Comparative therapeutic efficacy and safety of type-II collagen (UC-II), glucosamine and chondroitin in arthritic dogs: pain evaluation by ground force plate.

The investigation was conducted on client-owned moderately arthritic dogs with two objectives: (i) to evaluate therapeutic efficacy of type-II collagen (UC-II) alone or in combination with glucosamine hydrochloride (GLU) and chondroitin sulphate (CHO), and (ii) to determine their tolerability and safety. Dogs in four groups (n = 7-10), were treated daily for a period of 150 days with placebo (Group-I), 10 mg active UC-II (Group-II), 2000 mg GLU + 1600 mg CHO (Group-III), and UC-II + GLU + CHO (Group-IV). On a monthly basis, dogs were evaluated for observational pain (overall pain, pain upon limb manipulation, and pain after physical exertion) using different numeric scales. Pain level was also measured objectively using piezoelectric sensor-based GFP for peak vertical force and impulse area. Dogs were also examined every month for physical, hepatic (ALP, ALT and bilirubin) and renal (BUN and creatinine) functions. Based on observations, significant (p < 0.05) reduction in pain was noted in Group-II, III, and IV dogs. Using GFP, significant increases in peak vertical force (N/kg body wt) and impulse area (N s/kg body wt), indicative of a decrease in arthritis associated pain, were observed in Group-II dogs only. None of the dogs in any group showed changes in physical, hepatic or renal functions. In conclusion, based on GFP data, moderately arthritic dogs treated with UC-II (10 mg) showed a marked reduction in arthritic pain with maximum improvement by day 150. UC-II, GLU and CHO operate through different mechanisms of action, and were well tolerated over a period of 150 days.

Therapeutic efficacy of undenatured type-II collagen (UC-II) in comparison to glucosamine and chondroitin in arthritic horses.

The present investigation evaluated arthritic pain in horses receiving daily placebo, undenatured type II collagen (UC-II) at 320, 480, or 640 mg (providing 80, 120, and 160 mg active UC-II, respectively), and glucosamine and chondroitin (5.4 and 1.8 g, respectively, bid for the first month, and thereafter once daily) for 150 days. Horses were evaluated for overall pain, pain upon limb manipulation, physical examination, and liver and kidney functions. Evaluation of overall pain was based upon a consistent observation of all subjects during a walk and a trot in the same pattern on the same surface. Pain upon limb manipulation was conducted after the walk and trot. It consisted of placing the affected joint in severe flexion for a period of 60 sec. The limb was then placed to the ground and the animal trotted off. The response to the flexion test was then noted with the first couple of strides the animal took. Flexion test was consistent with determining clinically the degree of osteoarthritis in a joint. Horses receiving placebo showed no change in arthritic condition, while those receiving 320 or 480 or 640 mg UC-II exhibited significant reduction in arthritic pain (P < 0.05). UC-II at 480 or 640 mg dose provided equal effects, and therefore, 480 mg dose was considered optimal. With this dose, reduction in overall pain was from 5.7 +/- 0.42 (100%) to 0.7 +/- 0.42 (12%); and in pain upon limb manipulation from 2.35 +/- 0.37 (100%) to 0.52 +/- 0.18 (22%). Although glucosamine and chondroitin treated group showed significant (P < 0.05) reduction in pain compared with pretreated values, the efficacy was less compared with that observed with UC-II. In fact, UC-II at 480 or 640 mg dose was found to be more effective than glucosamine and chondroitin in arthritic horses. Clinical condition (body weight, body temperature, respiration rate, and pulse rate), and liver (bilirubin, GGT, and ALP) and kidney (BUN and creatinine) functions remained unchanged, suggesting that these supplements were well tolerated.

Therapeutic Efficacy and Safety of Undenatured Type II Collagen Singly or in Combination with Glucosamine and Chondroitin in Arthritic Dogs.

ABSTRACT This investigation was undertaken to evaluate the therapeutic efficacy and safety of glycosylated undenatured type II collagen (UC-II) alone or in combination with glucosamine HCl and chondroitin sulfate in arthritic dogs. Twenty dogs divided into four groups (n = 5) were daily treated orally for 120 days: group I, placebo; group II, 10 mg UC-II; group III, 2,000 mg glucosamine + 1,600 mg chondroitin; group IV, UC-II (10 mg) + glucosamine (2,000 mg) + chondroitin (1,600 mg), followed by a 30-day withdrawal period. On a monthly basis, dogs were examined for overall pain, pain upon limb manipulation, and exercise-associated lameness. Serum samples were analyzed for markers of liver function (ALT and bilirubin) and renal function (BUN and creatinine). Body weight was also measured at a monthly interval. Dogs in group I exhibited no change in arthritic conditions. Dogs receiving UC-II alone showed significant reductions in overall pain within 30 days (33%) and pain upon limb manipulation and exercise-associated lameness after 60 days (66% and 44%, respectively) of treatment. Maximum reductions in pain were noted after 120 days of treatment (overall pain reduction, 62%; pain reduction upon limb manipulation, 91%; and reduction in exercise-associated lameness, 78%). The overall activity of the dogs in the UC-II supplemented with glucosamine and chondroitin group (group IV) was significantly better than the glucosamine + chondroitin-supplemented group (group III). Glucosamine and chondroitin alleviated some pain, but in combination with UC-II (group IV) provided significant reductions in overall pain (57%), pain upon limb manipulation (53%), and exercise-associated lameness (53%). Following withdrawal of supplements, all dogs (groups II to IV) experienced a relapse of pain. None of the dogs in any groups showed any adverse effects or change in liver or kidney function markers or body weight. Data of this placebo-controlled study demonstrate that daily treatment of arthritic dogs with UC-II alone or in combination with glucosamine and chondroitin markedly alleviates arthritic-associated pain, and these supplements are well tolerated as no side effects were noted.

Efficacy and safety of glycosylated undenatured type-II collagen (UC-II) in therapy of arthritic dogs.

DeParle L. A., Gupta R. C., Canerdy T. D., Goad J. T., D'Altilio M., Bagchi M., Bagchi D. Efficacy and safety of glycosylated undenatured type-II collagen (UC-II) in therapy of arthritic dogs. J. vet. Pharmacol. Therap.28, 385-390. In large breed dogs, arthritis is very common because of obesity, injury, aging, immune disorder, or genetic predispositions. This study was therefore undertaken to evaluate clinical efficacy and safety of undenatured type-II collagen (UC-II) in obese-arthritic dogs. Fifteen dogs in three groups received either no UC-II (Group I) or UC-II with 1 mg/day (Group II) or 10 mg/day (Group III) for 90 days. Lameness and pain were measured on a weekly basis for 120 days (90 days treatment plus 30 days post-treatment). Blood samples were assayed for creatinine and blood urea nitrogen (markers of renal injury); and alanine aminotransferase and aspartate aminotransferase (evidence of hepatic injury). Dogs receiving 1 mg or 10 mg UC-II/day for 90 days showed significant declines in overall pain and pain during limb manipulation and lameness after physical exertion, with 10 mg showed greater improvement. At either dose of UC-II, no adverse effects were noted and no significant changes were noted in serum chemistry, suggesting that UC-II was well tolerated. In addition, dogs receiving UC-II for 90 days showed increased physical activity level. Following UC-II withdrawal for a period of 30 days, all dogs experienced a relapse of overall pain, exercise-associated lameness, and pain upon limb manipulation. These results suggest that daily treatment of arthritic dogs with UC-II ameliorates signs and symptoms of arthritis, and UC-II is well tolerated as no adverse effects were noted.

Cholinergic and noncholinergic brain biomarkers of insecticide exposure and effects.

The objective of this investigation was to determine the distribution of cholinergic and noncholinergic biomarkers in discrete brain regions (cortex, stem, striatum, hippocampus, and cerebellum) of rats treated with dimethyl sulfoxide (DMSO, controls), and insecticides such as carbofuran (CARB, 1.5 mg/kg, sc), or methyl parathion (MPTH, 5 mg/kg, ip). Both insecticides produced characteristic signs of anticholinesterase nature within 5-7 min after injection. In controls, analyses of the brain regions revealed a wide variability in the values of cholinergic (acetylcholinesterase, AChE) and noncholinergic (creatine kinase, CK; and lactic dehydrogenase, LDH, and their isoenzymes) biomarkers. The highest activities of AChE and LDH were found in the striatum (1661+/-23 micromol/g/h and 57,720+/-478 IU/l, respectively) and lowest in the cerebellum (118+/-6 micromol/g/h) and 39,480+/-918 IU/l, respectively). However, the activity of CK was found highest in the cerebellum (742,560+/-798 IU/l) and lowest in the hippocampus (353,400+/-11,696 IU/l). Each brain region showed a characteristic profile of CK and LDH isoenzymes. Among the CK isoenzymes, activity of CK-BB was highest (77.5-89.3%), followed by CK-MM (6.7-15.6%), and least CK-MB (0-6.9%). The cerebellum had no CK-MB activity. In all brain regions, CK-MM isoenzyme had only the CK-MM3 subform. Among the LDH isoenzymes, activity of LDH-4 was highest in all brain regions (23-40%), except the cerebellum in which LDH-1 was highest (29%). Compared to the brain, control serum contained very little CK and LDH activity, but serum had three distinct CK and five distinct LDH isoenzymes. Unlike brain regions, serum had three CK-MM subforms. Each insecticide induced characteristic alterations in brain biomarkers. AChE activity was maximally inactivated in cortex (90. 6%) with CARB, and in cerebellum (95.3%) with MPTH. With either insecticide, the least inhibition of AChE occurred in the striatum. Unlike AChE, carboxylesterase (CarbE) did not show brain regional variability in controls, and its activity was uniformly inhibited in all brain regions by CARB and comparatively greater by MPTH. CARB- or MPTH-induced characteristic alterations in CK, LDH, and their isoenzymes in the brain, which were also reflected in serum, as a result of their leakage from the brain by increased permeability due to depletion of ATP (38-57% and 33-47%, respectively) and phosphocreatine (PCr, 23-42% and 56-65%, respectively).

Role of high-energy phosphates and their metabolites in protection of carbofuran-induced biochemical changes in diaphragm muscle by memantine.

A combined antidotal treatment with memantine HCI (MEM, 18 mg/kg, s.c.) and atropine sulfate (ATS, 16 mg/kg, s.c.) provided complete protection against acute carbofuran toxicity (1.5 mg/kg, s.c.) in rats by multiple mechanisms. Carbofuran, in addition to inhibiting serine-containing esterases, also perturbed the activities of mitochondrial/cytoplasmic biomarker enzymes (creatine kinase, CK; and lactic dehydrogenase, LDH) in diaphragm muscle. The observed changes in the activity of biomarker enzymes were reflected in serum as a result of their leakage from the diaphragm due to a depletion of high-energy phosphates, such as adenosine triphosphate (ATP, 27%) and phosphocreatine (PCr, 33%) and their metabolites (ADP, 36%; AMP, 35%; and Cr, 38%). Combined treatment with MEM and ATS provided protection and reversal of the induced changes in biomarkers by preventing depletion of high-energy phosphates and thus maintaining normal cell membrane characteristics, including permeability and integrity. These results, along with those reported previously, indicate that MEM antagonizes carbofuran toxicity by multiple mechanisms.

Cholinergic and noncholinergic changes in skeletal muscles by carbofuran and methyl parathion.

The objective of this investigation was to determine the distribution of cholinergic (acetyl-cholinesterase, AChE) and noncholinergic markers in slow-, fast-, and mixed-fiber containing muscles (soleus, SOL; extensor digitorum longus, EDL; and diaphragm, DIA, respectively). Noncholinergic markers included high-energy phosphates (adenosine triphosphate, ATP; phosphocreatine, PCr; and their metabolites), and the activity of creatine kinase (CK) and lactate dehydrogenase (LDH) and their isoenzymes and subforms. All three types of muscles had only one CK isoenzyme, CK-MM, which totally consisted of MM3 subform. Levels of these determinants were highest in EDL followed by DIA and least in SOL. Another objective was to determine alterations of these markers under the influence of acute carbofuran (1.5 mg/kg) or methyl parathion (MPTH, 5 mg/kg) toxicity. Rats receiving either insecticide showed cholinergic signs with maximal severity including muscle fasciculations and convulsions within 15-30 min that lasted for about 2 h. At 1 h postinsecticide injection, when AChE was maximally inhibited (81-96%), significant depletion of ATP and PCr was evident in muscles (DIA > SOL > EDL), and activities of CK-MM and LDH were elevated in muscles and consequently in serum. Serum CK-MM3 activity was markedly reduced with sequential increase in MM2 and MM1 subforms, probably due to induced higher carboxypeptidase activity. These findings suggested that (1) the differences in levels of biochemical constituents in muscles depend upon the fiber type, (2) anticholinesterase insecticide-induced increased muscle activity produces characteristic changes in CK and LDH isoenzymes patterns, and (3) leakage of these enzymes/isoenzymes into serum is due to depletion of ATP and PCr, which are required to maintain the cell membrane permeability.

In vivo acute effects of carbofuran on protein, lipid, and lipoproteins in rat liver and serum.

The objective of this investigation was to determine the changes in proteins, lipids, and lipoproteins in liver and serum of rats acutely intoxicated with carbofuran (1.5 mg/kg sc). Under the influence of carbofuran acute intoxication, analysis of globulin fractions revealed remarkable changes: In liver, the levels of alpha-2, alpha-3, and gamma were significantly elevated while alpha-1 was reduced; in serum, alpha-1 and alpha-3 fractions were elevated while alpha-2, beta, and gamma remained unchanged. A transient increase in total protein and albumin was noted only in liver. Carbofuran produced significant increases in triglycerides and cholesterol in liver that were also seen in serum. In both the liver and serum the levels of low-density-lipoprotein cholesterol (LDL-C) were reduced while the values of very-low-density-lipoprotein cholesterol (VLDL-C) were elevated. The concentration of high-density-lipoprotein cholesterol (HDL-C) was drastically reduced in liver (23% of control) with a proportional rise in serum (176%). In liver, carbofuran caused marked depletion of adenosine triphosphate (ATP) and phosphocreatine (PCr) (38% and 22% of controls, respectively), resulting in increased cell membrane permeability, thereby allowing leakage of cell constituents. It was concluded that carbofuran, directly or indirectly, produced perturbations in lipoprotein metabolism.

Energy related metabolic alterations in diaphragm muscle resulting from acute methomyl toxicity.

Male Sprague-Dawley rats receiving an acute dose of methomyl (5 mg/Kg, sc) developed overt signs of toxicity within 2 min. The maximum severity, including muscle fasciculations and convulsions, was attained within 7-10 min and lasted for about 30 min. A very rapid recovery followed and by 90 min rats were free from obvious toxicity. During intoxication, the body temperature was significantly below normal. In diaphragm, when the activity of acetylcholinesterase (AChE) was markedly depressed (82%), the levels of high-energy phosphates, adenosine triphosphate (ATP) and phosphocreatine (PCr) were also significantly lowered (27% and 54%, respectively). Significant decreases in the levels of adenosine diphosphate (ADP, 19%), total adenine nucleotides (TAN, 27%), creatine (Cr, 27%), and total creatine compounds (TCrC, 29%) were noted at various intervals. The ratio of PCr/Cr was reduced by 53%. The adenylate energy charge [(ATP + 1/2 ADP)/(ATP + ADP + AMP)], an indicator of high-energy phosphate bond availability, remained unchanged throughout the time course. More than twofold elevation in the activity of Mg(2+)-facilitated creatine kinase (reverse Lohmann reaction) in diaphragm (CK-MM) and more than twofold increase in the levels of glucose in serum, were suggestive of greater synthesis of ATP. Higher activity of CK-MM was also noted in the serum. That high-energy phosphates were partially depleted suggested that the rate of ATP utilization was far greater than its synthesis. Methomyl intoxication also resulted in higher activity of LDH and its isoenzymes in muscle as a result of induced greater synthesis. Elevation of CK and LDH and their isoenzymes in the serum was probably a result of their leakage from the tissues due to loss of membrane permeability caused by significant depletion of ATP and PCr.

In vivo alterations in lactate dehydrogenase (LDH) and LDH isoenzymes patterns by acute carbofuran intoxication.

Intoxication with an acute dose of carbofuran (1.5 mg/kg, sc) in male Sprague-Dawley rats evoked severe toxic manifestations characteristic of hypercholinergic preponderance with profound muscle fasciculations and convulsions during 30-60 min, lasting for about 2 h. Lactate dehydrogenase, a "biomarker" cytoplasmic enzyme catalyzing the reversible reaction of lactate-pyruvate, was represented by five electrophoretically distinct isoenzymes in the serum and tissues. The amounts of each isoenzyme in different tissues were widely varied and consequently the patterns were tissue specific. A 24-h time-course following carbofuran administration indicated a two-fold increase in the activity of total LDH in serum and more than 30% in hemidiaphragm and liver. The patterns of LDH isoenzymes in serum revealed a significant (P less than 0.01) increase in all the isoenzymes except LDH-4 (64% decrease). Analysis of each tissue revealed characteristic changes in LDH isoenzyme patterns indicating organ-specific tissue damage. These alterations in LDH and its isoenzymes, in addition to acetylcholinesterase inhibition, may be directly or indirectly related to the mechanism(s) of the toxic action, and also provide insight into the site/organ(s) of toxic injury, thus providing an early prognostic indicator.

Carbofuran-induced alterations (in vivo) in high-energy phosphates, creatine kinase (CK) and CK isoenzymes.

Male Sprague-Dawley rats administered with an acute sublethal dose of carbofuran (1.5 mg/kg, s.c.) developed the signs of peak hypercholinergic activity during 30-60 min. At this time, in hemidiaphragm muscle, a significant decrease in ATP (28%) and phosphocreatine (PC) (29%) occurred without concurrent change in AMP and creatine (CR). A significant decrease in the levels of total adenine nucleotides (ATP + ADP + AMP) (20%) and total creatine compounds (PC + CR) (17%) was evident. The decline in the corresponding ratios of ATP/ADP (26%), ATP/AMP (39%), and PC/CR (20%) was therefore suggestive of greater utilization of ATP and PC in response to their increased demand for high-frequency muscle fasciculations. The energy charge = ATP + 1/2 ADP/(ATP + ADP + AMP), an index of high-energy phosphate adequacy in hemidiaphragm, remained unchanged. A significant (p less than 0.01) increase in serum magnesium with no concurrent change in calcium was also evident. The observed higher activity (152%) of total CK (EC 2.7.3.2) in the serum induced by carbofuran was possibly a reflection of more than a twofold increase in CK-BB isoenzyme (CK-1) and 141% increase in CK-MM isoenzyme (CK-3), which also strengthens our findings of enhanced synthesis of ATP and PC. Increased levels of CK-MM isoenzyme in the brain (253%) and hemidiaphragm (195%); and depletion of CK-BB isoenzyme in the hemidiaphragm (0%), heart (42%), and brain (77%), and of CK-MB isoenzyme (CK-2) in the brain (4%) and hemidiaphragm (14%), appeared to be the major contributory factors leading to enhanced serum CK activity.