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New therapeutic option for decreasing ART- related toxicity in HIV/HCV coinfected patients

R. Chehitiani1, B. Pylypchuk2, E. Zagaydanova1, O. Ordzanova3, L. Urchenko3 1State Medical University , AIDS- center, Luhansk, Ukraine; 2Scientist association , Ekomed, Kiev, Ukraine; 3 Tuberculosis hospital, Pulmonal tuberculosis, Lisichansk, Ukraine

   Background: Hepatic toxicity greatly complicates the management of HIV treatment - experienced patients. In several large trials of HIV/HCV co-infected patients, more participants stopped their assigned therapy due to toxicity than virological failure. In the Ukraine, most HIV -infected patients have few antiretroviral options due to limited resources. Significant metabolic disorders and underlying hepatic injury caused by chronic alcohol, drug addiction and hepatitis co-infection are common. Preliminary results of the immunomodulating agent Dzherelo suggested hepatic anti-inflammatory activity. We hypothesized that the addition of Dzherelo may attenuate hepatic toxicity in patients receiving antiretroviral therapy (ART). Methods: We report the 24-week results of an ongoing study initiated March 2005. 35 HIV/HCV co-infected patients suffering from alcohol abuse or/and drug addict were enrolled. ART including AZT/3TC/NVP was initiated. Dzherelo was added to the therapy a median of 8-10 weeks after starting ART. ART- related hepatotoxicity was defined as an increase in ALT/AST levels up to five times the upper limit of normal, or 3,5 fold increase if baseline level was abnormal.
Results: 25/35 (71%) patients had baseline elevations in liver function tests (LFT) Mean baseline values were AST - 49 U/L , ALT - 62 U/L before starting ART. At week 8, prior to starting dzherelo, LFT has increased to niean ALT -109 U/L, mean AST- 47 U/L. The number of patients with protocol-defined ART related hepatotoxicity was 6/35 (18 %). Among them 3 died and one refused further therapy due gastrointestinal disorders. ART therapy needed to be interrupted and therapy changed in 2 patients due to hepatotoxicity. After dzherelo was added to the treatment regimen, LFT values declined to mean ALT- 43U/L, mean AST- 45 U/L and hepatotoxicity was not seen in any patients at week 24. No new cases of hepatotoxicity were reported after starting Dzherelo, and no clinical signs of hepatic inflammation were seen. Bilirubin levels were in the normal range at week 24.
Conclusions: Dzherelo appeared to attenuate hepatotoxicity from ART (NRTI + NNRTI) in this population with underlying liver pathology. This may lead to improved hepatotoxicity and reduced toxicity related therapy interruptions. Further study of this agent in this role is merited.

Influence of novel immunomodulator to the prevalence of new events opportunistic infections in untreated HIV- infected individuals.

G. Kutsvna1, R. Chehitiany1, I. Bascacov1, E. Zagaydanova1, I. Zaharova1 Luhansk AIDS-center, Department of Infectious Disease of Krasnodon, Sverdlovsk, Severodonetsk, - Lisichansk, Rubezhnoye, Ukraine.

Access to combination antiretroviral therapy is unobtainable for the majority of patients in Ukraine. In this situation opportunistic infections (O.I.) are the main factor of mortality patients. We studied the influence of immunomodulate agent of Dzerelo to the frequencies of new events 0.I. and markers of HIV disease progression in untreated HIV infected patients. 150 HIV infected patients received Dzerelo (50 drops - twice daily, thirty minutes prior to a meal) for 12 weeks. The control group of 120 patients received no therapy. Patients were followed for an average of 12 months, Conventional HIV RNA and CD4 cell counts could not be monitored due to financial limitations, therefore a decline in the total lymphocyte counts (TLC) and hemoglobin (Hgb) concentration in the blood were used to monitor the patient's disease status (Johns Hopkins Bloomberg School of Public Health 1984-1991). Frequencies of new events O.I and clinical progression to HIV/AIDS was observed. Patients were aged 18 to 42. Baseline demographic, immunological and anthropometric characteristics were balance between the two groups. 20/150 therapy and 20/120 control patients had AIDS at baseline. After 8 weeks, significant changes in TLC were seen: +63,+ 101,+ 275,+453,+ 624, + 710 and -24,-15,-31,-40,-64,-119 cells/mm3 from baseline level by 8,12,16, 20, 24, 48 weeks for therapy and control groups respectively. In the both the therapy and control groups were patients with clinical and laboratory parameters of anemia. In the therapy group 34 patients had a mean baseline Hgb level of 8.9 g/dl. (P = 0.05), and in the control group 28 patients had a Hgb level of 9.3 g/dl (P = 0.03). From week 8 - 24 Hgb increased in the therapy group to a mean of 11.7g/dl (P= 0.04).Compared to a decrease to 9.0 g/dl in the control group (P = 0.03). During 48 week follow up period new events O.I were observed in 14(9,3%) and 29(31,2%) patients for therapy and control groups respectively . Among them were oral or esophageal candidacies 3(2%) and 9 (7,2%) , herpes zoster 2(1,3%) and 8(6,4%), bacterial pneumonia 5 (3,3%) and 7(5,6%), tuberculosis 1(0,7%) and 4 (3,2%), TORCH- Infection 3(2%) and 8(6,4%), herpes zoster meningitis any case and 3(2,4%) patients in the therapy and control groups respectively. New events of HIV disease progression to AIDS occurred in 1(1.5%)patient in the therapy group and 8(8 %) patients in the control group for study period. Our results suggest a beneficial effect of Dzerelo as a potential immunoenhancer in HIV infected patients. Improvements in both laboratory markers (TLC, Hgb), decrease the frequencies of O.I. and clinical progression were seen. This compound merits further study combination this immunomodulate agent with ARV for improve immunological effect and decrease the risk of develop O.I.

         Effects of a novel immunomodulator in HIV infected individuals

   K. Galina, Z. Etena, Z. Iryna
  Medical Institute Kiev, Luhansk AIDS-Center, Department of Infectious Disease of Krasnodon, Sverdlovsk, Kremennoye, Severodonetsk, Rubezhnoye, Lisichansk, Ukraine.
   The Ukraine shares with other resource limited European countries the great challenge of HIV infection. Access to combination antiretroviral therapy is unobtainable for the majority of patients and novel, less expensive options are urgently needed. To this end we have been investigating a naturally derived agent Dzerelo, which may contain immunoenhancing qualities beneficial to HIV infected pntients.
    150 HIV infected patients received Dzerelo (50 drops — twice daily, thirty minutes prior to a meal) for 12 weeks. The control group of 120 patients riceived no therapy. Patients were followed for an average of 12 months. Patients were not receiving other ARV therapy and had not in the past. Conventional HIV RNA and CD-4 cell counts could not be monitored due to financial limitations, therefore a decline in the total lymphocyte counts (TLC) and hemoglobin (Hgb) concentration in the blood were used to monitor the patient's disease status (Johns Hopkins Bloomberg School of Public Health 1984-1991). Clinical progression to HIV/AIDS was also assessed.
   Patients were aged 18 to -42. Baseline demographic, immunological and anthropometric characteristics were balance between the two groups. 20/150 therapy and 20/ 120 control patients had AIDS at baseline. No differences were detected between the groups during the first 4 weeks in total lymphocyte or total leukocyte counts. After 8 weeks, significant changes in TLC were seen: +63. +101, +275, +453. +624. +710 and -24, -15, -31,- 40, -64, -119 cclls/mm3 from baseline level by 8, 12, 16, 20, 24, 48 weeks for therapy and control groups respectively. Increases in TLC in the therapy group appeared dependent on baseline levels. In treated patients with TLC <1300 cells/mm3 at baseline, only 49,3 % returned to normal levels. Those with TLC >1300 cells/mm3 at baseline 84.3 % returned to normal levels during continued follow up. In the control group TLC declined by a mean 4,2% during the 24-week study period, and 7,8% during a 48-week follow up. In the both the therapy and control groups were patients with clinical arid laboratory parameters of anemia. In the therapy group 34 patients had a mean baseline Hgb level of 8.9 g/dl. (P=0.05), and in the control group 28 patients had a Hgb level of 9.3 g/dl (P=0.03). From week 8-24 Hgb increased in the therapy group to a mean of 11.7 g/dl (P=0.04). Compared to a decrease to 9.0 g/ dl  in the control group. (P=0.03). New events of HIV disease progression to AIDS occurred in 1 patient (1.5%.) in the therapy group and 8 patients (8%) in the control group, during the 48-week follow up period.
     Our results suggest a beneficial effect of Dzerelo as a potential immunoen-hancer in HIV infected patients. Improvements in both laboratory markers (TLC, Hgb) and clinical progression were seen. This compound merits further study as an inexpensive agent for HIV infected individuals in resource poor settings. It will be perspective use combination ARV and this imunomodulate therapy for improve immunological effects.

Effect of Immunomodulator Dzherelo on CD4 + T-Lymphocyte Counts and Viral Load in HIV Infected Patients Receiving Anti-Retroviral Therapy

Research Journal of Pharmacology 2 (1): 8-12, 2008 ISSN: 1815-9362
© Medwell Journals, 2008

1Lyudmila G. Nikolaeva, 1Tatyana V. Maystat, 2Volodymyr S. Pylypchuk, 3Yuri L. Volyanskii and 4Galyna A. Kutsyna 'Kharkov Regional AIDS Prophylaxis and Prevention Center, Kharkov Medical Academy of Postgraduate Education, 6 Bor'by Street, Kharkov 61044, Ukraine 2Ekomed LLC., Prospect Pravdy 80-A, Kiev 04208, Ukraine 3I.I. Mechnikov Institute of Microbiology and Immunology, Kharkov 61057, Ukraine 4Luhansk Regional AIDS Center, Luhansk 91045, Ukraine.

Abstract: The phase II, randomized, clinical trial was conducted in 40 HIV infected patients to evaluate the effect of oral immunomodulator Dzherelo on immune and viral parameters. The arm A (n = 20), received standard Anti-Retroviral Therapy (ART) consisting of zidovudine, lamivudine and efavirenz (AZT/3TC/EFV) and arm B (n = 20) received 50 drops of Dzherelo twice per day in addition to ART. After 2 months the total CD3 T-lymphocytes increased in ART recipients from 664 to 819 cells |±L«1 (p = 0.06), whereas inDzherelo recipients they rose from 595 to 785 cells |±L«1 (p = 0.03). The population of CD4 T-cells expanded by 57.3% in ART (218-343; p = 0.002) and by 93.5% in Dzherelo arms (184-356; p = 0.004). The accrual in absolute and relative number of CD8+ lymphocytes in ART and Dzherelo recipients was 43.2% (2.7%) and 50.4% (-0.5%) , respectively. The CD4/CD8 ratio in Dzherelo recipients had increased from 1.495 to 1.940 (p = 0.03) but in the control group the increase was not significant, i.e., 1.418-1.613 (p = 0.14). About three-quarters (14/19) of patients on ART displayed the decrease in viral load (1718-1419 copies mL«1; p = 0.008), while 95% of patients on Dzherelo had a reduction in the number of viral copies (1793-1368 copies; p = 0.001). Dzherelo has a favorable effect on T-lymphocyte subsets and viral burden in HIV patients when given as an immunomodulating adjunct to ART.
Key words: AIDS, antiviral, ART, HAART, immunotherapy, phytotherapy.
INTRODUCTION
The antiretroviral drug resistance, drug toxicity and adherence are major concerns in clinical management of HIV infection (Pokrovskii, 2001). The combination of antiviral drugs, such as Highly Active Antiretroviral Therapy (HAART), may prevent HIV from mutating and spreading, allowing patients to rebuild their immune system to the same levels as in normal individuals (Mocroft et al., 2007). On the other hand, the immune activation caused by enhanced immune reaction to HIV, is now recognized as a cause of depletion of CD4 T-cells and resulting immunodeficiency (Appay et al., 2005). These problems are driving force for research on new therapies that can provide an answer. Most optimal therapeutic solution is an effective and safe immunotherapy that could regulate the immune response in a manner favorable to a host. There are many types of immune modulators that have been used clinically for viral infections, but for HIV the choice of immune interventions is limited (Ershov, 2003). Ukraine has the highest prevalence of HIV infection in Eastern Europe (Kelly and Amirkhanian, 2003; Vander werf et al., 2006). Oral immunomodulator Dzherelo is used in Ukraine for the management of HIV infections, including patients co-infected with TB (Chkhetiany et al., 2006, 2007; Kutsyna et al., 2003, 2005; Prihoda et al., 2006; Zaitseva, 2006). Clinical studies have indicated that Dzherelo can significantly increase CD3 and CD4 T-lymphocyte populations and helps to achieve better clinical response when combined with standard Anti-Retroviral Therapy (ART) consisting of zidovudine (AZT); lamivudine (3TC) and Efavirenz (EFV) (Chkhetiany et al., 2007, 2006; Kutsyna et al, 2003, 2005; Prihoda et al, 2006). Dzherelo has been found to decrease the incidence of opportunistic infections and reverse AIDS-associated wasting (Chkhetiany et al., 2007; Kutsyna et al., 2003,
2005).
Corresponding Author: A. Galyna Kutsyna, Luhansk Regional AIDS Center, Luhansk 91045, Ukraine
Dzherelo  has also been found to decrease the hepatotoxicity associated with ART (Chkhetiany et al., 2006, 2007; Kutsyna et al, 2003, 2005; Prihoda et al,
2006; Zaitseva, 2006).
Dzherelo was approved in 1997 by the Ministry of Health of Ukraine as immunomodulatory preparation, which so far has been used by over 150,000 individuals for various indications including chronic bacterial and viral infections such as TB and HIV, autoimmune diseases and malignancy. Dzherelo contains concentrated aqueous-alcohol extract from medicinal plants such as Aloe, Common knotgrass, Yarrow, Purple coneflower, St. John's Wort, Centaury, Snowball tree berries, Nettle, Dandelion, Sweet-sedge, Oregano, Marigold, Seabuckthorn fruit, Elecampane, Tormentil, Greater plantain, Wormwood, Siberian golden root, Cottonweed, Licorice, Fennel, Birch tree fungus, Thyme, Three-lobe Beggarticks, Sage, Dog rose fruit and Juniper fruit. Our study was aimed at evaluating the effect of Dzherelo on immune cell subsets and viral load among HIV patients treated with standard ART in comparison to a control population which received ART alone.
MATERIALS AND METHODS
Patients: The patients, aged 20-59 years, have been selected and divided into arms A and B, each consisting of 20 patients randomized by their disease progression. The average (median) age in arms A and B was 33.9 (31.5) and 32.8 (31) years. The proportion of males and females was 15/5 and 17/3 in arms A and B, respectively. Patients were in advanced clinical stage of HIV infection with average baseline CD4+ T-cell count below 200 cells/microliter. Another inclusion criterion was the lack of any form of anti-retroviral therapy prior to the trial. The diagnosis of HIV infection was established by standard ELISA test further confirmed by Western blot analysis. The participation in this trial was voluntary and patients were enrolled only after signing the written consent indicating that they were free to withdraw from the study at any time. The conduct of the trial was approved by the advisory board of the AIDS center.
Treatment regimens : None of the patients received anti-retroviral therapy prior to the trial. After initial screening, qualifying patients were randomly divided in two arms: Arm A was prescribed: zidovudine (AZT) at 300 mg doses twice-daily; lamivudine (3TC) 150 mg tablets twice-daily and Efavirenz (EFV) 600 mg dose once-daily. The arm B received, in addition to ART, twice per day dose of Dzherelo which was given as 50 drops diluted in 100 mL of water.
Immunophenotyping oflymphocyte subpopulations: The samples of peripheral blood of patients with HIV were analyzed using commercially available Clonospectr panel of monoclonal antibodies against surface antigens of lymphocytes (MedBioSpectr, Moscow, Russia). Assays were carried out at study entry and after 1 and 2 months on the therapy. The absolute and percent values of the following subpopulations were assessed in a blinded fashion by fluorescent microscopy: Total T lymphocytes (CD3+), helper T lymphocytes (CD3+CD4+) and cytotoxic T lymphocytes (CD3+CD8+). In addition the changes in the ratio between CD4 and CD8 cells were evaluated as a part of assessment of the immune status of patients. The samples of the blood from 19 healthy blood donors were analyzed as a reference for normal values.
PCR analysis: Stored frozen samples of plasma were processed in bulk by using commercially available PCR kit (AmpliSense HIV-1, Central Research Institute of Epidemiology, Moscow, Russia) designed for quantitative analysis of HIV-RNA copies. Tests were carried out at baseline and after two months of the therapy.
Statistical analysis: The obtained results were analyzed with the aid of statistical software STATMOST (Datamost, South Sandy, UT). The baseline cell numbers relative to 1st and 2nd months of follow-up were evaluated by paired Student t-test. The non-parametric values of viral load were analyzed by Wilcoxon signed-rank test. All statistical calculations were per intent-to-treat basis or the total number of available patients without subgrouping them into responders and non-responders. The resulting probability values were considered as significant at the cut-off levels of p« 0.05.
RESULTS
After one month on the therapy there was a clear distinction between recipients of ART alone and those who received ART along with the daily dose of Dzherelo . This disparity became even more evident at the end of 2nd month of therapy. The changes in viral load among HIV patients of both groups have also reached statistical significance. These findings are described in detail below.
CD3+ total T Lymphocytes: After one month on ART alone the absolute and percent (%) values of total CD3+ lymphocytes per microliter of blood have changed in a statistically discordant manner, i.e., 664 (36.5%) vs 743(38.4%) with p = 0.13 (p = 0.03), as analyzed by paired Student t-test. Similarly, at the end of the first month, in the group receiving Dzherelo there was a statistical discordance between absolute and percent CD3+ values: 595(34.2%) vs 664(40.5%); p = 0.14(p = 0.0003). After 2 months the number of total CD3+ lymphocytes increased further to 785 (43.9%) in arm B, i.e., p = 0.034 (p = 0.0002), whereas in the control it increased to 819 (39.8%) cells, with probability values p = 0.06 (p = 0.03) , respectively. The accrual in total lymphocytes from baseline to the end of follow-up, was 23.3 and 31.9% for absolute and 9 and 28.4% for relative numbers in control and Dzherelo arms, respectively (Fig. 1).
CD4+ T Lymphocytes: The trends similar to those of total CD3+ lymphocytes were observed when CD3+CD4+ lymphocyte subsets were analyzed. Significant changes were seen in ART alone arm after one month, i.e., 218(30.3%)-295(35.2%); p = 0.007 (p = 0.02). Similarly in Dzherelo arm the helper T-cell counts have risen in a significant manner from 184 (28.4%)-254 (34.8%) cells; p = 0.03 (p = 0.0002). At the end of 2nd month lymphocyte subsets have risen to 343 (35.7%) and 356 (38%) with probability values p = 0.002 (p = 0.01) and p = 0.004(p = 0.0005) for arms A and B , respectively. When study completion results of ART and Dzherelo recipients were calculated in terms of accrual in CD4+ lymphocytes relative to entry levels there was an increase of 57.3% (17.8%) and 93.5% (33.8%) in absolute and relative values.
CD8+ T Lymphocytes: The changes observed in CD3+CD8+ cytotoxic T-cell population are different from those seen with helper cells. In ART alone group absolute but not relative numbers of CD8+ cells increased in a significant manner from 155 (22.6%)-203(24%); p = 0.014 (p = 0.21), while in Dzherelo group the changes were insignificant in both categories, i.e., from 123 (19.8%)-152 (20.3%); p = 0.08 (p = 0.28). At the end of the 2nd month the CTL population in ART group was still above baseline, i.e., 222 cells (23.2%), an accrual that was statistically significant for absolute numbers p = 0.009 but not significant when relative numbers were evaluated (p = 0.39). Similarly, among Dzherelo recipients the 2nd month absolute but not relative numbers of CD8 cells have also increased in a significant manner, from baseline levels 123(19.8%)-185(19.7%), with P values being 0.013 and 0.39, respectively. When study completion results of ART and Dzherelo recipients were calculated in terms of accrual in CD8 + lymphocytes as compared to baseline levels there was an accrual corresponding to 43.2% (2.7%) and 50.4% (-0.5%) of absolute and relative values, respectively.
Fig. 1: Changes in absolute and relative numbers of T-lymphocyte subsets at 2 months post-therapy as expressed in percentage values relative to their respective baseline levels.
CD4/CD8 ratio: The differential changes in CD4 and CD8 lymphocyte numbers had affected the CD4/CD8 ratio in patients on ART alone regimen as early as one month after treatment initiation. Their ratio had increased from baseline value 1.418-1.532 but without reaching significance (p = 0.23). The CD4/CD8 ratio among Dzherelo recipients had increased from 1.495-1.671, which was also above the cut-off value (p = 0.09). The disparity between CD4 and CD8 lymphocytes had progressed further by the end of 2nd month. Among ART alone patients the ratio had increased to 1.613(p = 0.14), while in Dzherelo group the ratio had risen to 1.940 (p = 0.03). When study end results of ART and Dzherelo recipients were calculated in terms of relative accrual from baseline levels there was a gain of 13.8 and 29.3% , respectively.
Lymphocyte subsets in normal blood donors: Samples of the peripheral blood of 19 healthy individuals were analyzed to obtain the normal distribution values of peripheral blood subsets. The average number of absolute and relative (%) CD3 lymphocytes were 1,370±169 cells nL»1 (52.9±6.8). The values of CD4 and CD8 lymphocytes were 622±89 (35.9±4.3) and 349±42(19.9±2.1), respectively, with ratio being 1.76±0.19.
Viral load: The viral load, as measured by plasma RNA-PCR at baseline and at the end of 2nd month, decreased in ART group (1718-1419 copies mL*1, p = 0.008), as analyzed by Wilcoxon signed rank test. In Dzherelo arm the viral load decreased from 1793-1368 copies; p = 0.001). About three-quarters (14/19) of patients on ART alone had displayed the decrease in viral load, while the 18 out of 19 of patients on Dzherelo (95%) had a reduction in their number of viral copies (Table 1).
Table 1: Effect of 2-month ART without or with Dzherelo on HIV-RNA plasma levels
DISCUSSION
In the prior studies Dzherelo has been shown to influence positively CD3 and CD4 lymphocyte numbers (Chkhetiany et al, 2006, 2007; Kutsyna et al, 2003). Dzherelo reduced the incidence of opportunistic infections and reversed body weight loss associated with HIV (Chkhetiany et al, 2006, 2007; Kutsyna et al, 2003, 2005). It had also reduced the toxic side effects of ART, the hepatotoxicity in particular (Chkhetiany et al., 2006, 2007; Zaitseva, 2006). For example, elevated liver aminopeptidase ALT and AST levels caused by ART have been shown to return back to normal levels. However, these studies have not dealt with the effect of Dzherelo on other immune markers and viral load.
Our 2-month study conducted in prior anti-retroviral drug-naive population reveals that when Dzherelo is added to ART there are significant benefits associated with this intervention. In our hands Dzherelo appears to display the same effect as reported by independent investigators. Our results indicate that administration of Dzherelo along with ART can produce significant increase in total CD3+ lymphocytes, CD4+ helper cells, better
CD4/CD8 ratio, higher number of CD3+HLA-DR+ activated lymphocytes and NK cells. Dzherelo appears to increase absolute but not relative numbers of CD8+ T lymphocytes and reduce significantly CD20+ B lymphocyte subpopulation. Furthermore, Dzherelo appears to contribute to inhibitory effect of ART on viral replication resulting in statistically significant lower viral load in a higher proportion of patients (Table 1).
It is well established that elevated CD3 and CD4 counts and higher CD4/CD8 ratio are associated with better prognosis in patients with HIV (Bonger and Goebel, 1991). For this reason Dzherelo is likely to influence positively the outcome of treatment and disease progression in our study population. Similarly, the viral load is a predictor of HIV disease progression, its persistent elevation in HIV infected patients is indicative of poor prognosis (Arduino et al., 2001; Dybul et al., 2002). While, there were earlier indications that Dzherelo may reduce the viral burden, our study is the first to report this phenomenon in a systemic fashion. Despite the fact that the HIV RNA levels had decreased by less than a log the difference between baseline and outcome levels was highly significant (Table 1). It is likely that the observed effect on viral load is mediated by immune cells since Dzherelo does not have the direct effect on HIV replication (Chkhetiany et al., 2007).
Many studies have been conducted aimed at determining the phenotype of immune cells in HIV infection. While there is a consensus that the immune response plays a critical role in determining the clinical outcome much more has to be learned in order to have a clear picture of cellular events during the course of disease. The understanding of the immune mechanism controlling HIV may result in design of better vaccines and immunotherapies. Currently available anti-retroviral therapy is far from ideal, requiring multiple drugs to be taken in combination for the rest of life of a patient (Pokrovskii, 2001). The extended duration of therapy, coupled with the side effects, often results in poor patient adherence, treatment failure and the emergence of drug resistance with major social and economic implications. We believe that the immunotherapy is the indispensable part of therapeutic strategies against HIV. The development of novel immune-based therapies is an urgent objective for anti-HIV drug discovery. Many immune interventions are available against bacteria, protozoa, fungi and viruses (Ershov, 2003). While often effective the mechanism of many immunomodulators is poorly understood. This downside should be balanced against clinically confirmed benefits. Our study provides an early glimpse into the putative immune mechanism of Dzherelo, which has been successfully used as an immune adjunct to HIV therapy in Ukraine during last ten years (Chkhetiany et al, 2006, 2007; Kutsyna et al, 2003, 2005; Prihoda et al., 2006; Zaitseva, 2006) Additional studies need to be conducted to develop better understanding of Dzherelo  properties and to enlarge the current arsenal of HIV therapies.
ACKNOWLEDGEMENT
We thank all participants who volunteered in this study. The generosity of Ekomed in supplying Dzherelo  is appreciated very much. The tireless support of clinical staff and technicians who contributed to this study has been of tremendous help to bring this study to fruition. The discussion with other investigators of Dzherelo who shared their insight and provided helpful suggestions has guided our study and we are thankful to them all.
REFERENCES
Appay, V., F. Boutboul and B. Autran, 2003. The HIV infection and immune activation: To fight and burn. Curr. Infect. Dis. Rep., 7: 473-479.
Arduino, J.M., M.A. Fischl, K. Stanley, A.C. Collier and D. Spiegelman, 2001. Do HIV type 1 RNA levels provide additional prognostic value to CD4(+) T lymphocyte counts in patients with advanced HIV type 1 infection? AIDS Res. Hum. Retroviruses, 17: 1099-105.
Bogner, J.R. and F.D. Goebel, 1991. Lymphocyte subsets as surrogate markers in antiretroviral therapy. Infection, 19: 103-108.
Chehitiany, R., V. Pylipchuk, O. Argzanova, N. Prihoda, I. Vihrova, E. Zagaidanova and G. Kutsyna, 2006. Influence of an agent with immunomodulating activities, Dzherelo , on immune response and metabolic parameters in combination therapy with NRTI+NNRTI and as a monotherapy in untreated HIV-infected individuals. Probl. Ecol. Med. Gen. Clin. Immunol., 71-72: 307-318.
Chkhetiany, R., V. Pylipchuk, O. Argzanova, N. Prihoda, L. Vihrova, E. Zagaynova and G. Kutsyna, 2007. Comparative effect of an immunomodulator Immunoxel (Dzherelo) when used alone or in combination with antiretroviral therapy in drug-naive HIV infected individuals. Int. J. Biotechnol., 9: 267-276.
Dybul, M., A.S. Fauci, J.G. Bartlett, J.E. Kaplan and A.K. Pau, 2002. Panel on Clinical Practices for Treatment of HIV. Guidelines for using antiretroviral agents among HIV-infected adults and adolescents. Ann. Int. Med., 137: 381-433.
Ershov, F.I., 2003. Use of immunomodulators in viral infections. Antibiot Khimioter, 48: 27-32.
Kelly, J.A. and Y.A. Amirkhanian, 2003. The newest epidemic: A review of HIV/AIDS in Central and Eastern Europe. Int. J. STD AIDS, 14: 361-71.
Kutsyna, G., R. Chechitiany, I. Bascacov, E. Zagaydanova and I. Zaharova, 2005. Influence of a novel immunomodulator on the prevalence of new events of opportunistic infections in untreated HIV-infected individuals. 3rd European HIV Drug Resistance Workshop. Athens, Greece., 4-7. Abst 36.
Mocroft, A., A.N. Phillips, J. Gatell, B. Ledergerber, M. Fisher, N. Clumeck, M. Losso, A. Lazzarin, G. Fatkenheuer and J.D. Lundgren, 2007. EuroSIDA study group. Normalisation of CD4 counts in patients with HIV-1 infection and maximum virological suppression who are taking combination antiretroviral  therapy:  An  observational  cohort study. Lancet, 370: 407-13.
Pokrovskii, V.V., 2001. Treatment of HIV-infections: Success or crisis?. Ter. Arkh, 73: 52-54.
Prihoda, N.D., O.V. Arjanova, N.I. Sokolenko and L.A. Vihrova, 2006. Clinical efficacy of phyto preparation Dzherelo in patients with co-morbid pathology: Pulmonary tuberculosis in combination with HIV infection. Probl. Ecol. Med. Gen. Clin. Immunol., 71-72: 151-161.
Van der Werf, M.J., O.B. Yegorova, N. Chentsova, Y. Chechulin, E. Hasker, V.I. Petrenko, J. Veen and L.V. Turchenko, 2006. Tuberculosis-HIV co-infection in Kiev City, Ukraine. Emerg. Infect. Dis., 12: 766-768.
Zaitseva, S.I., 2006. Clinical efficacy of phytopreparation Dzherelo and its influence on the functional status of liver in patients with destructive forms of tuberculosis. Probl. Ecol. Med. Gen. Clin. Immunol., 71-72: 132-140.

Effect of Immunomodulating Adjuvant Dzherelo (Immunoxel) in HIV Infected Patients Receiving Standard Antiretroviral Therapy

Lyudmila G. Nikolaeva1, Tatiana V. Maystat1, Yuri L. Volyanskii2, Volodymyr S. Pylypchuk3, Valery M. Frolov and Galyna A. Kutsyna 'Kharkov Regional AIDS Prophylaxis and Prevention Center, Kharkov Medical Academy of Postgraduate Education, 6 Bor'by Street, Kharkov 61044, Ukraine 2I.I. Mechnikov Institute of Microbiology and Immunology, Kharkov 61057, Ukraine 3EkomedLLC., Prospect Pravdy 80-A, Kiev 04208, Ukraine 4Luhansk State Medical University and Regional AIDS Center, Luhansk 91045, Ukraine
Abstract: Open-label, matched-case, comparative trial was conducted in 40 HIV-infected patients to evaluate the adjunct effect of Dzherelo (Immunoxel) on immune and viral parameters. Arm A (n=20) received anti-retroviral therapy (ART) consisting of zidovudine, lamivudine, and efavirenz and arm B (n=20) received ART with Dzherelo. After 2 months total T-lymphocytes increased in ART recipients from 664 to 819 cells/|il (P=0.06), whereas in Dzherelo recipients they rose from 595 to 785(P=0.03). The CD4 T-cells expanded by 57.3% (218 to 343; P=0.002) in the ART arm and by 93.5% (184 to 356; P=0.004) in the Dzherelo arm. The accrual in absolute and relative number of CD8+ lymphocytes in ART and in the Dzherelo recipients was 43.2% (2.7%) and 50.4% (-0.5%) respectively. The CD4/CD8 ratio in Dzherelo recipients in­creased from 1.495 to 1.940 (P=0.03) but insignificant in the control: 1.418 to 1.613 (P=0.14). Activated CD3+ HLA-DR+ T-cells increased from 209 to 264 (P=0.02) and from 161 to 348 (P=0.0007) in ART and Dzherelo recipients respec­tively. No changes in CD20+ B-lymphocytes were seen in the control, but in Dzherelo patients they declined from 509 to 333 (P=0.00008). The proportion of CD3- CD16+CD56+ NK cells was not affected by ART but addition of Dzhereloraised NK cells from 11.2% to 17.1% (P=0.0001). About three-quarters (14/19) of patients on ART displayed decrease in viral load (1718 to 1419 copies/ml; P=0.008), while 95% of patients on Dzherelo had a decrease (1793 to 1368; P=0.001). Dzherelo has a favorable effect on the immune status and viral burden when given as an immunomodulating adjunct to
ART.
Keywords: AIDS, antiviral, HAART, herbal, immunotherapy, phytotherapy.
BACKGROUND
Antiretroviral drug resistance, drug toxicity and adher­ence are major concerns in clinical management of HIV in­fection [1]. The combination of antiviral drugs, such as highly active antiretroviral therapy (HAART), may prevent HIV from mutating and spreading, allowing patients to re­build their immune system to the same levels as in normal individuals [2]. On the other hand, the immune activation caused by enhanced immune reaction to HIV is now recog­nized as a cause of depletion of CD4 T-cells and resulting immunodeficiency [3]. These problems are a driving force for research on new therapies that can provide suitable solu­tions. The most optimal therapeutic solution is an effective and safe immunotherapy that could regulate the immune response in a manner favorable to the host. There are many types of immune modulators that have been used clinically for viral infections, but for HIV the choice of immune inter­ventions is limited [4].
*Address correspondence to this author at the Department of Infectious Diseases and Epidemiology, Luhansk State Medical University, Luhansk 91045, Ukraine; Tel/Fax: +38-057-392-09-08; E-mail: kutsyna@list.ru
Ukraine has the highest prevalence of HIV infection in Eastern Europe [5, 6]. Oral immunomodulator Dzherelo is used in Ukraine for the management of HIV infections, in­cluding patients co-infected with TB [7-12]. Clinical studies have indicated that Dzherelo can significantly increase CD3 and CD4 T-lymphocyte populations and helps to achieve better clinical response when combined with standard anti-retroviral therapy (ART) consisting of zidovudine (AZT); lamivudine (3TC), and efavirenz (EFV) [7-9]. Dzherelo has been found to decrease the incidence of opportunistic infec­tions and reverse AIDS-associated wasting [7-9]. Dzherelo has also been found to decrease the hepatotoxicity associated with ART [7-9, 12].
Dzherelo was approved in 1997 by the Ministry of Health of Ukraine as an immunomodulating supplement, which so far has been used by over 500,000 individuals for various indications including chronic bacterial and viral infections such as TB and HIV, autoimmune diseases, and malignancy [7]. Dzherelo contains concentrated aqueous-alcohol extract from medicinal plants such as Aloe, Common knotgrass, Yarrow, Purple coneflower, Tutsan, Centaury, Snowball tree berries, Nettle, Dandelion, Sweet-sedge, Oregano, Marigold, Seabuckthorn fruit, Elecampane, Tormentil, Greater plantain, Wormwood, Siberian golden root, Cottonweed, Licorice, Fennel, Birch tree fungus, Thyme, Three-lobe Beggarticks, Sage, Dog rose fruit, and Juniper fruit. Our study was aimed at evaluating the effect of Dzherelo on immune cell subsets and viral load among HIV patients treated with standard ART in comparison to a control population which received ART alone.
MATERIALS AND METHODS
Patients
The design of this study is matched case comparative study. The patients, aged 20-59 years, have been selected and divided into arms A (ART alone) and B (Dzherelo added), each consisting of 20 patients matched by their aver­age initial CD4 counts, gender ratio and age. The average (median) age in arms A and B was 33.9 (31.5) and 32.8 (31) years. The proportion of males and females was 15/5 and 17/3 in arms A and B respectively. Patients were in ad­vanced clinical stage of HIV infection with average baseline CD4+ T-cell count being below 200 cells/microliter. Another inclusion criterion was the lack of any form of anti-retroviral therapy prior to the trial. The diagnosis of HIV infection was established by standard ELISA test further confirmed by Western blot analysis. The participation in this trial was vol­untary and patients were enrolled only after signing the writ­ten consent indicating that they were free to withdraw from the study at any time. The conduct of the trial was approved by the Ethics board of the AIDS center in compliance with the Helsinki declaration.
Treatment Regimens
None of the patients received anti-retroviral therapy prior to the trial. After initial screening, qualifying patients were randomly divided in two arms: arm A was prescribed: zi-dovudine (AZT) at 300 mg doses twice-daily; lamivudine (3TC) 150 mg tablets twice-daily, and efavirenz (EFV) 600 mg dose once-daily. The arm B received, in addition to the same three-drug ART, twice per day dose of Dzherelo which was given as 50 drops diluted in 100 ml of water.
Immunophenotyping of Lymphocyte Subpopulations
The samples of peripheral blood of patients with HIV were analyzed using commercially available Clonospectr panel of monoclonal antibodies against surface antigens of lymphocytes (MedBioSpectr, Moscow, Russia). Assays were carried out at study entry and after 1 and 2 months on the therapy. The absolute and percent values of the following subpopulations were assessed in a blinded fashion by fluo­rescent microscopy: total T lymphocytes (CD3+); helper T lymphocytes (CD3+CD4+); cytotoxic T lymphocytes (CD3+CD8+); B lymphocytes (CD20+); natural killer or NK cells (CD3-CD16+CD56+); and activated T lymphocytes
(CD3+HLA-DR+). In addition the changes in the ratio be­tween CD4 and CD8 cells were evaluated as a part of as­sessment of the immune status of patients. The samples of the blood from 19 healthy blood donors were analyzed as a reference for normal values.
PCR Analysis
Stored frozen samples of plasma were processed in bulk by using commercially available PCR kit (AmpliSense HIV-1, Central Research Institute of Epidemiology, Mos­cow, Russia) designed for quantitative analysis of HIV-RNA copies. Tests were carried out at baseline and after two months of the therapy.
Statistical Analysis
The obtained results were analyzed with the aid of statis­tical software STATMOST (Datamost, South Sandy, UT). The baseline cell numbers relative to 1st and 2nd months of follow-up were evaluated by paired Student t-test. The non-parametric values such as viral load were analyzed by Wil-coxon signed-rank test. All statistical calculations were per intent-to-treat basis or the total number of available patients without subgrouping them into responders and non-responders. The resulting probability values were considered as significant at the cut-off levels of P<0.05.
RESULTS
After one month on the therapy there was a clear distinc­tion between recipients of ART alone and those who re­ceived ART along with the daily dose of Dzherelo. This dis­parity became even more evident at the end of 2nd month of therapy. Some but not all immune markers of lymphocyte subsets were impacted in a statistically significant manner. The changes in viral load among HIV patients of both groups also reached statistical significance. These findings are de­scribed in detail below.
CD3+ Total T Lymphocytes
After one month on ART alone the absolute and percent (%) values of total CD3+ lymphocytes per microliter of blood changed in a statistically discordant manner i.e. from a baseline 664(36.5%) to 743(38.4%) with P=0.13(P=0.03), as analyzed by paired Student t-test. Similarly, after one month in the group receiving Dzherelo there was a statistical dis­cordance between absolute and percent CD3+ values: from 595(34.2%) to 664(40.5%); P=0.14(P=0.0003). After 2 months the number of total CD3+ lymphocytes increased further to 785 (43.9%) in arm B (Dzherelo) i.e. P=0.034 (P=0.0002), whereas in the control it increased to 819 (39.8%) cells, with probability values P=0.06 (P=0.03) re­spectively. The accrual in total lymphocytes from baseline to the end of follow-up, was 23.3% and 31.9% for absolute and 9% and 28.4% for relative numbers in control and Dzherelo arms respectively (Fig. 1).
CD4+ T Lymphocytes
The trends similar to those of total CD3+ lymphocytes were observed when CD3+CD4+ lymphocyte subsets were analyzed. Significant changes were seen in the ART alone arm after one month i.e.  218(30.3%) to 295(35.2%);
P=0.007(P=0.02). Similarly in the Dzherelo arm the helper T-cell counts rose in a significant manner from 184(28.4%) to 254(34.8%) cells; P=0.03(P=0.0002). At the end of 2nd month lymphocyte subsets rose to 343(35.7%) and 356(38%) with probability values P=0.002(P=0.01) and P=0.004 (P=0.0005) for arms A and B respectively. When study completion results of ART and Dzherelo recipients were calculated in terms of accrual in CD4+ lymphocytes relative to entry levels there was an increase of 57.3%(17.8%) and 93.5%(33.8%) in absolute and relative values.
 
Fig.(1). Changes in absolute and relative numbers of peripheral blood cell subpopulations at 2 months post-therapy as expressed in percent­age values (Y-axis) relative to their respective baseline levels.
 CD8+ T Lymphocytes
The changes observed in CD3+CD8+ cytotoxic T-cell population are different from those seen with helper cells. In the ART alone group, absolute but not relative numbers of CD8+ cells increased in a significant manner from 155(22.6%) to 203(24%); P=0.014 (P=0.21), while in the Dzherelo group the changes were insignificant in both cate­gories i.e. from 123(19.8%) to 152(20.3%); P=0.08(P=0.28).
At the end of the 2nd month the CTL population in the ART
group was still above baseline i.e. 222 cells (23.2%), an ac­crual that was statistically significant for absolute numbers P=0.009 but not significant when relative numbers were evaluated (P=0.39). Similarly, among Dzherelo recipients the 2nd month absolute but not relative numbers of CD8 cells also increased in a significant manner, from baseline levels 123(19.8%) to 185(19.7%), with P values being 0.013 and 0.39 respectively. When study completion results of ART and Dzherelo recipients were calculated in terms of accrual in CD8+ lymphocytes as compared to baseline levels there was an accrual corresponding to 43.2%(2.7%) and 50.4%(-0.5%) of absolute and relative values respectively.
CD4/CD8 Ratio
The differential changes in CD4 and CD8 lymphocyte numbers affected the CD4/CD8 ratio in patients on ART alone regimen as early as one month after treatment initia­tion. The ratio increased from baseline value 1.418 to 1.532 but without reaching significance (P=0.23). The CD4/CD8 ratio among Dzherelo recipients increased from 1.495 to 1.671, which was also above the cut-off value (P=0.09). The disparity between CD4 and CD8 lymphocytes had pro­gressed further by the end of 2nd month. Among the ART alone patients the ratio increased to 1.613(P=0.14), while in Dzherelo group the ratio rose to 1.940 (P=0.03). When study end results of ART and Dzherelo recipients were calculated in terms of relative accrual from baseline levels there was a gain of 13.8% and 29.3% respectively.
 CD3+HLA-DR+ Activated Lymphocytes
The absolute and percent numbers of activated CD3+HLA-DR+ T cells increased steadily in ART group from 209 (24.7%) to 231 (26.6%), and then to 264 (27.8%) with P=0.13(P=0.04) and P=0.02(P=0.02) at the end of first and second months respectively. In Dzherelo recipients this subpopulation increased at higher incremental rate from 161(25.9%) to 247(35.1%) and then to 348(36.5%) with P=0.01(P=0.0004) and P=0.0007(P=0.0003) respectively. The relative accrual in absolute and relative numbers in ART group was 26.3%(12.6%) at the end of the 2nd month. During the same time period the patients who received Dzherelo experienced 216%(40.9%) increase over baseline in absolute and relative numbers respectively.
CD20+ B Lymphocytes
In patients who received ART alone B-cell counts moved up slightly but differences were not statistically significant at either time point. At the end of the 1st month they rose from 522 (28.3%) to 526 (28.2%) cells P=0.43 (P=0.37) and at study conclusion they reached 542 (27.3%); P=0.47 (P=0.22). However, patients who received Dzherelo dis­played a significant decrease in CD20+ lymphocyte num­bers. The entry value of 509 (31%) cells went down to 367 (24.9%) at the 1st month and then to 333 (23.3%) toward end of the study with probability values P=0.003 (P=0.0002) and P=0.00008 (P=0.00002) respectively. At this time point the number of CD20-expressing cells became indistinguishable from the value of normal donors i.e. 337 cells (22.2%).
CD3-CD16+CD56+ NK Cells
The relative numbers of NK cells identified as CD3-negative but CD16+CD56+ population were not significantly affected by ART alone therapy: from baseline level of 19.3% they rose to 21.2% (P=0.17), and then to 22.3% at study conclusion (P=0.12). The addition of daily dose of Dzherelo, however, had a strong effect on gain of NK cells. The patients who started with an average 11.2% NK cells had their numbers increased to 13.7% (P=0.004), which further increased to 17.1% (P=0.0001). Thus, at the end of 2-month of therapy, the patients on Dzherelo had experienced 52.7% increase in NK cells as compared to baseline levels.
Lymphocyte Subsets in Normal Blood Donors
Samples of the peripheral blood of 19 healthy individuals were analyzed to obtain the normal distribution values of peripheral blood subsets. The average number of absolute and relative (%) CD3 lymphocytes were 1,370±169 cells/xl (52.9±6.8). The values of CD4 and CD8 lymphocytes were 622±89 (35.9±4.3) and 349±42 (19.9±2.1) respectively, with the ratio being 1.76±0.19. The HLA-DR+ expressing acti­vated T-cells represented 585±131 cells (35.2±3.5). The ab­solute and percent figures of CD20+ B-lymphocytes per ul of blood were 337±52 (22.2±4.3). The percentage of NK cells was 12.2±2.9%.
Viral Load
The viral load, as measured by plasma RNA-PCR at baseline and at the end of 2nd month, decreased in the ART group (1718 to 1419 copies/ml; P=0.008), as analyzed by Wilcoxon signed rank test. In the Dzherelo arm the viral load decreased from 1793 to 1368 copies; P=0.001). About three-quarters (14/19) of patients on ART alone displayed the de­crease in viral load, while 18 out of 19 of patients on Dzherelo (95%) had a reduction in their number of viral cop­ies (Table 1).
Table 1.  Effect of 2-Month ART without or with Dzherelo on HIV-RNA Plasma Levels

DISCUSSION
In prior studies Dzherelo has been shown to influence positively CD3 and CD4 lymphocyte numbers [7-9]. Dzherelo reduced the incidence of opportunistic infections and reversed body weight loss associated with HIV [7, 10]. It has also been shown to reduce the toxic side effects of ART, hepatotoxicity in particular [7, 9, 12]. For example, elevated liver aminopeptidase ALT and AST levels caused by ART have been shown to return back to normal levels when Dzherelo is used. However, these studies have not dealt with the effect of Dzherelo on other immune markers and viral load.
Our 2-month study conducted in prior anti-retroviral drug-naive population reveals that when Dzherelo is added to ART there are significant benefits associated with this intervention. In our hands Dzherelo appears to display the same effect as reported by independent investigators. Our results indicate that administration of Dzherelo along with ART can produce significant increase in total CD3+ lym­phocytes, CD4+ helper cells, better CD4/CD8 ratio, higher number of CD3+HLA-DR+ activated lymphocytes, and NK cells. Dzherelo appears to increase absolute but not relative numbers of CD8+ T lymphocytes and reduce significantly CD20+ B lymphocyte subpopulation. Furthermore, Dzherelo appears to contribute to the inhibitory effect of ART on viral replication resulting in statistically significant lower viral load in a higher proportion of patients (Table 1). These ef­fects could not be attributed to heterogeneity of patient popu­lations in two tightly matched treatment groups in which none of the initial characteristics in terms of age, gender, disease stage and cell counts were statistically different at baseline.
It is well established that elevated CD3 and CD4 counts and higher CD4/CD8 ratio are associated with better progno­sis in patients with HIV [13]. For this reason Dzherelo is likely to influence positively the outcome of treatment and disease progression in our study population. Similarly, viral load is a predictor of HIV disease progression, its persistent elevation in HIV infected patients being indicative of poor prognosis [14, 15]. While there were earlier indications that Dzherelo may reduce the viral burden, our study is the first to report this phenomenon in a systematic fashion. Despite the fact that the HIV RNA levels had decreased by less than a log the difference between baseline and outcome levels was highly significant (Table 1). While baseline and out­come viral burden appeared unusually low, this may have been due to the PCR kit we have used in this study; since we have seen similar low numbers in our study dealing with TB/HIV dually infected patients [10]. Nevertheless, it is likely that the observed effect on viral load is mediated by immune cells since Dzherelo does not have a direct effect on HIV replication [7].
Our study reveals that at the end of study the patients on Dzherelo had absolute and relative numbers of CD3+HLA-DR+ cells equal to 348 (36.5%) or 216% and 40.9% more compared to baseline, whereas patients on ART had their numbers increased to a maximum 26.3%. Compared to nor­mal blood donors the relative numbers in Dzherelo group were comparable but absolute values were lower i.e. 35.2% and 585 respectively. It has been shown that HIV+ patients usually have significantly higher HLA-DR expressing cells, which may negatively affect T cell immune responses and facilitate the disease progression [16]. Others suggested that the increase in HLA-DR-reactive T lymphocytes was not predictive of clinical outcome [17]. We do not know to which subpopulation of T lymphocytes the CD3+HLA-DR+ activated cells belong. Judging from relative changes in CD4 and CD8 subsets it is possible that the activation marker is expressed on CD4 cells. However, due to limitations of the commercially available panel of antibodies preventing simul­taneous staining with multiple fluorescent markers, we have not been able to verify this possibility. This needs to be as­certained in future studies.
Although a great deal of information is available about the role of T lymphocytes in the immune response against HIV comparatively little is understood regarding the in volvement of B lymphocytes. Our results show very little variation in CD20+ B-cell numbers in the ART-treated arm but a highly significant decrease in Dzherelo recipients to levels that were identical to those seen in normal individuals i.e. 333 (23.3%) vs 337 (22.2%). Contrarily, the absolute and relative numbers of B-cells were more than double in ART recipients. We do not know what the significance of this observation is. It has been reported that the expression of CD20 is consistently higher when B cells from HIV-infected individuals are compared with those from uninfected sub­jects [18]. Thus, we can only assert that Dzherelo is highly effective in restoring the normal balance of CD20 expressing B-cells. We have observed the drastic difference between two arms in relative number of NK cells after 2 months of ther­apy. When compared to the entry levels the patients on Dzherelo had 52.7% more of NK cells whereas the popula­tion of these cells had increased by only 15.5% in ART alone group. The role of NK cells in HIV remains unclear. While some indicated that higher number of NK cells is critical for controlling HIV, others indicated that the quality but not the number of cells is critical in defending the host against HIV infection [19, 20]. This discrepancy probably stems from differences in the design of studies some of which were based on measurement of functional activity but others were based on the enumeration of cells at various stages of the disease. Since we have not measured the functional activity of NK cells we do not know what is the significance of this phenomenon in regard to the immunopathogenesis of HIV. Based on the evidence that Dzherelo is highly effective as an immune adjuvant to ART we can only speculate that in our case the increase in NK numbers might be beneficial to the host.
Many studies have been conducted aimed at determining the phenotype of immune cells in HIV infection. While there is a consensus that the immune response plays a critical role in determining the clinical outcome much more has to be learned in order to have a clear picture of cellular events during the course of disease. The understanding of the im­mune mechanism controlling HIV may result in design of better vaccines and immunotherapies. Therefore, high prior­ity should be given to efforts of prevention and treatment of infectious complications in these patients.
Currently available chemotherapy for the treatment of HIV is not ideal, requiring multiple drugs to be taken in combination for the rest of life of a patient [1]. The extended duration of therapy, coupled with the side effects, often re­sults in poor patient adherence, treatment failure, and the emergence of drug resistance with major social and eco­nomic implications. We believe that immunotherapy is an indispensable part of therapeutic strategies against HIV. The development of novel immune-based therapies is an urgent objective for anti-HIV drug discovery. Many immune inter­ventions are available against bacteria, protozoa, fungi and viruses. While often effective the mechanism of many im­munomodulators is poorly understood. This downside should be balanced against clinically confirmed benefits. Our study provides an early glimpse into the putative immune mecha­nism of Dzherelo, which has been successfully used as an immune adjunct to HIV therapy in Ukraine during last ten years [7-12]. Additional studies are needed to develop better understanding of Dzherelo's properties and to supplement the current arsenal of HIV therapies.
ACKNOWLEDGEMENTS
We thank all participants who volunteered in this study. The generosity of Ekomed in supplying Dzherelo is appreci­ated very much. The tireless support of clinical staff and technicians who contributed to this study has been of tre­mendous help to bring this study to fruition. The discussion with other investigators of Dzherelo who shared their insight and provided helpful suggestions has guided our study and we are thankful to them all. This work was presented in part at the Keystone Symposia on HIV Pathogenesis and HIV Vaccines, March 27 - Apr 1, 2008, Banff, Alberta, Canada, through a grant from Bill and Melinda Gates Foundation's Global Health Travel Award, which is gratefully acknowl­edged.
CONFLICT OF INTEREST
All authors, except Volodymyr Pylypchuk, declare no conflict of interest. Pylypchuk is the inventor of Dzherelo (Immunoxel) and Director of Ekomed company.
ABBREVIATIONS
ART = Antiretroviral therapy
EFV = Efavirenz
HAART = Highly active antiretroviral therapy
HLA = Human leukocyte antigen
3TC = Lamivudine
NK = Natural killer
PCR = Polymerase chain reaction
RNA = Ribonucleic acid
TB = Tuberculosis
AZT = Zidovudine
REFERENCES
[1] Pokrovskii VV. Treatment of HIV-infections: success or crisis? Ter Arkh 2001; 73: 52-4.
[2] Mocroft A, Phillips AN, Gatell J, et al. EuroSIDA study group. Normalisation of CD4 counts in patients with HIV-1 infection and maximum virological suppression who are taking combination antiretroviral therapy: an observational cohort study. Lancet 2007; 370: 407-13.
[3] Appay V, Boutboul F, Autran B. The HIV infection and immune activation: "to fight and burn". Curr Infect Dis Rep 2005; 7: 473-9.
4] Ershov FI. Use of immunomodulators in viral infections. Antibiot Khimioter 2003; 48: 27-32. [5]       Kelly JA, Amirkhanian YA. The newest epidemic: a review of HIV/AIDS in Central and Eastern Europe. Int J STD AIDS 2003; 14: 361-71.
[6] van der Werf MJ, Yegorova OB, Chentsova N, et al. Tuberculosis-HIV co-infection in Kiev City, Ukraine. Emerg Infect Dis 2006; 12: 766-8.
[7] Chkhetiany R, Pylipchuk V, Argzanova O, et al. Comparative effect of an immunomodulator Immunoxel (Dzherelo) when used alone or in combination with antiretroviral therapy in drug-naïve HIV infected individuals. Int J Biotechnol 2007; 9: 267-76.
[8] Kutsyna G, Chechitiany R, Bascacov I, Zagaydanova E, Zaharova I. Influence of a novel immunomodulator on the prevalence of new events of opportunistic infections in untreated HIV-infected indi­viduals. 3rd European HIV Drug Resistance Workshop. Athens, Greece. April 4-7, 2005. Abstract #36.
[9] Prihoda ND, Arjanova OV, Yurchenko LV, et al. Adjuvant immu-notherapy of tuberculosis in drug-resistant TB and TB/HIV co-infected patients. Int J Biomed Pharm Sci 2008; 2: 59-64.
[10] Nikolaeva LG, Maystat TV, Pylypchuk VS, Volyanskii YuL, Masyuk LA, Kutsyna GA. Effect of oral immunomodulator Dzherelo (Immunoxel) in TB/HIV co-infected patients receiving anti-tuberculosis therapy under DOTS. Int Immunopharmacol 2008; 8: 845-51.
[11] Nikolaeva LG, Maystat TV, Pylypchuk VS, Volyanskii YL, Masyuk LA, Kutsyna GA. Changes in CD4+ T-cells and HIV RNA resulting from combination of anti-TB therapy with Dzherelo in TB/HIV dually infected patients. Drug Des Dev Ther 2008; 2: 87­93.
[12] Zaitseva SI. Clinical efficacy of phytopreparation Dzherelo and its influence on the functional status of liver in patients with destruc­tive forms of tuberculosis. Probl Ecol Med Gen Clin Immunol 2006; 71-72: 132-40.
[13] Bogner JR, Goebel FD. Lymphocyte subsets as surrogate markers in antiretroviral therapy. Infection 1991; 19: S103-8.
[14] Arduino JM, Fischl MA, Stanley K, Collier AC, Spiegelman D. Do HIV type 1 RNA levels provide additional prognostic value to CD4(+) T lymphocyte counts in patients with advanced HIV type 1 infection? AIDS Res Hum Retroviruses 2001; 17: 1099-105.
[15] Dybul M, Fauci AS, Bartlett JG, Kaplan JE, Pau AK. Panel on clinical practices for treatment of HIV. Guidelines for using antiret-roviral agents among HIV-infected adults and adolescents. Ann In­tern Med 2002; 137: 381-433.
[16] Mahalingam M, Peakman M, Davies ET, Pozniak A, McManus TJ, Vergani D. T cell activation and disease severity in HIV infection. Clin Exp Immunol 1993; 93: 337-43.
[17] Levacher M, Tallet S, Dazza MC, Dournon E, Rouveix B, Pocidalo JJ. Comparison with CD4+ lymphocyte count in non-progressors and progressors towards AIDS. Clin Exp Immunol 1990; 81: 177­82.
[18] Ginaldi L, De Martinis M, D'Ostilio A, Marini L, Quaglino D. Changes in antigen expression on B lymphocytes during HIV in­fection. Pathobiology 1998; 66: 17-23.
[19] Bruunsgaard H, Pedersen C, Skinhoj P, Pedersen BK. Clinical progression of HIV infection: role of NK cells. Scand J Immunol 1997; 46: 91-5.
[20] Alter G, Malenfant JM, Delabre RM, et al. Increased natural killer cell activity in viremic HIV-1 infection. J Immunol 2004; 173: 5305-11.
Received: February 19, 2009 Revised: March 19, 2009 Accepted: March 26, 2009
© Nikolaeva et al.; Licensee Bentham Open.
This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http: //creativecommons.org/licenses/by-nc/ 3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

New therapeutic option  for decreasing ART-related toxicity in HIV/HCV    co-infected  atients. 

 

                                                             

            

V Frolov. V.Pylypchuk. G.Kutsyna. R. Chehiliany. L Vihrova. E. Zagaydaiova O. Ofdzanova. L. Urchenko State Medical University. Luhansk . Ukraine Regional AIDS- center. Luhansk , Ukraine

 

MORE INFORMATION:

Galina Kulsyna

e-mail:galina@krem.Ig.ua

 htth-y/www.ekomed.com.ua e-mail: ekomed@itpak.com

 

 

 

 

 

Background

Hepatic toxicity greatly complicates the manage­ment of HIV treatment - experienced patients. In several large trials of HIV/HCV co-infected pa­tients, more participants stopped their assigned therapy due to toxicity than virological failure. In the Ukraine, most HIV -infected patients have few antiretroviral options due to limited resources. Significant metabolic disorders and underlying hepatic injury caused by chronic alcohol, drug ad­diction and hepatitis co-infection are common. Preliminary results of the immunomodulating agent dzherelo suggested hepatic anti-inflamma­tory activity. We hypothesized that the addition of dzherelo may attenuate hepatic toxicity in pa­tients receiving antiretroviral therapy (ART).
Freaquently meet features in the HIV/HCV -infected individuals in east Ukraine:
- drug addiction and alcohol dependency
- poor diet
- bad conditions of life
- financial limited for HCV therapies(interferon and antiviral).

Method

Now we report the 32 - week results of an ongo­ing study initiated March 2005. 35 HIV/HCV co-infected patients suffering from alcohol abuse or/and drug addict were enrolled. ART including AZT/3TC/NVP was initiated. Dzherelo was added to the therapy a median of 8-10 weeks after start­ing ART. ART- related hepatotoxicity was defined as an increase in ALT/AST levels up to five times the upper limit of normal, or 3,5 fold increase if baseline level was abnormal.

Summary of patients characteristic at base­line intention to treat population.

 Result

Evaluation of IFT under AZT/3TC/NVP ther­apy regime in continue first 8 weeks.
mean                                     mean values
baseline values                    at week 8
ALT-62 U/L             <=>            109 U/L
AST- 49 U/L           =>             47 U/L
ART- related hepatotoxicity
In continuer first 8 week therapy 6/35 (18 %) pa­tients was with protocol-defined ART related hepatotoxicity. Among them:
-3 died
-2 need ART interrupted
-1 refused from ART due to side effect (fatigue, vomiting, gastrointestinal disorders).
What therapy strategies need in this situation ?
Exist in study cohort of significant metabolic disorders and underlying myltifactorial liver injury caused by chronic alcohol, drug addiction and not antiviral treated hepatitis co-infection not give the optimism that new (change) therapy regime will be successful.


Preliminary results of the immunomodulating agent dzherelo suggested hepatic anti-inflammatory activity. We hypothesized that the addition of dzherelo may at­tenuate hepatic toxicity in patients receiving antiretro­viral therapy.
Immunomodulator Dzherelo was added to the therapy a median of 8-10 weeks after starting ART
  • For prevention further elevating of LFT under ART
  • For facilitate of immune responses to HIV/HCV- inf.:
 - recruitment of immune cells
- induct of potent cytokines secretion (INFs, lis)
                                                   
 acceleration the phase immune control under HCV infection

Change the LFT under two consecutive therapy regimes

No new cases of hepatotoxicity were reported after starting dzhere­lo, and no clinical signs ot hepatic inflammation were seen.

Repopulation of the CD4 cells count was corre­lated with normalization of LFT.

 Hypothetical mechanism of decreasing the baseline evaluate ALT in HIV/HCV infected individuals after added dzherelo to ART

 Hypothetical mechanism of decreasing the ART-related evaluate ALT in HIV/HCV infec­tion under AZT/3TC/NVP +dzherelo therapy regime 
• HCV- infection associate with increasing toxicity of HAART 
• Shown decreasing ALT values to normal range can caused the suppression of HCV replication
    inflammatory process in liver
the basis for decreasing hepatotoxicity of HAART

Conclution

This and previous clinical trials results suggest a beneficial effect of dzherlo as a potential immu-noenhancer in HIV infected patients. Also Dzherelo appeared to attenuate hepatotoxicity from ART ther­apy (NRTI + NNRTI) in population with underlying liver pathology. This may lead to improved hepato­toxicity and reduced toxicity related therapy inter­ruptions.
The new options for successful HIV Regimes -using immunomodulate agent dzherelo In Combination with ART for: 
• Decreasing ART- related toxicity in HIV/HCV co-infected patients 
• Restoring weak and transient Immune Responses to HIV/HCV in chronic HIV/HCV co-infection 
• Accelerating CD4 Reconstitution under ART - Restoring CD4 cell numbers in failures of immune reconstitution with ART -CD4 reconstitution in late AIDS
• Decrease  the risk of prevalence immune resto­
ration disease after ART

Reference

1. Lyahovaya T. Influence of Immunomodulate agenl of dzherelo lo the synthesis of serum interferon in the patients with chronical HCV-infection. )// Problems of ecological and medical genetics and clinical immunology. - 2005. -1* 5(68) - P.74-81. 
2.KutsinaG.A.. Chetiani R.8.. ZagaidanovaY G Effects of immuno­modulator  Dzherelo on Immune signs among HIV-infectedlndinlduals // Problems of ecological and medical genetics and immunology- 2003. -1*7(53) -P.51-60.
3.Chhetianl R.B.. Influence of plant derivative drug Dzherelo on circu­lating immune complexes and their molecular content among patients with chronical persistant infection of mixed genesis (Epstain-Barr and herpetic)// Problems of ecological and medical genetics and clinical im­munology. - 2004. - № 61. - P.130-134.

 

Comparative effect of an immunomodulator Immunoxel (Dzherelo™) when used alone or in combination with antiretroviral therapy in drug-naive HIV-infected individuals.

 Revaz Chkhetiany                                                          
Regional AIDS Center, Luhansk, Ukraine                  
 Vladimir Pylipchuk                                                            
EKOMED Company Ltd., Kiev, Ukraine                                 
 Olga Argzanova and Nathalia Prihoda
Tuberculosis Hospital, Lisichansk, Ukraine
 Ludmila Vihrova
Regional Hospital, Lisichansk, Ukraine
 Elena Zagaynova and Galina Kutsyna*
Luhansk Regional AIDS Center, 50-years of Defense of Luhansk Street, Luhansk 91045, Ukraine Fax: +00380-6454-347-106
Corresponding author

Abstract: Immunomodulating agent Immunoxel (Dzherelo) has been evaluated in 70 HIV-positive individuals divided into three arms: first, control arm, received standard antiretroviral therapy zidovudine/lamivudine/efavirenz (AZT/3TC/EFV); second-AZT/3TC/EFV+Immunoxel and third, Immunoxel alone, given orally, twice daily. At 32 weeks of follow-up CD4 cell counts increased in all arms, reaching + 102, +190 and +175 cells/mm', respectively. The proportion of patients who experienced adverse events attributable to study medication was 65%, 24% and 5%. Immunoxel attenuated hepatic toxicity in patients receiving ART as determined by liver function test. Baseline values for ALT aminotransferase were 36, 62 and 72 U/L, which at study conclusion have Copyright © XXXX Inderscience Enterprises Ltd. R. Chechitiany et al. risen to 78 U/L in arm A, but declined to 38 and 31 U/L in arms B and C. Immunoxel also reversed AIDS-associated wasting. The average weight gain was 1.4, 6.9 and 5.1kg. The results indicate that Immunoxel is safe and exerts beneficial effect in AIDS patients.

Keywords: Author please provide keywords.

Reference to this paper should be made as follows: Chkhetiany, R., Pylipchuk, V., Argzanova, O., Prihoda, N., Vihrova, L., Zagaynova, E. and Kutsyna, G. (XXXX) 'Comparative effect of an immunomodulator Immunoxel (Dzherelo™) when used alone or in combination with antiretroviral therapy in drug-naive HIV-infected individuals', Int. J. Biotechnology, Vol. X, No. Y, pp.XXX-XXX.

Biographical notes: Revaz Chkhetiany is the Head of the Regional AIDS Center, Luhansk, Ukraine, his area of expertise is viral diseases.
Vladimir Pylipchuk, PhD is the Head of Scientific Board of EKOMED, Kiev, Ukraine, his area of expertise is molecular biology, he is the author of exclusive technology relating to fluid crystal structures from organic molecules.
Olga Argzanova is the Chief of the Lungs and Tuberculosis Department at the Tuberculosis Hospital, Lisichansk, Ukraine.
Nathalia Prihoda is the Head of the Tuberculosis Hospital, Lisichansk, Ukraine.
Ludmila Vihrova is the Principal Epidemiologist of the Regional Hospital, Lisichansk.
Elena Zagaynova is a Specialist in HIV infection transmissions. Regional AIDS Center, Luhansk, Ukraine.
Galina Kutsyna is with the State Medical University and AIDS Regional Center, Luhansk, her area of expertise is immunopathogenesis of HIV infection, she has more than 40 publications to her credit, which were published in Ukraine and EU.

  1.   Introduction

The antiretroviral drug resistance, drug toxicity and adherence are major concerns in clinical management of HIV infection. On the other hand, the immune activation caused by immune reaction to HIV, is now recognised as a major cause of depletion of CD4 T-cells and resulting immunodeficiency (Appay et al., 2005). In fact, African monkeys, the natural hosts of simian immunodeficiency virus have adapted to this retrovirus by blocking immune activation and thus remaining healthy. These problems are driving force for research on new therapies that can provide an answer. Most optimal therapeutic solution is an effective and safe immunotherapy that could regulate the immune response in a manner favourable to a host.
Immunoxel is an oral immunomodulating agent produced in Ukraine by Ekomed company. It contains concentrated extracts from medicinal plants. In vitro studies on cultured thymocytes and epithelial thymic cells have shown that Immunoxel can induce synthesis of serum thymic factor and other substances with thymus like activity (Grinevich, 2001). Experiments on laboratory animals demonstrated the restoration of endocrine function and increase of thymus weight after partial thymectomia (Grinevich, 2001). It has been successfully used in the past for the therapy of various infectious diseases of viral origin such as herpes and Epstein-Barr viruses. The choice of the given immunomodulator for our study was made on the basis of the analysis of prior evidence indicating the tendency to restore suppressed immunity characteristic to chronic bacterial infections and malignant diseases (Barabai et al., 2004; Chechitiany, 2003, 2004; Grinevich, 2001; Shapovalovi, 2004; Zeleniy, 2003). Furthermore, Immunoxel has shown the efficacy in treatment of autoimmune diseases (Bodnar et al., 2002).

Preliminary data from pilot trials conducted by us in HIV-infected individuals have been very encouraging (Kutsyna et al., 2003, 2004, 2005). The results have shown that Immunoxel is safe, can increase the total and CD4 lymphocyte counts and reduce the incidence of Opportunistic Infections (OI). In addition it appeared to have favourable effect in diminishing the hepatoxicity of antiretroviral drugs. In #order to further evaluate the clinical benefit of Immunoxel in comparison to standard ART we initiated the multicentre trial conducted at four regional hospitals in Ukraine. Here we present data from 32-week, open label, three-arm trial in 70 HIV-infected individuals who were treated either with zidovudine (AZT), lamividine (3TC) and efavirenz (EFV), (AZT/3TC/EFV) in combination with Immunoxel or Immunoxel alone, assigned to arms A, B and C, respectively.

 2.   Materials and methods

 2.2   Treatment regimen

After initial screening, qualifying patients were randomly divided in three arms: arm A was prescribed: zidovudine (AZT) in 300 mg doses twice-daily; lamividine (3TC) 150 mg tablets twice-daily and efavirenz (EFV) 600mg once daily. The arm B received the same ART plus Immunoxel given as 50 drops added to a glass of water, twice-daily. The arm C received immunomodulator Immunoxel monotherapy in same 50 drops twice-daily dose. Immunoxel (Dzherelo) was provided by Ekomed company, Kiev, Ukraine. It contains aqueous alcohol extract of elecampane rhizome (Inula helenium); fennel fruit (Foenkulum vulgare); juniper berry (Juniperus communis); licorice root
(Glycyrrhiza glabra); oregano herb (Oreganum majorana): marigold flowers (Calendula officinalis); rose hips (Rosa canina) and thyme (Thymus serpyllum). These herbs are considered by the FDA as Generally Regarded As Safe (GRAS) substances. Immunoxel (Dzherelo) has been approved in 1997 by the Ministry of Health of Ukraine as a dietary, immunomodulating supplement.

2.3     Evaluation

Parameters such as CD4 cell counts, AIDS defining events, relapse and new events of Opportunistic Infection (01), adverse events and laboratory parameters were assessed at baseline and at weeks 12, 20, 32. For patients with clinical signs of adverse events laboratory tests were performed every two weeks. Baseline values for CD4 cells counts were defined as the average of the last screening and last baseline values. Adherence to treatment was assessed at each visit. Adverse events were graded by intensity and their relationship to the study medications. AIDS defining adverse events (as per 1993 Center for Disease Control definitions of AIDS) were recoded in the same way as all other adverse events.

 2.4     Statistical analysis

The primary outcome measure was CD4 cells count. The secondary endpoints were levels of adherence, incidence of HIV related events, adverse events, laboratory abnormalities, liver function test, incidence of opportunistic infections or coinfections, clinical improvement and changes in body weight. The trial was designed to compare arm A with arm B and C at weeks 12, 20, 32 and 48, using the proportion of patients. All statistical tests were performed as on the Intent-To-Treat (ITT) population, which included all patients who took at least one dose of study medications; patients who discontinued for any reason were considered as treatment failures. Tests were performed on the On-Treatment (OT) population, which included only those patients with available evaluation at that time point. The safety evaluation included all patients who had at least one post-baseline safety assessment.

 2.7   Patients' population

In this trial 70 male or female adults with HIV/AIDS were enrolled. Most of them were hepatitis virus C (HCV) coinfected, with significant proportion of them suffering from alcohol abuse or/and drug addiction. Significant metabolic disorders and underlying hepatic injury caused by chronic alcohol, drug addiction and hepatitis coinfection were common for study cohort. At study initiation 21% had active OI and 71% had grade three or more laboratory or clinical abnormalities. Patients were divided into three arms, their baseline characteristics were comparable. All participants were antiretroviral drug nai've. Each participant provided an informed consent and was free to withdraw from the study at any time. All patients were given symptomatic therapy for OI if required. The trial is designed to continue until the last enrolled patient reaches 48 weeks on therapy.

3.   Results

3.1   Change in CD4 cell counts

Significant increases from baseline were seen in all three arms at every assessed time point. By the end of first 12 weeks of treatment, CD4 cell counts changed as follows: in arm A reaching +122 cells/mm3; in arm B decreasing but not significantly -10 cells/mm' and in arm C increasing +36 cells/mm3 but not in a significant manner. On the follow-up CD4 counts increased progressively in all arms, reaching +108, +85 and +103 cells/mm' by 20 weeks and +102, +190 and +175 cells/mm'by 32 weeks, for arms A, B and C, respectively (OT analysis).
There was a significant difference between arm A compared with arms B and C in terms of change in CD4 counts from baseline to week 12. In arm A CD4 cell counts increased significantly compared with arm B, in which cell counts decreased
(A versus B; P < 0.04) and with arm C, where increasing CD4 count was not significant from baseline level (A versus C; P < 0.05).
The first statistically significant increase in CD4 counts from baseline was observed in groups B and C at week 20. By week 32 progressive increase in CD4 cells in these two groups was higher than in arm A (B versus A; P < 0.05 and C versus A; P < 0.05).
The arm C had three patients in terminal stage of AIDS and who had very low levels of CD4 cells count at baseline 65, 64 and 128 cells/mm'. After treatment initiation CD4 cells have ris.en, reaching +15,+111 and +194 cells/mm'by 12 weeks and +85, +103 and +1171 cells/mm'by 20 weeks. In parallel with rising CD4 cell counts these patients experienced progressive clinical improvement (Table 1).

3.2  Adverse events

Over 32 weeks of treatment significant differences were observed between arm A as compared with arms B and C in terms of overall incidents of adverse events. At least one adverse event, which possibly and probably was related to the study medications, was reported in 65, 24 and 5% of patients in arms A, B and C, respectively. Most clinical adverse events were mild - gastrointestinal symptoms were the most frequently reported adverse events. Over 32 weeks period, the most common adverse events considered as possibly related to study medication as well as those of moderate or severe intensity are shown in Table 2. There were clear differences between arms in a pattern or incidence of adverse events: the levels of diarrhoea and nausea were significantly higher in arm.
A compared with arm B. In contrast in arm C gastrointestinal symptoms were not observed throughout the study period. Occurrences of headache and fatigue were also significantly higher in arm A when compared with arm B and these symptoms were not observed in arm C. Seven patients (35%) in arm A experienced serious adverse events, which were considered to be possibly or probably related to study medication. In this arm 1(5%) patient refused further therapy and 6(30%) patients needed replacement therapy due to progressive fatigue, vomiting and gastrointestinal disorders and/or hepatotoxicity. In arms B and C no incidents of interruption or changing therapy were observed. There were also significant differences between three arms in respect to laboratory findings (Table 2).

 

*Defined as a fall from 1000-1500/mm1 to <5000/mm'or from >1500/mm'to <749/mm\ **Defined as a rise from 1.25-2.5 ULN to >10 ULN or from 1.25 ULN to >5 ULN. ***Defined as a rise from 1.25-2.5 ULN to >10 ULN or from 1.25 ULN to >5 ULN. **** Defined as a reduction from normal levels < 9.0 g/dl.

*****Defined as a rise from normal levels >6.3 mmol/1.

3.3  Opportunistic or coinfection defined events

During 32-week reporting period, 01 were observed in all three arms under therapy. 01 were observed in 6 (30%), 4 (13%) and 4(20%) patients for A, B and C arms, respectively (Table 3). There was a clear distinction between treatment arms in terms of frequency OI and co-infection events. These events were significantly higher in arm A compared with arms B or C.

Among most frequently reported relapses of OI and coinfection were oral or oesophageal candidiasis 4(20%), 2(7%) and 2(10%), herpetic infections 4(20%), 4(13%) and 3(15%) patients in the arms A, B and C, respectively. Acute tuberculosis was observed in 2(10%), 2(7%) patients in arms A and B, respectively. Similarly, the relapses of hepatitis C were observed only in arms A and B, 2(10%) and 1(3%), respectively.

 3.4  Effect on liver function

Most patients enrolled in study had underlying liver pathology. Signs of progressing chronic inflammatory process in liver caused by persistent viral infection, toxicity and alcohol were registered as mean values of standard Liver Function Test (LFT). Baseline ALT or/and AST were elevated in 7/20 (30%), 13/30 (43%), 11/20 (55%) patients in arms A, B and C, respectively. Mean baseline values for ALT were 36, 62 and 72 ULN, respectively. The median cumulative change from baseline ALT values were + 22, +18 and -16ULN at 12 weeks, +30, -36 and -34ULN by week 20 and + 42, -24, -41 ULN by week 32 for arms A, B and C, respectively. At week 32 ALT values have increased to a mean 78ULN in arm A and declined to 38 and 31 ULN in arms B and C, respectively. The difference between arms B and C compared with arm A in terms of ALT values (B versus A; P < 0.05, C versus A; P < 0.03). In arm C ALT values were decreasing progressively under therapy and became significant lower than baseline levels (P < 0.01). Similar trends were observed with AST marker. The mean baseline values of AST were 42, 49, 58ULN for arms A, B and C, respectively. At week 32 AST values increased to a mean 68ULN in arm A and decreased to 42, 26 ULN in arms B and C, respectively. The number of patients with ART related hepatotoxicity was 6(30%), 2(10%) patients in arm A and B, respectively. In arm C no cases of drug-related hepatotoxicity were noted.
3.5  Effect on body weight
Mean values for baseline body weight were 64.2 ± 8.9; 68.9 ± 7.6; 67.3 ± 8.4 kg for arms A, B and C. Among arms A, B and C 7(35%), 11(33%), 6(30%) patients had cachexia. The body mass steadily increased during therapy, gaining additional 1.4; 6.9 and 5.1 kg at week 32 for arms A, B and C, respectively. Weight gain varied for every patient ranging from 0.5-2 kg up to 6-9 kg during the therapy period. At the end of study, the mean weight values were 65.6 ± 7.3; 75.8 ± 6.9; 72.4 ± 8.6 kg for arms A, B and C respectively.

 4.  Discussion

AIDS is a significant threat to the mankind and the search for effective anti-HIV therapies is of paramount importance. Several chemical anti-HIV agents have been developed. However, besides the high cost, there are adverse effects and^toxicity associated with use of chemotherapy. The herbal medicines have frequently been used as alternative or adjunct means of therapy by HIV positive individuals and AIDS patients. Except few instances, there is insufficient evidence to support the benefit of plant-derived medicines (Liu et al., 2005). Potential beneficial effects of medicinal plants need to be confirmed by rigorous clinical trials, preferably by comparing them to standard ART.
In this study we compared the safety and efficacy of immunomodulator Immunoxel with standard ART AZT/3TC/EFV or combination of AZT/3TC7EFV with Immunoxel using as the primary endpoint the change in CD4 lymphocyte counts. The increase from baseline in CD4 cells was seen in all three arms at every assessed time point, except for arm B at 12 weeks. However, patients on standard ART therapy gained lowest number of cells when compared to B and C arms, that is, +102, +190 and +175 cells/mm3.
Additional parameters under consideration were the incidence of adverse events, frequency of opportunistic infections and coinfections, liver function test and effect on body weight. The incidence of adverse events which possibly and probably were related to the study medications was lowest in Immunoxel arm (5%) when compared to standard AZT/3TC/EFV tri-therapy (65%). Interestingly, patients in arm B who were treated with AZT/3TC/EFV and Immunoxel had significantly lower incidence (24%) of adverse events and hepatotoxicity despite exposure to the same ART dose as in arm A.
Immunoxel seems to normalise elevated liver enzyme levels. At treatment initiation baseline values for ALT were 36, 62 and 72 ULN but at week 32 AST values have increased to a mean 78 ULN in arm A but declined to 38 and 31 ULN in B and C arms. These properties of Immunoxel are of major consequence to management of ART toxicity. In addition to iatrogenic hepatotoxicity that ranges from mild hepatitis to liver failure there is a significant threat in form of chronic viral hepatitis, that is, HCV and HBV with higher risk of morbidity and mortality. Almost half of patients participating in this trial had confirmed HCV infection. However, despite the lack of hepatitis-specific treatment, patients who were given Immunoxel, experienced normalisation of initially high ALT and AST levels without a single incident of relapse. In contrast patients on AZT/3TC/EFV had higher incidence of hepatitis relapses. The patients on the same ART regimen supplemented with Immunoxel had lower number of outbreaks and normalised liver function test. These observations suggest that Immunoxel possesses anti-inflammatory activity.                                  
AIDS-associated wasting is the major factor that contributes to morbidity and mortality. Currently, there is no standard treatment for this condition, which remains poorly treatable even in countries with advanced medical care. Some of nutritional regimens did yield positive results, however their success was unpredictable. Weight gain observed with Immunoxel was significantly higher when compared to such supplements or ART therapy. This weight-correcting property alone represents a significant achievement that greatly augments the choice of available treatment options.
Obtained findings support our earlier open-label trials conducted on more than 200 patients. In prior studies we have observed significant increase in body weight, reduced anemia and leucopenia processes and the increase of total lymphocytes and their CD4+ subpopulation (Kutsyna et al., 2003, 2004, 2005). These data taken together with present findings indicate that Immunoxel is safe and effective for clinical management of HIV infection.
At this stage we are not certain as to what is the precise mechanism of Immunoxel action. It is possible that Immunoxel may affect directly viral replication. However, this has not been verified by in vitro studies. The broad-spectrum antiviral, antimicrobial and anti-tumor activities shown in the clinical setting also preclude such a conclusion. Preliminary results suggest that Immunoxel can correct autoimmune disorders in a variety of diseases (Barabai et al., 2004; Bodnar et al., 2002;
Chechitiany, 2003, 2004; Grinevich, 2001; Shapovalovi, 2004; Zeleniy, 2003). If we consider that the immunopathogenesis of HIV-infection is inherently an autoimmune process, then the role of Immunoxel as a modulator of HIV-caused self-destructive immunity makes a sense (Appay et al., 2005; Bourinbaiar et al., 2006).
Currently, several immunotherapeutic approaches are available, which operate on the premise that AIDS is an autoimmune disease. So far most of these therapies are in the domain of so-called therapeutic vaccines and related immunotherapies (Bourinbaiar et al., 2006). Very few validated, immune-based interventions are available when it comes to products of plant origin. Most studies in this area concern Oriental medicinal plants some of which were used for treatment of autoimmune disorders such as habitual abortion (Chen et al., 2003). However, we are not aware of any examples of application of autoimmunity-regulating herbs in infectious diseases, especially HIV infection. Not every herbal preparation can possess the right property suitable for such an indication. Indeed, some herbal supplements were shown to exacerbate autoimmunity - a property contrary to the intended action of Immunoxel (Lee and Werth, 2004). On the other hand it will be a mistake to classify Immunoxel as an immunosuppressant. The decrease in the frequency of opportunistic infections and absence of new episodes of OI as demonstrated in this trial support our prior observations, indicating that Immunoxel does not compromise the native immunity. These considerations are intriguing not only from the immunological point-of-view but are also important in finding effective therapeutic solutions for diseases which so far have been refractory to existing choices of treatment.
In conclusion, Immunoxel displays a broad-spectrum clinical activity that has far-reaching implications. It reduces drug toxicity and improves ART efficacy when used in combination with standard ART. Further studies are required to identify the mechanism of action and additional benefits associated with its use.
Acknowledgements
We thank all patients who participated in this study. The assistance of Ekomed in generously providing Immunoxel is very much appreciated.
 References
Appay, V., Boutboul, F., Autran, B. (2005) 'The HIV infection and immune activation: "to fight and burn", Current Infectious Disease Report, Vol. 7, pp.473^4-79.
Bodnar, P.M., Mykhal'chyshyn, H.P., Reznichenko, V.M., Moshchych, O.P. and Pylypchuk, V.S. (2002) 'Phytoconcentrates "Dzherelo" and "lizorm" in therapy of autoimmune thyroiditis', LikSprava, Vol. 8, pp.127-129.
Bourinbaiar, A.S., Root-Bernstein, R.S., Abulafia-Lapid, R., Rytik, P.G., Kanev, A.N., Jirathitikal, V. and Orlovsky, V.G. (2006) "Therapeutic AIDS vaccines', Current Pharmaceutical Design, Vol. 12, pp.2017-2030.
Lee, A.N. and Werth, V.P. (2004) 'Activation of autoimmunity following * use of immunostimulatory herbal supplements', Archives Dermatology, Vol. 140, pp.723-727.
Liu, J.P., Manheimer, E. and Yang, M. (2005) 'Herbal medicines for treating HIV infection and AIDS', Cochrane Database Systematic Reviews, 20 July, Vol. 3, CD003937.
Barabai, V.A., Grinevich, Y.A. and Zhinchenko, V.A. (2004) 'Influence of immunotherapy on effectiveness of radiation treatment of tumor on immunomodulator Dzherelo example', Problems of Ecological and Medical Genetics and Clinical Immunology, Vol. 61, pp.67-70.
Chechitiany, R.B. (2003) 'Influence of plant derivative drug Dzherelo on cell signs of immunity among patients with chronically persistence herpetic infection with chronical fatigue syndrome', Problems of Ecological and Medical Genetics and Clinical Immunology, Vol. 53, pp.92-99.
Chechitiany, R.B. (2004) 'Influence of plant derivative drug Dzherelo on circulating immune complexes and their molecular content among patients with chronic persistent infection of mixed genesis (Epstein-Barr and herpetic)', Problems of Ecological and Medical Genetics and Clinked Immunology, Vol. 61, pp. 130-134.
Chen, Z., Li, Y. and Li, Y. (2003) 'Review of study on mechanism of traditional Chinese medicine in treating autoimmunity disease', Zhong Yao Cai, Vol. 26, pp.218—221.
Grinevich, Y.A. (2001) 'Influence of phytoconcentrate "EcoMed" on immune system and body antitumor resistance', Collection of Works: Modulated Medical Preventive Methods, Their Use in Medical Practice, Vol. 1, pp. 13-33.
Kutsyna, G., Chechitiany, R., Bascacov, I., Zagaynova, E. and Zaharova, I. (2005) 'Influence of a novel immunomodulator to the prevalence of new events opportunistic infections in untreated HIV- infected individuals', Third European HIV Drug Resistance Workshop, Athens, Greece, 4-7 April, Abstract #36.
Kutsyna, G., Zagaynova, E. and Zaharova, I. (2004) 'Effects of a novel immunomodulator in HIV infected individuals', Seventh International Congress on Drug Therapy in HIV Infection, Glasgow, UK, 14-18 November, Abstract #224.
Kutsyna, G.A., Chechitiany, R.B. and Zagaydanova, Y.G. (2003) 'Effects of immunomodulator Dzherelo on immune signs among HIV-infected', Problems of Ecological and Medical Genetics and Clinical Immunology, Vol. 53, p.56.
Shapovalovi, I.O. (2004) 'Influence of plant derivative drug Dzherelo on signs of macrophage phagocyte system among teenagers, who had acute tonsillitis of viral etiology', Problems of Ecological and Medical Genetics and Clinical Immunology, Vol. 61, pp.135-144.
Zeleniy, I.I. (2003) 'Effectiveness of phytoconcentrate Dzherelo on immune rehabilitation of patients with erysipiloid pustulous-necrotic complications', Problems of Ecological and Medical Genetics and Clinical Immunology, Vol. 53, pp.34-49.

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