Experimental dog model for assessment of fasting and postprandial fatty acid metabolism: pitfalls and feasibility.

The dog is a widely-used model for conducting metabolic studies. This is mainly due to its large size and its physiology which is relatively similar to that of humans. Here, we attempted to optimize a postprandial metabolic study protocol used in dogs. Following acclimatization, female mongrel dogs underwent 9 h profiling for time-course baseline plasma data on triglyceride, adrenocorticotropic hormone (ACTH) and cortisol levels. One week later, carotid and jugular catheters were surgically inserted for sampling and infusions. Initial post-operative care, based on the literature (Protocol 1), consisted of analgesia (buprenorphine every 8-12 h and 2-3 doses/day of acepromazine), restriction by Pavlov harness within cages, and a two- to three-day recovery period. Throughout the experiment, dogs received a lipid tracer diluted in 5% bovine serum albumin (BSA). Compared with baseline, animals vomited (n = 6/6) and exhibited high ACTH + cortisol levels (stress biomarkers), resulting in blunted triglyceride peak levels. To avoid these undesirable effects, post-operative care was modified (Protocol 2) as follows: animals (n = 19) were given a single dose of buprenorphine and no acepromazine, were unrestrained and free to move within cages, the recovery period was extended to seven days, and the lipid tracer was diluted in 0.002% versus 5% BSA. Using this modified protocol, postprandial plasma-triglyceride and ACTH/cortisol patterns were similar to baseline values. Controlling for stressors, as well as for factors which may alter proper digestion, is critical for all postprandial metabolic studies. Our results show that an optimized postprandial metabolic protocol used in dogs reduces experimental variability, while improving animal care and comfort.

Lasting alterations of the sodium current by short-term hyperlipidemia as a mechanism for initiation of cardiac remodeling.

Clinical and animal studies indicate that increased fatty acid delivery to lean tissues induces cardiac electrical remodeling and alterations of cellular calcium homeostasis. Since this may represent a mechanism initiating cardiac dysfunction during establishment of insulin resistance and diabetes or anaerobic cardiac metabolism (ischemia), we sought to determine if short-term exposure to high plasma concentration of fatty acid in vivo was sufficient to alter the cardiac sodium current (INa) in dog ventricular myocytes. Our results show that delivery of triglycerides and nonesterified fatty acids by infusion of Intralipid + heparin (IH) for 8 h increased the amplitude of INa by 43% and shifted its activation threshold by -5 mV, closer to the resting membrane potential. Steady-state inactivation (availability) of the channels was reduced by IH with no changes in recovery from inactivation. As a consequence, INa "window" current, a strong determinant of intracellular Na+ and Ca2+ concentrations, was significantly increased. The results indicate that increased circulating fatty acids alter INa gating in manners consistent with an increased cardiac excitability and augmentation of intracellular calcium. Moreover, these changes could still be measured after the dogs were left to recover for 12 h after IH perfusion, suggesting lasting changes in INa. Our results indicate that fatty acids rapidly induce cardiac remodeling and suggest that this process may be involved in the development of cardiac dysfunctions associated to insulin resistance and diabetes.

The first preclinical pharmacotoxicological safety assessment of CGS 35601, a triple vasopeptidase inhibitor, in chronically instrumented, conscious, and unrestrained spontaneously hypertensive rats.

CGS 35601 is a triple vasopeptidase inhibitor (VPI) of angiotensin-converting enzyme, neutral endopeptidase, and endothelin-converting enzyme-1 with respective IC50 values of 22, 2, and 55 nM. We characterized the safety profile and toxicity of escalating doses of CGS 35601 over a 20-day period in chronically instrumented, unrestrained, conscious, male, spontaneously hypertensive rats (SHR). Once instrumented with an arterial catheter, the SHR were placed in metabolic cages allowing daily assessment of hemodynamics and blood sampling for biochemical and hematological measurements. After a 7-day stabilization period, the SHR were divided into 2 groups: Gr. 1, (n = 13 to 18) receiving CGS 35601 at 0.01, 0.1, 1 and 5 mg kg(-1) day(-1) (continuous i.a. infusion) for 5 consecutive days/dose, followed by a 5-day washout; and Gr. 2, (n = 10) receiving vehicle (saline). The highest dose of CGS 35601 dose-dependently reduced MABP from 156 +/- 4 up to 94 +/- 5 mm Hg, whereas heart rate, metabolic, electrolytic, and hematological profiles, growth, diuresis, and renal activity were unaffected, and no hepatic or liver toxicities were observed. These results suggest that this novel triple VPI presents no safety concerns at this stage and may become of interest for the treatment of hypertension and other cardiovascular disorders. Long-term chronic experiments are needed to assess possible angioedema and increases in vascular permeability.

Purine signaling and potential new therapeutic approach: possible outcomes of NTPDase inhibition.

Interest for extracellular nucleotides has increased since the pioneer work of Burnstock in the early seventies. Research on cellular functions modulated by purines and pyrimidines has led to the identification and characterization of the different components of purine signaling, namely purinoceptors and ecto-nucleotidases. Receptors for tri- and diphosphonucleosides, known as P2 nucleotide receptors, are designated either P2Y receptors, for those coupled to G-proteins, or P2X for those which are ligand gated-ion channels. Ecto-nucleoside triphosphate diphosphohydrolase (NTPDase; EC 3.6.1.5), previously identified as ecto-ATPase, ecto-ATPDase or CD39, is now considered as the main ecto-nucleotidase responsible for the sequential hydrolysis of beta and gamma phosphates of tri- and diphosphonucleosides. More recently, research has been focused on the development of specific agonists and antagonists to P2 purinoceptors. The need to develop specific inhibitors for NTPDase to understand the role of this enzyme has clearly emerged. This paper covers the development of specific molecules targeting purinergic signaling, more specifically the inhibition of NTPDase and their impact on the different physiological systems.

Characterization and immunohistochemical localization of nucleoside triphosphate diphosphohydrolase (NTPDase) in pig adrenal glands (presence of a non-sedimentable isoform).

Considering that adrenal glands possess a variety of purinoceptors associated with various cell types and that some of these cells (chromaffin cells) secrete large amounts of adenine nucleotides, it was of interest to localize nucleoside triphosphate diphosphohydrolase (NTPDase) in these glands and to define the biochemical characteristics of this ectonucleotidase. Immunolocalization produced a moderate reaction in capsula and medulla, with no signal in zona glomerulosa and zona reticularis. In contrast, a very strong reaction was found in zona fasciculata. Biochemical analysis of particulate fractions isolated from whole glands revealed high levels of ATPase and ADPase activities. This appeared to be attributable to the NTPDase since the level of ADPase was as high as ATPase. Both ATPase and ADPase activities were similarly inhibited by sodium azide. Additionally electrophoretograms with these two substrates showed comparable patterns. Western blots with 'Ringo', an antibody that recognizes the different isoforms of mammalian NTPDases, showed the presence of isoforms of NTPDases at 54 and 78 kDa, respectively. Interestingly, the 54 kDa isoform remains in the supernatant of a chromaffin granule lysate after ultracentrifugation. Up until now little interest has been given to the relationship between adrenal medulla and cortex. Presence of purinoceptors and ectonucleotidases in both these regions together with the effects of ATP in vivo and in vitro in different species indicate that purines play a significant role in adrenal glands.

Distribution, cloning, and characterization of porcine nucleoside triphosphate diphosphohydrolase-1.

In this study, we have investigated the distribution of the enzyme nucleoside triphosphate diphosphohydrolase-1 (NTPDase1; EC 3.6.1.5) in a subset of pig tissues by biochemical activity and Western blotting with antibodies against porcine NTPDase1. The highest expression of this enzyme was found in vascular endothelium, smooth muscle, spleen and lung. The complete cDNA of NTPDase1 from aorta endothelial cells was sequenced using primer walking. The protein consists of 510 amino acids, with a calculated molecular mass of 57 756 Da. The amino-acid sequence indicated seven putative N-glycosylation sites and one potential intracellular cGMP- and cAMP-dependent protein kinase phosphorylation site. As expected, the protein has a very high homology to other known mammalian ATPDases and CD39 molecules, and includes all five apyrase conserved regions. Expression of the complete cDNA in COS-7 cells confirmed that NTPDase1 codes for a transmembrane glycoprotein with ecto-ATPase and ecto-ADPase activities. Two proteolytic products of NTPDase1, with molecular mass of 54 and 27 kDa, respectively, were consistently present in proteins from transfected COS-7 cells and in particulate fractions from different tissues. A trypsin cleavage site, giving rise to these two cleavage products, was identified. In order to remain enzymatically active, the two cleavage products have to interact by non-covalent interactions.

Identification and immunolocalization of two isoforms of ATP-diphosphohydrolase (ATPDase) in the pig immune system.

The occurrence of a variety of purine receptors in the immune system indicates that extracellular purines play important functional roles. Extracellular purine concentrations are, in great part, determined by ectonucleotidases, namely, the ATP diphosphohydrolase, also identified as CD39, a lymphocyte cell surface marker. The latter enzyme converts triphospho- and diphosphonucleosides to nucleoside monophosphates. In this study, high levels of ATPase and ADPase activities have been found in homogenates of the different pig lymphoid organs. Specific activities decreased in the following order: spleen > bone marrow > thymus > lymph glands. The parallel decrease in ATPase and ADPase activities, in the presence of sodium azide, indicated that an ATP diphosphohydrolase (ATPDase) was responsible for these activities. Particulate fractions, prepared from the different lymphoid organs by ultracentrifugation on a sucrose cushion, showed about a 10-fold enrichment of ATPDase activity. Identity of ATPDase was confirmed by electrophoretograms of the particulate fractions and Western immunoblots, with an antibody that recognizes ATPDases from different sources. Two isoforms of ATPDase were found (I and II), corresponding to molecular masses of 78,000 and 54,000, respectively, as estimated by SDS-PAGE. Immunohistochemical localization was carried out on these different organs: In spleen, reaction was found in both white and red pulps. A particularly intense reaction was put in evidence in nervous fibers of this organ. Immunolocalization also showed positive reactions with tonsilar lymphoid structures, diffuse lymphoid tissues, and nodules associated with stomach, duodenum, jejunum, and ileum. In addition, our observations establish the presence of ATPDase in lymphocytes and macrophages of the pig immune system.

Pig pancreatic acinar cells possess predominantly the CCK-B receptor subtype.

Studies performed on pig indicated that its pancreas was insensitive to the gastrointestinal hormone cholecystokinin (CCK) and suggested that its secretions were rather under the control of the neurotransmitter acetylcholine. This study was performed to determine reasons for this insensitivity by comparing secretory responses to different secretagogues and establishing the dominant CCK receptor type. Pancreatic acini prepared from weaned piglets were evaluated for their sensitivity to carbamylcholine (Cch), caerulein, JMV-180, and secretin. RNA were extracted for CCK-A and CCK-B receptor expression using specific cRNA probes. Results indicate that pig pancreatic acini are sensitive to Cch and relatively insensitive to caerulein with no response to JMV-180, a CCKA agonist, or secretin; MK-329, a CCK-A receptor antagonist, significantly inhibited caerulein-induced enzyme secretion from 10(-8) M. The pig pancreas expresses few CCK-A mRNA receptors but a majority of CCK-B. These data demonstrate that the pig pancreas expresses a majority of CCK-B receptors. In conclusion, the pig pancreas possesses a large majority of CCK-B receptors responsible for their low sensitivity to CCK.

PEG-coated poly(lactic acid) nanoparticles for the delivery of hexadecafluoro zinc phthalocyanine to EMT-6 mouse mammary tumours.

Hexadecafluoro zinc phthalocyanine (ZnPcF16), a second generation sensitizer for the photodynamic therapy of cancer, was incorporated in three vehicles: poly(D,L-lactic acid) (PLA) nanoparticles, polyethylene glycol (PEG)-coated nanoparticles and a Cremophor EL (CRM) oil-water emulsion. Nanoparticles were prepared by the salting-out procedure. Biodistribution of the dye was assessed by fluorescence in EMT-6 mammary tumour bearing mice after intravenous injection of 1 mumol kg-1 ZnPcF16. Plain nanoparticles were rapidly retained by the reticuloendothelial system (RES) as reflected by the low area under the blood concentration-time curve (AUC0-168, 57 micrograms h g-1). Little tumour uptake of the dye was observed with this formulation. In contrast, PEG-coated nanoparticles displayed a reduced RES uptake, leading to significantly higher blood levels over an extended period (t1/2 30 h; AUC 0-168 227 micrograms h g-1) and enhanced tumour uptake. At 48 h post injection, tumour to skin and tumour to muscle concentration ratios reached 3.5 and 10.8, respectively. Blood levels of ZnPcF16 after administration as a CRM emulsion decreased faster than with PEG-coated nanoparticles (t1/2 12 h), but since no early liver uptake was observed, the AUC0-168 and the tumour uptake were only slightly lower. However, with the CRM formulation, a late liver uptake was observed, reaching 51% of the injected dose after 7 days.

Alterations of pancreatic amylase secretion in the reserpinized rat model of cystic fibrosis. Effects of cerulein and EGF.

Reserpine treatment resulted in altered enzyme secretion from rat pancreatic acini in response to carbamylcholine and secretin (1,2). This study was undertaken: (1) To evaluate if the alterations caused by reserpine can be prevented by EGF and/or cerulein treatments; (2) To determine the time-course of secretion recovery after reserpine treatment; and (3) To establish if EGF and/or cerulein treatments can accelerate such a recovery after the reserpine treatment. Male Sprague-Dawley rats (250-265 g) were used in these experiments. In experiment I, rats divided into three groups received either reserpine (R) or the reserpine vehicle for the controls (C) and the pair-fed controls (PF) for 7 d. During treatment, PF and R rats were given SC, twice a day, saline, EGF (10 micrograms/kg), cerulein (1 microgram/kg), or both at the same dose. C rats received saline in gelatin. In experiment II, rats were treated for 7 d with reserpine or the vehicle as described in experiment I, were allowed a 30-d recovery period and then were killed. In experiment III, C, PF, and R rats were treated for 7 d as described in experiment I; on the 8th d and for the next 6 d, reserpine rats received saline (reserpine-saline), cerulein, EGF, or both cerulein +EGF at the same dose as indicated in experiment I. C and PF rats received saline in gelatin. After sacrifice, acini were prepared, and amylase dose-response curves to carbamylcholine (Cch) and secretin were established. EGF, cerulein, or their combination given to R rats did not improve the desensitized secretory response to Cch.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations of the pancreatic secretory responses to secretin and to the ionophore A23187 by reserpine: a calcium-mediated phenomenon?

This study was undertaken to further characterize the secretory response of the rat pancreas after reserpine treatment. Rats were given reserpine (1 mg kg-1 day-1 i.p.) or vehicle for 7 days. To distinguish between specific effects of reserpine and those related to secondary malnutrition caused by the drug, the secretory response of a group of pair-fed (PF) animals to reserpine was also investigated. Amylase release from dispersed pancreatic acini, prepared from control (C), PF and reserpine-treated (R) rats were used to evaluate functional secretory capacity. Reserpine and pair-feeding caused reduced responses of pancreatic acini to secretin. The pair-feeding-altered secretin response was greatly improved by increasing extracellular Ca2+ concentration, whereas a slight improvement was noticed in the R group. Reserpine significantly reduced the secretory response to the ionophore A23187 at concentrations above 5 x 10(-7) M in 1.25 mM Ca2+; in 2.5 mM Ca2+, the response to the ionophore was significantly higher in the R group than in C at all ionophore concentrations. Furthermore, at 2 x 10(-7) M ionophore, the secretory response to secretin in the R group became significantly higher than that in the C group but comparable to that of the control+ionophore. In conclusion, reserpine affects the secretory response to secretin as did pre-exposure of pancreatic acini to a high concentration of carbamylcholine. The modified secretory response to the ionophore following reserpine treatment indicates that reserpine may act as a 'Ca2+ entry mechanism' antagonist which may explain the partial reduction in the secretin response.

Effects of cerulein and epidermal growth factor on pancreatic growth in the reserpinized rat model.

This study was undertaken to determine the effect of reserpine on rat pancreatic growth, to evaluate if reserpine-caused alterations can be prevented by epidermal growth factor (EGF) and/or cerulein treatment, to evaluate the time course of rat pancreas recovery after reserpine, and to determine if EGF and/or cerulein treatment can accelerate such a recovery. In the first experiment, three groups of male Sprague-Dawley rats (250-265 g) were used. Ad libitum-fed control animals received the reserpine vehicle, and one experimental group received reserpine (1 mg kg-1 day-1 for 7 days) while the other, pair-fed group received the reserpine vehicle with a reduced amount of food to result in malnourishment. Rats from each of these three groups were also assigned to one of four treatments consisting of saline, EGF (10 micrograms kg-1), cerulein (1 microgram kg-1), or a combination (same doses) twice a day for 7 days. In the morning of the 8th day, after an overnight fat, rats were killed. In the second experiment, rats were selected and treated with reserpine or the vehicle as described in experiment 1; after the 7-day treatment, a first cohort of animals was allowed a 30-day recovery period. Three other groups (an ad libitum-fed control, a pair-fed, and a reserpine group) were allowed a 6-day recovery period during which they were treated subcutaneously, twice a day, with either saline, EGF (10 micrograms kg-1), cerulein (1 microgram kg-1), or a combination (same doses). On the morning of the 31st or 7th day, after an overnight fat, rats were killed. After death, all pancreata were examined for weight and protein, amylase, chymotrypsinogen, RNA, and DNA content. In the ad libitum-fed control group, EGF caused pancreatic hypertrophy, whereas cerulein was associated with hypertrophy and hyperplasia. In the pair-fed malnourished group, the EGF effect was limited to slight increases in pancreatic weight and cell mass whereas cerulein caused hypertrophy; EGF plus cerulein caused pancreatic hyperplasia. In the reserpine group, EGF had no effect, whereas cerulein caused pancreatic hypertrophy and an increase in DNA content above the reserpine control. After 30 days of recovery, pancreata of pair-fed animals and those of reserpine-treated animals were comparable with those of the ad libitum-fed control rats with the exception of amylase levels, which remained reduced in the reserpine group.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of protein kinase C activity by palmitoyl esters of maltose.

Mixtures of maltose palmitates containing predominantly maltose tetrapalmitate (designated MTP) possess immune potentiating and antitumor properties. Immune potentiation derives from macrophage activation and B lymphocyte mitogenicity and antitumor action from anti-angiogenic activity. Their mode of action at the cellular level is not known. Since high performance liquid chromatography (HPLC) provided purified maltose palmitates, we tested whether they individually and as a mixture could modulate activity of protein Kinase C (PKC), an enzyme implicated in mitogenic and release reactions. MTP activated crude lymphocyte and purified brain PKC in the absence of phosphatidyl serine (PS). It also augmented labeled dibutyryl phorbol (PDBu) binding to the brain enzyme in the absence of phospholipid. HPLC purified maltose tetrapalmitates (two isomers) were insoluble in aqueous solvent, and activated PKC slightly after incorporation into PS liposomes. Purified maltose di- and tri-palmitates were inhibitory to the enzyme. The activation of PKC was, therefore, due to higher saturated maltose palmitates, well dispersed by less substituted maltose palmitates acting as emulsifiers.

Alterations of amylase secretion in the chronically reserpinized rat: an acetylcholine-mediated phenomenon.

Chronic reserpine treatment of animals, an experimental model for cystic fibrosis (CF), results in generalized exocrinopathy, impaired secretion, and decreased pancreatic content of amylase. However, the mechanisms of altered acinar pancreatic function in both CF patients and reserpine-treated rats are still unknown. The present study was designed to investigate the secretory response of the rat pancreas after reserpine treatment. Rats were given reserpine (1 mg kg-1 day-1 ip) or vehicle, and killed 2, 4, or 7 days later. To distinguish between specific effects of reserpine and those related to secondary malnutrition caused by the drug, secretory response of a group of pair-fed animals to reserpine was also investigated. During reserpine treatment, body weight and food intake were significantly reduced from the 2nd day on. Amylase release from dispersed pancreatic acini, prepared from control pair-fed and from reserpine-treated rats were used to evaluate functional secretory capacity. After 7 days, both chronic reserpine and pair-feeding significantly decreased pancreatic amylase concentration to 75 and 50% of controls, respectively. Reserpine caused desensitization of the pancreatic acini secretory response to carbamylcholine, without altering maximal amylase release. Desensitization occurred gradually and reached a maximum after 7 days. Secretory responses to caerulein and to the phorbol ester 12.0-tetradecanoyl phorbol-13 acetate (TPA) were not altered. These findings indicate that desensitization of amylase release after reserpine was specific to this muscarinic agonist. It may result from increased endogenous acetylcholine release in the course of treatment.

The ability of staurosporine to modulate pancreatic acinar cell desensitization by TPA, carbamylcholine and caerulein.

The implication of protein kinase C in the phenomenon of pancreatic acinar cell desensitization to carbamylcholine, caerulein and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was investigated using a potent PKC inhibitor, staurosporine. At a concentration of 1 microM, staurosporine caused a maximum 64% inhibition of amylase release from rat pancreatic acini stimulated by 100 nM TPA. At 100 nM, staurosporine reduced by 50 to 55% amylase secretion elicited by maximal concentrations of carbamylcholine or caerulein without affecting their potency. Staurosporine was also able to prevent completely desensitization by TPA of the subsequent secretory response to carbamylcholine and caerulein. Furthermore, staurosporine also totally prevented desensitization by caerulein of the subsequent secretory response to caerulein. In contrast, staurosporine only partially prevented desensitization by carbamylcholine of the subsequent secretory response to carbamylcholine. These results indicate that staurosporine is a potent inhibitor of protein kinase C as it inhibited the secretory response to carbamylcholine, caerulein and TPA. They also suggest that desensitization of the secretory response induced by TPA and caerulein used a common pathway involving protein kinase C activation. Finally, desensitization by carbamylcholine is more complex as it is only partially prevented at staurosporine; therefore, protein kinase C activation seems to be one of the factors involved.

Effect of maltose tetrapalmitate on tumor vascularization and tumor growth.

C3HBA mammary tumor (H-2k) was implanted s.c. by trocar in MTP responder C3H/HeN (H-2k) and non-responder C3H/HeJ (H-2k) female mice. One half of the animals received MTP i.p. The size of the tumor was measured everyday. Tumor growth was slightly slower in the C3H/HeJ than in the C3H/HeN. MTP treatment was effective only for the tumor implanted in the C3H/HeN. Microscopic and microscopic visualization of the tumor 1, 2, 3, and 15 days after tumor implantation and MTP treatment revealed poor vascular development in the MTP-treated C3H/HeN compared to untreated controls at days 2 and 3.

Evaluation of cortisone-heparin and cortisone-maltose tetrapalmitate therapies against rodent tumors. I. Biological studies.

The antitumor activity of either cortisone-heparin or cortisone-maltose tetrapalmitate combination or both was tested against two animal tumor models. The first model was orthotopically implanted bladder tumor established in syngeneic Fisher 344 rats. Shrinkage and growth arrest of the tumors were induced by cortisone and amplified by its combination with either heparin or maltose tetrapalmitate (MTP). The second model was trocar implanted C3HBA mammary tumor piece s.c. in syngeneic LPS and MTP responder C3H/HeN and non responder C3H/HeJ mice. The tumor was sensitive to growth inhibition by cortisone-MTP in the C3H/HeN but not by cortisone alone or cortisone-heparin. Tumor implanted in C3H/HeJ was much less sensitive to cortisone-MTP. Cortisone could be replaced by 17-alpha-hydroxyprogesterone, but not by cortexolone.

Evaluation of cortisone-heparin and cortisone-maltose tetrapalmitate therapies against rodent tumors. II. Pathological studies.

Pathological studies were undertaken in three tumor-host models which were subjected to cortisone based treatments. The first model was Fisher 344 rats with established orthotopically implanted syngeneic bladder tumor. Cortisone-herapin and cortisone-maltose tetrapalmitate (MTP) treatments induced focal areas of tumor necrosis and necrobiosis, whereas cortisone alone caused necrobiosis. The second model was C3HBA mammary tumor fragments implanted subcutaneously in syngeneic MTP responder C3H/HeN and MTP non-responder C3H/HeJ female mice. Only cortisone-MTP treatment led to an absence of capillary extension from surrounding blood vessels into the scant tumor stroma. The third model, ethyl carbamate induced primary lung cancer in AJ mice, was tested only with cortisone-herapin combination. The treatment caused central zones of necrosis.

Prophylactic antiangiogenic tumor treatment.

Complementary antitumor treatments are required to increase the cure rate achieved by surgery and/or radiotherapy by avoiding future recurrences and metastases. The growth of most solid tumors, particularly carcinomas, depends upon the simultaneous development of internal tumor vasculature to allow the proliferation of tumor cells. Inhibition of tumor vascularization is an indirect means of limiting tumor expansion. Daily administration of cortisone and maltose tetrapalmitate (MTP) abolished growth of implanted syngeneic C3HBA mammary tumor. Gross and macroscopic examination of these tumors revealed that tumor growth was prevented. Histological examination demonstrated lack of vascularization within the neoplastic tissue. We believe that this combination in an appropriate form could provide a prophylactic treatment regimen after conventional antitumor treatments in humans.

Antitumor efficacies of maltose tetrapalmitate immunotherapy alone and in combinations with radiotherapy and with cyclophosphamide chemotherapy against dimethylhydrazine induced colon and anal cancers in CDI mice.

Treatment of human colonic cancer in early stages when the process is still limited to the colonic wall is primarily surgery. We wished to see if maltose tetrapalmitate (MTP) immunotherapy alone or in combination with radiotherapy (R) and cyclophosphamide (C) chemotherapy would be effective against primary colon cancer in a fashion similar to that reported by us for primary liver cancer (Anticancer Research 6: 245-250, 1986). One hundred female CD1 mice were subjected to dimethylhydrazine (DMH) treatment once a week for 26 weeks, a period one week before which, colon cancer was histologically documented in each animal of a group that was sacrificed. Surprisingly, many of the animals harboured early anal cancer as well. At 28 weeks, 85 of the available animals were divided into 6 groups that received: Gr. 1, no treatment; Gr. 2, MTP alone (M); Gr. 3, radiotherapy alone (R); Gr. 4, cyclosphophamide alone (C); Gr. 5, R + C; Gr. 6, M + R + C. Criteria of treatment efficacy were: number, size and staging of colorectal tumors and the incidence and the size of anal tumors at death. Mean survival time was also determined although it remained a questionable criterium since most animals died due to complication (hepatic toxicity, pyelonephritis, thrombose) elicited by DMH, R and C toxicities and not as a result of colonic tumor size or metastases. As a single therapy, M appeared to be superior to either R or C alone. However, R + C combination was effective and was further improved upon by its association with M. With the triple combination, (M + R + C), lesions of both cancers decreased in size and/or number and the colon cancer histologically eclipsed from 46% of the treated animals.