Pieter Vermeij

Skeletal retention of bisphosphonate (pamidronate) and its relation to the rate of bone resorption in patients with breast cancer and bone metastases

Bisphosphonate pharmacokinetics may affect individual responses. Skeletal retention of pamidronate infused monthly to patients with bone metastases was highly variable (12‐98%) and did not diminish with time, showing the capacity of the skeleton to retain large amounts of bisphosphonate. Relationships between skeletal retention of pamidronate and rate of bone resorption are complex and depend on previous treatment and the total amount of retained bisphosphonate.

The Effects of Nitrogen-Containing Bisphosphonates on Human Epithelial (Caco-2) Cells, an In Vitro Model for Intestinal Epithelium

Nitrogen‐containing bisphosphonates (N‐PCP) are bisphosphonates with an increased antiresorptive potency. Aminobisphosphonates, N‐PCPs with an amino group, can cause nonspecific gastrointestinal complaints. It is not known whether these side effects are specific for these bisphosphonates or for the whole class of N‐PCPs. In this study, we investigated the effects of two aminobisphosphonates (pamidronate and alendronate) and a structurally similar N‐PCP (olpadronate) and their three respective calcium complexes on the viability and the intracellular calcium concentration ([Ca2+]i) of cultured Caco‐2 cells a model for intestinal epithelium. These cells were also examined for apoptosis or necrosis. In the presence of calcium, pamidronate and alendronate were toxic to the cells, with pamidronate being more toxic than alendronate. Olpadronate induced toxicity only at concentrations more than ten times higher than the toxic concentrations of pamidronate. In the absence of calcium definite signs of toxicity were observed only with pamidronate at clinically relevant concentrations. The complexes of pamidronate and alendronate with calcium were considerably less soluble than the olpadronate calcium complex. There were no signs of apoptosis. [Ca2+]i was transiently raised after treatment with the N‐PCPs. Doses at which responses were seen were, respectively, 0.02 mM (pamidronate), 0.3 mM (alendronate), and 2 mM (olpadronate). The peak of response was slightly greater after pamidronate treatment than after alendronate or olpadronate, respectively. In conclusion pamidronate, either as an ion or as a calcium complex, is the most toxic of the bisphosphonates tested for Caco‐2 cells. Alendronate was less toxic while olpadronate was the least toxic in presence of calcium. The solubility of the bisphosphonate complexes with calcium may account for these differences in toxicity.

The value of carbamazepine in the treatment of tinnitus

A double-blind controlled trial on the use of oral carbamazepine in the treatment of tinnitus is reported. The effects of carbamazepine and intravenous lidocaine on tinnitus were compared. Carbamazepine had less effect than the placebo.

High-performance ion-exchange chromatography with in-line complexation of bisphosphonates and their quality control in pharmaceutical preparations

An ion-exchange chromatographic method using an anion-exchange column was developed for the analysis of a number of bisphosphonates. The bisphosphonates were in-line complexed by copper(II) ions and added to the acidic eluent, to yield an UV-absorbing complex. Chromatographic parameters were studied for eight different bisphosphonates, particularly amino-1-hydroxyalkyl-1,1-bisphosphonates; special attention was paid to the relationship between retention and complex formation. The method was applied to the quality control of pamidronate injection concentrate and olpadronate tablets. The lower detection limit was 8 ng of disodium pamidronate, and the inter-assay precision was 1.0% for both pamidronate and olpadronate standard solutions and 1.8% for a 3 mg ml-1 disodium pamidronate injection concentrate. The method was compared with a previously described ion-exchange chromatographic method with conductivity detection, without copper(II) ions in the eluent.

Tinnitus suppression by intravenous lidocaine in relation to its plasma concentration

In a double‐blind controlled trial in nine patients with tinnitus we measured the lidocaine plasma concentrations during and after intravenous administration of lidocaine or placebo and scored the level of tinnitus on a visual analog scale. No patient showed any effect during the placebo infusion. Administration of lidocaine resulted in total suppression or suppression to a non‐annoying level of tinnitus in five patients, slight suppression but still annoying tinnitus in two patients, and worsening tinnitus in one patient. No effect of lidocaine was observed in one patient. Most relief was obtained at plasma concentrations between 1.5 and 2.5 µg/ml. In this concentration range a significant (p < 0.05) effect of lidocaine on tinnitus was observed. However, notable side effects were observed at plasma concentrations greater than 2.0 μg/ml. The effect persisted until plasma levels of about 0.5 μg/ml were reached. A large variability in the effects existed because of variations in lidocaine kinetics and because of the presumed psychologic components of tinnitus.

Comparative Tolerability and Efficacy of Treatments for Impotence

Modern pharmacological treatment of impotence is determined by the presenting symptoms. Since this involves symptomatology with a heterogenous aetiology, many different drugs are involved in the treatment of impotence.Drugs used for libido and arousal problems include testosterone, yohimbine, trazodone and apomorphine. Since patient self-assessment is the only parameter that can be used to measure the result of treatment and positive results are seldom affirmed, no positive benefit of these agents can be assumed at present.Oral medications for erectile dysfunction include yohimbine, trazodone, apo-morphine, phentolamine, arginine and sildenafil. Of these drugs, sildenafil has been the most systematically studied for effectiveness, but long term safety data await the results of post-marketing surveillance.Of the ejaculation disorder therapies, treatments for premature ejaculation are the best studied. Favourable results have been obtained with clomipramine, paroxetine and fluoxetine. The safety of these medications has been assessed through their long term use in psychiatry.Intracavernous self-injections for erectile disorders are performed using a variety of drugs and drug mixtures. Only alprostadil and the combination of papav-erine with phentolamine are widely used. Alprostadil is very well tolerated; however, penile pain is a serious problem in a significant proportion of patients. Papaverine in combination with phentolamine is effective, but penile fibrosis and priapism occur more often than with the use of alprostadil. Several new developments in this area are currently under way.Alternative routes for medication for erectile dysfunction include ointments and patches to the penile skin and the glans. Only transurethral alprostadil, ‘MUSE’ (medicated urethral system for erection) has been shown to be effective in large trials. Long term safety still has to be demonstrated, but the 1-year safety profile is encouraging.In general, the end points of impotence treatment studies are very diverse so efficacy data can only be assessed in comparative studies. However, long term comparison studies have not been performed. Safety demands must be set very high for this type of treatment since the disorders being treated present no threat to the patient’s health.

Rapid enrichment of human papillomavirus (HPV)-specific polyclonal T cell populations for adoptive immunotherapy of cervical cancer.

The majority of cervical cancers are caused by human papillomavirus type 16 (HPV16). Cervical cancer is associated with an ineffective host immune response against the HPV16 oncoproteins, characterized by the lack of the strong E6‐specific T‐helper type 1 (Th1) immunity that is generally present in healthy individuals, the presence of improperly polarized HPV16E6‐ and E7‐specific CD4+ T cells and increased numbers of regulatory T cells. Therefore, immunotherapeutic intervention is likely to require a modality that deletes the regulatory T cell component and enhances the HPV16‐specific Type 1 T cell response. HLA‐matched allogeneic stem cell transplantation may offer such a modality, because it involves the eradication of host immune cells and enables the transfer of donor derived tumor‐specific T cells to the patient. As a first step in the development of such a treatment, we evaluated the success rate of a protocol for enrichment of HPV16E6‐specific CD4+ T cells from healthy donor PBMC on the basis of their IFNγ secretion. After a short in vitro stimulation with overlapping 30 amino acid long HPV16E6 peptides, we enriched the IFNγ secreting cells by magnetic cell sorting. The obtained polyclonal CD4+ T cell populations recognized distinct epitopes within HPV16E6, as well as E6 protein, processed and presented by autologous professional antigen presenting cells. The described protocol proved successful in PBMC from more than half of the healthy adult blood donors. These HPV16E6‐specific CD4+ T cells may turn out to be an essential component of future adoptive T cell therapy for advanced cervical cancer, by orchestrating CTL dependent and independent tumoricidal mechanisms.

Relationships between pharmacokinetics and rate of bone turnover after intravenous bisphosphonate (olpadronate) in patients with Paget's disease of bone

Bisphosphonates are the treatment of choice of Pagets disease, but variable responses have been reported, and despite the availability of potent bisphosphonates, biochemical remission is not achieved in a substantial number of patients. This may, in part at least, be because of the influence of pharmacokinetics of bisphosphonates on their pharmacodynamics. That is the response of bone turnover to treatment. To address this issue, we examined the pharmacokinetics and pharmacodynamics of the bisphosphonate olpadronate given intravenously to 75 patients with Pagets disease, using a specific assay for olpadronate concentrations in serum and urine. The skeletal uptake of olpadronate varied greatly among patients and ranged between 10% and 90% of the administered dose. The two major determinants of skeletal uptake were renal function and prevalent rate of bone turnover. Serum and urinary data were well described by a physiology‐based four‐compartment pharmacokinetic model that takes into account the distribution of the bisphosphonate in the bone and its subsequent elimination. Bone turnover was suppressed to well within the normal range in virtually all patients. This, together with the absence of resolution of effect during 1 year, does not allow the construction of an adequate integrated pharmacokinetic/pharmacodynamic model. However, the pharmacokinetic model, described for the first time in Pagets disease, can accurately simulate the amount of bisphosphonate delivered to the skeleton with different dose regimens as well as the amount still present in bone after 1 year. Such approaches can lead to improved patient care and individualization of treatment of Pagets disease with bisphosphonates.

Ion-exchange liquid chromatographic analysis of bisphosphonates in pharmaceutical preparations

A simple, fast and uniform method has been developed for the quantitative determination of bisphosphonates in the quality control of pharmaceutical preparations. The method is based on ion-exchange liquid chromatography with conductivity detection. Separation is performed on a Waters IC-PAK Anion column using 2 mM nitric acid or 25 mM succinic acid as the mobile phase. Retention of the bisphosphonates can be influenced by pH and the anion concentration of the mobile phase. Sensitivity and selectivity are sufficient for the assay of bisphosphonates in bulk drug and pharmaceutical preparations. Sample preparation comprises dissolution or dilution of the sample in the mobile phase followed, if necessary, by filtration prior to HPLC analysis. Since the method is stability indicating, it is also well suited for shelf-life studies of bisphosphonate pharmaceutical preparations. Validation of the analytical method for the assay of pamidronate injection indicated an intra-day reproducibility of 1.7% (n = 6) and an inter-day reproducibility of 2.7% (n = 6). A linear relationship between response and concentration was found in the concentration range studied from 200 ng to 10 micrograms pamidronate per 20 microliters injected. The lower limit of detection (signal-to-noise ratio = 3) of pamidronate was about 100 ng.

Semi-automatic liquid chromatographic analysis of olpadronate in urine and serum using derivatization with (9-fluorenylmethyl)chloroformate.

The semi-automatic bioanalytical assays for olpadronate [(3-dimethylamino-1-hydroxypropylidene)bisphosphonate] involves a protein precipitation with trichloroacetic acid and a double co-precipitation with calcium phosphate for serum samples and a triple calcium co-precipitation for urine samples. These manual procedures are followed by an automated solid-phase extraction on a cation-exchange phase. The procedure is continued either directly, at high olpadronate levels in urine, or after off-line evaporation under nitrogen and reconstitution in water on the same robotic workstation. The continued automatic procedure comprehends derivatization with (9-fluorenylmethyl)chloroformate, ion-pair liquid-liquid extraction and ion-pair HPLC with fluorescence detection at 274/307 nm. The intra- and inter-day precisions for urine and serum samples are typically in the 5-8% range for different olpadronate concentrations [levels near the lower limit of quantification (LLQ) excluded]. The LLQ is 5 ng/ml olpadronate for a 2.5-ml urine sample and 10 ng/ml for a 1-ml serum sample, respectively.