bone at the level of (L1-L4), increases drastically of 9,5%, and the only biological parameter controlling mass bone which was drastically disturbed(PTH), was normalized.

Conclusion : Supplementation of DHEA in an HIV+ patient to restore a normal level leads to a drastic increase of CD4, and moreover by inducing bone remineralisation allows to couterbalance a well known side effect of HAART. This significant clinical case justify to undertake a large clinical trial

2.b. MOLECULAR MIMICRY BETWEEN HIV 1 NEF AND HUMAN MDM2 (MOUSE DOUBLE MINUTE 2),
A LIGAND OF P53, THE MAJOR TUMOR SUPPRESSOR PROTEIN IN ONCOLOGY.

* Association Positifs, BP 230, 75865 Paris 18, France. E-mail : positifs@positifs.org.
¹Université Paris-Sud XI, Chimie Thérapeutique, Faculté de Pharmacie, 92290 Châtenay-Malabry, France.
Correspondance : 31, Av du Bois 92290 Chatenay Malabry, France. E-mail : mkg_tran@yahoo.fr.
² Université Paris Jussieu, Tour 33. Association Positifs, Paris (E-mail : Positifs@positifs.org). E-mail : caprani@ccr.jussieu.fr
³ Centre Médical Europe, 44, rue d'Amsterdam, 75009 Paris, France. E-mail : maurisson@club-internet.fr

Background :

HIV 1 Nef is a kind of complex mosaic protein, endowed with many unrelated functions. One of the recent discovery (Greenway A.L., 2002) is that Nef has the same properties than mdm2, namely Nef binds to and inhibits p53, the major tumor suppressor protein, implicated in half of all the cancers.. Our objective is to analyse the Nef NH2 terminus [amino acids (AA) 1-57] to see whether it contains a similarity with mdm2. The method used is a comparison of the HIV 1 Nef AA sequences (in the 2002 Los Alamos databank) with the various human mdm2 isotypes. 

Result : 

There is a complete perfect molecular homology spanning an hexapeptide GAVTSS (Gly Ala Val Thr Ser Ser) common to many HIV 1 Nef and mdm2. Furthermore, the homology extended downstream of this hexapeptide (VSQDLD is read in reverse sense) :

Our alignment is strengthened by a similar homology in the same region between mdm2 (28-DKEESVE-34) and human polyoma T antigen (6-NREESME-12), a known p53 ligand.

In conclusion, HIV 1 Nef NH2 terminus is mimicking a viral mdm2 and inhibits p53. Thus Nef is a new member of the large p53 viral ligand family (papillomavirus E6, polyomavirus T antigen, adenovirus E1B, hepatitis B virus protein X). Nef role may be important in AIDS oncogenesis, in Kaposi sarcoma, lymphoma and cervical cancer. The deciphering of the precise p53 binding sequence around the hexapeptide GAVTSS may be an opportunity to synthetise short peptidomimetic antagonists or retroinverso peptides designed as anticancer drugs.

© Copyright association POSITIFS, 2003.