AIDS is one of the diseases which most heavily affects humankind. It is chiefly caused by a drop in the levels of T CD4+ lymphocytes: these cells play a leading role in orchestrating the response of the immune system.
The result is an actual loss of regulation within the immune system.
On one side this loss in regulation makes the subject vulnerable to opportunistic infections (pneumocystosis, cryptococcosis, deep candidiasis, infections caused by atypical mycobacteria) and to specific types of tumours (Kaposi’s sarcoma, lymphomas).
On the other side, the patient experiences a hyperactivation of the immune system with detrimental consequences for the body as a whole. AIDS is brought about by the human immunodeficiency viruses, HIV-1 and HIV-2, which are in turn divided into a number of subtypes. These chiefly infect T CD4+ lymphocytes.
The cellular damage caused bythe virus replication is the main cause for the numerical drop detected among these cells during the course of the infection.
HIV viruses also infect the monocytes/macrophages, certain glial cells and other types of cells expressing the CD4 surface antigen. HIV viruses are retroviruses, i.e. RNA viruses which need to go through a DNA phase in order to complete a replicative cycle. (Fig. 1).
This consists of pre-integration and post-integration phases.
The pre-integration phases provide for the attachment of the virus to the target-cell (by means of the gp120 viral glycoprotein), entrance into the cell of the material contained in the viral involucre, reverse transcription of viral RNA into proviral DNA, and integration of the latter into the DNA of the host cell. The post-integration phases provide for the transcription of viral DNA into messenger and genomic RNA (which will become part of the new virions), the translation of the viral messenger RNAs, the post-translational modifications of the viral proteins (intervention of the protease enzyme!) and the assembly and gemmation of the new virions. The action of the reverse transcriptase and protease enzymes is the chief target of the antiretroviral drugs used inclinical practice. Thanks to these drugs, in Western countries a significant decrease in the number of AIDS cases, and in the number of AIDS-related deaths, has been achieved over the last few years. Unfortunately, the extremely high cost of many of such, combined with the difficulties experienced in monitoring their effects, make it very difficult to employ them in developing countries, where the epidemic has unfortunately reached apocalyptic levels.
Within living memory there has never been such a vast epidemic and such a huge number of victims. Indeed, experts reckon that, since its appearance, AIDS has caused approximately twenty million deaths.
By way of an example, the tragic plague of Athens described by Thucydides (which was in fact exanthematous typhus), which hit part of the Mediterranean area in the 5th century B.C., and the plague epidemic which devastated Europe in the 17th century, whom we are all familiar with through A. Manzoni’s “Promessi Sposi”, produced a moderate number of deaths if compared to the victims of HIV-infection.
According to the latest epidemiological data (Tab. 1), in Sub-Saharan Africa alone, the number of people infected by the HIV virus exceeds twenty-eight million. Certain prevention strategies are attempting to hold backthe staggering increase in the number of new infection cases. However, experts forecast that, in certain areas in which seroprevalence reaches peaks up to 30%, there will be whole generations of working age people that are doomed to be literally wiped out, unless immediate therapeutic action is taken. This picture is particularly serious in that it relates to financial and living conditions which are already miserable to start with.
This death sentence weighing upon the working age population, may wipe outwhole generations of parents and teachers, thus causing the living conditions of a huge number of children to become disastrous, if not impossible.
Besides affecting Sub-Saharan Africa, AIDS heavily affects other poor countries, among which India and Thailand. Furthermore, data recently made available indicate that HIV-infection is spreading in a worrying manner through Central Asia, where it risks worsening particularly difficult conditions, due to the war and unstable political situations.

Tab1

Effects of Chloroquine on HIV Infection

The Historical Background
Chloroquine is a 9-aminoquinoline which has been known since 1934. Specifically synthesised to be employed as an antimalarial agent, it subsequently revealed to have interesting immunomodulating properties which have encouraged its application in the treatment of self-immune diseases, such as rheumatoid arthritis. For this specific pathology, the drug has represented a valid contribution to the available pharmacological aids, since it proved able toslow down the progress of the disease whilst showing limited toxicity.
The good tolerability, the low cost and the immunomodulating proprieties of chloroquine have suggested its application in fighting HIV infection. The earliest documentation relating to an anti-HIV effect of chloroquine dates back to 1990 (Tsai et al., AIDS Res Hum Retroviruses 1990). With this study, researchers proved that it was possible to reduce the infectivity of virions produced by cells infected through laboratory strains.
The studies conducted on chloroquine as an antiretroviral drug were subsequently abandoned to pursue studies on gene therapy, which unfortunately has not yet proved successful in producing applicable results, as well as studies on the inhibitors of reverse transcriptase and integrase viral enzymes, which have proved more effective but much more expensive and difficult to handle. The dramatic spreading of the epidemic in the Third World has however caused the scientific community to resume studies on possible low-cost therapies, since many of the countries which are more heavily hit by the epidemic are absolutely unable to bear the prohibitive costs of the Highly Active AntiRetroviral Therapy (HAART).
For these applications, chloroquine appeared to be an ideal candidate. Studies conducted by Johan Boelaert in Belgium and by Kirk Sperber in the United States have shown that chloroquine and its hydroxyl-derivative, hydroxychloroquine, have inhibitory effects that are synergic with certain less expensive antiretroviral drugs, such as zidovudine (AZT) and the combination didanosine (ddI) + hydroxyurea (HU) (Boelaert et al., AIDS Res Hum Retroviruses 1999). Furthermore, in clinical trials conducted at the Mount Sinai School of Medicine in New York, hydroxychloroquine has proved capable of reducing the viral load (Sperber et al., Clin Ther 1995).
A study recently conducted in conjunction with Johan Boelaert’s group has shown that chloroquine is provided in vitro with wide-spectrum antiretroviral properties, which can be observed on numerous African isolated viruses (Savarino et al., AIDS 2001). The same study further clarifies the manner in which chloroquine inhibits virus replication.

Action Mechanism
Both chloroquine and hydroxychloroquine are weak bases that accumulate in the acid vesicles and lead to the dysfunction of the enzymes required for the post-transduction modifications in the proteins. It has been demonstrated that weak bases, by increasing the pH of acid vesicles, inhibit the functionality of numerous enzymes, among which certain acid hydrolases, which inhibit the post-transcriptional modifications of the synthesised proteins (Savarino et al., J Clin Virol 2001).
We have confirmed that chloroquine and hydroxychloroquine increase the pH in the acid vesicles in a dose-dependent manner and have demonstrated that the virus produced by the cells treated with chloroquine or with hydroxychloroquine displayed reduced infectivity (Chiang et al., Clin Ther 1996).
As a further confirmation of the effect of chloroquine and hydroxychloroquine on the production of gp120, Tsai et al. and Chiang et al. have demonstrated that the production of gp120 is altered in the T-cells and in the isolated virions after the treatment with chloroquine or with hydroxychloroquine (Tsai et al.,1990; Chiang et al., Clin Ther 1996). Pal et al. have also demonstrated that monensin, an ionophore that increases endosome pH, inhibits the production of gp 120 in a T-cell line (Pal et al., Proc Natl Acad Sci USA 1988). By carrying out an overall review of such results, we find that chloroquine and hydroxychloroquine influence cellular factors that are essential for an adequate production of gp120.
Recent data indicate that chloroquine may act as an inhibitor of gp120 glycosylation (Savarino et al., AIDS 2001). This mechanism is compatible with the intravesicular pH rise induced by the drug, since certain mechanisms of glycosylation are pH-dependent. Certain glycosylated portions of the gp120 glycoprotein also have a significant effect on the infectivity of the virus. This would explain the drop in infectivity displayed by the virus produced in the presence of chloroquine (Fig. 2).

Antiviral Activity
Studies conducted in vitro show that chloroquine is capable of inhibiting the HIV virus under testing conditions mimicking physiological conditions. Certain studies have demonstrated an inhibition of HIV replication by overloading the cells with high concentrations of chloroquine and hydroxychloroquine before the infection, so as to “mimic” the drug build-up taking place in the tissues of patients subject to chronic treatment.
Other researchers have observed significant anti-viral effects by keeping cells infected with HIV under constant incubation with concentrations of chloroquine detected in the plasma of patients chronically treated with this drug, that is approximately 1-5 mM (Savarino et al., 2001). The antiviral activity of hydroxychloroquine has been confirmed in vivo by two clinical trials (Sperber et al., Clin Ther 1995, 1997). In a drug vs placebo pilot study conducted on 38 pharmacologically-naïve adults affected by HIV-1, Sperber et al. reported reduced plasmatic levels of viral RNA and IL-6 (a cytokine involved in the HIV-related immune disorders), as well as a stabilisation of the immune system, in patients who had been administered hydroxychloroquine for 8 weeks. On the other hand, no significant reduction in viral RNA levels was detectable in the patients treated with placebo. No side-effects were reported in connection with this drug, and patients appeared to have well tolerated the therapy (Sperber et al., Clin Ther 1995).
A second clinical trial was conducted on 72 HIV-positive asymptomatic subjects, with the purpose of comparing the effectiveness of hydroxychloroquine to that of zidovudine (AZT). Hydroxychloroquine significantly reduced the HIV-1 RNA levels in the plasma, although the drop proved less dramatic than the one obtained with AZT.
Despite the general trend, eight patients enrolled in the AZT group showed an increase in the HIV-1 RNA levels and in the cultured virus levels during the therapy with AZT. This suggested the onset of resistant strains. On the other hand, the anti-HIV-1 effect of hydroxychloroquine appeared more stable in time and no subject who had been administered hydroxychloroquine showed an increase in HIV-1 RNA and cultured virus levels during the 8-or 16-week therapy. These data suggest that resistance to hydroxychloroquine may not develop, or possibly that it develops more slowly (Sperber et al., Clin Ther 1997). Monotherapy is no longer adopted nowadays to treat patients affected by HIV-1.
Since chloroquine and hydroxychloroquine appear to have a new site of action (post-transcriptional inhibition of gp120), these drugs may prove particularly useful in combination with other anti-retroviral agents (e.g.: AZT, ddI and hydroxyurea). In addition, researchers have provided evidence as to an additive effect of AZT and hydroxychloroquine in patients affected by HIV-1 with inflammatory arthritis. Chiang et al. compared the anti-HIV-1 effect of hydroxychloroquine and AZT both on T-cell lines and on monocyte cell lines and demonstrated that the inhibitory effect of hydroxychloroquine on virus replication in vitro was not as remarkable as that obtained with AZT (Chiang et al., Clin Ther 1996). However the two drugs showed a synergistic effect. It has been known since 1993 that the hydroxyurea anti-metabolite has an anti-HIV-1 activity (Lori et al., Science 1994). This effect is significantly intensified when this drug is associated with didanosine (ddI). Hydroxyurea strengthens the anti-HIV-1 activity of ddI by targeting ribonucleotide reductase and depleting the supplies of dATP. This depletion causes an increase in the amounts of ddATP produced by the ddI incorporated in the HIV-1 DNA (Lori, AIDS 1999).
Researches have therefore evaluated whether the addition of chloroquine could strengthen the hydroxyurea - ddI combination to suppress the replication of HIV-1 in T-cell lines and in monocytes, as well as in primary T-cells (Boelaert and Sperber, Lancet 1998; Boelaert et al., AIDS Res Hum Retroviruses 1999). The data available indicate that the addition of chloroquine to the hydroxyurea + ddI combination is capable of further inhibiting viral replication in vitro.
This synergistic effect of chloroquine in combination with hydroxyurea + ddI supports the idea that this triple regimen ought to be tested through clinical trials. This could acquire special interest for people affected by HIV-1 in the Third World, taking into account the low cost of both chloroquine and hydroxyurea. To further support this hypothesis, researchers have found that the anti-HIV-1 effect of chloroquine is not only confined to the HIV strains that have spread throughout the Western countries (which belong to the HIV-1 B subtype). It has been demonstrated that chloroquine inhibits viral isolates belonging to various HIV-1 subtypes spread throughout Africa and the Far East and to HIV type-2. These antiviral effects could be detected in lymphocyte and monocyte cells treated with clinically reachable drug concentrations (Savarino et al., AIDS 2001) The effect of chloroquine on the subtype C of HIV-1 could prove particularly interesting, in view of the fact that the viral strains belonging to this subtype are responsible, on their own, for 50% of the cases of HIV infection worldwide. These prevail in Sub-Saharan Africa, in countries in which the economic conditions are particularly miserable.

Toxicity Studies
Having been used for quite a long time, chloroquine has a toxicity profile which is well known.In the therapy for acute malarial attacks, the drug may cause problems involving the central nervous system (loss of consciousness, convulsions); however these effects are very rare when the drug is administered according to the dosage advised for the antimalarial and rheumatoid arthritis prophylaxis (up to 500 mg/day). In these cases, the most dangerous toxic effect relates to the eyes, and it depends on the cumulative dosage rather than on the daily dosage. It consists of macular retinopathy.
Chloroquine maculopathy provides for two phases: one is early and reversible, and from a symptomatological point of view reveals itself as a dyschromia and visual field disorders; the other one appears later and is irreversible, and results in loss of vision. In any case, it is possible to avoid the occurrence of this effect, byhaving the patient treated with this drug undergo a regular colour vision test, which can be easily conducted, also by non-specialised staff performing low-level medicalization activities. Should dyschromia be detected, administration should be immediately interrupted, so as to prevent maculopathy to progress towards its irreversible stage.
During chronic treatment with chloroquine, other undesired effects have been reported, among which: gastrointestinal disorders, depigmentation, hair loss and cutaneous rash. However, these effects occur rarely and do not represent a serious danger for the health of the subject under treatment. As regards toxicity during pregnancy, normal clinical practice provides for administration of the drug to pregnant women in malaria prophylaxis. At these dosages it is well tolerated and may be deemed safe, both for the mother and for the foetus. On the other hand, we do not have an equivalent amount of information as to the effects brought about by the dosage advised for rheumatoidarthritis, which should in theory also apply for women suffering from HIV infection. Certain cases of foetus retinopathy have been reported but, also in this case, the toxicity would appear to depend on the cumulative dosage rather than on the daily dosage. An opportunity would therefore appear to open for brief treatments with chloroquine at 500 mg/day to prevent mother/foetus transmission.
A recent study published by the journal Lancet (Klinger et al.., Lancet 2001) seems to confirm the safety in administering the drug during pregnancy at such dosages.

Concluding Remarks

Potential Advantages
In summary, chloroquine displays many qualities which encourage further studies as to its potential employment as an anti-HIV drug.
a) Low Cost: Chloroquine is by far the most inexpensive drug among those displaying anti-HIV properties.
b) Tolerability: Having been known for almost seventy years, chloroquine has a toxicity profile which has been very well analysed. Any toxic effects may be easily checked without the aid of complex clinical facilities.
c) Compliance: Low toxicity, combined with accumulation phenomena, may hopefully reduce the therapy compliance problems which are often observed in non-Western countries.
A further factor whichencourages administration is chloroquine’s known antimalarial properties. The fact that the drug is usually employed for this purpose may protect HIV- positive individuals from the social stigmatization which unfortunately troubles many people who have to take known antiretroviral drugs.
d) Antiviral Activity: Chloroquine displays in vitro antiretroviral properties against numerous HIV subtypes (Savarino et al., AIDS 2001), whereas some among the most widely employed antiretroviral drugs have not shown great effectiveness against African isolated viruses, in particular against isolated viruses belonging to the C subtype.
e)Action Mechanism: Chloroquine appears to exert its anti-HIV effects through an entirely new mechanism (Savarino et al., AIDS 2001). The drugs which are currently used inhibit the virus with respect to proviral DNA synthesis and to the protease action. Further studies on the in vivo effectiveness of the drug may supply an additional means for fighting the virus at several levels of its replicative cycle.
f) Immunomodulating Properties: In addition to its direct action on the virus, chloroquine may involve interesting applications in the therapy of HIV infection, thanks to its immunomodulating properties. It has been demonstrated that chloroquine reduces lymphocyte activation. This property may prove interesting, since AIDS, especially in the event of infections supported by the HIV-1 C subtype, is associated with a generalised lymphocytic activation, which produces unpropitious effects. In addition, chloroquine reduces the production of IL-6 and TNF-a, whose action aids viral replication (Boelaert et al., J Clin Virol 2001).

Potential Applications
Although the chloroquine dosage advised for HIV infection cannot be borne for more than a few years, owing its cumulative toxicity, the confirmed effectiveness of chloroquine makes it in any case possible to delay the progression of the infection in many areas of the Third World whilst awaiting a more targeted therapeutic action.
The application of the drug in the prevention of mother-foetus transmission represents a particularly interesting issue.
If the data relating to in vivo anti-HIV activity and the data supplied by Kinger et al. (which support safety of chloroquine in pregnancy) were confirmed, chloroquine could be used in short therapeutic cycles to reduce intra partum contagion, which is the time at which most cases of mother-foetal contagion take place.
Another potential application could be designed for the post partum period. It has been recently hypothesised that chloroquine could be takenat lower dosages in conjunction with antiretroviral drugs and thus represent a new possible therapeutic option for HIV-positive patients (Boelaert et al., AIDS Res Hum Retroviruses 1999).

Kirk Sperber
Department of Clinical Immunology,
Mount Sinai School of Medicine, New York, NY, USA

Andrea Savarino
Department of Clinical Immunology,
Mount Sinai School of Medicine, New York, NY, USA
Istituto Clinica delle Malattie Infettive,
Università Cattolica del Sacro Cuore, Roma

LINKS CONSIGLIATI
http://www.unaids.org/epidemic_update/report_dec01/index.html Data relating to the prevalence of AIDS cases in the various regions of the world (as of December 2001) http://www.allafrica.com
Collection of online articles on Africa’s problems (including AIDS) http://www.aidsmeds.com Website describing antiretroviral drugs in general http://www.aidsmap.com/treatments/ixdata/english/70332B87-4B96-4BE5-B412-F89C293C7785.htm
Description of the anti-HIV effects of Chloroquine. http://www.aegis.com/news/ads/2001/AD012143.html
Article describing the potential use of Chloroquine in HIV transmission through mother’s milk