Highly Active AntiRetroviral Therapy and opportunistic protozoan infections

Opportunistic parasite infections (OPIs) are an important cause of morbidity and mortality in persons infected with HIV. In industrialised countries, the use of Highly Active AntiRetroviral Therapy (HAART) results to be effective in suppressing the HIV viral load, with a quantitative and qualitative improvement in the CD4+ T-cell count followed by a strong reduction of opportunistic infections including those caused by parasites. These successes have been mainly attributed to the reconstitution of the cell immunity, which play the most important role in controlling OPIs. However, there are many clinical reports and several laboratory results, which suggest that the control of OPIs in HIV-positive persons under HAART is also induced by the anti-HIV protease inhibitors (PIs), which inhibit the aspartyl proteases of the parasites. The non-conventional use of HIV-PIs seems to be an alternative way for the treatment of parasitic infections, which should be deeply investigated. Of five longitudinal studies carried out before and after the introduction of HAART, four studies showed a strong reduction of toxoplasmic encephalitis (TE) in HIV-positive persons under HAART, whereas in another study, no difference was observed in the incidence rate of TE before and after the introduction of HAART. The influence of HAART in reducing TE has been also confirmed in a randomised, controlled clinical trial, which showed that there is no increase in the risk of developing TE after beginning HAART, even though HIV-infected persons with TE had a discontinuing prophylaxis for Toxoplasma gondii. Four HIV protease inhibitors were tested against the T. gondii virulent RH strain in vitro, alone or in association with pyrimethamine or sulfadiazine. Ritonavir and nelfinavir were highly inhibitory for the parasite growth. Furthermore, none of the antiviral drugs negatively affected the anti-Toxoplasma activity of pyrimethamine or sulfadiazine. In HIV-Leishmania co-infections, a changing pattern has been observed in the HAART era, characterised by a high rate of relapses, which could be explained by the increased survival rate resulting from the effective antiretroviral therapy. A 64.8% decrease of the visceral leishmaniasis incidence was detected after HAART began to be used extensively in Spain. In a large cohort study carried out in ten European countries and in Australia, the relative risk to contract cryptosporidiosis as the first AIDS defining disease was reduced by 96% in the HAART era. In Italy, the relative risk of death for cryptosporidiosis reduced of 74% in the period 1997-98, when HIV-positive persons received HAART. In a large study carried out in Italy, isosporiasis was included in the group of opportunistic infections, of which the relative hazards showed a reduction of 95% in the HAART era. Since 1997, there was the evidence that the use of HAART in persons with advanced HIV infection can improve chronic diarrhoea and lead to disappearance of Enterocytozoon bieneusi from the stools. Although the reconstitution of the cellular immunity seems to be the main factor influencing the reduction of OPIs in persons with AIDS who undergo HAART, there are clinical and microbiological evidences, as well as in vitro and in vivo results, which indicate direct effects of HIV-PIs on the proteases of opportunistic parasites. These findings stress the existence of non-conventional unexpected benefits of PIs in HAART against protozoa. In addition, this benefit of PIs has been demonstrated also for Candida albicans secreted aspartyl proteases and for P. carinii acid proteases. In spite of these important results, HIV PIs are still very toxic for humans, specially in cases of very long treatment, and no clinical trial has been carried out for persons at risk, such as children and pregnant women, because the priority was to reduce the severity of HIV and not the evaluation of possible side effects of the therapy. It follows that further researches are needed to establish the non-conventional use of HIV PIs. Furthermore, the study of PIs against specific aspartyl proteases of those opportunistic protozoa that cause severe and intractable diseases, could be considered an alternative way towards the development of new drugs that may prove effective against these infections.     

Highly Active AntiRetroviral Therapy and cryptosporidiosis

In HIV infected persons, Cryptosporidium parvum causes chronic diarrhoea, which can be life-threatening in persons with AIDS and with a low CD4+ T cell count. However, a specific and effective therapy for this opportunistic infection does not yet exist. Since the use of a combination therapy with a highly active antiretroviral therapy (HAART), the prevalence of C. parvum infection in persons with AIDS has been strongly reduced. This favorable outcome was usually attributed to the recovery of the host immunity, however improvements from this opportunistic infection have been demonstrated even in the absence of immunological recovery. The aim of the present study was to determine whether HIV protease inhibitors (PIs) exert an anti-C. parvum activity. We selected the indinavir (an aspartyl protease inhibitor included in HAART) for our experiments, since a resolution of cryptosporidial enteritis in a person with AIDS after treatment with this drug has been reported. Human ileocecal adenocarcinoma tumor cells (HCT-8) were used as in vitro model. To determine whether or not indinavir had an effect on the parasite attachment to, or invasion of the HCT-8 cells, indinavir was added to the cultures at the same time as the infective dose (3 oocysts/cell) at one of the following concentrations: 0.1, 0.5, 5, 10, 20, and 50 microM (maximum DMSO content 0.5% vol/vol). To determine whether or not indinavir had an effect on established C. parvum infection, HCT-8 cells were infected with excysted oocysts at a ratio of 3 oocysts/cell at day 0, and then indinavir at a concentration of 50 microM was added to the cultures every 24 h for 4 days. The infection level was evaluated at 2, 3, 4 and 5 days p.i. using a flowcytometric assay. Three-day-old Balb/c mice were used as animal model, animals were infected per os with 50 microl of PBS containing 10(5) oocysts. The infected mice were divided into two groups (Group A and Group B), both of which received per os indinavir diluted in PBS with 0.1% DMSO at a concentration of 10 microM (24 mg/kg). For Group A, which consisted of 15 mice (3 litters), indinavir was administered at the same time that experimental infection was performed and then every day until the mice were sacrificed (i.e., 5 days p.i.), to determine the effect of indinavir on the attachment/invasion of the enterocytes. For Group B, which also consisted of 15 mice (3 litters), indinavir was administered after the infection was established (i.e., 72 h p.i.) and every day until being sacrificed, to determine the effect of indinavir on established infection. The mice of Group B were sacrificed 7, 10, 11 and 13 days p.i., corresponding to 4, 7, 8, and 10 days of treatment with indinavir. In vitro, the treatment of the excystated oocysts with different concentrations of indinavir reduced the percentage of HCT-8 infected cells in a dose-dependent manner. For established infection, the treatment with 50 microM of indinavir decreased the percentage of infected cells in a time-dependent manner. Treatment for 48 h resulted in a 40.1% reduction in infected cells (from 90% to 53%). After 72 h of treatment, the percentage of infected cells did not substantially differ from that observed after 48 h. Treatment for 96 h resulted in a 57.8% reduction (from 90 to 38%). In vivo, mice treated with indinavir at the same time they were infected with the oocysts showed a 93% reduction in the number of oocysts present in the entire intestinal contents and a 91% reduction in the number of intracellular parasites in the ileum. For established infection, indinavir treatment reduced the number of oocysts in the entire intestinal content by about 50% and the number of intracellular parasites in the ileum by about 70%. These data demonstrate that PIs directly exert an inhibitory effect on C. parvum and the extent of this effect depended on the specific dose and the duration of treatment. Although there are no reports of aspartyl proteases in C. parvum, the inhibitory effect of PIs on C. parvum growth in vitro suggests that aspartyl proteases could have some important functions for this parasite. In fact, proteolytic activities have been demonstrated during peak periods of excystation in C. parvum oocysts and cysteine and serine protease classes have been functionally associated with this process. Moreover, we identified several different C. parvum sequences that showed homology with a protein family related to aspartyl proteases. In prospect, PIs could be valuable for the chemotherapy of cryptosporidiosis.     

The design,synthesis and evaluation of novel HIV-1 protease inhibitors with high potency against PI-resistant viral strains

Replacement of the pyridylmethyl moiety in indinavir with a pyridyl oxazole yielded HIV-1 protease inhibitors (PI) with greatly improved potency against PI-resistant HIV-1 strains. A meta-methoxy group on the pyridyl ring and a gem-dimethyl methyl linkage afforded compound 10 with notable in vitro antiviral activity against HIV-1 viral strains with reduced susceptibility to the clinically available PIs. Compound 10 also demonstrated favorable in vivo pharmacokinetics in animal models.     

HIV aspartyl protease inhibitors as promising compounds against Candida albicans André Luis Souza dos Santos

Cells of Candida albicans (C. albicans) can invade humans and may lead to mucosal and skin infections or to deep-seated mycoses of almost all inner organs, especially in immunocompromised patients. In this context, both the host immune status and the ability of C. albicans to modulate the expression of its virulence factors are relevant aspects that drive the candidal susceptibility or resistance; in this last case, culminating in the establishment of successful infection known as candidiasis. C. albicans possesses a potent armamentarium consisting of several virulence molecules that help the fungal cells to escape of the host immune responses. There is no doubt that the secretion of aspartyl-type proteases, designated as Saps, are one of the major virulence attributes produced by C. albicans cells, since these hydrolytic enzymes participate in a wide range of fungal physiological processes as well as in different facets of the fungal-host interactions. For these reasons, Saps clearly hold promise as new potential drug targets. Corroborating this hypothesis, the introduction of new anti-human immunodeficiency virus drugs of the aspartyl protease inhibitor-type (HIV PIs) have emerged as new agents for the inhibition of Saps. The introduction of HIV PIs has revolutionized the treatment of HIV disease, reducing opportunistic infections, especially candidiasis. The attenuation of candidal infections in HIV-infected individuals might not solely have resulted from improved immunological status, but also as a result of direct inhibition of C. albicans Saps. In this article, we review updates on the beneficial effects of HIV PIs against the human fungal pathogen C. albicans, focusing on the effects of these compounds on Sap activity, growth behavior, morphological architecture, cellular differentiation, fungal adhesion to animal cells and abiotic materials, modulation of virulence factors, experimental candidiasis infection, and their synergistic actions with classical antifungal agents.     

Viral protease inhibitors affect the production of virulence factors in Cryptococcus neoformans

The effects of the protease inhibitors saquinavir, darunavir, ritonavir, and indinavir on growth inhibition, protease and phospholipase activities, as well as capsule thickness of Cryptococcus neoformans were investigated. Viral protease inhibitors did not reduce fungal growth when tested in concentrations ranging from 0.001 to 1.000 mg/L. A tendency toward increasing phospholipase activity was observed with the highest tested drug concentration in a strain-specific pattern. However, these drugs reduced protease activity as well as capsule production. Our results confirm a previous finding that antiretroviral drugs affect the production of important virulence factors of C. neoformans.     

In vitro evidence of inhibition of mitochondrial protease processing by HIV-1 protease inhibitors in yeast: a possible contribution to lipodystrophy syndrome

Highly active antiretroviral therapy has been associated with the emergence of lipodystrophy syndromes that have clinical features commonly seen in patients with mitochondrial dysfunction. The effect of therapeutic protease inhibitors (PIs) on mitochondrial function is unknown. Mitochondrial matrix space proteins possess an amino-terminal leader peptide that is removed by the mitochondrial processing protease (MPP). Lack of cleavage could result in non- or dysfunctional mitochondrial proteins. The effects of different PIs on protease processing using pure MPP or yeast mitochondria, recognized models for mammalian counterparts, were examined in vitro. Multiple PIs were found to inhibit MPP, evidenced by accumulation of immature pALDH and decreased levels of processed ALDH. Both indinavir and amprenavir at 5.0 mg/ml resulted in significant inhibition of MPP. Although inhibition of MPP was also observed with ritonavir and saquinavir, the inhibition was difficult to quantify due to background inhibition of MPP by DMSO that was required to solubilize the drugs for the in vitro studies. Indinavir was also shown to inhibit MPP within yeast mitochondria. Lack of processing may impair mitochondrial function and contribute to the observed mitochondrial dysfunctions in patients receiving HAART and implicated in antiretroviral-associated lipodystrophy.     

Stimulation of Cryptococcus neoformans isolated from skin lesion of AIDS patient matures dendritic cells and promotes HIV-1 trans-infection

Dendritic cells (DCs) play a pivotal role in host defense against invaded pathogens including fungi, while DCs are targeted by fungi for deleterious regulation of the host immune response. A few studies have reported fungal modulation of DC function in these immunocompromised AIDS patients. Cryptococcus neoformans (C. neoformans) is referred as one of the opportunistic fungi of AIDS. Here, we isolated native C. neoformans from an AIDS patient and investigated its effects on DC activation and function. Stimulation of C. neoformans matured DCs, and enhanced DC-mediated HIV-1 trans-infection; moreover, C. neoformans-stimulated DCs promoted the activation of resting T cells and provided more susceptible targets for HIV-1 infection. Microbial translocation has been proposed as the cause of systemic immune activation in chronic HIV-1 infection. Understanding the potential effects of pathogens on HIV-1-DC interactions could help elucidate viral pathogenesis and provide a new insight for against the spread of HIV.     

Dectin-1 is not required for the host defense to Cryptococcus neoformans

Dectin-1 is known as a sole receptor for beta-glucan, a major cell wall component of fungal microorganisms. In the current study, we examined the role of this molecule in the host defense to Cryptococcus neoformans, an opportunistic fungal pathogen in AIDS patients. There was no significant difference in the clinical course and cytokine production between dectin-1 gene-deficient and control mice. These results indicate that dectin-1 is not likely essential for the development of host protective responses to C. neoformans.     

Phenotyping of Cryptococcus neoformans strains in Bergamo,Italy (1985-2000)

Cryptococcus neoformans is the cause of the most common life-threatening fungal infection in patients with AIDS. Thirty strains of C. neoformans were collected from inpatients and typied evaluating activity, morphotyping, serotyping, chemosensitivity and adhesivity. Cryptococcus neoformans strains showed different aspectotype profile, the sole presence of serotypes A and D, good susceptibility to azoles and Amphotericin B. Phenotypic epidemiologic markers can be used: characterization of clinical strains excludes a common source.     

Immune stimulating and therapeutic potential of tuftsin-incorporated nystatin liposomes against Cryptococcus neoformans in leukopenic BALB/C mice

Cryptococcus neoformans infection is a common fungal infection in persons infected with human immune deficiency virus (HIV) or those with defective cell-mediated immunity. Since treatment of cryptococcal meningitis poses a big challenge, the present study aimed to develop a novel liposomal therapeutic formulation against cryptococcosis. Treatment with tuftsin-incorporated liposomes increased the anti-cryptococcal activity of murine peritoneal macrophages. Prophylactic treatment of mice with tuftsin-incorporated liposomes reduced the dissemination of C.?neoformans to brain tissues. Moreover, the co-administration of tuftsin with nystatin liposomes augmented the anti-cryptococcal activity of nystatin, as mice treated with tuftsin-incorporated nystatin liposomes showed the highest survival and least fungal burden in their brain tissues. The results of the present study favour the use of immune-stimulating molecules along with antifungal agents in the treatment of opportunistic fungal infections.     

A synthetic peptide as a novel anticryptococcal agent

An engineered, killer decapeptide (KP) has been synthesized based on the sequence of a recombinant, single-chain anti-idiotypic antibody (KT-scFv) acting as a functional internal image of a yeast killer toxin. Killer decapeptide exerted a strong fungicidal activity against Candida albicans, which was attributed to peptide interaction with beta-glucan. As this polysaccharide is also a critical component of the cryptococcal cell wall, we wondered whether KP was also active against Cryptococcus neoformans, a human pathogen of increasing medical importance. We found that KP was able to kill both capsular and acapsular C. neoformans cells in vitro. Furthermore, KP impaired the production of specific C. neoformans virulence factors including protease and urease activity and capsule formation, rendering the fungus more susceptible to natural effector cells. In vivo treatment with KP significantly reduced fungal burden in mice with cryptococcosis and, importantly, protected the majority of immunosuppressed animals from an otherwise lethal infection. Given the relevance of cryptococcosis in immunocompromised individuals and the inability of conventional drugs to completely resolve the infection, the results of the present study indicate KP as an ideal candidate for further studies on novel anticryptococcal agents.     

“In vitro” Antifungal activity of protease inhibitors

In the last five years, as HAART has become standard therapy in HIV seropositive or AIDS patients, changes have been noted in the numbers and types of opportunistic fungal infections in these cohorts of patients. Particularly, oropharyngeal candidiasis have become rare in HIV infected patients since the introduction of new anti-HIV drugs of the protease inhibitors type. At the Immunology Institute of the Universidad Central de Venezuela the most frequent protease inhibitors (PIs) used for the treatment of these patients have been: Nelfinavir (Viracept^TM, Roche),Indinavir (Crixivan^® Merck),Ritonavir (Norvir^®, Abbott),Saquinavir (Fortovase^®, Roche).Recently, we observed that recurrent candidiasis was less frequent and no Candidacould be isolated in our patients. A direct relation to the PIs was suspected. In order to assess the in vitro antifungal activity of the afore mentioned protease inhibitors on Candida sp., we used both the well diffusion test and the NCCLS broth microdilution test to assay 100 Candida sp. isolates from HIV seropositive or AIDS patients with syntomatic oropharyngeal Candida infection. In general, the data obtained with the well diffusion test were in agreement with those obtained by the broth microdilution test. All 100 isolates were susceptible to Saquinavir and 32 were susceptible to Indinavir using the NCCLS microdilution test,while 97 were susceptible to Saquinavir and 52 to Indinavir by the well diffusion test. From 17 C. albicans resistant to fluconazole, all were susceptible to Saquinavir by the NCCLS micro method and 16 by the well diffusion test. Our results showed anticandidal activity in vitro of PIs, mainly Saquinavir.This revised version was published online in October 2005 with corrections to the Cover Date.     

Anti-fungal therapy at the HAART of viral therapy

HIV-positive patients receiving combination therapy (highly active anti-retroviral treatment, HAART) suffer significantly fewer oral infections with the opportunistic fungal pathogen Candida albicans than non-HAART-treated patients. One component of HAART is an inhibitor of the HIV proteinase, the enzyme required for correct processing of retroviral precursor proteins. It would appear that HIV proteinase inhibitors also have a direct effect on one of the key virulence factors of C. albicans, the secreted aspartic proteinases (Saps). This suggests that the reduction in C. albicans infections in HIV-positive patients might not be solely the result of improved immunological status but could also be caused by the HAART treatment directly inhibiting Candida proteinases.     

A structural basis for the acute effects of HIV protease inhibitors on GLUT4 intrinsic activity

Human immunodeficiency virus (HIV) protease inhibitors (PIs) act as reversible noncompetitive inhibitors of GLUT4 with binding affinities in the low micromolar range and are known to contribute to alterations in glucose homeostasis during treatment of HIV infection. As aspartyl protease inhibitors, these compounds all possess a core peptidomimetic structure together with flanking hydrophobic moieties. To determine the molecular basis for GLUT4 inhibition, a family of related oligopeptides containing structural elements found in PIs was screened for their ability to inhibit 2-deoxyglucose transport in primary rat adipocytes. The peptide oxybenzylcarbonyl-His-Phe-Phe-O-ethyl ester (zHFFe) was identified as a potent inhibitor of zero-trans glucose flux with a K(i) of 26 mum. Similar to PIs, transport inhibition by this peptide was acute, noncompetitive, and reversible. Within a Xenopus oocyte expression system, zHFFe acutely and reversibly inhibited GLUT4-mediated glucose uptake, whereas GLUT1 activity was unaffected at concentrations as high as 1 mm. The related photoactivatable peptide zHFF-p-benzoylphenylalanine-[(125)I]Tyr-O-ethyl ester selectively labeled GLUT4 in rat adipocytes and indinavir effectively protected against photolabeling. Furthermore, GLUT4 bound to a peptide affinity column containing the zHFF sequence and was eluted by indinavir. These data establish a structural basis for PI effects on GLUT4 activity and support the direct binding of PIs to the transport protein as the mechanism for acute inhibition of insulin-stimulated glucose uptake.     

Evaluation of Systemic Antifungal Agents in X-Irradiated Mice

The effect of X irradiation on the survival time of animals experimentally infected with pathogenic fungi was studied, and the activity of antifungal agents in pre-irradiated hosts was evaluated. A 24-hr preinfection dose of X irradiation decreased the survival time of mice infected with Cryptococcus neoformans and Histoplasma capsulatum to a greater extent than Candida albicans or Blastomyces dermatitidis infections. Exposure to 400 r caused a significant reduction in the variation (S(2)) survival time of C. albicans or H. capsulatum mouse infections. A single 100-mg/kg dose of 5-fluorocytosine or amphotericin B administered within 24 hr postinfection significantly extended the survival time of mice infected with C. albicans. Delayed treatment with amphotericin B was effective against C. neoformans infections. Four 50-mg/kg doses of 5-fluorocytosine were more effective than a single 200-mg/kg dose against C. neoformans infections. A single dose of amphotericin B provided significant protection when administered 48 hr postinfection against B. dermatitidis in preirradiated mice. A single dose of saramycetin 48 hr postinfection was highly effective against H. capsulatum mouse infections. A 100-mg/kg dose of amphotericin B was only effective against this fungal pathogen when administered within 8 hr postinfection. In vivo activity of the antifungal agents studied was detected within 8 to 14 days. The relative in vivo activity of several antifungal agents indicated the importance of considering their individual pharmacological properties for optimum effectiveness. The experimental model used in this study should be useful for the detection and for the preclinical evaluation of new antifungal agents.     

Cytochrome c peroxidase contributes to the antioxidant defense of Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic pathogen and the leading cause of fungal meningitis. To survive within the host, this organism must be able to protect itself from oxidative stress. Cytochrome c peroxidase (Ccp1) is a mitochondrial antioxidant that catalyzes the degradation of hydrogen peroxide. In the present study, we characterized the contribution of the C. neoformans Ccp1 to antioxidant defense and for virulence. Consistent with studies of Ccp1 function in Sacchromyces cerevisiae, we found that Ccp1 contributes to resistance against exogenous oxidative stress in vitro. However, the oxidative stress phenotype does not diminish the virulence of ccp1 mutant strains in a murine model of C. neoformans disease. These results suggest that Ccp1 is involved in a complex system of protection against exogenous oxidative stress and that the elimination of this component of the antioxidant defense system does not diminish the virulence of C. neoformans.     

Synthesis and in vitro biological evaluation of mannose-containing prodrugs derived from clinically used HIV-protease inhibitors with improved transepithelial transport

In an approach to improve the pharmacological properties, safety and pharmacokinetic profiles, and their penetration into HIV reservoirs or sanctuaries, and consequently, the therapeutic potential of the current protease inhibitors (PIs) used in clinics, we investigated the synthesis of various mannose-substituted saquinavir, nelfinavir, and indinavir prodrugs, their in vitro stability with respect to hydrolysis, anti-HIV activity, cytotoxicity, and permeation through a monolayer of Caco-2 cells used as a model of the intestinal barrier. Mannose-derived conjugates were prepared in two steps, in good yields, by condensing an acid derivative of a protected mannose with the PIs, followed by deprotection of the sugar protecting group. With respect to hydrolysis, these PI prodrugs are chemically stable with half-life times in the 50-60 h range that are compatible with an in vivo utilization aimed at improving the absorption/penetration or accumulation of the prodrug in specific cells/tissues and liberation of the active free drug inside HIV-infected cells. These stabilities correlate closely with the low in vitro anti-HIV activity measured for those prodrugs wherein the coupling of mannose to the PIs was performed through the peptidomimetic PI's hydroxyl. Importantly, mannose conjugation to the PIs was further found to improve the absorptive transepithelial transport of saquinavir and indinavir but not of nelfinavir across Caco-2 cell monolayers, by contrast to glucose conjugation which had the opposite effect. The mannose-linked prodrugs of saquinavir and indinavir display therefore a most promising therapeutic potential provided that bioavailability, penetration into the HIV infected macrophages, and HIV-reservoirs of these PIs are improved.     

Enantioselectivity of inhibition of cytochrome P450 3A4 (CYP3A4) by ketoconazole: Testosterone and methadone as substrates

Racemic ketoconazole (KTZ) was the first orally active azole antifungal agent used in clinical practice and has become widely used in the treatment of mucosal fungal infections associated with AIDS immunosuppression and cancer chemotherapy. However, the use of KTZ has been limited because of adverse drug-drug interactions. KTZ blocks ergosterol biosynthesis by inhibiting the fungal cytochrome P450 (CYP51). KTZ is also a potent inhibitor of human cytochrome P450 3A4 (CYP3A4) enzyme, the major drug-metabolizing CYP isozyme in the human liver. We examined the enantioselective differences of KTZ in the inhibition of human CYP3A4 and in antifungal action. Dextro- and levo-KTZ exhibited modest enantioselective differences with respect to CYP3A4 inhibition of testosterone and methadone metabolism. For both substrates levo-KTZ was approximately a 2-fold more potent inhibitor. We examined the enantioselective differences in the in vitro activity of KTZ against medically relevant species of Candida and Aspergillus, as well as Cryptococcus neoformans. Overall, levo-KTZ was 2-4-fold more active than dextro-KTZ. Therefore, levo-KTZ is a more potent inhibitor of CYP3A4 and has stronger in vitro antifungal activity. Chirality 16:79-85, 2004.