Cholesterol sulfate inhibits osteoclast differentiation and survival by regulating the AMPK-Sirt1-NF-κB pathway.

Cholesterol sulfate (CS) is an activator of retinoic acid-related orphan receptor α (RORα). CS treatment or RORα overexpression attenuates osteoclastogenesis in a collagen-induced arthritis mouse model. However, the mechanism by which CS and RORα regulate osteoclast differentiation remains largely unknown. Thus, we aimed to investigate the role of CS and RORα in osteoclastogenesis and their underlying mechanism. CS inhibited osteoclast differentiation, but RORα deficiency did not affect osteoclast differentiation and CS-mediated inhibition of osteoclastogenesis. CS enhanced adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and sirtuin1 (Sirt1) activity, leading to nuclear factor-κB (NF-κB) inhibition by decreasing acetylation at Lys310 of p65. The NF-κB inhibition was restored by AMPK inhibitor, but the effects of CS on AMPK and NF-κB were not altered by RORα deficiency. CS also induced osteoclast apoptosis, which may be due to sustained AMPK activation and consequent NF-κB inhibition, and the effects of CS were significantly reversed by interleukin-1ß treatment. Collectively, these results indicate that CS inhibits osteoclast differentiation and survival by suppressing NF-κB via the AMPK-Sirt1 axis in a RORα-independent manner. Furthermore, CS protects against bone destruction in lipopolysaccharide- and ovariectomy-mediated bone loss mouse models, suggesting that CS is a useful therapeutic candidate for treating inflammation-induced bone diseases and postmenopausal osteoporosis.

ACAT-1-Regulated Cholesteryl Ester Accumulation Modulates Gemcitabine Resistance in Biliary Tract Cancer.

BACKGROUND: Biliary tract cancer (BTC) has few choices of chemotherapy, including gemcitabine, therefore exploring the mechanisms of gemcitabine resistance is important. We focused on lipid metabolism because biliary tract epithelial cells are essential in cholesterol and bile acid metabolism and the messenger RNA (mRNA) microarray analysis showed high acyl coenzyme A: cholesterol acyltransferase 1 (ACAT-1) expression in BTC gemcitabine-resistant (GR) cell lines. We hypothesized that aberrant accumulation of cholesteryl ester (CE) regulated by ACAT-1 could modulate GR in BTC. METHODS: CE accumulations were measured in human BTC cell lines, and the relationships between CE levels, ACAT-1 expressions, and gemcitabine sensitivity were analyzed. We performed a small-interfering RNA (siRNA)-mediated knockdown and biochemical inhibition of ACAT-1 in BTC cell lines and alterations of gemcitabine sensitivity were evaluated. To evaluate the clinical significance of ACAT-1 in regard to GR, immunohistochemistry was performed and ACAT-1 expressions were analyzed in resected BTC specimens. RESULTS: CE levels were correlated with ACAT-1 expressions and GR in four human BTC cell lines. siRNA-mediated knockdown of ACAT-1 in two independent GR cell clones as well as ACAT-1 inhibitor treatment significantly increased gemcitabine sensitivity; knockdown of ACAT-1: 5.63- and 8.02-fold; ACAT-1 inhibitor: 8.75- and 9.13-fold, respectively. ACAT-1 expression in resected BTC specimens revealed that the disease-free survival of the ACAT-1 low-intensity group (median 2.3 years) had a significantly better outcome than that of the ACAT-1 high-intensity group (median 1.1 years) under gemcitabine treatment after surgery (*p < 0.05). CONCLUSIONS: Our findings suggest that CE and ACAT-1 might be a novel therapeutic target for GR in BTC.

25-Hydroxycholesterol 3-Sulfate Recovers Acetaminophen Induced Acute Liver Injury via Stabilizing Mitochondria in Mouse Models.

Acetaminophen (APAP) overdose is one of the most frequent causes of acute liver failure (ALF). N-acetylcysteine (NAC) is currently being used as part of the standard care in the clinic but its usage has been limited in severe cases, in which liver transplantation becomes the only treatment option. Therefore, there still is a need for a specific and effective therapy for APAP induced ALF. In the current study, we have demonstrated that treatment with 25-Hydroxycholesterol 3-Sulfate (25HC3S) not only significantly reduced mortality but also decreased the plasma levels of liver injury markers, including LDH, AST, and ALT, in APAP overdosed mouse models. 25HC3S also decreased the expression of those genes involved in cell apoptosis, stabilized mitochondrial polarization, and significantly decreased the levels of oxidants, malondialdehyde (MDA), and reactive oxygen species (ROS). Whole genome bisulfite sequencing analysis showed that 25HC3S increased demethylation of 5mCpG in key promoter regions and thereby increased the expression of those genes involved in MAPK-ERK and PI3K-Akt signaling pathways. We concluded that 25HC3S may alleviate APAP induced liver injury via up-regulating the master signaling pathways and maintaining mitochondrial membrane polarization. The results suggest that 25HC3S treatment facilitates the recovery and significantly decreases the mortality of APAP induced acute liver injury and has a synergistic effect with NAC in propylene glycol (PG) for the injury.

Cholesterol metabolites alleviate injured liver function and decrease mortality in an LPS-induced mouse model.

BACKGROUND: Oxysterol sulfation plays a fundamental role in the regulation of many biological events. Its products, 25-hydroxycholesterol 3-sulfate (25HC3S) and 25-hydroxycholesterol 3, 25-disulfate (25HCDS), have been demonstrated to be potent regulators of lipid metabolism, inflammatory response, cell apoptosis, and cell survival. In the present study, we tested these products' potential to treat LPS-induced acute liver failure in a mouse model. METHODS: Acute liver failure mouse model was established by intravenous injection with LPS. The injured liver function was treated with intraperitoneal administration of 25HC, 25HC3S or 25HCDS. Serum enzymatic activities were determined in our clinic laboratory. ELISA assays were used to detect pro-inflammatory factor levels in sera. Western blot, Real-time Quantitative PCR and RT2 Profiler PCR Array analysis were used to determine levels of gene expression. RESULTS: Administration of 25HC3S/25HCDS decreased serum liver-impaired markers; suppressed secretion of pro-inflammatory factors; alleviated liver, lung, and kidney injury; and subsequently increased the survival rate in the LPS-induced mouse model. These effects resulted from the inhibition of the expression of genes involved in the pro-inflammatory response and apoptosis and the simultaneous induction of the expression of genes involved in cell survival. Compared to 25HC, 25HC3S and 25HCDS exhibited significantly stronger effects in these activities, indicating that the cholesterol metabolites play an important role in the inflammatory response, cell apoptosis, and cell survival in vivo. CONCLUSIONS: 25HC3S/25HCDS has potential to serve as novel biomedicines in the therapy of acute liver failure and acute multiple organ failure.

Pre-medication practices and incidence of infusion-related reactions in patients receiving AMPHOTEC: data from the Patient Registry of Amphotericin B Cholesteryl Sulfate Complex for Injection Clinical Tolerability (PRoACT) registry.

BACKGROUND: Clinical studies have suggested that rates of infusion-related reactions (IRRs) may be higher with amphotericin B colloidal dispersion (ABCD) versus other forms of amphotericin B. However, these studies did not permit the use of pre-medications upfront, which are now commonly used. Objectives To describe the use of pre-medications and determine the rate of IRRs in the real-world setting. METHODS: PRoACT, a multicentre, worldwide observational registry, captured real-world data about pre-medication practices and IRRs in patients receiving ABCD. Eligible patients were those beginning treatment with ABCD; treatment was according to the site's standard treatment practice. Incidence of IRRs was collected during the first 10 days of ABCD therapy. Clinical response data were collected 12 weeks after treatment start. RESULTS: One hundred and seventy patients from 21 worldwide sites were included (median age 37 years; 52% male). There were a total of 1230 ABCD infusions (mean dose 2.8 mg/kg/day); 90% of the infusions (1105/1230) had pre-medication. Common pre-medications included corticosteroids, antihistamines, paracetamol (acetaminophen) and metamizole. The overall IRR rate was 12% (147/1230) and was lower in infusions with pre-medication (11%) versus no pre-medication (22%), P < 0.001. Corticosteroids were associated with a decreased incidence of IRRs (P < 0.05), while paracetamol and antihistamines were not. The most common IRRs were chills (7%), fever (7%) and rigors (5%). Clearance of the fungal infection occurred in 52% of the participants. CONCLUSIONS: These data suggest a lower rate of IRRs with ABCD than previously reported. Pre-medication is associated with decreased IRR incidence. Corticosteroids in particular appear to decrease IRRs while paracetamol and antihistamines, though commonly used, do not.

A novel substrate mimetic inhibitor of PKB/Akt inhibits prostate cancer tumor growth in mice by blocking the PKB pathway.

We describe a novel, potent peptide substrate mimetic inhibitor of protein kinase B (PKB/Akt). The compound selectively kills prostate cancer cells, in which PKB is highly activated, but not normal cells, or cancer cells in which PKB is not activated. The inhibitor induces apoptosis and inhibits the phosphorylation of PKB substrates in prostate cancer cell lines and significantly increases the efficacy of chemotherapy agents to induce prostate cancer cell death, when given in combination. In vivo, the inhibitor exhibits a strong antitumor effect in two prostate cancer mouse models. Moreover, treated animals develop significantly less lung metastases compared to untreated ones, and the effect is accompanied by a significant decrease in blood PSA [prostate-specific antigen] levels in treated animals. This compound and its potential analogues may be developed into novel, potent, and safe anticancer agents, both as stand-alone treatment and in combination with other chemotherapy agents.

Amphotericin B colloidal dispersion: an improved antifungal therapy.

Amphotericin B colloidal dispersion (ABCD) is a near 1:1 discoidal complex of amphotericin B (AMB) and sodium cholesteryl sulfate (SCS) arranged as a bilayer of SCS interspersed with AMB via noncovalent interactions. The complex is stable in blood and plasma with minimal dissociation. In vitro and in vivo studies show that ABCD is as effective and four to five times safer than conventional AMB (CAB) for fungal infection. Compared with CAB treatment, ABCD demonstrates reduced peak plasma levels, prolonged residence time, and lowered AMB levels in most tissues including kidney, the major target of toxicity for CAB. In 572 patients with systemic fungal infections secondary to severe underlying disease, ABCD doses < or = 6 mg/kg/day were well tolerated, even in those who failed to tolerate or respond to CAB. Mild-to-moderate, dose-dependent, infusion-related adverse events typically seen with CAB were also observed with ABCD, with no sign of renal or hepatic toxicity. Complete or partial recovery was seen in 57.3%. Therefore, ABCD should be considered as an alternative treatment of systemic fungal infections.

Cholesteryl hemisuccinate treatment protects rodents from the toxic effects of acetaminophen, adriamycin, carbon tetrachloride, chloroform and galactosamine.

In addition to its use as a stabilizer/rigidifier of membranes, cholesteryl hemisuccinate, tris salt (CS) administration has also been shown to protect rats from the hepatotoxic effects of carbon tetrachloride (CCl4). To further our understanding of the mechanism of CS cytoprotection, we examined in rats and mice the protective abilities of CS and the non-hydrolyzable ether form of CS, gamma-cholesteryloxybutyric acid, tris salt (CSE) against acetaminophen-, adriamycin-, carbon tetrachloride-, chloroform- and galactosamine-induced toxicity. The results of these studies demonstrated that CS-mediated protection is not selective for a particular species, organ system or toxic chemical. A 24-h pretreatment of both rats and mice with a single dose of CS (100mg/kg, i.p.), resulted in significant protection against the hepatotoxic effects of CCl4, CHCl3, acetaminophen and galactosamine and against the lethal (and presumably cardiotoxic) effect of adriamycin administration. Maximal CS-mediated protection was observed in experimental animals pretreated 24 h prior to the toxic insult. These data suggest that CS intervenes in a critical cellular event that is an important common pathway to toxic cell death. The mechanism of CS protection does not appear to be dependent on the inhibition of chemical bioactivation to a toxic reactive intermediate (in light of the protection observed against galactosamine hepatotoxicity). However, based on the data presented, we can not exclude the possibility that CS administration inhibits chemical bioactivation. Our findings do suggest that CS-mediated protection is dependent on the action of the intact anionic CS molecule (non-hydrolyzable CSE was as protective as CS), whose mechanism has yet to be defined.

Phase I study of amphotericin B colloidal dispersion for the treatment of invasive fungal infections after marrow transplant.

Amphotericin B colloidal dispersion (ABCD; Amphocil) was evaluated in a phase I dose-escalation study in 75 marrow transplant patients with invasive fungal infections (primarily Aspergillus or Candida species) to determine the toxicity profile, maximum tolerated dose, and clinical response. Escalating doses of 0.5-8.0 mg/kg in 0.5-mg/kg/patient increments were given up to 6 weeks. No infusion-related toxicities were observed in 32% of the patients; 52% had grade 2 and 5% had grade 3 toxicity. No appreciable renal toxicity was observed at any dose level. The estimated maximum tolerated dose was 7.5 mg/kg, defined by rigors and chills and hypotension in 3 of 5 patients at 8.0 mg/kg. The complete or partial response rate across dose levels and infection types was 52%. For specific types of infections, 53% of patients with fungemia had complete responses, and 52% of patients with pneumonia had complete or partial responses. ABCD was safe at doses to 7.5 mg/kg and had tolerable-infusion-related toxicity and demonstrable antifungal activity.

Cholesterol sulfate, a second messenger for the eta isoform of protein kinase C, inhibits promotional phase in mouse skin carcinogenesis.

Cholesterol sulfate is a second messenger for the eta isoform of protein kinase C mediating squamous differentiation. We found that cholesterol sulfate inhibited the promotional phase of skin carcinogenesis in female CD-1 mice, which was initiated by 100 micrograms 7,12-dimethylbenz[a]-anthracene and promoted by a single application of 10 micrograms 12-O-tetradecanoylphorbol-13-acetate, followed by repeated applications of 10 micrograms mezerein once a week for 19 weeks. Cholesterol sulfate, when applied topically at a dose of 400 micrograms (820 mumol) 10 min before treatment with the promoters, markedly suppressed tumor formation, resulting in decrease of 56% in the incidence of tumor-bearing mice, 81% in the number of tumors/mouse, and 60% in the size of tumors at 20 weeks of the promotion. This inhibition was not due to elimination of the initiated cells. Treatment with the parental cholesterol at a dose of 320 micrograms (820 mumol), which does not activate the eta isoform, did not inhibit tumor promotion. Repeated treatment with cholesterol sulfate induced scaling of skin at the site of application. Cholesterol sulfate, unlike most inhibitors of tumor promotion, did not inhibit induction of ornithine decarboxylase and hyperplasia in mouse epidermis caused by topical treatment with 12-O-tetradecanoylphorbol-13-acetate. These findings suggest that cholesterol sulfate inhibits tumor promotion by stimulating a differentiation pathway mediated by the eta isoform of protein kinase C.

Treatment of kala-azar in Brazil with Amphocil (amphotericin B cholesterol dispersion) for 5 days.

We have treated 10 patients suffering from kala-azar in Brazil with Amphocil (amphotericin B cholesterol dispersion) at a dose of 2 mg/kg/d for 5 d, following an earlier study in which this dosage for 7 d was found to cure all of 9 patients, with no relapse during 12 months. In the present study, all patients demonstrated initial resolution of disease. Parasites were absent upon bone marrow re-aspiration 2 weeks after therapy; no spleen extended beyond the costal margin 2 months after therapy; white blood cell counts, platelet counts, and serum levels of albumin rapidly returned to normal. Although one patient relapsed at 5 months, 8 of the other 9 patients had spleens of normal size (undetectable on deep palpation) at 12 months after therapy. Fever, sometimes accompanied by increased respiratory rate, occurred on the first day of drug infusion in 8 of 10 patients and was more severe in patients < 6 years old. Pre-medication with a non-steroidal anti-inflammatory agent (diclofenac potassium) before the next 4 infusions protected against this side effect in 5 of 6 patients. The results of this and our previous study suggest that the most appropriate regimen of Amphocil for kala-azar is 2 mg/kg/d for 7 d, with pre-medication each day, in patients aged > 5 years.

Treatment of invasive aspergillosis.

Invasive aspergillosis is generally a life-threatening invasive opportunistic mycosis affecting principally the upper and lower respiratory tract. Therapeutic response rates vary considerably from one host group to another with particularly high mortality rates in bone marrow transplant, liver transplant and patients with aplastic anaemia or AIDS. Only two drugs are useful for therapy, amphotericin and itraconazole. Recent advances in the formulation of amphoterin B (AmBisome and Amphocil) have resulted in intravenous preparations with lower toxicity, particularly nephrotoxicity, but it has yet to be shown that they have an increased therapeutic index for the treatment of invasive aspergillosis. Itraconazole can only be used orally and in some particularly high-risk or critically ill patients adequate serum concentrations cannot be achieved. The addition of flucytosine or rifampicin to amphotericin B therapy has, at best, only a marginal benefit. Surgery is essential for some manifestations of invasive aspergillosis. This article reviews therapeutic strategies including criteria for initiation of therapy, combination and sequential therapy, duration of therapy and secondary prophylaxis and indications for surgery in invasive aspergillosis.

Overview of amphotericin B colloidal dispersion (amphocil).

Amphotericin B colloidal dispersion (ABCD) is an equimolar mixture of amphotericin B and cholesteryl sulphate with desirable preparation and stability characteristics. It allows the intravenous delivery of amphotericin B in doses up to 7 mg/kg daily. Peak serum concentrations of amphotericin B, given as ABCD, are lower, AUC0-infinity similar and half-life longer than deoxycholate amphotericin B. In vitro activity may be altered with respect to the deoxycholate preparation, some isolates being more resistant and others more susceptible. Preclinical toxicology with ABCD revealed a safety factor of five to 19-fold compared with deoxycholate amphotericin B. Animal models of coccidioidomycosis, disseminated cryptococcosis, candidiasis and invasive aspergillosis indicated a better therapeutic ratio, especially in cryptococcosis. Phase I/II studies in humans demonstrate efficacy against coccidioidomycosis, candidiasis and aspergillosis at doses from 1-7 mg/kg/day in at least 100 patients. Renal toxicity was acceptable but infusion-related side effects and anaemia were common. Side effects appear to decrease on therapy. Comparative studies with deoxycholate amphotericin B are needed.

Treatment of Brazilian kala-azar with a short course of amphocil (amphotericin B cholesterol dispersion).

Amphotericin B is an effective but toxic antileishmanial agent. Lipid-encapsulated amphotericin B should have a high therapeutic index for visceral leishmaniasis because reticuloendothelial cells, the sole site in which Leishmania is found, will phagocytize and concentrate the complex. Amphotericin B cholesterol dispersion (Amphocil; 2 mg/[kg.d] intravenously) was administered to 10 Brazilians with kala-azar for 10 days (cohort 1) and to 10 Brazilians with kala-azar for 7 days (cohort 2). All patients were successfully treated: 19 of the 20 patients were without visible parasites in the bone marrow; the mean time to afebrility was 4.2 days; spleen size regressed by a mean of 79% 2 months after therapy; and no patient had clinical or laboratory abnormalities by the end of 6-12 months of follow-up. Side effects were fever and chills accompanied by respiratory distress, but not nephrotoxicity, in children < 3 years of age.

Protection against carbon tetrachloride-induced hepatotoxicity by pretreating rats with the hemisuccinate esters of tocopherol and cholesterol.

Previous studies have demonstrated that alpha-tocopheryl hemisuccinate (TS) protects hepatocyte suspensions from chemical-induced toxicity. It has been suggested that TS cytoprotection is related to unique properties of the TS molecule or is dependent on the cellular release and activity of unesterified alpha-tocopherol (T). To test the unique cytoprotective nature of TS in vivo, the protective ability of T and tocopherol esters against carbon tetrachloride (CCl4)-induced hepatotoxicity in rats was examined. Hepatoprotection [determined by serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and histopathology] was not observed after T (or tocopheryl acetate and tocopheryl nicotinate) administration, even though this treatment resulted in a fivefold elevation in hepatic T content. Only pretreatment with TS (100 mg/kg, intraperitoneally) resulted in partial hepatoprotection against CCl4 (2.9 g/kg, orally) toxicity. These findings suggest that hepatoprotection results not from the cellular accumulation of T but rather from the intact TS molecule. To test this hypothesis, the hepatoprotective capacity of cholesteryl hemisuccinate (CS), unesterified cholesterol, and cholesteryl acetate (CA) was examined against CCl4 toxicity. As observed with the tocopherol derivatives, pretreatment with unesterified cholesterol or CA demonstrated no protective ability. However, when rats were pretreated with CS (100 mg/kg), the hepatotoxic effects of CCl4 (elevated serum AST and ALT levels and centrilobular necrosis) were completely prevented. The prevention of CCl4-induced hepatotoxicity by CS and TS do not appear to result from an alteration in hepatic drug metabolism. These data clearly demonstrate that CS and TS are unique and powerful cytoprotective agents against CCl4 hepatotoxicity in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Activity of amphotericin B cholesterol dispersion (Amphocil) in experimental visceral leishmaniasis.

Standard therapy of human visceral leishmaniasis with parenteral pentavalent antimonial agents is generally curative but has the disadvantages of a 28-day treatment course, occasional treatment failures, and toxicity. The antifungal and antileishmanial agent amphotericin B has been complexed with lipids to develop a less toxic formulation of amphotericin B. Because lipid particles are phagocytized by the reticuloendothelial system, lipid-associated amphotericin B should be concentrated in infected macrophages and be very effective against visceral leishmaniasis. One formulation, amphotericin B cholesterol dispersion (ABCD) (Amphocil), was tested for antileishmanial activity in Leishmania donovani-infected hamsters. In the first experiment, hamsters were infected, administered with the drug 3 days later, and then sacrificed after a further 4 days. ABCD (dose needed to suppress 99% of hepatic parasites compared with controls [SD (99)], 0.4 mg/kg of body weight) was 15 times as effective as conventional amphotericin B [SD (99), 6.0 mg/kg]. Pentavalent antimony in the form of meglumine antimonate had an SD (84) of 416 mg/kg. In a second experiment in which animals were allowed to become more heavily infected, the drug was administered 10 days after infection and the animals were sacrificed after a further 2, 7, or 11 days. ABCD was approximately four times as active as conventional amphotericin B. These experiments suggest that ABCD is at least four times as active as conventional amphotericin B against visceral leishmaniasis and that clinical trials are warranted.

Enhanced antigenicity of autologous leukemia cells enriched with cholesterylhemisuccinate.

Cholesterylhemisuccinate (CHS) has been used by different authors to enhance the antigenicity of tumor cells in human and in animals. In the present study, leukemic cells isolated mainly from patients with chronic myeloid and chronic lymphocytic leukemia were incubated for 90 min at 37 degrees in PBS containing 3.5% polyvinylpyrrolidone, 0.5% glucose, 1% human serum albumin and 150 micrograms/ml CHS 5 x 10(6) pretreated, irradiated, autologous leukemic cells were tested for their ability to elicit a delayed type hypersensitivity skin reaction. The negative controls included CHS-treated and untreated non-leukemic autologous blood cells as well as substance controls. Recall antigens such as tetanus, diphtheria and tuberculine included in the Multitest Mérieux served as positive controls. Results from 28 patients and 44 skin tests are presented. 19 of 23 patients tested under the best of experimental conditions generated a positive skin reaction with the CHS-treated autologous leukemic cells. Less than 12% of the patients reacted with any of the above negative controls. Under the same conditions, more than 90% of the CLL patients were reactive. 5 CLL patients were selected for a treatment including 6 weekly injections of 5 x 10(6) to 5 x 10(8) autologous CLL cells. 3 patients experienced an up to 60% reduction of their initial leukocyte counts. In one patient, the leukocyte counts continued to decrease more than 6 months after the treatment was completed. One patient showed no response and in one patient with increasing leukocytes this increase could not be reversed. The results are discussed in terms of earlier observations that membrane sterol content influences the presentation of cell surface proteins.

Treatment of experimental invasive aspergillosis with novel amphotericin B/cholesterol-sulfate complexes.

An immunosuppressed rabbit model of invasive aspergillosis was used to evaluate a novel micellar preparation of cholesterol sulfate complexed to amphotericin B. The acute LD50 of amphotericin B-deoxycholate was 5.1 mg/kg versus 20 mg/kg for the amphotericin/cholesterol-sulfate complexes. Amphotericin B-deoxycholate given iv at a dose of 1.5 mg/kg was more effective in sterilizing liver and kidney than the amphotericin/cholesterol-sulfate complexes given iv at 1.5-4.5 mg/kg, but infection persisted in the lungs of all rabbits treated with those doses. Infection persisted even when the rabbits were given a lethal dose of amphotericin B-deoxycholate (4.5 mg/kg), but a dose of 15 mg/kg of the amphotericin/cholesterol-sulfate complexes sterilized tissues and was associated with no acute lethality. Equivalent doses of the amphotericin/cholesterol-sulfate complexes were less effective than amphotericin B-deoxycholate, but a fourfold decrease in acute lethality improved the therapeutic index of amphotericin B. The amphotericin/cholesterol-sulfate complexes appear to be an improved means of amphotericin B delivery and may improve therapy for invasive aspergillosis.

Active immunotherapy of human solid tumor with autologous cells treated with cholesteryl hemisuccinate. A Phase I study.

A marked increase in specific immunogenicity of tumor cells is induced upon incorporation of cholesteryl hemisuccinate (CHS) into the cell membrane, which presumably promotes the expression of latent tumor-associated antigens. Immunotherapy with CHS-treated and irradiated tumor cells as vaccine was found to be very effective in various murine experimental tumors and in eliciting delayed-type hypersensitivity in cancer patients. Based on these findings, we have carried out a Phase I study on 21 patients with solid tumor who had exhausted standard therapeutic options. All participating patients were examined by conventional physical and clinical tests prior and during the study. The immunotherapy regimen for most patients consisted of an intramuscular injection of 2 X 10(7) CHS treated and irradiated autologous tumor cells given at 2-week intervals. Variations on this regimen were mostly due to the lack of sufficient number of cells. None of the patients displayed evidence of toxicity or any other local or systemic adverse effects. In seven patients, regression of tumor mass was observed. In six of nine patients who were tested for delayed-type hypersensitivity against their CHS treated tumor cells, a significant increase in skin reaction was observed after immunotherapy. The lack of any adverse reaction in this treatment, in addition to the observed positive clinical and immunologic response in advanced cancer patients, indicate a safe therapeutic potency which is planned to be investigated in the subsequent clinical studies.