A new nucleoside analogue, di(thymidine-3'-azido-2',3'-dideoxy-D-riboside)-5'-5'- p(1)-p(2)-pyrophosphate (AZTp(2)AZT), was designed and synthesized to overcome some limitations of conventional antiretroviral chemotherapy based upon nucleoside analogues (AZT, ddC, ddI). Indeed, the triphosphorylated form of these nucleoside analogues is able to inhibit the infectivity and replication of Human Immunodeficiency Virus (HIV-1), but the main problems in the use of these drugs concern their limited phosphorylation in some cells (e.g., macrophages) and their cytotoxic side effects. AZTp(2)AZT was encapsulated in human erythrocytes according to a conservative procedure of hypotonic shock-isotonic resealing and reannealing and showed a remarkable stability and a slow conversion to 5'-monophosphate (AZT-MP) and to AZT. This azidothymidine homodinucleotide seems to have chemical and biochemical properties enabling its profitable utilization in the erythrocyte-encapsulated form.

CD38, a lymphocyte differentiation antigen, is also a bifunctional enzyme catalyzing the synthesis of cyclic ADP-ribose (cADPR) from NAD+ and its hydrolysis to ADP-ribose (ADPR). An additional enzymatic activity of CD38 shared by monofunctional ADP-ribosyl cyclase from Aplysia californicais the exchange of the base group of NAD+ (nicotinamide) with various nucleophiles. Both human CD38 (either recombinant or purified from erythrocyte membranes) and Aplysia cyclase were found to catalyze the exchange of ADPR with the nicotinamide group of NAD+ leading to the formation of a dimeric ADPR ((ADPR)2). The dimeric structure of the enzymatic product, which was generated by recombinant CD38 and by CD38+Namalwa cells from as low as 10 μm NAD+, was demonstrated using specific enzyme treatments (dinucleotide pyrophosphatase and 5′-nucleotidase) and mass spectrometry analyses of the resulting products. The linkage between the two ADPR units of (ADPR)2 was identified as that between the N1of the adenine nucleus of one ADPR unit and the anomeric carbon of the terminal ribose of the second ADPR molecule by enzymatic analyses and by comparison with patterns of cADPR cleavage with Me2SO:tert-butoxide. Although (ADPR)2 itself did not release Ca2+ from sea urchin egg microsomal vesicles, it specifically potentiated the Ca2+-releasing activity of subthreshold concentrations of cADPR. Therefore, (ADPR)2 is a new product of CD38 that amplifies the Ca2+-mobilizing activity of cADPR.

Macrophages are important target cells for human immunodeficiency virus type 1 (HIV-1) infection. We have developed a drug targeting system for the selective delivery of phosphorylated nucleoside analogues to these phagocytosing cells. This system is based on the possibility of encapsulating the phosphorylated drugs into autologous erythrocytes and on the subsequent selective modification of their membranes to promote macrophage recognition and phagocytosis. Targeted delivery of phosphorylated nucleoside analogues to human, feline, and murine macrophages inhibits the infectivity of HIV-1, feline immunodeficiency virus, and LP-BM5 viruses more efficiently than the administration of the corresponding nucleoside analogues. In vivo administration of 2',3'-dideoxycytidine 5'-triphosphate (ddCTP) encapsulated into autologous erythrocytes to LP-BM5-infected mice was found to reduce infectivity and disease progression. Furthermore, the simultaneous administration of AZT or ddC produced additive antiviral effects. The possibility of using red cells as drug targeting systems was useful for the design, synthesis, and delivery of new antiviral nucleoside analogues. As a prototype of these new drugs, di-(thymidine-3'-azido-2',3'-dideoxy-D-riboside)-5'-5'-p1-p2-pyrophospha te (AZTp2AZT) was prepared. Although this drug in solution has the same antiviral activity as AZT, when administered encapsulated into erythrocytes it was several times more efficient in inhibiting the infectivity of human, feline, and murine immunodeficiency viruses. Thus, the availability of a drug targeting system for the selective delivery of antivirals to macrophages offers an additional possibility for the development of new drugs and of new combination antiviral therapies.

Mononuclear phagocytes, representing a known reservoir of HIV-1, feature a poor response to the nucleoside analogues currently used in AIDS therapy. The main reason is a weak phosphorylating activity to start metabolic activation that terminates with the intracellular synthesis of the retroviral reverse transcriptase inhibitors, i.e. the corresponding nucleoside triphosphates. The azidothymidine homodinucleotide (AZT(p2)AZT) recently designed and synthesized in our laboratories proved to be suitable to overcome this limitation. AZT(p2)AZT was encapsulated in erythrocytes where it showed a remarkable stability. This allowed to selectively perturb the membrane of the AZT(p2)AZT-loaded erythrocytes, thereby triggering subsequent opsonization and uptake by macrophage cultures. Macrophages exposed to carrier erythrocytes showed a significant resistance to infection by immunodeficiency viruses. Protection was observed with human, feline and murine macrophages against infection by HIV-1, FIV and LP-BM5 retroviral strains, respectively. These data demonstrate that AZT(p2)AZT-loaded and opsonized erythrocytes represent an efficient ''trojan horse'' resulting in significant protection of macrophages from retroviral infection.

Co-cultures of the murine macrophagic cell line RAW 264.7 with the L929 fibrosarcoma cell line, but not with the leukemia L1210 cell line, showed enhanced NO production over control RAW 264.7 cells. This potentiating effect, which was observed in detectably mycoplasma-free conditions and required low concentrations of recombinant murine IFN-γ, was due to soluble factors released from L929 cells and not to physical contact between the two cell types. The soluble factors were able to induce TNF-α in the macrophages and to potentiate the TNF-α release induced by IFN-γ. Increased generation of NO in RAW 264.7 cells co-cultured with L929 cells was prevented by a neutralizing anti-TNF-α antibody, suggesting that TNF-α is an autocrine factor for iNOS expression in these conditions. Also the L929 cell line showed a 4- to 5-fold enhanced NO production following co-culture with RAW 264.7 cells, thus indicating that exposure of tumor cells to macrophages can lead to an increased iNOS expression in tumor cells themselves.

The infectivity and replication of human (HIV-1), feline (FIV), and murine (LP-BM5) immunodeficiency viruses are all inhibited by several nucleoside analogues after intracellular conversion to their triphosphorylated derivatives. At the cellular level, the main problems in the use of these drugs concern their limited phosphorylation in some cells (e.g., macrophages) and the cytotoxic side effects of nucleoside analogue triphosphates. To overcome these limitations a new nucleoside analogue homodinucleotide, di(thymidine-3'-azido-2',3'-dideoxy-D-riboside)-5'-5'-p1-p2-pyrophosphat e (AZTp2AZT), was designed and synthesized. AZTp2AZT was a poor in vitro inhibitor of HIV reverse transcriptase, although it showed antiviral and cytotoxic activities comparable to those of the parent AZT when added to cultures of a HTLV-1 transformed cell line. AZTp2AZT encapsulated into erythrocytes was remarkably stable. Induction of erythrocyte-membrane protein clusterization and subsequent phagocytosis of AZTp2AZT-loaded cells allowed the targeted delivery of this impermeant drug to macrophages where its metabolic activation occurs. The addition of AZTp2AZT-loaded erythrocytes to human, feline, and murine macrophages afforded almost complete in vitro protection of these cells from infection by HIVBa-L, FIV, and LP-BM5, respectively. Therefore, AZTp2AZT, unlike the membrane-diffusing azidothymidine, acts as a very efficient antiretroviral prodrug following selective targeting to macrophages by means of loaded erythrocytes.

CD38 is a transmembrane glycoprotein involved as an orphan receptor in many physiological processes of lymphocytes. It is also a bifunctional enzyme that catalyzes at its ectocellular domain the synthesis from NAD+ (cyclase) and the hydrolysis (hydrolase) of the calcium-mobilizing metabolite cyclic ADP-ribose (cADPR). A still unexplained paradox concerns the relationship between ectocellular localization of CD38 and intracellular calcium-releasing activity of its intermediate product cADPR. Incubation of CD38+ human Namalwa B cells with external NAD+ elicited extensive membrane down-regulation of CD38 and its internalization in non-clathrin-coated vesicles. Since the internalized CD38 was demonstrated to be enzymatically active, this NAD+-dependent process is a hitherto unrecognized means for shifting cADPR metabolism from the cell surface to the intracellular environment.

CD38, a type II transmembrane glycoprotein predominantly expressed in blood cells, is a bifunctional ectoenzyme directly involved in the metabolism of cADP-ribose (cADPR). This is a potent Ca2+ mobilizer in several types of cells. The relationship between the ectocellular site of cADPR production and its intracellular calcium-related functions is poorly understood. Cultured rat cerebellar granule cells showed both enzymic activities of CD38, ADP-ribosyl cyclase and cADPR hydrolase, at a ratio of 16 to 1 respectively, and were immunostained by the anti-(human CD38) monoclonal antibody IB4. In these cells externally added cADPR and beta-NAD+ (the precursor of cADPR), but not alpha-NAD+ or ADP-ribose, enhanced the peak of the depolarization-induced rise in intracellular Ca2+ concentration. This effect was inhibited by 1 microM ryanodine, suggesting a potentiation of calcium-induced calcium release by cADPR. CD38 ectoenzyme activities, ADP-ribosyl cyclase and cADPR hydrolase, were also demonstrated in vivo by microdialysis of adult rat cerebellum, where IB4 bound to granule neurons selectively. Trace amounts (11.5 +/- 3.8 nM) of NAD+ were detected by microdialysis sampling and sensitive assays in the basal interstitial fluid of the cerebellum. These results provide a link between ectocellular cADPR turnover and intracellular calcium mobilization in cerebellum.

Human CD38 is a transmembrane glycoprotein involved in lymphocyte activation and adhesion to endothelium, The ectocellular domain of the molecule possesses properties of a bifunctional enzyme catalyzing both the synthesis from NAD(+) and the hydrolysis of the calcium-releasing metabolite cyclic ADP-ribose (cADPR), Surface expression of CD38 (mCD38) is rapidly and almost completely down-modulated upon ligation by specific mAb in cells from different lineages, The data presented here also show that, in addition to the existence of a mCD38, a soluble form of CD38 (sCD38) is detectable in the cell culture supernatant of allo-activated T lymphocytes and of several tumor cell lines. sCD38 is also present in vivo and is assayable in normal (fetal serum and amniotic fluid) and pathological (serum and ascites from patients with multiple myeloma, and serum from patients with AIDS) biological fluids, Immunoaffinity chromatography, SDS-PAGE and Western blot analyses with mAb and polyclonal antibodies, along with metabolic labeling, yield a body of data concerning the structure of sCD38, which displays a M(r) of 39 kDa, Native sCD38 maintains the ability to inhibit the binding activity of different anti-CD38 mAb and still catalyzes the synthesis and the hydrolysis of cADPR at the same ratio observed with mCD38. Furthermore, cross-linking experiments indicate that the purified soluble molecule binds a 120 kDa molecule expressed by monocytoid cells and identified as a candidate ligand for human mCD38.

A new Azidothymidine derivative, di-(thymidine-3′-azido-2′,3′-dideoxy-D-riboside)-5′-5′-p1-p2-pyrophosphate (AZTp2AZT), was encapsulated in human erythrocytes according to a conservative procedure of hypotonic shock-isotonic resealing and reannealing. Like in erythrocyte lysates supplemented with 1 mM ATP, intact red cells too were found to convert AZTp2AZT to 3′-Azido-3′-deoxythymidine which was then released linearly in plasma. The major metabolic pathway involved in this conversion was the symmetrical hydrolysis of AZTp2AZT to yield two 3′-Azido-3′-deoxythymidine-5′-phosphate molecules which were then dephosphorylated to 3′-Azido-3′-deoxythymidine. At late times of incubation, also a limited asymmetrical hydrolysis of AZTp2AZT became apparent in the intact erythrocytes, yielding 3′-Azido-3′-deoxythymidine-5′-diphosphate that was then converted to the triphosphorylated derivative. Therefore, erythrocytes loaded with AZTp2AZT act“in vitro” as bioreactors ensuring sustained and potentially useful release of 3′-Azido-3′-deoxythymi-dine.

Human CD38, a type II cell surface glycoprotein, is a bifunctional ectoenzyme catalyzing both ADP-ribosyl cyclase and cyclic ADP-ribose (cADPR) hydrolase reactions. It shares a high degree of sequence homology with the cyclase from Aplysia species and studies of site-directed mutagenesis have recently demonstrated the importance, but not elucidated the role, of several cysteine residues highly conserved between these proteins. N-Ethylmaleimide, iodoacetamide and thiol-oxidizing reagents failed to affect either the cyclase or the weaker hydrolase activity of the Aplysia californica protein. Likewise, these reagents did not impair the two activities of CD38 purified from human erythrocytes. beta-mercaptoethanol had no effect on the Aplysia enzyme activities, while it inactivated both the cyclase and the cADPR hydrolase of CD38 by inducing its extensive oligomerization. In intact erythrocytes the beta-mercaptoethanol-dependent enzyme inactivation was completely prevented by prior cross-linking of the membrane proteins with glutaraldehyde. These data demonstrate that none of the cysteine residues plays any direct catalytic role in CD38 and Aplysia proteins, and that disulfide bridges are essential for maintaining the monomeric, catalytically active structure of CD38.

Human erythrocytes contain a nicotinamide adenine dinucleotide phosphate (NADP[H])-binding protein, FX, whose levels are significantly increased in erythrocytes from glucose-6-phosphate dehydrogenase (G6PD)- deficient individuals bearing the mediterranean variant of G6PD. Elucidation of the still unknown biologic functions of FX was approached by means of amino acid sequencing of its 25 tryptic peptides. Searching in the EMBL data bank allowed identification of extensive homology between these tryptic peptides and all sequence- aligned regions encompassing the complete structure of a putative protein encoded by the P35B gene in the mouse. This gene, which differs from the normal allele by a point mutation, has been previously cloned from a tum- variant of the murine tumor cell line P815, so defined because it is associated with low tumorigenicity compared with the progenitor P815. The reported P35B cDNA contains an open reading frame (ORF) of 813 bp and encodes a putative protein of 271 amino acids (30 kD), whereas FX protein is 320 amino acids in length (35.81 kD, in good agreement with previous studies). However, a single base shift at position 4,752 of the P35B gene suppresses the stop codon after Phe 271 and allows continuation of the ORF for up to 320 amino acids to reach the same length as FX. The remarkably high extent (92%) of homology indicates that erythrocyte FX protein is the human homolog of the P35B gene product.

Extracellular ATP potentiates, by activation of P2y-type purinergic receptors, the production of NO induced by low doses of lipopolysaccharide (LPS) in the murine macrophagic cell line RAW 264.7 (Tonetti et al. (1994) Biochem. Biophys. Res. Commun. 203, 430-434). Release of TNF-α, known to be an autocrine factor for iNOS expression, was enhanced, too, following exposure of either LPS-induced or uninduced cells to externally added micromolar ATP. Reverse transcription-PCR experiments showed that extracellular ATP increases mRNA levels of both inducible NO synthase (iNOS) and of TNF-α to extents comparable to those of enzymatic and biological activities, respectively. These data demonstrate that activation of purinergic receptors by extracellular ATP results in an enhanced expression of the iNOS and TNF-α genes.

Carboplatin (CBDCA) and the pyrimidine aglycone divicine displayed cytotoxic effects of murine erythroleukemic cells (MELC), with ID50 values of 158 and 37 microM, respectively. Combination of CBDCA and divicine, at a 2:1 ratio, increased cytotoxicity considerably. Under specific conditions of time schedule of administration, the association of CBDCA and divicine resulted in a clear synergistic activity. Alkaline elution studies on both unirradiated and gamma-irradiated MELC demonstrated opposite patterns of DNA damage with the two molecules. Thus, CBDCA elicited DNA interstrand crosslinks (ISC), while divicine resulted in DNA single strand breaks (SSB). Association of both molecules led in the unirradiated cells to a higher SSB frequency than recorded with divicine alone. Accordingly, intracellular activation of CDBCA by redox cycling of divicine seems not to be involved. Rather, intracellular platinum appears to enhance cytotoxicity of divicine.

Inducible nitric oxide synthase (iNOS) activity in the murine macrophage cell line RAW 264.7 was increased from two- to four-fold after co-exposure of the cells to low doses of bacterial lipopolysaccharide (LPS) and micromolar ATP, compared to LPS alone. Extracellular ATP and its analogs "per se", i.e. without LPS, were not able to induce iNOS activity. The stimulating effect of UTP too, the concentration range of activity (1-100 mM nucleotides) and the rank of potency (ATP-γ-S = AMP-PNP > ATP = ADP ⪢ AMP-CPP = UTP) seem to indicate an involvement of P2y-type purinergic receptors. GTP, CTP and adenosine were virtually ineffective. These data suggest that binding of extracellular nucleotides to purinergic receptors may increase nitric oxide production by macrophages. This effect might occur in pathological conditions (i.e. inflammation/infection or trauma) where significant amounts of intracellular ATP can be released due to cellular damage.