Chronopharmacologic aspects of continuous AcSDKP infusion in mice.

Inconsistent results characterized N-acetyl-Ser-Asp-Lys-Pro (AcSDKP or Goralatide) effects upon hematologic proliferation, possibly because its circadian organization had been overlooked. We investigated the circadian changes in AcSDKP disposition in plasma and in bone marrow during continuous infusion and AcSDKP effects upon the circadian rhythms in bone marrow granulomonocytic precursors (CFU-GM) and circulating blood cell counts. One hundred ninety-six male B6D2F1 mice received a constant infusion of AcSDKP (24 microg/ day) or 0.9% NaCl for 7 days, using an osmotic minipump. All mice were synchronized with an alternation of 12 hours of light and 12 hours of darkness for 3 weeks prior to study. Mice were sacrificed on the fifth or seventh infusional day at 3, 9, 15, or 21 hours after light onset (HALO) in order to assess plasma and bone marrow AcSDKP concentrations, CFU-GM, and/or circulating blood cell counts. In control mice, plasma and bone marrow AcSDKP concentrations displayed a circadian rhythm with a maximum level during the dark span, at 21 and 15 HALO respectively, while CFU-GM, leukocyte, lymphocyte, and monocyte counts peaked during early light. Continuous AcSDKP infusion increased fivefold mean plasma AcSDKP level at 3 or 9 HALO, thus inverted its physiologic rhythm and suppressed the CFU-GM peak that normally occurs at these times. This inhibition however, was indirect, because the rhythms in bone marrow AcSDKP concentration were similar with or without AcSDKP infusion. Conversely, mean leukocyte and lymphocyte counts were significantly reduced with AcSDKP infusion, while their circadian rhythms remained unaffected and were amplified. The results indicate that AcSDKP pharmacology displays circadian rhythmicity and warrant the exploration of chronopharmacologic schedules of AcSDKP delivery for further protecting bone marrow against chemotherapy insults.

Polyadenylic: polyuridylic acid-induced determinants of host resistance to cytomegalovirus and their potentiation by hyperthermia.

The ability of spleen cells from poly A:poly U-treated mice to inhibit murine cytomegalovirus (MCMV) replication in confluent monolayer cells of secondary mouse embryo fibroblasts (MEFs) cultured at 37 and 40 degrees C was investigated. When spleen cells from BALB/c mice injected 48 h earlier with poly A:poly U were added to MEFs infected 2 h previously with MCMV, 37% less plaques were observed than in cultures containing control cells. Of interest, the poly A:poly U-induced antiviral activity at the elevated temperature (40 degrees C) resulted in a further drop to 61% in MCMV-induced plaques compared to those of the normothermic (37 degrees C) cultures. The antiviral function of spleen cells induced by poly A:poly U was evident in the supernatant fluid when cultured for 48 h at 37 degrees C. MCMV-induced plaques were reduced to 52 and 5% of controls in the plaque assays performed at 37 and 40 degrees C, respectively. Supernatant fluids generated at 40 degrees C, however, inhibited MCMV replication only when incubated at 40 degrees C. No direct inhibitory effect of the supernatant fluids on MCMV was evident; rather, inhibition was effected directly on the MEFs. The NK cell fraction of spleen cells from poly A:poly U-treated mice alone showed only a slight inhibitory effect at 40 degrees C. However, in the presence of the supernatant fluid from poly A:poly U-exposed spleen cells, the antiviral activity of NK cells was significantly increased both at 37 and 40 degrees C. The cellular source of the culture fluid showing poly A:poly U-induced antiviral activity appeared to be in the T-cell population. It was completely neutralized by monoclonal anti-IFN gamma antibody but not by anti-IFN beta, anti-IL4, anti-transforming growth factor, or anti-prostaglandin E2. In conclusion, these data document the ability of spleen cells from poly A:poly U-treated mice to inhibit MCMV replication and this activity is potentiated by hyperthermic conditions. The antiviral function of poly A:poly U-treated spleen cells appeared to be due mainly to the action of IFN gamma produced by T cells. The enhanced antiviral activity by hyperthermia appeared to be related to the action of IFN gamma rather than its production.

Antiviral action of polyadenylic-polyuridylic acid against HIV in cell cultures.

Polyadenylic-polyuridylic acid referred to as poly(A).poly(U) is a synthetic double-stranded RNA which has been shown to manifest both antitumoral and immunomodulatory activities. Here we used this agent to demonstrate its antiviral activity against the human immunodeficiency virus (HIV-1 and HIV-2). Treatment of cells with poly(A).poly(U) resulted in a significant delay in the development of the HIV-specific cytopathic effect characterized by the formation of syncytia and cell lysis. Furthermore, the production of virus measured by the concentration of the HIV major core protein was reduced by 90-95%. Under these experimental conditions, the synthesis of HIV proteins was reduced at least tenfold whereas the metabolism and proliferation of cells apparently were not affected. The inhibitory action of poly(A).poly(U) seems to be at the level of viral entry into cells. Combined treatment of infected cells with poly(A).poly(U) and azidothymidine (AZT) resulted in a 4-5-fold synergistic inhibitory effect. Previously, no toxicity has been observed in cancer patients with long-term treatment with poly(A).poly(U). In view of this and the significant anti-HIV effect, poly(A).poly(U) provides a potential candidate as a therapeutic drug in AIDS disease.

[Cicletanine administered with other antihypertensive agents]. / Etude du ciclétanine administré en association avec d'autres antihypertenseurs.

The antihypertensive effectiveness and the clinical and biochemical safety of cicletanine were evaluated in 84 patients (28 women, 56 men) presenting with permanent essential hypertension without severe cardiovascular complications. The hypertension was insufficiently controlled by a beta-blocker, a centrally acting antihypertensive drug or nifedipine. After 3 months of treatment during which cicletanine was added to each of these three classes of drugs, there was a significant fall of systolic arterial pressure (-18 mmHg with beta-blockers, -17 mmHg with central agents and -26 mmHg with nifedipine) and diastolic arterial pressure (-22, -21 and -28 mmHg respectively), resulting in normalization of blood pressure (less than 160/95 mmHg) in 63%, 43% and 50% respectively of patients in each therapeutic group. The fall of blood pressure was accompanied by a significant decrease of functional symptoms (headache, palpitations, dizziness); in the nifedipine group, the addition of cicletanine resulted in complete regression of anginal attacks. The therapeutic combinations were well tolerated; only two patients were excluded from the study for undesirable effects unascribable to cicletanine. Otherwise, the side-effects observed were minor. The biochemical values measured did not significantly vary, and the variations noted were of small amplitude.

Polyadenylic:polyuridylic acid-induced protection of BALB/c mice against acute murine cytomegalovirus infection.

Treatment of BALB/c mice with poly(A):poly(U) 18 h prior to infection with a lethal dose of murine cytomegalovirus (MCMV) increased survival. In parallel with increased survival, a 10- to 100-fold reduction of plaque-forming MCMV was found in the liver and spleen of mice 4 days post-infection with a sublethal dose of MCMV. Poly(A):poly(U) did not significantly increase natural killer cell activity or prolong the duration of elevated cytotoxic activity in infected animals. The possible role of interferon in the poly(A):poly(U)-induced protection of BALB/c mice is discussed.

Circadian-based effects of AcSDKP, with or without rhG-CSF on hematologic toxicity of chemotherapy in mice.

The hematologic toxicity of arabinosylcytosine (Ara-C) and carboplatin (CBDCA) as well as the stimulating effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on murine bone marrow vary according to their dosing time along the 24-h time scale. In the present study, we investigated whether the tolerability of Ara-C or CBDCA, given at their least toxic circadian time, could be improved further with AcSDKP, a negative regulator of hemopoiesis, rhG-CSF or both. A total of 228 B6D2F1 mice received once-daily injection of either Ara-C (42 mg/kg/d s.c.) for 7 d (d 0-6) at 8 hours after light onset - HALO) or CBDCA (40 mg/kg/d i.p.) for 5 d (d 2-6) at 16 HALO. AcSDKP (24 microg/d) was continuously infused for 7 d (d 0-6), using an osmotic minipump. rhG-CSF (400 microg/kg/d s.c.) was injected for 4 d (d 9-12) at 9 HALO. Subgroups of mice were sacrificed at 3 HALO on various days following treatment. AcSDKP significantly increased CFU-GM count on d 7 and leukocyte, neutrophil and monocyte counts on d 13 and d 16 compared to Ara-C alone. Also, rhG-CSF produced similar protective effects to those of AcSDKP with regard to leukocyte and CFU-GM counts. The combination of AcSDKP with rhG-CSF induced a further increase in total leukocytes and their subsets as compared to either agent alone, but did not alter the CFU-GM counts. Neither AcSDKP nor rhG-CSF nor their combination reduced CBD CA-induced hematological toxicity. In conclusion, AcSDKP or rhG-CSF administration further improved the tolerability of Ara-C beyond that already achieved with optimal circadian timing, while no such effect was observed in mice receiving CBDCA at the dose used. The results warrant further exploration of chronopharmacologic delivery schedules combining Ara-C with AcSDKP.

A classification of essential hypertensive patients according to the erythrocyte Na transport abnormalities: an application for monitoring the antihypertensive response to cicletanide.

We have studied the kinetic properties of the interaction of internal Na with four different Na transport pathways (ouabain-sensitive Na, K pump, bumetanide-sensitive Na,K cotransport, Li:Na countertransport and passive Na permeability) in erythrocytes from 197 essential hypertensive patients. This study permitted us to classify 168 of the 197 hypertensives studied into the following groups: Pump - hypertensives-six patients showing low pump affinity for internal Na and high maximal pump rate; Co - hypertensives -95 patients characterized by low cotransport affinity for internal Na and low or normal maximal outward cotransport rate. Counter + hypertensives - 35 patients characterized by low countertransport affinity for internal Na and high maximal countertransport rate; and Leak + hypertensives - 32 patients showing increased passive Na permeability. Having studied the apparent affinity of the Na,K cotransport system for external K we divided the co - hypertensives into three subgroups (symmetric, asymmetric or antisymmetric) according to the presence of low, normal or high K affinity. Forty-five hypertensive patients were submitted to antihypertensive treatment with a new diuretic and antihypertensive drug, cicletanide. Preliminary results indicate that one of the factors involved in the antihypertensive efficiency of the compound is the subgroup to which the hypertensive belongs.

Pharmcokinetics in healthy volunteers and patients of NAc-SDKP (seraspenide), a negative regulator of hematopoiesis.

NAc-SDKP is a peptide being tested as a bone marrow hematopoiesis protector in chemotherapy trials in cancer patients. We studied the pharmacokinetics of NAc-SDKP in six healthy human volunteers and in five patients undergoing chemotherapy. Plasma concentrations of NAc-SDKP were monitored using a specific enzyme immunoassay. Because NAc-SDKP is an endogenous compound, a preliminary study was undertaken to determine intra- and interday baseline variations in healthy subjects. The baseline value (range 1.7-3.2 nM) differed between subjects, but was constant over time. The influence of the route of administration was studied in six healthy volunteers with 128 nmol/kg given as a 12-hr intravenous infusion or as a single subcutaneous or intramuscular injection. After cessation of intravenous infusion in healthy volunteers, NAc-SDKP was characterized by a quick elimination phase, with a mean half-life of 4.5 min. The volume distribution was 117 ml/kg and the area under the curve was 117 nM hr. After subcutaneous and intramuscular administrations, peak plasma drug concentrations occurred at 0.26 and 0.28 hr, with Cmax values of 156 and 110 nM, respectively. The bioavailabilities determined after subcutaneous and intramuscular administrations were 100 and 81%, respectively. NAc-SDKP pharmacokinetics was studied in patients after intravenous infusion over 48 hr of a dose of between 51.3 and 513 nmol/kg. Area under the curve values increased proportionately with the dose. Mean clearance was lower in patients than in healthy volunteers: 524 vs. 1120 ml/hr/kg, respectively.

Goralatide (AcSDKP), a negative growth regulator, protects the stem cell compartment during chemotherapy, enhancing the myelopoietic response to GM-CSF.

The aim of our study was to investigate the protection afforded to the bone marrow by Goralatide (AcSDKP), an inhibitor of hemopoietic stem cell proliferation, when administered alone or in combination with a growth factor (granulocyte/macrophage colony-stimulating factor [GM-CSF]) during iterative cycles of Ara-C (cytarabine) treatment. In control mice receiving the inhibitor alone without Ara-C, the number of granulocytes was reduced during treatment, and a surge in number of peripheral blood cells was observed after its completion. Peripheral hematological responses were monitored during 3 consecutive cycles of Ara-C chemotherapy and the resultant nadir and recoveries. Analysis of variance of the treatment effects pooled over the 3 cycles showed that a treatment regimen in which the inhibitor was administered during the myelotoxic periods of chemotherapy confirmed the existence of a surge after completion of administration of the inhibitor and showed a significant protective effect. When the cycles of chemotherapy plus Goralatide were followed by GM-CSF, the recovery from leukopenic nadirs was accelerated and the white blood cells and granulocyte levels were markedly increased over those observed in control mice and in mice treated either with Goralatide alone or with GM-CSF alone. The differences were highly significant. A consistent and significant increase (p < 0.001) in platelet count was also noted in animals given Goralatide in conjunction with Ara-C or Ara-C + GM-CSF. After three treatment cycles, this response to the CSF was far better in mice treated by the inhibitor than when CSF was given alone, suggesting a protection of the stem cell pool.

Efficacy of polyadenylic.polyuridylic acid in the treatment of chronic active hepatitis B.

In order to assess whether polyadenylic.polyuridylic acid [poly(A).poly(U)] can be used as a new therapeutic agent for the treatment of chronic hepatitis B, 19 patients with histologically proven chronic active hepatitis B were injected intravenously with 100-150 mg of poly(A).poly(U) weekly for six weeks. Changes in alanine aminotransferase (ALT) levels, 2',5'-oligoadenylate synthetase (2'.5'-AS) activities and HBV markers were sequentially checked during and after treatments. Serum ALT levels were decreased gradually and 2'.5'-AS activities were significantly increased after initiation of poly(A).poly(U) injections. At the end of this trial (24th week) we have observed the normalizations of elevated ALT levels in 14 (73.7%), negative conversion of HBeAg in 11 (57.9%) and loss of HBV-DNA in 12 out of 19 patients (63.1%). Complete responses which had both normalization of ALT levels and negative conversion of HBeAg were noted in 11 patients (57.9%) and partial responses showing either normalization of ALT levels or negative conversion of HBeAg alone were in four out of 19 patients (21.1%). No notable adverse effects were observed during the treatments and follow-up period. It can be concluded that poly(A).poly(U) seems to be effective in the treatment of chronic active hepatitis B and has an advantage of being free of significant side effects.

The tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (Goralatide) protects from doxorubicin-induced toxicity: improvement in mice survival and protection of bone marrow stem cells and progenitors.

The tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP or Goralatide), a physiological regulator of hematopoiesis, inhibits the entry into the S-phase of murine and human hematopoietic stem cells. It has been shown to reduce the damage to specific compartments in the bone marrow resulting from treatment with chemotherapeutic agents, ionizing radiations, hyperthermy, or phototherapy. The present study was performed to assess the therapeutic potential of AcSDKP in vivo in reducing both the toxicity and the hematopoietic damage induced by fractionated administration of doxorubicin (DOX), a widely used anticancer drug. Here we showed that AcSDKP could reduce DOX-induced mortality in mice and could protect particularly the long-term reconstituting cells (LTRCs) in addition to colony forming units-spleen, high proliferative potential colony-forming cells, and colony-forming units-granulocyte-macrophage (CFU-GM) from DOX toxicity. The protection against DOX-induced mortality in mice was improved when AcSDKP was administered for 3 days, at a dose of 2.4 micrograms/d, by continuous subcutaneous (SC) infusion or fractionated s.c. injections starting 48 hours before DOX treatment. Moreover, the recovery of the CFU-GM population in the AcSDKP-DOX-treated mice was optimized by the subsequent administration of granulocyte colony-stimulating factor (G-CSF). The coadministration of AcSDKP with DOX may improve its therapeutic index by reducing both acute hematotoxicity on late stem cells and progenitors and long-term toxicity on LTRCs. Optimization of these treatments combined with G-CSF may provide an additional approach to facilitate hematopoietic recovery after cancer chemotherapy.