Indomethacin microspheres based on a combination of ethylcellulose and polyethyleneglycol were pr... more Indomethacin microspheres based on a combination of ethylcellulose and polyethyleneglycol were prepared using the solvent evaporation process. Release profiles of ensemble and individual microspheres were measured. Both were found to follow first-order kinetics, in contrast to what was expected. This was attributed to the fact that all the particles showed the same kinetic pattern and had a greater degree of
Contrary to human, porcine mucosa of the inner side of the lip is parakeratinized. Thus, although... more Contrary to human, porcine mucosa of the inner side of the lip is parakeratinized. Thus, although desirable due to its large surface area, it does not closely resemble human buccal mucosa to be considered a suitable model for systemic drug delivery research. Nevertheless, it can be utilized for comparative screening of topical or systemic delivery of bioactive agents, mostly lipophilic such as cannabinoids.
American Journal of Physiology - Endocrinology And Metabolism, 2017
Insulin-dependent type-1 diabetes (T1D) is driven by autoimmune β-cell failure, whereas systemic ... more Insulin-dependent type-1 diabetes (T1D) is driven by autoimmune β-cell failure, whereas systemic resistance to insulin is considered the hallmark of insulin-independent type-2 diabetes (T2D). In contrast to this canonical dichotomy, insulin resistance appears to precede the overt diabetic stage of T1D and predict its progression, implying that insulin sensitizers may change the course of T1D. However, previous attempts to ameliorate T1D in animal models or patients by insulin sensitizers have largely failed. Sensitization to insulin by MEthyl-substituted long-chain DICArboxylic acid (MEDICA) analogs in T2D animal models surpasses that of current insulin sensitizers, thus prompting our interest in probing MEDICA in the T1D context. MEDICA efficacy in modulating the course of T1D was verified in streptozotocin (STZ) diabetic rats and autoimmune nonobese diabetic (NOD) mice. MEDICA treatment normalizes overt diabetes in STZ diabetic rats when added on to subtherapeutic insulin, and pre...
Peptides are promising candidates as therapeutic agents due to their wide involvement in physiolo... more Peptides are promising candidates as therapeutic agents due to their wide involvement in physiological processes. However, their often non-selective activity and their poor drug-like properties, mainly their inherent low stability to enzymatic degradation and poor oral bioavailability, limit their clinical potential. Somatostatin is a peptide hormone involved in many different biological functions. The role of its five different receptor subtypes and their interplay in medicinal processes is only partially understood. In addition, it suffers from poor drug-like properties. We review several promising chemical modifications, including head-to-tail and backbone cyclization as well as N-methylation, which were applied throughout the years in the development of various somatostatin analogs. These modifications led to enhanced metabolic stability and intestinal permeability. In addition, several analogs exhibited specific receptor subtype activation. The results presented in this review suggest a potential use of these chemical modifications in order to achieve required characteristics for a bioactive peptide, mainly for clinical usage.
JS403, Carbamoylphosphonate molecule, is a promising drug candidate with anti-metastatic activity... more JS403, Carbamoylphosphonate molecule, is a promising drug candidate with anti-metastatic activity. The polarity and water solubility of JS403 evolve from the phosphonate moiety that is ionized at physiologic pH. JS403 is regarded as a BCS class III molecule, and exhibit poor absolute oral bioavailability of less than 1%. The aim of this investigation is to identify proper chitosan based absorption enhancer compound(s) that upon oral co-administration with JS403 which would enhance its bioavailability. To determine the optimal properties of the chitosan derivative (CD), 11 relevant compounds were synthesized, each with different attribute, and their impact on the intestinal permeability of JS403 was examined in-vitro using the CaCO2 monolayer model. Then, the oral bioavailability of JS403 coadministered with the selected CD was examined in the freely moving rat model. The in-vitro study identified two leading trimethyl CD, KC13, (85% deacetylation, MW 20,000 g/mol and 66% trimethylat...
In the current work, prolonged systemic delivery of apomorphine via buccal mucosa was shown to be... more In the current work, prolonged systemic delivery of apomorphine via buccal mucosa was shown to be a promising treatment for Parkinson`s disease as a substitute for clinically utilized subcutaneous infusions. Due to extensive 'first-pass' metabolism, apomorphine is administered parenterally to bypass liver metabolism. Drawbacks of parenteral administration cause low patient compliance and adherence to treatment. On the other hand, while also bypassing the liver, delivery through buccal mucosa has a superior safety profile, is less costly, lacks pain and discomfort, and possesses excellent accessibility, overall augmenting patient compliance. Current in vivo study in pigs showed: (1) steady plateau levels of apomorphine in plasma were obtained 30 min following administration and remained constant for eight hours until a delivery device was removed, (2) bioavailability of apomorphine was 55%-80% as opposed to <2% peroral and (3) simulation of the pharmacokinetic profile obtained in pigs predicted therapeutically relevant levels of apomorphine in human. Furthermore, antipyrine was incorporated as a permeation marker to enable mechanistic investigation of apomorphine release from the delivery device and its permeation through the buccal mucosa. In addition, limitations of an Ussing diffusion chamber as an ex vivo research tool were also discussed.
ABSTRACT Introduction: Sativex® spray is clinically utilized to deliver delta9-tetrahydrocannabin... more ABSTRACT Introduction: Sativex® spray is clinically utilized to deliver delta9-tetrahydrocannabinol and cannabidiol to oral mucosa for systemic absorption. We challenge the consensus that the mechanism of absorption following the oro-mucosal application occurs via the buccal tissue. Areas covered: Correctness of the consensus of this absorption pathway arose when reviewing publications regarding the influence fed versus fasting states have on pharmacokinetics of these cannabinoids administered to the oral mucosa. This finding is more suitable for peroral administration, where stomach content affects the absorption profile. We hypothesize that these cannabinoids are ingested and absorbed in the gastrointestinal tract. Expert opinion: Although clinical importance of Sativex® is not disputed, the wide acceptance of its being a successful example of drug delivery through oral mucosa is questionable. Sativex® acts as an example for other drugs delivered to oral mucosa for systemic absorption and unintentionally washed by the saliva flow into the gastrointestinal tract. Delivery of each medicine through oral mucosa should be validated in-vivo to ensure this route to be the predominant one. Revealing the underlying absorption mechanisms would enable predicting the impact of different physiological parameters such as saliva flow and fed/fasting states on the pharmacokinetics of the delivered medication.
Among numerous reported biochemical effects the lithium-inhibitable enzyme inositol-monophosphata... more Among numerous reported biochemical effects the lithium-inhibitable enzyme inositol-monophosphatase (IMPase) remains a viable target for lithium's therapeutic mechanism of action. Calbindin-D28k (calbindin) interacts with IMPase enhancing its activity. In the present study in silico modeling of IMPase-calbindin binding using the program MolFit indicated that the 55-66 amino acid segment of IMPase anchors calbindin via Lys59 and Lys61 with a glutamate in between (Lys-Glu-Lys motif). The model further suggested that the Lys-Glu-Lys motif interacts with residues Asp24 and Asp26 of calbindin. Indeed, we found that differently from wildtype calbindin, IMPase was not activated by mutated calbindin in which Asp24 and Asp26 were replaced by alanine. Calbindin's effect was significantly reduced by a peptide with the sequence of amino acids 58-63 of IMPase (peptide 1) and by six amino-acid peptides including at least part of the Lys-Glu-Lys motif. The three amino-acid peptide Lys-Glu-...
American Journal of Physiology-Heart and Circulatory Physiology
A new approach to assess autonomic nervous system (ANS) activity and its response to drug action ... more A new approach to assess autonomic nervous system (ANS) activity and its response to drug action is presented. Our approach is based on the use of a cumulative plot of data obtained by power spectral analysis of heart rate variability, in defined frequency bands, during short time epochs (e.g., 2 min in rats). The substantial temporal variability in power evolving from the constant balancing nature of the ANS activity is minimized by this approach and produces a measurable index of ANS activity vs. time. The cumulative plot emphasizes the temporal response pattern of different components of the ANS and thereby facilitates the investigation of the kinetics of action of drugs affecting the ANS. We used this method to measure the activity of cholinergic drugs in freely moving Sabra rats. Bolus atropine doses between 0.5 and 2 mg/kg produced a similar magnitude of effect, reduction of the ascending slope by 0.003 power units/h, whereas the duration of this effect was dose dependent. A l...
Hydrophilic peptides constitute most of the active peptides. They mostly permeate via tight junct... more Hydrophilic peptides constitute most of the active peptides. They mostly permeate via tight junctions (paracellular pathway) in the intestine. This permeability mechanism restricts the magnitude of their oral absorption and bioavailability. We hypothesized that concealing the hydrophilic residues of the peptide using the lipophilic prodrug charge masking approach (LPCM) can improve the bioavailability of hydrophilic peptides. To test this hypothesis, a cyclic N-methylated hexapeptide containing Arg-Gly-Asp (RGD) and its prodrug derivatives, masking the Arg and Asp charged side chains, were synthesized. The library was evaluated for intestinal permeability in vitro using the Caco-2 model. Further investigation of metabolic stability ex vivo models in rat plasma, brush border membrane vesicles (BBMVs), and isolated CYP3A4 microsomes and pharmacokinetic studies was performed on a selected peptide and its prodrug (peptide 12). The parent drug analogues were found to have a low permeability rate in vitro, corresponding to atenolol, a marker for paracellular permeability. Moreover, palmitoyl carnitine increased the Papp of peptide 12 by 4-fold, indicating paracellular permeability. The Papp of the prodrug derivatives was much higher than that of their parent peptides. For instance, the Papp of the prodrug 12P was 20-fold higher than the Papp of peptide 12 in the apical to basolateral (AB) direction. Whereas the permeability in the opposite direction (BA of the Caco-2 model) was significantly faster than the Papp AB, indicating the involvement of an efflux system. These results were corroborated when verapamil, a P-gp inhibitor, was added to the Caco-2 model and increased the Papp AB of prodrug 12P by 3-fold. The prodrug 12P was stable in the BBMVs environment, yet degraded quickly (less than 5 min) in the plasma into the parent peptide 12. Pharmacokinetic studies in rats showed an increase in the bioavailability of peptide 12 > 70-fold (from 0.58 ± 0.11% to 43.8 ± 14.9%) after applying the LPCM method to peptide 12 and converting it to the prodrug 12P. To conclude, the LPCM approach converted the absorption mechanism of the polar peptides from a paracellular to transcellular pathway that tremendously affects their oral bioavailability. The LPCM method provides a solution for the poor bioavailability of RGD cyclohexapeptides and paves the way for other active hydrophilic and charged peptides with poor oral bioavailability.
Angewandte Chemie (International ed. in English), Jan 18, 2017
A highly systematic approach for the development of both orally bioavailable and bioactive cyclic... more A highly systematic approach for the development of both orally bioavailable and bioactive cyclic N-methylated hexapeptides as high affinity ligands for the integrin αvβ3 is based on two concepts: a) screening of systematically designed libraries with spatial diversity and b) masking of the peptide charge with a lipophilic protecting group. The key steps of the method are 1) initial design of a combinatorial library of N-methylated analogues of the stem peptide cyclo(d-Ala-Ala5 ); 2) selection of cyclic peptides with the highest intestinal permeability; 3) design of sublibraries with the bioactive RGD sequence in all possible positions; 4) selection of the best ligands for RGD-recognizing integrin subtypes; 5) fine-tuning of the affinity and selectivity by additional Ala to Xaa substitutions; 6) protection of the charged functional groups according to the prodrug concept to regain intestinal and oral permeability; 7) proof of biological effects in mice after oral administration.
Indomethacin microspheres based on a combination of ethylcellulose and polyethyleneglycol were pr... more Indomethacin microspheres based on a combination of ethylcellulose and polyethyleneglycol were prepared using the solvent evaporation process. Release profiles of ensemble and individual microspheres were measured. Both were found to follow first-order kinetics, in contrast to what was expected. This was attributed to the fact that all the particles showed the same kinetic pattern and had a greater degree of
Contrary to human, porcine mucosa of the inner side of the lip is parakeratinized. Thus, although... more Contrary to human, porcine mucosa of the inner side of the lip is parakeratinized. Thus, although desirable due to its large surface area, it does not closely resemble human buccal mucosa to be considered a suitable model for systemic drug delivery research. Nevertheless, it can be utilized for comparative screening of topical or systemic delivery of bioactive agents, mostly lipophilic such as cannabinoids.
American Journal of Physiology - Endocrinology And Metabolism, 2017
Insulin-dependent type-1 diabetes (T1D) is driven by autoimmune β-cell failure, whereas systemic ... more Insulin-dependent type-1 diabetes (T1D) is driven by autoimmune β-cell failure, whereas systemic resistance to insulin is considered the hallmark of insulin-independent type-2 diabetes (T2D). In contrast to this canonical dichotomy, insulin resistance appears to precede the overt diabetic stage of T1D and predict its progression, implying that insulin sensitizers may change the course of T1D. However, previous attempts to ameliorate T1D in animal models or patients by insulin sensitizers have largely failed. Sensitization to insulin by MEthyl-substituted long-chain DICArboxylic acid (MEDICA) analogs in T2D animal models surpasses that of current insulin sensitizers, thus prompting our interest in probing MEDICA in the T1D context. MEDICA efficacy in modulating the course of T1D was verified in streptozotocin (STZ) diabetic rats and autoimmune nonobese diabetic (NOD) mice. MEDICA treatment normalizes overt diabetes in STZ diabetic rats when added on to subtherapeutic insulin, and pre...
Peptides are promising candidates as therapeutic agents due to their wide involvement in physiolo... more Peptides are promising candidates as therapeutic agents due to their wide involvement in physiological processes. However, their often non-selective activity and their poor drug-like properties, mainly their inherent low stability to enzymatic degradation and poor oral bioavailability, limit their clinical potential. Somatostatin is a peptide hormone involved in many different biological functions. The role of its five different receptor subtypes and their interplay in medicinal processes is only partially understood. In addition, it suffers from poor drug-like properties. We review several promising chemical modifications, including head-to-tail and backbone cyclization as well as N-methylation, which were applied throughout the years in the development of various somatostatin analogs. These modifications led to enhanced metabolic stability and intestinal permeability. In addition, several analogs exhibited specific receptor subtype activation. The results presented in this review suggest a potential use of these chemical modifications in order to achieve required characteristics for a bioactive peptide, mainly for clinical usage.
JS403, Carbamoylphosphonate molecule, is a promising drug candidate with anti-metastatic activity... more JS403, Carbamoylphosphonate molecule, is a promising drug candidate with anti-metastatic activity. The polarity and water solubility of JS403 evolve from the phosphonate moiety that is ionized at physiologic pH. JS403 is regarded as a BCS class III molecule, and exhibit poor absolute oral bioavailability of less than 1%. The aim of this investigation is to identify proper chitosan based absorption enhancer compound(s) that upon oral co-administration with JS403 which would enhance its bioavailability. To determine the optimal properties of the chitosan derivative (CD), 11 relevant compounds were synthesized, each with different attribute, and their impact on the intestinal permeability of JS403 was examined in-vitro using the CaCO2 monolayer model. Then, the oral bioavailability of JS403 coadministered with the selected CD was examined in the freely moving rat model. The in-vitro study identified two leading trimethyl CD, KC13, (85% deacetylation, MW 20,000 g/mol and 66% trimethylat...
In the current work, prolonged systemic delivery of apomorphine via buccal mucosa was shown to be... more In the current work, prolonged systemic delivery of apomorphine via buccal mucosa was shown to be a promising treatment for Parkinson`s disease as a substitute for clinically utilized subcutaneous infusions. Due to extensive 'first-pass' metabolism, apomorphine is administered parenterally to bypass liver metabolism. Drawbacks of parenteral administration cause low patient compliance and adherence to treatment. On the other hand, while also bypassing the liver, delivery through buccal mucosa has a superior safety profile, is less costly, lacks pain and discomfort, and possesses excellent accessibility, overall augmenting patient compliance. Current in vivo study in pigs showed: (1) steady plateau levels of apomorphine in plasma were obtained 30 min following administration and remained constant for eight hours until a delivery device was removed, (2) bioavailability of apomorphine was 55%-80% as opposed to <2% peroral and (3) simulation of the pharmacokinetic profile obtained in pigs predicted therapeutically relevant levels of apomorphine in human. Furthermore, antipyrine was incorporated as a permeation marker to enable mechanistic investigation of apomorphine release from the delivery device and its permeation through the buccal mucosa. In addition, limitations of an Ussing diffusion chamber as an ex vivo research tool were also discussed.
ABSTRACT Introduction: Sativex® spray is clinically utilized to deliver delta9-tetrahydrocannabin... more ABSTRACT Introduction: Sativex® spray is clinically utilized to deliver delta9-tetrahydrocannabinol and cannabidiol to oral mucosa for systemic absorption. We challenge the consensus that the mechanism of absorption following the oro-mucosal application occurs via the buccal tissue. Areas covered: Correctness of the consensus of this absorption pathway arose when reviewing publications regarding the influence fed versus fasting states have on pharmacokinetics of these cannabinoids administered to the oral mucosa. This finding is more suitable for peroral administration, where stomach content affects the absorption profile. We hypothesize that these cannabinoids are ingested and absorbed in the gastrointestinal tract. Expert opinion: Although clinical importance of Sativex® is not disputed, the wide acceptance of its being a successful example of drug delivery through oral mucosa is questionable. Sativex® acts as an example for other drugs delivered to oral mucosa for systemic absorption and unintentionally washed by the saliva flow into the gastrointestinal tract. Delivery of each medicine through oral mucosa should be validated in-vivo to ensure this route to be the predominant one. Revealing the underlying absorption mechanisms would enable predicting the impact of different physiological parameters such as saliva flow and fed/fasting states on the pharmacokinetics of the delivered medication.
Among numerous reported biochemical effects the lithium-inhibitable enzyme inositol-monophosphata... more Among numerous reported biochemical effects the lithium-inhibitable enzyme inositol-monophosphatase (IMPase) remains a viable target for lithium's therapeutic mechanism of action. Calbindin-D28k (calbindin) interacts with IMPase enhancing its activity. In the present study in silico modeling of IMPase-calbindin binding using the program MolFit indicated that the 55-66 amino acid segment of IMPase anchors calbindin via Lys59 and Lys61 with a glutamate in between (Lys-Glu-Lys motif). The model further suggested that the Lys-Glu-Lys motif interacts with residues Asp24 and Asp26 of calbindin. Indeed, we found that differently from wildtype calbindin, IMPase was not activated by mutated calbindin in which Asp24 and Asp26 were replaced by alanine. Calbindin's effect was significantly reduced by a peptide with the sequence of amino acids 58-63 of IMPase (peptide 1) and by six amino-acid peptides including at least part of the Lys-Glu-Lys motif. The three amino-acid peptide Lys-Glu-...
American Journal of Physiology-Heart and Circulatory Physiology
A new approach to assess autonomic nervous system (ANS) activity and its response to drug action ... more A new approach to assess autonomic nervous system (ANS) activity and its response to drug action is presented. Our approach is based on the use of a cumulative plot of data obtained by power spectral analysis of heart rate variability, in defined frequency bands, during short time epochs (e.g., 2 min in rats). The substantial temporal variability in power evolving from the constant balancing nature of the ANS activity is minimized by this approach and produces a measurable index of ANS activity vs. time. The cumulative plot emphasizes the temporal response pattern of different components of the ANS and thereby facilitates the investigation of the kinetics of action of drugs affecting the ANS. We used this method to measure the activity of cholinergic drugs in freely moving Sabra rats. Bolus atropine doses between 0.5 and 2 mg/kg produced a similar magnitude of effect, reduction of the ascending slope by 0.003 power units/h, whereas the duration of this effect was dose dependent. A l...
Hydrophilic peptides constitute most of the active peptides. They mostly permeate via tight junct... more Hydrophilic peptides constitute most of the active peptides. They mostly permeate via tight junctions (paracellular pathway) in the intestine. This permeability mechanism restricts the magnitude of their oral absorption and bioavailability. We hypothesized that concealing the hydrophilic residues of the peptide using the lipophilic prodrug charge masking approach (LPCM) can improve the bioavailability of hydrophilic peptides. To test this hypothesis, a cyclic N-methylated hexapeptide containing Arg-Gly-Asp (RGD) and its prodrug derivatives, masking the Arg and Asp charged side chains, were synthesized. The library was evaluated for intestinal permeability in vitro using the Caco-2 model. Further investigation of metabolic stability ex vivo models in rat plasma, brush border membrane vesicles (BBMVs), and isolated CYP3A4 microsomes and pharmacokinetic studies was performed on a selected peptide and its prodrug (peptide 12). The parent drug analogues were found to have a low permeability rate in vitro, corresponding to atenolol, a marker for paracellular permeability. Moreover, palmitoyl carnitine increased the Papp of peptide 12 by 4-fold, indicating paracellular permeability. The Papp of the prodrug derivatives was much higher than that of their parent peptides. For instance, the Papp of the prodrug 12P was 20-fold higher than the Papp of peptide 12 in the apical to basolateral (AB) direction. Whereas the permeability in the opposite direction (BA of the Caco-2 model) was significantly faster than the Papp AB, indicating the involvement of an efflux system. These results were corroborated when verapamil, a P-gp inhibitor, was added to the Caco-2 model and increased the Papp AB of prodrug 12P by 3-fold. The prodrug 12P was stable in the BBMVs environment, yet degraded quickly (less than 5 min) in the plasma into the parent peptide 12. Pharmacokinetic studies in rats showed an increase in the bioavailability of peptide 12 > 70-fold (from 0.58 ± 0.11% to 43.8 ± 14.9%) after applying the LPCM method to peptide 12 and converting it to the prodrug 12P. To conclude, the LPCM approach converted the absorption mechanism of the polar peptides from a paracellular to transcellular pathway that tremendously affects their oral bioavailability. The LPCM method provides a solution for the poor bioavailability of RGD cyclohexapeptides and paves the way for other active hydrophilic and charged peptides with poor oral bioavailability.
Angewandte Chemie (International ed. in English), Jan 18, 2017
A highly systematic approach for the development of both orally bioavailable and bioactive cyclic... more A highly systematic approach for the development of both orally bioavailable and bioactive cyclic N-methylated hexapeptides as high affinity ligands for the integrin αvβ3 is based on two concepts: a) screening of systematically designed libraries with spatial diversity and b) masking of the peptide charge with a lipophilic protecting group. The key steps of the method are 1) initial design of a combinatorial library of N-methylated analogues of the stem peptide cyclo(d-Ala-Ala5 ); 2) selection of cyclic peptides with the highest intestinal permeability; 3) design of sublibraries with the bioactive RGD sequence in all possible positions; 4) selection of the best ligands for RGD-recognizing integrin subtypes; 5) fine-tuning of the affinity and selectivity by additional Ala to Xaa substitutions; 6) protection of the charged functional groups according to the prodrug concept to regain intestinal and oral permeability; 7) proof of biological effects in mice after oral administration.
Uploads
Papers by Amnon Hoffman