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Recent developments in the HIV neuropathies.

PURPOSE OF REVIEW: With the introduction of highly active antiretroviral therapy peripheral neuropathies have become the most common neurological complications in HIV infection. The frequency and spectrum of these neuropathies are changing, as the various toxic and immune factors are modified by new treatment strategies. Recent studies have provided a better understanding of the risk factors, markers and relevant pathogenic mechanisms, and a thorough review of these is critical for an improved understanding of this important and increasingly common complication. RECENT FINDINGS: The combined use of dideoxynucleosides, in association with immune-mediated mechanisms triggered by HIV infection, are critical in the development of distal sensory polyneuropathy. Valuable markers of neuropathy such as intraepidermal nerve fiber density from skin biopsies have been validated and promise to be a valuable tool in the detection and monitoring of distal sensory polyneuropathy. Markers of virological activity have also been associated with the severity of neuropathic pain in distal sensory polyneuropathy. In some instances, the enhanced viral suppression from antiretroviral agents may actually improve or decrease the frequency of certain types of neuropathy. New evidence supports mitochondrial toxicity as a principal mechanism for dideoxynucleoside-associated sensory neuropathy, and questions arise about enhanced risk with pre-existing mitochondrial defects. Confirmed treatments are limited to the reduction of symptoms, with a need for the further investigation of corrective therapies. SUMMARY: Increased and improved surveillance for HIV-associated neuropathy will allow earlier interventions to improve quality of life and prevent severe toxicities. A better understanding of the prevailing mechanisms will allow for more effective interventions.

Biomarkers↗

An alpha-like DNA polymerase from Halobacterium halobium.

Two DNA polymerases have been isolated from extracts of Halobacterium halobium, one having a sedimentation coefficient of 11 S, designated as alpha-like polymerase and possessing the following characteristics. It is sensitive to both aphidicolin and N-ethylmaleimide but indifferent to the presence of a dideoxynucleoside triphosphate. Therefore this polymerase is very similar to the alpha DNA polymerase of eukaryotes. The enzyme requires 5 M NaCl for maximum activity. The other polymerase has a sedimentation coefficient of 4.4 S and is resistant to both aphidicolin and N-ethylmaleimide. However, it is inhibited by a dideoxynucleoside triphosphate.

Aphidicolin↗

Didanosine and zidovudine resistance patterns in clinical isolates of human immunodeficiency virus type 1 as determined by a replication endpoint concentration assay.

Reports of in vitro resistance of human immunodeficiency virus type 1 (HIV-1) to zidovudine (AZT) have raised concerns about the development of resistance to other dideoxynucleosides in clinical use. To address this, we have developed a screening assay which supports the growth of clinical isolates and have applied this to a series of paired isolates from patients entered into a phase I trial of didanosine (DDI). Thirteen patients (10 with AIDS, 3 with AIDS-related complex) who had been exposed to AZT for a mean of 6.5 months (range, 1 to 13 months) were treated with DDI at 750 mg/day. Paired isolates were obtained pretherapy and after a mean of 58 weeks (range, 21 to 90) of DDI therapy by coculture of peripheral blood mononuclear leukocytes (PBLs) with phytohemagglutinin-stimulated donor PBLs. Isolates were passaged only one additional time in PBLs and then tested in parallel in a microtiter assay with phytohemagglutinin-stimulated donor PBLs as targets. PBLs were infected with 10(5) 50% tissue culture infectious doses per 10(7) cells and exposed to DDI (1 to 50 microM) or AZT (0.01 to 100 microM), and supernatants were assayed for the HIV p24 antigen at 7 days postinfection. Control AZT-susceptible and resistant isolates were included. The median pre- and posttherapy DDI susceptibilities of the 13 pairs of isolates were 10.0 microM (range, 1 to 25 microM) and 17.5 microM (range, 2.5 to 50 microM), respectively (P = 0.036; Wilcoxon signed-rank test). These studies thus indicated that (i) the susceptibility to DDI tends to mildly decrease with drug exposure; (ii) the susceptibility to AZT improves with time off AZT; (iii) baseline susceptibilities to DDI have a wide range, and the CD4 response may correlate with the initial susceptibility; and (iv) a PBL-based microtiter assay is useful for screening clinical isolated for dideoxynucleoside susceptibility profiles.

Didanosine↗

In vitro induction of human immunodeficiency virus type 1 variants resistant to 2'-beta-Fluoro-2',3'-dideoxyadenosine.

2'-beta-Fluoro-2',3'-dideoxyadenosine (F-ddA) is an acid-stable purine dideoxynucleoside analog active against a wide spectrum of human immunodeficiency virus type 1 (HIV-1) and HIV-2 strains in vitro. F-ddA is presently undergoing a phase I clinical trial at the National Cancer Institute. We induced HIV-1 variants resistant to F-ddA by exposing wild-type HIV-1 (HIV-1LAI) to increasing concentrations of F-ddA in vitro. After 18 passages, the virus was fourfold less sensitive to F-ddA than HIV-1LAI. Sequence analyses of the passage 18 virus revealed changes in three amino acids in the reverse transcriptase (RT)-encoding region of the pol gene: P to S at codon 119 (P119S; present in 3 of 13 and 28 of 28 molecular clones before and after F-ddA exposure, respectively), V179D (0 of 13 and 9 of 28, respectively), and L214F (9 of 13 and 28 of 28, respectively). Drug sensitivity assays using recombinant infectious clones confirmed that P119S was directly responsible for the reduced sensitivity of HIV-1 to F-ddA. Various infectious clones with single or multiple amino acid substitutions conferring viral resistance against nucleoside RT inhibitors, including HIV-1 variants with multi-dideoxynucleoside resistance, were generally sensitive to F-ddA. The moderate level of resistance of HIV-1 to F-ddA, together with the lack of conferment of significant cross-resistance by the F-ddA-associated amino acid substitutions, warrants further investigation of F-ddA as a potential antiviral agent for use in treatment of HIV-1 infection.

Animals↗

Pharmacological inhibition of in vitro infectivity of human T lymphotropic virus type I.

Human T lymphotropic virus type I (HTLV-I) is an exogenous RNA tumor virus etiologically linked to adult T cell leukemia and related diseases. In this paper, we describe that two 2',3'-dideoxynucleoside analogues, erythro 3'-azido-2',3'-dideoxythymidine (also called azidothymidine) and 2',3'-dideoxycytidine can inhibit the infectivity of HTLV-I against helper/inducer T cells in vitro. Both 2',3'-dideoxynucleoside analogues inhibited the overgrowth of target T cells, which was a consequence of virally mediated transformation, when they were exposed to the virus and cultured with the compounds. A profound decrease in the expression of HTLV-I gag-proteins was also observed. Moreover, we observed that the amount of proviral DNA detected in cellular DNA from the target T cells was substantially reduced when the cells were protected by the compounds against the virus and that at certain concentrations of the compounds the synthesis of viral DNA was completely suppressed. These results may be of value in developing a new pharmacological strategy for preventing the replication and possibly blocking the transmission of HTLV-I and related retroviruses in human beings.

DNA, Viral↗

Establishment of a rodent model of HIV-associated sensory neuropathy.

Human immunodeficiency virus (HIV)-associated sensory neuropathy (SN) is the most common neurological complication of HIV infection in the current highly active antiretroviral therapy era. The painful sensory neuropathy is associated with the use of dideoxynucleoside antiretrovirals, and its development limits the choice of antiretroviral drugs in affected patients. There are presently no effective therapies for HIV-SN, and moreover there has been no robust animal model of HIV-SN in which candidate therapeutic agents can be tested. In this paper, we show that we have established a rodent model of HIV-SN by oral administration of a dideoxynucleoside drug, didanosine, to transgenic mice expressing the HIV coat protein gp120 under a GFAP promoter. The neuropathy in these rodents is characterized by distal degeneration of unmyelinated sensory axons, similar to the "dying back" pattern of C-fiber loss seen in patients with HIV-SN. This model will be useful in examining mechanisms of distal axonal degeneration and testing potential neuroprotective compounds that may prevent development of the sensory neuropathy.

Animals↗

New synthetic approach to nucleoside phosphorofluoridates and fluoridites. Novel type of phosphitylating reagents containing 4-nitrophenoxy group.

New DNA analogues which have -OP(O)F-O internucleotide linkage are prepared by two methods. One route is based on transformation of a deoxynucleoside phosphoroimidazolidate by benzoylfluoride into the corresponding phosphorofluoridate. Alternative route begins with condensing of a new type of phosphoroamidites containing 4-nitrophenoxy group: N,N,N',-tetraisopropyl-O-4-nitrophenylphosphorodiamidite 9 to yield the dideoxynucleoside-4-nitrophenylphosphites which are readily converted into the corresponding dideoxynucleoside phosphorofluoridates and their sulfur analogues. Second part of this paper illustrates application of the N,N-diisopropyl-O-methyl-O-4-nitrophenylphosphoroamidite 12 and N,N-diisopropyl-O-4-nitrophenyl-P-methylphosphonoamidite 16 in efficient synthesis of dideoxynucleotides and their thio- and selenoanalogues.

Amides↗

[A new drug in a new role: dipyridamole in the treatment of HIV-1 infections?].

Dipyridamole (DP) is a widely used coronary vasodilator and antithrombotic drug. The presented experiments demonstrate that DP inhibits the replication of HIV-1 and markedly potentiates the anti-HIV activity of azidothymidine (AZT) and dideoxycytidine in CD4-positive cells (monocyte-macrophages and T-lymphocytes). At the same time DP does not potentiate the bone marrow toxicity of AZT, and, in CEM-ss lymphoblastoid cells, it significantly suppresses the cytotoxicity of AZT. Studies on the mechanism of these effects suggest that DP inhibits the intracellular phosphorylation of physiological nucleosides, whereas it does not affect phosphorylation of AZT and other antiviral dideoxynucleoside drugs. This may lead to relative enhancement of the metabolic activation of dideoxynucleoside drugs and the inhibitory action of their active, triphosphate form on HIV reverse transcriptase. Studies comparing the binding of DP to proteins in tissue culture media and in human plasma suggest that in order to achieve significant antiviral potentiation in vivo, high doses of DP will be required.

Acquired Immunodeficiency Syndrome↗

Perspectives for the chemotherapy of AIDS.

In the design of selective inhibitors of the human immunodeficiency virus (HIV), the etiologic agent of AIDS, various steps of the virus replicative cycle could be envisaged as targets, i.e. virus adsorption to its cellular receptor (or another early event in virus replication such as penetration or uncoating), transcription of the viral RNA genome to proviral DNA (reverse transcription), trans-activation of viral mRNA transcription and translation, and, finally, virus release ("budding", or another late event in virus replication such as the assembly process). Although some potent HIV inhibitors such as heparin and dextran sulfate may interfere with an early step of the virus replicative cycle (adsorption) and others (interferon and interferon inducers) are assumed to act at a late step (budding), the majority of the anti-HIV agents appear to act at the reverse transcriptase level. Most of these reverse transcriptase inhibitors belong to the class of the 2',3'-dideoxynucleosides (ddN), and within this class of compounds a variety of 2',3'-dideoxy-, 2',3'-didehydro-2',3'-dideoxy-, 3'-azido-2',3'-dideoxy- and 3'-fluoro-2',3'-dideoxyribosides of both purines and pyrimidines have been described as potent and selective anti-HIV agents. Akin to 3'-azido-2',3'-dideoxythymidine (AZT), the sole anti-HIV compound that has so far been licensed for clinical use in the treatment of AIDS, all other ddN analogues are postulated to interact as competitive inhibitors (with respect to the natural substrates) and/or chain terminators of the HIV reverse transcriptase. To do so, the ddN analogues need first to be phosphorylated by cellular kinases to the corresponding 5'-triphosphates (ddNTPs), and together with the affinity of the ddNTPs for the HIV reverse transcriptase (relative to their affinity for the cellular DNA polymerases), the extent by which the ddNs are phosphorylated to the ddNTPs are critical determinants of their potency and selectivity as anti-HIV agents. Much more remains to be learned about the in vivo efficacy of the 2',3'-dideoxynucleoside analogues, and their pharmacokinetic and toxicological properties, before their true potential in the treatment of AIDS can be fully assessed.

Acquired Immunodeficiency Syndrome↗

[Inhibition of various stages of DNA replication in sea urchin embryos].

The embryos of the sea urchin Strongylocentrotus intermedius possess the ability to incorporate into their DNAs 2'-deoxynucleosides together with all their bases, i.e., adenine, guanine, cytosine and thymine. This incorporation is inhibited by 3'-amino-2',3'-dideoxynucleosides with the same bases. 5'-Amino-5'-deoxynucleosides and 5'-amino-2',5'-dideoxynucleosides moderately inhibit the incorporation of [3H]2'-deoxynucleosides into the DNAs by competing with the latter, presumably at the phosphorylation stage. The most potent inhibiting effect is exerted by 2'-amino-2'-deoxynucleosides and 2'-asido-2'-deoxynucleosides; the mechanism of this inhibition is still obscure, however.

Animals↗

Characterization of 2',3'-dideoxycytidine diphosphocholine and 2',3'-dideoxycytidine diphosphoethanolamine. Prominent phosphodiester metabolites of the anti-HIV nucleoside 2',3'-dideoxycytidine.

2',3'-Dideoxycytidine (ddCyd) is among the most potent of the anti-human immunodeficiency virus (HIV) agents of the dideoxynucleoside class. Its pharmacologically active metabolite 2',3'-dideoxycytidine 5'-triphosphate (ddCTP) is an effective inhibitor of HIV reverse transcriptase and thus of HIV replication. ddCyd differs, however, from other dideoxynucleoside agents such as 3'-azido-3'-deoxythymidine and 2',3'-dideoxyinosine in its capacity to generate phosphodiester metabolites (i.e. ddCDP choline and ddCDP ethanolamine). We have synthesized and characterized these two diesters, and established their identity with the metabolites formed in ddCyd-treated Molt-4 cells. Toward this end, the biologically generated metabolites have been isolated on a preparative scale and compared with the synthetic compounds mass spectroscopically, chromatographically, and enzymatically (i.e. their relative susceptibility to the catabolic enzymes alkaline phosphatase and venom phosphodiesterase). The concentration reached by each of these two phosphodiesters within cells can, under certain conditions, equal or exceed that of ddCTP, and their half-times of disappearance are long, indicating that they may serve as depot forms of ddCyd. The possible role of these phosphodiesters in contributing to the unusual toxicity of ddCyd is discussed.

Cells, Cultured↗

5-Amino-4-imidazolecarboxamide riboside potentiates the metabolism and anti-human immunodeficiency virus activity of 2',3'-dideoxyinosine.

The antiviral activity of the purine dideoxynucleosides 2',3'-dideoxyadenosine (ddA) and 2',3'-dideoxyinosine (ddI) is dependent on their conversion into ddA triphosphate in vivo. 5-Amino-4-imidazolecarboxamide riboside (AICA riboside), a natural metabolite in purine biosynthetic pathways, is converted into IMP, a substrate for the biosynthesis of adenine and guanine nucleotides, and enhances the intracellular purine nucleotide pools. Because IMP also serves as a phosphate donor in the anabolic phosphorylation of ddI (and ddA) into ddI monophosphate by the cytosolic enzyme 5'-nucleotidase, we investigated the effects of AICA riboside on the phosphorylation and antiretroviral activity of these purine nucleoside analogs. At an AICA riboside concentration of 0.5 mM, there was a approximately 2-fold increase in the intracellular ATP and GTP levels, whereas a nearly 8-fold increase was observed for the phosphorylation of ddA (or ddI). A marked reduction in intracellular pools of the pyrimidine nucleotides CTP and UTP was observed in AICA riboside-treated cells and inhibited cell proliferation. However, this growth inhibition was prevented by the addition of uridine to the cultures. Cells pretreated with AICA riboside and ddI were less susceptible to human immunodeficiency virus (HIV) infection and synthesized reduced levels of HIV proviral DNA. A 10-fold potentiation of the effectiveness of ddI against both wild-type HIV (HIVIIIB) and a ddI-resistant variant HIV was observed in the presence of 0.5 mM AICA riboside. These results show that AICA riboside modulates the anabolism and antiviral activity of ddI, and they have implications for possible therapies with dideoxynucleosides.

Aminoimidazole Carboxamide↗

Uptake and distribution of 2',3'-dideoxyinosine and its derivatives in a human monocytoid cell line.

The dideoxynucleoside analogue 2',3'-dideoxyinosine (ddI) has been used in the clinic as an alternative drug to zidovudine (AZT) in the treatment of patients with the acquired immunodeficiency syndrome (AIDS). However, it shows significant and variable toxicity in patients. It is known that various dideoxynucleoside analogues can cause the termination of the DNA chain following incorporation of the corresponding triphosphate metabolite by the polymerases. In the case of ddI, the presumed active metabolite is 2',3'-dideoxyadenosine 5'-triphosphate (ddATP). In order to understand the molecular basis for the toxicity of ddI, we evaluated the relationship between the intracellular formation of ddATP, its incorporation into cellular DNA and the effects on the growth of U937 cells, a human monocytoid cell line. Dideoxyinosine was not significantly toxic to U937 cells at concentrations as high as 500 microM in a 72 hrs. growth inhibition assay. The results of the uptake of 3HddI in this cell line showed a proportional increase in total metabolites with increasing concentrations of the drug (1-20 microM) after a 24 hrs. exposure. Incubation with 10 microM 3HddI resulted in the formation of low levels of ddATP within a period of 2 hrs. A significant amount of ddI-derived radioactivity was found in both DNA and RNA after exposure to 10 microM 3HddI for 24 to 72 hrs. However, no evidence of incorporation of ddATP into the cellular DNA fraction was obtained in these experimental conditions. Therefore, the lack of significant toxicity of ddI to U937 cells can be explained, at least in part, by its inability to incorporate ddATP into its cellular DNA at the doses studied.

Biological Transport↗

Lack of pharmacokinetic interaction between dipyridamole and zalcitabine in rats.

Resistance usually manifests following long-term dideoxynucleoside therapy of HIV-1 infection. This period appears to coincide with reduced dosage regimens. Resistance that is associated with long-term monotherapy may, in part, be due to decreased intracellular drug concentrations. It has been reported that intracellular uptake of the dideoxynucleosides is enhanced by dipyridamole. Hence, dipyridamole may potentially be used to optimize the effects of zalcitabine in HIV-1 antiretroviral "cocktail". The purpose of this study was to characterize the pharmacokinetics of zalcitabine when administered alone and concomitantly with dipyridamole. Also, we determined, indirectly, whether dipyridamole modulated the intracellular uptake of zalcitabine. Rats were intravenously administered either zalcitabine 100 mg/kg alone or with dipyridamole 15 mg/kg. Except renal clearance (CIR), there were no statistically significant differences in the pharmacokinetic parameters including the steady-state volume of distribution and distribution coefficient. Zalcitabine plasma concentrations declined rapidly in a bi-exponential fashion, with a terminal half-life of 1.03 +/- 0.18 hr. alone versus 1.08 +/- 0.22 hr. with dipyridamole. The area under the concentration-time curve was not significantly different with or without dipyridamole. ClR, was 1.42 +/- 0.37 l/hr./kg for zalcitabine alone versus 1.09 +/- 0.28 l/hr./kg with dipyridamole. Our single dose study show that zalcitabine disposition kinetics were not significantly modulated by dipyridamole.

Animals↗

A novel Raman spectrophotometric method for quantitative measurement of nucleoside triphosphate hydrolysis.

A novel spectrophotometric method, based upon Raman spectroscopy, has been developed for accurate quantitative determination of nucleoside triphosphate phosphohydrolase (NTPase) activity. The method relies upon simultaneous measurement in real time of the intensities of Raman marker bands diagnostic of the triphosphate (1115 cm(-1)) and diphosphate (1085 cm(-1)) moieties of the NTPase substrate and product, respectively. The reliability of the method is demonstrated for the NTPase-active RNA-packaging enzyme (protein P4) of bacteriophage phi6, for which comparative NTPase activities have been estimated independently by radiolabeling assays. The Raman-determined rate for adenosine triphosphate substrate (8.6 +/- 1.3 micromol x mg(-1) x min(-1) at 40 degrees C) is in good agreement with previous estimates. The versatility of the Raman method is demonstrated by its applicability to a variety of nucleotide substrates of P4, including the natural ribonucleoside triphosphates (ATP, GTP) and dideoxynucleoside triphosphates (ddATP, ddGTP). Advantages of the present protocol include conservative sample requirements (approximately 10(-6) g enzyme/protocol) and relative ease of data collection and analysis. The latter conveniences are particularly advantageous for the measurement of activation energies of phosphohydrolase activity.

Acid Anhydride Hydrolases↗

Separation of dideoxyribonucleosides in trace amounts by automated liquid chromatography and capillary electrophoresis.

No satisfactory high-performance liquid chromatographic (HPLC) method is currently available for the separation of the major dideoxyribonucleosides (ddNs) and their derivatives. A method involving HPLC has been developed for the separation of five major ddNs [ddA, ddC, ddI, azT and 2',3'-dideoxy-2',3'-didehydrothymidine (d4T)]. Elution of the common and modified components of DNA was also examined under the selected separation conditions of HPLC. The elution characteristics of these compounds were studied using serum plasma samples spiked with ddN derivatives. In addition, capillary electrophoresis (CE) was investigated for the separation of ddNs and their derivatives. Picomolar amounts of the five major ddNs and the metabolic product of azT [5'-O-glucuronide-3'-azido-3'-deoxythymidine (Glo-azT)] were satisfactorily resolved in 10 min by using a modification of CE. The spectral properties of the ddNs were characterized under different pH conditions and compared with those of their parent deoxyribonucleosides (dNs) because these compounds are commonly measured in HPLC by their spectral properties. The spectra of ddC and ddT derivatives resemble very closely those of dC and dT, but those of ddA and ddI differ to some extent from their parent dNs. The HPLC method was extensively examined for satisfactory resolutions of these compounds. For example, an isocratic elution method, although simple, failed to resolve these compounds and ion-pair chromatography did not offer any advantage. Gradient elution involving buffered solutions and increasing amounts of an organic modifier yielded satisfactory results. Methanol appeared to be the organic modifier of choice. A reversed-phase matrix with smaller than octadecyl alkyl chains did not produce the necessary interactions. Uniform spherical beads of smaller diameter produced superior resolutions. The separation of these compounds on three commercially available columns is discussed. The separation of human plasma samples spiked with dideoxynucleoside derivatives by HPLC was accomplished in ca. 16 min. The presence of the dNs did not interfere in their separations.

Capillary Action↗

Carbovir: the (-) enantiomer is a potent and selective antiviral agent against human immunodeficiency virus in vitro.

In this paper we describe the in vitro antiviral activity of the (-) enantiomer of carbocyclic 2',3'-deoxydidehydroguanosine, (-) carbovir, a nucleoside analogue that has selective and potent anti-HIV activity in a series of lymphocyte culture systems. The cellular cytotoxicity of this compound has also been evaluated in a number of systems and compared to the saturated dideoxynucleoside analogues AZT and ddC.

CD4-Positive T-Lymphocytes↗

Lack of synergy in the inhibition of HIV-1 reverse transcriptase by combinations of the 5'-triphosphates of various anti-HIV nucleoside analogs.

3'-Deoxy-3'-azidothymidine (AZT) has been shown to synergistically inhibit the replication of human immunodeficiency virus type 1 (HIV-1) in cell culture when combined with several other 2',3'-dideoxynucleoside analogs. In an effort to understand the biochemical mechanism of this synergy, we have examined the effect of combinations of the 5'-triphosphate of AZT (AZT-TP) with either ddCTP, ddATP, or the 5'-triphosphate of the carbocyclic analog of 2',3'-didehydro-2',3'-dideoxyguanosine (carbovir) on both the RNA-directed and DNA-directed DNA polymerase activity of HIV-1 reverse transcriptase. Kinetic studies, which evaluated the ability of these combinations to competitively inhibit the enzyme, showed that AZT-TP could not bind to the enzyme with either the RNA or DNA template at the same time as either of the other three inhibitors. None of these analogs could affect the incorporation of another analog into the DNA chain by the HIV-1 reverse transcriptase. These results indicated that synergistic inhibition of the HIV-1 reverse transcriptase is not responsible for the synergistic antiviral activity seen in cell culture with combinations of these nucleoside analogs.

Antiviral Agents↗