Bicyclams, in which the cyclam (1,4,8,11-tetraazacyclotetradecane) moieties are tethered via an aliphatic bridge (i.e., propylene, as in JM2763) are potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) (E. De Clercq, N. Yamamoto, R. Pauwels, M. Baba, D. Schols, H. Nakashima, J. Balzarini, Z. Debyser, B. A. Murrer, D. Schwartz, D. Thornton, G. Bridger, S. Fricker, G. Henson, M. Abrams, and D. Picker, Proc. Natl. Acad. Sci. USA 89:5286-5290, 1992). We have now found that the bicyclam JM3100, in which the cyclam moieties are tethered by an aromatic bridge [i.e., phenylenebis(methylene)], inhibits the replication of various HIV-1 and HIV-2 strains in various cell lines at a 50% effective concentration (EC50) of 1 to 10 ng/ml, which is about 100-fold lower than the concentration required for JM2763 to inhibit HIV replication and at least 100,000-fold lower than the cytotoxic concentration (> 500 micrograms/ml). In primary T4 lymphocytes or primary monocytes, JM3100 proved inhibitory to HIV-1(IIIB) and several clinical HIV-1 isolates at an EC50 of less than 1 ng/ml. On the basis of time-of-addition experiments, JM3100 appeared to interact with a viral uncoating event, and this was further corroborated by an uncoating assay in which RNase sensitivity of [5-3H]uridine-labeled virions was monitored. In addition, but possibly mechanistically related, JM3100 blocks formation of infectious particles. JM3100 was also found to interfere directly with virus-induced syncytium formation, albeit at a higher concentration (1 to 2 microgram/ml) than that required for inhibition of viral replication. Following subcutaneous injection of 10 mg of JM3100 per kg of body weight to rabbits, anti-HIV activity was detected in serum corresponding to serum drug levels exceeding for at least 6 h by >100-fold the EC(50) required to inhibit HIV replication in vitro. When combined with either 3'-azido-2',3' -dideoxythymidine or 2',3' -dideoxyinosine, JM3100 achieved a additive inhibition of HIV replication, and when repeatedly subcultivated in the presence of JM3100, the virus remained sensitive to the compound for at least 30 passages (120 days) in cell culture.

A series of bicyclams have been shown to be potent and selective inhibitors of human immunodeficiency virus (HIV). The compounds are inhibitory to the replication of various HIV-1 and HIV-2 strains in various human T-cell systems, including peripheral blood lymphocytes, at 0.14-1.4 microM, without being toxic to the host cells at 2.2 mM. The bicyclam JM2763 is active against 3'-azido-3'-deoxythymidine (zidovudine; AZT)-resistant HIV-1 strains and acts additively with AZT. Mechanism of action studies revealed that the bicyclams (i.e., JM2763) interact with an early event of the retrovirus replicative cycle, which could be tentatively identified as a viral uncoating event.

We have previously described the potent and selective inhibition of several strains of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) by JM2763, an n-propyl-linked dimer of the 1,4,8,11-tetraazamacrocyclic (cyclam) ring system. Upon further investigation, we have also found that incorporating an aromatic rather than aliphatic linker leads to analogs with higher antiviral potency. The prototype, JM3100 (19a, isolated as the octahydrochloride salt), which contains a p-phenylenebis(methylene) moiety linking the cyclam rings, inhibited the replication of HIV-1 (IIIB) and HIV-2 (ROD) at EC50's of 4.2 and 5.9 nM, respectively, while remaining nontoxic to MT-4 cells at concentrations exceeding 421 microM. In order to identify the structural features of bis-tetraazamacrocycles required for potent activity, we have prepared a novel series of phenylenebis(methylene)-linked analogs, in which the macrocyclic ring size was varied from 12 to 16 ring members. Depending upon the substitution of the phenylenebis(methylene) linker (para or meta), sub-micromolar anti-HIV activity was exhibited by analogs bearing macrocycles of 12-14 ring members but with varying cytotoxicity to MT-4 cells. Furthermore, while we found that identical macrocyclic rings are not required for activity, substituting an acyclic polyamine equivalent for one of the cyclam rings in 19a resulted in a substantial reduction in anti-HIV potency, clearly establishing the importance of the constrained macrocyclic structure. A short series of transition metal complexes of 19a were also prepared and evaluated. Complexes of low kinetic stability such as the bis-zinc complex retained activity comparable to that of the parent compound. Finally, the activity of bicyclam analogs appears to be insensitive to the electron-withdrawing or -donating properties of substituents introduced onto the linker, but sterically hindering groups such as phenyl markedly reduced activity. As a result, several analogs with anti-HIV potency comparable to that of 19a have been identified.

Among the 10 subtypes of the M group of human immunodeficiency virus type 1, subtype C is the most prevalent in India and may dominate worldwide in the near future; however, there has been no report on the infectious DNA clone of this subtype. We have isolated an infectious DNA clone of the 93IN101 strain of HIV-1 subtype C, which was isolated in India in 1993. MAGIC5 cells, which are derived from HeLa-CD4-LTR-beta-gal (MAGI) cells and express CCR5, were inoculated with the 93IN101 strain of HIV-1 subtype C. The genomic DNA of the infected cells was used as a template for amplification of the HIV-1 genome. The genome DNA obtained was subcloned into pBR322, and the resulting plasmid was designated as pIndie-C1. The insert of pIndie-C1 was 9680 bp in length and had an intact genomic organization with open reading frames of all structural, regulatory, and accessory proteins. Phylogenetic analysis confirmed that the nucleotide sequence of pIndie-C1 is closely related to those of HIV-1 subtype C isolated in India. Transfection of pIndie-C1 into 293T cells yielded as much virus as did pNL432, one of the most widely used HIV DNA clones. The recovered Indie-C1 virus infected MAGIC5 but not the parent MAGI cells, indicating that Indie-C1 is CCR5 tropic. Expressed Env protein was reacted efficiently with the sera of HIV-1-infected patients of India, but not of Japan. Expression of Nef and Vpr was also confirmed by immunoblotting.

The prevalence of hepatitis B virus (HBV), hepatitis C virus (HCV), and GB virus C or hepatitis G virus (GBV-C/HGV) infections was determined in 289 patients with liver disease in Ho Chi Minh City and 890 healthy inhabitants of its rural area, Dalat City, Vietnam, respectively. Serum HCV RNA and GBV-C/HGV RNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). HBsAg, HCV antibodies, and GBV-C/HGV RNA were detected in 139 (47%), 69 (23%), and ten (3%) subjects, respectively, often accompanied by elevated serum levels of alanine aminotransferase. HBsAg and HCV antibodies or HCV antibodies and GBV-C/HGV RNA were detectable simultaneously in 8% and 2% of the patients, respectively. In the inhabitants, HBsAg, HCV antibodies, and GBV-C/HGV RNA were found in 51 (5.7%), nine (1.0%), and 11 (1.2%) subjects, respectively. Thus, the prevalence of HBsAg, HCV antibodies, and GBV-C/HGV RNA was significantly higher in liver disease patients than those in the general population. In the samples from 69 patients and nine inhabitants who were seropositive for HCV antibodies, HCV RNA was detectable in 42 (61%) and 4 (44%), respectively. In patients with liver disease, ten belonged to HCV genotype 1a, ten to HCV 1b, three to HCV 2a, four to HCV 2b, and two to HCV 3a by PCR with genotype-specific primers. Nine patients had mixed genotypes, and the remaining four were not classified. Of the GBV-C/HGV RNA-positive individuals, two patients and two inhabitants were positive for HBsAg, while none of the residents had HCV antibodies, although six HCV antibodies (60%) and four HCV RNA (40%) were found in patients. When a phylogenetic tree of GBV-C/HGV was constructed based on the nucleotide sequences, the 21 isolates were classified into at least two genotypes; four isolates belonged to G2, and 17 to G3. The results indicate that in Ho Chi Minh HCV infection prevails with broad distribution of genotypes together with HBV infection among patients with liver disease. This study suggests that GBV-C/HGV infection occurs independently in the two different districts in association with HCV infection.

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Among the many mutations found in the hepatitis B virus (HBV) genome, some have been associated with fulminant hepatitis, as exemplified by precore-defective mutations. The aim of this study was to determine whether such mutations also are found in Vietnamese cases of fulminant hepatitis B. The full-genome nucleotide sequence of HBV in three patients with fulminant hepatitis (F-2, F-3, and F-6) and one with acute hepatitis (A-3), who were admitted to Cho Ray Hospital, Ho Chi Minh City, Vietnam was ascertained. Additionally, two patients with fulminant hepatitis (F-1 and F-7) and three with acute hepatitis (A-1, A-2, and A-5) were examined only for the precore/core region of HBV. Remarkably, the nonsense mutation at precore codon 28 (Trp82Stop) was found in four of the five patients with fulminant hepatitis, while all the acute hepatitis patients harbored wild type (one had a mixture of wild and mutant types). The missense mutations within the core region, Ile97Leu and Pro130Ile/Thr/Ser, were also remarkable in fulminant hepatitis. Only F-2 was free from these precore/core mutations, but F-2 was unique in that it possessed a chimeric genotype: it could be classified into genotype C as a whole, but its X region was of genotype B, like the other four fulminant hepatitis isolates (F-1, F-3, F-6, and F-7). The codon 41 of the X protein was Pro in all three fulminant hepatitis cases examined for this region, while it was Ser in the wild-type isolates of genotype B. Of note as negative data, the mutations C1653T and T1753M of the enhancer II (Enh II) and A1762T and G1764A of the precore/core promoter regions, once reported to be relevant to severe or fulminant hepatitis, were not found in the present cases. The results with the Vietnamese cases of fulminant hepatitis corroborated results of previous studies with respect to the mutations Trp28Stop of precore and Ile97Leu and Pro130Ile/Thr/Ser of core, but not for the mutations within Enh II and precore/core promoter region. Whether the Ser41Pro mutation in the X region of genotype B HBV is Vietnam-specific or disease-specific deserves further investigation.