Renal protective effect of ellipticine against streptozotocin induced diabetic nephropathy in rats via suppression of oxidative stress and inflammatory mediator.

PURPOSE: Diabetes mellitus is a serious health problem worldwide, and diabetic nephropathy is the complication. The diabetic nephropathy considerably enhances the oxidative stress, glycation, lipid parameters and inflammatory reaction. Ellipticine has potent free radical scavenging and anti-inflammatory effect. METHODS: In the current study, our objectives were to thoroughly examine the renal protective effects of ellipticine in a rat model of streptozotocin (STZ)-induced diabetic nephropathy (DN) and to elucidate the underlying mechanisms involved. For the induction of diabetic nephropathy, streptozotocin (50 mg/kg) was used, and rats were separated into groups and given varying doses of ellipticine (2.5, 5 and 7.5 mg/kg). The body weight, and renal weight were estimated. The inflammatory cytokines, renal biomarkers, inflammatory antioxidant, and urine parameters were estimated. RESULTS: Result showed that ellipticine considerably enhanced the body weight and reduced the renal tissue weight. Ellipticine treatment significantly (P < 0.001) repressed the level of blood urea nitrogen, serum creatinine, uric acid, blood glucose and altered the lipid parameters. Ellipticine significantly (P < 0.001) repressed the level of malonaldehyde and boosted the glutathione, catalase, superoxide dismutase, and glutathione peroxidase. Ellipticine treatment significantly (P < 0.001) reduced the inflammatory cytokines and inflammatory mediators. CONCLUSIONS: Ellipticine could be a renal protective drug via attenuating the inflammatory reaction, fibrosis and oxidative stress in streptozotocin induced rats.

Effective Antibacterial and Antihemolysin Activities of Ellipticine Hydrochloride against Streptococcus suis in a Mouse Model.

Streptococcal toxic shock-like syndrome (STSLS) caused by the epidemic strain of Streptococcus suis leads to severe inflammation and high mortality. The life and health of humans and animals are also threatened by the increasingly severe antimicrobial resistance in Streptococcus suis There is an urgent need to discover novel strategies for the treatment of S. suis infection. Suilysin (SLY) is considered to be an important virulence factor in the pathogenesis of S. suis In this study, ellipticine hydrochloride (EH) was reported as a compound that antagonizes the hemolytic activity of SLY. In vitro, EH was found to effectively inhibit SLY-mediated hemolytic activity. Furthermore, EH had a strong affinity for SLY, thereby directly binding to SLY to interfere with the hemolytic activity. Meanwhile, it was worth noting that EH was also found to have a significant antibacterial activity. In vivo, compared with traditional ampicillin, EH not only significantly improved the survival rate of mice infected with S. suis 2 strain Sc19 but also relieved lung pathological damage. Furthermore, EH effectively decreased the levels of inflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor alpha [TNF-α]) and blood biochemistry enzymes (alanine transaminase [ALT], aspartate transaminase [AST], creatine kinase [CK]) in Sc19-infected mice. Additionally, EH markedly reduced the bacterial load of tissues in Sc19-infected mice. In conclusion, our findings suggest that EH can be a potential compound for treating S. suis infection in view of its antibacterial and antihemolysin activity.IMPORTANCE In recent years, the inappropriate use of antibiotics has unnecessarily caused the continuous emergence of resistant bacteria. The antimicrobial resistance of Streptococcus suis has also become an increasingly serious problem. Targeting virulence can reduce the selective pressure of bacteria on antibiotics, thereby alleviating the development of bacterial resistance to a certain extent. Meanwhile, the excessive inflammatory response caused by S. suis infection is considered the primary cause of acute death. Here, we found that ellipticine hydrochloride (EH) exhibited effective antibacterial and antihemolysin activities against S. suisin vitroIn vivo, compared with ampicillin, EH had a significant protective effect on S. suis serotype 2 strain Sc19-infected mice. Our results indicated that EH, with dual antibacterial and antivirulence effects, will contribute to treating S. suis infections and alleviating the antimicrobial resistance of S. suis to a certain extent. More importantly, EH may develop into a promising drug for the prevention of acute death caused by excessive inflammation.

In vitro activity of a G-quadruplex-stabilizing small molecule that synergizes with Navitoclax to induce cytotoxicity in acute myeloid leukemia cells.

BACKGROUND: Acute Myeloid Leukemia (AML) is a malignancy of myeloid precursor cells that arise from genomic alterations in the expression of key growth regulatory genes causing cells to assume an undifferentiated state and continue to proliferate. Recent efforts have focused on developing therapies that target specific protein products of aberrantly expressed genes. However, many of the identified proteins are difficult to target and thought to be "undrugable" because of structural challenges, protein overexpression, or mutations that confer resistance to therapy. A novel technology that circumvents some of these issues is the use of small molecules that stabilize secondary DNA structures present in the promoters of many potential oncogenes and modulate their transcription. METHODS: This study characterizes the in vitro activity of the G-quadruplex-stabilizing small molecule GQC-05 in AML cells. The effect of GQC-05 on three AML cell lines was analyzed using viability and apoptosis assays. GQC-05 has been shown to down-regulate MYC through G-quadruplex stabilization in Burkitt's lymphoma cell lines. MYC expression was evaluated through qPCR and immunoblotting in the three AML cell lines following the treatment of GQC-05. In order to identify other therapeutic agents that potentiate the activity of GQC-05, combination drug screening was performed. The drug combinations were validated using in vitro cytotoxicity assays and compared to other commonly used chemotherapeutic agents. RESULTS: GQC-05 treatment of KG-1a, CMK and TF-1 cells decreased cell viability and resulted in increased DNA damage and apoptosis. Additionally, treatment of KG-1a, CMK and TF-1 with GQC-05 resulted in decreased expression of MYC mRNA and protein, with a more pronounced effect in KG-1a cells. Combination drug screening identified the Bcl-2/Bcl-XL inhibitor Navitoclax as a compound that potentiated GQC-05 activity. Co-treatment with GQC-05 and Navitoclax showed a synergistic decrease in cell viability of AML cells as determined by Chou-Talalay analysis, and induced more DNA damage, apoptosis, and rapid cytotoxicity. The cytotoxicity induced by GQC-05 and Navitoclax was more potent than that of Navitoclax combined with either cytarabine or doxorubicin. CONCLUSION: These results suggest that the G-quadruplex stabilizing small molecule GQC-05 induces down regulated MYC expression and DNA damage in AML cells. Treatment with both GQC-05 with a Bcl-2/Bcl-XL inhibitor Navitoclax results in increased cytotoxic activity, which is more pronounced than Navitoclax or GQC-05 alone, and more significant than Navitoclax in combination with cytarabine and doxorubicin that are currently being used clinically.

Identification of an IKKß inhibitor for inhibition of inflammation in vivo and in vitro.

Targeting on the IKKß to discover anti-inflammatory drugs has been launched for ten years, due to its predominant role in canonical NF-κB signaling. In the current study, we identified a novel IKKß inhibitor, ellipticine (ELL), an alkaloid isolated from Ochrosia elliptica and Rauvolfia sandwicensis. We found that ELL reduced the secretion and mRNA expression of TNF-α and IL-6 and decreased the protein expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in bone marrow derived macrophages (BMDMs) stimulated with LPS. In coincided with the results, ELL suppressed PGE2 and NO production in BMDMs. Underlying mechanistic study showed that ELL inhibited IκBα phosphorylation and degradation as well as NF-κB nuclear translocation, which was attributed to suppression of IKKα/ß activation. Furthermore, kinase assay and binding assay results indicated that ELL inhibited IKKß activity via directly binding to IKKß and in turn resulted in suppression of NF-κB signaling. To identify the binding sites of ELL on IKKß, IKKßC46A plasmid was prepared and the kinase assay was performed. The results demonstrated that the inhibitory effect of ELL on IKKß activity was impaired in the mutation, implying that anti-inflammatory effect of ELL was partially attributed to binding on cysteine 46. Furthermore, ELL up-regulated LC3 II expression and reduced p62 expression, suggesting that autophagy induction contributed to the anti-inflammatory effect of ELL as well. In coincided with the in vitro results, ELL increased the survival and antagonized the hypothermia in the mice with LPS-induced septic shock. Consistently, ELL reduced TNF-α and IL-6 production in the serum of the mice treated with LPS. Collectively, our study provides evidence that ELL is an IKKß inhibitor and has potential to be developed as a lead compound for treatment inflammatory diseases in the future.

Simultaneous Drug Targeting of the Promoter MYC G-Quadruplex and BCL2 i-Motif in Diffuse Large B-Cell Lymphoma Delays Tumor Growth.

Secondary DNA structures are uniquely poised as therapeutic targets due to their molecular switch function in turning gene expression on or off and scaffold-like properties for protein and small molecule interaction. Strategies to alter gene transcription through these structures thus far involve targeting single DNA conformations. Here we investigate the feasibility of simultaneously targeting different secondary DNA structures to modulate two key oncogenes, cellular-myelocytomatosis (MYC) and B-cell lymphoma gene-2 (BCL2), in diffuse large B-cell lymphoma (DLBCL). Cotreatment with previously identified ellipticine and pregnanol derivatives that recognize the MYC G-quadruplex and BCL2 i-motif promoter DNA structures lowered mRNA levels and subsequently enhanced sensitivity to a standard chemotherapy drug, cyclophosphamide, in DLBCL cell lines. In vivo repression of MYC and BCL2 in combination with cyclophosphamide also significantly slowed tumor growth in DLBCL xenograft mice. Our findings demonstrate concurrent targeting of different DNA secondary structures offers an effective, precise, medicine-based approach to directly impede transcription and overcome aberrant pathways in aggressive malignancies.

Fluorescence Characterization of Gold Modified Liposomes with Antisense N-myc DNA Bound to the Magnetisable Particles with Encapsulated Anticancer Drugs (Doxorubicin, Ellipticine and Etoposide).

Liposome-based drug delivery systems hold great potential for cancer therapy. The aim of this study was to design a nanodevice for targeted anchoring of liposomes (with and without cholesterol) with encapsulated anticancer drugs and antisense N-myc gene oligonucleotide attached to its surface. To meet this main aim, liposomes with encapsulated doxorubicin, ellipticine and etoposide were prepared. They were further characterized by measuring their fluorescence intensity, whereas the encapsulation efficiency was estimated to be 16%. The hybridization process of individual oligonucleotides forming the nanoconstruct was investigated spectrophotometrically and electrochemically. The concentrations of ellipticine, doxorubicin and etoposide attached to the nanoconstruct in gold nanoparticle-modified liposomes were found to be 14, 5 and 2 µg·mL(-1), respectively. The study succeeded in demonstrating that liposomes are suitable for the transport of anticancer drugs and the antisense oligonucleotide, which can block the expression of the N-myc gene.

Multi-responsive polymer micelles as ellipticine delivery carriers for cancer therapy.

In the present study, we describe the synthesis and physicochemical properties of a novel pH- and thermoresponsive micellar drug delivery system for an anticancer ellipticinium derivative based on the triblock copolymer poly(ethylene oxide)-block-[tert-butylacrylamide-co-6-(N-methacryloylamino)hexanoic acid hydrazide]-block-poly(ethylene oxide). The system was designed to meet the basic criteria required for drug carrier systems, namely, solubility in water (overcoming the insolubility of ellipticine), satisfactory drug loading, particle size suitable for an efficient enhanced permeability and retention effect and adequate stability in blood plasma (pH 7.4) followed by rapid drug release in tumors or tumor cell endosomes (pH<6.5). The copolymer in the form of a unimer can be eliminated by kidneys because the weight-average molecular weight of 21 kDa is sufficiently below the renal threshold. The half-life of drug release in a pH 5.0 buffer solution (pH of a late endosome) was ~45 h, but a negligible amount of the free ellipticine derivative was detected at pH 7.4 (pH of blood). Consequently, this supramolecular polymer conjugate is a good candidate for the delivery of ellipticine-based drugs and will therefore be subjected to more detailed studies.

Ellipticines as DNA-targeted chemotherapeutics.

The anti-tumor therapeutic ellipticine and its derivatives act as potent anticancer agents via a combined mechanism involving cell cycle arrest and induction of apoptosis. Cell death induced by ellipticine has been shown to engage a p53-dependent pathway, cell cycle arrest, interaction with several kinases and induction of the mitochondrial pathway of apoptotic cell death. Cell cycle arrest was shown to result from DNA damage caused by a variety of tumor chemotherapeutic agents; this is also the case for ellipticines. The prevalent DNA-mediated mechanisms of anti-tumor, mutagenic and cytotoxic activities of ellipticine are (i) intercalation into DNA, (ii) inhibition of DNA topoisomerase II activity, and (iii) covalent binding to DNA in vitro and in vivo after enzymatic activation by cytochrome P450 and/or peroxidase enzymes The mechanism leading to apoptosis by ellipticine is thought to also be associated with DNA damage, by inhibition of topoisomerase II and the covalent modification of DNA. In addition, the formation of ellipticine-DNA adducts ultimately can mutate cancer cells or initiate cell death. The aim of this review is to summarize our knowledge on the molecular mechanisms with the aim to explain the effectiveness of ellipticines as DNA-targeted chemotherapeutics in cancer cells.

Comparative in vitro and in vivo antimalarial activity of the indole alkaloids ellipticine, olivacine, cryptolepine and a synthetic cryptolepine analog.

Indole alkaloids ellipticine (1), cryptolepine triflate (2a), rationally designed 11-(4-piperidinamino)cryptolepine hydrogen dichloride (2b) and olivacine (3) (an isomer of 1) were evaluated in vitro against Plasmodium falciparum and in vivo in Plasmodium berghei-infected mice. 1-3 inhibited P. falciparum (IC50≤1.4 µM, order of activity: 2b>1>2a>3). In vitro toxicity to murine macrophages was evaluated and revealed selectivity indices (SI) of 10-12 for 2a and SI>2.8×10² for 1, 2b and 3. 1 administered orally at 50mg/kg/day was highly active against P. berghei (in vivo inhibition compared to untreated control (IVI)=100%, mean survival time (MST)>40 days, comparable activity to chloroquine control). 1 administered orally and subcutaneously was active at 10 mg/kg/day (IVI=70-77%; MST=27-29 days). 3 exhibited high oral activity at ≥50 mg/kg/day (IVI=90-97%, MST=23-27 days). Cryptolepine (2a) administered orally and subcutaneously exhibited moderate activity at 50mg/kg/day (IVI=43-63%, MST=24-25 days). At 50 mg/kg/day, 2b administered subcutaneously was lethal to infected mice (MST=3 days) and moderately active when administered orally (IVI=45-55%, MST=25 days). 1 and 3 are promising compounds for development of antimalarials.

Anthracyclines and ellipticines as DNA-damaging anticancer drugs: recent advances.

Over the past forty years, anthracyclines and ellipticines have attracted attention as promising cytostatics. In this review, we focus on their mechanisms of cytoxicity, DNA-damaging effects and adverse side-effects. We also summarize ways to enhance the therapeutic effects of these drugs together with a decrease in their adverse effects. Current drug design strategies are focused on drug bioavailability and their tissue targeting, whereas drug delivery to specific intracellular compartments is rarely addressed. Therefore, therapies utilizing the antineoplastic activities of anthracyclines and ellipticines combined with novel strategies such as nanotechnologies for safer drug delivery, as well as strategies based on gene therapy, could significantly contribute to medical practice.

Demonstration that drug-targeted down-regulation of MYC in non-Hodgkins lymphoma is directly mediated through the promoter G-quadruplex.

Most transcription of the MYC proto-oncogene initiates in the near upstream promoter, within which lies the nuclease hypersensitive element (NHE) III(1) region containing the CT-element. This dynamic stretch of DNA can form at least three different topologies: single-stranded DNA, double-stranded DNA, or higher order secondary structures that silence transcription. In the current report, we identify the ellipticine analog GQC-05 (NSC338258) as a high affinity, potent, and selective stabilizer of the MYC G-quadruplex (G4). In cells, GQC-05 induced cytotoxicity with corresponding decreased MYC mRNA and altered protein binding to the NHE III(1) region, in agreement with a G4 stabilizing compound. We further describe a unique feature of the Burkitt's lymphoma cell line CA46 that allowed us to clearly demonstrate the mechanism and location of action of GQC-05 within this region of DNA and through the G4. Most importantly, these data present, as far as we are aware, the most direct evidence of intracellular G4-mediated control of a particular promoter.

Non-genotoxic anti-neoplastic effects of ellipticine derivative NSC176327 in p53-deficient human colon carcinoma cells involve stimulation of p73.

We previously performed a high-throughput screen using real-time noninvasive bioluminescence imaging of p53 transcriptional activity and identified a group of small molecules that trigger p53-like transcriptional responses in p53-deficient tumor cells. Here we further examined the anti-tumor effects of selected compounds in vitro and showed that NSC176327, a derivative of the cytotoxic plant alkaloid ellipticine, exhibited strong anti-neoplastic effect sin wild-type p53, p53-mutant or p53-deficient human colon cancer cells. NSC176327 was more potent at inhibiting tumor cell growth as compared to chemotherapeutic drugs and other ellipticine derivatives and induced cell cycle arrest and apoptosis. Surprisingly, unlike what is observed with the parent compound ellipticine, a DNA damage signaling response was not observed with NSC176327 as evidenced by lack of phosphorylated histone H2AX foci in NSC176327-treated tumor cells. NSC176327 treatment caused a significant increase in p53-activated reporter signal in HCT116, SW620 and HCT116 p53-/- cells and upregulated DR5 and p21 protein expression. NSC176327 treatment also resulted in increased p73 protein expression and knockdown of transactivating isoforms of p73 in HCT116 p53-/- cells showed significant resistance to drug treatment. These results demonstrate an important role of p73 in the anti-tumor effects of NSC176327,and suggest that a close analogue of ellipticine may act by a non-genotoxic mechanism targeting the p53/p73 pathway as compared to the original parent compound that targets the same pathway.

Ellipticine derivative NSC 338258 represents a potential new antineoplastic agent for the treatment of multiple myeloma.

High-risk multiple myeloma can be correlated with amplification and overexpression of the cell cycle regulator CKS1B. Herein, we used the COMPARE algorithm to correlate high expression of CKS1B mRNA in the NCI-60 cell line panel with the concentration causing 50% growth inhibition (GI(50)) of >40,000 synthetic compounds. This led to the identification of NSC 338258 (EPED3), a highly stable, hydrophilic derivative of the plant alkaloid ellipticine. In vitro, this synthetic anticancer compound exhibits dramatic cytotoxic activity against myeloma cells grown in suspension or in coculture with stromal cells. EPED3-induced cell cycle arrest and an apoptotic progression that appear to be a consequence of the instantaneous effect of the drug on cytoplasmic organelles, particularly mitochondria. Disruption of mitochondria and cytoplasmic distribution of cytochrome c initiated the intracellular proteolytic cascade through the intrinsic apoptotic pathway. EPED3 is able to induce apoptosis in myeloma cells with de novo or acquired resistance to commonly administered antimyeloma agents. Collectively, our data suggest that EPED3 targets mitochondrial function to rapidly deplete chemical energy and initiate apoptosis in myeloma cells at nanomolar concentrations while leaving stromal cells unharmed.

Restoration of p53 to limit tumor growth.

PURPOSE OF REVIEW: p53 mutation occurs in over half of all human tumors. Among the remaining tumors, although they may process a wild-type p53, the pathways of p53-induced cell-cycle arrest and apoptosis are deficient. Therefore, p53 serves as a unique molecular target for cancer therapy. This review focuses on the current progress regarding restoration of p53 function in human tumors for molecularly targeted therapy. RECENT FINDINGS: Targeting p53 for cancer therapy has been intensively pursued. CP-31398 was the first small molecule identified with the ability to restore the wild-type conformation to mutant p53. Subsequently, PRIMA-1 and ellipticine were found to be able to induce mutant p53-dependent cell death. Nutlin was developed to rescue wild-type p53 from degradation mediated by MDM2. More recently, p53 family members can be activated and therefore serve as substitutes of p53 in tumor cells and induce cell death. SUMMARY: Loss of p53 function is a characteristic of almost all human tumors. Recent advances demonstrate that reconstitution of p53 function is possible and practical as a promising antitumor strategy.

Extending nature's leads: the anticancer agent ellipticine.

The natural plant product ellipticine was isolated in 1959 from the Australian evergreen tree of the Apocynaceae family. This compound was found to be an extremely promising anticancer drug. The planar polycyclic structure was found to interact with DNA through intercalation, exhibiting a high DNA binding affinity (10(6) M(-1)). The presence of protonatable ring nitrogens distinguished ellipticine from other simple intercalators. Both monocationic and uncharged species were found to be present under physiological conditions. The positive charge stabilized the binding of ellipticine to nucleic acids, while the more lipophilic uncharged compound was shown to readily penetrate membrane barriers. The structural nature of these compounds offers a plausible basis for the implication of multiple modes of action, including DNA binding, interactions with membrane barriers, oxidative bioactivation and modification of enzyme function; most notably that of topoisomerase II and telomerase. Pharmacologically, a number of toxic side effects have been shown to be problematic, but the amenability of ellipticine towards systematic structural modification has permitted the extensive application of rational drug design. A number of successful ellipticine analogs have been designed and synthesized with improved toxicities and anticancer activities. More recently the synthetic focus has broadened to include the design of hybrid compounds, as well as drug delivery conjugates. Considerable research efforts have been directed towards gaining a greater understanding of the mechanism of action of these drugs that will aid further in the optimization of drug design.

Small molecules that reactivate mutant p53.

Around half of all human tumours carry mutant p53. This allows escape from p53-induced cell cycle arrest and apoptosis. Many tumours express mutant p53 proteins at elevated levels. Restoration of wild-type p53 function should trigger massive apoptosis in tumour cells and thus eradicate tumours. Various types of small molecules have been identified that can restore native conformation and wild-type function to mutant p53. Such molecules may serve as leads for the development of novel efficient anticancer drugs.

Comparison of the pharmacological profile of an olivacine derivative and a potential prodrug.

BACKGROUND: The new olivacine derivative S 16020-2 (NSC-659687) has entered clinical trials on the basis of a marked antitumor activity in experimental models. Amongst the analogues which were synthesized to improve both therapeutic index and antitumor activity, the most active ones were those esterified on the 9-OH group such as S 30972-1, the glutaric acid monoester derivative. PURPOSE: To compare the pharmacological profile of S 30972-1 and S 16020-2 in vitro and in vivo and to investigate whether S 30972-1 could act as a prodrug of S 16020-2. METHODS: The two compounds were compared in vitro in terms of their activity in inhibiting cellular proliferation and perturbing the cell cycle and in vivo in terms of their antitumor activity in murine transplantable tumors and human orthotopic models. The plasma concentrations of S 16020-2 and S 30972-1 were determined in mice, in a comparative pharmacokinetic study after i.v. administration, using an HPLC assay. RESULTS: Although tumor cell proliferation and accumulation of cells in the G2 phase of the cell cycle were similarly affected by the two compounds after a continuous exposure (IC50 values of 30-50 n M), S 30972-1 was about tenfold less potent than S 16020-2 after short exposures. In vivo, S 30972-1 induced more long-term survivors than S 16020-2 among mice with Lewis lung carcinoma and sensitive or multidrug resistant P388 leukemias. The growth of Colon 38 carcinoma was slightly more inhibited by S 30972-1 than S 16020-2. In the more relevant human orthotopic models, using the optimal doses of each drug, 160 mg/kg S 30972-1 was significantly more active than 80 mg/kg S 16020-2 in the NCI-H460 lung carcinoma. The two compounds were significantly active in A549 lung carcinoma, moderately active in the NIH:OVCAR-3 ovary carcinoma and inactive in the NCI-H125 lung and DU145 prostate carcinomas. Pharmacokinetic study demonstrated that S 30972-1 is a prodrug of S 16020-2: the conversion was rapid and complete within 1 h of the administration of S 30972-1. CONCLUSIONS: The in vivo profile of these two compounds appeared very similar, although S 30972-1 exhibited globally a wider therapeutic index. The rapid conversion of S 30972-1 to S 16020-2 shows that S 30972-1 acts mainly as a prodrug of S 16020-2. This should be taken into account before considering S 30972-1 as a valuable back-up of S 16020-2.

Cardioprotective effects of 9-hydroxyellipticine on ischemia and reperfusion in isolated rat heart.

We determined the effect of 9-hydroxyellipticine (9HE) on ryanodine receptor (RyR) and cardiac function after global ischemia in isolated rat hearts. The binding of [3H]-ryanodine in rabbit cardiac sarcoplasmic reticulum was displaced by 9HE in a biphasic manner corresponding to the two sites model with IC50 values of 6.1 microM and 55 mM. The increase of the intracellular Ca2+ concentration induced by caffeine in CHO cells expressing cardiac-type RyR was suppressed by 9HE in a concentration-dependent manner. Pretreatment of the heart with 9HE decreased the total duration of reperfusion-induced ventricular fibrillation (VF) and delayed the onset of VF. There was also a significant recovery of contractile force of ischemic hearts following 9HE. Unlike nifedipine, an L-type Ca2+-channel blocker, 9HE did not suppress the contraction of rat papillary muscles. Thus, 9HE exerts the cardioprotective effects against ischemia /reperfusion injury without changing hemodynamic indices.

Viral and immunologic follow up of 4 to 9 years of AIDS treatments by quadruple combinations of virostatics including integrase inhibitors applied in short sequences differing by drug rotation.

AIM: To present the 4 to 9 years (median: 6 years) treatment follow up of 10 HIV1-AIDS patients, 9 at AIDS and 1 at A3 stages. METHODS: We have applied from 1992 to 1994, AZT combined with 2 integrase inhibitors, acriflavine and hydroxy-methyl-ellipticine. We could shift, in 1994, to combinations of 3 drugs including two more retrotranscriptase inhibitors (RTI), ddI and ddC, and, after 1995, to combinations of 4 drugs including also two other RTI, d4T and 3TC, and 3 protease inhibitors (PI), indinavir, ritonavir, and saquinavir. In 1998, as cobalamine was shown by an in vitro test, to act as integrase inhibitor, vitamin B12 was added in cycles of various lengths. Every three weeks, not only the investigations were repeated, but the virostatics were changed. RESULTS: No grade 2 virostatics toxicity has been registered. The viral loads (VL) decreased according to exponential curves. Their initial parts obeyed first order kinetics. The second parts were and still are asymptotic. The first parts could be rectilinear or sinuous. The sinuosities were associated to cofactors present before treatment (chimerism, UV irradiation, hepatitis C or B and C, brain toxoplasmosis). The asymptotic parts, whose VL were below PCR detectable levels, presented discrete, reversible HIV1 rebounds, associated to other cofactors (such as herpes zoster, herpes 6, CMV, flat condyloma, and influenza). Among immunologic parameters, the monocyte and CTL numbers increased and presented, during the rapidly decreasing part of VL curve, a significant inverse correlation with it. Neither CD4+ nor suppressor T-cell (STC) numbers presented such correlation. Near 100 % of CTL were CD28+. Later, vitamin B12 applications increased monocyte and CD28+ CTL numbers, and appeared to reinforce VL stabilization. CONCLUSION: The combinations of inhibitors affecting 3 retrovirus targets, retrotranscriptase, integrase, and protease have given to 10 out of 10 AIDS patients survivals varying today between 4 to 9 years, in excellent conditions. The UVA-pretreated patient is the only one presenting a not maximally reduced asymptotic VL, while his CD4+ and STC have been absent for 8 years. Other patient VL regressed exponentially to become asymptotic, below PCR detectable levels.