ALLN-177, oral enzyme therapy for hyperoxaluria.

PURPOSE: To evaluate the potential of ALLN-177, an orally administered, oxalate-specific enzyme therapy to reduce urine oxalate (UOx) excretion in patients with secondary hyperoxaluria. METHODS: Sixteen male and female subjects with both hyperoxaluria and a kidney stone history were enrolled in an open-label study. Subjects continued their usual diets and therapies. During a 3-day baseline period, two 24-h (24-h) urines were collected, followed by a 4-day treatment period with ALLN-177 (7,500 units/meal, 3 × day) when three 24-h urines were collected. The primary endpoint was the change in mean 24-h UOx from baseline. Safety assessments and 24-h dietary recalls were performed throughout. RESULTS: The study enrolled 5 subjects with enteric hyperoxaluria and 11 with idiopathic hyperoxaluria. ALLN-177 was well tolerated. Overall mean (SD) UOx decreased from 77.7 (55.9) at baseline to 63.7 (40.1) mg/24 h while on ALLN-177 therapy, with the mean reduction of 14 mg/24 h, (95% CI - 23.71, - 4.13). The calcium oxalate-relative urinary supersaturation ratio in the overall population decreased from a mean of 11.3 (5.7) to 8.8 (3.8) (- 2.8; 95% CI - 4.9, - 0.79). This difference was driven by oxalate reduction alone, but not any other urinary parameters. Mean daily dietary oxalate, calcium, and fluid intake recorded by frequent diet recall did not differ by study periods. CONCLUSION: ALLN-177 reduced 24-h UOx excretion, and was well tolerated. The results of this pilot study provided justification for further investigation of ALLN-177 in patients with secondary hyperoxaluria. TRIAL REGISTRATION: Clinicaltrials.gov NCT02289755.

A Double-Blind, Placebo Controlled, Randomized Phase 1 Cross-Over Study with ALLN-177, an Orally Administered Oxalate Degrading Enzyme.

BACKGROUND: Hyperoxaluria may result from increased endogenous production or overabsorption of dietary oxalate in the gastrointestinal tract leading to nephrolithiasis and, in some, to oxalate nephropathy and chronic kidney disease. ALLN-177 is an oral formulation of a recombinant, oxalate specific, microbial enzyme oxalate decarboxylase intended to treat secondary hyperoxaluria by degrading dietary oxalate in the gastrointestinal tract, thereby reducing its absorption and subsequent excretion in the urine. METHODS: This double-blind, placebo controlled, randomized, cross-over, phase 1 study of ALLN-177 evaluated the tolerability of ALLN-177 and its effect on urinary oxalate excretion in 30 healthy volunteers with hyperoxaluria induced by ingestion of a high oxalate, low calcium (HOLC) diet. The primary end point was the difference in the mean 24-hour urinary oxalate excretion during the ALLN-177 treatment period compared with the placebo treatment period. RESULTS: The daily urinary oxalate excretion increased in the study population from 27.2 ± 9.5 mg/day during screening to 80.8 ± 24.1 mg/day (mean ± SD) on the HOLC diet before introducing ALLN-177 or placebo therapy for 7 days. Compared to placebo, ALLN-177 treatment reduced urinary oxalate by 11.6 ± 2.7 mg/day, p = 0.0002 (least squares mean ± SD). CONCLUSIONS: In healthy volunteers, with diet-induced hyperoxaluria treatment with ALLN-177, when compared to placebo, significantly reduced urinary oxalate excretion by degrading dietary oxalate in the gastrointestinal tract and thereby reducing its absorption. ALLN-177 may represent a new approach for managing secondary hyperoxaluria and its complications.

Recombinant hexahistidine arginine decarboxylase (hisADC) induced endogenous agmatine synthesis during stress.

The arginine decarboxylase (ADC) is a significant functional enzyme, synthesizes agmatine through arginine metabolism, and agmatine was reported to posses protective properties in various tissues. This study first optimized the conditions for efficient hexahistidine tagged human ADC (hisADC) gene delivery into mouse fibroblast cell line (NIH3T3) using retroviral vector (pLXSN). Later, the functionality of the delivered hisADC gene in synthesizing agmatine during H(2)O(2) injury in NIH3T3 was also elucidated. Amplification of hisADC gene was performed using hisADC specific primers under specified conditions. The hisADC PCR product (1.4 kb) was ligated with pLXSN considering the restriction enzyme sites. The complete hisADC pLXSN clone was transfected into PT67 cell line following CalPhos Mammalian transfection method. RT-PCR and western blot results showed the specific and strong detection of hisADC genes in hisADC PT67 transfected cells compared with normal control and pLXSN transfected PT67 cells. The retrovirus containing hisADC gene (vhisADC) was infected into NIH3T3 (vhisADC NIH) using polybrene reagent. Immunocytochemical results showed hisADC expression in the cytoplasm of vhisADC NIH. HPLC analysis revealed high agmatine concentration in the vhisADC NIH, and the induced agmatine synthesized from the retroviral gene delivery prevented vhisADC NIH from H(2)O(2) injury which is evident by the decrease in lactate dehydrogenase (P < 0.05) leakage into the medium and less number of propidium iodide positive cells during injury compared to control group. The obtained results provide compelling evidence that higher level of hisADC transgene expression completely triggered the endogenous agmatine synthesis during H(2)O(2) injury thus protecting NIH3T3 cells against cytotoxicity.

14-day repeat-dose oral toxicity evaluation of oxazyme in rats and dogs.

Oxazyme (OC4) is an orally administered formulation that has as an active component a recombinant mutant form of Bacillus subtilis oxalate decarboxylase (OxDC) enzyme C383S, designed to degrade dietary oxalate in the stomach. Fourteen-day repeat-dose studies were conducted in rats and dogs to evaluate toxicity and determine a no observed adverse effect level (NOAEL). Animals were administered OC4 by oral gavage twice daily for 14 consecutive days. Reversibility, progression, and delayed appearance of any observed changes were evaluated in a subset of animals that underwent a recovery of 7 days following 14 days of control or test-article. There were no test-article-related adverse effects or deaths in either species. Results indicate that the NOAEL under the conditions used in the studies was 720.8 mg/kg/d in rats and 187.2 mg/kg/d in dogs, the high dose tested in each species.

Cross-reactivity between related sequences found in Acinetobacter sp., Pseudomonas aeruginosa, myelin basic protein and myelin oligodendrocyte glycoprotein in multiple sclerosis.

To investigate the possible role of molecular mimicry to bacterial components in multiple sclerosis (MS) pathogenesis we examined antibody responses to mimicry peptide sequences of Acinetobacter, Pseudomonas aeruginosa and myelin components. Antibodies to mimicry peptides from Acinetobacter (p<0.001), P. aeruginosa (p<0.001), myelin basic protein (MBP) (p<0.001) and myelin oligodendrocyte glycoprotein (MOG) (p<0.001) were significantly elevated in MS patients compared to controls. Antisera against MBP (residues 110-124) reacted with both Acinetobacter and Pseudomonas peptides from 4- and gamma-carboxymuconolactone decarboxylase, respectively. MOG (residues 43-57) antisera reacted with Acinetobacter peptide from 3-oxo-adipate-CoA-transferase subunit A. The role of these bacteria in MS is unclear but demonstrates that molecular mimicry is not restricted to viruses suggesting bacterial infections could play a role in MS pathogenesis. Further work is required to evaluate the relevance of these cross-reactive antibodies to the neuropathology of MS.

Fatal propionic acidemia in mice lacking propionyl-CoA carboxylase and its rescue by postnatal, liver-specific supplementation via a transgene.

Propionic acidemia (PA) is an inborn error of metabolism caused by the genetic deficiency of propionyl-CoA carboxylase (PCC). By disrupting the alpha-subunit gene of PCC, we created a mouse model of PA (PCCA(-/-)), which died in 24-36 h after birth due to accelerated ketoacidosis. A postnatal, liver-specific PCC expression via a transgene in a far lower level than that in wild-type liver, allowed PCCA(-/-) mice to survive the newborn and early infant periods, preventing a lethal fit of ketoacidosis (SAP(+)PCCA(-/-) mice). Interestingly, SAP(+)PCCA(-/-) mice, in which the transgene expression increased after the late infant period, continued to grow normally while mice harboring a persistent low level of PCC died in the late infant period due to severe ketoacidosis, clearly suggesting the requirement of increased PCC supplementation in proportion to the animal growth. Based on these results, we propose a two-step strategy to achieve an efficient PA prevention in human patients: a partial PCC supplementation in the liver during the newborn and early infant periods, followed by a larger amount of supplementation in the late infant period.

Induction of aromatic-L-amino acid decarboxylase by decarboxylase inhibitors in idiopathic parkinsonism.

We evaluated the effect of administration of L-dopa, alone or in combination with a peripheral decarboxylase inhibitor, on plasma levels of aromatic-L-amino acid decarboxylase (ALAAD). After single-dose administration of L-dopa plus benserazide (Madopar) in healthy subjects and in chronically treated patients with parkinsonism, plasma ALAAD followed for 2 to 3 hours fell, but returned to predosing levels within 90 minutes. Four groups of patients with idiopathic parkinsonism were studied during chronic treatment: Group I, no L-dopa treatment (n = 31); Group II, L-dopa alone (n = 15); Group III, L-dopa plus benserazide (n = 28); and Group IV, L-dopa plus carbidopa (Sinemet, n = 30). Plasma ALAAD 2 hours after dosing was normal in Groups I and II. ALAAD was increased threefold in Groups III and IV, suggesting induction of ALAAD by the coadministration of a peripheral decarboxylase inhibitor. In a study of 3 patients in whom L-dopa/benserazide was started, plasma ALAAD rose gradually over 3 to 4 weeks. Further detailed pharmacokinetic studies of L-dopa, dopamine, and ALAAD in plasma and cerebrospinal fluid are required to determine if the apparent ALAAD induction by a peripheral decarboxylase inhibitor may be related to the loss of clinical efficacy of combination therapy in some patients and how it is related to end-of-dose deterioration and on-off phenomena.

Treatment of idiopathic parkinsonism with L-dopa in the absence and presence of decarboxylase inhibitors: effects on plasma levels of L-dopa, dopa decarboxylase, catecholamines and 3-O-methyl-dopa.

The effect of levodopa (L-dopa), alone or in combination with a peripheral decarboxylase inhibitor (PDI), on plasma levels of aromatic-L-amino acid decarboxylase (ALAAD, = dopa decarboxylase), L-dopa, 3-O-methyl-dopa (3-OMD), dopamine (DA), noradrenaline, adrenaline and dopamine beta-hydroxylase has been studied. In healthy subjects and in patients with parkinsonism plasma ALAAD level fell after administration of L-dopa + benserazide, but returned to previous levels within 90 min. In a cross-sectional study blood was obtained, 2 h after dosing, from 104 patients with idiopathic parkinsonism, divided into four groups: no L-dopa treatment (group 1), L-dopa alone (group 2), L-dopa + benserazide (Madopar) (group 3) and L-dopa + carbidopa (Sinemet) (group 4). Plasma ALAAD, which was normal in groups 1 and 2, was increased 3-fold in groups 3 and 4, indicating that there was induction of ALAAD by the co-administration of PDI. Despite this induction of ALAAD, in groups 3 and 4, with half the daily L-dopa dose compared with group 2, plasma L-dopa and 3-OMD levels were 5 times higher, while plasma DA levels were not different. The DA/L-dopa ratio was decreased 5-fold in group 2 and 16-fold in groups 3 and 4 as compared with group 1. Neither 3-OMD levels nor 3-OMD/L-dopa ratios correlated with the occurrence of on-off fluctuations. In a longitudinal study of three patients started on Madopar treatment the induction of plasma ALAAD was found to occur gradually over 3-4 weeks. Further detailed pharmacokinetic studies in plasma and cerebrospinal fluid are required in order to elucidate whether the ALAAD induction by PDI may be related to the loss of clinical efficacy of combination therapy in some patients and how it is related to end-of-dose deterioration and on-off phenomena.

Microinjection of purified ornithine decarboxylase into Xenopus oocytes selectively stimulates ribosomal RNA synthesis.

This study has utilized stage VI oocytes of Xenopus laevis which have amplified the rDNA gene 1,000-fold to assess whether the microinjection of ornithine decarboxylase (OrnDCase) would stimulate [alpha-32P]guanosine incorporation into 45S and 18S/28S RNA selectively. The injection of purified OrnDCase into individual oocytes resulted in a greater than 2-fold increase in the incorporation of [32P]guanosine into 45S RNA and 18S/28S RNA with no increased incorporation into low molecular weight RNA. Further, an irreversible inhibitor of OrnDCase, alpha-difluoromethylornithine (CHF2-Orn), rapidly inhibited the endogenous activity of OrnDCase when added to the buffered Hepes solution bathing the oocytes and also inhibited the incorporation of [32P]guanosine into rRNA. The inhibitory effect of CHF2-Orn could not be reversed totally by addition of 10 microM putrescine to the oocytes. OrnDCase injected into oocytes in the presence of CHF2-Orn in the media did not stimulate incorporation of [32P]guanosine label into rRNA. However, when CHF2-Orn was removed from the buffered medium at the time of the injection of label and enzyme, a 3-fold increase of 32P incorporation into 18S/28S RNA occurred. Therefore, in an in vivo model in which amplified extrachromosomal rDNA gene copies are present, the microinjection of OrnDCase was capable of specifically stimulating rRNA synthesis. CHF2-Orn, a suicide enzyme inactivator of OrnDCase, was able to inhibit rRNA synthesis and, after washout, there was a more marked stimulation of rRNA synthesis than occurred after only the injection of OrnDCase alone. These data suggest further that OrnDCase is the labile protein that regulates the initiation of RNA synthesis.

Treatment of parkinsonism with l-dopa and a decarboxylase inhibitor. An electrophysiological and clinical study.

Twelve parkinsonian patients with an unsatisfactory therapeutic result on L-Dopa alone due to nausea, vomiting and involuntary movements were treated WITH L-Dopa and decarboxylase inhibitor. The daily dose reached 800mg L-Dopa and 200 mg decarboxylase inhibitor. Single doses of each of the components were also given. Electrophysiological examination of hypokinesia, tremor and rigidity, and clinical observation revealed clear evidence of rapid improvement on small doses of L-Dopa combined with decarboxylase inhibitor. Most of the improvement occurred during the 1st week before the maximal dose was reached. A single oral dose of decarboxylase inhibitor resulted in an improvement, suggesting the presence in the organism of a small AMOUNT OF L-Dopa. This work also shows the absence of liver toxicity of the drug used. Elimination of the extracerebral side effects nausea and vomiting in our opinion is a principle advantage of the compound compared to L-Dopa alone, wheras abnormal involuntary movements, which were found in all patients, remain the limiting adverse side effect.