Junji Shibata

Hepatic arterial injection chemotherapy with cisplatin suspended in an oily lymphographic agent for hepatocellular carcinoma

A method to prepare cisplatin suspended in an oily lymphographic agent, Lipiodol (LPS), has been established to deliver cisplatin to hepatocellular carcinoma (HCC) by the hepatic artery. Seventy‐one patients, one Stage I, 16 Stage II, 16 Stage III, and 38 Stage IV, were treated with LPS therapy. A partial response was obtained in 33 cases (46.5%), a minor response in 20 cases (28.2%), and no change in 18 cases (25.3%). In 34 patients whose serum alpha‐fetoprotein (AFP) levels were greater than 400 ng/ml, the serum AFP levels decreased in 31 patients (91.2%). The AFP decreased by more than 50% in 25 cases (73.5%) and more than 75% in 19 cases (55.9%). The plasma des‐γ‐carboxy prothrombin (DCP) levels decreased in all of the 26 DCP‐positive patients. The survival rate was 77% at 6 months and the 1‐year survival rate was estimated to be 55%. The patients treated with LPS therapy survived longer compared with patients given Lipiodol containing neocarzinostatin by the hepatic artery. Complications such as acute gastroduodenal mucosal lesions (24%), cholecystitis (2.8%), pancreatitis (7%), delayed jaundice (7%), and hepatic encephalopathy (4.2%) were observed after therapy. The peak plasma platinum (Pt) concentrations determined as ultrafilterable Pt occurred 5 to 20 minutes, and 5 to 60 minutes as total Pt after the end of LPS injection. The Pt concentrations in the tumor tissues were 42 times higher in four operated cases and 7.1 times higher in six autopsy cases than those in the nontumorous tissue. These results suggest that LPS selectively accumulates in the HCC, is long‐lasting and gradually releases the drug. In addition it is effective as a new anti‐cancer therapy for hepatocellular carcinoma.

Transcatheter arterial chemotherapy with and without embolization in patients with hepatocellular carcinoma.

Objective: This study compared the antitumor effect, adverse effects and survival between transcatheter arterial embolization (TAE) and transcatheter arterial infusion chemotherapy (TAI) in patients with hepatocellular carcinoma (HCC). Methods: The study population consisted of 168 consecutive patients with advanced HCC treated with transcatheter arterial treatments using cisplatin suspended in lipiodol. Among these, 74 patients were treated with TAE, and the remaining 94 patients were treated with TAI. Results: There were no significant differences in any baseline characteristics except hemoglobin, platelets, albumin, and glutamic pyruvic transaminase. Complete or partial tumor response was achieved in 54 patients (73%) in the TAE group and in 48 patients (51%) in the TAI group (p < 0.01). There were two treatment-related deaths caused by acute hepatic failure and acute renal failure in the TAE group. Nausea and deterioration of serum transaminase after TAE were significantly more severe than after TAI. Median survival time and survival rates at 5 years were 3.1 years and 25% in the TAE group, and 2.5 years and 18% in the TAI group (p = 0.37). Conclusion: TAE has a higher antitumor effect than TAI, but does not significantly improve the survival of patients with HCC.

Recurrent HIV-1 integration at the BACH2 locus in resting CD4+ T cell populations during effective highly active antiretroviral therapy

The persistence of latent human immunodeficiency virus type 1 (HIV-1) has been considered one of the major obstacles for eradication of the virus in infected individuals receiving successful antiretroviral therapy. To determine the contribution of integration sites to viral latency within clinical settings, an inverse polymerase chain reaction method was used to analyze integration sites in CD4(+) T cells from patients showing long-term undetectable plasma viral RNA. Of 457 sites identified in 7 patients, almost all (96%) resided within transcriptional units, usually in introns of the human genome. Studies of 18 genes in which HIV-1 integrates found them to be actively expressed in resting CD4(+) T cells. On the other hand, integration sites in the alpha satellite region was also identified in some patients, albeit at low frequency. Of particular interest, HIV-1-infected cells with multiple identical integration sites were detected in longitudinal analysis of samples from 3 patients, suggesting that these cells persist for long periods and that clonal expansion may occur. Furthermore, strong integration clusters in the BACH2 gene were observed in 2 patients (31% in patient 1 and 5% in patient 3). Our findings not only raise the possibility of biased target-site integration but also provide mechanistic insights into the long-term persistence of HIV-1.

Generation of High-Affinity Antibody against T Cell-Dependent Antigen in the Ganp Gene-Transgenic Mouse

Generation of high-affinity Ab is impaired in mice lacking germinal center-associated DNA primase (GANP) in B cells. In this study, we examined the effect of its overexpression in ganp transgenic C57BL/6 mice (GanpTg). GanpTg displayed normal phenotype in B cell development, serum Ig levels, and responses against T cell-independent Ag; however, it generated the Ab with much higher affinity against nitrophenyl-chicken gammaglobulin in comparison with C57BL/6. To further examine the affinity increase, we established hybridomas producing high-affinity mAbs and compared their affinities using BIAcore. C57BL/6 generated high-affinity anti-nitrophenyl mAbs (KD ∼ 2.50 × 10−7 M) of IgG1/λ1 and contained the VH186.2 region with W33L mutation. GanpTg generated much higher affinity (KD > 1.57 × 10−9 M) by usage of VH186.2 as well as noncanonical VH7183 regions. GanpTg also generated exceptionally high-affinity anti-HIV-1 (V3 peptide) mAbs (KD > 9.90 × 10−11 M) with neutralizing activity. These results demonstrated that GANP is involved in V region alteration generating high-affinity Ab.

Enhanced exposure of human immunodeficiency virus type 1 primary isolate neutralization epitopes through binding of CD4 mimetic compounds

ABSTRACT N-(4-Chlorophenyl)-N′-(2,2,6,6-tetramethyl-piperidin-4-yl)-oxalamide (NBD-556) is a low-molecular-weight compound that reportedly blocks the interaction between human immunodeficiency virus type 1 (HIV-1) gp120 and its receptor CD4. We investigated whether the enhancement of binding of anti-gp120 monoclonal antibodies (MAbs) toward envelope (Env) protein with NBD-556 are similar to those of soluble CD4 (sCD4) by comparing the binding profiles of the individual MAbs to Env-expressing cell surfaces. In flow cytometric analyses, the binding profiles of anti-CD4-induced epitope (CD4i) MAbs toward NBD-556-pretreated Env-expressing cell surfaces were similar to the binding profiles toward sCD4-pretreated cell surfaces. To investigate the binding position of NBD-556 on gp120, we induced HIV-1 variants that were resistant to NBD-556 and sCD4 in vitro. At passage 21 in the presence of 50 μM NBD-556, two amino acid substitutions (S375N in C3 and A433T in C4) were identified. On the other hand, in the selection with sCD4, seven mutations (E211G, P212L, V255E, N280K, S375N, G380R, and G431E) appeared during the passages. The profiles of the mutations after the selections with NBD-556 and sCD4 were very similar in their three-dimensional positions. Moreover, combinations of NBD-556 with anti-gp120 MAbs showed highly synergistic interactions against HIV-1. We further found that after enhancing the neutralizing activity by adding NBD-556, the contemporaneous virus became highly sensitive to antibodies in the patients plasma. These findings suggest that small compounds such as NBDs may enhance the neutralizing activities of CD4i and anti-V3 antibodies in vivo.

Clinical usefulness of des-γ-carboxy prothrombin assay in early diagnosis of hepatocellular carcinoma

Des-γ-carboxy prothrombin (DCP) was evaluated as a serological marker for hepatocellular carcinoma (HCC), particularly in patients with early HCC. In 1192 patients with various diseases, plasma DCP levels were measured by a newly developed enzyme immunoassay method using an anti-DCP monoclonal antibody. Of the 254 patients with HCC, 143 (56%) had abnormal DCP levels of greater than 0.1 AU/ml. In contrast, elevated DCP levels were rarely observed in patients with chronic hepatitis, liver cirrhosis, metastatic liver cancer, and other malignant tumors. Because no correlation was observed between DCP and α-fetoprotein (AFP), the combined measurement of these two complementary markers appears to be useful in the diagnosis of HCC. Since normal levels were observed in 29 of 30 patients (97%) with small liver tumors measuring 2 cm or less in diameter, the diagnostic application of the DCP assay to small liver tumors is limited. However, in patients with tumors larger than 2 cm, the plasma DCP assay may even be more useful than AFP. Among 46 patients with liver cirrhosis or chronic hepatitis who subsequently developed HCC, significantly increased DCP and AFP levels were observed in nine patients (20%) and 14 patients (30%), respectively, when a tumor was detected. When the results of both assays were combined, 19 patients (41%) had elevated levels of one or both markers. Although the plasma DCP assay alone is not sensitive enough to detect early small liver cancers, it could be applied as a complementary tumor marker together with AFP. The use of both these markers together with imaging modalities in the regular follow-up of patients with chronic liver disease who are at high risk for HCC might be of great benefit.

Phase I clinical study of a novel lipophilic platinum complex (SM-11355) in patients with hepatocellular carcinoma refractory to cisplatin/lipiodol

SM-11355 is a platinum complex developed to treat hepatocellular carcinoma (HCC). It is administered via the hepatic artery, using a carrier, lipiodol, that consists of ethyl esters of iodized poppy seed oil. We have performed a phase I clinical trial of an SM-11355-lipiodol formulation in 11 HCC patients, in order to investigate the maximum allowable dose and to maximize the efficacy and safety of the drug in the treatment of HCC. The SM-11355 arterial infusion suspension was administered at doses of 6, 12 and 20 mg ml−1 in a maximum lipiodol volume of 6 ml. An antitumour efficacy rating of complete response was achieved for one patient and a partial response rating was achieved for a second patient, giving an overall response rate of 18.2%. Anorexia, nausea and vomiting, pyrexia, thrombocytopenia and increases in AST, ALT and total bilirubin were observed as adverse effects, but each was transient and each patient had recovered completely by 4 weeks after drug administration. Hence, we concluded that the maximum allowable dose was not reached in this study. Overall, our results suggest that SM-11355 is effective in treating HCC and we suggest that the dose for early phase II trials should be 20 mg ml−1.

Impact of V2 Mutations on Escape from a Potent Neutralizing Anti-V3 Monoclonal Antibody during In Vitro Selection of a Primary Human Immunodeficiency Virus Type 1 Isolate

ABSTRACT KD-247, a humanized monoclonal antibody to an epitope of gp120-V3 tip, has potent cross-neutralizing activity against subtype B primary human immunodeficiency virus type 1 (HIV-1) isolates. To assess how KD-247 escape mutants can be generated, we induced escape variants by exposing bulked primary R5 virus, MOKW, to increasing concentrations of KD-247 in vitro. In the presence of relatively low concentrations of KD-247, viruses with two amino acid mutations (R166K/D167N) in V2 expanded, and under high KD-247 pressure, a V3 tip substitution (P313L) emerged in addition to the V2 mutations. However, a virus with a V2 175P mutation dominated during passaging in the absence of KD-247. Using domain swapping analysis, we demonstrated that the V2 mutations and the P313L mutation in V3 contribute to partial and complete resistance phenotypes against KD-247, respectively. To identify the V2 mutation responsible for the resistance to KD-247, we constructed pseudoviruses with single or double amino acid mutations in V2 and measured their sensitivity to neutralization. Interestingly, the neutralization phenotypes were switched, so that amino acid residue 175 (Pro or Leu) located in the center of V2 was exchanged, indicating that the amino acid at position 175 has a crucial role, dramatically changing the Env oligomeric state on the membrane surface and affecting the neutralization phenotype against not only anti-V3 antibody but also recombinant soluble CD4. These data suggested that HIV-1 can escape from anti-V3 antibody attack by changing the conformation of the functional envelope oligomer by acquiring mutations in the V2 region in environments with relatively low antibody concentrations.

Resistance profile of a neutralizing anti-HIV monoclonal antibody, KD-247, that shows favourable synergism with anti-CCR5 inhibitors

Background:The high-affinity humanized monoclonal antibody (MAb) KD-247 reacts with a tip region in gp120-V3 and cross-neutralizes primary isolates with a matching neutralization sequence motif. Methods:We induced an HIV-1 variant that was resistant to KD-247 by exposing the JR-FL virus to increasing concentrations of KD-247 in PM1/CCR5 cells, which expressed high levels of CCR5 in vitro. We determined the amino acid sequence of the gp120-encoding region of the JR-FL escape mutant from KD-247. To confirm that this substitution was responsible for the KD-247-resistance, a single-round replication assay was performed. We further evaluated the anti-HIV-1 interactions between KD-247 and various CCR5 inhibitors in vitro. Results:At passage 8 of the culture in the presence of 1000 μg/ml KD-247, one amino acid substitution, Gly to Glu at position 314 (G314E), was identified in the V3-tip of gp120. A pseudotyped virus with the G314E mutation was highly resistant to KD-247. Unexpectedly, this mutant virus was sensitive to CCR5 inhibitors, RANTES, recombinant human soluble CD4 (rsCD4) and an anti-CCR5 MAb, but resistant to an anti-CD4 MAb, compared with the wild-type virus. We also found that combinations of KD-247 and CCR5 inhibitors were highly synergistic. Conclusions:The present data suggest that KD-247 has certain advantages for possible passive immunotherapy. They are: high concentrations of KD-247 are needed for viral acquisition of KD-247 resistance; the escape variants are more sensitive to CCR5 inhibitors and rsCD4; and there are high levels of synergism between KD-247 and CCR5 inhibitors at all concentrations tested.

Combination therapy with ursodeoxycholic acid and colchicine for primary biliary cirrhosis

Twelve patients with primary biliary cirrhosis (PBC), stages I to III, received long‐term therapy with a combination of 600 mg ursodeoxycholic acid (UDCA) and 1 mg colchicine given daily for more than 2 years. Drug toxicity was mild; one patient experienced diarrhoea that was probably due to colchicine. Serum levels of bilirubin, alkaline phosphatase (ALPase), γ‐glutamyl transpeptidase and alanine aminotransferase decreased by more than 50% of the initial values. Serum albumin and cholesterol levels also improved, but immunoglobulins and anti‐mitochondrial antibody titre did not change. Histologic features in the eight patients who received serial liver biopsies before and 2 years after the beginning of treatment were evaluated. Piecemeal necrosis and portal inflammation were improved, but there was no change in portal fibrosis. Patients were divided into two groups; the first received both drugs from the outset, and the second group were started on UDCA for 3 months followed by the addition of colchicine. After 3 months, the improvement in serum bilirubin and ALPase in the first group was greater than in the second. However, in the second group, the ALPase levels had decreased significantly when measured at 6 and 9 months after the treatment compared with the levels at 3 months. These findings suggest that UDCA and colchicine may have a synergistic effect. This combination therapy appears to be safe and effective, both clinically and histologically, for treating PBC.